Synthesis and photolytic activation of 6″-O-2-nitrobenzyl uridine-5′-diphosphogalactose: a 'caged' UDP-Gal derivative

Article abstract of DOI:10.1016/j.carres.2008.01.021

Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-5′-diphosphogalactose (UDP-Gal) gives 6″-O-2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for β-(1→4)-galactosyltransferase (GalT). On irradiation at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the expected βGal-(1→4)-βGlcNAc disaccharidic linkage. 6″-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a 'caged' UDP-Gal for application in time-resolved crystallographic studies of β-(1→4)-GalT.

Full text of DOI:10.1016/j.carres.2008.01.021

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 875–881
Synthesis and photolytic activation of 600-O-2-nitrobenzyl
uridine-50-diphosphogalactose: a ‘caged’ UDP-Gal derivative
Karin Mannerstedt and Ole Hindsgaul*
Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark
Received 19 December 2007; received in revised form 14 January 2008; accepted 17 January 2008
Available online 26 January 2008
Abstract—Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-50-diphosphogalactose (UDP-Gal) gives 600-O-
2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for b-(1?4)-galactosyltransferase (GalT). On irradiation
at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the
expected bGal-(1?4)-bGlcNAc disaccharidic linkage. 600-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a
‘caged’ UDP-Gal for application in time-resolved crystallographic studies of b-(1?4)-GalT.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Galactosyltransferases; Sugar nucleotide analogue; Caged compound; Enzyme structure and mechanism
1. Introduction
HO
HO
O
OH
O
NH
O
P
O
O
P
O
Mammalian glycosyltransferases catalyze the transfer of
glycosyl-residues from their activated glycosyl-donors,
usually sugar nucleotide diphosphates, to OH-groups
on acceptor molecules. Hundreds of glycosyltransferases
are known to be involved in the biosynthesis of the oligo-
saccharide chains of mammalian glycoproteins and
glycolipids.1 These enzymes are broadly classified into
two main families, termed either ‘retaining’ or ‘invert-
ing’, depending on whether the stereochemistry of
the glycosidic bond in the donor is retained or inverted
in the product glycoside. The extensively studied b-
(1?4)-galactosyltransferase (b-(1?4)-GalT),2 for exam-
ple, is an inverting enzyme transferring galactose from
a-linked UDP-galactose (1, UDP-Gal) to OH-4 of
N-acetylglucosamine (GlcNAc) residues (2) to give
N-acetyllactosamine terminated structures (3) (Chart
1). This enzyme has been cloned and extensively studied
kinetically, through mutagenesis experiments and struc-
turally by crystallography.2 Comparable systematic
investigations have been performed on several retain-
ing galactosyltransferases, most notably the b-(1?3)-
+
N
O
OH
HO
O
O
O
O
O
OR
HO
HO
NHAc
OH
HO
UDP-Gal (1)
2
β(1,4)GalT
OH
HO
OH
O
O
OR
O
HO
NHAc
HO
HO
3 R = (CH2)8CO2Me
Chart 1. The reaction catalyzed by b-(1?4)-GalT, an inverting
enzyme.
galactosyltransferase3 and the human blood-group B
galactosyltransferase.4
The structures of the galactosyltransferases noted
above have been studied by X-ray crystallography as
the native enzymes, the native enzymes with either the
donor or acceptor in the active sites or with analogues
of the donor and/or the acceptor. Complexes of mutant
*
Corresponding author. E-mail: hindsgaul@crc.dk
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.01.021

876
K. Mannerstedt, O. Hindsgaul / Carbohydrate Research 343 (2008) 875–881
enzymes have also been solved. The general mechanistic
picture that has emerged5 is that after binding the
donor, one or more flexible peptide loops undergo con-
formational changes from open to closed forms creating
acceptor binding sites thus enabling the transfer reac-
tion. It has, of course, not been possible to get crystal
structures of the native enzymes in the presence of both
the acceptor and the donor as these react to give the
product glycoside. We report here the initiation of a
new research project aimed at providing ‘caged’ sub-
strate analogues for galactosyltransferases that we hope
will enable time-resolved crystallographic experiments6
to yield atomic details of the native transfer reaction.
