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A recent investigation into the reason why the trans-
sialidase from T. cruzi is more ecient in transferring
rather than hydrolyzing terminal sialic acid residues
centred on the crystal structure of the closely related
sialidase from T. rangeli.12 The two enzymes have over
70% sequence identity and comparison of the X-ray
crystal structure of T. rangeli and a homology model of
T. cruzi revealed similar three-dimensional structures in
the catalytic and lectin domains.12 Signi®cant dier-
ences were however, noted between the active site of
T. rangeli sialidase and that of T. cruzi trans-sialidase.
The target sialoside Neu5Aca(2,3)Lacb1Me (3) was
readily prepared by a similar method to that previously
described for 1.20 Thus, TMSOTf-mediated coupl-
ing21,22 between the sialosyl phosphite 4 and the lacto-
side 5, gave the desired sialyllactoside
deprotection (Scheme 1).
3 after
Another study has used NMR spectroscopy to investi-
gate the sialidase activity of recombinant T. cruzi trans-
sialidase.13 The stereoselectivity of sialoside hydrolysis
of two substrates, namely 4-methyl-umbelliferyl N-ace-
tyl-neuraminoside and a(2,3)-sialyl-lactose, was investi-
gated. The initial product of release in the case of both
substrates was found to be the thermodynamically less
stable a-anomer of sialic acid (Neu5Ac) that subse-
quently mutarotated to the b-anomer.13 This is con-
sistent with previous studies on other sialidases,14 19
which have all been identi®ed as retaining enzymes.
Scheme 1. Reagents and Conditions: (a) 5 (2.5 equiv), THF, TMSOTf
(0.2 equiv), 4 A sieves, 40 to 0ꢀC, 3 h, 56%; (b) NaOMe then NaOH
(1 M), 77%.
Since the sialic acid transfer functionality of T. cruzi
trans-sialidase is believed to play the more signi®cant
role in the pathogenesis of this microorganism,4,7 we
decided to investigate the sialic acid transfer function of
this enzyme. In line with our previous studies of other
sialidases17 19 and with the studies on the sialidase
Each of the three possible components of the proposed
T. cruzi trans-sialidase reaction, i.e. compounds 1, 2 and
3, were dissolved separately in deuterated phosphate
buered saline (PBS) (pH 7.4) and the 1H NMR spectra
recorded at 600 MHz and 25 ꢀC. This spectroscopic
examination revealed that each of the compounds could
be readily identi®ed from their respective anomeric
proton resonances. This is displayed in Figure 2 which
1
activity of T. cruzi trans-sialidase,13 we chose to use H
NMR spectroscopy to investigate the transferase activ-
ity of this enzyme. To the best of our knowledge this is the
®rst time that a trans-sialidase catalyzed sialic acid trans-
fer reaction has been probed using NMR spectroscopy.
1
shows the anomeric regions (d 4.2±4.7 ppm) of the H
As shown in Figure 1, T. cruzi trans-sialidase utilizes a
Neu5Aca(2,3)-galactoside based donor and an asialo-
galactoside as an acceptor. For the successful H NMR
NMR spectra of the sialyl donor 1 (Fig. 2a), the sialyl
acceptor 2 (Fig. 2b) and the anticipated product 3 of
Neu5Ac transfer (Fig. 2c). As is clearly shown, the
presence of each of the three components of the
transfer reaction can be easily monitored by examin-
ing the anomeric region of the H NMR spectrum of
a trans-sialidase catalyzed transfer reaction as it
occurs.
1
analysis of the T. cruzi trans-sialidase catalyzed transfer
of Neu5Ac from one galactoside to another, we needed
to choose two galactosides that would appear dierent
1
1
by H NMR spectroscopy when they were linked in an
a(2,3) manner to Neu5Ac. From a related synthetic
project, we felt that the use of Neu5Aca(2,3)Galb1Me (1)20
as donor and methyl b-d-lactoside (2) as acceptor would
be suitable for this investigation. However, it was ®rst
necessary to ascertain if the product of Neu5Ac transfer
from 1 to 2, namely Neu5Aca(2,3)Lacb1Me (3), could be
dierentiated from 1 and 2 by 1H NMR spectroscopy.
With the appropriate sialyl donor and acceptor mol-
ecules in hand, our attention turned to the trans-siali-
dase from T. cruzi. Partially puri®ed enzyme was
obtained from the culture supernatants utilizing a sialic
acid-based anity chromatographic medium23 as
previously described.24
The 1H NMR monitored trans-sialidase catalyzed
transfer reactions were recorded in deuterated phos-
phate buered saline (PBS) (pH 7.4) at 600 MHz and
25 ꢀC. In a typical experiment, a mixture of the sialyl
donor 1 and acceptor 2 (molar ratio 1:2) were dissolved
1
in deuterated PBS and the H NMR spectrum recorded
(t=0 min). T. cruzi trans-sialidase was then added to the
1
mixture of donor and acceptor and H NMR spectra
recorded at regular intervals over a period of 2 days. As
shown in Figure 2d, after 46 h it is evident that the
transfer reaction is being successfully catalyzed by