Phosgene-free synthesis of polypeptides using activated urethane derivatives of α-amino acids: An efficient synthetic approach to hydrophilic polypeptides

Article abstract of DOI:10.1039/c4ra03315a

A series of polypeptides have been synthesized from the corresponding N-phenoxycarbonyl derivatives of hydroxyl- or amino-functionalized α-amino acids such as l-serine, l-cysteine, l-threonine, l-tyrosine, l-glutamine, and l-asparagine. These derivatives are prepared by N-carbamylation of tetrabutylammonium salts of α-amino acids with diphenyl carbonate (DPC). The synthesis of polypeptides can be achieved by heating these N-phenoxycarbonyl derivatives at 60 °C in N,N-dimethylacetamide (DMAc) in the presence of n-butylamine through the polycondensation of the derivatives with accompanying elimination of phenol and CO₂. The NMR and MALDI-TOF mass analyses of the resulting polypeptides revealed that n-butylamine was successfully incorporated into the chain end of the polypeptides. Molecular weights of the polypeptides were controlled by varying the feed ratio of the urethane derivatives to n-butylamine. The employment of an amine-terminated poly(ethylene glycol) in place of n-butylamine permitted the successful synthesis of the corresponding diblock copolymers composed of polyether and polypeptide segments.

Full text of DOI:10.1039/c4ra03315a

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PAPER
Phosgene-free synthesis of polypeptides using
activated urethane derivatives of a-amino acids: an
efficient synthetic approach to hydrophilic
polypeptides†
Cite this: RSC Adv., 2014, 4, 29890
a
b
a
a
*
Shuhei Yamada, Atsushi Sudo, Mitsuaki Goto and Takeshi Endo
A series of polypeptides have been synthesized from the corresponding N-phenoxycarbonyl derivatives of
hydroxyl- or amino-functionalized a-amino acids such as L-serine, L-cysteine, L-threonine, L-tyrosine, L-
glutamine, and L-asparagine. These derivatives are prepared by N-carbamylation of tetrabutylammonium
salts of a-amino acids with diphenyl carbonate (DPC). The synthesis of polypeptides can be achieved by
heating these N-phenoxycarbonyl derivatives at 60 ꢀC in N,N-dimethylacetamide (DMAc) in the
presence of n-butylamine through the polycondensation of the derivatives with accompanying
elimination of phenol and CO2. The NMR and MALDI-TOF mass analyses of the resulting polypeptides
revealed that n-butylamine was successfully incorporated into the chain end of the polypeptides.
Molecular weights of the polypeptides were controlled by varying the feed ratio of the urethane
derivatives to n-butylamine. The employment of an amine-terminated poly(ethylene glycol) in place of
n-butylamine permitted the successful synthesis of the corresponding diblock copolymers composed of
polyether and polypeptide segments.
Received 12th April 2014
Accepted 25th June 2014
DOI: 10.1039/c4ra03315a
www.rsc.org/advances
cyclization of N-phenoxycarbonyl a-amino acids that can be
easily synthesized by N-carbamylation of onium salts of a-
Introduction
amino acid with DPC. These urethane derivatives can be used
not only as precursors of NCAs but also as useful monomers
for a more straightforward synthesis of polypeptides, where
heating these urethane derivatives in the presence of amines
gives the corresponding polypeptides through the in situ
formation of NCAs and their polycondensation along with the
elimination of phenol and CO2.5 The advantages of this
synthetic approach are (1) the successful incorporation of
amine residue in the chain end, (2) the control of molecular
weight by varying feed ratio between the urethane derivative
and amine, and (3) the easy handling and simple procedure
without the use and formation of any toxic compounds. In
the previous study, we have synthesized poly(Z-L-lysine) and
hydrophilic polypeptides such as poly(L-alanine), poly-
(L-valine), poly(L-methionine) and poly(L-tryptophan), by
utilizing polycondensation of the corresponding urethane
derivative.5
Herein, we report the applicability of the poly-
condensation system to the synthesis of polypeptides from
a-amino acids with functional groups such as L-serine, L-
cysteine, L-threonine, L-tyrosine, L-glutamine, and L-aspara-
gine. In addition to the resulting well-dened synthesis of
those polypeptides, a facile synthesis of diblock copolymers
composed of both polyether and polypeptide segment is
also reported.
