Peptide synthesis in water IV. Preparation of N- ethanesulfonylethoxycarbonyl (Esc) amino acids and their application to solid phase peptide synthesis

Article abstract of DOI:10.1248/cpb.52.422

A new N-protecting group, ethanesulfonylethoxycarbonyl (Esc), was designed to perform peptide synthesis in both aqueous and organic solvents. Esc-amino acids were prepared by the reaction of Esc-Cl and amino acids. Although Esc-Cl was a highly reactive re

Full text of DOI:10.1248/cpb.52.422

422
Chem. Pharm. Bull. 52(4) 422—427 (2004)
Vol. 52, No. 4
Peptide Synthesis in Water IV. Preparation of
N-Ethanesulfonylethoxycarbonyl (Esc) Amino Acids and Their
Application to Solid Phase Peptide Synthesis
Keiko HOJO,a Mitsuko MAEDA,a Timothy J. SMITH,b Eriko KITA,a Fumie YAMAGUCHI,a
a
,a
*
Sachiko YAMAMOTO, and Koichi KAWASAKI
a Faculty of Pharmaceutical Sciences, Kobe Gakuin University; Nishi-ku, Kobe 651–2180, Japan: and b School of
Pharmacy, University of Pacific; 3601, Pacific Avenue, Stockton, CA 95211, U.S.A.
Received November 25, 2003; accepted January 16, 2004
A new N-protecting group, ethanesulfonylethoxycarbonyl (Esc), was designed to perform peptide synthesis
in both aqueous and organic solvents. Esc-amino acids were prepared by the reaction of Esc-Cl and amino acids.
Although Esc-Cl was a highly reactive reagent, it was not stable and decomposed during the purification proce-
dure. A more stable reagent, ethanesulfonylethyl-4-nitrophenyl carbonate (Esc-ONp), was designed for prepara-
tion of Esc-amino acids. Esc-ONp was a stable reagent and could be purified by silica gel column chromatogra-
phy or recrystallization. Esc-amino acids were prepared by the reaction of Esc-ONp and amino acids in good
yield. To evaluate Esc-amino acids, Leu-enkephalin amide was synthesized using Esc-amino acids by the solid
phase method in water. Removal of the Esc group was performed with 0.025 mol/l NaOH in 50% aqueous
ethanol. Leu-enkephalin amide was successfully synthesized on a poly(ethylene glycol)-grafted polystyrene resin.
Esc-amino acids have moderate solubility in organic solvents (such as dimethylformamide and acetonitrile). Leu-
enkephalin amide was synthesized using Esc-amino acids by the solid phase method in dimethylformamide. Re-
moval of the Esc group was performed with 0.05 mol/l tetrabutylammonium fluoride in dimethylformamide. Syn-
thesis of Leu-enkephalin amide using Esc-amino acids in dimethylformamide was also successful. The yields of
synthesis of Leu-enkephalin amide in water and dimethylformamide were 71% and 67%, respectively.
Key words water-soluble protecting group; ethanesulfonylethoxycarbonyl; synthesis in water
Synthesis of peptides was an arduous task before develop- aqueous and organic solvents). The Esc group was intro-
ment of the solid phase method. The solid phase method has duced onto amino acids by the reaction of 2-ethanesul-
facilitated automated peptide synthesis and the development fonylethyl chloroformate (Esc-Cl) as shown in Chart 1
of combinatorial chemistry. It has many advantages com- (Route 1). 2-Ethanesulfonylethanol was converted to Esc-Cl
pared with classical solution peptide synthesis, but requires a by treatment with phosgene (prepared from triphosgene)7) in
large amount of organic solvents. Disposal of organic sol- dichloromethane. Since Esc-Cl was labile and decomposed
vents is one of many important environmental problems, during purification, it was used without purification for the
therefore we decided to perform peptide synthesis in water. next acylating reaction. Esc-Cl, H-Phe-OH and triethylamine
To perform synthesis in water, protected amino acids must be were allowed to react in aqueous 50% acetonitrile to give
water-soluble. We have studied water-soluble N-protecting Esc-Phe-OH. HPLC analysis of the crude product exhibited
groups and we developed 2-[phenyl(methyl)sulfonio]ethoxy- impurities and the product could not be purified by silica gel
carbonyl (Pms)1,2) as a water-soluble N-protecting group. column chromatography and recrystallization. Pure Esc-Phe-
Since the Pms group is an onium salt, it is rather unstable in OH was obtained by RP-HPLC purification and the yield was
comparison with N-protecting groups which are in general 38%. To improve the reaction yield and purification proce-
use (such as t-butoxycarbonyl group,3) benzyloxycarbonyl dure, another synthetic route (Route 2 in Chart 1) to the Esc-
group4) and 9-fluorenylmethoxycarbonyl (Fmoc) group5)) for amino acid was studied. 2-Ethanesulfonylethanol was reacted
peptide synthesis. Here we report preparation of the ethane- with 4-nitrophenyl chloroformate to give ethanesulfonyl-
sufonylethoxycarbonyl (Esc) group and its application to ethyl-4-nitrophenyl carbonate (Esc-ONp). The crude Esc-
peptide synthesis in water.
