The absolute configuration of gambieric acids A-D, potent antifungal polyethers, isolated from the marine dinoflagellate Gambierdiscus toxicus

Article abstract of DOI:10.1016/S0040-4020(00)00753-5

The absolute configurations of potent antifungal polyether compounds, gambieric acids A-D, isolated from the marine dinoflagellate Gambierdiscus toxicus were determined by combining the modified Mosher method, NMR analysis, and chiral fluorimetric HPLC. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00753-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 8995±9001
The Absolute Con®guration of Gambieric Acids A±D, Potent
Antifungal Polyethers, Isolated from the Marine Dino¯agellate
Gambierdiscus toxicus
Akio Morohashi,a Masayuki Satake,a Hiroshi Nagai,b Yasukatsu Oshimaa and
Takeshi Yasumotoa,
*
aGraduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
bSuntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, Japan
Received 15 May 2000; accepted 23 June 2000
AbstractÐThe absolute con®gurations of potent antifungal polyether compounds, gambieric acids A±D, isolated from the marine dino-
¯agellate Gambierdiscus toxicus were determined by combining the modi®ed Mosher method, NMR analysis, and chiral ¯uorimetric HPLC.
q 2000 Elsevier Science Ltd. All rights reserved.
A large number of polyether compounds having potent
biological activities have been isolated from marine
organisms.1 Their unique and intriguing structures have
been elucidated mainly by NMR spectroscopy. However,
their extremely limited amounts and non-crystalline proper-
ties hampered elucidation of their stereostructures by X-ray
crystallography. As the continuation of our efforts to
elucidate the absolute con®gurations of signi®cant polyether
compounds, e.g. ciguatoxin, maitotoxin, pectenotoxin,
dinophysistoxin-1, yessotoxin, and gambierol, by com-
bining CD, NMR and synthetic methods,2±7 we undertook
a stereochemical study of gambieric acids.
C11 was determined by NMR analysis after introducing
anisotropic reagents to the carboxyl group at C1 or 9-OH
in GAB. To determine the absolute con®guration at C48, we
protected 49-OH in GAB, oxidatively cleaved the C46±C47
double bond, coupled the resultant carboxylic acid to a
chiral ¯uorescent reagent, and compared the ¯uorescent
derivative by HPLC with synthetic reference compounds.
Similarly, chiral ¯uorescent reagents for HPLC were used to
determine the stereochemistry of 3-methylglutaric acid in
GAC and GAD. In this paper, we report the absolute con-
®guration of gambieric acids A±D thus determined (see
Fig. 1).
Gambieric acids A±D (GA A±D, 1±4) were potent anti-
fungal compounds isolated from the culture medium of
the marine dino¯agellate Gambierdiscus toxicus that
produces ciguatoxin precursors and maitotoxin.8 Their anti-
fungal activities against Aspergillus niger by the paper disk
method were 2000 times greater than that of amphotericin
B. The acids 1±4 are endogenous growth enhancers of the
dino¯agellate.9 Therefore, GAs are fascinating compounds
not only in structural features but also in their biological
activities. Previously we have determined the relative
stereochemistry of the segment composed of fused ether
rings. For synthesis of the whole molecule and understand-
ing of the mode of actions, however, elucidation of the
absolute con®guration of the whole molecule is essential.
In the present study, the absolute con®guration from C1 to
Results and Discussions
Absolute con®guration at C9
The relative stereostructure of the cyclic ether segment was
determined previously and found to be the same in all
gambieric acids. Thus, determination of the absolute con-
®guration at C12 or at C36 by the modi®ed Mosher's
method should lead to elucidation of all absolute stereo-
structure of the cyclic ether segment.10 To avoid the possi-
bility of having two MTPAs in vicinity, C9 and C12, 2 was
chosen for esteri®cation with (R)- and (S)-MTPA chloride.
ESI MS of the main product revealed an ion at m/z 1581
(M1pyridine)1 corresponding to bis-MTPA esters (5 and
6). Based on the 1H NMR spectra, one MTPA resided at C9
and the other at C49. Thus, the two MTPA esters were well
separated from each other to exert interaction. We ®rst
determined the absolute con®guration at C9 in 2. By analyz-
ing the COSY, TOCSY and NOESY spectra of 5 and 6, Dd
Keywords: polyether; absolute con®guration; modi®ed Mosher method;
chiral ¯uorimetric HPLC.
