Biotinylation of a Propargylated Cyclic (3′‐5′) Diguanylic Acid and of Its Mono‐6‐Thioated Analog Under “Click” Conditions

Andrzej Grajkowski1, Jacek Cieślak1, Christian Schindler2, Serge L. Beaucage1

1 Food and Drug Administration, Bethesda, Maryland, 2 Department of Microbiology & Immunology and Department of Medicine, Columbia University, New York, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 14.9
DOI:  10.1002/0471142700.nc1409s52
Online Posting Date:  March, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Commercial N2‐isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(propargyl)guanosine is converted to its 3′‐O‐levulinyl ester in a yield of 91%. The reaction of commercial N2‐isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐Otert‐butyldimethylsilyl‐3′‐O‐[(2‐cyanoethyl)‐N,N‐diisopropylaminophosphinyl]guanosine with N2‐isobutyryl‐2′‐O‐propargyl‐3′‐O‐(levulinyl)guanosine provides, after P(III) oxidation, 3′‐/5′‐deprotection, and purification, the 2′‐O‐propargylated guanylyl(3′‐5′)guanosine 2‐cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2‐cyanoethyl tetraisopropylphosphordiamidite and 1H‐tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2′‐Otert‐butyldimethylsilyl protecting groups leads to the desired propargylated c‐di‐GMP diester. Cycloaddition of a biotinylated azide with the propargylated c‐di‐GMP diester under click conditions provides the biotinylated c‐di‐GMP conjugate in an isolated yield of 62%. Replacement of the 6‐oxo function of N2‐isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐levulinyl‐2′‐O‐(propargyl)guanosine with a 2‐cyanoethylthio group is effected by treatment with 2,4,6‐triisopropybenzenesulfonyl chloride and triethylamine to give a 6‐(2,4,6‐triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3‐mercaptoproprionitrile and triethylamine, followed by 5′‐dedimethoxytritylation, affords the 6‐(2‐cyanoethylthio)guanosine derivative in a yield of 70%. The 5′‐hydroxy function of this derivative is reacted with commercial N2‐isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐Otert‐butyldimethylsilyl‐3′‐O‐[(2‐cyanoethyl)‐N,N‐diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono‐6‐thioated c‐di‐ GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c‐di‐GMP conjugate, and isolated in similar yields. Curr. Protoc. Nucleic Acid Chem. 52:14.9.1‐14.9.20. © 2013 by John Wiley & Sons, Inc.

Keywords: propargylated c‐di‐GMP; biotinylated azide; click conjugation reaction; biotinylated c‐di‐GMP conjugate; 6‐(2‐cyanoethylthio)guanosine; c‐di‐GMP mono‐6‐thioated analog

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis, Purification, and Characterization of the Mono‐6‐Thioated Analog of 2′‐O‐Propargylated Cyclic‐DI‐GMP
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(propargyl)guanosine ( 1, ChemGenes)
  • Pyridine (Acros)
  • Levulinic acid (Aldrich)
  • N,N′‐Dicyclohexylcarbodiimide (Aldrich)
  • Chloroform (CHCl 3, Fisher)
  • Acetic acid (AcOH, Acros)
  • Silica gel (60 Å, 230 to 400 mesh, Merck)
  • Methanol (MeOH, Fisher)
  • 1H‐Tetrazole (Glen Research)
  • Anhydrous acetonitrile (MeCN, Glen Research)
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐propargyl‐3′‐O‐[(N,N‐diisopropylamino) 2‐cyanoethyloxyphosphinyl]guanosine ( 3, ChemGenes)
  • 5.5 M tert‐butyl hydroperoxide in decane (Aldrich)
  • Hydrazine hydrate (Aldrich)
  • 2,4‐Pentanedione (Aldrich)
  • Hexane (Fisher)
  • Methylene chloride (CH 2Cl 2, Fisher)
  • 2‐Cyanoethyl tetraisopropylphosphordiamidite (Aldrich)
  • Dry argon gas cylinder (Matheson)
  • Concentrated aqueous ammonia (Aldrich)
  • Triethylamine trihydrofluoride (Aldrich)
  • 2 M triethylammonium acetate, pH 7.0 (Applied Biosystems)
  • Stir bars (VWR)
