Synthesis of biodegradable multiblock copolymers by click coupling of RAFT-generated heterotelechelic polyHPMA conjugates

Article abstract of DOI:10.1016/j.reactfunctpolym.2010.10.005

A new strategy for the synthesis of biodegradable high molecular weight N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymeric carriers has been designed. An enzyme-sensitive, alkyne-functionalized, chain transfer agent (CTA-GFLG-alkyne; N^α-(4-pentynoyl)-N^δ-(4- cyano-4-(phenylcarbonothioylthio)pentanoyl-glycylphenylalanylleucylglycyl) -lysine) was synthesized and used to mediate the reversible addition- fragmentation chain transfer (RAFT) polymerization and copolymerization of HPMA. Post-polymerization modification with 4,4′-azobis(azidopropyl 4-cyanopentanoate) resulted in the formation of heterotelechelic HPMA copolymers containing terminal alkyne and azide groups. Chain extension via click reaction resulted in high molecular weight multiblock copolymers. Upon exposure to papain, these copolymers degraded into the initial blocks. Similar results were obtained for copolymers of HPMA with N- methacryloylglycylphenylalanylleucylglycyl thiazolidine-2-thione and N-methacryloylglycylphenylalanylleucylglycyl-gemcitabine. The new synthetic method presented permits the synthesis of biocompatible, biodegradable high molecular weight HPMA copolymer-anticancer drug conjugates that possess long-circulation times and augmented accumulation in solid tumor tissue due to the enhanced permeability and retention effect.

Full text of DOI:10.1016/j.reactfunctpolym.2010.10.005

Reactive & Functional Polymers 71 (2011) 294–302
Contents lists available at ScienceDirect
Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
Synthesis of biodegradable multiblock copolymers by click coupling
of RAFT-generated heterotelechelic polyHPMA conjugates
a
a
a
a
a,b,
⇑
ˇ
ˇ
ˇ
Jiyuan Yang , Kui Luo , Huaizhong Pan , Pavla Kopecková , Jindrich Kopecek
a Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City, UT 84112-9452, USA
b Department of Bioengineering, University of Utah, Salt Lake City, UT 84112-9452, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 14 October 2010
A new strategy for the synthesis of biodegradable high molecular weight N-(2-hydroxypropyl)meth-
acrylamide (HPMA)-based polymeric carriers has been designed. An enzyme-sensitive, alkyne-function-
a
alized, chain transfer agent (CTA-GFLG-alkyne; N -(4-pentynoyl)-Nd-(4-cyano-4-(phenylcarbonothio-
Keywords:
N-(2-hydroxypropyl)methacrylamide
(HPMA)
Biodegradable multiblock copolymer
Telechelic
RAFT polymerization
Click reaction
ylthio)pentanoyl–glycylphenylalanylleucylglycyl)-lysine) was synthesized and used to mediate the
reversible addition–fragmentation chain transfer (RAFT) polymerization and copolymerization of HPMA.
Post-polymerization modification with 4,40-azobis(azidopropyl 4-cyanopentanoate) resulted in the for-
mation of heterotelechelic HPMA copolymers containing terminal alkyne and azide groups. Chain exten-
sion via click reaction resulted in high molecular weight multiblock copolymers. Upon exposure to
papain, these copolymers degraded into the initial blocks. Similar results were obtained for copolymers
of HPMA with N-methacryloylglycylphenylalanylleucylglycyl thiazolidine-2-thione and N-methacryloyl-
glycylphenylalanylleucylglycyl-gemcitabine. The new synthetic method presented permits the synthesis
of biocompatible, biodegradable high molecular weight HPMA copolymer–anticancer drug conjugates
that possess long-circulation times and augmented accumulation in solid tumor tissue due to the
enhanced permeability and retention effect.
Drug carrier
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
defined structures [8] as well as to introduce functional end-
groups that can act as attachment points for various biomacromol-
Poly[N-(2-hydroxypropyl)methacrylamide] (polyHPMA) is
a
ecules [9].
Herein we describe the synthesis of biodegradable, biocompati-
ble high molecular weight HPMA copolymer drug carriers that will
be long circulating in the vasculature leading to enhanced tumor
nonimmunogenic, neutral, hydrophilic polymer currently em-
ployed in the delivery of anticancer drugs. Its physical properties
and the synthetic flexibility have been proven to be very useful
when combined with biological active agents [1,2]. HPMA copoly-
mer–anticancer drug conjugates have been evaluated in numerous
cancer models [3,4] and in clinical trials [5]. The necessity to limit
the molecular weight distribution below the renal threshold (in or-
der to secure elimination from the organism) resulted in short
intravascular half-life and limited accumulation in the tumor. It
is well known that accumulation of macromolecules in solid tumor
is molecular weight-dependent [6]. Experimental evidence
suggests that the higher the molecular weight of HPMA copoly-
mer–drug conjugates, the higher the tumor accumulation with
concomitant increase in therapeutic efficacy [7].
accumulation. To this end, we synthesized
a-alkyne, x-azido
heterotelechelic polyHPMA using RAFT polymerization. A newly de-
signed chain transfer agent (CTA-GFLG-alkyne) contains a degrad-
able oligopeptide sequence, GFLG, and an alkyne group, which
enabled a direct synthesis of alkyne-functionalized polyHPMA as
well as HPMA copolymers with functional comonomers (N-methac-
ryloylglycylphenylalanylleucylglycyl thiazolidine-2-thione) or
polymerizable derivatives of anticancer drugs (N-methacryloylgly-
cylphenylalanylleucylglycyl-gemcitabine).
