Photocaging strategy for functionalisation of oligonucleotides and its applications for oligonucleotide labelling and cyclisation

Article abstract of DOI:10.1002/chem.201103833

We have used a photocaging strategy to develop novel phosphoramidites and expand the repertoire of protecting groups for modification of oligonucleotides by solid-phase synthesis. We synthesised five photolabile phosphoramidites and four new photolabile c

Full text of DOI:10.1002/chem.201103833

DOI: 10.1002/chem.201103833
Photocaging Strategy for Functionalisation of Oligonucleotides and Its
Applications for Oligonucleotide Labelling and Cyclisation
Meng Su, Jie Wang, and XinJing Tang*[a]
Abstract: We have used a photocaging
strategy to develop novel phosphorami-
dites and expand the repertoire of pro-
tecting groups for modification of oli-
gonucleotides by solid-phase synthesis.
We synthesised five photolabile phos-
phoramidites and four new photolabile
controlled pore glasses (CPGs). By
using these photolabile phosphorami-
dites and CPGs, modified oligodeoxy-
nucleotides (ODNs) with phosphate,
amine, acid, thiol and carbonyl moiet-
ies at 5’ and/or 3’ ends were readily
synthesised. To the best of our knowl-
edge, this is the first report of introduc-
ing a carbonyl at the 5’ end and thiol
groups at both ends of ODNs with
photolabile modifiers. Terminal label-
ling was also easily realised in solution
or by on-column solid-phase synthesis.
By using the photolabile amine modifi-
er and the photolabile acid CPG, cycli-
sation of an oligodeoxynucleotide was
achieved with good yields. This study
provides an alternative way to intro-
duce functional groups into oligonucle-
otides and expand the scope of oligo-
nucleotide bio-orthogonal labelling.
Keywords: caged compounds · oli-
gonucleotide modifiers · oligonucle-
otides · photoactivation · photola-
bile phosphoramidites
Introduction
ties and potential applications of such conjugates.[23–25] The
current widely used conjugation approaches of deprotected
oligonucleotides with peptides suffer from moderate yields
and laborious purification,[26,27] especially with positively
charged peptides. The solution-phase conjugation of protect-
ed oligonucleotides has advantages over the above meth-
ods.[28] 3) Oligonucleotides with attachments of both amine
and acid moieties are usually achieved in multiple steps. In
addition, cyclisation of these deprotected oligonucleotides
through amide bond formation always requires large excess-
es of reagents and long reaction times, produces low product
yields and results in significant amounts of non-specific co-
valent modification.[29] Photolabile moieties, also known as
caged compounds, have shown significance and wide appli-
cations in chemistry and biology.[30–35] Photocaged phos-
phate,[36] amino[28,37,38] and carboxyl groups[39,40] for modified
oligonucleotides were previously achieved by Greenberg
et al. They showed promising applications for bioconju-
gation with biotin, peptide and other small molecules. How-
ever, a long irradiation time may be needed for photolysis
of the 3, 4-dimethoxy-2-nitrobenzyl (DMNB) moiety due to
the slow photocleavage kinetics.[41,42] Here we report the
synthesis of modified oligodeoxynucleotides with photola-
bile functional groups that include phosphate, amine, acid,
thiol and carbonyl moieties at 5’ and/or 3’ ends to expand
the repertoire for the functionality of oligodeoxynucleotides.
Terminal labelling with fluorophores at 5’ or 3’ ends was suc-
Oligonucleotides altered by the attachment of functional
groups are very useful for oligonucleotide labelling and con-
jugation. These modified oligonucleotides display a variety
of applications in many fields, including antisense drug de-
velopment, DNA microarray[1] and sequence analysis,[2]
nanotechnology[3] and in vivo imaging.[4] Many functional
groups such as amine, acid and thiol groups have been incor-
porated into oligonucleotides by using corresponding modi-
fiers, and some of them are commercially available and
widely used.[5–22] Despite these successful methods for oligo-
nucleotide modification, there is still room to improve and
expand the development of protected functional groups for
oligonucleotide solid-phase synthesis, especially in the fol-
lowing circumstances: 1) When an oligonucleotide is la-
belled with multiple moieties through consecutive deprotec-
tion of amine protecting groups and formation of amide
bonds, a photocaging strategy may become a good choice in
addition to 4-methoxytriphenylmethyl (MMTr) and fluore-
nylmethyloxycarbonyl
(Fmoc)/trifluoactate
2) Conjugation of oligonucleotides with larger molecules,
such as peptides, is of significant interest due to the proper-
chemistry.
[a] M. Su, J. Wang, Prof. Dr. X. Tang
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University
cessfully achieved, respectively. Additionally,
a circular
oligodeoxynucleotide was synthesised after photodeprotec-
tion of photolabile amine and acid groups at 5’ and 3’ ends,
respectively, followed by coupling of the protected oligode-
oxynucleotide in DMF and deprotection of the cyclised
product by ammonium hydroxide.
No. 38 Xueyuan Rd., Beijing 100191 (P. R. China)
Fax : (+86)10-82805635
E-mail: xinjingt@bjmu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103833.
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FULL PAPER
Results and Discussion
(Scheme 1) was synthesised according to a previous
report[44] and then incorporated as the last coupling X of an
oligodeoxynucleotide sequence (5’ X TTT TTC TCT CTC
TCT G-3’, ODN 1, Table 1). After deprotection and cleav-
age from CPG, the oligodeoxynucleotide with the hydropho-
bic caging group has a longer retention time during purifica-
tion by RP-HPLC. HPLC traces of ODN 1 show two peaks
Functionalisation of oligodeoxynucleotides with caged 5’-
phosphate, amine, acid, thiol and carbonyl moieties: Photo-
labile moieties are widely used for temporarily controlled
protection of functional groups in many applications. To be
useful for oligonucleotide modification, photolabile moieties
should be stable during DNA synthesis cycles. The 2-nitro-
phenyl ethyl moiety is capable of withstanding the condi-
tions of DNA solid-phase synthesis and is a good candidate
for protecting functional groups on olignucleotides because
it has reasonable photocleavage efficiency and faster cleav-
age kinetics than the 3,4-dimethoxy-2-nitrobenzyl moiety.[43]
To make direct attachments of functional moieties to the 5’
end of oligonucleotides by solid-phase synthesis, phosphora-
midites are usually used. Photolabile phosphoramidite mon-
omers with phosphate, amine, acid, thiol and carbonyl moi-
eties at the 5’ end were synthesised by using photolabile
groups as temporary protecting groups, followed by phos-
phoramidition as shown in Scheme 1.
Table 1. Oligonucleotide sequences and modifications used in this study.
5’ X TTT TTC TCT CTC TCT G 3’
ODN 1
ODN 2
ODN 3
ODN 4
ODN 5
ODN 6
X=phosphate
X=amine
X=carboxylic acid
X=thiol (via sulfoether)
X=thiol (via carbonothioate)
X=ketone
1b
3b
5b
6b
7b
8b
5’ TTT TTC TCT CTC TCT Y 3’
ODN 7
ODN 8
ODN 9
ODN 10
Y=phosphate
Y=amine
Y=carboxylic acid
Y=thiol (via carbonothioate)
11b
12b
13
14b
5’ X TTT TTC TCT CTC TCT Y 3’
ODN 11 X=amino Y=carboxylic acid
To introduce a phosphate at the 5’ end of an oligodeoxy-
nucleotide, 1-(2-nitrophenyl) ethyl phosphoramidite (1b)
Scheme 1. Syntheses of photolabile protecting groups and photolabile phosphoramidites for 5’-end modification of oligonucleotides. Reagents and condi-
tions for 1a/3a/5a/6a/7a/8a to 1b/3b/5b/6b/7b/8b: P(OCH2CH2CN)NiPr2Cl, TEA, dichloromethane, 08C, 54 to 74%. DMAP=4-dimethylaminopyri-
dine, DSC=N,N’-disuccinimidyl carbonate, TBAB=tetrabutylammonium bromide, TBAF= tetrabutylammonium fluoride, TEA=triethylamine.
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X. Tang et al.
close to each other with similar peak intensity (see Figure S1
in the Supporting Information). Further MS results indicate
that these two fractions have the same molecular weight.
After irradiation of both oligodeoxynucleotide fractions
with UV light for 10 min (360 nm, 11 mWcmÀ2) to remove
the photolabile chiral 1-(2-nitrophenyl) ethyl moiety, fol-
lowed by HPLC separation, both samples showed the same
retention time and same molecular weight. The separation
of the two oligodeoxynucleotide isomers on the RP-HPLC
column may be due to the short distance between the chiral
1-(2-nitrophenyl) ethyl moiety and the helix of DNA oligo-
deoxynucleotide, as evidenced by two peaks at 147.7 and
147.3 ppm in the P31 NMR spectra of 1b. The chiral effect of
the 1-(2-nitrophenyl) ethyl moiety disappeared with elonga-
tion of the distance between the chiral moiety and an oligo-
deoxynucleotide (caged ODN 2), and only a single peak was
observed on HPLC traces for caged ODN 2.
In the case of the caged amine phosphoramidite 3b, we
first synthesised 3a with the reaction of 1-(2-nitrophenyl)
ethanol and DSC, followed by the addition of hydroxyhexyl
amine by using a similar synthetic strategy as literature re-
ports.[45] After phosphoramidition of 3a, the obtained 3b
was incorporated into the oligodeoxynucleotide sequence
(ODN 2, Table 1) with the same coupling conditions as
normal phosphoramidites. After ammonium hydroxide hy-
drolysis, HPLC traces show a main single peak, which was
proved to be the expected caged amine modified oligode-
oxynucleotide by MS (Table 2). Upon photolysis, the caging
group was removed and a new peak with the shorter reten-
tion time than caged ODN 2 on HPLC traces appeared. MS
confirmed that the new fraction was the uncaged amine-
modified oligodeoxynucleotide ODN 2, as shown in Table 2.
