Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis- (difluoromethylene)triphosphoric

G. K. Surya Prakasha,1, Mikhail Zibinskya, Thomas G. Uptona, Boris A. Kashemirova, Charles E. McKennaa, Keriann Oertella, Myron F. Goodmana, Vinod K. Batrab, Lars C. Pedersenb, William A. Beardb, David D. Shockb, Samuel H. Wilsonb, and George A. Olaha,1

aLoker Hydrocarbon Research Institute, Department of Chemistry and Department of Biology, University of Southern California, 837 Bloom Walk, Los Angeles, CA 90089-1661; and bLaboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

Contributed by George A. Olah, June 3, 2010 (sent for review April 28, 2010)

It is difficult to overestimate the importance of nucleoside tri- the altered chemical properties conferred on the compound by in cellular chemistry: They are the building blocks the fluorine substituent. The van der Waals’ radius of the fluorine for DNA and RNA and important sources of energy. Modifications atom (1.47 Å) is close to the size of (1.2 Å). Most of the of biologically important organic molecules with fluorine are of other substituent groups often used to replace hydrogen in the great interest to chemists and biologists because the size and creation of analogs are much larger. Thus fluorine is of electronegativity of the fluorine atom can be used to make defined unique value in the design of analogs, which can very closely ap- structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been proach the natural biochemical intermediate. Good analogs of around for some time, the progress in the area of modified tripho- this kind can be useful therapeutically, but they can also be ex- sphates was limited by the lack of synthetic methods allowing to tremely valuable in defining critical sizes that contribute to — CHEMISTRY access bisCF2-substituted nucleotide analogs one of the most structural considerations in biochemically important molecules. interesting classes of nonhydrolyzable nucleotides. These com- Further, fluorinated compounds are more hydrophobic than their pounds have “correct” polarity and the smallest possible steric hydrogen counterparts and are found to provide increase in perturbation compared to natural nucleotides. No other known bioavailability. nucleotides have these advantages, making bisCF2-substituted Recently, as a result of a comprehensive effort to prepare new analogs unique. Herein, we report a concise route for the prepara- nonhydrolyzable nucleotides, we developed a synthetic method tion of hitherto unknown highly acidic and polybasic bis(difluoro- for the preparation of the hitherto unknown bis(difluoromethy- methylene)triphosphoric acid 1 using a phosphorous(III)/phos- lene)triphosphoric acid 1 (BMF4TPA, Fig. 1), a highly acidic pen- phorous(V) interconversion approach. The analog 1 compared to “ ” triphosphoric acid is enzymatically nonhydrolyzable due to substi- tabasic acid. Because of the biostericity and similar polarity of the CF2 groups to the bridging oxygen atoms (3–5), this com- tution of two bridging oxygen atoms with CF2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the pound is a nonhydrolyzable analog of triphosphoric acid, albeit triphosphate polyanion. The fluorinated triphosphoric acid 1 was with lower pKaS (vide infra), a vital part of nucleotides containing used for the preparation of the corresponding fluorinated deoxy- both oxy- and deoxyriboses. It is important to emphasize that no nucleotides (dNTPs). One of these dNTP analogs (dT) was demon- other known (α,β),(β,γ)-bis-substituted triphosphate analog re- strated to fit into DNA polymerase beta (DNA pol β) binding pocket tains the “right” polarity and steric features of the natural tripho- by obtaining a 2.5 Å resolution crystal structure of a ternary sphate. This makes the preparation of the corresponding complex with the enzyme. Unexpected dominating effect of deoxynucleotide analogs with 1 of high scientific interest and im- triphosphate∕Mg2þ interaction over Watson–Crick hydrogen bond- portance. We report the synthesis and purification procedures for ing was found and discussed. deoxynucleotide analogs where CF2 groups are in both the (α,β)- β γ DNA polymerase beta ∣ nonhydrolyzable nucleotides ∣ fluorinated and ( , )-positions of the triphosphate group. Owing to the 1 triphosphate ∣ pentabasic acid ∣ isopolarity and bioisotericity unique properties of , these nucleotides should offer the best electronic and stereochemical mimicking of the natural deoxy- luorine is in group VII of the periodic system, and this element nucleoside triphosphates. The prepared analogs are (α,β),(β,γ)- α β β γ α β β γ Fshould be considered, according to Pauling, a “superhalogen” bisCF2 dATP, ( , ),( , )-bisCF2 dTTP, ( , ),( , )-bisCF2 dCTP, (1). Fluorine is considerably more electronegative than the other and (α,β),(β,γ)-bisCF2 dGTP. A 2.5 Å crystallographic structure halogens, and for this