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Synthesis of Mercaptoalkyl Glucosides with Potential Application as Bioassay Platform to Study

Carbohydrate-Protein Interaction

Marina V. Reconose1, Der-Lii Mike Tzou2, Susan D. Arco1*


1
Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101, Philippines
2
Insitute of Chemistry, Academia Sinica, Nankang, Taiwan
*
Corresponding Author Email Address: sdrarco@yahoo.com

Abstract

Three mercaptoalkyl glucosides having different length of alkanethiol spacer were synthesized via
trichloroacetimidate method. Self-assembled monolayer (SAM) of a representative glucoside was
created to serve as a potential bioassay platform to study the binding affinity of glucose/galactose
protein (GGBP) to the glucose moiety using surface plasmon resonance (SPR). SPR results showed that
the SAM of 8--D-mercaptooctyl glucoside can serve as a bioassay platform to study the interaction
between glucose moiety and GGBP. The affinity parameters obtained in phosphate buffer saline (PBS)
(pH 7.4), were KA: 1.17x1010 M-1; KD: 8.57x 10-11 M, clearly demonstrating strong binding.
Furthermore, 8--D-mercaptooctyl glucoside was characterized in situ on 10-nm gold nanoparticles
using uv-vis spectroscopy and transmission electron microscopy (TEM). Results showed the potential of
8--D-mercaptooctyl glucoside to create 8--D-mercaptooctyl glucoside-gold nanopartciles (AuNPs)
which can be useful in inhibition study. With the created SAM using the synthesized mercaptoalkyl
glucoside, this study sheds some new insights on the behaviour of the protein which might be important
when designing compounds to study carbohydrate-protein interaction specifically that of glucoside-
GGBP.

Keywords: Mercaptoalkyl Glucosides, Self-assembled Monolayer, Glucose/Galactose Protein

1. Introduction

Carbohydrates are one of the major components of the cell membrane. Arranged in a diverse and
heterogeneous manner on the surface, these biomolecules play important roles in various biological
processes and are crucial for the interactions of cells with one another, and with pathogens such as
viruses, bacteria and fungi [1-5]. Carbohydrates on the cell membrane surface are the key components of
glycoconjugates, specifically the glycoproteins and glycolipids. Since the cell surface serves as a site
for docking of other cells and molecules, the carbohydrate layer function as multivalent ligands for
receptors or target cells or molecules [1].Multivalency presentation of these ligands on the cell surface
strengthens their interaction and specificity with the receptor [6]. Since glycoconjugates are problematic
to isolate from natural sources and their total synthesis is often difficult to complete due to the
complexities of their structures, the interest of the glyco-scientists has been to synthesize simple
structured glycoconjugates from commercially available saccharides [7-8].
In the recent years, many different types of glycoconjugates have been synthesized by employing
different carbohydrate epitopes to serve as ligand and different spacer molecules of different lengths to
link the ligands with the immobilization surface. Glycoconjugates have been assembled to form different
architectures like micelles, clusters, dendrimers, and polymers or as self-assembled monolayers (SAM)
either in 2D using gold chip or 3D using gold nanoparticles or carbon nanotubes [8-27]. All these
structures can be used to create simplified model systems to mimic the complex natural structures of
glycoconjugates on cell membrane environment.

In this study, simple structured glycoconjugates specifically mercaptoalkyl glucosides were


synthesized. Three different mercaptoalkyl glucosides, each consisting of a glucose headgroup and an
alkane chain terminated in a thiol group (alkanethiol) were prepared via glycosyltrichloroacetimidate
with acetyl group as protecting group and trimethylsilyltrifluoromethanesulfonatetriflate (TMSOTf) as
activator. Given the self-assembly property of the alkanethiols [28-29], the glucosides were immobilized
onto a gold chip. The length of the alkanethiol spacer consisted of 8, 12 and 14 carbons since long-
chain (n 8) was found to yield higher packing density and more ordered SAMs [30]. The binding
affinity of the glucose/galactose–binding protein (GGBP) from E. coli with mercaptoalkyl glucoside
SAM was then analyzed by SPR.

2. Experimental

2.1 Materials, Reagents and Instrumentations


All chemicals were purchased from Sigma, Fluka or Mallinkcrodt Chemicals. All reagents and
chemicals were reagent grade and used without further purification unless otherwise stated. All solvents
were anhydrous grade unless otherwise noted. Solvents were removed between 20C and 40C (bath
temperature) using rotary evaporator unless otherwise indicated. Molecular sieves were heated (>170C)
and cooled in vacuum prior to use. All reactions were carried out in oven-dried glasswares under an
atmosphere of nitrogen unless otherwise specified.

1
H and 13C NMR spectra were recorded using AVANCE-500. Proton chemical shifts () are
reported in parts per million (ppm) relative to the methine singlet at 7.24 ppm for the residual CHCl3 in
the deuteriochloroform, or the methyl pentet at 3.33 ppm for the residual CHD2OD in the methanol – d4.
Carbon chemical shifts are reported in parts per million relative to the internal 13C signals in CDCl3
(77.23 ppm) and CD3OD-d4 (49.15 ppm). Mass spectra were obtained with a FAB JMS-700 double
focusing mass spectrometer (JEOL, Tokyo, Japan), MALDI Voyager DE-PRO (Applied Biosystem
Houston, USA) and ESI Finnigan LCQ mass spectrometer (ThermoFinnigan, San Jose, CA, United
States) in negative and positive mode. UV–vis absorption spectra were recorded on a Jasco V-
670spectrophotometer. TEM micrographs were taken on a JOEL 2000 FX transmission electron
microscope. Real time SPR analyses were performed on a BIAcore Biosensor 3000 system (BIAcore,
Uppsala, Sweden).

