Spontaneous hydrolysis of vasoactive intestinal peptide in neutral aqueous solution

Hydrolysis of radioiodinated vasoactive intestinal peptide (VIP) was observed in buffered aqueous solution at neutral pH and 38 °C. The reaction displayed apparent first-order kinetics at initial peptide concentrations below 3 nM (K_obs= 1.5 × 10^?5s^?1), but the rate deviated below predicted values at higher peptide concentrations. The rate constant derived from the reaction progress curve over three half lives, starting at a concentration of 82 PM peptide, was also consistent with a first-order process. The reaction results in several products that were isolated and characterized as peptide fragments. Based on the identity of these fragments, we deduced hydrolysis at five different peptide bonds clustered between residues 17–25 of VIP. Control experiments were devised to eliminate trivial explanations for the peptide hydrolysis. Peptides representing the C-terminal segment 15–28 and the internal segment 14–22 assayed by analogous methods and under identical conditions were not degraded at a measurable rate. Sodium dodecyl sulfate and acetonitrile, agents known to influence the secondary structure of VIP, inhibited its spontaneous hydrolysis, as did chloride salts of sodium and calcium, albumin and a peptide unrelated to VIP. The rate and product distribution are inconsistent with known pathways of peptide degradation involving cyclic imide or anhydride formation at asparagine or aspartate residues. We suggest that the breakdown of VIP in dilute solutions represents an autolytic process.     

SOLID-PHASE PEPTIDE SYNTHESIS OF SOMATOSTATIN USING MILD BASE CLEAVAGE OF Nα-9- FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS

Experimental details for the “Fmoc solid phase peptide synthesis” of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. Nα-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55–60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91% yield). Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.     

Synthesis of a tripeptide with a C-terminal nitrile moiety and the inhibition of proteinases

The synthesis of the tripeptide d -Phe-Pro-Arg with the nitrite group instead of the carboxylgroup is described. Initially, the corresponding peptide amide was synthesized by conventional methods in solution using Boc and Fmoc as the protecting group for d -Phe. The dehydration in order to create the nitrite moiety was achieved by treating the peptide amide with phosphorus oxichloride or trifluoroacetic anhydride. Best results were obtained by the use of phosphorus oxichloride in pyridine as the solvent in the presence of imidazole. After deprotection of the N-terminal amino acid the crude product was purified by chromatography on Butyl-Fractogel® HW-40 (S). The purity of the final product was checked on a RP18 phase by hplc. The existence of the nitrite group was demonstrated by i.r. and ¹³C-n.m.r. spectra. The peptide nitrite exhibited a strong inhibition of thrombin compared to the tripeptide amide.     

STUDIES ON PEPTIDES

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken VIP was synthesized in a conventional manner, using a new arginine derivative, N^G -mesitylene-2-sulfonylarginine, Arg(Mts). Treatment of a fragment, Z(OMe)-Thr-Asp-Asn-Tvr-NHNH₂ with methanesulfonic acid or HBr was found to give a product with a low recovery of Asp, after aminopeptidase digestion. Ring closure of the Asp-Asn unit seemed to be responsible for this phenomenon. Deprotection with HF or TFA exhibited definitely less such a tendency. In the final step of the synthesis, all protecting groups, including the Mts group, were removed by HF in the presence of m-cresol and the deprotected peptide was purified by ion-exchange chromatography on CM-cellulose followed by isoelectric focusing in Ampholine pH 9–11. Synthetic peptide exhibited the identical Rf value with that of natural chicken VIP and was active as the natural peptide.     

