18,19-Dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450(11) beta. Chemical synthesis and structural analysis by 1H NMR

A new metabolite was produced from 18-hydroxydeoxycorticosterone by the cytochrome P-450(11) beta linked hydroxylase system purified from bovine adrenocortical mitochondria. It was identified as 18,19-dihydroxydeoxycorticosterone by chemical synthesis on the basis of high-performance liquid chromatography, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance (1H NMR) spectroscopy, and detailed structural analysis of it was performed by 1H NMR spectroscopy. The methylene protons at the C-19 position of the steroid were nonequivalent and coupled with each other, having a coupling constant of 10.6 Hz. These protons had different coupling constants, 6.7 and 3.4 Hz, for the hydroxy proton at the C-19 position. Due to these couplings, the signals of the methylene protons were observed around 3.9 ppm as two double doublets. The methylene protons at the C-21 position were also nonequivalent, having a coupling constant of 11.1 Hz. Coupling constants between these methylene protons and the hydroxy proton at the C-21 position were 8.2 and 4.2 Hz, respectively. These results indicate that both hydroxymethyl groups at the C-19 and C-21 positions do not freely rotate in chloroform solution. The signals of hydroxy protons at the C-19 and C-21 positions were found at 1.25 and 1.87 ppm, respectively, by means of decoupling of the corresponding methylene protons. The hydroxy proton at the C-18 position was found to scarcely couple with any proton. This fact suggests that this hydroxy group is linked to the C-20 position, making a hemiketal bridge between the C-18 and the C-20.     

Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.     

Stereoselective reduction of C-2 substituted steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride

The effect of C-2 substitution on the stereoselective reduction of steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride was investigated. The C-2 mono- and di-substituted chloro and methyl derivatives were predominantly reduced to one of the epimeric alcohols. The 2 alpha-chloro and 2 alpha-methyl derivatives of 17 beta-acetoxy-5 alpha-androstan-3-one undergo stereoselective reduction with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride to the axial (3 alpha) alcohol as observed in the unsubstituted compound, whereas sodium borohydride gives predominantly the equatorial (3 beta) alcohol. The 2 beta-chloro, 2 beta-methyl, 2,2-dichloro, and 2,2-dimethyl derivatives are reduced predominantly to the equatorial (3 beta) alcohol by both reagents.     

Amorpha-4,11-diene synthase: mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate

Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-(2)H]FPP, (1S)-[1-(2)H]FPP, and [1,1-(2)H2]FPP. GC-MS analysis of amorpha-4,11-diene formed from the deuterated FPPs shows that the deuterium atoms are retained in the product. Furthermore, analysis of the MS-spectra obtained with the differently labeled substrate indicates that the H-1si-proton of FPP is transferred during the cyclization reaction to carbon 10 of amorphadiene while the H-1re-proton of FPP is retained on C-6 of the product. Proton NMR and COSY experiments proved that the original H-1si-proton of FPP is located at C-10 of amorpha-4,11-diene as a result of a 1,3-hydride shift following initial 1,6-ring closure. The results obtained support the previously suggested mechanism for the cyclization of farnesyl diphosphate by amorph-4,11-diene synthase involving isomerization of FPP to (R)-nerolidyl diphosphate (NPP), ionization of NPP, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure to generate the amorphane skeleton, and deprotonation at either C-12 or C-13 to afford the final product (1S,6R,7R,10R)-amorpha-4,11-diene.     

1,5-Hydride shift in Wolff-Kishner reduction of (20R)-3beta,20, 26-trihydroxy-27-norcholest-5-en-22-one: synthetic, quantum chemical, and NMR studies.

