C‐C Bond Formation: Synthesis of C5 Substituted Pyrimidine and C8 Substituted Purine Nucleosides Using Water Soluble Pd‐imidate Complex

Vijay Gayakhe1, Ajaykumar V. Ardhapure1, Anant R. Kapdi1, Yogesh S. Sanghvi2, Jose Luis Serrano3, Carola Schulzke4

1 Institute of Chemical Technology, Mumbai, 2 Rasayan, Encinitas, California, 3 Departamento de Ingeniería Minera, Geológica, y Cartográfica, Área de Química Inorgánica, Regional Campus of International Excellence “Campus Mare Nostrum,” Universidad Politécnica de Cartagena, Cartagena, 4 Ernst‐Moritz‐Arndt‐Universität Greifswald, Institut für Biochemie, Greifswald
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.37
DOI:  10.1002/cpnc.1
Online Posting Date:  June, 2016
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The synthesis of a highly efficient, water soluble [Pd(Sacc)2(TPA)2] complex for C‐C bond formation is described. Additionally, application of the [Pd(Sacc)2(TPA)2] complex for Suzuki‐Miyaura arylation of all four nucleosides (5‐iodo‐2′‐deoxyuridine [5‐IdU], 5‐iodo‐2′‐deoxycytidine [5‐IdC], 8‐bromo‐2′‐deoxyadenosine, and 8‐bromo‐2′‐deoxyguanosine) with various aryl/heteroaryl boronic acids in plain water under milder conditions is demonstrated. © 2016 by John Wiley & Sons, Inc.

Keywords: Suzuki‐Miyaura cross‐coupling; base‐modified nucleosides; nucleotides; palladium catalyst

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Water Soluble [Pd(Sacc)2(TPA)2]
  • Basic Protocol 2: Suzuki‐Miyaura Cross‐Coupling of 2′‐Deoxypyrimidine and 2′‐Deoxypurine Nucleosides
  • Basic Protocol 3: Synthesis of the Phosphoramidite Reagent
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Synthesis of Water Soluble [Pd(Sacc)2(TPA)2]

  • Palladium(II) acetate (e.g., Sigma Aldrich)
  • Dimethyl sulfide, >99% pure (Me 2S; e.g., ACROS Organics)
  • Saccharin, ≥98% pure (e.g., Sigma Aldrich)
  • Methanol
  • Diethyl ether, anhydrous
  • Dichloromethane (DCM), anhydrous
  • 1,3,5‐Triaza‐7‐phosphaadamantane (TPA; Phillips et al., )
  • 50‐mL single‐neck round‐bottom flask
  • Magnetic stir plate with stir bar
  • Vacuum pump
  • Dropping funnel
  • 25‐mL Schlenk tube
  • Additional reagents and equipment for mass spectrometry (unit 7.2; Castleberry et al., ), infrared spectroscopy, and 1H and 31P NMR spectroscopy (unit 7.2; James, )

Basic Protocol 2: Suzuki‐Miyaura Cross‐Coupling of 2′‐Deoxypyrimidine and 2′‐Deoxypurine Nucleosides

  • Nitrogen gas, ultra‐high purity
  • [Pd(Sacc) 2(PTA) 2] (see protocol 1)
  • Halonucleoside:
  • 5‐Iodo‐2′‐deoxyuridine (5‐IdU), ≥99% purity
  • 5‐Iodo‐2′‐deoxycytidine (5‐IdC), ≥99% purity
  • 8‐Bromo‐2′‐deoxyadenosine (8‐BrdA), ≥99% purity
  • 8‐Bromo‐2′‐deoxyguanosine (8‐BrdG), ≥99% purity
  • Triethylamine
  • Boronic acids (available from Combi‐Blocks):
  • Phenyl boronic acid
  • 4‐Methyl phenyl boronic acid
  • 4‐Methoxy phenyl boronic acid
  • 4‐Thiomethyl phenyl boronic acid
  • 4‐Formyl phenyl boronic acid
  • Napthalene‐2‐boronic acid
  • Benzofuran‐2‐boronic acid
  • Furan‐2‐boronic acid
  • Thiophene‐3‐boronic acid
  • 3,4‐Dimethoxy phenyl boronic acid
  • 3,5‐Dimethyl phenyl boronic acid
  • 3,4‐Methylenedioxy phenyl boronic acid
  • Ethyl acetate
  • Silica gel
  • Dichloromethane (DCM)
  • Methanol
  • 25‐mL Schlenk tube equipped with a glass stopper
  • Magnetic stir plate with stir bar
  • Vacuum pump
  • 1‐mL and 5‐mL syringe
  • 20‐G stainless steel needles
  • 80°C oil bath
  • Thread seal tape (e.g., Teflon tape)
  • Rotary evaporator equipped with a water aspirator
  • Additional reagents and equipment for performing thin‐layer chromatography ( appendix 3D; Meyers and Meyers, ), column chromatography ( appendix 3E; Meyers, ), mass spectrometry (unit 10.2; Castleberry et al., ), and 1H and 13C NMR spectroscopy (unit 7.2; James, )

