Photo-triggered generation of a free thiol group on DNA: Application to DNA conjugation

Article abstract of DOI:10.1016/j.tetlet.2011.10.150

DNA oligomers possessing a 2-nitrobenzyl (NB) protected thiol group have been prepared. The photo-remove of the NB to generate a free thiol group in DNA has been analyzed by using reverse-phase HPLC and denaturing gel electrophoresis. The photo-triggered

Full text of DOI:10.1016/j.tetlet.2011.10.150

Tetrahedron Letters 53 (2012) 78–81
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Photo-triggered generation of a free thiol group on DNA: application
to DNA conjugation
⇑
⇑
Tadao Takada , Yuta Kawano, Mitsunobu Nakamura, Kazushige Yamana
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA oligomers possessing a 2-nitrobenzyl (NB) protected thiol group have been prepared. The photo-
remove of the NB to generate a free thiol group in DNA has been analyzed by using reverse-phase HPLC
and denaturing gel electrophoresis. The photo-triggered generation of the thiol function in DNA was
applicable in the light-initiated ligation of thiol-modified DNA oligomers and Au–DNA conjugation.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 September 2011
Accepted 28 October 2011
Available online 4 November 2011
Keywords:
DNA
Thiols
Nitrobenzyl group
Photoreaction
DNA conjugation
DNA oligomers containing a thiol group have been used in con-
jugations between DNA–DNA and between DNA–protein.1–4 Small
molecule attachment to thiol containing DNA is a useful mean to
prepare several types of DNA probes and functional DNAs.5 The
chemical bonding between a thiol group and metal has enabled
to prepare a self-assembled DNA monolayer on a metal surface.6
Oligonucleotides modified with photoresponsive molecules have
been developed to achieve the template-directed ligation of DNA
strands induced by light excitation,7–9 and control the conforma-
tion and function of nucleic acids.10–14 We have anticipated that,
if a thiol group in DNA could be generated by light-irradiation,
the light generated thiol-DNA would be attractive for several appli-
cations in DNA conjugations.15–19 Here, we describe the synthesis
of a DNA oligomer possessing a 2-nitrobenzyl (NB) protected thiol
group. The photo-removal of the NB to generate a free thiol group
in DNA has been accomplished by UV-light irradiation. It is demon-
strated that the photo-triggered generation is applicable in the
light-initiated ligation of thiol-modified DNA oligomers and
DNA–Au conjugation.
A 2-nitrobenzyl (NB) group was selected to cap a thiol group
since it is known that this protecting group can be removed by
UV-light and the produced nitrosobenzene is much less reac-
tive.17,20,21 Single-strand DNA possessing NB capped thiol (NBS) at
the 50-teminus was synthesized in order to study the UV-light in-
duced thiol generation (Fig. 1a). The DNA sequences to demonstrate
the applicability of the photoreaction in DNA conjugation are
shown in Figure 1b. The DNA modified with NBS at the 50-terminus
Figure 1. (a) Light-triggered generation of a free thiol on DNA by removing a
protecting group (2-nitrobenzyl group) by UV irradiation. (b) Sequences of DNA and
chemical structures of 2-nitrobenzyl protected thiol (NBS) and dithiopyridine (SSP).
NBS and SSP are tethered at the 50-terminus of S1 and 30-terminus of S2,
respectively. DNA templates (T1 and T2) are designed so that he NBS moiety is
close to SSP when the two strands (S1 and S2) hybridize with T1 or T2.
(S1) and dithiopyridine (SSP)22,23 at the 30-terminus (S2) and tem-
plate DNAs (T1 and T2) have been prepared for this purpose.
NBS-modified DNA (S1) was synthesized using a standard
phosphoramidite technique for oligonucleotide synthesis. As
⇑
Corresponding authors.
E-mail address: takada@eng.u-hyogo.ac.jp (T. Takada).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.150

