Solid-phase peptide synthesis in water. Part 3: A water-soluble N-protecting group, 2-[phenyl(methyl)sulfonio]ethoxycarbonyl tetrafluoroborate, and its application to solid phase peptide synthesis in water

Article abstract of DOI:10.1016/j.tet.2003.12.036

Chemical synthesis of peptides has been performed in various organic solvents, but the safe disposal of organic solvents is now an important environmental issue. Our aim is to be able to perform solid-phase peptide synthesis in water. For this, we have designed a new water-soluble N-protecting group, 2-[phenyl(methyl)sulfonio]ethoxycarbonyl (Pms), and have studied its introduction onto amino acids. Pms-amino acids were prepared by treating 2-(phenylthio)ethoxycarbonyl amino acids with methyl iodide in the presence of silver tetrafluoroborate. Because sulfur-containing amino acids, such as Met and Cys, were modified by the reaction, we designed a new reagent, 2-[phenyl(methyl)sulfonio]ethyl-4-nitrophenyl carbonate, to introduce the Pms group on amino acids. This reagent is a stable crystalline material and its introduction onto amino acids (including sulfur-containing amino acids) was successful. The solid-phase synthesis of Leu- and Met-enkephalin amides using Pms-protected amino acids was successfully achieved in water.

Full text of DOI:10.1016/j.tet.2003.12.036

Tetrahedron 60 (2004) 1875–1886
Solid-phase peptide synthesis in water. Part 3: A water-soluble
N-protecting group, 2-[phenyl(methyl)sulfonio]ethoxycarbonyl
tetrafluoroborate, and its application to solid phase peptide
synthesis in waterq
*
Keiko Hojo, Mitsuko Maeda and Koichi Kawasaki
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan
Received 26 September 2003; accepted 11 December 2003
Abstract—Chemical synthesis of peptides has been performed in various organic solvents, but the safe disposal of organic solvents is now an
important environmental issue. Our aim is to be able to perform solid-phase peptide synthesis in water. For this, we have designed a new
water-soluble N-protecting group, 2-[phenyl(methyl)sulfonio]ethoxycarbonyl (Pms), and have studied its introduction onto amino acids.
Pms-amino acids were prepared by treating 2-(phenylthio)ethoxycarbonyl amino acids with methyl iodide in the presence of silver
tetrafluoroborate. Because sulfur-containing amino acids, such as Met and Cys, were modified by the reaction, we designed a new reagent,
2-[phenyl(methyl)sulfonio]ethyl-4-nitrophenyl carbonate, to introduce the Pms group on amino acids. This reagent is a stable crystalline
material and its introduction onto amino acids (including sulfur-containing amino acids) was successful. The solid-phase synthesis of Leu-
and Met-enkephalin amides using Pms-protected amino acids was successfully achieved in water.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
mechanism. Kunz also reported the use of 2-
(methylthio)ethoxycarbonyl5 and 2-(4-pyridyl)ethoxycar-
bonyl6 as a two-step-protecting group (Zweistufen–Schutz-
gruppe), which is removable under mild basic conditions
after its methylation or oxidation. In 1978, Kunz7 reported
preparation of a tripeptide, 2-[diphenyl(methyl)phospho-
nio]ethoxycarbonyl-Leu-Phe-Phe-OtBu, by the solution
method in water.
Chemical synthesis of peptides has been performed in
various organic solvents by both solid-phase and solution
methods. Advances in HPLC accelerated the development
of the solid-phase method, which in turn accelerated the
development of combinatorial chemistry. The solid-phase
method is now the principal method for peptide synthesis,
but it requires a large amount of organic solvent. Because
the safe disposal of organic solvent waste is an important
environmental issue, a method for peptide synthesis in water
using low toxic reagents would be desirable.
In previous papers, we reported the preparation of water-
soluble active esters (4-trimethylammoniophenyl ester and
sulfophenyl ester) and their application to peptide synthesis
by the solution method.8 Here, with the aim of achieving
solid phase peptide synthesis in water, we have designed
a new water-soluble N-protecting group. We designed the
2-[phenyl(methyl)sulfonio]ethoxycarbonyl tetrafluoroborate
(Pms) group (Fig. 1) as a water-soluble and easily
removable N-protecting group. Introduction of the Pms
group onto amino acids with various reagents was studied,
and Leu- and Met-enkephalin amides were synthesized by
the solid-phase method in water to evaluate the utility of
Pms-amino acids.
To perform the coupling reaction in water, protected amino
acids that are soluble in water are needed. Various polar
N-protecting groups that enhance solubility in polar
solvents including water have been reported, including a
methylsulfonylethoxycarbonyl group by Tesser and Bal-
vert-Geers,2 a 2-(triphenylphosphonio)ethoxycarbonyl
group by Kunz,3 and a 9-(2-sulfo)fluorenylmethoxycarbo-
nyl group by Merrifield and Bach.4 These protecting groups
are removable under basic conditions by a b-elimination
q See Ref. 1.
Keywords: 2-[Phenyl(methyl)sulfonio]ethoxycarbonyl group; Synthesis in
water; Water-soluble protecting group; Peptide synthesis; Solid phase
synthesis.
*
Corresponding author. Tel.: þ81-78-974-4794; fax: þ81-78-974-5689;
e-mail address: kawasaki@pharm.kobegakuin.ac.jp
Figure 1. Structure of Pms group.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.12.036

1876
K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
2. Results and discussion
and silver tetrafluoroborate. To overcome these limitations,
we designed preparation Routes 2–5 using various N-Pms
acylating agents, 2-[phenyl(methyl)sulfonio]ethyl chloro-
formate (Pms-Cl) 7, 2-[phenyl(methyl)sulfonio]-ethoxycar-
bonylsuccinimide (Pms-OSu) 9, 2-[phenyl(methyl)
sulfonio]ethoxycarbonyl-5-norbornene-endo-2,3-dicarboxy-
imide (Pms-ONB) 11, and 2-[phenyl(methyl)sulfonio]ethyl-
4-nitrophenyl carbonate (Pms-ONp) 13.1b
Pms-amino acids were prepared according to Route 1, as
shown in Scheme 1. 2-(Phenylthio)ethyl chloroformate 2
was prepared from 2-(phenylthio)ethanol 1 and phosgene
(prepared from triphosgen9) in dichloromethane. Com-
pound 2 was reacted with an amino acid to give
2-(phenylthio)ethoxycarbonyl (Pte) amino acid 3, which
was extracted by ethyl acetate from the reaction mixture and
showed a single spot on TLC. Compound 3 was used
without further purification and was treated with methyl
iodide and silver tetrafluoroborate to give Pms-amino acid
4. Pms-amino acids 4a–j (Table 1) were synthesized by
Route 1. These Pms-amino acids were obtained as
amorphous materials and identified by time-of-flight mass
spectrometry (Tof-MS) and NMR spectra (see Section 4). In
the 1H NMR spectra of Pms-amino acid 4, the methyl proton
adjacent to the sulfonium moiety showed a clear singlet at d
3.30. The signals due to the methylene region of the Pms
group appeared as highly complex multiplets at d 4.00–
4.40, which is much lower than this region in 3.
Pms-Cl 7 was prepared from phosgene9 and the onium salt
alcohol 2-[phenyl(methyl)sulfonio]ethanol 6, which was
converted from 1 by treatment with methyl iodide and silver
tetrafluoroborate. The resulting Pms-Cl 7 was unstable even
at low temperature, so it was used without purification to
prepare Pms-amino acid (Route 2). Introduction of the Pms
group onto Phe by using 7 at 210 8C in aqueous acetonitrile
resulted in Pms-Phe-OH 4a in low yield (27%) (Table 2).
HPLC analysis of the reaction mixture showed major
contaminant peaks of 2-[phenyl(methyl)sulfonio]ethanol 6
and its dehydrated product 5 (both might be derived from
decomposition of 7). These results indicate that 7 is so
unstable that it decomposes readily before reaction in
aqueous media such as aqueous acetonitrile. Thus, efficient
Pms acylating agents with a more stable leaving group than
chloride were required.
As expected, elimination of the Pms group from Pms-amino
acids proceeded according to a base-promoted b-elimin-
ation mechanism. The electron-withdrawing sulfonium
region of the Pms group renders hydrogens on the ethylene
portion labile, and thus the hydrogens are susceptible to
removal with weak bases to form the 2-[phenyl(methyl)-
sulfonio]ethylene 5. The mechanism of Pms elimination
from a Pms-amino acid was studied by treating Pms-Phe-
OH 4a with a 5% aqueous solution of NaHCO3 for 30 min.
The reaction mixture was checked by HPLC and Tof-MS
(Fig. 2). New peaks corresponding to 2-[phenyl(methyl)-
sulfonio]ethylene 5 and its water adduct, 2-[phenyl
(methyl)sulfonio]ethanol 6, were detected by Tof-MS and
HPLC, which verified that the Pms group was removed
through a b-elimination mechanism.
