Photoswitchable Oligonucleotides Containing Different Diarylethene

Jul 15, 2019 - For the 2-naphthyl-modified oligonucleotide, 50% closed-ring isomers were detected, while 47% were measured for the methyl-substituted ...
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Article Cite This: ACS Omega 2019, 4, 12125−12129

Photoswitchable Oligonucleotides Containing Different Diarylethene-Modified Nucleotides Christopher Sarter,† Surjendu Dey,† and Andres Jäschke* Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, 69120 Heidelberg, Germany

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S Supporting Information *

ABSTRACT: Diarylethenes are a well-studied class of photoswitches and have often been linked to partner molecules to render them photoresponsive. Earlier, our lab developed a new type of diarylethenes in which the purine or pyrimidine base of a nucleoside or oligonucleotide serves as one of the two aryl residues of the photochromic system. Here, we report the synthesis of three different diarylethenedeoxyuridine phosphoramidites and their site-specific incorporation into oligodeoxynucleotides by solid-phase synthesis. Various DNA sequences carrying single or multiple, identical or different photoswitchable moieties are synthesized with high yield and purity. Upon UV irradiation, these DNA strands form a colored closed-ring isomer. The combination of different diarylethenes within one strand leads to an additive absorption spectrum. The photochromic DNA oligonucleotides are thermostable and photoreversible.

INTRODUCTION The control and manipulation of biological systems using photoactive small molecules is an attractive approach in chemical and synthetic biology.1−4 Photoswitches are molecules that undergo distinct structural changes upon irradiation with light, which is noninvasive and offers high spatio-temporal resolution. A common approach to make biopolymers, such as proteins or nucleic acids, sensitive toward photochemical stimuli is their covalent linkage to photoswitchable entities. Most reported applications use azobenzenes because of their comparatively simple synthesis and generally good reversibility.5−8 In comparison to azobenzene-photoswitches, diarylethenes,9−12 spiropyrans,13,14 fulgides,15,16 and hemithioindigos17 have been applied less frequently in biomolecular systems. Diarylethenes were originally developed for optical information storage in the 1980s but found a variety of applications in materials sciences, nanotechnology, and biomimetic chemistry.3,18−22 Their most common architecture is shown in Figure 1A: two heteroaromatic, most often five-membered, rings are connected via a (perhydrogenated or perfluorinated) cyclopentenyl bridge. They undergo a reversible photochemical electrocyclic ring-closure reaction. The photophysical and chemical properties, such as absorption and isomerization wavelengths, quantum yields, extinction coefficients, thermal stability, and fatigue resistance can be tuned by attaching variable substituents R at the aryl rings.18,23 Our group developed the first photochromic nucleosidebased diarylethenes in which the nucleobase is actively taking part in the switching process (Figure 1B,C).24−27 This design warrants a stronger influence of light on the structure and function of the nucleoside, compared to examples in which a typical photoswitch was linked to (or separated from) the nucleoside by a spacer.10,28,29 A strong influence of substituents © 2019 American Chemical Society

Figure 1. Diarylethene-based photoswitches. (A) Isomerization reaction of typical diarylethenes. Only one of two diastereomers is shown for the closed-ring isomer. (B) Deazapurine nucleoside diarylethenes.24−26 (C) Pyrimidine nucleoside diarylethenes.27

on the photophysical properties could be demonstrated for these compounds.25,30,31 For the pyrimidine switches (Figure 1C), we also demonstrated the synthesis of oligonucleotides carrying the same diarylethene system by Suzuki cross-coupling of a substituted thiophenyl-cyclopentenyl boronic acid to 5iodouridine- and 5-iodocytidine-containing oligonucleotides.27 However, yields were hardly satisfying (35% at best for a single coupling), and this method did not allow at all the incorporation of two or more chemically different diarylethenes into a DNA strand in a site-specific manner. Recently, we reported the Received: April 15, 2019 Accepted: June 18, 2019 Published: July 15, 2019 12125

DOI: 10.1021/acsomega.9b01070 ACS Omega 2019, 4, 12125−12129

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Scheme 1. Synthesis of the Three Different Phosphoramidites

Table 1. Synthesized Photoswitchable DNA Strands (Ph = Phenyl, Me = Methyl, and 2-Np = 2-Naphthyl)a


Masses determined by mass spectrometry. For HPLC chromatograms, see Figure S1.

