1452
D. Lazarevic et al. / Carbohydrate Research 344 (2009) 1449–1452
0
0
0
0
0
0
0
0
Gel P2 with aq NH4HCO3 (250 mM). After freeze drying the prod-
J5 ,6b 2.0, J4 ,5 5.6, J3,4 J4,5 9.6, J5,6a 4.4, J5,6b 2.2, J2a ,2e 12.4, J2a ,3
uct, 10 was obtained as a colorless, very hygroscopic powder:
12.5 Hz.
1
24 mg (32%). H NMR (400 MHz, D2O): d 8.01 (d, 1H, H-600); 6.03
(d, 1H, H-10); 6.02 (d, 1H, H-500); 5.48 (dd, 1H, H-1); 4.46 (m, 2H,
H-20, H-30); 4.37 (m, 1H, H-40); 4.26 (m, 2H, H-5a0, H-5b0); 4.01
(dd, 1H, H-3); 3.95 (dd, 1H, H-4); 3.88 (br t, 1H, H-5); 3.68 (m,
2H, H-6a, H-6b); 2.11 (ddd, 1H, H-2a); 2.05 (ddd, 1H, H-2e); J1,P
7.1, J1,2a 3.5, J1,2e < 1, J2a,2e 13.5, J2a,3 11.7, J2e,3 5.7, J3,4 3.2, J4,5 < 1,
3.8. 2-Deoxy-
a
-
D
-lyxo-hexopyranosyl-1-phosphate (6)
-lyxo-hexopyranosyl)-
Dibenzyl (3,4,6-tri-O-benzyl-2-deoxy-
a-D
1-phosphate (15, 145 mg, 0.21 mmol) was dissolved in 1:2:1
water–MeOH–EtOAc (10 mL) and treated with Pd-on-charcoal
(10%, 10 mg) under hydrogen (50 bar) at room temperature for
10 h. After filtration over Celite and evaporation of the solvent,
the residue was lyophilized. The raw material was purified on
Bio-Gel P2 with deionized water. After lyophilization 33 mg
(65%) of the free-acid monoester 6 was obtained as a colorless
J5,6a 5.6, J5,6b 6.1, J6a,6b 10.9, J1 ,2 4.9, J5 ,6 8.1 Hz. 13C NMR
(100 MHz, D2O): d 166.6 (C-400); 152.5 (C-200); 142.0 (C-600); 103.0
(C-500); 92.7 (d, C-1); 88.8 (C-10); 83.6 (d, C-40); 74.1 (C-20); 72.0
(C-5); 70.0 (C-30); 68.9 (C-4); 66.5 (C-3); 65.3 (d, C-50); 61.9 (C-
0
0
00 00
6); 32.7 (d, C-2); JC-1,P 5.9, JC-2,P 8.3, JC-4 ,P 9.1, JC-5 ,P 5.3 Hz. 31P
NMR (202.5 MHz, D2O): d ꢀ10.5, ꢀ12.7.
0
0
amorphous foam: ½a D20
ꢂ
17.5 (c 1.0, D2O); 1H NMR (400 MHz,
D2O): d 5.47 (m, 1H, H-1); 3.98 (ddd, 1H, H-3); 3.92 (dd, 1H, H-
4); 3.84 (dt, 1H, H-5); 3.67 (m, 2H, H-6a, H-6b); 2.14 (ddd, 1H,
H-2a); 2.08 (ddd, 1H, H-2e); J1,P 7.3, J1,2a 3.6, J1,2e < 1, J2a,2e 13.7,
J2a,3 11.7, J2e,3 5.6, J4,5 < 1, J5,6a 6.1, J5,6b 6.1, J6a,6b 10.7 Hz. 13C NMR
(100 MHz, D2O): d 92.3 (d, C-1); 71.5 (C-5); 68.6 (C-4); 66.2 (C-
3); 61.9 (C-6); 32.1 (d, C-2); JC-1,P 5.7, JC-2,P 8.3 Hz. 31P NMR
(202.5 MHz, acetone-d6): d ꢀ0.21.
3.6. 2-Deoxy-b-
D-lyxo-hexopyranosyl-(1?4)-2-acetamido-2-
deoxy- -glucopyranose (12)
D
2-Deoxy-
1.22 mmol) was dissolved together with KCl (91 mg, 1.22 mmol),
PEP (554 mg, 2.7 mmol), ATP (37 mg, 61 mol), DTT (20 mg,
126 mol), UTP (36 mg, 61 mol), UDP-Glc (38 g, 61 mol), and
GlcNAc (300 mg, 1.34 mol) in 50 mM cacodylate buffer
(12.2 mL, pH 7.5) and degassed with argon for 10 min. Then
MgCl2ꢁ6 H2O (12 mg, 61 mol), BSA (12 mg), NaN3 (microspatula-
ful), MnCl2 (12 mg, 61 mol), GK (2U), PK (50 U) inorganic pyro-
D-lyxo-hexose (2-deoxy-D-galactose, 1, 200 mg,
l
l
l
l
l
l
Acknowledgments
l
Support of this study by the Fonds der Chemischen Industrie,
the Max-Buchner-Forschungsstiftung, and the Deutsche Fors-
chungsgemeinschaft (SFB 470, A5) is gratefully acknowledged.
l
phosphatase (50 U), UDP-glucose pyrophosphorylase (10 U),
galactose-1-phosphate uridyltransferase (5 U), and galactosyl-
transferase (3 U) were added. The reaction proceeded at 30 °C for
seven days under shaking, and the turnover was monitored by
measuring the amounts of both phosphate28 and pyruvate that
formed.29 Workup as above was followed by ion-exchange chro-
matography. Further purification was done by gel-permeation
chromatography on Bio-Gel P2 with water as eluent. After freeze
drying colorless amorphous compound 12 was obtained as an ano-
meric mixture: yield 131 mg (29%). 13C NMR (63 MHz, D2O): d
References
1. Sharan, N.; Lis, H. Eur. J. Biochem. 1993, 218, 1–27.
2. Varki, A. Glycobiology 1993, 3, 97–130.
3. Kobata, A. Acc. Chem. Res. 1993, 26, 319–324.
4. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
5. Gamblin, D. P.; Scoulan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131–163.
6. Paulsen, H. Angew. Chem., Int. Ed. Engl. 1990, 29, 823–839.
7. Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1503–1531.
8. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed.
Engl. 1995, 34, 412–432.
173.31 (NHCOCH3); 99.42 (C-10); 93.93 (C-1, b); 89.59 (C-1,
77.87 (C-4,
); 77.44 (C-4, b); 74.65 (C-50); 73.79 (C-5, b); 71.48
(C-3, b); 69.17 (C-5, ); 68.30 (C-3,
); 66.72 (C-30); 65.73 (C-40);
); 32.54 (C-
). FABMS: m/z 368
a);
9. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed.
Engl. 1995, 34, 521–546.
a
a
a
10. Bock, K.; Lundt, I.; Pedersen, C. Acta Chem. Scand. 1981, 35, 155–162.
11. Angyal, S. J.; Pickles, V. A. Aust. J. Chem. 1972, 25, 1711–1718.
12. Leon, B.; Liemann, S.; Klaffke, W. J. Carbohydr. Chem. 1993, 12, 597–610.
13. Nunez, H. A.; Barker, R. Biochemistry 1980, 19, 489–495.
14. Heidlas, J. E.; Lees, W. J.; Whitesides, G. M. J. Org. Chem. 1992, 57, 152–157.
15. Thiem, J.; Wiemann, T. Angew. Chem., Int. Ed. Engl. 1991, 30, 1163–1164.
16. Wong, C.-H.; Wang, R.; Ichikawa, Y. J. Org. Chem. 1992, 57, 4343–4344.
17. Thiem, J.; Wiemann, T. Synthesis 1992, 141–145.
60.43 (C-60); 59.18 (C-6); 55.29 (C-2, b); 52.80 (C-2,
a
20); 21.25 (NHCOCH3, b); 20.97 (NHCOCH3,
a
(M++1), 390 (M++Na).
3.7. 3,4,6-Tri-O-acetyl-2-deoxy-b-D-lyxo-hexopyranosyl-(1?4)-
1,3,6-tri-O-acetyl-2-acetamido-2-deoxy-a/b-D-glucopyranose
(13)
18. Berliner, L. J.; Robinson, R. D. Biochemistry 1982, 21, 6340–6343.
19. Hindsgaul, O.; Kaur, K. J.; Gokhale, U. B.; Srivastava, G.; Alton, G.; Palcic, M. M.
ACS Symp. Series 1991, 466, 38–50.
20. Lazarevic, D.; Thiem, J. Carbohydr. Res. 2006, 341, 569–576.
21. Srivastava, G.; Hindsgaul, O.; Palcic, M. M. Carbohydr. Res. 1993, 245, 137–144.
22. Perich, J. W.; Johns, R. B. Tetrahedron Lett. 1987, 28, 101–102.
23. Perich, J. W.; Johns, R. B. Synth. Commun. 1988, 142–144.
24. Fukase, K.; Kamikawa, T.; Iwai, Y. Bull. Chem. Soc. Jpn. 1991, 238, 287–306.
25. Hansen, R. G.; Freedland, R. A. J. Biol. Chem. 1955, 216, 303–307.
26. Roseman, S.; Distler, J. J.; Moffat, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83,
659–664.
By peracetylation of 12 (Ac2O, pyridine room temperature) and
workup, the heptaacetate 13 was obtained as an amorphous color-
less material. 1H NMR (400 MHz, (CD3)2CO): d 7.15 (d, 1H, NH,
a
);
); 5.77 (d, 1H, 1-Hb); 5.23 (m,
1H, 3-H); 5.23 (m, 1H, 40-H); 5.04 (ddd, 1H, 30-H); 4.83 (dd, 1H, 10-
7.11 (d, 1H, NH, b); 6.06 (d, 1H, 1-H
a
H); 4.34 (dd, 1H, 6a-H); 4.33 (ddd, 1H, 2-Ha); 4.23 (dd, 1H, 6b-H);
27. Moffat, J. G. Methods Enzymol. 1966, 8, 136–141.
4.20 (dd, 1H, 6a0-H); 4.11 (dd, 1H, 6b0-H); 4.07 (m, 1H, 2-Hb); 4.00
28. Fiske, C. H.; Subbarow, Y. P. J. Biol. Chem. 1925, 66, 375–400.
29. Raynard, A. M.; Hass, L. F.; Jacobsen, D. D.; Boyer, P. D. J. Biol. Chem. 1961, 236,
2277–2283.
(ddd, 1H, 50-H); 3.95 (t, 1H, 4-H); 3.83 (ddd, 1H, 5-H); 1.95 (m, 1H,
2e0-H); 1.76 (ddd, 1H, 2a0-H); JNH,2 9.4 (
a
, b), J1e,2 3.6 (
a), J1a,2 8.8 (b),
0
0
0
0
0
0
0
0
0
0
J2e ,3 3.2, J3 ,4 5.0, J1 ,2a 9.6, J1 ,2e 2.2, J6a,6b 12.2, J2,3 10.4, J5 ,6a 3.6,