Polypeptides have been widely employed to prepare a wide
variety of functional biomaterials for use in drug delivery
systems (DDSs) and tissue engineering, because of their
excellent biocompatibility and biodegradability.1 One of the
most efficient routes for the synthesis of polypeptides is the
ring-opening polymerization of a-amino acid N-carboxy-
anhydrides (NCAs).2 The polymerization gives polypeptides
with well-dened structures in terms of molecular weights
and terminal structures and thus is widely used as a highly
reliable method for the precision synthesis of polypeptide-
containing materials. However, the synthesis of NCAs usually
requires highly toxic phosgene and its derivatives.3 In addi-
tion, the sensitive nature of NCAs to moisture and heat has
prevented the production and the utilization of NCAs on an
industrial scale.
We have developed a phosgene-free synthesis of NCA using
diphenyl carbonate (DPC) as an alternative of phosgene.4 The
synthesis has been achieved by the selective intramolecular
aMolecular Engineering Institute, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka
820-8555, Japan. E-mail: tendo@moleng.fuk.kindai.ac.jp; Fax: +81-948-22-7210; Tel:
+81-948-22-7210
bDepartment of Applied Chemistry, Faculty of Science and Engineering, Kinki
University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra03315a
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Results and discussion
In this study, six functionalized amino acids, L-serine bearing
OH group, L-cysteine bearing SH group, L-threonine bearing OH
group, L-tyrosine bearing phenolic OH group, L-glutamine
bearing amide NH2, and L-asparagine bearing amide NH2, were
used as the starting materials. These amino acids were derived
into their protected forms 1 by the established methods for O-
alkylation, S-alkylation, and tritylation of amide group.6–9
Scheme 1 shows the process of the derivation of the protected
amino acids 1 into the corresponding urethanes 2, which was
conducted according to the reported procedure for the
synthesis of the urethane derivatives of O-benzyl-L-serine, O-
benzyl-L-cysteine, O-benzyl-L-threonine, O-benzyl-L-tyrosine, Nd-
trityl-L-glutamine, and Ng-trityl-L-asparagine.5 This synthetic
route to urethane derivatives 2 consists of two step reactions: in
the rst step, the amino acids were treated with tetrabuty-
lammonium hydroxide to convert them into the corresponding
ammonium salts. In the second step, to the ammonium salts
diphenyl carbonate (DPC) was added to achieve N-carbamoyla-
tion. The resulting urethane derivatives were isolated by column
chromatography and/or recrystallization. Their molecular
structures were conrmed by 1H NMR, 13C NMR, FT-IR,
elemental analysis, and high-resolution mass spectrometry.
These urethane derivatives are storable for at least two months
under ambient conditions without special caution to air and
moisture.
Previously, we have reported that analogous urethane
derivatives of various a-amino acids were efficiently converted
into the corresponding polypeptides by heating them in N,N-
dimethylacetamide (DMAc) at 60 ꢀC in the presence of amines.5
The addition of primary amine to this system gave the corre-
sponding polypeptide with primary amine-incorporated
terminal end. With following the procedure, we investigated the
polymerization of 2 (Scheme 2). First, that of L-serine-derivative
2a was performed by heating it in DMAc (0.5 mol Lꢁ1) in the
presence of n-BuNH2 (2 to 20 mol%). The progress of poly-
condensation was tracked by monitoring the amount of phenol
eliminated from the urethane derivative. The urethane deriva-
tive was completely consumed within 24 h. At that time, white
precipitates were observed, suggesting the formation of
Scheme
2
Synthesis of polypeptide (P2a–P2f) through poly-
condensation of urethane derivative (2a–2f).
polypeptide insoluble in DMAc. The resulting polypeptides were
isolated as ether-insoluble fractions. The FT-IR spectrum of this
product displays that the signal of urethane linkage (1716 cmꢁ1
)
was completely disappeared, while absorbance at 1628 cmꢁ1
and 1506 cmꢁ1, which are the characteristic signals from the
vibration of the peptide linkage (Fig. 1), was observed. The
spectrum indicates also that the P2a maintains the b-sheet form
in the solid state.
In order to conrm the structure of P2a, it was analyzed with
1H NMR. As shown in Fig. 2, 1H NMR spectrum of P2a in
deuterated TFA indicated the singles assignable to the terminal
n-BuNH2 residue at d ¼ 0.80, 1.08–1.46, 2.85–3.27 ppm. In
addition, degree of polymerization (DP) of P2a (Table 1, Entry 2)
was determined to be about 10 from the integrated intensity of
peak between the terminal n-BuNH2 residue and methine
proton at around d ¼ 4.83 ppm. This DP is close to the predicted
one based on the feed ratio. The resulting polypeptides P2a were
insoluble in acetone, CH2Cl2, THF, EtOAc and toluene, but
soluble in TFA. It was partially soluble in DMF. SEC analysis of
the only soluble part in DMF containing 10 mM LiBr was per-
formed to estimate the molecular weight of P2a. We found that
Scheme 1 Synthetic route to urethane derivatives of the protected Fig. 1 FT-IR spectra of (a) urethane derivative 2a and (b) polypeptides
a-amino acids (2a–2f).
