Polymers for Biomedicine

Polymers for Biomedicine

Synthesis, Characterization, and Applications

Edited byCarmen Scholz

University of Alabama in HuntsvilleAlabama, USA

This edition first published 2017© 2017 John Wiley & Sons, Inc

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

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Library of Congress Cataloging‐in‐Publication DataNames: Scholz, Carmen, 1963– editor.Title: Polymers for biomedicine : synthesis, characterization, and applications /

edited by Carmen Scholz.Description: Hoboken, New Jersey : John Wiley & Sons, Inc., 2017. | Includes bibliographical

references and index.Identifiers: LCCN 2017012622 (print) | LCCN 2017005426 (ebook) | ISBN 9781118966570 (cloth) |

ISBN 1118966570 (cloth) | ISBN 9781118967935 (Adobe PDF) | ISBN 9781118967881 (ePub)Subjects: LCSH: Polymers. | Polymerization. | Polymers in medicine. | Macromolecules.Classification: LCC QD381 .P61244 2017 (ebook) | LCC QD381 (print) | DDC 610.28/4–dc23LC record available at https://lccn.loc.gov/2017012622

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10 9 8 7 6 5 4 3 2 1

v

List of Contributors ix

Part I Pseudo‐Peptides, Polyamino Acids, and Polyoxazolines 1

1 Characterization of Polypeptides and Polypeptoides – Methods and Challenges 3David Huesmann and Matthias Barz

2 Poly(2‐Oxazoline): The Structurally Diverse Biocompatibilizing Polymer 31Rodolphe Obeid

3 Poly(2‐Oxazoline) Polymers – Synthesis, Characterization, and Applications in Development of POZ Therapeutics 51Randall W. Moreadith and Tacey X. Viegas

4 Polypeptoid Polymers: Synthesis, Characterization, and Properties 77Brandon A. Chan, Sunting Xuan, Ang Li, Jessica M. Simpson, Garrett L. Sternhagen, and Donghui Zhang

Part II Advanced Polycondensates 121

5 Polyanhydrides: Synthesis and Characterization 123Rohan Ghadi, Eameema Muntimadugu, Wahid Khan, and Abraham J. Domb

6 New Routes to Tailor‐Made Polyesters 149Kazuki Fukushima and Tomoko Fujiwara

7 Polyphosphoesters: An Old Biopolymer in a New Light 191Kristin N. Bauer, Hisaschi T.C. Tee, Evandro M. Alexandrino, and Frederik R. Wurm

Contents

Contentsvi

Part III Cationically Charged Macromolecules 243

8 Design and Synthesis of Amphiphilic Vinyl Copolymers with Antimicrobial Activity 245Leanna L. Foster, Masato Mizutani, Yukari Oda, Edmund F. Palermo, and Kenichi Kuroda

9 Enhanced Polyethylenimine‐Based Delivery of Nucleic Acids 273Jeff Sparks, Tooba Anwer, and Khursheed Anwer

10 Cationic Graft Copolymers for DNA Engineering 297Atsushi Maruyama and Naohiko Shimada

Part IV Biorelated Polymers by Controlled Radical Polymerization 313

11 Synthesis of (Bio)degradable Polymers by Controlled/“Living” Radical Polymerization 315Shannon R. Woodruff and Nicolay V. Tsarevsky

Part V Polydrugs and Polyprodrugs 355

12 Polymerized Drugs – A Novel Approach to Controlled Release Systems 357Bahar Demirdirek, Jonathan J. Faig, Ruslan Guliyev, and Kathryn E. Uhrich

13 Structural Design and Synthesis of Polymer Prodrugs 391Petr Chytil, Libor Kostka, and Tomáš Etrych

Part VI Biocompatibilization of Surfaces 421

14 Polymeric Ultrathin Films for Surface Modifications 423Henning Menzel

15 Surface Functionalization of Biomaterials by Poly(2‐oxazoline)s 457Giulia Morgese and Edmondo M. Benetti

16 Biorelated Polymer Brushes by Surface Initiated Reversible Deactivation Radical Polymerization 487Rueben Pfukwa, Lebohang Hlalele, and Bert Klumperman

Contents vii

Part VII Self‐Assembled Structures and Formulations 525

17 Synthesis of Amphiphilic Invertible Polymers for Biomedical Applications 527Ananiy M. Kohut, Ivan O. Hevus, Stanislaw A. Voronov, and Andriy S. Voronov

18 Bioadhesive Polymers for Drug Delivery 559Eneko Larrañeta and Ryan F. Donnelly

Index 603

ix

Evandro M. AlexandrinoMax Planck Institute for Polymer Research (MPIP)Mainz, Germany

Khursheed AnwerCelsion CorporationHuntsville, AL, USA

Tooba AnwerUniversity of Alabama at BirminghamBirmingham, AL, USA

Matthias BarzInstitute of Organic ChemistryJohannes Gutenberg‐Universität MainzMainz, Germany