The term ‘caged’ compound is used to refer to a bio-
logically unreactive molecule that can be released from
its ‘cage’ at the will of the experimenter to instanta-
neously produce the biologically-active compound.7
Most commonly, a caged compound is blocked with a
photolabile group so that irradiation can cleave this
group. The ATP derivative 4, functionalized with an
2-nitrobenzyl group, was the first reported caged com-
pound that was cleaved to release free ATP on irradia-
tion at 340 nm (Chart 2).8
To extend this concept to study galactosyltransferases,
we needed to establish a system where the enzyme could
bind to the acceptor and caged-UDP-Gal donor without
any reaction occurring, and to demonstrate the reaction
after cleavage of the caging group. We chose the readily
available b-(1?4)-GalT, the inverting enzyme catalysing
the reaction shown in Chart 1. b-(1?4)-GalT has been
shown to have a relaxed specificity towards the sugar
being transferred from the UDP-sugar with measurable
rates for glucose, 2-deoxy-galactose, some 6-O-substi-
tuted galactose derivatives and even GalNAc.9 Nishi-
mura’s group,10 however, has shown that O-6 of
UDP-Gal can be functionalized with a 10-atom long
PEG-based tether terminating in a naphtyl group and
NO2
O
N
HO
HO
O
O
NH
O
P
O
O
P
O
O
HO
O
O
O
O
OH
HO
Caged-UDP-Gal (8)
Chart 3. Structure of the target caged UDP-Gal derivative 8.
that a bromomethyl-naphthyl group (instead of a
naphthyl group) at the end of the tether successfully
affinity-labelled a tryptophan residue in the crystallo-
graphically-flexible loop, suggesting that this position
would be ideal for installing a photocleavable caging
group.
The target for the synthesis was therefore UDP-Gal
with a photolabile group protecting O-600, namely 8
(Chart 3). While many caging groups are known, we
elected to first make the simplest one: the 2-nitrobenzyl
ether. This group is not ideal, particularly as the photo-
lytic product 2-nitrobenzaldehyde (6, Chart 2) has been
reported to undergo chemical side-reactions with pro-
teins.7 For this reason, the 2-nitrophenylethyl caging
group shown in 5 is often used, yielding a more stable
ketone product (7). We therefore view 8 as a prototype
caged UDP-Gal designed to test the feasibility of
this approach, with superior photolabile groups to be
selected later if required.
2. Results and discussion
The synthesis of 8 (Scheme 1) started from commercially
available 1,2:3,4-di-O-isopropylidene-D-galactopyranose
(9), which was 2-nitrobenzylated using NaH and 2-nitro-
benzyl bromide in MeCN, assisted by microwave heating
at 180 °C for 4 min,11 to give 10 in 88% yield. Removal of
the isopropylidene protecting groups with 80% HOAc in
80 °C gave 11 (93%) which, on acetylation, gave 12
(89%). Selective anomeric deacetylation using hydrazine
acetate in DMF afforded 13 in 83% yield.
that resulting molecule is
a potent inhibitor of
b-(1?4)-GalT. In the same report, they demonstrated
O
O
O
P
O
P
O
P
N
H
A
O
O
O
O
O
R
OH OH
OH
The phosphate group was introduced by treatment of
13 with PCl3–imidazole–Et3N12 to give the a-anomeric
H-phosphonate 14 (62%) on hydrolytic work-up. The
H-phosphonate was converted into the fluorenyl-9-
methyl phosphodiester13 with 9-fluorenemethanol and
pivaloyl chloride in pyridine, followed by the oxidation
using iodine in pyridine–water to give 15. The fluorene-
9-methyl protecting group was cleaved with piperidine
in CH2Cl2 to give phosphomonoester 16 in 84% yield
(from 14). Coupling of 16 with UMP-morpholidate in
the presence of 1H-tetrazole in pyridine,14 followed by
deacetylation using Et3N in MeOH–H2O provided tar-
get compound 8 in 44% yield.
OH
HO
4 R=H
"Caged ATP"
5 R=Me
hν 340 nm
O
O
O
P
O
P
O
N
A
P
R
HO
O
O
O
O
OH OH
OH
+
OH
6 R=H
7 R=Me
HO
Free ATP
Chart 2. Photolytic removal of nitrobenzyl groups from caged ATP
(Ref. 8).

