STRUCTURE ODOUR RELATIONSHIP STUDY OF ......antiseptic, and anticancer agents; or elicit other...

STRUCTURE ODOUR RELATIONSHIP STUDY OF ACYCLIC MONOTERPENE ALCOHOLS, THEIR ACETATES AND SYNTHESIZED OXYGENATED

DERIVATIVES

STRUKTUR-GERUCH-BEZIEHUNGSSTUDIE ZU ACYCLISCHEN MONOTERPENALKOHOLEN, IHREN ACETATEN UND

SYNTHETISIERTEN OXYGENIERTEN DERIVATEN

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur Erlangung des Doktorgrades Dr. rer. nat.

Vorgelegt von

Shaimaa Awadain Elsharif

aus Ägypten

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 30.11.2017

Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer

Gutachter/in: Prof. Dr. Andrea Büttner Prof. Dr. Matthias Wüst

Acknowledgment

The completion of this thesis would not have been possible without Prof. Andrea Büttner’s

support and patience, as she guided me throughout my doctoral studies with her

unwavering enthusiasm, for which I am extremely grateful.

Many thanks go to Prof. Monika Pischetsrieder and Prof. Thomas Göen for assuming the role

of the examiners. Also, I would like to thank Dr. Ashutosh Banerjee for supporting me

especially at the beginning of my thesis work.

Moreover, I want to acknowledge the support of my colleagues in our research group at the

University of Erlangen. It was a great pleasure working with you all. Thank you for your

support, guidance and for answering my never ending questions with pleasure.

Last but not least, I would like to thank my family especially my parents; Ebtisam Saad and

Awadain Elsharif. They have shown unequivocal support from the very beginning and

throughout the process of completing this thesis as well as my sweethearts; Hazem and

Salma. No matter what I say would never suffice to express my gratitude towards you. Thank

you for your understanding, caring and for your presence in my life.

Table of Contents List of Abbreviations ....................................................................................................................... ii

Abstract .......................................................................................................................................... iv

Zusammenfassung ......................................................................................................................... vi

1 Introduction ........................................................................................................................... 1 1.1 Structure-Odour Activity Relationships ........................................................................ 1

1.2 Terpenes ......................................................................................................................... 3

1.2.1 Introduction to Terpenes ........................................................................................ 3

1.2.2 Biosynthetic Pathways Leading to Terpenes ......................................................... 4

1.3 Monoterpenes ............................................................................................................. 10

1.3.1 Definition ............................................................................................................... 10

1.3.2 Subgroups .............................................................................................................. 10

1.3.3 Occurrence in Plants ............................................................................................. 11

1.3.4 Industrial Use as Flavour, Fragrant, and Cosmetics Constituents ...................... 11

1.3.5 Pharmacological Effects ........................................................................................ 13

1.4 Acyclic Monoterpene Alcohols–The Monoterpenols ................................................ 15

1.4.1 Introduction to Monoterpenols ........................................................................... 15

1.4.2 Linalool – the Scent of Relaxation ........................................................................ 15

1.4.3 Geraniol – the Geranium Rose-Like Fragrant ...................................................... 18

1.4.4 Nerol– just an Isomer? .......................................................................................... 20

1.4.5 β-Citronellol – The Lemongrass Aroma................................................................ 22

1.5 Characterization of the Smell Properties of the Investigated Compounds .............. 24

1.5.1 High Resolution Gas Chromatography-Olfactometry ......................................... 24

1.6 References .................................................................................................................... 27

2 Aims and Outline.................................................................................................................. 37

3 Structure-Odor Relationships of Linalool, Linalyl Acetate and Their Corresponding Oxygenated Derivatives. .............................................................................................................. 39

4 Structure-Odor Relationship Study on Geraniol, Nerol and Their Synthesized Oxygenated Derivatives. ................................................................................................................................... 51

5 Influence of the Chemical Structure on the Odor Characters of β-Citronellol and Its Oxygenated Derivatives ............................................................................................................... 63

6 Metabolic Products of Linalool and Modulation of GABAA Receptors ............................. 73

7 Proceedings of the XV Weurman Flavour Research Symposium 2017: Structure-Odor Relationship Study of C-6 Unsaturated Acyclic Monoterpene Alcohols: A Comparative Approach ...................................................................................................................................... 83

ii

List of Abbreviations A. Aspergillus

AAC Acetoacetyl CoA

ADV Adenoviruses

C. Candida

CDP-ME Methylerythritolcytidyl diphosphate

CTP Cytidine triphosphate

DMAPP Dimethylallyl diphosphate

DXP 1-Deoxy-D-xylulose 5-phosphate

DXS 1-Deoxy-D-xylulose-5-phosphate synthase

FPP Farnesyl diphosphate

FPPS Farnesyl diphosphate synthase

G3P D-Glyceraldehyde 3-phosphate

GABAA γ-Aminobutanoic acid receptor A

GC-O Gas chromatography-olfactometry

GGPPS Geranyl geranyl diphosphate synthase

GPP Geranyl diphosphate

GPPS Geranyl diphosphate synthase

HMBPP 4-Hydroxy-3-methylbut-2-enyl-diphosphate

HMG-CoA 3-Hydroxy-3-methylglutaryl-CoA

IDI Isopentenyl diphosphate isomerase

IPP Isopentyl diphosphate

MEcPP 2-C-Methyl-D-erythritol-2,4-cyclodiphosphate

MEP 2-C-Methyl-D-erythritol 4-phosphate

MK Mevalonate kinase

MVA Mevalonic acid

MVD Mevalonate-5-diphosphate decarboxylase

NO Nitric oxide

PMK Phosphomevalonate kinase

S. Staphylococcus

iv

Abstract The replacement of synthetic conventional compounds by natural ingredients;

whether in medicine, food, or cosmetics; has been increasingly requested by consumers,

especially since the last decade. Terpenes in general and monoterpenes in particular are

secondary metabolites in plants, and they may be a promising natural alternative.

Monoterpenes, the main constituents of plants’ essential oils, are odorous compounds that

play a significant ecological role in plant evolution. They are primarily utilized by the flavour

and fragrance industries due to their characteristic aroma. In addition, a series of

representatives belonging to this substance class are antimicrobial, anti-inflammatory,

antiseptic, and anticancer agents; or elicit other therapeutic effects. Thereby, acyclic

monoterpene alcohols, mainly linalool, geraniol, nerol, and citronellol, are primarily in the

focus of scientific research. Besides their aromatic character and their role in aromatherapy,

they induce a series of pharmacological and physiological effects. In view of the latter, their

metabolic pathways have been previously investigated in both plants and animals. Linalool

and geraniol, for example, are metabolized giving 8-hydroxy and 8-carboxy derivatives; i.e.

undergoing oxidation at C-8. However, these metabolites have not been tested in terms of

odour or other physiological activities. Furthermore, no studies are at hand elucidating which

structural features of these substances are responsible for specific odour qualities and

potencies of these monoterpenes.

In the frame of this doctoral thesis, a comparison between chemical structure and

odour character of selected monoterpenes relating to linalool, geraniol, nerol, and β-

citronellol has been conducted, complemented by investigations on their acetate derivatives

and previously identified oxygenated metabolites. To achieve this aim, a series of oxygenated

derivatives, bearing an aldehyde, an alcohol, or an acid functional group at C-8, were

synthesized from the aforementioned terpene alcohols and acetates yielding 24 compounds,

yielding a comprehensive substance library for future elucidation of the substances’

presence in nature and evaluation of their further potential physiological properties. Within

this study, however, the focus lies on a comprehensive characterization of the compounds’

olfactory properties. Accordingly, all compounds were tested in relation to their odour

qualities and relative odour thresholds (OTs) in air, as well as potential inter-individual

variations in sensory perception for each single substance. Overall, the results show that

v

almost all investigated parent monoterpene alcohols and their acetates exhibited closely

related odour characters; ranging between citrus-like, fresh, fruity, floral-sweet, and fatty.

Amongst others, linalool was demonstrated to be the most potent monoterpene of the

group of investigated compounds, eliciting an OT of 3.2 ng/Lair. According to this study, the

presence of an OH group at C-3 in the linalool basic structure is the main contributor to its

characteristic odour quality and high potency. On the other hand, the occurrence of this OH

at C-1 in geraniol, nerol, and citronellol does not alter their odour quality but increases their

odour threshold levels, with values of 40, 60 and 10 ng/Lair, respectively. Esterification of this

OH-group to the respective acetate barely affected the odour quality, but provoked a decline

in odour potency. Substitution at C-8 of either the parent monoterpeneols or their acetates

by another OH-group retained the smell of the parent compounds but led to a dramatic

decrease in the potency. However, the smell potency was only retained when replacing the

alcoholic group at C-8 by an aldehyde or an acid group. It is worth mentioning that among

the acetate derivatives 8-oxolinalyl acetate elicits similar smell impressions as linalool, thus

exhibiting a citrus-like, fresh odour with an OT of 5.9 ng/Lair. Apart from that, further

oxidation of C-8 of linalool, geraniol, citronellol, and citronellyl acetate to their corresponding

acids led to a total odour loss.

To summarize, the aim of this thesis was to evaluate if the target acyclic

monoterpene alcohols and their acetates are the only odourous compounds in this

substance class or if related derivatives also bear the potential of eliciting interesting

olfactory effects. Finally, data of this substance library regarding smell properties was

complemented by retention index data (RI values) as well as mass spectrometric and nuclear

magnetic resonance data to aid researchers in future analytical studies when aiming at

identifying the target molecules in nature.

vi

Zusammenfassung In der Kosmetik- und Lebensmittelindustrie aber auch in der Medizin geht der Trend dahin,

dass konventionelle synthetisch hergestellte Inhaltstoffe durch natürliche ersetzt werden.

Terpene, insbesondere Monoterpene, welche zu der Gruppe der natürlich vorkommenden

sekundären Pflanzenmetabolite gehören, stellen dabei eine vielversprechende Alternative

dar. Wegen ihrer angenehmen Geruchseigenschaften werden sie seit langem in der Aroma-

und Parfumindustrie eingesetzt, zusätzlich weisen sie antimikrobielle, antiinflammatorische,

antiseptische und Krebs vorbeugende Eigenschaften auf. Von besonderer Bedeutung sind

hierbei vor allem azyklische Monoterpenalkohole wie Linalool, Geraniol, Nerol und

Citronellol. Sie werden geschätzt wegen ihres Geruchs und finden auch in der Aromatherapie

Anwendung, verfügen aber auch zusätzlich über einige positive pharmakologische und

physiologische Eigenschaften. Die Stoffwechselwege dieser Substanzen sind in Pflanzen und

Tieren intensiv erforscht. Linalool und Geraniol zum Beispiel werden zu 8-Hydroxy- und 8-

Carboxyderivaten metabolisiert. Diese Derivate wurden jedoch noch nicht systematisch

hinsichtlich ihres Geruchs oder ihrer physiologischen Aktivität untersucht. Insbesondere ist

nicht vollumfänglich geklärt, welche Strukturmerkmale für den spezifischen Geruch und die

Potenz dieser Monoterpene verantwortlich sind. Im Rahmen dieser Doktorarbeit wurde

deswegen die Beziehung zwischen der chemischen Struktur und den Geruchseigenschaften

ausgewählter Monoterpene und ihrer Acetate, sowie davon abgeleiteter Metabolite

untersucht. Dafür wurde eine Serie oxidierter Derivate von Linalool, Geraniol, Nerol und

Citronellol und deren Acetaten synthetisiert mit dem Ziel eine Substanzbibliothek für die

Untersuchung potentieller physiologischer Eigenschaften dieser Gruppe zu erstellen. Der

Fokus dieser Arbeit lag dabei auf der anschließenden Charakterisierung der

Geruchseigenschaften der synthetisierten Verbindungen.

Insgesamt wurden 24 Derivate mit einer Aldehyd-, Alkohol- oder Säurefunktion am C8-

Kohlenstoffatom synthetisiert und die Geruchsqualitäten und relativen Geruchsschwellen

dieser Substanzen in Luft bestimmt. Ein besonderes Augenmerk lag dabei zudem auf der

Erfassung möglicher interindividueller Unterschiede in der sensorischen Wahrnehmung

dieser Substanzen. Generell wurden die Gerüche der Monoterpenalkohole und deren

Acetate weitgehend ähnlich als frisch, fruchtig, blumig, süß und fettig beschrieben. Linalool

stellte sich dabei als das potenteste Monoterpen heraus mit einer Geruchsschwelle von 3,2

ng/L Luft. Dabei zeigen die Ergebnisse dieser Studie, dass die OH-Funktion am C3-

vii

Kohlenstoffatom in der Linalool-Basisstruktur für den charakteristischen Geruch und die

hohe Potenz verantwortlich ist. Die Einführung einer OH-Gruppe am C-1 Kohlenstoffatom

wie in Geraniol, Nerol oder Citronellol veränderte nicht den Geruch, führte allerdings im

Vergleich zu Linalool zu einem Anstieg der Geruchsschwellen. So betrugen die

Geruchsschwellen dieser Substanzen 40, 60 und 10 ng/L Luft. Veresterung der OH-Gruppe zu

den entsprechenden Acetaten beeinflusste kaum die Geruchsqualität, reduzierte jedoch die

Potenz der Substanzen. Substitution durch eine weitere OH-Gruppe an C-8 bei den

Monoterpenolen oder deren Acetaten führte ebenfalls zu einer starken Erhöhung der

Geruchsschwelle, während sich auch hier der Geruch der Substanzen kaum veränderte. So

wies zum Beispiel das 8-Oxolionalylacetat einen ähnlichen Geruch wie Linalool auf, jedoch

bei einer Geruchsschwelle von 5,9 ng/L Luft. Eine weitere Oxidation an C8 zu den

entsprechenden Säuren von Linalool, Geraniol, Citronellol und Citronellolacetat führte dazu,

dass diese Substanzen komplett geruchslos waren.

Zusammenfassend lässt sich sagen: Ziel dieser Doktorarbeit war herauszufinden, ob die

Monoterpenalkohole und deren Acetate die einzigen geruchsaktiven Substanzen in dieser

Substanzklasse sind oder ob auch andere Derivate, synthetisch hergestellt oder natürlich

vorkommend, über interessante Geruchseigenschaften verfügen. Diese Substanzbibliothek

wurde zum Schluss ergänzt mit den Retention Indizes sowie den Massenspektren und NMR-

Spektren der Substanzen, um zukünftig analytischen Studien in diesem Forschungsbereich zu

unterstützen.

1

1 Introduction 1.1 Structure-Odour Activity Relationships

The concept of correlating the chemical structure of a molecule to its biological activity

has been widely used in the field of chemistry, specifically medicinal and computational

chemistry. In the past few decades, several studies have been carried out regarding the

relationships between molecular structure and odour in order to design a new fragrant

compound [1].These structure-odour relationship studies (SOR), based on the odour qualities

and potencies of the compounds of interest, are, nowadays, a guidance for scientists in

understanding more about the mechanisms of human olfaction [2-4].

In the present doctoral work, a structure-odour relationship study was carried out on

acyclic monoterpene alcohols, which are extremely important in the field of aroma as they

are essential constituents in many fragrances and products of everyday use, such as food

aroma constituents, and due to the fact that they have been reported to impart various

positive pharmacological effects.

The monoterpenes of interest are linalool, geraniol, nerol, and citronellol. These

substances have in common that they are acyclic monoterpene alcohols with an alcoholic

functional group at C3 (linalool) or C1 (geraniol, nerol, citronellol). Linalool, geraniol, and

nerol share the same molecular formula (C10H18O) whereas citronellol has the molecular

formula C10H20O, being the dihydrogeraniol. Their metabolites and derivatives, many of

which have been previously reported in plants or in animals [5-7], have not been

systematically investigated with regard to their odour properties let alone other physiological

effects. A likely reason might be the lack of commercially available references for these

compounds and the limited published analytical data. Accordingly, to establish a SOR study of

these monoterpenes and their derivatives, bearing an alcohol, an aldehyde, or an acid at C8

of the parent monoterpene, it was first necessary to synthesize the required structurally

related compounds partially based on previously described methods but also employing

specifically adapted synthetic strategies. Structural confirmation of the synthesized

substances was accomplished by means of gas chromatography-mass spectrometry (GC-MS)

and nuclear magnetic resonance (1HNMR, 13CNMR). Furthermore, a comprehensive odour-

analytical database was created within this study, comprising the main smell attributes of the

respective derivatives as evaluated by an expert sensory panel together with determination

2

of their absolute odour threshold (OT) values in air using gas chromatography-olfactometry

(GC-O). Simultaneously, the retention indices (RI) on two capillary columns of different

polarities (such as DB-5 and DB-FFAP) were determined. Consequently, a comprehensive

substance library was established compiling chemo- and odour-analytical data of the

aforementioned compounds and derivatives.

Based on these findings, the impact of systematic variation of the chemical base

structure was studied regarding effects on the smell quality and/or potency of the resulting

derivatives, thereby elaborating the main requirements that are responsible for the odour of

this class of compounds.

The following sections will provide an overview on the chemical class of terpenes in

general (representing the parent class of monoterpenes) (section 1.2.1); their synthesis

(section 1.2.2); their occurrence in nature (section 1.3.3); their use as aroma compounds

(section 1.3.4); and their main pharmacological effects (section 1.3.5). These paragraphs are

complemented by a brief introduction of each acyclic monoterpene base structure

investigated in this study, thereby compiling an overview of their properties and applications

(sections 1.4.2 - 1.4.5). Finally, the concept of gas chromatography-olfactometry in odour

analysis is described (section 1.5.1). The resulting substance library comprising the analytical

database of these monoterpenes and their synthesized oxygenated derivatives is then

provided within the respective peer-reviewed publications.

3

1.2 Terpenes 1.2.1 Introduction to Terpenes

During the past few decades, intense scientific research focused on the most

abundant secondary metabolites in all living organisms, the terpenes. More than 55,000

terpenoic substances are widely distributed among different families of natural products

found in all biological kingdoms [8, 9].

In the scientific literature, the term terpene is frequently used interchangeably with

terpenoids, although they have different meanings. Whereas the term terpenes comprises

the hydrocarbons only, being composed of carbon and hydrogen, the term terpenoids refers

to terpenes containing additional functional groups, resulting in derivatives such as alcohols,

aldehydes, ketones, and acids. In the frame of this thesis, however, the term terpenes,

comprising both hydrocarbons and their functionalized derivatives, is mostly used.

Terpenes are secondary metabolites as they are commonly, not primarily, essential

for growth, development, or reproduction of any organism. However, this classification does

not expand on the broad additional effects of these secondary metabolites that keep the

ecosystem functioning [10]. These substances play important roles and may provide plants

with evolutionary advantages in relation to their distinct chemosensory properties such as

smell. Amongst others, they may exert insecticidal effects thus protecting plants and crops

against pests and pathogens [11], or may act as pollinator attractants in reproductive

processes [8].

Many terpenoids are renowned for their economic importance being widely used as

base structural moiety in the production of drugs, flavours, fragrances, pigments, and

disinfectants [12]. To mention but some examples, the monoterpene alcohol linalool, the

main essential oil constituent of rosewood, Aniba rosaeodora, is among the most frequently

used ingredients in perfume production [13]. In addition, the sesquiterpene lactone,

artemisinin, extracted from the shrub Artemisia annua, is used in the first-line treatment of

malaria. Last but not least, the tricyclic diterpenetaxol, isolated from the bark of the Pacific

yew tree, Taxus brevifolia, and its structural analogs, are potent anticancer agents [14].

4

1.2.2 Biosynthetic Pathways Leading to Terpenes

Terpenes are primarily synthesized in plants via common biosynthetic routes. In spite

of their diverse structures and functions, all terpenes are built up of isoprene units (five-

carbon atoms) following the isoprene rule [15]. According to the number of isoprene units in

their structure; which are connected through head-to-tail addition, terpenes are classified

according to their number of carbon atoms or sesquiterpenoid moieties, respectively:

monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), or

polyterpenes having up to 30,000 connected isoprene units [16, 17]. Just like terpenes,

terpenoids are likewise classified according to the number of isoprene units they are

constituted of and are further named with the suffix -oids, as in monoterpenoids (C10),

sesquiterpenoids (C15).

Thereby, isopentyl diphosphate (IPP) and its electrophilic isomer, dimethylallyl

diphosphate (DMAPP), are the universal precursors in the biosynthesis of terpenes. Starting

from these two building blocks (Figure 1), linear prenyl diphosphates are synthesized by a

group of enzymes belonging to the prenyltransferases. IPP and DMAP are condensed by the

catalytic effect of the prenyltransferase geranyl diphosphate synthase to give the C10 geranyl

diphosphate (GPP), the intermediate that can be converted to cyclic or linear end-products

representing the group of monoterpenes.

Similarly, sesquiterpenes are generated via the addition of a third isoprene unit to

GPP forming the C15 farnesyl diphosphate (FPP), the biosynthetic precursor of common

sesquiterpenes. Further polymerization of IPP and DMAP produces longer prenyl

diphosphates forming different classes of terpenes named according to the number of

contained isoprene units [18, 19].

5

Figure 1: Synthesis scheme showing different classes of terpenes adapted from Rehman, Hanif [20]

IPP and DMAPP biosynthesis is accomplished via two independent pathways: the mevalonic

acid (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway [21].

6

Although the MVA pathway was considered the universal route in the synthesis of

terpenes, it was found to be less prominent in plant secondary metabolites than the MEP

during the last decades [22]. MVA is dominant in most eukaryotes, archaea, few eubacteria

as well as the cytosol and mitochondria of plants, and generates the precursors for

sesquiterpenes (C15) and multiplied analogues such as triterpenes (C30), within the

cytoplasm [23]. On the other hand, the MEP pathway is the primary route in chloroplasts of

higher plants, cyanobacteria, eubacteria, and algae [24]. With its biosynthetic location in the

plastids, MEP leads to monoterpenes (C10), diterpenes (C20) and carotenoids (C40) [25].

1.2.2.1 Mevalonic Acid Pathway (MVA)

The MVA pathway, also known as mevalonate pathway, isoprenoid pathway, or 3-

hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase pathway, was discovered in yeasts and

animals in the 1950s [26]. It was assumed, for some time, that this pathway is responsible for

the production of both IPP and DMAP in all organisms although it is absent in most bacteria.

As shown in (Figure 2), the MVA pathway starts with the Claisen condensation of two acetyl

CoA molecules to form the acetoacetyl CoA through the catalytic action of the acetoacetyl

CoA transferase enzyme. Acetoacetyl CoA is converted, via an aldol reaction with another

acetyl CoA, to HMG-CoA by HMG synthase. In the next two reduction steps, two nicotinamide

adenine dinucleotide phosphate molecules are required to convert HMG-CoA to mevalonic

acid (MVA) with the help of HMG-CoA reductase. Subsequent phosphorylation of MVA gives

mevalonate-5-diphosphate (MVAPP) via two reactions catalyzed by mevalonic acid kinase

(MK) and phosphomevalonate kinase (PMK), respectively. Finally, IPP is produced from

decarboxylation of MVAPP by an ATP-coupled decarboxylation reaction catalyzed by

mevalonate-5-diphosphate decarboxylase (MVD). The IPP: DMAPP isomerase (IDI) then

catalyzes the interconversion between IPP and DMAPP [9, 12].

7

MVA pathway

Figure 2: Biosynthesis of IPP and DMAPP via mevalonic acid pathway (adapted from Vranová, Coman [12], Dubey, Bhalla [17]).

1.2.2.2 Methylerythritol Phosphate Pathway (MEP)

The MEP pathway, or the MVA-independent pathway, was discovered in bacteria and

the chloroplasts of green algae and higher plants by Rohmer, Arigoni, and others in the late

1990s and early 2000s [27-29]. This pathway starts with two different precursors, namely

pyruvate and D-glyceraldehyde 3-phosphate (G3P) (Figure 3). Both molecules undergo

condensation catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) yielding the 1-

deoxy-D-xylulose 5-phosphate (DXP), using thiamine pyrophosphate as a cofactor [24]. In the

next step, DXP is isomerized by DXP reducto-isomerase (DXR) to MEP. 4-Diphosphocytidyl-2-

C-methyl-D-erythritol (CDP-ME) synthase catalyzes, consequently, the coupling between MEP

and cytidine triphosphate (CTP) producing methylerythritolcytidyl diphosphate (CDP-ME). In

8

an ATP dependent reaction, CDP-ME kinase phosphorylates CDP-ME to 4-diphosphocytidyl-2-

C-methyl-D-erythritol-2-phosphate (CDP-MEP). Subsequently, the latter undergoes

cyclization to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) in a reaction catalyzed

by MEcPP synthase, releasing cytidine monophosphate (CMP). The pathway ends up by ring

opening of the cyclic pyrophosphate and the reductive dehydration of MEcPP to 4-hydroxy-3-

methylbut-2-enyl-diphosphate (HMBPP) being catalyzed by HMBPP synthase; HMBPP is

finally converted by HMBPP reductase to a mixture of IPP and DMAPP [9, 12, 22, 29].

9

MEP pathway

Figure 3: Biosynthesis of IPP and DMAPP via the methylerythritol phosphate pathway (adapted from Vranová, Coman[12], Dubey, Bhalla [17]).

10

1.3 Monoterpenes 1.3.1 Definition

Monoterpenes are secondary metabolites in plants and the main constituents of

essential oils. Thereby, they contribute to the specific smell characters of plants [30].

Monoterpenes are characterized by their lipophilicity, low-molecular weight, and volatility. In

plant tissue, they are primarily stored in the oil glands, glandular hairs, and trichomes of leafs

[31]. As mentioned in the previous section, GPP is the direct precursor in the formation of

monoterpenes comprising a series of consecutive reactions including hydrolysis, cyclizations,

and oxidoreductions.

1.3.2 Subgroups

There are two main types of monoterpenes:

acyclic (or linear) and cyclic which can be mono- or

bicyclic. Acyclic monoterpenes (Figure 4), such as cis-

α-ocimene and β-myrcene are 2,6-dimethyloctane

derivatives. Typical monocyclic monoterpenes, as

limonene and cymene (Figure 5), are, in principle,

cyclohexane derivatives with an isopropyl substituent,

commonly containing variable double bond moieties.

α-Pinene and β-pinene are, on the other hand, the

common types of bicyclic monoterpenes .(Figure 7).

Oxidation or rearrangement of the

monoterpene structure is a biochemical modification

that produces the so-called monoterpenoids. They may

possess additional functional groups leading to

substances with aldehydic, ketonic, ester, or alcohol

base structures [16, 32].

Figure 4: Acyclic monoterpenes

Figure 5: Monocyclic monoterpenes

11

1.3.3 Occurrence in Plants

Monoterpenes, as most secondary metabolites, are crucial for the evolution of plants.

Amongst others, they play an ecological role in plants in their defense against herbivores,

bacteria, and fungi via monoterpene emission which attract, in return, the natural enemies of

these herbivores [33]. In addition, they mediate interactions between plants and their

environment when acting as pollinator attractants or allelopathic agents [32, 34].

In aromatic plants, isoprenes and especially monoterpenes, may provide a thermo-

tolerance to their plants. Thus, plants secreting these terpenes can better tolerate short high-

temperature periods of sunlight than non-terpene-emitting plants. Thereby, those emitting

plants maintain high rates of photosynthesis compared to the non-emitting ones [20].

Volatilized monoterpenes can further protect plants against oxidative damage due to their

capability to react with oxidizing agents from the atmosphere [35]. Monoterpenes are also

found in oleoresins. Together with diterpene resin acids and sesquiterpenes, they act as

chemical and physical defense compounds. This complex mixture, synthesized by plants,

repels insects via intoxication and quenching. Moreover, they cure the plant wounds by

forming a band-aid secure layer [20].

1.3.4 Industrial Use as Flavour, Fragrant, and Cosmetics Constituents

In ancient times, but also in our modern world, fragrances and aromas are used as

essential additives enhancing the final quality of foods and beverages, as well as in body care

and other hygienic products. In recent time, there is a raising demand, however, for products

of natural origin. Therefore, natural flavour compounds that can improve the sensory appeal

of these products gained larger value and became more expensive than their artificial

counterparts [36]. Essential oils and their monoterpene constituents, originating from plants,

are the main sources of flavours and fragrances used in industry [37]. One of the first known

plants used in cosmetics is the peppermint plant, or Mentha piperita, which contains menthol

as its main essential oil constituent. It is primarily applied because of its anti-stress, tension-

relieving, and cooling effects.

12

Menthol (Figure 6), one of the most renowned

monocyclic monoterpenes, is used in mouth washes, soaps,

toothpastes, and other cleansing formulations [38, 39]. Linalool,

another well-known acyclic monoterpene alcohol, is among the

commonly used ingredients added in perfumes and household

cleaning agents. Due to its citrusy, fresh, and sweet odour, it is

applied as a flavour enhancer in processed food and beverages

[40]. Citral, an acyclic monoterpene aldehyde, is the principle

odourant of lemon oil. Owing to its citrusy and lemon-like smell that is strongly linked to the

subjective impression of freshness and cleanliness, citral is also widely used in household

products [41].

α- and β-pinene (Figure 7), two bicyclic monoterpenes found

in turpentine, are fragrance substances used to improve the

odour of industrial products. Moreover, they are precursors

of several flavour compounds such as citronellol, geraniol,

menthol, and verbenol [42].

Apart from that, a mixture of monoterpenes has also been

described in a formulation for the prevention and treatment of hair loss as patented by

Verona [43]. Among the main constituents of this mixture are: β-pinene, limonene, cineole,

linalool, and camphor. In general, these compounds are among the most important and

widely used monoterpenes. Verona [43] showed that the addition of this mixture with

salicylic acid and fatty acids (saturated or unsaturated) resulted in reduction of hair loss

together with a remarkable improvement in the growth and strength of hair.

Figure 6: Chemical

structure of menthol

Figure 7: α- (left) and β- (right) pinene molecules as an example of bicyclic monoterpenes.