2.3 Synthetic Procedures

2.3.1Synthesis of 1, 2, 3, 4, 6 – Penta-O-acetyl-/ -D-glucopyranose (3)


To a solution of 500 mg (2.77 mmol) -D-glucose (1) in 3.5 mL pyridine, 2.5 mL (26.45 mmol)
acetic anhydride was added and allowed to react overnight under N2 atmosphere. The completion of the
reaction was monitored by thin layer chromatography (TLC). Pyridine was removed using high vacuum.
Work-up: The residue was dissolved in ethyl acetate and the ethyl acetate layer was washed with water
twice. The organic layer was collected and dried over anhydrous MgSO4. The solvent was removed
under reduced pressure and the crude compound was purified by column chromatography on silica gel
using hexane/ethyl acetate. The product was further dried in high vacuum to afford 950 mg (88% based
on the expected yield of 1, 2, 3, 4, 6 – penta-O-acetyl-/ -D-glucopyranose) of pure 3 as white powder.
TLC (ethyl acetate); Rf = 0.8; 1H NMR (400 MHz, CDCl3) : (ppm) 6.56 (d, J=3.3 Hz, 1H), 5.97–5.592
(m, 2H), 5.44–5.36 (m, 3H), 5.18 (d, J=9.3 Hz, 2H), 5.10–5.01 (m, 6H), 4.24–4.18 (m, 4H), 4.06–4.01
(t, J= , 6H), 3.80–3.77 (m, 2H), 2.12 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H), 1.95 (s, 3H).

2.3.2 Synthesis of 2, 3, 4, 6 – Tetra-O-acetyl-/ -D-glucopyranose (4)


Compound 3 (1000 mg, 2.56 mmol) was dissolved in 0.1 M solution of hydrazine acetate in
dimethyl formamide (DMF). The mixture was allowed to react at 40C-50C under N2 atmosphere. The
completion of the reaction was monitored by TLC. DMF was removed using high vacuum. Work-up:
The residue was diluted with ethyl acetate. The solution was washed with water and saturated NaHCO 3.
The organic layer was collected and dried with MgSO4. The solvent was removed by rotary evaporation
and the residue was purified by column chromatography on silica gel using ethyl acetate/hexane. The
product was further dried in high vacuum to afford 860 mg (96% based on the expected yield of 2, 3, 4,
6 – tetra-O-acetyl-/ -D-glucopyranose) of pure 4 as viscous colorless liquid. TLC (hexane: ethyl
acetate = 3:1 v/v): Rf= 0.17; 1H NMR (400 MHz, CDCl3): (ppm) 5.49 (t, J=9.8 Hz, 1H), 5.40 (d, J=2.8
Hz, 1H), 5.03 (t, J=9.8 Hz, 1H), 4.88 – 4.83 (m, 1H), 4.71 (d, J=8, 1H), 4.21–4.17 (m, 2H), 4.12–4.06
(m, 1H), 2.10–1.90 (4s, 12H).

2.3.3 Synthesis of 2, 3, 4, 6 - Tetra-O-acetyl-/ -D-glucopyranosyltrichloroacetimidate (5)


To a solution of 648 mg (1.86 mmol) of compound 4 in 20 mL dichloromethane (DCM), 0.95
mL (9.30 mmol) trichloroacetonitrile with 242 mg (0.74 mmol) Cs2CO3 as catalyst was added. Cs2CO3
mediated the deprotonation of the anomeric hydroxy group. Work- up: After the completion of the
reaction, the mixture was filtered and the filtrate was washed twice with NH4Cl solution. The organic
layer was collected and dried. The solvent was removed by rotary evaporation and the residue was
purified by column chromatography on silica gel using ethyl acetate/hexane (1:1 v/v). The product was
further dried in high vacuum to afford 400 mg of crude compound 5. Since compound 5 is air sensitive
[1] and its purification by silica gel flash chromatography using a mixture of ethyl acetate and hexane as
solvent system leads to extensive degradation resulting in low yields [28], glycosylimidate was used in
the succeeding reaction without further purification. TLC confirmed that the reaction was completed and
only one product was formed. TLC (hexane: ethyl acetate = 3:1 v/v): Rf=0.4.

2.3.4 Synthesis of 8-Bromo-1-octanol (6a)


To a solution of 3000 mg (20.52 mmol) of 2ain 50 mL of cyclohexane, 50 mL (442 mmol) of
48% HBr was added. The reaction mixture was stirred at reflux for 6 hours. Work-up: The reaction
mixture was extracted with 100 mL of hexane three times, washed with saturated solution of NaHCO3,
brine and water three times each. The organic layer was collected, dried with MgSO4and filtered. The
solvents were removed by rotary evaporation and the resulting residue was purified by column
chromatography on silica gel using ethyl acetate/hexane (1:1 v/v). The product was further dried in high
vacuum to afford 2390 mg (56% based on the expected yield of 8-bromo-1-octanol) of pure 6aas pale
yellow solid. TLC (hexane: ethyl acetate = 8:2 v/v): Rf= 0.31; 1H NMR (400 MHz, CDCl3): (ppm) 3.61
(t, J=6.6 Hz, 2H, –CH2OH), 3.37 (t, J=7.1 Hz, 2H, –CH2Br), 1.85–1.78 (m, 2H, –CH2CH2Br), 1.54–1.50
(m, 2H, –CH2CH2OH), 1.40 (s large, 8H, –CH2–); 13C NMR (100 MHz, CDCl3): (ppm) 63.06 (C-O),
34.09, 32.95, 32.86, 29.37, 28.87, 28.25, 25.80.
2.3.5 Synthesis of 8-Thioacetyl-1-octanol (7a)

