Site Of Alkylation Research Articles

Direct Activation of Human Phospholipase C by Its Well Known Inhibitor U73122

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Designing New β-Lactams: Implications from Their Targets, Resistance Factors and Synthesizing Enzymes

Penicillins and cephalosporins are β-lactam antibiotics widely used to treat bacterial infectious diseases. They mainly target the cell wall biosynthesis pathway to inhibit bacterial growth. The targets, known as penicillin-binding proteins, are enzymes involved in the polymerization of glycan chains, cross-linking them during bacterial cell wall formation. However, the dispensation of these antibiotics has been concomitant with increasing incidence of resistance to them. Reportedly, this is due to the evolvement of two resistance mechanisms in the bacterial pathogens. One is the production of β -lactamases that cleave the β -lactam rings of penicillin and cephalosporin antibiotics, rendering them ineffective against the pathogens. Another is the modification of the targets, resulting in their inability to bind β -lactam antibiotics. Nevertheless, β -lactam antibiotics remain clinically relevant due to their high target specificity in bacteria and low toxicity to humans. Thus, to overcome the continuing emergence of resistance in pathogens, more efficacious β -lactams have to be developed and cephalosporins are often preferred over penicillins due to two alkyl sites in the cephalosporin core structure amenable for modification. Transformed β -lactams are expected to have improved antimicrobial spectra and pharmacokinetics. This is illustrated by the development of two cephalosporins, namely ceftobiprole and ceftaroline, which have shown good antimicrobial activities and are currently undergoing clinical trials. This review will discuss computer-aided studies of three enzymes closely related to cephalosporins: (1) its synthesizing enzyme, deacetoxycephalosporin C synthase, (2) its targets, the penicillin-binding proteins, and (3) its degrading enzyme, the β -lactamases, and their implications in the development of new cephalosporins.

Ionic liquid crystals derived from 4-hydroxypyridine

Monoalkylation of 4-hydroxypyridine gave either neutral N-alkyl-4-pyridones or 4-alkoxypyridines depending on the site (N or O) of alkylation. Further alkylation of these two neutral compounds yielded ionic liquid crystals (ILCs) of N-alkyl-4-alkoxypyridinium bromide, having two long alkyl chains. Alternatively, simple protonation of the two neutral compounds with HCl also induced the formation of ILCs of N-alkyl-4-hydroxypyridinium chloride or 4-alkoxypyridinium hydrochloride. Crystal structures of two different types of ionic liquid crystals and one neutral compound were determined via single crystal X-ray diffraction. Mesophase properties were examined by the technique of differential scanning calorimetry, polarized optical microscopy, and powder X-ray diffractometry. Anions influence the structure and stability of the mesophase in the dialkylated compounds. The self-assembly behavior of some typical compounds in solution was studied using the technique of NMR diffusion order spectroscopy. Finally, stable Au nanoparticles stabilized by a pyridinium salt were prepared and characterized.

Reductive Alkylation Causes the Formation of a Molten Globule-Like Intermediate Structure in Geobacillus zalihae Strain T1 Thermostable Lipase

A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T(m)) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated.

Cell Cycle Arrest and Apoptosis Induction by an Anticancer Chalcone Epoxide

Safe and effective chemotherapeutic agents for the treatment of pancreatic cancer remain elusive. We found that chalcone epoxides (1,3-diaryl-2,3-epoxypropanones) inhibited growth in two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2. Three compounds were active, with GI(50) values of 5.6 to 15.8 microM. Compound 4a, 1,3-bis-(3,4,5-trimethoxyphenyl)-2,3-epoxypropanone, had an average GI(50) of 14.1 microM in the NCI 60-cell-line panel. To investigate the mode of action, cell cycle analyses of BxPC-3 cells were carried out. Treatment of cells with 50 microM 4a resulted in dramatic accumulation at G2/M (61% after 12 h for 4a vs. 15% for untreated cells). The cells rapidly entered apoptosis. After 12 h, 26% of cells treated with 50 microM 4a had entered apoptosis vs. 4% for cells treated with 100 microM etoposide and 2% for untreated cells. Compound 4a interfered with paclitaxel enhancement of tubulin polymerization, suggesting microtubules as the site of action. Reaction of thiol nucleophiles with 4a under basic conditions resulted in epoxide ring-opening and retroaldol fragmentation, yielding alkylated thiol. MALDI mass spectrometry showed that retroaldol reaction occurred upon treatment of beta-tubulin with 4a. The site of alkylation was identified as Cys(354). Chalcone epoxides warrant further study as potential agents for treatment of cancer.

