8968 Chem. Commun., 2010, 46, 8968–8970 This journal is c The Royal Society of Chemistry 2010

Ionic-liquid-based catch and release mass spectroscopy tags for enzyme

monitoringw

M. Carmen Galan,* Anh Tuan Tran and Claire Bernard

Received 4th October 2010, Accepted 21st September 2010

DOI: 10.1039/c0cc04224b

A novel, inexpensive and versatile ionic-liquid-based catch and

release mass spectrometry tag (I-Tag) that facilitates substrate

purification, fast, robust and sensitive enzymatic reaction

monitoring and quantitative kinetic analysis has been developed.

The applicability of the system has been demonstrated in an

enzymatic assay with b-1,4-galactosyltransferase.

Proteins play a critical role in a variety of cellular events,

therefore, the study of small-molecule interactions with

proteins becomes crucial for understanding the sophisticated

processes of life. In order to visualize enzyme activity, a large

variety of assays have been developed over the years. The

majority of these assays are based on the use of expensive

synthetic substrates that release a radioactive, colored or

fluorescent product upon reaction. Alternatively, enzyme

reactions may also be followed using external indicators

that respond indirectly to product formation or substrate

consumption.1–4 Mass spectroscopy (MS) is a powerful analytical

technique that offers a fast, robust and sensitive method for

reaction monitoring, particularly for systems without straight-

forward alternatives or in high-throughput screenings.2

Ionic liquids (ILs) have emerged as a popular new class of

solvents in organic chemistry due to their unique physical and

chemical properties. ILs are particularly useful as new vehicles

for the immobilization of reagents in a number of synthetic

applications including oligosaccharide synthesis3 and more

recently in enzymatic transformations.4 Moreover, ILs are

ideal as MS probes for fast analysis because of their greater

spectral peak intensities and lower limits of detection.5

Protein glycosylation is more abundant and structurally

diverse than all other types of post-translational modification

combined.6 Protein- and lipid-bound oligosaccharides are

involved in a diverse range of biological processes such as

protein folding, cell–cell communication, bacterial adhesion,

viral infection and masking of immunological epitopes.7

Glycan assembly is primarily mediated by glycosyltransferases,

which typically act by adding monosaccharide residues from

mono- or diphosphate sugar nucleotides to growing oligo-

saccharide chains in a specific fashion, resulting in remarkably

complex structures.8 It has been estimated that mammalian

cells require well over 100 different glycosyltransferases to

biosynthesize all known oligosaccharide structures.9 Thus, these

enzymes represent an important target for the development of

potent inhibitors that could lead to drug discovery. However,

direct methods for rapid and simple kinetic or mechanistic

analysis of glycosyltransferase reactions are lacking.

Herein, we report the design, synthesis and application of an

inexpensive and versatile ionic-liquid-based catch and release

MS tag (I-Tag) that facilitates substrate purification, fast,

robust and sensitive enzymatic reaction monitoring and

quantitative kinetic analysis.

The probes are designed for easy attachment to substrates

and simple product release that is amenable to conjugation

to array platforms for further high-throughput biological

screening. Furthermore, as proof of concept, the I-Tag-based

strategy is validated successfully with an important enzyme

involved in glycan biosynthesis.

To this end, a trifunctional cross-linker was developed for

orthogonal attachment to the enzyme substrate (Fig. 1). The

linker contains an alkyne group for facile coupling to

azide-containing substrates, which are easily formed from

their halide-containing precursors, via the Cu(I)-catalyzed

1,3-dipolar cycloaddition reaction to form the corresponding

triazole adducts10 and an alkyl halide for incorporation of the

ionic component3a (Fig. 1). The triazole moiety and ionic head

provide an UV handle for alternative substrate detection. In

addition to those, the linker also contains a cleavage site to

facilitate the release of the I-Tag and the direct attachment of

the product to array platforms for high-throughput screening.

Among the various options, we selected a disulfide bond,

which is stable to many biological applications but can be

easily cleaved under reductive conditions to release a free

thiol.11 Thus, linkers 1 (Tag) and 2 (I-Tag) were synthesized

in 4 and 5 steps, respectively from mono-Boc-protected

cystamine in 79% and 75% overall yields (see ESIw for

experimental details).

To test the applicability of our methodology to enzymatic

assays, a model reaction was devised using b-1,4-galactosyl-transferase from bovine milk (b-1,4-GalT, EC 2.4.1.22) as

Fig. 1 General methodology for the ionic-liquid-based tag

methodology. Reaction monitoring and product release.

School of Chemistry, University of Bristol, Cantock’s Close, Bristol,BS8 1TS, UK. E-mail: M.C.Galan@bristol.ac.uk;Fax: +44 (0)117 929 8611; Tel: +44 (0)117 928 7654w Electronic supplementary information (ESI) available: Experimentalprocedures, characterization data for new compounds and NMRtraces. See DOI: 10.1039/c0cc04224b

COMMUNICATION www.rsc.org/chemcomm | ChemComm

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This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 8968–8970 8969

the enzyme catalyst. b-1,4-GalT transfers galactose from

UDP-galactose (UDP-Gal) to OH-4 of terminal b-linkedN-acetylglucosamine (GlcNAc). Studies on acceptor specificities

for b-1,4-GalT have revealed that the enzyme is quite tolerant

of modifications at the aglycone, a common feature shared by

other mammalian glycosyltranferases.12 Thus, it is to be

expected that the anomeric position of the glycoside substrate

is ideally suited for attachment of the ionic-based MS probe.

