Total synthesis of miraziridine A and identification of its major reaction site for cathepsin B

Article abstract of DOI:10.1016/j.tet.2007.06.082

The synthesis of miraziridine A, a pentapeptide derivative isolated from marine sponge, and its truncated analogs has been achieved. To construct the backbone of miraziridine A, a side-chain-unprotected vinylogous arginine was condensed with an aziridine-

Full text of DOI:10.1016/j.tet.2007.06.082

Tetrahedron 63 (2007) 9502–9513
Total synthesis of miraziridine A and identification
of its major reaction site for cathepsin B
b
b
c
Hiroyuki Konno,a, Kanako Kubo, Hidefumi Makabe, Emi Toshiro,
*
Naoyuki Hinoda,c Kazuto Nosakaa and Kenichi Akajia
aDepartment of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine,
Kita-ku, Kyoto 603-8334, Japan
bSciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Minamiminowa,
Kamiina, Nagano 399-4598, Japan
cDepartment of Biological Science and Technology, Faculty of Engineeing, University of Tokushima,
Minamijosanjima, Tokushima 770-8506, Japan
Received 4 June 2007; revised 25 June 2007; accepted 25 June 2007
Available online 4 July 2007
Abstract—The synthesis of miraziridine A, a pentapeptide derivative isolated from marine sponge, and its truncated analogs has been
achieved. To construct the backbone of miraziridine A, a side-chain-unprotected vinylogous arginine was condensed with an aziridine-con-
taining fragment prepared by a conventional solid-phase procedure. An analog lacking the vinylogous arginine site showed comparable in-
hibitory activity with miraziridine A, whereas an analog lacking the aziridine site showed remarkably weak inhibitory activity for cathepsin B.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
In the course of our recent research regarding cysteine pro-
tease inhibitors, we have conducted studies on the synthesis
of miraziridine A (1), which contains a peptidyl unsaturated
carboxylic acid and an aziridine structure,3,4 supposed to be
efficient reactive groups for the thiol functional group of cys-
teine protease. In this paper, we report procedures for syn-
thesizing miraziridine A (1) and its analogs in detail.
Toward the total synthesis of miraziridine A (1), we adopted
a route introducing a side-chain-unprotected vArg-OEt at
the late stage of the backbone construction, which makes it
possible to adopt a convenient solid-phase procedure for
the fragment preparation. Three truncated analogs (2–4)
(Fig. 1) were also synthesized to estimate the major reactive
site of miraziridine A (1) for cathepsin B.
Miraziridine A (1)1 is a pentapeptide isolated from the
marine sponge Theonella aff. mirabilis by Fusetani et al.
in 2000. This natural peptide is composed of (2R,3R)-
aziridine-2,3-dicarboxylic acid (Azd), L-leucine (Leu),
(3S,4S)-statine (Sta), (S)-a-aminobutyric acid (Abu), and
(S)-vinylogous arginine (vArg). Miraziridine A (1) is re-
ported to inhibit the action of cathepsin B with an IC50 value
of 1.4 mg/ml (2.1 mM). The first total synthesis of miraziri-
dine A (1) was described by Schaschke in 2004.2 All conden-
sation reactions for the backbone construction were
conducted by the classical solution procedure, which is little
applicable to the construction of libraries. N-terminal Azd
was introduced at the final stage of the backbone construc-
tion, since the aziridine moiety is known to be sensitive to
nucleophilic ring opening. Thus, a rather laborious process
of combining protecting groups, Bpoc/Boc combination,
was selected since it is compatible with the double bond
present at the C-terminal vArg(Boc)2 moiety. In addition,
two deprotection procedures were necessary at the final
stage to remove Boc from vArg(Boc)2 using acid and ethyl
esters by enzymatic hydrolysis.
2. Results and discussion
The route for the synthesis of miraziridine A (1) is shown in
Scheme 1. The backbone is constructed by the condensation
of a N-terminal tetrapeptide derivative (5) and C-terminal
H-vArg-OEt (6). An N-terminal tetrapeptide containing
EtO-Azd moiety is prepared by conventional Fmoc-based
solid-phase peptide synthesis (SPPS).
Keywords: Miraziridine A; Cysteine protease inhibitor; Cathepsin B; Azir-
idine; Vinylogous arginine.
* Corresponding author. Tel.: +81 (0)75 465 7659; fax: +81 (0)75 465 7660;
e-mail: konno@koto.kpu-m.ac.jp
The necessary EtO-Azd-OH (7) was prepared starting with
(2S,3S)-tartaric acid diethyl ester.5,6 To conduct the final de-
protection by single-step enzymatic hydrolysis, H-vArg-OEt
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.06.082

H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
9503
O
O
OH
O
O
H
N
H
N
HO
N
H
N
H
OH
HO-Azd-Leu-Sta-Abu-vArg-OH
N
H
O
O
Miraziridine A (1)
NH
HN
NH2
O
O
OH
OH
O
O
H
N
OH
HO
N
H
N
H
HO-Azd-Leu-Sta-Abu-OH (2)
N
H
O
O
O
O
O
H
N
H
N
AcHN
N
H
OH
Ac-Leu-Sta-Abu-vArg-OH (3)
NH
HN
NH2
OH
O
H
N
OH
H-Leu-Sta-Abu-OH (4)
H2N
N
H
O
O
Figure 1. Miraziridine A (1) and its analogs (2–4).
(6) containing no protecting groups at the side-chain is
employed.
(9b). Weinreb amidation of 9a and 9b gave 10a and 10b in
90 and 92% yield, respectively, without difficulty. Treatment
of each product with LiAlH4 afforded a cyclic hemiaminal
11a or 11b via an aldehyde in moderate yield. However,
the succeeding Horner–Emmons reaction of 11a and 11b
did not give the desired protected (S)-vinylogous arginines
12a and 12b, probably due to the unusual stability of the
cyclic hemiaminals 11a and 11b (Scheme 2).7
We first tried to synthesize (S)-vinylogous arginine start-
ing with Fmoc-Arg(4-methyl-2,3,6-trimethyl-benzenesul-
fonyl)-OH (Fmoc-Arg(Mtr)-OH, 9a) or Boc-Arg(Mtr)-OH
HO-Azd-Leu-Sta-Abu-vArg-OH
1
To suppress the hemiaminal formation, we examined the
effects of various protecting groups for the guanidine
functional group (Table 1), i.e., Mtr, Boc, Cbz, and Alloc
groups. Boc-Arg(Mtr,Boc)-OH (15a) was prepared by the
N-u-Boc protection of Boc-Arg(Mtr)-OH (9b) using
(Boc)2O/NaH. Weinreb amidation of 15a gave Boc-
Arg(Mtr,Boc)-N(OMe)Me (16a) with 48% yield. Boc-Arg
(diCbz)-N(OMe)Me (16b) was similarly prepared from
commercially available Boc-Arg(diCbz)-OH (15b) with
80% yield. Boc-Arg(diAlloc)-N(OMe)Me (16c) was ob-
tained with 50% yield from Boc-Arg(diAlloc)-OH (15c),
which was synthesized by Goodmann’s guanidination8,9 of
Boc-Orn-OH (13) in the presence of Et3N (Scheme 3). Other
N-u,N-u0-di-protected arginine Weinreb amides, 16d and
16e, were similarly prepared using the corresponding Good-
mann’s reagent, 14d and 14e.
O
EtO-Azd-Leu-Sta-Abu-OH
+
H2N
OEt
5
Fmoc-based
SPPS
NH
HN
NH2
O
O
H-vArg-OEt (6)
EtO
OH
Fmoc
OH
N
H
N
H
O
EtO-Azd-OH (7)
Fmoc-Leu-OH
OH
O
H
N
Fmoc
OH
Fmoc
OH
N
H
Each protected arginine Weinreb amide was then converted
to the corresponding vinylogous arginine by treatment with
LiAlH4 followed by triethenyl phosphonoacetate/NaH
(Table 1). Boc-Arg(Mtr,Boc)-N(OMe)Me (16a) gave the
O
Fmoc-Abu-OH
Fmoc-Sta-OH (8)
Scheme 1. Synthetic plan for miraziridine A (1).

9504
H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
O
O
P1HN
P1HN
OMe
OH
NH
N
NH(OMe)Me
BOP, Et3N
Me
LiAlH4
THF
CH2Cl2
NH
HN
NHMtr
HN
NHMtr
9a: P1 = Fmoc
9b: P1 = Boc
10a: 90%
10b: 92%
O
O
P1HN
P1HN
OEt
H
OH NH
N
P1HN
NHMtr
NH
NHMtr
NH
HN
HN
NHMtr
11a: 67%
11b: 76%
12a or 12b
Scheme 2.
desired compound Boc-vArg(Mtr,Boc)-OEt (17a) with only
14% yield in two steps (entry 1). Though the Cbz-protected
guanidine derivative (16b) was converted to Boc-vArg
(diCbz)-OEt (17b) in good yield (entry 2), these Cbz groups
were found to be little deprotected. N-u-Alloc-protected ar-
ginine derivatives 16c and 16d gave the desired product 17c
and 17d in low yield due to the instability of the guanidine
Alloc group (entry 3 and 4). Boc-Arg(diBoc)-N(OMe)Me
(16e) gave the corresponding aldehyde derivative as
a main product, which was converted to Boc-vArg(diBoc)-
OEt (17e) with 86% yield (entry 5). TFA-mediated deprotec-
tion of Boc groups in 17e gave the desired H-vArg-OEt (6)
quantitatively.
Fmoc protection afforded the desired Fmoc-statine (8) in
75% yield (Scheme 4).
The synthesis of miraziridine A (1) was conducted according
to the route shown in Scheme 5. The N-terminal tetrapeptide
derivative, EtO-Azd-Leu-Sta-Abu-OH (5), was prepared by
Fmoc-based SPPS. As a solid support, 2-chlorotrityl chlo-
ride resin was selected, because (3R,4R)-aziridine dicarb-
oxylic acid is unstable in the presence of strong acid.
