Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.     

Structure and activity of insulin, XIII. Specificity of the arginine-guanidino group in biologically active tetrapeptides of the insulin sequence B 22-25 (Arg-Gly-Phe-Phe).

The biologically active partial sequence Arg-Gly-Phe-Phe (position B 22-25 of the insulin B chain) in the form of the synthetic tetrapeptidamide, was compared in several bioassays with the following analogous synthetic peptides: homoarginyl-, ornithyl-, lysyl-, citrullyl-, alanyl- and NG-nitroarginyl-Gly-Phe-Phe-NH2. The syntheses of the lysyl- and alanyl-tetrapeptidamides are described. After intraperitoneal injection of the peptides in doses of 3-100 mumol per 100 g rat, together with [U-14C]glucose, the natural sequence Arg-Gly-Phe-Phe showed the highest insulin like activity (incorporation of labeled carbon into the diaphragm). The activity of the homoarginyl peptide was a little weaker. The ornithyl- and the lysyl-peptide, however, showed a remarkably diminished activity. The activity of the citrullyl-peptide was even lower and the alanyl-peptide was inactive. In vitro assays with rat diaphragm showed the same range of effects for the elevation of glucose uptake and glycogen content of the diaphragm. The activity decreased in the following order: Arg- greater than Har- greater than Orn- greater than Cit-Gly-Phe-Phe-NH2. Alanyl- and Nitroarginyl-Gly-Phe-Phe-NH2 were without effect. In isolated fat cells the glucose oxidation was enhanced significantly only by the arginyl-peptide. The results show that among the structures examined the guanidino group carried by the C5 chain of arginine is the most effective. The results are in accordance with our preceding work [1] using semisynthetic insulins obtained from natural A-chain and synthetic B-chain variants. In these products the replacement of Arg B 22 by ornithine or lysine also led to drastically diminished activity and after replacement of Arg B 22 by alanine the activity also disappeared.     

Studies on trypsin inhbitors. Part II. Synthesis of the protected tetrapeptide (sequence 11-14) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.     

Studies on trypsin inhibitors. Part IV, Synthesis of the protected undecapeptide (sequence 25-35) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the protected undecapeptide tert-butyloxycarbonylisoleucyl-threonyltyrosylserylasparaginyl-gamma-tert-butylglutamyl-S-acetamidomethylcysteinyl-valylleucyl-S-acetamidomethylcysteinylseriny hydrazide corresponding to positions 25-35 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal). The hepatapeptide free base methyl asparaginyl-gamma-tert-butylglutamyl-S-acetamidomethylcysteinylvalylleucyl-S-acetamidomethylcysteinylserinate (sequency 29-35) was acylated, by the azide procedure, with the tetrapeptide tert-butyloxycarbonl-isoleucylthreonyltyrosylserine hydrazide /sequence 25-28) and the resulting tert-butyloxycarbonylundecapeptide methyl ester was transformed into the corresponding hydrazide by hydrazinolysis. The stereochemical homogeneity of the final product was assessed, after partial deprotection with aqueous 90% trifuoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.     

Methyl 4-O-(4-alpha-D-glucopyranosyloxy-4-methoxybutyl)-alpha-D-+ ++glucopyranosi de, a modified oligosaccharide for studying interactions of carbohydrates with multisite proteins

Methyl 4-O-(4-alpha-D-glucopyranosyloxy-4-methoxybutyl)-alpha-D-glu copyranoside (9) was synthesised by transacetalation from methyl 2,3,6-tri-O-acetyl-4-O-(4,4-dimethoxybutyl)-alpha-D-glucopyranosid e and trimethylsilyl 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranoside followed by removal of the blocking groups. Compound 9, which is methyl alpha-maltotrioside modified by replacing the middle D-glucosyl residue with an acyclic spacer, competitively inhibits the hydrolysis of p-nitrophenyl alpha-maltotrioside by porcine alpha-amylase.     

