synthesis and characterization of dye-labeled copolymers by … · 2013-11-14 · ii synthesis and...

Synthesis and Characterization of Dye-labeled Copolymers by Reversible Addition-Fragmentation

Transfer (RAFT) Polymerization

by

Binxin Li

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Binxin Li (2008)

ii

Synthesis and Characterization of Dye-labeled Copolymers by

Reversible Addition-Fragmentation Transfer (RAFT)

Polymerization

Binxin Li

Master of Science

Department of Chemistry University of Toronto

2008

Abstract

Copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-hydroxysuccinimide

methacrylate (NMS) were synthesized by reversible addition-fragmentation transfer (RAFT)

polymerization using a semi-batch method. The copolymers were prepared in a wide range of

molecular weights (Mn= 4,200-14,000 g/mol) with narrow polydispersities (1.2-1.4). A new

approach was developed to prepare a modified RAFT chain transfer agent, a naphthalimide-dye-

labeled dithiobenzoate. It was used to prepare a naphthalimide-dye end-labeled poly(HPMA-co-

NMS). The copolymer was characterized by four different methods, 1H NMR spectroscopy via

end group characterization and using 3-(trimethylsilyl)propionic acid-d4 sodium salt (TSP)

external standard, end group analysis by UV-Vis spectroscopy and by GPC. The results obtained

from these measurements are in good agreement.

iii

Acknowledgments

I am very grateful to my research supervisor, Professor Mitchell A. Winnik, at University

of Toronto. He offered me great opportunity to be involved in this great research project. I learnt

many things from him, not only scientific skills, but also writing and communication skills. I

would like to thank him for his supervision, guidance and support.

My thanks also go to the members of the project “Metal Tagging of Antibodies for Early

Detection of Cancer Cells by ICP-MS”: Professor Mark Nitz, Dr. Xudong Lou, Dr. Conrael

Siegers, Dr. Vladimir Baranov, Dr. Olga Ornatsky, Isaac Herrera, Daniel Majonis, Ahmed I.

Abdelrahman. We had so many valuable discussions in the past two years. It was such enjoyable

experience to work with them.

I would also like to thank Professor William Reynolds and Dr. David McNally for their

kind discussions about NMR measurements, and Mr. Letian Wang for his constructive

suggestions on the synthesis of naphthalimide derivatives.

My special thanks go to all current and former members in Professor Winnik’s group and

their families: Dr. Gerald Guerin and Dr. Sophie Lun Sin, Dr. Jeff Haley and Heather Haley, Dr.

Pablo Froimowicz, Graeme Cambridge, Wanjuan Lin and Ruirui Huang, Yuanqing Liu and Ying

Sun, Lisa zur Borg, Vania Freire and Sergio Freire, Mohsen Soleimani, Neda Felorzabihi,

Daniele Fava, Syed Nawazish Ali, Lei Shen, Maren Schulze, Feng He, Dr. Walter Schroeder, Dr.

John Spiro, Dr. Hai Wang, Mingfeng Wang and Yishan Wang. They made my life in Toronto

much colorful. Thanks for their encouragement and strong support.

I dedicate my thesis to my families and dear friends for their support and unconditional love.

iv

Table of Contents

1 Introduction 1

1.1 Background 1

1.2 Objectives of the project 7

1.3 The system to be studied 13

1.4 Overview of the thesis 14

1.5 References 15

2 Homopolymerization of N-(2-hydroxypropyl)methacrylamide by

RAFT Polymerization 18

2.1 Introduction 18

2.2 Experimental Section 23

2.2.1 General Information 23

2.2.2 Synthesis and Characterization of HPMA and the Chain Transfer

Agent (CTA) 24

2.2.3 Synthesis and Characterization of Poly(HPMA) 26

2.3 Results and Discussion 28

2.4 Conclusions 40

2.5 References 41

3 Copolymerization of N-(2-hydroxypropyl)methacrylamide and

N-hydroxysuccinimide methacrylate by RAFT Polymerization 43

3.1 Introduction 43



3.2.2 Synthesis and Characterization of NMS 49

3.2.3 Synthesis and Characterization of Copolymers of HPMA and NMS 50


3.4 Conclusions 63

3.5 References 64

v

4 Synthesis and Characterization of Naphthalimide-dye-labeled

Poly(HPMA-co-NMS) by RAFT Polymerization 66

4.1 Introduction 66



4.2.2 Synthesis and Characterization of the Naphthalimide-dye-labeled

CTA 74

4.2.3 Synthesis and Characterization of the Naphthalimide-dye-labeled

Copolymer of HPMA and NMS 78


4.4 Conclusions 94

4.5 References 96

vi

List of Tables

Table 1.1 HPMA-based copolymer conjugates in clinical development 3

Table 1.2 Comparison of NMP, ATRP and RAFT 12

Table 2.1 Experimental results of the homopolymerization of HPMA 31

Table 2.2 Kinetics of the homopolymerization of HPMA at [M]/[CTA] =

80

34


160

37


320

38

Table 3.1 Experimental results of the RAFT polymerization of N-

hydroxysuccinimide methacrylate (NMS) using three different

CTAs

46

Table 3.2 Experimental results of the copolymerization of HPMA and

NMS

53

Table 3.3 Experimental results of the kinetics of the copolymerization of

HPMA and NMS

62

Table 4.1 Preparation of the naphthlimide-dye-labeled poly(HPMA-co-

NMS) by RAFT

82

Table 4.2 Experimental results of water content of the naphthalimide-dye

end-labeled poly(HPMA-co-NMS)

82

Table 4.3 GPC results of the naphthalimide-dye end-labeled poly(HPMA-

co-NMS)

84

Table 4.4 The characterization results of the naphthalimide-dye end-

labeled poly(HPMA-co-NMS) by 1H NMR

89

vii

Table 4.5 The determination of the absolute number-average molecular

weight of the naphthalimide-dye end-labeled poly(HPMA-co-

NMS)

93

Table 4.6 Summary of number-average molecular weight values of the

naphthalimide-dye end-labeled poly(HPMA-co-NMS)

determined with different methods

94

viii

List of Figures

Figure 1.1 Schematic representation of polymer-based nano-medicines 2

Figure 1.2 Current understanding of the mechanism of action of polymer-

drug conjugates

4

Figure 1.3 The structures of polymer–drug conjugates PK1 and PK2 8

Figure 1.4 The structure of the polymer–drug conjugate HPMA-TNP-470 10

Figure 2.1 Mechanism of reversible addition-fragmentation transfer

polymerization

19

Figure 2.2 Structure features of the thiocarbonylthio CTA and the related

intermediate compound

20

Figure 2.3 Guidelines of CTA design for various polymerizations 21

Figure 2.4 Synthesis of N-(2-Hydroxypropyl)methacrylamide 24

Figure 2.5 Synthesis of 4-cyanopentanoic acid dithiobenzoate 25

Figure 2.6 Synthesis pathway of poly(HPMA) 26

Figure 2.7 1H NMR spectrum of the homopolymer of HPMA 29

Figure 2.8 Kinetics (a) and evolution of the molecular weight and

polydispersity with conversion (b) during the polymerization of

HPMA (1 M in t-BuOH) performed at 80 °C in the presence of 4-

cyanopentanoic acid dithiobenzoate (CIDB 0.5 M in DMF) as the

chain transfer agent and AIBN as the initiator. The ratio of

HPMA/CIDB= 80

33






HPMA/CIDB= 160

36

ix






HPMA/CIDB= 320

39

Figure 3.1 Synthetic pathways of block copolymers of HPMA and

DMAPMA by RAFT polymerization

44

Figure 3.2 Synthesis strategies of poly(HPMA-co-NMS) 45

Figure 3.3 Synthesis of N-methacryloyloxysuccinimide 49

Figure 3.4 Synthesis pathway of poly(HPMA-co-NMS) 50

Figure 3.5 1H NMR spectrum of the copolymer of HPMA and NMS 54

Figure 3.6 1H NMR spectrum of conversion of HPMA and NMS in RAFT

copolymerization

58

Figure 3.7 Remaining of HPMA and NMS with reaction time in RAFT

copolymerization using a semi-batch method

59

Figure 3.8 The variation of composition of poly(HPMA-co-NMS) with

reaction time

60

Figure 3.9 Evolution of the molecular weight and polydispersity with time

(a), GPC traces of copolymers at various time intervals (b) during

the semi-batch RAFT copolymerization

61

Figure 4.1 Synthetic pathway of the reduction of a trithiocarbonate end

polymer and conjugation with N-(1-pyrenyl)maleimide

68

Figure 4.2 Synthetic pathway of the amine fictionalization and fluorescent

labeling of the polymer with a fluorescein N-hydroxysuccinimide

ester

69

Figure 4.3 Synthetic pathway of functional RAFT CTAs from the same

precursor 1

70

Figure 4.4 Synthetic pathway of biotinylated end-functionalized polymers 71

Figure 4.5 Synthesis of 9-isobutyl-4-ethylenediamino-1,8-naphthalimide 74

x

Figure 4.6 Synthesis of the naphthalimide-dye-labeled chain transfer agent 76

Figure 4.7 Synthesis pathway of the naphthalimide-dye end-labeled

Poly(HPMA-co-NMS)

78

Figure 4.8 Structure of the naphthalimide-dye end-labeled poly(HPMA-co-

NMS)

81

Figure 4.9 GPC traces of the naphthalimide-dye end-labeled poly(HPMA-

co-NMS) ([HPMA+NMS]/[CTA]/[AIBN]=320/2/1).

83

Figure 4.10 1H NMR spectrum of the naphthalimide-dye end-labeled

poly(HPMA-co-NMS)

86

Figure 4.11 Absorbance of 9-isobutyl-4-ethylenediamino-1,8-naphthalimide

at different concentrations in DMF

90

Figure 4.12 Absorbance at 440 nm versus concentration in DMF for 9-

isobutyl-4-ethylenediamino-1,8-naphthalimide (■),the

naphthalimide-dye end-labeled poly(HPMA-co-NMS) (1.13 g/L)

(●), and the fitted calibration curve (―)

91

Figure 4.13 Absorbance of the naphthalimide-dye end-labeled poly(HPMA-

co-NMS) (1.13 g/L) in DMF. The arrow points out the

absorbance at 440 nm

92

1

Chapter 1

Introduction

1 Introduction

1.1 Background

For many decades academia and pharmaceutical industry have put great effort

and money to fight against cancer, which is one of leading causes of premature death

worldwide. Great success has been achieved in cancer drug development since the end

of last century. Over thirty new cancer drugs have been approved by the U.S. Food and

Drug Administration (FDA) since 2001, such as Gleevec, Avastin and Sutent.

However, the overall success rate of cancer drugs is extremely low. Only 5% of drugs

entering clinical trials obtained marketing approval during 1990-2000.1 The main

reasons for the failure of candidate drugs have been identified. These include poor

pharmacokinetics (10%), insufficient therapeutic activity (30%) and toxicity (30%).2,3

Recently, a new therapy has been developed to address the issues that lead to failure of

candidate drugs. It uses polymers to conjugate candidate drugs, antibodies or DNA to

form anti-cancer nano-medicines (Figure 1.1).4 It integrates polymer chemistry,

biology, pharmaceutical science and nanotechnologies to accelerate the discovery and

development of cancer drugs.

2

Figure 1.1: Schematic representation of polymer-based nano-medicines4

(The figure is reproduced with the permission of the Nature Publishing Group)

Several polyethylene glycol (PEG)-based drugs have successfully entered into

clinical trials or the market for cancer therapy, such as PEG-adenosine deaminase and

PEG-α-interferon 2a.4,5 Moreover, PEGylation has already become a well-established

and powerful bio-technique in drug development.6 However, due to the nature of PEG,

the conjugation can only be made at the two ends of PEG, and this limits the loading

efficiency of functionalities. Comparing with PEGylated drugs, one advantage of

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates is that they have

potentially higher drug loading capability. In recent years, many HPMA-based

polymer conjugates as anticancer drugs also entered into clinical trials (Table 1.2).

3

Examples include HPMA copolymer-doxorubicin (PK1) and HPMA

copolymer-doxorubicin-galactosamine (PK2).4,5,7

Table 1.1: HPMA-based copolymer conjugates in clinical development4,5

Compound Name Status Indication

HPMA copolymer-doxorubicin PK1 Phase � Various cancers HPMA

copolymer-doxorubicin-galactosamine PK2 Phase � Particularly

hepatocellular carcinoma

HPMA copolymer-paclitzxel PNU166945 Phase � Various cancers HPMA copolymer-camptothecin MAG-CPT Phase � Various cancers HPMA copolymer-carboplatin

platinate AP5280 Phase �/� Various cancers

HPMA copolymer- diaminocyclohexane platinate

ProLindac Phase �/� Various cancers

The recent promising results from clinical trials and the market indicate that

polymers used in these conjugates can improve a drug/protein’s solubility, stability

and plasma half-life, and meanwhile reduce its toxicity/immunogenicity.5 The

mechanism of action of polymer-drug conjugates reveals reasons for the success of

these nano-medicines (Figure 1.1).5 The hydrophilic conjugates administered

intravenously can be designed to remain in circulation, because the clearance rate

depends on the molecular weight of conjugates and drugs covalently bound to

polymers are largely prevented from accessing normal tissues. The conjugates can

accumulate in tumor sites by passive targeting or active targeting. Passive targeting is

mainly due to the enhanced permeability and retention effect (EPR effect). Active

targeting can be introduced by binding cell-specific functionalities to the conjugates.

4

Depending on the linker used, drugs are usually released intracellular via lysosomal

enzymes or a lower pH environment.

