Chemoenzymatic total synthesis of (+)-conagenin, a low-molecular-weight immunomodulator

Article abstract of DOI:10.1016/S0040-4039(01)00649-9

Total synthesis of (+)-conagenin, a low-molecular-weight immunomodulator, was achieved based on enantioselective enzymatic reactions followed by chemoselective reductions.

Full text of DOI:10.1016/S0040-4039(01)00649-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4029–4031
Chemoenzymatic total synthesis of (+)-conagenin, a
low-molecular-weight immunomodulator
Shigeki Sano, Toshio Miwa, Kazuhiko Hayashi, Kazuo Nozaki, Yohei Ozaki and
Yoshimitsu Nagao*
Faculty of Pharmaceutical Sciences, The University of Tokushima, Sho-machi, Tokushima 770-8505, Japan
Received 19 March 2001; revised 10 April 2001; accepted 13 April 2001
Abstract—Total synthesis of (+)-conagenin, a low-molecular-weight immunomodulator, was achieved based on enantioselective
enzymatic reactions followed by chemoselective reductions. © 2001 Elsevier Science Ltd. All rights reserved.
(+)-Conagenin (CNG, 1), a natural product originally
isolated from fermentation broths of Streptomyces
roseosporus, has been found to stimulate activated T
cells by producing lymphokines as a low-molecular-
weight immunomodulator.1–3 In addition, the improve-
ment of efficacy of antitumor agents, such as
mitomycin C and adriamycin, by CNG (1) has also
been reported.4–9 These biological activities of CNG (1),
possessing an a-substituted serine moiety, has made this
compound an intriguing target for total synthesis. An
asymmetric total synthesis10 and an asymmetric formal
synthesis11 of CNG (1) have been reported, as have
investigations of further syntheses12,13 of diastereoiso-
mers of CNG (1). We report herein a successful conver-
gent method for the total synthesis of CNG (1) based
on enantioselective enzymatic reactions followed by
chemoselective reductions.
amorphous powder from s-symmetric prochiral diethyl
a-aminomalonate (4) by utilizing our chemoenzymatic
methodology based on enantioselective enzymatic
hydrolysis followed by chemoselective enantiodivergent
reduction.14 Methylation of 5 with (trimethylsilyl)-
diazomethane gave the (S)-Z-a-methylserine methyl-
ester (6) in 66% yield. Hydrogenolytic deprotection of
the N-Z group of 6, followed transesterification of the
resultant (S)-a-methylserine methylester in BnOH and
Et3N (10:1) at 60°C, afforded (S)-a-methylserine benzyl
ester (7) {colorless oil, [h]2D6 −9.0° (c 0.66, CHCl3)} in
44% yield from 6, as shown in Scheme 2.
Pentanoic acid 13 was also prepared by the chemoenzy-
matic sequence illustrated in Scheme 3. Photosensitized
oxygenation of 5-methylcyclopentadiene (8),15,16 fol-
lowed by in situ reduction with thiourea, furnished
s-symmetric prochiral 5-methyl-2-cyclopentene-1,4-diol
(2) in 35% yield.17 Enzymatic acetylation of 2 with
lipase AK (Amano Pharmaceutical Co. Ltd) in the
The retrosynthetic analysis of CNG (1) is outlined in
Scheme 1. (S)-Z-a-Methylserine (5) was synthesized in
94% ee {[h]2D6 +10.1° (c 0.88, EtOH)} as a colorless
Scheme 1. Retrosynthetic analysis of (+)-conagenin (CNG, 1).
Keywords: amino acids and derivatives; asymmetric synthesis; enzymes and enzyme reactions; natural products; reduction.
* Corresponding author. Fax: +81-88-633-9503; e-mail: ynagao@ph2.tokushima-u.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00649-9