Because N-hydroxysuccinimide (HOSu) ester10 is hydro-
philic, has good reactivity and might be more stable than
chloride, the preparation of Pms-OSu 9 was designed in two
different ways, as shown in Scheme 2. In method A, the
preparation of 9 from 7 and HOSu was attempted, but 9
could not be obtained because of the instability of 7. In
method B, 9 was prepared via Pte-OSu 8, which was easily
purified by column chromatography. Next, 8 was treated
with methyl iodide and silver tetrafluoroborate to give 9.
Although crude 9 was obtained in over 90% purity, further
purification was difficult because of its instability. The 9 was
used without purification to react with Phe in aqueous
acetonitrile to give Pms-Phe-OH 4a in 68? yield. (Route 3).
Route 1 is an easy and efficient method by which to prepare
Pms-amino acids; however, Pms sulfur-containing amino
acids (Met and Cys) cannot be prepared, because the sulfurs
of Met and Cys are converted to the onium salt by treatment
with methyl iodide. In addition, it is not possible to prepare
Pms-amino acids with an acid-labile group [such as t-butyl
(tBu) group, trityl (Trt) group and 2,2,5,7,8-pentamethyl-
chroman-6-sulfonyl (Pmc) group] at their side chain could
not be prepared by Route 1, because the acid labile group
was cleaved by methylation procedure with methyl iodide
Next, the N-hydroxy-5-norbornene-endo-2,3-dicarboximide
(HONB)11 carbonate (Pms-ONB) 11 (Fig. 3) was examined
as an acylating agent (Route 4 in Scheme 3). Compound 11
was prepared via Pte-ONB 10 in the same way as the
preparation of 9. Compound 11 was obtained in over 90%
purity but, like 9, it could not be purified further without its
decomposition. The crude 11 was reacted with Phe (Route
4) in aqueous acetonitrile to give Pms-Phe-OH 4a in 66%
Scheme 1. Preparation of Pms-amino acid (Route 1).

K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1877
Table 1. Analytical data for Pms amino acids
Amino acid
MW.
Tof-Ms (m/z)
[a]2D4a
HPLCb room temperature (min)
Yield (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
14a
14b
14c
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
Phe
Gly
Ala
Val
Ile
Leu
Ser
Asp
360.45
270.33
284.35
312.41
326.43
326.43
300.35
328.36
310.39
376.45
344.47
387.50
558.73
432.55
356.46
384.47
398.49
370.48
569.69
583.72
592.23
499.60
635.82
441.53
360.4
270.2
284.4
312.4
326.5
326.5
300.3
328.6
310.1
376.4
344.3
387.6
558.9
432.9
356.9
384.4
398.16
370.3
570.0
583.5
592.3
499.7
635.8
442.3
29.8
—
23.4
14.3d
17.8d
16.7
19.2
20.4
18.9d
17.6
20.1
12.2d
21.2
18.7
32.7f
24.7f
22.8
24.6
28.1
25.9
30.1f
30.7f
25.5f
36.5f
40.9f
28.3
81c
73c
217.5
þ5.7
þ2.1
29.8
þ3.0
þ11.4
219.2
22.3
233.5
220.1
þ7.3
23.8
þ2.1
þ12
63c
73c
68c
72c
56c
46c
Pro
Tyr
Met
69c
86c
65e
Cys(Acm)
Cys(Trt)
Tyr(tBu)
Ser(tBu)
Asp(OtBu)
Glu(OtBu)
Thr(tBu)
Asn(Trt)
Gln(Trt)
His(Trt)
Trp(Boc)
Arg(Pmc)
Lys(Boc)
47e
68e
69,e 65g
61,e 86g
49,e 50g
62e
25.8
þ4.9
213.2
29.2
23.6
24.3
27.3
23.8
74e
69,e 61g
55,e 59g
72,e 42g
59,e 62g
63,e 41g
57,e 43g
a
c¼1.0, CH3CN.
b
c
d
e
f
Column, DAISOPAK SP-120-5-ODS-B (2.5£250 mm). Flow rate, 1 ml/min. Eluent, CH3CN/H2O containing 0.05% TFA. Gradient: 10/90–50/50 (40 min).
Route 1.
Gradient: 1/99–50/50 (49 min).
Route 5.
Gradient: 10/90–70/30 (30 min).
Route 4.
g
Table 2. Yields and reaction times of Routes 1–5 for preparation of Pms-Phe-OH
Route 1
Route 2, Pms-Cl
Route 3, Pms-Osu
Route 4, Pms-ONB
Route 5, Pms-Onp
Yield (%)
Reaction time
81
—
27
,5 min
68
4 h
66
4 h
88
.1 day
Figure 2. Tof-MS spectrum of the deprotection mixture of Pms-Phe-OH.

1878
K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
Scheme 2. Synthesis of Pms-OSu.
Figure 3. Reagents for preparing Pms-amino acids.
Scheme 3. Preparation of Pms-amino acid (Routes 3 and 4). Route 3: 9, Pms-OSu. Route 4: 11, Pms-ONB.
yield. Compound 11 has a high reactivity, similar to that of
9.
compared to that with 9 and 11. The acylation with 13 took
more than 1 day at room temperature, while the acylation
with 9 and 11 took 4 h at room temperature. However, the
yield of 4a was 88% by acylation with 13, which was better
than that obtained with either 9 (68%) or 11 (66%).
Acylation of Phe with 7, 9, 11 and 13 (Routes 1–5) is
summarized in Table 2. The three acylating agents 9, 11 and
13, but not 7, gave a satisfactory coupling yield.
Looking for a more stable acylating agent, we examined the
Pms-4-nitrophenyl carbonate (Pms-ONp) 13 (Route 5 in
Scheme 4). Compound 6 was reacted with 4-nitrophenyl
chloroformate 12 at room temperature in acetonitrile to give
13 in 71% yield. Compound 13 was obtained as colorless
crystal and could be stored for a few months in a
refrigerator. Acylation of Phe with 13 in the presence of
pyridine in aqueous acetonitrile (Route 5) was slow as
The procedure for introducing the Pms group onto sulfur-
containing amino acids, such as Met, Cys(Trt) and

K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1879
Scheme 4. Preparation of Pms-amino acid (Route 5).
Cys(Acm), was examined through Route 5. Pms-Met-OH
14a was prepared using 13 in a mixture of aqueous 0.1%
Triton X-100 solution and acetonitrile (1/1) in the presence
of pyridine with a yield of 65% (Table 1). Pms-Cys(Acm)-
OH 14b and Pms-Cys(Trt)-OH 14c were also prepared by
the same procedure. The yields of 14b and 14c were 47 and
68%, respectively (Table 1). Whereas 14a and 14b were
readily soluble, 14c was sparingly soluble in water.
Compound 14c was soluble in aqueous organic solvents,
such as 50% acetonitrile and 50% dimethylformamide
(DMF), and also soluble in aqueous 5% Triton X-100
solution. Thus the hydrophobicity of the trityl group is
greater than the hydrophilicity of the Pms group in 14c.
negative result in the Kaiser test13 (ninhydrin test). The
resulting Fmoc-Gly-Phe-Leu-TentaGel resin was treated
with 20% piperidine/DMF to remove the Fmoc group. The
resin was hydrolyzed, and the amino acids in the acid
hydrolysate were analyzed. Removal of the Pms group from
the Pms-Phe-Leu-TentaGel resin was calculated from the
amino acid ratio of Gly and Phe in the acid hydrolysate. The
Pms group was removable with mild bases, such as an
aqueous solution of either 5% NaHCO3 or 5% Na2CO3, as
shown in Table 3. Treatment with an aqueous solution of
5% NaHCO3 for 30 min was not strong enough to remove
the Pms group completely, but a double treatment
(30 min£2) gave a satisfactory result.
Pms-amino acids with an acid-labile group [such as t-butyl
(tBu) group, trityl (Trt) group and 2,2,5,7,8-pentamethyl-
chroman-6-sulfonyl (Pmc) group] at their side chain could
not be prepared by Route 1, 11 and 13 were used to prepare
these Pms-amino acids (Schemes 3 and 4) and results were
passable as shown in Table 1.
An important factor to consider in solid-phase synthesis is
the swelling ability of the resin. To perform solid-phase
synthesis in water, a resin is required to swell in water.
However, the most common core resin, polystyrene resin,
does not swell in polar solvents such as methanol and water.
