formation of monosaccharide 1-phosphate from the cor-
responding monosaccharide and ATP; (2) a pyropho-
sphorylase-catalyzed formation of sugar-nucleotide and
pyrophosphate byproduct from nucleotide triphosphate
and the monosaccharide 1-phosphate. Taking advantage
of promiscuous enzymes involved in these pathways,
efficient chemoenzymatic approaches were developed
for preparative-scale synthesis of sugar-nucleotides and
their non-natural derivatives. For example, a bifunctional
L-fucose 1-kinase/GDP-Fuc pyrophosphorylase (FKP)
from Bacteroides fragilis was applied successfully for
the synthesis of GDP-Fuc and derivatives.6 In addition,
monosaccharide 1-kinases and a promiscuous UDP-sugar
pyrophosphorylase (BLUSP) were used efficiently for one-
pot enzymatic synthesis of UDP-hexose and derivatives
from simple hexose and derivatives.7 Furthermore, a panel
of UDP-HexNAc and derivatives were chemoenzymati-
cally prepared by combining an N-acetylhexosamine
1-kinase (NahK) and an UDP-N-acetylglucosomine pyr-
ophosphorylase (GlmU or AGX1) in either a one-pot or a
sequenctial manner.8,9
Nevertheless, such a simple synthetic route has not
yet been developed for the synthesis of GDP-Man and
other GDP-sugars, mainly due to the lack of suitable
monosaccharide 1-kinases. As a result, chemically pre-
pared or commercially available mannose 1-phosphate
and derivatives were generally used in the formation
of GDP-sugars.10ꢀ12 We recently found that a NahK from
Bifidobacterium infantis ATCC15697 (NahK_15697) could
phosphorylate a number of monosaccharides including
mannose and derivatives.13 Taking advantage of this
and the promiscuity of NahK_15697 and a GDP-Man
pyrophosphorylase from Pyrococcus furiosus DSM3638
(PFManC),12 we present here an efficient one-pot three-
enzyme system for quick preparative-scale synthesis of
GDP-sugars and their derivatives.
from monosaccharide 1-phosphates and guanosine 50-
triphosphate (GTP). The last enzyme was an inorganic
pyrophosphatase cloned from Escherichia coli (EcPpA).14
It drove the reaction toward the formation of GDP-sugars
by hydrolyzing the pyrophosphate byproduct.
Scheme 1. One-Pot Three-Enzyme Synthesis of GDP-Sugars
Genetic analysis showed that the DNA sequence of the
archaeal enzyme PFManC contains numerous rare co-
dons. To increase the heterologous protein expression level
in E. coli, the DNA sequence of PFManC was codon
optimized. The synthetic gene obtained by custom synthesis
was cloned into the pET22b(þ) vector. The protein was
overexpressed in E. coli BL21(DE3), yielding over 80 mg of
PFManC per liter of cell culture after purification.15
Besides GTP, it was reported that PFManC could
also utilize ATP to form ADP-sugars.12 In order to avoid
unexpected byproduct formation in the one-pot system,
GTP, instead of ATP, was used as the phosphate donor
for NahK_15697 (Scheme 1). To our delight, GTP was a
suitable substrate for NahK_15697. As shown in Table S1
and Figure S2, except for Man4N3 (6) which had a
relatively low yield of 36%, NahK_15697 was able to use
GTP as a phosphate donor for high-yield (>53%) phos-
phorylation of all other monosaccharides and derivatives
tested including mannose (1) and its derivatives (2ꢀ5),
talose (7), and glucose (8) as well as its C2-derivatives
(9ꢀ12). The results confirmed previously reported broad
substrate specificity of NahK toward both monosacchar-
ides and phosphate donors.8,13,16 We also tested a number
of C6 modified substrates, including Rha (25), Rha4N3
(26), PerNAc (27), 6-deoxyTal (28), and ManA (29), but
none was a suitable substrate (Table S1 and Figure S2) for
NahK_15697 when either ATP or GTP was used as the
phosphate donor. The results imply that the C6 hydroxyl
group may play essential roles in substrate recognition by
NahK_15697.
