Synthesis of tokaramide A, a cysteine protease inhibitor from marine sponge Theonella aff. mirabilis

Article abstract of DOI:10.1016/j.tet.2011.09.116

The first synthesis of tokaramide A (1) is described. Tokaramide A (1) was synthesized via the peptide with a side-chain unprotected arginine residue on Weinreb AM resin by reductive cleavage.

Full text of DOI:10.1016/j.tet.2011.09.116

Tetrahedron 67 (2011) 9067e9071
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of tokaramide A, a cysteine protease inhibitor from marine sponge
Theonella aff. mirabilis
,
b
c
Hiroyuki Konno a , Kazuto Nosaka , Kenichi Akaji
*
a Department of Biochemical Engineering, Graduate School of Science and Technology, Yamagata University, Yonezawa, Yamagata 992-8510, Japan
b Department of Chemistry, Hyogo Collage of Medicine, Nishinomiya, Hyogo 663-8501, Japan
c Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 August 2011
Received in revised form 23 September 2011
Accepted 24 September 2011
Available online 1 October 2011
The first synthesis of tokaramide A (1) is described. Tokaramide A (1) was synthesized via the peptide
with a side-chain unprotected arginine residue on Weinreb AM resin by reductive cleavage.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Tokaramide A
Cysteine protease inhibitor
Marine sponge
Unusual amino acid
Cathepsin B
1. Introduction
our program for the design and synthesis of cysteine protease
inhibitors,6 we herein describe the first synthesis of tokaramide
Tokaramide A (1) was isolated by Fusetani et al. from the marine
sponge Theonella aff. mirabilis and was shown to highly inhibit
cathepsin B with an IC50 value of 29 ng/mL.1 Cathepsin B, the most
extensively studied enzyme of the group, is not only a lysosomal
cysteine protease that plays a role in a large number of physio-
logical processes, but is also known to be involved in various dis-
ease stages, such as inflammation, trauma, muscular dystrophy, and
tumors.2 In particular, its possible roles in cancer metastasis are of
major concern in cancer chemotherapy as cathepsin B inhibitors are
potential anticancer drugs.3 Moreover, Hook’s group reported that
A (1) (Fig. 1).
cathepsin B cleaved the b-secretase site of the wild-type amyloid
Fig. 1. Structure of tokaramide A (1) from marine sponge.
precursor protein (APP) in the brain to produce amyloid beta
peptide, whose aggregation causes Alzheimer’s disease.4 Tokar-
amide A (1) is composed of two L-valine (Val), L-argininal (Argal),
2. Results and discussion
and p-hydroxybenzoyl (HBA) residues and is believed to inhibit the
enzyme by forming a tetrahedral hemithioacetate between the
aldehyde of the argininal residue and the thiolate of the enzyme’s
active site. Recently, peptide aldehyde has been used as an enzyme
inhibitor, a probe of peptide structureefunction relationships, and
as a precursor for the preparation of other compounds.5 As part of
We first examined the construction of C-terminus aldehyde via
the reduction of Weinreb amide on a solid support. Although sev-
eral methods for solid-phase synthesis of peptide aldehyde have
been reported,7 the reactions of Weinreb-type amides on solid
support have more potential to enable the synthesis of aldehyde
and ketone libraries using commercially available Fmoc-amino
acids.8 The condensation/deprotection procedure was repeated
for the introduction of Fmoc-Arg(Mtr)eOH, Fmoc-ValeOH, and
p-MEMOPhCO2H (6) to give the tripeptide resin (2). Reduction of 2
* Corresponding author. Tel./fax: þ81 238 26 3131; e-mail address: konno@
yz.yamagata-u.ac.jp (H. Konno).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.09.116

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H. Konno et al. / Tetrahedron 67 (2011) 9067e9071
with LiAlH4 in THF and deprotection with TFA afforded tokaramide
A (1) as a cyclized hemi-aminal structure in a trace amount of yield
(Scheme 1).
