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Sequence-Controlled Polymers

Sequence-Controlled Polymers

Edited by Jean-François Lutz

The Editor

Dr. Jean-François LutzUniversité de Strasbourg, CNRSInstitut Charles Sadron UPR2223 rue du Loess67034 Strasbourg Cedex 2France

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Contents

1 Defining the Field of Sequence-Controlled Polymers 1Jean-François Lutz

1.1 Introduction 11.2 Glossary 41.3 Sequence Regulation in Biopolymers 71.3.1 Nucleic Acids 71.3.2 Proteins 71.4 Bio-Inspired Sequence-Regulated Approaches 81.5 Sequence Regulation in Synthetic Macromolecules 91.5.1 Step-Growth Polymerizations 111.5.2 Chain-Growth Polymerizations 111.5.3 Multistep Growth Polymerizations 131.6 Characterization of SCPs 151.7 Impact in Materials Science 171.8 Some Words About the Future 19

References 20

2 Kinetics and Thermodynamics of Sequence Regulation 27Pierre Gaspard

2.1 Introduction 272.2 Generalities 282.2.1 Characterization of Sequences and Information 282.2.1.1 Single-Molecule Level of Description 282.2.1.2 Many-Molecule Level of Description 292.2.2 Precise or Loose Sequence Control during Copolymerization 302.2.3 Conditions for Growth or Dissolution 312.2.4 Kinetic Equations 322.3 Thermodynamics 332.3.1 Free Copolymerization 342.3.2 Template-Directed Copolymerization 352.3.3 Depolymerization 352.4 Kinetics Yielding Bernoulli Chains 362.5 Kinetics Yielding Markov Chains 362.6 Kinetics Yielding Non-Markovian Chains 40

vi Contents

2.7 Effect of Sequence Disorder on Ceiling and Floor Temperatures 402.8 Mechanical Power of Sequence Disorder 432.9 Template-Directed Copolymerization 442.10 Conclusion 45

Acknowledgments 45References 46

3 Nucleic Acid-Templated Synthesis of Sequence-DefinedSynthetic Polymers 49Zhen Chen and David R. Liu

3.1 Introduction 493.2 Enzymatic Templated Syntheses of Non-Natural Nucleic Acids 503.2.1 Polymerase-Catalyzed Syntheses of Backbone-Modified Nucleic

Acids 503.2.2 Polymerase-Catalyzed Syntheses of Nucleobase-Modified Nucleic

Acids 523.2.3 Polymerase-Catalyzed Syntheses of Sugar-Modified Nucleic Acids 543.2.4 Ligase-Catalyzed Syntheses of Non-Natural Nucleic Acids 583.3 Ribosomal Synthesis of Non-Natural Peptides 593.4 Nonenzymatic Polymerization of Nucleic Acids 613.5 Nonenzymatic Polymerization of Non-Nucleic Acid Polymers 673.6 Conclusion and Outlook 71


4 Design of Sequence-Specific Polymers by GeneticEngineering 91Davoud Mozhdehi, Kelli M. Luginbuhl, Stefan Roberts, and Ashutosh Chilkoti

4.1 Introduction 914.2 Design of Repetitive Protein Polymers 934.3 Methods for the Genetic Synthesis of Repetitive Protein Polymers 964.4 Expression of Repetitive Protein Polymers 1004.5 Expanding the Chemical Repertoire of Protein Polymers 1004.5.1 Chemo-Enzymatic Modification 1014.5.2 Incorporation of Noncanonical Amino Acids 1044.5.3 Post-Translational Modifications 1054.6 Summary and Outlook 107

References 108

5 Peptide Synthesis and Beyond the Use of Sequence-DefinedSegments for Materials Science 117Niels ten Brummelhuis, Patrick Wilke, and Hans G. Börner

5.1 Introduction 1175.2 The History of Solid-Phase-Supported Peptide Synthesis 1185.3 Supports for the Chemical Synthesis of Peptides 1205.4 Synthesis of Peptide–Polymer Conjugates 122

Contents vii

5.5 Identification of Functional Sequences 1255.5.1 Phage Display 1255.5.2 Split-and-Mix Libraries and SPOT Synthesis 1305.5.3 Applications of Libraries 1345.5.4 Dynamic Covalent (Pseudo)Peptide Libraries 1365.6 Sequence–Property Relationships 1365.7 Translation of Sequence to Synthetic Precision Polymer

Platforms 1375.8 Conclusion 141

References 141

6 Iterative Synthetic Methods for the Assembly ofSequence-Controlled Non-Natural Polymers 159Christopher Alabi

6.1 Introduction 1596.2 The Solid-Phase Approach 1616.2.1 Type of Solid Supports 1616.2.2 Iterative Assembly using Single Heterobifunctional Monomers 1626.2.3 Iterative Assembly using Multiple Heterobifunctional Monomers 1636.3 The Liquid-Phase Approach 1646.3.1 Requirements for Liquid-Phase Supports 1656.3.2 Examples of Iterative Liquid-Phase Methodologies 1656.3.3 The Fluorous Liquid-Phase Approach 1676.4 The Template Approach 1686.5 A Support-Free Approach 1706.6 Outlook 175

References 176

7 Sequence-Controlled Peptoid Polymers: Bridging the Gapbetween Biology and Synthetic Polymers 183Mark A. Kline, Li Guo, and Ronald N. Zuckermann

7.1 Introduction 1837.1.1 Closing the Gap between Biological Polymers and Synthetic

Polymers 1847.1.2 Enhancing Synthetic Polymers with Sequence Control 1847.2 Peptoids – Bridging the Gap 1877.3 Polypeptoid Synthesis 1897.3.1 Solution Polymerization Method 1897.3.2 Solid-Phase Synthesis Method 1907.3.2.1 Solid-Phase Peptide Synthesis 1907.3.2.2 Solid-Phase Peptoid Synthesis 1927.3.2.3 Solid-Phase Submonomer Synthesis Method 1927.3.3 Combinatorial Synthesis 1977.3.4 Polypeptoid Analysis 1977.4 Discovering Peptoid Properties Derived from Sequence Control 1987.4.1 Peptoids as Potential Therapeutics 199

viii Contents

7.4.2 Peptoids with Controlled Conformation 1997.4.2.1 Peptoid Properties Dominated by Side Chains 2017.4.2.2 The Effect of Bulky Side Chains 2017.4.2.3 The Peptoid Backbone Differs from a Peptide Backbone 2027.4.2.4 Cyclic Peptoids 2057.4.3 Peptoids That Function as Biomaterials 2057.4.3.1 Antimicrobial and Antifouling Peptoids 2067.4.3.2 Lipidated Peptoids for Drug Delivery 2067.4.4 Ordered Supramolecular Assemblies: Toward Hierarchal Structures

with Function 2067.4.4.1 Supramolecular Self-Assembly from Uncharged Amphiphilic Diblock

