STUDIES ON PEPTIDES

The heptacosapeptide amide corresponding to the entire amino acid sequence of gastrin-releasing peptide (GRP) was synthesized by assembling seven peptide fragments followed by deprotection with a new reagent system, IM trifluoro-methanesulfonic acid-thioanisole in TFA. The deprotected peptide was purified by ion-exchange chromatography on CM-cellulose followed by partition chromatography on Sephadex G-25. The latter was found to remove effectively the Met (O) derivative of GRP. The highly purified synthetic GRP was active as synthetic bombesin on the molar basis. A new carboxyl-activating reagent, thiazoline-2-thione, was employed for preparation of the necessary fragments.     

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.     

STEREOCHEMICAL STUDIES ON CYCLIC PEPTIDES

Conformational analysis of cyclic pentapeptides having two intra-ring 3 1 hydrogen bonds has been carried out. It is found that the structure can easily be formed with trans planar peptide units without causing significant angular strain at the α-carbon atoms. Four different types of conformations designated Types I-IV are possible for the backbone structure. Details of these four types of conformations and also the accommodating possibility of these types for all-glycyl and all-alanyl residues are presented. Three of the four types have relatively low energies for glycyl residues whereas the other one has a slightly higher energy. When alanyl residues are introduced at the five α-carbon atoms, the types that are energetically favourable depend upon the sequence of isomers. Energy calculations have also been carried out for the combinations of glycyl, L- and D-alanyl residues. The theoretical results are compared with available experimental observations both from solution and solid state studies.     

含磷缩合剂在药物和肽合成中的应用

综述了DPPA，DEPC等9种含磷化合物作为缩合剂在药物和多肽合成中的应用，其多数效果比DCC为优，消旋化程度小，产物容易分离。     

STUDIES ON THE SYNTHESIS OF PROTEINASE INHIBITORS

Several heterodetic cyclic nonapeptides modeled after the disulfide loops of Bowman-Birk inhibitor which involve either the anti-tryptic or anti-chymotryptic region have been synthesized by the conventional method. The inhibitory properties and the stabilities of these peptides toward trypsin and chymotrypsin, as well as the properties of dimers of the above nonapeptides, were examined.     

Solid-phase synthesis of (tyrosyl-alanyl-glutamyl)n,by segment condensation

(Tyr-Ala-Glu)_n, n= 1–9, were synthesized by segment condensation using the Fmoc/tert-butyl protection strategy and solid-phase techniques. The C-terminal residue was coupled to the resin and the peptides were built out by adding Fmoc-Glu(O-r-Bu)-Tyr(t-Bu)-Ala-OH units. When the desired lengths were reached the peptides were capped with Fmoc-Tyr(t-Bu)-Ala-OH units. Fmoc-Tyr(t-Bu)-Ala-OH and Fmoc-Glu(O-t-Bu)-Tyr(t-Bu)-Ala-OH were synthesized in aqueous solution by the successive addition of N-hydroxysuccinimide esters of Fmoc-Tyr(t-Bu) and Fnioc-Glu(0-t-Bu) to the growing chain. Neither sequential amino acid addition or segment condensation techniques were successful on polystyrene supports. However, the segment condensations were highly successful on kieselguhr-supported polydimethylacrylamide based resins. (Tyr-Ala-Glu)_n, n= 1–9, were tested as inhibitors of the protein [yosine kinase, pp60C^c-src. Inhibition, as measured by IC₅₀ values, increased with increasing size of the peptide.     

STUDIES ON TRYPSIN INHIBITORS

The synthesis by fragment condensation of protected peptides corresponding to the amino acid sequences 15–35, 25–52 and 15–52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The Rudinger modification of the azide procedure was used in the fragment coupling steps. The tert-butyloxycarbonylheptapeptide hydrazide (sequence 22–28) was reacted with the heptapeptide methyl ester free base (sequence 29–35) and the resulting tert-butyloxycarbonyltetradecapeptide methyl ester after selective deprotection, coupled with the benzyloxycarbonylheptapeptide hydrazide (sequence 15–21) to give the protected peptide methyl ester corresponding to the 15–35 sequence which was then converted to the corresponding hydrazide. The synthesis of the 25–52 sequence was achieved by assembling the protected peptide hydrazide corresponding to the amino acid residues 25–35, with the C-terminal heptadecapeptide 36–52. The resulting protected octaeicosapeptide (sequence 25–52) was selectively deblocked with trifluoroacetic acid and acylated with the benzyloxycarbonyldecapeptide hydrazide 15–24 to give the desired octatriacontapeptide corresponding to sequence 15–52 of the inhibitor. An attempt to prepare the 15–52 sequence through the condensation of fragments corresponding to 15–35 and 36–52 sequences was unsuccessful. The identity and purity of the synthetized peptide derivatives were established by elemental analysis (in some cases), amino acid analysis, optical rotation, and thin-layer chromatography in two solvent systems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values.     

