Nucleoside-based diarylethene photoswitches and their facile incorporation into photoswitchable DNA

Article abstract of DOI:10.1002/anie.201209943

Come into the light: Pyrimidine nucleosides were found to undergo fast and reversible ring closure upon irradiation with light when covalently attached to 2-[2-methyl-5-phenylthien-3-yl]cyclopent-1-ene (see scheme). Synthesis using an aqueous-phase Suzuki

Full text of DOI:10.1002/anie.201209943

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DOI: 10.1002/anie.201209943
DNA Photoswitches
Nucleoside-Based Diarylethene Photoswitches and Their Facile
Incorporation into Photoswitchable DNA**
Hana Cahovꢀ and Andres Jꢁschke*
The artificial control of DNA
structure and function is an
attractive field in chemical
and synthetic biology,[1] and
light is a powerful and conven-
ient trigger: it is non-invasive,
provides high spatio-temporal
resolution, and offers the
option of orthogonality. DNA
is reactive to light: the UV-
light-induced cyclodimeriza-
tion of pyrimidine nucleosides
is an important type of DNA
damage and has been studied
intensively.[2] Interestingly, this
Scheme 1. a) Isomerization of typical diarylethene photoswitches. b) A diarylethene photoswitch involving
7-deazadeoxyadenosine.[8] c) New photoswitchable DNA where a pyrimidine nucleotide is part of the
photoswitch.
chemical property has never
been exploited for the con-
struction of DNA-based rever-
sible photoswitches, and all
published work in this field is based on the covalent
functionalization of DNAwith small autonomous photoactive
molecules.[3] Different classes of such photoactive molecules
have been studied for DNA photoregulation;[4] azobenzenes
have been used most frequently and were, for example,
employed for modulating oligonucleotide duplex and triplex
formation and DNA transcription.[5] Other substance classes
studied in this context include arylvinyl derivatives,[6] spiro-
pyranes,[3a] and recently also diarylethenes.[3a,7] While a certain
influence of photoisomerization on the properties of the
nucleic acid was always observed, these approaches share one
common limitation: because an autonomous photoswitch was
attached to the DNA, either as an appendage or substituting
for nucleosides, the rearrangement of chemical bonds upon
encountering a photon (i.e., the photochemical reaction) was
strictly confined to this non-nucleosidic moiety.
tuted cyclopentenyl boronic ester was reacted with protected
7-iodo-8-methyl-7-deazadeoxyadenosine by Suzuki cross-
coupling, followed by deprotection. Upon irradiation with
light, these compounds were found to undergo a highly
efficient, reversible, electrocyclic rearrangement and the
switching wavelength could be tuned by the chemical nature
of substituents. Switching was found to be near-quantitative in
aprotic solvents, and the compounds retained the key proper-
ties of nucleotides, such as their capability to form Watson–
Crick base-pairs. Unfortunately, the photoisomerization was
found to proceed with low efficiency in aqueous solvents, and
the demanding synthesis involved limited the application of
these photoswitches to oligonucleotides.
To develop straight-forward access to truly photoswitch-
able DNA, we reconsidered our design approach; in contrast
to 7-iodo-8-methyl-7-deazadeoxyadenosine, the 5-iodo-sub-
stituted pyrimidine nucleosides 5I-dU and 5I-dC represent
oligonucleotide modifications readily available from com-
mercial suppliers, and offer the desired reactivity for different
cross-coupling reactions.[9] This could allow for the postsyn-
thetic conversion of an iodo-modified oligonucleotide into
a photoswitch, leading to the target compounds shown in
Scheme 1c. We note that this design challenges some of the
common design principles for diarylethene photoswitches:[10]
neither unsubstituted nor substituted pyrimidines have ever
been applied as components of diarylethene photoswitches,
and our target compounds contain just one (rather than two)
alkyl groups attached to the carbon atoms that form the new
bond in the cyclization reaction, a feature that is thought to be
important for the reversibility of photoswitching. This design
was first tested on the nucleoside level employing 5-iodo-
Recently, our lab reported a new type of diarylethene
photoswitches in which one of the two aryl moieties was
replaced by a nucleoside, namely 7-deaza-8-methyldeoxya-
denosine (Scheme 1).[8] These photoswitches were synthe-
sized in a convergent multi-step approach in which a substi-
[*] Dr. H. Cahovꢀ, Prof. Dr. A. Jꢁschke
Institut fꢂr Pharmazie und Molekulare Biotechnologie
Universitꢁt Heidelberg, 69120 Heidelberg (Germany)
E-mail: jaeschke@uni-hd.de
Homepage: http://www.jaeschke.uni-hd.de
[**] H.C. is grateful for a postdoctoral fellowship from the Alexander von
Humboldt foundation.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201209943.