To a solution of 2000 mg (9.56 mmol) of 6a in 10 mL dry DMF, 1310 mg (11.48 mmol) of


potassium thioacetate was added and allowed to react under N2 atmosphere at room temperature. Work –
up: After all the starting material was consumed, the mixture was extracted with 100 mL of ethyl acetate
and washed with water three times. The organic layer was collected, dried with MgSO 4 and filtered. The
organic solvents were removed by rotary evaporation and the resulting residue was purified by column
chromatography on silica gel using ethyl acetate/hexane (1:1 v/v). The product was further dried in high
vacuum to afford 1600 mg (82% based on the expected yield of 8-thioacetyl-1-octanol) of pure 7a as
white solid. TLC (hexane: ethyl acetate = 8:2 v/v): Rf = 0.26; 1H NMR (400 MHz, CDCl3): (ppm) 3.57
(t, J=6.6 Hz, 2H, –CH2OH), 2. 81 (t, J=7.4 Hz, 2H, –CH2S), 2.27 (s, 3H; SCOCH3), 1.53–1.48 (m, 4H;
CH2CH2), 1.37–1.27 (m, 4H; CH2CH2);13C NMR (100 MHz, CDCl3): (ppm) 196.23 (C=O), 63.05 (C-
OH), 32.87, 30.74, 29.60, 29.36, 29.27, 29.17, 28.84 (CH2), 25.81(CH3-C=O); HR-APCI calculated
227.1082 for C10H20O2SNa [M+Na]+; found 227.1076.

2.3.6 Synthesis of 2, 3,4,6-O-Tetraacetate-(8– thioacetyloctyl)-1-O- -D-glucopyranoside (8a)


To a flask containing 1180 mg (240 mmol) 5and 587 mg (2.87 mmol) 7ain dry DCM, 400 mg of
3Å molecular sieves was added. The mixture was stirred at room temperature under N2 atmosphere for 1
hour. The solution was then cooled to -20C and 0.65 mL TMSOTf was added slowly at stirring. Work-
up: After 15 minutes, the reaction was quenched with 500 mg NaHCO3, washed with saturated NaHCO3
and brine. The molecular sieves were removed by vacuum filtration. The organic layer was collected,
dried with MgSO4 and filtered. The solvents were removed by rotary evaporation and the resulting
residue was purified by column chromatography on silica gel using ethyl acetate/hexane (1:1 v/v). The
product was further dried in high vacuum to af 700 mg (55% in two steps, based on the expected yield
of 2, 3, 4, 6-O-tetraacetate-(8-thioacetyloctyl)-1-O--D-glucopyranoside) of pure compound 8a as white
powder. TLC (hexane: ethyl acetate = 7:3 v/v): Rf= 0.26;1H NMR (400 MHz, CDCl3): (ppm) 5.18 (t,
J=9.4 Hz, 1H, H-3), 5.06 (t, J=9.6 Hz, 1H, H-4), 4.96 (t, J=8.8 Hz, 1H, H-2), 4.46 (d, J=7.6 Hz, 1H, H-
1), 4.24 (dd, J=12.4, 4.8 Hz , 1H, H-6a), 4.11 (dd, J=12.2, 2.2 Hz, 1H, H-6b), 3.85–3.82 (m, 1H, –
CH2O–) , 3.68–3.67 ( m, 1H, H-5) , 3.45–3.43 ( m , 1H, –CH2O–), 2.82 (d, J=7.2 Hz, 2H, –CH2S–),
2.30 (s, 3H, –SCOCH3), 2.06–1.98 (4s, 3H, –CCOCH3 ) , 1.57–1.50 (m, 6H, CH2’s ) , 1.32–1.23 (m, 6H,
CH2’s); 13C NMR (100 MHz, CDCl3): (ppm) 196.20, 170.89, 170.53, 169.62, 169.47, 101.07, 73.15,
72.02, 71.64, 70.37, 68.80, 62.28, 30.83, 29.69, 29.58, 29.33, 29.24, 28.93, 25.94, 20.94, 20.86, 20.82;
HR-ESI calculated 557.2033 for C24H38O11SNa [M+Na]+; found 557.2035.

2.3.7 Synthesis of 8-Mercaptooctyl-1-O- -D-glucopyranoside (9a)


To a solution of 600 mg (1.12 mmol) of 8a in 10 mL HPLC grade MeOH, 30 mg (0.56 mmol)
NaOMewas added and the reaction was stirred for 1 hour. Work-up: Acidic resin was added to neutralize
the reaction and the solution was filtered off. MeOH was removed by rotary evaporation and the
resulting residue was purified by column chromatography on silica gel using ethyl acetate/hexane (1:1
v/v). The product was further dried in high vacuum to afford 150 mg (41% based on the expected yield
of 8-mercaptooctyl-1-O--D-glucopyranoside) of pure 9a as whitesolid. TLC (MeOH: DCM = 1:9 v/v);
Rf = 0.3; 1HNMR (400 MHz, CD3OD) : (ppm) 4.21 (d, J=7.6 Hz, 1H, H-1), 3.87–3.81 (m, 2H, H-6a, –
CH2O–), 3.6 (dd, J=12, 5.2 Hz, 1H, H-6b), 3.53–3.49 (m, 1H, –CH2O–), 3.31–3.23 (m, 3H, H-3, H-4,
H-5), 3.107–3.149 (m,1H, H-2), 2.45 (t, J=7.2 Hz, 2H, –CH2S–), 1.61–1.52 (m, 4H), 1.38–1.28 (m, 8H);
13
C NMR (100 MHz, CD3OD): (ppm) 104.53 (C-1), 78.32 (C-3), 78.06 (C-5), 75.30 (C-2), 71.88 (C-
4), 71.02 (C-6), 62.98 (-OCH2), 35.32, 30.90, 30.59, 30.27, 29.48, 27.16, 25.08 (-CH2’s); HR-ESI
calculated 347.1504 for C14H28O6SNa [M+Na]+; found 347.1500.

