Absolute configuration of a polyol fragment of blasticidin A, a specific inhibitor of aflatoxin production

Article abstract of DOI:10.1271/bbb.68.407

Blasticidin A (1) and aflastatin A (2), Streptomyces metabolites with similar structures, are specific inhibitors of aflatoxin production by Aspergillus parasiticus. The stereochemistry of the polyol fragment of 1 (3a) containing ten chiral centers was el

Full text of DOI:10.1271/bbb.68.407

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Absolute Configuration of a Polyol Fragment of
Blasticidin A, a Specific Inhibitor of Aflatoxin
Production
Shohei SAKUDAa, Hiroyuki IKEDAa, Takefumi NAKAMURAa & Hiromichi NAGASAWAa
a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
Sciences, The University of Tokyo
Published online: 22 May 2014.
To cite this article: Shohei SAKUDA, Hiroyuki IKEDA, Takefumi NAKAMURA & Hiromichi NAGASAWA (2004) Absolute
Configuration of a Polyol Fragment of Blasticidin A, a Specific Inhibitor of Aflatoxin Production, Bioscience, Biotechnology,
and Biochemistry, 68:2, 407-412, DOI: 10.1271/bbb.68.407
To link to this article: http://dx.doi.org/10.1271/bbb.68.407
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Biosci. Biotechnol. Biochem., 68 (2), 407–412, 2004
Absolute Configuration of a Polyol Fragment of Blasticidin A,
a Specific Inhibitor of Aflatoxin Production
Shohei SAKUDA,y Hiroyuki IKEDA, Takefumi NAKAMURA, and Hiromichi NAGASAWA
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Received September 10, 2003; Accepted October 10, 2003
Blasticidin A (1) and aflastatin A (2), Streptomyces
metabolites with similar structures, are specific inhib-
itors of aflatoxin production by Aspergillus parasiticus.
The stereochemistry of the polyol fragment of 1 (3a)
containing ten chiral centers was elucidated by applying
acetonide and MTPA methods to a variety of acetonide
derivatives of 3a, which determined the absolute
configuration of 3a. By using the similar methods, the
absolute configuration of the polyol fragment of 2 (4a)
was determined, which was the same as that elucidated
by J-based and other chemical methods previously.
structures, which are tetramic acid derivatives with
long, highly oxygenated alkyl chains (Fig. 1).4–6) We
found that aflastatin A could also inhibit the production
of other polyketide metabolites produced by fungi such
as pentaketide-derived melanin and patulin.7,8) The
detailed molecular mechanism for inhibiting the pro-
duction of aflatoxin or melanin by aflastatin A or
blasticidin A has not been verified, but we have clarified
that aflastatin A inhibited a very early step prior to the
expression of genes encoding the enzymes involved in
the aflatoxin or melanin biosynthetic pathway.8–10)
With respect to the stereochemistry of 1 and 2, we
have chemically assigned the total absolute configura-
tion of 2 and absolute configurations at C4, C6, C31,
C32, C33, C34, C35 and C37 of 1.3,11) To assign the
absolute configuration of 2, we prepared a variety of
fragment molecules and used the following strategy: (1)
the absolute configurations at C50, C4, C6, C33 and C39
were clarified, (2) the relative configurations at the
moieties from C6 to C10, from C10 to C25, from C25 to
C33 and from C33 to C37 were separately assigned, and
(3) by connecting the absolute configurations at C6 and
C33 with the relative configurations from C6 to C37, the
total absolute configuration of 2 was proposed.11) In this
procedure, the relative configuration of the polyol
fragment (4a) was assigned by the J-based method,12)
and this was used for assignment of the relative structure
Key words: blasticidin A; aflastatin A; aflatoxin; Asper-
gillus parasiticus; Streptomyces
Aflatoxins, a group of mycotoxins, are potent carci-
nogens in mammals and can be found as contaminants in
a wide variety of food and feed commodities.1)
A
specific inhibitor of aflatoxin biosynthesis may be a
good candidate for a useful drug to protect foods and
feeds from aflatoxin contamination, and is expected to
depress aflatoxin contamination without incurring rapid
spread of drug-resistant strains. We have recently found
that aflastatin A (2) and blasticidin A (1) showed specific
inhibitory activity toward aflatoxin production by
Aspergillus parasiticus.2,3) These compounds are all
Streptomyces metabolites and have similar unique
Fig. 1. Structures of Blasticidin A (1) and Aflastatin A (2).
