Article

pubs.acs.org/Biomac

Thiol-Mediated Controlled Ring-Opening Polymerization of Cysteine-

Derived β‑Thiolactone and Unique Features of Product Polythioester
Masato Suzuki,* Kazumasa Makimura, and Shin-ichi Matsuoka
Department of Materials Science and Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan
*
S Supporting Information

ABSTRACT: The controlled ring-opening polymerization of

the β-thiolactone derived from N-Boc cysteine was achieved
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using N-Boc-L-cysteine methyl ester as the initiator in NMP at

room temperature. The propagating end is the thiol group,
which attacks the carbonyl to open the monomer ring by
the C(O)−S bond scission. A thiol−ene click reaction
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demonstrated the utility of the thiol group at the propagating

terminal. The block copolymer was efficiently produced by the
terminal coupling of the polythioester with the norbornene
terminated PEG. As another interesting reaction, the
polythioester underwent the main chain transformation to polycysteine through the intramolecular S-to-N acyl migration,
triggered by the deprotection of the pendant Boc groups. The polythioester from L-cysteine showed Cotton effects between
200 and 300 nm in the circular dichroism (CD) spectrum. Although the CD pattern resembled that produced by the α-helix of
polypeptide, it was ascribable not to the second structure but to the relative orientation of the thioester and carbamate carbonyls
in the repeating unit.

■ INTRODUCTION
The chemistry of thioesters (S-alkyl carboxylic thioesters) is
essential in metabolic pathways, such as those including
coenzyme A, and sometimes employed in biorelated fields.
However, it is rarely covered in organic chemistry textbooks
and less familiar to chemists working in other fields, which
include polymer science. While there have been a huge number
of studies of polyesters and polyamides, polythioesters have
been much less reported with a single yet excellent review
article.1 Polythioesters have potentials as bio- and chemical
degradable materials that are different in property from
polyesters and polyamides. Polythioesters are prepared by
ring-opening polymerization as well as by polycondensation
in a manner similar to polyesters. Thiolactones are generally
polymerizable for 4-, 6-, 7-, and 18-membered rings,1−4 as are
consistent with lactones.
This article deals with the ring-opening polymerization of
β-thiolactone derived from cysteine and the unique features of
the product polythioester. A detailed literature survey revealed
that there are four reports that discuss the ring-opening
polymerizations of β-thiolactones. They are performed with
nucleophilic initiators such as amine, thiol, ammonium
carboxylate,5−7 as well as transition metal catalysts.8 However,
these polymerizations are uncontrollable. The monomers
studied therein involved the β-thiolactones derived from
N-(para-substituted benzenesulfonyl)-L-cysteine.5,6 In this
study, we employed the cysteine-derived β-thiolactone because
we are interested not only in the controlled polymerization of
thiolactone but also in the characteristic features of the product
polythioester. One such feature includes the easily exchangeable
characteristic of thioester with nucleophiles, which works in
metabolic pathways and is employed in artificial systems as well.
The typical example is Native Chemical Ligation,9 which is an
efficient method for connecting two peptides by the amide
formation between the thioester C-terminal and the cysteine
N-terminal. The intermolecular acyl transfer from the terminal
thioester to the terminal thiol is followed by the intramolecular
acyl migration from the thioester to the amino group in the
cysteine unit. We planned to apply the intramolecular S-to-N
acyl migration to the exchange reaction between the main
and side chains of the polythioester, which would be prepared
from the cysteine-derived β-thiolactone. In this context, the
t-butoxycarbonyl (Boc) group was employed for the amino
group protection of the monomer since the deprotection could
be performed under an acidic condition, which would hardly
affect the thioester linkage forming the polymer main chain.
Another feature of the optically active polythioester has been
also investigated using circular dichroism (CD) spectroscopy.
Accordingly, the studied subjects are outlined as follows: (1)
the ring-opening polymerization of the cysteine-derived
β-thiolactone and its mechanism, (2) two types of reactions
of the product polythioesters, i.e., the thiol−ene coupling
reaction of the terminal thiol for the block copolymer synthesis
and the transformation of the polythioester to polycysteine by
the S-to-N acyl migration, and (3) the CD spectroscopic study
of the optically active polythioester.
Received: December 28, 2015
Revised: February 3, 2016
Published: February 4, 2016

