SIDE REACTIONS IN PEPTIDE SYNTHESIS

The base-catalyzed ring closure ofβ-benzylaspartyl peptides was efficiently suppressed by the addition of phenols (with electron-withdrawing substituents) to the reaction mixtures. From a series of compounds tested, 2,4-dinitrophenol and pentachlorophenol were the most effective. No direct relationship was found between the acidity of the additives and their ability to suppress the formation of aminosuccinyl peptides. The applicability of 2,4-dinitrophenol and pentachlorophenol in practical syntheses was also examined.     

SIDE REACTIONS IN PEPTIDE SYNTHESIS. VI.

The acid catalyzed O → C migration of the benzyl group in the side chain of tyrosine could be reduced by applying HBr in a mixture of phenol and p-cresol instead of HBr in trifluoroacetic acid for acidolytic deprotection. This side reaction occurs also during the removal of Boc groups. The loss of O-benzyl protection and the formation of 3-benzyltyrosine residues could be suppressed by the application of a 7:3 mixture of trifluoroacetic acid and acetic acid. The acid- and base-catalyzed ring closure of β-benzylaspartyl residues to aminosuccinyl derivatives was also studied. In this case HBr in trifluoroacetic acid was found to be relatively harmless. Deprotection with HBr in a mixture of trifluoroacetic acid and p-cresol can be applied for peptides that contain both β-benzylaspartyl and O-benzyltyrosyl residues. An attempt to reduce the rate of the base-catalyzed side reaction by application of hindered tertiary amines was abandoned because the tertiary amines which were effective in this respect led to significant reduction of the rate of the desired reaction, the aminolysis of active esters, as well. A satisfactory solution for the problem was found in the selective catalysis of the active ester reaction with 1-hydroxybenzotriazole o 4-dimethylaminopyridine. These catalysts do not enhance the rate of ring closure and in their presence essentially pure β-benzylaspartyl peptides can be produced in good yield.     

β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF SHORTENED PEPTIDE CHAINS

Three analogs of β-endorphin have been synthesized by the solid-phase method: β_c-endorphin-(1–5)-(28–31), β_c-endorphin-(6–31) and β_h-endorphin-(1–5)-(16–31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than β-endorphin. These data suggest that the entire β-endorphin molecule is necessary for full in vivo analgesic activity.     

SYNTHESIS OF γ-L-GLUTAMYL-TAURINE*

Glu(Tau), a bioactive substance previously isolated from the protein free aqueous extract of bovine parathyroid powder, has been synthesized. The intermediate derivative Z-Glu(Tau)-OBzl was prepared in three different ways from Z-Glu-OBzl and (1) cystamine by using the mixed anhydride method followed by oxidation, (2) Tau by the active ester procedure via Z-Glu(ONp)-OBzl, (3) Tau applying mixed anhydride coupling. The protecting groups were removed by hydrogenolysis.     

PREPARATIVE MERITS OF THE MIXED ANHYDRIDE (MA) METHOD IN THE EXCESS USE OF DDZ-AMINO ACIDS IN THE PEPTIDE SYNTHESIS OF BIOLOGICALLY ACTIVE NEW ANTAMANIDE ANALOGUES*

The mixed anhydride (MA) method of peptide synthesis is further simplified by the repetitive excess use of Ddz-amino acids. In six comparative preparations of decapeptides this is demonstrated by the ease and speed of the synthetic manipulations, the efficiency of the monitoring of the reactions and purifications, and by the complete recycling of excess amino acid derivatives. In using of Ddz-amino acyl isobutylformate mixed anhydrides, side reactions are effectively suppressed, as proved by good coupling of Ddz-proline on to prolyl-peptides. Hydrophilic, crystalline and biological active antamanide analogues are obtained containing various functional side groups. The known cis-conformation of the antamanide prolyl-prolyl bonds is also established in the analogues by ¹³C-n.m.r. measurements. All new compounds are characterized by molecular peaks in the mass spectra.     

