Efficient preparation of Fmoc-aminoacyl-N-ethylcysteine unit, a key device for the synthesis of peptide thioesters

Article abstract of DOI:10.1039/c1ob05831b

The synthesis of Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteine, an N- to S-acyl migratory device for the preparation of peptide thioesters by Fmoc-SPPS (solid-phase peptide synthesis) is described. Condensation of Fmoc-aminoacyl pentafluorophenyl ester and N-ethyl-S-triphenylmethylcysteine was efficiently performed in the presence of HOOBt (3-hydroxy-3,4-dihydro-4-oxo-1,2,3- benzotriazine) in DMF. A small amount of diastereomer yielded during the reaction was easily separated by HPLC purification and the highly pure devices were obtained for most of the proteinogenic amino acids.

Full text of DOI:10.1039/c1ob05831b

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PAPER
Efficient preparation of Fmoc-aminoacyl-N-ethylcysteine unit, a key device
for the synthesis of peptide thioesters
Hironobu Hojo,*a Hajime Kobayashi,a Risa Ubagai,a Yuya Asahina,a Yuko Nakahara,a Hidekazu Katayama,a
Yukishige Itob and Yoshiaki Nakahara*a
Received 26th May 2011, Accepted 4th July 2011
DOI: 10.1039/c1ob05831b
The synthesis of Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteine, an N- to S-acyl migratory device
for the preparation of peptide thioesters by Fmoc-SPPS (solid-phase peptide synthesis) is described.
Condensation of Fmoc-aminoacyl pentafluorophenyl ester and N-ethyl-S-triphenylmethylcysteine was
efficiently performed in the presence of HOOBt (3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine) in
DMF. A small amount of diastereomer yielded during the reaction was easily separated by HPLC
purification and the highly pure devices were obtained for most of the proteinogenic amino acids.
Introduction
Peptide thioesters have been widely used as key intermedi-
ates for the synthesis of (glyco)proteins by ligation methods,
such as the thioester method1 and the native chemical ligation
method.2 This key intermediate was originally synthesized by
the Boc (t-butoxycarbonyl) method, considering the instability
of the thioester linkage to piperidine used for the Fmoc (9-
fluorenylmethoxycarbonyl) method.1 However, peptide thioester
preparation by the Fmoc method is attractive for the synthesis
of post-translationally modified proteins, such as glycoproteins
and phosphorylated proteins, since the linkages that exist in
Scheme 1 Post-SPPS thioesterification using N-alkylcysteine device.
these modifications are generally acid-sensitive. Due to this fact,
various methods have been developed to realize the peptide
of peptide thioesters having chiral amino acids at the C-terminus
thioester synthesis by the Fmoc method.3 Recently, we developed
was significantly increased. In the method, the dipeptide unit was
a promising strategy for peptide thioester preparation, in which
obtained by two-step reaction: high-pressure-promoted Fmoc-
N-alkylcysteine (NAC) in the C-terminus of the peptide is used
aminoacylation of N-ethyl-S-triphenylmethylcysteine allyl ester
by Fmoc-amino acid fluoride, followed by deallylation using Pd(0)
catalyst. However, the complexity in this method is that the first
as an N–S acyl migration device as shown in Scheme 1.4 This
procedure is fully compatible with conventional Fmoc strategy
and gives the peptide thioester in excellent yield. In addition,
step requires special apparatus for a high-pressure reaction, which
this method provides epimerization-free peptide thioesters. The
limits the general use of the procedure. Here, we want to report a
efficiency of this method has been demonstrated by its application
more general and one-pot procedure for the preparation of Fmoc-
aminoacyl N-ethylcysteines, which is a significant improvement for
to (glyco)protein syntheses.5
The remaining problem in this method was the low yield of
the efficient preparation of peptide thioesters by the NAC method.
peptide thioesters with a chiral amino acid at the C-terminus,
which was due to the incomplete coupling of the C-terminal
amino acid onto the sterically hindered N-alkylcysteine residue.
Results and discussion
As a solution to this problem, we recently reported loading
of C-terminal amino acids as preformed Fmoc-aminoacyl N-
ethylcysteines to the resin.4b Due to this improvement, the yield
Synthesis of the dipeptide unit using fluoride
In a previous paper, we initially examined the direct coupling of
Fmoc-amino acid activated by HATU (O-(7-azabenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and
DIEA (N,N-diisopropylethylamine) with N-ethyl-S-triphenyl-
methylcysteine [H-(N-Et)Cys(Trt)-OH].4b Although the reaction
proceeded quickly, a complex mixture containing the desired
dipeptide and the tripeptide by-product, with two N-ethylcysteine
aDepartment of Applied Biochemistry, Institute of Glycoscience, Tokai
University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan.
E-mail: hojo@keyaki.cc.u-tokai.ac.jp
bRIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-
0198, Japan. E-mail: yukito@riken.jp
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Table 1 Dipeptide preparation using Fmoc-Leu-F
residues, was obtained. In this reaction, it seemed that the
obtained dipeptide further acylated H-(N-Et)Cys(Trt)-OH by
transesterification from the Fmoc-amino acid OAt ester. Thus, we
speculated that carboxy-protection of the H-(N-Et)Cys(Trt)-OH
is essential to avoid this side reaction. In contrast to our result,
however, Brown and Schafmeister succeeded in the acylation of
N-methylamino acid with free carboxylic acid using Fmoc-amino
acid fluorides in HFIP (1,1,1,3,3,3-hexafluoro-2-propanol).6
According to their report, the reaction proceeds through the
formation of a mixed anhydride between N-methylamino acid
and Fmoc-amino acid fluoride, followed by intramolecular
aminolysis by the imino group of the N-methylamino acid. The
efficiency of the reaction was demonstrated by the syntheses
of dipeptides containing N-methylamino acids in high yield,
typically over 75%. Under their coupling conditions, no base was
added to avoid the esterification with the solvent, HFIP. However,
we speculated that this base-free condition also prevented the
further acylation reaction leading to the tripeptide formation.
Thus, we attempted to apply the same method for the synthesis
of the dipeptide using Fmoc-Leu fluoride with N-ethylcysteine
as a model, as shown in Table 1. The application of the exact
conditions of Brown’s report gave the desired dipeptide in good
yield (entry 1), though it took a longer time for the completion of
the reaction. We observed some diastereomer formation during
the reaction, but this side product was easily separated by RP
(reversed-phase) HPLC using an ODS (octadecylsilyl) column.
