Solid‐Phase Synthesis of Oligo‐ADP‐Ribose

Hans A.V. Kistemaker1, Nico J. Meeuwenoord1, Herman S. Overkleeft1, Gijsbert A. van der Marel1, Dmitri V. Filippov1

1 Department of Bio‐organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.68
DOI:  10.1002/0471142700.nc0468s64
Online Posting Date:  March, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Solid‐phase methodology for synthesis of adenosine diphosphate ribose oligomers (ADPr‐oligomers) of defined length is described using an advanced 2‐ribosyl adenosine phosphoramidite building block that is prepared in solution via an expeditious and high‐yielding method. The methodology is based on phosphitylation of a phosphomonoester as the key condensation step in the solid‐phase protocol. As ADP‐ribosylation appears to be an important yet poorly understood post‐translational modification of proteins, the presented methodology for rapid synthesis of ADPr‐fragments of exactly defined length should be of interest for researchers in the field of structural biology and cell biology. © 2016 by John Wiley & Sons, Inc.

Keywords: solid‐phase synthesis; poly(ADP‐ribose); phosphorylation; pyrophosphate

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Phosphoramidite of 2′‐Ribosyladenosine
  • Support Protocol 1: Synthesis of N‐(Phenyl)Trifluoroacetimidate Ribosyl Donor
  • Support Protocol 2: Preparation of HClO4‐SiO2
  • Basic Protocol 2: Solid‐Phase Synthesis of Oligo‐ADP‐Ribose
  • Support Protocol 3: Immobilization of 5′‐O‐Phosphate Methyl Ribofuranoside on CPG via The Q‐Linker
  • Support Protocol 4: Synthesis of Adenosine Phosphoramidite 12
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Phosphoramidite of 2′‐Ribosyladenosine

  Materials
  • Trifluoroacetimidate donor (1, see protocol 2)
  • 1,3,5‐Tri‐O‐benzoyl‐α‐D‐ribofuranose (2, >97.0% HPLC, TCI Europe)
  • 1,4‐Dioxane (Sigma‐Aldrich)
  • 1,2‐Dichloroethane (DCE)
  • Argon (or nitrogen) gas
  • Dichloromethane (DCM)
  • 3‐Å molecular sieves (Sigma‐Aldrich)
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 99%)
  • Triethylamine (Et 3N)
  • Pentane
  • Ethyl acetate (EtOAc)
  • tert‐Butanol (tBuOH, 5.5 M in nonane)
  • Acetic acid (AcOH)
  • Palladium on carbon (Pd/C, 10 w% Pd)
  • Hydrogen (H 2) gas
  • Celite
  • Toluene
  • Pyridine
  • 4‐(Dimethylamino)pyridine (DMAP)
  • Acetic anhydride (Ac 2O)
  • Sodium hydrogen carbonate (sat. aq. NaHCO 3)
  • Brine
  • Magnesium sulfate (MgSO 4)
  • N6‐Benzoyladenine
  • Acetonitrile (MeCN)
  • N,O‐Bis(trimethylsilyl)trifluoroacetamide (BSTFA)
  • HClO 4‐SiO 2 (see protocol 3)
  • Ethanol (EtOH)
  • 1 M aq. sodium hydroxide (NaOH)
  • Amberlite‐H+
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Acetone
  • Triethylamine trihydrofluoride (Et 3N·3HF)
  • Methanol (MeOH)
  • 1‐Methylimidazole hydrochloride (1‐MeIm·HCl)
  • 1‐Methylimidazole (1‐MeIm)
  • Dimethylformamide (DMF)
  • Di‐tert‐butyl‐N,N‐diisopropylphosphoramidite
  • tert‐Butyl hydroperoxide (tBuOOH, 5.5 M in nonane)
  • Trifluoroacetic acid (TFA)
  • N,N‐Diisopropylethylamine (DIPEA)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite
  • 25‐, 50‐, 100‐, and 250‐mL round‐bottom flasks with rubber septa
  • Rotary evaporator equipped with a membrane pump (or water aspirator)
  • Sintered glass filters (P3)
  • Automated silica gel chromatography system (e.g., Biotage Isolera Spektra Four with Biotage ZIP flash cartridges)
  • Vacuum oil pump
  • Reflux condenser
  • TLC plate: silica‐coated aluminum with fluorescent indicator (Merck silica 60 F 254)
  • 254‐nm UV lamp

