Syntheses of methyl 2,6-diacetamido-2,3,6-trideoxy-α-d-ribo-hexopyranoside (methyl di-N-acetyl-α-d-tobrosaminide) and methyl 2-acetamido-2,3,6-trideoxy-β-l-lyxo-hexopyranoside

The title glycosides were synthesised from d-glucose, via the common intermediate methyl 2-acetamido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-α-d-ribo-hexopyranoside.     

Addition of 1-nitroalkanes to methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside and N6-benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde

Various 1-nitroalkanes reacted with methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside to yield methyl 6-alkyl-6-deoxy-2,3-O-isopropylidene-6-nitro-β-d-ribofuranosides in 64–79% yield. Similarly, nitromethane and 1-nitropentane reacted with N⁶-benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde, to yield the corresponding 9-[6-alkyl-6-deoxy-2,3-O-isopropylidene-6-nitro-α-l-talo(β-d-allo)furanosyl]-N⁶-benzoyladenines in 74 and 44% yield, respectively. The potential utility of this nitroalkane addition for the synthesis of nucleosides having a C-5′C-6′ bond is discussed.     

Metal complexes of 3-acetamido-5-methylpyrazole

In this paper, we present the complete synthesis of the 3-Acetamido-5-methylpyrazole (3-Ac-AMP) from 3-amino-5-methylpyrazole and acetic acid anhydride, including its full spectroscopic characterization. The solid-state structure shows extensive H-bonding involving the acyl and pyrazole moieties. Upon coordination to Co²⁺, Zn²⁺, and Cd²⁺, the system adopts a geometry that allows it to bind to metal centres as a O,N-chelate. 3-Ac-AMP coordination to Zn and Cd was monitored by ¹H NMR showing the formation of presumably tetrahedral 2:1 complexes. In the solid state, Co and Zn complexes are centrosymmetric and octahedral having two 3-Ac-AMP ligands in the equatorial plane and two methanol ligands occupying axial positions. The systems form a layered structure in which the ClO₄ ⁻ counter ion links the layers via H-bonding.     

Studies on dehydro-l-ascorbic acid 3-oxime 2-phenylhydrazone: Conversion into substituted triazoles

l-threo-2,3-Hexodiulosono-1,4-lactone 3-oxime 2-(phenylhydrazone) (1) gave 2-(p-bromophenyl)-4-(l-threo-1,2,3-trihydroxypropyl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone (2), and this gave a diacetyl and a dibenzoyl derivative. On treatment of 2 with liquid ammonia, methylamine, or dimethylamine, the corresponding triazole-5-carboxamides (5–7) were obtained. Periodate oxidation of 5 gave 2-(p-bromophenyl)-4-formyl-1,2,3-triazole-5-carboxamide (10), and, on reduction, 10 gave 2-(p-bromophenyl)-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamide, characterized as its monoacetate. Condensation of 10 with phenylhydrazine gave the triazole hydrazone. Acetonation of 2 gave the isopropylidene derivative. Reaction of 2 with HBr-HOAc gave 4-(l-threo-2-O-acetyl-3-bromo-1,2-dihydroxypropyl)-2-(p-bromophenyl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone. Similar treatment of 1 with HBr-HOAc gave 5-O-acetyl-5-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 3-oxime 2-(phenylhydrazone). This was converted into 4-(l-threo-2-O-acetyl-3-bromo-1,2-dihydroxypropyl)-2-phenyl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone on treatment with boiling acetic anhydride. On reaction of 1 with benzoyl chloride in pyridine, dehydrative cyclization occurred, with the formation of 4-(l-threo-2,3-dibenzoyloxy-1-hydroxypropyl)-2-phenyl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone, which was converted into the amide on treatment with ammonia.     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

The synthesis of evernitrose and 3-epi-evernitrose

Evernitrose (2,3,6-trideoxy-3-C-methyl-4-O-methyl-3-nitro-L-arabino-hexopyranose) was synthesized from methyl 2,6-dideoxy-4-O-methyl-α-L-erythro-hexopyranosid-3-ulose (2) through introduction of an amino group attached to the tertiary branching carbon by the method of Bourgeois, and subsequent oxidation of the amino group by m-chloroperoxybenzoic acid to a nitro group. 3-Cyano-3-O-mesylation of 2 by Bourgeois's method gave exclusively the desired product having the L-ribo configuration; furthermore, the β anomer of 2 gave the L-ribo and L-arabino products in the ratio of 1:2. The latter compound was converted into 3-epi-evernitrose by a similar sequence of reactions.     

