Article Cite This: J. Org. Chem. 2017, 82, 11431-11439
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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*,†,‡ †
Institute of Organic Chemistry and Biochemistry of the 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 S Supporting Information *
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 were synthesized through aqueous Suzuki-Miyaura crosscoupling of halogenated nucleosides or nucleotides with 4-(trifluoroacetyl)phenylboronic acid. 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. biomolecules.14 Many types of fluorine-containing nucleotides have been developed15−17 and used for construction of ON or DNA probes which were mostly used for sensing DNA hybridization or DNA secondary structures by 19F NMR.15−17 However, there were only two examples of the use 19F NMR for detection of DNA−protein interactions: study of flipping of 5-fluorocytosine by HhaI methyltransferase18 and study of fluorine-labeled DNA polymerase in complex with DNA.19 Trifluoromethylketones are highly reactive toward nucleophilic addition20 and were repeatedly reported to specifically react with serine.21,22 Several inhibitors of serine proteases21 or reactive probes for other serine-containing proteins22 based on this reactive group were developed. Taking into account the desired serine-reactivity and the presence of 3 equivalent fluorine atoms (which increase the signal in 19F 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.
B
ase-modified DNA and oligo-2′-deoxyribonucleotides (ONs) find diverse applications in bioanalysis or chemical biology.1 Apart from classical chemical synthesis on solidsupport, they can efficiently be prepared by polymerase incorporation of modified nucleotides.2 5-Substituted pyrimidine or 7-substituted 7-deazapurine 2′-deoxyribonucleoside 5′O-triphosphates are usually good substrates for polymerases,3,4 in some cases even better than natural dNTPs.5 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 cross-linking with proteins. Recently we reported several environment-sensitive fluorophores which changed the color6 or lifetime7 on interactions of the DNA probes with proteins, as well as reactive modifications, i.e., vinylsulfonamide8 or chloroacetamide,9 which formed covalent cross-links with cysteine and or histidine. Others have also reported cross-linking of DNA modifications including diazirines for photo-cross-linking,10 thiols for disulfide formation,11 or (oxo)aldehydes12 or phenylselenyloxy-alkene electrophiles13 for cross-linking to lysine. So far, no DNA-reactive group specific for serine has been reported. Due to the absence of fluorine in most biomolecules, specific fluorine-labeling and 19F NMR is a powerful and sensitive approach for studying secondary structures or interactions of © 2017 American Chemical Society
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RESULTS AND DISCUSSION Synthesis. The synthesis of the desired 2,2,2-trifluoroacetophenone- (TAP-) modified nucleosides, nucleoside monoReceived: July 31, 2017 Published: October 9, 2017 11431
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
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The Journal of Organic Chemistry and triphosphates was achieved through the direct attachment of the aryl group by aqueous Suzuki-Miyaura cross-coupling reactions.23 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
also successfully arylated to furnish the dNTAPTPs in good yields. The NMR analysis of all TAP-modified nucleosides dNTAPs and nucleotides dNTAPMPs and dNTAPTPs 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 1. Synthesis of TAP-Modified Nucleosides and Nucleotidesa
Scheme 2. Equilibrium between the Ketone and Hydrate in Aqueous Solution
Incorporation of the TAP-Modified Nucleotides by DNA Polymerases. We tested the modified dNTAPTPs as building blocks for the enzymatic synthesis of the TAPmodified 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 (19ON_1XTAP) or four modifications into a 31-nt strand (31ON_4X TAP ). All modified nucleoside triphosphates dNTAPTPs 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−
a
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
product
yield (%)
1 2 3 4 5 6 7 8 9 10 11 12
dCI dUI dAI dGI dCIMP dUIMP dAIMP dGIMP dCITP dUITP dAITP dGITP
dCTAP dUTAP dATAP dGTAP dCTAPMP dUTAPMP dATAPMP dGTAPMP dCTAPTP dUTAPTP dATAPTP dGTAPTP
85 70 62 45 88 75 56 26 74 58 52 37
Figure 1. Primer extension using KOD XL polymerase. (A) 31 mer template (temp31). 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 4 (CTAP): dCTAPTP, dTTP, dGTP, dATP; lane 6 (UTAP): dUTAPTP, dATP, dGTP, dCTP; lane 8 (ATAP): dATAPTP, dTTP, dGTP, dCTP; lane 10 (GTAP): dGTAPTP, dTTP, dCTP, dATP. B) 19 mer template temp19X. 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, dTTP, dATP or dGTP; lane 4 (CTAP): dCTAPTP, dTTP, dGTP, dATP; lane 7 (UTAP): dUTAPTP, dATP, dGTP, dCTP; lane 10 (ATAP): dATAPTP, dTTP, dGTP, dCTP; lane 14 (GTAP): dGTAPTP, dTTP, dCTP, dATP.
