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• W1968547228 abstract "It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12Kennedy I.A. Hemscheidt T. Britten J.F. Spenser I.D. Can. J. Chem. 1995; 73: 1329-1337Crossref Google Scholar) and 4-hydroxy-L-threonine (13Bain A.D. Wolf E. Hughes D.W. Spenser I.D. Croat. Chem. Acta. 1996; 69: 681-688Google Scholar), serve as precursors of pyridoxol (vitamin B6) (1Hill R.E. Spenser I.D. Science. 1970; 169: 773-775Crossref PubMed Scopus (17) Google Scholar) in Escherichia coli. Together, these two compounds account for the entire C8N skeleton of the vitamin. 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2′,2,3,4,4′ of pyridoxol. 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5′. Both precursors are ultimately derived from glucose. The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4′) with pyruvic acid (which decarboxylates to yield C-2′,2). D-Erythroate (11Wolf E. Spenser I.D. J. Org. Chem. 1995; 60: 6937-6940Crossref Scopus (15) Google Scholar) enters the C3 unit, C-6,5,5′, and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine. It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12Kennedy I.A. Hemscheidt T. Britten J.F. Spenser I.D. Can. J. Chem. 1995; 73: 1329-1337Crossref Google Scholar) and 4-hydroxy-L-threonine (13Bain A.D. Wolf E. Hughes D.W. Spenser I.D. Croat. Chem. Acta. 1996; 69: 681-688Google Scholar), serve as precursors of pyridoxol (vitamin B6) (1Hill R.E. Spenser I.D. Science. 1970; 169: 773-775Crossref PubMed Scopus (17) Google Scholar) in Escherichia coli. Together, these two compounds account for the entire C8N skeleton of the vitamin. 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2′,2,3,4,4′ of pyridoxol. 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5′. Both precursors are ultimately derived from glucose. The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4′) with pyruvic acid (which decarboxylates to yield C-2′,2). D-Erythroate (11Wolf E. Spenser I.D. J. Org. Chem. 1995; 60: 6937-6940Crossref Scopus (15) Google Scholar) enters the C3 unit, C-6,5,5′, and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine. INTRODUCTIONOur early work on the biosynthesis of vitamin B6 in Escherichia coli mutants WG2 and WG3, based on studies originally using substrates labeled with 14C and 3H (1Hill R.E. Spenser I.D. Science. 1970; 169: 773-775Crossref PubMed Scopus (17) Google Scholar, 2Hill R.E. Gupta R.N. Rowell F.J. Spenser I.D. J. Am. Chem. Soc. 1971; 93: 518-520Crossref PubMed Scopus (0) Google Scholar, 3Hill R.E. Rowell F.J Gupta R.N. Spenser I.D. J. Biol. Chem. 1972; 247: 1869-1882Abstract Full Text PDF PubMed Google Scholar, 4Hill R.E. Spenser I.D. Can. J. Biochem. 1973; 51 (Correction (1974) Can. J. Biochem. 52, 271): 1412-1416Crossref PubMed Scopus (15) Google Scholar), and later substrates singly labeled with 13C (5Hill R.E. Miura I. Spenser I.D. J. Am. Chem. Soc. 1977; 99: 4179-4181Crossref PubMed Scopus (17) Google Scholar), established the pattern of incorporation into the skeleton of pyridoxol (1; see Structure 1) of the carbon atoms of glycerol, glucose, and pyruvic acid. From these results it was inferred that pyridoxol is constructed from three glucose-derived triose phosphates, two of which enter intact, supplying the C3 fragments, C-3,4,4′ and C-6,5,5′ of pyridoxol, with the carbon atom holding the phosphate ester group yielding C-4′ and C-5′, while the third triose phosphate unit yields a C2 unit, by decarboxylation of pyruvate, whose CH3-CO moiety then supplies the C2 unit, C-2′,2, i.e. the CH3 group and the adjacent ring carbon of pyridoxol.We now furnish definitive