Identification of lactose ureide, a urea derivative of lactose, in milk and milk products.

With the widespread consumption of milk, the complete characterization of the constituents of milk and milk products is important in terms of functionality and safety. In this study, a novel nonreducing carbohydrate was separated from powdered skim milk and was identified using electron spray ionization-mass spectrometry (m/z 385.1[M + H(+)]), ¹H, ¹³C, ¹H¹H-correlation spectroscopy, and heteronuclear single quantum-nuclear magnetic resonance spectra. The carbohydrate was identified as a lactose derivative of urea, N-carbamoyl-o-ß-D-galactopyranosyl-(1-4)-D-glucopyranosylamine (lactose ureide, LU). For the HPLC analysis of LU in milk and milk products, benzoylated LU, hepta-o-benzoyl lactose ureide (melting point 137-139°C; m/z 1,113 [M + H⁺]; wavelength of maximum absorption, λ(max), 229 nm; molar extinction coefficient, ε, 8.1037 × 107), was used as a standard. The crude nonreducing carbohydrate fraction from raw milk, thermally processed milk, and milk products such as powdered milks were directly benzoylated and subjected to HPLC analysis using an octadecylsilyl column to determine the quantity of LU. The content of LU in 10% solutions of powdered skim milk and powdered infant formula (5.0±1.1 and 4.9±1.5 mg/L, respectively) were almost 3-fold higher than that of UHT milk (1.6±0.5 mg/L) and higher than that of low-temperature, long-time-processed (pasteurized at 65°C for 30 min) milk (1.2±0.3 mg/L) and the fresh raw milk sample (0.3±0.1 mg/L). A time-course of the LU content in raw milk during heating at 110°C revealed that LU increased with time. From these results, it is likely that LU is formed by the Maillard-type reaction between the lactose and urea in milk and milk products. Because the concentration of LU in milk increased with the degree of processing heat treatment, it could serve as an indicator of the thermal deterioration of milk. Although it is known that the human intestine is unable to digest LU, the gastrointestinal bacteria in human subjects are able to digest and utilize urea nitrogen in formation of essential amino acids that are available to the host human. These findings suggest that LU in milk might have a functional role in human health.

Synthesis of (-)-methyl shikimate via enzymatic resolution of (1S*, 4R*, 5R*)-4-hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one.

The synthesis of methyl (-)-shikimate [(-)-2] was achieved via lipase-catalyzed optical resolution of (1S*, 4R*, 5R*)-4-hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one (3). Transesterification of (+/-)-3 and vinyl acetate with lipase MY and subsequent hydrolysis gave optically pure (-)-3. This compound was converted to (-)-2 in two steps.

Synthesis of macrotetrolide alpha, a designed polynactin analog composed of (+)- and (-)-bishomononactic acids.

Macrotetrolide alpha, a designed analog of polynactin composed of (+)- and (-)-bishomononactic acids, was synthesized. These monomers were prepared via optical resolution of the corresponding (S)-O-acetylmandelates. Assembly of the monomers to macrotetrolide alpha took seven steps without any loss of the intermediates.

Three new taxoids from the leaves of the Japanese yew, Taxus cuspidata.

Three new taxane diterpenoids were isolated from the leaves of the Japanese yew, Taxus cuspidta. Their structures were established as 5 alpha,13 alpha-diacetoxy-taxa-4(20),11-diene-9 alpha,10 beta-diol (1), 7 beta,13 alpha-diacetoxy-5 alpha-cinnamyloxy-2(3-->20)-abeo-taxa-4(20),11-diene-2 alpha,10 beta-diol (2), and 2 alpha,10 beta,13 alpha-triacetoxy-taxa-4(20),11-diene-5 alpha,7 beta,9 alpha-triol (3) respectively on the basis of 1D and 2D NMR data.

Four polyoxygenated cyclohexenes from the Chinese tree, Uvaria purpurea.

Two novel polyoxygenated cyclohexenes, 6-methoxyzeylenol (3) and 2-acetoxyzeylenone (4), together with two known compounds, zeylenol (1) and zeylenone (2), were isolated from the heartwood of the Chinese tree, Uvaria purpurea. Zeylenol (1) and zeylenone (2) both showed inhibitory activity toward the root growth of Lactuca sativa. Their structures were established by spectroscopic and chemical methods.

Identification of 2,3-dihydro-gamma-ionylideneethanol in Cercospora cruenta.

During our scrutiny of GC-EI-MS date for C15 alcohols as putative intermediates on the ABA biosynthetic pathway in Cercospora cruenta, a trace amount of 5-[2',2'-dimethyl-6'-methylene-1'-cyclohexyl]-3-methyl-4-penten-1-ol (2,3-dihydro-gamma-ionylideneethanol) was identified. Feeding experiments indicated that this compound was not an intermediate to ABA, but a catabolite that originated from gamma-ionylideneacetaldehyde. The stereochemistry of 2,3-dihydro-gamma-ionylideneethanol was deduced to be (3R,1'S) from a comparison with an authentic specimen prepared via baker's yeast asymmetric reduction.