Post-polymerization
modification of the other (non-alkynyl) chain end with an azido-
group was achieved by the reaction of the polymer with azido-
modified initiator V-501 (diazido-V-501; 4,40-azobis(azidopropyl
4-cyanopentanoate)). Unlike the reported ‘‘clickable’’ RAFT agents
[10], the new CTA contains an enzyme-sensitive oligopeptide (GFLG)
sequence [11]. This feature makes the heterotelechelic polyHPMA a
potentially biodegradable, long-circulation drug carrier. Addition of
catalyst Cu(I) resulted in chain extension and formation of high
molecular weight multiblock biodegradable HPMA copolymers.
Exposure of the multiblock copolymer to papain, an enzyme with
The emergence of living radical polymerization, especially
reversible addition–fragmentation chain transfer (RAFT) polymeri-
zation, provided a powerful tool to prepare polyHPMAs with well-
⇑
Corresponding author at: Department of Pharmaceutics and Pharmaceutical
Chemistry/CCCD, University of Utah, 20 S, 2030 E, Rm. 205B, Salt Lake City, UT
84112-9452, USA. Tel.: +1 801 581 7211; fax: +1 801 581 7848.
ˇ
E-mail address: jindrich.kopecek@utah.edu (J. Kopecek).
1381-5148/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.reactfunctpolym.2010.10.005

J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
295
similar specificity as lysosomal cathepsin B, resulted in total biodeg-
radation and formation of macromolecules with initial molecular
weight.
DIPEA (285 lL 1.61 mmol) were dissolved in DCM (10 mL) and
added to 2-chlorotrityl chloride resin (1.0 g, 1.29 mmol/g). The vial
was kept gently shaking for 1.5 h (Step a). The resin was transferred
to a polypropylene tube, rinsed with mixture of DCM:MeOH:DIPEA
(17:2:1) (20 mL ꢀ 4), followed by washing three times with DCM
and DMF, respectively. After removal of Fmoc-group with 10 mL
20% piperidine in DMF (5 min ꢀ 3), 4-pentynoic acid (2.5ꢀ,
2. Experimental
2.1. Materials
1.6 mmol) was reacted with
1.55 mmol, 586 mg) and DIPEA (5ꢀ, 3.2 mmol, 570
a
-NH2 of Lys using HBTU (2.4ꢀ,
l
L) as coupling
Side-chain protected Fmoc-amino acids, 2-(7-aza-1H-ben-
zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
agents to introduce the alkyne group (Step b). Ninhydrin test (also
known as Kaiser test) was used to confirm the completion of each
coupling step. Dde group was removed with 10 mL 3% hydrazine
in DMF (5 min ꢀ 3), and elongation of tetrapeptide GFLG was
accomplished by sequentially coupling Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Phe-OH, and Fmoc-Gly-OH using the HBTU/DIPEA
procedure, (Step c). The N-terminus of the peptide was capped with
4-cyanopentanoic acid dithiobenzoate in the presence of DIC/HOBt
(Step d). The product was cleaved from the resin with TFE/DCM (3:7
v/v) at room temperature for 2 h (Step e). The resin was removed by
filtration. The filtrate was condensed under reduced pressure, puri-
fied by precipitation of the solution in methanol into ether. Pink
powder was obtained. Yield: 339 mg (61%). The structure was ver-
ified by matrix assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS). [M + H]+ 862.38. 1H NMR
(400 MHz, Methanol-d4, d, ppm): 7.84–7.32 (m, 5 H, Ph-H),
(HATU),
O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexa-
fluoro-phosphate (HBTU), 4-dimethylaminopyridine (DMAP),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) were
from AAPPTec (Louisville, KY). Diisopropylcarbodiimide (DIC),
4,40-azobis(4-cyanovaleric acid) (V-501) and 2-mercaptothiazo-
line were from Fluka, 1-hydroxybenzotriazole (HOBt) was from
AK Scientific (Mountain View, CA), and gemcitabine hydrochlo-
ride was from NetQem (Durham, NC). N,N-diisopropylethylamine
(DIPEA; 99%) was from Alfa Aesar (Ward Hill, MA), diethyl ether
and dichloromethane (DCM) were purchased from Mallinckrodt
Baker (Phillipsburg, NJ). Trifluoroacetic acid (TFA; 99%) was from
Acros Organics (Morris Plains, NJ). 2,20-azobis(isobutyronitrile)
(AIBN) was from sigma–Aldrich (St. Louis, MO) and recrystallized
in ethanol. HPMA [12], N-methacryloylglycylphenylalanylleucyl-
glycyl thiazolidine-2-thione (MA-GFLG-TT) [13], and 4-cyanopen-
tanoic acid dithiobenzoate [14] were synthesized as previously
described. All other solvents were purchased from Sigma-Aldrich
as the highest purity available and used as received.
