Facile synthetic method of alkanethiol spacer for biointerface

Article abstract of DOI:10.1055/s-0032-1317728

An alkanethiol spacer which is suitable for biointerface was synthesized on 2-chlorotritylchloride (CTC) resin using solid-phase synthesis method. The triethylene glycol (TEG) unit containing building block was immobilized on CTC resin followed by a coupling alkanethiol building block. After dithiothreitol (DTT) treatment and cleavage of the resulting alkanethiol spacer from the resin, the spacer was obtained in a pure form without any purification steps. The TEG-alkanethiol spacer was applied for the DNA hybridization assay on a gold surface, and proved to work well when compared to the commercial spacer. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0032-1317728

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LETTER
letter
Facile Synthetic Method of Alkanethiol Spacer for Biointerface
Facile Synthetic Method of Alkanethiol Spacer
Hong-Jun Cho,a Sung Jun Park,a Jung Won Kim,b Sang-Myung Lee,*c Yoon-Sik Lee*a
a
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea
Fax +82(2)8801604; E-mail: yslee@snu.ac.kr
b
Department of Chemical Engineering, Kangwon National University, Kangwon-Do 245-711, Republic of Korea
c
Department of Chemical Engineering, Kangwon National University, Kangwon-Do 200-701, Republic of Korea
Fax +82(33)2513658; E-mail: sangmyung@kangwon.ac.kr
Received: 11.09.2012; Accepted after revision: 12.11.2012
been made to reduce the reaction steps or to propose effi-
cient methodology for the synthesis of alkanethiols,8,11,12
some limitations still exist including both complicated pu-
rification steps and low yields.
Abstract: An alkanethiol spacer which is suitable for biointerface
was synthesized on 2-chlorotritylchloride (CTC) resin using solid-
phase synthesis method. The triethylene glycol (TEG) unit contain-
ing building block was immobilized on CTC resin followed by a
coupling alkanethiol building block. After dithiothreitol (DTT)
treatment and cleavage of the resulting alkanethiol spacer from the
resin, the spacer was obtained in a pure form without any purifica-
tion steps. The TEG-alkanethiol spacer was applied for the DNA
hybridization assay on a gold surface, and proved to work well
when compared to the commercial spacer.
Here, we report a novel synthetic route for a TEG-alkane-
thiol spacer by solid-phase organic synthesis (SPOS)
method utilizing building blocks. Our method provides a
simple synthetic process, easy purification, as well as both
high yield and purity. Using the SAMs achieved on the
gold surface with the TEG-alkanethiol spacers, a DNA
hybridization assay was performed to evaluate the perfor-
mance of TEG-alkanethiol spacer in terms of nonspecific
adsorption and the results were compared with those of
11-mercaptoundecanoic acid (MUA) and the commercial
OEG spacers.
Key words: alkanethiol, solid-phase synthesis, self-assembled
monolayer, biointerface, nonspecific adsorption
Self-assembled monolayers (SAMs) are a renowned tool
for both modifying and functionalizing metal or nanoma-
terial surfaces. Because SAM formation provides a well-
appointed planar interface, it has opened the doors to fun-
damental studies of interfacial chemistry about the behav-
ior of biomaterial or catalyst on solid surfaces.1 Thus, the
functionalized SAM formation of alkanethiol on gold sur-
face has been employed for biosensing interface using
DNA,2 protein3 and cells4 and for catalytic reaction.5
The synthetic route for TEG-alkanethiol spacers is de-
scribed in Scheme 1. Before synthesis of alkanethiol spac-
er via SPOS, Fmoc-TEG-COOH (1), a building block
which contains triethylene glycol, was easily synthesized
without the need for column chromatography purifica-
tion.16 Another building block, 11,11′-dithiodiundecanoic
acid (3), was prepared as a thiol-protected alkyl spacer. A
disulfide protection method for thiol groups was chosen to
obtain free thiol under mild conditions. Then, the C-termi-
nal of compound 1 was introduced on 2-chlorotrityl chlo-
ride (CTC) resin with DIPEA, followed by Fmoc
deprotection. The existence of TEG spacer on the CTC
resin was identified by FT-IR [carbonyl band: 1737 cm–1,
amide band: 1664 cm–1, see Figure 1(b) in Supporting In-
formation]. The amount of compound 1 loaded on the res-
in was found to be 0.32 mmol/g by Fmoc titration and
elemental analysis. To couple the alkyl chain to the TEG
spacer, resin 2 was treated with an excess amount of com-
pound 3 and coupling reagents. Reduction of the resulting
resin with dithiothreitol (DTT) resulted in resin 4, regard-
less of partial cross-linking of resin 2 with compound 3.
Attachment of the alkyl spacer was monitored by ninhy-
drin test and was verified by the increase in the relative in-
tensity of amide peak in the FT-IR [amide band: 1661 cm–
1, see Figure 1(c) in Supporting Information] from resin 2.
Finally, the TEG-alkanethiol spacer 5 was cleaved from
resin 4 and was analyzed by ESI–MS (see Figure 2 in Sup-
porting Information). An oily product was obtained in
87% yield based on the initial loading of the TEG spacer.
This strategy did not require any purification steps during
synthesis, other than extraction and filtration steps. In ad-
As these interfaces of solid surfaces are becoming increas-
ingly important in bioanalysis, SAM molecules are re-
quired to be applicable for biocompatible surfaces. Since
Whiteside et al. first introduced SAMs of oligo(ethylene
glycol) (OEG)-terminated alkanethiols on gold surface,6,7
a wide variety of OEG alkanethiol spacers have been syn-
thesized.2b,8–12 Due to the presence of ethylene glycol
units, OEG-terminated SAMs possess high protein resis-
tance and the ability to reduce the nonspecific adsorption
of biomolecules, even in the case of tri(ethylene glycol)
(TEG)-terminated alkanethiol.6,13–15
However, it is not easy to synthesize these OEG alkaneth-
iol derivatives. Synthesis of OEG spacers by conventional
organic synthesis requires a minimum of four to five reac-
tion steps, complicated purifications in every step and
complex reaction conditions.2b,8–10 Moreover, additional
reaction process and purification steps are required to
elongate peptides or small bioactive materials from the
terminal alkanethiol. Although various attempts have
SYNLETT 2013, 24, 0020–0023
Advanced online publication: 10.12.2012
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DOI: 10.1055/s-0032-1317728; Art ID: ST-2012-U0763-L
© Georg Thieme Verlag Stuttgart · New York

