N-(3-Triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC): A heterobifunctional reagent for immobilization of biomolecules on glass surface

Article abstract of DOI:10.1016/j.bmcl.2008.08.075

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide (TPICC) has been developed by a simple 'two step reaction' for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides and proteins on the glass surface. The isothiocyanate functionality at one end of the reagent, TPICC was found specific for the ligands having either aminoalkyl (RNH₂) or mercaptoalkyl (R-SH) functionality. The synthesis of bioconjugates with the reagent was achieved through its isothiocyanate functionality at one end via the formation of stable thiourea linkage with aminoalkyl and dithiocarbamate linkage with mercaptoalkyl derivatives. The triethoxysilyl functionality of the reagent has been utilized for specific coupling with the virgin glass surface by a very simple methodology.

Full text of DOI:10.1016/j.bmcl.2008.08.075

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5217–5221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-(3-Triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-
carboxamide (TPICC): A heterobifunctional reagent for immobilization
of biomolecules on glass surface
*
Arvind Misra , Mohammad Shahid, Pratibha Dwivedi
Nucleic Acids Research Laboratory, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2008
Revised 2 August 2008
Accepted 21 August 2008
Available online 26 August 2008
An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohex-
ane-1-carboxamide (TPICC) has been developed by a simple ‘two step reaction’ for the preparation of bio-
conjugates and immobilization of biomolecules such as oligonucleotides, peptides and proteins on the
glass surface. The isothiocyanate functionality at one end of the reagent, TPICC was found specific for
the ligands having either aminoalkyl (RNH2) or me]rcaptoalkyl (R-SH) functionality. The synthesis of bio-
conjugates with the reagent was achieved through its isothiocyanate functionality at one end via the for-
mation of stable thiourea linkage with aminoalkyl and dithiocarbamate linkage with mercaptoalkyl
derivatives. The triethoxysilyl functionality of the reagent has been utilized for specific coupling with
the virgin glass surface by a very simple methodology.
Keywords:
Microarrays
Bioconjugates
Fluorescence
Ó 2008 Elsevier Ltd. All rights reserved.
Nucleic acids based detection and quantification methods play
an important role in the medical diagnostics and drug discovery.
The development of reliable, fast and inexpensive detection meth-
ods is important. The measurement of nucleic acid hybridization
under heterogeneous condition provides a variety of advantages
such convenience, real time and accurate quantification for diverse
applications. Recently, microarray technology (biochip) due to its
high flexibility and versatility has emerged as an indispensable
promising tool for gene discovery1, genome analysis,2 medical
diagnostics for genetic diseases,3 detection of single nucleotide
polymorphism,4 nucleic acid–ligand interaction,5 DNA sequencing
by hybridization6 and DNA computing.7
Two well established methodologies are available for construc-
tion of microarrays. The first methodology8 involves the direct on
surface synthesis (in situ) whereas the second9 one, popularly
known as ‘deposition method’ involves the immobilization of pre-
fabricated modified biomolecules with either covalent10–13 or non-
covalent14 bond formation on the surface. The later method utilizes
‘spotting technique’ and is more popular to construct low to med-
ium density microarrays on account of added advantages such as
affordability and flexibility. The microarrays constructed through
deposition method is also useful in the fact that modified/unmod-
ified biomolecules prepared chemically or enzymatically can be
fully purified and characterized prior to immobilization by spotting
method.
The construction of quality microarrays requires a suitable
chemical method for functionalization of the surface to facilitate
an efficient and unperturbed immobilization of biomolecules with
a well defined stable and specific architecture. Heterobifunctional
reagents which contain two different reactive functional groups at
either ends have been extensively used as a cross-linking reagents
and have also gained importance for surface immobilization and
pattering of biomolecules on various kind of solid supports.15–23
Recently, glass/silicon surfaces especially, glass microslide has been
used extensively in microarray technology for construction of oligo-
nucleotide, peptide, protein and other biomolecules microarrays.
