Protein & Peptide Letters, 2006, 13, 189-192 189

Solid Phase Peptide Synthesis in Water VI: Evaluation of Water-Soluble

Coupling Reagents for Solid Phase Peptide Synthesis in Aqueous Media

Keiko Hojo, Mitsuko Maeda, Naoko Tanakamaru, Kayo Mochida and Koichi Kawasaki *

Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan

Abstract: Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an
important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents
the disposal problem. Our ultimate aim is to develop an “environment-friendly” solid phase peptide synthesis (SPPS)
methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must
be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling
reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully
synthesized Leu-enkephalin amide on a solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4-
sulfophenylsulfonyl)ethoxycarbonylamino acids.

Keywords: 2-(4-Sulfophenylsulfonyl)ethoxycarbonyl group, Solid phase peptide synthesis in water, 4-(4,6-Dimethoxy-1,3,5-

triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM).

Solid phase peptide synthesis (SPPS) is a well- tertiary base and now is widely used during SPPS performed
established technique for the facile and rapid preparation of in organic solvents [9-11]. The other water-soluble in situ
peptides, but it requires large amounts of organic solvents. activating reagent, DMT-MM, is stable in water and alcohol
Since the safe disposal of organic solvents is an important [12]. We first examined the aqueous coupling of Sps-Phe-
environmental issue, the ability to synthesize peptides in an OH [4] to Leu-Rink amide-TentaGel resin mediated by
aqueous medium would be preferable. In order to do so, TNTU or DMT-MM. Previously, we found that the aqueous
water-soluble N-protected amino acids are necessary. We SPPS coupling of the two sequential hydrophobic amino
have successfully used the water-soluble N-protecting acids (i.e. Phe-Leu) of Leu-enkephaline amide occurred
groups, 2-[phenyl(methyl)sulfonio]ethoxycarbonyl (Pms) [1- slowly [1,2]. For SPPS in water, the resin must swell
3] and 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps) [4], significantly when mixed with water. Therefore, TentaGel
(Fig. 1) for the SPPS of Leu-enkephalin amide in water. For resin [poly(ethylene glycol)-grafted polystyrene resin] [18],
this paper, we focused on identifying novel coupling which swells well in various solvents, including water, was
reagents that are suitable for SPPS in water. To be useful, a used. H-Leu-Rink amide-TentaGel resin was prepared by
water-soluble coupling reagents must be not only water- coupling 9-fluorenylmethoxycarbonyl (Fmoc)–Leu-OH [19]
soluble, but also stable in water, and highly reactivity as a
coupling reagent. Previously we used the water-soluble NaO3S
carbodiimide [(WSCD), 1-ethyl-3-(3-dimethylaminopropyl) CH3 O
O O
carbodiimide] [5, 6], as the coupling reagent in conjunction S+
with the additive, N -hydroxy-5-norbornene-2,3- O S
BF4 O
dicarboximide (HONB) [7,8] during SPPS in water [1,2]. O
Recently, two in situ activating reagents, 2-(5-norbornene-
2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoro- Pms group Sps group
borate (TNTU) [9-11] and 4-(4,6-dimethoxy-1,3,5-triazin-2-
yl)-4-methylmorpholinium chloride (DMT-MM) [12-17]
(Fig. 2) were reported to be efficient coupling reagents. Figure 1. Water-soluble N-protecting groups, Pms and Sps groups
Since these reagents are also water-soluble, we tested their
potentials to act as coupling reagents during SPPS in water.
TNTU, an aminium salt and an in situ activating reagent, O MeO
converts protected amino acids to the corresponding Me
N
protected amino acid HONB esters in the presence of a N(CH3)2
N O + N N O
N(CH3)2 N Cl
BF4
O MeO
*Address correspondence to this author at the Faculty of TNTU DMT-MM
Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-
ku, Kobe 651-2180, Japan; Tel: +81 78 974 4794; Fax: +81 78 974 5689; E-
mail: kawasaki@pharm.kobegakuin.ac.jp Figure 2. Structures of TNTU and DMT-MM.