LETTER
Facile Synthetic Method of Alkanethiol Spacer
21
O
(a)
i, ii
HO
O
O
N
O
O
N
Fmoc
H2N
O
O
NH2
H
H
O
1
O
iii, iv
O
Cl
N
O
NH2
3
H
O
2
(b)
(c)
O
O
O
i
HS
S
10
S
OH
HO
OH
10
10
3
O
O
O
i
2
O
S
S
N
O
N
H
OH
3
10
10
H
O
4
O
O
ii, iii
HO
SH
N
O
N
3
10
H
H
O
5
Scheme 1Reagents and conditions: (a) Preparation of TEG-loaded resin 2: (i) succinic anhydride in MeCN, 2 h, r.t.; (ii) Fmoc-OSu, DIPEA in
MeCN and H2O, 2 h, r.t.; (iii) compound 1, DIPEA in CH2Cl2, 2 h, r.t.; (iv) 20% piperidine (v/v) in DMF, 1 h, r.t.; (b) Synthesis of 11,11′-
dithiodiundecanoic acid (3): (i) I2, DMSO in THF; (c) Synthesis of alkylthiol linker (5): (i) compound 3, DIC, HOBt, DIPEA in NMP, 12 h, 40
°C; (ii) DTT in NMP, 12 h, r.t.; (iii) TFA, TIS in CH2Cl2, 1 h, r.t.
dition, by applying a very mild cleavage condition (1% face. These results prove that TEG-alkanethiol spacer
TFA–CH2Cl2), the final compound was recovered from forms the hydrophilic SAM surface due to the carboxyl
CTC resin without oxidation or other side reactions. From group and ethylene glycol moiety of the spacer.
the result of TEG-alkanethiol spacer synthesis, our syn-
thetic strategy, based on solid-phase organic synthesis,
proved to be a simple and efficient method compared to
others. Furthermore, we can easily modify the terminal
functional group of the spacer by conjugating peptides or
small molecules for bioassay.
Next, the TEG-alkanethiol spacer was applied for SAM
formation on a gold surface. The SAM on gold surface
was achieved by immersing a gold-coated silicon chip in
an ethanolic solution of TEG-alkanethiol spacer. As a
control experiment, another SAM on gold surface was
prepared by using MUA. The SAM surfaces were charac-
terized by X-ray photoelectron spectroscopy (XPS) and
water contact angle (CA) measurement (Figure 1). Ele-
mental analysis on the gold surface by XPS confirmed the
Scheme 2 DNA assay for nonspecific binding test: (a) bare gold sur-
face; (b) SAMs of spacers on gold spots; (c) probe DNA (5′-NH2-
TCCTAATAACAAT-3′)-grafted surface using EDC and NHS; (d)
hybridization with fluorescent labeled target DNA (Cy3-5′-ATTGT-
existence of MUA (S contents) and TEG-alkanethiol (S
and N contents). The change of hydrophilic state on SAM
surfaces was measured by water contact angle. The SAM
of MUA (CA: 39.4°) and TEG-alkanethiol spacer (CA:
36.7°) were more hydrophilic than a bare gold surface
(CA: 63.5°) due to the negative charge on the terminal
carboxyl group. Furthermore, we found that the decrease
of the contact angle of the SAMs of TEG-alkanethiol
spacer was attributable to ethylene glycol units through
comparison with the water contact angle of the MUA sur-
TATTAGGA-3′) on the surface.
In order for the TEG-alkanethiol spacer to have proper
properties for biosensor application, whether or not it ex-
hibits the stable sensing signal and low nonspecific ad-
sorption of the biomolecule is important. TEG-alkanethiol
spacer was assessed with a DNA hybridization assay and
the results were compared to those of MUA and HS-C11-
© Georg Thieme Verlag Stuttgart · New York
Synlett 2013, 24, 20–23