K. Mannerstedt, O. Hindsgaul / Carbohydrate Research 343 (2008) 875–881
877
OBnoNO2
O
OBnoNO2
O
O
HO
HO
OBnoNO2
O
OH
AcO
AcO
O
o-NO2BnBr
NaH, MeCN
Ac2O
80% HOAc
80 o
O
Pyridine
C
O
O
O
O
O
HO
OH
AcO
OAc
O
9
10 (88%)
11 (93%)
12 (89%)
a) 9-Fluorene-
OBnoNO2
O
Imidazole,
AcO
AcO
methanol
Hydrazine-
acetate
OBnoNO2
AcO
AcO
PCl3, Et3N
MeCN, 0 oC
Piv-Cl, Pyridine
b) I2, Pyridine-H2O
-40 oC
O
DMF, 50 o
C
O
P
H
AcO
OH
AcO
13 (83%)
O
O
14 (62%)
Et3NH+
OBnoNO2
O
AcO
AcO
OBnoNO2
O
AcO
AcO
1. UMP-morpholidate,
1H-tetrazole, pyridine
2. a) Et3N, MeOH, H2O
b) Dowex H+,Dowex Na+
H
H
Piperidine
CH2Cl2
8 (44%)
N
O
P
O
O
AcO
AcO
P
2
O
O
O
O
O
16 (84%)
15
Et3NH+
Scheme 1. Synthesis of 8.
The photolability of the 2-nitrobenzyl group in 8 was
confirmed by irradiation of an aqueous solution of 8
(0.5 mg/0.5 mL) at 365 nm with a 400 W Hg(Xe) high-
pressure mercury lamp, while monitoring the disappear-
ance of 8 (m/z 700.1) and the appearance of UDP-Gal
(1) with m/z 564.8 in the negative-ion-mode electrospray
mass spectrum (Fig. 1). Irradiation for sequential
periods of 30 s showed that a total of 2 min of irradia-
tion were required to complete the conversion of 8 to
1 under these irradiation conditions. This uncaging reac-
tion is summarized in Scheme 2, Eq 1.
The results of the preliminary evaluation of 8 as a
caged donor for b-(1?4)-GalT are summarized in
Scheme 2. 8-Methoxycarbonyloctyl 2-acetamido-2-deoxy-
b-D-glucopyranoside (2), a known acceptor for b-(1?4)-
(GalT),15 was incubated in parallel with UDP-Gal (1)
and caged-UDP-gal (8) in the presence of the enzyme.
The progress of the reaction was monitored by mass-spec-
trometry where the known disaccharide product 315 could
be seen at m/z 554 [M+H]+ and m/z 576 [M+Na]+ (see
Supplementary data). Acceptor 2 was completely con-
verted to 3 within 2 h in the presence of UDP-Gal (Eq
Intens.
x106
1.5
Start
700.1
700.1
700.1
1.0
0.5
0.0
x105
30 s
6
4
564.8
2
0
x105
1 min
3
2
1
564.8
0
x105
1 min 30s
2 min
564.8
564.8
3
2
1
0
x105
4
700.1
3
2
1
0
m/z
450
500
550
600
650
700
750
Figure 1. ESIMS analysis of the time course of irradiation of 8 (m/z 700.1) to give 1 (m/z 564.8).

878
K. Mannerstedt, O. Hindsgaul / Carbohydrate Research 343 (2008) 875–881
like hydroxylamine or hydrazides may suppress this
hν
8
1 (>95%) (eq. 1)
side-reaction. Alternatively, the use of the methylbenzyl
group (shown in 5) or even more promising new-gener-
ation photolabile groups19 will be explored.
In summary, we synthesized the caged-UDP-Gal com-
pound 8 and confirmed that it is not a substrate for
b-(1?4)-GalT. After brief irradiation, however, 8
becomes uncaged to give UDP-Gal (1) and the enzy-
matic reaction proceeds to produce disaccharide 3. This
system therefore fulfills the minimum requirements for
time-resolved crystallography experiments.
2 min
β(1,4)GalT
1 + 2
8 + 2
8 + 2
3 (>95%) (eq. 2)
2 h
β(1,4)GalT
no reaction (eq. 3)
2 h
β(1,4)GalT
3 (50%)
(eq. 4)
hν (2 min),
then 15h
Scheme 2. Evaluation of 8 as a caged donor for b-(1?4)-GalT.