2.3.8 Synthesis of 12-Bromo-1-dodecanol (6b)


As for 6a, 2000 mg (9.88 mmol) of 2bwith 25mL (217 mmol) of 48% HBr in 25 mL
cyclohexane, was converted into crude product that was flash chromatographed on a column of silica gel
packed dry, eluted with hexane/ethyl acetate (1:1 v/v) and high vacuum-dried to afford 1960 mg (75%
based on the expected yield of 12-bromo-1-dodecanol) of pure 6b as pale yellow powder.TLC (hexane:
ethyl acetate = 8:2 v/v): Rf= 0.43; 1H NMR (400 MHz, CDCl3): (ppm) 3.62 (t, J=6.8 Hz, 2H, –
CH2OH), 3.38 (t, J=6.8 Hz, 2H, –CH2Br), 1.87–1.79 (m, 2H, –CH2CH2Br), 1.58–1.51 (m, 2H, –
CH2CH2OH), 1.34 (s large, 16 H); 13C NMR (100 MHz, CDCl3): (ppm) 63.32 (C–O), 34.20, 33.07,
29.73, 29.64, 28.98, 28.41, 25.97 (CH2).

2.3.9 Synthesis of 12-Thioacetyl-1-dodecanol (7b)


As for 7a, 1500 mg (5.66 mmol) of 6b with 774 mg (6.79 mmol) potassium thioacetate in 10 mL
DMF, was converted into a crude product that was flash chromatographed on a column of silica gel
packed dry and eluted with hexane-ethyl acetate (1:1 v/v) and high vacuum-dried to afford 1300 mg
(88% based on the expected yield of 12-thioacetyl-1-dodecanol) of pure 7b as white solid. TLC (hexane:
ethyl acetate = 8:2 v/v): Rf= 0.34; 1H NMR (400 MHz, CDCl3): (ppm) 3.62 (t, J=6.4 Hz, 2H, –
CH2OH), 2.84 (t, J=7.2 Hz, 2H, –CH2S), 2.30 (s, 3H; SCOCH3), 1.56–1.50 (m, 4H; CH2CH2), 1.34–1.24
(m, 18H); 13C NMR (100 MHz, CDCl3): (ppm) 196.27 (C=O), 63.33 (C–OH), 33.05, 30.83, 29.78,
29.74, 29.71, 29.65, 29.63, 29.40, 29.31, 29.04 (CH2), 25.96 (CH3–C=O); HR-ESI calculated 283.1708
for C14H28O2SNa [M+Na]+; found 283.1704.

2.3.10 Synthesis of 2, 3,4,6-O-Tetraacetate-(12– thioacetyldodecyl)-1-O- -D-glucopyranoside (8b)


The above procedure for 8a was used to convert the mixture of 1300 mg (5 mmol) of 7b and 600
mg (1.22 mmol) of crude compound 5 with 0.33 mL TMSOTf into 650 mg (47% in two steps, based on
the expected yield of 2, 3, 4, 6-O-tetraacetate-(12– thioacetyldodecyl)-1-O--D-glucopyranoside) of 8b
which was obtained as white powder. TLC (hexane: ethyl acetate = 6:4 v/v): Rf= 0.60; 1H NMR (400
MHz, CDCl3) : (ppm) 5.18 (t, J=9.6 Hz, 1H, H-3), 5.06 (t, J=9.6 Hz, 1H, H-4), 4.96 (t, J=5.9 Hz, 1H,
H-2), 4.47 (d, J=8 Hz, 1H, H-1), 4.24 (dd, J=12.2, 4.6 Hz, 1H, H-6a), 4.11 (dd, J=12.2, 2.2 Hz, 1H, H-
6b), 3.85–3.83 ( m, 1H, –CH2O–) , 3.68–3.65 ( m, H-5) , 3.46–3.43 ( m, 1H, –CH2O–), 2.84 (t, J=7.4
Hz, 2H, –CH2S), 2.30 (s, 3H, –SCOCH3), 2.06–1.98 (4s, 12H, –CCOCH3) , 1.57–1.50 (m, –CH2’s),
1.23 (m, –CH2’s); 13C NMR (100 MHz, CDCl3) : (ppm) 196.23, 170.89, 170.53, 169.62, 169.47,
101.09, 73.17, 72.02, 71.66, 70.46, 68.82, 62.30, 30.84, 29.77, 29.73, 29.68, 29.64, 29.54, 29.39, 29.33,
29.04, 26.04, 20.94, 20.83; HR-ESI calculated 613.2659 for C28H46O11SNa [M+Na]+; found 613.2648.

2.3.11 Synthesis of 12-Mercaptododecyl-1-O--D-glucopyranoside (9b)


As above for 8a, 600 mg (1.02 mmol) of 8b was deacetylated with 82.27 mg (1.52 mmol) of
NaOMe in 10 mL MeOH to afford 120 mg (31% based on the expected yield of 12-mercaptododecyl-1-
O--D-glucopyranoside) of pure 9b as white wax-like solid. TLC (MeOH: DCM =1:9 v/v); Rf = 0.3; 1H
NMR (400 MHz, CD3OD) : (ppm) 4.18 (d, J=7.6 Hz, 1H, H-1), 3.87 – 3.79 (m, 2H, H-6a, –CH2O–),
3.60 (dd, J=11.8, 5.4 Hz, 1H, H-6b), 3.50–3.45 (m, 1H, –CH2O–), 3.28–3.22 (m, 3H, H-3, H-5, H-4),
3.11 (dd, J=8.8, 8 Hz, 1H, H-2), 2.43 (t, J=7.2 Hz, 2H, –CH2S–), 1.58–1.49 (m, 4H), 1.25 (m, 2H); 13
C NMR (100 MHz, CD3OD): (ppm) 104.54 78.32, 78.07, 75.31, 71.87, 71.10, 62.97, 35.35, 30.95,
30.83, 30.78, 30.74, 30.34, 29.54, 27.24, 25.11; HR-ESI calculated 403.2130 for C18H36O6SNa
[M+Na]+; found 403.2130.

