Characterization of the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminal valine of mouse and rat hemoglobin.

Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselectivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In the studies presented here, L-valinamide was used as a model for the N-terminal valine of hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the reaction both in aqueous solution and within a protein microenvironment. Four products produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and characterized by (1)H NMR and GC/MS. These four reaction products were used as analytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 degrees C). The results show that the adducts formed by reaction at C-2 were formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The stereoisomers of each respective regioisomer were produced with similar rates of formation. The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using intact erythrocytes from Sprague-Dawley rats and B6C3F1 mice. After cleavage of the N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenylisothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitation of the four expected products by silylation of the PFPTH-valine-BMO derivatives. The hemoglobin results contrast with the valinamide results in that the reaction of BMO with the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts. The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse hemoglobin or with valinamide. These results, and the finding that the reaction with rat hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with mouse hemoglobin, indicate the importance of measuring all four adducts when comparing the relative rates of adduct formation both with model compounds and among different species.     

A comprehensive structural analysis of hemoglobin adducts formed after in vitro exposure of erythrocytes to butadiene monoxide

A widely used method for assessing occupational and environmental exposure to 1,3-butadiene involves the detection of hemoglobin adducts formed by the reactive metabolite butadiene monoxide (BMO). This assay employs the N-alkyl Edman method, which was developed to determine adducts formed at the amine group of the N-terminal valine of hemoglobin. Disadvantages of this procedure include its limitation to detecting only one adduct per globin chain, despite the presence of numerous other, and potentially more reactive, nucleophilic amino acids in hemoglobin. The method is also not suitable for determining whether the reaction of BMO occurs at the N-terminal valine of alpha- or beta-globin. The primary goals of the current research are to determine the degree of modification of alpha- and beta-globin chains by BMO and to localize the reactive residues to specific regions of the globin polypeptides. The reaction products after in vitro incubation of C57Bl/6 mouse erythrocytes with BMO were isolated by acid extraction of heme and microprecipitation of globin, followed by the determination of the number and location of adducts by mass spectrometry. The modification degree was monitored by electrospray mass spectrometry, which was used to measure the time- and concentration-dependent formation of BMO-hemoglobin adducts (< or =10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with alpha-globin than with beta-globin. Structural analysis was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography/mass spectrometry. These experiments allowed the localization of BMO-hemoglobin adducts to specific regions within alpha- and beta-globin, and also provided information about their relative reactivity. Interestingly, the initial site of adduct formation on alpha-globin is located near the N-terminal peptide, whereas the initial site on beta-globin is located at the C-terminal region. Collectively, the results establish differences in the reactivities of alpha- and beta-globin toward BMO, demonstrate the formation of multiple adducts at several alpha- and beta-globin sites, and show that the N-terminal valine residues are not the first to be modified by BMO.     

Procedure for monitoring exposure to sulfur mustard based on modified edman degradation of globin

A procedure for the modified Edman degradation of globin for determination of sulfur mustard adducts to the N-terminal valine residue in human hemoglobin has been developed for use under field laboratory conditions. The minimum detectable exposure level of human blood (in vitro) to sulfur mustard using this procedure is 100 nM. The interindividual and intraindividual variabilities of the procedure were acceptable (standard deviation < 10% and < 20%, respectively). The procedure could be properly set up and carried out in another laboratory within one working day, demonstrating its robustness.     