Kristin N. BauerMax Planck Institute for Polymer Research (MPIP)Mainz, Germany

Edmondo M. BenettiLaboratory for Surface Science and TechnologyDepartment of MaterialsETH ZürichZürich, Switzerland

Brandon A. ChanDepartment of Chemistry and Macromolecular Studies GroupLouisiana State UniversityBaton Rouge, LA, USA

Petr ChytilInstitute of Macromolecular ChemistryAcademy of Sciences of the Czech RepublicPrague, Czech Republic

Bahar DemirdirekDepartment of Chemistry and Chemical BiologyRutgers UniversityPiscataway, NJ, USA

Abraham J. DombSchool of Pharmacy‐Faculty of MedicineThe Hebrew University of Jerusalem, and Jerusalem College of Engineering (JCE)Jerusalem, Israel

Ryan F. DonnellySchool of PharmacyQueen’s University BelfastBelfast, UK

List of Contributors

List of Contributorsx

Tomáš EtrychInstitute of Macromolecular ChemistryAcademy of Sciences of the Czech RepublicPrague, Czech Republic

Jonathan J. FaigDepartment of Chemistry and Chemical BiologyRutgers UniversityPiscataway, NJ, USA

Leanna L. FosterMacromolecular Science and Engineering CenterUniversity of MichiganAnn Arbor, MI, USA

Tomoko FujiwaraDepartment of ChemistryUniversity of MemphisMemphis, TN, USA

Kazuki FukushimaDepartment of Polymeric and Organic Materials EngineeringYamagata University, Yonezawa, Yamagata, Japan

Rohan GhadiDepartment of PharmaceuticsNational Institute of Pharmaceutical Education and Research (NIPER)Hyderabad, India

Ruslan GuliyevDepartment of Chemistry and Chemical BiologyRutgers UniversityPiscataway, NJ, USA

Ivan O. HevusDepartment of Coatings and Polymeric MaterialsNorth Dakota State UniversityFargo, ND, USA

Lebohang HlaleleDepartment of Chemistry andPolymer ScienceStellenbosch UniversityMatieland, South Africa

David HuesmannInstitute of Organic ChemistryJohannes Gutenberg‐ Universität MainzMainzGermany

Wahid KhanDepartment of PharmaceuticsNational Institute of Pharmaceutical Education and Research (NIPER)Hyderabad, India

Bert KlumpermanDepartment of Chemistry and Polymer ScienceStellenbosch UniversityMatieland, South Africa

Ananiy M. KohutDepartment of Organic ChemistryLviv Polytechnic National UniversityLviv, Ukraine

Libor KostkaInstitute of Macromolecular ChemistryAcademy of Sciences of the Czech RepublicPrague, Czech Republic

List of Contributors xi

Kenichi KurodaMacromolecular Science and Engineering Center andDepartment of Biologic and Materials SciencesSchool of DentistryUniversity of MichiganAnn Arbor, MI, USA

Eneko LarrañetaSchool of PharmacyQueen’s University BelfastBelfast, UK

Ang LiDepartment of Chemistry and Macromolecular Studies GroupLouisiana State UniversityBaton RougeLA, USA

Atsushi MaruyamaDepartment of Life Science and TechnologyTokyo Institute of Technology Yokohama, Japan

Henning MenzelInstitut für Technische ChemieTechnische Universität BraunschweigBraunschweig, Germany

Masato MizutaniDepartment of Chemistry and Chemical BiologyBaker LaboratoryCornell UniversityIthaca, NY, USA

Randall W. MoreadithSerina TherapeuticsHuntsville, AL, USA

Giulia MorgeseLaboratory for Surface Science and TechnologyDepartment of MaterialsETH ZürichZürich, Switzerland

Eameema MuntimaduguDepartment of PharmaceuticsNational Institute of Pharmaceutical Education and Research (NIPER)Hyderabad, India

Rodolphe ObeidR&D/Process Development & Manufacturing Scale‐Up, IntelGenx Corp.St. LaurentQuebecCanada

Yukari OdaDepartment of Applied ChemistryKyushu UniversityFukuoka, Japan

Edmund F. PalermoDepartment of Materials Science and EngineeringRensselaer Polytechnic InstituteTroy, NY, USA

Rueben PfukwaDepartment of Chemistry and Polymer ScienceStellenbosch UniversityMatieland, South Africa

List of Contributorsxii

Naohiko ShimadaDepartment of Life Science and TechnologyTokyo Institute of TechnologyYokohama, Japan

Jessica M. SimpsonDepartment of Chemistry and Macromolecular Studies GroupLouisiana State UniversityBaton Rouge, LA, USA

Jeff SparksCelsion CorporationHuntsville, AL, USA

Garrett L. Sternhagen Department of Chemistry and Macromolecular Studies GroupLouisiana State UniversityBaton Rouge, LA, USA