Fmoc-Abu-OH was reacted with the 2-chlorotrityl chloride
i
resin in DMF/CH2Cl2 in the presence of Pr2NEt. The
Fmoc group of resulting resin was removed with 20% piper-
idine/DMF, and Fmoc-Sta-OH (8) was condensed by DIPC/
HOBt in the presence of Pr2NEt. The same deprotection/
i
(3S,4S)-Fmoc-statine (8)10 was prepared by a stereo-inver-
sion of the b-hydroxy group of (3R,4S)-Boc-statine-OEt
(19), which was easily prepared by diastereoselective reduc-
tion of the corresponding b-keto ester.11,12 Diastereoselec-
tive reduction of the b-keto ester to yield the desired
3S-hydroxy group is known to be hard to achieve.12 We
found that a stereo-inversion of the 3R-alcohol of 19 can
be achieved by using a mesyl leaving group to give the de-
sired oxazolidinone (20) in 68% yield. Hydrolysis and
condensation procedure was repeated for the introduction
of Fmoc-Leu-OH to afford tripeptide resin (21). EtO-Azd-
OH (7) was then condensed by diphenylphosphoryl azide
(DPPA)13/Et3N to give the tetrapeptide resin (22). The resin
was treated with HFIP (hexafluoroisopropanol)14/CH2Cl2
(1:4) to cleave the tetrapeptide derivative from the resin.
The product showed a single major peak on HPLC and
was purified by preparative HPLC to afford a homogeneous
tetrapeptide, EtO-Azd-Leu-Sta-Abu-OH (5), in 32% overall
Table 1. Synthesis of vinylogous arginine
O
O
BocHN
OMe
BocHN
N
OH NP2
NHP1
OEt
Me
1) LiAlH4, THF
BocHN
N
2) triethyl phosphonoacetate
NaH, THF
NH
NH
NHP1
P2N
NHP1
P2N
18
17
16
Entry
Substrate
P1
P2
Vinyl arginine (%)
Product Yield (%)
Hemiaminal
Yield (%)
Product
1
2
3
4
5
16a
16b
16c
16d
16e
Mtr
Boc
Cbz
Alloc
Alloc
Boc
17a
17b
17c
17d
17e
14
76
10
21
86
18a
18b
18c
18d
18e
ND
ND
75
0
Cbz
Alloc
Boc
Boc
0

H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
9505
O
O
N
O
N
BocHN
BocHN
OMe
14
BocHN
OH
NH
N
NH(OMe)Me
BOP, Et3N
OH
P2N
NHP1
Me
Et3N
CH2Cl2
CH2Cl2
NH
NH2
P2N
NHP1
P2N
NHP1
13
15
16
14c: P1, P2 = Alloc
14d: P1 = Boc, P2 = Alloc
14e: P1 = Boc, P2 = Boc
15c: P1, P2 = Alloc; 33%
15d: P1 = Boc, P2 = Alloc; 62%
15e: P1 = Boc, P2 = Boc; 26%
16c: P1, P2 = Alloc; 50%
16d: P1 = Boc, P2 = Alloc; 72%
16e: P1 = Boc, P2 = Boc; 65%
Scheme 3. Synthesis of protected arginine Weinreb amides 16c, 16d, and 16e.
O
OH
O
OH O
1) LiOH
EtOH/H2O
MsCl
Et3N
O
O
BocHN
FmocHN
HN
OEt
OH
OEt
2) Fmoc-OSu
Et3N, MeCN
H2O
68%
8
19
20
75%
Scheme 4. Synthesis of (3S,4S)-Fmoc-statine (8).
1) Fmoc-Abu-OH, iPr2NEt, DMF, CH2Cl2
2) piperidine, DMF
3) Fmoc-Sta-OH (8), DIPC, HOBt, iPr2NEt, DMF
resin
Cl
H-Leu-Sta-Abu-O-
21
4) piperidine, DMF
5) Fmoc-Leu-OH, DIPC, HOBt, iPr2NEt, DMF
6) piperidine, DMF
Cl
2-Chloro trityl
chloride resin
Cl
HFIP
CH2Cl2
EtO-Azd-OH (7)
DPPA, Et3N
EtO-Azd-Leu-Sta-Abu-OH
EtO-Azd-Leu-Sta-Abu-O-
DMF
overall 32%
5
22
H-vArg-OEt (6)
Porcine Liver
HATU, HOAt
Esterase
miraziridine A
1
EtO-Azd-Leu-Sta-Abu-vArg-OEt
23
iPr2NEt, DMF
Tris-HCl buffer (pH6.5)
92%
32%
Scheme 5. Total synthesis of miraziridine A (1).
yield. Coupling of the tetrapeptide (5) with H-vArg-OEt (6)
was achieved by a 5 min reaction using O-(7-azabenzotri-
azole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU)15/1-hydroxy-7-azabenzotriazole (HOAt)16 (Fig. 2a).
Subsequent HPLC-based purification afforded the desired
pentapeptide derivative, miraziridine A diethyl ester (23),
in 32% yield. For final deprotection, enzyme-assisted hydro-
lysis of the ester (23) in the presence of porcine liver esterase
inTris/HClbuffer(pH6.5)wasconductedaccordingtoapub-
lished procedure.2 By monitoring with HPLC, it was found
that the N-terminal ester function could be removed within
15 h, whereas 132 h was necessary for the saponification of
C-terminal ethyl ester (Fig. 2b). The crude product was puri-
fied by preparative HPLC to afford miraziridine A (1) in 92%
yield. The spectroscopic data (1H NMR, 13C NMR, IR,
FABMS, and TOFMS) on synthetic 1 were identical to those
of the natural product reported by Fusetani et al., within the
normal error limits.
Using the synthetic intermediates described above, truncated
analogs of miraziridine A, i.e., compounds 2–4, were pre-
pared. H-Leu-Sta-Abu-OH (4) and Ac-Leu-Sta-Abu-vArg-
OH (3) were prepared from intermediate tripeptide resin
(21). Analog 4 was prepared by the cleavage of 21 with
HFIP as above in 45% overall yield. Analog 3 was prepared
according to the route shown in Scheme 6. After acetylation
of 21 by Ac2O/pyridine/DMAP, Ac-Leu-Sta-Abu-OH (24)
was cleaved from the resin by HFIP with 33% overall yield.
Coupling of the tripeptide (24) and H-vArg-OEt (6) with
HATU/HOAt proceeded within 10 min. Subsequent purifi-
cation by HPLC afforded a tetrapeptide derivative (25) in
42% yield. Enzymatic hydrolysis of the ester (25) in the
presence of porcine liver esterase in Tris/HCl buffer (pH
6.5) gave Ac-Leu-Sta-Abu-vArg-OH (3) in 71% yield. The
truncated analog 2, HO-Azd-Leu-Sta-Abu-OH, was pre-
pared by enzymatic hydrolysis of EtO-Azd-Leu-Sta-Abu-
OH (5) using porcine liver esterase in 79% yield.

9506
H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
(a)
(b)
Mono ester
0 min
After 15 h
1
5
23
After 5 min
After 132 h
1
23
0
10
20
30
40
(min)
0
10
20
30
40
(min)
Figure 2. HPLC profiles for the total synthesis of miraziridine A (1): (a) coupling of 5 and 6; (b) saponification of 23 to give miraziridine A (1) via mono ester.
The inhibitory activity toward cathepsin B was determined
with an assay using a Z-Arg-Arg-MCA substrate developed
by Hiwasa et al.17,18 The inhibitory activities of miraziridine
A (1) and its truncated analogs (2–4), were evaluated using
suggested that the inhibitory activity is attributable mainly
to the aziridine site of miraziridine A (1). The results are
consistent with the structural data on CA-074 in a complex
with cathepsin B, which suggests that the N-terminal part
of miraziridine A (1) can easily adopt a conformation that
is similar to the binding mode of CA-074.20 Though the
vinylogous arginine site had a rather weak effect compared
with the aziridine site, the inhibitory activity of Ac-Leu-
Sta-Abu-vArg-OH (3) was about 10 times that of H-Leu-
Sta-Abu-OH (4). Further investigations are now underway
to clarify the inhibitory mechanism.
1) Ac2O
py.,DMAP
H-vArg-OEt (6)
HATU, HOAt
H-Leu-Sta-Abu-O-
21
Ac-Leu-Sta-Abu-OH
2) HFIP
CH2Cl2
33%
iPr2NEt, DMF
24
42%
Porcine Liver Esterase
In conclusion, we achieved the total synthesis of miraziridine
A (1) via the coupling of a side-chain-unprotected vArg-OEt
(6) and Azd-containing tetrapeptide (5). The major reaction
site of miraziridine A (1) for cathepsin B was estimated
to be the N-terminal aziridine site. The structure–activity
relationship of cathepsin B inhibitors will be reported in
due course.
Ac-Leu-Sta-Abu-vArg-OEt
25
Ac-Leu-Sta-Abu-vArg-OH
3
Tris-HCl buffer (pH6.5)
71%
Scheme 6. Synthesis of Ac-Leu-Sta-Abu-vArg-OH (3).
the corresponding IC50 and Ki19 values (Table 2). The IC50
value of the synthetic miraziridine A (2 mM) was very sim-
ilar to that reported for the natural product (2.1 mM). Com-
paring IC50 and Ki values of HO-Azd-Leu-Sta-Abu-OH
(2) and Ac-Leu-Sta-Abu-vArg-OH (3), it was strongly
3. Experimental
3.1. General
Table 2. Inhibitory activity of cathepsin B
Amino acids and coupling reagents were purchased from
Novabiochem or Watanabe Chemical Industries. All manip-
ulations were conducted under an inert atmosphere (N2). All
solvents were of reagent grade. THF was distilled from so-
dium and benzophenone ketyl. CH2Cl2 was distilled from
CaH2. All commercial reagents were of the highest purity
Inhibitor
IC50 (mM)
Ki (mM)
HO-Azd-Leu-Sta-Abu-vArg-OH (1)
HO-Azd-Leu-Sta-Abu-OH (2)
H-Leu-Sta-Abu-vArg-OH (3)
H-Leu-Sta-Abu-OH (4)
2
9
100
950
3
6.5
83
1000

H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
9507
available. Analytical TLC was performed on silica gel (60 F-
254, plates 0.25 mm). Column chromatography was carried
out on Wakogel 60 (particle size, 0.063–0.200 mm).