proof that these inferences were indeed correct. This direct evidence comes from the results of incorporation studies with substrates labeled with 13C at contiguous sites, so called “bond-labeled” compounds, whose mode of incorporation into pyridoxol was determined by 13C nuclear magnetic resonance spectroscopy (13C NMR).The power of this method lies in the circumstance that, when contiguous carbon atoms within a compound are 100% enriched in 13C, this fact is indicated by the presence, within the 13C NMR spectrum of the compound, of 13C-13C coupling, which is indicated by the appearance of peaks that are not present in the spectrum of a natural abundance sample or of a singly 13C-enriched sample. These new peaks are detectable even if the fully 13C enriched sample is a minor component of a mixture that consists mainly of the unenriched compound.The appearance of peaks due to 13C-13C coupling, in the signals of the 13C NMR spectrum of a biosynthetic product, as a result of incorporation of contiguously 100%13C-13C-enriched (i.e. bond-labeled) substrates, provides direct evidence for the transfer from substrate into the biosynthetic product of an intact C-C unit. The application of bond-labeled samples thus constitutes a powerful tool for the demonstration of the transfer of intact multi-carbon fragments in biosynthetic investigations; provided adequate incorporation can be achieved in a tracer experiment with a 13C bond-labeled substrate, these are the tracers of choice. Neither radioactive tracer methods nor the use of substrates that are enriched with stable isotopes at single sites can show incorporation of intact multi-carbon units. Furthermore, 13C NMR does not only detect the site of labeling, but at the same time confirms the identity of the labeled sample and determines its degree of chemical purity. A precondition of the application of NMR methods for the analysis of biosynthetic incorporation patterns is the reliable assignment of each spectral signal to the individual atom that gives rise to it.Support for the inferences drawn from our tracer studies with 14C-labeled substrates, that the pyridoxine skeleton was constructed from three glucose-derived subunits, came from an experiment with [1,2,3,4,5,6-13C6]D-glucose (referred to as Experiment 1 in Table I) (6Hill R.E. Iwanow A. Sayer B.G. Wysocka W. Spenser I.D. J. Biol. Chem. 1987; 262: 7463-7471Abstract Full Text PDF PubMed Google Scholar, 7Hill R.E. Sayer B.G. Spenser I.D. J. Chem. Soc. Chem. Commun. 1986; : 612-614Crossref Google Scholar), which demonstrated that, as predicted, only two carbon-carbon bonds, those between C-2 and C-3 and between C-4 and C-5, are newly formed in the course of the biosynthetic derivation of pyridoxol from glucose, and that glucose does indeed supply three intact multicarbon units, as the building blocks of the three fragments, C-2′,2, C-3,4,4′, and C-6,5,5′, of pyridoxol (Fig. 1, A).TABLE I.Experimental detailsExp. no.SubstratesWeight13C NMR spectrum of isolated pyridoxol HClmg/liter (mmol)1[1,2,3,4,5,6-13C6]-D-Glucose200 (5.6)A (Fig. 1; 6Hill R.E. Iwanow A. Sayer B.G. Wysocka W. Spenser I.D. J. Biol. Chem. 1987; 262: 7463-7471Abstract Full Text PDF PubMed Google Scholar and 7Hill R.E. Sayer B.G. Spenser I.D. J. Chem. Soc. Chem. Commun. 