a FriedeleCrafts-type reaction between the 2,4,6-trimethyl-4-
methoxyphenyl-sulfonyl (Mtr) protecting group and aldehyde
(Scheme 3). The initial attack of the electron-rich aromatic ring (10)
on the aldehyde could occur either before or after desulfonation to
form transient intermediate 11 followed by 12 with mass 134 Da
higher than tokaramide A (1). Subsequently dehydration, cycliza-
tion of the guanidine group, and elimination of the aromatic ring
results in the proposed modified peptide 9 with mass 16 Da smaller
than tokaramide A (1). It shall be assumed to give the precursor of
the compound 9 as a mixture in the ratio 4:3 via the elimination of
the
a-hydrogen in the C-terminal residue. TFA-mediated depro-
tection in the presence of several scavengers was also not effective.
For these reasons, TFA mediated the deprotection of 7 and fol-
lowing purification by RP-HPLC gave tripeptide (13) in 55% yield.
Subsequently, reduction of 13 with LiAlH4 in THF afforded tokar-
amide A (1) as a cyclic structure in moderate yield (Scheme 4).
As an alternative route, tripeptide (2) was prepared using Fmoc-
Arg(Mtr)eOH on Weinreb AM resin. Deprotection of 2 and sub-
sequent cleavage from resin with LiAlH4 gave tokaramide A (1) as
a cyclized hemi-aminal structure in 12% overall yield (Scheme 5).
Thus, we have completed the first synthesis of tokaramide A (1)
as a cyclic hemi-aminal structure on Weinreb AM resin. Peptide C-
terminus Weinreb amide with a side-chain unprotected arginine
residue could be easily converted to peptide aldehyde to avoid the
side reaction. The method will be used in solid-phase synthesis for
investigation of cysteine protease inhibitors in our laboratory.
Scheme 1.
In order to confirm the discouraging results of solid-phase
synthesis, it was attempted to synthesize the required peptide al-
dehyde by solution-phase peptide synthesis using Fmoc chemistry.
Fmoc-Arg(Mtr)eOH was coupled with N,N-methoxymethylamine
in the presence of BOP9/HOBt to give Weinreb amide (3) in 90%
yield. Sequential addition of the two valine residues gave protected
tripeptide (5). After removal of the Fmoc group with 20% piperidine
in MeCN, coupling of p-MEMOPhCO2H (6) using HATU10/HOAt11
afforded Weinreb amide-containing peptide (7) in 90% yield. Re-
duction of Weinreb amide (7) with LiAlH4 in THF gave the desired
hemi-aminal (8) in 60% yield. Finally, the hemi-aminal (8) was
treated with TFA; however, the 1H NMR and mass spectra of the
major product were not identical to those recorded for tokaramide
A (1) as a cyclic aminal structure and the determined structure 9
(Scheme 2).
3. Experimental
3.1. General
Solvents were reagent grade and dried prior to use. Fmoc-amino
acid derivatives and solid supports were obtained from Nova-
Scheme 2.
In addition to these anticipated issues, an expected problem
arose when the tripeptide (8) was cleaved, namely, the observation
of two abundant by-products with masses 134 Da greater and 16 Da
smaller than the desired product.12 The side reaction occurred in-
dependently of the cleavage cocktail and the abundance of the by-
products increased over time. This unique structure likely results in
biochem (Merck) or Watanabe Chemical, and were used without
further purification. Optical rotations were measured with a JASCO
DIP-7 spectrometer, and IR spectra were measured with a Horiba
FT-710 infrared spectrometer. 1H and 13C NMR spectra were mea-
sured with a JEOL JMN-400 or Bruker AM-300 spectrometer.
Chemical shifts (ppm) were relative to tetramethylsilane as an

H. Konno et al. / Tetrahedron 67 (2011) 9067e9071
9069
Scheme 3. Proposed mechanism for the formation of byproduct 9.
