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Natural Products aNalysis

Natural Products aNalysisInstrumentatIon methods and applIcatIons

edited by

Vladimiacuter havliacutec ekLaboratory of Molecular Structure CharacterizationInstitute of MicrobiologyAcademy of Sciences of the Czech RepublicPrague Czech Republic

Jaroslav spiacutežekLaboratory of Physiology and Genetics of ActinomycetesInstitute of MicrobiologyAcademy of Sciences of the Czech RepublicPrague Czech Republic

Copyright copy 2014 by John Wiley amp Sons Inc All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750-8400 fax (978) 750-4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748-6011 fax (201) 748-6008 or online at httpwwwwileycomgopermission

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

For general information on our other products and services or for technical support please contact our Customer Care Department within the United States at (800) 762-2974 outside the United States at (317) 572-3993 or fax (317) 572-4002

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Library of Congress Cataloging-in-Publication Data

Natural products analysis instrumentation methods and applications edited by Vladimiacuter Havliacutecek Laboratory of Molecular Structure Characterization Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic Jaroslav Spiacutezek Laboratory of Physiology and Genetics of Actinomycetes Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic pages cm Includes bibliographical references and index ISBN 978-1-118-46661-2 (hardback)1 Natural productsndashAnalysis 2 Chemistry Analytic I Havliacutecek Vladimiacuter editor II Spiacutezek Jaroslav editor QD7522N38 2014 543prime19ndashdc23

2014008371

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

To our families colleagues students and our advisors

Petr Sedmera and Zdenko Vanek

vii

Preface ix

Contributors xi

1 Natural Products Analysis Instrumentation Methods and Applications 1Vladimiacuter Havliacuteček and Jaroslav Spiacutežek

2 The Need for New Antifungal and Antimalarial Compounds 9Jaroslav Spiacutežek and Arnold L Demain

3 Emerging Instrumental Methods for Antimicrobial Resistance and Virulence Testing 25Plamen A Demirev

4 Plant and Marine Sources Biological Activity of Natural Products and Therapeutic Use 43Amedeo Amedei and Elena Niccolai

5 Emerging Trends for Stimulating the Discovery of Natural Products 115Navid Adnani Gregory A Ellis Thomas P Wyche Tim S Bugni Jason C Kwan and Eric W Schmidt

6 Advances and Challenges in Optical Molecular Spectroscopy Including Surface Plasmon Resonance-Based Methods for Bioanalysis 163Pavel Matějka Blanka Vlčkovaacute Lucie Bednaacuterovaacute and Petr Maloň

7 Advanced Techniques for NMR Analysis of Complex Biological MixturesmdashFrom Simple NMR to Hyphenated Techniques 239Helena Pelantovaacute Simona Baacutertovaacute and Marek Kuzma

8 Advances in X-Ray Diffraction Implications to the Pharmaceutical Industry 285Alexandr Jegorov and Michal Hušaacutek

Contents

viii Contents

9 Laser Ablation Inductively Coupled Plasma Mass Spectrometry as a Tool in Biological Sciences 313Michaela Vašinovaacute Galiovaacute Jan Havliš and Viktor Kanickyacute

10 Imaging Mass Spectrometry Metabolism and New Views of the Microbial World 349B Christopher Hoefler and Paul D Straight

11 Structural Separations for Natural Product Characterization by Ion MobilityndashMass Spectrometry Fundamental Theory to Emerging Applications 397Sarah M Stow Nichole M Lareau Kelly M Hines C Ruth McNees Cody R Goodwin Brian O Bachmann and John A McLean

12 High-Resolution Tandem Mass Spectrometry for Nonribosomal Peptide and Polyketide Analysis 433Rebecca H Wills Manuela Tosin and Peter B OrsquoConnor

13 Natural Product Drug Discovery and Analysis Using Mass Spectrometry and Affinity-Based Technologies 475Evelyn H Wang and Kevin A Schug

14 Glycosylated Ribosomally Synthesized Peptide Toxins Discovery Characterization and Applications 507Gillian E Norris and Mark L Patchett

15 Using Ultrahigh-Resolution Mass Spectrometry to Unravel the Chemical Space of Complex Natural Product Mixtures 545Constanze Muumlller Mourad Harir Norbert Hertkorn Basem Kanawati Dimitrios Tziotis and Philippe Schmitt-Kopplin

16 Functional Amyloid Fibrils Lessons from Microbes 571Sally L Gras and Dennis Claessen

Index 601

With 16 chapters written by a diverse group of scientists working in the forefront of natural product analysis and applications this book provides an overview of both the current status and future research directions of plant human microbial and animal metabolome analysis We hope that it will serve as a useful resource and an inspira-tion for scientists graduate students and undergraduate students in a wide variety of research fields including chemistry biology microbiology biochemistry and ana-lytical chemistry The common analytical fields such as X-ray mass spectrometry and nuclear magnetic resonance spectroscopy are viewed through their modern applications New emerging application areas such as chemical genetics and pepti-dogenomics are also covered We predict that in five years all these approaches may become standard tools providing new important drugs screened from a large natural products family

It would make us particularly happy if this book would find its way to visionaries and fans of this field of science

Prague Vladimiacuter Havliacuteček 2014 Jaroslav Spiacutežek

Preface

ix

xi

Navid Adnani School of Pharmacy University of WisconsinndashMadison Madison WI USA

Amedeo Amedei Department of Experimental and Clinical Medicine University of Florence Florence Italy

Brian O Bachmann Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

Simona Baacutertovaacute Department of Analytical Chemistry Institute of Chemical Technology Prague Prague Czech Republic