y
To whom correspondence should be addressed. Fax: +81-3-5841-8022; E-mail: asakuda@mail.ecc.u-tokyo.ac.jp

408
S. SAKUDA et al.
from C10 to C25 of 2. Therefore, the absolute config-
uration of 4a had not been directly confirmed with the
molecule itself. In this present work, we attempted to
determine the absolute configuration of the polyol
fragment of 1 (3a) by using the acetonide and MTPA
methods to obtain more information on the stereo-
chemistry of 1, and also attempted to confirm the
absolute configuration of 4a by these methods. We
describe the determination of the absolute configuration
of the polyol fragment of 1 (3a) and confirmation of the
stereochemistry of 4a.
HR-FABMS (NBA matrix) m=z 851.5044 (M+Na)þ
(calcd for C51H72O9Na, 851.5074); ꢀH (benzene-d6,
500 MHz) 7.60 (6H, ph), 7.16 (6H, ph), 7.07 (3H, ph),
4.51 (ddd, J ¼ 6, 6, 2 Hz, H-3), 4.21 (H-13), 4.17 (H-
11), 4.13 (H-15), 4.00 (H-9), 3.97 (dd, J ¼ 11:5, 2 Hz,
H-1a), 3.66 (dd, J ¼ 10, 2 Hz, H-7), 3.53 (dd, J ¼ 11:5,
1 Hz, H-1b), 3.49 (H-5), 3.46 (H-17a), 3.27 (H-17b),
2.22 (H-12a), 1.87 (H-8), 1.85 (H-4a), 1.80 (2H, H-16),
1.77 (H-10a), 1.77 (H-6), 1.76 (H-4b), 1.69 (H-12b),
1.41 (H-14a), 1.40 (H-10b), 1.31 (H-2), 1.27 (d,
J ¼ 7 Hz, H-20), 1.25 (H-14b), 1.19 (d, J ¼ 7 Hz, H-
18), 0.60 (d, J ¼ 6 Hz, H-19), 1.59, 1.55, 1.54, 1.54,
1.43, 1.40, 1.37. 1.33 (CH3 of isopropylidene moieties);
ꢀC (benzene-d6, 125 MHz) 73.0 (C7), 71.8 (C5), 71.7
(C9), 67.8 (C3), 66.8 (C1), 66.4 (C15), 65.6 (C13 or
C11), 65.5 (C11 or C13)), 59.9 (C17), 44.1 (C12), 39.8
(C8), 37.6 (C14), 37.6 (C16 or C4), 36.4 (C4 or C16),
35.1 (C6), 35.0 (C10), 31.5 (C2), 11.5 (C19), 10.9
(C18), 9.6 (C20), 145:0 ꢀ 3, 129:1 ꢀ 6, 127:9 ꢀ 6,
127:1 ꢀ 3, 87.0 (trityl moiety), 98:5 ꢀ 4 (quaternary
carbons of isopropylidene moieties), 30:7 ꢀ 2, 30.5,
30.3, 20:0 ꢀ 2, 19.6, 19.1 (CH3 of isopropylidene
moieties). Signals were assigned by 2D NMR.
JH-1a,H-2, JH-1b,H-2 and JH-6,H-7 values were determined
by decoupling experiments.
