Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches

Article abstract of DOI:10.1002/anie.202014878

Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine- and deoxycytidine-derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high-performance photoswitches with near-quantitative, fully reversible back-and-forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4-fold turn-off factor, demonstrating the potential for optochemical control of biological processes.

Full text of DOI:10.1002/anie.202014878

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 8164–8173
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Photoswitches
Hot Paper
Development of High-Performance Pyrimidine Nucleoside and
Oligonucleotide Diarylethene Photoswitches
Theresa Kolmar+, Simon M. Büllmann+, Christopher Sarter+, Katharina Hçfer, and
Andres Jäschke*
Abstract: Nucleosidic and oligonucleotidic diarylethenes
(DAEs) are an emerging class of photochromes with high
application potential. However, their further development is
hampered by the poor understanding of how the chemical
structure modulates the photochromic properties. Here we
synthesized 26 systematically varied deoxyuridine- and deoxy-
cytidine-derived DAEs and analyzed reaction quantum yields,
composition of the photostationary states, thermal and photo-
chemical stability, and reversibility. This analysis identified two
high-performance photoswitches with near-quantitative, fully
reversible back-and-forth switching and no detectable thermal
or photochemical deterioration. When incorporated into an
oligonucleotide with the sequence of a promotor, the nucleo-
tides maintained their photochromism and allowed the mod-
ulation of the transcription activity of T7 RNA polymerase
with an up to 2.4-fold turn-off factor, demonstrating the
potential for optochemical control of biological processes.
ical stimuli. Azobenzenes[8–14] and diarylethenes (DAEs)[15–19]
have most commonly been used for this purpose, while
spiropyrans,[20–23] fulgides,[24,25] and hemithioindigo-based
switches[26] were applied less frequently.
The core structure of the classical DAE photoswitches
shown in Figure 1A evolved over the years from the stilbenes.
It is characterized by a conjugated hexatriene system and was
originally developed as an optical information storage
medium in materials sciences.[27–30] Recently, such systems
have also been used in biological applications, such as
photopharmacology[31] and single molecule localization mi-
croscopy.[32] DAEs undergo a reversible photochemical elec-
trocyclic ring closure, which enlarges the conjugated p
electron system, rendering the compound colored in the
closed form and colorless in the open.
The photophysical and photochemical properties of DAE
photoswitches can be modulated and optimized by variation
of the substitution pattern (R).[36–38]
Introduction
In 2010, our group reported the first DAE-based photo-
chromic nucleosides in which a purine nucleobase (of
a nucleoside) represented one of the two aryl systems of the
diarylethene (Figure 1B). Importantly and different from
most other approaches towards photoswitchable nucleic
acids, the nucleobase formed an active constituent of the
photoswitch (rather than an appendage), changing bonding
and hybridization upon switching.[33,34] This new class of
purine nucleoside DAEs, and especially the deoxyadenosine-
The development of tools for the selective manipulation
and control of biomolecules is critical for the progress in many
areas of the life sciences. Among those, photochromic
molecules are of major interest, as they use light as
modulation channel.[1–5] The non-invasive nature of light
and the high spatio-temporal resolution offer key advantages
over electrical or chemical stimuli.[6,7] However, biomole-
cules, and in particular nucleic acids, usually do not possess
photochromic properties. Therefore, nucleic acids need to be
modified with suitable photoactive moieties in order to gain
remote control over their structure and function. A covalent
linkage to photoswitchable entities is a common approach to
make biomolecules sensitive towards reversible photochem-
[*] T. Kolmar,[+] S. M. Büllmann,[+] C. Sarter,[+] K. Hçfer, A. Jäschke
Institute of Pharmacy and Molecular Biotechnology, Heidelberg
University
Im Neuenheimer Feld 364, 69120 Heidelberg (Germany)
E-mail: jaeschke@uni-hd.de
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202014878.
ꢀ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution Non-Commercial
License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used
for commercial purposes.
Figure 1. DAE photoswitches. A) Classical design and isomerization
reaction. The reactive a carbon atoms are marked. B) Deazapurine
nucleoside-based photoswitches.[33,34] C) Pyrimidine nucleoside-based
photoswitches.[35]
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derived ones, turned out to have favorable switching charac-
cycloreversion, intensive color (i.e., high emax, vis) of the closed-
teristics with respect to their photostationary states (PSS),
ring form, thermal stability of the closed isomer, reversible
switching and fatigue resistance. The most promising pyrimi-
dine nucleoside DAEs should then be incorporated into
oligonucleotides and their properties compared to the
previous generation.[40,41] As the control of biological struc-
ture and function by light would benefit tremendously from
near-quantitative on/off switching, we aimed at generating
high-performance photochromic DAE oligonucleotides for
future applications in synthetic biology and nanotechnology.
