Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 605

_____________________________ _____________________________

Labelling of Various Macromolecules Using Positron Emitting 76Br and 68Ga

Synthesis and Characterisation

BY

ULRIKA YNGVE

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosophy in Organic Chemistry presentedat Uppsala University in 2001

ABSTRACT

Yngve, U. 2001. Labelling of Various Macromolecules Using Positron Emitting 76Brand 68Ga. Synthesis and Characterisation. Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 605. 58pp. Uppsala. ISBN 91-554-4939-5

Different prosthetic groups containing a trialkylstannyl- and an electrophilic grouphave been synthesised and labelled with the accelerator produced 76Br (T1/2=16 h)through oxidative bromination. The labelled prosthetic groups were conjugated toamino-containing macromolecules such as proteins and 5´-modified oligonucleotides.

N-Succinimidyl 4-[76Br]bromobenzoate 14 was synthesised in 65 % radio-chemical yield and was conjugated to 5´-hexylamino-modified phosphodiester andphosphorothioate oligonucleotides in 12-19 % isolated radiochemical yield. Thestability of the 76Br-oligonucleotide-conjugates in vivo in rats was investigated. Nodegradation from the 5´-end, resulting in labelled, low molecular weight compoundswas detected. Compound 14 has also been used for labelling of different proteins in23-61% radiochemical yield.

N-Succinimidyl-5-[76Br]bromo-3-pyridinecarboxylate 17 and methyl-4-[76Br]bromobenzimidate 15 were synthesised from the corresponding trimethyl-stannyl-compound in 25% and 40 % yield respectively. Compounds 14 and 17 wereconjugated to ε-Boc-octreotide in 55 and 50% isolated radiochemical yieldrespectively after microwave heating. Compound 15 did not react with octreotideunder the conditions investigated. The two 76Br-labelled octreotide derivativesshowed different lipophilicity and different binding-properties to tissue frommeningiomas.

Hyaluronic acid, a polysaccharide, was modified with tyramine and labelledby oxidative bromination using 76Br in 10% radiochemical yield.

The generator produced 68Ga (T1/2=68 min) was used to label octreotide andoligonucleotides modified with the metal chelating group 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA). 68Ga-DOTA-octreotide was isolated in65% radiochemical yield and a phosphorothioate 68Ga-DOTA-oligonucleotide wasisolated in 35% radiochemical yield after 30 min synthesis time.

Compound 14 was reacted with 3-aminomethylbenzylamine to give compound18. The specific radioactivity of 18 was determined to be 36 GBq/µmol by measuringthe ratio between the mass-peaks for the 76Br and 79Br-compounds using packed-capillary LC-MS.

Ulrika Yngve, Department of Organic Chemistry, Institute of Chemistry, UppsalaUniversity, Box 531, SE-751 21 Uppsala, Sweden

Ulrika Yngve 2001

ISSN 1104-232XISBN 91-554-4939-5

Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2001

Till Jonas

Papers included in this thesis:

This thesis is based on the following papers, which are referred to in the text by theirroman numerals.

I Synthesis of N-Succinimidyl 4-[76Br]Bromobenzoate and its Use inConjugation Labelling of MacromoleculesYngve, U.; Hedberg, E.; Lövqvist, V.; Tolmachev, V.; Långström, B. ActaChem. Scand. 1999, 53, 508-512.

II Synthesis and Characterization of 76Br-Labeled Phosphodiester andPhosphorothioate OligonucleotidesYngve, U.; Hedberg, E.; Wu, F.; Lendvai, G.; Bergström, M.; Långström, B.Submitted to Bioconj. Chem., Unpublished work copyright (2001) AmericanChemical Society

III Determination of Specific Radioactivity for 76Br Labelled CompoundsMeasuring the Ratio between 76Br and 79Br Using Packed CapillaryLiquid Chromatography Mass SpectrometryForngren, B. H.; Yngve, U.; Forngren, T.; Långström, B. Nucl. Med. Biol.2000, 27, 851-853.

IV Distribution of 76Br-Labeled Antisense Oligonucleotides of DifferentLength Determined Ex Vivo in RatsWu, F.; Yngve, U.; Hedberg, E.; Honda, M.; Lu, L.; Eriksson, B.; Watanabe,Y.; Bergström, M.; Långström, B. Eur. J. Pharm. Sci. 2000, 10, 179-186.

V 68Ga-Labelling of DOTA-OligonucleotidesYngve, U.; Långström, B. Preliminary manuscript

VI Labelling of Octreotide using 76Br-Prosthetic GroupsYngve,U.; Khan, T. S.; Bergström, M.; Långström, B. Submitted to J.Labelled Compd. Radiopharm. 2001

VII 68Ga-labelling of DOTA-OctreotideYngve, U.; Mäcke, H. R.; Långström, B. Submitted

VIII Appendix: Supplementary MaterialYngve, U.

Reprints were made with permission from the publishers.

I have been responsible for planning and carrying out all synthetic and labellingchemistry, including the chemical characterization of the labelled products, in paperI-VII. In papers I and II this work was done in collaboration with E. Hedberg. TheLC-MS analyses in paper III were carried out by B. Forngren. I wrote papers I-IIIand V-VII, paper II together with E. Hedberg and paper III together with B.Forngren. My contribution to paper IV was, except for the labelling synthesis, toperform the metabolic studies and I took part in writing the paper.

Contents:

Abstract 2Papers Included In the Thesis 4List of Abbreviations 6

1. Introduction 7Imaging technology in vivo and in vitro 7Macromolecules as tracers 8

2. Oligonucleotides 9Antisense oligonucleotides 10

Backbone modified oligonucleotides 11

3. Radiolabelling Synthesis for Use with Macromolecules 12Radionuclides used in PET 12Specific radioactivity 13Biological aspects 14Experimental considerations 14Labelling of macromolecules using halogens 15Labelling using metal chelates 18Oligonucleotides 19Octreotide 21Hyaluronic acid 22

4. Radiolabelling Using 76Br 23Production of [76Br]bromide 23Synthesis of stannyl-compounds as prosthetic groups I, VI, VIIIA 23Oxidative bromination of stannylated precursors I, VI, VIIIA 26Determination of specific radioactivity I, III 28Conjugation labelling 30Oligonucleotides I, II, VIIIA 31Proteins I, VI, VIIIC 33Peptides VII 35Labelling of Hyaluronic Acid VIIIB 37

5. Labelling Using 68Ga 39Production of 68Ga 39Chelators used with Ga 39DOTA-OctreotideVII 40DOTA-OligonucleotidesV 41

6. Characterisation of Labelled Oligonucleotides 43Analysis using LC-MS II 43Metabolite analysis IV 44Distribution of 76Br-oligonucleotides in vivo and in vitro IV 46Characterisation of hybridisation properties of labelledoligonucleotidesIV

47

7. Conclusions 49

Acknowledgements 50References 52

Abbreviations

β+ PositronBoc tert-ButyloxycarbonylBSA Bovine serum albuminChloramine-T N-Chloro-p-toluenesulphonamide sodium salt

CPB Cetylpyridinium bromide, 1-hexadecylpyridinium bromideDMF N,N-DimethylformamideDMSO Dimethyl sulphoxideDNA Deoxyribonucleic acid

DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

DOTATOC 4,7,10-Tricarboxymethyl-1,4,7,10-tetraazacyclododecan-1-yl-acetyl-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-L-threoninol (DOTA-D-Phe1-Tyr3-Octreotide)

EC Electron captureEDC 1-Ethyl-3(3-dimethylaminopropyl)carbodiimideESI Electrospray ionisationHPLC High-performance liquid chromatographyHSA Human serum albuminLC Liquid chromatographyMRI Magnetic resonance imagingmRNA Messenger-RNAMS Mass spectrometryODN OligodeoxynucleotidePCR Polymerase chain reactionPd(PPh3)4 Tetrakis(triphenylphosphine)palladium(0)PET Positron emission tomographyRNA Ribonucleic acidSODN Phosphorothioate oligodeoxynucleotideSPE Solid phase extractionSPECT Single photon emission tomographySulfo-NHS 1-Hydroxy-2,5-dioxo-3-pyrrolidinesulfonic acidTEAA Triethylammonium acetateTFA Trifluoroacetic acidTyr TyrosineUV Ultraviolet

7

1. Introduction

Imaging technology in vivo and in vitro

The first tracer-experiment was performed by Hevesy in 1923 using a radioactive lead

salt to detect the uptake in plants.1 Since then the technology has developed in for

example the areas of detection devices and synthesis of tracer compounds.

One detection technique is autoradiographic imaging where the samples, for example

slices of tissues or slices of small animals injected with a radiolabelled tracer, are

placed on photographic films or reusable phosphor imager screens to image the

distribution of radioactivity.2 This technique is suitable for the detection of β-particles

with high precision and sensitivity. An alternative way to investigate the distribution

of radioactivity is to take out samples, for example different organs from a rat, and

measure the γ- radiation in a well-counter.

Imaging of living subjects requires non-invasive approaches where the distribution of

a substance can be measured by external detectors. Magnetic Resonance Imaging3

(MRI) is based on the detection of stable nuclides while techniques such as Positron

Emission Tomography4 (PET) and Single Photon Emission Computed Tomography5

(SPECT) detect the decay of radioactive nuclides. A main differences between these

techniques are related to resolution and sensitivity.3

The PET technique is based on the use of positron emitting radionuclides that are

neutron deficient and decays by the emission of a positron, the anti-matter of an

electron. After a few millimetres in tissue, the positron encounters an electron, the

particles are annihilated and two 511 keV photons (γ-radiation) are emitted in

opposite directions. These anti-parallel photons are detected in coincidence by the

PET-camera and the distribution of radioactivity in a living subject can be measured

with high precision and quantified as a function of time and region.

8

Macromolecules as tracers

Proteins and peptides play an important role in many diseases and can therefore be

used as probes or diagnostic tools if radiolabelled. This technique has been exploited

in the radiolabelling of antibodies and analogues of the peptide somatostatin for the

diagnosis and treatment of tumours. Oligonucleotides are currently investigated for

treatment of cancers and viral infections.6

There are a limited number of biogenic radionuclides available (such as 3H, 11C, 13N,14C, 15O, 32P, 33P, 35S) and their usefulness in synthesis of tracer compounds are set by

chemical properties, half-life, decay-properties and availability. Common

radionuclides for imaging processes involving macromolecules, such as antibodies

and proteins, are isotopes of iodine7 or various radiometals (e.g. 99mTc) attached via

chelating groups8 and mostly using SPECT as imaging modality. Usually, analogues

of the molecule of interest are prepared to introduce the label since these elements are

not frequently found in biomolecules. Halogens can be introduced in molecules

containing activated aromatic rings, for example the amino acid tyrosine, by oxidative

halogenation. Another option is to conjugate a labelled molecule, a so-called

prosthetic group, to the macromolecule. As a part of the characterisation, biological

assays or other tests need to be performed to confirm that the properties of the

analogue do not differ substantially from the original molecule.