THE SYNTHESIS AND OPIATE ACTIVITY OF HUMAN β-ENDORPHIN ANALOGS

Four analogs of human β-endorphin have been synthesized and their opiate activities have been determined. One of the analogs, [27-phenylalanine, 31-glycine]-β_h-endorphin, was shown to possess an analgesic potency greater than that of the natural peptide.     

STABLE ISOLATED SYMMETRICAL ANHYDRIDES OF Nα-9-FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS IN SOLID-PHASE PEPTIDE SYNTHESIS:

The synthesis and isolation of symmetrical anhydrides of N^α-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids using water soluble carbodiimide is described. These compounds were used in a solid phase peptide synthesis of methionine-enkephalin on a p-benzyloxybenzyl ester polystyrene 1% divinylbenzene resin support. Homogeneous free pentapeptide was obtained in 42% overall yield. The Fmoc amino acid symmetrical anhydrides were stable during prolonged storage (2 years at 0°) and offer advantages over present “Fmoc solid phase” methods which use anhydrides formed in situ.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

‘100 years of peptide synthesis’: ligation methods for peptide and protein synthesis with applications to β‐peptide assemblies*

Abstract: A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, ‘ Synthesis of Proteins by Chemical Ligation ’ a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo‐prolines, ‘unrestricted’ amino‐acid residues (using the Staudinger reaction), as well as solid‐phase segment coupling by thioligation of unprotected peptides. In another section, ‘ Synthesis of β‐peptides by Thioligation ’, couplings involving β²‐ and β³‐peptides are described (with experimental details).     

SYNTHESIS AND ANALGESIC ACTIVITY OF HUMAN β-ENDORPHIN ANALOGS SUBSTITUTED AT POSITIONS 17, 18, OR 19

Three peptide analogs of β-endorphin which are substituted in positions 17, 18 or 19 have been synthesized and their analgesic potencies have been measured by the tail-flick method in mice. The results showed that the replacement of Phe-18 or Lys-19 by alanine reduced the potency to 15% whereas the replacement of Leu-17 by alanine reduced the analgesic potency to 68%.     

β-ENDORPHIN: SYNTHESIS AND RADIOLIGAND BINDING ACTIVITY OF ANALOGS CONTAINING CYSTINE BRIDGES

Two analogs of human β-endorphin containing cystine bridges between positions 21 and 26 or 14 and 26 have been synthesized and their radioligand binding activity using rat brain membranes has been determined. Both peptides showed three to four times the binding activity of human β-endorphin.     

HUMAN β-ENDORPHIN: SYNTHESIS AND CHARACTERIZATION OF ANALOGS IODINATED AND TRITIATED AT TRYOSINE RESIDUES 1 AND 27

Three tritiated analogs of human β-endorphin have been prepared from their corresponding iodinated analogs. The iodinated analogs (diiodotyrosine at positions 1, 27 or 1 and 27) were synthesized by the solid-phase method and were found to have biological and physical properties which were altered when compared with the native molecule. Catalytic exchange of these iodinated analogs in the presence of tritium yielded tritiated human β-endorphins having full biological activity and specific activity of 50–100 Ci/mmol. Both the iodinated and tritiated β-endorphin analogs were shown to be homogeneous by chromatography on carboxymethylcellulose, partition chromatography, paper chromatography, amino acid analysis, electrophoresis, high performance liquid chromatography, and isoelectric focusing on polyacrylamide.     

STUDIES ON RACEMIZATION DURING COUPLINGS USING A SERIES OF MODEL TRIPEPTIDES INVOLVING ACTIVATED RESIDUES WITH UNFUNCTIONALIZED SIDE CHAINS

Racemization studies have been carried out using as model tests couplings of N-benzyloxycarbonylglycyl-l -χ with benzyl N^e-benzyloxycarbonyl-l -lysinate followed by deprotection and analysis for the diastereomeric tripeptides with an amino acid analyzer, for χ = alanine, leucine, phenylalanine, valine and isoleucine. The order of susceptibility to racemization of residues during these segment couplings depends on whether the solvent is polar or apolar, with isoleucyl and valyl, followed by phenylalanyl, being the most susceptible ones in polar solvents. The racemization depressing effect of some additives on carbodiimide-mediated couplings has been examined. Reconciliation of apparent discrepancies in data in the literature on the relative merits of l-hydroxy-benzotriazole and N-hydroxysuccinimide is made on the basis of the nature of the model tests used in acquiring the data.     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

