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Paclitaxel (PTX) is a widely used chemotherapeutic agent in the clinic. However, its clinical benefit is limited due to its low water solubility, off-target toxicity, and for being a multidrug-resistant (MDR) substrate. To overcome these limitations in this study, a tumor-targeting peptide (CRGDK peptide, a ligand for NRP-1 receptor) conjugate of α-tocopheryl succinate (α-TOS) was synthesized and modified on PTX-loaded lipid aggregate (TL-PTX) to leverage the benefits of α-TOS, which include a) anti-cancer activity, b) increased PTX loading, and c) inhibition of MDR activity. Use of peptide conjugate of α-TOS (α-TOS-CRGDK) in lipid aggregate increased PTX entrapment efficiency by 20%, helped in NRP-1 specific cellular uptake and significantly enhanced apoptotic and cell killing activity (p0.01) of PTX compared to control formulation (CL-PTX) by inhibiting MDR-activity in melanoma resulting in ~70% increment in overall survival of melanoma tumor-bearing mice. In conclusion, CRGDK- α-TOS conjugate in association with PTX-loaded liposome provided a unique NRP-1 targeted, drug-resistant, reversing anticancer regimen for treating aggressive melanoma.

Rationale Nintedanib, an oral, triple angiokinase inhibitor, is used alongside docetaxel in the management of locally recurrent non-small-cell lung cancer and idiopathic pulmonary fibrosis. The present study deals with the identification and characterization of in vitro and in vivo stable and reactive (if any) metabolites of nintedanib and sheds light on some novel metabolites of the drug which have not been reported previously. Methods The study involved an oral administration of the drug to male Wistar rats, followed by collection of the biological matrices (urine, plasma and feces) at specific intervals for determination of in vivo metabolites. In addition, in vitro studies were performed on human and rat liver microsomes in the presence of appropriate co-factors. The samples were subjected to protein precipitation and nitrogen evaporation prior to ultra-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry analysis. The toxicities of all the metabolites were assessed in silico, employing ADMET Predictor™. Results A total of 18 metabolites of nintedanib were identified in all the matrices, of which nine were found to be novel and unreported previously. The unreported metabolites were elucidated as oxidative, demethylated and glucuronide conjugates of nintedanib. Interestingly, acetonitrile adducts of a few metabolites (low concentration) were also observed. No reactive metabolites were observed in this study. Conclusions Characterization of hitherto unknown in vitro and in vivo metabolites of nintedanib adds to the existing knowledge on the metabolism of the drug. Identification on the basis of the solvated adducts can be a useful approach for characterization of minor metabolites, which remain undetected owing to sensitivity issues.

The detection of post-translational modifications of proteins is an important comprehensive research area. Over the years, proteomic studies involving protein acetylation have attracted a great deal of attention. In the present study, we have focussed on the acetylation of histidine and the intrinsic stability of b1-ion of oxazolone ring and/or with side chain imidazole bicyclic product. The formation of oxazolone structure may occur when an amino moiety undergoes acetylation reaction and when it is present in the vicinity of the side chain imidazole moiety. Tryptic peptides generated from the proteins of Acenitobacter radioresistens MMC5-containing N-terminal histidine were explored in a standard proteomic workflow. Formation of [Formula: see text] ion with an oxazolone ring in these peptides has been supported by a tandem mass spectrometric study of a synthetic peptide and density functional theory calculations. The results obtained from this study have implications in understanding the fragmentation of the peptides generated in the proteomic workflows.

Guanine rich regions in DNA, which can form highly stable secondary structures, namely, G-quadruplex or G4 DNA structures, affect DNA replication and transcription. Molecules that stabilize G4 DNA have become important in recent years. In this study, G4 DNA stabilization, inhibition of telomerase, and anticancer activity of synthetic β-carboline–benzimidazole derivatives (5a, 5d, 5h, and 5r) were studied. Among them, derivatives containing a 4-methoxyphenyl ring at C1 and a 6-methoxy-substituted benzimidazole at C3 (5a) were found to stabilize telomeric G-quadruplex DNA efficiently. The stoichiometry and interaction of a synthetic, β-carboline–benzimidazole derivative, namely, 3-(6-methoxy-1H-benzo[d]imidazol-2-yl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole (5a), with human intermolecular G-quadruplex DNA at low concentrations were examined using electrospray ionization mass spectrometry. Spectroscopy techniques indicate that 5a may intercalate between the two stacks of G-quadruplex DNA. This model is sup...

