Structural determination of sulfoquinovosyldiacylglycerol by chiral syntheses

Article abstract of DOI:10.1016/S0040-4039(00)00638-9

Chiral sulfoquinovosyldiacylglycerols (SQDGs) have been synthesized to determine the absolute stereochemistry and the biological activities. The ¹ H NMR spectrum of a natural SQDG is comparable to that of synthetic (2S)-SQDG rather than that of the (2R) analogue. The biological activity of the respective isomers for DNA polymerase α and β inhibition was not distinguishable in the enzymatic assay.

Full text of DOI:10.1016/S0040-4039(00)00638-9

Tetrahedron Letters 41 (2000) 4403±4407
Structural determination of sulfoquinovosyldiacylglycerol by
chiral syntheses
Shinya Hanashima,a Yoshiyuki Mizushina,a Takayuki Yamazaki,a Keisuke Ohta,a
Shunya Takahashi,b Hiroyuki Koshino,b Hiroeki Sahara,c Kengo Sakaguchia and
Fumio Sugawaraa,*
aDepartment of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan
bThe Institute of Physical and Chemical Research, Wako, Saitama 351-9800, Japan
cMarine Biomedical Institute, Sapporo Medical University School of Medicine, Rishirifuji, Hokkaido 097-0101, Japan
Received 3 March 2000; revised 13 April 2000; accepted 14 April 2000
Abstract
Chiral sulfoquinovosyldiacylglycerols (SQDGs) have been synthesized to determine the absolute stereo-
chemistry and the biological activities. The 1H NMR spectrum of a natural SQDG is comparable to that of
synthetic (2S)-SQDG rather than that of the (2R) analogue. The biological activity of the respective isomers
for DNA polymerase a and b inhibition was not distinguishable in the enzymatic assay. # 2000 Elsevier
Science Ltd. All rights reserved.
Recently, DNA polymerase (pol) inhibitors, e.g. sulfolipids, fatty acids, sphingolipids, ceramides,
tepenoids and aromatic compounds, have been isolated from natural resources.1 12 The sulfolipids,
sulfoquinovosyldiacylglycerol (SQDG) and sulfoquinovosylmonoacylglycerol (SQMG), are sul-
fonic analogs of d-glucose bound to glycerol bearing fatty acids. We reported that sulfolipids in
the class of SQDG and SQMG from a fern,1 an alga2 and a marine invertebrate3 are potent
inhibitors of DNA pol a and b in vitro and human lung cancer in vivo. DNA polymerases are
essential for DNA replication and repair, and hence, enzymatic inhibitors lead to cell death.
SQDGs possess extensive biological activities such as anti-tumor e€ect,3 P-selectin receptor
inhibition,13 HIV-RT inhibition,2,14 AIDS-antiviral,15 and so on.
The stereochemistry of cyanobacterial SQDG has been determined to be (2S) by the comparison
of the optical rotation values.16 Although (2S)-SQDG was synthesized,16,17 the (2R) analogue has
not been prepared so far. We modi®ed our chiral synthetic method as follows to obtain both (2S)
* Corresponding author. Fax: +81 (0)471-23-9767; e-mail: sugawara@rs.noda.sut.ac.jp
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00638-9

