Enzymatic synthesis of colorimetric substrates to determine α-2,3- and α-2,6-specific neuraminidase activity

Article abstract of DOI:10.1039/c3ra44791j

Glycoconjugates containing either terminal α-2,3- or α-2,6-Neu5Ac-Gal disaccharides are found on cell surfaces of many animal glycans. Each linkage can be specifically recognized by lectins and enzymes such as neuraminidases. Here we describe a one-step enzymatic synthesis of two colorimetric substrates that allow for fast distinction of specific neuraminidase activity. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra44791j

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Enzymatic synthesis of colorimetric substrates to
determine a-2,3- and a-2,6-specific neuraminidase
activity†
Cite this: RSC Adv., 2013, 3, 21335
a
a
b
Received 30th August 2013
ˇ
´
´
Juana Elizabeth Reyes Martınez, Robert Sardzık, Josef Voglmeir
and Sabine L. Flitsch
Accepted 11th September 2013
a
*
DOI: 10.1039/c3ra44791j
www.rsc.org/advances
Glycoconjugates containing either terminal a-2,3- or a-2,6-Neu5Ac- 60SiaLac (Neu5Ac-a2,6Gal-b1,4Glc, 2) as substrates and the
Gal disaccharides are found on cell surfaces of many animal glycans. amount of Neu5Ac released was determined using a thio-
Each linkage can be specifically recognized by lectins and enzymes barbituric acid assay.10 More recent and sensitive methods rely
such as neuraminidases. Here we describe a one-step enzymatic on uorescently labelled sialyloligosaccharides e.g. BODIPY
synthesis of two colorimetric substrates that allow for fast distinction (4,4-diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pro-
of specific neuraminidase activity.
pionic acid) derivatives,11 however these methods are expensive.
Glycoarray technologies using immobilised substrates have also
been employed to detect the specicity and activity of inuenza
neuraminidases.12 However, the complexity of the readout
systems using lectins or mass spectrometry can make them
unsuitable for quantitative studies so colorimetric assays are an
attractive complementary tool to array techniques.
In the early 90's a colorimetric assay was reported by
H. Kodama and co-workers;13 the synthesis of chromogenic
sialylated compounds using 4-nitrophenyl b-D-galactopyrano-
side as a substrate was achieved using an enzymatic approach.
However, the high costs of the materials and enzymes required
for the assay made this an unsuitable method to determine
neuraminidase specic activity.
Sialic acids (e.g. Neu5Ac) are a family of monosaccharides which
are commonly found on the terminus of glycans present on
animal cell surfaces and are responsible for regulating a
number of biological recognition processes.1,2 Inuenza virus
hemaglutinin3 and numerous lectins such as Siglecs,4,5 which
are involved in immune responses have been shown to bind
sialic acids present on cell surfaces.6 In animal cell surface
glycans, sialic acids are oen linked to galactose with either
a-2,3- or a-2,6 glycosidic bonds depending on the tissue and
organism (as in 1 and 2; Scheme 1).7 Linkage specicity of lec-
tins and enzymes can have signicant biological impact. For
example, inuenza viral infection depends on the host sialic
acid containing ligands:3,8 whilst bird u viruses recognises
a-2,3-linked Neu5Ac, seasonal u viruses are specic for a-2,6-
linkage. Pandemic u strains can recognize both a-2,6- and
a-2,3-linked Neu5Ac.9 Many organisms, such as the inuenza
virus, express neuraminidases, which hydrolyse the sialic acid
linkages in glycoconjugates. Viral neuraminidases are therefore
therapeutic targets for anti-u treatment and high-throughput
assays to determine their specicity are important tools in the
diagnostics and development of new drugs.
In the past, the linkage selectivity of neuraminidases was
determined using 30SiaLac (Neu5Ac-a2,3Gal-b1,4Glc, 1) and
aMIB & School of Chemistry, The University of Manchester, 131 Princess Street,
Manchester, M1 7DN, UK. E-mail: sabine.itsch@manchester.ac.uk; Fax: +44 (0)161
2751311; Tel: +44 (0)161 3065172; +44 (0)161 3065173
bGlycomics and Glycan Bioengineering Research Center, Nanjing Agricultural
University, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra44791j
Scheme 1 Structures of a-2,3- (1) and a-2,6-sialyllactoside (2), which are
commonly found on animal cell surfaces.
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enzymatic glycosylations using either a-2,3- or a-2,6-selective
sialyltransferases or trans-sialidases.
Using an enzymatic approach we prepared a-2,3- and a-2,6-
Neu5Ac modied 5-bromo-4-chloro-3-indolyl-b-D-galacto-pyran-
osides 4 and 5 as novel substrates to test for sialidase activity
assays. Both glycoconjugates (Scheme 2) were synthesised
according to previously reported strategies using trans-sialidase
from Trypanosoma cruzi (TcTS) for a-2,3-linked Neu5Ac6 and
sialyl-transferase from Photobacterium damsela (ST6Pd060) for a-
2,6-linked Neu5Ac.19 TcTS does not require CMP-Neu5Ac donor
and can utilise sialoproteins such as fetuin. Due to reversibility
of the trans-glycosylation by TcTS, an excess of fetuin was used
for the synthesis of conjugate 4. Formation of both 4 and 5 was
conrmed by mass spectrometry (Fig. S1†, peak at m/z 722.76 for
4 and peak at m/z 722.80 for 5) and HPLC (Fig. S4†).
To distinguish the glycosidic linkage in 4 and 5, the amida-
tion/lactonisation method was used. This method is based on
the reaction of the sialylated compound with ammonium
chloride in the presence of DMT-MM. Upon activation of the
carboxylic group with DMT-MM an amide is formed in the case
of 2,6-linked Neu5Ac. In the case of 2,3-linked Neu5Ac a lactone
with the 2-OH group of the galactose residue is formed.20 Upon
permethylation, the two species give products that can be
