Synthesis of well-defined conjugated copolymers by RAFT polymerization using cysteine and glutathione-based chain transfer agents

Article abstract of DOI:10.1039/b708293b

Novel cysteine and glutathione-based chain transfer agents were synthesized and successfully applied to the straightforward synthesis of well-defined conjugates via a RAFT process. The Royal Society of Chemistry.

Full text of DOI:10.1039/b708293b

Supporting Information
Synthesis of well-defined conjugated copolymers by RAFT polymerization using
cysteine and glutathione-based chain transfer agents
Youliang Zhao and Sébastien Perrier*
Department of Colour and Polymer Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT,
UK
Tel.: +44 113 343 2932; Fax: +44 113 343 2947. E-mail: s.perrier@leeds.ac.uk
Experimental Section
Materials. All solvents, monomers, and other chemicals were purchased from Aldrich at the highest purity
available unless otherwise stated. N-acetyl glutathione was synthesized according to a literature method (see
Levy, E. J.; Anderson, M. E.; Meister, A. Anal. Biochem. 1993, 214, 135). Methyl acrylate (MA), butyl acrylate
(BA), methyl methacrylate (MMA), N,N-dimethylacrylamide (DMA) and styrene (St) were passed through a
basic alumina (Brockmann I) column to remove the inhibitor before use. N-isopropylacrylamide (NIPAM) was
recrystallized twice from hexane/toluene mixture prior to use. Dioxane was distilled over CaH2. 2,2’-
Azobisisobutyronitrile (AIBN) was recrystallized twice from ethanol. Other chemicals were of analytical grade
and used as received.
Synthesis of Chain Transfer Agents. To a stirred solution of N-acetyl glutathione (1.40 g, 4.0 mmol) in 50 mL
of methanol was added dropwise a solution of sodium methoxide in methanol (25 wt%, 2.59 g, 12 mmol) under
nitrogen. After stirring for 30 min, CS2 (0.45 g, 5.9 mmol) was added dropwise to the solution, and the mixture
was then stirred at ambient temperature for 5 h. To the yellow solution was added benzyl bromide (98%, 0.75 g,
4.3 mmol), and the mixture was stirred overnight under nitrogen. The mixture was concentrated, partitioned
between water (150 mL) and dichloromethane (DCM), and acidified by concentrated hydrochloric acid. After
standing for 10 min, three layers were formed, and 2-acetamido-S-(benzylsulfanylthiocarbonyl)glutathione
(ABSG) was precipitated as an intermediate layer due to its poor solubility in both water and DCM. The yellow
solid was collected by filtration, washed thoroughly with water and DCM, and dried in vacuum oven under
o
reduced pressure at 40 C until constant weight. ABSG (2.06 g, 4.0 mmol) was obtained as a yellow solid in
1
quantitative yield. H NMR (DMSO-d6), δ: 8.40 (d, J8.4, 1H, NH), 8.34 (t, J5.8, 1H, NH), 8.16 (d, J7.8, 1H,
NH), 7.2-7.4 (m, 5H, PhH), 4.67 (s, 2H, CH2S), 4.62 (m, 1H, CH, Hα-cys), 4.16 (m, 1H, CH, Hα-glu), 3.87 and 3.49
(m, each 1H, CH2, Hβ-cys), 3.75 (m, 2H, CH2, Hgly), 2.19 (t, J8.0, 2H, CH2, Hγ-glu), 1.95 and 1.76 (m, each 1H, CH2,
H
β-glu), 1.84 (s, 3H, CH3CO). 13C NMR (DMSO-d6), δ: 223.2 (C=S), 173.6, 171.8, 171.0, 169.9, 169.6 (C=O),
135.2, 129.4, 128.8, 127.9 (PhC), 51.6, 50.6, 41.0, 40.6, 38.7, 31.8, 27.0, 22.5. FT-IR (cm−1): 3279, 3060, 3030,
2979, 2918, 1711, 1640, 1526, 1495, 1452, 1415, 1370, 1346, 1215, 1192, 1137, 1064, 985, 927, 826, 798, 773,
694. Anal. Calcd for C20H25N3O7S3: C, 46.59%; H, 4.89%; N, 8.15%; S, 18.66%. Found: C, 46.56%; H, 4.92%;
1
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