Aims Doxorubicin is a widely used anthracycline derivative anticancer drug. Unfortunately, the clinical use of doxorubicin has the serious drawback of cardiotoxicity. In this study, we investigated whether baicalein, a bioflavonoid, can prevent doxorubicin-induced cardiotoxicity in vivo and we delineated the possible underlying mechanisms. Main methods Male BALB/c mice were treated with either intraperitoneal doxorubicin (15 mg/kg divided into three equal doses for 15 days) and/or oral baicalein (25 and 50 mg/kg for 15 days). Serum markers of cardiac injury, histology of heart, parameters related to myocardial oxidative stress, apoptosis and inflammation were investigated. Key findings Treatment with baicalein reduced doxorubicin-induced elevation of serum creatine kinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and ameliorated the histopathological damage. Baicalein restored the doxorubicin-induced decrease in both enzymatic and non-enzymatic myocardial antioxidants and increased the myocardial expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Further studies showed that baicalein could inverse the Bax/Bcl-2 ratio, suppress doxorubicin-induced p53, cleaved caspase-3 and PARP expression and prevented doxorubicin-induced DNA damage. Baicalein treatment also interferes with doxorubicin-induced myocardial NF-κB signaling through inhibition of IκBα phosphorylation and nuclear translocation of p65 subunit. Doxorubicin elevated iNOS and nitrites levels were also significantly decreased in baicalein treated mice. However, we did not find any significant change (p > 0.05) in the myocardial TNF-α and IL-6 levels in control and treated animals. Significance Our finding suggests that baicalein might be a promising molecule for the prevention of doxorubicin-induced cardiotoxicity.

Rationale The collisional-induced dissociations (CID) of the [M+H]+ ions of molecules having benzyl groups attached to N-atoms have been proposed to involve migration of the benzyl group through the intermediacy of ion/neutral complexes (INCs). We report the investigation of the mechanism of dissociation of protonated N-benzyl- and N-(1-phenylethyl)tyrosine amides by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and density functional theory (DFT) calculations. Methods The amides were synthesized from the corresponding amino acids and amines. The ESI-MS/MS spectra were recorded using an Agilent QTOF 6540 mass spectrometer. The DFT calculations were performed by using Gaussian 09 software. The structures of the [M+H]+ ions, intermediates, products and transition states (TS) were optimized at the B3LYP/6-31G(d,p) level of theory. Results CID of the [M+H]+ ions of N-benzyltyrosine amide yields two product ions due to rearrangements: (i) the [M+H–74]+ ion (m/z 197) due to benzyl migration to the hydroxyphenyl ring and (ii) the [M+H–45]+ ion (m/z 226) due to benzyl migration to the NH2 group. DFT calculations suggest that the rearrangements occur through an INC in which the benzyl cation is the cation partner. The [M+H]+ion of N-(1-phenylethyl)tyrosine amide rearranges to an INC of the 1-phenylethyl cation. Subsequent elimination of styrene occurs by transfer of a proton from the 1-phenylethyl cation to the neutral partner. Conclusions The [M+H]+ ions of both N-benzyl (1) and N-(1-phenylethyl) (2) tyrosine amide rearrange into INCs. The dissociation of [M+H]+ ion of 1 yields the benzyl cation and [M+H–74]+ and [M+H–45]+ due to benzyl migration to the hydroxyphenyl ring and NH2 group, respectively. However, the formation of the [M+H–74]+ ion is not observed when the aromatic ring is deactivated. The [M+H]+ ion of 2 either dissociates to form the 1-phenylethyl cation or [M+H–styrene]+. Copyright © 2015 John Wiley & Sons, Ltd.

Ondansetron hydrochloride was subjected to forced degradation studies under various conditions of hydrolysis (acidic, basic, and neutral), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A simple, selective, precise, and accurate high-performance liquid chromatography method was developed on a Waters Xterra C18 (150 × 4.6 mm id, 3.5 μm) column using 10 mM ammonium formate (pH 3.0)/methanol as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. The method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry for identification and structural characterization of stress degradation products of ondansetron. The drug showed significant degradation in base hydrolytic and photolytic stress conditions in the liquid state, while it was found to be stable in neutral, acidic, thermal, and oxidative stress conditions. A total of five degradation products were characterized and most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H](+) ions of the drug and its degradation products. Finally, the developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonisation guideline Q2 (R1).