Analytical and preparative HPLC was performed on a HITA-
CHI ELITE LaChrom system (OD, 220 nm) equipped with
the Nacalai tesque COSMOSIL 5C18-AR-II (4.6ꢀ150 mm
added LiAlH4 (15 mg, 392 mmol) at 0 ꢂC and the mixture
was stirred for 30 min at room temperature. To the mixture
were added H2O (25 ml) and 1 M NaOH (50 ml), and
filtered on a Celite pad. The organic layer was dried over
MgSO4 and concentrated in vacuo. The product was purified
with silica gel column chromatography (CHCl3/MeOH¼
98:2) to give hemiaminal 11a (32 mg, 54 mmol, 67%) as
a colorless oil. [a]2D7 ꢁ1.8 (c 0.4, CHCl3). IR (film) n max
cmꢁ1: 3423, 3338, 3016, 2939, 2854, 1716, 1635, 1252,
1
or 10ꢀ250 mm). H (300 MHz) and 13C (75 MHz) NMR
spectograms were recorded on a Bruker AM-300. Chemical
shifts are expressed in parts per million relative to TMS
1
1
(0 ppm) or CHCl3 (7.28 ppm for H and 77.0 ppm for 13C)
1120, 756. H NMR (CDCl3) d: 1.60–1.85 (4H, m), 2.11
or MeOH (3.30 ppm for 1H and 49.0 ppm for 13C). IR spec-
tograms were obtained on a HORIBA FREEXACT-II FT-
710 spectrometer. Optical rotations were recorded on a
HORIBA SEPA-200 or SEPA-300 polarimeter at the sodium
D line. Low-resolution mass spectra (LRMS) and high-
resolution mass spectra (HRMS) were obtained on either
a JOEL JMS-HX-211A or a JMS-HX-110A (EI or FAB)
and Bruker Autoflex-II (MALDI-TOF). Fluorescent inten-
sity was obtained on a Shimadzu RF-1500 under a xenon
lamp (Ex 380 nm, Em 460 nm).
(3H, s), 2.54 (2.1H, s), 2.58 (0.4H, s), 2.60 (0.5H, s),
2.63 (2.1H, s), 2.67 (0.9H, s), 3.13–3.31 (1.4H, m), 3.48–
3.58 (0.6H, m), 3.81 (3H, s), 3.63–3.76 (2H, m), 4.17 (1H,
m), 4.33 (0.3H, m), 4.42 (1H, m), 4.76 (0.7H, m), 5.17
(0.3H, d, J¼9.3 Hz), 5.55 (0.7H, d, J¼9.9 Hz), 5.66
(0.3H, br s), 5.71 (0.7H, br s), 6.47 (1H, s), 6.55 (2H, br
s), 7.28 (2H, m), 7.35 (2H, m), 7.52–7.76 (4H, m). 13C
NMR (CDCl3) d: 11.9, 18.3, 23.5, 24.0, 24.4, 29.6,
50.7, 55.4, 70.0, 75.6, 80.8, 93.6, 111.8, 120.0, 120.1,
124.4, 125.0, 127.1, 127.7, 128.0, 129.6, 132.9, 136.8,
138.6, 139.7, 141.3, 143.9, 145.9, 156.1, 158.7. MALDI-
TOFMS (M+H)+ calcd for C31H37N4O6S1: 593.243, found:
593.083.
3.2. Synthetic chemistry
3.2.1. N-a-Fmoc-N0-u0-Mtr-arginine-N,O-dimethyl-
hydroxyamine (10a). To a solution of Fmoc-Arg(Mtr)-OH
(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-diisopro-
pylethyl amine (4.29 ml, 24.6 mmol) at room temperature.
After being stirred for 2 h, the reaction mixture was poured
into H2O and extracted with ethyl acetate. Drying over
MgSO4 and subsequent evaporating gave crude Weinreb
amide 10a, which was chromatographed over silica gel
(CH3Cl/MeOH¼9:1) to give 10a (4.81 g, 7.39 mmol,
90%) as a colorless oil. [a]2D2 ꢁ1.8 (c 4.0, CHCl3). IR
(film) n max cmꢁ1: 3421, 2942, 1716, 1637, 1120, 838,
755. 1H NMR (CDCl3) d: 1.61 (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 (2.5H, s), 3.77 (0.5H, 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, J¼7.7 Hz). 13C NMR (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, 141.2, 143.6, 143.8, 156.6, 158.3,
183.9, 220.2. MALDI-TOFMS (M+H)+ calcd for
C33H42N5O7S1: 652.273, found: 652.342.
N-a-Boc-N-u-Mtr-hemiaminal (11b): [a]2D8 +13.4 (c 0.4,
CHCl3). IR (film) n max cmꢁ1: 3423, 3338, 3006, 2976,
2939, 1697, 1635, 1518, 1122, 987, 756. 1H NMR (CDCl3)
d: 1.43 (9H, s), 1.51–1.77 (4H, m), 2.14 (3H, m), 2.58 (3H,
s), 2.67 (3H, s), 3.13 (1H, m), 3.45–3.72 (2H, m), 3.83 (3H,
s), 4.67 (1H, br s), 4.98 (1H, d, J¼9.3 Hz), 5.58 (1H, t,
J¼3.6 Hz), 6.54 (1H, s), 6.63 (2H, br s). 13C NMR (CDCl3)
d: 11.9, 18.2, 23.6, 24.0, 24.5, 28.3, 36.7, 39.2, 50.2,
55.4, 58.3, 75.9, 79.6, 111.8, 124.9, 133.1, 136.7, 138.5,
155.2, 156.0, 158.6. MALDI-TOFMS (M+H)+ calcd for
C21H34N4O6S1Na1: 493.210, found: 493.192.
3.2.3. 1H-Pyrazole-N-Alloc-1-carboxamidine. To a solu-
tion of 1H-pyrazole-1-carboxamidine hydrochloride
(7.80 g, 53.2 mmol) in CH2Cl2 (100 ml) and DMF
(170 ml) were added DIEA (11.0 ml, 63.8 mmol) and
AllocCl (6.80 ml, 63.8 mmol) and the mixture was stirred for
4 h at room temperature. To the mixture were added satd
aq NaCl and ether, and the organic layer was evaporated
in vacuo to give 1H-pyrazole-N-Alloc-1-carboxamidine
(10.3 g, 53.1 mmol, 99%) as a colorless oil. IR (film)
n max cmꢁ1: 3448, 3307, 3153, 3136, 2956, 2921, 1774,
1755, 1668, 1635, 1533, 1508, 1429, 1419, 1392, 1371,
1
1338, 1284, 1265, 1213, 1188, 1178, 1036, 937, 764. H
NMR (CDCl3) d: 4.96 (2H, tt, J¼6.0, 1.2 Hz), 5.39 (1H,
dq, J¼10.5, 6.0 Hz), 5.50 (1H, dq, J¼17.1, 1.2 Hz), 6.10
(1H, ddt, J¼17.1, 10.5, 6.0 Hz), 6.45 (1H, dd, J¼2.7,
1.5 Hz), 7.77 (1H, d, J¼0.9 Hz), 8.19 (1H, dd, J¼2.7,
0.6 Hz). 13C NMR (CDCl3) d: 65.5, 68.8, 109.2, 120.4,
130.7, 130.9, 144.5, 162.3. HRFABMS (M+H)+ calcd for
C31H15N6O4: 195.0882, found: 195.0886.
N-a-Boc-N0-u0-Mtr-arginine-N,O-dimethylhydroxyamine
(10b): [a]2D8 ꢁ3.9 (c 0.3, CHCl3). IR (film) n max cmꢁ1
:
3431, 3321, 2929, 1709, 1655, 1558, 1464, 1300, 985,
748. H NMR (CDCl3) d: 1.42 (9H, s), 1.49–1.69 (4H, m),
1
2.12 (3H, s), 2.64 (3H, s), 2.70 (3H, s), 3.16 (3H, s), 3.19
(2H, m), 3.72 (3H, s), 3.82 (3H, s), 4.59 (1H, br s), 5.27
(1H, m), 6.52 (1H, s), 6.62 (2H, br s), 6.72 (1H, br s). 13C
NMR (CDCl3) d: 11.5, 17.9, 23.7, 25.2, 27.9, 36.4, 40.1,
55.0, 61.1, 79.1, 111.2, 124.0, 134.1, 135.9, 138.1, 155.2,
156.5, 157.7, 172.4. MALDI-TOFMS (M+H)+ calcd for
C23H40N5O7S1: 529.257, found: 529.296.
3.2.4. 1H-Pyrazole-N,N0-di-Alloc-1-carboxamidine (14c).
To a solution of 1H-pyrazole-N-Alloc-1-carboxamidine
(10.3 g, 53.2 mmol) in THF (160 ml) were added NaH
(4.50 g, 186 mmol) and AllocCl (19.8 ml, 186 mmol) at
0 ꢂC. The mixture was stirred for 6 h at room temperature.
To the mixture were added H2O and ether. The organic layer
was dried over MgSO4 and evaporated in vacuo. The product
3.2.2. N-a-Fmoc-N-u-Mtr-hemiaminal (11a). To a solu-
tion of amide (10a) (52 mg, 80 mmol) in THF (3 ml) was

9508
H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
was purified with silica gel column chromatography (hex-
ane/AcOEt¼2:1) to give 1H-pyrazole-N,N0-diAlloc-1-
carboxamidine (14c) (8.90 g, 32.0 mmol, 60%) as a colorless
oil. IR (film) n max cmꢁ1: 3153, 3136, 3089, 2951, 1819,
1784, 1747, 1685, 1649, 1435, 1398, 1356, 1261, 1205,
11.8, 14.9, 18.0, 18.1, 18.2, 23.77, 23.83, 24.4, 24.7, 27.4,
27.8, 28.1, 29.4, 29.6, 30.7, 40.6, 44.9, 52.8, 55.3, 60.9,
65.7, 80.0, 83.9, 84.7, 111.6, 120.0, 124.8, 132.2, 132.5,
136.6, 136.8, 138.5, 150.3, 152.4, 154.4, 154.9, 155.5,
158.6, 175.4. HRFABMS (M+H)+ calcd for C26H43N9O9S:
587.2751, found: 587.2721.