The aim of the work presented in this thesis is the development of synthetic methods

for radioactive labelling of macromolecules. Oligonucleotides, proteins and peptides

have been labelled with the accelerator produced positron emitting nuclide 76Br

(T1/2=16 h) by the use of prosthetic groups. The study also includes labelling of

oligonucleotides and a peptide using a chelating agent for the generator produced 68Ga

(T1/2=68 min).

The synthesis of the prosthetic groups, radiolabelling and conjugation conditions are

described. With the emphasis on the oligonucleotides, the labelled molecules have

been subjected to chemical characterisation as well as characterisation of the

behaviour in vivo or in vitro.

9

2. Oligonucleotides

Recently the human genetic code has been resolved and a revolution in drug design

based on the genetic code can be expected within the pharmaceutical industry. A

following step would be the identification and understanding of the proteins expressed

in organisms, a more problematic work due to the complexity of the proteins as

compared to nucleic acids.9

The DNA consists of sugar moieties of deoxy-D-ribose linked by a phosphate

backbone with purine and pyrimidine bases attached via the sugar. There are four

different bases in DNA, adenine (A), cytosine (C), guanine (G) and thymine (T).

RNA has the same type of structure where the sugar deoxyribose is replaced by ribose

and the base thymine is replaced by uracil (U) (Figure 1).

O

OPOO

O

N

N

N

N

O

NH2

ON

N

O

OPOO-

O

NH2

ON

NH

O

O

H3C

OPOO-

O

OPOO-

O

O

OPOO-

O

N

N

N

N

NH2O

N

NH

O

OPOO

O

O

O

OH

Thymine

Adenine

Cytosine

Guanine

Uracil

5´-end

3´-end

DNA RNA

Figure 1. The composition of RNA and DNA.

Hydrogen bonds can be formed between adenine and thymine (uracil in RNA) or

guanine and cytosine respectively, resulting in a double helix strand of DNA.10 The

sequence of bases in the DNA creates a gene that is coding for the synthesis of

10

proteins. The double helix partly uncoils and a complementary RNA-string,

pre-messenger-RNA, is built and transformed into messenger-RNA (mRNA). The

mRNA migrates into the cell where the ribosomes can read the encoded information

and assemble proteins from amino acids, in the so-called translation.

Antisense oligonucleotides

An antisense oligonucleotide is a synthetic single-stranded DNA that has a base

composition that is complementary to a part of DNA or RNA (the so called sense

strand).11 An oligonucleotide that is antisense to the mRNA can hybridise selectively

through Watson-Crick base pairing with the mRNA to form a double helix and inhibit

the protein synthesis that is encoded by that mRNA. There are two possible suggested

mechanisms, one is where this double-strand is prevented from transferring

information to the ribosome, thus protein synthesis is inhibited (Figure 2). The other

mode of action of the antisense oligonucleotide is that the mRNA-oligonucleotide

duplex can be recognised by degrading enzymes, RNase H, which destroy the mRNA.

The oligonucleotide is then released and may hybridise with the next mRNA molecule

(Figure 2).

Figure 2. Proposed mechanisms for inhibition of protein-synthesis by antisenseoligonucleotides.

Pro-messenger RNA

mRNA

Antisense oligonucleotide

RibosomeTranslation Protein-

synthesis

No translation

DNA

Enzymes

Breakdown of mRNA, release ofantisense oligonucleotide No translation

Ribosome

11

Antisense drugs are expected to inhibit the synthesis of the disease-causing protein

encoded by the target mRNA, and thereby intervene in an early stage of the disease-

causing process. There is a potential for antisense drugs to be more selective than

traditional drugs due to the multiple points of interaction between the mRNA and the

antisense oligonucleotide. When the sequence of the mRNA is known and

understood, the design of the drug should be straightforward. Statistically a 15-17

base sequence is enough to avoid unintentional complete match of the mRNA.12

Backbone modified oligonucleotides

In reality, there are many factors that complicate the use of antisense drugs.

Phosphodiester oligonucleotides are readily degraded by nuclease enzymes and have a

half-life of less than 30 minutes in the cell.11 One strategy of avoiding degradation

and to achieve more stable oligonucleotides would be to modify the phosphate

backbone (Figure 3). Replacing one of the non-bridging oxygens with sulphur gives

phosphorothioate oligonucleotides which are more stable towards attack by nucleases

and increases the half-life in serum to up to 24 hours. However, phosphorothioate

oligonucleotides show higher unspecific binding than phosphodiester oligonucleotides

to proteins.13 Methylphosphonate oligonucleotides are compounds where a methyl

group has replaced one of the non-bridging oxygens (Figure 3). The success of

phosphorothioate oligonucleotides as potential therapeutic agents is due to the fact

that they can be recognised by RNase H, while this mechanism does not work for

methylphosphonate oligonucleotides (Figure 2).14 Phosphorothioate and

methylphosphonate oligonucleotides are reported to hybridise with lower affinity than

phosphodiester oligonucleotides.15 One explanation is that phosphorothioate and

methylphosphonate oligonucleotides are synthesised as a mixture of disasteromers.11

O

O

HO Base

PX OO

X= O-, Phosphodiester DNAX= S-, Phosphorothioate DNAX= CH3, Methylphosphonate DNA

Figure 3. Backbone modified oligonucleotides.

12

3. Radiolabelling Synthesis for Use with Macromolecules

Radionuclides used in PET

The most commonly used nuclides for PET-studies are 11C, 15O and 18F, where 11C

and 18F are interesting from a synthetic point of view.16 The half-life of 11C allows for

investigations up to 2 hours whilst tracers containing 18F 17 can be studied for a longer

period of time. For some applications a longer half-life may be necessary. In the study

of biological processes involving macromolecules, such as proteins and

oligonucleotides, 76Br is of interest due to its half-life (16 h) allowing for studies up to

2 days with the PET camera. Another candidate for PET-applications is 124I which has

a lower amount of β+-emission than 76Br (Table 1). The chemistry for labelling with

iodine is well established, and similar chemistry can be applied for labelling with

bromine.

Another β+-emitter of interest is 68Ga which is produced from a 68Ge/68Ga-generator.

It is attractive since it does not require a cyclotron for production giving a readily

accessible nuclide that form stable complexes with several chelators.

Table 1. Some Positron Emitting Radionuclides.

Radionuclide Half-life NuclearReaction

Theoretical SpecificRadioactivity (GBq/µmol)

% β+

Decay11C 20.3 min 14N(p,α)11C 3.4×105 9915O 2.07 min 14N(d,n)15O 3.4×106 10018F 110 min 18O(p,n)18F 6.3×104 9761Cu 18 3.4 h 60Ni(d,n)61Cu 3.4×104 6168Ga 68 min 68Ge→68Gaa 1.0×105 8976Br 16.2 h 76Se(p,n)76Br 7.2×103 54110In 19 69 min 110Cd(p,n)110In 1.0×105 69124I 20 4.2 d 124Te(d,2n)124I 1.2×103 25a Generator produced, 68Ge [T1/2=270 d]/ 68Ga [T1/2=68 min]

Means of production

• Accelerators: Targets are bombarded with charged particles to produce

neutron deficient radionuclides. High specific radioactivity (vide infra) can

13

often be achieved since the production of many radionuclides is no carrier

added.21

• Reactors: In a reactor the target is bombarded by neutrons to form

radionuclides with an excess of neutrons.22

• Generators: Generators consist of a parent nuclide (produced by reactors or

accelerators) that decays to form the daughter radioisotope. One way to

separate the nuclides is to adsorb the parent nuclide on to a solid support in

such a fashion that the daughter nuclide can be eluted from the support.23

Specific radioactivity

Specific radioactivity is defined as the amount of radioactivity per unit mass and can

also be expressed as radioactivity per mole. The theoretical specific radioactivity can

be calculated from the half-life of the nuclide, N=A0× T1/2/ln2. Due to the isotopic

dilution from for example equipment, target materials and chemicals used, the

specific radioactivity achieved is usually not equal to the theoretical value. In order to

avoid isotopic dilution, the reaction conditions should be considered to minimise the

used of contaminating chemicals and materials. Examples of contaminating sources

are glass ware when working with radiometals or the atmosphere when working with

[11C]CO2. Also, in order to maximise the specific radioactivity, the synthesis time

should be minimised.

Effective specific radioactivity can be discussed when there are chemical impurities in

the final product-preparation that have biological activity towards the same system as

the desired product. Receptor binding assays have been designed for the

determination of the effective specific radioactivity. This has previously been

described for 16α-[77Br]-bromoestradiol, a steroid, where the effective specific

radioactivity was found to be about five times lower than the specific radioactivity. 24

Unlabelled impurities with similar biological properties as the labelled substance can

also be referred to as pseudo carriers, for example when Cl is incorporated instead of76Br and not removed in the purification process. Another example, discussed later in

this thesis, is the specific radioactivity of labelled proteins. In this case, the specific

radioactivity is determined by the amount of protein used in the labelling reaction,

since it is difficult to separate the unlabelled protein from the labelled by

chromatographic means.

14

Biological aspects

Some biological aspects are important to take into consideration when designing the

tracer molecules.

• To minimise the radiation dose to the subject undergoing a PET-investigation

and maximise the signal measured, the half-life of the radionuclide used

should match the biological half-life of the process studied.

• To obtain sufficient radioactivity for measurements at low concentrations of

the tracer, the specific radioactivity has to be high. For oligonucleotides, the

specific radioactivity required has been estimated to be 100 µCi/µg12, which

for a 30 mer oligonucleotide correspond to approximately 40 MBq/nmol.

• The measurement of radioactive decay does not discriminate between different

chemical species. Therefore the position of the label should be considered

regarding the metabolism of the molecule. Alternatively the position of the

label can be selected so that the cleavage of the label visualises a metabolic

process.25, 26 Analysis of plasma using chromatographic techniques can be of

value for interpretation of the PET-data if the tracer is metabolised.27, 28

• When prosthetic groups are used to introduce the label, the prosthetic group

should be chosen so that the biological activity is altered as little as possible.