Heating (20R)-3beta,20,26-trihydroxy-27-norcholest-5-en-22-one (1) with hydrazine and KOH at 160 degrees C completely converted the steroid to a diastereoisomeric mixture of the new (20R,22RS)-27-norcholest-5-ene-3beta,20,22-triols (2). Exclusive formation of 2 suggests that the expected Wolff-Kishner reduction to a methylene group at the C-22 ketone in 1 was diverted to the C-26 position by a 1,5-hydride shift. All attempts under acid conditions failed to produce a C-22 phenyl hydrazone from 1. However, reaction of 1 was reacted with phenylhydrazine in hot KOH, gave the C-26 phenylhydrazone 4 as the sole product. Evidently, under alkaline conditions, first a hydride ion undergoes an intramolecular transfer from the C-26 CH(2)OH group to the C-22 ketone in 1, and then the phenylhydrazine traps the newly formed aldehyde. To examine this hypothesis, we constructed computer-simulated transition state models from quantum chemical calculations and then compared data from these models with NMR measurements of the reaction mixtures containing 2. The NMR data showed that the C-22 diastereoisomers of 2 are formed in a nearly 1:1 ratio exactly as predicted from the energy-optimized transition states, which were calculated for intramolecular 1,5-hydride shifts that produced each of the two C-22 diastereoisomers. Accordingly, these results support the hypothesis that an intramolecular 1,5-hydride shift mechanism promotes complete conversion of 1 to 2 under classical Wolff-Kishner reduction conditions.     

Potential bile acid metabolites. 9. 3,12-Dihydroxy- and 12 beta-hydroxy-5 alpha-cholanoic acids

Syntheses of the heretofore unreported 3 alpha, 12 beta-, 3 beta, 12 beta-dihydroxy-, and 12 beta-hydroxy-5 alpha-cholanic acids of the 5 alpha-series, their methyl esters, and some related derivatives are described. In addition, allodeoxycholic (3 alpha, 12 alpha-dihydroxy) acid was prepared by a new route. The principal reactions involved were the stereoselective reduction of C-12 ketones with an amino-borane reagent and of a C-3 ketone with K-Selectride, and inversion of a 3 beta-tosylate derivative with N,N-dimethylformamide.     

A method to determine 18 O kinetic isotope effects in the hydrolysis of nucleotide triphosphates

A method to determine 18 O kinetic isotope effects (KIEs) in the hydrolysis of GTP that is generally applicable to reactions involving other nucleotide triphosphates is described. Internal competition, where the substrate of the reaction is a mixture of 18 O-labeled and unlabeled nucleotides, is employed, and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18 O at sites of mechanistic interest also contains 13C at all carbon positions, whereas the 16 O-labeled nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by the use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink interface. Carbon isotope ratios can be determined with accuracy and precision greater than 0.04% and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1(333)-catalyzed hydrolysis of [beta18 O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (<0.1%). A single KIE measurement can be conducted in 25 min with less than 5 microg nucleotide reaction product.     

Advanced NMR approaches for a detailed structure analysis of natural products

Some new nuclear magnetic resonance (NMR) approaches to elucidate chemical structures, which have not been determined by routine NMR methods, are presented. Selective detection of methine (CH), methylene (CH(2)), or methyl (CH(3)) signals in each subspectrum by editing NMR methods was utilized to reduce the complexity in crowded spectra. It also increased the peak separation and enhanced the sensitivity by limiting the measuring area of the 2D spectra. Several 2D methods to measure (2,3)J(CH) values, which are useful for stereochemical assignment are then introduced. To determine the structure of a highly hydrogen-deficient molecule, efficient correlation methods for long-range (13)C-(13)C coupling and (1)H-(15)N HMBC are also described.     

Biosynthetic origin of hydrogen atoms in the lipase inhibitor lipstatin

The lipase inhibitor lipstatin is biosynthesized in Streptomyces toxytricini via condensation of a C(14) precursor and a C(8) precursor, which are both obtained from fatty acid catabolism. To study the mechanism of this reaction in more detail, S. toxytricini was grown in medium containing a mixture of U-(13)C,U-(2)H-lipids and unlabeled sunflower oil or in a medium containing 70% D(2)O. Lipstatin was isolated and analyzed by (1)H,(2)H, and (13)C NMR spectroscopy. Hydrogen atoms at C-2, C-3, and C-4 of lipstatin were found to be derived from solvent protons. The formation of the lipstatin precursor 3-hydroxy-Delta(5,8)-tetradecadienoyl-CoA by beta oxidation of linoleic acid explains the incorporation of solvent hydrogen into the 4 position of lipstatin. The hydrogen in position 3 of lipstatin is most probably introduced from solvent by proton/deuterium exchange of a redox cofactor involved in the reduction of the keto group in the branched chain beta keto acid arising by a decarboxylative condensation. The incorporation of solvent hydrogen at position 2 can be explained by epimerization of a chiral intermediate at C-2 and C-3. Epimerization may involve a dehydration-rehydration mechanism.     