ONp was stable and could be purified by silica gel column
Tesser and Balvert-Geers6) reported the methanesul- chromatography or recrystallization from a mixture of ether
fonylethoxycarbonyl (Msc) group as a readily removable N- and hexane. The yield was 78%. Other Esc-amino acids
protecting group by treatment with 0.1 or 0.2 N NaOH solu- (Esc-Leu-OH, Esc-Gly-OH, Esc-Tyr-OH) were also prepared
tion. This protecting group is hydrophilic rather than hy- using Esc-ONp and their analytical data are shown in Table
drophobic. We designed the Esc group (Fig. 1), expecting it 1. Esc-Gly-OH and Esc-Tyr-OH were soluble in water and
to display both hydrophilic and hydrophobic character (i.e. Esc-Leu-OH and Esc-Phe-OH were not readily soluble in
we expect Esc-amino acids to exhibit reasonable solubility in water. Esc-Leu-OH and Esc-Phe-OH were soluble in aqueous
2% Triton X. To evaluate Esc-amino acids, Leu-enkephalin
amide was synthesized by the solid phase method in water
using Esc-amino acids. For solid phase synthesis in water, a
resin which swells appreciably well in water is necessary. Re-
cently, poly(ethylene glycol)-grafted polystyrene resins8)
were developed to increase the swelling ability of the resin in
Fig. 1. Structure of the Esc Group
To whom correspondence should be addressed. e-mail: kawasaki@pharm.kobegakuin.ac.jp
© 2004 Pharmaceutical Society of Japan

April 2004
423
Chart 1. Preparation of Esc-Amino Acids
Table 1. Analytical Data of Synthetic Esc-Amino Acids
TOF-MS
Elemental anal. Calcd (Found)
H
[a]D20
Route 1
yield (%)
Route 2
yield (%)
Amino acid
MW
(m/z)
(cϭ1.0, CH3CN)
C
N
(MϩNa)ϩ
318.26
44.73
(44.50)
51.05
(50.80)
35.14
(35.02)
48.69
(47.98)
7.17
(6.88)
5.81
(5.67)
5.48
(5.29)
5.55
(5.23)
4.74
(4.74)
4.25
(4.22)
5.85
(5.88)
4.06
(4.06)
Leu
Phe
Gly
Tyr
281.37
329.37
239.25
368.29
Ϫ9.2
15.5
—
—
39.3
—
68.2
78.3
57.0
43.1
(MϩNa)ϩ
352.12
(MϩNa)ϩ
232.23
(MϩNa)ϩ
368.29
Ϫ9.2
—
various solvents, including water. The present synthesis was Table 2. Coupling Reaction of Esc-Phe-OH on H-Leu-Rink-PEG Amide
Resin in Water
performed on the poly(ethylene glycol)-grafted Rink amide
polystyrene resin (Rink-PEG amide resin)9) which was re-
Coupling
reagents
Additive
agents
Yield
(%)
Entry
Base
Time
ported to have good swelling ability in water. Coupling reac-
tions were performed with a water-soluble carbodiimide
1
2
3
4
5
6
WSCD
WSCD
WSCD
WSCD
WSCD
WSCD
HOBt
HOAt
HOSu
HONB
Imidazole
HONB
—
—
—
—
—
1.5 h
1.5 h
1.5 h
1.5 h
1.5 h
14.6
3.5
41.6
78.6
11.7
93.4
[WSCD,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride] in aqueous 2% Triton X. Triton X increases
the solubility of Esc-amino acids and increases the swelling
ability of the resin. An additive was used to accelerate cou-
pling reactions. Introduction of Esc-Leu-OH on the resin was
performed with WSCD in the presence of N-hydroxy-5-nor-
bornene-2,3-dicarboximide (HONB)10) for 2 h. The yield of
the reaction was satisfactory judging from the Leu-content
on the resin. The next coupling reaction (introduction of Esc-
Phe-OH on the H-Leu-Rink-PEG amide resin) was per-
formed under the same conditions, but the reaction was slow.