Rationale McLafferty rearrangements occur in radical cations of molecules containing a carbonyl group and a γ hydrogen atom but are not common in the [M+H]+ ions of carbonyl compounds. We propose to investigate the collision-induced dissociation (CID) of the [M+H]+ ions of nicotinoyl and picolinoyl amides of 1- and 2-phenylethylamines to explore the possibility of McLafferty-type rearrangement. Methods The compounds for study were synthesized by the reaction of methyl nicotinate or methyl picolinate with 1- and 2-phenylethylamines. The CID mass spectra of electrospray ionization (ESI)-generated protonated molecules were obtained using a QSTAR XL quadrupole time-of-flight (QTOF) mass spectrometer, and density functional theory (DFT) calculations using the B3LYP method were employed to elucidate the fragmentation mechanisms. The total electronic and thermal energies of intermediate transition states (TSs) and product ions are reported relative to those of the [M+H]+ ions. Results CID of the [M+H]+ ions of N-[nicotinoyl]-2-phenylethylamine (1) yielded product ions of m/z 105 (1-phenylethyl cation) and 123 ([M+H–styrene]+ cation). The competitive formation of the ions of m/z 123 and 105 is proposed to involve a McLafferty-type rearrangement. Similarly, the [M+H]+ ions of the isomeric compound 2 and the N-[picolinoyl] phenylethyl amines (3 and 4) dissociate to yield ions of m/z 123 and 105. Conclusions A molecule of styrene was eliminated from the ESI-generated [M+H]+ ions of N-[nicotinoyl]phenylethylamines and the isomeric N-[picolinoyl]phenylethylamines, through a mechanism involving a McLafferty-type 1,5-H shift. The transition state energy for the 1,5-H shift is less for the amides of 1-phenylethylamine than for the amides of 2-phenylethylamine. The process occurs as a charge remote process and the presence of the pyridine ring is essential for the process. Copyright © 2015 John Wiley & Sons, Ltd.

DPP-4 inhibitors (gliptins) have shown better glycaemic control even in more feeble cases such as elderly people, patients with high cardiovascular and hypoglycaemic risk and individuals with renal impairment. However, the plasma concentrations of selected drugs has to be estimated to correlate those results with various uncertainties during clinical studies. A simple and sensitive LC-QTOF/MS method was developed and validated to measure the human plasma concentrations of vildagliptin, saxagliptin, sitagliptin, linagliptin and teneligliptin, using pioglitazone as an internal standard. Chromatographic separation of five gliptins was achieved on a Zorbax Eclipse Plus C-18 column Rapid Resolution HD (50 × 2.1 mm, 1.8 μm) using a mobile phase consisting of 20 mM ammonium formate and acetonitrile in gradient mode. Detection was performed with positive ion electrospray ionization mass spectrometry using target ions in selective ion mode. Simpler protein precipitation was employed for sample extraction from human plasma. The low recovery of gliptins observed due to non-specific binding to glass was surpassed by using polypropylene. The mean recovery was found to be 96.82 ± 1.03% (VIL), 94.32 ± 0.74% (SAX), 95.37 ± 2.09% (SIT), 91.67 ± 3.14% (LIN) and 93.29 ± 1.03% (TEN) respectively. The proposed method will serve as an excellent tool for various clinical studies like therapeutic drug monitoring, pharmacokinetics, toxicological and protein binding studies.

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality-by-design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters--column temperature, gradient time, and flow rate using eight experiments--to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused-core C18 (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry.

In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T2AG3) and d(T2AG3)2 sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV-Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T2AG3) and d(T2AG3)2 G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.

Gentamicin-induced nephrotoxicity has been well documented, although its underlying mechanisms and preventive strategies remain to be investigated. The present study was designed to investigate the protective effect of naringin, a bioflavonoid, on gentamicin-induced nephrotoxicity and to elucidate the potential mechanism. Serum specific renal function parameters (blood urea nitrogen and creatinine) and histopathology of kidney tissues were evaluated to assess the gentamicin-induced nephrotoxicity. Renal oxidative stress (lipid peroxidation, protein carbonylation, enzymatic and non-enzymatic antioxidants), inflammatory (NF-kB [p65], TNF-α, IL-6 and MPO) and apoptotic (caspase 3, caspase 9, Bax, Bcl-2, p53 and DNA fragmentation) markers were also evaluated. Significant decrease in mitochondrial NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity indicated the gentamicin-induced mitochondrial dysfunction. Naringin (100mg/kg) treatment along with gentamicin restored the mitochondrial function and increased the renal endogenous antioxidant status. Gentamicin induced increased renal inflammatory cytokines (TNF-α and IL-6), nuclear protein expression of NF-κB (p65) and NF-κB-DNA binding activity and myeloperoxidase (MPO) activity were significantly decreased upon naringin treatment. In addition, naringin treatment significantly decreased the amount of cleaved caspase 3, Bax, and p53 protein expression and increased the Bcl-2 protein expression. Naringin treatment also ameliorated the extent of histologic injury and reduced inflammatory infiltration in renal tubules. U-HPLS-MS data revealed that naringin co-administration along with gentamicin did not alter the renal uptake and/or accumulation of gentamicin in kidney tissues. These findings suggest that naringin treatment attenuates renal dysfunction and structural damage through the reduction of oxidative stress, mitochondrial dysfunction, inflammation and apoptosis in the kidney.