Figure 1.2: Current understanding of the mechanism of action of polymer-drug

conjugates5

A: Hydrophilic polymer-drug conjugates administered intravenously can be designed to remain

in the circulation — their clearance rate depends on conjugate molecular weight, which governs

the rate of renal elimination. a: Drug that is covalently bound by a linker that is stable in the

circulation is largely prevented fom accessing normal tissues (including sites of potential

toxicity), and biodistribution is initially limited to the blood pool. b: The blood concentration of

drug conjugate drives tumour targeting due to the increased permeability of angiogenic tumour

vasculature (compared with normal vessels), providing the opportunity for passive targeting

due to the enhanced permeability and retention effect (EPR effect). c: Through the

incorporation of cell-specific recognition ligands it is possible to bring about the added benefit of

receptor-mediated targeting of tumour cells. d: It has also been suggested that circulating low

levels of conjugate (slow drug release) might additonally lead to immunostimulation. e: If the

polymer–drug linker is stable in the circulation, for example, N-(2-hydrox

ypropyl)methacrylamide (HPMA) copolymer–Gly-Phe-Leu-Gly–doxorubicin, the relatively high

5

level of renal elimination (whole body t1/2 clearance >50% in 24 h) compared with free drug (t1/2

clearance 50% in 4 days) can increase the elimination rate. B: On arrival in the tumour

interstitium, polymer-conjugated drug is internalized by tumour cells through either fluid-phase

pinocytosis (in solution), receptor-mediated pinocytosis following non-specific membrane

binding (due to hydrophobic or charge interactions) or ligand–receptor docking. Depending on

the linkers used, the drug will usually be released intracellularly on exposure to lysosomal

enzymes (for example, Gly-Phe-Leu-Gly and polyglutamic acid (PGA) are cleaved by cathepsin

B) or lower pH (for example, a hydrazone linker degrades in endosomes and lysosomes (pH

6.5–<4.0). The active or passive transport of drugs such as doxorubicin and paciltaxel out of

these vesicular compartments ensures exposure to their pharmacological targets. Intracellular

delivery can bypass mechanisms of resistance associated with membrane efflux pumps such

as p-glycoprotein. If >10-fold, EPR-mediated targeting will also enable the circumvention of

other mechanisms of drug resistance. Non-biodegradable polymeric platforms must eventually

be eliminated from the cell by exocytosis. Rapid exocytic elimination of the conjugated drug

before release would be detrimental and prevent access to the therapeutic target. In general,

polymeric carriers do not access the cytosol. MRP, multidrug resistance protein. (The figure is

reproduced with the permission of the Nature Publishing Group)

With extensive laboratory research and clinical trials, several facts and rules

have been obtained in the discovery and development of polymer conjugates.

Polymers used in formation of polymer conjugates must be water-soluble and

biocompatible. Biocompatibility was defined by Prof. David F. Williams as “the

ability of a material to perform with an appropriate host response in a specific

application”.8 Moreover, many properties of polymer conjugates are strongly

molecular weight dependent, including biocompatibility, pharmacokinetics, and

immunocompatibility.9 Unfortunately, most current polymer conjugates are

6

synthesized by traditional free radical polymerization and therefore have broad

polydispersity. Three examples are PK1, PK2 and HPMA-TNP-470, whose structures

are shown in Figure 1.3 and Figure 1.4. Thus, it becomes critical to prepare polymers

and corresponding conjugates with a controlled molecular weight and a narrow

polydispersity. It is also essential to develop strategies and methods to characterize

these polymers and corresponding conjugates, in order to obtain a proper

understanding of the properties of polymer conjugates and to optimize their

performance.

7

1.2 Objectives of the project

I am particularly interested in the synthesis of biocompatible polymers with a

controlled molecular weight, narrow polydispersity and designed architecture.

Moreover, I also tried to develop a strategy and methodology to characterize these

kinds of polymers. The information will be useful and important for the

characterization of corresponding polymer conjugates.

As a potential bio-material, HPMA-based polymers are attracting great

attention in academia and in the pharmaceutical industry because of their hydrophilic

and immunocompatible properties. The homopolymer of HPMA has been used as a

blood plasma expander candidate.10 Poly(HPMA) with a molecular weight below 30

kD has been reported not to cause any defense reaction in vivo.11 14C-Poly(HPMA)

shows no mitogenicity, hematotoxcity or immunogenicity. When non-radioactive

poly(HPMA) was tested in vivo, the host’s immune response was not provoked.12

These promising results of poly(HPMA) have promoted HPMA-based copolymers to

be studied in the discovery and development of cancer drugs.

8

Figure 1.3: The structures of polymer–drug conjugates PK1 and PK2

a: N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (PK1; FCE28068). b:

HPMA copolymer–doxorubicin containing galactosamine (PK2; FCE 28069) to promote liver

targeting via the asialoglycoprotein receptor.

HPMA copolymer-Gly-Phe-Leu-Gly-doxorubicin (PK1) and HPMA

copolymer-Gly-Phe-Leu-Gly-doxorubicin containing galactosamine (PK2) were

prepared and tested in clinical trials to treat various cancers.7,13 Doxorubicin

hydrochloride is a very effective drug to treat several types of tumors. However, it has

high organ toxicities, which limits its applications in cancer treatments. PK1 and PK2

were designed to decrease doxorubicin dose-limiting toxicities and increase their

elimination half-life. The tetrapeptide linker is stable in blood circulation but can be

cleaved by lysosomal thiol-dependent proteases. Impressively, the maximum tolerated

dose of the polymer drug was ten times larger than that of free doxorubicin. The

9

prolonged plasma half-life was also confirmed by HPLC and gamma camera imaging.7

PK2 is the first active targeting polymer conjugate to enter into clinical trial. It contains

galactosamine to target the hepatocyte and hepatocellular carcinoma ASGP receptor

for liver cancer treatment. The clinical trials of PK1 and PK2 establish their anti-tumor

activities without any polymer related toxicity/immunogenicity observed.7,13

Later, a conjugate of a HPMA copolymer and TNP-470 was developed to

target angiogenesis of tumors. In clinical trials, TNP-470 itself slowed/inhibited tumor

growth of patients with metastatic cancer. However, its severe neurotoxicity prevents it

from entering the market.14 Thus, further modification of TNP-470 was highly

desirable. The anti-tumor activities of HPMA copolymer-TNP-470 were established

by experiments that showed that it inhibited melanoma and Lewis lung carcinoma

growth in animals. The conjugation of HPMA polymer and TNP-470 improved the

circulating half-life and the solubility of TNP-470, and it promoted selective

accumulation of the drug in tumors. Meanwhile, the conjugation prevented TNP-470

from crossing through the blood-brain barrier, and reduced its distribution in normal

organs. Thus, the drug-related toxicities were greatly decreased.15,16

10

Figure 1.4: The structure of the polymer–drug conjugate HPMA-TNP-470

The performance of HPMA copolymer conjugates after administration has

been investigated with various methods and techniques.9,17 This research showed that

the molecular weight of these synthetic polymer conjugates had a significant influence

on their properties, including toxicity, biocompatibility and pharmacokinetics.

Unfortunately, most of current polymer conjugates are synthesized by traditional free

radical polymerization and therefore have broad polydispersity. Thus, it becomes

highly desirable to control over the molecular weight and polydispersity of the

polymers.

11

Controlled free radical polymerization is a convenient and versatile technique

to synthesize polymers with a controlled molecular weight, narrow polydispersity and

designed architecture. Currently, three methods are well-developed and appear to be

most efficient for commercial applications. There are nitroxide-mediated

polymerization (NMP), atom transfer radical polymerization (ATRP), and reversible

addition-fragmentation transfer (RAFT) polymerization. The differences among NMP,

ATRP and RAFT are briefly compared in Table 1.2.18 RAFT polymerization is a

relatively simple methodology that can be used to polymerize a wide range of

monomers under various conditions. Thus, I selected RAFT polymerization to use in

this project to achieve the controlled polymerization of HPMA copolymers. More

details about RAFT polymerization will be discussed in the following chapters.

12

Table 1.2: Comparison of NMP, ATRP and RAFT18

Features NMP ATRP RAFT Monomers Styrenes for TEMPO

Acrylate & acrylamides NO methacrylates

Nearly all monomers with activated double bonds NO vinyl acetate

Nearly all monomers

Conditions Elevated temp (>120 °C TEMPO); Water-borne systems; Sensitive to O2

Large rang of temp (-30 - 150 °C); Water-borne systems; Tolerance to O2 and inhibitor with Mt0

Elevated temp for less reactive monomers; Water-borne systems; Sensitive to O2

End Groups Alkoxyamines Requires radical chemistry for transformations Relatively expensive Thermally unstable

Alkyl (pseudo) halides Either SN, E, or radical chemistry for transformations; Inexpensive & available; Thermally and photostable; Halogen exchange for enhanced cross propagation

Dithioesters, iodides & methacrylates Radical chemistry for transformations; Relatively expensive; Thermally and photo less stable Color/odor

Additives None NMP may be accelerated with acyl compounds

Transition metal catalyst Should be removed/recycled

Conventional radical initiator

Dye-labeled polymers have many applications in academia and industry, such

as coating, single chain characterization, biosensor, and imaging.19-22 There are two

different strategies to label a polymer. One of them is to attach dyes at random along

the polymer backbone. The second is to attach the dye to one or both ends of a polymer

chain. Both types of polymers can be obtained by preparing polymers with dye-labeled

functional molecules, such as monomers, initiators and chain transfer agents (in

RAFT).19 They can also be obtained by incorporating dyes into an existing polymer

chain via post modification reactions, such as activated ester substitution, Michael

addition, or “click” chemistry.20 In this project, I am interested in developing a strategy

of synthesis of end-labeled polymers by RAFT polymerization via a dye-labeled chain

13

transfer agent. The label can be used to characterize the polymers and the

corresponding polymer conjugates, and also used to monitor the action of polymer

conjugates in vivo.

1.3 The system to be studied

In this project, I describe the synthesis of naphthalimide-dye-labeled

poly(N-(2-hydroxypropyl)methacrylamide-co-N-hydroxysuccinimide methacrylate)

(poly(HPMA-co-NMS)) by RAFT polymerization, using a designed naphthalimide

dye-labeled CTA. Because HPMA and NMS have very different reactivity ratios (0.12

and 3.46 respectively), a semi-batch method was utilized for the copolymerization of

HPMA and NMS. The proportion of NMS in poly(HPMA-co-NMS) was adjusted by

changing the ratio of HPMA to NMS in the polymerization. The effect of

[HPMA+NMS]/[CTA] on the molecular weight of copolymers was investigated.

Several methods were employed to characterize the naphthalimide-dye-labeled

poly(HPMA-co-NMS), including nuclear magnetic resonance spectroscopy (NMR),

ultraviolet-visible spectroscopy (UV-Vis), gel permeation chromatography (GPC).

14

1.4 Overview of the thesis

The mechanism of RAFT polymerization is described in Chapter 2.

Poly(HPMA) was synthesized by RAFT polymerization, using 4-cyanopentanoic acid

dithiobenzoate as the chain transfer agent (CTA). The kinetics of the

homopolymerization of HPMA was studied, as well as the effect of the ratio of HPMA

to CTA on the molecular weight of poly(HPMA).

Chapter 3 describes how a semi-batch method was employed to synthesize

poly(HPMA-co-NMS), in order to achieve the controlled copolymerization of HPMA

and NMS. The kinetics of the copolymerization and the effect of the ratio of monomers

(HPMA+NMS) to CTA were also investigated to obtain a proper understanding of the

semi-batch copolymerization process.

A new approach to the synthesis of a labeled CTA is presented in Chapter 4.

The desired end-labeled poly(HPMA-co-NMS) was synthesized by RAFT

polymerization in presence of a naphthalimide dye-labeled CTA. The dye-labeled

copolymer was characterized by 1H NMR spectroscopy, GPC and UV-Vis

spectroscopy.

15

1.5 References

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2. Langer, R. Drug delivery and targeting. Nature 392, 5-10. 1998.

3. Allen, T. M. Ligand-targeted therapeutics in anticancer therapy. Nature

Reviews Cancer 2, 750-763. 2002.

4. Duncan, R. The dawning era of polymer therapeutics. Nature Reviews Drug

Discovery 2, 347-360. 2003.

5. Duncan, R. Polymer conjugates as anticancer nanomedicines. Nature Reviews

Cancer 6, 688-701. 2006.

6. Davis, F. F. The origin of pegnology. Adv.Drug Delivery Rev. 54, 457-458.

2002.

7. Vasey, Paul A.; Kaye, Stan B.; Morrison, Rosemary; Twelves, Chris; Wilson,

Peter; Duncan, Ruth; Thomson, Alison H.; Murray, Lilian S.; Hilditch, Tom E.;

Murray, Tom; Burtles, Sally; Fraier,D.; Frigerio, E.; Cassidy, Jim. Phase I

clinical and pharmacokinetic study of PK1

[N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: First member

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8. Williams, D. F. On the mechanisms of biocompatibility. Biomaterials 29,

2941-2953. 2008.

9. Seymour, L. W., Duncan, R., Strohalm, J., Kopecek, J. Effect of molecular

weight of N-(2-hydroxypropyl)methacrylamide copolymers on body

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distribution and rate of excretion after subcutaneous, intraperitoneal, and

intravenous administration to rats. J.Biomed.Mater.Res. 21, 1341-1358. 1987.

10. Rihova, B. Biocompatibility and immunocompatibility of water-soluble

polymers based on HPMA. Composites, Part B, 38, 386-397. 2007.

11. Kopecek, J., Sprincl, L., Lim, D. New types of synthetic infusion solutions. I.

Effect of solutions of some hydrophilic polymers on blood.

J.Biomed.Mater.Res. 7, 179-191. 1973.

12. Sprincl, L., Exner, J., Sterba, O., Kopecek, J. New types of synthetic infusion

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Biomedical Materials Research 10, 953-963. 1976.

13. Hopewell, J. W., Duncan, R., Wilding, D., Chakrabarti, K. Preclinical

evaluation of the cardiotoxicity of PK2: A novel HPMA

copolymer-doxorubicin-galactosamine conjugate antitumour agent.

Hum.Exp.Toxicol. 20, 461-470. 2001.

14. Bhargava, P.; Marshall, J.L.; Rizvi, N.; Dahut, W.; Yoe, J.; Figuera, M.; Phipps,

K.; Ong, V.S.; Kato, A.; Hawkins, M.J. A Phase I and pharmacokinetic study

of TNP-470 administered weekly to patients with advanced cancer. Clin

Cancer Res 5, 1989-1995. 1999.

15. Seymour, L. W., Ulbrich, K., Strohalm, J., Kopecek, J., Duncan, R. The

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39, 1125-1131. 1990.

16. Satchi-Fainaro, Ronit; Puder, Mark; Davies, John W.; Tran, Hai T.; Sampson,

David A.; Greene, Arin K.; Corfas, Gabriel; Folkman, Judah. Targeting

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Medicine, 10, 255-261. 2004.

17

17. Kissel, Maria; Peschke, Peter; Subr, Vladimir; Ulbrich, Karel; Strunz, Anke M.;

Kuhnlein, Rainer; Debus, Jurgen; Friedrich, Eckhard Detection and cellular

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18. Matyjaszewski, K. Controlled/Living Radical Polymerization. Progress in

ATRP, NMP, and RAFT. (Proceedings of a Symposium on Controlled Radical

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19. Fleming, Craig; Maldjian, Andre; Da Costa, Daniel; Rullay, Attvinder K.;

Haddleton, David M.; John, Justin; Penny, Paul; Noble, Raymond C.; Cameron,

Neil R.; Davis, Benjamin G. A carbohydrate-antioxidant hybrid polymer

reduces oxidative damage in spermatozoa and enhances fertility.

Nat.Chem.Biol. 1, 270-274. 2005.

20. Tao, L., Mantovani, G., Lecolley, F., Haddleton, D. M. alpha -Aldehyde

Terminally Functional Methacrylic Polymers from Living Radical

Polymerization: Application in Protein Conjugation "Pegylation".

J.Am.Chem.Soc. 126, 13220-13221. 2004.