The design of the prosthetic group can also be considered to increase the

stability of the molecule against degrading processes29 or to facilitate

excretion30.14, 31

Experimental considerations

The short half-life and the damaging radiation from the radionuclides used, put some

demands on the experimental methods used.

• From a radiation safety point of view, the time the chemist is exposed to the

radiation should be minimised. Considering the radiation dose to the chemist

and the possible application for the labelled compound in clinical

investigations, automatisation of the synthesis and purification procedures is

desirable.

• Large excess of reagents are used compared to the labelled precursor, giving

pseudo-first-order kinetics and fast reactions.32 The label should be introduced

15

as late as possible in the synthetic scheme to maximise the specific

radioactivity in the final product.

• Liquid chromatography (LC) and solid phase extraction (SPE) are routinely

used for purification of the labelled products. LC is also used for identification

and concentration determinations using UV-detection and radiometric

detection to match the retention time of the labelled compound with the

retention time of characterised standards. LC-MS is an important method for

characterisation of the products because of the high sensitivity and selectivity

and the possibility to identify a compound according to the molecular

weight.33

Labelling of macromolecules using halogens

Different radioactive isotopes of iodine are commonly used for the labelling of

proteins and antibodies because of its availability and range of decay properties. So

far these tracers have mostly been applied to SPECT and in vitro detection techniques.

Radiohalogens form chemically stable covalent bonds with the aromatic groups in the

proteins and high specific radioactivity can be achieved.31

OH

CH2

NHNHR'

OR

O

OH

CH2

NHNHR'

OR

O

X*

NaX*oxidant

X* CO

LG

NHNHR'''

O

NH2

R''

O

NHNHR'''

O

NH

R''

O

O*X

a)

b)

Figure 4. X*= Br, I, At, LG = leaving groupa) Direct halogenation of tyrosine-residue. b) Conjugation labelling at lysine residue.

16

A straightforward and well-established method to label proteins with iodine is through

oxidative halogenation on the tyrosine residues (Figure 4). 34-36 Bromine has a higher

oxidation potential than iodine but oxidative bromination of proteins have been

performed using enzymes as oxidants37, 38 or by the use of chloramine-T 39. The

methods usually gives high incorporation yields of the halogen but the oxidative

conditions can affect the activity of some proteins and it might be difficult to separate

the enzymes from the labelled product.40 Proteins labelled by oxidative iodination on

tyrosine can sometimes suffer from dehalogenation in vivo.

In the labelling of oligonucleotides and carbohydrates, for example hyaluronic acid,

there is no established method for direct halogenation so the molecules therefore have

to be derivatised prior to labelling.41 Conjugation labelling by the use of prosthetic

groups can be beneficial when direct labelling is not possible due to the lack of

tyrosine-residues or when the compound is sensitive to oxidative conditions. The use

of prosthetic groups is an established method for radiolabelling of proteins31,

derivatisation of oligonucleotides14 and it has also been used for the labelling of

peptides42, 43.

One drawback with the use of conjugation labelling is that the reaction is performed

in two steps, often leading to lower radiochemical yields compared to the direct

labelling. The advantage with conjugate labelling, however, is the mild reaction

conditions and also the increased stability towards in vivo dehalogenation caused by

enzymes. The labelling method is also applicable to a wide range of substrates.

Prosthetic groups for conjugation labelling

In the design of prosthetic groups, metal-halogen exchange reactions are reliable and

can be used for introducing a radiohalogen in aromatic or vinylic substrates under

oxidative conditions.44-48 Trialkylstannyl compounds are commonly used for this

metal-halogen exchange reaction due to high yields and good regiospecificity in the

halogenations.47 However, other metallic compounds such as organosilanes,

organogermanes, organomercurials and organothallates have also been used for the

introduction of radiohalogens.48

17

Activated esters of N-hydroxysuccinimide are good electrophiles for conjugation to

nucleophiles such as amines, forming stable amide-bonds with for example lysine-

residues or the N-terminal end in proteins and peptides or with amino-modified

oligonucleotides (Figure 5c).49 The first prosthetic group used for conjugation

labelling, iodinated 3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester,

also referred to as the Bolton-Hunter reagent, is now commercially available.50

NH CS

NH RNH CNH

R

R CO

O N

O

OR NCS

R CNH

OCH3

NH2

NH CO

R

a b c

Figure 5. Reaction of an amino-containing molecule with a) methyl-iminoesters, b)isothiocyanates and c) N-hydroxysuccinimidyl esters.

Iminoesters (imidates) are reported to form amidine bonds when reacted with amines

(Figure 5a) and may therefore be useful as prosthetic groups for labelling of

proteins.31, 51, 52, 53-55 Iminoesters are reported to be less reactive towards amines

than succinimidyl-esters but they are also less sensitive to hydrolysis. One advantage

with amidines is that they are protonated at physiological pH and that the original

charge on the protein is thereby retained.

Isothiocyanates are commonly used electrophiles for reaction with amines (Figure

5b). The thiourea bond formed may be more stable than that of the amide bond

formed from reaction of amines with activated esters (Figure 5c).56

Apart from the electrophile, the “R-group” (Figure 5) can also be varied in the

prosthetic group. This feature has been used in the design of N-succinimidyl 5-iodo-3-

pyridine-carboxylate as a prosthetic group57, which has been proven to be

advantageous in that the metabolite iodonicotinic acid is cleared from the tissue faster

than iodobenzoic acid (from the use of N-succinimidyl iodobenzoate as prosthetic

group) due to its reduced lipophilicity.58

18

Labelling using metal chelates

Many different radiometals with a large spectrum of decay-properties are available

from either generator systems or by cyclotron production.59, 60 These metals can be

used in the synthesis of radiopharmaceuticals containing a chelating functionality. A

few positron-emitting radiometals are presented in Table 1.

A chelating agent should be covalently attached to the molecule of interest prior to

reaction with the metal. Alternatively the chelating agent is labelled before

conjugation.61 Some commonly used chelating agents are presented in Figure 6. The

chelator can be attached to the molecule of interest via the carboxylate-group or the

chelator can be functionalised at a carbon for further reaction (SCN-TETA, see

Figure 6).8 Functionalisation of the carbon-skeleton requires a multiple step synthetic

scheme, while the carboxyl groups can be coupled to amines in one step using

carbodiimide chemistry.62

DFO

(CH2)2 CO

NH (CH2)5 NOH

C (CH2)2 C NHO O

(CH2)5 NOH

CO

CH3CO

N(CH2)5H2NOH

N N

CO2H

HO2C

HO2CN

CO2H

CO2H

CPTA

N

N N

N

OH

O

DOTA

NN

N N

CO2H

CO2H

HO2C

HO2C

N NCO2H

CO2H

HO2C

HO2C EDTA

DTPA

N

N N

N

CO2H

CO2HHO2C

HO2C

SCN

SCN-TETA

Figure 6. Some metal-chelators used for radiopharmaceuticals.

19

For many applications it would be advantageous with a labelling protocol that can be

applied for kit-type preparations.41 Due to the high efficacy of many metal chelators,

a small amount of the precursor is mixed with the radiometal giving high yields. It is

often sufficient to use solid phase extraction or gel-filtration to separate the unreacted

metal, if any, from the product. Since only small amounts of the chelator can be used

in order to achieve high specific radioactivity, the reactions are very sensitive to

contamination from other metals often found in trace amounts in buffers, glassware

etc.

For use in vivo and in vitro the metal chelates have to be stable towards translocation

of the metal to serum proteins, e.g. transferrin, or to degradation at physiological pH.

The complexes also have to be stable to competition form the cations found in serum

(Ca2+, Zn2+, Mg2+).63 The stability of the metal complex is to a larger extent

determined by the kinetic inertness (rate of dissociation) rather than of its

thermodynamic stability.64 The stability of the complex can be measured by

incubation in plasma, and samples can be analysed for degradation products. Cyclic

chelators often form more stable metal complexes than open chain ligands, which can

be explained by the high degree of preorganisation.64 Macrocyclic chelators (DOTA,

SCN-TETA) form complexes with high stability with a variety of metal ions.

Sometimes the incorporation of the metal is slow and trace metals can compete in the

reaction.65

Oligonucleotides

Oligonucleotides can be modified in the purine or pyrimidine bases, sugar-moiety or

in the 5´- or 3´-end to give a handle for introduction of a radioactive or stable labelled

group (Figure 7). End-modifications might also increase the stability of the

oligonucleotide from degradation by exonucleases.66

The label can be introduced by a conjugate technique where the oligonucleotide has

been modified with a linker that can be reacted with a radioactive labelled prosthetic

group.41 Other groups can be used to trace the behaviour of the oligonucleotide, for

example fluorescent groups67 or biotin68. Many prosthetic groups used in the

labelling of proteins contain an electrophilic group. The same approach as used for

20

labelling of peptides and proteins can then be used for oligonucleotides containing a

nucleophilic group (Figure 7), regardless of chain length, base composition or

backbone modifications.

O

O

O Base

P-X OO

PO

X-

X = O, S

H2N

Figure 7. 5´-Hexylamino-modified oligonucleotide.

Radiohalogens

Oligonucleotides with different backbone modifications containing a

phosphorothioate monoester in the 3´-position have been conjugated to N-(4-

[18F]fluorobenzyl)-2-bromoacetamide.69, 70 The method has since been modified to

include 76Br and 125I-labelling.71 Labelling of 5´-aminohexyl-oligonucleotides has

been achieved using 18F using N-succinimidyl [18F]fluorobenzoate72 or 4-

([18F]fluoromethyl)phenyl isothiocyanate73 and it also has been radioiodinated using

4-methoxyphenyl isothiocyanate56. N-Succinimidyl 4-tributylstannylbenzoate has

been preconjugated to an amino-modified oligonucleotide followed by labelling of the

conjugate with 125I.74 Oxidative iodination has also been performed on

oligonucleotides modified with a tyramine-group in the 5´-position.75 A stannyl-

derivative of uracil has been utilised in the automated synthesis of oligonucleotides to

produce a stannylated oligonucleotide derivative, which could be radioiodinated.76

Enzymatic methods have been also been used to incorporate 125I-labelled cytosine.77

Radiometals

Oligonucleotides (ODN) have been labelled with 99mTc by conjugating a chelating

agent to the 5´-position.78, 79. The chelator CPTA has been attached to the 5´-end of

oligonucleotides using phosphoramidite chemistry on an automated DNA-synthesiser

and the CPTA-oligonucleotide was labelled with 64Cu or 99mTc.80 Using DTPA as a

chelator, oligonucleotides with amino-modifications in different positions have been

labelled with 111In.81, 82 The DTPA-ODN has also been used for labelling with 67Ga

21

83, 99mTc and 153Sm.84 Other approaches for labelling of oligonucleotides with 111In is

the linkage of isothiocyanobenzyl-EDTA to oligonucleotides where deoxyuracil

containing an amino-linker was used as a substitute for deoxythymine.85 This allowed

for oligonucleotides with more than one linker per molecule, which could be used to

achieve higher specific radioactivity.

