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Carbohydrate-Protein Interaction
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.
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.
2. Experimental
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).
To immobilize the reference, it was first dissolved in 20% acetonitrile and injected at the rate of
5L/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 5L/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.
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 84C 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.
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.
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
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
5M 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 5M, 10M and 30M
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
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 5M, 10M and 30M 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
References
[1] Lindhorst TKL, Artificial Multivalent Sugar Ligands to Understand and Manipulate Carbohydrate-
Protein Interactions, Topics in Current Chemistry, 2002, 218, 201-235.
[2] Halcomb RL and Wong CH, Synthesis of oligosaccharides, glycopeptides and glycolipids, Current
Opinion in Structural Biology, 1993, 3, 694-700.
[3] Lim YB and Lee M, Self-assembled multivalent carbohydrate ligands, Organic & Biomolecular
Chemistry, 2007, 5, 401-405.
[4] Paulson JC, Blixt O and Collins BE, Sweet spots in functional glycomics, Nature Chemical Biology,
2006, 2(5), 238-248.
[5] Porcelli SA, Cutting glycolipids to size, Nature Immunology, 2001, 2(3), 191-192.
[6] Larsen K, Thygesen MB, Guillaumie F, Willats WGT and Jensen KJ, Solid-phase tools for
glycobiology, Carbohydrate Research, 2006, 341, 1209-1234.
[7] Schmidt M, Chatterjee SK, Dobner B and Nuhn DP, New modified single chained glycolipids. Part
1: Synthesis of deoxy and partially O-methylated glycolipids with or without a sulphur containing
spacer, Chemistry and Physics of Lipids, 2002, 114, 139-147.
[8] Lienemann M, Paanane A, Boer H, de la Fuente JM, Garcia I, Penades S and Koivula A.
Characterization of the wheat germ agglutinin binding to self- assembled monolayers of
neoglycoconjugates by AFM and SPR, Glycobiology, 2009, 19 (6), 633-643.
[9] Fukuda T, Onogi S and Miura Y, Dendritic sugar-microarrays by click chemistry, Thin Solid Films,
2009, 518, 880-888.
[11] Kadalbajoo M, Park J, Opdhal A, Suda H, Kitchens CA, Garno JC, Batteas JD, Tarlov MJ and
DeShong P, Synthesis and Structural Characterization of Glucopyranosylamide Films on Gold,
Langmuir, 2007, 23, 700-707.
[12] Zhang Y, Luo S, Tang Y, Yu L, Hou KY, Cheng JP, Zeng X and Wang PG, Carbohydrate-Protein
Interactions by “Clicked” Carbohydrate Self-Assembled Monolayers, Analytical Chemistry, 2006, 76,
2001-2008.
[13] Guo C, Boullanger P, Jiang L and Tiu T, Highly sensitive gold nanoparticles biosensor chips
modified with a self-assembled bilayer for detection of Con A, Biosensors and Bioelectronics, 2007, 22,
1830-1834.
[14] Song EH and Pohl NLB, Carbohydrate arrays: recent developments in fabrication and detection
methods with applications, Current Opinion in Chemical Biology, 2009, 13, 626-632
[15] Munoz JF, Andre S, Gabius HJ, Sinisterra JV, Hernaiz MJ and Linhardt, Green glycosylation using
ionic liquid to prepare alkyl glycosides for studying carbohydrate-protein interactions by SPR, Green
Chemistry, 2009, 11, 373-379.
[16] Kamiya S, Minamikawa H, Jung JW, Yang B, Masuda M and Shimizu T, Molecular Structure of
Glucopyranosylamide Lipid and Nanotube Morphology, Langmuir, 2005, 21, 743-750.
[17] Besenicar M, Macek P, Lakey JH and Anderluh, Surface plasmon resonance in protein-membrane
interactions, Chemistry and Physics of Lipids, 2006, 141, 169-178.
[18] Jungar C and Mandenius CF, Neoglycoconjugates as a ffinity ligands in surface plasmon resonance
analysis, Analytica Chimica Acta, 2001, 449, 51-58.
