The Practice of Peptide Synthesis (Springer Lab Manuals)

During the years 1980-81, as guests of the Deutsches Wollforschungsinstitut in Aachen, Germany, we were working on a small book entitled, "Principles of Peptide Synthesis". In the library of the Institute we noted that the volumes of Houben-Weyl's Handbuch der Organischen Chemie dealing with peptide synthesis were so much in use that they were ready to fall apart because the researchers of the Institute consulted them with amazing regularity. They were looking for references, but even more for experimental details which could be adapted to the particular problem they happened to face. In planning a new synthetic endeavor they tried to lean on the experience of others in analogous situations. This suggested to us that a smaller and hence more tractable book may be needed, a volume which can be kept on or near the bench to make examples of fundamental methods readily available in the laboratory. Such a collection could save numerous short trips to the library, a point particularly important where a library well equipped with the sources of the literature of peptide synthesis is not near at hand. Also, we thought that the envisaged book may be welcome by those who are more versed in English than in German. To the best of our knowledge no similar publication is available.

I Introduction.- II Protecting Groups.- 1 Introduction of Amine Protecting Groups.- 1.1 The p-Toluenesulfonyl Group.- p-Toluenesulfonyl-l-isoleucine.- 1.2 Phthalyl (Phthaloyl, Pht) Amino Acids.- Phthalyl-l-leucine.- 1.3 The Benzyloxycarbonyl Group.- Benzyloxycarbonyl-l-proline.- Benzyloxycarbonyl-l-leucine.- N?-Benzyloxycarbonyl-l-arginine 12.- Benzyloxycarbonyl-l-aspartic Acid ?-Benzyl Ester.- 1.4 4-Methoxybenzyloxycarbonylamino Acids.- 1.5 The tert-Butyloxycarbonyl (Boc) Group.- 1.5.1 Introduction of the tert-Butyloxycarbonyl Group with 2-tert-Butyloxycarbonyloximino-2-phenylacetonitrile.- tert-Butyloxycarbonyl-glycine.- tert-Butyloxycarbonyl-l-tryptophan.- 1.5.2 Introduction of the tert-Butyloxycarbonyl Group with tert-Butyl Pyrocarbonate.- 1.6 The Biphenylylisopropyloxycarbonyl (Bpoc) Group.- Bpoc-l-leucine.- Bpoc-l-leucine.- 1.7 The 9-Fluorenylmethyloxycarbonyl (Fmoc) Group.- 9-Fluorenylmethyl Chlorocarbonate.- 9-Fluorenylmethyloxycarbonyl-l-tryptophan.- 1.8 o-Nitrophenylsulfenylamino Acids.- 1.9 2-Trimethylsilylethyloxycarbonylamino Acids.- 4-Nitropheny1-2-Trimethylsilylethyl Carbonate.- 2-Trimethylsilylethyloxycarbonyl-l-proline.- 1.10 Maleoylamino Acids and Maleoyl-Peptides.- N-Methoxycarbonylmaleimide.- Maleoyl-glycine.- Maleoyl Peptides.- 1.11 Triphenylmethyl-amino Acids.- Trityl-l-leucine.- N-Trityl Amino Acids.- 2 Blocking of the ?