Imaging Active Infection in vivo Using D-Amino Acid Derived PET Radiotracers.

Occult bacterial infections represent a worldwide health problem. Differentiating active bacterial infection from sterile inflammation can be difficult using current imaging tools. Present clinically viable methodologies either detect morphologic changes (CT/ MR), recruitment of immune cells (111In-WBC SPECT), or enhanced glycolytic flux seen in inflammatory cells (18F-FDG PET). However, these strategies are often inadequate to detect bacterial infection and are not specific for living bacteria. Recent approaches have taken advantage of key metabolic differences between prokaryotic and eukaryotic organisms, allowing easier distinction between bacteria and their host. In this report, we exploited one key difference, bacterial cell wall biosynthesis, to detect living bacteria using a positron-labeled D-amino acid. After screening several 14C D-amino acids for their incorporation into E. coli in culture, we identified D-methionine as a probe with outstanding radiopharmaceutical potential. Based on an analogous procedure to that used for L-[methyl-11C]methionine ([11C] L-Met), we developed an enhanced asymmetric synthesis of D-[methyl-11C]methionine ([11C] D-Met), and showed that it can rapidly and selectively differentiate both E. coli and S. aureus infections from sterile inflammation in vivo. We believe that the ease of [11C] D-Met radiosynthesis, coupled with its rapid and specific in vivo bacterial accumulation, make it an attractive radiotracer for infection imaging in clinical practice

Fluorescence2D: Software for Accelerated Acquisition and Analysis of Two-Dimensional Fluorescence Spectra

The Fluorescence2D is free software that allows analysis of two-dimensional fluorescence spectra obtained using the accelerated “triangular” acquisition schemes. The software is a combination of Python and MATLAB-based programs that perform conversion of the triangular data, display of the two-dimensional spectra, extraction of 1D slices at different wavelengths, and output in various graphic formats

Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like the juvenile hormone in some insects. The role of these enzymes in some organisms is still not fully understood.Epoxides are highly reactive valuable intermediates used by the pharmaceutical industry. Enantiopure epoxides are of high value in the production of pharmaceuticals like pain-killers or protease-inhibitors. There are a number of ways to produce enantiopure epoxides, but nowadays an environmentally friendly manner has a high preference. One such environmentally friendly method is the use of the epoxide hydrolases. These enzymes are able to enantioselectively hydrolyze one epoxide-enantiomer to its vicinal diol. By this so-called kinetic resolution, it is possible to obtain both the epoxide and diol enantiopure. Enantiopure diols are also of high value in the fine and pharmaceutical chemistry.The first goal of this project is achieved: the epoxide hydrolase from Rhodotorula glutinis has been isolated and purified. The second goal (optimization of the reaction conditions) has been performed but it is still favorable to further optimize them. Initial experiments of enzyme stability towards temperature and pH has been performed with crude enzyme extracts, not with the purified enzyme. With respect to the third goal (a suitable method for the isolation and separation of epoxide and diol), the use of a recently described membrane reactor is recommended. The performance of this reactor, however, has not been verified for the EH studied.The enzyme is partially characterized. The EH was found to be a membrane associated enzyme. Whether or not it is actually a membrane bound enzyme (and how many times it passes the membrane) is still unknown. The enzyme consists of two (most probably) identical subunits with a molecular mass of 45 kDa. Amino acid analysis revealed that the enzyme belongs to thea/b-hydrolase fold family, because the characteristic catalytic histidine motive (G H F) can be found in the amino acid sequence. The N-terminal sequence, however, could not be detected. The amount of purified enzyme was too low to establish its the three-dimensional structure.With partially purified enzyme sample, the specific activity and enantioselectivity could be enhanced when detergents were added. Non-ionic detergents had the largest positive effects, e.g. the specific activity for 1,2-epoxyhexane and styrene oxide was enhanced three and eight times, respectively. In the same way, the enantioselectivity for 1,2-epoxyhexane and styrene oxide could be enhanced over 10 and nearly 5 times, respectively. In addition, non-ionic detergents had an enzyme stabilizing effect. Anionic detergents had a very clear negative effect: enzyme activities were reduced to 20%.Another method investigated to influence the stability, the activity and the enantioselectivity consists of polymerizing the epoxide hydrolase in a network. The enantioselective conversion of (±)-1,2-epoxyoctane was reversed from a preference for ( R )-1,2-epoxyoctane to ( S )-1,2-epoxyoctane when the enzyme had been imprinted with ( S )-1,2-epoxyoctane prior to co-polymerization. This is the first time that the above mentioned method was successfully performed with a membrane-associated enzyme of thea/b-hydrolase fold family to which EH belongs. The half-life of the immobilized and imprinted biocatalyst was enhanced at least 7-fold. Most remarkable was that washing the immobilized EH with HCl, followed by washing it with buffer, resulted in about 50% of the residual activity, while native EH completely lost its activityThe effect of increasing epoxide amounts (up to 10 mmol per 10 mL of water, leading to phase separations) on both the activity and enantioselectivity has been studied, including the effect of detergents on such two-phase enzymatic conversions. It appeared that cell-free extracts without detergents gave the highest activity at 10 mmol epoxide per 10 mL of water added, without loss of enantioselectivity as compared to 1 mmol epoxide per 10 mL of water emulsions.It is recommended either to use whole cells (overexpressing EH or not) in a bioreactor to produce enantiopure epoxides and diols in large quantities or to use an immobilized-imprinted enzyme polymer for the conversion of smaller quantities of epoxides in an enantioselectivity of your choice. Further investigations to improve both methods (whole cells, the enzyme properties and the immobilization and imprinting procedures) are required to optimize this type of conversions for practical applications