SOLID PHASE SYNTHESIS WITHOUT REPETITIVE ACIDOLYSIS

The utility of repetitive nonhydrolytic base cleavage of α-amino protective groups in solid phase peptide synthesis is shown by a preparation of the model tetrapeptide leucyl-alanyl-glycyl-valine on a p-benzyloxybenzyl ester polystyrene–1% divinylbenzene resin support. Nα-9-Fluorenylmethyloxycarbonyl (Fmoc: Carpino & Han, 1970, 1972) amino acids were coupled by the symmetrical anhydride procedure, followed by Fmoc group cleavage using 50% piperidine in methylene chloride. Quantitative removal of the Fmoc-tetrapeptide from the solid support was effected by treatment with 55% trifluoroacetic acid in methylene chloride. Homogeneous free tetrapeptide was obtained in 87% overall yield. The procedure is proposed to offer advantages over present solid phase methods which use acidolysis for repetitive α-amino group deblocking.     

Chemical synthesis of α-inhibin-92 by the thiocarboxyl segment coupling method

The 92 amino acid residue peptide, α-inhibin-92 (α-IB-92), has been synthesized by the thiocarboxyl segment strategy. Three segments were synthesized by the solid phase method, purified, and characterized: [GlyS³⁴]-α-IB-92-(l-34) (I), CF₃CO-[GlyS⁶⁵]-α-IB-92-(35–65) (II), and Msc-α-IB-92-(66–92) (III). All were reacted with citraconic anhydride followed by removal of the Msc group in III to give Ia, IIa, and IIIa, respectively. Peptide IIIa was coupled to IIa by the silver nitrate/N-hydroxysuccinimide procedure and, after removal of uncoupled segments and the trifluoroacetyl group, Ia was coupled followed again by removal of uncoupled segments. Final deblocking to remove citraconyl groups was accomplished under exceptionally mild conditions in aqueous acetic acid. The synthetic product was identical to natural α-IB-92 in amino acid analysis, HPLC, gel electrophoresis, and tryptic mapping. The synthetic peptide was indistinguishable from natural α-IB-92 in a radioimmunoassay and in an in vitro mouse pituitary assay for measuring suppression of FSH release in the presence of LHRH.     

4-Chloromethylphenoxyacetyl polystyrene and polyamide supports for solid-phase peptide synthesis

Two functionalised supports for the solid-phase synthesis of peptides under mild reaction conditions were prepared: 4-chloromethylphenoxyacetamidomethylcopoly (styrene-1%-divinylbenzene) and 4-chloromethylphenoxyacetyl-norleucylpoly(dimethylacrylamide). They were devised in order to avoid the danger of racemization which exists during base-catalyzed esterification of the first protected amino acid to the 4-alkoxybenzyl alcohol resins formerly employed in combination with N^α-9-fluorenylmethoxycarbonyl and tert.-butyl side-chain protecting groups. Esterification of N^α-protected amino acids to the new resins can be achieved easily and without significant levels of racemization by means of their caesium salts, while cleavage from the supports is possible by treatment with trifluoroacetic acid. The 4-chloromethylphenoxyacetyl polystyrene resin was tested by the synthesis of Leu-enkephalin which was cleaved, at the end of the synthesis, from the solid support in 91% yield by 60% trifluoroacetic acid in methylene chloride, and was shown to be more than 99% pure by ion-exchange chromatography and reverse phase high pressure liquid chromatography.     

Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-C～S⁶,D-Cys¹¹,Lys¹²,Nle¹⁷,Val²⁶,Thr²⁸]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys¹²,Cys¹⁷,Val²⁶,Cys²⁸]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (>40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor–active conformation of VIP.     

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.     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH₂及其类似物Phe-Pro-Arg-Phe-NH₂,并观察了此二种肽对大鼠血压和心率的影响。     

Synthesis,conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(Bu^t)-Ala-Thr(Bu^t)-Thr(Bu^t)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO₂)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO₂)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

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.     