2.3.12 Synthesis of 14-Bromo-1-tetradecanol (6c)


As for 6a, 500 mg (15.62 mmol) of 2c with 10 mL (88 mmol) of 48% HBr in 10 mL of
cyclohexane, was converted into crude product that was flash chromatographed on a column of silica gel
packed dry and eluted with hexane-ethyl acetate (1:1 v/v) and high vacuum-dried to afford 620 mg (97
% based on the expected yield of 14-bromo-1-tetradecanol) of pure 6c as a pale yellow powder. TLC
(hexane: ethyl acetate = 7:2 v/v): Rf = 0.44;1H NMR (400 MHz, CDCl3): (ppm) 3.61 (t, J=6.6 Hz, 2H,
–CH2OH), 3.38 (t, J=6.8 Hz, 2H, –CH2Br), 1.86–1.79 (m, 2H, –CH2CH2Br), 1.57–1.50 (m, 2H, –
CH2CH2OH), 1.41–1.27 (m, 8H, –CH2–); 13C NMR (100 MHz, CDCl3): (ppm) 63.23 (C–O), 34.15,
33.02, 32.95, 29.65, 29.55, 28.93, 28.35, 25.91 (CH2).

2.3.13 Synthesis of 14-Thioacetyl-1-tetradecanol (7c)


As for 7a, 600 mg (2.05 mmol) of 6c with 280 mg (2.46 mmol) potassium thioacetate in 10 mL
DMF, was converted into a crude product that was flash chromatographed on a column of silica gel
packed dry, eluted with hexane-ethyl acetate (1:1 v/v) and high vacuum-dried to afford 425 mg (72 %
based on the expected yield of 14-thioacetyl-1-tetradecanol) of pure 7c as a white powder. TLC (hexane:
ethyl acetate = 8:2 v/v): Rf= 0.38; 1H NMR (400 MHz, CDCl3): (ppm) 3.62 (t, J=6.4, 2H, –CH2OH),
2.84 (t, J=7.4 Hz, 2H, –CH2S), 2.30 (large s, 3H; SCOCH3), 1.51 (m, 4H; CH2CH2), 1.24 (m, H); 13C
NMR (100 MHz, CDCl3):  (ppm) 196.20 (C=O), 63.32 (C–OH), 30.83, 29.77, 29.73 (overlapping
peaks), 29.65, 29. 39, 29.30, 29.02, 25.95 (CH3–C=O); HR-ESI calculated 311.2021 for C16H32O2SNa
[M+Na]+; found 311.2013.

2.3.14 Synthesis of 2, 3,4,6-O-Tetraacetate-(14 – thioacetyltetradecyl)-1-O- -D-glucopyranoside


(8c)
The above procedure for 8a was adapted to convert the mixture of 350 mg (1.21 mmol) of 7c and
550 mg (1.22 mmol) of 5 with 0.31 mL TMSOTf into 380 mg (55% in two steps, based on the expected
yield of 2, 3, 4, 6-O-tetraacetate-(14-thioacetyltetradecyl)-1-O--D-glucopyranoside) of pure 8cas white
powder. TLC (hexane: ethyl acetate = 7:3 v/v): Rf = 0.31; 1H NMR (400 MHz, CDCl3): (ppm) 5.17 (t,
J=9.6 Hz, 1H, H-3), 5.06 (t, J=9.6 Hz, 1H, H-4), 4.95 (t, J=8.8 Hz, 1H, H-2), 4.46 (d, J=8 Hz, 1H, H-1),
4.24 (dd, J=12.4, 4.8 Hz, 1H, H-6a), 4.11 (dd, J=12.2, 2.6 Hz, 1H, H-6b), 4.04–3.81 (m, 1H, –CH2O–),
3.68–3.64 (m, 1H, H-5), 3.45–3.43 (m, 1H, –CH2O–), 2.83 (t, J=7.4 Hz, 2H, –CH2S), 2.29 (s, 3H, –
SCOCH3), 2.06–1.98 (4s, 12H, –CCOCH3), 1.61–1.49 (m, –CH2’s), 1.34–1.22 (m, –CH2’s); 13C NMR
(100 MHz, CDCl3): (ppm) 196.20, 170.86, 170.50, 169.59, 169.45, 101.07, 73.15, 72.01, 71.65, 70.44,
68.81, 62.28, 30.81, 29.80, 29.71, 29.63, 29.53, 29.37, 29.32, 29.02, 26.03, 20.91, 20.79; HR-ESI
calculated 641.2972 for C30H50O11SNa [M+Na]+; found 641.2963.