Hisaschi T.C. TeeMax Planck Institute for Polymer Research (MPIP)Mainz, Germany

Nicolay V. TsarevskyDepartment of Chemistry and Center for Drug Discovery, Design, and Delivery, Southern Methodist UniversityDallas, TX, USA

Kathryn E. UhrichDepartment of Chemistry and Chemical BiologyRutgers UniversityPiscataway, NJ, USA

Andriy S. VoronovDepartment of Coatings and Polymeric MaterialsNorth Dakota State UniversityFargo, ND, USA

Stanislaw A. VoronovDepartment of Organic ChemistryLviv Polytechnic National UniversityLviv, Ukraine

Tacey X. ViegasSerina TherapeuticsHuntsville, AL, USA

Shannon R. WoodruffDepartment of Chemistry and Center for Drug Discovery, Design, and Delivery, Southern Methodist UniversityDallas, TX, USA

Frederik R. WurmMax Planck Institute for Polymer Research (MPIP)Mainz, Germany

Sunting XuanDepartment of Chemistry and Macromolecular Studies GroupLouisiana State UniversityBaton Rouge, LA, USA

Donghui ZhangDepartment of Chemistry and Macromolecular Studies GroupLouisiana State UniversityBaton Rouge, LA, USA

1

Part I

Pseudo‐Peptides, Polyamino Acids, and Polyoxazolines

Polymers for Biomedicine: Synthesis, Characterization, and Applications, First Edition. Edited by Carmen Scholz. © 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

3

1

1.1 Introduction

Materials made from polypeptides, and recently also polypeptoids, have received considerable and growing attention in recent years. Since synthetic polypeptides, just like natural proteins, are made up of amino acids, they can be non‐toxic, biocompatible, and degradable in the body while they remain stable in aqueous solution. The multitude of different side chains enables the design of peptidic superstructures like polyion complexes [1,2], polymer micelles [3,4], polymer vesicles [5,6], nanofibers or ‐tubes [7], and hydrogels [8].

Apart from exactly defined polypeptides (i.e., proteins) that show a defined sequence of amino acids, there are also natural polypeptides that resemble less defined classic synthetic polymers. One of these polypeptides is poly(ɣ‐glutamic acid) [9,10], which is produced by bacteria and cnidaria [11]. It is the major constituent of nattō (Japanese food from fermented soy beans) and approved by the FDA for cosmetic applications.

1.2 Synthesis of Poly(peptide)s

Synthetic polypeptides were first described by Leuchs in the beginning of the twentieth century, although their polymeric nature was not acknowledged at that time [12–14]. Many researchers have explored synthetic polypeptides through the twentieth century [15,16] partially with poor results regarding polymerization kinetics, end‐group integrity, or dispersity, in particular with more complex systems such as block copolypeptides, star‐like polypeptides, or bottle‐brush polymers. The reasons for this are manifold, including monomer

Characterization of Polypeptides and Polypeptoides – Methods and ChallengesDavid Huesmann and Matthias Barz

Institute of Organic Chemistry, Johannes Gutenberg‐Universität Mainz, Mainz, Germany

Polymers for Biomedicine4

purity, monomer instability over prolonged periods of time and the fact that the polymerization does not necessarily follow a single mechanism (Figure 1.1). The most prominent competing pathways are the normal amine mechanism (NAM, which leads to a classical chain growth) and the activated amine mech-anism (AMM, which leads to undefined polymers through condensation of polymer chains). Further, addition of an N‐carboxyanhydride, NCA, monomer

NH

OO O

R

H2NR′

AMM

NAM

N–

OO O

R

R′NH

OHN

R

OH

O

NH

OO O

R

–R′NH3+

R′NH

O

NH2

R

R′NH

O

NH2

R

N

OHN

R

O

O

OR

N

O

NH–

R

O

O

OR

O–

O

N

O

NH2

R

O

O

OR

N–

OO O

R

N

O–O

RC O

R′NH

O HN

R

O

O O

NH

R

OH

O

+NCA+NCA

+NCA

n

Isocyanate

Carbamate

NCA

RNH2

CondensationProducts

N

OHN

R

O

O

OR

+

+

n

H

R′NH

OHN

R

HN

O

OH

O

RUrea Derivative

Rearrangement–CO2

–CO2

–CO2

Figure 1.1 Mechanisms of NCA polymerization: Normal amine mechanism (NAM) and activated monomer mechanism (AMM).

Characterization of Polypeptides and Polypeptoides 5

before decarboxylation can lead to carbamates, which can rearrange into urea units, while a deprotonated NCA can open to form an isocyanate. A more in‐depth discussion of the reaction mechanism is outside of the focus of this chapter and can be found in excellent reviews and books [15–17].