7.20–7.11 (m, 5 H, Ph-H), 4.49 (m, 1 H,
-Leu-H), 4.15 (m, 1 H, -Lys-H), 3.85–3.70 (m, 4 H, NH–CH2–CO),
3.20–3.03 (m, 3 H, CHCH2CH2CH2CH2NH, Ph-CHH), 2.85 (m, 1 H,
Ph-CHH), 2.50–2.24 (m, H, COCH2CH2C, COCH2CH2C and
a-Phe-H), 4.30 (m, 1 H,
a
a
8
HCCCH2CH2CO), 2.15 (s, HCCCH2), 1.84 (s, 3 H, (CN)C–CH3), 1.80–
1.27 (m, 9 H, CHCH2CH2CH2CH2NH, CHCH2CH(CH3)2), 0.83–0.77
(m,
2.2. Instrumentation
6
H, Leu-(CH3)2); 13C NMR (100 MHz, Methanol-d4, d,
Mass spectra of all synthesized compounds were obtained using
a mass spectrometer Voyager-DE (STR Biospectrometry Worksta-
tion, PerSeptiveBiosystems, Framingham, MA). 1H NMR and 13C
NMR spectra were recorded on a Mercury400 spectrometer in
CDCl3. Chemical shifts were reported in ppm (d) relative to CDCl3
(7.26 ppm for 1H and 77.0 ppm for 13C). Monomer conversion dur-
ing polymerization was determined from the concentration of
residual monomer using HPLC (Agilent Technologies 1100 series,
Zorbax C8 column 4.6 ꢀ 150 mm) with gradient elution from 2%
to 90% of Buffer B within 20 min and flowrate 1.0 mL/min (Buffer
A: deionized water (DI H2O) with 0.1% TFA, Buffer B: acetonitrile
containing 0.1% TFA). Size-exclusion chromatography (SEC) was
carried out on an ÄKTA FPLC system (Pharmacia) equipped with
miniDAWN TREOS and OptilabEX detectors (Wyatt Technology,
Santa Barbara, CA) with PBS (pH 7) as mobile phase. Superose 6/
Superose 12 HR10/30 columns and Superose 6 HR16/60 column
(Pharmacia) were used as needed. The molecular weight and
molecular weight distribution were calculated using ASTRA soft-
ware and calibration with polyHPMA fractions. UV–vis spectra
were measured on a Varian Cary 400 Bio UV–visible spectropho-
tometer. FTIR spectra were obtained on Bio-Rad FTS 6000.
ppm):225.04, 175.30, 174.88, 174.20, 173.89, 173.81, 172.13,
171.38, 146.10, 138.16, 134.27, 130.32, 129.78, 127.91, 119.84,
83.58, 70.40, 56.42, 53.78, 53.51, 47.39, 47.37, 43.93, 43.89, 43.51,
40.92, 40.15, 38.21, 36.06, 34.76, 32.23, 31.83, 29.82, 25.66, 24.34,
24.31, 24.15, 23.64, 21.83, 15.79. The purity was determined by
RP-HPLC (>95%).
2.4. Synthesis of 4,40-azobis(azidopropyl 4-cyanopentanoate) (2,
diazido-V-501)
4,40-Azobis(4-cyanovaleric acid) (V-501, 1 g, 3.6 mmol), 3-azi-
dopropanol (1.08 g, 10.7 mmol) and DMAP (0.35 g, 2.9 mmol) were
dissolved in DCM/THF (1:1 v/v, 25 mL) and cooled to 4 °C. DCC
(1.62 g, 7.9 mmol) in 10 mL of DCM was added dropwise. The reac-
tion mixture was stirred at 4 °C overnight, then at room tempera-
ture for 1 h. After completion of the reaction, two drops of acetic
acid were added to the reaction mixture, and stirring continued
for 30 min. Dicyclohexylurea (DCU) was removed by filtration
and the solvent was removed by rotary evaporation. The residue
was purified by silica gel chromatography (silica gel 60 Å, 200–
400 mesh, ethyl acetate/hexane 1/1); yield 1.05 g (66%). The struc-
ture was confirmed by 1H NMR and 13C NMR spectroscopy. 1H
NMR (CDCl3, d, ppm): 4.17 (m, 4H, –COOCH2); 3.37 (m, 4H,
N3CH2); 2.60–2.30 (m, 8H, CO–CH2CH2–C); 1.89 (m, 4H, C–CH2–
C); 1.68 (d, 6H, CH3). 13C NMR (CDCl3, d, ppm): 171.16; 117.42;
71.76; 62.01; 48.07; 33.05; 29.01; 27.97; 23.86.
a
2.3. Synthesis of N -(4-pentynoyl)-Nd-(4-cyano-4-
(phenylcarbonothioylthio)pentanoyl-glycylphenylalanylleucylglycyl)-
lysine (1, CTA-GFLG-alkyne)
The CTA-GFLG-alkyne was synthesized in several steps
(Scheme 1) using solid phase peptide synthesis (SPPS) methodology
and manual Fmoc/tBu strategy. Dde-Lys(Fmoc)-OH was chosen as a
linker in which two protecting groups (Fmoc- and Dde-) can be re-
moved selectively: one deprotected amino group was used to intro-
duce the alkyne into the peptide structure, the other functioned in
constructing the peptide sequence. A detailed experimental proce-
dure is shown below. Dde-Lys(Fmoc)-OH (351 mg, 0.64 mmol) and
2.5. Synthesis of N-methacryloylglycylphenylalanylleucylglycyl-
gemcitabine (3, MA-GFLG-gemcitabine)
MA-GFLG-TT (90 mg, 0.16 mmol) and gemcitabine hydrochlo-
ride (44.95 mg, 0.15 mmol) were dissolved in pyridine (5 mL) under
nitrogen atmosphere. The solution was stirred at 50 °C for 12 h and
then the solvent was removed by rotary evaporation. The crude

296
J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
a
Scheme 1. Synthesis of N -(4-pentynoyl)-Nd-(4-cyano-4-(phenylcarbonothioylthio)pentanoyl-glycylphenylalanylleucylglycyl)-lysine (CTA-GFLG-alkyne).
