Absolute stereo-structure of kendarimide A, a novelMDR modulator, from a marine sponge

Article abstract of DOI:10.3987/COM-04-10287

The absolute stereostructure of two N-methylcysteines in kendarimideA (1), a novel linear peptide reversing multidrug resistance (MDR) mediated by P-glycoprotein, was determined by comparison with synthetic model compounds. For this purpose, four diastere

Full text of DOI:10.3987/COM-04-10287

HETEROCYCLES, Vol. 65, No. 3, 2005
563
HETEROCYCLES, Vol. 65, No. 3, 2005, pp. 563 - 578
Received, 18th November, 2004, Accepted, 14th January, 2005, Published online, 21st January, 2005
ABSOLUTE STEREO-STRUCTURE OF KENDARIMIDE A, A NOVEL
MDR MODULATOR, FROM A MARINE SPONGE
Naoyuki Kotoku, Liwei Cao, Shunji Aoki, and Motomasa Kobayashi*
Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6,
Suita, Osaka 565-0871, Japan; E-mail: kobayasi@phs.osaka-u.ac.jp.
Abstract - The absolute stereostructure of two N-methylcysteines in kendarimide
A (1), a novel linear peptide reversing multidrug resistance (MDR) mediated by
P-glycoprotein, was determined by comparison with synthetic model compounds.
For this purpose, four diastereomers (17a-d) of the C-terminal tetrapeptides in
kendarimide A (1), containing L,L-, L,D-, D,L-, D,D-N-methylcysteinyl-N-
methylcysteine, were synthesized. The absolute configuration of both the two
adjacent N-methylcysteines in 1, which form an eight-membered disulfide ring
(ox-[MeCys-MeCys]), was elucidated to be L-configuration by comparison of the
NMR spectral data of the four model tetrapeptides with that of kendarimide A (1).
INTRODUCTION
As a part of our study of biologically active substances from marine organisms, we have been searching
for new modulators of multidrug resistance (MDR) in tumor cells and have isolated kendarimide A (1), a
novel modulator of P-glycoprotein (P-gp)-mediated MDR, from a marine sponge of Haliclona sp.
collected at Sulawesi Island, Indonesia.1 The chemical structure of 1 was characterized to be a linear
tetradeca-peptide composed of N-methylpyroglutamic acid (pyroMeGlu), an eight-membered disulfide-
containing N-methylcysteinyl-N-methylcysteine (ox-[MeCys-MeCys]), and many N-methylamino acid
residues (Figure 1). The plane structure of 1 was elucidated by analyses of 1D, 2D-NMR spectra and the
fragmentation in FAB MS spectrum. The absolute configurations of twelve amino acid residues in 1 were
determined to be all L-configuration by Marfey’s method except for two N-methylcysteines, which
decomposed during acidic hydrolysis of 1. So far, kendarimide A (1) was the first example of peptide
which have two adjacent N-methylated cysteines forming an eight-membered disulfide ring (ox-[MeCys-
MeCys]). A few examples of peptides or proteins, such as malformin A2 and the nicotinic acetylcholine

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HETEROCYCLES, Vol. 65, No. 3, 2005
receptor unit,3 are known as cysteinyl-cysteine forming an eight-membered disulfide ring (ox-[Cys-Cys]).
The eight-membered disulfide ring (ox-[Cys-Cys]) is known to be able to exist in different conformational
states,4,5 since it is a highly constrained structure element. A complex NMR spectrum has been observed
for the ox-[Cys-Cys] part of the nicotinic acetylcholine receptor unit, while kendarimide A (1) showed a
simple NMR spectrum assignable as a single conformation. Then, with the aim of determining the
configurations of the two N-methylcysteines in 1, four tetrapeptide diastereomers (17a-d) of the C-
terminus, containing an eight-membered L,L-, L,D-, D,L-, D,D-N-methylcysteinyl-N-methylcysteine ring,
were synthesized (Figure 1). Herein, we report full details of the synthesis of the model tetrapeptides
(17a-d) and the determination of the absolute configuration of N-methylcysteines in 1 by comparison of
their NMR spectral data with those of 1.
O
O
N
N
S
1
2
N
MeCys MeCys
N
N
S
O
*
L
-MeVal
O
O
L-Pha
*
S
S
N
N
O
O
O
N
H
N
N
*
*
O
HN
H
N
OR
O
O
N
OH
O
N
O
1
2
H
N
O
16a:
16b:
16c:
16d:
L
-MeVal-
-MeVal-
-MeVal-
L
-MeCys -
1
L
-MeCys -
2
L
-Pha , R= Ac
N
O
L
L
-MeCys -
1
D
-MeCys -
2
L
-Pha , R= Ac
N
N
L
L
D
-MeCys -
1
L
-MeCys -
2
L
-Pha , R= Ac
O
O
-MeVal-
D
-MeCys -D-MeCys -
L
-Pha , R= Ac
1
2
17a:
17b:
17c:
17d:
L
-MeVal-
-MeVal-
-MeVal-
-MeVal-
L
-MeCys -
1
L
-MeCys -
2
L
-Pha , R= H
L
L
-MeCys -
1
D
-MeCys -
2
L
-Pha , R= H
kendarimide A (1)
L
D-MeCys -L-MeCys -L-Pha , R= H
1
2
L
D-MeCys -D-MeCys -L-Pha , R= H
Figure 1. Chemical structures of kendarimide A and model tetrapeptides.
RESULTS AND DISCUSSION
The objective tetrapeptides at the C-terminus of kendarimide A (1) were synthesized in order from L-
phenylalaninol to L-N-methylvaline by one-by-one coupling. We started from the synthesis of compound
(17a) (L-MeVal-ox-[L-MeCys-L-MeCys]-L-Pha). N-Methylcysteine could be prepared by Birch reduction
of thiazolidine-4-carboxylic acid,6 which was easily synthesized from cysteine hydrochloride and 37%
formaldehyde.7 Sequential S- and N-protections with an acetamidomethyl (Acm) group and a tert-
butoxycarbonyl (Boc) group, respectively, gave N-Boc-MeCys(Acm)-OH (2a), an important precursor to
form a disulfide-bridge of the MeCys residue (Scheme 1). Coupling of 2a with 3, which was obtained
from L-phenylalaninol, by diethyl phosphorocyanidate (DEPC) in the presence of triethylamine (TEA)
provided a dipeptide (4a) in high yield (96%). Following removal of the N-protecting Boc group gave an