SYNTHESIS OF PHOTOSWITCHABLE DNA OLIGONUCLEOTIDES Phosphoramidites 4a−c were applied in solid-phase synthesis to synthesize 13 different photoswitchable deoxyoligonucleotide (PS-ODN) sequences as shown in Table 1 using the standard 1 μmol DNA synthesis and deprotection protocol. All oligonucleotides were analyzed (Figure S1) and purified by reversedphase HPLC and their correct molecular weight confirmed by mass spectrometry (Table 1). All oligonucleotides have the same nucleotide sequence, which represents one strand of a commonly used promoter of the T7 RNA polymerase. This sequence was chosen to allow future studies on photomodulation of RNA polymerase activity. Three internal thymidine positions were selected and replaced with the corresponding deoxyuridine-based diarylethenes. First, we introduced one phenyl-modified diarylethene at a time in the marked positions (Table 1, PS-ODN 1−3). The modified phosphoramidite was incorporated as efficient as the standard building blocks. Then, we introduced two and three phenylmodified photoswitches at different positions (Table 1, PSODN 4−7). Similar to the phenyl switch, single incorporation worked efficient also for the methyl (PS-ODN 8−9) and for the 2-naphthyl phosphoramidite (PS-ODN 10−11). Finally, we incorporated two or three different photoswitches into the same DNA strand (PS-ODN 12, 13).

synthesis of a diarylethene deoxyuridine phosphoramidite (with R = phenyl), its incorporation at up to three positions into an oligonucleotide by solid-phase synthesis, and the application of the resulting fully reversible photochromic oligonucleotides as a conditional fluorescence quencher in an all-optical excitonic switch designed for next-generation information processing.32 These oligonucleotides switched reversibly up to 200 times without detectable fatigue both in solution and absorbed on a dry solid surface, and increasing the number of diarylethenes per strand was found to linearly increase the extinction coefficient and hence, the efficiency of fluorescence quenching.32 Herein, we take the next step toward orthogonal photoswitches by synthesizing oligonucleotides that carry up to three identical or different diarylethenes as part of their sequence. Three different phosphoramidites are synthesized, incorporated into DNA by solid-phase synthesis, and the resulting photochromic oligonucleotides subjected to an initial photophysical and chemical characterization (Scheme 1).


5-Iodo-2′-deoxyuridine was 5′-DMT-protected using a standard procedure.33 The resulting compound 1 was coupled with three different boronic acid esters 2a−c (Ph = phenyl, Me = methyl, and 2-Np = 2-naphthyl) by Suzuki cross-coupling to obtain the photochromic nucleosides 3a−c in good (73−82%) yields. Phosphoramidites 4a−c were furnished by adding 2-cyanoethylN,N-diisopropylchlorophosphoramidite. For details and analytical characterization, see Supporting Information.

PHOTOPHYSICAL PROPERTIES OF THE SYNTHESIZED PHOTOSWITCHABLE DNA The synthesized DNA-sequences (PS-ODN 1−13) were subjected to an initial photophysical characterization. First, oligonucleotides were irradiated by UV light and the ring12126

DOI: 10.1021/acsomega.9b01070 ACS Omega 2019, 4, 12125−12129

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Table 2. Photophysical Properties of the Synthesized DNA Sequences



λmax,vis [nm]a

ε@λmax,vis [M−1 cm−1]b

Δ(λmax,vis) [nm]c

1 2 3 4 5 6 7 8 9 10 11 12 13


480 480 480 480 480 480 480 450 450 490 490 475 480

6200 6000 5500 11800 11250 11150 15250 2750 2800 3300 3400 5000 7700

10 10 10 10 10 10 10 5 5 8 8 n.a.d n.a.d

a Wavelength of the absorption maximum in the visible range of the photoswitchable DNA. bMolar extinction coefficient at λmax,vis at the photostationary state after UV irradiation. cDifference between λmax,vis of the photoswitchable DNA and the corresponding photoswitchable nucleoside. dNot applicable.

Figure 2. Comparison of photochromic and photophysical properties of photoswitchable DNA. (A) R = Ph, PS-ODN 1; (B) R = Me, PS-ODN 8; (C) R = 2-Np, PS-ODN 10; (D) R = Me, Ph, 2-Np, PS-ODN 13. Inset (a) absorption at λmax,vis, inset (b) thermostability measurements of the closed-ring isomer at different temperatures (25, 50, and 70 °C).

closure reaction monitored by UV−vis spectroscopy. Table 2

visible range. The three strands with a single phenyl-modified diarylethene at different positions have the same maximum (480 nm, Table 2, entries 1−3, Figures 2A and S2), while their molar extinction coefficients at the photostationary state vary slightly.

shows a summary of the photophysical properties. As observed for the nucleosides, electrocyclic ring closure leads to the appearance of a new absorption maximum in the 12127

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The same λmax,vis was observed after the incorporation of two or three phenyl-modified photoswitches (Table 2, entries 4−7, Figure S3), and the molar extinction coefficients increased almost linearly with the number of modifications (Figure S4). The phenyl-modified photoswitches turned out to be highly thermostable (insets in Figure 2); even at 70 °C the thermal ring opening occurred with a first-order rate constant kthermal of 0.011 ± 0.002 h−1 (average over all single-modified ODNs), corresponding to a half-life τ of 63 h. For DNA strands containing the methyl- or 2-naphthylmodified diarylethenes (Table 2, entries 8−11; Figures 2B,C and S5) similar trends were observed. Both strands carrying the methyl-modified photoswitch showed a maximum at 450 nm (Table 2, entries 8, 9), while the maximum at 490 nm was noticed for the oligonucleotides bearing the 2-naphthylmodified photoswitch (Table 2, entries 10, 11). The observed bathochromic shift of λmax,vis from methyl to phenyl to 2naphthyl is due to the increased conjugation of these residues with the neighboring thiophene ring and the extension of the conjugated system. In all cases, a 5−10 nm bathochromic shift of λmax,vis of the photoswitchable DNA compared to the free nucleoside is noticed (Table 2, entries 1−11). The naphthyl photoswitch showed a slightly reduced thermal stability (kthermal,70°C = 0.028 ± 0.005 h−1, τ70°C = 25 h), whereas the methyl photoswitches ring-opened much faster (kthermal,70°C = 0.25 ± 0.04 h−1, τ70°C = 2.8 h). At room temperature, however, even this switch had excellent long-term stability (τ25°C = 48 h). For PS-ODN 8 and 10, we succeeded in separating the openand closed-ring form by HPLC, allowing the determination of the composition of the photostationary state. For the 2naphthyl-modified oligonucleotide, 50% closed-ring isomers were detected, while 47% were measured for the methylsubstituted strand (Figure S6). Similarly, we recently determined a value of 50% for an oligonucleotide with a single phenyl-substituted diarylethene.32 For the photoswitchable DNA carrying different diarylethene moieties simultaneously, the absorption bands of the individual chromophores overlapped, yielding a single absorption band with a λmax,vis that was roughly the mean value of the maxima of the individual chromophores (Figure S7). Upon irradiation with visible (broad-band white) light, the absorbance of all oligonucleotides in the visible range was reduced to zero, and the UV−vis spectra were indistinguishable from those prior to UV irradiation, indicating reversibility and near-quantitative ring opening. All DNA strands showed good to excellent thermostability at 25, 50, and 70 °C (see insets in Figure 2).

UV irradiation leads to a photostationary state that contains only ∼50% of the closed-ring forms, thereby limiting the dynamic range of responses. Second, for orthogonal switches operating at different wavelengths, substituents for the diarylethene system must be identified that induce larger spectral shifts. In the current system, the absorbance maxima differ only by 40 nm (naphthyl vs methyl). Because the absorption bands are very broad (full widths at half-maximum ≥ 100 nm), the current combination of substituents does not offer suitable wavelengths for orthogonal switching. However, the synthetic methodology described here should be applicable to a wide range of substituted deoxyuridine diarylethenes.


S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b01070. General experimental details, synthesis of phosphoramidites, synthesis and purification of photochromic oligonucleotides, analytical HPLC chromatograms; spectral changes; absorption spectra; and HPLC time trace (PDF)


Corresponding Author

*E-mail: [email protected]. Web: ORCID

Andres Jäschke: 0000-0002-4625-2655 Author Contributions †

C.S. and S.D. contributed equally to this work.


The authors declare no competing financial interest.

ACKNOWLEDGMENTS The Authors thank Sandra Suhm, Heiko Rudy and Tobias Timmermann for technical assistance and Philipp Werther and Simon Büllmann for their assistance in the early stage of the experiments.


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CONCLUSIONS In conclusion, we synthesized three deoxyuridine-based diarylethene phosphoramidites, in which different substituents (Ph, Me, and 2-Np) are attached to the diarylethene moiety, and incorporated them into DNA with an incorporation efficiency indistinguishable from standard phosphoramidites. Applying this method, we could show for the first time the incorporation of three chemically different photoswitches into a single DNA strand. Furthermore, tuning of the photoinduced electrocyclic ring closure/ring-opening process is possible by changing the chemical nature of the substituents, as larger conjugated systems and push−pull systems tend to shift the absorption wavelength of the closed-ring form to longer wavelengths. However, for applications in biological or nanotechnology systems, at least two properties of the current system are not yet optimal: first, 12128

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DOI: 10.1021/acsomega.9b01070 ACS Omega 2019, 4, 12125−12129