We next tested the coupling in a more common solvent, DMSO.
As a result, the product was obtained in an acceptable yield,
as shown in entry 2. The increase in temperature accelerated
Solvent
T (◦C)
Time (h)
Product (%)
D/L (%)a
1
2
3
4
5
HFIP
55
55
65
75
85
20
24
20
4.5
2
69
61
46
42
40
1.4
3.3
8.7
9.5
12.5
DMSO
DMSO
DMSO
DMSO
a The diastereomer yield was expressed as a ratio of the peak area of the
diastereomer and the product on HPLC.
the reaction, but the yield was decreased, as shown in entries
3 to 5.
Synthesis of dipeptides using Pfp ester
If the acylation of N-alkylcysteine proceeds through mixed anhy-
dride formation followed by intramolecular aminolysis,6 a milder
acylation reagent would also be used for this synthesis, as the
actual acylation step proceeds intramolecularly, irrespective of the
acylation reagent. Thus, we next examined the use of more stable
Fmoc-amino acid Pfp (pentafluorophenyl) esters supplemented
with one equivalent of HOOBt (3-hydroxy-3,4-dihydro-4-oxo-
1,2,3-benzotriazine) as an additive to increase the reactivity.7 The
results are shown in Table 2. In HFIP, the reaction did not proceed
Table 2 Dipeptide preparation using Fmoc-AA-OPfp in the presence of HOOBt
Fmoc-AA-OPfp
HOOBt (eq)
Solvent
T (◦C)
Time (h)
Product (%)
D/L (%)a
1
2
3
4
5
6
7
8
Leu
Leu
Leu
Leu
Leu
Ala
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
HFIP
DMSO
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
55
rt
22
22
22
3
3
3
3
5
3
3
3
10
5
3
3
10
5
3
3
8
0
36
70
78
47
75
77
73
74
84
66
67
72
73
78
85
79
54
78
69
70
79
81
—
2.0
0.8
1.6
1.4
1.4
1.2
1.4
1.3
—
rt
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
Asp(OBut)
Glu(OBut)
Phe
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Gly
His(Trt)
Ile
Lys(Boc)
Met
Asn(Trt)
Pro
Gln(Trt)
Arg(Pbf)
Ser(But)
Thr(But)
Val
6.6b
0.7
1.5
1.4
N.D.
0.9
1.2
1.4
1.2
0.8
1.0
2.0
1.6
10
3
3
Trp(Boc)
Tyr(But)
a The diastereomer yield was expressed as a ratio of the peak area of the diastereomer and the product on HPLC. b Crude Pfp ester was directly used for
dipeptide synthesis.
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Table 3 Synthesis of model peptide thioesters
at all. However, in DMSO, the reaction proceeded and the product
was obtained in a moderate yield at room temperature. When
DMF was used as a solvent, the yield of the product was further
improved, even at room temperature, as shown in entry 3. The
increase in temperature to 50 ◦C accelerated the reaction and
the product was obtained in 78% isolated yield within 3 h. The
addition of HOOBt was essential to increase the yield of the
product, as in its absence the yield was significantly decreased,
as shown in entry 5. Thus, we decided to use the conditions used
for entry 4 for the preparation of other dipeptides. As shown
in Table 2 entries 6 to 23, the dipeptide units were obtained in
good to moderate yield, except for Arg. Fmoc-Arg(Pbf)-OPfp was
easily converted to an inactive lactam form, which resulted in this
low yield. In the case of Asn, the separation of the product with
its epimer, was not accomplished even with RP-HPLC. Thus, we
could not determine epimerization ratio at this stage. However, the
ratio was not significant, which is shown in the following section.
In the synthesis of dipeptides with amino acids with bulky side
chains (Ile, Pro, Thr and Val), the reaction was not complete within
3 h. In these cases, the elongation of the reaction was effective to
increase the yield of the product. Thus, we successfully completed
most of the dipeptide units useful for the thioester preparation for
segment coupling.
Peptide thioester
Yield (%)
Epimer/product (%)
QKTEF-SCH2CH2COOH
MGTAN-SCH2CH2COOH
FKVDS-SCH2CH2COOH
39
46
32
< 1
1.8
< 1
previous syntheses, two peaks appeared on the RP-HPLC at T =
0 (Fig. 2), which corresponds to the thioester form (compound
A) and the amide form (B). These peaks gradually converted
to a new peak (C), which corresponds to the desired thioester.
After leaving overnight, the reaction was almost complete, without
serious side reactions as shown in Fig. 2. After the reaction mixture
was left to stand for another day, the desired peptide thioester
was purified by RP-HPLC. As shown in Table 3, the yields were
highly increased compared to the previous report, in which the
C-terminal chiral amino acids were directly loaded to the resin-
bound N-ethylcysteine residue by HATU.4a
Model peptide thioester synthesis using dipeptide units
In order to demonstrate the usefulness of dipeptide units for
peptide thioester synthesis, model pentapeptide thioesters were
prepared by the NAC method. As C-terminal amino acids, Asn,
Phe and Ser were selected as examples. The synthetic procedure
in the case of Asn is shown in Fig. 1. Fmoc-Arg(Pbf)-OH was
introduced to Rink amide resin twice. After Fmoc removal, Fmoc-
Asn(Trt)-(N-Et)Cys(Trt)-OH was introduced using the DIC
(N,N¢-diisopropylcarbodiimide)–HOBt (1-hydroxybenzotriazole)
method. Then, the chain elongation was carried out using a
peptide synthesizer by the Fmoc method. After deprotection by
TFA, the crude peptide was dissolved in 50% aq. acetonitrile
containing 6 M urea and 5% (v/v) 3-mercaptopropionic acid
(MPA), and the solution was kept at 37 ◦C. As observed in
Fig. 2 RP-HPLC profile of the crude peptide thioesterification reaction
in Fig. 1. Elution conditions: column, Mightysil RP-18 GP (4.6 ¥ 150 mm,
Kanto, Japan) at a flow rate of 1 mL min-1; eluent, A, 0.1% aqueous TFA,
B, 0.1% TFA in acetonitrile. Asterisked peaks in the chromatogram (T =
24 h) are non-peptides. Peak D indicates the epimer of compound C.
