UDP-6-deoxy-6-fluoro-α-D-galactose binds to two different galactosyltransferases, but neither can effectively catalyze transfer of the modified galactose to the appropriate acceptor

Article abstract of DOI:10.1016/S0008-6215(99)00104-4

The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of α-(1→3)-galactosyltransferase (EC 2.4.1.151) and β-(1→4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [β-D-Gal-(1→4)-D-GlcNAc] for the β-(1→4)-galactosyltransferase and α-D-Gal-(1→3)-β-D-Gal-(1→4)-D-GlcNAc for the α-(1→3)-D-galactosyltransferase] when UDP-α-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-α-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-α-D-galactopyranoside, synthesized by the selective fluorination of methyl α-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-β-D-galactopyranose, which was converted to the corresponding 1-α-phosphate and used for the synthesis of 6. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00104-4

www.elsevier.nl/locate/carres
Carbohydrate Research 319 (1999) 24–28
UDP-6-deoxy-6-fluoro-a-
D-galactose binds to two
different galactosyltransferases, but neither can
effectively catalyze transfer of the modified galactose to
the appropriate acceptor
Cara-Lynne Schengrund a,*, Pavol Kova´c b
'
a Department of Biochemistry and Molecular Biology, The Pennsyl6ania State Uni6ersity College of Medicine,
Hershey, PA 17033, USA
b National Institute of Diabetes, Digesti6e and Kidney Diseases, National Institutes of Health, Building 8A,
Bethesda, MD 20892-0815, USA
Received 1 February 1999; accepted 22 April 1999
Abstract
The effect of substitution of the HO-6 of
(EC 2.4.1.151) and b-(1“4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate
acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the
D
-galactose with fluorine on the ability of a-(1“3)-galactosyltransferase
expected product [b-
(1“4)- -GlcNAc for the a-(1“3)-
oxy-6-fluoro-a- -galactose (6) was used in conjunction with each transferase, no product indicative of transfer of
6-deoxy-6-fluoro- -galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro- -galactose (3) was
obtained by hydrolysis of methyl 6-deoxy-6-fluoro-a- -galactopyranoside, synthesized by the selective fluorination of
methyl a- -galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetyl-
-galactopyranose, which was converted to the
D
-Gal-(1“4)-
D
-GlcNAc] for the b-(1“4)-galactosyltransferase and a-
D
-Gal-(1“3)-b-
D-Gal-
D
D
-galactosyltransferase] when UDP-a- -Gal was the substrate. When UDP-6-de-
D
D
D
D
D
D
ation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-b-
D
corresponding 1-a-phosphate and used for the synthesis of 6. © 1999 Published by Elsevier Science Ltd. All rights
reserved.
Keywords: Methyl 6-deoxy-6-fluoro-a-
D
-galactopyranoside; 6-Deoxy-6-fluoro-
D-galactose; 1,2,3,4-Tetra-O-acetyl-6-deoxy-6-
fluoro-b- -galactopyranose; UDP-6-deoxy-6-fluoro-
D
D
-galactose; Galactosyltransferase
1. Introduction
glycosidase [3], have been reported. The effect
of substituting a fluorine for the HO-6 of
galactose on the activity of a galactosyl trans-
ferase has also been studied using the appro-
priate nucleotide phosphate derivative [4].
Recently, a number of galactosyltrans-
ferases, enzymes that catalyze the transfer of
The effects of replacing a specific hydroxyl
group of a sugar with a fluorine atom on the
ability of the sugar to function as either a
ligand for a lectin [1], a molecule transported
by a specific carrier [2], or a substrate for a
galactose from UDP-a- -Gal to appropriate
D
acceptors, have been cloned and sequenced.
Breton et al. [5] analyzed cDNA sequences for
these enzymes and classified them into five
* Corresponding author. Tel.: +1-717-531-8048; fax: +1-
717-531-7072.
