Amino acid starter unit in the biosynthesis of macrolactam polyketide antitumor antibiotic vicenistatin

Article abstract of DOI:10.1016/S0040-4020(01)00820-1

Biosynthetic studies on the starter unit of antitumor antibiotic vicenistatin were undertaken by feeding experiments with (2S,3R)- and (2S,3S)-3-[²H₃]methylaspartate, and 3-amino-2-[²H₃]methylpropionate. The starter unit of the macrolactam part of vicenistatin was found to be derived from (2S,3S)-3-methylaspartate, but not from the (2S,3R)-isomer. The present as well as the previous results suggest that the starter is formed from L-glutamate through structural rearrangement catalyzed by glutamate mutase to (2S,3S)-3-methylaspartate, which in turn is loaded to polyketide synthase. Additional epimerization and decarboxylation may take place in the process either of activation or condensation to give the final C-18 stereochemistry.

Full text of DOI:10.1016/S0040-4020(01)00820-1

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 8237±8242
Amino acid starter unit in the biosynthesis of macrolactam
polyketide antitumor antibiotic vicenistatin
Hiroshi Nishida,a Tadashi Eguchib and Katsumi Kakinumaa,p
aDepartment of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
bDepartment of Chemistry andMaterials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Received 26 June 2001; accepted 30 July 2001
AbstractÐBiosynthetic studies on the starter unit of antitumor antibiotic vicenistatin were undertaken by feeding experiments with (2S,3R)-
and (2S,3S)-3-[2H3]methylaspartate, and 3-amino-2-[2H3]methylpropionate. The starter unit of the macrolactam part of vicenistatin was
found to be derived from (2S,3S)-3-methylaspartate, but not from the (2S,3R)-isomer. The present as well as the previous results suggest that
the starter is formed from l-glutamate through structural rearrangement catalyzed by glutamate mutase to (2S,3S)-3-methylaspartate, which
in turn is loaded to polyketide synthase. Additional epimerization and decarboxylation may take place in the process either of activation or
condensation to give the ®nal C-18 stereochemistry. q 2001 Elsevier Science Ltd. All rights reserved.
Vicenistatin is an antitumor antibiotic produced by
Streptomyces halstedii HC-34, and unique features are
found in its polyketide structure of the macrocyclic 20-
membered lactam aglycon and an aminosugar vicenisamine
as shown in Fig. 1. Its biological activity is also intriguing
since antitumor activities were shown particularly against
xenographed models of certain human colon cancers.1 We
have been interested in the biosynthesis of vicenistatin
because, while the lactam aglycon appears to be mostly
derived from the polyketide pathway, the starter portion is
irrelevant to the acetate±propionate theory. The same is
actually true in most of the macrolactam antibiotics and
related microbial metabolites including ansamycins,2
hitachimycin,3 lankacidins,4 Antibiotic TA,5 epothilone,6
and the cytochalacin group of antibiotics.7
and its related compounds, and the feeding experiments of
these substrates.
1. Results and discussion
Firstly, the labeled substrates, (2S,3S)- and (2S,3R)-3-
[2H3]methylaspartate were prepared according to the
method of Chamberlin et al.,11 as shown in Scheme 1. The
protected l-aspartic acid 1 was treated with lithium hexa-
methyldisilazide (LHMDS) in THF at 2238C to convert to
an intermediary enolate, which was then alkylated with
2
[2H3]methyl iodide (99 atom% H) to give, after chromato-
graphic puri®cation and recrystallization, highly diastereo-
selectively (2S,3R)-3-[2H3]methylaspartate derivative 2a in
87% yield. On the other hand, its diastereomer, (2S,3S)-
derivative 2b, was prepared by epimerization of 2a. Thus,
treatment of 2a with potassium hexamethyldisilazide
(KHMDS) in THF at 2238C, and subsequent aqueous
quenching gave a separable mixture of (2S,3R)- and
(2S,3S)-3-[2H3]methylaspartate derivative 2a and 2b in a
ratio of 11:10. The desired diastereomer 2b was obtained
after chromatographic separation and crystallization in 36%
yield, together with the starting 2a (29%). Deprotection of
2a and 2b was separately carried out by hydrogenation in
In the previous papers,8,9 we reported that the elongating
units of the vicenistatin aglycon were derived from acetate
and propionate in a standard polyketide biosynthetic
manner, whereas the starter unit appeared to be derived
from 3-methylaspartate or its equivalent, probably formed
by the reactions of glutamate mutase and decarboxylase.