Since by-products which did not contain Phe and Gly were
DIEA
30 min
7
WSCD
HONB
DIEA
1.0 h
100
8
9
WSCD
WSCD
HONB
HOBt
Pyridine
DIEA
1.5 h
1.5 h
97.2
26.1
Coupling reaction was performed with WSCD in the presence of an additive in
water. Details of the experiments are described in Experimental.
obtained after completing the synthesis of Leu-enkephalin with that of HONB. Fujino et al.10) reported that HONB sup-
amide, we speculated that introduction of Esc-Phe-OH and pressed a side reaction (N-acylurea formation, C→N re-
the reaction for the removal of the Esc group on Phe were arrangement reaction) of the carbodiimide method. HONB
slow. We examined the coupling reaction conditions for in- may suppress such side reaction, resulting in a better cou-
troduction of Esc-Phe-OH on the H-Leu-Rink-PEG amide pling yield. Addition of HONB gave a satisfactory result and
resin and the deprotection conditions for the Esc-Phe-Leu- further addition of equi-molar base [diisopropylethylamine
Rink-PEG amide resin. First, various additives [HONB, 1- (DIEA) or pyridine] to Leu on the resin resulted in the best
hydroxybenzotriazole (HOBt),11) 1-hydroxy-7-azabenzotria- yield (Table 2).
zole (HOAt),12) 1-hydroxysuccimide (HOSu)13) and imida-
Removal of the Esc group by various base treatments was
zole] were examined to stimulate the coupling reaction by examined using Esc-Phe-Leu-Rink-PEG amide resin. Be-
the WSCD method. Equi-molar additive to Esc-Phe-OH was cause removal of the Esc group on Phe was slower than that
used. After the reaction, the Esc-group was removed and the of other synthetic Esc-amino acids, Esc-Phe-Leu-Rink-PEG
resin was hydrolyzed. Phe and Leu content in an acid hy- amide resin was preferentially used as the test compound.
drolysate was analyzed and the yield was calculated from the The resin was treated with aqueous 0.01 mol/l NaOH (3
amino acid ratio of Phe and Leu. Addition of HOBt, HOAt, times, a few minutes each) and washed repeatedly with
imidazole and HOSu did not give better results compared water, followed by washing with DMF repeatedly. Then the

424
Vol. 52, No. 4
resin was swelled in DMF and reacted in DMF with Fmoc- (TFA), thioanisole and ethanedithiol (94/3/3) for 2 h. The re-
Gly-OH using diisopropylcarbodiimide in the presence of sulting Leu-enkephalin amide, H-Tyr-Gly-Gly-Phe-Leu-NH2,
HOBt until the resin gave a negative result in the Kaiser test was purified by HPLC and the HPLC profile is shown in Fig.
(ninhydrin test).14) The resulting resin was treated with 20% 2. The yield of purified Leu-enkephalin amide was 71%.
piperidine in DMF to remove the Fmoc group, and hy-
Since Esc-amino acids also exhibited moderate solubility
drolyzed. The amino acid ratio of Gly, Phe and Leu in the in organic solvents (such as DMF and acetonitrile), Leu-
acid hydrolysate was analyzed and the yield of the deprotec- enkephalin amide was synthesized on Rink amide resin using
tion procedure with the Esc-Phe-Leu-Rink-PEG amide resin Esc-amino acids in DMF. The Msc group was reported to be
was calculated. From the results shown in Table 3, complete highly base labile and we expected that the Esc group was re-
removal of the Esc group by treatment with 0.01 mol/l NaOH movable with same basic conditions for deprotection of the
and 0.025 mol/l NaOH was difficult. Even with 0.05 mol/l Fmoc group. Deprotection of the Esc group was performed
NaOH treatment (3 times, 3 or 10 min each), complete re- by treatment with 20% piperidine/DMF for 30 min. After
moval of Esc group could not be observed. Next, the depro- completion of the synthesis, we purified the product by
tection reaction was examined in a mixture of water and HPLC, but Leu-enkephalin amide was not obtained. During
EtOH. Addition of EtOH accelerated the deprotection reac- the synthesis, deprotection procedures were checked by the
tion and gave satisfactory results. The Esc group of the Esc- Kaiser test, but the tests did not give clear positive Kaiser
Phe-Leu-Rink-PEG amide resin was removed completely by test. To validate a deprotection procedure for removal of the
treatment with 0.025 mol/l NaOH (3 times, 3 min each) in Esc group in DMF, the Esc-Phe-Leu-Rink amide resin was
aqueous 50% EtOH. The Esc group was also removable with treated with various bases in DMF. As described above, re-
2.5% Na2CO3 (3 times, 5 min each) in 50% aqueous EtOH. moval of the Esc group on Phe with base was slower than
Considering the results of these coupling and deprotection that on other synthetic Esc-amino acids; Esc-Phe-Leu-Rink
studies, Leu-enkephalin amide was synthesized in water ac- amide resin was preferentially used as the test compound. To
cording to the procedure shown in Table 4. Synthetic Esc- compare with the Msc group, Msc-Phe-Leu-Rink amide
Tyr-Gly-Gly-Phe-Leu-Rink-PEG amide resin was treated resin was treated under the same conditions and the results
with 0.025 mol/l NaOH in 50% aqueous EtOH to remove Esc are shown in Table 5. Fmoc-Leu-Rink amide resin was
group, and then treated with a mixture of trifluoroacetic acid treated with 20% piperidine/DMF and the resulting resin was
coupled with Esc-Phe-OH with diisopropylcarbodiimide/
HOBt to give the Esc-Phe-Leu-Rink amide resin. The resin
was treated with various bases [20% piperidine/DMF, 2%
Table 3. Deprotection Studies with Esc-Phe-Leu-Rink-PEG Amide Resin
and Various Bases in Water
Time
(min)
Yield
(%)
Entry
Reagents
1
2
3
4
5
6
7
0.025 mol/l NaOH aq.