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RPâHPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C18 column (250 mmÃ4.6 mm i.d., 5 µm) with a mobile phase containing a gradient mixture of solvents, A (0.05% trifluoroacetic acid (TFA), pH=3.0) and B (acetonitrile). The eluted compounds were monitored at 254 nm; the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of >1.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LCâMS/MS and their fragmentation pathways were proposed. Keywords: Zotepine, Stability-indicating RPâHPLC method, Characterization, ESI-Q-TOF-MS, Bulk drugs and formulations

An isocratic reversed phase ultra performance liquid chromatography (RP-UPLC) method was developed for screening counterfeit medicines with UV detection at 210 nm. Chromatographic separation was performed on a Waters BEH C-18 column (50 × 2.1 mm, i.d., 1.7 μm particle size) with isocratic elution of a mobile phase containing a mixture of acetonitrile–methanol–ammonium formate buffer (0.01 M) (31 : 30.5 : 38.5 v/v/v) with a flow rate of 0.4 mL min−1 and pH 3 (adjusted with formic acid). A mixture design methodology was selected for the optimization and validation of the mobile phase composition. It was a trade-off between the experimental designs by graphical optimization of the technique using an overlay plot. In addition, the method validation was done as per the ICH guidelines using linearity, accuracy, precision, system suitability and robustness as parameters. The developed method was found to be sensitive, simple and highly robust for routine analysis and counterfeit detection of four selected drugs.

The electrospray ionization collisionally activated dissociation (CAD) mass spectra of protonated 2,4,6-tris(benzylamino)-1,3,5-triazine (1) and 2,4,6-tris(benzyloxy)-1,3,5-triazine (6) show abundant product ion of m/z 181 (C14H13+). The likely structure for C14H13+ is α-[2-methylphenyl]benzyl cation, indicating that one of the benzyl groups must migrate to another prior to dissociation of the protonated molecule. The collision energy is high for the ‘N’ analog (1) but low for the ‘O’ analog (6) indicating that the fragmentation processes of 1 requires high energy. The other major fragmentations are [M + H-toluene]+ and [M + H-benzene]+ for compounds 1 and 6, respectively. The protonated 2,4,6-tris(4-methylbenzylamino)-1,3,5-triazine (4) exhibits competitive eliminations of p-xylene and 3,6-dimethylenecyclohexa-1,4-diene. Moreover, protonated 2,4,6-tris(1-phenylethylamino)-1,3,5-triazine (5) dissociates via three successive losses of styrene. Density functional theory (DFT) calculations indicate that an ion/neutral complex (INC) between benzyl cation and the rest of the molecule is unstable, but the protonated molecules of 1 and 6 rearrange to an intermediate by the migration of a benzyl group to the ring ‘N’. Subsequent shift of a second benzyl group generates an INC for the protonated molecule of 1 and its product ions can be explained from this intermediate. The shift of a second benzyl group to the ring carbon of the first benzyl group followed by an H-shift from ring carbon to ‘O’ generates the key intermediate for the formation of the ion of m/z 181 from the protonated molecule of 6. The proposed mechanisms are supported by high resolution mass spectrometry data, deuterium-labeling and CAD experiments combined with DFT calculations. Copyright © 2012 John Wiley & Sons, Ltd.

A highly sensitive, selective and rapid liquid chromatography–electrospray ionization mass spectrometry (LC-MS) method has been developed and validated for simultaneous determination of moxifloxacin (MFX) and ketorolac (KTC) in rat plasma. Gemifloxacin (GFX) was used as an internal standard (IS). A simple protein precipitation method was used for the extraction of analytes from rat plasma. Effective chromatographic separation of MFX, KTC and GFX was achieved on a Kromasil C18 column (100 × 4.6 mm, 5 µm) using a mobile phase consisting of acetonitrile–10 mm ammonium acetate (pH 2.5)–0.1% formic acid (50:25:25) in an isocratic elution, followed by detection with positive ion electrospray ionization mass spectrometry using target ions of [M + H]+ at m/z 402 for MFX, m/z 256 for KTC and m/z 390 for GFX in selective ion recording mode. The method was validated over the calibration range of 5–100 ng/mL for MFX and 10–6000 ng/mL for KTC. The method demonstrated good performances in terms of intra- and inter-day precision (0.97–5.33%) and accuracy (93.91–101.58%) for both MFX and KTC, including lower and upper limits of quantification. The recoveries from spiked control samples were >75% for MFX and >79% for KTC. The matrix effect was found to be negligible and the stability data were within acceptable limits. Further, the method was also successfully applied to a single-dose pharmacokinetic study in rats. This method can be extended to measure plasma concentrations of both drugs in human to understand drug interaction and adverse effects. Copyright © 2012 John Wiley & Sons, Ltd.