There was a possibility that Fmoc-Asn(Trt)-(N-Et)Cys(Trt)-
OH used for the synthesis of compound C was contaminated
with a small amount of diastereomer, since it was not separated
by RP-HPLC. However, the epimer/product ratio of MGTAN-
SCH2CH2COOH was only 1.8%, indicating that the epimerization
of Asn during the dipeptide synthesis was at most this level, which
is comparable to other amino acids. In addition, we confirmed
that the post-SPPS thioesterification reaction in the NAC method
proceeds essentially epimerization-free.
Conclusion
We successfully established an efficient one-pot procedure for the
preparation of Fmoc-aminoacyl N-ethylcysteine, a key device for
the synthesis of peptide thioesters by the NAC method. This
Fig. 1 Synthetic route for peptide thioesters using the dipeptide unit.
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procedure is mild and simple compared to the previous method
using the high pressure promoted method, and thus can be
generally applicable for the synthesis of peptide thioesters by the
NAC method. The dipeptide units obtained will be efficiently used
for the synthesis of (glyco)proteins by ligation methods.
-CH2CH3), 3.09 (m, 1H, Cys-bH), 2.91 (m, 2H, -CH2CH3, Cys-
bH), 2.75 (m, 1H, Cys-aH), 1.33 (d, 2H, J = 6.8 Hz, Ala-CH3),
1.02 (brt, 3H, J = 7.01 Hz, -CH2CH3). 13C-NMR: d 60.2 (Cys-aC),
47.0 (Ala-aC), 45.0 (-CH2CH3), 30.2 (Cys-bC), 19.4 (Ala-CH3),
13.9 (-CH2CH3). Anal. calcd for C42H40N2O5S·1/2H2O: C, 72.70;
H, 5.96; N, 4.04; S, 4.62. Found: C, 72.88; H, 6.01; N, 3.92; S,
4.48%. MALDI TOF MS, calcd for C42H40N2O5S (M + Na)+:
707.26. Found: m/z 707.21.
Experimental
General
N -(9-Fluorenylmethoxycarbonyl)-O4 -tert-butyl-L -aspartyl-N-
ethyl-S-triphenylmethyl-L-cysteine
Specific rotation values were determined with a Jasco P-2200
polarimeter at 20
2
◦C for solutions in CHCl3. Column
chromatography and flash chromatography were performed on
silica gel PSQ 100B (Fuji Silysia) and Wakogel C-500HG (Wako),
respectively. TLC and HPTLC were performed on silica gel 60
F254 (E. Merck). 1H- and 13C-NMR spectra were recorded with a
Jeol AL400 spectrometer. Chemical shifts are expressed in ppm
downfield from the signal for internal Me4Si for solutions in
CDCl3. Peptide synthesis was carried out by a Peptide synthesizer
433A (Applied Biosystems) using the FastMoc protocol. HPLC
was performed with a recycling preparative HPLC model 9201
(Japan Analytical Industry Co.) on Inertsil ODS-SP using 80%
aq acetonitrile containing 0.1% TFA as an eluent. Amino acid
composition was determined using a LaChrom amino acid
analyzer (Hitachi) after hydrolysis with a 6 M HCl solution at
150 ◦C for 2 h in an vacuum-sealed tube. Yields of peptides were
calculated based on the amino acid analysis data.
Rf 0.55 (9 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -65.8 (c, 1.2).
1H-NMR: d 7.65 (d, 2H, J = 7.3 Hz, Ar), 7.45 (t, 2H, J = 6.8 Hz,
Ar), 7.35–7.09 (m, 19H, Ar), 5.95 (d, 1H, J = 9.3 Hz, Asp-NH),
4.83 (m, 1H, Asp-aH), 4.18 (m, 2H, -CH2CHAr2), 4.07 (m, 1H,
-CHAr2), 3.47 (m, 1H, -CH2CH3), 3.05 (dd, 1H, J = 5.9, 13.7
Hz, Cys-bH), 2.77 (m, 1H, Cys-bH), 2.71–2.61 (m, 3H, Cys-aH,
Asp-bH, -CH2CH3), 2.44 (dd, 1H, J = 6.3 15.6 Hz, Asp-bH), 1.31
(s, 9H, t-Bu), 0.89 (brt, 3H, J 6.8 Hz, -CH2CH3). 13C-NMR: d
60.8 (Cys-aC), 47.7 (Asp-aC), 44.9 (-CH2CH3), 39.1 (Asp-bC),
29.9 (Cys-bC), 13.8 (-CH2CH3). Anal. calcd for C47H48N2O7S: C,
71.92; H, 6.16; N, 3.57; S, 4.08. Found: C, 71.69; H, 6.44; N, 3.37;
S, 3.91% MALDI TOF MS, calcd for C47H48N2O7S (M + Na)+:
807.31. Found: m/z 807.29.
N -(9-Fluorenylmethoxycarbonyl)-O5 -tert-butyl-L-glutamyl-N-
ethyl-S-triphenylmethyl-L-cysteine
Synthesis of Fmoc-aminoacyl N-ethyl-S-triphenylmethyl-L-cystei-
ne using fluoride
1
Rf 0.39 (9 : 1 CHCl3–CH3OH). [a]D -29.6 (c, 1.3). H-NMR: d
7.74 (d, 2H, J = 7.3 Hz, Ar), 7.54 (dd, 2H, J = 7.8, 13.2 Hz, Ar),
7.44–7.17 (m, 19H, Ar), 5.77 (d, 1H, J = 8.8 Hz, Glu-NH), 4.57
(m, 1H, Glu-aH), 4.39 (m, 2H, -CH2CHAr2), 4.17 (t, 1H, J =
7.3 Hz, -CHAr2), 3.59 (m, 1H, -CH2CH3), 3.04 (m, 1H, Cys-bH),
2.88 (m, 2H, -CH2CH3, Cys-bH), 2.79 (m, 1H, Cys-aH), 2.30 (m,
2H, Glu-g H), 1.99 (m, 1H, Glu-bH), 1.72 (m, 1H, Glu-bH), 1.42
(s, 9H, t-Bu), 1.01 (brt, 3H, J 7.1 Hz, -CH2CH3). 13C NMR: d 60.2
(Cys-aC), 50.1 (Glu-aC), 47.0 (-CH2CH3), 30.1 (Cys-bC), 13.9
(-CH2CH3). Anal. calcd for C48H50N2O7S·1/2H2O: C, 71.35; H,
6.36; N, 3.47; S, 3.97. Found: C, 71.29; H, 6.47; N, 3.39; S, 3.79%
MALDI TOF MS, calcd for C48H50N2O7S (M + Na)+: 821.32.
