Synthetic studies of the cyclic depsipeptides bearing the 3-amino-6-hydroxy-2-piperidone (Ahp) unit. Total synthesis of the proposed structure of micropeptin T-20

Article abstract of DOI:10.1016/j.tet.2004.12.009

The first total synthesis of a cyclic depsipeptide possessing the 3-amino-6-hydroxy-2-piperidone (Ahp) unit was successfully achieved in a convergent manner by the oxidative construction of the Ahp unit at the later stage of the synthesis. This synthetic work provides data indicating that the structure of the target Ahp-depsipeptide, micropeptin T-20, should be re-examined.

Full text of DOI:10.1016/j.tet.2004.12.009

Tetrahedron 61 (2005) 1459–1480
Synthetic studies of the cyclic depsipeptides bearing the
3-amino-6-hydroxy-2-piperidone (Ahp) unit. Total synthesis of the
proposed structure of micropeptin T-20
Fumiaki Yokokawa,a,† Akiko Inaizumia and Takayuki Shioirib,
*
aGraduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
bGraduate School of Environmental and Human Sciences, Meijo University, Shiogamaguchi, Tempaku, Nagoya 468-8502, Japan
Received 14 October 2004; revised 5 November 2004; accepted 2 December 2004
Available online 18 December 2004
Abstract—The first total synthesis of a cyclic depsipeptide possessing the 3-amino-6-hydroxy-2-piperidone (Ahp) unit was successfully
achieved in a convergent manner by the oxidative construction of the Ahp unit at the later stage of the synthesis. This synthetic work provides
data indicating that the structure of the target Ahp-depsipeptide, micropeptin T-20, should be re-examined.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Micropeptin T-20 was isolated from the cyanobacterium
Microcyctis aeruginosa collected from the freshwater Kang
Krachan Dam in Thailand, and its structure was determined
to be 1 (Fig. 1) by Kaya and co-workers.1b This cyclic
depsipeptide strongly inhibits chymotrypsin with an IC50 of
2.5!10K9 M, but weakly inhibits tyrosinase with an IC50 of
5!10K3 M.
The 3-amino-6-hydroxy-2-piperidone (Ahp) unit has been
recently characterized as a constituent in more than 60
cyclic depsipeptides derived from cyanobacteria.1 These
cyclic depsipeptides commonly consist of the 19-membered
ring, and generally exhibit an interesting and significant
inhibiting action against peptidic proteases. The unique
unprecedented Ahp moiety will be biosynthetically derived
from glutamate, and probably play an important role to
exhibit biological activity because it will participate in
converting the cyclic depsipeptide into a bioactive confor-
mation due to the conformationally restricted structure and
hydrogen bonding with the free hydroxyl group.
As a continuation of our interests in the synthetic studies of
biologically active aquatic natural products,2 we have
investigated methods used for the synthesis of the Ahp
peptides. Herein, we wish to report the total synthesis of
micropeptin T-20 having the proposed structure 1,3 which
revealed that natural micropeptin T-20 was not identical
Figure 1. The proposed structure of micropeptin T-20 and the structure of the Ahp unit.
Keywords: Total synthesis; Cyclic depsipeptide; Macrolactamization; Hemiaminal; Cyanobacteria.
* Corresponding author. Tel./fax: C81 52 832 1555; e-mail: shioiri@ccmfs.meijo-u.ac.jp
† Present address: Tsukuba Research Institute, Novartis Pharma K. K., Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.12.009

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F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
Scheme 1.
with the synthetic 1. Thus the structure of micropeptin T-20
should be re-examined.
two ways to give the 6-hydroxy-2-piperidone ring A (path A
in Scheme 2) or 5-hydroxypyrrolidine ring B (path B).
Control of the stereochemistry of the newly formed
hydroxyl group will be another problem to solve. Further-
more, the hemiaminal ring is generally labile under both
strong acidic and basic conditions, and it will easily undergo
dehydration to produce the dehydro derivatives C or D,
respectively.4 Thus the construction of the Ahp unit should
be done during the later stage of the synthesis.
2. Synthetic strategy
Our synthetic plan for constructing micropeptin T-20 (1) is
depicted in Scheme 1: (1) Preparation of the depsipeptide 5
and the tripeptide 6, (2) fragment condensation of the
depsipeptide 5 with the tripeptide 6, (3) macrolactamization,
(4) attachment of the side chain 3, (5) oxidative cyclization
to form the Ahp unit, and (6) deprotection. One of the most
important problems to overcome in this synthesis lies in the
formation of the hemiaminal-Ahp ring. The obvious starting
material to construct the Ahp unit will be the aldehyde X
prepared from glutamate, and aldehyde X will cyclize in
As shown in the structure of micropeptin T-20 (1), it is a
very polar compound because it has three hydroxyl groups
in addition to the sodium phosphate function. Selective
protection and deprotection of these polar groups will be
another point to overcome. One of the most important
problems to construct macrocyclic depsipeptides will be
Scheme 2.

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1461
Scheme 3.
where the macrocyclization should be done and which will
be better: macrolactamization or macrolactonization.
Because the nucleophilicity of the amino group is generally
higher than that of the hydroxyl group, macrolactamization
will be preferred. An N-methylamino acid part in a linear
peptide will form a bent structure, and it should be situated
in the middle part of a cyclization precursor. This led us to
synthesize the depsipeptide 5 and the tripeptide 6. Coupling
of these peptide fragments and then macrolactamization of
the linear peptide 4 will give the 19-membered cyclic
Scheme 4.

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F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
depsipeptide 2. After addition of the side chain fragment 3,
construction of the Ahp moiety followed by deprotection
will finally afford micropeptin T-20 (1).
smoothly coupled with the carboxylic acid from 19 to
give the tripeptide 6 in 60% yield,9 as shown in Scheme 4.
Preparation of the hexapeptide, the cyclization precursor,
was first tried by coupling of the depsipeptide 5 with the
O-TBS derivative 20. After deprotection of each compound
as shown in Scheme 5, coupling by the DEPC method
afforded the hexapeptide 21 without the TBS function in
40% yield. It should be noted that removal of the TBS group
again occurred in this case.9 On the other hand, the coupling
of 5 with the phenolic derivative 6 smoothly proceeded to
give the hexapeptide 21 in 77% yield, which was converted
to the O-TBS derivative 4.
3. Results and discussions
The synthesis of the depsipeptide 5 started from Boc-(S)-
phenylalanine (7) by conversion to the corresponding allyl
ester 8, as shown in Scheme 3. After acidic deprotection of
the tert-butoxycarbonyl (Boc) group of 8, the resulting de-
Boc derivative was coupled with trichloroethoxycarbonyl
(Troc)-(S)-threonine (10) using diethyl phosphorocyanidate
(DEPC, (EtO)2P(O)CN) in the presence of triethylamine5 to
quantitatively give the dipeptide 11. Esterification of 11
with Boc-(S)-isoleucine with N-ethyl-N0-(3-(dimethyl-
amino)propyl)carbodiimide hydrochloride (EDCI$HCl)
afforded the depsipeptide 5.
We are now at the stage of macrolactamization. After
removal of the allyl group from the ester part of 4, the Boc
group was removed under acidic conditions. The resulting
deprotected peptide underwent cyclization under high
dilution conditions, as summarized in Table 1. So far,
pentafluorophenyl diphenylphosphinate (FDPP, (C6H5)2-
P(O)OC6F5)10 in the presence of diisopropylethylamine in
dichloromethane furnished best result, giving the macro-
lactam 2 in 84% yield. Unexpectedly, the yield of 2 using
the corresponding pentafluorophenyl ester was low (23%).
The required N-methyl-(S)-tyrosine derivative (13) was
prepared from Boc-(S)-tyrosine (12) by conversion to its
O-tert-butyldimethylsilyl (TBS) derivative, which afforded
the N-methyl derivative 13 according to our synthesis of the
corresponding (R)-derivative,6 as shown in Scheme 4.
Esterification of the acid 13 gave the methyl ester 14,
whose Boc group was removed under acidic conditions.
Coupling of the de-Boc derivative with Boc-(S)-phenyl-
alanine (7) using bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BopCl)7 gave the dipeptide 15.
Since there is no precedent on the construction of the Ahp
ring in peptides, some model experiments were carried out,
as shown in Scheme 6. First, the tripeptide 20 underwent a
catalytic debenzylation to give the corresponding alcohol,
which was subjected to oxidation with the Dess–Martin
periodinane.11 Neither the aldehyde 22 nor Ahp derivative
was detected in the products. Next, the macrocyclic
depsipeptide 2 underwent the catalytic removal of the
benzyl function and then the product was analogously
oxidized as described above. Again the aldehyde 23 was
very labile and immediately transformed into a complex
mixture. It was thought that the stability of the aldehyde
might depend on the presence of the side chain which is
attached at the N-terminal of threonine in the micropeptins
and affects the conformation. Thus the construction of the
The pentahomoserine derivative 19, which will become a
constituent of the Ahp unit, was prepared from Boc-(S)-
glutamic acid benzyl ester (17) by its conversion to the
mixed anhydride, reduction to the alcohol 18,8 and then
benzylation. After the acidic deprotection of the Boc group
of 15 and saponification of 19, respectively, coupling by the
DEPC method afforded a mixture of the tripeptides 20 and 6
in 16 and 24% yields, respectively. In contrast, the dipeptide
16, obtained by removal of the TBS group from 15 with
TBAF (Bu4NF), underwent acidic deprotection and
Scheme 5.

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
Table 1. Macrolactamization of the linear precursor 4
1463
Entry
Coupling conditions
Time (h)
Yield (%)
1
2
3
4
5
DPPA, NaHCO3, DMF, 4 8C
DEPC, NaHCO3, DMF, 4 8C
HATU, Pri2NEt, DMF, rt
HATU, Pri2NEt, DMF, 4 8C
FDPP, Pri2NEt, DMF, rt
72
96
14
96
14
52
44
38
59
57
6
FDPP, Pri2NEt, CH2Cl2, rt
17
84
DPPA, (C6H5O)2P(O)N3; FDPP, (C6H5)2P(O)OC6F5; HATU,
.
Ahp unit was postponed after the introduction of the side
chain.
in low yield. Finally, the primary alcohol 30 was treated
with dibenzyl N,N-diisopropyl phosphoramidite (31) to give
the phosphite, which was oxidized with m-chloroperbenzoic
acid (m-CPBA)17 to produce the desired side chain fragment
3a in quantitative yield.
To prepare the side chain, (S)-glyceric acid allyl ester (25)
obtained from (S)-serine (24)12 was converted to the bis-
TBS ester 26, as shown in Scheme 7. The attempted
selective deprotection of the bis-TBS ester 26 with HF-
pyridine13 afforded a mixture of the primary alcohol 27,
secondary alcohol 28, and diol 25. Thus the diol 25 was first
converted to the monosilylated compound 28,14 which was
transformed to the benzyl derivative 29. Removal of the
TBS group afforded the primary alcohol 30. Phosphoryl-
ation of 30 failed using o-xylene N,N-diethylphosphor-
amidite15 or the Mitsunobu reaction with dibenzyl
phosphate.16 The Mitsunobu reaction of the diol 25 with
dibenzyl phosphate proceeded to give the mono phosphate
Since both the hydroxyl and phosphate residues of the side
chain 3a were protected by the benzyl group, the benzyl
group of the homoserine part of the cyclic depsipeptide 2
should be removed before the introduction of the side chain.
Although the hydrogenolytic removal of the benzyl group
from 2 proceeded using 20% Pd(OH)2, attempted
removal of the Troc group using zinc in acetic acid failed.
In contrast, removal of the Troc group from 2 rapidly
proceeded to give the corresponding amino derivative using
zinc in acetic acid, but hydrogenolysis did not give the
Scheme 6.

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F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
Scheme 7.
Scheme 8.

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1465
Scheme 9.
required N,O-deprotected compound under a variety of
conditions. This might be due to the high polarity of the
de-Troc product.
equilibrium between the aldehyde 35 and the hemiaminal
36 was detected, and attempted cyclization of 35 using
p-toluenesulfonic acid or pyridinium p-toluenesulfonate
(PPTS) caused the gradual decomposition of the starting
aldehyde 35.
To overcome this difficulty, replacement of the Troc group
with the less polar Boc group was undertaken. Thus, after
removal of the Troc group of 2 with zinc in acetic acid as
described above, the crude product was converted to the Boc
derivative 32, as shown in Scheme 8. The Boc derivative 32
smoothly underwent catalytic hydrogenolysis to give the
Boc alcohol 33, from which the Boc group was removed
under acidic conditions. After removal of the allyl group
from 3a, the side chain fragment was coupled with the
amine from 33 to give the Ahp precursor 34.
We now returned to the model experiments on the
cyclization of the aldehyde to the hemiaminal. Thus, 1,5-
pentanediol (37) was transformed into the monobenzyl ether
38,19 which was converted to the carboxylic acid and
coupled with phenylalanine methyl ester from 39, as shown
in Scheme 10. The resulting amide 40 was converted to the
aldehyde 41 through catalytic debenzylation followed by
the Parikh–Doering oxidation. Cyclization of the aldehyde
41 to 42 was carried out under a variety of conditions, as
summarized in Table 2. Many Lewis acids were not
effective and resulted in no reaction or decomposition of
the starting aldehyde 41 (entries 1–6 in Table 2). Alumina
was also not useful, and cyclization followed by
dehydration occurred using silica gel at higher temperature
to give the dehydro derivative 43 (entries 7–9). Both
p-toluenesulfonic acid and PPTS were ineffective (entries
The Ahp precursor 34 was oxidized with Dess–Martin
periodinane to give the aldehyde 35 in 50% yield, as shown
in Scheme 9. Alternatively, the use of 1-hydroxy-1,2-
benziodoxol-3(1H)-one 1-oxide (IBX), a milder oxidant,18
afforded 35 in 80% yield, while oxidation with pyridinium
dichromate resulted in the formation of a complex mixture.
The aldehyde 35 was relatively stable compared to 23. No

1466
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
Scheme 10.
Table 2. Hemiaminalization of the model compound 41
Entry
Reagents
Solvents
Temperature (time)
Results
1
2
3
4
5
6
7
8
ZnCl2
MgBr2
SnCl4
Sc(OTf)3
TiCl4
BF3$Et2O
Al2O3
Al2O3
SiO2
p-TsOH
PPTS
CH2Cl2
Et2O
K78 8C (0.5 h) to rt (24 h)
0 8C (1 h) to rt (48 h)
0 8C (1 h) to rt (1 h)
0 8C (2.5 h)
No reaction
Trace
Complex mixture
Trace
Trace
Trace
No reaction
Unknown product
Dehydro product 43 (62%)
Trace (dehydro product 43: major)
Trace
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
Toluene
Toluene
CH2Cl2
CH2Cl2
0 8C (1 h)
K78 8C (4 h) to rt (2 h)
0 8C (1 h) to rt (2 h)
80 8C (18 h)
rt (1 h) to 80 8C (12 h)
0 8C (1 h)
0 8C (1 h) to rt (2.5 h)
9
10
11
12
0.1 M pH 6 phosphate buffer
THF
0 8C (0.5 h) to rt (20 h)
6-Hydroxy-2-piperidone 42 (66%)
10–11). We thought at this stage that the addition of water to
the solvent system might prevent the dehydration. In fact,
treatment of the aldehyde 41 in tetrahydrofuran containing
0.1 M phosphate buffer (pH 6) afforded the desired
piperidone 42 as a single isomer in 66% yield (entry 12).
In the synthetic route to micropeptin T-20, removal of both
the TBS and benzyl groups will be necessary after the
construction of the Ahp unit. Thus, the stability of the model
compound 42 toward hydrogenolysis and TBAF was
examined. The hemiaminal 42 was stable to hydrogenolysis
but treatment with TBAF caused dehydration to give the
dehydro derivative 43 in only 30 min, which suggested the
requirement of the careful treatment with TBAF.
derivative 44 as a single isomer, and the 1H NMR spectrum
of its Ahp part was completely identical with that of natural
micropeptin T-20 (1), proving its stereochemistry as
depicted. The conformational effect of the cyclic depsi-
peptide will govern the stereochemistry. The above
experiments revealed that treatment of the aldehyde 35
with TBAF would give rise to the Ahp ring formation
accompanied by desilylation. In fact, after oxidation of 34
with IBX, treatment with TBAF afforded the Ahp derivative
44 as a single isomer in good yield.
The stability of the Ahp structure will be due to cooperation
of the resonance effect between the nitrogen and carbonyl
group in the Ahp ring in addition to the anomeric and
substituent effects. Since the dehydration in the Ahp nucleus
will occur when the C5-axial hydrogen and the hydroxyl
group are situated antiperiplanar, the freedom of the
6-membered chair conformation will affect the stability.
This will be the reason for the different reactivities between
the model compound 42 and the real substrate 44.
The results of the above experiments indicated that both
aldehyde and hemiaminal structures were labile to acidic
conditions and the strong oxidative conditions were not
necessary. After oxidation of the alcohol 34 with IBX,
treatment of the aldehyde 35 with a pH 6 phosphate buffer
afforded a 1:1 mixture of the starting aldehyde 35 and the
Ahp derivative 36 in low yield, as shown in Scheme 9. Since
the mixture was unseparable and labile, desilylation of the
mixture was carried out with TBAF to give the Ahp
Debenzylation at the side chain of 44 was attempted under a
hydrogen atmosphere by using 10% Pd–C or 20% Pd(OH)2

