5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.

Medicinal plants and their uses by the people in the Region of Randa, Djibouti.

ETHNOPHARMACOLOGICAL RELEVANCE: The article presents the local knowledge on medicinal plants and their relevance in managing health problems. Important ethnobotanical leads are given with priority species and disease categories, casting insight on future phytochemical and pharmacological studies. AIM OF THE STUDY: The use of traditional medicinal plants has been an integral part of the traditional healthcare systems in Djibouti. However, scientific studies on the traditional herbal healing systems of the various cultural groups have never been undertaken. This study has, therefore, aimed at assessing plant-related ethnomedicinal knowledge of the people in Randa Region; prioritising the plants with respect to common disease categories and inferring about prospects of new pharmacological products. MATERIALS AND METHODS: Interview-based ethnobotanical field study was carried out to document the plant-based ethnomedicinal knowledge handed down to the present by the oral tradition of people living in 24 villages in Tadjourah District of Randa Region (north Djibouti). Informant Consensus Factors (ICF) and Fidelity Level (FL) values of the medicinal plants were calculated to check the level of informant agreement and the healing potentials of the species. RESULTS: A total of 91 plant species that belong to 72 genera and 40 families were documented. Most of these species (92%) were collected from non-cultivated areas. Their local names and traditional uses in medicine were also studied. The plant family Fabaceae was represented by the highest number of taxa (17 species). Strong informant agreements hinted at good healing potentials of some species as shown by high values of consensus factors for eye diseases (0.98), mouth diseases (0.93), kidney problems (0.89) and microbial infections (0.84). Dodonea angustifolia, Solanum cordatum, Grewia erythraea, Acalypha indica, Acacia etbaica, Fagonia schweinfurthii, Solanum coagulans, Senna alexandrina and Grewia tembensis scored high FL values emerging as promising priority species for future pharmacological screening against microbial infections. CONCLUSION: The results of this study may inspire further ethnobotanical and ethnopharmacological research and investigations toward drug discovery in Djibouti and beyond.

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations.

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.

Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis.

FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.

Application of phosphoramidate ProTide technology significantly improves antiviral potency of carbocyclic adenosine derivatives.

We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal S9 fractions.

Role of the covalent flavin linkage in monomeric sarcosine oxidase.

Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.

Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia.

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional enzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine) and transfer of the oxidized methyl group into the one-carbon metabolic pool. In addition to four different subunits, TSOX contains three coenzymes (FAD, FMN, and NAD) and a binding site for tetrahydrofolate, the coenzyme acceptor of the oxidized methyl group from sarcosine. Based on preliminary success in crystallization of the natural enzyme, the genes encoding the subunits for TSOX from Pseudomonas maltophilia (pTSOX) were cloned by functional screening of a genomic library. Recombinant enzyme exhibiting the same specific activity as natural pTSOX could not be isolated using a similar or identical purification procedure. This difficulty was overcome by affinity purification of recombinant pTSOX containing a C-terminal (His)(6) tag on the subunit (gamma) encoded by soxG, the gene located at the 3' end of the pTSOX operon. Affinity-purified pTSOX could not be crystallized, a problem traced to microheterogeneity in the recombinant enzyme where about half of the FMN is present in a modified form that is not found in the natural enzyme and may be a biosynthetic intermediate. The modified flavin was eliminated by expression of the recombinant enzyme in the presence of sarcosine, the same reagent used to induce expression of the natural enzyme. Homogenous recombinant pTSOX was isolated from cells grown in the presence of sarcosine by chromatography on affinity and hydrophobic interaction matrices. High quality crystals that diffract to 1.85 A resolution have been obtained.

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein.

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.

Improved antiviral activity of the aryloxymethoxyalaninyl phosphoramidate (APA) prodrug of abacavir (ABC) is due to the formation of markedly increased carbovir 5'-triphosphate metabolite levels.

The anti-human immunodeficiency virus (HIV) activity of abacavir (ABC; 1-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol) could be markedly enhanced by administering the aryloxymethoxyalaninyl phosphoramidate prodrug derivative of ABC (pro-ABC-MP) to virus-infected cell cultures. Metabolic studies with radiolabeled ABC and pro-ABC-MP in human T-lymphocyte and primary macrophage cell cultures revealed a significantly increased delivery of the activated (phosphorylated) metabolite of ABC (ABC-MP) by pro-ABC-MP, and the concomittant appearance of markedly higher intracellular levels of carbovir 5'-triphosphate (CBV-TP), which represents the eventual antivirally active metabolite of ABC. The intracellular amounts of ABC-MP and appearance of CBV-TP closely correlated with the extracellular pro-ABC-MP concentrations that were administered to the cell cultures within a concentration range between 0.5 and 100 microM. The highest amounts of CBV-TP were observed within 6-24 h after drug administration. The improved delivery of ABC-MP and metabolic conversion to CBV-TP explain the markedly enhanced antiviral activity of the prodrug of ABC, and warrant further exploration of this prodrug technology on ABC and related compounds to further enhance and optimize their antiviral efficacy.