ideas in chemistry and molecular sciences€¦ · tomorrow’s chemistry today. concepts in...

Ideas in Chemistry and Molecular Sciences

Where Chemistry Meets Life

Edited byBruno Pignataro

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Ideas in Chemistry and Molecular

Sciences


Related Titles

Pignataro, Bruno (ed.)

Ideas in Chemistry andMolecular SciencesAdvances in Synthetic Chemistry

2010

ISBN: 978-3-527-32539-9


Ideas in Chemistry andMolecular SciencesAdvances in Nanotechnology, Materialsand Devices

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ISBN: 978-3-527-32543-6

Carta, Giorgio/Jungbauer, Alois

Protein ChromatographyProcess Development and Scale-Up

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Tomorrow’s Chemistry TodayConcepts in Nanoscience, OrganicMaterials and Environmental Chemistry2nd edition

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Chemical BiologyLearning through Case Studies

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Redox Signaling and Regulationin Biology and Medicine

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ISBN: 978-3-527-31925-1

Gjerde, Douglas T./Hoang, Lee/Hornby, David

RNA Purification and AnalysisSample Preparation, Extraction,Chromatography

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Ideas in Chemistry and Molecular Sciences

Where Chemistry Meets Life


The Editor

Prof. Bruno PignataroUniversity of PalermoDepartment of Physical ChemistryViale delle Scienze90128 PalermoItaly

Cover

We would like to thank Dr. AdrianaPietropaolo (ETH Zürich) for providing uswith the graphic material used in the coverillustration.

All books published by Wiley-VCH arecarefully produced. Nevertheless, authors,editors, and publisher do not warrant theinformation contained in these books,including this book, to be free of errors.Readers are advised to keep in mind thatstatements, data, illustrations, proceduraldetails or other items may inadvertently beinaccurate.

Library of Congress Card No.: applied for

British Library Cataloguing-in-PublicationDataA catalogue record for this book is availablefrom the British Library.

Bibliographic information published bythe Deutsche NationalbibliothekThe Deutsche Nationalbibliotheklists this publication in the DeutscheNationalbibliografie; detailed bibliographicdata are available on the Internet at.

2010 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

All rights reserved (including those oftranslation into other languages). No partof this book may be reproduced in anyform – by photoprinting, microfilm, or anyother means – nor transmitted or translatedinto a machine language without writtenpermission from the publishers. Registerednames, trademarks, etc. used in this book,even when not specifically marked as such,are not to be considered unprotected by law.

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Printed in the Federal Republic of GermanyPrinted on acid-free paper

ISBN: 978-3-527-32541-2

Set ISBN: 978-3-527-32875-8

V

Contents

Preface XIIIList of Contributors XIX

Part I Biochemical Studies 1

1 The Role of Copper Ion and the Ubiquitin System in NeurodegenerativeDisorders 3Fabio Arnesano

1.1 Introduction 31.2 Metal Ions in the Brain 41.3 Brain Copper Homeostasis 51.4 Brain Copper and Neurodegenerative Disorders 81.5 The Role of Ubiquitin in Protein Degradation 91.6 Failure of the Ubiquitin System in Neurodegenerative Disorders 131.7 Interaction of Ubiquitin with Metal Ions 151.7.1 Thermal Stability of Ubiquitin 151.7.2 Spectroscopic Characterization of CuII Binding 151.7.3 Possible Implications for the Polyubiquitination Process 171.7.4 CuII-Induced Self-Oligomerization of Ub 171.7.5 Cooperativity between CuII-Binding and Solvent Polarity 181.7.6 Comparison with Other Metal Ions 191.8 Biological Implications 211.8.1 The Redox State of Cellular Copper 211.8.2 Ubiquitin and Phospholipids 221.9 Conclusions and Perspectives 23

Acknowledgments 24References 24

2 The Bioinorganic and Organometallic Chemistry of Copper(III) 31Xavi Ribas and Alicia Casitas

2.1 Introduction 312.2 Bioinorganic Implications of Copper(III) 33

Ideas in Chemistry and Molecular Sciences: Where Chemistry Meets Life. Edited by Bruno PignataroCopyright 2010 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32541-2

VI Contents

2.2.1 Dinuclear Type-3 Copper Enzymes 332.2.2 Particulate Methano Monooxygenase (pMMO) 362.2.3 Mononuclear Monooxygenating Copper-based Enzymes 382.2.4 Trinuclear Copper Models for Laccase 402.3 Organometallic CuIII Species in Organic Transformations 412.3.1 C–C Bond Formation in Organocuprate(I) Catalysis 422.3.1.1 Conjugate Addition to α-Enones 422.3.1.2 Acetylene Carbocupration 432.3.1.3 SN2 and SN2′ Alkylations 432.3.2 Aryl–Heteroatom Bond Formation in Cu-mediated Cross-coupling

Processes 442.3.3 Aromatic and Aliphatic C–H Bond Organometallic

Functionalizations 452.3.3.1 Catalytic Systems 452.3.3.2 Stoichiometric Systems 472.4 Miscellany: Cuprate Superconducting Materials 512.5 Overview and Future Targets 51

References 52

3 Chemical Protein Modification 59Gonçalo J. L. Bernardes, Justin M. Chalker, and Benjamin G. Davis

3.1 Introducing Diversity by Posttranslational Modification 593.2 Chemistry: A Route to Modified Proteins 603.3 Challenges in Chemical Protein Modification 613.4 Traditional Methods for Protein Modification 613.4.1 Lysine Modification 623.4.1.1 Activated Esters 623.4.1.2 Isocyanates and Isothiocyanates 623.4.1.3 Reductive Alkylation 623.4.1.4 IME Reagents 633.4.2 Glutamic and Aspartic Acid Modification 643.4.3 Cysteine 643.4.3.1 Alkylation 653.4.3.2 Disulfides 663.4.3.3 Desulfurization at Cysteine 673.5 Recent Innovations in Site-Selective Protein Modification 703.5.1 Dehydroalanine: A Useful Chemical Handle for Protein