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deoxyuridine and 5-iododeoxycytidine. The reaction of these
compounds with 2-[2-methyl-5-phenylthien-3-yl]cyclopent-1-
ene boronic acid pinacol ester,[8] under Suzuki–Miyaura cross-
coupling conditions that were previously optimized,[11] gave
the corresponding products in yields of 44–61% (Figure 1a).
The photochromic behavior of these compounds was
observed on TLC plates under UV light during the prepara-
tion. Moreover, an intense orange to red color change
occurred upon UV irradiation in chloroform. To test their
performance in polar protic solvents, the compounds were
dissolved to a concentration of 300 mm in water/ethanol 9:1,
and irradiated at 254 nm. A rapid photochromic reaction took
place in this solvent system, too, yielding a change from
colorless to orange (dUPS) or red (dCPS; Figure 1a).
irradiation at 366 nm. The UV absorption spectra of the
closed-ring forms of dUPS and dCPS showed new maxima at
470 nm and 507 nm, respectively (Figure 1b; Supporting
Information, Figure S1). In 30 mm solution, the photosta-
tionary state was reached after five minutes of irradiation
(UV hand lamp, 8 W, 6 cm distance), and prolonging the
irradiation did not lead to further spectral changes. The ring-
opening reverse reaction occurred more rapidly and was
complete after two minutes of irradiation by visible light
(Intralux-600-1 lamp, low intensity light, 150 W, 6 cm dis-
tance). The process was highly reversible and consistent
switching behavior was verified over seven cycles of irradi-
ation of 60 mm solutions by UV light for ten minutes and
visible light for ten minutes (Figure 1c, Figure S1b). For any
practical application, it is important that the photoisomers are
thermally stable and do not revert over time without
irradiation. Surprisingly, we found a significant difference in
the thermal stability between dUPS and dCPS. Although dUPS
Because irradiation by 254 nm UV light is known to cause
DNA damage, we tested whether irradiation at 366 nm would
also induce the photochromic reaction, which turned out to be
the case, and all further measurements were performed with
Figure 1. a) Aqueous Suzuki–Miyaura cross-coupling of 5-iododeoxyuridine and 5-iododeoxycytidine with boronic acid pinacol ester and
photoisomerization reaction of the nucleosides dUPS and dCPS. TPPTS=triphenylphosphine-3,3’,3’’-trisulfonic acid trisodium salt. b) Absorption
spectrum of a dUPS solution after irradiation by 366 nm UV light (band at 470 nm increases) and subsequent irradiation with visible light (band at
470 nm decreases). Inset: time trace at the absorbance maximum. c) Reversibility of the photoisomerization measured in a solution of dUPS
(60 mm) over seven cycles of 10 min irradiation with 366 nm and 10 min with visible light. d) Thermal stability of the closed-ring form measured
in solutions of dUPS and dCPS (30 mm) over 60 min at 258C, 508C, and 908C.
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Table 1: Sequences of oligonucleotide substrates for Suzuki–Miyaura
cross-coupling reaction.
showed high stability and displayed very little degradation
even when stored for 60 minutes at 908C, dCPS reverted to the
open-ring form rapidly already at room temperature (Fig-
ure 1d).
1
By combining H NMR spectroscopy and UV/Vis spec-
trometry, the photostationary states (i.e., the amount of
opened- and closed-ring forms) after UV irradiation were
determined to contain 83% closed-ring isomer for dUPS
(Figure S2) and 98% for dCPS (Figure S3). The ring-opening
reaction upon visible light irradiation was found to be
quantitative (less than 0.5% closed-ring form, Figure S4).
