Phosgene-free synthesis of polypeptides: Useful synthesis for hydrophobic polypeptides through polycondensation of activated urethane derivatives of α-amino acids

Article abstract of DOI:10.1002/pola.26775

We report a useful synthetic method of polypeptides using a series of urethane derivative of α-amino acids (l-leucine, l-phenylalanine, l-valine, l-alanine, l-isoleucine, l-methionine), which are readily synthesized by N-carbamoylation of tetrabutylammoni

Full text of DOI:10.1002/pola.26775

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Phosgene-Free Synthesis of Polypeptides: Useful Synthesis for
Hydrophobic Polypeptides through Polycondensation of Activated
Urethane Derivatives of a-Amino Acids
Shuhei Yamada, Koichi Koga, Atsushi Sudo, Mitsuaki Goto, Takeshi Endo
Molecular Engineering Institute, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan
Correspondence to: T. Endo (E-mail: tendo@moleng.fuk.kindai.ac.jp)
Received 15 February 2013; accepted 7 May 2013; published online
DOI: 10.1002/pola.26775
ABSTRACT: We report a useful synthetic method of polypep-
tides using a series of urethane derivative of a-amino acids (L-
leucine, L-phenylalanine, L-valine, L-alanine, L-isoleucine, L-
methionine), which are readily synthesized by N-carbamoyla-
tion of tetrabutylammonium salts of a-amino acids with
diphenyl carbonate. Heating these urethane derivatives in N,N-
dimethylacetamide in the presence of n-butylamine success-
fully gave the corresponding polypeptides with well-defined
structures through polycondensation with the elimination of
phenol and CO2. The matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry investigation
showed that the resulting polypeptides had an n-BuNH2-incor-
porated initiating end and an amino group at propagating end.
These results strongly indicated that primary amines served as
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an initiator in this polycondensation system. 2013 Wiley Peri-
odicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 00, 000–
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KEYWORDS: amino acid; monomers; polycondensation; poly-
peptide; ring-opening polymerization
used N,N0-carbonyldiimidazole for the polycondensation of a-
amino acids in aqueous solution.7 They proposed the in-situ
formation of NCAs through intramolecular cyclization of N-
(imidazolylcarbonyl) amino acids. Krichelforf et al. reported
the utilization of a more stable compound, a-(N-aryloxycar-
bonyl)amino-x-carboxylalkane, as a monomer for polyamide
synthesis.8 The proposed reaction mechanism involved the
thermally induced elimination of ArOH from the urethane
moiety to produce a-isocyanato-x-carboxylalkane, which
underwent the polyaddition with the elimination of CO2.
INTRODUCTION Synthetic polypeptides have been widely
used as important and essential components to design drug
delivery system1 and tissue engineering,2 due to their desira-
ble properties such as biocompatibility and biodegradability.
For their applications, most polypeptides have been prepared
by the ring-opening polymerization of a-amino acid N-car-
boxyanhydrides (NCAs). Polymerization of NCAs is generally
initiated by various nucleophiles, such as water, alcohols, or
primary amines. The initiation with primary amines is the
most frequently used one to construct various architectures
including block copolymers,3 star-shaped polymers,4 and
hyperbranched polymers.5 Furthermore, the remarkable fea-
ture of their polymerization is that molecular weight of poly-
peptides is precisely controlled by varying the feed ratio
between monomers and initiators. However, in spite of the
aforementioned advantages of NCAs, the sensitive nature of
NCAs to moisture and heat has limited their use to the
small-scale synthesis of polypeptides
We have previously reported a useful synthetic method for
NCAs and polypeptides via intramolecular cyclization of N-
aryloxycarbonyla-amino acids into corresponding NCAs.9
Among their activated urethane-type derivatives of a-amino
acids, N-(phenyloxycarbonyl) amino acids would be the most
promising monomers toward practical use of polypeptide-
based functional materials, because of easy handling of ure-
thane derivatives and its simple procedure for polypeptides
synthesis without the use and production of any toxic com-
pounds. Recently, we showed polypeptides synthesis of poly-
L-lysine with well-defined terminal structure using these ure-
thane derivatives of amino acid in the presence of amine,
and approaching for the synthesis of block copolypeptides.10
In this article, we demonstrate a facile polypeptide synthesis
from various amino acids with hydrophobic side chains
Development of alternative procedures for polypeptide syn-
thesis using more accessible monomers is a great challenge
in this research area. To date, some researchers have
reported various useful methods for polypeptide synthesis.