To synthesise caged acid and thiol phosphoramidites (5b,
6b), 6-(tert-butyldimethylsilyloxy) hexanoic acid and 6-mer-
capto-1-hexanol were used to react with 1-(1-bromoethyl)-2-
nitrobenzene by substitution of the bromo group of 2 to
obtain 5a and 6a as shown in Scheme 1. After phosphorami-
dition of 5a and 6a, 5b and 6b were obtained and attached
to the 5’ end of oligodeoxynucleotides on solid phase ac-
cording to the similar procedure for the synthesis of the
caged amino-modified oligodeoxynucleotide. To obtain the
acid-functionalised oligodeoxynucleotide ODN 3, photolysis
is needed to release the acid group. The solid CPG with the
modified oligodeoxynucleotide of caged ODN 3 suspended
in acetonitrile/water (4:1) was first irradiated by UV lamp.
Then the irradiated CPG can be directly used for further
conjugation on the solid phase with other molecules, such as
fluorophores and peptides. To further characterise ODN 3,
we hydrolysed the irradiated CPG of ODN 3 with ammoni-
um hydroxide. The obtained ODN 3 was separated by
HPLC and confirmed by MS (Table 2). Oligodeoxynucleoti-
des with an acid group at the 5’ end could also be used for
coupling with amine-containing base-sensitive molecules or
proteins. Direct uncaging of a protected carboxylic acid on
oligodeoxynucleotides has advantages to previous efforts of
acid functionalisation of oligodeoxynucleotides through two-
step coupling by using an amine phosphoramidite and suc-
cinic anhydride.[29]
Using standard DNA synthesis cycles for the caged thiol
modified ODN with 6b for the last coupling, we failed to
obtain a main peak for the caged thiol-modified oligodeoxy-
nucleotide ODN 4 on HPLC separation traces. Previous lit-
erature reported that the oxidation step with iodine caused
the removal of the S-trityl group and dimerisation of free
thiol-oligodeoxynucleotides.[46] However reduction of the
obtained oligodeoxynucleotide with dithiothrietol (DTT)
was ineffective and could not restore the activity of free
thiol groups. MS results of the separated fractions from
HPLC showed a mix of molecule weights of 5100 Da, which
is the expected caged ODN 4, and 5132 Da, which is in
agreement with the oxidation of sulfoether to sulfone by I2/
pyridine/H2O/THF. A shorter oxidation time or replacement
of the oxidant mixture by carbon tetrachloride/triethyla-
mine/N-methylimidazole/H2O lowered the possibility of oxi-
dation of sulfoether. Photoirradiation of purified caged
ODN 4 produced a new single peak on HPLC trace with
shorter retention time (see Figure S2 in the Supporting In-
formation). However, MS results indicated the MW of the
new fraction was the same as the MW of caged ODN 4. Fur-
ther treatment of this new irradiated product with DTT
failed to recover thiol-modified oligodeoxynucleotide ODN
4. According to the literature report,[47] the free thiol moiety
Table 2. ESI-TOF/MALDI-TOF results and HPLC retention times for
the modified oligodeoxynucleotides.
Expected
MW [Da]
Measured
MW [Da]
Retention
time [min]
ODN 1
ODN 2
ODN 3
ODN 4
ODN 5
ODN 6
4981
5124
–
5097
–
4984
5127
–
5100
–
21.98
24.62
–
24.12
–
5’-end caged
5067
5071
21.85
ODN 1
ODN 2
ODN 3
ODN 4
ODN 5
4832
4931
4946
4949
4948
4834
4934
4949
5099[a]
4949
10.14
12.39
9.59
17.07
15.28
5’-end
irradiated
ODN 6
ODN 7
ODN 8
ODN 9
ODN 10
4902
4503
4602
4617
4619
4907
4504
4607
4619
4617
11.75
10.28
11.53
9.79
3’-end
irradiated
12.04
ODN 2-TAMRA
ODN 3-pyrene
ODN 8-pyrene
ODN 11 linear
ODN 11 vircular
5343
5159
4872
4796
4778
5345
5163
4874
4799
4783
21.45
28.25
28.06
À
14.29[b]
14.67[b]
can react with a nitroso group to form an N S bond, which
may also be occurring with this oligodeoxynucleotide This
observation is different from previous reports of the caged
cysteine moiety in proteins that showed full recovery of bio-
[a] Photoirradiated immediate was obtained. [b] These results were per-
formed on analysis HPLC column.
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Photocaging Strategy for Functionalisation of Oligonucleotides
FULL PAPER
logical activities after photouncaging.[48] To overcome the
problem with our thiol modification, 7b was synthesised
after phosphoramidition of 7a (Scheme 1) for the synthesis
of carbonothioate protected oligodeoxynucleotide ODN 5.
Direct treatment by ammonium hydroxide generated a main
peak on HPLC trace with a molecular weight of 5002 Da by
MALDI TOF, which is probably the oligodeoxynucleotide
with thiol-acrylonitrile adduct (calcd MW 5001=4948
(ODN 4)+53 (CH2=CHCN)). With the addition of DTT to
ammonium hydroxide during deprotection, the expected
thiol-modified oligodeoxynucleotide (MW=4949 Da) was
obtained (Table 2 and Figure S4 in the Supporting Informa-
tion).
Aldehyde and ketone functional groups are occasionally
incorporated into oligonucleotides for use in conjugation to
specific moieties such as peptides containing nucleophilic
groups (aminooxy, aminothiol, or hydrazine, etc.).[49,50] These
conjugation reactions are efficient but suffer from poor sta-
bility of aldehyde or ketone on oligonucleotides due to
a known propensity to react with purine bases.[17] Previously,
the aldehyde moiety has been incorporated into oligonucle-
otides through post-transformation. Usually acetate-protect-
ed diol or protected serine residue is first coupled to an oli-
gonucleotide, followed by careful oxidation after deprotec-
tion. Here, we applied
a photolabile 1-(2-nitrophenyl)
ethane-1,2-diol as the protecting group for 7-hydroxybutan-
2-one and synthesised phosphoramidite 8b (Scheme 1).
Caged ODN 6 with protected ketone at the 5’ end was then
synthesised with 8b under standard DNA “DMT-ON’ꢁ solid-
phase synthesis cycles. After ammonium hydroxide depro-
tection and HPLC purification, caged ODN 6 was confirmed
by ESI (Table 2 and Figure S4 in the Supporting Informa-
tion). Upon photolysis, a new oligodeoxynucleotide product
with shorter retention time was collected and was confirmed
to be ketone-modified oligodeoxynucleotide ODN 6 by
MALDI TOF (Table 2 and Figure S4 in the Supporting In-
formation).
Figure 1. a) HPLC traces of the photocleavage of caged ODN 2 versus ir-
&
radiation time. b) Photocleavage efficiencies of ODN 1 ( ) and ODN 2
À1
*
(
). The concentration of the oligodeoxynucleotide was 20 nmolmL
and the photolysis was conducted with UV light (365 nm, 11 mWcmÀ2).
To study the photocleavage kinetics of aqueous caged
oligodeoxynucleotide solutions, caged phosphate ODN
1 and amine modified ODN 2 (20 mm) were irradiated with
light (365 nm, 11 mWcmÀ2) and the formation of resulting
uncaged modified oligodeoxynucleotides was monitored by
HPLC. As seen in Figure 1a, photodeprotection of caged
ODN 1 and ODN 2 in aqueous solutions was fast and the
uncaging efficiencies were over 95% in 15 min based on
HPLC traces under our photolysis conditions (Figure 1b).
Photocleavage of both caged oligodeoxynucleotides satisfied
first-order reaction kinetics with k=0.358 minÀ1 (ODN 1,
R2 =0.975) and 0.293 minÀ1 (ODN 2, R2 =0.986).
zyl (DMNB) derivatives (CPG-As, Figure 2) have been re-
ported.[36,37,39] The desired oligodeoxynucleotides can be ob-
tained upon photolysis at 365 nm. Here we synthesised
a series of new photocleavable solid-phase supports (CPG
11–14, Figure 2) based on 2-amino-1-(2-nitrophenyl) ethanol
moiety 9, which can be readily synthesised from o-nitroace-
tophenone in four steps. As shown in Scheme 2, 9 coupled
with 4-pentyoic acid to obtain 11a with the introduction of
an alkyne group that is used to couple with azide-functional-
ised LCAA-CPG through a copper-catalysed click reaction.
Product 11a serves as an intermediate for the preparation of
11b, 12b, 13 and 14b for further reaction with an azide
group for 3’-end modification of solid-phase supports. By
using these photolabile supports, oligodeoxynucleotides with
3’-phosphate, amine, carboxylic acid and thiol groups were
synthesised.
Functionalisation of oligodeoxynucleotides with caged 3’-
phosphate, amine, acid and thiol moieties: To synthesise 3’-
end modified oligonucleotides with different functional
groups by using photolabile protecting moieties, new con-
trolled pore glass supports needed to be synthesised. Previ-
ously, photolabile solid-phase supports modified with caged
acid and amine groups based on 4,5-dimethoxy-2-nitroben-
For the phosphate-group modification of oligonucleotides
at the 3’ end, only DMTr-Cl was needed for the reaction
with a hydroxyl group of 11a to give 11b, which can be di-
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X. Tang et al.
rectly used for the synthesis of the photolabile solid support.