13

1.3.5 Pharmacological Effects

Although the use of essential oils has been primarily related to food flavourings,

cosmetics, and perfumes due to their aroma, research demonstrates the high potential of the

use of volatile monoterpene constituents to cure and prevent human diseases [36, 44].

Natural monoterpenes and their synthetic derivatives have repeatedly been described in

relation to their various pharmacological effects: studies showed that monoterpenes have

antimicrobial [45], anti-inflammatory [46], antispasmodic [47], analgesic [48], and anaesthetic

[49] effects. Moreover, in the last few years, several monoterpenes have been shown to be

potential chemotherapeutic agents [32, 50]. Among them, the monocyclic monoterpene

limonene was found to inhibit the development of several types of cancer, such as liver, skin,

and lung cancer [32]. Geraniol, a monoterpene alcohol found in lemon grass, has been

demonstrated to inhibit the growth of hepatoma, leukaemia, and colon cancer cells [50].

Nevertheless, monoterpenes have also been studied while highlighting the prevention

and treatment of cardiovascular diseases owing to their vasodilation and hypotension effects.

Carvacrol, limonene, citronellol, myrtenol, and linalool are among these monoterpenes

showing in-vivo and in-vitro cardiovascular effects in both humans and animals [51]. Apart

from that, other monoterpenoids, like thymol and terpineol, exerted potent antioxidant and

free radical scavenging activities as reported by Mata, Proença [52]. Monoterpenes have also

been shown to be anti-inflammatory and to positively impact several respiratory disorders. In

particular, 1,8-cineol and menthol are among the most effective compounds for alleviating

bronchopulmonary disorders [53]. A recent study showed that both linalool and linalyl

acetate, as anti-inflammatory compounds, were capable of decreasing the edema in the

carrageenan-induced paw edema rat model following systemic administration [50].

Several aromatic plants are known to be multifunctional, thus exhibiting different

pharmacological activities. For instance, fennel, an aromatic medicinal plant rich in limonene

and fenchone, is used as carminative, digestive, lactogogue, and diuretic agent. Moreover, it

has been shown to exert beneficial effects in the treatment of respiratory and

gastrointestinal disorders [54].

As mentioned earlier in this section, monoterpenes are effective antimicrobial agents.

Specifically, some monoterpeneoids such as thymol, menthol, and linalyl acetate are effective

14

against several gram-positive bacteria such as Staphylococcus aureus and gram-negative

bacteria such as Escherichia coli [55]. Other monoterpenes, namely geraniol and citral, exert

antifungal activity against a human fungal pathogen called Cryptococcus neoformans, which

usually infects lungs or the central nervous system [50, 56]. Moreover, citral is not only an

antifungal agent but is also capable of reducing the infectivity of Herpes simplex virus (HSV),

thus exihibiting a significant anti-HSV activity [50].

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1.4 Acyclic Monoterpene Alcohols–The Monoterpenols 1.4.1 Introduction to Monoterpenols

Monoterpene alcohols, or monoterpenols as sometimes referred to in literature, are

2,6-dimethyloctane derivatives containing variable double bond moieties and a hydroxyl-

function. The most important substances of this class are linalool, geraniol, nerol, citronellol,

myrcenol, and dihydromyrcenol [57]. They are used in perfumery because of their pleasant

olfactory properties since ancient times. Due to their exceptional smell properties and other

physiological and pharmacological effects, most studies were mainly focused on linalool,

geraniol, nerol, and citronellol [58-60].

1.4.2 Linalool – the Scent of Relaxation

1.4.2.1 A Brief Introduction to Linalool

Linalool, or 3,7-dimethyl-1,6-octadien-3-ol, is an

acyclic, unsaturated, tertiary monoterpene alcohol, comprising

two enantiomers that are both found in plants: (3S)-(+)-

linalool and (3R)-(-)-linalool (Figure 8). Thereby, the latter is

more common in nature [13]. It is one of the main ingredients

of essential oils of over 200 plant species belonging to over

50% of plant families. Among the most well-known plants that

are rich in linalool are rosewood (Aniba rosaeodora Ducke,

Lauraceae), Ho leaf (Cinnamomum camphora Nees,Lauraceae), fruits of coriander

(Coriandrumsativum L., Apiaceae), and flowering tops of lavender (Lavendula officinalis Chaix

sin., L.angustifolia Mill., Lamiaceae) (Figure 9) [13, 61].

In 1875, a French scientist isolated linalool from Cayenne Bois de Rose oil (rosewood

oil), thereby accomplishing early industrial production of linalool. Due to the high subsequent

industrial demand, production of linalool from natural oils was more and more compensated

by the supply of its synthetic analog [61].

Although synthetic linalool was initially met with great resistance by perfumers due to

the presence of trace impurities altering the odour, it later became the main product owing

to its price and availability, compared to the natural linalool.

Figure 8: (S)-(+)- and (R)-(-)-Linalool

16

1.4.2.2 An Overview of the Properties and Uses of Linalool

Linalool is a colourless liquid of low molecular

weight (154.25 g/mol), comprising a branched structure,

and thus being a highly volatile compound [62]. It is

characterized by a fresh, clean, and floral odour with a

citrus impression [61]. However, its enantiomers possess

different odour notes, with the odour of (3S)-(+)-linalool

being perceived as sweet, floral with a citrus note, while

(3R)-(-)-linalool elicits a woody, lavender-like smell. These

fragrant properties make linalool one of the top

ingredients in perfume industry. Linalool is also added, as a fragrant and flavouring

substance, to many cosmetic products, household agents, as well as processed food and

beverages [13].

Since ancient times, linalool is a renowned essential oil component (especially of

rosewood and lavender plants) that is primarily used in aromatherapy to induce sedative

effects [63]. In several studies, sleep-inducing, hypnotic, hypothermic, and anticonvulsant

actions of linalool have been proven in both animals and humans [64-66]. It is reported that

these anxiolytic effects of linalool may be due to its potentiating effect on the γ-

aminobutanoic acid receptor A (GABAA)-receptor response in the central nervous system [67,

68].

Together with its ester linalyl acetate, linalool was also found to possess anti-

inflammatory properties [69]. Scientists showed that linalool inhibits significantly and dose-

dependently the lipopolysaccharide-induced production of tumour necrosis factor-α and

Interleukin-6 cytokines in vitro [70]. Moreover, in vivo studies revealed that linalool causes

significant inhibition of carrageenan-induced edema in rats [69]. The mechanisms suggested

by in vitro and in vivo studies show that linalool may act by inhibiting the production of

inflammatory cytokines via blocking nuclear factor NF-κB and mitogen-activated protein

kinases pathways, antagonizing the N-methyl-D-aspartate effects and reducing the synthesis

or release of nitric oxide (NO).

Figure 9: Flowering tops of lavender are rich in linalool

(Source: pixabay.com)

17

Linalool has also been reported with antibacterial, antiviral, or antiparastic activity.

Despite the fact that essential oils have commonly been shown to exert higher antimicrobial

activity than their major components, it was recently found that the antimicrobial potential

of linalool is stronger than that of the pure essential oils of Lavandula angustifolia (lavender)

and Citrus paradisi (grape fruit). This effect was observed against Staphylococcus aureus

(gram positive bacteria), Escherichia coli (gram negative bacteria), and Candida albicans

(dimorphic fungus) [71]. In addition, the authors of the same study [71] observed that linalool

had higher antimicrobial activity against E. coli than Juniperus communis, Citrus bergamia,

and Pelargonium graveolens essential oils.

Apart from that, a study on the antifungal activity of linalool reported the ability of

this monoterpenol to inhibit the formation of new C. albicans biofilms and reduce existing C.

albicans biofilms. This study suggests that linalool may have potential clinical applications in

treating biofilm-associated C. albicans infections, which show high resistance to traditional

antifungal agents [72]. Chiang, Ng [73] showed that linalool, being a component of Ocimum

basilicum L.; has an anti-adenoviral activity against three types of adenovirus (ADV), namely

ADV-3, ADV-8, and ADV-11. Last but not least, linalool, derived from Cinnamomum camphora

L., was discovered to be a promising chemotherapeutic agent against schistosomiasis-causing

parasites. It showed extreme molluscicidal and cercaricidal properties against Oncomelania

hupensis and Schistosoma japonicium, respectively, by damaging the gills and hepato-

pancreas of the snails, as well as disrupting their cercarial tegument [74].

Among the life-threatening diseases to the human race is cancer. Scientists worldwide

keep searching for chemical and natural anti-cancer agents that may act as promising anti-

cancer drugs. Linalool possesses an anti-angiogenic activity thus having a role in treating or

even preventing the progression of cancer cells [75]. It has been shown to be a promising

agent against prostate cancer [76], endothelial ovarian carcinoma [77], colon cancer [78],

leukaemia and cervical cancer [79]. Moreover, Ravizza, Gariboldi [80] reported that linalool

reverses the doxorubicin resistance in human breast adenocarcinoma cells via enhancing the

sensitivity of these carcinogenic cells towards the cytotoxic activity of doxorubicin.

To sum up, linalool is a plant derived monoterpene alcohol that can be considered a

lead compound in the aroma and flavour industry and a future natural alternative or

supporting agent in medical applications.

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1.4.3 Geraniol – the Geranium Rose-Like Fragrant

1.4.3.1 Discovery of Geraniol

Geraniol, or 2-trans-3,7-dimethyl-2,6-octadien-1-ol, is an

unsaturated, acyclic, primary allylic monoterpene alcohol

found originally in geranium oil (Figure 11), from where it

received its name [81].

In 1871, Oscar Jacobsen discovered geraniol as a geraniol-

nerol mixture in palmarosa oil, and could successfully

isolate it as a pure compound [82]. In fact, the so-called geraniol is a mixture of two cis-trans

isomers (Figure 10). Later, the trans isomer, geraniol, was isolated from palmarosa oil while

the cis isomer, nerol, was isolated from neroli oil [81].

1.4.3.2 An overview on the properties and uses of geraniol

Geraniol is a clear to pale-yellow oil, soluble in

most organic solvents but insoluble in water.

Although it is the allylic isomer of linalool, its

sweet, fruity, berry-like, floral and rose-like odour

is more notable and less common than that of

linalool [81, 83, 84]. Owing to its low molecular

weight (154.25 g/mol) and its pleasant odour. This

monoterpenol is responsible for the odour of many

essential oils and is an industrially important, widely used fragrance material [85]. It is

present in about i) 76% of deodorants, ii) 41% of household detergents and iii) 33% of natural

cosmetic formulations sold in the European market [86-88].

Geraniol is the most active tick and mosquito repellent among the other compounds

occurring in geranium and citrus essential oils [89, 90]. In addition, it is used as a natural pest

control agent exhibiting low toxicity [91]. Additionally, antibacterial and antifungal activities

were reported for this substance against a large number of microorganisms [92]. Andoğan,

Baydar [93] showed that geraniol inhibits Staphylococcus aureus and Escherichia coli strains.

Besides, geraniol shows significant antifungal activity against Candida krusei and Aspergillus

fumigates at low concentration [94].

Figure 11: Geranium flower rich in geraniol(Source: pixabay.com)

Figure 10: trans-(left) and cis-(right)isomers of geraniol

19

Many studies reported the in vivo and in vitro anticancer properties of geraniol. It inhibits the

growth of leukaemia and melanoma cells, hepatoma cells and pancreatic cancer cells [95].

Furthermore, this monoterpenol elicits mainly cytostatic effects against human colon cancer

cells, with no cytotoxic activity being reported [96]. An in vivo study done by Carnesecchi,

Bras-Gonçalves [97], showed that geraniol was able, as an adjuvant drug, to potentiate the

antitumor effect of 5-flurouracil and to increase the survival time of nude mice grafted with

the human colorectal tumour cells. Moreover, the ester derivative of geraniol, the geranyl

acetate, has also been reported to have anticancer properties against human hepatoma cell

lines [98].

Several other pharmacological effects have been reported for geraniol. Among them is its

anti-inflammatory property. Immunosuppressive activity of geraniol has been observed both

in vivo and in vitro [99, 100] with fewer side effects than other conventional anti-

inflammatory drugs [101]. In addition, geraniol has been shown to have anti-oxidative

potential, acting either via an increase of the glutathione content, an inhibition of NO release

and decreasing lipid peroxidation as reported by Tiwari and Kakkar [102], or through

enhancement of the production of antioxidants by activating the nuclear factor E2-related

factor 2 (Nrf2) as shown by Jayachandran, Chandrasekaran [103]. Geraniol also exerts

neuroprotective [104] as well as hepatoprotective effects [105].

Being an abundant constituent in a large number of plants with various biological properties

and minor toxicity, geraniol is considered a promising natural compound in consumer health-

promoting products.

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1.4.4 Nerol– just an Isomer?

1.4.4.1 Definition and Occurrence in Nature

Being the cis-isomer of geraniol, nerol

(cis-2,6-dimethyl-2,6-octadien-8-ol) is one of the

well-known acyclic monoterpene alcohols (Figure

10). It is found in various plants such as Melissa

officinalis L [106], Lippia spp [107], Cymbopogen

flexuosus, Wisteria brachybotrys, and Rosa

damascaena Mill (Figure 12) [108]. In addition,

nerol and its ester, neryl acetate, are found

among the oxygenated constituents of the

Helichrysum italicum (Roth) G. Don essential oil

[109]. Interestingly, nerol has also been reported in the mandibular glands of the stingless

bee Trigona fulviventris (Hymenoptera: Apidae) composing more than 50% of its volatile

components, where it acts as an alarm substance and a marking pheromone [110].

1.4.4.2 An overview on the properties and uses of nerol

Nerol, a colourless volatile liquid having a molecular weight of 154.25 g/mol, is a

widely used fragrant ingredient in several fragrances. The odour of nerol is described in

literature as floral, citrus-like, and sweet [84]. Accordingly, it is used in the composition of

cosmetic products such as fine fragrances, shampoos, toiletries, as well as in non-cosmetic

products like household cleaners and detergents [111].

In line with other monoterpenes, nerol has several therapeutic effects. It is among the

monoterpenes possessing antibacterial activity, especially against resistant S. aureus strains

[112]. Nevertheless, it has the highest antifungal activity against some Aspergillus strains,

namely A. flavus and A. ochraceus, indicating its capability to be a food preservative in food

products [113]. In addition, it is also among the natural compounds that exert significant

activity against Candida albicans [114].

In a recent study, nerol has been shown to have antioxidant activity [115]. Moreover,

an anti-inflammatory effect has been demonstrated for nerol, especially in relation to

gastrointestinal disorders such as ulcerative colitis and gastric ulcer. It alleviates the

Figure 12: Nerol is among the odourous constituents of Rosa damascaena Mill

flower. (Source: pixabay.com)

21

pathological features of colitis in the oxazolone model, protects from gastric damage, and has

an immunomodulatory effect [116]. Thus, it has been postulated that nerol can be a potential

alternative for controlling ulcerative colitis.

22

1.4.5 β-Citronellol – The Lemongrass Aroma

1.4.5.1 Definition and Occurrence in Nature

β-Citronellol, 3,7-dimethyloct-6-en-1-ol, or

dihydrogeraniol, is a natural acyclic monoterpenol

found in the essential oils of several aromatic plants

such as Cymbopogen citratus [117], Cymbopogen

winterianus [118], and Lippia alba (Mill.) [119]. In

addition, citronellol is one of the major constituents of

geranium and Pelargonium graveolens L'Her flowers, composing 27% [90] and 36.4% [120]

of their volatile oils, respectively (Figure 14). There are two isomeric forms of citronellol

found in nature, the R-(+) - and the S-(-)- isomers (Figure 13). The R-(+) - citronellol, the more

prevalent isomer, is mainly found in the essential oils of the family of Rutaceae. On the other

hand, the S-(-)- citronellol, present mainly in geranium and citronella oils, is the less common

isomer [121].

1.4.5.2 Properties and Uses of Citronellol

Citronellol is a fragrant ingredient that is

characterized as being a colourless, oily liquid, having a

molecular weight of 156.27 g/mol [122]. Its odour has

been described in literature as sweet, rose-like, and

floral [83, 84, 123]. Owing to these odour notes, it is a

widely used fragrant material especially in floral

compositions [124].

In a study analyzing the fragrance chemicals in

domestic and occupational products, it is interesting to

note that citronellol is among the five most frequently

used fragrances in products like soaps, fabric

conditioners, furniture polish, and detergents [125].

Moreover, it is used as a flavouring agent in food and beverages such as alcoholic and non-

alcoholic beverages, ice creams, candies, and baked goods.

Figure 13: R-(+) - (left) and S-(-) - (right) isomers of citronellol.

Figure 14: Citronellol composes 36.4% of the volatile oil of Pelargonium graveolens L’Her flower. (Source: pixabay.com)

23

During the last few years, studies aimed to show that β-citronellol elicits different

pharmacological properties. Among these effects is its hypotensive potential acting through

its vasodilator effect [126, 127]. Citronellol is the main component of common medicinal

herbs, among them different lemongrass species such as Cymbopogen citratus and

Cymbopogen winterianus, that are still used, since ancient times in the context of folk

medicine, as antihypertensive drugs, and further studies confirmed their hypotension and

vasorelaxation effects [128, 129].

Another important pharmacological property of β-citronellol is its anti-inflammatory

activity [130]: Su and his coauthors showed that citronellol inhibited NO and prostaglandin E2

production, which are involved in various inflammatory disorders. In a strategy to develop

natural products with potent efficacy and minimal side effects, citronellol was among a group

of compounds tested for their antinociceptive behavior on orofacial pain in mice [131].

Moreover, citronellol exerts modest anticonvulsant activity when being compared to

standard drugs like diazepam, as shown by de Sousa, Gonçalves [121]. Apart from that,

citronellol is an antimicrobial agent. Of a group of monoterpenoids such as carveol, carvone,

and citronellal, it exhibited the highest inhibitory and bactericidal activity against E. coli and S.

aureus [132]. Accordingly, it can be a natural alternative to conventional antimicrobial drugs

treating for example bacterial topical infections. Last but not least, it is interesting to note

that citronellol has been proposed to be a promising nutraceutical antidiabetic agent due to

its capability of enhancing insulin production, thus, decreasing glucose level in

Streptozotocin-induced diabetic rats [133].

24

1.5 Characterization of the Smell Properties of the Investigated Compounds The structure-odour relationship study carried out in the present work was

accomplished through the following strategy: 1) structurally related compounds, in this case

the corresponding acetates and metabolites of the aforementioned monoterpenols, were

either purchased or synthesized; and 2) evaluated regarding their odour qualities and odour

thresholds (OT) in air using the state-of-the-art odorant analytical methods, i.e. gas

chromatography-olfactometry (GC-O). Data of the investigated compounds in addition to the

complete procedures of their synthesis and analysis are discussed in details in chapters 3,

4 and 5.

1.5.1 High Resolution Gas Chromatography-Olfactometry

A key strategy in odorant research is the use of the human nose as a highly sensitive

and selective detector, coupled with gas chromatographic analysis in the so-called gas

chromatography-olfactometry (GC-O). The principle of GC-O is based on connecting a

standard GC with an odour port in addition to conventional detectors such as a mass

spectrometer (MS) or flame ionization detector (FID) [134]. In particular, the gas effluent is

split after elution from the separation column into two gas streams, one being then

transferred to the analytical detector (MS or FID) and the other to the sniffing port, also

called olfactory detection port (ODP), as illustrated in Figure 15. This coupling of olfactive

detection with a chemo-analytical device is crucial for obtaining an analytical signal

Figure 15: Gas chromatography-olfactometry with an

olfactory detection port and a flame ionization detector.

25

representing not only the absolute quantitative presence of a single compound, even trace

concentrations, but also its chemosensory power.

This technique offers the advantage that the assessor can distinguish between

odorous and non-odorous substances, thus, detecting the odour qualities and rating the

intensities of the perceived odours [135]. In addition, odour-active impurities that may

accompany the target substance are separated therefore during the chromatographic

process. Accordingly, their influence on the olfactometric results is ruled out. Various GC-O

techniques have been developed for the evaluation of the relative odour impact of the single

odorous constituents. They can be classified into three groups based on the method of

determination: 1) detection frequency methods, 2) dilution to threshold methods, and 3)

direct intensity methods [136].

In the first type, the frequency detection method, a panel of 6-12 assessors analyse

the same sample. At a given retention time, the proportion of panellists who detect an

odorant is counted. Each assessor records the duration of the peak of the respective odorant.

The individual responses are combined to produce an aromagram where the peak height is

the number of panellists who could detect a certain odour. The obtained results are

described as the nasal impact frequency (NIF). The NIF value is set to a value of one or zero

depending on whether all the assessors perceived a given odour or not at a given retention

time, respectively. The surface of the NIF (SNIF) value describes the peak areas (frequency x

duration) taking into consideration the olfactory stimulation time. Simplicity is the main

advantage of this method as it does not require too much training and is the least time

consuming. On the other hand, its main limitation is that the result obtained is only related to

the odour intensity sensed at a certain concentration of the sample. This means that if the

sample is detected at higher concentrations than the detection threshold, therefore the

results might be the same regardless of its concentration [135, 136].

The second type of analysis is the dilution to threshold method, which is used in this

work. Two different analytical procedures, namely combined hedonic response measurement

(CHARM) analysis and aroma extract dilution analysis (AEDA) as introduced by Acree, Barnard

[137], and Grosch [138-140], respectively, have been developed to detect the odour potency

of volatiles in air. Both analytical types follow comparable but somewhat divergent evaluation

procedures. After stepwise dilution of the sample, usually as a series of 1:1 or 1:2 dilutions,

26

each dilution is applied to the GC-O for analysis by the assessors. These dilutions are sniffed

and odour impressions are noted until no odour is perceivable.

In case of AEDA, the maximum dilution in which the odour is still detectable by the

GC-O is known as flavour dilution (FD) factor [139-141]. On the other hand, in CHARM

analysis, the duration of the odorant during the GC separation is recorded and signals from

each dilution step are evaluated [137]. Consequently, the peak height in CHARM analysis is

equivalent to the FD factor in AEDA but gets an additional dimension due to the temporal

aspect.

Accordingly, the main difference between both dilution to threshold methods is that

the duration of odorant perception in CHARM analysis is taken into account with the final

detected dilution value, while in AEDA the time during which an aroma is perceivable has no

influence on this value [142]. Validation of the final result offers the advantage of obtaining

reliable odour descriptions due to the multiple detection of the same odour. However, some

major drawbacks are addressed for these screening methods; among them: 1) several

dilutions should be sniffed by more than one assessor which is time-consuming, and 2) inter-

individual changes arising regarding each person’s detection threshold. So, to overcome this

issue, repetitive identical runs under the same conditions should be applied for each

assessor.

Direct intensity methods, the third type of determination methods, monitors the

intensity of the smell and its duration during GC-O analysis and is not only limited to the

presence or absence of a stimulus as in the other two types [136]. Different types of

quantitative scales are used to measure the intensity of the odour of the eluting compound.

These scales can be a single, time-averaged measurement after the elution of the analyte

(posterior intensity evaluation method), or a dynamic measurement, which is more

frequently used, in which continuous tracing of the perceived intensity takes place producing

a chromatogram-like chart with intensity over time (OSME). The height of the peak obtained

represents the maximum odour intensity of the given odorant while the width corresponds

to the odour duration. The main drawback of the direct intensity is the intensive training

required for the assessors to obtain reproducibility and agreement with one another which is

time consuming. However, a trained panel will be able to give consistent and precise

discrimination [135].

27

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2 Aims and Outline Studies focusing on linalool and other structurally related acyclic monoterpene alcohols,

namely geraniol, nerol, citronellol, and their acetates, are being limited to their use as odour

enhancers or as pharmaceutical products (chapter 1.4). To date, no scientific research was

done to reveal the influence of the chemical structure of this interesting group of compounds

on their odour properties. Moreover, their metabolic products, which are found in either

plants or animals, have not been investigated in terms of physiological or pharmacological

properties. This is, most probably, due to the difficulty of obtaining these compounds in pure

form either commercially or through chemical synthesis. Therefore, the first aim of this thesis

was to synthesize these metabolites by novel or previously known synthetic methods and

evaluate their odour qualities and odour thresholds in air using the state-of-the-art methods,

i.e. gas chromatography-olfactometry (chapter 1.5). A further aim of the present study was to

find out which structural features are responsible for the odour characters of this group of

monoterpene alcohols. This was accomplished by comparing the odour qualities and

potencies of the respective synthesized compounds to their parent molecules (chapters

3,4,5). In this regard, metabolites having similar or more pleasant odour characters, with

respect to their parent monoterpenes, are revealed. Simultaneously, synthesized derivatives

of linalool and its acetate were tested for their pharmacological effects especially on GABA

receptors in a collaborative study comparing the positive allosteric potential of linalool at

GABAA receptors with its metabolites (chapter 6).

This study is mainly focused on the effect of adding oxygen-bearing functional groups to

the parent molecules as the most commonly found metabolites, in literature, are carbon-8

oxygenated derivatives such as 8-hydroxy- or 8-carboxy-linalool. Therefore, the synthesized

compounds have either an aldehyde, an alcohol, or an acid functional group at C-8 position.

Structure elucidation of these compounds was established through GC-MS and NMR analyses

(chapters 3,4,5). Comparison of the odour properties of the metabolites with those of the

parent monoterpenes indicates that the OH group already existing at C-1 (geraniol, nerol and

citronellol) or C-3 (linalool) of the monoterpenes is the main contributor to these potent

pleasant odours. As shown in this thesis, an additional aldehyde, alcohol, or acid functional

group leads to either change in the odour quality, or potency, or both or even an odour loss.

Apart from that, the linalool metabolite bearing an aldehyde group at C-8 (8-oxolinalool) is

the only derivative among the hydroxylated and carboxylated at carbon 8 that positively

38

affects GABAergic currents, whereas the linalyl acetate derivatives do not show any

significant changes of GABAergic currents (chapter 6).

Therefore, the aim of this doctoral thesis was to introduce a structure-odour relationship

study of these acyclic monoterpene alcohols by systematic variation of the chemical basic

structure. In addition, data on the synthetic procedures leading to their derivatives as well as

their analytical parameters (including their retention indices (RI), mass spectroscopy (MS),

and nuclear magnetic resonance (NMR) data is compiled in a comprehensive odour-analytical

substance library.

Overall, the presented work aims to propose the structural features responsible for the

odour qualities and potencies of the aforementioned acyclic monoterpene alcohols by the

aid of their acetates and synthesized derivatives. Thereby, the established data library can be

a beneficial tool in future analytical research; for example in determination of olfactory

receptors binding pockets, understanding the human olfaction mechanisms, and designing

new natural fragrant compounds.

39

3 Structure-Odor Relationships of Linalool, Linalyl Acetate and Their Corresponding Oxygenated Derivatives.

Frontiers in chemistry, 2015, 3, article 57.

Shaimaa A. Elsharif 1, Ashutosh Banerjee 2 and Andrea Buettner 1, 3*

1 Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, [email protected]

2 Department of Chemistry and Pharmacy, Pharmaceutical Chemistry, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

3 Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging, Freising, Germany.

* corresponding author

Reprinted from Frontiers in chemistry, 2015, 3. Elsharif SA, Banerjee A and Buettner A, Structure-odor relationships of linalool, linalyl acetate and their corresponding oxygenated derivatives, article 57.

Copyright © 2015 Elsharif, Banerjee and Buettner, with permission from the authors.

Copyright © 2015 Elsharif, Banerjee and Buettner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

ORIGINAL RESEARCHpublished: 06 October 2015

doi: 10.3389/fchem.2015.00057

Frontiers in Chemistry | www.frontiersin.org 1 October 2015 | Volume 3 | Article 57

Edited by:

Dejian Huang,

National University of Singapore,

Singapore

Reviewed by:

Muhammad Safder,

University of Karachi, Pakistan

Marcin Szymanski,

Poznan University of Medical

Sciences, Poland

*Correspondence:

Andrea Buettner,

Erlangen, Germany

[email protected]

Specialty section:

This article was submitted to

Food Chemistry,

a section of the journal

Frontiers in Chemistry

Received: 31 July 2015

Accepted: 14 September 2015

Published: 06 October 2015

Citation:

Elsharif SA, Banerjee A and

Buettner A (2015) Structure-odor

relationships of linalool, linalyl acetate

and their corresponding oxygenated

derivatives. Front. Chem. 3:57.

doi: 10.3389/fchem.2015.00057

Structure-odor relationships oflinalool, linalyl acetate and theircorresponding oxygenatedderivativesShaimaa A. Elsharif 1, Ashutosh Banerjee 2 and Andrea Buettner 1, 3*

1Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander-Universität

Erlangen-Nürnberg, Erlangen, Germany, 2Department of Chemistry and Pharmacy, Pharmaceutical Chemistry, Emil Fischer

Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, 3Department of Sensory Analytics,

Fraunhofer Institute for Process Engineering and Packaging, Freising, Germany

Linalool 1 is an odorant that is commonly perceived as having a pleasant odor, but is also

known to elicit physiological effects such as inducing calmness and enhancing sleep.

However, no comprehensive studies are at hand to show which structural features are

responsible for these prominent effects. Therefore, a total of six oxygenated derivatives

were synthesized from both 1 and linalyl acetate 2, and were tested for their odor

qualities and relative odor thresholds (OTs) in air. Linalool was found to be the most potent

odorant among the investigated compounds, with an average OT of 3.2 ng/L, while the

8-hydroxylinalool derivative was the least odorous compound with an OT of 160 ng/L;

8-carboxylinalool was found to be odorless. The odorant 8-oxolinalyl acetate, which has

very similar odor properties to linalool, was the most potent odorant besides linalool,

exhibiting an OT of 5.9 ng/L. By comparison, 8-carboxylinalyl acetate had a similar OT

(6.1 ng/L) as its corresponding 8-oxo derivative but exhibited divergent odor properties

(fatty, greasy, musty). Overall, oxygenation on carbon 8 had a substantial effect on the

aroma profiles of structural derivatives of linalool and linalyl acetate.