N, 8.12%; S, 18.61%. ESI-MS: m/z = 514.0786 [M − H]−, theoretical value = 514.0782 (Calcd for
C20H24N3O7S3).
Other CTAs were synthesized according to a similar method and obtained in almost quantitative yield.
2-Acetamido-3-(benzylsulfanylthiocarbonylsulfanyl)propionic acid (ABSPA): purified by silica flash column
chloromatography using a gradient eluent of 1:1 hexane/ethyl acetate to pure ethyl acetate to give a yellow solid
1
product in 99.2% of isolated yield. H NMR (DMSO-d6), δ: 8.43 (d, J8.1, 1H, NH), 7.32 (m, 5H, PhH), 4.68 (s,
2H, CH2S), 4.51 (m, 1H, CH), 3.92 and 3.54 (m, each 1H, CH2), 1.83 (s, 3H, CH3CO). 13C NMR (DMSO-d6), δ:
222.9 (C=S), 171.5, 169.5 (C=O), 135.1, 129.3, 128.7, 127.8 (PhC), 50.5, 40.5, 37.7, 22.4. FT-IR (cm−1): 3355,
3313, 3066, 3029, 2954, 2912, 1706, 1615, 1549, 1494, 1455, 1415, 1376, 1340, 1310, 1225, 1144, 1112, 1068,
1042, 1012, 976, 940, 916, 896, 860, 826, 800, 768, 700, 660. Anal. Calcd for C13H15NO3S3: C, 47.39%; H,
4.59%; N, 4.25%; S, 29.20%. Found: C, 47.42%; H, 4.60%; N, 4.23%; S, 29.15%. ESI-MS: m/z = 328.0147 [M −
H]−, theoretical value = 328.0141 (Calcd for C13H14NO3S3).
2-Acetamido-3-(methoxycarbonylphenylmethylsulfanylthiocarbonylsulfanyl)propionic acid (AMSPA): purified
by silica flash column chloromatography using ethyl acetate as an eluent to give a yellow solid product in 98.5%
of isolated yield. 1H NMR (DMSO-d6), δ: 8.45 (t, J7, 1H, NH), 7.41 (m, 5H, PhH), 5.87 (d, J2.4, 1H, CHS), 4.49
(m, 1H, CH), 3.88 and 3.56 (m, each 1H, CH2), 3.68 (s, 3H, CH3O), 1.83 (s, 3H, CH3CO). 13C NMR (DMSO-d6),
δ: 221.3 (C=S), 171.6, 169.9, 168.8 (C=O), 132.6, 129.5, 129.4, 128.9 (PhC), 57.6, 53.4, 50.7, 38.2, 22.5. FT-IR
(cm−1): 3337, 3322, 3062, 3030, 2999, 2950, 1728, 1619, 1529, 1496, 1453, 1432, 1373, 1333, 1280, 1209, 1152,
1070, 1004, 922, 900, 799, 727, 694, 664. Anal. Calcd for C15H17NO5S3: C, 46.49%; H, 4.42%; N, 3.61%; S,
24.83%. Found: C, 46.55%; H, 4.43%; N, 3.60%; S, 24.78%. ESI-MS: m/z = 386.0201 [M − H]−, theoretical
value = 386.0196 (Calcd for C15H16NO5S3).
2-Acetamido-S-(methoxycarbonylphenylmethylsulfanylthiocarbonyl)glutathione (AMSG): purified by phase
separation between water and DCM, filtration, and washed with water and DCM to afford a yellow solid product
in 98.2% of isolated yield. 1H NMR (DMSO-d6), δ: 8.40 (d, J8.4, 1H, NH), 8.35 (m, 1H, NH), 8.16 (d, J7.7, 1H,
NH), 7.3-7.5 (m, 5H, PhH), 5.86 (d, J2.5, 1H, CH), 4.62 (m, 1H, CH, Hα-cys), 4.16 (m, 1H, CH, Hα-glu), 3.84 and
3.49 (m, each 1H, CH2, Hβ-cys), 3.75 (m, 2H, CH2, Hgly), 3.68 (s, 3H, CH3O), 2.19 (t, J8.0, 2H, CH2, Hγ-glu), 1.95
and 1.76 (m, each 1H, CH2, Hβ-glu), 1.84 (s, 3H, CH3CO). 13C NMR (DMSO-d6), δ: 221.2 (C=S), 173.6, 171.7,
170.9, 169.7, 169.5, 168.7 (C=O), 132.5, 129.3, 128.7, 128.4 (PhC), 57.3, 53.3, 51.5, 50.5, 40.9, 38.9, 31.7, 27.0,
22.4. FT-IR (cm−1): 3288, 3062, 3031, 2953, 2923, 2856, 1729, 1645, 1524, 1452, 1433, 1412, 1375, 1336, 1279,
1255, 1208, 1162, 1118, 1071, 1046, 1006, 923, 889, 870, 801, 729, 695. Anal. Calcd for C22H27N3O9S3: C,
46.06%; H, 4.74%; N, 7.32%; S, 16.77%. Found: C, 46.02%; H, 4.74%; N, 7.30%; S, 16.75%. ESI-MS: m/z =
572.0850 [M − H]−, theoretical value = 572.0837 (Calcd for C22H26N3O9S3).
General Procedure for RAFT Polymerization. Polymerizations were conducted in dioxane using AIBN as the
primary radical source and ABSPA, AMSPA, ABSG, AMSG or GSH-PA macro CTA as the chain transfer agent.
In a typical polymerization, DMA (1.983 g, 20.0 mmol), ABSG (0.103 g, 0.20 mmol), and AIBN (6.6 mg, 0.040
mmol) was added to a glass tube with a magnetic stirring bar, and dioxane was added until the total volume was
12.5 mL. The tube was sealed with a rubber septum, and the contents were degassed with nitrogen for 10 min.
2
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