-Carboxyl Group.- 2.1 Methyl Esters.- 2.2 Ethyl Esters.- 2.2.1 Esterification with the Help of Gaseous HCl.- l-Tyrosine Ethyl Ester.- 2.2.2 Esterification Catalyzed by p-Toluenesulfonic Acid.- l-Methionine Ethyl Ester p-Toluenesulfonate.- 2.3 Benzyl Esters.- Glycine Benzyl Ester p-Toluenesulfonate.- tert-Butyloxycarbonyl-l-asparagine Benzyl Ester.- l-Glutamic Acid ?-Benzyl Ester.- 2.3.1 Polymeric Benzyl Esters.- Hydroxymethyl-Polymer.- tert-Butyloxycarbonyl-glycyl Resin.- Benzyloxycarbonyl-l-isoleucyl-Polymer.- 2.4 4-Nitrobenzyl Esters.- l-Alanine-4-Nitrobenzyl Ester p-Toluenesulfonate.- 2.5 4-Methoxybenzyl Esters of N?-Protected Amino Acids.- Benzyloxycarbonylglycine 4-Methoxybenzyl Ester.- 2.6 Benzhydryl Esters (Diphenylmethyl Esters).- 2.7 Phthalimidomethyl Esters.- 2.8 tert-Butyl Esters.- Addition of N?-Acylamino Acids to Isobutylene.- l-Proline tert-Butyl Ester.- Transesterification with tert-Butyl Acetate.- Esterification of Free Amino Acids through Acid Catalyzed Addition to Isobutylene.- l-Tyrosine tert-Butyl Ester.- 2.9 Phenacyl Esters.- Benzyloxycarbonylglycine Phenacyl Ester.- 2.10 2-Trimethylsilylethyl Esters.- 2.11 4-Picolyl Esters.- Benzyloxycarbonyl-l-phenylalanine 4-Picolyl Ester.- Benzyloxycarbonyl-l-valine 4-Picoly1 Ester.- 2.12 9-Fluorenylmethyl Esters.- tert-Butyloxycarbonyl-l-phenylalanine 9-Fluorenylmethyl Ester.- 2.13 2-Benzyloxyphenyl Esters.- l-Alanine 2-Benzyloxyphenyl Ester Hydrochloride.- 2.14 Phenyl Esters.- 2.15 Allyl Esters.- 3 Protection of Side Chain Functions.- 3.1 Serine Ethers.- N-tert-Butyloxycarbonyl-O-benzyl-l-Serine Cyclohexylammonium Salt.- 3.2 Ethers of Threonine.- O-tert-Butyl-l-threonine.- 3.3 Tyrosine Ethers.- O-Benzyl-l-tyrosine.- 3.4 Acylation of the ?-Amino Group of Lysine.- N?-Trifluoroacetyl-lysine 51.- N?-p-Toluenesulfonyl-l-lysine 52.- 3.5 Protection of the Guanidino Group of Arginine.- Nitro-l-arginine.- N?-Benzyloxycarbonyl-NG-p-toluenesulfonyl-l-arginine 54.- N?-Benzyloxycarbonyl-N?, N?-bis-(1-adamantyloxycarbonyl)-l-arginine 56.- N?-4-Methoxybenzyloxycarbonyl-NG-mesitylene-sulfonyl-l-arginine Cyclohexylammonium Salt 57.- NG-4-Methoxy-2, 3,6-trimethylbenzenesulfonyl-l-arginine 57.- 3.6 Masking the Imidazole in Histidine.- 3.6.1 Nim-p-Toluenesulfonyl-l-histidine.- N?-Benzyloxycarbonyl-Nim-tosyl-l-histidine 59.- 3.6.2 N?-tent-Butyloxycarbonyl-N?-benzyloxymethyl-l-histidine.- 3.6.3 Nim-Trityl-l-histidine.- 3.7 Blocking of the Indole Nitrogen in Tryptophan.- Nim-Formyl-l-tryptophan 62.- 3.8 Protection of Side Chain Carboxyl Groups.- l-Aspartic Acid ?-Benzyl Ester (?-Benzyl-l-aspartate).- l-Glutamic Acid ?-tert-Butyl Ester.- 3.9 Protection of the Carboxamide Function.- of the 4,4?-Dimethoxybenzhydryl Group.