Use of the 3-nitro-2-pyridine sulfenyl protecting group to introduce Nε-branching at lysine during solid-phase peptide synthesis I. Application to the synthesis of a peptide template containing two addressable sites

TASPs (template-assembled synthetic peptides) are generated by the covalent attachment of linear peptides to a common peptide backbone, thus generating larger synthetic peptides/proteins with prefolded structure. In this work we present a strategy for the synthesis of a heterotemplate-assembled synthetic peptide containing two addressable sites. This orthogonal protection strategy would allow the selective introduction of different peptide chains via the ε-amino functions of template lysines being protected by either fluorenylmethoxycarbonyl (Fmoc) or 3-nitro-2-pyridine sulfenyl (Npys) groups. The N^α-Boc-N^ε-Npys-l -lysine required for solid-phase peptide synthesis (SPPS) is not readily available at a reasonable cost. To facilitate the more widespread use of this reagent we have compared the two published procedures for synthesizing this protected amino acid and evaluated the suitability of the products for SPPS. Two resin-bound peptides, a tripeptide Ac-G-K-Npys)-G-resin and an octapeptide template Ac-P₁-K₂-K₃-L₄-K_s-K₆-P₇-G₈-resin, were synthesized by SPPS. The ε-amino functions of lysines K₂ & K₆ and K₃ & K₅ of the octapeptide were protected by Fmoc and Npys groups, respectively. Secondly, these peptides were used to evaluate various reagents and reaction conditions for the deprotection of the ε-amino function of lysines bearing the N_ε-Npys protecting group. A procedure for the optimized selective and quantitative deprotection of the Npys group from the ε-amino function of lysine in a resin-bound peptide using 2-mercaptopyridine-N-oxide is described. © Munksgaard 1995.     

Peptide formation in the presence of metal ion protecting groups

A quantitative study of the degree of racemization induced by the [(NH₃)₅Co-(III)-] protecting group when bound to the C-terminal of the amino acids Leu, Phe, and His, as has been carried out. Racemization was determined by forming the diastereomeric cobalt dipeptides [(Leu)(AA)Co(III)(NH3)₅] where AA = L-Leu, L-Phe, and L-His; after cobalt removal (using NaBH₄), the peptide diastereomers were analyzed quantitatively using an amino acid analyzer. No racemization was observed within experimental error (0.3%) as a result of the substitution of the [(NH3)₅Co(III)-] group on the amino acids and peptides studied.     

β-Endorphin: synthesis and properties of human β-endorphin-(1–28) and its analogs

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–28) (II), [D-Ala², Gln⁸] - β_h-EP-(1–28) (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 85; II, 380; III, 146. Relative potencies in an analgesic assay were: β_h-EP; 100; I, 18; II, 36; III, 13.     

New mild acid-labile protecting groups for the guanidino function of Nα-fluorenylmethoxycarbonyl-l-arginine in solid-phase peptide synthesis: 10, 11-dihydro-5H-dibenzo[a,d]. cyclohepten-5-yl, 2-methoxy-10, 11-dihydoro-5H-dibenzo[a,d]. cyclohepten-5-yl and 5H-dibenzo[a,d]. cyclohepten-5-yl groups

10, 11-Dihydro-5H-dibenzo[a, d]. cyclohepten-5-yl [5-dibenzosuberyl]. and 5H-dibenzo[a, d]. -cyclohepten-5-yl [5-dibenzosuberenyl]. groups have been found to be useful protecting groups for the guanidino function of arginine in solid-phase peptide synthesis on Fmoc chemistry. The arginine derivatives ( 4a, b, c ) derivatized with these groups were easily deprotected with mild acid (less than 30 min with 25% trifluoroacetic acid). Tryptophan-containing peptide sequences, two hexapeptides ( 6 ) and (8), were synthesized in good yield by mild acid treatment (50% trifluoroacetic acid in 1 h) of the peptide resins ( 5a, c-f and 7a, c, d ) assembled via 4a, b, c using benzotriazol-1 -yl-oxy-tris-(pyrrolidino)-phosphonium hexafluorophosphate-1-hydroxybenzotriazole mediated coupling.     

Allyl ester as temporary protecting group for the β-carboxy function of aspartic acid

The use of the allyl ester as a temporary protecting function in aspartyl peptides is described. The β-allyl ester was introduced into Boc-Asp-Phe-NH₂ 4a, Boc-Asp-Gly-NH₂ 4b and Boc-Asp-Ser-NH₂ 4c by means of the caesium salt method. It was removed from these molecules 5 using noble metal catalysts under weakly basic, acidic or even neutral conditions likewise in high yield. The formation of an aminosuccinyl derivative during the deblocking reactions was not observed. Treatment of the dipeptide Boc-Asp(OAll)-AS-NH₂ with an excess of triethylamine at room temperature induced the ring closure to the cyclic imide.     

β-Endorphin

Ostrich β-endorphin has been synthesized by the solid-phase method. Opiate activity in a radioreceptor binding assay is about seven times that of human β-endorphin. Structural differences within positions 6–15 account for the increased binding potency.     

