Red light-activated phosphorothioate oligodeoxyribonucleotides

Article abstract of DOI:10.1016/j.bmcl.2008.06.079

Hairpin-structured phosphorothioate oligodeoxyribonucleotides containing a singlet oxygen-sensitive linker in the loop were prepared. These compounds do not bind complementary nucleic acids in the dark. Upon irradiation with red light in the presence of chlorine e6 the linker within these compounds is cleaved and a single-stranded oligodeoxyribonucleotide is produced. The latter compound is an efficient binder of complementary nucleic acids. This is the first example of 'caged' phosphorothioate oligodeoxyribonucleotides, whose nucleic acid binding ability is triggered by red light.

Full text of DOI:10.1016/j.bmcl.2008.06.079

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4336–4338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Red light-activated phosphorothioate oligodeoxyribonucleotides
*
Alexandru Rotaru, János Kovács, Andriy Mokhir
Inorganic Chemistry Institute, University of Heidelberg, In Neuenheimer Feld 270, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Hairpin-structured phosphorothioate oligodeoxyribonucleotides containing a singlet oxygen-sensitive
linker in the loop were prepared. These compounds do not bind complementary nucleic acids in the dark.
Upon irradiation with red light in the presence of chlorine e6 the linker within these compounds
is cleaved and a single-stranded oligodeoxyribonucleotide is produced. The latter compound is an
efficient binder of complementary nucleic acids. This is the first example of ‘caged’ phosphorothioate
oligodeoxyribonucleotides, whose nucleic acid binding ability is triggered by red light.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 8 February 2008
Revised 23 June 2008
Accepted 25 June 2008
Available online 28 June 2008
Keywords:
Phosphorothioate oligodeoxyribonucleotide
Red light activation
Singlet oxygen
Nucleic acid binder
‘Caged’ nucleic acids are compounds, which are not biologically
active until uncaged by light. They may be used for spatially and
temporally controlled photoregulation of biological processes.
With few exceptions all reported ‘caged’ antisense oligodeoxyribo-
nucleotides (ODNs), siRNAs, and plasmids, are activated by UV-
light.1 Light of this type is strongly absorbed by cellular compo-
nents and is highly toxic to cells.2 Haselton and co-workers have
demonstrated that UV-light itself may inhibit gene expression.3
This limits applications of ‘caged’ agents in cells and in vivo. We
have recently reported ‘caged’ ODNs, which can be activated by
light in any chosen spectral region, including, for example, red
light.4 Red light is significantly less toxic than UV-light and can
deeply permeate into tissues.5
Herein, we expand our original concept to ‘caged’ phosphoro-
thioate ODNs (PT-ODNs). In contrast to their natural counterparts,
PT-ODNs are more suitable for cellular applications. These com-
pounds are formally obtained by substitution of one oxygen atom
in the phosphodiester group of the natural backbone for a sulfur
atom. PT-ODNs are stable in the presence of cellular hydrolytic en-
zymes and permeate the cellular membrane better than ODNs.6
These compounds are used as antisense agents and immunostimu-
lants.7 Correspondingly, ‘caged’ PT-ODNs may be potentially used
for light-controlled regulation of gene expression and activity of
immune system.
is produced.8 It induces cleavage of the linker that leads to forma-
tion of an unstable intermolecular duplex. The latter duplex disso-
ciates forming the biologically active A strand.
We have used p-hydroquinone ether and 1,2-dithioethylene
fragments as linkers. These moieties were expected to be sensitive
to 1O2.4,9 They were introduced within PT-ODNs using phospho-
ramidites L1 and L2. L1 was synthesized in accordance with
Scheme 1. First, 4,40-dihydroxydiphenyl ether was alkylated by 3-
bromopropan-1-ol, K2CO3 mixture in acetone to obtain 4,40-(3-
hydroxypropan-1-oxy)diphenyl ether. One of the hydroxyl groups
of the latter compound was protected with 4,40-dimethoxytrityl
group and another one was phosphatylated to obtain phospho-
ramidite L1. Phosphoramidite L2 was prepared as described else-
where.4 PT-ODNs 1 and 2 were prepared on an automated DNA
synthesizer. Beaucage sulfurizing reagent was used in place of
the oxidizer solution.
