Stepwise Solid‐Phase Synthesis of Nucleopeptides

Anna Grandas1, Vicente Marchán1, Laurent Debéthune1, Enrique Pedroso1

1 Institut de Biomedicina de la Universitat de Barcelona, Barcelona
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.22
DOI:  10.1002/0471142700.nc0422s31
Online Posting Date:  December, 2007
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Abstract

Phosphodiester‐linked peptide‐oligonucleotide conjugates (nucleopeptides) are obtained by stepwise solid‐phase procedures. The peptide is first assembled on a suitably derivatized solid matrix and the oligonucleotide is subsequently elongated at the free hydroxyl group of the linking amino acid. Temporary acid‐labile and permanent base‐labile protecting groups are combined. Careful choice of the protection scheme is required to prevent and minimize side reactions that may degrade the target molecule. Curr. Protoc. Nucleic Acid Chem. 31:4.22.1‐4.22.54. © 2007 by John Wiley & Sons, Inc.

Keywords: nucleopeptides; stepwise solid‐phase synthesis; protecting groups; peptide‐oligonucleotide conjugates

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Stepwise Manual Solid‐Phase Synthesis of the Peptide
  • Support Protocol 1: Preparation of the Solid Support (H‐HMFS‐IRAA‐MBHA‐PS)
  • Support Protocol 2: Preparation of Boc‐Arg(Fmoc)2‐OH
  • Support Protocol 3: Preparation of Boc‐Cys(S‐tBu)‐OH
  • Support Protocol 4: Preparation of Boc‐Hse(DMTr)‐O−HTEA+
  • Support Protocol 5: Preparation of Boc‐Thr(Ac)‐OH
  • Basic Protocol 2: Elongation of the Oligonucleotide Chain and Cleavage and Deprotection of the Nucleopeptide
  • Basic Protocol 3: Analysis, Purification, and Characterization of Nucleopeptides
  • Support Protocol 6: Reduction of Sulfoxide‐Protected Methionine‐Containing Nucleopeptides
  • Support Protocol 7: Deprotection and Purification of Cysteine‐Containing Nucleopeptides
  • Basic Protocol 4: Analysis and Purification of Nucleopeptides by Polyacrylamide Gel Electrophoresis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Stepwise Manual Solid‐Phase Synthesis of the Peptide

  Materials
  • Derivatized p‐methylbenzhydrylamine solid support (H‐HMFS‐IRAA‐MBHA‐PS; see protocol 2)
  • Dichloromethane (DCM; peptide synthesis grade)
  • N,N‐Dimethylformamide (DMF; peptide synthesis grade)
  • t‐Butoxycarbonyl‐protected amino acids (Boc‐aa‐OH):
    • Ala, Arg·HCl, Asn, Asp(OFm), Gln, Glu(OFm), Gly, His(Dnp), His(Tos), Ile, Leu, Lys(Fmoc), Lys(Tfa), Met(O), Phe, Pro, Ser, Ser(Ac), Thr, Trp(For), Tyr, Tyr(Ac), and Val (Novabiochem, Bachem, Neosystem)
    • Boc‐Arg(Fmoc) 2‐OH (see protocol 3)
    • Boc‐Cys(S‐tBu)‐OH (Fluka; also see protocol 4)
    • Boc‐Hse(DMTr)‐OHNEt 3+ (see protocol 5)
    • Boc‐Thr(Ac)‐OH (see protocol 6)
  • N,N′‐Dicyclohexylcarbodiimide (DCC) or N,N′‐diisopropylcarbodiimide (DiPC)
  • 4‐Dimethylaminopyridine (DMAP)
  • Methanol (MeOH; HPLC grade)
  • Concentrated HCl, analytical grade
  • Propionic acid
  • Phenol, crystalline
  • 3 M aqueous p‐toluenesulfonic acid containing 0.2% tryptamine
  • 0.06 M citrate buffer, pH 2
  • Acetic anhydride
  • Benzoyl chloride (optional)
  • Pyridine, synthesis grade
  • 30% (v/v) trifluoroacetic acid (TFA; peptide synthesis grade) in DCM
  • 5% (v/v) N,N‐diisopropylethylamine (DIPEA) in DCM
  • 1‐Hydroxybentotriazole (HOBt; for coupling unprotected Arg, Asn, Gln, Hse)
  • 0.5 M HOBt in DCM (for coupling unprotected Arg)
  • (Azabenzotriazole‐1‐yl‐oxy‐tris‐pyrrolidino)phosphonium hexafluorophosphate (PyAOP; for coupling any aa onto Pro)
  • N,N‐Diisopropylethylamine (DIPEA)
  • Ethyl acetate (EtOAc)
  • 1 M sulfuric acid
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Ninhydrin reagents A and B (see reciperecipes)
  • Saturated chloranil solution: ∼0.75 g of 2,3,5,6‐tetrachloro‐1,4‐benzoquinone in 25 mL toluene
  • Acetone
  • Peroxide‐free dioxane (see recipe)
  • 1 M 1,4‐dithiothreitol (DTT) in peroxide‐free dioxane (for S‐tBu‐protected Cys)
  • 32% (v/v) ammonium hydroxide (store at 4°C)
  • HPLC mobile phase A: 0.045% TFA in water (HPLC grade)
  • HPLC mobile phase B: 0.036% TFA in acetonitrile (HPLC grade)
  • α‐Cyano‐4‐hydroxycinnamic acid (CHA) or dihydroxybenzoic acid (DHB) matrix
  • 0.1% TFA in 1:1 (v/v) acetonitrile/water (HPLC grade)
  • 2‐ to 50‐mL disposable polypropylene syringes with porous polyethylene discs and Teflon two‐way stop cocks (RTV SF2, Shimadzu Scientific Research)
  • Vacuum filtration system
  • Teflon stir rod
  • Desiccator
  • Pyrex tubes for amino acid analyses
  • Methane/oxygen flame
  • 110° and 155°C heating block
  • Rotary evaporator equipped with a water aspirator or vacuum pump (use aspirator unless pump is indicated)
  • 0.45‐µm nylon filters
  • Automatic amino acid analyzer (Beckman System 6300)
  • 25‐mL separatory funnel
  • Filter paper
  • 25‐ and 50‐mL round‐bottom flasks
  • Small glass tubes for ninhydrin or chloranil tests
  • 4‐mL screw‐cap pressure tubes, O‐ring seal preferred
  • Additional reagents for HPLC and MALDI‐TOF‐MS (see protocol 8 and Chapter 10)
CAUTION: Trifluoroacetic acid is a very corrosive acid. Breathing the vapors is very harmful and it is possible to be quickly overcome by them. Always manipulate with extreme caution in a well‐ventilated fume hood and wear appropriate protective clothing.