SYNTHESIS OF SOMATOSTATIN AND [D-TRP8]- SOMATOSTATIN

Experimental details for practical syntheses of somatostatin and D-Trp⁸-somatostatin are described. The peptides were assembled from three fragments which permit further syntheses of analogs with modifications at positions 1, 2 or 8. N^α-Bpoc protecting groups were used for the two major fragments and these were selectively removed in the presence of the tert-butyl derived amino acid side chain functionalities. The two cysteine residues were protected by acetamidomethyl groups. All the peptide intermediates were fully characterized and a 10-g synthesis of the protected tetradecapeptide is reported. Major fragments were coupled by the azide method in good yield. Dihydrosomatostatin and D-Trp⁸-dihydrosomatostatin were isolated, purified, characterized and cyclized. Polymeric side-product was successfully recycled (by reduction with dithiothreitol and reoxidation) to give an overall yield for the oxidation of 52%. Somatostatin and D-Trp⁸-somatostatin were purified by gel filtration or countercurrent distribution and the final products were fully characterized and determined to be>97% pure by reversed phase high performance liquid chromatography.     

CHOLECYSTOKININ (PANCREOZYMIN)

The acetyl-derivative of the biologically active C-terminal 7-peptide portion of cholecystokinin (CCK), N-acetyl-O-sulfate-L-tyrosyl-L-methionyl-glycyl-L-tryptophyl-L-methionyl-L-aspanyl-L-phenylalanine amide was prepared by the condensation of 2-peptide segments with 1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline as coupling reagent. The N-terminal residue, tyrosine, was incorporated by the active ester method. The same 7-peptide was prepared also by stepwise chain-lengthening, starting with the C-terminal residue. The 9-fluorenylmethyloxycarbonyl group was applied for the protection of α-amino functions. In the release of amylase from acinar cells of the pancreas of guinea pigs, the acetyl-7-peptide amide was about 3 times more potent than CCK 27–33 and equal in potency to CCK 26–33. The new derivative strongly stimulated the contraction of the in situ guinea pig gall bladder.     

PHOTOLABILE MULTI-DETACHABLE p-ALKOXYBENZYL ALCOHOL RESIN SUPPORTS FOR PEPTIDE FRAGMENT OR SEMI-SYNTHESIS

Two photolabile multi-detachable alkoxybenzyl alcohol resins, 2-[4-(oxymethyl)phenoxy]propionyl-resin 4 and 4-[4-(oxymethyl)phenoxymethyl]-3-nitrobenzamidomethyl-resin 5 have been synthesized. Bpoc-peptide attached to resin 4 or 5 when treated with 50% trifluoroacetic acid provided the free, unprotected peptide, but on photolysis gave Bpoc-peptide p-hydroxybenzyl ester. Removal of the p-hydroxybenzyl ester in aqueous base or by oxidative work up gave a protected Bpoc-peptide suitable for fragment synthesis at its C-terminus. However, methylation of the ester to Bpoc-peptide p-methoxybenzyl ester followed by removal of the Bpoc-group gave a protected peptide p-methoxybenzyl ester suitable for fragment coupling at its N-terminus. The efficacies of these resins were evaluated in the syntheses of a model tetrapeptide and an octapeptide by using N^α-Bpoc-, Fmoc- and Nps-amino acids. The use of 2-thiopyridine with pyridinium hydrochloride as a new and efficient thiolytic reagent for the deprotection of the Nps-group was studied.     

BIDIRECTIONAL SYNTHESIS OF [5-ASPARTIC ACID] ARGININE VASOPRESSIN ON POLY-N-ACRYLYLPYRROLIDINE RESIN

In the proposed biologically active conformation of vasopressin at its antidiuretic receptor, the side-chain carboxamide moiety of the 5-position asparaginyl residue has been suggested to be an active element for the initiation of the antidiuretic response. [5-Aspartic acid] arginine vasopressin, the analog in which the -NH₂ portion of the primary amide has been replaced by an -OH group, has been synthesized and tested for some of the pharmacological activities of vasopressin. The partially protected nonapeptide intermediate was assembled bidirectionally on a poly-N-acrylylpyrrolidine resin. The 6-position cysteinyl residue was attached to the resin via its side-chain through an S-carbamoyl linkage. First the COOH-terminus was extended by coupling with Pro-Arg(Tos)-Gly-NH₂, then the NH₂-terminus was extended in a stepwise manner. [5-Aspartic acid] arginine vasopressin was found to possess 86.5 ± 4.8 units/mg of antidiuretic potency, 17% of the parent hormone. In addition, the analog possesses rat pressor and rat uterotonic potencies of 6.93 ± 0.15 and 0.38 ± 0.03 units/mg, respectively. This result suggests that a carboxylic acid moiety on the 5-position aspartyl residue retains sufficient steric features and hydrophilicity in common with the carboxamide moiety present in the hormone to substitute for it as an active element at the antidiuretic receptor.     