Lucie Bednaacuterovaacute Institute of Organic Chemistry and Biochemistry AS CR Laboratory of Molecular Spectroscopy Prague Czech Republic

Tim S Bugni School of Pharmacy University of WisconsinndashMadison Madison WI USA

Dennis Claessen Microbial Biotechnology Leiden University Institute Biology Leiden Leiden The Netherlands

Arnold L Demain Charles A Dana Research Institute for Scientists Emeriti (RISE) Drew University Madison NJ USA

Plamen A Demirev Johns Hopkins University Applied Physics Laboratory MD USA

Gregory A Ellis School of Pharmacy University of WisconsinndashMadison Madison WI USA

Michaela Vašinovaacute Galiovaacute Department of Chemistry Faculty of Science and Central European Institute of Technology Masaryk University Brno Czech Republic

Cody R Goodwin Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

Sally L Gras Microbial Biotechnology Leiden University Institute Biology Leiden Leiden The Netherlands

Contributors

xii Contributors

Mourad Harir Research Unit Analytical BioGeoChemistry Helmholtz Center Munich ndash German Research Center for Environmental Health Neuherberg Germany

Vladimiacuter Havliacutec ek Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Jan Havliš National Centre for Biomolecular Research Faculty of Science and Central European Institute of Technology Masaryk University Brno Czech Republic

Norbert Hertkorn Research Unit Analytical BioGeoChemistry Helmholtz Center Munich ndash German Research Center for Environmental Health Neuherberg Germany

Kelly M Hines Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

B Christopher Hoefler Department of Biochemistry and Biophysics Texas A amp M University College of Agriculture and Life Sciences College Station TX USA

Michal Hušaacutek Institute of Chemical Technology Prague Czech Republic

Alexandr Jegorov Teva Czech Industries Ceskeacute Budejovice Czech Republic

Basem Kanawati Research Unit Analytical BioGeoChemistry Helmholtz Center Munich ndash German Research Center for Environmental Health Neuherberg Germany

Viktor Kanickyacute Department of Chemistry Faculty of Science and Central European Institute of Technology Masaryk University Brno Czech Republic

Marek Kuzma Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Jason C Kwan Department of Medicinal Chemistry College of Pharmacy University of Utah Utah USA

Nichole M Lareau Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

Petr Maloň Institute of Organic Chemistry and Biochemistry AS CR Laboratory of Molecular Spectroscopy Prague Czech Republic

Pavel Matějka Institute of Chemical Technology Department of Physical Chemistry Prague Czech Republic

John A McLean Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

C Ruth McNees Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

Contributors xiii

Constanze Muumlller Research Unit Analytical BioGeoChemistry Helmholtz Center Munich ndash German Research Center for Environmental Health Neuherberg Germany

Elena Niccolai Department of Experimental and Clinical Medicine University of Florence Florence Italy

Gillian E Norris Institute of Fundamental Sciences Massey University New Zealand

Peter B OrsquoConnor Department of Chemistry University of Warwick Coventry UK

Mark L Patchett Institute of Fundamental Sciences Massey University New Zealand

Helena Pelantovaacute Department of Analytical Chemistry Faculty of Science Palacky University Olomouc Czech Republic

Eric W Schmidt Department of Medicinal Chemistry College of Pharmacy University of Utah Utah USA

Philippe Schmitt-Kopplin Research Unit Analytical BioGeoChemistry Helmholtz Center Munich ndash German Research Center for Environmental Health Neuherberg Germany

Kevin A Schug Department of Chemistry and Biochemistry The University of Texas at Arlington Arlington TX USA

Jaroslav Spiacutežek Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Sarah M Stow Department of Chemistry Vanderbilt Institute of Chemical Biology Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University Nashville TN USA

Paul D Straight Department of Biochemistry and Biophysics Texas A amp M University College of Agriculture and Life Sciences College Station TX USA

Manuela Tosin Department of Chemistry University of Warwick Coventry UK

Dimitrios Tziotis Research Unit Analytical BioGeoChemistry Helmholtz Center Munich ndash German Research Center for Environmental Health Neuherberg Germany

Blanka Vlčkovaacute Department of Physical and Macromolecular Chemistry Charles University Prague Czech Republic

Evelyn H Wang Department of Chemistry and Biochemistry The University of Texas at Arlington Arlington TX USA

Rebecca H Wills Department of Chemistry University of Warwick Coventry UK

Thomas P Wyche School of Pharmacy University of WisconsinndashMadison Madison WI USA

Natural Products Analysis Instrumentation Methods and Applications First Edition Edited by Vladimiacuter Havliacuteček and Jaroslav Spiacutežek copy 2014 John Wiley amp Sons Inc Published 2014 by John Wiley amp Sons Inc

1

Chapter 1

This book aims at highlighting the newest trends in analytical chemistry that have recently been or soon will be employed in the analysis of natural products and their complex mixtures All contributing authors were motivated to stress the innovative aspects in emerging natural product chemistries and were asked to formulate their own personal visions clearly indicating which milestones can be achieved in their fields of expertise in a five-year frame The book is structured according to analytical instrumental approaches used either routinely or experimentally for structure characterization andor determination of both low- and high-molecular-weight natural products

11 BOOK MOtIVatION

This book enumerates the most recent and cutting-edge analytical approaches including those that have not yet been commercialized into the rejuvenated natural products field For example less-traditional applications of synchrotron irradiation to small molecules are reported when referring to standard X-ray diffraction Likewise examples of the newest hyphenation techniques with impact on screening and secondary metabolism studies are described in cases in which well-known mul-tidimensional NMR spectroscopy is discussed