E-mail address: cschengrund@psu.edu (C.-L. Schengrund)
0008-6215/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00104-4

C.-L. Schengrund, P. Ko6a´c
'
/ Carbohydrate Research 319 (1999) 24–28
25
families, four of which have a DxD motif.
This motif was proposed to function either in
binding of UDP or in the catalytic process. As
a first step in determining whether similarities
exist in the interaction of different galactosyl-
not as good as the overall yield (ꢀ42%)
obtained by converting methyl a- -galacto-
pyranoside via four synthetic steps [8], it is a
much less laborious, more efficient procedure.
The conversion of 2“3 was conveniently
achieved by hydrolysis with aqueous tri-
fluoroacetic acid (TFA).
D
transferases with UDP-a-
the ability of two galactosyl-transferases to
catalyze the transfer of 6-deoxy-6-fluoro-
galactopyranose from UDP-6-deoxy-6-fluoro-
a- -galactose (6) to the appropriate acceptor.
D-Gal, we studied
The two commonly used O-acetylation pro-
cedures [9] (using acetic anhydride with either
sodium acetate or pyridine) were used to
D
-
D
acetylate
D
-galactose, and the distribution of
isomeric acetates formed was evaluated by
TLC and NMR spectroscopy. The products in
the crude material obtained by either method
produced two bands when examined by TLC.
NMR spectroscopy indicated that the sodium
acetate–acetic anhydride procedure produced
the highest percentage of b-pyranose; there-
fore, it was used for the synthesis of 1,2,3,4-
2. Results and discussion
Previous syntheses [4,6] of 6-deoxy-6-fluoro-
a- -galactopyranosyl phosphate (5) started
D
with anomeric mixtures of per-O-acetyl-6-de-
oxy-6-fluoro- -galactopyranose and galacto-
furanose, formed by acetylation of 6-deoxy-6-
fluoro- -galactose (3). In this work, to obtain
6, we used crystalline 1,2,3,4-tetra-O-acetyl-6-
deoxy-6-fluoro-b- -galactopyranose (4), ob-
-galacto-
D
D
tetra-O-acetyl-6-deoxy-6-fluoro-b- -galactose
D
(4). Material highly enriched in 4 was ob-
tained from the first zone following chro-
matography of the per-O-acetylated products.
Crystallization from ethanol then gave pure 4.
The anomeric configuration in 4 followed
from the observed J1,2 and JC-1,H-1 coupling
constants.
D
tained as follows. Methyl a-
D
pyranoside (1) was fluorinated with diethyl-
aminosulfur trifluoride (DAST), as previously
described for the fluorination of methyl b- -
D
galactopyranoside [7]. A mixture of products
was formed, with methyl 6-deoxy-6-fluoro-a-
Initial reaction rates for galactosyltrans-
ferase-catalyzed transfer of galactose from
UDP-3H-Gal to the appropriate acceptor were
determined by monitoring the time over which
a linear increase in label associated specifically
with the neutral sugar fraction was obtained.