Conjugation 713.5.2 Metal-Mediated Protein Modification 713.5.2.1 Modification at Natural Residues 723.5.2.2 Iridium-Catalyzed Reductive Alkylation of Lysine 743.5.2.3 Modification of Unnatural Residues 743.5.2.4 Olefin Metathesis at S-Allyl Cysteine 773.5.3 Metal-Free Methods for Modifying Unnatural Amino Acids 783.5.3.1 Oxime Ligation at Aldehydes and Ketones 78

Contents VII

3.5.3.2 Azide and Alkyne Modification 793.5.3.3 Selective Modification of Tetrazole-Containing Proteins 803.5.3.4 Tetrazine Ligation 813.5.4 Dual Modification 813.6 Conclusion and Outlook 81

References 82

Part II Drug Delivery 93

4 Vitamin B12: A Potential Targeting Molecule for Therapeutic DrugDelivery 95Pilar Ruiz-Sánchez

4.1 Introduction 954.2 Transport Mechanism 964.3 Metabolism of B12 974.3.1 Adenosylcobalamin-Dependent Reactions 974.3.2 Methylcobalamin-Dependent Reactions 994.4 Vitamin B12 Derivatives 994.4.1 Structure 994.4.2 b-, d-, e-Cobalamin Derivatives 994.4.3 Modifications on the Ribose Moiety 1014.4.4 β-Axial Position 1024.4.4.1 Cobalamin Alkylation 1024.4.4.2 Heterodinuclear Concept 1034.5 Outlook 108


5 Strategies for Microsphere-Mediated Cellular Delivery 117Rosario M. Sanchez-Martin, Lois M. Alexander, Juan ManuelCardenas-Maestre, and Mark Bradley

5.1 Introduction 1175.1.1 Cellular Delivery 1175.1.2 Delivery Devices 1175.1.2.1 Liposomes 1175.1.2.2 Cell-penetrating Peptides 1185.1.2.3 Dendrimers 1185.1.2.4 Nanomaterials 1185.2 Microspheres 1195.2.1 Biodegradable Microspheres 1195.2.1.1 Preparation 1195.2.2 Biostable Microspheres 1205.2.2.1 Applications of Biostable Microspheres 1205.2.2.2 Preparation 1205.2.3 Microspheres and Solid-phase Chemistry 121

VIII Contents

5.2.3.1 Preparation of Microspheres 1215.2.3.2 Fmoc Chemistry on Microspheres 1215.2.3.3 Dual Functionality of Microspheres 1225.2.3.4 Coupling Agents 1245.2.4 Noncleavable Link to Microspheres 1265.2.4.1 Microsphere-based Intracellular Sensing 1265.2.4.2 siRNA Delivery 1275.2.5 Cleavable Linkers 1305.2.5.1 Ester Bonds 1305.2.5.2 Disulfide Bonds 1325.2.6 Bioconjugation 1335.2.6.1 Streptavidin–Biotin 1345.3 Future Perspectives 135


Part III Research in Therapeutics 141

6 Fundamental Processes in Radiation Damage to DNA: How Low-EnergyElectrons Damage Biomolecules 143Ilko Bald

6.1 Radiation Damage and the Role of Low-Energy Electrons 1436.1.1 How Chemical Bonds are Broken by Low-energy Electrons 1456.1.2 DEA Studies of Gas-Phase DNA Building Blocks: The

Nucleobases 1476.2 DEA Studies on Model Compounds for the DNA Backbone 1486.2.1 Electron Attachment to d-Ribose 1486.2.2 Cross-Ring Cleavage of d-Ribose Proceeds with Selective Charge

Retention 1506.2.3 The Nature of the Transient Negative d-Ribose Anions 1546.2.4 One Step Further: Tetraacetyl-d-Ribose 1556.2.5 The Use of Laser-Induced Acoustic Desorption (LIAD) to Study DEA to

Larger Biomolecules 1596.2.6 Sugar–Phosphate Cleavage Induced by 0 eV Electrons: DEA to

d-Ribose-5′-Phosphate 1596.3 Outlook and Future Prospects 161


7 Structure-Based Design on the Way to New Anti-infectives 167Anna Katharina Herta Hirsch

7.1 Introduction 1677.2 Isoprenoids and the Nonmevalonate Pathway 1697.2.1 4-Diphosphocytidyl-2C-methyl-d-erythritol Kinase (IspE) 1707.2.2 Structure of IspE 170

Contents IX

7.2.3 Active Site of IspE 1707.3 Targeting the CDP-Binding Pocket of IspE 1747.3.1 Design 1747.3.1.1 Possible Ribose Analogues 1757.3.1.2 Design of the Vector 1767.3.2 Optimization of the Ribose Analogue 1767.3.3 Importance of the Vector 1787.3.4 Optimization of the Filling of the Small, Hydrophobic Pocket 1797.3.4.1 The ‘‘55% Rule’’ 1797.3.4.2 Evaluation of Inhibitors Featuring Different Sulfone

Substituents 1807.3.5 Summary of the First-Generation Inhibitors 1827.4 X-ray Cocrystal Structure Analysis 1827.4.1 Design of Water-Soluble Inhibitors 1827.4.2 Enzyme Assays of Inhibitors Designed to be Water Soluble 1837.4.3 Structural Analysis 1847.4.4 Lessons Learnt from the Cocrystal Structure 1857.5 Conclusions and Outlook 1857.5.1 Conclusions 1857.5.2 Outlook 186