A poly(ethylene glycol)-grafted polystyrene resin, TentaGel
resin,14 has been reported as a resin designed to have
increased swelling ability in both polar and non-polar
solvents. This TentaGel resin was used for synthesis of Leu-
enkephalin amide using a water-soluble carbodiimide
[WSCD, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride15] as a coupling reagent in water. Because
the introduction of Phe onto the resin and the subsequent
coupling reaction for peptide synthesis was slow under the
above conditions, an aqueous solution of 0.2% Triton X-100
was used as a solvent to increase the swelling ability of the
resin and to increase the solubility of reactants. Further-
more, HOSu, which is soluble in water, was used as an
additive to accelerate the coupling reactions.16 Although the
coupling reactions were accelerated under this condition, b-
Ala was found in the acid hydrolysate of the synthetic Leu-
enkephalin amide. Pure synthetic Leu-enkephalin amide
could not be separated from by-products which contained b-
Ala by HPLC. The amino acid ratios in the acid hydrolysate
To evaluate Pms-amino acids, Leu-enkephalin amide (H-
Tyr-Gly-Gly-Phe-Leu-NH2) was synthesized by the solid-
phase method in water. Before the synthesis, removal of the
Pms group by various base treatments was examined using
Pms-Phe-Leu-TentaGel resin. Because removal of the Pms
group on Phe-Leu-TentaGel resin with base was slower than
that on Phe itself, the Pms-Phe-Leu-TentaGel resin was
preferentially used as the test compound. Hydrophobic or
bulky sequence like Phe-Leu may have influence on
removal of the Pms group in water. The resin was treated
with various base solutions to remove the Pms group in
water and then reacted with 9-fluorenylmethoxycarbonyl
glycine (Fmoc-Gly-OH) with diisopropylcarbodiimide/1-
hydroxybenzotriazole12 in DMF until the resin gave a
Table 3. Deprotection studies on the Pms-Phe-Leu-TentaGel resin
Entry
Reagents
Time (min) Yield (%)
Table 4. Synthetic protocol for the solid phase peptide synthesis in water
1
2
5% NaHCO3/H2O
5% NaHCO3/H2O
30
30
80
96
Step
Reagents
Time
3
4
5
6
7
8
9
10
0.01 mol/l NaOH/H2O
0.01 mol/l NaOH/H2O
0.005 mol/l NaOH/EtOH–H2O (1/1) 3, 3, 3
0.001 mol/l NaOH/EtOH–H2O (1/1) 3, 3, 3
2.5% NaHCO3/EtOH–H2O (1/1)
2.5% NaHCO3/EtOH–H2O (1/1)
2.5% Na2CO3/EtOH–H2O (1/1)
2.5% Na2CO3/EtOH–H2O (1/1)
1, 1, 1
3, 3, 3
100
100
86
77
81
100
75
100
1
2
3
4
5
H2O
3 min£2
Aq. 5.0% NaHCO3 or aq. 0.01 mol/l NaOH 30 min£2 or 3 min£2
H2O
3 min£2
3 min£3
3 h
3, 3, 3
5, 5, 5
3, 3, 3
5, 5, 5
Aq. 0.2% Triton X
Pms-amino acid, WSCD, HONB, in aq.
0.2% Triton X
6
Aq. 0.2% Triton X
3 min£3

1880
K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
Figure 4. HPLC profiles of synthetic Leu-enkephalin amide. (A)
Preparative HPLC Column, DAISOPAK SP-120-5-ODS-B
Figure 5. HPLC profile of synthetic crude Met-enkephalin amide. (A)
Crude Met-enkephalin amide prepared on CLEAR resin. (B) Crude Met-
enkephalin amide prepared on TentaGel resin. Column, DAISOPAK SP-
120-5-ODS-B (2.5£250 mm). Flow rate, 1 ml/min. Eluent, CH3CN/H2O
containing 0.05% TFA. Gradient: 10/90–50/50 (20 min).
(20£250 mm). Flow rate, 10 ml/min. Eluent, CH3CN/H2O containing
0.05% TFA. Gradient: 10/90–50/50 (40 min). (B) Analytical HPLC of the
purified sample. Column, DAISOPAK SP-120-5-ODS-B (2.5£250 mm).
Flow rate, 1 ml/min. Eluent, CH3CN/H2O containing 0.05% TFA.
Gradient: 10/90–50/50 (40 min).
and after the main peak of Met-enkephalin amide, which
corresponded to that of Met-enkephalin amide prepared by
the Fmoc-based solid-phase method using organic solvents.
The early peaks contained deletion peptides (such as H-Tyr-
Gly-Phe-Met-NH2 and H-Gly-Gly-Phe-Met-NH2) and oxi-
dized peptide (such as H-Tyr-Gly-Gly-Phe-Met(O)-NH2
and H-Tyr-Gly-Phe-Met(O)-NH2), whereas the later peaks
contained non-peptide compounds that probably derived
from the used resin.
of synthetic crude Leu-enkephalin separated by HPLC was;
Tyr 0.82, Gly 1.92, Phe 1.06, b-Ala 4.69. Formation of b-
Ala has been reported as a product of a side reaction (Lossen
rearrangement reaction17) when HOSu is used as an
additive. We did not examine this side reaction (Lossen
rearrangement reaction) further, but it might occur more
easily in aqueous media than in non-polar solvents.
Next, HONB,11 which is also water-soluble, was used as an
additive instead of HOSu and Leu-enkephalin was syn-
thesized according to the protocol shown in Table 4. The
Pms group was removed by treatment with an aqueous
solution of 5% NaHCO3 for 30 min, or by two lots of this
treatment for Phe, because removal of the Pms group on Phe
was slower than that on other Pms-amino acids, as described
above. The synthetic Pms-Tyr-Gly-Gly-Phe-Leu-TentaGel
resin was treated with an aqueous solution of 5% NaHCO3
and then treated with trifluoroacetic acid (TFA) to cleave the
peptide from the resin. The product was purified by HPLC
on an ODS column. The HPLC profile of the synthetic Leu-
enkephalin amide is shown in Figure 4. The retention time
of the synthetic Leu-enkephalin amide was identical to that
of Leu-enkephalin amide prepared by the Fmoc-based solid-
phase method using organic solvents. The total yield
calculated from the amino group content of the starting
TentaGel resin was 61%.
For comparison, Met-enkephalin amide was also syn-
thesized in water using TentaGel resin, and the HPLC
profile of the crude product is shown in Figure 5B. Again,
minor peaks of deletion peptides (such as H-Tyr-Gly-Phe-
Met-NH2 and H-Gly-Gly-Phe-Met-NH2) and oxidized
peptides (such as H-Tyr-Gly-Gly-Phe-Met(O)-NH2 and H-
Tyr-Gly-Phe-Met(O)-NH2) were observed before and after
the main peak of Met-enkephalin amide. The yield of Met-
enkephalin amide prepared on TentaGel resin was 32% and
was just a little better than that of Met-enkephalin amide
prepared on CLEAR resin (29%).
The yield of Leu-enkephalin and Met-enkephalin using
TentaGel resin was 61 and 32%, respectively. Synthetic
protocol for preparation of these 2 enkephalins was different
(deprotection with 5% NaHCO3 and 0.01 mol/l NaOH), but
this difference might be mainly derived from properties of
Leu and Met. The synthesis of both Met and Leu-enkephalin
amide was performed under atmosphere (i.e. not under inert
gas). Oxidation of Met during synthesis was observed in the
profile of HPLC and might cause the lower yield.
In addition to the TentaGel resin, a cross-linked ethoxylate
acrylate resin (CLEAR resin which has been also reported18
to swell not only with organic solvents but also with water)
was examined as a solid support for synthesis in water. Met-
enkephalin amide was synthesized using CLEAR resin in
water according to the same procedure (shown in Table 4)
except for the base treatment. The Pms group was removed
by two 3-min treatments with an aqueous solution of
0.01 mol/l NaOH, which yielded a more rapid and complete
removal of the Pms group. The synthetic H-Tyr-Gly-Gly-
Phe-Met-CLEAR-resin was treated with TFA to cleave the
peptide from the resin, and then purified by HPLC on an
ODS column. The crude HPLC profile of the synthetic Met-
enkephalin amide is shown in Figure 5A. The yield
calculated from amino group content of the starting
CLEAR resin was 29%. Minor peaks were observed before
3. Conclusion
Pms, a new water-soluble N-protecting group with high base
lability and high polarity, has been developed and its
application to solid-phase peptide synthesis in water has
been evaluated. The derivative Pms-ONp was designed to
prepare all types of Pms-amino acids including sulfur-
containing amino acids. This reagent is a crystalline
compound and can be kept stable in a refrigerator for a
few months. Leu- and Met-enkephalin amides were
successfully synthesized by the solid-phase method in

K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1881
water using Pms-amino acids and, as such, this study may be
the first to report the successful synthesis of peptides by the
solid-phase method in water. In addition, we evaluated the
potential of CLEAR resin and TentaGel resin for solid-
phase synthesis in water. Future work should aim to develop
a new resin that swells more than these two resins.
(400 MHz, CD3CN) d 7.40 (d, J¼7.4 Hz, 2H), 7.29 (t,
J¼7.4 Hz, 2H), 7.20 (t, J¼7.4 Hz, 1H), 4.30 (s, 1H), 4.17 (t,
J¼6.9 Hz, 2H), 3.16 (t, J¼6.9 Hz, 2H), 1.37 (d, J¼7.2 Hz,
3H) Rf1 0.28.
4.1.4. Pte-Val-OH (3d). Oily material. Yield 82%. 1H NMR
(400 MHz, CD3CN) d 7.39 (d, J¼7.4 Hz, 2H), 7.29 (t,
J¼7.4 Hz, 2H), 7.19 (t, J¼7.4 Hz, 1H), 4.19 (t, J¼6.9 Hz,
2H), 4.06 (s, 1H), 3.16 (t, J¼6.9 Hz, 3H), 2.16 (m, 1H), 0.97
(d, J¼6.8 Hz, 3H), 0.94 (d, J¼6.8 Hz, 3H). Rf1 0.51.