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1467
Table 3. Selective deprotection of 26
Entry
Conditions
Temperature (time)
Results
Reference
1
2
3
4
3.5 M HF-pyridine, THF
AcOH-H2O-THF (13:7:3)
CF3CO2H-H2O-THF (9:1:40)
Oxone, MeOH-H2O (1:1) C
rt (5 h)
Complex mixture
13
rt (9 h) K40 8C (4.5 h) to rt (11 h)
0 8C (0.5 h) to rt (6 h)
rt (6 h)
28%
54%
Trace
21a
21a
21b
5
CeCl3$7H2O, NaI, MeCN
rt (15 h) to reflux (3 h)
62%
21c
Scheme 11.

1468
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
in 90% aqueous ethanol.17,20 The product was revealed to be
the monobenzylated derivative 45 (Scheme 9). Although
both of the benzyl functions at the phosphate part were
removed, the benzyl group at the ether function could not be
removed at all. The addition of acetic acid with a longer
reaction time resulted in cleavage of the side chain.
not the same compound reported by Kaya and co-workers.1b
Comparison of the NMR spectra leads us to conclude that
our synthetic compound has the structure 1 and it will be
configurationally isomeric to natural micropeptin T-20. A
complete similarity was observed in the spectral data of
their cyclic depsipeptide part. However, a difference was
seen at the methine proton at C2 of the glyceric acid part and
1
We now needed to renew the protective group at the
glyceric phosphate side chain, therefore, the protection with
the TBS group was reinvestigated, the results of which are
shown in Table 3. So far, a combination of cerium chloride
and sodium iodide21c toward 26 gave the best result that
furnished the primary alcohol 27 in 62% yield. The alcohol
27 was analogously converted to the phosphate 3b as the
conversion of 30 to 3a (See Scheme 7).
methylene protons at C3; the H signal of the synthetic
material was 0.2 ppm higher than that of the natural one and
the difference of 3 ppm was observed in their 13C NMR
spectra. This obvious spectral difference at the side chain
was also observed after treatment with a pH 7 phosphate
buffer followed by purification on an ODS column.
Furthermore, pH and concentration proved not to influence
the chemical shift of the NMR spectra. This structural
discrepancy between the synthetic and natural micropeptin
T-20 led us to synthesize another micropeptin T-20 having
the (R)-glyceric acid phosphate residue.
After introduction of the side chain 3b to the cyclic
depsipeptide 33, oxidation of the alcohol 46 with IBX
followed by treatment with TBAF afforded the Ahp peptide
47 as a single isomer. Catalytic removal of the benzyl
function was easily accomplished over 10% Pd–C in 90%
aqueous ethanol, as shown in Scheme 11. From the NMR
spectral data, our synthetic micropeptin T-20 is apparently
The alcohol 48 was submitted to oxidation, allylation, and
then acidic treatment to give the (R)-glyceric acid allyl ester
(49), which was converted to the (R)-glyceric acid
phosphate 50, as shown in Scheme 12. After removal of
Scheme 12.

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1469
the allyl group, coupling with the amine from the cyclic
depsipeptide 34 afforded 51 with the (R)-side chain. IBX
oxidation and treatment with TBAF afforded 52, which was
converted to micropeptin T-20 (53) having the (R)-side
chain as described above. However, this compound 53 was
again not identical with natural micropeptin T-20.
1167; 1H NMR (270 MHz, CDCl3) d 1.41 (9H, s, tBu), 3.02–
3.14 (2H, m, Phe-3-H), 4.58–4.61 (3H, m, Phe-2-H,
CO2CH2), 4.98 (1H, br, NH), 5.22–5.33 (2H, m, CO2CH2-
CHCH2), 5.78–5.93 (1H, m, CO2CH2CHCH2), 7.12–7.23
(5H, m, C6H5); 13C NMR (67.8 MHz, CDCl3) d 28.1, 38.2,
54.3, 65.7, 79.7, 118.7, 126.8, 128.0, 129.0, 131.4, 135.9,
154.9, 171.4; Anal. calcd for C17H23NO4: C, 66.86, H, 7.59,
N, 4.59. Found: C, 66.72, H, 7.63, N, 4.65.
Based on the preceding analysis and synthesis, we conclude
that our synthesis proceeded as intended to correctly provide
molecules possessing structures 1 and 53. Although the
method for the synthesis of the Ahp-containing cyclic
depsipeptide was established,22 this work strongly indicates
that the structure of micropeptin T-20 should be revised.
Unfortunately, however, since no natural micropeptin T-20
is presently available, structural re-assignment will require
re-isolation.
4.1.2. Troc-(S)-Thr-OH (10). To a solution of (S)-Thr-OH
(9) (15.0 g, 0.12 mol) in THF (120 ml) and 2.5 N aqueous
NaOH (120 ml) was added 2,2,2-trichloroethoxycarbonyl
chloride (10 ml!3, total 0.19 mol) three times every 1 h at
0 8C. The mixture was stirred at 0 8C for 6 h, and then
concentrated. The residue was acidified with 1 M aqueous
KHSO4 and extracted with EtOAc (!3). The extracts were
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (BW-820 MH, hexane–EtOAcZ1:1 to 1:3
then EtOAc only) to give 10 (23.8 g, 65%) as a colorless
amorphous solid: [a]2D3ZC0.67 (c 1.0, CHCl3); IR nmax
4. Experimental
4.1. General information
1
(CHCl3) cmK1 3424, 1717, 1525, 1408, 1113; H NMR
(270 MHz, CDCl3) d 1.23–1.35 (3H, m, Thr-4-H), 4.37–
4.42 (1H, m, Thr-3-H), 4.48–4.51 (1H, m, Thr-2-H), 4.69
(1H, d, JZ12.2 Hz, OCH2CCl3), 4.76 (1H, d, JZ11.8 Hz,
OCH2CCl3), 6.22 (1H, br, NH); 13C NMR (67.8 MHz,
CDCl3) d 19.2, 59.1, 65.7, 68.0, 74.6, 95.1, 155.4, 173.9;
Anal. calcd for C7H10C3NO5$1/4EtOAc$1/2H2O: C, 29.34,
H, 3.91, N, 4.39. Found: C, 29.13, H, 3.73, N, 4.70.
Infrared spectra were recorded on a SHIMADZU FT IR-
8100 spectrometer. Optical rotations were measured on a
JASCO DIP-1000 digital polarimeter with a sodium lump
(lZ589 nm, D line) and are recorded as follows: [a]TD (C
1
g/100 ml, solvent). H NMR spectra were recorded on a
JEOL EX-270 or a-500 spectrometer in deuterio solvent
using tetramethylsilane (TMS) or CHCl3 (d 7.26 ppm) or
CH3OH (d 3.30 ppm) as an internal standard. Data are
described as follows: chemical shift, integration, multi-
plicity (sZsinglet, dZdoublet, tZtriplet, qZquartet, brZ
broad, mZmultiplet), coupling constants (Hz), and assign-
ment. 13C NMR spectra were recorded on a JEOL EX-270
(67.8 MHz) spectrometer with complete proton decoupling.
Chemical shifts are described in ppm with the solvent as the
internal standard (CDCl3: d 77.0 ppm, CD3OD: d
49.0 ppm). Analytical thin layer chromatography was
performed on Merck Art. 5715, Kiselgel 60 F245/0.25 mm
thickness plates. Visualization was accomplished with UV
light, phosphomolybdic acid, or ninhydrin solution followed
by heating. Mass spectra were obtained on a JEOL JMS-SX
102A (EI) and JMS-AX 505HA (FAB) spectrometer.
Column chromatography was performed with silica gel
BW-820 MH or BW-200 MH (Fuji Silysia Co.). Solvents
for extraction and chromatography were reagent grade.
Tetrahydrofuran (THF) was distilled from sodium/benzo-
phenone ketyl. Diethyl ether (Et2O) was distilled from
lithium aluminum hydride (LiAlH4). Dichloromethane
(CH2Cl2) was distilled from calcium hydride. All other
commercially available reagents were used as received.
4.1.3. Troc-(S)-Thr-(S)-Phe-OAllyl (11). Boc-(S)-Phe-
OAllyl (8) (1.00 g, 3.29 mmol) was treated with 4 N HCl–
EtOAc (5 ml) at 0 8C and the mixture was stirred for 1.5 h.
Removal of the solvent under reduced pressure afforded the
crude hydrochloride salt as a colorless solid.
To a solution of the above crude amine salt and Troc-(S)-
Thr-OH (10) (880 mg, 2.99 mmol) in DMF (6 ml) was
added DEPC (0.72 ml, 4.74 mmol) and Et3N (1.04 ml,
7.47 mmol) at 0 8C. The mixture was stirred at 0 8C for 1 h,
then at room temperature for 19 h. After dilution with
EtOAc, the whole mixture was successively washed with
1 M aqueous KHSO4, H2O, saturated aqueous NaHCO3,
H2O, and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (BW-820 MH, hexane–EtOAcZ2:1 to
1:1) to give 11 (1.37 g, quant.) as a colorless amorphous
solid: [a]2D4ZK8.8 (c 1.0, CHCl3); IR nmax (CHCl3) cmK1
3324, 1740, 1662, 1538; 1H NMR (270 MHz, CDCl3) d 1.17
(3H, d, JZ6.2 Hz, Thr-4-H), 2.79 (1H, br, exchangeable
with D2O, OH), 3.05 (1H, dd, JZ7.0, 13.8 Hz, Phe-3-H),
3.20 (1H, dd, JZ5.9, 14.0 Hz, Phe-3-H), 4.08–4.15 (1H, m,
Thr-3-H), 4.25–4.35 (1H, m, Thr-2-H), 4.60–4.80 (4H, m,
CO2CH2CH, OCH2CCl3), 4.84–4.92 (1H, m, Phe-2-H),
5.25–5.36 (2H, m, CO2CH2CHCH2), 5.81–5.95 (1H, br,
CO2CH2CHCH2), 7.10–7.33 (5H, m, C6H5); 13C NMR
(67.8 MHz, CDCl3) d 17.9, 37.6, 53.3, 58.7, 66.2, 66.8, 74.7,
95.2, 119.2, 127.2, 128.5, 129.1, 131.2, 135.5, 154.9, 169.9,
170.0; Anal. calcd for C19H24Cl3N2O6: C, 47.37, H, 4.81, N,
5.81. Found: C, 47.54, H, 4.84, N, 5.51.
4.1.1. Boc-(S)-Phe-OAllyl (8). To a solution of Boc-(S)-
Phe-OH (7) (3.0 g, 11.3 mmol) in DMF (30 ml) was added
KHCO3 (2.3 g, 23.0 mmol) and allyl bromide (1.4 ml,
16.2 mmol) at 0 8C. The mixture was stirred at 0 8C for 1 h,
then at room temperature for 14 h. The reaction was
quenched with 1 M aqueous KHSO4 and the mixture was
extracted with EtOAc. The extracts were successively
washed with H2O, saturated aqueous NaHCO3, H2O, and
brine, dried over Na2SO4, and concentrated in vacuo to give
8 (3.6 g, quant.) as a colorless oil: [a]2D3ZC27.9 (c 1.0,
CHCl3); IR nnmeaaxt cmK1 3375, 2978, 1743, 1716, 1498, 1367,
4.1.4. Troc-(S)-Thr[Boc-(S)-Ile]-(S)-Phe-OAllyl (5). To
a solution of dipeptide 11 (50 mg, 0.10 mmol) and

