1H-Tetrazole as Catalyst in Phosphomorpholidate Coupling
Reactions: Efficient Synthesis of GDP-Fucose, GDP-Mannose, and
UDP-Galactose
Valentin Wittmann and Chi-Huey Wong*
The Scripps Research Institute, Department of Chemistry, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received October 28, 1996
X
An improved procedure is described for the efficient and high-yield (76-91%) synthesis of nucleoside
diphosphate sugars from the readily available nucleoside 5′-monophosphomorpholidate and sugar
1-phosphate in the presence of 1H-tetrazole. Comparative kinetic investigations by means of
31
P
NMR spectroscopy with different additives (1,2,4-triazole, acetic acid, N-hydroxysuccinimide,
4-(dimethylamino)pyridine hydrochloride, perchloric acid) and mass spectrometric analysis suggest
that tetrazole acts as an acid and as a nucleophilic catalyst in the pyrophosphate bond formation.
Complex carbohydrates and their conjugates are in-
volved in various types of biochemical recognition pro-
cesses.
1
Glycosyltransferase-catalyzed synthesis of these
important structures is attractive since these reactions
proceed regio- and stereoselectively in aqueous media
without requiring complicated manipulations of protect-
ing groups.
2,3
As glycosyl donors, the glycosyltransferases
of the Leloir pathway
4,5
in mammalian systems employ
primarily eight sugar nucleotides: UDP-Glc, UDP-
GlcNAc, UDP-Gal, UDP-GalNAc, GDP-Man, GDP-Fuc,
UDP-GlcUA, and CMP-NeuAc. For an efficient use of
glycosyltransferases in the synthesis of oligosaccharides
a practicable (high yield) preparation of these cosub-
strates is demanded.
Most of the chemical syntheses of sugar diphosphate
nucleosides
6,7
involve the coupling of a glycosyl phosphate
1 with an activated nucleoside monophosphate (NMP)
(Scheme 1).
8-14
Of the commonly used activated NMP
derivatives, phosphoramidates such as phosphorimida-
zolidates
8-10
and especially phosphomorpholidates 2
11-14
are the most popular, the latter being introduced in 1959
by Moffatt and Khorana.
15,16
Nevertheless, the reaction
between a sugar 1-phosphate and an NMP-morpholidate
is very slow (reaction times of 5 days are usual), and
yields rarely exeed 70%. GDP-Fuc, in particular, is
obtained in only 20-50% yield.
17-22
Recently, a new
approach, involving the reaction of glycosylbromides with
UDP and GDP, was reported.
23
In the enzymatic prepa-
ration of sugar diphosphate nucleosides, a glycosyl
phosphate is reacted with a nucleoside triphosphate
(NTP), catalyzed by a nucleoside diphosphate sugar
pyrophosphorylase (Scheme 1).
10,24-29
The NTP may also
be generated in situ in coupled enzyme reactions. In the
case of GDP-Fuc, the enzymatic preparation has been
* To whom correspondence should be addressed. Tel.: (619) 784-
2487. Fax: (619) 784-2409. E-mail: Wong@Scripps.edu.
X
Abstract published in Advance ACS Abstracts, March 1, 1997.
(1) Varki, A. Glycobiology 1993, 3,97-130.
(2) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic
Chemistry; Pergamon: Oxford, 1994.
(3) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew.
Chem., Int. Ed. Engl. 1995, 34, 521-546.
(4) Leloir, L. F. Science 1971, 172, 1299-1303.
(5) Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631-
664.
(6) Heidlas, J. E.; Williams, K. W.; Whitesides, G. M. Acc. Chem.
Res. 1992, 25, 307-314.
(7) Kochetkov, N. K.; Shibaev, V. N. Adv. Carbohydr. Chem.
Biochem. 1973, 28, 307-399.
(8) Cramer, F.; Neunhoeffer, H. Chem. Ber. 1962, 95, 1664-1669.
(9) Hoard, D. E.; Ott, D. G. J. Am. Chem. Soc. 1965, 87, 1785-1788.
(10) Simon, E. S.; Grabowski, S.; Whitesides, G. M. J. Org. Chem.
