Synthesis of 25-13c-amphotericin B methyl ester: A molecular probe for solid-state NMR measurements

Article abstract of DOI:10.1246/cl.2009.114

A ¹³C-labeled amphotericin B (AmB) derivative was synthesized based on a hybrid strategy combining chemical synthesis with degradation of a natural product through successive cross-coupling reactions and macrolactonization. The specimen regiosp

Full text of DOI:10.1246/cl.2009.114

114
Chemistry Letters Vol.38, No.2 (2009)
Synthesis of 25-13C-Amphotericin B Methyl Ester:
A Molecular Probe for Solid-state NMR Measurements
Naohiro Matsushita, Yukiko Matsuo, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata, and Tohru OishiÃ
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043
(Received November 6, 2008; CL-081048; E-mail: oishi@chem.sci.osaka-u.ac.jp)
A
13C-labeled amphotericin B (AmB) derivative was syn-
Macrolactonization
OH
thesized based on a hybrid strategy combining chemical synthe-
sis with degradation of a natural product through successive
cross-coupling reactions and macrolactonization. The specimen
regiospecifically 13C-labeled (99% enrichment) at C25 position
corresponding to the polyene moiety would be a powerful tool
for structural analysis of the molecular assembly formed by
AmB based on solid-state NMR measurements.
OH
O
37
Me
O
OH
1
41
HO
O
OH OH
OH OH
Me
CO2R
22
39
28
H
O
Me
21
25
40
O
Me
OH
Cross-
coupling
1: R = H (AmB)
2: R = Me, 25-13
C
HO
NH2
Figure 1. Structures of AmB and 25-13C-AmB methyl ester.
Amphotericin B (AmB, 1) is a polyene macrolide antibiotic
produced by Streptomyces nodosus, which has been used as a
standard drug for treatment of deep-seated systemic fungal in-
fections for over 40 years.1 Although it is widely accepted that
AmB associates with sterols in the phospholipid bilayer mem-
brane of the target cell to form barrel–stave type pores,2 details
of the molecular architecture of the ion-channel assembly re-
main unclear.3 During the course of our studies on the structure
of the assembly based on solid-state NMR measurements,4 we
have investigated the interaction between AmB and sterols using
an AmB–sterol conjugate,5 in which the AmB moiety was uni-
formly enriched with 13C (ꢀ50% average labeling index) pre-
pared by fermentation in the presence of U–13C glucose.6 How-
ever, specimens labeled at specific positions are required to esti-
mate the accurate inter-atomic distances between 13C and 19F
nuclei by solid-state NMR. Although it is known that 13C-
enriched AmB selectively labeled at C39, C40, and C41 corre-
sponding to the terminal of the molecule is produced by feeding
with 3-13C-propionate (7–10% labeling index),6 selective incor-
poration of 13C at the middle of the molecule is unachievable by
fermentation. Therefore, chemical synthesis7 would be a practi-
cal way to provide regioselectively labeled specimens with high
labeling index (ꢀ99%), since we have already developed a ver-
satile method for synthesizing the AmB derivative in which H28
was substituted with 19F.8 Herein we report a synthesis of 25-
13C-AmB methyl ester 2 labeled at the central part of the polyene
moiety based on the hybrid synthetic strategy combining chemi-
cal synthesis and degradation of the natural product.
The 13C-labeled derivative 2 was to be constructed by the
Stille coupling of the C1–C21 and C22–C37 segments followed
by macrolactonization (Figure 1). Synthesis of the polyene part
corresponding to the C22–C37 segment 9 commenced with the
Stille coupling of the iodoolefin 38,9 and stannane 410 to afford
phosphonate 5 in 86% yield (Scheme 1). On the other hand,
the Wittig reaction of aldehyde 711 with 13C-labeled ylide 612
prepared from commercially available 2-13C-bromoacetic acid
(99% labeling index), followed by reduction of the resulting
ꢀ,ꢁ-unsaturated ester with DIBAL-H (85%, 2 steps) and the