Support Protocol 1: Preparation of the Solid Support (H‐HMFS‐IRAA‐MBHA‐PS)

  • p‐Methylbenzhydrylamine resin: (α‐amino‐α‐xylyl)‐polystyrene reticulated with 1% divinylbenzene (f = 0.5 to 0.6 mmol/g; Novabiochem)
  • 9‐Fluorenylmethoxycarbonyl‐protected internal reference amino acid (Fmoc‐aa‐OH; Novabiochem, Bachem, Neosystem)
  • 20% (v/v) piperidine in DMF
  • N‐[9‐(Hydroxymethyl)‐2‐fluorenyl]succinamic acid (H‐HMFS‐OH; Albericio et al., )
  • 2‐ and 10‐mL polypropylene syringes fitted with polyethylene discs
  • Teflon two‐way stopcocks
  • 50‐mL volumetric flasks
  • Double‐beam UV spectrophotometer, calibrated, and quartz cuvettes

Support Protocol 2: Preparation of Boc‐Arg(Fmoc)2‐OH

  Materials
  • N‐(tert‐Butoxycarbonyl)‐L‐arginine hydrochloride (Novabiochem)
  • Acetonitrile, anhydrous (see recipe)
  • Argon (Ar), dry
  • Dichloromethane (DCM, anhydrous; see recipe) in a septum‐sealed distillation collection bulb
  • N,N‐Diisopropylethylamine (DIPEA), anhydrous (see recipe)
  • Chlorotrimethylsilane
  • Dichloromethane (DCM; synthesis grade)
  • 9‐Fluorenylmethyl chloroformate (Fmoc‐Cl), dried overnight under vacuum in a desiccator containing P 2O 5
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Methanol (MeOH)
  • Acetic acid (AcOH)
  • Hexanes
  • 230‐ to 400‐mesh silica gel
  • 250‐mL oven‐dried round‐bottom flask and rubber septum
  • Desiccator containing P 2O 5
  • Glass syringe and needle, oven dried
  • 40°C heat block
  • 250‐mL separatory funnel
  • Gravity and vacuum filtration devices and filter paper
  • 500‐mL round‐bottom flasks
  • 2 × 5–cm silica‐coated thin‐layer chromatography (TLC) plates
  • 254‐nm UV light source
  • Rotary evaporator with a vacuum aspirator
  • 5 × 25–cm glass chromatography column with solvent reservoir bulb
  • Additional reagents and equipment for TLC ( appendix 3D), column chromatography ( appendix 3E), NMR, IR, and mass spectrometry