Synthesis,radiolabeling and biological activity of peptide oostatic hormone and its analogues

A series of Pro peptides containing the sequence of the oostatic hormone 3d and its shorter analogues 3a-3c differing in a number of the C-terminal Pro residues was prepared for a study of its effect on oogenesis in Sarcophaga bullata Parker (Diptera). Peptides 3a-3d were synthesized in solution by the fragment condensation of Boc-Tyr-Asp(OtBu)-Pro-Ala-Pro-OH (2f) with Pro oligopeptides H-(Pro)_2-5-OtBu. The ammo-terminal protected pentapeptide acid 2f was prepared by a stepwise procedure from TFA-H-Ala-Pro-OMe using Boc-Pro-OH, Z-Asp(OtBu)-OSu and Boc-Tyr-OSu. The H(Z)-(Pro)2-5-OtBu oligopeptides 1a-1h were synthesized from Z-Pro-OH and H-Pro-OtBu by a combination of stepwise procedure and fragment condensation. The ₁₂₅I-labeled molecules of the octapeptide 3b and decapeptide 3d were used for radiotracer distribution studies. Evidence of content of the labeled peptide material in various parts of the insect body (ovaries, head, intestine) is presented. The time distribution of the labeled material in the insect organs was correlated with results of histological analysis of ovaries treated by nonlabeled peptides. The peptides assayed affected processes of egg development in 20–60% of ovarioles. The decapeptide 3d caused changes consisting in some resorbed egg chambers and normal appearance of vitellogenic eggs, whereas the octapeptide 3b caused abnormal yolk deposition and formation of big eggs with irregular yolk granules, proliferation of follicular epithelium in some egg chambers and about the same amount of resorbed egg chambers as decapeptide. These structural differences are complementary to the different values of organ radioactivities. © Munkagaard 1997.     

新皮啡肽I(DELI)类似物的合成及构效关系研究

用固相多肽合成法合成了类阿片肽新皮啡肽I(DELI)及其三个类似物(将Asp4从5位到7位逐一后移)。实验发现DELI在离体条件(浓度范围10^-14～10mol·L^-1)和在体条件(剂量范围0.5～5μg·kg^-1)下能提高红细胞玫瑰花环形成细胞百分率(E-RFC)和红细胞C3b受体花环百分率(RBC-CR₁),且可被纳洛酮阻断。这些结果表明DELI能提高大鼠免疫功能。实验还发现DELI及其类似物的镇痛和免疫活性次序(由大到小)分别是Asp⁴,Asp⁷,Asp⁵,Asp⁶和Asp⁴,Asp⁷,Asp⁶,Asp⁵。     

STUDIES OF PEPTIDE ANTIBIOTICS:

Tyrocidine A (TA) is an antibiotic cyclic decapeptide with the sequence of cyclo (-L-Val¹ -L-Orn²-L-Leu³-D-Phe⁴-L-Pro⁵-L-Phe⁶-D-Phe⁷-L-Asn⁸-L-Gln⁹-L-Tyr¹⁰-). Gramicidin S (GS) regarded as a homolog of TA is also a cyclic decapeptide with the sequence of cyclo (-L-Val¹-L-Orn²-L-Leu³-D-Phe⁴-L-Pro⁵- L-Val⁶-L-Orn⁷-L-Leu⁸ -D-Phe⁹ -L-Pro¹⁰ -). GS shows higher antibacterial activity, whereas TA exhibits inhibitory activity on the biosynthesis of RNA. Two analogs of TA, [L-Val⁶]-TA (12a) and [L-Orn⁷]-TA (12b), were synthesized by the conventional method in order to study the interrelationships between the two related antibiotics TA and GS. Antibacterial activities of 12a and TA are nearly the same, but the activity of 12b is significantly lower. The optical rotatory dispersion spectra of 12a, 12b, and TA showed a trough at 233 nm region; the troughs of 12a and TA are nearly the same in depth, but the trough of 12b is shallower. Relationships between structure and activity of 12a and 12b compared with TA and GS were discussed.     