The revitalization of the natural product field is documented by an increase in the number of peer-reviewed articles illustrated by a Web of Science search (Figure 11)

Natural products analysis Instrumentation Methods and applications

Vladimiacuter HaVliacutecek and JaroslaV spiacuteŽek

institute of microbiology academy of sciences of the czech republic prague czech republic

2 Natural products aNalysis

The number of hits is seen to have increased threefold if the term ldquonatural product activityrdquo is evaluated Antibacterial antifungal antineoplastic anti-inflammatory and other activities are also reported in patent literature SciFinder returned constant data for the 2007ndash2013 period oscillating between 60 and 80 patent applications published annually Diverse applications of natural products are also subjects of many review articles and book chapters Interestingly no monograph focused on instrumentation used for identification of natural products has been published in the past decade This market gap was identified by Wiley senior editor Jonathan T Rose ldquoIn my opinion given that plants and natural products are major sources for current and potential drugs there is need for a book geared to researchers and professionals to facilitate natural product analysis synthesis and drug discovery This type of book could explain the basics of natural products as pharmaceuticals analytical tools and techniques methods for isolation and elucidation and applications for library design and in drug discovery Such a book will find a welcome audience in organic and medicinal chemists biochemists analytical and medicinal chemists microbiologists and biomedical researchersrdquo

In this book the instrumentation represents the common denominator The con-tributors were motivated to make a very brief introduction to physicochemical principles of their methods and give an up-to-date overview of the most important applications relevant to natural products In a limited number of chapters the tutorial part was extended giving the reader the opportunity to get acquainted with both the fundamentals and future trends in one place Personal views and mutual instrumental evaluations will help the newcomers to find a suitable technique For instance whereas nuclear magnetic resonance spectroscopy is nonselective and less sensitive (ldquoalways tells the truthrdquo) mass spectrometry is selectively sensitive (ldquotells you what you want to hearrdquo)

0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012

Figure 11 Report of published items accessed from the Web of Knowledge (Thomson Reuters) on December 31 2013 illustrates the number of papers published annually in the field of ldquoNatural Product Structurerdquo

discoVery pHases 3

12 the BrOaD FIeLD OF NatUraL prODUCtS

Chapters 2ndash4 represent medically oriented introductory chapters Chapter 2 focuses specifically on fungi and malaria and defines the current microbiology challenges in the field of natural product discovery These two application areas were deliberately selected because they are rather underestimated in the review literature The importance of tackling antimicrobial resistance and the application of standardized combination therapies is stressed Drug degradation products arising from enzyme-specific reactions drug target reprogramming or ejecting the drug out of the bacterial or fungal cells belong to known mechanisms by which microbes fight against antimicrobial drugs In the field of drug resistance cultivation of microorganisms in drug-containing stable isotope-labeled media are particularly promising Mass spectrometry (MS) is then used for the determination of natural isotope shift reflecting the viability of the microorganism and its ability to consume and metabolize the labeled nutrients The potential and limitations of NextGen or NextNext sequencing methods are briefly described in the perspective section in Chapter 3 The importance of peptidogenomic methods for the determination of virulence mechanisms of pathogens is accentuated by means of imaging mass spectrometry in Chapter 10

The introductory segment of this book is terminated by Chapter 4 in which the major fractionation and isolation procedures of natural products are briefly outlined Major attention is dedicated to the respective biological activities of natural products The chapter is subdivided according to plant and marine origin of most important metabolites that have found significant medical applications The authors faced a difficult task to select the clinically most important active principles of both marine and plant origin and align their pharmacokinetic and biological properties with medical applications Attention was paid to organic compounds in different phases of biological trials Most important applications of natural compounds in cardiovascular infectious cancer and other areas are summarized

13 DISCOVerY phaSeS

Recent applications of metabolomics proteomics mutagenomics and genomics in exploiting bacterial natural products are summarized in Chapter 5 In mass spectrometry-based metabolomics the problem of silent or cryptic NP biosynthesis pathways (the ldquosilent parvomerdquo) is discussed in the context of the quest for novel chemistries Mass spectral alignment strategies are outlined (XCMS MZMine commercial products) and supported by principal component analysis program packs (SIMCA MATLAB etc) the importance of which is documented (for example) on strain prioritization Two proteomic approaches in natural product discoveries are reviewed The first is the Kelleher group proteomic investigation of secondary metabolism (PrISM) utilizing the phosphopantetheinyl ejection assay [1] The second proteomic technique is represented by an Orthogonal Active Site Identification System (OASIS) [2] In the (meta)genomic part the amplicon sequencing shotgun


librariesmetagenomics and single-cell genomics methods are outlined and supported by success stories Genome annotation pipelines are provided (CloVR-microbe AntiSMASH NCBI SMART) The importance of concerted application of density functional theory and 2D NMR spectroscopy for absolute structure determi-nation in natural products is stressed in the final part of the chapter The applications of residual dipolar couplings in nuclear magnetic resonance (NMR) circular dichroism and classical chemistry are also emphasized and create the bridge to molecular tools