P2a obtained by polycondensation of 2a in the presence of n-BuNH2.
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Fig. 2 1H NMR spectrum of P2a obtained from polycondensation of
urethane derivative in the presence of n-BuNH2 ([2a]0/[amine]0 ¼ 10).
Fig.
3
MALDI-TOF mass spectrum of P2a obtained from poly-
condensation of urethane derivatives in the presence of n-butylamine
([2a]0/[amine]0 ¼ 10).
the molecular weight of the soluble part increased as the feed
ratio of 2a to n-BuNH2 increased in the range from 5 to 20 (Table
1, Entries 1–3), while the polydispersity index remained rela-
tively narrow (1.12–1.17). However, in all case, poly-
condensation of 2a gave a lower molecular weight than
theoretical one estimated with the feed ratio, due to the
precipitation of P2a from the reaction mixture.
A MALDI-TOF mass spectrum of P2a exhibited one prom-
inent series of mass signals with D177, which is good agreement
with the monomer unit of P2a and the respective peaks is
assigned to the polymer structure possessing n-BuNH2-incor-
porated initiating end and amino group at the propagating end
(Fig. 3). The detected signals are distributed in the range of DP
from 7 to 16. From these result, we have conrmed that n-
BuNH2 serves as an initiator at the initial stage of the
polycondensation system to give the well-dened terminal
structure.
Likewise, other polypeptides (P2b–P2f) were also obtained
under same reaction condition and their results are summa-
rized in Table 1. The heating of 2b–2f/DMAc solution in the
presence of n-BuNH2 successfully afforded the corresponding
polypeptides in the excellent yield (>88%). In the case of poly(O-
benzyl-L-cysteine) P2b and poly(O-benzyl-L-tyrosine) P2d, their
polypeptides were immediately precipitated from the reaction
mixture due to quite low solubility in DMAc solution. Therefore,
higher molecular weight polypeptides (Mn ¼ >2000) are not
obtained at all. On the other hand, poly(O-benzyl-L-threonine)
a
Table 1 Synthesis of polypeptides by polycondensation of urethane derivatives 2a–2f in the presence of n-BuNH2
Feed ratio of [2]0 to
[–NH2]0
Reaction time
(h)
Entry
Urethane derivative
Polypeptide
Conv.b (%)
Yieldc (%)
Mnd (PDI)
1
2
3
4
5
6
7
8
2a
2a
2a
2a
2b
2b
2b
2c
2c
2c
2c
2d
2d
2e
2e
2e
2e
2f
P2a
P2a
P2a
P2a
P2b
P2b
P2b
P2c
P2c
P2c
P2c
P2d
P2d
P2e
P2e
P2e
P2e
P2f
5
10
20
50
5
10
20
5
10
25
50
10
20
5
10
25
50
5
5
12
24
24
5
12
12
12
24
40
72
12
24
5
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
79
84
80
87
88
95
90
96
85
91
89
93
91
96
93
98
93
96
97
700 (1.12)
1000 (1.13)
1970 (1.17)
N.d.
910 (1.13)
1700 (1.16)
1890 (1.33)
840 (1.18)
2000 (1.23)
3500 (1.26)
6500 (1.52)
1360 (1.10)
N.d.
1290 (1.12)
2500 (1.13)
4400 (1.15)
5400 (1.45)
700 (1.16)
2300 (1.15)
9
10
11
12
13
14
15
16
17
18
19
10
18
24
5
2f
P2f
10
16
a
Polycondensation condition: conc. ¼ 0.5 M, at 60 ꢀC in DMAc. b Calculated by 1H NMR spectra in CDCl3 or DMSO-d6. c Isolated yield. d Estimated
by SEC (eluent: DMF solution with LiBr (10 mM), calibrated by polystyrene standards).