The complex reaction mechanism has led to the development of controlled NCA polymerization methods starting in the end of the last century. In the late 1990s, the group of Timothy Deming was the first to demonstrate that the NCA polymerization using transition metal catalysts proceeds in a living man-ner and yields well‐defined polypeptides (Figure 1.2) [18]. While this approach has been very successful for the preparation of well‐defined and complex poly-peptide architectures [5,6,19], it has the need for a transition metal catalyst. Additionally, the synthesis of hybrid structures remains challenging since the transition metal catalyst needs to be modified [20].

As a complementary approach, Cheng and coworkers have reported silylated amine initiators, which allow control over NCA polymerization [22,23]. The trimethylsilyl residue remains at the polymer terminus over the course of the polymerization, allowing the preparation of defined polypeptides (Figure 1.3). The rate of polymerization is not slowed down by this polymerization tech-nique as the polymerization (M/I = 300) was reported to be completed within 24 h or less. Amine initiated polymerization has been reported to be complete

R

O

HNO

O

(L)nM

M = Co, Ni

+–CO

(L)n MR

HNO

O

NCA

–2 CO2

–CO2

NH

HNR

O

RNCA

HN

NHM(L)n

M(L)n

HN

R

O

O

R R

proton

migrationHNM(L)n

N

R O

NH

O

R

R

HNM(L)n

M(L)n

N

R O

Polymer

NCAM(L)n

N

NH

HN

R

R

O

O

Polymer

protonmigration HN N

R O

NH

O

Polymer

R

Initiation:

Propagation:

Figure 1.2 Initiation and propagation of metal catalyzed NCA polymerization. Source: Deming 2000 [21]. Reproduced with permission of American Chemical Society.

Polymers for Biomedicine6

within the same time frame (17 h for Xn = 438, poly(benzyl glutamic acid), (PGlu(OBn)) [24].

On the other hand, several approaches have been investigated to optimize the conditions of conventional amine initiated NCA polymerization. Vayaboury et al. used non‐aqueous capillary electrophoresis to show a dramatic increase of living chain ends by lowering the polymerization temperature to 0 °C [25]. Unfortunately, no GPC plots and polymer dispersities of the obtained poly-mers were presented. Heise and coworkers investigated the influence of reduced temperature further [26] and used vacuum for the removal of CO2 from the reaction to increase its speed [27]. CO2 liberation is a step in NCA polymerization, which depends highly on the pressure in the reaction vessel. Wooley and coworkers reported the removal of CO2 by nitrogen flow through the reaction mixture, thereby increasing also the polymerization speed [28]. Both findings are surprising since theoretical studies and experiments have shown that CO2 liberation is not the rate determining step of the polymeriza-tion [15,29–31]. However, the performed control polymerizations (no nitrogen flow) yielded polypeptides with high dispersities of 1.38 and 2.19 for Xn of 50 and 100, respectively, while dispersities of PGlu(OBn) initiated by primary amines are usually well below 1.2 [32].

Scholz and Vayaboury tackled the issue of different secondary structures in the growing peptide by introducing thiourea to suppress hydrogen‐bond for-mation [33]. It was found that the dispersity of polypeptides decreased mark-edly, independent of whether macroinitiators (PEG‐NH2) or low molar mass initiators (hexylamine) were used.

TMSTMS TMS

TMS

TMSTMS

NCA

SiMe3

Si

Si

Si

Si

HN

HN

HN

HN

R

O

O

O O

O

O

HN

O

OR

OO

OO

R O

O

ONH2

H2N

O

R

Si

O

O

O

O

R

R NH

NH

n

NH

NH

N

HN

HN

O

Figure 1.3 Mechanism of trimethylsilyl‐mediated NCA polymerization. Source: Lu 2007 [22]. Reproduced with permission of American Chemical Society.

Characterization of Polypeptides and Polypeptoides 7

In a different approach, Schlaad and coworkers introduced HCl salts of primary amines as initiators, lowering the reactivity of the growing chain end [34]. Elevated temperatures (40–80 °C) were used to counteract the slow polymeri-zation. This method was complemented by other ammonium salts, namely different acetates [35] and recently the non‐nucleophilic tetraflouroborates by Vicent and coworkers [36].

Finally, Hadjichristidis and coworkers reported on the use of highly puri-fied  monomers, solvents and reagents under high vacuum techniques [24]. Interestingly, these results suggest that all the previously mentioned potential side reactions are impurity related and that control can be achieved by working with highly pure solvents, monomers, and initiators.

1.3 Characterization of Poly(peptide)s

The aim for better and better control over the NCA polymerization over the last century was also accompanied by the development of analytical methods that allowed for a better characterization of polypeptidic materials. As with many classes of polymers, polypeptides are often characterized by the most widely used analytical techniques NMR and GPC to determine composition, size and dispersity of the polymers. However, due to the periodical peptide bond in the polypeptide backbone, these polymers are often not in a random coil conformation – as is usually the case for other polymers. This leads to two major challenges in the characterization of polypeptides: (1) The secondary structures must be characterized using for example NMR, IR, CD spectros-copy, or X‐ray diffraction and (2) the different secondary structures lead to a change in the hydrodynamic radius of the polymers, limiting the usefulness of  methods that rely on the hydrodynamic radius to deduce other physical parameters (e.g., GPC). It is worth noting, that these challenges do not apply to most polypeptoids, since they lack the free hydrogen at the amide bond and therefore do usually not form secondary structures.