Mutation spectra induced by adozelesin in the supF gene of human XP-A fibroblasts

Adozelesin is a synthetic analog of the antitumor antibiotic, CC-1065, which alkylates N3 of adenine in the minor DNA groove in a sequence-specific manner. Here we tested the mutation spectra induced by adozelesin in the supF gene of human XP-A fibroblasts using a shuttle vector assay. Adozelesin primarily induces mutations via an A --> T transversion and a single base insertion. The A --> T transversion (43/59) was observed at the adenine alkylation site in the 5'-ATTTA* sequence (A* is the site of alkylation). The single base insertion (5/59) was observed at the 3'-side of the covalently modified adenine in the 5'-CTAAA* sequence. The results of this study suggest that the DNA alkylating sequence of adozelesin influences the type of DNA mutation.

Fluorine Plasma Treatments of Poly(propylene) Films, 2 – Modeling Reaction Mechanisms and Scaling

AbstractThe surface properties of commodity hydrocarbon polymers such as poly(propylene) (PP) can be modified by functionalization with plasma‐generated radicals and ions. For example, affixing fluorine to a hydrocarbon surface lowers surface energy and increases hydrophobicity. One such process is treatment of PP films in low‐pressure, capacitively coupled plasmas (CCPs) sustained in F2‐containing gas mixtures. F atoms produced in the plasma abstract H atoms from the hydrocarbon and passivate the resulting alkyl sites producing CFn sites. Energetic ion and photon fluxes sputter and initiate crosslinking. In this paper, the plasma fluorination of PP in a CCP sustained in Ar/F2 is discussed with results from a two‐dimensional plasma hydrodynamics model. The surface reaction mechanism includes a hierarchy of H abstraction and F/F2 passivation reactions, as well as crosslinking, and ion and photon‐activated processes. Predictions for surface composition were compared to experiments. We found that the lack of total fluorination with long plasma exposure is likely caused by crosslinking, which creates CC bonds that might otherwise be passivated by F atoms. Increasing steric hindrances as fluorination proceeds also contribute to lower F/C ratios.magnified image

Molecular design of sequence specific DNA alkylating agents

Sequence-specific DNA alkylating agents have great interest for novel approach to cancer chemotherapy. We designed the conjugates between pyrrole (Py)-imidazole (Im) polyamides and DNA alkylating chlorambucil moiety possessing at different positions. The sequence-specific DNA alkylation by conjugates was investigated by using high-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The results showed that polyamide chlorambucil conjugates alkylate DNA at flanking adenines in recognition sequences of Py-Im polyamides, however, the reactivities and alkylation sites were influenced by the positions of conjugation. In addition, we synthesized conjugate between Py-Im polyamide and another alkylating agent, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). DNA alkylation reactivies by both alkylating polyamides were almost comparable. In contrast, cytotoxicities against cell lines differed greatly. These comparative studies would promote development of appropriate sequence-specific DNA alkylating polyamides against specific cancer cells.