© 2016 American Chemical Society 1135 DOI: 10.1021/acs.biomac.5b01748

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Figure 1. 1H NMR spectrum (400 MHz, DMSO-d6) of the polythioester prepared from L-1 under the conditions of entry 1 in Table 1. The inset
shows the expanded area around 3 ppm after a drop of CF3CO2H was added. The N−H and methine protons show major and minor signal pairs
due to the stereoisomerism of the Boc amide group.
■ EXPERIMENTAL SECTION
General Methods and Materials. Instruments and measurement
conditions are described in the Supporting Information. All reactions
except for the N-Boc formation were conducted in dry solvents under
nitrogen atmosphere. The procedures from the previous literatures
were modified to prepare the monomers, L- and DL-3-(tert-butoxy-
carbonylamino)thietan-2-one,10 as well as norbornene-terminated
poly(ethylene glycol).11 N-Boc-L-cysteine methyl ester was prepared
from L-cysteine methyl ester hydrochloride. The detailed synthetic
procedures for these materials are seen in the Supporting Information.
Typical Procedure of the Ring-Opening Polymerization of
Monomer 1. To L-1 (200 mg, 0.98 mmol) dissolved in N-methyl-
pyrrolidone (NMP) (1 mL) was added the initiator (1−10 mol %).
The reaction mixture was stirred for 2 h at room temperature (r.t.) and
then added dropwise into diethyl ether (60−70 mL). The precipitated
polythioester was collected by centrifugation, washed with diethyl
ether 3 times, and dried under vacuum. 1H NMR (400 MHz, DMSO-
d6, Figure 1) δ 7.8−7.6 (br, NH), 7.3−7.45 (br, NH), 4.2−3.9 (m,
N−CH), 3.65 (s, terminal O−CH3), 3.4−3.2 (appeared by the
addition of a drop of CF3CO2H, m, S−CH2), 2.95−2.8 (m, S−CH2),
1.6−1.2 (m, C−CH3 and terminal SH). 13C NMR (100 MHz, DMSO-
d6, Figure S2) δ 199.9, 155.2, 79.5, 79.0, 78.6, 59.8, 52.2, 29.7, 28.1,
27.8. IR (ATR, Figure S4) 3219, 1683, 1515, 1164 cm−1.
Synthesis of the Dimer and Trimer Methyl Esters. To N-Boc-
L-Cysteine methyl ester (380 mg, 1.61 mmol) in NMP (0.3 mL) was
added a solution of DL-1 (102 mg, 0.5 mmol) in NMP (0.3 mL). After
3 h with stirring at r.t., diethyl ether was added to the reaction mixture.
The organic layer was washed with water, dried over MgSO4, and
concentrated under vacuum. The crude material was subjected to silica
gel column chromatography (CH2Cl2/Acetone, from 20/1 to 1/1),
giving the diastereomeric mixtures, DL,L-dimer (122 mg, 55% yield,
Rf = 0.53) and DL,DL,L-trimer (57 mg, 35% yield, Rf = 0.38) methyl
esters. The DL,L-dimer methyl ester: 1H NMR (400 MHz, CDCl3,
Figure S9) δ 5.53 (t, J = 10.3 Hz, 1H), 5.23 (t, J = 7.1 Hz, 1H), 4.72−
4.49 (m, 2H), 3.77 (s, 1.5H), 3.76 (s, 1.5H), 3.49 (dd, J = 4.6, 13.9 Hz,
0.5H), 3.37 (d, J = 5.4 Hz, 1H), 3.30−3.13 (m, 1.5H), 2.83−2.68
(m, 1H), 1.55 (dt, J = 7.2, 10.7 Hz, 1H), 1.485 (s, 4.5H), 1.479 (s,
4.5H), 1.444 (s, 4.5H), 1.439 (s, 4.5H). 13C NMR (100 MHz, CDCl3,
Figure S10) δ 199.7, 199.1, 170.9, 170.8, 154.9, 80.8, 80.3, 60.7, 60.6,
52.72, 52.68, 52.6, 31.2, 28.2, 27.1. IR (Figure S25). ESI-HRMS m/z
calcd for C17H30N2O7S2 [M + Na]+, 461.1392; found, 461.1402. The
DL,DL,L-trimer methyl ester: 1H NMR (400 MHz, CDCl3, Figure S11)
δ 5.56 (br, 1H), 5.39−5.12 (m, 2H), 4.72−4.60 (m, 1H), 4.60−4.41
(br, 2H), 3.85−3.68 (overlapped singlets, 3H), 3.55−3.04 (m, 5H),
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2.89−2.65 (m, 1H), 1.55−1.50 (m, 1H), 1.57−1.35 (overlapped singlets,
27H). IR (Figure S25). ESI-HRMS m/z calcd for C17H30N2O7S2
[M + Na]+, 664.2008; found, 664.2015.
In the same manner, the reaction of N-Boc-L-Cysteine methyl ester
(906 mg, 3.9 mmol, contaminated with a small amount of (Boc)2O)
with L-1 (314 mg, 1.5 mmol) in NMP (1.5 mL) afforded the L,L-
dimer (82 mg, 13 yield, contaminated with 13 mol % of (Boc)2O) and
L,L,L-trimer (124 mg, 25% yield, contaminated with 2 mol % of
(Boc)2O) methyl ester. The L,L-dimer methyl ester: 1H NMR
(400 MHz, CDCl3, Figure S12) δ 5.54 (d, J = 8.8 Hz, 1H), 5.24 (d, J =
7.8 Hz, 1H), 4.68−4.59 (m, 1H), 4.59−4.49 (m, 1H), 3.76 (s, 3 H),
3.37 (d, J = 5.4 Hz, 2H), 3.27−3.14 (m, 1H), 2.82−2.66 (m, 1H),
1.55−1.50 (m, 1H), 1.48 (s, 9H), 1.44 (s, 9H). 13C NMR (100 MHz,
CDCl3, Figure S13) δ 199.7, 170.9, 154.9, 80.7, 80.2, 60.6, 52.7, 52.6,
31.2, 28.2, 27.3, 27.1. IR (Figure S25). ESI-HRMS m/z calcd for
C17H30N2O7S2 [M + Na]+, 461.1392; found, 461.1398. The L,L,L-
trimer methyl ester: 1H NMR (400 MHz, CDCl3, Figure S15) δ 5.55
(d, J = 8.8 Hz, 1 H), 5.26 (d, J = 7.8 Hz, 2H), 4.72−4.60 (m, 1H),
4.60−4.38 (m, 2H), 3.75 (s, 3H), 3.48−3.13 (m, 5H), 2.83−2.68 (m,
1H), 1.55−1.50 (m, 1H), 1.49 (s, 9H), 1.45 (s, 18H). 13C NMR
(100 MHz, CDCl3, Figure S16) δ 200.8, 199.1, 170.8, 154.9, 80.8,