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P2c showed a good solubility in DMAc solution, leading to the the formation of oligopeptides as main product. Additionally,
homogeneous system without any precipitations during poly- each molecular weight (P2e and P2f) estimated from SEC
condensation. The molecular weight increased up to Mn ¼ 6500, analysis was much lower than that predicted from the feed
when the feed ratio ([2c]0/[n-BuNH2]0) was varied in the range of ratio monomer to initiator. This might be due to the aggre-
5 to 50. As shown by the SEC trace in Fig. 4, that of P2c (Table 1, gations of polypeptides containing the bulky trityl group in
Entries 8 and 9) are unimodal and the PDI remained relatively DMF with 10 mM LiBr when compared with polystyrene
low. However, the PDI became gradually broad for the synthesis standards. The MALDI-TOF mass analysis of all polypeptides
of higher molecular weight of P2c ([2c]0/[n-BuNH2]0 ¼ 50), indicated that the incorporation of n-BuNH2 residue at the
suggesting that small amount of the side reactions might be terminal end of polypeptides and therefore it acts as an initi-
present. It is noteworthy the polycondensation of P2c required ator during the polycondensation system (for the spectra of all
much longer reaction time than those of other urethane deriv- polypeptides see the ESI†).
atives to reach the high conversion. This difference in the
Based on the above results, we have attempted a facile
reaction time is likely due to the difference in the rate of NCA synthesis of diblock copolymer using poly(ethylene glycol)
formation of P2c that depends on steric hindrance of the containing amino group at the terminal (PEG–NH2) as a source
substituent. The prolonged reaction time might increase prob- of polyether segment (Scheme 3). According to the procedure
ability of undesired side reaction.
described above, 2a and PEG–NH2 (Mn ¼ 2100 and PDI ¼ 1.05)
We next investigated the polypeptide synthesis of poly(N- were dissolved in DMAc solution ([2a]0/[amine]0 ¼ 10) and
trityl-L-glutamine) P2e and poly(N-trityl-L-asparagine) P2f then the mixture was stirred at 60 ꢀC for 12 h. Aer the reaction
1
through polycondensation of urethane derivative. In all case, for 12 h, H NMR analysis showed that 2a was converted into
the urethane derivatives of 2e and 2f were completely polypeptide through polycondensation without any precipita-
ꢀ
consumed within 24 h at 60 C in DMAc solution to give the tion from the reaction mixture. Pouring the reaction mixture
corresponding polypeptide, even though the bulky trityl group into diethyl ether gave white powder in a high yield (89%). The
is introduced as protecting group on the side chain. For formation of polypeptide was veried by 1H NMR analysis (see
synthesis of P2e with low molecular weight, the obtained ESI†) and the SEC prole in DMF appeared a unimodal and
polypeptides possessed a good solubility in DMAc solution. All narrow peak, which is clearly shied to a higher molecular
reactions were homogeneous and gave polypeptides with well- weight region from that of the original PEG–NH2. Compared
controlled molecular weight and narrow PDI (1.12–1.16). with a original poly(O-benzyl-L-serine), the solubility of diblock
However, for the synthesis of polypeptide with higher molec- copolymer was improved by the conjugation of PEG chain
ular weight ([2e]0/[n-BuNH2]0 ¼ 50), precipitation was observed possessing high solubility in common organic solvent and
in DMAc solution, and this polycondensation showed the therefore it is soluble in DMF, DMAc, acetone, THF, and
formation of polypeptide with a high PDI. Polycondensation of CHCl3. These results clearly indicated the successful conju-
2f was also investigated. For polycondensation of P2f with low gation of poly(O-benzyl-L-serine) segment into PEG chain.
molecular weight, polypeptide with well-controlled manner Also, we further investigated the synthesis of diblock copol-
was observed ([2f]0/[n-BuNH2]0 ¼ 5 and 10), but higher ymer by using other urethane derivative such as 2b and 2f to
molecular weight (Mn ¼ >2300) was not obtained, because of demonstrate the versatility of our polycondensation system
restricted chain extension caused by steric hindrance of bulky and the results are summarized in Table 2. Polycondensation
trityl group close to main chain. Their polycondensation gave of 2b with PEG–NH2 successfully gave the desired diblock
copolymer in high yield (91%). Using a polypeptide having
high solubility; poly(N-trityl-L-glutamine), we could nd the
adjustment of polypeptide chain length by varying the feed
ratio between 2e and amine in PEG ([2e]0/[amine]0 ¼ 10 and
20), while the PDI remains narrow even aer polycondensation
(Fig. 5). With the utilization of other functional amines,
synthesis of polypeptide-based material with functional group
on the side chain and their application to biomedical engi-
neering is now on progress.