Some of them are further structured in tutorial Chapter 6 referring to the applications of electronic and vibrational spectroscopies Advances and challenges in optical molecular spectroscopy of biomolecules and natural products are supported by chiroptic methods and placed in the context with surface-enhanced techniques and surface plasmon resonance (SPR) sensing NMR users and fans will appreciate Chapter 7 a substantial part of which is dedicated to sample preparation and handling Attention is also paid to LC-NMR setup with most common instrumental variants and practical recipes (on-flow stop-flow and the combination of solid-phase extraction and MS) Their properties in terms of sensitivity sample concentration and sample nature are discussed in detail Similarly both supervised and unsupervised methods of statistical data evaluation are reported Differential analysis is addressed in specialized subchapters dedicated to statistical heterospectroscopy statistical total correlation spectroscopy and other methods The reader can benefit from public databases of NMR spectra and web servers dedicated to NMR metabolomics Covariance NMR data processing of TOCSY and NOESY spectra is described Virtual NMR chromatography (including its 3D variant) is used for distinguishing signals coming from small or large molecules Food adulteration plant extract analysis and tens of other NMR applications in metabolomics are presented

14 aBSOLUte StrUCtUre

Chapter 8 describes the general technique of X-ray diffraction including the single-crystal and powder methods and it covers advances in the instrumentation currently in use The central argument that X-ray diffraction has a great potential and plays an increasingly important role in the structure determination of natural products is well documented and supported by the possibility to provide absolute structure determination packing of molecules in the crystal and structure determination in the presence of solvents in the crystal unit Public academic software programs Sir2011 SuperFlip CRYSTALS and checkCIF are referred to and Cambridge Structural Database is stressed The chapter discusses in a reader-friendly manner the common myths of X-ray diffraction (excessive time and high amount of samples needed for analyses samples do not crystallize) It also provides a set of examples showing cases of natural product whose stereochemistry or absolute configuration originally suggested by other tools was completely revised or reassigned by X-ray Practical comparison of what can be achieved with both laboratory or synchrotron sources and what can also be achieved with given crystal size and quality is reported

mass spectral applicatioNs iN coNcert 5

as well The chapter is concluded with a belief that the number of research groups producing a mere ldquoamorphous white solidrdquo will steadily decrease Neutron diffraction and electron diffraction are briefly outlined A short notice on the analysis of NRPSPKS domains by crystallography is also presented in Chapter 12

15 MaSS SpeCtraL appLICatIONS IN CONCert

Although mass spectrometry is a mature technique celebrating its 100-year anniversary some of its newer applications have revolutionized the emerging fields of peptidoge-nomics and metabolomics and also significantly contributed to revitalization of the natural product field Chapters 9 to 15 are thus dedicated to both the instrumentation (inductively coupled plasma imaging ion mobility affinity ultrahigh resolution) and applications of mass spectrometry (ribosomal and nonribosomal natural products) In Chapter 9 solid or semisolid samples are probed by inductively coupled plasma (ICP) mass spectrometry with special attention to heteroelementsmdashthat are metals metalloids and nonmetals For beginners in the field the instrumental setup is briefly outlined with numerous applications to inorganic and organic matter analysis including proteins separated by native polyacrylamide gel electrophoresis Particular attention is paid to laser ablation also when combined with 2D or 3D bioimaging approaches The importance of elemental fractionation phenomena in quantitative determination is described and key variables defined (eg sample planarity aerosol transport vaporization or ionization efficiency) Suppression of spectral interferences by collisional or dynamic reactive interactions in the gas phase is placed in context with the resolving power of a mass analyzer The analytical limitations of ICP-MS are defined in a fair manner The part on laser ablation ICP-MS comparison to other techniques of surface analysis will also be of interest to the reader EMPA XRD XFA XRA XRF PIXE NAA and some other instrumental tools are mutually compared and appropriate applications defined (including imaging) The chapter concludes with a critical personal view of quantitation in selected peer-reviewed papers reporting misleading results

Imaging mass spectrometry is addressed in Chapter 10 In the introductory part ionization techniques (SIMS MALDI and DESI) used for mass spectrometry imaging are reviewed in a tutorial manner while their practical limits and prospects for future technical development are described in the final visionary part of the chapter nanoDESI is defined as a central technique for bacterial imaging mass spectrometry Ionization enrichment by derivatization and labeling strategies are outlined with a special attention to analysis of carbohydrates oligonucleotides and other less common molecules Experimental considerations are defined with respect to applications in microbiology Particular emphasis is devoted to ecology and elemental analysis with submicron spatial resolution Biosynthesis secretion exchange symbiotic interaction or competitions are described Many topics make this chapter interesting not only for analytical chemists and natural product fans but also for (micro)biologists and biochemists in general for instance the role of siderophores in iron piracy is outlined The importance of peptidogenomic approaches is documented for example by


characterization of cannibalistic phenomena in bacteria and other bacterial or inter-kingdom interactions

Chapter 11 is devoted to the specific exploration of primary and secondary metabolites by ion mobility mass spectrometry (IM-MS) In addition to a historical overview fundamentals and instrumentation approaches introduce the reader into the field of natural product prioritization and dereplication without chromatographic separation IM-MS offers a 10ndash4 lower peak capacity but is much faster (10 ms) com-pared to LC-FTMS separations Different classes of biomolecules are separated in the order of increasing gas-phase packing efficiencies or densities lipids lt peptidesproteins lt carbohydrates lt oligonucleotides IM structural separation can readily resolve isobaric species resulting from conformational isomers Various arrangements for performing tandem mass spectrometry on IM instruments as well as the com-putational approaches for collision cross sections are provided Contemporary efforts are underway to construct an atlas of conformation space to direct the rapid molecule identification Future trends are targeted to peak resolution improvement and development of ion mobility imaging area