reversibility and fatigue resistance.[39]
In 2013 our group developed a 2nd type of DAE-based
photochromic nucleosides, which incorporated two rare
features: a six-membered heterocyclic pyrimidine ring acted
as one of the aryls, and only one rather than the common two
alkyl group was present at the reactive a carbon atoms
(Figure 1C). Such a structural setup of a DAE was unprece-
dented at the time. To our surprise, these pyrimidine-based
DAEs showed significant photochromism, high reversibility
and thermal stability of the closed-ring isomer. However,
a major drawback of this system was that UV-irradiation led Results and Discussion
to a PSSUV that contained ꢀ 50% of the closed-ring form
under the conditions used at that time, thereby limiting the
use of these compounds in applications where a higher
dynamic range of response is required.[35] Additionally, only
a few examples were synthesized (phenyl-, naphthyl- and
methyl-substituted at the 5-position of the thiophene, as
shown in Figure 1C), and a comprehensive photophysical
characterization was lacking. Therefore, the previously gath-
ered data are not sufficient to understand and generalize the
properties of these pyrimidine nucleoside-based DAEs. In
order to address these shortcomings, we undertook a system-
atic investigation of structure–activity relationships by chem-
ical variation. Our primary goal was to understand which
structural features render a candidate a good photochrome,
including the following key properties: quantitative switching
in both directions, high quantum yields for cyclization and
In general, the absorption of chromophores can be
modulated either by extending the conjugated p-system, or
by incorporating auxochromic groups, i.e., electron-with-
drawing or electron-donating groups.[42,43] Both approaches
have been applied extensively to (non-nucleosidic)
DAEs.[27,44] Based on literature precedents and synthetic
feasibility, we selected 13 substituents for incorporation into
2’-deoxyuridine (dU) and 2’-deoxycytidine (dC) DAE photo-
switches (Scheme 1A).
Synthesis of the Nucleosidic DAEs
Position 5 of the thiophene ring was chosen as the primary
site of structural variation, since the nucleobase, including its
Scheme 1. Design and synthesis of dU-and dC-based DAE switches. A) Arrangement of the substituents on the thiophene moiety from electron
donating (EDG, red) to electron withdrawing (EWG, blue). B) General synthetic approach involving a Suzuki coupling. C) Synthesis of the boronic
acid pinacolate esters with residues that are inert against n-BuLi (Top: Non-sensitive R) and residues that require borylation via Miyaura
borylation (Bottom: sensitive R). a) Ar-X, Na2CO3 (aq.), Pd(dppf)Cl2, 808C, THF; b) n-BuLi, B(OBu)3, À788C, THF; c) dibromocyclopentene,
Na2CO3 (aq.), Pd(dppf)Cl2, 808C, THF; d) n-BuLi, BPinOiPr, À788C, THF; e) B2Pin2, KOAc, Pd(dppf)Cl2, 1208C, dioxane; f) dibromocyclopentene,
Na2CO3 (aq.), Pd(dppf)Cl2, 808C, THF; g) B2Pin2, KOAc, Pd(dppf)Cl2, 1208C, dioxane.
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ability to base-pair, should be kept as “natural” as possible (as
demonstrated previously).[33] Specifically, we introduced the
electron-donating conjugated benzodithiole (Bdt), N-N-di-
methylamino-naphthalene (NpNMe2) and N-N-dimethylani-
line (PhNMe2) groups. We varied the length of the conjugated
system from methyl (Me) via phenyl (Ph) and 2-naphthyl
(Np) to anthracenyl (An) and introduced electron-withdraw-
ing 4-pyridyl (4Py), 2-pyridyl (2Py), bis(trifluormethyl)-
phenyl (Ph(CF3)2), tert-butylester-phenyl (PhtBu), p-nitro-
phenyl (PhNO2) and cyano (CN) groups. This panel allows
a comprehensive investigation of p-system lengths and
auxochromic effects on various photochemical and photo-
physical parameters. The general synthetic approach is based
on our earlier work.[33] First, the modified boronic acid
pinacolate esters of the cyclopentene-bridged thiophene 4R
had to be synthesized. These compounds were coupled to 5-
iodo-2’-deoxyuridine and 5-iodo-2’-deoxycytidine, respective-
ly, in a Suzuki cross-coupling reaction (Scheme 1B). Two
main routes were established for the synthesis of the 13
boronic acid pinacolate esters (Scheme 1C):
between the electronic character of the substituent at the
thiophene moiety and the newly formed absorption maximum
(Figure 3B). (Modified) Hammett parameters s’ were ob-
tained from 13C shifts of the carbon in para position to the
substituents (Figure 3A).[46] On this scale, a value of s’ > 1
implies an electron-withdrawing character of the substituent,
while s’ < 1 indicates an electron-donating character. Aux-
ochromes like PhNMe2, PhtBu (Figure 2E/F, Figure 3B), or
PhNO2 caused a shift of the absorption maximum to higher
wavelength due to their ÀM or + M (mesomeric) effects,
while more electron-neutral substituents like Ph showed
a hypsochromic shift of the absorption maximum (Figure 2C/
D). Similar tendencies have been observed earlier for sym-
metrical dithienylethenes.[37,38] Only dUAn and dCAn did not
show any photochromicity, while dUBdt showed a biphasic
behavior where first a broad new band with a maximum at
550 nm appeared, which then gradually disappeared in favor
of another intensive band at 525 nm, suggesting rapid
decomposition of the initially formed closed-ring form (Fig-
ure S1).
For residues that can withstand treatment with n-BuLi,
the synthetic procedure described by Singer et al.[33] was used,
in which the thiophene derivatives 2R were furnished by
Suzuki cross-coupling of (4-bromo-5-methylthiophen-2-yl)
boronic acid 1 with the corresponding aryl halides (Ar-X).
Then, in a one-pot reaction, 2R was activated (as boronic acid)
in situ, and the cyclopentene bridge was attached in a second
Suzuki reaction with 1,2-dibromocyclopentene to give 3R. In
the last step, 3R was converted to the boronic acid pinacolate
ester 4R.
For more sensitive residues R (CN, PhNO2, Ph(CF3)2,
PhtBu and Bdt), pinacolate ester 4R had to be synthesized via
a different route, namely by Miyaura borylation and subse-
quent Suzuki cross-coupling.[45] For synthetic details and
standard analytical characterization, see Supporting Informa-
tion.