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S. Sano et al. / Tetrahedron Letters 42 (2001) 4029–4031
methylation with MeI gave methyl ester 12 {colorless
oil, [h]2D6 +89.5° (c 1.00, CHCl3)} in 85% yield. After
benzylation of 12 with benzyl 2,2,2-trichloroacetimidate
in the presence of BF3·Et2O,22 the resulting ester was
subjected to alkaline hydrolysis to give pentanoic acid
13 {colorless oil, [h]2D6 +3.1° (c 1.04, CHCl3)} in 63%
yield from 12.
Scheme 2. (a) Porcine liver esterase (PLE)/1/15 M phosphate
buffer (pH 7.0)–MeCN (10:1)/rt/12 h; (b) LiBH4/Et2O–THF
(3:1)/reflux/1.5 h; (c) TMSCHN2/MeOH–benzene (2:7)/rt/4 h;
(d) H2/10% Pd–C/MeOH/rt/12 h; (e) BnOH–Et3N (10:1)/
60°C/2 days.
Condensation of a-methylserine derivative 7 with pen-
tanoic acid 13 accompanied by 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDC·HCl)
in the presence of N-methylmorpholine (NMM) and
1-hydroxybenzotriazole (HOBt) in DMF furnished the
protected CNG 14 {colorless oil, [h]2D6 +22.7° (c 0.52,
CHCl3)} as an enantiomerically pure form in 57% yield
(Scheme 4). Finally, compound 14 was submitted to
catalytic hydrogenolysis on Pd–C to give CNG (1)
{colorless powder, [h]2D5 +48.4° (c 0.40, MeOH), lit.1
[h]D27 +55.4°, lit.10 [h]2D6 +48.7° (c 0.43, MeOH)} in 95%
yield. The accomplishment of this total asymmetric
synthesis was confirmed by the identity of all physico-
chemical data of the synthesized and the natural CNG
(1).23
presence of vinyl acetate as an acyl donor afforded a
chiral mono ester 9 {colorless oil, [h]2D5 −76.3° (c 0.98,
CHCl3)} in 94% yield with high enantiomeric excess
(99% ee).18 The absolute configuration of 9 was deter-
1
mined by H NMR analysis (400 MHz, CDCl3) of the
Mosher
ester
[(R)-
and
(S)-methoxy(trifl-
uoromethyl)phenylacetate (MTPA)] derivatives of 9.19
After benzylation of 9 with BnBr in the presence of
Ag2O in 87% yield, oxidative cleavage of the double
bond of the resulting benzyl ether 10 {colorless oil, [h]D26
−4.1° (c 1.01, CHCl3)}, followed by alkaline hydrolysis
of the acetoxy group, lactonization with cyanuric chlo-
ride, and chemoselective reduction of the activated
carboxyl group,20 provided the desired primary alcohol
11 {colorless column from Et2O, mp 61–63°C, [h]D26
+182.4° (c 1.02, CHCl3)} without isolation of each
intermediate (23% yield, four steps). Radical-induced
deoxygenation of 11 was performed using Barton’s
method21 to give the corresponding chiral lactone
3 {colorless powder from Et2O, mp 91–94°C, [h]D25
+146.7° (c 1.00, CHCl3)} (46% yield, two steps). The
desired stereochemistry of 3 was revealed by NOE
analysis (300 MHz, CDCl3) as follows. When C3-H of
3 was irradiated, an NOE enhancement of the C4- and
C5-Me protons was recognized, as shown in Fig. 1. A
similar NOE enhancement between the C4- and C5-Me
protons of 3 was also observed. Treatment of the
lactone 3 with aqueous NaOH in MeOH followed by
In summary, we have described a convergent and
chemoenzymatic total synthesis of (+)-conagenin
(CNG, 1). This methodology will also be applicable for
the synthesis of a variety of stereoisomers of CNG (1).
O
2
BnO
3
1
O
H
H
4
5
H
3
Me
Me
1
1
Figure 1. Selected H–1H NOE enhancements (300 MHz H
NMR, CDCl3) for 3.
Scheme 3. (a) O2/hw/rose bengal/AcONa/MeOH/−78°C/80 min; (b) (NH2)2CS/MeOH/rt/13 h; (c) lipase AK/CH2ꢀCHOAc/THF/
rt/7 h; (d) Ag2O/BnBr/KI/toluene/rt/15.5 h; (e) NaIO4/KMnO4/Na2CO3/acetone–H2O (5:6)/rt/1 h; (f) 1N NaOH/MeOH/rt/1 h;
(g) H+; (h) cyanuric chloride/NMM/DME/rt/3 h; (i) NaBH4/0°C/10 min; (j) PhOCSCl/DMAP/CH2Cl2/rt/4 h; (k) AIBN/n-
Bu3SnH/toluene/75°C/1 h; (l) 1N NaOH/MeOH/rt/1 h; (m) H+ (pH 6–7); (n) K2CO3/MeI/acetone/rt/3.5 h; (o) BnOCNHCCl3/
BF3·Et2O/cyclopentane/rt/2 h; (p) 1N K2CO3/MeOH/reflux/2 h.

