A chemoenzymatic synthesis of 5a-carba-α-D-mannose-6-phosphate

Article abstract of DOI:10.1016/j.carres.2011.04.011

An efficient chemical synthesis of 5a-carba-α-D-mannose and its enzymatic elaboration to 5a-carba-α-D-mannose-6-phosphate, using yeast hexokinase, is described.

Full text of DOI:10.1016/j.carres.2011.04.011

Carbohydrate Research 346 (2011) 1257–1261
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
A chemoenzymatic synthesis of 5a-carba-a-D-mannose-6-phosphate
Paula M. Williams a, Gary N. Sheldrake a, Simon J. F. Macdonald b, Chris J. Hamilton c,
⇑
a School of Chemistry and Chemical Engineering, David Kerr Building, Queen’s University Belfast, Stranmillis Rd, Belfast BT9 5DG, United Kingdom
b Glaxo SmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom
c School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient chemical synthesis of 5a-carba-a-D-mannose and its enzymatic elaboration to 5a-carba-a-D-
mannose-6-phosphate, using yeast hexokinase, is described.
Received 9 February 2011
Received in revised form 2 April 2011
Accepted 6 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Available online 12 April 2011
Keywords:
Carba sugar
Pseudo-sugar
Hexokinase
Biotransformation
1. Introduction
tracted by the additional protection/deprotection steps required
for installation of the phosphate moiety via chemical means.2,3
Carba sugars, where the ring oxygen (i.e., O-5) is replaced by a
methylene group, are of significant interest as non-hydrolysable
carbohydrate mimetics and as non-reducing analogues of reducing
sugars with fixed anomeric configuration. Whilst the syntheses
and applications of numerous carba sugar derivatives have been
widely reported (e.g., analogues of mono-, di-, tri-saccharides and
sugar nucleotides as glycosidase or glycosyltransferase inhibi-
tors),1 little attention has so far been given to carba-hexose-6-
5a-Carba-a-D-mannose-6 phosphate (2) (Fig. 1) could have po-
tential applications as a substrate analogue for structural and/or ki-
netic studies of mannose-6-phosphate utilising enzymes such as
KDN-9-phosphate synthetase (KDN biosynthesis)5,6 and bifunc-
tional phosphomannose isomerase/GDP-mannose-pyrophospho-
rylases (GDP-Man biosynthesis).7
Within this context, we were interested in developing a more
efficient synthetic route to 5a-carba- -mannose (1) and its enzy-
a-D
phosphates. Both anomers of 5a-carba-
D
-glucose-6-phosphate
matic elaboration to 5a-carba-a-D-mannose-6-phosphate (2)
have previously been employed as inert substrate mimics for inhi-
bition studies of myo-inositol-1-phosphate synthase2 and struc-
tural studies of 2-deoxy-scyllo-inosose synthase.3,4 However,
syntheses of carba sugars are often lengthy, and the preparation
of these carba-glucose-6-phosphates derivatives are further pro-
(Fig. 1) using yeast hexokinase (EC 2.7.1.2).
2. Results and discussion
Most of the previously reported syntheses of 1 are convoluted
(>20 steps).8,9,13 To date, the most succinct route involves a 11-step
synthesis of 1 in ꢀ6% overall yield from tri-O-acetyl-
D
-glucal.10
However, this route was only reported in a communication format
and the experimental procedures and characterisation data have
never been published. In our hands this route was not readily
reproducible, which prompted us to develop a more amenable syn-
thetic strategy. Our preparation of 1 involved the facile synthetic
elaboration of monosilylated triol 10, which has previously been
prepared as a key intermediate in the total synthesis of (ꢁ)-eutip-
oxide (Scheme 1).11 Starting from 3-hydroxy-2-pyrone (4) the cin-
chona base-catalysed asymmetric Diels–Alder reaction with the
chiral (S)-(+)-3-acroyl-4-phenyl-2-oxazolidinone (3) yielded
bridged bicyclolactone 5. Methanolysis of both the bridgehead lac-
tone and the oxazolidinone chiral auxiliary, followed by selective
R
R
4
6
5
OH
O
OH
2
5a
HO
HO
HO
HO
1
OH
3
OH
R = OH 1
R = OP(O)(OH )2
R = OH Man
R = OP(O)(OH )2 Man-6-P
2
Figure 1. D-Mannose, D-mannose-6-phosphate and their carba sugar derivatives 1
and 2.