* Corresponding author. Tel.: 181-22-717-8815; fax: 181-22-717-8817;
e-mail: satake@biochem.tohoku.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00753-5

8996
A. Morohashi et al. / Tetrahedron 56 (2000) 8995±9001
Figure 1. Structures of gambieric acids A±D.
dicarboximide)-1-propyl tri¯uoromethanesulfonate] as a
chiral ¯uorigenic reagent.11 As shown in Scheme 1, 1 mg
of the bis-(R)-MTPA ester (6) was cleaved at the C46±C47
double bond with NaIO4/RuCl3, and the resultant carboxylic
acid was esteri®ed to give a ¯uorescent (R)-2A1P derivative
(7). Reference 48S- and 48R- 2A1P-esters (7) were prepared
from 3-hydroxy-2(R or S)-methylpropionate (8) as shown in
Scheme 1, and their structures were con®rmed by ESI MS
(MH1, m/z 608) and NMR spectra. The two reference
diastereomers showed clearly different retention times (R:
14.72, S: 16.36 min) on a chiral HPLC column (Chiracel
OJ-R, 4.6£250 mm) with 65% MeCN. The retention time
of the derivative prepared from 2 agreed well with that of
the reference 2A1P-ester having 2S con®guration. Conse-
quently, C48 con®guration in 2 before C46±C47 bond
cleavage was unambiguously determined to be R.
Figure 2. The partial structure of 5 and 6 indicating the Dd (dS2dR) values
(ppm).
(dS2dR) values of the protons around C9 were obtained, as
depicted in Fig. 2. The signs of Dd values were clearly
distributed symmetrically around C9. The signs of Dd
values for protons from H4 to H2-8 were positive, while
those for protons from H2-10 to H16 were negative. The
distribution of the signs of protons well re¯ected the aniso-
tropic effects of the MTPA ester at C9 and allowed us to
assign R con®guration at C9 in 2.
Absolute con®gurations at C3 and C4
The absolute con®guration at C3 was determined by the
modi®ed Mosher's method using a chiral anisotropic
reagent, phenylglycine methyl ester (PGME), originally
developed to identify the absolute con®guration of a chiral
carboxylic acids.12 GAB (2) was condensed, respectively,
with (S)- and (R)-PGME in DMF to give GAB (S)-PGME
Absolute con®guration at C48
The absolute con®guration at C48 in 2 was determined by
HPLC by using (R)-2A1P-OTf [(R)-2-(anthracene-2,3-
Scheme 1.

A. Morohashi et al. / Tetrahedron 56 (2000) 8995±9001
8997
signs, while those of H5 and Me51 showed positive signs.
In 1D-HOHAHA spectra irradiated at Me50, the high-®eld
proton at C2 was observed as a triplet having 13 Hz
coupling, the low-®eld proton as a doublet having 13 Hz
coupling, and H4 as a double-doublet having 9 and
4 Hz couplings. These results indicated that H3 was anti
to both the high-®eld proton at C2 and H4, and was gauche
to the low-®eld proton at C2. Observed NOE correlations
Me50/H4, the low-®eld proton H2/H5, and H3/Me51
indicated that Me50 and C5 were in anti conformation.
These Dd values, coupling constants, and NOE correlations
unanimously supported the conformation from PGME to C7
as shown in Fig. 3. From the conformation and the Dd
values, the con®gurations at both C3 and C4 were deter-
mined to be S.