2.3.15 Synthesis of 14-Mercaptotetradecyl-1-O--D-glucopyranoside (9c)


As above for 9a, 300 mg (0.48 mmol) of 7c was deacetylated with 39 mg (0.73 mmol) of NaOMe in 10
mL MeOH to afford 70 mg (35 % based on the expected yield of 14-mercaptotetradecyl-1-O--D-
glucopyranoside) of pure 9c as pale yellow waxy compound. TLC (MeOH: DCM = 1:9 v/v); Rf = 0.3;
1
H NMR (400 MHz, CD3OD) : (ppm) 4.19 (d, J=7.6 Hz, 1H, H-1), 3.85–3.79 (m, 2H, H-6a, –CH2O–
), 3.61 (dd, J=12 , 5.2 Hz, 1H, H-6b), 3.49–3.42 (m, 1H, –CH2O–), 3.26–3.24 (m, 3H, H-3, H-5, H-4),
3.10 (dd, J=15.2, 6.4 Hz, 1H, H-2), 3.128–3.074 (t, J= Hz, 2H, –CH2S– ), 2.449 – 2.414 (m, 4H),
1.639–1.495 (m, 2H); 13C NMR (100 MHz, CD3OD): (ppm) 104.53, 78.30, 78.07, 75.29, 71.83, 71.08,
62.93, 35.38, 33.22, 30.91, 30.89, 30.85, 30.81, 30.77, 30.44, 30.36, 29.56, 27.26, 25.11, 23.88; HR-ESI
calculated 431.2 for C20H40O6SNa [M+Na]+; found 431.2.

2.4 Preparation of Gold Nanoparticles (AuNPs)


AuNPs was used to investigate the binding events of the synthesized mercaptoalkyl glucosides in
situ and their subsequent interaction with GGBP using UV-vis spectroscopy.

In a typical preparation, a hydrosol containing 10 monodispersed AuNPs was prepared by


reduction of chloroaurate ions using a weak reducing agent, trisodium citrate (TSC). An aqueous
solution of 0.25 mM HAuCl4 (50 mL) was heated to boiling using reflux set-up and then added with 1.6
mL of 1% TSC. In about 5 min, the boiling solution turned faintly blue (nucleation). After a few more
minutes, the blue color gradually changed into a brilliant red, indicating the formation of AuNPs. The
resulting solution was allowed to cool down to room temperature [31].

2.5 BIAcore Experiments

2.5.1 Immobilization of mercaptoalkyl glucosides onto the gold chip


The following procedure was carried out on a BIAcore Biosensor 3000 system automatically
carrying out the steps on an autorun program. Before starting the SPR analysis, the flow system was first
cleaned by 0.5% SDS and 50 mM glycine at pH 9.5. The SIA gold chip was then mounted in the
instrument. The syringe and compartments were then filled with immobilization buffer. The buffer was
then allowed to flow into the system including all the channels on the chip. The sensogram was then
started. The flow cell, which will be used for the immobilization of the reference and the flow cells,
which will be used for the immobilization of mercaptoalkyl glucoside were then set. Each SIA gold chip
has flow cells (i.e. four channels). The reference (unbound alkanethiol) was used to subtract the possible
nonspecific binding of the GGBP.

To immobilize the reference, it was first dissolved in 20% acetonitrile and injected at the rate of
5L/min. This was done three times followed by the injection of wash solution composed of 0.05%
SDS, also three times. This is to wash the unbound compounds. The reference solution and wash
solution were injected alternately until the surface of the gold chip was saturated with the compound as
indicated by the stable response (no change in RU value).

For the immobilization of mercaptoalkyl glucoside, the same procedure was done as that of the
reference. Mercaptoalkyl glucoside was first dissolved in 20% acetonitrile. The resulting solution was
injected at the rate of 5L/min. Wash solution was injected to remove the excess compound.
Alternately, the solution of mercaptoalkyl glucoside and wash solution were injected until no change in
response was observed.

2.5.2 Measurement of binding affinity


The standard baseline buffer, phosphate buffer saline (PBS, 136.9 mMNaCl, 2.7 mMKCl, 10.2
mM Na2HPO4 and 1.8mM KH2PO4, pH 7.4 at 25C was used as running buffer. The GGBP at different
concentrations in this buffer was then allowed to flow over the immobilized reference and mercaptooctyl
glucoside. Each concentration of GGBP was injected at the rate of 30 L/min. A total volume of 150 L
was used for each run. A series of sensogram was then obtained. After the run, the chip was washed with
wash solution containing 0.05% SDS. This is to remove the GGBP from the SAM.
The KA and KD values for the interaction of GGBP with the SAMs of mercaptoalkyl glucosides
were automatically calculated by the BIAcore evaluation software fitting program.

3. Results and Discussion

3.1Synthesis

The methodology for this experiment was divided into two major parts; 1) the synthesis of the
spacers (Scheme 1) and 2) the synthesis of mercaptoalkyl glucosides (Scheme 2). The synthesis of
spacers involved two steps: synthesis of -bromo-1-alkanol and synthesis of -thioacetyl-1-alkanol.

In the synthesis of -bromo-1-alkanol, the corresponding diol was heated with aqueous HBr
(48% in water) at 84C in a continuous extraction apparatus with stirring to form -bromo-1-alkanol.
The second step is the thioacetylation of the -bromo-1-alkanol. Bromide is a good leaving group and
usually gives fast reaction [32]. In this study, each -bromo-1-alkanol was reacted with potassium
thioacetate (KSCOCH3) in dry DMF under N2 [26]. The reaction involved the nucleophilic displacement
of bromide by the thioacetyl ion affording -thioacetyl-1-alkanol. This procedure gave high conversion
of -bromo-1-alkanol to -thioacetyl-1-alkanol.