  • 50‐ and 250‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • 1‐, 3‐, and 10‐mL plastic syringes (BD)
  • 21‐G stainless steel syringe needles
  • Magnetic stirrer (VWR)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • 50‐ and 100‐mL separatory funnels (Kontes)
  • 2.5 × 7.5–cm TLC plates pre‐coated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Chromaflex TLC developing jars (Kontes)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 250‐mL Erlenmeyer flasks (Kimax)
  • High vacuum oil pump (Savant)
  • 9‐in. disposable Pasteur pipets (Fisher)
  • 30‐mL sintered‐glass funnels (coarse porosity, Kontes)
  • 4‐mL screw‐cap glass vials (Wheaton)
  • 5‐µm Supelcosil LC‐18S HPLC column (25 cm × 10 mm; Supelco)

Basic Protocol 2:

  Materials
  • N‐D‐(+)‐Biotinyl‐4,7,10,13,16‐pentaoxa‐1,19‐diaminononadecane ( 10, Berry & Associates)
  • Acetonitrile (MeCN, Acros)
  • Pyridine (C 5H 5N, Acros)
  • Azidoacetic anhydride: bromoacetic acid (Aldrich) and sodium azide (Aldrich); Dyke et al. ( )
  • N,N′‐Dicyclohexylcarbodiimide (Aldrich)
  • Dry argon gas cylinder (Matheson)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Chloroform (Fisher)
  • Methanol (Fisher)
  • Propargylated c‐di‐GMP ( 9, see protocol 1)
  • Copper(II) sulfate pentahydrate (Aldrich)
  • tris‐(Benzyltriazolylmethyl)amine (TBTA): benzyl azide (Aldrich) and tripropargylamine (Aldrich), prepared according to Chan et al. ( )
  • Dimethyl sulfoxide (Acros)
  • tert‐Butanol (Aldrich)
  • Sodium L‐ascorbate (Aldrich)
  • 4‐mL screw‐cap glass vials (Wheaton)
  • Stir bars (VWR)
  • Magnetic stirrer (VWR)
  • 0.9 × 8–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates pre‐coated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Chromaflex TLC developing jars (Kontes)
  • 100‐mL round‐bottom flasks (Kontes)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)

Basic Protocol 3: Synthesis, Purification, and Characterization of the Mono‐6‐Thioated Analog of 2′‐O‐Propargylated Cyclic‐DI‐GMP

  Materials
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytr ityl)‐2′‐O‐propargyl guanosine ( 1, ChemGenes)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Methanol (Fisher)
  • Methylene chloride (Fisher)
  • Chloroform (Fisher)
  • N‐D‐(+)‐Biotinyl‐4,7,10,13,16‐pentaoxa‐1,19‐diaminononadecane ( 10, Berry and Associates)
  • 4‐(Dimethylamino)pyridine
  • Triethylamine (Aldrich)
  • 2,4,6‐Triisopropylbenzenesulfonyl chloride (Aldrich)
  • Anhydrous sodium sulfate (Fisher)
  • 1‐Methylpyrrolidine (Aldrich)
  • 2‐Cyanoethanethiol (Gerber et al., )
  • Acetic acid (Acros)
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐propargyl‐3′‐O‐[(N,N‐diisopropylamino)2‐cyanoethyloxy)phosphinyl] guanosine ( 3, ChemGenes)
  • Anhydrous acetonitrile (Glen Research)
  • Sodium hydrosulfide hydrate (Aldrich)
  • Concentrated aqueous ammonia (Aldrich)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates pre‐coated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Chromaflex TLC developing jars (Kontes)
  • 25‐ 50‐, 100‐, and 250‐mL round‐bottom flasks (Kontes)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • 1‐, 3‐, and 10‐mL syringes (B‐D)
  • 21‐G stainless steel syringe needles
  • 100‐mL Erlenmeyer flasks (Kimax)
  • 50‐ and 100‐mL separatory funnels (Kontes)
  • Magnetic stirrer (VWR)
  • 30‐mL sintered‐glass funnels (coarse porosity, Kontes)
  • High vacuum oil pump (Savant)
  • 4‐mL screw‐cap glass vials (Wheaton)
  • Additional reagents and equipment (see protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abdul‐Sater, A.A., Said‐Sadier, N., Ojcius, D.M., Yilmaz, O., and Kelly, K.A. 2009. Inflammasomes bridge signaling between pathogen identification and the immune response. Drugs Today 45:105‐112.
