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Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-protein Interactions by 19F NMR Spectroscopy Agata Olszewska, Radek Pohl, and Michal Hocek J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01920 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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The Journal of Organic Chemistry

Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-protein Interactions by 19F NMR Spectroscopy Agata Olszewska†, Radek Pohl† and Michal Hocek†‡* †

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, 160 00 Prague 6, Czech Republic



Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 12843 Prague 2, Czech Republic

19

KEYWORDS: Nucleosides, Nucleotides, F NMR Spectroscopy, DNA-protein interactions, modified DNA

ABSTRACT: A series of 7-[4-(trifluoroacetyl)phenyl]-7-deazaadenine and -7-deazaguanine as well as 5-substituted uracil and cytosine 2'-deoxyribonucleosides and mono- and triphosphates through aqueous Suzuki-Miyaura crosscoupling of halogenated nucleosides or nucleotides with 4-(trifluoroacetyl)phenylboronic acid were synthesized. The modified nucleoside triphosphates were good substrates for DNA polymerases applicable in primer extension or PCR synthesis of modified oligonucleotides or DNA. Attempted cross-linking with a serine-containing protein did not proceed, however the trifluoroacetophenone group was a sensitive probe for the study of DNA-protein interactions by 19F NMR.

Base-modified DNA and oligo-2'deoxyribonucleotides (ONs) find diverse applications 1 in bioanalysis or chemical biology. Apart from classical chemical synthesis on solid-support, they can efficiently be prepared by polymerase incorporation of modi2 fied nucleotides. 5-Substituted pyrimidine or 7substituted 7-deazapurine 2'-deoxyribonucleoside 5'O-triphosphates are usually good substrates for poly3,4 merases, in some cases even better than natural 5 dNTPs. As a result of our long-standing interest in DNA-protein interactions, we became interested in DNA modifications capable of detecting these interactions by changes of some spectral properties or in modifications capable of selective covalent crosslinking with proteins. Recently we reported several environment-sensitive fluorophores which changed 6 7 the color or life-time on interactions of the DNA probes with proteins, as well as reactive modifications, 8 9 i.e. vinylsulfonamide or chloroacetamide which formed covalent cross-links with cystein and or histidine. Others have also reported cross-linking of DNA modifications including diazirines for photocross10 11 linking, thiols for disulfide formation, or 12 (oxo)aldehydes or phenylselenyloxy-alkene electro13 philes for cross-linking to lysine. So far, no DNAreactive group specific for serine has been reported.

Due to the absence of fluorine in most biomolecules, 19 specific fluorine-labelling and F NMR is a powerful and sensitive approach for studying secondary struc14 tures or interactions of biomolecules. Many types of 15fluorine-containing nucleotides have been developed 17 and used for construction of ON or DNA probes which were mostly used for sensing DNA hybridization 19 15-17 or DNA secondary structures by F NMR. . However, 19 there were only two examples of the use F NMR for detection of DNA-protein interactions: study of flip18 ping of 5-fluorocytosine by HhaI methyltransferase and study of fluorine-labelled DNA polymerase in 19 complex with DNA. Trifluoromethylketones are highly reactive toward 18 nucleophilic addition and were repeatedly reported to 19,20 specifically react with serine. Several inhibitors of 19 serine proteases or reactive probes for other serine20 containing proteins based on this reactive groups were developed. Taking into account the desired serine-reactivity and the presence of 3 equivalent fluorine 19 atoms (which increase the signal in F NMR), we designed trifluoroacetylphenyl-linked nucleosides and nucleotides as promising building blocks for the synthesis of ONs or DNA probes and report here on their synthesis, polymerase incorporation into DNA and applications.

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3

RESULTS AND DISCUSSION.

4

Synthesis The synthesis of the desired 2,2,2trifluoroacetophenone- (TAP-) modified nucleosides, nucleoside mono- and triphosphates was achieved through the direct attachment of the aryl group by 21 aqueous Suzuki-Miyaura cross-coupling reactions. At first the reaction was tested on nucleosides. Starting from 7-iodopurine or 5-iodopyrimidine 2'deoxyribonucleosides, the reactions with 4(trifluoroacetyl)phenylboronic acid pinacol ester in the presence of the Pd(OAc)2/TPPTS catalytic system and K2CO3 in H2O/CH3CN (Scheme 1, Table 1) gave the TAP TAP desired TAP-substituted nucleosides (dC , dU or TAP dA ) in good yields of 62-85%, whereas the 7TAP deazaguanine derivative dG was obtained in moderate 45% yield, probably due to the limited stability of 7-iodo-7-deazaguanine. Similarly, the iodinated nucleI osides monophosphates dN MPs reacted with the same boronate under the same conditions to give the TAP TAP-substituted nucleotides dN MPs in yields comparable to nucleosides. The most difficult (hydrolytiI cally unstable) triphosphates dN TPs were also sucTAP cessfully arylated to furnish the dN TPs in good yields.

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dA dG I

I

62

TAP

45

dG

TAP

5

dC MP

dC

6

dU MP

I

dU

7

I

dA MP

8

I

dG MP

9

I

dC TP

10

I

dU TP

11

I

dA TP

12

I

dG TP

TAP

dA

MP

88

TAP

MP

75

TAP

MP

56

dA

dG

TAP

MP

TAP

dC

TP

26 74

TAP

TP

58

TAP

TP

52

dU dA

dG

TAP

TP

37

The NMR analysis of all TAP-modified nucleosides TAP TAP TAP dN s and nucleotides dN MPs and dN TPs showed that they are predominantly (or exclusively) present in the form of hydrates (Scheme 2) confirming the high susceptibility of the trifluoromethyl substituted oxo group to nucleophilic addition.

Scheme 2. Equilibrium between the ketone and hydrate in aqueous solution.

Scheme 1. Synthesis of TAP-modified nucleosides and nucleotides. Reagents and conditions i) Pd(OAc)2 (4 mol%), TPPTS (8 mol%), K2CO3 (3 equiv.), MeCN/H2O. Table 1. Synthesis of Trifluoroacetophenone Modified Nucleosides and Nucleotides entry

starting compound

1

dC

2

I

dU

I

product

Yield (%)

TAP

85

TAP

70

dC

dU

Incorporation of the TAP-modified nucleotides by DNA polymerases. We tested the modified TAP dN TPs as building blocks for the enzymatic synthesis of the TAP-modified DNA using different polymerases. At first we studied primer extension (PEX) reactions catalyzed by KOD XL, Vent(exo-) or Pwo polymerases. The templates and primer (for sequences see Table S1 in the SI) were chosen in order to introduce one modification into a 19-nt extended primer strand TAP (19ON_1X ) or four modifications into a 31-nt strand TAP (31ON_4X ). All modified nucleoside triphosphates TAP dN TPs were found to be good substrates for all of the tested enzymes and were successfully incorporated into the DNA bearing one or four modifications. Figure 1 shows the PAGE analyses of the PEX reactions using KOD XL polymerase (for gels with other polymerases, see figure S1-S2 in the SI) revealing clean strong bands of desired full-length products in all cases. The PEX products were also characterized by MALDI-TOF analysis (see Table 2) to confirm the correct length and mass of the products.

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The Journal of Organic Chemistry ladder; lanes 2, 5, 8 (+): natural dNTPs; lanes 3, 6, 9 (C-/A-/T/G-): negative controls without dCTP,dTTP or dATP; Lane 4 TAP TAP TAP TAP (C ): dC TP, dTTP, dGTP, dATP; lane 7 (A ): dA TP, TAP TAP dCTP, dGTP, dTTP; lane 10 (U ): dU TP, dATP, dTTP, dGTP; 1.3 % (2%) agarose gel for 339-mer (98-mer) stained with GelRed

Figure 1. Primer extension using KOD XL polymerase. 31 A) 31 mer template (temp ). Lane 1 (P): Primer; lane 2 (+): natural dNTPs; lanes 3, 5, 7, 9 (C-/T-/A-/G-): negative control without dCTP, dTTP, dATP or dGTP; lane TAP TAP TAP 4 (C ): dC TP, dTTP, dGTP, dATP; lane 6 (U ): TAP TAP TAP dU TP, dATP, dGTP, dCTP; lane 8 (A ): dA TP, TAP TAP dTTP, dGTP, dCTP; lane 10 (dG TP): dG TP, dTTP, 19X dCTP, dATP. B) 19 mer template temp . Lanes 1, 8 (P): Primer; lanes 2, 5, 9, 12 (+): natural dNTPs; lanes 3, 6, 10, 13 (C-/T-/A-/G-): negative control without dCTP, TAP TAP dTTP, dATP or dGTP; lane 4 (C ): dC TP, dTTP, TAP TAP dGTP, dATP; lane 7 (U ): dU TP, dATP, dGTP, TAP TAP dCTP; lane 10 (A ): dA TP, dTTP, dGTP, dCTP; TAP TAP lane 14 (G ): dG TP, dTTP, dCTP, dATP.