Figure 3. Presumable conformation and the Dd (dS2dR) values (ppm) of
C1±C7 portion of 12 and 13.
amide (12) and GAB (R)-PGME amide (13). The Dd values
(dS2dR) are indicated in Fig. 3. To apply the PGME method
to a b chiral carboxylic acid, conformation analysis is
essential. The Dd value of the high-®eld proton at C2
showed little shift, while that of the low-®eld proton showed
positive sign. The NOE correlation from the amide proton
was observed only to the high-®eld proton at C2. Thus, the
high-®eld proton was positioned on the PGME plane and the
low-®eld proton had pro-R position (Fig. 3). Moreover,
the Dd values of H3, Me50, and H4 showed negative
Con®guration at C11 and C12
The absolute con®guration at C11 was determined by eluci-
dating the conformation between C7 and C11 based on 3JH,H
and NOE data.13 The same signal shapes of H7, H9 and H11
in 2, 12 and 13 indicated that the conformation from C7 to
C11 in 2 was kept unchanged by amidation. The amide 12
was chosen for analysis, because signals for H7, H9 and
Figure 4. The plausible conformation of C7±C11 segment of 12 and 13 based on NMR experiments data. For clarity, C3, C17, and the ether oxygen at C15
were omitted in the models.
Scheme 2.

8998
A. Morohashi et al. / Tetrahedron 56 (2000) 8995±9001
H11 were well isolated in the 1D 1H NMR spectrum.
Decoupling difference spectra irradiated at H7, H9 and
H11 in 12 were measured to elucidate coupling constants
between these oxymethine protons and interposed
methylenes. In the spectra the low-®eld proton (H8-b) at
C8 had a large coupling constant (10 Hz) and thus was
anti to H7. The high-®eld proton (H8-a) at C8 was deduced
to be gauche to H7 and anti to H9. Similarly, the low-®eld
proton (H10-a) at C10 having a large coupling constant
(10 Hz) was deduced to be anti to H9, while a high-®eld
proton (H10-b) at C10 was gauche to H9 and anti to H11.
NOE correlations were observed between H8-b/H6-b,
H8-b/H10-b, H8-a/H6-a, H8-a/H10-a, H9/H7, H9/H11,
H10-a/Me on C12, H10-a/H11, and H10-b/Me on C12.
amides 14. The ester bond in 14 was then hydrolyzed with
0.2 N NaOH in 90% MeOH. The resultant carboxylic acid
15 was reacted with a chiral ¯uorigenic reagent (S)-1A2P-
OTf [(S)-1-(anthracen-2,3-dicarboximide)-2-propyl tri¯uoro-
methanesulfonate] to produce a ¯uorescent derivative 16.
References for 16 in HPLC analysis were prepared from
(R)-3-methylsuccinic acid 1-monomethyl ester 17 accord-
ing to Scheme 3. The carboxylic acid 17 was reduced to
aldehyde 19 (2 steps). Wittig reaction of the aldehyde 19
with (Ph3PMe)Br produced ole®n 20. After hydrolysis of 20
under alkaline condition, 21 was reacted with (R)-naphthyl-
ethylamine to generate amide 22. Hydroboration of 22 with
9-BBN and H2O2 produced alcohol 23. The resultant
alcohol 23 was oxidized to carboxylic acid 25, followed
by esteri®cation with (S)-1A2P-OTf to produce an HPLC
standard (R)-16. A diastereomixture of (R)-16 and (S)-16
was prepared from 3-methylglutaric acid 26 by amidation
with (R)-naphthylethylamine and esteri®cation with
(S)-1A2P-OH. These diastereomers were separated on a
reversed phase column (Cosmosil 5C18 AR, 4.6£250 mm)
with 60% MeCN. The retention time of the ¯uorescent
derivative 16 prepared from 3 and 4 agreed with that of
(30S) standard (S)-16. Further con®rmation was accom-
plished by LC/MS analysis by selected ion monitoring at
m/z 587. The derivative having 30R con®guration was
detected in a ratio of S to R in 10:1. However, based on
the predominance of S con®guration at C30 in the products,
we assigned S con®guration to C30. The absolute con®gura-
tion of entire molecule of gambieric acids A±D was thus
elucidated by using extremely small amounts of samples.
2
Based on the dependence of JC,H on the dihedral angle of
an oxygen function, the HMBC correlation from H10-a to
C9 suggested that H10-a was gauche to C9-OH.14 These
data pointed to the conformation from C7 to C11 as shown
in Fig. 4. As the absolute con®guration at C9 had been
already determined as R, the absolute con®guration at C11
was determined to be S. As the relative con®gurations of
other asymmetric centers had been established in previous
experiment,8 the present result led to the absolute con®gura-
tion of GAB as shown in 2. The R con®guration at C12 was
supported by the absence of NOE correlation H11/Me on
C12 in the ROESY spectrum.
