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 Experimental Section 48
3.2.1 General Information 48
3.2.2 Synthesis and Characterization of NMS 49
3.2.3 Synthesis and Characterization of Copolymers of HPMA and NMS 50
3.3 Results and Discussion 52
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 Experimental Section 73
4.2.1 General Information 73
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
4.3 Results and Discussion 80
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
Table 2.3 Kinetics of the homopolymerization of HPMA at [M]/[CTA] =
160
37
Table 2.4 Kinetics of the homopolymerization of HPMA at [M]/[CTA] =
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
Figure 2.9 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= 160
36
ix
Figure 2.10 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= 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
1. Collins, I., Workman, P. New approaches to molecular cancer therapeutics.
Nat.Chem.Biol. 2, 689-700. 2006.
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
of a new class of chemotherapeutic agents-drug-polymer conjugates.
Clin.Cancer Res. 5, 83-94. 1999.
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
16
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
solutions. III. Elimination and retention of
poly-[N-(2-hydroxypropyl)methacrylamide] in a test organism. Journal of
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
pharmacokinetics of polymer-bound adriamycin. Biochemical Pharmacology
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
angiogenesis with a conjugate of HPMA copolymer and TNP-470. Nature
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
localisation of the synthetic soluble macromolecular drug carrier pHPMA. Eur
J Nucl Med Mol Imaging 29, 1055-1062. 2002.
18. Matyjaszewski, K. Controlled/Living Radical Polymerization. Progress in
ATRP, NMP, and RAFT. (Proceedings of a Symposium on Controlled Radical
Polymerization held on 22-24 August 1999, in New Orleans.) [In: ACS Symp.
Ser., 2000; 768]. 484. 2000.
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
a: from 1H NMR in CDCl3;
b: Mn = 143.18×80;
c: from GPC using NMP (containing 0.2 wt% LiCl) as the eluent and PMMA standards.
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
Figure 2.9: 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= 160.
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.
Table 2.3: Kinetics of the homopolymerization of HPMA at [M]/[CTA] = 160
[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
a: from 1H NMR in CDCl3;
b: Mn = 143.18×160;
c: from GPC using NMP (containing 0.2 wt% LiCl) as the eluent and PMMA standards.
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.
Table 2.4: Kinetics of the homopolymerization of HPMA at [M]/[CTA] = 320
[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
a: from 1H NMR in CDCl3;
b: Mn = 143.18×320;
c: from GPC using NMP (containing 0.2 wt% LiCl) as the eluent and PMMA standards.
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
Figure 2.10: 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= 320.
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.
3. Moad, G., Rizzardo, E., Thang, S. H. Living Radical Polymerization by the
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
3.2 Experimental Section
3.2.1 General Information
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.
1H NMR: The measurements were performed with a Varian Mercury 400 spectrometer.
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.
Elemental Analysis: The measurements were performed with a 2400 Series II CHNS
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
3.3 Results and Discussion
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
a: from 1H NMR in CDCl3;
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
(The figure is reproduced with the permission of the American Chemical Society)
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
(The figure is reproduced with the permission of the American Chemical Society)
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
(The figure is reproduced with the permission of the American Chemical Society)
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
(The figure is reproduced with the permission of the American Chemical Society)
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
4.2 Experimental Section
4.2.1 General Information
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.
1H NMR: The measurements were performed with a Varian Mercury 400 spectrometer.
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.
Elemental Analysis: The measurements were performed with a 2400 Series II CHNS
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
4.3 Results and Discussion
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
Retention volume (ml)
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
Retention volume (ml)
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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