Table 2. MALDI data of TAP modified oligonucleotides. ssDNA

M (calcd.) (Da)

M (found) (Da)

19ON_1C TAP 19ON_1A

TAP

6124.0 6150.0

6127.0 [M+3H] 6151.4 [M+1H]

TAP

19ON_1U

-

6125.3

6127.8 [M+2H]

TAP

6090.2

6091.0 [M+1H]

-

TAP

9377.2

9378.7 [M+1H]

TAP

9957.5

9958.0 [M+1H]

19ON_1G

30ON_1C 31ON_1U

-

-

Figure 2. Agarose gel analysis of PCR products amplified by KOD XL DNA polymerases. A) 98 mer B) 339 mer; Lane 1 (L):

In order to prepare long double-stranded DNA (dsDNA) containing a high density of modifications on TAP both strands, we subjected the modified dN TPs in polymerase chain reaction (PCR). PCR amplification was assayed using three different thermostable DNA polymeases, KOD XL, Vent (exo-) or Pwo, using 98mer or 339-mer templates and the agarose gels were visualized by staining with GelRed. From these polymerases, the best results were achieved by KOD XL which readily gave the corresponding full-length PCR TAP TAP TAP products using dA TP, dC TP or dU TP as substrates in the presence of the other three non-modified dNTPs with both templates (Figure 2). In the case of TAP dG TP, we used 5′-6-FAM-labeled primers and fluorescence detection to visualize the gels, because 7deazaguanosine bases are known to quench fluores22 cence of DNA staining intercalators. However, no products of PCR amplification with this nucleotide were observed (Figure S7 in the SI). Testing of cross-linking of TAP-modified DNA using p53 mutant. As mentioned above, trifluoromethyl ketones are known to react with serine protease to 19,20 form hemiacetals. This reaction is inherently reversible and the stability of the products varies depending on many factors. Firstly, we tested the possible cross-linking on model reactions of TAP-linked TAP nucleoside monophosphate (dC MP) with Nacetylserine (1.5 - 50 equiv.) in triethylammonium acetate (TEAA) buffer (pH 8.4) or in acetone in ratio 2:1 with 0.1 M NaOH. Unfortunately we did not observe any formation of the desired cross-linked product by TLC, HPLC, or mass spectrometry (Scheme S1 in the 9 SI). As a result of our previously published data , we knew that proximity effect can facilitate DNA-protein cross-linking reactions which do not proceed on model small molecules. Thus we prepared several DNA probes containing two modified nucleotides, either TAP TAP TAP TAP dC , dA , dU or dG in the consensus sequence for binding of the p53 protein. The DNAprotein interactions were studied using the GSTtagged core domain of a tumor suppressor p53 (GSTp53CD) mutant, we prepared mutant of the 23,24 GSTp53CD with C277 replaced by serine. The ability of the GSTp53CD_C277S mutant to recognize modified DNA was analyzed by incubation of the particular DNA probes with protein for 30 min on ice and the outcome was monitored by 5% EMSA (see Figure S9 A in the SI) which confirmed that the p53 mutant recognizes and binds to the modified DNA. Then, the potential covalent cross-link formation was tested by incuba-

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tion of the modified DNA with the p53 mutant (see Figure 3) for 2 h at r.t. and monitored by denaturing SDS PAGE (see Figure S9 B in the SI). Unfortunately, we did not observe formation of the covalent crosslink. Either even the proximity effect was not enough to facilitate the reaction or the hemiketal was too unstable to survive the denaturating gel electrophoresis conditions.

19

TAP

Figure 4. F NMR spectra of dU modified ssDNA TAP TAP (31ON_U ) and DNA_1U (duplex). Conditions: 1.1 nM of oligonucleotide, in 20mM phosphate buffer at pH=7

Figure 3. Attempted unsuccessful cross-linking of TAPmodified DNA with GST p53CD_C277S, 19

F NMR spectroscopy study of TAP-modified DNA and its interactions with proteins. To test the 19 applicability of the TAP group in F NMR spectroscopy of nucleic acids, we first focused on the detection of changes in secondary structure. Model hairpin-forming TAP ON which contained the dU modification in the TAP loop of the hairpin (31ON_1U ) was synthesized by PEX using a biotinylated template followed by magne19 toseparation of the modified template strand. The F NMR chemical shift of the TAP group (-84.45 ppm) in the hairpin ON was almost the same as the shift of the TAP TAP-modified triphosphate dU TP (-84.18 ppm) in water. Subsequently the hairpin was treated with the 31_1U complementary ON (temp ) in water at a molar ratio of 1:1 (final DNA concentration of 1.1 nM) and the mixture was heated to 95 °C and slowly cooled down to 25 °C after 95 min. leading to formation of DNA duplex in the same way as our previous studies on fluorinated 15 aminobenzoxazole-modified DNA. The resulting du19 19 plex was analyzed by F NMR. The F chemical shift of the TAP group in the duplex DNA was −85.11 ppm (Figure 4), which is only a small change of 0.67 ppm compared to the hairpin. Apparently the TAP-label is less sensitive to secondary structural changes than the 15 previously reported fluorinated aminobenzoxazole, which showed a change of 9.04 ppm in a similar hybridization experiment.

We next endeavored to exploit the potential of the 19 TAP-label for F NMR spectroscopic analysis of DNAprotein interactions, which has been underexplored in 18,19 the past. Double stranded DNA bearing one modiTAP fied dC in the p53 consensus sequence was prepared 19 TAP by PEX. Our initial F NMR experiment, DNA_1C was measured at a concentration of 0.5 nM in 20 mM phosphate buffer at pH=7, VP/DTT/KCl (1:1:1 v/v/v). 19 TAP We observed a F chemical shift of dC -modified TAP DNA at -83.20 ppm (Figure 5a). Then, the DNA_1C was tested by incubation with protein for 30 min on ice and the outcome was monitored by 5% EMSA (see Figure S10 A in the SI) which confirmed that p53 recognizes and binds to the modified DNA. After the incubation on ice the DNA-protein sample was used for 19 another F NMR measurement which revealed a new signal at -74.68 ppm with a significant shift of 8.52 ppm (Figure 5b). Then the sample was denaturated at 19 55°C for 1 h and the F NMR spectrum showed again only the original shift of free TAP-DNA at -83.20 (Figure 5c). This unambigously proves that the additional signal at -74.68 ppm is due to the interaction with p53 protein, although we cannot say whether it is just shielding of the CF3 group by protein or whether it is an equilibrium of ketone and hydrate form of TAP in the complex with protein. In a negative TAP control experiment, the incubation of the DNA_1C with bovine serum albumin (BSA) did not show any 19 changes in F chemical shift (NMR spectra S15).

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The Journal of Organic Chemistry boronic acid pinacol ester, palladium(II) acetate, tris(3sulfonatophenyl)phosphine hydrate sodium salt from, 5iodo-2'-deoxycytidine and 5-iodo-2'-deoxyuridine were purchased from commercial suppliers. The water used for synthesis was of HPLC quality.

Preparation of trifluoroacetophenone modified TAP nucleosides (dN ) General procedure I: Nucleoside I analogue (dN ) (1 equiv.), 2,2,2-Trifluoroacetophenone4-boronic acid pinacol ester (1.3 equiv.), K2CO3 (3 equiv.), TPPTS (8%) and Pd(OAc)2 (4%) were dissolved in mixture water/acetonitrile (1:2, 2 ml) under argon atmosphere. The reaction mixture was stirred at 80 °C overnight then evaporated in vacuo. The products were purified by column chromatography. 19

