Ninhydrin as a reversible protecting group of amino‐terminal cysteine

Abstract: The proximity of the α‐amine and β‐thiol of α‐amino terminal‐cysteine (NT‐Cys) residues in peptides imparts unique chemical properties that have been exploited for inter‐ and intra‐molecular ligation of unprotected peptides obtained through both synthetic and biological means. A reversible protecting group orthogonal to other protection strategies and reversible under mild conditions would be useful in simplifying the synthesis, cleavage, purification and handling of such NT‐Cys peptides. It could also be useful for the sequential ligation of peptides. To this end, we explored tri‐one chemistry and found that ninhydrin (indane‐1,2,3 trione) reacted readily with cysteine or an NT‐Cys‐containing peptide on‐ or off‐resin at pH 2–5 to form Ninhydrin‐protected Cys (Nin‐Cys) as a thiazolidine (Thz). The Thz ring, protecting both the amino and thiol groups in Nin‐Cys, completely avoids the formylation and Thz side reactions found during hydrofluoric acid (HF) cleavage when N‐π‐benzyloxymethyl histidine groups are present. Nin‐Cys is stable during coupling reactions and various cleavage conditions with trifluoroacetic acid or HF, but is deprotected under thiolytic or reducing conditions. These properties enable a facile one‐step deprotection and end‐to‐end‐cyclization reaction of Nin‐Cys peptides containing C‐terminal thioesters.     

Synthetic peptide derived from α‐fetoprotein inhibits growth of human breast cancer: investigation of the pharmacophore and synthesis optimization

Abstract: A synthetic peptide that inhibits the growth of estrogen receptor positive (ER+) human breast cancers, growing as xenografts in mice, has been reported. The cyclic 9‐mer peptide, cyclo[EMTOVNOGQ], is derived from α‐fetoprotein (AFP), a safe, naturally occurring human protein produced during pregnancy, which itself has anti‐estrogenic and anti‐breast cancer activity. To determine the pharmacophore of the peptide, a series of analogs was prepared using solid‐phase peptide synthesis. Analogs were screened in a 1‐day bioassay, which assessed their ability to inhibit the estrogen‐stimulated growth of uterus in immature mice. Deletion of glutamic acid, Glu1, abolished activity of the peptide, but glutamine (Gln) or asparagine (Asn) could be substituted for Glu1 without loss of activity. Methionine (Met2) was replaced with lysine (Lys) or tyrosine (Tyr) with retention of activity. Substitution of Lys for Met2 in the cyclic molecule resulted in a compound with activity comparable with the Met2‐containing cyclic molecule, but with a greater than twofold increase in purity and corresponding increase in yield. This Lys analog demonstrated anti‐breast cancer activity equivalent to that of the original Met‐containing peptide. Therefore, Met2 is not essential for biologic activity and substitution of Lys is synthetically advantageous. Threonine (Thr3) is a nonessential site, and can be substituted with serine (Ser), valine (Val), or alanine (Ala) without significant loss of activity. Hydroxyproline (Hyp), substituted in place of the naturally occurring prolines (Pro4, Pro7), allowed retention of activity and increased stability of the peptide during storage. Replacement of the first Pro (Pro4) with Ser maintains the activity of the peptide, but substitution of Ser for the second Pro (Pro7) abolishes the activity of the peptide. This suggests that the imino acid at residue 7 is important for conformation of the peptide, and the backbone atoms are part of the pharmacophore, but Pro4 is not essential. Valine (Val5) can be substituted only with branched‐chain amino acids (isoleucine, leucine or Thr); replacement by d ‐valine or Ala resulted in loss of biologic activity. Thus, for this site, the bulky branched side chain is essential. Asparagine (Asn6) is essential for activity. Substitution with Gln or aspartic acid (Asp), resulted in reduction of biologic activity. Removal of glycine (Gly8) resulted in a loss of activity but nonconservative substitutions can be made at this site without a loss of activity indicating that it is not part of the pharmacophore. Cyclization of the peptide is facilitated by addition of Gln9, but this residue does not occur in AFP nor is it necessary for activity. Gln9 can be replaced with Asn, resulting in a molecule with similar activity. These data indicate that the pharmacophore of the peptide includes side chains of Val5 and Asn6 and backbone atoms contributed by Thr3, Val5, Asn6, Hyp7 and Gly8. Met2 and Gln9 can be modified or replaced. Glu1 can be replaced with charged amino acids, and is not likely to be part of the binding site of the peptide. The results of this study provide information that will be helpful in the rational modification of cyclo[EMTOVNOGQ] to yield peptide analogs and peptidomimetics with advantages in synthesis, pharmacologic properties, and biologic activity.     

Synthesis and physico‐chemical properties of peptides in soil humic substances

Abstract: Soil humic substances (HS) are heterologous, polydispersive, and multi‐functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with ¹³C‐glucose or ¹⁵N‐nitrate for 8.5 months. Evidence for random‐coil peptidic structures in the labeled HS was obtained from 2‐D nuclear magnetic resonance (NMR), pyrolysis gas chromatography‐mass spectrometry (pyro‐GC‐MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid‐state NMR data. Detailed ¹³C and ¹⁵N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC‐MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre‐existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.     