A series of positional isomeric pairs of Fmoc-protected dipeptides, Fmoc-Gly-Xxx-OY/Fmoc-Xxx-Gly-OY (Xxx = Ala, Val, Leu, Phe) and Fmoc-Ala-Xxx-OY/Fmoc-Xxx-Ala-OY (Xxx = Leu, Phe) (Fmoc = (9-fluorenylmethyl) oxycarbonyl) and Y = CH3/H), have been characterized and differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MSn). In contrast to the behavior of reported unprotected dipeptide isomers which mainly produce y 1 + and/or a1 + ions, the protonated Fmoc-Xxx-Gly-OY, Fmoc-Ala-Xxx-OY and Fmoc-Xxx-Ala-OY yield significant b 1 + ions. These ions are formed, presumably with stable protonated aziridinone structures. However, the peptides with Gly- at the N-terminus do not form b1 + ions. The M + H+ ions of all the peptides undergo a McLafferty-type rearrangement followed by loss of CO2 to form M + H-Fmoc + H+. The MS3 collision-induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this work. Further, negative ion MS 3 CID has also been found to be useful for differentiating these isomeric peptide acids. The MS3 of M-H-Fmoc + H- of isomeric peptide acids produce c1 -, z1 - and y1 - ions. Thus the present study of Fmoc-protected peptides provides additional information on mass spectral characterization of the dipeptides and distinguishes the positional isomers. Copyright © 2011 John Wiley & Sons, Ltd.

Four pairs of positional isomers of ureidopeptides, FmocNH-CH(R(1))-ϕ(NH-CO-NH)-CH(R(2))-OY and FmocNH-CH(R(2))-ϕ(NH-CO-NH)-CH(R(1))-OY (Fmoc = [(9-fluorenyl methyl)oxy]carbonyl; R(1) = H, alkyl; R(2) = alkyl, H and Y = CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). The major fragmentation noticed in MS/MS of all these compounds is due to --N--CH(R)--N--bond cleavage to form the characteristic N- and C-terminus fragment ions. The protonated ureidopeptide acids derived from glycine at the N-terminus form protonated (9H-fluoren-9-yl)methyl carbamate ion at m/z 240 which is absent for the corresponding esters. Another interesting fragmentation noticed in ureidopeptides derived from glycine at the N-terminus is an unusual loss of 61 units from an intermediate fragment ion FmocNH = CH(2) (+) (m/z 252). A mechanism involving an ion-neutral complex and a direct loss of NH(3) and CO(2) is proposed for this process. Whereas ureidopeptides derived from alanine, leucine and phenylalanine at the N-terminus eliminate CO(2) followed by corresponding imine to form (9H-fluoren-9-yl)methyl cation (C(14)H(11) (+)) from FmocNH = CHR(+). In addition, characteristic immonium ions are also observed. The deprotonated ureidopeptide acids dissociate differently from the protonated ureidopeptides. The [M - H](-) ions of ureidopeptide acids undergo a McLafferty-type rearrangement followed by the loss of CO(2) to form an abundant [M - H - Fmoc + H](-) which is absent for protonated ureidopeptides. Thus, the present study provides information on mass spectral characterization of ureidopeptides and distinguishes the positional isomers.

A cyclic tetrapeptide is prepared from alternating (S)-beta-Caa (C-linked carbo-beta-amino acid) and (R)-Ama (alpha-aminoxy acid). Extensive NMR (in CDCl(3) solution) and mass spectral (MS) studies show its halide binding capacity, with a special affinity to the chloride ion. At higher concentration it was found to form molecular aggregates as evidenced from transmission electron microscopic and atomic force microscopic analysis, confirming the formation of nanorods.