80.2, 60.7, 60.4, 52.7, 52.65, 31.2, 31.1, 28.2, 27.3. IR (Figure S25).
ESI-HRMS m/z calcd for C17H30N2O7S2 [M + Na]+, 664.2008; found,
664.2015.
Synthesis of the Block Polymer by the Thiol−ene Coupling
Reaction at the Polymer Terminals. The typical procedure is as
follows. The NMP (0.8 mL) solution of L-1 (204 mg, 1.0 mmol) was
degassed through three freeze−thaw cycles and placed under N2
atmosphere. N-Boc-L-cysteine methyl ester (101 mg) was dissolved in
NMP (1010 mg), and a part (253 mg) of this initiator solution was
added to the above monomer solution, resultantly feeding the initiator
(23.0 mg, 0.097 mmoL) in 10 mol % for the monomer. After 1 h, a
half of the reaction mixture was added into diethyl ether (50 mL) to
precipitate the polythioester (101 mg, 89%), which was washed twice
with diethyl ether. Other half was added to norbornene-terminated
PEG-750 (100 mg, ∼ 2 equiv) and 2,2-dimethoxy-2-phenyl-
acetophenone (DMPA, 5 mg, 0.02 mmol) in a test tube. This mixture
was subjected to the irradiation by a high pressure Hg lamp (100 W)
for 4 h. The resultant solution was poured into diethyl ether (50 mL).
The precipitate was collected by centrifugation, washed twice with diethyl
ether, and dried under vacuum to give the block copolymer (114 mg,
72%). 1H NMR spectrum (200 MHz, DMSO-d6): see Figure S19.
Transformation of the Polythioester to Polycysteine by
S-to-N Acyl Migration. Trifluoroacetic acid (0.5 mL) was dropwise
DOI: 10.1021/acs.biomac.5b01748
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added to the suspension of the polythioester (103 mg) in dichloro-
methane (1.0 mL) and stirred for 4 h at 0 °C. The resultant
homogeneous solution under ice-cooling was neutralized by the slow
addition of a saturated NaHCO3 solution. The generated precipitate
was collected by centrifugation, washed successively with water and
methanol, and dried under vacuum to give polycysteine (25 mg, 49%
yield). Poly(DL-cysteine): 1H NMR (400 MHz, DMSO-d6, Figure 3)
δ 8.31 (br, 1H), 4.47 (br, 1H), 3.17−2.62 (m, 2H), 2.37 (br, 1H). 13C
NMR (100 MHz, DMSO-d6, Figure S20) δ 169.7, 55.0, 26.3. IR (ATR,
Figure S21) 3284, 2552, 1635, 1507 cm−1. Poly(L-cysteine): 1H NMR
(Figure S23), IR (Figure S24).
■
RESULTS AND DISCUSSION
Ring-Opening Polymerization of β-Thiolactone. The
β-thiolactones derived from L- and DL-cysteine, 3-(tert-butoxy-
carbonylamino)thietan-2-one (L- and DL-1), were found to
undergo the ring-opening polymerization at r.t. under neutral
conditions, where N-Boc-L-cysteine methyl ester acted as
the initiator in NMP (Scheme 1). Results from both NMR and
Scheme 1. Ring-Opening Polymerization of β-Thiolactone 1
matrix assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectroscopy indicated that the corre-
sponding polythioester was produced. Figure 1 shows the 1H
NMR spectrum of the product polymer from L-1 at entry 1 in
Table 1. The signal due to one of the diastereomeric methylene
protons was hidden in the large water signal but appeared
through the addition of a drop of CF3CO2H. The stereo-
isomerism of the Boc amide group causes the N−H and
methine protons to show major and minor signal pairs,
respectively. The signal assignment was assisted by the COSY
spectrum (Figure S1). The IR spectrum (Figure S4) shows the
overlapped peak at 1683 cm−1 for the stretching vibration of
two different carbonyl bonds, thioester and carbamate. The
MALDI-TOF mass spectrum (Figure 2a) indicates that the
product polythioester has a defined repeating unit and terminal
structures. The molecular weights of the product polythioesters
were successfully controlled by the feed ratios of the initiator
(entries 1−4). GPC elution curves were shifted to the higher
molecular weight region with decreasing initiator feed ratios,
Table 1. Ring-Opening Polymerization of β-Thiolactone 1a
entry monomerb initiator initiator (mol %) yield (%)c
f
1 L N-Boc-L-Cys-OMe 10 96
2 L N-Boc-L-Cys-OMe 3 77
3 L N-Boc-L-Cys-OMe 2 84
4 L N-Boc-L-Cys-OMe 1 76
5 L benzyl amine 10 89
6 DL N-Boc-L-Cys-OMe 3 62
7 DL N-Boc-L-Cys-OMe 2 57
a
In NMP at r.t. bChirality of the monomer. cAn Et2O-insoluble part. dEvaluated by GPC (PSt Std.) using NMP (LiBr 0.25 g/L) or THF as the
eluent. Values in the parentheses are evaluated using THF. eDegree of polymerization calculated from the peak intensity ratio between the methine
proton in the repeating unit and the methyl ester protons from the N-Boc-L-Cys-OMe (or the phenyl protons from the benzylamine). The
calculated DP value involves the cysteine unit from the N-Boc-L-Cys-OMe. fN-Boc-L-cysteine methyl ester.
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and then, the degree of polymerization (DP) values, which