- 3.10 Blocking the Sulfhydryl Group in Cysteine.- S-Benzyl-l-cysteine.- S-Ethylcarbamoyl-l-cysteine.- S-Acetamidomethyl-l-cysteine.- S-Triphenylmethyl-l-cysteine (S-Trityl-l-cysteine).- N-tert-Butyloxycarbonyl-S-(3-nitro-2-pyridinesulfenyl-l-cysteine.- S-tert-Butyl-l-cysteine hydrochloride.- 3.11 Protection of the Thioether in Methionine.- l-Methionine Sulfoxide.- N-tert-Butyloxycarbonyl-S-methyl-l-methionine p-Nitrophenyl Ester p-Toluenesulfonate.- III Activation and Coupling.- 1 The Acid Chloride Procedure.- p-Toluenesulfonyl-l-isoleucyl-glycine Ethyl Ester.- p-Toluenesulfonyl-l-isoleucyl-l-glutaminyl-l-asparagine.- 2 The Azide Process.- 2.1 Peptide Hydrazides Through Hydrazinolysis of Peptide Esters.- N-Benzyloxycarbonyl-S-benzyl-l-cysteinyl-l-seryl-l-histidine Hydrazide.- 2.2 Preparation of Peptide Hydrazides from Carboxylic Acids.- tert-Butyloxycarbonyl-?-benzyl-l-aspartyl-l-leucyl-l-N?-benzyloxycarbonyl-lysine Hydrazide.- 2.3 Protected Hydrazides.- N-Benzyloxycarbonyl-l-prolyl-glycyl-N’-tert-butyloxycarbonyl-hydrazine.- N-Benzyloxycarbonyl-glycyl-glycyl-N’-tert-butyloxycarbonyl-hydrazine.- Glycine Benzyloxycarbonylhydrazide Trifluoroacetate.- 2.4 Conversion of Hydrazides to Azides and Coupling with Azides.- 2.4.1 Generation of the Azide with the Aid of Sodium Nitrite and Coupling.- Benzyloxycarbonyl-l-seryl-l-tyrosyl-l-seryl-l-me thionyl-?-benzyl-l-glutamic Acid.- 2.4.2 Conversion of a Hydrazide to the Azide with the Aid of Butyl Nitrite and Acylation with the Azide “In Situ”.- Benzyloxycarbonyl-l-tyrosyl-l-isoleucyl-l-glutaminyl-l-asparaginyl-S-benzyl-l-cysteinyl-l-prolyl-l-leucyl-glycinamide.- 2.4.3 Conversion of Protected Amino Acid or Peptide Hydrazides to the Azides Followed by Acylation with the Isolated Azide.- 3 Symmetrical Anhydrides.- tert-Butyloxycarbonyl-l-phenylalanine Anhydride.- 4 Mixed Anhydrides.- 4.1 The Isovaleric Acid Mixed Anhydride Method.- Benzyloxycarbonyl-l-leucyl-glycine Ethyl Ester.- 4.2 Trimethylacetic Acid (Pivalic Acid) Mixed Anhydrides.- Benzyloxycarbonyl-?-methylalanyl-?-methylalanine Methyl Ester.- N?-Benzyloxycarbonyl-N?-p-toluenesulfonyl-l-lysylglycine Ethyl Ester 91.- 4.3 The Ethyl Carbonate Mixed Anhydride Method.- Benzyloxycarbonyl-l-leucyl-glycine Ethyl Ester.- 4.4 Isobutylcarbonic Acid Mixed Anhydrides.- Benzyloxycarbonyl-glycyl-l-phenylalanyl-glycine Ethyl Ester.- 4.5 Coupling with the Aid of o-Phenylene Phosphorochloridite.- Preparation of N-Benzyloxycarbonyl-S-benzyl-l-cysteinyl-l-tyrosyl-l-isoleucyl-l-glutaminyl-l-asparaginyl-S-benzyl-l-cysteinyl-l-prolyl-l-leucyl-glycine Amide.- 5 Preparation of Active Esters.- 5.1 Cyanomethyl Esters.- N-Benzyloxycarbonyl-S-benzyl-l-cysteine Cyanomethyl Ester.- 5.2 Preparation of p-Nitrophenyl Esters.