Similar to the synthesis of 3a, preparation of 12a was ach-
ieved via reaction of 11a with DSC, followed by the addi-
tion of 6-amino-1-hexanol. Synthesis of 13 was accomplished
directly by the coupling of 11a and 10 with DCC as the cou-
pling reagent. In the case of caged thiol-functionalised CPG,
we did not try to synthesise the sulfoether due to its inability
to survive the oxidation cycle of DNA synthesis and photoir-
radiation, as mentioned before. Instead, the carbonothioate
14a was synthesised by two-step coupling 11a and 6-mer-
capto-1-hexanol in one-pot in the presence of DSC. The re-
spective photolabile nitrobenzyl derivatives (12a, 14a) were
carried onto 12b and 14b by standard dimethoxytritylation.
Solid supports of CPG 11-14 were prepared by coupling
the alkyne group of 11b, 12b, 13 and 14b with the azide
preloaded long-chain alkylamine-controlled pore-glass sup-
port (LCAA-CPG, 99 mmolgÀ1), followed by capping with
acetic anhydrite (Figure 2). First, LCAA-CPG reacted with
4-azidobutanoic acid in DMF solution at the presence of
HBTU and DMAP. Photolabile 11b, 12b, 13 and 14b were
then coupled with the azide-loaded CPG through Huisgen
cycloaddition. The loading efficiencies were determined by
the absorbance of DMTr cation on a certain amount CPG in
methylene chloride containing 3% trifluoroacectic acid. The
Figure 2. Structure and synthesis of modified CPGs in this study.
DIPEA=N,N-diisopropylethylamine, HBTU=O-(1H-benzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate.
Scheme 2. Syntheses of photolabile modifiers for modified CPGs. Reagents and conditions for 11a to 11b: DMTr-Cl, Py, RT, 78%; for 12/14a to 12/14b:
DMTr-Cl, TEA, dichloromethane, RT, 78/76%. DCC=dicyclohexylcarbodiimide, DMTr-Cl: 4,4’-dimethoxytrityl chloride, EDC=1-ethyl-3-(2-dimethyl-
aminopropyl)carbodiimide, HOBt = N-hydroxybenzotriazole.
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Photocaging Strategy for Functionalisation of Oligonucleotides
FULL PAPER
amount of DMTr was then calculated to prove the effective-
ness of two-step coupling (azide loading and click coupling).
Loading capacities of these photolabile CPGs over two
steps was determined to be over 55 mmolgÀ1 (see Table S1 in
the Supporting Information). To determine the photocleav-
age efficiencies, a certain amount of these CPGs was irradi-
ated for 30 min in acetonitrile/water, and the DMTr cation
of irradiated CPGs and total DMTr cation of the same
amount of unirradiated CPGs were then measured. The per-
centage of lost DMTr cation after photoirradiation was de-
fined as the photocleavage efficiency. Table S1 in the Sup-
porting Information data also lists photocleavage efficiencies
for all four photolabile CPGs in 30 min irradiation.
For functionalisation of the 3’-end of oligodeoxynucleoti-
des, 15-mer ODN 7/8/9/10 (5’-TTT TTC TCT CTC TCT Y-
3’) were synthesised with the corresponding photolabile
CPGs under standard DNA synthesis cycles. The photolysis
was conducted on solid-phase suspension (acetonitrile/water
v/v 4/1, 6 mgmLÀ1) to cleave the oligodeoxynucleotides
from the corresponding CPGs. The photocleaved oligodeox-
ynucleotides were then subject to deprotection with concen-
trated aqueous ammonium hydroxide. By following HPLC
purification and DMTr removal with aqueous acetic acid,
identification of the modified oligodeoxynucleotides was
confirmed by MS (Table 2).
caged ODN 11 on CPG was irradiated under the same pho-
tolysis conditions mentioned above and the supernatant was
collected as protected ODN 11. After removing all solvents
and washing with ether, coupling reagents (BOP, HBTU
and EDC) for amide bond formation were added to the resi-
due. The oligodeoxynucleotide was then deprotected with
concentrated ammonium hydroxide and was further purified
with RP-HPLC. HPLC traces showed two main peaks (see
Figure S3 in the Supporting Information). One small peak
was confirmed by MS to be the uncyclised linear oligode-
oxynucleotide and the other peak with a slightly longer re-
tention time was the expected cyclised oligodeoxynucleo-
tide, the mass of which was confirmed by MS (Table 2).
After optimisation of coupling conditions, the combination
of BOP/HOBt/DIPEA produced a cyclised product with
a better coupling yield (80%) based on integration of peaks
corresponding to the linear and circular oligodeoxynucleoti-
des on HPLC traces, as shown in Table S2 in the Supporting
Information.
Conclusion
We have developed novel phosphoramidites and CPGs with
photolabile protecting groups for modification of oligonucle-
otides by solid-phase synthesis. We synthesised five photola-
bile blocks to protect phosphate, amine, thiol, acid and car-
bonyl groups for the 5’ end of ODNs, and four new photola-
bile solid-phase CPGs to generate phosphate, amino, thiol
and acid groups at the 3’ end of ODNs. To the best of our
knowledge, this is the first report of the addition of a carbon-
yl group at the 5’ end and thiol groups at both ends of
ODNs with photolabile modifiers. Furthermore, cyclisation
of a protected oligodeoxynucleotide was achieved by using
the photolabile amine phosphoramidite modifier and photo-
labile acid CPG during solid-phase synthesis. These studies
provide an alternative way to introduce functional groups
into oligonucleotides, and expand the scope of oligonucleo-
tide bio-orthogonal labelling.
Labelling modified oligodeoxynucleotides with uncaged
functional groups: To prove the activities of uncaged func-
tional groups, different dyes were used for the conjugation
with modified oligodeoxynucleotides. ODN 2, ODN 3 and
ODN 8 were selected for labelling reactions. ODN 2 with
the amine group was attached to tetramethylrhodamine
(TAMRA) NHS ester in DMF/aqueous NaHCO3, and ODN
3 with the acid group was coupled with 1-pyrenemethyla-
mine, whereas protected ODN 8 with the amine group was
linked to 1-pyrenebutyric acid. After ammonium hydroxide
hydrolysis and HPLC purification, we obtained the expected
fluorophore-labelled ODNs with the anticipated MS as
listed in Table 2. The labelling efficiencies were usually
higher in comparison to the coupling of deprotected oligo-
deoxynucleotides in aqueous solutions. For ODN 3, the effi-
ciency of on-column coupling with pyrenemethylamine by
using benzotriazol-1-yloxytris(dimethylamino)-phosphonium
hexafluorophosphate (BOP) reagent reached 82% based on
HPLC results, whereas there was very low yield of the la-
belled oligodeoxynucleotide product under aqueous condi-
tions due to low solubility of pyrenemethylamine in water.
For the 3’-end modified oligodeoxynucleotide ODN 8, the
uncaged amino group could efficiently couple with 1-pyrene-
butyric acid in DMF solution (85% yield), which is better
than the yields from oligodeoxynucleotide bioconjugation in
aqueous solutions.
Experimental Section
General methods for organic synthesis: All solvents used are dried and
distilled by using standard methods. Dichloromethane, TEA, THF and
acetonitrile are dried over CaH2 and are freshly distilled before every
use. All reagents were purchased from Alfa Aesar, Sigma Aldrich or
J&K and used without further purification. All mixed solvent systems are
reported as v/v solutions. All reactions are monitored by TLC using com-
mercial Merck Plates coated with silica gel GF254 (0.24 mm thick). Flash
column chromatography is performed with silica gel purchased from
Qingdao Haiyang Chemical Company (200–300 mesh). 1H (400 MHz),
13C (100 MHz) and 31P NMR (162 MHz) spectra are recorded on
a Bruker spectrometer at 258C. Chemical shifts (d, ppm) are quoted rela-
tive to the residual solvent and coupling constants (J) are corrected and
quoted to the nearest 1 Hz. MS are measured on MALDI TOF or a Q-
TOF spectrometer by using electrospray ionisation (ESI). When necessa-
ry, the reactions are conducted in a dark room.
Furthermore, circular oligodeoxynucleotides could also be
synthesised by using a similar approach. Caged ODN 11 was
synthesised under normal solid-phase synthesis with photo-
labile acid CPG 13 as the 3’-end modification and photola-
bile amine modifier 3b as the 5’-end modification. The
Chem. Eur. J. 2012, 18, 9628 – 9637
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9633

X. Tang et al.
1-(2-Nitrophenyl) ethanol (1a): 1-(2-Nitrophenyl) ethanol was synthes-
ised according to the literature procedure with minor modification.[51] o-
Nitroacetophenone (3.51 g, 21.3 mmol) was dissolved in 1,4-dioxane
(15 mL) and methanol (9 mL) and stirred for 10 min at 08C. Then
NaBH4 (1.60 g, 42.3 mmol) was slowly added. The mixture was left to
warm to room temperature whilst stirring for 1 h and was then quenched
with acetone (15 mL). Solvents were removed in vacuo. The residue was
dissolved in ethyl acetate (70 mL) and washed with H2O (3ꢂ50 mL). The
organic layer was then dried over Na2SO4, filtered and concentrated
in ether (80 mL) and the ether solution was washed with H2O (3ꢂ
80 mL) and dried over Na2SO4. After removal of ether, the residue was
purified by flash column chromatography with 10:1 petroleum ether/
ethyl acetate as the eluent to give yellow oil [1-(2-nitrophenyl) ethyl 6-
(tert-butyldimethylsilyloxy)hexanoate], which was further dissolved in
TBAF (1m in THF, 20 mL). After stirring for 1 h at room temperature,
the solution was concentrated and the obtained residue was purified by
flash column chromatography with 1:1 petroleum ether/ethyl acetate as
the eluent to give 5a as a clear, yellow oil (748 mg, 2.66 mmol, 66% yield
for 3 steps from ethyl 6-hydroxyhexanoate). 1H NMR (400 MHz, CDCl3)
d=7.92 (d, 1H, J=8 Hz), 7.62 (d, 2H, J=4 Hz), 7.42 (m, 1H), 6.32 (m,
1H), 3.62 (t, 2H, J=6 Hz), 2.34 (m, 2H), 1.64 (d, 3H, J=3 Hz), 1.62–
1.32 ppm (m, 6H); 13C NMR (100 MHz, CDCl3) d=172.4, 138.0, 133.4,
128.3, 127.1, 124.4, 67.9, 62.6, 34.2, 32.2, 25.2, 24.5, 21.9 ppm; MS (ESI-
TOF+): m/z: calcd for C14H21NO3: 304.12; found: 304.08 [M]+.
under reduced pressure to give 1a as
a clear yellow oil (3.52 g,
21.1 mmol, 99% yield); 1H NMR (400 MHz, CDCl3): d=7.84 (dd, 2H,
J=8, 11 Hz), 7.63 (t, 1H, J=7 Hz), 7.40 (t, 1H, J=8 Hz), 5.39 (q, 1H,
J=6 Hz), 1.54 ppm (d, 3H, J=6 Hz); 13C NMR (100 MHz, CDCl3): d=
147.8, 141.0, 133.5, 128.0, 127.5, 124.2, 65.5, 24.2 ppm.
