Biomolecular

The syntheses of 5-arylsulfanyl-or 5-arylselanylpyrimidine and 7-arylsulfanyl-or 7-arylselanyl-7-deazapurine nucleosides and nucleotides were developed by the Cu-mediated sulfanylations or selanylations of the corresponding 5-iodopyrimidine or 7-iodo-7-deazapurine nucleosides or nucleotides with diaryldisul ﬁ des or -diselenides. The reactions were also applicable for direct modi ﬁ cations of 2 ’ -deoxycytidine triphosphate and the resulting 5-arylsulfanyl or 5-arylselanyl-dCTP served as substrates for the polymerase synthesis of modi ﬁ ed DNA bearing arylsulfanyl or arylselanyl groups in the major groove.


Introduction
DNA molecules bearing modifications in the major groove have found diverse applications mainly in bioanalysis and chemical biology. 1 5-Substituted pyrimidine and 7-substituted 7-deazapurine 2′-deoxyribonucleoside 5′-O-triphosphates (dNTPs) are good substrates for DNA polymerases in the enzymatic synthesis of base-modified DNA. 2,3Modified dNTPs are mostly synthesized by the triphosphorylation of the corresponding modified nucleosides 4 but this approach can fail in the case of some reactive modifications not compatible with the triphosphorylation methodology.Therefore, direct methods of functionalization of dNTPs are desirable but are inherently difficult due to the lability of dNTPs which are prone to hydrolysis.So far, the only reported reactions suitable for modification of dNTPs have been the aqueous cross-coupling reactions of halogenated dNTPs, 3,5 thiol-maleimide addition, 6 some amide-forming reactions of 5-aminoalkylethynyl-dUTP, 7 hydrazone-formation, 8 Diels-Alder 9 and the CuAAC click reaction. 10The Suzuki-Miyaura cross-coupling reaction with arylboronic acids 11 and the Sonogashira reactions with terminal acetylenes 12 are the most general and useful reactions used in the synthesis of base-modified dNTPs.In addition, several examples of the Heck coupling 13 with acrylates, as well as the Stille reaction 14 with aryl-or alkenylstannanes were recently reported.To the best of our knowledge, no method for direct attachment of a heteroatom to dNTPs has been published.
5-Alkylsulfanyl-or arylsulfanyl-pyrimidine nucleosides were reported to inhibit thymidylate kinase 15 and slightly destabilized DNA duplexes, 16 whereas saturated 5-phenylsulfanylthymidine analogues were used 17 as radical precursors for photochemical generation of thymine in DNA.Some 7-arylsulfanyl-7deazaadenosine analogues displayed 18 weak cytostatic effects, while the corresponding 7-S-substituted 7-deazaguanine derivatives have never been reported.5-Selenylated pyrimidine nucleotides inhibit thymidylate synthase 19 and have been utilized 20 for modification of DNA or RNA for X-ray crystallography and 5-( phenylselenylmethyl)uracil was used 21 as a T radical precursor for DNA crosslinking.Also the related 5-( phenyltelluranyl)uracil nucleoside has been prepared 22 and, after incorporation into DNA, it was used for X-ray and STM imaging.However, no selenylated 7-deazapurines have been known so far.Therefore, we report here the synthesis of the arylsulfanyl and arylselanyl derivatives of pyrimidine and 7-deazapurine nucleosides and nucleotides and their potential for polymerase incorporation into DNA.