Octreotide

Due to the interest in the role of

somatostatin receptors for

tumour diagnosis and treatment,

methods have been developed to

radiolabel analogues of

octreotide, a synthetic

octapeptide that has shown high

selectivity for the somatostatin

receptor. 111In-DTPA-(D)Phe1-

octreotide (Octreoscan ) and 123I-Tyr3-octreotide are routinely used for clinical

applications.86

Radiohalogens

Both N-succinimidyl 3-iodobenzoate 42 and N-succinimidyl 4-[18F]fluorobenzoate 43

have been used to label peptides. The [18F]fluoro-, [131I]iodo- and [211At]astato-

analogues of octreotide have previously been prepared by conjugation to the

corresponding N-succinimidyl 4-halobenzoate 87, N-succinimidyl 5-halopyridine-3-

carboxylate 87, 4-nitrophenyl 2-[18F]fluoropropionate 88 or 4-[18F]fluorobenzoic

acid 89. Direct iodination on Tyr-3-octreotide has also been performed.90

Radiometals

Different metal chelating ligands have been coupled to different derivatives of

octreotide and subsequently radiolabelled using 64Cu,91 67Ga,92 68Ga,93 111In,94

99mTc,95 86Y,96 and 90Y 97, 98.

NN

NHO

HOO

O

O

S

H2NN

NN

O

O

ON

OS

NH2

HO

NH

Octreotide

22

Hyaluronic acid

Hyaluronic acid is a linear polysaccharide composed of disaccharide units of N-

acetyl-D-glucosamine and D-glucuronic acid. Hyaluronic acid has been

radioiodinated by reacting tyramine with the aldehyde terminal followed by oxidative

iodination of the tyramine-group.99 The specific radioactivity obtained is limited by

the 1:1 molar ratio of tyramine to hyaluronic acid, yielding a maximum of one

radioiodine per polysaccharide. The high molecular weight hyaluronic acid has been

labelled with iodine after cyanogen bromide activation of the polysaccharide followed

by conjugation to tyrosine and radioiodination.100 Due to the possibility of multiple

sites of labelling in one molecule, higher specific radioactivity can be obtained.

Hydrazinolytic N- deacetylation of the amide-functionality gave access to an amine

that could be derivatised with tyrosine-cellobiose and subsequently iodinated.101

Hyaluronic acid has also been labelled with the short-lived isotope 11C (T1/2= 20 min)

using [11C]CNBr.102

Preconjugation of a prosthetic group was the only option available for the high-

molecular weight hyaluronic acid. Coupling of diamines to the carboxylic acid moiety

of the hyaluronic acid to by carbodiimide activation to introduce a nucleophile in the

molecule has proven to be unsuccessful.103 Hydrazides could be coupled to fragments

of hyaluronic acid due to their better nucleophilicity at a pH around 4-5.104 For

reasonable yields in conjugation reactions with a labelled electrophile about 70 nmol

amine/100 µl is required. This corresponds to 230 amino-modifications/ hyaluronic

acid molecules (Mw=1,5×106) at a concentration of 5 mg/ml.

23

4. Radiolabelling using 76Br

Due to its partial decay by positrons and its half-life of 16 h, 76Br is an attractive

nuclide for labelling of oligonucleotides and proteins for possible applications in PET.

Electrophilic bromine has been used for bromination of activated aromatic rings such

as tyrosine. The use of halogen-demetalation reactions are advantageous due to high

regioselectivity and its efficacy even on deactivated systems. Bromine-labelling by

nucleophilic substitution can be used, but there can, however, be a problem with

decreasing effective specific activity if the leaving group is a halogen. For aromatic

substrates, other reactions can used, for example the Sandmeyer reaction.105

Production of [76Br]bromide

The radionuclide 76Br is produced by proton irradiation of a [76Se]selenium enriched

Cu2Se-pellet using a low-energy cyclotron.106 The 76Br is recovered from the target-

pellet using a thermo-chromatographic method and it is obtained in water or ethanol.

In order to obtain reproducible yields in the labelling reaction, the water solution of

[76Br]bromide is purified using a C-18 SPE-disc to remove lipophilic components.76Br has previously been produced indirectly by the EC-decay of cyclotron produced76Kr (T1/2= 14.6 h)107, 108 or by a more direct route using the 75As(3He, 2n)76Br

nuclear reaction109.

Synthesis of stannyl-compounds as prosthetic groups I, VI, VIIIA

There are two major routes for the preparation of aromatic stannylated compounds,

either by nucleophilic attack of a lithiated compound on trialkylstannyl chloride or by

using a palladium(0)-catalysed coupling reaction between an aromatic or vinylic

halide and hexaalkyldistannane (Figure 8).

R' = CH3, (CH2)3CH3

ClSnR'3

BuLi

Sn2R'6, Pd(0)

R SnR'3

R LiX R

Figure 8. Two routes to aromatic stannyl-compounds.

24

Due to the milder reaction conditions, the route using palladium(0) catalysis was

preferred for introducing the stannyl functionality when synthesising the prosthetic

groups used in paper I, VI and VIIIA. However in the synthesis of N-succinimidyl-5-

trimethylstannyl-3-pyridinecarboxylate 13, the published method using n-butyl

lithium was applied.57

N-Succinimidyl 4-trialkylstannylbenzoate 4 I

N-Succinimidyl trialkylstannylbenzoates have previously been labelled with 211At 110,77Br 111 and different isotopes of iodine 112, 113. Several procedures have been

published for the preparation of N-succinimidyl trialkylstannylbenzoates.29-30, 112-114

These methods were modified in the synthesis of succinimidyl 4-trimethylstannyl-

benzoate 4a. Recently, a two-step route to 4a has been described, where iodobenzoic

acid was stannylated followed by esterfication.115

R3Sn CO

OCH2CH3I C

O

OCH2CH3

Sn2R6Pd(PPh3)4toluene

NaOH (aq)Ethanol

R3Sn CO

OHDisuccinimidylcarbonatePyridineAcetonitrile

R3Sn CO

O N

O

O

1 2

3 4a) R= Me4b) R= Bu

Scheme 1.

As described in paper I, ethyl 4-iodobenzoate was stannylated using hexamethylditin

and tetrakis(triphenyl-phosphine)palladium(0) [Pd(PPh3)4] as a catalyst to give

compound 2 in 93% yield.113, 114 The stannylbenzoate 2 was hydrolysed followed by

esterfication with N,N’-disuccinimidylcarbonate to give the desired 4a (16%)

(Scheme 1).30 The tributylstannyl analogue 4b was synthesised using the same

method.

Methyl-4-trimethylstannylbenzimidate 7VI

For halogenation of proteins, methyl-4-trimethylstannylbenzimidate has been

suggested as prosthetic group.116 Methyl-4-bromobenzimidate 6 was prepared from

25

4-bromobenzonitrile using the Pinner reaction where methanol was used both as a

solvent and a reactant (Scheme 2) and isolated in 74 % yield.117 When diethyl ether

was used as solvent in the Pinner reaction a lower yield was obtained.118 Base

catalysed conversion of bromobenzonitrile to the iminoester119 also resulted in low

yields. The methyl-4-bromobenzimidate was stannylated using the same conditions as

for compound 2 giving methyl-4-trimethylstannylbenzimidate 7 in 40% yield.

Sn2(CH3)6Pd(PPh3)4tolueneBr C N (CH3)3Sn C

NH

OCH3

5 7

Br CNH

OCH3

6

1) HCl(g)MeOH2) K2CO3

Scheme 2.

4-Tributylstannylphenylisothiocyanate 10VIIIA

Synthesis of 4-tributylstannylaniline 9 was achieved using triethylamine as solvent

and Pd(0) as catalyst in a low yield (16%) (Scheme 3).120 The 4-tributylstannylanilin

was transformed to the corresponding isothiocyanate 10 using thiocarbonyl-

diimidazole, a thiophosgene equivalent, in 83% yield (Scheme 3).121 Attempts were

made to synthesise the 4-trimethylstannylaniline using the same method as for the 4-

tributylstannylaniline but without success.

CHCl3

N NNN

S

Sn2Bu6Pd(PPh3)4Et3N

Bu3Sn NCSBu3Sn NH2Br NH2

9 108

Scheme 3.

N-Succinimidyl-5-trimethylstannyl-3-pyridinecarboxylate 13VI

Preparation of N-succinimidyl-5-trimethylstannyl-3-pyridinecarboxylate 13 was

carried out using n-butyl lithium and trimethylstannyl chloride (Scheme 4).57

26

CH3 SO2N-Na+

Cl

Chloramine-T

N-HydroxysuccinimideDCCCH2Cl2

1) n-BuLi (2 eq)2) (CH3)3SnCl (2.2 eq)

N

(CH3)3Sn CO

OSn(CH3)3

N

CO

OHBr

N

(CH3)3Sn CO

O N

O

O

11 12

13

Scheme 4.

Oxidative bromination of stannylated precursors I, VI, VIIIA

[76Br]Bromide can be oxidised to electrophilic bromine using

for example chloramine-T or peracetic acid as the

oxidant.105 The stannyl-compounds 4, 7, 10 and 13 were

brominated with 76Br, using chloramine-T as the oxidant.

N-Succinimidyl 4-[76Br]bromobenzoate 14 I

Bromination of 4a using chloramine-T as oxidant and ethanol as solvent gave isolated

yields of 76BrNHS 14 around 65% (Scheme 5). Other oxidants, such as N-

chlorosuccinimide, tert-butyl hydroperoxide and IODO-BEADS (polymer-bound

chloramine-T) were tested using 4a as the substrate for bromination, but these

oxidants gave lower yields than chloramine-T.

CO

O N

O

O

76BrMe3Sn CO

O N

O

O

4a 1476BrNHS

[76Br]Br-

Chloramine-T HOAC, EtOH70°C, 10 min

Scheme 5.

