Enzymatic synthesis of dehydro cyclo(His-Phe)s, analogs of the potent cell cycle inhibitor, dehydrophenylahistin, and their inhibitory activities toward cell division

Cyclo(His-Phe) was effectively converted to its dehydro derivatives by the enzyme of Streptomyces albulus KO-23, an albonoursin-producing actinomycete. Two types of dehydro derivatives were isolated from the reaction mixture and identified as cyclo(DeltaHis-DeltaPhe) and cyclo(His-DeltaPhe). This is the first report on cyclo(His-DeltaPhe) and the enzymatic preparation of both compounds. Cyclo(DeltaHis-DeltaPhe), a tetradehydro cyclic dipeptide, exhibited a minimum inhibitory concentration of 0.78 mumol/ml inhibitory activity toward the first cleavage of sea urchin embryos, in contrast to cyclo(His-DeltaPhe) that had no activity. The finding that the isoprenylated derivative of cyclo(DeltaHis-DeltaPhe), dehydrophyenylahistin, had 2,000 times higher activity than cyclo(DeltaHis-DeltaPhe) indicates that an isoprenyl group attached to an imidazole ring of the compound was essential for the inhibitory activity.     

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues.

Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget's disease. Our studies on the conformational features of human calcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21-[Asp17, Lys21]hCt (1), which had a 5-10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836-847]. We hypothesized that a stabilized, possibly type I beta turn/beta sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19-member to 17-member ring-size analogues of 1 with the structure cyclo17,21-[Asp17,XX21]hCt with XX = Orn (2), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19-member cyclo17,21-[Glu17,Dab21]hCt (6). Analyses of the far-UV CD spectra indicated increased type I beta turn and antiparallel beta sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30-fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in 'freezing' hCt in a bioactive conformation. Our findings provide evidence for the first time that a beta turn/beta sheet conformation in region 17-21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead structure for a hCt-based drug for the treatment of osteoporosis and other bone-disorder-related diseases.     

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial,anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d ‐Tyr‐d ‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d ‐Tyr‐d ‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d ‐Tyr‐d ‐Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d ‐Tyr‐d ‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d ‐Tyr‐d ‐Phe). Flow cytometry analysis showed that the cyclo(d ‐Tyr‐d ‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d ‐Tyr‐d ‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d ‐Tyr‐d ‐Phe) with synthetic cyclo(d ‐Tyr‐d ‐Phe) and cyclo(l ‐Tyr‐l ‐Phe). Natural and synthetic cyclo(d ‐Tyr‐d ‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded significant activity than natural and synthetic cyclo(d ‐Tyr‐d ‐Phe). The results of the present study reveals that cyclo(d ‐Tyr‐d ‐Phe) is more bioactive than cyclo(l ‐Tyr‐l ‐Phe). To the best of our knowledge, this is the first time that cyclo(d ‐Tyr‐d ‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d ‐Tyr‐d ‐Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.     

Enzymatic Synthesis of Dehydro Cyclo(His–Phe)s,Analogs of the Potent Cell Cycle Inhibitor,Dehydrophenylahistin, and Their Inhibitory Activities toward Cell Division

Cyclo(His–Phe) was effectively converted to its dehydro derivatives by the enzyme of Streptomyces albulus KO-23, an albonoursin-producing actinomycete. Two types of dehydro derivatives were isolated from the reaction mixture and identified as cyclo(ΔHis–ΔPhe) and cyclo(His–ΔPhe). This is the first report on cyclo(His–ΔPhe) and the enzymatic preparation of both compounds. Cyclo(ΔHis–ΔPhe), a tetradehydro cyclic dipeptide, exhibited a minimum inhibitory concentration of 0.78 μmol/ml inhibitory activity toward the first cleavage of sea urchin embryos, in contrast to cyclo(His–ΔPhe) that had no activity. The finding that the isoprenylated derivative of cyclo(ΔHis–ΔPhe), dehydrophyenylahistin, had 2,000 times higher activity than cyclo(ΔHis–ΔPhe) indicates that an isoprenyl group attached to an imidazole ring of the compound was essential for the inhibitory activity.     

Cyclic dipeptide oxidase from Streptomyces noursei. Isolation, purification and partial characterization of a novel, amino acyl alpha,beta-dehydrogenase.

Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of alpha,beta-dehydro-Phe (Delta Phe) and alpha,beta-dehydro-Leu (Delta Leu) residues during the biosynthesis of albonoursin, cyclo(Delta Phe-Delta Leu), an antibiotic produced by Streptomyces noursei. It was purified 600-fold with a 30% overall recovery, and consists of the association of a single type of subunit with a relative molecular mass of 21,066 resulting in a large homopolymer of relative molecular mass over 2,000,000. The enzyme exhibits a typical flavoprotein spectrum with maxima at 343.5 and 447.5 nm, the flavin prosthetic group being covalently bound to the protein. The catalytic reaction of the natural substrate cyclo(L-Phe-L-Leu) occurs in a two-step sequential reaction leading first to cyclo(alpha,beta-dehydro-Phe-L-Leu) and finally to albonoursin. Kinetic parameters for the first step were determined (K(m) = 53 microM; k = 0.69 s(-1)). The enzyme was shown to catalyze the conversion of a variety of cyclo(dipeptides) and can be reoxidized at the expense of molecular oxygen by producing H(2)O(2). This reaction mechanism, which differs from those already described for the formation of alpha,beta-dehydro-amino acids, might consist of the transient formation of an intermediate imine followed by its rearrangement into an alpha,beta-dehydro-residue.     

Isolation, structure determination, and phytotoxicity of unusual dioxopiperazines from the phytopathogenic fungus Phoma lingam.

The isolation, chemical structure elucidation, and bioactivity of polanrazines B-F, five new dioxopiperazines produced by isolates of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.] originating from Poland, are reported. Polanrazines C and E showed moderate but selective toxicity, causing necrotic and chlorotic lesions (1-3 mm diameter) on brown mustard leaves.     

Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites

Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid (1) and asperfumin (2), together with six known bioactive compounds including monomethylsulochrin, fumigaclavine C, fumitremorgin C, physcion, helvolic acid and 5alpha,8alpha-epidioxy-ergosta-6,22-diene-3beta-ol as well as other four known compounds ergosta-4,22-diene-3beta-ol, ergosterol, cyclo(Ala-Leu) and cyclo(Ala-Ile). Through detailed spectroscopic analyses including HRESI-MS, homo- and hetero-nuclear correlation NMR experiments (HMQC, COSY, NOESY and HMBC), the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3')-5'-methoxyl-7'-methyl-(1'H, 2'H, 4'H)-quinoline-2',4'-dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in vitro bioactive assays against three human pathogenic fungi Candida albicans, Tricophyton rubrum and Aspergillus niger. As a result, asperfumoid, fumigaclavine C, fumitremorgin C, physcion and helvolic acid were shown to inhibit C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 microg/mL, respectively.     

Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.     

Effect of cycloheximide [correction of cyclohexamide] injected at proestrus on ovarian protein synthesis, peptide and steroid hormone levels, and ovulation in the hamster

Injecting 2 or 4 mg of cycloheximide (cyclo) at the onset of the proestrous release of gonadotropins prolongs the estrogen (E2) surge, diminishes progesterone (P4) secretion, and prevents ovulation by 0900 h of the next morning (Saidapur and Greenwald, 1981). The present study was designed to determine the effects of 0, 2, 4, or 8 mg cyclo injected at 1400 h proestrus (Day 4) on ovarian protein synthesis and other parameters. Ovulation was delayed until 1400 h estrus by 2 mg cyclo or prevented by 8 mg, and the latter treatment resulted in the death of all animals by 48 h. After 4 mg cyclo, ovulation was delayed in some animals, but the most characteristic feature was the development of large cystic follicles that ultimately transformed into corpora hemorrhagica. All animals lived after the injection of 4 mg cyclo. Ovaries collected 2, 8, 16, or 24 h after treatment were incubated with [3H]leucine for 1 h to assess the effects of cyclo on protein synthesis. Injection of phenobarbital at 1300 h proestrus, which blocks follicle-stimulating hormone (FSH) and luteinizing hormone (LH) surges, reduced ovarian protein synthesis at 1600 h to 61% of the control value. The incorporation of [3H]leucine was reduced to 75%, 37%, and 35% of the 1600-h control value by 2, 4, and 8 mg cyclo, respectively, but without affecting surge levels of FSH and LH. However, by 0600 h estrus, protein synthesis was increased significantly in all the cyclo-treated groups, which provides insight into the half-life of the compound (approximately equal to 8 h for 2-4 mg cyclo). At 1600 and 2200 h proestrus cyclo resulted in serum FSH and LH levels similar to controls, but increased serum prolactin and prolonged E2 levels at Day 4 of 2200 h and decreased serum P4 at both times. The second surge in FSH, which is in progress by 0600 h estrus, was abolished by 4 or 8 mg cyclo but not by the 2-mg dose. This is the first time for any species that ovarian protein synthesis has been measured in the proestrous normal or cyclo-treated animal. We conclude for the hamster that 4 mg cyclo is the optimal dose for blocking ovarian protein synthesis and ovulation and inducing formation of cystic follicles.     

