Substrate specificity of N-acetylhexosamine kinase towards N-acetylgalactosamine derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.07.104

We report herein a bacterial N-acetylhexosamine kinase, NahK, with broad substrate specificity towards structurally modified GalNAc analogues, and the production of a GalNAc-1-phosphate library using this kinase.

Full text of DOI:10.1016/j.bmcl.2009.07.104

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5433–5435
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substrate specificity of N-acetylhexosamine kinase towards
N-acetylgalactosamine derivatives
Li Cai a, , Wanyi Guan a,b, , Wenjun Wang c, Wei Zhao c, Motomitsu Kitaoka d, Jie Shen c,
Crystal O’Neil a, Peng George Wang a,
*
a Departments of Chemistry and Biochemistry, The Ohio State University, 484 w. 12th Ave. Columbus, OH 43210, USA
b National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China
c College of Pharmacy, Nankai University, Tianjin 300071, China
d National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein a bacterial N-acetylhexosamine kinase, NahK, with broad substrate specificity towards
Received 26 June 2009
Revised 21 July 2009
Accepted 22 July 2009
Available online 25 July 2009
structurally modified GalNAc analogues, and the production of a GalNAc-1-phosphate library using this
kinase.
Ó 2009 Published by Elsevier Ltd.
Keywords:
Kinase
Phosphorylation
Specificity
As an important sugar residue, N-acetylgalactosamine (GalNAc)
widely exists in living systems and involves many biological pro-
cesses: It is a main component of some glycosaminoglycans1 and
also exists as the initial sugar residues in mucin-type O-glycans.2
Efficient construction of these clinically significant GalNAc-con-
taining glycoconjugates enzymatically requires uridine 50-diphos-
and the construction of a library of the corresponding GalNAc-1-
P analogues.
To determine the substrate specificity of NahK behaving as a
‘GalNAc kinase’, we designed and synthesized eight GalNAc deriv-
atives with modifications on the C-2, 4- or 6-positions. C-2 deriva-
tives 2–5 were easily obtained through one step N-acylation as
described before.9 Synthesis of the 6-deoxy analogue 7 from inter-
mediate 13 utilized the regioselective mesylation and reduction
method as described by Bundle and co-workers10 (Scheme 1, Part
I). However, poor yield was observed for this reaction, contrary
to our expectations based on the reasonable yield obtained with
this method using GlcNAc as the substrate.9 Anomeric deprotec-
tion using N-bromosuccinimide furnished 6-deoxy GalNAc 7.11 To
improve the yield for preparing compound 7, a second strategy
was used as shown in Scheme 1, Part II. Starting from diol 17,12
6-tosylated 18 was obtained by selective tosylation followed by
acetylation. Deoxygenation at the 6-position of 18 was performed
by iodination and subsequent radical reduction.13 Deprotection of
triacetate 19 by sodium methoxide afforded 7 in good overall yield.
The 6-azido compound 6 was simply constructed either by selec-
tive tosylation of 13 followed by displacement by sodium azide
to give intermediate 14 or direct substitution at C-6 of tosylated
18. Similar deprotection procedures by NBS or sodium methoxide
provided analogue 6.
pho-GalNAc (UDP-GalNAc) as
a sugar donor by Leloir-type
glycosyltransferases.3 UDP-GalNAc can be obtained by enzymatic
epimerization of UDP-GlcNAc, however the yield is limited,4
restricting its use in biological areas. A salvage pathway, which
reutilizes GalNAc released from degraded glycoconjugates, pre-
sents a more promising alternative: GalNAc is first activated by a
GalNAc kinase to form GalNAc-1-P, then converted to UDP-GalNAc
by a pyrophosphorylase.5 Such GalNAc kinases have been discov-
ered in mammals and for the past decade have been employed in
in vitro synthesis6 and in situ regeneration7 of UDP-GalNAc; how-
ever, no bacterial GalNAc kinase has yet been reported. Our group
has previously found that an N-acetylhexosamine kinase (NahK)
from Bifidobacterium longum could well accept GalNAc as its sub-
strate;8 thereby GalNAc-1-P was synthesized on a gram scale.9
Here, we report the investigation of the substrate specificity of this
bacterial kinase toward structurally modified GalNAc analogues
* Corresponding author. Tel.: +1 614 292 9884; fax: +1 614 688 3106.
E-mail address: wang.892@osu.edu (P.G. Wang).
Contributed equally to this work.
For the synthesis of 4-modified analogues (Scheme 2), 4,6-phe-
nylmethylene protected N-acetyl glucosamine 2014 was deprotec-
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.07.104