4404
and (2R) analogues to determine the C-2 stereochemistry of natural SQDG (1) by comparison of
the 1H NMR data and the active isomer involved in in vitro DNA pol inhibition.
(1) d-Glucose was chosen as the glycosyl donor instead of d-glucal used in the syntheses of
three anti-HIV-1 sulfolipids.16 (2) As the glycosyl acceptor, enantiomerically pure (2S)-iso-
propylideneglycerol was used;17 19 however, the fatty acid esters of the chiral glycerols were
employed to avoid racemization under the acidic conditions of glycosylation.16
Compound 2 was prepared from d-glucose in several steps including the selective reduction of
40,60-O-benzylidene derivative with AlCl3±LiAlH4 in Et2O±CH2Cl2.20 Tosylation of 2 with TsCl
a€orded 3, followed by deallylation with PdCl2 in MeOH to give 4 (Scheme 1). The tosyloxy
group was then converted into a thioacetyl group with potassium thioacetate in DMF to yield 5.
The reaction of 5 with CCl3CN and DBU in CH2Cl2 selectively gave a-anomer 6 (H-10, ꢀ 6.41,
J=3.48 Hz),21 while no b-anomer was found. The glycosyl acceptors (9, 12) were prepared from
(2S)- and (2R)-isopropylidene glycerols by the following reactions: benzylation, deprotection,
esteri®cation (8, 11) and debenzylation (9, 12). The imidates (6) were treated with (2S)- or (2R)-
1,2-bis-O-stearylglycerol (9 or 12) under the presence of TMSOTf as Lewis acid. Two glycosylations
(6 and 9, 6 and 12) proceeded a-selectively and a€orded only a-anomer 13 and 14, respectively.21
Oxydation of 13 and 14 with OXONE16 gave sodium sulfonate derivatives (15, 16), and then the
Scheme 1. (a) TsCl, DMAP, C5H5N, rt, 15hr, 92% (3). (b) PdCl2, MeOH, rt, 18 h, 80% (4). (c) CH3COSK, DMF,
80ꢀC, 3 h, 86% (5). (d) CCl3CN, DBU, CH2Cl2, rt, 5.5 h, 70% (6). (e) TMSOTf, CH2Cl2, MS4A, rt, 6 h, 62% (13),
67% (14). (f) OXONE, AcOH, AcOK, rt, 4 h, 68.8% (15), 55.1% (16). (g) Pd/C, H2, rt, 18 h, 42.5% (17), 51.5% (18).
(h) BnBr, NaH, DMF, rt, 3 h. (i) TsOH, MeOH, rt, 16 h. (j) C17H35COOH, EDCI, DMAP, C5H5N, rt, 6 h, 37.5%
(three steps) (8), 84.8% (three steps) (11). (k) Pd/C, H2, rt, 4 h, 94.5% (9), 80.7% (12).

4405
1
catalytic hydrogenation with Pd±C a€orded chiral SQDGs 17 and 18, respectively.21 The H
NMR spectra of (2R)-SQDG (17), (2S)-SQDG (18) and the natural product (1) in DMSO-d6
indicated that the stereochemistry at C-2 of the natural product (1) (ꢀ 4.33(H-1a)/4.11(H-1b)) was
con®rmed to be S-form, since the chemical shifts of glycerol H-1 were ꢀ 4.30(H-1a)/4.22(H-1b) of
17 and 4.33(H-1a)/4.11(H-1b) of 18 (Table 1).
Table 1
1H NMR data (600 MHz) of the SQDGs in DMSO-d6
The synthetic compounds 17 and 18 inhibited eukaryotic DNA pol a and b1 similarly as illustrated
in Fig. 1. Since the IC50 values of the two compounds were not distinguishable, the chirality at C-2
of glycerol may not be essential for in vitro DNA pol inhibition. This observation may be useful
for further research and development of SQDGs for clinical usage.
Figure 1. Inhibition of DNA polymerases a and b by synthetic SQDGs 17 and 1822