Materials and Methods
Preparation of 5 and 6. Trityl chloride (20 mg) and a
catalytic amount of 4-dimethylaminopyridine were
added to a solution of 3a (10.0 mg) in a mixture of dry
CH3CN (0.9 ml) and dry pyridine (0.6 ml), and the
mixture was stirred at room temperature for 24 h. The
reaction mixture was purified by reverse-phase HPLC
(column: Capcell Pak C18, 10 ꢀ 250 mm, Shiseido;
mobile phase: gradient elution of 20–90% CH3CN in
water in 30 min; flow rate: 5 ml/min) to afford 3b
(2.5 mg) and 3c (1.9 mg). 3b and 3c: FABMS (NBA
matrix) m=z 691 (M+Na)þ. Dimethoxypropane (0.5 ml)
and a trace amount of p-TsOH were added to a solution
of 3b (2.5 mg) in dry DMF (0.5 ml), and the mixture was
stirred at ꢁ20 ꢂC for 4 h. After adding triethylamine
(0.5 ml) to the solution, the reaction mixture was
purified by reverse-phase HPLC under the same con-
ditions as those used for the isolation of 3b and 3c to
afford 5 (2.2 mg). Compound 3c (1.9 mg) was converted
to 6 (1.5 mg) by using the same procedure. 5: FABMS
(NBA matrix) m=z 851 (M+Na)þ; HR-FABMS (NBA
matrix) m=z 851.5103 (M+Na)þ (calcd for
C51H72O9Na, 851.5074); ꢀH (benzene-d6, 500 MHz)
7.62 (6H, ph), 7.18 (6H, ph), 7.09 (3H, ph), 4.42 (ddd,
J ¼ 12, 3, 3 Hz, H-5), 4.23 (H-9), 4.09 (H-15), 4.09 (H-
13), 4.07 (H-11), 3.98 (ddd, J ¼ 11, 5, 2 Hz, H-3), 3.73
(dd, J ¼ 10, 2 Hz, H-7), 3.73 (2H, H-17), 3.37 (dd,
J ¼ 10, 7 Hz, H-1a), 3.25 (dd, J ¼ 10, 5 Hz, H-1b), 2.22
(H-6), 2.15 (H-10a), 2.11 (H-14a), 2.05 (H-2), 1.70 (H-
4a), 1.59 (H-16a), 1.58 (H-10b), 1.58 (H-14b), 1.45 (H-
12a), 1.37 (H-8), 1.36 (H-4b), 1.34 (H-12b), 1.14 (H-
16b), 1.12 (d, J ¼ 7 Hz, H-18), 1.00 (d, J ¼ 7 Hz, H-20),
0.92 (d, J ¼ 7 Hz, H-19), 1.59, 1.58, 1.55, 1.54, 1.41,
1.36. 1.34. 1.33 (CH3 of isopropylidene moieties); ꢀC
(benzene-d6, 125 MHz) 73.9 (C7), 70.9 (C3), 69.7 (C9),
69.2 (C5), 66.1 (C1), 66.0 (C11), 65.5 (C13), 65.5
(C15), 59.8 (C17), 43.6 (C14), 40.0 (C2), 40.0 (C10),
38.5 (C6), 37.1 (C12), 32.8 (C8), 31.5 (C16), 29.1 (C4),
13.1 (C18), 9.6 (C19), 5.0 (C20), 145:1 ꢀ 3, 129:2 ꢀ 6,
128:0 ꢀ 6, 127:2 ꢀ 3, 87.1 (trityl moiety), 98:5 ꢀ 4
(quaternary carbons of isopropylidene moieties),
30:7 ꢀ 2, 30:5 ꢀ 2, 19:9 ꢀ 3, 19.3 (CH3 of isopropyli-
dene moieties). Signals were assigned by 2D NMR, and
the JH-7,H-8 value was determined by decoupling experi-
ments. 6: FABMS (NBA matrix) m=z 851 (M+Na)þ;
Preparation of 7a, 7b and 7c. Acetonidation of 3a
was carried out by the same procedure as that used for