SYNTHESIS OF γ-METHYL-l-LEUCINE (NEOPENTYLGLYCINE,NEO) AND DERIVATIVES SUITABLE FOR PEPTIDE SYNTHESIS

The modified Strecker synthesis of Patel & Worsley was combined with an enzymatic deamidation in the final step, to produce the title compound. The amino acid is a member of the family of the “fat” amino-acids with high lipophilicity and space-filling properties, as estimated by its Hansch hydrophobicity constant and its Charton steric parameter v. It can be used for qualitative and quantitative studies of structure-activity relationships in biologically active peptides. Three of its derivatives suitable for peptide synthesis are also described.     

SOLID-PHASE PEPTIDE SYNTHESIS USING MILD BASE CLEAVAGE OF NαFLUORENYLMETHYLOXYCARBONYLAMINO ACIDS,EXEMPLIFIED BY A SYNTHESIS OF DIHYDROSOMATOSTATIN

N ^α-9-Fluorenylmethyloxycarbonyl (Fmoc) amino acids will be of advantage in solid phase peptide synthesis. The Fmoc-group is quantitatively cleaved by mild base (piperidine). This permits the use of tert-butyl-type side chain blocking and of peptide-to-resin linkage cleavable by mild acidolysis. Side reactions arising from repetitive acid deprotection and final HF cleavage in contemporary solid phase synthesis are avoided. Fully bioactive and homogeneous dihydrosomatostatin was obtained in 53% overall yield.     

SYNTHESIS OF γ-CARBOXYGLUTAMIC ACID AND ITS DERIVATIVES VIA MANNICH-BASE CONDENSATION

This report describes the synthesis of N^α-formyl and N^α-benzyloxycarbonyl-γγ-di-t-butyl-γ-carboxyglutamic acid via Mannich-base condensation starting from di-t-butyl malonate and diethyl formamido or benzyloxycarbonylamidomalonate.     

β-ENDORPHIN: SYNTHESIS AND RADIORECEPTOR BINDING ACTIVITY OF βh-ENDORPHIN-(1–27) AND ITS ANALOGS

β_h-Endorphin-(1–27) (I), [Ac-Tyr¹]-β_h-endorphin-(1–27) (II), [Gln⁸]-β_h-endor-phin-(1–27) (III), and [Ac-Tyr¹, Gln⁸]-β_h-endorphin-(1–27) (IV) were synthe sized by the solid-phase method. The binding potency of I-IV to rat brain membrane preparations was measured by radioreceptor binding assay. The relative potencies were: β_h-endorphin, 100; I, 30; II, 0.04; III, 90; IV, 0.07.     

ALTERNATIVE SYNTHESIS OF CORTICOTROPIN-LIKE INTERMEDIATE LOBE PEPTIDE (HUMAN CLIP)

A novel synthesis of human CLIP, a peptide corresponding to the sequence of human ACTH-(18–39) is described. The dodecapeptide chain was assembled by a combination of fragment condensation and stepwise synthesis while using N^α-benzyloxycarbonyl and side chain tert.-butyl-derived protective groups combination. The final deprotection was performed by acidolysis in trifluoroacetic acid. The end product was purified by ion exchange chromatography on carboxy-methyl cellulose.     

HUMAN β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF ANALOGS WITH SUBSTITUTIONS IN POSITIONS 2, 9, 18, 27 AND 31

Four analogs of the opioid peptide human β-endorphin (β_h-EP) have been synthesized: [d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, [d -Phe¹⁸,Phe²⁷,Gly³¹)-β_h-EP, [d -Thr²,d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, and [d -Thr²,d -Phe¹⁸,Phe²⁷,Gly³¹]-β_h-EP. All are practically indistinguishable from β_h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.