Synthesis and properties of dermorphin and an analog of β-endorphin containing the dermorphin sequence

Dermorphin (I) and [D-Ala², Phe³, Gly⁴, Tyr⁵, Pro⁶]-β_c-EP (II) have been synthesized by the solid-phase method (β-EP, camel β-endorphin). Positions I through 7 of II correspond to the sequence of I. Relative potencies of synthetic peptides in the mouse tail-flick test for analgesia by the intracerebroventricular route were: human β-endorphin, 100; camel β-endorphin, 164; I, 450; II, 440. The dermorphin was about 670 times more potent than morphine in the assay. Peptide II represents a rare instance where the enkephalin moiety of β-endorphin has been altered to produce a more potent analgesic.     

β-Endorphin: synthesis and properties of analogs modified in positions 8 and 31

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: [Gln^8.31]-β_h-EP(I), [Arg⁸, Gln³¹] -β_h-EP(II), [Ala⁸, Gln³¹] -β_h-EP (III), and [Val⁸, Gln³¹]-β_h-EP(IV). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 200; II, 150; III, 150; IV, 120. Relative potencies in an analgesic assay were: β_h-EP, 100; 1,236; II, 254;III, 116; IV, 121. The side-chain of Glu-8 in β_h-EP can be replaced by a variety of structures without diminishing biological activity.     

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.     

Synthesis of a metal binding protein designed on the α/β scaffold of charybdotoxin*

The α/β scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid-phase synthesis and HBTU (2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupling of Fmoc-protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post-synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2°, yield. Circular dichroism analysis showed that the newly designed protein is folded in a α/β structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu²⁺ and Zn²⁺ ions bind with high affinity to the newly engineered protein. These results demonstrate that the α/β fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins. © Munksgaard 1995.     

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.     

β-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human β-endorphin (β-EP) which contain cystine bridges, [Cys¹⁵-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (I) and [Cys¹⁶-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2–2.5 times the opiate receptor binding activity of β-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg^{9,19,24,28,29}Cys(Cam)^11,26, Phe²⁷,Gly³¹] and [Arg^{9,19,24,28,29},Cys-(Cam)^12,26, Phe²⁷, Gly³¹], were shown to have approximately the same opiate receptor activity as β-endorphin.     

Formation of aminosuccinyl derivative from β-phenacyl aspartyl peptides catalyzed by sodium thiophenoxide

In the solid-phase synthesis of cholecystokinin 30–33, Trp-Met-Asp-Phe- amide, the β-phenacyl ester was used to protect the β-carboxyl of aspartyl residue. The ester was cleaved, on the solid support, with a 1 M solution of sodium thiophenoxide in DMF, prior to ammonolysis. The product, after purification by countercurrent distribution, was identified as a mixture of isoasparaginyl and aspartyl peptides. A study of the deprotection step, with sodium thiophenoxide, on a model peptide (t-butyloxycarbonyl-β-phenacyl-aspartyl-phenylalanineamide) showed the rapid formation of the aminosuccinyl derivative, catalyzed by this reagent.     

Investigation on the stability of the Dde protecting group used in peptide synthesis: migration to an unprotected lysine1

An investigation of the stability of the Dde protecting group for amines, used in solid-phase peptide synthesis, shows that an unprotected ε-NH₂ group of lysine can acquire the Dde protection from another ε-NH₂ group or from an α-NH₂ group. An unprotected α-NH₂, however, cannot remove Dde from an ε-NH₂ function. This migration takes place during Fmoc removal from the ε-NH₂ with piperidine and/or during the subsequent washing steps. The Dde migration is also possible in neat dimethylformamide by a direct nucleophilic attack of the free ε-NH₂ group. Addition of piperidine to the reaction medium accelerates the side reaction, probably because of the formation of an unstable piperidine-Dde adduct. Dde migration can he prevented if the 9-fluorenylmethyloxycarbonyl is cleaved with 1,8-diazabicyclo[5,4,0]undec-7-ene for a short reaction time (2%, 3 × 3 min). Finally, this rearrangement is shown to occur both as an intra- and intermolecular reaction between peptides on the same resin bead.     

Studies of peptide antibiotics. XLVI. Syntheses of gramicidin S analogs containing D-α, β-diaminopropionic acid or α,β-dehydroalanine*

A gramicidin S (GS) analog ([d -Dpr^4,4] GS) containing d -α,β-diaminopropionic acid (D-Dpr) in place of D-Phe at 4,4′ positions was derived from [l -Orn(δ-formyl)^2,2, d -Dpr(β-Z)^4,4′]GS, which was synthesized by conventional method in solution. An analog [ΔAla^4,4′]GS was synthesized from [l -Orn(δ-Boc)^2,2′, d -Dpr^4,4′]GS through Hofmann degradation of the d -Dpr residues. Antimicrobial activities of these analogs were tested; [d -Dpr(β-Z)^4,4′]GS and [ΔAla^4,4′]GS showed high antimicrobial activities against Gram-positive bacteria. [d -Dpr^4,4′]-GS showed an appreciable activity against Gram-negative bacteria such as Escherichia coli. Four semigramicidin S (semiGS) analogs such as [ΔAla⁴]semiGS were synthesized; these had no antimicrobial activity. Analogs containing ΔAla residues were hydrogenated, and the formation of l -Ala or d -Ala residues was determined. The ΔAla residues in [ΔAla^4,4′] GS were reduced to dl -Ala, and ΔAla in [ΔAla⁴]semiGS mostly to l -Ala. The relationships of the antimicrobial activity, CD curves and asymmetric hydrogenation to the structure were discussed.     