1-(1-Bromoethyl)-2-nitrobenzene (2): 1-(2-Nitrophenyl) ethanol (1a)
(425 mg, 2.50 mmol), triphenylphosphine (984 mg, 3.75 mmol) and CBr4
(1.25 g, 3.75 mmol) were dissolved in THF (10 mL) and stirred for 30 min
at room temperature. The solution was filtered and concentrated and
then purified by flash column chromatography by eluting with 4:1 petro-
leum ether/ethyl acetate to give 2 as a brown oil (558 mg, 2.43 mmol,
97% yield); 1H NMR (400 MHz, CDCl3) d=7.88 (dd, 1H, J=1, 4 Hz),
7.81 (dd, 1H, J=1, 4 Hz), 7.65 (dd, 1H, J=1, 4 Hz), 7.42 (dd, 1H, J=1,
7 Hz), 5.80 (q, 1H, J=7 Hz), 2.07 ppm (d, 3H, J=7 Hz); 13C NMR
(100 MHz, CDCl3) d=147.5, 137.7, 133.3, 129.8, 128.8, 124.2, 41.7,
27.1 ppm.
6-[1-(2-Nitrophenyl)ethylthio]hexan-1-ol
(6a):
Cs2CO3
(438 mg,
1.34 mmol), TBAB (432 mg, 1.34 mmol) and NaI (227 mg, 1.51 mmol)
were dissolved in DMF (6 mL) under nitrogen. 6-Mercapto-1-hexanol
(183 mL, 2.07 mmol) was added to above solution and the mixture was
stirred for 1 h at room temperature and was then cooled to 08C. 1-(1-
Bromoethyl)-2-nitrobenzene (2) (302 mg, 1.31 mmol) dissolved in DMF
(5 mL) was added dropwise. After stirring for 11 h, the reaction mixture
was diluted in ethyl acetate (50 mL) and the ethyl acetate solution was
then washed with H2O (3ꢂ50 mL). The combined organic solution was
dried over Na2SO4. After the removal of ethyl acetate, the residue was
purified by flash column chromatography with 2:1 petroleum ether/ethyl
acetate as the eluent to give 6a as a clear yellow oil (297 mg, 1.05 mmol,
80% yield); 1H NMR (400 MHz, CDCl3) d=7.81 (d, 1H, J=8 Hz), 7.69
(d, 1H, J=8 Hz), 7.56 (t, 1H, J=8 Hz), 7.32 (t, 1H, J=8 Hz), 4.55 (q,
1H, J=7 Hz), 3.55 (t, 2H, J=7 Hz), 2.29 (m, 2H), 1.56 (d, 3H, J=7 Hz),
1.45 (m, 4H), 1.26 ppm (m, 4H); 13C NMR (100 MHz, CDCl3): d=149.3,
139.1, 132.8, 129.4, 127.4, 123.5, 62.6, 38.1, 32.4, 31.5, 29.2, 28.4, 25.1,
22.8 ppm; MS (ESI-TOF+): m/z: calcd for C14H21NO3S: 306.11; found:
306.08 [M]+.
1-(2-Nitrophenyl) ethyl 6-hydroxyhexylcarbamate (3a): 1-(2-Nitrophenyl)
ethanol (1a) (1.03 g, 6.17 mmol) and DSC (2.37 g, 9.26 mmol) were dis-
solved in CH3CN (20 mL). TEA (2.08 mL, 15.0 mmol) was added to the
mixture and the solution was left stirring for 3 h at room temperature. 6-
Amino-1-hexanol (1.45 g, 12.4 mmol) dissolved in dichloromethane
(15 mL) was then added. After stirring for another 30 min at room tem-
perature, the reaction was stopped. The solution was concentrated and
the residue was then diluted with dichloromethane (50 mL). The organic
layer was washed with H2O (3ꢂ50 mL) and dried over Na2SO4. After fil-
tration and concentration, the obtained residue was further purified by
flash column chromatography with 1:2 petroleum ether/ethyl acetate as
the eluent to give 3a as a clear yellow oil (1.78 g, 5.74 mmol, 93% yield);
1H NMR (400 MHz, CDCl3) d=7.90 (d, 1H, J=8 Hz), 7.60 (d, 2H, J=
3 Hz), 7.39(d, 1H, J=4 Hz), 6.20 (d, 1H, J=6 Hz), 4.90 (s, 1H), 3.56 (t,
2H, J=6 Hz), 3.09 (t, 2H, J=6 Hz), 1.83 (s, 1H) 1.59 (d, 3H, J=6 Hz),
1.57–1.24 ppm (m, 8H); 13C NMR (100 MHz, CDCl3) d=155.3, 147.6,
138.7, 133.4, 128.1, 127.0, 124.3, 68.4, 62.5, 40.7, 32.4, 29.8, 26.2, 25.2,
22.2 ppm; MS (ESI-TOF+): m/z: calcd for C15H22N2O5: 333.14; found:
333.10 [M]+.
(S)-6-Hydroxyhexyl O-1-(2-nitrophenyl)ethyl carbonothioate (7a): 1-(2-
Nitrophenyl) ethanol (1a) (1.28 g, 7.66 mmol), DSC (2.94 g, 11.5 mmol)
and TEA (1.59 mL, 11.5 mmol) were dissolved in acetonitrile (20 mL)
under nitrogen. After stirring at room temperature for 1 h, DMAP
(1.40 g, 11.5 mmol) and 6-mercapto-1-hexanol (1.45 mL, 11.5 mmol) were
added. H2O (80 mL) was added to stop the reaction over another 15 min
whilst stirring. The water layer was exacted with dichloromethane (3ꢂ
50 mL). The organic phase was collected, dried and concentrated. The
residue was further purified by flash column chromatography with 2:1 pe-
troleum ether/ethyl acetate as the eluent to give 7a as a clear yellow oil
(1.32 g, 4.04 mmol, 57% yield). 1H NMR (400 MHz, CDCl3): d=7.94 (d,
1H, J=8 Hz), 7.64 (d, 2H, J=4 Hz), 7.42 (m, 1H), 6.41 (q, 1H, J=
6 Hz), 3.58 (t, 2H, J=6 Hz), 2.78 (m, 2H), 1.78 (brs, 1H), 1.65 (d, 3H,
J=7 Hz), 1.60–1.49 (m, 4H), 1.33 ppm (m, 4H); 13C NMR (100 MHz,
CDCl3): d=170.3, 147.2, 137.4, 133.8, 128.5, 127.0, 124.4, 70.9, 62.5, 32.3,
30.7, 29.5, 28.2, 25.1, 22.0 ppm; MS (ESI-TOF): m/z: calcd for
C14H21NO3S: 350.10; found: 350.15 [M]+.
6-(tert-Butyldimethylsilyloxy)hexanoic acid (4): tert-Butyldimethylsilyl
chloride (1.84 g, 12.2 mmol) and imidazole (800 mg, 11.8 mmol) were dis-
solved in DMF (10 mL). Ethyl 6-hydroxyhexanoate (650 mL, 4.00 mmol)
was added to the mixture with stirring under nitrogen at room tempera-
ture. After 24 h, TLC confirmed the reaction was completed. The reac-
tion mixture was then diluted with ether (50 mL). The organic layer was
washed with H2O (3ꢂ50 mL) and dried over Na2SO4. The solution was
then concentrated to give the residue as a yellow oil, which was then dis-
solved in a methanol solution of Triton B (40% w/w, 10 mL) and stirred
for 1 h at room temperature. Methanol was then removed and the resi-
due was diluted with H2O (20 mL), followed by the adjustment of solu-
tion acidity to pH 4 with 1m HCl. The aqueous phase was extracted by
ether (5ꢂ20 mL) and the combined ether solution was dried over
Na2SO4. Ether was removed to give the residue 4 as a yellow oil;
1H NMR (400 MHz, CDCl3): d=3.60 (t, 2H, J=6 Hz), 2.36 (t, 2H, J=
7 Hz), 1.65 (m, 2H), 1.53 (m, 2H), 1.40 (dd, 2H, J=4, 6 Hz), 0.89 (s,
9H), 0.04 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=179.1, 62.9, 33.9,
32.4, 26.0, 25.3, 24.5, 18.3, À5.3 ppm.