The combination of a complex polymerization mechanism with many potential side reactions on one hand and challenging characterization on the  other calls for extremely careful interpretation of obtained data. In the following sections, we will introduce different analytical methods for analyzing polypeptides highlighting their advantages and limitations.

1.4 Gel Permeation Chromatography (GPC)

Gel permeation chromatography is certainly one of the most important analy-sis methods in polymer chemistry yielding not only average molecular weights, but also a value for polymer dispersity, describing the width of the molecular

Polymers for Biomedicine8

weight distribution. However, these molecular weight distributions are often not obtained directly, but indirectly using a calibration by polymer standards.

The separation in the GPC column itself is enabled by polymer beads with different pore sizes. Large molecules cannot enter the pores and elute first from the columns, while smaller molecules can diffuse into the pores, thus remaining in the column for a longer time.

However, the separation does not occur by molecular weight, but by polymer size (i.e., hydrodynamic volume) and molecular weight is only inferred from calibration. To obtain correct molecular weights from this method, two condi-tions should be fulfilled: (1) The polypeptide has to be in one conformation and (2) the standards for the calibration have to be the same polymer (or at least very similar in structure) and have to exhibit the same secondary structure as the polymer measured.

Both conditions are virtually never fulfilled when working with polypeptides. Even very good solvents for polypeptides like dimethylformamide (DMF), dimethylacetamide (DMAc), N‐methylpyrolidone (NMP), and hexafluoroiso-propanol (HFIP) are usually not able to suppress secondary structures in pro-tected polypeptides, leading to a strong change in hydrodynamic volume [37]. Further, the standards used for calibration are in many cases poly(ethylene glycol) (PEG) or poly(methyl methacrylate) (PMMA), both molecular struc-tures are far from that of polypeptides (Figure 1.4).

Therefore, it appears reasonable to produce GPC standards for each class of polypeptides, as done by Hadjichristidis and coworkers. They synthesized nar-rowly distributed PGlu(OBn) and determined their molecular weight (Mn) by membrane osmometry. They then used these samples for the calibration of their GPC [24]. Using a calibration with polypeptide standards, the molecular weights from GPC measurements can be used to create a meaningful kinetic plot (Figure 1.5). Unfortunately, calibration with polypeptides is seldom per-formed, since polypeptide standards are not commercially available.

In some cases, bimodal molecular weight distributions can be observed due to a change in secondary structure, which results in a pronounced change in hydrodynamic volume. This can, for example, be seen in growing poly(N‐ ε‐benzyloxycarbonyl‐L‐lysine) (PLys(Z)) chains that undergo a transition in secondary structure at a degree of polymerization around 15 [37]. Observing the polymer at different time points of the polymerization shows a monomodal distribution (random coil) changing to a bimodal distribution when two secondary structures (random coil and α‐helix) are present (Figure 1.6). Once

O

PMMA PEG

OHN

R

O

Polypeptide

OH3C

Figure 1.4 Molecular structures of common GPC standards compared to polypeptides.

Characterization of Polypeptides and Polypeptoides 9

[M]/[I] = 52

[M]/[I] = 110

[M]/[I] = 223

[M]/[I] = 438

1,6 × 105

2.4 × 10–2

2.0 × 10–2

1.6 × 10–2

1.2 × 10–2

8.0 × 10–3

4.0 × 10–3

9.0 × 10–3 1.8 × 10–2 2.7 × 10–2 3.6 × 10–2 4.5 × 10–2 5.4 × 10–2

kp, o

bs (

1 m

ol–1

min

–1)

0.00.0

1,4 × 105

1,2 × 105

1,0 × 105

8,0 × 104

6,0 × 104

4,0 × 104

2,0 × 104

0,0

[M]/[I] = 743

[I], mol–1

10 20 30 40 50

Polymer conversion

60 70 80 90 100

Figure 1.5 Kinetic plot from the amine initiated polymerization of poly(glutamic acid). Molecular weights were obtained from GPC data, calibrated with poly(glutamic acid) standards. Source: Aliferis 2004 [24]. Reproduced with permission of American Chemical Society.

16 18 20

Vol/mL

Polymerizationtime1

0.8

0.6

0.4

0.2

0

RID

22 24 26 28

Figure 1.6 DMF GPC of a growing PLys(Z) chain showing the transition from random coil to α‐helix. Source: Huesmann 2014 [37]. Reproduced with permission of American Chemical Society.

Polymers for Biomedicine10

all chains have reached the appropriate length to form only α‐helices, the dis-tribution becomes monomodal again.