Synthetic fragments and analogues of elastin. I. The synthesis

The synthesis of some repetitive sequences of elastin and their simplified analogues, all comprising the structural unit Gly-X-Gly (X = Val, Leu, Ala), is described. In particular, the following peptides and polypeptides were synthesized and characterized: Boc-Gly-Val-Gly-Gly-Leu-OMe, Boc-Gly-Leu-Gly-Gly-Val-OMe, Boc-(Gly-Val-Gly-Gly-Leu)2-OMe, Boc-(Gly-Val-Gly-Gly-Leu)3-OMe, Boc-Gly-Val-Gly-Gly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-Val-Gly-Gly-Val-OMe, poly(Ala-Gly-Gly), poly(Val-Gly-Gly), and poly(Leu-Gly-Gly). In every case, the synthesis was accomplished by classical procedures in solution, by using the p-nitrophenyl ester method for the polycondensation step, and the mixed anhydride or the azide methods for the coupling steps.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Synthesis of two diastereoisomeric p-nitrophenyl phosphodiesters of 2'',3''-secouridine and their affinity for phosphodiesterases.

The synthesis of the p-nitrophenyl esters of the 5'- and 3'-phosphates of the nucleoside analogue 2',3'-secouridine are described. Unlike the corresponding diesters of thymidine, these two compounds are diastereoisomers. Their affinity for phosphodiesterases types I and II were investigated. Both analogues were hydrolysed very slowly by snake venom phosphodiesterase but their affinity for the enzyme was similar to that of the p-nitrophenyl ester of thymidine 5'-monophosphate of which they were both competitive inhibitors with Ki approximately Km. Neither compound was hydrolysed by spleen phosphodiesterase but both competitively inhibited the p-nitrophenyl ester of thymidine 3'-monophosphate, with Ki's slightly higher than the Km. Although for each enzyme the Ki of the correct analogue phosphodiester (i.e. the 5'-derivative for snake venom and the 3'-derivative for spleen) was the lower, the absolute specificity seen for the normal substrates had been lost.     

Some amino sugars structurally related to 6-deoxymannojirimycin precursors prepared from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose and methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside

Methyl (5S)-5-C-amino-5-cyano-5-deoxy-2,3-O-isopropylidene-alpha-D-lyxofuranoside has been synthesised from methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside, applying the Strecker synthesis. Analogously, methyl (5S) and (5R)-5-C-amino-5-cyano-5,6-dideoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosides were prepared from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose. The 5-S configuration was unambiguously determined by single-crystal X-ray diffraction analysis of corresponding N-acetyl derivatives. Conformations of five-membered rings are discussed. The conversion of N-acetylated amino nitriles to N-acetylamino acid ethyl ester and amide, respectively, is also described.     

Synthesis and anti-aggregatory activity of linear retro-inverso RGD peptides.

Six retro-inverso tri- and tetrapeptide analogues of RGD were prepared and their anti-aggregatory activity was determined by platelet aggregation tests in comparison with the corresponding parent peptides. An efficient method for the introduction of a malonyl-aspartic residue into a peptide chain is described for the first time. A 2-3-fold decrease in potency or total loss of bioactivity was observed with the new peptides; structure-activity relationships are discussed.     

Synthesis of a new carrier for immunization: polytuftsin. Two examples of its use with peptides selected in the hepatitis B surface antigen

Sequential poly(Arg-Thr-Lys-Pro) consisting mainly of the repeat of tuftsin Thr-Lys-Pro-Arg was synthesized by condensing the p-nitrophenyl ester of Arg(HCl)-Thr-Lys-(2-Cl-Z)-Pro in the presence of HOBt. Two haptenic sequences of the Pre-S region of hepatitis B virus antigen (10-26 and 39-55) were prepared by solid phase and coupled to polytuftsin via glutaraldehyde. The peptides, either free or coupled to polytuftsin, were administrated to mice and the antisera were assayed by ELISA. Coupling the peptides to the polypeptide significantly improved the anti-peptide antibody titer in Freund complete adjuvant or in NaCl 0.9%. Cross-reaction between antibodies induced by the peptides and the native protein was also improved. Polytuftsin alone is very poorly immunogenic.     

The acetates of p-nitrophenyl alpha-L-arabinofuranoside--regioselective preparation by action of lipases

All possible di-O-acetates and mono-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitrophenyl alpha-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di-O-acetates were obtained by acetylation of p-nitrophenyl alpha-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents. The 5-O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl alpha-L-arabinofuranoside catalysed by porcine pancreas lipase. Finally, the 2- and 3-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were obtained in two steps. The enzymatic di-O-acetylation of p-nitrophenyl alpha-L-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis of the primary acetyl group by CCL or LAY.     