Copolypeptoids 2077.4.4.2 Structures from Amphiphilic, Ionic-Aromatic Diblock

Copolypeptoids 2077.4.4.3 Free-Floating Two-Dimensional Peptoid Nanosheets with Crystalline

Order 2117.5 Conclusion 214


8 Sequence and Architectural Control in GlycopolymerSynthesis 229Yamin Abdouni, Gokhan Yilmaz, and C. Remzi Becer

8.1 Introduction: Glycopolymer–Lectin Binding 2298.2 Sequence-Controlled Glycopolymers 2308.2.1 Sequence-Defined Glycooligomers 2318.2.2 Sequence Control via Time-Regulated Additions 2348.2.3 Sequence Control via Time-Regulated Chain Extensions 2358.2.4 Sequence Control via Orthogonal Reactions 2378.3 Self-Assembly of Glycopolymers 2388.3.1 Self-Assembly Based on Amphiphilicity 2388.3.2 Temperature-Triggered Self-Assemblies 2428.3.3 pH-Responsive Self-Assemblies 2438.3.4 Self-Assembly Based on Electrostatic Interactions 2458.4 Single-Chain Folding of Glycopolymers: The Future? 2488.4.1 Selective Point Folding 2498.4.2 Repeat Unit Folding 2498.5 General Conclusion and Future Outlook 251


9 Sequence Regulation in Chain-Growth Polymerizations 257Makoto Ouchi

9.1 Introduction 2579.2 Alternating Copolymerization 2599.2.1 Addition Polymerization 2599.2.2 Alternating ROMP 261

Contents ix

9.3 Iterative Single-Unit Addition with Living Polymerization 2629.3.1 Iterative Process along with Purification via Peak Separation 2639.3.2 Iterative Process along with Transformation of Pendant Group 2669.4 Template-Assisted Polymerization 2679.4.1 Template Initiator 2689.4.2 Template Inimer 2699.5 Cyclopolymerization 2709.6 Ring-Opening Polymerization of Sequence-Programmed

Monomer 2729.7 Conclusion 274

References 274

10 Sequence-Controlled Polymers by Chain Polymerization 281Junpo He, Jie Ren, and Erlita Mastan

10.1 Introduction 28110.2 Sequence-Controlled Polymers by Various Polymerization

Mechanisms 28210.2.1 Anionic Polymerization 28210.2.2 Cationic Polymerization 28910.2.3 Ring-Opening Polymerization (ROP) 29010.2.4 Ring-Opening Metathesis Polymerization (ROMP) 29210.2.4.1 Regioselective ROMP of Substituted Cyclooctene 29210.2.4.2 Regioselective ROMP of Macrocyclic Compounds 29410.2.4.3 Alternating Copolymerization 29610.2.4.4 Kinetic Control for Polymers with Sequence-Defined

Functionalities 29910.2.5 Radical Polymerization 30010.2.5.1 Polymers with Alternating AB Sequence 30110.2.5.2 Polymer with ABB (1 : 2) Sequence 30510.2.5.3 Polymers with Site-Specific Functionalization 30710.2.5.4 Polymers with Precisely Controlled Sequence at Monomer Level 30910.2.5.5 Other Sequence-Controlled Polymers 31210.2.6 Coordination Polymerization 31510.3 Concluding Remarks 316

References 317

11 Sequence-Controlled Polymers via CationicPolymerization 327Sadahito Aoshima and Arihiro Kanazawa

11.1 Introduction 32711.2 Recent Developments in Living Cationic Polymerization 32811.2.1 Design of Initiating Systems for Living Polymerization 32811.2.2 Base-Assisting Living Systems with Various Metal Halides 32911.2.3 New Monomers for Cationic Polymerization 33011.3 Sequence-Regulated Functional Polymers 33111.3.1 Synthesis of New Block, Gradient, and End-Functionalized

Polymers 331

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11.3.2 Synthesis of Various Alternating Polymers by Controlled CationicPolymerization 334

11.3.3 Synthesis of New Ring Polymers 33611.4 Sequence Control Based on the Cationic Copolymerization of Vinyl

and Cyclic Monomers 33711.4.1 Strategy for Sequence Control by Copolymerizing Different Types of

Monomers 33711.4.2 Concurrent Cationic Vinyl-Addition and Ring-Opening

Copolymerization of VEs and Oxiranes 33811.4.3 Terpolymerization via the Exclusive One-way Cycle of Crossover

Propagation Reactions 34111.4.4 Concurrent Cationic Vinyl-Addition and Ring-Opening

Copolymerization Mediated by Long-Lived Species 344References 345

12 Periodic Copolymers by Step-Growth Polymerization 349Zi-Long Li and Zi-Chen Li

12.1 Introduction 34912.2 Carbon-Chain Periodic Polymers 35212.2.1 Acyclic Diene Metathesis Polymerization 35212.2.2 Atom Transfer Radical Coupling 35512.2.3 C(sp3)–C(sp3) Coupling 35512.2.4 Atom Transfer Radical Polyaddition 35612.3 Hetero-Chain Periodic Polymers 35712.3.1 Polycondensation or Polyaddition of Oligomonomers 35712.3.1.1 Polycondensation 35712.3.1.2 Polyaddition via Click Reactions 35912.3.1.3 Radical Addition–Coupling Polymerization 36212.3.2 One-Pot Sequential Monomer Addition and Polymerization 36412.3.3 Multicomponent Polymerizations 36412.4 Conclusions and Outlook 369

References 372

13 Click and Click-Inspired Chemistry for the Design ofSequence-Controlled Polymers 379Steven Martens, Joshua O. Holloway, and Filip E. Du Prez

13.1 Introduction to “Click” and Click-Inspired Reactions Within the Areaof Sequence-Controlled Polymers 379

13.2 Click and Click-Inspired Reactions for Sequence Building 38013.2.1 Copper(I)-Catalyzed Azide/Alkyne Cycloaddition 38013.2.2 Thiol–X and Thiolactone Chemistries 38613.2.3 Diels–Alder: Photo-Triggered and Thermally Induced Reactions 39513.3 Conclusions and Outlook 400

References 400

Contents xi

14 One-Pot Sequence-Controlled (SC) Multiblock Copolymers viaCopper-Mediated Polymerization 417Athina Anastasaki, Richard Whitfield, Vasiliki Nikolaou, Nghia P. Truong,Glen R. Jones, Nikolaos G. Engelis, Evelina Liarou, Michael R. Whittaker,and David M. Haddleton