Buess et al. developed a-carboxy hydroxamic acids that rear-
ranged into isocyanate carboxylic acids with a potential
capability to give corresponding polypeptides.6 Orgel et al.
Additional Supporting Information may be found in the online version of this article.
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SCHEME 1 Synthetic procedure for activated urethane derivatives of a-amino acids (1a–1f).
(L-leucine, L-phenylalanine, L-valine, L-alanine, L-isoleucine,
L-methionine) to expand the scope of our polycondensation
method with using activated urethane derivatives.
acceleration effect of n-butylamine was observed also in the
previous study on the synthesis of poly-L-lysine.10 Therefore,
under same conditions, the polycondensation of urethane
derivatives 1b–1f were performed. The results are summar-
ized in Table 2. During the polycondensation of 1a–1f, precip-
itation or gelation was observed to suggest the formation of
polypeptides in a b-sheet structure, which are generally insol-
uble in solvents. After pouring the reaction mixture into
diethyl ether, the polypeptides were isolated as white pow-
ders in high yields. All the obtained polypeptides 2a–2f were
insoluble in common organic solvents, such as DMF, acetone,
THF, and CHCl3, but soluble in CF3COOH (TFA). The insolubil-
ity of the obtained polypeptides in common organic solvents
prevented the characterization with size exclusion chromatog-
raphy. For the polycondensation of L-valine derivative 1c, and
L-isoleucine derivative 1e, longer reaction times (over 50 h)
were required for their complete consumption, compared to
the other a-amino acids. The difference in polymerization
time is likely due to the difference in rate of NCA formation
that depends on steric hindrance of the substituent and the
difference in solubility of the formed polypeptide.
RESULTS AND DISCUSSION
A series of activated urethane derivatives (1a–1f) for polypep-
tide synthesis were readily prepared by N-carbamoylation of
tetrabutylammonium salts of a-amino acids with diphenyl car-
bonate (DPC) in acetonitrile, according to the method previ-
ously reported (Scheme 1).10 As shown in Table 1, after
purification with column chromatography and/or recrystalliza-
tion, all urethane derivatives were successfully obtained in rel-
atively high yields. These urethane derivatives are stable so
that they could be stored at room temperature for several
months without any caution to moisture and heat. The molec-
ular structures of the obtained urethane derivatives were
1
identified by H NMR, 13C NMR, and FTIR analyses.
In the previous report, we described that the activated ure-
thane derivative of L-lysine efficiently gave polypeptide
through the in situ formation of NCA and subsequent ring-
opening polymerization in high polar solvents such as N,N-
dimethylacetamide (DMAc), along with releasing phenol and
carbon dioxide.10 Furthermore, the addition of n-butylamine
to the system gave the corresponding polypeptides with n-
butylamine-incorporated initiating end (Scheme 2). Accord-
ingly, we first investigated the polycondensation behavior of
the urethane derivatives of L-leucine 1a by heating their
DMAc solution (0.5 M) at 60 ꢀC in the absence of n-butyla-
mine under argon atmosphere (entry 1 in Table 1). Aliquots
of the reaction mixture were taken for monitoring the forma-
Finally, we performed matrix-assisted laser desorption/ioni-
zation time-of-flight mass spectrometry (MALDI-TOF MS)
analysis of the polypeptide obtained by the polymerization
from urethane derivatives in the presence of n-butylamine to
confirm their molecular weight and the terminal structure.
For the analysis, the polypeptide was dissolved in TFA, and
mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and sodium
trifluoroacetate (NaTFA) as a matrix and a cationization
agent, respectively. The MALDI-TOF MS spectrum of poly-L-
valine 2c is shown in Figure 2 (for the spectra of other
1
tion of phenol by H NMR spectroscopy. After heating for 48
h, a small amount of precipitate was formed from the reaction
mixture to suggest the formation of the target polypeptide.
The resulting product was obtained as ether-insoluble parts
TABLE 1 Synthesis of Urethane Derivatives from N-Carbamoy-
lation of Tetrabutylammonium Salts of a-Amino Acids with
Diphenyl Carbonates (DPC)
1
(yield 5 10%). The H NMR spectrum measured at this point
revealed low consumption of 1a (conversion 5 35%). Its
FTIR spectrum (Fig. 1) indicated two characteristic peaks to
amide linkage at 1648 and 1536 cm21, which are attributed
to C@O stretching and NAH bending vibrations. Next, the
effect of n-butylamine on this polycondensation system was
investigated. In the presence of n-butylamine (2 mol %), the
consumption of 1a was significantly accelerated. 1H NMR
analysis showed the complete consumption of the urethane
derivative within 28 h (entry 2 in Table 1) and the corre-
sponding polypeptide was obtained in high yield (89%). This
Urethane
Entries
a-Amino Acid
Derivative
Yield (%)
1
2
3
4
5
6
Leucine
1a
1b
1c
1d
1e
1f
70
60
58
62
65
75
Phenylalanine
Valine
Alanine
Isoleucine
Methionine
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SCHEME 2 Polypeptides synthesis from urethane derivatives of a-amino acids in the presence of n-butylamine.
