2-(4-Sulfophenylsulfonyl)ethoxycarbonyl group: A new water-soluble N-protecting group and its application to solid phase peptide synthesis in water

Article abstract of DOI:10.1016/j.tetlet.2004.10.095

Solid phase peptide synthesis is carried out in organic solvents, creating environmental problems after disposal. To avoid this problem, we aimed to perform solid phase peptide synthesis in water. A new water-soluble N-protecting group, 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps) group, was designed and Sps-amino acids were prepared. To evaluate the utility of this technique, Leu-enkephalin amide was prepared by solid phase synthesis using Sps-amino acids in water.

Full text of DOI:10.1016/j.tetlet.2004.10.095

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 9293–9295
2-(4-Sulfophenylsulfonyl)ethoxycarbonyl group: a new
water-soluble N-protecting group and its application to
solid phase peptide synthesis in water
Keiko Hojo, Mitsuko Maeda and Koichi Kawasaki*
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan
Received 24 September 2004; revised 6 October 2004; accepted 8 October 2004
Abstract—Solid phase peptide synthesis is carried out in organic solvents, creating environmental problems after disposal. To avoid
this problem, we aimed to perform solid phase peptide synthesis in water. A new water-soluble N-protecting group, 2-(4-sulfo-
phenylsulfonyl)ethoxycarbonyl (Sps) group, was designed and Sps-amino acids were prepared. To evaluate the utility of this tech-
nique, Leu-enkephalin amide was prepared by solid phase synthesis using Sps-amino acids in water.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Solid phase synthetic methods have made peptide syn-
thesis simple, rapid, and easily subject to automation.
However this procedure requires a large amount of
organic solvents, raising environmental concerns over
disposal. In this report we have investigated how to
perform peptide synthesis in water. To carry out peptide
synthesis in water, water-soluble protected amino acids
are needed. Various water-soluble N-protecting groups
have been reported, including the methylsulfonylethoxy-
carbonyl group described by Tesser and Balvert-Geers,1
and the 2-(triphenylphosphonio)ethoxycarbonyl,2 2-(tri-
phenylphosphonio)isopropyloxycarbonyl,3 and 2-(4-
pyridyl)ethoxycarbonyl4 groups reported by Kunz
et al. Kunz has prepared a tripeptide, 2-[diphen-
yl(methyl)phosphonio]ethoxycarbonyl-Leu-Phe-Phe-O-
t-Bu by a solution method in water.2 Kunz also reported
that the 2-(methylthio)ethoxycarbonyl group could be
removed under mild basic conditions after treatment
with methyl iodide.5 We previously reported the prepa-
ration of water-soluble N-protected amino acids,
2-[phenyl(methyl)sulfonio]ethoxycarbonylamino acids
(Pms-amino acids)6 and 2-ethanesulfonylethoxycarbon-
ylamino acids (Esc-amino acids)7 and their application
to solid phase peptide synthesis in water. We also re-
ported the preparation of water-soluble active esters,
4-sulfophenyl ester derivatives.8 Here, we have designed
a new water-soluble N-protecting group, 2-(4-sulfo-
phenylsulfonyl)ethoxycarbonyl (Sps) group, based on
this water-soluble active ester (Fig. 1).
Sps-amino acids were prepared as shown in Figure 2. 2-
Phenylthioethanol (1) was reacted with 4-nitrophenyl
chloroformate to form 2-phenylthioethyl 4-nitrophenyl
carbonate (Pte-ONp, 2),9a which was then subjected to
sulfonation to give 2-(4-sulfophenylthio)ethyl 4-nitro-
phenyl carbonate (Spt-ONp, 3).9b 3 was converted to
its sodium salt and then reacted with an amino acid to
form 2-(4-sulfophenylthio)ethoxycarbonylamino acid
(Spt-amino acid), which was then subjected to oxidation
with hydrogen peroxide/trifluoroacetic acid to form Sps-
amino acid (Route 1). Since methionine and cysteine
contain sulfur, Sps-derivative of these amino acids
cannot be obtained via Route 1. Alternatively, 3 was
oxidized to form 2-(4-sulfophenylsulfonyl)ethyloxycar-
bonyl 4-nitrophenyl carbonate (6)9c which was con-
verted to its sodium salt, and then coupled with an
amino acid to give the Sps-amino acid (Route 2).
NaO3S
NaO3S
O
O
O
S
O
BF4
S
O
O
H3C
Keywords: 2-(4-Sulfophenylsulfonyl)ethoxycarbonyl group; Water-sol-
uble N-protecting group; Peptide synthesis in water.
Pms group
4-sulfophenyl group
Figure 1. Pms-,6 4-sulfophenyl-8 and Sps-groups.
Sps group
*
Corresponding author. Tel.: +81 78 974 4794; fax: +81 78 974
5689; e-mail: kawasaki@pharm.kobegakuin.ac.jp
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.10.095

9294
K. Hojo et al. / Tetrahedron Letters 45 (2004) 9293–9295
NO2
NO2
O
O
S
S
Cl
O
O
O
OH
Et3N
2 : Pte-ONp
1
1) conc. H2SO4 / Ac2O
2) NaCl
Route 1
COOH
R
NO2
O
R
O
S
H2N
S
O
N
H
COOH
O
O
Et3N
NaO3S
4
3 : Spt-ONp
NaO3S
NaO3S
1) H2O2-TFA
2) NaCl
1) H2O2-TFA
2) NaCl
Route 2
R
NaO3S
NO2
O
R
O
O
O
O
S
O
H2N
COOH
S
O
O
N
H
COOH
O
6 : Sps-ONp
5 : Sps-amino acid
N
Figure 2. Synthetic scheme for Sps-amino acids.
Sps-Phe-OH,10a Sps-Gly-OH,10b and Sps-Leu-OH10c
were prepared via Route 1, and Sps-Tyr(t-Bu)-OH10d
was prepared via Route 2. All synthetic Sps-amino acids
and their corresponding sodium salts are soluble in
water. The sodium salts are stable crystalline solids.
We previously reported the preparation of water-soluble
N-protected amino acids, Pms-6 and Esc-amino acids,7
and their application to solid phase peptide synthesis
in water. However, Pms-amino acids were rather unsta-
ble due to their onium form and Esc-amino acids (except
Esc aromatic amino acids) were not detectable by meas-
urement of optical absorption. In contrast, the Sps
group is stable and can be detected by UV absorption.
Therefore Sps-amino acids display characteristics suit-
able for automatic solid phase peptide synthesis in water.
100
80
60
40
20
0
5.0% NaHCO3 aq.
5.0% Na2CO3 aq.
0.025 mol/L NaOH
(50% EtOH aq.)
0
25
50
75
100
time (min)
Figure 3. Deprotection of Sps-Phe-OH. Sps-Phe-OH (4.8mg, 10lmol)
was dissolved in aqueous 5.0% NaHCO3 (400lL) or aqueous 5.0%
Na2CO3 or aqueous 0.025M NaOH and the solution was stirred at
room temperature. An aliquot (10lL) was taken periodically for
analysis by HPLC. The rate of deprotection was calculated by
measurement of the peak area of Sps-Phe-OH.
The Sps group can be removed by treatment with a mild
base, such as aqueous 5% Na2CO3, in water. Deprotec-
tion of Sps-amino acids was tested by treating Sps-Phe-
OH either with aqueous 5% Na2CO3 or aqueous 5%
NaHCO3 or aqueous 0.025M NaOH at room tempera-
ture, as shown in Figure 3. The Sps group on Phe was
totally removed within 5min by treatment with 5%
Na2CO3 or 0.025M NaOH, whereas removal was
incomplete using 5% NaHCO3 even after 40min.
Tyr(t-Bu)-Gly-Gly-Phe-Leu-TentaGel resin was treated
with trifluoroacetic acid to cleave the peptide from the
resin. The liberated peptide was purified by HPLC using
To evaluate the utility of Sps-amino acids, Leu-enkeph-
alin amide (H-Tyr-Gly-Gly-Phe-Leu-NH2) was pre-
pared according to the protocol shown in Table 1 on a
TentaGel resin [poly(ethylene glycol) grafted polysty-
rene resin]11 that swelled well in water. Sps-amino acids
were used as their sodium salt and coupling reactions
were performed with a water-soluble carbodiimide
[WSCD, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide
hydrochloride]12 in the presence of N-hydroxy-5-nor-
bornene-2,3-dicarboximide (HONB)13 to accelerate the
reaction. Aqueous 0.2% Triton X solution was used as
a solvent to increase swelling ability of the resin and sol-
ubility of Sps-amino acids. Removal of the Sps protec-
tion group was performed with 0.025M NaOH in
aqueous 50% ethanol for 3min(· 2). The synthetic H-
Table 1. Synthetic protocol for the solid phase synthesis of Leu-
enkephalin amide in water
Step
Reagents
Time
1
2
Wash
Deprotection
H2O
3min · 2
3min · 2
0.025M NaOH/aq
50% EtOH
H2O
3
4
5
Wash
Wash
3min · 2
3min · 3
Aq 0.2% Triton X
Coupling reaction Sps-amino acid, WSCD, 2h · 2
HONB, DIEA in aq
0.2% Triton X
6
Wash
Aq 0.2% Triton X
3min · 3
Four times molar quantity of each reagent (Sps-amino acid, WSC,
DIEA and HONB) calculated from amino content of the used resin
was used.

K. Hojo et al. / Tetrahedron Letters 45 (2004) 9293–9295
9295
8. Kawasaki, K.; Tsuji, T.; Maeda, M.; Matsumoto, T.;
Hirase, K. Chem. Pharm. Bull. 1987, 35, 1044–1048.
9. (a) Pte-ONp: Yield 85%. mp 43–45ꢁC. 1H NMR d
(CD3OD): 8.29 (2H, d-like, J = 9.3Hz), 7.44 (2H, d-like,
J = 7.1Hz), 7.43 (2H, d-like, J = 9.3Hz), 7.31 (2H, dd, J =
7.3, 7.1Hz), 7.22 (1H, t, J = 7.3), 4.40 82H, t, J = 6.7Hz),
3.28 (2H, t, J = 6.7Hz). ESI-Mass m/z: Calcd for
C15H17N2O5S ([M + NH4]+) 337.09, Found: 337.1.
(b) Spt-ONp: Yield: 79.7%. mp 193–194ꢁC (dec.) amor-
phous material. 1H NMR
d (CD3OD): 8.30 (2H,
d, J = 9.3Hz), 7.77 (2H, d, J = 8.4Hz), 7.47 (2H, d,
J = 8.3Hz), 7.43 (2H, d, J = 9.3Hz), 4.45 (2H, t, J =
6.6Hz), 3.70 (2H, t, J = 6.6Hz), TOF-MS m/z: Calcd
C15H12NO8S2 ([M ꢀ H]ꢀ) 398.39. Found: 398.48.
(c) Sps-ONp: Yield: 80.3%. mp 171–174ꢁC (dec.). 1H
NMR d (CD3OD): 8.29 (2H, d, J = 8.2Hz), 8.15 (2H, m),
8.03 (2H, m), 7.31 (d, J = 8.2Hz), 4.70 (2H, t-like), 3.96
(2H, t-like). TOF-MS m/z: Calcd C15H12NO10S2
([M ꢀ H]ꢀ) 430.40. Found: 430.57.
Figure 4. HPLC profiles of synthetic Leu-enkephalin amide. (A) Crude
Leu-enkephalin amide. Column, DAISOPAK SP-120-5-ODS-B
(20 · 250mm). Flow rate, 10mL/min. Eluent, CH3CN/H2O containing
0.05% TFA. Gradient: 10/90–50/50 (40min). (B) Analytical HPLC of
purified sample. Column, DAISOPAK SP-120-5-ODS-B (2.5 ·
250mm). Flow rate, 1mL/min. Eluent, CH3CN/H2O containing
0.05% TFA. Gradient: 10/90–50/50 (40min). OD at 220nm.
26
10. (a) Sps-Phe-OH: Yield 77%. mp 242–245ꢁC (dec.). ½aꢁD
1
ꢀ5.2 (c = 1.0, H2O). H NMR d (CD3OD): 8.04 (2H, dd,
J = 8.5, 1.1Hz), 7.95 (2H, dd, J = 8.5, 1.1Hz), 7.23 (5H,
m), 4.34 (1H, m), 4.28 (1H, m), 4.28 (1H, m), 3.25 (2H, t-
like), 3.25 (1H, m), 2.90 (1H, m). TOF-MS m/z: Calcd for
C18H18NO9S2 ([M ꢀ H]ꢀ) 456.48. Found: 456.37.