4406
Acknowledgements
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science,
Sports and Culture of Japan 681-8049-09672167 to Sugawara, and also supported in part by
Sasakawa Scienti®c Research Grant to Mizushina from The Japan Science Society.
References
1. Mizushina, Y.; Watanabe, I.; Ohta, K.; Takemura, M.; Sahara, H.; Takahashi, N.; Gasa, S.; Sugawara, F.;
Matsukage, A.; Yoshida, S.; Sakaguchi, K. Biochem. Pharmacol. 1998, 55, 537±541.
2. Ohta, K.; Mizushina, Y.; Hirata, N.; Takemura, M.; Sugawara, F.; Matsukage, A.; Yoshida, S.; Sakaguchi, K.
Chem. Pharm. Bull. 1998, 46, 684; 1999, 22, 111±116.
3. Sahara, H.; Ishikawa, M.; Takahashi, N.; Ohtani, S.; Sato, N.; Gasa, S.; Akino, T.; Kikuchi, K. Br. J. Cancer
1997, 75, 324±332.
4. Ishiyama, H.; Ishibashi, M.; Ogawa, A.; Yoshida, S.; Kobayashi, J. J. Org. Chem. 1997, 62, 3831±3836.
5. Sun, D. A.; Deng, J. Z.; Shelley, R. S.; Sidney, M. H. J. Am. Chem. Soc. 1999, 121, 6120±6124.
6. Deng, J. Z.; Sun, D. A.; Shelley, R. S.; Sidney, M. H.; Ronald, L. C.; John, R. E. J. Chem. Soc., Perkin Trans. 1
1999, 1147±1149.
7. Chen, J.; Zhang, Y. H.; Wang, L. K.; Steven, J. S.; Angela, M. S.; Sidney, M. H. J. Chem. Soc., Chem. Commun.
1998, 2769±2770.
8. Ma, J.; Shelley, R. S.; Sidney, M. H. J. Nat. Prod. 1999, 62, 1660±1663.
9. Deng, J. Z.; Shelly, R. S.; Sidney, M. H. J. Nat. Prod. 1999, 62, 1624±1626.
10. Sun, D. A.; Shelly, R. S.; Edward, P. L.; Sidney, M. H. J. Nat. Prod. 1999, 62, 1110±1113.
11. Deng, J. Z.; Shelly, R. S.; Sidney, M. H.; Carl, F. I.; Mark, E. H. J. Nat. Prod. 1999, 62, 1000±1002.
12. Deng, J. Z.; Shelly, R. S.; Sidney, M. H. J. Nat. Prod. 1999, 62, 477±480.
13. Golik, J.; Dickey, J. K.; Todderud, G.; Lee, D.; Alford, J.; Huang, S.; Klohr, S.; Eustice, D.; Aru€o, A.; Agler,
M. L. J. Nat. Prod. 1997, 60, 387±389.
14. Loya, S.; Reshef, V.; Mizrachi, E.; Silberstein, C.; Rachamin, Y.; Carmeli, S.; Hizi, A. J. Nat. Prod. 1998, 61, 891±
895.
15. Gustafson, K. R.; Cardellina II, J. H.; Fuller, R. W.; Weislow, O. S.; Kiser, R. F.; Snader, K. M.; Patterson,
G. M. L.; Boyd, M. R. J. Nat. Can. Inst. 1989, 81, 1254±1258.
16. Gordon, D. M.; Danishefsky, S. J. J. Am. Chem. Soc. 1992, 114, 659±663.
17. Gigg, R.; Penglis, A. A. E.; Conant, R. J. Chem. Soc., Perkin Trans. 1 1980, 2490±2493.
18. Sugawara, F.; Nakayama, H.; Ogawa, T. Carbohydr. Res. 1982, 108, C5; 1982, 123, C25±C28.
19. Sugawara, F.; Nakayama, H.; Strobel, G. A.; Ogawa, T. Agric. Biol. Chem. 1986, 50, 2251±2259, 1986, 50, 2261±
2271.
20. Peter, F.; Andras, L.; Pal, N. Carbohydr. Res. 1982, 55±67.
21. Physicochemical data: Compound 6. [ꢁ]D +56.0ꢀ(c 1.17, CHCl3), IR (cm^1): 3250, 3000, 2950, 2850, 1930, 1860,
1
1650, 1430, 1340, 1200, 1090±930, 880, 770, H NMR (300 MHz, CDCl3): ꢀ 8.60 (1H, s, CˆNH), 7.32 (15H, m,
ArH), 6.40 (1H, d, J=3.5 Hz, anomeric H), 5.00 (1H, d, J=13.6 Hz, ArCH2O), 4.90 (1H, d, J=13.7 Hz,
ArCH2O), 4.82 (1H, d, J=10.7 Hz, ArCH2O), 4.74 (1H, d, J=11.7 Hz, ArCH2O), 4.67 (1H, d, J=11.7 Hz,
ArCH2O), 4.62 (1H, d, J=10.6 Hz, ArCH2O), 4.04 (2H, m), 3.71 (1H, dd, J=9.6 and 3.5 Hz), 3.42 (2H, m), 3.13
(1H, dd, J=13.9 and 6.7 Hz), 2.31 (3H, s, SCOCH3), LRFABMS: m/z 654.1 [M+H]+, HRFABMS: calcd m/z
654.1094 for C31H3353Cl237ClNO6S [M+H]+, found m/z 654.1071. Compound 13. [ꢁ]D +23.4ꢀ(c 1.16, CHCl3),
1H NMR (300 MHz, CDCl3): ꢀ 7.24±7.34 (15H, m, ArH), 5.25 (1H, m), 4.59±4.98 (7H, m, anomeric H and
ArCH2O), 4.37 (1H, dd, J=12.0 and 3.8Hz), 4.21 (1H, dd, J=11.9 and 6.0 Hz), 3.93 (1H, t, J=9.2 Hz), 3.73±3.81
(2H, m), 3.48±3.56 (2H, m), 3.37 (1H, dd, J=12.4 and 2.9 Hz), 3.30 (1H, t, J=9.2 Hz), 3.03 (1H, dd, J=13.7 and
7.5 Hz), 2.33 (3H, s, SCOCH3), 2.27 (4H, m, OCOCH2), 1.59 (4H, m, OCOCCH2), 1.25 (64H, br, CH2), 0.86 (6H,
t, J=6.9 Hz, CH3), 13C NMR (75 MHz, CDCl3): ꢀ 194.7, 173.3, 172.9, 138.5, 138.1, 137.9, 127.5±128.5, 96.7, 81.5,
80.2, 79.9, 75.7, 75.1, 72.9, 69.7, 69.7, 65.7, 62.4, 34.2, 34.1, 29.0±30.7, 24.8, 22.6, 14.1. Compound 14. [ꢁ]D
+32.5ꢀ(c 0.85, CHCl3), 1H NMR (300 MHz, CDCl3): ꢀ 7.32 (15H, m, ArH), 5.23 (1H, m), 4.96 (1H, d, J=10.8 Hz,
ArCH2O), 4.89 (d, 1H, J=10.7 Hz, ArCH2O), 4.79 (d, 1H, J=11.0 Hz, ArCH2O), 4.74 (d, 1H, J=12.0 Hz,