polypeptides see the Supporting Information). These signals
were regularly located with an interval of 99 Da, which cor-
responds to the formula weight of L-valine, and polymeriza-
tion degree of 2c was confirmed to be distributed in the
wide range from 10 to 40, presumably, due to the precipita-
tion of polypeptides during the polycondensation. Further-
more, these mass numbers give close agreement with Na1-
cationized polymer structure, possessing n-butylamine-incor-
porated initiating end and –NH2 group at propagating end.
These polypeptide structures were completely similar to that
synthesized by butylamine-initiated ring-opening polymeriza-
tion of NCAs. In addition, 1H NMR spectrum of 2c (TFA-d1)
also provided the evidence for the existence of the butyla-
mine reside on the terminal of polypeptide (Fig. 3). The
butylamine residue was identified in the spectrum at 1.40,
1.61, and 3.45 ppm. These results strongly indicated n-butyl-
amines serves as initiator in this polycondensation system.
Thus, this polycondensation system would be a more facile
alternative approach for synthesis of polypeptides by the ini-
tiation with amino groups. With this useful synthetic method
for polypeptides synthesis, development of biocompatible
materials is now in progress.
received without further purification, unless stated other-
wise. Butylamine (n-BuNH2) and N,N-dimethylacetamide
(DMAc) were purified by distillation over calcium hydride
(CaH2), before use. 1H and 13C NMR spectra were recorded
with a JEOL ECS-400 (400 MHz) spectrometer, and chemical
shifts were recorded in ppm units using tetramethylsilane as
an internal standard. Fourier transform infrared spectros-
copy (FTIR) analysis was recorded on Thermo Scientific
Nicolet iS10 over a range from 600 to 4000 cm21. MALDI-
TOF MS analysis was performed on a PerSeptive Biosystems
Voyager DE Pro Bio Spectrometry workstation. The MALDI-
TOF MS samples were prepared using 2,5-DHB and NaTFA,
as a matrix material and a cationization agent, respectively.
The polypeptides in TFA solution (5 mg mL21) was mixed
with 2,5-DHB/THF solution (10 mg mL21) and NaTFA/THF
solution (1 mg mL21) at a ratio of 1:2:1. The final mixed sol-
utions were deposited onto a sample plate and allowed to
air dry. The resulting samples were irradiated with 337 nm
of nitrogen laser, and detected on positive mode at 20 kV.
Melting point was measured on Bibby Stuart scientific melt-
ing point apparatus SMP3.
CONCLUSIONS
In this article, we proved that a series of urethane derivative
of a-amino acids (L-leucine, L-phenylalanine, L-valine, L-alanine,
L-isoleucine, L-methionine) has a potential to be used as mono-
mers to facile synthesis of polypeptides with well-defined
structure. These urethane derivatives were readily synthesized
by two-step reactions, that is, (1) transformation of a-amino
acids into the corresponding ammonium salts and (2) N-carba-
moylation of the salts with DPC, and could be stored without
any signs of decomposition for several months at room tem-
perature. The polycondensation of these urethane derivatives
was successfully carried out by heating them in DMAc in the
presence of n-butylamine. The MALDI-TOF MS investigation
showed that the resulting polypeptides have n-BuNH2-incorpo-
rated initiating end and ANH2 group at propagating end,
implying that primary amines can serve as an initiator in this
polycondensation system. Therefore, this polycondensation sys-
tem would be conveniently used as a synthetic method of
polypeptides with well-defined terminal structure.
EXPERIMENTAL
Materials and Instruments
All the reagents were purchased from Aldrich, Tokyo Chemi-
cal Industry, and Watanabe Chemical Industry, and used as
FIGURE 1 FT-IR spectrum of ether-insoluble part obtained by
polycondensation of urethane derivatives 1a (entry 1 in Table )
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TABLE 2 Synthesis of Polypeptides from Urethane Derivatives (1a–1f) in the Presence of n-Butylamine (2 mol %)a
a-Amino
Urethane
Derivative
Reaction
Time (h)
Entries
Acid
Polypeptide
Conv. (%)c
Yield (%)d
1b
2
Leucine
1a
1a
1b
1c
1d
1e
1f
2a
2a
2b
2c
2d
2e
2f
48
28
12
50
36
72
18
35
>99
>99
>99
>99
>99
>99
10
89
85
78
80
69
91
Leucine
3
Phenylalanine
Valine
4
5
Alanine
6
Isoleucine
Methionine
7
a
c
Polycondensation condition: Conc. 5 0.5 M, at 60 ꢀC in DMAc.