distinguished by MS analysis. Using lactose as an acceptor the
characteristic peaks at m/z 838.19 m/z for Neu5Ac-a2,3Gal-
b1,4Glc and 851.22 m/z for Neu5Ac-a2,6Gal-b1,4Glc were
observed in MALDI-ToF MS spectra (Fig. S2†).
It is well known that sialic acids readily undergo fragmen-
tation under the desorption–ionization process during MALDI
MS analysis. To stabilise the sialylated analytes and signicantly
increase the signal intensity, a permethylation step prior to
analysis is commonly used. The most widely used technique in
glycomics is the NaOH and methyl iodide method. Powell et al.21
developed a simple methyl esterication of the free carboxylic
acid in sialosides which has also been shown to prevent
cleavage. In our hands it has been found that also non-
methylated Neu5Ac containing glycans can be analysed by
MALDI MS when DHB (2,5-dihydroxybenzoic acid) is used as a
matrix in positive, reectron mode. For the analysis of both 4
and 5 respectively, no permethylation was needed.
Scheme 2 Enzymatic synthesis of sialylated 5-bromo-4-chloro-3-indolyl-b-D-
galactopyranosides 4 and 5.
Here we describe the synthesis of two new chromogenic
substrates, 4 and 5, that allow us to determine the activity and
linkage specicity of a diverse range of neuraminidases. The
compounds were designed to release 5-bromo-4-chloro-indolyl-
b-D-galacto-pyranoside (X-Gal 3) upon neuraminidase activity.
The chemical synthesis of sialosides such as 4 and 5 is a
lengthy process requiring multistep protection, activation and
coupling procedures.14,15 As an alternative route, we investigated
the use of biocatalysis for the synthesis of 4 and 5, which offers a
more effective and time efficient synthetic strategy. Oligosac-
charides including sialosides have been synthesised in the past
with high efficiency, regio- and stereo-specicity.16–18
For targets 4 and 5 the enzymatic route was particularly
attractive since both regioisomers would potentially be acces-
sible from commercially available X-Gal (3) through a one-step
Scheme 3 Two step activity assay: in the first step neuraminidase releases 6 (Neu5Ac) from the substrate 4 or 5. In the following step b-galactosidase releases
5-bromo-4-chloro-indol from X-Gal 3 which quickly oxidises to form blue indigo derivative 8.
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Table 1 Bacteria with specific neuraminidase activity
Activity against a-2,3- Activity against a-2,6-
Neu5Ac linkage (4)
Neu5Ac linkage (5)
Bacillus pumilus
Arthrobacter aurescens
+
+
ꢀ
ꢀ
Fig. 1 Colorimetric determination of neuraminidase activity with a-2,3- (4) and
a-2,6-sialylated 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside 5 as substrates.
(A) Samples with bacterial neuraminidases (a) S. typhimorium neuraminidase, (b)
C. perfringens neuraminidase, (c) A. ureafaciens neuraminidase, (d) TcTS (purified
enzyme); (B) activity of three bacterial sialidases determined at 655 nm.
CAZy database.27 Genomic studies have shown that sialidase
family sequences share a common domain of four or ve copies
of an aspartate box well known as the BNR/Asp-box motif.
The bacterial genomes of B. pumilus and A. aurescens have
recently been sequenced. The sequence analysis revealed the
Using substrates 4 and 5, we have established a colorimetric presence of BNR/Asp-box motif suggesting the possible exis-
assay based on an indigo-derivative formation for a quick and tence of neuraminidases in this species, but the linkage speci-
easy detection of specic hydrolytic activity of neuraminidases. city was unknown. We were interested in using our probes, 4
Upon incubation of compounds 4 and 5 with an active neur- and 5, to conrm activity and determine specicity of any sia-
aminidase, an indolyl compound is released in the presence of lidase activity in these two bacteria. Activity was tested on crude
b-galactosidase which quickly undergoes oxidation to form an cell extracts. Interestingly, both bacteria showed neuraminidase
indigo derivative (Scheme 3). The activity can easily be detected activity, which was selective for a-2,3 (4) but not for a-2,6 (5)
by blue coloration of the sample without need of any expensive linkage (Table 1).
equipment and complicated technical skills. Both compounds 4
In conclusion, we have synthesised two new sialylated
and 5 respectively were used to test the neuraminidase activity compounds 4 and 5 as substrates for specic a-2,3 or a-2,6
of various bacterial sialidases in this two steps reaction neuraminidase activity. Both compounds are accessible by a
sequence (Scheme 3). As the highly specic b-galactosidase only one step enzymatic synthesis from commercially available
cleaves terminal Gal residues, compounds 4 and 5 are not materials. 4 and 5 could be used to detect novel neuraminidase
accepted. Aer incubation of all reaction mixtures with bacte- specicity in crude bacterial extracts and determine linkage
rial neuraminidases, the blue coloration in positive samples specicity at the same time in a simple colorimetric assay.
could be observed by naked eye aer the addition of b-galac- Compounds such as 4 and 5 will be useful to develop diagnostic
tosidase (Fig. 1A). For quantication of the enzymatic activity, tools for detecting neuraminidase activity and for developing
maximum absorbance of the indigo derivative 8 was deter- screens for inhibitors of these enzymes, particularly in the
mined (Fig. S3†). The commercial neuraminidase from S. context of viral and some parasitic infections.
typhimorium with activity towards a-2,3-linked Neu5Ac and
neuraminidase from A. ureafaciens with activity towards a-2,6-
linked Neu5Ac were used as standards. Spectrophotometric
analysis allowed us to determinate the activity and specicity of
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