Absolute con®guration at C30
The absolute con®guration at C30 in 3 and 4 was determined
by a chiral HPLC method as shown in Scheme 2. As
chromatographic separation of 3 and 4 was impossible,
5 mg of a mixture containing 3 and 4 in a ratio 3:2 was
used for experiments. The carboxylic moiety in 3 and 4
was protected with (R)-naphthylethylamine to generate
The stereochemical information obtained in this study is
expected to enhance synthetic efforts toward these
intriguing molecules.
Scheme 3.

A. Morohashi et al. / Tetrahedron 56 (2000) 8995±9001
8999
Experimental
reacted with (R)-2A1P-OTf in the presence of (Et4N)2CO3
in CH2Cl2. The reaction mixture was chromatographed on
silica gel with hexane/EtOAc (5:2) to give the ¯uorescent
reference 7.
ESI MS and NMR spectra measurements
ESI MS spectra were measured with a Finnigan mat TSQ
700 spectrometer. NMR spectra of GAB-MTPA esters and
GAB-PGME amides were measured with a Varian Unity
INOVA 600 spectrometer in pyridine-d5 at 208C.
1
(R)-7: H NMR (600 MHz, CDCl3): d 1.12 (3H, d), 1.55
(3H, d), 2.78 (1H, m), 3.42 (3H, S), 4.27(1H, dd), 4.40
(1H,), 4.43 (1H, dd), 4.51(1H, t), 4.70 (1H, m), 7.29, 7.39,
7.61, 8.07, 8.48, 8.61 (phe and anthracene); HR-FAB MS
[M1H]1 608.1899; calcd for C33H29F3NO7 608.1897.
Preparation of GAB-9,49-bis-MTPA esters (5 and 6)
1
(S)-7: H NMR (600 MHz, CDCl3): d 1.06 (3H, d), 1.55
To 200 mg of 2 dissolved in pyridine were added DMAP
(500 mg), TEA and (R)- or (S)-MTPA chloride. The mixture
was kept under stirring at rt for 5 h. The reaction was
stopped by adding MeOH and the residue extracted with
CHCl3 was puri®ed on a Capcell Pak C8 UG120 column
with linear gradient elution from 65% MeCN to 100%
MeCN.
(3H, d), 2.80 (1H, m), 3.44 (3H, s), 4.28 (1H, dd), 4.33
(1H, dd), 4.40 (1H, dd), 4.62 (1H, t), 4.69 (1H, m), 7.35,
7.42, 7.61, 8.09, 8.50, 8.63 (phe and anthracene); HR-FAB
MS [M1H]1 608.1895.
Fluorimetric HPLC analysis for 7
GAB-9,49-bis-(S)-MTPA esters (5). dH (ppm): 2 (2.22,
2.37), 3 (2.51), Me50 (1.30), 4 (3.68), 5 (2.16), Me51
(0.92), 6 (1.59, 1.65), 7 (4.48), 8 (2.05, 2.15), 9 (6.01), 10
(2.23, 2.52), 11 (3.87), 14 (2.03, 2.16), 15 (3.75), 16 (3.60),
17 (1.92, 2.54).
The ¯uorimetric HPLC analysis was performed under the
following conditions: column, Chiracel OJ-R (4.6£250 mm,
Daicel Kogyo); solvent, 65% MeCN; ¯ow, 1.2 ml/min;
detection, excitation at 298 nm and emission at 462 nm.
The retention time was 14.72 min for R- and 16.36 min
for S-ester.
GAB-9,49-bis-(R)-MTPA esters (6). dH (ppm): 2 (2.22,
2.37), 3 (2.51), Me50 (1.31), 4 (3.60), 5 (2.11), Me51
(0.86), 6 (1.48, 1.54), 7 (4.02), 8 (1.90, 2.03), 9 (6.04), 10
(2.23, 2.55), 11 (3.95), 14 (2.05, 2.19), 15 (3.75), 16 (3.65),
17 (1.95, 2.55).