14.1 Introduction 41714.2 Criteria for the Successful Synthesis of SC Multiblock

Copolymers 41914.3 Historical Background toward the Development of One-Pot SC

Multiblocks 41914.4 Access to SC Acrylic Multiblock Copolymers 42014.4.1 The Cu(0)-Wire-Mediated RDRP Approach 42014.4.1.1 When to Use Cu(0)-Wire-Mediated RDRP 42214.4.1.2 When Not to Use This Technique 42214.4.1.3 Protocol for the Synthesis of Acrylic Multiblock Copolymers via

Cu(0)-Wire-Mediated RDRP 42314.4.2 Light-Mediated Copper Polymerization for the Synthesis of Acrylic

Multiblock Copolymers 42414.4.2.1 Attributes of the Light-Mediated Copper Polymerization

Technique 42614.4.2.2 Reasons Not to Select This Technique 42614.4.2.3 Protocol for the Synthesis of Acrylic Multiblock Copolymers via

Light-Mediated Copper Polymerization 42614.5 Access to SC Acrylamide Multiblock Copolymers (The CuBr/Me6Tren

Disproportionation Technique) 42714.5.1 Why Use the CuBr/Me6Tren Disproportionation Technique 42814.5.2 Reasons Not to Select This Technique 42814.5.3 Protocol for the Synthesis of Acrylic Multiblock Copolymers via

CuBr/Me6Tren Disproportionation Technique 42914.6 Perspective and Outlook 429

References 430

15 Properties and Applications of Sequence-ControlledPolymers 435Jordan H. Swisher, Jamie A. Nowalk, Michael A. Washington, and Tara Y. Meyer

15.1 Introduction 43515.1.1 Definitions 43615.1.2 Types of Sequence-Dependent Properties 43715.1.3 Categories of Sequence Comparison Studies 43815.2 Molecular Properties 43915.2.1 Monomer Order 43915.2.2 Electronic/Vibrational Properties and Reactivity 43915.3 Solution-Phase Properties 43915.3.1 Folding 44115.3.2 Recognition 443

xii Contents

15.3.3 Aggregation 44415.4 Sequence Dependence of Bulk-Phase Properties 44515.4.1 Category I – Block Composition 44615.4.1.1 Block Dispersity 44615.4.1.2 Block Frequency 44615.4.2 Category II – Monomer Distribution 44915.4.2.1 Tacticity 44915.4.2.2 Alternating versus Random (and Block) 45315.4.2.3 Gradient Copolymers 45415.4.3 Category III – Precision Placement 45415.4.4 Category IV – Side-Chain Sequence 45815.4.5 Category V – Complex Sequences 45815.5 Conclusions and Outlook 46115.5.1 Solution-Phase Properties 46215.5.2 Bulk-Phase Properties 46415.5.3 The Future 466

References 466

16 Tandem Mass Spectrometry Sequencing ofSequence-Controlled and Sequence-Defined SyntheticPolymers 479Laurence Charles

16.1 Introduction 47916.2 MS/MS Principle 48016.3 MS/MS of Sequence-Controlled Copolymers 48216.4 MS/MS of Sequence-Defined Polymers 48516.4.1 Biomimetics 48516.4.2 Sequence-Defined Copolymers for Information Storage 49016.5 Conclusions and Perspectives 498

References 500

Index 505

1

1

Defining the Field of Sequence-Controlled PolymersJean-François Lutz

Université de Strasbourg, CNRS, Institut Charles Sadron UPR22, 23 rue du Loess, 67034 Strasbourg Cedex 2,France

1.1 Introduction

This book is fully devoted to an exploding area in fundamental and appliedpolymer science, namely the synthesis, characterization, and utilization ofsequence-controlled polymers (SCPs) [1]. This topic is at the same time very oldand very new: old because monomer sequence regulation is a central conceptin biology and has therefore been thoroughly studied in biochemistry and bio-physics for several decades. Thus, efficient methods are available for the synthesisand characterization of sequence-defined biopolymers such as deoxyribonucleicacid (DNA), ribonucleic acid (RNA), and proteins [2]. In comparison, however,the development of man-made polymers with controlled monomer sequences isa much more recent area of research. As pointed out in early essays and reviews[3–5], very little research efforts have been devoted to that topic until about10 years ago. However, the subject has recently gained significant academicattention, in particular during the last 5 years. This recent emerging trend isactually what is described in the present book, and all the authors of the fol-lowing chapters have been actively participating in the development of that newdiscipline. Yet, it should be clearly stated that the field of SCPs was not started5 years ago. Studies on monomer sequences began with the development of thefirst synthetic copolymers and can therefore be tracked back to the early days ofpolymer science [6, 7]. However, as described in a recent historical summary [8],the subject has been stuck for more than 50 years and was mostly limited to thesynthesis of classical copolymers such as statistical, alternating, periodic, andblock copolymers. More complex degrees of sequence regulation were regardedby the polymer science community as difficult to achieve or as the preserveof biochemists. This state of mind is exactly what has changed during the lastfew years, which have witnessed the emergence of a large number of originalideas and concepts for making SCPs [1]. As a consequence, the descriptionof monomer sequences in man-made polymers has started to get outdated inclassical textbooks. In particular, many copolymers that have been reported inrecent years are difficult to describe using current polymer nomenclature and

Sequence-Controlled Polymers, First Edition. Edited by Jean-François Lutz.© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.

2 Sequence-Controlled Polymers

terminology [9]. In this context, the objective of the present volume is to define,rationalize, and categorize that burgeoning new field of research.

As a key to decode the present volume, the reader should always keep inmind that the field of SCPs is actually a hybrid discipline located at the interfacebetween biology and polymer science, as depicted in Figure 1.1. Biologicalpolymers, such as nucleic acids and proteins, are indeed archetypal examplesof macromolecules with perfectly controlled monomer sequences. Thus, oneimportant trend in the field consists in using biological principles to synthesizeSCPs. Protein engineering [10] and DNA-templated polymerizations [11] aregood examples of strategies following this school of thought (see Chapters 3 and4 of this volume for details). However, a second important trend in the field ofSCPs aims to synthesize non-natural macromolecules that are as structurallydefined as biopolymers [4]. In such approaches, man-made chemistry conceptsare used to synthesize the polymers. These tools can be classical polymerizationapproaches such as chain-growth and step-growth polymerizations but also toolsthat are imported from other areas of chemistry such a solid-phase synthesisand molecular machines [12, 13]. Solid-phase iterative chemistry was first intro-duced for the chemical synthesis of biopolymers such as proteins [14, 15] andnucleic acids [16, 17]. However, it was later demonstrated that such approachescan also be used for preparing a wide variety of synthetic sequence-definedpolymers (see Chapters 5–7 for details), which includes peptide–polymerbio-hybrid structures [18], peptidomimetics such as peptoids [19], as wellas fully unnatural macromolecules such as information-containing polymers[20, 21]. Yet, such multistep growth approaches remain overall experimentallydemanding as well as time consuming, and consequently more straightforwardsequence-controlled polymerization strategies have been also investigated in thepast years. For instance, living or pseudo-living chain-growth polymerizationssuch as carbanionic polymerization, cationic polymerization, and controlledradical polymerization have been extensively studied for monomer sequenceregulation (see Chapters 8–11 for details). Original step-growth polymerizationconcepts have also been recently examined for the preparation of periodic SCPs(see Chapter 12 for details). Furthermore, efficient chemical transformationsknown as “click” reactions, which have become quite popular in syntheticpolymer chemistry during the last decade [22, 23], have also been shown tobe useful tools for the preparation and modification of sequence-controlledmacromolecules (see Chapter 13).