0929-8665/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

190 Protein & Peptide Letters, 2006, Vol. 13, No. 2 Hojo et al.

to the resin by reaction with 1,3-diisopropylcarbodiimide in
dimethylformamide (DMF), and then the Fmoc group was
removed by treatment with 20% piperidine/DMF. Sps-Phe-
OH, which is a colorless crystalline solid and readily soluble
in water, was prepared as previously reported [2]. An
aqueous 2.0% Triton X100 solution was the solvent because
it increases the swelling ability of the resin and the
solubilities of the reactants. After coupling Sps-Phe-OH to
the H-Leu-Rink amide-TentaGel resin, the Sps group was
removed by treatment with aqueous 0.025 M NaOH in
aqueous 50% EtOH and the resin was hydrolyzed with 6 N
HCl. The Phe and Leu content of the acid hydrolysate was
determined and the coupling yield calculated using Phe to
Leu ratio. The results of the coupling reactions using TNTU
are summarized in Table 1. Entry 1 is a control experiment
using WSCD/HONB. This reaction was performed with
N,N-diisopropylethylamine (DIEA) present for 3 hr and went
to completion. Conversely, the reaction using TNTU in the
presence of DIEA did not give a satisfactory result. The
coupling yield was 6% (entry 2). The TNTU reaction in the
presence of HONB was also examined, but the coupling
yield did not improve significantly (entry 3). TNTU is an “in
situ” reagent that reacts with a carboxyl group to give a 5-
norbornene-2,3-dicarboximide ester. We compared the
extent of TNTU-derived HONB ester formation using Sps-D-
Ala-OH (prepared according to the procedure reported in
26
Reference 2. Yield 61%. mp 205-208°C (dec.). [
] D=
+23.4° (c 1.0, H2O). H NMR  (D2O): 8.05 (2H, d, J = 8.2
1
Hz), 8.01(2H, d, J = 8.2 Hz), 4.36 (2H, m), 4.29 (1H, m),
3.62 (2H, t, J = 5.8 Hz), 1.31 (3H, m). TOF-MS m/z: Calcd.
for C15H20NO9S2 ([M-H]-) 380.37. Found: 380.81) with
DMF and water as solvents. Sps-D-Ala-OH, HONB, TNTU
and DIEA were dissolved in water or DMF and the reaction
products were identified using HPLC. After 10 minutes,
quantitative ester formation was observed for the DMF
system, but only a trace amount of the ester was found in the
water mixture. HONB ester formation from
ethanesufonylethoxycarbonylphenylalanine (Esc-Phe-OH)
[20], HONB, DIEA and TNTU in DMF and in 50%
DMF/water was also characterized using HPLC. Quantitative
ester formation occurred when DMF was the solvent, but
only a trace amount of the ester was detected with 50%
DMF/water as the solvent. Therefore, TNTU is not a suitable
coupling reagent in a water milieu.
Falchi et al. tested the effectiveness of DMT-MM as the
coupling reagent during SPPS, with tetrahydrofuran or N-
methyl-2-pyrrolidone (NMP) as the solvent [21]. They
recommended NMP as the solvent for SPPS when DMT-
MM is to be used as the coupling reagent. Kunishima et al.
reported successful formation of carboxamides using DMT-
MM in alcohols and aqueous media by the solution method
[14]. We examined DMT-MM for use as a coupling reagent
during SPPS in aqueous media.
The results of coupling reactions using DMT-MM in the
presence of N-methylmorpholine (NMM) are summarized in
Table 2. First, we coupled Sps-Phe-OH to H-Leu amide-
TentaGel in aqueous 2.0% Triton X100 solution (entry 1),

Sps Phe OH
H Leu Rink PEG Sps Phe Leu Rink PEG
TNTU

Table 1. Coupling of Sps-Phe-OH to H-Leu-TentaGel resin using TNTU

Entry Reagent Additive Base Time (hr) Yield (%)