The purity of the stannyl-compound is of importance for the labelling yield since a

large excess of stannyl-compound is used compared to the halogen. Even small

amounts of impurities can give large amounts of labelled side-products. For example,

iodination of tributylstannyl 4-benzoic acid is reported to be much faster than the

iodination of the succinimidylester.116

27

The N-succinimidyl 4-trimethylstannylbenzoate 4a was chosen over N-succinimidyl

4-tributylstannylbenzoate 4b as substrate for radiobromination since recrystallisation

was possible, giving higher purity and higher labelling yields. It has also been

reported that the trimethylstannyl compound is more reactive than the tributylstannyl

compound in halogenation reactions.112

The [76Br]bromide-solution was purified in order to remove lipophilic compounds that

interfered in the labelling reaction. The labelling yields in the synthesis of 14 could be

increased from 10-35% to 65% by purification of the [76Br]bromide-solution through

a C-18 SPE disc.

N

76Br CO

O N

O

O1776BrNNHS

76Br NCS

1676BrNCS

76Br C

OCH3

NH

1576BrIm

Methyl-4-[76Br]bromobenzimidate 15 VI

Methyl-4-trimethylstannylbenzimidate 7 was brominated using chloramine-T in

methanol/ acetic acid at 40°C for 20 min to give 76BrIm 15 in 40 % yield after LC-

purification. Due to the low reactivity of 76BrIm 15 in conjugation to amines, the

labelling conditions were not further optimised. The identity of 76BrIm was confirmed

using LC-MS after a synthesis with carrier added bromide.

4-[76Br]Bromophenylisothiocyanate 16 VIIIA

The compound 76BrNCS 16 was synthesised using the same conditions as in the

synthesis of 76BrNHS 14 (Scheme 5) and could be isolated in 32% yield using

reversed phase chromatography. The compound was poorly retained on a silica

column using hexane as the eluent and the peaks from the stannyl-compound and 16

overlapped to some extent. The amount of radioactivity in the vessel decreased when

the aqueous solvent was removed from purified 16 by evaporation. It has been

reported that radioiodinated 4-methoxyphenylisothiocyanate is volatile when heating

above 45°C.56

28

N-Succinimidyl-5-[76Br]bromo-3-pyridinecarboxylate 17 VI

Radiobromination of N-succinimidyl-5-trimethylstannyl-3-pyridinecarboxylate 13

gave a 25% yield using chloramine-T as the oxidant. The reaction proceeded at room

temperature but with a lower yield compared to compound 4a. The [76Br]bromide was

consumed and a lipophilic labelled by-product was formed. This by-product was not

formed in the synthesis of 14 using the same batch of [76Br]bromide, indicating

impurities in compound 13. Compound 17 was isolated using straight phase HPLC

with either a mobile phase containing ethyl acetate: hexane: acetic acid 30:70:2 or 2%

methanol in dichloromethane. A drawback with the buffer containing acetic acid was

the difficulty in removing all acid when evaporating the solvent, giving a lower yield

in the following conjugation reaction. An increased amount of bromonicotinic acid

due to hydrolysis of 17, was observed.

Determination of specific radioactivity I, III

The bromide produced in the Cu276Se-target is expected to be of high specific

radioactivity due to the assumed carrier free production. The carrier bromide in the

labelling reaction most likely has its origin from the solvents and chemicals used.

Ethanol and chloramine-T are for example reported to contain varying amounts of

bromide.45, 122

The specific radioactivity of [76Br]bromide was determined using ion-exchange

chromatography with conductivity detection and was compared to the specific

radioactivity of the labelled 76BrNHS 14 determined by UV-detection.I It was found

that the specific radioactivity of the 76Br-ethanol solution was higher than the specific

radioactivity for 76BrNHS 14 as expected (180 vs. 86 GBq/µmol in this case). In the

determination of the specific radioactivity of 76BrNHS, the amount of pseudo-carrier

N-succinimidyl 4-chlorobenzoate in the purified product was included because of its

similar reactivity towards amines as 14. Chlorination can be a side-reaction when

using chlorine-containing oxidants.46, 123

The effective specific radioactivity of the 76Br-labeled oligonucleotides was assumed

to be of the same magnitude as the specific radioactivity of 76BrNHS. This is based on

the assumption that the chemical purity of isolated 76Br-labelled oligonucleotides is of

the same magnitude, with respect to unlabelled analogues, as the chemical purity of

29

the BrNHS since unconjugated oligonucleotides are efficiently separated from the

labelled oligonucleotides (see further chapter 6.)

LC-MS in determination of specific radioactivity III

For substances synthesised with high specific radioactivity and for compounds that

show low UV-absorption, the sensitivity obtained by UV-detection is not always

enough for determination of the specific radioactivity. Electrospray ionisation mass

spectrometry (ESI-MS) can be used as a complementary technique to UV-detection to

improve the sensitivity and selectivity in determination of specific radioactivity, for

substances that are readily ionised.33 Overlapping peaks in the UV-trace can be

resolved using the mass spectrometer as a detector since it is possible to detect one

mass at a time. When there is a limited amount of sample available, sensitivity can be

gained if columns with small inner diameters are used in combination with large

injection volumes and on-column focusing of the analyte since the mass spectrometer

behaves as a concentration sensitive detector.

To investigate whether the sensitivity obtained by LC-ESI-MS was sufficient to

measure the the mass for a 76Br-compound, 76BrNHS was conjugated to

3-aminomethyl-benzylamine to get a compound 18 that was readily ionised by ESI.

N-(3-Aminomethylbenzyl)-4-[76Br]bromobenzamide 18 was synthesised in 90%

radiochemical yield (Scheme 7). Analysis of 2 MBq of the crude product of 18 made

it possible to detect the mass for the 76Br and the 79Br-compound respectively. On-

column focusing was obtained by injecting the sample in a non-eluting solvent

containing reduced amounts of acid and organic modifier.

CH2Cl2, RT 40 min18

14

NC

O

O O

O

76BrCH2NH2

H2NCH2

76Br C NHCH2

CH2NH2O

Scheme 7.

The specific radioactivity was determined by calculating the peak area ratios of the

isotopes of compound 18 at m/z 316 and m/z 319, corresponding to the protonated

30

molecules [M+H]+of the 76Br and 79Br analogues, respectively (Figure 9). The

specific radioactivity in this case was determined to be 36 GBq/µmol. As a control

experiment, the sample was analysed after 22 h and 66 h and the ratio between the

m/z 316 and m/z 319 had changed in accordance with the decay of 76Br.

Figure 9. 76Br and 79Br analoguesof 18 in their respective masschromatograms with ESI-MSdetection of the protonatedmolecules [M+H]+ at m/z 316 andm/z 319 respectively. The analytewas detected at 18.5 minutes. They-axis in the chromatogram form/z 316 is magnified 24 times inthe figure.

The amount of 76Br-compound detected was 2 MBq, which was equal to 0.3 pmol.

The compound detected at 17.4 min in the chromatogram m/z 316 is an unidentified

substance present at low concentrations. Due to the base-line separation from the

analyte peak at 18.5 min together with the control experiment made to confirm the

decay of the bromine labelled product, it was concluded that the unidentified peak did

not affect the experiment.

Conjugation labelling

An excess of the nucleophile to be labelled has to be used in order to obtain

acceptable radiochemical yields in the conjugation reaction due to competing

hydrolysis of the labelling reagent in aqueous solutions. The labelling of peptides and

oligonucleotides can be performed in organic solvents or mixtures of organic solvents

and buffers depending on solubility. Labelled peptides and oligonucleotides can often

be separated from their unlabelled counterparts using HPLC in order to achieve a

higher effective specific radioactivity. Working with proteins, the specific

radioactivity is determined by the amount of added protein since it is difficult to

separate the labelled protein from the unlabelled.

5.00 10.00 15.00 20.00 25.00rt0

100

%

0

100

%

18.5

x24

17.4

18.5

m/z 319

m/z 316

31

Oligonucleotides I, II

Labelling of oligonucleotides using N-succinimidyl 4-[76Br]bromobenzoateI, II

The labelled oligonucleotide conjugates were synthesized according to Scheme 8. The

solvent was removed from the purified compound 14 and the oligonucleotide was

added to the residue in a buffer-DMF solution. The mixture was heated at 50-70°C for

30 min, when all the 76BrNHS was consumed.

76Br CO

O N

O

OODN(CH2)6NH2Buffer pH 9, DMF50°C or 70°C, 30 min

76Br C NH(CH2)6ODNO14

Scheme 8.

The effect of pH and buffer on the conjugation reaction was investigated using 50

mM borate buffer pH 7.5-9.0 and 0.5 M carbonate buffer pH 9.0-10.5 and oligomer

SODN20 (Table 2) as a model compound. Based on the results (Figure 10), carbonate

buffer pH 9.0 was chosen for further experiments.

05

10152025

7 8 9 10 11

pH

Rad

ioch

emic

al y

ield

, R

CY,

(%)

RCY(%) borateRCY(%) carbonate

Figure 10. pH-Dependence of the conjugation yield using SODN20 and 50 mMborate or 0.5 M carbonate buffer. Yields are determined by size exclusionchromatography.

The effect of the amount of DMF was also investigated where 20-60% DMF gave

similar results, but increasing the amount of DMF to 80% resulted in lower yields,

possibly due to poor solubility of the oligonucleotide.

The difference in lipophilicity between the labelled oligonucleotide conjugate and the

amino-modified oligonucleotide made it possible to separate the two compounds

using reverse phase liquid chromatography, giving a better effective specific

32

radioactivity. Characterisation of the labelled oligonucleotides is further discussed in

chapter 6. The use of a large excess of oligonucleotide was necessary in order to get

an acceptable yield in the conjugation reaction. Higher concentration of

oligonucleotide gave higher conjugation yields using the 18F-analogue of 14.72

Table 2. Sequences of oligodeoxynucleotides used.Length Sequence, X = hexylamino-linker Abbreviation

Phosphorothioate oligonucleotides

6-mer 5´-XCACCTT-3´ SODN6

12-mer 5´-XCACCTTAGTGTC-3´ SODN12

20-mer 5´-XCACCTTAGTGTCCCCTTTTG-3´ SODN20

30-mer 5´- XCACCTTAGTGTCCCCTTTTGTCATAGGGCT-3´ SODN30

Phosphodiester oligonucleotides

20-mer 5´-XCACCTTAGTGTCCCCTTTTG-3´ ODN20

30-mer 5´- XCACCTTAGTGTCCCCTTTTGTCATAGGGCT-3´ ODN30

30-mer 5´-AGCCCTATGACAAAAGGGGACACTAAGGTG-3´ ODNsense

The conjugation yields were found to vary with the length of the oligomers (Table 3).