1986; : 612-614Crossref Google Scholar)D-Glucose8002[1,2-13C2]-D-Glucose300 (5.6)B (Fig. 2)D-Glucose7003Sodium [2,3-13C2]Pyruvate150 (1.3)C (Fig. 2)D-Glucose1000 (5.6)4Sodium [2,3-13C2]Pyruvate200 (1.8)Not shownD-XyloseaIn Experiments 4, 6, and 9, D-xylose in place of D-glucose was used as the general carbon source in order to avert the possibility that the presence of glucose might limit the uptake and incorporation of the labeled carbohydrate substrates, [2,3-13C2]-1-deoxy-D-xylulose (Experiment 6) and [2,3-13C2]-D-erythroate (Experiment 9). Experiment 4 served as a test of D-xylose as the general carbon source. This change in the general carbon source was made after consideration of the results of the D-glucose displacement experiments, Experiments 5 and 7, and after failure to observe 13C incorporation in an early experiment with [2,3-13C2]-1-deoxy-D-xylulose. Whereas unlabeled 4-hydroxy-L-threonine completely suppressed the incorporation, into C-6,5,5′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 7), unlabled 1-deoxy-D-xylulose only partially suppressed the incorporation, into C-2′,2,3,4,4′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 5). Furthermore, in an experiment with [2,3-13C2]-1-deoxy-D-xylulose in which D-glucose served as the general carbon source, no 13C enrichment was detectable in the pyridoxol that was isolated. We reasoned that these results may have been the consequence of an “inducer exclusion effect,” a phenomenon that occurs in bacterial systems, whereby certain carbohydrates (so-called “PTS-carbohydrates”), e.g. glucose, inhibit the transport and metabolism of other carbohydrates (so-called “class I non-PTS carbohydrates”) (PTS = phosphoenolpyruvate:carbohydrate phosphotransferase system) (16) of which 1-deoxy-D-xululose might be one. We surmised that in our short term (6 h) incubations, D-glucose might have inhibited the uptake of 1-deoxy-D-xululose, whereas entry of the amino acid, 4-hydroxy-L-threonine, had not been affected. If this reasoning were correct, the problem might be overcome by using as the general carbon source in this experiment a non-PTS-carbohydrate such as D-xylose, in place of D-glucose, a PTS-carbohydrate. Cultures of E. coli mutant WG2 were established on the minimal medium, with D-xylose as the general carbon source, and in a test experiment (Experiment 4) it was found that under these conditions incorporation of label from sodium [2,3-13C2]pyruvate matched the result obtained when D-glucose served as the general carbon source (Experiment 3). Having thereby established that D-xylose could replace D-glucose as the general carbon source without measurable impairment of pyridoxine biosynthesis, we proceeded with Experiments 6 and 9 (Table I).500 (3.3)(similar to C)5[1,2,3,4,5,6-13C6]-D-Glucose200 (5.6)D (Fig. 3)D-Glucose8001-Deoxy-D-xylulose750 (5.6)6[2,3-13C2]-1-Deoxy-D-xylulose200 (1.5)F (Fig. 4)D-XyloseaIn Experiments 4, 6, and 9, D-xylose in place of D-glucose was used as the general carbon source in order to avert the possibility that the presence of glucose might limit the uptake and incorporation of the labeled carbohydrate substrates, [2,3-13C2]-1-deoxy-D-xylulose (Experiment 6) and [2,3-13C2]-D-erythroate (Experiment 9). Experiment 4 served as a test of D-xylose as the general carbon source. This change in the general carbon source was made after consideration of the results of the D-glucose displacement experiments, Experiments 5 and 7, and after failure to observe 13C incorporation in an early experiment with [2,3-13C2]-1-deoxy-D-xylulose. Whereas unlabeled 4-hydroxy-L-threonine completely suppressed the incorporation, into C-6,5,5′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 7), unlabled 1-deoxy-D-xylulose only partially suppressed the incorporation, into C-2′,2,3,4,4′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 5). Furthermore, in an experiment with [2,3-13C2]-1-deoxy-D-xylulose in which D-glucose served as the general carbon source, no 13C