Pathways of inclusion of isotopes 2H and 13C into exometabolites in course of glucose utilization by medusomycete]

The inclusion of 2H and 13C isotopes into the products of glucose utilization by medusomycete during its growth on deuterated media was studied by high-resolution NMR spectroscopy. Both unlabeled and 13C-labeled (in positions 1, 2, 6) glucose was used. It was shown that the glucose utilization proceeds by the classical Embden-Meyerhof-Parnas pathway. The incorporation of deuterium to the methyl group of ethanol can occur only during glucose-fructose-6-phosphate and phosphoenolpyruvate--pyruvate conversion. None of these stages by themselves is responsible for the existing distribution of deuterium atoms. The maximum inclusion of deuterium to the methyl group is no more than two atoms for the first glucose fragment (C1-C2-C3) and no more than one, for the second fragment (C4-C5-C6). The methylene group of ethanol is more accessible for deuterons because the proton surroundings of carbon atoms C2 and C5 completely changes. It was concluded that the maximum proton exchange occurs at positions C2 and C5; at positions C1, the proton exchange is lesser, and at position C6 it is the least. It was also shown that about 10% C1-C3 of triose leave the glycolysis cycle and are used in other processes.     

Analysis of 1H NMR-detectable mobile lipid domains for assessment of apoptosis induced by inhibitors of DNA synthesis and replication

Cell membrane rearrangements coincident with apoptosis may contribute to the increase in the ratio of methylene (CH(2) at 1.3 ppm) to methyl (CH(3) at 0.9 ppm) resonance signal intensity as observed by proton nuclear magnetic resonance ((1)H NMR). We studied CH(2) and CH(3) resonances in cultured cell lines treated with etoposide and fludarabine or bioflavonoid quercetin. Etoposide treatment (10 microM, 18 h) resulted in 3.3 fold increase of the CH(2)/CH(3) signal intensity ratio and 6.4 fold decrease in choline signal of MT4 cells. Incubation of Namalwa cells with fludarabine (3 microM, 72 h) increased the CH(2)/CH(3) signal intensity ratio by 2.4 fold and choline resonance intensity was unchanged. Quercetin treatment (30 microM, 1.5 month) increased CH(2)/CH(3) ratio by 2.1 fold. Necrotic cell death upon ethanol (20%) or DMSO (30%) treatment did not change the CH(2)/CH(3) signal intensity ratio. (1)H NMR-based study of mobile lipid domains is sensitive for detection of early engagement into apoptosis.     

Studies on the stereoselective synthesis of a protected α-D-Gal-(1→2)-D-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-D-Gal-(1→2)-D-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-D-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-D-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-D-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH(3) at C-1 and benzoyl groups at C-4 and C-6.     

Detection of early apoptotic changes in cells in vitro using nuclear magnetic resonance spectroscopy

The technique of proton magnetic resonance spectroscopy (1H MRS) was used as the sensitive express method for early specific detection of the apoptotic cells. The technique allows recognition of the changes in signal intensities corresponding to methylene (CH2) and methyl (CH3) protons of the mobile lipid domains (MLD) and choline, which are characteristic of apoptotic rather than of necrotic cells. A strong linear correlation between MLD content (calculated as CH2/CH3 signal intensity ratio) and the number of apoptotic cells in Namalwa or MT4 cell lines has been shown for any inducer of apoptosis used in the study. MLD content estimated by 1H MRS technique correlated significantly with apoptotic cells numbers (r = 0.992) recorded by conventional techniques. The increase in MLD content was registered as early as 60 min after the addition of etoposide coinciding with the time course of caspase-3 activation.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