Hemoglobin adduct levels in rat and mouse treated with 1,2:3,4-diepoxybutane

For cancer risk assessment of 1,3-butadiene from rodent cancer test data, the in vivo doses of formed 1,2:3,4-diepoxybutane (DEB) should be known. In vivo doses of DEB were measured through a specific reaction product with hemoglobin (Hb), a ring-closed adduct, N,N-(2,3-dihydroxy-1,4-butadiyl)valine (Pyr-Val), to N-terminal valines. An analytical method based on tryptic digestion of Hb and quantification of Pyr-modified heptapeptides by LC-MS/MS has been further developed and applied in vivo to DEB-treated rats. Furthermore, N-(2,3,4-trihydroxybutyl)valine adducts (THB-Val) to the N-terminal valine in Hb were measured in rats and mice treated with DEB and in a complementary experiment with 1,2-epoxy-3,4-butanediol (EBdiol), using a modified Edman degradation method and GC-MS/MS. In vitro reactions of hemolysate with DEB and EBdiol were used to measure reaction rates for adduct formation needed for calculation of doses and rates elimination in vivo. The results showed that the level of the Pyr-Val adduct per administered dose of DEB was approximately the same in rats as had earlier been observed in mice [Kautiainen et al. (2000) Rapid Commun. Mass Spectrom. 14, 1848-1853]. Levels of the THB-Val adduct after DEB treatment were 3-4 times higher in rat than in mouse, probably reflecting an enhanced hydrolysis of DEB to EBdiol catalyzed by epoxide hydrolase. After EBdiol treatment, the THB-Val adduct levels were about the same in rat and mouse. Calculations from in vitro data show that the Pyr-Val adduct is a relevant monitor for the in vivo dose of DEB and that THB-Val primarily reflects doses to EBdiol. The calculated rates of formation of adducts and rates of elimination agree with expectations. Procedures for quantification of Hb adducts as modified peptides as well as preparation and characterization of peptide standards have been evaluated.     

Haemoglobin adducts from isoprene and isoprene monoepoxides

1. Isoprene is metabolised in vitro by oxygenation of either double bond to 2-ethenyl2-methyloxirane (epoxide A) and 2-(1-methylethenyl)oxirane (epoxide B). The reactivity in vitro and formation in vivo of the monoepoxides of isoprene were studied by the formation of adducts to N-terminal valines in haemoglobin (Hb). These adducts were analysed by mass spectrometry after cleavage and derivatization by a modified Edman degradation method. 2. When red blood cells were incubated with commercial isoprene oxide (about 95% epoxide A, 5% epoxide B) adducts from both epoxides were formed. 3. It is confirmed that epoxide A is hydrolysed much faster than epoxide B. The rates are enhanced by phosphate buffer (epoxide A), probably through acid catalysis, and by the presence of red blood cells (both epoxides), due to enzymatic detoxification. 4. Comparison of total valine adduct levels in Hb from isoprene and isoprene oxide injected i.p. led to the conclusion that 23 and 1% of injected isoprene was metabolized to the epoxides in mouse and rat, respectively.     

Hydroxyethylation of hemoglobin by 1-(2-chloroethyl)-1-nitrosoureas

Following treatment of cancer patients with three 1-(2-chloroethyl)-1-nitrosoureas, hemoglobin was isolated and analyzed by GC-MS for N-(2-hydroxyethyl)-N-terminal valine. This alkylated amino acid was liberated as a (pentafluorophenyl)thiohydantoin from the hemoglobin by a modified Edman degradation procedure. Following intravenous infusion of fotemustine [diethyl[1-[3-(2-chloroethyl)-3-nitrosoureido]ethylphosphonate] (ca. 90 mg/m2) the levels of (hydroxyethyl)valine in two patients increased steadily, reaching a peak of ca. 300 pmol/g after 6 h. In a further five patients receiving fotemustine (100 mg/m2) alkylation levels 24 h after treatment ranged from 132 to 1524 pmol/g of globin. Treatment with TCNU [1-(2-chloroethyl)-3-[2-[(dimethylamino)sulfonyl]ethyl]-1- nitrosourea] or ACNU [1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea] resulted in similar increases in (hydroxyethyl)valine in hemoglobin, although the amounts (as with fotemustine) showed considerable interindividual variation. It appears that the measurement of (hydroxyethyl)valine in hemoglobin may be a suitable monitor of exposure to hydroxyethylating agents during (chloroethyl)nitrosourea chemotherapy.     

