A new reagent, 2-[phenyl(methyl)sulfonio]ethyl-4-nitro-phenylcarbonate tetrafluoroborate (Pms-ONp), for preparing water-soluble N-protected amino acids

Article abstract of DOI:10.1016/S0040-4039(03)00470-2

Peptide syntheses are performed in various organic solvents, the disposal of which is an environmental problem. To avoid this problem, peptide synthesis in water using reagents of low toxicity is desirable. For peptide synthesis in water, we previously re

Full text of DOI:10.1016/S0040-4039(03)00470-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 2849–2851
A new reagent,
2
-[phenyl(methyl)sulfonio]ethyl-4-nitro-phenylcarbonate
tetrafluoroborate (Pms-ONp), for preparing water-soluble
N-protected amino acids
Keiko Hojo, Mitsuko Maeda, Yuka Takahara, Sachiko Yamamoto and Koichi Kawasaki*
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
Received 16 January 2003; revised 12 February 2003; accepted 14 February 2003
Abstract—Peptide syntheses are performed in various organic solvents, the disposal of which is an environmental problem. To
avoid this problem, peptide synthesis in water using reagents of low toxicity is desirable. For peptide synthesis in water, we
previously reported the design of a water-soluble N-protecting group, 2-[phenyl(methyl)sulfonio]ethoxycarbonyl tetrafluoroborate
(
2
Pms) group, but the introduction of this group onto sulfur-containing amino acids was problematic. Here, a new reagent,
-[phenyl(methyl)sulfonio]ethyl-4-nitrophenylcarbonate tetrafluoroborate (Pms-ONp), has been designed and used to prepare Pms
derivatives of sulfur-containing amino acids. Pms-Met was prepared and tested for the solid-phase synthesis of Met-enkephalin
amide in water using a crosslinked ethoxylate acrylate resin. © 2003 Elsevier Science Ltd. All rights reserved.
Solid-phase peptide synthesis was originally devised by
Merrifield, and its many advantages have led not only
silver tetrafluoroborate to give the Pms amino acid 4.
Pms-Met-OH and Pms-Cys-OH can not be prepared by
this method, however, because the sulfurs of Met and
Cys are converted to the onium salt by treatment with
methyl iodide.
1
to automatic peptide synthesis but also to the develop-
ment of combinatorial chemistry. The synthetic proce-
dure of solid-phase synthesis is simple but it requires a
large amount of organic solvent. Because the safe dis-
posal of organic solvents is an important environmental
issue, our aim is to perform peptide synthesis in water.
For this purpose, protected amino acids that are solu-
To prepare Pms-amino acids including Pms-Met-OH
and Pms-Cys-OH derivatives, we therefore designed the
activated acylating onium reagent, 2-[phenyl-
2
ble in water must be generated. Previously, we
(
methyl)sulfonio]ethyl-4-nitrophenylcarbonate
tetra-
reported
2-[phenyl(methyl)sulfonio]ethoxycarbonyl
fluoroborate 6, Pms-ONp. 1 was treated with methyl
iodide and silver tertafluoroborate in acetonitrile to give
(
Pms) group as a water-soluble N-protecting group.
Pms-amino acid derivatives are readily soluble in water
and the Pms group is easily removable by treatment
with mild bases. Pms amino acids were prepared
according to route A, as shown in Scheme 1. 2-
2
-[phenyl(methyl)sufonio]ethanol tertafluoroborate
5
(
yield 88%). 4-Nitrophenylchloroformate was reacted
with 5 in acetonitrile to give 6, Pms-ONp , in 71%
4
4
yield. Pms-Met-OH 8 was prepared using 6 in a mix-
(
Phenylthio)ethyl chloroformate 2 was prepared from
-(phenylthio)ethanol 1 and phosgene (prepared from
ture of 0.1% Triton X/water and acetonitrile (1/1) in the
presence of pyridine with a yield of 61%. We also
examined another synthetic route for Pms-Met-OH by
the reaction of 2-[phenyl(methyl)sulfonio]ethoxy-
carbonyl chloride tetrafluoroborate(Pms-Cl) 7, but the
preparation of 7 by the reaction of 5 and phosgene
failed because of the poor solubility of 5 in organic
solvents. In addition, 7 could not be prepared in
aqueous solvents such as aqueous acetonitrile and
aqueous tetrahydrofuran because 7 and phosgene are
unstable in aqueous media.
2
3
triphosgen) in dichloromethane. 2 was reacted with an
amino acid to give the phenylthioethoxycarbonyl (Pte)
amino acid 3, and then treated with methyl iodide and
Keywords:
2-[phenyl(methyl)sulfonio]ethyl-4-nitrophenylcarbonate
tetrafluoroborate; water-soluble N-protecting group; solid-phase syn-
thesis; Met-enkephalin amide.
*
Corresponding author. Tel.:+81-78-974-1551; fax:+81-78-974-5689;
e-mail: kawasaki@pharm.kobegakuin.ac.jp
0
040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00470-2