TAP

Figure 5. F NMR shifts of (a) DNA_1C , (b) TAP DNA_1C in presence of GST p53CD, (c) after denaturation at 55°C. Conditions: 0.5 nM of oligonucleotide, 2.5 equiv. of GSTp53CD. For full spectra, see Figure S12 in the SI. Conclusions. We have described a short and efficient synthesis of a series of TAP-modified nucleosides, nucleotide mono- and triphosphates by the direct attachment of the aryl group by the Suzuki-Miyaura cross-coupling reaction. All of the modified triphosphates were found to be good substrates for various DNA polymerases and were efficiently incorporated TAP TAP into ssONs and dsDNA by PEX. dC TP, dA TP and TAP dU TP were found to be a good substrates for KOD TAP XL polymerase in PCR, whereas dG TP was found to be inefficient in that experiment. The attempted experiments with p53 mutant did not show any formation of a stable covalent cross-link of TAP-modified DNA with serine in the protein. On the other hand, the TAP 19 group shows potential as a label for F NMR spectroscopy. The sensitivity to changes of secondary structure was rather weak, however, it showed a pronounced sensitivity to DNA-protein interactions. EXPERIMENTAL SECTION NMR spectra were recorded on a 600 MHz (600.1 MHz for 1 13 1 H, 150.9 MHz for C) or a 500 (500.0 MHz for H, 470.4 MHz 19 31 13 for F, 202.3 MHz for P, 125.7 MHz for C) spectrometers from sample solutions in D2O, or CD3OD. Chemical shifts (in ppm, δ scale) were referenced as follows: D2O (referenced to 1 dioxane as internal standard: 3.75 ppm for H NMR and 69.30 13 ppm C NMR); CD3OD (referenced to solvent signal: 3.31 1 13 31 ppm for H NMR and 49.00 ppm for C NMR); P chemical shifts were referenced to H3PO4 as external reference (0 ppm). Coupling constants (J) are given in Hz. NMR spectra of dNTPs were measured in phosphate buffer at pH 7.1. Complete assignment of all NMR signals was achieved by using a combination of H, H-COSY, H,C-HSQC and H,C-HMBC experiments. Mass spectra and high resolution mass spectra were measured on LTQ Orbitrap XL mass spectrometer with a linear ion trap MS and the Orbitrap mass analyzer, using ESI ionization technique. 2,2,2-Trifluoroacetophenone-4-

5-[4-(Trifluoroacetyl)phenyl]-2'-deoxycytidine TAP (dC ) I

Prepared according to General procedure I, from dC (50 mg, 0.14 mmol), 2,2,2-Trifluoroacetophenone-4boronic acid pinacol ester (55 mg, 0.18 mmol), K2CO3 (58 mg, 0.42 mmol), TPPTS (6.4 mg, 0.011 mmol) and Pd(OAc)2 (1.3 mg, 5.6 µmol)were heated overnight. The crude product was purified by column chromatography using DCM/methanol (9/2) as a mobile phase. TAP 1 dC was isolated as a yellow powder (48 mg, 85%). H NMR (500.0 MHz, CD3OD): 2.22, 2.24 (2 × ddd, 2 × 1H, Jgem = 13.6, J2ʹb,1ʹ = 6.3, J2ʹb,3ʹ = 2.8, H-2′b); 2.40 (ddd, 2H, Jgem = 13.6, J2ʹa,1ʹ = 6.3, J2ʹa,3ʹ = 4.2, H-2′a); 3.70 (dd, 2H, Jgem = 12.0, J5ʹb,4ʹ = 3.5, H-5ʹb); 3.792, 3.795 (2 × dd, 2 × 1H, Jgem = 12.0, J5ʹa,4ʹ = 3.0, H-5ʹa); 3.93 (ddd, 2H, J4ʹ,5ʹ = 3.5, 3.0, J4ʹ,3ʹ = 4.0, H-4ʹ); 4.36 – 4.41 (m, 2H, H-3′); 6.30 (t, 2H, J1′,2′ = 6.3, H-1′); 7.43 – 7.47 (m, 4H, H-ophenylene); 7.69 – 7.73 (m, 4H, H-m-phenylene); 8.128, 13 8.131 (2 × s, 2 × 1H, H-6). C NMR (150.9 MHz, CD3OD): 42.3 (CH2-2′); 62.3 (CH2-5′); 71.6 (CH-3′); 87.7 (CH1′); 88.9 (CH-4′); 98.6 (q, JC,F = 29.08, C(OH)2CF3); 110.2 (C-5); 124.7 (q, JC,F = 288.3, CF3); 129.7 (CH-ophenylene); 130.4 (CH-m-phenylene); 135.6 (C-iphenylene); 137.5 (C-p-phenylene); 141.9 (CH-6); 157.7 19 (C-2); 165.5 (C-4); 203.1 (COCF3, from HMBC). F NMR (470.4 MHz, CD3OD): -80.27.MS MS (ESI ): m/z (%) 398.1 (100) [M-H], 418.1 (64) [M+K], 400.1 (35) [M+H]. + HRMS (ESI-IT) m/z: [M+H] calcd for C17H17F3N3O5 400.1115; found 400.1114. 5-(4-(Trifluoroacetyl)phenyl)-2'-deoxyuridine TAP (dU ) I

Prepared according to General procedure I, from dU (50 mg, 0.14 mmol), 2,2,2-Trifluoroacetophenone-4boronic acid pinacol ester (55 mg, 0.18 mmol), K2CO3 (58 mg, 0.42 mmol), TPPTS (6.4 mg, 0.011 mmol) and Pd(OAc)2 (1.3 mg, 5.6 µmol)were heated overnight. The crude product was purified by column chromatography using DCM/methanol (8/2) as a mobile phase. TAP 1 dU was isolated as a yellow powder (39 mg, 70%) H NMR (500.0 MHz, CD3OD): 2.30 – 2.39 (m, 4H, H-2′); 3.75 (dd, 2H, Jgem = 12.0, J5ʹb,4ʹ = 3.0, H-5ʹb); 3.830, 3.833

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(2 × dd, 2 × 1H, Jgem = 12.0, J5ʹa,4ʹ = 3.0, H-5ʹa); 3.96 (q, 2H, J4ʹ,5ʹ = J4ʹ,3ʹ = 3.0, H-4ʹ); 4.43 – 4.47 (m, 2H, H-3′); 6.36 (t, 2H, J1′,2′ = 6.5, H-1′); 7.59 – 7.65 (m, 8H, H13 o,m-phenylene); 8.36 (s, 2H, H-6). C NMR (150.9 MHz, CD3OD): 41.7 (CH2-2′); 62.4 (CH2-5′); 72.0, 72.0 (CH-3′); 86.8 (CH-1′); 89.0 (CH-4′); 97.8 (q, JC,F = 31.3, C(OH)2CF3); 115.4, 115.4 (C-5); 124.4 (q, JC,F = 286.9, CF3); 129.0 (CH-o-phenylene); 129.2 (CH-mphenylene); 135.2 (C-p-phenylene); 135.6 (C-i19 phenylene); 140.2 (CH-6); 151.9 (C-2); 164.6 (C-4). F NMR (470.4 MHz, CD3OD): -80.53. MS (ESI-IT ): m/z (%) 399.3 (100) [M]. HRMS (ESI-IT) m/z: [M-H] calcd for C17H14F3N2O6 399.0802; found 399.0809. 5-[4-(Trifluoroacetyl)phenyl)-2'-deoxy-7TAP deazaadenosine (dA ) I

Prepared according to General procedure I, from dA (50 mg, 0.13 mmol), 2,2,2-Trifluoroacetophenone-4boronic acid pinacol ester (51 mg, 0.17 mmol), K2CO3 (54 mg, 0.39 mmol), TPPTS (6 mg, 0.010 mmol) and Pd(OAc)2 (1.1 mg, 5.2 µmol)were heated overnight. The crude product was purified by column chromatography using DCM/methanol (8/2) as a mobile phase. TAP 1 dA was isolated as a yellow powder (35 mg, 62%) H NMR (500.0 MHz, CD3OD): 2.36 (ddd, 1H, Jgem = 13.4, J2′b,1′ = 6.0, J2′b,3′ = 2.7, H-2′b); 2.72 (dddd, 1H, Jgem = 13.4, J2′a,1′ = 8.2, J2′a,3′ = 6.0, J2′a,4′ = 1.2, H-2′a); 3.74 (dd, 1H, Jgem = 12.1, J5ʹb,4ʹ = 3.7, H-5′b); 3.81 (dd, 1H, Jgem = 12.1, J5ʹa,4ʹ = 3.3, H-5′b); 4.03 (m, 1H, H-4′); 4.55 (dt, 1H, J3ʹ,2ʹ = 6.0, 2.7, J3ʹ,4ʹ = 2.7, H-3′); 6.60 (dd, 1H, J1′,2′ = 8.2, 6.0, H-1′); 7.49 (s, 1H, H-6); 7.56 (m, 2H, H-o-phenylene); 13 7.72 (m, 2H, H-m-phenylene); 8.14 (s, 1H, H-2). C NMR (125.7 MHz, CD3OD): 41.5 (CH2-2′); 63.6 (CH25′); 73.0 (CH-3′); 86.5 (CH-1′); 89.1 (CH-4′); 98.1 (q, JC,F = 31.5, CCF3); 102.6 (C-4a); 118.1 (C-5); 123.1 (CH-6); 124.5 (q, JC,F = 287.3, CF3); 129.5 (CH-o-phenylene); 130.1 (CH-m-phenylene); 135.7 (C-p-phenylene); 136.8 (C-i19 phenylene); 151.2 (C-7a); 152.3 (CH-2); 158.9 (C-4). F + NMR (470.4 MHz, CD3OD): -80.39. MS (ESI ): m/z (%) + 441.1 (100) [M+K]. HRMS (ESI-IT) m/z: [M+Na] calcd for C19H17F3N4O4Na 445.1095; found 445.1094. 5-[4-(Trifluoroacetyl)phenyl]-2'-deoxy-7TAP deazaguanosine (dG ) I