21. Liu, Y., Haley, J. C., Deng, K., Lau, W., Winnik, M. A. Effect of polymer

composition on polymer diffusion in poly(butyl acrylate-co-methyl

methacrylate latex films. Macromolecules, 40, 6422-6431. 2007.

22. Carvell, M., Robb, I. D., Small, P. W. The influence of labeled mechanisms on

the fluorescence behavior of polymers bearing fluorescein labels. Polymer 39,

393-398. 1997.

18

Chapter 2

Homopolymerization of N-(2-hydroxypropyl)methacrylamide

by RAFT Polymerization

2 Homopolymerization of

N-(2-hydroxypropyl)methacrylamide by RAFT

Polymerization

2.1 Introduction

This chapter describes the synthesis of homopolymers of

N-(2-hydroxypropyl)methacrylamide (HPMA) by reversible addition-fragmentation

transfer (RAFT) polymerization. RAFT has attracted great interest in academia and in

industry since the first patent was claimed by the CSIRO in 1998.1 As one of the most

versatile techniques, RAFT is carried out by simply adding chain transfer agents (CTAs,

such as a thiocarbonylthio compound ZC(=S)SR) into a conventional free radical

polymerization system. The key feature of RAFT is the sequence of

addition-fragmentation equilibration involving a chain transfer agent. The mechanism of

RAFT polymerization is shown in Figure 2.1. Initiation and termination occur as in a

conventional free radical polymerization. Typically, thermal initiators such as

2,2'-azodiisobutyronitrile (AIBN) are used in RAFT polymerization. In the early stages

of the polymerization, the propagating radical (Pn•) reacts with the thiocarbonylthio

compound (1) to form the intermediate radical (2), followed by the fragmentation to

19

produce a new radical (R•) and a polymeric thiocarbonylthio compound (3). Then, this

new radical (R•) reacts with monomers to form a new propagating radical (Pm•).

Consequently, the equilibration between active species (Pn• and Pm•) and dormant

polymeric thiocarbonylthio compounds (3) leads to a simultaneous growth of all chains

with minimal termination reactions. Finally, a polymer can be obtained with a

well-controlled molecular weight and narrow polydispersity, as well as R and Z groups

at each end of the polymer chain respectively.

Figure 2.1: Mechanism of reversible addition-fragmentation transfer polymerization2

(The figure is reproduced with the permission of the CSIRO)

20

There are several factors affecting the success of RAFT polymerization,

including the reaction temperature, the solvent, the nature of monomers and ratios of

monomer/CTA and CTA/initiator.2,3 Among all these factors, the chain transfer agent

plays an essential role to accomplish a successful RAFT polymerization (Figure 2.2).

The addition rate of radicals to the C=S double bond is strongly influenced by the

substituent Z.4 The relative effectiveness of a CTA can be rationalized, in terms of the

interaction between the substituent Z and the C=S double bond.

Figure 2.2: Structure features of the thiocarbonylthio CTA and the related intermediate

compound2

(The figure is reproduced with the permission of the CSIRO)

Meanwhile, the substituent R must be a good homolytic leaving group and able

to reinitiate the polymerization efficiently.5 In the polymerization of methyl

methacrylate (MMA) or its derivatives, the efficiency of a CTA depends strongly on the

nature of the substituent R. Although the rate constant of addition to the thiocarbonyl

group does not strongly depend on R, the transfer coefficient is determined by the

relative leaving ability of R and the propagating polymer Pn(m). The stability, polarity

21

and steric factor of R• are essential in determining the transfer coefficient and the

effectiveness of a CTA. In order to guide the selection and design of CTAs for

polymerizations of various monomers, the relative reactivities of different functional

groups as R and Z substituents were investigated by scientists at the CSIRO in

Australia.4,5 These results are summarized in Figure 2.3.

Figure 2.3: Guidelines of CTA design for various polymerizations2

AM, acrylamide; AN, acrylonitrile; MA, methyl acrylate; MMA, methyl methacrylate; NVP,

N-vinylpyrrolidone; S, styrene; VAc, vinyl acetate. (The figure is reproduced with the permission

of the CSIRO)

The first report of the homopolymerization of

N-(2-hydroxypropyl)methacrylamide (HPMA) by RAFT polymerization described

reactions in aqueous media. McCormicks’ group synthesized poly(HPMA) using

4-cyanopentanoic acid dithiobenzoate as the chain transfer agent (CTA) and

4,4'-azobis(4-cyanopentanoic acid) as the initiator in an acetic acid buffer solution at 70

°C.6 They found that a lower ratio of CTA to initiator resulted in a faster rate of

polymerization, affording high monomer conversion and well-defined polymers (i.e., Mn

22

= 97000 g/mol, Mw/Mn = 1.07). Moreover, in order to demonstrate the retention of the

dithioester end group and “living” nature of the polymerization, they also used

poly(HPMA)-marcoCTA (Mn = 36100 g/mol, Mw/Mn = 1.05) to produce corresponding

poly(HPMA-b-HPMA) (Mn = 98800 g/mol, Mw/Mn = 1.08) under similar

polymerization conditions. Later, Kane et al. demonstrated a synthesis of poly(HPMA)

in tert-butyl alcohol by RAFT polymerization, which opens the door to copolymerize

HPMA with other hydrolytic sensitive monomers in this solvent.7

In this chapter, I describe the synthesis of poly(HPMA) by RAFT polymerization.

The poly(HPMA) was characterized by 1H NMR spectroscopy and gel permeation

chromatography (GPC). The kinetics of the homopolymerization of HPMA was also

studied with different ratios of HPMA to CTA. The results from the kinetics study were

used to determine the appropriate experimental parameters for the copolymerization of

HPMA and N-hydroxysuccinimide methacrylate (NMS) described in the next chapter.

23

2.2 Experimental Section

2.2.1 General Information

N-(2-hydroxypropyl)methacrylamide (HPMA) and 4-cyanopentanoic acid

dithiobenzoate (CIDB) were synthesized as described below. 1-Amino-2-propanol

(≥98.0%, Fluka), methacryloyl chloride (≥97.0% (GC), contains ~0.02%

2,6-di-tert-butyl-4-methylphenol as stabilizer, Fluka), bromobenzene (≥99%, ACP

Chemicals Inc.), carbon disulfide (≥99.9%, ACP Chemicals Inc.),

4,4’-azobis(4-cyanovaleric acid) (≥98.0%, Fluka), 2,2’-azobis(2-methylpropionitrile)

(AIBN) (98%, Aldrich), 1,3,5-trioxane (≥99%, Aldrich), tert-Butyl alcohol (t-BuOH)

(≥99.5%, Aldrich), N,N-dimethylformamide (DMF) (99.8%, Aldrich),

1-methyl-2-pyrrolidinone (NMP) (≥99%, Aldrich) and other chemicals were used as

received.

1H NMR: The measurements were performed with a Varian Mercury 300 spectrometer.

CDCl3 and d6-DMSO are purchased from Cambridge Isotope Laboratories, Inc.

GPC: Polymers were analyzed by GPC using 1-methyl-2-pyrrolidinone (NMP)

containing 0.2 wt% LiCl as the eluent and PMMA standards. GPC analysis was carried

out at 80 °C at a flow rate of 0.6 ml/min with a refractive index detector.

Elemental Analysis: The measurements were performed with a 2400 Series II CHNS

24

Analyzer.

2.2.2 Synthesis and Characterization of HPMA and the Chain

Transfer Agent (CTA)

Figure 2.4: Synthesis of N-(2-Hydroxypropyl)methacrylamide

Synthesis and characterization of N-(2-Hydroxypropyl)methacrylamide (HPMA)

The preparation basically follows the procedure published by Ulbrich et al.8 A

suspension of anhydrous sodium bicarbonate (18.0 g) and 1-amino-2-propanol (24.40 g)

in 85 ml freshly distilled dichloromethane was cooled to 0 °C, and followed by the

dropwise addition over 1 hour of a solution of methacryloyl chloride (31.36 g) in 40 ml

dichloromethane via an additional funnel under cooling and vigorous stirring. The

reaction mixture was then stirred overnight at room temperature, after which anhydrous

sodium sulfate (10 g) was added. The mixture was filtered, and the filtrate was

concentrated to half of the original volume with a rotaevaporator. The monomer was

obtained by crystallization from dichloromethane at -20 °C and purified by

recrystallization from acetone, and dried under vacuum at room temperature over night.

Yield: 9.54 g (23%); m.p.: 65-66 °C; 1H NMR (CDCl3): δ(ppm) 1.19 (d, 3H, CH3),

OCl

H2N OHO

NH

OHNaHCO3

CH2Cl2+

25

1.97(s, 3H, CH3), 3.20-3.96 (m, 2H, CH2), 3.95 (m, H, CH), 5.35 (s, H, H2C=C), 5.73 (s,

H, H2C=C); Elemental Analysis (calculated/measured): C (58.72/58.44), H (9.15/8.68),

N (9.78/9.67).

Figure 2.5: Synthesis of 4-cyanopentanoic acid dithiobenzoate

Synthesis and characterization of 4-cyanopentanoic acid dithiobenzoate (CIDB)

Magnesium turnings (3.00 g) were placed into a round-bottom flask with a

catalytic amount of iodine. Bromobenzene (18.84 g) was mixed with dry THF (90 ml).

Then a 10 ml mixture of bromobenzene and THF was added to the flask and heated

slightly. The remaining mixture was added slowly, while the temperature of reaction

remained below 40 ºC. The reaction was then stirred at room temperature for one hour,

after which the flask was cooled to 0 ºC. Carbon disulfide (9.15 g) was added to the

Grignard mixture at 0 ºC. When the reaction finished after two hours, deionized water

(350 ml) was added, and the salts were removed by filtration. Concentrated HCl (~10 ml)

was added to the filtrate and the mixture was extracted with diethyl ether. After

evaporating the solvent with a rotaevaporator, absolute ethanol (100 ml) was added into

2.HCl

1.H2O

THF, < 40 ºC

Mg, I2 Br MgBr BrMgS SCS2

0 ºC

N N CNOH

OCN

HO

OS

S S

S+

Ethyl acetate

80 ºC2 S

S CNOH

O

HS S DMSO, I2

Ethanol

S

S S

S

26

the dithiobenzoic acid with DMSO (18.75 g) and a catalytic amount of iodine. The

reaction proceeded at room temperature for two hours, and then the mixture was filtered.

The purple solid (bis(thiocarbonyl) disulfide) was dried under vacuum at room

temperature overnight. Yield: 11.04 g (60%); m.p.: 96-98 ºC; 1H NMR (CDCl3): δ(ppm)

7.45 (t, 4H), 7.61 (t, 2H), 8.07 (t, 4H).

A solution of bis(thiocarbonyl) disulfide (0.306 g) and

4,4’-azobis(4-cyanovaleric acid) (0.420 g) in ethyl acetate was degassed with nitrogen

and heated at 80 ºC for 20 hours. The solvent was removed with a rotaevaporator, and

the residue (product) was purified by chromatography on silica with ethyl

acetate/hexane as the eluent. Yield: 0.490 g (88%); m.p.: 81-84 ºC; 1H NMR (CDCl3):

δ(ppm) 1.95 (s, 3H, CH3), 2.40-2.78 (m, 4H, CH2CH2), 7.40 (m, 2H, m-ArH), 7.58 (t,

1H, p-ArH), 7.90 (dd, 2H, o-ArH); Elemental Analysis (calculated/measured): C

(55.89/55.86), H (4.69/4.82), N (5.01/5.05).

2.2.3 Synthesis and Characterization of Poly(HPMA)

Figure 2.6: Synthesis pathway of poly(HPMA)

OHN

OH AIBN, t- BuOH, 80 ºC

S

S CNOH

OmS OH

S

OHN

CN

O

OH

27

The polymerizations were carried out under an Argon (Ar) atmosphere using the

Schlenk technique. A typical polymerization procedure is described below. The stock

solution was prepared comprising AIBN (140.08 mg), CIDB (41.08 mg), and

1,3,5-trioxane (internal standard, 449 mg) in degassed DMF (5 ml). HPMA (0.458 g)

were evacuated and back-filled with Ar three times. The degassed t-BuOH (3.2 ml) was

injected into a round-bottom flask containing HPMA to form a 1 M solution, and a

solution of AIBN, CIDB and 1,3,5-trioxane in DMF (200 ul) was transferred into the

same flask. The reaction was carried out at 80 °C immediately and finally quenched in

an acetone-dry ice bath. Aliquots (0.1 ml) were taken out for NMR analysis throughout

the reaction. The final polymer was precipitated using a mixture of anhydrous diethyl

ether and anhydrous acetone (v:v=1:1), recovered by centrifugation, and then

lyophilized overnight for further analysis by NMR and GPC.

28

2.3 Results and Discussion

The polymerization of N-(2-Hydroxypropyl)methacrylamide (HPMA) was

carried out by RAFT polymerization, using 4-cyanopentanoic acid dithiobenzoate

(CIDB) as the chain transfer agent and AIBN as the initiator at 80 °C (Table 2.1). The

choice of chain transfer agents is crucial to the success of a RAFT polymerization.

Considering the nature of HPMA, a chain transfer agent with phenyl as the substituent Z

and cyanoalkyl as the substituent R will be favored (Figure 2.3). 4-cyanopentanoic acid

dithiobenzoate was preferred as the CTA because it fits the RAFT polymerization of

various monomers and the carboxy group can be utilized to incorporate various

functionalities into the CTA. tert-Butyl alcohol was chosen as a useful solvent for the

polymerization of HPMA, and DMF was used to dissolve the initiator and the chain

transfer agent. The conversion of HPMA in the reaction was monitored by 1H NMR (in

CDCl3), using 1,3,5-trioxane as an internal standard. The final polymer was dissolved in

d6-DMSO for the NMR experiment.

The 1H NMR spectrum of poly(HPMA) is shown in Figure 2.7. The main peaks

are assigned to the corresponding protons of the structure drawn in the figure. The

characteristic resonances for poly(HPMA) are clearly evident. The peak at 0.4-1.4 ppm

is ascribed to the methyl protons (d,f). The peak at 1.4-2.2 ppm is ascribed to the

methylene group (e). The peaks at 2.9 ppm and 3.7 ppm are ascribed to the protons of

the amide methylene (g) and to the proton of the alcohol methine (h) respectively. The

29

signals of hydroxyl proton and amine proton appear at 4.7 ppm and 7.2 ppm respectively.

It is also easy to identify some signals of the phenyl group from the CTA, such as the

proton (a) at 7.8 ppm. The peak at 3.3 ppm is due to water absorbed by the deuterated

DMSO used in the NMR experiment.