Found: m/z 821.21.
Fmoc-amino acid fluoride (1.2 eq to cysteine) and N-ethyl-S-
triphenylmethyl-L-cysteine were dissolved in HFIP or DMSO at a
concentration of 0.1 mol l-1 and the solution was stirred at various
temperatures and times as shown in Table 1. After the solvent was
removed in vacuo, the residue was purified by silica gel column
chromatography, followed by a recycling HPLC system to obtain
the desired dipeptide units.
General procedure for the synthesis of Fmoc-aminoacyl N-ethyl-S-
triphenylmethyl-L-cysteine using Pfp ester in the presence of
HOOBt
Equimolar amounts of Fmoc-amino acid Pfp ester, N-ethyl-S-
triphenylmethyl-L-cysteine, and HOOBt were dissolved in DMF
at a concentration of 0.25 mol l-1 and the solution was stirred at
50 ◦C. After the solvent was removed in vacuo, the residue was
purified by silica gel column chromatography, followed by flash
chromatography or recycled HPLC to obtain the desired dipeptide
units. Usually, the recycling mode in the HPLC system was not
necessary, but in the case of Asn, the diastereomer, if any, was not
separated even with the recycling mode.
N-(9-Fluorenylmethoxycarbonyl)-L-phenylalanyl-N-ethyl-S-trip-
henylmethyl-L-cysteine
Rf 0.52 (9 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -55.7 (c, 1.1).
1H-NMR:d 7.68 (d, 2H, J = 7.3 Hz, Ar), 7.50 (t, 2H, J = 7.8
Hz, Ar), 7.37–6.98 (m, 24H, Ar), 6.24 (d, 1H, J = 8.8 Hz, Phe-
NH), 4.61 (dd, 1H, J = 8.8, 14.6 Hz, Phe-aH), 4.23–4.11 (m, 3H,
-CH2CHAr2, -CHAr2), 3.15 (dd, 1H, J = 7.3, 14.2 Hz, Cys-bH),
2.92 (m, 2H, Phe-bH), 2.79 (m, 1H, -CH2CH3), 2.47 (t, 1H, J = 6.8
Hz, Cys-aH), 2.35 (dd, 1H, J = 6.3, 14.1 Hz, Cys-bH), 1.96 (m, 1H,
-CH2CH3), 0.73 (t, 3H, J = 7.3 Hz, -CH2CH3). 13C-NMR: d 61.2
(Cys-aC), 51.9 (Phe-aC), 44.3 (-CH2CH3), 40.0 (Phe-bC), 29.7
(Cys-bC), 13.6 (-CH2CH3). Anal. calcd for C48H44N2O5S H2O: C,
74.01; H, 5.95; N, 3.60; S, 4.12. Found: C, 74.11; H, 6.19; N, 3.35;
S, 3.77% MALDI TOF MS, calcd for C48H44N2O5S (M + Na)+:
783.29. Found: m/z 783.25.
N -(9-Fluorenylmethoxycarbonyl)-L-alanyl-N -ethyl-S-triphenyl-
methyl-L-cysteine
Rf 0.46 (17 : 3 CHCl3–MeOH). [a]D -28.9 (c, 1.3). 1H-NMR: d 7.74
(d, 2H, J = 7.3 Hz, Ar), 7.57 (t, 2H, J = 7.8 Hz, Ar), 7.44–7.18 (m,
19H, Ar), 5.83 (d, 1H, J = 7.3 Hz, Ala-NH), 4.53 (m, 1H, Ala-aH),
4.29 (m, 2H, -CH2CHAr2), 4.17 (m, 1H, -CHAr2), 3.41 (m, 1H,
6810 | Org. Biomol. Chem., 2011, 9, 6807–6813
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Na -(9-Fluorenylmethoxycarbonyl)-Ns -triphenylmethyl-L-histidyl-
N-ethyl-S-triphenylmethyl-L-cysteine
6.6 Hz, Leu-dH), 0.94 (d, 3H, J = 6.6 Hz, Leu-dH). 13C-NMR: d
60.3 (Cys-aC), 24.5 (Leu-g C), 23.4 (Leu-dC), 21.7 (Leu-dC), 14.0
(-CH2CH3). Anal. calcd for C45H46N2O5S·1/3H2O: C, 73.74; H,
6.42; N, 3.82; S, 4.37. Found: C, 73.78; H, 6.71; N, 3.66; S, 4.10%
MALDI TOF MS, calcd for C45H46N2O5S (M + Na)+: 749.30.
Found: m/z 749.21.
Rf 0.40 (9 : 1 CHCl3–MeOH, 1% AcOH). [a]D -19.6 (c, 1.0). 1H-
NMR: d 7.88 (d, 2H, J = 7.3 Hz, Ar), 7.79 (d, 0.5 H, J = 8.8
Hz, His-NH of conformer 1), 7.65 (t, 2H, J = 7.6 Hz, Ar), 7.43–
7.22 (m, 32H, Ar), 7.06 (m, 2H, Ar), 4.73 (m, 0.5H, His-aH of
conformer 1), 4.62 (m, 0.5H, His-aH of conformer 2), 4.16 (m, 3H,
-CH2CHAr2), 3.61 (m, 0.5H, -CH2CH3), 2.94 (m, 3H, His-bHx2,
-CH2CH3), 1.03 (m, 3H, -CH2CH3). 13C-NMR: d 59.4 (Cys-aC),
14.2 (-CH2CH3). Anal. calcd for C64H56N4O5S 2·5H2O: C, 74.04;
H, 5.92; N, 5.40. Found: C, 74.21; H, 5.70; N, 5.28% MALDI
TOF MS, calcd for C64H56N4O5S (M + Na)+: 1015.39. Found:
m/z 1015.62.