- Benzyloxycarbonyl-l-phenylalanine p-Nitrophenyl Ester.- 5.3 o-Nitrophenyl Esters.- tert-Butyloxycarbonyl-glycine o-Nitrophenyl Ester.- 5.4 2,4-Dinitrophenyl Esters.- N-Benzyloxycarbonyl-l-threonine 2,4-Dinitrophenyl Ester.- 5.5 2,4,5-Trichlorophenyl Esters.- 5.6 Pentachlorophenyl Esters.- Benzyloxycarbonyl-glycyl-l-phenylalanine Pentachlorophenyl Esters.- 5.7 Pentafluorophenyl Esters.- Benzyloxycarbonyl-glycyl-l-phenylalanine Pentafluorophenyl Ester.- tert-Butyloxycarbonylamino Acid Pentafluorophenyl Esters.- 5.8 N-Hydroxyphthalimide Esters.- 5.9 N-Hydroxysuccinimide Esters.- tert-Butyloxycarbonyl-l-alanine N-Hydroxysuccinimide Ester.- 5.10 1-Hydroxypiperidine Esters.- Benzyloxycarbonyl-l-leucine 1-Hydroxypiperidine Ester.- 5.11 Esters of 5-Chloro-8-Hydroxy-Quinoline.- 6 Coupling with Active Esters.- 6.1 Coupling in Organic Solvents.- 6.1.1 Acylation with Cyanomethyl Esters.- N-Benzyloxycarbonyl-S-benzyl-l-cysteinyl-l-tyrosine.- 6.1.2 Chain Lengthening with p-Nitrophenyl Esters.- Benzyloxycarbonyl-l-asparaginyl-S-benzyl-Lcysteinyl-l-prolyl-l-leucyl-glycinamide.- 6.1.3 Acylation with N-Hydroxysuccinimide Esters.- tert-Butyloxycarbonyl-glycyl-l-phenylalanyl-glycine Ethyl Ester.- Benzyloxycarbonyl-l-phenylalanyl-l-tyrosine Ethyl Ester.- 6.1.4 Coupling with 2,4-Dinitrophenyl Esters.- Benzyloxycarbonyl-l-threonyl-l-alanyl-l-alanyl-l-alanine Ethyl Ester.- 6.2 Coupling in Organic-Aqueous Media.- 6.2.1 Acylation with p-Nitrophenyl Esters.- Benzyloxycarbonyl-l-phenylalanyl-glycyl-l-proline.- 6.2.2 Acylation with N-Hydroxysuccinimide Esters.- Benzyloxycarbonyl-glycyl-l-proline.- Benzyloxycarbonyl-l-prolyl-glycine.- 6.3 Catalysis of Active Ester Reactions.- 6.3.1 Catalysis with Imidazole.- Benzyloxycarbonyl-l-prolyl-l-tyrosine Methyl Ester.- 6.3.2 Catalysis with 1-Hydroxybenzotriazole.- Benzyloxycarbonyl-l-leucyl-l-valinamide.- 6.3.3 Catalysis with 3-hydroxy-3,4-dihydro-quinazoline-4-one.- Benzyloxycarbonyl-l-isoleucyl-glycinamide.- 7 Peptide Bond Formation with the Aid of Coupling Reagents.- 7.1 The Dicyclohexylcarbodiimide (DCC, DCCI) Method.- 7.1.1 Coupling with DCC.- N-Phthaloyl-l-threonyl-l-phenylalanine Methyl Ester.- 7.1.2 Coupling with Carbodiimides in the Presence of 1-Hydroxybenzotriazole.- tert-Butyloxycarbonyl-l-leucyl-l-phenylalanine Methyl Ester.- 7.1.3 Coupling with Dicyclohexylcarbodiimide in the Presence of N-Hydroxysuccinimide.- Phthalyl-l-phenylalanyl-l-valyl-l-glutaminyl-l-tryptophyl-l-leucyl-l-methionyl-l-asparaginyl-O-tert-butyl-l-threonine tert-Butyl Ester.- N-Benzyloxycarbonyl-O-tert-butyl-l-tyrosyl-l-leucyl-?-tert-butyl-l-aspartic Acid Methyl Ester.- 7.2 Coupling with 1-Isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ).- Benzyloxycarbonyl-l-alanyl-l-tyrosine Methyl Ester.- 7.3 The Carbonyldiimidazole Method.- Benzyloxycarbonyl-glycyl-l-tyrosine Ethyl Ester.- 7.4 Coupling with N-Ethyl-5-phenylisoxazolium-3?-sulfonate.- N?, N?-Dibenzyloxycarbonyl-l-lysyl-glycine Ethyl Ester 123.- 7.5 Coupling with 1-Benzotriazolyl-tri-dimethylamino-phosphonium Hexafluorophosphate (BOP-Reagent).- 7.6 O-Benzotriazolyl-tetramethyluronium Hexafluorophosphate.- Preparation of the HBTU-Reagent.- Coupling with HBTU.- IV Removal of Protecting Groups.- 1 Hydrogenation.- 1.1 Hydrogenolysis of Benzyl Esters, Benzyl Ethers and of the Benzyloxycarbonyl Group.- l-Prolyl-l-leucyl-glycinamide.- 1.2 Catalytic Hydrogenation of Methionine Containing Peptides.- Benzyloxycarbonyl-glycyl-l-tryptophyl-l-methionyl-?-tert-butyl-l-aspartyl-l-phenylalanine Amide.- 1.3 Transfer Hydrogenation with 1,4-Cyclohexadiene as Hydrogen Donor.- N?-tert-Butyloxycarbonyl-l-lysy1-O-tert-butyl-l-threonine Methyl Ester 133.- 2 Reduction with Sodium in Liquid Ammonia.- l-Glutaminyl-l-asparagine.- 3 Removal of the Phthalyl (Phthaloyl) Group by Hydrazinolysis.- Glycyl-glycine.- 4 Acidolysis.- 4.1 Hydrobromic Acid in Acetic Acid.- Glycyl-l-phenylalanine Benzyl Ester Hydrobromide.- S-Benzyl-l-cysteinyl-l-prolyl-l-leucyl-glycinamide.- 4.2 Hydrobromic Acid in Trifluoroacetic Acid.- ?-Melanotropin.- 4.3 Hydrochloric Acid in Acetic Acid.- Nitro-l-arginyl-copolymer.- 4.4 Trifluoroacetic Acid.- l-Methionyl-l-glutaminyl-l-histidyl-l-phenylalanyl-Larginyl-l-tryptophyl-glycinamide Acetate.- 4.5 Removal of Protecting Groups with Trifluoroacetic Acid-Thioanisole.- Porcine Vasoactive Intestinal Peptide.- 4.6 Liquid Hydrogen Fluoride.- Oxytocin.- 5 Hydrolysis.- 5.1 Base Catalyzed Hydrolysis of Alkyl Esters (Saponification with Alkali).- N-Benzyloxycarbonyl-S-benzyl-l-cysteinyl-l-tyrosine.- 5.2 Acid Catalyzed Hydrolysis.- Phthalyl-l-phenylalanyl-glycine.- 5.3 Enzyme Catalyzed Hydrolysis.- 5.3.1 Enzymatic Removal of the Phenylacetyl Group from the ?-Amino Group of Lysine Residues.- Partial Deprotection of a Dipeptide Derivative.- Desamino Lysine Vasopressin.- 5.3.2 Chymotrypsin Catalyzed Hydrolysis of Alkyl Esters.- l-Prolyl-l-phenylalanine.- Benzyloxycarbonyl-l-valyl-l-tyrosine.- 6 Base Catalyzed Elimination.- 6.1 Removal of the 9-Fluorenylmethyloxycarbonyl (Fmoc) Group.- 9-Fluorenylmethyloxycarbonyl-l-asparaginyl-S-benzyl-l-cysteinyl-l-prolyl-l-tryptophyl-glycinamide.- 6.2 Cleavage of 9-Fluorenylmethyl Esters.- l-Leucyl-l-phenylalanine.- 7 Nucleophilic Displacement.- 7.1 Removal of the o-Nitrobenzenesulfonyl (Nps) Group.- N?-tert-butyloxycarbonyl-l-lysyl-l-leucyl-l-phenylalanyl-N?-tert-butyloxycarbonyl-l-lysyl-N?-tert-butyloxycarbonyl-l-lysine 156.