   Abdul‐Sater, A.A., Grajkowski, A., Erdjument‐Bromage, H., Plumlee, C., Levi, A., Schreiber, M.T., Lee, C., Shuman, H., Beaucage, S.L., and Schindler, C. 2012a. The overlapping host responses to bacterial cyclic dinucleotides. Microbes Infect. 14:188‐197.
   Abdul‐Sater, A., Jin, L., Grajkowski, A., Beaucage, S.L, Allen, I.C., Ting, J.P.‐Y., Cambier, J.C., and Schindler, C. 2012b. Cyclic‐diGMP and cyclic diAMP activate the NLRP3 inflammasome EMBO Reports. Submitted.
   Adams, C.J., Murray, J.B., Farrow, M.A., Arnold, J.R.P., and Stockley, P.G. 1995. Incorporation of 6‐thioguanosine into oligoribonucleotides. Tetrahedron Lett. 36:5421‐5424.
   Amiot, N., Heintz, K., and Giese, B. 2006. New approach for the synthesis of c‐di‐GMP and its analogues. Synthesis 24:4230‐4236.
   Barber, G.N. 2011. Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses. Curr. Opin. Immunol. 23:10‐20.
   Berndl, S., Herzig, N., Kele, P., Lachmann, D., Li, X., Wolfbeis, O.S., and Wagenknecht, H.‐A. 2009. Comparison of a nucleosidic vs non‐nucleosidic postsynthetic “click” modification of DNA with base‐labile fluorescent probes. Bioconjug. Chem. 20:558‐564.
   Burdette, D.L., Monroe, K.M., Sotelo‐Troha, K., Iwig, J.S., Eckert, B., Hyodo, M., Hayakawa, Y., and Vance, R.E. 2011. STING is a direct innate immune sensor of cyclic di‐GMP. Nature 478:515‐519.
   Chan, T.R., Hilgraf, R., Sharpless, K.B., and Fokin, V.V. 2004. Polytriazoles as copper(I)‐stabilizing ligands in catalysis. Org. Lett. 6:2853‐2855.
   Dyke, J.M., Groves, A.P., Morris, A., Ogden, J.S., Dias, A.A., Oliveira, A.M.S., Costa, M.L., Barros, M.T., Cabral, M.H., and Moutinho, A.M.C. 1997. Study of the thermal decomposition of 2‐azidoacetic acid by photoelectron and matrix isolation infrared spectroscopy. J. Am. Chem. Soc. 119:6883‐6887.
   Galperin, M.Y. 2005. A census of membrane‐bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts. BMC Microbiol. 5:35.
   Gerber, R.E, Hasbun, C., Dubenko, L.G., Fong King, M., and Bierer, D.E. 2000. β‐Mercaptopropionitrile. In Organic Syntheses, Vol. 77. (D. J. Hart, ed.) pp. 186‐197, John Wiley & Sons, New York.
   Grajkowski, A., Cieślak, J., Gapeev, A., Schindler, C., and Beaucage, S.L. 2010. Convenient synthesis of a propargylated cyclic (3′‐5′) diguanylic acid and its “click” conjugation to a biotinylated azide. Bioconjug. Chem. 21:2147‐2152.
