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Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA

Soňa Boháčováa, Lucie Ludvíkováb, Lenka Poštová Slavětínskáa, Zuzana Vaníkováac, Petr Klánb and Michal Hocek*ac
aInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, CZ-16610 Prague 6, Czech Republic. E-mail: hocek@uochb.cas.cz
bDepartment of Chemistry and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic
cDepartment of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 2, CZ-12843, Czech Republic

Received 19th January 2018 , Accepted 1st February 2018

First published on 2nd February 2018


Nucleosides, nucleotides and 2′-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).


Introduction

Photocleavable protecting groups1 are gaining growing attention due to their applications in organic synthesis, chemical biology and many other areas. In biomolecules, photocaging1,2 and cleavage of the photoremovable protective groups by light, which is both biocompatible and bioorthogonal, is often used for triggering or switching biological processes. Photocaging of nucleic acids3 is a particularly active field. The photocaging on the nucleobase (mostly at N3 or exocyclic O4/N4 atoms of pyrimidines) is used4 to prevent the base-pairing of oligonucleotides which only after photodeprotection can hybridize with the complementary strand. On the other hand, photocaging of the phosphate backbone5 increases the resistance to hydrolysis and influences the interactions with other biomolecules. Photochemical deprotection can therefore either trigger or block biological processes, i.e., primer extension, transcription, antisense effect, RNA interference, translation, etc. It is important to mention that all these types of caged oligonucleotides and nucleic acids can only be synthesized chemically because the corresponding caged (deoxy)nucleoside triphosphates (NTPs or dNTPs) are not suitable substrates for polymerases.3–5 On the other hand, photocaging in the major-groove site of nucleotides and nucleic acids has been studied less frequently. Photocaged 5-(aminopropargyl)-pyrimidine dNTPs were used for polymerase synthesis of DNA and photochemical release of the reactive amino functions in the major groove.6 Nitrobenzyl- (NB), and -phenylethyl-caged 5-(hydroxymethyl)pyrimidine or 7-hydroxymethyl-7-deazapurine 2′-deoxyribonucleoside triphosphates (dNTPs) were used as reversible terminators of primer extension in sequencing.7 On the other hand, 6-O-(2-nitrobenzyl)guanosine and 4-O-(2-nitrobenzyl)uridine triphosphates8 were not substrates for RNA polymerases until photochemical decaging.

5-(Hydroxymethyl)uracil (5hmU) is a rare DNA base previously found in bacteriophages,9 microorganisms,10 and also eukarytotic genomes.11 The Schultz group has even evolved12 a bacterium which replaced thymines with 5hmU in genome showing that this base is biocompatible. Unlike the related 5-(hydroxymethyl)cytosine (5hmC),13 which is a known epigenetic mark and intermediate in active demethylation of DNA,14 the biological role of 5hmU is not known so far.15 In order to create an artificial epigenetic system for the regulation or switching of gene expression, we have systematically studied16 the influence of chemical modifications in the major groove of DNA on bacterial transcription and found that some small modifications are tolerated by the bacterial RNA polymerase and transcription factor and the modified DNA still serves as a template for transcription. Obviously, any bulkier modifications in the major groove inhibited the transcription.16 Recently we found17 that the presence of 5hmU or 5hmC in template DNA can even enhance (up to 3.5 times) or inhibit bacterial transcription (depending on the promoter) and thus even hmU could be an epigenetic mark.

Previously, we have also studied the influence of major-groove modifications on recognition and cleavage of DNA by restriction endonucleases (REs) and found that some small modifications at T/U18 or A19 are tolerated (and the REs cleave the modified DNA), whereas all bulky modifications or any modifications at G20 or C18 bases inhibit the cleavage. We also developed21 transient chemical protection of DNA from RE cleavage by polymerase incorporation of bulky 7-(triethylsilylethynyl)-7-deazaadenine which, after desilylation, released 7-ethynyl-7-deazaadenine-modified DNA being fully cleavable by several REs. In order to develop a more biocompatible and bioorthogonal switch, we recently reported22 the use of 5-[(2-nitrobenzyl)oxymethyl]uracil dNTP as a substrate for DNA polymerases leading to the enzymatic synthesis of DNA photocaged in the major-groove. The presence of the bulky NB group in the major groove blocked specific interactions and cleavage by restriction endonucleases (REs) but, after photodeprotection by irradiation using UV light (365 nm), the 5-hydroxymethyluracil-containing DNA was recognized and cut by REs. It was a good proof of the principle, but for any in cellulo or in vivo applications, the use of UV light is not desirable1b because of its toxicity and limited penetration through tissues. Therefore, it is desirable to develop alternative photocaging groups for 5hmU which would be compatible with the synthesis of modified dNTPs, polymerase incorporation and would be cleavable by visible light. Major challenges in the design and development of new photocaging groups for nucleoside triphosphates are (i) limited chemical stability of some groups (e.g. carbonates) during the triphosphorylation steps and (ii) insufficient substrate activities of some very bulky dNTPs for polymerase incorporations to DNA (in particular by PCR). For example, the nitrophenethyl-caged dNTPs were incorporated by polymerases but terminated the primer extension.7 Here we report on the synthesis of 5hmU dNTPs protected with 6-nitropiperonyl (NP)23 and anthryl-9-methyl (An)24 groups and comparison of substrate activities and photocleavage reactions with parent NB-caged nucleotides.

Results and discussion

Synthesis

Although there were some reports on selective modification of hydroxymethyl groups in 5-hydroxymethyl-2′-deoxyuridine,25 for the desired ether formation in the introduction of the photocaging groups, we chose an opposite approach based on nucleophilic substitution of a suitably protected 5-bromomethyl-2′-deoxyuridine derivative (Scheme 1). In analogy to the previous related studies,7,22,26 we started from 3′,5′-bis-O-(TBDMS)-3-Boc-protected 5-bromomethyl-2′-deoxyuridine 1 and performed substitution reactions with the corresponding benzyl-type alcohol in the presence of 2,2,6,6-tetramethylpiperidine in CH3CN at 50 °C. The addition of AgOTf was important for the precipitation of AgBr and thus shifting the equilibrium towards products. The desired fully protected caged nucleosides 2a and 2b were isolated in moderate yields (10 and 21%). The An-caged intermediate 2c could not be isolated in pure form and was used in the next step in the crude form. Subsequently, the BOC group was removed using sodium bicarbonate27 in MeOH at 50 °C, whereas the common reagents such as CF3COOH or TBAF failed. The TBDMS group was then removed using Et3N·3HF28 in THF. The desired photocaged nucleosides dUNB and dUNP were isolated in good yields of 64 and 57%, whereas the dUAn was isolated in low 9% overall yield.
image file: c8ob00160j-s1.tif
Scheme 1 Synthesis of photocaged 2′-deoxyuridine monophosphates and triphosphates (i) ROH, 2,2,6,6-tetramethylpiperidine, silver triflate, CH3CN (ii) NaHCO3, MeOH (iii) Et3N.3HF, THF (iv) POCl3, PO(OMe)3, (NHBu3)2H2P2O7 (v) POCl3, PO(OMe)3, 2 M TEAB. *Prepared according to the literature.22