HETEROCYCLES, Vol. 65, No. 3, 2005
565
amine (5a).
SH
SAcm
COOH
2a
a
H
Boc
N
COOH
N
SAcm
Me
Me
H
N
d, e
R
L
-N-methylcysteine
N
OAc
CH Ph
Me
O
b, c
2
H N
2
H N
2
OH
CH Ph
OAc
CH Ph
4a, R = Boc
5a, R = H
2
2
3
L-phenylalaninol
Scheme 1. Reagents and conditions: a) i. acetamidomethanol, conc. HCl; ii. (Boc)2O, 1N NaOH, 42% in
2 steps. b) i. (Boc)2O, 1N NaOH; ii. Ac2O, pyridine, 91% in 2 steps. c) TFA, CH2Cl2, 97%. d) DEPC, Et3N,
DMF, 96%. e) TFA, CH2Cl2, quant.
Coupling reaction of 5a with 2a could not proceed under the above conditions or by 1-(3-
8
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt).
Exchange of the additive from HOBt to 1-hydroxy-7-azabenzotriazole (HOAt) 9 and the solvent from
CH2Cl2 to CH2Cl2-DMF (2:1) led to successful synthesis of a tripeptide (6a) (Scheme 2). Subsequent
treatment with iodine10 afforded compound (7a) through oxidative S-deprotection and disulfide bond
formation. Boc deprotection of 7a with TFA led to an amine (8a). However, condensation of 8a with N-
Boc-MeVal-OH could not proceed to give the desired tetrapeptide (9a) under the condition of
EDCI/HOAt. Condensation of 10a, a deprotected form of the tripeptide (6a), with N-Boc-MeVal-OH was
also unsuccessful.
SAcm
S
S
Me
N
O
H
N
b
H
N
a
R
2a + 5a
R
N
OAc
CH Ph
N
N
OAc
CH Ph
Me
O
O
2
Me
Me
O
2
AcmS
6a, R = Boc
10a, R = H
7a, R = Boc
8a, R = H
c
c
d
d
SAcm
S
S
Me
O
Me
N
O
H
H
N
Boc
N
N
N
N
OAc
CH Ph
Boc
N
N
OAc
CH Ph
Me
O
Me
O
2
O
Me
Me
O
2
AcmS
11a
9a
Scheme 2. Reagents and conditions: a) EDCI, HOAt, CH2Cl2/DMF, 58%. b) I2, CH2Cl2/MeOH, 86%. c)
TFA, CH2Cl2, quant. d) N-Boc-L-MeVal-OH, EDCI•HCl, HOAt, CH2Cl2/DMF.

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HETEROCYCLES, Vol. 65, No. 3, 2005
N-Methylated amino acid residues are found in many biologically active peptides, such as cyclosporins,11
theonellapeptolides12 and didemnins.13 Formation of a peptide bond between N-methylated amino acids is
difficult, and many general reagents for peptide bond-formation are ineffective. To resolve this problem
and meet the demand for efficient synthesis of these sterically hindered peptides, novel coupling reagents
have been developed.14 Bis(2-oxo-3-oxazolidinyl)phosphinic chloride15 (BOP-Cl), which was successfully
employed in the synthesis of cyclosporin A, and bromotris(dimethylamino)phosphonium
hexafluorophosphate16 (Brop), which was effective even in the case of N-alkylated amino acids, attracted
our attention. Choice of the protecting group of N-H is also important for successful coupling, and 9-
fluorenylmethoxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz) groups were effective in the synthesis of
cyclosporin A.17 On the basis of this information, we carried out a model synthesis to find an effective
method as shown in Scheme 3. The coupling reaction of Fmoc- or Cbz-protected N-methylvaline with L-
MeCys(Acm)-OMe (12) by Brop and diisopropylethylamine (DIEA) proceeded to give the corresponding
dipeptide (13), while the coupling reaction using BOP-Cl failed regardless of the N-protecting group of N-
methylvaline.
SAcm
SAcm
Me
N
Me
N
O
coupling reagent
COOH
+
H
R
R
N
COOMe
N
COOMe
DIEA, CH Cl
2
rt, 1 h
2
Me
Me
N-R-L-MeVal-OH
12
13
Yield of 13 (%)
Brop
Entry
R
BOP-Cl
-
-
1
2
3
Boc
Cbz
-
-
34
30
Fmoc
Scheme 3.
Taking the convenience of deprotection into consideration, N-Fmoc-protected N-methylvaline was
selected for the coupling unit with tripeptide (10a), and the desired tetrapeptide (14a) was obtained in
77% yield (Scheme 4). Following formation of a disulfide bond by oxidation with iodine, compound
(14a) was readily converted to an eight-membered disulfide (18a). However, Fmoc deprotection of 18a in
the presence of diethylamine gave a complex mixture instead of the desired tetrapeptide (16a). Therefore,
removal of the Fmoc group of 14a was executed beforehand to give 15a, and subsequent treatment with
iodine led to the desired tetrapeptide acetate (16a). Finally, Na2CO3 treatment of compound (16a) in 70%
EtOH gave the desired tetrapeptide (17a).