A new class of diastereomeric pairs of non-natural amino acid peptides derived from butyloxycarbonyl (Boc-)protected cis-(2S,3R)- and trans-(2S,3S)-beta-norbornene amino acids including a monomeric pair have been investigated by electrospray ionization (ESI) tandem mass spectrometry using quadrupole time-of-flight (Q-TOF) and ion-trap mass spectrometers. The protonated cis-BocN-beta-nbaa (2S,3R) (1) (betanbaa = beta-norbornene amino acid) eliminates the Boc group to form [M+H-Boc+H](+), whereas an additional ion [M+H-C(4)H(8)](+) is formed from trans-BocN-beta-nbaa (2S,3S) (2). Similarly, it is observed that the peptide diastereomers (di-, tri- and tetra-), with cis-BocN-beta-nbaa (2S,3R)- at the N-terminus, initially eliminate the Boc group to form [M+H-Boc+H](+) which undergo further fragmentation to give a set of product ions that are different for the peptides with trans-BocN-beta-nbaa (2S,3S)- at the N-terminus. Thus the Boc group fragments differently depending on the configuration of the amino acid present at the N-terminus. It is also observed that the peptide bond cleavage in these peptides is less favoured and most of the product ions are formed due to retro-Diels-Alder fragmentation. Interestingly, sodium-cationized peptide diastereomers mainly yield a series of retro-Diels-Alder fragment ions which are different for each diastereomer as they are formed starting from [M+Na-Boc+H](+) in peptides with cis-BocN-beta-nbaa (2S,3R)- at the N-terminus, and [M+Na-C(4)H(8)](+) in peptides with trans-BocN-beta-nbaa (2S,3S)- at the N-terminus. All these results clearly indicate that these diastereomeric pairs of peptides yield characteristic product ions which help distinguish the isomers.

Upon CA, ESI generated [M + H](+) ions of chalcone (benzalacetophenone) and 3-phenyl-indanone both undergo losses of H(2)O, CO, and the elements of benzene. CA of the [M + H](+) ions of 2-methoxy and 2-hydroxychalcone, however, prompts instead a dominant loss of ketene. In addition, CA of the [M + H](+) ions of 2-methoxy-beta-methylchalcone produces an analogous loss of methylketene instead. Furthermore, the [M + D](+) ion of 2-methoxychalcone upon CA eliminates only unlabeled ketene, and the resultant product, the [M + D - ketene](+) ion, yields only the benzyl-d(1) cation upon CA. We propose that the 2-methoxy and 2-hydroxy (ortho) substituents facilitate a Nazarov cyclization to the corresponding protonated 3-aryl-indanones by mediating a critical proton transfer. The resultant protonated indanones then undergo a second proton transport catalysis facilitated by the same ortho substituents producing intermediates that eliminate ketene to yield 2-methoxy- or 2-hydroxyphenyl-phenyl-methylcarbocations, respectively. The basicity of the ortho substituent is important; for example, replacement of the ortho function with a chloro substituent does not provide an efficient catalyst for the proton transports. The Nazarov cyclization must compete with an alternate cyclization, driven by the protonated carbonyl group of the chalcone that results in losses of H(2)O and CO. The assisted proton transfer mediated by the ortho substituent shifts the competition in favor of the Nazarov cyclization. The proposed mechanisms for cyclization and fragmentation are supported by high-mass resolving power data, tandem mass spectra, deuterium labeling, and molecular orbital calculations.

The fragmentations of protonated and deprotonated ions of a new class of N-blocked hybrid Boc-carbopeptides containing repeats of gamma-Caa/gammaAbu- and beta-Caa/gammaAbu- (Caa==C-linked carbo gamma(4)-/beta(3)- amino acids derived from D-xylose, gammaAbu = gamma-aminobutyric acid) have been studied using electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS/MS of a pair of these protonated diastereomers produces distinct fragmentation of the Boc group. The formation of [M + H-56](+) corresponding to loss of isobutylene is more pronounced for Boc-NH-(R)-gamma-Caa-gammaAbu-OH (2) whereas it is of low abundance for Boc-NH-(S)-gamma-Caa-gammaAbu--OH (1). Similarly, MS(2) of [M--H](-) of 2 produces an abundant [M--H--C(CH(3))(3)OH--CO(2)](-) ion, which is absent for its diastereomeric isomer 1. From this, it can be suggested that MS/MS of N-blocked Boc-protected carbopeptides may be helpful in distinguishing the stereochemistry of the N-terminus Caa. MS(3) of [M + H-Boc + H](+) ions of peptides with a gamma-amino acid (gamma-Caa/gammaAbu) at the N-terminus produces only abundant y(n) (+) ions. On the other hand, characteristic fragmentations involving the peptide backbone (b(n) (+) and y(n) (+)) and the side chain are seen when beta-Caa is at the N-terminus of the peptides. MS(3) of the [M--H--C(CH(3))(3)OH](-) ion of peptides containing gamma-Caa/gammaAbu at the N-terminus gave y(n) (-) and [M--H--C(CH(3))(3)OH--CO(2)](-) ions, whereas the presence of beta-Caa at the N-terminus yielded predominantly [M--H--C(CH(3))(3)OH--HNCO](-). Thus, on the basis of our previous study and that presented here we propose that the fragmentation of these hybrid carbopeptides is highly influenced by the type of carbo amino acid present at the N-terminus.