Phosphorylation reactions were carried out with POCl3 in trimethyl phosphate at 0 °C. Quenching of the reaction with 2 M TEAB gave the desired monophosphates dUNBMP, dUNPMP and dUAnMP in moderate yields after isolation by HPLC. To form dNTPs, phosphorylation was carried out in the same way followed by the addition of pyrophosphate and tributyl amine in DMF. The targets dUNBTP, dUNPTP and dUAnTP were obtained in moderate yields after isolation by HPLC. The uncaged 5-hydroxymethyl-2′-deoxyuridine triphosphate dUhmTP was prepared in 27% yield by the photolysis of dUNPTP (with 59% recovery of the starting material) in water using irradiation by UV LEDs at either 375 or 385 nm followed by the separation on HPLC (Scheme 2).


image file: c8ob00160j-s2.tif
Scheme 2 Synthesis of dUhmTP.

Photophysical properties of modified monophosphates and quantum yields of uncaging reactions

Initially, we recorded the absorption spectra of the model caged monophosphates dURMP in water and calculated the molar absorption coefficients for selected wavelengths in the range of 355–437 nm (Table 1 and Table S1 and Fig. S1–S7 in the ESI). The parent dUNBMP exerted an absorption maximum at 264 nm and showed a very low absorption above 300 nm. At 365 nm (under which the NB group is cleaved22), the molar absorption coefficient is only 238 M−1 cm−1 (Table 1, Table S1 and Fig. S3 in the ESI). On the other hand, the nitropiperonyl-caged nucleotide dUNPMP has a strong absorption maximum at 358 nm and still a relatively significant absorption at 400 nm (1940 M−1 cm−1, Table 1 and Fig. S4 in the ESI). dUAnMP showed three absorption maxima at 349, 367 and 386 nm as expected for an anthracene moiety. The spectra of dUNPMP and dUAnMP (Table S1, Fig. S4 and S6 in the ESI) indicate that photochemical deprotection can be possible using light of wavelengths up to 425 nm.
Table 1 Absorption maxima of caged dURMPs in water
Compound dUNBMP dUNPMP dUAnMP
a Determined only for relevant maxima >300 nm.
λmax (nm) [εmax, (M−1 cm−1)]a 264 [n.d.] 252 [n.d.] 241 [n.d.]
358 [5798] 349 [4006]
367 [5527]
386 [5050]


Then we determined the quantum yields (QY) of uncaging. The measurements were carried out at 365 nm in water with a ferrioxalate actinometer29 according to published procedures30 giving an absolute value of QY. The QY of photocleavage of dUNPMP was moderate with the value of 4.4 ± 0.8%, whereas the QY value for dUAnMP was negligible (in the range of an experimental error). Our attempts to measure QYs of previously published NB-group-protected dUNBMP failed because of two factors. (a) The molar absorption coefficient of dUNBMP at 365 nm was too low and the measurements had to be carried out in too concentrated solutions, and (b) the nitrosobenzene photoproduct formed during the reaction strongly absorbs at 365 nm. The fluorescence quantum yields of modified monophosphates in water were also determined at the excitation wavelength of 365 nm using quinine sulfate dissolved in 0.5 M H2SO4 as a standard31 giving relative values of QYs. UAnMP is moderately fluorescent with the quantum yield of 4.48 ± 0.15%; UNBMP and UNPMP are not fluorescent.

Incorporation of caged nucleotides into DNA

Caged triphosphates dUNBTP, dUNPTP and dUAnTP as well as the uncaged dUhmTP were tested as substrates for DNA polymerases.32 At first, the primer extension (PEX) reaction was tested using a 19-mer template (for sequences of ONs see Table S4 in the ESI) encoding for the incorporation of one modified dUR followed by three guanines. Fig. 1a shows the successful incorporation of all modified dUR nucleotides using KOD XL (for successful PEX using Vent (exo-) polymerase, see Fig. S12 in the ESI). The extended modified oligonucleotide (ON) products were confirmed by the MALDI-TOF analysis showing masses corresponding to Uhm-containing ONs (Fig. S13 in the ESI) because the laser irradiation using in MALDI cleaves the photoremovable groups. In the case of An modification, both caged and uncaged products were detected (Fig. S14 in the ESI). We also performed a simple kinetics of single nucleotide extension (Fig. S15 and S16 in the ESI) showing that the incorporation of modified dURTPs was only slightly slower compared to that of TTP. Then, the PEX reaction was performed with a more demanding 31-mer template designed for the incorporation of 4 dUR modifications (Fig. 1b). This PEX proceeded successfully with dUhmTP, dUNBTP and dUNPTP giving a clean fully extended product. UANTP appeared to be a slightly worse substrate for DNA polymerase giving the mixture of extended products also containing truncated ONs. The worse outcome was probably due to the bulkiness and hydrophobicity of the anthracene group.
image file: c8ob00160j-f1.tif
Fig. 1 Denaturing PAGE analysis of KOD XL DNA polymerase catalyzed (a) PEX with oligo1T 19-mer template and (b) PEX with prb4basII 31-mer template: lane 1, P: primer; lane 2 T+: product of PEX with natural dNTPs; lane 3, T: products of PEX in the absence of dTTP; lane 4, hm: product of PEX with dUhmTP and three natural dNTPs; lane 5, NB: product of PEX with dUNBTP and three natural dNTPs; lane 6, NP: product of PEX with dUNPTP and three natural dNTPs; lane 7, An: product of PEX with dUAnTP and three natural dNTPs. Agarose gel analysis of PCR with (c) FVL-A 98-mer or (d) 297-mer template: lane 1, L: 100 bp ladder; lane 2, +: product of PCR with natural dNTPs; lane 3, −: product of PCR in the absence of dTTP; lane 4, hm: product of PCR with dUhmTP and three natural dNTPs; lane 5, NB: product of PCR with dUNBTP and three natural dNTPs; lane 6, NP: product of PCR with dUNPTP and three natural dNTPs; lane 7, An: product of PCR with dUAnTP and three natural dNTPs.