Acknowledgments 186List of Abbreviations 186References 187

8 Drug–Membrane Interactions: Molecular Mechanisms UnderlyingTherapeutic and Toxic Effects of Drugs 191Marlene Lúcio, José L. F. C. Lima, and Salette Reis

8.1 Biological Membranes 1918.1.1 Role of Membranes in Life Maintenance 1918.1.2 Structure and Composition of Membranes 1918.1.3 Dynamic Molecular Organization of Membranes 1938.2 Drug–Membrane Interactions 1958.2.1 Possible Effects of Drugs on Membranes 1958.2.2 Clinical Relevance of the Drug–Membrane Interaction Studies 1958.2.2.1 Contribution for Drug Development 1958.2.2.2 Understanding Therapeutic and Toxic Effect of Drugs 1978.2.2.3 Understanding Mechanisms of Multidrug Resistance 1988.2.2.4 Controlling Enzymatic Inhibition 1988.3 Analysis and Quantification of Drug–Membrane Interactions 1998.3.1 Membrane Model Systems 1998.3.2 Experimental Techniques 2008.4 Drug–Membrane Interactions Applied to the Study of Nonsteroidal

Anti-inflammatory Drugs (NSAIDs) 2018.4.1 Drug Fundamental Physical–Chemical Studies 2028.4.2 Membrane Structural and Dynamic Studies 203

X Contents

8.4.3 Results 2058.5 Conclusions and Future Research Directions 206


9 Targeting Disease with Small Molecule Inhibitors of Protein–ProteinInteractions 215Fedor Forafonov, Elena Miranda, Ida Karin Nordgren, and Ali Tavassoli

9.1 Introduction 2159.2 High-Throughput Screening of Chemical Libraries 2169.3 High-Throughput Screening of Biosynthesized Libraries 2209.3.1 Cyclic Peptide Inhibitors of AICAR Transformylase Activity 2229.3.2 Cyclic Peptide Inhibitors of HIV Budding 2249.4 Future Direction 2269.4.1 Small Molecule Inhibitors of Tumor Hypoxia Response

Network 2269.4.2 Targeting Protein–Protein Interactions in Asthma 2289.4.3 Targeting the Protein Interaction Networks of Influenza Virus 230


10 Cracking the Glycocode: Recent Developments in Glycomics 239Lars Hillringhaus and Jürgen Seibel

10.1 State of the Art 23910.1.1 Introduction 23910.1.2 Carbohydrate-Based Drugs 23910.1.3 Carbohydrate Synthesis 24010.1.3.1 Chemical Synthesis 24110.1.3.2 Enzymatic Synthesis 24310.1.3.3 Glycoprotein Synthesis 24410.1.4 Glycomics 24510.1.4.1 Mass Spectrometry 24510.1.4.2 Microarrays 24610.1.4.3 Cell, Tissue, and Metabolic Labeling 24610.1.4.4 Bioinformatics 24710.2 Some New Insights in Glycomics 24710.2.1 Microwave-Assisted Glycosylation for the Synthesis of

Glycopeptides 24710.2.2 Highly Efficient Chemoenzymatic Synthesis of Novel Branched

Thiooligosaccharides by Substrate Direction with Glucansucrases 25110.2.3 Identification of New Acceptor Specificities of Glycosyltransferase R

with the Aid of Substrate Microarrays 25710.3 Future Perspectives 259


Contents XI

Part IV Enzyme Chemistry 265

11 O2 Reactivity at Model Copper Systems: Mimicking TyrosinaseActivity 267Anna Company

11.1 General Introduction: O2 Activation and Model Systems 26711.2 Copper Proteins Involved in O2 Activation 26811.2.1 Hemocyanin 26911.2.2 Tyrosinase 27011.3 O2 Binding and Activation at Biomimetic Cu Complexes 27211.3.1 Copper–Dioxygen Adducts 27211.3.2 Ligand Architecture: Influence on Reactivity toward O2 27511.3.3 Hydroxylation of Aromatic Rings: Mimicking Tyrosinase Activity 27811.3.3.1 Intramolecular Aromatic Hydroxylation 27811.3.3.2 Intermolecular ortho-Hydroxylation of Phenolic Compounds 28011.4 Concluding Remarks 285

References 286

Part V Structure–Property Relationship and Biosensing 291

12 Chirality in Biochemistry: A Computational Approach for InvestigatingBiomolecule Conformations 293Adriana Pietropaolo

12.1 Introduction 29312.1.1 Molecular Chirality in Living Systems 29312.1.2 Protein Secondary Structures 29512.1.3 Protein Secondary Structure Assignment 29612.1.4 Intrinsic Chirality and Protein Secondary Structures 29712.2 Computational Techniques for Studying Protein Dynamics 29712.3 Employing Chirality to Analyze Protein Motions 29812.3.1 The Chirality Index 29812.3.2 Using Chirality to Understand Protein Structure 30012.3.3 Chirality Index as a Tool for Monitoring Protein Dynamics 30212.3.4 Chirality and Circular Dichroism 30512.4 Perspectives 308


13 Collisional Mechanism–Based E-DNA Sensors: A General Platformfor Label-Free Electrochemical Detection of Hybridization andDNA Binding Proteins 313Francesco Ricci

13.1 Introduction 31313.2 E-DNA Signaling Mechanism 31513.3 E-DNA Sensor for DNA Binding Proteins Detection 319

XII Contents

13.4 Conclusions and Future Perspectives 32113.5 Acknowledgments 324

References 324

Index 327

XIII

Preface

The idea of publishing books based on contributions given by emerging youngchemists arose during the preparation of the first EuCheMs (European Associationfor Chemical and Molecular Sciences) Conference in Budapest. In this conferenceI cochaired the competition for the first European Young Chemist Award aimedat showcasing and recognizing the excellent research being carried out by youngscientists working in the field of chemical sciences. I then proposed to collect in abook the best contributions from researchers competing for the Award.