Keywords: Linalool, linalyl acetate, gas chromatography-olfactometry, odor threshold in air, 8-oxolinalyl acetate,

8-carboxylinalyl acetate, odor qualities, retention index

Introduction

In folk medicines as well as aroma therapy, essential oils and fragrance compounds are being usedas therapeutic agents for relieving pain, anxiety reduction and energy enhancement (Lahlou, 2004;Kako et al., 2008; Kiecolt-Glaser et al., 2008). Among them, due to their high volatility, the acyclicmonoterpenes are a valuable class of compounds useful for the flavor and fragrance industries (Kingand Dickinson, 2003). One of the most important acyclic monoterpene substances is linalool 1which represents about 70% of the terpenoids of floral scents (Stashenko and Martinez, 2008). Inperfumery, linalool is a commonly used fragrant ingredient being a component of many perfumestop notes and being found in 60–90% of cosmetic products (Cal and Krzyzaniak, 2006). Its odor isdescribed in literature as floral, citric, fresh and sweet (d’Acampora Zellner et al., 2007). It is alsoadded to household cleaning agents, furniture care products, waxes, as well as to processed foodand beverages, as a fragrance and flavor agent. Linalool is found in the essential oils of over 200plant species, belonging to different families (Stashenko and Martinez, 2008). For example, linalool

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Elsharif et al. Structure-odor relationships of linalool and derivatives

FIGURE 1 | Lavender oil main constituents.

and its ester form, linalyl acetate 2, are the lavender oil mainconstituents (Figure 1; Buchbauer et al., 1991). The odor oflinalyl acetate is described as floral, sweet and citric, andadditionally as minty and slightly caraway-like (d’AcamporaZellner et al., 2007).

Lavender oils are widely used to enhance sleep. Thereby, ithas been demonstrated that lavender aromatics can improvesleep in the elderly (Hudson, 1996) and infants (Field et al.,2008). Furthermore, exposure to lavender odors during sleepresults in increased duration of deep slow-wave stage sleep(Goel et al., 2005). A closely related therapeutic effect is anxietyreduction, having also been reported for lavender essentialoil usage (Tasev et al., 1969). In view of this, linalool hasbeen demonstrated to not only activate olfactory receptors butalso to modulate ion channel receptor potentials such as thetransient receptor potential channels (TRP) and to potentiateγ-aminobutanoic acid receptor A (GABAA)-receptor responsein the central nervous system (Kessler et al., 2012, 2014); thelatter receptor system has been shown to be strongly involvedin sedative, anxiolytic and calming processes. TRP channels,on the other hand, are involved in numerous physiologicalconditions and diseases, and their potential modulation by aromacompounds such as linalool is discussed comprehensively inFriedland and Harteneck (2015). Based on these observationsthere is a general understanding that linalool plays a validrole in the calming response in humans. However, metabolicside products of linalool, both in plants as well as animals orhumans have not been regarded comprehensively in view ofeither smell or other physiological effects. For gaining deeperinsights into the metabolic origin and further fate of linalooland its derivatives, studies were carried out to investigate themetabolism of these substances both in plants (Luan et al.,2006) and animals (Chadha andMadyastha, 1984). Experimentalstudies on rats using 14C-labeled linalool showed that it is rapidlyabsorbed from the intestinal tract after oral administration.The major part of linalool is metabolized by the liver to polarcompounds which are mainly excreted in urine as free formor conjugates; only minor amounts are excreted via the feces.Allylic oxidation becomes an important pathway upon repeatedadministration, being mediated by the cytochrome P-450 system.8-Hydroxylinalool and 8-carboxylinalool were detected as majormetabolites after 20 days administration of linalool in rats. Aminor part undergoes partial ring closure to α-terpineol, withthe generation of small amounts of geraniol and nerol. These

metabolites are also excreted in urine as free forms or conjugates.Products of linalool reduction (dihydro-, tetrahydrolinalool)were also identified in rodent urine (Aprotosoaie et al., 2014).A significant proportion of orally administered linalool followsintermediary metabolic pathways as shown in Scheme 1 (schememodified from Aprotosoaie et al., 2014).

8-Hydroxylinalool was not only found as a metabolite inmammalian species, but also as an oxidation product isolatedfrom the grape berry mesocarp after linalool was applied to it(Luan et al., 2006). 8-Carboxylinalool was found to be among theconstituents of the fruits of Euterpe oleracea (Chin et al., 2008)and the flower of Albizia julibrissin (Yahagi et al., 2012). Linalylacetate metabolism was also studied in Pseudomonas incognita(Renganathan and Madyastha, 1983), where it was shown thatthe C-8-methyl moiety is subjected to selective oxidation, giving8-hydroxylinalyl acetate which is then oxidized to 8-oxo and 8-carboxylinalyl acetate, respectively. Apart from that, 8-oxolinalylacetate was first isolated from lavandin oil and hence reported as aconstituent of a natural product (Mookherjee and Trenkle, 1973).8-Carboxylinalyl acetate was found in trace amounts (<0.01%) inJabara (Citrus jabara Hort ex. Tanaka) peel extract (Mookherjeeand Trenkle, 1973; Table 1).

Therefore, we conclude that the carbonyl, the hydroxyl andthe carboxylic acid functional groups in α-position to thedouble bond are very common in nature. These metaboliteshave been previously synthesized as regio-selectively deuteratedcompounds for the investigation of their bioconversion intolilac during an in vivo feeding experiment to Syringa vulgarisL., Oleaceae, to study the metabolic pathway of linalool andits derivatives (Kreck et al., 2003). Non-deuterated derivativeswere used as reference substances for elucidation of compoundsin essential oils isolated from plants to reveal their structuraland organoleptic properties (Van Dort et al., 1993). However,the latter study does not contain any explanation of accuratemethods of smell determination, nor discuss any further potentialphysiological impact on humans. Accordingly, neither the odorqualities and odor thresholds of these substances are investigatedsystematically, nor is it clear what makes linalool so unique for itsodor but also other physiological effects.

Based on these considerations we synthesized, starting from1 and 2, previously reported metabolites and hypotheticalderivatives of linalool and its related ester in order to determinetheir respective odor qualities and thresholds. We thereby aimedat elucidating if linalool itself represents the most potent andcharacteristic member of this substance group or if any otherpotent compounds are promising natural physiological chemo-stimuli in humans. Finally, the aim was to provide a substancelibrary that should further aid in future analytical studies, withcompiled data on Retention Indices (RI-values) as well as massspectrometric and nuclear magnetic resonance data.

Materials and Methods

ChemicalsThe following chemicals were purchased from the suppliers givenin parentheses: linalool, linalyl acetate, selenium dioxide, sodiumborohydride, methanol, methanol anhydrous, ethanol, dioxane,






SCHEME 1 | Main linalool metabolic pathway in mammals (scheme modified from Aprotosoaie et al., 2014).

tert-butyl alcohol, 2-methyl-2-butene, petroleum ether, sodiumchlorite, sodium dihydrogen phosphate, ethyl acetate, hexane,magnesium sulfate (Aldrich, Steinheim, Germany), diethyl ether(Fisher Scientific, Loughborough).

General MethodsAll reactions requiring anhydrous conditions were carriedout under nitrogen and the solvents were dried before useto remove moisture using appropriate drying solvents. Allreactions were monitored by TLC using Kieselgel 60 F254plates. Visualization of the reaction components was achievedusing UV fluorescence (254 nm) and KMnO4 stain. Columnchromatography was carried out over silica gel 60. The yieldsreported are after purification.1H and 13C NMR spectra wererecorded in deuterated solvents and chemical shifts (δ) arequoted in parts per million (ppm) calibrated to TMS (1H and13C). Coupling constants (J) were measured in Hertz (Hz).The following abbreviations are used to describe multiplicities:

s = singlet, d = doublet, t = triplet, q = quartet, b = broad,m = multiplet. The identity of all intermediates and syntheticproducts was determined by MS/EI.

Nuclear Magnetic Resonance (NMR) Spectra1H and 13C NMR spectra were recorded in CDCl3 on an Avance360 spectrometer, 360 MHz, and Avance 600, 600 MHz (BrukerBiospin, Rheinstetten, Germany) at room temperature operatedat 360 or 600 MHz (1H) and 90 or 150 MHz (13C), withtetramethylsilane (TMS) as internal standard.

Gas chromatography-olfactometry (GC/O) andGC-electron Impact-mass Spectrometry(GC-EI-MS)GC-O analyses were performed with a Trace GC Ultra (ThermoFisher Scientific GmbH, Dreieich, Germany) by using thefollowing capillaries: FFAP (30m × 0.32mm fused silicacapillary, free fatty acid phase FFAP, 0.25µm; Chrompack,






TABLE 1 | Retention indices and occurrence of linalool and its derivatives.

Entry Odorant RIa Previously identified in

DB5 FFAP

1 Linalool 1108 1550 Some examples: Wood of Aniba rosaeodora Ducke, Lauraceaeb, Flowering tops of Lavandula officinalis, L.

angustifolia Mill., Lamiaceaec, Coriandrum sativum L., Apiaceaed, Flowers of Citrus sinensis Osbeck,

Rutaceaee

2 Linalyl acetate 1264 1563 Lavandula angustifolia Millerf , Micromeria kerneri and Micromeria juliana Lamiaceaeg, Origanum vulgare

Lamiaceaeh.

3 8-Oxolinalool 1350 2150 Narcissus trevithian and Narcissus geranium Amaryllidaceaei

4 8-Oxolinalyl acetate 1490 2133 Lavandin oilj

5 8-Hydroxylinalyl acetate 1530 2333 As a linalyl acetate metabolite by pseudomonas incognitak

6 8-Hydroxylinalool 1380 2320 Chamaecyparis Obtusel, Vitis vinifera (muscat grape skins)m, Pluchea indican, As a linalool metabolite in urine

of ratso, and as a linalool oxidation product in grape berry mesocarpp.

7 8-Carboxylinalool 1540 1929 Fruits of Euterpe oleraceaq, flower of Albizia julibrissinr.

8 8-Carboxylinalyl acetate 1650 1957 Trace amounts in Jabara (Citrus jabara Hort ex. Tanaka)s

aretention indices were determined as described by Van Den Dool and Kratz (1963).bChantraine et al. (2009).cOzek et al. (2010).dTsagkli et al. (2012).eMiguel et al. (2008).fBuchbauer et al. (1991).gKremer et al. (2014).hAndi et al. (2012).iVan Dort et al. (1993).jMookherjee and Trenkle (1973).kRenganathan and Madyastha (1983).lMatsubara et al. (1990).mStrauss et al. (1988).nUchiyama et al. (1989).oChadha and Madyastha (1984).pLuan et al. (2006).qChin et al. (2008).rYahagi et al. (2012).sOmori et al. (2011).

Mühlheim, Germany) and DB5 (30m × 0.32mm fused silicacapillary DB-5, 0.25µm; J&W Scientific, Fisons Instruments).The helium carrier gas flow was set at 2.0mL/min. Thecompounds eluting at the end of the capillaries were split witha Y-splitter (J&W Scientific; ratio 1:1 v/v) and transferred viatwo deactivated capillaries (0.5m × 0.2mm, J &W Scientific)to a flame ionization detector and a heated sniffing port(temperature: 250◦C). The samples were applied onto thecapillary using a cold-on-column injector at 40◦C. After 2min,the oven was heated at a rate of 15◦C/min to 240◦C andheld for 2min. GC-EI MS analyses were performed withan Agilent MSD 5975C (Agilent Technologies, Waldbronn,Germany) and a Thermo ITQ 900 (Thermo Fisher Scientific,Dreieich, Germany) with the capillaries described above. Massspectra in the electron impact mode (EI-MS) were generatedat 70 eV.

Retention Indices (RI)Retention indices (Table 1) were determined by the methodpreviously described by Van Den Dool and Kratz (1963).

Evaluation of Odor QualityThe odor qualities were determined during GC-O evaluationby the aid of panelists who were trained volunteers from

the University of Erlangen (Erlangen, Germany), exhibiting noknown illness at the time of examination and with auditedolfactory function. In preceding weekly training sessions theassessors were trained for at least half a year in recognizingorthonasally about 90 selected known odorants at differentconcentrations according to their odor qualities and innaming these according to an in-house developed flavorlanguage.

Determination of Odor ThresholdsOdor thresholds were determined in air following the proceduredescribed by Czerny et al. (2011) using (E)-dec-2-enal as aninternal odor standard. This procedure offers the advantage thatcompounds that might be present as odor-active impurities inthe reference compound are separated from the target odorantsduring the chromatographic separation step. In consequence, aninfluence of such components on the results is avoided. Also,odor thresholds can be compared to each other on an absolutebasis without interference with any matrix system as would bethe case e.g., when determining odor thresholds in water. Thedetection odor thresholds of the panel were calculated as thegeometric mean of the individual thresholds according to Czernyet al. (2008).






Synthesis, General ProceduresGeneral Procedure 1(GP1)Generally, the method of Wakayama et al. (1973) was used(Scheme 2). Compounds 1 and 2 and selenium dioxide (1eq.) were dissolved in dioxane/ethanol 9:1 (v/v), and thesolution was heated at 80◦C for 5 h. After removal of seleniumdeposit by filtration, the solvent was removed under reducedpressure using a rotary evaporator. The residue was treated withdiethyl ether/petroleum ether 1:1 (v/v), and after removal ofthe solvent, the residue was purified by flash chromatographyon silica gel 60 (Merck), with a mobile phase of petroleumether/diethyl ether, affording the crude compounds 3 and 4,

respectively.

(E)-3,7-dimethyl-8-oxoocta-1,6-dien-3-ol (3),8-oxolinaloolFollowing GP1, from 1 (4.8 g, 31.1mmol) and seleniumdioxide (3.4 g, 30.4mmol) in 30ml dioxane/ethanol 9:1 (v/v),compound 3 was prepared. Flash chromatographic purificationwith petroleum ether/diethyl ether 1:4 (v/v) yielded 1.4 g (29%)of 3 as orange oil.1HNMR (600 MHz, CHLOROFORM-d) δ ppm9.38 (1 H, s), 6.42–6.56 (1 H, m), 5.92 (1 H, dd, J = 17.26,10.67Hz), 5.25 (1 H, dd, J = 17.26, 0.91Hz), 5.11 (1 H, dd, J =

10.90, 0.91Hz), 2.35–2.45 (2 H, m), 1.74 (3 H, s), 1.61–1.71 (2H, m), 1.31–1.35 (3 H, m).13C NMR (91 MHz, CHLOROFORM-d) δ ppm 195.2., 154.6, 144.3, 139.2, 112.4, 72.9, 40.3, 28.1, 23.8,9.1.MS (EI) m/z (%) (rel.int.): 168 [M+] (1), 98(15), 87(27),82(24), 71(100), 55(33), 43(58), 41(23).

(E)-3,7-dimethyl-8-oxoocta-1,6-dien-3-yl-acetate(4), 8-oxolinalyl AcetateFollowing GP1, from 2 (5 g, 25mmol) and selenium dioxide (2.7g, 25mmol) in 15ml dioxane/ethanol 9:1 (v/v), compound 4 wasprepared. Flash chromatographic purification with petroleumether/diethyl ether 3:2(v/v) yielded 1.4 g (29%) of 4 as orangeoil.1H NMR (600 MHz, CHLOROFORM-d) δ ppm 9.39 (1 H, s),6.44–6.50 (1 H, m), 5.96 (1 H, dd, J = 17.56, 11.14Hz), 5.15–5.26(2 H, m), 2.37 (2 H, q, J = 7.93Hz), 2.06–2.12 (1 H, m), 2.02 (3H, s), 1.87–1.95 (1 H, m), 1.74 (3 H, s), 1.59 (3 H, s).13C NMR(151 MHz, CHLOROFORM-d)δ ppm 195.1, 169.9, 153.7, 141.1,139.5, 113.8, 82.3, 38.1, 23.79, 23.79, 22.1, 9.12. MS (EI) m/z (%)(rel.int.): 210 [M+] (1), 150(18.38), 135(14), 121(19), 107(18.05),93(26), 82(41), 71(46), 55(29), 43(100).

Procedure 2(E)-8-hydroxy-3,7-dimethylocta-1,6-dien-3-yl-acetate

(5), 8-hydroxylinalyl AcetateCompound 4 (800mg, 3.81mmol) was dissolved in dry methanol(40ml) and sodium borohydride (NaBH4; 1.8 g, 4.72mmol)was added (Liu et al., 2003; Scheme 2). The solution wasallowed to stir at −10◦C. After 1 h, water was added and thereaction mixture was extracted with dichloromethane (DCM).The organic layer was dried over sodium sulfate. After removalof the solvent, the residue was subjected to flash chromatographyeluted with petroleum ether/diethyl ether 2:3 (v/v) and yielded626mg (77%) of 5 as light yellow oil.1H NMR (360 MHz,CHLOROFORM-d) δ ppm 5.97 (1 H, dd, J = 17.48, 10.90Hz),

SCHEME 2 | Synthetic pathways for the synthesis of linalool and linalyl acetate oxygenated derivatives following procedures 1-4.






5.36–5.43 (1 H, m), 5.15 (2 H, dd, J = 17.48, 11.13Hz), 3.99(2 H, d, J = 5.45Hz), 2.03–2.09 (2 H, m), 2.01 (3 H, s),1.75–1.96 (2 H, m), 1.66 (3 H, s), 1.55 (3 H, s). 13C NMR (91MHz, CHLOROFORM-d) δ ppm 169.9, 141.7, 135.2, 125.4, 113.3,82.8, 68.8, 39.4, 23.7, 22.2, 21.9, 13.6. MS (EI) m/z (%) (rel.int.):211 [M+-1] (1), 134(7), 119(27), 93(46), 79(35), 67(30), 55(24),43(100).

Procedure 3(E)-2, 6-dimethylocta-2,7-diene-1,6-diol (6),

8-hydroxylinaloolCompound 5 (311 mg, 1.46mmol) was dissolved in methanol(50ml) and 0.1M KOH (50ml) was added (Hasegawa, 1983;Scheme 2). The reaction mixture was allowed to stir at 60◦C.After 4 h, the solution was extracted with DCM and theorganic layer was dried over sodium sulfate. The solvent wasremoved under reduced pressure and flash chromatographicpurification with petroleum ether/diethyl ether 1:4 (v/v) yielded203.8mg (82%) of 6 as transparent oil.1H NMR (600 MHz,CHLOROFORM-d) δ ppm 5.93 (1 H, dd, J = 17.19, 10.76Hz),5.41–5.45 (1 H, m), 5.16 (2 H, dd, J = 17.75, 10.95Hz), 4.00 (2H, s), 2.03–2.16 (2 H, m), 1.68 (3 H, s), 1.59–1.65 (2 H, m), 1.31(3 H, s). 13C NMR (151 MHz, CHLOROFORM-d) δ ppm 144.9,135.0, 125.9, 111.8, 73.3, 68.9, 41.7, 27.9, 22.3, 13.6. MS (EI) m/z(%) (rel.int.): 170 [M+] (1), 150(16), 135(13), 131(18), 107(17),95(25), 82(39), 71(44), 55(28), 43(100).

General Procedure 4 (GP4)Pinnick oxidation was used for the following syntheses(Pinnick et al., 1981; Scheme 2). The aldehydes 3 and 4 weredissolved in 25ml of tert-butyl alcohol and 6ml 2-methyl-2-butene. A solution of sodium chlorite (9.2 eq.) and sodiumdihydrogenphosphate (6.9 eq.) in 10ml water was added dropwise over a 10min period. The reaction mixture was stirredat room temperature overnight. Volatile components were thenremoved under vacuum, the residue was dissolved in 30ml waterand this was extracted with two 15ml portions of hexane. Theaqueous layer was acidified to pH 3 with HCl and extracted withthree 20ml portions of ether. The combined ether layers werewashed with 50ml cold water dried and concentrated to give 7and 8, respectively.

(E)-6-hydroxy-2,6-dimethylocta-2,7-dienoic-acid(7), 8-carboxylinaloolFollowing GP4, Compound 3 (800 mg, 4.75mmol) was dissolvedin 25ml tert-butyl alcohol and 6ml 2-methyl-2-butene. Asolution of sodium chlorite (3.95 gm, 43.7mmol) and sodiumdihydrogenphosphate (3.93 gm, 32.7mmol) in 10ml water wasadded dropwise over a 10min period, compound 7was prepared.Flash chromatographic purification with ethyl acetate/methanol9.5:0.5 (v/v) yielded 373.7mg (42.6%) of 7 as white solidspecks.1HNMR (600MHz, CHLOROFORM-d) δ ppm 6.76–6.94(1 H, m), 5.89 (1 H, dd, J = 17.37, 10.58Hz), 5.22 (1 H, dd, J =17.37, 1.13Hz), 5.08 (1 H, dd, J = 10.76, 0.94Hz), 2.14–2.33 (2H, m), 1.81 (3 H, s), 1.64 (2 H, m, J = 18.70, 10.60Hz), 1.30(3 H, s). 13C NMR (151 MHz, CHLOROFORM-d) δ ppm 172.1,144.5, 144.4, 127.1, 112.2, 72.9, 40.4, 27.9, 23.6, 12.0. MS (EI)m/z

182 [M+-2] (1), 151(4), 138(7), 121(15), 111(14), 103(16), 95(16),82(11), 71(100), 67(18), 55(24).

(E)-6-acetoxy-2,6-dimethylocta-2,7-dienoic-acid(8), 8-carboxylinalyl AcetateFollowing GP4, compound 4 (0.3 gm, 1.24mmol) was dissolvedin 25ml tert-butyl alcohol and 6ml 2-methyl-2-butene. Asolution of sodium chlorite (1.08 gm, 11.4mmol) and sodiumdihydrogenphosphate (1.05 gm, 8.55mmol) in 10ml water wasadded dropwise over a 10min period, compound 8was prepared.Flash chromatographic purification with ethyl acetate/methanol9.5:0.5 (v/v) yielded 131.8mg (47%) of 8 as light yellow oil.1HNMR (600 MHz, CHLOROFORM-d) δ ppm 6.87–6.92 (1 H, m),5.95 (1 H, dd, J = 17.37, 10.95Hz), 5.17 (2 H, dd, J = 17.37,10.95Hz), 2.17–2.24 (2 H, m), 2.02 (3 H, s), 1.84–2.00 (2 H, m),1.83 (3 H, s), 1.57 (3 H, s). 13CNMR (91 MHz, CHLOROFORM-d) δ ppm 173.1, 169.8, 144.1, 141.1, 127.2, 113.5, 82.3, 38.1, 23.6,23.3, 22.0, 11.8. MS (EI) m/z 226 [M+] (2), 166(15), 148(19),121(100), 105(84), 91(91), 79(98), 67(89), 55(42).

Results

Odor qualities for 1 and 2 and their C-8 oxygenated synthesizedderivatives were investigated by trained panelists using GC-O,the obtained attributes are shown in Table 2. It was found thatlinalool, 8-oxolinalool and 8-hydroxylinalool exert the same orat least closely related odor qualities. Odor attributes namedby the panelists were citrus-like, sweet, soapy, and lemon-like,whereas no odor was perceived at the sniffing port in caseof 8-carboxylinalool. It is worth mentioning that the lattercompound was odorless for all panelists in the concentrationlevels evaluated, that means at a concentration up to about200µg/ml.

The odor of linalyl acetate and its derivatives was described ascitrus-like, soapy, fatty, and fresh similarly to linalool with the soleexception of 8-carboxylinalyl acetate; the smell of this substancewas described as waxy, fatty, musty, rancid, and greasy. Panelistsdid not mention in any case the attributes citrus-like, soapy, fresh,or lemon-like for the latter compound.

As shown in Table 2, it is worth mentioning that the attributesprovided by 60–70% of the panelists in case of linalool werecitrus-like and flowery, whereas soapy was selected by only 37%of the panel as descriptor. Only one panelist described linaloolodor as balsamic. Regarding the 8-oxolinalool odor, all panelistsagreed on the attributes fatty and citrus-like, while 25% ofthe panel named the attribute soapy. In contrast to the lattertwo compounds, exhibiting intense odor, 8-hydroxylinalool wasperceived by most panelists as low in odor intensity even in aconcentration of 390µg/ml at the sniffing port. About 88% of thepanelists provided the attribute citrus-like for description of thelatter compound, while only one panelist perceived the substanceas soapy and flowery; unlike for linalool and the 8-oxolinalool, nopanelist mentioned fatty as a descriptor for 8-hydroxylinalool.

In view of linalyl acetate, the citrus-like impression wasreported by 60% of the panelists; fatty and soapy were namedby 25% while lemon-like and melissa-like attributes were givenby only one panelist. Furthermore, for the 8-oxolinalyl acetate






TABLE 2 | Odor qualities of all eight panelists (P1 to P8) and median of the odor threshold of all compounds.

Entrya Structure Odor qualitiesb OT [ng/L(air)]

P1 P2 P3 P4 P5 P6 P7 P8 Range Median

1 OH Citrus,

soapy,

fresh

Citrus,

soapy,

lemon-

like

Flowery,

balsamic

Citrus,

sweet

Citrus,

flower

Citrus,

soapy,

flower

Lemon-

like,

green,

fatty

Citrus,

flowery

2.1–8.4 2.1

2 OAc Citrus,

fatty,

sweet

Citrus,

fatty

Citrus,

fresh,

acidic

Sweet,

fatty

Citrus Soapy,

fatty

Lemon-

like,

Melissa

Citrus 12.7–407 152.5

3 OH

O

Fatty Lemon-

like,

sweet,

citrus,

soapy

Fatty,

fruity,

balsamic

Fatty,

citrus

Lemon-

like,

citrus

Fatty,

citrus

Fresh,

citrus,

lemon-

like

Fatty,

citrus,

soapy

4.8–305 28.55

4 OAc

O

Citrus,

fatty,

soapy

Lemon-

like,

fatty,

sweet

Citrus,

fatty,

linalool-

like,

soapy,

balsamic

Sweet,

fatty

Lemon-

like,

flower

Soapy,

fatty

Lemon-

like,

green,

fresh

Citrus,

fatty

0.6–79 4.9

5 OAc

OH

Citrus,

fatty

Citrus,

soapy

Citrus,

orange,

flowery,

balsamic

Citrus,

fatty,

fresh,

fruity

Fresh,

fruity

Citrus,

fatty

Lemon-

like,

fresh

Citrus,

flowery

4.9–634 198

6 OH

OH

Citrus Lemon-

like,

sweet,

flowery

Citrus,

fresh

Citrus,

sweet

Fresh Citrus,

soapy,

sweet

Lemon-

like,

orange

Citrus 7.7–989 247

7 OH

OH

O

Odorless Odorless Odorless Odorless Odorless Odorless Odorless Odorless – –

(Continued)






TABLE 2 | Continued

Entrya Structure Odor qualitiesb OT [ng/L(air)]

P1 P2 P3 P4 P5 P6 P7 P8 Range Median

8 OAc

OH

O

Fatty,

musty,

rancid

Sweet,

musty

Fatty,

greasy

Fatty,

greasy

Fatty Fatty Fatty Fatty,

waxy

1.5–24 6.1

aNumbering refer to Table 1.bOdor qualities as perceived at the sniffing port.

TABLE 3 | Odor thresholds OT (GC-O) of all eight panelists (P1 to P8) of all compounds.

Entrya Odorant OT [ng/L(air)]b Groupc Literatured

P1 P2 P3 P4 P5 P6 P7 P8

1 Linalool 8.4 8.4 2.1 2.1 2.1 2.1 4.2 2.1 3.2 n.r.

2 Linalyl acetate 407 203 51 102 12.7 203 407 51 110.9 n.r.

3 8-Oxolinalool 305 38 19 76 4.8 9.5 305 19.1 38.1 n.r.

4 8-Oxolinalyl acetate 2.8 4.9 4.9 79 0.6 4.9 10 10 5.9 n.r.

5 8-Hydroxylinalyl acetate 634 634 40 79 4.9 317 317 20 102.8 n.r.

6 8-Hydroxylinalool 989 989 247 247 7.7 31 494 62 160.3 n.r.

7 8-Carboxylinalool – – – – – – – – – n.r.

8 8-Carboxylinalyl acetate 3.1 3.1 6.1 6.1 1.5 24 24 6.1 6.1 n.r.

aNumbering refer to Table 1.bOdor thresholds in air were determined as described by Ullrich and Grosch (1987).cGroup odor threshold was calculated as a geometric mean of the individual thresholds of panelists.dn.r.: Odor threshold was not reported previously.

odor 75% of the panelists concordantly reported a fatty attribute.About 40% of the panel further described the odor as lemon-likeand citrus-like while only one panelist gave the attribute balsamicand linalool-like. Nevertheless, the main 8-hydroxylinalyl acetateodor attributes were citrus-like, being named by most of thepanelists (70–80%), while fresh and fruity were given by only20% of the panel. In contrast to all the previously mentionedodors, the 8-carboxylinalyl acetate smell was described asfatty and additionally as musty, rancid and greasy, but notas soapy.

When evaluating more closely the individual odor thresholdresults of the panelists, it becomes evident that there aresome inter-individual differences that do not only vary from acompound to another but also for a specific substance. Overall,all compounds were perceived with an intense to mediumintense odor with the sole exception of 8-hydroxylinaloolwhich imparted weak odor intensity. Thereby, one panelist wasexceptionally sensitive to all compounds, recording a thresholdvalue as low as 0.6 ng/L for the 8-oxolinalyl acetate, thus,being the lowest threshold value determined within this study(Table 3). In case of linalool, 60% of the panelists achieved athreshold value of 2.1 ng/L while in case of the other compoundsnot more than two panelists concordantly displayed the same

odor threshold. To name but one example, the highest thresholdwas recorded for two panelists for 8-hydroxylinalool with 989ng/L; this value is by a factor of 128 higher than the lowestrecorded threshold (7.7 ng/L), in this case again achieved by thesensitive panelist who was discussed before.