Melanocyte-Directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma.

Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28 was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase. The more sterically demanding phenyl mustard prodrugs 9 and 10 were oxidised by mushroom tyrosinase at a similar rate to tyrosine methyl ester. In contrast, tyramine chain elongation via heteroatom insertion was detrimental and the rate of mushroom tyrosinase oxidation of phenyl mustard prodrugs 21 and 22 decreased by 10 nanomol/min.     

Synthesis of ester-linked lithocholic acid dimers

Four lithocholic acid dimers were synthesised via esterification. The ester-linked dimer, 3-oxo-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), was obtained by condensation of methyl lithocholate with 3-oxo-5beta-cholan-24-oic acid. Borohydride reduction of this ester-linked dimer gave 3alpha-hydroxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), which was acetylated to 3alpha-acetoxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta). Reaction of methyl lithocholate with oxalyl chloride yielded the oxalate dimer, bis(5beta-cholan-24-oic acid methyl ester)-3alpha-yl oxalate.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.     

A circular dichroism study of DNA-basic peptides associations in the absence or in the presence of Ca++.

The interaction of DNA with basic peptides (Lys methyl ester*, Lys2, (Lys)2methyl ester) has been studied by circular dichroism. The changes of the DNA CD spectra in the presence of peptides are interpreted as a transconformation from the B form to the C form of DNA. The presence of Ca++ in the mixture induces a supplementary transconformation. These observations suggest Ca++-basic peptides-DNA complexes as a structural model for chromatin.     

Protein labeling and biotinylation of peptides during spot synthesis using biotin p-nitrophenyl ester (biotin-ONp)

Biotin-labeled peptides are used for numerous biochemical and microbiological applications. Due to the strong affinity of biotin to streptavidin, the detection of biotinylated molecules is very sensitive. A powerful technique for parallel synthesis and high-throughput screening of peptides is the spot synthesis. One example for the use of spot synthesis is the screening of biotinylated peptides synthesized on cellulose membranes, which is particularly favorable for the investigation of protease cleavage sites. Additionally, in combination with biotinylated protein samples, the spot technique can be used for investigations of peptide-protein and protein-protein interactions. Here, we present our results of the use biotin p-nitrophenyl ester (biotin-ONp) in spot synthesis and as a reagent for biotin-labeling of protein samples.     

The nature of differences in amidase and esterase activities of some acyltrypsins]

Specific trypsin substrates (esters, anilides, amides, peptides) were shown to accelerate deacetylation of monoacetylated trypsin. The amidase activity of monoacetyl-, monopropyonyl-, and tetraformyl-trypsin was not manifested if the amidase activity of native enzyme was suppressed in these preparations by the ester substrates (benzoylarginine ethyl ester or p-nitrophenyl acetate). Therefore the differences in the residual amidase and esterase activities of these acylated trypsin preparations found earlier did not contradict the universality of the acylenzyme mechanism. These differences are due to the strong deacylating effect of specific substrate in its complex with the enzyme modified with nonspecific acyl residue. The latter fact is suggested to be an experimental confirmation of the "induced fit" hypothesis.     

Enzymatic Oligomerization of the Tetrapeptide Ester Allylglycine-Phenylalanine-Phenylalanine-Allylglycine Ethyl Ester

The native hydrolytic action of subtilisin Carlsberg was reversed to oligomerize the tetrapeptide ester L-Ag-L-Phe-L-Phe-L-Ag-OEt, containing the unnatural amino acid allyglycine (Ag, where R = -CH₂-CH = CH₂), in several miscible aqueous/organic solvent systems. Mass spectrometry indicated that the octapeptide ester (Ag-Phe-Phe-Ag)² OEt was formed in all cases and that the dodecapeptide ester (Ag-Phe-Phe-Ag)₃OEt was formed in one case. Additional mass spectrometry peaks indicated that a number of other peptides larger than the octapeptide ester (Ag-Phe-Phe-A)²OEt may also have been formed. Infrared analysis revealed that the oligopeptide products exhibit P-sheet peptide folding.