(b) Sps-Gly-OH: Yield 68%. mp 219–222ꢁC (dec.). 1H
NMR d (CD3OD): 8.04 (2H, d, J = 8.6Hz), 8.00 (2H, d,
J = 8.6Hz), 4.38 (2H, t, J = 5.7Hz), 3.70 (2H, s), 3.64 (2H,
t, J = 5.7Hz). TOF-MS m/z: Calcd for C11H12NO9S2
([M ꢀ H]ꢀ) 366.35. Found: 366.24.
an ODS column. The yield of synthetic Leu-enkephalin
amide14 was 61% as calculated from the amino group
content of the starting resin (Fig. 4).
In conclusion, we have designed a new water-soluble
protecting group, the Sps group, and verified its utility
by successful solid phase synthesis of Leu-enkephalin
amide in water. We have compared the Sps group with
our previously reported water-soluble N-protecting
groups. The Sps group is more stable than the Pms
group and the UV absorption properties are preferable
to those of the Esc group for detection. Further work
should aim to develop application of Sps-amino acids
to preparation of large peptides.
26
(c) Sps-Leu-OH: Yield 70%. mp 238–242ꢁC (dec.). ½aꢁD
1
ꢀ22.7 (c = 1.0, H2O). H NMR d (CD3OD): 8.05 (2H, d,
J = 8.2Hz), 8.01 (2H, d, J = 8.2Hz), 4.37 (2H, m), 4.10
(1H, m), 3.62 (2H, t, J = 5.8Hz), 1.67 (1H, m), 1.55 (1H,
m), 0.92 (6H, m). TOF-MS m/z: Calcd for C15H20NO9S2
([M ꢀ H]ꢀ) 422.46. Found: 422.29.
(d) Sps-Tyr(t-Bu)-OH: Yield 68%. mp 248–250ꢁC (dec.).
26
½aꢁD ꢀ5.6 (c = 1.0, H2O). 1H NMR d : 8.04 (1H, dd,
J = 8.4, 1.1Hz), 7.95 (2H, d, J = 8.4Hz), 7.03 (2H, J = 8.5,
1.1Hz), 6.71 (2H, J = 8.5, 1.1Hz), 4.26 (1H, m), 4.24 (2H,
m), 3.55 (2H, m), 3.03 (1H, m), 2.80 (1H, m), 1.25 (9H, m).
TOF-MS m/z: Calcd for C22H26NO10S2 ([M ꢀ H]ꢀ)
528.59. Found: 528.56.
References and notes
1. Tesser, G. I.; Balvert-Geers, I. C. Int. J. Pept. Protein Res.
1975, 7, 295–305.
2. Kunz, H. Chem. Ber. 1976, 109, 2670–2683.
3. Kunz, H.; Schaumloffel, G. Liebigs Ann. Chem. 1985,
1784–1793.
4. Kunz, H.; Birnbach, S. Tetrahedron Lett. 1984, 25, 3567–
3570.
5. Kunz, H. Chem. Ber. 1976, 109, 3693–3706.
6. Hojo, K.; Maeda, M.; Kawasaki, K. J. Pept. Sci. 2001, 7,
615–618.
11. Bayer, E.; Rapp, W. In Chem. Peptides and Proteins;
Volter, E., Bayer, E., Ovchinikov, Y. A., Ivanov, V. T.,
¨
Eds.; Walter de Gruyter: Berlin, 1986; pp 3–8.
12. Sheehan, J.; Cruickshunk, P. A.; Boshrt, G. L. J. Org.
Chem. 1961, 26, 2525–2528.
13. Fujino, M.; Kobayashi, S.; Obayashi, M.; Fukuda, T.;
Shinagawa, S.; Nishimura, O. Chem. Pharm. Bull. 1974,
22, 1857–1863.
24
14. ½aꢁD +8.2 (c = 0.2, H2O). TOF-MS m/z 577.0 ([M + Na]+,
7. Hojo, K.; Maeda, M.; Smith, T. J.; Kita, E.; Yamaguchi,
F.; Yamamoto, S.; Kawasaki, K. Chem. Pharm. Bull.
2004, 52, 422–427.
C28H39N6O6Na requires 577.64). Amino acid ratios in an
acid hydrolysate: Tyr, 0.98; Gly, 2.00, Phe, 0.96; Leu, 1.01.
(average recovery: 88%).