Mixed SAMs and MALDI-ToF MS: Preparation of N-glycosylamine derivative and thioctic acid methyl ester bearing 1,2-dithiolane groups and detection of enzymatic reaction on Au

Article abstract of DOI:10.1016/j.bioorg.2011.10.004

Herein, we report an enzymatic galactosylation reaction of β-glucopyranosylamide 4 and thioctic acid methyl ester 5 bearing 1,2-dithiolane groups to form a new system of mixed self-assembled monolayers (SAMs) on gold. Characterization of the enzymatic activity was conveniently achieved by mass spectrometry.

Full text of DOI:10.1016/j.bioorg.2011.10.004

Bioorganic Chemistry 40 (2012) 6–9
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Preliminary Communications
Mixed SAMs and MALDI–ToF MS: Preparation of N-glycosylamine derivative
and thioctic acid methyl ester bearing 1,2-dithiolane groups and detection
of enzymatic reaction on Au
⇑
Hani Mutlak A. Hassan , Beatrice A. Maltman
Manchester Interdisciplinary Biocentre (MIB), University of Manchester, 131 Princess Street, Manchester M1 7DN, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 October 2011
Available online 9 November 2011
Herein, we report an enzymatic galactosylation reaction of b-glucopyranosylamide 4 and thioctic acid
methyl ester 5 bearing 1,2-dithiolane groups to form a new system of mixed self-assembled monolayers
(SAMs) on gold. Characterization of the enzymatic activity was conveniently achieved by mass
spectrometry.
Keywords:
Ó 2011 Elsevier Inc. All rights reserved.
Mixed self-assembled monolayers
Gold
Enzymatic reaction
Mass spectrometry
1
. Introduction
In 1983, Nuzzo and Allara discovered the formation of self-
2. Experimental
Synthesis of 1-amino-1-deoxy-b-D-N-acetylglucoside (2). To a
microwave reaction tube was added N-acetyl-D-glucosamine 1
(250 mg, 1.1 mmol), ammonium carbonate (1.25 g, 13 mmol),
assembled monolayers (SAMs) of alkanethiolates on gold and are
pioneers of this field [1]. Since then, SAMs have been employed
in various arenas of chemical science; interfacial chemical reac-
tions, detection of ligand–protein interactions, drug discovery, to
mention a few [2]. Matrix-assisted laser desorption/ionization
and time-of-flight mass spectrometry (MALDI–ToF MS) has previ-
ously been utilized to confirm molecules adsorbed to a gold sur-
face. While, unfortunately, the majority of applications have used
SAMs with alkanethiolate structures for biological studies [3], we
were attracted in using the 1,2-dithiolane group for anchoring b-
glucopyranosylamide 4 along with thioctic acid methyl ester 5
on gold, to form mixed SAMs via the 1,2-dithiolane rings, and
examining whether the desired galactosylation enzymatic reaction
of these mixed SAMs (see Fig. 1) could be verified by mass spec-
trometry. The 1,2-dithiolane group tends to form stable SAMs than
their thiol counterpart [4], presumably because of the cyclic disul-
fide structure, and thus could offer favorable substrate conforma-
tion for binding to biomolecules. Herein, we show the use of
mixed SAMs and MALDI–ToF MS analysis allowed a rapid and sim-
ple characterization of a galactosylation reaction of 1,2-dithiolane-
based carbohydrate 4 on a gold surface.
3 2
and anhydrous (CH ) SO (900 lL). The tube was sealed and irradi-
ated under microwave conditions (10 W, 250 psi, 40 °C) for 1.5 h.
After lyophilization, b-glycosylamine 2 was obtained as a white so-
lid in 91% yield. The crude product 2 was used in the next step
without purification. Analytical data were consistent with the liter-
ature [6]. Synthesis of 2,3,4,6-tetrahydroxyl-1-N-thioctoyl-b-D-gluco-
pyranosylamide (4). To an oven dried round-bottomed flask was
added thioctic acid 3 (1,2-dithiolane-3-pentanoic acid) (247 mg,
1.2 mmol), HBTU (1.36 g, 3.6 mmol), HOBt (162 mg, 1.2 mmol),
and anhydrous (CH ) NCHO (9 mL). The reaction mixture was stir-
3 2
red for 15 min and transferred to a round-bottomed flask contain-
ing b-glycosylamine 2 (0.220 g, 1 mmol) and stirring continued at
rt overnight, under N
der reduced pressure and the crude product was purified via silica
gel column chromatography (5 ? 10% CH OH in CHCl ) to give 4 in
an oil form. The product was then azeotropically dried with tolu-
ene to afford a yellow solid (270 mg, 66%). Analytical data were
consistent with the literature [8]. Synthesis of methyl 5-(1,2-dithio-
lan-3-yl)pentanoate (5). To a stirred solution of thioctic acid 3
2
atmosphere. The solvent was removed un-
3
3
(
206 mg, 1 mmol) in anhydrous CH
lytic amount of conc. H SO . The reaction mixture was stirred at
rt overnight. After workup, 5 was obtained as a yellow oil
218 mg, 99%) and used for SAMs studies without purification.
3
OH (100 mL) was added cata-
2
4
(
⇑
Corresponding author.
E-mail address: hani.mutlak.a.hassan@gmail.com (H.M.A. Hassan).
Analytical data were consistent with the literature [5]. Preparation
procedure of self-assembled monolayers. A gold substrate was
0
045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.10.004

