Rapid measurement of hydrogen sulphide in human blood plasma using a microfluidic method.

Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H2S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H2S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H2S (range 0-100 µM). We have measured the baseline level of endogenous H2S in healthy volunteers which was found to lie in the range of 70 µM - 125 µM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H2S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H2S in health and disease.

Broad spectrum anti-infective properties of benzisothiazolones and the parallels in their anti-bacterial and anti-fungal effects.

Various mono- and bis-benzisothiazolone derivatives were synthesized and screened against different strains of bacteria and fungi in order to understand the effect of multiple electrophilic sulfur atoms and substitution pattern in the immediate vicinity of reactive sulfur. Staphyllococcus aureus-ATCC 7000699, MRSA and S. aureus-ATCC 29213 (Quality Control strain) were more susceptible to this class of compounds, and the most potent derivative 1.15 had MIC50 of 0.4µg/mL (cf. Gentamicin=0.78µg/mL). CLogP value, optimally in the range of 2.5-3.5, appeared to contribute more to the activity than the steric and electronic effects of groups attached at nitrogen. By and large, their anti-fungal activities also followed a similar trend with respect to the structure and CLogP values. The best potency of IC50=0.1µg/mL was shown by N-benzyl derivative (1.7) against Aspergillus fumigatus; it was also potent against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, and Candida parapsilosis with IC50 values ranging from 0.4 to 1.3µg/mL. Preliminary studies also showed that this class of compounds have the ability to target malaria parasite with IC50 values in low micromolar range, and improvement of selectivity is possible through structure optimization.

Regio-selective lipase catalyzed hydrolysis of oxanorbornane-based sugar-like amphiphiles at air-water interface: A polarized FT-IRRAS study.

Interfacial hydrolysis of oxanorbornane-based amphiphile (Triol C16) by Candida rugosa lipase was investigated using real-time polarized Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS). The kinetics of hydrolysis was studied by analyzing the ester carbonyl ν(CO) stretching vibration band across the two dimensional (2D) array of molecules at the confined interface. In particular, we demonstrate Triol C16 to form Michaelis-Menten type complex, like that of lipid-substrate analogues, where the Triol C16 head group remained accessible to the catalytic triad of the lipase. The enzyme-induced selective cleavage of the ester bond was spectroscopically monitored by the disappearance of the intense ν(CO) resonance at 1736cm-1. Consequently, the in situ spectroscopic measurements evidenced selective ester hydrolysis of Triol C16 yielding Tetrol C2OH and Palmitic acid, which remained predominantly in the undissociated form at the interface. The conformation sensitive amide I (majorly ν(CO)) and the interfacial water reorganization suggested 2D ordering of the enzyme molecules following which interfacial reactions were employed towards probing the enzyme kinetics at the air/water interface. The investigation demonstrated further the potential of IRRAS spectroscopy for real-time monitoring the hydrolytic product formation and selectivity at biomimetic interfaces.

A modular approach towards drug delivery vehicles using oxanorbornane-based non-ionic amphiphiles.

The self-assembly of non-ionic amphiphiles with a hydroxylated oxanorbornane head-group was controlled using amino acid units as spacers between hydrophilic and lipophilic domains to get spherical supramolecular aggregates. The ability of these systems to harbour therapeutic agents like ibuprofen, and their drug-release profiles were evaluated. Apart from directing the assembly, the intervening amino acid unit was found to help in drug entrapment as well. The presence of cholesterol improved their drug-loading ability, and an encapsulation efficiency of up to 66% was shown by the formulation containing the phenylalanine residue as the spacer (NC1c). There was no burst release, and 45% drug release was observed at the end of 24 h in this case (cf. soyaphosphatidylcholine based formulation = 49%). The results from SEM, Cryo-TEM, PXRD and confocal microscopic studies with some insights into molecular packing in this class of aggregates are also included.

Determination of antimalarial compound, ARB-89 (7ß-hydroxy-artemisinin carbamate) in rat serum by UPLC/MS/MS and its application in pharmacokinetics.