reason it is the only halogen that is extre- of a ternary complex of DNA pol β and (α,β),(β,γ)-bisCF2 dTTP mely unlikely to form the positive ion. The bond energy of the is also reported and discussed. C-F bond is among the highest found in natural products and is difficult to be broken enzymatically (2). Recent advances in or- ganofluorine chemistry have been responsible for the develop- Author contributions: G.K.S.P., C.E.M., M.F.G., and G.A.O. designed research; M.Z., T.G.U., ment of a large number of new compounds of importance in B.A.K., K.O., V.K.B., L.C.P., W.A.B., and D.D.S. performed research and analyzed data; G.K.S.P. and M.Z. contributed new reagents; and G.K.S.P., M.Z., K.O., V.K.B., and W.A.B. biology and medicine. The knowledge gained in the synthesis wrote the paper. of organofluorine compounds has also provided the pharmacol- The authors declare no conflict of interest. ogist with selective inhibitors of biological processes and has Data deposition: The crystal structure 10 data have been deposited in the Cambridge given the medicinal chemist the opportunity to design more active Structural Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United therapeutic agents. The use of fluoro compounds in studies of Kingdom (CSD reference no. 778743). enzyme and pharmacological mechanisms has advantages not 1To whom correspondence may be addressed: E-mail: [email protected] or [email protected]. found with many other analogs because insight into the biochemi- This article contains supporting information online at www.pnas.org/lookup/suppl/ cal phenomenon can often be gained from an understanding of doi:10.1073/pnas.1007430107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1007430107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 24, 2021 controlled by two factors: the rate of deprotonation and steric hindrance of the base. Generally, in the case of slow deprotona- tion only a small amount of (difluoromethyl) anion was available for reaction with phosphorous electrophile, while the major part of 4 simply remained in the reaction mixture unchanged. In such a case, the majority of the base reacted with phosphorous electrophile producing the phosphoramide. These observations suggested that a sterically hindered lithium base would work well in the reaction, providing relatively fast de- protonation of 4 and suppression of direct reaction of the base with phosphorous electrophile. After a brief screening of several bases, it was found that use of lithium 2,2,6,6-tetramethylpiperi- dine amide (LTMPA) as a base gave access to compound 8 in 93% yield as determined by NMR analysis. A doublet of triplet at þ6.5 ppm (2P) and a multiplet at þ54.0 ppm (1P) were observed in the 31P NMR of the crude mixture indicating the formation of 8 Fig. 1. Polarities of bridging units in tiphosphate anion and its CX2-substi- compound . The fluorine NMR spectrum showed two nonequi- tuted analogs. valent fluorine atoms as doublets of triplets at −116.8 and −118.5 ppm, respectively. Interestingly, the bulky lithium hexa- methyldisilazide (LHMDS) did not provide positive results; a Results and Discussion complex mixture was formed instead. 4 Synthesis of BMF TPA. Although the synthesis of bis(methylene)tri- In situ oxidation of compound 8 with 2.5 equivalents of meta- 3 has been known for a long time (6, 7), all at- chloroperbenzoic acid (m-CPBA) in dichloromethane followed tempts at its direct preparation from dialkyl methylphosphonate by purification on silica gave access to BMF4TPA amido-ester – have failed (8). Michaelis Arbuzov-type reactions have been 9 in 75% overall isolated yield (starting from phosphonate 4). shown to be useful in the preparation of nonfluorinated analogs Treatment of compound 9 with TMSBr (10) followed by hydro- (6, 9). Although bis(difluoromethylene)triphosphoric acid 1 has a lysis gave access to the bis(difloromethylene)phosphoric acid 1, very simple structure, it remained unknown, most likely due to which was quantitatively converted to the ammonium salt by the failure of conventional approaches mentioned above. We passing through DOWEX ion-exchange resin in the ammonium 4 have now discovered that BMF TPA can be accessed starting form. The overall yield of salt 10 was 61%. directly from diethyl (difluoromethyl)phosphonate 4 (Scheme 1) As was discussed above, the introduction of fluorine atoms into employing phosphorous (III)/phosphorous(V) interconversion a molecule has an impact on the physical and chemical properties protocol. of the molecule; therefore we intended to compare bond lengths 4 Our initial approach to the synthesis of BMF TPA began with and angles of the fully fluorinated analog to those of sodium tri- the direct reaction between 2 eq. of (diethylphosphinyl)difluoro- . X-ray structure of salt 10 (Fig. 2) revealed that the methyllithium (generated by LDA) with dichlorophosphate 5 length of the P-O bridging bond in the original triphosphate 4 (Scheme 1). This reaction did not provide BBMF TPA ester (1.61 and 1.68 Å) is expectedly shorter than the length of the 6; a complex mixture was formed instead. All attempts to prepare corresponding P-C bond (1.86–1.87 Å) in the fluorinated analog 6 with a variety of bases were unsuccessful. However, it was found of the ammonium salt. Terminal P-O bond lengths for BMF4TPA that in most of the cases, the base reacted faster with the phos- salt were approximately the same as in phorus electrophile than with phosphonate 4. This could be (around 1.5 Å) indicating excellent isostericity for the crucial