3. Experimental
2), but no disaccharide product (either with or without a
nitrobenzyl group) could be detected in the corresponding
incubation with 8 (Eq 3). The latter solution was then irra-
diated at 365 nm for 2 min and incubated overnight after
which the mass-spectrum suggested an approximately
50% yield of product 3 (Eq 4). The presence of the hydro-
phobic aglycone in 2 and 3 allowed their isolation from
the reaction mixture by reverse-phase extraction car-
tridges. The 1H NMR spectrum of this retrieved material
confirmed the presence of 2 and 3 in a ratio of near 1:1
(see Supplementary data).
The above results confirmed our hope that 8 would
not be a substrate for the enzyme, but it remained to
demonstrate that it did in fact bind to the enzyme which
is a requirement for its use as a caged donor in time-
resolved protein crystallography. To demonstrate this,
8 was evaluated as an inhibitor for the transfer reaction
shown in Chart 1 and in Eq 2 (Scheme 2) using a
reported radioactive assay,16 where it could be com-
pared to the known inhibitor uridine 50-diphosphate
(UDP). Assuming 8 to be a competitive inhibitor
allowed the estimation of the Ki-values for 8 (42 lM)
and UDP (29 lM) using the equation i = [I]/
([I] + Ki{1 + [S]/Km}),17 where i is the fractional inhi-
bition, [I] the inhibitor concentration and [S] the substrate
concentration. The Km-value for UDP-Gal was set to
43 lM.18 Compound 8 was thereby shown to be an
inhibitor of b-(1?4)-GalT and only slightly less potent
than the known inhibitor UDP.
3.1. General methods
Organic solvents were concentrated under diminished
pressure at <40 °C (bath temperature). TLC was carried
out on silica plates (Merck 60 F254 aluminium plates)
with detection by UV-light or 8% sulfuric acid. Column
chromatography was performed on silica gel (Merck,
˚
230–400, 60 A) and reversed phase chromatography
˚
was performed on silica gel (Bondapak C18 125 A 37–
55 lm, Waters). Photolysis reactions were carried out
under a 400 W Hg(Xe) high-pressure mercury lamp.20
ESIMS was recorded on a Bruker Esquire 3000-Plus
Ion Trap instrument with samples injected as solutions
in 1:1 MeCN–water mixtures. HRESIMS was recorded
on a Q-Tof Ultima instrument from Micromass. 1H, 13
C
and 31P NMR spectra were recorded at 20 °C on a Bru-
ker DPX 250 MHz instrument, Varian Unity 500 MHz
(compounds 3 and 8) or Bruker Avance 600 MHz
instrument (compound 16) using CDCl3 (7.26 ppm for
1H and 77.16 for 13C), D2O (4.79 ppm for H and ace-
1
tone 31 ppm for 13C) or CD3OD (3.31 ppm for H and
1
49.0 ppm for 13C) as the internal standards. 31P NMR
spectra were recorded in CDCl3, CD3OD or D2O using
2% H3PO4 in D2O as the external standard.
3.2. 1,2:3,4-Di-O-isopropylidene-6-O-2-nitrobenzyl-a-D-
galactopyranose (10)
It was disappointing to see that the enzyme looses
substantial activity as a result of the irradiation. This
is most likely due to the reaction of the released alde-
hyde 6 with the enzyme, as discussed above. Further evi-
dence for this proposal comes from the observation that
addition of a pre-irradiated solution of 8 to the enzyme
and acceptor 2 led to the same reduced yield of disaccha-
ride formation.