Prepared according to General procedure I, from dG (50 mg, 0.127 mmol), 2,2,2-Trifluoroacetophenone-4boronic acid pinacol ester (50 mg, 0.165 mmol), K2CO3 (52 mg, 0.38 mmol), TPPTS (6 mg, 0.010 mmol) and Pd(OAc)2 (1.1 mg, 5.1 µmol)were heated overnight. The crude product was purified by column chromatography using DCM/methanol (7/3) as a mobile phase. TAP 1 dG was isolated as a yellow powder (25 mg, 45%) H NMR (500.0 MHz, CD3OD): 2.29 (ddd, 1H, Jgem = 13.4, J2′b,1′ = 6.0, J2′b,3′ = 2.9, H-2′b); 2.59 (ddd, 1H, Jgem = 13.4, J2′a,1′ = 8.0, J2′a,3′ = 6.2, H-2′a); 3.73 (dd, 1H, Jgem = 12.0, J5ʹb,4ʹ = 4.1, H-5′b); 3.79 (dd, 1H, Jgem = 12.0, J5ʹa,4ʹ = 3.7, H5′b); 3.96 (ddd, 1H, J4ʹ,5ʹ = 4.1, 3.7, J4ʹ,3ʹ = 2.9, H-4′); 4.51 (dt, 1H, J3ʹ,2ʹ = 6.2, 2.9, J3ʹ,4ʹ = 2.9, H-3′); 6.47 (dd, 1H, J1′,2′

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= 8.0, 6.0, H-1′); 7.25 (s, 1H, H-6); 7.54 (m, 2H, H-m13 phenylene); 7.87 (m, 2H, H-o-phenylene). C NMR (125.7 MHz, CD3OD): 41.2 (CH2-2′); 63.6 (CH2-5′); 72.9 (CH-3′); 85.3 (CH-1′); 88.6 (CH-4′); 98.0 (q, JC,F = 31.0, CCF3); 99.4 (C-4a); 117.9 (CH-6); 121.1 (C-5); 124.5 (q, JC,F = 286.8, CF3); 128.8 (CH-o-phenylene); 128.9 (CH-mphenylene); 133.3 (C-p-phenylene); 136.9 (C-i19 phenylene); 153.6 (C-7a); 154.0 (C-2); 161.8 (C-4). F NMR (470.4 MHz, CD3OD): -80.58. MS (ESI ): m/z (%) 437.3 (84) [M-H]. HRMS (ESI-IT) m/z: [M-H] calcd for C19H16F3N4O5 437.1086; found 437.1078. Preparation of trifluoroacetophenone modified TAP nucleoside monophosphates (dN MP) General procedure II: Nucleoside monophosphate analogue I (dN MP) (1 equiv.), trifluoroacetophenone (1.3 equiv.), K2CO3 (3 equiv.), TPPTS (8%) and Pd(OAc)2 (4%) were dissolved in mixture water/acetonitrile (1:1, 2 ml) under argon atmosphere. The reaction mixture was stirred at 80 °C for 4h then evaporated in vacuo. The products were purified by C18 reversed-phase HPLC using water/methanol (15 to 100%) containing 0.1 M TEAB buffer as eluent. Several codistillations with water and conversion to sodium salt (Dowex 50WX8 in Na+ cycle) followed by freeze-drying from water gave the TAP desired dN MPs as white solids. 5-[4-(Trifluoroacetyl)phenyl]-2'-deoxycytidine 5'TAP O-phosphate (dC MP) Prepared according to General procedure II, from I dC MP (50 mg, 0.109 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (43 mg, 0.14 mmol), K2CO3 (45 mg, 0.33 mmol), TPPTS (4.95 mg, 8.72 µmol) and Pd(OAc)2 (1 mg, 4.36 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TEAB buffTAP er as a eluent , product dC MP was isolated as a 1 yellow powder (49 mg, 88%). H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.35 (ddd, 1H, Jgem = 14.0, J2ʹb,1ʹ = 7.7, J2ʹb,3ʹ = 6.4, H-2′b); 2.44 (ddd, 1H, Jgem = 14.0, J2ʹa,1ʹ = 6.2, J2ʹa,3ʹ = 3.4, H-2′a); 3.81 – 3.93 (m, 2H, H-5′); 4.15 (td, 1H, J4ʹ,5ʹ = 5.1, J4ʹ,3ʹ = 3.4, H-2′b); 4.51 (dt, 1H, J3′,2′ = 6.4, 3.4, J3′,4′ = 3.4, H-3′); 6.34 (dd, 1H, J1′,2′ = 7.7, 6.2, H-1′); 7.52 (m, 2H, H-o-phenylene); 7.74 13 (s, 1H, H-6); 7.80 (m, 2H, H-m-phenylene). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 41.5 (CH2-2′); 66.5 (d, JC,P = 4.5, CH2-5′); 73.9 (CH-3′); 88.6 (d, JC,P = 8.3, CH-4′); 88.7 (CH-1′); 95.9 (q, JC,F = 32.3, CCF3); 112.7 (C-5); 125.6 (q, JC,F = 286.5, CF3); 130.7 (CH-m-phenylene); 132.2 (CH-o-phenylene); 136.7 (C-iphenylene); 139.4 (C-p-phenylene); 142.7 (CH-6); 159.8 31 1 (C-2); 167.2 (C-4). P{ H} NMR (202.3 MHz, D2O): 3.46. 19 F NMR (470.4 MHz, D2O): -84.20. MS (ESI ): m/z (%) 478 (100) [M]. HRMS (ESI-IT) m/z: [M] calcd for C17H16F3N3O8P 478.0633; found 478.0665.

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The Journal of Organic Chemistry -

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5-[4-(Trifluoroacetyl)phenyl]-2'-deoxyuridine 5'TAP O-phosphate (dU MP)

IT) m/z: [M] calcd for C19H17F3N4O7P 501.0793; found 501.0792.

Prepared according to General procedure II, from I dU MP (50 mg, 0.109 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (43 mg, 0.14 mmol), K2CO3 (45 mg, 0.33 mmol), TPPTS (4.95 mg, 8.72 µmol) and Pd(OAc)2 (1 mg, 4.36 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TEAB buffTAP er as a eluent , product dU MP was isolated as a 1 yellow powder (43 mg, 75%) H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.40 (ddd, 1H, Jgem = 14.1, J2ʹb,1ʹ = 6.4, J2ʹb,3ʹ = 3.4, H-2′b); 2.46 (ddd, 1H, Jgem = 14.1, J2ʹa,1ʹ = 7.7, J2ʹa,3ʹ = 6.4, H-2′a); 3.83 – 3.95 (m, 2H, H5′); 4.15 (td, 1H, J4ʹ,5ʹ = 5.1, J4ʹ,3ʹ = 3.4, H-2′b); 4.55 (dt, 1H, J3′,2′ = 6.4, 3.4, J3′,4′ = 3.4, H-3′); 6.34 (dd, 1H, J1′,2′ = 7.7, 6.4, H-1′); 7.59 (m, 2H, H-o-phenylene); 7.75 (m, 13 2H, H-m-phenylene); 7.86 (s, 1H, H-6). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 40.8 (CH2-2′); 66.5 (d, JC,P = 4.5, CH2-5′); 74.0 (CH-3′); 88.3 (CH-1′); 88.7 (d, JC,P = 8.2, CH-4′); 96.0 (q, JC,F = 32.3, CCF3); 118.1 (C-5); 125.6 (q, JC,F = 286.6, CF3); 130.1 (CHm-phenylene); 131.5 (CH-o-phenylene); 136.5 (C-iphenylene); 138.9 (C-p-phenylene); 141.9 (CH-6); 154.5 31 1 (C-2); 168.0 (C-4). P{ H} NMR (202.3 MHz, D2O): 3.83. 19 F NMR (470.4 MHz, D2O): -84.18. . MS (ESI ): m/z (%) 479 (100) [M]. HRMS (ESI-IT) m/z: [M] calcd for C17H15F3N2O9P 479.0470; found 479.0472.