Dereplicationmdashthat is elimination of already known compounds from further investigationmdashis also depicted in Chapter 12 High-resolution tandem mass spec-trometry combined with collisionally activated dissociation andor electron-induced dissociation was used for the purpose In its introductory part the chapter gives basic information on the biosynthesis of nonribosomal peptides and polyketides providing the reader with the initial orientation in the enzyme systems and corresponding natural products subsequently ldquotargetedrdquo by other analytical tools (mainly NMR and X-ray) and strategies (Ppant ejection OASIS PrISM) [3] The second part of the chapter shows representative success stories where tandem mass spectrometry was used as the main tool

The direct and indirect affinity mass spectrometry assays for drug discovery are addressed in Chapter 10 Techniques such as fragment-based lead assembly nanoelectrospray ionization multitarget affinity specificity screening detection of oligonucleotide-ligand complexes by electrospray ionization (ESI) and ESI-electron capture dissociation are included in the section of direct affinity mass spectrometry assays (MASS and DOLCE involving nucleic acid binding) Frontal affinity chromatography affinity capillary electrophoresis ultrafiltration gel permeation chromatography size exclusion chromatography and automated ligand identification system are also reported

16 COMpLeX StrUCtUreS aND COMpLeX MIXtUreS

The last part of the book contains three application chapters that show the complexity of natural product structures and indicate that state-of-the-art equipment is a prereq-uisite but not a guarantee for successful structure elucidation or even characteriza-tion Chapter 13 is dedicated to ribosomally synthesized peptide toxins Because these peptide natural products have been overviewed elsewhere [4] G Norris and M Patchett focused on characterizing glycosylated ribosomal products and began with

refereNces 7

Richard Phillips Feynmanrsquos principle of Science ldquoThe first principle is that you must not fool yourself and you are the easiest person to foolrdquo (Caltechrsquos commencement address 1974) This principle was then applied to the exciting story of glycocin reflecting its long-lasting development and final contributions of 2D NMR circular dichroism spectroscopy Edman sequencing and Fourier transform ion cyclotron resonance mass spectrometry The chapter also refers to useful specific or broader antibacterial peptide and prokaryotic glycoprotein repositories including manually curated ones Non-ribosomally synthesized and glycosylated antimicrobial peptides as well as potential biological functions and roles of glycosylation in the discussed organisms are also reviewed The authors conclude with applications of venom glycopeptides bacteriocins and glycocins in biotechnology and the food industry

Chapter 14 is dedicated to the description of the organic chemical diversity within complex biological and geochemical systems studied by ultrahigh-resolution mass spectrometry Basic principles of ion cyclotron resonance are reported and various metabolome databases useful for searches based on elemental composition are described The chapter also reports on basic statistics and mathematical tools for data visualization The utility of Van Krevelen diagrams or Kendrick mass defect plots for selective displaying of compounds of interest including endogenous small molecules or drugs and their metabolites is demonstrated by the success story of Chlamydia-infected human cells

The whole book is concluded with an application chapter (Chapter 15) in which the analytical armory is represented by electron and atomic force field microscopies Basic principles and experimental setups in both techniques are briefly discussed in the introductory part Amyloid fibrils represent the central subject of structural studies supported by recent literature including patents Although these protein aggregates have been associated with more than 30 serious illnesses including Alzheimerrsquos Parkinsonrsquos Huntingtonrsquos prion diseases or atherosclerosis the chapter also highlights the important nontoxic biological functions of amyloids Potential and proven properties of most important polypeptides (eg chaplins rod-lins or hydrophobins) are reported in the context of recent structural studies shed-ding light on processes like biofilm formation microbial adhesion initiation of aerial growth and so on Important applications include coating of catheters improving biocompatibility of implants detergent-resistant glass coatings stainless steel lubri-cation to reduce friction drug delivery systems and many others The fibrils may also be important in infection They interfere with blood clotting and activate the immune system Conversely fibril disruption and detachment by d-amino acids can define new emerging applications in this fascinating field

reFereNCeS

1 Dorrestein P C Bumpus S B Calderone C T Garneau-Tsodikova S Aron Z D Straight P D Kolter R Walsh C T Kelleher N L (2006) Facile detection of acyl and peptidyl intermediates on thiotemplate carrier domains via phosphopantetheinyl elimina-tion reactions during tandem mass spectrometry Biochemistry 45 12756ndash12766


2 Meier J L Niessen S Hoover H S Foley T L Cravatt B F Burkart M D (2009) An orthogonal active site identification system (OASIS) for proteomic profiling of natural product ACS Chemical Biology 4 948ndash957

3 Chen Y Q Ntai I Ju K S Unger M Zamdborg L Robinson S J Doroghazi J R Labeda D P Metcalf W W Kelleher N L (2012) A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria J Proteome Res 11 85ndash94

4 Arnison P G Bibb M J Bierbaum G Bowers A A Bugni T S Bulaj G Camarero J A Campopiano D J Challis G L Clardy J Cotter P D Craik D J Dawson M Dittmann E Donadio S Dorrestein P C Entian K-D Fischbach M A Garavelli J S Goeransson U Gruber C W Haft D H Hemscheidt T K Hertweck C Hill C Horswill A R Jaspars M Kelly W L Klinman J P Kuipers O P Link A J Liu W Marahiel M A Mitchell D A Moll G N Moore B S Mueller R Nair S K Nes I F Norris G E Olivera B M Onaka H Patchett M L Piel J Reaney M J T Rebuffat S Ross R P Sahl H-G Schmidt E W Selsted M E Severinov K Shen B Sivonen K Smith L Stein T Suessmuth R D Tagg J R Tang G-L Truman A W Vederas J C Walsh C T Walton J D Wenzel S C Willey J M van der Donk W A (2013) Ribosomally synthesized and post-translationally modified peptide natural prod-ucts Overview and recommendations for a universal nomenclature Natural Product Reports 30 108ndash160