Next, we tested dUhmTP, dUNBTP, dUNPTP and dUAnTP in PCR using either a 98-mer or (a more demanding) 297-mer template. In agreement with our previous communications,17,22 the PCR reactions with dUhmTP and dUNBTP, as well as with the new nitropiperonyl-caged dUNPTP, proceeded but the products of amplification were rather weak. The agarose gel of the 98-mer PCR (Fig. 1c) shows the bands of the modified PCR products only after the concentration of the sample, whereas the longer PCR (Fig. 1d) showed only very weak bands with somewhat faster mobility even after the concentration of the sample. However, in all cases, the identity of the modified amplicons was confirmed by sequencing. The bulky and hydrophobic dUAnTP did not give any amplification products in either of the PCR reaction.

Photochemical deprotection of caged DNA

In order to study the photocleavage of caged DNA, we used the previously described22 approach utilizing the cleavage of modified DNA by RsaI restriction endonuclease for monitoring the photochemical deprotection of DNA. The caged DNA containing bulky protecting groups is not cleaved by RE, whereas the DNA containing small hydroxymethyluracil modification is fully cleaved (Scheme 3). Therefore, we prepared 30-mer PEX products containing a dUR modification in the recognition site for RsaI. We tested the cleavage of the DNA by RE before and after irradiation using different LED diodes. In our previous work, we showed22 that the NB-caged DNA can be efficiently liberated by irradiation at 355 nm. Now, we tested the photoremoval of all three caging groups with UV or visible light at different wavelengths (355, 375, 385, 400 and 425 nm). Uncaging experiments at 437 nm and higher wavelengths could not be tested because of the photo-bleaching of the 6-FAM used for labeling and visualization of ONs on gels (Fig. S17). Considering the negligible absorbances of dURMPs at 437 nm (see Table S1 in the ESI for molar absorption coefficients at 437 nm), we do not expect that liberation would proceed at 437 nm and higher wavelengths.
image file: c8ob00160j-s3.tif
Scheme 3 Caging group prevents the cleavage of DNA by restriction enzymes. When the cage is removed by UV light, DNA cleavage by restriction enzymes is restored.

Fig. 2 shows the results of photochemical deprotection reactions of modified DNA at 375 and 425 nm (b), whereas Table 1 (and Fig. S22–S27 in the ESI) gives complete data at all tested wavelengths. ImageJ software was used to quantify the ratios of uncaged DNA (Table 1). Because of different optical power of the different LEDs, the reaction times differed; we tested at least 3 different irradiation times to reach (almost) complete conversion when possible. In all cases, the observed photochemical cleavage of the An group was very inefficient (even at 355 nm) and the conversions were very low (up to 15% even with prolonged time). The cleavage of NB and NP groups proceeded with comparable efficiencies at lower wavelengths (355–385 nm) reaching good conversions (92–95%) in reasonable reaction times (15–120 min). Only at higher wavelengths (400 and 425 nm), the nitropiperonyl group was cleaved significantly faster. At the highest wavelength of 425 nm, the cleavage of the nitrobenzyl group did not reach full conversion, whereas the cleavage of the NP group was still almost quantitative. Taking into account the almost negligible absorbance of the nitrobenzyl group at 400 and 425 nm, it was rather surprising that the NB group was still at least partly cleavable. However, as expected, uncaging of NPs was much faster and more efficient at these wavelengths, and we conclude that this group can be removed with visible light (Table 2).


image file: c8ob00160j-f2.tif
Fig. 2 Denaturing PAGE analysis of PEX with RsT 30-mer template followed by uncaging with the UV LED (a) 375 nm (2.5 mW) (b) 425 nm (10–16 mW): lane 1, P: primer; lane 2, −: product of PEX in the absence of dTTP; lane 3, +: product of PEX with natural dNTPs; lane 4, +: products of PEX with natural NTPs followed by the reaction with RsaI; lane 5, hm: product of PEX with dUhmTP and three natural dNTPs; lane 6, hm: product of PEX with dUhmTP and three natural dNTPs followed by the reaction with RsaI; lane 7,11,15, products of PEX with dUNBTP, dUNPTP or dUAnTP and three natural dNTPs; lanes 8–10, 12–14 and 16–18, product of PEX with dUNBTP, dUNPTP or dUAnTP and three natural dNTPs after irradiation with the UV lamp (irradiation time in minutes) followed by reaction with RsaI.
Table 2 DNA uncaging conversions quantification evaluated from gels using ImageJ software
Caging group Λ (nm) Optical powera (mW) Time 1 (min) Conv. (%) Time 2 (min) Conv. (%) Time 3 (min) Conv. (%)
a Optical power of the LED diodes given by the manufacturer.
NB 355 0.8–1.2 10 52 60 78 120 95
NP 355 0.8–1.2 10 59 60 79 120 93
An 355 0.8–1.2 60 6 240 10 480 13
NB 375 2.5 5 43 20 82 40 93
NP 375 2.5 5 64 20 90 40 93
An 375 2.5 10 4 60 9 120 10
NB 385 11.0 2 49 5 73 15 92
NP 385 11.0 2 76 5 85 15 95
An 385 11.0 5 9 20 13 60 15
NB 400 21–29 2 32 5 45 20 78
NP 400 21–29 2 59 5 81 20 92
An 400 21–29 10 8 60 11 120 11
NB 425 10–16 30 34 120 56 180 63
NP 425 10–16 30 68 120 86 180 92
An 425 10–16 30 5 60 7 120 7


Conclusions

We prepared three types of photocaged derivatives of 2′-deoxy-5-(hydroxymethyl)uridine nucleoside, mono- and triphosphates protected by 2-nitrobenzyl-, 6-nitropiperonyl- and anthryl-9-methyl groups, and tested their enzymatic incorporations to DNA and photochemical deprotections. The NB- and NP-caged dNTPs (dUNBTP and dUNPTP) were good substrates for DNA polymerases and worked in both PEX and PCR reactions. The An-linked dUAnTP was a less efficient substrate but it was still possible to incorporate it to at least some sequences using PEX. The liberation of photocaged DNA was tested by monitoring the cleavage of DNA with REs. We show that both NB- and NP-caged DNA can be efficiently deprotected by UV irradiation, whereas the nitropiperonyl group is suitable for uncaging with visible light at 400 or 425 nm. Therefore, the NP-caging has a better potential for in cellulo or in vivo photocaging of 5-(hydroxymethyl)uracil in DNA. The An group does not apparently have much potential in DNA photocaging but still the An-substituted dNTP can find some use in polymerase synthesis of DNA bearing a fluorescent extended aromatic system for applications in imaging or selection of hydrophobic aptamers.