After demonstrating the function of these photoswitches
at the nucleoside level, the next task was to develop
a synthetic route to photoswitch-modified DNA, preferably
also using Suzuki cross-coupling, because this would allow for
the use of the same intermediates. To our knowledge, there is
only one example of the Suzuki cross-coupling of oligonucle-
otides, namely the reaction of short (2-mer to 15-mer) 8-Br-
dG-modified oligonucleotides with various phenyl boronic
acids and one benzothiophene boronic acid.[12] Neither Suzuki
cross-coupling of other halogenated oligonucleotides nor the
attachment of more sterically demanding boronic acids has
been reported. Application of the conditions elaborated by
Omumi et al. (708C for 24 hours with Na2CO3 as a base in
aqueous solution)[12] to our problem (5-iodinated pyrimidine
oligonucleotides, combined with sterically demanding bor-
onic acid esters) however, did not yield detectable amounts of
product. We therefore tested conditions developed for the
derivatization of sensitive nucleoside triphosphates with
a variety of boronic acids (CsCO3, water/acetonitrile mixture,
argon atmosphere, 1208C, 60 minutes).[13] The reactions were
performed on a seven nanomole scale and the products
immediately purified by HPLC (Figures S5,S6). Nine differ-
ent modified oligonucleotides (15- and 19-mers, Table 1;
Table S1) bearing one or two photoswitchable groups were
prepared by this method and the yields of the isolated
products were between 16% and 35%. Cross-coupling at
terminal positions (3’ or 5’) was generally more efficient than
at internal ones. All products were characterized by ESI mass
spectrometry (Figures S7,S8). Dehalogenation is known to be
the major side reaction under conditions of steric hin-
drance,[14] and we could confirm for several oligonucleotides
that the major undesired product was indeed the dehalo-
genated oligonucleotide (Table 1).
Oligo
Substrate[a]
Yield[b] Dehalogenation[c]
25%
15mer-
dUPS1
5’-AGCAACAIUCGATCGG-3’
15mer-
5’-AGCAACAICCGATCGG-3’
5’-IUGCAACATCGATCGG-3’
5’-ICGCAACATCGATCGG-3’
5’-AGCAACATCGATCGIU-3’
5’-AGCAACATCGATCGIC-3’
22%
26%
34%
25%
35%
dCPS1
15mer-
dUPS2
15mer-
dCPS2
15mer-
dUPS3
15mer-
dCPS3
19mer-
dUPS1
5’-TCTAATACGACTCACIUATA-3’ 20%
5’-TCTAATACGACTCACTAIUA-3’ 19%
5’-TCTAATACGACTCACIUAIUA-3’ 16%
45%
19mer-
48%
dUPS2
19mer-
dUPS3
30%, 45%
[a] Substrate of the Suzuki-Miyaura cross-coupling reaction. [b] Yield of
the isolated product. [c] Yield of the isolated product of dehalogenation.
lowest for internal incorporation, suggesting that the location
of the photostationary state after UV irradiation varies with
the structural context. As part of a DNA oligonucleotide, the
diarylethene chromophore showed highly reversible photo-
switching (Figure 2b) and high thermal stability (Figure 2c).
Because the dCPS photoswitch was found not to be
thermally stable in its closed form, we decided to focus our
studies on the properties of double-stranded DNA (dsDNA)
containing dUPS nucleotides. The properties of the modified
dsDNA before and after irradiation by 366 nm UV light were
compared with those of unmodified DNA. First, the influence
of the modification on duplex stability was analyzed by
thermal denaturation analysis (Figure S9, Table S2). One
internal modification (oligonucleotide 15mer-dUPS1) caused
a decrease in the melting temperature (Tm) of 2.38C, both in
the opened- and closed-ring form, compared to the unmodi-
fied duplex, while 3’- or 5’-terminal modifications were found
to have a negligible effect on the stability in the open-ring
form. On the other hand, these modifications were found to
have a stabilizing effect after ring closure (DTm = 0.98C for 5’-
terminal and DTm = 1.68C for 3’-terminal incorporation). To
investigate the effect of the photoswitch modification on the
overall helical structure, CD spectra were recorded prior to
and following UV irradiation (Figure S9). The spectra were
almost identical to unmodified DNA when the modification
was terminal. Only when the dUPS nucleotide was in center of
the dsDNA, an apparent shift to a more A-like (i.e., RNA-
like) conformation was observed in comparison with natural
The photoswitch-modified oligonucleotides were then
tested for photochromicity. Solutions of single-stranded
oligonucleotides 15mer-dUPS1–15mer-dCPS3 (25 mm) were
irradiated for five minutes at 366 nm, and then their UV/Vis
spectra were measured (Figure 2). All prepared oligonucle-
otides exhibited photoswitching properties. Compared to the
free nucleosides, the absorbance maxima of dUPS and dCPS
incorporated into DNA showed a bathochromic shift from
470 nm to 477 nm for dUPS and from 507 nm to 540 nm for
dCPS.