enrichment was detectable in the pyridoxol that was isolated. We reasoned that these results may have been the consequence of an “inducer exclusion effect,” a phenomenon that occurs in bacterial systems, whereby certain carbohydrates (so-called “PTS-carbohydrates”), e.g. glucose, inhibit the transport and metabolism of other carbohydrates (so-called “class I non-PTS carbohydrates”) (PTS = phosphoenolpyruvate:carbohydrate phosphotransferase system) (16) of which 1-deoxy-D-xululose might be one. We surmised that in our short term (6 h) incubations, D-glucose might have inhibited the uptake of 1-deoxy-D-xululose, whereas entry of the amino acid, 4-hydroxy-L-threonine, had not been affected. If this reasoning were correct, the problem might be overcome by using as the general carbon source in this experiment a non-PTS-carbohydrate such as D-xylose, in place of D-glucose, a PTS-carbohydrate. Cultures of E. coli mutant WG2 were established on the minimal medium, with D-xylose as the general carbon source, and in a test experiment (Experiment 4) it was found that under these conditions incorporation of label from sodium [2,3-13C2]pyruvate matched the result obtained when D-glucose served as the general carbon source (Experiment 3). Having thereby established that D-xylose could replace D-glucose as the general carbon source without measurable impairment of pyridoxine biosynthesis, we proceeded with Experiments 6 and 9 (Table I).500(3.3)4-Hydroxy-L-threoninebWhen this experiment (Experiment 6) was performed, it was already known, from Experiments 7 and 8, that 4-hydroxy-L-threonine serves as a direct precursor of pyridoxol. A sample of the amino acid was added since it was observed that this stimulates pyridoxol biosynthesis.100 (0.74)L-Threoninec4-Hydroxy-L-threonine shows antimetabolite properties in E. coli, inhibiting the growth of E. coli B when cultured on a pyridoxine-supplemented growth medium. Addition of L-threonine (20 mg/liter) permits the mutant to grow in the presence of the amounts of 4-hydroxy-L-threonine that were added to the medium in Experiments 6, 7, and 8 (100, 750, and 160 mg/liter, respectively).20 (0.17)7[1,2,3,4,5,6-13C6]-D-Glucose200 (5.6)E (Fig. 3)D-Glucose8004-Hydroxy-L-threonine750 (5.5)L-Threoninec4-Hydroxy-L-threonine shows antimetabolite properties in E. coli, inhibiting the growth of E. coli B when cultured on a pyridoxine-supplemented growth medium. Addition of L-threonine (20 mg/liter) permits the mutant to grow in the presence of the amounts of 4-hydroxy-L-threonine that were added to the medium in Experiments 6, 7, and 8 (100, 750, and 160 mg/liter, respectively).20 (0.17)8[2,3-13C2]-4-Hydroxy-L-threonine160 (1.2)G (Fig. 5)D-Glucose500 (2.8)L-Threoninec4-Hydroxy-L-threonine shows antimetabolite properties in E. coli, inhibiting the growth of E. coli B when cultured on a pyridoxine-supplemented growth medium. Addition of L-threonine (20 mg/liter) permits the mutant to grow in the presence of the amounts of 4-hydroxy-L-threonine that were added to the medium in Experiments 6, 7, and 8 (100, 750, and 160 mg/liter, respectively).20 (0.17)9dOnly three 1 liter cultures (rather than five) were employed in this experiment.Potassium [2,3-13C2]-D-erythroate200 (1.1)H (Fig. 5)D-XyloseaIn Experiments 4, 6, and 9, D-xylose in place of D-glucose was used as the general carbon source in order to avert the possibility that the presence of glucose might limit the uptake and incorporation of the labeled carbohydrate substrates, [2,3-13C2]-1-deoxy-D-xylulose (Experiment 6) and [2,3-13C2]-D-erythroate (Experiment 9). Experiment 4 served as a test of D-xylose as the general carbon