3.2. Synthesis
3.2.1. p-MEMOPhCO2H (6). To a solution of p-hydroxybenzalde-
hyde (50.0 g, 409 mmol) in CH2Cl2 (700 ml) were added N,N-dii-
sopropylethylamine (107 ml, 614 mmol) and MEMCl (51.4 ml,
450 mmol) at 0 ꢁC. The mixture was warmed to room temperature
and stirred for 10 h. Reaction mixture was partitioned between H2O
(500 ml) and CH2Cl2 (500 ml). The organic layer was washed with
brine (300 ml), dried over MgSO4, filtered, and concentrated in
vacuo. To a solution of the crude residue in acetone (500 ml) was
added Jones’ reagent at 0 ꢁC. After 30 min, the mixture was added to
Celite, filtered using a Celite pad, and concentrated in vacuo. The
residue was crystallized (hexane/AcOEt) to give carboxylic acid
(92.0 g, quant.) as a white solid. IR (film) nmax cmꢂ1: 2928, 2888,
1687,1610,1517,1419,1303,1267,1172,1149, 958, 846, 771, 667, 549.
1H NMR (400 MHz, CDCl3)
d
: 3.38 (3H, s), 3.56 (2H, t, J¼4.6 Hz), 3.84
Scheme 4. Solution phase synthesis of tokaramide A (1).
(1H, t, J¼4.6 Hz), 5.34 (2H, s), 7.11 (2H, d, J¼9.0 Hz), 8.06 (2H, d,
J¼8.8 Hz). 13C NMR (100 MHz, CDCl3)
d: 59.1, 68.0, 71.6, 93.1, 115.8,
122.8, 132.3, 161.4, 171.8. FABHRMS [MþH]þ calcd for C11H15O5:
227.0919, found: 227.0923.
3.2.2. Fmoc-Arg(Mtr)eN(OMe)Me (3). To
a solution of Fmoc-
Arg(Mtr)eOH (5.00 g, 8.21 mmol) in CH2Cl2 (50 ml) were added
NH(OMe)Me$HCl (1.20 g, 12.3 mmol), BOP (5.44 g, 12.3 mmol),
HOBt (1.66 g, 12.3 mmol), and N,N-diisopropylethylamine
(4.29 ml, 24.6 mmol) at room temperature. After stirring for 2 h,
the reaction mixture was poured into H2O and extracted with
AcOEt. Drying over MgSO4 and subsequent evaporation in vacuo
gave crude Weinreb amide 3, which was chromatographed over
silica gel (CH3Cl/MeOH¼9:1) to give 3 (4.81 g, 7.39 mmol, 90%) as
Scheme 5. Solid-phase synthesis of tokaramide A (1) using Weinreb AM resin.
internal standard. Mass spectra were recorded by Bruker autoflex II
(MALDI-TOFMS) and JEOL JMS-HX211A (FABMS) instruments. HPLC
was carried out on a Cosmosil 5C18 AR-II column (4.6ꢀ150 mm or
10ꢀ250 mm), which was eluted with MeCN in 0.1% aqueous TFA
and detected at OD 220 nm.
a colorless oil. ½a D22
ꢃ
ꢂ1.8 (c 4.0, CHCl3). IR (film) nmax cmꢂ1: 3421,
2942, 1716, 1637, 1120, 838, 755. 1H NMR (400 MHz, CDCl3)
d: 1.61

9070
H. Konno et al. / Tetrahedron 67 (2011) 9067e9071
(3H, m), 1.74 (2H, m), 2.10 (3H, s), 2.59 (1H, s), 2.61 (3H, s), 2.69
(3H, s), 3.18 (3H, s), 3.30 (1H, m), 3.71 (2H, s), 3.71e3.77 (1H, s),
3.80 (3H, s), 4.17 (1H, t, J¼6.8 Hz), 4.37 (2H, m), 4.70 (1H, br s),
5.77 (1H, d, J¼8.1 Hz), 6.03 (2H, s), 6.50 (1H, s), 7.28 (2H, t,
J¼6.1 Hz), 7.36 (2H, t, J¼7.6 Hz), 7.56 (2H, t, J¼6.1 Hz), 7.76 (2H, d,
3.2.5. (MEM)HBA-Val-Val-Arg(Mtr)eN(OMe)Me (7). To a solution of
Fmoc-Val-Val-Arg(Mtr)eN(OMe)Me (5) (660 mg, 1.05 mmol) in
MeCN (10 ml) was added piperidine (2 ml) at room temperature.
