Potent in vivo inhibitors of rat renin: Analogues of human and rat angiotensinogen sequences containing different classes of pseudodipeptides at the scissile site*

Using solid-phase methodology we have synthesised peptides based on the 8–14 or 6–14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P₁-P₁’cleavage site: Leu-Ψ[CH₂NH]Leu; Leu-[CH(OH)CH₂]Val; Leu-Ψ[CH(OH)CH₂]Leu and Leu-Ψ[CH(NH₂)CH₂]Val. In vitro, peptide Piv-His-Pro-Phe-His-Leu-Ψ[CH(OH)CH₂]Leu-Tyr-Tyr-Ser-NH₂( XXI ) is the most potent inhibitor of rat plasma renin reported having an IC₅₀ of 0.21 nM; it is a much weaker inhibitor of human renin (IC₅₀ 45 nM). Peptide Boc-His-Pro-Phe-His-Leu-Ψ[CH(OH)CH₂] Leu-Val-Ile-His-NH₂ ( XX ) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two-kidney, one-clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.     

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-2

The [Tyr⁴⁰]preprorenin (40–50) peptide methyl ester, an undecapeptide related to the human renin prosegment, has been synthesized using a stepwise strategy with hydrogenolisable protections on the side chains. The final deprotection was very difficult as observed by ¹ H NMR and reversed phase HPLC. 2D ¹ H NMR spectroscopy of the purified peptide allowed the assignment of all protons.     

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-1

The [Tyr⁴⁷, Nle⁵³] preprorenin (47–60) peptide methyl ester, a tetradecapeptide related to the human renin prosegment, has been synthesized using a three-segment coupling strategy. Selective deprotection of the segments before coupling allowed an easy removal of the final tetradecapeptide side chain-protecting groups by acidolysis and an easy purification. Antibodies raised against this peptide bound the plasmatic inactive renin.     

Synthesis and properties of human β-endorphin-(1–17) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–17) (I), [D-Ala²]-β_h-EP-(1–17) (II), [Gln⁸]-β_h-EP-(1–17) (III) and [D-Ala², Gln⁸]-β_h-EP-(1–17) (IV). Measurement in a radio-receptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 33; II, 47; III, 889; IV, 123; β_h-endorphin, 2253.     

Synthesis of HC toxin and related cyclopeptides containing the (L-Ala-D-Ala-L-Ada-D-Pro) sequence

In studies leading to HC toxin synthesis, a phytotoxic cyclic tetrapeptide with the sequence cyclo (L-Ala-D-Ala-L-Aoe-D-Pro), we have determined optimal conditions for the cyclization which constitutes one of the most important steps in the synthesis of the toxin. All four possible sequences containing an optically active precursor, i.e. L-Ada = (2 S)-2-amino-9-decenoic acid instead of Aoe, have been prepared and subjected to cyclization. Owing to the differences in racemization risk during activation of the terminal carboxyl aminoacid different cyclization procedures have been applied. Cyclopeptide yields and selectivity between cyclomonomer and dimer both containing the title sequence are mainly controlled by the linear precursor sequence. The cyclic tetrapeptide is only obtained with D-proline in the C-terminal position, the best yield reached by the -ONSu activation method. Starting from the peptide, the (9S, 9R) HC toxin epimer on the epoxidic carbon atom has been further synthesized in two steps.     

Synthesis of peptide inhibitors of human spleen fibrinolytic proteinase (SFP) and human leukocyte elastase-like proteinase (ELP)

Stereoisomers of specific substrates for SFP and ELP, Suc-L-Tyr-L-Leu-L-Val-pNA and Suc-L-Ala-L-Tyr-L-Leu-L-Val-pNA, were synthesized by the conventional solution method. Suc-L-Tyr-D-Leu-D-Val-pNA was found to be an effective and specific inhibitor of SFP and ELP. Suc-L-Tyr-L-Leu-L-Val-Pipe and Suc-L-Tyr-D-Leu-D-Val-Pipe inhibited only SFP very slightly and showed no inhibitory effect on ELP. Both Dan-L-Tyr-L-Leu-L-Val-pNA and Dan-L-Tyr-D-Leu-D-Val-pNA exhibited an inhibitory effect on both SFP and ELP.     

Synthesis and biological evaluation of human preproenkephalin (100-111) and its analogs*

The dodecapeptide sequence, Tyr-Gly-Gly-Phe-Met-Lys-Arg-Tyr-Gly-Gly-Phe-Met (BI), which is totally conserved in the primary structures of human, bovine, rat, and toad preproenkephalins, has been synthesized by the solid-phase method. Coupling reactions were achieved by using symmetrical anhydrides of tert.-butyloxycarbonylamino acids preformed with N-tert.-butyl,N′-methylcarbodiimide. 6-Arg and 7-Lys analogs have also been obtained. The peptides show opiate activity in both GPI and MVD assay, and possess antinociceptive properties as estimated by the hot-plate test in mice when applied intracisternally.     