Scheme 1. Synthetic route to BMF4TPA.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1007430107 Surya Prakash et al. Downloaded by guest on September 24, 2021 known (14, 15). These methods include the Mitsunobu reaction (8, 16–18), couplings promoted by DCC and other dehydrating agents (19, 20), electrophilic phosphorylation developed by Yoshikawa, Ludwig, and Eckstein employing 2-chloro-4H-1,3, 2-benzodioxaphosphorin-4-one (21–24), nucleophilic cleavage of phosphoryl anhydride (25), reactions involving phosphorami- dates (19, 26–30), Blackburn’s method employing nucleophilic substitution at 5′ position (31–33), and enzymatic phosphoryla- tion (20, 34, 35). All of these methods lack versatility and their success depends on many factors, such as nucleophilicity/electro- philicity of the phosphate source, steric bulkiness, stability of the product under acidic or basic conditions, etc. After comprehensive screening of several known protocols for coupling nucleosides with phosphoric, phosphonic, and car- boxylic , we found that Balckburn’s method of nucleophilic ′ 4 substitution of toslylate at 5 -position with tetrabutylammonium Fig. 2. X-ray structure of BMF TPA pentaamonium salt dihydrate 10. salt 11 was the only suitable method among those screened. In the cases of deoxyadenosine, deoxythymidine, and protected deoxy- metal-binding site of the triphosphate analog. The P(2)-C(2)-P cytidine, conversions to 12 were 85%, 91%, and 88%, respec- (3) angle in BMF4TPA was only slightly different compared to tively. However, the protected deoxyguanosine 5′-tosylate re- the triphosphate: 123.8° vs. 121°. However, the P(2)-C(1)-P(1) acted with 11 very slowly, and the maximum conversion that could angle was significantly smaller: 114.7°; the C(1)-P(2)-C(2) angle be achieved was 10% (Scheme 2). Interestingly, tributylammo- was 106.8° vs. 98° in triphosphate. The O-P(1)-O and O-P(3)-O nium salt of BMF4TPA 13 showed absolutely no reactivity in angles in BMF4TPA were approximately 114°; the O-P(1)-C(1) Blackburn’s method, although similar nucleophilic substitution and O-P(3)-C(2) angles are around 102–107°, close to the value with tributylammonium salt of difluoromethylene bisphosphonic for triphosphate (106°) (11). All these data indicate that substitu- acid proceeds with high yields (31–35). This could be due to 4

tion of bridging oxygen atoms in triphosphate with CF2 groups intramolecular hydrogen bonding in BMF TPA tributylammo- CHEMISTRY has only moderate impact on the overall spatial arrangement of nium salt, which may significantly lower nucleophilicity of the triphosphate 10. anionic species. Salt 10 can be converted back to the pentabasic acid 1 and re- lated alkyl ammonium salts; the latter are soluble in organic Biological Tests. Having analogs 12 in hand, we demonstrated the solvents. We expected the lower basicity of the fluorinated analog nonhydrolyzable nature of these compounds on (α,β),(β,γ)- compared to triphosphate. Indeed, the determined at 25 °C pKa4 bisCF2 dTTP (12b) analog as a representative example. A (5.33) and pKa5 (7.23) of the new acid were lower than the cor- single-turnover gap-filling assay was performed, for correct (op- responding pKas of triphosphoric acid (5.83–6.50 and 8.73–9.24 posite template base dA) and incorrect (opposite template base respectively) (12, 13) with the potential to stretch the isoacidity dG) pairings (Fig. 3). After reacting for 1 min at concentrations of model to new levels with the corresponding dNTP analogs. dNTP well above the Kd, the hydrolyzable (β,γ)-CF2 dTTP analog (35) was incorporated to essentially 100% primer extension Synthesis of dNTP Analogs. Only a few methods for attaching low under both correct and mispairing conditions (lanes 4 and 8). nucleophilicity phosphates to the 5′-carbon of a nucleoside are However, no incorporation was observed after 10 min for either

Scheme 2. Synthesis of deoxynucleotide analogs 12 via tosylate substitution at the 5′ position:

Surya Prakash et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 24, 2021 Fig. 4. Superposition of the active site of the ternary complex of DNA pol β with incoming 12b,(α,β), (β,γ)-bisCF2 dTTP (gray), and the corresponding tern- ary complex with (α,β)-NH dUTP (cyan) (PDB ID code 2FMS).