Microbial hydroxylation of (+/-)- and (-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid into (+/-)- and (-)-xanthoxin acid by Cunninghamella echinulata.

Microbial hydroxylation of (+/-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid (3a) with Cercospora cruenta, a fungus producing (+)-abscisic acid, gave a four-stereoisomeric mixture consisting of (+)- and (-)-xanthoxin acid (4a), and (+)- and (-)-epi-xanthoxin acid (5a) by an HPLC analysis with a chiral column. Screening of the microorganisms capable of oxidizing (+/-)-3a showed that Cunninghamella echinulata stereoselectively oxidized (+/-)-3a to xanthoxin acid (4a) with the some degree of enantioselectivity as (-)-3a to (-)-4a.

New 11(15-->1)abeotaxane, 11(15-->1),11(10-->9)bisabeotaxane and 3,11-cyclotaxanes from Taxus yunnanensis.

Chemical examination of the seeds of Chinese yew, Taxus yunnanensis Cheng et L. K. Fu resulted in the isolation of an 11(15-->1)abeotaxane, an 11(15-->1), 11(10-->9)bisabeotaxane and two 3,11-cyclotaxanes. The structures of these new taxoids were established as 13alpha-acetoxy-5alpha-cinnamoyloxy-11(15-->1)abeotaxa-4(20),11-diene-9alpha,10beta,15-triol (1), 20-acetoxy-2alpha-benzoyloxy-4alpha, 5alpha, 7beta, 9alpha, 13alpha-pentahydroxy-11(15-->1), 11(10-->9) bisabeotax-11-eno-10,15-lactone (2), 2alpha,10beta-diacetoxy-5alpha-cinnamoyloxy-9alpha-hydroxy-3,11 -cyclotax-4(20)-en-13-one (3) and 10beta-acetoxy-2alpha,5alpha,9alpha-trihydroxy-3,11-cyclotax-4(20)-en-13-one (4) on the basis of spectral analyses.

Synthesis of southern (C1'-C11') and eastern (C8-C18) fragments of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger.

Synthesis of the southern C1'-C11' and eastern C8-C18 fragments of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger, was achieved. The southern fragment was synthesized by using the Evans aldol reaction and cis-selective iodoetherification as the key steps in a 9.6% overall yield (7 steps). The eastern fragment was constructed via the Julia coupling reaction and cis-selective iodoetherification in a 3.0% overall yield (8 steps from the known epoxide).

Early biosynthetic pathway to abscisic acid in Cercospora cruenta.

A new biosynthetic intermediate of ABA, (2Z,4E)-gamma-ionylideneacetaldehyde, was isolated from young mycelia of Cercospora cruenta. Under an (18)O2 atmosphere, an oxygen atom of this endogenous aldehyde was exclusively labeled. Similarly, three (18)O atoms were incorporated into the ABA molecule recovered after prolonged incubation; selectively labeled were one of the carboxyl oxygen atoms and the two on the ring portion of ABA. A feeding experiment with [1-(13)C]glucose proved the exclusive operation of the mevalonate pathway for the formation of both ABA and beta-carotene. These results suggest that (2Z,4E)-gamma-ionylideneacetaldehyde can be a key ABA biosynthetic intermediate formed by the oxidative cleavage of a carotenoid precursor.

The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves.

Abscisic acid (ABA) is a plant hormone involved in seed development and germination and in responses to various environmental stresses. The last step of ABA biosynthesis involves oxidation of abscisic aldehyde, and aldehyde oxidase (EC ) is thought to catalyze this reaction. An aldehyde oxidase isoform, AOdelta, encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3, and AAO4), is the most likely candidate for the enzyme, because it can efficiently catalyze the oxidation of abscisic aldehyde to ABA. Here, we report the isolation and characterization of an ABA-deficient Arabidopsis mutant that maps at the AAO3 locus. The mutant exhibits a wilty phenotype in rosette leaves, but seed dormancy is not affected. ABA levels were significantly reduced in the mutant leaves, explaining the wilty phenotype in rosettes, whereas the level in the mutant seeds was less reduced. No AOdelta activity could be detected in the rosette leaves of the mutant. Sequence data showed that the mutant contains a G to A substitution in the AAO3 gene. The mutation causes incorrect splicing of the ninth intron of AAO3 mRNA. We thus conclude that the ABA-deficient mutant is impaired in the AAO3 gene and that the gene product, AOdelta, is an aldehyde oxidase that catalyzes the last step of ABA biosynthesis in Arabidopsis, specifically in rosette leaves. Other aldehyde oxidases may be involved in ABA biosynthesis in other organs.

Taxane diterpenoids from Taxus yunnanensis and Taxus cuspidata.