ODNs attached to controlled pore glass (CPG) were cleaved from
the support and deprotected using aqueous ammonia (20%, 24 h,
L
red light
PS
A concept of photo-activated PT-ODNs is presented in Figure 1.
In the ‘caged’ PT-ODN sequence A is blocked by sequence B. The
loop of this compound contains a 1O2-sensitive linker. In the pres-
ence of a photosensitizer (PS) and upon illumination with light, 1O2
A
B
Active PT-ODN
"Caged" PT-ODN
Figure 1. A concept of red light-activated PT-ODNs: PS is a red light-absorbing
photosensitizer; L is a 1O2-sensitive linker; A is a biologically active sequence; B is a
blocker sequence.
* Corresponding author. Tel.: +49 6221 548441; fax: +49 6221 548439.
E-mail address: andriy.mokhir@urz.uni-heidelberg.de (A. Mokhir).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.079

A. Rotaru et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4336–4338
4337
a
O
O
HO
O
3
OH
57 %
O
OH
3
HO
b
S
S
O
3
3
O
DMT
O
3
O
P
(i-Pr)2N
L2
O
OCNE
DMT
O
3
Figure 3. Photocleavage of PT-ODN2 in the presence of chlorine e6 monitored by
MALDI-TOF mass spectrometry; spectrum 1, irradiated probe; spectrum 2, probe
kept in the dark; a mixture of 6-aza-2-thiothymine (ATT, saturated solution in
CH3CN) with diammonium citrate (0.1 M in water), 2/1 (v/v) was used as a matrix,
matrix/probe 2/1 (v/v); IS is an internal standard, PT-ODN4. Other experimental
details are given in the text. Nature of fragments A and B is clear from Fig. 1.
14 %
(i-Pr)2N
O
P
L1
OCNE
PT-ODN1: TCG AGC GTT CTC L1 ACG CTC GA
PT-ODN2: TCG AGC GTT CTC L2 ACG CTC GA
PT-ODN3: TCG AGC GTT CTC
conditions. Therefore, it was not further studied. In contrast, PT-
ODN2 containing 1,2-dithioethylene moiety is cleanly cleaved
forming expected fragments A and B (Figs. 1 and 3). Additional
peaks in the mass spectrum of the irradiated mixture correspond
to gas phase adducts of the PT-ODNs with ATT matrix (A-ATT, B-
ATT, Fig. 3). When PT-ODN2 is kept in the dark it is stable for at
least 48 h.
PT-ODN4: ACG CTC GA
c_ODN: TAMRA~GAG AAC GCT CGA
c_RNA: TAMRA~UUU UGA GAA CGC UCG AUU UU
Scheme 1. Synthesis of phosphoramidite L1 and sequences of PT-ODNs and
complementary to them c_ODN; TAMRA
N,N,N0,N0-tetramethylrhodamine.
Reagents and conditions: (a) Br(CH2)3OH, K2CO3, acetone; (b) 1—DMT-Cl, pyridine,
Length of the blocking sequence B was optimized to achieve
that (a) PT-ODN2 exists in solution in the hairpin form and (b)
products of PT-ODN2 photocleavage (compounds A and B) do
not bind to each other at 22 °C. PT-ODN2 with an 8-mer blocking
sequence exhibited the optimal properties. In particular, melting
point (Tm) of PT-ODN2 in phosphate buffer (10 mM) containing
NaCl (150 mM) is well above 22 °C: 65.0 0.4 °C. Moreover, it is
not dependent on the concentration of the PT-ODN. The latter fact
excludes the possibility of formation of intermolecular associates
in the PT-ODN2 solution, while the former one indicates that
ꢀ100% of PT-ODN2 exists in the hairpin form at 22 °C. Furtheron,
products of PT-ODN2 photocleavage do not bind to each other at
room temperature, since the duplex formed between PT-ODN3
(sequence A) and PT-ODN4 (sequence B) melts below 20 °C. Thus,
–
2—ClP(OCH2CH2CN)N(i-Pr)2, DIEA; CNE: 2-cyanoethyl.