⇑
Corresponding author. Tel.: +44 (0)1603 591449; fax: +44 (0)1603 592003.
E-mail address: c.hamilton@uea.ac.uk (C.J. Hamilton).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.04.011

1258
P. M. Williams et al. / Carbohydrate Research 346 (2011) 1257–1261
X
O
O
OR2
R1
R1
X
(i)
(ii)
+
O
6 R1 = CO2Me,
HO
O
R2 = H
O
(iii)
(iv)
OH
3
4
OH
R1 = CO2Me,
7
5
O
R
2 = TBS
8 R1 = CH2OH,
2 = TBS
R
OR1
OH
1
OBn
R1O
(v)
10
R
= H
OTBS
OTBS
(vi)
(vii)
BnO
R1O
R1O
(viii)
(ix)
13
R1 = Bn
R2 = TBS
O
11 R1 = Bn
9
OR2
R
1 = H
12
R2 = TBS
OR1
R1O
O
(vii)
R1O
R1O
N
R1 = H
(x)
X =
O
1
14
R
R
1 = Bn
2 = H
Ph
OR1
15 R1 = Ac
Scheme 1. Reagents and conditions: (i) cinchonine (0.3 equiv), 95:5 2-PrOH–H2O, 0 °C, 2 h, 89%; (ii) NaOMe, MeOH, 20 min; (iii) TBSCl, imidazole, DMF, 60 °C, 16 h, 75% over
two-steps; (iv) LiAlH4, THF, 0 °C, 3 h, 94%; (v) NaIO4, THF–H2O, 30 min, 68%; (vi) NaBH(OAc)3, AcOH, 30 min, 91%; (vii) BnBr, NaH, Bu4NI, THF 0 °C to rt 16 h, 69% for 11, 73% for
13; (viii) K2OsO4, NMO, Acetone–H2O, 16 h, 70%; (ix) TBAF, THF, 16 h, 80%; (x) Pd(OH)2, 2-PrOH–H2O, H2, 2 days, 100%; (xi) Ac2O, pyridine, 16 h, 100%.
silylation of the secondary alcohol and reduction of the methyl es-
ter groups, provided triol 8. Periodate-mediated oxidative cleavage
of the vicinal diol moiety of 8 yielded ketone 9, which was then
stereoselectively reduced by treatment with sodium triacetoxy-
borohydride to provide 10. To install the remaining stereocentres,
the bulky tert-butyldimethylsilyl ether at C-1 and the benzyl ether
at C-4 were sufficient to direct the stereospecific osmium tetrox-
ide-catalysed dihydroxylation of 1112 as confirmed by NOE cou-
pling between H-4 and both the hydroxyl groups in the purified
reaction product 12. The newly formed diol motif was temporarily
benzylated prior to removal of the silyl protecting group at the
pseudo-anomeric position to also obtain selectively deprotected
The synthetic utility of yeast hexokinase has previously been
demonstrated for the preparation of a number of pyranose and
furanose analogues of glucose, including a gram-scale preparation
of mannose-6-phosphate.15 Yeast hexokinase was, therefore, em-
ployed for a multi-milligram scale enzymatic conversion of 1 to
its 6-phosphate derivative 2 using phosphoenol pyruvate and
pyruvate kinase to regenerate the sub-stoichiometric quantities
of ATP in situ (Scheme 2). Assays were first performed to compare
the relative substrate efficiencies of 1 and
buffer (pH 7.5), an anomeric mixture of -mannose (
1H NMR spectroscopy) exhibited a Km value of 43
M (Table 1). The
D
-mannose. In phosphate
D
a:b = 66:34 by
l
Km value for 1 could not be determined, because the Michaelis–
5a-carba-
a-
D-mannose derivative 14, which could potentially
Menten plot was still linear up to the maximum concentration
serve as a useful intermediate for the chemical synthesis of car-