Structure of 2-acetamido-3-amino-2,3-dideoxy-d-glucofuranurono-6,3-lactam

An X-ray crystallographic analysis of the title compound, an N-acetyl derivative of the 2,3-diamino-2,3-dideoxy-d-glucofuranurono-6,3-lactam found in the hydrolyzate of Pseudomonas aeruginosa P14 lipopolysaccharide, was performed. The crystals are monoclinic, space group P2₁, a = 11.704(2), b = 5.333(1), c = 7.399(2) Å, β = 91.63(2)°, and Z = 2. The structure was solved by direct methods and refined by the block-diagonal least-squares method to a final R value of 0.046 for 796 independent reflections. The dihedral angle between the mean plane through the furanose ring and that through the γ-lactam ring is 106.2(2)°, the furanose ring is ¹T₂, and the C-3, C-4, C-6, and N-3 atoms of the γ-lactam ring are nearly coplanar. The conformation in aqueous solution is discussed, based on the ¹H-n.m.r. data.     

Lactose as an inexpensive starting material for the preparation of aldohexos-5-uloses: synthesis of l-ribo and d-lyxo derivatives

Partially protected derivatives of l-ribo- and d-lyxo-aldohexos-5-ulose have been prepared starting from triacetonlactose dimethyl acetal derivatives. Key steps of the synthetic sequences are (a) the synthesis of 4′-deoxy-4′-eno- and 6′-deoxy-5′-eno lactose derivatives, and (b) the epoxidation-methanolysis of the above-mentioned enol ethers to give 1,5-bis-glycopyranosides, masked form of the target 1,5-dicarbonyl hexoses.     

Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39

l-allo-Threonine aldolase (l-allo-threonine acetaldehyde-lyase), which exhibited specificity for l-allo-threonine but not for l-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of l-allo-threonine (K_m=1.45 mM, V_max=45.2 μmol min⁻¹ mg⁻¹). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5′-phosphate participates in the enzymatic reaction. The enzyme does not act on either l-serine or l-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (l-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or l-threonine aldolase (EC 4.1.2.5).     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

Synthesis of 3-amino-2,3,6-trideoxy-d-ribo- and -l-lyxo-hexofuranoses suitable for nucleoside synthesis

N-Acetylepidaunosamine (3-acetamido-2,3,6-trideoxy-d-ribo-hexopyranose) was converted into the diethyl dithioacetal and this was cyclized with HgCi₂, HgO, and MeOH, to give methyl 3-acetamido-2,3,6-trideoxy-α- and -β-d-ribo-hexofuranoside (4 and 5). These anomers were acetylated or (p-nitrobenzoyl)ated, and the esters were subjected to acetolysis, to afford 3-acetamido-1,5-di-O-acetyl-2,3,6-trideoxy-d-ribo-hexofuranose and 3-acetamido-1-O-acetyl-2,3,6-trideoxy-5-O-(p-nitrobenzoyl)-d-ribo-hexofuranose, respectively. Alternatively, compounds 4 and 5 were hydrolyzed to the free bases with barium hydroxide, and these were converted into the trifluoroacetamido derivatives which, on (p-nitrobenzoyl)ation and acetolysis, afforded 1-O-acetyl-2,3,6-trideoxy-5-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-d-ribo-hexofuranose. To prepare the corresponding daunosamine derivative, 2,3,6-trideoxy-3-(trifluoroacetamido)-l-lyxo-hexopyranose was converted into the diethyl dithioacetal, and this was cyclized in the same way, to afford methyl 2,3,6-trideoxy-3-(trifluoroacetamido)-α- and -β-l-lyxo-hexofuranoside. On (p-nitrobenzoyl)ation and acetolysis, both afforded 1-O-acetyl-2,3,6-trideoxy-5-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexofuranose.     

Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C)

Short-chain dehydrogenase/reductase homologues from Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C) show high sequence similarity to serine dehydrogenase from Agrobacterium tumefaciens. We cloned each gene encoding YdfG and YMR226C into E. coli JM109 and purified them to homogeneity from the E. coli clones. YdfG and YMR226C consist of four identical subunits with a molecular mass of 27 and 29 kDa, respectively. Both enzymes require NADP⁺ as a coenzyme and use l-serine as a substrate. Both enzymes show maximum activity at about pH 8.5 for the oxidation of l-serine. They also catalyze the oxidation of d-serine, l-allo-threonine, d-threonine, 3-hydroxyisobutyrate, and 3-hydroxybutyrate. The k_cat/K_m values of YdfG for l-serine, d-serine, l-allo-threonine, d-threonine, l-3-hydroxyisobutyrate, and d-3-hydroxyisobutyrate are 105, 29, 199, 109, 67, and 62 M^?1 s^?1, and those of YMR226C are 116, 110, 14600, 7540, 558, and 151 M^?1 s^?1, respectively. Thus, YdfG and YMR226C are NADP⁺-dependent dehydrogenases acting on 3-hydroxy acids with a three- or four-carbon chain, and l-allo-threonine is the best substrate for both enzymes.     

