Counter-current chromatography with off-line detection by ultra high performance liquid chromatography/high resolution mass spectrometry in the study of the phenolic profile of Lippia origanoides.

Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMSn) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMSn and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMSn studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each technique's potential.

Comparative evaluation of aspheric toric intraocular lens implantation and limbal relaxing incisions in eyes with cataracts and ≤3 dioptres of astigmatism.

PURPOSE: To compare the visual outcomes of aspheric toric intraocular lens (IOL) implantation and limbal relaxing incisions (LRI) for management of coexisting age-related cataracts and astigmatism. METHODS: In this prospective study, sixty eyes of 60 patients with visually significant cataract and coexisting corneal astigmatism ≤3 dioptres (D) were randomised to undergo phacoemulsification with either aspheric toric IOL or aspheric monofocal IOL with LRI. The main outcome measures were postoperative 3-month uncorrected visual acuity (UCVA), contrast sensitivity, rotational stability of the toric IOL and spectacle independence. RESULTS: The postoperative UCVA, contrast sensitivity and refractive astigmatism were significantly better than the baseline measurements for both groups (p≤0.001). There was no significant difference detected for these parameters between LRI and toric IOL groups postoperatively (p≥0.119). At both postoperative month 1 and 3, the percentages of eyes in need of spectacles were lower in toric group than LRI group (p≤0.030). IOL misalignment was noted in three eyes in the toric IOL group (mean misalignment 7.67±4.04°). On vector analysis, magnitude of error (ME) was negative in the LRI group indicating undercorrection, whereas the ME was close to zero for toric group. CONCLUSIONS: Both toric IOL implantation and LRI were effective in correcting corneal astigmatism ≤3 D during phacoemulsification, while LRI tended to undercorrect astigmatism.

Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism.

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.

Inhibition by glutathione derivatives of bovine liver glyoxalase II (hydroxyacylglutathione hydrolase) as a probe of the N- and S-sites for substrate binding.

The nature of the binding determinants used in the interaction of glutathione-based derivatives and bovine liver glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) has been investigated. Linear competitive inhibition was observed for S-blocked and S,N-blocked glutathiones with bovine liver glyoxalase II (molecular weight 22 500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis; pI = 7.48 by analytical isoelectric focussing). There is a significant hydrophobic region on the enzyme to bind substituents around the sulphydryl-derived moiety of the substrate--a hydrophobic S-site. However, there is no evidence for binding of the N-site of the substrate (or inhibitor) to glyoxalase II. In contrast to glyoxalase I, there is no linkage between binding forces used at the S- and N-sites. Binding of S,N-dicarbobenzoxyglutathione is pH-dependent, showing dependence on an ionisation with pKapp approximately equal to 7.2 (binding more tightly at higher pH), as is the kcat value (pKapp approximately equal to 7.8) for S-D-lactoylglutathione.

Organothallium(III) reagents for modification of biomacromolecules: interaction of thallium derivatives with transfer RNA.

As an extension of work on the inhibition of enzymes by arylthallium(III) reagents, the thallium analogues of the organomercurials, we have studied the interactions of these molecules with transfer RNA. In contrast to thallous acetate, thallium(III) derivatives (thallic trifluoroacetate, p-methylphenylthallium(III) bis-trifluoroacetate (MPT) and o-carboxyphenylthallium(III) bis-trifluoroacetate) bound to Escherichia coli tRNA. The interaction was fully reversible upon Sephadex G-25 gel filtration, and binding constants and stoichiometries were evaluated by a number of procedures. The likely site of interaction was shown to be the thiouridine residue (s4U8) based on changes induced by MPT on the absorbance around 330 nm. No changes in stacking interactions could be detected from the absorption or circular dichroic spectra. The detailed structure of the groups on thallium(III) affected the interaction with tRNA. Thalliation at s4U8 affects the absorbance at 335 nm and the amino-acid uptake capacity of E. coli tRNAPhe in parallel, the latter being progressively inhibited by increasing amounts of MPT. In a model nucleoside system, uridine disulphide is probably formed from reduced thiouridine by the oxidative action of the Tl(III) reagents. No evidence of cross-linking of E. coli tRNA molecules under gel electrophoretic conditions was obtained in contrast to the model nucleoside. The easily reversible interaction of MPT with sulphur sites in E. coli tRNA contrasts with the stable (to gel filtration) bonds formed between MPT and (thiol) sites in enzymes.

pH Dependence of the inhibition of yeast glyoxalase I by porphyrins.