preparing 5 from 3b to afford tetraacetonide derivatives
of 3a. The crude reaction mixture was dissolved in
dichloromethane (0.5 ml), and 4-dimethylaminopyridine
(150 mg) and acetic anhydride (0.1 ml) were added to
the solution. After being stirred for 18 h at room
temperature, the reaction mixture was purified by HPLC
(column: Capcell Pak C18, 10 ꢀ 250 mm, Shiseido;
mobile phase: gradient elution of 30–100% CH3CN in
water in 30 min; flow rate: 5 ml/min) to afford 7a
(2.0 mg). 7a: FABMS (NBA matrix) m=z 629 (M+H)þ;
HR-FABMS (NBA matrix) m=z 629.4251 (M+H)þ
(calcd for C34H61O10, 629.4265); ꢀH (benzene-d6,
500 MHz) 5.45 (H-9), 4.45 (H-3), 4.07 (H-13), 4.02
(H-15), 4.02 (H-11), 3.91 (dd, J ¼ 11, 2.5 Hz, H-1a),
3.68 (H-7), 3.66 (2H, H-17), 3.48 (b.d, J ¼ 11:5 Hz, H-
1b), 3.43 (H-5), 2.04 (H-10a), 2.02 (H-14a), 1.90 (H-8),
1.78 (H-4a), 1.78 (H-10b), 1.69 (H-4b), 1.63 (H-12a),
1.62 (H-6), 1.52 (H-14b), 1.51 (H-16a), 1.29 (H-12b),
1.25 (H-2), 1.14 (d, J ¼ 7 Hz, H-18), 1.03 (d, J ¼ 7 Hz,
H-20), 1.03 (H-16b), 0.55 (d, J ¼ 7 Hz, H-19), 1.83
(Ac), 1.53, 1.52, 1.50, 1.43, 1:36 ꢀ 2, 1.35, 1.31 (CH3 of
isopropylidene moieties); ꢀC (benzene-d6, 125 MHz)
75.0 (C7), 73.8 (C9), 71.6 (C5), 67.7 (C3), 67.1 (C11),
66.8 (C1), 65.4 (C15), 59.8 (C17), 43.5 (C14), 39.4
(C10), 37.7 (C8), 37.5 (C12), 36.4 (C4), 35.2 (C6), 31.4
(C2), 31.4 (C16), 11.6 (C19), 10.8 (C18), 8.7 (C20),
169.9 (Ac), 21.0 (Ac), 98:5 ꢀ 3, 97.8 (quaternary
carbons of isopropylidene moieties), 31.4, 30:7 ꢀ 2,
30.4, 19.7, 19:3 ꢀ 2, 19.1 (CH3 of isopropylidene
moieties). Signals were assigned by 2D NMR.

Absolute Configuration of a Polyol Fragment of Blasticidin A
409
To a solution of 7a (2.0 mg) in absolute MeOH, a
small piece of Na was added, and the mixture was stirred
at room temperature for 5 h. Dichloromethane (20 ml)
was added to the reaction mixture, and the solution was
washed with water, dried, and evaporated to afford a
deacetylated product (1.2 mg). A half of the product
(0.6 mg) was dissolved in dry dichloromethane (0.5 ml),
and 4-dimethylaminopyridine (6 mg), triethylamine
(3 ꢁl) and (S)-(+)-ꢂ-methoxy-ꢂ-(trifluoromethyl)phen-
ylacetyl chloride (MTPA-Cl, 5 ꢁl) were added to the
solution. After being stirred at room temperature for 5 h,
3-dimethylaminopropylamine (3.2 ꢁl) was added to the
reaction mixture, and the solution was stirred for a
further 10 min. Dichloromethane (20 ml) was added to
the reaction mixture, and the solution was washed with