product was purified by column chromatography (silica gel 60 Å,
200–400 mesh, DCM: CH3OH = 6:1) to give white solid in 66.1%
yield. The structure was confirmed by 1H NMR spectroscopy and
MALDI-TOF MS. MALDI-TOF MS: m/z 706.22 (M + H)+, 728.21
(M + Na)+, 744.19 (M + K)+. 1H NMR: 8.20 (d, 1 H, J = 7.6 Hz, H-6),
7.84 (s, 2 H, NH), 7.31 (d, 1 H, J = 7.6 Hz, H-5), 7.06 (m, 5 H, Ph-H),
6.97 (s, 1 H, NH), 6.12 (t, J = 7.6 Hz, H-10), 5.64 (s, 1 H, CH3–CH(R)–
Similarly, the polymerization in DI H2O was conducted at 70 °C
using V-501 as the initiator.
For kinetic studies, stock solutions were prepared as described
above but in a vial equipped with a septum. Aliquots were trans-
ferred via syringe to six different ampoules; each one was attached
to the Schlenk-line and had been purged with nitrogen. The
ampoules were sealed under N2 atmosphere followed by place-
ment in a 60 °C oil bath. At different time points, the samples were
taken out and quenched in liquid nitrogen. The ampoule was
opened just prior to measurement. Twenty microliter polymer
solution was diluted into 1 mL and analyzed by HPLC. The mono-
mer conversion was calculated by comparison of the remaining
monomer concentration with initial concentration. The molecular
weight of the sample was measured using SEC after precipitation
of the polymer solution into cold anhydrous ether.
CH2–Ha), 5.24 (s, 1 H, CH3–CH(R)–CH2–Hb), 4.52 (m, 1 H,
a
-H), 4.23
(m, 1 H, H-30), 4.15 (m, 1 H,
a
-H), 3.97 (m, 1 H, H-40), 3.83 (m, 4 H,
NH–CH2–CO), 3.66–3.80 (m, 2 H, H-50, H-500), 3.06 (m, 2 H, Ph-CH2),
2.04 (s, 1 H, OH), 1.85 (s, 1 H, OH), 1.78 (s, 3 H, CH3), 1.52 (m, 3 H,
CHCH2CH(CH3)2), 0.79 (m, 6 H, CHCH2CH(CH3)2).
2.6. RAFT polymerization of HPMA mediated by CTA-GFLG-alkyne
HPMA homopolymerizations were performed in either DI H2O at
70 °C using V-501 as the initiator or methanol at 60 °C using AIBN as
the initiator. A typical polymerization procedure in methanol was
as follows: An ampoule containing HPMA (0.5 g, 3.5 mmol) and
CTA-GFLG-alkyne (8.6 mg, 0.01 mmol) was attached to the
Schlenk-line. After three vacuum–nitrogen cycles to remove oxy-
gen in the ampoule, 2.5 mL degassed methanol was added, followed
by addition of AIBN solution in methanol (0.55 mg in 0.1 mL) via
syringe. The ampoule was sealed and polymerization was per-
formed at 60 °C for 20 h. The polymer was obtained by precipitation
into acetone and purified by re-dissolving in methanol and precip-
itation in acetone two more times. PolyHPMA with pink color was
dried under vacuum. Yield 70%.
2.7. Synthesis of HPMA copolymer containing thiazolidine-2-thione
(TT) reactive groups (P-TT)
HPMA copolymer containing side-chain reactive group was pro-
duced by copolymerization of HPMA with MA-GFLG-TT. An am-
poule containing HPMA (164.5 mg, 1.15 mmol), MA-GFLG-TT
(34 mg, 0.06 mmol), CTA-GFLG-alkyne (3 mg, 3.46 lmol) was at-
tached to the Schlenk-line. After three vacuum–nitrogen cycles to
remove oxygen, 1 mL degassed methanol was added, followed by
addition of AIBN solution in methanol (0.2 mg in 0.1 mL, 1.12 lmol)
via syringe. The ampoule was sealed and copolymerization was
performed at 60 °C for 24 h. The polymer was obtained by precipi-
tation into acetone and purified by re-dissolving in methanol and

J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
297
precipitation in acetone two more times. The copolymer (P-TT) was
dried under vacuum. Yield: 108 mg (54%). The content of TT in the
copolymer was determined by UV spectrophotometry in methanol
was fractionated by size exclusion chromatography on a Superose
6 preparative column. The fractions were dialyzed against H2O to
remove salts using a 6–8 kDa cutoff membrane and freeze-dried.
(e
305 = 10,800 L molꢁ1 cmꢁ1).
2.12. Cleavage of multiblock polyHPMA and multiblock HPMA
copolymer containing anticancer drug gemcitabine
2.8. Synthesis of HPMA copolymer containing anticancer drug
gemcitabine (P-gemcitabine)
The degradation of multiblock polyHPMA and multiblock HPMA
copolymer–gemcitabine conjugate was performed in McIlvaine’s
buffer (50 mM citrate/0.1 M phosphate) at pH 6.0,37 °C using pa-
pain as model enzyme. Papain, at concentration of 0.29 mg/mL
(determined by UV at 280 nm, with absorbance of 25 at 1%), was
reduced with 5 mM glutathione for 5 min before addition to poly-
mer (3 mg polymer/mL enzyme solution). The medium was ana-
lyzed with size-exclusion chromatography using a Superose 6
HR/10/30 column with PBS (pH 7) as the mobile phase. In addition,
for HPMA copolymer–gemcitabine conjugate, the medium was also
analyzed with HPLC to detect the cleaved gemcitabine.