A number of porphyrin derivatives have been found to inhibit yeast glyoxalase I (EC 4.4.1.5) at 25 degrees C, including haemin, protoporphyrin IX, coproporphyrin III, haematoporphyrin, deuteroporphyrin as well as meso-(tetrasubstituted) porphines. Bilirubin and chlorophyllin were also inhibitory, but not cobalamin, adipic, pimelic or suberic acids. Whilst the Ki value for linear competitive inhibition by meso-tetra(4-methylpyridyl)porphine was pH-dependent, analogous Ki values for meso-tetra(4-carboxyphenyl)- and meso-tetra(4-sulphonatophenyl)porphines followed the Henderson-Hasselbalch equation with pKapp values of 7.10 and 6.50, respectively. Protoporphyrin showed similar behaviour (pKapp 7.06) with a deviation at lower pH. The haemin pH profile for Ki showed a maximum at approx. pH 6.5. The redox reaction between haemin and glutathione did not interfere in the inhibition studies. The Ki value for S-(p-bromobenzyl)glutathione was pH-independent. A detailed analysis of porphyrin binding modes was undertaken.

Carboxypeptidase Y from Saccharomyces cerevisiae. Conformational differences reflected in kinetic behaviour in water and deuterium oxide.

The glycoenzyme carboxypeptidase Y (peptidyl--amino-acid hydrolase, EC 3.4.16.1), from baker's yeast (British Fermentation Products Strain, Ng 72), of molecular weight 60 000, had a protein portion closely similar to those in the literature for carboxypeptidase Y isolations from other yeast sources, but was 25.3 wt% carbohydrate (mannose 83.3% by wt., glucosamine 10.3% by wt. with traces of galactose and galactosamine). Circular dichroic spectra indicated that the enzyme lost its beta-structure as the pH was lowered from 8.08 to 4.16. At p2H 8.22 in 2H2O media the conformation of this enzyme was different from that observed at pH 8.08. A tyrosine residue appeared to be perturbed by lowering the pH of the medium. Carboxypeptidase Y was rapidly, and essentially irreversibly, inactivated at low p2H. The pH profile of kcat for the carboxypeptidase Y-catalysed hydrolysis of 4-nitrophenyltrimethylacetate showed two inflections at 45 degrees C: one at pKapp approximately 3.7 insensitive to temperature variation (ascribed to a carboxyl group), and one of pKapp approximately 5.7 markedly temperature-dependent and possibly caused by a histidine residue.

Organothallium(III) reagents for modification of biomacromolecules: irreversible labelling of phosphoglycerate kinase from rabbit muscle.

Organothallium(III) reagents, by analogy with organomercurials, have been found to rapidly label phosphoglycerate kinase from rabbit muscle. By use of a radio-labelled version of p-methylphenylthallium(III) bis-trifluoroacetate (MPT) the inhibition was shown to be irreversible by the criterion of gel filtration desalting. The rate of labelling was shown to depend on the temperature, enzyme and thallium reagent concentrations, and the presence or absence of the various substrates of the enzyme. The structure and oxidation state of the thallium reagent used affected the extent of modification by the compounds MPT, o-carboxyphenylthallium(III) bis-trifluoroacetate, thallic trifluoroacetate and thallous acetate. A number of pieces of evidence implicate cysteine residues in the labelling, including changes in the free thiol titre of the enzyme on thalliation, model studies on the interaction of thiols (e.g. glutathione) with thallium(III) and thallous materials, the lack of inactivation of phosphoglycerate kinase from yeast (which has only one thiol residue distant from the active site), and the partial restoration of enzymic activity by treatment of thalliated enzyme with sulphydryl reducing agents. Substrate protection studies showed that modification of rabbit muscle phosphoglycerate kinase by MPT was fully prevented by 3-phosphoglycerate and partially by MgATP. The latter protected only against the fast phase of thallic modification, the slower phase being unaffected. The presence of MgADP potentiated the labelling by MPT. No evidence of an MgADP-induced conformational change in the enzyme could be obtained from fluorescence or circular dichroic spectroscopies, although changes of the native spectra were noted on thalliation by MPT alone. The cross-linking potential of these arylthallium(III) reagents is discussed along with conformational changes required to trigger the hinge-movement between the N- and C-domains of the protein.