Support Protocol 1: Synthesis of N‐(Phenyl)Trifluoroacetimidate Ribosyl Donor

  Additional materials (also see protocol 1)
  • D‐Ribose
  • Allyl alcohol
  • Acetyl chloride
  • Imidazole
  • Triisopropylsilyl chloride (TIPS‐Cl)
  • Diethyl ether (Et 2O)
  • Sodium hydride (NaH, 60% in mineral oil)
  • Benzyl bromide (BnBr)
  • Tetrahydrofuran (THF)
  • (1,5‐Cyclooctadiene)(pyridine)(tricyclohexylphosphine)‐iridium hexafluorophosphate (Ir(COD)(Ph2MeP)2PF6)
  • Iodine (I 2)
  • Sodium thiosulfate (sat. aq. Na 2S 2O 3)
  • Cesium carbonate (Cs 2CO 3)
  • 2,2,2‐Trifluoro‐N‐phenylacetimidoyl chloride (TCI Europe)

Support Protocol 2: Preparation of HClO4‐SiO2

  Materials
  • Silica gel (200 mesh)
  • Diethyl ether (Et 2O)
  • 70% aqueous perchloric acid (HClO 4)
  • Argon gas
  • Rotary evaporator equipped with a membrane pump (or water aspirator)
  • Vacuum oil pump

Basic Protocol 2: Solid‐Phase Synthesis of Oligo‐ADP‐Ribose

  Materials
  • Adenosine phosphoramidites 10 and 12 (see protocol 1 and protocol 6)
  • 1,8‐Diazabicycloundec‐7‐ene (DBU)
  • Dimethylformamide (DMF)
  • (1S)‐(+)‐(10‐Camphorsulfonyl)‐oxaziridine (CSO)
  • Acetonitrile (MeCN, anhydrous, Biosolve)
  • Hydrochloric acid (HCl), concentrated
  • Hexafluoroisopropanol (HFIP)
  • Pyridine
  • 5‐(Ethylthio)‐1H‐tetrazole (ETT)
  • 3‐Å molecular sieves
  • Preloaded CPG resin 11 (see protocol 5)
  • Argon gas
  • 35% aqueous ammonium hydroxide (NH 4OH, Across)
  • 0.15 M triethylammonium acetate (TEAA) in Milli‐Q‐purified water
  • Mobile phase A: 20 mM ammonium acetate (NH 4OAc) in Milli‐Q‐purified water
  • Mobile phase B: 1 M NH 4OAc in Milli‐Q‐purified water
  • Mermade‐6 oligonucleotide synthesizer (Bioautomation corporation)
  • Screw‐cap tube
  • AKTA‐Explorer‐10 FPLC equipped with Frac‐950 Fraction Collector (GE Healthcare) and:
  • Superdex‐30‐HR column (16 × 600 mm)
  • Source‐15Q column (16 × 100 mm; GE Healthcare)
  • Lyophilizer (Christ Alpha 2‐4 LDplus)

Support Protocol 3: Immobilization of 5′‐O‐Phosphate Methyl Ribofuranoside on CPG via The Q‐Linker