This recent progress in the synthesis of SCPs has also raised a large numberof new questions and challenges in the broad field of polymer science. Indeed,the emerging domain of SCPs is not restricted to polymer synthesis but alsoopens up new directions in polymer physics, analytical chemistry, engineering,materials science, and nanotechnology. For instance, the characterization ofSCPs requires the development of new analytical approaches [24]. Besidesstandard polymer analytics such a nuclear magnetic resonance spectroscopy andsize-exclusion chromatography, SCPs call for more specific sequencing methodsthat permit the full characterization of monomer sequences (see Chapter 16).As highlighted in Figure 1.1, engineering aspects will also probably play anincreasing role in the field of SCPs in the near future. Indeed, although SCPs are

Enzyme and ribozyme synthesis

Replication/transcription/translation

Recombinant protein production

Gene editing

Automated solid-phase synthesis

Non-canonical monomers

PCR amplification

Sequencing technologies

Biochemistry

Molecular biology Polymer physics Polymer chemistry

Engineering

Analytical chemistry

Sequence-controlled

polymers

High chemical diversity

Step-growth/chain-growth

Scalable polymerization processes

Externally-regulated processes

Flow chemistry, microfluidics

Combinatorial chemistry

Iterative polymer synthesis

Polymer modification

AA AB

BB

NH2

O

N

NH

Figure 1.1 The field of sequence-controlled polymers is at the crossroads between biology (red) and polymer science (blue). One trend in the field consists inimporting sequence-defined biopolymers in materials science and nanotechnology applications, whereas the opposite one consists in rendering syntheticmacromolecules as structurally defined as natural ones. In that regard, sequence-controlled polymers constitute a new category of macromolecules that fillsthe gap between evolution-optimized biopolymers and conventional plastics.


not foreseen to be used as large-scale commodity polymers, the developmentof automated protocols and combinatorial approaches seem mandatory forfacilitating their synthesis and processing. Last but not least, SCPs have alreadyopened up many new opportunities for the design of applied polymer materials.The range of applications of SCPs is particularly broad and includes areas thatare as diverse as data storage, catalysis, anti-counterfeiting technologies, infor-mation technologies, microelectronics, automotive industry, nanomedicine, andbiotechnology (see Chapter 15).

The field of SCPs is therefore an exciting multidisciplinary domain withits own terminology, topics, challenges, and frontiers. In order to guide thereaders through this new scientific universe, this chapter provides a succinctgeneral overview about SCPs. In particular, the following sections introduce,in a few words, the main aspects and topics that will be developed in moredetail throughout the whole book. At first, some general terms and definitionsare briefly presented. Afterward, the different mechanisms, either biological orman-made, that permit the synthesis of SCPs are presented and categorized.Biological sequence-regulated polymerizations that occur in living organismsare briefly described, but the reader is guided to specialized biology textbooksfor more detailed information [2]. Man-made approaches are presented morecomprehensively and ranked in subcategories. Finally, some general considera-tions about the sequencing and properties of SCPs are presented at the end ofthis chapter. Overall, this introductory chapter should serve as a useful startingpoint for understanding what SCPs are.

1.2 Glossary

As mentioned in the introduction, the field of SCPs is still relatively new andtherefore definitions and terminologies are still malleable. However, it is note-worthy that traditional polymer vocabulary is not sufficient for describing thedifferent kinds of SCPs that have been reported in recent years [9]. For instance,only standard copolymers such as block, alternating, random, statistical, andperiodic copolymers are described in official IUPAC polymer terminology [25].This language comes mostly from chain-growth copolymerizations (i.e., reactiv-ity ratios driven copolymerizations) and is limited for describing other situations.Thus, some broader definitions are proposed below. These definitions are notyet official (unless noted) and may still evolve.

Sequence-controlled polymer. A sequence-controlled polymer is a macro-molecule in which monomer sequences are controlled to some degree. Thiscontrol can be absolute but is not necessary. For example, an alternating copoly-mer prepared by radical chain-growth polymerization is a sequence-controlledpolymer, although it is also a nonuniform polymer in which chains have differentlengths and slightly different compositions.

Sequence-defined polymer. Syn. sequence-specific polymer, sequence-orderedpolymer. A sequence-defined polymer is a uniform macromolecule with an exactchain length and a perfectly defined sequence of monomers. In other words, eachmonomer unit is at a defined position in the chain. Sequence-defined polymersconstitute therefore a subclass of the field of sequence-controlled polymers, asproposed in Figure 1.2.

(Multi)block

copolymers

Gradient

copolymers

Periodic

copolymers

Nonuniform (polydisperse), Ð > 1.0 Uniform (monodisperse), Ð ~ 1.0

Aperiodic

copolymers

Alternating

copolymers

Chain-

positioning Biopolymers Foldamers

Unnatural

biopolymers

Information-containing

macromolecules

3′A C T A G

0 0 0 0 001 1 1

X Y

α ω α ω α ω

α ωα ω α ω α ω

5′

Sequence-controlled polymers

Sequence-defined polymers

Figure 1.2 Schematic representation of the meaning of the terms “sequence-controlled polymers” and “sequence-defined polymers.” “Sequence-controlledpolymers” is a broad term that denotes all kind of polymers in which sequences are controlled to some degree. “Sequence-defined polymers” is a morerestrictive term that denotes uniform polymers with perfectly controlled monomer sequences. A sequence-defined polymer is also sequence-controlled.A sequence-controlled polymer is not necessarily sequence-defined.


Primary structure. Syn. microstructure. [IUPAC] In the context ofmacromolecules such as proteins, constitutional formula, usually abbreviatedto a statement of the sequence and if appropriate cross-linking of chains [26].Additional information from the author: although traditionally belonging to theglossary of biology, the term “primary structure” may also be used to denote thesequence distribution of non-natural sequence-defined polymers.

Periodic copolymer. [IUPAC] A copolymer consisting of macromoleculescomprising more than two species of monomeric units in regular sequence [25].Additional information from the author: a periodic copolymer is a macro-molecule containing minimal motifs that repeat periodically in the chain. Forexample, polymers with repeating motifs [AB]n (i.e., alternating), [ABC]n, or[ABCD]n are periodic copolymers. A periodic copolymer can be uniform (i.e.sequence-defined) or non-uniform (i.e. sequence-controlled).

