M. C. Galan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2205–2208
2207
Table 2. Apparent kinetic parameters for the transfer of N-acetyl-
neuraminic acid to glycosyl acceptors 1–13 by a-1,3-fucosyltransferase
VI
with methanol in the presence of methyl iodide in DMF
to give 35. Compounds 36 and 37 were obtained by
displacement of the pentafluorophenyl ester of 34
by ammonia or methylamine, respectively. Catalytic
hydrogenation over Pd/C of 35–37 gave the target
compounds 11, 12 and 13, respectively.
Acceptor
Km (lM)
Rel (Vmax
)
Rel (Vmax/Km)
1
350 50
390 50
290 50
120 30
250 30
400 50
190 20
115 10
140 10
1.0
1.2
2.8
3.1
4.5
6.7
3.6
2
3
1.3
0.8
4
The apparent kinetic parameters of transfer of CMP-
[14C]-Neu5Ac and GDP-[14C]-Fucose to acceptors 1–13
catalysed by rat liver recombinant a-2,6-sialyltransferase
(purchased from Calbiochem) and human recombinant
a-1,3-fucosyltransferases VI (purchased from Calbio-
chem), respectively, were determined by reported
assays.16;18;20;21 In each case, the Km for 1 was in close
agreement with previous data and the Vmax values were
set at 1.
5
0.9
6
0.61.5
1.3
1.0
7
6.8
8.7
7.3
9.3
8
9
1.0
0.7
10
11
12
13
75
7
450 70
N.a
1540 50
0.61.2
N.a.
0.60.4
N.a.
N.a. not active.
The apparent kinetic parameters of the sialylation of
compounds 1–4 by a-2,6-sialyltransferase showed that
the methyl groups at C-6and C-2 hydroxyls signifi-
0
cantly lowered the catalytic efficiency (Vmax/Km) of the
transfer due to a notable increased Km and a somewhat
smaller Vmax (Table 1). Furthermore, the data shows that
the both methyl groups induce similar and additive
effects. Interestingly, compound 5, which has a more
bulky methanesulfonyl group at C-6, displayed a slightly
improved catalytic efficiency indicating that electronic
and not steric effects are probably important for
favourable interactions with the periphery of the
enzymesÕ binding pocket.
The fact that different enzymes respond differently to
chemical modification of LacNAc was also born out by
the observation that the sialyltransferase did not tolerate
the introduction of a free amine at C-6(compound 6)
whereas this modification had only minor effect on the
fucosyltransferase. Interestingly, for both enzymes
acylation of the amine by an acetyl (7), sulfonyl (8) or
formyl group (9) gave compounds that were equally or
slightly better acceptors than LacNAc and the data of
compounds 6–10 indicate that electronic and not steric
factors are important determinants for the observed
modulation of catalytic efficiencies. The introduction of
The apparent kinetic data for a-1,3-fucosyltransferases
VI (Table 2) demonstrate that this enzyme responds
differently to the introduced chemical modifications
and for compounds 2–5 slightly improved catalytic
efficiencies were measured. In particular, the effect of
di-O-methylation was dramatic and in the case of the
a-2,6-sialyltransferase a 15-fold loss of catalytic effi-
ciency was observed whereas a 2.5-fold increase was
measured for the a-1,3-fucosyltransferases VI. It can be
concluded that the architecture of the binding sites of
the two enzymes differ significantly, which can be
exploited for the preparation of selective substrates.
0
a formamide at C-6and methyl ether at C-2 gave
compound 10, which was a remarkable good substrate
for the fucosyltransferase and displayed the lowest Km of
all compounds tested. On the other hand, these modi-
fications resulted in a small loss of catalytic efficiency for
the sialyltransferase. For both enzymes, methyl ester 11
was a slightly poorer substrate than LacNAc, whereas
introduction of an amide (12) and methyl amide (13)
resulted in large reductions in catalytic efficiency.
Comparing the data of compound 7, which is modified
by a NHC(O)CH3 function and compound 13 which has
a C(O)NHCH3 group, shows that the arrangement of
atoms has a dramatic effect on catalytic efficiencies and
this observation indicates that these functional groups
make direct interactions with the binding site of the
enzymes.
Table 1. Apparent kinetic parameters for the transfer of N-acetyl-
neuraminic acid to glycosyl acceptors 1–13 by a-2,6-sialyltransferase
Acceptor
Km (mM)
Rel (Vmax
)
Rel (Vmax/Km)
Previous studies have identified key polar functionalities
of a glycosyl acceptor by selective methylation or
deoxygenation of hydroxyls.16;18 For some other hy-
droxyls, these modifications lead to a reduction of
acceptor activity and it has been proposed that these
functionalities interact with the periphery of the binding
site of the enzyme. The results described in this com-
munication show that by employing a wider range of
functionalities, a more detailed picture of the architec-
ture of a binding site can be obtained. Furthermore, it is
shown that the introduction of these functionalities may
lead to compounds that display high selectivity for
particular transferases. Such analogues will be devel-
oped as primers for selective metabolic inhibition of
1
1.7 0.2
4.1 0.8
4.3 0.4
11.2 0.8
0.7 0.1
5.8 0.2
1.4 0.2
0.8 0.1
1.1 0.3
3.1 0.61.0
N.d.
1.0
0.8
0.6
0.2
2
3
0.8
0.5
0.2
4
0.04
1.3
5
0.9
0.5
6
0.08
7
0.60.4
0.7
1.61.5
8
0.9
9
10
11
12
13
0.3
N.d.
<0.2
0.2
0.2
>10
2.4 0.2
0.02
0.08
N.d. not determined.