1470
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
Boc-(S)-Ile-OH (35 mg, 0.15 mmol) in THF (0.5 ml) was
added DMAP (1.2 mg, 0.01 mmol) and EDCI$HCl (28 mg,
0.15 mmol) at 0 8C. The mixture was stirred at 0 8C for 1 h,
then at room temperature for 12 h. After dilution with
EtOAc, the whole mixture was washed with 1 M aqueous
KHSO4, saturated aqueous NaHCO3, and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-200
MH, hexane–EtOAcZ4:1) to give 5 (63 mg, 80%) as a
colorless amorphous powder: [a]2D4ZC18.7 (c 1.0, CHC3);
IR nmax (CHCl3) cmK1 3437, 1747, 1716, 1697, 1684, 1506;
1H NMR (270 MHz, CDCl3) d 0.88–0.93 (6H, m, Ile-5,6-
H), 1.18–1.28 (5H, m, Ile-4-H, Thr-4-H), 1.49 (9H, s, tBu),
1.67–1.80 (1H, m, Ile-3-H), 3.06–3.23 (2H, m, Phe-3-H),
4.02–4.10 (1H, m, Thr-2-H), 4.32–4.37 (1H, m, Ile-2-H),
4.59 (2H, d, JZ5.6 Hz, CO2CH2CH), 4.73 (2H, s,
OCH2Cl3), 4.84–4.92 (1H, m, Phe-2-H), 4.95–5.05 (1H,
br, NH), 5.20–5.38 (3H, m, Thr-3-H, CO2CH2CHCH2),
5.78–5.89 (1H, m, CO2CH2CHCH2), 5.90–6.00 (1H, br,
NH), 7.15–7.30 (6H, m, C6H5, NH); 13C NMR (67.8 MHz,
CDCl3) d 11.2, 14.9, 15.2, 25.2, 28.3, 36.7, 37.7, 53.7, 56.7,
58.4, 65.9, 69.7, 74.6, 80.2, 95.2, 118.9, 127.0, 128.5, 129.1,
131.3, 135.9, 153.9, 156.0, 167.3, 170.7, 171.2; Anal. calcd
for C30H42Cl3N3O9: C, 51.84, H, 6.09, N, 6.05. Found: C,
51.55, H, 6.07, N, 5.77.
washed with 1 M aqueous KHSO4 and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ5:1 to 3:1) to give 13 (607 mg, 84%)
as a pale yellow amorphous solid: [a]2D3ZK55.1 (c 1.0,
CHCl3) ([a]2D5ZC57.7 (c 1.1, CHCl3) for (R)-isomer6); IR
cmK1 3200, 1705, 1512, 1257; H NMR (270 MHz,
neat
1
n
max
CDCl3) d 0.17 (6H, s, SiMe2), 0.97 (9H, s, Si-tBu), 1.36,
1.41 (9H, s!2, O-tBu), 2.65, 2.73 (3H, s!2, NMe), 2.98–
3.25 (2H, m, Tyr-3-H), 4.41–4.72 (1H, m, Tyr-2-H), 6.76
(2H, d, JZ8.6 Hz, Tyr-6,8-H), 7.05 (2H, d, JZ7.6 Hz, Tyr-
5,9-H); 13C NMR (67.8 MHz, CDCl3) d K4.5, 18.0, 25.5,
28.0, 28.1, 32.4, 32.6, 33.8, 34.3, 60.0, 60.6, 80.3, 80.6,
120.0, 129.8, 154.2, 155.1, 156.1, 175.6; Anal. calcd for
C21H35NO5Si: C, 61.58; H, 8.61; N, 3.42. Found: C, 61.67;
H 8.68; N; 3.21.
4.1.7. Boc-(S)-N-Me-Tyr(TBS)-OMe (14). To a solution of
Boc-(S)-N-Me-Tyr(TBS)-OH (13) (1.22 g, 2.98 mmol) in
DMF (10 ml) was added KHCO3 (0.60 g, 5.96 mmol) and
MeI (0.26 ml, 4.17 mmol) at 0 8C. The mixture was stirred
at 0 8C for 1 h, then at room temperature for 12 h. The
reaction was quenched with 1 M aqueous KHSO4 and the
mixture was extracted with EtOAc. The extracts were
successively washed with H2O, saturated aqueous NaHCO3,
H2O, and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (BW-820 MH, hexane–EtOAcZ10:1 to
4.1.5. Boc-(S)-Tyr(TBS)-OH. To a solution of Boc-(S)-
Tyr-OH (12) (3.0 g, 10.6 mmol) in DMF (20 ml) was added
TBSCl (4.8 g, 31.9 mmol) and imidazole (4.3 g, 63.6 mmol)
at 0 8C. The solution was stirred at 0 8C for 1 h, then at room
temperature for 12 h. After dilution with EtOAc, the whole
mixture was washed with 1 M aqueous KHSO4, saturated
aqueous NaHCO3, brine, dried over Na2SO4, and concen-
trated in vacuo. K2CO3 (2.1 g, 15 mmol) in H2O (30 ml)
was added to a solution of the above crude disilylate in THF
(60 ml) and MeOH (30 ml). The mixture was stirred at room
temperature for 48 h. After dilution with Et2O, the whole
mixture was successively washed with 1 M aqueous
KHSO4, H2O, and brine, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
3:1) to give Boc-(S)-Tyr(TBS)-OH (3.6 g, 86%) as a
5:1) to give 14 (1.19 g, 94%) as a colorless oil: [a]2D3Z
neat
max
K46.5 (c 1.0, CHCl3); IR n
cmK1 1747, 1699, 1512,
1
1257, 1170; H NMR (270 MHz, CDCl3) d 0.16 (6H, s,
SiMe2), 0.96 (9H, s, Si-tBu), 1.36, 1.38 (9H, m, O-tBu), 2.67
(3H, s, NMe), 2.89–3.30 (2H, m, Tyr-3-H), 3.73 (3H, s,
CO2Me), 4.40–4.44, 4.83–4.87 (1H, m, Tyr-2-H), 6.75 (2H,
d, JZ7.9 Hz, Tyr-6,8-H), 6.95–7.06 (2H, m, Tyr-5,9-H);
13C NMR (67.8 MHz, CDCl3) d K4.5, 18.0, 25.5, 28.1,
31.8, 32.7, 34.1, 34.6, 51.9, 59.5, 61.8, 79.7, 80.1, 120.0,
129.8, 154.3, 154.8, 171.5, 171.8; Anal. calcd for
C22H37NO5Si: C, 62.38; H, 8.80; N, 3.31. Found: C,
62.02; H, 8.80; N, 3.33.
4.1.8. Boc-(S)-Phe-(S)-N-Me-Tyr(TBS)-OMe (15). Boc-
(S)-N-Me-Tyr(TBS)-OMe (14) (300 mg, 0.73 mmol) was
treated with 4 N HCl–EtOAc (2.5 ml) at 0 8C and the
mixture was stirred for 1 h. Removal of the solvent under
reduced pressure afforded the crude hydrochloride salt as a
colorless solid.
colorless amorphous solid: [a]2D0ZC30.2 (c 1.1, CHCl3)
neat
max
([a]2D5ZK30.4 (c 1.5, CHCl3) for (R)-isomer6); IR n
1
cmK1 3367, 1716, 1512, 1257, 1167; H NMR (270 MHz,
CDCl3) d 0.18 (6H, s, SiMe2), 0.97 (9H, s, Si-tBu), 1.41 (9H,
s, O-tBu), 3.01–3.14 (2H, m, Tyr-3-H), 4.50–4.60 (1H, br,
Tyr-2-H), 4.85–4.95 (1H, br, NH), 6.77 (2H, d, JZ8.6 Hz,
Tyr-6,8-H), 7.04 (2H, d, JZ8.2 Hz, Tyr-5,9-H); 13C NMR
(67.8 MHz, CDCl3) d K4.5, 18.1, 25.5, 28.2, 37.0, 54.2,
80.0, 120.0, 128.6, 130.3, 154.6, 155.3, 175.4; Anal. calcd
for C20H33NO5Si: C, 60.73; H, 8.41; N, 3.54. Found: C,
60.58; H 8.31; N; 3.52.
To a solution of the above crude amine salt and Boc-(S)-
Phe-OH (7)(230 mg, 0.87 mmol) in CH2Cl2 (3 ml) was
added Et3N (0.25 ml, 1.82 mmol) and BopCl (220 mg,
0.87 mmol) at 0 8C. The reaction mixture was stirred at 4 8C
for 15 h. After dilution with EtOAc, the whole mixture was
successively washed with 1 M aqueous KHSO4, saturated
aqueous NaHCO3, and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-200 MH, hexane–EtOAcZ
5:1) to give 15 (300 mg, 72%) as a colorless amorphous
solid: [a]2D4ZK47.4 (c 1.0, CHCl3); IR nmax (CHCl3) cmK1
3324, 1743, 1713, 1645, 1510, 1255, 1170; 1H NMR
(270 MHz, CDCl3) d 0.15 (6H, s, SiMe2), 0.95 (9H, s, Si-
tBu), 1.38 (9H, s, O-tBu), 2.73 (3H, s, NMe), 2.80–2.90 (2H,
m, Phe-3-H), 2.90–3.05 (1H, m, Tyr-3-H), 3.15–3.25 (1H,
4.1.6. Boc-(S)-N-Me-Tyr(TBS)-OH (13). To a solution of
Boc-(S)-Tyr(TBS)-OH (700 mg, 1.77 mmol) in THF
(5.5 ml) was carefully added 1.54 N t-BuLi in pentane
(2.5 ml, 3.89 mmol) at K78 8C. The solution was stirred at
K78 8C for 30 min, then MeI (3.3 ml, 53.2 mmol) was
added at 0 8C. After 1 h, the reaction mixture was allowed to
warm to room temperature and stirred for 18 h. The reaction
was quenched with saturated aqueous NaHCO3 and the
mixture was extracted with EtOAc. The extracts were

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1471
1
m, Tyr-3-H), 3.68 (3H, s, CO2Me), 4.68–4.78 (1H, m, Tyr-
2-H), 5.08–5.15 (2H, m, Phe-2-H), 6.68–6.80 (2H, m, Tyr-
6,8-H), 6.93–6.99 (2H, m, Tyr-5,9-H) 7.15–7.30 (5H, m,
C6H5); 13C NMR (67.8 MHz, CDCl3) d K3.8, 18.7, 26.2,
28.9, 33.4, 34.4, 39.8, 52.2, 52.7, 59.6, 80.1, 120.7, 127.8,
128.9, 130.0, 130.2, 130.4, 137.0, 155.0, 155.5, 171.4,
172.6; Anal. calcd for C31H46N2O6Si$1/2H2O: C, 64.22; H,
8.17; N, 4.83. Found: C, 64.57; H, 7.99; N, 5.13.
cmK1 3367, 1743, 1714, 1498, 1161; H NMR (270 MHz,
CDCl3) d 1.42 (9H, s, tBu), 1.58–2.05 (4H, m, Phs-3,4-H),
3.45 (2H, t, JZ6.0 Hz, Phs-5-H), 4.30–4.40 (1H, m, Phs-2-
H), 4.46 (2H, s, CH2OCH2C6H5), 5.10–5.22 (3H, m,
CO2CH2C6H5, NH), 7.29–7.34 (10H, m, C6H5!2); 13C
NMR (67.8 MHz, CDCl3) d 25.4, 28.2, 29.3, 53.2, 66.8,
69.3, 72.7, 79.6, 127.4, 128.0, 128.2, 128.4, 135.3, 138.2,
155.3, 172.5; Anal. calcd for C24H31NO5: C, 69.71; H, 7.56;
N, 3.39. Found: C, 69.63; H, 7.68; N, 3.19.
4.1.9. Boc-(S)-Phe-(S)-N-Me-Tyr-OMe (16). To a solution
of dipeptide 15 (1.0 g, 1.75 mmol) in THF (1 ml) was added
1 N TBAF in THF (5.3 ml, 5.3 mmol) at 0 8C. The mixture
was stirred at 0 8C for 1 h. After dilution with EtOAc, the
whole mixture was washed with H2O and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ2:1) to give 16 (797 mg, quant.) as a
4.1.12. Boc-(S)-Phs(Bn)-(S)-Phe-(S)-N-Me-Tyr-OMe (6)
(PhsZpentahomoserine). Dipeptide 16 (700 mg,
1.22 mmol) was treated with 4 N HCl–dioxane (5 ml) at
0 8C and the mixture was stirred for 30 min. Removal of the
solvent under reduced pressure afforded the crude hydro-
chloride salt as a colorless solid.
colorless amorphous powder: [a]2D4ZK19.6 (c 1.1, CHCl3);
To a solution of the pentahomoserine derivative 19 (560 mg,
1.35 mmol) in THF (5 ml) was added 0.5 N aqueous LiOH
(5 ml) at 0 8C. The mixture was stirred at room temperature
for 12 h, then Et2O was added. The separated aqueous layer
was acidified with 1 M aqueous KHSO4, salted out, and
extracted with EtOAc (!2). The extracts were dried over
Na2SO4, and concentrated in vacuo to give a crude
carboxylic acid as a colorless oil which was used for the
next step without further purification.
neat
max
IR n
cmK1 3324, 1741, 1698, 1646, 1516, 1250, 1169;
1H NMR (270 MHz, CDCl3) d 1.37 (9H, s, tBu), 1.70–1.90
(1H, br, OH), 2.19–2.26 (1H, m, Tyr-3-H), 2.75 (3H, s,
NMe), 2.79–2.88 (2H, m, Tyr-3-H, Phe-3-H), 2.91–3.04
(1H, m, Phe-3-H), 3.67 (3H, s, CO2Me), 4.73–4.81 (1H, m,
Tyr-2-H), 5.13–5.24 (2H, m, Phe-2-H, NH), 6.66–6.72 (2H,
m, Tyr-6,8-H), 6.82–7.00 (2H, m, Tyr-5,9-H), 7.17–7.28
(5H, m, C6H5); 13C NMR (67.8 MHz, CDCl3) d 15.1, 28.2,
31.5, 32.5, 33.7, 39.0, 51.5, 52.2, 59.0, 65.8, 79.9, 115.4,
126.7, 128.3, 129.4, 129.8, 136.1, 155.2, 170.8, 172.5.
To a solution of the above crude amine salt and carboxylic
acid in DMF (5 ml) was added DEPC (0.22 ml, 1.42 mmol)
and Et3N (0.42 ml, 3.05 mmol) at 0 8C. The mixture was
stirred at 0 8C for 1 h, then at room temperature for 18 h.
After dilution with EtOAc, the whole mixture was
successively washed with 1 M aqueous KHSO4, H2O,
saturated aqueous NaHCO3, H2O, and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ1:1) to give 6 (477 mg, 60%) as a
colorless amorphous powder: [a]2D4ZK2.6 (c 1.0, CHCl3);
IR nmax (CHCl3) cmK1 3303, 1741, 1693, 1518, 1228, 1170;
1H NMR (270 MHz, CDCl3) d 1.49 (9H, m, tBu), 1.50–1.85
(5H, m, Phe-3,4-H, OH), 2.57 (3H, s, NMe), 2.68–2.95 (3H,
m, Tyr-3-H, Phe-3-H), 3.20–3.27 (1H, m, Phe-3-H), 3.46–
3.60 (2H, m, Phs-5-H), 3.70 (3H, s, CO2Me), 3.90–4.05 (1H,
m, Phs-2-H), 4.57 (2H, s, OCH2C6H5), 4.80–4.89 (1H, m,
Tyr-2-H), 5.00–5.10 (1H, m, Phe-2-H), 5.50–5.65 (1H, br,
NH), 6.35–6.45 (1H, br, NH), 6.57 (2H, d, JZ8.2 Hz, Tyr-
6,8-H), 6.88 (2H, d, JZ8.2 Hz, Tyr-5,9-H), 7.12–7.36 (10H,
m, C6H5!2); 13C NMR (67.8 MHz, CDCl3) d 15.1, 25.4,
28.2, 30.0, 31.7, 33.6, 38.9, 50.0, 52.1, 54.0, 57.5, 65.7,
70.0, 73.1, 80.1, 115.4, 126.8, 127.1, 127.7, 127.9, 128.0,
128.2, 128.4, 129.4, 129.7, 130.2, 135.7, 137.4, 155.2,
170.7, 170.9, 171.4; Anal. calcd for C37H47N3O8$1/
2EtOAc$ 1/2H2O: C, 65.53; H, 7.33; N, 5.88. Found: C,
65.80; H, 7.07; N, 6.13.
4.1.10. Benzyl (S)-2-tert-butoxycarbonylamino-5-
hydroxypentanoate (18). To a solution of Boc-(S)-Glu-
OBn (17) (4.0 g, 11.8 mmol) in THF (60 ml) was added
Et3N (5.0 ml, 35.5 mmol) and ethyl chloroformate (3.4 ml,
35.5 mmol) at K10 8C under an argon atmosphere. The
mixture was stirred at K10 8C for 1 h, and NaBH4 (1.78 g,
47.2 mmol) in H2O (60 ml) was added dropwise at K10 8C.
The mixture was stirred at K10 8C for 1 h, then at room
temperature for 2 h. The reaction was quenched with 1 M
aqueous KHSO4, and the mixture was extracted with EtOAc
(!3). The extracts were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ1:1) to give 18 (2.86 g, 76%) as a
neat
max
colorless oil: [a]2D4ZK3.9 (c 1.0, CHCl3); IR n
cmK1
3367, 1738, 1713, 1517, 1165; 1H NMR (270 MHz, CDCl3)
d 1.43 (9H, s, tBu), 1.32–1.93 (5H, m, Phs-3,4-H, OH), 3.63
(2H, t, JZ6.1 Hz, Phs-5-H), 4.32–4.45 (1H, m, Phs-2-H),
5.11–5.23 (3H, m, CH2C6H5, NH), 7.35 (5H, s, C6H5); 13
C
NMR (67.8 MHz, CDCl3) d 28.1, 29.1, 53.1, 61.7, 65.7,
66.9, 79.8, 128.3, 135.2, 155.4, 172.5; Anal. calcd for
C17H25NO5: C, 63.14; H, 7.79; N, 4.33. Found: C, 62.96; H,
7.63; N, 4.49.
4.1.11. Benzyl (S)-2-tert-butoxycarbonylamino-5-benzyl-
oxypentanoate (19). To a solution of the pentahomoserine
derivative 18 (1.4 g, 4.31 mmol) in Et2O (14 ml) was added
Ag2O (4.0 g, 17.2 mmol) and BnBr (3.6 ml, 30.1 mmol) at
room temperature under an argon atmosphere. The mixture
was stirred for 14 h. The mixture was filtered through a pad
of celite and the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography
4.1.13. Troc-(S)-Thr[Boc-(S)-Phs(Bn)-(S)-Phe-(S)-N-
Me-Tyr-(S)-Ile]-(S)-Phe-OAllyl (21). Depsipeptide 6
(482 mg, 0.69 mmol) was treated with 4 N HCl–dioxane
(3 ml) at 0 8C. The mixture was stirred at 0 8C for 1 h, then
at room temperature for 2 h. Removal of the solvent under
reduced pressure afforded the crude hydrochloride salt as a
colorless amorphous solid.
(BW-200 MH, hexane–EtOAcZ3:1) to give 19 (1.46 g,
max
neat
82%) as a colorless oil: [a]2D2ZK4.2 (c 1.0, CHCl3); IR n