source. This change in the general carbon source was made after consideration of the results of the D-glucose displacement experiments, Experiments 5 and 7, and after failure to observe 13C incorporation in an early experiment with [2,3-13C2]-1-deoxy-D-xylulose. Whereas unlabeled 4-hydroxy-L-threonine completely suppressed the incorporation, into C-6,5,5′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 7), unlabled 1-deoxy-D-xylulose only partially suppressed the incorporation, into C-2′,2,3,4,4′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 5). Furthermore, in an experiment with [2,3-13C2]-1-deoxy-D-xylulose in which D-glucose served as the general carbon source, no 13C enrichment was detectable in the pyridoxol that was isolated. We reasoned that these results may have been the consequence of an “inducer exclusion effect,” a phenomenon that occurs in bacterial systems, whereby certain carbohydrates (so-called “PTS-carbohydrates”), e.g. glucose, inhibit the transport and metabolism of other carbohydrates (so-called “class I non-PTS carbohydrates”) (PTS = phosphoenolpyruvate:carbohydrate phosphotransferase system) (16) of which 1-deoxy-D-xululose might be one. We surmised that in our short term (6 h) incubations, D-glucose might have inhibited the uptake of 1-deoxy-D-xululose, whereas entry of the amino acid, 4-hydroxy-L-threonine, had not been affected. If this reasoning were correct, the problem might be overcome by using as the general carbon source in this experiment a non-PTS-carbohydrate such as D-xylose, in place of D-glucose, a PTS-carbohydrate. Cultures of E. coli mutant WG2 were established on the minimal medium, with D-xylose as the general carbon source, and in a test experiment (Experiment 4) it was found that under these conditions incorporation of label from sodium [2,3-13C2]pyruvate matched the result obtained when D-glucose served as the general carbon source (Experiment 3). Having thereby established that D-xylose could replace D-glucose as the general carbon source without measurable impairment of pyridoxine biosynthesis, we proceeded with Experiments 6 and 9 (Table I).500 (3.3)a In Experiments 4, 6, and 9, D-xylose in place of D-glucose was used as the general carbon source in order to avert the possibility that the presence of glucose might limit the uptake and incorporation of the labeled carbohydrate substrates, [2,3-13C2]-1-deoxy-D-xylulose (Experiment 6) and [2,3-13C2]-D-erythroate (Experiment 9). Experiment 4 served as a test of D-xylose as the general carbon source. This change in the general carbon source was made after consideration of the results of the D-glucose displacement experiments, Experiments 5 and 7, and after failure to observe 13C incorporation in an early experiment with [2,3-13C2]-1-deoxy-D-xylulose. Whereas unlabeled 4-hydroxy-L-threonine completely suppressed the incorporation, into C-6,5,5′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 7), unlabled 1-deoxy-D-xylulose only partially suppressed the incorporation, into C-2′,2,3,4,4′ of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 5). Furthermore, in an experiment with [2,3-13C2]-1-deoxy-D-xylulose in which D-glucose served as the general carbon source, no 13C enrichment was detectable in the pyridoxol that was isolated. We reasoned that these results may have been the consequence of an “inducer exclusion effect,” a phenomenon that occurs in bacterial systems, whereby certain carbohydrates (so-called “PTS-carbohydrates”), e.g. glucose, inhibit the transport and metabolism of other carbohydrates (so-called “class I non-PTS carbohydrates”) (PTS = phosphoenolpyruvate:carbohydrate