4-Hydroxybutan-2-one 1-(2-nitrophenyl)-1,2-diyl ketal (8a): A THF solu-
tion (15 mL) of 1-(2-nitrophenyl)ethane-1,2-diol (7, 183 mg, 1.0 mmol,
prepared by following
a
previous report[29, 52]), 4-hydroxybutan-2-one
(86 mL, 1.0 mmol) and p-toluenesulfonic acid (3.8 mg, 0.02 mmol) was re-
fluxed with molecular sieves in a Dean–Stark setup for 6 h. The reaction
solution was filtered and concentrated. The obtained residue was then
purified by flash column chromatography by eluting with 3:1 petroleum
ether/ethyl acetate to give 8a as a yellow oil (100 mg, 0.4 mmol, 40%
yield). 1H NMR (400 MHz, CDCl3): d=8.07 (d, 1H, J=8 Hz), 7.95 (d,
1H, J=8 Hz), 7.69 (t, 1H, J=8 Hz), 7.47 (dt, 1H, J=1,8 Hz), 5.66 (t,
1H, J=7 Hz), 4.75 (dd, 0.1H, J=7, 8 Hz), 4.68 (dd, 0.9H, J=7,8 Hz),
3.96 (m, 1H), 3.86 (m, 1H), 3.76 (m, 1H), 2.18 (m, 2H), 1.61 (s, 0.4H),
1.50 ppm (s, 2.6H); 13C NMR (100 MHz, CDCl3): d=147.3, 136.5, 134.2,
128.6, 127.6, 127.4, 124.8, 111.4, 73.7, 71.2, 59.0, 41.1, 23.0 ppm; MS (ESI-
TOF+): m/z: calcd: C15H22N2O5: 276.08; found: 276.06 [M]+.
1-(2-Nitrophenyl) ethyl 6-hydroxyhexanoate (5a): 1-(1-Bromoethyl)-2-ni-
trobenzene (2), Cs2CO3 (1.30 g, 3.99 mmol) and NaI (1.20 g, 8.00 mmol)
were dissolved in acetone (15 mL). tert-Butyldimethylsilyl chloride (4)
(1.25 g, 5.42 mmol) dissolved in anhydrous acetone (20 mL) was added to
the mixture with stirring. After refluxing for 16 h, the reaction was
stopped and the solvent was removed in vacuo. The residue was dissolved
General procedure for 1b, 3b, 5b–8b: Alcohol (1a, 3a, 5a, 6a, 8a)
(0.5 mmol, 1.0 equiv) was weighted into a round-bottomed flask. The
9634
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 9628 – 9637

Photocaging Strategy for Functionalisation of Oligonucleotides
FULL PAPER
flask was purged with nitrogen and dichloromethane (10 mL) and trie-
thylamine (3.0 equiv) were added to yield a clear colourless solution, fol-
lowed by the addition of 2-cyanoethyl diisopropylchlorophosphoramidite
(1.5 equiv). The reaction mixture was stirred at room temperature. After
disappearance of the alcohol, the reaction was stopped. The reaction so-
lution was then diluted with dichloromethane and washed with 0.1m
NaHCO3 (2ꢂ30 mL). The organic layer was collected and dried over
Na2SO4. The solvent was removed and the residue was purified by flash
column chromatography with petroleum ether/dichloromethane contain-
ing 3% triethylamine as the eluent to give 1b, 3b, 5b, 6b, 8b as a yellow
oil.
Product 1b: Yield: 71%; 1H NMR (400 MHz, CDCl3): d=7.80 (m, 2H),
7.59 (m, 1H), 7.35 (m, 1H), 5.46 (m, 1H), 3.81 (m, 1H), 3.60 (m, 2H),
3.45 (m, 1H), 2.62–2.46 (m, 2H), 1.52 (m, 3H), 1.15–0.86 ppm (m, 12H);
13C NMR (100 MHz, CDCl3): d=146.9 (m), 140.0 (m), 133.2 (m), 128.2
(m), 127.6 (m), 123.6 (m), 117.2 (m), 67.0 (dd, J=10 Hz), 58.2 (dd, J=
20 Hz), 42.9 (m), 24.9–24.0 (m), 23.8 (m), 19.9 ppm (m); 31P NMR
(162 MHz, CDCl3): d=147.7 (s), 147.3 ppm (s).
Product 3b: Yield: 59%; 1H NMR (400 MHz, CDCl3): d=7.92 (d, 1H,
J=8 Hz), 7.61 (d, 2H, J=4 Hz), 7.40 (m, 1H), 6.22 (q, 1H, J=6 Hz),
4.75 (br, 1H), 3.82 (m, 2H), 3.58 (m, 4H), 3.12 (m, 2H), 2.63 (t, 2H),
1.60 (d, 3H, J=6 Hz), 1.58 (m, 2H), 1.47 (m, 2H), 1.34 (m, 4H),
1.17 ppm (t, 12H, J=4 Hz); 13C NMR (100 MHz, CDCl3): d=155.2,
147.5, 138.7, 133.3, 128.0, 127.0, 124.2, 117.6, 68.3, 63.3 (d, J=17 Hz), 58.1
(d, J=19 Hz), 42.9 (d, J=12 Hz), 40.8, 30.9 (d, J=7 Hz), 29.7, 26.2, 25.4,
24.4 (d, J=8 Hz), 22.1, 20.2 ppm (d, J=7 Hz); 31P NMR (162 MHz,
CDCl3) d=147.2 ppm (s).
Product 5b: Yield: 63%; 1H NMR (400 MHz, CDCl3): d=7.91 (d, 1H,
J=8 Hz), 7.61 (d, 2H, J=4 Hz), 7.42 (m, 1H), 6.30 (t, 1H, J=6 Hz),
3.85–3.75 (m, 2H), 3.66–3.53 (m, 4H), 2.62 (t, 2H, J=6 Hz), 2.32 (m,
2H), 1.64–1.34 (m, 11H), 1.16 ppm (m, 12H); 13C NMR (100 MHz,
CDCl3): d=172.3, 147.8, 138.0, 133.4, 128.3, 127.1, 117.6, 67.8, 63.4, 63.2
(d, J=17 Hz), 58.2 (d, J=19 Hz), 43.0(d, J=12 Hz), 34.2, 30.8 (d, J=
8 Hz), 25.4, 24.5 (d, J=8 Hz), 21.9, 20.3 ppm (d, J=7 Hz); 31P NMR
(162 MHz, CDCl3) d=147.3 ppm (s).
Product 6b: Yield: 54%; 1H NMR (400 MHz, CDCl3): d=7.84 (d, 1H,
J=8 Hz), 7.71 (d, 1H, J=8 Hz), 7.58 (t, 1H, J=8 Hz), 7.34 (t, 1H, J=
8 Hz), 4.56 (q, 1H, J=7 Hz), 3.80 (m, 2H), 3.58 (m, 4H), 2.62 (t, 2H, J=
6 Hz), 2.30 (m, 2H0), 1.59 (d, 3H, J=7 Hz), 1.54 (m, 2 Hz), 1.44 (m,
2 Hz), 1.28 (m, 4 Hz), 1.16 ppm (t, 12H, J=7 Hz); 13C NMR (100 MHz,
CDCl3): d=149.4, 139.2, 132.8, 129.4, 127.5, 123.6, 117.6, 63.5 (d, J=
17 Hz), 58.3 (d, J=18 Hz), 43.0 (d, J=12 Hz), 38.2, 31.6, 30.9 (d, J=
7 Hz), 29.1, 28.4, 25.4, 24.5 (t, J=8 Hz), 22.9, 20.3 ppm (d, J=7 Hz);
31P NMR (162 MHz, CDCl3): d=147.3 ppm (s).
Product 7b: Yield: 73%; 1H NMR (400 MHz, CDCl3): d=7.95 (d, 1H,
J=8 Hz), 7.64 (d, 2H, J=4 Hz), 7.43 (m, 1H), 6.40 (q, 1H, J=8 Hz),
3.80 (m, 2H), 3.58 (m, 4H), 2.77 (m, 2H), 2.61 (t, 2H, J=10 Hz), 1.65 (d,
3H, J=8 Hz), 1.57 (m, 4H), 1.34 (m, 2H), 1.15 ppm (t, 12H, J=8 Hz);
13C NMR (100 MHz, CDCl3): d=170.3, 147.3, 137.5, 133.8, 128.5, 127.1,
124.4, 117.6, 70.9, 63.4 (d, J=17 Hz), 58.2 (d, J=18 Hz), 42.9 (d, J=
12 Hz), 30.9 (d, J=7 Hz), 30.8, 29.5, 28.2, 25.3, 24.5 (t, J=8 Hz), 24.4,
22.0, 20.3 ppm (d, J=7 Hz); 31P NMR (162 MHz, CDCl3): d=147.2 ppm
(s).
Product 8b: Yield: 74%; 1H NMR (400 MHz, CDCl3): d=8.04 (d, 1H,
J=8 Hz), 7.92 (d, 1H, J=8 Hz), 7.67 (t, 1H, J=8 Hz), 7.45(t, 1H, J=
8 Hz), 5.59 (m, 1H), 4.60 (t, 1H, J=8 Hz), 3.88–3.59 (m, 7H), 2.64 (t,
2H, J=3 Hz), 2.24 (t, 0.9H, J=7 Hz), 2.12 (t, 1.1H, J=7 Hz), 1.60 (s,
1.7H), 1.48 (s, 1.3H), 1.19 ppm (dd, 12H, J=4,6 Hz); 13C NMR
(100 MHz, CDCl3): d=147.3, 137.0, 136.6, 134.0, 128.4, 127.6, 124.6,
117.5, 110.5, 110.2, 74.3, 71.3, 59.6, 58.3, 43.1, 40.9, 39.5, 24.6, 23.5,
20.3 ppm; 31P NMR (162 MHz, CDCl3): d=147.7 ppm (s).
125.1, 49.6 ppm; MS (ESI-TOF+): m/z: calcd for C8H10N2O3: 205.06;
found: 205.06 [M]+.