  Materials
  • D‐Ribose
  • Methanol (MeOH)
  • Acetyl chloride
  • Pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Argon (or nitrogen) gas
  • Ethyl acetate (EtOAc)
  • Sodium hydrogen carbonate (sat. aq. NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Pentane
  • Triethylamine (Et 3N)
  • Acetic anhydride (Ac 2O)
  • 4‐(Dimethylamino)pyridine (DMAP)
  • 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC)
  • Hydroquinone‐O,O′‐diacetic acid (Q‐linker)
  • Chloroform (CHCl 3)
  • Dichloromethane (DCM)
  • Ethanol (EtOH)
  • Long‐chain alkyl amino controlled‐pore glass (LCAA‐CPG, 120‐200 mesh, 500 Å)
  • Acetonitrile (MeCN)
  • 1‐Hydroxybenzotriazole (HOBT)
  • N,N′‐Diisopropylcarbodiimide (DIC)
  • N,N′‐Diisopropylethylamine (DIPEA)
  • Dimethylformamide (DMF)
  • Cap A: 80:10:10 (v/v/v) tetrahydrofuran/acetic anhydride/2,6‐lutidine (Sigma‐Aldrich, cat. no. 555312)
  • Cap B: 84:16 (v/v) tetrahydrofuran/N‐methylimidazole (Sigma‐Aldrich, cat. no. L050040)
  • Trichloroacetic acid (TCA)
  • Trifluoroacetic acid (TFA)
  • 1‐Methylimidazolium chloride
  • Di‐tert‐butyl‐N,N‐diisopropylphosphoramidite
  • (1S)‐(+)‐(10‐Camphorsulfonyl)‐oxaziridine (CSO)
  • 35% aqueous ammonia (NH 4OH)
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Rotary evaporator equipped with a membrane pump (or water aspirator)
  • Vacuum oil pump
  • Automated silica gel chromatography system (e.g., Biotage Isolera Spektra Four with Biotage ZIP flash cartridges)
  • Fritted columns (20‐mL syringe with frit, cap, plunger or stopper, and septum)
  • UV‐vis spectrophotometer

