Comparative Characterization of Immune Reagents Based on Hemiacetals of Aflatoxin B1 and Sterigmatocystine

Aflatoxin B₁ and sterigmatocystine hemiacetal derivatives were synthesized, and their conjugation to albumins and gelatin and also spectral and immunochemical characteristics of reaction products were studied. Data on the specificity and analytical properties of the antibodies produced by immunization with conjugated antigens are given. The possible mechanism of hemiacetal interaction with proteins is discussed. Based on immune reagents to sterigmatocystine hemiacetal, a test system was developed for determination of sterigmatocystine at the sensitivity of 0.1 ng/ml.     

Evidence for hemiacetal formation between N-acyl-L-phenylalaninals and alpha-chymotrypsin by cross-saturation nuclear magnetic resonance spectroscopy.

N-Acetyl-L-phenylalaninal exists predominantly in its hydrated form in aqueous solution, but the aldehyde and not the hydrate is shown by nuclear magnetic resonance (NMR) spectroscopy to be the effective inhibitor of alpha-chymotrypsin. NMR spectroscopy also indicates that the initial alpha-chymotrypsin-N-acetyl-L-phenylalaninal complex is in equilibrium with a hemiacetal formed between the aldehyde and the active site serine residue. The rate of the latter equilibration is slow on the NMR time scale but the hemiacetal can be detected by cross-saturation NMR spectroscopy. N-Benzoyl-L-phenylalaninal is a more potent inhibitor of alpha-chymotrypsin than the N-acetyl derivative and both the formation of the enzyme-inhibitor complex and the hemiacetal are slow on the NMR time scale, but the hemiacetal in the enzyme can be detected by cross-saturation NMR spectroscopy. The N-acyl-L-phenylalaninals also bind to N-methylhistidinyl-57-alpha-chymotrypsin, but clear evidence for hemiacetal formation was not obtained by cross-saturation NMR spectroscopy either because the hemiacetal was not formed or more probably because the rate of dissociation was slow compared with the rate of relaxation of the hemiacetal proton. The dissociation constant of N-benzoyl-L-phenylalaninal to dehydroalaninyl-195-alpha-chymotrypsin was found to be high relative to the dissociation constant to native alpha-chymotrypsin, supporting the NMR evidence that a hemiacetal with the Ser-195 is formed on association of N-benzoyl-L-phenylalaninal with alpha-chymotrypsin.     

Enzymatic formation of the bisfuran structure in aflatoxin biosynthesis.

A relatively stable enzyme system that converts versiconal hemiacetal acetate to versicolorin A was isolated from the soluble fraction of the homogenized cells of Aspergillus parasiticus ATCC 15517. The cell-free preparation did not require oxygen or oxidized nicotinamide adenine dinucleotide phosphate for activity, nor did it require dithiothreitol, polyclar (polyvinyl pyrrolidone), or glycerol for stabilization of activity. It was susceptible to inhibition by dichlorvos and cysteine. Isotope tracer studies revealed involvement of several intermediates in the conversion of versiconal hemiacetal acetate to versicolorin A. These findings confirm the biogenetic relationship of versiconal hemiacetal acetate and versicolorin A, and they confirm that the bisfuran ring structure in aflatoxins and related fungal metabolites is derived from the hemiacetal structure of versiconal hemiacetal acetate.     

Role of versicolorin A and its derivatives in aflatoxin biosynthesis.

The involvement of various anthraquinone metabolites in the biosynthesis of aflatoxin B1 was investigated by using a labeled double-substrate technique in a cell-free system. The results showed that both versicolorin A hemiacetal and versicolorin A hemiacetal acetate were converted to aflatoxin B1, whereas versicolorin A was not, even though it was added to the same cell-free system. Thus, versicolorin A hemiacetal, versicolorin A hemiacetal acetate, or both were implicated as key intermediates, whereas versicolorin A and C became side shunt metabolites. These latter compounds reentered the pathway depending on the availability of the appropriate enzymes and suitability of conditions. Dichlorvos, a specific inhibitor of aflatoxin biosynthesis, is considered to have its primary action on either an oxygenase or dehydrogenase involved in the pathway and to act in a secondary capacity as an inhibitor of an esterase which may also be involved in the pathway.     