Synthesis,receptor binding activity and fluorescence property of fluorescent enkephalin analogs containing L-1-pyrenylalanine

The novel fluorescent amino acid, l -1-pyrenylalanine (l -Pya), was prepared by the asymmetric hydrogenation of cyclic dehydrodipeptide. Fluorescent enkephalins containing one or two Pya residues at position 1, 4 or 5 of [d -Ala², Leu⁵]enkephalin were synthesized by the solution method. Mono-Pya-enkephalins showed strong fluorescence intensities and potent binding affinities with specificity and selectivity for opiate receptors. However, di-Pya-enkephalins showed markedly decreased receptor binding affinities. These results indicate that the incorporation of two Pya residues into enkephalin makes the peptide unable to interact with the opiate receptors, although introduction of one Pya residue is effective to elicit a specific receptor interaction. Di-Pya-enkephalins showed intramolecular excimer spectra, indicating that the peptides are able to take possible folded conformations.     

人甲状旁腺素(1-34)的合成与聚乙二醇化修饰

目的合成人甲状旁腺素(1 34) [hPTH(1-34) ]并进行聚乙二醇化修饰。方法应用固相多肽合成方法合成并经高效液相色谱纯化得hPTH(1-34) ,Cys-hPTH(1- 34)和hPTH(1-34)-Cys-NH2 。Cys hPTH(1-34)和hPTH(1-34)-Cys-NH2 在水溶液(pH 7～8)中分别与平均相对分子质量为5 0 0 0的单甲氧基马来酰亚胺基聚乙二醇(mPEG50 0 0 MAL)反应,经高效液相色谱纯化得Cys(mPEG50 0 0 MAL)- hPTH(1-34)和hPTH(1-34)-Cys(mPEG50 0 0- MAL) -NH2 。结果hPTH(1-34) ,Cys(mPEG50 0 0 MAL) hPTH(1 34)和hPTH(1 34) Cys(mPEG50 0 0 MAL) NH2 的质谱和氨基酸组成分析结果均与理论值一致。hPTH(1-34) -Cys(mPEG50 0 0-MAL) NH2 保持了较好的体外活性,Cys(mPEG50 0 0- MAL) hPTH(1-34)保持了较好的体内活性。结论采用一种简便的方法成功实现了对hPTH(1-34)的N端或C端的聚乙二醇化修饰     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.     

1-(1H-1,2,4-三唑-1-基)-(2,4-二氟苯基)-3-[N-环丙基-N-(4-取代苄基)]-2-丙醇的合成及抗真菌活性

目的：以氟康唑为先导化合物,设计合成新的三唑醇类化合物,并研究其抗真菌活性。方法：引入4位羧酸酯取代的苄基侧链结构,合成一系列目标化合物,所有化合物结构均经MS、^1H-NMR等谱确证;选择8种真菌为实验菌株,测定其体外抗真菌活性。结果：合成了15个未见文献报道的目标化合物;所有化合物对所选真菌均表现出了一定的抑菌活性,其中化合物（1）,（2）和（3）对除薰烟曲霉菌外的7种菌都表现出了较好的抑菌活性。结论：4位羧酸酯取代的苄基侧链结构的引入对目标化合物的抗菌活性有一定的影响,侧链越短．抑菌活性越好.     

1-(1H-1,2,4-三唑-1-基)-(2,4-二氟苯基)-3-(N-异丙基-N-取代氨基)-2-丙醇的合成及抗真菌活性

目的：以氟康唑为先导化合物,设计合成新的三唑醇类化合物,并研究其抗真菌活性。方法：引入异丙基及取代氨基侧链结构,合成一系列目标化合物,所有化合物结构均经MS、^1H—NMR等谱确证;选择8种真菌为实验菌株,测定其体外抗真菌活性。结果：合成了15个目标化合物;所有化合物对所选真菌均表现出了一定的抑菌活性,其中化合物（6c）,（6d）,（6e）,（6f）和（6g）对除薰烟曲霉菌外的7种菌株都表现出了较好的抑菌活性。结论：取代氨基侧链结构的引入对目标化合物的抗菌活性有一定的影响,侧链为取代苄基活性较好,且取代苄基侧链越短,抑菌活性越好。     

1-(1,2,4-三唑-1H -1-基)-2-(2,4-二氟苯基)-3-(4-取代苄基-1-哌嗪基)-2-丙醇的合成及抗真菌活性

目的寻找广谱、高效、低毒的新一代三唑类抗真菌药物。方法根据靶酶活性位点的空腔大小、各种力场和关键残基分布，设计并合成了19个1-(1,2,4-三唑-1H -1-基)-2-(2,4-二氟苯基)-3-(4-取代苄基-1-哌嗪基)-2-丙醇类化合物，测定了体外抗真菌活性。结果所有化合物对8种致病真菌均有较强的抗真菌活性，对深部真菌的活性明显优于浅部真菌。结论绝大部分化合物的抗真菌活性明显高于氟康唑和特比萘芬，其中化合物VIII-1，10，12，17具有广谱、高活性的优点，值得进一步深入研究。     