Experimental

Full experimental part is given in the ESI. Selected typical experiments are given below.

3′,5′-Bis-O-(tert-butyldimethylsilyl)-3-tert-butoxycarbonyl-5-(6-nitropiperonyloxy)methyl-2′-deoxyuridine (2b)

6-Nitropiperonyl alcohol (240 mg, 1.22 mmol) and silver triflate (308 mg, 1.20 mmol) were suspended in dry acetonitrile and 2,2,6,6-tetramethylpiperidine (205 μL, 1.21 mmol) was added. The mixture was stirred for 10 min at 50 °C. Then solution of 1 in acetonitrile was added and the reaction was stirred for another 2 h at 50 °C. The solvent was removed under reduced pressure and the mixture was separated by chromatography (hexane[thin space (1/6-em)]:[thin space (1/6-em)]ethyl acetate 5[thin space (1/6-em)]:[thin space (1/6-em)]1) to give yellow oil (97 mg, 21%). 1H NMR (500 MHz, DMSO-d6): 0.03, 0.04, 0.078 and 0.081 (4 × s, 4 × 3H, CH3Si); 0.84 and 0.87 (2 × s, 2 × 9H, (CH3)3C); 1.51 (s, 9H, (CH3)3COCO); 2.18 (ddd, 1H, Jgem = 13.5 Hz, J2′a,1′ = 6.2 Hz, J2′a,3′ = 3.2 Hz, H-2′a); 2.31 (ddd, 1H, Jgem = 13.5 Hz, J2′b,1′ = 7.3 Hz, J2′b,3′ = 5.9 Hz, H-2′b); 3.70 (dd, 1H, Jgem = 11.4 Hz, J5′a,4′ = 4.4 Hz, H-5′a); 3.74 (dd, 1H, Jgem = 11.4 Hz, J5′b,4′ = 4.6 Hz, H-5′b); 3.84 (td, 1H, J4′,5′a = J4′,5′b = 4.5 Hz, J4′,3′ = 3.0 Hz, H-4′); 4.26 (s, 2H, B-CH2O); 4.35 (dt, 1H, J3′,2′b = 5.8 Hz, J3′,4′ = J3′,2′a = 3.1 Hz, H-3′); 4.80 (s, 2H, OCH2Ph); 6.10 (dd, 1H, J1′,2′b = 7.3 Hz, J1′,2′a = 6.2 Hz, H-1′); 6.24 (s, 2H, OCH2O); 7.26 (s, 1H, H-6′′); 7.70 (s, 1H, H-3′′); 7.79 (s, 1H, H-6). 13C NMR (125.7 MHz, DMSO-d6): −5.31, −5.28, −4.73 and −4.56 (CH3Si); 17.94 and 18.23 ((CH3)3C); 25.89 and 25.98 ((CH3)3C); 27.24 ((CH3)3COCO); 39.5 (CH2-2′); 62.78 (CH2-5′); 65.02 (B-CH2O); 68.79 (OCH2Ph); 72.11 (CH-3′); 85.56 (CH-1′); 86.71 ((CH3)3COCO); 87.46 (CH-4′); 103.69 (OCH2O); 105.51 (CH-3′′); 107.36 (CH-6′′); 110.03 (C-5); 132.28 (CH-1′′); 139.86 (CH-6); 141.05 (C-2′′); 147.05 (C-4′′); 147.82 ((CH3)3COCO); 148.06 (C-2); 152.49 (C-5′′); 159.98 (C-4). MS (ESI): m/z (%):789 (50)[M + Na + H]2+, 788 (100) [M + Na]+; HRMS (ESI): m/z calcd for C35H55N3O12NaSi2: 788.32195 [M + Na]+; found: 788.32165.

3′,5′-Bis-O-(tert-butyldimethylsilyl)-5-(6-nitropiperonyloxy)methyl-2′-deoxyuridine (3b)

Compound 2b (131 mg, 0.17 mmol) was dissolved in methanol, NaHCO3 (144 mg, 1.70 mmol) was added and the suspension was stirred at 56 °C overnight. The solvent was removed under vacuum and the residue was extracted with ethyl acetate and purified by column chromatography (hexane[thin space (1/6-em)]:[thin space (1/6-em)]EtOAc 3[thin space (1/6-em)]:[thin space (1/6-em)]2) to give 3b as yellow powder (81 mg, 71%). 1H NMR (500 MHz, DMSO-d6): 0.039, 0.044, 0.080 and 0.082 (4 × s, 4 × 3H, CH3Si); 0.85 and 0.87 (2 × s, 2 × 9H, (CH3)3C); 2.12 (ddd, 1H, Jgem = 13.4 Hz, J2′a,1′ = 6.2 Hz, J2′a,3′ = 3.1 Hz, H-2′a); 2.21 (ddd, 1H, Jgem = 13.4 Hz, J2′b,1′ = 7.8 Hz, J2′b,3′ = 6.0 Hz, H-2′b); 3.69 (dd, 1H, Jgem = 11.3 Hz, J5′a,4′ = 4.3 Hz, H-5′a); 3.74 (dd, 1H, Jgem = 11.3 Hz, J5′b,4′ = 4.6 Hz, H-5′b); 3.80 (td, 1H, J4′,5′a = J4′,5′b = 4.5 Hz, J4′,3′ = 2.9 Hz, H-4′); 4.20 and 4.23 (2 × dd, 2 × 1H, Jgem = 11.6 Hz, JCH2,CH2 = 0.7 Hz, B-CH2O); 4.35 (dt, 1H, J3′,2′b = 5.9 Hz, J3′,4′ = J3′,2′a = 3.0 Hz, H-3′); 4.78 (d, 2H, JCH2,CH2 = 0.7 Hz, OCH2Ph); 6.14 (dd, 1H, J1′,2′b = 7.8 Hz, J1′,2′a = 6.2 Hz, H-1′); 6.23 (s, 2H, OCH2O); 7.28 (s, 1H, H-6′′); 7.65 (s, 1H, H-6); 7.68 (s, 1H, H-3′′); 11.50 (bs, 1H, NH). 13C NMR (125.7 MHz, DMSO-d6): −5.35, −5.34, −4.75 and −4.59 (CH3Si); 17.91 and 18.18 ((CH3)3C); 25.86 and 25.94 ((CH3)3C); 39.42 (CH2-2′); 62.90 (CH2-5′); 65.15 (B-CH2O); 68.57 (OCH2Ph); 72.25 (CH-3′); 84.38 (CH-1′); 87.03 (CH-4′); 103.59 (OCH2O); 105.39 (CH-3′′); 107.41 (CH-6′′); 110.55 (C-5); 132.43 (CH-1′′); 139.32 (CH-6); 141.02 (C-2′′); 146.95 (C-4′′); 150.61 (C-2); 152.40 (C-5′′); 163.13 (C-4). MS (ESI): m/z (%): 688 (100) [M + Na]+, 689 (42)[M + Na + 2H]3+, 1353 (22) [2M + Na]+; HRMS (ESI): m/z calcd for C30H48N3O10Si2: 666.28748 [M + H]+; found: 666.28727.