In direct comparison with the dU switch carrying the same
substituent R, the absorption maximum of the dC switch is
bathochromically shifted, for example, 475 nm for dUNp vs.
494 nm for dCNp (see Figure 2A/B, Table 1). This bathochro-
mic shift is likely caused by the electronic differences between
the two nucleobases. The carbonyl function in uridine exerts
a ÀI (inductive) and ÀM effect, whereas the amino group of
cytidine has a ÀI and + M effect. Both groups act as
auxochromes, however the amino group of cytidine seems
to have a stronger influence on the absorption spectrum. An
exception from this rule is the Ph(CF3)2 substituent where
lmax, vis of the dC switch is hypsochromically shifted, relative to
the dU switch.
Ring Opening and Reversibility
Upon irradiation with Vis light matching the absorption
maximum of the closed-ring form, complete discoloration was
observed for 8 of the 12 photochromic dU switches and for 3
of the 12 dC switches, indicating that for these compounds, the
PSSvis was close to 100% open-ring isomer (Figure 2A/C/D/
Photochromism
A first assessment of the photochromism of the synthe-
sized compounds was performed by UV/Vis spectroscopy of
each nucleosidic DAE upon UV light irradiation (310– E, Figure S3,S4). These compounds could be switched back
340 nm, Figure 2, Figure S1,S2). In both the dUR and the
dCR series, all compounds except the anthracene derivative
showed photochromism, i.e., the formation of a new absorp-
tion band in the visible wavelength range (460–510 nm) upon
irradiation with UV-light, indicative of the electrocyclic ring
closure reaction. For nearly all compounds, this irradiation
resulted in a PSSUV (for exceptions see below). In both classes
of switches (dUR and dCR) there was a clear influence of the
substituent on the absorption spectrum. Variation of R
allowed to shift the closed-ring absorption maximum over
a range of 85 nm (for dU switches) and 70 nm (for dC
switches) (Table 1).
and forth by alternating UV and Vis light irradiation over
several cycles, essentially reaching the same absorption
maxima and minima after each cycle. This behavior indicates
the absence of significant irreversible side reactions. The
remaining compounds exhibited different patterns of irrever-
sibility and fatigue (Figure 2B/F, Figure S3,S4). These pat-
terns were particularly pronounced for the electron-donating
substituents Bdt, NpNMe2, PhNMe2 (Figure S3,S4). In gen-
eral, the members of the dC series exhibited decreased
reversibility and increased fatigue, compared to the dU series
(Figure 2A/B, E/F). The introduction of electron-withdraw-
ing trifluormethyl substituents at aromatic rings, which was
reported to significantly improve the fatigue resistance of
dithienylethenes,[47] surprisingly reduced the reversibility in
our nucleosidic DAEs (Figure S5, compare dCPh and
Residues that enlarge the conjugated system, for example,
from methyl over phenyl to naphthyl, shifted the closed-ring
absorption maximum to longer wavelengths (Figure S1,S2).
Hammett parameter correlations indicated a relationship
dCPh CF ).
3
2
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Figure 2. Absorption spectra, reversibility and thermal stability of dU-based (A, C, E) and dC-based (B, D, F) photoswitches. This selection
includes switches with electron donating (A, B), electron neutral (C, D) and electron withdrawing (E, F) substituents. The absorption spectra of
the open (black line) and calculated closed (red line) isomer and the PSSUV (dashed blue line) are shown. In each panel the reversibility (top
t
right) and the thermal stability (low right) are shown. The yellow area indicates the spectral separation of dUPh Bu. For obtaining the PSSUV
absorption spectra, the samples were irradiated with their optimal wavelength (see footnotes of Table 1) at 50–70 mM in water/ethanol mixtures.
Thermal Stability
Switching Yields— Composition of the PSSUV
DAEs are known to undergo not only photochemical, but
also thermal reactions. In particular, the closed-ring form may
revert via a thermal pathway back to the open-ring isomer.
This is an undesired property for some applications while it is
beneficial for others. We investigated the thermal stability of
all photochromic nucleosides by monitoring the absorbance
at the closed-ring maximum over one hour at 258C, 508C and
708C (Figure S6,S7). Most dU switches showed < 5% de-
crease in absorbance over 1 h even at 708C (Figure 2A/C/E),
while many dC switches deteriorated significantly at 508C
(Figure 2B/D/F). The thermal stability of DAEs is known to
correlate with the aromatic stabilization energy of the
heterocycles. The superiority of uridine switches in terms of
thermal stability is based on their lower aromatic stabilization
energy.[48]
After this first qualitative assessment of the photochromic
properties we selected those compounds that were sufficiently
thermostable and fatigue-resistant for a quantitative charac-
terization. For 11 dU and 10 dC switches, the composition of
the PSSUV induced by UV irradiation could be determined by
HPLC (Table 1, Figure S8,S9). The dU switches generally
showed higher switching yields than the dC switches (see
dotted blue spectra in Figure 2). The percentage of closed-
ring form in the PSSUV varied in the dU series from 14% for
t
dUPhNO to 93% for the dUPh Bu. Substituents without a heter-
2
oatom (dUMe, dUPh, dUNp) and thus without significant
electron-donating or -withdrawing effects generally showed
switching yields between 50–78% (Table 1). Our best photo-
t
chromic nucleosides (dUPh Bu, dU2Py, dU4Py) showed an almost
quantitative conversion to the closed form (Table 1), which is
comparable to many non-nucleosidic DAEs.[44] Based on the
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Table 1: Photophysical properties of the dU and dC photoswitches.