4. Experimental
Optical rotations were determined with an automatic
polarimeter, model DIP-360 (Japan Spectroscopic Co.)
Tof-Mass spectra were measured with a KRATOS-MALDI
mass spectrometer (SiMADZU Co.) and ESI-Mass spectra
were measured with a Waters ZQ2000 mass spectrometer.
Reversed phase HPLC was performed using a Waters model
600 equipment with a DISOPAK column and gradient
system of acetonitrile/water containing 0.05% TFA. Open
column chromatography was performed on Silica gel 60
(BW-127ZH, Fuji Silicia Chemical Co.). Solvent system for
ascending thin-layer chromatography on Silica gel G (type
60, Merck) is indicated as follows: Rf1¼CHCl3–MeOH–
4.1.5. Pte-Ile-OH (3e). Oily material. Yield 76%. 1H NMR
(400 MHz, CD3CN) d 7.39 (d, J¼7.4 Hz, 2H), 7.29 (t,
J¼7.4 Hz, 2H), 7.19 (t, J¼7.4 Hz, 1H), 4.18 (t, J¼6.9 Hz,
2H), 4.08 (s, 1H), 3.16 (t, J¼6.9 Hz, 2H), 1.87 (m, 1H), 1.52
(m, 1H), 1.25 (m, 1H), 0.95 (d, J¼7.0 Hz, 3H), 0.92 (t,
J¼7.0 Hz, 3H). Rf1 0.57.
4.1.6. Pte-Leu-OH (3f). Oily material. Yield 80%. 1H NMR
(400 MHz, CD3CN) d 7.40 (d, J¼7.4 Hz, 2H), 7.29 (t,
J¼7.4 Hz, 2H),7.19(t, J¼7.4 Hz, 1H),4.17(t, J¼6.9 Hz, 2H),
4.16 (s, 1H), 3.16 (t, J¼6.9 Hz, 2H), 1.72 (m, 1H), 1.59 (m,
1H), 0.95 (d, J¼5.3 Hz, 3H), 0.93 (t, J¼5.3 Hz, 3H). Rf1 0.56.
4.1.7. Pte-Ser-OH (3g). Oily material. Yield 65%. 1H NMR
(400 MHz, CD3CN) d 7.40 (d, J¼7.3 Hz, 2H), 7.28 (t,
J¼7.3 Hz, 2H), 7.20 (t, J¼7.3 Hz, 1H), 4.53 (br s, 1H), 4.20
(t, J¼6.9 Hz, 2H), 3.89 (dd-like, 1H), 3.83 (dd-like, 1H),
3.16 (t, J¼6.9 Hz, 2H). Rf1 0.24.
1
H2O (8/3/1, lower phase). H NMR spectra were recorded
on a Bruker 400 MHz spectrometer. 2-(phenylthio)ethanol
and 4-nitorphenyl chloroformate were purchased from
Tokyo Kasei Co., Ltd, Japan. HOSu, HONB and amino
acid derivatives were purchased from Watanabe Chemical
Industries, Ltd, Japan.
4.1. Route 1. Preparation of Pte-amino acids
1
4.1.8. Pte-Asp-OH (3h). Colorless solid. Yield 66%. H
NMR (400 MHz, CD3CN) d 7.41 (d, J¼7.4 Hz, 2H), 7.29 (t,
J¼7.4 Hz, 2H), 7.21 (t, J¼7.4 Hz, 1H), 4.50 (s, 1H), 4.19 (t,
J¼6.9 Hz, 2H), 3.16 (t, J¼6.9 Hz, 2H), 2.75 (m, 1H). Rf1 0.12.
4.1.9. Pte-Pro-OH (3i). Colorless solid. Yield 78%. 1H
NMR (400 MHz, CD3CN) d 7.39 (d, J¼7.3 Hz, 2H), 7.30 (t,
J¼7.3 Hz, 2H), 7.20 (t, J¼7.3 Hz, 1H), 4.24 (m, 1H), 4.20
(m, 2H), 3.47 (m, 1H), 3.20 (m, 1H), 3.18 (t, J¼6.9 Hz, 2H)
2.43 (m, 1H), 2.00 (m, 1H), 1.89 (m, 2H). Rf1 0.51.
4.1.1. Pte-Phe-OH (3a). To a solution of 2-(phenyl-
thio)ethanol (4.4 ml, 30 mmol) and triphosgen (2.96 g,
10 mmol) in tetrahydrofuran (100 ml), Et3N (4.18 ml,
30 mmol) in tetrahydrofuran (40 ml) was added dropwise
at room temperature and the reaction mixture was stirred for
1.5 h. The mixture was filtered and the solvent was
evaporated. The residue was dissolved in acetonitrile
(30 ml) and added to a solution of Phe (4.95 g, 30 mmol)
and Et3N (4.18 ml, 30 mmol) in a mixture of acetonitrile
and H2O (1/1, 100 ml) at 0 8C. The mixture was stirred for
2 h at room temperature keeping its pH at 8 by addition of
Et3N and the solvent was removed in vacuo. The residue
was dissolved in AcOEt and extracted with aqueous 5%
NaHCO3. The aqueous layer was acidified with 1.0 mol/l
HCl, and the resulting precipitate was extracted with
AcOEt. The extract was washed with saturated NaCl
solution and concentrated in vacuo to leave an oily material.
Yield 890 mg, 86%. 1H NMR (400 MHz, CD3CN) d 7.41 (d,
J¼7.4 Hz, 2H), 7.29 (t, J¼7.4 Hz, 2H), 7.25 (m, 5H), 7.21
(t, J¼7.4 Hz, 1H), 4.43 (dd, J¼5, 14 Hz, 1H), 4.18 (t,
J¼6.9 Hz, 2H) 3.18 (t, J¼6.9 Hz, 3H), 3.05 (m, 1H), 2.92
(m, 1H). Rf1 0.60.
4.1.10. Pte-Tyr-OH (3j). Oily material. Yield 68%. 1H
NMR (400 MHz, CD3CN) d 7.40 (d, J¼7.4 Hz, 2H), 7.32 (t,
J¼7.4 Hz, 2H), 7.22 (t, J¼7.4 Hz, 1H), 7.07 (d, J¼8.6 Hz,
2H), 6.74 (d, J¼8.6 Hz, 2H), 4.32 (m, 1H), 4.14 (t,
J¼6.9 Hz, 2H), 3.16 (t, J¼6.9 Hz, 2H), 3.13 (m, 1H), 2.83
(m, 1H). Rf1 0.34.
4.2. Preparation of Pms-amino acids
Yields, MS spectra data and rotations of synthetic Pms-
amino acids are shown in Table 1.
4.2.1. Pms-Phe-OH (4a). To an acetonitrile (15 ml)
solution of 3a (400 mg, 1.15 mmol) and silver tetrafluoro-
borate (389 mg, 2.0 mmol), methyl iodide (0.8 ml,
2.0 mmol) was added and the mixture was refluxed over-
night at 40 8C. After cooling, the yellow precipitates were
filtered off and the solvent was removed in vacuo. The
residue was purified by preparative HPLC. Amorphous
The following Pte-amino acids were prepared according to
the procedure described above.
1
4.1.2. Pte-Gly-OH (3b). Colorless solid. Yield 78%. H
NMR (400 MHz, D2O) d 7.39 (d, J¼7.4 Hz, 2H), 7.29 (t,
J¼7.4 Hz, 2H), 7.20 (t, J¼7.4 Hz, 1H), 4.17 (t, J¼6.9 Hz,
2H), 3.76 (s, 2H), 3.16 (t, J¼6.9 Hz, 3H). Rf1 0.20.
1
material. H NMR (400 MHz, D2O) d 7.95 (d, J¼7.5 Hz,
2H), 7.81 (t, J¼7.3 Hz, 1H), 7.71 (dd, J¼7.5, 7.3 Hz, 2H),
4.1.3. Pte-Ala-OH (3c). Oily material. Yield 70% 1H NMR
7.25 (m, 5H), 4.45 (m, 1H), 4.39 (m, 1H), 4.20 (m, 1H), 3.99

1882
K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1
(m, 2H), 3.34 (s, 3H), 3.23 (m, 1H), 2.91 (m, 1H). Anal.
Calcd for C19H22BF4NO4S·1/3TFA: C, 48.68; H, 4.64; N,
2.89. Found: C, 48.62; H, 4.32; N, 2.89.
4.2.9. Pms-Pro-OH (4i). Amorphous material. H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.5 Hz, 2H), 7.84 (t,
J¼7.3 Hz, 1H), 7.75 (dd, J¼7.5, 7.3 Hz, 2H), 4.57 (m,
1H), 4.38 (m, 1H), 4.16 (m, 1H), 4.00 (m, 2H), 3.30 (s, 3H),
3.47–3.01 (m, 2H), 2.22 (m, 1H), 2.00 (m, 1H), 1.84 (m,
2H). Anal. Calcd for C15H20BF4NO4S·TFA: C, 39.86; H,
4.33; N, 2.73. Found: C, 39.54; H, 4.10; N, 2.74.