Figure 2.7: 1H NMR spectrum of the homopolymer of HPMA

The polymerization of HPMA (1 M in t-BuOH) was performed at 80 °C in the presence of

4-cyanopentanoic acid dithiobenzoate (CIDB 0.5 M in DMF) as the chain transfer agent and AIBN

as the initiator. The ratio of HPMA/CIDB was 160. The insert is the enlarged 1H NMR region

between 6.8 and 8.1 ppm. The main peaks are assigned to the corresponding protons of the

structure drawn in the figure. The solvent was d6-DMSO. The protons (labeled as “a”) of the

phenyl end group of the polymer chain are used to characterize the molecular weight of

poly(HPMA).

30

The 1H NMR spectrum of poly(HPMA) with the presence of the phenyl group

proves that the 4-cyanopentanoic acid dithiobenzoate (CIDB) was successfully

incorporated into the polymerization of HPMA (Figure 2.7). Moreover, 1H NMR (in

d6-DMSO) was also employed to estimate the degree of polymerization (DP) of HPMA

as well as the absolute number-average molecular weight of poly(HPMA). The degree

of polymerization of HPMA is evaluated by the ratio of the integral of peak (h) over the

integral of peak (a), using Equation 2.1:

(2.1)

Here Ih and Ia are the integrals of peak (h) and peak (a), respectively. nh and na are the

number of protons (h) and protons (a), respectively. The absolute number-average

molecular weight of poly(HPMA) was evaluated from the degree of polymerization of

HPMA. However, due to the signal noise of the phenyl group, the molecular weight of

poly(HPMA) obtained from NMR has a significant error.

The molecular weight and polydispersity of poly(HPMA) were also

characterized by GPC. Poly(HPMA) was dissolved in 1-methyl-2-pyrrolidinone (NMP)

to form a 2 mg/ml solution. This solution (100 μl) was injected into the GPC instrument.

PMMA standards with narrow PDIs were used to construct a calibration curve, and a

small amount of LiCl was added into the eluent to prevent aggregation of the polymers.

All of the results are listed in Table 2.1.

DPHPMA= Ia×nh

Ih×na

31

Table 2.1: Experimental results of the homopolymerization of HPMA

NMR results b GPC results c [HPMA]/[CTA]

/[AIBN]

reaction time

(hours)

conversion a

%

Mn

(theory)

(×103 g/mol )

DP Mn (×103 g/mol )

Mw (×103 g/mol )

Mn (×103 g/mol )

PDI

320/2/1 8.25 75 23 289 41 41 29 1.4

a: from 1H NMR in CDCl3;

b: from 1H NMR in d6-DMSO, Mn = 143.18×DPHPMA;

c: from GPC using NMP (containing 0.2 wt% LiCl) as the eluent and PMMA standards.

The GPC results show that poly(HPMA) was obtained with reasonable control of

the molecular weight and a narrow polydispersity. It indicates that poly(HPMA) was

successfully synthesized by RAFT polymerization. The molecular weight of

poly(HPMA) obtained from NMR is higher than the theoretical one. This result may be

a consequence of the poor signal quality of the phenyl group, as mentioned previously.

The molecular weight of poly(HPMA) obtained by GPC is also not an accurate value,

because it is determined from a calibration curve constructed with PMMA standards.

However, comparing with the result from NMR, the results from GPC are more reliable

in this experiment.

In order to obtain a better understanding of the polymerization of HPMA, the

polymerization kinetics was studied under the same reaction conditions. The

polymerization of HPMA was carried out with three different ratios of HPMA to CIDB

including 80:1, 160:1 and 320:1. The ratio of CIDB to AIBN remained 2:1 in all the

cases. Aliquots were taken out throughout the polymerization for NMR and GPC

32

analysis. The conversion of HPMA was determined by 1H NMR (in CDCl3). The

molecular weight and polydispersity of poly(HPMA) were analyzed by GPC using

PMMA standards and NMP containing 0.2 wt% LiCl as the eluent.

The kinetics of the polymerization of HPMA was first studied with

[HPMA]/[CIDB]= 80. All of results are listed in Table 2.2. The relationship of

conversion and ln([M]0/[M]t) with polymerization time is plotted as shown in Figure 2.8

(a). ln([M]0/[M]t) increased linearly with reaction time at the first four hours of the

polymerization. Later, the rate of the polymerization became slower. The conversion of

HPMA finally reached 79% after 7 hours. The linear increase of ln([M]0/[M]t) with

polymerization time indicates that there was a constant concentration of active species in

the polymerization, and the rate of the polymerization was first order with respect to

HPMA monomer. This result is consistent with the polymerization of HPMA being a

controlled radical polymerization. The rate of the polymerization decreased gradually

after 4 hours because most of HPMA had been consumed.

33

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

Time (h)

Con

vers

ion

(%)

0.0

0.5

1.0

1.5

2.0

mSOH

S

OHN

CN

O

OH

ln([M]0 /[M

]t )

a

0 10 20 30 40 50 60 70 80 900

2

4

6

8

10

12

14

16

18

Conversion (%)

Mn (

∗103 g

/mol

)

1.0

1.2

1.4

1.6

1.8

2.0b

Mw/M

n

Figure 2.8: Kinetics (a) and evolution of the molecular weight and polydispersity with

conversion (b) during the polymerization of HPMA (1 M in t-BuOH) performed at 80 °C in

the presence of 4-cyanopentanoic acid dithiobenzoate (CIDB 0.5 M in DMF) as the chain

transfer agent and AIBN as the initiator. The ratio of HPMA/CIDB= 80.

34

Figure 2.8 (b) shows the relationship of the molecular weight of poly(HPMA)

and polydispersity with conversion in the polymerization of HPMA with

[HPMA]/[CIDB]=80. There is a typical linear increase of the molecular weight with

the conversion of HPMA until the conversion reached 72%. The molecular weight

distribution of poly(HPMA) remained quite narrow throughout the polymerization.

These results establish that the poly(HPMA) can be synthesized with a controlled

molecular weight and narrow polydispersity in the presence of CIDB as the CTA.

Table 2.2: Kinetics of the homopolymerization of HPMA at [M]/[CTA] = 80

[HPMA]/[CTA]/[AIBN]

reaction time

(hours)

conversion a %

Mn

(theory) b

(×103 g/mol )

Mn (experiment) c

(×103 g/mol )

PDI c

1 21 6.3 1.2 2 46 12 1.2 3 61 14 1.2

160/2/1 4 72 11 16 1.1 5 75 14 1.3 6 76 16 1.2 7 79 16 1.2


b: Mn = 143.18×80;


The kinetics of the polymerization of HPMA was also studied with

[HPMA]/[CIDB]=160. All of results are listed in Table 2.3. The relationship of

conversion and ln([M]0/[M]t) with polymerization time is plotted as shown in Figure 2.9

(a). ln([M]0/[M]t) increased linearly with reaction time at the first four hours of the

35

polymerization. Later the rate of the polymerization decreased gradually. The

conversion of HPMA finally reached 76% after 8 hours. The linear increase of

ln([M]0/[M]t) with polymerization time indicates that there was a constant concentration

of active species in the polymerization, and the rate of the polymerization was first order

with respect to HPMA monomer. As in the case of [HPMA]/[CIDB]=80, the rate of the

polymerization decreased gradually after 4 hours because most of HPMA had been

consumed.

36

0 1 2 3 4 5 6 7 8 90

10

20

30

40

50

60

70

80

Time (h)

Con

vers

ion

(%)

0.0

0.5

1.0

1.5

2.0

a

ln([M]0 /[M

]t )

0 10 20 30 40 50 60 700

10

20

30

40

Conversion (%)

Mn (

∗103 g

/mol

)

1.0

1.2

1.4

1.6

1.8

2.0b

Mw/M

n





37

Figure 2.9 (b) shows the relationship of the molecular weight of poly(HPMA)

and polydispersity with conversion in the polymerization of HPMA with

[HPMA]/[CIDB]=160. There is also a linear increase of the molecular weight with the

conversion of HPMA until the conversion reached 71%. The molecular weight

distribution of poly(HPMA) remained around 1.3 throughout the polymerization. These

results establish that at this monomer/CTA ratio, poly(HPMA) can be synthesized with a

controlled molecular weight and narrow polydispersity in the presence of CIDB as the

CTA and AIBN as the initiator.


[HPMA]/[CTA]/[AIBN] reaction

time (hours)

conversion a %

Mn

(theory) b

(×103 g/mol )

Mn ( experiment ) c

(×103 g/mol )

PDI c

1 11 10 1.2 2 42 18 1.2 3 6 23 1.3

320/2/1 4 68 23 25 1.3 5 71 27 1.3 6 72 29 1.3 7 73 29 1.3 8 76 31 1.3


b: Mn = 143.18×160;


38

Similar phenomena are observed in the homopolymerization of HPMA with

[HPMA]/[CIDB]=320 (Table 2.4). Figure 2.10 (a) shows that ln([M]0/[M]t) increased

linearly with reaction time at the first 3 hours of the polymerization. Later the rate of the

polymerization decreased gradually. The conversion of HPMA finally reached 74% after

7 hours. As the conversion of HPMA increased, the molecular weight of poly(HPMA)

increased linearly until the conversion reached 67% (Figure 2.10 (b)).The polydispersity

remained narrow in the polymerization. These results confirm that the polymerization of

HPMA with [HPMA]/[CIDB]=320 was successfully carried out in the presence of CIDB

as the CTA and AIBN as the initiator.


[HPMA]/[CTA]/[AIBN]

reaction time

(hours)

conversion a %

Mn

(theory) b

(×103 g/mol )

Mn (experiment) c

(×103 g/mol )

PDI c

2 30 21 1.2 3 43 30 1.3 4 58 32 1.3

640/2/1 5 67 46 33 1.3 6 71 35 1.4 7 74 37 1.4


b: Mn = 143.18×320;


39

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

Time (h)

Con

vers

ion

(%)

0.0

0.5

1.0

1.5

2.0a

ln([M]0 /[M

]t )

0 10 20 30 40 50 60 70 800

5

10

15

20

25

30

35

40

Conversion (%)

Mn (

∗103 g

/mol

)

1.0

1.2

1.4

1.6

1.8

2.0b

Mw/M

n





40

Comparing the plots in Figure 2.8 (a), Figure 2.9 (a) and Figure 2.10 (a), it shows

that as the ratio of HPMA to CIDB increased, the rate of the polymerization decreased.

The GPC results (Table 2.2, Table 2.3 and Table 2.4) also confirm that the molecular

weight of poly(HPMA) can be tuned by changing the ratio of HPMA to CIDB in the

polymerization. The polymer had a higher polydispersity index when it was prepared

with a higher ratio of HPMA to CIDB. This result suggests that there was higher relative

concentration of radical species compared to one of dormant species, when the

polymerization was carried out with a high ratio of HPMA to CIDB.

2.4 Conclusions

Homopolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were successfully

synthesized by RAFT polymerization with a wide range of controlled molecular weights

and narrow polydispersities. 4-Cyanopentanoic acid dithiobenzoate (CIDB) is an

effective chain transfer agent for the polymerization of HPMA. The polymers were

characterized by 1H NMR and GPC. The kinetics of the homopolymerization of HPMA

gave results consistent with a controlled radical polymerization. There was a linear

consumption of HPMA within four hours from the start of the polymerization. These

results provide important parameter that I used to determine the addition rate of

N-hydroxysuccinimide methacrylate (NMS) for the copolymerization of HPMA and

NMS. This topic is described in the next chapter.

41

2.5 References

1. Chiefari, John; Chong, Y.K.; Ercole, Frances; Krstina, Julia; Jeffery, Justine; Le,

Tam P.T.; Mayadunne, Roshan T.A.; Meijs, Gordon F.; Moad, Catherine L.;

Moad, Graeme; Rizzardo, Ezio; Thang, San H. Living Free-Radical

Polymerization by Reversible Addition-Fragmentation Chain Transfer: The

RAFT Process. Macromolecules 31, 5559-5562. 1998.

2. Moad, G., Rizzardo, E., Thang, S. H. Living Radical Polymerization by the

RAFT Process-A First Update. Australian Journal of Chemistry 59, 669-692.

2006.


RAFT Process. Australian Journal of Chemistry 58, 379-410. 2005.

4. Chiefari, John; Mayadunne, Roshan T.A.; Moad, Catherine L.; Moad, Graeme;

Rizzardo, Ezio; Postma, Almar; Skidmore, Melissa A.; Thang, San H.

Thiocarbonylthio Compounds (S:C(Z)S-R) in Free Radical Polymerization with

Reversible Addition-Fragmentation Chain Transfer (RAFT Polymerization).

Effect of the Activating Group Z. Macromolecules 36, 2273-2283. 2003.

5. Chong, Y.K.; Krstina, Julia; Le, Tam P.T.; Moad, Graeme; Postma, Almar;

Rizzardo, Ezio; Thang, San H. Thiocarbonylthio Compounds [S:C(Ph)S-R] in

Free Radical Polymerization with Reversible Addition-Fragmentation Chain

Transfer (RAFT Polymerization). Role of the Free-Radical Leaving Group (R).

Macromolecules 36, 2256-2272. 2003.

6. Scales, C. W., Vasilieva, Y. A., Convertine, A. J., Lowe, A. B., McCormick, C. L.

Direct, controlled synthesis of the nonimmunogenic, hydrophilic polymer,

poly(N-(2-hydroxypropyl)methacrylamide) via RAFT in aqueous media.

Biomacromolecules 6, 1846-1850. 2005.

42

7. Yanjarappa, M. J., Gujraty, K., V, Joshi, A., Saraph, A., Kane, R. S. Synthesis of

copolymers containing an active ester of methacrylic acid by RAFT: controlled

molecular weight scaffolds for biofunctionalization. Biomacromolecules 7,

1665-1670. 2006.

8. Ulbrich, K.; Subr, V.; Strohalm, J.; Plocova, D.; Jelinkova, M.; Rihova, B.

Polymeric drugs based on conjugates of synthetic and natural macromolecules. I.

Synthesis and physico-chemical characterisation. J Control Release 64, 63-79.

2000.

43

Chapter 3

Copolymerization of N-(2-hydroxypropyl)methacrylamide

and N-hydroxysuccinimide methacrylate by RAFT

Polymerization

3 Copolymerization of

N-(2-hydroxypropyl)methacrylamide and

N-hydroxysuccinimide methacrylate by RAFT

Polymerization

3.1 Introduction

In recent years, several attempts have been made to synthesize

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers by RAFT polymerization.