5-[4-(Trifluoroacetyl)phenyl]-2'-deoxy-7TAP deazaguanosine-5'-O-phosphate (dG MP)

5-[4-(Trifluoroacetyl)phenyl]-2'-deoxy-7TAP deazaadenosine 5'-O-phosphate (dA MP) Prepared according to General procedure II, from I dA MP (50 mg, 0.104 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (41 mg, 0.135 mmol), K2CO3 (43 mg, 0.31 mmol), TPPTS (4.7 mg, 8.32 µmol) and Pd(OAc)2 (0.9 mg, 4.16 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TEAB buffTAP er as a eluent , product dA MP was isolated as a 1 yellow powder (31 mg, 56%). H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.34 (ddd, 1H, Jgem = 14.0, J2′b,1′ = 6.1, J2′b,3′ = 3.1, H-2′b); 2.61 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 8.2, J2′a,3′ = 6.3, H-2′a); 3.75 (t, 2H, JH,P = J5ʹ,4ʹ = 5.5, H-5′); 4.11 (td, 1H, J4ʹ,5ʹ = 5.5, J4ʹ,3ʹ = 3.1, H-4′); 4.61 (dt, 1H, J3ʹ,2ʹ = 6.3, 3.1, J3ʹ,4ʹ = 3.1, H-3′); 6.49 (dd, 1H, J1′,2′ = 8.2, 6.1, H-1′); 7.38 (s, 1H, H-6); 7.40 (m, 2H, H-o-phenylene); 7.73 (m, 2H, H-m-phenylene); 13 8.03 (s, 1H, H-2). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 40.6 (CH2-2′); 66.6 (d, JC,P = 4.4, CH2-5′); 74.4 (CH-3′); 85.2 (CH-1′); 88.1 (d, JC,P = 8.4, CH-4′); 96.0 (q, JC,F = 32.2, CCF3); 103.2 (C-4a); 119.2 (C-5); 123.2 (CH-6); 125.7 (q, JC,F = 286.6, CF3); 130.6 (CH-m-phenylene); 131.1 (CH-o-phenylene); 137.8 (C-i-phenylene); 138.3 (C-p-phenylene); 152.5 (C31 1 7a); 153.9 (CH-2); 159.6 (C-4). P{ H} NMR (202.3 MHz, 19 D2O): 3.77. F NMR (377.3 MHz, D2O): -84.14.MS (ESI ): m/z (%) 501.1 (100) [M], 523.1 (20) [M+Na]. HRMS (ESI-

Prepared according to General procedure II, from I dG MP (50 mg, 0.101 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (39 mg, 0.131 mmol), K2CO3 (42 mg, 0.30 mmol), TPPTS (4.59 mg, 8.08 µmol) and Pd(OAc)2 (0.9 mg, 4.04 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TEAB TAP buffer as a eluent , product dC MP was isolated as a 1 yellow powder (14 mg, 26%). H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.39 (ddd, 1H, Jgem = 14.0, J2′b,1′ = 6.2, J2′b,3′ = 3.0, H-2′b); 2.71 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 8.3, J2′a,3′ = 6.3, H-2′a); 3.87 (dd, 2H, JH,P = 6.1, J5ʹ,4ʹ = 5.4, H-5′); 4.15 (td, 1H, J4ʹ,5ʹ = 5.4, J4ʹ,3ʹ = 3.0, H4′); 4.66 (dt, 1H, J3ʹ,2ʹ = 6.3, 3.0, J3ʹ,4ʹ = 3.0, H-3′); 6.47 (dd, 1H, J1′,2′ = 8.3, 6.2, H-1′); 7.31 (s, 1H, H-6); 7.70 (m, 13 2H, H-m-phenylene); 7.79 (m, 2H, H-o-phenylene). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 40.5 (CH2-2′); 66.7 (d, JC,P = 4.0, CH2-5′); 74.5 (CH-3′); 85.4 (CH-1′); 88.3 (d, JC,P = 8.5, CH-4′); 96.1 (q, JC,F = 32.2, CCF3); 100.7 (C-4a); 119.5 (CH-6); 123.0 (C-5); 125.7 (q, JC,F = 287.1, CF3); 129.8 (CH-mphenylene); 130.9 (CH-o-phenylene); 137.4 (C-pphenylene); 137.8 (C-i-phenylene); 155.0 (C-7a); 155.7 31 1 (C-2); 163.8 (C-4). P{ H} NMR (202.3 MHz, D2O): 3.90. 19 F NMR (470.4 MHz, D2O): -84.18. MS (ESI ): m/z (%) 517 (100) [M] 539 (39) [M+Na-H]. HRMS (ESI-IT) m/z: [M] calcd for C19H17F3N4O8P 517.0733; found 517.0741. Preparation of trifluoroacetophenone modified TAP nucleoside triphosphates (dN TP) General proceI dure III: Nucleoside triphosphate analogue (dN TP) (1 equiv.), trifluoroacetophenone (1.3 equiv.), K2CO3 (3 equiv.), TPPTS (8%) and Pd(OAc)2 (4%) were dissolved in mixture water/acetonitrile (2:1, 2.5 ml) under argon atmosphere. The reaction mixture was stirred at 80 °C for 2h then evaporated in vacuo. The products were purified by C18 reversed-phase HPLC using water/methanol (15 to 100%) containing 0.1 M TEAB buffer as eluent. Several codistillations with water and conversion to sodium salt (Dowex 50WX8 in Na+ cycle) followed by freeze-drying from water gave the TAP desired dN TPs as white solids. 5-[4-(Trifluoroacetyl)phenyl]-2'-deoxycytidine 5'TAP O-triphosphate (dC TP) Prepared according to General procedure III, from I dC TP (50 mg, 0.076 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (29 mg, 0.095 mmol), K2CO3 (30 mg, 0.22 mmol), TPPTS (3.3 mg, 5.87 µmol) and Pd(OAc)2 (0.66 mg, 2.93 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M

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The Journal of Organic Chemistry TAP

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TEAB buffer as a eluent , product dC TP was isolated 1 as a yellow powder (40 mg, 74%). H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.36 (ddd, 1H, Jgem = 14.0, J2ʹb,1ʹ = 7.4, J2ʹb,3ʹ = 6.4, H-2′b); 2.45 (ddd, 1H, Jgem = 14.0, J2ʹa,1ʹ = 6.3, J2ʹa,3ʹ = 3.7, H-2′a); 4.09 – 4.21 (m, 2H, H-5′); 4.22 (qd, 1H, J4′,3′ = J4′,5′ = 3.7, JH,P = 1.7, H-4′); 4.61 (dt, 1H, J3′,2′ = 6.4,3.7, J3′,4′ = 3.7, H-3′); 6.36 (dd, 1H, J1′,2′ = 7.4, 6.3, H-1′); 7.54 (m, 2H, H-o-phenylene); 7.80 13 (m, 2H, H-m-phenylene); 7.84 (s, 1H, H-6). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 42.0 (CH2-2′); 68.0 (d, JC,P = 5.5, CH2-5′); 73.4 (CH-3′); 88.3 (d, JC,P = 9.1, CH-4′); 88.8 (CH-1′); 96.0 (q, JC,F = 32.2, CCF3); 112.8 (C-5); 125.6 (q, JC,F = 286.4, CF3); 130.8 (CH-m-phenylene); 132.2 (CH-o-phenylene); 136.7 (C-iphenylene); 139.3 (C-p-phenylene); 142.8 (CH-6); 159.8 31 1 (C-2); 167.2 (C-4). P{ H} NMR (202.3 MHz, D2O): 22.56 (t, J = 19.6, Pβ); -11.57 (d, J = 19.6, Pα); -8.46 (bd, J = 19 19.6, Pγ). F NMR (470.4 MHz, D2O): -84.22. MS (ESI ): m/z (%) 558 (100) [M-PO3 ]. HRMS (ESI-IT) m/z: [M+3H] calcd for C17H18F3N3O14P3 637.9953; found 637.9959. 5-[4-(Trifluoroacetyl)phenyl]-2'-deoxyuridine 5'TAP O-triphosphate (dU TP) Prepared according to General procedure III, from I dU TP (50 mg, 0.076 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (30 mg, 0.098 mmol), K2CO3 (31 mg, 0.22 mmol), TPPTS (3.45 mg, 6.07 µmol) and Pd(OAc)2 (0.7 mg, 3.03 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TAP TEAB buffer as a eluent , product dU TP was isolat1 ed as a yellow powder (31 mg, 58%). H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.34-2.48 (m, 2H, H-2′); 4.12-4.24 (m, 3H, H-4′,5′); 4.77 (dt, 1H, J3′,2′ = 6.6,3.6, J3′,4′ = 3.6, H-3′); 6.41 (t, 1H, J1′,2′ = 6.9, H-1′); 7.55 (m, 2H, H-o-phenylene); 7.73 (m, 2H, H-m13 phenylene); 7.78 (s, 1H, H-6). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 41.2 (CH2-2′); 68.1 (d, JC,P = 5.5, CH2-5′); 73.4 (CH-3′); 87.9 (d, JC,P = 9.0, CH-4′); 88.2 (CH-1′); 97.0 (q, JC,F = 31.9, CCF3); 118.9 (C-5); 126.3 (q, JC,F = 288.3, CF3); 130.0 (CH-mphenylene); 131.4 (CH-o-phenylene); 138.0 (C-iphenylene); 140.1 (C-p-phenylene); 141.0 (CH-6); 159.7 31 1 (C-2); 174.4 (C-4). P{ H} NMR (202.3 MHz, D2O): 21.57 (dd, J = 19.9, 19.1, Pβ); -11.04 (d, J = 19.1, Pα); -5.71 19 (d, J = 19.9, Pγ). F NMR (470.4 MHz, D2O): -84.17. MS (ESI ): m/z (%) 559.0 (100) [M-PO3 +2H]. HRMS (ESIIT) m/z: [M+3H] calcd for C17H17F3N3O15P3 638.9792; found 638.9799. 5-[4-(Trifluoroacetyl)phenyl]-2'-deoxy-7TAP deazaadenosine 5'-O-triphosphate (dA TP) Prepared according to General procedure III, from I dA TP (50 mg, 0.0734 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (29 mg, 0.095 mmol), K2CO3 (30 mg, 0.22 mmol),