ba-mannose-derived oligosaccharides. Herein, compound 14 was
globally deprotected under hydrogenation conditions to provide
Table 1
Substrate kinetics of D-mannose and 1 with yeast hexokinase
5a-carba-
was obtained compared to a previously reported value of +1.9.13
The -manno-configuration of 1 was further confirmed by anal-
ysis of the 1H NMR coupling constants (J1,2 = J2,3 = 3.0 Hz,
3,4 = 9.6 Hz, J4,5 = 9.0 Hz). A sample of 1 was also per-acetylated
to give penta-O-acetyl-5a-carba- -mannose (15) and a larger
a-D-mannose (1). An agreeable optical rotation of +0.94
Substrate
Km
Rel. Vmax/Km
a-D
43
>24 mMa
l
M
100
7.3 ꢂ 10ꢁ5b
D-Mannose
5a-Carba-
a-
D
-mannose (1)
J
a
The rate was still linear with respect to 1 up to the maximum concentration
assayed (24 mM).
a-D
optical rotation of +30.6 was recorded, which was also comparable
b
Relative to D-mannose normalised to 100.
with previously reported values (+27.1,13 +27.814).
HO
HO
hexokinase
NAD+
pyruvate
kinase
NADH
CO2H
HO
HO
CO2H
ATP
ADP
O
lactate
dehydrogenase
1
OH
OH
OH
D-lactate
pyruvate
CO2H
H2O3PO
(B)
HO
HO
HO
OPO3H2
2
Phosphoenol
pyruvate
(A)
Scheme 2. (A) Hexokinase-catalysed conversion of 1 to 2 coupled with enzymatic regeneration of ATP in situ; (B) NADH-dependent lactate dehydrogenase was included in
kinetic assays to monitor the reaction progress.

P. M. Williams et al. / Carbohydrate Research 346 (2011) 1257–1261
1259
assayed (24 mM). However, the substrate efficiency (derived from
the slope of the Lineweaver–Burk plot) was approximately six or-
4.2. 5a-Carba-a-D-mannopyranose (1)
ders of magnitude lower than that of
noteworthy that a comparably large increase in Km and reduction
in substrate efficiency has previously been reported between
glucose and 5-deoxy-5-thio-
-glucose for the same enzyme.16,17
Evidently the anomeric ring oxygen is important, but not essential
for hexose substrate recognition and efficient turnover by yeast
hexokinase.
D
-mannose (Table 1). It is
To a solution of 14 (50 mg 0.092 mmol) in 5:1 2-PrOH–H2O
(3 mL) was added a catalytic amount of Pd(OH)2. The reaction
mixture was allowed to stir under an atmosphere of H2 for
2 days. The mixture was filtered through Celite, the 2-PrOH was
removed in vacuo and the aqueous solution was freeze-dried to
D
-
D
yield 1 as a white solid (16.3 mg, 100%). ½a D20
ꢃ
+0.94 (c 1.5, H2O)
(Lit.13 +1.9); 1H NMR (500 MHz CDCl3): dH 4.04 (1H, dd, J 3.0,
3.1, H-1), 3.98 (1H, dd, J 2.9, 3.0, H-2), 3.77 (1H, dd, J 11.2, 3.7,
H-6a), 3.75 (1H, dd, J 9.6, 3.2, H-3) 3.69 (1H, dd, J 11.2, 6.0, H-
6b), 3.59 (1H, t, J 9.9, H-4), 1.91–1.83 (1H, m, H-5), 1.81–1.69
(2H, m, H-5a); 13C NMR (125 MHz, CDCl3): dC 73.14 (C-2), 72.90
(C-3), 70.82 (C-4), 69.55 (C-1), 63.08 (C-6), 39.26 (C-5), 28.85
(C-5a); HRESIMS (ꢁ ion): calcd for C7H13O5 [MꢁH]ꢁ: m/z
177.0763; found: 177.0753.