1) gave the desired TAP-substituted nucleosides (dCTAP, dUTAP, or dATAP) in good yields of 62−85%, whereas the 7deazaguanine derivative dGTAP was obtained in moderate 45% yield, probably due to the limited stability of 7-iodo-7deazaguanine. Similarly, the iodinated nucleosides monophosphates dNIMPs reacted with the same boronate under the same conditions to give the TAP-substituted nucleotides dNTAPMPs in yields comparable to nucleosides. The most difficult (hydrolytically unstable) triphosphates dNITPs were 11432
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
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The Journal of Organic Chemistry
Unfortunately we did not observe any formation of the desired cross-linked product by TLC, HPLC, or mass spectrometry (Scheme S1 in the SI). As a result of our previously published data,9 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 dCTAP, dATAP, dUTAP, or dGTAP in the consensus sequence for binding of the p53 protein. The DNA−protein interactions were studied using the GST-tagged core domain of a tumor suppressor p53 (GSTp53CD) mutant, we prepared mutant of the GSTp53CD with C277 replaced by serine.25,26 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 incubation of the modified DNA with the p53 mutant (see Figure 3) for 2 h at r.t. and monitored by denaturing SDS
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. Table 2. MALDI Data of TAP-Modified Oligonucleotides ssDNA
M (calcd.) (Da)
19ON_1CTAP 19ON_1ATAP 19ON_1UTAP 19ON_1GTAP 30ON_1CTAP 31ON_1UTAP
6124.0 6150.0 6125.3 6090.2 9377.2 9957.5
M (found) (Da) 6127.0 6151.4 6127.8 6091.0 9378.7 9958.0
[M+3H]− [M+1H]− [M+2H]− [M+1H]− [M+1H]− [M+1H]−
In order to prepare long double-stranded DNA (dsDNA) containing a high density of modifications on both strands, we tested the modified dNTAPTPs in polymerase chain reaction (PCR). PCR amplification was assayed using three different thermostable DNA polymeases, KOD XL, Vent (exo-), or Pwo, using 98-mer 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 products using dATAPTP, dCTAPTP, or dUTAPTP as substrates in the presence of the other three nonmodified dNTPs with both templates (Figure 2). In the case of dGTAPTP, we used 5′-6-FAM-labeled primers
Figure 3. Attempted unsuccessful cross-linking of TAP-modified DNA with GST p53CD_C277S,.
PAGE (see Figure S9 B in the SI). Unfortunately, we did not observe formation of the covalent cross-link. 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 F NMR Spectroscopy Study of TAP-Modified DNA and Its Interactions with Proteins. To test the applicability of the TAP group in 19F NMR spectroscopy of nucleic acids, we first focused on the detection of changes in secondary structure. Model hairpin-forming ON which contained the dUTAP modification in the loop of the hairpin (31ON_1UTAP) was synthesized by PEX using a biotinylated template followed by magnetoseparation of the modified template strand. The 19F NMR chemical shift of the TAP group (−84.45 ppm) in the hairpin ON was almost the same as the shift of the TAPmodified triphosphate dUTAPTP (−84.18 ppm) in water. Subsequently the hairpin was treated with the complementary ON (temp31_1U) 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 aminobenzoxazole-modified DNA.15 The resulting duplex was analyzed by 19F NMR. The 19F 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
Figure 2. Agarose gel analysis of PCR products amplified by KOD XL DNA polymerases. (A) 98 mer (B) 339 mer; Lane 1 (L): ladder; lanes 2, 5, 8 (+): natural dNTPs; lanes 3, 6, 9 (C-/A-/T-/G-): negative controls without dCTP,dTTP or dATP; Lane 4 (CTAP): dCTAPTP, dTTP, dGTP, dATP; lane 7 (ATAP): dATAPTP, dCTP, dGTP, dTTP; lane 10 (UTAP): dUTAPTP, dATP, dTTP, dGTP; 1.3% (2%) agarose gel for 339-mer (98-mer) stained with GelRed.