Purification and characterization of ostrich pancreatic secretory trypsin inhibitor

Ostrich pancreatic secretory trypsin inhibitor was isolated and purified using acid extraction, salt fractionation, SP-Sephadex C-50 and QAE-Sephadex A-25 chromatography and RP-HPLC. The amino acid sequence of ostrich PSTI showed it is a single peptide chain containing 69 amino acid residues with the highest homology between ostrich and chicken PSTI. The molecular weight, as determined by electronspray mass spectrometry and from amino acid sequence data, is 7650 Da. The isoelectric point of ostrich PSTI was found to be 5.7. Ostrich PSTI specifically inhibited ostrich and commercial bovine trypsin with K_i values of 8.0 × 10^?9 and 2.4 × 10^?7M, respectively, while no inhibitory effects were observed with other serine proteases. © Munksgaard 1996.     

SYNTHESIS OF THE DODECAPEPTIDE-α MATING FACTOR OF SACCHAROMYCES CEREVISIAE

The synthesis of His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, the dodeca-peptide α-mating factor from Saccharomyces cerevisiae, and its Ala²-and Cha²-(β-cyclohexylalanine) analogs are reported. Peptides were synthesized in solution using a combination of mixed anhydride and 1-hydroxybenzotriazole accelerated active ester coupling procedures. Dilute methanesulfonic acid (0.1–0.2 M) in methylene chloride-formic acid solution was employed to specifically remove the tert.-butoxycarbonyl group in the presence of the benzyloxycarbonyl group. Free peptides were obtained using catalytic transfer hydrogenation with formic acid as the hydrogen donor followed by mild acidolysis with trifluoroacetic acid. The α-factor and the Cha²-analog exhibited almost equal ability to cause “shmooing” of a-mating types of S. cerevisiae whereas the Ala²-analog exhibited no activity in this assay. These results differ with structure-activity studies reported on the tridecapeptide α-factor.     

Synthesis and photolytic cleavage of bovine insulin B22–30 on a nitrobenzoylglycyl-poly (ethylene glycol) support

The synthesis of the C -terminal nonapeptide of bovine insulin B-chain is desscribed. 4-(Bromomethyl)-3-nitrobenzoylglycyl-poly(ethylene glycol) (Mr = 15 000) was used as soluble support. The C -terminal alanine was first converted to Boc-Ala-O-(2-nitro-4-carboxy) benzyl ester which was then coupled to Gly-PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH-20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion-exchange chromatography on CM-Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.     

Fmoe SPPS using Perloza™ beaded cellulose

Perloza? beaded cellulose was functionalised by a cyanoethylation reduction procedure to give aminopropyl Perloza. Fmoc-amino acids were anchored to aminopropyl Perloza beaded cellulose via the TFA labile 4-oxymethylphenoxyacetyl (HMPA) linker. Using Fmoc-aminoacyl-4-oxymethylphenoxyacetyl-2,4-dichloro-phenyl esters, all 20 amino acids were anchored at substitution levels ranging from 0.37 to 0.65 mmol/g. Fmoc-amino acids were also anchored using the peptide-amide linker 4-[(R,S)-1-[1-(9H-fluoren-9-yl)-methoxycarbonylamino]-(2′,4′dimethoxy-benzyl]phenoxyacetic acid. The Fmoc-aminoacyl resins were used for SPPS using Fmoc chemistry. SPPS was carried out using either an LKB Biolynx 4175 low-pressure pumped column continuous-flow peptide synthesiser or an ABI 430A automated vortexing batchwise instrument. Comparison of peptides made using each synthesiser showed little difference in quality of the crude peptides. Different Fmoc-amino acid activation methods (DIC/HOB_t/DMF, HBTU, DIC/HOB_t/DCM) were found to be equally useful with Perloza. Peptides were cleaved using TFA plus scavengers; however, the TFA-swollen resin was not readily separated from the TFA peptide solution by simple filtration. Therefore alternative cleavage workup procedures were used with Perloza. Peptides were purified by HPLC and characterised by HPLC and amino acid analysis, and in some cases by FAB-MS. Successful syntheses ranged from 5 to 34 amino acids in length. Some of the peptides were also synthesized using a polystyrene support and standardised (ABI Fastmoc?) SPPS protocols. The crude cleaved peptides from each synthesis were compared by HPLC analysis. The overall aim of our work with Perloza is synthesis of resin-bound peptide ligands for affinity chromatography and antibody generation. HPLC analysis of crude peptides showed that peptides up to 20 amino acids in length were of reasonable purity when synthesised using Perloza, thus encouraging us to continue with investigations of SPPS of resin-bound ligands.     