phosphotransferase system) (16Postma, P. W., Lengeler, J. W., Jacobson, G. R., (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology, (Neidhardt, F. C., ed), 2nd Ed., Vol. 1, pp. 1149–1174, ASM Press, Washington, D.C..Google Scholar) of which 1-deoxy-D-xululose might be one. We surmised that in our short term (6 h) incubations, D-glucose might have inhibited the uptake of 1-deoxy-D-xululose, whereas entry of the amino acid, 4-hydroxy-L-threonine, had not been affected. If this reasoning were correct, the problem might be overcome by using as the general carbon source in this experiment a non-PTS-carbohydrate such as D-xylose, in place of D-glucose, a PTS-carbohydrate. Cultures of E. coli mutant WG2 were established on the minimal medium, with D-xylose as the general carbon source, and in a test experiment (Experiment 4) it was found that under these conditions incorporation of label from sodium [2,3-13C2]pyruvate matched the result obtained when D-glucose served as the general carbon source (Experiment 3). Having thereby established that D-xylose could replace D-glucose as the general carbon source without measurable impairment of pyridoxine biosynthesis, we proceeded with Experiments 6 and 9 (Table I).b When this experiment (Experiment 6) was performed, it was already known, from Experiments 7 and 8, that 4-hydroxy-L-threonine serves as a direct precursor of pyridoxol. A sample of the amino acid was added since it was observed that this stimulates pyridoxol biosynthesis.c 4-Hydroxy-L-threonine shows antimetabolite properties in E. coli, inhibiting the growth of E. coli B when cultured on a pyridoxine-supplemented growth medium. Addition of L-threonine (20 mg/liter) permits the mutant to grow in the presence of the amounts of 4-hydroxy-L-threonine that were added to the medium in Experiments 6, 7, and 8 (100, 750, and 160 mg/liter, respectively).d Only three 1 liter cultures (rather than five) were employed in this experiment. Open table in a new tab The results of the tracer studies with 13C bond-labeled substrates that are here presented define more precisely the mode of incorporation of glucose carbon atoms into the glucose-derived subunits, C-2′,2, C-3,4,4′ and C-6,5,5′, of pyridoxol. They show, further, that an intact C2 unit, derived from C-3,2 of pyruvic acid, serves as the precursor of the C2-unit, C-2′,2, and that the C5 chain, C-2′,2,3,4,4′ of pyridoxol, originates by linear combination of the pyruvate-derived C2 unit and one of the glucose-derived C3 units. Furthermore, the results establish the identities of the two multicarbon precursors whose union accounts for the formation of the complete skeleton of pyridoxine. It is shown that 1-deoxy-D-xylulose (12) supplies the intact C5 chain, C-2′,2,3,4,4′, while the C3N unit, N-1,C-6,5,5′, is derived intact from 4-hydroxy-L-threonine (13), which is thereby proven to be an intermediate of the biosynthetic route. D-Erythroate (11) is shown to serve as an intermediate between glucose and 4-hydroxy-L-threonine. Preliminary reports of part of this work have appeared elsewhere (8Kennedy I.A. Hill R.E. Pauloski R.M. Sayer B.G. Spenser I.D. J. Am. Chem. Soc. 1995; 117: 1661-1662Crossref Scopus (37) Google Scholar, 9Wolf E. Hill R.E. Sayer B.G. Spenser I.D. J. Chem. Soc. Chem. Commun. 1995; : 1339-1340Crossref Google Scholar, 10Himmeldirk K. Kennedy I.A. Hill R.E. Sayer B.G. Spenser I.D. J. Chem. Soc. Chem. Commun. 