After stirring for 30 min, the reaction mixture was evaporated to
remove piperidine and the residue was poured into Et2O. Depro-
tected residue was decanted and the solvent was removed to give
crude HeVal-Val-Arg(Mtr)eN(OMe)Me without further purifica-
tion. To a solution of crude HeVal-Val-Arg(Mtr)eN(OMe)Me in
CH2Cl2 (10 ml) were added p-MEMOPhCO2H (6) (356 mg,
1.58 mmol), HATU (599 mg, 1.58 mmol), HOAt (214 mg, 1.58 mmol),
and N,N-diisopropylethylamine (0.150 ml, 1.58 mmol) at room
temperature. After stirring for 2 h, the reaction mixture was poured
into H2O and extracted with AcOEt. Drying over MgSO4 and sub-
sequent evaporation gave crude tripeptide 7, which was chroma-
tographed over silica gel (CH3Cl/MeOH¼9:1) to give 7 (790 mg,
J¼7.6 Hz). 13C NMR (100 MHz, CDCl3)
d: 11.9, 18.3, 24.1, 36.6, 36.7,
40.9, 47.1, 55.4, 67.1, 77.2, 111.7, 119.9, 124.7, 125.1, 127.1, 127.69,
127.73, 133.7, 136.5, 138.5, 141.2, 143.6, 143.8, 156.6, 158.3, 183.9,
220.2. MALDI-TOFMS [MþH]þ calcd for C33H42N5O7S: 652.273,
found: 652.342.
3.2.3. Fmoc-Val-Arg(Mtr)eN(OMe)Me (4). To a solution of Fmoc-
Arg(Mtr)eN(OMe)Me (3) (300 mg, 0.460 mmol) in MeCN (5 ml)
was added piperidine (1 ml) at room temperature. After stirring for
30 min, the reaction mixture was evaporated to remove piperidine
and the residue was poured into Et2O. The mixture was decanted
and the solvent was removed to give crude HeArg(Mtr)eN(OMe)
Me. To a solution of crude HeArg(Mtr)eN(OMe)Me in CH2Cl2 (5 ml)
were added Fmoc-ValeOH (312 mg, 0.624 mmol), EDCI (175 mg,
0.920 mmol), HOBt (124 mg, 0.920 mmol), and N,N-diisopropyle-
thylamine (0.160 ml, 0.920 mmol) at room temperature. After
stirring for 2 h, the reaction mixture was poured into H2O and
extracted with AcOEt. Drying over MgSO4 and subsequent evapo-
ration in vacuo gave crude dipeptide 4, which was chromato-
graphed over silica gel (CH3Cl/MeOH¼9:1) to give 4 (180 mg,
0.945 mmol, 90%) as a colorless oil. ½a D22
ꢂ16.2 (c 0.4, CHCl3). IR
ꢃ
(film) nmax cmꢂ1: 3311, 1635, 1558, 1234, 1120, 955, 755. 1H NMR
(400 MHz, CDCl3)
d
: 0.81 (3H, d, J¼6.8 Hz), 0.84 (3H, d, J¼6.6 Hz),
0.97 (3H, d, J¼6.8 Hz), 0.99 (3H, d, J¼6.6 Hz), 1.61 (4H, m), 1.80 (1H,
m), 1.94 (1H, m), 2.11 (3H, s), 2.26 (1H, m), 2.60 (3H, s), 2.67 (3H, s),
3.18 (3H, br s), 3.36 (5H, br s), 3.54 (2H, m), 3.71 (3H, s), 3.80 (2H,
m), 3.81 (3H, s), 4.39 (1H, t, J¼8.1 Hz), 4.44 (1H, t, J¼8.1 Hz), 5.02
(1H, br s), 5.29 (1H, s), 6.30 (1H, br s), 6.51 (1H, s), 7.02 (2H, d,
J¼8.8 Hz), 7.12 (1H, br s), 7.34 (1H, br s), 7.61 (1H, br s), 7.79 (2H, d,
J¼8.8 Hz). 13C NMR (100 MHz, CDCl3)
d: 12.0, 18.4, 18.6, 18.8, 19.2,
0.240 mmol, 53%) as a colorless oil. ½a D22
ꢃ
ꢂ10.4 (c 2.0, CHCl3). IR
19.4, 24.1, 24.9, 29.9, 30.67, 30.7, 55.5, 59.0, 60.0, 67.9, 71.6, 76.7,
77.3, 93.2, 111.7, 115.8, 124.7, 127.2, 129.3, 133.9, 136.5, 138.6, 156.5,
158.4, 160.0, 167.5, 172.4. MALDI-TOFMS [MþH]þ calcd for
C39H63N7O11S: 836.423, found, 836.429.