Biological activity of silylated amino acid containing substance P analogues

Abstract: The need to replace natural amino acids in peptides with nonproteinogenic counterparts to obtain new medicinal agents has stimulated a great deal of innovation on synthetic methods. Here, we report the incorporation of non‐natural silylated amino acids in substance P (SP), the binding affinity for the two hNK‐1 binding sites and, the potency to stimulate phospholipase C (PLC) and adenylate cyclase of the resulting peptide. We also assess the improvement of their stability towards enzyme degradation. Altogether, we found that replacing glycine with silaproline (Sip) in position 9 of SP leads to a potent analogue exhibiting an increased resistance to angiotensin‐converting enzyme hydrolysis.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

New indolicidin analogues with potent antibacterial activity*

Abstract: Indolicidin is a 13‐residue antimicrobial peptide amide, ILPWKWPWWPWRR‐NH₂, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two‐fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR‐NH₂ in which Trp‐residues 4,6,8,9,11 were replaced in all positions with X = a single non‐natural building block; N‐substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non‐natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N‐terminus with a hydrophobic non‐natural building block. We prepared 22 analogues of indolicidin and [Phe^4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non‐natural building block attached to the N‐terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non‐natural building blocks, are promising lead structures for developing future therapeutics.     

多发性骨折后全身炎症反应综合征的监控

目的　分析多发性骨折后全身炎症反应综合征 (SIRS)的监测因素。方法　将 87例多发性骨折患者分为SIRS组、非SIRS组和死亡组 ,比较各组血清肿瘤坏死因子 (TNF -α)、粒细胞集落刺激因子 (G -CSF)、补体C3 、免疫球蛋白 (IgG)等的变化。同时观察失血量分级与SIRS的关系。结果　SIRS组G -CSF和IgG水平明显低于非SIRS组 ,TNF -α水平明显高于非SIRS组 (P <0 .0 5 ) ;死亡组G -CSF、补体C3 及IgG水平较SIRS组进一步降低 ,TNF -α水平进一步升高 (P <0 .0 5 )。随失血量增大 ,SIRS发生率、病死率升高 (P <0 .0 5 )。结论　G -CSF ,IgG ,TNF -α水平及失血量可作为SIRS的监控指标。     

特异性真菌作用于龙血树材质形成血竭的研究

目的 探讨剑叶龙血树血竭的形成与微生物活动之间的关系。方法 用分离自剑叶龙血树根部的内生真菌9568D镰孢霉接种于剑叶龙血树材质(经灭活处理)。结果 保湿培养4～5个月后，在接种部位有红色血脂颗粒形成，经UV、IR光谱分析及抗菌活性实验，初步证实该血脂与来自剑叶龙血树的天然血竭无本质差异。结论 特异性真菌作用于龙血树材质可促成血竭的形成。     

Über Brückenvenenverletzungen bei tödlich verunglückten Kraftfahrzeuginsassen

Experimental data and clinical as well as postmortem experiences have indicated that subdural hematomas are less frequent in acceleration injuries in traffic accidents compared to falls or assaults. The present report demonstrates that this does not hold true in the same way for bridging vein ruptures (one of the predominant causes for subdural bleedings). Ruptures of these vessels without subdural bleeding (SDB) are only seldom mentioned in the literature. However, if no SDB is present, no one will look for these structures. In our institute a systematic analysis of the bridging veins in all cases of lethal blunt head injury is made: prior to the careful morphological preparation we investigate these vessels by radiographic imaging after filling with contrast medium. 6 car passengers (age between 4 and 31 years) which suffered a lethal head injury were examined in the last year. 2 victims had impressed fractures with cerebral compression injuries. In 1 case the base of the skull was broken and in 3 cases no skull fracture was present; no serious focal brain injury had occurred in these 4 cases, but 3 victims had signs of diffuse brain injury. In 5 cases a direct impact of the head against the interior of the car was obvious. In 5 cases ruptures of several bridging veins could be demonstrated. In one case (survival for 3 days) a minor SDB (20 ml) was present and the ruptures had been closed by thrombosis; another victim died at the scene. The other 3 victims survived between 4 and 15 hours without developing SDB and without closing of the ruptures by thrombosis. This combination is surprising and shows that our knowledge concerning the relationship between bridging vein ruptures and SDB is restricted. The frequency of bridging vein lesions in severe head injuries is likely underestimated in the clinical as well as in the postmortem literature. A rapid increase of intracranial pressure after the accident resulting in a collapse of the cerebral circulation is probably responsible for the absence of the SDB in the presented cases.     

Platten- und Schraubenosteosynthese bei Femurschaftfrakturen Retrospektive Studie von 500 Femurschaftfrakturen

The authors collected 500 cases of breaks of the thighbone shaft which were treated using osteosynthesis with screws, between 1st January 1992 and 31st of December 1996 at the Department for Traumatology of the University Medical Center in Ljubljana. Most patients were male (70.6%), the average age was 36.6 years, and the majority of patients had been injured in traffic accidents. In 50% of cases, only the thighbone was broken, the rest had suffered multiple injuries, or were polytraumatised. Their stay in hospital averaged 17 days, while the average time to union was 21 weeks. In most cases (85%), treatment proceeded with no complications. Chronical post-traumatic osteitis occurred in 9 of the patients with open breaks (1.8%), and only in 2 patients (0.4%) with closed breaks, which is within the limits of tolerance. With respect to our experience and results with osteosynthesis with plates and screws in the thighbone shaft, we believe that the mentioned method is good and gives results which are comparable to those achieved using other methods.