and fluorescence detection to visualize the gels, because 7deazaguanosine bases are known to quench fluorescence of DNA staining intercalators.24 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 form hemiacetals.21,22 This reaction is inherently reversible and the stability of the products varies depending on many factors. First, we tested the possible cross-linking on model reactions of TAP-linked nucleoside monophosphate (dCTAPMP) with N-acetylserine (1.5−50 equiv) in triethylammonium acetate (TEAA) buffer (pH 8.4) or in acetone in ratio 2:1 with 0.1 M NaOH. 11433
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
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The Journal of Organic Chemistry
ketone and hydrate form of TAP in the complex with protein. In a negative control experiment, the incubation of the DNA_1CTAP with bovine serum albumin (BSA) did not show any changes in 19F chemical shift (NMR spectra S15).
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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 SuzukiMiyaura cross-coupling reaction. All of the modified triphosphates were found to be good substrates for various DNA polymerases and were efficiently incorporated into ssONs and dsDNA by PEX. dCTAPTP, dATAPTP, and dUTAPTP were found to be a good substrates for KOD XL polymerase in PCR, whereas dGTAPTP 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 TAPmodified DNA with serine in the protein. On the other hand, the TAP group shows potential as a label for 19F NMR spectroscopy. The sensitivity to changes of secondary structure was rather weak, however, it showed a pronounced sensitivity to DNA−protein interactions.
Figure 4. 19F NMR spectra of dUTAP-modified ssDNA (31ON_UTAP) and DNA_1UTAP (duplex). Conditions: 1.1 nM of oligonucleotide, in 20 mM phosphate buffer at pH = 7.
secondary structural changes than the previously reported fluorinated aminobenzoxazole,15 which showed a change of 9.04 ppm in a similar hybridization experiment. We next endeavored to exploit the potential of the TAP-label for 19 F NMR spectroscopic analysis of DNA−protein interactions, which has been underexplored in the past.18,19 Double stranded DNA bearing one modified dCTAP in the p53 consensus sequence was prepared by PEX. Our initial 19F NMR experiment, DNA_1CTAP 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). We observed a 19F chemical shift of dCTAPmodified DNA at −83.20 ppm (Figure 5a). Then, the
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EXPERIMENTAL SECTION
NMR spectra were recorded on a 600 MHz (600.1 MHz for 1H, 150.9 MHz for 13C) or a 500 (500.0 MHz for 1H, 470.4 MHz for 19F, 202.3 MHz for 31P, 125.7 MHz for 13C) spectrometers from sample solutions in D2O, or CD3OD. Chemical shifts (in ppm, δ scale) were referenced as follows: D2O (referenced to dioxane as internal standard: 3.75 ppm for 1H NMR and 69.30 ppm 13C NMR); CD3OD (referenced to solvent signal: 3.31 ppm for 1H NMR and 49.00 ppm for 13C NMR); 31P 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. 2,2,2Trifluoroacetophenone-4-boronic acid pinacol ester, palladium(II) acetate, tris(3-sulfonatophenyl)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 Nucleosides (dNTAP). General procedure I: Nucleoside analogue (dNI) (1 equiv), 2,2,2-trifluoroacetophenone-4-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. 5-[4-(Trifluoroacetyl)phenyl]-2′-deoxycytidine (dCTAP). Prepared according to general procedure I, from dCI (50 mg, 0.14 mmol), 2,2,2-trifluoroacetophenone-4-boronic 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. dCTAP was isolated as a yellow powder (48 mg, 85%).1H NMR (500.0 MHz, CD3OD): 2.22, 2.24 (2 × ddd, 2 × 1H, Jgem = 13.6, J2′b,1′ = 6.3, J2′b,3′ = 2.8, H-2′b); 2.40 (ddd, 2H, Jgem = 13.6, J2′a,1′ = 6.3, J2′a,3′ = 4.2, H-2′a); 3.70 (dd, 2H, Jgem = 12.0, J5′b,4′ = 3.5, H-5′b); 3.792, 3.795 (2 × dd, 2 × 1H, Jgem = 12.0, J5′a,4′ = 3.0, H-5′a); 3.93 (ddd, 2H, J4′,5′ = 3.5, 3.0, J4′,3′ = 4.0, H-4′); 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-o-phenylene); 7.69−7.73 (m, 4H, H-m-phenylene); 8.128, 8.131 (2 × s, 2 × 1H, H-6).13C NMR (150.9 MHz, CD3OD): 42.3
Figure 5. 19F NMR shifts of (a) DNA_1CTAP, (b) DNA_1CTAP in the 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.