The following Pms-amino acids were prepared according to
the procedure described above (Route 1).
1
4.2.2. Pms-Gly-OH (4b). Amorphous material. H NMR
1
(400 MHz, D2O) d 7.95 (d, J¼7.8 Hz, 2H), 7.82 (t,
J¼7.2 Hz, 1H), 7.72 (dd, J¼7.8, 7.2 Hz, 2H), 4.52 (dt,
J¼13, 5.1 Hz, 1H), 4.32 (dt, J¼13, 5.1 Hz, 1H), 4.00 (t,
J¼5.1 Hz, 2H), 3.74 (s, 1H), 3.31 (s, 3H). Anal. Calcd for
C12H16BF4NO4S·1/2TFA: C, 37.70; H, 4.02; N, 3.38.
Found: C, 37.92; H, 4.23; N, 3.36.
4.2.10. Pms-Tyr-OH (4j). Amorphous material. H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.5 Hz, 2H), 7.80 (t, J¼7.3 Hz,
1H), 7.71 (dd, J¼7.5, 7.3 Hz, 2H), 7.07 (d, J¼8.4 Hz, 2H),
6.73 (d, J¼8.4 Hz, 2H), 4.41 (m, 1H), 4.34 (m, 1H), 4.19 (m,
1H), 3.97 (m, 1H), 3.88 (m, 1H), 3.31 (s, 3H), 3.13 (m, 1H),
2.81 (m, 1H). Anal. Calcd for C19H22BF4NO5S·1/2TFA: C,
46.17; H, 4.36; N, 2.69. Found: C, 46.43; H, 4.52; N, 2.60.
1
4.2.3. Pms-Ala-OH (4c). Amorphous material. H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.8 Hz, 2H), 7.84 (t,
J¼7.2 Hz, 1H), 7.74 (dd, J¼7.8, 7.2 Hz, 2H), 4.50 (m,
1H), 4.34 (m, 1H), 4.30 (m, 1H), 4.01 (m, 2H), 3.31 (s, 3H),
1.31 (m, 3H). Anal. Calcd for C13H18BF4NO4S·1/
2TFA·CH3CN: C, 40.15; H, 4.49; N, 4.68. Found: C,
40.41; H, 4.22; N, 4.29.
4.3. Route 2
4.3.1. 2-[Phenyl(methyl)sulfonio]ethanol (6). To a solu-
tion of 2-(phenylthio)ethanol (1.34 ml, 10 mmol) and silver
tetrafluoroborate (2.32 g, 12 mmol) in acetonitrile (60 ml),
methyl iodide (1.24 ml, 20 mmol) was added and the
mixture was stirred overnight at 40 8C. After cooling, the
yellow precipitate was filtered off and the solvent was
evaporated in vacuo. The residue was flash chromato-
graphed on silica gel, eluted with CH3Cl–MeOH–H2O (8/
3/1, lower phase). The collected fractions were combined
and concentrated in vacuo to leave a colorless oil. Yield
1
4.2.4. Pms-Val-OH (4d). Amorphous material. H NMR
(400 MHz, D2O) d 7.97 (d, J¼7.5 Hz, 2H), 7.84 (t,
J¼7.3 Hz, 1H), 7.75 (dd, J¼7.5, 7.3 Hz, 2H), 4.51 (m,
1H), 4.34 (m, 1H), 4.01 (t, J¼5.2 Hz, 2H), 3.92 (m, 1H),
3.33 (s, 3H), 2.11 (m, 1H), 0.93 (d-like, 3H), 0.92 (d-like,
3H). Anal. Calcd for C15H22NBF4O4S·2/5TFA: C, 40.59; H,
4.69; N, 3.68. Found: C, 40.97; H, 4.21; N, 3.56.
1
2.26 g, 88%. H NMR (400 MHz, D2O) d 7.97 (dd-like,
J¼8.0, 1.7 Hz, 2H), 7.84 (tt, J¼7.5, 1.7 Hz, 1H), 7.75 (dd-
like, J¼8.0, 7.5 Hz, 2H), 4.03 (m, 1H), 4.34 (m, 1H), 3.90
(m, 2H), 3.82 (s, 3H), 3.78 (m, 1H), 3.31 (s, 3H). Tof-MS
m/z 169.14 (Mþ, C9H13OS requires 169.26).
4.2.5. Pms-Ile-OH (4e). Amorphous material. 1H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.5 Hz, 2H), 7.83 (t,
J¼7.3 Hz, 1H), 7.73 (dd, J¼7.5, 7.3 Hz, 2H), 4.54 (m,
1H), 4.34 (m, 1H), 4.01 (br s, 2H), 3.95 (m, 1H), 3.31 (s,
3H), 1.82 (m, 1H), 1.38 (dquint., J¼14.0, 7.0 Hz, 1H), 1.17
(dquint., J¼14.0, 7.0 Hz, 1H), 0.90 (3H, d, J¼7.0 Hz, bC–
CH3), 0.89 (3H, t, J¼7.0 Hz, gC–CH3). Anal. Calcd for
C16H24BF4NO4S·2/3TFA: C, 39.06; H, 4.40; N, 2.40.
Found: C, 38.67; H, 4.20; N, 2.22.
4.3.2. Pms-Phe-OH (4a). To a suspension of 6 (513 mg,
2.0 mmol) and triphosgen (394 mg, 1.33 mmol) in dichloro-
methane (30 ml), Et3N (0.56 ml, 4.0 mmol) in dichloro-
methane (10 ml) was added dropwise at 210 8C, and the
mixture was stirred at 210 8C for 1.5 h. After evaporation of
the solvent in vacuo at 10 8C, the residue was dissolved in
acetonitrile (15 ml), and this solution was added to a solution
of Phe (330 mg, 2.0 mmol) and Et3N (0.35 ml, 2.5 mmol) in a
mixture of acetonitrile and water (1/1, 50 ml) at 0 8C. The
reaction mixture was stirred at 0 8C for 2 h and the solvent was
removed in vacuo. The product was purified by preparative
HPLC. Yield: 241 mg. The product was identified with Pms-
Phe-OH prepared through Route 1 by NMR and Mass spectra.
1
4.2.6. Pms-Leu-OH (4f). Amorphous material. H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.5 Hz, 2H), 7.83 (t,
J¼7.3 Hz, 1H), 7.73 (dd, J¼7.5, 7.3 Hz, 2H), 4.54 (m,
1H), 4.34 (m, 1H), 4.01 (br s, 2H), 3.98 (m, 1H), 3.31 (s, 3H),
1.62 (m, 1H), 1.48 (m, 2H), 0.93 (d, J¼5.5 Hz, 3H), 0.90 (t,
J¼5.5 Hz, 3H). Anal. Calcd for C16H24BF4NO4S·TFA: C,
41.51; H, 4.68; N, 2.59. Found: C, 41.00; H, 4.78; N, 2.66.
4.4. Route 3
1
4.2.7. Pms-Ser-OH (4g). Amorphous material. H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.5 Hz, 2H), 7.83 (t,
J¼7.3 Hz, 1H), 7.73 (dd, J¼7.5, 7.3 Hz, 2H), 4.54 (m, 1H),
4.34 (m, 1H), 4.22 (m, 1H), 4.01 (br s, 2H), 3.31 (s, 3H), 3.94
(m, 1H), 3.86 (m, 1H). Anal. Calcd for C13H18BF4NO5S: C,
40.33; H, 4.69; N, 3.62. Found: C41.01; H, 4.36; N, 3.59.
4.4.1. Pte-OSu (8). To a solution of 2-(phenylthio)ethanol
(1.34 ml, 10 mmol) and triphosgen (1.96 g, 6.6 mmol) in
tetrahydrofuran (40 ml), Et3N (2.79 ml, 20 mmol) in tetra-
hydrofuran (15 ml) was added dropwise at 0 8C. The
reaction mixture was stirred at 0 8C for 1.5 h and the
mixture was filtered. The solvent was evaporated off in
vacuo and the residue was dissolved in acetonitrile (15 ml).
The solution was added to a solution of HOSu (1.15 g,
10 mmol) and Et3N (1.39 ml, 10 mmol) in a mixture of
acetonitrile (30 ml) at 0 8C, and the mixture was stirred at
room temperature for 3 h keeping its pH at 8. The solvent
was removed in vacuo and the residue was purified by flash
chromatography on silica gel, using hexane–ethyl acetate
1
4.2.8. Pms-Asp-OH (4h). Amorphous material. H NMR
(400 MHz, D2O) d 7.96 (d, J¼7.7 Hz, 2H), 7.83 (t,
J¼7.5 Hz, 1H), 7.73 (dd, J¼7.7, 7.5 Hz, 2H), 4.50 (m,
1H), 4.47 (m, 1H), 4.34 (m, 1H), 4.01 (br s, 2H), 3.32 (s,
3H), 2.78 (m, 2H). Anal. Calcd for C14H18BF4NO6S·2/
3TFA·1/2CH3CN: C, 35.94; H, 3.42; N, 3.40. Found: C,
35.63; H, 3.49; N, 3.46.

