27.14 The Strategy of Peptide Synthesis The Challenge of Peptide Synthesis •Making peptide bonds between amino acids is not difficult: there are numerous methods to make amides from amines and carboxylic acids. •The challenge is connecting amino acids in the correct sequence. •Random peptide bond formation in a mixture of phenylalanine and glycine, for example, will give four dipeptides. •Phe—Phe Gly—Gly Phe—Gly Gly—Phe Amino Acids are Structurally Bifunctional H O N H OH H H O N H OH Glycine L-Phenylalanine Possible Products from the Condensation of Phenylalanine and Glycine O H2N H OH H O N N H H2N O Gly-Gly OH O Phe-Gly O H N H2N OH O Gly-Phe O H H N H2N H OH O Phe-Phe H Step 1 of Peptide Synthesis: Protection • 1.Limit the number of possible reactions by "protecting" the nitrogen of one amino acid and the carboxyl group of the other. H O N PG OH H H O PG N H O O-Protected Glycine N-Protected L-Phenylalanine C-Electrophile PG N-Nucleophile protecting group Step 2 of Peptide Synthesis: Coupling • 2. Couple the two protected amino acids. H O N OH PG H H O PG N H O Peptide Coupling H O N PG O H N H PG O PG-Phe-Gly-PG Peptide Synthesis, Step 3: Global Deprotection • 3.Deprotect the amino group at the Nterminus and the carboxyl group at the Cterminus. H O N PG O H N H PG O Deprotection O H3N O H N H O Phe-Gly Does the Requirement for Three Extra Steps Outweigh the Need to Purify a Mixture? O H2N H OH H O N N H H2N O Gly-Gly OH O Phe-Gly O H N H2N OH O Gly-Phe O H H N H2N H OH O H Phe-Phe Yes - synthesis is easier than purification 27.15 Amino Group Protection What are we Trying to Achieve by Protecting the Amino Group? Amino groups can behave as nucleophiles and undergo reaction with carboxylic acid derivatives. The nitrogen atom in amides is much less nucleophilic. As a result, amide derivatives of amines can be viewed as protecting groups. Amine Amide Derivative O H2N O OR H N R2 OR O Nucleophilic Atom Non-Nucleophilic Atom Peptide Synthesis: Amine Protecting Groups 1. Amino groups are normally converting them to amides. atom in an amide does not nucleophile and will not react groups. protected by The nitrogen behave as a with carboxyl 2. Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z or Cbz. 3. Cbz-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride. Amine Protecting Groups: Benzyloxycarbonyl O O R O Cl Benzyloxycarbonyl chloride (Cbz-Cl) O N H Benzyloxycarbonyl group Amine Protecting Groups: Introduction of Benzyloxycarbonyl Protecting Groups O O O H3N O 1. NaOH H2O H Cl 2. H3O+ O H N O OH O (85%) H Benzyloxycarbonyl is Abbreviated to Cbz or Z O O H N H N O Cbz OH O H H N OH H Cbz-Phe H O Z OH Cleavage of Cbz Groups •An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by: •a)catalytic hydrogenolysis under extremely mild conditions •b) cleavage with HBr in acetic acid •Both reagents cleave the relatively weak benzylic carbon-oxygen ether bond, albeit by Hydrogenolysis of Cbz Groups O H N O OEt H O N H O ethyl ester is stable to hydrogenolysis H2, Pd/C solvent O H N HO H2C Toluene (volatile) H O OEt H N H O Carbamic Acid (Very Unstable) Hydrogenolysis of Cbz Groups O H N HO O OEt H N H O Spontaneous decarboxylation O H2N OEt H N H O O C O (100%) Acid-Mediated Cleavage of Cbz Groups O H N O OEt H O N H O HBr acetic acid O Br H3N H2C Benzyl bromide (volatile) Br OEt H N H O O C O (82%) Amine Protecting Groups: tertButyloxycarbonyl O O O O R O N H O tert-Butyloxycarbonyl group O O O Di-tert-butyl dicarbonate (Boc 'anhydride') O O Cl tert-Butyl chloride (instablity limits use) tert-Butyloxycarbonyl is Abbreviated to Boc O O H N O H N OH O H Boc OH H Boc-Phe Cleavage of Boc Groups The tert-butyloxycabonyl protecting group is readily removed by treatment wit strong, anhydrous BrØnsted acids: a) cleavage with trifluoroacetic acid in methylene chloride b) cleavage with HBr in acetic acid Both reagents cleave the quaternary carbonoxygen ether bond by an acid-mediated elimination reaction. Acid-Mediated Cleavage of Boc Groups O H N O OEt H O N H O O F3C OH O F3C Butene (volatile) H H CH3 O O OEt H3N H N H O O C H3C trifluoroacetic acid O (high yield) 27.16 Carboxyl Group Protection Peptide Synthesis: Carboxyl Protecting Groups Carboxyl groups are normally protected as esters. Deprotection of methyl and ethyl esters is by hydrolysis in base. Benzyl esters hydrogenolysis. can be cleaved by Simultaneous Hydrogenolysis of Cbz Group and Benzyl Ester O H N O O H O N H O H2, Pd/C solvent O H N HO H2C Toluene (volatile) H O OH H N H O Carbamic Acid (Very Unstable) Simultaneous Hydrogenolysis of Cbz Group and Benzyl Ester O H N HO O OEt H N H O Spontaneous decarboxylation O H2N OEt H N H O O C O (87%) 27.17 Peptide Bond Formation