Investigation of the transformations of a novel anti-cancer agent combining HPLC, HPLC–MS and direct ESI–HRMS analyses

One of the main problems of anti-cancer therapy is an insufficient differentiation between normal and malignant cells by the known anti-proliferant agents. The antibody-directed enzyme prodrug therapy is a promising approach for a selective treatment of cancer, in which a non-toxic prodrug is enzymatically converted into a highly cytotoxic drug at the surface of malignant cells by a targeted antibody–enzyme conjugate. The transformations and the stability of a very promising novel prodrug and its corresponding cytotoxic derivative were now investigated in detail by high-performance liquid chromatography (HPLC)–mass spectrometry (MS). In order to determine the time-dependent DNA alkylation efficiency and the sequence selectivity of the novel compounds, DNA binding studies using direct electrospray–Fourier transform ion cyclotron resonance–MS (ESI–FTICR–MS) have been performed. These measurements were accompanied by HPLC analyses followed by MS of the separated species to confirm the results of the direct ESI–FTICR–MS measurements. The sites of DNA alkylation could be identified unambiguously by the mass spectrometric fragmentation pattern of the alkylated oligodeoxynucleotides as well as by the results of HPLC followed by MS. A combination of all techniques applied led to a better understanding of the mode of action of the new therapeutics and might be used for an estimation of the cytotoxicity of different prodrugs and drugs since the alkylation efficiency correlates with the bioactivity of the compounds in cell culture investigations.After enzymatic cleavage of the sugar moiety, the untoxic prodrug is converted rapidly into the corresponding highly cytotoxic drug that alkylates DNA with high efficiencyElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-009-2963-x) contains supplementary material, which is available to authorized users.

Role of the Sulfur Atom on the Reactivity of Methionine toward OH Radicals: Comparison with Norleucine

Density functional theory has been used to model the OH reaction with Gly-Met-Gly and Gly-Nle-Gly tripeptides. The first one is predicted to be about 100 times faster than the second one. Therefore, if a methionine fragment is replaced by norleucine, the overall reactivity of the peptide toward free radicals is expected to be significantly reduced, which is in agreement with previous experimental findings. Since the most reactive sites were found to be located in the central backbone for Nle and in the terminal fragment of the side chain for Met, this decrease is expected to be even more critical for large-sized free radicals. The S atom seems to activate not only those alkyl sites next to it but also those located an odd number of bonds apart. In addition the viability of different paths explaining the formation of methionine sulfoxide has been tested, and it is proposed that this process involves the formation of R-SO radical and formaldehyde. The results from the present work offer an explanation to the role of sulfur atom on the reactivity of methionine toward free radicals. They also support the preponderant role of Met35 on the development of the Alzheimer disease.

Synthesis, Cytotoxic Activity, and Mechanism of Action of Furo[2,3-c]acridin-6-one and Benzo[b]furo[3,2-h]acridin-6-one Analogues of Psorospermin and Acronycine

Compounds possessing the epoxyfuran system present in the natural cytotoxic dihydrofuroxanthone psorospermin (4) fused onto the acridone or benzo[b]acridone chromophores present in the antitumor acronycine (1) and S23906-1 (3) were prepared. The basic furoacridone and benzofuroacridone cores bearing an isopropenyl substituent at a convenient position were synthesized by condensation of 1,3-dihydroxyacridone (7) or 1,3-dihydroxybenz[b]acridin-12(5H)-one (9) with (E)-1,4-dibromo-2-methylbut-2-ene. In both series, the (2R*,1'S*) epoxides, with the same relative configuration as psorospermin, were the most active compounds, exhibiting cytotoxic properties with IC50 values in the 10-100 nM range. As in the acronycine and psorospermin series, the new compounds act through alkylation of the DNA guanine units. However, a strong difference was noted in the DNA alkylation site between the benzopyranoacridone S23906-1, which alkylates DNA guanine units at position N-2 in the minor groove, and the new 13H-benzo[b]furo[3,2-h]acridin-6-one derived epoxide 21, which alkylates DNA guanine units at position N-7 in the major groove.