When comparing linalool with its oxygen-containing analogs,we found that linalool is the most potent odorant having anodor threshold of 3.2 ng/L in air (Table 3). All other compoundsinvestigated within this study exhibited an odor threshold ofat least a factor of 2 higher than the threshold of linalool. Toanalyze the secret beyond this intensive odor, it is feasible to havea closer look at the respective substituents on the monoterpenestructure.

Addition of an aldehyde group at C-8 of linalool increases thethreshold by a factor of 12 (38.1 ng/L). Reduction of this aldehydeto the corresponding alcohol, giving the 8-hydroxylinalool,results in a dramatic decrease in the potency, and a large increasein the threshold value (160.31 ng/L). Upon oxidation of the8-oxolinalool to the corresponding 8-carboxylinalool the odortotally disappears. This means that the C-3 hydroxy group is theonly substituent responsible for the linalool high potency and lowthreshold value, whereas any substituents on C-8, referring tothis study (see Figure 2), especially the aldehydic or the alcoholic






FIGURE 2 | Influence of oxygenated functional groups on the odor

threshold of odorants.

functional groups, can still own the same linalool pleasant smellbut lack its potency.

For linalyl acetate, the acetate ester of linalool, the odorthreshold was determined to be 110.9 ng/L, which is the highestvalue in relation to linalool despite its sweet, citrus fresh odor.Surprisingly, 8-oxolinalyl acetate, the linalyl acetate-8-aldehyde,was found to be the most potent compound of its correspondingester derivatives (see Figure 2) with an odor threshold of 5.9 ng/Lwhich is close to the odor threshold of linalool itself. Its odorquality was also described to be linalool-like and very intensecompared to that of its parent substance, the linalyl acetate.Again, the reduction of the C-8 aldehyde to the respective alcoholgives the 8-hydroxylinalyl acetate with an odor threshold of 102.8ng/L which is comparatively lower than that of linalyl acetateitself. Interestingly, the 8-carboxylinalyl acetate, the oxidationproduct of the 8-oxolinalyl acetate, retained the odor threshold(6.1 ng/L) to be nearly the same as for the 8-oxolinalyl acetate(5.9 ng/L) but displayed a complete change in the odor qualityto reveal greasy, rancid, and musty attributes rather than the

citrus-like, soapy, and lemon-like qualities of the 8-oxolinalylacetate (Table 2).

Conclusion

From the previous results, one can deduce first insightsinto structure-odor relationships for the investigated linaloolderivatives. Amongst others, the presence of a hydroxy groupat C-3 in linalool is the main contributor to both odor qualityand potency of all mentioned compounds in this study; thereby,the C-8 position does not contain any functionality in case oflinalool. On the contrary, the acetate derivative of this hydroxygroup, linalyl acetate, displayed low odor potency. However, wecould show that this is compensated by C-8 oxidation yielding8-oxolinalyl acetate and the 8-carboxylinalyl acetate with lowthresholds that are in a comparable range as the threshold oflinalool but eliciting different odor attributes. On the other handwe could show that the reduced moiety at the C-8 oxidationproducts yielding the corresponding hydroxy function, does not

positively contribute to odor potency, irrespective of whetherthe C-3 bares a hydroxy or an ester function; this structuralmodification resulted in the highest odor thresholds determinedwithin this study. To sum up, it can be concluded that in view ofthe investigated substances predominantly the C-3 substitutionwith a hydroxy group, a relatively non-voluminous and polarligand, is important for high odor potency and the characteristicsmell properties that are related to linalool. If this hydroxygroup is esterified, then C-8 substitution with either an aldehydeor a carboxyl group is crucial to maintain the odor threshold,albeit, thereby losing the specific odor character. Any otherstructural changes investigated within this study led to eitherdrastic decrease in the potency or even total odor loss.

Acknowledgments

We thank the members of our working group for theirparticipation in the sensory analyses.

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51

4 Structure-Odor Relationship Study on Geraniol, Nerol and Their Synthesized Oxygenated Derivatives.

Journal of Agricultural and Food Chemistry, 2016.

Special Issue: 11th Wartburg Symposium on Flavor Chemistry and Biology

Shaimaa Awadain Elsharif† and Andrea Buettner*,†,§

†Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander-Universitat Erlangen-Nürnberg, Henkestrasse 9, 91054 Erlangen, Germany, *(A.B.) Phone: +49-9131-85 22739. E-mail: [email protected] §Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Strasse 35, 85354 Freising, Germany


Reprinted with permission from (Elsharif, S.A. and A. Buettner, Structure–Odor Relationship Study on Geraniol, Nerol, and Their Synthesized Oxygenated Derivatives. Journal of Agricultural and Food Chemistry, 2016.).

Copyright © 2016 American Chemical Society


Structure−Odor Relationship Study on Geraniol, Nerol, and TheirSynthesized Oxygenated DerivativesShaimaa Awadain Elsharif† and Andrea Buettner*,†,§

†Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander-Universitat Erlangen-Nurnberg,Henkestrasse 9, 91054 Erlangen, Germany§Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Strasse 35,85354 Freising, Germany

ABSTRACT: Despite being isomers having the same citrus-like, floral odor, geraniol, 1, and nerol, 3, show different odorthresholds. To date, no systematic studies are at hand elucidating the structural features required for their specific odorproperties. Therefore, starting from these two basic structures and their corresponding esters, namely, geranyl acetate, 2, andneryl acetate, 4, a total of 12 oxygenated compounds were synthesized and characterized regarding retention indices (RI), massspectrometric (MS), and nuclear magnetic resonance (NMR) data. All compounds were individually tested for their odorqualities and odor thresholds in air (OT). Geraniol, the Z-isomer, with an OT of 14 ng/L, was found to be more potent than itsE-isomer, nerol, which has an OT of 60 ng/L. However, 8-oxoneryl acetate was the most potent derivative within this study,exhibiting an OT of 8.8 ng/L, whereas 8-oxonerol was the least potent with an OT of 493 ng/L. Interestingly, the 8-oxoderivatives smell musty and fatty, whereas the 8-hydroxy derivatives show odor impressions similar to those of 1 and 3. 8-Carboxygeraniol was found to be odorless, whereas its E-isomer, 8-carboxynerol, showed fatty, waxy, and greasy impressions.Overall, we observed that oxygenation on C-8 affects mainly the odor quality, whereas the E/Z position of the functional groupon C-1 affects the odor potency.

KEYWORDS: 8-hydroxygeraniol, odor threshold in air, retention indices, gas chromatography−olfactometry, mass spectrometry

■ INTRODUCTION

Geraniol, 1, or (2E)-3,7-dimethylocta-2,6-dien-1-ol, and its Z-isomer, nerol, are fragrant substances of great value.1 The smellof geraniol has previously been described as sweet, fruity, andberry-like, whereas nerol has been reported as having floral,citrus-like, and sweet-smelling properties.2 Geraniol, 1, andgeranyl acetate, 2, are major components of the essential oil ofCymbopogon martinii Roxb. Wats. var. motia, known aspalmarosa, which is commonly obtained from its leaves andflowering tops.3 Besides being used as a scenting substance insoaps, perfumes, and cosmetics, palmarosa oil is a potentmosquito repellent and antiseptic and has also pain-relievingproperties.3 Various studies further show that 1 has antitumoractivity against various cancer cells, both in vitro and in vivo.4,5

Geraniol, 1, has been reported to exert several otherpharmacological effects such as antimicrobial, antibacterial,anti-inflammatory, and antioxidant effects, as well as neuro-protectivity and hepatoprotectivity.6 1 has also been shown toexert an anticonvulsant effect, which has been proven to beindependent of benzodiazepine receptors.7 Nerol, 3, or (2Z)-3,7-dimethylocta-2,6-dien-1-ol, is a monoterpene that can befound in various medicinal plants such as Lippia spp. andMelissa officinalis L. These species are renowned for theirantimicrobial, antioxidant, and antiviral properties due to thepresence of nerol.8−10 It was also shown that 3, just like severalother monoterpene alcohols, exerts an anxiolytic effect in miceand can also be used for the treatment of anxiety andrestlessness.11 In addition, nerol is not only applied as anodorant in fragrances, soaps, and shampoos but also added tohousehold cleaners and detergents.12 Neryl acetate, 4, was

found as one of the major compounds isolated from theessential oil of the curry plant, Helichrysum italicum (Asteraceae), which is known for its anti-inflammatory,antioxidant, fungicidal, and astringent effects.13 Moreover, theessential oil of H. italicum is used as an emollient and as afixative in perfumes due to its intense odor.13 Still, the smell ofneryl acetate is described in the literature as rose-like, not spicyand curry-like as one might assume from its dominant presencein H. italicum essential oil.14

Apart from the natural occurrence of the above-mentionedcompounds 1−4, a series of derivatives have been observed inboth plants and animals. After oral administration of geraniol,8-hydroxygeraniol and 8-carboxygeraniol were isolated asmetabolic products from the urine of rats.15 Interestingly, thecarbon 8-hydroxylation of geraniol and nerol is also the firststep in the biosynthesis of indole alkaloids in the higher plantC. roseus.15 8-Hydroxygeraniol and 8-hydroxynerol were furtherfound as glycosidically bound monoterpenes in Shiraz andMuscat of Alexandria leaves and berries16 and as aglycones innectarines.17 8-Carboxygeraniol, also known as foliamenthoicacid, was isolated in small amounts from Tongling white gingerand was found as a secoiridoid derivative, foliamenthin, in

Special Issue: 11th Wartburg Symposium on Flavor Chemistry andBiology

Received: October 11, 2016Revised: October 27, 2016Accepted: October 29, 2016Published: October 29, 2016

Article

pubs.acs.org/JAFC

© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.6b04534J. Agric. Food Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/JAFC

http://dx.doi.org/10.1021/acs.jafc.6b04534

Menyanthes trifoliata.18 Geranyl and neryl acetates, 2 and 4, arealso present in the peel and leaf oils of lemon and lime speciesbelonging to the family Rutaceae,19 as well as in the peel,flowers (neroli), and leaves (petitgrain) of bitter orange (Citrusaurantium L.).20 Geranyl acetate can be further found in sweetflag leaves at different growing phases.21 Its odor was describedin the literature as woody, floral, and rosy.2 Among theoxidation products of the acetates, 2 and 4, the 8-oxogeranylacetate is the only compound that was found as a constituent ofthe attractant pheromone blend that is secreted by theAustralian predaceous bug, Oechalia schellenbergii22 (Table 1).Apart from that, no systematic data are available on the odor

characteristics of these metabolites or derivatives or of anyother physiological effects of these compounds on humans oranimals. To close this gap, the aim of our study was tosystematically evaluate the odor qualities and odor thresholdsof these substances as we previously did in a comparativeapproach for the related derivatives of linalool and linalylacetate.23 To achieve this aim, we synthesized, starting fromgeraniol, nerol, and their acetates, reported metabolites andhypothetical derivatives of these acyclic monoterpenesfollowing the same study design as in our previousinvestigation. The aim was to allow for a comprehensiveanalytical and olfactory evaluation and to establish an extendedsubstance library aiding future analytical research, therebycomprising data on retention indices (RI values) as well as massspectrometric and nuclear magnetic resonance data.

■ MATERIALS AND METHODSChemicals. Geraniol, nerol, geranyl acetate, neryl acetate, selenium

dioxide, sodium borohydride, methanol anhydrous, ethanol, dioxane,

tert-butyl alcohol, 2-methyl-2-butene, sodium chlorite, sodiumdihydrogen phosphate, ethyl acetate, hexane, and magnesium sulfatewere purchased from Aldrich (Steinheim, Germany), and diethyl etherwas from Fisher Scientific (Loughborough, UK).

General Methods. All reactions requiring anhydrous conditionswere carried out under nitrogen, and the solvents were dried beforeuse to remove moisture using appropriate drying agents. All reactionswere monitored by thin-layer chromatography (TLC) using silica gel60 F254 plates (Merck, Darmstadt, Germany). Visualization of thereaction components was achieved using ultraviolet (UV) fluorescence(254 nm) and potassium permanganate (KMnO4) staining. Columnchromatography was carried out over silica gel 60. The yields reportedare after purification. 1H and 13C nuclear magnetic resonance (NMR)spectra were recorded in deuterated solvents, and chemical shifts (δ)are quoted in parts per million (ppm) calibrated to tetramethylsilane(TMS) (1H and 13C). Coupling constants (J) were measured in hertz(Hz). The identity of all intermediates and synthetic products wasdetermined by electron ionization mass spectrometry (EI-MS).

Nuclear Magnetic Resonance (NMR) Spectra. 1H and 13CNMR spectra were recorded in deuterated chloroform (CDCl3) onAvance 360 and Avance 600 spectrometers (Bruker Biospin,Rheinstetten, Germany) at room temperature operated at 360 or600 MHz (1H) and 90 or 150 MHz (13C), respectively, with TMS asinternal standard.

Gas Chromatography−Olfactometry (GC-O) and GC−Elec-tron Ionization−Mass Spectrometry (GC-EI-MS). GC-O analyseswere performed with a Trace GC Ultra (Thermo Fisher ScientificGmbH, Dreieich, Germany) by using the following capillaries: FFAP(30 m × 0.32 mm i.d. fused silica capillary, free fatty acid phase FFAP,film thickness = 0.25 μm; Chrompack, Muhlheim, Germany) and DB5(30 m × 0.32 mm i.d. fused silica capillary DB-5, film thickness = 0.25μm; J&W Scientific, Fisons Instruments, Mainz-Kastel, Germany). Thehelium carrier gas flow was set for FFAP and DB5 capillary columns at2.5 and 1.2 mL/min, respectively. The compounds eluting at the end

Table 1. Retention Indices and Occurrence of the Acyclic Monoterpenes and Their Derivatives

RIa

compound odorant DB5 FFAP previously identified

1 geraniol 1260 1850 examples: palmarosa (Cymbopogon martinii (Roxb.) Poaceae,3,39 Ocimum gratissimum Lamiaceae,40 lemon balm(Melissa of f icinalis L.) Laminaceae41

2 geranyl acetate 1382 1768 examples: palmarosa (Cymbopogon martinii (Roxb.) Poaceae,3,39 Citrus limon (L.) Burm. and Citrus aurantifolia(Christm.) Swing (family Rutaceae),19 Sweet Flag (Acorus calamus L.) Araceae21

3 nerol 1233 1766 Lippia spp.9 and Melissa of f icinalis L.8 Warionea saharae Benth & Coss. (Asteraceae),42 Xeranthemumcylindraceum and X. annum Astraceae,43 sweet orange essential oil (Citrus sinensis (L.) Osbeck)44

4 neryl acetate 1363 1720 Helichrysum italicum (Roth) G.Don subsp. italicum (Asteraceae),13 Citrus limon (L.) Burm. and Citrusaurantifolia (Christm.) Swing (family Rutaceae),19 Citrus aurantium L. (Rutaceae)20

5 8-oxogeraniol 1516 2400 fruits of Amomum tsao-ko Zingiberaceae,45 component of the secretion of male scent organs of Africanmilkweed butterflies (Danainae)46

6 8-oxogeranylacetate

1620 2350 pheromone blend from Australasian predaceous bug, Oechalia schellenbergii (Heteroptera: Pentatomidae)22

7 8-oxonerol 1494 2462 not reported8 8-oxoneryl acetate 1600 2315 not reported9 8-hydroxygeraniol 1531 2642 fruits of Euterpe oleracea Mart. (Arecaceae),47 leaves and grape berries from Vitis vinifera Muscat of Alexandria

and Shiraz cultivars,16 yellow-fleshed nectarines (Prunus persica L. cv. Springbright),17 as a geraniol metabolitein urine of rats15

10 8-hydroxygeranylacetate

1631 2512 not reported

11 8-hydroxynerol 1505 2600 yellow-fleshed nectarines (Prunus persica L. cv. Springbright),17 leaves and grape berries from Vitis viniferaMuscat of Alexandria and Shiraz cultivars16

12 8-hydroxynerylacetate


13 8-carboxygeraniol 1665 3228 from tongling white ginger Zingiber of f icinale Roscoe, Zingiberaceae,48 flowers of Osmanthus f ragrans var.aurantiacus,49 leaves of Tecoma chrysantha JACQ,18 as a geraniol metabolite in urine of rats15

14 8-carboxygeranylacetate


15 8-carboxynerol 1659 3207 not reported16 8-carboxyneryl

acetate1765 3088 not reported

aRetention indices were determined as described by van den Dool and Kratz.24

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b04534J. Agric. Food Chem. XXXX, XXX, XXX−XXX

B


of the capillaries were split with a Y-splitter, ratio 1:1 (v/v) (J&WScientific), and transferred via two deactivated capillaries (0.5 m × 0.2mm) (J&W Scientific) to a flame ionization detector and a heatedsniffing port (temperature = 250 °C). The samples were applied ontothe capillary using a cold-on-column injector at 40 °C. After 2 min, theoven was heated at a rate of 10 °C/min to 240 °C and held for 7 min(DB-FFAP) or to 280 °C and held for 5 min (DB-5), respectively.GC-EI MS analyses were performed with an MSD 5975C (Agilent

Technologies, Waldbronn, Germany) and an ITQ 900 (ThermoFisher Scientific, Dreieich, Germany) with the capillaries describedabove. Mass spectra in the electron impact mode (EI-MS) weregenerated at 70 eV.Retention Indices (RI). Linear retention indices (Table 1) were

determined by GC-O using mixtures of individual odorants dissolvedin dichloromethane together with a series of n-hydrocarbons (C6−C34) dissolved in n-pentane, according to the method previouslydescribed.24

Evaluation of Odor Quality. The odor qualities were determinedduring GC-O evaluation with the aid of seven panelists (one male, sixfemales, aged 28−33 years) who were trained volunteers from theUniversity of Erlangen, exhibiting no known illness at the time ofexamination and with audited olfactory function.In preceding weekly training sessions, the assessors were trained for

at least half a year in recognizing orthonasally about 140 selectedknown odorants such as linalool, benzaldehyde, E-2-nonenal, andvanillin at different concentrations according to their odor qualitiesand in naming these according to an in-house-developed flavorlanguage. The odorants were presented to the panel in 140 mL Weckjars (J. Weck GmbH u. Co. KG, Wehr-Oflingen, Germany) as aqueoussolutions that were prepared following two protocols: (1) Either anethanolic stock solution of the odorant was prepared by dissolving thereference compounds in ethanol abs in given concentrations, followedby dilution of the odorant stock solution with tap water to reach abouta 10-fold concentration of the odorant above its respective odorthreshold in water, so that clear smell perception was ensured, therebytaking care that ethanol content remained limited to a minimum, or(2) the pure odorant was directly weighed and dissolved in tap water,again with the goal of reaching about a 10-fold concentration of theodor threshold value in water of the respective compound.With regard to the GC-O experiments that were carried out on the

synthesized compounds, samples were evaluated by each panelistrepeatedly on different days, as they performed dilution analyses, aswell as on different capillary columns (DB-FFAP and DB-5). Thereby,panelists were informed about elution times of the compounds toensure that inhalation corresponded with substance elution from thesniffing port and were asked to relate the naming of their sensoryimpressions to smell impressions that they recalled from training, ifpossible.Determination of Odor Thresholds. Odor thresholds were

determined in air following the procedure described by Czerny et al.25

using E-dec-2-enal as an internal odor standard. According to thisprocedure, E-dec-2-enal and the respective target compound weredissolved in dichloromethane at known concentrations, in each caseproviding one stock solution comprising both odorants. This stocksolution was diluted stepwise by a factor of 1:2 (v/v), and all dilutionswere applied to HRGC-O until no odor was perceivable. From theflavor dilution (FD) factors and the relative concentrations of standardand target compound, the odor threshold values in air were thencalculated as previously described.25 The detection odor thresholds ofthe panel were calculated as the geometric mean of the individualthresholds according to the method of Czerny et al.26

Syntheses, General Procedures. Procedure 1. Generally, themethod of Wakayama et al.27 was used (Figure 1). Compounds 1−4and selenium dioxide (SeO2) (1 equiv) were dissolved in dioxane/ethanol (9:1, v/v), and the solution was heated at 80 °C for 5 h. Afterremoval of selenium deposit by double filtration, the solvent wasremoved under reduced pressure using a rotary evaporator. Theresidue was purified twice by flash chromatography on silica gel 60(Merck), with a mobile phase of hexane/ethyl acetate, affording thecrude compounds 5−8.

(2E,6E)-8-Hydroxy-2,6-dimethylocta-2,6-dienal (5), 8-Oxogera-niol. Compound 5 was prepared following procedure 1 from 1 (1.9g, 12.9 mmol) and SeO2 (1.4 g, 12.9 mmol) in 30 mL of dioxane/ethanol (9:1, v/v). Flash chromatographic purification with hexane/ethyl acetate (1:1, v/v) yielded 268 mg (12.4%) of 5 as a yellow oil:1H NMR (360 MHz, CDCl3) δ 9.38 (s, 1 H), 6.46 (t, J = 6.70 Hz, 1H), 5.45 (t, J = 6.24 Hz, 1 H), 4.11 (d, J = 7.04 Hz, 2 H), 2.49 (q, J =7.57 Hz, 2 H), 2.10−2.15 (m, 2 H), 1.75 (s, 3 H), 1.70 (s, 3 H); 13CNMR (90 MHz, CDCl3) δ 194.7, 153.2, 139.1, 137.3, 124.1, 40.1, 27.0,16.0, 8.8; MS (EI) m/z (%) (rel int) 168 [M+] (1), 136 (8), 135 (81),121 (88), 105 (66), 91 (88), 79 (100), 77 (73), 67 (26), 55 (45).

(2E,6E)-3,7-Dimethyl-8-oxoocta-2,6-dien-1-yl acetate (6), 8-Ox-ogeranyl Acetate. Compound 6 was prepared following procedure 1from 2 (2.1 g, 10.5 mmol) and SeO2 (1.2 g, 10.5 mmol) in 30 mL ofdioxane/ethanol (9:1, v/v). Flash chromatographic purification withhexane/ethyl acetate (3:1, v/v) yielded 438 mg (20%) of 6 as a paleyellow oil 1H NMR (360 MHz, CDCl3) δ 9.37 (s, 1 H), 6.43 (t, J =7.20 Hz, 1 H), 5.37 (t, J = 7.04 Hz, 1 H), 4.58 (d, J = 7.04 Hz, 2 H),2.48 (q, J = 7.34 Hz, 2 H), 2.22 (t, J = 7.72 Hz, 2 H), 2.02−2.04 (m, 3H), 1.73 (s, 6 H); 13C NMR (150 MHz, CDCl3) δ 195.1, 171.0, 153.3,140.3, 140.0, 119.6, 61.1, 37.7, 27.0, 21.0, 16.4, 9.2; MS (EI) m/z (%)(rel int) 210 [M+] (1), 150 (3), 135 (56), 121 (100), 107 (39), 91(72), 79 (55), 77 (48), 67 (16), 55 (18).

(2E,6Z)-8-Hydroxy-2,6-dimethylocta-2,6-dienal (7), 8-Oxonerol.Compound 7 was prepared following procedure 1 from 3 (2.16 g, 14mmol) and SeO2 (3 g, 27 mmol) in 30 mL of dioxane/ethanol (9:1, v/v). Flash chromatographic purification with hexane/ethyl acetate (6:1to 1:1, v/v) yielded 200 mg (8.5%) of 7 as a yellow oil: 1H NMR (360MHz, CDCl3) δ 9.39 (s, 1 H), 6.46 (td, J = 7.27, 1.14 Hz, 1 H), 5.50(t, J = 6.93 Hz, 1 H), 4.14 (d, J = 7.00 Hz, 2 H), 2.45−2.48 (m, 2 H),2.26−2.31 (m, 2 H), 1.78 (s, 3 H), 1.75 (s, 3 H); 13C NMR (90 MHz,CDCl3) δ 195.1, 153.2, 139.8, 138.4, 125.5, 58.9, 30.5, 27.5, 23.2, 9.2;MS (EI) m/z (%) (rel int ) 168 [M+] (1), 149 (8), 135 (38), 121(46), 107 (33), 105 (41), 91 (93), 79 (100), 77 (89), 67 (33), 55 (55).

(2Z,6E)-3,7-Dimethyl-8-oxoocta-2,6-dien-1-yl Acetate (8), 8-Ox-oneryl Acetate. Compound 8 was prepared following procedure 1from 4 (2 g, 10.1 mmol) and SeO2 (1.2 g, 10.3 mmol) in 20 mL ofdioxane/ethanol (9:1, v/v). Flash chromatographic purification withhexane/ethyl acetate (6:1 to 1:1, v/v) yielded 171 mg (8%) of 4 as ayellow oil: 1H NMR (600 MHz, CDCl3) δ 9.39 (s, 1 H), 6.33−6.62(m, 1 H), 5.43 (t, J = 7.18 Hz, 1 H), 4.48−4.64 (m, 2 H), 2.48 (q, J =7.55 Hz, 2 H), 2.26−2.35 (m, 2 H), 2.04 (s, 3 H), 1.80 (s, 3 H), 1.75(s, 3 H); 13C NMR (150 MHz, CDCl3) δ 195.1, 170.9, 152.9, 140.8,139.8, 120.5, 60.7, 30.5, 27.3, 23.2, 21.0, 9.2. MS (EI) m/z (%) (rel int

Figure 1. Synthetic pathways leading to the target oxygenatedcompounds of geraniol, nerol, and their acetates.



C


) 210 [M+] (1), 150 (4), 135 (63), 121 (100), 105 (45), 91 (74), 79(67), 77 (55), 67 (16), 55 (19).Procedure 2. Generally, the method of Liu et al.28 was used (Figure

1). Compounds 5−8 and sodium borohydride (NaBH4) (2 equiv)were dissolved in dry methanol. The solution was stirred at −10 °C.After 1 h, water was added and the reaction mixture was extracted withdichloromethane. The organic layer was dried over sodium sulfate.After removal of the solvent, the residue was purified by flashchromatography on silica gel 60 (Merck), with a mobile phase ofhexane/ethyl acetate, affording the crude compounds 9−12. As thisapproach has been previously reported as rather a mild-selectiveprocedure,29 all target compounds were specifically analyzed by GC-MS and NMR and compared to data reported in the literature toexclude potential side reactions.(2E,6E)-2,6-Dimethylocta-2,6-diene-1,8-diol (9), 8-Hydroxygera-

niol. Compound 9 was prepared following procedure 2 from 5 (117mg, 0.69 mmol) and NaBH4 (78 mg, 2.1 mmol) in 10 mL of drymethanol. Flash chromatographic purification with hexane/ethylacetate (3:1, v/v) yielded 83.6 mg (70%) of 9 as a colorless oil: 1HNMR (360 MHz, CDCl3) δ 5.34−5.46 (m, 2 H), 4.14 (d, J = 6.81 Hz,2 H), 3.99 (s, 2 H), 2.12−2.19 (m, 4 H), 1.68 (s, 3 H), 1.58 (s, 3 H);13C NMR (90 MHz, CDCl3) δ 139.1, 135.2, 125.6, 124.0, 69.0, 59.3,39.1, 27.1, 16.2, 13.8; MS (EI) m/z (%) (rel int) 169 [M+ − 1] (1),151 (5), 137 (9), 121 (20), 119 (35), 105 (24), 93 (40), 91 (100), 67(99), 55 (22). The 1H NMR data fully agreed with an earlier paper.30

(2E,6E)-8-Hydroxy-3,7-dimethylocta-2,6-dien-1-yl Acetate (10),8-Hydroxygeranyl Acetate. Compound 10 was prepared followingprocedure 2 from 6 (17.5 mg, 0.083 mmol) and NaBH4 (41 mg, 1.1mmol) in 10 mL of dry methanol. Flash chromatographic purificationwith hexane/ethyl acetate (3:1, v/v) yielded 11.4 mg (64.5%) of 10 asa colorless oil: 1H NMR (600 MHz, CDCl3) δ 5.31−5.39 (m, 2 H),4.58 (d, J = 7.18 Hz, 2 H), 3.99 (d, J = 4.91 Hz, 2 H), 2.17 (q, J = 7.43Hz, 2 H), 2.07−2.12 (m, 2 H), 2.05 (s, 3 H), 1.70 (s, 3 H), 1.66 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 171.2, 141.7, 135.3, 125.2, 118.7,

68.9, 61.4, 39.0, 25.6, 21.0, 16.4, 13.7; MS (EI) m/z (%) (rel int ) 212[M+] (1), 152 (5), 134 (14), 121 (8), 119 (76), 105 (24), 93 (30), 91(100), 79 (69), 67 (82), 55 (19). The NMR spectral data agreed withHolsclaw et al.31

(2E,6Z)-2,6-Dimethylocta-2,6-diene-1,8-diol (11), 8-Hydroxyner-ol. Compound 11 was prepared following procedure 2 from 7 (25 mg,0.15 mmol) and NaBH4 (97 mg, 2.5 mmol) in 20 mL of dry methanol.Flash chromatographic purification with hexane/ethyl acetate (1:1, v/v) yielded 18.6 mg (72.9%) of 11 as a colorless oil: 1H NMR (360MHz, CDCl3) δ 5.34−5.51 (m, 1 H), 4.83−4.91 (m, 1 H), 4.05−4.08(m, 2 H), 3.98 (brs, 2 H), 2.12−2.17 (m, 4 H), 1.73 (s, 3 H), 1.65 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 138.6, 135.6, 125.0, 124.8, 68.6,58.9, 25.3, 23.3, 13.7; MS (EI) m/z (%) (rel int) 171 [M+ + 1] (1),151 (2), 137 (10), 121 (7), 119 (26), 105 (15), 93 (28), 91 (100), 79(88), 67 (71), 55 (16). The 1H NMR data agreed with Ono et al.32

(2Z,6E)-8-Hydroxy-3,7-dimethylocta-2,6-dien-1-yl Acetate (12),8-Hydroxyneryl Acetate. Compound 12 was prepared followingprocedure 2 from 8 (66 mg, 0.3 mmol) and NaBH4 (29 mg, 0.7mmol) in 10 mL of dry methanol. Flash chromatographic purificationwith hexane/ethyl acetate (2:1, v/v) yielded 32.5 mg (48%) of 12 as acolorless oil: 1H NMR (600 MHz, CDCl3) δ 5.39 (brs, 1 H), 5.34−5.38 (m, 2 H), 4.57 (d, J = 7.18 Hz, 2 H), 3.99 (s, 2 H), 2.13−2.18 (m,4 H), 2.05 (s, 3 H), 1.77 (s, 3 H), 1.67 (s, 3 H); 13C NMR (150 MHz,CDCl3) δ 171.1, 142.0, 135.6, 125.0, 119.6, 68.7, 61.2, 31.7, 26.0, 23.4,21.0, 13.63; MS (EI) m/z (%) (rel int ) 212 [M+] (1), 137 (10), 121(7), 119 (74), 105 (23), 93 (26), 91 (100), 79 (67), 67 (77), 55 (14),51 (11). The NMR data fully agreed with Tomooka et al.33

Procedure 3. Pinnick oxidation was used for the followingsyntheses34 (Figure 1). The aldehydes 5−8 were dissolved in tert-butyl alcohol and 2-methyl-2-butene. A solution of sodium chlorite(NaClO2) (9.2 equiv, 80%) and sodium dihydrogen phosphate(NaH2PO4) (6.9 equiv) in water was added dropwise over a 10 minperiod. The reaction mixture was stirred at room temperature (25−30°C) overnight. Volatile components were then removed under

Table 2. Odor Qualities Defined by Panelists and Median of the Odor Threshold of All Compounds

odor qualitiesa OT (ng/Lair)

compound PA PB PC PD PE PF PG range median

1 citrus, fresh,floral, sweet

citrus, fresh,fatty

citrus, floral citrus, orange-peel,neroli

citrus, fresh,floral, sweet

fatty, sweet,fruity

citrus, soapy 2.8−23.1 23.1

2 fruity fatty, sweet citrus, floral citrus, fresh citrus, sweet,floral

soapy lemon-like 18.8−150.7 75.3

3 citrus, fresh floral, sweet,fresh

citrus, floral lemon-peel,balsamic, fruity

citrus, fresh,lemon-like

soapy, citrus lemon, soapy 11.3−180.2 89.8

4 anosmia phenolic clove, floral clove, floral clove, sweet,floral

fatty, dusty phenolic,sweet

27−432 121.5

5 fatty, musty,old

sweet-sour,herb

fatty, musty floral, soapy, fresh fatty, musty fatty, musty citrus, fresh 39.8−637.2 318.6

6 fatty, musty sweet, fresh orange-peel,dusty, fatty

cumin, spicy fatty, musty,lemon-like

fatty, soapy,dusty

citrus, greasy 6.8−108 13.5

7 fatty, musty sweet, green,herb

fatty, musty sandal-wood,balsamic

fatty, musty fatty, dusty fatty, citrus 50.6−6480 405

8 citrus, fatty,musty

sweet wax, plastic balsamic, cumin citrus, fatty,musty

fatty, citrus citrus, sweet,fatty

0.41−26.2 13.1

9 fatty, soapy fresh, herb anosmia floral, citrus, sweet fatty, citrus fatty, fruity citrus 135−540 40510 fatty, citrus grassy, fresh floral, body-

lotionorange, fruity citrus, soapy citrus, soapy citrus, soapy 27−108 54

11 citrus, sweet,fresh

sweet, vanilla citrus balsamic, woody vanilla, sweet vanilla, sweet citrus 112.7−1803 450.9

12 citrus, floral,soapy

citrus, sweet citrus citrus citrus anosmia citrus 27−216 81

13 odorless odorless odorless odorless odorless odorless odorless14 floral, sweet,

coconutgrassy, dill plasticine anosmia plasticine,

greasycoconut,sweet

sweet 14−112.3 42.1

15 anosmia green, dill plasticine fatty, greasy fatty, waxy,greasy

fatty, waxy pungent 74.3−4752 74.3

16 sweet, roasted dill, green plasticine,waxy

acidic plasticine,waxy, greasy

coconut, fatty herbal, green 6.8−216 27

aOdor qualities as perceived at the sniffing port.