Enantiomeric separation of homocysteine and cysteine by electrokinetic chromatography using mixtures of gamma-cyclodextrin and carnitine-based ionic liquids

Two new chiral ionic liquids based on the ester of the non-protein amino acid carnitine as cationic counterpart were synthesized, characterized and evaluated as chiral selectors for the enantiomeric separation of homocysteine and cysteine derivatized with 9-fluorenylmethoxycarbonyl chloride by electrokinetic chromatography. The use of both ionic liquids, L-carnitine methyl ester bis(trifluoromethane)sulfonimide and L-carnitine methyl ester L-(+)-lactate, as sole chiral selectors in the separation buffer, did not allow the enantiomeric separation of the studied amino acids. However, the combined use of L-carnitine methyl ester bis(trifluoromethane)sulfonimide ionic liquid with gamma-CD forming a dual system led to the enantiomeric separation of both analytes and showed the existence of a strong synergistic effect. On the contrary, the dual system gamma-CD plus L-carnitine methyl ester L-(+)-lactate did not improve the enantiomeric separations with respect to those obtained using the CD alone. The influence of different experimental variables such as buffer composition and pH, and ionic liquid concentration was investigated. Also, the nature of the anionic moiety was evaluated by comparing the results obtained with both ionic liquids when combined with gamma-CD. The use of 2 mM gamma-CD combined with 5 mM L-carnitine methyl ester bis(trifluoromethane)sulfonimide in a 50 mM phosphate buffer at pH 7.0 enabled to achieve the simultaneous enantiomeric separation of homocysteine and cysteine with high resolution values (> 6.0) in analysis times close to 12 min

Determination of Tobacco Alkaloid Enantiomers Using Reversed Phase UPLC/MS/MS

Nʹ-Nitrosonornicotine (NNN), a carcinogenic tobacco-specific Nʹ-nitrosamine (TSNA), is on the FDA list of harmful and potentially harmful constituents (HPHCs). Nornicotine, a product of the demethylation of nicotine, is the immediate alkaloid precursor for NNN formation. Nicotine, nornicotine and NNN are optically active. The accumulation of the isomers of nicotine, nornicotine, and NNN impacts their biological activity. In this paper, we report the determination of tobacco alkaloid enantiomers (including nicotine, nornicotine, anabasine, and anatabine) in samples of different tobacco lines using a reversed phase ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS) method. Current method demonstates excellent detection capability for all alkaloid enantiomers, with correlation coefficients (r2) \u3e 0.996 within their linear dynamic ranges. The limit of detection (LOD) and limit of quantitation (LOQ) of all analytes are less than 10 ng/mL and 30 ng/mL, respectively. In addition, their recovery and coefficient of variation (CV%) are within 100–115% and 0.2–3.7%, respectively. The method validated in this paper is simple, fast, and sensitive for the quantification of alkaloid enantiomers in tobacco leaf and has been applied to investigations of tobacco alkaloid enantiomer ratios in different tobacco lines and tobacco products

Enantiomeric separation of non-protein amino acids by Electrokinetic Chromatography