Phosphorylation of a 66:34
went to >90% completion within 10 s of adding 10 units of hexoki-
nase. This is much faster than the rate of mutarotation for -man-
nose,18,19 demonstrating that yeast hexokinase readily
phosphorylates both - and b- -mannose under these assay condi-
tions. These observations suggest that it is the replacement of the
ring oxygen rather than the fixed -configuration of 1 that ac-
counts for its dramatically diminished substrate properties com-
pared to -mannose. Despite this, it was still possible to utilise
a:b anomeric mixture of D-mannose
D
a
D
a
D
4.3. 5a-Carba-a-D-mannopyranose-6-phosphate (2)
hexokinase for a multi-milligram scale preparation of 2. For the
purpose of the biotransformation, the phosphate buffer (pH 7.5)
was replaced with aqueous ammonium hydrogen carbonate (pH
8.3), which could easily be removed by freeze drying during puri-
fication. This also negated the need for K+ which is required to
A solution of yeast hexokinase (1000 U), 1 (50 mg, 0.28 mmol)
pyruvate kinase (50 U) phosphoenolpyruvate (47 mg, 0.28 mmol)
and ATP (0.05 mmol) in 5 mL of buffer (50 mM NH4HCO3, 1 mM
MgCl2, pH 8.3), was incubated at 30 °C for 18 h. The reaction mix-
ture was concentrated on a rotary evaporator and then freeze-
dried before being purified by anion-exchange chromatography
eluting with a 10–50 mM gradient of NH4HCO3) to yield 2 as a
maintain pyruvate kinase activity,20 because NH4 can confer the
þ
same effect.21,22 Under these conditions the turnover rate for
D-
mannose under saturating conditions increased 1.6-fold. Under
these conditions, when 50 mg of 1 (56 mM) was incubated with
1000 units of yeast hexokinase for 18 h, 10 mg of 2 was obtained
(17% yield). The unconsumed substrate 1 was also isolated readily
during purification by anion-exchange chromatography.
white powder (12 mg, 17%). ½a D20
ꢃ
+9.3 (c 1.0, H2O); 1H NMR
(500 MHz D2O): dH 3.92 (1H, d, J 2.3, H-1), 3.85 (1H, dd, J 3.1, 2.3,
H-2), 3.81 (1H, dd, J 10.5, 5.0, H-6a), 3.70–3.66 (1H, m, H-6b),
3.65–3.62 (1H, m, H-3) 3.57 (1H, t, J 9.8, H-4), 1.80–1.75 (1H, m,
H-5), 1.73–1.63 (2H, m, H-5a); 13C NMR(125 MHz, D2O): dC 73.19
(C-2), 72.55 (C-3), 70.44 (C-4), 69.74 (C-1), 65.20 (d, J 4.5 C-6),
38.68 (d, J 6.7 C-5), 29.10 (C-5a); 31P NMR (121 MHz D2O): dP
5.38; HRESIMS (ꢁ ion): calcd for C7H15O8P [MꢁH]ꢁ: m/z
257.0426; found: m/z 257.0426.
3. Conclusions
An improved synthetic route to 5a-carba-a-D-mannose (1) has
been achieved in 11% yield over 11 steps from 3-hydroxy-2-pyrone
(4). Subsequent enzymatic elaboration to the 6-phosphate deriva-
tive 2 has also been demonstrated using commercially available
yeast hexokinase. Considering the broad pyranose/furanose sub-
strate tolerance of yeast hexokinase,15 the work herein might also
be employed in a simple extension of established chemical synthe-
ses of other carba sugars to readily access their 6-phosphate
derivatives.