Preparation of GAB-(S)- and (R)-PGME amides (12
and 13)
To GAB (200 mg) and (S)- or (R)-PGME-HCl in DMF
cooled to 08C were added TEA, PyBOP and HOBt. After
stirring for 17 h at rt, the mixture was added with CHCl3 and
washed with H2O. The organic layer was chromatographed
on a Capcell pak C8 (4.6£150 mm, Shiseido) with 50%
MeCN to give 10 and 11 (m/z 1219.6 for [M1H]1).
Preparation of ¯uorescent derivative (7) from GAB-
9,49-bis-(R)-MTPA ester (6)
To the ester 6 (1 mg) in CCl4/MeCN (1:1), NaIO4 and RuCl3
in 0.25 M phosphate buffer (pH 6.9) were added and the
solution was stirred for 1 h at rt. After adding NaHCO3
the solution was washed with CH2Cl2. The aqueous layer
was acidi®ed with 6N HCl, extracted with CH2Cl2, and the
organic layer was dried over MgSO4. The extract was
reacted with (R)-2A1P-OTf and (Et4N)CO3 in CH2Cl2 by
stirring for 7 h at 408C. The reaction mixture was puri®ed
on a Chiracel OJ-R column with 65% MeCN to give 7.
GAB-(S)-PGME amide (12). dH (ppm): 2 (2.36, 2.58), 3
(2.46), Me50 (1.35), 4 (3.61), 5 (2.18), Me51 (0.873).
GAB-(R)-PGME amide (13). dH (ppm): 2 (2.36, 2.55), 3
(2.48), Me50 (1.43), 4 (3.62), 5 (2.17), Me51 (0.868).
Preparation of ¯uorescent reference compounds (7)
from methyl 3-hydroxy-2(R and S)-methylpropionates
(8)
Preparation of ¯uorescent derivative 16 from GAC (3)
and GAD (4) mixture
A mixture of 3 and 4 (3:2, 5 mg), EDC-HCl (170 mg) and
(R)-naphthylethylamine (810 mg) were dissolved in CH2Cl2
(100 ml) and stirred for 13 h. The reaction mixture was
diluted with CH2Cl2 (4 ml), washed with NH4Cl, and
dried over MgSO4. The amide 14 was hydrolyzed in 0.2N
NaOH/90% MeOH (200 ml) for 6 h. The reaction mixture
was diluted with 0.1N NaOH (2 ml) and washed with
EtOAc (2 ml). The aqueous layer acidi®ed with HCl was
extracted with EtOAc (2 ml), and the organic layer was
dried over MgSO4. The resultant glutaric acid derivative
15 was reacted with (S)-1A2P-OTf (300 mg) in CH2Cl2
(100 ml) in the presence of (Et4N)2CO3 (300 mg) for 15 h
at 408C. The reaction mixture was developed on a silica gel
TLC plate with hexane/EtOAc (1:1) and the ¯uorescent
derivative 16 was extracted from the plate.
A mixture of 8 (26.5 mg), benzyl alcohol (70 ml), p-toluene
sulfonic acid (13 mg), and molecular sieves in toluene
(500 ml) was stirred for 10 h at 808C. The reaction mixture
was diluted with EtOAc, washed with NaHCO3 solution,
dried over MgSO4, and puri®ed on silica gel with hexane/
EtOAc (3:2) to give benzyl ester 9 (R 21 mg, S 24 mg). The
ester 9 (5 mg) was reacted with (S)-MTPA chloride in
pyridine (90 ml) and TEA (10 ml). The reaction mixture
was diluted with EtOAc (4 ml), washed with NaHCO3 and
NH4Cl solutions, dried over MgSO4 and puri®ed on silica
gel with hexane/EtOAc (6:1) to give (R)-MTPA ester 10.
The (R)-MTPA ester 10 in MeOH was reduced with 10%
Pd(OH)2 under an H2 atmosphere by stirring for 1 h at rt.