N-(9-Fluorenylmethoxycarbonyl)-L-methionyl-N-ethyl-S-triphen-
ylmethyl-L-cysteine
Rf 0.30 (19 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -22.3 (c, 1.0).
1H-NMR:d 7.74 (d, 2H, J = 7.3 Hz, Ar), 7.56 (brt, 2H, J =
8.5 Hz, Ar), 7.43–7.18 (m, 19H, Ar), 5.85 (d, 1H, J = 8.3 Hz,
Met-NH), 4.70 (m, 1H, Met-aH), 4.30 (d, 2H, -CH2CHAr2),
4.17 (t, 1H, -CHAr2), 3.51 (m, 1H, -CH2CH3), 3.06 (dd, 1H,
J = 9.8, 14.1 Hz, Cys-bH), 2.91 (m, 2H, -CH2CH3, Cys-bH),
2.78 (m, 1H, Cys-aH), 2.06 (s, 3H, -SCH3), 1.95 (m, 1H, Met-
bH), 1.86 (m, 1H, Met-bH), 1.02 (t, 3H, J = 6.8 Hz, -CH2CH3).
13C-NMR: d 60.4 (Cys-aC), 49.9 (Met-aC), 45.3 (-CH2CH3),
30.1 (Cys-bC), 29.9 (Met-g C), 15.6 (Met-CH3), 14.0 (-CH2CH3).
Anal. calcd for C44H44N2O5S2·1/2H2O: C, 70.09; H, 6.02; N,
3.72; S, 8.51% Found: C, 70.13; H, 6.19; N, 3.58; S, 8.05.
MALDI TOF MS, calcd for C44H44N2O5S2 (M + Na)+: 767.26.
Found: m/z 767.33.
N-(9-Fluorenylmethoxycarbonyl)-L-isoleucyl-N-ethyl-S-triphenyl-
methyl-L-cysteine
Rf 0.43 (9 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -23.4 (c, 1.1).
1H-NMR:d 7.74 (d, 2H, J = 7.8 Hz, Ar), 7.56 (brt, 2H, J =
8.5 Hz, Ar), 7.43–7.16 (m, 19H, Ar), 5.79 (d, 1H, J = 9.3, Ile-
NH), 4.39 (dd, 1H, J = 7.3, 9.3 Hz, Ile-aH), 4.30 (m, 2H,
-CH2CHAr2), 4.17 (t, 1H, J = 7.3 Hz, -CHAr2), 3.48 (m, 1H,
-CH2CH3), 3.13 (dd, 1H, J = 9.8, 14.6 Hz, Cys-bH), 2.92 (m, 2H,
-CH2CH3, Cys-bH), 2.59 (dd, 1H, J = 4.9, 9.3 Hz, Cys-aH), 1.73
(m, 1H, Ile-bH), 1.53 (m, 1H, Ile-g H), 1.10 (m, 1H, Ile-g H), 0.99
(t, 3H, J = 6.8 Hz, -CH2CH3), 0.90 (m, 6H, Ile-CH3). 13C-NMR:
d 60.5 (Cys-aC), 55.0 (Ile-aC), 45.6 (-CH2CH3), 38.4 (Ile-bC),
31.9 (Cys-bC), 24.0 (Ile-g C), 15.7 (Ile-CH3), 13.9 (-CH2CH3),
11.3 (Ile-CH3). Anal. calcd for C45H46N2O5S·1/2H2O: C, 73.44;
H, 6.44; N, 3.81; S, 4.36. Found: C, 73.55; H, 6.54; N, 3.79%
MALDI TOF MS, calcd for C45H46N2O5S (M + Na)+: 749.30.
Found: m/z 749.71.
N2 -(9-Fluorenylmethoxycarbonyl)-N4 -triphenylmethyl-L-asparag-
inyl-N-ethyl-S-triphenylmethyl-L-cysteine
1
Rf 0.46 (19 : 1 CHCl3–CH3OH 0.5% AcOH). H-NMR: d 7.64
(d, 2H, J = 7.3 Hz, Ar), 7.42 (brt, 2H, J = 7.6 Hz, Ar), 7.33–
7.08 (m, 34H, Ar), 6.13 (brs, 1H, Asn-NH), 4.81 (brd, 1H, J =
6.3 Hz, Asn-aH), 4.18 (m, 1H, -CH2CHAr), 4.08 (m, 1H,
-CH2CHAr), 4.00 (m, 1H, -CHAr), 3.23 (m, 1H, -CH2CH3),
3.05 (m, 1H, Cys-bH), 2.67–2.45 (m, 4H, Cys-a, bH, Asn-
bHx2), 0.81 (brs, 3H, -CH2CH3). 13C-NMR: d 59.9 (Cys-aC), 48.7
(Asn-aC), 43.6 (-CH2CH3), 39.8 (Asn-bC), 29.8 (Cys-bC), 13.9
(-CH2CH3). Anal. calcd for C62H55N3O6S·2H2O: C, 74.01; H, 5.91;
N, 4.18; S, 3.19. Found: C, 73.95; H, 5.78; N, 4.01; S, 3.05%
MALDI TOF MS, calcd for C62H55N3O6S (M + Na)+: 992.37.
Found: m/z 992.26.
N2 - (9 - Fluorenylmethoxycarbonyl) - N6 - tert - butoxycarbonyl - L-
lysyl-N-ethyl-S-triphenylmethyl-L-cysteine
Rf 0.48 (17 : 3 CHCl3–CH3OH). [a]D -23.0 (c, 1.0).1H-NMR: d
7.73 (d, 2H, J = 7.3 Hz, Ar), 7.56 (m, 2H, Ar), 7.44–7.16 (m, 19H,
Ar), 5.93 (d, 1H, J = 7.8 Hz, Lys-NH), 4.54 (m, 1H, Lys-aH),
4.29 (m, 2H, -CH2CHAr2), 4.16 (m, 1H, -CHAr2), 3.41 (m, 1H,
-CH2CH3), 2.79 (m, 1H, Cys-aH), 1.40 (s, 9H, t-Bu), 1.33 (d,
2H, J = 6.8 Hz, Lys-bH), 1.01 (brt, 3H, J 6.6 Hz, -CH2CH3).