D


vacuum, the residue was dissolved in 30 mL of water, and this wasextracted twice with 15 mL portions of hexane. The aqueous layer wasacidified to pH 3 with HCl and extracted three times with 20 mL ofether. The combined ether layers were washed with 50 mL of coldwater, dried, purified by flash chromatography on silica gel 60(Merck), with a mobile phase of hexane/ethyl acetate, andconcentrated to give 13−16.(2E,6E)-8-Hydroxy-2,6-dimethylocta-2,6-dienoic Acid (13), 8-

Carboxygeraniol. Compound 13 was prepared following procedure3, where compound 5 (2.3 g, 14.1 mmol) was dissolved in 50 mL oftert-butyl alcohol and 20 mL of 2-methyl-2-butene. A solution ofNaClO2 (11.7 g, 161 mmol) and NaH2PO4 (11.6 g, 97 mmol) in 40mL of water was added dropwise over a 10 min period. Flashchromatographic purification with hexane/ethyl acetate (6:1 to 1.5:2,v/v) yielded 832 mg (32%) of 13 as a pale yellow oil: 1H NMR (600MHz, CDCl3) δ 6.76 (td, J = 7.25, 1.50 Hz, 1 H), 5.43 (td, J = 7.00,1.50 Hz, 1 H), 4.16 (d, J = 7.00 Hz, 2H), 2.33 (q, J = 7.50 Hz, 2H),2.16 (t, J = 7.50 Hz, 2H), 1.81 (s, 3H), 1.64 (s, 3H); 13C NMR (150MHz, CDCl3) δ 172.1, 146.5, 139.4, 127.1, 124.6, 58.9, 40.4, 27.3,16.4, 12.8; MS (EI) m/z (%) (rel int ) 166 [M+ − H2O] (1), 151 (19),148 (100), 121 (17), 117 (16), 105 (42), 91 (77), 79 (50), 77 (61), 67(23), 65 (30).(2E,6E)-8-Acetoxy-2,6-dimethylocta-2,6-dienoic Acid (14), 8-

Carboxygeranyl Acetate. Compound 14 was prepared followingprocedure 3, where compound 6 (175 mg, 0.83 mmol) was dissolvedin 25 mL of tert-butyl alcohol and 6 mL of 2-methyl-2-butene. Asolution of NaClO2 (694 mg, 9.5 mmol) and NaH2PO4 (691 mg, 5.7mmol) in 10 mL of water was added dropwise over a 10 min period.Flash chromatographic purification with hexane/ethyl acetate (3:1, v/v) yielded 75.4 mg (40%) of 14 as a pale yellow oil: 1H NMR (600MHz, CDCl3) δ 6.86 (td, J = 7.27, 0.94 Hz, 1 H), 5.30−5.42 (m, 1 H),4.59 (d, J = 7.18 Hz, 2 H), 2.34 (q, J = 7.43 Hz, 2 H), 2.14−2.22 (m, 2H), 2.05 (s, 3 H), 1.83 (s, 3 H), 1.72 (s, 3 H); 13C NMR (150 MHz,CDCl3) δ 173.4, 171.2, 143.9, 140.8, 127.5, 119.2, 61.2, 37.8, 27.0,20.9, 16.4, 12.0; MS (EI) m/z (%) (rel int) 226 [M+] (1), 166 (8), 150(7), 122 (11), 121 (100), 105 (46), 91 (37), 77 (26), 67 (10), 55 (5).(2E,6Z)-8-Hydroxy-2,6-dimethylocta-2,6-dienoic Acid (15), 8-

Carboxynerol. Compound 15 was prepared following procedure 3,where compound 7 (25 mg, 0.15 mmol) was dissolved in 20 mL oftert-butyl alcohol and 5 mL of 2-methyl-2-butene. A solution ofNaClO2 (174 mg, 2.4 mmol) and NaH2PO4 (210 mg, 2.3 mmol) in 10mL of water was added dropwise over a 10 min period. Flashchromatographic purification with hexane/ethyl acetate (1:1, v/v)yielded 10 mg (37%) of 15 as a yellow oil: 1H NMR (360 MHz,CDCl3) δ 6.83 (br dt, J = 6.70, 1.20, 1.20 Hz, 1 H), 5.48 (br t, J = 7.50,7.50 Hz, 1 H), 4.12 (d, J = 7.50 Hz, 2 H), 2.28−2.36 (m, 2 H), 2.20−2.27 (m, 2 H), 1.84 (s, 3 H), 1.73−1.78 (m, 3 H); 13C NMR (150MHz, CDCl3) δ 168.2, 141.4, 140.7, 127.1, 119.2, 60.5, 30.8, 27.2,23.2, 12.4; MS (EI) m/z (%) (rel int) 166 [M+ − H2O] (1), 151 (3),148 (100), 133 (3), 121 (8), 105 (10), 91 (12), 79 (8), 77 (11), 67(4), 65 (5).(2E,6Z)-8-Acetoxy-2,6-dimethylocta-2,6-dienoic Acid (16), 8-

Carboxyneryl Acetate. Compound 16 was prepared followingprocedure 3, where compound 8 (65 mg, 0.31 mmol) was dissolvedin 15 mL of tert-butyl alcohol and 5 mL of 2-methyl-2-butene. Asolution of NaClO2 (290 mg, 4.0 mmol) and NaH2PO4 (290 mg, 2.4mmol) in 10 mL of water was added dropwise over a 10 min period.Flash chromatographic purification with hexane/ethyl acetate (2:1, v/v) yielded 13.3 mg (19%) of 16 as a yellow oil: 1H NMR (600 MHz,CDCl3) δ 6.87 (t, J = 6.99 Hz, 1 H), 5.42 (t, J = 7.37 Hz, 1 H), 4.56(d, J = 7.18 Hz, 2 H), 2.33 (q, J = 7.30 Hz, 2 H), 2.24−2.28 (m, 2 H),2.05 (s, 3 H), 1.85 (s, 3 H), 1.79 (s, 3 H); 13C NMR (150 MHz,CDCl3) δ 173.0, 171.1, 144.0, 141.2, 128.0, 120.2, 61.0, 31.2, 27.3,23.3, 21.0, 12.0; MS (EI) m/z (%) (rel int) 226 [M+] (1), 166 (8), 151(7), 148 (5), 121 (100), 105 (48), 91 (40), 77 (33), 67 (11), 55 (6).

■ RESULTS AND DISCUSSION

The odor qualities of 1−4 and their synthesized oxygenatedderivatives were investigated by trained panelists using GC-O.

The obtained odor attributes are shown in Table 2. Thisprocedure offers the advantage that compounds that might bepresent as odor-active impurities accompanying the referencecompound are separated from the target odorants during thechromatographic separation step. Consequently, an influence ofsuch components on the results is ruled out. Also, odorthresholds can be compared to each other on an absolute basiswithout interference with any matrix system as would be thecase, for example, when odor thresholds are determined inwater, oil, or solid matrices. Potential pitfalls may arise in GC-Oif panelists do not take care to inhale at the time point ofodorant elution from the GC column, so they specifically needto be instructed on elution times as done in the present study.Moreover, the chromatographic shape of the substances mayvary between different columns of different polarities.Accordingly, it is recommendable to perform the experimentson capillaries with different polarities, as is done in the presentstudy. Results obtained were reproducible and consistent as thecomponents were evaluated more than once on differentcolumns on different days. In addition, no changes in odor wereobserved when panelists olfactorily evaluated different steps ofconcentration. It is worth mentioning that any impurity thatmight be still present with our synthesized compounds did notinterfere in the odor property owing to the fact that elution byGC-O on different capillary columns revealed the purecompounds without any interferences from even minuteamounts that may occur, for example, from organoseleniumbyproducts.In general, geraniol, 1, geranyl acetate, 2, and nerol, 3, shared

the main attributes citrus-like, floral, sweet, and fruity with thesole exception of neryl acetate, 4, which was described withunexpected odor attributes, namely, clove-like and phenolic.Panelists (three of seven) specifically reported an olfactorysimilarity to the characteristic clove constituent 4-eugenol.Geraniol, 1, was categorized by our panel as smelling citrus-

like as in previous studies.35 However, this odor descriptor wasmore clearly specified by some individuals to smell like orangepeel and neroli. Additional odor attributes given for 1 werefresh and fatty as perceived by 43% of the subjects, whereasonly 29% gave the attributes soapy and sweet. The odor ofgeranyl acetate, 2, was further described as sweet and floral by28% of the panel, whereas only two panelists used the termssoapy and fruity. On the other hand, other odor attributes suchas soapy and floral were given to nerol, 3, by 29% of the panel.Apart from that, 44−55% of the panel used the attribute lemon-like as a more precise descriptor for 3. The odor of nerylacetate, 4, was perceived by 42% of the panel as floral and sweetand by 29% as clove-like and phenolic as previously mentioned.Only one panelist perceived 4 as fatty-dusty. Despite beingrated by the panelists as an intense odorant, one panelist wasanosmic to 4 even when directly smelling the substance in itspure oil form from the bottle.With regard to the odor qualities of the oxygenated

derivatives, most of the 8-oxo derivatives were predominantlydescribed by our panel as fatty and musty, whereas the 8-oxogeranyl acetate, 6, and the 8-oxoneryl acetate, 8, were ratedas citrus-like by 42 and 57% of the panel, respectively. On theother hand, the 8-hydroxy derivatives showed odor attributescomparable to those of the respective parent alcohols oracetates. Accordingly, these attributes comprised the mainimpressions citrus-like, floral, soapy, and fruity. In addition tothese smell impressions, half of the panel rated the smell of 8-hydroxynerol, 11, rather unexpectedly, as sweet and vanilla-like.



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Apart from that, it is interesting to note that 8-hydroxynerylacetate, 12, was the sole substance that all panelistsunequivocally agreed on as being citrus-like, with the exceptionof only one panelist who was anosmic to this substance.In contrast to this, all 8-carboxy derivatives showed

plasticine-like, greasy, and waxy smells, with the sole exceptionof 8-carboxygeraniol, 13, which was unequivocally perceived asodorless by all panelists, even at a concentration of 500 μg/mL.However, this plasticine-like odor could not be further specifiedby the panel, as they could not relate this impression to specificodor references. Nevertheless, this is most likely due to the factthat the smell of plasticine as a product of daily use is rather acomplex odorant mixture than a single compound.Interestingly, one panelist described the smell of both 8-

carboxynerol, 15, and 8-carboxyneryl acetate, 16, as green anddill-like. Moreover, these two attributes were consistentlyreported by this panelist whenever smelling the two substances,even at different concentrations and on different days. On theother hand, sweet and coconut-like were recorded as attributesby 43% of the panel for the 8-carboxygeranyl acetate, 14,whereas only one subject named coconut-like for its isomer, 16.The same panelist who was anosmic to neryl acetate, 4, asmentioned earlier, was also anosmic to the 8-carboxynerol 15 atthe concentrations evaluated in this study, that is, aconcentration of 220 μg/mL.

With regard to the odor thresholds of the investigatedcompounds, it became clear, as shown in Figure 2A, thatgeraniol is the most potent compound (14 ng/Lair) comparedto its acetate, 2, and its corresponding oxygenated derivatives 5,6, 9, 10, and 14. Thereby, 8-hydroxygeraniol, 9, with an OT of340 ng/Lair, was the least potent compound in this substancegroup. In general, the OT values of geranyl acetate, 2, and itsoxygenated derivatives ranged between these OT values andpredominantly between 22 and 66 ng/Lair.It can be observed that oxidation of C-8 of geraniol to 8-

oxogeraniol led to an increase in the odor threshold by a factorof 13 (194 ng/Lair), thereby turning the citrus-like odor ofgeraniol to a fatty-musty smell. Reduction of the aldehydegroup to the corresponding alcohol, the 8-hydroxygeraniol,retained the citrus-like, fresh odor but led to a further decline inodor potency (340 ng/Lair). Further oxidation of the 8-oxogeraniol to the 8-carboxygeraniol resulted in total odor loss.This demonstrates that an increase in polarity at C-8 obviouslyreduces odor potency. This finding is in agreement with ourobservations in our previous structure−odor relationship studyof linalool and its C-8 oxygenated derivatives.23 Accordingly, itappears that not only does the presence of the OH group at C-1 in geraniol or at C-3 in linalool define the citrusy characterand high odor potency but that modification at C-8 alsoinfluences both odor quality and potency.

Figure 2. Influence of the oxygenated functional groups on the odor thresholds of (A) geraniol, geranyl acetate, and their oxygenated derivatives and(B) nerol, neryl acetate, and their oxygenated derivatives. Displayed are individual odor thresholds as reported by seven panelists.



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Despite being isomers and having comparable smell, nerol, 3,was found to be less potent than 1 by a factor of 4 (odorthreshold = 60.5 ng/Lair). Still, it is the most potent substanceamong its oxygenated derivatives, comparable to what weobserved for geraniol. Introduction of the aldehyde function atC-8 of nerol, giving 8-oxonerol, not only changed the smellfrom citrus-like to musty but also increased the threshold to493 ng/Lair. On the other hand, a hydroxy function at C-8,giving 8-hydroxynerol, retained the citrus-like main impressionbut additionally provoked a sweet, vanilla-like note that wasperceived by almost half of the panel. The 8-carboxynerol wasfound to be the most potent odorant among the neroloxidation derivatives with a threshold value of 268 ng/Lair;however, it exhibited a greasy, waxy odor. Apparently, increaseof the polarity of the C-8 oxygenated functional group in nerolresults in an increase of potency, which is opposite theobservations made for linalool and geraniol and theirderivatives. This consideration is, however, made regardless ofthe odor quality of the respective compounds, which alsochanged as discussed before.With regard to the acetate derivatives, 8-oxogeranyl acetate

was found to be the most potent compound (22 ng/Lair)among the geranyl acetate derivatives; it was dominated by amusty, dusty odor character. The alcoholic derivative, 8-hydroxygeranyl acetate, retained the citrus-like odor but with a3-fold higher odor threshold value (65 ng/Lair). Thus, the odorquality and threshold value of the 8-hydroxygeranyl acetate arenearly similar to those of the geranyl acetate. An acid moiety atC-8 in the 8-carboxygeranyl acetate produced a decrease in theodor threshold value to 39 ng/Lair; however, the smell turnedfrom citrus-like to sweet-coconut and plasticine-like.Overall, the term musty was used as a main descriptor for the

odor of all 8-oxo derivatives, mainly 8-oxogeraniol, 8-oxogeranyl acetate, and 8-oxonerol. The panel was not ableto further specify this musty odor by any other sensoryterminology but additionally reported the terms citrus-like (twopanelists) in the case of 8-oxoneryl acetate and fatty (allpanelists) for all 8-oxo derivatives. It has been previouslyreported that the term musty is a general term comprising suchdivergent impressions as dusty, papery, earthy, or moldy.36

Interestingly, and in line with these previous studies, the termmusty was further specified by two panelists by the additionalterm dusty in the case of the 8-oxogeranyl acetate and by onlyone panelist in the case of 8-oxonerol. Although geosmin and 2-methylisoborneol are compounds that are most commonlyassociated with musty odor,36 there are no common structuralfeatures between these substances and the odorants inves-tigated within the present study. According to Jong andBirmingham37 2-octanone and 3-octanone are two compoundsproduced by mushrooms that also possess a musty odor.However, none of these substances elicits a comparable mustyimpression as perceived for the compounds of the presentstudy. Nevertheless, one might postulate that the carbonylmoiety is a common functional group of these 8-carbon chainketones and our 8-oxo derivatives and might be responsible forthe musty odor perceived by the panel. On the other hand, thesame structural feature did not affect the pleasant odors of the8-oxolinalool and 8-oxolinalyl acetate as no musty smell wasrecorded for these compounds.23

As discussed above, neryl acetate, with a relatively high odorthreshold among its derivatives (96 ng/Lair), showedunexpected odor attributes, namely, clove-like and phenolic.These descriptors are not common among this substance group

and have never been reported before for this substance.Interestingly, the smell of neryl acetate has been previouslydescribed as rose-like.14 However, the method of smelldetermination was not detailed in this study, so it is unclearwhere these differences originate. Potentially, the differingimpressions of the previous study arose from other sideproducts or contaminations, as application of GC-O asanalytical strategy was not mentioned in the experimentalpart of the study. Interestingly, clove-like and phenolic wouldrelate to a completely different substance class, namely, guaiacoland phenol derivatives such as 4-vinylguaiacol.38 One mighthypothesize that the presence of a 4-carbon chain with a doublebond at C-2 and a methoxy or an acetoxy group at C-1 mightbe the common structural feature responsible for this smellimpression as illustrated in Figure 3.

From the data compiled in Table 3, where all individualthreshold values of the participants are shown, it becomesevident that interindividual variation in sensitivity to thesesubstances was pronounced. For example, 8-oxogeraniol wasfound to have the highest individual OT value (637 ng/Lair);yet, on average, it was not the least potent compound amongthe geraniol derivatives. It is interesting to note that no singlepanelist was especially sensitive to all compounds; however,one panelist recorded the lowest OT values for threecompounds, namely, geraniol (2.8 ng/Lair), geranyl acetate(18.8 ng/Lair), and 8-hydroxygeranyl acetate (27 ng/Lair).As can be seen from the data shown in Figure 2B, the lowest

OT values were determined in the nerol substance group with8.8 ng/Lair for 8-oxoneryl acetate and 27 ng/Lair for 8-carboxyneryl acetate, followed by nerol with a threshold valueof 60 ng/Lair. On the other hand, 8-oxonerol was found to bethe least potent compound in this study, with a threshold valueof 493 ng/Lair.When the data of individual panelists are analyzed, the

absolute lowest and highest individual OT values were recordedfor two nerol derivatives, with 0.41 ng/Lair for 8-oxonerylacetate and 6480 ng/Lair for 8-oxonerol. Interestingly, these twovalues were obtained from the same panelist; moreover, thispanelist was also exceptional in other terms, that is, anosmic to8-hydroxyneryl acetate. Overall, the odor threshold valuesshowed that, for each substance, at least two panelists showedcomparable OT values regardless of the odor quality perceived.In addition, not more than one subject was anosmic to eachcompound as previously mentioned, with a total of five caseswithin the whole study.From the above results, several insights into structure−odor

relationships can be deduced for the investigated geraniol andnerol derivatives. With the exception of 8-oxo and 8-carboxygeranyl acetate, all E-isomer derivatives showed lowerodor threshold values than their respective Z-analogues.Accordingly, the E/Z configuration greatly affects the potency

Figure 3. Common structure features that are potentially responsiblefor the common clove-like odor of both compounds.



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of the compounds regardless of the odor quality. An aldehydegroup at C-8 changed the odor completely from citrus-like tomusty, with 8-oxoneryl acetate showing somewhat exceptionalbehavior as this substance retained the citrus-like odor but was,in the case of two panelists, associated with a musty nuance. Bycomparison of the results of this investigation with our previousstudy on linalool and its derivatives,23 some links in structure−odor relationships become obvious. First, substituting C-8 withan OH group enhances the dominance of the citrus-likecharacter; this is especially true for compounds bearing anacetate moiety at C-1. In addition, 8-hydroxy C-1 acetates aremore potent than their respective 8-hydroxy C-1 alcohols. Next,a carboxyl group at C-8 is responsible for plasticine-like, greasyimpressions as these were named for all odor-active acidderivatives. Last, it could be demonstrated that an increase ofthe polarity of the oxygen-bearing group at C-8 of geraniol orlinalool23 decreases the odor potency until the odor disappears.In contrast to this, the polarity is directly related to the odorpotency for the oxygenated derivatives of nerol. Morespecifically, in this case a carboxyl function at C-8 of nerol ismore potent than an aldehyde group, regardless of the odorquality.To sum up, our study shows that geraniol, 1, nerol, 3, their

acetates (2 and 4), and their corresponding oxygenatedderivatives, 5−16, were found to be odor active with the soleexception of 8-carboxygeraniol. Odor threshold and odorquality determinations revealed that structural changes on thebasic skeleton of these monoterpenes led to derivatives that canbe more potent or more pleasant with regard to their smellcharacter than the parent monoterpene itself. The 8-oxo and 8-hydroxy derivatives of geraniol, geranyl acetate, and nerylacetate showed comparable smells for all panelists, whereasoxygenated derivatives of nerol showed very divergent odorcharacteristics among panelists, with, for example, highvariations in odor thresholds for 8-oxonerol and very divergentassignment of odor attributes for 8-carboxynerol. Furthermore,cases of anosmia were recorded five times for four panelists,three panelists for either 8-hydroxygeraniol, 8-carboxygeranylacetate, or 8-hydroxyneryl acetate and one panelist showedanosmia to both 8-carboxynerol and neryl acetate.

Overall, the structure−odor relationship of these mono-terpene alcohols can be summarized as follows: The OH groupat C1 is the sole contributor to the odor quality and potency ofboth isomers 1 and 3. Replacing this OH by an acetate groupled only to a remarkable decrease in the potency but not theodor quality, with the sole exception of neryl acetate. For thissubstance the odor turned from the characteristic citrus-,lemon-like impression of nerol to a clove-like, phenolic note.On the other hand, an OH at C-8 preserved the commoncitrus-like smell of the parent monoterpenes, but not thepotency, irrespective of whether C-1 bears an OH or an acetategroup. However, a carbonyl group at C-8 led to the appearanceof a musty note that was either relatively pure (8-oxogeraniol)or accompanied by other odor notes (8-oxoneryl acetate). Lastbut not least, substitution of C-8 by a highly polar functionalgroup, the carboxy group, turned the odor of all derivatives towaxy, greasy, and plasticine-like.Accordingly, our study demonstrates that potentially

naturally occurring nerol and geraniol derivatives might be ofsensory relevance as was previously demonstrated for thecorresponding linalool derivatives. Future studies will need toelucidate if these substances contribute to certain smells innature or if they can impart other physiological effects, apartfrom pure smell sensation.

■ AUTHOR INFORMATIONCorresponding Author*(A.B.) Phone: +49-9131-85 22739. E-mail: [email protected].

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the members of our working group for theirparticipation in the sensory analyses.

■ REFERENCES(1) Semikolenov, V. A.; Ilyna, I. I.; Maksimovskaya, R. I. Linalool togeraniol/nerol isomerization catalyzed by (RO)3VO complexes:

Table 3. Odor Thresholds of Panelists for All Compounds

OTa,b (ng/Lair)

compound odorant PAc PB PC PD PE PF PG groupd

1 geraniol 23.1 23.1 23.1 23.1 11.53 11.53 2.88 14.052 geranyl acetate 75.33 18.83 150.66 37.66 150.66 150.66 18.83 61.793 nerol 179.50 22.52 180.15 11.26 89.77 180.15 22.52 60.554 neryl acetate n.o. 432 216 27 27 27 432 96.215 8-oxogeraniol 637.2 39.82 637.2 318.6 39.82 79.65 637.2 194.186 8-oxogeranyl acetate 6.75 13.5 54 13.5 27 13.5 108 22.147 8-oxonerol 1620 50.62 810 202.5 202.5 6480 405 493.758 8-oxoneryl acetate 13.09 26.19 13.09 26.19 3.27 0.41 26.19 8.819 8-hydroxygeraniol 540 270 n.o. 540 135 270 540 340.1710 8-hydroxygeranyl acetate 54 54 108 54 108 108 27 65.8211 8-hydroxynerol 901 112.72 901 225.45 450.9 1803 225.45 450.7612 8-hydroxyneryl acetate 216 54 54 216 27 n.o. 108 85.7213 8-carboxygeraniol14 8-carboxygeranyl acetate 14.04 56.16 56.16 n.o. 28.08 28.08 112.32 39.7115 8-carboxynerol n.o. 74.25 594 74.25 74.25 4752 74.25 21016 8-carboxyneryl acetate 27 13.5 6.75 216 6.75 54 54 27

an.o., no odor perceived by the panelist. bOdor thresholds in air were determined as described by Czerny et al.25 cP specifies the respective panelistsA, B, C, D, E, F, and G. dGroup odor threshold was calculated as a geometric mean of the individual thresholds of panelists.



H




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5 Influence of the Chemical Structure on the Odor Characters of β-Citronellol and Its Oxygenated Derivatives

Food Chemistry, 2017. 232: p. 704-711.

Shaimaa Awadain Elsharif a, Andrea Buettner a,b,*

a Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 9, 91054 Erlangen, Germany, E-mail addresses: [email protected] (S.A. Elsharif), [email protected] (A. Buettner).

b Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Strasse 35, 85354 Freising, Germany, [email protected]


Reprinted from Food Chemistry, 232, Elsharif, S.A., A. Buettner, Influence of the chemical structure on the odor characters of β-citronellol and its oxygenated derivatives, pages. 704-711.

Copyright © 2017, with permission from Elsevier



Food Chemistry 232 (2017) 704–711

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Food Chemistry

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Influence of the chemical structure on the odor charactersof b-citronellol and its oxygenated derivatives

http://dx.doi.org/10.1016/j.foodchem.2017.04.0530308-8146/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Chemistry and Pharmacy, FoodChemistry, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürn-berg, Henkestrasse 9, 91054 Erlangen, Germany.

E-mail addresses: [email protected] (S.A. Elsharif), [email protected],[email protected] (A. Buettner).