HETEROCYCLES, Vol. 65, No. 3, 2005
567
SAcm
S
S
Me
N
O
Me
N
O
H
N
d
H
N
a
R
10a
N
N
OAc
H
N
N
OR
CH Ph
Me
O
Me
O
CH Ph
2
O
Me
Me
O
2
AcmS
16a, R = Ac
17a, R = H
14a, R = Fmoc
15a, R = H
e
c
S
S
Me
N
O
b
H
N
14a
Fmoc
N
N
OAc
O
Me
Me
O
CH Ph
2
18a
Scheme 4. Reagents and conditions: a) N-Fmoc-L-MeVal-OH, Brop, DIEA, CH2Cl2, 77%. b) I2,
CH2Cl2/MeOH, 88%. c) Et2NH, CH2Cl2, 81%. d) I2, CH2Cl2/MeOH, 67%. e) Na2CO3, 70% EtOH, 74%.
Following the procedure of the synthesis of the tetrapeptide acetate (16a) (ox-LLLL-OAc (= ox-L-MeVal-
XX-L- Pha-OAc type; XX= L,L-N-methylcysteinyl-N-methylcysteine)) and its deacetylated analog (17a,
ox-LLLL-OH) , other diastereomeric tetrapeptide acetates (16b (ox-LLDL-OAc), 16c (ox-LDLL-OAc), and
16d (ox-LDDL-OAc)) and their deacetylated analogs (17b (ox-LLDL-OH), 17c (ox-LDLL-OH), and 17d (ox-
LDDL-OH)) were also prepared. All the proton and carbon signals of four acetates (16a – 16d) were
assigned by analysis of 1D- and 2D-NMR (COSY, HMQC, and HMBC) spectra as shown in Tables 1 and
2. Each of the tetrapeptide acetates (16a-16d) showed a similar 1H-NMR spectrum except for N-H proton
in phenylalaninol moiety and N-Me proton in MeCys2 moiety. Thus, the N-H proton signals of
phenylalaninol moiety for 16b and 16c and the N-Me proton signals of MeCys2 for 16a and 16d appeared
at higher fields. On the other hand, the 13C-NMR spectra of those tetrapeptide acetates were classified into
two groups (group A for 16a and 16d; group B for 16b and 16c). Thus, the carbon signals of C-1 and C-3
in MeCys1 and C-2 in MeCys2 for 16b and 16c were observed at lower fields. The chemical shifts of the
characteristic proton and carbon signals for 16a and 16d resembled to those for 1. However, the presence
of an acetyl group in 16a-16d made difficult to further comparison with the 1H-NMR spectrum of 1.
The deacetylated tetrapeptides (17a-17d) were unstable, in particular, compounds 17b (ox-LLDL-OH) and
17c (ox-LDLL-OH) were readily decomposed even when kept in a freezer. For this reason, we could
compare only the respective 1H-NMR spectral data for 17a-17d (Table 3). Fortunately, each of the four
diastereomers (17a-17d) showed the characteristic 1H-NMR spectrum, respectively. The chemical shifts
of some signals assignable to the L-phenylalaninol in 17b, 17c, and 17d were significantly different with
those of 1. For example, the N-H proton signals in 17b and 17c appeared at higher field than that of 1,
and the each hydroxymethyl proton of the phenylalaninol moiety in 17c and 17d is overlapped and
indistinguishable. The proton signals assignable to the terminal tetrapeptide in kendarimide A (1) were
closely similar to those of 17a. Thus, both N-methylcysteines in 1 were determined to be L-configuration,

568
HETEROCYCLES, Vol. 65, No. 3, 2005
Table 1. 1H-NMR spectral data for the C-terminal portion of kendarimide A and model tetrapeptide acetates
(ox-LXXL-OAc type, CDCl3, 500 MHz).
dH (J in Hz)
C/no.
kendarimide A
ox-LLLL-OAc
(16a)
ox-LLDL-OAc
(16b)
ox-LDLL-OAc
(16c)
ox-LDDL-OAc
(16d)
MeVal
2
3
5.28, d (11.0)
2.27#
3.27#
1.98, m
3.26#
1.92, m
3.49, d-like
1.97, m
3.27#
2.43
3-Me
0.96, d (6.5)
0.77, d (6.5)
2.96, s
1.04, t-like
1.04, t-like
2.37, s
1.02, d (6.7)
0.99, d (6.7)
2.34, s
1.02, d (6.7)
1.00, d (6.7)
2.42, s
1.13
1.11
2.54, s
4
NMe
MeCys1
2
3a
3b
NMe
MeCys2
2
3a
3b
NMe
Pha*
1a
5.56, dd like
3.23, dd (13.6, 11.7)
2.86#
5.65, d-like
3.25#
2.90#
3.23, s
5.43#
3.60, t-like
3.22, t-like
3.25, s
5.44#
3.35#
3.09#
2.94, s
5.54, d-like
3.22#
2.98#
3.24, s
3.29, s
5.23, dd like
3.33, dd (14.2, 4.0)
2.85#
5.13, dd (11.6, 4.3)
3.23#
2.95#
2.14, s
5.40#
3.40#
3.12, dd-like
3.27, s
5.42#
3.59, t-like
3.12, t-like
3.27, s
5.11, dd (11.6, 3.7)
2.96#
2.93#
2.68, s
2.34, s
3.77, dd (11.7, 3.0)
3.23, dd (11.7, 4.3)
4.28, m
2.95#
2.83#
7.20-7.26, m
7.37, d (8.7)
-
4.25, dd (11.3, 4.6)
4.15, dd (11.3, 5.8)
4.45, m
2.95#
2.74#
7.13-7.26, m
7.40, d (8.5)
2.10, s
4.11, dd-like
4.02, dd (11.3, 5.8)
4.38, m
2.86#
2.82#
7.16-7.31, m
6.66, d (8.5)
2.07, s
4.10#
4.10#
4.43, m
2.91, dd-like
2.78, dd-like
7.17-7.36, m
6.34
4.17, dd (11.0, 4.0)
4.02, dd (11.0, 6.7)
4.42, m
2.98#
2.90#
7.23-7.28, m
7.63, d (7.9)
2.01, s
1b
2
3a
3b
ArH
NH
COCH3
2.08, s
* phenylalaninol; # overlapped
and the total absolute stereostructure of kendarimide A (1) was clarified.
EXPERIMENTAL
The following instruments were used to obtain physical data: JEOL JMS SX-102 for FABMS spectra;
1
13
JEOL JNM Lambda-500 (500 MHz) and Varian unity inova 600 (600 MHz) for H-NMR and C-NMR
spectra. Silica gel (Fuji silysia BW-200) was used for column chromatography. Pre-coated thin layer
chromatography (TLC) plates (Merck, Kieselgel, 60F254) were used for TLC. Spots on TLC plates were
detected by spraying acidic p-anisaldehyde solution (p-anisaldehyde 25 ml, c-H2SO4 25 ml, acetic acid 5
ml, EtOH 425 ml) and ninhydrin solution (ninhydrin 2 g in sat. aq. n-BuOH) with subsequent heating. All
1
new compounds were determined to be >95% pure by H NMR spectroscopy. All the reaction solvents
were distilled prior to use according to standard procedure. N-Boc-MeCys(Acm)-OH (2) was prepared by
literature procedure.8
Abbreviations Acm; acetamidomethyl, Boc; tert-butoxycarbonyl, Brop; bromotris(dimethylamino)-