(film) nmax cmꢂ1: 3423, 3320, 1710, 1650, 1120, 844, 757. 1H NMR
(400 MHz, CDCl3)
d
: 0.91 (3H, d, J¼8.1 Hz), 0.93 (3H, d, J¼7.3 Hz),
1.53 (3H, m), 1.74 (2H, m), 2.08 (3H, s), 2.58 (3H, s), 2.65 (3H, s), 2.79
(1H, s), 3.17 (3H, s), 3.72 (1H, s), 3.77 (3H, s), 4.18 (1H, m), 4.37e4.40
(1H, m), 4.95e5.79 (1H, br s), 6.30 (2H, s), 6.48 (2H, s), 7.25 (2H, m),
7.35 (2H, q, J¼7.6 Hz), 7.54 (2H, t, J¼7.6 Hz), 7.72 (2H, dd, J¼4.4,
3.2.6. (MEM)HBA-Val-Val-Argal(Mtr) (8). To a solution of tripeptide
7 (14 mg, 0.017 mmol) in THF (1 ml) was added LiAlH4 (5 mg,
0.13 mmol) at 0 ꢁC. After stirring for 1 h, the mixture was added to
H2O and 1 M NaOH. The crude residue was filtered through a Celite
pad and evaporated in vacuo. The residue was purified by chro-
matography on silica gel to give 8 (8 mg, 0.010 mmol, 60%) as
3.2 Hz). 13C NMR (100 MHz, CDCl3)
d: 11.8, 17.7, 18.2, 19.1, 23.9, 25.1,
29.4, 31.3, 36.55, 36.59, 38.4, 40.5, 46.9, 47.0, 55.2, 59.9, 61.4, 67.0,
77.3, 111.5, 120.0, 124.4, 124.9, 126.9, 127.5, 133.8, 136.2, 138.3, 141.0,
141.1, 143.5, 143.7, 156.4, 156.5, 158.1, 171.8. MALDI-TOFMS [MþH]þ
calcd for C38H52N6O8S: 751.349, found 751.355.
a yellow oil. ½a 2D2
ꢃ
ꢂ15.0 (c 0.2, MeOH). IR (film) nmax cmꢂ1: 3318,
2962, 2927, 1708, 1631, 1504, 1234, 1120, 991, 752. 1H NMR
3.2.4. Fmoc-Val-Val-Arg(Mtr)eN(OMe)Me (5). To
a
solution of
(400 MHz, CDCl3)
d
: 0.87 (3H, d, J¼6.8 Hz), 0.89 (3H, d, J¼7.1 Hz),
Fmoc-Val-Arg(Mtr)eN(OMe)Me (4) (660 mg, 0.878 mmol) in
MeCN (5 ml) was added piperidine (1 ml) at room temperature.
After stirring for 30 min, the reaction mixture was evaporated to
remove piperidine and the residue was poured into Et2O. Depro-
tected residue was decanted and the solvent was removed to give
crude HeVal-Arg(Mtr)eN(OMe)Me without further purification. To
a solution of crude amine in CH2Cl2 (5 ml) were added Fmoc-
ValeOH (600 mg, 1.77 mmol), HATU (600 mg, 1.58 mmol), HOAt
(200 mg, 1.77 mmol), and N,N-diisopropylethylamine (0.300 ml,
1.72 mmol) at room temperature. After stirring for 2 h, the reaction
mixture was poured into H2O and extracted with AcOEt. Drying
over MgSO4 and subsequent evaporation in vacuo gave crude tri-
peptide 5, which was chromatographed over silica gel (CH3Cl/
MeOH¼9:1) to give 5 (490 mg, 576 mmol, 66%) as a colorless oil.