1
1180, 1115, 935, 766. H NMR (CDCl3) d: 4.69–4.75 (4H,
m), 5.19–5.44 (4H, m), 5.75–6.03 (2H, m), 6.51 (1H, dd,
J¼1.8, 1.2 Hz), 7.54 (1H, dd, J¼1.5, 0.6 Hz), 8.30 (1H, d,
J¼3.0 Hz). 13C NMR (CDCl3) d: 67.9, 68.3, 110.9, 119.1,
119.6, 129.6, 130.5, 131.2, 144.7, 148.9, 157.2. HRFABMS
(M+H)+ calcd for C12H15N4O4: 279.1093, found: 279.1089.
3.2.7. N-a-Boc-N-u,N0-u0-di-Alloc-arginine (15c). To
a solution of Boc-Orn-OH (2.70 g, 11.6 mmol) in CH2Cl2
(58.0 ml) were added Et3N (1.60 ml, 11.6 mmol) and 1H-
pyrazole-N,N0-di-Alloc-1-carboxamidine (14c) (3.20 g,
11.6 mmol) and the mixture was stirred for 18 h at room
temperature. To the mixture were added 1 M HCl and ether.
The organic layer was dried over MgSO4 and evaporated
in vacuo. The product was purified with silica gel column
chromatography (CHCl3) to give N-a-Boc-N-u,N0-u0-di-
Alloc-arginine (15c) (1.70 g, 3.80 mmol, 33%) as a colorless
oil. [a]2D0 +15.0 (c 1.1, CHCl3). IR (film) n max cmꢁ1: 3336,
3140, 2978, 2935, 1732, 1716, 1637, 1626, 1417, 1369,
1323, 1259, 1211, 1144, 1051, 769. 1H NMR (CDCl3)
d: 1.44 (9H, s), 1.71 (3H, s), 1.94 (1H, s), 3.47 (2H, d,
J¼3.3 Hz), 4.35 (1H, s), 4.62 (4H, d, J¼5.7 Hz), 5.18–
5.39 (4H, m), 5.83–6.03 (2H, m), 7.62 (1H, s), 8.36 (1H,
s), 11.20 (2H, br s). 13C NMR (CDCl3) d: 25.0, 28.3, 29.7,
40.5, 53.0, 66.2, 66.9, 80.0, 105.5, 117.8, 119.4, 130.9,
132.9, 132.9, 153.7, 156.0, 163.5, 175.8. HRFABMS
(M+H)+ calcd for C19H31N4O8: 443.2142, found: 443.2148.
3.2.5. 1H-Pyrazole-N-Boc-N0-Alloc-1-carboxamidine
(14d). To a solution of 1H-pyrazole-1-carboxamidine
(666 mg, 4.54 mmol) in CH2Cl2 (8.50 ml) and DMF
(14.2 ml) were added DIEA (949 ml, 5.45 mmol) and
Boc2O (1.19 g, 5.45 mmol) at 0 ꢂC. The mixture was stirred
for 5 h at room temperature. To a mixture were added satd aq
NaCl and ether. The organic layer was dried over MgSO4
and evaporated in vacuo. The product was purified with
column chromatography (hexane/AcOEt¼4:1) to give 1H-
pyrazole-N-Boc-1-carboxamidine (895 mg, 4.26 mmol) as
a colorless oil. IR (film) n max cmꢁ1: 3436, 3319, 3145,
3126, 2966, 1658, 1610, 1363, 1313, 1173, 980, 760. To a
solution of 1H-pyrazole-N-Boc-1-carboxamidine (895 mg,
4.26 mmol) in THF (21.0 ml) were added NaH (358 mg,
14.9 mmol) and AllocCl (1.58 ml, 14.9 mmol) at 0 ꢂC. The
mixture was stirred for 11 h at room temperature. To the
mixture were added satd aq NH4Cl and AcOEt. The organic
layer was dried over MgSO4 and evaporated in vacuo. The
product was purified with silica gel column chromatography
(hexane/AcOEt¼4:1) to give 1H-pyrazole-N-Boc-N0-Alloc-
1-carboxamidine (14d) (1.10 g, 3.73 mmol, 82%) as a color-
less oil. IR (film) n max cmꢁ1: 3149, 3140, 2981, 2945, 1817,
1780, 1751, 1685, 1431, 1392, 1371, 1338, 1265, 1180,
1149, 1117, 1036, 937, 766. 1H NMR (CDCl3) d: 1.40
(4H, s), 1.53 (4H, s), 1.60 (1H, s), 4.68–4.75 (1.75H, m),
4.95–4.98 (0.25H, m), 5.20–5.50 (2H, m), 5.75–6.13 (1H,
m), 6.45–6.51 (1H, m), 7.73 (0.75H, dd, J¼8.7, 0.9 Hz),
7.76 (0.25H, dd, J¼9.3, 0.9 Hz), 8.19 (0.25H, d,
J¼3.0 Hz), 8.28 (0.75H, d, J¼2.4 Hz). 13C NMR (CDCl3)
d: 27.5, 27.8, 27.9, 68.8, 109.2, 118.9, 120.3, 129.3, 130.7,
130.9, 144.5, 149.1, 157.4. HRFABMS (M+H)+ calcd for
C13H19N4O4: 295.1406, found: 295.1413.
3.2.8. N-a,N-u-di-Boc-N0-u0-Alloc-arginine (15d). To
a solution of Boc-Orn-OH (1.92 g, 8.29 mmol) in CH2Cl2
(41.4 ml) were added Et3N (1.16 mmol) and 1H-pyrazole-N-
Boc-N0-Alloc-1-carboxamidine (14d) (2.44 g, 8.29 mmol)
and the mixture was stirred for 15 h at room temperature.
To the mixture was added 1 M HCl and ether. The organic
layer was dried over MgSO4 and evaporated in vacuo. The
product was purified with silica gel column chromatography
(CHCl3) to give diBoc-Alloc-arginine (15d) (2.36 g,
5.16 mmol, 62%) as a colorless oil. [a]2D0 +17.0 (c 1.2,
CHCl3). IR (film) n max cmꢁ1: 3329, 3145, 2979, 2935,
1718, 1639, 1621, 1417, 1369, 1269, 1255, 1157, 1142,
1
1053, 754. H NMR (CDCl3) d: 1.44 (9H, s), 1.48 (9H, s),
1.71 (3H, s), 1.93 (1H, s), 3.45 (2H, d, J¼4.8 Hz), 4.34
(1H, s), 4.59 (2H, dd, J¼5.7, 1.2 Hz), 5.19–5.39 (2H, m),
5.90–6.03 (1H, m), 8.48 (1H, s). 13C NMR (CDCl3) d:
25.1, 28.0, 28.1, 28.3, 29.7, 40.4, 53.0, 66.1, 66.9, 117.6,
131.0, 132.9, 153.0, 153.7, 155.7, 156.6, 163.4, 175.8.
HRFABMS (M+H)+ calcd for C20H35N4O8: 459.2455,
found: 459.2462.
3.2.6. N-a,N0-u0-di-Boc-N-u-Mtr-arginine (15a). To a so-
lution of Boc-Arg(Mtr)-OH (256 mg, 437 mmol) in CH2Cl2
(5 ml) were added NaH (88 mg, 2.19 mmol) and (Boc)2O
(191 mg, 874 mmol) and the mixture was stirred for 18 h at
room temperature. To the mixture were added 1 M HCl
and ether. The organic layer was dried over MgSO4 and
evaporated in vacuo. The product was purified with silica
gel column chromatography (CHCl3) to give N-a-N0-u0-di-
Boc-N-u-Mtr-arginine (15a) (154 mg, 262 mmol, 66%) as
a colorless oil. [a]2D7 +13.1 (c 1.4, CHCl3). IR (film) n max
cmꢁ1: 3394, 3329, 3286, 2927, 2937, 1716, 1620, 1564,
3.2.9. N-a,N-u,N0-u0-tri-Boc-L-arginine (15e). Procedure
A: to a solution of Boc-Arg-OH (12.0 g, 36.7 mmol) in
2 N NaOH (210 ml) and 1,4-dioxane (180 ml) at 0 ꢂC was
added Boc2O (16.5 g, 75.6 mmol) and the mixture was
stirred for 3 h. To the mixture were added Boc2O (8.20 g,
37.6 mmol) and 1,4-dioxane (90 ml). After 39 h, 3 M HCl
(pH¼7) and AcOEt were added. The organic layer was dried
over MgSO4 and evaporated in vacuo. The product was
purified with silica gel column chromatography (CHCl3/
MeOH¼99:1) to give N-a,N-u,N0-u0-tri-Boc-L-arginine
(15e) (4.99 g, 10.5 mmol, 28%) as a colorless oil.
1
1523, 1369, 1275, 1151, 1122, 912, 808, 733. H NMR
(CDCl3) d: 1.42 (9H, s), 1.48 (9H, s), 1.55–1.95 (4H, m),
2.12 (3H, s), 2.60 (1H, s), 2.61 (2H, s), 2.68 (3H, s), 3.29
(1.32H m), 3.74 (0.68H, m), 3.81 (3H, s), 4.16 (0.68H, m),
4.26 (1.32H, m), 5.24 (0.68H, m), 6.40 (0.32H, m), 6.54
(1H, s), 7.56 (1H, br s), 7.83 (0.32H, br s), 8.34 (0.68H, br
s), 9.13 (0.32H, s), 9.78 (0.68H, s). 13C NMR (CDCl3) d:
Procedure B: to a solution of Boc-Orn-OH (2.00 g,
8.81 mmol) in CH2Cl2 (20.0 ml) were added Et3N

H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
9509
(1.20 mmol, 8.80 mmol) and 1H-pyrazole-N,N0-di-Boc-1-
carboxamidine (14e)8 (2.73 g, 8.81 mmol) and the mixture
was stirred for 20 h at room temperature. To the mixture
were added 1 M HCl and ether. The organic layer was dried
over MgSO4 and evaporated in vacuo. The product was
purified with silica gel column chromatography (CHCl3/
MeOH¼100:1) to give N-a,N-u,N0-u0-tri-Boc-L-arginine
(15e) (1.08 g, 2.29 mmol, 26%) as a colorless oil. [a]D27
+18.0 (c 1.06, CHCl3). IR (film) n max cmꢁ1: 3327, 3286,
3153, 2979, 2933, 1720, 1643, 1616, 1415, 1367, 1333,
N-a-Boc-N-u,N0-u0-di-Alloc-arginine-N,O-dimethylhydroxy-
amine (16c): [a]2D0 ꢁ2.7 (c 0.69, CHCl3). IR (film) n max
cmꢁ1: 3336, 2981, 2943, 1728, 1712, 1639, 1367, 1323,
1255, 1211, 1169, 1140, 1049, 995. 1H NMR (CDCl3)
d: 1.42 (9H, s), 1.67 (4H, s), 3.20 (3H, s), 3.45 (2H, q,
J¼6.3 Hz), 3.76 (3H, s), 4.61 (4H, dd, J¼16.5, 5.7 Hz),
4.68 (1H, d, J¼1.2 Hz), 5.18–5.38 (4H, m), 5.83–6.05
(2H, m), 8.30 (1H, s). 13C NMR (CDCl3) d: 25.0, 28.3,
30.1, 32.0, 40.5, 49.9, 61.6, 66.2, 66.9, 79.6, 117.7, 119.3,
131.0, 133.0, 153.7, 156.0, 163.5, 172.6. HRFABMS
(M+H)+ calcd for C21H36N5O8: 486.2564, found: 486.2566.