短瓣花中的微量新环肽成分

从中国民间草药短瓣花（Brachystemma calycinum D.Don)干燥根的乙醇提取物中分离得到4个新的微量环八肽,命名为短瓣花环肽A、B、C和D。经光谱分析鉴定。它们的结构分别为cyclo(Pro^1-Phe-Leu-Ala^1-Thr-Pro^2-Ala^2-Gly)(1)、cyclo(Pro^1-Ala-Phe-Trp-Asp-Pro^2-Leu-Gly)(2)、cyclo(Pro^1-Ⅱe-Gly-Pro^2-Val-Ala^1-Ala^2-Tyr)(3)和cyclo(Pro-OMet-Trp-Ⅱe-Gly-Ala-Leu-Asp)(4)。     

Solution-phase synthesis of Aib-containing cyclic hexapeptides

The 18-membered Aib-containing cyclohexapeptides, cyclo(-Gly-Aib-Aib-Gly-Aib-Phe-) (22), cyclo(-Gly-Aib-Phe(2Me)-Gly-Aib-Aib-) (24a), cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Aib-) (24b), and cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Phe-) (25), have efficiently been synthesized by solution-phase techniques. The linear precursors 1a-1d were prepared by combining the 'azirine/oxazolone method' for incorporation of alpha,alpha-disubstituted alpha-amino acids (Aib, Phe(2Me)) into the peptide chains by classical peptide coupling methods for segment condensations. Deprotection of the amino and carboxy termini of 1a-1d, followed by cyclization with DEPC as the coupling reagent, gave the above-mentioned cyclic hexapeptides 22, 24a, 24b, and 25 in good yields (26-57%). The solid-state conformations of the linear hexapeptides 1d, 16 and 27, and of the cyclohexapeptides 22 and 25 have been established by X-ray crystallography.     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

Isolation of cyclo(His-Pro)-like immunoreactivity from human urine and demonstration of its immunologic, pharmacologic, and physico-chemical identity with the synthetic peptide

Measurements of cyclo(His-Pro) levels in human urine were carried out by specific radioimmunoassay. Cyclo(His-Pro)-like immunoreactivity in Human urine was found to be immunologically, pharmacologically, and physico-chemically identical to that of synthetic cyclo(His-Pro). The concentration of urinary cyclo(His-Pro) in 24-h collection was 1133.8 +/- 122.5 nmol/L, with a range of 606 to 1865 nmol/L. The daily excretion rate of cyclo(His-Pro) was 1812 +/- 248 nmol cyclo(His-Pro)/g creatinine, or 1814 +/- 199 nmol cyclo(His-Pro/day.     

Poly-(cyclo)dextrins as ethoxzolamide carriers in ophthalmic solutions and in contact lenses

Efficient ophthalmic therapy requires the development of strategies that can provide sufficiently high drug levels in the ocular structures for a prolonged time. This work focuses on the suitability of poly-(cyclo)dextrins as carriers able to solubilize the carbonic anhydrase inhibitor (CAI) ethoxzolamide (ETOX), which is so far used for oral treatment of glaucoma. Topical ocular treatment should notably enhance the efficiency/safety profile of the drug. Natural α-, β- and γ-cyclodextrins and a maltodextrin were separately polymerized using citric acid as cross-linker agent under mild conditions. The resultant hydrophilic polymers exhibited larger capability to solubilize ETOX than the pristine (cyclo)dextrins. Moreover, they provided sustained drug diffusion in artificial lachrymal fluid. Interestingly the poly-(cyclo)dextrins solutions facilitate the loading of remarkably high doses of ETOX in poly(2-hydroxyethyl methacrylate)-based contact lenses. Exploiting ionic interactions between functional groups in the contact lenses and remnant free carboxylic acids in the citric acid linkers of poly-(cyclo)dextrins led to the retention of the drug-loaded poly-(cyclo)dextrins and, in turn, to sustained release for several weeks.     