- 7.2 Cleavage of 2-Trimethylsilylethyl Esters with Fluorides.- N-Benzyloxycarbonyl-O-tert-butyl-l-threonine.- 8 Iodolysis.- Removal of the S-Trityl Group by Iodolysis with Concomitant Oxidation to the Disulfide.- 9 Reduction of Methionine Sulfoxide Containing Peptides.- l-Prolyl-l-tyrosyl-N?-tosyl-l-lysyl-l-methionine.- V Special Procedures.- 1 Ammonolysis of Esters.- 1.1 Conversion of a Peptide Ethyl Ester to the Corresponding Amide.- Benzyloxycarbonyl-l-prolyl-l-leucyl-glycinamide.- 1.2 Ammonolysis of Active Esters.- Benzyloxycarbonyl-l-asparagine amide.- 2 Transesterification.- 2.1 Cleavage of the Ester Bond Between Peptide and Polymeric Support by Transesterification Followed by Hydrolysis.- Benzyloxycarbonyl-l-alanyl-l-phenylalanine.- 3 Cyclization.- 3.1 Cyclization Through the Formation of a Disulfide Bond.- Desamino-oxytocin.- 3.2 Ring Closure Through an Amide Bond.- Ditosyl-gramicidin S.- Desthiomalformin.- 4 Polycondensation.- 4.1 N-Carboxyanhydrides.- N?-Benzyloxycarbonyl-N?-carboxy-l-lysine Anhydride 173.- 4.2 Poly-N?-benzyloxycarbonyl-l-lysine.- 4.3 Synthesis of Sequential Peptides by Polycondensation.- Poly (l-lysyl-l-alanyl-l-alanine) Trifluoroacetate.- 5 Partial Synthesis.- Human Insulin.- Zinc Free Insulin.- NA1, NB1-Di-tert-butyloxycarbonyl-B(23-30)-desocta-peptide Insulin, (Boc)2-DOI 179.- Human Insulin Through Condensation with Dicyclohexylcarbodiimide.- Human Insulin via Enzyme-Catalyzed Coupling.- VI Models for the Study of Racemization.- 1 Benzoyl-leucyl-glycine Ethyl Ester.- 2 The Anderson-Callahan Test.- Benzyloxycarbonyl-glycyl-l-phenylalanyl-glycine Ethyl Ester.- 3 Acylation with Acetyl-l-isoleucine.- Acetyl-l-isoleucine.- Acetyl-l-isoleucyl-glycine Ethyl Ester and Acetyl-D-alloisoleucyl-glycine Ethyl Ester.- 4 The Izumiya-Muraoka Model.- Glycyl-alanyl-leucine.- 5 Synthesis and Enantioselective Enzymic Hydrolysis of Tetraalanine.- VII Reagents for Peptide Synthesis.- 1 tert-Butyl Azidoformate.- tert-Butyl Phenyl Carbonate.- tert-Butyl Carbazate.- tert-Butyl Azidoformate.- 2 1-Adamantyl Chlorocarbonate.- 3 1-Isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ).- 4 Diphenyl Phosphorazidate (Diphenylphosphoryl Azide, DPPA).- 5 3-Hydroxy-3,4-dihydroquinazoline-4-one (4-Hydroxyquinazoline 3-oxide).- 2-Aminobenzhydroxamic Acid.- 3-Hydroxy-3,4-dihydroquinazoline-4-one.- 6 1-Hydroxybenzotriazole.- 7 Ethyl 2-hydroximino-2-cyanoacetate.- 8 1-Guanyl-3,5-dimethyl-pyrazole Nitrate.- VIII Appendix.- 1 Conversion of Dicyclohexylammonium Salts of Protected Amino Acids to the Free Acids.- N-o-Nitrobenzenesulfonyl-O-tert-butyl-l-threonine N-hydroxysuccinimide Ester.- 2 Preparation pf Analytical Samples.- 2.1 Elemental Analysis.- 2.2 Amino Acid Analysis.- 2.3 NMR Spectra.