o
The tube was subsequently immersed into an oil bath preheated to 60 C. After 45 min, the polymerization was
1
quenched by ice water. The monomer conversion was determined to be 20.5% by H NMR. The polymer was
recovered by precipitating into a large amount of cold hexane thrice from its dioxane solution, and molecular
weight and polydispersity of GSH-PNIPAM were obtained by GPC: Mn = 2910, PDI = 1.11. Other polymers
were synthesized according to a similar approach, and obtained by precipitation into cold hexane, diethyl ether or
methanol.
Characterization. The number-average molecular weight (Mn) and polydispersity index (PDI) of polymer
samples were determined by GPC at ambient temperature using THF as an eluent at a flow rate of 1.0 mL/min
and toluene as a flow rate marker. PSt samples were calibrated with PSt standard samples with Mn value in the
range of 7500000-580 g/mol, other samples were calibrated using PMMA standard samples with Mn value in the
1
range of 1944000-1020 g/mol. H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker 400
UltraShield spectrometer in CDCl3 or DMSO-d6. C, H and N contents were determined by combustion followed
by chromatographic separation and thermal conductivity detection using a Carlo Erba 1108 Elemental Analyzer.
Sulfur analyses were conducted using the Schoniger Oxygen Flask combustion method followed by the relevant
titration. Fourier Transform Infrared (FT-IR) spectra were recorded on a Perkin-Elmer Spectrum One FT-IR
spectrometer using a single reflection horizontal ATR accessory. The electrospray ionization mass spectrometry
(ESI-MS) was performed on a Bruker Daltonics micrOTOF mass spectrometer.
Major ('living')
X
a.
X
Minor
(termination, chain transfer)
R1
R2
R1
N
R2
S
S
R
X =
O
Br
Z
RAFT
(R-group)
NMP
ATRP
b.
R
Major ('living')
S
R
Z
S
Minor
(termination, chain transfer)
Scheme S1. Conjugates and side products obtained from living radical polymerisation initiated by
polypeptide sequences.
3
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