were calculated from the content of the terminal methyl ester in
the 1H NMR spectra, were consistent with the feed ratios of the
initiator. The oligomeric polythioester from L-1 (entry 1) was
soluble in THF, whereas the higher molecular weight products
(entries 2−4) had limited solubility. The most suitable solvents
were DMSO, NMP, and hexafluoroisopropanol.
Benzylamine also initiated the polymerization, and the 1H
NMR spectra of the product (Figure S5) showed the formation
of the polyester incorporating an N-benzyl amide moiety,
whose content was in good agreement with the initiator−
monomer feed ratio (entry 5). However, the GPC measure-
ment showed that it had a higher molecular weight than the
polyester prepared by using N-Boc-L-cysteine methyl ester in
the same initiator−monomer feed ratio (entry 1). In addition,
the MALDI-TOF mass spectrum (Figure 2b) shows unidentified
peak series, revealing that some side reactions were involved. The
observed values as the peak series B have differences by 1 mass
unit from the calculated ones for the proton adducts of the cyclic
products. Instead, the peak series C was correctly assigned to the
polythioester having the benzyl amine residue as the initiation
terminal. Thiol is known to conduct exchange reactions between
polythioesters in the presence of Et(i-Pr)2N3 and to undergo
facile oxidation to disulfide under basic conditions. The latter
side reaction consequently leads to an increase in the molecular
weight by the polymer terminal coupling. Such side reactions
were likely involved in the polymerization in the presence of
benzylamine.
Et(i-Pr)2N (10 mol %), which has almost no nucleophilicity,
was found to conduct very fast polymerization of L-1 in NMP
at r.t. A very viscous solution was formed several minutes after
the addition of Et(i-Pr)2N and then gradually reduced in
viscosity. GPC analyses indicated that the molecular weights of
the reaction mixtures decreased with time from Mn = 13 600
(10 min) to 5900 (7 days) (Figure S8). A trace of water or
impurity possibly worked to initiate the polymerization and to
partially cleave the polythioester in the presence of Et(i-Pr)2N
catalyst, which accelerated the polymerization. The steric
hindrance of Et(i-Pr)2N allows exclusion of a zwitterionic
mechanism involving the nucleophilic monomer activation,
which is representatively proposed for the pyridine-catalyzed
ring-opening polymerization of oxathiolane-2,5-dione.1
In contrast to L-1, DL-1 produced a polythioester with higher
solubility in THF and hot CHCl3 (entries 6 and 7, Table 1).
Thus, THF was also examined as a reaction solvent for the
polymerization of DL-1 using N-Boc-L-cysteine methyl ester
(10 mol %) as the initiator. No polymerization was found to
time (h) Mn (×103)d Mw (×103)d PDId DPe
1 2.9 (3.4) 4.7 (4.0) 1.6 (1.2) 12
2 4.9 9.8 2 28
2 6 12.7 2.1 44
2 8.8 20.7 2.4 -
1 (5.4) (9.7) (1.8) 10
2 5.0 (11.0) 9.0 (14.0) 1.7 (1.3) 23
2 5.5 (14.0) 10.2 (17.0) 1.9 (1.2) 45
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Figure 2. MALDI-TOF-MS spectra (matrix: 2,5-dihydroxybenzoic acid) of the polythioesters prepared from L-1 under the conditions of entries 1
(a) and 5 (b) in Table 1.
have taken place even under reflux of THF, suggesting that NMP
has an essential effect to promote the polymerization. Its high
polarity with small basicity increases the nucleophilicity of the
thiol group as the active end (vide infra).
Polymerization Mechanism of β-Thiolactone. As is
widely known, the anionic ring-opening polymerization of
β-lactone uniquely occurs via the CH2−O bond scission, whereas
other lactones are usually polymerized via the C(O)−O bond
scission. Thus, these two ring-opening modes have different
propagating ends, which are the carboxylate anion for the former
and the alkoxide for the latter. Similar mechanistic study is
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required for the ring-opening polymerization of β-thiolactone to
determine whether the CH2−S or the C(O)−S bond scission
ring-opening mode is selected. We performed the reactions of
DL- and L-1 with an excess amount of N-Boc-L-cysteine methyl
ester in NMP and successfully isolated the 1:1 and 2:1 ring-
opening products, whose identification revealed that the ring
undergoes C(O)−S bond scission and that the thiol group is
the propagating end (Scheme 2). There are three previous
studies on the related reactions.10,12,13 Two of them report that
alkyl thiols attack the carbonyl carbons of β-thiolactones in
CHCl3 containing Et3N catalyst12 and in CH3CN at 75 °C,13
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Scheme 2. Reactions of β-Thiolactone 1 with An Excess Amount of N-Boc-L-Cys-OMe

