Some non-anomerically C-C-linked carbohydrate amino acids related to leucine-synthesis and structure determination

(5'R)-5'-Isobutyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione was synthesised starting from methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside via methyl 6-deoxy-6-isopropyl-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose applying the Bucherer-Bergs reaction. Its 5'-R configuration was confirmed by X-ray crystallography. Corresponding alpha-amino acid-methyl (5R)-5-amino-5-C-carboxy-5,6-dideoxy-6-isopropyl-alpha-D-lyxo-hexofuranoside (alternative name: 2-[methyl (4R)-beta-L-erythrofuranosid-4-C-yl]-D-leucine) was obtained from the above hydantoin by acid hydrolysis of the isopropylidene group followed by basic hydrolysis of the hydantoin ring. Analogous derivatives with 5S configuration, formed in a minority, were also isolated and characterised.     

Chemical synthesis of methyl 6'-alpha-maltosyl-alpha-maltotrioside and its use for investigation of the action of starch synthase II

The branched pentasaccharide methyl 6'-alpha-maltosyl-alpha-maltotrioside was chemically synthesised and investigated as a primer for particulate starch synthase II (SSII) using starch granules prepared from the low-amylose pea mutant lam as the enzyme source. For chemical synthesis, the trichloroacetimidate activation method was used to synthesise methyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->6)-O-[(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl-(1-->4)]-O-(2,3-di-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-glucopyranoside, which was then debenzylated to provide the desired branched pentasaccharide methyl 6'-alpha-maltosyl-alpha-maltotrioside as documented by 1H and 13C NMR spectroscopy. Using a large excess of the maltoside, the pentasaccharide was tested as a substrate for starch synthase II (SSII). Both of the non-reducing ends of methyl 6'-alpha-maltosyl-alpha-maltotrioside were extended equally resulting in two hexasaccharide products in nearly equal amounts. Thus, SSII catalyses an equimolar and non-processive elongation reaction of this substrate. Accordingly, the presence of the alpha-1,6 linkages does not dictate a specific structure of the pentasaccharide in which only one of the two non-reducing ends are available for extension.     

Induction of pilocarpine formation in jaborandi leaves by salicylic acid and methyljasmonate

Jaborandi seedlings were subjected to different treatments in order to study the induction of pilocarpine in the leaves. In addition four extraction methods were assessed to extract the alkaloid from dried leaves. The highest yielding extraction and recovery was observed when dried leaves were first treated with base and then extracted with chloroform. Salt stress (NaCl), wounding, hypoxia, and N and K omission of the nutrient soln caused reductions in pilocarpine contents. Whereas complete nutrient soln and P omission maintained normal levels of the alkaloid. Salicylic acid and methyljasmonate induced a 4-fold increase of pilocarpine, but this increase was dependent on the concentration and time after exposure.     

Biosynthesis of 14,15-dehydro-12-oxo-phytodienoic acid and related cyclopentenones via the phytoprostane D(1) pathway

A novel group of cyclopentenone prostaglandin-like compounds, deoxy phytoprostanes J(1), together with their precursors, phytoprostanes D(1), were identified in tobacco, tomato and Arabidopsis. Previously, it was thought that 14,15-dehydro-12-oxo-phytodienoic acid, a member of the deoxy phytoprostanes J(1) family, is derived from either 12-oxo-phytodienoic acid or diketols via the allene oxide synthase pathway. Results suggest that 14,15-dehydro-12-oxo-phytodienoic acid as well as structurally related cyclopentenones of the chromomoric acid family are synthesized via the phytoprostane D(1) pathway in planta. Notably, 14,15-dehydro-12-oxo-phytodienoic acid is more abundant than 12-oxo-phytodienoic acid in all three species so far analyzed.     

X-ray and conformational analysis of methyl 3-amino-2,3-dideoxy-alpha-D-arabino-hexopyranoside

The structure, conformation and configuration of methyl 3-amino-2,3-dideoxy-alpha-d-arabino-hexopyranoside were confirmed by (1)H NMR, (13)C NMR and IR spectroscopy, as well as by optical rotation. The structure of the compound studied was also determined by single crystal X-ray crystallography at 293 K and refined to a final R=0.0521 based on 1798 independent reflections. The title compound crystallized in the tetragonal space group P4(3) with a=6.572(1) angstrom, b=6.572(1) angstrom, c=41.161(8) angstrom, D(c)=1.324 Mgcm(-3) and V=1777.8(5) angstrom(3) for Z=8. The packing arrangement in the unit cell displayed a stratified structure. Moreover, medium-strength N-H. . .O and O-H. . .O hydrogen bonds, which stabilized the 3-D structure of compound I, were observed.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

Synthesis and biological evaluation of a radiolabeled analog of methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside directed towards influencing cellular glycosaminoglycan biosynthesis.