For the synthesis of mercaptoalkyl glucosides, synthesis via glycosyltrichloroacetimidate was


employed. In the glycosidation using glycosyltrichloroacetimidate as glycosyl donor, the first step was
the acetylation of glucose (protection of the hydroxyl groups). -D- Glucose was reacted with acetic
anhydride in dry pyridine to produce D-glucose pentaacetate either in - or - anomer form. The
peracetylated glucose was analyzed by 1H NMR. The anomeric proton is the only signal displayed as a
doublet in the 1H NMR spectrum. Using the Karplus equation, where protons in a conformationally
stable six-membered ring having an axial-axial relationship have a relatively large coupling constant
(approximately 10 Hz) and protons in an axial-equatorial relationship have a smaller coupling constant
(approximately 3 Hz), the configuration at carbon 1 can be assigned as either - anomer ( 6.29, J 3.7
Hz) or -anomer ( 5.64, J 8.1 Hz) [33]. Based on the obtained 1H NMR data, compound 3was mostly
- anomer ( 6.56, J= 3.3 Hz). The reaction was carried out under thermodynamic control (i.e. reaction
was allowed to react overnight).

The second step involved selective anomeric deacetylation to produce compound 4. Hydrazine
acetate in dry DMF was added into peracetylated glucose and allowed to react under N2 to afford
compound 4 mostly -anomer with the yield of 96% ( 4.71, J= 8 Hz). The reaction was carried out
under kinetic control.The third step was the formation of glycosyltrichloroacetimidate that served as
glycosyl donor. This was done by reacting 4 with trichloroacetonitrile in the presence of base catalysts
such as K2CO3, sodium hydride, DBU or Cs2CO3[34]. In this study Cs2CO3 was used because it was
readily available. Cs2CO3 mediated the deprotonation of the anomeric hydroxy group. The anomeric
oxide ion formed adds readily to the nitrile group of trichloroacetonitrile giving off O-
glycosyltrichloroacetimidate, 5[34].

Since O- glycosyltrichloroacetimidate is air sensitive [1] and that the purification of this
compound by silica gel chromatography using a mixture of ethyl acetate and hexane as solvent system
leads to extensive degradation resulting to low yield [35], O-glycosyltrichloroacetimidate was used
immediately without further purification. TLC showed only one spot with an Rf value of 0.4 (hexane:
ethyl acetate = 3:1 v/v) indicating that trichloroacetimidate was the only product formed. After the
formation of trichloroacetimidate, 5, the synthesized spacer, -thioacetyl-1-alkanol, was mixed with 5 in
dry DCM and stirred for one hour under N2. Since this synthesis required an anhydrous environment,
molecular sieves were also added.

The final step of the synthesis is the deacetylation with the use of NaOMe in HPLC grade
MeOH. The deacetylation was based on the previous work of Lin CC and colleagues [23, 24]. The final
product obtained was mostly -anomer in modest yield (41%, 31% and 35% for compounds 9a, 9b and
9c, respectively) as indicated by the presence of doublet peak at 4.21 ppm (J = 7.6 Hz), 4.18 ppm ( J =
7.6 Hz) and 4.19 ppm ( J= 7.6 Hz), respectively.

Scheme 2. Synthesis of mercaptoalkyl glucosides


3.2 Characterization of Mercaptoalkyl Glucosides

3.2.1 Characterization of Au-nanoparticles modified with 8-mercaptooctyl glucosides using uv-vis


spectroscopy

In order to investigate the binding events of the synthesized mercaptoalkyl glucosides in situ at
the surface of the 10 nm-AuNPs and their subsequent interaction with GGBP, we used uv-vis
spectroscopy. This procedure was based on the characterization of 12-mercaptododecyl- -maltoside-
modified AuNPs and its interaction with Concanavalin A done by Sato Y and colleagues [36]. First, the
absorbance of the solution of the unmodified gold nanoparticles (AuNPs) was measure. Second, into the
solution of unmodified AuNPs was added the 8-mercaptooctyl glucoside. The color of the solution and
absorbance were observed, measured and compared to that of the unmodified AuNP. The investigation
of the color change of the modified AuNP in a particular buffer is important especially when doing a
competition or inhibition assay using SPR. The solution containing the modified AuNP should have a
negligible change in color. In other words, the modified AuNP must be stable in the buffer that is to be
used. Third, into the modified AuNPs solution was added the GGBP. Likewise, the color and
absorbance of the resulting solution were observed and measured and compared to that of the
unmodified AuNP and modified AuNP without GGBP. To check that the GGBP does not bind with the
AuNPs, GGBP was added to the unmodified AuNPs. No aggregation due to the presence of AuNP-
GGBP complex should be observed as shown by the insignificant change in color of the solution
suggesting that there is no effect of the salt content of the buffer used.

Figure 1shows the uv-vis spectrum of 10-nm AuNPs. The peak absorption spectrum (max) is
518 nm as shown in Figure 1a. A colloidal solution of AuNPs generally exhibits a reddish orange color,
because such nanoparticles show an optical absorption peak caused by localized surface plasmon
resonance [13]. When 8-mercaptooctyl glucoside was added to this nanoparticle solution (total
concentration of 10 M), the color of the solution changed from orange red to pinkish red (Figure 1b).
The absorbance spectrum of colloidal AuNPs exhibited a red shift in the peak wavelength along with an
increase in the absorbance at 598 nm. This observation resulted from the change in the refractive index
on the surface of the gold caused by the modification of their surfaces by 8--D-mercaptooctyl
glucosides. The 8--D-mercaptooctyl glucosides self-assembled onto the AuNP surface due to the thiol
group interacting with the Au forming stable Au – S bonds.Figure 1c shows the spectrum of 8--D-
mercaptooctyl glucoside-AuNP after adding GGBP. The spectrum became much broader compared with
the spectrum before adding GGBP. This shows that the GGBP recognizes the D-glucose part of the
glucoside group on the modified AuNPs.
Figure 1. UV-vis spectra of unmodified and modified Figure 2. TEM of unmodified and modified AuNPs
AuNPs in PBS. (a) Unmodified AuNPs; (b) 8--D- in PBS. (a) Unmodified AuNPs; (b) 8--D-
mercaptooctyl glucoside-modified AuNPs; (c) 8--D- mercaptooctyl glucoside-modified AuNPs; (c) 8--D-
mercaptooctyl glucoside-modified AuNPs after mercaptooctyl glucoside-modified AuNPs after
addition of GGBP; (d) unmodified AuNPs with GGBP addition of GGBP