and found to be similar to (α,β)-CF2-dTTP analog (Ki ¼ 1.4 mM) (35). Likewise, the (α,β),(β,γ)-bisCF2 dATP bound weakly (Ki ¼ 0.7 mM). The inhibition constants were deter- mined by competing with the corresponding natural nucleoside β γ Fig. 3. Control experiment: gap-filling assay of 12b and ( , )-CF2 dTTP 15. triphosphate (i.e., dTTP insertion opposite a templating dA in the case of bisCF2 dTTP). When the binding constant for correct pairing or mispairing (lanes 3 and 7) when 12b was used at bisCF2 dATP 12a was determined by competing with dTTP inser- the same concentrations as (β,γ)-CF2 dTTP. This result verifies tion opposite a templating dA, the binding affinity was very simi- that 12b is indeed nonhydrolyzable and therefore cannot be lar (Ki ¼ 1.2 mM). Because Watson–Crick hydrogen bonding will utilized by the polymerase for DNA primer extension. not occur with the analog in this situation (i.e., would create an The interesting property of dNTP analogs 12 would be their A–A mismatch), this result suggests that only the triphosphate behavior in the catalytic site of a dNTP utilizing enzymes. The portion of the analog and natural nucleotide are competing. This crystal structure of the ternary complex of DNA pol β with the is also consistent with the weak binding affinity observed when incoming analog 12b opposite dA represents the precatalytic the bridging Oα;β of the incoming nucleotide is substituted with state of the nucleotidyl transfer reaction for correct incorpo- CF2 (35). Thus, although the crystal structure indicates that the ration, containing all atoms required for catalysis including two bisCF2-substituted analog is well tolerated within the polymerase active site Mg2þ ions (PDB ID code 3LK9). As expected, the sub- active site, the primary conformation in solution does not permit stitution of CF2 for the (α,β)-bridging oxygen prevented dissocia- Watson–Crick type hydrogen bonding. tion of the pyrophosphate leaving group, trapping the enzyme Properties of the new set of dNTP analogs may have interest- complex prior to catalysis. ing applications in biomedical research. Although these com- Besides the intended effect of Oα;β and Oβ;γ replacement on pounds are weak inhibitors, complete nonhydrolyzability of the the overall basicity of the nucleotide analog, one must also con- analogs allows very important structural evaluation of dNTP uti- sider additional electrostatic and steric effects that may affect the lizing enzymes via X-ray crystallography by trapping their cata- active site of the enzyme. Earlier structures of DNA pol β with lytic complexes. Low pKa values of 1 and relatively low basicity incoming nucleoside triphosphate analogs with CF2 substituted of 12 open an opportunity for one to investigate binding pockets for Oα;β or Oβ;γ indicated that this substitution is well tolerated of other enzymes in terms of phosphate–metal interactions, which 2þ (35, 36). Recently, even Oβ;γ has been substituted by CXY group should be different especially for other than Mg metal ions. (X;Y ¼ H, F, Cl, Br, and /or CH3) (37, 38). The structure of the Such studies are under way. DNA pol β precatalytic complex where both bridging oxygens are In conclusion, an efficient synthesis of bis(difluoromethylene) substituted with CF2, determined to 2.5 Å resolution, superim- triphosphoric acid—an interesting analog of naturally abundant pose well with previously determined ternary complex structures triphosphoric acid—was carried out. The alterations in the pro- of DNA pol β Fig. 4) (35, 39). This clearly indicates that minimal perties of triphosphate that can often be predicted from a fluor- structural perturbation is caused by substitution of both phos- ine substituent are valuable in the development of correlations phoanhydride oxygen atoms with CF2 groups. between structure and function. As probes for the mechanism Although phosphate mimics bearing difluoromethylene and of polymerase action and its relationship to polymerase fidelity, fluoromethylene groups are also reported to bind in a number methylene-substituted dNTP analogs permit exploration of of other enzyme active sites (40, 41), an interesting observation stereoelectronic effects on active site interactions. A particularly was made when we measured the binding constants for the new challenging area with potentially wide practical application is analogs. The binding constant for the (α,β),(β,γ)-bisCF2 dTTP the use of fluorinated analogs as probes to elucidate subtle analog was estimated through a steady-state inhibition analysis differences in the catalytic activity of different enzymes. The

