Synthesis and bioactivity of 6alpha- and 6beta-hydroxy analogues of castasterone

The reduction of castasterone with sodium in ethanol produced chiefly the known 6alpha-hydroxy stereoisomer, whereas reduction with sodium orohydride in methanol afforded mainly the novel 6beta-epimer. Both compounds showed variable bioactivity through four separate assays via the rice leaf lamina inclination bioassay. However, when treated with an appropriate statistical program to remove outliers, the averaged results clearly indicated that the two 6-hydroxy epimers possess comparable and significant bioactivity, which is, however, lower than that of castasterone or brassinolide. When applied together with 1000 ng of the auxin, indole-3-acetic acid (IAA), the activity of both the 6alpha and 6beta hydroxy epimers was enhanced by ca. one order of magnitude across a wide range of doses.     

28-Norcastasterone is biosynthesized from castasterone

Metabolic experiments with deuterium-labeled castasterone in seedlings of Arabidopsis thaliana, Oryza saliva and Lycopersicon esculentum, and cultured cells of Catharanthus roseus were performed, and the metabolites were analyzed by GC-MS. In all the plant species examined, [2H3]28-norcastasterone was identified as a metabolite of [26,28-2H6]castasterone, indicating that castasterone is the biosynthetic origin of 28-norcastasterone. Moreover, the natural occurrence of 28-norcastasterone and 28-nortyphasterol in seedlings of A. thaliana has been demonstrated. This is the first report of the natural occurrence of 28-nortyphasterol in plants.     

Spirostanic diosgenin precursors from Dioscorea composita tubers

The main saponin from the fresh tuber of Dioscorea composita was dioscin and from the fermented material 3-O-[α-l-rhamnopyranosyl(1→4)-β-d-glucopyranosyl]diosgenin. The ¹³C NMR chemical shifts of saponins were used in the determination of their structure. No free sapogenin was isolated from the fresh tuber.     

Novel biosynthetic pathway of castasterone from cholesterol in tomato

Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol --> cholestanol and 6-deoxo-28-norteasterone <--> 6-deoxo-28-nor-3-dehydroteasterone <--> 6-deoxo-28-nortyphasterol --> 6-deoxo-28-norcastasterone --> 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.     

Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum

An in vitro enzyme assay using radioisotope‐labeled ³H‐castasterone (³H‐CS) or ³²P‐ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography‐mass spectrometry (GC‐MS) analysis using non‐isotope‐labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C‐23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay. However, the fractions showed increased bioactivity after treatment with wheat germ acidic phosphatase (WGAP). Additionally, CS was identified from the hydrolysate by WGAP using GC‐MS analysis in both plants. In contrast, the polar fractions obtained from BR‐deficient mutants, Arabidopsis cyp85a2 and tomato d^x, did not show an increase in biological activity with WGAP treatment, and no free BRs, including CS, were detected in the hydrolysate. This suggests that CS phosphate is a naturally occurring biologically inactive conjugate that is generated when CS is normally synthesized in Arabidopsis and tomato plants. Taken together, these results suggest that phosphorylation of CS is an important conjugation process for the maintenance of the homeostatic level of an active BR and thus the regulation of the growth and development of plants.     

Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus

Feeding experiments with tritium- and deuterium-labeled castasterone (CS) were conducted with three cell lines of Catharanthus roseus, including crown gall cells and nontransformed cells. In all three cell lines, the conversion of CS to brassinolide (BL) was observed and unequivocally confirmed by gas chromatography/mass spectrometry (GC/MS). This is the first conclusive evidence that CS is the biosynthetic precursor of BL.Biosynthesis of brassinosteroids in Catharanthus roseus. Part II. Part I of this series: Yokota et al. (1990a).     

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.     

Regulation of castasterone level in primary roots of maize, Zea mays

Gas chromatography–mass spectrometry analysis revealed that primary roots of maize contain 28-norcastasterone (28-norCS) and its biosynthetic precursors, cholesterol, and cholestanol, which suggests that the C₂₇-brassinosteroid (C₂₇-BR) biosynthetic pathway to generate 28-norCS is operative in the roots. A cell-free enzyme solution prepared from maize roots successfully mediated C24-methylation of 28-norCS to produce castasterone (CS) with the aid of S-adenosyl- l -methionine, which indicates that CS can be generated through C₂₇-BR biosynthesis, as well as C₂₈-BR biosynthesis, in maize roots. Enzymatic conversion study using the cell-free enzyme solution demonstrated that CS is converted into 26-norCS in the enzyme solution. Exogenously applied 28-norCS and 26-norCS showed less activity than CS in the activation of gravitropic curvature and inhibition of root elongation. Taken together, a steady-state level of CS, the active BR in maize roots, seems to be strictly controlled by complicated processes such as C₂₈- and C₂₇-BR biosynthesis and biodegradation by C26-demethylation to exert its biological activity.     

2,3-Epoxybrassinosteroids are intermediates in the biosynthesis of castasterone in seedlings of Secale cereale

The involvement of the 2,3-epoxybrassinosteroids secasterone and 2,3-diepisecasterone in the biosynthesis of castasterone has been demonstrated in seedlings of Secale cereale by LC-ESI-MS. Deuterated secasterone, upon administration to rye seedlings, was incorporated into castasterone and its 2beta- and 3beta-epimers. Administration of deuterated 2,3-diepisecasterone resulted in castasterone and 2-epicastasterone. A biosynthetic subpathway from typhasterol/teasterone via 2,3-epoxybrassinosteroid intermediates to castasterone is discussed.     

Biosynthesis of brassinolide from teasterone via typhasterol and castasterone in cultured cells of Catharanthus roseus

The biosynthesis of brassinolide (BL) in crown gall and nontransformed cells of Catharanthus roseus in which BL, castasterone (CS), typhasterol (TY), and teasterone (TE) are endogenous was investigated using deuterated TY and TE as substrates. The metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and/or GC-selected ion monitoring (SIM). It was found that these cells converted TY to CS and BL, as well as TE to TY and CS. Because the pathway from CS to BL in the cells has already been confirmed, a biosynthetic sequence of TE TY CS BL was established. Reversible conversion between TE and TY was observed.Biosynthesis of brassinosteroids in Catharanthus roseus. Part III. Part II of this series: Suzuki et al. (1993).     

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

Glucosylation of cytokinin analogues

Glucosylation of adenine and 6-methylaminopurine was not detected in derooted 10-day-old radish seedlings. However, 4-(purin-6-ylamino)butanoic amide and 6-(3,4-dimethoxybenzylamino)purine (N⁶-substituted adenines with negligible cytokinin activity), like the highly active cytokinin 6-benzylaminopurine, were converted to 7-glucopyranosides. The N²-substituted guanine, 2-benzylaminopurin-6-one, and 6-benzylamino-2-(2-hydroxy-ethylamino)purine were also metabolized to glucosides which were probably 7-glucopyranosides. Hence glucosylation of purines is not restricted to N⁶-substituted adenines with strong cytokinin activity. Although only ca 1.6% of 6-benzylamino-9-(4-chlorobutyl)purine taken up by the derooted seedlings could be accounted for as 7- and 9-glucosides, a considerable proportion was metabolized to these glucosides in cotyledons excised from 2-day-old radish seedlings. The high cytokinin activity of this 9-substituted compound may be a consequence of cleavage of the 4-chlorobutyl groud at N-9.     

Covalent analogues of nucleobase-pairs

Covalently linked base pairs analogues consisting of purine-purine or purine-pyrimidine conjugates linked by carbon linkages of diverse length and configuration (ethylene, vinylene, acetylene and phenylene) were prepared.     

Synthesis of sorbistin analogues

D-Galactose was converted into the glycosylating agents 4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alpha-D-glucopyranosyl chloride (11) and the methyl beta-D-thiopyranoside 19. Condensation of 11 with 2,5-diazido-1,6-di-O-benzoyl-2,5-di-deoxy-L-iditol in the presence of mercury salts gave 24% of 2,5-diazido-3-O-(4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alp ha-D- glucopyranosyl)-1,6-di-O-benzoyl-2,5-dideoxy-L-iditol. Methyl trifluoromethanesulfonate-promoted glycosylation of 1,3-diazido-2-O-benzyl-1,3-dideoxy-5,6-O-isopropylidene-D-gulit ol with 19 in the presence of 2,6-di-tert-butyl-4-methylpyridine gave 1,3-diazido-4-O-(4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alp ha-D- glucopyranosyl)-2-O-benzyl-1,3-dideoxy-5,6-O-isopropylidene-D-gulitol (42), whereas, in the absence of base, migration of the O-isopropylidene group occurred, affording 1,3-diazido-6-O-(4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alp ha-D- glucopyranosyl)-2-O-benzyl-1,3-dideoxy-4,5-O-isopropylidene-D-gulitol in addition to 42.     

Evaluation of hadacidin analogues

Several derivatives of hadacidin have been developed and evaluated for activity against adenylosuccinate synthetase.     

Genotoxicity of alpha-asarone analogues

The role of cholesterol in the formation of atherosclerotic lesions during hypercholesterolemia has been confirmed. alpha-Asarone is a substance of a potent hypolipidemic activity which is isolated from plants. We previously described the synthesis of several alpha-asarone analogues exhibiting hypolipidemic and antiplatelet activity. Genotoxic activity of four selected alpha-asarone analogues was theoretically evaluated based on quantum-mechanical method for calculation of enthalpy of carbocations formation (DeltaH(R)) according to the Testa's method. In the present paper, we evaluated the mutagenic and genotoxic activity of alpha-asarone isomers 2-5 based on the reference Ames test and micronucleus test. Results obtained in the study show that tested isomers were non-mutagenic, however, they exhibited growing cytotoxic activity. Relationship between the heat of formation of their putative metabolic intermediates and mutagenic/genotoxic activity was not confirmed.     

New analogues of leucine-methionine-enkephalin

Nine analogues of the opioid pentapeptides leucine-/ methionine-enkephalinamide, involving replacement of amino acid at position 5 or amino acids at positions 2 and 5, have been synthesized by the solid phase method using mainly 9-fluorenylmethyloxycarbonyl amino acid trichlorophenyl esters in the presence of 1-hydroxybenzotriazole, the solid support being the Merrifield resin. All the analogues were effective in inhibiting the electri cally stimulated contractions of the guinea pig ileum (in vitro) and one of them, tyrosyl-Dnorvalyl-glycyl-phenylalanyl-methioninamide was found to be about 82 times more active than morphine. They also exhibited analgesic activity as well as antidiarrhoeal activity in mice (in vivo). Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.     

Synthesis of chacotriose analogues

We report here the synthesis of three chacotriose analogues, namely beta-L-fucopyranosyl-(1-->2)-[beta-L-fucopyranosyl-(1-->4)]-D-glucopyranose, beta-L-fucopyranosyl-(1-->2)-[beta-L-fucopyranosyl-(1-->4)]-d-galactopyranose, and alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->4)]-alpha-D-galactopyranose.     

Deletion analogues of transportan

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.