For b-(1“4)-galactosyltransferase it was at
least 120 min, while for the a-(1“3)-galacto-
syltransferase it was at least 45 min. Inhibition
studies indicated that a five-fold molar excess
of 6 relative to UDP-Gal reduced the amount
of tritiated galactose transferred from UDP-
3H-Gal by more than 50% for each enzyme
(Table 1). HPLC analyses indicated that both
transferases catalyzed formation of the ex-
D-galactopyranoside (2) having the slowest
mobility upon TLC. The 20–25% yield of
pure 2 obtained after silica gel chromatogra-
phy was comparable to the yield of methyl
6-deoxy-6-fluoro-b- -galactopyranoside pro-
D
duced, in the same way, from methyl b- -
D
galactopyranoside [7] as well as the ꢀ20%
yield obtained by fluoride ion displacement of
a sulfonyloxy group at C-6 of 1,2:3,4-di-O-iso-
propylidene-a- -galactopyranose [8]. While
D
pected product, b-
for the b-(1“4)-galactosyltransferase and a-
-Gal-(1“3)-b- -Gal-(1“4)- -GlcNAc for
the a-(1“3)-galactosyltransferase, when
D
-Gal-(1“4)-
D
-GlcNAc
D
D
D
UDP-Gal was the substrate. However, when 6
was used as the substrate, no peaks indicative
of product synthesis were seen by HPLC anal-
ysis. The results obtained with the b-(1“4)-
galactosyltransferase are in agreement with
the previously reported observation that when

26
C.-L. Schengrund, P. Ko6a´c
'
/ Carbohydrate Research 319 (1999) 24–28
Table 1
ported by homonuclear decoupling experi-
Inhibition by 6 of b-(1“4)- and a-(1“3)-galactosyltrans-
ferase-catalyzed transfer of galactose from UDP-Gal to ac-
ceptor
ments or homonuclear and heteronuclear
2-dimensional correlation spectroscopy, run
with the software supplied with the spectrome-
ter. Chemical-ionization mass spectra (CIMS)
were measured with a Finnegan 4600 Spec-
trometer, and optical rotations with a Perkin–
Elmer Automatic Polarimeter, model 341.
Reactions requiring anhydrous conditions
were carried out under nitrogen. Porcine, re-
combinant a-(1“3)-galactosyltransferase, b-
(1“4)-galactosyltransferase from bovine
milk, linear B-2 trisaccharide, and N-acetyl-
lactosamine were purchased from Calbiochem
(San Diego, CA), UDP-Gal [Gal-6-3H, 60 Ci/
mM] from American Radiolabeled Chemicals,
Inc. (St. Louis, MO), 1,2,3,4,6-penta-O-acetyl-
% Inhibition
[6]/[UDP-Gal]
b-(1“4)
a-(1“3)
0
1
5
10
0
0
a
2494
39910
7694
7594
55916
7492
a Values given are percent inhibition plus and minus the
standard deviation of the percent inhibition in replicate mea-
surements.
6 was the substrate, it catalyzed the release of
a negligible amount (0.3%) of UDP [4]. The
lack of detectable product in this study proba-
bly reflects (1) use of a much shorter reaction
time (1 h in this study compared with 48 h for
the synthesis of 6%-deoxy-6%-fluorolactosamine
in [4]), and (2) use of different amounts of
b-(1“4)-galactosyltransferase (0.25 mU were
used in this study compared to the 7 mU used
in initial reaction rate studies and 4 U used to
obtain 6%-deoxy-6%-fluorolactosamine in [4]).
The inhibition studies indicate for the first
time that, while the presence of fluorine at C-6
b-
D
-galactopyranose from Aldrich Chemical
-galactopy-
Co. (Milwaukee, WI), methyl a-
D
ranoside from Sigma Chemical Co., and alka-
line phosphatase from calf intestine was from
Boehringer Mannheim (Indianapolis, IN). So-
lutions in organic solvents were concentrated
at 40 °C/2 kPa.
Preparation of methyl 6-deoxy-6-fluoro-h- -
D
galactopyranoside (2).—DAST (3.75 mL, 28
mmol) was added with stirring over 30 min, at
in -galactose has little effect on the ability of
D
−40 °C, to a suspension of methyl a- -galac-
D
each enzyme to bind to 6, it apparently
topyranoside (1, 1 g, 5 mmol) in dry CH2Cl2
(20 mL). The mixture was stirred at −40 °C
for another 30 min and then at room tempera-
ture for 3 h. TLC (8:1 CH2Cl2 –CH3OH) then
showed that essentially all of 1 was consumed
and that several products had been formed.
After cooling to −20 °C, excess reagent was
destroyed by the sequential addition of
methanol (10 mL), and NaHCO3 (3 g). The
cooling bath was removed, and when the ef-
fervescence ceased, the mixture was filtered,
and the filtrate was concentrated. The residue
was chromatographed (silica gel using a step-
gradient of 15:1“8:1 CH2Cl2–CH3OH), to
give the product having the slowest mobility,
pure 2: mp 139–141 °C (from acetone); lit.