As shown in Scheme 1, three enzymes were used in one
pot to synthesize GDP-Man, GDP-Glc, their derivatives,
and GDP-Tal. The first enzyme was NahK_15697, which
catalyzed the formation of monosaccharide 1-phosphates.
The second enzyme was PFManC, which catalyzed the
reversible formation of GDP-sugars and pyrophosphate
(6) (a) Yi, W.; Liu, X.; Li, Y.; Li, J.; Xia, C.; Zhou, G.; Zhang, W.;
Zhao, W.; Chen, X.; Wang, P. G. Proc. Natl. Acad. Sci. U.S.A. 2009,
106, 4207–12. (b) Wang, W.; Hu, T.; Frantom, P. A.; Zheng, T.; Gerwe,
B.; Del Amo, D. S.; Garret, S.; Deidel, R. D., III; Wu, P. Proc. Natl.
Acad. Sci. U.S.A. 2009, 106, 16096–101.
(7) Muthana, M. M.; Qu, J.; Li, Y.; Zhang, L.; Yu, H.; Ding, L.;
Halekan, H.; Chen, X. Chem. Commun. 2012, 48, 2728–30.
(8) (a) Cai, L.; Guan, W.; Kitaoka, M.; Shen, J.; Xia, C.; Chen, W.;
Wang, P. G. Chem. Commun. 2009, 45, 2944–6. (b) Cai, L.; Guan, W.;
Wang, W.; Zhao, W.; Kitaoka, M.; Shen, J.; O’Neil, C.; Wang, P. G.
Bioorg. Med. Chem. Lett. 2009, 19, 5433–5.
The synthesis of GDP-sugars was carried out using
the one-pot three-enzyme system shown in Scheme 1.17
As listed in Table 1,18 the system was quite efficient in
synthesizing GDP-Man (13, 94%), GDP-ManNH2 (14, 75%),
(9) (a) Guan, W.; Cai, L.; Fang, J.; Wu, B.; Wang, P. G. Chem.
Commun. 2009, 45, 6976–8. (b) Chen, Y.; Thon, V.; Li, Y.; Yu, H.; Ding,
L.; Lau, K.; Qu, J.; Hie, L.; Chen, X. Chem. Commun. 2011, 47, 10815–7.
(10) (a) Watt, G. M.; Flitsch, S. L.; Fey, S.; Elling, L.; Kragl, U.
Tetrahedron: Asymmetry 2000, 11, 621–8. (b) Zou, L.; Zheng, R. B.;
Lowary, T. L. Beilstein J. Org. Chem. 2012, 8, 1219–26.
(14) (a) Lahti, R.; Pitkaeranta, T.; Valve, E.; Ilta, I.; Kukko-kalske,
E.; Heinonen, J. J. Bacteriol. 1988, 170, 5901–7. (b) See Supporting
Information for details about cloning, overexpression, and purification.
(15) See Supporting Information for details about cloning, over-
expression, and purification of PFManC.
(11) Marchesan, S.; Macmillan, D. Chem. Commun. 2008, 44, 4321–
4323.
(16) Nishimoto, M.; Kitaoka, M. Appl. Environ. Microb. 2007, 73,
6444–9.
(12) Mizanur, R. M.; Pohl, N. L. Org. Biomol. Chem. 2009, 7, 2135–9.
(13) Li, Y.; Yu, H.; Chen, Y.; Lau, K.; Cai, L.; Cao, H.; Tiwari, V. K.;
Qu, J.; Thon, V.; Wang, P. G.; Chen, X. Molecules 2011, 16, 6396–407.
(17) See Supporting Information for reaction details.
(18) All NMR and MS data are available in the Supporting
Information.
Org. Lett., Vol. 15, No. 21, 2013
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