0.025 mol/l NaOH aq.
0.05 mol/l NaOH aq.
0.05 mol/l NaOH aq.
0.01 mol/l NaOH (EtOH–H2O)
0.01 mol/l NaOH (EtOH–H2O)
0.025 mol/l NaOH (EtOH–H2O)
3, 3, 3
10, 10, 10
3, 3, 3
10, 10, 10
1, 1, 1
21
36
96
98
29
77
89
3, 3, 3
1, 1, 1
8
0.025 mol/l NaOH (EtOH–H2O)
3, 3, 3
100
9
10
11
12
2.5% NaHCO3 (EtOH–H2O)
2.5% NaHCO3 (EtOH–H2O)
2.5% Na2CO3 (EtOH–H2O)
2.5% Na2CO3 (EtOH–H2O)
3, 3, 3
5, 5, 5
3, 3, 3
5, 5, 5
16
20
87
Fig. 2. HPLC Profiles of Synthetic Crude Leu-Enkephalin Amide Synthe-
sized by the Solid Phase Method in Water
100
A: Crude Leu-enkephalin amide. B: Purified Leu-enkephalin amide. Column,
DAISOPAK SP-120-5-ODS-B (4.6ϫ250 mm). Flow rate, 1 ml/min. Eluent,
CH3CN/H2O containing 0.05% TFA. Gradient: 10/90→50/50 (20 min).
Esc-Phe-Leu-Rink-PEG amide resin was treated with various bases in water and the
resulting resin was coupled with Fmoc-Gly-OH in DMF. After removal of the Fmoc
group, the resin was hydrolyzed. From amino acid analysis of the acid hydrolysate, the
deprotection rate of Phe was calculated.
Table 5. Deprotection Studies with Esc- and Msc-Phe-Leu-Rink Amide
Resins and Various Bases in DMF
Table 4. Protocol for the Solid Phase Synthesis Using Esc-Amino Acids
in Water
Protecting
group
Time
(min)
Yield
(%)
Entry
Reagents
Step
Reagents
Time
1
2
3
4
5
6
Msc
Msc
Msc
Esc
Esc
Esc
20% piperidine–DMF
2.0% DBU–DMF
0.05 mol/l TBAF–DMF
20% piperidine–DMF
2.0% DBU–DMF
20
20
20
20
20
20
Ͻ2.0
30
100
Ͻ2.0
38
1
2
H2O
3 minϫ2
3 minϫ3
0.025 mol/l NaOH
(H2O–EtOH)
H2O
2.0% Triton X aq.
Esc-amino acid
WSCD
3
4
5
3 minϫ2
3 minϫ3
1 h
0.05 mol/l TBAF–DMF
100
7
Esc
0.05 mol/l TBAF–DMF
5
100
HONB, DIEA
in 2.0% Triton X aq.
2.0% Triton X aq.
Esc-Phe-Leu-Rink amide resin was treated with various bases in DMF and the result-
ing resin was coupled with Fmoc-Gly-OH in DMF. After removal of Fmoc group, the
resin was hydrolyzed. From amino acid analysis of the acid hydrolysate, the deprotec-
tion rate of the Esc group from the Phe residue was calculated.
6
3 minϫ3

April 2004
425
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU)15) and 0.05 mol/l
tetrabutylammonium fluoride (TBAF)16)] for 20 min to re-
move the Esc group and then reacted with Fmoc-Gly-OH and
with diisopropylcarbodiimide/HOBt in DMF until the resin
gave a negative result with the Kaiser test (ninhydrin test).14)
The resulting resin (Fmoc-Gly-Phe-Leu-Rink amide resin)
was treated with 20% piperidine in DMF to remove the Fmoc
group, and hydrolyzed. The amino acid ratio of Gly, Phe and
Leu in the acid hydrolysate was analyzed and the yield of the
deprotection procedure with the Esc-Phe-Leu-Rink amide
resin was calculated from the amino acid ratios. Contrary to
our expectations, Esc and Msc groups were rather stable to
20% piperidine treatment. Furthermore, Esc and Msc groups
could not be removed completely by 2% DBU/DMF treat-
ment for 20 min. Since Wade et al.15) reported that the Fmoc
group was removable by 2% DBU/DMF treatment for 5 min,
further study regarding the deprotection of Esc and Msc
groups was done. Esc-Phe-OH and Msc-Phe-OH were
treated with 2% DBU/DMF and the reaction mixture was ex-
amined by HPLC. As shown in Fig. 3, complete removal of
Msc and Esc groups on Phe could not be achieved by 2%
DBU/DMF treatment, even after 220 min. The Esc group
tended to be more labile to piperidine and DBU treatment
when compared with the Msc group. Esc and Msc groups
could be removed completely by 0.05 mol/l TBAF/DMF
treatment for 5 min.