S. Sano et al. / Tetrahedron Letters 42 (2001) 4029–4031
J. Antibiot. 1999, 52, 548–551.
4031
10. Hatakeyama, S.; Fukuyama, H.; Mukugi, Y.; Irie, H.
Tetrahedron Lett. 1996, 37, 4047–4050.
11. Enders, D.; Bartsch, M.; Runsink, J. Synthesis 1999,
243–248.
´
12. Kova´cs-Kulya´ssa, A.; Herczegh, P.; Sztaricskai, F. Tetra-
Scheme 4. (a) EDC·HCl/NMM/HOBt/DMF/rt/6 h; (b) H2/
10% Pd–C/MeOH/rt/5 days.
hedron Lett. 1996, 37, 2499–2502.
´
13. Kova´cs-Kulya´ssa, A.; Herczegh, P.; Sztaricskai, F. Tetra-
hedron 1997, 53, 13883–13896.
Acknowledgements
14. Sano, S.; Hayashi, K.; Miwa, T.; Ishii, T.; Fujii, M.;
Mima, H.; Nagao, Y. Tetrahedron Lett. 1998, 39, 5571–
5574.
This work was partially supported by a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Science, Sports and Culture, Japan. The authors are
grateful to Professor S. Hakakeyama (Nagasaki Uni-
versity) for useful discussions and for kindly providing
(+)-conagenin and its spectral data. They would also
like to thank Amano Pharmaceutical Co. Ltd for the
supply of lipase AK.
15. McLean, S.; Haynes, P. Tetrahedron 1965, 21, 2313–2327.
16. McLean, S.; Haynes, P. Tetrahedron 1965, 21, 2329–2342.
17. Kaneko, C.; Sugimoto, A.; Tanaka, S. Synthesis 1974,
876–877.
18. Enantiomeric excess was determined by CSP HPLC anal-
ysis (Chiralcel OD, hexane–propan-2-ol).
19. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J.
Am. Chem. Soc. 1991, 113, 4092–4096.
20. Falorni, M.; Porcheddu, A.; Taddei, M. Tetrahedron
Lett. 1999, 40, 4395–4396.
21. Barton, D. H. R.; Motherwell, W. B.; Stange, A. Synthe-
sis 1981, 743–745.
References
1. Yamashita, T.; Iijima, M.; Nakamura, H.; Isshiki, K.;
Naganawa, H.; Hattori, S.; Hamada, M.; Ishizuka, M.;
Takeuchi, T.; Iitaka, Y. J. Antibiot. 1991, 44, 557–559.
2. Kawatsu, M.; Yamashita, T.; Osono, M.; Ishizuka, M.;
Takeuchi, T. J. Antibiot. 1993, 46, 1687–1691.
3. Ishizuka, M.; Kawatsu, M.; Yamashita, T.; Ueno, M.;
Takeuchi, T. Int. J. Immunopharmacol. 1995, 17, 133–139.
4. Kawatsu, M.; Yamashita, T.; Osono, M.; Masuda, T.;
Ishizuka, M.; Takeuchi, T. J. Antibiot. 1993, 46, 1692–
1698.
5. Kawatsu, M.; Yamashita, T.; Ishizuka, M.; Takeuchi, T.
J. Antibiot. 1994, 47, 1123–1129.
6. Kawatsu, M.; Yamashita, T.; Ishizuka, M.; Takeuchi, T.
J. Antibiot. 1995, 48, 222–225.
7. Kawatsu, M.; Yamashita, T.; Ishizuka, M.; Takeuchi, T.
Anticancer Res. 1996, 16, 2937–2941.
22. Iversen, T.; Bundle, D. R. J. Chem. Soc., Chem. Com-
mun. 1981, 1240–1241.
23. Synthesized (+)-conagenin (CNG, 1): 1H NMR (400
MHz, CD3OD) l 0.93 (3 H, d, J=7.1 Hz), 1.22 (3 H, d,
J=6.3 Hz), 1.50 (3 H, s), 1.89 (1 H, dq, J=2.4, 6.3 Hz),
3.84 (1 H, d, J=11.0 Hz), 3.85 (1 H, q, J=6.1 Hz), 4.04
(1 H, d, J=11.0 Hz), 4.15 (1 H, d, J=2.4 Hz); 13C NMR
(75 MHz, CD3OD) l 8.4, 20.0, 21.2, 43.7, 62.8, 66.1,
71.2, 75.1, 175.8, 176.9. The synthesized methyl ester of
(+)-conagenin (CNG, 1) also had spectroscopic properties
identical to the authentic sample:10 1H NMR (400 MHz,
CDCl3) l 0.92 (3 H, d, J=7.1 Hz), 1.26 (3 H, d, J=6.3
Hz), 1.55 (3 H, s), 1.8 (2 H, brs), 2.09 (1 H, tq, J=2.1, 7.1
Hz), 2.40 (1 H, brs), 3.81 (3 H, s), 3.84 (1 H, d, J=11.2
Hz), 3.9 (1 H, brs), 4.06 (1 H, d, J=11.2 Hz), 4.35 (1 H,
d, J=2.2 Hz), 7.51 (1 H, brs); 13C NMR (75 MHz,
CDCl3) l 4.9, 20.5, 21.9, 40.3, 53.0, 61.9, 66.8, 72.2, 77.2,
173.0, 173.3
8. Kawatsu, M.; Yamashita, T.; Ishizuka, M.; Takeuchi, T.
Anticancer Res. 1997, 17, 917–922.
9. Hamada, M.; Sonotake, E.; Yamamoto, S.; Moriguchi, S.
.