[19] Linman MJ, Taylor JD, Yu H, Chen X and Cheng, Q, Surface Plasmon Resonance Study of
Protein-Carbohydrate Interactions Using Biotinylated Sialosides, Analytical Chemistry, 2008, 80, 4007-
4013.
[20] Ratner DM, Adams EW, Su J, O’Keefe BR, Mrksich M and Seeberger PH, Probing Protein-
Carbohydrate Interactions with Microarrays of Synthetic Oligosaccharides, Chemistry & Biochemistry,
2004, 5, 379-383.
[21] Deniaud D, Julienne K and Gouin SG, Insights in the rational design of synthetic multivalent
glycoconjugates as lectin ligands, Organic and Biomolecular Chemistry, 2011, 9, 966-979.
[22] Ojeda R, de Paz JL, Barrientos AG, Martin-Lomas M and Penades S, Preparation of
multifunctional glyconanoparticles as a platform for potential carbohydrate-based anticancer vaccines,
Carbohydrate Research, 2007, 342, 448-459.
[23] Lin CC, Yeh YC, Yang CY, Cheng GF, Chen YC, Wu YC and Chen CC, Quantitative analysis of
multivalent interactions of carbohydrate-encapsulated gold nanoparticles with Concanvalin A,
Chemistry Communication, 2003, 2920-2921.
[24] Lin CC, Yeh YC, Yang CY, Chen CL, Chen GF, Chen CC and Wu YC, Selective Binding of
Mannose Encapsulated Gold Nanoparticles to Type 1 Pili in Escherichia coli, Journal of American
Chemical Society, 2002, 124, 3508-3509.
[25] Liang, CH, Wang CC, Lin YC, Chen CH, Wong CH and Wu CY, Iron Oxide/Gold Core/Shell
Nanoparticles for Ultrasensitive Detection of Carbohydrate-Protein Interactions, Analytical Chemistry,
2009, 81, 7750-7756.
[26] Barrientos AG, de la Fuente JM, Rojas TC, Fernandez A and Penades S, Gold Glyconanoparticles :
Synthetic Polyvalent Ligands Mimicking Glycocalyx-Like Surfaces as Tools for Glycobiological
Studies, Chemistry European Journal, 2003, 9,
1909-1921.
[27] Thomas GB, Rader LH, Park J, Abezgauz L, Danino Dganit, DeShong P and English DS,
Carbohydrate Modified Cationic Vesicles: Probing Multivalent Binding at the Bilayer Interface, Journal
of American Chemical Society, 2009, 131, 5471-5477.
[28] Love JC, Estroff LA, Kriebel JK, Nuzzo RG, and Whitesides GM, Self-Assembled Monolayers of
Thiolates on Metals as a Form of Nanotechnology, Chemical Reviews ,2005, 105 (4), 1103−1169.
[29] Delamarche, E and Michel B, Structure and stability of self-assembled monolayers, Thin Solid
Films, 1996, 273, 54-60.
[30] Hu ZG. Prunici P, Patzner P ,and Hess P, Infrared Spectroscopic Ellipsometry of n-Alkylthiol (C5-
C18) Self-Assembled Monolayers on Gold, Journal of Physical Chemistry B, 2006, 110, 14824-14831.
[31] Obliosca JM, Wang PC and Tseng FG, Probing quenched dye fluorescence of Cy3-DNA-Au-
nanoparticle hybrid conjugates using solution and array platforms, Journal of Colloid and Interface
Science, 2012, 371, 34-41.
[33] Batchelor R, Northcote PT, Harvey JE, Dangerfield EM and Stocker BL, Stereochemical Control in
Carbohydrate Chemistry, Journal of Chemical Education, 2008, 85 (5), 689-691.
[35] Cheng A, Hendel JL, Colangelo K, Bonin M and Auzanneau FI, Convenient Temporary Methyl
Imidate Protection of N-Acetylglucosamine and Glycosylation at O-4, Journal of Organic Chemistry,
2008, 73, 7574-7579.
[37] Schasfoort RBM and Tudos AJ, Handbook of Surface Plasmon Resonance, 2008, Royal Society of
Chemistry.