Triethylammonium 6-[bis(4-methoxyphenyl)ACTHNUTRGNE(NUG phenyl)methoxy]hexanoate
(10): Ethyl 6-hydroxyhexanoate (200 mL, 1.23 mmol) and triethylamine
(430 mL, 3.08 mmol) were added to a solution of DMTr-Cl (500 mg,
1.48 mmol) in dichloromethane (10 mL). After stirring at room tempera-
ture for 1 h, the reaction mixture was washed with saturated aqueous
NaHCO3 (2ꢂ30 mL). The combined organic layer was dried over
Na2SO4. The solvent was removed and the residue was then dissolved in
the mixture of NaOH aqueous solution (6 molLÀ1, 25 mL) and ethanol
(4 mL). The mixture was stirred at room temperature and the reaction
was monitored by the disappearance of the ethyl ester by TLC analysis.
After completion, the mixture was diluted with CHCl3 (50 mL) and
washed with water (3ꢂ50 mL). The organic layer was concentrated and
the residue was purified by flash column chromatography with 3:7:1
methanol/dichloromethane/triethylamine as the eluent to give 10 as
a
white solid (550 mg, 1.08 mmol, 87% yield). 1H NMR (400 MHz,
CDCl3): d=7.42–7.18 (m, 10H), 6.80 (d, 4H, J=9 Hz), 3.78 (s, 6H), 3.47
(s, 1H), 3.01 (t, 2H, J=7 Hz), 2.84 (q, 6H, J=7 Hz), 2.21 (t, 2H, J=
8 Hz), 1.60 (m, 4H, J=8 Hz), 1.38 (dd, 2H, J=7,12 Hz), 1.16 ppm (t,
3H, J=7 Hz); 13C NMR (100 MHz, CDCl3): d=179.2, 158.3, 145.5, 136.8,
130.2, 128.2, 127.6, 126.5, 113.0, 85.6, 63.6, 55.2, 45.1, 36.5, 30.1, 26.4, 26.0,
9.5 ppm.
N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide (11a): 4-Pentynoic
acid (640 mg, 6.51 mmol), EDC·HCl (1.24 g, 6.47 mmol) and HOBt
(996 mg, 6.50 mmol) were dissolved in DMF (5 mL) and stirred at room
temperature for 1 h. Then 9 (988 mg, 5.40 mmol) in DMF (3 mL) was
added dropwise. The reaction mixture was stirred at room temperature
for another 3 h and was then diluted with ethyl acetate (50 mL). The
ethyl acetate solution was washed with H2O (3ꢂ50 mL) and dried over
Na2SO4. The solution was concentrated and the residue was purified by
flash column chromatography with 1:1 petroleum ether/ethyl acetate as
the eluent to give 11a as a faint yellow solid (1.13 g, 4.31 mmol, 80%
yield). 1H NMR (400 MHz, CDCl3): d=7.95 (m, 2H), 7.67
ACHTUNGTRENNUNG(m, 1H),
7.45(m, 1H, J=1, 8 Hz), 6.22 (s, 1H), 5.38 (t, 1H, J=4 Hz), 3.70 (dd,
2H, J=6 Hz). 2.55 (m, 2H), 2.46 (m, 2H), 2.02 ppm (m, 1H); 13C NMR
(100 MHz, CDCl3): d=173.8, 147.7, 137.5, 133.6, 128.8, 128.5, 124.5, 82.6,
70.8, 69.7, 47.8, 35.1, 14.9 ppm; MS (ESI-TOF+): m/z: calcd for
C13H14N2O4: 285.09; found: 285.08 [M]+.
N-{2-[Bis(4-methoxyphenyl)ACTHNUGTRNEUNG(phenyl)methoxy]-2-(2-nitrophenyl)ethyl}-4-
pentynamide (11b): N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide
(11a) (92 mg, 351 mmol) and DMTr-Cl (150 mg, 443 mmol) were dissolved
in pyridine (5 mL) and the reaction mixture was stirred at room tempera-
ture overnight. The solvent was removed and the residue was purified by
flash column chromatography with 1:1 petroleum ether/ethyl acetate con-
taining 1% triethylamine as the eluent to give 11b as a colourless oil
(154 mg, 273 mmol, 78% yield). 1H NMR (400 MHz, CDCl3) d=7.77 (d,
1H, J=8 Hz), 7.66 (d, 1H, J=8 Hz), 7.49–7.18 (m, 11H), 6.68 (dd, 4H,
J=9 Hz), 5.38 (t, 1H, J=5 Hz), 3.78 (d, 6H, J=8 Hz), 2.43 (m, 2H), 2.32
(m, 2H), 1.96 ppm (t, 1H, J=2 Hz); 13C NMR (100 MHz, CDCl3) d=
170.5, 158.6, 158.5, 146.8, 145.1, 138.1, 135.6, 135.3, 132.7, 130.2, 130.1,
130.0, 127.9, 127.8, 127.3, 126.9, 123.7, 113.2, 113.0, 87.6, 82.9, 69.8, 69.2,
46.2, 45.1, 35.3, 14.6 ppm; MS (ESI-TOF+): m/z: calcd for C34H32N2O6:
587.22; found: 587.26 [M]+.
1-(2-Nitrophenyl)-2-(4-pentynamido)ethyl
(12a):
6-hydroxyhexylcarbamate
N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide
(11a)
(131 mg, 500 mmol), DSC (154 mg, 600 mmol) and DMAP (74 mg,
600 mmol) were dissolved in CH3CN (7 mL) under nitrogen. The mixture
was stirred at room temperature for 30 min. The solution of 6-amino-1-
hexanol (70 mg, 600 mmol) in dichloromethane (5 mL) was then added
dropwise. After stirring at room temperature for another 10 min, the mix-
ture was concentrated and the residue was purified by flash column chro-
matography with 10:1 dichloromethane/triethylamine as the eluent to
give 12a as a yellow solid (150 mg, 370 mmol, 74% yield). 1H NMR
(400 MHz, CDCl3): d=7.93 (d, 1H, J=8 Hz), 7.63 (m, 2H), 7.42 (m,
1H), 6.65 (br, 1H), 6.19 (m,1H), 5.50 (br, 1H), 3.85 (m, 1H), 3.55 (m,
3H), 3.05 (dd, 2H, J=6,12 Hz), 2.49 (s, 1H), 2.43 (m, 2H), 2.35 (d, 2H,
J=14 Hz), 1.96 (s, 1H), 1.51–1.41 (m, 4H), 1.31–1.28 ppm (m, 4H);
2-Amino-1-(2-nitrophenyl) ethanol (9): 2-Amino-1-(2-nitrophenyl) etha-
nol (9) was synthesised according to the protocol published before with
modification[53] from o-nitroacetophenone as a faint yellow solid (40%
yield over 3 steps). 1H NMR (400 MHz, [D4]MeOH) d=7.80 (dd, 2H,
J=8,11 Hz), 7.62 (t, 1H, J=7 Hz), 7.40 (t, 1H, J=8 Hz), 5.06 (d, 1H, J=
3,8 Hz), 2.90 (dd, 1H, J=2,13 Hz), 2.64 ppm (dd, 1H, J=8,13 Hz);
13C NMR (100 MHz, [D4]MeOH) d=149.4, 139.7, 134.3, 129.5, 129.4,
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X. Tang et al.
13C NMR (100 MHz, CDCl3): d=171.4, 155.4, 134.4, 133.5, 128.8, 128.0,
124.6, 82.6, 71.0, 69.2, 62.3, 43.8, 40.8, 35.1, 32.3, 29.5, 26.2, 25.2,
14.7 ppm; MS (ESI-TOF+): m/z: calcd for C20H27N3O6: 428.18; found:
428.13 [M]+.
d=8.03–7.25 (m, 13H), 6.81 (d, 4H, J=9 Hz), 6.47 (dd, 1H, J=4,7 Hz),
6.08 (br, 1H), 3.88–3.81 (m, 2H), 3.77 (s, 6H), 3.02 (t, 2H, J=7 Hz), 2.78
(t, 2H, J=7 Hz), 2.45 (t, 2H, J=2 Hz), 2.44 (m, 2H), 1.99 (t, 2H, J=
3 Hz), 1.59–1.55 (m, 4H), 1.35–1.30 ppm (m, 4H); 13C NMR (100 MHz,
CDCl3) d=170.8, 170.6, 158.3, 147.7, 145.3, 136.6, 133.7, 133.2, 130.0,
129.2, 128.1, 127.9, 127.6, 126.5, 124.9, 112.9, 85.6, 82.8, 73.1, 69.5, 63.1,
55.1, 43.3, 35.2, 31.1, 29.8, 29.5, 28.5, 25.7, 14.7 ppm; MS (ESI-TOF+): m/
z: calcd for C41H44N2O8S: 747.28; found: 747.27 [M]+.