DNA_1CTAP 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 another 19F 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 55 °C for 1 h and the 19F 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 11434
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
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The Journal of Organic Chemistry
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 Nucleoside Monophosphates (dNTAPMP). General procedure II: Nucleoside monophosphate analogue (dNIMP) (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 reversedphase 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 desired dNTAPMPs as white solids. 5-[4-(Trifluoroacetyl)phenyl]-2′-deoxycytidine 5′-O-phosphate (dCTAPMP). Prepared according to general procedure II, from dCIMP (50 mg, 0.109 mmol), 2,2,2-trifluoroacetophenone-4boronic 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 buffer as a eluent, product dCTAPMP was isolated as a yellow powder (49 mg, 88%).1H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.35 (ddd, 1H, Jgem = 14.0, J2′b,1′ = 7.7, J2′b,3′ = 6.4, H-2′b); 2.44 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 6.2, J2′a,3′ = 3.4, H-2′a); 3.81−3.93 (m, 2H, H-5′); 4.15 (td, 1H, J4′,5′ = 5.1, J4′,3′ = 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 (s, 1H, H-6); 7.80 (m, 2H, H-m-phenylene). 13C 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-ophenylene); 136.7 (C-i-phenylene); 139.4 (C-p-phenylene); 142.7 (CH-6); 159.8 (C-2); 167.2 (C-4). 31P{1H} NMR (202.3 MHz, D2O): 3.46. 19F 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. 5-[4-(Trifluoroacetyl)phenyl]-2′-deoxyuridine 5′-O-phosphate (dUTAPMP). Prepared according to general procedure II, from dUIMP (50 mg, 0.109 mmol), 2,2,2-trifluoroacetophenone-4boronic 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 buffer as a eluent, product dUTAPMP was isolated as a yellow powder (43 mg, 75%)1H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.40 (ddd, 1H, Jgem = 14.1, J2′b,1′ = 6.4, J2′b,3′ = 3.4, H-2′b); 2.46 (ddd, 1H, Jgem = 14.1, J2′a,1′ = 7.7, J2′a,3′ = 6.4, H-2′a); 3.83−3.95 (m, 2H, H-5′); 4.15 (td, 1H, J4′,5′ = 5.1, J4′,3′ = 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, 2H, H-m-phenylene); 7.86 (s, 1H, H-6). 13C 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 (CH-m-phenylene); 131.5 (CH-ophenylene); 136.5 (C-i-phenylene); 138.9 (C-p-phenylene); 141.9 (CH-6); 154.5 (C-2); 168.0 (C-4). 31P{1H} NMR (202.3 MHz, D2O): 3.83. 19F 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-7-deazaadenosine 5′-O-phosphate (dATAPMP). Prepared according to general procedure II, from dAIMP (50 mg, 0.104 mmol), 2,2,2-trifluoroacetophenone-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 buffer as a eluent, product dATAPMP was isolated as a yellow powder (31 mg, 56%). 1H 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,
(CH2−2′); 62.3 (CH2−5′); 71.6 (CH-3′); 87.7 (CH-1′); 88.9 (CH4′); 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-o-phenylene); 130.4 (CH-m-phenylene); 135.6 (C-i-phenylene); 137.5 (C-p-phenylene); 141.9 (CH-6); 157.7 (C-2); 165.5 (C-4); 203.1 (COCF3, from HMBC).19F 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 (dUTAP). Prepared according to general procedure I, from dUI (50 mg, 0.14 mmol), 2,2,2-trifluoroacetophenone-4-boronic 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. dUTAP was isolated as a yellow powder (39 mg, 70%)1H NMR (500.0 MHz, CD3OD): 2.30−2.39 (m, 4H, H-2′); 3.75 (dd, 2H, Jgem = 12.0, J5′b,4′ = 3.0, H-5′b); 3.830, 3.833 (2 × dd, 2 × 1H, Jgem = 12.0, J5′a,4′ = 3.0, H-5′a); 3.96 (q, 2H, J4′,5′ = J4′,3′ = 3.0, H-4′); 4.43−4.47 (m, 2H, H-3′); 6.36 (t, 2H, J1′,2′ = 6.5, H1′); 7.59−7.65 (m, 8H, H-o,m-phenylene); 8.36 (s, 2H, H-6). 