1472
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
To a solution of tripeptide 6 (409 mg, 0.63 mmol) in THF
(3 ml) was added 0.5 N aqueous LiOH (3 ml) at 0 8C. The
mixture was stirred at 0 8C for 1 h, then at room temperature
for 1 h. After acidification with 1 M aqueous KHSO4, the
mixture was extracted with EtOAc (!3). The extracts were
dried over Na2SO4, concentrated in vacuo to give a crude
carboxylic acid as a colorless oil which was used for the
next step without further purification.
4.40 (3H, m, Phs-2-H, Thr-2-H, Ile-2-H), 4.40–4.70 (6H, m,
OCH2Cl3, OCH2C6H5, CO2CH2CH), 4.70–5.00 (3H, m,
Phe(1)-2-H, Phe(2)-2-H, Tyr-2-H), 5.19–5.30 (3H, m, Thr-
3-H, CO2CH2CHCH2), 5.70–5.90 (1H, m, CO2CH2-
CHCH2), 6.65–6.73 (2H, m, Tyr-6,8-H), 6.93–7.04 (2H,
m, Tyr-5,9-H), 7.15–7.43 (15H, m, C6H5!3); 13C NMR
(67.8 MHz, CDCl3) d K4.72, K4.62, 10.9, 15.2, 18.0, 25.5,
25.7, 28.3, 33.2, 36.6, 37.7, 49.8, 53.9, 58.1, 62.1, 65.8,
69.6, 70.5, 73.1, 74.6, 79.5, 95.3, 118.6, 120.1, 126.8, 126.9,
127.6, 127.7, 128.3, 128.5, 128.7, 129.0, 129.1, 129.6,
130.1, 131.3, 135.7, 136.1, 137.6, 153.8, 154.3, 155.0,
168.4, 169.9, 170.5, 171.6, 172.9; Anal. calcd for C67H91-
Cl3N6O14Si$EtOAc: C, 59.76; H, 6.99; N, 5.89. Found: C,
59.86; H, 6.98; N, 5.62.
To a solution of the above crude amine salt and carboxylic
acid in DMF (4 ml) was added DEPC (0.11 ml, 0.76 mmol)
and Et3N (0.22 ml, 1.58 mmol) at 0 8C. The mixture was
stirred at 0 8C for 1 h, then at room temperature for 14 h.
After dilution with EtOAc, the whole mixture was
successively washed with 1 M aqueous KHSO4, H2O,
saturated aqueous NaHCO3, H2O, and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ1:1) to give 21 (600 mg, 77%) as a
colorless amorphous powder: [a]2D0ZK13.4 (c 1.0, CHCl3);
4.1.15. Cyclo[Troc-(S)-Thr-(S)-Phe-(S)-Phs(Bn)-(S)-
Phe-(S)-N-Me-Tyr(TBS)-(S)-Ile] (2) (entry
6 in
Table 1). To a solution of hexapeptide 4 (2.07 g,
1.54 mmol) in THF (10 ml) was added (PPh3)4Pd (0.18 g,
0.15 mmol) and morpholine (0.20 ml, 2.25 mmol) at room
temperature. The mixture was stirred for 2 h. After dilution
with EtOAc, the whole mixture was washed with 1 M
aqueous KHSO4, and brine, dried over Na2SO4, and
concentrated in vacuo to give a crude carboxylic acid as a
yellow amorphous powder which was used for the next step
without further purification.
1
IR nmax (CHCl3) cmK1 3303, 1741, 1645, 1516, 1243; H
NMR (500 MHz, CDCl3) d 0.81–0.96 (6H, m, Ile-5,6-H),
1.11–1.27 (5H, m, Ile-4-H, Thr-4-H), 1.38, 1.44 (9H, s!2,
tBu), 1.58–1.90 (5H, m, Phs-3,4-H, OH), 1.95–2.06 (1H, br,
Ile-3-H), 2.34–2.56 (1H, m, Tyr-3-H), 2.55–2.85 (5H, m,
Phe(2)-3-H, NMe), 2.91–3.01 (1H, m, Tyr-3-H), 3.04–3.15
(2H, m, Phe(1)-3-H), 3.46–3.55 (2H, m, Phs-5-H), 3.96–
4.05 (1H, br, Phs-2-H), 4.09–4.22 (1H, br, Thr-2-H), 4.37–
4.51 (1H, br, Ile-2-H), 4.54–4.62 (6H, m, OCH2Cl3,
OCH2C6H5, CO2CH2CH), 4.65–4.89 (3H, m, Phe(1)-2-H,
Phe(2)-2-H, Tyr-2-H), 5.20–5.28 (3H, m, Thr-3-H, CO2-
CH2CHCH2), 5.79–5.84 (1H, m, CO2CH2CHCH2), 6.64–
6.73 (2H, m, Tyr-6,8-H), 6.87–6.91 (2H, m, Tyr-5,9-H),
7.11–7.35 (15H, m, C6H5!3); 13C NMR (67.8 MHz,
CDCl3) d 10.8, 14.1, 15.1, 20.9, 25.0, 28.2, 33.0, 35.8,
36.4, 37.5, 37.8, 50.1, 53.2, 53.8, 56.5, 57.3, 57.7, 65.7,
65.8, 69.9, 70.5, 74.5, 79.2, 95.3, 115.4, 118.5, 126.7, 127.5,
127.7, 127.8, 128.1, 128.2, 128.3, 128.6, 128.9, 129.0,
130.0, 131.2, 135.6, 135.8, 137.5, 154.0, 155.0, 169.5,
170.5, 170.9, 171.8, 173.1; Anal. calcd for C61H77Cl3N6-
O14$EtOAc$H2O: C, 58.66; H, 6.59; N, 6.32. Found: C,
58.56; H, 6.38; N, 6.25.
The above carboxylic acid was treated with 4 N HCl–
dioxane (20 ml) at 0 8C for 1.5 h. Removal of the solvent
under reduced pressure afforded the crude hydrochloride
salt as a yellow amorphous powder.
To a solution of the above deprotected hexapeptide in
CH2Cl2 (700 ml) was added DIEA (1.30 ml, 7.50 mmol),
and FDPP (1.15 g, 3.00 mmol) in CH2Cl2 (20 ml) at room
temperature. The mixture was stirred at room temperature
for 17 h, then concentrated. The residue was diluted with
EtOAc, washed with 1 M aqueous KHSO4, saturated
aqueous NaHCO3, and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by
silica gel column chromatography (BW-820 MH, hexane–
EtOAcZ1:1) to give cyclic peptide 2 (1.52 g, 84%) as a
pale yellow amorphous powder: [a]2D4ZK27.1 (c 0.9,
CHCl3); IR nmax (CHCl3) cmK1 3303, 1739, 1645, 1512,
4.1.14. Troc-(S)-Thr[Boc-(S)-Phs(Bn)-(S)-Phe-(S)-N-
Me-Tyr(TBS)-(S)-Ile]-(S)-Phe-OAllyl (4). To a solution
of hexapeptide 21 (960 mg, 0.78 mmol) in DMF (3 ml) was
added imidazole (462 mg, 6.27 mmol) and TBSCl (473 mg,
3.13 mmol) at 0 8C. The mixture was stirred at 0 8C for 1 h,
then at room temperature for 12 h. After dilution with
EtOAc, the whole mixture was successively washed with
1 M aqueous KHSO4, H2O, saturated aqueous NaHCO3,
H2O, and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (BW-820 MH, hexane–EtOAcZ2:1) to
give 4 (788 mg, 75%) as a colorless amorphous powder:
[a]2D0ZK2.6 (c 0.9, CHCl3); IR nmax (CHCl3) cmK1 3302,
1
1259; H NMR (270 MHz, CDCl3) d 0.01, 0.04 (6H, s,
SiMe2), 0.69–0.95 (6H, m, Ile-5,6-H), 0.88, 0.95 (9H, s!2,
tBu), 1.10–1.50 (5H, m, Ile-4-H, Thr-4-H), 1.50–1.90 (5H,
br, Ile-3-H, Phs-3,4-H), 2.08–2.55 (2H, m, Phe(2)-3-H),
2.67, 2.80 (3H, s!2, NMe), 2.90–3.20 (2H, m, Tyr-3-H),
3.30–3.70 (4H, m, Phe(1)-3-H, Phs-5-H), 4.12–4.40 (3H, m,
Phs-2-H, Thr-2-H, Ile-2-H), 4.40–4.60 (4H, m, OCH2Cl3,
OCH2C6H5), 4.60–4.80 (2H, m, Tyr-2-H, Phe(1)-2-H),
4.95–5.05 (1H, m, Phe(2)-2-H), 5.25–5.35 (1H, m, Thr-3-
H), 6.65–6.72 (2H, m, Tyr-6,8-H), 6.88–6.93 (2H, m, Tyr-
5,9-H), 7.01–7.34 (15H, m, C6H5!3); 13C NMR
(67.8 MHz, CDCl3) d K4.7, K4.5, 10.7, 10.9, 14.9, 15.2,
17.9, 25.5, 25.6, 29.9, 30.7, 33.3, 36.5, 50.6, 51.3, 57.0,
57.5, 70.0, 70.4, 72.2, 74.7, 95.3, 120.1, 127.0, 127.2, 127.9,
128.5, 128.6, 128.9, 129.1, 129.7, 129.8, 130.0, 130.2,
135.9, 136.0, 137.6, 154.3, 154.4, 154.8, 168.7, 169.2,
170.2, 171.8, 172.1; Anal. calcd for C59H77N6O11Si$H2O:
C, 59.11; H, 6.64; N, 7.01. Found: C, 58.97; H 6.62; N; 7.01.
1
1741, 1682, 1645, 1510, 1255, 910; H NMR (270 MHz,
CDCl3) d 0.00, 0.10 (6H, s!2, SiMe2), 0.83–0.97 (6H, m,
Ile-5,6-H), 0.86, 0.93 (9H, s!2, Si-tBu), 1.14–1.28 (5H, m,
Ile-4-H, Thr-4-H), 1.36, 1.39 (9H, s!2, O-tBu), 1.60–2.05
(5H, br, Ile-3-H, Phs-3,4-H), 2.20–2.65 (2H, m, Phe(2)-3-
H), 2.70–2.90 (4H, m, Tyr-3-H, NMe), 3.00–3.30 (3H, m,
Tyr-3-H, Phe(1)-3-H), 3.38–3.70 (2H, m, Phs-5-H), 4.08–