1990, 55, 1834-1841.
(11) Moffatt, J. G. Methods Enzymol. 1966, 8, 136-142.
(12) Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G. J.
Am. Chem. Soc. 1961, 83, 659-663.
(13) Clark, V. M.; Hutchinson, D. W.; Kirby, A. J.; Warren, S. G.
Angew. Chem. 1964, 76, 704-712.
(14) Scheit, K. H. Nucleotides Analogs, Synthesis and Biological
Function; Wiley: New York, 1980.
(15) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1959, 81, 1265.
(16) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 649-
658.
(17) Nunez, H. A.; O’Connor, J. V.; Rosevear, P. R.; Barker, R. Can.
J. Chem. 1981, 59, 2086-2095.
(18) Gokhale, U. B.; Hindsgaul, O.; Palcic, M. M. Can. J. Chem.
1990, 68, 1063-1071.
(19) Schmidt, R. R.; Wegmann, B.; Jung, K.-H. Liebigs Ann. Chem.
1991, 121-124.
(20) Veeneman, G. H.; Broxterman, H. J. G.; Marel, G. A. v. d.;
Boom, J. H. v. Tetrahedron Lett. 1991, 32, 6175-6178.
(21) Ichikawa, Y.; Sim, M. M.; Wong, C.-H. J. Org. Chem. 1992, 57,
2943-2946.
(22) Adelhorst, K.; Whitesides, G. M. Carbohydr. Res. 1993, 242,
69-76.
(23) Arlt, M.; Hindsgaul, O. J. Org. Chem. 1995, 60,14-15.
(24) Wong, C.-H.; Haynie, S. L.; Whitesides, G. M. J. Org. Chem.
1982, 47, 5416-5418.
(25) Kawaguchi, K.; Kawai, H.; Tochikura, T. Mehtods Carbohydr.
Chem. 1980, 8, 261-269.
(26) Tochikura, T.; Kawaguchi, K.; Kawai, H.; Mugibayashi, Y.;
Ogata, K. J. Fermentl. Technol. 1968, 46, 970.
(27) Tochikura, T.; Kawai, H.; Tobe, S.; Kawaguchi, K.; Osugi, M.;
Ogata, K. J. Fermentl. Technol. 1968, 46, 957.
(28) Korf, U.; Thimm, J.; Thiem, J. Synlett 1991, 313-314.
(29) Ginsburg, V. Adv. Enzymol. 1964, 26, 35.
Scheme 1
2144 J. Org. Chem. 1997, 62, 2144-2147
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First publ. in: Journal of Organic Chemistry 62 (1997), pp. 2144-2147
Konstanzer Online-Publikations-System (KOPS) - URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/4408/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-44085
carried out only on an analytical scale.
30,31
Here, we
report the use of 1H-tetrazole as catalyst in phospho-
morpholidate coupling reactions, giving rise to shorter
reaction times (1-2 days) and higher yields (76-91%).
32
Results and Discussion
The best results for the reaction of a glycosyl phosphate
1 in its trialkylammonium form and an NMP-morpholi-
date 2 are usually obtained if the reaction is carried out
in pyridine.
33
However, in the case of GDP-Fuc, even
after a reaction time of 5 days, we were able to detect
large amaounts of both starting materials, as judged by
TLC. As the morpholino group in 2 must be protonated
before acting as a leaving group and the trialkylammo-
nium counterion of 1 (aqueous pK
a
ca. 10-11)
34
is the
only proton source present, we felt that the addition of
an acidic catalyst might improve the outcome of the
reaction. 1H-Tetrazole (pK
a
4.9) is commonly used for
the activation of phosphoramidites
35,36
and accelerates
coupling during oligonucleotide synthesis by the phos-
photriester method.