Dess–Martin oxidation afforded aldehyde 8 (87%). The Horner–
Emmons reaction of 8 with phosphonate 5 resulted in the for-
mation of heptaene 9 as a single isomer (68%).
OEE OTBS
*
I
Ph3P CHCOOEt
Me
Me Me
6
* = 13
C
3
+
O
P
+
Bu3Sn
OMe
OHC
SnBu3
OMe
4
7
a
b-d
Me
TBSO
OEE
Me
O
P
25
OHC
+
SnBu3
OMe
Me
*
8
OMe
5
e
37
Me
TBSO
OEE
Me
22
25
Me
SnBu3
*
9
Scheme 1. Synthesis of the C22–C37 segment 9. Reagents and
.
conditions: a) [Pd2(dba)3] CHCl3, (i-Pr)2NEt, DMF, rt, 86%;
b) toluene, 65 ꢁC; c) DIBAL-H, CH2Cl2, À78 ꢁC, 85% (2 steps);
d) Dess–Martin periodinane, pyridine, CH2Cl2, 0 ꢁC to rt, 87%;
e) LHMDS, THF, 0 ꢁC, 68%.
Synthesis of the C1–C21 segment 10 and coupling with the
C22–C37 segment 9 was carried out as shown in Scheme 2. Deg-
radation of natural AmB8,13 via (i) protection of the amino group
with an Fmoc group and the carboxylic acid as methyl ester
(86%, 2 steps), (ii) selective protection of the 1,3-diols (3,5-
and 9,11-positions) as p-methoxybenzylidene (MP) acetals and
remaining hydroxy groups as TBS ethers (68%, 2 steps), (iii) ex-
haustive ozonolysis of the heptaene moiety (65%) and the subse-
quent Takai olefination14,15 of the resulting aldehyde (63%), and
(iv) selective saponification in the presence of methyl ester and
Copyright Ó 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.2 (2009)
115
acetals by treating with HCl in MeOH (86 mM) at 0 ꢁC for 1 h,
and (iv) hydrolysis of the methyl ketal with aqueous HCl
(86 mM) at 0 ꢁC for 5 h, to afford 25-13C-amphotericin B methyl
ester 2 (53%, 2 steps), which was further purified by HPLC.
The 13C NMR spectra of 2 showed a high intensity signal at
134.23 ppm corresponding to C25.18
AmB (1)
a-h
OTBS
9
OMe
1
11
3
5
HO
OTBS
8
In summary, we have developed a practical method for syn-
thesizing regioselectively 13C-labeled AmB derivatives in high
labeling index. Preparation of AmB derivatives labeled at other
positions and solid-state NMR measurements using 2 are cur-
rently in progress in our laboratory.
O
O
O
O
O
O
CO2Me
H
O
MP
MP
10
I
21
O
Me
MP = p-MeOC6H4
TBSO
OTBS
This work was supported by Grant-in-Aids for Scientific Re-
search (S), and for Scientific Research on Priority Areas from
MEXT, Japan. N.M. expresses his special thanks for The Global
COE Programs of Osaka University.
NHFmoc
i
HO
O
O
OTBS
O
OMe
OTBS
Me
TBSO
OR
O
O
O
O
CO2Me
References and Notes
H
O
Me
MP
MP
1
2
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*
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Me
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11: R = EE
12: R = H
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NHFmoc
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O
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TBSO
CO2Me
H
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4
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MP
*
25
Me
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13
TBSO
OTBS
l-o
NHFmoc
2
Scheme 2. Synthesis of 25-13C-AmB methyl ester 2. Reagents
and conditions: a) Fmoc-OSu, pyridine, DMF, rt; b) CH3I,
Na2CO3, DMF, rt, 86% (2 steps); c) CSA, p-MeOC6H4CH(OMe)2,
MeOH, rt; d) TBSOTf, 2,6-lutidine, CH2Cl2, 0 ꢁC, 68% (2 steps);
e) O3, CH2Cl2, MeOH, À78 ꢁC, 2 h, then PPh3, rt, 65%; f) CrCl2,
CHI3, THF, rt, 63%; g) LiOH, THF, H2O, MeOH, rt; h) Fmoc-
5
6
7
.
OSu, pyridine, DMF, rt, 63% (2 steps); i) 9, [Pd2(dba)3] CHCl3,
Ph3As, (i-Pr)2NEt, THF, rt; j) PPTS, p-MeOC6H4CH(OMe)2,
MeOH, rt; k) 2-methyl-6-nitrobenzoic anhydride, DMAP, CH2Cl2,
rt, 26% (3 steps); l) HFÁpyridine, MeOH, 50 ꢁC; m) piperidine,
MeOH, rt, 64% (2 steps); n) HCl, MeOH, 0 ꢁC, 1 h, quenched with
NaHCO3 (solid); o) solvent exchange to t-BuOH, H2O, then HCl,
0 ꢁC, 5 h, 53% (2 steps); then HPLC purification (16%).
8
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Published on the web (Advance View) December 27, 2008; doi:10.1246/cl.2009.114