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1007430107 Surya Prakash et al. Downloaded by guest on September 24, 2021 triphosphate group is the part of the nucleotide molecule that gradually warmed to 0 °C. After stirring the solution for 2 h at undergoes catalytic cleavage; therefore its modifications may re- 0 °C, a white precipitate formed that was filtered out and washed veal additional nuances of some enzyme actions. It is hoped that with a small amount of dichloromethane. The organic layers were our recent finding of unique fluronated deoxynucleotide analogs combined together, washed with saturated NaHCO3 solution, will give more insight into the biochemical properties of fluori- dried over Na2SO4, and subjected to SiO2 column chromatogra- nated compounds and also contribute to the understanding of phy (gradient conditions: EtOAc∕Hexane ¼ 1∶1, then EtOAc, the mechanism of certain types of enzyme catalysis. It seems clear then EtOAc∕MeOH ¼ 9.5∶0.5). The yield of compound 9 was that the availability of judiciously designed fluorinated nucleotide 75%. The purity of 9 as a yellow oil may vary from 92 to 1 analogs is of considerable value in the development of such 98%. H NMR (CDCl3): 1.38 (dt, J ¼ 6.8 Hz, J ¼ 0.8, 12H), approaches. Moreover, a rare example of strong nonpolymeric 2.83 (s, 3H), 2.86 (s, 3H), 4.28–4.44 (m, J ¼ 6.8 Hz, 8H) ppm. 1 13 multibasic acid shall find applications in other areas of scientific C NMR (CDCl3): 16.5(s), 16.6(s), 35.8 (s,), 65.7 (d, J ¼ research such as catalysis, analytical chemistry, fuel cell research, 6.8 Hz), 65.9 (d, J ¼ 6.8 Hz), 119.1 (m) ppm. 19F NMR and medicinal chemistry. −116 8 J ¼ 382 0 J ¼ 82 3 (CDCl3): . (td, FF . Hz, FP . Hz) ppm, −118 5 J ¼ 382 0 J ¼ 82 3 31 . (td, FF . , FP . Hz). P NMR (CDCl3): Experimental Methods – J ¼ 82 3 J ¼ 50 0 – 2.2 4.0 (dt, PF . Hz, PP . Hz, 2P), 15.8 18.4 (m, General Remarks. Unless otherwise mentioned, all reagents were 1H) ppm. HRMS (FABþ): 466.0929 found, 466.0936 calculated purchased from commercial sources. Diethyl (difluoromethyl) for C12H27F4NO7P3. phosphonate was prepared from chlorodifluoromethane and tri- ethylphsophite according to a known procedure (42). Absolute Synthesis of Bis(difluoromethylene)triphosphoric Acid Pentaammo- THF was obtained by distillation from sodium. All NMR spectra nium Salt 10. 2 g (4.3 mmol) of amino-ester 9 was placed in a round were recorded on a 400 MHz instrument with tetramethylsilane bottom flask equipped with a magnetic stirrer. 5.5 mL (6.5 g, as the 1H standard, chlorotrifluoromethane as the 19F standard, 31 10 eq., 43 mmol) of bromotrimethylsilane was added under an and phosphoric acid as a standard for P experiments. The Ar atmosphere, the flask was closed, covered with parafilm, residual solvent signal (unless otherwise mentioned) was used 13 and left at room temperature with stirring. After 5 d, the reaction as the standard for C NMR spectra. All reported chemical shifts mixture was cooled down to 0 °C and 10 mL of water was added are in ppm. Low- and high-resolution MS spectra and elemental dropwise within 2 min. The resulting biphasic solution was stirred microanalysis were obtained from independent commercial enti-

at 0 °C for 30 min and extracted with Et2O (3 times, 15 mL each). CHEMISTRY ties. HPLC analysis and purification of the deoxynucleotide ana- Then water was partially removed in vacuum, and the remaining logs 12a and 12b were performed on a Varian ProStar 210 solvent ∕ aqueous solution was passed through DOWEX 50WX8-200 delivery module with Shimadzu SPD-10A VP UV Vis detector. (NH þ form) ion-exchange resin. Water was removed from the Varian PureGel SAX 10 mm × 100 mm−7 μm column was used 4 resulting solution of ammonium salt in vacuum, and the remain- for analysis (0–50% 0.5 M LiCl). Macherey-Nagel Nucleogel ing solid was recrystallized from H2O∕NH3 ·H2O ¼ 9∶1. Addi- SAX 1000-10 25 mm × 150 mm column (0–100% 0.5 M TEAB; tion of small quantities of MeOH usually helps the crystallization pH ¼ 8.0) and Dynamax 100A C-18 21.4 mm × 250 mm column process. Salt 10 was obtained as white crystals in 81% yield (after (0.1 M TEAB, 4% CH3CN, pH >¼ 7.4) were used for dual-pass two additional collections of crystals upon evaporation of the sol- preparative isolation. HPLC analysis and purification of the 4 19 12cBz 12c 12dibu 12d vent) and 61% overall yield starting from phosphonate . F deoxynucleotide analogs , , , and were per- −118 3 J ¼ 80 5 31 NMR (D2O): . ppm (t, FP . Hz). P NMR (D2O): formed on a Shimadzu HPLC system (SCL-10A VP, SPD-10A J ¼ 80 5 J ¼ 40 3 – VP, and LC-8A). Tosoh Bioscience DEAE-5PW 21.5 × 15 cm, 4.59 ppm (dt, PF . Hz, PP . Hz, 2P), 15.60 17.30 (m, 1P). MS (ESI-): 324.9 (corresponds to monodissociated 13 μm (100%-40% 1 M TEAB, pH ¼ 8.0) and Shimadzu Premier 4 μ 250 × 23 BMF TPA). CHN-analysis: C 5.39 (calculated 5.37). H 5.44 (cal- C18 5 mm preparative columns were used for purifi- SI Appendix cation (100%-40% 1 M TEAB, pH ¼ 8.0). Tosoh Biosciense culated 5.41), N 15.68 (calculated 15.66). X-ray: see . analytical DEAE-5PW 7.5 mm × 7.5 cm (100%-40% 1 M NaCl) μ 250 × 4 6 General Procedure for the Preparation of 12a–d. 0.4 mmol of 2′- and Shimadzu Premier C18 5 . mm columns were used ′ 13 ð Þ for analysis. deoxy-5 -tosylnucleoside and 307 mg (0.2 mmol) of Bu4N 5 BMF4TPA salt 11 were added to a sealable reaction vessel. 