The application of microslides is attributable to ease of modification
by silane chemistry, low intrinsic fluorescence, homogeneous sur-
face, resistance to heat, favorable optical properties for sensitive
fluorescence imaging and cost effectiveness.
The morphology of the constructed quality microarrays basi-
cally depends upon the nature of surfaces selected as well as on
employed chemical methods. Generally, solid surface is first, acti-
vated through a chemical method using suitable reagents/activa-
tors, to make surface functional/reactive corresponding to the
respective modified biomolecules. The functional groups on the
surface generated can be of either electrophilic nature such as
aldehyde, epoxy, isocyanate, isothiocyanate, N-hydroxy-
succinimide ester (NHS) or of nucleophilic nature such as semi-
carbazide and amino.22–24 Electrophilic nature of surfaces are
found highly reactive to amino, thiol and hydroxyl reactive func-
tional groups and can be used potentially for efficient covalent
immobilization of different classes of compounds such as oligonu-
cleotides, peptides and proteins under milder and ambient reaction
* Corresponding author. Tel.: +91 0542 2307321x104; fax: +91 0542 2368127/
2368175.
E-mail address: arvindmisra2003@yahoo.com (A. Misra).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.075

5218
A. Misra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5217–5221
conditions. A large number of combinations, including different
functional group containing surfaces and biomolecules have been
in practice and most of them popularly include maleimide-
thiol,25,26 alkyne-azide,27 hydrazide-aldehyde,28 aminooxy-alde-
hyde,20 epoxy-glycol,23,29 gold-thiol, isothiocyanate-amino,22 etc.
Recently, a trifluoroethanesulphonate ester approach, using hete-
robifunctional reagent N-(2-trifluoroethanesulfonatoethyl)-N-tri-
ethoxysilylpropyl-3-amine (NTMTA) has been reported30 to
immobilize and construct microarrays of amino and mercaptoalkyl
derivative of biomolecules (e.g., oligonucleotides). Consequently,
the design and development of an alternative chemical approach
for immobilization of aminoalkyl/mercaptoalkyl modified biomol-
ecules on the solid surface, such as glass/silicon is significantly
important and still elusive in the area of microarray technology.
Here, we describe the design and synthesis of a new heterobi-
functional reagent N-(3-triethoxysilylpropyl)-4-(isothiocyanatom-
and dried to get NHS ester of trans-4-(aminomethyl)cyclohexane
carboxylic acid hydrochloride in 71% yield. Finally, the NHS ester
of trans-4-(aminomethyl)cyclohexane carboxylic acid hydrochlo-
ride was taken in anhydrous DMF (10 ml) and kept in an ice-bath,
filtered and to the filtrate, 3-aminopropyltriethoxy silane (APTS)
(1.6 mmol) and triethylamine (TEA) (1.1 mmol) were added drop-
wise with continuous stirring for 1 h. The reaction mixture was al-
lowed to come at room temperature and left for overnight stirring.
After the completion of reaction, (monitored on TLC) solvents were
evaporated in vacuum. The crude product was taken in hexane/
petroleum ether and washed comprehensively (3 times) with 3%
NaHCO3 solution (25 ml), containing triethylamine (TEA). The or-
ganic layer was pooled and evaporated to dryness to afford N-(3-
triethoxysilylpropyl)-4-(aminomethyl) cyclohexane-1-carboxam-
ide, 2 in 75% yield. The hydrochloride salt of unreacted amino acid,
N-hydroxysuccinimide and triethylamine hydrochloride were re-
moved in washing step and subsequently amino functionality be-
come free for further reaction.