Synthesis of the 3- and 4-methyl, 3,4-dimethyl, and 3,4,6-trimethyl ethers of methyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside

The methyl ethers of 2-amino-2-deoxy-D-mannose are reference compounds in studies, by the methylation procedure, of the chemical structure of polysaccharides containing 2-amino-2-deoxy-D-mannose and 2-amino-2-deoxy-D-mannuronic acid residues. Methylation of methyl 2-acetamido-2-deoxy-α-D-mannopyranoside (1) gave the 3,4,6-trimethyl ether. Methylation of the 6-trityl ether of 1, followed by detritylation, gave the 3,4-dimethyl ether of 1. Methylation of the 4,6-O-benzylidene derivative (6) of 1, followed by removal of the benzylidene group, gave the 3-methyl ether of 1. Benzoylation of 6, followed by removal of the benzylidene group and monobenzoylation, gave the 3,6-dibenzoate of 1, which was methylated, and the product saponified, to give the 4-methyl ether of 1; the latter compound was also obtained by a similar route via the 3-O-acetyl-6-O-benzoyl derivative.     

Biosynthesis of myo-inositol in rat mammary gland. Isolation and properties of the enzymes

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) and 1l-myo-inositol 1-phosphatase (EC 3.1.3.25) were isolated and partially purified from lactating rat mammary gland. The synthase had an apparent molecular weight of 290,000 as determined by gel filtration; its pH optimum was 7.2, and the K_m for glucose 6-phosphate was 0.5 mm. No other compound could act as a substrate, but the synthase was inhibited 100% by d-gluconic acid 6-phosphate, 54% by d-fructose 6-phosphate, 31.8% by d-galactose 6-phosphate, and 29.6% by d-mannose 6-phosphate each at 5mm. Activity was stimulated 2-fold by the addition of 1 mm NAD⁺ and 40% by 14 mm ammonium ions, whereas it was inhibited by 30% in the presence of 1 mm NADH and by 93.6% when incubated with 1 mmp-mercuribenzoate. Reagents which interfere with Schiff-base formation, pyridoxal 5′-phosphate and trinitrobenzenesulfonate, inhibited the enzyme, but EDTA was without effect.The 1l-myo-inositol 1-phosphatase from rat mammary tissue appears to exist in a native tetrameric form of 210,000 as determined by gel filtration which, upon heating at 70 °C for 15 min, is converted into a stable monomer of approximately 52,000. Mg²⁺ (1.5 mm) was an absolute requirement for activity though Mn²⁺ gave 17% of the activity provided by Mg²⁺. Sodium, potassium, or ammonium ions were stimulatory, but lithium ions were strongly inhibitory. 1l-myo-Inositol 1-phosphatase specifically cleaved 1l-myo-inositol 1-phosphate and was 60% as active toward l-α-glycerol phosphate with only minor activity toward other phosphorylated compounds. The pH optimum was 8.0 and the K_m for 1l-myo-inositol 1-phosphate was 0.8 mm.     

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-d-glucosamine

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[¹⁸F]fluoroacetyl-d-glucosamine (¹⁸F-GlcNFAc). The uptake of ¹⁸F-GlcNFAc was slightly higher (P < 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. ¹⁸F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-d-[l-¹⁴C]glucosamine, 2-deoxy-d-[l-¹⁴C]glucose and [6-³H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.     

Synthesis of gem-di-C-substituted derivatives of carbohydrates by way of nucleophilic-addition reactions of aldohexofuranoid 3-C-methylene derivatives

Nucleophilic Michael-type additions to aldohexofuranoid 3-C-methylene derivatives, namely, 3-deoxy-1,2:5,6-di-O-isopropylidene-3-C-nitromethylene-α-d-ribo-hexofuranose and 3-C-[cyano(ethoxycarbonyl)methylene]-3-deoxy-1,2:5,6-di-O-isopropylidene-α-d-ribo-hexofuranose employing phase-transfer catalysis, afforded novel gem-di-C-substituted sugars. The conversion of 3-deoxy-1,2:5,6-di-O-isopropylidene-3-C-methyl-3-C-nitromethyl-α-d-allo-hexofuranose into a 3-C-hydroxymethyl-3-C-methyl derivative with titanium trichloride, and that of the nitromethyl groups of 3-deoxy-1,2:5,6-di-O-isopropylidene-3,3-di-C-nitromethyl-α-d-ribo-hexofuranose, and 3-deoxy-1,2:5,6-di-O-isopropylidene-3-C-methyl-3-C-nitromethyl- and -3-C-nitromethyl-α-d-allo-hexofuranose into cyano groups with phosphorus trichloride in pyridine is also described.     