Peptide Synthesis: Forming Peptide Bonds The two major methods are: 1. coupling of suitably protected amino acids using N,N'dicyclohexylcarbodiimide (DCC) 2. via an active ester of the N-terminal amino acid. N,N'-Dicyclohexylcarbodiimide (DCC) is a Powerful Dehydrating Agent H O N H R1 H O R2 C N N,N'-dicyclohexylcarbodiimide H O N H N R1 O H Very high temps R2 -H2O O N H O R1 'H2O' N H R2 Amide N H N,N'-dicyclohexylurea Peptide Coupling is a Condensation Reaction • 2. Couple the two protected amino acids. H O N OH PG H H O PG N H O Peptide Coupling H O N PG -H2O O H N H PG O DCC-Mediated Peptide Coupling • 2. Couple the two protected amino acids. H O H N Cbz OH O N Et H O H DCC, CHCl3 H O N O Cbz H N H Et O (83%) Mechanism of DCC-Promoted Coupling H O N Cbz OH N C N H 1,2-Addition H O N N Cbz O H N H O-Acylisourea derivative O-Acylisoureas are Powerful Acylating Agents The O-acylisourea intermediate formed by addition of the Cbz-protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent. H O N N Cbz O H N H O-Acylisourea Derivative O H3C O O CH3 Acid Anhydride Mechanism of DCC-Promoted Coupling H O N N Cbz O N H H O N Attack by the amine function of the carboxylprotected amino acid on the carbonyl group leads to nucleophilic acyl substitution. H 1,2-Addition then Proton Transfer OEt H OH N N Cbz O N H O N H Unstable Intermediate OEt Mechanism of DCC-Promoted Coupling H OH N Unstable Intermediate N Cbz O N H N H Attack by the amine function of the carboxylprotected amino acid on the carbonyl group leads to nucleophilic acyl substitution. Elimination O OEt H O N OEt Cbz O N H H N H N,N'-dicyclohexylurea N H O Dipeptide Peptide Synthesis: Forming Peptide Bonds The two major methods are: 1. coupling of suitably protected amino acids using N,N'dicyclohexylcarbodiimide (DCCI) 2. via an active ester of the N-terminal amino acid. Peptide Synthesis: Active Ester Method A p-nitrophenyl ester is an example of an "active ester.” p-Nitrophenyl is a better leaving group than methyl or ethyl, and p-nitrophenyl esters are more reactive in nucleophilic acyl substitution. O N+ O O– O is a more powerful acylating agent than...... Alkyl O O Alkyl Ester 4-Nitrophenyl (PNP) Ester Peptide Synthesis: Active Ester Method H NO2 O N Cbz O H O N H 1,2-Addition then Proton Transfer OEt H NO2 OH N Cbz O N H O Unstable Intermediate OEt Peptide Synthesis: Active Ester Method H NO2 OH N Cbz Unstable Intermediate O N H Elimination O OEt H O N OEt Cbz H O2N N H O OH para-Nitrophenol Dipeptide 27.18 Solid-Phase Peptide Synthesis: The Merrifield Method Solid Phase Peptide Synthesis In solid-phase synthesis, the starting material is bonded to an inert solid support. Reactants are added in solution. Reaction occurs at the interface between the solid and the solution. Because the starting material is bonded to the solid, any product from the starting material remains bonded as well. Purification involves simply washing the byproducts from the solid support. Polystyrene is the Basis for the Solid Support H H H C H H C C H H H C C H H H C C H H C C C H •The solid support is a copolymer of styrene and divinylbenzene. It is represented above as if it were polystyrene. Cross-linking with divinylbenzene simply provides a more rigid polymer. Functionalization of Polystyrene •Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings. Chloromethylation of Polystyrene Cl O C H2 Cl CH2 Cl CH2 Me SnCl4 Solid Phase Peptide Synthesis Cl CH2 Cl CH2 The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2). Solid Phase Peptide Synthesis Cl CH2 Cl CH2 The chloromethylated resin is treated with the Boc-protected C-terminal amino acid. Nucleophilic substitution occurs, and the Bocprotected amino acid is bound to the resin as an ester. Merrifield Procedure CH2 CH CH2 CH CH2 O – BocNHCHCO R CH CH2 CH2Cl CH Merrifield Procedure CH2 CH CH2 CH CH2 O BocNHCHCO Next, the Boc protecting group is removed with HCl. R CH CH2 CH2 CH Merrifield Procedure CH2 CH CH2 CH CH2 O DCCI-promoted coupling adds the second amino acid H2NCHCO R CH CH2 CH2 CH Merrifield Procedure CH2 CH CH2 CH CH2 O BocNHCHC R' O CH CH2 CH CH2 NHCHCO R Remove the Boc protecting group. Merrifield Procedure CH2 CH CH2 CH CH2 O H2NCHC R' O CH2 CH CH CH2 NHCHCO R Add the next amino acid and repeat. Merrifield Procedure CH2 CH CH2 O CH CH2 O + H3N peptide C NHCHC R' CH O CH2 CH CH2 NHCHCO R • Remove the peptide from the resin with Merrifield Procedure CH2 CH CH2 CH CH2 CH CH2 CH2Br O O + H3N peptide C NHCHC R' O – NHCHCO R CH Merrifield Procedure •Merrifield automated his solid-phase method. •Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield. •Synthesized ribonuclease (124 amino acids) in 1969. 369 reactions; 11,391 steps