However, the C-18 stereochemistry of vicenistatin is
opposite to the usual product of glutamate mutase, i.e.
(2S,3S)-3-methylaspartate.10 Therefore, we undertook
feeding experiments of deuterium labeled 3-methylaspartate
diastereomers in order to con®rm whether or not the
standard glutamate mutase reaction is involved in the bio-
synthesis of vicenistatin. In this paper, we describe the
synthesis of deuterium labeled 3-methylaspartate isomers
Keywords: antibiotics; biosynthesis; amino acid; glutamate mutase.
Corresponding author. Tel.: 181-3-5734-2227; fax: 181-3-5734-3713;
e-mail: kakinuma@chem.titech.ac.jp
p
Figure 1. Structure of vicenistatin.
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00820-1

8238
H. Nishida et al. / Tetrahedron 57 *2001) 8237±8242
Scheme 1.
the presence of 10% Pd±C catalyst under high pressure of
hydrogen (30 atm), and subsequent acidic hydrolysis
afforded (2S,3R)- and (2S,3S)-3-[2H3]methylaspartic acid
(3a and 3b) in 82 and 79% yield, respectively.
reverse reaction of glutamate mutase, and the labeled
l-glutamate is deaminated into 2-oxoglutarate, which in
turn is decarboxylated into succinyl-CoA in the TCA
cycle. The well-established rearrangement pathway from
succinyl-CoA into methylmalonyl-CoA may give rise to
10,12
The feeding experiments using these substrates were carried
out as reported previously,8,9 and the labeled vicenistatin
was puri®ed. The H NMR spectra of labeled vicenistatin
the elongating C3unit,
mediate derived from propionate.
being equivalent to an inter-
2
are shown in Fig. 2A and B. No deuterium incorporation
was observed in the feeding experiment of (2S,3R)-3-
[2H3]methylaspartate 3a (Fig. 2B). In contrast, when
(2S,3S)-3-[2H3]methylaspartate 3b was used as substrate,
deuterium was ef®ciently incorporated into all the methyl
groups of the aglycon including the C-23methyl group
(Fig. 2A). The incorporation of deuterium into the C-23
methyl group was in fact much higher than that of
deuterated glutamate in the previous experiments.8,9 This
observation strongly suggested that (2S,3S)-3-methyl-
aspartate is the actual precursor of the starter unit of the
aglycon. It appears therefore that the starter unit is
biosynthesized through (2S,3S)-3-methylaspartate derived
from l-glutamate by the catalysis of glutamate mutase in
the usual stereochemistry.
Further manipulation for the starter unit of the aglycon
include epimerization at the C-3position and decarboxyl-
ation of (2S,3S)-3-methylaspartate. To get some insight into
the stage and mechanism of epimerization, the feeding
experiment of (2S,3S)-3-[2H3]methyl-[3-2H1]aspartate 5
was undertaken. This substrate was synthesized by quench-
ing the enolate derived from 2a with deuterium oxide and
deprotection of the resulting 4b as described above. The
feeding experiment of the synthesized 5 was carried out as
described above, and the obtained vicenistatin was analyzed
by 2H NMR as also shown in Fig. 2C. As was expected, the
resulted spectrum was quite similar to that of the feeding
experiment of (2S,3S)-3-[2H3]methylaspartate. The high
incorporation of deuterium into the C-23methyl group
was observed and no deuterium signal was shown at the
C-18 position (dH 1.82). These results clearly demonstrated
that the epimerization took place after the glutamate mutase,
probably via enolate intermediate. However, consecutive
epimerization at the C-3position immediately after the
glutamate mutase reaction must be ruled out, because
Additional less ef®cient incorporation of deuterium into the
methyl groups at C-20, C-21, and C-22 can be rationalized
straightforwardly. Thus, (2S,3S)-3-[2H3]methylaspartate is
partially transformed into labeled l-glutamate by the

H. Nishida et al. / Tetrahedron 57 *2001) 8237±8242
8239
Figure 2. Partial 1H and 2H NMR spectra of non-labeled and deuterated vicenistatins: (A); 2H NMR spectrum (60 MHz, pyridine) of labeled vicenistatin with
(2S,3S)-3-[2H3]methylaspartate 3b, (B); 2H NMR spectrum of labeled vicenistatin with (2S,3R)-3-[2H3]methylaspartate 3a, (C); 2H NMR spectrum of labeled
vicenistatin with (2S,3S)-3-[2H3]methyl-[3-2H1]aspartate 5, (D); 2H NMR spectrum of labeled vicenistatin with 3-amino-2-[2H3]methylpropionate 9, (E); 1H
NMR spectrum (400 MHz, pyridine-d5) of non-labeled vicenistatin.