4.4. (1S,4S,5R)-4-O-Benzyl-5-benzyloxymethyl-1-O-(tert-
butyldimethylsilyl)cyclohex-2-en-1,4-diol (11)
A solution of sodium hydride (0.25 g, 10.5 mmol) and Bu4NI
(0.30 g, 0.84 mmol) in anhyd THF (10 mL) was cooled to 0 °C under
an atmosphere of Ar. A solution of 10 (1.1 g, 4.2 mmol) in anhyd
THF (20 mL) was added, and the mixture was allowed to stir for
2 h at 0 °C. Benzyl bromide (1.15 mL, 10.5 mmol) was then added
and the reaction mixture was allowed to warm to room tempera-
ture and stir for 16 h. The reaction was quenched with water
(10 mL) and the products were extracted into Et2O (3 ꢂ 10 mL).
The combined organic layers were dried (MgSO4), concentrated
and purified by column chromatography (1:4 EtOAc–hexane) to
4. Experimental
4.1. General
All chemicals were obtained commercially from Acros, Avocado,
Fluka, and Sigma–Aldrich. Pyrone 4 was either purchased from Ty-
ger Scientific.
yield 11 as a colourless oil (1.27 g, 69%); ½a D20
ꢁ53.43 (c 1.1,
ꢃ
Rabbit muscle pyruvate kinase, and rabbit muscle lactate dehy-
drogenase were purchased from Sigma and yeast hexokinase (Cat.
No. 376811) from Calbiochem. 1H and 13C NMR spectra were mea-
sured on either a Bruker AV 300, DPX 300 or DRX 500 spectrome-
ter. 1H and 13C NMR spectra were referenced to the NMR solvent as
an internal standard. Samples analysed in D2O were spiked with
acetonitrile to provide an internal standard. 31P NMR spectra were
referenced to triethyl phosphate as an internal standard. Mass
spectra were recorded on a VG Autospec, a Waters LCT Premier
or a VG Quattro mass spectrometer. Optical rotations were ob-
tained with a Perkin–Elmer Model 341 polarimeter. Automated
flash chromatography was performed on an Argonaut Flashmaster
II using pre-packed silica cartridges. Anion-exchange chromatogra-
phy was performed using a Bio-Rad Bio-Scale Mini Macro-prep
HighQ cartridge on a Bio-Rad LP system.
CH2Cl2); 1H NMR (500 MHz CDCl3): dH 7.31–7.18 (10H, m, Ar-H),
5.38 (1H, dd, J 10.1, 2.3, H-2), 5.70 (1H, m, H-3), 4.55 (1H, d, J
11.6, PhCHAHB), 4.44 (1H, d, J 12.0, PhCHAHB), 4.41 (1H, d, J 11.6
PhCHAHB), 4.38 (1H, d, J 12.0, PhCHAHB), 4.12 (1H, dd, J 3.9, 3.8,
H-4), 3.87 (1H, ddd, J 8.5, 2.9, 2.0 H-1), 3.57 (1H, dd, J 9.2, 5.4,
CHAHBOBn), 3.44 (1H, dd, J 9.2, 3.9, CHAHBOBn), 2.31–2.20 (1H,
m, H-5), 1.79 (1H, dddd, J 13.6, 3.6, 3.5, 1.0, H-6a), 1.70 (1H, ddd,
J 13.7, 11.0, 4.3, H-6b), 0.83 (9H, s, 3 ꢂ CH3), 0.04 (3H, s, CH3)
0.03 (3H, s, CH3); 13C NMR (125 MHz, CDCl3): dC 138.64, 131.43
(C-2), 129.29 (C-3), 128.19, 128.15, 127.77, 127.36, 127.28, 74.33
(C-1), 72.81 (CH2), 70.85 (CH2), 70.28 (CH2), 64.07 (C-4), 35.07
(C-5), 33.28 (C-6), 25.76 (CH3), 18.04 (C(CH3), ꢁ4.61 (CH3), ꢁ4.79
(CH3); HRESIMS (+-ion): calcd for C27H38O3Si [M+Na]+: m/z
461.2488; found: m/z 461.2497.