Hani Mutlak A. Hassan, B.A. Maltman / Bioorganic Chemistry 40 (2012) 6–9
7
3
Scheme 2. Reagents and conditions: (a) CH OH, conc. H
2
SO
4
, 0.08 M, rt, overnight;
(
3 2 4
b) CH OH, conc. H SO , 0.01 M, rt, overnight. Formation of ester 5 and anhydride 6
observed by TLC and MS and its exclusive formation under different esterification
conditions.
Fig. 1. Schematic representation of immobilization and galactosylation process on
Au.
immersed into a solution of 4 and 5 (1:25 ratio) in ethanol (1 mL;
1
mM final concentration) and left overnight at rt. The substrate
was then washed thoroughly with ethanol and dried with N . Enzy-
matic galactosylation procedure. The gold substrate was immersed
into a solution of b-1,4-galactosyltransferase (10 L, 0.06 units),
% UDP-Gal (50 L), MOPS buffer (100 L, pH 7.5, 500 mM), MnCl
50 L, 200 mM), and water (790 L). After the solution was sha-
ken gently at 30 °C overnight, the substrate was washed thor-
2
l
1
(
l
l
2
l
l
oughly with ethanol and water then dried with N
2
and analyzed
by MALDI–ToF MS.
3
. Results and discussion
The study commenced by first preparing b-glycosylamine 2
which was obtained in good yield (91%) by microwave irradiation
of N-acetylglucosamine 1 with ammonium carbonate (modifica-
tion of the Kochetkov amination) [6]. Glycosylamines are an
important motif in carbohydrate science, particularly in glycopep-
tides and glycoproteins [7]. Coupling of 2 with the commercially
available thioctic acid 3 using HOBt and HBTU as peptide coupling
Scheme 1. Synthesis of b-glucopyranosylamide 4 bearing 1,2-dithiolane group for
immobilization on Au.
Fig. 2. MALDI–ToF MS spectrum displaying desired molecular mass of 4 plus sodium and potassium adducts (at 431 and 447 m/z, respectively) after immobilization on Au.

8
Hani Mutlak A. Hassan, B.A. Maltman / Bioorganic Chemistry 40 (2012) 6–9
Fig. 3. Illustration of immobilization of 4 and 5 on Au and galactosylation reaction to give disaccharide 7.
Fig. 4. MALDI–ToF MS confirmed successful galactosylation product (593 m/z, MNa+).

Hani Mutlak A. Hassan, B.A. Maltman / Bioorganic Chemistry 40 (2012) 6–9
9
reagents delivered the desired b-glucopyranosylamide 4 [8] in 66%
4. Conclusion
isolated yield in a short two-step synthetic pathway (Scheme 1).
This straightforward route benefits in obtaining only the b-anomer
directly from 1. Because the employment of mixed monolayers on
gold aids recognition of biological events [2], we decided to synthe-
size thioctic acid methyl ester 5 as it was envisioned that this com-
pound (as apposed to acid 3) would not pose unwanted
interference with the enzymatic reaction once co-immobilized on
gold. The first attempted generation of ester 5 was performed by
In this communication, we have shown that the use of mixed
SAMs and MALDI–ToF MS provided a highly efficient detection of
an enzymatic activity, employing b-glucopyranosylamide 4 to-
gether with thioctic acid methyl ester 5 (both bearing 1,2-dithio-
lane groups) to form a new system of mixed monolayers on gold.
We wish to extend the use of the thioctic acid structure for other
endeavors such as discovering inhibitors of enzymes using SAMs
and MALDI–ToF MS technology.
subjecting thioctic acid 3 to esterification conditions (CH
conc. H SO , rt, overnight, 0.08 M). Analysis by TLC and MS indi-
cated the formation of the desired product 5 along with anhydride
(two molecules of thioctic acid 3 underwent a condensation
reaction).
We then exclusively generated 5 under higher dilution condi-
tions (CH OH, cat. conc. H SO , rt, overnight, 0.01 M) in 99% yield.
3
OH, cat.
2
4
Acknowledgment
6
We thank Dr. N. Mullin for biological assistance and Prof. S.
Flitch for helpful discussion on this work. The BBSRC is gratefully
acknowledged.
3
2
4
The modified reaction conditions prevented self-condensation to
arise and gave 5 as the sole product (Scheme 2).
References
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2
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(
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+
5
93 m/z (MNa ), therefore giving robust evidence that the enzy-
1
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was not fully converted to 7, the combination of 4 and 5 to form
mixed SAMs seems to have conveniently worked well, despite
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drate ligand and the gold surface.
[
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