(2S,3R)-3-[2H3]methylaspartate was not incorporated at all
as described above. Therefore, the subsequent reaction
could be decarboxylation reaction, probably with the aid
of pyridoxal phosphate coenzyme, and a decarboxylated
3-methylaspartate, i.e. 3-amino-2-methylpropionate, seemed
to be a candidate for the starter unit in the polyketide
elongation.
into the methyl groups at the C-20, C-21, and C-22
positions. The results clearly demonstrated that 3-amino-
2-methylpropionate was not a precursor of the starter unit
and the fed 3-amino-2-methylpropionate was instead
converted into the methylmalonyl-CoA. The latter
deuterium incorporation into the methyl groups at C-20,
C-21, and C-22 can be explainable as follows. Deuterated
3-amino-2-methylpropionate appeared to be deaminated
into labeled methylmalonate semialdehyde. The labeled
methylmalonate semialdehyde was then converted into
methylmalonyl-CoA through methylmalonate or propio-
nate.13 In any event, since 3-amino-2-methylpropionate is
not a precursor of the starter unit of the aglycon. Chain
elongation reaction may be the plausible step after the
formation of (2S,3R)-3-methylaspartate by the glutamate
mutase reaction.
To examine this possibility, we next undertook a feeding
experiment of dl-3-amino-2-[2H3]methylpropionate, which
was prepared as also shown in Scheme 1. The protected
b-alanine 6 was treated with LHMDS in THF at 2788C to
convert to an intermediary enolate, which was quenched
with [2H3]methyl iodide to give 3-amino-2[2H3]methyl-
propionate derivative 7 in 83% yield. Hydrogenation,
followed by acid treatment of the resulting acid 8, gave
the desired dl-3-amino-2-[2H3]methylpropionate 9 in high
yield. The feeding experiment using this substrate was
carried as described above, and the 2H NMR of the obtained
vicenistatin was also shown in Fig. 2D. To our surprise, no
deuterium incorporation into the C-23methyl group was
observed. Instead, deuterium was apparently incorporated
Several macrolactam antibiotics and related microbial
metabolites are considered to be biosynthesized by the poly-
ketide pathway with an amino acid starter unit.3±7 In
rifamycin biosynthesis, the amino acid starter 3-amino-5-
hydroxybenzoic acid is directly loaded on a polyketide

8240
H. Nishida et al. / Tetrahedron 57 *2001) 8237±8242
Figure 3.
synthase (PKS) having an amino acid loading domain.14 In
Antibiotic TA biosynthesis, one of the polyketide synthase
(PKS) was reported to contain the sequences similar to the
large family of non-ribosomal peptide synthethases
(NRPS).5 Further, epimerization of amino acids during the
NRPS reactions was reported in several biosynthetic
systems.15 Accordingly, in addition to these precedence,
the present study strongly suggests that the glutamate
mutase reaction product, (2S,3S)-3-methylaspartate, may
be directly loaded into the corresponding PKS, and the
appropriate epimerization and decarboxylation of (2S,3S)-
3-methylaspartate unit take place in the PKS reaction as
shown in Fig. 3. While a post-PKS modi®cation cannot be
ruled out for these reactions at the moment, genetic as well
as enzymatic investigation of vicenistatin biosynthesis
should shed light on this interesting biosynthetic problem.