Aperiodic copolymer. Aperiodic copolymers are copolymers in which themonomer sequence distribution is not regular but follows the same arrangementin all chains [9, 27]. An aperiodic copolymer shall be distinguished from randomor statistical copolymers, in which sequences are uncontrolled. For example, anon-natural information-containing macromolecule contains, in most cases, anaperiodic sequence-coded message.

Block copolymer. [IUPAC] A copolymer that is a block polymer. In theconstituent macromolecules of a block copolymer, adjacent blocks are constitu-tionally different, that is, adjacent blocks comprise constitutional units derivedfrom different species of monomer or from the same species of monomer butwith a different composition or sequence distribution of constitutional units [25].Additional information from the author: multiblock copolymers contain severalsuccessive blocks that are arranged in a defined block sequence.

Gradient copolymer. A gradient copolymer is a macromolecule in whichcomonomer distribution follows a gradual change in composition along thechains. Gradient copolymers are often nonuniform macromolecules preparedby a controlled or living chain-growth process [28]. Although rarely describedin the literature, sequence-defined uniform gradient copolymer may also beconceived and achieved.

Irregular copolymer. Related terms: random copolymers, statistical copoly-mers. The term irregular copolymer could be used to denote all copolymers inwhich monomer sequences are uncontrolled. It could include different types ofuncontrolled sequences that follow Bernoullian or Markovian statistics. It shall,however, be noted that the official IUPAC term “irregular polymer” depicts verydifferent situations that are related to the polymer architecture [25].

Sequencing. The term “sequencing” denotes an analytical techniquethat permits the comprehensive deciphering of monomer sequences of asequence-defined polymer [24]. It should be noted that sequencing meth-ods are restricted to the analysis of uniform sequence-defined polymers.Although non-official, the term “sequence analysis” is proposed for the analyticalcharacterization of nonuniform sequence-controlled polymers.

Defining the Field of Sequence-Controlled Polymers 7

1.3 Sequence Regulation in Biopolymers

Sequence control is a key feature in biology. Indeed, in DNA the geneticinformation is stored in controlled A, T, G, and C nucleotide sequences,following Watson–Crick principles [29]. In addition, the high complexity andadvanced properties of protein-based biomaterials are determined by the pri-mary structure of sequence-defined peptide chains [30, 31]. The polymerizationmechanisms that lead to monomer sequence regulation in biological polymersare complex and enabled by large biocatalysts such as enzymes and ribozymes.The reader is therefore guided to comprehensive biology textbooks for detailedinformation on these processes [2]. A condensed description of biologicalpolymerizations is described in the following subsections.

1.3.1 Nucleic Acids

Two main types of nucleic acids are found in biology, DNA and RNA. Theformer is found in cell nuclei and stores the genome of living organisms,whereas the latter has different types and functions, such as messenger RNA(mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA). Sequence-definednucleic acids are synthesized in vivo via two main polymerization processes,namely DNA replication (DNA→DNA) and transcription (DNA→mRNA).Small noncoding RNAs are also obtained via other mechanisms, which areoutside the scope of this chapter [2]. Replication is a template polymerizationin which the sequence-defined double-stranded DNA is copied into two newnascent strands, namely the leading and the lagging strands. The synthesis ofthe leading strand is an interesting and inspirational mechanism for the field ofSCPs [3]. In this part of the replication process, the nascent sequence-definedDNA strand is formed with the help of DNA polymerases, which catalyze theformation of phosphodiester bonds between the growing 3′-OH terminus ofthe leading DNA chain and a nucleoside triphosphate monomer. In particular,the polymerase provides an optimal environment for confining the templatestrand, the leading nascent strand, and the upcoming monomer, thus enablingsequence regulation. In particular, this confined environment prevents anyuncontrolled polymerization of the nucleoside triphosphate and also allowserror correction in the rare event of an erroneous monomer incorporation.Furthermore, this synthesis is directional, and after each monomer attachment,the polymerase walks along the template strand, thus allowing its sequence tobe accurately copied. The DNA→mRNA transcription mechanism is overallvery similar to leading strand synthesis in replication but involves specific RNApolymerases that unwind double-stranded DNA and promotes phosphodiesterbond formation [2]. Furthermore, the monomers are not deoxyribose nucleosidetriphosphates as in DNA replication but ribose-based equivalents.

1.3.2 Proteins

Proteins are sequence-defined biopolymers that are built using 22 differentα-amino acid monomers (21 in eukaryotes). The primary structure of proteins


(i.e., the sequence of amino acids in a peptide chain) influences chain-foldinginto defined secondary, tertiary, and eventually quaternary structures, whichgovern their biological properties and functions. In biological organisms,proteins are synthesized via the translation process (mRNA→proteins), inwhich the nucleotide sequences of mRNA are transcripted into amino acidsequences [32]. From a polymer chemistry point of view, translation is probablythe most achieved polymerization mechanism identified to date, since it allowsinformation transfer between two totally different types of polymeric back-bones, namely from a poly(phosphodiester) to a polyamide. This fascinatingmechanism is enabled by the ribosome, which is a large and complex catalyticnucleoprotein. In particular, the ribosome is a machinery that provides an idealconfined environment for bringing together an mRNA template chain, a growingprotein chain, and tRNAs, which are molecular adaptors allowing translation.Indeed, tRNAs contain both a nucleic acid an anticodon trimer that can bindto a codon on the mRNA template and a polymerizable amino acid monomer.After installation of a tRNA on the mRNA template, the ribosome catalyzesthe reaction of the amino acid with the nascent protein and liberates afterwardthe adaptor. Following the basic codon/anticodon rules of the genetic code, theinformation of the mRNA strand is translated into a protein primary structure.Furthermore, after each amino acid attachment, the ribosome moves forward onthe mRNA template, thus allowing complete translation of the genetic sequence.