Shaimaa Awadain Elsharif a, Andrea Buettner a,b,⇑aDepartment of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 9, 91054 Erlangen, GermanybDepartment of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Strasse 35, 85354 Freising, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 January 2017Received in revised form 29 March 2017Accepted 10 April 2017Available online 11 April 2017

Keywords:8-Oxocitronellol8-HydroxycitronellolOdor threshold in airGas chromatography-olfactometryRetention indicesMass spectrometry

b-Citronellol, 1, and citronellyl acetate, 2, are renowned fragrant constituents in perfumes and flavoringagents in foods and beverages. Both substances smell citrussy, fresh and floral. To elucidate the structuralfeatures required for these sensory effects, six C-8 oxygenated derivatives of 1 and 2 were synthesizedand analytically characterized. All compounds were tested for their odor qualities and odor thresholdsin air, revealing that there were no significant differences in odor impressions from the parent monoter-penes and their derivatives in most cases; however, substantial differences in their odor threshold valueswere observed, with b-citronellol as the most potent (10 ng/Lair) and 8-hydroxycitronellyl acetate as theleast potent odorant (1261 ng/Lair). 8-Oxocitronellyl acetate was the only compound that was describedwith divergent odor attributes, namely musty, rotten and coconut-like. 8-Carboxycitronellol and8-carboxycitronellyl acetate were found to be odorless.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

b-Citronellol, 1 (3,7-dimethyloct-6-en-1-ol), an essential oilcomponent of lemon grass or Cymbopogon citratus (Abegaz,Yohannes, & Dieter, 1983), citronella grass or Cymbopogon winteri-anus (Quintans-Júnior et al., 2008) and bushy matgrass or Lippiaalba (Tavares et al., 2005), is used as a flavoring agent in foodand beverages (Dziala, Peplonski, Klopotek, Lisicki, & Tecza, 2000;Murakami, Furukawa, Kawasaki, & Ota, 2013). The odor of citronel-lol has previously been described as being sweet, rose-like(Hognadottir & Rouseff, 2003; Zhao et al., 2016), while citronellylacetate, 2, has been reported to elicit a fresh, fruity smell associatedwith a lemon scent (Zhao et al., 2016). Due to their pleasant smellproperties, 1 and 2 are fragrant compounds that are widely used inperfumes with citrus, floral notes (Bartsch, Uhde, & Salthammer,2016; Rios et al., 2013). Owing to the chiral center at carbon 3,b-citronellol has two isomeric forms that both occur in nature.The R-(+)-isomer, a slight oily light rosy-leafy, petal-like odorouscompound (Yamamoto et al., 2004), is the most commonisomer found in the Rutaceae family. On the other hand, theS-(�)-isomer with very fresh light and clean, rosy, leafy, petal-like

odor, is less common, and is mainly found in geranium andcitronella oils (de Sousa et al., 2006).

Apart from that, 1 came into focus as potential treatment in car-diovascular diseases based on the consideration that the decoctionof lemon grass leaves, being rich in 1, has previously been used as ahypotensive agent in folk medicine (Carbajal, Casaco, Arruzazabala,Gonzalez, & Tolon, 1989). Besides, 1 has been demonstrated toexert some other pharmacological effects such as antibacterial,antifungal, antispasmodic as well as anticonvulsant activity (Britoet al., 2012). Apart from that, this odorant is one of the most effec-tive natural tick repellants against nymphs of Amblyomma ameri-canum (L.) or lone star tick (Tabanca et al., 2013), the mostcommon tick reported to bite humans in the southeast and south-central USA which can cause people to develop an allergy to redmeat proteins (Wolver, Sun, Commins, & Schwartz, 2013). In addi-tion, 1 has been reported as a fragrance allergen, i.e. in the contextof scented consumer products for indoor application (Bartsch et al.,2016). In animal studies it has been shown that oral administrationof citronellol to streptozotocin (STZ)-induced diabetic ratsenhanced the levels of insulin, hemoglobin and hepatic glycogen,but also ameliorated the production of important carbohydrate-metabolizing enzymes (Srinivasan & Muruganathan, 2016). Thesefindings suggest that 1 may be a beneficial agent in the treatmentof diabetes mellitus.

Citronellyl acetate 2 has previously been identified as the mainodor compound in fresh rhizomes of ginger where the smell of the

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substance has been described as rose-like (Nishimura, 1995). Inaddition, 2 has been described as one of the most important aromacompounds in kumquat peel oil; in that study its odor wasdescribed as citrus, kumquat-like (Choi, 2005). Interestingly, in astudy regarding the smell characteristics of both enantiomers of2, the odor of the S-isomer was described as fresh, lime- andcitrus-like with a camphoraceous note, whereas the additional noteof the R-isomer, apart from fresh, lime- and citrus-like, was reportedas being dirty aldehydic, without any further specification of thisannotation. However, these smell character assignments were pro-vided in the course of an odor threshold (OT) determination of thetwo enantiomers of 2 in an aqueous ethanol solution applying a tri-angular test (Yamamoto et al., 2004). Accordingly, the additionalsensory influence of ethanol cannot be excluded.

Apart from that, citronellyl acetate has been shown to act as anantinociceptive agent, like citronellol. 2 is also present as majorconstituent in Corymbia citriodora (Rios et al., 2013). This plant,also known as Eucalyptus citriodora resin, has been demonstratedto be a potent antihepatoma agent with pro-apoptotic activity inHepG2 cells (human hepatoma cells); moreover, it also exerts anti-fungal, antibacterial and insect-repellent activity (Shen, Chen, &Duh, 2012).

Recent studies of our group showed that oxygenated derivativesof potent odorous terpenoid compounds can exert interesting andpleasant odor effects. Apart from that, our comprehensive revisionof reports of such substances on their occurrence in nature, and ofother potential effects of these derivatives in plant and animalkingdom, revealed that such derivatives may be underrated, andthat some of these compounds might still await their future dis-covery. For that reason, we previously established a comprehen-sive database on oxygenated derivatives of linalool, geraniol,nerol and their acetates (Elsharif, Banerjee, & Buettner, 2015;Elsharif & Buettner, 2016). The aim of the present study was to fur-ther expand this database, now focusing on derivatives of citronel-lol and its acetate.

Among all oxygenated derivatives of 1 and 2 (Fig. 1), only twocompounds have previously been identified in nature, namely

Fig. 1. Synthetic pathways leading to the target oxygenat

8-hydroxycitronellol, 5, as one of the diol constituents of the polarextract of the rose flowers (Rosa damascena Mill.) (Knapp et al.,1998), and the R-isomer of 8-carboxycitronellol, 7 (Table 1). Thelatter substance has been isolated from a Chinese herb calledCistanche salsa, and has further been reported to be an antiosteo-porotic compound (Yamaguchi, Shinohara, Kojima, Sodeoka, &Tsuji, 1999). It is important to note that compounds 8-oxocitronellol, 3, 8-oxocitronellyl acetate, 4, and 8-hydroxycitronellyl acetate, 6, have not been previously identifiedin nature as constituents in plants or animals, yet they were syn-thesized in some studies for different purposes. To mention butsome examples, compound 3 was synthesized in a study on theselective oxidation of primary allylic alcohols to their correspond-ing aldehydes (Kalsi, Chhabra, Singh, & Vig, 1992), whereas com-pound 4 was synthesized as an intermediate in the synthesis ofthe minor sex pheromone component of two Brazilian soybeanstink bugs (Het.: Pentatomidae) (Zarbin et al., 2000). Similarly,compound 6 was synthesized in a study showing the exclusive1,2 reduction of functionalized conjugated aldehydes containingother reducible and acid-sensitive functionalities with sodium tri-acetoxyborohydride to obtain allylic alcohols (Singh, Sharma, Kaur,& Kad, 2000). As for 8-carboxycitronellyl acetate, 8, this substancehas neither been identified in nature nor synthesized before, thus,is a newly reported odorant of this study.

Apart from that, no systematic data are to date available on theodor characteristics of these oxygenated derivatives of citronelloland citronellyl acetate, let alone their potential further effects onhumans or other animals. To close this gap, the aim of our studywas to systematically evaluate the odor qualities and OTs of thesesubstances as we previously did in a comparative approach for therelated derivatives of linalool (Elsharif et al., 2015) and geraniol(Elsharif & Buettner, 2016). To achieve this aim we synthesized,starting from 1 and 2, derivatives with oxygenated functionalgroups at C-8, and determined their respective odor qualities andOTs in air. This substance library should provide us with the oppor-tunity of evaluating if b-citronellol and its ester are the only odor-ous compounds in this substance class, or if related compounds

ed derivatives of b-citronellol and citronellyl acetate.

Table 1Retention indices and occurrence of the acyclic monoterpenes and their derivatives.

Compound Odorant RIa Previously identified in

FFAP DB5

1 b-Citronellol 1763 1230 Cymbopogon citratusb, Cymbopogon winterianus b, Lippia alba b and Pelargonium graveolens (Geraniaceae)c

2 Citronellyl acetate 2220 1352 Grapefruit oil d, kumquat (Fortunella japonica Swingle) peel oil e

3 8-Oxocitronellol 2350 1494 Not reported4 8-Oxocitronellyl acetate 2267 1598 Not reported5 8-Hydroxycitronellol 2550 1502 Rose flowers (Rosa damascena Mill.) f

6 8-Hydroxycitronellyl acetate 2450 1610 Not reported7 8-Carboxycitronellol 3140 1656 Cistanche salsa, R-enantiomer only g

8 8-Carboxycitronellyl acetate 3013 1745 Not reported

a Retention indices were determined as described by van den Dool and Kratz (1963).b Brito et al. (2012).c Tabanca et al. (2013).d Moshonas (1971).e Choi (2005).f Knapp et al., 1998.g Yamaguchi et al. (1999).

706 S.A. Elsharif, A. Buettner / Food Chemistry 232 (2017) 704–711

also bear the potential of eliciting interesting olfactory effects. Inaddition, we aimed at providing a comprehensive substance libraryincluding the respective mass spectrometric data, a compilation ofretention indices on two commonly used chromatographic capil-laries of different polarity as well as their nuclear magnetic reso-nance spectra.

2. Material and methods

2.1. Chemicals

b-Citronellol, citronellyl acetate, selenium dioxide, sodiumborohydride, anhydrous methanol, ethanol, dioxane, tert-butylalcohol, 2-methyl-2-butene, sodium chlorite, sodium dihydrogenphosphate, ethyl acetate, hexane, and magnesium sulfate werepurchased from Aldrich (Steinheim, Germany). Diethyl ether wasfrom Fisher Scientific (Loughborough, UK).

2.2. General methods

All reactions requiring anhydrous conditions were carried outunder dry nitrogen, and the solvents were dried before use toremove moisture using appropriate drying agents. All reactionswere monitored by thin-layer chromatography (TLC) using silicagel 60 F254 plates (Merck, Darmstadt, Germany). Visualization ofthe reaction components was achieved using UV fluorescence(254 nm) and potassium permanganate (KMnO4) staining. Columnchromatography was carried out over silica gel 60. The yieldsreported are after purification. 1H and 13C nuclear magnetic reso-nance (NMR) spectra were recorded in deuterated solvents, andchemical shifts (d) are quoted in parts per million (ppm) calibratedto tetramethylsilane (TMS) (1H and 13C). Coupling constants (J)were measured in hertz (Hz). The identity of all intermediatesand synthetic products was determined by electron ionizationmass spectrometry (EI-MS).

2.3. Nuclear Magnetic Resonance (NMR) spectra

1H and 13C NMR spectra were recorded in deuterated chloro-form (CDCl3) on Avance 360 and Avance 600 spectrometers (BrukerBiospin, Rheinstetten, Germany) at room temperature, at 360 or600 MHz (1H) and 90 or 150 MHz (13C), respectively, with TMS asinternal standard.

2.4. High Resolution gas Chromatography-Olfactometry (HRGC-O)

HRGC-O was performed with a Trace Ultra GC (Thermo FisherScientific GmbH, Dreieich, Germany) using the following capillar-ies: DB-FFAP (30 m � 0.32 mm i.d. fused silica capillary, free fattyacid phase FFAP, film thickness = 0.25 mm; Chrompack, Mühlheim,Germany) and DB5 (30 m � 0.32 mm i.d. fused silica capillary DB-5, film thickness = 0.25 mm; J&W Scientific, Santa Clara, CA). Thehelium carrier gas flow was set for FFAP and DB5 capillary columnsto 2.5 and 1.2 mL/min, respectively. The compounds eluting at theend of the capillaries were split using a Y-splitter, ratio 1:1 (J&WScientific), and were transferred via two deactivated capillaries(0.5 m x 0.2 mm) (J&W Scientific) to a flame ionization detectorand a heated sniffing port (temperature = 250 �C). The sampleswere applied to the capillary column using a cold-on-column injec-tor set to 40 �C. After 2 min the oven temperature was raised at10 �C/min to 240 �C (DB-FFAP) or 8 �C/min to 280 �C (DB-5), andheld for 7 min. The pre-column was changed regularly to avoidaccumulation of any contaminants.

The synthesized substances were additionally subjected to chi-ral chromatography, employing the following chiral capillary col-umns: Rt-yDEXsa, Rt-bDEXse, Rt-bDEXsa and Rt-bDEXcst (Restek)using the following temperature program: 40 �C as initial temper-ature for 2 min, then raised at 8 �C/min to 230 �C and held for7 min, with a flow rate of 1.0 mL/min, using the GC-FID systemas described above. However, separation turned out to be ineffec-tive so that this approach was not pursued further.

2.5. High Resolution Gas Chromatography-Mass Spectrometry(HRGC-MS)

MS spectra were recorded using a 5975C MSD quadrupole sys-tem in connection with a 7890A GC system (Agilent Technologies,Waldbronn, Germany). The system was equipped with a GerstelCIS 4 injection system and Gerstel MPS 2 autosampler (Gerstel,Duisburg, Germany). The analytical capillaries used were DB-FFAP and DB-5 (30 m, 0.25 mm, film thickness = 0.25 mm; AgilentJ&W Scientific, Santa Clara, USA). An uncoated, deactivated fusedsilica capillary was used as pre-column (2–3 m, 0.53 mm). Thetemperature program used was as follows: 40 �C for 2 min, thenfrom 8 �C/min to 240 �C and 250 �C for DB-FFAP and DB-5, respec-tively, and held for 7 min. The carrier gas was helium and the flowrate was 1.0 mL/min. Injection volumes were 1 mL in each case. EI-mass spectra were generated in full scan mode, (m/z range 30–350)at 70 eV ionization energy.


2.6. Retention indices (RI)

Linear retention indices (Table 1) were determined by GC-Ousing mixtures of individual odorants dissolved in dichloro-methane together with a series of n-hydrocarbons (C6–C34) dis-solved in n-pentane, according to the method of van den Dooland Kratz (1963).

2.7. Evaluation of odor quality

The odor qualities were determined during GC-O evaluationwith the aid of five panelists (five females, aged 28–33 years)who were trained volunteers from the University of Erlangen(Erlangen, Germany), exhibiting no known illness at the time ofexamination and with audited olfactory function. The assessorswere trained for at least half a year in recognizing orthonasallyabout 140 selected known odorants at different concentrationsaccording to their odor qualities and in naming these accordingto an in-house developed flavor language. The detailed procedureis described in our previous publication (Elsharif & Buettner, 2016).

With regards to the GC-O experiments that were carried out onthe synthesized compounds, samples were evaluated by each pan-elist repeatedly on different days (in the course of performing therespective dilution analyses), and additionally on different capil-lary columns (DB-FFAP and DB-5). Thereby, panelists wereinformed about elution times of the compounds to ensure thatinhalation corresponded with substance elution from the sniffingport, and were asked to relate the naming of their sensory impres-sions to smell impressions that they recalled from training, ifpossible.

2.8. Odor threshold values (OT)

OTs in air were determined using (E)-2-decenal as an internalodor standard (Czerny, Brueckner, Kirchhoff, Schmitt, & Buettner,2011; Ullrich & Grosch, 1987). According to this procedure, (E)-2-decenal and the respective target compound were dissolved indichloromethane at known concentrations, in each case providingone stock solution comprising both odorants. This stock solutionwas diluted stepwise by a factor of 1:2 (v/v), and all dilutions wereapplied to HRGC-O until no odor was perceivable. From the flavordilution factors (FD) and the relative concentrations of standardand target compound, the OT values in air were then calculatedas previously described in Czerny et al. (2011). The detection OTsof the panel were calculated as the geometric mean of the individ-ual thresholds, according to the method of Czerny et al. (2008).

2.9. Syntheses, general procedures

2.9.1. Procedure 1Generally, the method of Wakayama, Namba, Hosoi, and Ohno

(1973) was used (Fig. 1, P1). Compounds 1 and 2 and seleniumdioxide (SeO2) (1 equiv.) were dissolved in dioxane/ethanol 9:1(v/v), and the solution was heated at 80 �C for 5 h. After removalof selenium deposit by double filtration, the solvent was removedunder reduced pressure using a rotary evaporator. The residue waspurified twice by flash chromatography on silica gel 60 (Merck),with an appropriate mobile phase mixture of petroleum ether/diethyl ether or hexane/ethyl acetate to afford the crude com-pounds 3 and 4, respectively.

2.9.1.1. (E)-(±)-8-Hydroxy-2,6-dimethyloct-2-enal (3), 8-Oxocitronel-lol. CAS Registry Number 194092-52-9. Compound 3 was preparedfollowing procedure 1, starting from 1 (4.9 g, 31.3 mmol) andSeO2 (3.4 g, 31.3 mmol) in 25 mL of dioxane/ethanol 9:1 (v/v).Flash chromatographic purification with petroleum ether/diethyl

ether 1:4 (v/v) yielded 1.182 g (22.2%) of 3 as a reddish-orangeoil: 1H NMR (600 MHz, CDCl3-d) d 9.34 (s, 1 H), 6.47(t, J = 6.00 Hz, 1 H), 3.60–3.71 (m, 2 H), 2.27–2.40 (m, 2 H), 1.71(s, 3 H), 1.56–1.67 (m, 2 H), 1.45–1.56 (m, 1 H), 1.27–1.45 (m, 2H), 0.91 (d, J = 7.00 Hz, 3 H); 13C NMR (90 MHz, CDCl3) d 195.40,155.13, 139.08, 60.54, 39.45, 35.47, 29.21, 26.46, 19.21, 9.04; MS(EI) m/z (%) (rel. int.) 170 [M+] (1), 152 (3), 137 (6), 123 (10), 97(100), 95 (62), 84 (41), 71 (26), 67 (44), 55 (46), 41 (50).

2.9.1.2. (E)-(±)-3,7-Dimethyl-8-oxooct-6-en-1-yl acetate (4), 8-Oxoc-itronellyl Acetate. CAS Registry Number 79763-09-0. Compound 4was prepared following procedure 1, starting from 2 (2.96 g,15 mmol) and SeO2 (1.7 g, 15 mmol) in 20 mL of dioxane/ethanol9:1 (v/v). Flash chromatographic purification with hexane/ethylacetate 5:1(v/v) yielded 0.44 g (14%) of 4 as a reddish-orange oil:1H NMR (360 MHz, CDCl3) d 9.38 (s, 1 H), 6.45 (td, J = 7.27,1.14 Hz, 1 H), 4.06–4.15 (m, 2 H), 2.36 (dt, J = 14.99, 7.27 Hz, 2H), 2.02 (s, 3 H), 1.74 (s, 3 H), 1.42–1.72 (m, 5 H), 0.95 (d,J = 6.36 Hz, 3 H); 13C NMR (90 MHz, CDCl3) d 195.19, 171.11,154.42, 139.41, 62.63, 35.37, 35.31, 29.68, 26.45, 21.01, 19.25,9.20; MS (EI) m/z (%) (rel. int.) 212 [M+] (1), 152 (18), 137 (14),126 (19), 109 (19), 95 (72), 81 (45), 69 (29), 67 (41), 55 (41),43 (100).

2.9.2. Procedure 2Generally, the method of Liu, Chen, and Huang (2003) was used

with some modifications (Fig. 1, P2). Compounds 3 and 4 andsodium borohydride (NaBH4) (2 equiv.) were dissolved in drymethanol. The solution was allowed to stir at –10 �C. After 1 h,water was added and the reaction mixture was extracted withdichloromethane. The organic layer was dried over sodium sulfate.After removal of the solvent, the residue was purified by flash chro-matography on silica gel 60 (Merck), with an appropriate mobilephase mixture of petroleum ether/diethyl ether or hexane/ethylacetate, affording the crude compounds 5 and 6, respectively. Asthis approach has been previously reported as a rather mild-selective procedure (Ward & Rhee, 1989), all target compoundswere specifically analyzed by GC-MS and NMR and compared todata reported in the literature to exclude potential side reactions.

2.9.2.1. (E)-(±)-2,6-Dimethyloct-2-ene-1,8-diol (5), 8-Hydroxyc-itronellol. CAS Registry Number 23062-07-9. Compound 5 was pre-pared following procedure 2, starting from 3 (314 mg,1.84 mmol) and NaBH4 (79 mg, 2.1 mmol) in 20 mL of dry metha-nol. Flash chromatographic purification with petroleum ether/diethyl ether 1:4 (v/v) yielded 262.2 mg (82.7 %) of 5 as a yellowoil: 1H NMR (360 MHz, CDCl3) d 5.29–5.45 (m, 1 H), 3.96 (br. s.,2 H), 3.58–3.72 (m, 2 H), 1.92–2.11 (m, 2 H), 1.65 (s, 3 H), 1.15–1.59 (m, 5 H), 0.89 (d, J = 6.59 Hz, 3 H); 13C NMR (90 MHz, CDCl3)d 134.56, 126.21, 68.76, 60.83, 39.66, 36.65, 28.89, 24.91, 19.44,13.59; MS (EI) m/z (%) (rel. int.) 172 [M+] (1), 154 (5), 139 (9),121 (90), 109 (70), 93 (55), 81 (100), 69 (76), 55 (97), 43 (98).The 1H NMR data fully agreed with Madyastha and Murthy (1988).

2.9.2.2. (E)-(±)-8-Hydroxy-3,7-dimethyloct-6-en-1-yl acetate (6), 8-Hydroxycitronellyl Acetate. CAS Registry Number 95576-02-6. Com-pound 6 was prepared following procedure 2, starting from 4(180.3 mg, 0.85 mmol) and NaBH4 (65 mg, 1.73 mmol) in 12 mLof dry methanol. Flash chromatographic purification with hex-ane/ethyl acetate 5:1 (v/v) yielded 98.6 mg (54 %) of 6 as a yellowoil: 1H NMR (600 MHz, CDCl3) d 5.37 (t, J = 1.00 Hz, 1 H), 4.04–4.13(m, 2 H), 3.98 (br. s., 2 H), 1.97–2.09 (m, 5 H), 1.65 (s, 3 H), 1.16–1.58 (m, 5 H), 0.91 (d, J = 6.42 Hz, 3 H); 13C NMR (150 MHz, CDCl3)d 171.11, 134.61, 125.93, 68.72, 62.80, 36.60, 36.41, 35.23, 29.32,27.64, 24.79, 20.90, 19.26, 13.49; MS (EI) m/z (%) (rel. int.) 214


[M+] (1), 136 (5), 121 (100), 107 (18), 93 (27), 81 (27), 69 (19), 55(28), 43 (56). The 1H NMR data agreed with Singh et al. (2000).

2.9.3. Procedure 3Pinnick oxidation was used for the following syntheses (Pinnick,

Bal, & Childers, 1981) (Fig. 1). The aldehydes 3 and 4 were dis-solved in 25 mL tert-butyl alcohol and 6 mL 2-methyl-2-butene.A solution of sodium chlorite (NaClO2) (9.2 equiv., 80%) and sodiumdihydrogen phosphate (NaH2PO4) (6.9 equiv.) in water was addeddropwise over a 10-min period. The reaction mixture was stirred atroom temperature (20–23 �C) overnight. Volatile components werethen removed under vacuum, the residue was dissolved in 30 mLof water, and this mixture was then extracted twice with 15 mLportions of hexane in each case. The aqueous layer was acidifiedto pH 3 with hydrochloric acid (HCl) and extracted thrice with20-mL portions of ether in each case. The combined ethereal layerswere washed with 50 mL of cold water, then dried over sodiumsulfate, purified by flash chromatography on silica gel 60 (Merck),with an appropriate mobile phase mixture of petroleum ether/diethyl ether or hexane/ethyl acetate, and concentrated to give 7and 8, respectively.

2.9.3.1. (E)-(±)-8-Hydroxy-2,6-dimethyloct-2-enoic acid (7), 8-Car-boxycitronellol. Compound 7 was prepared following procedure 3,where compound 3 (0.19 g, 1.11 mmol) was dissolved in 25 mLof tert-butyl alcohol and 6 mL of 2-methyl-2-butene. A solutionof NaClO2 (1 g, 13.8 mmol) and NaH2PO4 (1 g, 8.33 mmol) in10 mL of water was added dropwise over a 10-min period. Thereaction was further completed and the work up was done asdescribed in procedure 3. Flash chromatographic purification withpetroleum ether/diethyl ether 1:4 (v/v) yielded 162.5 mg (78.2 %)of 7 as a transparent oil: 1H NMR (600 MHz, CDCl3) d 6.88(t, J = 7.90 Hz, 1 H), 3.65–3.74 (m, 2 H), 2.15–2.25 (m, 2 H), 1.83(s, 3 H), 1.59–1.66 (m, 2 H), 1.48–1.53 (m, 1 H), 1.29–1.35 (m, 2 H),0.93 (d, J = 5.70 Hz, 3 H); 13C NMR (150 MHz, CDCl3) d 172.70,144.82, 127.02, 60.88, 39.54, 35.61, 29.27, 26.35, 19.32, 11.99;MS (EI) m/z (%) (rel. int.) 184 [M+ – 2] (1), 168 (30), 139 (5), 112(35), 111 (54), 95 (100), 87 (40), 82 (64), 69 (55), 55 (45).

2.9.3.2. (E)-(±)-8-Acetoxy-2,6-dimethyloct-2-enoic acid (8), 8-Car-boxycitronellyl acetate. Compound 8was prepared following proce-dure 3, where compound 4 (0.185 g, 0.87 mmol) was dissolved in25 mL of tert-butyl alcohol and 6 mL of 2-methyl-2-butene. A solu-tion of NaClO2 (0.73 g, 10.1 mmol) and NaH2PO4 (0.82 g, 6.8 mmol)in 10 mL of water was added dropwise over a 10-min period. Thereaction was further completed and the work up was done asdescribed in procedure 3. Flash chromatographic purification withhexane/ethyl acetate 4:1 (v/v) yielded 36.4 mg (18.3 %) of 8 as apale yellow oil: 1H NMR (600 MHz, CDCl3) d 6.89 (td, J = 7.55,1.13 Hz, 1 H), 4.03–4.21 (m, 2 H), 2.23 (dd, J = 15.49, 8.31 Hz, 2H), 2.05 (s, 3 H), 1.85 (s, 3 H), 1.30–1.72 (m, 5 H), 0.95 (d,J = 6.42 Hz, 3 H); 13C NMR (150 MHz, CDCl3) d 172.83, 171.08,144.80, 126.85, 62.59, 53.27, 35.25, 35.13, 29.45, 26.16, 20.87,19.09, 11.86; MS (EI) m/z (%) (rel. int.) 228 [M+] (1), 210 (40),168 (30), 150 (20), 135 (26), 111 (39), 95 (100), 82 (45), 67 (55),55 (45), 43 (78). This compound is synthesized here for the firsttime.

3. Results and discussion

b-Citronellol, citronellyl acetate and their C-8 synthesized oxy-genated derivatives were investigated in terms of their odor qual-ity and OTs in air by our trained panel using GC-O; the obtainedodor attributes are shown in Table 2. The advantage of this proce-dure is that potential odor-active side products are separated from

the target odorants during the chromatographic separation step.The influence of such components on the results, therefore, is ruledout. Moreover, the absolute OTs can be compared to each otherwithout interference with any matrix system as for example whenOTs are determined in water, oil or solid matrices. Apart from that,using GC-O may provoke the risk of lack of inhalation at therequired elution time of the odorant of interest from the GC col-umn. Accordingly, panelists are clearly instructed and trained toinhale exactly at the specified elution times as done in the presentstudy. Moreover, it is recommended to perform the experimentson capillary columns with different polarities, as in this study,since the chromatographic shape of the substances may varybetween different columns of different polarities. To ensure thereproducibility and consistency of the results, the componentswere evaluated more than once on different days. In addition, nochanges in odor were observed when panelists olfactorily evalu-ated different steps of concentration. It is worth mentioning thatany impurity that might have still been associated with our syn-thesized compounds, such as potential organo-selenium byprod-ucts, could be shown to not interfere with odor detection of ourtarget molecules as GC-O on different capillary columns revealedthe pure compounds without any interferences from even minuteamounts of side-products.

In the following, the odor character of the respective com-pounds will be presented first. In general, it was observed thatthe 8-oxo and 8-hydroxy derivatives of 1 exerted the same or atleast similar odor qualities as their parent molecule. The odor attri-butes named by the panel were mainly citrus-like, fresh and soapy.On the other hand, citronellyl acetate 2, and its 8-hydroxy deriva-tive 6, shared comparable citrus-like and soapy notes, whereas the8-oxocitronellyl acetate 4 elicited different odor impressionsaccording to some panelists. These odor notes were musty as givenby four panelists, and coconut-like as reported by two panelists. Inaddition, only two out of five panelists mentioned that the odor of4 imparted a citrus nuance beside the musty note. It is worth men-tioning that 8-carboxycitronellol 7, as well as 8-carboxycitronellylacetate 8, evoked no odor at the sniffing port for all panelists up toconcentrations of 440 and 300 mg/L, respectively.

When specifically analyzing the odor qualities as named by theindividual panelists, it was found that, generally, all panelistsagreed upon the term citrus-like as a descriptor for b-citronellol1, whereas 40% of the panel perceived this substance as fresh andfloral. In addition, other smell attributes given to 1were fatty, sweetand perfume-like. 80% of the panel described the odor of 8-oxocitronellol 3 as citrus-like, and 60% as being fresh, whereas pan-elist B described the substance as being fresh and sweet withoutspecifically mentioning the term citrus. On the other hand, thewhole panel agreed on the attributes citrus-like and fresh as thepredominant descriptors for the odor of 8-hydroxycitronellol, 5,with the sole exception of panelist D who found this odor to bepungent instead of fresh.