Support Protocol 3: Preparation of Boc‐Cys(S‐tBu)‐OH

  Materials
  • S‐(tert‐Butylthio)‐L‐cysteine (H‐Cys(S‐tBu)‐OH; Novabiochem)
  • 2:1 (v/v) dioxane/water
  • 1 M NaOH
  • Di‐tert‐butyldicarbonate (Boc 2O)
  • Hexanes
  • 1 M HCl
  • Ethyl acetate (EtOAc)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 50‐mL and 250‐mL round‐bottom flasks
  • 100‐mL separatory funnel
  • Gravity filtration device and filter paper
  • Rotary evaporator equipped with vacuum aspirator
  • Additional reagents and equipment for TLC ( appendix 3D), 1H NMR, and mass spectrometry

Support Protocol 4: Preparation of Boc‐Hse(DMTr)‐O−HTEA+

  Materials
  • L‐Homoserine (Novabiochem)
  • Pyridine, anhydrous (see recipe)
  • Argon (Ar), dry
  • Chlorotrimethylsilane
  • Triethylamine
  • Di‐tert‐butyldicarbonate (Boc 2O)
  • 32% ammonium hydroxide
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Dichloromethane (DCM), neutralized (see recipe)
  • Methanol (MeOH)
  • Triethylamine
  • Acetonitrile, anhydrous (see recipe)
  • 250‐mL oven‐dried round‐bottom flask with rubber septum
  • Oven‐dried glass syringes and needles
  • Rotary evaporator equipped with a water aspirator
  • 500‐mL round‐bottom flask
  • Additional reagents and equipment for column chromatography (see protocol 3 and appendix 3E), TLC ( appendix 3D), 1H and 13C NMR, and mass spectrometry

Support Protocol 5: Preparation of Boc‐Thr(Ac)‐OH

  Materials
  • N‐(tert‐Butoxycarbonyl)‐L‐threonine (Novabiochem)
  • Ethyl acetate (EtOAc)
  • 4‐Nitrobenzyl bromide
  • Triethylamine
  • 1 M HCl
  • 5% (w/v) aqueous sodium hydrogencarbonate (NaHCO 3) solution
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Dichloromethane (DCM)
  • Methanol (MeOH)
  • Acetic anhydride
  • 4‐Dimethylaminopyridine (DMAP)
  • Argon (Ar), dry
  • 5% (dry basis) palladium on activated carbon
  • Hydrogen gas
  • Hexanes
  • Diethyl ether
  • Dicyclohexylamine
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Reflux condenser
  • Gravity and vacuum filtration devices and filter paper
  • 250‐ and 500‐mL separatory funnels
  • Rotary evaporator with a vacuum aspirator
  • 25‐mL oven‐dried round‐bottom flask with rubber septum
  • Additional reagents and equipment for column chromatography (see protocol 3 and appendix 3E), TLC ( appendix 3E), 1H and 13C NMR, IR, and mass spectrometry

Basic Protocol 2: Elongation of the Oligonucleotide Chain and Cleavage and Deprotection of the Nucleopeptide