Kinetic isotope effects of proton transfer in aqueous and methanol containing solutions,and in gramicidin A channels

The electrochemical conductivities of HCL and DCI were measured in: H(2)O and D(2)O; in methanol and fully deuterated methanol; and in water-methanol solutions. The single channel conductances to H(+) (g(H)) and D(+) (g(D)) in various gramicidin A (gA) ion channels incorporated in glycerylmonooleate planar bilayers were also measured. Kinetic isotope effects (KIE) were estimated from the ratio of conductivity measurements. In 1 and 5 M HCl aqueous solutions and in 1 M HCl+3.7 M methanol, the KIE ( approximately 1.35) is not different from values previously determined in dilute acid solutions. This suggests that the mobility of protons in those solutions is largely determined by proton transfer. In 10 M HCl, however, where the mobility of protons is likely to be determined by hydrodynamic diffusion, the measured KIE is considerably larger (1.47). Possible causes for this effect are discussed. The KIE of proton conductivities in 5 and 50 mM HCl in methanol and d-methanol is approximately 1.15. This is considerably smaller than the ratio between conductivities of 5 mM KCl in methanol and d-methanol (1.24). The KIE values (1.22-1.37) for g(H) in gA channels in 1 M HCl are significantly larger than for other monovalent cations and consistent with H(+) transfer. Methanol reduces g(H) in gA channels. The KIE of this effect is not different from the one measured in the absence of methanol. Possible mechanisms for the methanol-induced block of H(+) conductivities in solution and gA channels are discussed.     

Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.     

Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.     

Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates

Solavetivone, a potent antifungal phytoalexin, is derived from a vetispirane-type sesquiterpene, premnaspirodiene, by a putative regio- and stereo-specific hydroxylation, followed by a second oxidation to yield the alpha,beta-unsaturated ketone. Mechanistically, these reactions could occur via a single, multifunctional cytochrome P450 or some combination of cytochrome P450s and a dehydrogenase. We report here the characterization of a single cytochrome P450 enzyme, Hyoscyamus muticus premnaspirodiene oxygenase (HPO), that catalyzes these successive reactions at carbon 2 (C-2) of the spirane substrate. HPO also catalyzes the equivalent regio-specific (C-2) hydroxylation of several eremophilane-type (decalin ring system) sesquiterpenes, such as with 5-epi-aristolochene. Moreover, HPO displays interesting comparisons to other sesquiterpene hydroxylases. 5-Epi-aristolochene di-hydroxylase (EAH) differs catalytically from HPO by introducing hydroxyl groups first at C-1, then C-3 of 5-epi-aristolochene. HPO and EAH also differ from one another by 91-amino acid differences, with four of these differences mapping to putative substrate recognition regions 5 and 6. These four positions were mutagenized alone and in various combinations in both HPO and EAH and the mutant enzymes were characterized for changes in substrate selectivity, reaction product specificity, and kinetic properties. These mutations did not alter the regio- or stereo-specificity of either HPO or EAH, but specific combinations of the mutations did improve the catalytic efficiencies 10-15-fold. Molecular models and comparisons between HPO and EAH provide insights into the catalytic properties of these enzymes of specialized metabolism in plants.     

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.     

Stereoselective synthesis of some methyl-substituted steroid hormones and their in vitro cytotoxic activity against human gastric cancer cell line MGC-803

A series of 3-, 7-, 15-, and 16-methyl-substituted steroid analogs were synthesized via a highly stereoselective 1,6-conjugate addition. Under the catalysis of CuBr, AlMe₃ reacted with four steroid dienone precursors to afford either the corresponding α-epimer of C-3 and C-7 methyl-substituted steroids as the major products, and the ratio of α/β was up to 10/1. No β-epimer has been detected for methyl addition at C-16. However, under the same reaction conditions, enantioselective methyl addition at C-15 afforded the 15β-epimer as the major product. The preliminary SAR analysis showed that the methyl substituents at C-7α and C-15β positions lead to a dramatical increase in potency against human gastric cancer cell line MGC-803.