0.96 (3H, d, J¼8.3 Hz), 0.98 (3H, d, J¼7.1 Hz), 1.35 (4H, m), 1.71 (2H,
m), 2.10 (3H, s), 2.24 (1H, m), 2.57 (3H, s), 2.65 (3H, s), 3.13 (1H, m),
3.36 (3H, s), 3.53 (2H, m), 3.70 (1H, m), 3.82 (5H, m), 3.96 (1H, br s),
4.26 (1H, br s), 4.54 (1H, br s), 5.28 (2H, s), 5.52e5.62 (1H, br s), 6.51
(1H, s), 6.65 (1H, br s), 6.81 (1H, br s), 6.99e7.22 (1H, br s), 7.02 (2H,
d, J¼9.0 Hz), 7.80 (2H, d, J¼8.8 Hz). 13C NMR (100 MHz, CDCl3)
d:
12.0, 18.2, 18.4, 18.8, 19.3, 19.4, 24.1, 24.2, 29.8, 30.5, 31.0, 55.5, 59.1,
59.7, 67.96, 68.0, 71.6, 71.7, 77.3, 86.3, 93.2, 111.8, 116.0, 125.0, 129.1,
129.4, 136.8, 138.6, 158.7, 165.4, 199.9. FABHRMS [MþH]þ calcd for
C37H57N6O10S: 777.3857, found: 777.3866.
3.2.7. HBA-Val-Val-Argal [tokaramide A (1)] and compound 9. A
solution of 8 (8 mg, 0.010 mmol) in TFA was stirred for 2 h. The
reaction mixture was evaporated to remove TFA. The residue was
purified by preparative RP-HPLC (CH3CN/H2O) to give tokaramide A
(1) (0.2 mg, 0.0004 mmol, 4%) and 9 (4 mg, 0.009 mmol, 56%) as
½
a 2D2
ꢃ
ꢂ21.4 (c 1.5, CHCl3). IR (film) nmax cmꢂ1: 3286, 2962, 1716,
1623, 1450, 1241, 1029, 757, 740. 1H NMR (400 MHz, CDCl3)
d: 0.87
(6H, d, J¼6.8 Hz), 0.93 (6H, br s), 1.52 (2H, br s), 1.76 (1H, br s), 1.98
(1H, br s), 2.03 (1H, m), 2.11 (3H, s), 2.59 (3H, s), 2.67 (3H, s), 3.16
(4H, br s), 3.68 (3H, s), 3.80 (3H, s), 4.05 (1H, m), 4.16 (1H, m) 4.27
(1H, m), 4.40 (2H, m), 5.01 (1H, br s), 6.07 (1H, br s), 6.25 (2H, br s),
6.50 (1H, s), 6.92 (1H, br s), 7.25 (2H, m), 7.35 (2H, m), 7.55 (2H, t,
J¼8.1 Hz), 7.57 (1H, br s), 7.72 (2H, d, J¼7.6 Hz). 13C NMR (100 MHz,
a colorless oil. Tokaramide A (1): 1H NMR (300 MHz, CD3OD)
d: 0.97
(3H, d, J¼6.3 Hz), 0.98 (3H, d, J¼6.6 Hz), 1.02 (3H, d, J¼6.9 Hz), 1.03
(3H, d, J¼6.6 Hz), 1.62e1.77 (4H, m), 2.05 (1H, m), 2.16 (1H, m), 3.20
(2H, m), 3.89 (1H, m), 4.16 (1H, m), 4.34 (1H, t, J¼8.4 Hz), 4.70 (1H,
m), 6.84 (2H, d, J¼8.7 Hz), 7.73 (2H, d, J¼8.7 Hz), 8.00 (1H, m), 8.01
(1H, m). 13C NMR (75 MHz, CDCl3)
d: 20.1, 29.1, 31.1, 39.6, 55.3, 59.4,
CDCl3)
d
: 11.9, 18.3, 18.4, 18.7, 19.1, 19.2, 24.1, 24.9, 29.8, 30.9, 31.2,
95.9, 114.8, 124.9, 129.3, 159.4, 162.3, 169.3, 176.1. MALDI-TOFMS
[MþH]þ calcd for C23H36N6O5: 477.283, found: 477.388. Com-
pound 9: IR (film) nmax cmꢂ1: 3398, 2966, 2924, 1662, 1203, 1138,
32.1, 47.1, 55.4, 58.7, 60.7, 61.7, 67.1, 77.3, 111.7, 119.9, 124.7, 125.2,
127.0, 127.6, 133.7, 136.4, 138.4, 141.2, 143.8, 143.9, 156.4, 156.7,
158.3, 172.4. MALDI-TOFMS [MþH]þ calcd for C43H60N7O9S:
850.417, found, 850.275.