This was further encouraged by EuCheMs, SCI (Italian Chemical Society),RSC (Royal Society of Chemistry), GDCh (Gesellschaft Deutscher Chemiker), andWiley-VCH and brought out in the book ‘‘Tomorrow’s Chemistry Today’’ edited bymyself and published by Wiley-VCH.

The motivation gained by the organization from the above initiatives was, tome, the trampoline for co-organizing the second edition of the award during thesecond EuCheMs Conference in Torino. Under the patronage of EuCheMs, SCI,RSC, GDCh, the Consiglio Nazionale dei Chimici (CNC), and the European YoungChemists Network (EYCN), the European Young Chemist Award 2008 was againfunded by the Italian Chemical Society.

In Torino, once again, I personally learned a lot and received important inputsfrom the participants about how this event can serve as a source of new ideas andinnovations for the research work of many scientists. This is also related to the factthat the areas of interest for the applicants cover many of the frontier issues ofchemistry and molecular sciences (see also Chem. Eur. J. 2008, 14, 11252–11256).But, more importantly, I was left with the increasing feeling that our future needsof new concepts and new technologies should be largely in the hands of the newscientific generation of chemists.

In Torino, we received about 90 applications from scientists (22 to 35 yearsold) from 30 different countries all around the world (Chem. Eur. J. 2008, 14,11252–11256).

Most of the applicants were from Spain, Italy, and Germany (about 15 fromeach of these countries). United Kingdom, Japan, Australia, United States, Brazil,Morocco, Vietnam, as well as Macedonia, Rumania, Slovenia, Russia, Ukraine,and most of the other European countries were also represented. In terms ofapplicants, 63% were male and about 35% were PhD students; the number of


XIV Preface

postdoctoral researchers was only a small percentage, and only a couple of themcame from industry. Among the oldest participants, mainly born between 1974 and1975, several were associate professors or researchers at Universities or ResearchInstitutes and others are lecturers, assistant professors, or research assistants.

The scientific standing of the applicants was undoubtedly very high and many ofthem made important contributions to the various symposia of the 2nd EuCheMsCongress. A few figures help to substantiate this point. The, let me say, ‘‘h index’’of the competitors was 20, in the sense that more than 20 applicants coauthoredmore than 20 publications. Some patents were also presented. Five participantshad more than 35 publications, and, h indexes, average number of citationsper publication, and number of citations, were as high as 16, 35.6, and 549,respectively. Several of the papers achieved further recognition as they were quotedin the reference lists of the young chemists who where featured on the covers oftop journals. The publication lists of most applicants proudly noted the appearanceof their work in the leading general chemistry journals such as Science, Nature,Angewandte Chemie, Journal of the American Chemical Society, or the best nichejournals of organic, inorganic, organometallic, physical, analytical, environmental,and medicinal chemistry.

All of this supported the idea of publishing a second book with the contributionsof these talented chemists.

However, in order to have more homogeneous publications and in connectionto the great number of interesting papers presented during the competition, wedecided to publish three volumes.

This volume represents indeed one of the three edited by inviting a selectionof young researchers who participated in the European Young Chemist Award2008. The other two volumes concern the two different areas of synthetic chemistryand nanotechnology/materials-science and are, respectively, entitled ‘‘Ideas inChemistry and Molecular Science: Advances in Synthetic Chemistry’’ and ‘‘Ideasin Chemistry and Molecular Sciences: Advances in Nanotechnology, Materials, andDevices’’.

It is important to mention that the contents of the books are a result of the workcarried out in several topmost laboratories around the world both by researchers whoalready lead their own group and by researchers who worked under a supervisor.I would like to take this occasion to acknowledge all the supervisors of the invitedyoung researchers for their implicit or explicit support to this initiative that I hopecould also serve to highlight the important results of their research groups.

The prospect of excellence of the authors was evident from the very effusiverecommendation letters sent by top scientists supporting the applicants for theAward.

A flavor of these letters is given by the extracts from some of the sentences below:‘‘The candidate creativity in polymer design for therapeutic impact is extremely

impressive. I believe that he is an excellent example of an outstanding youngEuropean chemist’’; ‘‘Is an energetic, enthusiastic, articulate, bright researcher’’;‘‘Talented and enthusiastic scientist who has a lot of determination and persistence.I was impressed by the strong preparation, enthusiasm and motivation. First rate