Enhancement of peptide coupling reactions by 4-dimethylaminopyridine

4-Dimethylaminopyridine (DMAP) was found to be useful in the enhancement of peptide coupling reactions mediated by dicyclohexylcarbodiimide or symmetrical anhydrides. In an automated synthesis of the model heptapeptide Boc-Ala-Cle-Ile-Val-Pro-Arg(Tos)-Gly-OCH₂-Resin (Cle, cycloleucine), the efficiencies of various coupling methods such as dicyclohexylcarbodiimide, dicyclohexylcarbodiimide plus 1-hydroxybenzotriazole, and symmetrical anhydride were compared with that of dicyclohexylcarbodiimide plus 4-dimethylaminopyridine. Based on the amino acid composition of the peptide-resin samples and high pressure liquid chromatographic analyses of the protected heptapeptide amide obtained from the ammonolytic cleavage of the peptideresin samples, it was concluded that only dicyclohexylcarbodiimide plus 4-dimethylaminopyridine gave the desired near quantitative couplings in those cycles involving the sterically hindered amino acid residues. Observations were also made that 4-dimethylaminopyridine was a useful additive in a modified symmetrical anhydride method of coupling. In the synthesis of the model tetrapeptide Leu-Ala-Gly-Val on a Pam resin, the anhydride couplings were accelerated by DMAP and the product was equivalent in homogeneity to that obtained by the best previous methods. In addition, no racemization was detectable by a sensitive chromatographic method. There also was no detectable racemization found in a DCC-DMAP coupling of Boc-Ile-OH with H-Val-OCH₂-resin. However, significant racemization was observed during the coupling of Boc-Phe-OH with H-Glu(OBzl)-OCH₂-resin. DMAP is recommended as an additive for coupling hindered amino acids, particularly C^α-substituted residues, where little or no racemization is expected.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-α-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C₁₈ reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H₂O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

Weak acid-catalyzed pyrrolidone carboxylic acid formation from glutamine during solid phase peptide synthesis

Formation of pyrrolidone carboxylic acid (pyroglutamic acid) residues from amino-terminal glutaminyl residues in peptides was shown to be catalyzed by weak acids, but not by strong acids. During dicyclohexylcarbodiimide-mediated coupling reactions the N^α-protected amino acid reagent accelerated this cyclization and resulted in a significant amount of chain termination. The side reaction could be minimized by accelerating the coupling reaction and simultaneously reducing the time of exposure to weak acids. The most effective procedure was to couple in dimethylformamide with the preformed symmetric anhydride of the amino acid.     

Partially modified retro-inverso peptides

Several synthetic routes are reported to prepare the hetero diprotected 1,1-diaminoalkanes from N-acylated amino acids or peptides for incorporation into partially modified retro-inverso peptides. The Curtius rearrangement was carried out on the N-protected aminoacyl azides obtained from the N-protected aminoacyl hydrazide by nitrosyl chloride or by sodium azide reaction with an appropriate mixed carboxylic carbonic acid anhydride. The resulting isocyanate was allowed to react with alcohol to give a urethane-type protecting group or, via a “one-pot” approach, directly with a carboxyl carrying component to yield the modified (reversed) peptide bond. The carboxyl component can be either an N-acylated amino acid or a malonic acid. The more standard route involves selective deprotection of the 1,1-diaminoalkane residue followed immediately by coupling with a carboxyl component to yield the same modified peptide derivative.