One‐Flask Synthesis of Cyclic Diguanosine Monophosphate (c‐di‐GMP)

Barbara L. Gaffney1, Roger A. Jones1

1 Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 14.8
DOI:  10.1002/0471142700.nc1408s48
Online Posting Date:  March, 2012
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The bacterial signaling molecule, cyclic diguanosine monophosphate (c‐di‐GMP), plays a key role in controlling biofilm formation and pathogenic virulence, among many other functions. It has widespread consequences for human health, and current research is actively exploring its molecular mechanisms. The convenient one‐flask, gram‐scale synthesis of c‐di‐GMP described here will facilitate these efforts. Curr. Protoc. Nucleic Acid Chem. 48:14.8.1‐14.8.7. © 2012 by John Wiley & Sons, Inc.

Keywords: c‐di‐GMP; bacterial signaling; chemical synthesis

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • S.1 3′‐O‐[(diisopropylamino)(2‐cyanoethoxy)phosphino]‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐Otert‐butyldimethylsilyl‐N2‐isobutyrylguanosine
  • Dry acetonitrile (CH 3CN)
  • 3 Å molecular sieves
  • Pyridinium trifluoroacetate
  • tert–Butyl amine
  • Dichloroacetic acid (DCA)
  • Pyridine
  • Dry nitrogen or argon
  • 5.5 M tert‐Butyl hydroperoxide in decane
  • Ice bath
  • Sodium bisulfite (NaHSO 3)
  • Dichloromethane (CH 2Cl 2)
  • 95% 5,5‐Dimethyl‐2‐oxo‐2‐chloro‐1,3,2‐dioxaphosphinane (DMOCP)
  • Iodine (I 2)
  • Sodium bicarbonate (NaHCO 3)
  • Ethyl acetate (EtOAc)
  • Diethyl ether (Et 2O)
  • Methanol (CH 3OH)
  • Potassium hydroxide (KOH)
  • 33% methylamine (CH 3NH 2) in anhydrous ethanol
  • Triethylamine (TEA; Et 3N)
  • Triethylamine trihydrogen fluoride (Et 3N⋅3HF)
  • HPLC‐grade acetone
  • 100‐mL pear‐shaped flask
  • Rotary evaporator
  • Vacuum pump
  • Septa
  • 100‐mL, 250‐mL, 500‐mL, and 2‐L round‐bottom flasks
  • Stir bar
  • 16‐G double‐tipped needle
  • Disposable syringes and needles
  • 2‐L Erlenmeyer flasks
  • 2‐L separatory funnels
  • Spatula
  • Filter paper
  • Sintered glass funnels
  • Desiccator
  • 50°C oil bath
  • Additional reagents and equipment for analytical high‐performance liquid chromatography (HPLC; unit 10.5)
NOTE: Reagents and solvents are available from Sigma‐Aldrich, and the apparatus from Fisher.
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Literature Cited

Literature Cited
   Amiot, N., Heintz, K., and Giese, B. 2006. New approach for the synthesis of c‐di‐GMP and its analogues. Synthesis 24:4230‐4236.
   Cotter, P.A. and Stibitz, S. 2007. c‐di‐GMP‐mediated regulation of virulence and biofilm formation. Curr. Opin. Microbiol. 10:17‐23.
   Gaffney, B.L., Veliath, E., Zhao, J., and Jones, R.A. 2010. One‐flask syntheses of c‐di‐GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogues. Org. Lett. 12:3269‐3271.
   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 signalling in bacteria. Nat. Rev. Microbiol. 7:263‐273.
   Hyodo, M. and Hayakawa, Y. 2004. An improved method for synthesizing cyclic bis (3′‐5′)diguanlic 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.
   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.
   Ko, J., Ryu, K.‐S., Kim, H., Shin, J.‐S., Lee, J.‐O., Cheong, C., and Choi, B.‐S. 2010. Structure of PP4397 reveals the molecular basis for different c‐di‐GMP binding modes by Pilz domain proteins. J. Mol. Biol. 398:97‐110.
   Kulshina, N., Baird, N.J., and Ferré‐D'Amaré, A.R. 2009. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch, Nat. Struct. Mol. Biol. 16:1212‐1217.
   Lee, E.R., Baker, J.L., Weinberg, Z., Sudarsan, N., and Breaker, R.R. 2010. An allosteric self‐splicing ribozyme triggered by a bacterial second messenger Science 329:845‐848.
   Romling, U. and Amikam, D. 2006. Cyclic di‐GMP as a second messenger. Curr. Opin. Microbiol. 9:218‐228.
   Ross, P., Mayer, R., Weinhouse, H., Amikam, D., Huggirat, Y., Benziman, M., de Vroom, E., Fidder, A., Paus, P.d., 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.
   Schirmer, T. and Jenal, U. 2009. Structural and mechanistic determinants of c‐di‐GMP signalling. Nat. Rev. Micro. 7:724‐735.
   Smith, K.D., Lipchock, S.V., Ames, T.D., Wang, J., Breaker, R R., and Strobel, S.A. 2009. Structural basis of ligand binding by a c‐di‐GMP riboswitch. Nat. Struct. Mol. Biol. 16:1218‐1223.
   Smith, K.D., Shanahan, C.A., Moore, E.L., Simon, A.C., and Strobel, S.A. 2011. Structural basis of differential ligand recognition by two classes of bis‐(3′‐5′)‐cyclic dimeric guanosine monophosphate‐binding riboswitches. Proc. Natl. Acad. Sci. U.S.A. 108:7757‐7762.
   Sudarsan, N., Lee, E.R., Weinberg, Z., Moy, R.H., Kim, J.N., Link, K.H., and Breaker, R.R. 2008. Riboswitches in eubacteria sense the second messenger cyclic di‐GMP. Science 321:411‐413.
   Tamayo, R., Pratt, J.T., and Camilli, A. 2007. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis, Ann. Rev. Microbiol. 61:131‐148.
   Yan, H. and Aguilar, A.L. 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.
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