Isosteric conversion of protein carboxyl groups into carboxamide groups. I. Examination of alkyl and aryl esters as model compounds

With a view to substituting protein carboxyl groups by carboxamide groups the properties of a number of model compounds have been examined. The methyl ester of acetylglycine can be 97% converted into amide in 7 M ammonium acetate/ 7 M ammonia in 10 min. Amidation of the N-ethylsalicylamide ester of benzoyl-glycine, prepared by reaction with N-ethylbenzisoxazolium fluoroborate (EBI), is 100% complete in 2 M ammonium acetate/ammonia, pH 9.2, within 1 min; no rearrangement into imide occurred at all. Under the conditions of protein modification (0.1 M EBI, pH 4.2, 0°) acetyltyrosinamide is 13% converted into a compound, presumably containing the N-ethylsalicylimidate group, exhibiting an absorption maximum at 309 nm (e = 3300). This compound decomposes in ammonium acetate/ammonia, pH 9.1, within 1 min. Nα-acetylhistidinamide is not converted at all. Reaction with aminogroups can either be avoided by protection of these groups or be analyzed by spectroscopy of the amidine produced. Spectral data required for analysis have been measured for N-ethylsalicylamide (Λ^max295 nm, e = 3200 at pH 6, Λ_max324 nm, e = 5100 at pH 10, pK 8.15), for O-acetyl-N-ethylsalicylamide (e = 1200 at 250 nm, e = 180 at 280 nm) and for O-diethylsalicylamidine (Δ_max277nm, e = 2330 at pH 6, Λ_max302 nm, e = 3230 at pH 10, pK 7.90).     

Denaturation studies on lysozyme by n.m.r. measurements of its inhibitors

A method for studying denaturation of an enzyme by n.m.r. measurements of its specific inhibitor is reported and discussed for the case of lysozyme and N-acetyl glucose amine. It was shown that in the presence of bound inhibitor molecules, the enzyme is more resistant to the denaturant agent as compared to the free enzyme.     

N.m.r. study of conformational changes in lysozyme around the thermal transition point

Natural abundance carbon-13 n.m.r. at 50.3 MHz has been used to further document the thermal transition that hen egg-white lysozyme undergoes in solution between 20° and 30°. The study focuses on the temperature sensitivity of more than 50 carboxylic, aromatic and aliphatic single carbon resonances for which unambiguous assignments to specific residues are known. The analysis of selective perturbations in chemical shifts indicates that residues located on both edges of the active site cleft and in the hydrophobic box are primarily involved in the temperature-induced conformational transition. N.m.r. results are compared with crystallographic data on low temperature (form A) and high temperature (form B) interconverting lysozyme crystals, taking advantage of the recent availability of quality high resolution maps for B form orthorhombic crystals. In most cases, a good correlation is found at the atomic level between residues involved in the thermal transition in solution and in the crystalline state. Discrete discrepancies are noted for some residues such as Trp-62 and His-15.     

Enzymatic conversion of morphine to pseudomorphine.

The interaction of morphine with an enzymatic oxidation-reduction system was investigated. It has been reported that morphine is converted to a highly water-soluble metabolite by incubation with horseradish peroxidase (HRP), albumin and H₂O₂ (Misra and Mitchell, Experientia 27, 1442 (1971)). After incubating [¹⁴C]morphine with HRP and H₂O₂, we detected two radioactive compounds with t.l.c. One compound corresponded to morphine. Using spectral analysis, the other, ¹⁴C-labeled compound, was identified as pseudomorphine, a dimerized oxidation product of two molecules of morphine. Using compounds structurally related to morphine, it was determined that: (1) a free phenolic hydroxyl group in position 3 was necessary for the enzymatic oxidation of morphine to pseudomorphine; (2) a carbonyl group in position 6 of the morphinan ring prevents the formation of the dimer; and (3) substitution of other functional groups on the morphine molecule did not affect the peroxidase-catalyzed dimerization. By optimizing reaction conditions, measurable rates of peroxidase-catalyzed pseudomorphine formation were observed at morphine concentrations as low as 5 × 10^?7 M. The nature of peroxidase enzymes and the phenolic character of morphine suggest that the formation of pseudomorphine proceeds through a morphine-free radical intermediate.     