Whether or not this inactivation side-reaction will
turn out to be detrimental in a short-duration (millisec-
ond) crystallographic experiment using a rapid pulsed-
laser remains to be seen. Should that turn out to be
the case, the addition of aldehyde-sequestering agents
2-Nitrobenzyl bromide (83 mg, 0.384 mmol) and NaH
(9 mg, 0.384 mmol) were added to a soln of 1,2:3,4-
di-O-isopropylidene-D-galactopyranose (9) (50 mg,
0.192 mmol) in MeCN (1 mL) and irradiated (Biotage
Initiator microwave synthesizer) in a closed vessel for
4 min at 180 °C. The reaction mixture was quenched
with MeOH, concentrated and purified by silica gel
chromatography (10:1 toluene–EtOAc) to give 10
1
(67 mg, 0.169 mmol, 88%). H NMR (CDCl3, d ppm):
1.33 (s, 6H, CCH3), 1.43 (s, 3H, CCH3), 1.55 (s, 3H,
CCH3), 3.75 (m, 2H, H-6a, H-6b), 4.07 (m, 1H, J4,5
1.8 Hz, H-5), 4.28 (dd, 1H, J4,5 1.9 Hz, J4,3 7.9 Hz,
H-4), 4.32 (dd, 1H, J2,3 2.4 Hz, H-2), 4.62 (dd, 1H, J2,3

K. Mannerstedt, O. Hindsgaul / Carbohydrate Research 343 (2008) 875–881
879
2.4 Hz, J3,4 7.9 Hz, H-3), 4.95 (s, 2H, ArCH2), 5.54 (d,
1H, J1,2 5.0 Hz, H-1), 7.41 (dt, 1H, J 1.4 Hz, J 8.2 Hz,
Ar-H), 7.62 (dt, 1H, J 1.2 Hz, J 7.6 Hz, Ar-H), 7.82 (d,
1H, J 7.8 Hz, Ar-H), 8.03 (dd, 1H, J 1.2 Hz, 8.2 Hz,
Ar-H); 13C NMR (CDCl3, d ppm): 24.6, 25.0, 26.1,
26.2, 66.8, 69.9, 70.0, 70.7, 70.8, 71.3, 96.5, 108.7, 109.5,
124.7, 127.9, 128.9, 129.2, 133.7; HRESIMS: [M+H]+
calcd for C19H25NO8, 396.1658; found, 396.1656.
6.3 Hz, H-5a), 4.72 (br s, 0.25H, H-1b), 4.88 (m, 2H,
ArCH2), 5.09 (d, 0.5H, H-2b, H-3b), 5.16 (dd, 0.75H,
J2,3 10.8 Hz, J3,4 3.6 Hz, H-2a), 5.43 (dd, 0.75H, J2,3
10.7 Hz, J3,4 3.2 Hz, H-3a), 5.51 (m, 1.75H, H-4a,
H-4b, H-1a), 7.44 (m, 1H, Ar-H), 7.67 (m, 2H, Ar-H),
8.07 (m, 1H, Ar-H); 13C NMR (CDCl3, d ppm): 20.8,
20.8, 21.0, 67.5, 67.6, 67.8, 68.6, 69.0, 69.2, 69.0, 69.6,
70.1, 70.2, 70.7, 71.3, 73.2, 90.9, 96.2, 124.8, 124.9,
128.3, 128.9, 128.9, 133.9, 134.0, 170.2, 170.4, 170.5;
HRESIMS: [M+Na]+ calcd for C19H23NO11,
464.1169; found, 464.1168.
3.3. 1,2,3,4-Tetra-O-acetyl-6-O-2-nitrobenzyl-a-D-galacto-
pyranose (12)
Compound 10 (85 mg, 0.215 mmol) was dissolved in
80% HOAc, heated to 80 °C overnight, concentrated
3.5. 2,3,4-Tri-O-acetyl-6-O-2-nitrobenzyl-a-D-galacto-
pyranosyl hydrogen-phosphonate triethylammonium salt
(14)
1
to give 11 (63 mg, 200 mmol, 93%). H NMR (D2O,
d ppm): 3.45 (dd, 0.6H, H-2b), 3.60 (dd, 0.6H, J3,2
3.4 Hz, J3,4 9.9 Hz, H-3b), 3.70–3.93 (m, 4.4H, H-2a,
H-3a, H-4a, H-4b, H-5b, H-6a, H-6b), 4.21 (t, 0.4H,
