Phototriggered drug release from functionalized oligonucleotides by a molecular beacon strategy

Article abstract of DOI:10.1002/anie.200250832

More than just a light switch: A phototriggered molecule-releasing system controlled through intramolecular triplet quenching by using a molecular beacon strategy with photoactive probe oligodeoxynucleotides (ODNs) has been developed (see picture).

Full text of DOI:10.1002/anie.200250832

Communications
Phototriggered Molecular Release
were incorporated into the 3’- and 5’-ends of the ODN,
respectively. The stem of the closed-form ODN keeps a
phenacyl ester and a naphthalene ring in close proximity to
each other, causing the efficient triplet quenching, as in the
case of a molecular beacon. In contrast, when the probe ODN
is transformed to the open form by hybridization with target
DNA,[1] photocleavage of the phenacyl ester proceeds very
smoothly under irradiation, because the photoactive phenacyl
ester is far away from the naphthalene quencher.
Phototriggered Drug Release from Functional-
ized Oligonucleotides by a Molecular Beacon
Strategy
Akimitsu Okamoto, Kazuhito Tanabe, Takeshi Inasaki,
and Isao Saito*
The synthetic route for the functionalized ODN is shown
in Scheme 1. The photocleavable phenacyl ester 3, which has
both a maleimide functional group to be connected to thiol-
DNA hybridization biosensors offer considerable promise for
obtaining sequence information of genes in a fast and simple
manner. Various DNA probes that give signals in a sequence-
specific fashion, as represented by molecular beacons, have
been widely used.[1–3] However, there are very few DNA
probes that release functional molecules on recognition of the
target sequence.[4,5] The sequence-specific molecule-releasing
system that is triggered by external stimulation, such as
irradiation would be a very powerful tool for gene analysis.
Herein we report for the first time a molecule-releasing
system controllable by an intramolecular quenching based on
a molecular-beacon strategy by using photoactive probe
oligodeoxynucleotides (ODNs). The photoreaction of the
probe ODN containing a photoactive group and a triplet
quencher at both ends of the strand was very inefficient when
it was in the closed-form ODN (a stem-and-loop structure),
whereas irradiation of the open-form ODN hybridized with
the complementary DNA resulted in a rapid release of the
functional molecule from the ODN (see Figure 1).
ODN sequences for the molecule-releasing system were
designed according to the molecular-beacon strategy.[1] Phe-
nacyl ester[6–8] as a photocleavable group via a triplet excited
state, and substituted naphthalene[9,10] as a triplet quencher
Scheme 1. Reagents and conditions: a) K2CO3, N-(2-bromoethyl)phthal-
imide, acetone, reflux, 10 h, 23%; b) 40% methylamine, 2:1 chloroform-
methanol, RT, 5 h, 64%; c) maleic anhydride, toluene, reflux, 5 h;
d) sodium acetate, acetic acid, reflux, 1.5 h, 49% in two steps;
e) PhMe3NBr3, THF, RT, 3 h, 35%; f) (þ)-biotin, triethylamine, DMF,
RT, 2 h, 76%; g) thionyl chloride, reflux, 2 h; h) TBDMSO(CH2)2NH2,
THF, RT, 12 h, 43% in two steps; i) TBAF, THF, RT, 3 h, 82%;
j) (iPr2N)2PO(CH2)2CN, 1H-tetrazole, acetonitrile, RT, 1.5 h, quant.;
k) DNA autosynthesizer, then 28% ammonia, 558C, 12 h; l) 5 mm
dithiothreitol, 25 mm sodium phosphate (pH 8.4), 238C, 12 h, quant.;
m) 0.5 mm 3, 250 mm sodium phosphate (pH 7.0), 238C, overnight,
quant.
modified DNA and a biotin unit as a leaving functional
molecule, was prepared from 4-hydroxyacetophenone (1).
Naphthalene phosphoramidite, 6, was prepared from 1-
naphthylacetic acid (4) in four steps. ODNs containing both
a naphthalene unit at the 5’ end and a disulfide unit at the
3’ end were synthesized by using a DNA synthesizer by a
conventional phosphoramidite method. The disulfide group
of the given ODNs 7 was reduced to thiol by dithiothreitol,
and then coupled with 3 to give ODNs 9a and 9b that were
equipped with a photoactive group and a quencher at each
end.
We initially investigated the efficiency of the photoreac-
tion of ODN 9a by irradiation at 312 nm before and after
hybridization with the complementary target DNA 5’-d(CAT-
AGGTCTTAACTT)-3’. The irradiation of a solution of 9a in
1 5 mm sodium cacodylate (pH 7.0) and 2-propanol (2:1) was
Figure 1. Outline for the phototriggered drug release from functional-
ized oligonucleotides by a molecular beacon strategy.
[*] Prof. I. Saito, Dr. A. Okamoto, K. Tanabe, T. Inasaki
Department of Synthetic Chemistry and Biological Chemistry
Faculty of Engineering
Kyoto University and
SORST, Japan Science and Technology Corporation
Kyoto 606–8501 (Japan)
Fax : (+81)75-753-5676
E-mail: saito@sbchem.kyoto-u.ac.jp
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200250832
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Angewandte
Chemie
conducted at 08C for 10 seconds, and the disappearance of 9a
was monitored by HPLC. The conversion of closed-form 9a[11]
was relatively inefficient (5% conversion, kobs = 0.03 mm sꢀ1),
whereas irradiation of open form 9a hybridized with the
complementary strand resulted in an efficient degradation
(25% conversion, kobs = 0.17 mm sꢀ1). These results clearly
indicate that a structural change caused by hybridization of 9a