T. Takada et al. / Tetrahedron Letters 53 (2012) 78–81
79
Figure 2. (a) HPLC profiles of S1 before (upper panel) and after UV irradiation
(5 min, lower panel). A transilluminator with a UV filter (365 nm) was used for the
sample irradiation. The reaction mixtures were analyzed by HPLC on an ODS
column (4.6 mm ꢀ 150 mm) and detected at 260 nm using 50 mM ammonium
formate/acetonitrile gradient (4–20% in 20 min). (b) Plots of conversion yield
obtained from the area of the new peak at 7 min as a function of the irradiation
time.
Scheme 1. Synthesis of phosphoramidite derivatives (2). Reagents: (i) 3-mercapto-
1-propanol, 1 N NaOH, dioxane. (ii) 2-cyanoethyl-N,N,N0,N0-tetraisopropylphospho-
rodiamidite, 1H-tetrazole, acetonitrile.
shown in Scheme 1, the preparation of phosphoramidite (2)
began with 3-mercapto-1-propanol (1) that was reacted with 2-
nitrobenzylchloride,24 and then the hydroxyl group was con-
verted to the phosphoramidite.25 The 30 min coupling time was
used for 2 and the oligonucleotide was treated with conc. ammo-
nium hydroxide at 55 °C for 12 h. The deprotected oligomer was
purified by reverse phase HPLC, and the purified oligomer was
verified by MALDI TOF-MS.25
The photoreaction was carried out for S1 (10
lM) in a 20 mM Na
phosphate buffer (pH 7.0) at room temperature by illumination of
Figure 3. (a) Photo-induced ligation of oligonucleotides via a thiol-disulfide exchange reaction. (b) HPLC profiles of S1 by UV irradiation (5 min) under aerobic (left panel) and
anaerobic conditions (right panel). HPLC analysis of photoreaction under aerobic condition at 55 °C. The ligated product is denoted by the asterisk. (c) Denaturing PAGE of the
photoligated product. Samples of the DNA assembly (S1/S2/T2) were irradiated for various times. Lane 1: no irradiation. Lane 2–6: irradiation for 0–240 s. Lane 7: incubation
with DTT for 2 h after irradiation for 240 s.