The same behavior is also visible for PLys(Z) of different degrees of polym-erization (Figure 1.7).

An estimation of the hydrodynamic radius (Rh) of a PLys(Z) random coil (worm‐like chain model) and α‐helix (cylinder) with DP = 15 shows an increase in hydrodynamic volume of α‐helix over random coil [37].

Taking this into account, it is clear that molecular weights of polypeptides from GPC have to be treated with extreme care. A molecular weight obtained with PMMA standards should never be treated as the real molecular weight of polypeptides, nor should every broadening or bimodal molecular weight dis-tributions be directly related to poorly defined polymers. Bimodal molecular weight distributions are not necessarily caused by side reactions but might be attributed to the coexistence of different secondary structures. Vice versa, it would not be wise to infer the coexistence of different secondary structures just from bimodal GPC distributions. Thus, in addition to standard GPC analytics it seems highly beneficial to investigate solution conformation by complementary characterization methods.

1

0.8

0.6

0.4

0.2

0

16 17 18 19 20 21

Vol/mL

PLys(Z)25

PLys(Z)50

PLys(Z)100

PLys(Z)200

RID

Figure 1.7 HFIP GPCs of PLys(Z), from right to left: P(Lys(Z)25, P(Lys(Z)50, P(Lys(Z)100, P(Lys(Z)200. Source: Huesmann 2014 [37]. Reproduced with permission of American Chemical Society.

Characterization of Polypeptides and Polypeptoides 11

1.5 Infrared (IR) Spectroscopy

Fourier transformed infrared (FT‐IR) spectroscopy is one of the oldest analyti-cal methods in organic chemistry and has become an established tool for the structural characterization of proteins. Using infrared light (wave numbers 4000–400 cm−1) vibrational modes in molecules are exited, leading to the absorption of light of a characteristic wavelength. Samples can be measured in solution or in the solid state. The use of attenuated total reflection (ATR) units allows easy measurements of small sample volumes.

For the analysis of polypeptides, the absorption of the amide I band (CO stretching, around 1650 cm−1) and amide II band (NH bending, CN stretching, around 1550 cm−1) are typically used. The typical amide I frequencies of protein secondary structures are shown in Table 1.1. α‐Helices show absorption between 1650–1657 cm−1, while random coils shift the absorption to lower wave numbers (1640–1651 cm−1) and turns show an adsorption at higher wave numbers (1655–1675 cm−1, 1680–1696 cm−1). The distinction of parallel and anti‐parallel β‐sheets is not easy, since their absorption is very similar. Parallel β‐sheets absorb at 1626–1640 cm−1 and anti‐parallel β‐sheets at 1612–1640 cm−1. However, anti‐parallel β‐sheets have a second, weak band at 1670–1690 cm−1, which allows discrimination between the two conformations. It should be stressed that these numbers are guidelines and may vary with changing amino acids [38,39].

IR spectra can of course only provide a qualitative picture of the secondary structures present in the polypeptide. Further, the bands are sensitive to the applied solvent and its water content. For the characterization of synthetic polypeptides organic solvents are often necessary to dissolve protected poly-peptides like DMF or DMAc. These solvents have strong amide bond

Table 1.1 Typical amide I frequencies of different secondary structures. Source: Pelton 2000 [40]. Reproduced with permission of Elsevier.

Conformation Amide I/cm−1

α‐helix 1650–1657

β‐sheet (anti‐parallel) 1612–1640,1670–1690 (weak)

β‐sheet (parallel) 1626–1640turn 1655–1675,

1680–1696random coil 1640–1651

Polymers for Biomedicine12

absorption bands themselves and can therefore not be used when looking at secondary structures. Water is also problematic, due to absorption at 1650 cm−1, which can be suppressed using high peptide concentrations, short path length and careful baseline subtraction. Urea and thiourea absorb between 1600–1700 cm−1 and also trifluoroacetates are problematic due to their absorption at 1673 cm−1. HFIP on the other hand, does not absorb between 1500–3000 cm−1 and can easily dissolve most polypeptides. Whenever measurements are per-formed in dry state, it should be kept in mind that there might be a pronounced change in secondary structure due to the absence of interaction with the solvent, which leads to a partial or complete collapse of the polypeptide.

Zhang and coworkers synthesized a mannose modified poly(glutamic acid) which, like its precursors, adopts an α‐helical conformation [41]. Among other techniques, IR spectroscopy was used to confirm this conformation in the solid state (Figure 1.8).

amide II amide I

D

D

C

1000 1500 2000 2500 3000 3500

Wave number (cm–1)

Abs

orba

nce

(a.u

.)

A BO O

ONaN3, DMF

60 °C, 48 hn

HN

Cl O O

O

PMEDTA, CuBrDMF, rt, 24 h

n

HN

N3 O O O

O

OH OH

OH

OH

OO

OOHHO

HOHO

n

HN

NN N

C

B

A

νC=O

νalkyne

νN=N=N

νC-H νO/N-H

νO-H

Figure 1.8 IR spectrum of mannose modified poly(glutamic acid) as well as its precursors, showing typical amide I and II bands of an α‐helical conformation. Source: Tang 2010 [41]. Reproduced with permission of American Chemical Society.