HETEROCYCLES, Vol. 65, No. 3, 2005
569
Table 2. 13C-NMR spectral data for the C-terminal portion of kendarimide A and model tetrapeptide acetates
(ox-LXXL-OAc type, CDCl3, 150 MHz).
dc
C/no.
ox-LLLL-OAc
(16a)
ox-LLDL-OAc
(16b)
ox-LDLL-OAc
(16c)
ox-LDDL-OAc
(16d)
kendarimide A
MeVal
1
2
3
172.88
57.78
27.72
19.50
17.98
30.30
174.52
64.93
23.40
18.41
16.45
34.56
175.64
65.54
31.13
19.89
17.99
35.05
172.71
64.48
30.77
19.17
18.45
34.04
172.84
63.81
30.68
18.86
18.54
33.52
3-Me
4
NMe
MeCys1
1
2
173.00
56.63
38.80
32.50
172.95
55.98
38.70
32.04
179.43
53.08
47.23
31.49
179.00
53.45
47.10
32.20
172.84
57.24
38.51
33.06
3
NMe
MeCys2
1
2
167.91
59.25
37.17
28.01
167.97
58.84
37.39a
27.43
168.57
62.87
38.84
32.65
168.52
62.75
38.90
32.52
167.73
58.93
37.39b
28.02
3
NMe
Pha*
1
2
3
4
5/9
6/8
7
CO
64.60
52.94
37.02
137.83
129.20
128.60
126.55
-
65.95
50.12
64.46
49.98
37.55
136.72
129.14
128.67
126.85
170.78
20.66
65.15
49.88
37.32
136.95
129.14
128.85
126.88
170.95
20.84
65.19
50.62
37.51a
137.32
129.03
128.56
126.64
171.00
20.89
37.45b
137.48
129.34
128.37
126.55
170.81
20.85
CH3
* phenylalaninol
-
a, b : Assignment may be interchangeable.
phosphonium
hexafluorophospate,
Cbz;
benzyloxycarbonyl,
BOP-Cl;
bis(2-oxo-3-
oxazolidinyl)phosphinic chloride, DIEA; diisopropylethylamine, DMF; N,N-dimethylformamide, EDCI;
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, TEA; triethylamine, EtOAc; ethyl acetate,
Et2NH; diethylamine, Fmoc; 9-fluorenylmethoxycarbonyl, HOAt; 1-hydroxy-7-azabenzotriazole, HOBt;
1-hydroxybenzotriazole, MeCys; N-methylcysteine, MeOH; methanol, MeVal; N-methylvaline, Pha;
phenylalaninol.
L-Pha-OAc (3). L-Phenylalaninol (0.8 g, 5.3 mmol) was dissolved in 1 N aqueous NaOH (6.4 mL, 6.4
mmol), and di-tert-butyl dicarbonate (1.28 g, 5.8 mmol) was added. The reaction mixture was stirred at
25 °C for 12 h and then brought to pH 3 by adding 1 N aqueous KHSO4. The resulting solution was
extracted with EtOAc (3 x 30 mL), and the combined organic phase was washed with brine, dried over
Na2SO4, filtered and concentrated in vacuo to give a crude N-Boc-L-Pha-OH. To the pyridine solution

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HETEROCYCLES, Vol. 65, No. 3, 2005
571
(10.6 mL) of the above crude product, acetic anhydride (4.4 mL, 47 mmol) was added at 0 °C and stirred at
25 °C for 12 h. The reaction mixture was poured into ice-cold water (30 mL) and extracted with Et2O (3 x
30 mL). The combined organic phase was washed with 0.5 N aqueous HCl, saturated aqueous NaHCO3,
brine, dried over Na2SO4, filtered and concentrated in vacuo. Flash chromatography (25% EtOAc–n-
hexane eluent) of the crude product afforded N-Boc-L-Pha-OAc (1.41 g, two steps, 91%) as a white
1
powder. H-NMR (500 MHz, CDCl3, d): 7.19-7.33 (5H, m), 4.68 (1H, m), 4.05 (2H, t-like), 2.82 (2H, dd-
like), 2.11 (3H, s), 1.43 (9H, s).
To a solution of N-Boc-L-Pha-OAc (0.70 g, 2.4 mmol) in CH2Cl2 (7.0 mL), TFA (7.0 mL) was added
dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 3 h and then concentrated in vacuo. The
residue was dissolved in CH2Cl2 (7.0 mL) and benzene (20 mL), and evaporated in vacuo to give a TFA
1
salt of 3 (0.71 g, 97%) as a white amorphous solid. H-NMR (500 MHz, CDCl3, d): 7.20-7.37 (5H, m),
4.32 (1H, dd, J = 12.5, 2.8 Hz), 4.12 (1H, dd, J = 12.5, 6.4 Hz), 3.70 (1H, m), 3.10 (1H, dd, J = 13.7, 6.4
Hz), 2.95 (1H, dd, J = 13.7, 8.9 Hz), 2.07 (3H, s).
N-Boc-L-MeCys(Acm)-L-Pha-OAc (4a). TFA salt of 3 (61 mg, 0.20 mmol) and 2a (64 mg, 0.21 mmol)
were dissolved in DMF (0.87 mL), and DEPC (30 µL, 0.24 mmol) was added at 0 °C. After 15 min, TEA
(45 µL, 0.40 mmol) was added and the reaction mixture was stirred at 0 °C for 2 h, and then at 25 °C for
12 h. The reaction solution was diluted with EtOAc/benzene (v/v, 3:1, 20 ml) and washed with 1M aqueous
KHSO4, saturated aqueous NaHCO3, brine, dried over Na2SO4, filtered and concentrated in vacuo. Flash
chromatography (EtOAc eluent) of the crude product gave dipeptide (4a) (96 mg, 96%) as a white
amorphous solid. FAB MS: m/z 482 (M+H)+. HR-FAB MS: m/z 482.2301, calcd for C23H36N3O6S, Found
482.2313. 1H-NMR (500 MHz, CDCl3, d): 7.16-7.31 (5H, m), 6.66 (1H, br s), 6.38 (1H, d-like), 4.80 (1H,
t-like), 4.57 (1H, dd-like), 4.41 (1H, m), 4.10 (3H, m), 3.05 (1H, dd, J = 14.6, 6.1 Hz), 2.86 (1H, dd, J =
14.0, 6.7 Hz), 2.73-2.80 (2H, m), 2.72, 2.50 (total 3H, both br s), 2.10 (3H, s), 2.00 (3H, s), 1.49 (9H, s).
N-Boc-L-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (6a). To a solution of 4a (96 mg, 0.19 mmol) in
CH2Cl2 (1.5 mL) at 0 °C, TFA (1.5 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 3
h and then concentrated in vacuo. The residue was dissolved in CHCl3 (30 mL), and the organic layer was
washed with saturated aqueous NaHCO3, brine, dried over Na2SO4, filtered and concentrated in vacuo to
give 5a (69 mg, quant.) as a white amorphous solid. FAB MS: m/z 382 (M+H)+. HR-FAB MS: m/z
382.1805, calcd for C18H28N3O4S, Found 382.1803. 1H-NMR (500 MHz, CDCl3, d): 7.53 (1H, d, J= 8.5 Hz),
7.16-7.29 (5H, m), 6.69 (1H, br s), 4.39 (1H, m), 4.26 (1H, dd, J = 14.0, 6.7 Hz), 4.11 (1H, dd, J = 11.3,
4.6 Hz), 4.05 (1H, dd, J = 11.3, 5.8 Hz), 3.84 (1H, dd, J = 14.0, 5.5 Hz), 3.14 (1H, t-like), 2.88 (1H, dd, J =