known composition of the PSSUV, we calculated the spectra of
the pure closed-ring isomers. The extinction coefficients e at
the absorption maximum of closed-ring form of the dU series
were generally higher than those of the dC series (Figure 2,
Table 1). As expected, DAE switches without aromatic
substituents generally had the lowest e values, followed by
one-ring aromatic and heteroaromatic substituents, and the
highest values were determined for larger conjugated and
of the quantum yields of ring opening and ring closing plays
a significant role for the composition of the PSSUV as
illustrated by Equation (1), which holds true in the absence
of side reactions:
ꢀ
ꢁ
CŠ
elO FlOC
el
1
C FlCO
OŠ
PSS
push-pull-systems. For our two best compounds (in terms of
t
PSSUV composition), dUPh Bu and dU2Py, the strong absorption
band in the UV-region was hypsochromically shifted upon
irradiation with UV light (by 29 nm and 19 nm, respectively),
causing an increased spectral separation between the open
and closed isomers in the region around 340 nm (see yellow
area in Figure 2E). This results in a favorable situation for the
cyclization reaction, since in this case the open isomer has
a higher extinction coefficient and therefore absorbs stronger
than the closed one, driving ring closure more strongly than
ring opening and significantly effecting the composition of the
PSSUV. Such spectral shifts were less pronounced in the dC
series, explaining their lower switching efficiency (Figure 2B/
D/F). Besides the spectral separation, which is reflected in the
ratio of the extinction coefficients of the two isomers, the ratio
Quantum yields
Quantum yields were determined using a modified quan-
tum yield determination setup by Megerle et al.[49] In order to
determine reaction quantum yields, the excited state of the
photochromic nucleosides should not enter additional irre-
versible reaction pathways. Therefore, quantum yields were
determined for 16 selected compounds (Table 1). UV-induced
cyclization quantum yields were generally high for both the
dU and dC series, ranging from 0.23 (dUPhNMe ) to 0.65 (dU
)
2Py
2
with the exception of dUPhNO (fOC = 0.02). These values
agree well with those for classical DAEs.[30,37] Additionally, we
observed a correlation between the Hammett parameter (s’)
2
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Buckup et al. by transient absorption experiments of some dU
switches.[50] This overall high quantum yield for the ring
opening reaction is especially interesting for applications
where a fast back-and-forth switching of the two isomers is
desired. Considering the most important properties for
applications in biology (near-quantitative light-induced
switching in both directions, reversibility, thermal stability)
combined, our investigation revealed two dU switches that
were excellent with respect to all three properties, namely
t
dU2Py and dUPh Bu, while dUPh, dUMe, dU4Py and dUPh CF had
3
2
t
a slightly lower overall performance. dU2Py and dUPh Bu not
only rival many non-nucleosidic DAEs,[27] but beat most
azobenzene-based photochromic nucleosides.[14,31,51] The most
important difference to the literature-known azobenzene C-
nucleosides[12] and C5-azobenzene-substituted dU switches[52]
is the quantitative switching upon Vis irradiation (ring
opening for DAE, cis ! trans for azobenzenes), which allows
for a higher dynamic range. Our high performance dU
switches also show an increased thermal stability compared to
ortho methylated azobenzene switches, for example, 4-
carboxy-2’,6’-dimethylazobenzene (2’,6’-Me-Azo), which
were incorporated into DNA via the phosphate-backbone.
The thermal isomerization half-life of dU2Py at 708C is more
than 3 times higher (123 h) than that of 2’,6’-Me-Azo (36 h)
which was determined at only 608C.[53] The 2 excellent and the
4 good photochromic dU derivatives all have visible absorp-
tion maxima of the closed-ring form ꢀ 475 nm, indicating that
our strategies for bathochromic shifting (extension of the p-
system, addition of auxochromic groups) achieved the desired
goal only at the cost of a reduced overall performance. The
members of the dC series typically perform worse than the dU
derivatives in all three criteria. dC2Py is the only promising
candidate, if experiments at or slightly above room temper-
ature are planned.
Figure 3. Dependence of selected photophysical properties on the
electronic character of the substituents of dU switches. A) Schematic
arrangement of the electron withdrawing (EWG) and electron donating
(EDG) substituents on the thiophene moiety according to their 13C-
NMR-derived Hammett parameters (s’) for all derivatives that can be
analyzed by this formalism. The carbon atom C4 that was used for the
calculation of the Hammett parameters is marked with a star. B) Ham-
mett correlation of s’ with the absorption maximum in the visible
wavelength range of the closed-ring form. C) Hammett correlation of
s’ with the ring-closing quantum yields (FOC).
and fOC (Figure 3C). Particularly high cyclization quantum
yields were found for switches with electron-neutral substitu-
ents, while strong auxochromes such as PhNMe2 or PhNO2 led
to a much lower quantum yield fOC. For most compounds, UV
irradiation stimulated both cyclization and ring opening. The
ratio of cyclization and cycloreversion quantum yields
measured upon UV irradiation was found to differ signifi-
cantly between the dU and the dC series. While it is usually
> 1 for the dU switches, it is smaller for the dC switches. This
shows that for uridine-based photochromes the cyclization is
preferred over the cycloreversion, which is not the case for the
cytidines, furthermore explaining their different PSSUV com-
positions. Those uridine switches that do not have a phenyl
ring at the periphery of the thiophene seem to be an exception
in this respect, since their quantum yields of the ring opening
Photochromic Oligonucleotides
For use in automated solid-phase synthesis, phosphor-
t
amidites containing the dU2Py and dUPh Bu structures had to be
synthesized. Starting from commercial 5-iodo-2’-deoxyuri-
dine, the 5’-hydroxyl group was protected as the dimethoxy-
trityl ether, followed by a Suzuki cross-coupling reaction with
the corresponding boronic acid pinacolate ester 4R and
reaction with 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite (Figure 4A), generating phosphoramidites 10R. In-
corporation of 10R into 19mer oligonucleotides was accom-
plished with high incorporation efficiency indistinguishable
from commercial standard phosphoramidites (Figure S10).