9

Chapter 2

21 INtrODUCtION

Infectious diseases were the leading cause of death in 1900s and today they are the second most important killer in the world number three in developed nations and fourth in the United States [1] Although not all statistics may be absolutely correct and some may even be misleading it has been clearly demonstrated that infectious diseases are the leading causes of mortality and morbidity [2] It is particularly drastic in elderly and debilitated populations Americans are infected with bacteria at a rate of 25 million people per year resulting in 100000 deaths In our previous reviews [3 4] we concentrated mainly on bacterial infections In the present review we focus on fungal diseases and malaria

Antibiotic resistance has rapidly developed and this fact was originally inter-preted as a relatively modern phenomenon This assumption has long been supported by the fact that collections of microorganisms from the pre-antibiotic period are highly sensitive to antibiotics Relatively recent molecular-biological studies how-ever indicated that the occurrence of resistance genes in the environment is much more frequent than originally thought [5] In a recent paper DrsquoCosta et al [6] showed that antibiotic resistance precedes the use of antibiotics and propose that it is an ancient phenomenon commonly detected in the environment This is in agreement with the facts that antibiotic resistance is so often observed and occurs at a high frequency

the Need for New antifungal and antimalarial Compounds

Jaroslav spiacuteŽEk

Institute of Microbiology academy of sciences of the Czech republic prague Czech republic

Charles a Dana research Institute for scientists Emeriti (rIsE) Drew University Madison NJ Usa

arNolD l DEMaIN

10 ThE NEED for NEw aNTIfUNgal aND aNTIMalarIal CoMpoUNDs

This indicates that new antibiotics select preexisting resistance determinants that had circulated inside the microbial pangenome for millennia This should be kept in mind in management of the presently used and new antibiotics In addition the pipeline for new antibiotics is running dry and new antibiotics are desperately needed Many excellent reviews on the uses of antibiotics and antibiotic resistance have recently been published [7 8] We previously concentrated on bacterial infections [3 4] In this review resistant microorganisms are classified as those that are not inhibited by antibiotics or antimicrobials or antimicrobial compounds (these words will be used interchangeably) Resistance has steadily increased since systemic anti-biotics were introduced in the 1930s and 1940s In the present review we focus on fungal infections and malaria since these diseases appear to be among the most serious threats to the human population We will primarily but not always discuss com-pounds described as antibiotics according to the definition of Selman Waksman the Nobel Prize laureate in 1949 Waksman suggested the use of the word ldquoantibioticsrdquo classifying these compounds as ldquochemical substances that are produced by micro-organisms and that have the capacity in dilute solution to selectively inhibit the growth and even to destroy other microorganismsrdquo However this definition has been modified many times In this review we discuss natural compounds produced by microorganisms and plants as well as semisynthetic and synthetic compounds that have antimicrobial activity

22 FUNGaL INFeCtIONS

221 Fungal Diseases

Fungal infections have continued to increase due to population aging increases in immu-nocompromized individuals and use of central venous catheters and broad-spectrum antibacterials In an excellent review Calderone et al [9] classified fungal diseases as cutaneous and subcutaneous infections mucosal invasions and bloodstream infections They stated that of all fungal diseases dermatophytosis is probably the most prevalent but also the least studied in regard to hostndashfungus interactions Also according to the authors fungal diseases are endemic (histoplasmosis blastomycosis coccidiomycosis penicilliosis paracoccidiomycosis) or pandemic (invasive candidiasis aspergillosis including invasive aspergillosis cryptococcosis fusariosis mucormycoses dermato-phytosis) It is probable that the greatest threats to life today include those pathogens that cause common bloodstream infections

Treatment of patients with fungal diseases has become a serious burden Candidaemia is estimated to involve about 300000 cases per year worldwide and mortality can be as high as 30ndash55 In the United States Candida albicans kills between 3000 and 11000 people per year due to nosocomial candidemia Invasive aspergillosis can occur in different patient groups It is estimated that around 10 of new leukemia cases develop invasive aspergillosismdashthat is 30000 per yearmdashof stem cell transplants Fifty-four thousand patients in the United States the United Kingdom and Europe will need treatment for Aspergillus infections In chronic destructive pulmonary disease roughly 12 will need antifungals for aspergillosismdashthat is roughly

fUNgal INfECTIoNs 11

216000 per year Over 50 of invasive aspergillosis patients will die from their infection even if treated In AIDS patients 1 million contract cryptococcal menin-gitis resulting in 600000 deaths 70 of which are in sub-Saharan Africa Less fatal infections affecting large numbers of people worldwide include cutaneous fungal infectionsmdashnamely nail infections and athletersquos footmdashaffecting some 15 billion peo-ple or 25 of the world population Hair infection which is common in young chil-dren is predicted to affect some 200 million people worldwide In addition according to Vandenputte et al [10] resistance to antifungals rapidly increases The authors propose how to cope with this problem According to them it is estimated that 1728 billion people contract a fungal disease with 1 million deaths occurring

222 antifungal therapy

In the paper by Waksman et al [11] the authors stated that

The great majority of antibiotics that have been tested in the numerous screening programmes concerned with the search for new chemotherapeutic agents have been tested primarily for their activity against different bacteria Only limited consideration has been given to those antibiotics which possess mainly antifungal properties With the growing importance of various antibiotics in clinical medicine however especially in the treatment of diseases caused by bacteria and some larger viruses the need for substances with antifungal properties especially substances that are not too toxic and which offer promise in human and animal therapy has become of great importance