HPMA copolymer containing anticancer drug gemcitabine (P-
gemcitabine) was produced by copolymerization of HPMA with a
polymerizable derivative of the drug. An ampoule containing HPMA
(160 mg, 1.12 mmol), MA-GFLG-gemcitabine (40 mg, 0.057 mmol),
and CTA-GFLG-alkyne (3 mg, 3.46 lmol) was attached to the
Schlenk-line. After three vacuum–nitrogen cycles to remove oxy-
gen, 1 mL degassed MeOH were added, followed by addition of ini-
tiator AIBN solution in methanol (0.2 mg in 0.1 mL, 1.12 lmol) via
syringe. The ampoule was sealed, and copolymerization was per-
formed at 60 °C for 24 h. The polymer was obtained by precipitation
into acetone and purified by re-dissolving in methanol and precip-
itation in acetone two more times. The copolymer (P-gemcitabine)
was isolated as a pink powder and dried under vacuum. Yield:
136 mg (68%). The content of gemcitabine was estimated by UV
3. Results and discussion
in methanol (e
300 = 5710 L molꢁ1 cmꢁ1).
3.1. Design and synthesis of functional biodegradable RAFT agent, CTA-
GFLG-alkyne
2.9. Post-polymerization chain end modification
In RAFT polymerizations, chain transfer agents play a crucial
part, so they must be chosen carefully [15]. 4-Cyanopentanoic acid
dithiobenzoate was the first chain transfer agent used for HPMA
RAFT polymerization [8]. Due to the high activation of the thiocar-
bonyl function by the phenyl substituent, it has been one of the
most commonly used RAFT agents [16]. Numerous publications
indicated that modifications of carboxy group in this chain transfer
agent could be made without loss of fragmentation ability. Reac-
tive or potentially reactive groups such as azido-, trimethylsilane
The dithiobenzoate group was replaced with an azido group by
the reaction of the RAFT-generated polymer with diazido-V-501
(Scheme 2). Briefly, polyHPMA (125 mg, Mn = 36 kDa, 3.5
was added into an ampoule. Diazido-V-501 (20 ꢀ , 32 mg,
70 mol) solution in methanol was then added via syringe. The
lmol)
l
solution was bubbled with N2 for 30 min, sealed and kept stirring
at 70 °C for 3 h. The polymer, heterotelechelic N3–polyHPMA–
C„CH, was purified by precipitation into ether and dried under
vacuum (yield 104 mg).
protected alkyne-, TT-,
duced into the chain transfer agent structure; this provided a path-
way for direct synthesis of -functionalized polymers [9,10,17–20].
a-pentafluorophenyl ester have been intro-
a
2.10. Synthesis of biodegradable multiblock polyHPMA by click
reaction
In this study, a new functional dithiobenzoate derivative was
designed and synthesized as shown in Scheme 1. An alkyne group
was incorporated at the R-group (S@C(Z)-SR) without protection.
The important feature of the new chain transfer agent is the inser-
tion of an enzyme-degradable peptide linker (GFLG), which makes
it a versatile tool for the synthesis of polymers with biodegradable
backbones. The successful synthesis of the new CTA-GFLG-alkyne
was validated by MALDI-ToF-MS, 1H NMR, and 13C NMR. The
Heterotelechelic N3–polyHPMA–C„CH (40 mg, 1.1
sodium ascorbate (4.4 mg, 22 mol) were added to a vial and then
attached to Schlenk-line. The vial was evacuated and refilled with
nitrogen three times before adding 200 L of degassed DI H2O. The
solution was bubbled with N2 for 30 min. The click reaction was
initiated by adding 50 L CuSO4 (1.8 mg, 11 mol) aqueous solu-
lmol) and
l
l
l
l
molar absorption coefficient in methanol was measured as e302
=
tion via syringe. The reaction mixture was kept at RT for 12 h.
The polymer was precipitated into ether and dried under vacuum.
Yield: 32 mg (80%). The removal of copper salts was accomplished
when the product was fractionated by size-exclusion chromatogra-
phy on a Superose 6 preparative column using PBS as the eluent.
The fractions were dialyzed against H2O to remove salts using a
6–8 kDa cutoff membrane and freeze-dried.
15340 Mꢁ1 cmꢁ1
.
3.2. RAFT polymerization of HPMA mediated by CTA-GFLG-alkyne
The results of the RAFT polymerization of HPMA mediated by
CTA-GFLG-alkyne are shown in Table 1 and Fig. 1. The agreement
between theoretical and experimental molecular weights over
the course of polymerization and the narrow molecular weight dis-
tribution (Table 1) indicated that a well-defined polymer structure
was achieved. Fig. 1A shows the kinetic plots of HPMA polymeriza-
tion in water and methanol. In both solvents pseudo-first-order
kinetics was observed. Fig. 1B presents the evolution of the molec-
ular weight distribution with time for HPMA homopolymerization
in methanol at 60 °C. The peaks are unimodal; their shift toward
higher molecular weights with time further indicates the con-
trolled nature of the polymerization process. Fig. 1C shows that
the polymerization proceeded in a controlled manner in the whole
range of evaluated conversions, i.e. up to 60%.