After HPLC purification, the identity of the modified
oligonucleotides was confirmed by mass spectrometry (Fig-
ure 4B). Both oligonucleotides showed photochromism, and
a 6 nm bathochromic shift of lmax,vis of the photoswitchable
DNA compared to the free nucleoside (Figure 5). The
reaction are significantly increased (dUMe
f
CO = 0.38, dUCN
f
CO = 0.78). The photochromic nucleosides dUPh, dUNp
,
t
dUPh Bu and dU2Py showed a clear preference for the cycliza-
tion reaction with ratios between 1.35 and 2.14. Thus, together
with a large spectral separation in the UV range, a high ratio
of cyclization and cycloreversion quantum yields is respon-
sible for the almost quantitative switching of dU2Py and
t
dUPh Bu, which makes them our best-performing candidates
for the incorporation into oligonucleotides. An enormous
difference in comparison to classical DAEs is apparent for the
cycloreversion quantum yields fCO when irradiated at the
absorption band in the visible wavelength range (470 nm– percentage of closed-ring form in the PSSUV after UV
505 nm). There, the quantum yields of the nucleoside-based
DAEs are more than one order of magnitude larger than
those of non-nucleosidic DAEs (which are typically only a few
percent).[27] A similar tendency has already been observed by
irradiation was only slightly decreased (by 5–6%), compared
to the nucleoside (Figure S11). However, the photochromic
oligonucleotides showed strongly reduced quantum yields (4-
t
fold for dU2Py and 2-fold for dUPh Bu). These effects were
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Figure 4. Design, synthesis and properties of photochromic oligonucleotides. A) General synthetic approach. a) DMT-Cl, DMAP, rt, pyridine;
b) 4R, Cs2CO3 (aq.), Pd(dppf)Cl2, 1208C, acetonitrile/water (2/1); c) CEP-Cl, N(Et)(iPr)2, rt, DCM; d) complementary strand, 958C, buffer.
B) Synthesized photochromic DNA strands with determined masses. C) Photophysical properties and melting temperatures of the synthesized
DNA strands.
Figure 5. Absorption spectra, reversibility and thermal stability of T7-2Py-(ss) (A) and T7-PhtBu-(ss) (B). The absorption spectra of the open (black
line) and calculated closed (red line) isomer and the PSSUV (dashed blue line) are shown. The inset shows the zoomed-in range between 300 and
750 nm and the spectral separation is highlighted in yellow. In each panel, the reversibility (top right) and the thermal stability (low right) are
shown. For obtaining the PSSUV absorption spectra, the samples were irradiated with 340 nm at 35–40 mM in TRIS-buffer (40 mM, pH 7) with
22 mM MgCl2.
primarily due to the different steric and electronic environ-
ment of a nucleoside DAE inside an oligonucleotide, while
the effects of solvents and buffers[54] were less important
(Figure S12,13). Since the ratio of ring closure and ring
opening quantum yield upon UV irradiation in the oligonu-
cleotide was still > 1, only minor changes in the PSSUV
composition were observed. Due to the reduced quantum
yields, slightly longer irradiation times were necessary to
reach the PSSUV, compared to the nucleosides. Both modified
oligonucleotides showed excellent thermal stability of their
closed-ring isomer at 258C and 378C and high reversibility
(Figure 5). Upon hybridization with a fully complementary
19mer strand, the duplexes retained their photochromicity.
Duplex formation, however, influenced the composition of
the PSSUV differently for the two modifications (Figure 4C).
t
In the dUPh Bu-containing duplex, the percentage of closed-
ring isomer was reduced by 17%, compared to the single-
strand. No significant decrease in the quantum yields of the
ring opening reactions (upon UV and Vis irradiation) was
detected. However, the quantum yield for ring closure was
reduced by a factor of 2.5, explaining the altered PSSUV
composition. For the dU2Py-containing strand, duplex forma-
tion led to an PSSUV after UV-irradiation containing only
20% closed-ring isomer, which could be explained by the
strong decrease of the ring closing quantum yield (0.17 (ss)!
0.02 (ds)) and the concurrent increase of the ring opening
quantum yield (0.12 (ss)!0.21 (ds), Figure 4C). Compared to
Azo- and DM-Azo-derivatized oligonucleotides,[14,31,51] our
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photochromic T7-PhtBu oligonucleotide has a higher switch-
ing efficiency upon UV irradiation (88% vs. 77% in the single
strand and 70% vs. 30% in the duplex).
Upon Vis irradiation, our oligonucleotides exhibit quan-
titative switching, while this cannot be achieved for the
azobenzenes due to a spectral overlap of the two isomers. In
addition, our photochromic oligonucleotides show an in-
creased thermal stability (half-lives at 378C: 88.5 h for T7-
PhtBu (ss) and 67 h for T7-2Py (ss) vs. 36.5 h for S-Azo-DM in
the single strand).
To analyze the influence of the bulky DAE substitution on
DNA duplex stability, melting curves were recorded. In the
open form, the incorporation of dU2Py did not alter the
t
melting temperature, whereas dUPh Bu induced a destabiliza-
tion of the duplex (DT= À3.78C). This could be due to the
steric demand of the tert-butyl ester group. The cyclization of
the DAE unit caused a further destabilization of the duplex
by approximately 18C for both compounds (Figure S14).
Figure 6. Photocontrol of transcription A) Design of the in vitro tran-
scription assay. The DAE-modified strand (red) is hybridized to
a complementary strand (blue) that is linked to the sequence of the
malachite green aptamer (MGA). Transcription by T7 RNA polymerase
generates the aptamer which binds to its ligand malachite green
(MG), causing a fluorescence increase. B) Time course of the in vitro
transcription visualizing the increase of fluorescence intensity. The
photoregulation efficiency was defined as a and the relative activity
compared to the positive control was defined as b. C) Measured rate
constants for transcriptions.
Photocontrol of Transcription In Vitro
The sequence of the 19mer photochromic oligonucleo-
tides was chosen to represent the non-templating strand of
a promoter for the DNA-dependent RNA polymerase of
bacteriophage T7, an enzyme commonly used for RNA
synthesis by in vitro transcription. After combination with
a templating strand, a photochromic promoter is formed. Our
was defined as the ratio of initial transcription rates (kobs) of
irradiated and non-irradiated template. In the open-ring form,
both modified promoters showed almost the same activity as
the unmodified T7 promotor (relative activity b of 96% and
92%, respectively, Figure 6B), indicating that modifications
at this position do not perturb the interaction with the
enzyme. However, in the PSSUV after UV irradiation, a strong
goal was to test whether an “optochemical” control of
t
enzymatic RNA synthesis is possible with dU2Py- or dUPh Bu
-
modified promoters. There are— to our knowledge— only
very few reports on transcription control by incorporation of
a single reversible photoswitch. Asanuma and co-workers
inserted a threoninol-linked azobenzene at various positions
of both templating and non-templating strand of a T7
promotor, and monitored the enzymatic synthesis before
and after UV irradiation of the DNA duplex.[31] At different
positions, transcription was suppressed in the trans-form but
proceeded faster in the cis-form. The activation factors for
single modifications were mostly between 1.5- and 2.3-fold,
decrease of the transcription rate up to 2.39 fold for the
t
dUPh Bu and 1.66 fold for dU2Py was observed. These results
demonstrate that photoregulation of transcription is possible
using our optimized nucleosidic DAEs and suggest that
reversible photoswitching in situ can be achieved after further
optimization.
and the activated form was at least 30% less active than the Conclusion
unmodified promoter. Cahovµ and Jäschke observed that
replacement of dT at position À2 by dUPh inhibited tran-
scription activity by ꢁ 50%, while UV irradiation restored
activity almost quantitatively.[35] Based on the crystal struc-
In this work we provided the first systematic structure–
activity study of pyrimidine nucleoside-based DAE photo-
switches. A collection of 26 different dU- and dC-based DAEs
was synthesized of which 24 showed the typical photoinduced
electrocyclic ring closure and opening. While we could shift
the visible absorption maximum of the closed-ring form
considerably into the bathochromic direction by addition of
auxochromes or extension of the p-system, none of these red-
shifted compounds had the desired combination of properties
required for biological applications (near-quantitative switch-
ing in both directions, full reversibility, thermal stability). We
could establish a correlation between the electronic character
of the substituents reflected in the Hammett parameter and
the absorption maximum of the closed-ring isomer. Our
winning DAEs all had rather simple, small, and electronically
mostly neutral substituents, such as methyl, pyridyl, or
(substituted) phenyl, indicating that the options for tuning
ture of the T7 polymerase[55] we decided to incorporate the
t
photochromic nucleosides dU2Py and dUPh Bu at position À8 of
the non-template strand, one of the most responsive positions
for photoregulation observed in the azobenzene study.[31] The
modified non-template strand was hybridized with the
template strand consisting of the complementary promotor
sequence linked to the sequence encoding the malachite
green aptamer (MGA).[56] Transcribed MGA forms a fluores-
cent complex with its target malachite green (MG), so that
progress of the transcription can be monitored continuously
in real time by fluorescence (Figure 6A).[57–59] After a short
initiation phase, a linear increase in fluorescence was
observed, from which we calculated the initial transcription
rates. The photoregulation efficiency (a) of the transcription
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[9] J. Broichhagen, M. Schçnberger, S. C. Cork, J. A. Frank, P.
Marchetti, M. Bugliani, A. M. J. Shapiro, S. Trapp, G. A. Rutter,
D. J. Hodson, D. Trauner, Nat. Commun. 2014, 5, 5116.
[10] A. Damijonaitis, J. Broichhagen, T. Urushima, K. Hüll, J.
Nagpal, L. Laprell, M. Schçnberger, D. H. Woodmansee, A.
Rafiq, M. P. Sumser, W. Kummer, A. Gottschalk, D. Trauner,
ACS Chem. Neurosci. 2015, 6, 701 –707.
the absorption spectrum of the closed-ring form via thiophene
modification are more limited than we thought. Our inves-
tigation revealed further that it is much easier to generate
high-performance dU-switches than dC switches.