water, dried, evaporated, and purified by HPLC (col-
umn: Capcell Pak C18, 10 ꢀ 250 mm, Shiseido; mobile
phase: gradient elution of 50–100% CH3CN in water in
20 min; flow rate: 4 ml/min) to afford an (R)-MTPA
ester (7b, 0.3 mg). By the same procedure, the (S)-
MTPA ester (7c, 0.3 mg) was prepared with (R)-(ꢁ)-
MTPA-Cl from deacetylated 7a (0.6 mg). 7b: FABMS
(NBA matrix) m=z 803 (M+H)þ; HR-FABMS (NBA
matrix) m=z 803.4556 (M+H)þ (calcd for C42H66F3O11,
803.4557); ꢀH (CDCl3, 500 MHz) 7.56 (2H, ph), 7.41
(3H, ph), 5.17 (H-9), 4.24 (ddd, J ¼ 8, 6, 2 Hz, H-3),
4.12 (dd, J ¼ 11:5, 2.5 Hz, H-1a), 4.02 (H-15), 3.98 (dt,
J ¼ 12, 3 Hz, H-17a), 3.85 (H-13), 3.84 (H-17b), 3.72
(H-7), 3.58 (dd, J ¼ 11:5, 1 Hz, H-1b), 3.58 (3H,
OCH3), 3.54 (H-11), 3.46 (H-5), 2.03 (H-8), 1.82 (H-
10a), 1.77 (H-14a), 1.69 (H-10b), 1.66 (H-4a), 1.61 (H-
16a), 1.58 (H-4b), 1.51 (H-6), 1.44 (H-12a), 1.41 (H-
14b), 1.41 (H-16b), 1.41 (H-2), 1.00 (H-12b), 1.05 (d,
J ¼ 7 Hz, H-18), 0.87 (d, J ¼ 7 Hz, H-20), 0.68 (d,
J ¼ 7 Hz, H-19), 1.48, 1.44, 1.39, 1:36 ꢀ 2, 1.31, 1.26,
1.13 (CH3 of isopropylidene moieties). Signals were
assigned by the DQF-COSY spectrum. 7c: FABMS
(NBA matrix) m=z 803 (M+H)þ; HR-FABMS (NBA
matrix) m=z 803.4565 (M+H)þ (calcd for C42H66F3O11,
803.4557); ꢀH (CDCl3, 500 MHz) 7.60 (2H, ph), 7.40
(3H, ph), 5.17 (H-9), 4.23 (ddd, J ¼ 8, 6, 2 Hz, H-3),
4.12 (dd, J ¼ 11:5, 2.5 Hz, H-1a), 4.02 (H-15), 3.97 (dt,
J ¼ 12, 3 Hz, H-17a), 3.91 (H-13), 3.83 (H-17b), 3.79
(H-11), 3.65 (dd, J ¼ 10:5, 2 Hz, H-7), 3.58 (dd,
J ¼ 11:5, 1 Hz, H-1b), 3.53 (3H, OCH3), 3.43 (H-5),
1.99 (H-8), 1.84 (H-10a), 1.77 (H-14a), 1.77 (H-10b),
1.64 (H-4a), 1.60 (H-16a), 1.58 (H-4b), 1.51 (H-12a),
1.49 (H-6), 1.43 (H-14b), 1.42 (H-16b), 1.40 (H-2), 1.06
(H-12b), 1.05 (d, J ¼ 7 Hz, H-18), 0.78 (d, J ¼ 7 Hz, H-
20), 0.62 (d, J ¼ 7 Hz, H-19), 1.44, 1.43, 1.36, 1.35,
1.34, 1.32, 1.30, 1.29 (CH3 of isopropylidene moieties).
Signals were assigned by the DQF-COSY spectrum.
Trityl chloride (20 mg) and 4-dimethylaminopyridine
(3 mg) were added to a solution of 4a (9.6 mg) in a
mixture of dry CH3CN (0.9 ml) and dry pyridine
(0.6 ml), and the mixture was stirred at room temper-
ature for 48 h. The reaction mixture was purified by
reverse-phase HPLC (column: Capcell Pak C18,
10 ꢀ 250 mm, Shiseido; mobile phase: gradient elution
of 10–90% CH3CN in water in 30 min; flow rate: 5 ml/
min) to afford 4b (1.4 mg), 4c (1.4 mg) and 4d (0.3 mg).
4b and 4c: FABMS (NBA matrix) m=z 741 (M+H)þ.
