Click Chemistry: The “click” reaction is readily performed using

“Click Solution”. The solution is comprised of 0.1 M Copper (I)

Bromide (CuBr) / 0.1 M Tris[(1-benzyl-1H-1,2,3-triazol-4-

yl)methyl]amine (TBTA) in ratio of 1:2 in mixture of Dimethyl

Sulfoxide (DMSO) and tert-butanol (3:1). This solution can then be

used to ligate alkyne with azide. Reaction consists of alkyne boronic

acid (0.1M), azide-biotin (0.1M) and freshly prepared Click Solution.

The reaction mixture is then mixed and is shaken at room

temperature for 3-4 hours. The product can then be used directly.

BLI: A kinetic study of the ELISA-like protocol was performed

using Forte Bio’s “Octet Red 96” biolayer interferometer. For this,

gly-HSA, was covalently immobilized to amine-reactive second

generation biosensors (AR2G) using standard EDC/s-NHS coupling.

After optimization of the pH for immobilization, the selected pH was

used to immobilize three different concentrations of gly-HSA and

controls. Borotin™ probe as produced above was then applied to

each sensor. The sensors were then briefly washed in buffer and

subsequently dipped in an equal concentration of streptavidin-HRP

conjugate and finally, DAB, a common HRP substrate.

Plate assay: A similar ELISA-like assay was also done with gly-

HSA immobilized in a standard 96-well plate. The only difference in

this fluorescent, end-point assay is that an HRP substrate (QuantaRed

HRP substrate) producing a fluorescent signal, was used.

Boronates have been used in affinity chromatography to

separate glycosylated proteins, enzymes and RNA [1]. All of

these separations rely on the highly specific, reversible-

covalent interaction between boronic acid and cis-diols of

sugars. It is well-known that certain glycosylation patterns on

proteins serve as the distinguishing markers for many cancers

[2–7]. Unfortunately, due to the rather poor diversity of

chemical structure among carbohydrates, the development of

high affinity, high-selectivity reagents to such groups has been

difficult [8].

Here we describe the creation of a chemical boronate-biotin

probe or “Borotin™” for readily determining, quantitatively,

the total cis-diol (i.e. glycation or glycosylation) content in a

protein or complex mixture of proteins. In this demonstration,

glycated human serum albumin (gly-HSA) was immobilized

directly, however the specificity of the Borotin™ probe for a

particular target in a complex mixture can be readily provided

by using the probe in conjunction with a standard affinity

reagent (capture antibody or aptamer).

“BorotinTM”: A dual function boronate-biotin molecular probe for measuring glycation/glycosylation of proteins Ilavarasi Gandhi, Mithil Soni, Hardik Jani, Kaushik Narendran, Mark Morris and George W. Jackson

Base Pair Biotechnologies, Houston, TX, USA

.

We have developed a patent-pending “BorotinTM” dual-

function probe as a convenient molecule for any application in

which a cis-diol (i.e. saccharide) group is desired to be

quantified in an ELISA-like setting. The probe can be used

alone to quantify total cis-diol levels or used in conjunction

with any capture affinity reagent (aptamer or antibody) for

greater assay specificity. Herein we describe the facile creation

of the probe from two commercially available starting reagents

using high-yield click chemistry and usage of the probe in a

prototype assay using biolayer interferometry for readout. The

resulting probe leverages the reversible, pH-dependent

covalent binding of boronic acid to cis-diols for specific

molecular recognition of glyco-groups on proteins and the

convenient biotin “handle” for subsequent signal amplification

via commonly available streptavidin-enzyme conjugates.

References 1. Hageman JH, Kuehn GD: Boronic Acid Matrices for the Affinity Purification of Glycoproteins and Enzymes. In

Practical Protein Chromatography. New Jersey: Humana Press; , 11:45–72.

2. Shore DA, Wilson IA, Dwek RA, Rudd PM: Glycosylation and the function of the T cell co-receptor CD8. Adv.

Exp. Med. Biol. 2005, 564:71–84.

3. König R, Ashwell G, Hanover JA: Glycosylation of CD4. Tunicamycin inhibits surface expression. J. Biol. Chem.

1988, 263:9502–9507.