Compound 2 (1 equiv) and TEA (4 equiv) were taken in THF
(10 ml), cooled to 0 °C and CS2 (1.5 equiv) was added dropwise
(2 h) with continuous stirring. After complete addition, reaction
mixture was allowed to come at room temperature and tosyl chlo-
ride (1.5 equiv) was added and stirred the reaction mixture further
for 20–30 min to generate isothiocyanate functionality. The sol-
vents were evaporated in vacuum and to the crude product acidic
water (20 ml) was added. The desired product was extracted with
diethylether (3ꢀ 20 ml). The combined organic layer was dried
over anhydrous sodium sulphate, filtered and concentrated in vac-
uum. The desired reagent TPICC 3 was eluted with hexane by pass-
ing over short silica column as a semisolid mass in 82% yield.32
The proposed reagent was synthesized keeping in mind the
objective of a versatile heterobifunctional reagent, specific for ami-
ethyl)cyclohexane-1-carboxamide
(TPICC)
with
some
modification in our previously reported thiol specific reagent31 to
generate isothiocyanate functionality. The applicability of the re-
agent on the glass beads and microslides has been tested for the
immobilization of aminoalkyl/mercaptoalkyl functional group con-
taining ligands and biomolecules such as oligonucleotides and
smaller peptides.
The reagent was synthesized at room temperature by stirring
suspension of trans-4-(aminomethyl)cyclohexane carboxylic acid
hydrochloride, 1 (1.5 mmol) in THF (15 ml), N-hydroxysuccinimide
(NHS) (2.0 mmol) and dicylohexylcarbodiimide (DCC) (1.2 mmol)
for 2 h under argon atmosphere (Scheme 1). After the completion
of reaction (monitored on TLC), the reaction mixture was kept at
0 °C for 1 h to precipitate out dicyclohexylurea as a byproduct
and concentrated in vacuum to leave a solid which was redis-
solved, filtered (three times to remove further dicyclohexylurea)
HOOC
N
S
R
C
NH2.HCl
(1)
L
NH2
L
SH
(i)
H
N
H
N
H
N
O
S
L
R
R
L
EtO
EtO Si
EtO
N
H
S
S
NH2
Thiourea
Dithiocarbamate
Approach
(2)
(ii)
Approach
O
EtO
EtO Si
EtO
C
N
N
H
S
(3)
(Glass)
PATH-2
L
X
PATH-1
O
O
S
OH OEt
Si O Si
OH OEt
EtO
EtO Si
EtO
C
NH
N
S
N
H
N
H
Z
L
(5)
(4)
(Glass)
L
X
O
NH
S
OH OEt
Si O Si
OH OEt
N
H
Z
L
(6)
L = Ligands, peptides, oligonucleotides; X = -SH, -NH2, Z = S, N.
Scheme 1. Synthesis of heterobifunctional reagent TPICC. Reagents and conditions: (i) N-hydroxysuccinimide (NHS)/3-Aminopropyltriethoxysilane (APTS)/N,N’-
dicyclohexylcarbodiimide (DCC)/dimethylformamide (DMF); (ii) CS2/TEA/Tosyl chloride/0 °C/tetrahydrofuron (THF).

A. Misra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5217–5221
5219
noalkyl/mercaptoalkyl ligands and biomolecules through
a
straightforward simple, cost effective and short chemical method-
ology. The stable isothiocyanate functionality of the reagent, TPICC
is expected to form stable thiourea/dithiocarbamate type of link-
ages with amino/thiol derivatives in aqueous and organic medium
at a suitable pH range 6.5–8.5. While on the other hand the pres-
ence of triethoxysilane group further, makes the reagent specific
for glass/silicon surfaces through the formation of stable silyl-ethe-
ral (–Si-O-Si–) linkages.