1
1159, 1138, 1053, 1026, 750. H NMR (CDCl3) d: 1.45
(9H, s), 1.48 (9H, s), 1.49 (9H, s), 1.69 (3H, s), 1.90 (1H,
s), 3.48 (2H, s), 4.32 (1H, s), 5.33 (1H, d, J¼7.2 Hz). 13C
NMR (CDCl3) d: 25.3, 28.1, 28.3, 28.4, 29.9, 40.5, 53.2,
79.7, 80.0, 83.4, 153.3, 156.0, 156.4, 163.1, 175.3.
HRFABMS (M+H)+ calcd for C21H39N4O8: 475.2768,
found: 475.2775.
N-a,N-u-di-Boc-N0-u0-Alloc-(4R)-arginine-N,O-dimethyl-
hydroxyamine (16d): [a]2D0 ꢁ0.41 (c 0.67, CHCl3). IR (film)
n max cmꢁ1: 3336, 2978, 2937, 1720, 1639, 1369, 1269,
1
1255, 1159, 1138, 1051. H NMR (CDCl3) d: 1.45 (9H, s),
1.51 (9H, s), 1.53–1.79 (4H, m), 3.21 (3H, s), 3.45 (2H, q,
J¼6.2 Hz), 3.77 (3H, s), 4.59 (2H, dd, J¼5.7, 1.5 Hz),
4.63–4.69 (1H, m), 5.21 (1H, dd, J¼10.5, 1.4 Hz), 5.32
(1H, dd, J¼17.1, 1.4 Hz), 5.92–6.05 (1H, m), 8.42 (1H, s),
11.36 (1H, s). 13C NMR (CDCl3) d: 25.0, 28.0, 28.2, 28.3,
30.1, 32.0, 40.4, 50.0, 66.1, 66.2, 66.8, 79.6, 83.3, 117.6,
119.3, 133.1, 153.1, 155.5, 156.5, 162.3, 163.5, 172.6.
HRFABMS (M+H)+ calcd for C22H40N5O8: 502.2877,
found: 502.2871.
3.2.10. N-a,N0-u0-di-Boc-N-u-Mtr-arginine-N,O-dime-
thylhydroxyamine (16a). To a solution of N-a-Boc-N-
u,N0-u0-diCbz-arginine (15a) (154 mg, 262 mmol) in
CH2Cl2 (5 ml) were added Et3N (68 ml, 524 mmol), BOP
(151 mg, 341 mmol), and NH(OMe)Me-HCl (33 mg,
341 mmol) and then the mixture was stirred for 3 h at
room temperature. To the mixture were added 1 M HCl
(pH¼5) and ether. The organic layer was washed with satd
aq NaHCO3, dried over MgSO4, and evaporated in vacuo.
The product was purified with silica gel column chromato-
graphy (hexane/AcOEt¼2:1) to give amide (16a) (109 mg,
173 mmol, 66%) as a colorless oil. [a]2D7 +5.8 (c 0.85,
CHCl3). IR (film) n max cmꢁ1: 3405, 3317, 3294, 1712,
1660, 1618, 1460, 1369, 1279, 1151, 1122, 916, 732, 675.
1H NMR (CDCl3) d: 1.42 (3.6H, s), 1.43 (5.4H, s), 1.49
(5.4H, s), 1.50 (3.6H, s), 1.52–1.80 (4H, m), 2.14 (3H, s),
2.62 (1.2H, s), 2.63 (1.8H, s), 2.70 (3H, s), 3.16 (1.2H, s),
3.18 (1.8H, s), 3.29 (1H, q, J¼6.6 Hz), 3.72 (1.2H, s), 3.74
(1.8H, s), 3.84 (3H, s), 4.62 (1H, m), 5.18 (1H, t,
J¼9.0 Hz), 6.55 (1H, s), 7.85 (0.4H, s), 8.31 (0.6H, s),
9.10 (0.4H, s), 9.80 (0.6H, s). 13C NMR (CDCl3) d: 11.9,
18.3, 23.9, 24.5, 24.8, 27.88, 27.93, 28.3, 30.1, 30.2, 32.0,
40.1, 45.2, 49.9, 50.2, 55.4, 61.5, 79.5, 79.6, 83.9, 84.5,
111.6, 124.8, 132.9, 133.1, 136.6, 136.9, 138.7, 150.3,
152.5, 154.6, 154.9, 155.4, 158.6, 158.6, 172.5. HRFABMS
(M+H)+ calcd for C28H48N5O9S1: 630.3173, found:
690.3204.
N-a,N-u,N0-u0-tri-Boc-L-arginine-(4R)-arginine-O,N-dime-
thylhydroxyamine (16e): [a]2D0 +0.64 (c 1.5, CHCl3). IR
(film) n max cmꢁ1: 3329, 2978, 2935, 1718, 1641, 1618,
1
1367, 1331, 1169, 1134, 1051, 756. H NMR (CDCl3) d:
1.37 (9H, s), 1.43 (18H, s), 1.51–1.71 (4H, m), 3.14 (3H,
s), 3.37 (2H, d, J¼5.7 Hz), 3.70 (3H, s), 4.62 (1H, s), 5.19
(1H, d, J¼9.0 Hz), 8.26 (1H, s), 11.43 (1H, s). 13C NMR
(CDCl3) d: 25.0, 27.9, 28.2, 28.2, 29.9, 31.9, 40.2, 50.0,
61.4, 79.0, 79.4, 82.9, 153.1, 155.4, 156.6, 162.3, 163.4,
172.6. HRFABMS (M+H)+ calcd for C23H44N5O8:
518.3193, found: 518.3193.
3.2.11. N-a,N0-u0-di-Boc-N-u-Mtr-vinylogous arginine
ethyl ester (17a). To a solution of amide (16a) (109 mg,
173 mmol) in THF (5 ml) was added LiAlH4 (25 mg,
658 mmol) at 0 ꢂC and the mixture was stirred for 30 min
at room temperature. To the mixture were added H2O
(25 ml) and 1 M NaOH (50 ml), and filtered on a Celite
pad. The organic layer was dried over MgSO4 and concen-
trated in vacuo. The crude product was used for the next re-
action without further purification. To a solution of triethyl
phosphonoacetate (250 mg, 661 mmol) in THF (5 ml) was
added NaH (25 mg, 735 mmol) at 0 ꢂC. The mixture was
stirred for 1 h and the crude product was added dropwise
into it. After 20 min, satd aq NH4Cl and AcOEt were added,
and the organic layer was washed with brine, dried over
MgSO4, and evaporated in vacuo. The product was purified
with silica gel column chromatography (hexane/
AcOEt¼3:1) to give ethyl ester (17a) (16 mg, 24 mmol,
14%) as a colorless oil. [a]2D0 +5.3 (c 0.65, CHCl3). IR
(film) n max cmꢁ1: 3329, 3286, 2979, 2870, 1720, 1618,
1369, 1279, 1244, 1153, 1122, 916, 810, 733, 677. 1H
NMR (CDCl3) d: 1.29 (3H, s, J¼7.2 Hz), 1.44 (9H, s),
1.50 (9H, s), 1.55–1.78 (4H, m), 2.15 (3H, s), 2.63 (3H, s),
2.70 (3H, s), 3.30 (2H, q, J¼6.0 Hz), 3.84 (3H, s), 4.18
(2H, q, J¼7.2 Hz), 4.21 (1H, m), 4.45 (1H, m), 5.88 (1H,
dd, J¼15.6, 1.5 Hz), 6.55 (1H, s), 6.77 (1H, dd, J¼15.6,
The following compounds (16b–16e) were prepared in
a manner similar to 16a.
N-a-Boc-N-u,N0-u0-di-Cbz-arginine-N,O-dimethylhydroxy-
amine (16b): [a]2D4 +0.95 (c 0.55, CHCl3). IR (film) n max
cmꢁ1: 3389, 3292, 3059, 3032, 2972, 2934, 2370, 2322,
1716, 1662, 1610, 1510, 1456, 1379, 1255, 1199, 1170,
1
1097, 1055, 1005. H NMR (CDCl3) d: 1.42 (9H, s), 1.52
(2H, s), 1.68 (2H, s), 3.13 (3H, s), 3.67 (3H, s), 3.98 (2H,
t, J¼6.6 Hz), 4.64 (1H, s), 5.14 (2H, s), 5.23 (2H, s), 7.29–
7.41 (10H, m), 9.29 (1H, br s), 9.46 (1H, s). 13C NMR
(CDCl3) d: 24.8, 28.3, 30.0, 32.0, 44.4, 50.2, 61.4, 66.9,
68.8, 79.4, 127.7, 127.8, 128.1, 128.2, 128.6, 134.6, 136.8,
155.3, 155.6, 160.3, 163.7, 172.5. HRFABMS (M+H)+ calcd
for C29H40N5O8: 586.2879, found: 586.2877. Anal. Calcd
C29H39N5O8: C, 59.47; H, 6.71; N, 11.96. Found: C,
59.62; H, 6.64; N, 11.99.

9510
H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
5.4 Hz), 8.32 (1H, t, J¼5.1 Hz), 9.81 (1H, s). 13C NMR
(CDCl3) d: 11.9, 14.2, 18.3, 23.9, 25.4, 28.0, 28.3, 32.0,
55.5, 60.5, 84.1, 111.7, 121.2, 124.9, 133.0, 136.8, 138.7,
147.6, 150.4, 152.7, 155.0, 158.8, 166.1. HRFABMS
(M+H)+ calcd for C30H49N4O9S: 641.3220, found: 641.3218.
