The reaction of enol(ate)s with electrophiles is used extensively in organic synthesis for stereoselective C–C bond formation. Protein-based catalysts have had comparatively limited application for the stereoselective formation of C–C bonds of choice via enolate chemistry. We describe protein engineering studies on 5-carboxymethylproline synthases, members of the crotonase superfamily, aimed at enabling stereoselective C–C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates. Active site substitutions, including at the oxyanion binding site, enable the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The results reveal the potential of the ubiquitous crotonase superfamily as adaptable catalysts for the control of enolate chemistry.

Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme–substrate and protein–protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system.

Amino acid analyses reveal that JMJD6-catalysed hydroxylation of RNA-splicing regulatory protein fragments occurs to give hydroxylysine products with 5S stereochemistry. This contrasts with collagen lysyl hydroxylases, which give 5R-hydroxylated products. The work suggests that more than one subfamily of lysyl hydroxylases has evolved and illustrates the importance of stereochemical assignments in proteomic analyses.

This study was designed to evaluate the suitability of chitosan polymer as a vehicle for topical delivery system. celecoxib, which is a nonsteroidal anti-inflammatory drug, was incorporated into the gel vehicles in a concentration of 0.5 % w/v. Gels were prepared using three different concentrations and different molecular weights of chitosan. Viscosity, drug release from gels, permeation of drug through rat skin and anti-inflammatory activity of the drug were studied. In vitro release characteristics of the drug from different gels were carried out using dialysis membrane in phosphate buffer using a pH of 6.8. The results showed that, the gel form containing 1.0 % w/v medium molecular weight chitosan has superior drug release than other forms, whilst the gel form containing 2.0 % w/v high molecular weight chitosan shows the lowest amount of drug release. The release data were treated with various kinetic principles to assess the relevant parameters. The results revealed an inverse correlation between the percent drug release and the polymer concentration used. The results also showed that the release of drug from the prepared gels obeyed the Higuchi’s diffusion model. The permeation of drug through rat skin was carried out. The flux of drug is independent on the viscosity of the formulae. The anti-inflammatory activity of the drug in different gel formulations was studied using carrageenan-induced rat paw edema method. The results obtained show that there is excellent anti-inflammatory activity of the gel forms on rat paw edema.

In the design of oral delivery, alginates (Alg) have attracted increasing attention. However, due to their hydrophilic character incorporation of small hydrophilic drugs such as tolmetin sodium (TOL) into Alg beads will not provide the desired regular shape as well as delayed drug release. There is no study investigating the effect of methylcellulose (MC) and dual cross-linking (CaCl2 and glutaraldehyde, GA) on their shape, swelling and release behaviors. Hence this study aimed to evaluate the influence of MC and cross-linking agents on these behaviors of Alg microbeads prepared using the ionotropic gelation method compared with Alg microspheres prepared with w/o emulsion method. The results obtained display some interesting information. Both concentration of MC and type of cross-linking agent had dramatic effects on shape as well as swelling, erosion and release behaviors of the prepared microbeads. Swelling through ion-exchange process of Alg/MC blend single cross-linked microbeads was hindered in the case of dual cross-linked microbeads. The release of the drug from Alg/MC dual cross-linked microbeads was extended for up to 12 h and the release mechanism was shifted from erosion type release to time-independent release process. Analgesic activity study indicated significantly different response patterns compared with plain TOL solution.

Factor-inhibiting hypoxia-inducible factor (FIH) is an Fe(II) ⁄ 2-oxoglutarate-dependent dioxygenase that acts as a negative regulator of the hypoxiainducible factor (HIF) by catalysing b-hydroxylation of an asparaginyl residue in its C-terminal transcriptional activation domain (CAD). In addition to the hypoxia-inducible factor C-terminal transcriptional activation domain (HIF-CAD), FIH also catalyses asparaginyl hydroxylation of many ankyrin repeat domain-containing proteins, revealing a broad sequence selectivity. However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues. Here, we report that histidinyl residues within the ankyrin repeat domain of tankyrase-2 can be hydroxylated by FIH. NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the b-position. The results further expand the scope of FIH-catalysed hydroxylations.

The objective of this work was to develop suitable film formulations of ketorolac tromethamine (KT) for transdermal use and to investigate the effect of film composition and permeation enhancers on the in-vitro release and skin permeation of the drug. Polyvinyl alcohol (PVA), sodium carboxymethylcellulose (NaCMC), and chitosan were used as film-forming polymers. The adhesive hydrophilic polymers plastoid® E35L (PL E35) and polyvinyl pyrrolidone (PVP) were added to improve bioadhesion. The permeation enhancers used were oleyl alcohol (OA), sodium glycocholate (NaGC) and propylene glycol (PG). Formulated films were characterized by measuring their mean thickness, mass, drug content, folding endurance and bioadhesion. In-vitro release was studied using the USP XXIII rotating paddle method and in-vitro permeation across hairless rat skin was studied using an in-vitro diffusion cell. Addition of PVP enhanced the drug release and permeation especially in case of chitosan, while Plastoid® E35L improved permeation only. Skin permeation of the drug was greatly improved by the addition of permeation enhancers, the rank of their effectiveness was: sodium glycocholate (Na GC) > oleyl alcohol (OA) > propylene glycol (PG). The results obtained showed that these polymeric films can be a promising therapeutic system for the transdermal delivery of ketorolac.

The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1– 3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia-inducible factor catalyses the hydroxylation of an asparagine-residue in hypoxia-inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia-inducible factor hydroxylases negatively regulate hypoxia-inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen-sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical -2- quench ⁄MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal oxygendependent degradation domain of hypoxia-inducible factor-a with oxygen to form hydroxylated C-terminal oxygen-dependent degradation domain and succinate is much slower (approximately 100-fold) than for other similarly studied 2-oxoglutarate oxygenases. Stopped flow⁄ UV-visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mo¨ ssbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2-oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2- oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor.

Vitamin K is a fat-soluble vitamin involved in blood coagulation and bone metabolism. The detection and monitoring of vitamin K homologues in rheumatoid arthritis (RA) patients is a challenging problem due to the smaller concentrations of vitamin K and the presence of several interfering medications. Therefore, this study aimed to develop a new highly sensitive and selective chemiluminescence (CL) method designated to quantify vitamin K homologues in plasma of RA patients including phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4, vitamin K2) and menaquinone-7 (MK-7, vitamin K2). The method was based on the unique photochemical properties of vitamin K homologues that were exploited for selective luminol CL reaction. The correlation coefficients of 0.998 or more were obtained in the concentration ranges of 0.1–100 ngmL−1 vitamin K homologues. The detection limits were 0.03–0.1 ngmL−1 in human plasma for vitamin K homologues. The developed HPLC-CL system was successfully applied for selective determination of vitamin K homologues in plasma of RA patients. The developed method may provide a useful tool for monitoring vitamin K homologues in different clinical studies such as RA, osteoporosis and hepatocellular carcinoma in which vitamin K is intervented.