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1473
(Entry 1 in Table 1) To a solution of the deprotected peptide
from 4 (70 mg, 0.054 mmol) in DMF (25 ml) was added
DPPA (23 ml, 0.11 mml) in DMF (2.5 ml) and NaHCO3
(45 mg, 0.54 mmol) at 0 8C. The mixture was stirred at 4 8C
for 72 h, then concentrated. The residue was purified as
described above to give 2 (33 mg, 52%).
CO2CH2CHCH2), 5.86–6.00 (1H, m, CO2CH2CHCH2);
13C NMR (67.8 MHz, CDCl3) d 64.0, 66.1, 71.8, 118.8,
131.3, 172.6; Anal. calcd for C6H10O4: C, 49.31; H, 6.90.
Found: C, 49.05; H 6.92.
4.1.17. Allyl (S)-2-hydroxy-3-(tert-butyldimethylsiloxy)-
propanoate (28). To a solution of the allyl propanoate 25
(1.9 g, 13 mmol) in CH2Cl2 (40 ml) was added Et3N
(1.47 ml, 15.6 mmol), DMAP (63 mg, 0.52 mmol), and
TBSCl (2.15 g, 14.3 mmol) at 0 8C. The mixture was stirred
at 0 8C for 2 h, then at room temperature for 11 h. After
dilution with Et2O, the whole mixture was washed with 1 M
aqueous KHSO4, and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (BW-820 MH, hexane–
(Entry 2 in Table 1) To a solution of the deprotected peptide
from 4 (77 mg, 0.056 mmol) in DMF (25 ml) was added
DEPC (17 ml, 0.12 mmol) in DMF (3 ml) and NaHCO3
(47 mg, 0.56 mmol) at 0 8C. The mixture was stirred at 4 8C
for 96 h, then concentrated. The residue was purified as
described above to give 2 (29 mg, 44%).
(Entry 3 in Table 1) To a solution of the deprotected peptide
from 4 (76 mg, 0.058 mmol) in DMF (30 ml) was added
HATU (67 mg, 0.18 mmol) and DIEA (50 ml, 0.29 mmol) at
0 8C. The mixture was stirred at 0 8C for 3 h, and at room
temperature for 10 h, then concentrated. The residue was
purified as described above to give 2 (26 mg, 38%).
EtOAcZ10:1) to give 28 (2.29 g, 68%) as a colorless oil:
[a]2D5ZK7.6 (c 1.0, CHCl3); IR n
cmK1 3496, 2930,
neat
max
1747, 1254, 1128; 1H NMR (270 MHz, CDCl3) d 0.03, 0.05
(6H, s!2, SiMe2), 0.86 (9H, s, tBu), 3.04 (1H, d, JZ7.9 Hz,
OH), 3.83–3.98 (2H, m, CH2OSi), 4.20–4.26 (1H, m,
CHOH), 4.65–4.69 (2H, m, CO2CH2), 5.23–5.38 (2H, m,
CO2CH2CHCH2), 5.85–5.99 (1H, m, CO2CH2CHCH2); 13
C
(Entry 4 in Table 1) To a solution of the deprotected peptide
from 4 (53 mg, 0.04 mmol) in DMF (20 ml) was added
HATU (31 mg, 0.08 mmol) and DIEA (28 ml, 0.16 mmol) at
0 8C. The mixture was stirred at 4 8C for 96 h, then
concentrated. The residue was purified as described above
to give 2 (28 mg, 59%).
NMR (67.8 MHz, CDCl3) d K5.5, 25.6, 65.0, 65.9, 71.9,
118.7, 131.4, 172.3; Anal. calcd for C12H24O4Si: C, 55.35;
H, 9.29. Found: C, 55.17; H, 9.24.
4.1.18. Allyl (S)-2-benzyloxy-3-(tert-butyldimethyl-
siloxy)propanoate (29). To a solution of the 2-hydroxy-
propanoate 28 (1.22 g, 4.70 mmol) in Et2O (20 ml) was
added Ag2O (3.27 g, 14.1 mmol) and BnBr (2.8 ml,
23.5 mmol) at room temperature under argon atmosphere.
The mixture was stirred for 4 h. The mixture was filtered
through a pad of celite and the filtrate was concentrated in
vacuo. The residue was purified by silica gel column
chromatography (BW-200 MH, hexane–Et2OZ20:1) to
(Entry 5 in Table 1) To a solution of the deprotected peptide
from 4 (208 mg, 0.16 mmol) in DMF (80 ml) was added
FDPP (74 mg, 0.19 mmol) in DMF (5 ml) and DIEA
(0.14 ml, 0.80 mmol) at room temperature. The mixture
was stirred at room temperature for 14 h, then concentrated.
The residue was purified as described above to give 2
(107 mg, 57%).
give 29 (1.24 g, 75%) as a colorless oil: [a]2D5ZK37.9 (c
neat
max
1.1, CHCl3); IR n
cmK1 2955, 1751, 1450, 1257, 1132,
4.1.16. Allyl (S)-2,3-dihydroxypropanoate (25). To a
solution of (S)-Ser-OH (24) (4.20 g, 40 mmol) in 0.4 M
hydrochloric acid (200 ml) was added NaNO2 (5.53 g,
80 mmol) in H2O (100 ml) slowly at K10 8C. After
addition, the mixture was allowed to warm to room
temperature and stirred for 24 h, then concentrated. The
residue was filtered to remove inorganic salt. Acetone–
CHCl3 (1:1) was added to the filtrate and the mixture was
concentrated in vacuo. This work-up was repeated three
times to remove H2O completely. The mixture was filtered
through a pad of celite and the filtrate was concentrated in
vacuo to give crude (S)-glyceric acid (9.92 g) as a yellow oil
which was used for the next step without further
purification.
1
837; H NMR (270 MHz, CDCl3) d 0.04 (6H, s, SiMe2),
0.87 (9H, s, tBu), 3.91 (2H, t, JZ5.2 Hz, CH2OSi), 4.08
(1H, dd, JZ4.7, 5.8 Hz, COCH), 4.53 (1H, d, JZ11.8 Hz,
CH2C6H5), 4.62–4.66 (2H, m, CO2CH2), 4.76 (1H, d, JZ
11.9 Hz, CH2C6H5), 5.17–5.38 (2H, m, CO2CH2CHCH2),
5.85–5.99 (1H, m, CO2CH2CHCH2), 7.27–7.38 (5H, m,
C6H5); 13C NMR (67.8 MHz, CDCl3) d K5.4, 18.1, 25.7,
64.1, 65.3, 72.4, 79.4, 118.5, 127.7, 127.9, 128.3, 131.7,
137.4, 170.5; Anal. calcd for C19H30O4Si$1/20 hexane$1/20
Et2O: C, 65.32; H, 8.77. Found: C, 65.68; H, 8.38.
4.1.19. Allyl (S)-2-benzyloxy-3-hydroxypropanoate (30).
To a solution of the protected allyl propanoate 29 (460 mg,
1.31 mmol) in THF (2 ml) was added 1 N TBAF in THF
(2.6 ml, 2.6 mmol) at 0 8C. The mixture was stirred at 0 8C
for 1 h. After dilution with EtOAc, the whole mixture was
washed with H2O and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
To a solution of the above crude carboxylic acid (3.0 g) in
allyl alcohol–CHCl3 (1:2, 40 ml) was added p-TsOH
(114 mg, 0.6 mmol) at room temperature. The mixture
was refluxed for 3 h, then concentrated in vacuo. The
residue was purified by silica gel column chromatography
(BW-820 MH, hexane–EtOAcZ1:2) to give 25 (957 mg,
54% in 2 steps) as a colorless oil: [a]2D5ZK21.1 (c 1.2,
3:1) to give 30 (251 mg, 79%) as a colorless oil: [a]2D5Z
neat
max
K85.1 (c 1.1, CHCl3); IR n
cmK1 3453, 1747, 1454,
neat
max
1
CHCl3); IR n
cmK1 3389, 1740, 1207, 1120; H NMR
1190, 1122; 1H NMR (270 MHz, CDCl3) d 2.10–2.25 (1H,
br, OH), 3.90–3.95 (2H, m, CH2OH), 4.12 (1H, dd, JZ3.6,
5.1 Hz, COCH), 4.52 (1H, d, JZ11.2 Hz, CH2C6H5), 4.69
(2H, d, JZ5.9 Hz, CO2CH2), 4.84 (1H, d, JZ11.6 Hz,
CH2C6H5), 5.24–5.39 (2H, m, CO2CH2CHCH2), 5.86–6.00
(270 MHz, CDCl3) d 2.40–2.70 (1H, br, exchangeable with
D2O, OH), 3.30–3.50 (1H, br, exchangeable with D2O, OH),
3.80–3.95 (2H, br, CH2OH), 4.30 (1H, br, CHOH),
4.70–4.73 (2H, m, CO2CH2), 5.26–5.40 (2H, m,

1474
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
(1H, m, CO2CH2CHCH2), 7.25–7.39 (5H, m, C6H5); 13C
NMR (67.8 MHz, CDCl3) d 63.3, 65.5, 72.6, 78.6, 118.6,
128.0, 128.1, 128.4, 131.5, 136.9, 170.1; Anal. calcd for
C13H16O4$1/5 EtOAc: C, 65.28; H, 6.99. Found: C, 65.49;
H, 6.80.
H), 7.01–7.40 (15H, m, C6H5!3); 13C NMR (67.8 MHz,
CD3OD) d K4.5, K4.4, 11.3, 12.0, 15.1, 15.8, 17.6, 18.8,
26.1, 28.7, 30.4, 37.8, 51.2, 53.0, 56.7, 58.3, 72.8, 73.7,
80.7, 121.2, 127.5, 127.7, 128.5, 128.6, 128.7, 129.3, 129.4,
129.5, 129.9, 130.0, 130.3, 131.6, 137.5, 138.6, 139.6,
155.6, 157.3, 170.4, 171.6, 172.0, 173.2, 174.1, 174.3; Anal.
calcd for C61H84N6O11Si$H2O: C, 65.22; H, 7.72; N, 7.48.
Found: C, 65.61; H 7.64; N; 7.49.
4.1.20. 2-Propenyl (S)-3-[(dibenzyloxyphosphinyl)oxy]-
2-benzyloxypropanoate (3a). To a solution of the
3-hydroxypropanate 30 (20 mg, 0.08 mmol) in CH2Cl2
(1 ml) was added 1H-tetrazole (17 mg, 0.24 mmol) and
N,N-diisopropyl dibenzyl phosphoramidite (31) (5.7 ml,
0.17 mmol) at room temperature. The mixture was stirred
at room temperature for 1 h, then cooled to K30 8C.
m-CPBA (70%, 40 mg, 0.16 mmol) was added and the
mixture was stirred at K30 8C for 40 min. The reaction was
quenched with saturated aqueous NaHCO3 and the mixture
was extracted with Et2O. The extracts were successively
washed with 1 M aqueous KHSO4, H2O, and brine, dried
over MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ10:1 to 5:1 then 3:1) to give 3a
(40 mg, quant.) as a colorless oil: [a]2D2ZK28.4 (c 1.1,
4.1.22. Cyclo[Boc-(S)-Thr-(S)-Phe-(S)-Phs-(S)-Phe-(S)-
N-Me-Tyr(TBS)-(S)-Ile] (33). To a solution of Boc
protected cyclic peptide 32 (330 mg, 0.3 mmol) in EtOAc
(2 ml) was added 20% Pd(OH)2 (100 mg) under an argon
atmosphere. The black slurry was stirred under 1 atom of H2
at room temperature for 3 h. The mixture was filtered
through a pad of celite and the filtrate was concentrated in
vacuo to give 33 (312 mg, quant.) as a colorless amorphous
powder which was used for the next step without further
purification. IR nmax (CHCl3) cmK1 3303, 1730, 1682, 1651,
1
1645, 1634, 1512, 1454, 1257, 1170; H NMR (270 MHz,
CDCl3) d K0.02, 0.04 (6H, s!2, SiMe2), 0.65–0.95 (6H, m,
Ile-5,6-H), 0.87, 0.94 (9H, s!2, Si-tBu), 1.18–1.30 (5H, br,
Ile-3-H, Phs-3,4-H), 1.44 (9H, s, O-tBu), 1.70–2.00 (5H, br,
Ile-3-H, Phs-3,4-H), 2.46–2.57 (2H, m, Phe(2)-3-H), 2.80,
2.85 (3H, s!2, NMe), 2.95–3.20 (2H, m, Tyr-3-H), 3.36–
3.40 (2H, m, Phe(1)-3-H), 3.45–3.48 (2H, br, Phs-5-H),
4.07–4.23 (1H, m, Phs-2-H), 4.25–4.65 (4H, m, Thr-2-H,
Ile-2-H, Phe(1)-2-H, Tyr-2-H), 5.22–5.45 (2H, m, Phe(2)-2-
H, Thr-3-H), 6.66–6.72 (2H, m, Tyr-6,8-H), 6.90–6.98 (2H,
m, Tyr-5,9-H), 7.00–7.35 (10H, m, C6H5!2).
neat
max
1
CHCl3); IR n
cmK1 1755, 1456, 1280, 1136, 1021; H
NMR (270 MHz, CDCl3) d 4.15–4.18 (1H, m, COCH),
4.21–4.36 (2H, m, CHCH2OP), 4.53 (1H, d, JZ11.7 Hz,
CHOCH2C6H5), 4.61–4.64 (2H, m, CO2CH2), 4.76 (1H, d,
JZ11.6 Hz, CHOCH2C6H5), 4.99, 5.02 (4H, s!2,
P(OCH2C6H5)!2), 5.21–5.35 (2H, m, CO2CH2CHCH2),
5.81–5.93 (1H, m, CO2CH2CHCH2), 7.26–7.36 (15H, m,
C6H5!3); 13C NMR (67.8 MHz, CDCl3) d 56.9, 67.2, 69.3,
72.7, 76.8, 119.0, 127.9, 128.0, 128.1, 128.4, 128.5, 131.4,
135.7, 136.9, 168.9; Anal. calcd for C27H29O7P: C, 65.32;
H, 5.89. Found: C, 65.05; H, 5.94.
4.1.23. (S)-20-Benzyloxy-10-cyclo[(S)-Thr-(S)-Phe-(S)-
Phs-(S)-Phe-(S)-N-Me-Tyr(TBS)-(S)-Ile]-propanoate 30-
dibenzyl phosphate (34). Cyclic peptide 33 (30 mg,
30 mmol) was treated with 4 N HCl–dioxane (1 ml) at 0 8C
and the mixture was stirred at 0 8C for 2 h, then at room
temperature for 3 h. Removal of the solvent under reduced
pressure afforded the crude hydrochloride salt as a colorless
solid.
4.1.21. Cyclo[Boc-(S)-Thr-(S)-Phe-(S)-Phs(Bn)-(S)-Phe-
(S)-N-Me-Tyr(TBS)-(S)-Ile] (32). To a solution of cyclic
peptide 2 (610 mg, 0.52 mmol) in THF (3 ml) was added Zn
(900 mg) and AcOH (0.5 ml) at room temperature. The
mixture was stirred for 4.5 h, and filtered through a pad of
celite. The filtrate was concentrated in vacuo to give a crude
amine salt as a colorless solid.
To a solution of phosphate 3a (22 mg, 45 mmol) in THF
(0.3 ml) was added (PPh3)4Pd (5 mg, 4,5 mmol) and
morpholine (6 ml, 68 mmol) at room temperature. The
mixture was stirred for 3.5 h. After dilution with EtOAc,
the whole mixture was washed with 1 M aqueous KHSO4,
and brine, dried over Na2SO4, and concentrated in vacuo to
give a crude carboxylic acid as a yellow oil which was used
for the next step without further purification.
To a solution of the above crude amine salt in THF (3 ml)
was added Boc2O (340 mg, 1.55 mmol) and Et3N (0.22 ml,
1.55 mmol) at 0 8C. The mixture was stirred at 0 8C for 1 h,
then at room temperature for 5 h. After dilution with EtOAc,
the whole mixture was successively washed with 1 M
aqueous KHSO4, H2O, and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
1:1) to give 32 (438 mg, 77%) as a colorless solid:
[a]2D3ZK42.0 (c 0.5, CHCl3); IR nmax (CHCl3) cmK1
To a solution of the above crude amine salt and carboxylic
acid in DMF (0.5 ml) was added DEPC (7 ml, 45 mmol) and
Et3N (10 ml, 75 mmol) at 0 8C. The mixture was stirred at
0 8C for 1 h, then at room temperature for 12 h. After
dilution with EtOAc, the whole mixture was successively
washed with 1 M aqueous KHSO4, H2O, saturated aqueous
NaHCO3, H2O, and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by thin
layer chromatography (hexane–EtOAcZ1:4) to give 34
(21 mg, 52%) as a colorless solid: [a]2D4ZK11.1 (c 1.0,
CHCl3); IR nmax (CHCl3) cmK1 3303, 1738, 1651, 1518,
1
3303, 1732, 1682, 1654, 1645, 1510, 1253, 914; H NMR
(270 MHz, CDCl3) d 0.01, 0.03, 0.07 (6H, s!3, SiMe2),
0.65–0.90 (6H, m, Ile-5,6-H), 0.88, 0.95 (9H, s!2, Si-tBu),
1.15–1.30 (5H, m, Ile-4-H, Thr-4-H), 1.41 (9H, s, O-tBu),
1.55–1.90 (5H, br, Ile-3-H, Phs-3,4-H), 2.35–2.75 (2H, m,
Phe(2)-3-H), 2.66, 2.81 (3H, s!2, NMe), 2.86–3.18 (2H, m,
Tyr-3-H), 3.35–3.50 (2H, m, Phe(1)-3-H), 3.35–3.70 (2H,
m, Phs-5-H), 4.03–4.12 (1H, br, Phs-2-H), 4.30–4.60 (4H,
br, Thr-2-H, Ile-2-H, Phe(1)-2-H, Tyr-2-H), 4.46, 4.48 (2H,
s!2, CH2C6H5), 4.85–5.05 (2H, br, Phe(2)-2-H, Thr-3-H),
6.64–6.72 (2H, m, Tyr-6,8-H), 6.85–6.96 (2H, m, Tyr-5,9-
1
1263, 1020; H NMR (270 MHz, CDCl3) d K0.05, K0.01
(6H, s!2, SiMe2), 0.68–0.98 (6H, m, Ile-5,6-H), 0.87, 0.91
(9H, s!2, Si-tBu), 1.00–1.20 (3H, m, Thr-4-H), 1.20–1.55