污损生物膜细菌Pseudoalteromonas issachenkonii YT1305-1的鉴定及其代谢物化学成分分析

【目的】对污损生物膜细菌YT1305-1进行菌种鉴定;研究其作为污损生物膜优势菌之一的代谢产物。【方法】通过16S rRNA基因序列分析,结合系统进化树和细菌生理生化实验对菌种进行鉴定,通过硅胶柱层析分离方法和核磁共振检测技术分析其代谢物的化学成分。【结果】发现生物膜中存在明显的优势菌株,假交替单胞菌属为优势菌属之一。16S rRNA序列比对分析表明Pseudoalteromonas issachenkonii为优势菌种之一,将目标菌种命名为Pseudoalteromonas issachenkonii YT1305-1,对其代谢物化学成分进行分析,共得到10个化合物,其中包括5个二酮哌嗪(DKPs)类信号分子,环(甘氨酸-丙氨酸)(1)、环(脯氨酸-甘氨酸)(2)、环(脯氨酸-酪氨酸)(3)、环(4-羟基-脯氨酸-亮氨酸)(4)和环(4-羟基-脯氨酸-丙氨酸)(5),以及尿嘧啶(6)、胸腺嘧啶(7)、胸腺嘧啶脱氧核苷(8)、己二酸二(2-乙基己)酯(9)和邻苯二甲酸二(2-乙基己)酯(10)。【结论】污损生物膜中存在明显的优势菌,其中之一为P.issachenkonii YT1305-1,在其代谢产物中发现了疑似信号分子的物质DKPs,有研究表明该物质能调控生物膜的形成,进而影响生物污损的形成,为探究生物污损现象奠定了物质基础。     

Staphylopeptide A,a new cyclic tetrapeptide from culture broth of Staphylococcus sp.

Re-investigation of the culture broth of the marine bacterium Staphylococcus sp. (no. P-100826-4-6) afforded a new cyclic tetrapeptide namely staphylopeptide A (1), along with five known compounds, cyclo (l-prolyl-l-valine) (2), cyclo (l-prolyl-l-tyrosine), (3), cyclo (l-prolyl-l-alanine) (4), l-phenylalanine (5), and l-tryptophan (6). The structures of the isolated compounds were determined by extensive IR, 1D (¹H and ¹³C) and 2D (¹H-¹H COSY, HSQC, HMBC, and NOESY) NMR and HRFABMS spectral measurements. The antimicrobial activity of the isolated compounds (1–6) was evaluated.     

Cyclo(Dehydrohistidyl-L-tryptophyl), an inhibitor of nitric oxide production from a fungal strain, Fb956

In the course of screening for nitric oxide inhibitors in activated microglial BV-2 cells, cyclo(dehydrohistidyl-Ltryptophyl) was isolated from solid-state fermentation cultures of an unidentified fungal strain, Fb956. Its structure was determined by spectroscopic methods including 2D NMR and chiral TLC analyses. Cyclo(dehydrohistidyl-L-tryptophyl) was found to have an inhibitory activity on nitric oxide production with an IC50 of 6.5 muM in activated BV-2 cells. The structure determination and biological activity of cyclo(dehydrohistidyl- L-tryptophyl) was reported for the first time in this study.     

Determination of molecular weights by differential cryoscopy on small volumes of dilute solutions of oligopeptides

A differential cryoscope of the equilibrium type is described. It requires only 1 ml of sample for a molecular-weight determination of a solute in a solvent that freezes below room temperature. The instrument is sensitive to +/- 0.0002 degrees C, which corresponds to +/- 0.0001 mol of solute per 1000 g of water. The apparatus was evaluated by measuring the freezing point depressions of 0.015 M urea and 0.01 M alanine in water, the measured molecular weights being accurate to within +/- 5%. The molecular weights of the following oligopeptides were then measured to determine their states of aggregation in the cited solvents: Ac-Asn-Pro-Tyr-NHMe in H2O and dimethyl sulfoxide and cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl), cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl), cyclo(L-alanyl-L-analyl-omega-capryl), cyclo(L-alanyl-D-alanyl-omega-capryl), and Ac-Tyr-Pro-Asn-NHMe in dimethyl sulfoxide.