Attempts have been made to characterize ionic and neutral C2H2S +/o isomers, viz., thioketene (1 +), ethynylthiol (2 +), and thiirene (3 +) using various tandem mass spectrometric methodologies. Dissociative electron ionization of thiophene, 1-propane thiol and thiirane were used to produce the C2H2S + (m/z 58) ions. The collisional activation (CA) mass spectrum of m/z 58 ions from thiophene is highly compatible with thioketene ions which has been supported by the CA mass spectrum of metastably generated m/z 58 ions and by comparison with the previously reported CA mass spectrum of thioketene ions. Theoretical calculations at the B3LYP/6-311G(d,p), and G2/G2(MP2) levels predict that low energy thioketene ions equilibrate with other isomeric ions viz., ethynylthiol and thiirene prior to dissociation by loss of H (m/z 57) and CH (m/z 45). The CA spectra of C2H2S + ions from 1-propane thiol and thiirane are nearly identical indicating a facile isomerization of the initial structures at energies lower than the dissociative threshold. The low energy CID spectra of the m/z 58 ions from three precursors are found to be similar. However, significant differences are noticed in the relative abundances of product ions in the NR spectra of m/z 58 ions from the three precursors. While the NR mass spectra of m/z 58 ions from thiophene and thiirane confirms the stability of thioketene neutrals, the NR spectra of m/z 58 ions from 1-propane thiol suggests formation of a mixture of ethynylthiol and thioketene ions. Calculations predict a different thermo-chemistry for 1 +–3 +, but, due to a mixture of reacting isomeric ions whatever the precursor molecule is, similar results were obtained for the associative ion/molecule reactions between the C2H2S + ions with methylisocyanide.

The collisional-induced dissociations (CID) of the [M+H]The amides were synthesized from the corresponding amino acids and amines. The ESI-MS/MS spectra were recorded using an Agilent QTOF 6540 mass spectrometer. The DFT calculations were performed by using Gaussian 09 software. The structures of the [M+H]CID of the [M+H]The [M+H]

Protonated silanoic acid, HSi(OH) 2 +, 1a + , is cleanly generated by the dissociative electron ionization of triethoxysilane, HSi(OC 2 H 5 ) 3 , and tetraethoxysilane, Si(OC 2 H 5 ) 4 . This follows from tandem mass spectrometric experiments and CBS-QB3 model chemistry calculations. The calculations predict that 1a + (ΔH f (298 K) = 205 kJ mol -1 ) is separated by high barriers from its isomers HOSiOH 2 +, 1b + and HSi(=O)OH 2 +, 1c + . Low-energy (metastable) ions 1a + dissociate by loss of H 2 O via the pathway 1a + → 1b + → SiOH + +H 2 O. Analysis of the metastable peak for this process confirms that the isomerization step 1a + → 1b + is rate determining. The calculations further predict that the incipient ions 1b + communicate via a low barrier with the proton-bound dimer Si=O...H...OH 2 +, 1d + . This dimer ion is much lower in energy than its counterpart O=Si...H...OH 2 +, 1e + , which is calculated to be only marginally stable. A comparison of the potential energy diagram for the silicon-containing ions 1a + -1e + with that of their carbon analogues reveals that the dissociation chemistries of HSi(OH) 2 + and HC(OH) 2 + are only superficially similar. Neutralization-reionization experiments confirm the theoretical prediction that the HSi(OH) 2 * radical (ΔH f (298 K) = -455 kJ mol -1 ) is a stable species in the rarefied gas phase. However, owing to a mismatch of Franck-Condon factors a large fraction of the neutralized ions dissociates by loss of H* yielding Si(OH) 2 .

The reaction of 1:1 zwitterionic intermediate generated in situ from dimethyl acetylenedicarboxylate (DMAD) and cyclohexyl isocyanide with aldehydes and quinones is described. The reaction of stoichiometric amounts of DMAD, isocyanide and aldehydes afforded 2-aminofurans in good yields, while the reaction with quinones gave iminolactones.

The zwitterion generated from diisopropylaminoisocyanide and dimethyl acetylenedicarboxylate (DMAD) reacts with a variety of carbonyl and dicarbonyl compounds affording substituted 1-aminopyrrolin-2-ones.