13C 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-m-phenylene); 135.2 (C-p-phenylene); 135.6 (C-i-phenylene); 140.2 (CH-6); 151.9 (C-2); 164.6 (C-4). 19F 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-7-deazaadenosine (dATAP). Prepared according to general procedure I, from dAI (50 mg, 0.13 mmol), 2,2,2-trifluoroacetophenone-4-boronic 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. dATAP was isolated as a yellow powder (35 mg, 62%) 1H 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, J5′b,4′ = 3.7, H-5′b); 3.81 (dd, 1H, Jgem = 12.1, J5′a,4′ = 3.3, H-5′b); 4.03 (m, 1H, H-4′); 4.55 (dt, 1H, J3′,2′ = 6.0, 2.7, J3′,4′ = 2.7, H3′); 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); 7.72 (m, 2H, H-m-phenylene); 8.14 (s, 1H, H2). 13C NMR (125.7 MHz, CD3OD): 41.5 (CH2−2′); 63.6 (CH2− 5′); 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-i-phenylene); 151.2 (C-7a); 152.3 (CH-2); 158.9 (C-4). 19F 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-7-deazaguanosine (dGTAP). Prepared according to general procedure I, from dGI (50 mg, 0.127 mmol), 2,2,2-trifluoroacetophenone-4-boronic 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. dGTAP was isolated as a yellow powder (25 mg, 45%) 1H 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, J5′b,4′ = 4.1, H-5′b); 3.79 (dd, 1H, Jgem = 12.0, J5′a,4′ = 3.7, H-5′b); 3.96 (ddd, 1H, J4′,5′ = 4.1, 3.7, J4′,3′ = 2.9, H-4′); 4.51 (dt, 1H, J3′,2′ = 6.2, 2.9, J3′,4′ = 2.9, H-3′); 6.47 (dd, 1H, J1′,2′ = 8.0, 6.0, H-1′); 7.25 (s, 1H, H6); 7.54 (m, 2H, H-m-phenylene); 7.87 (m, 2H, H-o-phenylene). 13C 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-m-phenylene); 133.3 (C-pphenylene); 136.9 (C-i-phenylene); 153.6 (C-7a); 154.0 (C-2); 161.8 (C-4). 19F NMR (470.4 MHz, CD3OD): −80.58. MS (ESI−): 11435
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
Article
The Journal of Organic Chemistry
−22.56 (t, J = 19.6, Pβ); −11.57 (d, J = 19.6, Pα); −8.46 (bd, J = 19.6, Pγ). 19F 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′-O-triphosphate (dUTAPTP). Prepared according to general procedure III, from dUITP (50 mg, 0.076 mmol), 2,2,2-trifluoroacetophenone-4boronic 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 TEAB buffer as a eluent, product dUTAPTP was isolated as a yellow powder (31 mg, 58%). 1H 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-m-phenylene); 7.78 (s, 1H, H-6). 13C 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-m-phenylene); 131.4 (CH-ophenylene); 138.0 (C-i-phenylene); 140.1 (C-p-phenylene); 141.0 (CH-6); 159.7 (C-2); 174.4 (C-4). 31P{1H} NMR (202.3 MHz, D2O): −21.57 (dd, J = 19.9, 19.1, Pβ); −11.04 (d, J = 19.1, Pα); −5.71 (d, J = 19.9, Pγ). 19F NMR (470.4 MHz, D2O): −84.17. MS (ESI−): m/z (%) 559.0 (100) [M-PO3−+2H]. HRMS (ESI-IT) m/z: [M+3H]− calcd for C17H17F3N3O15P3− 638.9792; found 638.9799. 5-[4-(Trifluoroacetyl)phenyl]-2′-deoxy-7-deazaadenosine 5′-O-triphosphate (dATAPTP). Prepared according to general procedure III, from dA I 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), TPPTS (3.3 mg, 5.87 μmol), and Pd(OAc)2 (0.66 mg, 2.93 μmol). After purification by C18 reversephase HPLC with water/methanol (15−100%) containing 0.1 M TEAB buffer as a eluent, product dATAPTP was isolated as a yellow powder (31 mg, 52%).1H 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, H-4′); 4.77 (m, 1H, H-3′); 6.65 (dd, 1H, J1′,2′ = 7.8, 6.5, H-1′); 7.54 (s, 1H, H-6); 7.57 (m, 2H, H-o-phenylene); 7.78 (m, 2H, H-m-phenylene); 8.15 (s, 1H, H-2). 13C 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 (Ci,p-phenylene); 152.7 (C-7a); 154.1 (CH-2); 159.9 (C-4). 