Characterization of Polypeptides and Polypeptoides 13

Scholz’s group investigated the secondary structure of oligopeptides [42]. IR was used to identify the different secondary structures of 20‐mers of protected lysine, cysteine, and glutamic acid. A shift of the amide I band can be seen between the α‐helical lysine and glutamic acid on one hand and the cysteine in a β‐sheet conformation on the other hand (Figure 1.9).

Another very useful application of IR spectroscopy in NCA polymerization lies in measuring the progress of the reaction. The monomer conversion can be easily visualized by the disappearance of the strong absorption band of the NCA at around 1860 and 1785 cm−1 (in DMF) (Figure 1.10).

Like FT‐IR spectroscopy, Raman spectra can be recorded by measuring the scattered instead of the absorbed light. In this case, different selection rules apply and different bands might be visible.

In conclusion, IR‐spectroscopy is a very powerful and easy to use tool to get a qualitative insight into the secondary structure of polypeptides, while quan-titative interpretation needs further treatment of the raw data by peak decon-volution and is strictly only applicable to natural proteins. When measuring in solution, the absorption of the solvent must be kept in mind, while the dry state may not represent the conformation of the polypeptide in solution.

N-hexylamino-p(L-Lys)

N-hexylamino-p(L-Cys)

N-hexylamino-p(L-Glu)

α-helixLysine

Cysteine

Glutamate

1850

3650 3150 2650 2150 1650 1150 650

95

85

75

65

55

45

1650 1450

α-helixβ-sheet

Figure 1.9 IR spectrum of protected lysine, cysteine, and glutamic acid 20‐mers. The arrows indicate the amide I band. Source: Ulkostki 2013 [42]. Reproduced with permission of American Chemical Society.

Polymers for Biomedicine14

1.6 Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectroscopy is a complementary technique commonly used in polymer synthesis, as well as protein structure determination. The technique is based on the absorption of electromagnetic radiation by nuclei with a spin different from zero (1H, 13C, 15N) in a strong magnetic field. In this section, only the application of relatively simple 1D NMR experiments for the determination of chain length, composition, and secondary structure will be discussed. More complex or multidimensional NMR techniques for the determination of pro-tein tertiary structures like NOESY will not be discussed here but can be found in literature [43,44].

The simplest application is the determination of chain length, by comparing protons of the initiator or end group to protons of the repeating unit. To receive accurate results from this method, all chains need to carry the reference groups. To reference to the initiator, all chains should be started by the initiator and not by impurities in the solvent (e.g., water). For the end group the post polymeri-zation modification reaction performed needs to be quantitative. Further, the reference signals have to be well separated from the signals of the repeat unit to allow separate integration. To receive an accurate measurement even for larger polypeptides, a strong initiator signal is beneficial. Neopentylamine has proven to be a very reliable initiator that features nine equivalent protons,

Wavenumber (cm–1)

DMFMonomerin DMF

Polymerin DMF

Tra

nsm

ittan

ce

2000 1800 1600 1400 1200 1000 600800

1

0.8

0.6

0.4

0.2

1.2

1800 175018501900 900 8509501000

1.05

1

0.95

0.9

0.85

1.1

1.05

1

1.1

Figure 1.10 Useful IR NCA peaks to control monomer conversion (Lys(Z) in DMF).

Characterization of Polypeptides and Polypeptoides 15

which do not overlap with any amino acid signals [37]. Using this initiator, polypeptides with length of more then 420 repeating units can be identified by NMR as can be seen in Figure 1.11.

NMR spectroscopy can further be used to determine the secondary struc-ture of polypeptides in solution. Especially the α‐position of amino acids is very sensitive to changes in the secondary structure. This can be seen in the chemical shift of the α‐proton as well as the α‐carbon in 1H and 13C NMR spectroscopy, respectively [45–47]. The chemical shifts for 13Cα, 13Cβ, 13C’, 15N, 1HN, and 1Hα in α‐helix, β‐sheet, and random coil conformation have been collected and can be used for easy reference (Table 1.2) [47]. These chemical shifts given in Table 1.2 are attributed to unprotected amino acids in water, but  only shift slightly when measuring protected polypeptides in organic solvents [37].

The effect of secondary structure in a growing PLys(Z) chain can be observed in Figure 1.12 (left). While the NMR spectrum shows two Hα peaks for short PLys(Z) the peak of the random coil disappears with increasing degree of polymerization.

13C‐NMR spectroscopy was extensively applied for the study of secondary structures in acetonitrile, TFA and in the solid state [48–50], as well as for sequence analysis [51] by Kricheldorf and coworkers. Figure 1.12 (right) shows a solid state 13C CP/MAS (cross polarization/magic angle spinning) NMR spectrum of poly(norvaline). The peaks corresponding to Cα, Cβ, and C’ show split peaks indicating the coexistence of α‐helix and β‐sheets.