Recently, McCormick et al. prepared hydrophilic/cationic block copolymers of HPMA

and DMAPMA (N-[3-(dimethylamino)propyl]methacrylamide) by RAFT

polymerizaiton.1 The synthetic pathways are presented in Figure 3.1. First, they used

the same CTA (4-cyanopentanoic acid dithiobenzoate) to prepare homopolymers of

HPMA and DMAPMA by RAFT polymerization. Then, these RAFT-generated

poly(HPMA) and poly(DMAPMA) were used as macroCTAs to synthesize

corresponding block polymers of HPMA and DMAPMA. Recently, Hong et al. also

reported the synthesis of block copolymers of HPMA and NIPAM

(N-isopropylacrylamide) by RAFT polymerization.2 The block polymers of HPMA

44

and NIPAM were obtained with molecular weights in a wide range of 7,800 – 26,300

g/mol. The PDIs of these block polymers were very narrow (1.15-1.29). However,

these copolymers, such as poly(HPMA-b-DAMPMA) and poly(HPMA-b-NIPAM),

can not be used to incorporate ligands or drugs into the polymers. In order to form

HPMA-based polymer conjugates, activated polymers need to be synthesized by

RAFT polymerization. The activated polymers provide a versatile scaffold to be

conjugated with a variety of ligands via post-polymerization modifications.

Figure 3.1: Synthetic pathways of block copolymers of HPMA and DMAPMA by RAFT

polymerization1

(The figure is reproduced with the permission of the American Chemical Society)

N-hydroxysuccinimide methacrylate (NMS) has been widely used to

synthesize activated copolymers, such as poly(NMS-co-HPMA) and

45

poly(NMS-co-NIPAM).3-5 The succinimide ester group of NMS can react with amines

to form amides. Thus, a variety of functionalities with primary amine groups can be

incorporated into copolymers containing NMS via related substitution reactions. Two

strategies are presented in Figure 3.1 to synthesize copolymers of HPMA and NMS.

OHN

OH

ON OO

O +n m

OONO O

OHN

OH

ON OO

On

OONO O

H2N OHn m

OONO O

OHN

OH

a

b

Figure 3.2: Synthesis strategies of poly(HPMA-co-NMS)

As shown in Figure 3.2 (a), one strategy is to first prepare a NMS

homopolymer by a controlled radical polymerization. The obtained poly(NMS) can be

transformed to a copolymer of HPMA and NMS by reacting with isopropanolamine.6

The advantage of this strategy is that the composition of poly(HPMA-co-NMS) can be

tailored easily by changing addition amount of isopropanolamine in the substitution

reaction. Müller and his colleagues first investigated the possibility of the

homopolymerization of NMS by RAFT.7 Three different CTAs were examined for the

polymerization of NMS, including 1-cyanoisopropyl dithiobenzoate, benzyl

1-pyrrolecarbodithioate and cumyl 1-pyrrolecarbodithioate. Unfortunately, the

46

experimental results (in Table 3.1) showed that the chain transfer agents did not work

at all in the polymerization process. The polymerization turned to be an uncontrolled

free radical polymerization. The results indicate that RAFT polymerization is not

suitable to prepare the homopolymer of NMS. The reason is still under investigation

by them.

Table 3.1: Experimental results of the RAFT polymerization of N-hydroxysuccinimide methacrylate (NMS) using three different CTAs7

Monomer

CTA reaction

time (hours)

conversion %

Mn

(theory)

(×103 g/mol )

Mn (experiment)

(×103 g/mol )

PDI

16 81 3.1 24.5 1.52

1-cyanoisopropyl dithiobenzoate 10 70 2.7 24.2 1.47

NMS 16 89 3.1 43.5 2.11

benzyl 1- pyrrolecarbodithioate 10 74 2.6 41.3 2.34

16 83 3.1 24.1 1.71

cumyl 1- pyrrolecarbodithioate 10 60 2.3 22.4 1.78

Recently, another strategy was developed by Kane et al.8 to synthesize a

copolymer of HPMA and NMS directly by RAFT polymerization (Figure 3.2 (b)). Due

to different reactivity ratios of HPMA and NMS (0.12 and 3.46 respectively),

conventional copolymerization will lead to the preferential incorporation of NMS in a

copolymer chain, and even form a block polymer.8 Thus, a semi-batch method was

employed to prepare copolymers of HPMA and NMS by RAFT polymerization in

Kane’s group.8 A series of poly(HPMA-co-NMS) were synthesized with molecular

weights over a range of 4.3-53.6 kDa and with narrow polydispersities (1.1-1.2).

Moreover, Kane’s group established that the semi-batch method enabled the

47

composition of poly(HPMA-co-NMS) to remian constant during the polymerization.

However, the incorporation of NMS in the copolymer was only around 20 mol%. For

medicine-delivery applications, the composition of poly(HPMA-co-NMS) need to be

tailored over a wide range, in order to load various amount of drugs or other

functionalities. Thus, the preparation of poly(HPMA-co-NMS) with various

compositions is a worthwhile objective.

In this chapter, a semi-batch method is described to synthesize

poly(HPMA-co-NMS) by RAFT polymerization. The copolymers of HPMA and NMS

were characterized by GPC and 1H NMR spectroscopy. The effect of

[HPMA+NMS]/[CIDB] on the molecular weight of the copolymers was investigated,

as well as the effect of [HPMA]/[NMS] on the composition of the copolymers. The

kinetics of the copolymerization of HPMA and NMS was also studied in order to

obtain a better understanding about the semi-batch copolymerization method.

48



N-methacryloxysuccinimide (NMS) was synthesized as described below.

N-hydroxysuccinimide (98%, Aldrich), methacryloyl chloride (≥97.0% (GC), contains

~0.02% 2,6-di-tert-butyl-4-methylphenol as a stabilizer, Fluka),

2,2’-azobis(2-methylpropionitrile) (AIBN) (98%, Aldrich), 1,3,5-trioxane (≥99%,

Aldrich), tert-butanol (t-BuOH) (≥99.5%, Aldrich), N,N-dimethylformamide (DMF)

(99.8%, Aldrich), 1-methyl-2-pyrrolidinone (NMP) (≥99%, Aldrich) and other

chemicals were used as received.


CDCl3 and d6-DMSO were purchased from Cambridge Isotope Laboratories, Inc.

GPC: Polymers were analyzed by GPC using NMP as the eluent (0.2% LiCl) and

PMMA standards. GPC analysis was carried out at 80 °C at a flow rate of 0.6 ml/min

with a refractive index detector.


Analyzer.

49

3.2.2 Synthesis and Characterization of NMS

Figure 3.3: Synthesis of N-methacryloyloxysuccinimide

The preparation basically follows the procedure published by Shunmugam et al.

(Figure 3.3).4 A solution of N-hydroxysuccinimide (7.08 g) and triethylamine (9.45 g)

in 50 mL tetrahydrofuran (THF) was cooled to 0 °C, and followed by the dropwise

addition of methyl methacryloyl chloride (6 mL) via an additional funnel. The reaction

was stirred for 12 hours at room temperature and then concentrated with a

rotaevaporator. The mixture was dissolved in dichloromethane, and washed with

deionized water, saturated sodium bicarbonate solution and finally water. The organic

layer was dried by magnesium sulfate, filtered, and evaporated with a rotaevaporator.

The product was purified by recrystallization with an ethyl acetate/hexane mixture,

and dried under vacuum at 45 °C overnight. Yield: 2.73 g (25%); m.p.: 98.5-100.5 °C;

1H NMR (CDCl3): δ(ppm) 2.06 (s, 3H, CH3), 2.86 (s, 4H, CH2), 5.88 (s, H, H2C=C),

6.42 (s, H, H2C=C); Elemental Analysis (calculated/measured): C (52.46/52.59), H

(4.95/5.00), N (7.65/7.63).

O

N

O

O

ON OO

OH

OCl

Triethylamine

THF+

50

3.2.3 Synthesis and Characterization of Copolymers of HPMA and

NMS

Figure 3.4: Synthesis pathway of poly(HPMA-co-NMS)

The copolymerization reactions were carried out under an Argon (Ar)

atmosphere using the Schlenk technique (Figure 3.4). A typical copolymerization

procedure (entry 3 in Table 3.2) is described below. A stock solution was prepared

consisting of AIBN (140.08 mg), CIDB (41.08 mg) and 1,3,5-trioxane (internal

standard, 449 mg) in degassed DMF (5 ml). The monomers were evacuated and

back-filled with Ar three times. The solvents (t-BuOH and DMF) were degassed with

Ar. t-BuOH (1.9 ml) was injected into a round-bottom flask containing HPMA (0.275

g) to form a 1 M solution, and then the solution of AIBN, CIDB and 1,3,5-trioxane in

DMF (200 μl) was transferred into the flask. The mixture was heated at 80 °C for 30

minutes, and then a solution of NMS in DMF (0.5 M) was added continuously into the

reaction at 0.43 ml/h through an airtight syringe by a syringe pump (KD Scientific,

Model 780100). After the addition of the NMS solution, the reaction was kept at 80 °C

for 30 more minutes and was then quenched in an acetone-dry ice bath. Aliquots (0.1

+ OHN

OH

ON OO

O

AIBN, t- BuOH/DMF, 80 ºC

S

S CNOH

O n mS OHS

OONO O

OHN

CN

O

OH

51

ml) were taken out for NMR analysis throughout the polymerization. The final

polymer was precipitated within a mixture of anhydrous diethyl ether and anhydrous

acetone (v:v=1:1), recovered by centrifugation, and then lyophilized overnight. The

polymer product (106 mg) was stored at 4 °C.

52


The copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) and

N-hydroxysuccinimide methacrylate (NMS) was synthesized by RAFT polymerization

at 80 °C, in the presence of 4-cyanopentanoic acid dithiobenzoate (CIDB) as the CTA

and AIBN as the initiator. t-Butanol is a good solvent for the polymerization of HPMA,

and DMF was used to dissolve NMS. A semi-batch method was utilized to perform the

copolymerization because of the different reactivity ratios (0.12 and 3.46 respectively

based on the Kelen-Tudos method) of HPMA and NMS.8 The more reactive monomer

(NMS) was continuously added into the reaction mixture to form a “random”

copolymer of HPMA and NMS. The addition rate of NMS was determined by the rate

of homopolymerization of HPMA, which was described in the previous chapter. In

order to avoid initiating NMS at the beginning of the polymerization, NMS was added

into the reaction 30 minutes after the homopolymerization of HPMA was initiated. The

copolymerization was allowed to proceed for 30 minutes after the addition of NMS to

consume the rest of NMS monomer in the reaction mixture. The conversion of HPMA

and NMS was determined by 1H NMR (CDCl3). The purified polymers were analyzed

by 1H NMR and GPC. The copolymerization was carried out with two ratios of

monomers (HPMA+NMS) to CIDB (80:1 and 160:1), in order to study the effect of

[HPMA+NMS]/[CIDB] on the molecular weight of the copolymers. Two ratios of

HPMA to NMS (3:1 and 8:1) were used to prepare copolymers with different

compositions. The experimental results are summarized in Table 3.2.

53

Table 3.2: Experimental results of the copolymerization of HPMA and NMS


b: from 1H NMR in d6-DMSO, Mn = 143.18×DPHPMA+183.16×DPNMS;

c: from GPC using NMP (0.2% LiCl) as the eluent and PMMA standards.

A typical 1H NMR spectrum of poly(HPMA-co-NMS) (entry 2 in Table 3.2) is

shown in Figure 3.5. The main peaks are assigned to the corresponding protons of the

structure drawn in the figure. The characteristic resonances for poly(HPMA-co-NMS)

are clearly evident. The peak at 0.4-1.4 ppm is ascribed to the methyl protons (c, e, f).

The peak at 1.4-2.2 ppm is ascribed to the methylene groups (b, d) of both monomer

units. The peaks at 2.8 ppm and 3.0 ppm are ascribed to the protons of the succinimide

(g) and to the protons of the amide methylene (h) respectively. The signal of the

alcohol methine (i) appears at 3.7 ppm. The signals of hydroxyl proton and amine

proton appear at 4.7 ppm and 7.3 ppm respectively. The signals of the phenyl end

group are also very clear and easily identified, such as the protons (a) at 7.8 ppm. The

peak at 3.3 ppm is due to water absorbed by the deuterated DMSO used in the NMR

experiment.

conversion a %

composition b %

GPC results c entry no.

[HPMA+NMS]/[CIDB]/[AIBN]

HPMA:NMS

(mol:mol)

NMS addition

rate (ml/h)

HPMA

NMS

HPMA

NMS

Mn (experiment) b (×103 g/mol )

Mn

(×103 g/mol )

PDI 1 160/2/1 3:1 0.32 44 34 70 30 4.2 11 1.4 2 160/2/1 8:1 0.32 55 49 89 11 6.6 17 1.2 3 320/2/1 3:1 0.43 42 24 69 31 12 18 1.4 4 320/2/1 8:1 0.21 48 39 90 10 14 28 1.2

54

Figure 3.5: 1H NMR spectrum of the copolymer of HPMA and NMS

The copolymerization of HPMA and NMS was performed at 80 °C in the presence of

4-cyanopentanoic acid dithiobenzoate (CIDB) as the chain transfer agent and AIBN as the

initiator. [HPMA+NMS]/[CIDB]/[AIBN] was 160/2/1. The ratio of HPMA to NMS was 8:1. The

main peaks are assigned to the corresponding protons of the structure drawn in the figure. The

insert is the enlarged 1H NMR region between 6.8 and 8.3 ppm. The solvent was d6-DMSO. The

relaxation delay time was 25 seconds, and the number of scans was 64. The protons (labeled

as “a”) of the phenyl end group of the polymer chain are used to characterize the molecular

weight of poly(HPMA-co-NMS).

The absolute number-average molecular weight of poly(HPMA-co-NMS) was

determined by 1H NMR (in d6-DMSO) (Figure 3.5). The degrees of polymerization of

HPMA and NMS are evaluated by the ratio of the integrals of peak (g), peak (h) and

peak (i) over the integral of peak (a), using Equation 3.1 and Equation 3.2 respectively:

55

(3.1)

(3.2)

Here Ia is the integral of peak (a). Ii, Ig and Ih are the integrals of peak (i), peak (g) and

peak (h), respectively. na is the number of protons (a). ni, ng and nh are the number of

proton (i), protons (g) and protons (h), respectively. The absolute number-average

molecular weight of poly(HPMA-co-NMS) was calculated from the degrees of

polymerization of monomers (HPMA and NMS), as well as the composition of

poly(HPMA-co-NMS) (Table 3.2).

Two ratios of HPMA to NMS (3:1 and 8:1) were used in the copolymerization

to demonstrate the tunability of composition of poly(HPMA-co-NMS). From the

results in Table 3.2 (entry 1, 3 and entry 2, 4), as the ratio of NMS to HPMA increased

from 1:8 to 1:3 in the copolymerization, the percentage of NMS in

poly(HPMA-co-NMS) increased from 10% to 30%. This result establishes that the

composition of poly(HPMA-co-NMS) can be adjusted by using different ratios of

HPMA to NMS. The actual incorporation of NMS in the copolymer was slightly

higher than the ratio of NMS to HPMA in the copolymerization. It probably reflects

the higher reactivity of NMS. From the comparison of entry 1 and 2 as well as entry 3

and 4 in Table 3.2, one can see that poly(HPMA-co-NMS) has a relatively broad

polydispersity (PDI=1.4) when it was prepared with a higher ratio of NMS to HPMA.