To that end, functionalized linker 1, displaying the activated

alkyne, was reacted with azide propyl N-acetylglucosamine 3,

that had been previously prepared from commercial

D-glucosamine in 4 steps in a 61% overall yield, in the presence

of Cu(I) to give triazole adduct 4 that was subsequently labeled

then labeled by reaction with 1-methylimidazole in the

presence of KBF4 to yield ionic-labeled substrate 5 in 75%

yield over the 2 steps after purification. Compound 5 was

purified by simple biphasic extractions since the non-I-Tag

materials can be washed away in solvent such as hexanes

and diethyl ether (Method A, see experimental in ESIw).Alternatively, linker 2 could be directly coupled to 3 to afford

substrate 5 in 84% yield (Method B in ESIw) (Scheme 1). In

order to validate that the incorporation of the I-Tag was

compatible with the enzymatic process, initial transfer of

galactose from UDP-Gal to modified acceptor 5 catalysed

by b-1,4-GalT was first performed on a preparative scale and

the reaction monitored by LC-MS using reported procedures

(Fig. 2).12 The structure of the resulting I-Tag-tethered

disaccharide 6 (96% yield) was confirmed by 1H- and 13C-NMR

and high-resolution MS analyses, demonstrating that the IL

containing MS probe was tolerated by the enzyme. To further

demonstrate the versatility of the MS probe and that the ionic

component could easily be cleaved under mild conditions,

the dithiol functionality in 6 was reduced using an excess of

tris(2-carboxyethyl)phosphine in water resulting in free thiol 7

being isolated and analyzed by 1H-NMR and MS (see ESIw for

details).

Subsequently, we determined the apparent kinetic parameters

for 5 to provide a relative measure of substrate binding affinity

with respect to the natural acceptor N-acetylglucosamine, as

well as demonstrating the usefulness of the IL based MS probe

for monitoring and quantifying enzyme kinetics. Following

reported assay conditions12 (Fig. 3), the depletion of 5

was monitored by LC-MS, where I-Tag linker 2 was used

as internal standard. The apparent Km value for 5 was

2.7 � 0.5 mM with a catalytic efficiency (Vmax/Km) of the

enzyme of 0.05 � 0.01 min�1 under the assayed conditions.

The kinetic parameters obtained for IL bound 5 are similar to

those reported for free GlcNAc13 (Km 1.7 mM and (Vmax/Km)

of 0.02 � 0.01 min�1), which shows that the presence of the

MS probe at the anomeric position of the glycoside acceptors

does not interfere significantly with the enzyme binding site.

In summary, this work shows a new and efficient strategy

for both qualitative and quantitative enzyme characterization.

The use of a versatile ionic-liquid-based MS probe, featuring

easy substrate attachment and product release, allows for

fast and sensitive enzyme monitoring by mass spectroscopy

without the need for expensive radioactive or fluorescence labeled

substrates. Furthermore, the presence of a UV chromophore

in the linker provides an alternative mode of detection.

Enzyme studies with b-1,4-GalT have proven that this

methodology can be applied to biological screening of

glycosyltranseferases. Our group is currently screening other

glycosyltransferases which will be the topic of a full paper in

due course. Ionic liquid based tagged substrates are purified by

simple biphasic extractions from the non-I-Tagged moieties,

upon covalent attachment of the IL-based tag, since the non-

I-Tag materials can be washed away in solvents in which the

ionic components are insoluble. Moreover, the ability to

selectively cleave the I-Tag to release products bearing a free

thiol that could be used for direct attachment to array platforms

will be very useful for subsequent high throughput biological

screening.14 We believe that this new class of IL based MS

probes will be very valuable for general and fast enzyme

monitoring in a variety of biological systems.Scheme 1 (i) Ascorbic acid, CuSO4�5H2O; (ii) 1-methylimidazole,

KBF4; (iii) b-1,4-GalT, UDP-Gal, pH 8.2; (iv) TCEP (50 mM), H2O.

Fig. 2 LC-MS chromatogram of preparative enzymatic reaction after

24 h. (A) UV spectrum of preparative enzymatic reaction. (B) TIC

trace for disaccharide product 6 [M+] 821. (C) TIC trace for

I-Tagged-starting material 5 [M+] 659.

Fig. 3 Michaelis–Menten plot for I-Tagged compound 5.Dow

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8970 Chem. Commun., 2010, 46, 8968–8970 This journal is c The Royal Society of Chemistry 2010

We gratefully acknowledge financial support from EPSRC

and The Royal Society and we thank Prof. M. M. Palcic

(Carlsberg Res. Lab.) for advice on the b-1,4-GalT assays and

the gift of UDP-Galactose, Prof. R. J. Cox for use of the

LC-MS and Prof. R. A. Field for useful discussions.

Notes and references

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