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1475
(2H, m, Ile-4-H), 1.60–2.05 (5H, br, Ile-3-H, Phs-3,4-H),
2.50–2.90 (4H, m, Tyr-3-H, Phe(2)-H), 2.86 (3H, s, NMe),
3.00–3.35 (4H, m, Phs-5-H, Phe(1)-3-H), 3.50–3.70 (1H, br,
OH), 3.90–4.20 (2H, br, Phs-2-H, Ga-2-H), 4.20–4.80 (6H,
m, Thr-2-H, Ile-2-H, Phe(1)-2-H, Tyr-2-H, Ga-3-H), 4.52,
4.59 (2H, s!2, CHOCH2C6H5), 4.80-5.15 (6H, m, Phe(2)-
2-H, Thr-3-H, P(OCH2C6H5)!2), 6.67–7.05 (4H, m, Tyr-
5,6,8,9-H), 7.05–7.40 (20H, m, C6H5!4) 7.40–7.70 (5H, m,
C6H5); 13C NMR (67.8 MHz, CD3OD) d K4.4, K4.3, 12.2,
14.5, 15.0, 18.0, 18.9, 20.9, 26.1, 28.5, 29.7, 30.1, 30.5,
34.4, 37.0, 38.2, 51.4, 53.1, 56.2, 58.6, 61.5, 62.1, 63.7,
68.3. 70.7, 70.8, 70.9, 72.8, 74.0, 79.5, 79.7, 121.2, 127.5,
127.7, 129.0, 129.2, 129.3, 129.4, 129.5, 129.9, 130.0,
130.3, 131.0, 131.2, 131.6, 132.2, 136.8, 136.9, 137.6,
137.9, 138.4, 138.7, 155.6, 170.3, 170.6, 170.7, 171.5,
173.4, 174.1, 174.5; HRFABMS (m-nitrobenzyl alcohol)
calcd for C73H93N6O15PSi [MCH]C: 1353.6284. Found:
1353.6309.
4.1.25. 5-Benzyloxy-1-pentanol (38). To a solution of 1,5-
pentanediol (37) (5.2 g, 50 mmol) in THF (100 ml) was
added 15-crown-5-ether (0.3 ml, 3 mmol), finely grinded
NaOH (12 g, 0.3 mol), and BnBr (6.0 ml, 50 mmol) at
10 8C. The mixture was stirred at 10 8C for 4 h. After
dilution with EtOAc, the whole mixture was washed with
brine (!3), dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromato-
graphy (BW-820 MH, hexane–EtOAcZ2:1 to 1:1) to give
38 (4.0 g, 41%) as a colorless oil:1H NMR (270 MHz,
CDCl3) d 1.41–1.67 (7H, m, CH2!3, OH), 3.48 (2H, t, JZ
6.3 Hz, CH2OCH2C6H5), 3.62 (2H, d, JZ6.3 Hz, CH2OH),
4.50 (2H, s, CH2C6H5), 7.25–7.39 (5H, m, C6H5).
4.1.26. N-(5-Benzyloxypentanoyl)-(S)-phenylalanine
methyl ester (41). To a solution of pentanol 38 (2.5 g,
13 mmol) in CH2Cl2 (15 mmol) was added Et3N (9.1 ml,
65 mmol), and pyridine$SO3 (10.3 g, 65 mmol) in DMSO
(15 ml) slowly at 0 8C. The mixture was stirred at 0 8C for
20 min. The reaction was quenched with saturated aqueous
NaHCO3 and the mixture was extracted with Et2O. The
extracts were washed with H2O and brine, dried over
MgSO4, and concentrated in vacuo to give a crude aldehyde
as a colorless oil which was used for the next step without
further purification.
4.1.24. (S)-20-Benzyloxy-10-cyclo[(S)-Thr-(S)-Phe-(S)-
Ahp-(S)-Phe-(S)-N-Me-Tyr(TBS)-(S)-Ile]-propanoate
30-dibenzyl phosphate (44). To a solution of cyclic peptide
34 (35 mg, 26 mmol) in DMSO (0.7 ml) was added IBX
(30 mg, 0.10 mmol) at room temperature. The mixture was
stirred for 3 h. After dilution with EtOAc, the whole mixture
was washed with H2O (!2) and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by
silica gel column chromatography (BW-820 MH, EtOAc
only) to give aldehyde 35 (28 mg, 80%) as a colorless oil.
To a solution of the above crude aldehyde in H2O (7 ml) and
t-BuOH (28 ml) was added 2-methyl-2-butene (6.9 ml,
65 mmol), NaH2PO4 (2.34 g, 19.5 mmol), and NaClO2
(1.76 g, 19.5 mmol) at 0 8C. The mixture was stirred at
0 8C for 1 h. The reaction was quenched with 1 M KHSO4
and the mixture was extracted with EtOAc. The extracts
were washed with brine, dried over Na2SO4, and concen-
trated in vacuo to give a crude carboxylic acid as a colorless
oil which was used for the next step without further
purification.
To a solution of the above aldehyde in THF (1 ml) was
added 1 N TBAF in THF (20 ml) at 0 8C. The mixture was
stirred at 0 8C for 20 min. After dilution with EtOAc, the
whole mixture was washed with H2O and brine, dried over
Na2SO4, and concentrated in vacuo to give 44 (25 mg, 85%)
as a colorless oil: [a]2D7ZK130.1 (c 0.1, CHCl3); IR nmax
(CHCl3) cmK1 3389, 1732, 1675, 1670, 1667, 1651, 1634,
Boc-(S)-Phe-OMe (39) (4.2 g, 15 mmol) was treated with
4 N HCl–dioxane at 0 8C and the mixture was stirred for 2 h.
Removal of the solvent under reduced pressure afforded the
crude hydrochloride salt as a colorless amorphous powder.
1
1517, 1456, 1261, 1020; H NMR (270 MHz, CDCl3) 0.74
(3H, t, JZ6.6 Hz, Ile-5-H), 0.75 (3H, d, JZ7.3 Hz, Ile-6-
H), 0.88–0.95 (1H, m, Ile-4-H), 1.07 (3H, d, JZ6.4 Hz, Thr-
4-H), 1.40–1.55 (1H, m, Ile-4-H), 1.60–1.80 (2H, Ile-3-H,
Ahp-5-H), 1.83–2.00 (2H, m, Ahp-4,5-H), 2.13–2.35 (1H,
m, Tyr-3-H), 2.55–2.63 (1H, m, Ahp-4-H), 2.86 (3H, s,
NMe), 2.88–3.00 (1H, m, Tyr-3-H), 3.05–3.45 (4H, m,
Phe(1)-3-H, Phe(2)-3-H), 3.80 (1H, br, exchangeable with
D2O, OH), 3.85–3.95 (1H, m, Ahp-3-H), 4.00 (1H, br, Ga-2-
H), 4.20–4.35 (2H, m, Ga-3-H), 4.38 (1H, br, exchangeable
with D2O, OH), 4.45 (1H, d, JZ9.9 Hz, Thr-2-H), 4.56 (2H,
s, CHOCH2C6H5), 4.56–4.60 (1H, br, Ile-2-H), 4.78–4.90
(1H, br, Phe(1)-2-H), 4.90–4.95 (1H, br, Tyr-2-H), 5.00,
5.03 (4H, s!2, P(OCH2C6H5)!2), 5.34 (1H, s, Ahp-6-H),
5.20–5.40 (2H, m, Phe(2)-2-H, Thr-3-H), 6.76 (2H, d, JZ
8.4 Hz, Tyr-6,8-H), 7.05 (2H, d, JZ8.3 Hz, Tyr-5,9-H),
7.10–7.34 (25H, m, C6H5!5); 13C NMR (67.8 MHz,
CD3OD) d 11.5, 14.5, 16.5, 18.3, 18.9, 20.9, 26.2, 30.7,
31.6, 34.3, 36.4, 37.4, 38.8, 50.7, 52.5, 56.0, 56.2, 57.4,
61.5, 63.1, 70.7, 70.8, 70.9, 71.0, 74.0, 75.7, 116.6, 127.3,
127.5, 128.8, 129.0, 129.1, 129.2, 129.3, 129.5, 129.6,
130.0, 130.5, 131.7, 136.9, 137.0, 137.5, 138.0, 139.0,
157.5, 170.3, 170.8, 171.1, 172.8, 173.0, 174.5; HRFABMS
(m-nitrobenzyl alcohol) calcd for C67H77N6O15P [MCH]C:
1237.5263. Found: 1237.5337.
To a solution of the above crude carboxylic acid and amine
salt in DMF (40 ml) was added DEPC (2.4 ml, 15.6 mmol)
and Et3N (4.2 ml, 32.5 mmol) at 0 8C. The mixture was
stirred at 0 8C for 1 h, then at room temperature for 2 h.
After dilution with EtOAc, the whole mixture was
successively washed with 1 M KHSO4, H2O, saturated
aqueous NaHCO3, H2O, and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
1
2:1 to 1:1) to give 40 (3.73 g, 80%) as a colorless oil: H
NMR (270 MHz, CDCl3) d 1.58–1.73 (4H, m, CH2!2),
2.19 (2H, t, JZ7.2 Hz, COCH2), 3.00–3.17 (2H, m, Phe-3-
H), 3.46 (2H, t, JZ6.1 Hz, CH2CH2O), 3.71 (3H, s,
CO2Me), 4.47 (2H, s, OCH2C6H5), 4.84–4.92 (1H, m,
Phe-2-H), 5.90–5.95 (1H, br, NH), 7.06–7.33 (10H, m,
C6H5!2).
4.1.27. N-(5-Formylbutanoyl)-(S)-phenylalanine methyl
ester (41). To a solution of 40 (2.5 g, 6.8 mmol) in EtOAc
(20 ml) was added 20% Pd(OH)2 (500 mg) under an argon
atmosphere. The black slurry was stirred under 1 atom of H2

1476
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
at room temperature for 3 h. The mixture was filtered
through a pad of celite and the filtrate was concentrated in
vacuo to give the debenzylated alcohol (2.01 g, quant.) as a
4.1.30. Allyl (S)-2,3-bis(tert-butyldimethylsiloxy)pro-
panoate (26). To a solution of allyl propanoate (25)
(1.55 g, 10.6 mmol) in CH2Cl2 (20 ml) was added 2,6-
lutidine (5.0 ml, 42.4 mmol) and TBSOTf (7.3 g,
31.8 mmol) at 0 8C. The mixture was stirred at 0 8C for
1 h. After dilution with Et2O, the whole mixture was
successively washed with 1 M aqueous KHSO4, H2O, and
brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(BW-820 MH, hexane–Et2OZ15:1) to give 26 (3.78 g,
quant.) as a colorless oil: [a]2D3ZK16.6 (c 1.1, CHCl3); IR
nnmeaaxt cmK1 2955, 2930, 2858, 1759, 1472, 1257, 1148, 1128;
1H NMR (270 MHz, CDCl3) d 0.05 (6H, s, SiMe2), 0.08,
0.09 (6H, s!2, SiMe2), 0.88 (9H, s, tBu), 0.90 (9H, s, tBu),
3.73–3.87 (2H, m, CH2OSi), 4.23 (1H, t, JZ5.3 Hz,
CHOSi), 4.62 (2H, d, JZ5.6 Hz, CO2CH2), 5.22–5.37
(2H, m, CO2CH2CHCH2), 5.85–5.97 (1H, m, CO2CH2-
CHCH2); 13C NMR (67.8 MHz, CDCl3) d K5.3, K4.7,
18.4, 25.8, 65.0, 65.7, 72.7, 118.7, 131.5, 172.3; Anal. calcd
for C12H24O4Si$1/2H2O: C, 56.35; H, 10.25. Found: C,
56.72; H, 10.07.
colorless oil which was used for the next step without
cmK1 3306, 1745, 1649, 1534,
neat
max
further purification: IR n
1
1219; H NMR (270 MHz, CDCl3) d 1.51–1.58 (2H, m,
CH2), 1.64–1.75 (2H, m, CH2), 2.23 (2H, t, JZ7.2 Hz,
COCH2), 2.10–2.30 (1H, br, OH), 3.03–3.19 (2H, m, Phe-3-
H), 3.60 (2H, t, JZ6.1 Hz, CH2OH), 3.73 (3H, s, CO2Me),
4.86–4.93 (1H, m, Phe-2-H), 6.00–6.10 (1H, br, NH), 7.08–
7.31 (5H, m, C6H5).
To a solution of the above alcohol (260 mg, 0.93 mmol) in
CH2Cl2 (1.5 ml) was added Et3N (0.65 ml, 4.65 mmol), and
pyridine$SO3 (740 mg, 4.65 mmol) in DMSO (1.5 ml)
slowly at 0 8C. The mixture was stirred at 0 8C for 1 h.
The reaction was quenched with saturated aqueous NaHCO3
and the mixture was extracted with Et2O. The extracts were
washed with H2O and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
neat
max
1:2) to give 41 (140 mg, 54%) as a colorless oil: IR n
cmK1 3302, 1743, 1724, 1651, 1539, 1217; 1H NMR
(270 MHz, CDCl3) d 1.88–1.96 (2H, m, CH2CH2CH2), 2.22
(2H, t, JZ7.2 Hz, COCH2), 2.45 (2H, t, JZ7.1 Hz,
CH2CHO), 3.10–3.20 (2H, m, Phe-3-H), 3.76 (3H, s,
CO2Me), 4.85–4.92 (1H, m, Phe-2-H), 5.85–5.92 (1H, br,
NH), 7.07–7.32 (5H, m, C6H5), 9.72 (1H, s, CHO).
4.1.31. Allyl (S)-2-(tert-butyldimethylsiloxy)-3-hydroxy-
propanoate (27). To a solution of bis(TBS)propanoate 26
(100 mg, 0.26 mmol) in CH3CN (2.5 ml) was added
CeCl3$7H2O (150 mg, 0.39 mmol) and NaI (40 mg,
0.26 mmol) at room temperature. The mixture was stirred
at room temperature for 15 h, and refluxed for 3 h. The
reaction was quenched with 1 N aqueous HCl, then
concentrated. After dilution with EtOAc, the whole mixture
was washed with H2O and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
5:1) to give 27 (43 mg, 62%) as a colorless oil: IR nnmeaaxt cmK1
4.1.28. Methyl (2S)-2-(6-hydroxy-2-piperidone-1-yl)-3-
phenylpropanoate (42). To a solution of aldehyde 41
(15 mg, 54 mmol) in THF (0.3 ml) was added 0.1 M pH 6
phosphate buffer (0.3 ml) at 0 8C. The mixture was stirred at
0 8C for 30 min, then at room temperature for 20 h. After
dilution with EtOAc, the whole mixture was washed with
H2O and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (BW-820 MH, hexane–EtOAcZ1:1 to
1:3) to give 42 (10 mg, 66%) as a colorless oil: [a]2D2Z
K78.4 (c 0.82, CHCl3); IR nmax (CHCl3) cmK1 3389, 1738,
1629, 1300, 1234; 1H NMR (270 MHz, CDCl3) d 1.45–1.73
(2H, m, piperidone-4-H), 1.86–2.10 (2H, m, piperidone-5-
H), 2.20–2.50 (2H, m, piperidone-3-H), 3.10–3.25 (1H, br,
exchangeable with D2O, OH), 3.30–3.50 (2H, m, Phe-3-H),
3.81 (3H, s, CO2Me), 3.98 (1H, dd, JZ4.7, 9.1 Hz,
piperidine-6-H), 4.07–4.15 (1H, m, Phe-2-H), 7.11–7.31
(5H, m, C6H5); 13C NMR (67.8 MHz, CD3OD) d 16.4, 31.3,
33.1, 35.6, 52.6, 62.4, 83.0, 127.8, 129.6, 130.5, 139.5,
172.5, 172.8; Anal. calcd for C15H19NO4: C, 64.97, H,6.91,
N, 5.05. Found: C, 65.24, H, 6.88, N, 5.02.
1
3496, 2958, 1759, 1615, 1255, 1140; H NMR (270 MHz,
CDCl3) d 0.10, 0.14 (6H, s!2, SiMe2), 0.92 (9H, s, tBu),
2.15–2.24 (1H, br, OH), 3.80–3.86 (2H, br, CH2OH), 4.33
(1H, t, JZ4.6 Hz, CHOSi), 4.65 (2H, d, JZ5.6 Hz,
CO2CH2), 5.24–5.38 (2H, m, CO2CH2CHCH2), 5.85–5.99
(1H, m, CO2CH2CHCH2).
4.1.32. Allyl (S)-2-(tert-butyldimethylsiloxy)propanoate
3-dibenzyl phosphate (3b). To a solution of 3-hydroxy-
propanate 27 (150 mg, 0.57 mmol) in CH2Cl2 (2.0 ml) was
added 1H-tetrazole (120 mg, 1.71 mmol) and N,N-diiso-
propyl dibenzyl phosphoramidite (0.23 ml, 0.69 mmol) at
room temperature. The mixture was stirred at room
temperature for 1.5 h, then cooled K30 8C. m-CPBA
(70%, 280 mg, 1.14 mmol) was added and the mixture
was stirred at K30 8C for 1 h. The reaction was quenched
with saturated aqueous NaHCO3 and the mixture was
extracted with Et2O. The extracts were washed with 1 M
aqueous KHSO4, H2O, and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (BW-820 MH, hexane–EtOAcZ
5:1 to 3:1) to give 3b (303 mg, quant.) as a colorless oil:
[a]2D1ZK12.8 (c 1.2, CHCl3); IR nmax (CHCl3) cmK1 2955,
2930, 1759, 1456, 1283, 1260, 1153, 1018; 1H NMR
(270 MHz, CDCl3) d 0.07, 0.09 (6H, s!2, SiMe2), 0.89
(9H, s, tBu), 4.11–4.18, 4.24–4.32 (2H, m, CH2OP), 4.37–
4.40 (1H, m, CHOSi), 4.60 (2H, d, JZ5.6 Hz, CO2CH2),
5.01, 5.03 (2H, s!2, CH2C6H5), 5.04, 5.06 (2H, s!2,
CH2C6H5), 5.21–5.34 (2H, m, CO2CH2CHCH2), 5.83–5.95
4.1.29. Methyl (2S)-2-(3,4-dihydro-2-pyridone-1-yl)-3-
phenylpropanoate (43). A colorless oil: [a]2D4ZK55.4 (c
0.17, CHCl3); IR nmax (CHCl3) cmK1 1741, 1670, 1378,
1
1215; H NMR (270 MHz, CDCl3) d 2.00–2.50 (4H, m,
pyridone-3,4-H), 2.99–3.08 (1H, m, Phe-3-H), 3.35–3.43
(1H, m, Phe-3-H), 3.73 (3H, s, CO2Me), 5.13–5.17 (1H, m,
Phe-2-H), 5.27–5.34 (1H, m, pyridone-5-H), 6.04 (1H, d,
JZ7.8 Hz, pyridone-6-H), 7.15–7.28 (5H, m, C6H5); 13C
NMR (67.8 MHz, CDCl3) d 19.8, 31.3, 35.8, 52.5, 56.6,
77.2, 106.8, 126.7, 127.0, 128.3, 128.9, 136.4, 159.9, 169.2,
170.7. HRMS calcd for C15H17O3N: 259.1208. Found:
259.1210.