Fig. 3. Deprotection of Esc- and Msc-Groups on Phe by 2% DBU/DMF
Treatment
Table 6. Protocol for the Solid Phase Synthesis Using Esc-Amino Acids
in DMF
Step
Reagents
Time
1
2
3
4
DMF
0.05 mol/l TBAF–DMF
DMF
3 minϫ2
3 min
3 minϫ2
1 h
Esc-amino acid
DIC, HONB
DMF
5
3 minϫ3
Leu-enkephalin amide was synthesized using Esc-amino
acids on Rink amide resin in DMF according to a procedure
shown in Table 6. Removal of the Esc group was accom-
plished by 2 consecutive 3 min treatments with 0.05 mol/l
TBAF/DMF. Synthetic Esc-Tyr-Gly-Gly-Phe-Leu-Rink amide
resin was treated with 0.05 mol/l TBAF/DMF and then
treated with a mixture of TFA, thioanisole and ethanedithiol
(94/3/3). The resulting crude Leu-enkephalin amide was pu-
rified by HPLC and the HPLC profile is shown in Fig. 4A. In
addition to the desired product (YGGFL), by-products which
were deletion peptides of Phe, Gly and Tyr were detected and
the yield of the Leu-enkephalin amide was 23%. As shown in
Fig. 4A, the major deletion peptide was YGFL and the area
ratio of the YGGFL peak and YGFL peak was 1.00 : 0.81.
The Esc group of Esc-Phe-OH was completely removable by
2 consecutive 3 min treatments with 0.05 mol/l TBAF/DMF,
but the removal of the Esc-group from the Esc-Phe-Leu-Rink
amide resin may be incomplete. Since the by-products were
derived from incomplete removal of the Esc group, Leu-
enkephalin amide was synthesized again employing a longer
TBAF treatment step to improve the deprotection procedure.
The Esc group was removed by 3 treatments (3 min, 3 min,
10 min) with 0.05 mol/l TBAF/DMF during synthesis and the
HPLC profile of synthetic crude Leu-enkephalin is shown in
Fig. 4B. The yield of the purified Leu-enkephalin was 67%
(calculated from the used resin). The area ratio of the
YGGFL peak and YGFL peak was 1.00 : 0.09.
Fig. 4. HPLC Profiles of Synthetic Crude Leu-Enkephalin Amide
Leu-enkephalin amide was prepared by deblocking the Esc group through treatment
with 0.05 mol/l TBAF/DMF [A: 3 min (3 times). B: 3 min (2 times) plus 10 min].
moval of the Esc group in water is accelerated with addition
of alcohol. The Esc group was removable completely by
treatment with 0.025 mol/l NaOH in aqueous 50% EtOH and
removable even with 2.5% Na2CO3 in aqueous 50% EtOH.
Removal of Esc and Msc groups in DMF with piperidine and
DBU was rather difficult, but were removable with 0.05 mol/l
TBAF/DMF. The Esc group will be a useful protecting group
in synthesis of peptides and we were successful in synthesiz-
ing Leu-enkephalin amide on a resin in both aqueous and or-
ganic solvents.
Experimental
In the preceding papers1,2) we reported Pms-amino acids as
water-soluble N-protected amino acids. They are readily sol-
uble in water, but sparingly soluble in organic solvents. Since
Esc-amino acids have moderate solubility in both aqueous
and organic solvents, they may be used for coupling reac-
tions in various solvents. Pms-amino acids are onium salts
and they are rather unstable, but Esc-amino acids are stable
compounds. The Esc group is removable with base and re-
RP-HPLC was conducted with a Waters 600 on a DAISOPAK column
using gradient systems of CH3CN/H2O containing 0.05% trifluoroacetic
acid. TOF-MS spectra were measured with a Shimadzu/Kratos Kompact
MALDI IV mass spectrometer. Synthetic peptides were hydrolyzed with 6 N
HCl and amino acid compositions of acid hydrolysates were determined
with a Waters Pico·TAG amino acid analyzer. Optical rotations were mea-
sured with a JASCO DIP-360 polarimeter.