In the case of SODN30 conjugation yields increased somewhat (from 6-8% to 12-

15% radiochemical yield) when the oligomer was denatured at 90°C for 10 min prior

to conjugation or if the reaction mixture was heated at 70°C in the conjugation

reaction (Table 3).II This increase in conjugation yield might be due to the possibility

for the 30 mer to form a cyclic dimer at lower temperatures and thereby limiting the

access to the amino-linker.

In Table 3, there are large discrepancies between the isolated yields and the analytical

yields in for example entry 1 and 2. The concentration of DMF was higher in the

samples used for preparative LC compared to the analytical samples. When the

concentration of organic modifier in the injecting solvent is higher than in the mobile

phase, retention can be poor for parts of the injecting plug. The use of larger injection

volumes eliminated this problem as more buffer could be added to the sample.

33

Table 3. Radiochemical yields of labelled ODNs and SODNs using 14.a

Entry Sequence Radiochemical

yield (%)b

Radiochemical

purity (%)

n

1 76Br-SODN6 37-40 (18-19) >99 2

2 76Br-SODN12 33-40 (5-18) >99 3

3 76Br-SODN20 23-27 (15-19) >99 4

5 76Br-SODN30 (10-12) >99 10

6 76Br-ODN20 18(12) >99 1

7 76Br-ODN30 (6-12) >99 2aReaction conditions: 100 µl 30:35:35 DMF:0.5 M carbonate buffer pH 9.0:H2O,50°C (70° for ODN30 and SODN 30), 30 min. bParentheses refer to isolated radiochemical yieldswhereas other yields are based on analyses of the crude reaction mixture by analytical LC.

Labelling of oligonucleotides using 4-[76Br]bromophenylisothiocyanate VIIIA

An 18 mer oligonucleotide (5´-X ACG GAG GCA TCG CTT AGC (X= hexylamino-

linker)) (10 OD, 70 nmol) was dissolved in DMF and borate buffer pH 8 and added to

the dried compound 16. The mixture was heated at 40°C for 30 min and isolated in 14

% radiochemical yield using gel filtration liquid chromatography. Most of the

remaining radioactivity was stuck to walls of the reaction vial, probably due to poor

water-solubility of compound 16.

Proteins

Labelling of proteins has to be performed in aqueous solution in order not to denature

the protein and at a pH where the lysine-residues are partly deprotonated

Since a protein can contain many lysine-residues, there can be many differently

labelled species in the product. For imaging purposes it is important that the labelled

molecules behave in the same manner as the unlabelled. Therefore the function of the

labelled protein-conjugate has to be investigated as a part of the characterisation.

Conjugation to a lysine-residue in the active site of the molecule might affect the

activity of the protein or peptide.

34

Table 4. Conjugation-labelling of proteinsa.

Protein Concentration

(mg/ml)

Prosthetic group Conjugation yield

(%)

Chromogranin A 1 76BrNHS 14 23-28

Hyaluronidase 5 76BrNHS 14 36

Human Serum Albumin 1 76BrNHS 14 39-61

Bovine Serum Albumin 10 76BrIm 15 10aReactions performed in borate buffer, pH 8-8.5.

Chromogranin AI

Chromogranin A is a protein produced in high amounts in some patients with

neuroendocrine tumours.124 The protein was labelled with 76Br by the use of the

prosthetic group 14. The conjugation reaction was performed at pH 8.5 and 35°C for

30-40 min and the product was isolated in 23-28 % radiochemical yield using gel

filtration (Table 4).

HyaluronidaseVIIIC

Hyaluronidase is an enzyme that is responsible for the cleavage of the polysaccharide

hyaluronic acid. To understand the role of hyaluronic acid and hyaluronidase in

rejection of transplanted organs 125, hyaluronidase was labelled by conjugation to76BrNHS and isolated in 36% radiochemical yield. The enzyme concentration used

was 5 mg/ml since higher specific radioactivity was not required for these

experiments.

To test whether the activity of the enzyme remained after labelling synthesis a series

of labelling experiments were conducted where different amounts of carrier BrNHS

was added. The amount of carrier ranged from 0-4 equivalents of BrNHS compared to

the hyaluronidase to give up to one modification per protein molecule (25%

radiochemical yield). After the decay of the radioactivity, the activity of the

hyaluronidase was tested.126 It was found that about 70% of the initial enzymatic

activity remained for the protein-batch with one modification/molecule. This was

considered sufficient for further studies using the brominated hyaluronidase.

35

AlbuminI, VI

76BrIm 15 was used to label bovine serum albumin (BSA). Using a high concentration

of BSA (10 mg/ml in borate buffer pH 8.7) the yield, as determined by size exclusion

chromatography, was 10%. This showed that the reactivity of 76BrIm 15 is lower

towards amines than that of 76BrNHS 14 which gave conjugation yields of 39-61%

using a tenfold lower protein concentration (Table 4).

Peptides

The resemblance of proteins and peptides makes it possible to use the same

methodology for labelling. Due to the smaller size of the peptide the effect of the

conjugate group might be larger than that for proteins. Protective groups can be

introduced in the peptide to direct the positioning of the label, avoiding conjugation to

amino acids in the active part of the peptide. Conjugation labelling of peptides can be

performed in organic solvents or mixtures of organic solvents and buffer depending

on solubility to decrease the competing hydrolysis of the prosthetic group. As a

consequence of the change in properties of the peptides by conjugate labelling, such

as lipophilicity, it is often possible to separate the labelled peptide-conjugate and the

peptide by liquid chromatography, thus giving a higher effective specific radioactivity

(Figure 11).

Figure 11. LC-chromatogram showing the analysis of the crude product of 76Br-labelled octreotide 19 using a C-18 column and a gradient of 10 mM TFA in waterand acetonitrile.

OctreotideVI

Protection of the amine-group in the ε-lysine-residue of octreotide to avoid

conjugation to this position is necessary since it is in the active site of the molecule.

Reaction conditions can be optimised to ensure selective conjugation to the N-

terminal amine in presence of lysine-residues due to the difference in pKa.49, 127

0 5 10 15 20 25 30 35

76Br-octreotide 19octreotide

UV-trace

radioactivity

retention time (min)

36

Since ε-Boc-protected octreotide was available and the deprotection could be

performed quantitatively without additional purification steps, the ε-Boc-octreotide

was used.

Labelling of octreotide using N-succinimidyl 4-[76Br]bromobenzoate 14

Conjugation of Boc-Octreotide and 76BrNHS 14 was performed in CH2Cl2, to

facilitate removal of the Boc-group by the use of TFA. In combination with

microwave heating (10 W, 30 min) a radiochemical yield of 70-90 % was achieved as

determined by HPLC-analysis (from 76BrNHS). After deprotection in TFA/CH2Cl2,

[76Br]bromobenzoyl-octreotide 19 was isolated in 55 % radiochemical yield by HPLC

calculated from 14 (Scheme 10).

Conjugation of 76BrNHS to ε-Boc-Octreotide using the same conditions as for

oligonucleotides (carbonate or borate buffer pH 9, DMF, 50°C 30 min) gave low

conjugation yields even at relatively high concentrations of Octreotide. A

concentration of 10 mg/ml (8 µmol/ml) was used, which is a tenfold increase of the

nucleophile-concentration as compared to the oligonucleotide-concentration in

corresponding reactions. Conjugation in pure organic solvents such as DMF and

DMSO also gave yields below 10 %. Even though organic bases were added to ensure

deprotonation of the free amine, the conjugation yields did not exceed 25 %.

Conventional heating as well as microwave heating was applied using DMF as

solvent.

Labelling of octreotide using N-succinimidyl-5-[76Br]bromo-3-pyridinecarboxylate 17

Since the same leaving group is present in compounds 17 and 14 (Scheme 9), similar

conditions for conjugation to octreotide were used and the octreotide-conjugate 20

was isolated in a 50 % radiochemical yield. The pyridine-moiety in compound 17

makes the conjugation-product 20 less lipophilic than 19, which is expected to

improve the binding-properties.

37

N

76Br CO

NH Octreotide

CO

76Br NH Octreotide

1) Boc-octreotide in CH2Cl2MW 10W, 30 min

2) TFA, RT, 30 min

N

76Br CO

O N

O

O

CO

O N

O

O

76Br

17

14 19

20

Scheme 9.

In preliminary experiments, the binding properties of octreotide conjugates 19 and 20

to meningioma tissue were investigated using autoradiography. High specific binding

was observed for 20, while 19 mostly showed unspecific binding. This might be

explained by the different lipophilicity of the peptide-conjugates.

Octreotide labelling using methyl-4-[76Br]bromobenzimidate 15

The compound 15 was investigated for conjugation to octreotide as an alternative to

17. The aim was to obtain a less lipophilic octreotide-conjugate than 19. The

conditions used for conjugating 76BrNHS 14 to octreotide (dichloromethane as solvent

and microwave heating) were investigated but no product was formed and most of the76BrIm 15 was left unreacted. Other solvents such as DMF, DMSO and combinations

of organic solvents and buffers were investigated without positive results. Using an

excess of stable BrIm 6 in methanol/dichloromethane and heating at 70° over night

gave conjugation to octreotide as identified by LC-ESI-MS. The use of these solvents

in the labelling reaction,however, proved unsuccessful.

Hyaluronic acid VIIIB

Labelled high-molecular weight hyaluronic acid (mean Mw=1.5×106g/mol) was

needed for distribution studies over 2-3 days using PET. For this application, the

specific radioactivity was of minor importance. Labelling of a hyaluronic acid-

tyramine conjugate was explored due to the simplicity of the method.99 The method

38

has the drawback of low specific radioactivity but the advantage that the hyaluronic

acid is subjected to minimum derivatisation.

n

OHO

HOOCO

OH

OHOCH2

HONH

O

O

OO

HOOC

OH

OHO

HOCH2

HOOH

NH

O

H

O

n

OHO

HOOCO

OH

OHOCH2

HONH

O

O

OO

HOOC

OH

OHO

HOCH2

HOOH

NH

O

OHNHCH2CH2

1)

2) NaCNBH3

OHH2NCH2CH2

Scheme 10. Derivatisation of hyaluronic acid using tyramine.

The aldehyde-end of hyaluronic acid was subjected to reductive amination using

tyramine and sodium cyanoborohydride (Scheme 10). The reaction was monitored by

size exclusion chromatography and UV-detection.