1 WSCD HONB DIEA 3 100

2 TNTU - DIEA 3 6

3 TNTU HONB DIEA 3 10

Sps Phe OH
H Leu Rink PEG Sps Phe Leu Rink PEG
DMT-MM

Table. 2. Coupling of Sps-Phe-OH to H-Leu-TentaGel resin using DMT-MM

Entry Solvent Base Time (hr) Yield (%)

1 aqueous 2.0% Triton X-100 NMM 3 32

2 aqueous 2.0% Triton X-100 NMM 24 44

3 aqueous 5% EtOH NMM 3 41

4 aqueous 10% EtOH NMM 3 51

5 aqueous 50% EtOH NMM 3 100

6 aqueous 50% EtOH NMM 1 47

SPPS in Water Protein & Peptide Letters, 2006, Vol. 13, No. 2 191

but the yield was not satisfactory (32%). The yield increased
to 44% when the reaction time was extended to 24 hr.
Kunishima et al. reported that condensation reactions using
DMT-MM when dissolved in various alcohols, including
EtOH and MeOH, gave satisfactory results (~99% yield)
[12]. Therefore, we tried the coupling reaction in aqueous
EtOH. (entries 3-6) as EtOH has low toxicity. The reaction
in aqueous 50% EtOH proceeded smoothly and resulted in a
satisfactory coupling yield (entry 5).
To evaluate our synthetic strategy, which uses Sps-amino
acids, DMT-MM, and a solid support suspended in aqueous
50% EtOH, we attempted the synthesis of Leu-enkephalin
amide. Sps-amino acids [Sps-Phe-OH, Sps-Gly-OH, Sps-
Leu-OH, and Sps-Tyr(tBu)-OH] [4] were coupled
Figure 3. HPLC profiles of synthetic Leu-enkephaline amide A:
successively to Rink amide-TentaGel resin using DMT-
Crude Leu-enkephalin amide. B: Purified Leu-enkephalin amide.
MM/NMM according to the protocol of Table 3. Coupling Column: DAISOPAK SP-120-5-ODS-B (4.6 x 250 mm). Flow rate:
reactions were performed for 3 hr and the Sps groups were 1 mL/min. Eluent: CH3CN/H2O containing 0.05% trifluoroacetic
removed with two consecutive 3 min treatments of 0.025 M acid. Gradient: 10/9050/50. OD at 220 nm.
NaOH in aqueous 50% EtOH. Synthetic Sps-Tyr(tBu)-Gly-
Gly-Phe-Leu-Rink amide-TentaGel resin was treated with coupling reagent when peptide synthesis is carried out using
0.025 M NaOH in aqueous 50% EtOH and then treated with Esc-amino acids. We have successfully synthesized Leu-
trifluoroacetic acid to cleave the peptide from the resin. The enkephalin amide in aqueous 50% EtOH using DMT-MM.
resulting crude Leu-enkephalin amide, H-Tyr-Gly-Gly-Phe- Our method is efficient and “environment-friendly” when
Leu-NH2, was purified by HPLC and the HPLC profile is compared with the usual solid phase methods performed in
shown in Fig. 3. The yield of purified Leu-enkephalin amide organic solvents.

Table 3. Protocol for the SPPS of Leu-enkephalin amide using Sps-amino acids and DMT-MM in aqueous 50% EtOH