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14.0, 6.7 Hz), 2.82 (1H, dd, J = 14.5, 4.0 Hz), 2.78 (1H, dd, J = 14.0, 4.6 Hz), 2.71 (1H, dd, J = 14.5, 7.6
Hz), 2.30 (3H, s), 2.07 (3H, s), 1.97 (3H, s).
A solution of 2a (61 mg, 0.20 mmol) in DMF/CH2Cl2 (v/v, 1:2, 1.0 mL) was treated with HOAt (270 mg,
0.40 mmol) and EDCI (380 mg, 0.40 mmol) at 0 °C for 15 min under stirring. To the reaction mixture, a
DMF/CH2Cl2 solution (v/v, 1:2, 1.0 mL) of 5a (74 mg, 0.19 mmol) was added, and the reaction mixture
was stirred at 25 °C for additional 12 h. The reaction mixture was poured into 1 N aqueous HCl and
extracted with EtOAc (3 x 30 mL). The combined organic phases were washed with saturated aqueous
NaHCO3, brine, dried over Na2SO4, filtered and concentrated in vacuo. Flash chromatography (9%
MeOH–EtOAc eluent) of crude products afforded 6a (77 mg, two steps, 58 %) as a white amorphous solid.
FAB MS: m/z 670 (M+H)+. HR-FAB MS: m/z 670.2944, calcd for C30H48N5O8S2, Found 670.2975. A
1
mixture of multiple conformers. H-NMR (500 MHz, CDCl3, d): 7.13-7.24 (5H, m), 6.38-7.06 (3H, d, t-
like), 5.23, 5.18 (1H, dd-like), 5.06, 5.11 (1H, dd-like), 4.67 (1H, dd, J = 11.0, 4.9 Hz), 4.51 (1H, dd-like),
4.36 (1H, m), 3.93-4.24 (4H, m), 2.45-3.06 (12H, m), 1.94-2.03 (9H, series of s), 1.41-1.45 (9H, series of
s).
N-Boc-ox-[L-MeCys-L-MeCys]-L-Pha-OAc (7a). To a solution of iodine (55 mg, 0.216 mmol) in CH2Cl2-
MeOH (10:1, 22 mL), 6a (14 mg, 0.021 mmol) in CH2Cl2 (6.6 mL) was added dropwise, and the reaction
mixture was stirred at 25 °C for 12 h. The reaction mixture was cooled in an ice bath, and 5% aqueous
Na2S2O3 was added until the color of iodine disappeared. The reaction mixture was washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo. Flash chromatography (50% EtOAc–n-hexane
eluent) of the crude product afforded 7a (9.5 mg, 86%) as a white amorphous solid. FAB MS: m/z 526
1
(M+H)+. HR-FAB MS: m/z 526.2046, calcd for C24H36N3O6S2, Found 526.2071. H-NMR (500 MHz,
CDCl3, d): 7.14-7.28 (5H, m), 5.33 (1H, d, J = 11.0 Hz), 5.10 (1H, dd, J = 11.6, 4.3 Hz), 4.43 (1H, m), 4.28
(1H, dd, J = 11.3, 4.6 Hz), 4.05 (1H, dd, J = 11.3, 5.2 Hz), 3.74 (1H, br s), 3.18 (2H, m), 3.03 (3H, s), 2.83-
2.97 (2H, m), 2.69 (1H, dd, J = 13.4, 10.4 Hz), 2.20 (3H, s), 2.09 (3H, s), 1.50 (9H, s).
N-Fmoc-L-MeVal-L-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (14a). To a solution of the compound
(6a) (67 mg, 0.10 mmol) in CH2Cl2 (0.75 mL) at 0 °C, TFA (0.75 mL) was added dropwise. The mixture
was stirred at 0 °C for 3 h, and then concentrated in vacuo. The residue was dissolved in CHCl3 (20 mL),
and the organic layer was washed with saturated aqueous NaHCO3, brine, dried over Na2SO4, filtered and
concentrated in vacuo to give 10a as a white amorphous solid. To a solution of Fmoc-L-MeVal (35 mg,
0.10 mmol), 10a (57 mg, 0.10 mmol), Brop (47 mg, 0.12 mmol) in CH2Cl2 (0.75 mL), and DIEA (47 µL,
0.24 mmol) was added. The reaction mixture was stirred at 25 °C for 12 h, and then CH2Cl2 was