   Gross, O., Thomas, C.J., Guarda, G., and Tschopp, J. 2011. The inflammasome: An integrated view. Immunol. Rev. 243:136‐151.
   Gu, C. and Wang, Y. 2007. In vitro replication and thermodynamic studies of methylation and oxidation modifications of 6‐thioguanine. Nucleic Acids Res. 35:3693‐3704.
   Guo, Z. and Xue, J. 2009. Levulinic anhydride. In Encyclopedia of Reagents for Organic Synthesis, 2nd Edition (L.A. Paquette, D. Crich, P.L. Fuchs, and G. Molander, eds.) pp 5961‐5963. John Wiley & Sons, Hoboken.
   Hassner, A., Strand, G., Rubinstein, M., and Patchornik, A. 1975. Levulinic esters. Alcohol protecting group applicable to some nucleosides. J. Am. Chem. Soc. 97:1614‐1615.
   Hayakawa, Y., Nagata, R., Hirata, A., Hyodo, M., and Kawai, R. 2003. A facile synthesis of cyclic bis(3′→5′)diguanylic acid. Tetrahedron 59:6465‐6471.
   Hengge, R. 2009. Principles of c‐di‐GMP signaling in bacteria. Nat. Rev. Microbiol. 7:263‐273.
   Hu, D.‐L., Narita, K., Hyodo, M., Hayakawa, Y., Nakane, A., and Karaolis, D.K.R. 2009. c‐di‐GMP as a vaccine adjuvant enhances protection against systemic methicillin‐resistant Staphylococcus aureus (MRSA) infection. Vaccine 27:4867‐4873.
   Humes, E. and Yan, H. 2006. Convenient synthesis of 3′,5′‐cyclic diguanylic acid (cdiGMP). Nucleic Acids Symp. Ser. 50:5‐6.
   Hyodo, M. and Hayakawa, Y. 2004. An improved method for synthesizing cyclic bis(3′‐5′)diguanylic acid (c‐di‐GMP). Bull. Chem. Soc. Jpn. 77:2089‐2093.
   Hyodo, M., Sato, Y., and Hayakawa, Y. 2006. Synthesis of cyclic bis(3′‐5′)diguanylic acid (c‐di‐GMP) analogs. Tetrahedron 62:3089‐3094.
   Iwasaki, A. and Medzhitov, R. 2010. Regulation of adaptive immunity by the innate immune system. Science 327:291‐295.
   Karaolis, D.K.R., Means, T.K., Yang, D., Takahashi, M., Yoshimura, T., Muraille, E., Philpott, D., Schroeder, J.T., Hyodo, M., Hayakawa, Y., Talbot, B.G., Brouillette, E., and Malouin, F. 2007a. Bacterial c‐di‐GMP is an immunostimulatory molecule. J. Immunol. 178:2171‐2181.
   Karaolis, D.K.R., Newstead, M.W., Zeng, X., Hyodo, M., Hayakawa, Y., Bhan, U., Liang, H., and Standiford, T.J. 2007b. Cyclic di‐GMP stimulates protective innate immunity in bacterial pneumonia. Infect. Immun. 75:4942‐4950.
   Kiburu, I., Shurer, A., Yan, L., and Sintim, H.O. 2008. A simple solid‐phase synthesis of the ubiquitous bacterial signaling molecule, c‐di‐GMP and analogues. Mol. BioSyst. 4:518‐520.
   McWhirter, S.M., Barbalat, R., Monroe, K.M., Fontana, M.F., Hyodo, M., Joncker, N.T., Ishii, K.J., Akira, S., Colonna, M., Chen, Z.J., Fitzgerald, K.A., Hayakawa, Y., and Vance, R.E. 2009. A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic‐di‐GMP. J. Exp. Med.206:1899‐1911.