THE PURIFICATION OF PEPTIDES BY PARTITION CHROMATOGRAPHY BASED ON A HYDROPHOBICITY SCALE*

A study of the efficiency of partition chromatography for the purification of peptides as a function of structure has been undertaken. A series of 19 omission analogs of camel β-endorphin and of some of its partial sequences have been synthesized with each analog missing only a single amino acid. Their chromatographic properties have been examined with use of the Martin hypothesis and the R_M concept, and a hydrophobicity scale for the amino acid side chains was obtained. To a first approximation a correlation with the Tanford hydrophobicity scale for amino acids was found. A decrease in hydrophobicity has been observed with increasing chain length and is discussed in terms of column efficiencies required for the purification of synthetic peptides.     

Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH₂Tyr¹,D-Ala²,Ala¹⁵]-GRF(1-29)-NH₂ ( 4 ), from the precursor, [Ala^15,29]-GRF(4-29)-OH ( 1 ). C-Terminal amidation of 1 to form [Ala¹⁵]-GRF(4-29)-NH₂ ( 2 ) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala²⁹-OH for Arg-NH₂. The target analog 4 was then obtained by acylation of segment 2 with desNH₂Tyr-D-Ala-Asp(OH)-OR ( 3 ) (R = CH₃CH₂ or 4-NO₂C₆H 4 CH₂) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3 . GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 °C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp³-Ala⁴ and Asp²⁵-Ile²⁶. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH₂Tyr-D-Ala-Asp(OH)-OR ( 3 ) [R = CH₃CH_2- ( 3a ), CH_3- ( 3b ), ClCH₂CH_2- ( 3c ), C₆H₅CH_2- ( 3d ), 4-No₂C₆H₄CH_2- ( 3e )] revealed that the rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4 , increase with increasing specificity (V_max/K_m) of GSE for the tripeptide ester. The specificity varies in the order 3e>3d>3c>3b>3a and appears to depend on an increase in the maximum turnover rate (V_max) with increasing basicity of R. This work demonstrates the coupling of a small peptide segment containing unnatural amino acids to the N-terminus of an intermediate-length polypeptide, without side-chain protection, by GSE, a potentially less costly and more stable alternative to the V8 protease.     

重组人内抑素的抗肿瘤活性

目的观察重组人内抑素抗肿瘤活性及对血管内皮细胞增殖的抑制作用。方法用人癌裸鼠移植性肿瘤及小鼠移植性肿瘤模型对重组人内抑素进行抗肿瘤药效学观察,用MTT法观察其对血管内皮细胞及肿瘤细胞增殖的抑制作用。结果重组人内抑素,可明显抑制人胃癌BGC803和人乳腺癌B37裸鼠移植性肿瘤的生长,对小鼠肝癌H22实体瘤亦呈一定的抑制作用。MTT试验结果显示重组人内抑素可抑制人胎脐静脉血管内皮细胞ECV304的增殖,对人结肠癌HCT-8等肿瘤细胞的增殖无影响。结论重组人内抑素具有较强的抗肿瘤作用,其作用机理可能与抑制血管内皮细胞增殖、抑制肿瘤新生血管形成有关。     

Solution synthesis and biological activity of human pleiotrophin,a novel heparin‐binding neurotrophic factor consisting of 136 amino acid residues with five disulfide bonds

Abstract: Human pleiotrophin (hPTN), a novel heparin‐binding neurotrophic factor consisting of 136 amino acid residues with five intramoleular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility of our strategy using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermediates, Boc‐(1‐64)‐OH and H‐(6 5‐136)‐OBzl, in CHL/TFE (3 : 1; v/v) using 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC) in the presence of 3,4‐dihydro‐3‐hydroxy‐4‐oxo‐1,2,3‐benzotriazine (HOOBt). After removal of all protecting groups using the HF procedure followed by treatment with Hg(OAc)₂, the fully deprotected peptide was subjected to an oxidative folding reaction. The product was confirmed as having the correct disulfide structure by examining the cystine peptides obtained by enzymatic digestions, and as possessing the same biological activities as those of the natural product. The N‐ and C‐terminal half domains (1‐64 and 65‐136) were also synthesized, and measurement of their biological activities indicated that the C‐terminal half domain displays almost all the activities of the full‐length molecule, whereas the N‐terminal half domain shows almost no activity. From these results, we were able to confirm that the C‐terminal half domain is responsible for the expression of biological activities in the same manner as human midkine (hMK), another heparin‐binding neurotrophic growth factor.