Basic Protocol 3: Synthesis of the Phosphoramidite Reagent

  • 5‐Phenyl‐2′‐deoxyuridine (see protocol 2)
  • Pyridine, anhydrous
  • Toluene, anhydrous
  • Nitrogen gas, ultra‐high purity
  • 4‐Dimethylaminopyridine (DMAP)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Dichloromethane (DCM)
  • 5% (w/v) NaHCO 3
  • Brine
  • Na 2SO 4
  • Hexane, anhydrous
  • Acetonitrile
  • Bis(N,N‐disopropylamine)‐2‐cyanoethoxyphosphoramidite (Phos reagent)
  • 1,1′‐Carbonyldiimidazole
  • Ethyl acetate
  • 50‐mL single‐neck round‐bottom flask
  • Vacuum pump
  • Magnetic stir plate with stir bar
  • Rotary evaporator
  • Stainless steel spatula
  • C18 column
  • –20°C freezer
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D; Meyers and Meyers, ), HPLC (unit 10.5; Sinha and Jung, ), and 1H and 13C NMR spectroscopy (unit 7.2; James, )
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Literature Cited

Literature Cited
  Amann, N., Pandurski, E., Fiebig, T., and Wagenknecht, H.A. 2002. Electron injection into DNA: Synthesis and spectroscopic properties of pyrenyl‐modified oligonucleotides. Chem. Eur. J. 8:4877‐4883. doi: 10.1002/1521‐3765(20021104)8:21%3c4877::AID‐CHEM4877%3e3.0.CO;2‐P.
  Capek, P. and Hocek, M. 2005. Efficient one‐step synthesis of optically pure (adenin‐8‐yl)phenylalanine nucleosides. Synlett 19:3005‐3007. doi: 10.1055/s‐2005‐921896.
  Capek, P., Pohl, R., and Hocek, M. 2006. Cross‐coupling reactions of unprotected halopurine bases, nucleosides, nucleotides and nucleoside triphosphates with 4‐boronophenylalanine in water. Synthesis of (purin‐8‐yl)‐ and (purin‐6‐yl)phenylalanines. Org. Biomol. Chem. 4:2278‐2284.
  Castleberry, C.M., Rodicio, L.P., and Limbach, P.A. 2008. Electrospray ionization mass spectrometry of oligonucleotides. Curr. Protoc. Nucl. Acid Chem. 33:10.2.1‐10.2.19. doi: 10.1002/0471142700.nc1002s35.
  Chanda, A. and Fokin, V.V. 2009. Organic synthesis “On water.” Chem. Rev. 109:725‐748. doi: 10.1021/cr800448q.
  Cho, J.H. and Shaughnessy, K.H. 2012. Aqueous‐phase Sonogashira alkynylation to synthesize 5‐substituted pyrimidine and 8‐substituted purine nucleosides. Curr. Protoc. Nucl. Acid. Chem. 49:1.27.1‐1.27.10. doi: 10.1002/0471142700.nc0127s49.
  Fresneau, N., Hiebel, M.A., Agrofoglio, L.A., and Berteina‐Raboin, S. 2012. Efficient synthesis of unprotected C‐5‐Aryl/Heteroaryl‐2′‐deoxyuridine via a Suzuki‐Miyaura reaction in aqueous media. Molecules 17:14409‐14417. doi: 10.3390/molecules171214409.
  Greco, N.J. and Tor, Y. 2005. Simple fluorescent pyrimidine analogues detect the presence of DNA abasic sites. J. Am. Chem. Soc. 127:10784‐10785. doi: 10.1021/ja052000a.
  Hocek, M. and Silhar, P. 2007. Palladium‐catalyzed cross‐coupling reactions in C6 modifications of purine nucleosides. Curr. Protoc. Nucl. Acid Chem. 1:16.1‐1.16.17. doi: 10.1002/0471142700.nc0116s28.
  James, T.L. 2000. NMR Determination of oligonucleotide structure. Curr. Protoc. Nucl. Acid Chem. 00:7.2.1‐7.2.16. doi: 10.1002/0471142700.nc0702s00.
  Kapdi, A.R., Gayakhe, V., Sanghvi, Y.S., Garcia, J., Lozano, P., da Silva, I., Perez, J., and Serrano, J.L. 2014. New water soluble Pd‐imidate complexes as highly efficient catalysts for the synthesis of C5‐arylated pyrimidine nucleosides. RSC Adv. 4:17567‐17572. doi: 10.1039/c4ra01326c.