Preface XV

researcher’’; ‘‘. . .outstanding, very broad and far reaching research achievements’’;‘‘The personality and the scientific research achievements will certainly paveto the candidate the way as a future leader of an independent and creativeresearch group. Due to his original and uncommon approach together with thehigh quality of results, he will become well known in the field of medicinalchemistry. All this exciting work relied on his ability as a chemist and on hisbright intellect, which allows him to move across disciplines seamlessly’’; ‘‘Thecandidate is a very articulate, knowledgeable, driven, clear minded, and extremelypersonable young scientist’’; ‘‘Application in this work has been outstanding,strong, powerful and very dedicated. The candidate will be in a very good positionto make tremendous impact in the area of therapeutics and to become a leaderin the area of organic chemistry in the next generation’’; ‘‘As one of the mosttalented graduate students to have emerged for our group he has the vision,skill and drive to make a real difference to the national research community’’;‘‘. . .outstanding young scientist and a charming person’’; ‘‘. . .has an astonishinglevel of productivity’’; ‘‘. . .work of very high caliber’’; ‘‘Such deep and insightfulinvestigations are refreshing and invigorating in my view especially in times thattend to favor simple, phenomenological results’’; ‘‘The candidate has been theintellectual driver and forged the necessary collaborations to address this complexproblem. He deft integration of the various experimental and theoretic disciplinesneeded for the work is impressive’’; ‘‘The candidate performed outstanding researchprojects’’; ‘‘He should be a compelling and deserving candidate of the EuropeanYoung Chemist award’’; ‘‘The candidate is for sure one of the most outstandingand promising young researchers that I know in the field of molecular science.Despite being young, he has a lot of experience and high motivation. He isexcellent in the lab and extremely smart person’’; ‘‘The candidate has performedwork of top quality and originality and of great breadth’’; ‘‘. . .a special individualwho ranks among the very best’’; ‘‘Exceptional in many respect. The candidatecommands superior experimental and intellectual skills and has shown greatchemical structure intuition in the project’’. ‘‘. . .outstanding scientific debater. Thepersonality is impeccable’’. ‘‘Extremely gifted young researcher who has alreadydeveloped an outstanding track record of achievement while working in one ofthe world’s premier laboratories in the USA’’; ‘‘Has the skills, expertise andleadership potential to discover new compounds that will revolutionize the therapyof some difficult diseases. The work is well-aligned to the University’s strategyof supporting cross disciplinary research of the highest quality’’; ‘‘. . .unusuallybroad dimension of experimental skills’’; ‘‘This is a rare combination of expertise’’;‘‘enables to undertake a diverse scope of key problems. In my opinion these arethe type of individuals who will drive the field forward’’; ‘‘. . .has an engaging,outgoing, attractive personality to which others instinctively respond’’; ‘‘Brilliantyoung chemist with a great potential’’.

The chapters written by the various contributors cover several areas of life scienceand range from more or less typical biochemical studies, to research relevant in thetherapeutic field, to the enzyme or drug activity, to drug delivery, to computationalstructure–activity relation or sensors.

XVI Preface

In the area of biochemical studies, the book collects a contribution in the fieldof inorganic chemistry of the brain with the chapter on the role of metal ions(Cu(II) in particular) and the ubiquitin–proteasome system on neurodegenerativedisorder (Arnesano). In another chapter, the strategies for chemical modificationof proteins as well as selective installation of biochemical probes and tethering oftherapeutic cargo to proteins are highlighted (Bernardes et al.). Another chapter(Ribas et al.) is centered on Cu(III) ion and its bioinorganic and organometallicchemistry. In particular, this contribution turns out to be relevant for understandingcopper-dependent metalloenzymes.

In the drug delivery domain, a chapter is dedicated to vitamin B12 as potentialtargeting molecule for therapeutic drug delivery (Ruiz Sánchez). This potentialityis vividly illustrated by the author with the following definition: vitamin B12 is anattractive ‘‘Trojan horse’’ for therapeutic drug delivery.

A second contribution starts from the consideration that the cellular membraneposes a formidable barrier to the intracellular flux of materials circulating extra-cellularly due to its selective permeability based predominantly on ionic charge,hydrophobicity, and size. As such, many drugs, nucleic acids, proteins, and otherinvestigative constructs are not able to translocate the cellular membrane aloneand often require a delivery vehicle for function and/or activity. After consid-ering various vehicles this contribution focuses on a highly competitive cellulardelivery technology based on polymeric microspheres-mediated cellular delivery(Sanchez-Martin). In particular, the author presents different strategies that canbe applied for the attachment of biomolecules to the microspheres and their ap-plications as a delivery system. Monodispersed populations of robust cross-linkedmicrospheres of defined sizes (from 200 nm to 2 µm) have been synthesized andstandard solid phase multistep protocols have been applied to them.

Several contributions are primarily dedicated to research that has relevance tothe therapeutic field.

The chapter (Lucio et al.) on molecular mechanisms, underlying therapeutic andtoxic effects of drugs, highlights the importance of membrane composition and thedynamic molecular organization of membranes in drug–membrane interaction.Also, the work aims to exemplify an application of drug–membrane interactionstudies in the evaluation of the nonsteroidal anti-inflammatory drug effect onmembranes. These studies may prove valuable in the design of novel drugformulations with increasing efficacy and reduced side effects.

A second contribution in the area deals with targeting diseases with smallmolecule inhibitors of protein–protein interactions (Tavassoli et al.). The chapterstarts from the consideration that there are several thousand protein–proteininteractions that control the majority of biological processes. There is therefore greatpotential for small molecule therapeutics that affect disease through modulation ofprotein–protein interactions. In this chapter, there is then a discussion on recentapproaches taken toward controlling protein interactions with small molecules,from logical design using structural data to high-throughput screening of chemicaland biological libraries. The authors go on to outline their own efforts in this

Preface XVII

field, and end with a detailed discussion of some of the important protein–proteininteractions currently being targeted.

Another chapter (Hirsh) is dedicated to the design and synthesis of inhibitors ofthe kinase IspE as potential antimalarians opening a number of research avenuessummarized in the chapter itself, while another contribution (Hillringhaus) dealswith recent developments in glicomics – a technology that can be anticipated thatit will strongly influence tomorrow’s therapy in the areas of various diseases.