yl)-3-[2H3]methylaspartate (2a). To a solution of dimethyl
l-N-benzyl-N-(9-phenyl¯uoren-9-yl)aspartate 111 (14.7 g,
29.9 mmol) in THF (80 ml) was added dropwise a solution
of LHMDS in THF (45.0 ml, 1.0 M solution, 45.0 mmol) at
2238C. After being stirred for 10 min, [2H3]methyl iodide
(6.52 g, 45.0 mmol) was added dropwise. The reaction was
kept at the same temperature for 1 h and was then quenched
with sat. aqueous NH4Cl. The layers were separated, and the
aqueous layer was extracted twice with ether. The combined
organic layer was washed with brine, dried over Na2SO4,
and evaporated. The residual solid was recrystallized from
EtOAc±hexane to give 2a (10.9 g, 71%). The mother liquor
was chromatographed on silica gel with hexane±EtOAc
(6:1) to give an additional 2a (2.4 g, 16%); total 13.3 g
(87%): mp 151±1538C (lit.11 154±1558C for non-labeled
1
2R,3S-compound); H NMR (CDCl3): d 2.55 (d, Jˆ11.2
Hz, 1H), 2.91 (s, 3H), 3.54 (s, 3H), 3.79 (d, Jˆ11.2 Hz,
1H), 4.35 (d, Jˆ14.4 Hz, 1H), 4.69 (d, Jˆ14.1 Hz, 1H),
7.21±8.02 (m, 18H); 13C NMR (CDCl3): d 14.9 (septet,
Jˆ18.9 Hz), 4.06, 50.4, 50.8, 51.1, 62.5, 80.2, 119.2,
119.8, 126.6, 127.19, 127.23, 127.3, 127.4, 127.5, 127.7,
128.0, 128.3, 129.0, 139.1, 141.4, 142.1, 144.3, 144.5,
147.2, 171.1, 174.1. Anal. Calcd for C33H282H3NO4: C,
77.93; H12H, 6.14; N; 2.75. Found: C, 77.61; H12H,
6.04; N; 2.62.
In summary, the present study has clearly demonstrated that
the starter unit of the macrolactam part of vicenistatin is
derived from (2S,3S)-3-methylaspartate through the
glutamate mutase reaction in the usual stereochemical
manner. The glutamate mutase reaction product, (2S,3S)-
3-methylaspartate, was suggested to be directly loaded
into the PKS.
2.1.2. Dimethyl (2S,3S)-N-benzyl-N-(9-phenyl¯uoren-9-
yl)-3-[2H3]methylaspartate (2b). To a solution of 2a
(80.0 mg, 0.158 mmol) in THF (2.0 ml) was added dropwise
a solution of KHMDS in THF (0.50 ml, 0.5 M solution,
0.25 mmol) at 2238C. The mixture was stirred at the
same temperature for 10 min, and then at 08C for 12 h.
The reaction was quenched with sat. aqueous NH4Cl. The
layers were separated, and the aqueous layer was extracted
twice with ether. The combined organic layer was washed
with brine, dried over Na2SO4, and evaporated. Flash silica
gel column chromatography with hexane±EtOAc (6:1) and
recrystallization from EtOAc±hexane gave the more polar
product 2a (23mg, 29%) and the less polar product 2b
(29 mg, 36%): mp 179±1818C (lit.11 184±1858C for non-
2. Experimental
2.1. General
Column chromatography was carried out with a Kieselgel
60 (70±230 mesh or 230±400 mesh, Merck). All reactions,
except for catalytic hydrogenation reactions, were carried
out in an inert (Ar or N2) atmosphere. THF was distilled
1
2
from sodium benzophenone ketyl. H-, 13C- and H NMR
spectra were recorded on a JEOL LA-300, or a LA-400
spectrometer. Data are reported as follows: chemical shift
(ppm), multiplicity (sˆsinglet, dˆdoublet, tˆtriplet, qˆ
quartet, mˆmultiplet, brˆbroad), coupling constant (Hz),
and integration. IR spectra were taken on a Horiba FT-710
Fourier-transform infrared spectrometer. Elemental
analyses were performed with a Perkin±Elmer 2400
1
labeled 2R,3R-compound); H NMR (CDCl3): d 2.26 (d,
Jˆ11.5 Hz, 1H), 2.96 (s, 3H), 3.35 (s, 3H), 3.54 (d, Jˆ
11.2 Hz, 1H), 4.16 (d, Jˆ13.4 Hz, 1H) 4.40 (d, Jˆ
13.4 Hz, 1H), 7.22±7.78 (m, 18H). Anal. Calcd for
C33H282H3NO4: C, 77.93; H12H, 6.14; N, 2.75. Found: C,
77.79; H12H, 6.19: N, 2.65.