Two methods are presented for the synthesis of methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside. The first method employs the Barton-McCombie deoxygenation methodology, and the second method utilizes an oxidation-beta-elimination methodology that allows for the incorporation of hydrogen isotopes into the title compound. Hence, methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside (4) and methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside-6-t (14) were synthesized and evaluated for their ability to inhibit hepatocyte, cell-surface glycosaminoglycan biosynthesis and to incorporate a [(3)H] radiolabel into isolated glycosaminoglycans, respectively. Compound 4, at a concentration of 1.0 mM, demonstrated a reduction of D-[(3)H]glucosamine and [(35)S]sulfate incorporation into isolated glycosaminoglycans by 69 and 59%, of the control cultures, respectively. At 10 and 20 mM, 4 demonstrated a maximum inhibition of incorporation of both radiolabels to approximately 10% of the control cultures. Compound 14 demonstrated a maximum incorporation of a [(3)H] radiolabel into isolated cell-surface glycosaminoglycans at 10 and 20 mM. The mechanism of inhibition of glycosaminoglycan biosynthesis is due, in part, to the incorporation of a 4-deoxy moiety into glycosaminoglycan chains resulting in premature chain termination.     

Model reactions of a carbonylation catalyst: phosphite induced migratory CO insertion in [MeIr(CO)2I3]

Carbonylation of the anionic iridium(III) methyl complex, [MeIr(CO)₂I₃]⁻ (1) is an important step in the new iridium-based process for acetic acid manufacture. A model study of the migratory insertion reactions of 1 with P-donor ligands is reported. Complex 1 reacts with phosphites to give neutral acetyl complexes, [Ir(COMe)(CO)I₂L₂] (L = P(OPh)₃ (2), P(OMe)₃ (3)). Complex 2 has been isolated and fully characterised from the reaction of Ph₄As[MeIr(CO)₂I₃] with AgBF₄ and P(OPh)₃; comparison of spectroscopic properties suggests an analogous formulation for 3. IR and ³¹P NMR spectroscopy indicate initial formation of unstable isomers of 2 which isomerise to the thermodynamic product with trans phosphite ligands. Kinetic measurements for the reactions of 1 with phosphites in CH₂Cl₂ show first order dependence on [1], only when the reactions are carried out in the presence of excess iodide. The rates exhibit a saturation dependence on [L] and are inhibited by iodide. The reactions are accelerated by addition of alcohols (e.g. 18× enhancement for L = P (OMe)₃ in 1:3 MeOH-CH₂Cl₂). A reaction mechanism is proposed which involves substitution of an iodide ligand by phosphite, prior to migratory CO insertion. The observed rate constants fit well to a rate law derived from this mechanism. Analysis of the kinetic data shows that k₁, the rate constant for iodide dissociation, is independent of L, but is increased by a factor of 18 on adding 25% MeOH to CH₂Cl₂. Activation parameters for the k₁ step are ΔH^≠ = 71 (±3) kJ mol⁻, ΔS^≠ = −81 (±9) J mol⁻¹ K⁻¹ in CH₂Cl₂ and ΔH^≠ = 60(±4) kJ mol⁻¹, ΔS^≠ = −93(± 12) J mol⁻¹ K⁻¹ in 1:3 MeOH-CH₂Cl₂. Solvent assistance of the iodide dissociation step gives the observed rate enhancement in protic solvents. The mechanism is similar to that proposed for the carbonylation of 1.     

Senescence of Flag Leaves and Ears of Wheat Hastened by Methyl Jasmonate

Treatment of flag leaves and ears of wheat plants with MJ (jasmonic acid methylester) (10⁻⁵ and 10⁻⁴ m) did not increase ethylene production, but it did accelerate senescence as indicated by the loss of chlorophyll. MJ also caused the closure of stomata, and consequently the rates of transpiration and photosynthesis decreased. Early maturity shortened the grain filling period, so the thousand grain weight was lower. Although ethylene elicited the same physiologic effects, the syndrome of senescence by MJ is independent of the former. We conclude that senescence and death in wheat are far from being elucidated; however, MJ and ethylene seem to participate in the phenomenon. Received July 10, 1997; accepted January 5, 1998