With regard to the odor of citronellyl acetate 2, 60% of the panelperceived it as citrus-like and soapy, while one panelist describedthis smell as being citrus-like but also floral. As mentioned earlierin this section, the main odor attributes given for the 8-hydroxycitronellyl acetate, 6, were citrus-like and soapy as reportedby all panelists with the sole exception of one subject who did notmention the term soapy. Interestingly, the panelist, who gave theattribute floral instead of soapy to citronellyl acetate, added thesame attribute to describe the smell of its 8-hydroxy derivative6. Last but not least, it is worth mentioning that the 8-oxocitronellyl acetate 4 was the only compound that elicitedcompletely different smell characters. This substance wasperceived as coconut-like or sweet by 40% of the subjects, whilethe majority of the panel (80%) found this smell to be musty. Thepanel was not able to further specify this musty odor by any other

Table 2Odor qualities defined by panelists and median of the odor threshold of all compounds.

Compound Odor qualitiesa,b OT [ng/L(air)]

PA PB PC PD PE Range Median

1 citrus, floral, perfume citrus, sweet, fresh citrus, fatty citrus, soapy citrus, fresh, floral 4.6–36.8 9.22 citrus fatty, soapy fatty, soapy citrus, soapy citrus, floral 219–1754 877.23 citrus fresh, sweet citrus, fresh, fatty citrus, fresh, flowery citrus, fatty 126–4039 504.94 musty, rotten, sweet, citrus coconut, sweet, musty meaty, fatty, coconut musty, citrus rotten, musty, fatty 173–1382 345.65 citrus, fresh citrus, fresh citrus, fresh, soapy citrus, pungent citrus, fresh 25.3–405 4056 citrus, soapy citrus, soapy citrus, soapy, fresh citrus citrus, soapy, floral 631–2523 1261.47 odorlessc odorless odorless odorless odorless8 odorlessd odorless odorless odorless odorless

a Odor qualities as perceived at the sniffing port.b PA–PE are the five panelists.c no odor observed up to a concentration of 440 mg/L.d no odor observed up to a concentration of 300 mg/L.


sensory terminology, with the sole exception of two panelists whofurther specified this odor with the term rotten. In another context,it has previously been reported that the term musty is a generalterm comprising such divergent impressions as dusty, papery,earthy, or moldy (Chambers IV, Smith, Seitz, & Sauer, 1998;Strube & Buettner, 2010). Interestingly, one single panelistdescribed the odor of 4 as being meat-like. However, substancesthat have previously been associated with meaty odor, like2-methyl-3-furanthiol and its dimer bis(2-methyl-3-furyl)disulfide, generally contain a sulfur atom in their structure(Dreher, Rouseff, & Naim, 2003; Yang, Song, Chen, & Zou, 2011).In view of this it is interesting to note that 4 was perceived asmusty with a citrusy background by two panelists, so that onemight speculate that the musty impression was interpreted as ameaty tone by this single panelist. Moreover, it is interesting tonote that in our previous study on geraniol and its oxygenatedderivatives (Elsharif & Buettner, 2016) comparable observationswere made for the 8-oxo-derivatives, where some panelistsreported the odor as being musty with a citrus-like nuance.

When regarding the averaged as well as the individual OT datashown in Table 2 and 3, it becomes evident that there were bothvariations in the threshold data between different substances butalso for the same compound, when taking into consideration theunderlying threshold data of individual panelists. Overall, all sub-stances were perceived as intense to medium intense odorantswith the sole exception of the 8-hydroxycitronellyl acetate, 6,which imparted a relatively weak odor intensity, and 8-carboxycitronellol, 7, as well as 8-carboxycitronellyl acetate, 8which were not perceived by any of the panelists.

Among the individual panelists, no one showed exceptionalsensitivity to all compounds. However, panelist B was especiallysensitive to b-citronellol 1 and its derivatives, whereas panelist C

Table 3Odor thresholds of panelists for all compounds.

Compound Odorant OT [ng/L(air)]a

PA P

1 b-Citronellol 9.2 42 Citronellyl acetate 877 83 8-Oxocitronellol 4040 14 8-Oxocitronellyl acetate 173 35 8-Hydroxycitronellol 405 26 8-Hydroxycitronellyl acetate 1260 17 8-Carboxycitronellol – –8 8-Carboxycitronellyl acetate – –

a Odor thresholds in air were determined as described by Czerny et al. (2011).b Group odor threshold was calculated as a geometric mean of the individual thresho

yielded the lowest OT values for citronellyl acetate 2 and itsderivatives.

Overall, b-citronellol 1was found to be the most potent odorantamong all compounds investigated in the present study, yielding athreshold value of 10.5 ng/Lair. Thereby, the variance in individualthreshold data was found to be within the range from 4.6 to36.8 ng/Lair, roughly covering one order of magnitude. On the otherhand, 8-hydroxycitronellyl acetate, 6, was the least potent odorsubstance, with a threshold value of 1261 ng/Lair. In view of thisit is interesting to note that the whole panel’s OT value of 8-oxocitronellol 3 did not rank as the highest (879 ng/Lair), despitethe fact that two subjects recorded the highest individual OT value(4039 ng/Lair) for this substance. It is also worth mentioning thatall panelists recorded the same OT value (405 ng/Lair) for 8-hydroxycitronellol 5, with the sole exception of panelist B, whowas exceptionally sensitive to 1 and its C-8 derivatives, as men-tioned before, recording a threshold value of 25.3 ng/Lair for 5.

Overall, the absolute lowest individual OT value was recordedfor b-citronellol with 4.6 ng/Lair (panelists B and D), whereas thehighest individual OT value was found for 8-oxocitronellol with4040 ng/Lair (panelists A and D). Nevertheless, this did not correlatewith a general exceptionally high or low sensitivity of the respec-tive panelists. Moreover, at least two panelists showed comparableOT values for each substance, regardless of the odor qualityperceived.

Apart from the two substances that were found to be generallyodorless, namely, 8-carboxycitronellol, 7, and 8-carboxycitronellylacetate, 8, no further cases of anosmia were observed for the com-pounds investigated in this study.

The OT of citronellyl acetate 2 was found to be in an intermedi-ate range between those of its two derivatives 4 and 6, with a valueof 665 ng/Lair; the OT values of 4 and 6 were 346 ng/Lair and

Group b

B PC PD PE

.6 18.4 4.6 36.8 10.577 219 1750 439 66526 505 4040 505 87946 173 1380 346 3465.3 405 405 405 233260 631 2520 1260 1260

– – – –– – – –

lds of panelists.


1260 ng/Lair, respectively. This is in line with our previousobservation that this type of oxidation of the parent acetates doesnot decrease odor potency, but that the parent molecules some-times exert a more pleasant smell, (Elsharif & Buettner, 2016;Elsharif et al., 2015).

From the above results, several insights into structure–odorrelationships can be deduced for the investigated b-citronelloland citronellyl acetate derivatives. Obviously, addition of an alde-hyde group at C-8 of b-citronellol led to a remarkable decrease inthe smell potency and intensity, resulting in an increase in OTvalue by a factor of 83, whereas the odor quality was not affected.Reduction of this C-8 aldehyde group to the corresponding alcoholled to an enhancement of the citrusy impression, thereby increas-ing both the smell potency of the resulting substance even further.In contrast to this, further oxidation of the C-8 aldehyde group,yielding an acid moiety, led to total odor loss.

A comparison of the results of this investigation with our previ-ous study on linalool and its derivatives (Elsharif et al., 2015)reveals that linalool, citronellol and their C-8 oxygenated deriva-tives are generally characterized by the same odor qualities; never-theless, they are obviously not equally potent odorants.Substituting C-8 with an OH-group enhances the dominance ofthe citrus-like character, regardless whether C-1 bears a hydroxyor an acetate group. An aldehyde group at C-8, on the other hand,mainly changes the odor from predominantly citrus-like to musty;this is especially true in case of the C-1 acetate derivatives. How-ever, a carboxyl group at C-8 could be shown to result in total odorloss.

With regard to the citronellyl acetate derivatives, it was foundthat an aldehyde group at C-8, yielding 8-oxocitronellyl acetate,increased odor potency but shifted the odor quality from citrusyto musty, rotten-like. The citrus-like, soapy impression was retainedonly when reducing the aldehyde function to the respective alco-hol, giving 8-hydroxycitronellyl acetate, but going along with amajor decrease in odor potency. Again, replacement by an acidmoiety, leading to 8-carboxycitronellyl acetate, caused total odorloss.

To summarize the main outcomes of our study, we could showthat b-citronellol, 1, citronellyl acetate, 2, and their correspondingoxygenated derivatives (3–6) were all found to be odor active withthe exception of 8-carboxycitronellol and 8-carboxycitronellylacetate (7 and 8). Odor quality and OT determinations revealedthat structural changes on the basic skeleton of these monoterpe-nes led to derivatives that can be more pleasant with regards tosmell character than the parent monoterpene but did not increaseodor potency. Moreover, all derivatives investigated within thisstudy showed comparable smells as reported by all panelists withthe sole exception of 8-oxocitronellyl acetate; in this case quitedivergent smell impressions were reported by the panelists. It isfurther worth mentioning that no cases of anosmia were recorded.

Overall, the structure–odor relationship of these monoterpenealcohols can be summarized as follows; the OH group at C1 isthe sole contributor to the odor quality and potency of 1. Replacingthis OH by an acetate group led to a remarkable decrease in odorpotency but did not change the odor quality. On the other hand,an OH at C-8 preserved the common citrus-like smell of the parentmonoterpenes, but not the potency, irrespective of whether C-1contained an OH or an acetate group. However, a carbonyl groupat C-8 together with an OH group at C-1 did not affect the odorquality but decreased the potency, while replacing the OH at C-1by an acetate moiety shifted the smell impression to a musty note,yet being accompanied by other odor tones such as citrusy andsweet. Last but not least, a carboxy function at C-8 led to odor loss.

Comparing these findings with those of our previous studies(Elsharif & Buettner, 2016; Elsharif et al., 2015), it can be summa-rized that the OH group at C-8 at the basic monoterpene structure

obviously does not affect the odor quality of the correspondingderivatives, but impacts the OT, irrespective of whether C-1 or C-3 bears an alcohol or an acetate moiety. Apart from that, an alde-hyde function at C-8 shifts the overall citrus-like impression to amusty note.

Accordingly, our study demonstrates that potentially naturallyoccurring b-citronellol and citronellyl acetate derivatives mightbe of sensory relevance as was previously demonstrated for thecorresponding linalool and geraniol derivatives. Future studies willneed to elucidate if these substances contribute to certain smells innature, or if they can impart other physiological effects, apart frompure smell sensation.

Funding statement

This research did not receive any specific grant from fundingagencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

No conflict of interest to be reported.

Acknowledgments

We thank the members of our working group for their partici-pation in the sensory analyses.

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73

6 Metabolic Products of Linalool and Modulation of GABAA Receptors

Frontiers in chemistry, 2017, 5, article 46.

Sinem Milanos 1, 2, Shaimaa A. Elsharif 2, Dieter Janzen 1, Andrea Buettner 2, 3 and Carmen Villmann 1*

1 Institute of Clinical Neurobiology, Julius-Maximilians-University of Würzburg, Würzburg, Germany, [email protected]

2 Department of Chemistry and Pharmacy, Food Chemistry, Emil-Fischer-Center, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.

3 Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging, Freising, Germany.


Reprinted from Frontiers in chemistry, 2017, 5. Milanos S, Elsharif SA, Janzen D, Buettner A and Villmann C, Metabolic Products of Linalool and Modulation of GABAA Receptors, article 46.

Copyright © 2017 Milanos, Elsharif, Janzen, Buettner and Villmann, with permission from the authors.

Copyright © 2017 Milanos, Elsharif, Janzen, Buettner and Villmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


ORIGINAL RESEARCHpublished: 21 June 2017

doi: 10.3389/fchem.2017.00046

Frontiers in Chemistry | www.frontiersin.org 1 June 2017 | Volume 5 | Article 46

Edited by:

Marcello Iriti,

Università degli Studi di Milano, Italy

Reviewed by:

Marcin Szymanski,

Poznan University of Medical

Sciences, Poland

Viduranga Y. Waisundara,

Rajarata University of Sri Lanka,

Sri Lanka

*Correspondence:

Carmen Villmann

[email protected]

Specialty section:

This article was submitted to

Food Chemistry,

a section of the journal

Frontiers in Chemistry

Received: 08 May 2017

Accepted: 12 June 2017

Published: 21 June 2017

Citation:

Milanos S, Elsharif SA, Janzen D,

Buettner A and Villmann C (2017)

Metabolic Products of Linalool and

Modulation of GABAA Receptors.

Front. Chem. 5:46.

doi: 10.3389/fchem.2017.00046

Metabolic Products of Linalool andModulation of GABAA ReceptorsSinem Milanos 1, 2, Shaimaa A. Elsharif 2, Dieter Janzen 1, Andrea Buettner 2, 3 and

Carmen Villmann 1*

1 Institute of Clinical Neurobiology, Julius-Maximilians-University of Würzburg, Würzburg, Germany, 2Department of Chemistry

and Pharmacy, Food Chemistry, Emil-Fischer-Center, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany,3Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging, Freising, Germany

Terpenoids are major subcomponents in aroma substances which harbor sedative

physiological potential. We have demonstrated that various monoterpenoids such as

the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon

overexpression of inhibitory α1β2 GABAA receptors in various expression systems.

However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool

undergoes metabolic modifications including oxygenation and acetylation, which may

affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the

modulatory potential of linalool derivatives at α1β2γ2 GABAA receptors upon transient

overexpression. Following receptor expression control, electrophysiological recordings in

a whole cell configuration were used to determine the chloride influx upon co-application

of GABA EC10−30 together with the modulatory substance. Our results show that only

oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas

derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated

linalool derivatives resulted in non-significant changes of GABAergic currents. We can

conclude that metabolism of linalool reduces its positive allosteric potential at GABAA

receptors compared to the significant potentiation effects of the parent molecule linalool

itself.

Keywords: Cys-loop receptor, GABAA receptor, linalool, linalyl acetate, oxygenation, patch-clamp

INTRODUCTION

Essential oils form a class of concentrated volatile compounds generated as secondary metabolitesin aromatic plants and are characterized by their strong and pleasant odor (Silva et al., 2009; Rowan,2011). These odorous volatile substances have been widely used in folk medicine and aromatherapy(de Almeida et al., 2004).

Linalool, an acyclic monoterpene, is an important odorous constituent in a series of plantaromas. Amongst others, it is the major component of essential oils (35–51%) obtained fromvarious types of lavender extracts and is widely used for production of fragrances, shampoos, soaps,and detergents (Mitic-Culafic et al., 2009; Carrasco et al., 2016). In addition to the high importanceof linalool in perfume industry, several studies on lavender oils have demonstrated the ability ofthese aromas to improve sleep in elderly people and infants (Hudson, 1996; Field et al., 2008). In linewith these observations on humans, a physiological effect on sedation and anxiety-related behaviorhas been shown in several animal studies following inhalation of linalool with comprehensiveanalysis of mice in relation to anxiety-related behavior, social interactions, and aggression behavior






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Milanos et al. Monoterpene Modulation of GABAA Receptors

(Buchbauer et al., 1993; Bradley et al., 2007; Linck et al.,2010; Souto-Maior et al., 2011). The molecular mechanismsbehind these actions of linalool as an anxiolytic agent however,remain unsolved. Studies focusing on ion channels were carriedout to understand the physiological action of linalool. Usingelectrophysiological measurements, a non-selective suppressionof voltage-gated sodium channels has been demonstratedin olfactory receptor neurons and Purkinje cells (Narusuyeet al., 2005). Furthermore, linalool suppressed the functionof excitatory glutamate receptors (Silva Brum et al., 2001;Ohkuma et al., 2002). In contrast to suppressive effects, apotentiation of inhibitory GABAA receptors has been previouslyreported (Kessler et al., 2012, 2014). The observed potentiationat inhibitory GABAA receptors is in agreement with the linalooleffect on sedation and anxiety-related behaviors in mice.

In nature or after uptake by ingestion or inhalation,lavender oil undergoes numerous chemical modifications,possibly because lavender oil lacks a natural protection againstautoxidation (Hagvall et al., 2008; Christensson et al., 2016). Afteradministration of lavender oil, the main constituent linalool isexposed to enzymatic activity of cytochrome P-450 (CYP76C1)in lung and liver (Chadha and Madyastha, 1984; Boachonet al., 2015). Linalool derivatives such as 8-oxolinalool, 8-hydroxylinalool, and 8-carboxylinalool are formed (Aprotosoaieet al., 2014). Recently, it has been shown that plant leaves alsocontain cytochromes in order to generate linalool metabolitesthemselves (Boachon et al., 2015).

Linalool also undergoes acetylation processes. Linalylacetate metabolism was studied before in Pseudomonasincognita (Renganathan and Madyastha, 1983). In additionto acetylation, the C8-moiety of linalyl acetate is susceptibleto further oxidation processes giving rise to 8-hydroxylinalylacetate, 8-oxolinalyl acetate, and 8-carboxylinalyl acetate(Renganathan and Madyastha, 1983). A recent study on thesmell characteristics of such structural derivatives of linaloolsuggested that oxygenation at C8 has a substantial impact onodor properties with 8-oxolinalyl acetate harvesting similar odorproperties compared to linalool (Elsharif et al., 2015). The furtherphysiological potential of these numerous linalool derivatives,besides smell, has not yet been investigated with the suggestedtarget molecules. Here, we set out an experimental series to closethis gap with the analysis of the modulatory activity of linaloolderivatives at GABAA receptors, which are involved in sedativeprocesses in the central nervous system.

GABAARs belong to the superfamily of Cys-loop receptorsand are ligand gated ion channels (Grenningloh et al., 1987).These receptors are mainly present in the mammalian centralnervous system (CNS) and are expressed throughout almost theentire brain. Their function in the adult organism is to inhibitincoming action potentials by enhancing the anion concentrationof the neuronal cytosol, leading to hyperpolarization of the cell.Binding of two GABA (4-aminobutanoic acid) neurotransmittermolecules to the extracellular domain of the receptor complex aresufficient to induce conformational changes that allow channel

Abbreviations: GABAAR, GABAA receptor; C3, carbon atom 3; C8, carbon atom

8; GABA, 4-aminobutanoic acid.

opening and a chloride ion influx (Sieghart, 2006). The structureof GABAA channels is determined by five subunits that arrangein a rosette-like structure to form the ion channel pore (Millerand Aricescu, 2014). There are 19 genes that encode for GABAARsubunits (α1–6, β1–4, γ1–3, δ, ε, θ, ρ1–2, π) that form GABAA

receptor channels (Sieghart, 2006).Previously, it was demonstrated that processes such as

sedation and anxiety are mediated by different GABAARconfigurations (Low et al., 2000). Thus, GABAARs representexcellent drug targets for anticonvulsant and sedative agents(Middendorp et al., 2014; Mendonca Junior et al., 2015).

In addition to anticonvulsants, GABAA receptorsare allosterically modulated by monoterpenes. Amongmonoterpenes, bicyclic monoterpenes, and/or carriers ofhydroxyl groups harbor the highest positively modulatingpotential (van Brederode et al., 2016). In a previous study, wehave shown that linalool and the bicyclic monoterpenes myrtenoland verbenol identified in Sideritis extracts enhanced GABAergiccurrents 2- to 7-fold (Kessler et al., 2012, 2014). Whereas,linalool is well-analyzed in terms of GABAA modulatorycapacity, linalool metabolites or degradation products have neverbeen investigated in the context of GABAA receptor modulation.

Here, we investigate linalool derivatives and theirpotential action on GABAARs by overexpressing the ionchannel in HEK293 cells. HEK293 cells are widely used forelectrophysiological measurements. They are easy to handleand to transfect with plasmid DNA of interest. Receptoroverexpression is enabled by transfection of these cells withvarious GABAA receptor subunits and represents a ratherconsistent readout to study the neurotropic action of linaloolderivatives.

GABAAR functionality was investigated upon applicationof the agonist GABA in the presence and absence oflinalool derivatives. The readouts were electrophysiologicalmeasurements (patch-clamp) to determine the allostericpotential of the derivatives in terms of modulation of ion channelfunctionality.

MATERIALS AND METHODS

ChemicalsLinalool derivatives were synthesized by Shaimaa Elsharif. Allcompounds except L5 have been characterized as described(Elsharif et al., 2015). GABA and linalool were purchased fromSigma-Aldrich (Taufkirchen, Germany). 1,2-Dihydrolinalool(TCI Europe Research Chemicals, Eschborn, Germany),selenium dioxide, ethanol, dioxane and petroleum ether (Sigma-Aldrich, Taufkirchen, Germany), and diethyl ether (Scientific,Loughborough, UK) were purchased for the synthesis of8-oxo-1,2-dihydrolinalool (L5).

Synthesis of 8-Oxo-1,2-DihydrolinaloolCompound 8-oxo-1,2-dihydrolinalool (L5) (Figure 1) wasprepared following the method of Wakayama et al. (1973). 1,2-Dihydrolinalool (4.9 g, 31.4 mmol) and selenium dioxide (SeO2;3.5 g, 31.4 mmol) were dissolved in 25 mL of dioxane/ethanol9:1 (v/v) and the solution was heated at 80◦C for 5 h. After






FIGURE 1 | Linalool and linalool metabolites. Linalool a monoterpene alcohol

(3,7-dimethyl-1,6-octadiene-3-ol), linalyl acetate and its derivatives.

Compounds L1-3 are acetylated at carbon 3. Compound L1 (8-hydroxylinalyl

acetate), compound L2 (8-oxolinalyl acetate), compound L3 (8-carboxylinalyl

acetate). Compounds L4-7 are linalool metabolites substituted at carbon atom

8. Compound L4 (8-hydroxylinalool), compound L5 (8-oxo-dihydrolinalool),

compound L6 (8-oxolinalool), compound L7 (8-carboxylinalool).

removal of selenium deposit by double filtration, the solvent wasremoved under reduced pressure using a rotary evaporator. Theresidue was purified twice by flash chromatography on silica gel60 (Merck, Darmstadt, Germany) with petroleum ether/diethylether 1:4 (v/v) to afford 1.182 g (22.2 %) of L5 as a reddish-orangeoil:

1HNMR (600MHz, CDCl3-d) δ 0.92 (t, J= 1.00 Hz, 3 H) 1.19(s, 3 H) 1.31–1.43 (m, 2 H) 1.53 (q, J = 7.30 Hz, 2 H) 1.59–1.64(m, 2 H) 1.75 (s, 3 H) 6.52 (t, J = 1.00 Hz, 1 H) 9.38 (s, 1 H); 13CNMR (151 MHz, CDCl3) δ 195.26, 154.94, 139.20, 72.54, 39.48,34.49, 26.18, 23.68, 9.11, 8.22; MS (EI)m/z (%) (rel int) 170 [M+](1), 155 (3), 141 (6), 123 (10), 95 (82), 83 (41), 73 (94), 67 (44), 55(46), 43 (100).

Cell LinesThe HEK293 cell line (Human embryonic kidney cells) waspurchased from ATCC (Wesel, Germany) and grown in Earle’sminimal essential medium (MEM) supplemented with 10% fetalcalf serum, 200 mM GlutaMAX, 100 mM sodium pyruvate and50 U/mL penicillin/streptomycin (Sigma-Aldrich, Taufkirchen,Germany) under standard growth conditions at 37◦C and 5%CO2.

Transfection of Cell LineHEK293 cells were transiently transfected using a modifiedcalcium-phosphate precipitation method. Plasmid DNAs ofGABAA α1, β2, γ2 subunits and GFP were mixed in a ratioof 1:1:2:1 and supplemented with 2.5 M CaCl2 and 2x HBSbuffer (50 mM HEPES, 12 mM glucose, 10 mM KCl, 280mM NaCl, 1.5 mM Na2HPO4, pH 6.98) followed by 20 minincubation at room temperature. The transfection solution wasapplied onto the cells for 6 h. Cell medium was replaced byfresh medium to reduce transfection stress. Electrophysiologicalrecordings were performed 48 h post-transfection. To controlfor transfection efficiency, a GFP plasmid was cotransfectedwhen cells were used for electrophysiological analysis. Using afluorescence microscope, green fluorescent cells were detectable.It is expected that cells transfected with GFP also have taken upother plasmid DNAs provided in the same transfection solution.Only green cells were used for electrophysiological analysis.

Immunocytochemical StainingsCells were transfected with GABAA α1 and β2 subunits togetherwith pDsRed-Monomer-Mem 1:1:0.5. pDsRed-Monomer-Mem(Clontech, Mountain View, CA, USA) encodes a fusion proteinconsisting of neuromodulin (GAP-43) and a red fluorescentprotein used as plasma membrane marker. To proof surfaceexpression, the α1 subunit was stained following fixation with4% PFA and three washing steps with PBS (phosphate-bufferedsaline). A blocking step was included for 1 h using 5%normal goat serum in PBS. Cells were not permeabilized toallow staining of surface membrane proteins only. As primaryantibody a rabbit-anti GABAAR α1 (ST-J93186-200, Biotrend,Köln, Germany) was used in a 1:500 dilution. After 1 h ofincubation, cells were washed three times with PBS beforethe secondary antibody goat-anti rabbit Alexa488 (Dianova,Hamburg, Germany) was applied for 30 min. After a finalwash, a 5 min incubation step with DAPI (1:20,000) wasdone before cover slips were mounted with Mowiol (Sigma-Aldrich, Taufkirchen, Germany). Immunocytochemical stainingswere imaged using the Olympus IX-81 confocal microscope(Olympus, Hamburg, Germany).

Electrophysiological MeasurementsForty-eight hours after transfection, electrophysiologicalmeasurements were carried out. The electrophysiologicalsetup is supplied with a fluorescence microscope, which easilyallowed detection of GFP positive cells. It is believed thatGFP positive cells also have taken up other plasmid DNAsprovided by the same transfection solution. All GFP positive cellsselected for electrophysiological recordings showed an inwardchloride current following application of the agonist GABAdemonstrating the successful cotransfection of GABAA receptorsubunits forming functional ion channels.

Whole cell recordings from transfected HEK293 cellswere performed by application of ligand (GABA) at variousconcentrations (0.3, 1, 3, 10, 30, 300, 1,000 µM) to estimate theconcentration where half-maximal channels responded (EC50).Current signals were amplified with an EPC-10 amplifier (HEKA,






Lambrecht, Germany). The aroma substances (2 mM) were co-applied for 500 ms together with GABA at EC10−30 to the samecell. Cells measured in the same experiment with GABA andGABA + compound were compared and taken for analysisand data presentation. Mean absolute current values of all cellsmeasured are shown in Table 1.

Recording pipettes were fabricated from borosilicatecapillaries with an open resistance of about 4 M�. Cells that weresealed with a resistance of 0.5–1 G� and leak currents below–300 pA were taken into the data pool. The capacity of recordedcells was in the range of 10–15 pF. All current responses weremeasured at a holding potential of –60 mV. The experimentswere carried out at room temperature. The extracellular bufferconsisted of 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mMMgCl2, 11 mM EGTA, 10 mM HEPES, with a pH adjusted to7.4 with NaOH. The internal buffer was 120 mM CsCl, 20 mMN(Et)4Cl, 1 mM CaCl2, 3 mM MgCl2, 11 mM EGTA, 10 mMHEPES with a pH adjusted to 7.2 with CsOH.

Statistical AnalysisConcentration-response curves were constructed from the peakcurrent amplitudes obtained with at least seven appropriatelyspaced concentrations in the range 0.3–1,000 µM GABA.Using a non-linear algorithm (Microcal Origin), concentration-response data were first analyzed using the following Hillequation IGABA/Isat = [GABA]nHill/[GABA]nHill + ECnHill

50 whereIGABA refers to the current amplitude at a given GABAconcentration, Isat is the current amplitude at saturatingconcentrations of GABA, EC50 is the GABA concentrationproducing half-maximal current responses, and nHill is the Hillcoefficient.

The significance levels for recorded absolute currents werecalculated using the students t-test. Data presented do alwaysshow mean values. The error bars refer to SEM (standard errorof the mean) values. Significance level are ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001.

TABLE 1 | Electrophysiological properties of modulated GABAA receptor of the

α1β2γ2 subtype.

Compound Iabs [nA]

± SEM

Irel [%]

± SEM

Iabs [nA]

± SEM

Irel [%]

± SEM

10 µM GABA 10 µM GABA 10 µM GABA

+ compound

10µM GABA

+ compound

Linalool 0.4 ± 0.07 100 ± 15 0.7 ± 0.1 160 ± 14

Linalyl

acetate

0.4 ± 0.07 100 ± 15 0.6 ± 0.3 136 ± 16

L1 1.5 ± 0.8 100 ± 53 1.0 ± 0.4 68 ± 42

L2 1.1 ± 0.4 100 ± 37 1.3 ± 0.4 122 ± 33

L3 0.6 ± 0.1 100 ± 12 0.5 ± 0.1 95 ± 16

L4 0.5 ± 0.1 100 ± 16 0.5 ± 0.1 82 ± 26

L5 0.8 ± 0.4 100 ± 37 1.3 ± 0.5 144 ± 58

L6 0.5 ± 0.2 100 ± 48 0.8 ± 0.3 166 ± 35

L7 1.1 ± 0.5 100 ± 35 1.1 ± 0.3 99 ± 29

RESULTS

GABAA receptors are modulated by terpenoid substances.Linalool, a major component of lavender oil harbors GABAA

receptor modulatory potential. Here we investigate derivativesof linalool that can be biosynthesized by plants or formed bybiotransformation processes of linalool in the liver or lungfollowing inhalation of linalool or food intake. These substanceshave already been characterized in the context of their structureodor-activity relationships (Elsharif et al., 2015).