4d: FABMS (NBA matrix) m=z 983 (M+H)þ. Com-
pound 4b was tritylated again by the same procedure to
obtain a further amount of 4d, which was converted to
the tetraacetonide (9, 0.9 mg) by the same procedure as
that described for the preparation of 5. 9: FABMS (NBA
matrix) m=z 1143 (M+H)þ; HR-ESIMS m=z 1143.6945
(M+H)þ (calcd for C74H95O10, 1143.6925); ꢀH (ben-
zene-d6, 500 MHz) 7.60 (12H, ph), 7.13 (12H, ph), 7.04
(6H, ph), 4.52 (H-9), 4.23 (H-13), 4.10 (H-17), 4.03 (H-
15), 4.01 (H-7), 4.00 (H-3), 3.82 (dd, J ¼ 9, 2 Hz, H-5),
3.80 (dd, J ¼ 9, 2 Hz, H-11), 3.46 (H-19a), 3.45 (t,
J ¼ 9 Hz, H-1a), 3.25 (H-19b), 3.22 (dd. J ¼ 9, 6, Hz,
H-1b), 2.29 (H-10), 2.08 (H-14a), 2.02 (H-2), 1.92 (H-
6), 1.83 (H-4), 1.80 (H-18), 1.55 (H-8), 1.51 (H-14b),
1.38 (H-12), 1.27 (d, J ¼ 7 Hz, H-22), 0.99 (d, J ¼ 7 Hz,
H-23), 0.99 (d, J ¼ 7 Hz, H-24), 0.87 (d, J ¼ 7 Hz, H-
20), 0.62 (d, J ¼ 7 Hz, H-21), 1.58, 1.55, 1.475, 1.465,
1:45 ꢀ 2, 1.35, 1.34 (CH3 of isopropylidene moieties);
ꢀC (benzene-d6, 125 MHz) 74.1, 74.0, 73.5, 72.0, 70.3,
69.2, 66.8, 66.3, 66.3, 60.2, 40.5, 40.3, 39.4, 38.0, 38.0,
35.4, 33.3, 32.2, 28.7, 11.8, 10.4, 10.1, 9.9, 5.4, 145.5
(trityl), 145.4 (trityl), 129.6 (trityl), 128.8 (trityl), 127.6
(trityl), 87.3 (trityl), 87.3 (trityl), 99.2, 99.0, 98.9, 98.1
(quaternary carbons of isopropylidene moieties), 31.2,
31.0, 30.8, 30.8, 20.6, 20.5, 20.4, 20.2 (CH3 of
isopropylidene moieties). Proton signals were assigned
by the DQF-COSY spectrum. JH-3,H-4, JH-4,H-5 and
JH-11,H-12 values were determined by decoupling experi-
ments. Carbon signals were not assigned by 2D NMR.
Acetonidation of 4c with dimethoxypropane by the
same procedure as that used for preparing 5 mainly
produced the tetraacetonide of 4c (10a, 0.7 mg). 10a:
FABMS (NBA matrix) m=z 901 (M+H)þ. Compound
10a was converted to (R)- and (S)-MTPA esters (10b,
0.15 mg, and 10c, 0.12 mg) by the same procedure as
that used for preparing 7b and 7c. 10b: FABMS (NBA
matrix) m=z 1117 (M+H)þ; HR-FABMS (NBA matrix)
m=z 875.5134 (M-trityl+2H)þ (calcd for C46H74F3O12,
875.5132); ꢀH (CDCl3, 500 MHz) 7.20–7.58 (20H, ph),
5.38 (dd, J ¼ 6, 3 Hz, H-5), 4.15 (H-9), 4.11 (H-17),
4.00 (dd, J ¼ 11, 2 Hz, H-1a), 4.00 (H-13), 3.93 (H-15),
3.83 (H-7), 3.69 (dd, J ¼ 10, 2 Hz, H-3), 3.60 (dd,
J ¼ 10, 2 Hz, H-11), 3.58 (dd, J ¼ 11, 1 Hz, H-1b), 3.50
(3H, OCH3), 3.24 (H-19a), 3.06 (H-19b), 2.11 (H-4),
1.92 (H-10), 1.90 (H-6), 1.80 (H-14a), 1.71 (H-18), 1.46
(H-16a), 1.38 (H-2), 1.37 (H-14b), 1.35 (H-12), 1.33 (H-
8a), 1.18 (H-8b), 1.14 (H-16b), 1.04 (d, J ¼ 7 Hz, H-20),
0.84 (d, J ¼ 7 Hz, H-24), 0.83 (d, J ¼ 7 Hz, H-22), 0.79
Preparation of 8, 9, 10b and 10c. The preparation of 8
has been reported previously.11) Since the J values
(JH-1a,H-2, JH-1b,H-2, JH-5,H-6 and JH-10,H-11) for 8 had not
been determined, decoupling experiments were done to
determine them in this study.