J4,5 6.0 Hz, H-5a), 4.54 (d, 0.6H, J1,2 7.8 Hz, H-1b),
4.90 (s, 2H, ArCH2), 5.22 (d, 0.4H, J1,2 3.3 Hz,
H-1a), 7.53 (m, 1H, ArH), 7.69 (m, 1H, ArH), 8.03
(d, 1H, J 8.2 Hz, ArH); 13C NMR (D2O, d ppm):
68.7, 69.1, 69.4, 69.5, 70.0, 70.2, 70.3, 70.5, 70.7,
72.2, 73.1, 73.8, 92.7, 96.8, 125.4, 129.6, 130.4, 134.6;
ESIMS: [M+Na]+ calcd for C13H17NO8, 338.1; found,
338.0. The residue was acetylated using Ac2O (1 mL)
and pyridine (2 mL) for 1 h. The reaction mixture
was concentrated and purified by silica gel chromato-
graphy (5:1 toluene–EtOAc) to give 12 (86 mg,
0.178 mmol, 89%). 1H NMR (CDCl3, d ppm): 1.96–
2.15 (m, 12H, CCH3), 3.53–3.75 (m, 2H, H-6a,
H-6b), 4.05 (t, 0.6H, J4,5 6.4 Hz, H-5b), 4.35 (t, 0.4H,
J 6.2 Hz, H-5a), 4.85 (m, 2H, ArCH2), 5.09 (dd,
0.6H, H-3b), 5.32 (m, 2H, H-3a, H-2a, H-2b), 5.51
(d, 0.6H, J3,4 3.3 Hz, H-4b), 5.57 (s, 0.4H, H-4a),
5.69 (d, 0.6H, J1,2 8.3 Hz, H-1b), 6.36 (d, 0.4H, J1,2
2.6 Hz, H-1a), 7.40 (t, 0.6H, J 7.3 Hz, Ar-H), 7.65
(m, 2H, Ar-H), 8.03 (d, 1H, J 8.1 Hz, Ar-H); 13C
NMR (CDCl3, d ppm): 20.6, 20.7, 20.9, 21.0, 66.7,
67.4, 67.7, 68.1, 68.6, 68.9, 70.0, 70.1, 71.1, 73.0, 89.9,
92.3, 124.8, 128.2, 128.2, 128.7, 128.8, 133.9, 134.0,
169.1, 169.5, 170.0, 170.2; HRESIMS: [M+Na]+ calcd
for C21H25NO12, 506.1274; found, 506.1268.
PCl3 (127 lL, 1.45 mmol) was added to a soln of
imidazole (125 mg, 1.84 mmol) and Et3N (275 lL,
1.97 mmol) in MeCN at 0 °C and stirred for 30 min.
Compound 13 (116 mg, 0.263 mmol) in MeCN was
added during 30 min at 0 °C. The reaction mixture
was stirred at rt for 15 min, quenched with TEAB (tri-
ethylammonium bicarbonate) (1 M) and concentrated.
The residue was dissolved in CHCl3, washed with
TEAB (triethylammonium hydrogen carbonate buffer,
1 M), filtered through cotton wool, concentrated and
purified by silica gel chromatography (1:0?9:1
CHCl3–MeOH containing 1% Et3N) to give 14
1
(98 mg, 0.162 mmol, 62%). H NMR (CDCl3, d ppm):
1.93 (s, 3H, CCH3), 2.00 (s, 3H, CCH3), 2.07 (s, 3H,
CCH3), 3.57 (m, 2H, H-6a, H-6b), 4.50 (t, J 6.3 Hz,
H-5), 4.80 (m, 2H, ArCH2), 5.20 (m, 1H, H-2), 5.42
(dd, 1H, J2,3 10.7 Hz, J3,4 3.2 Hz, H-3), 5.51 (m, 1H,
H-4), 5.81 (dd, 1H, J1,P 8.8 Hz, J1,2 3.4 Hz, H-1), 6.92
(d, 1H, JP,H 636 Hz, P–H), 7.38 (t, 1H, J 7.3 Hz,
Ar-H), 7.63 (m, 2H, Ar-H), 7.99 (d, 1H, J 8.2 Hz,
Ar-H); 13C NMR (CDCl3, d ppm): 20.7, 20.8, 67.8,
68.0, 68.1, 69.1, 70.0, 91.9, 92.0, 124.6, 128.1, 128.8,
134.0, 170.0, 170.2, 170.3; 31P NMR (CDCl3, d ppm):
1.81; HRESIMS: [M+Na]+ calcd for C19H23NO13P,
528.0883; found, 528.0864.