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TPPTS (3.3 mg, 5.87 µmol) and Pd(OAc)2 (0.66 mg, 2.93 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TAP TEAB buffer as a eluent , product dA TP was isolated 1 as a yellow powder (31 mg, 52%). H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.45 (ddd, 1H, Jgem = 14.0, J2′b,1′ = 6.5, J2′b,3′ = 3.3, H-2′b); 2.72 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 7.8, J2′a,3′ = 6.0, H-2′a); 4.11 (ddd, 1H, Jgem = 11.3, JH,P = 5.3, J5′b,4′ = 4.0, H-5′b); 4.17 (ddd, 1H, Jgem = 11.3, JH,P = 5.8, J5′a,4′ = 4.0, H-5′a); 4.23 (m, 1H, H4′); 4.77 (m, 1H, H-3′); 6.65 (dd, 1H, J1′,2′ = 7.8, 6.5, H1′); 7.54 (s, 1H, H-6); 7.57 (m, 2H, H-o-phenylene); 7.78 13 (m, 2H, H-m-phenylene); 8.15 (s, 1H, H-2). C NMR (125.7 MHz, D2O, ref(external dioxane) = 69.3 ppm): 41.0 (CH2-2′); 68.2 (d, JC,P = 5.4, CH2-5′); 73.8 (CH-3′); 85.5 (CH-1′); 87.8 (d, JC,P = 8.7, CH-4′); 96.0 (q, JC,F = 32.4, CCF3); 103.5 (C-4a); 120.2 (C-5); 123.3 (CH-6); 125.7 (q, JC,F = 286.9, CF3); 130.5 (CH-m-phenylene); 131.4 (CH-o-phenylene); 138.1 (C-i,p-phenylene); 152.7 (C-7a); 31 1 154.1 (CH-2); 159.9 (C-4). P{ H} NMR (202.3 MHz, D2O): -21.86 (bs, Pβ); -10.97 (d, J = 18.6, Pα); -6.43 (bd, J 19 = 17.4, Pγ). F NMR (470.4 MHz, D2O): -84.17. MS (ESI ): m/z (%) 581.1 (100) [M-PO3 +2H]. HRMS (ESI-IT) m/z: [M+3H] calcd for C19H19F3N4O13P3 661.0107; found 661.0119. 5-[4-(Trifluoroacetyl)phenyl]-2'-deoxy-7TAP deazaguanosine 5'-O-triphosphate (dG TP) Prepared according to General procedure III, from I dG TP (50 mg, 0.071 mmol), 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester (28 mg, 0.093 mmol), K2CO3 (30 mg, 0.22 mmol), TPPTS (3.3 mg, 5.7 µmol) and Pd(OAc)2 (0.6 mg, 2.9 µmol). After purification by C18 reverse-phase HPLC with water/methanol (15-100%) containing 0.1 M TEAB TAP buffer as a eluent , product dG TP was isolated as a 1 yellow powder (18 mg, 37%). H NMR (600.1 MHz, D2O, ref(dioxane) = 3.75 ppm): 2.32 (ddd, 1H, Jgem = 14.1, J2′b,1′ = 6.4, J2′b,3′ = 3.2, H-2′b); 2.63 (ddd, 1H, Jgem = 14.1, J2′a,1′ = 7.9, J2′a,3′ = 6.3, H-2′a); 4.10 – 4.18 (m, 2H, H-5′); 4.20 (m, 1H, H-4′); 4.71 (dt, 1H, J3ʹ,2ʹ = 6.3, 3.2, J3ʹ,4ʹ = 3.2, H3′); 6.38 (dd, 1H, J1′,2′ = 7.9, 6.4, H-1′); 7.24 (s, 1H, H-6); 7.68 (m, 2H, H-m-phenylene); 7.78 (m, 2H, H-o13 phenylene). C NMR (150.9 MHz, D2O, ref(dioxane) = 69.3 ppm): 40.7 (CH2-2′); 68.3 (d, JC,P = 5.4, CH2-5′); 73.8 (CH-3′); 85.6 (CH-1′); 87.6 (d, JC,P = 8.6, CH-4′); 96.1 (q, JC,F = 32.3, CCF3); 100.6 (C-4a); 119.3 (CH-6); 122.8 (C-5); 125.7 (q, JC,F = 286.5, CF3); 129.7 (CH-mphenylene); 130.7 (CH-o-phenylene); 137.1 (C-pphenylene); 137.8 (C-i-phenylene); 154.8 (C-7a); 155.4 31 1 (C-2); 163.5 (C-4). P{ H} NMR (202.3 MHz, D2O): 21.73 (t, J = 19.1, Pβ); -10.90 (d, J = 19.1, Pα); -6.27 (d, J = 19 19.1, Pγ). F NMR (470.4 MHz, D2O): -84.17. MS (ESI ): m/z (%) 597 (100) [M-PO3 +2H]. HRMS (ESI-IT) m/z: [M+3H] calcd for C19H19F3N4O14P3 677.0061; found 677.0068.

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Biochemistry General remarks: The MALDI-TOF TM spectra were measured on UltrafleXtreme MALDI-TOF/TOF mass spectrometer with 1 kHz smartbeam II laser. The measurements were done in reflectron mode by droplet technique, with the mass range up to 30 kDa. The matrix consisted of 3-hydroxypicolinic acid (HPA)/picolinic acid (PA)/ammonium tartrate in ratio 9/1/1. The matrix (1 μl) was applied on the target (ground steel) and dried down at room temperature. The sample (1 μl) and matrix (1 μl) were mixed and added on the top of dried matrix preparation spot and dried down at room temperature. UV-Vis spectra were measured on NanoDrop1000 at room temperature. Samples were concentrated on CentriVap Vacuum Concentrator system. Synthetic oligonucleotides (primers, templates and biotinylated templates; for sequences see Table S1) were purchased from commercial suppliers. Natural nucleoside triphosphates (dATP, dGTP, dTTP, dCTP) were obtained from Thermo Scientific, Vent(exo-) and Pwo DNA polymerases were purchased from New England Biolabs. KOD XL DNA polymerase from Merck, streptavidine magnetic particles from Roche, QI® Aquick Nucleotide Removal Kit from Qiagen. All solutions were prepared in MilliQ water. Other chemicals were of analytical grade. TAP

Incorporation of dN TP into 31-mer template by PEX: The reaction mixture (20 μl) contained primer B 31 (prim ) (0.5 μM), template (temp ) (0.75 μM), DNA polymerase (0.05 U KOD XL, dNTPs (either all natural or 3 natural and 1 modified, 20 μM) in 2 μl of enzyme reaction buffer supplied by the manufacturer. Primer was labelled on its 5´-end by 6-carboxyfluorescein (6FAM). The reaction mixture was incubated for 30 min at 60 °C in a thermal cycler. Primer extension was stopped by addition of stop solution (2×, 95% [v/v] formamide, 0.5 mM EDTA, 0.025% [w/v] bromophenol blue, 0.025% [w/v] xylene cyanol, SDS 0.025 [w/v]) and heated for 5 min at 95 °C. Samples were separated by 12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under denaturing conditions (TBE 1×, 42 mA, 1 hour). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S1-S2 in the SI). TAP