4407
ArCH2O), 4.69±4.58 (3H, m, ArCH2O, anomeric H), 4.41 (1H, dd, J=11.9 and 3.7 Hz), 4.21 (1H, dd, J=11.9 and
6.3 Hz), 3.94(1H, t, J=9.2 Hz), 3.77±3.75 (m, 2H), 3.53±3.46 (m, 2H), 3.40 (dd, 1H, J=13.7 and 3.0 Hz), 3.31 (t,
1H, J=9.2 Hz), 3.05 (dd, 1H, J=13.7 and 7.5 Hz), 2.33 (3H, s, SCOCH3), 2.31 (4H, m, OCOCH2), 1.60 (4H, m,
OCOCCH2), 1.25 (64H, br, CH2), 0.88 (6H, t, J=6.4 Hz, CH3), 13C NMR (75 MHz, CDCl3): ꢀ 194.7, 173.7,
173.0, 138.5, 138.1, 137.8, 128.4±127.6, 97.1, 81.5, 80.2, 80.1, 75.2, 73.0, 69.63, 69.59, 66.1, 62.4, 34.2, 34.1, 31.9,
30.7, 30.5, 29.7±29.1, 24.9, 22.6, 14.1. Compound 17. [ꢁ]D +23.6ꢀ(c 0.39, chloroform:methanol:water=65:25:4),
LRFABMS (negative mode) m/z 849.5 [M^H]^, 583.3 [M^C18:0]^, 565.5 [M^C18:0-H2O]^ HRFABMS calcd m/z
849.5762 for C45H85O12S [M^H]^, found m/z 849.5764, 13C NMR (125 MHz, DMSO-d6): ꢀ 172.48, 172.36, 98.30,
74.46, 72.83, 71.59, 69.77, 68.55, 64.95, 62.36, 33.56, 33.43, 31.28, 29.04, 29.00, 28.95, 28.81, 28.78, 28.70, 28.44,
24.47, 22.08, 13.93. Compound 18. [ꢁ]D +38.8ꢀ (c 0.42, chloroform:methanol:water=65:25:4), LRFABMS
(negative mode) m/z 849.5 [M^H]^, 583.3 [M^C18:0]^, 565.5 [M^C18:0^H2O]^, HRFABMS calcd m/z 849.5762
for C45H85O12S [M^H]^, found m/z 849.5694, 13C NMR (125 MHz, DMSO-d6): ꢀ 172.50, 172.35, 98.28, 74.34,
72.88, 71.60, 69.70, 68.53, 64.60, 62.62, 33.55, 33.42, 31.29, 29.05, 29.01, 28.83, 28.79, 28.71, 28.46, 24.45, 22.09,
13.92.
22. The standard reaction mixture for pol a puri®ed from calf thymus (or recombinant rat DNA pol b puri®ed from
E. coli JMpb5) and Taq pol contained 50 mM tris^HCl, 1 mM dithiothreitol, 1 mM MgCl2, 5 mM poly(dA)/
oligo(dT)12 18(2/1), 10 mM [3H]dTTP (100 cpm/pmol), 15% glycerol and 8 ml of an enzyme inhibitor solution in
DMSO. After incubation, the radioactive DNA product was collected on a DEAE-cellulose paper disc, and then
the radioactivity was measured in a scintillation counter.