A remarkable feature of the nucleoside-based DAEs
reported here is their exceptionally high cycloreversion
quantum yield, which is of great interest, e.g., for applications
as a short term information storage medium.[40,60,61] Based on
[11] J. A. Frank, M. Moroni, R. Moshourab, M. Sumser, G. R. Lewin,
D. Trauner, Nat. Commun. 2015, 6, 7118.
[12] T. Goldau, K. Murayama, C. Brieke, S. Steinwand, P. Mondal, M.
Biswas, I. Burghardt, J. Wachtveitl, H. Asanuma, A. Heckel,
Chem. Eur. J. 2015, 21, 2845 –2854.
[13] B. Horstmann, M. Korbus, T. Friedmann, C. Wolff, C. M. Thiele,
F. J. Meyer-Almes, Bioorg. Chem. 2014, 57, 155 –161.
[14] Y. Kamiya, T. Takagi, H. Ooi, H. Ito, X. Liang, H. Asanuma,
ACS Synth. Biol. 2015, 4, 365 –370.
our analysis of 26 nucleosidic DAEs, two dU switches
t
(dUPh Bu, dU2Py) were identified as promising candidates for
the incorporation into the T7 promoter to control the activity
of its polymerase. The photochromism of the monomers was
maintained in the environment of the oligonucleotide, and for
t
dUPh Bu even in the duplex. The transcription rate of the
[15] S. Barrois, H. A. Wagenknecht, Beilstein J. Org. Chem. 2012, 8,
905 –914.
[16] X. Chen, S. Wehle, N. Kuzmanovic, B. Merget, U. Holzgrabe, B.
Kçnig, C. A. Sotriffer, M. Decker, ACS Chem. Neurosci. 2014, 5,
377 –389.
polymerase was modulated with an up to 2.4-fold turn on/off-
factor with a single modification site. Future work will focus
on increasing the turn-off factor of the transcription rate by
modifying the template strand that is directly recognized by
the polymerase. This system offers great potential for the
reversible optochemical control over genes of interest.
[17] K. Fujimoto, M. Kajino, I. Sakaguchi, M. Inouye, Chem. Eur. J.
2012, 18, 9834 –9840.
[18] K. Fujimoto, T. Maruyama, Y. Okada, T. Itou, M. Inouye,
Tetrahedron 2013, 69, 6170 –6175.
[19] R. Gçstl, S. Hecht, Chem. Eur. J. 2015, 21, 4422 –4427.
[20] M. Lion-Dagan, I. Willner, J. Photochem. Photobiol. A 1997,
108, 247 –252.
[21] C. ÖzÅoban, T. Halbritter, S. Steinwand, L. M. Herzig, J. Kohl-
Landgraf, N. Askari, F. Groher, B. Fürtig, C. Richter, H.
Schwalbe, B. Suess, J. Wachtveitl, A. Heckel, Org. Lett. 2015,
17, 1517 –1520.
[22] T. Sakata, Y. Yan, G. Marriott, Proc. Natl. Acad. Sci. USA 2005,
102, 4759 –4764.
[23] Y. H. Tsai, S. Essig, J. R. James, K. Lang, J. W. Chin, Nat. Chem.
2015, 7, 554 –561.
Acknowledgements
The authors thank Dr. R. Wirth and D. Englert for discussion
of the manuscript. We acknowledge H. Rudy, D. Wolf and T.
Timmermann for technical assistance and K. Haubrich and V.
Haller for their assistance in the early stage of the experi-
ments (all Heidelberg University). We also acknowledge H.
Derondeau (LMU Munich) for her help in calculating the
quantum yields. Open access funding enabled and organized
by Projekt DEAL.
[24] I. Willner, S. Rubin, J. Wonner, F. Effenberger, P. Bäuerle, J. Am.
Chem. Soc. 1992, 114, 3150 –3151.
[25] Y. Yokoyama, Chem. Rev. 2000, 100, 1717 –1739.
[26] S. Wiedbrauk, H. Dube, Tetrahedron Lett. 2015, 56, 4266 –4274.
[27] M. Irie, Chem. Rev. 2000, 100, 1685 –1716.
[28] M. Irie, T. Lifka, S. Kobatake, N. Kato, J. Am. Chem. Soc. 2000,
122, 4871 –4876.
Conflict of interest
The authors declare no conflict of interest.
[29] M. Irie, T. Lifka, K. Uchida, S. Kobatake, Y. Shindo, Chem.
Commun. 1999, 747 –748.
[30] M. Irie, M. Mohri, J. Org. Chem. 1988, 53, 803 –808.
[31] M. Liu, H. Asanuma, M. Komiyama, J. Am. Chem. Soc. 2006,
128, 1009 –1015.
Keywords: diarylethenes ·in vitro transcription ·nucleic acids ·
photochromism ·photoresponsive DNA
[32] B. Roubinet, M. Weber, H. Shojaei, M. Bates, M. L. Bossi, V. N.
Belov, M. Irie, S. W. Hell, J. Am. Chem. Soc. 2017, 139, 6611 –
6620.
[33] M. Singer, A. Jäschke, J. Am. Chem. Soc. 2010, 132, 8372 –8377.
[34] M. Singer, A. Nierth, A. Jäschke, Eur. J. Org. Chem. 2013, 2766 –
2769.
[1] C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer, A. Heckel,
Angew. Chem. Int. Ed. 2012, 51, 8446 –8476; Angew. Chem.
2012, 124, 8572 –8604.
[2] A. Gautier, C. Gauron, M. Volovitch, D. Bensimon, L. Jullien, S.
Vriz, Nat. Chem. Biol. 2014, 10, 533 –541.