Figure 2. Purity of HPLC purified PT-ODN2; (A) MALDI-TOF mass spectrum; 2,4,6-
trihydroxyacetophenone (0.3 M in CH3CN), diammonium citrate (0.1 M in water),
2/1 (v/v) was used as a matrix, matrix/probe 2/1 (v/v) (B) HPLC profile; gradient and
other conditions are given in Ref. 10.
25 °C) and then purified by HPLC. Fractions containing >90% pure
PT-ODNs were combined and lyophilized (Fig. 2).10
Photodecomposition of the linkers within the PT-ODNs was
studied by using MALDI-TOF mass spectrometry and HPLC. In the
typical assay a mixture of PT-ODN (5 lM) and chlorine e6 (1 equiv)
in ammonium acetate buffer (150 mM, pH 7), which contained PT-
ODN4 as an internal standard (IS), was exposed to red light (mer-
cury lamp, red filter) for 1.5 h. Then the sample was treated with
dithiotreitol (DTT) (10 mM) and analyzed by mass spectrometry
(Fig. 3). Chlorine e6 was selected since this photosensitizer is effi-
ciently excited by red light (kmax ꢀ410 and 650 nm), is soluble in
water and has good membrane permeability. The latter property
is important for cellular applications of ‘caged’ PT-ODNs. PT-
ODN1 containing the hydroquinone ether moiety is stable at these
Figure 4. Gel-electrophoresis (native conditions, 20% acrylamide); in all lanes:
acetate buffer 100 mM, pH 7, DTT 10 mM, NaCl 1 M. Lane 1, c_RNA (1
negative control; lane 2, c_RNA (1 M), PT-ODN3 (10 M) – positive control; lane
3: c_RNA (1 M), PT-ODN2 (10 M); chlorine e6 (50 M), kept in the dark for 1.5 h;
lane 4: c_RNA (1 M), PT-ODN2 (10 M); chlorine e6 (50 M), irradiated with red
light for 1.5 h; lane 5, c_ODN (1 M), PT-ODN2 (10 M); chlorine e6 (50 M), kept
in the dark for 1.5 h; lane 6, c_ODN (1 M), PT-ODN2 (10 M); chlorine e6 (50 M),
irradiated with red light for 1.5 h; lane 7, c_ODN (1 M), PT-ODN3 (10 M) –
positive control; lane 8: c_ODN (1 M) – negative control.
lM) –
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l

4338
A. Rotaru et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4336–4338
References
O
O
1O2
1. Monroe, W. T.; Haselton, F. R. Light reversible suppression of DNA bioactivity
with cage compounds. In Dynamic Studies in Biology Phototriggers, Photoswitches
and Caged Biomolecules; Goeldner, M., Givens, R., Eds.; Wiley-VCH: New York,
2005; p 513.
S
S
S
S
2. Meunier, J.-R.; Sarasin, A.; Marrot, L. Photochem. Photobiol. 2002, 755, 437.
3. Monroe, W. T.; McQuain, M. M.; Chang, M. S.; Alexander, J. S.; Haselton, F. R. J.
Biol. Chem. 1999, 274(30), 20895.
4. Rotaru, A.; Mokhir, A. Angew. Chem., Int. Ed. 2007, 46(32), 6180.
5. Eichler, J.; Knof, J.; Lenz, H. Radiat. Environ. Biophys. 1977, 14(3), 239.
6. Eckstein, F. Ann. Rev. Biochem. 1985, 54, 367.
7. a Krieg, A. M. Annu. Rev. Immunol. 2002, 20, 709; b Agarwal, S.; Kandimalla, E. R.
Curr. Cancer Drug Targets 2001, 1, 197.
8. Szacilowski, K.; Macyk, W.; Drzewiecka-Matuszek, A.; Brindell, M.; Stochel, G.
Chem. Rev. 2005, 105, 2647.
H2O
or DTT
O
O
+
SH
HS
H
H
+
S
S
Scheme 2. A mechanism of singlet oxygen induced decomposition of ASCH@CHSA
group in PT-ODN2.
