Synthesis of Peptide‐Oligonucleotide Conjugates by Diels‐Alder Cycloaddition in Water

Vicente Marchán1, Anna Grandas1

1 Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.32
DOI:  10.1002/0471142700.nc0432s31
Online Posting Date:  December, 2007
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Abstract

Peptide‐oligonucleotide conjugates incorporating all the nucleobases and trifunctional amino acids are obtained by Diels‐Alder reaction between diene‐modified oligonucleotides (2′‐deoxyribo‐ or ribo‐) and malemide‐derivatized peptides. Both reagents are easily synthesized by on‐column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction is carried out under mild conditions, in aqueous solution at 37°C, affording the desired peptide‐oligonucleotide conjugate with high purity and yield. The speed of the reaction depends on the size and composition of both reagents, but it is accelerated by the presence of positively charged amino acids in the peptide fragment. However, a small excess of maleimide‐derivatized peptide may be required in some cases to complete the reaction within 8 to 10 hr. Curr. Protoc. Nucleic Acid Chem. 31:4.32.1‐4.32.31. © 2007 by John Wiley & Sons, Inc.

Keywords: peptide‐oligonucleotide conjugate; Diels‐Alder cycloaddition; oligonucleotide synthesis; peptide synthesis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Synthesis of 5′‐Diene‐Modified 2′‐Deoxyoligoribonucleotides
  • Support Protocol 1: Synthesis of Diene‐Phosphoramidite
  • Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligoribonucleotides
  • Basic Protocol 3: Synthesis of Maleimide‐Modified Peptides
  • Alternate Protocol 1: Synthesis of Maleimide‐Modified Peptides on a 2‐Chlorotrityl Chloride Resin
  • Basic Protocol 4: Preparation of Peptide‐Oligonucleotide Conjugates by Diels‐Alder Cycloaddition in Water
  • Alternate Protocol 2: Preparation of Cysteine‐Containing Peptide‐Oligonucleotide Conjugates
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐Diene‐Modified 2′‐Deoxyoligoribonucleotides

  Materials
  • DNA phosphoramidites (Glen Research): 5′‐O‐(4,4′‐dimethoxytrityl)‐N‐protected‐2′‐deoxyribonucleoside‐3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl)phosphoramidites, where the nucleobases are:
    • N6‐benzoyladenin‐9‐yl
    • N2‐isobutyrylguanin‐9‐yl
    • N4‐benzoylcytosin‐1‐yl
    • thymin‐1‐yl
  • Argon (Ar) gas, dry
  • Anhydrous acetonitrile (MeCN; DNA synthesis–grade; H 2O content <30 ppm, J.T. Baker)
  • Diene‐phosphoramidite (see protocol 2)
  • Dichloromethane (DCM, HPLC‐grade)
  • Deblocking solution: 3% (w/v) trichloroacetic acid (TCA) in dichloromethane (Glen Research)
  • Activator solution: 0.45 M 1H‐tetrazole in anhydrous acetonitrile (Glen Research)
  • Capping A solution: acetic anhydride in tetrahydrofuran (Glen Research)
  • Capping B solution: 10% (w/v) 1‐methylimidazole in tetrahydrofuran/pyridine (Glen Research)
  • Oxidizer solution: 0.02 M iodine in tetrahydrofuran/water/pyridine (Glen Research)
  • 32% (v/v) ammonium hydroxide (store at 4°C)
  • Deionized, Milli‐Q‐purified water (Millipore; 18 mΩ/cm)
  • Methanol (MeOH, HPLC‐grade)
  • Empty DNA synthesizer bottles, oven‐dried, with rubber septa
  • Glass syringes, cannulae and needles, oven‐dried
  • Vacuum desiccator containing P 2O 5 and KOH
  • 50‐mL round‐bottom flasks, oven‐dried, with rubber septa
  • Rotary evaporator equipped with a water aspirator or vacuum pump
  • Automatic DNA synthesizer (e.g., ABI 3400, Applied Biosystems)
  • Synthesis columns for 1‐µmol scale synthesis (Applied Biosystems or Glen Research), controlled‐pore glass (CPG, 500 Å)
  • 4‐mL screw‐cap pressure tubes, O‐ring seal preferred
  • 55°C oven
  • Disposable polypropylene syringes and polyethylene filters (cotton optional in some steps)
  • Microcentrifuge tubes
  • Lyophilizer or SpeedVac evaporator
  • Double‐beam UV spectrophotometer, calibrated, and quartz cuvettes
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C) and for analysis, purification, and characterization (units 10.1 10.7)