1996; : 1187-1188Crossref Scopus (47) Google Scholar).EXPERIMENTAL PROCEDURESOrganismScheme 1The synthesis of potassium [2,3-13C2]D-erythroate (11) from [1,2-13C2]acetylene (2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)MediaNutrient MediumThis was a nutrient broth medium (Oxoid Ltd., London, United Kingdom) prepared according to the supplier's instructions.Minimal Salts MediumThe minimal salts medium contained the following salts: 7 g/liter KH2PO4, 3 g/liter K2HPO4, 1 g/liter (NH4)2SO4, 0.1 g/liter MgSO4, and 0.01 g/liter CaCl2. D-Glucose (Experiments 1-3, 5, 7, and 8) or D-xylose (Experiments 4, 6, and 9) served as the general carbon source. Pyridoxal hydrochloride was added to a concentration of 6 × 10−7 M when the minimal medium was used to grow the pdxH− mutant.All media were prepared in distilled water and were sterilized by autoclaving. The pyridoxal hydrochloride supplement solution was sterilized by filtration.Stock CulturesStock cultures of E. coli B WG2 were maintained on monthly slants of the nutrient broth medium. After subculturing from the previous month's stock, fresh slants were incubated 24 h at 37°C and were then stored at 4°C. Every time fresh stock slants were prepared, slants of minimal salts medium, with and without pyridoxal supplementation, were inoculated and incubated 24 h at 37°C in order to monitor for the presence of wild-type revertants.Labeled CompoundsThe following labeled compounds were acquired from a commercial source (Cambridge Isotope Laboratories, Inc. (CIL)): [1,2,3,4,5,6-13C6]D-glucose (98%13C), [1,2-13C2]D-glucose (99%13C), and sodium [2,3-13C2]pyruvate (99%13C).The following labeled compounds were prepared from commercial starting materials by multi-step syntheses devised for the purpose. [2,3-13C2]4-Hydroxy-L-threonine was synthesized (11Wolf E. Spenser I.D. J. Org. Chem. 1995; 60: 6937-6940Crossref Scopus (15) Google Scholar) from [1,2-13C2]acetylene (99%13C) (CIL) in eight steps with an overall yield of 13%. [2,3-13C2]1-Deoxy-D-xylulose was synthesized (12Kennedy I.A. Hemscheidt T. Britten J.F. Spenser I.D. Can. J. Chem. 1995; 73: 1329-1337Crossref Google Scholar) from ethyl bromo[1,2-13C2]acetate (99%13C) (CIL) in 14 steps with an overall yield of 15%. Potassium [2,3-13C2]D-erythroate (11) was synthesized (Scheme 1) from [1,2-13C2]acetylene (99%13C) (2) (CIL) in nine steps with an overall yield of 20%.Molecules that are fully enriched with 13C at contiguous sites, particularly if such molecules have CS symmetry, yield strongly coupled 13C NMR spectra. The enriched carbon atoms and their neighbors form a “deceptively simple” ABX system (13Bain A.D. Wolf E. Hughes D.W. Spenser I.D. Croat. Chem. Acta. 1996; 69: 681-688Google Scholar). The coupling constants were determined by simulating the spectra using the XSIM computer program of Dr. Kirk Marat, Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.Potassium [2,3-13C2]D-erythroate {Potassium [2,3-13C2]-(2R,3R)-2,3,4-trihydroxybutanoate} (11). [2,3-13C2]1,4-Di(tert-butyldiphenylsilyloxy)-(Z)-but-2-ene (5)Scheme 1The synthesis of potassium [2,3-13C2]D-erythroate (11) from [1,2-13C2]acetylene (2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)1H NMR (200 MHz, CDCl3): δ 7.56-7.61 (m, 8H), 7.23-7.73 (m, 12H), 5.06-6.22 (dm, 1JC,H = 156 Hz, 2H), 4.07 (d, 3JH, H = 3.74 Hz, 4H), 0.97 (s, 18H).13C NMR (75.5 MHz, CDCl3): δ 135.5, 133.6, 129.9 (enriched), 128.8, 127.6, 60.5 (X part of a deceptively simple ABX system, 1JAB = 70 Hz, 1JAX = 45 Hz, 2JBX = 3 Hz), 26.8, 19.1.IR 1The abbreviations used are: IRinfrared spectrummsmass spectrumm.p.melting point. (film): 1111 (vmax) cm−1.ms (CI): m/z 584 (30%, m + NH4+), 311 (100%).