[10], mp 139 °C. Yields in various prepara-
tions were between 20 and 25%; 13C NMR
(D2O) l 101.6 (C-1), 84.2 (d, J6,F 168 Hz,
C-6), 71.2 (C-3), 70.6 (d, J4,F 6.8 Hz, C-4),
70.6 (d, J5,F 21.5 Hz, C-5), 70.1 (C-2).
negates their ability to catalyze the transfer of
the 6-deoxy-6-fluoro-a- -galactosyl moiety
D
from UDP to the acceptor sugar.
3. Experimental
General methods.—All reactions were moni-
tored by thin-layer chromatography (TLC),
on Silica Gel GHLF plates (Analtech, Inc.,
Newark, DE). Compounds were visualized us-
ing 5% sulfuric acid in 95% ethanol and/or
UV light. Column chromatography was car-
ried out using either E. Merck Silica Gel 60
[230–400 mesh ASTM (Bodman Industries,
Aston, PA)], Dowex-50W (H+) from Sigma
Chemical Co. (St. Louis, MO), or Bio-Gel
P-2, Fine (Bio-Rad Laboratories, Hercules,
CA), as appropriate. 1H and 13C NMR spectra
were obtained at 300 and 75 MHz, respec-
tively, using a Varian Mercury spectrometer.
When feasible, the assignments were sup-
Preparation of 6-deoxy-6-fluoro-D-galactose
(3).—Compound 2 (1.56 g, 7.6 mmol) was

C.-L. Schengrund, P. Ko6a´c
'
/ Carbohydrate Research 319 (1999) 24–28
27
J5,F 23.4 Hz, C-5), 70.68 (C-3), 67.70 (C-2),
and 66.70 (d, J4,F 5.8 Hz, C-4), 20.69, 20.56,
20.51, 20.46 (4 CH3). Anal. Calcd for
C14H19FO9: C, 48.00; H, 5.47. Found: C,
48.04; H, 5.46.
The mother liquor from crystallization of 4
was combined with material that was unre-
solved by chromatography, deacetylated with
NaOMe–MeOH (Zemple´n), and reprocessed
as described above.
treated with 50% aq trifluoroacetic acid (86
mL) for 5 h at 100 °C. After concentration
and co-evaporation with water to remove
TFA, chromatography on silica gel, using a
step gradient of 12:1“5:1 CH2Cl2–CH3OH,
gave first unchanged 2. Eluted next was 3.
Pure 3 was obtained upon crystallization
(H2O–EtOH, 1.1 g, 76% yield), mp 160–
1
162 °C, lit. [10], 160 °C; H NMR (D2O) for
the anomeric mixture (a:bꢀ1:1): l 5.28 (d,
J1,2 3.5 Hz, H-1a), 4.8–4.48 (m, H-6ab, incl. d
at 4.61, J1,2 7.9 Hz, H-1b), 4.32 (ddd, J4,5B1,
J5,6a 4.0, J5,6b 7.0, J5,F 16.6 Hz, H-5b), 4.04–
3.93 (m, H-4a,b,5a), 4.87 (dd, J2,3 10.1, J3,4 3.1
Hz, H-3a) 3.80 (dd, H-2a), 3.66 (dd, J2,3 10.1,
J3,4 3.8 Hz, H-3b), 3.50 (dd, J2,3 9.6 Hz, H-2b);
13C NMR (D2O) l 96.52 (C-1b), 92.45 (C-1a),
83.57, 83.22 (2d, J6,F 164.6 Hz, H-6a,b), 73.48
(d, J5,F 19.8 Hz, C-5b), 72.6 (C-3b), 71.8 (C-
2b), 69.06 (d, J5,F 18.9 Hz, C-5a), 69.06 (d, J4,F
7.4 Hz, C-4a), 68.94 (C-3a), 68.47 (d, J4,F 7.4
Hz, C-4b), and 68.29 (C-2a).