HRFABMS (M+H)+ calcd for C24H41N4O8: 513.2924,
found: 513.2933.
N-a,N-u,N0-u0-tri-Boc-(4R)-vinylogous arginine ethyl ester
(17e): [a]2D0 +2.4 (c 1.1, CHCl3). IR (film) n max cmꢁ1: 3384,
2979, 2933, 1716, 1610, 1512, 1367, 1275, 1254, 1165,
1149, 982, 756. 1H NMR (CDCl3) d: 1.27 (3H, t,
J¼7.1 Hz), 1.44 (9H, s), 1.51 (18H, s), 1.62 (4H, br s),
3.80 (1H, s), 3.91 (1H, s), 4.16 (2H, q, J¼3.4 Hz), 4.43
(1H, s), 5.70 (1H, d, J¼8.4 Hz), 6.01 (1H, dd, J¼15.9,
1.4 Hz), 6.86 (1H, dd, J¼15.6, 5.0 Hz), 9.20 (1H, s), 9.36
(1H, s). 13C NMR (CDCl3) d: 14.2, 24.7, 28.0, 28.2, 28.4,
29.9, 44.4, 51.5, 60.3, 78.9, 79.3, 83.8, 118.0, 120.7,
148.7, 150.0, 154.9, 155.4, 160.7, 162.3, 163.6, 166.4.
HRFABMS (M+H)+ calcd for C25H45N4O8: 529.3237,
found: 529.3228.
The following compounds (17b–17e) were prepared in
a manner similar to 17a.
N-a-Boc-N-u,N0-u0-di-Cbz-vinylogous arginine ethyl ester
(17b): mp 91–92 ꢂC. [a]D20 +14.5 (c 0.19, CHCl3). IR
(KBr) n max cmꢁ1: 3404, 3346, 3277, 3088, 3067, 3030,
2982, 2937, 2874, 2361, 2343, 1724, 1693, 1655, 1612,
1520, 1450, 1381, 1302, 1251, 1174, 1101, 1010. 1H NMR
(CDCl3) d: 1.28 (3H, t, J¼7.2 Hz), 1.41 (9H, s), 1.43–1.62
(2H, m), 1.66 (2H, d, J¼7.2 Hz), 3.93–4.12 (2H, m), 4.18
(2H, q, J¼7.1 Hz), 4.28 (1H, br s), 4.92 (1H, d, J¼7.8 Hz),
5.15 (2H, s), 5.24 (2H, s), 5.88 (1H, dt, J¼15.6, 1.5 Hz),
6.77 (1H, dt, J¼15.6, 4.9 Hz), 7.27–7.43 (10H, m), 9.26
(1H, br s), 9.45 (1H, br s). 13C NMR (CDCl3): 14.3, 25.2,
28.4, 30.8, 44.3, 51.4, 60.4, 67.0, 68.9, 79.5, 120.7, 127.7,
127.8, 128.3, 128.7, 128.8, 134.5, 136.8, 148.1, 155.1,
155.7, 160.4, 163.7, 166.1. HRFABMS (M+H)+ calcd for
C31H41N4O8: 597.2926, found: 597.2924. Anal. Calcd
C31H40N4O8: C, 62.40; H, 6.76; N, 9.39. Found: C, 62.35;
H, 6.71; N, 9.32.
3.2.12. (4R)-Vinylogous arginine ethyl ester (6). To a solu-
tion of N-a,N-u,N0-u0-tri-Boc-(4R)-vinylogous arginine
ethyl ester (17e) in CH2Cl2 (500 ml) was added TFA
(500 ml). The mixture was stirred for 2 h at 0 ꢂC. After evap-
oration of the TFA, the resulting precipitate was concen-
trated in vacuo to give (4R)-vinylogous arginine ethyl ester
TFA salt (6) (4.5 mg, 10.4 mmol, 100%) as a colorless oil.
[a]D20 +5.2 (c 1.3, CHCl3), IR (film) n max cmꢁ1: 3363,
1
3188, 2922, 1670, 1203, 1138, 839, 800, 723. H NMR
(CD3OD) d: 1.30 (3H, t, J¼6.9 Hz), 1.55–1.93 (4H, m),
3.24 (2H, t, J¼6.6 Hz), 3.33 (1H, s), 3.95–4.02 (1H, m),
4.23 (2H, q, J¼7.1 Hz), 6.19 (1H, d, J¼15.6 Hz), 6.83
(1H, dd, J¼15.8, 8.0 Hz), 7.88 (2H, s). 13C NMR
(CD3OD) d: 14.4, 25.8, 30.8, 41.7, 52.9, 62.1, 127.1,
142.5, 158.8, 166.6. HRFABMS (M+H)+ calcd for
C10H21N4O2: 229.1665, found: 229.1662.
N-a-Boc-N-u,N0-u0-di-Alloc-(4R)-vinylogous arginine eth-
yl ester (17c) and hemiaminal (18c): Compound 17c: [a]D20
+14.5 (c 0.19, CHCl3). IR (film) n max cmꢁ1: 3336, 2976,
2929, 2850, 1718, 1639, 1367, 1259, 1213, 1167, 1051. 1H
NMR (CDCl3) d: 1.27 (3H, m), 1.46 (9H, s), 1.68 (4H, d,
J¼5.1 Hz), 3.51 (2H, s), 4.21 (3H, q, J¼6.9 Hz), 4.34 (1H,
s), 4.53–4.69 (4H, m), 5.22–5.42 (4H, m), 5.87–6.07 (3H,
m), 6.83 (1H, dd, J¼15.8, 5.6 Hz). 13C NMR (CDCl3) d:
14.2, 25.6, 28.4, 29.7, 31.6, 40.5, 41.3, 41.9, 55.5, 60.5,
66.2, 67.0, 81.2, 117.8, 119.5, 121.2, 131.3, 133.0, 147.8,
155.3, 162.8, 166.5, 190.5. HRFABMS (M+H)+ calcd for
C23H37N4O8: 497.2611, found: 497.2606. Compound 18c:
[a]2D0 +4.4 (c 0.95, CHCl3). IR (film) n max cmꢁ1: 3342,
2976, 2943, 2873, 1772, 1716, 1618, 1522, 1508, 1456,
3.2.13. (4S,5S)-Ethyl (sec-butyl-2-oxo-ozazolidin-5-yl)-
acetate (20). To a solution of 19 (376 mg, 1.24 mmol) in
CH2Cl2 were added MsCl (96 ml, 1.24 mmol) and Et3N
(259 ml, 1.86 mmol) at ꢁ20 ꢂC and the mixture was stirred
for 3 h at room temperature. To the mixture were added H2O
and ether, and the organic layer was washed with satd aq
NaHCO3, dried over MgSO4, and evaporated in vacuo.
The product was purified with silica gel column chromato-
graphy (hexane/AcOEt¼2:1) to give oxazolidinone (20)
(184 mg, 792 mmol, 68%) as a colorless oil. [a]2D0 ꢁ42.0
(c 0.68, CHCl3), IR (film) n max cmꢁ1: 3217, 3104, 2958,
1
1363, 1327, 1246, 1167, 1120, 756. H NMR (CDCl3) d:
1.46 (9H, s), 1.67–1.86 (4H, m), 2.16–2.27 (1H, m), 2.79–
2.89 (1H, m), 3.61–3.68 (1H, m), 4.49 (1H, d, J¼13.8 Hz),
4.61 (2H, d, J¼8.4 Hz), 4.83 (1H, s), 5.26 (1H, dd,
J¼10.5, 0.9 Hz), 5.36 (1H, dd, J¼17.1, 0.9 Hz), 5.92–6.03
(1H, m), 11.33 (1H, s). 13C NMR (CDCl3) d: 14.2, 22.1,
28.2, 28.3, 28.5, 43.9, 52.3, 60.4, 66.8, 118.6, 132.3,
145.6, 154.8, 155.0, 162.9. HRFABMS (M+H)+ calcd for
C15H27N4O5: 343.1981, found: 343.1982.
1
2871, 1749, 1468, 1444, 1367, 1273, 1003, 968, 793. H
NMR (CDCl3) d: 0.93–0.96 (6H, m), 1.25–1.30 (3H, m),
1.41–1.67 (3H, m), 2.41 (1H, dd, J¼17.7, 4.6 Hz), 2.81
(1H, dd, J¼17.7, 6.9 Hz), 3.62 (1H, dt, J¼9.2, 4.6 Hz),
4.19 (2H, t, J¼7.1 Hz), 4.57 (1H, q, J¼4.6 Hz), 5.30 (1H,
s). 13C NMR (CD3OD) d: 21.7, 23.1, 24.9, 36.1, 43.3,
55.0, 58.3, 155.1, 174.2. HRFABMS (M+H)+ calcd for
C11H19N1O5: 299.1300, found: 299.1299.
N-a,N-u-di-Boc-N0-u0-Alloc-(4R)-vinylogous arginine ethyl
ester (17d): [a]2D0 +7.80 (c 0.92, CHCl3). IR (film) n max
cmꢁ1: 3329, 3153, 2979, 1720, 1639, 1576, 1518, 1456,
1
1419, 1369, 1306, 1271, 1255, 1161, 1140, 1053, 756. H
3.2.14. Fmoc-Sta-OH (8). To a solution of 20 (182 mg,
794 mmol) in MeOH was added aq LiOH. The mixture
was stirred for 1.5 h at room temperature. Et3N (152 ml,
1.09 mmol) and Fmoc-OSu (350 mg, 1.04 mmol) were
added and then the mixture was stirred for 15 min at room
temperature (pH¼8.5–9.0). The mixture was evaporated in
vacuo. The crude product was added to 1.5 N HCl and the
resulting precipitate was filtrated to give Fmoc-Sta-OH (8)
(316 mg, 794 mmol, 75%) as a white powder. All spectra
NMR (CDCl3) d: 1.29 (3H, t, J¼7.2 Hz), 1.44 (9H, s),
1.49 (9H, s), 1.63–1.67 (4H, br s), 3.45 (2H, m), 4.19
(2H, q, J¼7.1 Hz), 4.32 (1H, s), 4.60 (2H, dt, J¼5.7,
1.4 Hz), 5.19–5.36 (2H, m), 5.81–6.06 (2H, m), 6.82
(1H, dd, J¼15.6, 5.4 Hz), 8.43 (1H, s), 11.36 (1H, s). 13C
NMR (CDCl3) d: 14.2, 25.6, 28.0, 28.2, 28.4, 29.5, 31.7,
40.5, 51.5, 55.5, 60.5, 66.1, 83.5, 117.7, 119.5, 121.1,
133.1, 147.9, 153.2, 155.1, 156.6, 157.5, 163.5, 166.2.