847, 723. 1H NMR (300 MHz, CD3OD)
d: 1.04 (12H, m), 2.05 (2H, m),
2.17 (1H, m), 2,32 (1H, m), 2.34 (2H, t, J¼5.7 Hz), 3.59 (2H, m), 4.21

H. Konno et al. / Tetrahedron 67 (2011) 9067e9071
9071
(1H, t, J¼7.8 Hz), 4.37 (1H, t, J¼8.4 Hz), 6.85 (2H, d, J¼9.0 Hz), 7.40
(1H, s), 7.75 (2H, d, J¼9.0 Hz), 8.03 (1H, d, J¼8.1 Hz), 8.21 (1H, d,
J¼8.1 Hz). MALDI-TOFMS [MþH]þ calcd for C23H34N5O5: 459.272,
found: 459.258.
removed, the resin was rinsed with DMF and Et2O. LiAlH4 (20 mg,
0.53 mmol) in THF (2 ml) was added to the resultant resin. After
stirring for 30 min, the reaction mixture was poured into H2O, fil-
tered, and subsequent evaporation gave crude peptide. The residue
was purified by preparative RP-HPLC (CH3CN/H2O) to give 1 (11 mg,
0.024 mmol, 12%) as a colorless oil.
3.2.8. HBA-Val-Val-ArgeN(OMe)Me (13). A solution of 7 (17 mg,
0.022 mmol) in TFA (1 ml) was stirred for 2 h. The reaction mixture
was evaporated to remove TFA. The residue was purified by pre-
parative RP-HPLC (CH3CN/H2O) to give 13 (10 mg, 0.018 mmol, 82%)
Acknowledgements
as a white powder. ½a D22
ꢃ
ꢂ12.7 (c 0.6, CHCl3). IR (film) nmax cmꢂ1
:
3316, 2921, 1630, 1458, 1298, 1201, 985, 849, 750. 1H NMR
The authors thank Prof. Shigeki Matsunaga of the Graduate
School of Agriculture and Life Science, the University of Tokyo for
spectral data of the natural product. This work was supported in
part by a Grant-in-aid from the Japan Society for the Promotion of
Science (21689004) and Chisso Corporation Fund.
(400 MHz, CDCl3) d: 1.00 (12H, m), 1.67 (4H, m), 2.08 (1H, m), 2.17
(1H, m), 2.70 (2H, m), 3.33 (3H, s), 3.82 (3H, s), 4.21 (1H, m), 4.35
(1H, m), 4.85 (1H, m), 6.85 (2H, d, J¼8.4 Hz), 7.50 (1H, br s), 7.74 (2H,
d, J¼8.4 Hz), 7.99 (1H, br s), 8.09 (1H, br s), 8.31 (1H, br s). 13C NMR
(75 MHz, CDCl3) d: 19.0, 19.4, 19.7, 19.9, 26.1, 29.8, 31.8, 35.4, 37.0,
41.9, 60.4, 61.3, 116.17, 126.1, 130.5, 158.7, 162.3, 163.7, 170.2, 173.6,
173.7, 174.2, 174.3. MALDI-TOFMS [MþH]þ calcd for C25H42N7O6:
536.319, found: 536.287.
References and notes
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