Developmental conversion of inotropism by endothelin I and angiotensin II from positive to negative in mice.

Inotropic effects on isolated neonatal and adult mouse myocardium of endothelin I and angiotensin II were examined. Endothelin I produced a sustained positive inotropic response in the neonate but a sustained negative response in the adult. Both were concentration-dependent and were inhibited by the endothelin ETA receptor antagonist, BQ-123 (Cyclo(D-a-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)). Angiotensin II produced a sustained positive inotropic response in the neonate while a sustained negative response in the adult. Both were concentration-dependent and were inhibited by the angiotensin AT1 receptor antagonist, YM358 (2,7-diethyl-5-((2'-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl-5H-pyrazolo(1,5-b)(1,2,4)tria zole potassium salt monohydrate). These results indicate that inotropic responses of the mouse heart to cardioactive peptides are unique among experimental animal species and may be reversed during development.     

One-pot conversion of trimethylsilyl ethers into urethanes using chlorosulfonyl isocyanate: Application to the synthesis of a novel neuromodulator carisbamate

This paper reports a novel synthetic method for the preparation of various urethanes and the application to the synthesis of carisbamate. The reaction of primary (2a, 2e and 2f) or secondary (2g–2i) trimethylsilyl ethers with chlorosulfonyl isocyanate afforded the corresponding urethanes in good yields without affecting the olefin moiety. However, in the case of secondary benzylic trimethylsilyl ether 2j, the corresponding urethane 3j was obtained in low yield. From the difference in reactivity between the primary and secondary benzylic trimethylsilyl ethers, the one-pot synthesis of carisbamate 1 from bis-trimethylsilyl ether 2l was achieved.     

Antibacterial to antifungal conversion of neamine aminoglycosides through alkyl modification. Strategy for reviving old drugs into agrofungicides

Many Actinomycetes aminoglycosides are widely used antibiotics. Although mainly antibacterials, a few known aminoglycosides also inhibit yeasts, protozoans and important crop pathogenic fungal oomycetes. Here we show that attachment of a C8 alkyl chain to ring III of a neamine-based aminoglycoside specifically at the 4″-o position yields a broad-spectrum fungicide (FG08) without the antibacterial properties typical for aminoglycosides. Leaf infection assays and greenhouse studies show that FG08 is capable of suppressing wheat fungal infections by Fusarium graminearum-the causative agent of Fusarium head blight-at concentrations that are minimally phytotoxic. Unlike typical aminoglycoside action of ribosomal protein translation miscoding, FG08's antifungal action involves perturbation of the plasma membrane. This antibacterial to antifungal transformation could pave the way for the development of a new class of aminoglycoside-based fungicides suitable for use in crop disease applications. In addition, this strategy is an example of reviving a clinically obsolete drug by simple chemical modification to yield a new application.     

应用基质辅助激光解析电离飞行时间质谱源后衰变技术鉴定蛋白质

目的应用源后衰变(PSD)技术结合数据库检索鉴定二维蛋白质电泳(2DE)胶上的蛋白质斑点。方法 用已知多肽(ACTH)18~39肽段和TPA被胰酶降解后的1个肽段建立了PSD-MALDI-TOF-MS方法。结果应用已建立的PSD-MALDI-TOF-MS法分别将2DE分离后胶上的2个未知蛋白质斑点鉴定为40S核糖体蛋白S12和dnaK抑制蛋白。结论PSD技术在蛋白质组学研究中有较大的应用前景。     

Angiotensin I as a dipsogen: efficacy in brain independent of conversion to angiotensin II

Angiotensin I or II injected into preoptic or anterior hypothalamic areas, or intraventricularly, evoked water drinking in rats. Neither vehicle controls nor SQ 20,881 (angiotensin-converting enzyme inhibitor) produced drinking. SQ 20,881, which inhibits the conversion of angiotensin I to II, did not suppress the drinking evoked by either angiotensin I or II even at inhibitor/angiotensin ratios of 100:1. The results indicate the possibility that brain receptor sites underlying the dipsogenic response to angiotensin II are responsive to angiotensin I as well, or that additional sites are responsive to angiotensin I.     