K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1883
1
(2/1) as an eluate. The collected fractions were combined
and concentrated in vacuo to leave a colorless oil. Yield.
2.12 g, 72%. H NMR (400 MHz) d (CD3CN): 7.42 (d,
J¼7.1 Hz, 2H), 7.34 (dd, J¼7.3, 7.1 Hz, 2H), 7.25 (dd,
J¼7.3 Hz, 1H), 4.43 (t, J¼6.5 Hz, 2H), 3.27 (t, J¼6.5 Hz,
2H), 2.76 (s, 4H). ESI-MS m/z 313.2[(MþNH4)þ,
C13H13NO5·NH4 requires 313.3]. Rf [(hexane–ethyl acetate
(2/1)] 0.38.
oil. Yield 450 mg, 98%. H NMR (400 MHz, CD3CN) d
7.96 (d, J¼7.5 Hz, 2H), 7.84 (t, J¼7.4 Hz, 1H), 7.74 (dd,
J¼7.5, 7.4 Hz, 2H), 6.18 (s, 2H), 4.73 (m, 1H), 4.49 (m,
1H), 4.04 (m, 1H), 3.93 (m, 1H), 3.45 (d-like, 2H), 3.30
(d-like, 2H), 3.29 (s, 3H), 1.78 (dd, J¼9.0, 1.6 Hz, 1H), 1.52
(dd, J¼9.0, 1.6 Hz, 1H). Tof-MS m/z 374.5 (Mþ,
C19H20NO5S requires 374.43).
1
4.5.3. Pms-Phe-OH (4a). To a solution of Phe (83 mg,
0.5 mmol) and pyridine (40.43 ml, 0.5 mmol) in aqueous
0.1% Triton X-100 solution–acetonitrile (1/1, 20 ml), 11
[prepared from 10 (179 mg, 0.5 mmol)] in acetonitrile
(10 ml) was added at 0 8C and the mixture was stirred at
room temperature for 4 h. The solvent was removed in
vacuo and the residue was purified by preparative HPLC to
give a colorless oil. The product was identified with Pms-
Phe-OH prepared through Route 1 by NMR and Mass
spectra.
4.4.2. Pms-OSu (9). To an acetonitrile solution (15 ml) of 8
(295 mg, 1.0 mmol) and silver tetrafluoroborate (232 mg,
1.2 mmol), methyl iodide (1.24 ml, 20 mmol) was added,
and the mixture was stirred overnight at 40 8C. After
cooling, the yellow precipitate was filtered off and the
solvent was removed in vacuo to leave a colorless oil. Yield
384 mg, 97%. 1H NMR (400 MHz, CD3CN) d 7.96 (d,
J¼7.5 Hz, 2H), 7.84 (t, J¼7.4 Hz, 1H), 7.74 (dd, J¼7.5,
7.4 Hz, 2H), 4.73 (m, 1H), 4.49 (m, 1H), 4.04 (m, 1H), 3.93
(m, 1H), 3.29 (s, 3H), 2.76 (s, 4H). Tof-MS m/z 310.41 (Mþ,
C14H16NO5S requires 310.35).
The following Pms-amino acids were prepared from Pms-
ONB and a corresponding amino acid according to the
procedure described above (Route 4).
4.4.3. Pms-Phe-OH (4a). To a solution of Phe (83 mg,
0.5 mmol) and pyridine (40.43 ml, 0.5 mmol) in aqueous
0.1% Triton X-100 solution–acetonitrile (1/1, 20 ml), 9
[prepared from 8 (146 mg, 0.5 mmol)] in acetonitrile
(10 ml) was added at 0 8C, and the mixture was stirred at
room temperature for 4 h. The solvent was removed in
vacuo, the product was purified by preparative HPLC to
give a colorless oil. The product was identified with Pms-
Phe-OH prepared through Route 1 by NMR and Mass
spectra.
1
4.5.4. Pms-Tyr(tBu)-OH (15a). Amorphous material. H
NMR (400 MHz, CD3CN) d 7.86 (d, J¼7.6 Hz, 2H), 7.80 (t,
J¼7.6 Hz, 1H), 7.69 (dd, J¼7.5, 7.3 Hz, 2H), 7.14 (d,
J¼8.4 Hz, 2H), 6.93 (d, J¼8.4 Hz, 2H), 4.35 (m, 1H), 4.34
(m, 1H), 4.13 (m, 1H), 3.80 (m, 1H), 3.72 (m, 1H), 3.18 (s,
3H), 3.14 (m, 1H), 2.89 (m, 1H), 1.29 (s, 9H). Anal. Calcd
for C23H30BF4NO5S·TFA: C, 47.41; H, 4.93; N, 2.21.
Found: C, 47.85; H, 4.54; N, 2.40.
1
4.5. Route 4
4.5.5. Pms-Ser(tBu)-OH (15b). Amorphous material. H
NMR (400 MHz, CD3CN) d 7.96 (d, J¼7.7 Hz, 2H), 7.84 (t,
J¼7.4 Hz, 1H), 7.72 (dd, J¼7.7, 7.4 Hz, 2H), 4.54 (m, 1H),
4.34 (m, 1H), 4.28 (br m, 1H), 4.02 (br s, 2H), 3.77 (m, 1H),
3.68 (m, 1H), 3.32 (s, 3H), 1.19 (s, 9H). Anal. Calcd for
C17H26BF4NO5S·1/2TFA: C, 43.21; H, 5.34; N, 2.80.
Found: C, 43.35; H, 4.86; N, 2.66.
4.5.1. Pte-ONB (10). To a solution of 2-(phenylthio)ethanol
(1.34 ml, 10 mmol) and triphosgen (1.96 g, 6.6 mmol) in
tetrahydrofuran (40 ml), Et3N (2.79 ml, 20 mmol) in
tetrahydrofuran (15 ml) was added dropwise at 0 8C.
The reaction mixture was stirred at 0 8C for 1.5 h and
filtered. The solvent was removed filtrate in vacuo and the
residue was dissolved in acetonitrile (15 ml) and added to
a solution of HONB (1.79 g, 10 mmol) and Et3N
(1.39 ml, 10 mmol) in acetonitrile (30 ml) at 0 8C. The
reaction mixture was stirred at room temperature for 3 h
keeping it pH at 8. The solvent was removed in vacuo
and the residue was purified by flash chromatography on
silica gel using hexane–ethyl acetate (2/1) as an eluate.
The collected fractions were combined and concentrated
4.5.6. Pms-Asp(OtBu)-OH (15c). Amorphous material. 1H
NMR (400 MHz, CD3CN) d 7.96 (d, J¼7.7 Hz, 2H), 7.84 (t,
J¼7.5 Hz, 1H), 7.72 (dd, J¼7.7, 7.5 Hz, 2H), 4.53 (m, 1H),
4.45 (br m, 1H), 4.34 (m, 1H), 4.01 (br s, 2H), 3.32 (s, 3H),
2.79 (d, J¼5.5 Hz, 2H) 1.44 (s, 9H). Anal. Calcd for
C18H26BF4NO6S·1/2TFA: C, 43.20; H, 5.06; N, 2.65.
Found: C, 43.89; H, 4.78; N, 2.65.
1
in vacuo to leave a colorless solid. Yield 2.72 g, 76%. H
4.5.7. Pms-Glu(OtBu)-OH (15d). Amorphous material. 1H
NMR (400 MHz, CD3CN) d 7.92 (d, J¼7.7 Hz, 2H), 7.82 (t,
J¼7.5 Hz, 1H), 7.72 (dd, J¼7.7, 7.5 Hz, 2H), 4.43 (m, 1H),
4.21 (m, 1H), 4.11 (m, 1H), 3.87 (m, 1H), 3.78 (m, 1H), 3.23
(s, 3H), 2.29 (m, 2H), 2.07 (m, 1H), 1.86 (m, 1H), 1.43 (s,
9H). Anal. Calcd for C19H28BF4NO6S·1/5TFA: C, 45.86; H,
5.59; N, 2.76. Found: C, 46.15; H, 5.38; N, 2.63.
NMR (400 MHz, CD3CN) d 7.42 (d, J¼7.1 Hz, 2H), 7.34
(dd, J¼7.3, 7.1 Hz, 2H), 7.25 (dd, J¼7.3 Hz, 1H), 6.18
(s, 2H), 4.36 (t, J¼6.5 Hz, 2H), 3.45 (d-like, 2H), 3.30
(d-like, 2H) 3.18 (t, J¼6.5 Hz, 2H), 1.78 (dd, J¼9.0,
1.6 Hz, 1H), 1.52 (dd, J¼9.0, 1.6 Hz, 1H). ESI-MS m/z
377.3 [(MþNH4)þ, C18H17NO5S·NH4 requires 377.3]. Rf
[hexane–AcOEt (2/1)] 0.45.
1
4.5.8. Pms-Thr(tBu)-OH (15e). Amorphous material. H
4.5.2. Pms-ONB (11). To a solution of 10 (359 mg,
1.0 mmol) and silver tetrafluoroborate (232 mg, 1.2 mmol)
in acetonitrile (15 ml), methyl iodide (1.24 ml, 20 mmol)
was added and the mixture was stirred overnight at 40 8C.
The resulting yellow precipitate was removed by filtration
and the solvent was evaporated in vacuo to leave a colorless
NMR (400 MHz, CD3CN) d 7.96 (d, J¼7.7 Hz, 2H), 7.84 (t,
J¼7.5 Hz, 1H), 7.72 (dd, J¼7.7, 7.5 Hz, 2H), 4.46 (m, 1H),
4.23 (m, 1H), 4.22 (br m, 1H), 4.22 (m, 2H), 4.07 (m, 1H),
3.89 (m, 1H), 3.80 (m, 1H), 3.23 (s, 3H), 1.16 (s, 12H).
Anal. Calcd for C18H28BF4NO5S·1/2TFA: C, 44.37; H,
5.59; N, 2.72. Found: C, 44.35; H, 5.36; N, 2.51.

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K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1
4.5.9. Pms-Asn(Trt)-OH (15f). Amorphous material. H
NMR (400 MHz, CD3CN) d 7.87 (d, J¼7.7 Hz, 2H), 7.74 (t,
J¼7.5 Hz, 1H), 7.72 (br s, 1H), 7.63 (dd, J¼7.7, 7.5 Hz,
2H), 7.24 (m, 15H), 4.37 (m, 1H), 4.18 (m, 1H), 4.05 (m,
1H), 3.82 (m, 1H), 3.74 (m, 1H), 3.20 (s, 3H), 2.88 (m, 1H),
2.77 (m, 1H). Anal. Calcd for C34H35BF4N2O5S·1/
3TFA·CH3CN: C, 57.38; H, 4.95; N, 5.63. Found: C,
57.74; H, 4.91; N, 6.08.
washed with water and crystallized from ether-hexane to
give colorless crystal. Yield 298 mg, 71%. Mp 140 8C
(decomp.). 1H NMR (400 MHz, D2O) d 8.31 (dd, J¼9.4 Hz,
2H), 7.98 (d, J¼7.5 Hz, 2H), 7.86 (t, J¼7.3 Hz, 1H), 7.75
(dd, J¼7.5, 7.3 Hz, 2H), 7.43 (dd, J¼9.4 Hz, 2H), 4.67 (m,
1H), 4.45 (m, 1H), 4.04 (m, 1H), 3.95 (m, 1H), 3.29 (s, 3H).
Tof-MS m/z 334.5 (Mþ, C16H16NO5S requires 334.37).
Anal. Calcd for C16H16BF4NO5S: C, 45.63; H, 3.83; N,
3.33. Found: C, 45.68; H, 3.91; N, 3.39.
1
4.5.10. Pms-Gln(Trt)-OH (15g). Amorphous material. H
NMR (400 MHz, CD3CN) d 7.87 (d, J¼7.7 Hz, 2H), 7.77 (t,
J¼7.5 Hz, 1H), 7.66 (dd, J¼7.7, 7.5 Hz, 2H), 7.54 (br s, 1H),
7.25 (m, 15H), 4.37 (m, 1H), 4.18 (m, 1H), 4.05 (m, 1H),
3.79 (m, 1H), 3.74 (m, 1H), 3.20 (s, 3H), 2.42 (m, 2H), 2.11
(m, 1H), 1.81 (m, 1H). Anal. Calcd for C33H33BF4N2O5S·2/
3TFA·1/3CH3CN·1/3H2O: C, 56.43; H, 4.91; N, 4.27.
Found: C, 56.26; H, 4.67; N, 4.42.
4.6.2. Pms-Phe-OH (4a). To a solution of Phe (83 mg,
0.5 mmol) and pyridine (40.4 ml, 0.5 mmol) in aqueous
0.1% Triton X-100 solution–acetonitrile (1/1, 20 ml), 13
(211 mg, 0.5 mmol) in acetonitrile (10 ml) was added at
0 8C, and the mixture was stirred at room temperature for
4 h. After removal of the solvent, the residue was dissolved
in water and washed with AcOEt. The aqueous layer was
concentrated in vacuo and the residue was purified by
preparative HPLC to give a colorless oil. The product was
identified by NMR and Mass spectra with Pms-Phe-OH
prepared through Route 1.
1
4.5.11. Pms-His(Trt)-OH (15h). Amorphous material. H
NMR (400 MHz, CD3CN) d 8.34 (s, 1H), 7.87 (d, J¼7.7 Hz,
2H), 7.79 (m, 1H), 7.70 (dd, J¼7.7, 7.5 Hz, 2H), 7.43–7.15
(m, 15H), 7.08 (s, 1H), 4.44 (m, 1H), 4.34 (m, 1H), 4.18 (m,
1H), 3.87 (m, 1H), 3.75 (m, 1H), 3.29 (d, J¼14.7, 4.3 Hz),
3.21 (s, 3H), 3.06 (dd, J¼14.7, 9.0 Hz, 1H). Anal. Calcd for
C35H34BF4N3O4S·2TFA·H2O: C, 50.61; H, 4.14; N, 4.54.
Found: C, 50.47; H, 3.82; N, 4.41.
The following Pms-amino acids were prepared from Pms-
ONp and a corresponding amino acid according to the
procedure described above.
1
4.6.3. Pms-Met-OH (14a). Amorphous material. H NMR
1
4.5.12. Pms-Trp(Boc)-OH (15i). Amorphous material. H
(400 MHz, D2O) d 7.98 (d, J¼7.7 Hz, 2H), 7.85 (t,
J¼7.5 Hz, 1H), 7.75 (dd, J¼7.7, 7.5 Hz, 2H), 4.52 (m, 1H),
4.34(m, 1H), 4.34(m, 1H), 4.01(m, 2H), 3.32(s, 3H), 2.91(m,
2H), 2.21 (s, 3H). Anal. Calcd C14H19BF4NO4S2·TFA: C,
37.44; H, 4.25; N, 2.57. Found: C, 37.71; H, 4.43; N, 2.60.
NMR (400 MHz, CD3CN) d 8.09 (d, J¼8.3 Hz, 1H), 7.84–
7.72 (m, 3H), 7.67–7.61 (m, 3H), 7.53 (d-like, 1H), 7.33
(t-like, 1H), 7.26 (t-like, 1H), 4.49 (m, 1H), 4.35 (m, 1H),
4.14 (m, 1H), 3.78 (m, 1H), 3.71 (m, 1H), 3.32 (m, 1H), 3.15
(m, 1H), 3.12 (m, 1H), 1.63 (s, 9H). Anal. Calcd for
C26H31BF4N2O6S·1/2TFA: C, 51.29; H, 5.06; N, 4.49.
Found: C, 51.24; H, 5.04; N, 4.21.
1
4.6.4. Pms-Cys(Acm)-OH (14b). Amorphous powder. H
NMR (400 MHz, D2O) d 7.94 (d, J¼7.5 Hz, 2H), 7.80 (t,
J¼7.5 Hz, 1H), 7.72 (t, J¼7.5 Hz, 2H), 4.52 (m, 1H), 4.34
(m, 1H), 4.34, 4.27 (ABq, J¼14 Hz, 1H), 4.31 (m, 1H), 4.00
(m, 2H), 3.31 (s, 3H), 2.92 (dd, J¼14, 4.7 Hz, 1H), 2.88
(dd, J¼14, 4.7 Hz, 1H), 1.99 (s, 3H). Anal. Calcd for
C16H23BF4N2O5S2·1/2TFA: C, 38.43; H, 4.46; N, 5.27.
Found: C, 38.39; H, 4.21; N, 4.98.
4.5.13. Pms-Arg(Pmc)-OH (15j). Amorphous material. 1H
NMR (400 MHz, CD3CN) d 7.92 (d, J¼7.7 Hz, 2H), 7.78 (t,
J¼7.1 Hz, 1H), 7.70 (dd, J¼7.7, 7.1 Hz, 2H), 6.21 (br s,
1H), 4.43 (m, 1H), 4.21 (m, 1H), 4.04 (br m, 1H), 3.87 (m,
1H), 3.79 (m, 1H), 3.59 (m, 1H), 3.23 (s, 3H), 2.65 (t,
J¼6.8 Hz, 2H), 2.52 (s, 3H), 1.82 (t, J¼6.8 Hz, 2H), 1.78 (br
m, 1H), 1.62 (br m, 1H), 1.58 (br m, 2H), 1.30 (s, 6H). Anal.
Calcd for C30H43BF4N4O7S2·TFA·1/2CH3CN: C, 46.24; H,
5.35; N, 7.35. Found: C, 46.49; H, 5.17; N, 7.30.
4.6.5. Pms-Cys(Trt)-OH (14c). Amorphous powder. 1H
NMR (400 MHz, CD2CN) d 7.87 (d, J¼7.5 Hz, 2H), 7.75
(t, J¼7.5 Hz, 1H), 7.65 (t, J¼7.5 Hz, 2H), 7.26–7.39
(m, 15H), 4.40 (m, 1H), 20 (m, 1H), 3.94 (m, 1H), 3.85 (br
d, J¼14 Hz, 1H), 3.75 (br d, J¼14 Hz, 1H), 3.19 (s, 3H),
2.64 (dd like, 1H), 2.54 (dd like, 1H) Anal. Calcd for
C32H32BF4NO4S2: C, 59.54; H, 5.00; N, 2.17. Found: C,
59.79; H, 4.77; N, 1.88.