9. a Baugh, S. D. P.; Yang, Z.; Leung, D. K.; Wilson, D. M.; Breslow, R. J. Am. Chem.
Soc. 2001, 123, 12488; b Setsukinai, K. I.; Urano, Y.; Kikuchi, K.; Higuchi, T.;
Nagano, T. J. Chem. Soc., Perkin Trans 2 2000, 2453.
10. Synthesis of 4,4’-(3-hydroxypropan-1-oxy)diphenyl ether: A suspension of 4,40-
dihydroxydiphenyl ether (1.0 g, 4.9 mmol) and K2CO3 (1.4 g, 9.8 mmol) in
acetone (10 mL) was warmed up to 50 °C. Then solution of 3-brom-1-propanol
(0.9 mL, 10.3 mmol) in acetone (5 mL) was slowly added. The reaction mixture
was stirred overnight at reflux temperature. After addition of water (10 mL)
the reaction mixture was heated until everything was dissolved. The product
was crystallized from this solution upon cooling down to 22 °C. The white
crystals were filtered, washed with cold water, and dried in vacuo. Yield 0.89 g
(57%); 1H NMR (MeOD, 200 MHz) d 1.98 (m, 4H), 3.75 (t, 4H, J = 6.2), 4.06 (t, 4H,
J = 6.2), 6.88 (d, 8H, J = 0.6); 13C NMR (MeOD, 50 MHz) d33.5, 59.6, 66.3, 116.6,
120.5, 153.1, 156.3; Calcd. for C18H23O5 [M+H]+ 319.2; found (ESI-MS+): 319.2.
Synthesis of 3-[4-(4-{3-[Bis-(4-methoxyphenyl)-phenyl-methoxy]-propoxy}-
phenoxy)-phenoxy]-propan-1-ol: A mixture of 4,40-bis[(3-phenoxy-propan-1-
ol)] ether (0.70 g, 2.2 mmol) and 4-dimethylaminopyridine (DMAP, 0.18 g,
1.5 mmol) was dissolved in pyridine (25 mL) and diisopropylethylamine (DIEA,
compound A is expected to be in the single stranded, biologically
active form after photoactivation of PT-ODN2.
UV-melting data were corroborated by gel-electrophoresis
experiments (Fig. 4). One observes that sequence A in PT-ODN2
is blocked and can bind neither complementary RNA (c_RNA) nor
DNA (c_ODN), whereas the product of red light-induced PT-
ODN2 photocleavage forms a stable duplex with both RNA and
DNA targets.
The mechanism of L2 cleavage is illustrated in Scheme 2. 1O2 is
first produced in the result of red light-induced excitation of chlo-
rine e6 to the triplet state followed by its relaxation to the ground
state via energy transfer to 3O2. Singlet oxygen forms 2 + 2 addition
product with the ASCH@CHSA fragment of PT-ODN2. This product
is unstable. It decomposes to formic acid thioesters, which are
transformed in the presence of DTT or water into thiols.4 It has
been reported that singlet oxygen can be quenched by sulfur-
containing compounds.11 Therefore, photoactivation of ‘caged’
PT-ODNs could be slowed down due to interactions of the phosp-
horothioate groups with singlet oxygen. Fortunately, this effect
seems to be less important. For example, we observed that 1O2-in-
duced activation of ‘caged’ phosphorothioate ODNs is only 1.5
times slower than that of ‘caged’ natural ODNs.
In summary, we have prepared ‘caged’ phosphorothioate oligo-
deoxyribonucleotides. These compounds are inert in the dark,
whereas they are activated by red light in the presence of chlorine
e6. The ‘uncaged’ form of PT-ODNs is an efficient binder of single-
stranded nucleic acids. This is the first example of phosphorothio-
ate oligodeoxyribonucleotides, whose nucleic acid binding ability
is triggered by red light.
255 l
L, 1.5 mmol) was added. Then 4,40-dimethoxytrityl chloride (0.73 g,
2.2 mmol) in pyridine (15 mL) was added to the reaction mixture over 40 min.
The solution was stirred overnight at 22 °C. Volatile components were removed
and the residue was co-evaporated twice with toluene (15 mL, each portion).