37,38
It turned out that this hetero-
cycle is also an efficient catalyst for the phosphoramidate
coupling. Reaction of triethylammonium fucosyl phos-
phate 4 with guanosine 5′-monophosphomorpholidate (2,
B ) guanine) (GMP-morpholidate) and 1H-tetrazole in
pyridine was complete after 2 days. Size exclusion
chromatography on Bio-Gel P-2, using ammonium bicar-
bonate solution as eluent, gave spectroscopically pure
GDP-Fuc (5) after a single purification step. Under these
conditions, an almost complete (>98%) exchange of the
alkylammonium counterions occurred, and 5 was ob-
tained as its ammonium salt in 85% yield. This material
was conveniently stored and successfully used in the
fucosyltransferase-catalyzed synthesis of sialyl Lewis X.
In an analogous manner, mannosyl phosphate (6) and
galactosyl phosphate (8) were converted into GDP-Man
(7)
10,12,39
and UDP-Gal (9)
12,40
in 76 and 91% yield,
respectively (Scheme 2). The phosphates 6 and 8 were
used in the trioctylammonium forms, which gave faster
reactions than the triethylammonium forms. A common
problem in couplings with GMP-morpholidate is its low
solubility in pyridine.
12
Interestingly, addition of tetra-
zole to a mixture of GMP-morpholidate and pyridine
produced a homogeneous solution immediately.
When used to activate phosphoramidites, tetrazole is
known to act as both an acid and nucleophilic catalyst,
and tetrazolophosphane derivatives have been identified
as reactive intermediates.
41,42
In order to get information
about the essential features of tetrazole for activation of
phosphomorpholidates, we carried out the coupling reac-
tion with different additives and followed the course of
the reaction by
31
P NMR spectroscopy (Figure 1). As
solvent we used a 7:3 mixture of pyridine and DMSO-d
6
in order to prevent precipitation of GDP-Fuc or guanosine
5′-monophosphate (GMP). The additives used were 1H-
tetrazole, 1,2,4-triazole, acetic acid, N-hydroxysuccinim-
(30) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J.; Garcia-
Junceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.;
Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc. 1992, 114, 9283-9298.
(31) Stiller, R.; Thiem, J. Liebigs Ann. Chem. 1992, 467-471.
(32) A preliminary communication of the use of 1H-tetrazole in
phosphoromorpholidate coupling reactions is contained in: Murray,
B. W.; Wittmann, V.; Burkart, M. D.; Hung, S.-C.; Wong, C.-H.
Biochemistry 1997, 36, 823-831.
(33) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1958, 80,
3756-3761.
(34) All pK
a
data given in this paper correspond to aqueous systems.
(35) Barone, A. D.; Tang, J.-Y.; Caruthers, M. H. Nucl. Acids Res.
1984, 12, 4051-4061.
(36) Sim, M. M., Kondo, H., Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260-2267.
(37) Seth, A. K.; Jay, E. Nucleic Acids Res. 1980, 8, 5445-5459.
(38) Zarytova, V. F.; Knorre, D. G. Nucleic Acids Res. 1984, 4, 2091-
2110 and cited references.
(39) Pallanca, J. E.; Turner, N. J. J. Chem. Soc., Perkin Trans. 1
1993, 3017-3022.
(40) Heidlas, J. E.; Lees, W. J.; Whitesides, G. M. J. Org. Chem.
1992, 57, 152-157.
(41) Dahl, B. H.; Nielsen, J.; Dahl, O. Nucl. Acids Res. 1987, 15,
1729-1743.
(42) Berner, S.; Mu¨hlegger, K.; Seliger, H. Nucl. Acids Res. 1989,
17, 853-864.
Scheme 2
Figure 1.
31
P NMR monitoring of (A) GMP-morpholidate and
(B) GDP-Fuc (5) during reaction of β-
L-fucopyranosyl phos-
phate (4) with 1.3 equiv of GMP-morpholidate and 3.2 equiv
of an additive in 7:3 pyridine/DMSO-d
6
:(b) no additive; (0)
1,2,4-triazole; (2) acetic acid; (×) NHS; (4) DMAP‚HCl; (9)1H-
tetrazole; (O) perchloric acid.