Support Protocol 1: Synthesis of Diene‐Phosphoramidite

  Materials
  • Argon (Ar), dry
  • Tetrahydrofuran, anhydrous (unit 4.22)
  • N,N‐Diisopropylamine, anhydrous: place freshly distilled N,N‐diisopropylamine (Aldrich, b.p. 83° to 84°C) over 4‐Å molecular sieves and under Ar atmosphere overnight
  • Dry ice/acetone bath (reagent‐grade acetone)
  • 2.2 M n‐butyllithium solution in hexanes (Aldrich)
  • Hexamethylphosphoramide, anhydrous: keep over 4‐Å molecular sieves and under Ar atmosphere overnight
  • Ethyl (E)‐2,4‐hexadienoate (ethyl sorbate, Aldrich)
  • Glacial acetic acid
  • Diethyl ether, anhydrous when required (Aldrich)
  • 2.5% (w/v) aqueous sodium hydrogencarbonate (NaHCO 3) solution
  • Brine (saturated aqueous NaCl)
  • Magnesium sulfate (MgSO 4), anhydrous
  • Lithium aluminum hydride
  • 2 M aqueous sodium hydroxide (NaOH)
  • Acetonitrile (MeCN; HPLC‐grade)
  • Dichloromethane (DCM), anhydrous (unit 4.22)
  • Triethylamine (TEA), anhydrous: place under calcium hydride lumps under an Ar atmosphere for at least one night before use; prepare fresh before each use
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • Methanol (MeOH)
  • Iodine
  • Silver nitrate (AgNO 3), optional
  • Dichloromethane, neutralized (unit 4.22)
  • Hexanes
  • 230‐ to 400‐mesh silica gel
  • 25‐, 50‐, 100‐, 250‐, 500‐, and 1000‐mL round‐bottom flasks (oven‐dried when needed) and rubber septa
  • Glass syringes, needles, and cannulae, oven‐dried
  • 1‐L flasks
  • 250‐mL, 500‐mL, and 1‐L separatory funnels
  • Gravity filtration device and filter paper
  • Rotary evaporator equipped with a water aspirator or vacuum aspirator
  • 250‐mL three‐neck, oven‐dried, round‐bottom flask with rubber septa
  • 100‐mL oven‐dried addition funnel with pressure‐equalization arm and rubber septum
  • Oven‐dried reflux condenser closed by a calcium chloride guard tube
  • Vacuum distillation apparatus
  • 10 to 20 mmHg vacuum system
  • 2 × 5–cm silica‐coated thin‐layer chromatography (TLC) plates
  • 254‐nm UV light source
  • 5 × 25–cm glass chromatography column with solvent reservoir bulb
  • Additional reagents and equipment for TLC ( appendix 3C), column chromatography ( appendix 3E), NMR, and mass spectrometry

Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligoribonucleotides

  Materials
  • RNA phosphoramidites (Link Technologies): 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(methyl or tert‐butyldimethylsilyl)‐N‐protected‐ribonucleoside‐3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl)phosphoramidites, where the nucleobases are:
    • N6‐phenoxyacetyladenin‐9‐yl
    • N2‐4‐isopropylphenoxyacetylguanin‐9‐yl
    • N4‐acetylcytosin‐1‐yl
    • uridin‐1‐yl
  • Anhydrous acetonitrile (MeCN)
  • Activator solution: 0.3 M benzylthio‐1H‐tetrazole (BTT, Link Technologies) in anhydrous acetonitrile
  • Dichloromethane (DCM, HPLC‐grade)
  • Deblocking solution: 3% (w/v) trichloroacetic acid (TCA) in dichloromethane (Glen Research)
  • Capping A solution: acetic anhydride in tetrahydrofuran (Glen Research)
  • Capping B solution: 10% (w/v) 1‐methylimidazole in tetrahydrofuran/pyridine (Glen Research)
  • Oxidizer solution: 0.02 M iodine in tetrahydrofuran/water/pyridine (Glen Research)
  • Argon source (Ar)
  • 32% aqueous ammonium hydroxide
  • 33% (w/v) methylamine in anhydrous ethanol (Aldrich)
  • Ethanol, 1:1 (v/v) in water, and anhydrous
  • Dimethylsulfoxide, anhydrous (DMSO, Fluka)
  • Triethylamine tris(hydrofluoride) (Aldrich)
  • Isopropyl trimethylsilyl ether (prepare as described by Song and Jones, )
  • Diethyl ether, anhydrous
  • RNase‐free water: mix Milli‐Q water vigorously with 0.1% DEPC overnight and autoclave twice, or obtain RNase‐free water from Milli‐Q system equipped with a 5000‐Da ultrafiltration cartridge
  • 0.1 M ammonium bicarbonate in RNase‐free water (made from RNase‐free solid, Fluka)
  • ABI 3400 synthesizer (or equivalent)
  • Synthesis columns for 1‐µmol scale (Link Technologies or Glen Research), controlled‐pore glass (CPG, 500‐Å)
  • 4‐mL screw‐cap pressure tubes with O‐ring seals
  • 65°C oven or heat block
  • 15‐mL centrifuge tubes
  • Rotary evaporator
  • Lyophilizer
  • RNase‐free materials for manipulating diene‐oligoribonucleotides (e.g., microcentrifuge tubes, tips, HPLC bottles, and columns)
  • Additional reagents and equipment for automated oligonucleotide synthesis, analysis, purification, and characterization (see protocol 1, appendix 3C, units 3.5, 3.6, & 10.1 10.7)

Basic Protocol 3: Synthesis of Maleimide‐Modified Peptides

  Materials
  • Rink amide p‐methylbenzhydrylamine solid support (Novabiochem)
  • Dichloromethane (DCM; peptide synthesis–grade)
  • N,N‐Dimethylformamide (DMF; peptide synthesis–grade)
  • Nα‐(9‐Fluorenylmethoxycarbonyl)‐protected amino acids (Fmoc‐aa‐OH; Novabiochem, Bachem): e.g., for sequences illustrated here, Arg(Pbf), Gly, Leu, Phe, Ser(tBu), and Tyr(tBu) (tBu: tert‐butyl; Pbf: 2,2,4,6,7‐pentamethyldihydrobenzofuran‐5‐sulfonyl)
  • O‐(7‐Azabenzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium hexafluorophosphate (HATU)
  • N,N‐Dimethylformamide, anhydrous: place over 4‐Å molecular sieves overnight and bubble with nitrogen for 2 to 3 hr to remove volatile amines
  • N,N‐Diisopropylethylamine (DIPEA)
  • Methanol (MeOH; HPLC‐grade)
  • Acetic anhydride
  • 20% (v/v) piperidine in N,N‐dimethylformamide
  • N,N′‐Dicyclohexylcarbodiimide (DCC) or N,N′‐diisopropylcarbodiimide (DiPC)
  • 1‐Hydroxybenzotriazole (HOBt)
  • 3‐Maleimidepropanoic acid (Aldrich, Bachem)
  • Trifluoroacetic acid (TFA, HPLC‐grade)
  • Triisopropylsilane (TIS)
  • Phenol, crystalline
  • Diethyl ether, anhydrous, cold
  • Deionized, Milli‐Q‐purified water (Millipore, 18 mΩ/cm)
  • Concentrated HCl
  • Acetonitrile (MeCN, HPLC‐grade)
  • Buffer (e.g., citrate buffer, pH 2)
  • 2‐, 5‐, and 10‐mL disposable polypropylene syringes with porous polyethylene discs and Teflon two‐way stopcocks (RTV SF2, Shimadzu Scientific Research)
  • Vacuum filtration system
  • Teflon stir rod
  • Desiccator
  • 50‐mL centrifuge tubes
  • Lyophilizer
  • Pyrex tubes for amino acid analyses
  • Methane/oxygen flame
  • 110° and 155°C heating blocks
  • Rotary evaporator equipped with a water aspirator or vacuum pump
  • 0.45‐µm nylon filters
  • Automatic amino acid analyzer (e.g., Beckman)
  • Double‐beam UV spectrophotometer, calibrated, and quartz cuvettes
  • Additional reagents and equipment for manual solid‐phase peptide synthesis, analysis, purification, and characterization (units 4.22& 4.28)