3.6. 2,3,4-Tri-O-acetyl-6-O-2-nitrobenzyl-a-D-galacto-
pyranosyl phosphate bispiperidinium salt (16)
3.4. 2,3,4-Tri-O-acetyl-6-O-2-nitrobenzyl-D-galacto-
pyranose (13)
Pivaloyl chloride (24 lL, 0.191 mmol) was added to a
soln of compound 14 (28 mg, 0.0462 mmol) and 9-fluo-
renemethanol (23 mg, 0.120 mmol) in pyridine. The
reaction mixture was stirred at rt for 3 h and then
cooled to ꢀ40 °C and a soln of I2 (23 mg, 0.0924 mmol)
in pyridine–water (49:1, 2 mL) was added. The oxida-
tion was completed at 0 °C and the reaction was
diluted with CHCl3, washed with Na2S2O3 (10%) and
TEAB (1 M), filtered (cotton wool) and concentrated.
The residue was dissolved in CH2Cl2 and piperidine
(0.4 mL) was added and stirred at rt for 30 min,
Hydrazine acetate (14 mg, 0.155 mmol) was added to
compound 12 (50 mg, 0.103 mmol) in DMF and heated
to 50 °C for 30 min. The reaction mixture was diluted
with EtOAc and washed with NaCl (satd, aq), dried
(Na2SO4), filtered, concentrated and purified by silica
gel chromatography (5:1 toluene–EtOAc) to give 13
1
(38 mg, 0.086 mmol, 83%). H NMR (CDCl3, d ppm):
1.98–2.16 (m, 9H, CCH3), 3.64 (m, 2H, H-6a, H-6b),
3.97 (t, 0.25H, J4,5 6.3 Hz, H-5b), 4.51 (t, 0.75H, J4,5

880
K. Mannerstedt, O. Hindsgaul / Carbohydrate Research 343 (2008) 875–881
concentrated and purified by reversed phase chromato-
MOPS buffer (50 mM, 200 lL, pH 7.0). 400 mU b-
(1?4)-galactosyltransferase (from bovine milk, Fluka,
1 U in 1 mL MOPS buffer pH 7.0, containing BSA
1 mg/mL and 5 mM MnCl2) and alkaline phosphatase
(Merck, 10 U) were added and the reaction mixture
was left at rt. After 2 h the reaction was radiated
with a 400 W Hg(Xe) high-pressure mercury lamp at
365 nm for 2 min and left at room temperature over
night. The mixture was diluted with water and purified
on SepPak C-18 cartridges, which were pre-washed with
15 mL MeOH and 50 mL water. The SepPak cartridges
were washed with 50 mL water and the product was
eluted with 10 mL MeOH to give disaccharide 3 (50%)
and unreacted acceptor 2 (50%). The yields were deter-
graphy (1:0?9:1 water–MeOH) to give 16 (27 mg,
1
0.039 mmol, 84%). H NMR (CD3OD, d ppm): 1.96 (s,
3H, CCH3), 2.07 (s, 3H, CCH3), 2.11 (s, 3H, CCH3),
3.60 (t, 1H, H-6a), 3.70 (m, 1H, H-6b), 4.60 (t, 1H, J4,5
6.9 Hz, H-5), 4.90 (m, 2H, ArCH2), 5.11 (m, 1H, H-2),
5.43 (dd, 1H, J3,4 3.3 Hz, J2,3 10.9 Hz, H-3), 5.58 (d,
1H, J 2.1 Hz, H-4), 5.74 (dd, 1H, J1,P 7.5 Hz, J1,2
3.3 Hz, H-1), 7.50 (t, 1H, J 7.9 Hz, ArH), 7.72 (m, 1H,
ArH), 8.04 (d, 1H, J 8.0 Hz, ArH); 13C NMR (CD3OD,
d ppm): 20.6, 20.7, 20.9, 68.6, 69.2, 69.5, 69.9, 70.9, 93.4,
93.5, 125.6, 129.6, 130.4, 134.7, 171.7, 172.1, 172.2; 31P
NMR (CD3OD, d ppm): ꢀ0.93; ESIMS: [MꢀH]ꢀ calcd
for C19H24NO14P, 520.1; found, 520.1.
1
mined from the H NMR spectrum that was consistent
3.7. Uridine 50-(6-O-2-nitrobenzyl-a-D-galactopyranosyl)
diphosphate disodium salt (8)
with previously reported data.