1.4 Bio-Inspired Sequence-Regulated Approaches

As described in Section 1.3, evolution has selected optimal polymerizationprocesses for the preparation of sequence-defined nucleic acids and proteins.Thus, an obvious strategy for preparing artificial SCPs consists in exploitingthese biological mechanisms. For instance, a large number of studies havebeen devoted to artificial replication processes. These studies are depicted indetail in previous reviews [1, 11] as well as in Chapter 3 of this book. Recently,Holliger and coworkers have described non-natural genetic polymers that canreplicate [33], evolve, and catalyze simple reactions [34]. Such polymers containartificial backbones that differ from the deoxyribose and ribose backbones ofDNA and RNA. However, it was demonstrated that these polymers can performenzymatic replication and, in particular, be copied from a DNA template andeven transcribe back their sequence information to a DNA chain [33]. Inaddition, nucleotides containing non-natural nucleobases have been extensivelystudied in in vitro enzymatic replication [1, 35]. Romersberg and coworkers havereported recently that non-natural base pairs can be recognized by the biologicalreplication machinery [36]. These examples show that biology can be tweakedfor the preparation of non-natural SCPs. However, the aforementioned studiesdeal with synthetic biology and are a little removed from the classical field ofpolymer science. Therefore, enzyme-free artificial replication has also beenwidely investigated for the preparation of synthetic sequence-defined polymers


[11]. Although a large number of studies have turned out to be unsuccessful [3],promising results have been obtained by Liu and coworkers, for example,for the enzyme-free, DNA-templated synthesis of sequence-defined peptidenucleic acids [37]. More recently, the same group has described the enzyme-freetranslation of DNA templates into fully synthetic, sequence-defined polymers[38]. This interesting example emphasizes that bio-inspired (but non-biological)mechanisms can be successfully developed for the preparation of SCPs. Besidesreplication, the complementarity of DNA strands is also an interesting featurefor preparing SCPs. For instance, Turberfield and coworkers have studiedself-recognizing DNA supports for programming and guiding the synthesis ofnon-natural, sequence-defined segments [39, 40].

Ribosomal synthesis is also a tool and an inspiration for the field of SCPs.For instance, protein-based SCPs can be synthesized in vivo in bacteria. Insuch approaches, a gene coding for a specific protein is included in bacterialDNA using recombinant DNA techniques. This gene is then expressed bythe ribosomal machinery of the bacteria, and the targeted protein polymer isafterward extracted. This interesting methodology is described in reviews [10]and in Chapter 4 of this book. Most of the pioneering studies describing therelevance of tailored proteins made by genetic engineering in materials sciencehave been reported by Tirrell and coworkers [41–43]. In particular, they haveshown that non-canonical amino acids can be utilized by bacterial ribosomes,thus allowing the synthesis of non-natural, protein-based SCPs [44]. Yet, theseapproaches remain so far strictly restricted to α-amino acid-based proteins.In this context, Suga and coworkers have studied the ribosomal compatibilityof other monomers such as β-amino acids [45, 46]. Although still preliminary,such approaches could allow ribosomal synthesis of non-natural SCPs. Anotherinteresting challenge is the development of ribosome-inspired synthetic proces-sive catalysts [47]. A first convincing proof of concept was recently publishedby Leigh and coworkers [48, 49], who described a rotaxane-based molecularmachine allowing sequence-defined synthesis.

1.5 Sequence Regulation in Synthetic Macromolecules

Traditionally, synthetic polymerization methods are split in two main categories:that is, step-growth and chain-growth polymerization. However, in recent years,multistep growth approaches in which polymers are prepared using stepwisereactions have become more and more prominent in polymer chemistry, inparticular for the synthesis of SCPs. Thus, in a recent review [13], syntheticpolymerizations have now been ranked in three main categories, as depicted inFigure 1.3. This new chart will be followed in the present section for describingchemical approaches that have been reported in recent years for the synthesisof SCPs. In particular, these approaches will be presented starting with themethods leading to the less uniform polymers (i.e., step growth that typicallyleads to Ð ∼ 2) and finishing with the one leading to uniform structures (i.e.,multistep growth that typically leads to Ð ∼ 1).

Step-growth polymerizations

Bifunctional Multicomponent

Conventional

Ester Amide Urethane ADMET CuAAC

Living

step-growth

Living/controlled Uncontrolled

Ionic Radical ROMP ROP

Linear iterative synthesis Dendrimers

Liquid phase Solid phase Convergent Divergent

Single

insertion Protected Orthogonal

Anionic Cationic NMP ATRP RAFT

ARGET SARA ICAR

Others

Chain-growth polymerizations Multistep-growth synthesis

1.01 < Ð < 2Ð ~ 2 Ð ~ 1

X Y X Y X Y X Y

X Y

I I+n + 1n

* *

I X

X Y

X

X Y

YX

Y X Y X

Y

Figure 1.3 Synthetic polymers can be prepared via a step-growth, chain-growth, or multistep-growth mechanism [13]. Definition of the acronyms: acyclicdiene metathesis (ADMET), activators regenerated by electron transfer (ARGET), atom transfer radical polymerization (ATRP), copper-catalyzed azide–alkynecycloaddition (CuAAC), initiators for continuous activator regeneration (ICAR), nitroxide-mediated polymerization (NMP), reversible addition–fragmentationchain-transfer polymerization (RAFT), ring-opening polymerization (ROP), supplemental activator and reducing agent (SARA).


1.5.1 Step-Growth Polymerizations

In step-growth polymerizations, multifunctional monomers are reacted toafford linear, cyclic, or cross-linked architectures. For example, as shown inFigure 1.3, bifunctional monomers containing two reactive functions X andY usually lead to linear polymers. Alternatively, two monomers XX and YYcan be copolymerized. Step-growth polymerizations are overall easy methodsbut lead generally to a relatively poor control of chain length and molecularweight distribution. In terms of monomer sequence regulation, such poly-merizations are also limited by their simple mechanism. Yet, step-growthpolymerizations are very interesting options for the preparation of periodiccopolymers. Indeed, in many cases, it is possible to include long functionalspacers between the functionalities X and Y. Hence, the step-growth polymer-ization of sequence-controlled telechelic oligomers allows the synthesis of highmolecular weight macromolecules containing periodically repeating motifs.For instance, using such strategies, a given functional group can be includedperiodically in a polymer chain and the distance between the functional sitescan be precisely controlled. Among the pioneers in that area, Wagener andcoworkers have described the acyclic diene metathesis (ADMET) synthesis ofprecision polyolefins containing periodically spaced alkyl branches or polargroups [50–52]. Similar concepts have also reported by our group [53] as wellas that of Du Prez [54] using step-growth “click” polymerizations based oneither CuAAC or thiol–ene chemistry. Furthermore, step-growth strategiesare practical tools to polymerize functional, sequence-defined oligomers. Forexample, Kamigaito and coworkers have synthesized periodic ABCC-periodicvinyl copolymers using metal-catalyzed step-growth radical polymerization[55]. A comparable strategy was investigated by Meyer and coworker for thesynthesis of sequence-controlled poly(lactic-co-glycolic acid) (PLGA) [56].In this, approach, oligomers containing defined lactic/glycolic acid minimalsequences and reactive alcohol (X) and carboxylic acid (Y) terminal functionswere polymerized, thus affording periodic aliphatic polyesters.