  Lussier, T., Herve, G., Enderlin, G., and Len, C. 2014. Original access to 5‐aryluracils from 5‐iodo‐2′‐deoxyuridine via a microwave assisted Suzuki‐Miyaura cross‐coupling/deglycosylation sequence in pure water. RSC Adv. 4:46218‐46223. doi: 10.1039/C4RA04814H.
  Macickova‐Cahova, H., Pohl, R., Horakova, P., Havran, L., Spacek, J., Fojta, M., and Hocek, M. 2011. Alkylsulfanylphenyl derivatives of cytosine and 7‐deazaadenine nucleosides, nucleotides and nucleoside triphosphates: Synthesis, polymerase incorporation to DNA and electrochemical study. Chem. Eur. J. 17:5833‐5841. doi: 10.1002/chem.201003496.
  Meyers, C. 2000. Column chromatography. Curr. Protoc. Nucl. Acid Chem. 3:A.3E.1‐A.3E.7. doi: 10.1002/0471142700.nca03es03.
  Meyers, C. and Meyers, D. 2008. Thin‐layer chromatography. Curr. Protoc. Nucl. Acid Chem. 34:A.3D.1‐A.3D.13. doi: 10.1002/0471142700.nca03ds34.
  Mizuta, M., Banba, J.I., Kanamori, T., Tawarada, R., Ohkubo, A., Sekine, M., and Seio, K. 2008. New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction. J. Am. Chem. Soc. 130:9622‐9623. doi: 10.1021/ja800991m.
  Phillips, A.D., Gonsalvi, L., Romerosa, A., Vizza, F., and Peruzzini, M. 2004. Coordination chemistry of 1,3,5‐triaza‐7‐phosphaadamantane (PTA) transition metal complexes and related catalytic, medicinal and photoluminescent applications. Coord. Chem. Rev. 248:955‐993. doi: 10.1016/j.ccr.2004.03.010.
  Rankin, K.M., Sproviero, M., Rankin, K., Sharma, P., Wetmore, S.D., and Manderville, R.A. 2012. C8‐heteroaryl‐2′‐deoxyguanosine adducts as conformational fluorescent probes in the nari recognition sequence. J. Org. Chem. 77:10498‐10508. doi: 10.1021/jo302164c.
  Sanghvi, Y.S. 2014. Nucleoside amidites as building‐blocks for synthesis of therapeutic oligonucleotides: A mini‐review. Chimica Oggi‐Chem. Today 32:10‐15.
  Sartori, G., Enderlin, G., Herve, G., and Len, C. 2012. Highly effective synthesis of C‐5‐Substituted 2′‐deoxyuridine using Suzuki‐Miyaura cross‐coupling in water. Synthesis 44:767‐772. doi: 10.1055/s‐0031‐1289709.
  Shaughnessy, K.H. 2009. Hydrophilic ligands and their application in aqueous‐phase metal‐catalyzed reactions. Chem. Rev. 109:643‐710. doi: 10.1021/cr800403r.
  Shaughnessy, K.H. 2015. Palladium‐catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides. Molecules 20:9419‐9454. doi: 10.3390/molecules20059419.
  Sinha, N.D. and Jung, K.E. 2015. Analysis and purification of synthetic nucleic acids using HPLC. Curr. Protoc. Nucl. Acid Chem. 61:10.5.1‐10.5.39. doi: 10.1002/0471142700.nc1005s61.
  Tanpure, A.A. and Srivatsan, S.G.. 2012 Synthesis and photophysical characterisation of a fluorescent nucleoside analogue that signals the presence of an abasic site in RNA. Chembiochem. 13:2392‐2399. doi: 10.1002/cbic.201200408.
  Train, B.C., Bilgesü, S.A., Despeaux, E.C., Vongsutilers, V., and Gannett, P.M. 2014. Single C8‐arylguanine modifications render oligonucleotides in the Z‐DNA conformation under physiological conditions. Chem. Res. Toxicol. 27:1176‐1186. doi: 10.1021/tx5000798.
  Western, E.C., Daft, J.R., Johnson, II, E.M., Gannett, P.M., and Shaughnessy, K.H. 2003. Efficient one‐step Suzuki arylation of unprotected halonucleosides, using water‐soluble palladium catalysts. J. Org. Chem. 68:6767‐6774. doi: 10.1021/jo034289p.
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