Diversity-oriented synthesis of spiro-substituted 1,3-thiazine library via a one-pot, two-step, three-component reaction

A sequential one-pot, two-step, three-component reaction for efficient synthesis of spiro-substituted 1,3-thiazine library has been developed. The syntheses were achieved by reacting cyanoacetamide with isothiocyanate derivatives to give rise to 2-cyano-3-mercaptoacrylamides, which are trapped in situ by various cycloketones through cyclization, providing multifunctionalized spiro-substituted 1,3-thiazine analogues. This procedure features short reaction time, generally good to excellent yields, easily available starting materials, and operational simplicity. This chemistry provides an efficient and promising synthetic strategy to diversity-oriented construction of the 1,3-thiazine skeleton.     

Hemoglobin adducts in animals exposed to gasoline and diesel exhausts. 1. Alkenes

Blood samples from rats and hamsters exposed to automotive engine exhausts in the Committee of Common Market Automobile Constructors long-term inhalation study at Battelle-Geneva were analysed for the levels of 2-hydroxyethylvaline (HOEtVal) and 2-hydroxypropylvaline (HOPrVal) in hemoglobin (Hb). These adducts to the N-terminus of the Hb chains were determined by gas chromatography-mass spectrometry of derivatives obtained by a modified Edman degradation that specifically cleaves off alkylated N-terminal amino acids (valine in Hb). The adduct levels found correspond to the metabolic conversion of about 5-10% of inhaled ethene and propene to ethylene oxide and propylene oxide, respectively, in agreement with results from earlier studies on mice inhaling radio-labelled alkenes. It is concluded that the alkenes, via epoxides, are the main sources of the observed HOEtVal and HOPrVal. From calculated doses and estimates of genotoxic potency the contribution from ethene in urban air to human cancer risk is discussed.     

Structural characterization of bacitracin components by Frit-fast atom bombardment (FAB) liquid chromatography/mass spectrometry (LC/MS).

The structural characterization of minor components of bacitracin (BC) complex was carried out using a technique of liquid chromatography/mass spectrometry (LC/MS). Satisfactory total ion current chromatogram of BC complex and excellent mass spectra of many components were given by Frit-fast atom bombardment (FAB) LC/MS analytical system, and the structures of 13 minor components could be proposed. The 13 minor components were classified into two groups, bacitracin A (BC-A) related components and bacitracin F (BC-F) related components depending on their common N-terminal moieties. The structures of BC-A related components and BC-F related components were the same as those of BC-A and BC-F, respectively, except that one to three of isoleucine and leucine residues are replaced by valines. The BC-F related components were degradation products of BC-A related components through the same degradation process as that of BC-A.     

Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of β-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

3-N-Substituted aminomethyl derivatives of rifamycin SV. A convenient method of synthesis, cyclization of certain derivatives, and anticellular and antiviral activities of several derivatives.

A new synthesis of Mannich bases of rifamycin SV using the Borch2 procedure with rifaldehyde is described. This new synthesis offers two advantages over the previously published method. It provides a route to monoalkyl-aminomethylrifamycins (le-h) and to unsubstituted aminomethylrifamycins that were not accessible by the old procedure. The new method also offers a preparative route to Mannich bases 1a and 1b were needed in multigram quantities for biological testing. In addition, the cyclization of certain of the monoalkylaminomethylrifamycins to the novel N,15-didehydro-15-epi[methano(alkylimino)]rifamycin SV derivatives (2) is described. The anticellular and antiviral effects of representatives of both series of compounds against cultured mouse cells and murine oncornavirus are are discussed.     

Isolation of a heptapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln (valorphin) with some opiate activity

Bovine hypothalamic tissue was extracted and purified by solid phase extraction and several reversed-phase HPLC steps. The amino acid sequence of the purified peptide was determined by Edman degradation to be Val-Val-Tyr-Pro-Trp-Thr-Gln. This was confirmed by comparison of its chromatographic behavior with that of the synthetic peptide, and mass spectrometric analysis resulted in a mass identical to the calculated mass for this peptide. This heptapeptide shows homology with residues 32-38 of the beta-chain of bovine hemoglobin. The peptide inhibited the electrically induced contractions of the guinea pig ileum muscle preparation; this inhibition was reversible by naloxone. It also inhibited the binding of ¹²⁵I-DAMGO (selective for μ receptors) to rat brain with an IC₅₀ of 10 μm and the binding of ³H-DPDPE (selective for σ receptors) with an IC₅₀ of 185 μm . With two valines at the N-terminus and some opiate activity, valorphin seems a suitable name for this newly isolated peptide.     