5-(6-Nitropiperonyloxy)methyl-2′-deoxyuridine (dUNP)

Compound 3a (80 mg, 0.12 mmol) was dissolved in dry THF, triethylamine trihydrofluoride (171 μL, 1.05 mmol) was added and the reaction was stirred overnight at rt. The solvent was removed under reduced pressure and the mixture was separated by chromatography (DCM[thin space (1/6-em)]:[thin space (1/6-em)]MeOH 7[thin space (1/6-em)]:[thin space (1/6-em)]1) to give dUNP as yellow powder (42 mg, 80%). 1H NMR (500 MHz, DMSO-d6): 2.05–2.15 (m, 2H, H-2′); 3.55 (ddd, 1H, Jgem = 11.8 Hz, J5′a,OH = 5.1 Hz, J5′a,4′ = 3.9 Hz, H-5′a); 3.59 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH = 5.4 Hz, J5′b,4′ = 3.9 Hz, H-5′b); 3.79 (td, 1H, J4′,5′a = J4′,5′b = 3.9 Hz, J4′,3′ = 2.9 Hz, H-4′); 4.20 and 4.23 (2 × dd, 2 × 1H, Jgem = 11.6 Hz, JCH2,CH2 = 0.8 Hz, B-CH2O); 4.24 (m, 1H, H-3′); 4.79 (d, 2H, JCH2,CH2 = 0.8 Hz, OCH2Ph); 5.02 (t, 1H, JOH,5′a = JOH,5′b = 5.3 Hz, OH-5′); 5.25 (d, 1H, JOH,3′ = 4.3 Hz, OH-3′); 6.16 (t, 1H, J1′,2′b = J1′,2′a = 6.8 Hz, H-1′); 6.23 (s, 2H, OCH2O); 7.33 (s, 1H, H-6′′); 7.68 (s, 1H, H-3′′); 7.99 (s, 1H, H-6); 11.44 (bs, 1H, NH). 13C NMR (125.7 MHz, DMSO-d6): 39.85 (CH2-2′); 61.49 (CH2-5′); 65.28 (B-CH2O); 68.52 (OCH2Ph); 70.64 (CH-3′); 84.41 (CH-1′); 87.67 (CH-4′); 103.57 (OCH2O); 105.32 (CH-3′′); 107.41 (CH-6′′); 110.34 (C-5); 132.75 (CH-1′′); 139.82 (CH-6); 140.92 (C-2′′); 146.90 (C-4′′); 150.49 (C-2); 152.50 (C-5′′); 163.01 (C-4). MS (ESI): m/z (%): 460 (100) [M + Na]+, 461 (21)[M + Na + H]2+, 897 (39)[2M + Na]+, 898 (16) [2M + Na + H]2+; HRMS (ESI): m/z calcd for C18H20N3O10: 438.11432 [M + Na]+; found: 438.11433.

5-(6-Nitropiperonyloxy)methyl-2′-deoxyuridine monophosphate (dUNPMP)

Nucleoside dUNP (22 mg, 0.05 mmol) and proton sponge (22 mg, 0.10 mmol) were suspended in trimethyl phosphate (150 μL) in an argon purged flask and the suspension was cooled to 0 °C. Then, redistilled POCl3 (5 μL, 0.06 mmol) was added. The reaction mixture was stirred at 0 °C for 45 min and then aqueous solution of TEAB (2 M, 2 mL) was added and the mixture was evaporated under reduced pressure. The product was purified by HPLC chromatography (0.1 M aq. TEAB 0 → 50% methanol), co-evaporated several times with water and converted to sodium salt on Dowex. Monophosphate dUNPMP (6 mg, 21%) was obtained as a yellowish lyophilizate (water). 1H NMR (500 MHz, D2O): 2.35 (ddd, 1H, Jgem = 14.1 Hz, J2′a,1′ = 6.5 Hz, J2′a,3′ = 4.0 Hz, H-2′a); 2.39 (ddd, 1H, Jgem = 14.1 Hz, J2′b,1′ = 7.3 Hz, J2′b,3′ = 5.9 Hz, H-2′b); 3.91–3.97 (m, 2H, H-5′); 4.13 (m, 1H, H-4′); 4.44 (s, 2H, B-CH2O); 4.56 (dt, 1H, J3′,2′b = 5.8 Hz, J3′,2′a = J3′,4′ = 4.0 Hz, H-3′); 4.87 and 4.91 (2 × d, 2 × 1H, Jgem = 14.0 Hz, OCH2Ph); 6.15 (m, 2H, OCH2O); 6.27 (bt, 1H, J1′,2′b = J1′,2′a = 6.9 Hz, H-1′); 7.15 (s, 1H, H-6′′); 7.60 (s, 1H, H-3′′); 8.01 (s, 1H, H-6). 13C NMR (125.7 MHz, D2O): 39.13 (CH2-2′); 64.21 (d, JC,P = 4.6 Hz, CH2-5′); 66.25 (B-CH2O); 69.71 (OCH2Ph); 71.75 (CH-3′); 85.91 (CH-1′); 86.74 (d, JC,P = 8.3 Hz, CH-4′); 104.08 (OCH2O); 106.27 (CH-3′′); 109.67 (CH-6′′); 111.65 (C-5); 132.13 (CH-1′′); 142.10 (C-2′′); 142.33 (CH-6); 147.88 (C-4′′); 152.74 (C-2); 153.00 (C-5′′); 166.72 (C-4). 31P NMR (202.4 MHz, D2O): 4.55 (s, 1P, P-5′). MS (ESI): m/z (%): 516 (100) [M], 517 (23)[M + H]; HRMS (ESI): m/z calcd for C18H19N3O13P: 516.06610 [M]; found: 516.06531.