1-(1H-1,2,4-三唑-1-基)-(2,4-二氟苯基)-3-(N-环己基-N-取代氨基)-2-丙醇的合成及抗真菌活性

目的：以氟康唑为先导化合物,设计合成新的三唑醇类化合物,并研究其抗真菌活性。方法：引入环己基侧链结构,合成一系列目标化合物,所有化合物结构均经Ms、1H—NMR等谱确证;选择8种真菌为实验菌株,测定其体外抗真菌活性。结果：合成了11个未见文献报道的目标化合物,部分化合物对所选真菌均表现出了一定的抑菌活性。结论：引入环己基对抗真菌活性影响较大。     

Synthesis and opioid activity of 2–substituted dynorphin A-(1-13) amide analogues1

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH₂) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; K_i values for the analogues in the radioligand binding assays and IC₅₀ values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH₂, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys²]Dyn A-(1-13)NH₂ in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI)

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67–94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)^73,87] 67–94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.     

Synthesis,conformation, receptor binding and biological activities of monobiotinylated human insulin‐like peptide 3*

Abstract: Biotin‐avidin immobilization has been routinely used as a tool to study peptide–receptor and peptide–antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides’ receptor, and to analyse ligand‐receptor binding. Insulin‐like peptide 3 (INSL3) is a peptide hormone which contains A‐ and B‐chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein‐coupled receptor, we chemically synthesized N^α‐mono‐biotinylated human INSL3 (B‐hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with ³³P‐labelled relaxin H2 (B₃₃). The modified B‐hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B‐hINSL3 contains a higher α‐helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N‐terminal region of the A‐chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N‐terminus of the A‐chain promoted conformational stability which, in turn, permitted better receptor activation.     

Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis

S-4-methoxytrityl cysteine was synthesized and converted into the corresponding Fmoc-Cys(Mmt)-OH by its reaction with Fmoc-OSu. As compared to the corresponding Fmoc-Cys(Trt)-OH, the S-Mmt-function was found to be considerably more acid labile. Quantitative S-Mmt-removal occurs selectively in the presence of groups of the tert butyl type and S-Trt by treatment with 0.5–1.0% TFA. The new derivative was successfully utilized in the SPPS of Tyr¹-somatostatin on 2-chlorotrityl resin. In this synthesis groups of the Trt-type were exclusively used for amino acid side-chain protection. Quantitative cleavage from the resin and complete deprotection was performed by treatment with 3% TFA in DCM–TES (95:5) for 30 min at RT. We observed no reduction of tryptophan under these conditions. © Munksgaard 1996.     

Reinvestigation of the disulfide bridge arrangement in human pro-opiomelanocortin N-terminal segment (hNT 1–76)

The cystine bridge structure of the amino-terminal fragment of human proopiomelanocortin has been reinvestigated. Highly purified amino-terminal fragment 1–76 was rapidly isolated from human pituitaries using only reverse-phase liquid chromatography (RP-HPLC). This peptide was then subjected to trypsin and V8-protease digestion and the products separated by RP-HPLC> and subjected to amino acid and microsequence analysis. The results show that disulfide bridges link Cys-2 to Cys-24 and Cys-8 to Cys-20. Amino acid analysis and amino sugar determination confirm (i) the previously proposed sequence and (ii) the suggestion of the presence of two glycosylation sites in this molecule. These are most probably located at Thr-45 (O-glycosylation) and at Asn-65 (N-glycosylation).     

Structural characterizations of neuropeptide tyrosine (NPY) and its agonist analog [Ahx5-17]NPY by NMR and molecular modeling

The structures of human NPY and of its centrally truncated agonist analog [Ahx^5–17]NPY have been investigated in DMSO-d₆ by two-dimensional NMR and by molecular modeling. For both peptides, a complete resonance assignment was achieved and a large number (more than 200) of inter-residue NOE connectivities were observed, including long-range connectivities between the N- and C-terminal ends of the chain. Molecular models were calculated using NOE constraints by distance geometry, simulated annealing and conjugate gradient energy minimization. The results indicate that both peptides are folded in the center of their chain, NPY adopting the hairpin shape, whereas the central portion of [Ahx^5–17]NPY is characterized by relatively large loops. In contrast to previous models, practically no α-helical structure exists for these peptides under our conditions, but two β-turns are found in NPY and one in [Ahx^5–17]NPY. The proximity of the terminal ends could be the determinant factor for their activity. © Munksgaard 1995.