Role of versicolorin A and its derivatives in aflatoxin biosynthesis

The involvement of various anthraquinone metabolites in the biosynthesis of aflatoxin B1 was investigated by using a labeled double-substrate technique in a cell-free system. The results showed that both versicolorin A hemiacetal and versicolorin A hemiacetal acetate were converted to aflatoxin B1, whereas versicolorin A was not, even though it was added to the same cell-free system. Thus, versicolorin A hemiacetal, versicolorin A hemiacetal acetate, or both were implicated as key intermediates, whereas versicolorin A and C became side shunt metabolites. These latter compounds reentered the pathway depending on the availability of the appropriate enzymes and suitability of conditions. Dichlorvos, a specific inhibitor of aflatoxin biosynthesis, is considered to have its primary action on either an oxygenase or dehydrogenase involved in the pathway and to act in a secondary capacity as an inhibitor of an esterase which may also be involved in the pathway.     

The origin of the periodate-resistant D-glucose residues in starches and glycogens

Under ordinary analytical conditions, the proportion of periodate-resistant-D-glucose residues in starches and glycogens was consistently about one-third of the proportion of branching points. The resistant D-glucose residues became freely oxidisable after the limit-oxidised glucans had been reduced with sodium borohydride. The results can be explained by assuming that, when a D-glucose residue carrying a branch at position 6 is oxidised, the resulting two aldehyde groups both form 6-membered hemiacetal rings with the closest hydroxyl groups on neighbouring, unoxidised residues in the same, (1→4)-linked chain, whereas when the other D-glucose residues are oxidised, only one of the aldehyde groups shows a strong tendency to form a hemiacetal of this kind. It is suggested that, in the unbranched units, the other aldehyde group preferentially forms a hemiacetal with the primary hydroxyl group in the same unit.     

Recherches dans la série des acétals cycliques : XIII. Ouverture du 4-oxo et de 4-hydroxy-3,6,8-trioxabicyclo[3.2.1]octanes et du 3-oxo-2,5,7-trioxabicyclo[2.2.2]octane par l'aluminohydrure de lithium et l'iodure de méthylmagnésium

This study describes ring-opening reactions, by lithium aluminum hydride and by methylmagnesium iodide, on bicyclic, oxygenated heterocycles possessing a lactone or hemiacetal group. The reactions give rise to dihydroxy derivatives of 1,3-dioxolanes and 1,3-dioxanes; the latter are the first examples of compounds corresponding to 1,3-acetals of a ketone with glycerol. Evidence is presented for a hemiacetal intermediate in the reaction of magnesium at a lactone group.     

Stereoselective construction of tetra-substituted tetrahydrofuran compounds from benzylic hemiacetal in the presence of H2 and a Pd catalyst: stereoselective synthesis of a stereoisomer of (-)-virgatusin and its antimicrobiological activity

Tetra-substituted tetrahydrofuran compounds were stereoselectively prepared from benzylic hemiacetal in the neutral condition by employing the simple reagent, H(2), and a Pd catalyst. The stereoselective conversion of benzylic hemiacetal to two different stereoisomers of the tetrasubstituted tetrahydrofuran compound was observed. One of these tetrahydrofuran compounds was converted to the virgatusin stereoisomer to estimate its antimicrobiological activity.     

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.     

Structural analysis of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli: direct evidence of substrate binding and cofactor-induced conformational changes

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.     

The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy

Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9(S)?hydroperoxide (9(S)?HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60?s at 0?°C, pH?4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC–MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9?hydroxy?9?[(1′E,3′Z)?nonadienyloxy]?nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [¹³C₁₈]9(S)?HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name “hydroperoxide lyase”, which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with “hemiacetal synthase” (HAS).     

Membrane actions of male contraceptive gossypol tautomers

The role of different gossypol tautomers in the interaction of this molecule with membranes has been investigated using the isolated hemiacetal moiety of gossypol and the pH dependency of the keto-enol tautomeric equilibrium. Our results indicate that: the actions of the hemiacetal tautomer cannot explain the effects of gossypol on mitochondrial oxidative phosphorylation, lipid membrane interfacial potentials, and proton conductance of lipid bilayers; the enolate forms of gossypol are the species that bind to the membrane interface and decrease the electrostatic interfacial potential; and the uncharged (keto and/or enol) species in equilibrium with the enolate forms of gossypol give the molecule the ability to carry protons across biological membranes.     

Saundersiosides C-H, rearranged cholestane glycosides from the bulbs of Ornithogalum saundersiae and their cytostatic activity on HL-60 cells.