Irreversible inhibition of dihydrodipicolinate synthase by 4-oxo-heptenedioic acid analogues

We report the synthesis of (2 E,5 E)-4-oxoheptadienedioic acid and (2 E)-4-oxoheptenedioic acid and evaluation of both diester and diacid analogues as inhibitors of bacterial dihydrodipicolinate synthase. Enzyme kinetic studies allowed the determination of second-order rate constants of inactivation; and substrate co-incubation studies have shown the inhibitors act at the active-site. Mass spectrometric analyses have further explored the enzyme–inhibitor interaction and determined the sites of enzyme alkylation.

Unexpected Desilylative-alkylation of 3-O-tert-Butyl-dimethylsilyl Galangin

Biological activities of flavonoids have led to the creation of many therapeutic forms of plant flavonoids. Data on the chemical structures of a variety of flavonoids have been obtained, and the fundamental mechanisms of action of flavonoids as antioxidants, anti-inflammatories, cardiotonics, radioprotectors, antitumor agents and antiviral agents have been identified. However, little effort has been expended on synthetic flavonoid analogues presumably due to the difficulties in controlling the regiochemistry of the flavonoids. As a part of our ongoing efforts directed at the structureactivity relationship studies (SARs) of naturally occurring flavonoids, we have been interested in the regioselective alkylation of flavanones (naringenin, 1, Fig. 1) and flavones (apigenin, 2, Fig. 1). Herein, we report our recent attempts on the regioselective alkylation of a flavonol, galangin (3, Fig. 1). The flavonol has an additional hydroxyl group at the 3 position of the ring C (Fig. 1), which is known to be the primary site of alkylation. Thus, we envisaged that 3-Oprotected galangin would be selectively converted into the 7O-alkyl galangin under alkylating conditions, and set out to synthesize the key intermediate 4 starting from the commercially available chrysine 7 (Scheme 1). Treatment of chrysine 7 with Me2SO4 and K2CO3 in acetone provided the 5,7-di-O-methyl chrysine 8, which was subjected to the α-hydroxylation conditions to give the 5,7di-O-methyl galangin 9 in 60% yield. Protection of the 3-OH group with TBDMSCl and DMAP in anhydrous pyridine followed by Lewis acid-mediated demethylation provided the key intermediate 4, which was smoothly transformed into the alkylated product 5 upon treatment with substituted benzyl bromides (3-ClBnBr, 4-ClBnBr and 3-CNBnBr) and K2CO3 in acetone. However, under the alkylation conditions, the TBDMS protecting group was lost, and the NOESY analysis of 5 showed that there was no nOe correlation between the benzylic and A-ring protons (H6 and H8) (Fig. 2), which implied that the alkylation did not take place at the 7-O position. Instead, the benzylic protons of 5 showed strong nOe correlation with aromatic protons at the B-ring, which confirms that the alkylation site is 3-O rather than 7O. Protection of the 3-hydroxy group of 9 with TBDPSCl instead of TBDMSCl was attempted to provide more stable silyl ether but resulted in the same desilylative alkylation product 5 (data not shown). Based on this result, we presumed that the unexpected 3O-alkylated products were formed via desilylative-alkylation mechanism (Scheme 2). The flavonoid is deprotonated with K2CO3 to give an anion 11, which resonanced to the corresponding chromen-4-ol anion 12. The alkoxide ion then attacks the nearby TBDMS group to result in silyl migration (3-O to 4-O). The enolate anion at the 3-position 13, thus formed, attacks benzylic bromide to provide the 3-O-alkyl product 14, which resonances back to the stable aromatic form with concurrent loss of the TBDMS group upon aqueous work-up. In order to verify the desilylative-alkylation mechanism,

Synthesis of New Hydroxybenzo[a]carbazoles

An investigation of the alkylation sites of 4,7-dihydroxybenzo[a]pyrrolo[3,4-c]carbazole-1,3(2H,8H)-dione, as well as the site of reduction in the maleimide upper ring, was carried out which resulted in the synthesis of diversely substituted 4-hydroxybenzo[a]pyrrolo[3,4-c]carbazoles. Moreover, the synthesis of 5,6-dicyano-1,4-dihydroxy-11H-benzo[a]carbazole was performed in which the maleimide ring of the above scaffold is missing and is replaced by electron-withdrawing nitrile groups.