Phosphomorpholidate Coupling Reactions J. Org. Chem., Vol. 62, No. 7, 1997 2145
ide (NHS), 4-(dimethylamino)pyridine hydrochloride
(DMAP‚HCl), and perchloric acid. Figure 1A,B shows the
decrease of GMP-morpholidate and the increase of GDP-
Fuc, respectively, over time. Since the amount of hy-
drolysis differed in every reaction, the former gives a
more realistic picture of the degree of activation of the
morpholidate. Although tetrazole and acetic acid (pK
a
4.75) have almost identical pK
a
values, tetrazole acceler-
ates the reaction much more than acetic acid, suggesting
a mechanism in addition to simple acid catalysis, possibly
nucleophilic catalysis. On the other hand, 1,2,4-triazole
(pK
a
10.0), which is a stronger nucleophile in pyridine
than tetrazole,
43
has only little effect on the reaction rate
compared with the uncatalyzed coupling. The addition
of a nucleophile alone is obviously not sufficient; a proton
source is also needed. DMAP is a widely used hypernu-
cleophilic acylation catalyst. When applied as its hydro-
chloride (pK
a
6.1), it is almost as effective as tetrazole,
but due to its restricted solubility in the reaction medium
its practical importance is rather low. NHS (pK
a
6.1)
shows the same acceleration as acetic acid, suggesting
some enhancement due to nucleophilic catalysis, but it
is not as efficient as the equally acidic DMAP‚HCl. By
far the most strongly activating acid is perchloric acid.
Since perchlorate is a poor nucleophile, we conclude that
acid alone is sufficient for activating the morpholidate.
However, in addition to GDP-Fuc large amounts of GMP
were also formed, resulting in a lower yield of GDP-Fuc
than in the case with tetrazole. Addition of powdered 4
Å molecular sieves (Aldrich) (which react markedly as
base in an aqueous suspension) strongly inhibits the
coupling reaction even in the presence of tetrazole. From
these findings, we conclude that tetrazole activates GMP-
morpholidate by protonation of the leaving group nitro-
gen and presumably by nucleophilic catalysis via the
highly reactive phosphotetrazolide 10, which reacts with
fucosyl phosphate 4 to GDP-Fuc (5).
In accordance with the proposed intermediate 10 was
the observation of a new resonance at δ -12.2 ppm
44
in
a
31
P NMR spectrum from a solution of GMP-morpholi-
date in pyridine/DMSO-d
6
(7:3) 10 min after addition of
2 equiv of tetrazole. The integral of this signal cor-
responded to 4% of used morpholidate. When treated
with excess of methanol or ethanol, this signal disap-
peared immediately and new resonances at -1.4 and
-1.3 ppm, arising from the phosphodiesters 11 and 12,
respectively, showed up and increased slowly. In addi-
tion, we subjected a mixture of GMP-morpholidate and
tetrazole in pyridine to mass spectrometric analysis,
using electrospray ionization in the negative mode. A
spectrum taken after 20 min
45
showed, in addition to the
peaks of starting material (M - H
+
431, 100) and
hydrolysis product GMP (M - H
+
362, 5.3), a new peak
at m/z 414 (16), consistent with the proposed intermedi-
ate 10.
In conclusion, we have shown that 1H-tetrazole is an
efficient catalyst for phosphomorpholidate coupling reac-
tions. Qualitative kinetic investigations and mass spec-
trometric analysis suggest a mixture of general acid
catalysis and nucleophilic catalysis to be responsible for
the rate acceleration. Reaction times are shorter and
yields are higher than those reported previously. Since
a variety of NMP-morpholidates is commercially avail-
able, the tetrazole-activated coupling with glycosyl phos-
phates provides easy access to sugar nucleotides for
subsequent glycosyltransferase-catalyzed glycosylations.