5434
L. Cai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5433–5435
Part I
HO
HO
HO
HO
OMs
O
NBS
NaBH4, DMSO
O
O
SPh
85-90 oC
SPh
HO
acetone/H2O
90% Yield
HO
MsCl, Py
-15 o
60% Yield
OH
NHAc
NHAc
NHAc
15
23% Yield
C
16
7
i) TsCl, Py
ii) NaN3, DMF,
HO
HO
N3
RO
RO
OR
O
HO
HO
N3
NBS
acetone/H2O
O
O
15-crown-5
SPh
SPh
OH
34% totally
71% Yield
NHAc
NHAc
NHAc
12: R = Ac
14
6
NaOMe
100%
13: R = H
Part II
i) NaI, DME, 85 oC
ii) AIBN, Bu3SnH
Toluene, reflux
AcO
AcO
AcO
HO
OTs
O
OH
O
HO
i) p-TsCl, pyridine
ii) Ac2O, Pyridine
O
MeOH/MeONa
71% Yield
O
AcO
AcO
84% two steps
OAc
77% two steps
HO
OH
AcHN
AcHN
AcHN
AcHN
OAc
OAc
17
18
19
7
i) NaN3, DMF,
15-crown-5
ii) MeOH/MeONa
HO
HO
N3
O
OH
NHAc
6
Scheme 1. Synthesis of 6-azido and 6-deoxy analogues.
i) Tf2O, pyridine,CH 2Cl2
N3
OH
O
ii) NaN3, DMF, rt
iii) MeONa/MeOH
OAc
O
HO
65% totally
OH
i)MeOH/AcCl
CH2Cl2
Ph
O
O
NHAc
HO
AcO
O
8
S
i) AIBN Bu3SnH
Toluene, 60 oC
ii) MeOH MeONa
AcO
ii) Ac2O, pyridine
CH2Cl2, -20 o
65% two steps
S
AcHN
AcHN
OH
O
Ph
C
OAc
O
C
OAc
OAc
O
Cl
O
O
20
21
DMAP, CH 3CN
71%
62% totally
HO
OH
AcO
22
Scheme 2. Synthesis of 4-azido and 4-deoxy analogues.
Ph
NHAc
AcHN
OAc
9
ted by treatment with acetyl chloride in methanol;15 subsequent
selective acetylation at C-6 position gave the 4-hydroxyl com-
pound 21.16 Triflation of compound 21 followed by nucleophilic
substitution using sodium azide gave the triacetate which was
deprotected with sodium methoxide to afford the target 4-azido
GalNAc 8. Conversion of alcohol 21 to the corresponding thionocar-
bonate 22, followed by radical deoxygenation17 and deacetylation,
yielded desired analogue 9.18
The recombinant NahK was expressed and purified as previ-
ously described.9 The enzymatic production of GalNAc-1-P ana-
logues was carried out in a mixture containing 40 mM GalNAc or
its analogues, 50 mM ATP, 10 mM MgCl2, and 1.5 mg/mL NahK in
100 mM Tris–HCl buffer (pH 9.0). After incubation at 37 °C for
19 h, the mixture was briefly boiled and centrifuged to remove
protein. All GalNAc-1-P analogues synthesized here were purified
by normal phase silica gel chromatography and isolated yields
are shown in Table 1.19
relatively high yields of 2-modified analogues 1–5 and almost
unreactive galactose 10,8 that 2-amido group is necessary for en-
zyme activity. The bulkiness of R1 group exhibited a slight influ-
ence on enzyme activity (compounds 4 and 5). Significantly, the
enzyme displayed a wide tolerance of 4-modification (R3) includ-
ing axial hydroxy (1), equatorial hydroxy (GlcNAc),9 azido (8)
and deoxy (9), indicating 4-hydroxyl group may not be necessary
for enzyme recognition, a feature that could greatly expand our
prospective library of UDP-GlcNAc and UDP-GalNAc sugar donor
analogues. Conversely, the tolerance for 6-modification is still
quite limited: the reactions of both 6-azido (6) and 6-deoxy (7)
compounds produced dramatically decreased yields.
In summary, the bacterial N-acetylhexosamine kinase, NahK,
has broad substrate specificity towards structurally modified Gal-
NAc analogues. Tolerance for 2- and 4-modifications was high
while tolerance for 6-modification was relatively limited. Enzy-
matic conversion of the sugar-1-P compounds produced here to a
sugar donor library for glycosyltransferase-catalyzed reaction will
be reported in due course.
The results clearly indicate that most GalNAc analogues can be
phosphorylated in good yields (Table 1). We can conclude, from

L. Cai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5433–5435
5435
Table 1
Acknowledgements
Isolated yields for enzymatic 1-phosphorylation reactions
R3
R3
R2
O
R2
O
P. G. Wang acknowledges National Cancer Institute (R01
CA118208), NSF (CHE-0616892) for financial support. W. Guan
acknowledges China Scholarship Council for financial support. Mass
spectra data were collected on a Bruker micrOTOF instrument pro-
vided by a grant from the Ohio BioProducts Innovation Center.
NahK
ATP
HO
HO
OH
O
P
NH
HN
O
R1
R1
O
O
O
O
NH4
2
1-10
Supplementary data
Entry
Substrate structure
1-Phosphorylation yield (%)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.104.
OH
HO
78a
References and notes
O
1
HO
AcHN
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OH
OH
HO
HO
O
2
3
4
85a
86a
77a
NH
OH
OH
OH
O
Et
OH
HO
HO
O
NH
O
Pr
OH
HO
HO
O
NH
O
Ph
OH
HO
HO
O
19. NMR data for the 1-phosphate derivatives of compound 2, 3, 7 and 9: (a)
Lazarevic, D.; Thiem, J. Carbohydr. Res. 2002, 337, 2187; (b) Illarionov, P. A.;
Torgov, V. I.; Hancock, I. I.; Shibaev, V. N. Russ. Chem. Bull. 2001, 50, 1303; (c)
Srivastava, G.; Alton, G.; Hindsgaul, O. Carbohydr. Res. 1990, 207, 259.
5
65a
NH
OH
O
N3
N3
HO
HO
HO
6
7
8
9
42a
37a
73a
70a
<5b
O
AcHN
OH
OH
O
HO
AcHN
OH
O
N3
HO
AcHN
OH
O
OH
OH
OH
HO
AcHN
OH
O
HO
HO
10
HO
a
Isolated yield from silica gel column chromatography.
Detected by TLC.
b