3.2.2 Characterization of Au-nanoparticles modified with 8-mercaptooctyl glucosides using TEM

For TEM analysis, one to three drops of each of the following solutions: unmodified AuNPs, 8-
mercaptooctyl glucoside-modified AuNPs, 8--D-mercaptooctyl glucoside-modified AuNPs reacted
with GGBP, were carefully placed on a copper grid surface and allowed to dry. The TEM images were
taken and compared. The TEM image of the unmodified AuNPs shows the distribution of the
nanoparticles. It also shows that the AuNPs exhibit the same size (Figure 2a). After 8--D-
mercaptooctyl glucoside modification, some of the modified particles were observed to become closer
with the neighboring particles but were still well dispersed in general (Figure 2b). Figure 2c shows the
TEM micrographs after adding GGBP in glucoside-modified AuNP solution. The particles aggregated
after the addition of the protein (Figure 2c10-nm magnification); however, the distance between the
aggregated AuNPs became larger compared to the distance between the modified AuNPs without the
protein. This shows that the interaction between GGBP and carbohydrate moiety is dominant compared
to the intermolecular interactions between carbohydrate hydrocarbons.
3.2.3 SPR Analysis

The result of the SPR analysis of the interaction of GGBP to the SAM of 8--D-mercaptooctyl
glucoside can be seen from the sensograms (Figure 3). The optimized titration curve was obtained from
5M to 30 M of GGBP. Same set of concentrations was used for the reference.

Figure 3. Binding of GGBP to SAMs in PBS at 7.4; (a) 8-octanethiol as reference, and (b) 8--D-
mercaptooctyl glucoside

When GGBP solution was added to the mercaptooctyl SAM, a clear association and dissociation
phase was observed in the sensogram. Similar sensogram was obtained using the reference 1-octanethiol
SAM (Figure 3a). As can be seen from Figure 3b, the sensogram signal (for 5M, 10M and 30M
GGBP) increased rapidly at the beginning of the injection and then continued to rise at a slower rate
which does not reach a plateau phase within the injection time. After 5-minute injection time (300s), the
SAM was washed with PBS buffer to remove the protein. The sensogram signal started to decrease as
shown from the above figure. To completely remove the protein, 0.5 % SDS solution was used.

The fitting of the GGBP-8--D-mercaptooctyl glucoside SAM binding data was tested to one-
site binding model included in the evaluation software of the BIAcore instrument (Figure 4). Before the
data fitting, the reference curves were subtracted from the glucoside curves [8]. The use of reference to
subtract the nonspecific binding was based on the work of Lienemann and colleagues, in which they
used 11-mercaptoundecanol as reference for the study of wheat germ agglutinin binding to SAM of N-
acetyl-D-glucosamine (GlcNac) [8].
Figure 4. SPR data fitting of GGBP binding to 8--D-mercaptooctyl glucoside SAMin PBS at pH 7.4

GGBP conc (M) Rmax(RU) Req(RU) ka(1/M-s) Kd (1/s) KA (1/M) KD (M)


5 126 283 x 10-3
10 255 566 x 10-3 566 4.85 x 10-8 1.17 x 1010 8.57 x 10-11
30 804 0.017
Table 1. Kinetic parameters of the binding of GGBP to the SAM of 8--D-mercaptooctyl glucoside in PBS
at pH 7.4

The kinetic parameters namely, association rate constant (ka), dissociation rate constant (kd),
equilibrium association constant (KA), equilibrium dissociation constant (KD) and maximum responses
(Rmax) of the binding of GGBP to the 8--D-mercaptooctyl glucoside SAM were calculated from least-
square fits of a Langmuir binding model to equilibrium responses (Req) recorded at different GGBP
concentrations as shown in Figures 4 . The violet, red and blue curves correspond to the sensograms of
the binding of 5M, 10M and 30M GGBP, respectively to the SAMs of 8-mercaptooctyl glucosides.
It can be seen from the figure the in all of sensograms, the response increased at the beginning of the
injection and then continued to rise at a slower rate.

The high KAvalue (typical range of 105–1012[49]), indicates that the GGBP and the SAM of
mercaptooctyl glucosides has high affinity to associate. Its low KDvalue (typical range of 10-5 – 10-12
[37]), indicates the high stability of the complex as shown in Table 1.

4. Conclusion

The three glucosides, 8--D-mercaptooctyl glucoside, 12--D-mercaptododecyl glucoside and


14--D-mercaptotetradecyl glucoside were successfully synthesized in relatively good yield via the
trichloroacetimidate method. Of these compounds, 8--D-mercaptooctyl glucoside was evaluated as a
potential bioassay platform to study carbohydrate-protein interaction. Self-assembled monolayer (SAM)
of 8--D-mercaptooctyl glucoside on the gold chip was achieved and was used as platform assay to
study the interaction of glucose/galactose binding protein (GGBP) with the glucose moiety of the
glucosides using SPR. The affinity parameters obtained in PBS (pH 7.4), were KA: 1.17x1010 M-1; KD:
8.57x 10-11 M, clearly demonstrating strong binding. Furthermore, 8--D-mercaptooctyl glucoside was
characterized in situ on 10-nm gold nanoparticles using uv-vis and TEM demonstrating its ability to
form stable 8--D-mercaptooctyl glucoside-AuNP in PBS.

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