A last contribution (Bald) in this area deals with the important studies onthe electron-induced modification of biomolecules, which may have potentialimplications for the design of new chemotherapeutic and radiosensitizing drugsand for the development of more efficient protocols in cancer therapy.

Understanding how nature works in the area of enzyme chemistry is the goalof a chapter (Company) dedicated to the tyrosinase reactivity. In this chapter, inparticular, it is shown how the model chemistry approach has allowed to reproduceand to better understand the mechanisms by which O2 is activated in the dinuclearcopper protein just called tyrosinase.

The studies summarized in another chapter (Ricci) refer to electrochemicalbiosensors, termed E-DNA sensors, that are based on the target binding–inducedfolding of electrode-bound DNA probes and gives contribution on E-DNA signalingmechanisms. In addition, studies on E-DNA sensors for DNA binding proteindetection are also reported. The E-DNA sensing platform has then demonstrated tobe not only a promising and appealing approach for the sequence-specific detectionof DNA and RNA but also to be flexible enough to be adaptable for the detection ofDNA–protein interaction.

Taking into account that chirality selection is a key issue in many importantbiochemical phenomena such as protein folding and enzyme recognition, anotherchapter discusses the role of chirality in the chemistry of the life sciences. In particu-lar, a novel methodology for the study of local chirality is presented, which providesone with a deeper understanding of the connection between secondary structureand protein flexibility. This computational study for investigating biomolecule con-formation reported in the chapter (Pietropaolo) suggests that chirality is a centralorganizing principle in life that confers order at every length scale up to the fullcell.

Probably, everyone who reads this book will have their own opinion on what isrelevant for the future of life chemistry, and in this respect I would like to notifythat, due to its peculiar genesis, the book reflects the opinions of a select group ofyoung chemists and therefore does not pretend to cover the whole area.

The main aim is just to offer a variety of individual views that will provokethought possibly giving an attractive insight into the minds and research areas forthe next generation of chemical and molecular sciences, especially those relatedwith life sciences and technologies.

Starting from this point, I hope that the many ideas that can be grasped sailingthrough the various contributions by the young authors of the book should bevery useful for helping to make several step forward in the field of chemistry andmolecular sciences to solve problems related to our health or more generally to our

XVIII Preface

life as well as to discover new tools in medicinal chemistry needed for the presentand the future society.

I cannot end this preface without acknowledging all the authors and the personswho helped me in the book project together with all the societies (see the bookcover) that motivated and sponsored the book. I’m personally grateful to ProfessorsGiovanni Natile, Francesco De Angelis and Luigi Campanella for their motivationand support in this activity.

Palermo, October 2009 Bruno Pignataro

XIX

List of Contributors

Lois M. AlexanderUniversity of EdinburghChemical Biology SectionSchool of ChemistryJoseph Black BuildingWest Mains RoadEdinburgh EH9 3JJScotland

Fabio ArnesanoUniversity of Bari ‘‘A. Moro’’Department of Farmaco-ChimicoVia E. Orabona 470125 BariItaly

Ilko BaldFreie Universität BerlinInstitut für Chemie undBiochemie–Physikalische undTheoretische ChemieTakustraße 3D-14195 BerlinGermany

and

Interdisciplinary NanoscienceCenter (iNANO)Aarhus UniversityNy Munkegade8000 Aarhus CDenmark

Gonçalo J. L. BernardesUniversity of OxfordDepartment of ChemistryChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUnited Kingdom

Mark BradleyUniversity of EdinburghChemical Biology SectionSchool of ChemistryJoseph Black BuildingWest Mains RoadEdinburgh EH9 3JJScotland

Alicia CasitasUniversitat de GironaDepartment de Quı́micaGrup de Quı́mica Inorgànica iSupramolecular (QBIS)Campus Montilivi 17071Girona – CataloniaSpain

Justin M. ChalkerUniversity of OxfordDepartment of ChemistryChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUnited Kingdom


XX List of Contributors

Anna CompanyTechnische Universität BerlinInstitut für Chemie.Sekretariat C2Straße des 17.Juni 135D-10623 Berlin (Germany)

Benjamin G. DavisUniversity of OxfordDepartment of ChemistryChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUnited Kingdom

Fedor ForafonovUniversity of SouthamptonSchool of ChemistryUniversity Road, HighfieldSouthampton SO17 1BJUnited Kingdom

Lars HillringhausUniversity of OxfordDepartment of ChemistryChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUnited Kingdom

Ida Karin NordgrenUniversity of SouthamptonSchool of ChemistryUniversity Road, HighfieldSouthampton SO17 1BJUnited Kingdom

Anna Katharina Herta HirschUniversité de StrasbourgInstitut de Science et d’IngénierieSupramoléculaires8 Allée Gaspard MongeStrasbourg 67000France

José L. F. C. LimaUniversidade do PortoFaculdade de FarmáciaRequimte, Serviço deQuı́mica-Fı́sicaRua Anı́bal Cunha, 164Porto 4099-030Portugal

Marlene LúcioUniversidade do PortoFaculdade de FarmáciaRequimte, Serviço deQuı́mica-Fı́sicaRua Anı́bal Cunha, 164Porto 4099-030Portugal

Juan Manuel Cardenas-MaestreUniversity of EdinburghChemical Biology SectionSchool of ChemistryJoseph Black BuildingWest Mains RoadEdinburgh EH9 3JJScotland

Elena MirandaUniversity of SouthamptonSchool of ChemistryUniversity Road, HighfieldSouthampton SO17 1BJUnited Kingdom

List of Contributors XXI

Adriana PietropaoloUniversitá di CataniaDipartimento di ScienzeChimicheViale A. Doria 6Catania I-95125Italy

and

ETH ZúrichDepartment of Chemistry andApplied BiosciencesInstitute of ComputationalScienceUSI CampusVia Giuseppe Buffi 13CH-6900 LuganoSwitzerland