Experimental Section
General Methods. Anhydrous pyridine was purchased
from Aldrich and used without further purification. 4-Mor-
pholine-N,N′-dicyclohexylcarboxamidinium guanosine 5′-mono-
phosphomorpholidate, 4-morpholine-N,N′-dicyclohexylcarbox-
amidinium uridine 5′-monophosphomorpholidate, dipotassium
R-
D-mannosyl phosphate, and dipotassium R-D-galactosyl phos-
phate were purchased from Sigma. For the synthesis of bis-
(cyclohexylammonium) β-
L-fucopyranosyl phosphate, L-fucose
was converted into (2,3,4-tri-O-benzoyl-β-
L-fucopyranosyl) diben-
zyl phosphate (three steps, 95% yield)
21
and subsequently
deprotected (two steps, 87% yield)
22
according to published
procedures. Cation-exchange resin AG 50W-X2 (H
+
form,
strongly acidic) was purchased from Bio-Rad Laboratories and
converted to the appropriate salt form prior to its use. When
samples were coevaporated with dry pyridine in order to
remove residual water, argon was used to bring the pressure
back to normal. Analytical thin layer chromatography was
performed using silica gel 60 F
254
precoated glass plates
(Merck); compound spots were visualized by quenching of
fluorescence and/or by charring after treatment with cerium
molybdophosphate. Size-exclusion chromatography was per-
formed on Bio-Gel P-2 Gel, fine (Bio-Rad Laboratories).
31
P
NMR spectra were recorded at 162.0 MHz (Bruker AMX-400)
and referenced to internal triphenylphosphine oxide (δ
P
) 26.5
in 7:3 pyridine/DMSO-d
6
), which itself was referenced to 85%
H
3
PO
4
(δ
P
) 0.00) as external standard.
Monoammonium Guanosine 5′-Diphospho-β-
L-fucose
(5). Bis(cyclohexylammonium) β-
L-fucopyranosyl phosphate
(624 mg, 1.41 mmol) was dissolved in H
2
O (15 mL), applied to
a Bio-Rad AG 50W-X2 cation-exchange column (Et
3
N
+
, 2.5 ×
8 cm), and eluted with H
2
O (150 mL). The solution was
evaporated, coevaporated with MeOH (2 × 10 mL), and dried
for 3 d under vacuum to give triethylammonium β-
L-fucopy-
ranosyl phosphate (4) (512 mg). The content of triethylamine
was determined to be 1.16 equiv (
1
H NMR).
A mixture of 4 (57.5 mg, 159 µmol) and 4-morpholine-N,N′-
dicyclohexylcarboxamidinium guanosine 5′-monophosphomor-
pholidate (201 mg, 255 µmol) was coevaporated with dry
pyridine (3 × 1.5 mL). 1H-Tetrazole (33 mg, 477 µmol) and
dry pyridine (0.8 mL) were added, and the solution was stirred
at rt. After a while, product started to precipitate. The
reaction was monitored by TLC (2:1 i-PrOH/1 M NH
4
OAc).
After 2 d, the mixture was diluted with water (1.5 mL) to
become a clear solution, evaporated, and coevaporated with
water (2 × 1.5 mL). The residue was purified on a Bio-Gel
P-2 column (2.5 × 70 cm), eluted with 250 mM NH
4
HCO
3
,
to give 5 (82.4 mg, 85%) as a white solid after lyophilization
(R
f
0.43, 2:1 i-PrOH/1 M NH
4
OAc). The
1
H NMR spectral data
were in agreement with those published.
17,18
Monoammonium Guanosine 5′-Diphospho-r-D-man-
nose (7). Dipotassium R-
D-mannosyl phosphate (110 mg, 311
µmol) was dissolved in H
2
O (1 mL) and passed through a Bio-
Rad AG 50W-X2 cation-exchange column (pyridinium form,
1.5 × 5 cm). The solution was concentrated to a volume of 5
mL, and pyridine (15 mL) and tri-n-octylamine (136 µL, 311
µmol) were added. The mixture was evaporated and coevapo-
rated with dry pyridine (3 × 1.5 mL).
4-Morpholine-N,N′-dicyclohexylcarboxamidinium guanosine
5′-monophosphomorpholidate (392 mg, 497 µmol) was added,
and the mixture was coevaporated with dry pyridine (3 × 1.5
(43) Chandrasegaran, S.; Murakami, A.; Kan, L.-s. J. Org. Chem.
1984, 49, 4951-4957.
(44) A similar chemical shift (δ -11.24 ppm) for a comparable
phosphotetrazolide has been reported previously.