Improved method for the synthesis of Nα-9-fluorenylmethyloxycarbonyl-Nδ,ω bis-adamantyloxycarbonyl-L-arginine

A modified method is reported for the prepartion of N^α-9-fluorenylmethyloxycarbonyl-N^δ,ω bis-adamantyloxycarbonyl-L-arginine, giving an overall yield of 60% over three steps based on N^α-benzyloxycarbonyl-L-arginine. Commercially available adamantyl fluoroformate for guanidine function protection of N^α benzyloxycarbonyl-L-arginine, catalytic transfer hydrogenation with formic acid on palladium black for removal of the benzyloxycarbonyl protecting group, and fluorenylmethylsuccinimidyl carbonate for the final synthesis, were introduced to simplify and reduce the cost of preparation of this arginine derivative. The reaction conditions have been accurately studied at each step in order to optimize the yields.     

Solid-phase synthesis of peptides via α, β-unsaturated amino acids: Oxytocin,simultaneous incorporation of amide functions in COOH-terminal and endo-positions

Oxytocin was synthesized via the solid-phase method using dehydroalanine as pseudo-protecting group of the carboxyl-terminal as well as the ω-amide functions of asparagine and glutamine in endo-position. Starting with Boc-Gly-Dha-resin and using Boc-L-Asp(Dha-NHEt)-OH and Boc-L-Glu(Dha-NHEt)-OH as precursors of asparagine and glutamine, respectively, oxytocin was assembled in stepwise manner under solid phase synthesis conditions. Treatment of the protected [Glu(Dha-NHEt)⁴, Asp(Dha-NHEt)⁵]-oxytocin-Dha-resin with 1 N HCl in glacial acetic acid in the presence of 3 equiv. water removed the peptide from the support with the simultaneous formation of the asparagine and glutamine residues to give the protected nonapeptide amide: Cbz-Cys(Bzl)-Tyr(Bzl)-He-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH₂, which was deprotected with sodium in liquid ammonia and then oxidized with diiodoethane to give oxytocin. After purification by gel chromatography and countercurrent distribution, the product displayed the chemical and physical properties and oxytocic activity (533 ± 30 IU/mg) of a standard oxytocin preparation.     

Cleavage of aspartyl β-phenacyl esters by selenophenol under neutral conditions

Neutral selenophenol in DMF accomplished the removal of the β-phenacyl protecting group of aspartic acid in solution, on the peptide Boc-Asp(β-OPac)-Phe amide and on the resin peptide Boc-Trp(Nⁱⁿ-For)-Met-Asp(β-OPac)-Phe- (CCK 30–33) without α, β-rearrangement.     

Synthetic peptide models for protein secondary structures β-Sheet formation in acyclic cystine peptides

The conformations of the symmetrical cystine peptides Boc-Cys -(Val)_n -Trp-OMe Boc-Cys-(Val)_n -Trp-OMe (n = 1, 1; 2, 2; 3, 3) have been examined in solution, in order to evaluate the use of disulfide crosslinks in stabilizing extended β-strand conformations in acyclic sequences. NMR studies in (CD₃)₂ SO provide evidence for the solvent inaccessible nature of the Val (2) NH group in peptides 1 and 2. J_HNCHxH values are indicative of extended structures. Sequential interresidue nuclear Overhauser effects support the population of β-strand structures in both peptides. The fluorescence quantum yield of tryptophan determined in methanol follows the order 2 > 1 ñ 3. Reduction of the disulfides with NaBH₄ results in large enhancements of emission intensity, with the changes following the order 1 > 3 > > 2. The order of quenching is a function of the disposition of the indole and disulfide sidechains in an extended beta;-sheet structure.     

Synthesis,purification and characterization of thymosin α11, a new thymic peptide

A synthesis of thymosin α₁₁, a new 35 amino acid thymic peptide structurally related to thymosin α₁ (J. Caldarella, G.J. Goodall, A.M. Felix, E.P. Heimer, S.B. Salvin & B.L. Horecker (1983) Proc. Natl. Acad. Sci US 80, 7424–7427), is reported. The synthesis was accomplished by the solid phase method employing the 4-(aminoacyloxymethyl) phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. The synthetic material was purified by preparative high pressure liquid chromatography and was found to be equivalent to the natural compound in its chemical properties.