producing comparable products to our results. The other study
reports that β-thiolactones undergo CH2−S bond scission by aryl
thiols with Cs2CO3 in DMF.11 Aryl thiol has a higher acidity than
alkyl thiol and in situ generates the thiolate anion, which has
a different character from neutral alkyl thiol. Accordingly, it is
concluded that the ring-opening polymerization of β-thiolactone
1 takes place through the C(O)−S bond scission by the
neutral thiol group as the propagation end. It is recently reported
that strong organic base catalysts are required for the thiol to
mediate the ring-opening polymerization of ε-thiocaprolactone,
which is less reactive than β-thiolactone.4
Thiol−ene Coupling Reaction to Give Block Copoly-
mer. A thiol group has unique features compared with amino
and hydroxy groups. Essential examples are the thiol-mediated
molecular self-assembly on a gold surface, a thiol−disulfide redox
reaction in biosystems, and a thiol−ene click reaction in synthetic
chemistry. The above finding that the obtained polythioester has
the thiol group at the propagating end prompted us to utilize the
thiol−ene click reaction for block copolymer synthesis via
terminal coupling.14 Norbornene-terminated PEGs were used as
the coupling partners and were prepared from commercially
available poly(ethylene glycol) monomethyl ethers (MW = 750,
1000, 2000).11 The polythioesters were prepared from L-1 with
N-Boc-L-cysteine methyl ester (10 and 2 mol %), and the poly-
merization mixtures were directly subjected to the terminal
coupling reactions with norbornene-terminated PEGs using
DMPA as the radical photoinitiator (Scheme 3, Table 2). The
in diethyl ether, while that from PEG 2000 is insoluble in diethyl
ether but soluble in toluene. The reprecipitation solvents were
selected according to these characters. The moderate isolate yield
in entry 1 is ascribable to the higher solubility due to the lower
molecular weight of the block copolymer. The results of the
GPC analyses for entries 1 and 2 in Table 2 showed that the
unimodal elution profiles shifted to the higher molecular weight
region through the reaction (Figure S18). In entry 3, the higher
molecular weight thioester that was insoluble in THF became
THF-soluble through the incorporation into the block copoly-
mer with PEG. These findings suggest that all the polythioester
molecules have the thiol groups at their terminals and are
transformed to the block copolymers, supporting the suggestion
that the thiol group is the propagating end to perform the
controlled polymerization of 1.
Transformation of Polythioester to Polycysteine. As
mentioned in the Introduction, the intramolecular S-to-N acyl
migration in a cysteine skeleton10,15,16 was examined for the
polythioesters that were prepared from L- and DL-1 under the
conditions of entries 1 and 6 in Table 1, respectively. As shown
in Scheme 4, the pendant Boc groups were readily excluded by
Scheme 4. Transformation of Polythioester to Polycysteine
by S-to-N Acyl Migration