Polycondensation was performed in the absence of n-butylamine.
Calculated by 1H NMR spectra in CDCl3.
Ether-insoluble parts.
b
d
General Procedure for Synthesis of Various
Urethane Derivatives
N-Phenoxycarbonyl-L-leucine (1a)
then the reaction mixture was stirred for 3 h. The resulting
mixture was transferred into a separatory funnel containing
distilled water (30 mL) and ethyl acetate (30 mL). The aque-
ous layer was acidified to pH 2–3 with 1 M HCl and extracted
with ethyl acetate (3 3 30 mL). The organic fractions were
combined, washed with brine, dried over Na2SO4, filtrated,
and concentrated under reduced pressure. The crude products
were purified by flash column chromatography (eluting with a
gradient from 30 to 60% ethyl acetate in n-hexane), and then
recrystallization from ethyl acetate/n-hexane to give 3.5 g
(70%) of 1a as colorless oil.
To a stirred suspension of L-leucine (2.6 g, 20 mmol) in meth-
anol (30 mL), tetrabutylammonium hydroxide (37% in metha-
nol) (13.8 g, 20 mmol) was slowly added at room
temperature. After stirring for 1 h, the reaction mixture was
concentrated under reduced pressure. The resulting residues
were dissolved in acetonitrile (20 mL). The solution was
added dropwise over 10 min to a stirred solution of DPC (4.2
g, 20 mmol) in acetonitrile (25 mL) at ambient condition, and
FIGURE 2 MALDI-TOF MS spectrum of poly-L-valine 2c obtained from polycondensation of urethane derivatives in the presence of
n-butylamine (entry 4 in Table 2).
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1H NMR (400 MHz, CDCl3, d, ppm): 1.50–1.60 (m, 3H), 4.42–
4.54 (m, 1H), 5.66 (d, 1H, J 5 7.6 Hz), 7.08–7.15 (m, 2H),
7.16–7.24 (m, 1H), 7.31–7.39 (m, 2H). 13C NMR (100 MHz,
CDCl3, d, ppm): 18.47, 49.76, 121.67, 125.71, 129.46, 150.78,
154.27, 177.73. IR (neat, cm21): 3297, 2995, 1696, 1524,
1491, 1240, 1211, 1175, 1157, 687.
N-Phenoxycarbonyl-L-isoleucine (1e)
The urethane derivative 1e was prepared from L-isoleucine,
according to the procedure described above and was
obtained as colorless oil in 65% yield.
1H NMR (400 MHz, CDCl3, d, ppm): 0.90–1.09 (m, 6H), 1.19–
1.35 (m, 1H), 1.46–1.58 (m, 1H), 1.96–2.08 (m, 1H), 4.43
(dd, 1H, J 5 8.8 Hz, J 5 4.5 Hz), 5.64 (d, 1H, J 5 8.8 Hz),
7.06–7.25 (m, 3H), 7.29–7.39 (m, 2H). 13C NMR (100 MHz,
CDCl3, d, ppm): 11.74, 15.64, 24.99, 37.94, 58.50, 121.62,
125.62, 129.41, 150.88, 154.71, 176.89. IR (neat, cm21):
2964, 1707, 1523, 1487, 1384, 1200, 1149, 1023, 758, 686.
N-Phenoxycarbonyl-L-methionine (1f)
The urethane derivative 1f was prepared from L-methionine,
according to the procedure described above andꢀ was
obtained as white powder in 75% yield, mp: 69.5–70.3 C.
FIGURE 3 1H NMR spectrum of poly-L-valine 2c in TFA-d.
1H NMR (400 MHz, CDCl3, d, ppm): 0.86–1.09 (m, 6H), 1.55–
1.91 (m, 3H), 4.37–4.51 (m, 1H), 5.60 (d, 1H, J 5 8.6 Hz),
7.06–7.25 (m, 3H), 7.28–7.39 (m, 2H). 13C NMR (100 MHz,
CDCl3, d, ppm): 21.80, 22.49, 24.92, 41.50, 52.66, 121.66,
125.65, 129.41, 150.83, 154.70, 177.94. IR (neat, cm21):
3309, 2958, 1710, 1527, 1488, 1203, 1155, 1024, 686.