Table S1. RAFT Polymerization of Various Monomers Mediated by ABSPA, AMSPA, ABSG and
AMSG.a
run
1
2
3
4
5
6
7
8
CTA
M
NIPAM
DMA
MA
BA
St
MMA
[M]0/[CTA]0
200
200
200
200
200
200
200
200
200
200
200
200
300
300
300
300
300
300
300
300
300
300
300
300
t (h)
3
3
C%b
86.1
92.4
91.1
88.3
16.7
59.9
97.7
97.8
94.4
93.2
10.4
35.2
89.4
92.8
82.4
75.1
11.2
47.4
96.4
97.8
90.7
89.1
9.72
28.5
Mn(th)c
19800
18600
16000
23000
3810
12300
22500
19800
16600
24300
2550
Mn(GPC)d
23600
22200
21600
25900
4160
134000
27300
21100
20400
25900
2870
11400
38500
30200
28800
32000
4220
200000
39200
30800
29500
35600
4250
PDId
1.15
1.11
1.17
1.21
1.20
1.59
1.12
1.09
1.15
1.18
1.25
1.35
1.13
1.18
1.16
1.18
1.21
1.68
1.14
1.21
1.18
1.17
1.22
1.37
ABSPA
ABSPA
ABSPA
ABSPA
ABSPA
ABSPA
14
14
14
14
15
15
15
15
15
15
4
AMSPA NIPAM
AMSPA
AMSPA
AMSPA
AMSPA
AMSPA
ABSG
ABSG
ABSG
ABSG
ABSG
DMA
MA
BA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
St
MMA
NIPAM
DMA
MA
BA
St
MMA
NIPAM
DMA
MA
7440
30900
28100
21800
29400
4010
14800
33300
29700
24000
34800
3610
4
4
4
15
15
15
15
15
15
15
15
ABSG
AMSG
AMSG
AMSG
AMSG
AMSG
AMSG
BA
St
MMA
9130
16300
a Polymerization conditions: [CTA]0:[AIBN]0 = 5:1, [M]0 = 1.60 mol/L, in dioxane at 60 oC. b Monomer
1
c
conversion determined by H NMR.
Theoretical molecular weight, Mn(th) = MWm × C% ×
d
[M]0/[CTA]0 + MWCTA, where MWm and MWCTA stands for molecular weight of monomer and CTA.
Molecular weight and polydispersity determined by GPC.
4
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

(a)
9
8
7
6
5
4
3
2
1
Chemical shift (ppm)
(b)
240
200
160
120
80
40
0
Chemical shift (ppm)
Figure S1. 1H (a) and 13C (b) NMR spectra of ABSG.
5
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

(a)
9
8
7
6
5
4
3
2
1
Chemical shift (ppm)
(b)
240
200
160
120
80
40
0
Chemical shift (ppm)
Figure S2. 1H (a) and 13C (b) NMR spectra of AMSG.
6
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

(a) ABSPA
(b) AMSPA
(c) ABSG
(d) AMSG
4000 3500 3000 2500 2000 1500 1000
Wavenumber (cm-1)
500
Figure S3. ATR FT-IR spectra of various RAFT agents.
17.6%
37.9%
53.4%
66.0%
82.7%
89.4%
11.1%
20.3%
29.6%
47.4%
62.0%
74.1%
86.7%
92.8%
(a)
(b)
11
12
13
14
15
16
17
12
13
14
15
16
17
18
Retention time (min)
Retention time (min)
9.02%
14.8%
19.5%
28.3%
50.4%
75.1%
87.0%
10.1%
23.5%
34.0%
46.5%
57.5%
72.5%
82.4%
88.3%
(c)
(d)
12
13
14
15
16
17
18
11
12
13
14
15
16
17
18
Retention time (min)
Retention time (min)
Figure S4. GPC traces of GSH-PA conjugates obtained by ABSG-mediated RAFT polymerization at
different conversions: GSH-PNIPAM (a), GSH-PDMA (b), GSH-PMA (c) and GSH-PBA (d). Samples
were synthesized as described in Figure 1.
7
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

