Synthesis and evaluation of phosphoramidate amino acid-based inhibitors of sialyltransferases.

Article abstract of DOI:10.1016/S0960-894X(02)00735-7

Several phosphoramidate analogues of CMP-N-acetylneuraminic acid were prepared for evaluation as inhibitors of alpha-2,3- and alpha-2,6-sialyltransferase. Central to the synthesis was the oxidative coupling of an amino acid ester with an H-phosphonate to construct the phosphoramidate linkage. All compounds synthesized were weak inhibitors of both of the sialyltransferases as determined by an HPLC-based inhibition assay.

Full text of DOI:10.1016/S0960-894X(02)00735-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 301–304
Synthesis and Evaluation of Phosphoramidate Amino Acid-Based
Inhibitors of Sialyltransferases
Lisa J. Whalen, Kerry A. McEvoy and Randall L. Halcomb*
University of Colorado, Department of Chemistry and Biochemistry, UCB 215, Boulder, CO 80309-0215, USA
Received 31 May 2002;accepted 12 August 2002
Abstract—Several phosphoramidate analogues of CMP-N-acetylneuraminic acid were prepared for evaluation as inhibitors of
a-2,3- and a-2,6-sialyltransferase. Central to the synthesis was the oxidative coupling of an amino acid ester with an H-phosphonate
to construct the phosphoramidate linkage. All compounds synthesized were weak inhibitors of both of the sialyltransferases as
determined by an HPLC-based inhibition assay.
# 2002 Elsevier Science Ltd. All rights reserved.
The sialyltransferases are a family of membrane-bound
enzymes located in the endoplasmic reticulum and
Golgi apparatus of cells. As a subset of the glycosyl-
transferases, sialyltransferases catalyze the transfer of
sialic acid (N-acetylneuraminic acid, NeuAc) from cyti-
dinyl-50-monophospho-b-N-acetylneuraminic acid (1,
CMP-NeuAc) to acceptor hydroxyl groups located on
the carbohydrate regions of glycoproteins and glyco-
lipids (Scheme 1).1 The presence of sialic acid-contain-
ing oligosaccharides has been linked to biologically
relevant processes such as cell adhesion and inflamma-
tion.2 Furthermore, studies have shown a link between
sialyltransferase activity and the growth and metastasis
of certain tumor cells.3 As a result, there is considerable
interest in the synthesis and evaluation of inhibitors of
the sialyltransferases.
Although 1 appears to be the common donor of sialic
acid for all of the sialyltransferases, each enzyme varies
in regioselectivity and to some degree in acceptor speci-
ficity. We chose to evaluate two commercially available
enzymes, the a-2,3- and a-2,6-sialyltransferases. Limited
information has been collected on the structure and
active site composition of the sialyltransferases,
although they appear to share a common topography.4
However, one well-known distinction between the two
enzymes is the acceptor sequence recognized by each.
The a-2,6-sialyltransferase from rat liver (a-2,6-ST)
prefers the N-acetyllactosamine (LacNAc) sequence,
while the a-2,3-sialyltransferase from rat liver (a-2,3-ST)
recognizes either LacNAc or lactose.4b,5 Although
potential inhibitors of the a-2,6-ST have been pre-
pared,6 the a-2,3-ST remains much less studied. Most
efforts to inhibit the a-2,6-ST are centered on the
synthesis of analogues of 1 itself or of a proposed
transition state conformation of 1.
Our design for inhibitors of sialyltransferases included
analogues of 1 in which the natural phosphodiester
linkage is replaced with a phosphoramidate (Fig. 1).
The nitrogen of the phosphoramidate originates from
Scheme 1. Reaction catalyzed by the sialyltransferases.
*Corresponding author. Fax: +1-303-492-5894;e-mail: halcomb@
colorado.edu
Figure 1. CMP-NeuAc (1), the natural substrate of the sialyl-
transferases, and phosphoramidate analogues of 1.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00735-7