McLafferty rearrangements occur in radical cations of molecules containing a carbonyl group and a γ hydrogen atom but are not common in the [M+H](+) ions of carbonyl compounds. We propose to investigate the collision-induced dissociation (CID) of the [M+H](+) ions of nicotinoyl and picolinoyl amides of 1- and 2-phenylethylamines to explore the possibility of McLafferty-type rearrangement.The compounds for study were synthesized by the reaction of methyl nicotinate or methyl picolinate with 1- and 2-phenylethylamines. The CID mass spectra of electrospray ionization (ESI)-generated protonated molecules were obtained using a QSTAR XL quadrupole time-of-flight (QTOF) mass spectrometer, and density functional theory (DFT) calculations using the B3LYP method were employed to elucidate the fragmentation mechanisms. The total electronic and thermal energies of intermediate transition states (TSs) and product ions are reported relative to those of the [M+H](+) ions.CID of the [M+H](+) ions of N-[nicotinoyl]-2-phenylethylamine (1) yielded product ions of m/z 105 (1-phenylethyl cation) and 123 ([M+H-styrene](+) cation). The competitive formation of the ions of m/z 123 and 105 is proposed to involve a McLafferty-type rearrangement. Similarly, the [M+H](+) ions of the isomeric compound 2 and the N-[picolinoyl] phenylethyl amines (3 and 4) dissociate to yield ions of m/z 123 and 105.A molecule of styrene was eliminated from the ESI-generated [M+H](+) ions of N-[nicotinoyl]phenylethylamines and the isomeric N-[picolinoyl]phenylethylamines, through a mechanism involving a McLafferty-type 1,5-H shift. The transition state energy for the 1,5-H shift is less for the amides of 1-phenylethylamine than for the amides of 2-phenylethylamine. The process occurs as a charge remote process and the presence of the pyridine ring is essential for the process.

Background Telomeric and NHE III1, a c-MYC promoter region is abundant in guanine content and readily form G-quadruplex structures. Small molecules that stabilize G-quadruplex DNA were shown to reduce oncoprotein expression, initiate apoptosis and they may function as anticancer molecules. Methods Electrospray ionization mass spectrometry, spectroscopy, isothermal titration calorimetry, Taq DNA polymerase stop assay, real time PCR and luciferase reporter assay. Cell migration assay to find out the effect of derivatives on normal as well as cancer cell proliferation. Results Among three different dihydroindolizino indole derivatives, 4-cyanophenyl group attached derivative has shown maximum affinity, selective interaction and higher stability towards G-quadruplex DNA over dsDNA. Further, as a potential G-quadruplex DNA stabilizer, 4-cyanophenyl linked dihydroindolizino indole derivative was found to be more efficient in inhibiting in vitro DNA synthesis, c-MYC expression and cancer cell proliferation among human cancer cells. Conclusion The present study reveals that dihydroindolizino indole derivative having 4-cyanophenyl group has potential to stabilize G-quadruplex DNA and exhibit anticancer activity. General significance These studies are useful in the identification and synthesis of lead derivatives that will selectively stabilize G-quadruplex DNA and function as anticancer agents.

Background Cardiovascular complication due to diabetes has remained a major cause of death. There is an urgent need to intervene the cardiac complications in diabetes by nutritional or pharmacological agents. Thus the present study was designed to find out the effectiveness of garlic on cardiac complications in insulin-resistant diabetic rats. Methods and Results SD rats were fed high fructose (65%) diet alone or along with raw garlic homogenate (250 mg/kg/day) or nutrient-matched (65% corn starch) control diet for 8 weeks. Fructose-fed diabetic rats showed cardiac hypertrophy, increased NFkB activity and increased oxidative stress. Administration of garlic significantly decreased (p

ESI-protonated 1,5-bis-(2-methoxyphenyl)-1,4-pentadien-3-one (1) undergoes a gas-phase Nazarov cyclization and dissociates via expulsions of ketene and anisole. The dissociations of the [M + D](+) ions are accompanied by limited HD scrambling that supports the proposed cyclization. Solution cyclization of 1 was effected to yield the cyclic ketone, 2,3-bis-(2-methoxyphenyl)-cyclopent-2-ene-1-one, (2) on a time scale that is significantly shorter than the time for cyclization of dibenzalacetone. The dissociation characteristics of the ESI-generated [M + H](+) ion of the synthetic cyclic ketone closely resemble those of 1, suggesting that gas-phase and solution cyclization products are the same. Additional mechanistic studies by density functional theory (DFT) methods of the gas-phase reaction reveals that the initial cyclization is followed by two sequential 1,2-aryl migrations that account for the observed structure of the cyclic product in the gas phase and solution. Furthermore, the DFT calculations show that the methoxy group serves as a catalyst for the proton migrations necessary for both cyclization and fragmentation after aryl migration. An isomer formed by moving the 2-methoxy to the 4-position requires relatively higher collision energy for the elimination of anisole, as is consistent with DFT calculations. Replacement of the 2-methoxy group with an OH shows that the cyclization followed by aryl migration and elimination of phenol occurs from the [M + H](+) ion at low energy similar to that for 1.