Among all the antimalarial agents, artemisinin and its semi synthetic family of analogs are the most potent antimalarials available for the treatment of Plasmodium falciparum infections. But these analogs have a few issues such as shorter half-lives and low oral bioavailability values. In order to overcome these inherent problems, novel artemisinin analogs were synthesized from 7ß-hydroxy artemisinin by the Department of Medicinal Chemistry, University of Mississippi using a new synthesis mechanism. Out of all the 7ß-hydroxy artemisinin analogs synthesized, 7ß-hydroxy artemisinin carbamate (ARB-89) was chosen as a lead compound because of its high in vitro and in vivo activity. In this manuscript, a sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of ARB-89 in rat serum. The analysis was carried out on an Acquity™ UPLC BEH C(18) column (1.7 µm, 2.1 mm × 50 mm) with a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization (ESI) mode. The selected mass-to-charge (m/z) ratio transitions used in the multiple reaction monitoring (MRM) for ARB-89 and artemisinin (internal standard) were m/z 778.4>253.4 and m/z 283.4>151.1 respectively. The calibration curve was linear from 1.00 ng/mL to 10.0 µg/mL (r(2)=0.999). A simple protein precipitation method was used for extraction. Moreover, the inter-day and intra-day precision values were found to be less than 15%. The recoveries of the method ranged from 94.0% to 96.7% at three concentrations. ARB-89 in rat serum was found to be stable at room temperature for 12h. This method was successfully used to quantitate the novel antimalarial compound ARB-89 after intravenous and oral administration to rats.

Synthesis of beta-hydroxyphosphonate and 1,2-dihydroxy acyclic nucleoside analogs via 1,3-dipolar cycloaddition strategy.

A convenient synthetic approach toward nucleoside analogs where beta-hydroxyphosphonate- or 1,2-dihydroxy units are connected to the nucleic acid base through a triazole spacer is discussed.

Progress in the development of peroxide-based anti-parasitic agents.

Progress made in the past decade pertaining to the development of anti-parasitic agents based on artemisinin is presented. Apart from discussions on important derivatives obtained through functionalization at the C-3, C-9, C-10 and O-11 positions of artemisinin, an outline on its seco analogs and artemisinin bundles are given for a broader perspective on structure-activity relationships. Success with synthetic peroxides, drug-hybrid approaches, broad-spectrum anti-infective properties of peroxide compounds and a survey on peroxide-containing natural products other than artemisinin with available biological data are included to highlight recent trends and avenues for future research. A supplementary material with details on the biological properties of a larger collection of molecules belonging to the above structural classes is also given for reference.

Transformation of artemisinin by Cunninghamella elegans.

Semi-synthetic derivatives of the anti-malarial drug artemisinin hold great promise in the search for an effective and economical treatment of chloroquine-resistant forms of malaria. Unfortunately, synthetic functionalization of the artemisinin skeleton is often tedious and/or impractical. We seek to utilize 7beta-hydroxyartemisinin, obtained from microbial transformation, as a semi-synthetic precursor for the synthesis of novel 7beta-substituted artemisinin anti-malarial agents. Here we employ liquid cultures of Cunninghamella elegans as a means for the rational and economical bioconversion of artemisinin to 7beta-hydroxyartemisinin in 78.6% yield. In addition, there were three other bioconversion products: 7beta-hydroxy-9alpha-artemisinin (6.0%), 4alpha-hydroxy-1-deoxoartemisinin (5.4%), and 6beta-hydroxyartemisinin (6.5%).

Unusual cyclo-tetra and hexa peptidation of bis-boc-cystine with cystine-di-OMe: one step preparation of the novel 32- and 48-membered cyclotetracystine and cyclohexacystine.

The unprecedented formation of 32- and 48-membered macrocycles that inscribe 4 and 6 cystine units, in the peptidation of bis-Boc-cystine with cystine di-OMe is reported.

One-step transformation of tricyclopentabenzene (trindane, C(15)H(18)) to bicyclo(10.3.0)pentadec-1(12)ene- 2,6,7,11-tetrone (C(15)H(18)O(4)) and its aldol product, 12-hydroxy-16-oxatetracyclo(10.3.1.0.(1,5)0(7,11))hexadec-7(11)ene-2,6-dione (C(15)H(18)O(4)).

[reaction: see text] Ozonolysis of 1 largely results in 2 and 3, having features similar to several classes of natural products. The retention of the C(15) pericycle suggests preference for the cleavage of pi-bonds endo to the cyclopentane ring. This unique property of trindane offers opportunities for synthesis of complex natural products from this hydrocarbon that can be made in quantity by acid-catalyzed trimerization of cyclopentanone.