43
(45) The mass spectrum was recorded after dilution with dioxane.
2146 J. Org. Chem., Vol. 62, No. 7, 1997 Wittmann and Wong
mL). 1H-Tetrazole (70 mg, 994 µmol) and dry pyridine (1.55
mL) were added, and the solution was stirred at rt. The
reaction was monitored by TLC (2:1 i-PrOH/1 M NH
4
OAc).
After 2 d, the mixture was diluted with water (2 mL) and
evaporated. The residue was suspended in 100 mM NH
4
HCO
3
and extracted with ether to remove the trioctylamine. After
evaporation, the residue was purified on a Bio-Gel P-2 column
(2.5 × 95 cm), eluted with 250 mM NH
4
HCO
3
, lyophilized, and
precipitated from H
2
O/MeOH (1:2) by addition of acetone to
give 7 (147 mg, 76%) as a white solid after lyophilization (R
f
0.31, 2:1 i-PrOH/1 M NH
4
OAc). The
1
H NMR spectral data
were in agreement with those published.
39
Monoammonium Uridine 5′-Diphospho-r-D-galactose
(9). Dipotassium R-
D-galactosyl phosphate, containing 5.5 mol
of H
2
O (110 mg, 253 µmol), was converted into the trioctylam-
monium salt using 110 µL (253 µmol) trioctylamine and
reacted with 4-morpholine-N,N′-dicyclohexylcarboxamidinium
uridine 5′-monophosphomorpholidate (296 mg, 404 µmol) and
1H-tetrazole (57 mg, 808 µmol) in dry pyridine (1.25 mL) as
described for 7. Purification on a Bio-Gel P-2 column (2.5 ×
95 cm), eluted with 250 mM NH
4
HCO
3
, lyophilization, and
precipitation from H
2
O/MeOH (1:2) by addition of acetone gave
9 (134 mg, 91%) as a white solid after lyophilization (R
f
0.40,
2:1 i-PrOH/1 M NH
4
OAc). The
1
H NMR spectral data were
in agreement with those published.
40
31
P NMR Spectroscopical Monitoring of Morpholidate
Couplings. 4-Morpholine-N,N′-dicyclohexylcarboxamidinium
guanosine 5′-monophosphomorpholidate (1.01 g, 1.28 mmol)
and internal standard triphenylphosphine oxide (44.5 mg, 160
µmol) were coevaporated with dry pyridine (4 × 5 mL), dried
under vacuum for 2 d, and dissolved in pyridine/DMSO-d
6
(7:
3, 3.9 mL). This stock solution was the same for all of the
following reactions. Triethylammonium β-
L-fucopyranosyl
phosphate (4) (36.2 mg, 100 µmol) was coevaporated with dry
pyridine (3 × 1.5 mL) and dried under vacuum for 2 d. GMP-
morpholidate stock solution (500 µL) and 3.2 equiv (320 µmol)
of the additive, i.e., 1H-tetrazole (22 mg), 1,2,4-triazole (22 mg),
acetic acid (18 µL), NHS (37 mg), DMAP‚HCl (51 mg), or
anhydrous HClO
4
(19 µL), respectively, were added. The
resulting solution was transferred into a 5-mm NMR tube via
syringe, and the reaction was followed by proton-coupled
31
P
NMR spectroscopy using a pulse angle of 60° and a relaxation
delay of 5 s. Collection of 64 transients gave adequate spectra.
Signals at δ 4.95 (GMP-morpholidate), 0.29 (GMP), -0.73 (4),
and -13.38 ppm (5)
46
were integrated and referenced to the
integral of internal standard triphenylphosphine oxide, whose
concentration was assumed to stay constant.
Acknowledgment. This work was kindly supported
by the NSF. V.W. acknowledges a fellowship from the
Deutsche Forschungsgemeinschaft.
JO9620066
(46) Chemical shifts refer to the uncatalyzed reaction. Slightly
different values were observed with each additive.
Phosphomorpholidate Coupling Reactions J. Org. Chem., Vol. 62, No. 7, 1997 2147