80
T. Takada et al. / Tetrahedron Letters 53 (2012) 78–81
UV-light (365 nm). The reaction mixtures were analyzed by reverse
phase HPLC and the results are shown in Figure 2. The oligomer S1
shows the peak with a retention time of 9.0 min under the analyt-
ical conditions. After the UV irradiation, a new peak at 6.5 min ap-
peared. The photo-product was separated and isolated, and
analyzed by MALDI-TOF-MS. The result (MS observed: 3774.3) is
in agreement with the fact that this product is thiol-containing
DNA oligomer. In a separate experiment, the isolated product was
reacted with S-methylmethane thiosulfonate to convert thiol func-
tion into Me–S–S–. The product was analyzed by using the MS and
the result (MS observed: 3818.2) is consistent with that of DNA oli-
gomer having Me–S–S– function. From these analyses, we con-
firmed that the DNA obtained after the photoreaction indeed has
the free thiol group. Figure 2b indicates the time course of the pho-
toreactions. The photochemical conversion from S1 to thiol-DNA
reaches to 70% within 5 min irradiation.
We have tested the ligation of two thiol-containing DNAs on
template DNA via a thiol/disulfide exchange. The DNA assembly
used for this test consists of two short strands (S1 and S2) and a
template T1 (Fig. 3a). S1 has NBS at the 50-terminus and S2 has a
disulfide bond at the 30-terminus. S1 and S2 hybridize with T1 so
that the two functional groups are close together. A free thiol is
generated by photo-removal of the protecting group, and then re-
acts with neighboring disulfides to generate a new disulfide bond
between two strands, leading to ligation of the two strands on
the template (Fig. 3a, bottom).
Figure 4. (a) Conjugation of gold nanoparticles (AuNPs) with DNA induced by
photo-triggered generation of thiols via removal of 2-nitrobenzyl group. Surface
modification of AuNPs with DNA proceeds via the formation of Au–S bond. (b) UV–
Vis absorption spectra of AuNPs solution observed after the salt addition. Black:
AuNPs with irradiation, red: AuNPs without irradiation, blue: AuNPs and S1 with
irradiation, green: AuNPs and S1 without irradiation.
The ligation reaction of S1 and S2 was analyzed by RP-HPLC (
Fig. 3b). The HPLC was carried out at 55 °C to maintain the thermal
dehybridization of the assembly during the analysis. With irradia-
tion at 365 nm under aerobic conditions, the two DNAs (S1 with a
retention time of 12.5 min, S2 of 14.3 min,) decreased concomitant
with the appearance of a new peak (10.5 min) which was assigned
to the ligated product of S1 and S2. No formation of the ligated
product was observed in the absence of the template DNA. Under
anaerobic conditions, the yields for the ligation reaction were sig-
nificantly improved, indicating that any undesired spontaneous
oxidation of the thiol was suppressed by removing the molecular
oxygen in the solution.26
We have examined the ligation reaction of various irradiation
times by PAGE experiments (Fig. 3c). In these experiments, the
36-mer template (T2) was used to discriminate between the tem-
plate and the ligated product. Two 12-mer ODNs (S1 and S2) de-
creased with the increased irradiation time and a new band
corresponding to the 24-mer ODN appeared, which was attributed
to the ligated-product of S1 and S2 by photoirradiation. By adding
the reducing agent, 1,4-dithio-1-threitol (DTT), after the photoirra-
diation for 240 s, the band of the ligated product completely disap-
peared and was converted to 12-mer ODN, meaning that a
disulfide bond of the ligated product was cleaved by the reducing
agent. These results showed that two short strands on the template
DNA can be photochemically ligated via a disulfide bond formation
and the ligated products can be chemically cleaved by adding
reducing reagents.
In order to demonstrate further application of the photo-trig-
gered generation of the free thiols on DNA, immobilization of the
DNA on gold nanoparticles (AuNPs) via the formation of the Au–
S bond induced by irradiation was investigated (Fig. 4a and b).
Aqueous solutions containing AuNPs (20 nm diameter) mixed with
or without S1 were irradiated for 60 min, incubated for 16 h at
55 °C, and then a solution of Na phosphate buffer (pH 7.0) contain-
ing NaCl was added to the solution for adjusting the pH and salt
concentration (0.1 M NaCl) of the solution.27,28 When incubated
without S1, precipitation of the AuNPs occurred after the addition
of the salt irrespective of the irradiation. The color of the superna-
tant after centrifugation turned from red to colorless, and the solu-
tions of the AuNPs showed no strong absorption in the visible
region. These phenomena are due to salt-induced aggregation of
the AuNPs, which is consistent with the fact that water-soluble
AuNPs are normally unstable at a high salt concentration.29 In con-
trast, when the solution of the AuNPs mixed with S1 was irradiated
and incubated for many hours, no precipitation of the AuNPs was
observed after the salt addition. In addition, characteristic plasmon
absorption of the AuNPs at 530 nm was observed. This result
showed that surface modification of the AuNPs with DNA was
achieved by the reaction of the free thiols of S1 with the gold sur-
face, preventing the aggregation of the AuNPs by a salt effect.
Without irradiation, precipitation of the AuNPs occurred and a very
small plasmon absorption in visible region was observed due to the
poor reactivity of the protected thiols to the gold surface. This sur-
face modification method initiated by light irradiation has the
advantage of preparing the AuNPs–DNA conjugates because there
is no need for chemical reduction of the disulfide bonds to thiols
and a subsequent purification step before use to remove the reduc-
ing agent.28
In summary, we have prepared DNA possessing a thiol function
that is protected by a photoremovable 2-nitrobenzyl group. It was
demonstrated that the generation of a free thiol on DNA by light
irradiation and the ligation of two DNA sequences via the thiol-
disulfide exchange reaction was possible on a DNA template. We
have also shown that the light-initiated surface modification is a
convenient way to prepare Au nanoparticles–DNA conjugates.
Light-triggered generation of thiols on DNA may be used for vari-
ous types of DNA conjugations based on thiol chemistry.
Acknowledgment
This research was supported in part by a Grand-in-Aid for
Scientific Research from the Japan Society for the Promotion of
Science (JSPS).
References and notes
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