Preparation of 6-deoxy-6-fluoro-h- -galac-
D
topyranosyl phosphate (5).—The dipotassium
salt of 5 was prepared from 4 (414 mg, 1.18
mmol) as described by MacDonald [11] for
a-
purified by chromatography on silica gel using
step-gradient of 10:2:1“10:4:0.5 2-
D
-galactose 1-phosphate. The product was
a
propanol–water–CH3COOH. Yield, 354 mg,
88%: [h]D +74° (c 0.2 H2O), lit. [6], for the
analogous derivative of
D-galactose, +81° (c
1
0.2 H2O); H NMR (D2O): l 5.48 (dd, 1 H,
J1,2 3.6, J1,P 6.9 Hz, H-1), 4.67, 4.52 (2 m, 1 H
each, J6,F 46.6 Hz, H-6a,b), 4.33 (2 t, 1 H, J5,6
5.8, J5,F 17.2 Hz, H-5), 4.02 (dd, 1 H, J4,F 1.1
Hz, J3,4 3.3 Hz, H-4), 3.88 (dd, 1 H, J2,3 10.0
Hz, H-3), 3.74 (ddd, 1 H, J2,P 2.2 Hz, H-2);
13C NMR (D2O): l 94.4 (C-1, JC-1,P 5.7 Hz),
83.65 (C-6, JC-6,F 163.8 Hz), 69.67 (C-5, JC-5,F
19.5 Hz), 69.07 (C-3), 69.02 (C-4, JC-4,F 6.9
Hz), 68.45 (C-2, JC-2,P 8.0 Hz).
1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-i- -
D
galactopyranose (4).—Compound 3 (900 mg,
4.7 mmol) was added to a mixture of fused
sodium acetate (0.45 g) in acetic anhydride (9
mL) and stirred at 110 °C for 3 h, when TLC
(3.5:1 hexane–acetone) showed the reaction to
be complete. Excess Ac2O was destroyed by
stirring the mixture with aq NaHCO3. The
product was extracted into CH2Cl2, and the
organic phase was dried and concentrated.
TLC showed the presence of two major
Preparation of UDP-6-deoxy-6-fluoro-h-
galactose (6).—The dipotassium salt of 5 was
converted to UDP-6-deoxy-6-fluoro-a-
D-
1
D
-
bands. H NMR spectroscopy indicated the
galactose using the procedure described by
Wittmann and Wong [12] for the synthesis of
UDP-Gal. After purification on a Bio-Gel P-2
column with 0.25 M NH4HCO3 as the eluant,
the sample was lyophilized. Recovered UDP-
ratio of anomers present to be ap:bp:af:bf=
18.4:52.0:15.4:14.2. Upon chromatography on
silica gel (3.5:1 hexane–acetone), the second
zone eluted gave 4 in \90% purity (NMR,
730 mg). The material solidified upon standing
and crystallization from EtOH (twice) gave
pure 4, mp 94–96 °C, [h]D +27.6° (c 1.9
6-deoxy-6-fluoro-a- -galactose was dissolved
D
in water and converted to the disodium salt by
first adding Dowex 50W (H+) to adjust the
pH to ꢀ6, filtering to remove the Dowex,
and then adding NaOH to bring the pH to
ꢀ7. Excess salt was removed by filtration
through a YC05 membrane that had a molec-
1
CHCl3); H NMR (CDCl3) l: 5.74 (d, 1 H,
J1,2 8.3 Hz, H-1), 5.50 (bd, 1 H J3,4 ꢀ2.9 Hz,
H-4), 5.35 (dd, 1 H, J2,3 10.3 Hz, H-2), 5.10
(dd, 1 H, H-3), 4.53 (ddd, 1 H, J5,6a 6.3 Hz, 1
H, J6a,b 9.8 Hz, J6a,F 46.9 Hz, H-6a), 4.44
(ddd, 1 H, J5,6b 5.9 Hz, J6b,F 46.3 Hz, H-6b),
4.11 (m, 1 H, H-5), 2.17, 2.12, 2.05, 2.00 (4 s,
3 H each, 4 COCH3); 13C NMR (CDCl3) l:
169.97, 169.86, 169.33, 168.88 (4 CO), 92.05
(C-1), 80.19 (d, J6,F 171.9 Hz, C-6), 72.15 (d,
1
ular-weight cutoff of 500. 31P and H NMR
(D2O) spectral data agreed with those re-
ported previously [4]. Mass spectral analysis
of the disodium salt gave the anticipated m/z
peak at 613 [M+1]+.