Scheme 3. Thiol−Ene Coupling Reaction of Polythioester

with Norbornene-Terminated PEG at The Terminals

the treatment with CF3CO2H (TFA), yielding the correspond-

ing ammonium TFA salt. The neutralization with NaHCO3 aq.
block copolymers were isolated by reprecipitation into diethyl formed the free amino group, which spontaneously accepted
ether (entries 1 and 2) or toluene (entry 3). The norbonene- the acyl migration, in every repeating unit. The product
terminated PEGs prepared from PEG 750 and 1000 are soluble polymer precipitated rapidly after neutralization occurred and

Table 2. Synthesis of Block Copolymer by Thiol−Ene Coupling Reaction at The Terminals of Polythioester and Norbornene-
Terminated PEG
polythioester norbornene-terminated PEG block copolymer
a 3 b 3 b 3 b 3 b
entry feed raio L-1/initiator Mn (×10 ) Mw (×10 ) starting PEG Mn(×10 ) Mw (×10 ) yield (%) Mn (×103)b Mw (×103)b
c
1 10 3.3 4.0 PEG750 1.1 1.2 72 4.6 5.2
2 10 3.1 3.8 PEG1000 1.4 1.5 89c 5.0 5.7
3 50 THF-insoluble PEG2000 3.1 3.2 61d 12 16

a
Initiator: N-Boc-L-cysteine methyl ester. bGPC (THF, PSt Std.) . cA diethyl ether-insoluble part. dA toluene-insoluble part.

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Figure 3. 1H NMR spectrum (400 MHz, DMSO-d6) of poly(DL-cysteine) transformed from the polythioester that was prepared under the
conditions of entry 6 in Table 1.