Separation and identification of trinucleotide–melphalan adducts from enzymatically digested DNA using HPLC–ESI–MS

Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery of the modified species than C(18). The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified cross-link formation in DNA, even though at low abundance and only a few adduct types were detected.

Carbonyl Reductase Inactivation May Contribute to Mouse Lung Tumor Promotion by Electrophilic Metabolites of Butylated Hydroxytoluene: Protein Alkylation in Vivo and in Vitro

Promotion of lung tumors in mice by the food additive butylated hydroxytoluene (BHT) is mediated by electrophilic metabolites produced in the target organ. Identifying the proteins alkylated by these quinone methides (QMs) is a necessary step in understanding the underlying mechanisms. Covalent adducts of the antioxidant enzymes peroxiredoxin 6 and Cu,Zn superoxide dismutase were detected previously in lung cytosols from BALB/c mice injected with BHT, and complimentary in vitro studies demonstrated that QM alkylation causes inactivation and enhances oxidative stress. In the present work, adducts of another protective enzyme, carbonyl reductase (CBR), were detected by Western blotting and mass spectrometry in mitochondria from lungs of mice one day after a single injection of BHT and throughout a 28-day period of weekly injections required to achieve tumor promotion. BHT treatment was accompanied by the accumulation of protein carbonyls in lung cytosol from sustained oxidative stress. Studies in vitro demonstrated that CBR activity in lung homogenates was susceptible to concentration- and time-dependent inhibition by QMs. Recombinant CBR underwent irreversible inhibition during QM exposure, and mass spectrometry was utilized to identify alkylation sites at Cys 51, Lys 17, Lys 189, Lys 201, His 28, and His 204. Except for Lys 17, all of these adducts were eliminated as a cause of enzyme inhibition either by chemical modification (cysteine) or site-directed mutagenesis (lysines and histidines). The data demonstrated that Lys 17 is the critical alkylation target, consistent with the role of this basic residue in NADPH binding. These data support the possibility that CBR inhibition occurs in BHT-treated mice, thereby compromising one pathway for inactivating lipid peroxidation products, particularly 4-oxo-2-nonenal. These data, in concert with previous evidence for the inactivation of antioxidant enzymes, provide a molecular basis to explain lung inflammation leading to tumor promotion in this two-stage model for pulmonary carcinogenesis.

Structural and Mechanistic Studies of Arylalkylhydrazine Inhibition of Human Monoamine Oxidases A and B

The structure and mechanism of human monoamine oxidase B (MAO B) inhibition by hydrazines are investigated and compared with data on human monoamine oxidase A (MAO A). The inhibition properties of phenylethylhydrazine, benzylhydrazine, and phenylhydrazine are compared for both enzymes. Benzylhydrazine is bound more tightly to MAO B than to MAO A, and phenylhydrazine is bound weakly by either enzyme. Phenylethylhydrazine stoichiometrically reduces the covalent FAD moieties of MAO A and of MAO B. Molecular oxygen is required for the inhibition reactions, and the level of O2 consumption for phenylethylhydrazine is 6-7-fold higher with either MAO A or MAO B than for the corresponding reactions with benzylhydrazine or phenylhydrazine. Mass spectral analysis of either inhibited enzyme shows the major product is a single covalent addition of the hydrazine arylalkyl group, although lower levels of dialkylated species are detected. Absorption and mass spectral data of the inhibited enzymes show that the FAD is the major site of alkylation. The three-dimensional (2.3 A) structures of phenylethylhydrazine- and benzylhydrazine-inhibited MAO B show that alkylation occurs at the N(5) position on the re face of the covalent flavin with loss of the hydrazyl nitrogens. A mechanistic scheme is proposed to account for these data, which involves enzyme-catalyzed conversion of the hydrazine to the diazene. From literature data on the reactivities of diazenes, O2 then reacts with the bound diazene to form an alkyl radical, N2 and superoxide anion. The bound arylalkyl radical reacts with the N(5) of the flavin, while the dissociated diazene reacts nonspecifically with the enzyme through arylalkylradicals.