New analytical methodologies enabling the enantiomeric separation of a group of non-protein amino acids of interest in the pharmaceutical and food analysis fields were developed in this work using Electrokinetic Chromatography. The use of FMOC as derivatization reagent and the subsequent separation using acidic conditions (formate buffer at pH 2.0) and anionic cyclodextrins as chiral selectors allowed the chiral separation of eight from the ten non-protein amino acids studied. Pyroglutamic acid, norvaline, norleucine, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, and selenomethionine were enantiomericaly separated using sulfated-alpha-CD while sulfated-gamma-CD enabled the enantiomeric separation of norvaline, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, selenomethionie, citrulline, and pipecolic acid. Moreover, the potential of the developed methodologies was demonstrated in the analysis of citrulline and its enantiomeric impurity in food supplements. For that purpose, experimental and instrumental variables were optimized and the analytical characteristics of the proposed method were evaluated. LODs of 2.1 x 10(-7) and 1.8 x 10(-7) M for D-and L-citrulline, respectively, were obtained. D-Cit was not detectable in any of the six food supplement samples analyzed showing that the effect of storage time on the racemization of citrulline was negligible. (C) 2016 Elsevier B.V. All rights reserved

Development of an in-capillary derivatization method by CE for the determination of chiral amino acids in dietary supplements and wines

A fast in-capillary derivatization method by CE with 6-aminoquinolyl-N-hydroxysuccinimidyl\ud carbamate was developed for the first time for the determination of\ud amino acid enantiomers (arginine, lysine, and ornithine) in dietary supplements and\ud wines. Because of the initial current problems due to the formation of precipitates into\ud the capillary during the derivatization reaction, a washing step with an organic solvent as\ud DMSO between injections was necessary. Different approaches were also investigated to\ud enhance the sensitivity of detection. A derivatization procedure, where plugs of ACN,\ud derivatizing agent (10mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), and\ud sample in borate (1:1 v/v) were injected in tandem (2, 3, and 6 s, respectively, at 50 mbar),\ud was selected because it enabled to obtain the most sensitive and reproducible results.\ud Appropriate analytical characteristics (linearity, LOD and LOQ, precision, absence of\ud matrix interferences, and accuracy) were obtained for this method. Finally, the optimized\ud method was successfully applied to the determination of the enantiomers of arginine,\ud lysine, and ornithine in food samples of different complexities (dietary supplements and\ud wines).The authors thank the Ministry of Education and Science (Spain) and the Comunidad Autónoma de Madrid (Spain) for research projects CTQ2006-03849/BQU and S-0505/AGR- 0312, respectively. Carmen Garcı´a-Ruiz thanks the Ministry of Science and Technology (Spain) for the Ramon y Cajal program (RYC-2003-001). A.B.M.-G. thanks the University of Alcala for her pre-doctoral contract

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design

Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) analysis for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of experimentally measured kinetic isotope effects and hybrid QM/MM calculations. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, SN2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of positively charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogues to be used as, for example, chemical biology tools and pharmaceutical lead compounds

MIANN models in medicinal, physical and organic chemistry

[Abstract] Reducing costs in terms of time, animal sacrifice, and material resources with computational methods has become a promising goal in Medicinal, Biological, Physical and Organic Chemistry. There are many computational techniques that can be used in this sense. In any case, almost all these methods focus on few fundamental aspects including: type (1) methods to quantify the molecular structure, type (2) methods to link the structure with the biological activity, and others. In particular, MARCH-INSIDE (MI), acronym for Markov Chain Invariants for Networks Simulation and Design, is a well-known method for QSAR analysis useful in step (1). In addition, the bio-inspired Artificial-Intelligence (AI) algorithms called Artificial Neural Networks (ANNs) are among the most powerful type (2) methods. We can combine MI with ANNs in order to seek QSAR models, a strategy which is called herein MIANN (MI & ANN models). One of the first applications of the MIANN strategy was in the development of new QSAR models for drug discovery. MIANN strategy has been expanded to the QSAR study of proteins, protein-drug interactions, and protein-protein interaction networks. In this paper, we review for the first time many interesting aspects of the MIANN strategy including theoretical basis, implementation in web servers, and examples of applications in Medicinal and Biological chemistry. We also report new applications of the MIANN strategy in Medicinal chemistry and the first examples in Physical and Organic Chemistry, as well. In so doing, we developed new MIANN models for several self-assembly physicochemical properties of surfactants and large reaction networks in organic synthesis. In some of the new examples we also present experimental results which were not published up to date.Ministerio de Ciencia e Innovación; CTQ2009-07733Universidad del Pais Vasco; UFI11/22Universidad del Pais Vasco; GIU 094