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K. Hojo et al. / Tetrahedron Letters 44 (2003) 2849–2851
2
Scheme 1. Synthetic scheme for Pms-ONp and Pms-amino acids. Route A: Previous synthetic scheme for Pms-amino acids.
Route B: Present synthetic scheme.
N-Pms-S-acetoamidomethylcysteine [Pms-Cys(Acm)-
Met-enkephalin amide, H-Tyr-Gly-Gly-Phe-Met-NH2.
The HPLC profile of the crude peptide is shown in
Figure 1A. The peptide was purified by preparative
HPLC. The yield calculated from the used CLEAR
4
OH] 9 and N-Pms-S-tritylcysteine [Pms-Cys(Trt)-OH]
4
1
0 were also prepared in the same manner. The yields
of 9 and 10 were 25% and 68%, respectively. Whereas 8
and 9 were readily soluble, 10 was sparingly soluble in
water. 10 was soluble in aqueous organic solvents, such
as 50% acetonitrile and 50% dimethylforamide. Thus,
the hydrophobicity of the trityl group is greater than
the hydrophlicity of the Pms group in 10.
4
resin was 29%. Minor peaks were observed before and
after the main peak of Met-enkephalin amide. The
early peaks contained deletion peptides (such as Tyr-
Gly-Phe-Met and Gly-Gly-Phe-Met), whereas the later
peaks contained non-peptide compounds that might
have derived from the used resin. For comparison,
Met-Enkephalin amide was also prepared by the solid-
phase method in water using the poly(ethylene glycol)-
grafted Rink amide resin, and the HPLC profile of the
crude product is shown in Figure 1B. Again, minor
peaks were observed before and after the main peak of
Met-enkephalin amide. The early peaks contained dele-
tion peptides (such as Tyr-Gly-Phe-Met and Gly-Gly-
Phe-Met) and the later peak at 24 min contained
Recently, a few resins that swell with water have
become commercially available. To evaluate peptide
synthesis using Pms amino acids and such resins, Met-
enkephalin amide was prepared by the solid-phase
method in water according to the protocol in Table 1.
2
Previously, we studied preparation of Leu-enkephalin
amide on a poly(ethylene glycol)-grafted Rink amide
5
resin using Pms-amino acids in water. Here we selected
a crosslinked ethoxylate acrylate resin (CLEAR, pur-
chased from Peptides International) that has been
reported to swell not only with organic solvent, but also
Table 1. Synthetic protocol for the solid phase synthesis
of Met-enkephalin amide in water
6
with water. The water-soluble carbodiimide (WSC),
1
-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydro-
Steps
Reagents
Reaction time
7
chloride, was used as a coupling reagent and N-
hydroxy-5-norbornene-2,3-dicarboximide (HONB) was
8
1
2
0.2% Triton X/H O
Pms-amino acid (4 equiv.), WSC (4
equiv.) HONB (4 equiv.) in 0.2%
3 min×6
2 h×2
2
added as an additive to accelerate the coupling reac-
tion. We used 0.5% Triton X in water as the solvent
and removed the Pms group by treatment with 0.01 N
aq. NaOH. Synthetic Pms-Tyr-Gly-Gly-Phe-Met-
CLEAR resin was treated first with 0.01 N aq. NaOH,
and then by a mixture of trifluoroacetic acid, thioan-
isole and ethanedithiol (94/3/3) to liberate the synthetic
Triton X/H O
2
3
4
5
6
0.2% Triton X/H O
3 min×6
3 min×2
3 min×2
3 min×3
2
H O
2
0.01N aq. NaOH
H O
2