apparatus. [2H3]Methyl iodide (99 atom% H) and deuter-
2
2
ium oxide (99.8 atom% H) were purchased from Aldrich.
2.1.1. Dimethyl (2S,3R)-N-benzyl-N-(9-phenyl¯uoren-9-

H. Nishida et al. / Tetrahedron 57 *2001) 8237±8242
8241
2.1.3. (2S,3R)-3-[2H3]Methylaspartic acid hydrochloride
(3a). A mixture of 2a (4.0 g, 7.9 mmol), tri¯uoroacetic acid
(1.72 g, 15.1 mmol), and 10% Pd±C (1.0 g) in CH2Cl2±
methanol (1:2, 45 ml) was stirred for 13.5 h under 31 atm
pressure of hydrogen. The mixture was ®ltered through a
pad of Celite, which was then washed with methanol. The
®ltrate and washings were combined and concentrated. The
residue was chromatographed with CHCl3±methanol (5:1)
to give oily residue, which was used for the next step
without further puri®cation. A solution of the obtained
residue in 6 M HCl (30 ml) was stirred at 608C for 16 h.
After concentration of the mixture, the residue was
dissolved in a minimal amount of water and loaded to a
cation-exchange resin column (Dowex 50W-X2, H1
form). The column was washed with water, and eluted
with 5% aqueous pyridine. Ninhydrin-positive fractions
were combined and evaporated. To the residue, was added
4N HCl and the solution was evaporated to give 1.2 g of 3a
in the same manner as described for the preparation of 3a to
give 5 as a HCl salt (639 mg, 97%): mp 158±1628C; H
1
2
NMR (D2O) 4.21. Anal. Calcd for C5H6 H4NO4Cl: C,
32.01; H12H, 5.37; N, 7.41. Found: C, 32.22; H12H,
5.26; N, 7.31.
2.1.7. Benzyl 3-(N-tert-butoxycarbonyl)amino-2-[2H3]-
methylpropionate (7). To a solution of 616 (8.50 g,
30.4 mmol) in THF (130 ml) was added dropwise a solution
of LHMDS (67.0 ml, 1.0 M solution in THF, 67.0 mmol) at
2788C. After being stirred for 10 min, [2H3]methyl iodide
(4.84 g, 33.4 mmol) was added dropwise. The reaction was
kept at the same temperature for 10 min and was then
quenched with sat. aqueous NH4Cl. The layers were sepa-
rated, and the aqueous portion was extracted with EtOAc.
The combined organic layer was washed with brine, dried
over Na2SO4, and evaporated. Flash silica gel column
chromatography with hexane±EtOAc (4:1) provided
1
1
7.50 g of 7 (83%) as an oil: H NMR (CDCl3) 1.43(s,
as a HCl salt (82%, 2 steps): mp 149±1518C; H NMR
(D2O): d 3.20, (br d, Jˆ3.7 Hz, 1H), 4.17 (d, Jˆ4.1 Hz,
9H), 2.72 (br t, Jˆ5.9 Hz, 1H), 3.19±3.40 (m, 3H), 4.94
(br, 1H), 5.14 (br, 2H), 7.35 (m, 5H); 13C NMR (CDCl3)
13.4 (septet, Jˆ19.7 Hz), 27.9, 39.4, 42.6, 65.8, 78.6, 127.5,
1H). Anal. Calcd for C5H7 H3NO4Cl: C, 32.18; H12H,
2
5.41; N; 7.51. Found: C, 32.26; H12H, 5.45; N, 7.33.
2
127.7, 128.1, 135.5, 155.5, 174.6; H NMR (CHCl3) 1.16.