H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
9511
for the synthetic Fmoc-Sta-OH (8) were well consistent with
those of the authentic standard (purchased from Novabio-
7.50 (1H, d, J¼9.6 Hz), 8.42 (1H, d, J¼8.4 Hz). 13C NMR
(CD3OD) d: 10.9, 14.4, 14.6, 22.0, 22.5, 23.3, 23.7, 26.1,
26.2, 26.3, 31.8, 36.0, 37.7, 41.5, 41.5, 41.8, 41.9, 50.9,
52.2, 54.6, 57.0, 61.7, 63.0, 71.3, 122.3, 148.7, 158.6,
165.2, 167.8, 170.8, 171.3, 174.1, 174.5, 174.6. MALDI-
TOFMS (M+H)+ calcd for C34H61N8O9: 725.456, found:
725.344. HRFABMS (M+H)+ calcd for C34H61N8O9:
725.4562, found: 725.4556.
1
chem). H NMR (CDCl3) d: 0.58 (0.68H, d, J¼6.3 Hz),
0.73 (0.68H, d, J¼6.3 Hz), 0.88 (4.64H, d, J¼5.4 Hz),
1.16–1.32 (1H, m), 1.42–1.59 (2H, m), 1.86 (0.2H, d,
J¼5.4 Hz), 2.21 (0.2H, dd, J¼15.6, 9.9 Hz), 2.47–2.54
(1.6H, m), 2.98 (0.75H, d, J¼6.6 Hz), 3.15 (0.25H, br s),
3.64 (0.75H, m), 3.77 (0.25H, m), 3.98 (1H, m), 4.20 (1H,
m), 4.37 (2H, m), 4.51 (1H, d, J¼7.5 Hz), 5.36 (0.75H, d,
J¼9.3 Hz), 5.72 (0.25H, d, J¼9.3 Hz), 6.83 (1H, br s),
7.22–7.43 (4H, m), 7.53 (2H, m), 7.70 (2H, m).
3.2.17. Miraziridine A (1). To a solution of 23 (1.0 mg,
1.38 mmol) in DMSO (2.0 ml) were added H2O (8 ml) and
porcine liver esterase in Tris/HCl buffer solution (2.0 mg/
80 ml) and the mixture was allowed to stand for 132 h at
30 ꢂC. The mixture was filtered and the filtrate was purified
by preparative HPLC (CH3CN/H2O¼26:73) to give mirazir-
idine A (1) (0.85 mg, 12.7 mmol, 92%) as a white powder.
[a]3D0 ꢁ61.0 (c 0.10, MeOH). HPLC: tR¼25.52 min
(CH3CN, 2–40%/40 min). IR (film) n max cmꢁ1: 3359,
3.2.15. EtO-Azd-Leu-Sta-Abu-OH (5). To a suspension of
2-Cl-trityl chloride resin (192 mg, 1.31 mmol/g) in DMF
(1 ml) and CH2Cl2 (500 ml) were added Fmoc-Abu-OH
(245 mg, 755 mmol) and DIEA (132 ml, 755 mmol). The
mixture was stirred for 2.5 h at 25 ꢂC. To the resultant resin
was added 20% piperidine in DMF and the mixture was
stirred for 30 min at 25 ꢂC. Fmoc-Sta-OH (8) (300 mg,
755 mmol) and Fmoc-Leu-OH (267 mg, 755 mmol) were
successively condensed to this resin using DIPC (117 ml,
755 mmol)/HOBt (116 mg, 755 mmol) for 3 h. EtO-Azd-
OH (7) (120 mg, 755 mmol) was condensed with DPPA
(163 ml, 755 mmol) and Et3N (105 ml, 755 mmol) for 4 h.
The resulting resin was treated with HFIP/CH2Cl2 (1:3,
2 ml) for 2 h and the mixture was filtrated. After evaporation
of the HFIP, ether was added, and the resulting precipitate
was purified by preparative HPLC (CH3CN/H2O¼43:57)
to yield a tetrapeptide (5) (42 mg, 81.6 mmol, 32%) as a white
powder. [a]2D8 ꢁ40.9 (c 0.3, MeOH). HPLC: tR¼13.76 min
(CH3CN, 30–40%/30 min). IR (film) n max cmꢁ1: 3278,
1
3195, 2922, 2852, 1657, 1635, 1541, 1203, 1136, 721. H
NMR (CD3OH) d: 0.86 (3H, d, J¼6.3 Hz), 0.90 (3H, d,
J¼6.3 Hz), 0.92 (3H, d, J¼6.0 Hz), 0.97 (3H, d,
J¼6.3 Hz), 0.99 (3H, t, J¼7.2 Hz), 1.32 (1H, m), 1.55–
1.75 (10H, m), 1.81–1.92 (1H, m), 2.22–2.43 (2H, m),
2.68 (1H, d, J¼2.1 Hz), 2.84 (1H, d, J¼2.1 Hz), 3.11–3.24
(2H, m), 3.85–3.92 (1H, m), 3.98–4.03 (1H, m), 4.12–4.19
(1H, m), 4.26–4.33 (1H, m), 4.58 (1H, br s), 5.90 (1H, dd,
J¼15.5, 2.0 Hz), 6.83 (1H, dd, J¼15.5, 5.9 Hz), 7.43 (2H,
d, J¼8.4 Hz), 8.07 (1H, d, J¼6.9 Hz), 8.39 (1H, d,
J¼9.9 Hz), 8.46 (1H, d, J¼8.4 Hz). 13C NMR (CD3OH) d:
10.9, 21.9, 22.5, 23.3, 23.6, 26.0, 26.2, 26.4, 32.2, 37.2,
37.5, 41.5, 41.8, 42.1, 50.9, 52.2, 55.2, 57.5, 71.3, 82.6,
122.8, 148.9, 159.0, 170.0, 171.6, 174.0, 174.3. MALDI-
TOFMS (M+H)+ calcd for C30H53N8O9: 669.394, found:
669.268. HRFABMS (M+H)+ calcd for C30H53N9:
669.3936, found: 669.3945.
1
3062, 2958, 2871, 1732, 1653, 1539, 1201, 1142, 723. H
NMR (CD3OD) d: 0.94–1.03 (15H, m), 1.31 (4H, t,
J¼7.1 Hz), 1.33–1.38 (1H, m), 1.57–1.67 (4H, m), 1.69–
1.81 (2H, m), 1.86–1.96 (1H, m), 2.33 (2H, d, J¼6.6 Hz),
2.80 (1H, d, J¼2.7 Hz), 2.93 (1H, d, J¼2.4 Hz), 3.98 (1H,
m), 4.00 (1H, m), 4.19–4.29 (2H, m), 4.33 (1H, dd, J¼8.1,
5.1 Hz), 4.42 (1H, t, J¼7.4 Hz). 13C NMR (CD3OD) d:
9.1, 13.0, 20.5, 20.8, 21.9, 22.4, 24.4, 24.6, 24.6, 34.6,
36.2, 39.9, 40.3, 40.5, 51.4, 52.5, 53.7, 61.5, 70.1, 169.0,
169.8, 172.7, 173.2, 174.0. MALDI-TOFMS (M+H)+ calcd
for C43H27N4O8: 515.308, found: 515.238. HRFABMS
(M+H)+ calcd for C24H43N4O8: 515.3081, found: 515.3077.
3.2.18. HO-Azd-Leu-Sta-Abu-OH (2). To a solution of 5
(2.0 mg, 3.9 mmol) in DMSO (5 ml) were added H2O
(20 ml) and porcine liver esterase in Tris/HCl buffer solution
(4.0 mg/80 ml) and the mixture was allowed to stand for
132 h at 30 ꢂC. The mixture was filtered and the filtrate
was purified by preparative HPLC (CH3CN/H2O¼30:70)
to give HO-Azd-Leu-Sta-Abu-OH (2) (1.5 mg, 3.1 mmol,
79%) as a white powder. [a]2D8 ꢁ16.5 (c 0.02, MeOH).
HPLC: tR¼24.08 min (CH3CN, 2–40%/30 min). IR (film)
n max cmꢁ1: 3278, 3062, 2958, 2871, 1732, 1653, 1539,
3.2.16. EtO-Azd-Leu-Sta-Abu-vArg-OEt (23). To a solu-
tion of H-vArg-OEt (6) (1.1 mg, 4.70 mmol) and EtO-
Azd-Leu-Sta-Abu-OH (5) (2.4 mg, 4.70 mmol) in DMF
(20.0 ml) were added DIEA (0.8 ml, 4.70 mmol), HATU
(1.8 mg, 4.70 mmol), and HOAt (0.6 mg, 4.70 mmol) and
the mixture was stirred for 5 min at room temperature.
AcOH (0.8 ml) was added and the mixture was evaporated
in vacuo. The residue was purified with preparative HPLC
(CH3CN/H2O¼38:62) to give EtO-Azd-Leu-Sta-Abu-
vArg-OEt (23) (1.1 mg, 15.0 mmol, 32%) as a white powder.
[a]3D0 ꢁ33.9 (c 0.01, MeOH). HPLC: tR¼37.15 min
(CH3CN, 2–40%/40 min). IR (film) n max cmꢁ1: 3342,
2960, 1653, 1541, 1458, 1203, 1182, 1138, 1036, 800,
1
1201, 1142, 723. H NMR (CD3OD) d: 0.94–1.03 (15H,
m), 1.25–1.38 (1H, m), 1.55–1.68 (4H, m), 1.69–1.81 (2H,
m), 1.86–1.95 (1H, m), 2.32 (2H, d, J¼6.6 Hz), 2.70 (1H,
br s), 2.94 (1H, br s), 3.96 (2H, m), 4.32 (1H, dd, J¼8.1,
5.1 Hz), 4.41 (1H, t, J¼7.5 Hz), 7.58 (1H, d, J¼9.0 Hz).
13C NMR (CD3OD) d: 10.6, 21.9, 22.2, 23.4, 23.8, 25.9,
26.0, 26.1, 33.2, 37.1, 41.3, 41.7, 52.8, 53.9, 55.2, 71.5,
167.5, 169.0, 172.0, 174.7. MALDI-TOFMS (M+H)+ calcd
for C22H39N4O8: 487.276, found: 487.199.