Figure 4. UV (dotted lines) and CD (solid lines) spectra (in (CF3)2CHOH) of the polythioester prepared from L-1 under the conditions of entry 2
in Table 1 (a), N-Boc-S-acetyl-L-cysteine methyl ester (b), the L,L-dimer methyl ester (c), and the L,L,L-trimer methyl ester (d).

was identified as polycysteine by NMR and IR spectroscopy
and GPC. In the 1H NMR spectra (Figures 3 and S23), which
show the signals associated with polycysteine, a small signal due
to the residual tert-butyl group is observed at 1.46 ppm, but its
relative intensity shows that approximately 98% or more of the
Boc groups are deprotected in Figures 3 and S23, respectively.
The IR spectra (Figures S21 and S24) show the characteristic
peaks produced from the displacement of carbamate groups by
the amide groups, as compared with those of the starting
polythioesters (Figures S4 and S7). A weak peak that is attri-
butable to the thiol group is observed at 2552 or 2551 cm−1.
The GPC analyses were performed only for poly(DL-cysteine),
since even oligomeric poly(L-cysteine) (DP ∼ 10) was
insoluble in NMP as the GPC eluent. Based on the results in
Figure S22, poly(DL-cysteine) was observed to elute in a higher
molecular weight region compared with the starting poly-
thioester, although the exclusion of the Boc groups led to a
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decrease in the molecular weight. This is reasonably elucidated
by the higher rigidity of poly(DL-cysteine) to increase the
molecular size, since the amide group is more rigid than the
thioester group due to the larger resonance between the
carbonyl and the nitrogen. Another possibility is the inter-
molecular connection of poly(DL-cysteine) by the oxidative
S−S bond formation. Gelation was observed for the DMSO
solution of poly(DL-cysteine). The intermolecular S-to-N acyl
migration was probably involved to some extent, because N,S-
diacetyl-L-cysteine methyl ester was detected in the model
reaction using N-Boc-S-acetyl-L-cysteine methyl ester.
CD Spectra of Polythioester. It is interesting that
thioester absorbs much longer-wavelength UV than other
carbonyl compounds. The polythioester from L-1 has a λmax at
238 nm ([C(O)−S] = 1.5 × 10−4 M in (CF3)2CHOH, ε =
5000) due to the π−π* transition, indicating that thioester is a
good probe for CD spectroscopy. As observed in Figure 4a, the
DOI: 10.1021/acs.biomac.5b01748
Biomacromolecules 2016, 17, 1135−1141
Biomacromolecules
polythioester from L-1 showed evidence of Cotton effects in
the CD spectra. The largest negative band (minimum 279 nm)
is ascribed to the weak UV absorption by the n−π* forbidden
transition and is accompanied by a pair of positive and negative
bands (maximum 202 nm and minimum 243 nm) due to the
π−π* transition. It is interesting that this Cotton effects pattern
resembles that of α-helical polypeptide, which shows a large
positive band (maximum 190 nm) and two negative ones
(minimums 202 and 222 nm). These are due to the π−π* and
n−π* absorptions by the amide group at the shorter wave-
lengths. Although N-Boc-S-acetyl-L-Cysteine methyl ester
produced a different CD pattern (Figure 4b), the L,L-dimer
and the L,L,L-trimer methyl esters produced similar CD spectra
(Figures 4c and d). Accordingly, the Cotton effects of the
polythioester from L-1 are not caused by the secondary
structure of the polymer chain. Instead, a pair of carbonyl
groups of the thioester and the carbamate in the polythioester
repeating unit bears a geometrical resemblance to the neigh-
boring amide carbonyl groups in α-helical polypeptide, leading
to the similarity in the CD spectrum pattern between these
polymers. Both these carbonyl pairs in the polythioester and
polypeptide are located at a position three bonds away from
each other and have similar conformational orientations in the
presence of the interaction with the hexafluoroisopropanol
solvent.
■
CONCLUSION
The thiol under neutral conditions in NMP, which promoted
the nucleophilic reactivity of the thiol, successfully mediated
the controlled ring-polymerizations of β-thiolactones, L-1 and
DL-1. Since thioesters readily react with nucleophiles, the ring-
opening polymerizations of thiolactones are difficult to control