with the complementary DNA strongly affects the consump-
tion of 9a.[12]
To obtain photoproducts, we examined the irradiation of a
shorter single-stranded ODN 9b, and analyzed the reaction
products by mass spectrometry. With five minutes of irradi-
ation, 9b was completely consumed and afforded the product
mixture. The molecular weight [(MꢀH)ꢀ] of the reaction
products given by MALDI-TOF mass spectrometry was
2775.49, thus indicating the formation of acetophenone 10b
(calcd 2775.99) (Scheme 2). Such reductive cleavage of
phenacyl ester was also confirmed in the photolysis of a
model system, 4-methoxyphenacyl ester of biotin.[6,13,14]
We next quantified the biotin that was released from 9a by
the photolysis of the phenacyl ester. After irradiation at
312 nm of a solution of 9a (200 pmol), the photoproduct
mixture was isolated filtration through a centrifugal filter and
mixed with a solution of the complex of avidin and 2-((4’-
hydroxyphenyl)azo)benzoic acid (HABA).[15,16] Then, the
decrease of the absorbance at 500 nm (A500) originating
from the avidin-HABA complex was monitored. The
decrease of A500 is known to be caused by the displacement
of HABA from the avidin-HABA complex by the released
biotin.[16] The amount of biotin that was released from 9a was
Figure 2. Quantification of biotin released from 9a. Irradiation
(312 nm) of 200 pmol of 9a in the presence or absence of the comple-
mentary ODN 5’-d(CATAGGTCTTAACTT)-3’ was carried out in 15 mm
sodium cacodylate (pH 7.0)-iPrOH (2:1) at RT with a monochromator
(JASCO CRM-FD, 300 W Xe lamp, 40 counts; one count of irradiation
approximately corresponds to a surface energy of 0.02 Jcmꢀ2). After
the reaction mixture was filtered through a Microcon centrifugal filter
(YM-3, 3000 NMWL), the filtrate was mixed with a solution of avidin-
HABA complex (0.43 mg of ImmunoPure Avidin, PIERCE and 62.5 mg
of ImmunoPure HABA, PIERCE) in 86 mm sodium phosphate (pH 7.2)
and 130 mm sodium chloride, and then the absorbance at 500 nm
(A500) of the mixture was measured. The amount of released biotin
was quantified by using a linear fit (the blue solid line, y=ꢀ7.0ꢀ10ꢀ5
x
+ 0.9147; R=0.9966) based on A500 of an avidin-HABA solution in the
presence of 0, 100, and 200 pmol of biotin.
ular beacon strategy. Hybridization of the photoactive
probe ODN with the complementary target DNA
resulted in a rapid photolytic cleavage of phenacyl
ester with the release of biotin, although closed form
ODN before hybridization suppresses biotin release due
to the intramolecular triplet quenching. The drug
release occurs effectively by UV irradiation when a
specific sequence has been recognized. This new drug-
releasing system will facilitate the rational design of a
well-controllable prodrug for gene analysis.
Scheme 2.
Received: December 23, 2002 [Z50832]
Keywords: · drug delivery · molecular devices ·
oligonucleotides · photolysis
determined by using a calibration curve of A500 given from
control experiments (Figure 2). Irradiation of closed-form 9a
resulted in a slight decrease of A500, corresponding to 12%
biotin release. In contrast, a drastic decrease of A500 corre-
sponding to 84% biotin release was observed in the presence
of complementary ODN 5’-d(CATAGGTCTTAACTT)-3’.
The efficiency of biotin release was significantly altered by the
conformational change of 9a by hybridization with the target
DNA sequence. These results strongly suggest that naphtha-
lene attached to the 5’ end of 9a acts as an effective
intramolecular quencher in closed form 9a and suppresses
photodegradation of the phenacyl ester, which readily
occurred in open-form 9a.
.
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In summary, we have demonstrated for the first time a
phototriggered molecule-releasing system by using a molec-
Angew. Chem. Int. Ed. 2003, 42, 2502 – 2504
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ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
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[11] Closed-form 9a was stable at room temperature. The Tm was
40.48C in 1 0 mm sodium cacodylate (pH 7.0) and 1m m NaCl.
[12] A control ODN that does not have a 5’ triplet quencher 9a–naph
was also tested. Very rapid degradation was observed for both
closed form 9a–naph and open form 9a–naph hybridized with
the complementary strand (59% and 55% conversion, respec-
tively).
[13] A solution of 4-methoxyphenacyl ester of biotin (0.14 mmol) in
acetonitrile/2-propanol/water (1:4:2, v/v/v) was irradiated at
312 nm with a transilluminator for 3.5 h at room temperature.
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to give 4-methoxyacetophenone and biotin in 59% yield.
[14] Several peaks were detected by HPLC in the reaction mixture of
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in addition to product 10b as given by MALDI-TOF mass. For a
solution containing duplex ODN 5’-d(GCGAGAAGTTAA-
GACCTATGCTCGC)-3’/5’-d(CATAGGTCTTAACTT)-3’ and
4-methoxyphenacyl ester of biotin, 8% consumptions of ODN
were observed for 60 s irradiation. Further conversion of 10b by
irradiation was not observed.
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ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2003, 42, 2502 – 2504