O
S
bH
l
f
b
O
N
j
d
O
DMSO
OHi,i'
a
H
k
n
c
N
S
S
N
H
H2O
h,n
g
h
O
e
HO
O
O
m
nNH
b
o
a
o
(a)
l
j
i,e
f
d
m
b
c
h
k
e
g
i'
9
8
7
6
5
4
3
2
1
0
Chemical shift (ppm)
m
O
b H
l
f
O
N
H g
b
N
b
j
O
OH i,i'
d
a
H
N
k
c
S
S
n
i
e
O
h
S
HO
O
O
N
DMSO
H2O
m
(b)
j
j
a
k
d
h
f
l
c
e
g
b
i'
h
e
9
8
7
6
5
4
3
2
1
0
Chemical shift (ppm)
8
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

O
S
b H
l
f
O
N
j
b
DMSO
d,i
O
OHi,i'
a
d
H
N
k
n
c
S
S
a
N
H
g
e
O
h
O m
HO
O
O
b
H2O
m
(c)
e,j
j
f
k
l
h
b
g
i'
c
e
h
9
8
7
6
5
4
3
2
1
0
Chemical shift (ppm)
O
b
g
l
H
N
f
O
b
j
OHi,i'
O
d
a
H
N
k
n
DMSO
c
S
S
N
H
h
e
O
S
H2O
HO
O
O
O
b
j,n,o
p
m
n
o
p
(d)
h,m
a
d,i,k
e,j
f
c
h
e
l
i'
g
b
9
8
7
6
5
4
3
2
1
0
Chemical shift (ppm)
Figure S5. 1H NMR spectra of GSH-PNIPAM (a), GSH-PDMA (b), GSH-PMA (c) and GSH-PBA (d).
The GSH-PA samples were synthesized by runs 1-4 in Table S2.
9
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007

Table S2. Chain extension polymerization mediated by GSH-PA macro CTAs in dioxane at 60 oC.a
b
Mn(th)f
Mn(GPC)g
PDIg
macro CTA
run
M
C%
C%
Mn(th)c
Mn(GPC)d
PDId
1.11
Mn(NMR)e
1
2
3
4
20.5
2840
2910
2780
DMA
93.5
91.8
92.7
90.6
21300
25000
23700
23800
22200
29300
27500
25500
1.11
1.10
1.09
1.07
36.3
26.2
21.9
4110
2770
3320
4200
3880
3480
1.12
1.12
1.13
4220
2700
3270
NIPAM
NIPAM
NIPAM
a
Polymerization conditions: [M]0:[macro CTA]0 = 200:1, [M]0 = 1.00 mol L−1, [AIBN]0 = 1.20 mmol L−1, t = 6 h, macro-
CTA = GSH-PNIPAM (run 1), GSH-PDMA (run 2), GSH-PMA (run 3) and GSH-PBA (run 4). b Macro CTAs synthesized
o
by ABSG-mediated RAFT polymerization, [M]0:[ABSG]0:[AIBN]0 = 100:1:0.2, [M]0 = 1.60 mol L−1, in dioxane at 60 C. c
Theoretically calculated molecular weight using equation Mn(th) = MWM × C% × [M]0/[ABSG]0 + MWABSG, where MWM and
d
MWABSG mean the molecular weight of monomer and ABSG. Molecular weight and polydispersity determined by GPC
e
1
using PMMA standard. Molecular weight determined by H NMR by comparing the signal areas of protons in polymer
f
chains with NH (7.9-8.5 ppm) or CH (about 4.6 ppm, Hα-cys) protons in glutathione unit. Mn(th) = MWM × C% ×
[M]0/[macro CTA]0 + MWGSH-PA, where MWM and MWGSH-PA stand for the molecular weight of monomer and macro CTA. g
Molecular weight and polydispersity of block copolymers determined by GPC using PMMA standard.
10
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2007