1-(2-Nitrophenyl)-2(4-pentynamido)ethyl
6-[bis(4-methoxyphenyl)-
ACHTUNGTRENNUNG(phenyl) methoxy]hexylcarbamate (12b): 1-(2-Nitrophenyl)-2-(4-pentyna-
mido)ethyl 6-hydroxyhexylcarbamate (12a) (97 mg, 240 mmol) and
DMTr-Cl (100 mg, 261 mmol) were dissolved in dichloromethane (5 mL)
under nitrogen to yield a purplish-red solution. Triethylamine (82 mL,
590 mmol) was added and the mixture was stirred at room temperature
for 30 min to generate a yellow solution. The reaction mixture was dilut-
ed with dichloromethane (40 mL) and the organic solution was then
washed with saturated NaHCO3 solution (2ꢂ50 mL). The combined di-
chloromethane solution was dried over Na2SO4. The solvent was removed
and the residue was purified by flash column chromatography with 1:1
petroleum ether/ethyl acetate containing 1% triethylamine as the eluent
Modification for LCAA-CPG: HBTU (38 mg, 100 mmol), DMAP (12 mg,
100 mmol) and 4-azidobutanoic acid (43 mg, 330 mmol) were dissolved in
DMF (1 mL). The mixture was shaken for 30 min and then added to
LCAA-CPG (100 mg, 99 mmolgÀ1, 9.9 mmol). The mixture was shaken at
room temperature for 24 h. Afterwards the solid phase was washed with
methanol (3ꢂ1 mL), dichloromethane (3ꢂ1 mL) and ether (1 mL) se-
quentially. The solid phase was then dispersed in DMSO (1 mL) with
11b/12b/13/14b (200 mmol), l-ascorbic acid sodium salt (20 mg,
100 mmol), copper(II) sulfate (8 mg, 50 mmol) and DIPEA (8 mL,
50 mmol). The mixture was stirred at 258C for 24 h. The solid phase was
washed with DMSO (1 mL), 0.1m NaHCO3 aq (1 mL), methanol (3ꢂ
1 mL), dichloromethane (3ꢂ1 mL) and ether (1 mL). The beads were
capped with Cap A and Cap B for 30 min at room temperature.
to give 12b as
a colourless foam (127 mg, 187 mmol, 78% yield).
1H NMR (400 MHz, CDCl3) d=7.97–7.17 (m, 13H), 6.81 (d, 4H, J=
9 Hz), 6.26 (br, 1H), 6.16 (m, 1H), 4.92 (br, 1H), 3.89 (m, 1H), 3.78 (s,
6H), 3.70 (m, 1H), 3.09 (m, 2H), 3.02 (m, 2H, J=6 Hz), 2.47 (t, 2H, J=
6 Hz), 2.37 (t, 2H, J=7 Hz), 1.96 (d, 1H, J=2 Hz), 1.72 (s, 1H), 1.57 (t,
2H, J=7 Hz),1.44 (t, 2H, J=7 Hz), 1.35 (t, 2H, J=7 Hz), 1.25 ppm (t,
3H, J=7 Hz); 13C NMR (100 MHz, CDCl3): d=171.0, 158.3, 155.2, 145.4,
136.7, 133.6, 130.0, 129.0, 128.2, 128.0, 127.7, 126.6, 124.7, 113.0, 85.6,
82.8, 71.1, 69.3, 63.2, 55.2, 44.0, 41.1, 35.3, 29.9, 29.7, 26.6, 25.9, 14.8,
14.2 ppm; MS (ESI-TOF+): m/z: calcd for C41H45N3O8: 730.31; found:
730.21 [M]+.
Loading efficiency: Modified CPGs were treated with 3% TFA in di-
chloromethane for 3 min at room temperature for deblocking. The load-
ing efficiency was calculated by the amount of DMTr cation in dichloro-
methane (lobs. =504 nm) relative to the amino group on LCAA-CPG pur-
chased.
Cleavage efficiency: Modified CPGs were irradiated for 30 min (365 nm,
11 mWcmÀ2) in acetonitrile/water (v/v 4/1, 6 mgmLÀ1) while stirring. The
solid phase was dried and the solution phase was concentrated in vacuo.
The cleavage efficiency was determined by the amount of DMTr cation
cleaved relative to the total DMTr on CPG.
1-(2-Nitrophenyl)-2-(4-pentynamidoethyl
(phenyl)methoxy] hexanoate (13): Triethylammonium 6-[bis(4-
methoxyphenyl)(phenyl)methoxy] hexanoate (10) (270 mg, 504 mmol),
6-[bis(4-methoxyphenyl)-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
DCC (210 mg, 800 mmol) and DMAP (18 mg, 80 mmol) were dissolved in
DMF. After stirring at room temperature for 1 h, 11a (100 mg, 382 mmol)
was added and the reaction mixture was further stirred at 708C for an-
other 3 h. After the reaction was stopped, the reaction mixture was dilut-
ed with ethyl acetate (50 mL). The organic layer was washed with H2O
(3ꢂ50 mL) and dried over Na2SO4. After removal of solvent, the residue
was purified by flash column chromatography with 1:1 petroleum ether/
ethyl acetate containing 1% triethylamine as the eluent to give 13 as
General method for oligonucleotide synthesis: Oligonucleotides were
synthesised by using an Applied Biosystems Incorporated 394 automated
synthesiser. Long-chain alkylamine controlled pore glass support was pur-
chased from Millipore Corporation. CPG:dG and phosphoramidite mon-
omers were purchased from AuGCT Biotech. All oligodeoxynucleotides
were synthesised on a 1 mmol scale with standard DNA synthesis cycles
on CPG:dG (ODN 1–6) or modified LCAA-CPG (ODN 7–11). For the
terminal coupling of ODN 1–6, the coupling time was extended to 240 s.
1
a yellow oil (121 mg, 185 mmol, 48% yield). H NMR (400 MHz, CDCl3):
d=7.90–7.08 (m, 13H), 6.73 (d, 4H, J=8 Hz), 6.28 (m, 1H), 6.00 (br,
1H), 4.03 (q, 1H, J=7 Hz), 3.82 (m, 1H), 3.70 (s, 6H), 2.39–2.23 (m,
6H), 1.96 (s, 2H), 1.88 (s, 1H), 1.53–1.25 ppm (m, 6H); 13C NMR
(100 MHz, CDCl3): d=172.6, 170.9, 158.3, 148.0, 145.3, 136.5, 133.6,
133.5, 129.9, 129.1, 129.0, 128.1, 128.0, 127.8, 127.7, 127.6, 126.5, 124.6,
113.1, 112.9, 85.6, 82.8, 70.2, 69.3, 63.0, 60.3, 55.1, 43.5, 35.1, 34.0, 25.8,
25.1, 24.6, 24.3, 14.7, 14.1 ppm; MS (ESI-TOF+): m/z: calcd for
C40H42N2O8: 701.28; found: 701.21 [M]+.
Semi-preparation HPLC was carried out with Agilent C18 column (5 mm,
9.4ꢂ250 mm) on Varian Prostar. Conditions: solvent A, 0.05m TEAA
buffer; solvent B, acetonitrile. Started at 15% B; linear gradient to 50%
B over 35 min, flow rate: 1 mLminÀ1. HPLC analyses were performed
with Symmetry C18 column (5 mm, 9.4ꢂ250 mm) on Waters alliance
e2695 started at 0% B; linear gradient to 30% B over 30 min. MS was
measured either on ESI-Q-TOF or MALDI-TOF.
(S)-6-Hydroxyhexyl-O-1-(2-nitrophenyl)-2-(4-pentynamido)ethylcarbono-
Photolysis: All samples were irradiated in a quartz cell with a stirrer bar
by UVP high intensity inspection UV Lamp (11 mWcmÀ2) at 365 nm for
30 min or less. The irradiated samples were maintained at ꢀ308C with an
ice bag.
thioate
(14a):
N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide
(11a) (199 mg, 400 mmol), DSC (123 mg, 480 mmol) and DMAP (60 mg,
480 mmol) were dissolved in acetonitrile (10 mL) under nitrogen. The
mixture was stirred at room temperature for 30 min and then 6-mercap-
to-1-hexanol (65 mL, 480 mmol) was added. After another 1.5 h, the mix-
ture was concentrated and the residue was purified by flash column chro-
matography with 1:1 petroleum ether/ethyl acetate containing 1% trie-
thylamine as the eluent to give 14a as a yellow oil (123 mg, 292 mmol,
77% yield). 1H NMR (400 MHz, CDCl3) d=8.01 (d, 1H, J=8 Hz), 7.65
(m, 2H), 7.48 (dd, 1H, d=2,4 Hz), 6.45 (dd, 1H, J=4,7 Hz), 6.18 (br,
1H), 3.90–3.79 (m, 2H), 3.62 (dd, 2H, J=2,7 Hz), 2.81 (t, 2H, J=7 Hz),
2.44 (m, 2H), 2.37 (m, 2H), 1.72–1.45 ppm (m, 8H); 13C NMR (100 MHz,
CDCl3) d=171.0, 170.7, 147.7, 133.8, 133.1, 129.2, 127.9, 124.9, 82.8, 73.2,
69.4, 62.7, 43.3, 39.0, 35.2, 32.5, 31.0, 29.4, 28.2, 29.2, 25.5, 14.7 ppm; MS
(ESI-TOF+): m/z: calcd for C20H26N2O6S: 445.14; found: 445.15 [M]+.
Hydrolysis: Concentrated ammonium hydroxide (500 mL) was added to
dry ODNs or CPGs, shaken for 24 h at room temperature, and was then
concentrated by Thermo Savant SPD 2010 SpeedVac System.
DMTr removal: 80% aqueous acetic acid (500 mL) was added to dry
ODNs, shaken for 30 min at room temperature, and was then concentrat-
ed.
Attachment ODN 2 with 5-TAMRA-NHS: ODN 2 on CPG was sus-
pended in acetonitrile/water (v/v 4/1, 6 mgmLÀ1) in a quartz cell. After
365 nm UV irradiation for 30 min, the CPG was washed with acetonitrile,
dried and suspended in DMF (500 mL) and 0.1m NaHCO3, followed by
the addition of 5-TAMRA-NHS (10 equiv relative to the amount of
oligodeoxynucleotide on ODN approximately) for 24 h. The solid-phase
CPG was washed with DMF (3ꢂ500 mL) and acetonitrile (3ꢂ500 mL).
After deprotection and HPLC purification, the desired TAMRA conju-
gated ODN was obtained in 44% yield according to HPLC trace at
260 nm. MALDI-TOF: m/z: calcd: 5345; found: 5345.