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evaporated. The resulting residue was dissolved in EtOAc (30 mL) and washed with 5% aqueous KHSO4,
brine, 5% aqueous NaHCO3, brine, dried over Na2SO4, concentrated in vacuo. Flash chromatography (9%
MeOH-EtOAc eluent) of the crude product afforded 14a (69 mg, 77%) as a white amorphous solid. FAB
MS: m/z 905 (M+H)+. HR-FAB MS: m/z 905.3941, calcd for C46H61N6O9S2, Found 905.3921. A mixture
(3:1) of two conformers. 1H-NMR (500 MHz, CDCl3, d): 7.09-7.78 (14H, m), 6.41-6.90 (2H, d-like), 5.66,
5.69 (1H, both dd, J = 9.8, 4.3 Hz), 5.22, 5.28 (1H, both dd, J = 11.0, 4.9 Hz), 3.91-4.83 (11H, series of dd
or d), 2.35-3.21 (16H, series of dd or s), 1.99-2.09 (9H, series of s), 0.19-0.49, 0.81-0.86 (6H, series of d).
N-Fmoc-L-MeVal-ox-[L-MeCys-L-MeCys]-L-Pha-OAc (18a). Synthesis of 18a (88% yield) was
executed by the method described in the synthesis of 7a. FAB MS: m/z 761 (M+H)+. HR-FAB MS: m/z
1
761.3043, calcd for C40H49N4O7S2, Found 761.2987. A mixture (1:1) of two conformers. H-NMR (500
MHz, CDCl3, d): 7.79 (2H, m), 7.54-7.60 (2H, m), 7.01-7.43 (10H, m), 5.28, 5.50 (1H, both d, J = 11.0 Hz),
5.00, 5.12 (1H, both dd-like), 4.73, 4.94 (1H, both dd, J = 10.9, 3.7 Hz), 3.78, 4.85 (1H, both d, J = 10.7
Hz), 4.38, 4.56 (1H, both dd, J = 10.6, 6.7 Hz), 4.32-4.45 (1H, m), 4.12-4.24 (2H, series of dd), 4.04-4.18
(1H, both dd, J = 11.2, 4.9 Hz), 2.57-3.23 (6H, series of dd), 2.03, 2.05, 2.09, 2.12, 2.24, 2.72, 2.88, 3.27
(12H, series of s), 2.17, 2.30 (1H, m), 0.25, 0.53, 0.88, 0.99 (6H, series of d, J = 6.7 Hz).
L-MeVal-L-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (15a). To a solution of 14a (63 mg, 0.070 mmol)
in CH2Cl2 (0.7 mL), Et2NH (0.1 mL, 0.97 mmol) was added and stirred at 25 °C for 12 h. Reaction mixture
was concentrated in vacuo, and the resulting residue was purified by flash chromatography (25% MeOH-
EtOAc eluent) to provide 15a (39 mg, 81%) as a colorless amorphous. FAB MS: m/z 683 (M+H)+. HR-
FAB MS: m/z 683.3261, calcd for C31H51N6O7S2, Found 683.3245. 1H-NMR (500 MHz, CDCl3, d): 7.13-
7.31 (5H, m), 6.86 (2H, m), 6.60 (1H, t-like), 5.83 (1H, dd, J = 9.2, 4.9 Hz), 5.29 (1H, dd, J = 11.0, 4.9 Hz),
4.80 (1H, dd, J = 14.0, 7.3 Hz), 4.59 (1H, dd, J = 14.0, 7.3 Hz), 4.45 (1H, m), 4.05-4.16 (4H, m), 3.35 (1H,
d-like), 2.68-3.14 (6H, m), 2.92 (3H, s), 2.60 (3H, s), 2.41 (3H, s), 2.09 (3H, s), 2.07 (3H, s), 2.00 (3H, s),
1.05 (3H, d, J = 7.0 Hz), 0.91 (3H, d, J = 7.0 Hz).
L-MeVal-ox-[L-MeCys-L-MeCys]-L-Pha-OAc (16a). Synthesis of 16a (67% yield) was executed by the
method described in the synthesis of 7a. FAB MS: m/z 539 (M+H)+. HR-FAB MS: m/z 539.2362, calcd for
C25H39N4O5S2, Found 539.2368. 1H-NMR (500 MHz) and C-NMR (125 MHz) data in CDCl3 are given
13
in Table 1 and Table 2.
L-MeVal-ox-[L-MeCys-L-MeCys]-L-Pha-OH (17a). To a solution of 16a (4.6 mg, 8.6 µmol) in EtOH-
H2O (v/v, 7:3, 250 µL) Na2CO3 (2.9 mg, 27 µmol) was added. The reaction mixture was stirred at 25 °C for

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50 min and then neutralized with 1N aqueous HCl. The resulting solution was extracted with EtOAc,
washed with brine, dried over Na2SO4, filtered and concentrated, and the resulting residue was purified by
flash chromatography (30% MeOH-AcOEt eluent) to give 17a (3.1 mg, 74%) as a colorless powder. FAB
MS: m/z 497 (M+H)+. HR-FAB MS: m/z 497.2256, calcd for C23H37N4O4S2, Found 497.2242. 1H-NMR
(500 MHz) data in CDCl3 are given in Table 3.
N-Boc-D-MeCys(Acm)-L-Pha-OAc (4b). Colorless powder. FAB MS: m/z 482 (M+H)+. HR-FAB MS: m/z
1
482.2301, calcd for C23H36N3O6S, Found 482.2329. H-NMR (500 MHz, CDCl3, d): 7.17-7.31 (5H, m),
6.35-6.71 (2H, br s or d-like), 4.40-4.82 (3H, t-like, dd-like or m), 4.02-4.12 (3H, m), 3.03 (1H, dd, J =
14.6, 6.1 Hz), 2.71-2.89 (6H, m or s), 2.07 (3H, s), 1.99 (3H, s), 1.47 (9H, s).
D-MeCys(Acm)-L-Pha-OAc (5b). Colorless powder. FAB MS: m/z 382 (M+H)+. HR-FAB MS: m/z
1
382.1805, calcd for C18H28N3O4S, Found 382.1817. H-NMR (500 MHz, CDCl3, d): 7.56 (1H, d, J = 8.5
Hz), 7.19-7.31 (5H, m), 6.81 (1H, t-like), 4.42 (1H, m), 4.37 (1H, dd, J = 14.0, 6.7 Hz), 4.21 (1H, dd, J =
14.0, 6.1 Hz), 4.14 (1H, dd, J = 11.0, 4.3 Hz), 4.06 (1H, dd, J = 11.0, 5.8 Hz), 3.16 (1H, dd, J = 6.7, 4.3
Hz), 2.78-2.94 (4H, series of dd), 2.30 (3H, s), 2.08 (3H, s), 2.01 (3H, s).
N-Boc-L-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (6b). White amorphous solid. FAB MS: m/z 670
(M+H)+. HR-FAB MS: m/z 670.2944, calcd for C30H48N5O8S2, Found 670.2938. A mixture of multiple
conformers. 1H-NMR (500 MHz, CDCl3, d): 6.54-6.95, 7.91 (2H, series of d-like or t-like), 7.09-7.24 (6H,
m), 3.72-4.45, 4.73, 4.99, 5.11, 5.64, 6.29 (9H, series of dd or m), 2.42-3.06 (12H, series of dd or s), 1.86-
2.00 (9H, series of s), 1.35-1.43 (9H, series of s).
L-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (10b). White amorphous solid. FAB MS: m/z 570 (M+H)+.
HR-FAB MS: m/z 570.2420, calcd for C25H40N5O6S2, Found 570.2417. A mixture (5:4) of two conformers.
1H-NMR (500 MHz, CDCl3, d): 7.68 (1H, t-like), 7.16-7.28 (5H, m), 6.85 (1H, t-like), 6.78 (1H, d, J = 9.2
Hz), 5.28 (1H, dd, J = 11.0, 4.9 Hz), 4.49, 4.42 (2H, both dd, J = 14.0, 6.7 Hz), 4.34 (1H, m), 4.17 (1H, dd,
J = 14.0, 5.8 Hz), 4.11 (1H, dd, J = 14.0, 4.6 Hz), 4.06 (1H, dd, J = 11.6, 4.3 Hz), 3.98 (1H, dd, J = 11.6,
5.5 Hz), 3.64 (1H, dd, J = 7.3, 4.9 Hz), 2.98 (3H, s), 2.62-2.95 (6H, series of dd), 2.34 (3H, s), 2.04 (3H, s),
1.99 (3H, s), 1.97 (3H, s).
N-Fmoc-L-MeVal-L-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (14b). White amorphous solid. FAB MS:
m/z 905 (M+H)+. HR-FAB MS: m/z 905.3941, calcd for C46H61N6O9S2, Found 905.3949. A mixture of