DPNMS=

Ii×na

Ia×niDPHPMA=

Ia×ng

(Ig+h−Ii×nh/ni)×na

56

This result may be due to the incompatibility of NMS and RAFT polymerization,

which was mentioned in the introduction of this chapter.

GPC was employed to determine the molecular weights and polydispersities of

the poly(HPMA-co-NMS) samples relative to PMMA standards. As shown in Table

3.2, copolymers of HPMA and NMS were obtained with molecular weights ranging

from 11,000 to 28,000 g/mol. In all cases, polydispersities of the copolymers were

between 1.2 and 1.4. The GPC results indicate that the ratio of monomers

(HPMA+NMS) to CIDB has a significant influence on the molecular weight of the

copolymer obtained. A copolymer of HPMA and NMS with a higher molecular weight

can be obtained by increasing the ratio of monomers (HPMA+NMS) to CIDB in the

polymerization. The results of GPC confirm that RAFT polymerization has been

successfully applied to synthesize poly(HPMA-co-NMS) with a controlled molecular

weight and narrow polydispersity.

The kinetics of the copolymerization of HPMA and NMS was also studied to

obtain a better understanding of the copolymerization reaction. One typical case (entry

3 in Table 3.2) is described below. The ratio of monomers (HPMA+NMS) to CIDB

was 160:1, and the ratio of CIDB to AIBN remained 2:1. The ratio of HPMA to NMS

was 3:1. The copolymerization was carried out with the semi-batch method at 80 °C

over 4 hours. The solution of NMS was added into the reaction mixture beginning 30

minutes after the homopolymerization of HPMA was initiated. The copolymerization

57

was allowed to proceed for 30 minutes after the end of the addition of NMS to

consume the rest of NMS in the reaction mixture. Aliquots were taken out throughout

the polymerization for analysis.

The conversion of HPMA and NMS was determined by 1H NMR (in CDCl3)

(Figure 3.6). The peaks at 5.2 and 5.7 ppm are ascribed to the vinyl protons of HPMA,

and the peaks at 5.8 and 6.3 ppm are ascribed to the vinyl protons of NMS. The peak of

1,3,5-trioxane at 5.1 ppm is used as an internal standard to determine the remaining of

HPMA and NMS in the reaction mixture.

58

Figure 3.6: 1H NMR spectrum of conversion of HPMA and NMS in RAFT

copolymerization. The numbers in different color zones indicate the average value of

the vinyl protons of each corresponding monomer, comparing with the peak of the

internal standard.

As shown in Figure 3.7, the amount of HPMA in the reaction decreased linearly

with reaction time, and there was a small retention of NMS in the reaction mixture

during the polymerization. Finally, 42% of HPMA and 24% NMS were consumed after

four hours. The linear consumption of HPMA in the copolymerization indicates that

there was a constant concentration of active species in the reaction, and the rate of the

Norm

alized Intensity R

eact

ion

Tim

e

OHN

OH

Chemical Shift (ppm) 6.5 6.0 5.5 5.0

4 h

3.5 h

2.5 h

0.5 h

1.5 h

0 h

ON OO

O

O O

O

6.0

11.2

10.2

9.3

7.8

7.0

6.4 2.8

3.0

1.9

0.7

59

copolymerization was first order with respect to HPMA monomer.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

NMS

HPMAM

onom

er R

emai

ning

(mm

ol)

Reaction Time (h)

Figure 3.7: Remaining of HPMA and NMS with reaction time in RAFT copolymerization

using a semi-batch method

The copolymer was purified from each sample and analyzed by GPC and NMR

(Table 3.3). The composition of poly(HPMA-co-NMS) at different intervals of time

was determined by 1H NMR (in d6-DMSO) as described above. Figure 3.8 shows that

the percentage of HPMA in poly(HPMA-co-NMS) decreased slightly from 80% to

69% as the incorporation of NMS increased from 20% to 31%. The relatively high

percentage of HPMA in the copolymer at 1.5 hour of the polymerization results mainly

from the segment of poly(HPMA) formed during the initial 30 minutes of the

polymerization.

60

1.5 2.0 2.5 3.0 3.5 4.00

20

40

60

80

100

NMS

HPMA

Cop

olym

er C

ompo

sito

n

Reaction Time (h)

Figure 3.8: The variation of composition of poly(HPMA-co-NMS) with reaction time

GPC was employed to study the evolution of the molecular weight and

polydispersity of poly(HPMA-co-NMS) with reaction time in the copolymerization of

HPMA and NMS. As shown in Figure 3.9 (a), the molecular weight of

poly(HPMA-co-NMS) increased linearly from 10,000 to 18,000 g/mol during four

hours of the reaction. The polydispersity remained at 1.3-1.4 throughout the

polymerization. Moreover, Figure 3.9 (b) shows that all of GPC curves of copolymer

samples are symmetric. This result indicates that no irreversible termination could be

detected in the copolymerization. Finally, poly(HPMA-co-NMS) was obtained with

Mn= 18,000 g/mol and PDI= 1.4.

61

0 1 2 3 40

5

10

15

20

Mn

Time (h)

Mn

(103 g

/mol

)

1.0

1.1

1.2

1.3

1.4

1.5a

Mw /M

n

7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.50.0

0.2

0.4

0.6

0.8

1.0

RI (

a.u.

)

Retention Volume (ml)

1.5 h 2.5 h 3.5 h 4.0 h

b

Figure 3.9: Evolution of the molecular weight and polydispersity with time (a), GPC

traces of copolymers at various time intervals (b) during the semi-batch RAFT

copolymerization.

62

Table 3.3: Experimental results of the kinetics of the copolymerization of HPMA and

NMS

composition a GPC results b

[M]/[CTA] reaction

time (hours)

HPMA NMS Mw (×103 g/mol )

Mn (×103 g/mol )

PDI

1.5 80 20 13 10 1.3 160 2.5 72 28 20 15 1.3

3.5 71 29 25 18 1.4 4 69 31 26 18 1.4

a: from 1H NMR in d6-DMSO;

b: from GPC using NMP (0.2% LiCl) as the eluent and PMMA standards.

The results from NMR and GPC are summarized in Table 3.3. The composition

results from 1H NMR reveal that the incorporation of NMS in the copolymer increased

gradually as the copolymerization proceeded. The semi-batch method enabled the

distribution of NMS units to be nearly random in the copolymer, except for the

segment of poly(HPMA) formed at the beginning of the polymerization. The random

distribution of activated esters in the copolymer can avoid potential steric problems in

further substitution reactions of the copolymers with amines. The GPC results show

that the molecular weight of poly(HPMA-co-NMS) increased linearly with reaction

time, and the polydispersity remained relatively narrow (1.3-1.4). These results

confirm that the RAFT copolymerization of HPMA and NMS was successfully carried

out with the semi-batch method.

63

3.4 Conclusions

Copolymers of HPMA and NMS were synthesized by RAFT polymerization,

using 4-cyanopentanoic acid dithiobenzoate as the chain transfer agent and AIBN as

the initiator. Due to the different reactivity ratios of HPMA and NMS, a semi-batch

method was employed for the copolymerization. The more reactive monomer (NMS)

was continuously added into the reaction mixture containing HPMA. The molecular

weight of poly(HPMA-co-NMS) could be adjusted by the ratio of monomers (HPMA

+ NMS) to CTA. By changing the ratio of HPMA to NMS, I was able to vary the

composition of poly(HPMA-co-NMS) over a wide range. The copolymers of HPMA

and NMS were characterized by 1H NMR and GPC. The results show that the

copolymers were obtained with controlled molecular weights and narrow

polydispersities. The kinetics of the copolymerization of HPMA and NMS was studied

to obtain a better understanding of the copolymerization reaction. The results from

GPC and NMR reveal that the distribution of NMS was nearly random in the

copolymer chain. The molecular weight of the copolymer increased with reaction time,

and the polydispersity remained narrow. All of the results support the conclusion that

poly(HPMA-co-NMS) can be successfully synthesized by RAFT polymerization using

a semi-batch method.

64

3.5 References

1. Scales, Charles W.; Huang, Faqing; Li, Na; Vasilieva, Yulia A.; Ray, Jacob;

Convertine, Anthony J.; McCormick, Charles L. Corona-Stabilized

Interpolyelectrolyte Complexes of SiRNA with Nonimmunogenic,

Hydrophilic/Cationic Block Copolymers Prepared by Aqueous RAFT

Polymerization. Macromolecules 39, 6871-6881. 2006.

2. Hong, C. Y., Pan, C. Y. Direct Synthesis of Biotinylated Stimuli-Responsive

Polymer and Diblock Copolymer by RAFT Polymerization Using Biotinylated

Trithiocarbonate as RAFT Agent. Macromolecules 39, 3517-3524. 2006.

3. Savariar, E. N., Thayumanavan, S. Controlled polymerization of

N-isopropylacrylamide with an activated methacrylic ester. J.Polym.Sci., Part

A: Polym.Chem. 42, 6340-6345. 2004.

4. Shunmugam, R., Tew, G. N. Efficient route to well-characterized homo, block,

and statistical polymers containing terpyridine in the side chain. Journal of

Polymer Science, Part A: Polymer Chemistry 43, 5831-5843. 2005.

5. Monge, S., Haddleton, D. M. Synthesis of precursors of poly(acryl amides) by

copper mediated living radical polymerization in DMSO. European Polymer

Journal 40, 37-45. 2003.

6. Godwin, A., Hartenstein, M., Muller, A. H. E., Brocchini, S. Narrow molecular

weight distribution precursors for polymer-drug conjugates. Angewandte

Chemie, International Edition 40, 594-597. 2001.

7. Schilli, C., Mueller, A. H. E., Rizzardo, E., Thang, S. H., Chong, B. Y. K.

Controlled radical polymerization of N-isopropylacrylamide and of activated

esters for the synthesis of polymer-protein and polymer-drug conjugates.

Polymer Preprints (American Chemical Society, Division of Polymer

65

Chemistry) 43, 687-688. 2002.

8. Yanjarappa, M. J., Gujraty, K., V, Joshi, A., Saraph, A., Kane, R. S. Synthesis

of copolymers containing an active ester of methacrylic acid by RAFT:

controlled molecular weight scaffolds for biofunctionalization.

Biomacromolecules 7, 1665-1670. 2006.

66

Chapter 4

Synthesis and Characterization of

Naphthalimide-dye-labeled Poly(HPMA-co-NMS) by RAFT

Polymerization

4 Synthesis and Characterization of

Naphthalimide-dye-labeled

Poly(HPMA-co-NMS) by RAFT Polymerization

4.1 Introduction

There is a long history of the study of polymers by labeling them with various

dyes. This method has been used in the past to study a variety of different properties of

polymers, such as their behavior in solutions and their location within a

multicomponent system.1-6 Dye-labeled polymers have been used for various

applications, such as in coatings, for single chain characterization, for sensors, and for

imaging.1-4 Some of these polymers have dyes randomly located on the backbone.

Others have dyes attached specially to one end. I am particularly interested in the

synthesis of end-labeled polymers. There are two approaches to label the chain end of a

polymer. One is to synthesize a polymer with a reactive end group. Then the dye can be

attached to the polymer via certain reactions, such as Michael addition or “click”

chemistry.2,6 The coupling chemistry needs to be chosen carefully. Both polymer and

dye should have suitable active functionalities to form a covalent bond. The second

way is to introduce labeling moieties to a compound which will be used to initiate or

67

terminate the polymerization. For RAFT polymerization, one can use a dye-labeled

chain transfer agent (in RAFT).3,7 Using these strategies and methods, one can also

synthesize polymer derivatives containing other species of interest, such as proteins,

peptides, and nanopaticles.8-10

Both labeling strategies have been used previously to prepare dye end-labeled

polymers by RAFT polymerization. One recent example is shown in Figure 4.1.6

Poly(N-isopropylacrylamide) (PNIPAM) was prepared by RAFT polymerization,

using 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid as the CTA. The

PNIPAM had a thiocarbonylthio end group which resulted from the incorporation of

the CTA during the polymerization. The thiocarbonylthio end group of PNIPAM was

reduced to a thiol group with the reducing agent NaBH4. Then, a maleimide-thiol

coupling reaction was employed to label the RAFT-generated polymer with a pyrene

dye. Note that, the thiol end group of this type of polymer would also be available to

react with other thiols or maleimides, to bind a variety of molecules of interest to the

polymer chain.

68

Figure 4.1: Synthetic pathway of the reduction of a trithiocarbonate end polymer and

conjugation with N-(1-pyrenyl)maleimide6


However, polymeric thiols often have a relatively low reactivity, which limits

the labeling efficiency. Figure 4.2 presents one strategy to solve this problem.11 The

thiol at the end of poly(N-(2-hydroxypropyl)

methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) was converted to a

primary amine group via disulfide exchange reaction. Later, the reactivity of the amine

end was demonstrated by a ninhydrin assay. Although this polymer end group

modification increased the fraction of accessible end groups, the process of

modification is still complex, and the degree of dye modification could only reach

80%.11 Moreover, it was difficult to separate the modified polymer from the unlabeled

one.

69

Figure 4.2: Synthetic pathway of the amine fictionalization and fluorescent labeling of

the polymer with a fluorescein N-hydroxysuccinimide ester11


One feature of the RAFT polymerization is that the chain transfer agent (CTA),

such as a thiocarbonylthio compound ZC(=S)SR, lead to polymers containing the R

group at one end and the Z group at the other end of the chain.12 Based on this feature,

another strategy was developed to label polymers synthesized by RAFT

polymerization (Figure 4.3). Bathfield and his colleagues recently reported an

70

approach to synthesize several modified RAFT CTAs from a simple precursor,

succinimido-2-[[2-phenyl-1-thioxo]thio]-propanoate (compound 1, Figure 4.3). The

activated ester in the R group of the precursor was reacted with several amino

derivatives to form the functionalized CTAs, including N-aminoethylmorpholine (2a),

6-amino-6-desoxy-1,2:3,4-di-O-isopropylidene-6-α-D-galactopyranose (2b),

(+)-biotinyl-3,6-dioxaoctanediamine (2c). These modified CTAs can be used to

prepare bio-related end-functionalized polymers by RAFT polymerization.7 However,

amino derivatives can rapidly degrade the thiocarbonylthio function by thioamidation,

which competes with the reaction between the CTA precursor and amino derivatives in

the preparation of modified CTAs.

Figure 4.3: Synthetic pathway of functional RAFT CTAs from the same precursor 17


71

Meanwhile, Hong et al. synthesized a biotinylated trithiocarbonate CTA

(Figure 4.4).13 They used the biotinylated CTA to prepare bioresponsive polymers by

RAFT polymerization. One problem with this biotinylated polymer is that the ester

link between the biotin derivative and the polymer may undergo hydrolysis in aqueous

environments. Nevertheless, compared with post-polymerization modification, this

strategy is more simple and efficient to prepare end-functionalized polymers with

various moieties of interest.