Fig. 4 SEC profiles of P2c obtained by polycondensation of urethane
derivative 2c in the presence of n-BuNH2; (a) feed ratio ([2c]0/[amine]0 Scheme 3 Synthesis of diblock copolymer through polycondensation
¼ 5) and (b) feed ratio ([2c]0/[amine]0 ¼ 10).
of urethane derivative in the presence of PEG–NH2.
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Table 2 Synthesis of diblock copolymer by polycondensation of urethane derivatives in the presence of PEG–NH2 (Mn ¼ 2100, PDI ¼ 1.05)a
Entry
Urethane derivative
Feed ratio [2]0/[–NH2]0
Reaction time (h)
Conv.b (%)
Yieldc (%)
Mnd (PDI)
1
2
3
4
2a
2b
2e
2e
10
10
10
20
12
12
12
18
>99
>99
>99
>99
89
91
84
89
4600 (1.15)
5000 (1.12)
5400 (1.15)
8500 (1.10)
a
Polycondensation condition: conc. ¼ 0.5 M, at 60 ꢀC in DMAc. b Calculated by 1H NMR spectra in DMSO-d6. c Isolated yield. d Estimated by SEC
(eluent: DMF solution with LiBr (10 mM), calibrated by polystyrene standards).
Experimental section
Materials and Instruments
Polyethylene glycol containing amino group at terminal (SUN-
BRIGHT MEPA-20H, (MeO–PEG–(CH2)3–NH2), Mn ¼ 2100, PDI
¼ 1.05) was purchased from NOF Corporation. All other
chemicals and solvents were purchased from Tokyo Chemical
Industry and Watanabe Chemical Industry, and used without
further purication unless otherwise denoted below. A n-
butylamine (n-BuNH2) and N,N-dimethylacetamide (DMAc)
were puried by the distillation over calcium hydride (CaH2),
prior to use. For the protection of hydroxyl group of L-serine, we
have conducted benzyl protection for hydroxyl group using
benzyl bromide (see ESI†). The introduction of the protecting
group for other a-amino acids was carried out in accordance
with the published procedures to give the corresponding 1a–1f
(S-benzyl-L-cysteine,6 O-benzyl-L-threonine,7 O-benzyl-L-tyro-
sine,8 Nd-trityl-L-glutamine,9 and Ng-trityl-L-asparagine9).
Analytical TLC was performed on commercial Merck plates
coated with silica gel (TLC Silica gel 60 F254). Column chroma-
Fig.
5 SEC profiles of diblock copolymer obtained by poly-
tography was carried out by using Kanto Silica Gel 60N (spher-
ical, neutral, 63–210 mm). 1H (400 MHz) and 13C NMR (100 MHz)
spectra were recorded with a JEOL ECS-400 spectrometer, and
chemical shis were recorded in ppm units using tetrame-
thylsilane (TMS) as an internal standard. Fourier transform
infrared spectroscopy (FT-IR) analysis was recorded on Thermo
Scientic Nicolet iS10 over a range from 600 to 4000 cmꢁ1. Size
exclusion chromatography (SEC) was carried out on TOSOH
HLC-8220 system equipped with three consecutive polystyrene
gel columns [TSK-gels (bead size, exclusion limited molecular
weight); super-AW4000 (6 mm, >4 ꢂ 105), super-AW3000 (4 mm,
>6 ꢂ 104) and super-AW2500 (4 mm, >2 ꢂ 103)] and refractive
condensation of urethane derivative 2e in the presence of PEG–NH2.
Conclusion
Facile and phosgene-free approach to the synthesis of poly-
peptides with functional groups such as L-serine, L-cysteine, L-
threonine, L-tyrosine, L-glutamine, and L-asparagine has been
developed by polycondensation of their urethane derivative
with the elimination of phenol and CO2. The polycondensation
smoothly take place by heating urethane derivatives at 60 ꢀC
N,N-dimethylacetamide (DMAc) in the presence of n-BuNH2 to
give the corresponding polypeptides in high yield. The detailed
analysis with NMR and MALDI-TOF mass spectrometry
appeared that n-BuNH2 was successfully incorporated into the
chain end of polypeptide during the polycondensation system.
In addition, chain length of polypeptide was adjusted by feed
ratio between urethane derivative and amine. Based on
successful synthesis of polypeptides in the presence of amine,
we could prove the synthesis of a diblock copolymer by utilizing
amine-terminated poly(ethylene glycol) as a source of the poly-
ether segment.
ꢀ
index and ultraviolet detectors at 40 C. The system was oper-
ated using 10 mM LiBr in DMF as eluent at a ow rate of 0.5 mL
minꢁ1. Polystyrene standards were employed for calibration.