2.11. Synthesis of multiblock HPMA copolymers containing anticancer
drug gemcitabine
Multiblock HPMA copolymer–gemcitabine conjugates were
synthesized following a similar procedure as described in section
2.10. Briefly, N3–P-gemcitabine–C„CH (40 mg, Mn 28.5 kDa,
1.4
in 200
30 min; then, the click reaction was initiated by adding 50
CuSO4 (2.23 mg, 14 mol) aqueous solution via syringe. The reac-
lmol) and sodium ascorbate (5.5 mg, 28
lmol) were dissolved
l
L of degassed DI H2O. The solution was bubbled with N2 for
lL
l
tion mixture was kept stirring at RT for 12 h. The polymer was pre-
cipitated into ether and dried under vacuum. Yield: 29 mg (72%).
The removal of copper salts was accomplished when the product
The living feature of the system was further confirmed by evo-
lution of molecular weight when isolated polyHPMA from one

298
J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
Scheme 2. Schemes of: (a) synthesis of comonomers MA-GFLG-TT and MA-GFLG-gemcitabine; and (b) RAFT copolymerizations of HPMA with MA-GFLG-TT/MA-GFLG-
gemcitabine.
Table 1
Summary of HPMA polymerization mediated by CTA-GFLG-alkyne.
% conva
Mth
MnSEC
Mw/Mn
b
Entry
[M]0/[CTA]0/[l]0
Initiator
Solvent
Temp °C
Time h
1
2
3
4
5
6
350:1:0.2
350:1:0.2
350:1:0.2
350:1:0.2
350:1:0.33
350:1:0.33
V-501
V-501
V-501
V-501
AIBN
H2O
H2O
H2O
H2O
CH3OH
CH3OH
70
70
70
70
60
60
1.3
2.8
4.5
7
8
17
20.9
43.5
55.1
70.2
23.5
42.2
11,300
22,700
28,400
35,100
11,800
22,000
12,800
24,200
30,500
36,000
12,300
22,300
1.07
1.04
1.02
1.06
1.04
1.06
AIBN
a
The monomer conversion was measured by HPLC and calculated from HPMA calibration curve.
Theoretical value (Mth) calculated from the equation: Mth = {([M]0/[CTA]0)*conversion* wHPMA + MwCTA}.
b
experiment was used as macro-CTA to mediate a new polymeriza-
tion of HPMA (Fig. 1D). The obtained ‘‘diblock’’ polyHPMA showed
the expected value of molecular weight and narrow molecular
weight distribution, indicating that the dithiobenzoate end group
remained functional in polyHPMA as previously shown with other
chain transfer agents [8].
thelow polydispersity at all time pointsindicatethatthe copolymer-
ization was controlled similarly to the HPMA homopolymerization.
The copolymerization of HPMA with MA-GFLG-gemcitabine
also proceeded in a controlled manner (results not shown).
Gemcitabine hydrochloride shows absorbance maxima at 232
and 263 nm. These peaks shift to 248 nm and 300 nm, respectively,
after attachment to MA-GFLG-OH. No further changes in absor-
bance maxima have been detected after polymerization. Therefore,
the content of gemcitabine in the HPMA copolymers was estimated
by UV as previously reported [21] (Table 2).
3.3. RAFT copolymerization of HPMA
Next, we evaluated HPMA copolymerization using a functional
monomer (MA-GFLG-TT) that provided groups suitable for poly-
meranalogous attachment of anticancer drugs or a polymerizable
derivative of drug (3, MA-GFLG-gemcitabine) that incorporated
the drug directly by copolymerization (Scheme 2).
3.4. Synthesis of
(co)polymers
a-alkyne, x-azido heterotelechelic HPMA
The results of copolymerization of HPMA with MA-GFLG-TT in
methanol at 60 °C mediated by CTA-GFLG-alkyne are shown in
Fig. 2. The linear increase of molecular weight with conversion and
The polymers produced by RAFT polymerization mediated by
CTA-GFLG-alkyne contain an alkyne group at one chain end and
the dithiobenzoate group at the other end. To produce a-alkyne,

J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
299
Fig. 1. RAFT polymerization of HPMA mediated by CTA-GFLG-alkyne: (A) pseudo first-order rate plots for polymerizations in methanol at 60 °C and in water at 70 °C, (B)
evolution of molecular weight (RI detection; Superose 12 column) with time for HPMA polymerization in methanol at 60 °C, (C) plot of Mn versus conversion for HPMA
polymerization, (D) SEC chromatogram (RI detection; Superose 6 column) of RAFT polymerization of HPMA in water at 70 °C using polyHPMA as macro-CTA, [M]0/[macro-
CTA] = 500, Mth = 66,700 (q = 70%) and showing the evolution of molecular weight.
[20,22,23]. For example, Goldmann et al. have shown the replace-
ment of end dithiobenzoate groups on polystyrene with alkyne
using propargyl alcohol modified V-501 (an azo-compound)
resulted in total conversion. A heterotelechelic a-azido, x-alkyne
polystyrene was obtained. At low polymer concentrations, polysty-
rene macrocycles were produced via click reaction [20].
In this study, diazido-V-501 was synthesized and 20-fold molar
excess was used for end-modification. The complete removal of
dithiobenzoate group was demonstrated by UV/Vis spectroscopy
(Fig. 3A; no detectable dithiobenzoate group absorbancy at
300 nm) and by 1H NMR (absence of detectable dithiobenzoate
group in d = 7.3–7.9 ppm, 5H; not shown). The presence of azido
group was confirmed by the appearance of a new peak at
2100 cmꢁ1 (azide) [20] in FTIR spectra (Fig. 3B). SEC analysis
showed that there was no significant change in the molecular
weight and molecular weight distribution during end-group mod-
ification (Fig. 3C). Nonetheless, the appearance of a small higher
molecular weight shoulder (indicated by arrow) suggests that
recombination of radicals might be a minor side reaction.