2 mL Synthesis of [(Dimethylamino)phosphoryl] bis(difluoromethylene) of DMF was introduced as solvent and the vessel was sealed. The phosphonate 9. 50 mL of absolute THF was placed in a 200 mixture was heated at 110 °C for 1 hr and then left at 35 °C for mL round bottom flask equipped with magnetic stirrer under several days. Conversion was checked by analytical HPLC on Ar atmosphere. 2.84 mL (2.36 g, 16.8 mmol, 1.05 eq to the phos- DEAE-5PW weak ion-exchange column with 1M NaCl as eluent. phonate) of 2,2,6,6-tetramethylpiperidine was added to THF, and After satisfactory conversion was reached, DMF was removed un- the reaction mixture was cooled down to 0 °C. Then 6.72 mL der high vacuum and the remainder was dissolved in water. Un- (1.05 eq to the phosphonate) of 2.5 M n-BuLi solution in hexanes dissolved particles were filtered off and the filtrate was subjected was added to the solution dropwise, maintaining the tem- to HPLC purification on a preparative DEAE-5PW weak or SAX perature between 0 and 5 °C. The reaction mixture was stirred at ion-exchange column or with 1M Et3NHHCO3∕H2O as eluent. 0 °C for 30–40 min and then cooled down to −78 °C. 3 g (16 mmol) Second pass through reverse phase C18 HPLC column was per- Bz ibu of diethyl (difluoromethyl)phosphonate 4 in 10 mL of absolute formed for 12a,b. Compounds 12c and 12d were deprotected THF was added dropwise to the solution of LTMPA, maintained in H2O∕MeOH∕NH4OH ¼ 1∶1∶4 mixture at room temperature at −78 °C. The reaction mixture was stirred for 40–50 min at overnight. The next day, the reaction mixture was concentrated in −78 °C, and then a solution of 0.915 mL (1.16 g, 7.98 mmol, vacuo, redissolved in water and subjected for reverse phase 0.5 eq to the phosphonate) dimethylphosphoramidous dichloride HPLC purification. 7 in 5 mL of absolute THF was added dropwise, maintaining the Yield of (α,β),(β,γ)-bisCF2 dATP (12a) was 26.6 mg (24% at 1 temperature at −78 °C. The reaction mixture was stirred for 2 h at 100% conversion). H NMR (400 MHz, D2O): 1.09 (t, J ¼ þ −78 °C and then the temperature was slowly raised to ambient. 7.3 Hz, 27H, Et3NH ), 2.59–2.69 (m, 1H), 2.83–2.93 (m, 1H), þ THF was removed in vacuum, and 60 mL of dichloromethane 3.07 (q, J ¼ 7.3 Hz, 18H, Et3NH ), 4.25–4.42 (m, 2H), 4.85– was added. The gray heterogeneous mixture was cooled down 4.95 (m, 1H), 6.55 (t, J ¼ 6.8 Hz, 1H), 8.31 (s, 1H), 8.61 (s, 1H) 19 to −78 °C, and 7.3 g (2.5 eq) of 75% m-CPBA was added portion- ppm. F NMR (376 MHz, D2O): −116.9 (t, J ¼ 74.1 Hz), −118.3 31 wise. The solution was stirred for 15 min at −78 °C and was (t, J ¼ 74.0 Hz) ppm. P NMR (162 MHz, D2O): 2.5–4.5

Surya Prakash et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 24, 2021 − (m, 2P), 12.0–15.0 (m, 1P) ppm. HRMS: calculated for ppm. HRMS: calculated for C11H15F4N3O11P3 533.9861, found − C12H15F4N5O10P3 557.9973, found 557.9961. 533.9860. Yield of (α,β),(β,γ)-bisCF2 dTTP (12b) 19.6 mg (20% at 90% Yield of (α,β),(β,γ)-bisCF2 dGTP (12d) was 2.2 mg (19% at 1 J ¼ 7 3 1 conversion). H NMR (400 MHz, D2O): 1.24 (t, . Hz, 27H, 2 J ¼ 7 3 þ 10% conversion). H NMR (400 MHz, D O): 1.09 (t, . Hz, Et3NH ), 1.95 (s, 3H), 2.35–2.47 (m, 1H), 3.05 (q, J ¼ 7.3 Hz, þ – – – þ 27H, Et3NH ), 2.26 2.34 (m, 1H), 2.61 2.70 (m, 1H), 3.02 3.12 18H, Et3NH þ m, 1H), 4.20–4.45 (m, 3H), 4.76 (m, 1H), 6.48 19 (m, 1H), 4.04–4.10 (m, 3H), 6.13 (dd, J ¼ 7.8 Hz, J ¼ 6.2 Hz, (t, J ¼ 6.8 Hz, 1H), 7.77 (s, 1H) ppm. F NMR (376 MHz, 19 1H), 7.91 (s, 1H) ppm. F NMR (376 MHz, D2O): −118.1 (t, J ¼ D2O): −117.7 (t, J ¼ 74.4 Hz), −118.6 (dt, J ¼ 74.2 Hz, 31 31 73.0 Hz), −118.9 (t, J ¼ 72.9 Hz) ppm. P NMR (162 MHz, J ¼ 69.8 Hz) ppm. P NMR (162 MHz, D2O): 2.5–4.5 (m, – D2O): 3.6–5.4 (m, 2P), 13.0–15.0 (m, 1P) ppm. HRMS: calculated 2P), 12.5 15.0 (m, 1P) ppm. HRMS: calculated for − − 12 15 4 5 11 C12H16F4O12P3 548.9858, found 548.9857. for C H F N O P3 573.9923, found 573.9919. Yield of (α,β),(β,γ)-bisCF2 dCTP (12c) was 11.1 mg (15% at 1 95% conversion). H NMR (400 MHz, D2O): 1.06 (t, J ¼ ACKNOWLEDGMENTS. We are indebted to Dr. J. M. Krahn for his help in pre- þ paring the analog parameters and topology files for structure determination. 7.3 Hz, 27H, Et3NH ), 2.04–2.12 (m, 1H), 2.15–2.23(m, 1H), þ Financial support from the National Institutes of Health (NIH) and the Loker 2.97 (q, J ¼ 7.3 Hz, 18H, Et3NH ), 3.96 (m, 1H), 4.06 (m, Hydrocarbon Institute is greatly appreciated. This research was supported by 2H), 4.40 (m, 1H), 5.92 (d, J ¼ 7.6 Hz, 1H), 6.12 (t, J ¼ 6 9 J ¼ 7 6 19 NIH program grant project 5-U19-CA105010 and in part by Research Project . Hz, 1H), 7.79 (d, . Hz, 1H) ppm. F NMR (376 Numbers Z01 ES050158 and Z01 ES050161 to Dr. S.H. Wilson in the Intramural MHz, D2O): −118.8 (t, J ¼ 75.3 Hz), −119.3 (t, 77.9 Hz) ppm. Research Program of the NIH, National Institute of Environmental Health 31 P NMR (162 MHz, D2O): 1.0–2.7 (m, 2P), 10.4–12.6 (m, 1P) Sciences.