1260
P. M. Williams et al. / Carbohydrate Research 346 (2011) 1257–1261
4.5. 4,6-Di-O-benzyl 1-O-tert-butyldimethylsilyl-5a-carba-
mannopyranoside (12)
a
-
D
-
4.70 (1H, d, J 12.2, PhCHAHB), 4.63 (1H, d, J 11.8, PhCHAHB), 4.59
(1H, d, J 11.9, PhCHAHB), 4.56 (1H, d, J 12.7, PhCHAHB), 4.53 (1H,
d, J 12.1, PhCHAHB) 4.47 (1H, d, J 11.6, PhCHAHB), 4.44 (1H, d, J
12.1, PhCHAHB), 4.06–4.02 (1H, m, H-1), 3.81–3.83 (2H, m, H-3,
H-4), 3.72 (1H, dd, J 5.7, 2.4, H-2), 3.61 (1H, dd, J 9.0, 5.0, H-6a),
3.58 (1H, dd, J 9.0, 6.5, H-6b), 2.24–2.18 (1H, m H-5), 1.98 (1H,
ddd, J 13.5, 9.0, 3.4, H-5a), 1.77–1.73 (1H, m, H-5a0); 13C NMR
(125 MHz, CD3OD): dC 139.03, 138.86, 128.58, 128.54, 128.07,
127.96, 127.93, 127.82, 127.76, 127.72, 127.67, 79.85, 79.70,
77.33 (C-2, C-3, C-4), 74.01 (CH2), 73.22 (CH2), 72.87 (CH2), 72.65
(CH2), 71.09 (C-6), 67.44 (C-1), 37.86 (C-5), 30.50 (C-5a); HRESIMS
(+-ion): calcd for C38H39O5 [M+H]+: m/z 539.2820; found: m/z
539.2797.
To a stirred solution of 11 (1.2 g, 2.7 mmol) and N-methylmor-
pholine N-oxide (NMO, 1.1 g, 8.2 mmol) in 4:1 acetone–H2O
(35 mL) was added a catalytic amount of K2OsO4ꢄH2O. The mixture
was allowed to stir for 16 h at room temperature. The solvent was
removed in vacuo and the residue was dissolved in chloroform
(20 mL). The organic phase was washed with brine (3 ꢂ 5 mL),
dried (MgSO4), concentrated and purified by column chromatogra-
phy (3:7 EtOAc–hexane), to yield 12 as a yellow oil (893 mg, 70%);
½
a 2D0 +22.18 (c 2.1, CH2Cl2); 1H NMR (500 MHz CDCl3): dH 7.34–7.27
ꢃ
(10H, m, Ar-H), 4.66 (1H, d, J 11.3, Ph-CHAHB) 4.57 (1H, d, J 11.3, Ph-
CHAHB), 4.50–4.47 (2H, m, Ph-CH2), 3.99 (1H, dd, J 5.8, J 3.0, H-1),
3.84–3.79 (2H, m, H-2, H-3), 3.75 (1H, dd, J 9.2, 4.6, H-6a), 3.64
(1H, dd J 8.8, H-4), 3.48 (1H, dd, J 9.3, 2.9, H-6b) 2.36 (1H, br s, –
OH), 2.30 (1H, br s, –OH), 2.17–2.10 (1H, m, H-5), 2.03 (1H, dd J
12.3, 2.4 H-5a) 1.64–1.59 (1H, m, H-5a0), 0.82 (9H, s, 3 ꢂ CH3),
0.04 (3H, s, CH3), 0.03 (3H, s, CH3); 13C NMR (125 MHz, CDCl3):
dC 138.61, 138.50, 128.70, 128.36, 128.04, 127.97, 127.64, 127.54,
79.57 (C-4), 74.38 (CH2), 73.71 (CH), 73.08 (CH2), 72.83 (CH),
70.52 (C-6), 69.41 (C-1), 36.90 (C-5), 31.27 (C-5a), 25.73 (CH3),
17.95 (C(CH3)3), ꢁ4.88 (CH3), ꢁ4.99 (CH3); HRESIMS (+-ion): calcd
for C27H40O5SiNa [M+Na]+: m/z 495.2543; found: m/z 495.2578.