5-(6-Nitropiperonyloxy)methyl-2′-deoxyuridine triphosphate (dUNPTP)

Nucleoside dUNP (40 mg, 0.09 mmol) and proton sponge (40 mg, 0.18 mmol) were suspended in trimethyl phosphate (270 μL) in an argon purged flask and the suspension was cooled to 0 °C. Then, redistilled POCl3 (9 μL, 0.10 mmol) was added. The reaction mixture was stirred at 0 °C for 45 min and then ice-cold solution of (NHBu3)2H2P2O7 (208 mg, 0.46 mmol) and tributyl amine (90 μL, 0.38 mmol) in anhydrous DMF (4 mL) was added. The reaction mixture was stirred at 0 °C for another 1 h. Then aqueous solution of TEAB (2 M, 2 mL, 4 mmol) was added and the mixture was evaporated under reduced pressure. The residue was co-evaporated several times with water. The product was purified by HPLC chromatography (0.1 M aq. TEAB 0 → 50% methanol), co-evaporated several times with water and converted to sodium salt on Dowex. Triphosphate dUNPTP (17 mg, 24%) was obtained as a yellowish lyophilizate (water). 1H NMR (500 MHz, D2O): 2.34–2.40 (m, 2H, H-2′); 4.18 (qd, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.5 Hz, J4′,P = 2.0 Hz, H-4′); 4.22 (ddd, 1H, Jgem = 11.6 Hz, J5′a,P = 5.1 Hz, Jr = 3.2 Hz, H-5′a); 4.26 (ddd, 1H, Jgem = 11.6 Hz, J5′b,P = 6.1 Hz, J5′b,4′ = 3.6 Hz, H-5′b); 4.40 and 4.47 (2 × d, 2 × 1H, Jgem = 12.1 Hz, B-CH2O); 4.66 (td, 1H, J3′,2′a = J3′,2′b = 5.4 Hz, J3′,4′ = 3.7 Hz, H-3′); 4.84 and 4.92 (2 × d, 2 × 1H, Jgem = 14.0 Hz, OCH2Ph); 6.15 (t, 2H, JCH2,LR = 1.3 Hz, OCH2O); 6.26 (t, 1H, J1′,2′b = J1′,2′a = 6.7 Hz, H-1′); 7.13 (s, 1H, H-6′′); 7.58 (s, 1H, H-3′′); 7.96 (s, 1H, H-6). 13C NMR (125.7 MHz, D2O): 39.31 (CH2-2′); 65.65 (d, JC,P = 5.5 Hz, CH2-5′); 66.29 (B-CH2O); 69.77 (OCH2Ph); 70.73 (CH-3′); 85.91 (CH-1′); 86.20 (d, JC,P = 9.1 Hz, CH-4′); 104.11 (OCH2O); 106.27 (CH-3′′); 109.84 (CH-6′′); 111.55 (C-5); 132.11 (CH-1′′); 142.08 (CH-6); 142.15 (C-2′′); 147.90 (C-4′′); 152.47 (C-2); 152.94 (C-5′′); 166.06 (C-4). 31P NMR (202.4 MHz, D2O): −20.95 (t, 1P, Jβ,α = Jβ,γ = 19.8 Hz, Pβ); −10.57 (d, 1P, Jα,β = 19.5 Hz, Pα); −5.11 (bd, 1P, Jγ,β = 20.0 Hz, Pγ). MS (ESI): m/z (%): 516 (41) [M + H-HP2O6], 596 (100) [M + 2H-HPO3], 618 (60) [M + H + Na-HPO3]; HRMS (ESI): m/z calcd for C18H20N3O19P3Na: 697.98070 [M + H + Na]; found: 697.97937.

PEX – multiple modifications

The reaction mixture (20 μL) contained KOD XL DNA polymerase (2.5 U μL−1, 0.02 μL), mixture of dATP, dCTP and dGTP (1 mM, 1 μL), dTTP (1 mM, 1 μL) or modified dURTP (1 mM, 2 μL), 6-FAM labelled primer 248sh (3 μM, 1 μL), 31-mer template TINA-prb4basII (3 μM, 1.5 μL) and 10× buffer for KOD XL DNA polymerase (2 μL) supplied by the manufacturer. Reaction mixtures were incubated for 30 min at 60 °C in a thermal cycler. After the reaction samples were denatured by the addition of stop solution (20 μL, 80% [v/v] formamide, 20 mM EDTA, 0.025%, [w/v] bromophenol blue, 0.025% [w/v] xylene cyanol, PCR water) heating was followed for 5 min at 95 °C. The reaction mixtures were separated using 12.5% denaturing PAGE.

Uncaging of DNA

The reaction mixture (20 μL) contained KOD XL DNA polymerase (2.5 U μL−1, 0.2 μL), mixture of dATP, dCTP and dGTP (1 mM, 0.2 μL), dTTP or modified dUTP (1 mM, 0.3 μL) 6-FAM labelled primer 248sh (3 μM, 1 μL) and 30-mer template RsT (3 μM, 1.5 μL) and 10× buffer for KOD XL DNA polymerase (2 μL) supplied by the manufacturer. Reaction mixtures were incubated for 30 min at 60 °C in a thermal cycler.

After that, the reaction samples were either:

(a) denatured by the addition of the stop solution (23 μL, 80% [v/v] formamide, 20 mM EDTA, 0.025%, [w/v] bromophenol blue, 0.025% [w/v] xylene cyanol, PCR water) and water (3 μL) and heated at 95 °C for 5 min,

(b) incubated with RSaI (0.8 μL) in CutSmart buffer (2.2 μL) and denatured by addition of the stop solution (23 μL) and heated at 95 °C for 5 min, or

(c) irradiated with a UV LED followed by incubation with RSaI (0.8 μL) in CutSmart buffer (2.2 μL) and denatured by the addition of the stop solution (23 μL) and heating at 95 °C for 5 min.

Reaction mixtures were separated using 12.5% denaturing PAGE.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Czech Academy of Sciences (Praemium Academiae award to M. H.) and by the Czech Science Foundation (17-03419S to S. B., Z. V. and M. H.; GA13-25775S to P. K.). The RECETOX research infrastructure was supported by the projects of the Czech Ministry of Education (LO1214 and LM2011028; P. K.).