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L. J. Whalen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 301–304
Scheme 2. (a) H2O, CH3CN, tetrazole, rt (66%);(b) amino acid ester hydrochloride, CCl 4, Et3N, CH2Cl2, rt (30–78%);(c) (i) (Ph 3P)4Pd, Ph3P,
morpholine, CH2Cl2, rt;(ii) NaOMe, MeOH, 0.1 M NaOH, rt;NH 4HCO3 (80%).
Scheme 3. (a) (i) Methyl glycolate, tetrazole, CH3CN, 0 ꢁC;(ii) Dimethyl dioxirane (DMDO), CH Cl2, rt (78%);(b) (i) (Ph P)4Pd, Ph3P, morpho-
line, CH2Cl2, rt;(ii) NaOMe, MeOH, 0.1 M NaOH, rt;NH 4HCO3 (66%).
2
3
an amino acid, thereby allowing access to a diverse
array of side chains with different functional groups. It
was hypothesized that a side chain could be found that
has favorable interactions with amino acid residues in
the active site of the sialyltransferases.
phosphorus was obtained in each case;however, in the
synthesis of 4f a 2.2:1 ratio of diastereomers resulted. It is
interesting to note that coupling reactions attempted with
l-histidine methyl ester and N-e-Fmoc-l-lysine methyl
ester were not successful. A deprotection sequence of
deallylation, debenzoylation, and methyl ester saponifica-
tion completed the synthesis of phosphoramidates 5a–f.
The synthesis of the phosphoramidate conjugates began
with cytidine O-allyl phosphoramidite 27 as a 1:1 mix-
ture of diastereomers at the phosphorus stereocenter
(Scheme 2). Hydrolysis of 2 to H-phosphonate 3 as a 1:1
mixture of diastereomers was accomplished using tetra-
zole as a catalyst. Coupling of a variety of amino acid
esters to 3 using the Atherton–Todd reaction8 furnished
protected phosphoramidates 4a–f. In the syntheses of
compounds 4a–e, a 1:1 ratio of diastereomers at
An additional potential inhibitor, the CMP–glycolate
conjugate 7, was also prepared from 2 (Scheme 3).
Coupling of methyl glycolate to 2 produced a mixture of
phosphite and phosphate triesters which was then oxi-
dized9 with dimethyldioxirane (DMDO) to yield phos-
phate triester 6 as a 1:1 mixture of diastereomers.
Deprotection of the allyl and benzoyl esters and benza-
mide followed by saponification of the methyl ester
furnished compound 7.
An assay reported by Schmidt6d and coworkers was
used as the basis for inhibition studies. For this HPLC-
based enzyme inhibition assay, the modified dis-
accharide acceptors 8 and 10 (Scheme 4) that bear UV-
chromophores were needed. Lactose-based acceptor 8,
for use in the a-2,3-ST assay, was prepared from lactose
as previously reported.7 LacNAc-based acceptor 10, for
use in the a-2,6-ST assay, was prepared from tri-
chloroacetimidate 9.10 Standard borontrifluoride-cata-
lyzed glycosylation11 of
9 with 4,5-dimethoxy-2-
nitrobenzyl alcohol afforded exclusively the b-anomer.
Deprotection of the phthalimide, acetamide formation,
and deacylation of the alcohols gave the acceptor 10.
Scheme 4. (a) (i) BF3-Et2O, 4,5-dimethoxy-2-nitrobenzyl alcohol,
CH2Cl2, rt (82%);(b) (i) H 2N–NH2–H2O, MeOH, reflux;(ii) Ac 2O,
pyridine, rt;(iii) NaOMe, MeOH, reflux (60%).
Both 8 and 10 have a lmax value at 348 nm
(e348=6000 Mꢀ1 cmꢀ1), which is far removed from the