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1477
(1H, m, CO2CH2CHCH2), 7.33 (10H, s, C6H5!2); 13C
NMR (67.8 MHz, CDCl3) d K5.2, K4.9, 18.4, 25.7, 65.9,
68.9, 68.9, 69.3, 69.4, 71.6, 71.7, 118.8, 127.8, 128.3, 128.4,
131.4, 135.5, 135.6, 169.9; Anal. calcd for C26H37O7PSi: C,
59.98; H, 7.16. Found: C, 59.89; H, 7.26.
and concentrated in vacuo. The residue was purified by thin
layer chromatography (CHCl3–MeOHZ10:1) to give
aldehyde (9 mg, 36%) as a colorless solid.
To a solution of the above aldehyde in THF (0.4 ml) was
added 1 N TBAF in THF (20 ml, 20 mmol) at 0 8C. The
mixture was stirred at 0 8C for 1 h, then concentrated.
The residue was purified by thin layer chromatography
(CHCl3–MeOHZ15:1) to give Ahp product 47 (5.5 mg,
95%) as a colorless solid: [a]2D4ZK29.6 (c 0.21, CHCl3); IR
nmax(CHCl3) cmK1 3389, 2930, 1732, 1681, 1651, 1635,
1539, 1456, 1294, 1140, 1022; 1H NMR (270 MHz,
CD3OD) d 0.72–0.95 (6H, m, Ile-5,6-H), 1.18–1.28 (5H,
m, Ile-4-H, Thr-4-H), 1.57–1.90 (4H, m, Ile-3-H, Ahp-4,5-
H), 1.95–2.08 (1H, m, Tyr-3-H), 2.45–2.70 (2H, m, Tyr-3-
H, Ahp-4-H), 2.81 (3H, s, NMe), 2.85–3.05 (2H, m, Phe(2)-
3-H), 3.30–3.56 (2H, m, Phe(1)-3-H), 3.80–3.87 (1H, m,
Ahp-3-H), 4.20–4.25 (3H, br, Ga-2,3-H), 4.47 (1H, d, JZ
5.7 Hz, Ile-2-H), 4.53 (1H, br, Thr-2-H), 4.61–4.70 (1H, m,
Phe(1)-H), 4.95–5.15 (6H, m, Tyr-2-H, Phe(2)-H,
P(OCH2C6H5)!2), 5.15 (1H, br, Ahp-6-H), 5.44–5.53
(1H, br, Thr-3-H), 6.81 (2H, d, JZ8.2 Hz, Tyr-6,8-H),
6.88 (2H, d, JZ6.3 Hz, Tyr-5,9-H), 7.06–7.18 (10H, m,
C6H5!2), 7.35 (10H, s, P(OCH2C6H5)!2); 13C NMR
(67.8 MHz, CD3OD) d 11.4, 16.5, 18.4, 22.4, 26.2, 30.7,
31.7, 34.2, 36.4, 37.3, 38.7, 50.1, 50.7, 50.8, 52.6, 56.1,
57.5, 63.1, 70.7, 70.8, 70.9, 71.0, 72.0, 73.6, 75.7, 116.7,
127.4, 127.5, 128.9, 129.1, 129.3, 129.6, 129.8, 130.3,
130.5, 131.7, 136.9, 137.1, 137.5, 138.9, 157.7, 170.5,
170.7, 171.3, 172.7, 173.0, 174.6; HRFABMS (m-nitro-
benzyl alcohol) calcd for C60H71N6O15P [MCNa]C:
1169.4613. found 1169.4620.
4.1.33. (S)-20-(tert-Butyldimethylsiloxy)-10-cyclo[(S)-
Thr-(S)-Phe-(S)-Phs-(S)-Phe-(S)-N-Me-Tyr(TBS)-(S)-
Ile]-propanoate 30-dibenzyl phosphate (46). Cyclic
peptide 33 (270 mg, 0.27 mmol) was treated with 4 N
HCl–dioxane (2 ml) at 0 8C and the mixture was stirred at
0 8C for 2.5 h. Removal of the solvent under reduced
pressure afforded the crude hydrochloride salt as a colorless
solid.
To a solution of phosphate 3b (208 mg, 0.40 mmol) in THF
(1.5 ml) was added (PPh3)4Pd (46 mg, 0.04 mmol) and
morpholine (52 ml, 0.60 mmol) at room temperature. The
mixture was stirred for 1.5 h. After dilution with EtOAc, the
whole mixture was washed with 1 M aqueous KHSO4 and
brine, dried over Na2SO4, and concentrated in vacuo to give
a crude carboxylic acid as a yellow oil which was used for
the next step without further purification.
To a solution of the above crude amine salt and carboxylic
acid in DMF (2 ml) was added DEPC (62 ml, 0.4 mmol) and
Et3N (93 ml, 0.66 mmol) at 0 8C. The mixture was stirred at
0 8C for 1 h, then at room temperature for 17 h. After
dilution with EtOAc, the whole mixture was successively
washed with 1 M aqueous KHSO4, saturated aqueous
NaHCO3, and brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (hexane–EtOAcZ1:5 to CHCl3–MeOHZ
20:1) to give 46 (211 mg, 57%) as a colorless solid:
[a]2D2ZK20.3 (c 1.0, CHCl3); IR nmax(CHCl3) cmK1 3389,
3238, 2950, 2932, 1738, 1682, 1660, 1645, 1539, 1255,
1020; 1H NMR (270 MHz, CDCl3) d 0.01, 0.08 (12H, s!2,
SiMe2!2), 0.64–0.93 (6H, m, Ile-5,6-H), 0.82, 0.84 (18H,
s!2, tBu!2), 1.03–1.44 (5H, m, Ile-4-H, Thr-4-H), 1.60–
2.00 (5H, m, Ile-3-H, Phs-3,4-H), 2.50–2.95 (4H, m, Tyr-3-
H, Phe(2)-3-H), 2.78, 2.84 (3H, s!2, NMe), 3.05–3.45 (2H,
m, Phe(1)-3-H), 3.50–3.80 (2H, m, Phs-5-H), 4.00–4.80
(7H, m, Phs-2-H, Ga-2-H, Thr-2-H, Ile-2-H, Tyr-2-H, Ga-3-
H), 4.83–5.15 (6H, m, Phe(1)-H, Phe(2)-H,
P(OCH2C6H5)!2), 5.20–5.30 (1H, br, Thr-3-H), 6.68–
6.73 (2H, m, Tyr-6,8-H), 6.83–7.36 (22H, m, Tyr-5,9-H,
C6H5!4); 13C NMR (67.8 MHz, CD3OD) d K5.1, K4.6,
K4.5, K4.3, 12.1, 15.1, 17.9, 18.9, 26.1, 27.4, 29.7, 30.6,
34.4, 38.3, 51.3, 53.1, 56.1, 56.9, 58.6, 62.1, 63.7, 70.8,
70.9, 72.0, 72.8, 121.2, 127.6, 127.7, 127.8, 129.0, 129.2,
129.3, 129.4, 129.5, 129.6, 129.8, 130.1, 130.3, 131.3,
131.6, 136.9, 137.0, 137.7, 138.6, 155.7, 170.4, 170.5,
171.4, 171.5, 171.7, 173.4, 173.6, 174.6; HRFABMS
(m-nitrobenzyl alcohol) calcd for C72H101N6O15PSi2 [MC
H]C: 1377.6679. Found: 1377.6696.
4.1.35. Micropeptin T-20 (1). To a solution of Ahp product
47 (10 mg, 4.3 mmol) in 90% aqueous EtOH (1 ml) was
added 10% Pd–C (10 mg) under an argon atmosphere. The
black slurry was stirred under 1 atom of H2 at room
temperature for 1 h. The mixture was filtered through a pad
of celite and the filtrate was concentrated in vacuo to give a
deprotected product (7.8 mg, 89%) as a pale yellow solid.
The above deprotected product was treated with pH 7
phosphate buffer–MeOH (2:3, 1 ml) and the mixture was
stirred at room temperature for 1 h. After concentration, the
residue was purified by ODS silica gel column chromato-
graphy (BW-300 MH, H2O then MeOH) to give 1 (6.8 mg,
77%) as a pale yellow solid: [a]2D3ZK24.6 (c 0.15,
1
CH3OH); H NMR (500 MHz, CD3OD) d 0.87 (3H, t, JZ
7.4 Hz, Ile-5-H), 0.91 (3H, d, JZ6.7 Hz, Ile-6-H), 1.07–
1.15 (1H, m, Ile-4-H), 1.27 (3H, d, JZ6.4 Hz, Thr-4-H),
1.28–1.35 (1H, m, Ile-4-H), 1.60–1.66, 1.68–1.75 (2H, m,
Ahp-5-H), 1.77–1.89 (2H, m, Ahp-4-H, Ile-3-H), 1.98–2.04
(1H, m, Tyr-3-H), 2.56–2.66 (1H, m, Ahp-4-H), 2.66–2.80
(2H, m, Phe(1)-3-H, Phe(2)-3-H), 2.85 (3H, s, NMe), 2.92–
2.98 (1H, m, Tyr-3-H), 3.36–3.45 (2H, m, Phe(1)-3-H,
Phe(2)-3-H), 3.83 (1H, ddd, JZ6.7, 9.4, 12.2 Hz, Ahp-3-H),
4.00–4.07 (1H, m, Ga-3-H), 4.13–4.18 (2H, m, Ga-2,3-H),
4.53 (1H, br, Thr-2-H), 4.65–4.71 (2H, m, Ile-2-H, Phe(1)-
2-H), 4.90–4.96 (1H, m, Tyr-2-H), 5.03–5.06 (1H, m,
Phe(2)-2-H), 5.16 (1H, br, Ahp-6-H), 5.46–5.52 (1H, m,
Thr-3-H), 6.82 (2H, d, JZ8.5 Hz, Tyr-6,8-H), 6.89 (2H, d,
JZ7.1 Hz, Tyr-5,9-H), 7.08–7.21 (10H, m, C6H5!2); 13C
NMR (500 MHz, CD3OD) d 11.6, 16.7, 18.2, 22.4, 26.2,
4.1.34. (S)-20-Hydroxy-10-cyclo[(S)-Thr-(S)-Phe-(S)-
Ahp-(S)-Phe-(S)-N-Me-Tyr-(S)-Ile]-propanoate 30-
dibenzyl phosphate (47). To a solution of cyclic peptide
46 (25 mg, 18 mmol) in DMSO (0.4 ml) was added IBX
(21 mg, 75 mmol) at room temperature. The mixture was
stirred for 3 h. After dilution with EtOAc, the whole mixture
was washed with H2O (!2) and brine, dried over Na2SO4,