Six novel rearranged cholestane glycosides with a six-membered hemiacetal ring system, designated as saundersiosides C-H, were isolated from the bulbs of Ornithogalum saundersiae. Their structures were determined on the basis of spectroscopic analysis and the result of hydrolysis. The conformation of the six-membered hemiacetal ring of the rearranged cholestanes was shown to be almost a boat-form by molecular mechanics and molecular dynamics calculation studies. Among the isolated compounds, saundersioside E, F, G and H with an aromatic acid ester group at the glycoside moiety were found to be highly cytostatic to human leukemia HL-60 cells, showing IC50 values of 0.021, 0.019, 0.063 and 0.052 microM, respectively, which are as potent as those of the clinically applied anticancer agents, etoposide and methotrexate.     

The structure of glutaraldehyde in aqueous solution determined by ultraviolet absorption and light scattering.

The structure of glutaraldehyde (GA) in aqueous solutions has been the subject of much debate. Since there were fundamental problems in the experiments in the preceding studies, in this article, the structure of GA was investigated with uv absorption and light scattering to avoid those problems. It was discovered that 70% glutaraldehyde solution contains a large quantity of polymeric species with cyclic hemiacetal structure. On dilution, the polymerized glutaraldehyde slowly converted to monomers. In dilute solution, glutaraldehyde is almost monomeric at pH 3-8, the major portion taking the cyclic hemiacetal structure. The structure of GA in 20% solution is similar to that in more dilute solution. alpha, beta-Unsaturated structure does not exist in aqueous solution regardless of the concentration of glutaraldehyde.     

Biosynthesis of versicolorin A.

The incorporation of various potential intermediates into versicolorin A by a versicolorin A-accumulating mutant of Aspergillus parasiticus was studied. Both whole mycelium and cell-free extracts of this mutant were able to convert 14C-labeled versiconal hemiacetal acetate to versicolorin A. By the use of a labeled double substrate technique it was shown that two other compounds, versicolorin A hemiacetal and its acetate derivative, were also converted to versicolorin A. It is concluded that one or both of these compounds are intermediates in the biosynthesis of versicolorin A and therefore may possibly be involved in the biogenesis of the aflatoxins.     

The “heterolytic hydroperoxide lyase” is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10–60 s at 0 °C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1′-hydroxy-3′-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [¹⁸O₂]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [¹⁸O₂]13-HPOT retained two and one ¹⁸O atoms, respectively, whereas no ¹⁸O was incorporated from [¹⁸O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

Oligouronide signaling of proteinase inhibitor genes in plants: structure-activity relationships of Di- and trigalacturonic acids and their derivatives.

Polygalacturonic acid (DPave approximately 20), alpha-1,4-di- and trigalacturonic acids, delta 4,5-alpha-1,4-di- and delta 4,5-alpha-trigalacturonic acids, and several chemically modified derivatives of these oligomers were prepared. Their proteinase inhibitor-inducing activities were determined by supplying solutions of the compounds to young, excised tomato plants through their cut stems. Digalacturonic acid, on a molar basis, was the most active oligomer (ED50 approximately 1.5 micrograms/plant), being about three times more active than the parent oligogalacturonic acid (ED50 approximately 5.5 micrograms/plant). The specific inducing activity of trigalacturonic acid was about half that of digalacturonic acid. Both delta 4,5-di- and delta 4,5-trigalacturonic acids were about half as active as di- and trigalacturonic acids, respectively. Reduction of the hemiacetal (carbonyl) group of the di- and trigalacturonic acids with sodium borohydride completely destroyed proteinase inhibitor inducing activities, indicating that the inducing activity of both acids depends upon an intact hemiacetal at the reducing termini. Reduction of the double bonds of delta 4,5-di- and delta 4,5-trigalacturonic acids by catalytic hydrogenation with H2 (palladium catalyst) produced derivatives with specific inducing activities of approximately one-half that of the parent compounds. Thus, while the reducing termini of oligogalacturonides require an intact hemiacetal for proteinase inhibitor inducing activities, the nonreducing termini of the small oligouronides do not require a C4 hydroxyl nor a C5 proton to be active inducers.     

A convenient synthesis of glycosyl chlorides from sugar hemiacetals using triphosgene as the chlorine source

Treating partially protected sugar hemiacetals with triphosgene in THF results in the formation of glycosyl chlorides. The method is compatible with acid-sensitive isopropylidene protecting groups in the hemiacetal substrates.     

Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of fatty acid hydroperoxides into hemiacetals

The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.