1.5.2 Chain-Growth Polymerizations

In chain-growth polymerizations, the growing polymer chains containing atleast one active center react with the monomers one by one and incorporatethem successively [13]. Chain-growth polymerizations can proceed via radical,anionic, cationic, or ring-opening mechanisms, as shown in Figure 1.3. Althoughmonomers are incorporated successively in these polymerizations, the process isusually performed in one pot, and therefore chain-growth copolymerizations areusually not sequence-regulated [4]. When two monomers are copolymerized ina chain-growth process, the primary structure of the formed copolymer dependson the initiation/propagation kinetics (i.e., living, controlled, or steady-state)and on the difference in reactivity between the monomers, which is expressedby the reactivity ratios. In most known cases, the reactivity of the comonomersis not markedly different, and therefore statistical copolymers with irregularprimary structures are formed. However, in some particular cases, such as theradical copolymerization of styrene (donor monomer) and maleic anhydride


(acceptor monomer), sequence-regulated alternating copolymers are formed[57, 58]. Yet, as highlighted in earlier reviews [8], the mechanism of chain-growthcopolymerizations had already been known in the 1940s [59, 60], and since thenvery little progress had been made for several decades. As a result, until veryrecently, the main types of copolymers that were prepared by chain-growthcopolymerizations were statistical, alternating, and block copolymers [4]. Thissituation has changed with the development of living polymerizations [61] and,in particular, with the widespread utilization of controlled radical polymer-ization methods [62, 63]. Indeed, in a living or controlled/living chain-growthmechanism, all chains are initiated and grow simultaneously. As a consequence,in copolymerizations, the difference in reactivity of the comonomers can be“written” in the microstructure of the formed chains [64]. The first example ofthat was the description of gradient copolymers by Matyjaszewski et al. [28].Since then, more precise sequence regulation has been reported. For instance,in 2007, our group introduced a facile concept for placing functional monomersat precise chain locations in chain-growth radical copolymerizations [65]. Inthis approach, donor and acceptor comonomers are used. However, insteadof using equimolar amounts of comonomers as in standard alternating radicalcopolymerizations, a discrete amount of the acceptor comonomer (typicallyan N-substituted maleimide) is copolymerized with a large excess of thedonor monomer (generally styrene). As a consequence of the controlled/livingmechanism and of the marked reactivity differences of the comonomers, theN-substituted maleimide is usually incorporated into relatively narrow regionsof the growing chains [64]. Thus, using time-controlled monomer additions,it is possible to prepare complex functional microstructures using this simplemethodology [66–68]. Although not leading to perfectly defined microstruc-tures [69], such easy sequence-controlled copolymerizations open up interestingavenues for the design and folding of tailored functional polymers [70–73].Donor and acceptors comonomers have also been used for preparing othertypes of SCPs by controlled radical copolymerization. For instance, Kamigaitoand coworkers have described the synthesis of AAB-periodic polymers bycopolymerization of limonene and N-substituted maleimides [74, 75]. Periodiccopolymers can also be prepared by chain-growth cyclopolymerization ofmonomers containing minimal AB- or ABA-sequence motifs, as elegantlyproposed by Ouchi and coworkers [76, 77]. Another interesting recent trend inthe field of SCPs is the development of multiblock copolymers [78], as describedin Chapter 14 of this book. Although for several decades the synthesis of blockcopolymers has been mainly limited to diblock or triblock copolymers, it hasbeen shown in recent years that longer block sequences can be prepared. Inparticular, the groups of Coates [79], Perrier [80, 81], and Haddleton [82, 83]have made significant progress in that domain.

Attempts have also been made to achieve monomer single insertion in chainpolymerizations. In such strategies, the chain-growth process is decomposedinto a multistep growth polymerization (see Section 1.5.3), as underlined inFigure 1.3. The earliest example was reported by Higashimura and cowork-ers, who described the successive insertion of four vinyl ether monomers incationic polymerizations [84]. Attempts have also been made subsequently in


controlled radical polymerizations, although they are limited so far to a verysmall number of monomer insertions [85, 86]. Junkers and coworkers partiallybypassed that problem by implementing an inline chromatography purificationprotocol that permits the separation of sequence-defined oligomers from poly-disperse mixtures [87]. Using this approach, the synthesis of sequence-defined,acrylate-based pentamers has been reported [88]. Sawamoto and coworkers havealso investigated advanced chemical concepts for regulating single monomerinsertion in radical polymerizations. For instance, they have studied innovativetemplate initiators that permit the recognition and selective chain-incorporationof specific monomers [89, 90]. However, this elegant chemistry is limited so farto low molecular weight models. More recently, they have described the iterativeradical addition of monomer carrying bulky side chains [91]. This process allowssuccessive monomer insertions but requires three relatively time-consumingchemical steps for each monomer attachment. In a very recent publication, theseauthors have also depicted an elegant stepwise cyclopolymerization mechanism,inspired by ribosomal chemistry [92]. This process was, however, only validatedfor a small number of iterations.

1.5.3 Multistep Growth Polymerizations

In multistep growth approaches, uniform polymers are prepared using stepwisechemical steps [13]. For instance, the successive attachment of bifunctionalmonomers containing two reactive functions X and Y allows the synthesisof uniform polymers (Ð ∼ 1) [1]. In order to prevent these monomers frompolymerizing via a step-growth process (see Section 1.5.1), one of both reactivefunctions of the monomer is temporarily protected. For instance, a simplestrategy to synthesize uniform polymers is iterative exponential growth, inwhich a bifunctional XY monomer containing a protected X function is reactedwith its analog containing a protected Y function [93]. After coupling, theformed adduct is divided in two batches, which are selectively deprotected onone terminus and coupled again. The process is then repeated a certain numberof times until a desired length is reached. In other words, the chain length ofthe polymer is multiplied by 2 at each stage of the process (i.e., going throughsuccessive dimer, tetramer, octamer, 16mer, 32mer stages). Although simpleand scalable, this method remains, however, mostly limited to the synthesisof periodic primary structures. For attaining a wider variety of sequences(e.g., aperiodic coded sequences), iterative approaches in which the monomersare attached one by one are used, as depicted in Figure 1.3. In such approaches,a growing polymer chain containing a reactive X group is reacted with abifunctional XY monomer containing a protected X function. Hence, monomerscan be successively attached using stepwise coupling/deprotection cycles. Sincemonomers are assembled following a predetermined order, such a stepwisesynthesis is therefore a powerful strategy for preparing sequence-definedpolymers. Such approaches have been initially developed for the chemicalsynthesis of biopolymers, in particular peptides [3]. Yet, stepwise synthesis issomewhat difficult when performed in solution, because it requires intensivepurifications after each monomer attachment. One important breakthrough