We will use the above statement as a motto and will now concentrate our attention on antifungal compounds

The 2011 antifungal market amounted to $61 billion Antifungal compounds have different mechanisms of action in fungal cells yeast cells in particular [12] Flucytosine affects pyrimidine metabolism as well as RNA and DNA biosynthesis Azoles inhibit ergosterol biosynthesis pathway at different steps Echinocandins inhibit cell wall β-glucan synthesis in fungal cells and polyene antifungals target fungal membrane sterols

However there are similarities in the mode of action of some antifungal compounds due mainly to the fact that many fungi have their own form of cholesterol that is ergosterol The enzymes involved in biosynthesis of ergosterol differ from those of biosynthesis of cholesterol to such an extent that antifungal compounds that target the fungal enzymes practically do not affect those involved in human cell cholesterol biosynthesis Such compounds thus have a relatively broad spectrum

Treating and caring for patients with fungal diseases is becoming a serious burden It is commonly accepted that fungal infections are caused by two types of fungi (a) primary pathogens and (b) opportunistic pathogens Whereas primary pathogens can cause infections in healthy populations opportunistic pathogens could be in general commensal microorganisms in healthy humans that do not cause any problems and become pathogenic only under specific conditions namely in immunocompromised individuals Most primary pathogens are filamentous fungi whereas most of the opportunistic pathogens are yeasts


There are not too many classes of antifungal drugs In general we can recognize four main types of compounds that are used in the clinical practice fluoropyrimidine analogs polyenes azoles and echinocandins [10] As with any other drugs antifungal drugs should be nontoxic and preferably highly water-soluble their effect should be selective and resistance should not develop easily It is usually the case that not all of the above requirements are met simultaneously It should also be noted that a number of antifungal drugs are also used or have even been primarily used for the treatment of other diseasesmdashfor example different types of cancer Examples are the fluoropyrimidine drugs that are primarily used for the treatment of colorectal cancer

223 Fluoropyrimidines

Among fluoropyrimidines flucytosine is probably the most important antifungal compound Its mode of action is based on interference with pyrimidine metabolism and the synthesis of RNADNA and proteins Flucytosine is indicated for the treatment of infections caused by strains of Candida and Cryptococcus neoformans and some filamentous fungi [13] Some in vitro studies showed emerging resistance to flucytosine Papon et al [14] described the molecular mechanisms of flucytosine resistance Due to increased flucytosine resistance it has been suggested that flucy-tosine should always be used in combination with other antifungal agents as the standard of care for the treatment of fungal infections

224 azoles

Azoles are cyclic organic molecules that can be divided into two groups according to the number of nitrogen atoms in the azole ring Imidazoles contain two nitrogen atoms and triazoles contain three nitrogen atoms Azole drugs intervene with ergos-terol biosynthesis and their effect on fungal infections is thus selective Triazoles continue to be a critical component of therapy for most forms of invasive mycoses as well as the key to the successful management of patients at all levels of invasive mycoses [15] Of the azole compounds fluconazole itraconazole variconazole and posaconazole are the most important [16] As with other antimicrobial compounds their widespread use has resulted in increasing resistance of fungal pathogens and even worse in cross-resistance to commonly used triazoles and to a new generation of triazoles [10] In addition the relative toxicity of azoles complicates their use

Fungi are an important component of nosocomial bloodstream infections that kill 40 of the infected patients Since the azole compound ketoconazole (KTC) inhibits lanosterol 14-methylase blocking sterol synthesis in fungi and mammals it has been used against infections by Candida and molds However it is toxic causing hepatitis Studies were carried out by Zhang et al [17] to find compounds that synergize the effect of a low dose of KTC (ie 001μgmL) which produces about 20 of maximal antifungal activity Screening of 20000 microbial extracts resulted in 12 compounds with broad spectra of activity Of these seven showed only minor cytotoxicity against human hepatoma cells The most efficient was beauvericin (BEA) In mice infected with Candida parapsilosis the combination of BEA at 05 mgkg and


KTC at 05 mgkg prolonged survival and reduced fungal counts in their kidneys lungs and brains Such effects were not observed even at a very high dose of KTC alone (50 mgkg) Candida parapsilosis used in this study is the second most commonly isolated fungus in clinical laboratories following C albicans The antifungal effect of the combination was much better than for one of these alone in vitro or in an immuno-compromised mouse model BEA in the combination broadened the KTC spectrum on drug-resistant strains and reduced its side effects Although BEA has little antifungal activity the combination was fungicidal compared to the fungistatic activity of KTC The combination showed no negative effect on human liver HerpG2 cells