Hemoglobin adducts of epoxybutene in workers occupationally exposed to 1,3-butadiene

1,3-Butadiene (BD) is an important chemical widely used in the synthetic rubber industry. Hemoglobin adducts of two of its reactive metabolites have been already investigated as possible parameters for exposure assessment. In this study hemoglobin adducts of epoxybutene (EB) were analyzed in blood samples from 17 workers in a BD monomer production unit and 19 controls in a heat production unit of a petrochemical plant near Prague, Czech Republic. BD exposure was determined by personal air sampling. The median level of exposure was 440 microg/m3 (range < 11-17 mg/m3) for the exposed workers and < 6 microg/m3 (< 5-150 microg/m3) for the controls. The adduct N-(2-hydroxy-3-butenyl)valine (HBVal) formed by the reaction of the N-terminal valine of globin with carbon-1 of EB was measured. The N-alkylated amino acid was analyzed by gas chromatography/mass spectrometry (GC/MS) after degradation by the modified Edman procedure. Using published methods problems arose with high background levels, especially in the negative ion chemical ionization (NCI) mode. In the present study a limit of detection of 0.2 pmol/g globin was achieved by using 400 mg globin, a variation in extraction solvents, an additional purification step and a widely extended GC temperature program. The median hemoglobin adduct level of the Czech BD monomer production workers (0.7 pmol/g globin; n = 17) was significantly higher than that of the controls (0.2 pmol/g globin; n = 19; P<0.05). Smoking controls showed higher hemoglobin adduct levels than nonsmoking controls (P<0.1) and significantly higher BD exposure levels (P<0.01).     

Protected amino acid tetrafluorophenyl esters for peptide synthesis

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large k_coupling/k_racemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-N^im-benzyl-histidine.     

Structures of bacillomycin D and bacillomycin L peptidolipid antibiotics from Bacillus subtilis

The complete structures of bacillomycin D and bacillomycin L were revised by FAB mass spectrometry and by Edman degradation of the derivatives resulting from the N-bromosuccinimide reaction. The homologous components of both series of antibiotics were separated by HPLC and the beta-amino acids were identified by capillary gas chromatography.     

Comparison of the hemoglobin adducts formed by administration of N-methylolacrylamide and acrylamide to rats.

Acrylamide (AM) and N-methylolacrylamide (NMA) are used in the formulation of grouting materials. AM undergoes metabolism to a reactive epoxide, glycidamide (GA). Both AM and GA react with hemoglobin to form adducts that can be related to exposure to AM. The objective of this study was to evaluate the extent to which NMA could form the same adducts as AM. N-(2-carbamoylethyl)valine (AAVal derived from AM) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal derived from GA) were measured following a single oral dose of AM (50 mg/kg) or NMA (71 mg/kg) in male F344 rats. AAVal and GAVal were measured by a modified Edman degradation to produce phenylthiohydantoin derivatives and liquid chromatography/tandem mass spectrometry. In AM-treated rats, AAVal was 21 +/- 1.7-pmol/mg globin (mean +/- SD, n = 4), and GAVal was 7.9 +/- 0.8 pmol/mg. In NMA-treated rats, AAVal was 41 +/- 4.9 pmol/mg, and GAVal was 1.4 +/- 0.1 pmol/mg. Whether AAVal was derived from reaction of NMA with globin followed by loss of the hydroxymethyl group, or loss of the hydroxymethyl group to form AM prior to reaction with globin, is not known. However, the higher ratio of AAVal:GAVal in NMA-treated rats (29 vs. 2.6 in AM-treated rats) suggests that reaction of NMA with globin is the predominant route to AAVal in NMA-treated rats. The detection of GAVal in NMA-treated rats indicates oxidation of NMA, either directly or following conversion to AM. The lower levels of GAVal on NMA administration suggest that a much lower level of epoxide was formed than compared with AM treatment.     