Melting Points were determined with a Yanagimoto micro-melting point
apparatus. Column chromatography was performed on silica gel 60 (BW-

426
Vol. 52, No. 4
127ZH, Fuji Silica Chemical Co.). 1H-NMR spectra were recorded with a
Bruker 400 MHz spectrometer.
0.1 mmol) and the H-Leu-Rink-PEG amide resin (192 mg, 50 mmol) was re-
acted with diisopropylcarbodiimide (15.6 ml, 0.1 mmol) in the presence of
HOBt (13.5 mg, 0.1 mmol) in DMF until the resin gave a negative Kaiser
test. The resulting Esc-Phe-Leu-Rink-PEG-resin was washed with DMF and
dichloromethane and dried in vacuo. Yield 216 mg (99%). Esc-Phe-Leu-
Rink-PEG-resin (10 mg each) was treated with various base solutions
(0.01 mol/l NaOH in aqueous 50% EtOH, 0.025 mol/l NaOH in 50% aque-
ous EtOH, aqueous 0.05 mol/l NaOH solution, 2.5% Na2CO3 in aqueous
50% EtOH, 2.5% NaHCO3 in aqueous 50% EtOH). After base treatment,
the resin was washed with H2O and DMF. Then Fmoc-Gly-OH (3.0 mg,
10 mmol) was coupled on the resin with diisopropylcarbodiimide (1.6 ml,
10 mmol) and HOBt (1.4 mg, 10 mmol) in DMF. The resulting resin was
washed with DMF and dichloromethane, and dried in vacuo. The resin was
hydrolyzed and Gly, Phe and Leu content in the hydrolysate were analyzed.
Each deprotection yield was calculated from the amino acid ratio of Gly and
Leu. Results are summarized in Table 3.
Deprotection Studies Involving Esc and Msc Groups of Esc- and Msc-
Phe-Leu-Rink Amide Resins with Various Bases in DMF Esc-Phe-Leu-
Rink amide resin was prepared from Fmoc-Rink amide resin (100 mg,
62 mmol) via Fmoc-Leu-Rink amide resin using diisopropylcarbodiimide/
HOBt as a coupling reagent in DMF. Yield 104 mg (98%). The rseins (10 mg
each) were swelled with DMF and treated with 20% piperideine, 2%
DBU/DMF and 0.05 mol/l TBAF/DMF respectively. After base treatment,
the each resin was washed with DMF and coupled with Fmoc-Gly-OH
(9.0 mg, 30 mmol) using diisopropylcarbodiimide/HOBt in DMF until the re-
action mixture showed negative Kaiser test. The resulting resins were
washed with DMF and dichloromethane, and dried in vacuo. The resins
were hydrolyzed and Gly, Phe and Leu content in hydrolysates were ana-
lyzed. Each deprotection yield was calculated from the amino acid ratio of
Gly and Leu.
Esc-Phe-OH Prepared through Route 1 in Chart 1 To a solution of
2-ethansulfonylethanol (276 mg, 2.0 mmol) and triphosgen (394 mg,
1.33 mmol) in dichloromethane (30 ml), Et3N (0.56 ml, 4.0 mmol) in
dichloromethane (20 ml) was added at 0 °C and the mixture was stirred at
0 °C for 1.5 h. After removal of the solvent in vacuo at 10 °C, the residue
was dissolved in acetonitrile (15 ml). The solution was added to a solution of
Phe (330 mg, 2.0 mmol) and Et3N (0.35 ml, 2.5 mmol) in 50% aqueous ace-
tonitrile (30 ml) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h.
After evaporation in vacuo, the residue was purified by HPLC. Yield:
125 mg, 38%. 1H-NMR (CD3CN) d: 7.30—7.22 (5H, m), 5.97 (1H, br d),
4.40 (1H, m), 4.32 (2H, t, Jϭ5.3 Hz), 3.21 (2H, t, Jϭ5.3 Hz), 3.18 (1H, dd,
Jϭ14.0, 5.0 Hz), 3.06 (2H, q, Jϭ7.4 Hz), 2.92 (1H, dd, Jϭ14.0, 9.2 Hz),
1.25 (3H, t, Jϭ7.4 Hz). [a]D23 Ϫ15.54° (cϭ1.0, CH3CN). mp 78 °C. TOF-MS
m/z: 352.12 [MϩNa]ϩ (Calcd for C14H19NO6SNa: 352.36). Anal. Calcd for
C14H19NO6: C, 51.05; H, 5.81; N, 4.25. Found: C, 50.80; H, 5.67; N,4.22.