Figure 4.4: Synthetic pathway of biotinylated end-functionalized polymers13


72

The 1,8-naphthalimide chromophore and it derivatives have been studied for

many years.14-20 Many of these derivatives are easily synthesized from the

corresponding naphthalic anhydrides. They have relatively high fluorescent quantum

yields, high photostability and tunable spectroscopic properties.15,16,20 Many

1,8-naphthalimide derivatives have been incorporated covalently or non-covalently

into various polymeric materials to study the properties and behavior of the polymers.

A family of 1,8-naphthalimide derivatives such as

9-butyl-4-butylamino-1,8-naphthalimide have been used to monitor the diffusion of

polymers by Förster resonance energy transfer (FRET), both in our group3 and by May

et al.19 These type of molecules were also used to study the degradation of polymeric

materials.21 Polymers containing the 1,8-naphthalimide group have also been studied

for other applications such as sensors, probes, OLEDs, solar cells, as well as

photochemotherapy.22-27

This chapter describes the synthesis of a naphthalimide-dye-labeled

poly(HPMA-co-NMS) by RAFT polymerization, using a

9-isobutyl-4-ethylenediamino-1,8-naphthalimide labeled CTA. The labeled CTA was

prepared by a one-step reaction from a dye-labeled CTA precursor and

bis(thiocarbonyl) disulfide. The characterization of the naphthalimide-dye-labeled

polymer was carried out by UV-Vis spectroscopy, GPC and NMR spectroscopy. The

naphthalimide-dye-polymer conjugates can be used as fluorescent probes and labels

for various biological applications.

73



9-Isobutyl-4-ethylenediamino-1,8-naphthalimide (BEAN) was synthesized as

described below. 4-Bromo-1,8-naphthalimide anhydride (BNA) (95%, Aldrich),

isobutylamine (99%, Aldrich), ethylenediamine (99%, Aldrich), oxalyl chloride (98%,

Aldrich), 2,2’-azobis(2-methylpropionitrile) (AIBN) (98%, Aldrich), 1,3,5-trioxane

(≥99%, Aldrich), tert-butanol (t-BuOH) (≥99.5%, Aldrich), N,N-dimethylformamide

(DMF) (99.8%, Aldrich), 1-methyl-2-pyrrolidinone (NMP) (≥99%, Aldrich) and other

chemicals were used as received.


CDCl3 and d6-DMSO were purchased from Cambridge Isotope Laboratories, Inc.

3-(trimethylsilyl)propionic acid-d4 sodium salt (TSP) was purchased from Aldrich.

GPC: Polymers were analyzed by GPC using NMP containing 0.2 wt% LiCl as the

eluent and PMMA standards. GPC analysis was carried out at 80 °C at a flow rate of

0.6 ml/min with refractive index and UV absorbance detectors.


Analyzer.

74

4.2.2 Synthesis and Characterization of the

Naphthalimide-dye-labeled CTA

Figure 4.5: Synthesis of 9-isobutyl-4-ethylenediamino-1,8-naphthalimide

Synthesis and characterization of 9-isobutyl-4-ethylenediamino-1,8-naphthalimide

9-isobutyl-4-ethylenediamino-1,8-naphthalimide was synthesized in two steps

(Figure 4.5). 4-Bromo-1,8-naphthalic anhydride (BNA) (1.4 g) and isobutylamine (2

ml) were added to 1,4-dioxane (40 ml) at room temperature. The solution was stirred

for 4.5 hours at 100 ºC. The solvent was then evaporated with a rotaevaporator. The

yellow solid was purified by silica column chromatography using heptane:acetone

(12:1, v:v) as the eluent. Finally, the yellow solid product

9-isobutyl-4-bromo-1,8-naphthalimide (BBN) was dried under vacuum at 50 ºC over

night. Yield: 1.14 g (72%); m.p.: 130.0-131.5 ºC; 1H NMR (CDCl3): δ(ppm) 0.98 (d,

6H), 2.24 (m, 1H), 4.04 (d, 2H), 7.85 (dd, 1H), 8.04 (d, 1H), 8.42 (d, 1H), 8.58 (d, 1H),

BNA BEAN BBN

50 ºC, 2.5 h1, 4- Dioxane100 ºC, 4~5 hBr

OO O

HN

NO O

NH2

Br

NO ONH2 NH2H2N

75

8.66 (d, 1H); Elemental Analysis (calculated/measured): C (57.85/57.98), H

(4.25/4.32), N (4.22/4.50).

BBN (3.020 g) was added to ethylenediamine (50 ml). The solution was stirred

at 50 ºC for 2.5 hours. The reaction mixture was treated with toluene (200 ml), and

then the volatile liquids were evaporated with a rotaevaporator. The crude product was

dissolved in 1M aq. HCl (70 ml). The product was precipitated when the solution

became weakly basic (pH~8) by adding 1M aq. NaOH. The mixture was then filtered.

The yellow solid product 9-isobutyl-4-ethylenediamino-1,8-naphthalimide (BEAN)

was dried under vacuum at 50 ºC overnight. Yield: 2.662 g (94%); m.p.: 162-164 ºC;

1H NMR (CDCl3): δ(ppm) 0.98 (d, 6H), 1.24 (broad, 2H), 2.25 (m, 1H), 3.18 (t, 2H),

3.42 (m, 2H), 4.03 (d, 2H), 6.14 (broad, 1H), 6.72 (d, 1H), 7.62 (t, 1H), 8.16 (d, 1H),

8.45 (d, 1H), 8.58 (d, 1H); Elemental Analysis (calculated/measured): C (69.43/68.52),

H (6.80/6.66), N (13.49/13.66).

76

Figure 4.6: Synthesis of the naphthalimide-dye-labeled chain transfer agent

Synthesis and characterization of the naphthalimide-dye-labeled chain transfer agent

The synthesis route to the naphthalimide-dye-labeled CTA is shown in Figure

4.6. Oxalyl chloride (5.3 ml) was added into a stirred suspension of

4,4’-Azobis(4-cyanovaleric acid) (1.708 g) in anhydrous CH2Cl2 with a catalytic

amount of N,N-Dimethylformamide at room temperature. After 3 hours, the reaction

mixture turned clear, and was evaporated with a rotaevaporator to leave a yellow solid

of 4,4'-(diazene-1,2-diyl)bis(4-cyanopentanoyl chloride) (AVAC). Yield: 1.820 g

(95.6%); 1H NMR (CDCl3): δ(ppm) 1.72 (d, 6H), 2.62 (m, 4H), 3.14 (m, 4H).

N N CNCl

OCN

Cl

O

N N CNOH

OCN

HO

O

AVAC

Cl ClO

OCH2Cl2, DMF, r.t., 3h

HN

NO O

NH2

Dichloromethane

R=

N N CNR

OCN

R

O

Ethyl acetate

80 ºC+ N N CN

R

OCN

R

OS

S S

SS

S CNR

O2

NHN

O

OHN

N N CNCl

OCN

Cl

O

AVAN

NCTA

BEAN

77

BEAN (1.557g) was dissolved in 100 ml anhydrous CH2Cl2. The solution was

cooled to 0 °C. A solution of AVAC (0.632 g) and N,N-Diisopropylethylamine (2.29

mL) in 20 ml anhydrous CH2Cl2 was added dropwise to the dye solution via an

additional funnel under N2 protection. The reaction was then stirred for 12 hours at

room temperature and then concentrated with a rotaevaporator. The mixture was

washed with saturated sodium bicarbonate solution, 1% aq. HCl and finally 2 M aq.

NaCl. The organic layer was dried over magnesium sulfate, filtered, and concentrated.

The product (AVAN) was precipitated in diethyl ether, and lyophilized over night.

Yield: 1.36 g (79%); 1H NMR (CDCl3): δ(ppm) 0.98 (d, 12H, CH3), 1.69 (d, 6H, CH3),

2.22-2.52 (m, 8H, CH2CH2), 3.44 (m, 4H, CH2), 3.68 (t, 4H, CH2), 4.01 (d, 4H, CH2),

6.57 (d, 2H), 7.60 (t, 2H), 8.12 (d, 2H), 8.35 (d, 2H), 8.51 (d, 2H).

A solution of bis(thiocarbonyl) disulfide (0.156 g) and AVAN (0.213 g) in ethyl

acetate was degassed with nitrogen and heated at 80 ºC for 20 hours. The solvent was

removed with a rotaevaporator, and the residue was purified by silica column

chromatography using ethyl acetate/hexane as the eluent. Yield: 0.049 g (34%); m.p.:

90-93 ºC; 1H NMR (CDCl3): δ(ppm) 0.99 (d, 6H, CH3), 1.93 (s, 3H, CH3), 2.44-2.71

(m, 4H, CH2CH2), 3.54 (t, 2H, CH2), 3.77 (t, 2H, CH2), 4.04 (d, 2H, CH2), 6.17 (broad,

1H, NH), 6.60 (d, 1H, naphthilc-H), 6.91 (broad, 1H, NH)�7.66 (t, 1H), 7.35 (m, 2H,

m-ArH), 7.38 (t, 1H, p- ArH), 7.84 (dd, 2H, o-ArH), 8.24 (d, 1H), 8.45 (d, 1H), 8.57 (d,

1H); Elemental Analysis (calculated/measured): C (65.01/64.83), H (5.63/5.77), N

(9.78/9.23).

78

4.2.3 Synthesis and Characterization of the

Naphthalimide-dye-labeled Copolymer of HPMA and NMS

Figure 4.7: Synthesis pathway of the naphthalimide-dye end-labeled

poly(HPMA-co-NMS)

The copolymerization reactions were carried out under an Argon (Ar)

atmosphere using the Schlenk technique (Figure 4.7). The copolymerization procedure

is similar to the procedure described in the previous chapter. A stock solution was

prepared consisting of AIBN (6.6 mg), NCTA (45.7 mg) and 1,3,5-trioxane (internal

standard, 77.6 mg) in degassed DMF (1 ml). The monomers were evacuated and

back-filled with Ar three times. The solvents were degassed with Ar. t-BuOH (1.9 ml)

was added into a round-bottom flask containing HPMA (0.275 g) to form a 1 M

solution, and then the solution of AIBN, NCTA and 1,3,5-trioxane in DMF (200 μl)

was transferred into the flask. The mixture was heated at 80 °C for 30 minutes, and

then a solution of NMS in DMF (0.5 M) was added continuously into the reaction

mixture at 0.43 ml/h through an airtight syringe by a syringe pump (KD Scientific,

+ OHN

OH

ON OO

O

AIBN, t- BuOH/DMF, 80 ºC

R=

S

S CNR

O

NHN

O

OHN

Rn mS

S

OO

NO O

OHN

CN

O

OH

79

Model 780100). After the addition of the NMS solution, the reaction was kept at 80 °C

for 30 more minutes and was then quenched in an acetone-dry ice bath. Aliquots (0.1

ml) were taken out for NMR analysis throughout the reaction. The final polymer was

precipitated using a mixture of anhydrous diethyl ether and anhydrous acetone

(v:v=1:1), recovered by centrifugation, and then lyophilized overnight. The polymer

product (26 mg) was stored at 4 °C.

80


One feature of RAFT polymerization is that a chain transfer agent (such as a

thiocarbonylthio compound ZC(=S)SR) is used in the formation of polymers. As a

consequence, a polymer obtained by RAFT polymerization will have R and Z groups

at each end of the polymer chain. Since the substituent Z is prone to aminolysis, the

modification on the substituent R is a better strategy to introduce functionalities. A new

facile approach is presented here to prepare end-functionalized RAFT chain transfer

agents through the substituent R.

A naphthalimide-dye-labeled chain transfer agent was synthesized from a

modified CTA precursor and bis(thiocarbonyl) disulfide via a one-step reaction (Figure

4.6). Bis(thiocarbonyl) disulfide was synthesized from bromobenzene, which was

described in Chapter 2. The phenyl group will be the Z group of the labeled chain

transfer agent. The modified CTA precursor was synthesized by a reaction of the

naphthalimide-dye with an amine group and

4,4'-(diazene-1,2-diyl)bis(4-cyanopentanoyl chloride). The naphthalimide-dye

modified CTA precursor will provide the R group of the labeled chain transfer agent.

Finally the naphthalimide-dye-labeled CTA was synthesized by heating the reaction

mixture of the modified CTA precursor and bis(thiocarbonyl) disulfide. The yield was

only 34% after the chromatography purification, but the product was quite pure proved

by the results of NMR measurement and elemental analysis.

81

I chose an amide link between the dye and chain transfer agent for the synthesis

of end-labeled poly(HPMA-co-NMS) (Figure 4.8). The amide link prevents the

labeled polymer from side-reactions in substitution of activated ester group and

hydrolysis in aqueous environments.

HN

NH

N

O

O

n mS

S

OO

NO O

OHN

CN

O

OH

Figure 4.8: Structure of the naphthalimide-dye end-labeled poly(HPMA-co-NMS)

The copolymer of N-(2-Hydroxypropyl)methacrylamide (HPMA) and

N-hydroxysuccinimide methacrylate (NMS) was synthesized by RAFT polymerization,

using the same procedure described in the previous chapter. Instead of

4-cyanopentanoic acid dithiobenzoate (CIDB), the naphthalimide-dye-labeled chain

transfer agent (NCTA) was used to obtain the dye-labeled poly(HPMA-co-NMS)

(Figure 4.8). In order to obtain a polymer with a molecular weight around 20×103

g/mol, the ratio of monomers (HPMA+NMS) to NCTA was chosen as 160:1, and the

ratio of NCTA to AIBN remained 2:1. The ratio of HPMA to NMS was chosen as 3:1.

The copolymerization was carried out in the semi-batch mode at 80 °C over 4 hours.

The conversion of HPMA and NMS was determined by 1H NMR, using 1,3,5-trioxane

as an internal standard (Table 4.1).

82

Table 4.1: Preparation of the naphthlimide-dye-labeled poly(HPMA-co-NMS) by RAFT

a: the total molar feed ratio of HPMA and NMS;

b: from 1H NMR in CDCl3.

In order to determine the water content of the product, a certain amount of

copolymer sample was heated at 90 °C under vacuum for several days and weighed

from time to time until the weight of the copolymer sample became constant. The

results showed that there was 0.8 wt% water in the copolymer sample (Table 4.2).

