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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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:
- N ⁶ ‐benzoyladenin‐9‐yl
- N ² ‐isobutyrylguanin‐9‐yl
- N ⁴ ‐benzoylcytosin‐1‐yl
- thymin‐1‐yl
Argon (Ar) gas, dry
Anhydrous acetonitrile (MeCN; DNA synthesis–grade; H₂ O 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 P₂ O₅ 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 (NaHCO₃ ) solution
Brine (saturated aqueous NaCl)
Magnesium sulfate (MgSO₄ ), 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 (AgNO₃ ), 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:
- N ⁶ ‐phenoxyacetyladenin‐9‐yl
- N ² ‐4‐isopropylphenoxyacetylguanin‐9‐yl
- N ⁴ ‐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)
N ^α ‐(9‐Fluorenylmethoxycarbonyl)‐protected amino acids (Fmoc‐aa‐OH; Novabiochem, Bachem): e.g., for sequences illustrated here, Arg(Pbf), Gly, Leu, Phe, Ser(t Bu), and Tyr(t Bu) (t Bu: 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 )
N ^α ‐(9‐Fluorenylmethoxycarbonyl)‐protected amino acids (Fmoc‐aa‐OH; Novabiochem, Bachem): e.g., for sequences illustrated here, Ala, Arg(Pbf), Asp(Ot Bu), Gln(Trt), Glu(Ot Bu), His(Trt), Lys(Boc), Met, Phe, Ser(t Bu), and Thr(t Bu) (Pbf: 2,2,4,6,7‐pentamethyldihydrobenzofuran‐5‐sulfonyl; Trt: trityl; t Bu: 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

Figure 4.32.1 General scheme for the preparation of peptide‐oligonucleotide conjugates by Diels‐Alder cycloaddition in water.

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Figure 4.32.2 Solid‐phase synthesis of diene‐modified oligonucleotides (A ; see Basic Protocols 1 and 2) and maleimide‐derivatized peptides (B ; see and ). Deprotection of 2′‐deoxyoligoribonucleotides was carried out by treatment with conc. NH₄ OH. For oligoribonucleotides, treatment with conc. NH₄ OH and CH₃ NH₂ was followed by removal of 2′‐ O ‐TBDMS with TEA·3HF in DMSO. Abbreviations: CNE, 2‐cyanoethyl; DMSO, dimethylsulfoxide; Fmoc, 9‐fluorenylmethoxycarbonyl; HOBt, 1‐hydroxybenzotriazole; TBDMS, tert ‐butyldimethylsilyl; TEA, triethylamine; TFA, trifluoroacetic acid.

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Figure 4.32.3 Synthesis of ( E )‐3,5‐hexadienyl‐( N , N ‐diisopropyl)‐2‐cyanoethyl phosphoramidite (see ). Abbreviations: CNE, 2‐cyanoethyl; LDA, lithium diisopropylamide.

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Figure 4.32.4 General scheme for preparation of cysteine‐containing peptide‐oligonucleotide conjugates by Diels‐Alder reaction in water (see ). Abbreviations: Aa, amino acid; Fmoc, 9‐fluorenylmethoxycarbonyl; HOBt, 1‐hydroxybenzotriazole; PG, protecting group; TFA, trifluoroacetic acid.

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Figure 4.32.5 Reversed‐phase HPLC traces of diene‐^5′ dACGTTGAG (A ) and diene‐^5′ dAGCACGGCCATGCATGCTTTGATGA (C ), and their conjugation reactions with maleimide‐Gly‐Arg‐Arg‐Leu‐Ser‐Tyr‐Ser‐Arg‐Arg‐Arg‐Phe‐NH₂ after 30 min (B and D , respectively). See Basic Protocols 1 and 4 for gradient conditions, eluents, and column employed.

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Figure 4.32.6 Reversed‐phase HPLC traces of diene‐^5′ rundefinedUCAACGUGAGundefinedundefined (A ), its conjugation reaction with maleimide‐Gly‐Arg‐Arg‐Leu‐Ser‐Tyr‐Ser‐Arg‐Arg‐Arg‐Phe‐NH₂ after 30 min (B ), the conjugation reaction between diene‐^5′ dT₁₅ and maleimide‐Lys‐Glu‐Thr‐Ala‐Ala‐Ala‐Lys‐Phe‐Glu‐Arg‐Gln‐His‐Met‐Asp‐Ser‐Ser‐Thr‐Ser‐Ala‐Ala‐OH after 20 hr (C ), and 8 hr later after having added the amount of peptide equivalent to that of unreacted oligonucleotide (D ). See Basic Protocols 1, 2, and 4 for gradient conditions, eluents, and column employed. Panels C and D from Marchán et al. ().

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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.
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	Pirrung, M.C. 2006. Acceleration of organic reactions through aqueous solvent effects. Chem. Eur. J. 12:1312‐1317.
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	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.
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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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Synthesis of Peptide‐Oligonucleotide Conjugates by Diels‐Alder Cycloaddition in Water

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited