Bioactivity of 25-hydroxy-, 26-hydroxy, 25,26-dihydroxy- and 25,26-epoxybrassinolide.

The bioactivity of 25-hydroxybrassinolide, (25S)- and (25R)-26-hydroxybrassinolide, (25S)- and (25R)-25,26-dihydroxybrassinolide, and of (25R)-25,26-epoxybrassinolide was tested in the rice leaf lamina inclination assay. The 25- and (25S)-26-hydroxy derivatives are known metabolites of the naturally-occurring phytohormone brassinolide, whereas the other compounds are novel, but closely related, congeners. When tested alone, all showed either no activity or only weak activity at relatively high doses. When coapplied with indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids, enhanced bioactivity was observed for each compound. However, even when applied together with IAA, none of the compounds proved more bioactive than brassinolide with or without IAA. We conclude from these results that enzymatic hydroxylation of endogenous brassinolide at C-25 and/or C-26 does not enhance brassinosteroid activity, and so does not comprise an activation pathway in brassinolide biosynthesis. Instead, these hydroxylations result in modest to appreciable metabolic deactivation.     

Structure–Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity

A number of novel brassinosteroid analogues were synthesized and subjected to the rice leaf lamina inclination bioassay. Modified B-ring analogues included lactam, thiolactone, cyclic ether, ketone, hydroxyl, and exocyclic methylene derivatives of brassinolide. Those derivatives containing polar functional groups retained considerable bioactivity, whereas the exocyclic methylene compounds were devoid of activity. Analogues containing normal alkyl and cycloalkyl substituents at C-24 (in place of the isopropyl group of brassinolide) showed an inverse relationship between activity and chain length or ring size, respectively. The corresponding cyclopropyl and cyclobutyl derivatives were significantly more active than brassinolide and appear to be the most potent brassinosteroids reported to date. When synergized with the auxin indole-3-acetic acid (IAA), their bioactivity can be further enhanced by 1–2 orders of magnitude. The cyclopropyl derivative, when coapplied with the auxin naphthaleneacetic acid, gave a significant increase in yield of wheat in a field trial. Certain 25- and 26-hydroxy derivatives are known metabolites of brassinosteroids. All of the C-25 stereoisomers of 25-hydroxy, 26-hydroxy, and 25,26-dihydroxy derivatives of brassinolide were prepared and shown to be much less active than brassinolide. This indicates that they are likely metabolic deactivation products of the parent phytohormone. A series of methyl ethers of brassinolide was synthesized to block deactivation by glucosylation of the free hydroxyl groups. The most significant finding was that the compound where three of the four hydroxyl groups (at C-3, C-22, and C-23) had been converted to methyl ethers retained substantial bioactivity. This type of modification could, in theory, allow brassinolide or 24-epibrassinolide to resist deactivation and thus offer greater persistence in field applications. A series of nonsteroidal mimetics of brassinolide was designed and synthesized. Two of the mimetics showed significant bioactivity and one had bioactivity comparable to brassinolide, but only when formulated and coapplied with IAA. They thus represent the first nonsteroidal analogues possessing brassinosteroid activity.     

Synthesis and bioactivity of C-29 brassinosteroid analogues with different functional groups at C-6

In this paper, we report the synthesis and bioactivity of four synthetic analogues of 28-homobrassinosteroids, in order to evaluate the influence in bioactivity when the C-6 keto group is replaced by different functional groups. The synthetic analogues are 6-deoxo-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,22,23-tetraol], 6alpha-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,6alpha,22,23-pentaol], 6beta-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,6beta,22,23-pentaol], and [(22R,23R)-6alpha-fluorostigmasta-2alpha,3alpha,22,23-tetraol]. Results indicate that replacement of the 6-keto moiety by an beta or alpha hydroxyl group led to a decrease in activity, whereas the 6-deoxo analogue showed a very low activity, confirming the importance of an electronegative moiety at C-6 to observe hormonal potency. The 6alpha-fluorinated analogue elicited a low activity, similar to that of the 6-deoxo analogue.     

Chemical modification of the sugar part of methyl acarviosin: synthesis and inhibitory activities of nine analogues.

Nine analogues of methyl acarviosin (1), the core structure of acarbose and its homologues, the 6-hydroxy-(2), 6-azido-(3), 6-amino- (4), 6-acetamido-(5), 6-methoxy-(6), 6-hydroxy-2-O-methyl-(8), and 6-hydroxy-3-O-methyl derivatives (9), including the 5-methoxycarbonyl analogue (7) and 3,6-anhydro derivative (10) of 2, were synthesized by chemical modification of the sugar part of 2 derived by condensation of methyl 3,4-anhydro-alpha-D-galactopyranoside (17) and 4,7:5,6-di-O-isopropylidenevalienamine (26) or by direct coupling between 26 and the 6-substituted methyl 3,4-anhydro-alpha-D-galactopyranoside derivatives. Compounds 2 and 8 show notable inhibitory activity against yeast alpha-D-glucosidase almost comparable to that of 1. Introduction of a polar substituent at C-6 of 1 decreases the inhibitory activity. Interestingly, inversion of the conformation of the sugar part of 1 by introduction of the 3,6-anhydro bridge elicits almost no effect on the inhibitory activity.     

Brassinolide and [26, 28-2H6]brassinolide are differently demethylated by loss of C-26 and C-28, respectively, in Marchantia polymorpha

Metabolism of brassinolide in Marchantia polymorpha was investigated by use of in vivo suspension cultured cells. GC-MS analysis of metabolites derived from non-labelled brassinolide and [26, 28-2H6] brassinolide revealed that brassinolide was converted to 26-norbrassinolide while [26, 28-2H6]brassinolide to [26-2H3]28-norbrassinolide. It seems that Marchantia cells recognized [26, 28-2H6]brassinolide as a xenobiotic rather than brassinolide and deteriums attached to C-28 significantly affect demethylation reaction due to isotopic effect. Thus, demethylation of brassinolide in planta seems to proceed by loss of C-26 rather than C-28. The present finding is the first evidence for demethylation metabolism of brassinosteroids. The biological activity of 26-norbrassinolide was 10-fold reduced as shown by the rice lamina inclination test. However, because of its high biological activity, it remains difficult to conclude whether or not C-26 demethylation serves as an important deactivation process of brassinolide.     

Optimization of a rice lamina inclination assay for detection of brassinosteroids: I. effect of phytohormones on the inclination activity

Effect of ABA, GA₃, zeatin (Zea) and IAA on the inclination activity was examined by a rice lamina inclination assay using a Korean cultivar, Tongjin. Treatment with ABA, GA₃, Zea or IAA alone failed to increase the inclination response significantly at the concentration tested from 0.1 ppm to 10 ppm. However, treatment with 0.1 and 1 ppm ABA in the presence of brassinolide inhibited the inclination activity induced by treatment with brassinolide alone. And treatment with 0.1 and 1 ppm GA₃ or Zea in the presence of brassinolide strongly inhibited the inclination activity induced by treatment with brassinolide alone. On the other hand, the inclination activity by treatment with brassinolide alone was clearly promoted by treatment with 0.1 and 1 ppm IAA in the presence of brassinolide. Based on the synergistic effect induced by treatment with BR and IAA, we could develope an improved rice lamina inclination assay whose minimum detectable concentration of brassinolide is 0.00001 ppm /petri dish. The minimum detectable concentration in our assay was five times as low as that of the previous rice lamina inclination assay.     

Synthesis of 4-cyano- and 4-nitrophenyl 2,5-anhydro- 1,6-dithio-alpha-D-gluco- and -alpha-L-guloseptanosides carrying different substituents at C-3 and C-4

Treatment of 1,6:2,5-dianhydro-3,4-di-O-methanesulfonyl-1-thio-D-glucitol in methanol with sodium hydroxide afforded 1,6:2,5:3,4-trianhydro-1-thio-allitol, 1,4:2,5-dianhydro-6-methoxy-1-thio-D-galactitol, 1,6:2,5-dianhydro-4-O-methyl-1 -thio-D-glucitol, 1 ,6:2,5-dianhydro-3-O-methanesulfonyl-1 -thio-D-glucitol and 1 ,6:2,5-dianhydro-4-deoxy-1-thio-D-erythro-hex-3-ulose (14) in 5, 4, 28, 5.5 and 41% yield, respectively. Formation of these derivatives can be explained via a common sulfonium intermediate. Reduction of 14 with sodium borohydride and subsequent acetylation afforded 3-O-acetyl-1,6:2,5-dianhydro-4-deoxy-1-thio-D-xylo-hexitol, the absolute configuration of which was proved by X-ray crystallography. The 1,6:2,5-dianhydro-1-thio-D-hexitol derivatives in which the free OH groups were protected by acetylation, methylation or mesylation were converted by a Pummerer reaction of their sulfoxides into the corresponding 1-O-acetyl hexoseptanose derivatives which were used as donors for the glycosidation of 4-cyano- and 4-nitrobenzenethiol, respectively. The Pummerer reaction of 1,6:2,5-dianhydro-4-deoxy-3-O-methyl-1-thio-D-xylo-hexitol S-oxide gave, besides 1-O-acetyl-2,5-anhydro-3-deoxy-4-O-methyl-6-thio-alpha-L- (23) and 1-O-acetyl-2,5-anhydro-4-deoxy-3-O-methyl-6-thio-alpha-D-xylo-hexoseptanose (25), 1-O-acetyl-4-deoxy-2,6-thioanhydro-D-lyxo-hexopyranose, formed in a rearrangement reaction. The same rearrangement took place, when a mixture of 23 and 25 was used as donor in the glycosidation reaction with 4-cyanobenzenethiol, applying trimethylsilyl triflate as promoter. The oral antithrombotic activity of the obtained alpha-thioglycosides was determined in rats, using Pescador's model.     

C-25 epimeric 26-haloponasterone A: synthesis, absolute configuration and moulting activity

A convenient synthesis of inokosterone has been accomplished. Inokosterone exists as two C-25 epimers, which could be separated from each other through their diacetonide derivatives. The absolute configuration of these compounds was determined. Two C-25 epimers of 26-chloroponasterone A were synthesized from the respective C-25 epimeric inokosterone. Two epimeric 26-bromo and 26-iodoponasterone A compounds were also synthesized. Moulting activity of these compounds was evaluated using the Musca bioassay, and it was found that the (25S)-26-halo analogues were more active than the corresponding (25R)-26-halo analogues. Among the 25S series, an increase in activity with an increase in size of the halogen atom was observed, indicating that the steric factor was more important than the electronic factor in binding of these ecdysteroid analogues to the receptor. On the other hand, a decrease in activity with an increase in size of the halogen atom was noted in the 25R series, suggesting that the steric factor was less important than the electronic factor. The results indicated that the configuration at C-25 and the substituent at C-26 have significant influences on the interaction of ecdysteroids with their receptor.     

First derivatives of myo-inositol 1,4,6-trisphosphate modified at positions 2 and 3: structural analogues of D-myo-inositol 1,4,5-trisphosphate

Novel, structurally modified potential mimics of the second messenger D-myo-inositol 1,4,5-trisphosphate, based on the biologically active regioisomer D-myo-inositol 1,4,6-trisphosphate, were synthesised. DL-5-O-Benzyl-1,4,6-tri-O-p-methoxybenzyl-myo-inositol was the key intermediate for the preparation of the following compounds: DL-3-deoxy-, DL-3-deoxy-2-O-methyl-, DL-3-O-(2-hydroxyethyl)-, DL-3-O-(3-hydroxypropyl)- and DL-3-O-(4-hydroxybutyl)-myo-inositol 1,4,6-trisphosphate. DL-1,4,6-Tri-O -allyl-5-O-benzyl-myo-inositol was used to prepare DL-2-O-methyl-myo-inositol 1,4,6-trisphosphate. Deoxy-compounds were prepared by reduction of the corresponding tosylated intermediate using Super Hydride. The hydroxyalkyl groups were introduced at the C-3 of myo-inositol using the corresponding benzyl protected hydroxy alkyl bromide via the cis-2,3-O-dibutylstannylene acetal. Methylation and benzylation at C-2 was accomplished using methyl iodide and benzyl bromide, respectively, in the presence of sodium hydride. Deblocking of p-methoxybenzyl groups was accomplished with TFA in dichloromethane and the allyl groups were removed by isomerisation to the cis-prop-1-enyl derivative, which was hydrolysed under acidic conditions to give the corresponding 1,4,6-triol. The 1,4,6-triols were phosphitylated with the P(III) reagent bis(benzyloxy)(diisopropylamino)phosphine in the presence of 1H-tetrazole then oxidised with 3-chloroperoxybenzoic acid followed by deblocking by hydrogenolysis to give DL-2-O-methyl-, DL-3-O-deoxy-, DL-3-O-deoxy-2-O-methyl-, DL-3-O-(2-hydroxyethyl)-, DL-3-O-(3-hydroxypropyl)- and DL-3-O-(4-hydroxybutyl)-myo-inositol 1,4,6-trisphosphate, respectively.     

Preparation and antimicrobial activity of hydroxypropyl chitosan

Water-soluble hydroxypropyl chitosan (HPCS) derivatives with different degrees of substitution (DS) and weight-average molecular weight (Mw) were synthesized from chitosan and propylene epoxide under basic conditions. Their structure was characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis, which showed that both the OH groups at C-6 and C-3 and the NH2 group of chitosan were alkylated. The DS value of HPCS ranged from 1.5 to 3.1 and the Mw was between 2.1x10(4) and 9.2x10(4). In vitro antimicrobial activities of the HPCS derivatives were evaluated by the Kirby-Bauer disc diffusion method and the macrotube dilution broth method. The HPCS derivatives exhibited no inhibitory effect on two bacterial strains (Escherichia coli and Staphylococcus aureus); however, some inhibitory effect was found against four of the six pathogenic fruit fungi investigated. Some derivatives (HPCS1, HPCS2, HPCS3, HPCS3-1, and HPCS4) were effective against C. diplodiella and F. oxysporum. HPCS3-1 is the most effective one with MIC values of 5.0, 0.31, 0.31, and 0.16mg/mL against A. mali, C. diplodiella, F. oxysporum, and P. piricola, respectively. Antifungal effects were also observed for HPCS2 and HPCS3-1 against A. mali, as well as HPCS3 and HPCS3-1 against P. piricola. The results suggest that relatively lower DS and higher Mw value enhances the antifungal activity of HPCS derivatives.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Transglycosylation reaction of Mucor hiemalis endo-beta-N-acetylglucosaminidase using sugar derivatives modified at C-1 or C-2 as oligosaccharide acceptors

We investigated the transglycosylation reaction of the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii using such sugar derivatives as N-acylated d-glucosamines, C-glucosyl derivatives, and a 2-O-glycosylated disaccharide as acceptors. We found that a variety of sugar derivatives modified at C-1 or C-2 could be used as acceptors for transglycosylation by Endo-M to create novel oligosaccharides.     

Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

The structure of a galactan sulfate from the red seaweed Bostrychia montagnei

The sulfated, methylated galactan isolated from the red seaweed Bostrychia montagnei, showed an unusually narrow structural dispersion. This agaran has the defining linear backbone of alternating 3-linked beta-D-galactopyranosyl units and 4-linked alpha-L-galactopyranosyl and 3,6-anhydrogalactopyranosyl residues. The D-units have C-6 methylation, C-6 single stubs of xylopyranosyl and minor to trace amounts of (possible) C-6 linked single stubs of galactopyranosyl. These units are mainly sulfated on C-4 with lesser sulfation at C-6 and minor at C-2. The L-residues are mainly methylated on C-2 of the 3,6-anhydrogalactopyranosyl and sulfated on C-3 of the L-galactopyranosyl; minor amounts of 2,3- and 3,6-disulfated and 2-O-methyl or 2-O-glycosyl 3-sulfated L-galactopyranosyl were also found.     

Studies on structure-activity relationship of sphaeropsidins A-F, phytotoxins produced by Sphaeropsis sapinea f. sp. cupressi

Six forms of sphaeropsidins (SA-SF), three- and tetra-cyclic unrearranged pimarane diterpenes produced by Sphaeropsis sapinea f. sp. cupressi, as well as eight derivatives obtained by chemical modification of SA-SC, were assayed for their bioactivity. The effect of each compound on plants which are host or non-host of the pathogen was investigated. Activity on some plant pathogenic fungi was also tested. Some structure-activity relationships have been identified for both phytotoxic and antifungal activity. It appears that the integrity of the tricyclic pimarane system, the preservation of the double bond C(8)-C(14), the tertiary hydroxyl group at C-9, the vinyl group at C-13, and the carboxylic group at C-10 as well as the integrity of the A-ring provide these molecules with non selective phytotoxic and antimycotic activity.     

Effect of natural and synthetic benzyl benzoates on calmodulin

The present investigation describes the effect of the spasmolytic benzylbenzoates 1-9 from Brickellia veronicifolia on CaM using a functional in vitro enzymatic assay. Bovine brain PDE1 was used as a monitoring enzyme. The most active natural inhibitors of the system CaM-PDE1 were benzyl benzoates 3-5, which inhibited the activity of PDE1 in a concentration-dependent manner. In addition, three series of analogs of compound 4, compounds 10a-32a, were prepared and assayed. The benzyl benzoates from the first series, namely 10a-24a, possess no substituents on ring B but different number and position of hydroxyl or methoxy groups in ring A. The second group (25-32a), on the other hand, possesses an A ring identical to that on compound 4, but different substituents in Ring B. The most active compounds were 14a, 15a and 30a. These compounds were two to six times more potent than chlorpromazine, a well known CaM inhibitor. Benzyl benzoates 14a and 15a have methoxyl groups at C-2/C-4 and C-3/C-4 in ring A, respectively; while 30a, in addition to the methoxyl groups at C-2/C-6 of ring A, hold a benzoyloxy moiety at C-3' of ring B. Kinetic studies revealed that compounds 3, 4, 14a, 15a and 30a behave as competitive CaM antagonists.     

Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-glutamyl carboxylase activity than MK-4 both in vitro and in vivo

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.     

The structure of the agaran sulfate from Acanthophora spicifera (Rhodomelaceae, Ceramiales) and its antiviral activity. Relation between structure and antiviral activity in agarans

The sulfated agaran isolated by water extraction from the red seaweed, Acanthophora spicifera (Rhodomelaceae, Ceramiales), is made up of A-units highly substituted with sulfate groups on C-2 (28-30%), sulfates on C-2 and 4,6-O-(1'-carboxyethylidene) groups (9-15%), and only the C-2 sulfate groups (5-8%) with small amounts of C-6 sulfate, 6-O-methyl, and nonsubstituted residues. B-units are formed mainly by 3,6-anhydro-alpha-L-galactose (15-16%) and its precursor, alpha-L-galactose 6-sulfate (10-17%), together with lesser amounts of 3,6-anhydro-alpha-L-galactose 2-sulfate, alpha-L-galactose 2,6-disulfate, alpha-L-galactose 2,3,6-tri-sulfate, alpha-L-galactose 2,6-disulfate 3-xylose, 2-O-methyl-alpha-L-galactose, and unsubstituted alpha-L-galactose. Small, but significant quantities of beta-D-xylose were found in all the fractions, together with small amounts to traces of D-glucose. Some of the fractions have high antiviral activity. Attempts to correlate structure and antiviral activity in agarans are presented.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

New chiral selectors: design and synthesis of 6-TBDMS-2,3-methyl beta-cyclodextrin 2-2' thioureido dimer and 6-TBDMS-2,3-methyl (or 2-methyl-3-acetyl) beta-cyclodextrin bearing an (R) Mosher acid moiety

Cyclodextrin (CD) derivatives are important selectors for analytical chiral recognition. Their enantioselectivities and chemical properties depend on ring size and on nature, number and location of substituents. This paper describes the synthesis of 6-O-TBDMS-2,3-O-methyl beta-cyclodextrins bearing in position 2 either a single (R)-Mosher acid moiety or a second CD unit, in view of their possible application as chiral selectors. Most synthetic steps were successfully carried out under high-intensity ultrasound using a new sonochemical reactor developed in the authors' laboratory. 6-O-TBDMS-2-O-methyl-3-[(S)-2-methylbutyl]-beta-CD was also synthesized and tested with gas chromatography; the enantiorecognition power of the other CD derivatives is also being tested. A computational study of model structures to design these CD derivatives.