After removal of the catalyst by ®ltration, the product 11
was concentrated in vacuo. The carboxylic acid 11 was

9000
A. Morohashi et al. / Tetrahedron 56 (2000) 8995±9001
Synthesis of references for the ¯uorescent derivative 16
(8.8 mg) in a (Et4N)2CO3 (9.6 mg) in CH2Cl2 (1 ml) for
4 h under stirring at rt. The ¯uorescent ester 16 was obtained
by puri®cation on silica gel with CHCl3/MeOH (50:1).
To a solution of DMF (550 ml) in CH2Cl2 (8.6 ml) was
added (COCl)2 (900 ml) at 08C. After stirring for 1 h at
08C, MeCN (7.7 ml) and THF (13.7 ml) were added, and
the solution was cooled to 2308C. (R)-3-Methylsuccinic
acid 1-monomethyl ester 17 (1 g in 580 ml pyridine and
13.7 ml THF) was added dropwise for 30 min and the solu-
tion was stirred for 1 h at 2308C and for 30 min at 2208C.
The resultant acid chloride 18 was reduced by adding drop-
wise a THF solution of LiAlH(O-t-Bu)3 (2 g in 10 ml) in
30 min at 2788C. The solution was further stirred for
30 min at 2788C, acidi®ed with 2 M HCl (9.6 ml) brought
to rt, and washed with H2O (20 ml). The aqueous layer was
further extracted with diethylether (10 ml£2). The
combined organic layer was washed with saturated solution
of NaHCO3 and NH4Cl, and dried over MgSO4 to give
aldehyde 19. A mixture of (Ph3PMe)Br (13 g) in THF
(50 ml) (Me3Si)2NNa in THF/H2O (4:6, 15 ml) was stirred
for 15 min at 08C, and aldehyde 19 in THF (5 ml) was
added. The solution was further stirred for 30 min and
washed with saturated NH4Cl solution. The organic layer
was dried over MgSO4 to give ole®n 20. Unpuri®ed ole®n
20 was hydrolyzed in 1N NaOH/90% MeOH (50 ml) for
7 h. The hydrolyzate was diluted with H2O (50 ml) and
extracted ®rst with CH2Cl2 (100 ml£2) and, after acidi®ca-
tion with HCl (20 ml), with CHCl3 (50 ml£2) and diethyl-
ether (50 ml). The combined organic layer was dried over
MgSO4 and puri®ed on silica gel with CHCl3/MeOH (5:1) to
give carboxylic acid 21 (135 mg, m/z 127.2 for [M2H]2).
Carboxylic acid 21 (135 mg) and EDC-HCl (240 mg) were
dissolved in CH2Cl2 (2 ml) and mixed with (R)-naphthyl-
ethylamine (180 mg) for reaction under stirring for 11 h.
Puri®cation of the reaction mixture over silica gel with
hexane/EtOAc (3:1) yielded amide 22 (225 mg, m/z 268.0
for [M1H]1). To a solution of amide 22 (100 mg, in THF
5 ml) was added 0.5 M 9-BBN in THF (1.5 ml) at 08C. The
temperature was brought to rt and the solution was stirred
for 3.5 h at rt. The second addition of 9-BBN was made and
the solution was stirred for 1.5 h. The hydroborate adduct
was oxidized by adding 3N NaOH (1 ml) and H2O2 (1 ml) at
08C and by stirring the mixture for 10 h at rt. The reaction
mixture was diluted with H2O (20 ml) and extracted with
EtOAc (20 ml£2). The combined organic layer was washed
with saturated NaCl solution (10 ml), dried over MgSO4 and
the residue was puri®ed over silica gel with hexane/EtOAc
(1:10) to give alcohol 23 (m/z 286.0 for [M1H]1). Solution
of (COCl)2 (300 ml in 3 ml CH2Cl2) and DMSO (300 ml)
were mixed at 2788C and stirred for 10 min. Alcohol 23 in
CH2Cl2 (2 ml) was added and stirring continued for another
15 min. Triethylamine was added and the solution was
brought to rt under stirring. A saturated solution of NH4Cl
(20 ml) was added and the reaction mixture was extracted
with diethyl ether (20 ml). The organic layer was dried over
MgSO4 to give aldehyde 24 (m/z 284.0 [M1H]1). The alde-
hyde 24 was oxidized with NaClO2 (100 mg) in a solution
containing 2-methyl-2-butene (200 ml) and NaH2PO4
(60 mg) in 10 ml of t-BuOH/H2O (7:3). After 3 h, the reac-
tion mixture was acidi®ed with dil. HCl, extracted with
CHCl3 (10 ml), and dried over MgSO4. Puri®cation of the
residue on silica gel with CHCl3/MeOH (5:1) yielded
carboxylic acid 25 (90 mg) {m/z 298.0 [M1H]1}. The
carboxylic acid 25 was reacted with (S)-1A2P-OTf
1
(R)-16: H NMR (300 MHz, CDCl3): d 0.92 (3H, d), 1.23
(3H, d), 1.68 (3H, d), 2.04 (1H, dd), 2.23 (3H, m), 2.35 (1H,
dd), 3.82 (2H, d), 5.27 (1H, dd), 5.99 (1H, m), 6.48 (amide,
d), 7.42, 7.51, 7.54, 7.62, 7.78, 7.83, 8.09, 8.43, 8.60 (naph
and anthracene), ESIMS; 587.2 [M1H]1.