   Ogunniyi, A.D., Paton, J.C., Kirby, A.C., McCullers, J.A., Cook, J., Hyodo, M., Hayakawa, Y., and Karaolis, D.K.R. 2008. c‐di‐GMP is an effective immunomodulator and vaccine adjuvant against pneumococcal infection. Vaccine 26:4676‐4685.
   Ouyang, S., Song, X., Wang, Y., Ru, H., Shaw, N., Jiang, Y., Niu, F., Zhu, Y., Qiu, W., Parvatiyar, K., Li, Y., Zhang, R, Cheng, G., and Liu, Z.‐J. 2012. Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di‐GMP binding. Immunity 36:1073‐1086.
   Rao, F., Pasunooti, S., Ng, Y., Zhuo, W., Lim, L., Liu, A.W., and Liang, Z.–X. 2009. Enzymatic synthesis of c‐di‐GMP using a thermophilic diguanylate cyclase. Anal. Biochem. 389:138‐142.
   Ross, P., Weinhouse, H., Aloni, Y., Michaeli, D., Weinberger‐Ohana, P., Mayer, R., Braun, S., de Vroom, E., van der Marel, G.A., van Boom, J.H., and Benziman, M. 1987. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279‐281.
   Ross, P., Mayer, R., Weinhouse, H., Amikam, D., Huggirat, Y., Benziman, M., de Vroom, E., Fidder, A., de Paus, P., Sliedregt, L.A.J.M., van der Marel, G.A., and van Boom, J.H. 1990. The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. J. Biol. Chem. 265:18933‐18943.
   Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise Huisgen cycloaddition process: Copper(I)‐catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41:2596‐2599.
   Schroder, K., Muruve, D.A., and Tschopp, J. 2009. Innate immunity: Cytoplasmic DNA sensing by the AIM2 inflammasome. Curr. Biol. 19:R262‐R265.
   Simm, R., Morr, M., Remminghorst, U., Andersson, M., and Römling, U. 2009. Quantitative determination of cyclic diguanosine monophosphate concentrations in nucleotide extracts of bacteria by matrix‐assisted laser desorption/ionization–time‐of‐flight mass spectrometry. Anal. Biochem. 386:53‐58.
   Tornøe, C.W., Christensen, C., and Meldal, M. 2002. Peptidotriazoles on solid phase: [1,2,3]‐Triazoles by regiospecific copper(I)‐catalyzed 1,3‐dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67:3057‐3064.
   Trinchieri, G. 2010. Type I interferon: Friend or foe? J. Exp. Med. 207:2053‐2063.
   Trinchieri, G. and Sher, A. 2007. Cooperation of toll‐like receptor signals in innate immune defence. Nat. Rev. Immunol. 7:179‐190.
   Woodward, J.J., Iavarone, A.T., and Portnoy, D.A. 2010. c‐di‐AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science 328:1703‐1705.
   Yan, H. and Chen, W. 2010. 3′,5′‐Cyclic diguanylic acid: A small nucleotide that makes big impacts. Chem. Soc. Rev. 39:2914‐2924.
   Yan, H. and López Aguilar, A. 2007. Synthesis of 3′,5′‐cyclic diguanylic acid (cdiGMP) using 1‐(4‐chlorophenyl)‐4‐ethoxypiperidin‐4‐yl as a protecting group for 2′‐hydroxy functions of ribonucleosides. Nucleosides Nucleotides Nucleic Acids 26:189‐204.
   Zhang, Z., Gaffney, B.L., and Jones, R.A. 2004. c‐di‐GMP displays a monovalent metal ion‐dependent polymorphism. J. Am. Chem. Soc. 126:16700‐16701.
   Zheng, Q., Xu, Y‐Z., and Swann, P.F. 1997. Photochemical cross‐linking of λ‐cro repressor to operator DNA containing 4‐thiothymine or 6‐thioguanine. Nucleosides Nucleotides 16:1799‐1803.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library