Chemical examination of the seeds of the Chinese yew, Taxus runnanensis Cheng et L. K. Fu and the Japanese yew, Taxus cuspidata Sieb et Zucc, resulted in the isolation of four taxane diterpenoids. The structures of these taxoids were established as (12alpha)-2alpha-acetoxy-5alpha,9alpha, 10beta-trihydroxy-3,11-cyclotax-4(20)-en-13-one; 2alpha,7beta,13alpha-triacetoxy-5alpha, 9alpha-dihydroxy-2(3-->20)abeotaxa-4(20),11-dien-10-one; 9alpha,10beta-diacetoxy-5alpha-cinnamoyloxytaxa- 4(20),11-dien-13alpha-ol and the known 2alpha,7beta,9alpha,10beta,13-pentaacetoxytax a-4(20),12-diene-5alpha,11beta-diol on the basis of spectral analysis.

Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana.

Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOalpha, AObeta and AOgamma, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOdelta, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AO delta was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOdelta. These results indicate that AOdelta is encoded by AAO3. AOdelta produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 microM), and the oxidation product was determined by gas chromatography-mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOdelta, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.

Synthesis of (+/-)-methyl bishomononactate, a monomeric component of polynactin antibiotics.

The synthesis of (+/-)-methyl bishomononactate, one of the monomeric components of polynactin antibiotics (macrotetrolides), was achieved via cis-selective iodoetherification as the key step.

Enzymatic resolution of (+/-)-epoxy-beta-cyclogeraniol, a synthetic precursor for abscisic acid analogs.

Lipase-catalyzed optical resolution of (+/-)-epoxy-beta-cyclogeraniol (1), a key synthetic intermediate for epoxy-beta-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (-)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E = 1600).

Syntheses of (+/-)-methyl 6'alpha-demethyl-6'alpha-cyanoabscisate and (+/-)-methyl 6'alpha-demethyl-6'alpha-methoxycarbonylabscisate.

New abscisic acid analogs possessing a cyano or methoxycarbonyl group at the 6'alpha-position of methyl abscisate were synthesized by regioselective hydrocyanation. These compounds had weak activity in the rice second leaf sheath elongation test.

New taxane diterpenoid from seed of the Chinese yew, Taxus yunnanensis.

A novel taxane diterpenoid with a rearranged 5/7/6-membered ring system was isolated from seeds of the Chinese yew, Taxus yunnanensis. Its structure was established as 9alpha,13alpha-diacetoxy-10beta-benzoxy-5alpha- cinnamoyl-11(1 5-->1)-abeotaxa-4(20),11-dien-15-ol on the basis of a spectroscopic analysis. Its relative stereochemistry is proposed from the results of NOESY experiments.

Two novel taxane diterpenoids from the needles of Japanese yew, Taxus cuspidata.

Two novel taxane diterpenoids were isolated from the needles of Japanese yew, Taxus cuspidata, and their structures were determined to be lbeta-hydroxy-7beta-acetoxytaxinine (1) and lbeta,7beta-dihydroxytaxinine (2) on the basis of spectral analyses including 2D-NMR studies.

Three new taxoids from the seeds of Japanese yew, Taxus cuspidata.

Five taxane diterpenoids were isolated from the seeds of the Japanese yew, Taxus cuspidata Sieb. et Zucc. The structures were established as 2 alpha,7 beta,13 alpha-triacetoxy-5 alpha-(3'-dimethylamino-3'-phenyl)-propionyloxy-2(3-->20)-aboe+ ++-taxa-9,10-dione (1) and 2 alpha,7 beta,9 alpha,10 beta-tetraacetoxy-5 alpha-[(2R,3S),N,N-dimethyl-3-phenylisoseryloxy]-taxa-4(20),11- dien-13-one (2), 2 alpha, 9 alpha, 10 beta-triacetoxy-5 alpha-cinnamoxy-taxa-4(20),11-diene-13 alpha-ol (3), taxezopidine J (4), and taxuspine D (5) on the basis of 1D, 2D NMR, and MS spectral analysis. Compounds 1, 2, and 3 are new compounds.

Synthesis and biological evaluation of novel 9-functional heterocyclic coupled 7-deoxy-9-dihydropaclitaxel analogue.

Novel 9-functional heterocyclic coupled 7-deoxy-9-dihydropaclitaxel analogues 17 and 22-24 synthesized from a natural taxoid 5-cinnamoyltriacetyltaxicin-I (3) and their biological evaluation in tubulin assembly activity and cytotoxicity in vitro against several human tumor cell lines are first presented. The biologically tested results show that 17, 22 and 23 are inactive in tubulin assembly assay and have no more remarkable cytotoxicities against human tumor cell lines SK-OV3, WIDR and MCF-7, though 22 and 23 exhibit more potent cytotoxicity against human liver cancer and human esophagus cancer cell lines (BEL-7402 and ECa-109) than paclitaxel.