1478
F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
30.6, 31.6, 34.3, 36.4, 37.4, 39.1, 50.8, 52.5, 55.8, 56.1,
57.4, 63.2, 68.3, 73.5, 73.7, 75.8, 116.7, 127.5, 127.7, 129.1,
129.4, 129.5, 130.1, 130.7, 131.8, 137.7, 139.0, 157.7,
190.9, 171.2, 171.5, 172.9, 173.3, 174.3, 174.6; HRFABMS
(m-nitrobenzyl alcohol) calcd for C46H57N6Na2O15P [MC
H]C: 1011.3492. Found: 1011.3533.
cmK1 3474, 2932, 1755, 1615, 1257, 1142; 1H NMR
(270 MHz, CDCl3) d 0.10, 0.14 (6H, s!2, SiMe2), 0.92
(9H, s, tBu), 2.15–2.24 (1H, br, OH), 3.80–3.86 (2H, br,
CH2OH), 4.32 (1H, t, JZ4.6 Hz, CHOSi), 4.65 (2H, d, JZ
5.4 Hz, CO2CH2), 5.24–5.38 (2H, m, CO2CH2CHCH2),
5.85–5.97 (1H, m, CO2CH2CHCH2).
4.1.36. Allyl (R)-2,3-dihydroxypropanoate (49). To a
solution of alcohol 48 (1.0 g, 7.56 mmol) in H2O (30 ml)
was added KOH (1.0 g, 15.1 mmol) in H2O (20 ml) and
KMnO4 (1.8 g, 11.3 mmol) in H2O (30 ml) at 0 8C. The
mixture was stirred at 0 8C for 2 h. The mixture was filtered
through a pad of celite and the filtrate was concentrated in
vacuo. The residue was acidified to pH 4 with 1 M aqueous
KHSO4, salted out, and extracted with EtOAc (!3). The
extracts were dried over Na2SO4, concentrated in vacuo to
give a crude carboxylic acid as a colorless oil which was
used for the next step without further purification.
4.1.39. Allyl (R)-2-(tert-butyldimethylsiloxy)propanoate
3-dibenzyl phosphate (50). Obtained from the above
mono(TBS)propanoate according to the preparation of 3b.
A colorless oil: [a]2D2ZC12.6 (c 0.6, CHCl3); IR nmax
(CHCl3) cmK1 2955, 2930, 1759, 1456, 1283, 1260, 1153,
1
1018; H NMR (270 MHz, CDCl3) d 0.07, 0.09 (6H, s!2,
SiMe2), 0.89 (9H, s, tBu), 4.11–4.18 (1H, m, CH2OP), 4.24–
4.32 (1H, m, CH2OP), 4.37–4.40 (1H, m, CHOSi), 4.60 (2H,
d, JZ5.7 Hz, CO2CH2), 5.01, 5.03 (2H, s!2, CH2C6H5),
5.04, 5.06 (2H, s!2, CH2C6H5), 5.21–5.34 (2H, m,
CO2CH2CHCH2), 5.83–5.95 (1H, m, CO2CH2CHCH2),
7.33 (10H, s, C6H5!2); 13C NMR (67.8 MHz, CDCl3) d
K5.2, K4.9, 18.4, 25.7, 65.9, 68.9, 69.0, 69.3, 69.4, 71.6,
71.7, 118.8, 127.8, 128.3, 128.4, 131.4, 135.5, 135.6, 169.9.
Anal. calcd for C26H37O7PSi$1/2Et2O: C, 60.30; H, 7.59.
Found: C, 60.42; H, 7.45.
To a solution of the above crude carboxylic acid in DMF
(20 ml) was added KHCO3 (1.5 g, 15.0 mmol) and allyl
bromide (1.03 ml, 11.4 mmol) at 0 8C. The mixture was
stirred at 0 8C for 1 h, then at room temperature for 10 h.
After dilution with Et2O, the whole mixture was succes-
sively washed with 1 M aqueous KHSO4, H2O, saturated
aqueous NaHCO3, H2O, and brine, dried over MgSO4, and
concentrated in vacuo to give a crude allyl ester as a
colorless oil which was used for the next step without
further purification.
4.1.40. (R)-20-(tert-Butyldimethylsiloxy)-10-cyclo[(S)-
Thr-(S)-Phe-(S)-Phs-(S)-Phe-(S)-N-Me-Tyr(TBS)-(S)-
Ile]-propanoate 30-dibenzyl phosphate (51). Cyclic
peptide 34 (100 mg, 0.10 mmol) was treated with 4 N
HCl–dioxane (2 ml) at 0 8C and the mixture was stirred at
0 8C for 1 h. Removal of the solvent under reduced pressure
afforded the crude hydrochloride salt as a colorless solid.
The above crude ester was treated with 1 N aqueous HCl–
THF (1:1, 20 ml) at room temperature. The mixture was
stirred for 5.5 h, then concentrated. After dilution with
EtOAc, the whole mixture washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (BW-820
MH, hexane–EtOAcZ1:1) to give 49 (440 mg, 40% in 3
steps) as a colorless oil: [a]2D2ZC21.6 (c 1.1, CHCl3); IR
To a solution of phosphate 50 (80 mg, 0.15 mmol) in THF
(1 ml) was added (PPh3)4Pd (18 mg, 15 mmol) and morpho-
line (20 ml, 0.22 mmol) at room temperature. The mixture
was stirred for 1 h. After dilution with EtOAc, the whole
mixture was washed with 1 M aqueous KHSO4 and brine,
dried over Na2SO4, and concentrated in vacuo to give a
crude carboxylic acid as a yellow oil which was used for the
next step without further purification.
neat
max
1
n
cmK1 3389, 2945, 1748, 1615, 1205, 1120, 1067; H
NMR (270 MHz, CDCl3) d 2.60–2.74 (1H, br, OH), 3.47–
3.57 (1H, br, OH), 3.85–4.00 (2H, m, CH2OH), 4.27–4.37
(1H, m, CHOH), 4.72 (2H, d, JZ5.6 Hz, CO2CH2), 5.26–
5.39 (2H, m, CO2CH2CHCH2), 5.85–6.00 (1H, m, CO2-
CH2CHCH2); 13C NMR (67.8 MHz, CDCl3) d 64.0, 65.5,
71.6, 119.1, 131.1, 172.5; Anal. calcd for C6H10O4$1/6H2O:
C, 48.32; H, 6.98. Found: C, 48.43; H, 7.03.
To a solution of the above crude amine salt and carboxylic
acid in DMF (1 ml) was added DEPC (24 ml, 0.15 mmol)
and Et3N (35 ml, 0.25 mmol) at 0 8C. The mixture was
stirred at 0 8C for 1 h, then at room temperature for 13 h.
After dilution with EtOAc, the whole mixture was
successively washed with 1 M aqueous KHSO4, saturated
aqueous NaHCO3, and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane–EtOAcZ1:5) to give 51
(118 mg, 85%) as a colorless solid: [a]2D2ZK2.9 (c 0.3,
CHCl3); IR nmax(CHCl3) cmK1 3389, 3282, 2957, 2990,
4.1.37. Allyl (R)-2,3-bis(tert-butyldimethylsiloxy)pro-
panoate. Obtained from 49 according to the preparation
neat
max
of 26. A colorless oil: [a]2D3C16.4 (c 1.0, CHCl3); IR n
cmK1 2955, 2930, 2887, 1759, 1472, 1257, 1148, 1128; 1H
NMR (270 MHz, CDCl3) d 0.05 (6H, s, SiMe2), 0.08, 0.09
(6H, s!2, SiMe2), 0.88 (9H, s, tBu), 0.90 (9H, s, tBu), 3.73–
3.87 (2H, m, CH2OSi), 4.29 (1H, t, JZ5.3 Hz, CHOSi),
4.62 (2H, d, JZ5.6 Hz, CO2CH2), 5.22–5.37 (2H, m,
1
1735, 1682, 1649, 1510, 1259, 1021; H NMR (270 MHz,
CDCl3) d K0.06, K0.02 (6H, s!2, SiMe2), 0.10, 0.12 (6H,
s!2, SiMe2), 0.68–0.93 (6H, m, Ile-5,6-H), 0.83, 0.93
(18H, s!2, tBu!2), 1.00–1.44 (5H, m, Ile-4-H, Thr-4-H),
1.70–1.90 (5H, m, Ile-3-H, Phs-3,4-H), 2.55–2.67 (2H, m,
Tyr-3-H), 2.82, 2.86 (3H, s!2, NMe), 2.75–2.96 (2H, m,
Phe(2)-3-H), 3.03–3.28 (2H, m, Phe(1)-3-H), 3.58–3.68
(2H, m, Phs-5-H), 3.95–4.16 (1H, m, Phs-2-H), 4.18–4.33
(1H, m, Ga-2-H), 4.35–4.55 (4H, m, Ga-3-H, Ile-2-H, Thr-
2-H), 4.55–4.65 (1H, m, Phe(1)-2-H), 4.93–5.10 (6H, m,
Tyr-2-H, Phe(2)-2-H, P(OCH2C6H5)!2), 5.20–5.30 (1H,
CO2CH2CHCH2), 5.86–5.97 (1H, m, CO2CH2CHCH2); 13
C
NMR (67.8 MHz, CDCl3) d K5.3, K4.9, 18.4, 25.8, 25.9,
65.4, 66.0, 74.0, 118.4, 131.8, 172.5. Anal. calcd for
C12H24O4Si: C, 57.70; H, 10.22. Found: C, 57.46; H, 10.36.
4.1.38. Allyl (R)-(tert-butyldimethylsiloxy)-3-hydroxy-
propanoate. Obtained from the above bis(TBS)propanoate
according to the preparation of 27. A colorless oil: IR n
neat
max

F. Yokokawa et al. / Tetrahedron 61 (2005) 1459–1480
1479
br, Thr-3-H), 6.66 (2H, d, JZ8.3 Hz, Tyr-6,8-H), 6.94 (2H,
d, JZ8.4 Hz, Tyr-5,9-H), 7.15–7.31 (20H, m, C6H5!4);
13C NMR (67.8 MHz, CD3OD) d K5.0, K4.4, K4.3, 12.4,
14.9, 17.9, 18.3, 18.9, 19.0, 26.1, 26.4, 27.7, 29.8, 30.4,
34.4, 38.2, 38.5, 51.3, 52.9, 56.2, 56.6, 58.5, 62.1, 63.5,
70.8, 70.9, 73.2, 73.9, 121.2, 127.6, 129.1, 129.2, 129.3,
129.4, 129.6, 129.7, 129.9, 130.1, 131.1, 131.6, 133.0,
133.7, 136.9, 137.0, 137.7, 138.4, 155.6, 170.4, 170.5,
171.4, 172.0, 173.6, 174.3, 174.6; FABMS (m-nitrobenzyl
alcohol) m/z 1380 [MCH]C.
(3H, s, NMe), 2.92–2.98 (1H, m, Tyr-3-H), 3.35-3.44 (2H,
m, Phe(1)-3-H, Phe(2)-3-H), 3.80–3.87 (2H, m, Ahp-3-H,
Ga-3-H), 4.08–4.16 (1H, m, Ga-3-H), 4.30–4.33 (1H, m,
Ga-2-H), 4.52 (1H, br, Thr-2-H), 4.65 (1H, d, JZ5.5 Hz,
Ile-2-H), 4.70 (1H, dd, JZ4.2, 10.5 Hz, Phe(1)-2-H), 4.93–
4.96 (1H, m, Tyr-2-H), 5.02–5.07 (1H, m, Phe(2)-2-H), 5.16
(1H, br, Ahp-6-H), 5.46–5.50 (1H, m, Thr-3-H), 6.84 (2H, d,
JZ9.4 Hz, Tyr-6,8-H), 6.89 (2H, d, JZ7.0 Hz, Tyr-5,9-H),
7.09–7.20 (10H, m, C6H5!2); 13C NMR (500 MHz,
CD3OD) d 11.6, 16.6, 18.1, 22.3, 26.3, 30.6, 31.7, 34.2,
36.4, 37.3, 39.0, 50.8, 52.6, 55.8, 56.1, 57.4, 63.2, 68.3,
73.8, 73.9, 75.8, 116.7, 127.6, 127.7, 129.1, 129.4, 129.6,
130.0, 130.6, 131.8, 137.7, 138.9, 157.7, 170.9, 171.2,
171.6, 172.9, 173.3, 174.2, 174.6; FABMS (m-nitrobenzyl
alcohol) m/z 1011 [MCH]C.
4.1.41. (R)-20-Hydroxy-10-cyclo[(S)-Thr-(S)-Phe-(S)-Phs-
(S)-Phe-(S)-N-Me-Tyr(TBS)-(S)-Ile]-propanoate 30-
dibenzyl phosphate (52). To a solution of cyclic peptide
51 (20 mg, 14 mmol) in DMSO (0.4 ml) was added IBX
(16 mg, 58 mmol) at room temperature. The mixture was
stirred for 4 h. After dilution with EtOAc, the whole mixture
was washed with H2O (!2) and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by thin
layer chromatography (CHCl3–MeOHZ10:1) to give
aldehyde (9 mg, 45%) as a colorless solid.
Acknowledgements
This work was financially supported in part by a Grant-in-
Aid for Research at Nagoya City University (to F. Y.),
Uehara Memorial Foundation (to F. Y.), and Grant-in-Aids
from the ministry, Education, Science, Sports and Culture,
Japan. We thank Dr. Kunimitsu Kaya for providing us with
the 1H and 13C NMR spectra of micropeptin T-20. The high
resolution FAB mass spectrum was measured by Dr. Akito
Nagatsu (Nagoya City University), to whom the authors
would like to give thanks.
To a solution of the above aldehyde in THF (0.3 ml) was
added 1 N TBAF in THF (23 ml, 23 mmol) at 0 8C. The
mixture was stirred at 0 8C for 1 h, then concentrated. The
residue was purified by thin layer chromatography (CHCl3–
MeOHZ10:1) to give Ahp product 52 (5.0 mg, 75%) as a
1
colorless solid: H NMR (270 MHz, CD3OD) d 0.85–0.94
(6H, m, Ile-5,6-H), 1.03–1.20 (2H, m, Ile-4-H), 1.25 (3H, d,
JZ6.6 Hz, Thr-4-H), 1.58–1.90 (4H, m, Ile-3-H, Ahp-4,5-
H), 1.92–2.02 (1H, br, Tyr-3-H), 2.55–2.80 (3H, m, Ahp-4-
H, Phe(1)-3-H, Phe(2)-2-H), 2.84 (3H, s, NMe), 2.85–3.02
(1H, m, Tyr-3-H), 3.20–3.40 (2H, m, Phe(1)-3-H, Phe(2)-3-
H), 3.79–3.86 (1H, m, Ahp-3-H), 4.03–4.13, 4.18–4.27 (2H,
m, Ga-3-H), 4.28–4.45 (1H, br, Ga-2-H), 4.52 (1H, br, Thr-
2-H), 4.60 (1H, d, JZ6.0 Hz, Ile-2-H), 4.65–4.70 (1H, m,
Phe(1)-2-H), 4.95–5.13 (6H, m, Tyr-2-H, Phe(2)-2-H,
P(OCH2C6H5)!2), 5.16 (1H, br, Ahp-6-H), 5.46–5.53
(1H, br, Thr-3-H), 6.80 (2H, d, JZ8.4 Hz, Tyr-6,8-H),
6.88 (2H, d, JZ6.4 Hz, Tyr-5,9-H), 6.98–7.25 (10H, m,
C6H5!2), 7.37 (10H, s, P(OCH2C6H5)!2).
References and notes
1. For a few representative examples, see (a) Pettit, G. R.;
Kamano, Y.; Herald, C. L.; Dufresne, C.; Cerny, R. L.; Herald,
D. L.; Schmidt, J. M.; Kizu, H. J. Am. Chem. Soc. 1989, 111,
5015–5017. (b) Okano, T.; Sano, T.; Kaya, K. Tetrahedron
Lett. 1999, 40, 2379–2382 and references therein. (c) Nogle,
L. M.; Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2001,
64, 716–719. For a recent review on peptides from freshwater
cyanobacteria, see (d) Harada, K. Chem. Pharm. Bull. 2004,
52, 889–899. For a review on serine protease inhibitory
peptides derived from cyanobacteria, see (e) Radau, G.
Pharmazie 2000, 55, 555–560.
4.1.42. (20R)-Micropeptin T-20 (53). To a solution of Ahp
product 52 (5.0 mg, 4.3 mmol) in 90% aqueous EtOH
(0.4 ml) was added 10% Pd–C (4 mg) under an argon
atmosphere. The black slurry was stirred under 1 atom of H2
at room temperature for 1 h. The mixture was filtered
through a pad of celite and the filtrate was concentrated in
vacuo to give a deprotected product (4.5 mg, quant.) as a
colorless solid.
2. (a) For a review, see Shioiri, T.; Hamada, Y. Synlett 2001,
148–201. (b) For a recent achievement from our laboratories,
see Yokokawa, F.; Asano, T.; Okino, T.; Gerwick, W. H.;
Shioiri, T. Tetrahedron 2004, 60, 6859–6880.
3. Preliminary accounts of this work were already published:
(a) Yokokawa, F.; Inaizumi, A.; Shioiri, T. Tetrahedron Lett.
2001, 42, 5903–5908. (b) Yokokawa, F.; Inaizumi, A.; Shioiri,
T. Peptide Sci. 2002, 2001, 33–36.
The above deprotected product was treated with pH 7
phosphate buffer–MeOH (2:3, 0.5 ml) and the mixture was
stirred at room temperature for 0.5 h. After concentration,
the residue was purified by ODS silica gel column
chromatography (BW-300 MH, H2O then MeOH) to give
53 (3.5 mg, 81%) as a colorless solid: [a]2D4ZK11.9 (c 0.1,
4. Dehydrodolastatin 13 was also isolated together with
dolastatin 13, see Ref. 19.
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30, 3147–3153 and references cited therein.
1
CH3OH); H NMR (500 MHz, CD3OD) d 0.87 (3H, t, JZ
7.5 Hz, Ile-5-H), 0.91 (3H, d, JZ6.7 Hz, Ile-6-H), 1.07–
1.15 (1H, m, Ile-4-H), 1.24 (3H, d, JZ6.4 Hz, Thr-4-H),
1.30–1.37 (1H, m, Ile-4-H), 1.60–1.90 (5H, m, Ahp-4,5-H,
Ile-3-H), 1.98–2.05 (1H, m, Tyr-3-H), 2.56–2.62 (1H, m,
Ahp-4-H), 2.68–2.79 (2H, m, Phe(1)-3-H, Phe(2)-3-H), 2.85
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