Generation of Linalool DerivativesLinalool is a monoterpene alcohol (3,7-dimethyl-1,6-octadiene-3-ol), while linalyl acetate is substituted with an acetyl moietyat carbon atom 3 (C3) (Figure 1). The conjugated compoundscan be divided into two groups. The first group of compounds(L1-L3) (Figure 1) contains twomodifications within the linaloolstructure. (i) L1-L3 are acetylated at carbon atom 3 (C3). (ii)These acetylated compounds carry an additional modification oncarbon atom 8 (C8). Both modifications generate compoundsL1 (8-hydroxylinalyl acetate), L2 (8-oxolinalyl acetate), andL3 (8-carboxylinalyl acetate) (Figure 1). The second group oflinalool derivatives is oxygenated on C8, while the hydroxy-group at C3 remained. This series results in compoundsL4 (8-hydroxylinalool), L5 (8-oxo-1,2-dihydrolinalool), L6 (8-oxolinalool), and L7 (8-carboxylinalool) (Figure 1). L5 is the onlylinalool derivative with two substitutions, an oxygenation at C8and a reduction at C1-C2 generating 8-oxo-1,2-dihydrolinalool.

GABAA α1β2γ2 Receptor Complex Used forFunctional Analysis Is Expressed at theCell SurfaceThe GABAAR complex requires cell surface expression inorder to allow functional ion channel analysis. Functionalheteromeric GABAA receptor channels are formed by α1β2and α1β2γ2 subunits (Luger et al., 2015). Therefore, theexpression of the α1 subunit was tested by immunocytochemicalstainings (Figure 2). The cotransfection of GAP-43 allowedan estimation of the transfection efficiency in addition toits function as cellular membrane marker. The transfectionefficiency was determined to around 60% (Figure 2A, lowermagnification, right merged image). The GABAA α1 subunitis transported to the cellular surface and co-localized withthe membrane marker GAP-43 (Figures 2A,B). The enlargedpictures demonstrate the precise surface expression by theobserved ring-like structure at the outer cell surrounding(Figure 2B). This ring-like structure is only observed withoutpermeabilization of the cells during staining procedure.Since homomeric α1 receptors have never been shown toform functional GABAA receptors, the proof of heteromericreceptor functionality can only be given by electrophysiologicalanalysis.

GABAAR Modulation by LinaloolDerivativesAll measurements were carried out 48 h post-transfection. Toanalyze GABAAR functionality, a series of GABA concentrations






FIGURE 2 | HEK293 transiently expressing GABAA α1β2 receptors and GAP-43 fused with red fluorescent protein. (A) Upper lane demonstrates transfection

efficiency controlled by co-transfection of the membrane marker protein GAP-43 fused to dsRed (magenta). The α1 subunit of the GABAA receptor was stained

specifically at the cellular surface (green). The merged picture represents co-localization resulting in a white signal of both the GABAA α1 receptor subunit and GAP43

(magenta) at the plasma membrane. (B) The lower lane shows an enlarged image of a single stained cell. DAPI staining was used to mark the nucleus of the cell

(blue), α1 subunit is marked in green and GAP-43 in magenta.

(0.3–1,000 µM) was applied to patched GFP-positive transfectedHEK293 cells in an α1β2γ2 configuration to determine the EC50

value 37 ± 0.4 µM (Figure 3A). Most published data analyzingion channel modulators use EC5−10 concentrations of agonistto come up with highest effects observed for the modulatingagents (Khom et al., 2007). Higher GABA concentrations up tosaturating levels will highly reduce the potentiating effects ofthe compounds. We used an EC10−30 GABA concentration (10µM GABA) for physiological analysis (Figure 3B). The currentsobserved after 10 µM GABA application were normalized to100% (Figure 3B, black bar). Linalool (2 mM) or linalyl acetatewere co-applied with 10 µM GABA. The observed enhancedrelative currents were quantified to 160 ± 14% for linalool(white bar) and 136 ± 16% for linalyl acetate (striped bar;Figure 3B, Table 1). The 1.6-fold increase in Irel followinglinalool application corresponded to a significant elevation (∗P< 0.05). The data obtained for linalool are in line with thedescribed potency reported previously in the literature (Kessleret al., 2014).

The linalool derivatives (2 mM) were co-applied on the cellafter a first application of the agonist alone (10 µM) and resultedin an inward chloride current (Figure 4, Table 1). Applicationof the acetylated compounds L1–L3 together with GABA didnot result in significant changes of the GABAergic currents(Figure 4). Compound L1 (8-hydroxylinalyl acetate) resulted ina decreased relative current (Irel) of 68 ± 42%, while compoundL2 (8-oxolinalyl acetate) reached 122 ± 33% of a GABA-evoked current. Current values for L1 demonstrated that amongdifferent cells a large variability exists otherwise this derivativewould be considered a negative modulator. L3 (8-carboxylinalylacetate) was with 95 ± 16% almost indistinguishable from

GABA application only (Figure 4, for absolute current values seeTable 1).

In case of the oxygenated linalool products differencesin modulation were seen. Compound L4 (8-hydroxylinalool)reached 82 ± 26% of the relative currents observed afterGABA application alone. Again, a hydroxy group at C8 rathergenerated reduced GABAergic currents. In contrast, compoundsL5 (8-oxo-1,2-dihydrolinalool) and L6 (8-oxolinalool) showed apositive modulatory effect at GABAA receptors of the α1β2γ2configuration. This receptor configuration refers to 65% of allGABAergic neurons in vivo. L6 application generated GABAergiccurrents, which were 1.66-fold increased (166 ± 35% comparedto GABA alone, Figures 5A,B, Table 1). The modulatory potencyof L6 was comparable to linalool, but did not reach significance.Coapplication of GABA together with L5 had also a pronouncedeffect with 144 ± 58% of GABA-induced currents (Figure 5A).Compound L7 (8-carboxylinalool) had no significant effect with99± 29% of GABA activity when applied (Figures 5A,B).

DISCUSSION

Linalool is well-known for its odor potential in perfume industryand aromatherapy. Due to the calming effect of linalool, thisacyclic monoterpene has been investigated in studies on sedationand anxiety in animal models (Linck et al., 2010; Souto-Maioret al., 2011). It may be tempting to assume that smell potencyand pleasantness of a compound might be related to furtherphysiological action, especially balancing and relaxation effectsor anxiolytic potential. Well-being as induced by the sense ofsmell and odorous substances, however, most likely needs to be






FIGURE 3 | GABAA α1β2γ2 receptor EC50 determination for the agonist

GABA. (A) Calculation of half-maximal receptor activation using seven different

concentrations of the agonist (0.3–1,000 µM). The EC50 was determined at

37 ± 0.4 µM GABA, while EC10−30was calculated to 10 µM. (B) The black

bar illustrates the mean relative current of GABAA receptors α1β2γ2 at 10 µM

GABA. Potentiation of GABAergic currents by linalool (2 mM) at

EC10−30GABA concentrations (10 µM) refers to the white bar, *P < 0.05;

linalyl acetate (striped bar); n = number of cells recorded.

separated into different modes of action and temporally as wellmechanistically independent pathways.

While odorants act in humans first at the peripherywith activation of G-protein coupled odorant receptors, i.e.,metabotropic receptors, their mode of allosteric modulation atGABAA receptors differs as these ionotropic receptors formstructurally completely different physiological entities in higherbrain regions. Distinct GABAA receptor configurations in thecentral nervous system have been shown to mediate processes,such as sedation and anxiolytic effects (Low et al., 2000). Amodulation of GABAA receptors of the sedative α1β2γ2 subtypeby linalool was proven in Xenopus oocytes and an allostericmechanism for the observed interaction was postulated (Kessleret al., 2014). Here, the same GABAA receptor subtype wasused to analyze linalool and its derivatives after acetylationof linalool at C3 and further oxygenation at C8 of linalooland linalyl acetate. Following control of receptor expression,

FIGURE 4 | Acetylated linalool derivatives do not potentiate GABAergic

currents in HEK293 cells. Ten micromolars of GABA were applied to α1β2γ2

receptors and refers to 100% (100 ± 15% GABA application, black bar;

relative to GABA + linalyl acetate). Acetylated linalool compounds such as

linalyl acetate (striped bar), L1 (8-hydroxylinalyl acetate), L2 (8-oxolinalyl

acetate), L3 (8-carboxylinalyl acetate) used in a 2 mM concentration were

co-applied with GABA (gray bars), n = number of cells recorded.

the determination of the EC50-value for half-maximal receptoractivation was comparable to EC50-values described in theliterature (Sieghart, 2006).Maximal neurotransmitter applicationwill superpose the modulatory efficacy of terpenes at GABAA

receptors (Hossain et al., 2002). A GABA concentration of 10µM corresponding to EC10−30 was used to obtain maximalmodulation by the volatile compound used. At the usedGABAA receptor configuration α1β2γ2, linalool potentiated theGABAergic responses significantly by 1.7-fold similar to previousobservations (Kessler et al., 2014).

Oxidative and polymerization processes of essential oils mayresult in loss of quality and pharmacological properties (Turekand Stintzing, 2013). The linalool skeleton is characterized bydouble bonds and the C3 hydroxy group making it vulnerableto further modifications including derivatization (Raguso, 2016).The importance of C3 in linalool as a main contributor toodor quality has recently been shown (Elsharif et al., 2015).Acetylation at C3 resulted in low odor potency. This effect canbe compensated by a further modification of linalool at theC8 atom. Oxidation at C8 generating 8-oxolinalyl acetate (L2)and 8-carboxylinalyl acetate (L3) rescued odor potency whilehydroxylation at C8 did not positively contribute to odor potency(Elsharif et al., 2015). The modulation of the GABAA receptorfunctionality was also affected by modifications of the linaloolstructure. Although changes in GABAergic currents were smalland data did not reach significance, 8-oxolinalyl acetate (L2)showed a tendency to positively modulate GABAA receptors witha 1.2-fold increase whereas 8-carboxylinalyl acetate (L3) did notmodulate the receptor. Hydroxylation at C8 of linalyl acetateled to a reduction of the overall GABAergic responses. Thus,hydroxylation of linalyl acetate does not only give rise to lowodor quality but also to lack of modulating potential at GABAA

receptors.






FIGURE 5 | Oxygenated linalool derivatives potentiated GABAergic currents in

HEK293 cells. (A) 10 µM GABA applications are nominated to 100% (100 ±

15% GABA application relative to GABA + linalool, black bar), linalool (white

bar), *P < 0.05. Compounds L4 (8-hydroxylinalool) and L7 (8-carboxylinalool)

did not enhance or reduce GABA-evoked currents significantly. Compounds

L5 (8-oxo-dihydrolinalool) and L6 (8-oxolinalool) enhanced GABAergic

currents (gray bars). (B) Representative current traces detected from

transfected HEK293 cells with GABAA receptor subunits α1β2γ2 following an

application of 10 µM GABA for 500 ms (black trace) or a co-application on the

same cell of 10 µM GABA + L6 (dark gray trace, potentiating) or L7 (light gray

trace, not potentiating), small bars above the traces refer to the time the

agonist ± modulator were applied to the cell, n = number of cells recorded.

Oxidation via cytochrome P-450 of linalool occurs inthe liver of animals but similar oxygenation reactions havealso been depicted in plants (Chadha and Madyastha, 1984;Boachon et al., 2015). The determined oxygenated derivativesof linalool in animals or plants were 8-oxolinalool (L6), 8-hydroxylinalool (L4), and 8-carboxylinalool (L7) (Chadha andMadyastha, 1984; Aprotosoaie et al., 2014). Moreover, odorantsand other volatile substances can also be metabolized right at theperiphery within the nasal cavity in the frame of peri-receptorevents, thereby potentially generating novel substances anddegrading others. This process has been shown to go along withchanges in smell character and intensity of specific substances(Schilling, 2017). Terpenoid substances can also undergo heavybiotransformation prior to metabolism following ingestion andpassage of the gastro-intestinal tract (Heinlein and Buettner,2012). In the gut, chemical transformation processes areprimarily associated with the acidic conditions in the stomach.A series of other terpenoid derivatives has been determined

following these transformation pathways, e.g., linalool degradedto geraniol, nerol, and α-terpineol (Heinlein and Buettner, 2012).Accordingly, biotransformation and bioavailability are importantaspects when discussing potential physiological effects of aromasubstances.

The odor-function relationship analysis revealed thatoxygenation at C8 of linalool and 8-hydroxylinalool showedreduced odor potential. 8-Carboxylinalool was odorless (Elsharifet al., 2015). Yet, it needs to be kept in mind that additionalnasal biotransformation processes may occur in smell perceptionof humans, in some cases. It is not fully clear if these smelleffects do only relate to the respective substance or potentiallyalso some derivatives thereof. Nevertheless, it is interesting tonote that analysis of the allosteric modulation at the GABAA

receptor subtype α1β2γ2 demonstrated that 8-hydroxylinalool(L4) and 8-carboxylinalool (L7) lost the ability to potentiateGABAergic responses. In contrast, 8-oxolinalool (L6, 1.6-fold)and 8-oxo-1,2-dihydrolinalool (L5, 1.4-fold) kept at least inpart the ability to potentiate GABAA receptors but did notreach significance as observed for linalool. In summary, thehydroxy group at C3 in linalool is obviously important for thepositive allosteric modulation at inhibitory GABAA receptors.The structural modification at C8 by oxygenation might bepromising to further increase modulatory potency maybe bycombining this oxygenation with other structural modifications,e.g., at C1/C2. These data might also have an impact on structuralalterations of other monoterpenes or bicyclic terpenes, whichhave also been pointed out to allosterically interact with GABAA

receptors (Granger et al., 2005; Kessler et al., 2014). Our grouprecently investigated the effect of structural modification onsmell parameters of other related terpenoid substances, namelygeraniol, nerol, β-citronellol, and their related acetates (Elsharifand Buettner, 2016). Of these, β-citronellol has previously alsobeen shown to exert GABAergic modulatory activity (Kessleret al., 2014). Accordingly, it will be interesting to investigate iftheir related analogs show comparable effects in GABAergicmodulatory capacity. Processing of monoterpenes followingingestion or inhalation accordingly becomes an importantissue since these substances also target other ligand-gated ionchannels. The allosteric interaction between linalool and GABAA

receptors is hypothesized to occur by a direct interaction withthe transmembrane domains of the receptor embedded in thehydrophobic lipid bilayer of neuronal cells and thus, accessibleby the highly hydrophobic monoterpene (Khom et al., 2007). Theanalyzedmetabolic products of linalool increased solubility of thecompounds. We can conclude from our study that increasing thecompounds solubility results in a reduction of GABAA receptormodulation. To keep the balance between lipophilicity andsolubility is therefore important in novel screens for modulatorsmaintaining their natural odor and physiological potential.

In our previous study we could show that linalool and8-oxolinalyl acetate (L2) were both the most intense odorantsof all substances investigated, with odor thresholds as low as 3and 6 ng/L air, respectively (Elsharif et al., 2015). Interestingly,both compounds also showed comparable GABAA modulatoryactivity. However, in this context it is important to note thatsmell perception, i.e., the “peripheral” effect of such substances,






can be highly variable between individuals. Amongst others,we could show that such odorants are, at times, perceivedwith highly variable odor thresholds and, vice versa, odorintensities by different subjects, and can even elicit differentsmell impressions. It is hypothesized here that, in contrast tothis individually variable sensory effect, the pharmacologicaleffect of neurotropic action, as mediated via GABAA receptorexpression in transfected cells, rather represents a consistentreadout. Moreover, several in vivo studies have demonstratedcommon physiological effects on sedation and anxiety-relatedbehavior in mice following inhalation of linalool (Buchbaueret al., 1993; Bradley et al., 2007; Linck et al., 2010; Souto-Maior et al., 2011). Thus, our study suggests that a combinedapproach of physiological determination of odor and theirmetabolite neurotropic activities ideally by high-throughputtechniques combined with personalized odor perceptionprofiles represent a future perspective for aromatherapy andanxiety-related diseases. All these considerations, however,require much more detailed combined studies in the future,

likewise involving human studies, with specific focus on singleindividuals.

AUTHOR CONTRIBUTIONS

Participated in research design: CV, AB. Conducted experiments:SM, DJ. Substances synthesized by SE, Writing paper: CV, SM,AB.

FUNDING

This work was supported by DFG VI586 (CV).

ACKNOWLEDGMENTS

We would like to thank Lampros Milanos for critical readingof the manuscript and helpful comments. Gudrun Schell andNadine Vornberger are highly acknowledged for their excellenttechnical assistance.

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doi: 10.1246/bcsj.46.3183

Conflict of Interest Statement: The authors declare that the research was

conducted in the absence of any commercial or financial relationships that could

be construed as a potential conflict of interest.

Copyright © 2017 Milanos, Elsharif, Janzen, Buettner and Villmann. This is an

open-access article distributed under the terms of the Creative Commons Attribution

License (CC BY). The use, distribution or reproduction in other forums is permitted,

provided the original author(s) or licensor are credited and that the original

publication in this journal is cited, in accordance with accepted academic practice.

No use, distribution or reproduction is permitted which does not comply with these

terms.


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83

7 Proceedings of the XV Weurman Flavour Research Symposium 2017: Structure-Odor Relationship Study of C-6 Unsaturated Acyclic Monoterpene Alcohols: A Comparative Approach

Weurman Book: Flavour Science

Shaimaa Awadain Elsharif a and Andrea Buettner a,b

a Friedrich-Alexander-Universität Erlangen-Nürnberg, Professorship for Aroma Research, Emil Fischer Center, Department of Chemistry and Pharmacy, Henkestraße 9, 91054 Erlangen, Germany, [email protected], [email protected]

b Fraunhofer Institute for Process Engineering and Packaging, Giggenhauser Straße 35, 85354 Freising, Germany, [email protected]

The paper “Structure-odor relationship study of C-6 unsaturated acyclic monoterpene alcohols: a comparative approach” (S.A. Elsharif & A. Buettner) was submitted in July 2017 to be published in the book ‘Flavour Science – Proceedings of the 15th Weurman Flavour Research Symposium’.

Reprint for the purpose of this PhD thesis with permission of B. Siegmund et al. (Eds.) and Verlag der TU Graz (Publisher).




84

Structure-odor relationship study of C-6 unsaturated acyclic monoterpene

alcohols: a comparative approach

Shaimaa Awadain Elsharifa and Andrea Buettnera,b

a Friedrich-Alexander-Universität Erlangen-Nürnberg, Professorship for Aroma Research, Emil Fischer Center, Department of Chemistry

and Pharmacy, Henkestraße 9, 91054 Erlangen, Germany, [email protected], [email protected]

b Fraunhofer Institute for Process Engineering and Packaging, Giggenhauser Straße 35, 85354 Freising, Germany,

[email protected]

Abstract

Acyclic monoterpenes are a valuable class of compounds useful for the flavor and fragrance industries [1].

Among them are the C-6 unsaturated monoterpene alcohols, namely linalool, geraniol, nerol and β-citronellol.

These substances exhibit pleasant smell properties, are prevalent in the essential oils of many plants and are

pharmacologically and physiologically active. Thereby, it is interesting to note that linalool and geraniol,

specifically, do not only activate olfactory receptors, but have also other physiological activities, e.g. acting as

anti-cancerogens [2, 3]. Systematic elucidation of the sensory characteristics of metabolic derivatives of this

substance group, however, is very limited as most work, until today, focused on the basic acyclic monoterpene

compounds. Our studies demonstrated that a series of these metabolites are odor active compounds, at times

exhibiting exceptionally pleasant smells [4, 5]. In the course of our studies, we started from the respective

monoterpene alcohols and their corresponding acetates, yielding a total of 24 oxygenated derivatives via diverse

synthetic strategies, and characterized their olfactory properties. Specifically, these compounds were tested with

regards to their odor qualities, relative odor thresholds (OTs) in air, and potential inter-individual variations in

human sensory perception for each single substance. Finally, a comprehensive substance library was established

comprising the respective retention index data (RI values) as well as mass spectrometric and nuclear magnetic

resonance data, to aid in future analytical studies on this sensorially fascinating substance class.

Introduction

Apart from being fragrant compounds and main constituents of essential oils, linalool, geraniol, nerol and

citronellol (comprised within the group of monoterpeneols) are characterized by several pharmacological and

physiological properties. To mention but some examples, linalool, found in lavender plant, potentiates GABAA

receptor modulatory activity in the central nervous system; this mechanism is supposed to be the underlying

principle for sleep-inducing and balancing effects in humans [6, 7]. Similarly, nerol, present in lemon balm

(Melissa officinalis L.) and bushy matgrass (Lippia alba), showed to exhibit an anxiolytic effect in mice without

producing tremors or seizures [8]. Geraniol, the trans-isomer of nerol and being mainly found in palmarosa

(Cymbopogon martinii (Roxb.)), is a plant-based insect repellent especially active against mites [9], ticks [10]

and mosquitos [11]. Being a main component in various plants with antihypertensive properties, such as lemon

grass leaves (Cymbopogon citratus) and citronella grass (Cymbopogon winterianus), citronellol has been

reported to be used in the treatment of several cardiovascular diseases in folk medicine [12]. Recently, studies

showed that citronellol has a vasodilatory effect and therefore is claimed to be a hypotensive agent [13, 14].

Apart from that, these monoterpenes and their acetate esters have previously been studied in view of their odor

characters, without comprehensively correlating these smell properties with their chemical structure. In addition,

the metabolic derivatives of these compounds in plants, animals and microorganisms have been studied [15-17].

The main metabolic pathway includes C-8 hydroxylation of these monoterpenes yielding 8-hydroxy compounds

which are further oxidized to the corresponding 8-carboxy derivatives. Due to the lack of commercial

availability of these metabolites, the present work aimed at the synthesis of a total of 24 C-8 oxygenated

compounds, and the determination of their odor qualities and odor thresholds (OT) in air using gas

chromatography-olfactometry (GC-O). It was found that most of these derivatives elicited distinct smells [4, 5,

18]. Therefore, a structure-odor relationship study was established in a comparative approach comprising all the

aforementioned monoterpenes, their acetates and their derivatives, highlighting the main structural features and

functional groups that impact the odor quality and potency of this substance class.




85

Figure 1: General synthetic pathways leading to the

oxygenated derivatives

Experimental

Syntheses

General synthetic pathways are shown in Figure 1.

Chemicals required for synthesis were purchased from

Sigma-Aldrich or Fischer Scientific. Data comprising

nuclear magnetic resonance spectra (1H and 13C), mass

spectra as well as retention indices were recorded and are

described in Elsharif, Banerjee [4], Elsharif and Buettner

[5], Elsharif and Buettner [18].

Evaluation of odor quality

Odor qualities were determined using GC-O involving

five panelists who were trained volunteers from the

University of Erlangen. Compounds were evaluated by each

panelist repeatedly on different days on different capillary

columns (DB-FFAP and DB-5). Panelists were asked to

relate their sensory impression to an in-house developed

flavor language.

Odor threshold determination

Following the procedure of Czerny, Brueckner [19],

odor thresholds in air were determined using E-2-decenal as

an internal standard with aroma extract dilution analysis

(AEDA), where the flavor dilution (FD) factor and the

relative concentration of the compounds and the standard

are the basis for the calculation of the corresponding odor

threshold in air.

Results and discussion

Substances are regarded in the following two groups:

1) monoterpene alcohols (linalool, geraniol, nerol and

citronellol) and their oxygenated derivatives, and 2) the

corresponding monoterpene acetates (linalyl, geranyl, neryl and citronellyl acetates) and their oxygenated

derivatives. Synthesized compounds with aldehydic, alcoholic or acidic functional group at C-8, were termed

with the prefix 8-oxo, 8-hydroxy or 8-carboxy, respectively. Tables 1 (group 1) and 2 (group 2) show a

comparison of the odor attributes perceived by at least 60% of the panel and group odor thresholds calculated as

a geometric mean of the individual thresholds of panelists.

We found that parent monoterpene alcohols and their 8-hydroxy derivatives elicited citrus-like, fresh odor

attributes. Only two 8-oxo derivatives, 8-oxolinalool and 8-oxocitronellol showed similar odor attributes, i.e.

citrus-like and fresh. On the other hand, the two isomers, 8-oxogeraniol and 8-oxonerol exhibited a fatty, musty

odor. All 8-carboxy derivatives of this group were odorless with the sole exception of the 8-carboxynerol which

was found to have a fatty, waxy odor. Odor potency of the parent monoterpeneols was much higher than that of

the corresponding oxygenated derivatives. Linalool, with an OT of 3.2 ng/Lair, was the most potent odorant,

while 8-oxocitronellol was the least (OT: 879 ng/Lair). Geraniol and citronellol elicited almost equal OT values

(11.5, 11 ng/Lair, respectively), whereas the OT of nerol was 6-fold lower (68 ng/Lair). Although the additional

OH group at C-8 preserved the citrus-like odor of the parent monoterpeneols, it tremendously decreased their

potency. In case of 8-oxogeraniol and 8-oxonerol, the aldehyde group turned the odor to musty. A C-8 carboxy

group added to linalool, geraniol or citronellol yielded odorless substances. The overall findings can be

summarized as follows: 1) the OH-group at C-1 or C-3 is responsible for the citrus-like odor and the low OT of

the parent monoterpenes, 2) an additional OH at C-8 only retains the odor quality but not the potency, 3)

oxidation of the OH at C-8 to the corresponding aldehyde group commonly turns the odor to musty and fatty,

and 4) further oxidation of the aldehyde to the respective acid leads to odorless compounds.

The parent monoterpene acetates elicited similar odor characters closely related to their monoterpene

alcohols (citrus-like) with the sole exception of neryl acetate which smells sweet and phenolic (Table 2).

Similarly, the 8-hydroxy acetates provoked citrus-like, soapy smells. 8-Oxogeranyl and 8-oxocitronellyl acetates

were perceived as fatty, musty and rotten, musty. Interestingly, all 8-carboxy acetates were found to be odor

active compounds with the sole exception of 8-carboxycitronellyl acetate. The panel described their smells as

fatty for 8-carboxylinalyl acetate, sweet and coconut-like for 8-carboxygeranyl acetate, and green for 8-

carboxyneryl acetate.

86

Table 1: Odor qualities and thresholds for the acyclic monoterpene alcohols and their synthesized oxygenated derivatives

Name Odor qualitya % of panelists Odor thresholdb,c

ng/Lair

Linalool Citrus 80 3.2

Geraniol Citrus,

fresh, fatty

80

60

11.5

Nerol Citrus, fresh 60 68

β-Citronellol Citrus

Floral, fresh

100

40

11

8-Oxolinalool Citrus,

fatty

80

60

50

8-Oxogeraniol Fatty, musty 60 139

8-Oxonerol Fatty,

musty

80

60

534.4

8-Oxocitronellol Citrus,

fresh

80

60

879

8-Hydroxylinalool Citrus,

sweet

80

40

123.6

8-Hydroxygeraniol Citrus, fatty 60 310.2

8-Hydroxynerol Citrus,

sweet, vanilla

40

60

451

8-Hydroxycitronellol Citrus,

fresh

100

80

233

8-Carboxynerold Fatty, waxy 40 297

Table 2: Odor qualities and thresholds for the acyclic monoterpene acetates and their synthesized oxygenated derivatives

Name Odor qualitya % of panel Odor thresholdb,c

ng/Lair

Linalyl acetate Citrus, fatty 60 134

Geranyl acetate Citrus 60 57.1

Neryl acetated Phenolic, sweet 40 108

Citronellyl acetate Citrus, soapy 60 665

8-Hydroxylinalyl acetate Citrus,

fresh, fatty

80

60

120.3

8-Hydroxygeranyl acetate Citrus,

soapy

80

60

62

8-Hydroxyneryl acetated Citrus 80 92

8-Hydroxycitronellyl acetate Citrus,

soapy

100

80

1261

8-Oxolinalyl acetate Citrus, lemon-like,

fatty

60 6

8-Oxogeranyl acetate Fatty, musty 60 20.5

8-Oxoneryl acetate Citrus, fatty 80 26.1

8-Oxocitronellyl acetate Musty,

rotten

80

60

346

8-Carboxylinalyl acetate Fatty 100 7

8-Carboxygeranyl acetate Sweet,

coconut

60

40

37.1

8-Carboxyneryl acetate Green 40 24

aCommon odor attributes given by the panel as perceived at the sniffing port. bOdor thresholds in air were determined as described by Ullrich and Grosch [20]. cOdor threshold was calculated as a geometric mean of the individual thresholds of panelists. dAnosmia observed.

87

Despite having a 42-fold lower OT than linalool, linalyl acetate derivatives were the most potent

compounds in this group with an OT of 6 ng/Lair for 8-oxolinalyl acetate, followed by the 8-carboxy derivative

(OT: 7 ng/Lair). The OT value of 8-hydroxycitronellyl acetate (1261 ng/Lair), on the other hand, was the

highest threshold value recorded, thus being the least potent compound in this study. The findings can be

summarized as follows: 1) the acetate group at C-1 or C-3 decreases the odor potency at least by a factor of 5,

but preserves the citrusy odor of the parent monoterpeneols with the sole exception of neryl acetate, 2) addition

of an OH- group at C-8 enhances the citrus odor with an increase in potency, 3) the C-8 aldehyde group leads to

the appearance of a musty odor for 8-oxogeranyl and 8-oxocitronellyl acetates, and 4) an acid moiety at C-8 of

the acetates induces odor attributes other than citrusy, but with a further increase in odor potency. It is important

to note that single cases of anosmia were observed for individuals with the following compounds: 8-

hydroxynerol, neryl acetate and 8-hydroxyneryl acetate.

Overall, linalool was found to be the most potent odorant in this study, followed by geraniol and

citronellol. Interestingly, 8-oxolinalyl acetate shows similar odor properties as linalool. A hydroxy function at

C-8 positively contributes to the odor quality but not potency, irrespective whether C1 or C3 bear an alcohol or

an acetate group. Any other structural changes investigated within this study led to either changes in odor

character, a decrease in the potency, or even total odor loss.

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