3.9. Inhibition studies
UMP-morpholidate (50 mg, 0.0726 mmol) and 1H-tetra-
zole (10 mg, 0.145 mmol) were added to a soln of 16
(22 mg, 0.0363 mmol) in pyridine. After stirring at rt
for 2 days the reaction mixture was concentrated. The
residue was dissolved in 7:1 MeOH–water and Et3N
(252 lL, 1.81 mmol) was added. The reaction mixture
was stirred at room temperature for 48 h, concentrated
and purified by DEAE-Sephacel ion-exchange column
chromatography (Pharmacia, linear gradient from
30 mM to 400 mM NH4HCO3) and concentrated to
about 10 mL. Dowex 50W X 8 (H+, 200–400 mesh)
was added slowly until the pH reached 6.5. The resin
was removed by filtration and Dowex 50W X 8 (Na+,
20–50 mesh) was added and stirred for a couple of min-
utes. The resin was removed by filtration and the filtrate
lyophilized. The residue was passed through a gel filtra-
tion column (Sephadex G-10) eluting with water,
followed by reversed phase chromatography eluting
with water and lyophilization gave 8 (12 mg, 161 lmol,
44%). 1H NMR (D2O, d ppm): 3.71–3.76 (m, 3H,
H00-2, H00-6a, H00-6b), 3.88 (d, 1H, J 10.6 Hz, H00-3),
3.99 (s, 1H, H00-4), 4.11–4.18 (m, H0-2, H0-3, H0-4),
4.24 (m, H0-5a, H0-5b), 4.29 (t, 1H, J4,5 6.6 Hz, H00-5),
4.89 (q, 2H, J 14.4 Hz, J 6.7 Hz, ArCH2), 5.58 (dd,
1H, J1,P 7.3 Hz, J1,2 3.4 Hz, H00-1), 5.84 (m, 2H, H-6,
H0-1), 7.50 (m, 1H, H-5), 7.69 (m, 2H, ArH), 7.83 (d,
1H, ArH, J 8.2 Hz), 8.03 (d, 1H, ArH, J 8.1 Hz); 31P
NMR (D2O, d ppm): ꢀ11.2, ꢀ12.8; HRESIMS: [MꢀH]ꢀ
calcd for C22H29N3O19P2, 700.0792; found, 700.0795.
Inhibition studies were carried out using a reported
3
radiochemical assay.16 Briefly, H-labelled galactose is
enzymatically transferred from labelled UDP-Gal to
acceptor 2 giving the hydrophobic labelled product 3
(Chart 1) that can be isolated on a Sep-Pak C18 car-
tridge and eluted with MeOH for quantitation. The
assays were performed at 37 °C in a total volume of
15 lL containing 50 mM MOPS buffer pH 7.0, 20 mM
MnCl2, 1 mg/mL bovine serum albumin, 50 lM UDP-
Gal, UDP-[6-3H]Gal (corresponding to 50,000 dpm)
and 400 lM GlcNAc-(CH2)8COOMe (2). The concen-
tration of the b-(1?4)-GalT was chosen so that not
more than 10% of the donor was consumed in the reac-
tion. The reaction mixtures were then transferred to pre-
washed C18 Sep-Pak cartridges which were then washed
with water (50 mL) and the radiolabelled product was
eluted with MeOH (3.5 mL) and quantitated by liquid
scintillation.16 The Ki values for 8 and UDP were esti-
mated from incubations performed at inhibitor concen-
trations of 25 lM, 50 lM and 100 lM.
Acknowledgements
This work is a result of an ongoing research collabora-
tion with the groups of Anne Imberty (CERMAV,
CNRS, France) and Monica M. Palcic (Carlsberg
Laboratory, Denmark) who are acknowledged for the
valuable discussions in the planning stages of the project.
We thank Pia Breddam for recording the HRESIMS.
3.8. Photolysis of uridine 50-(6-O-2-nitrobenzyl-a-D-
galactopyranosyl) diphosphate disodium salt
Supplementary data
Uridine
50-(6-O-2-nitrobenzyl-a-D-galactopyranosyl)
diphosphate disodium salt (2.5 mg, 3.35 lmol) and
8-methoxycarbonyloctyl 2-acetamido-2-deoxy-b-D-gluco-
pyranoside (2) (0.7 mg, 1.79 lM) were dissolved in
1H and 13C NMR spectra of compounds 10–14 and 16,
1H NMR spectrum of 8 and the product described in Eq
4 (Scheme 2), ESIMS spectra of the products described

K. Mannerstedt, O. Hindsgaul / Carbohydrate Research 343 (2008) 875–881
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