The time kinetics for the formation of conjugates in between
heterobifunctional reagent, TPICC and aminoalkyl/mercaptoalkyl
ligands was carried out alternatively as reported previously.31 In
first alternative (PATH-1), reagent TPICC was treated with glass
beads CPG 500 Å through its triethoxysilyl functionality and then
covalently conjugated separately with ligands, viz., DMTr-(CH2)6-
NH2 and DMTr-(CH2)6-SH in dimethylformamide (DMF) and 1.5%
TEA. In the second alternative (PATH-2), first reagent TPICC was
conjugated with both the ligands through their amino and thiol
functionality to form reagent–ligand conjugates or bioconjugates
with the formation of stable thiourea and dithiocarbamate link-
ages, respectively. The reagent–ligand conjugates so formed were
subsequently attached on to unmodified glass beads, CPG 500 Å
surface through its triethoxysilyl function. The progress of the
reaction was monitored by withdrawing the reaction vials after a
regular time intervals and after proper washing and drying steps,
weighed amount of support (1–2 mg) was treated with 3% trichlo-
roacetic acid (TCA) in 1,2-dichloroethane for 5 min.33 The loading
on support (Table 1) was estimated by spectrophotometric deter-
mination of DMTr cation released at 505 nm wavelength. A graph
Figure 1. Time kinetics for aminoalkyl (1) and mercaptoalkyl (2) ligands, respec-
tively, to know optimum time requirement for immobilize biomolecules on the
glass surface (CPG 500Å), via both the alternating paths.
suitable for immobilization process on the glass support and can
be potentially useful for the immobilization of biomolecules on
the glass/silicon surfaces.
In order to test the potentiality of the newly developed hetero-
bifunctional reagent TPICC, derivatives of tripeptides having free
amino and thiol ends were prepared by coupling with the ligand
O-4,40-dimethoxytrityl-5-aminopentan-1-ol (DTAP) to bring dime-
thoxytrityl group at the other end.34 Tritylated (DMTr) smaller
peptides, dissolved in 1 M sodium borate buffer (pH 8.5) were sub-
jected to reaction conditions of PATH-1 and also for same time per-
iod to complete the immobilization process on glass beads. After
completion of reaction DMTr group was removed by treating glass
beads with 3% TCA in 1,2-dichloroethane and as mentioned above
loading was estimated in good yields (Table 1).
To validate further the current methodology of immobilization,
unmodified microslides were charged with heterobifunctional re-
agent TPICC (0.3 M in DMF) at 45 °C in a humid chamber for 1 h
with continues shaking. Washing of the microslides were carried
out in DMF followed by diethyl ether (15 ml, 3 times) and dried un-
der anhydrous condition to get isothiocyanate functionality on the
glass surface. 50-Aminoalkyl and mercaptoalkyl-oligonucleotides,
viz., H2N-(CH2)6OPO3-d(AAA AAA AAA AAA AAA) and HS-(CH2)6O-
was plotted between time (min) and loading (lmol/g) on the glass
beads support to estimate the extent of reaction and optimum time
requirement for complete chemical reaction of immobilization. For
the covalent bond formation between support and reagent TPICC,
or in between glass support and reagent–ligand conjugates the
reaction was allowed to proceed at 45 °C in an Eppendorf Ther-
momixer for 45–60 min.
From Figure 1 we observed that immobilization of merca-
ptoalkyl ligand on the support reached to saturation within
60 min with the formation of dithiocarbamate linkage while ami-
noalkyl ligand has taken much longer time (120 min), to complete
the reaction through the formation of thiourea linkage. These opti-
mum time periods were further utilized for the immobilization of
other biomolecules on the glass surface. The immobilization pro-
cess was found faster through dithiocarbamate approach however,
the extent of loading on the support (Table 1) was found greater in
case of thiourea approach. Further, as evident from kinetic data,
although the alternative paths are showing promising results how-
ever, comparatively, first alternative (PATH-1) in which reagent
TPICC was covalently attached with glass support, prior to immo-
bilization of aminoalkyl/mercaptoalkyl ligand, has been found
more favorable and feasible with greater extent of immobilization
of biomolecules. Thus, the reagent TPICC developed was found
PO3-d(AAA AAA AAA AAA AAA) (0.20 A260 unit) (100
in 0.1 M sodium phosphate buffer containing 1.5% TEA (pH 8.5),
respectively, were spotted manually26 (0.5
L) with the help of
lL), dissolved
l
pipette on the isothiocyanate activated microslides. The reaction
was allowed to proceed for respective optimum time periods. After
completion of reaction the microslides were washed and treated
with capping buffer solution containing ethanolamine (1%), to neu-
tralize the activated slides for further reaction. After proper wash-
ing and drying, immobilized microslide were hybridized with
complementary fluorescent oligonucleotide probe strand ODN1,
Table 1
Immobilization of ligand and biomolecules using heterobifunctional reagent, TPICC
S.No.