4. Matsui T, Kojima H, Suzuki H, Hamajima H, Nakazato H, Ito K, Nakao A, Sakamoto J: Sialyl Lewisa expression as

a predictor of the prognosis of colon carcinoma patients in a prospective randomized clinical trial. Jpn. J. Clin.

Oncol. 2004, 34:588–593.

5. Baldus SE, Mönig SP, Zirbes TK, Thakran J, Köthe D, Köppel M, Hanisch F-G, Thiele J, Schneider PM, Hölscher

AH, Dienes HP: Lewis(y) antigen (CD174) and apoptosis in gastric and colorectal carcinomas: correlations with

clinical and prognostic parameters. Histol. Histopathol. 2006, 21:503–510.

6. Rapoport E, Le Pendu J: Glycosylation alterations of cells in late phase apoptosis from colon carcinomas.

Glycobiology 1999, 9:1337 –1345.

7. Rye PD, Bovin NV, Vlasova EV, Walker RA: Monoclonal antibody LU-BCRU-G7 against a breast tumour-

associated glycoprotein recognizes the disaccharide Gal beta 1-3GlcNAc. Glycobiology 1995, 5:385–389.

8. Burroughs S, Wang B: Boronic acid-based lectin mimics (boronolectins) that can recognize cancer biomarker,

the Thomsen-Friedenrich antigen. Chembiochem 2010, 11:2245–2246.

9. Cheng Y, Dai C, Peng H, Zheng S, Jin S, Wang B: Design, Synthesis, and Polymerase‐Catalyzed Incorporation of

Click‐Modified Boronic Acid–TTP Analogues. Chemistry – An Asian Journal 2011, 6:2747–2752.

10. Dai C, Wang L, Sheng J, Peng H, Draganov AB, Huang Z, Wang B: The first chemical synthesis of boronic acid-

modified DNA through a copper-free click reaction. Chem. Commun. (Camb.) 2011, 47:3598–3600.

11. Dai C, Cheng Y, Cui J, Wang B: Click reactions and boronic acids: applications, issues, and potential solutions.

Molecules 2010, 15:5768–5781.

12. Li M, Lin N, Huang Z, Du L, Altier C, Fang H, Wang B: Selecting aptamers for a glycoprotein through the

incorporation of the boronic acid moiety. J. Am. Chem. Soc. 2008, 130:12636–12638.

Acknowledgements

Methods

Introduction

Discussion .

Results

Figure 2: Kinetic Forte Bio based assay set up for quantitation of glycated human serum albumin (gly-HSA) using Borotin™ probe.

The Aptamer Discovery Company

Figure 3: Quantitating the DAB substrate step of above kinetics assay

Linear trendline: y = 1051.5x + 42933 R² = 0.6894

Log (avg) trendline: y = 7442.9ln(x) + 45839 R² = 0.9777

0

10000

20000

30000

40000

50000

60000

70000

80000

0 5 10 15 20 25 30

Avg

flu

ore

sce

nce

val

ue

s

Protein concentration (ug/ml)

Figure1: Example of a click-chemistry reaction to create the boronate-biotin probe. “Click chemistry” is otherwise known as the copper-catalyzed, “azide - alkyne Huisgen cycloaddition”.

Figure 4: Endpoint fluorescence assay for gly-HSA in standard plate-reader using Borotin™ probe.

The results shown here demonstrate the feasibility of using a

novel, dual function chemical probe for totalizing the

amount of glycation on a particular target protein. When

used in conjunction with a standard capture antibody or

aptamer, we expect that similar assays could be developed

for numerous proteins. The most important clinical example

of such a test is likely the hemoglobin A1c test for assessing

retrospective blood glucose exposure in diabetes. Thus, we

believe the new Borotin™ probe could be utilized to

augment such testing to new proteins, and we are currently

working towards such assays as well as improving the

preliminary results here.

This study was supported in part by an SBIR grant from the

National Institutes of Health entitled, “Assay for Monitoring

Glycemic Control in Diabetics” to Dr. Ralph Ballerstadt.

Abstract