225 echinocandins

Echinocandins are antifungals introduced into practice within the last 15 years They were the first anifungals found to inhibit cell wall biosynthesis This group of com-pounds includes natural semisynthetic and synthetic antibiotics derived from lipopep-tides They are cyclic lipohexapeptides synthesized on nonribosomal peptide synthase complexes by different ascomycotic fungi Although weakly soluble and relatively narrow spectrum in activity echinocandins have been used against Candida and Aspergillus infections due to their unique mode of action Their mechanism of action is noncompetitive inhibition of the beta-13-glucan synthase complex targeting the cell wall It is worth noting that human cells do not contain 13-β-glucan thus avoiding direct human cell toxicity The first echinocandin-type antimycotic echinocandin B was discovered in the 1970s It was discovered independently at the Ciba-Geigy Sandoz and Eli Lilly companies from Asperillus nidulans var echinolatus A nidulans var roseus and Aspergillus rugulosus This was followed by the isolation of more than 20 natural echinocandins including aculeacins mulundocandins pneumocandins sporiofungins catechol-sulfate echinocandins and cryptocandin Of the echinocandins the semisynthetic cyclic lipohexapeptides caspofungin micafungin and anidulafungin are currently approved for use Since they show good fungicidal (vs Candida spp) or fungistatic (vs Aspergillus spp) activity against the most important human patho-genic fungi including azole-resistant strains they are important additions to the list of antifungal drugs [18] Natural echinocandins are produced by several fungal speciesmdashfor example Aspergillus spp [19] Echinocandin antifungals were compared by Eschenauer et al [20] Of the clinically used echinocandins the authors compared the pharmacokinetic parameters of caspofungin micafungin and anidulafungin in adults and in pediatrics What are still needed are new derivatives for oral administration or with better activity against Pneumocystis carnii a fungus of great importance Other desirable targets are C neoformans and Histoplasma capsulatum

226 Other peptides

With the increase in resistance to commercial antibiotics antimicrobial peptides are being considered for medical use [21] They contain 15 to nearly 50 amino acids are generally positively charged are synthesized by the ribosome and are modified post-translationally More than 2000 are known some of which are toxic


A lipopeptide-producing Bacillus amyloliquefaciens strain inhibits clinical isolates of C albicans by producing a cyclic lipopeptide containing a hexapeptide and a 3-hydroxy fatty acid with 15 carbon atoms [22] The purified lipopeptide kills drug-resistant C albicans and also inhibits other yeasts Candida albicans is the most virulent agent infecting immunocompromised patients with HIV infections Most of the available drugs against C albicans have toxicity problems Previous cyclic lipo-peptides included surfactin iturin and fengymycin Of the yeasts inhibited by the new compound Candida tropicalis is also a pathogen

Novexatin of Novabiotics (Aberdeen UK) is a cyclic and highly cationic (arginine-rich) peptide targeting fungal infections of toenails It is in Phase II clinical trials Another group under study are the peptoids which have a natural amino acid backbone with synthetic side-chain residues conferring protease resistance and increased hydrophobicity thus enhancing membrane permeability

227 polyenes

Polyenes are broad-spectrum antifungal antibiotics produced by soil actinomycetes especially the bacterial genus Streptomyces They are active against fungi parasites enveloped viruses and prion diseases This large family of polyketides includes nystatin amphotericins candicidin pimaricin and rimocidin Polyene antibiotics are formed as a macrolide ring with polyunsaturations closed by an ester or lactone [12] They act by interacting with ergosterol to form transmembrane channels that cause leakage of cellular K+ and Mg2+ leading to fungal death

Nystatin was isolated from the fermentation broth of Streptomyces noursei and is still used as a topical antifungal agent Amphotericin B and its analogs are produced by Streptomyces nodosus After the discovery of amphotericin B roughly 90 poly-enes were discovered but most of them were not used in clinical practice due to their poor solubility stability oral bioavailability andor toxicity [16] The effect of amphotericin B is based on the complex formation between the antifungal molecule and the ergosterol-containing membrane that results in altered permeability and leakage of important cell components thus killing the cell Nystatin binds to ergos-terol in the fungal membrane producing membrane permeability changes leading to release of K+ sugars and metabolites Disruption of the membrane is apparently rersponsible for death of the fungal cells however the modes of action of ampho-tericin B and nystatin are apparently different [12] As compared with the above-mentioned polyene antibiotics that form pores in the membrane the relatively recently described polyene antibiotic natamycin inhibits growth of yeasts and molds via inhibition of amino acid and glucose transport across the plasma membrane This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins [23]

Although polyene macrolide antibiotics including nystatin and amphotericin B possess fungicidal activity they show toxicity and poor distribution in tissues Thus their clinical applications are relatively limited With the aim of generating new less toxic nystatin derivatives Brautaset et al [24] using genetic engineering designed purified and tested four new nystatin derivatives with promising therapeutic effects

Natural Products aNalysis


edited by











2014008371


10 9 8 7 6 5 4 3 2 1



vii

Preface ix

Contributors xi









Contents

viii Contents









Index 601




Preface

ix

xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes





edited by











2014008371


10 9 8 7 6 5 4 3 2 1



vii

Preface ix

Contributors xi









Contents

viii Contents









Index 601




Preface

ix

xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes












2014008371


10 9 8 7 6 5 4 3 2 1



vii

Preface ix

Contributors xi









Contents

viii Contents









Index 601




Preface

ix

xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes






vii

Preface ix

Contributors xi









Contents

viii Contents









Index 601




Preface

ix

xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes




viii Contents









Index 601




Preface

ix

xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes







Preface

ix

xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes




xi














Contributors

xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes




xii Contributors


















Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes




Contributors xiii




















1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes





1

Chapter 1


11 BOOK MOtIVatION









0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes







0

500

1000

1500

2000

2500

2007 2008 2009 2010 2011 2012


discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes




discoVery pHases 3




13 DISCOVerY phaSeS



















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes





















refereNces 7




reFereNCeS







9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes









9

Chapter 2

21 INtrODUCtION







arNolD l DEMaIN




221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes







221 Fungal Diseases
















224 azoles





225 echinocandins


226 Other peptides





227 polyenes

















224 azoles





225 echinocandins


226 Other peptides





227 polyenes






225 echinocandins


226 Other peptides





227 polyenes







227 polyenes




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