Salette ReisUniversidade do PortoFaculdade de Farmácia,Requimte, Serviço deQuı́mica-Fı́sicaRua Anı́bal Cunha, 164Porto 4099-030Portugal

Xavi RibasUniversitat de GironaDepartment de Quı́micaGrup de Quı́mica Inorgànica iSupramolecular (QBIS)Campus Montilivi 17071Girona – CataloniaSpain

Francesco RicciUniversity of Rome Tor VergataDipartimento di Scienze eTecnologie ChimicheVia della Ricerca Scientifica00133 RomeItaly

Pilar Ruiz-SánchezUniversität ZürichAnorganisch-chemisches InstitutWinterthurerstr. 1908057 ZürichSwitzerland

Rosario M. Sanchez-MartinUniversity of EdinburghChemical Biology SectionSchool of ChemistryJoseph Black BuildingWest Mains RoadEdinburgh EH9 3JJScotland

Jürgen SeibelUniversity of WürzburgInstitute of Organic ChemistryAm HublandD-97074 WürzburgGermany

Ali TavassoliUniversity of SouthamptonSchool of ChemistryUniversity Road, HighfieldSouthampton SO17 1BJUnited Kingdom

1

Part IBiochemical Studies


3

1The Role of Copper Ion and the Ubiquitin System inNeurodegenerative DisordersFabio Arnesano

1.1Introduction

Ubiquitin (Ub) plays a crucial role in intracellular protein degradation via the pro-teasome and the autophagy–lysosome pathways [1]. Failure to eliminate misfoldedproteins can lead to the formation of toxic aggregates and cell death [2]. Insolubleprotein aggregates enriched with Ub are a hallmark of most neurodegenerativedisorders including Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis andprion diseases [3, 4]. All of these disorders have been linked to metal accumulationand disturbance of redox and metal homeostasis in the brain [5–7], and metalions have been implicated in the aggregation of disease-related, amyloidogenicproteins [8–12]. The potential role of metal ions in the aggregation of Ub hasrecently been examined [13, 14]. CuII is different from ZnII, NiII, AlIII, or CdII

in that it binds to the N-terminal end of Ub, destabilizes the protein, and pro-motes its oligomerization into spherical particles. By mimicking the condition oflow dielectric constant experienced near a membrane surface, the assembly ofspherical oligomers of Ub yields a series of intermediate species leading to anextended nonfibrillar filament network. Aggregate disassembly is triggered by CuII

chelation or reduction [14]. Intermediate annular and porelike structures, stabilizedby the interaction of CuII-induced Ub oligomers with lipid bilayers, resemble toxicprotofibrillar species produced by amyloidogenic proteins, which cause membranepermeabilization and disruption of metal homeostasis [15–19]. Susceptibility toaggregation of Ub represents a potential risk factor for disease onset or progressionwhile cells attempt to tag and process toxic substrates. CuII binding and proximityto biological membranes appear to dramatically increase the aggregation propen-sity of Ub and other disease-related proteins, thus emphasizing the importanceof preserving cellular compartmentalization and metal homeostasis for the correctfunctioning of protein degradation systems. Recent findings reinforce the visionof metal ions as key factors and promising therapeutic targets in protein confor-mational disorders [20, 21]. New strategies are being developed that will help toinvestigate their functional and pathogenic interactions in vivo.


4 1 The Role of Copper Ion and the Ubiquitin System in Neurodegenerative Disorders

1.2Metal Ions in the Brain

The brain is a specialized organ that controls cognitive and motor functions. Tocarry out its functions the brain requires the highest concentrations of metal ionsin the body and the highest per-weight consumption of body oxygen [22].

Metal ions in the brain fulfill catalytic and structural roles, which includethe stabilization of biomolecules (e.g., MgII in nucleic acids, ZnII in Zn-fingertranscription factors) or dynamic processes (e.g., NaI and KI in ion channels, CaII

in neuronal cell signaling) [23].The dynamic partitioning of these metal ions is controlled by ion-specific

channels that selectively allow passage of ions in and out of cells. In the brain, theuneven distribution of NaI and KI ions across a cell membrane creates a potentialthat enables transmission of nervous pulses. CaII is also a key modulator of molecu-lar information transfer within and between cells during neurotransmission; mosteukaryotic cells either export or store CaII within membrane-enclosed vesicles tomaintain cytosolic- free CaII levels at 100–200 nM, roughly 10 000-fold less than inthe extracellular space [23].

More recently, considerable attention has been directed to the role of transitionmetal ions in the brain [22, 24]. Zn, Fe, Cu, and related d-block metals are emergingas significant players in both neurophysiology and neuropathology, particularly withregard to aging and neurodegenerative diseases [25]. Relatively high concentrationsof these d-block metals are present within the different cellular compartments,the values ranging from 100 to 1000 µM [22]. The metal concentrations in braintissue are up to 10 000-fold higher than those in common neurotransmitters andneuropeptides. Not only do these metals serve as components of various proteinsand enzymes essential for normal brain function, but, in the labile form, arealso involved in specialized brain activities; therefore, if misregulation of theirhomeostasis occurs, toxicity, mediated also by oxidative stress in the case of Cu andFe [26], could ensue. Oxidative stress has been identified in many neurodegenerativediseases, and is commonly associated with increased levels of at least one of thesetransition metal ions in specific brain regions [27].