Alternate Protocol 1: Synthesis of Maleimide‐Modified Peptides on a 2‐Chlorotrityl Chloride Resin

  • 2‐Chlorotrityl chloride resin (Novabiochem)
  • Neutralized and anhydrous dichloromethane (DCM; unit 4.22)
  • Nα‐(9‐Fluorenylmethoxycarbonyl)‐protected amino acids (Fmoc‐aa‐OH; Novabiochem, Bachem): e.g., for sequences illustrated here, Ala, Arg(Pbf), Asp(OtBu), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Met, Phe, Ser(tBu), and Thr(tBu) (Pbf: 2,2,4,6,7‐pentamethyldihydrobenzofuran‐5‐sulfonyl; Trt: trityl; tBu: tert‐butyl; Boc: tert‐butoxycarbonyl)
  • Thioanisole
  • 50‐mL round‐bottom flask
  • Additional reagents and equipment for manual solid‐phase peptide synthesis, analysis, purification, and characterization (see protocol 4 and unit 4.22)

Basic Protocol 4: Preparation of Peptide‐Oligonucleotide Conjugates by Diels‐Alder Cycloaddition in Water

  Materials
  • Aqueous solution of diene‐modified oligonucleotide of known concentration (see Basic Protocols protocol 11 and protocol 32)
  • Aqueous solution of maleimide‐derivatized peptide of known concentration (see protocol 4 and protocol 5)
  • Milli‐Q‐purified water
  • 0.5‐ to 1.5‐mL microcentrifuge tubes
  • 37°C heating block
  • Additional reagents and equipment for analysis, purification, and characterization of oligonucleotides and analogs (units 4.18, 4.22, 4.28& 10.1 10.7)

Alternate Protocol 2: Preparation of Cysteine‐Containing Peptide‐Oligonucleotide Conjugates

  • Aqueous solution, of known concentration, of maleimide‐derivatized peptides with cysteine residues protected with the S‐tert‐butyl group (see protocol 4 and protocol 5)
  • 0.05 M aqueous ammonium acetate
  • n‐Propanol
  • Argon source (Ar)
  • Tris(n‐butyl)phosphane
  • Sephadex G‐10
  • 2‐mL glass flasks and rubber septa
  • Lyophilizer
  • Rotary evaporator equipped with a vacuum aspirator
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Figures