(CH2)2NHRHO

76Br

[76Br]Br-

Chloramine-T(CH2)2NHRHO

Scheme 11. R=hyaluronic acid.

The end-modified hyaluronic acid was radiobrominated using chloramine-T in a

radiochemical yield of 10 % (Scheme 11). Hyaluronic acid was precipitated by

addition of ethanol:methanol:i-propanol (90:5:5) saturated with sodium chloride and

isolated by centrifugation. The pellet was redissolved in water and analysed by size-

exclusion chromatography. The concentration of hyaluronic acid used in these

experiments was 5 mg/ml.

39

5. Labelling using 68Ga

There are currently two radioisotopes of gallium that are commonly used in nuclear

medicine, 67Ga (T1/2=78 h, decay by EC) and 68Ga (T1/2=68 min, decay by β+ 89%).

Another isotope, 66Ga (T1/2=9.4 h) decays to 57 % by β+ and has potential for use in

PET.

The oxidation state of Ga is +III, and the complexing ligands used are dominated by

those where oxygen, nitrogen or sulphur act as donor atoms. Insoluble Ga(OH)3 is the

predominant species in the pH-interval 3.0-9.5. The labelling reactions involving Ga3+

are therefore generally performed in a buffer containing weakly coordinating ligands

such as acetate and citrate to keep the Ga3+ in solution. Ga3+ can then be transferred to

complexes of higher stability.59 For many applications, the Ga-citrate-complex is too

stable for rapid transchelation. The Ga-complexes used as radiopharmaceuticals

should be stable towards hydrolysis and should also be more stable than the Ga(III)-

transferrin complex to avoid transchelation in vivo.59, 128

Production of 68Ga68Ga is available from 68Ge (T1/2=270.8 d) in a generator-system.128 The 68Ge is

attached to an ion-exchange column and the 68Ga can be eluted in 0.1 M hydrochloric

acid.129 67Ga is commercially available and is routinely used in nuclear medicine for

imaging. A method for producing 66Ga and 68Ga by bombardment of Zn-targets has

been proposed.130

Chelators used with Ga

The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, has

been shown to form stable complexes with Ga as shown in the labelling of DOTA0-D-

Phe1-Tyr3-octreotide (DOTATOC).92 The chelating agent, in this case DOTA, can be

attached to the molecule of interest by modification of the carbon skeleton or by using

the carboxyl groups. 131 Other chelators used are DTPA132 and DFO

(desferrioxamine) 93 (Figure 6). The chelator has to be tribasic in order to satisfy the

40

nuclear charge of gallium and it needs to be hexadentate in order to form

coordinatively saturated complexes.133

DOTA-OctreotideVII

DOTA-modified octreotide, DOTATOC, was labelled with 68Ga produced from a68Ge/68Ga-generator. Sodium acetate was added to achieve a 0.4 M acetate solution of68Ga. DOTATOC was added to the 68Ga-acetate-solution, the mixture was heated for

25 min and the product was isolated in 65 % decay corrected radiochemical yield and

>98% radiochemical purity using solid phase extraction (Scheme 12).

Problems due to colloid formation were encountered when changing the solvent of the68Ga eluate from 0.1 M HCl to 0.4 M acetate. The colloid formation could be

decreased if the generator was conditioned by 0.1 M HCl 4-5 hours before eluting the

batch used for labelling. The 68Ga-solution was passed through a C-18 SPEC before

adding sodium acetate.

NN

N NHOOC

HOOC

NN

NHO

HOO

O

O

S

HNN

NN

O

O

ON

OS

NH2

HO

NH

O

HOOC

OH

68Ga in 0.1 M HClNaOAc, pH 5.525 min, 100°C

68GaNN

N NHOOC

HOOC

NN

NHO

HOO

O

O

S

HNN

NN

O

O

ON

OS

NH2

HO

NH

O

HOOC

OH

Scheme 12.

41

The identity of the Ga-DOTATOC was confirmed by LC-MS after a synthesis with

carrier-added GaNO3. Peaks corresponding to Fe-DOTATOC and Zn-DOTATOC

could be detected from a no carrier added synthesis with 68Ga, due to contamination

from buffers and reaction vessels.

The 68Ga-DOTATOC showed high specific binding to somatostain-receptor positive

tissues such as meningiomas and brain cortex. This was demonstrated by blocking

experiments in frozen section autoradiography (Figure 13). The uptake in tissue-

samples from meningiomas and rat and pig brain could be blocked by 1 µM

octreotide in the incubation solution.

a)

b)

Figure 13. Frozen section autoradiography of a) uptake of 68Ga-DOTATOC in

selected tissues, b) uptake of 68Ga-DOTATOC with added octreotide (1 µM).

DOTA-OligonucleotidesV

The chelating agent DOTA was coupled to the phosphorothioate oligonucleotide

(SODN20 and SODN30, Table 2), using the method described by Lewis for coupling

of DOTA to proteins.62 The oligonucleotide was reacted with the N-hydroxy-

sulfosuccinimide ester of DOTA, prepared in situ using the water-soluble EDC

42

(1-ethyl-3(3-dimethylaminopropyl)carbodiimide) as coupling reagent to give DOTA-

SODN (Scheme 13).

NN

N N

COOH

HOOC

HOOC

COOH

NN

N N

COOH

HOOC

HOOC

C NH(CH2)6SODNO

DOTA

DOTA-SODN

NN

N N

COOH

HOOC

HOOC

C O NS O-O

O

O

O

O

2) SODN(CH2)6NH2

DOTA-NHS

1) EDC

NHO

SO

OONa

O

O

Scheme 13.

The purity of the DOTA-SODN was crucial for successful labelling, since free DOTA

would also chelate 68Ga. Purification by NAP-5 gelfiltration was not sufficient to

separate unreacted DOTA from the DOTA-SODN. The DOTA-SODN was retained

on a C-18 SPE column using 50 mM triethylammonium acetate buffer (TEAA). The

DOTA could then be washed away before the DOTA-SODN was eluted using

acetonitrile: water. No remaining DOTA or unmodified oligonucleotide could be

detected when analysing DOTA-SODN by electrospray mass spectrometry (direct

infusion).

The DOTA-SODN (SODN20 and SODN30) was labelled using 68Ga in 0.4 M acetate

and heating at 100°C for 25 min giving 35 % radiochemical yield. Purification by

NAP-5 gelfiltration did not give satisfactory purity of the product, whilst purification

by C-18 SPE gave a radiochemically pure product.

43

6. Characterisation of Labelled Oligonucleotides

Analysis using LC and ESI-MS II

In analysis of oligonucleotides with reverse phase chromatography, TEAA buffer is

used due to its ion-pairing properties, which increase the retention. In this study

polystyren divinylbenzene columns were used for separation and identification of the

oligonucleotides. Using this method, the 76Br-labelled oligomers could be separated

from the unlabelled oligomers.

Oligonucleotides can be analysed by mass spectrometry using electrospray ionisation.

Due to the polyanionic nature of the oligonucleotides, there is a distribution of

different charged species (M-nH+)n-. The polyanionic backbone can form adducts with

sodium and potassium and thereby decrease the sensitivity due to many different

charged species. The presences of a strong organic base such as triethylamine or

piperidine suppresses the formation of sodium and potassium adducts and increases

the sensitivity.134, 135 The TEAA buffer system, however, is not compatible with the

use of ESI-MS.134 Thus, to enable ESI-MS analysis, the ODN-samples had to be

desalted using gel filtration or dialysis. Buffer-systems compatible with ESI-MS have

been suggested where the LC-separation is good.134

In order to characterise the 76Br-labelled oligonucleotides, syntheses with carrier

added BrNHS were performed and the products were isolated according to the

radiotrace in the HPLC-chromatogram. The products were desalted, concentrated and

dissolved in piperidine/imidazole buffer and i-propanol prior to analysis by ESI-MS

using direct infusion. The distribution of ions with different charges could then be

used to calculate the molecular mass of the compound. An example is showed in

Figure 13. Oligonucleotides conjugated to the chelator DOTA were also identified by

the use of ESI-MS.

44

[M-9H+]9-

[M-10H+]10-

[M-8H+]8-

500 600 700 800 900 1000 1100 1200 1300 1400 1500Da/e 0

100

%

795

706

636

908

1060

Reconstituted mass: 6368 Da Expected mass: 6371 Da

[M-6H+]6-

[M-7H+]7-

Figure 13. ESI-MS spectrum of 76Br-ODN20 after synthesis with carrier-addedBrNHS. Peaks with z = -6 to z= -10 were detected.

Metabolite analysisIV

PET or autoradiographic techniques can be used to detect the distribution of

radioactivity in an object but it does not discriminate between the signals from the

originally labelled molecule and radiolabelled metabolites. Therefore it is important to

investigate the stability of the labelled tracer in vivo. Degradation of the labelled

oligonucleotides from the 5´-end would give radioactive low-molecular weight

species whereas degradation from the 3´-end would give labelled oligonucleotides of

different length. Degradation by exonucleases from the 3´-end is thought to be one of

the major pathways.66

Oligonucleotides longer than 15 mer can efficiently be precipitated by the cationic

detergent cetylpyridinium bromide, CPB.136-138 Carrier DNA has to be added for

effective precipitation of small amounts of oligonucleotides. A plausible mechanism

for the precipitation is the formation of a salt between the phosphates of the DNA and

the quaternary ammonium group of the detergent, which gives a thick hydrophobic

coat around the DNA, making it insoluble in water.137 The detergent can then be

removed by ethanol washing whilst the DNA and oligonucleotides remain insoluble.

Experiments using 76Br-labelled HSA as a substrate for CPB-precipitation indicated

that the proteins were not precipitated under the conditions used. High molecular

weight components (i.e. proteins or ODN>10 mer) could be separated from low

molecular weight components by the use of disposable NAP-5 size exclusion

gelfiltration columns.

45

Control experiments where the labelled oligonucleotides were homogenated with

kidney tissue were performed (referred to as “Control” in Figure 14) to test the

recovery of oligonucleotides from the CPB-precipitation and NAP gel filtration,

which was found to be about 80 %. Blank experiments without addition of kidney

gave the same results, indicating that the oligonucleotides were not metabolised in the

homogenation process.

Homogenated samples of kidney and liver from rats injected with 76Br-SODN20 and76Br-SODN30 were analysed using CPB precipitation and gel filtration. About 80 %

of the radioactivity from the homogenates was recovered in the precipitate (Figure

14). Gel filtration showed similar results as approximately 80 % of the radioactivity

was found in the high-molecular fraction (Figure 14).