Preparation of a diastereomixture standard from
3-methylglutaric acid (26)
3-Methylglutaric acid 26 (365 mg) was subjected to reaction
with (R)-naphthylethylamine (970 mg) and EDC-HCl
(479 mg) in CH2Cl2 (4 ml) for 10 h under stirring. The reac-
tion mixture was diluted with CHCl3 (30 ml) and washed
with 1N HCl (10 ml). The organic layer was dried over
MgSO4, and the residue was chromatographed on silica
gel with CHCl3/MeOH (10:1) to give half amide 27
(522 mg). The amide 27 (180 mg), EDC-HCl (120 mg),
and (R)-1A2P-OH (61 mg) were dissolved in CH2Cl2
(4 ml) and stirred for 10 h. The reaction mixture was diluted
with EtOAc (50 ml) and washed successively with 1N HCl
(30 ml), H2O (30 ml), and NaHCO3 (30 ml). The organic
layer was dried over MgSO4 and chromatographed on silica
gel with CHCl3/MeOH (50:1). The diastereomers of 16 were
separated on a Wakosil-II 5SIL-100 column with CHCl3.
1
(S)-16: H NMR (300 MHz, CDCl3): d 0.90 (3H, d), 1.30
(3H, d), 1.63 (3H, d), 1.97 (1H, dd), 2.23 (3H, m), 2.37 (1H,
dd), 3.86 (2H, d), 5.27 (1H, dd), 5.92 (1H, m), 6.15 (amide,
d), 7.39, 7.52, 7.63,7.83, 8.07, 8.44, 8.57 (naph and anthra-
cene), ESIMS; 587.3 [M1H]1.
Fluorimetric HPLC analysis of 16
The ¯uorimetric HPLC analysis was performed under the
following conditions: column, Cosmosil 5C18 (4.6£
250 mm, Nacalai tesque); solvent, 60% MeCN; ¯ow,
1.6 ml/min; detection, excitation at 298 nm and emission
at 462 nm. The retention time for (S)-16 was 18.30 min
and for (R)-16 19.67 min.
LC/MS analysis of 16
The LC/MS analysis was carried out under the following
conditions: column, Capcell pak (2.0£150 mm, Shiseido);
solvent, 60% MeCN; ¯ow, 0.4 ml/min; mass spectrometer,
Finnigan mat TSQ-700; ion mode, positive; capillary
celsius, 2508C; electron multiplier, 1200 V; sheath gas, 60
psi; auxiliary gas, 20 ¯ow-units; dwell time, 2.0 s. The
retention time 24.34 min for (S)-16 and 26.27 min for
(R)-16.
Acknowledgements
The authors acknowledge Professor H. Ohrui at Tohoku
University for his generous gift of AP-OTf and Professor
T. Kusumi at Tokushima University and Professor M.
Murata at Osaka University for discussions. This work
was supported by a Grant-in-Aid from the Ministry of

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