Support Protocol 4: Synthesis of Adenosine Phosphoramidite 12

  Materials
  • Adenosine
  • Pyridine
  • Argon (or nitrogen) gas
  • Trimethylsilyl chloride (TMS‐Cl)
  • Benzoyl chloride
  • 29% aqueous ammonia (NH 4OH)
  • Ethyl acetate (EtOAc )
  • Diethyl ether (Et 2O)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl)
  • Isobutyric anhydride
  • Dichloromethane (DCM)
  • 5% (v/v) aqueous citric acid
  • Magnesium sulfate (MgSO 4)
  • Acetonitrile (MeCN)
  • para‐Toluenesulfonic acid (p‐TsOH)
  • Petroleum ether
  • N,N‐Diisopropylethylamine (DIPEA)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite
  • Sodium hydrogen carbonate (sat. aq. NaHCO 3)
  • Pentane
  • 100‐mL round‐bottom flasks
  • Rotary evaporator equipped with a membrane pump (or water aspirator)
  • Vacuum oil pump
  • Automated silica gel chromatography system (e.g., Biotage Isolera Spektra Four with Biotage ZIP flash cartridges)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Agarwal, A., Rani, S., and Vankar, Y.D. 2004. Protic acid (HClO4 supported on silica gel)‐mediated synthesis of 2,3‐unsaturated‐O‐glucosides and a chiral furan diol from 2,3‐glycals. J. Org. Chem. 69:6137‐6140. doi: 10.1021/jo049415j.
  Chakraborti, A.K. and Gulhane, R. 2003. Perchloric acid adsorbed on silica gel as a new, highly efficient, and versatile catalyst for acetylation of phenols, thiols, alcohols, and amines. Chem. Commun. 2003:1896‐1897. doi: 10.1039/b304178f.
  Framski, G., Gdaniec, Z., Gdaniec, M., and Boryski, J. 2006. A reinvestigated mechanism of ribosylation of adenine under silylating conditions. Tetrahedron 62:10123‐10129. doi: 10.1016/j.tet.2006.08.046.
  Gibson, B.A. and Kraus, W.L. 2012. New insights into the molecular and cellular functions of poly(ADP‐ribose) and PARPs. Nat. Rev. Mol. Cell Bio. 13:411‐424. doi: 10.1038/nrm3376.
  Gold, H., van Delft, P., Meeuwenoord, N., Codee, J.D.C., Filippov, D.V., Eggink, G., Overkleeft, H.S., and van der Marel, G.A. 2008. Synthesis of sugar nucleotides by application of phosphoramidites. J. Org. Chem. 73:9458‐9460. doi: 10.1021/jo802021t.
  Hache, B., Brett, L., and Shakya, S. 2011. Phosphonate‐free phosphorylation of alcohols using bis‐(tert‐butyl) phosphoramidite with imidazole hydrochloride and imidazole as the activator. Tetrahedron Lett. 52:3625‐3629. doi: 10.1016/j.tetlet.2011.05.020.
  Kistemaker, H.A.V., van der Heden van Noort, G.J., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2013. Stereoselective ribosylation of amino acids. Org. Lett. 15:2306‐2309. doi: 10.1021/ol400929c.
  Kistemaker, H.A.V., Lameijer, L.N., Meeuwenoord, N.J., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2015a. Synthesis of well‐defined adenosine diphosphate ribose oligomers. Angew Chem. Int. Ed. Engl. 54:4915‐4918. doi: 10.1002/anie.201412283.
  Kistemaker, H.A.V., Meeuwenoord, N.J., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2015b. On the synthesis of oligonucleotides interconnected through pyrophosphate linkages. Eur. J. Org. Chem. 2015:6084‐6091. doi: 10.1002/ejoc.201500911.
  Kistemaker, H.A.V., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2015c. Branching of poly(ADP‐ribose): Synthesis of the core motif. Org. Lett. 17:4328‐4331. doi: 10.1021/acs.orglett.5b02143.
  Lambrecht, M.J., Brichacek, M., Barkauskaite, E., Ariza, A., Ahel, I., and Hergenrother, P.J. 2015. Synthesis of dimeric ADP‐ribose and its structure with human poly(ADP‐ribose) glycohydrolase. J. Am. Chem. Soc. 137:3558‐3564. doi: 10.1021/ja512528p.
  Mikhailov, S.N., Kulikova, I.V., Nauwelaerts, K., and Herdewijn, P. 2008. Synthesis of 2′‐O‐alpha‐D‐ribofuranosyladenosine, monomeric unit of poly(ADP‐ribose). Tetrahedron 64:2871‐2876. doi: 10.1016/j.tet.2008.01.028.
  Moyle, P.M. and Muir, T.W. 2010. Method for the synthesis of mono‐ADP‐ribose conjugated peptides. J. Am. Chem. Soc. 132:15878‐15880. doi: 10.1021/ja1064312.
  Napolitano, J.G., Gavin, J.A., Garcia, C., Norte, M., Fernandez, J.J., and Daranas, A.H. 2011. On the configuration of five‐membered rings: A spin‐spin coupling constant approach. Chem. Eur. J. 17:6338‐6347. doi: 10.1002/chem.201100412.
  Niedball, U. and Vorbrüggen, H. 1970. Synthesis of nucleosides. 3. A general synthesis of pyrimidine nucleosides. Angew. Chem. Int. Ed. 9:461‐462. doi: 10.1002/anie.197004612.
  Pon, R.T. and Yu, S.Y. 1997. Hydroquinone‐O,O′‐diacetic acid (`Q‐linker') as a replacement for succinyl and oxalyl linker arms in solid phase oligonucleotide synthesis. Nucleic. Acids Res. 25:3629‐3635. doi: 10.1093/nar/25.18.3629.
  Richert, C., Roughton, A.L., and Benner, S.A. 1996. Nonionic analogs of RNA with dimethylene sulfone bridges. J. Am. Chem. Soc. 118:4518‐4531. doi: 10.1021/ja952322m.
  Ryan, K.J., Acton, E.M., and Goodman, L. 1971. Synthesis of 2‐thio‐D‐ribose and 2′‐thioadenosine derivatives. J. Org. Chem. 36:2646‐2657. doi: 10.1021/jo00817a018.
  Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection—efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319. doi: 10.1021/ja00369a029.
  van der Heden van Noort, G.J., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2010a. Synthesis of nucleotidylated poliovirus VPg proteins. J . Org. Chem. 75:5733‐5736. doi: 10.1021/jo100757t.
  van der Heden van Noort, G.J., van der Horst, M.G., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2010b. Synthesis of mono‐ADP‐ribosylated oligopeptides using ribosylated amino acid building blocks. J. Am. Chem. Soc. 132:5236‐5240. doi: 10.1021/ja910940q.
  van der Heden van Noort, G.J., Overkleeft, H.S., van der Marel, G.A., and Filippov, D.V. 2011. Ribosylation of adenosine: An orthogonally protected building block for the synthesis of ADP‐ribosyl oligomers. Org. Lett. 13:2920‐2923. doi: 10.1021/ol200971z.
Key References
  Kistemaker et al., 2015a. See above.
  Basic Protocols 1 and 2 are based on this article.
  Kistemaker et al., 2015b. See above.
  This article questions the previous methodology for solid‐phase synthesis of oligonucleotides containing repetitive pyrophosphate bridges and proves it to be irreproducible.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library