Incorporation of dN TP into 19-mer template by PEX: PEX reactions with 19-mer template were performed in the same way as described above. The B reaction mixture (20 μl) contained primer (prim ) (0.2 19_X μM), template (temp ) (0.3 μM), DNA polymerase (0.05 U KOD XL, dGTP/or dTTP in the case of 19_1G temp (0.6 μM), either natural or TAP modified dNTPs (0.6 μM) in 2 μl of enzyme reaction buffer supplied by the manufacturer. Primer was labelled on its 5´-end by 6-carboxyfluorescein (6-FAM). The reaction mixture was incubated for 30 min at 60 °C in a thermal cycler. Primer extension was stopped by addition of

stop solution (2×, 95% [v/v] formamide, 0.5 mM EDTA, 0.025% [w/v] bromophenol blue, 0.025% [w/v] xylene cyanol, SDS 0.025 [w/v]) and heated for 5 min at 95 °C. Samples were separated by 12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under denaturing conditions (TBE 1×, 42 mA, 1 hour). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S1-S2 in the SI). TAP

31_1U

Incorporation of dU TP into the temp by PEX The reaction mixture (20 μl) contained primer C 31_1U (prim ) (0.5 μM), template (temp ) (0.5 μM), DNA polymerase (0.05 U 10x diluted KOD XL), dNTPs (either all natural or 3 natural and 1 modified, 0.15 μM) in 2 μl of enzyme reaction buffer supplied by the manufacturer. Primer was labelled on its 5´-end by 6carboxyfluorescein (6-FAM). The reaction mixture was incubated for 30 min at 60 °C in a thermal cycler. Primer extension was stopped by addition of stop solution (2×, 95% [v/v] formamide, 0.5 mM EDTA, 0.025% [w/v] bromophenol blue, 0.025% [w/v] xylene cyanol, SDS 0.025 [w/v]) and heated for 5 min at 95 °C. Samples were separated by 12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under denaturing conditions (TBE 1×, 42 mA, 1 hour). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S3 in the SI). PCR of trifluoroacetophenone modified dNTPs : Agarose gel electrophoresis PCR products containing 6X DNA loading dye (60 mM EDTA, 10 mM Tris-HCl (pH 7.6), 60 % glycerol, 0.03 % bromphenole blue, 0.03 % xylene cyanol FF, Thermo Scientific) were subjected to horizontal electrophoresis (Owl EasyCastB, Thermo Scientific) and analyzed on either 1.3 % or 2 % agarose gels (containing 0.5x TBE buffer, pH 8). The gels were run at 118 V for ca. 90–120 min. PCR products were visualized with GelRed (Biotium, 10 000X in H2O) using an electronic dual wave transilluminator equipped with GBox iChemi-XRQ Bio imaging system (Syngene, Life Technologies) or TyphoonTM FLA 9500 (GE Healthcare Life Sciences), respectively. 339-mer: 30 PCR cycles were run in PCR cycler, preheated to 80 °C, under the following conditions: preheating for 3 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 70 °C, extension for 2 min at 72 °C, followed by final extension step of 5 min at 72 °C. PCR products were analyzed on a 1.3 % agarose gel in 0.5 × TBE buffer (Figure S6).The PCR reaction mixture (10 µL) was prepared by mixing of Vent(exo)/KOD XL DNA Polymerase (1.6 U/µL), natural dNTPs (20 µM), functionalized dNTPs (4 mM, 40 µM for TAP TAP TAP TAP C /A and 100 µM for U /G ), primers (2 µM, FOR REV prim and 2 µM, prim , 5´-FAM labelled primers TAP were used in the case of G Figure S7) and template

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temp (0.255 µM) in Vent(exo-)/KOD XL reaction buffer (1 µL) supplied by the manufacturer. The PCR reaction mixture (10 µL) was prepared by mixing of Pwo DNA Polymerase (4 U), natural dNTPs (120 µM), TAP TAP functionalized dNTPs (400 µM for C /A and 1mM TAP TAP FOR for U /G ), primers (2 µM, prim and 2 µM, REV Pveg prim ) and template temp (0.255 µM) in Pwo reaction buffer (1 µL) supplied by the manufacturer. 98-mer: 30 PCR cycles were run in PCR cycler, preheated to 80 °C, under the following conditions: preheating for 3 min at 94 °C, denaturation for 1 min at 95 °C, annealing for 1 min at 53 °C, extension for 1 min at 72 °C, followed by final extension step of 3 min at 75 °C. PCR products were analyzed on a 2 % agarose gel in 0.5 × TBE buffer (Figure S5). The PCR reaction mixture (20 µL) was prepared by mixing of Vent(exo-)/KOD XL DNA Polymerase (2 U), natural dNTPs (40 µM), funcTAP TAP tionalized dNTPs (200 µM for C /A and 500 µM TAP TAP FOR-L20 for U /G ), primers (2 µM, prim and 2 µM, REV-LT25TH prim 5´-FAM labelled primers were used in TAP FVL-A the case of G Figure S7) and template temp (0.25 µM) in Vent(exo-)/KOD XL reaction buffer (2 µL) supplied by the manufacturer. The PCR reaction mixture (10 µL) was prepared by mixing of Pwo DNA Polymerase (4 U), natural dNTPs (120 µM), functionalized TAP TAP dNTPs (400 µM for C /A and 800 µM for TAP TAP FOR-L20 U /G ), primers (1 µM, prim and 1 µM, primREV-LT25TH FVL-A ) and template temp (0.25 µM) in PWO reaction buffer (2 µL) supplied by the manufacturer. MALDI-TOF analysis of TAP-modified oligonuTAP TAP TAP cleotides (19ON_1C , 19ON_1A , 19ON_1U , TAP TAP TAP 19ON_1G and 30ON_1C 31ON_1U ): The PEX solution (50 μl) contained KOD XL DNA polymerase B 19X A 30_1C (0.5 U), primer (prim for temp , prime for temp C 31_1U and prime for temp ) (4 μM), 5´-biotinylated tem19X 30_1C 31_1U plate (temp , temp or temp ) (4 μM), dNTPs (either natural or modified, 264 μM) in KOD XL reaction buffer supplied by the manufacturer. The reaction mixture was incubated for 40 min at 60 °C in a thermal cycler. The reaction was stopped by cooling to 4 °C. Streptavidine magnetic particles (Roche, 60 μl) were washed with binding buffer (3 × 200 μl, 10mM Tris, 1 mM EDTA, 100 mM NaCl, pH 7.5). The PEX solution and binding buffer (50 μl) were added into the streptavidine magnetic particles. The mixture was incubated for 30 min at 15 °C and 1200 rpm. The magnetic beads were collected on a magnet (DynaMagTM-2, Invitrogen) and washed with wash buffer (3 × 500 μl, 10 mM Tris, 1 mM EDTA, 500 mM NaCl, pH 7.5) and water (4 × 500 μl). Then water (50 μl) was added and the sample was denatured for 2 min at 55 °C and 900 rpm. The beads were collected on a magnet and the solution was transferred into a clean vial. The product was quantified on NanoDrop and then evaporated to dryness, then dissolved in the mixture water/acetonitrile (1:1, 5 µl) analyzed by MALDI-TOF mass spectrometry

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(the results are summarized in Table 2, for copies of mass spectra see Figures S13-S18 in the SI). TAP

Preparation of modified ssDNA (31ON_U ) for 19 F NMR studies. The sample was prepared as described above from the PEX solution (200 μl) and was 19 then measured by F NMR spectroscopy. Purification of the PEX products on QIAquick Nucleotide Removal Kit (QIAGEN). From the manufacturer protocol, 10 volumes of buffer PNI was added to 1 volume of PEX sample and mixed. The sample was applied to the QIAquick spin column and centrifuge for 1 min at 6000 rpm. 750 μl of buffer PE was added to the column and centrifuge for 1 min at 6000 rpm. For drying samples were centrifuged for an additional 1 min at 13000 rpm. For DNA elution: the QIAquick spin column was placed in a clean microcentrifuge tube and 50 μl of water was added to the center of the membrane and centrifuged for 1 min at 13000 rpm. Incubation of modified DNA with tumor supTAP pressor protein p53 mutants. Modified DNA_2C was prepared by PEX as described below. The products were purified on QIAquick Nucleotide Removal Kit (QIAGEN) and eluted with water. The reaction mixtures for GSTp53_C275S or GSTp53_C277S protein binding (20 μl) were prepared from purified PEX (10 μl, 6 ng/μl), KCl (500 mM, 2 μl), DTT (2 mM, 2 μl), VP buffer (50 mM Tris, 0.1% Triton-X100, pH 7.6, 2 μl) and GSTp53CD_C275S or GSTp53CD_C277S stock solution (700 ng/μl in 25 mM Hepes pH 7.6, 200 mM KCl, 10% glycerol, 1 mM DTT, 1 mM benzamidine; 2 μl or 3 μl ). Control sample was prepared analogously without GSTp53CD mutants. All samples were incubated for 30 min on ice, glycerol was added (80%, 2 μl) and a part of the reaction mixture (3.5 μl) was separated by use of a 5% EMSA (acrylamide/bisacrylamide 37.5:1; 0.5×TBE, 4 °C, 80 V / 1 hour). The rest in vials was incubated for 2 hours at 25 °C. Loading buffer (5×, 0.3 M Tris.HCl, 5% SDS, 50% glycerol, 2.5% β-mercaptoethanol, 0.05% bromphenol blue) was added and the mixture was heated at 65 °C for 10 minutes. The samples (10 μl) were separated by 10% SDS-PAGE (0.025 M Tris, 0.192 M glycine, 0.1 % SDS) at room temperature (100 V / 40 min then 150 V / 1.30 hour). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S8 in the SI). TAP