O

HN

O

O

9 H

840 H

NHb

agg

g

f f

f e

NH20.82n

b

c

� (ppm)

b

d

a,b gfec

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

d

bb

b

Figure 1.11 NMR of poly(N‐ε‐benzyloxycarbonyl‐L‐lysine) (PLys(Z)), degree of polymerization 420, initiator: neopentylamine.

Table 1.2 Statistically derived chemical shifts in secondary structures. Source: Wishart 2011 [47]. Reproduced with permission of Elsevier.

13Cα13Cβ

13C’ 15N 1HN1Hα

Residue Helix Sheet Coil Helix Sheet Coil Helix Sheet Coil Helix Sheet Coil Helix Sheet Coil Helix Sheet Coil

Ala 54.8 51.5 52.8 18.3 21.1 19.1 179.4 176.1 177.7 121.4 124.5 123.6 8.08 8.44 8.15 4.03 4.77 4.26Cys (ox) 58.0 55.0 55.6 39.4 43.9 41.0 176.2 173.6 174.9 117.7 121.0 118.0 8.20 8.80 8.25 4.15 5.15 4.65Cys (red) 61.3 56.9 57.5 27.8 30.2 29.4 176.2 173.6 174.9 117.7 121.0 118.0 8.20 8.80 8.25 4.15 5.15 4.65Asp 56.7 53.9 54.2 40.5 42.3 40.9 178.0 175.5 176.3 119.2 122.2 120.0 8.18 8.51 8.36 4.43 4.94 4.60Glu 59.1 55.5 56.9 29.4 32.0 30.2 178.6 175.4 176.4 119.0 122.1 120.4 8.22 8.53 8.37 4.01 4.78 4.28Phe 60.8 56.7 58.0 38.8 41.5 39.5 177.1 174.3 175.6 119.2 121.1 119.7 8.18 8.75 8.17 4.16 5.09 4.54Gly 46.9 45.2 45.5 N/A N/A N/A 175.5 172.6 173.9 107.5 109.3 109.1 8.29 8.34 8.33 3.81 4.20 3.96His 59.0 55.1 55.9 29.5 31.9 30.0 177.0 174.2 174.8 118.0 120.5 118.7 8.10 8.62 8.21 4.33 5.06 4.53Ile 64.6 60.1 61.0 37.6 39.9 38.7 177.7 174.9 175.6 119.7 122.8 120.9 8.02 8.68 7.98 3.67 4.68 4.15Lys 58.9 55.4 56.6 32.3 34.6 32.8 178.4 175.3 176.3 119.2 122.2 120.5 7.99 8.48 8.23 3.99 4.69 4.26Leu 57.5 54.1 54.9 41.6 43.8 42.4 178.5 175.7 176.9 119.6 124.1 121.5 8.05 8.60 8.08 4.00 4.82 4.36Met 58.1 54.6 55.7 32.3 35.1 33.4 178.0 174.8 175.4 118.2 121.7 119.7 8.09 8.64 8.18 4.07 4.96 4.38Asn 55.5 52.7 53.2 38.6 40.1 38.6 176.9 174.6 175.1 117.3 121.6 118.2 8.22 8.60 8.40 4.48 5.06 4.66Pro 65.5 62.6 63.5 31.5 32.3 31.9 178.3 176.2 176.9 N/A N/A N/A N/A N/A N/A 4.22 4.60 4.37Gln 58.5 54.8 56.1 28.5 31.3 29.1 178.0 174.9 175.9 118.4 121.1 119.5 8.04 8.48 8.23 3.99 4.80 4.26Arg 58.9 55.1 56.4 30.1 32.2 30.7 178.3 175.1 176.0 118.9 122.3 120.4 8.07 8.56 8.25 3.99 4.74 4.24Ser 60.9 57.5 58.4 63.1 65.2 64.0 175.9 173.6 174.5 114.9 116.9 115.6 8.14 8.50 8.23 4.25 4.91 4.47Thr 65.6 61.1 61.6 68.9 70.8 70.1 175.9 173.7 174.7 114.6 116.5 113.4 8.04 8.51 8.16 4.00 4.86 4.45Val 66.2 60.8 62.1 31.5 33.9 32.7 177.7 174.8 175.7 119.2 121.9 119.8 8.02 8.62 8.04 3.58 4.60 4.12Trp 60.0 56.4 57.8 29.3 31.5 29.7 178.1 175.4 176.2 119.8 122.1 120.2 8.12 8.59 7.92 4.38 5.19 4.55Tyr 61.0 56.8 58.0 38.3 41.0 39.0 177.4 174.5 175.4 119.2 121.4 119.5 8.07 8.68 8.06 4.09 5.10 4.52