Esc-ONp To a solution of ethylsulfonylethanol (249 mg, 1.8 mmol) and
Et3N (278 ml, 2.0 mmol) in dichloromethane (30 ml), 4-nitrophenylchloro-
formate (402 mg, 2.0 mmol) in dichloromethane (5 ml) was added, and the
mixture was refluxed at 40 °C for 4 h. After filtration, the solvent was re-
moved in vacuo and the residue was purified by column chromatography on
silica gel using hexane–ethyl acetate (2/1) as an eluent. Yield: 69%, color-
1
less crystal, mp 49 °C, H-NMR (CD3CN) d: 8.30 (2H, d, Jϭ9.3 Hz), 7.40
(2H, d, Jϭ9.3 Hz), 4.74 (2H, t, Jϭ5.8 Hz), 3.44 (2H, t, Jϭ5.8 Hz), 3.14 (3H,
q, Jϭ7.5 Hz), 1.46 (3H, t, Jϭ7.5 Hz). TOF-MS m/z: 326.61 [MϩNa]ϩ
(Calcd for C10H11NO7SNa: 326.29). Anal. Calcd for C11H13NO7S: C, 43.56;
H, 4.32; N, 4.62. Found: C, 43.34; H, 4.28; N, 4.62.
Esc-Phe-OH Prepared through Route 2 in Chart 1 To a solution of
Phe (82.5 mg, 0.5 mmol) and Et3N (70 ml, 0.5 mmol) in 50% aqueous ace-
tonitrile (30 ml), Esc-ONp (181 mg, 0.6 mmol) in acetonitrile (5 ml) was
added and the mixture was stirred overnight at room temperature. After re-
moval of the solvent in vacuo, the residue was dissolved in ethyl acetate. The
solution was extracted with 5% NaHCO3 and the aqueous layer was acidified
to pH 3 with 1 N HCl. The mixture was extracted with ethyl acetate and the
extract was washed with saturated sodium chloride solution and evaporated
in vacuo. The residue was crystallized from ether–hexane. Yield: 128 mg,
78%. The product was identified with Esc-Phe-OH prepared through Route
1 by NMR and MS.
Deprotection studies involving the Msc group of the Msc-Phe-Leu-Rink
amide resin with various bases in DMF were performed in a similar manner
as described above.
2% DBU/DMF Treatment of Esc-Phe-OH Esc-Phe-OH (2.0 mg,
6.1 mmol) was dissolved in 2% DBU/DMF (400 ml) and the solution was
stirred at room temperature. An aliquot (10 ml) was taken periodically for
analysis by HPLC and the rate of deprotection was calculated by measure-
ment of the peak area of Esc-Phe-OH. Results are shown in Fig. 3. Complete
removal of the Esc group was not observed even after 220 min.
The following Esc-amino acids were prepared according to the procedure
described above: Esc-Gly-OH: Yield: 57.0% (colorless solid). 1H-NMR
(CD3CN) d: 5.98 (1H, br s) 4.40 (2H, t, Jϭ5.3 Hz), 3.81 (2H, s), 3.29 (2H, t,
Jϭ5.3 Hz), 3.06 (3H, s), 1.31 (3H, t, Jϭ7.5 Hz). mp 73 °C, TOF-MS m/z:
262.36 [MϩNa]ϩ (Calcd for C7H13NO6SNa: 262.24). Anal. Calcd for
C6H11NO6S: C, 35.14; H, 5.48; N, 5.85. Found: C, 35.02; H, 5.29; N, 5.88.
Solid Phase Synthesis of Leu-Enkephalin Amide in Water Fmoc-
Rink-PEG amide resin (100 mg, amino content 25 mmol) was treated with
20% piperidine/DMF to remove the Fmoc group. After washing with DMF
and aqueous 2.0% Triton X, synthesis was performed according to a proto-
col shown in Table 4. Esc-Leu-OH (29.5 mg, 0.1 mmol), Esc-Phe-OH
(32.9 mg, 0.1 mmol), Esc-Gly-OH (24.7 mg, 0.1 mmol), and Esc-Tyr-OH
(34.5 mg, 0.1 mmol) was coupled in turn with WSCD (19.1 mg, 1.0 mmol)/
HONB 17.9 mg (1.0 mmol) in a presence of DIEA (17.4 ml, 0.1 mmol). Re-
moval of the Esc group was carried out with 0.025 mol/l NaOH in 50%
aqueous EtOH. Synthetic H-Tyr-Gly-Gly-Phe-Leu-Rink-PEG amide resin
(109 mg) was treated with TFA-thioanisole-ethandithiol (94-3-3, 5 ml) for
2 h at room temperature. After filtration, TFA was removed in vacuo and the
residue was dissolved in water. The solution was washed with ether and
lyophilized. The crude product was purified by HPLC. Yield (based on
amino group content of the resin) 12 mg (71%. Fluffy powder). [a]D24 ϩ9.0°
(cϭ0.2, H2O), TOF-MS m/z: 555.3 [Mϩ1]ϩ (Calcd for C28H39N6O6:
555.64). Amino acid ratios in an acid hydrolysate: Tyr 0.93; Gly 1.88; Phe
1.01; Leu 1.00. (average recovery: 97%).