Mitochondrial Protein Targets of Thiol-Reactive Electrophiles

Mitochondria serve a pivotal role in the regulation of apoptosis or programmed cell death. Recent studies have demonstrated that reactive electrophiles induce mitochondrion-dependent apoptosis. We hypothesize that covalent modification of specific mitochondrial proteins by reactive electrophiles serves as a trigger leading to the initiation of apoptosis. In this study, we identified protein targets of the model biotin-tagged electrophile probes N-iodoacetyl- N-biotinylhexylene-diamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]carboxamido)butane (BMCC) in HEK293 cell mitochondrial fractions by liquid chromatography-tandem mass spectrometry (LC-MS-MS). These electrophiles reproducibly adducted a total of 1693 cysteine residues that mapped to 809 proteins. Protein modifications were selective in that only 438 cysteine sites in 1255 cysteinyl peptide adducts (35%) and 362 of the 809 identified protein targets (45%) were adducted by both electrophiles. Of these, approximately one-third were annotated to the mitochondria following protein database analysis. IAB initiated apoptotic events including cytochrome c release, caspase-3 activation, and poly(ADP-ribose)polymerase (PARP) cleavage, whereas BMCC did not. Of the identified targets of IAB and BMCC, 44 were apoptosis-related proteins, and adduction site specificity on these targets differed between the two probes. Differences in sites of modification between these two electrophiles may reveal alkylation sites whose modification triggers apoptosis.

Preferential Protonation and Methylation Site of Thiopyrimidine Derivatives in Solution: NMR Data

Protonation (alkylation) sites of several thiopyrimidine derivatives were directly determined by 1H-15N (1H-13C) heteronuclear single quantum coherence/heteronuclear multiple bond correlation methods, and it was found that in all compounds, protonation (methylation) occurred at the N1 nitrogen. GIAO DFT chemical shifts were in full agreement with the determined tautomeric structures. According to ab initio calculations, the stability of the different protonated forms and methylated derivatives was favored due to thermodynamic control and not kinetic control.

Analysis for Plasma Protein Biomarkers Following an Accidental Human Exposure to Sulfur Mustard

Following an accidental human exposure to a vesicating agent, plasma samples were analyzed for specific biomarkers of sulfur mustard. One individual suffered chemical burns over 6.5% of the body surface area and required hospitalization; the second individual developed a single, small blister. Plasma specimens from both individuals were examined using two different assays. The first assay targeted sulfur mustard adducts to cysteine-34 of albumin using affinity chromatography, enzyme digestion, and analysis of the alkylated peptide fragment using liquid chromatography-tandem mass spectrometry. The second assay targeted alkylation sites of glutamic and aspartic acids of plasma proteins. Following precipitation of plasma proteins, the sulfur mustard adducts were cleaved from the protein using base, derivatized, and analyzed using gas chromatography-mass spectrometry. Samples obtained over a 42-day period from the individual requiring hospitalization produced positive results for sulfur mustard adducts using both assays. Observed levels of the sulfur mustard biomarker decreased by approximately 75% between days 2 and 42 for both assays. Samples obtained over a six-day period from the individual with a single, small blister produced positive results for the albumin adduct assay. Observed levels were much lower than levels from the hospitalized patient. Blood samples from suspected human exposures to sulfur mustard have only rarely been made available for analysis by sensitive and specific laboratory assays. The data presented here add significantly to the small database of information that currently exists on human biomarkers of sulfur mustard exposure, linking a well-documented exposure event with levels of plasma protein adducts.