13C-NMR: d 60.1 (Cys-aC), 50.7 (Lys-aC), 30.2 (Cys-bC),
28.4 (C(CH3)3), 19.4 (Lys-bC), 14.0 (-CH2CH3). Anal. calcd for
C50H55N3O7S·2H2O: C, 68.39; H, 6.77; N, 4.79; S, 3.65. Found:
C, 68.51; H, 6.51; N, 4.70; S, 3.58% MALDI TOF MS, calcd for
C50H55N3O7S (M + Na)+: 864.37. Found: m/z 864.35.
N -(9-Fluorenylmethoxycarbonyl)-L-prolyl-N -ethyl-S-triphenyl-
methyl-L-cysteine
Rf 0.30 (19 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -60.5 (c, 1.0).
1H-NMR (DMSO-d6): d 7.87 (m, 2H, Ar), 7.67–7.59 (m, 2H,
Ar), 7.40–7.21 (m, 19H, Ar), 4.67 (d, 0.6H, Pro-aH of conformer
1), 4.55 (d, 0.4 H, Pro-aH of conformer 2), 4.24–4.01 (m, 3H,
-CH2CHAr), 2.96 (m, 1H, -CH2CH3), 2.80 (brt 1H, J = 11.0
Hz, Cys-bH), 2.63 (m, 1H, Cys-bH), 2.30 (m, 0.6 H, Pro-bH of
conformer 1), 2.18 (m, 0.4 H, Pro-bH of conformer 2), 1.90–1.71
(3H, Pro-bH, g H x2), 1.08 (t, 3H x0.4, J = 6.6 Hz, -CH2CH3 of
conformer 2), 0.98 (t, 3H x0.6, J = 6.8 Hz, -CH2CH3 of conformer
1). 13C-NMR: d 59.1, 58.8 (Cys-aC), 43.8, 43.5 (-CH2CH3), 30.5
(Cys-bC), 14.3 14.1 (-CH2CH3). Anal. calcd for C44H42N2O5S
1·5H2O: C, 71.62; H, 6.15; N, 3.80; S, 4.35. Found: C, 71.97; H,
6.43; N, 3.47; S, 3.69% MALDI TOF MS, calcd for C44H42N2O5S
(M + Na)+: 733.27. Found: m/z 733.61.
N -(9-Fluorenylmethoxycarbonyl)-L -leucyl-N -ethyl-S -triphen-
ylmethyl-L-cysteine
Rf 0.32 (19 : 1 CHCl3–CH3OH 1% AcOH). [a]D -33.2 (c, 1.6).
1H-NMR: d 7.74 (d, 2H, J = 7.3 Hz, Ar), 7.56 (t, 2H, J = 7.3
Hz, Ar), 7.46–7.19 (m, 19 H, Ar), 5.58 (d, 1H, J = 9.3 Hz, Leu-
NH), 4.57 (m, 1H, Leu-aH), 4.31 (m, 2H, -CH2CHAr2), 4.18
(brt, 1H, J = 7.1 Hz, -CHAr2), 3.40 (m, 1H, -CH2CH3), 3.09–2.89
(m, 3H, Cys-bH ¥ 2, -CH2CH3), 2.74 (dd, 1H, 4.9, 9.3 Hz, Cys-
aH), 1.70 (m, 1H, Leu-g H), 1.52 (m, 1H, Leu-bH), 1.42 (m, 1H,
Leu-bH), 1.06 (brt, 3H, J = 7.1 Hz, -CH2CH3), 0.96 (d, 3H, J =
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N2 -(9-Fluorenylmethoxycarbonyl)-N5 -triphenylmethyl-L-glutam-
inyl-N-ethyl-S-triphenylmethyl-L-cysteine
-CH2CH3), 3.04 (dd, 1H, J = 5,4, 14.1 Hz, Cys-aH), 2.91 (dd, 1H,
J = 8.3, 14.1 Hz, Cys-bH), 2.78 (m, 2H, Cys-bH, -CH2CH3), 1.16
(s, 9H, t-Bu), 1.08 (d, 3H, J = 6.3 Hz, Thr-g H), 0.98 (t, 3H, J = 7.3
Hz, -CH2CH3). 13C-NMR: d 68.7 (Thr-bC), 60.7 (Cys-aC), 55.1
(Thr-aC), 45.3 (-CH2CH3), 30.3 (Cys-bC), 28.2 (C(CH3)3), 18.8
(Thr-CH3), 13.7 (-CH2CH3). Anal. calcd for C47H50N2O6S·H2O:
C, 71.55; H, 6.64; N, 3.55; O, 14.19; S, 4.06. Found: C, 71.37; H,
6.57; N, 3.41; S, 3.90% MALDI TOF MS, calcd for C47H50N2O6S
(M + Na)+: 793.33. Found: m/z 793.07.
Rf 0.45 (19 : 1 CHCl3–CH3OH 1% AcOH). [a]D -20.9 (c, 1.0). 1H-
NMR: d 7.73 (d, 2H, J = 7.3 Hz, Ar), 7.55 (dd, 2H, J = 7.8 15.6 Hz,
Ar), 7.40–7.15 (m, 34H, Ar), 5.85 (d, 1H, J = 7.8 Hz, Gln-NH),
4.37 (m, 1H, Gln-aH), 4.32 (d, 2H, J = 7.3 Hz, -CH2CHAr), 4.16
(t, 1H, J = 6.8 Hz, -CHAr), 3.39 (m, 1H, -CH2CH3), 2.95 (dd,
1H, J = 10.7 14.1 Hz, Cys-bH), 2.87 (m, 2H, Cys-a, bH), 2.69
(m, 1H, -CH2CH3), 2.32 (m, 2H, Gln-g H), 2.09 (m, 1H, Gln-
bH), 1.64 (m, 1H, Gln-bH), 0.87 (t, 3H, -CH2CH3). 13C-NMR:
d 59.8 (Cys-aC), 50.4 (Gln-aC), 44.5 (-CH2CH3), 32.6 (Gln-g C),
30.1 (Cys-bC), 29.5 (Gln-bC), 14.0 (-CH2CH3). Anal. calcd for
C63H57N3O6S·1.5H2O: C, 74.83; H, 5.98; N, 4.16; S, 3.17. Found:
C, 74.76; H, 6.14; N, 3.91; S, 2.96% MALDI TOF MS, calcd for
C63H57N3O6S (M + Na)+: 1006.39. Found: m/z 1006.29.