Inhibition of selected plant systems by stereoisomers of chloramphenicol

The stereospecificity of chloramphenicol isomers on the inhibition of several plant systems was investigated. l-Threo, d-erythro, l-erythro and the antibiotic d-threo-chloramphenicol were effective inhibitors of auxin-induced elongation, ¹⁴C-leucine uptake and ¹⁴C-leucine incorporation into the protein fraction of coleoptiles from Avena sativa and Triticum vulgare. The isomers also inhibited Avena coleoptile uptake of ¹⁴C-α-aminoisobutyric acid and the de novo synthesis of α-amylase by aleurone layers from Hordeum vulgare seeds. All four compounds inhibited these processes to about the same extent and over a similar high concentration range (5 × 10⁻⁴ to 5 × 10⁻³ M). Bioassay of extracts from Avena coleoptiles treated with the non-antibiotic isomers revealed no tissue conversion into d-threo-chloramphenicol.     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).     

Resolution of anomeric 2-amino-2-deoxy-d-glycofuranosides and -glycopyranosides by cation-exchange chromatography

Methyl 4,6-O-benzylidene-2-deoxy-α-D-ribo-hexopyranoside (1) is converted into methyl 3,4-di-O-benzoyl-6-bromo-2,6-dideoxy-α-D-ribo-hexopyranoside (3) via the 3-O-benzoyl derivative (2) of 1 by subsequent treatment with N-bromosuccinimide. Compound 3 is the key intermediate in high-yielding, preparative syntheses of the title dideoxy sugars, which are constituents of many antibiotics. Dehydrohalogenation of 3 affords the 5,6-unsaturated glycoside 7. which undergoes stereospecific reduction by hydrogen with net inversion at C-5 to give methyl 3,4-di-O-benzoyl-2,6-dideoxy-β-L-lyxo-hexopyranoside (8), whereas reductive dehalogenation of 3 provides the corresponding D-ribo derivative 4. The unprotected glycosides 9 (L-lyxo) and 5 (D-ribo) are readily obtained by catalytic transesterification, and mild, acid hydrolysis gives the crystalline title sugars 10 (L-lyxo) and 6 (D-ribo) in 45 and 57% overall yield from 1 without the necessity of chromatographic purification at any of the steps.     

Linking ascorbic acid production in Ribes nigrum with fruit development and changes in sources and sinks

Background and Aims

Understanding the synthesis of ascorbic acid (l-AsA) in green tissues in model species has advanced considerably; here we focus on its production and accumulation in fruit. In particular, our aim is to understand the links between organs which may be sources of l-AsA (leaves) and those which accumulate it (fruits). The work presented here tests the idea that changes in leaf and fruit number influence the accumulation of l-AsA. The aim was to understand the importance of leaf tissue in the production of l-AsA and to determine how this might provide routes for the manipulation of fruit tissue l-AsA.

Methods

The experiments used Ribes nigrum (blackcurrant), predominantly in field experiments, where the source–sink relationship was manipulated to alter potential leaf l-AsA production and fruit growth and accumulation of l-AsA. These manipulations included reductions in reproductive capacity, by raceme removal, and the availability of assimilates by leaf removal and branch phloem girdling. Natural variation in fruit growth and fruit abscission is also described as this influences subsequent experimental design and the interpretation of l-AsA data.

Key Results

Results show that fruit l-AsA concentration is conserved but total yield of l-AsA per plant is dependent on a number of innate factors many of which relate to raceme attributes. Leaf removal and phloem girdling reduced fruit weight, and a combination of both reduced fruit yields further. It appears that around 50 % of assimilates utilized for fruit growth came from apical leaves, while between 20 and 30 % came from raceme leaves, with the remainder from ‘storage’.

Conclusions

Despite being able to manipulate leaf area and therefore assimilate availability and stored carbohydrates, along with fruit yields, rarely were effects on fruit l-AsA concentration seen, indicating fruit l-AsA production in Ribes was not directly coupled to assimilate supply. There was no supporting evidence that l-AsA production occurred predominantly in green leaf tissue followed by its transfer to developing fruits. It is concluded that l-AsA production occurs predominantly in the fruit of Ribes nigrum.