The precipitates were redissolved and analysed by reversed phase HPLC. Fractions

were collected and measured for radioactivity. The samples from the liver and kidney

showed the same elution profile as the control sample. In the separation of

oligonucleotides that differed by only one mer in length, for example metabolites

degraded from the 3´-end, ion exchange chromatography has previously been reported

to be superior to reverse phase chromatography.139

Figure 14. Results fromanalysis of homogenatedtissue from rats injectedwith 76Br-SODN20 or76Br-SODN30.

CPB-precipitation

0255075

100

Kidney

20

Liver

20

Contro

l 20

Kidney

30

Liver

30

Contro

l 30%

of t

otat

raio

activ

ity

average stdev.

NAP-5 gelfiltration

0255075

100

Kidney

20

Liver

20

Contro

l 20

Kidney

30

Liver

30

Contro

l 30

% o

f tot

atl r

adio

activ

ity

average stdev

46

Distribution of 76Br-oligonucleotides in vivo and in vitroIV

Statistically a 15-17 mer oligonucleotide is necessary to avoid the oligonucleotide

from hybridising specifically to more than one target. Longer oligomers can improve

the affinity but can also lower specificity since parts of the oligonucleotides can

hybridise with other targets.12 The mechanisms for transport and excretion might also

change with length. The aim of this study was to explore the distribution of 76Br-

labelled oligonucleotides in normal rats. The sequences used were 6, 12, 20 and 30

mer SODN (Table 2) complementary to the mRNA for synthesis of chromogranin A,

a protein found in high concentrations in patients with neuroendocrine tumours.124

The oligonucleotides were labelled using 76BrNHS 14 and the 76Br-SODN was

injected in the rats. After 20 hours the animals were sacrificed (2 hours for 76Br-

ODN30 due to the rapid metabolism of phosphodiester oligonucleotides). Visualising

the distribution of 76Br-SODN using autoradiography, the uptake in the kidney cortex

dominated for the SODN6 and SODN12. For SODN12 a discrete uptake could be

noticed in the liver. Going to the larger SODN20 the kidney cortex still dominated the

image but the uptake in the liver was evident. Using SODN30 the highest uptake was

noted in the liver, spleen and red bone marrow and the uptake in the kidney cortex

was significantly lower (Figure 15). The result from the autoradiography was

confirmed using quantitative measurement of the radioactivity in the organs (Table 5).

With 76Br-ODN30 the uptake was measured after 2 hours and the highest uptake

could be noticed in the adrenals.

Table 5. Standardised Uptake Value (SUV) * of different lengths of labelled antisensephosphorothioate oligonucleotides in rats 20 hours after injection.

76Br-SODN6 76Br-SODN12 76Br-SODN20 76Br-SODN30

Liver <0.5 <0.5 8.6 12.3

Kidney 13.3 53.1 17.8 5.1

Spleen <0.5 <0.5 3.4 1.9

Adrenals <0.5 <0.5 1.1 1.6* SUV=(organ radioactivity/organ weight)/(total given radioactivity/rat body weight)

The SUV of heart, lungs, pancreas, intestines, stomach and blood was under 0.5.

47

Figure 15. Ex-vivo autoradiography of rat injected with 76Br-SODN30, 76Br-SODN20

and 76Br-SODN12.

Characterisation of the Hybridisation-Properties of Labelled OligonucleotidesIV

The impact of the labelling procedure on the oligonucleotide probes was investigated

using hybridisation to a complementary 30 mer oligonucleotide in solution. The

results were analysed using gel electrophoresis and the results were visualised by

ethidium bromide staining and autoradiography. It was shown that the labelled

phosphodiester 76Br-ODN30 was able to hybridise to its complementary ODNsense

(Table 2) whilst the phosphorothioate 76Br-SODN30 could not hybridise under the

conditions used (Figure 16).

To further confirm the ability of the probes to hybridise specifically to a longer

sequence single-stranded DNA, reverse-transcription-polymerase chain reaction (RT-

PCR) was used. RNA was isolated from tissue from adrenals, kidney, liver, heart and

lungs in rat. The RNA was transcribed using reverse transcriptase and parts of the

mRNA coding for chromogranin was amplified by PCR to obtain a single stranded

230 mer DNA-fragment with the same sequence as part of mRNA (except that uridine

is replaced by deoxythymine). Two oligonucleotide sequences were used as primers

in the PCR-reaction, one with the same sequence as the labelled oligonucleotides. The

48

length of the DNA-fragment (230 mer) was determined by the position of the

sequence of ODN30, which was located in the 3´-end of the DNA-fragment. The76Br-ODN30 and 76Br-SODN30 were then employed as forward primers to amplify

the sequence complementary to the DNA-fragment. In the case of 76Br-ODN30 in

combination with RNA isolated form adrenals, a 230 mer fragment was successfully

amplified and identified by gel electrophoresis followed by sequencing. The

incorporation of the label in the 230 mer fragment was verified by autoradiography of

the gel. No amplified product could be detected when 76Br-SODN30 was employed as

a forward primer.

a) b)

Figure 16. Gelelectrophoresis analysis of the hybridization reaction with ODNsense.Gel after: a) Staining with ethidium bromide, b) Autoradiography (the loding place oflane 1, 3 and 5 are marked). The different lanes contain the following: 1) Mw-markerin steps of 10 base-pairs, 2) 76Br-ODN30 and ODNsense, 3) ODN30 and ODNsense,4) 76Br-ODN30, 5) ODN30, 6) ODNsense. The 76Br-ODN30 –ODNsense duplex canbe detected in lane 2.

49

7. Conclusions

Macromolecules such as oligonucleotides, proteins and peptides could be labelled

with 76Br by conjugation to a labelled prosthetic group containing an electrophilic

functionality. The N-Succinimidylesters, isothiocyanates and iminoesters were

investigated as electrophiles in the conjugation reaction, where the best results were

obtained with the succinimidylesters. 76Br could be introduced by halogenation-

demetallation reactions using trimethylstannyl- or tributylstannyl-compounds as

substrates. For some of the labelled macromolecule-conjugates, biological properties

such as binding to selected tissue, enzymatic activity or ability to hybridise, have been

investigated to see whether the prosthetic group affected these properties.

The advantage of using 76Br instead of 18F is the possibility to, in a simple fasion,

oxidise bromine and obtain the labelled prosthetic group in a one step reaction, while

the use of nucleophilic fluorine often requires a multi-step synthesis.

Another option for labelling of macromolecules is to use a radiometal in combination

with a chelating agent. Using this method, oligonucleotides and a peptide containing

the chelating agent DOTA could be labelled with 68Ga. The advantage with this

labelling strategy is that the reaction is performed in one step and that the only

purification necessary is solid phase extraction. Since the 68Ga is obtained from a

generator-system and the protocol to label these DOTA-macromolecules is suitable

for kit type preparations.

50

Acknowledgement:

Efter de här åren så är det några jag skulle vilja tacka extra mycket för hjälp, stöd och

uppmuntran. Tack också till er som bidragit till den kunskap och möjlighet till utveckling som

jag fått ta del av. Ett sort TACK till:

Professor Bengt Långström för förmågan att alltid hitta nya lösningar och för en aldrig

sinande entusiasm.

Dr Elisabeth Hedberg, Lisa, för introduktion till oligonukleotider och brommärkningar samt

för det samarbete som följde. Din orädda attityd till det mesta och ditt goda humör var en

tillgång på labbet. Tack för visat intresse (både för våra oligosar och skvaller) även efter du

”blev doktor”.

Dr Benita Forngren för samarbete med att bestämma specifik radioaktivitet. Tack också för all

hjälp med mass spektrometern och kromatografi.

Dr Vladimir Tolmachev för alla sena kvällar och tidiga mornar du överfört dina kunskaper om

bromproduktion samt för de brombatcher du producerat. Tack även för alla konstruktiva

förslag och diskussioner rörande gallium-inmärkningar.

Dr Feng Wu for collaboration with the oligonucleotides. Thank you for your patience when

synthesis failed, was delayed or when the amount of radioactivity was not what you expected.

Doc. Mats Bergström, Michinari Honda, Shaheena Tanweera Khan och Gabor Lendvai för

gott samarbete.

Doc. Gunnar Antoni, dr Margareta Björkman, dr Elisabeth Hedberg, dr Koichi Kato, dr Jonas

Malmström och fil. lic. Linda Samuelsson för synpunkter på utkast till avhandlingen. Dr.

Nicholas Power for liguistic corrections.

51

Personal på Kemikum, framförallt Gunnar, Leffe, Vicke, Tomas, Tatti och Eva för assistans

med allehanda saker. Personal på PET-centrum: Jocke och Kalle för hjälp med cyklotronen

och bromproduktioner samt Lotta och Pernilla för diverse inköp. Och tack till er som tryckt

”beam off” så jag fått gå hem lite tidigare.

Tobias, Benita, Lisa, Sven Åke, Koichi, Pelle, Johanna och Per för mycket träskkaffe,

diskussionerom stort och smått, lunch och lite jobb också.

Föredetta och nuvarande medlemmar av grupp BLå för trevligt arbetsklimat. Samt alla ni

andra på Kemikum, Glunten och PET-centrum som bidragit till att arbetsdagarna blev lite

roligare.

C. F. Liljevalchs, Anna Whitlocks och Acta Chemica Scandinavica stipendiefonder samt

Sigurd och Elsa Goljes Minne och Knut och Alice Wallenbergs stiftelser för generösa bidrag

till konferensresor.

Till er som sett till att det funnits ett liv utanför Kemikum:

”Flickorna från V-Dala”: Ulrika, Emma, Eva och Sandra. Tack för allt kul på nationen (och

annat också) och för andra perspektiv på tillvaron. Ett speciellt tack till Ulrika för vänskap

och stöd under hela studietiden och för att sedan bli min referens till ”verkligheten”.

”Flickorna från Uppsala”: Eva-Lotta, Åsa och Stina, för en vänskap som förhoppningsvis

alltid kommer att finnas, även om det ibland blir långt mellan träffarna.

Mamma och Pappa för det sjävförtroende och den självständighet ni gett mig, det har alltid

varit bra att ha.

Liselott, min allra bästa lillasyster.

Farmor för att du ständigt bevisar att ingenting är omöjligt.

Jonas, för ditt orubbliga lugn i alla situationer. Tack för all kärlek, stöd och uppmuntran. De

här åren hade inte varit lika roliga om jag inte haft dig att dela dem med.

52

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