4.8. 1,2,3,4,6-Penta-O-acetyl-5a-carba-a-D-mannopyranose (15)
Ac2O (0.06 mL, 0.6 mmol) was added to a solution of 1 (10 mg,
0.56 mmol) in pyridine (0.7 mL), and the mixture was allowed to
stir at room temperature for 16 h. Solvent was removed in vacuo,
the residue was dissolved in CH2Cl2 (0.3 mL) and the solution
was washed with aq CuSO4 (3 ꢂ 0.3 mL) and water (3 ꢂ 0.3 mL)
to furnish pure 15 as an oil (21 mg, quantitative). ½a D20
ꢃ
+30.6 (c
2.1, CH2Cl2) (lit. +27.1,13 +27.814); 1H NMR (500 MHz CDCl3): dH
5.29 (1H, dd, J 2.3, 4.3, H-1), 5.22–5.15 (2H, m, H-3, H-4), 5.00
(1H, dd, J 3.0, 3.1, H-2), 4.08 (1H, dd, J 5.5, 11.4, H-6a), 3.92 (1H,
dd, J 3.9, 11.3, H-6b), 2.22 (1H, m, H-5), 2.11 (3H, s, CH3), 2.10
(3H, s, CH3), 2.04 (3H, s, CH3), 2.02 (3H, s, CH3), 1.95 (3H, s, CH3),
1.90 (1H, dd J 12.5, 8.8, H-5a), 1.85–1.81 (1H, m, H-5a0); 13C NMR
(125 MHz, CDCl3): dC 170.73 (C@O) 170.02 (C@O) 169.96 (C@O)
169.30 (C@O) 70.75 (C-3/C-4) 69.26 (C-1) 68.23 (C-2) 63.66 (C-5)
27.41 (C-5a) 21.00 (CH3) 20.82 (CH3) 20.74 (CH3) 20.70 (CH3)
20.62 (CH3).
4.6. 2,3,4,6-Tetra-O-benzyl 1-O-tert-butyldimethylsilyl-5a-
carba-a-D-mannopyranoside (13)
To a solution of NaH (0.16 g, 6.7 mmol) and Bu4NI (0.24 g,
0.67 mmol) in anhyd THF (10 mL) at 0 °C under an atmosphere of
Ar, a solution of 12 (0.8 g, 1.69 mmol) in anhyd THF (20 mL) was
added drop wise. The mixture was allowed to stir for 2 h. Benzyl
bromide (0.75 mL, 6.7 mmol) was then added at 0 °C and the mix-
ture was allowed to warm to room temperature and stirred for
16 h. Water (10 mL) was added and the products were extracted
into Et2O (3 ꢂ 10 mL). The combined organic layers were dried
(MgSO4), concentrated and purified by column chromatography
(1:4 EtOAc–hexane) to yield 13 as a colourless oil (3.2 g, 73%).