Ligand
S-R + Ligand
R-L + Support
1.
2.
3.
4.
5.
SH-(CH2)6-ODMTr
H2N-(CH2)6-ODMTr
H2N-(LysꢁGlyꢁbAla)-(CH2)5-ODMTr
HS-(CysꢁGlyꢁbAla)-(CH2)5-ODMTr
H2N-(CH2)6OPO3-d(AAA AAAAAA AAA AAA)
33.9
38.9
24.5
19.3
l
l
l
l
M/g of support
M/g of support
M/g of support
M/g of support
29.9
32.9
22.9
15.4
l
l
l
l
M/g of support
M/g of support
M/g of support
M/g of support
Hybridization with
Hybridization with
Fluorescent probe ODN1
Hybridization with
Fluorescent probe ODN1
Hybridization with
6.
HS-(CH2)6OPO3-d(AAA AAAAAA AAA AAA)
Fluorescent probe ODN1
Fluorescent probe ODN1
S, glass support (CPG 500Å and microslides); R, heterobifunctional reagent (TPICC). Values reported are within range of 2% percentage error.

5220
A. Misra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5217–5221
viz., FAM-(CH2)3-OPO3-d(TTT TTT TTT TTT TTT). ODN1 (40
l
l, 0.25
fluorescent oligonucleotide ODN1. After thorough washing with
phosphate buffer microslides were dried and visualized. No mea-
surable amount of fluorescence signal was obtained in case of
unmodified immobilized oligonucleotide duplexes (1, microslide
C and D) thereby, confirming the specificity of current methodol-
ogy for isothiocyanate and amino/mercaptoalkyl derivatives.
Conclusively, an efficient heterobifunctional reagent has been
developed for the immobilization of aminoalkyl/mercaptoalkyl li-
gands and biomolecules on a glass/silicon surfaces through a stable
thiourea/dithiocarbamate linkages. The further application of the
reagent, its stability, suitability for construction of good quality
of arrays of biomolecules and specificity of detection is under con-
sideration and will be presented in further communications.
A260 unit), dissolved in 0.1 M phosphate buffer containing 1.0 M
NaCl (pH 7.5) was charged on the spotted area and kept for 2 h
at 45 °C in a hybridization chamber and then at room temperature
overnight to facilitate complete hybridization between the com-
plementary oligonucleotide strands. After thorough washing with
phosphate buffer (15 ml, 3 times) the slides were dried and fluo-
rescence intensity of the duplexes on the microslides were visual-
ized under fluorescence microscopes (Fig. 2).
The minimum concentration of oligonucleotide required for the
immobilization on the glass surface was determined by reacting a
isothiocyanate-activated glass microslide (PATH-1) with a fluores-
cein labeled oligonucleotide strand d(AAA AAA AAA AAA AAA
AAA)-OPO3-(CH2)6SH dissolved in phosphate buffer, in four differ-
ent concentration (2.5, 5, 10, and 15
buffer and distilled water (15 ml) fluorescence intensity was mea-
sured. It has been observed that the spot of 10 M concentration
was sufficient for easy visualization (figure not given) and hence
was selected for immobilization of oligonucleotides and their
hybridization study.
lM) and after washing with
Acknowledgments
l
The financial support from DST, New Delhi, is gratefully
acknowledged. Authors also acknowledge the help of Dr. S. Kumar
for scanning microslides and RSIC, Lucknow, for spectral data.
To demonstrate the specificity (Fig. 3) of the current methodol-
ogy using newly developed heterobifunctional reagent, unmodified
oligonucleotides strand d(AAA AAA AAA AAA AAA) and 50-amino/
mercaptoalkyl-oligonucleotides, viz., H2N-(CH2)6OPO3-d(AAA AAA
AAA AAA AAA) and HS-(CH2)6OPO3-d(AAA AAA AAA AAA AAA)
References and notes
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Figure 2. Fluorescence images of immobilized oligonucleotides, H2N-(CH2)6OPO3-
d(AAA AAA AAA AAA AAA) (in microslide A) and HS-(CH2)6OPO3-d(AAA AAA AAA
AAA AAA) (in microslide B) (spotted in 0.5 lL, 10 lM) (PATH-1). Hybridization of
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Figure 3. Fluorescence images showing the specificity of immobilization: 1,
unmodified oligonucleotide d(AAA AAA AAA AAA AAA) (in both microslide C and
D); 2, amino modified oligonucleotide H2N-(CH2)6OPO3-d(AAA AAA AAA AAA
AAA) (in microslide C) and HS-(CH2)6OPO3-d(AAA AAA AAA AAA AAA) (in
31. Misra, A. Bioorg. Med. Chem. Lett. 2007, 17, 3749.
32. Characterization of 2: 1H NMR (DMSO-d6) (d ppm): 1.31–3.89 (m, 15H,
–CH2CH3), 1.2 (t, 2H, –CH2), 1.78 (m, 2H, –CH2), 3.33 (m, 2H, –CH2NH–), 8.1 (d,
1H, J = 6.9 Hz, –NHCO–), 1.3–1.62 (m, 8H, cyclohexane), 2.15 (m, 1H), 1.79
(m,1H), 2.65 (m, 2H, CH2NH2). MALDI-TOF: [M+H]+ 360.54 (calc.), 362.1
microslide D) (spotted in 0.5
lL, 10
lM) (PATH-1). Hybridization of both micro-
slides with fluorescent oligonucleotide probe, ODN1 (40
lL, 0.25 A260 unit).

A. Misra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5217–5221
5221
(found). Characterization of 3: 1H NMR (DMSO-d6) (d ppm): 1.35–3.91 (m, 15H,
–CH2CH3), 1.2 (t, 2H, –CH2), 1.8 (m, 2H, –CH2), 3.31 (m, 2H, –CH2NH–), 8.21 (d,
1H, J = 6.7 Hz, –NHCO–), 1.35–1.61 (m, 8H, cyclohexane), 2.12 (m, 1H). MALDI-
TOF: [M+H]+ 402.45 (calc.), 403.4 (found).
J2 = 7.12 Hz, –CH2CONH), 2.85 (t, 2H, J = 6.7 Hz, –CH2NH2), 3.25 (t, 2H,
J = 7.1 Hz, –CH2NH), 3.57 (t, –CH2O–), 3.38 (t, 2H, –CH2), 3.66 (t, 1H,
J = 8.15 Hz, –CHNH2), 3.83 (s, 6H, –OCH3), 3.97 (br, 4H, NH2), 4.19 (s, 2H,
–CH2), 7.18 (m, 13H, DMTr), 7.67 (br, 3H, –NH). Characterization of ligand 4: 1
H
33. Gait, M. J. Oligonucleotide Synthesis: A Practical Approach; IRL Press: Oxford,
1984.
NMR (DMSO-d6) (d ppm): 1.18 (m, 2H, –CH2), 1.49 (m, 2H, –CH2), 1.65 (m, 2H,
–CH2), 2.21 (s, 3H, –COCH3), 2.64 (t, 2H, –CH2), 3.26 (d, 2H, –CH2SH), 3.1 (t, 2H,
–CH2), 3.47 (t, 2H, –CH2O–), 3.41 (t, 2H, CH2), 4.19 (s, 2H, CH2), 3.63 (s, 6H,
OCH3), 4.89 (t, 1H, CHNH), 7.28 (m, 13H, DMTr), 7.57 (br, 4H, –NH).
34. Characterization of ligand 3: 1H NMR (DMSO-d6) (d ppm): 1.19 (m, 4H, –CH2–),
1.35 (m, 4H, –NHCH2), 1.41 (m, 2H, –CH2), 1.57 (m, 2H, –CH2), 2.47 (t, 2H, J1,