Fig. 2. RAFT copolymerization of HPMA with 5 mol% MA-GFLG-TT in methanol at
60 °C (([M]0/[CTA-GFLG-alkyne]0 = 350,[CTA-GFLG-alkyne]0/[AIBN]0 = 3). Evolution
of molecular weight and polydispersity (Mw/Mn) with conversion.
Table 2
Characterization of HPMA copolymersa.
TT- or gem-content % Yieldb MnSEC
Mw/Mn
3.5. Synthesis of biodegradable multiblock polymer by Cu (I) catalyzed
Entry Polymer
click reaction of a-alkyne, x-azido heterotelechelic polyHPMA
mo1%
2.35
wt.%
1
2
P-TT
N/A
4.12
53.8
68.0
33,900 1.03
28,500 1.02
To validate our hypothesis that the new design of polymeric
drug carriers will result in degradable mulitblock copolymers, the
P-gemcitabine 2.24
a
copperI-catalyzed Huisgen 1,3-dipolar cycloaddition of
a
-alkyne,
Polymerizations were carried out in CH3OH at 60 °C for 24 h, using AIBN as
initiator, [M]0 = 1 M, [M]0/[CTA]0/[AIBN] = 350:1:0.33.
x
-azido heterotelechelic polyHPMA into high molecular weight
b
Yield was determined gravimetrically.
multiblock polymers (Scheme 3) was studied, and their degrada-
tion into initial polymer that possess a molecular weight below
the renal threshold was also investigated. The advantage of this
system is the presence of two orthogonal end groups on each mac-
romolecule, which facilitates the stoichiometric azide–alkyne click
reaction. The effects of concentration, the type and ratio of catalyst,
x
-azido heterotelechelic polyHPMA, a radical reaction of the initial
polymer with an excess of diazido-V-501 was performed
(Scheme 3). The radical-induced removal of thiocarbonylthio end
group from RAFT-generated polymers has been studied previously

300
J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
Scheme 3. General approach to the synthesis of biodegradable multiblockpolyHPMA via RAFT polymerization and click chemistry.
Fig. 3. (A) UV spectra of polyHPMA containing an alkyne end group and a dithiobenzoate end-group (polyHPMA-before end-modification) and of heterotelechelicpolyHPMA
containing an azido and an alkyne end-group (polyHPMA after end-modification) – note the difference at absorbency at 300 nm; (B) FTIR spectra demonstrating the
appearance of the azido peak at 2100 cmꢁ1 in the end-modified polyHPMA; (C) SEC chromatograms of copolymer of HPMA with MA-GFLG-TT: comparison of profiles before
and after end-group modification.
and different solvents have been examined. To avoid formation of
cyclic polymers, high concentrations of functional groups and of
the initial polymer were used; the polymer was dissolved in the
minimal amount of solvent needed for dissolution. The click reac-
that the same click chemistry that was used for polyHPMA can also
be used to prepare multiblock HPMA copolymer–drug conjugates,
we chose to evaluate gemcitabine, an FDA approved anticancerdrug.
It is important that the chain extension and degradation of the
drug (gemcitabine)-containing HPMA copolymer proceeded simi-
larly to the HPMA homopolymer (Fig. 4). An aqueous solution of
P-gemcitabine (Mw 28.5 kDa; PDI 1.02) was exposed to copper cat-
alyst (CuSO4/sodium ascorbate) resulting in chain extension into
multiblock copolymer. As shown in Fig. 5A and B, about 20% of
the heterotelechelic polymer formed a dimer and 40% formed high-
er oligomers. The multiblock copolymer was fractionated on a
Superose 6 preparative column. The selected fraction of multiblock
P-gemcitabine (Mw 224 kDa; PDI 1.09) was exposed to a solution
of papain. The degraded product showed very narrow polydisper-
sity and the molecular weight close to the original heterotelechelic
copolymer (Mw 33 kDa, PDI 1.04) (Fig. 5C). Moreover, compared
with the free gemcitabine, the identical elution time of the cleaved
gemcitabine from both the monomer and the multiblock polymer–
drug conjugate upon exposure to papain indicated that conjuga-
tion and click reaction did not distort the structure of gemcitabine
(Fig. 5D). Detailed evaluation of drug release from such multiblock
conjugate and its biological effect is currently on progress.
tion was first conducted in DMF and methanol using CuBr/L-ascor-
bic acid as catalyst system as previously reported [24]. However,
polyHPMA is a water-soluble polymer and frequently it is conju-
gated to bioactive molecules. Consequently, we chose DI H2O as
solvent and CuSO4/sodium ascorbate as catalyst. There was no sig-
nificant difference between the solvent/catalyst systems used; as
expected, a high polymer concentration favored the click reaction
(data not shown). Typical results are shown in Fig. 4. An initial
polyHPMA (Mw 41.8 kDa; PDI 1.05) was exposed to in situ formed
copper catalyst (CuSO4/sodium ascorbate) resulting of chain exten-
sion into multiblock polymer. The multiblock polymer was
fractionated on a Superose 6 preparative column. The selected frac-
tion of multiblock polymer (Mw 291 kDa; PDI 1.11) was exposed to
a solution of papain, a thiol proteinase with specificity similar to
lysosomal cathepsin B. The degradation product was a polymer
with a molecular weight distribution close to the original one
(Mw 41.7 kDa, PDI 1.04). Thus the synthetic design which includes
the insertion of an enzymatically cleavable sequence into the chain
transfer agent, end-group modification and chain extension ap-
pears to be a new and efficient pathway for the synthesis of new
backbone degradable polymeric drug carriers.