31P{1H} NMR (202.3 MHz, D2O): −21.86 (bs, Pβ); −10.97 (d, J = 18.6, Pα); −6.43 (bd, J = 17.4, Pγ).19F 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-7-deazaguanosine 5′-O-triphosphate (dGTAPTP). Prepared according to general procedure III, from dGITP (50 mg, 0.071 mmol), 2,2,2-trifluoroacetophenone-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 buffer as a eluent, product dGTAPTP was isolated as a yellow powder (18 mg, 37%).1H 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, H5′); 4.20 (m, 1H, H-4′); 4.71 (dt, 1H, J3′,2′ = 6.3, 3.2, J3′,4′ = 3.2, H-3′); 6.38 (dd, 1H, J1′,2′ = 7.9, 6.4, H-1′); 7.24 (s, 1H, H-6); 7.68 (m, 2H, Hm-phenylene); 7.78 (m, 2H, H-o-phenylene). 13C 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-m-phenylene); 130.7 (CH-ophenylene); 137.1 (C-p-phenylene); 137.8 (C-i-phenylene); 154.8 (C-
1H, Jgem = 14.0, J2′a,1′ = 8.2, J2′a,3′ = 6.3, H-2′a); 3.75 (t, 2H, JH,P = J5′,4′ = 5.5, H-5′); 4.11 (td, 1H, J4′,5′ = 5.5, J4′,3′ = 3.1, H-4′); 4.61 (dt, 1H, J3′,2′ = 6.3, 3.1, J3′,4′ = 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-mphenylene); 8.03 (s, 1H, H-2). 13C 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, CH4′); 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 (C-7a); 153.9 (CH-2); 159.6 (C-4). 31P{1H} NMR (202.3 MHz, D2O): 3.77.19F NMR (377.3 MHz, D2O): −84.14.MS (ESI−): m/z (%) 501.1 (100) [M], 523.1 (20) [M+Na]. HRMS (ESI-IT) m/z: [M]− calcd for C19H17F3N4O7P− 501.0793; found 501.0792. 5-[4-(Trifluoroacetyl)phenyl]-2′-deoxy-7-deazaguanosine5′-O-phosphate (dGTAPMP). Prepared according to general procedure II, from dGIMP (50 mg, 0.101 mmol), 2,2,2-trifluoroacetophenone-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 reversephase HPLC with water/methanol (15−100%) containing 0.1 M TEAB buffer as a eluent, product dCTAPMP was isolated as a yellow powder (14 mg, 26%).1H 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, J5′,4′ = 5.4, H-5′); 4.15 (td, 1H, J4′,5′ = 5.4, J4′,3′ = 3.0, H-4′); 4.66 (dt, 1H, J3′,2′ = 6.3, 3.0, J3′,4′ = 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, 2H, H-mphenylene); 7.79 (m, 2H, H-o-phenylene). 13C 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, CH4′); 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-m-phenylene); 130.9 (CH-o-phenylene); 137.4 (C-p-phenylene); 137.8 (C-i-phenylene); 155.0 (C-7a); 155.7 (C-2); 163.8 (C-4). 31P{1H} NMR (202.3 MHz, D2O): 3.90. 19F 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 Nucleoside Triphosphates (dNTAPTP). General procedure III: Nucleoside triphosphate analogue (dNITP) (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 reversedphase 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 desired dNTAPTPs as white solids. 5-[4-(Trifluoroacetyl)phenyl]-2′-deoxycytidine 5′-O-triphosphate (dCTAPTP). Prepared according to general procedure III, from dCITP (50 mg, 0.076 mmol), 2,2,2-trifluoroacetophenone-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 TEAB buffer as a eluent, product dCTAPTP was isolated as a yellow powder (40 mg, 74%).1H NMR (500.0 MHz, D2O, ref(external dioxane) = 3.75 ppm): 2.36 (ddd, 1H, Jgem = 14.0, J2′b,1′ = 7.4, J2′b,3′ = 6.4, H-2′b); 2.45 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 6.3, J2′a,3′ = 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-ophenylene); 7.80 (m, 2H, H-m-phenylene); 7.84 (s, 1H, H-6). 13C 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-ophenylene); 136.7 (C-i-phenylene); 139.3 (C-p-phenylene); 142.8 (CH-6); 159.8 (C-2); 167.2 (C-4). 31P{1H} NMR (202.3 MHz, D2O): 11436
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
Article