[35] H. Cahovµ, A. Jäschke, Angew. Chem. Int. Ed. 2013, 52, 3186 –
3190; Angew. Chem. 2013, 125, 3268 –3272.
[3] A. Jäschke, FEBS Lett. 2012, 586, 2106 –2111.
´
[4] W. Szymanski, J. M. Beierle, H. A. V. Kistemaker, W. A. Vele-
[36] M. Irie, Photochem. Photobiol. Sci. 2010, 9, 1535 –1542.
[37] M. Irie, K. Sakemura, M. Okinaka, K. Uchida, J. Org. Chem.
1995, 60, 8305 –8309.
[38] S. L. Gilat, S. H. Kawai, J.-M. Lehn, Chem. Eur. J. 1995, 1, 275 –
284.
[39] C. Sarter, M. Heimes, A. Jäschke, Beilstein J. Org. Chem. 2016,
12, 1103 –1110.
[40] D. L. Kellis, C. Sarter, B. L. Cannon, P. H. Davis, E. Graugnard,
J. Lee, R. D. Pensack, T. Kolmar, A. Jäschke, B. Yurke, W. B.
Knowlton, ACS Nano 2019, 13, 2986 –2994.
ma, B. L. Feringa, Chem. Rev. 2013, 113, 6114 –6178.
[5] J. Bath, A. J. Turberfield, Nat. Nanotechnol. 2007, 2, 275 –284.
[6] V. I. Minkin in Molecular Switches (Eds.: B. L. Feringa, W. R.
Browne), Wiley-VCH, Weinheim, 2011, pp. 37 –74.
[7] S. Loudwig, H. Bayley in Dynamic Studies in Biology: Photo-
triggers, Photoswitches and Caged Biomolecules (Eds.: M.
Goeldner, R. Givens), Wiley-VCH, Weinheim, 2005, pp. 253 –
340.
[8] H. Asanuma, T. Ito, T. Yoshida, X. Liang, M. Komiyama, Angew.
Chem. Int. Ed. 1999, 38, 2393 –2395; Angew. Chem. 1999, 111,
2547 –2549.
[41] C. Sarter, S. Dey, A. Jäschke, ACS Omega 2019, 4, 12125 –12129.
8172
www.angewandte.org
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 8164 –8173

Angewandte
Research Articles
Chemie
[42] R. Wizinger, Chimia 1961, 15, 89 –105.
[54] Y. Ishibashi, T. Umesato, M. Fujiwara, K. Une, Y. Yoneda, H.
[43] H. Zollinger, Color Chemistry— Syntheses, Properties, and
Applications of Organic Dyes and Pigments, Wiley-VCH,
Weinheim, 2003, pp. 15 –20.
[44] M. Irie, T. Fukaminato, K. Matsuda, S. Kobatake, Chem. Rev.
2014, 114, 12174 –12277.
Sotome, T. Katayama, S. Kobatake, T. Asahi, M. Irie, H.
Miyasaka, J. Phys. Chem. C 2016, 120, 1170 –1177.
[55] G. M. Cheetham, D. Jeruzalmi, T. A. Steitz, Nature 1999, 399,
80 –83.
[56] J. R. Babendure, S. R. Adams, R. Y. Tsien, J. Am. Chem. Soc.
2003, 125, 14716 –14717.
[45] T. Ishiyama, M. Murata, N. Miyaura, J. Org. Chem. 1995, 60,
7508 –7510.
[57] K. Hçfer, L. V. Langejürgen, A. Jäschke, J. Am. Chem. Soc.
2013, 135, 13692 –13694.
[46] F. Gillanders, L. Giordano, S. A. Díaz, T. M. Jovin, E. A. Jares-
Erijman, Photochem. Photobiol. Sci. 2014, 13, 603 –612.
[47] M. Herder, B. M. Schmidt, L. Grubert, M. Pätzel, J. Schwarz, S.
Hecht, J. Am. Chem. Soc. 2015, 137, 2738 –2747.
[48] G. Sun, M. C. Nicklaus, Theor. Chem. Acc. 2007, 117, 323 –332.
[49] U. Megerle, R. Lechner, B. Kçnig, E. Riedle, Photochem.
Photobiol. Sci. 2010, 9, 1400 –1406.
[58] M. Ilgu, J. Ray, L. Bendickson, T. Wang, I. M. Geraskin, G. A.
Kraus, M. Nilsen-Hamilton, Methods 2016, 98, 26 –33.
[59] K. Furukawa, H. Abe, N. Abe, M. Harada, S. Tsuneda, Y. Ito,
Bioorg. Med. Chem. Lett. 2008, 18, 4562 –4565.
[60] C. L. Ward, C. G. Elles, J. Phys. Chem. Lett. 2012, 3, 2995 –3000.
[61] C. L. Ward, C. G. Elles, J. Phys. Chem. A 2014, 118, 10011 –
10019.
[50] T. Buckup, C. Sarter, H. R. Volpp, A. Jäschke, M. Motzkus, J.
Phys. Chem. Lett. 2015, 6, 4717 –4721.
[51] X. Liang, R. Wakuda, K. Fujioka, H. Asanuma, FEBS J. 2010,
277, 1551 –1561.
[52] S. Mori, K. Morihiro, S. Obika, Molecules 2014, 19, 5109 –5118.
[53] H. Nishioka, X. Liang, H. Asanuma, Chem. Eur. J. 2010, 16,
2054 –2062.
Manuscript received: November 6, 2020
Revised manuscript received: January 5, 2021
Accepted manuscript online: January 21, 2021
Version of record online: March 3, 2021
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