410
S. SAKUDA et al.
(d, J ¼ 7 Hz, H-23), 0.79 (d, J ¼ 7 Hz, H-21), 1.41,
1.35, 1:345 ꢀ 2, 1.34, 1.33, 1.325, 1.30 (CH3 of
isopropylidene moieties). Signals were assigned by the
DQF-COSY spectrum. 10c: FABMS (NBA matrix) m=z
1117 (M+H)þ; HR-FABMS (NBA matrix) m=z
875.5153 (M-trityl+2H)þ (calcd for C46H74F3O12,
875.5132); ꢀH (CDCl3, 500 MHz) 7.20–7.62 (20H, ph),
5.38 (dd, J ¼ 6, 3 Hz, H-5), 4.15 (H-9), 4.11 (H-17),
4.00 (H-1a), 3.99 (H-13), 3.96 (H-7), 3.93 (H-15), 3.71
(dd, J ¼ 10, 2 Hz, H-3), 3.59 (H-11), 3.58 (H-1b), 3.57
(3H, OCH3), 3.24 (H-19a), 3.06 (H-19b), 2.07 (H-4),
1.94 (H-6), 1.92 (H-10), 1.80 (H-14a), 1.71 (H-18), 1.46
(H-16a), 1.40 (H-8a), 1.37 (H-14b), 1.36 (H-2), 1.35 (H-
12), 1.14 (H-16b), 1.10 (H-8b), 1.03 (d, J ¼ 7 Hz, H-
20), 0.88 (d, J ¼ 7 Hz, H-22), 0.84 (d, J ¼ 7 Hz, H-24),
0.80 (d, J ¼ 7 Hz, H-23), 0.70 (d, J ¼ 7 Hz, H-21), 1.41,
1.39, 1.35, 1:33 ꢀ 2, 1.32, 1.315, 1.29 (CH3 of isopro-
pylidene moieties). Signals were assigned by the DQF-
COSY spectrum.
Tetraacetonides 5 and 6 were next used for determin-
ing the relative configurations of C2-C3, C6-C7 and C8-
C9 of 3a. The conformation of the acetonide ring
containing the C2-C3 moiety in 6 was confirmed as a
chair form, as already mentioned. Therefore, two small J
values (JH-1a,H-2 ¼ 1 Hz, JH-1b,H-2 ¼ 2 Hz) in the ring
system indicated the axial orientation of the methyl
group at C2, showing a threo relationship for C2-C3 in
3a (Fig. 2A). Similarly, the stereochemistry of the
acetonide ring containing C6-C7 in 6 and that containing
Results and Discussion
Absolute configuration of the polyol fragment of
blasticidin A
The polyol fragment of 1 (3a), which contained ten
chiral centers from C10 to C23 of 1, was prepared from
1 by NaIO4 oxidation, followed by NaBH4 reduction, as
reported previously.5) Since 3a had a typical structure
consisting of 1,3-diol systems, we applied the acetonide
method13) to analyze the relative stereochemistry of 3a.