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multiple conformers. 1H-NMR (500 MHz, CDCl3, d): 7.18-7.75 (14H, m), 8.10, 8.04, 6.81-7.11 (2H, d-like
or t-like), 5.18-6.58, 3.68-3.96 (13H, series of dd or m), 2.30, 2.44-3.06 (16H, series of dd, s or m), 1.86-
2.09 (9H, series of s), 0.21-0.26, 0.46-0.53, 0.80-0.95 (6H, series of d, J = 6.7 Hz).
L-MeVal-L-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (15b). White amorphous solid. FAB MS: m/z 683
(M+H)+. HR-FAB MS: m/z 683.3261, calcd for C31H51N6O7S2, Found 683.3258. A mixture (7:3) of two
1
conformers. H-NMR (500 MHz, CDCl3, d): 8.22 (1H, d, J = 9.2 Hz), 6.92-7.69 (total 8H, series of m),
3.88-4.97, 3.30, 4.77-5.55, 6.22 (9H, dd-like, t-like or m), 2.36, 2.53-3.14 (16H, series of dd or s), 1.92-2.9
(9H, series of s), 1.88 (1H, m), 0.88-0.94, 0.97-1.03 (6H, series of d, J = 6.7 Hz).
L-MeVal-ox-[L-MeCys-D-MeCys]-L-Pha-OAc (16b). Colorless powder. FAB MS: m/z 539 (M+H)+. HR-
FAB MS: m/z 539.2362, calcd for C25H39N4O5S2, Found 539.2364. 1H-NMR (500 MHz) and 13C-NMR (125
MHz) data in CDCl3 are given in Table 1 and Table 2.
L-MeVal-ox-[L-MeCys-D-MeCys]-L-Pha-OH (17b). Colorless powder. FAB MS: m/z 497 (M+H)+. HR-
FAB MS: m/z 497.2256, calcd for C23H37N4O4S2, Found 497.2230. 1H-NMR (500 MHz) data in CDCl3
are given in Table 3.
N-Boc-D-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (6c). White amorphous solid. FAB MS: m/z 670
(M+H)+. HR-FAB MS: m/z 670.2944, calcd for C30H48N5O8S2, Found 670.2933. A mixture of multiple
conformers. 1H-NMR (500 MHz, CDCl3, d): 7.02-7.23 (6H, m), 6.24, 6.53-6.85, 8.02 (2H, series of d or t-
like), 3.72-5.15, 5.52, 5.70 (9H, series of dd or m), 2.29-3.06 (12H, series of dd or s), 1.88-2.01 (9H, series
of s), 1.33-1.44 (9H, series of s).
D-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (10c). White amorphous solid. FAB MS: m/z 570 (M+H)+.
HR-FAB MS: m/z 570.2420, calcd for C25H40N5O6S2, Found 570.2408. A mixture (5:4) of two conformers.
1H-NMR (500 MHz, CDCl3, d): 7.73 (1H, t-like), 7.16-7.28 (5H, m), 6.90 (1H, t-like), 6.70 (1H, d, J = 8.5
Hz), 5.31, (1H, dd, J = 10.4, 4.9 Hz), 4.50, 4.53 (2H, both dd, J = 7.0, 4.6 Hz), 4.39 (1H, m), 4.02-4.17 (4H,
series of dd), 3.59 (1H, dd, J = 7.3, 4.3 Hz), 3.06 (1H, dd, J = 15.0, 5.2 Hz), 2.63-2.94 (8H, series of dd or
s), 2.36 (3H, s), 2.09 (3H, s), 2.00 (3H, s), 1.99 (3H, s).
N-Fmoc-L-MeVal-D-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (14c). White amorphous solid. FAB MS:
m/z 905 (M+H)+. HR-FAB MS: m/z 905.3941, calcd for C46H61N6O9S2, Found 905.3957. A mixture of