3.2.19. Ac-Leu-Sta-Abu-OH (24). The resin (21) (50 mg)
was stirred with Ac2O (50 ml)/pyridine (200 ml) in the pres-
ence of a catalytic amount of DMAP. The resulting resin was
treated with HFIP/CH2Cl2 (1:3, 1 ml) for 2 h. After evapora-
tion of the HFIP, ether was added, and the resulting preci-
pitate was purified with preparative HPLC (CH3CN/
H2O¼32:68) to yield tripeptide (24) (9.2 mg, 22.1 mmol,
1
721. H NMR (CD3OD) d: 0.88–1.06 (15H, m), 1.31 (6H,
q, J¼7.0 Hz), 1.34–1.38 (2H, m), 1.58–1.97 (10H, m),
2.27–2.49 (2H, m), 2.83 (1H, d, J¼2.4 Hz), 2.96 (1H, d,
J¼2.1 Hz), 3.17–3.29 (2H, m), 3.90–3.99 (1H, m), 4.02–
4.44 (1H, m), 4.12–4.44 (6H, m), 4.57–4.63 (1H, m), 5.99
(1H, dd, J¼15.8, 1.5 Hz), 6.90 (1H, dd, J¼15.8, 5.6 Hz),

9512
H. Konno et al. / Tetrahedron 63 (2007) 9502–9513
33%) as a white powder. [a]2D5 ꢁ11.2 (c 0.42, MeOH).
HPLC: tR¼28.31 min (CH3CN, 30–40%/40 min). IR (film)
n max cmꢁ1: 3286, 2956, 2871, 1720, 1643, 1541, 1369,
13C NMR (CD3OD) d: 10.9, 21.9, 22.5, 23.4, 23.7, 26.0,
26.3, 31.9, 41.5, 41.7, 42.0, 50.8, 52.1, 54.5, 57.0, 71.4, 79.2,
122.5, 148.9, 163.8, 174.3, 174.6, 175.1. MALDI-TOFMS
(M+H)+ calcd for C28H52N7O7: 598.393, found: 598.361.
1
1043, 754. H NMR (300 MHz, CD3OD) d: 0.91 (3H, d,
J¼6.3 Hz), 0.94 (3H, d, J¼6.3 Hz), 0.95 (3H, d, J¼
6.3 Hz), 0.99 (3H, d, J¼6.3 Hz), 1.01 (3H, t, J¼7.5 Hz),
1.33 (1H, m), 1.56–1.81 (6H, m), 1.89 (1H, m), 2.01 (3H,
s), 2.34 (2H, d, J¼6.6 Hz), 3.98 (2H, m), 4.32 (1H, dd,
J¼8.1, 5.1 Hz), 4.37 (1H, t, J¼7.5 Hz). 13C NMR
(75 MHz, CD3OD) d: 10.6, 21.9, 22.3, 22.4, 23.4, 23.8,
25.79, 25.83, 25.94, 26.0, 41.4, 41.57, 41.65, 41.71, 41.8,
52.6, 52.7, 53.8, 55.2, 55.3, 71.4, 71.5, 173.6, 174.1,
175.2, 175.4. MALDI-TOFMS (M+H)+ calcd for
C20H38N3O6: 416.276, found: 416.267.
3.2.22. H-Leu-Sta-Abu-OH (4). To the resin (21) (59 mg)
was added HFIP/CH2Cl2 (1:3, 1 ml) and the mixture was
stirred for 2 h. After evaporation of the HFIP, ether was
added, and the resulting precipitate was purified with prepar-
ative HPLC (CH3CN/H2O¼35:65) to yield a tripeptide
(4) (42 mg, 81.6 mmol, 32%) as a white powder. [a]2D0 ꢁ7.5
(c 0.04, MeOH), HPLC: tR¼32.00 min (CH3CN, 2–40%/
40 min). IR (film) n max cmꢁ1: 3278, 2956, 2929, 2870,
1
1716, 1651, 1522, 1458, 1201, 1140, 837, 800. H NMR
(CD3OD) d: 0.94 (3H, d, J¼6.6 Hz), 0.96 (3H, d,
J¼6.9 Hz), 1.00 (3H, d, J¼7.8 Hz), 1.02 (3H, d,
J¼7.8 Hz), 1.03 (3H, t, J¼5.3 Hz), 1.39 (1H, m), 1.53–
1.79 (1H, m), 1.90 (1H, m), 2.30 (1H, dd, J¼14.1, 7.5 Hz),
2.36 (1H, dd, J¼14.1, 5.7 Hz), 3.90 (1H, m), 3.99 (1H,
ddd, J¼7.5, 5.4, 2.4 Hz), 4.08 (1H, ddd, J¼9.9, 4.2,
2.7 Hz), 4.33 (1H, dd, J¼8.1, 5.1 Hz). 13C NMR (CD3OD)
d: 9.8, 21.6, 21.8, 22.5, 23.1, 24.5, 24.8, 25.0, 39.7, 40.5,
40.8, 52.2, 52.3, 53.8, 70.2, 169.9, 172.5, 174.6. MALDI-
TOFMS (M+H)+ calcd for C18H36N3O85: 374.266, found:
374.281.
3.2.20. Ac-Leu-Sta-Abu-vArg-OEt (25). To a solution of
H-vArg-OEt (6) (3.8 mg, 16.1 mmol) and Ac-Leu-Sta-Abu-
OH (24) (6.7 mg, 16.1 mmol) in DMF (20.0 ml) were added
DIEA (2.7 ml, 16.1 mmol), HATU (6.2 mg, 16.1 mmol), and
HOAt (2.1 mg, 16.1 mmol) and the mixture was stirred for
10 min at room temperature. AcOH (2 ml) was added and
the mixture was evaporated in vacuo. The residue was puri-
fied with preparative HPLC (CH3CN/H2O¼39:61) to give
Ac-Leu-Sta-Abu-vArg-OEt (25) (4.2 mg, 6.7 mmol, 42%)
as a white powder. [a]D20 ꢁ5.9 (c 0.01, MeOH). HPLC:
tR¼38.35 min (CH3CN, 2–40%/30 min). IR (film) n max
cmꢁ1: 3307, 3195, 2958, 2873, 1653, 1541, 1448, 1369,
3.3. Inhibitory activity
1
1284, 1203, 1182, 1095, 1041, 839, 802, 721. H NMR
(CD3OD) d: 0.86 (3H, d, J¼6.3 Hz), 0.91 (3H, d,
J¼6.0 Hz), 0.93 (3H, d, J¼6.3 Hz), 0.98 (3H, d, J¼
6.3 Hz), 1.01 (3H, d, J¼7.5 Hz), 1.27 (3H, t, J¼7.2 Hz),
1.31 (1H, m), 1.54–1.77 (10H, m), 1.89 (1H, m), 2.03 (3H,
s), 2.31 (1H, dd, J¼14.4, 6.9 Hz), 2.37 (1H, dd, J¼14.4,
7.2 Hz), 3.21 (2H, m), 3.90 (1H, m), 4.03 (1H, td, J¼7.2,
2.1 Hz), 4.13–4.24 (3H, m), 4.30 (1H, m), 4.59 (1H, m),
5.96 (1H, dd, J¼15.9, 1.8 Hz), 6.88 (1H, dd, J¼15.6,
5.4 Hz), 7.31 (1H, d, J¼9.6 Hz), 8.37 (1H, d, J¼8.7 Hz).
13C NMR (CD3OD) d: 10.9, 13.6, 21.9, 22.5, 23.4, 23.7,
26.0, 26.2, 26.3, 31.8, 41.4, 41.7, 41.8, 41.9, 50.8, 57.1,
61.7, 71.3, 122.2, 148.8, 167.8, 173.9, 174.1, 174.6.
MALDI-TOFMS (M+H)+ calcd for C30H56N7O7: 626.424,
found: 626.334.
Bovine spleen cathepsin B was purchased from Sigma
Chemical company (St. Louis, MO). Cathepsin B (10 mU)
was preincubated at 40 ꢂC for 10 min in 90 ml of 50 mM
MES (pH 6.0), 2 mM DTT, and 0.1% Brij-35. The solution
was then mixed with 20 mM Z-Arg-Arg-MCA and each in-
hibitor (1 nM to 1 mM) in DMSO (10 ml), and the mixture
was incubated at 40 ꢂC for 10 min. The reaction was stopped
by adding 100 ml of 100 mM sodium monochloroacetate,
30 mM sodium acetate, and 70 mM acetic acid (pH 4.3).
Ki values were determined according to the method of Dixon
and Webb.19
Acknowledgements
3.2.21. Ac-Leu-Sta-Abu-vArg-OH (3). To a solution of 25
(2.1 mg, 3.35 mmol) in DMSO (4.0 ml) was added H2O
(16 ml) and porcine liver esterase in Tris/HCl buffer solution
(4.0 mg/160 ml) and the mixture was allowed to stand for
72 h at 30 ꢂC. The mixture was filtered and the filtrate was
purified by preparative HPLC (CH3CN/H2O¼23:77) to
give Ac-Leu-Sta-Abu-vArg-OH (3) (1.5 mg, 2.39 mmol,
71%) as a white powder. [a]2D8 ꢁ18.3 (c 0.02, MeOH).
HPLC: tR¼22.62 min (CH3CN, 2–40%/40 min). IR (film)
n max cmꢁ1: 3329, 2958, 2866, 1653, 1639, 1541, 1458,
We thank Dr. Nobutaka Fujii and Dr. Shinya Oishi (Kyoto
University) for the measurement of mass spectra. This
work was supported in part by a grant from Chisso, Co.,
Ltd., Nissui Research Fundation and a Grant-in-aid for
Scientific research from the Japan Society for the Promotion
of Science (Grant 16790084 to H.K.).
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1
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