J. Am. Chem. Soc. 2000, 122, 6803-6804
6803
Scheme 1. (a) The Reaction Catalyzed by Ep and (b) the
Syntheses of R-D-Hexopyranosyl Phosphatesa
A General Enzymatic Method for the Synthesis of
Natural and “Unnatural” UDP- and TDP-Nucleotide
Sugars
Jiqing Jiang, John B. Biggins, and Jon S. Thorson*
Laboratory for Biosynthetic Chemistry
Memorial Sloan-Kettering Cancer Center
and the Sloan-Kettering DiVision
Joan and Sanford I. Weill Graduate School of
Medical Sciences, Cornell UniVersity
1275 York AVenue, Box 309, New York, New York 10021
ReceiVed April 26, 2000
Many bioactive metabolites possess unusual carbohydrates
required for molecular recognition.1 The glycosyltransferases
which incorporate these essential ligands are known to rely almost
exclusively upon UDP- and TDP-nucleotide sugars and some have
demonstrated promiscuity toward the sugar donor.2 These dis-
coveries have led to the exploitation of the carbohydrate biosyn-
thetic machinery to manipulate metabolite glycosylation,3 revi-
talizing interest in methods to expand the repertoire of available
UDP- and TDP-sugar nucleotides.4 We now report that a substrate
specificity reevaluation of Salmonella enterica LT2 R-D-glucopy-
ranosyl phosphate thymidylyltransferase (Ep) reveals this enzyme
can convert a wide array of R-D-hexopyranosyl phosphates to their
corresponding UDP- and TDP-nucleotide sugars. Thus, we present
a general chemoenzymatic method to rapidly generate these
reagents, the significance of which is in providing a substrate set
for developing in vitro glycosylation systems.5
The selected enzyme for this study is a member of the prevalent
nucleotidylyltransferase family responsible for the reversible
conversion of R-D-glucopyranosyl phosphate (Scheme 1a, 2) and
NTP (e.g. 1) to the corresponding NDP-sugar nucleotide (3) and
pyrophosphate (4). Of the many nucleotidylyltransferases studied,
the 3-forming thymidylyltransferases have received the least
attention.6 The best characterized thymidylyltransferase (Ep) is
from Salmonella in which substrate specificity studies were
limited to only a few available hexopyranosyl phosphates.6a To
extend these studies, we overexpressed the rmlA-encoded Ep in
E. coli to provide the desired Ep as >5% of the total soluble
protein.7 The corresponding Ep was purified to near homogeniety
a (a) Ph3P, CCl4; (b) Ac2O, pyr; (c) (i) LiAlH4, (ii) AcOH/HCl, (iii)
BzCl, pyr; (d) BzCl, pyr; (e) pFPTC-Cl, DMAP; (f) (n-Bu)3SnH; (g) (i)
NaH, imidazole; (ii) CS2; (iii) CH3I; (h) AIBN, (n-Bu)3SnH; (i) (i)
CF3CO2H, (ii) BzCl, pyr; (j) EtS-TMS, ZnI2; (k) (i) NaOMe; (ii) NaH,
BnBr; (l) (BnO)2P(O)OH, CF3SO3H, NIS; (m) H2, Pd/C; (n) (i) HBr; (ii)
(BnO)2P(O)OH, silver triflate, 2,4,6-collidine; (o) NaOH; (p) AcOH/HCl.
In each case, cation exchange provided the Na+ salt.
with a specific activity of 110 U mg-1, a 2-fold improvement
over the previously reported values.6a,8
* To whom correspondence should be addressed. E-mail: jthorson@
sbnmr1.ski.mskcc.org. Fax: (212) 717-3066.
(1) (a) Liu, H.-w.; Thorson, J. S. Annu. ReV. Microbiol. 1994, 48, 223-
256. (b) Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 14, 99-110. (c)
Omura, S., Ed. In Macrolide Antibiotics, Chemistry, Biology and Practice;
Academic Press: New York, 1984. (d) Johnson, D. A.; Liu, H.-w. Curr. Opin.
Chem. Biol. 1998, 2, 642-649. (e) Trefzer, A.; Salas, J. A.; Bechthold, A.
Nat. Prod. Rep. 1999, 16, 283-299.
Most of the R-D-hexopyranosyl phosphates examined were
synthesized from free sugars while 2, 56, and 57 were com-
mercially available. For most synthetically derived glycosyl
phosphates (Scheme 1b), a general phosphorylation strategy from
the appropriately protected precursor relied upon (i) anomeric
activation via the ethyl-thio-â-D-pyranoside (9, 17, 25, 30, 35,
and 409), (ii) deprotection/reprotection (10, 18, 26, 31, and 36),
(iii) phosphorylation (11, 19, 27, 32, 37, and 41), and (iv)
complete deprotection (12, 20, 28, 33, 38, and 43). The overall
yield of this four-step phosphorylation strategy ranged from 19
to 28% including the final ion exchange. Alternatively, phospho-
rylation (45, 49, and 53) via the glycosyl halide followed by
(2) Gal, D-galactose; Glc, D-glucose; Man, D-mannose; NTP, nucleotide
triphosphate; pFPTC, pentafluorophenoxythiocarbonyl; TDP, thymidine diphos-
phate; TMP, thymidine monophosphate; TTP, thymidine triphosphate; UDP,
uridine diphosphate.
(3) (a) Madduri, K.; Kennedy, J.; Rivola, G.; Inventi-Solari, A.; Filppini,
S.; Sanuso, G.; Colombo, A. L.; Gewain, K. M.; Occi, J. L.; MacNeil, D. J.;
Hutchinson, C. R. Nature Biotech. 1998, 16, 69-74. (b) Zhao, L.; Ahlert, J.;
Xue, Y.; Thorson, J. S.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc. 1999,
121, 9881-9882 and references therein.
(4) (a) Zhao, Y.; Thorson, J. S. J. Org. Chem. 1998, 63, 7568-7572. (b)
Elhalabi, J. M.; Rice, K. G. Curr. Med. Chem. 1999, 6, 93-116.
(5) Altreuter, D. H.; Clark, D. S. Curr. Opin. Biotech. 1999, 10, 130-
136.
(6) (a) Lindquist, L.; Kaiser, R.; Reeves, P. R.; Lindberg, A. A. Eur. J.
Biochem. 1993, 211, 763-770. (b) Gallo, M. A.; Ward J.; Hutchinson, C. R.
Microbiol. 1996, 142, 269-275.
(7) The gene rmlA was previously known as rfbA (Reeves et al. Trends
Microbiol. 1996, 4, 495-502).
(8) An (NH4)2SO4 precipitate of E. coli-prfbA-C crude extracts was dialyzed
against buffer B (20 mM Tris‚HCl, 1 mM EDTA, pH 7.5). The dialysate was
resolved by anion exchange (DE52, 3 × 15 cm, 50 mL buffer B wash followed
by a linear gradient of 0-500 mM NaCl, 1.0 mL min-1) and the Ep fractions
combined, concentrated, and further resolved by FPLC gel filtration (S-200,
2 × 70 cm, 50 mM Tris‚HCl, 200 mM NaCl, pH 7.5). The purified Ep was
stored in aliquots (-80 °C) until used.
10.1021/ja001444y CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/30/2000