Cloning and molecular characterization of BmHYA1, a novel hyaluronidase from the venom of Chinese red scorpion Buthus martensi Karsch

In this communication, the full protein sequence of a novel venom hyaluronidase BmHYA1 was reported. It is the first full hyaluronidase amino acid sequence from scorpion venom. It was deduced from nucleotide sequence by 3′ Rapid Amplification of cDNA Ends (RACE) PCR cloning, followed by alignment with the N-terminal amino acid sequence, which was obtained by Edman degradation. BmHYA1 has 385 amino acid residues containing five potential N-glycosylation sites. The phylogenetic analysis indicates early divergence and independent evolution of BmHYA1 from other hyaluronidases.     

Hydroxyethylvaline adduct formation in haemoglobin as a biological monitor of cigarette smoke intake

The ethylene oxide adduct formed on the N-terminal valine in haemoglobin was investigated as a biological monitor of tobacco smoke intake. The modified method developed for the determination of the hydroxyethylvaline adduct (HOEtVal) involved reaction of globin with pentafluorophenyl isothiocyanate, extraction of the HOEtVal thiohydantoin product, derivatization of this by trimethylsilylation and quantitation by capillary gas chromatography with selective ion monitoring mass spectrometry using a tetradeuterated internal standard. The method was applied to globin samples from 26 habitual cigarette smokers and 24 non-smokers. There was a significant correlation between cigarette smoke intake as measured by the average number of cigarettes smoked per day and HOEtVal levels (r=0.537, p<0.01). Background levels were found in non-smokers (mean 49.9 pmol/g Hb, range 22–106 pmol/g Hb). Smoking increased these levels by 71 pmol/g Hb/ 10 cigarettes per day.Cotinine levels in plasma of the smokers were determined by GC-NPD using 2-methyl-4-nitroaniline as internal standard. For non-smokers cotinine was determined by GC-MS selective ion monitoring using d₃-methylcotinine as internal standard. There was no correlation between number of cigarettes smoked per day and cotinine levels (r=0.297, p>0.05) although cotinine was correlated with HOEtVal (r=0.43, p<0.01).The HOEtVal adduct levels thus appear to be a suitable biomonitor for exposure to hydroxyethylating agents in cigarette smoke, reflecting an integrated dose over the erythrocyte lifetime. This is in contrast to plasma cotinine determinations which reflect only the previous day's exposure to nicotine in smoke.     

Effects of flecainide and quinidine on Kv4.2 currents: voltage dependence and role of S6 valines

1. The effects of flecainide and quinidine were studied on wild-type Kv4.2 channels (Kv4.2WT), channels with deletion of the N-terminal domain (N-del) and channels with mutations in the valine residues located at positions 402 and 404 in the presence (V[402,404]I) or in the absence (N-del/V[402,404]I) of the N-terminus. 2. The experiments were performed at 37 degrees C on COS7 cells using the whole-cell configuration of the patch-clamp technique. 3. Flecainide and quinidine inhibited Kv4.2WT currents in a concentration-dependent manner (IC(50)=23.6+/-1.1 and 12.0+/-1.4 microMat +50 mV, respectively), similar to their potency for the rest of the constructs at the same voltage. In Kv4.2WT channels, flecainide- and quinidine-induced block increased as channel inactivation increased. In addition, the inhibition produced by quinidine, but not by flecainide, increased significantly at positive test potentials. Similar effects were observed in N-del channels. However, in V[402,404]I and N-del/V[402,404]I channels, the voltage dependence of block by both quinidine and flecainide was lost, without significant modifications in potency at +50 mV. 4. These results point to an important role for S6 valines at positions 402 and 404 in mediating voltage-dependent block by quinidine and flecainide.