  Materials
  • Peptide‐derivatized support (see protocol 1)
  • 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
    • N2‐dimethylaminomethyleneguanin‐9‐yl
    • N4‐benzoylcytosin‐1‐yl
    • N4‐acetylcytosin‐1‐yl
    • thymin‐1‐yl
  • Argon (Ar) and nitrogen (N 2, optional) gas, dry
  • Anhydrous acetonitrile (see recipe) in a septum‐sealed distillation collection bulb
  • Activator solution (see recipe)
  • Cap A and B capping reagents (Glen Research)
  • Oxidizer solution (see recipe)
  • Peroxide‐free THF (see recipe)
  • Detritylation solution: 3% (w/v) trichloroacetic acid (TCA, 99%, Glen Research) in neutralized DCM
  • 2% (v/v) N,N‐diisopropylethylamine (DIPEA) in neutralized DCM
  • Neutralized DCM (see recipe)
  • 0.1 M p‐toluenesulfonic acid monohydrate in acetonitrile
  • 70% (v/v) perchloric acid
  • Absolute ethanol (EtOH)
  • Peroxide‐free dioxane (see recipe)
  • 32% ammonium hydroxide (store at 4°C)
  • Tetra‐n‐butylammonium fluoride (TBAF, Aldrich)
  • Anhydrous tetrahydrofuran (see recipe) in a septum‐sealed distillation collection bulb
  • Methanol (MeOH), HPLC grade
  • Glacial acetic acid
  • 200‐ to 400‐mesh Dowex 50WX4‐400 ion‐exchange resin (4.8 meq Na+/g; Fluka)
  • 1 M NaOH
  • Synthesis column for 5‐µmol‐scale synthesis: old empty OPC cartridge (ABI) with a body and two caps (13‐mm aluminum seals; Chromatographic Specialties)
  • Empty DNA synthesizer bottles, oven dried, with rubber septa
  • Vacuum desiccator containing P 2O 5
  • Glass syringes and needles, oven dried
  • ABI 380B automatic DNA synthesizer
  • External fraction collector and 15‐mL test tubes
  • Double‐beam UV spectrophotometer, calibrated, and quartz cuvettes
  • 20‐mL screw‐cap pressure tubes, O‐ring seal preferred
  • 55°C oven
  • Disposable polypropylene syringes and polyethylene filters (cotton optional in some steps)
  • 50‐mL round‐bottom flasks
  • Teflon two‐way stopcocks
  • Rotary evaporator with a vacuum aspirator
  • Lyophilizer
  • Vacuum filtration system
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C) and amino acid analysis (see protocol 1)
CAUTION: All solutions and reagents required for the DNA synthesizer should be manipulated and prepared in a well‐ventilated fume hood.NOTE: Oxidizer solution, DIPEA solution, neutralized DCM, and peroxide‐free THF should be filtered (Pro‐XR nylon filters, 0.25 µm, 25 mm) before they are added to the synthesizer bottles.

Basic Protocol 3: Analysis, Purification, and Characterization of Nucleopeptides

  Materials
  • Crude deprotected nucleopeptide (see protocol 7 or protocol 10)
  • 0.01 M ammonium acetate, pH 7.0
  • HPLC mobile phase A: 0.01 M ammonium acetate, pH 7.0
  • HPLC mobile phase B: 1:1 (v/v) acetonitrile/water
  • Methanol (MeOH), HPLC grade
  • MPLC mobile phase A: 0.05 M ammonium acetate, pH 7.0
  • MPLC mobile phase B: 70% (v/v) 0.05 M ammonium acetate solution, pH 7.0, in 1:1 (v/v) acetonitrile/water
  • Matrix: 2′,4′,6′‐trihydroxyacetophenone (THAP) or 3‐hydroxypicolinic acid (3‐HPA)
  • Ammonium citrate
  • Acetonitrile, HPLC grade
  • 0.1% (v/v) triethylamine in water
  • Isopropanol (optional)
  • 0.1 M Tris·Cl, pH 8.0 ( appendix 2A)
  • 0.1 M MgCl 2
  • 1.5 U/500 µL snake venom phosphodiesterase (SVDP; Boehringer‐Mannheim; EC 3.1.4.1)
  • 0.0337 U/µL bacterial alkaline phosphatase (AP; Sigma; EC 3.1.16.1)
  • 0.2 M ammonium acetate, pH 5.4 and 8.3
  • 0.23 U/µL calf spleen phosphodiesterase (SpPD; Sigma; EC 3.1.3.1)
  • Concentrated HCl
  • 0.1 M ammonium acetate, pH 5.3
  • 1:32 (v/v) 2‐mercaptoethanol/water
  • 20 mg/mL (286 U/mL) papaine (from Papaya latex; Sigma; EC 3.4.22.2)
  • Glacial acetic acid
  • 3 mg/mL (72 U/mL) microsomal leucine aminopeptidase (mLAP type VI‐S from pork kidney microsomes; Sigma; EC 3.4.11.2)
  • 0.025 M MnCl 2 buffer solution, pH 8.3
  • 5 mg/mL (875 U/mL) prolidase (from pork kidney; Sigma; EC 3.4.13.9)
  • High‐performance liquid chromatographic system (HPLC) with:
    • Injector (autosampler preferred), sample loop, and syringe (for manual loading)
    • 0.1 to 5 mL/min pumping system (binary)
    • UV/Vis detector, variable wavelength between 190 and 600 nm (preferred) or dual‐wavelength detection
    • Data integrating system
    • Gradient system: displays and stores for redisplay and reformatting (preferred) or programmable
    • Analytical column: reversed‐phase C18 column (i.e., Kromasil, 10 µm, 4.0 × 250 mm)
    • Semipreparative column: reversed‐phase C18 column (i.e., Kromasil, 10 µm, 10.0 × 250 mm)
    • Automatic fraction collector (optional)
  • Lyophilizer
  • Double‐beam UV spectrophotometer, calibrated
  • Quartz cuvettes
  • Preparative chromatographic system (medium‐pressure liquid chromatography, MPLC):
    • Piston pump
    • Reversed‐phase C18‐filled glass column (i.e., Vydac 15 to 20‐µm i.d., 300‐Å porosity, 22 × 2 cm)
    • Automatic fraction collector
    • UV/Vis detector with fixed‐wavelength detection
    • Teflon tubing connectors and adapters
    • Gradient‐forming device with two equal‐diameter cylinders and a Teflon stopcock between them
    • Chart recorder
  • Pyrex tubes
  • 37°C water bath or heating block
  • Benchtop centrifuge
  • Additional reagents and equipment for reduction of sulfoxide‐protected methionine (see protocol 9; optional), deprotection of tert‐butylthio‐protected cysteine (see protocol 10; optional), MALDI‐TOF‐MS (unit 10.1), ESI‐MS (unit 10.2), and amino acid analysis (see protocol 1)