22
H.-J. Cho et al.
LETTER
Figure 1 The characterization of the SAMs on gold surface by XPS and contact angle: (a) bare gold surface, (b) SAM surface of MUA, and (c)
SAM surface of TEG-alkanethiol spacer 5.
EG3-COOH commonly used in biointerface under identi-
cal condition. The assay procedures are summarized in
Scheme 2. The SAM on gold spot array for DNA detec-
tion was prepared by incubating it in ethanolic solution of
TEG-alkanethiol spacer. After EDC/NHS activation on
the terminal carboxyl group, the probe DNA (5′-NH2-
TCCTAATAACAAT-3′) was immobilized on the gold
spot surface. Then, fluorescence-labeled target DNA (5′-
Cy3-ATTGTTATTAGGA-3′) of different concentrations
(1 mM, 10 mM) which were fully complementary to the
probe DNA was hybridized on the surface. As a negative
control, the noncomplementary DNA (5′-Cy3-
GTACCTATTGCCT-3′, 10 mM) was treated on the sur-
face in the same manner. The specific hybridization of tar-
get DNA could be quantitatively analyzed by
Figure 2 The results of fluorescence-based DNA hybridization assay
from three kinds of SAMs (HS-C11-EG3-COOH, MUA and TEG-al-
kanethiol spacer from left to right) on gold spots. Noncomplementary
DNA (10 mM) as a control and complementary DNA (1 mM and 10
fluorescence intensity per unit area due to the terminal
Cy3 dyes on the target DNAs. After sufficient washing
steps, the fluorescence intensities from three kinds of
SAMs on gold spots were measured by a fluorescence
scanner.
mM) were used for the assay.
As shown in Figure 2, the fluorescence intensities on
SAMs of TEG-alkanethiol spacer and HS-C11-EG3-
COOH were well correlated with the concentrations of
target DNA. In contrast, the fluorescence intensity on
SAM of MUA displayed inconsistent patterns as the con-
centration of target DNA increased with wide error rang-
es. In control experiment, the amount of nonspecific target
DNA adsorption on SAM of MUA was much higher than
others. These results show that OEGs of TEG-alkanethiol
spacer and HS-C11-EG3-COOH contribute to stabilizing
the fluorescence signals by specific DNA binding and re-
ducing nonspecific adsorption on SAM surface. The per-
formance of SAMs of TEG-alkanethiol spacer was almost
similar to that of commercially available HS-C11-EG3-
COOH spacer. As a result, the TEG-alkanethiol spacer
that we facilely and efficiently synthesized is a reliable
material to form SAMs for biointerface.
Synlett 2013, 24, 20–23
© Georg Thieme Verlag Stuttgart · New York

LETTER
Facile Synthetic Method of Alkanethiol Spacer
23
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In conclusion, TEG-alkanethiol spacer for biointerface
was synthesized by a mild and simple synthetic method
based on SPOS. Using building blocks prepared by simple
procedures, and without complicated purification steps,
we efficiently obtained a TEG-alkanethiol spacer from
CTC resin. The results of DNA assay on SAM of TEG-al-
kanethiol demonstrate that the TEG-alkanethiol spacer
provided a biocompatible interface with reducing nonspe-
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