(S)-6-[Bis(4-methoxyphenyl)ACHTNUGTRENNUNG
2-(4-pentynamido)ethyl
methoxyphenyl)ACHTUNGTRENNUNG(phenyl)methoxy]hexyl-O-1-(2-nitrophenyl)-2-(4-penty-
namido)ethyl carbonothioate (14b) was obtained by a similar procedure
as 12a to 12b as a yellow oil (76% yield). 1H NMR (400 MHz, CDCl3):
9636
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 9628 – 9637

Photocaging Strategy for Functionalisation of Oligonucleotides
FULL PAPER
Attachment ODN 3 with pyrenemethylamine: ODN 3 on CPG was sus-
pended in acetonitrile/water (v/v 4/1, 6 mgmLÀ1) in a quartz cell. After
365 nm UV irradiation for 30 min, the CPG was washed with acetonitrile
and dried. The residue was suspended in a DMSO solution (100 mL) of
BOP, HOBt, DIPEA and pyrenemethylamine hydrochloride (10 equiv).
After having been shaken for 24 h at room temperature, the solid phase
was washed with DMF (3ꢂ500 mL), acetonitrile (3ꢂ500 mL) and ether
(500 mL). After hydrolysis and HPLC purification, the desired ODN con-
jugated with pyrene was obtained in 82% yield. MALDI-TOF m/z:
calcd: 5162; found: 5163.
[16] F.-G. Rong, A. H. Soloway, Nucleosides Nucleotides Nucleic Acids
1994, 13, 2021.
[17] J.-T. Hwang, K. A. Tallman, M. M. Greenberg, Nucleic Acids Res.
1999, 27, 3805.
[18] M. A. Podyminogin, E. A. Lukhtanov, M. W. Reed, Nucleic Acids
Res. 2001, 29, 5090.
[19] H. Urata, M. Akagi, Tetrahedron Lett. 1993, 34, 4015.
[20] K. Lu, Q. Duan, L. Ma, D. Zhao, Bioconjugate Chem. 2010, 21, 187.
[21] X. Wu, S. Pitsch, Bioconjugate Chem. 1999, 10, 921.
[22] G. P. Miller, E. T. Kool, J. Org. Chem. 2004, 69, 2404.
[23] E. Bonfils, C. Depierreux, P. Midoux, N. T. Thuong, M. Monsigny,
A. C. Roche, Nucleic Acids Res. 1992, 20, 4621.
[24] D. R. Corey, J. Am. Chem. Soc. 1995, 117, 9373.
[25] G. Tong, J. M. Lawlor, G. W. Tregear, J. Haralambidis, J. Org. Chem.
1993, 58, 2223.
[26] R. K. Bruick, P. E. Dawson, S. B. H. Kent, N. Usman, G. F. Joyce,
Chem. Biol. 1996, 3, 49.
[27] J.-C. Truffert, U. Asseline, A. Brack, N. T. Thuong, Tetrahedron
1996, 52, 3005.
[28] D. L. McMinn, M. M. Greenberg, J. Am. Chem. Soc. 1998, 120,
3289.
[29] X. Tang, M. Su, L. Yu, C. Lv, J. Wang, Z. Li, Nucleic Acids Res.
2010, 38, 3848.
[30] G. Mayer, A. Heckel, Angew. Chem. 2006, 118, 5020; Angew. Chem.
Int. Ed. 2006, 45, 4900.
[31] A. Deiters, Curr. Opin. Chem. Biol. 2009, 13, 678.
[32] X. Tang, I. J. Dmochowski, Mol. BioSyst. 2007, 3, 100.
[33] D. D. Young, A. Deiters, Org. Biomol. Chem. 2007, 5, 999.
[34] W. Lin, C. Albanese, R. G. Pestell, D. S. Lawrence, Chem. Biol.
2002, 9, 1347.
Attachment ODN 8 with 1-pyrenebutyric acid: ODN 8 on CPG was sus-
pended in acetonitrile/water (v/v 4/1, 6 mgmLÀ1) in a quartz cell. After
365 nm UV irradiation for 30 min, the solution was evaporated to dry-
ness. The solution of BOP, HOBt, DIPEA and 1-pyrenebutyric acid in
DMF (100 mL) was added to the dry power and the mixture was then
shaken for 24 h at room temperature. After evaporation, hydrolysis and
desalting, the HPLC trace showed 85% of labelling yield. After detrityla-
tion, MALDI-TOF confirmed the expected ODN: calcd: 4872; found:
4874.
Cyclisation of ODN 11: ODN 11 on CPG was suspended in acetonitrile/
water (v/v 4/1, 6 mgmLÀ1) in a quartz cell. After 365 nm UV irradiation
for 30 min, the solution was evaporated to dryness. The solution of BOP
(or HBTU/EDC·HCl for the comparison), HOBt and DIPEA in DMF
(100 mL) was added to the dry power,. The mixture was shaken for 24 h
at room temperature. After evaporation, hydrolysis, desalting and HPLC
purification, MALDI-TOF confirmed the desired fractions: linear: calcd:
4796; found: 4799; circular: calcd: 4779; found: 4783.
[35] B. K. Ruble, J. L. Richards, J. C. Cheung-Lau, I. J. Dmochowski,
Inorg. Chim. Acta 2012, 380, 386.
Acknowledgements
[36] D. L. McMinn, R. Hirsch, M. M. Greenberg, Tetrahedron Lett. 1998,
39, 4155.
[37] D. L. McMinn, M. M. Greenberg, Tetrahedron 1996, 52, 3827.
[38] J. D. Kahl, D. L. McMinn, M. M. Greenberg, J. Org. Chem. 1998, 63,
4870.
This work is supported by the National Natural Science Foundation of
China (grant no. 21072015), the National Basic Research Program of
China (973 Program; grant no. 2012CB720600 and Program for New
Century Excellent Talents in University (NCET-10–0203).
[39] D. J. Yoo, M. M. Greenberg, J. Org. Chem. 1995, 60, 3358.
[40] J. D. Kahl, M. M. Greenberg, J. Org. Chem. 1999, 64, 507.
[41] P. Pan, H. Bayley, FEBS Lett. 1997, 405, 81.
[42] C. Chang, T. Fernandez, R. Panchal, H. Bayley, J. Am. Chem. Soc.
1998, 120, 7661.
[43] Photosensitive Molecules for Controlling Biological Function (Eds.:
J. J. Chambers, R. H. Kramer), Humana Press, 2011, p. 66.
[44] D. M. Rothman, M. E. Vazquez, E. M. Vogel, B. Imperiali, Org.
Lett. 2002, 4, 2865.
[45] J. F. Cameron, J. M. J. Frechet, J. Am. Chem. Soc. 1991, 113, 4303.
[46] N. D. Sinha, R. M. Cook, Nucleic Acids Res. 1988, 16, 2659.
[47] A. Barth, J. E. T. Corrie, M. J. Gradwell, Y. Maeda, W. Maentele, T.
Meier, D. R. Trentham, J. Am. Chem. Soc. 1997, 119, 4149.
[48] M. Ghosh, I. Ichetovkin, X. Song, J. S. Condeelis, D. S. Lawrence, J.
Am. Chem. Soc. 2002, 124, 2440.
[1] N. Venkatesan, B. H. Kim, Chem. Rev. 2006, 106, 3712.
[2] A. Sassolas, B. D. Leca-Bouvie, L. J. Blum, Chem. Rev. 2008, 108,
109.
[3] N. L. Rosi, C. A. Mirkin, Chem. Rev. 2005, 105, 1547.
[4] A. San Juan, M. Bala, H. Hlawaty, P. Portes, R. Vranckx, L. J. Feld-
man, D. Letourneur, Biomacromolecules 2009, 10, 3074.
[5] T. Horn, M. S. Urdea, Tetrahedron Lett. 1986, 27, 4705.
[6] J. E. Celebuski, C. Chan, R. A. Jones, J. Org. Chem. 1992, 57, 5535.
[7] P. A. Guzaev, H. Salo, A. Azhayev, H. Lonnberg, Tetrahedron 1995,
51, 9375.
[8] N. Karino, Y. Ueno, A. Matsuda, Nucleic Acids Res. 2001, 29, 2456.
[9] B. A. Connolly, Nucleic Acids Res. 1987, 15, 3131.
[10] T. W. Greene, P. G. M. Wuts, 3rd ed., John Wiley & Sons Inc., New
York, 1999.
[11] B. A. Connolly, P. Rider, Nucleic Acids Res. 1985, 13, 4485.
[12] C. R. Petrie, M. W. Reed, A. D. Adams, J. R. B. Meyer, Bioconjugate
Chem. 1992, 3, 85.
[49] E. Trꢃvisiol, E. Defrancq, J. Lhomme, A. Laayoun, P. Cros, Tetrahe-
dron 2000, 56, 6501.
[50] S. Dey, T. L. Sheppard, Org. Lett. 2001, 3, 3983.
[51] X. Tang, I. J. Dmochowski, Org. Lett. 2005, 7, 279.
[52] D. Gravelj, J. Hebert, D. Thoraval, Can. J. Chem. 1983, 61, 400.
[53] S. R. Ram, K. P. Chary, D. S. Iyengar, Synth. Commun. 2000, 30,
4495.
[13] M. Antopolsky, A. Azhayev, Nucleosides Nucleotides Nucleic Acids
2001, 20, 539.
[14] J. Sharma, R. Chhabra, S. C. Andersen, V. K. Gothelf, H. Yan, Y.
Liu, J. Am. Chem. Soc. 2008, 130, 7820.
[15] J. R. Stokes, A. Macaskill, A. J. Dougan, G. P. Hargreaves, M. H.
Stanford, E. W. Smith, K. Faulds, D. Graham, Chem. Commun.
2007, 2811.
Received: December 6, 2011
Revised: May 14, 2012
Published online: July 5, 2012
Chem. Eur. J. 2012, 18, 9628 – 9637
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9637