These absorbance maxima did not depend on the
oligonucleotide sequence or position of the modification
within the oligonucleotide, however, the absolute absorbance
was highest when the photoswitch was incorporated at the 5’-
end, while it was lower for 3’-terminal incorporation and
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Figure 3. Denaturing polyacrylamide gel electrophoresis of in vitro
transcription reactions performed on photoswitch-modified dsDNA
a) 19mer-dUPS1, b) 19mer-dUPS2, or c) 19mer-dUPS3 without any irradi-
ation, labeled “o”, and after 10 min irradiation by 366 nm UV light,
labeled “c”. Transcription from an unmodified promoter, labeled “+”.
their closed-ring forms gave higher amounts of product
compared to the opened-ring form. Clear positional effects
could be observed: a single modification at position À4 had
only a small effect (Figure 3a), while at position À2 (Fig-
ure 3b) and in the doubly modified promoter (Figure 3c)
significant inactivation by the open-ring modified DNA was
observed, but UV irradiation fully restored activity. A more
comprehensive modification analysis could reveal other
promoter positions sensitive to photoswitchting and ulti-
mately result in higher switching factors.
Figure 2. a) UV spectra of single-stranded oligonucleotides 15mer-
dUPS1–15mer-dCPS3 after irradiation by 366 nm UV light for 5 min.
b) Reversibility the photoisomerization measured in solutions of
15mer-dUPS2 (10 mm) over five cycles of 10 min irradiation with
366 nm light and 10 min with visible light. c) Thermal stability of the
closed-ring form of 15mer-dUPS2 (10 mm solution) measured over
35 min at 258C, 508C, and 908C.
We report herein the first example of photoswitchable
oligonucleotides in which a structural constituent of DNA
itself, namely the nucleobase, is involved in the rearrange-
ment of chemical bonds, changes hybridization, bond order,
and geometry, and therefore forms an active part of the
photoswitch. The atomic positions involved are exactly the
same that take part in the UV-induced cyclodimerization
photo-damage reaction of DNA (C5 and C6 of the pyrimi-
dines). A simple one-step method was presented for the
conversion of iodo-modified oligonucleotides into highly
specific, fully reversible photoswitches based on Suzuki
chemistry. This new approach, combined with the well-
known acceptance of iodo-modified nucleotides by DNA
polymerases[16] and the recent development of mild and
biocompatible cross-coupling conditions,[17] may lead to new
applications of reversible DNA photoswitches in biology,
microscopy, and nanotechnology.
DNA. In all cases, the UV-induced DNA conformational
changes were small.
Photoregulation of fundamental biological processes like
replication or transcription could give new opportunities for
cell manipulation or bionanotechnology.[15] To investigate the
influence of the diarylethene photoswitch on transcription,
we incorporated one or two photoswitch moieties into the T7
promoter region of a partially double-stranded DNA tem-
plate. Three different modified promoters were prepared with
dUPS nucleotides in positions À2 and/or À4 of the non-
template strand. Each of these constructs was subjected to
transcription with or without prior irradiation at 366 nm for
ten minutes and compared with transcription from an
unmodified template. Aliquots were withdrawn after 1, 5,
10, and 20 minutes and analyzed by polyacrylamide gel
electrophoresis (Figure 3). The presence of the bulky photo-
switch modification was not found to be detrimental to
transcription; in all cases, the polymerase synthesized full-
length product. Generally, the photoswitchable promoters in
Received: December 12, 2012
Published online: February 12, 2013
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Keywords: DNA damage · nucleosides · photochemistry ·
photoswitchable DNA · Suzuki cross-coupling
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