Anal. Calcd for C16H202H3O4N: C, 64.85; H12H, 7.82; N,
4.73. Found: C, 64.55, H12H, 7.70; N, 4.68.
2.1.4. (2S,3S)-3-[2H3]Methylaspartic acid hydrochloride
(3b). Compound 2b (1.95 g, 3.83 mmol) was treated in the
same manner as described for the preparation of 3a to give
3b as a HCl salt (563mg, 79%): mp 155±160 8C; 1H NMR
(D2O): d 3.11, (br d, Jˆ2.4 Hz, 1H), 4.21 (d, Jˆ3.7 Hz,
2.1.8. 3-(N-tert-Butoxycarbonyl)amino-2-[2H3]methyl-
propionic acid (8). A mixture of 7 (6.96 g, 23.5 mmol)
and 10% Pd±C (427 mg) in dioxane (70 ml) was stirred
for 8 h under an atmospheric pressure of hydrogen. The
mixture was ®ltered through a pad of Celite and concen-
trated. Recrystallization from CHCl3±hexane provided
1H). Anal. Calcd for C5H7 H3NO4Cl: C, 32.18; H12H,
2
5.41; N, 7.51. Found: C, 32.32; H12H, 5.36; N, 7.21.
2.1.5. Dimethyl (2S,3R)-N-benzyl-N-(9-phenyl¯uoren-9-
yl)-3-[2H3]methyl-[3-2H]aspartate (4a) and dimethyl
(2S,3S)-N-benzyl-N-(9-phenyl¯uoren-9-yl)-3-[2H3]methyl-
[3-2H]aspartate (4b). To a solution of 2a (15.0 g,
29.5 mmol) in THF (450 ml) was added dropwise a solution
of KHMDS in THF (65.0 ml, 0.5 M solution, 32.5 mmol) at
2238C. After being stirred at the same temperature for
15 min, D2O (10 ml) was added. The mixture was warmed
to room temperature and sat. aqueous NH4Cl was added.
The layers were separated, and the aqueous portion was
extracted twice with ether. The combined organic layer
was washed with brine, dried over Na2SO4, and evaporated.
Silica gel column chromatography with hexane±EtOAc
(4:1) gave a mixture of 4a and 4b. This isomerization and
deuterium introduction procedure was repeated three times.
Flash silica gel column chromatography of the mixture of 4a
and 4b with hexane±EtOAc (6:1) gave the more polar
product 4a (4.05 g, 27%) and the less polar product 4b
1
4.28 g of 8 (88%): mp 88±908C; H NMR (CDCl3, ca. 2:1
mixture of conformers) major: 1.44 (s, 9H), 2.69 (br, 1H),
3.25 (br dd, Jˆ6.1, 13.4 Hz, 1H), 3.34 (br m, 1H), 5.05 (br,
1H), 10.7 (br 1H), minor: 1.47 (s, 9H), 2.65 (br, 1H), 3.26
2
(br, 1H), 3.39 (br m, 1H), 6.39 (br, 1H); H NMR (CHCl3)
1.18. Anal. Calcd for C9H142H3O4N: C, 52.42; H, 8.31; N,
6.79. Found: C, 52.39, H, 8.61; N, 6.99.
2.1.9. 3-Amino-2-[2H3]methylpropionic acid hydro-
chloride (9). A solution of 8 (1.20 g, 5.82 mmol) in 4 M
HCl±dioxane (2:1, 20 ml) was stirred for 1 h. The mixture
was evaporated to yield 824 mg of 9 as a HCl salt (quant.):
mp 119±1238C; 1H NMR (D2O) 2.68 (br dd, Jˆ5.0, 8.2 Hz,
1H), 2.91 (dd, Jˆ5.0, 13.0 Hz, 1H), 3.03 (dd, Jˆ8.2,
2
13.0 Hz, 1H). Anal. Calcd for C4H7 H3O4NCl: C, 33.69;
H12H, 7.07; N, 9.82. Found: C, 33.54, H12H, 7.00; N,
9.84.