Table 4.2: Experimental results of water content of the naphthalimide-dye end-labeled poly(HPMA-co-NMS)

Heating

time (hours)

Heating temperature

(°C)

Weight (mg)

Water content

% 0 1.334 10 1.328 22 90 1.323 0.8 36 1.323

Poly(HPMA-co-NMS) was obtained by RAFT polymerization at 80 °C, using the

naphthalimide-dye-labeled CTA. ([HPMA+NMS]/[NCTA]/[AIBN]=320/2/1, [HPMA]/[NMS]=3/1)

The nominal molecular weight and polydispersity of the dye-labeled

poly(HPMA-co-NMS) were determined by GPC using PMMA standards. As shown in

Figure 4.9, the GPC provides traces obtained from both the refractive index (RI)

conversion b %

[HPMA+NMS]/[NCTA]/[AIBN]

HPMA:NMS a

(mol:mol)

NMS addition rate

(ml/h)

Reaction Time

(hours)

Reaction Temperature

(°C) HPMA NMS 320/2/1 3:1 0.43 4 80 31 31

83

detector and the UV absorbance (λ= 430 nm) detector. There are two peaks observed in

the UV trace at different retention time (14.5 min and 16.7 min). They are ascribed to

the dye-labeled poly(HPMA-co-NMS) and free dye-labeled CTA respectively. The RI

signal of the dye-labeled poly(HPMA-co-NMS) appears at the same retention time

(14.5 min) as the UV signal of the copolymer. However, due to the lower sensitivity of

the RI detector, the signal of free dye-labeled CTA was considerably less intense in the

RI trace.

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5

0

10

20

30

Retention volume (ml)

RI (

a.u.

)

-5

0

5

10

15

20

UV 43

0 (a.u

.)

Polymer

Free dye

RI

UV

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5

0

10

20

30


RI (

a.u.

)

-5

0

5

10

15

20

UV 43

0 (a.u

.)

Polymer

Free dye

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5

0

10

20

30


RI (

a.u.

)

-5

0

5

10

15

20

UV 43

0 (a.u

.)

Polymer

Free dye

RI

UV

Figure 4.9: GPC traces of the naphthalimide-dye end-labeled poly(HPMA-co-NMS)

([HPMA+NMS]/[CTA]/[AIBN]=320/2/1). The trace exhibiting significant noise is from the

UV detector.

84

The GPC curves (UV and RI signals) of dye-labeled poly(HPMA-co-NMS) are

symmetric, which indicates that no irreversible termination could be detected in the

copolymerization. The RI and UV peaks appear at the same retention time (14.5 min),

which indicates that the dye is covalently bound to the polymer. The GPC trace from

the UV detector shows that there is a small amount of free dye-labeled chain transfer

agent in the polymer sample. The residual dye indicates incomplete polymer

purification. The content of free dye-labeled CTA is determined by the ratio of the

areas of the UV signals of the copolymer and the free dye-labeled CTA. The effect of

free dye-labeled CTA in the copolymer will also be considered in following

measurements. The GPC results indicate the success of the RAFT polymerization to

synthesize dye end-labeled poly(HPMA-co-NMS) with a controlled molecular weight

and narrow polydispersity (Table 4.3).

Table 4.3: GPC results of the naphthalimide-dye end-labeled poly(HPMA-co-NMS)

Mw (×103 g/mol )

Mn (×103 g/mol )

PDI

Free CTA Content (mol%)

31 21 1.4 16

GPC uses NMP (containing 0.2% LiCl) as the eluent, PMMA standards, refractive index detector and UV absorbance detector (λ= 430 nm).

The naphthalimide-dye end-labeled poly(HPMA-co-NMS) was also

characterized by 1H NMR (in d6-DMSO) (Figure 4.10). The main peaks in the

spectrum are assigned to the corresponding protons of the polymer structure drawn in

the figure. The characteristic resonances for poly(HPMA-co-NMS) are apparent in the

85

spectrum. The peak at 0.4-1.4 ppm is ascribed to the methyl protons (m, f, k). The peak

at 1.4-2.2 ppm is ascribed to the methylene groups (n, g) of both monomer units. The

peaks at 2.8 ppm and 3.0 ppm are ascribed to the protons of the succinimide (o) and to

the protons of the amide methylene (i), respectively. The signal of the alcohol methine

(j) appears at 3.7 ppm. The signals of hydroxyl proton and amine proton appear at 4.7

ppm and 7.3 ppm, respectively. The signals of the naphthalimide are also very clear

and easily identified, such as the proton (a) at 8.6 ppm, the proton (e) at 8.5 ppm and

the proton (c) at 8.3 ppm. The peak at 3.3 ppm is due to water absorbed by the

deuterated DMSO used in the NMR experiment.

86

Figure 4.10: 1H NMR spectrum of the naphthalimide-dye end-labeled

poly(HPMA-co-NMS)

The copolymerization of HPMA and NMS was performed at 80 °C in the presence of a

naphthalimide-dye-labeled chain transfer agent (NCTA) and AIBN.

[HPMA+NMS]/[NCTA]/[AIBN] was 320/2/1. The ratio of HPMA to NMS was 3:1. The main

peaks are assigned to the corresponding protons of the structure drawn in the figure. The insert

is the enlarged 1H NMR region between 6.6 and 8.8 ppm. The solvent was d6-DMSO. The

relaxation delay time was 25 seconds, and the number of scans was 1280. The protons

(labeled as “a”) of the naphthalimide end group of the polymer chain are used to characterize

the molecular weight of the dye end-labeled poly(HPMA-co-NMS).

87

The absolute number-average molecular weight of the dye end-labeled

poly(HPMA-co-NMS) was determined by 1H NMR using two different methods. One

utilized the proton signal of the naphthalimide end group at 8.65 ppm as a standard

(Figure 4.10). Then, the degrees of polymerization of HPMA and NMS were evaluated

by the ratio of the integrals of the peak (i), peak (j) and peak (o) over the integral of

peak (a), using Equation 4.1 and Equation 4.2 respectively:

(4.1)

(4.2)

Here Ia is the integral of peak (a). Ii, Ij and Io are the integrals of peak (i), peak (j) and

peak (o), respectively. na is the number of protons (a). ni, nj and no are the number of

protons (i), proton (j) and protons (o), respectively. The absolute number-average

molecular weight of poly(HPMA-co-NMS) was calculated from the degrees of

polymerization of monomers (HPMA and NMS), as well as the composition of the

dye-labeled poly(HPMA-co-NMS).

The other method used 3-(trimethylsilyl)propionic acid-d4 sodium salt (TSP) as

an external standard for the quantitative measurement by NMR. The procedure is

described below. The dye-labeled poly(HPMA-co-NMS) (6.17 mg) was dissolved in

d6-DMSO (0.6913 g). A solution (19.8 mg) of TSP in d6-DMSO (1mg/g) was added

into the polymer solution. Then the sample was measured by 1H NMR. The signals of

(Ii+o−Ij×ni/nj)×na

Ia×ni

DPNMS=

Ij×na DPHPMA=

Ia×nj

88

the naphthalimide-dye and TSP were integrated to obtain the mole ratio of the

naphthalimide-dye to TSP. Then, the amount of naphthalimide-dye could be calculated

quantitatively, because the amount of TSP in the sample is known. Thus, the absolute

number-average molecular weight of the dye-labeled poly(HPMA-co-NMS) is

determined from the weight of the polymer sample and the moles of the

naphthalimide-dye end group in the sample.

The results obtained from 1H NMR (d6-DMSO) are summarized in Table 4.4.

In order to obtain a spectrum of high quality, several parameters had to be optimized

for the NMR experiment. The relaxation delay time was set to 25 seconds to ensure

complete relaxation of the protons. The number of scans was 1280 to enhance the

signal-to-noise ratio. The NMR spectrum (Figure 4.10) shows that the signals of the

naphthalimide are very distinct and well separated from other peaks in the spectrum.

This improves the accuracy and precision of molecular weight determination using the

naphthalimide end group. TSP is a common external standard for quantitative NMR

measurements in aqueous media. Here, TSP external standard was employed to

measure directly the amount of naphthalimide-dye in the sample by 1H NMR. This

method also serves as a comparison to assess the accuracy of the molecular weight

obtained via the naphthalimide end group analysis. The Mn values determined with

both methods are in good agreement.

89

Table 4.4: The characterization results of the naphthalimide-dye end-labeled poly(HPMA-co-NMS) by 1H NMR a

Composition (%) DP

HPMA NMS HPMA NMS

Mn by the end group b

(×103 g/mol )

Mn by TSP c

(×103 g/mol )

69 31 54 24 16 18

a: The solvent was d6-DMSO. The relaxation delay time was 25 seconds and the number of

scans was 1280;

b: Mn = 143.18×DPHPMA+183.16×DPNMS;

c: Mn = [Mass of poly(HPMA-co-NMS)]/ [Mole of naphthalimide-dye].

Due to the presence of the naphthalimide-dye, the end-labeled

poly(HPMA-co-NMS) has a strong UV absorption at 440 nm (Figure 4.12). Because

each polymer contained one naphthalimide-dye, the absolute number-average

molecular weight of the dye-labeled polymer can also be determined by UV-Vis

spectroscopy. The calibration curve of UV absorbance of the naphthalimide-dye was

built using 9-isobutyl-4-ethylenediamino-1,8-naphthalimide (BEAN) as a model

compound (Figure 4.11). A series of solutions of BEAN in DMF were prepared with

accurately known concentrations. The absorbance of these solutions at 440 nm was

measured by UV-Vis spectrometry. The sharp feature of UV spectra at 420 nm is an

artifact from the spectrometer. The experimental extinction coefficient was obtained

from the calibration curve of UV absorbance of naphthalimide-dye as 1.29×104

M-1cm-1 (Figure 4.12). The calibration curve does not have a zero-intercept (-0.02). It

is mainly due to a slight systemic error in the measurement.

90

300 350 400 450 500 550 6000.0

0.5

1.0

1.5

2.0

2.5

Abs

orpt

ion

(a.u

.)

Wavelength (nm)

2.44*10-3 M 9.78*10-4 M 5.55*10-4 M 2.44*10-4 M 9.78*10-5 M 5.55*10-5 M

Figure 4.11: Absorbance of 9-isobutyl-4-ethylenediamino-1,8-naphthalimide at different

concentrations in DMF. The dash line in the figure is the position of 440 nm wavelength.

91

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Abs

orpt

ion

(a.u

.)

Concentration ( 10-5 mol/L)

Figure 4.12: Absorbance at 440 nm versus concentration in DMF for

9-isobutyl-4-ethylenediamino-1,8-naphthalimide (■), the naphthalimide-dye end-labeled

poly(HPMA-co-NMS) (1.13 g/L) (●), and the fitted calibration curve (―)

Considering the molecular weight determination via UV-Vis spectroscopy, the

dye-labeled poly(HPMA-co-NMS) (1.134mg) was dissolved in DMF to form a 1.13

g/L solution, and then the absorbance of the dye-labeled polymer at 440 nm was

measured by UV-Vis spectrometry (Figure 4.13). From the resulting spectrum, the

molarity of the dye-labeled poly(HPMA-co-NMS) was calculated using the

experimental extinction coefficient. The value of Mn was calculated according to

Equation 4.3:

92

(4.30)

Here [Polymer] is the concentration (g/L) of the dye-labeled poly(HPMA-co-NMS), ε

is the experimental extinction coefficient (M-1cm-1), l is the length of the cell (cm), and

A440 is the UV absorbance of the dye-labeled poly(HPMA-co-NMS) at 440 nm. The

absolute number-average molecular weight of the dye-labeled poly(HPMA-co-NMS)

is determined to be 18×103 g/mol (Table 4.5).

(440, 0.0937)

300 350 400 450 500 550 6000.00

0.02

0.04

0.06

0.08

0.10

0.12

Abs

orpt

ion

(a.u

.)

Wavelength (nm)

Figure 4.13: Absorbance of the naphthalimide-dye end-labeled poly(HPMA-co-NMS)

(1.13 g/L) in DMF. The arrow points out the absorbance at 440 nm

[Polymer]×ε×l

A440

Mn=

93

Table 4.5: The determination of the absolute number-average molecular weight of the naphthalimide-dye end-labeled poly(HPMA-co-NMS)

Weight of sample

(mg) Absorbance at

440 nm Extinction coefficient at 440 nm

(×104 M-1cm-1) Mn

(×103 g/mol )

1.134 0.094 1.29 18

The number-average molecular weight of the naphthalimide end-labeled

poly(HPMA-co-NMS) was determined using four independent methods. These

include 1H NMR via end group analysis and via TSP as an external standard, end group

analysis by UV-Vis spectroscopy, and nominal determination of Mn by GPC. These

values are collected in Table 4.6. The results obtained from quantitative NMR using

TSP external standard and UV-Vis spectroscopy are in excellent agreement. Both

methods give a value of Mn= 18×103 g/mol. The result from NMR end group analysis

is 16×103 g/mol, which is very close to 18×103 g/mol. The difference may result from

the error of integration of the characteristic peaks of poly(HPMA-co-NMS). GPC was

also employed to determine the molecular weight of the dye-labeled

poly(HPMA-co-NMS) relative to PMMA standards with narrow PDIs. This method

yielded a value of Mn= 21×103 g/mol. The divergence of this value to the one obtained

by NMR and UV-Vis is an indication of the different hydrodynamic volumes of the

dye-labeled poly(HPMA-co-NMS) and PMMA in NMP.

94

Table 4.6: Summary of number-average molecular weight values of the

naphthalimide-dye end-labeled poly(HPMA-co-NMS) determined with different methods

Mn (×103 g/mol )

GPC NMREnd Group NMRTSP UV-Vis

21 16 18 18

4.4 Conclusions

A new approach was developed to prepare a functionalized RAFT chain

transfer agent. Initially, a naphthalimide-dye with an amine group

(9-isobutyl-4-ethylenediamino-1,8-naphthalimide) was synthesized. Subsequently, the

naphthalimide-dye was attached to a CTA precursor

(4,4'-(diazene-1,2-diyl)bis(4-cyanopentanoyl chloride)) via an amide bond to form a

modified CTA precursor. Finally, a naphthalimide-dye-labeled CTA was prepared in a

one-step reaction, using this modified CTA precursor and bis(thiocarbonyl) disulfide.

The yield was only modest (34%) but the product was pure.

The naphthalimide-dye-labeled CTA was then successfully employed in the

synthesis of a dye end-labeled poly(HPMA-co-NMS). The GPC results show that the

copolymer of HPMA and NMS obtained had a narrow molecular weight distribution.

The absolute number-average molecular weight of the dye-labeled copolymer was

determined by four independent methods, 1H NMR spectroscopy via end group

characterization and using TSP external standard, end group analysis by UV-Vis

95

spectroscopy and by GPC. The results obtained from these measurements are in good

agreement. The copolymer of HPMA and NMS provides a scaffold to carry various

functional molecules such as drugs and proteins via the substitution of the pendant

activated esters. The dye label can be utilized to characterize the substitution efficiency

and the corresponding polymer conjugates. The dye end-labeled poly(HPMA-co-NMS)

has great potential applications in medicine, tissue engineering and other areas. The

end functionalization strategy described here can be broadly applied to prepare

end-functionalized polymers with various desired moieties such as drugs, proteins,

peptides and nanoparticles for a variety of applications in biochemistry, medicine and

material science.

96

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