Synthesis
Previously, 5-(alkylsulfanyl)pyrimidine bases or nucleosides were prepared by alkylation of 5-mercaptouracil, 23 reactions of toxic 5-(chloromercuri)pyrimidines with disulfides, 24 or more recently by Pd-catalyzed coupling of 5-bromopyrimidine † Electronic supplementary information (ESI) available: Experimental part, and copies of spectra.See DOI: 10.1039/c6ob01917j ‡ Passed away on July 11, 2016.a derivatives with thiols, 25 whereas 7-arylsulfanyl-7-deazapurines were prepared by Cu-mediated S-H sulfenylations. 18The 5-selenylated pyrimidines were prepared by Mn-mediated C-H selenylations 20 or electrophilic aromatic selenylation. 26nspired by the work of Taniguchi 27 on the copper-catalyzed reactions of diaryldisulfides or diaryldiselenides with iodoarenes, we started our study by testing the reactions of unprotected halogenated nucleosides. 5The reaction conditions were first tested on 5-iodo-2′-deoxycytidine (dC I ) in reaction with diphenyldisulfide.The Cu-catalyzed (10 mol% of CuI) reactions in the presence or absence of Mg 27 gave complex mixtures of products.Therefore, we used stoichiometric amounts of copper powder in the presence of 2,2′-bipyridine (bpy).The reactions were performed at 80-110 °C in DMF (Scheme 1).Under these conditions (Method A), the desired 5-phenylsulfanyl-2′-deoxycytidine was formed as the major product (in addition to small amounts of dehalogenated 2′-deoxycytidine) and isolated in a good yield of 58%.A similar conversion and yield were achieved when using pre-generated phenylsulfanylcuprate (Method B).
The same reaction of nucleoside dC I (Method A) was then performed with a small series of diaryldisulfides to obtain the Then we tested other iodinated nucleosides (Scheme 1).The reaction of 5-iodo-2′-deoxyuridine (dU I ) with PhSCu (Method B) provided the 5-substituted dU PhS nucleoside in 47% yield, whereas the reactions with dithienyldisulfide or diselenides (Method A) gave the other corresponding 5-arylsulfanyl-or phenyl-or methylselanyl uracil nucleosides (dU ThS , dU PhSe and dU MeSe ) in low yields.The reactions of 7-iodo-7-deazaadenine dA I and -7-deazaguanine dG I nucleosides with PhSCu (Method B) gave the 7-(phenylsulfanyl)deazapurine nucleosides dA PhS and dG PhS in acceptable 50 or 39% yields, whereas the reactions with diphenyldiselenide furnished the corresponding phenylselanyl nucleosides dA PhSe and dG PhSe in moderate yields.Again, the reaction with dimethyldiselenide gave very low conversion and dA MeSe was isolated only in 14% yield.Apparently the reactivity of dimethyldiselenide is very low and the methylsulfanylation is of very limited synthetic applicability.
Next, we tested the reactions of nucleotides and started with stable nucleoside 5′-O-monophosphates (dNMPs).The model iodinated dC I MP was tested in reactions with diaryldisulfides or diselenides (Scheme 2, Method A).Most of these reactions gave very low conversions and only two products, dC ThS MP and dC PhSe MP, were isolated in acceptable yields.On the other hand, the phosphorylation of the 5-arylsulfanyl-or arylselanyl-cytosine nucleosides gave the desired modified nucleotides in better yields (22-48%).
Finally, we tested the reactions for direct modification of hydrolytically labile dNTPs (Scheme 3).Thus the iodinated triphosphate dC I TP was reacted with PhSCu (Method B) to give the desired 5-( phenylsulfanyl)-dCTP (dC PhS TP) in low 7% yield.Better conversions were achieved when using reactions with diaryldisulfides or diselenides (Method A).The desired dC ThS TP and dC PhSe TP were obtained in good yields of 24 and 31% (which are fully comparable to the typical yields of the cross-coupling reactions of dNTPs [11][12][13][14] ).

Polymerase incorporation of modified nucleotides
The three new 5-S-or Se-linked dNTPs (dC PhS TP, dC ThS TP and dC PhSe TP) were then tested as substrates for DNA polymerases.At first we tested them in a primer extension (PEX) reaction with KOD XL, Vent(exo-) or Pwo polymerases, 15-mer primer 248-sh and a 19-mer template temp oligo1C (for sequences, see Table 1).Fig. 1 shows the PAGE analysis of the PEX reactions.While KOD XL and Vent(exo-) polymerases gave quite clean bands of the 19-mer oligonucleotide (ON) products bearing one modified dC RX nucleotide, Pwo gave a mixture of the full-lengths and a truncated product.
Then we tested the same nucleotides (dC PhS TP, dC ThS TP and dC PhSe TP) in a more challenging PEX reaction using a 31-mer template temp Prb4baseII (Fig. 2).This PEX reaction leads to a 31-mer DNA containing four modified dC RX nucleotides.KOD XL was found to be the best polymerase which gave clean full-length products in all three cases, whereas the other two enzymes gave less clean products containing minor amounts of truncated products.The PEX products were characterized by MALDI-TOF analysis (Table 2).
Finally, we tested the nucleotides (dC PhS TP, dC ThS TP and dC PhSe TP) in PCR amplification using a 98-mer template (temp FLV-A ).Fig. 3 shows that all three dNTPs were good substrates of KOD XL polymerase in PCR reaction and gave the corresponding full-length amplified products (double-stranded DNA with modification in both strands).The yield of PCR with dC PhSe TP was further improved by the addition of Mg 2+ or a Scheme 3 Sulfanylations and selanylations of dNTPs.

Conclusions
In conclusion, we developed a new method for the direct functionalization of 5-iodopyrimidine and 7-iodo-7-deazapurine nucleosides and nucleotides based on Cu-mediated arylsulfanylation or aryl/alkylselenylation.The reactions are even applicable for modification of fragile halogenated dNTPs.The S-or Se-modified dNTPs are good substrates for DNA polymerases and can be used as building blocks for the enzymatic synthesis of modified ONs or DNA.In this way, an aryl substituent can be attached to the nucleobase (in nucleoside, nucleotides or DNA) through a flexible sp 3 -hybridized sulfide or selenide linkage, which in principle offers a possibility for further transformations (e.g.oxidations).The arylsulfanyl or arylselenyl group can also serve as a radical precursor and the selenyl substituents can be used for crystallography of nucleic acids.The aryl group can be functionalized (e.g.NO 2 or MeO groups) so the approach can be potentially used for redox 28 or fluorescent 29 labelling of nucleic acids.Research along these lines is ongoing.

Experimental
For the full Experimental part, procedures and characterization of all compounds, see the ESI.†  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

Table 1
List of ON sequences used in this study

Table 2
MALDI-TOF data of modified oligodeoxyribonucleotides