under basic conditions, which accelerate not only the poly-
merizations of thiolactones but also the reactions of the product
polythioester main chains. In the previous literature, dealing
with the β-thiolactone derived from N-tosyl-L-cysteine, the
ring-opening polymerization examined in DMF was found to
occur spontaneously to some extent with no initiator.5 This
finding was not elucidated therein, but it might be caused by a
trace amount of base generated from DMF to promote the
polymerization. The same research group revealed that the
para-substituents on the N-benzenesulfonyl group influenced
the polymerizability of the cysteine-derived β-thiolactones in
DMF containing PhCH2SH as the initiator.6 The authors men-
tion that the N−H proton plays an important role, however,
which is not specified, in the polymerization. Thus, there is a
possibility that the Boc group has a preferable effect on the
controlled polymerizations of L- and DL-1. These kinds of
substituent effects are future problems to be studied.
The controlled polymerizations of L- and DL-1 take place via
the C(O)−S bond scission, and the thiol group as the
propagating end is efficient for further transformation, typically,
using a thiol−ene click reaction. We demonstrated it through
the block copolymer synthesis by the terminal coupling. Another
topic is the exchange reaction between the main and side chains
of the product polythioester. The N-Boc groups at the side
chains were successfully deprotected without damaging the
thioester groups in the main chain. Subsequently, the S-to-N acyl
migration took place from the thioester groups to the free amino
groups, transforming the polythioester into polycysteine. The
optically active polythioester from L-1 showed an interesting CD
spectrum pattern similar to that of α-helical polypeptide. There
were a large negative band (minimum 279 nm) due to the n−π*
1141
Article
absorption and a pair of positive and negative bands (maximum
202 nm and minimum 243 nm) due to the π−π* absorption.
These Cotton effects were attributable to the relative conforma-
tional orientation of the thioester and Boc carbonyl groups in the
repeating unit.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.biomac.5b01748.
Some of detailed experimental protocols, additional
NMR and IR spectra, and GPC profiles (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: suzuki.masahito@nitech.ac.jp.
Notes
The authors declare no competing financial interest.
■ REFERENCES
(1) Kricheldorf, H. R.; Schwarz, G. J. Macromol. Sci., Part A: Pure
Appl.Chem. 2007, 44, 625.
(2) Kato, M.; Toshima, K.; Matsumura, S. Biomacromolecules 2007, 8,
3590.
(3) Ura, Y.; Al-Sayah, M.; Montenegro, J.; Beierle, J. M.; Leman, L. J.;
Ghadiri, M. R. Org. Biomol. Chem. 2009, 7, 2878.
(4) Bannin, T. J.; Kiesewetter, M. K. Macromolecules 2015, 48, 5481.
(5) Fleš, D.; Tomašić, V. J. Polym. Sci., Part B: Polym. Lett. 1968, 6,
809.
(6) Fleš, D.; Tomašić, V.; Samsa, M.; Ahmetović, D.; Jerman, B.; Fleš,
M. J. Polym. Sci., Polym. Symp. 1973, 42, 321.
(7) Jerman, B.; Fleš, D. J. Polym. Sci., Polym. Chem. Ed. 1976, 14,
1117.
(8) Adams, R. D.; Huang, M.; Huang, W. Organometallics 1997, 16,
4479.
(9) Kent, S. H. B. Chem. Soc. Rev. 2009, 38, 338.
(10) Crich, D.; Sana, K. J. Org. Chem. 2009, 74, 3389.
(11) Liu, Y.; Piñoń , V., III; Weck, M. Polym. Chem. 2011, 2, 1964.
(12) Vasil’eva, T. P.; Bystrova, V. M.; Kil’disheva, O. V.; Knunyants, I.
L. Izv. Akad. Nauk SSSR, Ser. Khim. 1986, 1299.
(13) Noel, A.; Delpech, B.; Crich, D. Org. Biomol. Chem. 2012, 10,
6480 and the correction Org. Biomol. Chem. 2015, 13, 9324. The
original article reports that the β-thiolactone undergoes CH2−S bond
scission by the reaction with the alkyl thiol, but it has been recently
corrected..
(14) Review articles: Lowe, A. B. Polym. Chem. 2010, 1, 17. and
Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Chem. Soc. Rev. 2010, 39,
1355.
(15) Hiskey, R. G.; Mizoguchi, T.; Inui, T. J. Org. Chem. 1966, 31,
1192.
(16) Yoshiya, T.; Hasegawa, Y.; Kawamura, W.; Kawashima, H.;
Sohma, Y.; Kimura, T.; Kiso, Y. Biopolymers 2011, 96, 228.
DOI: 10.1021/acs.biomac.5b01748
Biomacromolecules 2016, 17, 1135−1141