Matrix-assisted laser desorption/ionization time-of-ight
(MALDI-TOF) mass spectrometry was performed at room
temperature on a Shimadzu Biotech AXIMA Condence using a-
cyano-4-hydroxycinnamic acid (CHCA) and sodium tri-
uoroacetate, as a matrix material and a cationization agent,
respectively. In order to prepare the sample for MALDI-TOF
mass analysis, the obtained polypeptide was dissolved in TFA (2
mg mLꢁ1), and then the solution was mixed with CHCA–THF
solution (5 mg mLꢁ1) and sodium triuoroacetate–THF solu-
tion (1 mg mLꢁ1) at a ratio of 1 : 2 : 1. The nal mixture was
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deposited onto sample plate and allowed to air dry. The CDCl3, d, ppm): 1.23–1.40 (m, 3H), 4.19–4.28 (m, 1H), 4.38–4.49
resulting sample were irradiated with 337 nm of nitrogen laser (m, 2H), 4.55–4.63 (m, 1H), 5.87 (d, 1H, J ¼ 9.2 Hz), 7.03–7.20
and detected on positive mode at 20 kV. Melting point was (m, 3H), 7.21–7.40 (m, 7H). 13C NMR (100 MHz, CDCl3, d, ppm):
measured on Bibby Stuart scientic melting point apparatus 16.41, 58.74, 71.32, 74.25, 121.61, 125.62, 127.87, 128.07,
SMP3. Elemental analysis was performed on LECO CHNS-932 128.55, 129.41, 137.49, 150.94, 155.28, 175.58. IR (neat, cmꢁ1):
analyzer. High-resolution mass spectrometry was conducted 2978, 1715, 1514, 1484, 1453, 1380, 1309, 1201, 1155, 1089,
using a JEOL JMS-700 mass spectrometer.
1051. HRMS (FAB, m/z): calcd for C18H19NO5, 329.1263; found,
330.1346 (M + H)+.
Synthesis of urethane derivative: N-(phenoxycarbonyl)-O-
benzyl-L-serine (2a)
To a stirred suspension of O-benzyl-L-serine (2.4 g, 12.5 mmol) Synthesis of urethane derivative: N-(phenoxycarbonyl)-O-
in methanol 15 mL, tetrabutylammonium hydroxide (37% in benzyl-L-tyrosine (2d)
methanol) (8.8 g, 12.5 mmol) was slowly added at room
According to the procedure described above, the urethane
temperature. Aer stirring for 1 h, the reaction mixture was
derivative was synthesized from O-benzyl-L-tyrosine and 2d was
concentrated under reduced pressure. The resulting residue
obtained as white powder in 41% yield, mp: 115.3–117.0 ꢀC. 1H
NMR (400 MHz, CDCl3, d, ppm): 2.93–3.27 (m, 2H), 4.63–4.72
was dissolved into acetonitrile (15 mL), and then the resulting
solution added dropwise to a stirred solution of diphenyl
carbonate (DPC) (2.7 g, 12.5 mmol) in acetonitrile (15 mL) at
(m, 1H), 5.01 (s, 2H), 5.50 (d, 1H, J ¼ 7.8 Hz), 6.87–6.96 (m, 2H),
7.04–7.20 (m, 5H), 7.26–7.43 (m, 7H). 13C NMR (100 MHz,
room temperature. Aer stirring the solution for 1 h, the
CDCl3, d, ppm): 36.97, 55.00, 70.16, 115.24, 121.63, 125.69,
distilled water (100 mL) was added into the resulting mixture.
127.62, 128.11, 128.70, 129.45, 130.45, 136.99, 150.81, 154.34,
The resulting mixture was acidied to pH 2–3 with 1 M HCl, and
158.23, 176.01. IR (neat, cmꢁ1): 3324, 3030, 1723, 1690, 1530,
1509, 1487, 1384, 1239, 1005, 807, 742, 693. Anal. calcd for
then extracted with ethyl acetate (3 ꢂ 20 mL). The combined
organic layer was dried over Na2SO4, ltrated, and concentrated
C
23H21NO5: C, 70.58; H, 5.41; N, 3.58%. Found: C, 70.88; H,
under reduced pressure. The crude products were puried with
by column chromatography (eluting with a gradient from 30–
70% ethyl acetate in n-hexane), and then recrystallization from
ethyl acetate/n-hexane. Yield: 3.1 g (78%) of 2a as white powder,
mp: 96.5–97.7 ꢀC. 1H NMR (400 MHz, CDCl3, d, ppm): 3.76–3.85
(m, 1H), 3.95–4.02 (m, 1H), 4.54–4.63 (m, 3H), 5.94 (d, 1H, J ¼
5.43; N, 3.73%.