1. Pauling L (1960) The Nature of the Chemical Bond (Cornell Univ Press, Ithaca, N.Y.), 24. Arzumanov AA, Dyatkina NB (1994) An alternative route for preparation of 3rd Ed. α-methylphosphonyl-β,γ-diphosphates of thymidine derivatives. Nucleos Nucleot 2. Goldman P (1969) The carbon-fluorine bond in compounds of biological interest. 13:1031–1037. Science 164:1123–1130. 25. Trowbridge DB, Yamamoto DM, Kenyon GL (1972) Ring openings of trimetaphospho- 3. Hirai G, Watanabe T, Yamaguchi K, Miyagi T, Sedeoka M (2007) Stereocontrolled and ric acid and its bismethylene analog Syntheses of adenosine 5′-bis(dihydroxyphosphi- convergent entry to CF2-sialosides: Synthesis of CF2-linked ganglioside GM4. JAm nylmethyl) and 5′-amino-5′-deoxyadensoine 5′-triphosphate. J Am Chem – Chem Soc 129:15420 15421. Soc 94:3816–3824. α α α 4. Blackburn GM, Turkmen H (2005) Synthesis of -fluoro- and , -difluoro-benzene- 26. Kadokura M, Wada T, Urashima C, Sekine M (1997) Efficient synthesis of γ-methyl- methanesulfonamides: New inhibitors of carbonic anhydrase. Org Biomol Chem capped guanosine 5′-triphosphate as a 5′-terminal unique structure of U6 RNA via – 3:225 226. a new triphosphate bond formation involving activation of methyl phosphorimidazo- 5. Filler R, Kobayashi Y (1983) Biomedical Aspects of Organofluorine Chemistry (Kodan- lidate using ZnCl2 as a catalyst in DMF under anhydrous conditions. Tetrahedron Lett sha and Elsevier Biomedical, Amsterdam). 38:8359–8362. 6. Maier L, Gredig R (1969) Organophosphorus compounds XXXVI. Preparation and 27. Hamilton CJ, Roberts SM, Shipitsin A (1998) Synthesis of a potent inhibitor of HIV properties of bis(dialkoxyphosphonylmethyl)-, bis(alkoxyphosphinylmethyl)-, and bis reverse transcriptase. J Chem Soc Chem Comm 1087–1088. (oxophosphoranylmethyl)phosphinic acid esters and the corresponding acids. Helv 28. Hamilton CJ, Roberts SM (1999) Synthesis of fluorinated phosphonoacetate derivatives Chim Acta 52:827–845. of carbocyclic nucleoside monophosphonates and activity as inhibitors of HIV reverse 7. Bel’skii VE, Zyablikova TA, Panteleeva AR, Shermergorn IM (1967) Synthesis and pro- transcriptase. J Chem Soc Perk T 1 1051–1056. perties of dimethylenetriphosphine ester. Doklady Akademii Nauk SSSR 177:340–343. 8. Klein E, Mons S, Valleix A, Mioskowski C, Lebeau L (2002) Synthesis of enzymatically 29. Mohamady S, Jakeman DL (2005) An improved method for the synthesis of nucleoside – and chemically non-hydrolyzable analogs of dinucleoside triphosphates Ap(3)A and triphosphate analogues. J Org Chem 70:10588 10591. β γ Gp(3)G. J Org Chem 67:146–153. 30. Blackburn GM, Kent DE, Kolkmann F (1981) Three new , -methylene analogues of – 9. Taylor SD, Mirzaei F, Bearne SL (2008) Bismethylene triphosphate nucleotides of . J Chem Soc Chem Comm 1188 1190. uridine 4-phosphate analogues: A new class of anionic pyrimidine nucleotide 31. Ma Q-F, Bathurst IC, Barr PJ, Kenyon GL (1992) New thymidine triphosphate analogue analogues. J Org Chem 73:1403–1412. inhibitors of human immunodeficiency virus-1 reverse transcriptase. J Med Chem 10. McKenna CE, Higa MT, Cheung NH, McKenna MC (1977) The facile dealkylation of 35:1938–1941. phosphonic acid dialkyl esters by bromotrimethylsilane. Tetrahedron Lett 155–158. 32. Blackburn GM, Guo M-J, Langston SP, Taylor GE (1990) Novel phosphonate and thio- 1 3 11. Davies DR, Corbridge DEC (1958) The crystal structure of sodium triphosphate, phosphate analogues of Ap3A, diadenosine 5′,5′′′-P ,P -triphosphate. Tetrahedron Na5P3O10, phase II. Acta Crystallogr 11:315–319. Lett 31:5637–5640. 