3408
C. Y. Zamora et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3406–3410
cells incubated with 9a for 2 h were washed thoroughly to remove
any free 4MU generated from enzymatic activity. The cells were re-
suspended in cell lysis buffer and the fluorescence intensity of the
resulting lysate read. The fluorescence intensity of the washed cell
samples was equal to that of empty wells (without cells or com-
pound), indicating that all conjugates (cleaved or uncleaved) had
been removed from the sample (Fig. S1). From this result, we con-
clude that neither the glycosides nor 4MU had diffused across the
membrane during the incubation time of these assays, and that
that the observed hydrolysis of the glycosides was likely the result
of activity of sialidase isoforms associated with the cell membrane.
Therefore, our results demonstrate the utility of 4MU sialosides as
probes of cell-surface localized sialidase activity toward unnatural
sialic acids.
To explore the substrate specificity of a bacterial sialidase, we
also assayed a commonly employed neuraminidase isolated from
Clostridium perfringens [EC 3.2.1.18] for in vitro activity against
our modified sialic acids. Many bacterial strains do not have innate
biosynthetic pathways for sialic acids and scavenge them from the
host.35 As a result, unnatural sialic acid substrates are readily
incorporated by the natural metabolism of many bacterial strains,
even in the presence of excess N-acetyl neuraminic acid.36 How-
ever, the activity of C. perfringens sialidase towards sialosides other
than N-acetyl or N-glycolyl has not been previously reported.
Accordingly, we measured the enzymatic activity of C. perfrin-
gens sialidase using compounds 9a–f as fluorogenic substrates.
Using 0.05 mU sialidase from C. perfringens, we found the cleavage
activity against glycosides 9b–f relative to 9a by the bacterial en-
zyme to be significantly lower than that observed in our mamma-
lian whole-cell assay (Fig. 2). Only glycolyl analog 9f and
monofluorinated analog 9c were cleaved by C. perfringens sialidase
in our assay with fluorescence intensities of only 6% of that ob-
served for the natural ligand 9a. It is interesting to note that con-
structs that were cleaved relatively well by HL60 sialidases, such
as 9b, were not cleaved at all by this enzyme, indicating greater
permissivity in the mammalian sialidase pockets than anticipated.
Our results suggest that sialidase-catalyzed removal of unnatural
sialic acids from metabolically engineered bacterial glycomes
Figure 1. Activity of endogenously-expressed sialidases in HL60 against N-acyl
neuraminic acid glycosides 9a–f. Whole HL60 cells in acetate buffer (pH 4.5) were
treated for 2 h with 0.125 mM of each compound (blue bars) at 37 °C. Grey bars
represent the same reaction in the presence of 0.1 mM of sialidase inhibitor DANA.
The pH of each reaction was adjusted to 10.7 with glycine buffer and fluorescence
was read with excitation wavelength of 365 nm and emission wavelength of
450 nm. Error bars are the standard deviations. All experiments were performed in
at least biological triplicate and are representative of multiple independent trials.
natural substrate 9a. Previously, Li et al. reported a slight increase
in the activity of sialidase NEU2 against p-nitrophenol constructs
displaying the N-glycolyl and N-azidoacetyl functionalities on sia-
lic acid.34 We hypothesized that a similar trend may hold for the
collective activities of cell-surface sialidases on our compound 9f.
In addition, we anticipated a decrease in the cleavage activity upon
derivatives 9b, 9c, and 9e as a result of fluorination, steric bulk or a
combination thereof.
The results are shown in Figure 1. As predicted the N-glycolyl
compound 9f showed 20% greater activity than 9a. We also ob-
served 50% more hydrolysis relative to the natural sugar for com-
pound 9c in which one hydrogen is replaced by fluorine.
However, when a trifluoromethyl group is present distal to the car-
bonyl, as in 9b and 9e, we observed a decrease in the ability of cell-
surface enzymes to cleave these constructs. In the case of the N-
isobutyryl neuraminic acid glycoside (9d), we observed a 60%
reduction in hydrolysis compared to 9a, possibly due to the in-
crease in steric bulk and hydrophobicity. We note the surprising
result obtained in the presence of compound 9e, wherein we ob-
served negligible cleavage of the glycoside. The mechanism of ac-
tion of this behavior is currently under investigation in our
laboratory.
In order to rule out hydrolysis mechanisms independent of sial-
idase activity as a significant source of fluorescence measured,
whole cells in acetate buffer were treated with 9a–f in the pres-
ence of 0.1 mM 2-dehydroxy-2-deoxyneuraminic acid (DANA), a
non-specific sialidase inhibitor.13,26 In every case, we observed
nearly complete abrogation of cleavage of the 4MU glycosides by
the cells in the presence of DANA (Fig. 1). This strongly indicates
that the hydrolysis observed for 9a–f by HL60 cells was due to sial-
idase activity. Based on the known cellular localization of sialidase
isoforms, we postulate that these compounds are primarily acted
upon by sialidase isoforms NEU1 and/or NEU3. Our results suggest
that these enzymes are likely capable of cleaving unnatural sialic
acids from cell surface glycoconjugates to varying degrees, includ-
ing those with longer N-acyl modifications.
Figure 2. Activity of bacterial sialidase against N-acyl neuraminic acid glycosides
9a–f. Purified recombinant sialidase from C. perfringens (0.05 mU) in acetate buffer
(pH 4.5) was treated for 1 h with 0.125 mM of each construct (blue bars) at 37 °C.
Grey bars represent the same reaction in the presence of 0.1 mM of sialidase
inhibitor DANA. The pH of each reaction was adjusted to 10.7 with glycine buffer
and fluorescence was read with excitation wavelength of 365 nm and emission
wavelength of 450 nm. Error bars are the standard deviations. All experiments were
performed in at least biological triplicate and are representative of multiple
independent trials.
We do not expect ionic and polyhydroxylated compounds 9 to
be cell permeable and thus accessible to cleavage by sialidase iso-
forms localized to the cell interior. To confirm this expectation,