Trapping of reactive intermediates in enzymology. Exogenous flavin reduction during catalytic turnover of substrate by glyoxalase I.

Although but weak inhibitors of glyoxalase I under steady-state conditions, flavins are reduced by yeast glyoxalase I (lactoyl-glutathione lyase, EC 4.4.1.5) plus its substrate (the hemithiolacetal from glutathione and phenylglyoxal) during catalytic turnover. Studies with 10-ethylisoalloxazine showed that this flavin reduction was peculiar not merely to glyoxalase I's substrate, but was characteristic of the complete system, enzyme plus substrate undergoing catalytic turnover. Flavins are poor hydride-ion acceptors and the reduction observed most likely represents an oxidative trap of a transient carbanion formed in the glyoxalase I mechanism of action. Hydrophobic flavins were more efficient traps than the hydrophilic ones, and values of the Km for the phenylglyoxal: glutathione hemithiolacetal adduct measured by the flavin-reaction and by normal steady-state kinetics were closely similar. This argues that trapping has occurred of an enediolate ion (an enzyme-generated carbanion) still bound to glyoxalase I.

The glutamyl binding site of trypanothione reductase from Crithidia fasciculata: enzyme kinetic properties of gamma-glutamyl-modified substrate analogues.

Trypanothione reductase, central to the redox defense systems of parasitic trypanosomes and leishmanias, is sufficiently different in its substrate-specificity from mammalian glutathione reductase to represent an attractive target for chemotherapeutic intervention. Previous studies of the physiological substrates trypanothione (N1,N8-bis(glutathionyl)spermidine) and N1-glutathionylspermidine disulphide established that the spermidine moiety of these substrates can be replaced by the 3-dimethyl-propylamide group (N1-glutathionyl-N3-dimethyl-propylamide). With this modification, the specificity for the gamma-glutamyl moiety of the substrate was examined. Kinetic analysis of a series of substrate analogues indicated that neither the alpha-carboxylate or alpha-amino functions of the L-gamma-glutamyl group is essential for recognition, since this group could be replaced by uncharged benzyloxycarbonyl or t-butyloxycarbonyl groups with relative catalytic efficiencies (kcat/Km) of 58 and 11%, respectively, of N1-glutathionyl-N3-dimethylpropylaminedisulphide. Other substitutions are less well tolerated (e.g., beta-L-aspartyl or aminobutyryl) or not at all (e.g., glutaryl). These findings are discussed in relation to the structural model of TR from Trypanosoma congolense. The successful structural replacements achieved have potential application for drug delivery.

Partial transition-state inhibitors of glyoxalase I from human erythrocytes, yeast and rat liver.

Glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) converts the hemithiolacetal of glutathione and an alpha-ketoaldehyde to S-D-lactoylglutathione which is hydrolysed under the catalytic influence of glyoxalase II to produce D-lactate and regenerate glutathione. There is much evidence that glyoxalase I operates via an enediol intermediate, and in this study a number of inhibitors are described which were designed based on the enediol moiety of this reactive intermediate. These enediol and paene-enediol moieties were combined with groups designed to make use of an adjacent hydrophobic site and can be described as partial transition-state analogues. Derivatives of lapachol and kojic acid were good competitive inhibitors of glyoxalase I from various sources unless the free hydroxy group was blocked or replaced. Flavones with strong inhibitors of glyoxalase I and gallocyanine (a dye) showed spectral changes on binding to glyoxalase I indicative of binding to a metal-ion site (probably Zn2+ or Mg2+). The use of the enediol-binding determinant to produce glyoxalase I inhibitors is discussed as a route to potential antitumour derivatives.

A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification.

The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homologous to a range of dehydrogenases including alcohol dehydrogenases, L-threonine-3-dehydrogenase, D-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dehydrogenase to provide the template, with additional modelling input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme. A range of potential ligands was designed based on the homology model and tested as inhibitors of CAD2 and horse liver alcohol dehydrogenase.

The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons.

O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.

Evidence for gene duplication forming similar binding folds for NAD(P)H and FAD in pyridine nucleotide-dependent flavoenzymes.