3.7. General comments
The combination of living radical polymerization and click reac-
tion for the synthesis of diblock and multiblock copolymers has been
studied extensively. Several approaches have been evaluated: the
coupling of telechelic copolymers with low molecular weight com-
pounds, such as the reaction of diazido terminated polystyrene
3.6. Chain extension and degradation of HPMA copolymers
Our ultimate goal is to synthesize relatively high molecular
weight, degradable polyHPMA–drug conjugates. To demonstrate

J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
301
Fig. 4. Chain extension of heterotelechelic polyHPMA (Mw = 41.8 kDa, Mw/Mn = 1.05) by copper catalyzed alkyne and azide 1,3-dipolar cycloaddition into a multi block
copolymer. Incubation of the multiblock copolymer (selected fraction; Mw = 291 kDa, Mw/Mn = 1.11) with 0.14 mg/mL papain in citrate–phosphate buffer (pH 6.0) at 37 °C
resulted in total degradation of the oligopeptide sequences in multiblock copolymer backbone and formation of the polymer with initial molecular weight (Mw = 41.7 kDa,
Mw/Mn = 1.04).
Fig. 5. (A) SEC chromatogram (RI detection; Superose 6 column) of initial P-gemcitabine conjugate (dashed line) and the multiblock copolymer after chain extension (full
line); (B) SEC chromatogram (RI detection; Superose 6 column) of the multiblock copolymer after chain extension after Astra software multipeak fitting; (C) chain extension
of heterotelechelic copolymer of HPMA with MA-GFLG-gemcitabine (Mw = 28 kDa, Mw/Mn = 1.06) by copper catalyzed alkyne and azide 1,3-dipolar cycloaddition into a
multi block copolymer (selected fraction of Mw = 224 kDa, Mw/Mn = 1.09). Incubation of the multiblock copolymer (selected fraction; Mw = 224 kDa, Mw/Mn = 1.09) with
0.14 mg/mL papain in citrate–phosphate buffer (pH 6.0) at 37 °C resulted in total degradation of the oligopeptide sequences in multiblock copolymer backbone and formation
of the polymer with close to initial molecular weight (Mw = 33 kDa, Mw/Mn = 1.04), (D) HPLC traces of: (a) multiblock high molecular weight copolymer of HPMA–
gemcitabine conjugate (P-gem, fraction of Mw = 224 kDa, Mw/Mn = 1.09; (b) gemcitabine cleaved from multiblock high molecular weight copolymer (P-gem); (c)
gemcitabine cleaved from monomer, MA-GFLG-gemcitabine, and (d) free gemcitabine.
[25,26] or diazido terminated triblock and pentablock copolymers
with propargyl alcohol [27]; the coupling of two polymers with dif-
ferent end functionalities such as coupling of azido- and alkyne-ter-
minated semitelechelic polymers [28]; and attachment of polymers
with dissimilar reactivities [10]. These studies clearly demonstrated
the feasibility to prepare multiblock copolymers using the combina-
tion of living radical polymerization and click reactions. Polymers
with different functionalities, different reactivities, and those pre-
pared by different polymerization techniques have been combined
into multiblock copolymers (reviewed in [29]). The contribution of
this study is to expand the chain extension to heterotelechelic poly-
mers and anticancer drugs-containing heterotelechelic copolymers,

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J. Yang et al. / Reactive & Functional Polymers 71 (2011) 294–302
ˇ
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We have presented a new design for the synthesis of high molec-
ular weight biodegradable multiblock HPMA (co)polymers that
consists of three steps: (a) RAFT (co)polymerization mediated by
an enzyme-sensitive RAFT agent in both aqueous and organic med-
ia; (b) post-polymerization modification; and (c) click reaction. The
versatility of this strategy permits facile incorporation of hydro-
philic/hydrophobic comonomers into the polyHPMA structure. As
an example, a heterotelechelic polyHPMA precursor with pendant
TT reactive groups was synthesized, which can be further conju-
gated with amine-containing bioactive agents. More importantly,
multiblock biodegradable HPMA copolymer conjugate bearing
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4455.
anti-cancer drug gemcitabine with
200 kDa (the renal threshold is 40–45 kDa) was obtained by click
coupling of heterotelechelic -alkyne, -azido HPMA copolymer–
a molecular weight over
a
x
gemcitabine conjugate (initial mol. wt. 28.5 kDa). Exposure to
papain demonstrated the degradability of the HPMA copolymer
backbone and the release of intact gemcitabine. The results indicate
the potential of this HPMA-based drug delivery system for en-
hanced intravascular half-life with concomitant improvement in
cancer therapeutic efficacy.
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3558–3561.
Acknowledgements
[26] P.L. Golas, N.V. Tsarevsky, B.S. Sumerlin, K. Matyjaszewski, Macromolecules 39
(2006) 6451–6457.
[27] P.L. Golas, N.V. Tsarevsky, B.S. Sumerlin, L.M. Walker, K. Matyjaszewski, Austr.
J. Chem. 60 (2007) 400–404.
The research was supported in part by NIH Grants GM69847,
CA51578, and CA132831.
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