in the field was the introduction by Merrifield of solid-phase synthesis, whichgreatly simplifies the purification in multistep growth approaches (see Chapter 5)[14, 15]. In this approach, the chains are grown on cross-linked polymer beadsthat can be filtered. Thus, after each chemical step, the growing polymers can beeasily separated from the excess reactants. Similar results can also be obtainedwith other types of supports, such as controlled-pore glass, solid nanoparticles,soluble polymer supports, or fluorescent tags. Although multistep growthsynthesis has been predominantly studied for peptides and oligonucleotides,it allows also the preparation of non-natural sequence-defined polymers. Forinstance, such approaches have been utilized for the design of a wide varietyof non-natural bio-oligomers, including peptidomimetics and foldamers [3].More recently, these strategies have also been investigated for preparing abroader variety of synthetic polymers [94], including, for example, non-naturalinformation-containing macromolecules (see Section 1.7) [20]. For example,phosphoramidite chemistry, which was initially developed for the synthesisof oligonucleotides [17], is also a practical chemistry for preparing syntheticsequence-defined polymers, as recently demonstrated by Sleiman and coworkers[95] and by our group [96, 97]. Using this approach, our group has reported theautomated synthesis of uniform, sequence-defined chains containing more than100 monomer units [98], thus demonstrating that multistep growth approachesare not restricted to oligomers. Yet, it should be clearly understood that suchlong chains can be obtained only if the yield of each individual reaction isnear-quantitative. Thus, highly efficient reactions should be selected for optimalmultistep growth synthesis. Another important parameter in those syntheses istime. Indeed, coupling steps that require hours or days for reaching high yieldsare unpractical for the synthesis of long polymers. Thus, chemical reactionsthat can be completed in less than 1 h are somewhat preferred. For example,an automated coupling/oxidation/deprotection phosphoramidite cycle usuallytakes ∼5–10 min. Thinking further out into the future of the field, ultrafast ormicrowave-assisted reactions, which would permit the coupling steps to proceedin less than 5 min, would be very interesting options. Significant efforts havealso been made for avoiding the use of protecting groups in iterative syntheses.For instance, interesting orthogonal iterative approaches have been recentlyreported. They are described in detail in recent reviews [12, 94] as well as inChapter 6 of this book. The first example of an efficient orthogonal iterative pro-cess was the so-called submonomer strategy reported by Zuckermann et al. forthe synthesis of peptoids (see Chapter 7) [99]. More recently, related approacheshave been reported by our group as well as by the groups of Alabi [100], DuPrez [101, 102], and Meier [103, 104]. For example, our group described theorthogonal synthesis of oligo(triazole amide)s [105, 106], oligo(alkoxyamineamide)s [107, 108], oligo(alkoxyamine phosphodiester)s [109], and oligocarba-mates [110]. These examples illustrate that a wide variety of uniform syntheticpolymers can now be synthesized by iterative chemistry, thus opening interestingnew perspectives for the design of new types of functional macromolecules(see Section 1.8).


1.6 Characterization of SCPs

Standard polymer analytical methods that are used for the characterization ofhomopolymers and simple copolymers are not always sufficient for examiningsequence-controlled microstructures. For instance, a diblock copolymerstructure can be relatively easily evidenced using size-exclusion chromatographyand NMR, but this analytical combination cannot be used for characterizingmore complex sequence-defined polymers. In a recent review, we proposeddifferent meanings for the terms “sequence analysis” and “sequencing” [24].The former denotes the examination of sequence tendencies in polydispersepolymers (e.g., analysis of dyads and triads by 13C NMR), while the latter refersto comprehensive deciphering of sequence-defined information, as performedin proteomics and genomics. For several decades, only sequence analysistools, in particular NMR [111, 112], have been used in polymer science forthe characterization of copolymers [7]. However, in very recent years, somesequencing methods have also been screened for characterizing non-natural,sequence-defined macromolecules. It should be mentioned that several analyt-ical techniques that are used for the sequencing of proteins and nucleic acidsutilize biological reactions (e.g., polymerase chain reaction or PCR amplification)that cannot be applied to synthetic polymers [24]. However, some techniquesare suitable for both biological and abiotic polymers (Figure 1.4). For instance,the most suitable tool for reading non-natural sequences is probably tandemmass spectrometry (MS/MS). In MS/MS measurements, macromoleculesare fragmented by collision-induced dissociation, and the mass differencesbetween the fragments are used to decode the monomer sequences. In closecollaboration with the group of Charles, we have shown in recent years thatMS/MS is a very practical method to read the sequences of a wide variety ofnon-natural information-containing polymers [110, 115–117]. In particular, itwas observed that the MS/MS of synthetic polymers is often even easier thanthat of biopolymers since the macromolecular structure of non-natural analytescan be optimized to favor fragmentation and sequencing [109, 110, 118]. Therecent findings are summarized in Chapter 16 of this book.

Besides MS/MS, many other analytical tools have shown promise for char-acterizing the sequences of non-natural SCPs. For instance, nanopore-basedanalytics (Figure 1.4) [119], which have been optimized for DNA sequencingduring the last few decades [113, 120], can also be interesting tools to readsynthetic sequences. In such measurements, sequence-defined polyelectrolytesare subjected to an ionic current, which thread through natural (e.g., porousmembrane proteins such as hemolysin or aerolysin) or non-natural (e.g., holesin graphene or molybdenum disulfide flat surfaces) pores of a defined diameter.The interactions of the monomers with the pore lead to voltage variations thatcan be correlated to the sequence. Such measurements are, however, not trivial,and DNA sequencing required almost 20 years of optimization. This is, in part,due to the fact that DNA nucleobases have closely related molecular structures.In comparison, the sequences of non-natural SCPs could be intentionallyengineered to interact to a higher or smaller extent with a pore, thus rendering

Destructive analysis Nondestructive analysis

Tandem mass spectrometry Nanopore sequencing Ion-beam deposition

400

35 eV

O

O

O N O N O N O N NH Br

H+O

OH

[11010 + H]+

NH

O

NH

O

NH

O

NH

226 Da

385.3

851.6

1012.6

m/z 385.3

c2.+

c2.+

m/z 1012.6y4.+ m/z 772.4y

3.+

m/z 851.6c4.+

c4.+

y4.+y

3.+

y2.+c

3.+

m/z 611.4c3.+

m/z 546.3y2.+

226 Da

70

7.5

240 Da

240 Da 144 Da

305 Da

1156.7

600 800 1000 m/z

CytC

Cu (001)

BSA

Cu (001)

10 nm

54

6.3

61

1.4

77

2.4

1 nm

Figure 1.4 Overview of some analytical techniques that are used for the characterization of SCPs. Some of these techniques lead to the destruction of theanalytes (shown in blue), while some other are nondestructive (shown in red). The nanopore sequencing scheme is reprinted from Ref. [113]. (Reproducedwith permission of Nature Publishing Group.) The scanning tunneling microscopy (STM) images obtained after electrospray ion beam surface deposition arereprinted from Ref. [114]. (Reproduced with permission of American Chemical Society.)

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