References

  1. (a) P. Klan, T. Solomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina, V. Popik, A. Kostikov and J. Wirz, Chem. Rev., 2013, 113, 119–191 CrossRef CAS PubMed; (b) T. Šolomek, J. Wirz and P. Klán, Acc. Chem. Res., 2015, 48, 3064–3072 CrossRef PubMed.
  2. Reviews: (a) I. Ahmed and L. Fruk, Mol. Biosyst., 2013, 9, 565–570 RSC; (b) C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer and A. Heckel, Angew. Chem., Int. Ed., 2012, 51, 8446–8476 CrossRef CAS PubMed; (c) C. W. Riggsbee and A. Deiters, Trends Biotechnol., 2010, 28, 468–475 CrossRef CAS PubMed; (d) H.-M. Lee, D. R. Larson and D. S. Lawrence, ACS Chem. Biol., 2009, 4, 409–427 CrossRef CAS PubMed; (e) A. Deiters, Curr. Opin. Chem. Biol., 2009, 13, 678–686 CrossRef CAS PubMed; (f) G. C. R. Ellis-Davies, Nat. Methods, 2007, 4, 619–628 CrossRef CAS PubMed; (g) D. D. Young and A. Deiters, Org. Biomol. Chem., 2007, 5, 999–1005 RSC.
  3. Reviews: (a) M. Ikeda and M. Kabumoto, Chem. Lett., 2017, 46, 634–640 CrossRef CAS; (b) Q. Liu and A. Deiters, Acc. Chem. Res., 2014, 47, 45–55 CrossRef CAS PubMed; (c) L. M. Ceo and J. T. Koh, ChemBioChem, 2012, 13, 511–513 CrossRef CAS PubMed; (d) X. Tang, J. Zhang, J. Sun, Y. Wang, J. Wu and L. Zhang, Org. Bimol. Chem., 2013, 11, 7814–7824 RSC; (e) Recent example: L. Anhauser, F. Muttach and A. Rentmeister, Chem. Commun., 2018, 54, 449–451 RSC.
  4. Examples of base-photocaged nucleic acids: (a) A. Deiters, R. A. Garner, H. Lusic, J. M. Goven, M. Dush, N. M. Nascone-Yoder and J. A. Yoder, J. Am. Chem. Soc., 2010, 132, 15644–15650 CrossRef CAS PubMed; (b) J. M. Govan, R. Uprety, J. Hemphill, M. O. Lively and A. Deiters, ACS Chem. Biol., 2012, 7, 1247–1256 CrossRef CAS PubMed; (c) J. Hemphill, J. Govan, R. Uprety, M. Tsang and A. Deiters, J. Am. Chem. Soc., 2014, 136, 7152–7158 CrossRef CAS PubMed; (d) A. Rodrigues-Correia, X. M. M. Weyel and A. Heckel, Org. Lett., 2013, 15, 5500–5503 CrossRef CAS PubMed; (e) M. A. H. Fichte, X. M. M. Weyel, S. Junek, F. Schäfer, C. Herbivo, M. Goeldner, A. Specht, J. Wachtveitl and A. Heckel, Angew. Chem., Int. Ed., 2016, 55, 8948–8952 CrossRef CAS PubMed.
  5. Examples of phosphate-photocaged nucleic acids: (a) W. T. Monroe, M. M. McQuain, M. S. Chang, J. S. Alexander and F. R. Haselton, J. Biol. Chem., 1999, 274, 20895–20900 CrossRef CAS PubMed; (b) S. Shah, S. Rangarajan and S. H. Friedman, Angew. Chem., Int. Ed., 2005, 44, 1328–1332 CrossRef CAS PubMed; (c) A. Teraoka, K. Murakoshi, K. Fukamauchi, A. Z. Suzuki, S. Watanabe and T. Furuta, Chem. Commun., 2014, 50, 664–666 RSC.
  6. J. Wu and X. Tang, Bioorg. Med. Chem., 2013, 21, 6205–6211 CrossRef CAS PubMed.
  7. (a) V. A. Litosh, W. D. Wu, B. P. Stupi, J. C. Wang, S. E. Morris, M. N. Hersh and M. L. Metzker, Nucleic Acids Res., 2011, 39, e39 CrossRef CAS PubMed; (b) B. P. Stupi, H. Li, J. C. Wang, W. D. Wu, S. E. Morris, V. A. Litosh, J. Muniz, M. N. Hersh and M. L. Metzker, Angew. Chem., Int. Ed., 2012, 51, 1724–1727 CrossRef CAS PubMed.
  8. K. Ohno, D. Sugiyama, L. Takeshita, T. Kanamori, Y. Masaki, M. Sekine and K. Seio, Bioorg. Med. Chem., 2017, 25, 6007–6015 CrossRef CAS PubMed.
  9. (a) H. Witmer, J. Virol., 1981, 39, 536–547 CAS; (b) R. G. Kallen, M. Simon and J. Marmur, J. Mol. Biol., 1962, 5, 248–250 CrossRef CAS PubMed.
  10. (a) P. M. M. Rae, Science, 1976, 194, 1062–1064 CAS; (b) F. Kawasaki, D. Beraldi, R. E. Hardisty, G. R. McInroy, P. van Delft and S. Balasubramanian, Genome Biol., 2017, 18, 23 CrossRef PubMed.
  11. (a) T. Pfaffeneder, F. Spada, M. Wagner, C. Brandmayr, S. K. Laube, D. Eisen, M. Truss, J. Steinbacher, B. Hackner, O. Kotljarova, D. Schuermann, S. Michalakis, O. Kosmatchev, S. Schiesser, B. Steigenberger, N. Raddaoui, G. Kashiwazaki, U. Müller, C. G. Spruijt, M. Vermeulen, H. Leonhardt, P. Schär, M. Müller and T. Carell, Nat. Chem. Biol., 2014, 10, 574–581 CrossRef CAS PubMed; (b) J. Guz, D. Gackowski, M. Foksinski, R. Rozalski and R. Olinski, Biol. Reprod., 2014, 91, 55 Search PubMed; (c) Z. Djuric, L. K. Heilbrun, M. S. Simon, D. Smith, D. A. Luongo, P. M. LoRusso and S. Martino, Cancer, 2006, 77, 691–696 CrossRef.
  12. A. P. Mehta, H. Li, S. A. Reed, L. Supekova, T. Javahishvili and P. G. Schultz, J. Am. Chem. Soc., 2016, 138, 7272–7275 CrossRef CAS PubMed.