Videos

Literature Cited

   Barlos, K., Chatzi, O., Gatos, D., and Stavropoulos, G. 1991. 2‐Chlorotrityl chloride resin. Studies on anchoring of Fmoc‐amino acids and peptide cleavage. Int. J. Peptide Protein Res. 37:513‐520.
   Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223‐2311.
   Breslow, R. 1991. Hydrophobic effects on simple organic reactions in water. Acc. Chem. Res. 24:159‐164.
   Chandrasekhar, J., Shariffskul, S., and Jorgensen, W.L. 2002. QM/MM Simulations for Diels‐Alder reactions in water: Contribution of enhanced hydrogen bonding at the transition state to the solvent effect. J. Phys. Chem. B 106:8078‐8085.
   Chollet, A. 1990. Selective attachment of oligonucleotides to interleukin‐1b and targeted delivery to cells. Nucleosides Nucleotides Nucleic Acids 9:957‐966.
   Dantas de Araújo, A., Palomo, J.M., Cramer, J., Koehn, M., Schroeder, H., Wacker, R., Niemeyer, C., Alexandrov, K., and Waldmann, H. 2006. Diels‐Alder ligation and surface immobilization of proteins. Angew. Chem. Int. Ed. 45:296‐301.
   Fields, G.B. and Noble, R.L. 1990. Solid‐phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161‐214.
   Gregory, J.D. 1955. The stability of N‐ethylmaleimide and its reaction with sulfhydryl groups. J. Am. Chem. Soc. 77:3922‐3923.
   Hill, K.W., Taunton‐Rigby, J., Carter, J.D., Kropp, E., Vagle, K., Pieken, W., McGee, D.P.C., Husar, G.M., Leuck, M., Anziano, D.J., and Sebesta, D.P. 2001. Diels‐Alder bioconjugation of diene‐modified oligonucleotides. J. Org. Chem. 66:5352‐5358.
   Ikeda, Y., Kawahara, S., Yoshinari, K., Fujita, S., and Taira, K. 2005. Specific 3′‐terminal modification of DNA with a novel nucleoside analogue that allows a covalent linkage of a nuclear localization signal and enhancement of DNA stability. ChemBioChem 6:297‐303.
   Latham‐Timmons, H.A., Wolter, A., Roach, J.S., Giare, R., and Leuck, M. 2003. Novel method for the covalent immobilization of oligonucleotides via Diels‐Alder bioconjugation. Nucleosides Nucleotides Nucleic Acids 22:1495‐1497.
   Li, C.J. 2005. Organic reactions in aqueous media with a focus on carbon‐carbon bond formations. A decade update. Chem. Rev. 105:3095‐3165.
   Marchán, V., Ortega, S., Pulido, D., Pedroso, E., and Grandas, A. 2006a. Diels‐Alder cycloadditions in water for the straightforward preparation of peptide‐oligonucleotides conjugates. Nucleic Acids Res. 34:1‐9.
   Marchán, V., Pulido, D., Pedroso, E., and Grandas, A. 2006b. Linking the 3′ ends of oligonucleotide duplexes with cystine disulfide bridges. Eur. J. Org. Chem. 958‐963.
   Miller, C.A. and Batey, R.A. 2004. Hetero Diels‐Alder reactions of nitrosoamidines: An efficient method for the synthesis of functionalized guanidines. Org. Lett. 6:699‐702.
   Otto, S. and Engberts, J.B.F.N. 2003. Hydrophobic interactions and chemical reactivity. Org. Biomol. Chem. 1:2809‐2820.
   Pirrung, M.C. 2006. Acceleration of organic reactions through aqueous solvent effects. Chem. Eur. J. 12:1312‐1317.
   Pozsgay, V., Vieira, N.E., and Yergey, A. 2002. A method for bioconjugation of carbohydrates using Diels‐Alder cycloaddition. Org. Lett. 4:3191‐3194.
   Pulido, D., López‐Alonso, J.P., Marchán, V., González, C., Grandas, A., and Laurents, D.V. Preparation of ribonuclease S domain–swapped dimers conjugated with DNA and RNA: Modulating the activity of ribonucleases. Bioconjug. Chem. In press.
   Rideout, D.C. and Breslow, R. 1980. Hydrophobic acceleration of Diels‐Alder reactions. J. Am. Chem. Soc. 102:7816‐7817.
   Rink, H. 1987. Solid‐phase synthesis of protected peptide fragments using a trialkoxy‐diphenyl‐methylester resin. Tetrahedron Lett. 28:3787‐3790.
   Song, Q. and Jones, R.A. 1999. Use of silyl ethers as fluoride scavengers in RNA synthesis. Tetrahedron Lett. 40:4653‐4654.
   Sproat, B.S. 2005. RNA synthesis using 2′‐O‐(tert‐butyldimethylsilyl) protection, Vol. 288. In Methods in Molecular Biology. Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed.) pp. 17‐31. Humana Press, Totowa, N.J.
   Sun, X.L., Stabler, C.L., Cazalis, C.S., and Chaikof, E.L. 2006. Carbohydrate and protein immobilization onto solid surfaces by sequential Diels‐Alder and azide‐alkyne cycloadditions. Bioconjug. Chem. 17:52‐57.
   Temsamani, J., Bonnafous, C., Rousselle, C., Fraisse, Y., Clair, P., Granier, L.A., Rees, A.R., Kaczorek, M., and Scherrmann, J.M. 2005. Improved brain uptake and pharmacological activity profile of morphine‐6‐glucuronide using a peptide vector‐mediated strategy. J. Pharmacol. Exp. Ther. 313:712‐719.
   Tung, C.H., Rudolph, M.J., and Stein, S. 1991. Preparation of oligonucleotide‐peptide conjugates. Bioconjug. Chem. 2:464‐465.
   Venkatesan, N. and Kim, B.H. 2006. Peptide conjugates of oligonucleotides: Synthesis and applications. Chem. Rev. 106:3712‐3761.
Key References
   Marchán et al., 2006a. See above.
  This paper describes for the first time the synthesis of peptide‐oligonucleotide conjugates by using the Diels‐Alder cycloaddition in water.
   Venkatesan and Kim, 2006. See above.
  Review covering synthesis and applications of peptide‐oligonucleotide conjugates.
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