For pyridine nucleotide-dependent flavoenzymes, binding both FAD and NAD(P)H on a single amino-acid chain, we have found a high degree of internal sequence similarity for certain regions of the FAD and NAD(P)H binding portions of the chain for any given protein. This was the case for a range of enzyme classes, including disulphide oxidoreductases (such as glutathione reductase, trypanothione reductase, lipoamide dehydrogenase, mercuric reductase), mono- and dioxygenases, nitrite reductase, alkyl hydroperoxidase and NADH dehydrogenase from E. coli. This provides strong support for gene duplication as the origin of at least part of the FAD and NAD(P)H recognising domains of such enzymes.

Use of an additional hydrophobic binding site, the Z site, in the rational drug design of a new class of stronger trypanothione reductase inhibitor, quaternary alkylammonium phenothiazines.

Improved rationally designed lead drug structures against African trypanosomiasis, Chagas disease, and leishmaniasis were obtained against trypanothione reductase from Trypanosoma cruzi. Substituted-benzyl [3-(2-chloro-4a, 10a-dihydrophenothiazin-10-yl)propyl]dimethylammonium salts, synthesized by Menschutkin quaternization of the tertiary alkylamine omega-nitrogen atom of chlorpromazine, were linear, competitive inhibitors of recombinant trypanothione reductase from T. cruzi, with either trypanothione disulfide or N-benzyloxycarbonyl-L-cysteinylglycyl 3-dimethylaminopropylamide disulfide as substrate. The permanent positive charge on the distal nitrogen atom of the tricyclic side chain contribution to binding was estimated as >/=5.6 kcal.mol(-1) by comparison with the analogue with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving K(i) values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl](3, 4-dichlorobenzyl)dimethylammonium derivative (K(i) 0.12 microM), was approximately 2 orders of magnitude more inhibitory than the parent chlorpromazine. Several of these quaternary phenothiazines completely inhibited T. brucei parasite growth in vitro at <1 microM. Antiparasite activity was not solely determined by inhibition strength against trypanothione reductase, there being a strong contribution from hydrophobicity (for example, benzhydryl-quaternized chlorpromazime had ED(50) < 1 microM). Although active against Leishmania donovani, none of the analogues showed major improvement in this activity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED(50) < 1 microM) growth of the amastigote stage of T. cruzi.

Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria.

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.

Phenothiazine inhibitors of trypanothione reductase as potential antitrypanosomal and antileishmanial drugs.

Given the role of trypanothione in the redox defenses of pathogenic trypanosomal and leishmanial parasites, in contrast to glutathione for their mammalian hosts, selective inhibitors of trypanothione reductase are potential drug leads against trypanosomiasis and leishmaniasis. In the present study, the rational drug design approach was used to discover tricyclic neuroleptic molecular frameworks as lead structures for the development of inhibitors, selective for trypanothione reductase over host glutathione reductase. From a homology-modeled structure for trypanothione reductase, replaced in the later stages of the study by the X-ray coordinates for the enzyme from Crithidia fasciculata, a series of inhibitors based on phenothiazine was designed. These were shown to be reversible inhibitors of trypanothione reductase from Trypanosoma cruzi, linearly competitive with trypanothione as substrate and noncompetitive with NADPH, consistent with ping-pong bi bi kinetics. Analogues, synthesized to define structure-activity relationships for the active site, included N-acylpromazines, 2-substituted phenothiazines, and trisubstituted promazines. Analysis of Ki and I50 data, on the basis of calculated log P and molar refractivity values, provided evidence of a specially favored fit of small 2-substituents (especially 2-chloro and 2-trifluoromethyl), with a remote hydrophobic patch on the enzyme accessible for larger, hydrophobic 2-substituents. There was also evidence of an additional hydrophobic enzymic region available to suitable N-substituents of the promazine nucleus. Ki data also indicated that the phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Selected compounds were tested for in vitro activity against Trypanosoma brucei, T. cruzi, and Leishmania donovani, with selective activities in the micromolar range being determined for a number of them.

Photolability of bleomycin and its complexes.

Bleomycin has been found to be very sensitive to photolysis and on irradiation with the full spectral range of a medium-pressure mercury lamp undergoes a number of photo-induced reactions; there is a process in which the absorbance at 310 nm decreases, and a slower process in which it increases with photolysis time. The faster process can be studied by irradiation at approximately 300-350 nm, is complete in approximately 8 min, obeys first order kinetics, but is itself biphasic. The Fe(II) and Cu(II) complexes of bleomycin are also photolabile, as is the bleomycin-DNA complex.