Synthesis of urethane derivative: Na-(phenoxycarbonyl)-Nd-
trityl-L-glutamine (2e)
8.4 Hz), 7.02–7.15 (m, 2H), 7.16–7.22 (m, 1H), 7.26–7.38 (m, 7H). According to the procedure described above, the urethane
13C NMR (100 MHz, CDCl3, d, ppm): 54.44, 69.46, 73.67, 121.66, derivative was synthesized from Nd-trityl-L-glutamine and 2e was
125.69, 127.90, 128.18, 128.67, 129.45, 137.19, 150.88, 154.62, obtained as white powder in 81% yield, mp: 121.2–122.6 ꢀC. 1H
174.99. IR (neat, cmꢁ1): 3326, 1716, 1659, 1528, 1490, 1202, NMR (400 MHz, DMSO-d6, d, ppm): 1.70–1.83 (m, 1H), 1.93–2.05
1140, 1107, 1063, 744, 698. Anal. calcd for C17H17NO5: C, 64.75; (m, 1H), 2.35–2.52 (m, 2H), 3.95–4.05 (m, 1H), 7.09 (d, 2H, J ¼
H, 5.43; N, 4.44%. Found: C, 64.68; H, 5.39; N, 4.40%.
7.4 Hz), 7.14–7.29 (m, 16H), 7.34–7.40 (m, 2H), 8.03 (d, 1H, J ¼
8.0 Hz), 8.64 (s, 1H). 13C NMR (100 MHz, DMSO-d6, d, ppm):
26.78, 32.50, 53.56, 69.24, 121.57, 124.99, 126.32, 127.45,
128.51, 129.28, 144.87, 150.98, 154.39, 171.13, 173.37. IR (neat,
cmꢁ1): 1746, 1715, 1636, 1535, 1488, 1205, 749, 699. Anal. calcd
for C31H28N2O5: C, 73.21; H, 5.55; N, 5.51%. Found: C, 73.51; H,
5.83; N, 5.27%.
Synthesis of urethane derivative: N-(phenoxycarbonyl)-S-
benzyl-L-cysteine (2b)
According to the procedure described above, the urethane
derivative was synthesized from S-benzyl-L-cysteine and 2b was
obtained as white powder in 70% yield, mp: 111.5–113.0 ꢀC. 1H
NMR (400 MHz, CDCl3, d, ppm): 2.87–3.03 (m, 2H), 3.76 (s, 2H),
4.65 (m, 1H), 5.80 (d, 1H, J ¼ 8.0 Hz), 7.08–7.15 (m, 2H), 7.17–
7.38 (m, 8H). 13C NMR (100 MHz, CDCl3, d, ppm): 33.35, 36.86, Synthesis of urethane derivative: Na-(phenoxycarbonyl)-
53.48, 121.64, 125.78, 127.53, 128.83, 129.09, 129.50, 137.51, Ng-trityl-L-asparagine (2f)
150.80, 154.45, 174.92. IR (neat, cmꢁ1): 3348, 3028, 1721, 1705,
1665, 1520, 1489, 1388, 1254, 1218, 1187, 767, 698, 685. Anal.
calcd for C17H17NO4S: C, 61.61; H, 5.17; N, 4.23%. Found: C,
61.51; H, 5.04; N, 4.18%.
According to the procedure described above, the urethane
derivative was synthesized from Ng-trityl-L-asparagine and 2f
was obtained as white powder in 56% yield, mp: 181.4–183.0 ꢀC.
1H NMR (400 MHz, DMSO-d6, d, ppm): 2.67–2.79 (m, 2H), 4.28–
4.39 (m, 1H), 7.05–7.10 (m, 2H), 7.12–7.30 (m, 16H), 7.35–7.42
(m, 2H), 8.05 (d, 1H, J ¼ 8.3 Hz), 8.68 (s, 1H). 13C NMR (100
Synthesis of urethane derivative: N-(phenoxycarbonyl)-O-
benzyl-L-threonine (2c)
MHz, DMSO-d6, d, ppm): 37.92, 51.16, 69.46, 121.46, 125.01,
According to the procedure described above, the urethane 126.38, 127.44, 128.56, 129.31, 144.71, 150.92, 153.97, 168.66,
derivative was synthesized from O-benzyl-L-threonine. Aer 172.87. IR (neat, cmꢁ1): 3030, 1702, 1640, 1520, 1490, 1445,
purifying the crude product with column chromatography, 2c 1293, 1214, 1028, 750, 698. Anal. calcd for C30H26N2O5: C, 72.86;
1
was obtained as colorless oil in 75% yield. H NMR (400 MHz, H, 5.30; N, 5.66%. Found: C, 72.61; H, 5.37; N, 5.58%.