The Journal of Organic Chemistry 7a); 155.4 (C-2); 163.5 (C-4). 31P{1H} NMR (202.3 MHz, D2O): −21.73 (t, J = 19.1, Pβ); −10.90 (d, J = 19.1, Pα); −6.27 (d, J = 19.1, Pγ).19F 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. Biochemistry General Remarks. The MALDI-TOF 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, QIAquick Nucleotide Removal Kit from Qiagen. All solutions were prepared in Milli-Q water. Other chemicals were of analytical grade. Incorporation of dNTAPTP into 31-mer Template by PEX. The reaction mixture (20 μL) contained primer (primB) (0.5 μM), template (temp31) (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 labeled 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/bis(acrylamide) 19:1, 25% urea) under denaturing conditions (TBE 1 × , 42 mA, 1 h). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S1−S2 in the SI). Incorporation of dNTAPTP into 19-mer Template by PEX. PEX reactions with 19-mer template were performed in the same way as described above. The reaction mixture (20 μL) contained primer (primB) (0.2 μM), template (temp19_X) (0.3 μM), DNA polymerase (0.05 U KOD XL, dGTP/or dTTP in the case of temp19_1G (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 labeled 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/bis(acrylamide) 19:1, 25% urea) under denaturing conditions (TBE 1 × , 42 mA, 1 h). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S1−S2 in the SI). Incorporation of dUTAPTP into the temp31_1U by PEX. The reaction mixture (20 μL) contained primer (primC) (0.5 μM), template (temp31_1U) (0.5 μM), DNA polymerase (0.05 U 10× 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 labeled 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/ bis(acrylamide) 19:1, 25% urea) under denaturing conditions (TBE 1 × , 42 mA, 1 h). 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. Thirty 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 CTAP/ATAP and 100 μM for UTAP/GTAP), primers (2 μM, primFOR and 2 μM, primREV, 5′-FAM labeled primers were used in the case of GTAP Figure S7) and template tempPveg (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), functionalized dNTPs (400 μM for CTAP/ATAP and 1 mM for UTAP/GTAP), primers (2 μM, primFOR and 2 μM, primREV) and template tempPveg (0.255 μM) in Pwo reaction buffer (1 μL) supplied by the manufacturer. 98-mer. Thirty 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), functionalized dNTPs (200 μM for CTAP/ ATAP, and 500 μM for UTAP/GTAP), primers (2 μM, primFOR‑L20, and 2 μM, primREV‑LT25TH 5′-FAM labeled primers were used in the case of GTAP Figure S7) and template tempFVL‑A (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 dNTPs (400 μM for CTAP/ATAP, and 800 μM for UTAP/GTAP), primers (1 μM, primFOR‑L20, and 1 μM, primREV‑LT25TH) and template tempFVL‑A (0.25 μM) in PWO reaction buffer (2 μL) supplied by the manufacturer. MALDI-TOF Analysis of TAP-Modified Oligonucleotides (19ON_1CTAP, 19ON_1ATAP, 19ON_1UTAP, 19ON_1GTAP, and 30ON_1CTAP 31ON_1UTAP). The PEX solution (50 μL) contained KOD XL DNA polymerase (0.5 U), primer (primB for temp19X, primeA for temp30_1C, and primeC for temp31_1U) (4 μM), 5′biotinylated template (temp19X, temp30_1C or temp31_1U) (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, 10 mM 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 (the results are summarized in Table 2, for copies of mass spectra see Figures S13−S18 in the SI). 11437
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
Article