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multiple conformers. 1H-NMR (500 MHz, CDCl3, d): 7.10-7.75 (14H, m), 8.29, 6.46-6.84 (2H, d-like or t-
like), 5.01-6.16, 3.64-4.90 (13H, series of dd or m), 2.15-3.07 (16H, series of dd, s or m), 1.93-2.10 (9H,
series of s), 0.14, 0.21, 0.51, 0.55, 0.81, 0.94 (6H, series of d, J = 6.7 Hz).
L-MeVal-D-MeCys(Acm)-L-MeCys(Acm)-L-Pha-OAc (15c). White amorphous solid. FAB MS: m/z 683
(M+H)+. HR-FAB MS: m/z 683.3261, calcd for C31H51N6O7S2, Found 683.3279. A mixture (1:1) of two
conformers. 1H-NMR (500 MHz, CDCl3, d): 8.22, 6.91 (1H, both d, J = 8.5 Hz), 7.10-7.29 (5.5H, m), 6.59,
6.83, 7.67 (1.5H, series of t-like), 4.83, 4.97, 5.27, 5.54, 6.24 (2.5H, dd-like or t-like), 3.81-4.61 (6.5H,
series of dd or m), 2.29-3.35 (7H, series of dd or d), 2.29, 2.36, 2.51, 2.83, 2.91, 2.99 (9H, series of s), 1.98,
2.03, 2.07, 2.08 (9H, series of s), 1.90 (1H, m), 0.88, 0.93, 1.04 (6H, series of d, J = 6.7 Hz).
L-MeVal-ox-[D-MeCys-L-MeCys]-L-Pha-OAc (16c). Colorless powder. FAB MS: m/z 539 (M+H)+.
HR-FAB MS: m/z 539.2362, calcd for C25H39N4O5S2, Found 539.2373. 1H-NMR (500 MHz) and 13C-NMR
(125 MHz) data in CDCl3 are given in Table 1 and Table 2.
L-MeVal-ox-[D-MeCys-L-MeCys]-L-Pha-OH (17c). Colorless powder. FAB MS: m/z 497 (M+H)+. HR-
FAB MS: m/z 497.2256, calcd for C23H37N4O4S2, Found 497.2216. 1H-NMR (500 MHz) data in CDCl3
are given in Table 3.
N-Boc-D-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (6d). White amorphous solid. FAB MS: m/z 670
(M+H)+. HR-FAB MS: m/z 670.2944, calcd for C30H48N5O8S2, Found 670.2968. A mixture of multiple
conformers. 1H-NMR (500 MHz, CDCl3, d): 7.13-7.23 (5H, m), 6.56-7.07 (2H, d-like or t-like), 5.08-5.59
(2H, d-like or m), 3.98-4.74 (5H, m), 2.62-3.04 (12H, series of dd or s), 1.89-2.08 (9H, series of s), 1.38-
1.42 (9H, series of s).
D-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (10d). White amorphous solid. FAB MS: m/z 570 (M+H)+.
HR-FAB MS: m/z 570.2420, calcd for C25H40N5O6S2, Found 570.2431. A mixture (5:4) of two conformers.
1H-NMR (500 MHz, CDCl3, d): 8.50, 6.91 (1H, both d, J = 8.5 Hz), 7.50-7.11 (1H, both t-like), 7.17-7.31
(5H, m), 6.72 (1H, m), 5.37, (0.5H, dd, J = 10.4, 4.9 Hz), 3.92-4.68 (8H, series of dd or m), 3.73 (0.5H, t, J
= 6.1 Hz), 2.68-3.07 (6H, series of dd), 2.78, 2.96 (3H, 2s), 2.20, 2.40 (3H, both s), 1.99-2.07 (9H, series of
s).
N-Fmoc-L-MeVal-D-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (14d). White amorphous solid. FAB MS:

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m/z 905 (M+H)+. HR-FAB MS: m/z 905.3941, calcd for C46H61N6O9S2, Found 905.3929. A mixture (3:2) of
1
two conformers. H-NMR (500 MHz, CDCl3, d): 7.17-7.78 (13H, m), 6.26, 6.86, 6.99, 7.05 (2H, t-like),
6.63, 6.81 (1H, both d, J = 8.5 Hz), 5.55, 5.76 (1H, both dd, J = 10.4, 4.3 Hz), 5.25, 5.10 (1H, both dd, J =
11.5, 4.9 Hz), 4.95 (0.6H, dd, J = 10.7, 3.4 Hz), 3.91-4.78 (10.4H, series of dd or m), 2.51-3.07 (6H, series
of dd), 2.61-3.02 (7.5H, series of s), 2.30, 1.93 (1H, m), 1.92-2.08 (10.5H, series of s), 0.12-0.97 (6H,
series of d, J = 6.7 Hz).
L-MeVal-D-MeCys(Acm)-D-MeCys(Acm)-L-Pha-OAc (15d). White amorphous solid. FAB MS: m/z 683
(M+H)+. HR-FAB MS: m/z 683.3261, calcd for C31H51N6O7S2, Found 683.3248. A mixture (4:1) of two
conformers. 1H-NMR (500 MHz, CDCl3, d): 7.59, 6.94 (1H, both d, J = 8.5 Hz), 7.18-7.30 (5H, m), 7.03,
7.39 (1H, both t-like), 6.73, 6.82 (1H, both t-like), 5.88, 5.69 (1H, both dd, J = 9.8, 4.9 Hz), 5.33, 4.23 (1H,
both dd, J = 11.0, 4.9 Hz), 4.66, 4.74 (1H, both dd, J = 14.0, 7.3 Hz), 4.53, 4.62 (1H, both dd, J = 14.0, 7.0
Hz), 4.36 (1H, m), 3.97-4.13 (4H, series of dd), 3.29 (1H, d-like), 2.62-3.08 (6H, series of dd), 2.86-3.00
(6H, series of s), 2.27, 2.28 (3H, both s), 1.98, 1.99, 2.00, 2.04, 2.08 (9H, series of s), 1.93 (1H, m), 0.90,
0.92, 1.01, 1.06 (6H, series of d, J = 6.7 Hz ).
L-MeVal-ox-[D-MeCys-D-MeCys]-L-Pha-OAc (16d). Colorless powder. FAB MS: m/z 539 (M+H)+. HR-
FAB MS: m/z 539.2362, calcd for C25H39N4O5S2, Found 539.2358. 1H-NMR (500 MHz) and 13C-NMR (125
MHz) data in CDCl3 are given in Table 1 and Table 2.
L-MeVal-ox-[D-MeCys-D-MeCys]-L-Pha-OH (17d). Colorless powder. FAB MS: m/z 497 (M+H)+. HR-
FAB MS: m/z 497.2256, calcd for C23H37N4O4S2, Found 497.2260. 1H-NMR (500 MHz) data in CDCl3 are
given in Table 3.
ACKNOWLEDGEMENTS
The authors are grateful to the Takeda Science Foundation, the Tokyo Biochemical Research Foundation,
and the Ministry of Education, Culture, Sports, Science, and Technology of Japan for financial supports.
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