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Paper
Other diblock copolymers were also prepared by same
procedure using urethane derivative 2b or 2e. All polymers were
collected as white powder. The physical data is listed below.
Synthesis of polypeptides by polycondensation of urethane
derivatives
Typical procedure: urethane derivatives of O-benzyl-L-serine 2a
(315 mg, 1 mmol) was dissolved in anhydrous DMAc (2 mL), and
the solution was added into a Schlenk tube. Aer the addition of
n-BuNH2–DMAc solution (25 ml, 2 ꢂ 10ꢁ3 mmol mLꢁ1), the
resulting mixture was stirred at 60 ꢀC for 24 h under argon
atmosphere. The reaction mixture was cooled to room temper-
ature, and poured into diethyl ether (100 mL). Precipitates were
isolated by the ltration and then dried under vacuum to yield
138 mg (78%) of P2a as white powder. 1H NMR (400 MHz, TFA-
d, d, ppm): 3.50–4.03 (br, 2H), 4.28–4.62 (br, 2H), 4.74–4.96 (br,
1H), 6.93–7.34 (br, 5H). IR (neat, cmꢁ1): 3281, 2860, 1628, 1506,
1452, 1100, 732, 693.
1
PEG-b-poly(S-benzyl-L-cysteine). H NMR (400 MHz, DMSO-
d6, d, ppm): 2.48–2.85 (br, 2H), 3.49 (s, 18H), 3.64–3.78 (br, 2H),
4.55–4.75 (br, 1H), 7.13–7.33 (br, 5H), 8.26–8.60 (br 1H). IR
(neat, cmꢁ1): 3268, 2876, 1624, 1520, 1341, 1100, 961, 841, 697.
PEG-b-poly(N-trityl-L-glutamine). 1H NMR (400 MHz, DMSO-
d6, d, ppm): 1.43–1.97 (br, 2H), 2.10–2.43 (br, 2H), 3.49 (s, 18H),
4.05–4.34 (br, 1H), 7.02–7.43 (br, 15H), 7.72–8.10 (br, 1H), 8.39–
8.74 (br, 1H). IR (neat, cmꢁ1): 3307, 2865, 1651, 1489, 1446,
1095, 752, 697.
Acknowledgements
Other polypeptides (P2b and P2d) were obtained by similar
procedure as white powder. For the polycondensation of 2c, 2e,
and 2f, the reaction mixture was poured into water–methanol
mixture (vol. 8 : 2) with stirring, and white precipitates were
collected by ltration. The physical data is listed below.
This work was nancially supported by JSR Corporation. High-
resolution mass spectrometry (HRMS) experiment was per-
formed under the Cooperative Research Program of “Network
Joint Research Center for Materials and Devices”.
1
P2b. H NMR (400 MHz, TFA-d, d, ppm): 2.59–2.99 (br, 2H),
3.65 (s, 2H), 4.35–4.63 (br, 1H), 7.02–7.15 (br, 5H). IR (neat,
cmꢁ1): 3269, 3026, 2914, 1623, 1513, 1492, 1229, 694.
1
References
P2c. H NMR (400 MHz, CDCl3, d, ppm): 0.70–1.35 (br, 3H),
3.61–4.20 (br, 1H), 4.25–4.86 (br, 3H), 7.05–7.35 (br, 5H). IR
(neat, cmꢁ1): 3286, 2975, 2868, 1633, 1494, 1452, 1379, 1345,
1208, 1091, 733, 694.
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P2d. H NMR (400 MHz, TFA-d, d, ppm): 2.71–3.10 (br, 2H),
4.62–5.11 (m, 3H), 6.70–7.40 (m, 9H). IR (neat, cmꢁ1): 3267,
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2H), 2.27 (br, 2H), 4.19 (br, 1H), 7.05–7.36 (br 15H), 7.50–7.83
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2H), 3.96–4.83 (br, 1H), 6.90–7.34 (br, 15H), 7.41 (s, 1H), 7.90–
8.82 (br, 1H). IR (neat, cmꢁ1): 3313, 3055, 1667, 1489, 749, 698,
633.
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29896 | RSC Adv., 2014, 4, 29890–29896
This journal is © The Royal Society of Chemistry 2014