K. Hojo et al. / Tetrahedron Letters 44 (2003) 2849–2851
2851
water on both the CLEAR resin and the poly(ethylene
glycol)-grafted Rink amide resin, although the yields
were lower than that of Met-enkephalin amide pre-
pared in organic solvents. We have examined the poten-
tial of the CLEAR resin and the poly(ethylene
glycol)-grafted Rink amide resin for solid-phase synthe-
sis in water, but neither resin swelled sufficiently in
water. Future work should aim to develop a new resin
that swells more than these two resins in water.
References
Figure 1. HPLC profiles of synthetic crude Met-enkephalin
amide synthesized by the solid-phase method in water. A:
Crude Met-enkephalin amide prepared on CLEAR resin. B:
Crude Met-enkephalin amide prepared on the poly(ethylene
glycol)-grafted Rink amide resin. The main peaks at 18 min in
the eluates in both A and B contained Met-enkephalin amide.
Column, DAISOPAK SP-120-5-ODS-B (4.6×250 mm). Flow
rate, 1 ml/min. Eluent, CH CN/H O containing 0.05% TFA.
1. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154.
2. Hojo, K.; Maeda, M.; Kawasaki, K. J. Peptide Sci. 2001,
7, 615–618.
3. Eckert, H.; Forster, B. Angew. Chem., Int. Ed. Engl. 1987,
26, 894–895.
4. Pms-ONp 6: mp. 134°C (decomp.), Tof-MS m/z: 334.2
+
1
(M , C H NO S requires 334.4), H NMR (400 MHz,
16
16
5
CD CN): l 8.30 (2H, d-like, J=9.3 Hz); 7.99 (2H, dd-like,
3
2
3
Gradient: 10/90“50/50 (20 min). OD at 220 nm.
J=8.5, 1.2 Hz); 7.85 (1H, tt-like, J=7.4, 1.2 Hz); 7.75
(
2H, dd-like, J=7.4, 8.5 Hz); 7.43 (2H, d-like, J=9.35
thioanisole. After purification by HPLC, the yield of
Met-enkephalin amide prepared on the poly(ethylene
glycol)-grafted Rink amide resin was 32%. The
poly(ethylene glycol)-grafted Rink amide resin swells
better than CLEAR resin in water, which might
account for the slightly higher yield of Met-enkephalin
amide prepared on the poly(ethylene glycol)-grafted
Rink amide resin. The two synthetic Met-enkephalin
amides (prepared on either type of resin) were verified
by comparison with the HPLC of authentic Met-
enkephalin amide prepared by the traditional fluorenyl-
Hz); 4.67 (1H, m); 4.45 (1H, m); 4.04 (1H, m); 3.96 (1H,
24
m); 3.32 (3H, s). Pms-Met-OH 8: [h] −33.6° (c=1.0,
D
+
CH CN), Tof-MS m/z: 344.3 (M , C H NO S requires
3
15 22
4 2
2
4
344.5). Pms-Cys(Acm)-OH 9: [h]
−20.1° (c=1.0,
D
+
CH CN), Tof-MS m/z: 387.5 (M , C H N O S requires
3
16 23
2
5 2
2
4
D
387.5). Pms-Cys(Trt)-OH 10: [h] +7.3° (c=1.0, CH CN),
3
+
Tof-MS m/z: 558.5 (M , C H NO S requires 558.7).
32
32
4 2
24
Met-enkephalin amide: [h]D −6.7° (c=0.8, 20% CH CN/
3
+
H O), Tof-MS m/z: 573.7 [(M+1) , C H N O S requires
2
27 36
6
6
572.7]. Amino acids analysis, Tyr 1.01; Gly 1.00; Phe 0.95;
Met 0.93 (average recovery 94%).
9
methoxycarbonyl (Fmoc) group-based solid-phase
5. Bayer, E.; Rapp, W. In Chemistry of Peptides and
Proteins; V o¨ lter, E.; Bayer, E.; Ovchinikov, Y. A.; Ivanov,
V. T., Eds.; Walter de Gruyter: Berlin, 1986; pp. 3–8.
6. Kempe, M.; Barany, G. J. Am. Chem. Soc. 1996, 118,
method using organic solvents. The yield of Met-
enkephalin amide prepared by the Fmoc group-based
solid-phase method was 76%.
1083–7093.
The new reagent, Pms-ONp, was designed to prepare
Pms-amino acids including sulfur-containing amino
acids. Because the reagent is a crystalline compound
and can be kept stable in a refrigerator, the preparation
of Pms-amino acids is much easier with Pms-ONp than
it is with the former method (route A, Scheme 1).
Met-Enkephalin amide was successfully synthesized in
7. Sheehan, J. C.; Cruickshank, P. A.; Boshrt, G. L. J. Org.
Chem. 1961, 26, 2525–2528.
8. Fujino, M.; Kobayashi, S.; Obayashi, M.; Fukuda, T.;
Shinagawa, S.; Nishimura, O. Chem. Pharm. Bull. 1974,
22, 1857–1863.
9. Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92,
5748–5749.