Step Reagents Time

1 H 2O 3 min X 2

2 0.025 M NaOH in aqueous 50% EtOH 3 min X 2

3 H 2O 3 min X 2

4 aqueous 2.0% Triton X-100 3 min X 3

5 Sps-amino acid, DMT-MM, NMM in aqueous 50% EtOH 3 hr

6 aqueous 2.0% Triton X-100 3 min X 3

which was calculated using the amino group content of the REFERENCES
26
used resin was 58%. {[
] D= +8.3 (c 0.2, H2O). TOF-MS [1]
m/z: Calcd for C28H39N6O6Na ([M+Na]+) 577.64, Found:
577.2. Amino Acid ratios in an acid hydrolysate: Tyr, 0.97; [2]
Gly, 2.00; Phe, 0.98; Leu, 1.05. (average recovery; 91%)}.
[3]
In conclusion, we have examined TNTU and DMT-MM
for their ability to perform as coupling reagents in aqueous [4]
media. Although TNTU was reported to be an efficient
[5]
coupling reagent, it did not work well in water. TNTU [6]
degraded in water, which resulted in poor coupling. DMT-
MM also was ineffective as a coupling reagent when the [7]
reaction was attempted in water alone, but was a satisfactory
[8]
reagent in aqueous 50% EtOH. The swelling ability of the [9]
resin for SPPS is an important factor for performing solid
phase synthesis, and the Rink amide-TentaGel resin swells [10]
better in aqueous 50% EtOH than in water alone. Recently, [11]
we reported the Esc group as a water-soluble N-protecting
[12]
group [20]. Esc-amino acids are more soluble in aqueous
50% EtOH than in water alone. DMT-MM will be a suitable
Hojo, K., Maeda, M. and Kawasaki, K. (2001) J. Peptide Sci., 7,
615.
Hojo, K., Maeda, M., Takahara, Y., Yamamoto, S. and Kawasaki,
K. (2003) Tetrahedron Lett., 44, 2849.
Hojo, K., Maeda, M. and Kawasaki, K. (2004) Tetrahedron, 60,
1875.
Hojo, K., Maeda, M. and Kawasaki, K. (2004) Tetrahedron Lett.,
45, 9293.
Sheehan, J. C. and Hlavka, J. J. (1956) J. Org. Chem., 21, 439.
Sheehan. J. C., Cruickshank, P. A. and Boshart, G. L. (1961) J.
Org. Chem., 26, 2525.
Fujino, M., Kobayashi, S., Fukuda, T., Shinagawa, S. and
Nishimura, O. (1974) Chem. Pharm. Bull., 22, 1857.
Kitada, C. and Fujino, M. (1978) Chem. Pharm. Bull., 26, 585.
Knorr, R., Trzeciak, A., Bannwarth, W. and Gillessen, D. (1989)
Tetrahedron Lett., 30, 1927.
Bannwarth, W. and Knorr, R. (1991) Tetrahedron Lett., 32, 1157.
Bannwarth, W., Schmidt, D., Stallard, B., Hornung, C., Knorr, R.
and Müller, F. (1988) Helv. Chim. Acta, 8, 2085.
Kunishima, M., Kawachi, C., Morita, J., Terao, K., Iawasaki, F.
and Tani, S. (1999) Tetrahedron, 55, 13159.
192 Protein & Peptide Letters, 2006, Vol. 13, No. 2 Hojo et al.

[13] Kunishima, M., Kawachi, C., Iwasaki, F., Terao, K. and Tani, S. [18] Bayer, E. (1991) Angew. Chem. Int. Ed., 30, 113.
(1999) Tetrahedron Lett., 40, 5327. [19] Fields, G. B. and Noble, R. L. (1990) Int. J. Peptide Protein Res.,
[14] Kunishima, M., Kawachi, C., Hioki, K., Terao, K. and Tani, S. 35, 161.
(2001) Tetrahedron, 57, 1550. [20] Hojo, K., Maeda, M., Smith, T. J., Kita, E., Yamaguchi, F.,
[15] Kaminski, Z. J., Paneth, P. and Rudzinski, J. (1998) J. Org. Chem., Yamamoto, S. and Kawasaki, K. (2004) Chem. Pharm. Bull. 52,
63, 4248. 422.
[16] Kaminski, Z. J. (1987) Synthesis, 917. [21] Falchi, A., Giacomelli, G., Prcheddu, A. and Taddei, M. (2000)
[17] Kaminski, Z. J. (1985) Tetrahedron Lett., 26, 2901. Synlett, 275.

Received: August 08, 2005 Accepted: September 16, 2005