Incorporation of dN TP into p53 consensus TAP DNA sequence by PEX: For dC TP: The reaction A mixture (72 μl) contained primer prim (0.17 μM), 30_2C temp (0.17 μM), DNA polymerase (0.05 U 10x diluted KOD XL), dNTPs (either all natural or 3 natural and 1 modified, 0.23 μM) in 6.2 μl of enzyme reaction buffer supplied by the manufacturer. TAP

For dA TP: The reaction mixture (65 μl) contained A 30_2A primer prim (0.19 μM), temp (0.14 μM), DNA polymerase (0.1 U KOD XL 10x diluted KOD XL),

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The Journal of Organic Chemistry

dNTPs (either all natural or 3 natural 0.05 μM and 1 modified 0.03 μM,) in 6.2 μl of enzyme reaction buffer supplied by the manufacturer. TAP

For dU TP: The reaction mixture (65 μl) contained A 30_2T primer prim (0.19μM), temp (0.19 μM), DNA polymerase (0.07 U KOD XL 10x diluted KOD XL), dNTPs (either all natural or 3 natural and 1 modified, 0.08 μM) in 6.2 μl of enzyme reaction buffer supplied by the manufacturer. TAP

For dG TP: The reaction mixture (65 μl) contained A 30_2G primer prim (0.19 μM), temp (0.19 μM), DNA polymerase (0.07 U KOD XL 10x diluted KOD XL), dNTPs (either all natural or 3 natural 0.06 μM and 1 modified 0.08 μM) in 6.2 μl of enzyme reaction buffer supplied by the manufacturer. Primer was labelled on its 5´-end by 6carboxyfluorescein (6-FAM). The reaction mixture was incubated for 30 min at 60 °C in a thermal cycler and a part of the samples (10 μl) were mixed with stop solution (2×, 95% [v/v] formamide, 0.5 mM EDTA, 0.025% [w/v] bromophenol blue, 0.025% [w/v] xylene cyanol, SDS 0.025 [w/v]) and heated for 5 min at 95 °C. Samples were separated by 12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under denaturing conditions (TBE 1×, 42 mA, 1 hour). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S4 in the SI). The rest in vials was used for DNA-protein studies. Incubation of modified DNA with GSTp53_C277S. TAP Modified DNA_1X with p53 consensus DNA sequence were prepared by PEX as described above. The products were purified on QIAquick Nucleotide Removal Kit (QIAGEN) and eluted with water. The reaction mixtures for GSTp53_C277S protein binding (20 μl) were prepared from purified PEX (10 μl, 6 ng/μl), KCl (500 mM, 2 μl), DTT (2 mM, 2 μl), VP buffer (50 mM Tris, 0.1% Triton-X100, pH 7.6, 2 μl) and GSTp53CD_C277S stock solution (700 ng/μl in 25mM Hepes pH 7.6, 200 mM KCl, 10% glycerol, 1 mM DTT, 1 mM benzamidine; 2 μl). Control sample was prepared analogously without GSTp53CD mutant. All samples were incubated for 30 min on ice, glycerol was added (80%, 2 μl) and a part of the reaction mixture (3.5 μl) was separated by use of a 5% EMSA (acrylamide/bisacrylamide 37.5:1; 0.5×TBE, 4 °C, 80 V / 1 hour). The rest in vials was incubated for 2 hours at 25 °C. Loading buffer (5×, 0.3M Tris.HCl, 5% SDS, 50% glycerol, 2.5% β-mercaptoethanol, 0.05% bromphenol blue) was added and the mixture was heated at 65 °C for 10 minutes. The samples (10 μl) were separated by 10% SDS-PAGE (0.025 M Tris, 0.192 M glycine, 0.1% SDS) at room temperature (100 V / 40 min then 150 V / 1.30 hour). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare

Incubation of modified DNA with tumor sup19 pressor protein p53 for F NMR studies. Modified TAP DNA_1C was prepared by PEX (300 μl) containing KOD XL DNA polymerase (0.5 U), 5´-FAM labelled A 30_1C primer prim (3.3 μM), template (temp ) (3.3 μM), dNTPs (either natural or modified, 264 μM) in KOD XL reaction buffer supplied by the manufacturer. The reaction mixture was incubated for 30 min at 60 °C in a thermal cycler. The products were purified on QIAquick Nucleotide Removal Kit (QIAGEN) and eluted with water. The reaction mixtures for GSTp53_CD protein binding (400 μl) were prepared from purified PEX (50 μl, 211 ng/μl), KCl (500 mM, 17 μl), DTT (2 mM, 17 μl), VP buffer (50 mM Tris, 0.1% Triton-X100, pH 7.6, 17 μl) and GSTp53CD stock solution (700 ng/μl in 25 mM Hepes pH 7.6, 200 mM KCl, 10% glycerol, 1 mM DTT, 1 mM benzamidine; 220 μl ). Control sample was prepared analogously without GSTp53CD. All samples were incubated for 30 min on ice, glycerol was added (80%, 2 μl) and a part of the reaction mixture (3.5 μl) was separated by use of a 5% EMSA (acrylamide/bisacrylamide 37.5:1; 0.5×TBE, 4 °C, 80 V / 1.5 19 hour). The rest in vials was measured by F NMR spectroscopy. Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Fig19 ure S10 A in SI), for the F NMR spectra see Copies of NMR spectra 13-14 in SI. After the measurement protein was denatured for 1 hour at 55 °C, and sample was 19 measured again by F NMR (Figure S11-S12 in the SI). 19

Incubation of modified DNA with BSA for F TAP NMR studies. Modified DNA_1C was prepared in the same way as for p53. Purified PEX product (50 μl, 199 ng/μl) was incubated with bovine serum albumin (BSA 20 mg/ml, 100 μl ) and KCl (500 mM, 17 μl), DTT (2 mM, 17 μl), VP buffer (50 mM Tris, 0.1% Triton-X100, pH 7.6, 17 μl) Control sample was prepared analogously without BSA. All samples were incubated for 30 min on ice, glycerol was added (80%, 2 μl) and a part of the reaction mixture (3.5 μl) was separated by use of a 5% EMSA (acrylamide/bisacrylamide 37.5:1; 0.5×TBE, 4 °C, 19 80 V / 1.5 hour). The rest in vials was measured by F NMR spectroscopy Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE 19 Healthcare (Figure S10 B in the SI), for the F NMR spectra see Copies of NMR spectra 15 in SI Single strand DNA annealing. The subsequent hyTAP bridization between the dU modified ssDNA TAP 31_1U (31ON_U ) and temp was performed in H2O at a TAP molar ratio of 1:1 to yield a final DNA_1U in concentration of 1.1 nM, and carried out by heating the mixture at 95 °C and slowly cooling it down to 25 °C for 95 minutes. The resulting duplex was analysed by gel 19 electrophoresis and F NMR measurement (Figure S12 in the SI). 19

F NMR Measurements of biomolecules

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TAP

TAP

F NMR spectra of 31ON_U , DNA_1U , TAP TAP DNA_1C , DNA_1C in complex with GSTp53CD or BSA were measured on 500MHz NMR spectrometer equipped with 5 mm BBO H&F CryoProbe in 20 mM phosphate buffer in H2O at 5 °C using acetone-d6 and C6F6 in 1 mm coaxial capillary for external lock and reference signal (-163 ppm), respectively. Data were acquired using acquisition time 0.5 s, relaxation delay 1 s and 30 000 scans.

ASSOCIATED CONTENT Supporting Information. Contains lists of sequences, additional gels and copies of MALDI and NMR spectra.

AUTHOR INFORMATION Corresponding Author * [email protected]

ACKNOWLEDGMENT The work was supported by the Czech Academy of Sciences (RVO: 61388963 and Praemium Academiae to M. H.), Czech Science Foundation (P206-12-G151). The authors thank Dr. Marie Brazdova (IBP Brno) for expression of p53 and its mutants.

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Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-Protein Interactions by 19F NMR Spectroscopy.

A series of 7-[4-(trifluoroacetyl)phenyl]-7-deazaadenine and -7-deazaguanine as well as 5-substituted uracil and cytosine 2'-deoxyribonucleosides and ...
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