Support Protocol 6: Reduction of Sulfoxide‐Protected Methionine‐Containing Nucleopeptides

  • Deprotected crude nucleopeptide (see protocol 7)
  • N‐Methylmercaptoacetamide (Aldrich)

Support Protocol 7: Deprotection and Purification of Cysteine‐Containing Nucleopeptides

  • Nucleopeptide‐support (see protocol 7, step 7)
  • Cysteine hydrochloride (Novabiochem)
  • 0.1 M 1,4‐dithiothreitol (DTT) in peroxide‐free dioxane (see recipe)
  • 40% (w/v) methylamine (Aldrich), aqueous
  • 0.05 M ammonium acetate buffer, pH 7.0
  • 30‐mL screw‐cap pressure tube, O‐ring seal preferred
  • Disposable polypropylene syringe with polyethylene filter (or cotton)
  • 50‐mL round‐bottom flask
  • Peristaltic pump
  • Sephadex G‐10 column (90 × 2 cm)

Basic Protocol 4: Analysis and Purification of Nucleopeptides by Polyacrylamide Gel Electrophoresis

  Materials
  • Urea, ultrapure
  • 38% (w/v) acrylamide/2% (w/v) bisacrylamide (see recipe)
  • 10× TBE buffer (1.3 M Tris, 0.45 M boric acid, 25 mM EDTA)
  • 10% (w/v) ammonium persulfate (store up to 1 week at 4°C)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED)
  • Bromphenol blue
  • Xylene cyanol
  • Glycerol
  • Deprotected crude or purified nucleopeptide (see Basic Protocols protocol 72 or protocol 83)
  • 50% (v/v) aqueous formamide
  • Stains‐all (Aldrich)
  • Formamide
  • Concentrated HCl
  • Isopropanol
  • 3 M Tris·Cl, pH 8.8 ( appendix 2A)
  • 2 M ammonium acetate buffer, pH 7.0
  • Acetonitrile, HPLC grade
  • 50% (v/v) aqueous methanol (MeOH)
  • 250‐mL Erlenmeyer flask
  • 20‐ and 60‐mL syringes
  • 95° and 37°C water baths or heating blocks
  • Sep‐Pak cartridges (Waters)
  • Polyethylene disc
  • Disposable tubes
  • Lyophilizer
  • Additional reagents and equipment for polyacrylamide electrophoresis (unit 10.4 and appendix 3B)
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Literature Cited

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Key References
   Dreef‐Tromp, C.M., van den Elst, HR., van den Boogaart, J.E., van der Marel, G.A., and van Boom, J.H. 1992. Solid‐phase synthesis of an RNA nucleopeptide fragment from the nucleoprotein of poliovirus. Nucl. Acids Res. 20:2435‐2439.
  Largest phosphodiester‐linked peptide‐oligoribonucleotide hybrid synthesized to date.
   Tung and Stein, 2000. See above.
  Reviews covering synthesis and applications of peptide‐oligonucleotide conjugates.
   Venkatesan and Kim, 2006. See above.
  Particularly interesting for the description of alternatives for preparing different kinds of peptide‐oligonucleotide conjugates, including nucleopeptides. Advantages and problems are analyzed.
   Zubin et al., 2002. See above
  The authors wish to acknowledge the contribution of Dr. Jordi Robles, who set up the basic nucleopeptide stepwise solid‐phase synthesis methodology, in addition to the contributions of many other people over the years.
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