N-(9-Fluorenylmethoxycarbonyl)-valyl-N-ethyl-S-triphenylmeth-
yl-L-cysteine
Rf 0.31 (19 : 1 CHCl3–CH3OH 0.5% Ac OH). [a]D -31.1 (c, 1.0).
1H-NMR:d 7.74 (d, 2H, J = 7.8 Hz, Ar), 7.56 (dd, 2H, J = 7.3, 10.2
Hz, Ar), 7.43–7.17 (m, 19H, Ar), 5.77 (d, 1H, J = 9.3 Hz, Val-NH),
4.37 (dd, 1H, 6.3, 9.3 Hz, Val-aH), 4.29 (d, 2H, -CH2CHAr2),
4.18 (brt, 1H, J = 7.3 Hz, -CHAr2), 3.49 (m, 1H, -CH2CH3),
3.09 (dd, 1H, J = 9.8, 14.6 Hz, Cys-bH), 2.94 (m, 2H, -CH2CH3,
Cys-bH), 2.61 (dd, 1H, J = 4.9, 8.8 Hz, Cys-aH), 1.98 (m, 1H,
Val-bH), 1.00 (brt, 1H, J = 7.1 Hz, -CH2CH3), 0.96–0.86 (m,
6H, Val-CH3). 13C-NMR: d 60.4 (Cys-aC), 55.6 (Val-aC), 45.6
(-CH2CH3), 30.3 (Cys-bC), 19.6 (Val-CH3), 17.4 (Val-CH3), 14.0
(-CH2CH3). Anal. calcd for C44H44N2O5S·1/2 H2O: C, 73.21; H,
6.28; N, 3.88; O, 12.19; S, 4.44. Found: C, 73.50; H, 6.44; N, 3.85;
S, 4.27% MALDI TOF MS, calcd for C44H44N2O5S (M + Na)+:
735.29. Found: m/z 735.52.
N-(9-Fluorenylmethoxycarbonyl)-Ng -(2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl)
Rf 0.38 (9 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -22.9 (c, 1.1). 1H-
NMR (DMSO-d6): d 7.89 (d, 2H, J = 7.8 Hz, Ar), 7.73 (dd, 2H,
J = 7.3, 12.7 Hz, Ar), 7.59 (d, 1H, J = 8.3 Hz, Arg-NH), 7.43–7.25
(m, 19 H, Ar), 4.34 (m, 1H, Arg-aH), 4.22 (m, 3H, -CH2CHAr2),
3.31 (m, Cys-aH), 3.05 (m, 3H, -CH2CH3, Arg-dH ¥ 2), 2.92 (s,
2H, Pbf: -CH2-), 2.52 (s, 3H, Pbf: -CH3), 2.44 (s, 3H, Pbf: -CH3),
1.99 (s, 3H, Pbf: -CH3), 1.54 (1H, Arg-bH), 1.48 (1H, Arg-bH),
1.39 (s, 6H, -CH3 x2), 1.03 (brt, 3H, J = 6.6 Hz, -CH2CH3). 13C-
NMR: d 59.3 (Cys-aC), 42.5 (Pbf: -CH2-), 28.3 (Pbf: -CH3), 19.0
(Pbf: -CH3), 17.6 (Pbf: -CH3), 14.3 (-CH2CH3) 12.3 (Pbf: -CH3).
Anal. calcd for C58H63N5O8S2·1.5H2O: C, 66.39; H, 6.34; N, 6.67.
Found: C, 66.33; H, 6.37; N, 6.31% MALDI TOF MS, calcd for
C58H63N5O8S2 (M + Na)+: 1044.4. Found: m/z 1044.40.
Na-(9-Fluorenylmethoxycarbonyl)-N1-tert-butoxycarbonyl-tryp-
tophyl-N-ethyl-S-triphenylmethyl-L-cysteine
Rf 0.39 (19 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -23.0 (c, 1.2).
1H-NMR:d 7.76 (t, 2H, J = 7.1 Hz, Ar), 7.61–7.03 (m, 26H, Ar),
6.60 (d, 1H, J = 8.8 Hz, Trp-NH), 4.92 (m, 1H, Trp-aH), 4.38 (m,
1H, -CH2CHAr2), 4.23 (m, 2H, -CH2CHAr2), 3.23 (dd, 1H, J =
8.8, 13.7 Hz, Trp-bH), 3.11–3.03 (m, 2H, Trp-bH, -CH2CH3),
2.93 (m, 1H, Cys-aH), 2.70 (m, 1H, Cys-bH), 2.36 (m, 1H,
-CH2CH3), 2.30 (m 1H, Cys-aH), 1.57 (s, 9H, But), 0.82 (t, 3H, J =
6.8 Hz, -CH2CH3). 13C-NMR: d 60.7 (Cys-aC), 50.9 (Trp-aC),
29.8 (Cys-bC), 28.2 (C(CH3)3), 13.7 (-CH2CH3). Anal. calcd for
C55H53N3O7S·2H2O: C, 70.57; H, 6.14; N, 4.49; O, 15.38; S, 3.43.
Found: C, 70.63; H, 5.92; N, 4.59; S, 3.38% MALDI TOF MS,
calcd for C55H53N3O7S (M + Na)+: 922.35. Found: m/z 922.39.
N-(9-Fluorenylmethoxycarbonyl)-O3-tert-butyl-L-seryl-N-ethyl-S-
triphenylmethyl-L-cysteine
Rf 0.43 (9 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -55.7 (c, 1.0).
1H-NMR:d 7.74 (d, 2H, J = 7.8 Hz, Ar), 7.58 (dd, 2H, J = 7.3,
15.1 Hz, Ar), 7.42–7.16 (m, 19H, Ar), 5.88 (d, 1H, J = 8.8 Hz,
Ser-NH), 4.66 (m, 1H, Ser-aH), 4.29 (m, 2H, -CH2CHAr2), 4.18
(m, 1H, -CHAr2), 3.66 (m, 1H, -CH2CH3), 3.51 (m, 1H, Ser-
bH), 3.38 (brt, 1H, J = 8.5 Hz, Ser-bH), 3.21 (dd, 1H, J = 6.3,
14.1 Hz, Cys-bH), 2.83–2.74 (m, 2H, Cys-a, bH), 2.52 (m, 1H,
-CH2CH3), 1.07 (s, 9H, t-Bu), 0.96 (t, 3H, J = 7.1 Hz, -CH2CH3).