To obtain the desired tetraacetonide derivatives of 3a
selectively, 3a was treated with trityl chloride to protect
a primary hydroxyl group at C1 or C17. Obtained 3b and
3c were respectively converted to 1- and 17-O-tritylte-
traacetonides (5 and 6) with dimethoxypropane in the
presence of p-TsOH. In the 13C NMR spectra of 5 and 6,
all signals of methyl carbons of the isopropylidene
moieties were observed around 20 ppm or 30 ppm, and
signals of the quaternary carbons of the moieties were
observed around 99 ppm. This indicates that each of the
6-membered acetonide rings in 5 and 6 was present in a
chair form,14) showing a syn relationship of each of the
corresponding 1,3-diol systems.
C8-C9 in 5 were confirmed from the J values [JH-6,H-7
¼
10 Hz in 6 (Fig. 2B) and JH-7,H-8 ¼ 2 Hz in 5 (Fig. 2C)].
Thus, the relative configurations for C6-C7 and C8-C9
of 3a were assigned as erythro and threo, respectively.
The total relative configuration of 3a was determined
from the results obtained.
When 3a was treated with dimethoxypropane in the
presence of p-TsOH, followed by acetylation, the 9-O-
acetyltetraacetonide of 3a (7a) was mainly obtained. To
determine the absolute configuration at C9 of 3a by
applying the MTPA [2-methoxy-2-phenyl-2-(trifluoro-
methyl)acetic acid] method,15) 7a was deacetylated and
converted to the (R)- and (S)-MTPA esters (7b and 7c).
1
The H NMR spectra of 7b and 7c were measured, and
ꢀꢀ (¼ ꢀS ꢁ ꢀR) values were obtained. As shown in
Figure 3, the pattern for the distribution of positive and
negative ꢀꢀ values on the molecule was typical for the
case of C9 with an absolute configuration of R. By
connecting this absolute configuration to the relative
configuration of 3a, the total absolute configuration of
Fig. 2. Relative Stereochemistry of the Acetonide Rings in 5, 6, 8 and 9.

Absolute Configuration of a Polyol Fragment of Blasticidin A
411
By mono-tritylation of 4a, followed by acetonidation,
the 19-trityltetraacetonide (10a) was mainly obtained.
This tetraacetonide was converted to (R)- and (S)-MTPA
esters (10b and 10c) and their 1H NMR spectra were
measured. The ꢀꢀ (¼ ꢀS ꢁ ꢀR) values obtained (Fig. 3)
indicated the 5R configuration, which confirmed the
total absolute configuration of 4a.
The absolute configurations at C4, C6, C31, C32,
C33, C34, C35 and C37 of 1 have been found to be the
same as those of the corresponding part of 2. In this
present work, the orientation of seven hydroxyl groups
at the moiety from C11 to C23 and a methyl group at
C10, which are commonly present in 1 and 2, are shown
to be the same. Work to assign the stereochemistry of
the remaining part of blasticidin A is now in progress.
Fig. 3. ꢀꢀ (¼ ꢀS ꢁ ꢀR) Values (ppm) Obtained for the MTPA Esters.
Acknowledgments
the polyol fragment of 1 was determined. Thus, the
absolute configurations at C10, C11, C13, C14, C15,
C16, C17, C19, C21 and C23 of 1 were clarified (Fig. 1).
This work was supported by Grant-aid for scientific
research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (No.
13024223) and by a grant from the JSPS Program for
Research for the Future.
Absolute configuration of the polyol fragment of
aflastatin A
We confirmed the relative configuration of 4a by
using a similar method to that used in the case of 3a. We
had previously prepared the pentaacetonide of 4a (8) to
confirm the relative stereochemistry of three 1,3-diol
systems at C5-C7, C9-C11 and C13-C15 of 4a.11) This
time, to confirm the remaining four 1,3-diol systems at
C3-C5, C7-C9, C11-C13 and C15-C17, the 1,19-
ditrityltetraacetonide (9) was prepared by tritylation of
4a, followed by acetonidation. From the 13C NMR
spectrum of 9, the syn relationship of all the four 1,3-
diol systems was confirmed. Similarly to the case of 5 or
6, the relative configurations of C2-C3, C4-C5, C6-C7,
C10-C11 and C12-C13 were also confirmed from the J
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