Transporters for Cu, Zn, Fe, and Mn play an important part in the intracellulardistribution of these metals [28], such that defects in their regulation, whichcould possibly occur with aging, may create an environment that could result inprotein misfolding and aggregation, thereby accelerating degenerative conditions[2, 29, 30]. Notably, brain homeostasis of metals is intertwined with changes in onemetal leading to changes in the levels of other metals [24]. This is well establishedfor Cu and Fe, where decreased Cu bioavailability may result in altered Fe levels,and for Fe and Mn, where Fe deficiency leads to a significant increase in brain Mn.

The following discussion will be focused on basic aspects of brain Cu home-ostasis. The widespread distribution and mobility of Cu required for normal brainfunction, along with the numerous correlations between Cu misregulation and avariety of neurodegenerative diseases, have prompted interest in studying its rolesin neurophysiology and neuropathology [26, 31].

1.3 Brain Copper Homeostasis 5

1.3Brain Copper Homeostasis

Copper is the third-most abundant transition metal in the brain, after Fe and Zn,with average neuronal Cu concentrations of ∼0.1 mM. This redox-active nutrientis distributed unevenly within brain tissue, as Cu levels in the gray matter are two-to threefold higher than those in the white matter. Cu is particularly abundantin the locus coeruleus (1.3 mM), the neural region responsible for physiologicalresponses to stress and panic, as well as the substantia nigra (0.4 mM), the centerfor dopamine production in the brain [32]. The major oxidation states for Cuions in biological systems are cuprous CuI and cupric CuII; the former is morecommon in the reducing intracellular environment, and the latter is dominantin the more oxidizing extracellular environment [33]. Levels of extracellular CuII

vary from 10−25 µM in blood serum, 0.5−2.5 µM in cerebrospinal fluid (CSF),and 30 µM in the synaptic cleft. Intracellular Cu levels within neurons canreach concentrations higher than 2–3 orders of magnitude [32]. Like Zn andFe, brain Cu is partitioned into tightly bound and labile pools. Owing to itsredox activity, Cu is an essential cofactor in numerous enzymes that handle thechemistry of oxygen or its metabolites, including cytochrome c oxidase (CcO),Cu, Zn superoxide dismutase (Cu,Zn-SOD1), ceruloplasmin (CP), dopamine βmonooxygenase (DβM), peptidylglycine α-hydroxylating monooxygenase (PHM),and tyrosinase [31].

Because of its propensity to trigger aberrant redox chemistry and oxidativestress when unregulated, the brain maintains strict control over its Cu levels anddistributions. An overview of homeostatic Cu pathways in the brain is given inFigure 1.1.

Many of the fundamental concepts for neuronal Cu homeostasis are derivedfrom studies in yeast, but the brain provides a more complex system with its ownunique and largely unexplored inorganic physiology. There is little ‘‘free’’ Cu in theyeast cytoplasm, which is due to the tight regulation of metallochaperones [35, 36];however, many open questions remain concerning the homeostasis of organelleCu stores, particularly in higher organisms with specialized tissues. Some datasuggest that yeast and mammals possess pools of labile Cu in the mitochondrialmatrix [37].

Uptake of Cu by the blood–brain barrier (BBB) is considered to occur throughthe P-type ATPase ATP7A, which can pump Cu into the brain [38]. Mutations inthe related gene lead to Menkes disease, an inherited neurodegenerative disorderthat is globally characterized by brain Cu deficiency. This phenotype is mirroredby Wilson disease, which involves mutations in the ATP7B gene responsible forexcretion of excess Cu from the liver into the bile. Loss of ATP7B function leads toabnormal increase of Cu in the liver [39].

The extracellular trafficking of brain Cu differs from that in the rest of the body.CSF, the extracellular medium of the brain and central nervous system, possessesa distinct Cu homeostasis from blood plasma, which carries Cu to organs in therest of the body. Cp, a multicopper oxidase that is essential for Fe metabolism, is

6 1 The Role of Copper Ion and the Ubiquitin System in Neurodegenerative Disorders

Cu+ Cu2+

Presynaptic cell

SOD1

CCS

hCtr1

Cox17

ATP7

A

CcO

ReductaseMT3

Atox1

Postsynaptic cell

APP

?

Synapse

?

Sco1/Cox11

Metalloprotein

PrPc

Endosome

Figure 1.1 A schematic model of neuronal copper home-ostasis. Reprinted with permission from [34]. Copyright 2008American Chemical Society.

the major carrier of CuII in the plasma, but houses less than 1% of Cu in CSF [32].The primary protein or small-molecule ligands for Cu in CSF remain unidentified.Uptake of Cu into brain cells requires reduction of CuII to CuI. Steap proteinsmay fulfill this role like the yeast ferric and cupric reductases Fre1 and Fre2 [40].Following reduction, CuI ions can be transported into cells through a variety oftrafficking pathways [41, 42].

A major class of proteins involved in cellular Cu uptake is the copper transport(Ctr) protein family. Ctr1 is a representative member that is ubiquitously expressed.It resides predominantly in the plasma membrane and is essential for the survival ofmammalian embryos and for Cu import into neurons and astrocytes [43]. ElevatedCu stimulates rapid endocytosis and degradation of Ctr [44]. Ctr1 contains threetransmembrane helices, an N-terminal extracellular domain, and a C-terminalcytosolic domain. Electron crystallography revealed that Ctr1 is trimeric andpossesses the type of radial symmetry associated with the structure of certain ionchannels [45]. A region of low protein density at the center of the trimer is consistentwith the existence of a Cu permeable pore. Mutagenesis studies have establishedthat a methionine(Met)-rich motif in the N-terminal domain and a Met-rich motifat the extracellular end of the second transmembrane helix of Ctr1 play a pivotal

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