Reações intramoleculares como modelos não miméticos de catálise enzimática

This review gives a critical idea on the importance of intramolecular reactions as models for enzymatic catalysis. Intramolecular lactonizations, ester and amide hydrolysis studies result in theories which try to explain the difference between intermolecular, intramolecular and enzyme reactions and rationalize the enhancement promoted by these biological catalyst.

Formation and decomposition of N-alkyinaphthalimides: experimental evidences and ab initio description of the reaction pathways

The kinetics of hydrolysis of 1,8-N-butyl-naphthalimide (1,8-NBN) to 1,8-N-butyl-naphthalamide (1,8-NBAmide) and of 2,3-N-butyl-naphthalimide (2,3-NBN) to 2,3-N-butyl-naphthalamide (2,3-NBAmide), as well as the formation of the respective anhydrides from the amides were investigated in a wide acidity range. 1,8-NBN equilibrates with 1,8-NBAmide in mild alkali. Under the same conditions 2,3-NBN quantitatively yields 2,3-NBAmide. Over a wide range of acidities the reactions of the 1,8- and 2,3-N-butyl-naphthalamides (or imides) yield similar products but with widely different rates and at distinct pH`s. Anhydride formation in acid was demonstrated for 1,8-NBAmide. The reactions mechanisms were rationalized in the manifold pathways of ab initio calculations. The differences in rates and pH ranges in the reactions of the 1,8- and 2,3-N-butyl-naphthalamides were attributed to differences in the stability of the tetrahedral intermediates in alkali as well as the relative stabilities of the five and six-membered ring intermediates. The rate of carboxylic acid assisted 1,8-N-Butyl-naphthalamide hydrolysis is one of the largest described for amide hydrolysis models. Copyright (C) 2010 John Wiley & Sons, Ltd.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Reactions of some non-enolisable chloroketones with amide ion and a new synthesis of acridones /|nGuo-shyoung John Chen. -- 260 St. Catharines, Ont. : [s. n.],

This research was directed mainly towards the investigation of the reacti.ons of· substituted chlorobenziophenones under strongly basi,c conditions. The work 'can be divided into two main sections. The Introduction deals mainly with historical studies on aryne chemistry and the Haller-Bauer reaction. Secti.on I i.s concerned with syntheses of 2-benzamido-2'chlorobenzophenone and 2-benzamido~3'-chlorobenzophenone,and with thei,r respective reactions wi.th potassium amide in ammonia. o-Chlorophenylacetic acid was converted to the acid chloride and then by Friedel-Craftsreaction with benzene to w-(o-chlorophenyl)acetophenone. Reaction wi.th phenylhydrazine and Fischer cyclization gave 3- (0chlorophenyl)- 2-phenylindole, which was ozonized to 2-benzamido-2'chlorobenzophenone. The isomeric 3' -chlor,..o ke: tone was similarly synthesised from m-chlorophenylacetic acid. Both the 2'- and 3' -ch.loroketones gave N-benzoylacridone on treatment with potassium amide in ammonia; an aryne mechanism is involved for the 3'-chloroketone but aryne and nucleophilic substitution mechanisms are possible for the 2'-chloroketone. Hydrolysis of the 2'- and 3'-chloroketones gave 2-amino-2'chlorobenzophenone and 2-amino-3'-chlorobenzophenone respectively. A second new acridone synthesis is given in the Appendix involving reactions of these two ketones with potassium t-butoxide in t-butylbenzene. i Section 2 deals with the investigation of the reaction of some tricyclic ch1orobenzophenones with potassium amide in liquid ammonia. These were 1-ch1orof1uorenone; which was pr~pared in several steps from f1uoranthene, and 1- and 2-ch1oroanthraquinones. 1-Ch1orof1uorenone gave 1-aminof1uorenone ; 1-ch1oroanthraquinone gave 1- and 2-aminoanthraquinones; 2-ch1oroanthraquinone was largely recovered from the attempted reaction.

Fragrance release from the surface of branched poly(amide)s

Enzymes are powerful tools in organic synthesis that are able to catalyse a wide variety of selective chemical transformations under mild and environmentally friendly conditions. Enzymes such as the lipases have also found applications in the synthesis and degradation of polymeric materials. However, the use of these natural catalysts in the synthesis and the post-synthetic modification of dendrimers and hyperbranched molecules is an application of chemistry yet to be explored extensively. In this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester groups situated on the peripheral layer of two families of branched polyamides. These branched polyamides were conjugated to simple fragrances citronellol and L-menthol via ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched polyamide support was carried out in aqueous buffered systems at slightly basic pH values under the optimum operative conditions for the enzymes used. These preliminary qualitative investigations revealed that partial cleavage of the ester functionalities from the branched polyamide support had occurred. However, the ability of the enzymes to interact with the substrates decreased considerably as the branching density, the rigidity of the structure and the bulkiness of the polyamide-fragrance conjugates increased.

Tripodal molecules for the promotion of phosphoester hydrolysis

A series of low molecular weight tripodal amide/histidine-containing compounds (1–2) have been synthesised and shown to increase the rate of bis-(p-nitrophenyl) phosphate (BNPP) and soman (GD) breakdown in buffered aqueous solution.