The crude product was purified by column chromatography (silica gel, ethyl
acetate:hexane:triethylamine (TEA) = 69:30:1; Rf = 0.35). Yield 0.26 g (19%). 1
H
NMR (CDCl3, 200 MHz) d 2.00 (m, 4H), 3.22 (t, 2H, J = 6.0), 3.73 (s, 6H), 3.82 (t,
2H, J = 6.0), 4.06 (t, 2H, J = 5.8), 4.09 (t, 2H, J = 7.2), 6.71–6.89 (m, 12H), 7.10–
7.32 (m, 7H), 7.34–7.42 (m, 2H).
Synthesis of L1 (all operations were conducted at anaerobic conditions): The
above described alcohol (0.26 g, 0.4 mmol) was dissolved in CH2Cl2
(10 mL) and DIEA (0.4 mL, 2.3 mmol) was added. 2-Cyanoethyldiisopropyl-
amidochloridophosphite (0.3 mL, 1.3 mmol) was added slowly. After 2 h the
reaction mixture was poured into saturated aqueous solution of NaHCO3
(15 mL) and extracted with CH2Cl2 (3ꢁ 15 mL). The combined organic layers
were dried over magnesium sulfate. Volatile components were removed. The
crude product was purified by column chromatography (silica gel, ethyl
acetate:hexane:TEA = 68:30:2; Rf = 0.71). Yield 0.26 g (76%). 31P NMR (CDCl3) d
147.7; 1H NMR (CDCl3, 400 MHz) d 1.16 (t, 12H, J = 6.4), 1.96–2.09 (m, 4H), 2.59
(t, 2H, J = 6.4), 3.25 (t, 2H, J = 6.0), 3.53–3.61 (m, 2H), 3.76 (s, 6H), 3.77–7.85 (m,
4H), 4.04 (t, 2H, J = 6.0), 4.11 (t, 2H, J = 6.4), 6.73–6.91 (m, 12H), 7.12–7.31 (m,
7H), 7.39–7.41 (m, 2H); 13C NMR (CDCl3, 100 MHz) d 20.3 (d, J = 6.9), 24.5 (d,
J = 7.6), 24.6 (d, J = 7.6), 30.0, 31.0 (d, J = 7.3), 43.0 (d, J = 12.3), 55.2, 58.3 (d,
J = 19.3), 59.7, 60.2 (d, J = 17.4), 65.0, 65.4, 85.9, 113.0, 115.4, 117.6, 119.4,
119.5, 126.6, 127.7, 128.2, 130.0, 136.4, 145.2, 151.4, 151.6, 154.6, 154.7, 158.3.
PT-ODN1: Yield 8.6%. HPLC (Column: Macherey-Nagel Nucleosil 300-5, C18,
250/4.6; solvent A: 0.1 M (NEt3H)(OAc) in water, pH 7.0; solvent B: CH3CN,
gradient: 0% B for 5 min, in 30 min to 25% B, in 10 min to 90% B, 90% B for
9 min) Rt = 32–35 min. MALDI-TOF MS: Calcd for C211H266N71O107P20S20 [MꢂH]ꢂ:
6765, found 6764.
These compounds can potentially exhibit antisense activity. We
are currently testing them for red light-controlled gene expression
in HeLa cells.
Acknowledgments
PT-ODN2: Yield 4.4%. HPLC (Column: Macherey-Nagel Nucleosil 100-5, C4,
125/2; gradient: 0% B for 0.5 min, in 9.5 min to 35% B, in 2 min to 100% B)
Rt = 8.53 min. MALDI-TOF MS: Calcd for C201H260N71O104P20S22 [MꢂH]ꢂ: 6655,
found 6659.
We thank Deutsche Forschungsgemeinschaft (MO1418-2/1),
University of Heidelberg (B22) and AvH Stiftung (postdoctoral fel-
lowship for J.K.) for financial support.
11. Devasagayam, T. P. A.; Sundquist, A. R.; Di Mascio, P.; Saiser, S.; Stes, H.
J. Photochem. Photobiol. B Biol. 1991, 9, 105.