Role of endogenous channels in red blood cells response to their exposure to the pore forming toxin Sticholysin II.

Sticholysin II (St II) is a highly hemolytic cytolysin isolated from the sea anemone Stichodactyla heliantus. The toxin hemolytic action takes place through the formation of channels that provoke an electrolyte unbalance leading to osmotic shock. The lytic event must involve the exchange of electrolytes and the entrance of water, leading to red blood cell disruption. These processes can occur through St II pores and/or the endogenous red blood cells transporters. In order to evaluate the contribution of these channels to water, anion and cation transport, we have measured the hemolysis and K+ efflux rates in the presence of several specific inhibitors. The results obtained in the presence of Hg, an AQP1 blocker, indicate that water transport through these channels is not essential for the occurrence of the lytic process induced by St II. The data also support a partial role of K+ and anion transporters. In particular, they are compatible with a preferential K+ efflux though the K(+)/Cl- co-transport as a response to the promoted swelling. Furthermore, they suggest that chloride influx, a process that can regulate both K+ efflux and lysis, is partially mediated by the endogenous cell transporters, in particular, band-3 anion exchange system being relevant at early stages of the lytic process.     

Small-molecule inhibitors binding to protein kinase. Part II: the novel pharmacophore approach of type II and type III inhibition

Background: Protein kinases are essential enzymes propagating cellular signal transduction processes and consequently emerged as central targets for drug discovery against a wide range of diseases with a strong historical focus on oncological disorders. Several high-resolution crystal structures of various ATP-competitive inhibitors in complex with their target protein kinases have been determined and represent a wealth of detailed information about binding modes, inhibition mechanisms, and associated structure– activity relationships of target-bound small molecules. Objective: In this second part of a two-part review, we discuss the binding mode of inhibitors that target protein kinases in their inactive state. Methods: The scope of this review covers inhibitors for which crystal structures in complex with their respective kinases in the inactive state are available. Results: Structural parameters of both inhibitors and kinases contribute to the complexity of designing kinase inhibitors. Kinase inhibitors that target the inactive state of a kinase have become a novel rule in the design of highly active and selective compounds. The combination of high-resolution structures of ligand-enzyme complexes with especially detailed kinetic studies will in the long-term help to develop new low-molecular weight type II inhibitors.     

Pyrimidoquinazoline-based antitumor agents. Design of topoisomerase II to DNA cross-linkers with activity against protein kinases

A series of pyrimidoquinazoline analogues, possessing either 4,5-g or 5,4-g fusion, were studied with respect to cytotoxicity, topoisomerase II inhibitory activity, in vivo activity, and DNA cleavage and DNA-protein cross-linking properties. These analogues were designed as electron-deficient anthraquinones with dual alkylating centers to cross-link DNA with topoisomerase II. Our studies verified the presence of DNA-protein cross-linking in vitro as well as topoisomerase II poisoning by pyrimidoquinazoline analogues. In addition, COMPARE analysis revealed that the pyrimidoquinazolines possess inhibitory activity against both topoisomerase II and protein kinases, such as the paullones, a dual property observed in other antineoplastic agents influencing phosphoester transfer.     

离子注入诱变育种技术在柔红霉素高产菌选育中的应用

应用低能N+离子对柔红霉素产生菌天蓝淡红链霉菌SIPI1482野生型菌株的孢子进行注入诱变选育,获得了柔红霉素产抗能力提高15倍的高产突变株.与亲株的菌落形态比较发现,高产突变株SIPI1482M2的菌落形态发生较大的变化.