12. Beukenkamp J, Reiman W, III, Lindenbaum S (1954) Behavior of condensed phosphates 33. Yanachkov I, Wright GE (1994) Synthesis of the P1,P2-methylene analog of N2-(p-n- in anion-exchange chromatography. Anal Chem 26:505–512. butylphenyl)-2′- deoxyguanosine 5′-triphosphate: a non-substrate inhibitor of DNA 13. Watters JI, Loughran ED, Lambert SM (1956) The acidity of triphosphoric acid. JAm polymerases. Nucleos Nucleot 13:339–350. Chem Soc 78:4855–4858. 34. Li R, Muscate A, Kenyon GL (1996) Synthesis, characterization, and inhibitory activities 14. Burgess K, Cook D (2000) Syntheses of nucleoside triphosphates. Chem Rev of nucleoside α,β-imido triphosphate analogues on human immunodeficiency virus-1 – 100:2047 2060. reverse transcriptase. Bioorg Chem 24:251–261. 15. McKenna CE, Kashemirov BA, Blazewska KM (2009) Product class 16: Phosphoric acid 35. Upton TG, et al. (2009) α,β-difluoromethylene deoxynucleoside 5′-triphosphates: A – and derivatives. Science of Synthesis 42:779 921. convenient synthesis of useful probes for DNA polymerase β structure and function. 16. Taylor SD, Mirzaei F, Bearne SL (2006) An unsymmetrical approach to the synthesis of Org Lett 11:1883–1886. bismethylene triphosphate analogues. Org Lett 8:4243–4246. 36. Sucato CA, et al. (2007) Modifying the β,γ leaving-group bridging oxygen alters 17. Saady M, Lebeau L, Mioskowski C (1995) Direct esterification of phosphonic acid salts nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA using the mitsunobu reaction. SYNLETT 643–644. polymerase β. Biochemistry 46:461–471. 18. Vincent S, et al. (1998) Synthesis of enzymatically stable analogues of GDP for binding 37. McKenna CE, et al. (2007) (R)-β,γ-Fluoromethylene-dGTP-DNA ternary complex with studies with transducin, the G-protein of the visual photoreceptor. J Org Chem DNA polymerase β. J Am Chem Soc 129:15412–15413. 63:7244–7257. 38. Batra VK, et al. (2010) Halogenated β,γ-mthylene- and ehylidene-dGTP-DNA ternary 19. Shipitsin AV, et al. (1999) New modified nucleoside 5′-triphosphates: Synthesis, complexes with DNA plymerase β: Structural eidence for serespecific bnding of the properties towards DNA polymerases, stability in blood serum and antiviral activity. – J Chem Soc Perk T 1 1039–1050. fuoromethylene aalogues. J Am Chem Soc 132:7617 7625. 20. Rezende MC, Vallejos G, Osorio-Olivares M, Sepulveda-Boza S (2001) Synthesis of some 39. Batra VK, et al. (2006) Magnesium-induced assembly of a complete DNA polymerase – modified nucleotides of cytidine. Synth Commun 31:3699–3705. catalytic complex. Structure 14:757 766. α 21. He K, Hasan A, Krzyzanowska B, Shaw BR (1998) Synthesis and separation of diaster- 40. Berkowitz DB, Bose M (2001) ( -Monofluoroalkyl): A class of isoacidic eomers of ribonucleoside 5′-(α-P-borano)triphosphates. J Org Chem 63:5769–5773. and “tunable” mimics of biological phosphates. J Fluorine Chem 112:13–33. 22. Li P, et al. (2005) Synthesis of alpha-P-modified nucleoside diphosphates with 41. Romanenko VD, Kukhar VD (2006) Fluorinated phosphonates: Synthesis and biome- ethylenediamine. J Am Chem Soc 127:16782–16783. dical application. Chem Rev 106:3868–3935. 23. Ludwig J, Eckstein F (1989) Rapid and efficient synthesis of nucleoside 5′-0-(1-thiotri- 42. Bergstrom DE, Shum PW (1988) Synthesis and characterization of a new fluorine- phosphates), 5′-triphosphates and 2′,3′-cyclophosphorothioates using 2-chloro-4H- substituted nonionic dinucleoside phosphonate analog. P-Deoxy-P-(difluoromethyl)- 1,3,2-benzodioxaphosphorin-4-one. J Org Chem 54:631–635. thymidylyl(30 → 50)thymidine. J Org Chem 53:3953–3958.

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