The Journal of Organic Chemistry Preparation of Modified ssDNA (31ON_UTAP) for 19F NMR Studies. The sample was prepared as described above from the PEX solution (200 μL) and was then measured by 19F 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. 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 Suppressor Protein p53 Mutants. Modified DNA_2CTAP 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/bis(acrylamide) 37.5:1; 0.5 × TBE, 4 °C, 80 V/1 h). The rest in vials was incubated for 2 h 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 min. 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 h). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S8 in the SI). Incorporation of dN TAP TP into p53 Consensus DNA Sequence by PEX. For dCTAPTP: The reaction mixture (72 μL) contained primer primA (0.17 μM), temp30_2C (0.17 μM), DNA polymerase (0.05 U 10× 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. For dATAPTP: The reaction mixture (65 μL) contained primer primA (0.19 μM), temp30_2A (0.14 μM), DNA polymerase (0.1 U KOD XL 10× diluted KOD XL), 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. For dUTAPTP: The reaction mixture (65 μL) contained primer primA (0.19 μM), temp30_2T (0.19 μM), DNA polymerase (0.07 U KOD XL 10× 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. For dGTAPTP: The reaction mixture (65 μL) contained primer primA (0.19 μM), temp30_2G (0.19 μM), DNA polymerase (0.07 U KOD XL 10× 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 labeled on its 5′-end by 6-carboxyfluorescein (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/bis(acrylamide) 19:1, 25% urea) under denaturing conditions (TBE 1 × , 42 mA, 1 h). 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. Modified DNA_1XTAP 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 25 mM 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/ bis(acrylamide) 37.5:1; 0.5 × TBE, 4 °C, 80 V/1 h). The rest in vials was incubated for 2 h 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 min. 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 h). Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare Incubation of Modified DNA with Tumor Suppressor Protein p53 for 19F NMR Studies. Modified DNA_1CTAP was prepared by PEX (300 μL) containing KOD XL DNA polymerase (0.5 U), 5′-FAM labeled primer primA (3.3 μM), template (temp30_1C) (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/ bis(acrylamide) 37.5:1; 0.5 × TBE, 4 °C, 80 V/1.5 h). The rest in vials was measured by 19F NMR spectroscopy. Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S10 A in SI), for the 19F NMR spectra see Copies of NMR spectra 13−14 in SI. After the measurement protein was denatured for 1 h at 55 °C, and sample was measured again by 19F NMR (Figure S11−S12 in the SI). Incubation of Modified DNA with BSA for 19F NMR Studies. Modified DNA_1CTAP 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/bis(acrylamide) 37.5:1; 0.5 × TBE, 4 °C, 80 V/1.5 h). The rest in vials was measured by 19F NMR spectroscopy. Visualization was performed by fluorescence imaging using Typhoon FLA 9500, GE Healthcare (Figure S10 B in the SI), for the 19F NMR spectra see Copies of NMR spectra 15 in SI Single Strand DNA Annealing. The subsequent hybridization between the dUTAP modified ssDNA (31ON_UTAP) and temp31_1U was performed in H2O at a molar ratio of 1:1 to yield a final DNA_1UTAP 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 min. The resulting duplex was analyzed by gel electrophoresis and 19F NMR measurement (Figure S12 in the SI). 19 F NMR Measurements of Biomolecules. 19F NMR spectra of 31ON_UTAP, DNA_1UTAP, DNA_1CTAP, DNA_1CTAP in complex with GSTp53CD or BSA were measured on 500 MHz 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. 11438
DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
Article
The Journal of Organic Chemistry
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b01920. Contains lists of sequences, additional gels, and copies of MALDI and NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Michal Hocek: 0000-0002-1113-2047 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS 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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DOI: 10.1021/acs.joc.7b01920 J. Org. Chem. 2017, 82, 11431−11439