13C-NMR: d 63.2 (Ser-bC), 61.0 (Cys-aC), 50.5 (Ser-aC), 44.5
(-CH2CH3), 29.9 (Cys-bC), 27.2 (C(CH3)3), 13.7 (-CH2CH3).
Anal. calcd for C46H48N2O6S: C, 72.99; H, 6.39; N, 3.70; S, 4.24.
Found: C, 72.65; H, 6.54; N, 3.68; S, 4.11% MALDI TOF MS,
calcd for C46H48N2O6S (M + Na)+: 779.31. Found: m/z 779.37.
N-(9-Fluorenylmethoxycarbonyl)-O4-tert-butyl-tyrosyl-N-ethyl-S-
triphenylmethyl-L-cysteine
Rf 0.38 (19 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -64.0 (c, 1.0).
1H-NMR: d 7.76 (d, 2H, J = 7.3 Hz, Ar), 7.59 (dd, 2H, J = 7.3,
13.7 Hz, Ar), 7.41–7.15 (m, 19H, Ar), 7.03 (d, 2H, J = 8.3 Hz, Ar),
6.67 (m, 2H, Ar), 6.19 (d, 1H, J = 8.3 Hz, Tyr-NH), 4.60 (m, 1H,
Tyr-aH), 4.34–4.20 (m, 3H, -CH2CHAr2, -CHAr2), 3.29 (m, 1H,
Cys-bH), 2.97 (m, 2H, Tyr-bH), 2.71 (m, 1H, -CH2CH3), 2.50 (m,
2H, Cys-a, bH), 1.94 (m, 1H, -CH2CH3), 1.19 (m, 9H, t-Bu), 0.76
(brt, 3H, J = 7.1 Hz, -CH2CH3). 13C-NMR: d 61.4 (Cys-aC), 52.3
(Tyr-aC), 44.1 (-CH2CH3), 39.6 (Tyr-bC), 29.7 (Cys-bC), 28.7
(C(CH3)3), 13.6 (-CH2CH3). Anal. calcd for C52H52N2O6S·0.7H2O:
C, 73.85; H, 6.36; N, 3.31; S, 3.79. Found: C, 73.81; H, 6.47; N,
3.25; S, 3.66% MALDI TOF MS, calcd for C52H52N2O6S (M +
Na)+: 855.34. Found: m/z 855.38.
N -(9-Fluorenylmethoxycarbonyl)-O3 -tert-butyl-L -threonyl-N-
ethyl-S-triphenylmethyl-L-cysteine
Rf 0.43 (19 : 1 CHCl3–CH3OH 0.5% AcOH). [a]D -27.8 (c, 1.0).
1H-NMR:d 7.74 (d, 2H, J = 7.3 Hz, Ar), 7.57 (dd, 2H, J = 7.3,
13.2 Hz, Ar), 7.44 (d, 4H, J = 7.3 Hz, Ar), 7.36 (t, 2H, J =
7.3 Hz, Ar), 7.29–7.17 (m, 13H, Ar), 5.76 (d, 1H, J = 8.3 Hz,
Thr-NH), 4.48 (dd, 1H, J = 5.4, 8.3 Hz, Thr-aH), 4.36–4.26 (m,
2H, -CH2CHAr2), 4.19 (m, 1H, -CHAr2), 3.84 (m, 2H, Thr-bH,
6812 | Org. Biomol. Chem., 2011, 9, 6807–6813
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Synthesis of peptide thioesters
sity for their support with a grant-in-aid for high technology
research.
Fmoc-Rink amide MBHA resin (74 mg, 25 mmol) was subjected
to automated synthesis by the FastMoc protocol to give Arg(Pbf)-
Arg(Pbf)-NH-resin. To this resin, Fmoc-amino acyl N-ethyl-S-
tritylcysteine (50 mmol), activated by DIC (11.6 ml, 75 mmol)
and HOBt (10.1 mg, 75 mmol) in dichloromethane for 30 min
at room temperature, was added. After the mixture was shaken
overnight, the peptide chain was elongated by the FastMoc
protocol. A part of the resin obtained (30 mg) was treated with
a TFA cocktail (TFA : H2O : triisopropylsilane 90 : 5 : 5, 400 ml) at
room temperature for 1 h. After removing TFA under a nitrogen
stream, the peptide was precipitated with ether. The precipitate
was washed twice with ether, dried in vacuo, and dissolved in 3 ml
of 50% aqueous acetonitrile containing 6 M urea and 5% (v/v)
3-mercaptopropionic acid (MPA). After the mixture was filtered,
the filtrate was kept at 37 ◦C for 2 d. The solution was loaded
on a RP-HPLC column (Mightysil 5C18, 10 ¥ 250 mm) and the
fraction containing the product was isolated and lyophilized to
give the desired peptide thioesters.
Notes and references
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Gln-Lys-Thr-Glu-Phe-SCH2CH2COOH
39% based on the amino group in the initial resin. MALDI-TOF
mass, found: m/z 740.5, calcd for (M + H)+: 740.3. Amino acid
analysis: Thr0.91Glu2.05Phe1Lys1.02
.
Met-Gly-Thr-Ala-Asn-SCH2CH2COOH
46% based on the amino group in the initial resin. MALDI-TOF
mass, found: m/z 581.0, calcd for (M + H)+: 581.2. Amino acid
analysis: Asp1.11Thr1.03Gly1Ala0.99Met1.00
.
Phe-Lys-Val-Asp-Ser-SCH2CH2COOH
32% based on the amino group in the initial resin. MALDI-TOF
mass, found: m/z 683.1, calcd for (M + H)+: 683.3. Amino acid
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analysis: Asp1.04Ser0.85Val1.01Phe1Lys1.02
.
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Acknowledgements
This work was supported by a grant-in-aid for creative scientific
research (17GS0420) from the Japan Society for the Promotion
of Science, and by a grant-in-aid for scientific research from
the Ministry of Education, Sports, Science and Technology of
Japan (20380069). We thank Dr H. Koshino for HRNMR, Dr
T. Nakamura for HRMS, and staff of the analysis laboratory
for elemental analysis at Riken. We also thank Tokai Univer-
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This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6807–6813 | 6813
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