  13. (a) X. Lu, B. S. Zhao and C. He, Chem. Rev., 2015, 115, 2225–2239 CrossRef CAS PubMed; (b) M. Wagner, J. Steinbacher, T. F. J. Kraus, S. Michalakis, B. Hackner, T. Pfaffeneder, A. Perera, M. Müller, A. Giese, H. A. Kretzschmar and T. Carell, Angew. Chem., Int. Ed., 2015, 54, 12511–12514 CrossRef CAS PubMed; (c) T. Pfaffeneder, B. Hackner, M. Truss, M. Münzel, M. Müller, C. A. Deiml, C. Hagemeier and T. Carell, Angew. Chem., Int. Ed., 2011, 50, 7008–7012 CrossRef CAS PubMed; (d) D. Globisch, M. Münzel, M. Müller, S. Michalakis, M. Wagner, S. Koch, T. Brückl, M. Biel and T. Carell, PLoS One, 2010, 5, e15367 CAS.
  14. (a) A. Perera, D. Eisen, M. Wagner, S. K. Laube, A. F. Künzel, S. Koch, J. Steinbacher, E. Schulze, V. Splith, N. Mittermeier, M. Muller, M. Biel, T. Carell and S. Michalakis, Cell Rep., 2015, 11, 283–294 CrossRef CAS PubMed; (b) C. M. Greco, P. Kunderfranco, M. Rubino, V. Larcher, P. Carullo, A. Anselmo, K. Kurz, T. Carell, A. Angius, M. V. G. Latronico, R. Papait and G. Condorelli, Nat. Commun., 2016, 7, 12418 CrossRef CAS PubMed; (c) C. You, D. Li, X. Dai and Y. Wang, Sci. Rep., 2014, 4, 7052 CrossRef CAS PubMed; (d) L. Wang, Y. Zhou, L. Xu, R. Xiao, X. Lu, L. Cheng, J. Chong, H. Li, C. He, X.-D. Fu and D. Wang, Nature, 2015, 523, 621–625 CrossRef CAS PubMed; (e) M. W. Kellinger, C.-X. Song, J. Chong, X.-Y. Lu, C. He and D. Wang, Nat. Struct. Mol. Biol., 2012, 19, 831–833 CrossRef CAS PubMed.
  15. R. Olinski, M. Starczak and D. Gackowski, Mutat. Res., 2016, 767, 59–66 CAS.
  16. V. Raindlová, M. Janoušková, M. Slavíčková, P. Perlíková, S. Boháčová, N. Milisavljevič, H. Šanderová, M. Benda, I. Barvík, L. Krásný and M. Hocek, Nucleic Acids Res., 2016, 44, 3000–3012 CrossRef PubMed.
  17. M. Janoušková, Z. Vaníková, F. Nici, S. Boháčová, D. Vítovská, H. Šanderová, M. Hocek and L. Krásný, Chem. Commun., 2017, 53, 13253–13255 RSC.
  18. H. Macíčková-Cahová, R. Pohl and M. Hocek, ChemBioChem, 2011, 12, 431–438 CrossRef PubMed.
  19. H. Macíčková-Cahová and M. Hocek, Nucleic Acids Res., 2009, 37, 7612–7622 CrossRef PubMed.
  20. M. Mačková, S. Boháčová, P. Perlíková, L. Poštová Slavětínská and M. Hocek, ChemBioChem, 2015, 16, 2225–2236 CrossRef PubMed.
  21. P. Kielkowski, H. Macíčková-Cahová, R. Pohl and M. Hocek, Angew. Chem., Int. Ed., 2011, 50, 8727–8730 CrossRef CAS PubMed.
  22. Z. Vaníková and M. Hocek, Angew. Chem., Int. Ed., 2014, 53, 6734–6737 CrossRef PubMed.
  23. D. D. Young and A. Deiters, Bioorg. Med. Chem. Lett., 2006, 16, 2658–2661 CrossRef CAS PubMed.
  24. A. K. Singh and P. K. Khade, Tetrahedron Lett., 2005, 46, 5563–5566 CrossRef CAS.
  25. (a) Q. Dai, C.-X. Song, T. Pan and C. He, J. Org. Chem., 2011, 76, 4182–4188 CrossRef CAS PubMed; (b) A. S. Hansem, A. Thalhammer, A. H. El-Sagheer, T. Brown and C. J. Schofield, Bioorg. Med. Chem. Lett., 2011, 21, 1181–1184 CrossRef PubMed; (c) L. C. Sowers and G. P. Beardsley, J. Org. Chem., 1993, 58, 1664–1665 CrossRef CAS; (d) G. T. Shiau, R. F. Schinazi, M. S. Chen and W. H. Prusoff, J. Med. Chem., 1980, 23, 127–133 CrossRef CAS PubMed; (e) M. Münzel, D. Globish, T. Brückl, M. Wagner, V. Welzmiller, S. Michalakis, M. Müller, M. Biel and T. Carell, Angew. Chem., Int. Ed., 2010, 49, 5375–5377 CrossRef PubMed.
  26. (a) M. Kort, E. Ebrahimi, E. R. Wijsman, G. A. Marel and J. H. Boom, Eur. J. Org. Chem., 1999, 2337–2344 CrossRef; (b) L. Simeone, L. De Napoli and D. Montesarchio, Chem. Biodiversity, 2012, 9, 589–597 CrossRef CAS PubMed.
  27. A. Porcheddu, G. Giacomelli, I. Piredda, M. Carta and G. Nieddu, Eur. J. Org. Chem., 2008, 5786–5797 CrossRef CAS.
  28. M. C. Pirrung, S. W. Shuey, D. C. Lever and L. Fallon, Bioorg. Med. Chem. Lett., 1994, 4, 1345–1346 CrossRef CAS.
  29. C. G. Hatchard and C. A. Parker, Proc. R. Soc. London, Ser. A, 1956, 235, 518–536 CrossRef CAS.
  30. P. Klan and J. Wirz, Photochemistry of Organic Compounds: From Concepts to Practice, John Wiley & Sons Ltd, Chichester, 1st edn, 2009 Search PubMed.
  31. C. Würth, M. Grabolle, J. Pauli, M. Spieles and U. Resch-Genger, Nat. Protoc., 2013, 8, 1535–1550 CrossRef PubMed.
  32. For reviews on polymerase incorporation of modified nucleotides, see: (a) M. Hocek, J. Org. Chem., 2014, 79, 9914–9921 CrossRef CAS PubMed; (b) A. Hottin and A. Marx, Acc. Chem. Res., 2016, 49, 418–427 CrossRef CAS PubMed; (c) M. Hollenstein, Molecules, 2012, 17, 13569–13591 CrossRef CAS PubMed; (d) M. Kuwahara and N. Sugimoto, Molecules, 2010, 15, 5423–5444 CrossRef CAS PubMed.

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ob00160j

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