1
(5.31 g, 35%): 4a; mp 152±1558C; H NMR (CDCl3): d
2.90 (s, 3H), 3.54 (s, 3H), 3.78 (s, 1H), 4.35 (d, Jˆ
2.2. Culture of Streptomyces halstedii HC34 for feeding
experiments
14.1 Hz, 1H) 4.68 (d, Jˆ14.4 Hz, 1H), 7.14±7.98 (m,
2
18H); H NMR (CHCl3): d 0.58, 2.53. Anal. Calcd for
C33H272H4NO4: C, 77.77; H12H, 6.13; N, 2.75. Found: C,
An autoclaved 100 ml seed medium (potato starch 3%, soya
¯ake 1.5%, yeast extract 0.2%, corn steep liquor 0.5%, NaCl
0.3%, MgSO4´7H2O 0.05%, CaCO3 0.3%, and CoCl2´6H2O
0.0005%, pH adjusted to 7.1 with 3M NaOH) in a 500 ml
baf¯ed ¯ask equipped with a cotton plug was inoculated
with a scraping from an agar slant of the producing St.
halstedii HC 34. The culture was grown on a shaker at
278C and 200 rpm for 48 h. Vegetative cultures (100 ml£
12) were initiated by inoculation of the vegetative medium
(potato starch 3%, soya ¯ake 1.5%, yeast extract 0.2%, corn
1
77.67; H12H, 6.19; N, 2.68. 4b; mp 180±1828C; H NMR
(CDCl3): d 2.96 (s, 3H), 3.35 (s, 3H), 3.49 (s, 1H), 4.16 (d,
Jˆ12.9 Hz, 1H) 4.41, (d, Jˆ13.4 Hz, 1H), 7.22±7.95 (m,
2
18H); H NMR (CHCl3): d 1.11, 2.27. Anal. Calcd for
C33H272H4NO4: C, 77.77; H12H, 6.13; N, 2.75. Found: C,
77.61; H12H, 6.04; N, 2.62.
2.1.6. (2S,3S)-3-[2H3]Methyl[3-2H]aspartic acid hydro-
chloride (5). Compound 4b (1.80 g, 3.53 mmol) was treated

8242
H. Nishida et al. / Tetrahedron 57 *2001) 8237±8242
4. (a) Uramoto, M.; Otake, N.; Cary, L.; Tanabe, M. J. Am.
Chem. Soc. 1978, 100, 3616. (b) Kakinuma, K.; Uzawa, J.;
Uramoto, M. Tetrahedron Lett. 1982, 23, 5303.
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steep liquor 0.5%, NaCl 0.3%, MgSO4´7H2O 0.05%, CaCO3
0.3%, and CoCl2´6H2O 0.0005%, pH adjusted to 7.1 with
3M NaOH) with 0.5 ml of a seed culture and were grown
under the same conditions for 72 h. Isotopically labeled
substrates were added twice to the culture at 24 and 48 h
after inoculation.
Â
6. Molnar, I.; Schupp, T.; Ono, M.; Zirkle, R. E.; Milnamow, M.;
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The fermentation broth (1.2 l) was centrifuged (7,000 rpm,
30 min) to give a mycelium cake. The mycelium cake was
extracted overnight with acetone (2.0 l). The extracts were
®ltered and concentrated in vacuo to an aqueous state. The
aqueous solution was extracted three times with EtOAc. The
combined organic layer was dried over Na2SO4 and
evaporated. The residue was applied to a silica gel column
chromatography with EtOAc, and then CHCl3±methanol
(5:1). Vicenistatin containing fractions were collected and
concentrated to dryness. The residue was further puri®ed by
¯ash silica gel column chromatography with CHCl3±MeOH
(9:1) to give a colorless powder of vicenistatin (ca 10 mg).
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È
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Â
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Acknowledgements
This work was supported by `Research for the Future'
Program of The Japan Society for the Promotion of Science
(JSPS-RFTF96I00302) and a Grant-in-Aid for Scienti®c
Research from the Ministry of Education, Science, Sports
and Culture.
È
14. August, R. R.; Tang, L.; Yoon, Y. J.; Ning, S.; Muller, R.; Yu,
T.-W.; Taylor, M.; Hoffmann, D.; Kim, C.-G.; Zhang, X.;
Hutchinson, C. R.; Floss, H. G. Chem. Biol. 1998, 5, 69.
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(b) Linne, U.; Marahiel, M. A. Biochemistry 2000, 39,
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