4.9. Hexokinase assays with
D-mannose
Assays were performed in a final volume of 1000
lL of buffer
(50 mM sodium phosphate, 20 mM KCl, 1 mM MgCl2, pH 7.5) in
disposable cuvettes containing pyruvate kinase (2.5 U) phospho-
½
a 2D0
ꢃ
+7.35 (c 4.3, CH2Cl2); 1H NMR (500 MHz CDCl3): dH 7.38–
enolpyruvate (2 mM), ATP (500
NADH (140 M) and varying concentrations of
1000 M) at 30 °C. Reactions were initiated by the addition of
lM), lactate dehydrogenase (5 U),
7.27 (20H, m, Ar-H), 5.08 (1H, d, J 10.6, PhCHAHB), 4.92 (1H, d, J
12.4, PhCHAHB), 4.83 (1H, d, J 11.9, PhCHAHB), 4.72 (1H, d, J 12.0,
PhCHAHB), 4.69 (1H, d, J 12.4, PhCHAHB), 4.62 (1H, d, J 10.6,
PhCHAHB), 4.58 (1H, d J 12.0, PhCHAHB), 4.55 (1H, d, J 12.0,
PhCHAHB), 3.94 (1H, dd, J 6.9, 3.4, H-1), 3.81–3.83 (2H, m, H-2, H-
3), 3.76 (1H, dd, J 8.9, 5.3, H-6a), 3.66–3.64 (1H, m, H-4), 3.59
(1H, dd, J 9.0, 2.5, H-6b), 2.24–2.18 (1H, m, H-5), 2.05–1.99 (1H,
m, H-5a), 1.67–1.22 (1H, m, H-5a0), 0.87 (9H, s, CH3), 0.00 (3H, s,
CH3), ꢁ0.07 (3H, s, CH3); 13C NMR (125 MHz, CDCl3): dC 139.02,
139.00, 138.90, 128.29, 128.26, 128.23, 127.87, 127.70, 127.46,
127.43, 127.39, 127.28, 81.91, 78.74, 78.60 (C-2, C-3, C-4), 75.88
(CH2), 72.83 (CH2), 72.80 (CH2), 70.74 (CH2), 70.74 (C-6), 68.34
(C-1), 37.42 (C-5), 30.95 (C-5a), 25.67 (CH3), 17.84 (C(CH3)3),
ꢁ5.07 (SiCH3), ꢁ5.07 (SiCH3); HRESIMS (+-ion): calcd for
l
D
-mannose (25–
l
hexokinase (100 mU) and progress was monitored by the decrease
in absorbance at 340 nm due to NADH consumption. Kinetic data
were analysed (by non-linear regression) using Grafit Version 5
(Erithacus Software Ltd).
4.10. Hexokinase assays with 5a-carba-D-mannose (1)
Assay mixtures composed of yeast hexokinase (20 U), pyruvate
kinase (0.25 U) phosphoenolpyruvate (4 mM), ATP (2 mM) and dif-
ferent concentrations of 1 (0–24 mM) in 100
dium phosphate, 20 mM KCl, 1 mM MgCl2, pH 7.5), were incubated
at 30 °C. At different time points 20 L aliquots of the assay mixture
were transferred into a cuvette containing lactate dehydrogenase
(1 U) and NADH (140 M) in a final volume of 1000 L of buffer
lL of buffer (50 mM so-
l
C
41H53O5Si [M+H]+: m/z 653.3662; found: m/z 653.3686.
l
l
4.7. 2,3,4,6-Tetra-O-benzyl-5a-carba- -mannopyranose (14)
a
-
D
(50 mM sodium phosphate, 20 mM KCl, and 1 mM MgCl2, pH 7.5)
and the reaction progress was determined from the decrease in
absorbance at 340 nm due to the consumption of NADH. Control as-
says, in the absence of 1, were also performed to generate a calibra-
tion curve so that NADH consumption due to background ATPase
activity of hexokinase could be subtracted from the above assays.
To a solution of 13 (0.81 g, 1.24 mmol) in THF (10 mL), was
added a 1 M solution of TBAF in THF (7.5 mL, 7.5 mmol). The mix-
ture was allowed to stir for 16 h at room temperature, and EtOAc
(10 mL) was then added. The reaction mixture was washed with
NaHCO3 (3 ꢂ 5 mL), H2O (3 ꢂ 5 mL) and brine (3 ꢂ 5 mL). The or-
ganic layer was dried (MgSO4), concentrated and purified by col-
umn chromatography (1:4 EtOAc–hexane) to yield 14 as a clear
Acknowledgements
oil (530 mg, 80%). ½a D20
ꢃ
+1.04 (c 3.0, CH2Cl2); 1H NMR (500 MHz
CD3OD): dH 7.37–7.23 (20H, m, Ar-H), 4.72 (1H, d, J 11.5, PhCHAHB),
This work was supported by a Marie Curie early stage research
training award. We also thank Graham Inglis and Ken Down for

P. M. Williams et al. / Carbohydrate Research 346 (2011) 1257–1261
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