28
C.-L. Schengrund, P. Ko6a´c' / Carbohydrate Research 319 (1999) 24–28
Enzyme assays.—To determine a-(1“3)-
galactosyltransferase. The ability of 6 to
inhibit the transfer of galactose from UDP-
3H-Gal to acceptor was monitored by carrying
out the reaction in the presence of inhibitor
and then determining the amount of label
associated with material eluted from the Bio-
Rad AG 1-X8 column with water. As de-
scribed above, the presence of nonspecific
label in the eluant was accounted for by sub-
tracting label recovered in reactions carried
out in the absence of acceptor sugar.
galactosyltransferase activity, the enzyme
(0.25 mU) was preincubated for about 15 min
at 37 °C with 20 mM N-acetyllactosamine in
0.1 M sodium cacodylate buffer, pH 6.5, con-
taining 20 mM MnCl2, 36 U of alkaline phos-
phatase, and 10 mg of bovine serum albumin
(BSA) in a total volume of 40 mL. At the end
of the preincubation, the solution was made 2
mM in UDP-Gal or 6 by adding 10 mL of a 10
mM solution of the UDP-Gal or 6 in 0.1 M
sodium cacodylate buffer, pH 6.5, containing
20 mM MnCl2, and incubated at 37 °C for
30–35 min. In experiments requiring UDP-
3H-Gal, an aliquot that gave ꢀ200,000 cpm
was added with the UDP-Gal or UDP-Gal
plus 6. The product was separated from unre-
acted UDP-sugar and UMP by ion-exchange
chromatography on Bio-Rad Ag 1-X8 [13].
When labeled substrate was used, the amount
of labeled galactose present in the product was
determined by counting in a scintillation
counter. The presence of nonspecific label in
the eluant was accounted for by subtracting
label recovered in reactions carried out in the
absence of acceptor sugar from that obtained
in its presence. When cold substrate was used,
the product was identified by chromatography
using a Dionex HPLC (Sunnyvale, CA) with a
Dionex CarboPacTM PA1 (4×250 mm)
column and NaOH (0.1 M) as the solvent.
NaOH (0.4 M) was added to the eluant prior
to passage through a pulsed amperometric
detector.
Acknowledgements
This work was supported in part by grant
NS35653 awarded by the National Institute of
Neurological Disorders and Stroke. The au-
thors would like to thank Mostafa Sheykjnan-
zari for assistance with the HPLC analyses,
and Noel Whitaker for mass spectral analyses.
References
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A similar procedure was used to determine
the activity of b-(1“4)-galactosyltransferase.
Enzyme (0.25 mU) was preincubated for 15
min at 37 °C with 20 mM 2-acetamido-2-de-
oxy- -glucose in 50 mM HEPES, pH 7.2,
D
containing 20 mM MnCl2, alkaline phos-
phatase (36 U) and BSA. Except for the use of
a 1 h incubation time, the rest of the proce-
dure was unchanged from that for a-(1“3)-
[13] E.E. Boeggeman, P.V. Balaji, N. Sethi, A.S. Masibay,
P.K. Qasta, Protein Eng., 6 (1993) 779–785.
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