Solid Phase Synthesis of Leu-Enkephalin Amide in DMF Synthesis
was carried out using Esc-amino acids on Fmoc-Rink amide resin (50 mg,
amino group content 33 mmol) in DMF according to the protocol shown in
Table 6. Coupling reactions of each Esc-amino acid (130 mmol) were carried
out with diisopropylcarbodiimide/HONB and the Esc group was removed
with 0.05 mol/l TBAF/DMF. Synthetic H-Tyr-Gly-Gly-Phe-Leu-Rink amide
resin was treated with TFA–thioanisole–ethanedithiol (94–3–3) and the re-
sulting peptide was purified by HPLC. Yield) 14.8 mg (67% based on the
amino group content of the starting resin. fluffy powder). [a]D24 ϩ9.0°
(cϭ0.2, H2O), TOF-MS m/z: 555.3 [Mϩ1]ϩ (Calcd for C28H39N6O6:
555.64). Amino acid ratios in an acid hydrolysate: Tyr 1.01; Gly 2.00, Phe
1.01; Leu 0.89. (average recovery: 98%).
1
Esc-Leu-OH: Yield: 68.2% (colorless solid). H-NMR (CD3CN) d: 5.98
(1H, br d), 4.38 (2H, t, Jϭ5.3 Hz), 4.13 (H, q, Jϭ7.1 Hz), 3.27 (2H, t,
Jϭ5.3 Hz), 3.06 (3H, q, Jϭ7.4 Hz), 1.70 (1H, m), 1.57 (2H, t, Jϭ7.1 Hz),
1.29 (3H, t, Jϭ7.4 Hz), 0.93 (3H, d, Jϭ7.1 Hz), 0.90 (3H, d, Jϭ7.1 Hz).
[a]D24 Ϫ14.73° (cϭ1.0, CH3CN). mp 50 °C. TOF-MS m/z: 318.26 [MϩNa]ϩ
(Calcd for C14H20NO6SNa: 318.34). Anal. Calcd for C14H20NO6S: C, 44.73;
H, 7.17; N, 4.74. Found: C, 44.50; H, 6.88; N, 4.74.
Esc-Tyr-OH: Yield: 56% (colorless solid). 1H-NMR (CD3CN) d: 7.07
(2H, d, Jϭ8.6 Hz), 6.74 (2H, d, Jϭ8.6 Hz, ArH 3, 5), 5.97 (1H, br d), 4.39
(2H, t, Jϭ5.3 Hz), 4.32 (H, m), 3.30 (2H, t, Jϭ5.3 Hz), 3.13 (1H, dd,
Jϭ14.0, 5.0 Hz), 3.06 (2H, q, Jϭ7.4 Hz), 2.83 (1H, dd, Jϭ14.0, 9.2 Hz),
1.25 (3H, t, Jϭ7.4 Hz), [a]D24 Ϫ9.2 (cϭ1.0, CH3CN). mp 75 °C. TOF-MS
m/z: 368.29 [MϩNa]ϩ (Calcd for C14H19NO7SNa: 368.36). Anal. Calcd for
C14H19NO7S: C, 48.69; H, 5.55; N, 4.06. Found: C, 47.98; H, 5.23; N, 4.06.
Coupling Reactions of Esc-Phe-OH on H-Leu-Rink-PEG Amide
Resin in Water Esc-Phe-OH (16 mg, 50 mmol) was coupled with H-Leu-
Rink-PEG amide resin (48 mg, 12.5 mmol. Prepared by Fmoc strategy in
DMF)) in aqueous 2% Triton X. WSCD (9.5 mg, 50 mmol) and an additive
were used for the coupling reaction. Various additives [HOBt (6.8 mg,
50 mmol), HOAt (6.8 mg, 50 mmol), HOSu (5.8 mg, 50 mmol), HONB
(9.0 mg, 50 mmol) and imidazole (3.4 mg, 50 mmol)] were tested to increase
the coupling yield. DIEA (8.7 ml, 50 mmol) or pyridine (4.0 ml, 50 mmol) was
added to keep reaction mixture at pH 8. After the coupling reaction, the Esc
group was removed with aqueous 0.025 mol/l NaOH and the resin was hy-
drolyzed. Phe and Leu content in a hydrolysate was analyzed and each cou-
pling yield was calculated from the amino acid ratio of Phe and Leu. Results References
are summarized in Table 2.
1) Hojo K., Maeda M., Kawasaki K., J. Peptide Sci., 7, 615—618 (2001).
Deprotection Studies Involving the Esc Group on Esc-Phe-Leu-Rink-
PEG Amide Resin with Various Bases in Water Esc-Phe-OH (32.9 mg,
2) Hojo K., Maeda M., Takahara Y., Yamamoto S., Kawasaki K., Tetrahe-
dron Lett., 44, 2849—2851 (2003).

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