Electrochemical behaviour of (N-R-4-cyanopyridinium)pentaammineruthenium(II) derivatives in acidic medium. Hydrolysis of coordinated nitriles

The electrochemical behaviour of N-R-4-cyanopyridinium (4-rcp) (R = methyl, decyl, dodecyl, or benzyl) coordinated to pentaammineruthenium(II) in CF3COOH-CF3COONa (μ = 0.1 M, pH 3) aqueous medium was studied by means of cyclic voltammetry and constant potential electrolysis. The electrochemical oxidation of the metallic centre (Ep ca 0.51 V/SCE) can be described as a reversible monoelectronic charge-transfer followed by an irreversible chemical reaction, which is the hydrolysis of N-R-4-cyanopyridiniumpentaammineruthenium(III) (A) to N-R-4-carboxamidepyridiniumruthenium (III) (B) with the kf1 values depending on the type of alkyl group. The E 1 2 values are not significantly influenced by the nature of the alkyl group. At more negative potential (ca -0.5 V/SCE), B undergoes an electrochemical reduction followed by an aquation reaction to produce aquopentaammineruthenium(II) and free N-R-4-carboxamidepyridinium. The amide was identified by comparison of its cyclic voltammogram and UV-vis spectrum with that of a sample prepared by chemical reaction. The results were also discussed by comparison with other systems, and show that nitrile-amide conversion catalysed by pentaammineruthenium(II) complexes is possible. © 1994.

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.

Esi(+)-ms And Gc-ms Study Of The Hydrolysis Of N-azobenzyl Derivatives Of Chitosan.

New N-p-chloro-, N-p-bromo-, and N-p-nitrophenylazobenzylchitosan derivatives, as well as the corresponding azophenyl and azophenyl-p-sulfonic acids, were synthesized by coupling N-benzylvchitosan with aryl diazonium salts. The synthesized molecules were analyzed by UV-Vis, FT-IR, 1H-NMR and 15N-NMR spectroscopy. The capacity of copper chelation by these materials was studied by AAS. Chitosan and the derivatives were subjected to hydrolysis and the products were analyzed by ESI(+)-MS and GC-MS, confirming the formation of N-benzyl chitosan. Furthermore, the MS results indicate that a nucleophilic aromatic substitution (SnAr) reaction occurs under hydrolysis conditions, yielding chloroaniline from N-p-bromo-, and N-p-nitrophenylazo-benzylchitosan as well as bromoaniline from N-p-chloro-, and N-p-nitrophenylazobenzyl-chitosan.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Hydrolysis of methyl benzoate from Piper arboreum by Naupactus bipes beetle

A new natural product was isolated from Piper arboreum (Piperaceae) leaves, the methyl 3-geranyl-4-hydroxybenzoate (1). The metabolism of P. arboreum leaves by Naupactus bipes beetle (Germar, 1824 - Coleoptera: Curculionidae) led to the hydrolysis of 1 to 3-geranyl-4-hydroxybenzoic acid (2). The structures of both compounds were determined based on spectroscopic analysis (¹H and 13C NMR, MS, and IR).

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background: Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions. Results: Vascular bundles were more abundant in the rind, whereas parenchyma cells predominated in the pith region. UV measurements of untreated fiber cell walls gave absorbance spectra typical of grass lignin, with a band at 278 nm and a pronounced shoulder at 315 nm, assigned to the presence of hydroxycinnamic acids linked to lignin and/or to arabino-methylglucurono-xylans. The cell walls of vessels had the highest level of lignification, followed by those of fibers and parenchyma. Pith parenchyma cell walls were characterized by very low absorbance values at 278 nm; however, a distinct peak at 315 nm indicated that pith parenchyma cells are not extensively lignified, but contain significant amounts of hydroxycinnamic acids. Cellular UV image profiles scanned with an absorbance intensity maximum of 278 nm identified the pattern of lignin distribution in the individual cell walls, with the highest concentration occurring in the middle lamella and cell corners. Chlorite treatment caused a rapid removal of hydroxycinnamic acids from parenchyma cell walls, whereas the thicker fiber cell walls were delignified only after a long treatment duration (4 hours). Untreated pith samples were promptly hydrolyzed by cellulases, reaching 63% of cellulose conversion after 72 hours of hydrolysis, whereas untreated rind samples achieved only 20% hydrolyzation. Conclusion: The low recalcitrance of pith cells correlated with the low UV-absorbance values seen in parenchyma cells. Chlorite treatment of pith cells did not enhance cellulose conversion. By contrast, application of the same treatment to rind cells led to significant removal of hydroxycinnamic acids and lignin, resulting in marked enhancement of cellulose conversion by cellulases.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background: Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings: In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance: These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer`s spent grain

Brewer`s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 degrees C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2(3) full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer`s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.

Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer`s spent grain

Enzymatic hydrolysis of brewer`s spent grain in three different forms: original (untreated), pretreated by dilute acid (cellulignin), and pretreated by a sequence of dilute acid and dilute alkali (cellulose pulp), was studied to verify the effect of hemicellulose and lignin on cellulose conversion into glucose. The hydrolysis was carried out using a commercial cellulase concentrate (Celluclast 1.5 L) in an enzyme/substrate ratio of 45 FPU/g, 2% (w/v) substrate concentration, 45 degrees C for 96 h. According to the results, the cellulose hydrolysis was affected by the presence of hemicellulose and/or lignin in the sample. The cellulose conversion ratio (defined as glucose yield + cellobiose yield) from cellulignin was 3.5-times higher than that from untreated sample, whereas from cellulose pulp such value was 4-times higher, correspondent to 91.8% (glucose yield of 85.6%). This best result was probably due to the strong modification in the material structure caused by the hemicellulose and lignin removal from the sample. As a consequence, the cellulose fibers were separated being more susceptible to the enzymatic attack. It was concluded that the lower the hemicellulose and lignin contents in the sample, the higher the efficiency of cellulose hydrolysis. (C) 2007 Elsevier Inc. All rights reserved.