4.5.14. Pms-Lys(Boc)-OH (15k). Amorphous material. 1H
NMR (400 MHz, CD3CN) d 7.93 (d, J¼7.7 Hz, 2H), 7.82 (t,
J¼7.5 Hz, 1H), 7.72 (dd, J¼7.7, 7.5 Hz, 2H), 4.43 (m, 1H),
4.21 (m, 1H), 4.06 (m, 1H), 3.81 (m, 1H), 3.24 (s, 3H), 3.02
(t-like, 2H), 1.79 (br m, 2H), 1.67 (br m, 2H), 1.39 (s, 11H).
Anal. Calcd for C21H33BF4N2O6S·4/3TFA: C, 42.11; H,
5.13; N, 4.71. Found: C, 42.11; H, 5.13; N, 4.75.
4.7. Examination in deprotection rate of Pms group on
Pms-Phe-Leu-TentaGel resin with various bases in
water
4.6. Route 5
Pms-Phe-OH (89.4 mg, 0.2 mmol) and the H-Leu-TentaGel
resin (192 mg, 50 mmol) was reacted with WSCD (38.2 mg,
0.2 mmol) in a presence of HONB (35.8 mg, 0.2 mmol) in
aqueous 0.2% Triton X solution. The resulting Pms-Phe-
Leu-TentaGel resin was washed with DMF and dichloro-
methane and dried in vacuo. Yield 210 mg (98%). Amino
acid ratio in an acid hydrolysate: Phe 1.00, Leu 0.96. The
resin (10 mg each) was treated with various base solutions
4.6.1. Pms-ONp (13). To a solution of 6 (256 mg,
1.0 mmol) in acetonitrile (15 ml, a solution of 4-nitrophenyl
chloroformate (402 mg, 2.0 mmol) in acetonitrile (10 ml)
and a solution of Et3N (279 ml, 2.0 mmol) in acetonitrile
(10 ml) were added alternately at 0 8C, and the mixture was
stirred at room temperature for 2 h. The reaction mixture
was filtered and concentrated in vacuo, and the residue was

K. Hojo et al. / Tetrahedron 60 (2004) 1875–1886
1885
(aqueous 5% NaHCO3, aqueous 0.01 mol/l NaOH,
0.005 mol/l NaOH in 50% aqueous EtOH, 2.5% NaHCO3
in aqueous 50% EtOH, 2.5% Na2CO3 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 until the
resin gave negative Kaiser test. The resulting resin was
washed with DMF and dichloromethane, and dried in vacuo.
After removal of the Fmoc group with 20% piperidine/DMF,
the resin was hydrolyzed and Gly, Phe and Leu contents 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.
product was purified by HPLC to give a white amorphous
powder. Yield 34 mg (based on amino group content of the
resin), 86%. [a]2D4¼þ9.08 (c¼0.2, H2O). Tof-MS m/z 555.3
[(Mþ1)þ, C28H39N6O6 requires 555.64]. Amino acid ratios
in an acid hydrolysate: Tyr 0.97, Gly 2.00, Phe 0.98, Leu,
1.01 (average recovery 97%).
4.7.3. Synthesis of Met-enkephalin amide on CLEAR
resin in water. CLEAR resin (86 mg, 25 mmol) was
swelled with dichloromethane and DMF and Fmoc group
on the resin was removed with 20% piperidine/DMF. After
washing with DMF and H2O, the resin was swelled with
aqueous 0.2% Triton X-100 solution. Synthetic reaction was
carried out according to the protocol shown in Table 4. Pms-
Met-OH 41.3 mg (0.1 mmol), Pms-Phe-OH 44.7 mg
(0.1 mmol), Pms-Gly-OH 35.7 mg (0.1 mmol), and Pms-
Tyr-OH 46.3 mg (0.1 mmol) were coupled in turn by
WSCD 19.1 mg (0.1 mmol) and HONB 17.9 mg
(0.1 mmol). Deprotection was carried out by treatment
(twice) with an aqueous solution of 0.01 mol/l NaOH for
3 min. After completion of the synthetic reaction, the
peptide resin (H-Tyr-Gly-Gly-Phe-Met-CLEAR resin) was
washed with H2O, DMF and dichloromethane, and dried in
vacuo. Yield 92 mg, 86%. The whole resin was treated with
TFA–thioanisole–ethanedithiol (94/3/3, 15 ml) for 2 h at
room temperature. The resin was filtered off and the TFA
was removed in vacuo to leave a yellowish oil. The residue
was dissolved in water, washed with ether, and lyophilized.
The crude product was purified by HPLC to give an
amorphous powder. Yield (based on amino group content of
the resin): Yield 4.9 mg, 29%. [a]2D4¼þ6.78 (c¼0.8, 20%
CH3CN/H2O). Tof-MS m/z: 573.7 [(Mþ1)þ, C27H36N6O6S
requires 573.68]. Amino acid ratios in an acid hydrolysate:
Tyr 1.01, Gly 2.00, Phe 0.93; Met 0.95 (average recovery:
94%).
4.7.1. Synthesis of Leu-enkephalin amide on TentaGel
resin in water. The Fmoc-Rink amide-TentaGel resin
(100 mg, 25 mmol) was swelled with dichloromethane and
DMF, and then Fmoc group on the resin was removed by
treatment with 20% piperidine/DMF. After washed with
DMF and H2O, the resin was swelled with aqueous 0.2%
Triton X-100 solution. The synthesis was carried out
according to the protocol shown in Table 4. Pms-Leu-OH
41.3 mg (0.1 mmol), Pms-Phe-OH 44.7 mg (0.1 mmol),
Pms-Gly-OH 35.7 mg (0.1 mmol), and Pms-Tyr-OH
46.3 mg (0.1 mmol) were coupled in turn with WSCD
19.1 mg (MW: 191.7, 0.1 mmol) and HONB 17.9 mg
(0.1 mmol). Deprotection was carried out with an aqueous
solution of 5.0% NaHCO3. After completion of the
synthetic reaction, the peptide resin (H-Tyr-Gly-Gly-Phe-
Leu-TentaGel resin) was washed with H2O, DMF and
dichloromethane, and dried in vacuo. Yield: 107.5 mg,
97%. The whole resin was treated with TFA–thioanisole–
ethanedithiol (94/3/3, 15 ml) for 2 h at room temperature.
The resin was removed by filtration and the TFA was
removed in vacuo to leave a yellowish oil. The residue was
dissolved in water, washed with ether, and lyophilized. The
crude product was purified by HPLC to give an amorphous
powder. Yield (based on amino group content of the resin):
Yield 10.3 mg, 61%. [a]2D4¼þ8.28 (c¼0.2, H2O). Tof-MS
m/z 555.0 [(Mþ1)þ, C28H39N6O6 requires 555.64]. Amino
acid ratios in an acid hydrolysate: Tyr 0.99, Gly 2.00, Phe
0.96, Leu 0.98 (average recovery: 83%).
4.7.4. Synthesis of Met-enkephalin amide on TentaGel
resin in water. Performed in the same manner as described
in Section 4.7.1. Yield (based on amino group content of the
resin): yield 5.8 mg, 32% (amorphous powder).
[a]2D4¼þ7.18 (c¼0.8, 20% CH3CN/H2O). Tof-MS m/z
573.4 [(Mþ1)þ, C27H36N6O6S requires 573.68]. Amino
acid ratios in an acid hydrolysate: Tyr 0.99, Gly 2.00, Phe
0.96, Met 0.98 (average recovery 92%).
4.7.2. Synthesis of Leu-enkepharin amide by the Fmoc
strategy. The Fmoc group on the Rinkamide resin (100 mg,
60 mmol) was removed by treatment with 20% piperidi-
ne/DMF. Synthesis was carried out according to a general
Fmoc strategy19 Fmoc-Leu-OH 63.6 mg (0.18 mmol),
Fmoc-Phe-OH 69.7 mg (0.18 mmol), Fmoc-Gly-OH
53.5 mg (0.18 mmol), and Fmoc-Tyr(tBu)-OH 82.7 mg
(0.18 mmol) were coupled in turn by diisopropylcarbodi-
imide (0.18 mmol) and 1-hydroxybenzotriazole 24.3 mg
(0.18 mmol). Deprotection of Fmoc group was carried out
with 20% piperidine/DMF. After completion of the
synthetic reaction, the peptide resin (H-Tyr(tBu)-Gly-Gly-
Phe-Leu-Rinkamide resin was washed with DMF and
dichloromethane, and dried in vacuo. Yield 123 mg, 99%.
The whole resin was treated with TFA–thioanisole–
ethanedithiol (94/3/3, 15 ml) for 2 h at room temperature.
The resin was filtered off, and the TFA was removed in
vacuo to leave a yellowish oil. The residue was dissolved in
water, washed with ether, and lyophilized. The crude
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