[2,3-13C2]1,4-Di-(O-tert-butyldiphenylsilyl)erythritol {[2,3-13C2]meso-1,4-Di-(O-tert-butyldiphenylsilyloxy)butane-2,3-diol} (6)[2,3-13C2]1,4-Di(tert-butyldiphenylsilyloxy)-(Z)-but-2-ene (5) (6.01 g, 10.6 mmol) was dissolved in tert-butanol (21 ml). Tetrahydrofuran (8 ml), water (4 ml), and N-methylmorpholine N-oxide (1.60 g, 13.7 mmol) were added, and the air above the mixture was displaced by nitrogen. An aqueous solution of osmium tetroxide (4% w/v, 4 ml) was added and the mixture stirred 4 h at room temperature, after which time no starting material remained (TLC: Silica gel 60, ethyl acetate/hexane 1:4). Sodium bisulfite (125 mg) in water (5 ml) was added, followed by Florisil® (5.25 g) and the mixture was stirred 10 min and was then filtered through silica gel and eluted with ethyl acetate (250 ml). Solvent was evaporated and the residue recrystallized from hexane (30 ml), yielding colorless crystals of the product (6), m.p. 90°C (3.48 g, 54.6%). The mother liquor was concentrated and the residue chromatographed (Silica gel 60, diethyl ether/petroleum ether 1:7, v/v), yielding a further 1.11 g (17.4%) of product, together with a mixed fraction from which an additional 189 mg of 6 was obtained by crystallization. Total yield was 4.78 g (75.0%).1H NMR (300 MHz, CDCl3): δ 7.69-7.73 (m, 8H), 7.26-7.50 (m, 12H), 3.50-4.13 (dm, 1JC, H = 141 Hz, 2H), 3.92 (s, 4H), 2.75 (s (br.) 2H), 1.11 (s, 18H).13C NMR (75.5 MHz, CDCl3): δ 135.5, 133.0, 129.8, 127.8, 71.9 (enriched), 65.1 (X part of a deceptively simple ABX system, 1JAB = 43 Hz, 1JAX = 36 Hz, 2JBX = 6 Hz), 26.9, 19.2.IR (KBr): 3511, 692 (vmax) cm−1.ms (CI): m/z 618 (15%, m + NH4+), 367 (100%), 131 (90%).[2,3-13C2]1,4-Di-(O-tert-butyldiphenylsilyl)-2,3-O-isopropylideneerythritol {[4,5-13C2]meso-4,5-Di-(O-tert-butyldiphenylsilyloxymethyl)-2,2-dimethyl-1,3-dioxolane} (7)Diol (6) (4.78 g, 7.95 mmol) was dissolved in dry benzene (100 ml). 2,2-Dimethoxypropane (15 ml, 122 mmol) was added and the mixture heated to 80°C. Pyridinium p-toluenesulfonate (275 mg) was added and methanol removed by azeotropic distillation (b.p. 54°C), until the reaction was complete (∼1 h). Benzene (∼75 ml) was distilled off, the mixture cooled to room temperature, and diethyl ether added. The solution was washed with water (2 × 50 ml), dried (MgSO4), and concentrated in vacuo to yield the dioxolane (7) (5.05 g, 100%).1H NMR (200 MHz, CDCl3): δ 7.57-7.64 (m, 8H), 7.24-7.39 (m, 12H), 4.45-4.75 (m, 1H), 3.62-4.00 (m, 5H), 1.36 (s 3H), 1.32 (s, 3H), 0.96 (s, 18H).13C NMR (75.5 MHz, CD2Cl2): δ 135.6, 135.5, 133.5, 133.4, 129.6, 127.7, 108.4, 77.4 (enriched), 63.2 (X part of a deceptively simple ABX system, 1JAB = 37 Hz, 1JAX = 40 Hz, 2JBX = 5 Hz), 27.8, 26.7, 25.3, 19.1.IR (film): 1112 (vmax) cm−1.ms (CI): m/z 658 (25%, m + NH4+), 565 (95%), 385 (100%).[2,3-13C2]2,3-O-Isopropylideneerythritol {[4,5-13C2]meso-4,5-Dihydroxymethyl-2,2-dimethyl-1,3-dioxolane} (8)Disilyl ether (7) (5.05 g, 7.95 mmol) was dissolved in tetrahydrofuran (40 ml). Water (0.5 ml, 28 mmol) and a solution of tetra-n-butylammonium fluoride in tetrahydrofuran (1 M, 24 ml) were added and the mixture kept 90 min at room temperature. Florisil® (8.5 g) was then added with stirring, which was continued for 10 min, and the mixture was then filtered through a pad of silica gel and eluted with ethyl acetate/methanol (1:1 v/v, 100 ml). The residue, obtained after evaporation of the solvent, was chromatographed on silica gel (ethyl acetate/hexane, 1:2 v/v, followed by ethyl acetate/methanol 1:1 v/v). The oily product was further purified by vacuum distillation (Kugelrohr, 1 mm Torr, 145°C). Diol (8" @default.
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• W1968547228 title "The Biogenetic Anatomy of Vitamin B6" @default.
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