1H NMR (400 MHz, CDCl3, d, ppm): 2.02–2.18 (m, 4H), 2.19–
2.31 (m, 1H), 2.55–2.69 (m, 2H), 4.58 (m, 1H), 5.85 (d, 1H, J
5 8.1 Hz), 7.08–7.15 (m, 2H), 7.17–7.23 (m, 1H), 7.31–7.40
(m, 2H). 13C NMR (100 MHz, CDCl3, d, ppm): 15.52, 30.04,
31.47, 53.29, 121.62, 125.75, 129.46, 150.75, 154.62, 176.57.
IR (neat, cm21): 3330, 2911, 1701, 1520, 1490, 1421, 1286,
1239, 1210, 1186, 1025, 777, 685.
N-Phenoxycarbonyl-L-phenylalanine (1b)
The urethane derivative 1b was prepared from L-phenylala-
nine, according to the procedure described above and was
Synthesis of Polypeptides by Polycondensation
of Urethane Derivatives
ꢀ
obtained as white powder in 60% yield, mp: 102.3–103.0 C.
1H NMR (400 MHz, CDCl3, d, ppm): 3.00–3.35 (m, 2H), 4.73
(m, 1H), 5.51 (d, 1H, J 5 8.2 Hz), 7.07 (d, 2H, J 5 7.7 Hz),
7.16–7.25 (m, 3H), 7.27–7.36 (m, 5H). 13C NMR (100 MHz,
CDCl3, d, ppm): 37.77, 54.84, 121.62, 125.73, 127.50, 128.90,
129.46, 129.48, 135.43, 150.74, 154.32, 176.32. IR (neat,
cm21): 3371, 3030, 1744, 1712, 1518, 1489, 1376, 1217,
1182, 751, 697.
Typical procedure is as follows: urethane derivatives of L-leu-
cine 1a (251 mg, 1.0 mmol) were dissolved in dry DMAc (2
mL), and added into a Schlenk tube. After addition of n-
BuNH2/DMAc solution (20 lL, 1.0 3 1023 mmol lL21), the
ꢀ
polycondensation was performed at 60 C under argon atmos-
phere. The reaction mixture was cooled to room temperature
and poured into diethyl ether. The precipitated polypeptides
were isolated by filtration, and then dried under vacuum to
yield 125 mg (85%), as white powder of 2a: IR (neat, cm21):
3291, 2955, 2869, 1648, 1536, 1467, 1365.
N-Phenoxycarbonyl-L-valine (1c)
The urethane derivative 1c was prepared from L-valine,
according to the procedure described above and was
obtained as colorless oil in 58% yield.
Other polypeptides were also obtained as a white powder
and the physical data of other polypeptides are listed below.
2b: IR (neat, cm21): 3282, 1659, 1630, 1519, 1494, 744,
696. 2c: IR (neat, cm21): 3269, 2961, 1628, 1547, 1390,
1226, 710. 2d: IR (neat, cm21): 3271, 2980, 2936, 1625,
1528, 1448, 1377, 1304, 1160, 1104, 1048, 964, 890. 2e: IR
(neat, cm21): 3265, 2961, 1624, 1544, 1222, 1154, 719. 2f:
IR (neat, cm21): 3280, 2912, 1645, 1536, 1433, 1282, 1111.
1H NMR (400 MHz, CDCl3, d, ppm): 0.94–1.13 (m, 6H), 2.24–
2.33 (m, 1H), 4.39 (m, 1H), 5.72 (d, 1H, J 5 9.0 Hz), 7.05–
7.22 (m, 3H), 7.31–7.38 (m, 2H). 13C NMR (100 MHz, CDCl3,
d, ppm): 17.51, 19.20, 31.25, 59.12, 121.63, 125.66, 129.44,
150.91, 154.85, 176.85. IR (neat, cm21): 3308, 2965, 1707,
1525, 1487, 1390, 1201, 1150, 760, 686.
N-Phenoxycarbonyl-L-alanine (1d)
The urethane derivative 1d was prepared from L-alanine,
according to the procedure described above and was
obtained as white powder in 62% yield, mp: 112.0–112.7 C.
ACKNOWLEDGEMENT
ꢀ
This work was financially supported by JSR Corporation.
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6 C. D. Hurd, C. M. Buess, J. Am. Chem. Soc. 1951, 73, 2409–
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