L. J. Whalen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 301–304
303
Scheme 5. Reactions of a-2,3-ST and a-2,6-ST with their specific acceptors 8 and 10 bearing UV-chromophores.
lmax of the cytosine chromophore (lmax=272 nm). The
transfer of sialic acid to either 8 or 10 was cleanly
Table 1. Ki values for compounds 5a–f and 7
Compd
a-2,3-ST
Ki (mM)a
a-2,6-ST
resolved with reverse-phase HPLC. The identities of the
sialylated products (Scheme 5), compounds 117 and 12,
Ki (mM)a
1
CMP
5a
5b
5c
5d
5e
0.065 (0.007)
5.3 (1.0)
2.3 (1.1)
0.90 (0.30)
2.3 (2.7)
5.1 (1.9)
0.064 (0.019)
2.4 (0.14)
1.7 (0.57)
2.5 (0.99)
3.8 (1.1)
2.2 (0.35)
0.73 (0.45)
0.37 (0.25)
were confirmed by H NMR and mass spectrometry of
preparative-scale reaction products.
The evaluation studies of 5a–f and 7 were performed in
an aqueous solution containing MES buffer (200 mM,
pH 6.0), sodium chloride (100 mM), disodium-EDTA
(0.5 mM), Triton X-100 (0.01%), and 8 or 10 (20 mM).
Once enzyme was added, the reactions were incubated
5f
7
0.30 (0.13)
0.71 (0.48)
aValues are means of three experiments, three to four determinations
each;standard deviation is given in parentheses.
ꢁ
in a water bath at 37 C until a detectable amount of
product 11 (a-2,3-ST) or 12 (a-2,6-ST) was produced.
At this time they were frozen in liquid nitrogen and
stored at ꢀ20 ꢁC until HPLC analysis, which was never
more than 1 or 2 days. Storing the reactions for a longer
period of time affected the reproducibility of results.
Detection and integration of either 8 and 11 or 10 and
12 permitted the calculation of percent product and thus
initial velocity. A Michaelis–Menten curve was used to
determine the Km of 1 for the a-2,3-ST and the a-2,6-ST
under the reaction conditions. The average Km deter-
mined from three runs was 0.3 mM for the a-2,3-ST, in
agreement with the value previously reported by this
laboratory.7 The average Km determined for the a-2,6-
ST was 0.1 mM, which is reasonably close to the value
of 0.046 mM reported by Schmidt and coworkers.6d
a small difference. Compounds 5a, 5b, 5d, and 5e
had significantly higher Ki’s and were relatively poor
inhibitors. Based on the similarity of 5a to 7, it is
apparent that a phosphoramidate is not a completely
adequate mimic of a phosphodiester. The data for
compound 5f suggest that aromatic groups can have
favorable interactions in the active site;however, the
simple presence of an aromatic group is not entirely
sufficient since compound 5d has a larger Ki.
Similar results were obtained with inhibition assays of
the a-2,6-ST;compounds 5f and 7 again possess the
lowest Ki values, reinforcing the significance of the
phosphodiester linkage and the importance of aromatic
groups. Again, 5d has a larger Ki than 5f, suggesting that
the indole ring of 5f has a more favorable interaction in
the active site than the phenyl ring of 5d.
Inhibition assays were carried out under identical reac-
tion conditions, except that the inhibitor was added to
the reaction;the concentrations of 5a–f and 7 ranged
from 0.1 to 10 mM in the experiments. A fixed con-
centration of 20 mM acceptor 8 or 10 was used. Line-
weaver–Burk plots were used to classify compounds 5a–
f and 7 as competitive inhibitors. For the a-2,3-ST, Km
and Vmax in conjunction with the Michaelis–Menten
equation for competitive inhibition were used to deter-
mine the Ki (Table 1). For the a-2,6-ST, Dixon plots
were used to determine the Ki values (Table 1). The Ki
for CMP, a known inhibitor of the sialyltransferases,12
was determined as a standard. The values of 65 mM and
64 mM are within agreement of the reported Ki of 50 mM
for human serum sialyltransferase.12b In a typical Ki
experiment, four different concentrations of 1 and four
different concentrations of inhibitor were used, resulting
in a total of 16 reactions per inhibition experiment.
To summarize, we synthesized a series of phosphor-
amidate analogues of 1 and evaluated their ability to
inhibit two members of the sialyltransferase family of
enzymes using an HPLC-based assay. All of the phos-
phoramidates were weak inhibitors of the a-2,3-ST and
the a-2,6-ST. However, two trends emerged in the ana-
lysis. First, the importance of the phosphate ester link-
age in 1 was demonstrated through the comparison of
compounds 5a and 7. Second, the relevance of aromatic
side chains in the phosphoramidates was shown through
compounds 5f and 5d. However, the location and type
of these aromatic side chains has a large impact on the
inhibitory effects. We hope to use these findings to
design stronger second-generation inhibitors.
As seen in Table 1, none of the phosphoramidate ana-
logues inhibits the a-2,3-ST as strongly as CMP;they
have inhibition constants that are at least an order of
magnitude higher. The Ki of 5f is similar to the Km of 1.
While the binding constants for 7 and 5c appear higher
than those of 5f and 1, they are less than 5-fold higher,
Acknowledgements
This research was supported by the NIH (GM55852).
R.L.H. also thanks the National Science Foundation
(Career Award), the Camille and Henry Dreyfus

304
L. J. Whalen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 301–304
P. N.;Giannis, A. Angew. Chem., Int. Ed. Engl. 1999, 38, 1379.
Foundation (Camille Dreyfus Teacher-Scholar Award),
Pfizer (Junior Faculty Award), and Novartis (Young
Investigator Award) for support. This work was greatly
facilitated by a 500 MHz NMR spectrometer that was
purchased partly with funds from an NSF Shared Instru-
mentation Grant (CHE-9523034). L.J.W. thanks the
National Science Foundation for a predoctoral fellowship.
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