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WO2010017526A1 - Nucléotides de purine marqués de manière isotopique dans la base purique, leur procédés de fabrication et leurs utilisations - Google Patents

Nucléotides de purine marqués de manière isotopique dans la base purique, leur procédés de fabrication et leurs utilisations Download PDF

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WO2010017526A1
WO2010017526A1 PCT/US2009/053225 US2009053225W WO2010017526A1 WO 2010017526 A1 WO2010017526 A1 WO 2010017526A1 US 2009053225 W US2009053225 W US 2009053225W WO 2010017526 A1 WO2010017526 A1 WO 2010017526A1
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adenine
guanine
enzymes
isotopically labeled
gguuaanniinnee
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James R. Williamson
Heather Schultheisz
Lincoln G. Scott
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide

Definitions

  • the present invention relates generally to purine nucleotides isotopically labeled in the purine base, methods of making thereof and uses thereof, for example, in biochemical and biophysical studies of nucleic acid structure and function. Also provided is a method of synthesizing 3 C- 10-formyl-tetrahydrofolate and a method of for immobilizing recombinant enzymes of the pentose phosphate pathway and the denovo purine synthesis pathway with fusion affinity domains attached to a metal chelate resin.
  • Biocatalysis is a viable alternative to the chemical synthesis of many biologically important compounds. Enzymatic synthesis may offer distinct advantages over chemical methods, including, for example, faster reaction times, superior product yields, increased product specificity, lower costs and less detrimental environmental impact. Accordingly, a wide variety of compounds, including, for example, vanillin, amylose, ⁇ -lactam antibiotics, glycopeptides, iminocyclitols and chiral amino acids have been made via enzymatic synthesis.
  • enzymatic synthesis has been expanded to include a combination of biochemical pathways, (i.e., multistep enzymatic synthesis) which provides access to a variety of complex molecules, including, for example, vitamin B 12, heparan sulfate pentasaccharide, enterocin polyketides and 3 C- and 2 H-labeled ribonucleotides while maintaining the above advantages of simple enzymatic syntheses.
  • biochemical pathways i.e., multistep enzymatic synthesis
  • RNA and DNA molecules which contain key structural information in local interactions including, for example, hydrogen bonding, protonation, ligand interactions, and stacking, remains limited to chemical synthesis and/or combinations of chemical and enzymatic synthesis.
  • Such synthetic methods and starting materials vary in implementation and complexity depending on the desired nucleotide and labeling pattern. Accordingly, uniformly or specifically labeled purine bases in ribonucleotides and deoxynucleotides are not commercially available and economical chemical synthesis of large amounts of labeled purines is typically challenging.
  • Uniformly or specifically labeled purine bases in ribonucleotides and deoxynucleotides and methods for making thereof may have applications, for example, in biochemical and biophysical studies of nucleic acid structure and function.
  • the present invention satisfies these and other needs by providing purine nucleotides isotopically labeled in the purine base, methods of making thereof and uses thereof, for example, in biochemical and biophysical studies of nucleic acid structure and function.
  • Z is hydrogen, -PO 3 , -
  • R 2 is H2" or 02'H; 02', 03', 04', 05', and 06 are independently 16 O, 17 O, or 18 O; Cl', CT, C3 ⁇ C4 ⁇ C5', are independently 11 C, 12 C, 13 C or 14 C; C2 and C8 are both either 11 C, 12 C, 13 C or 14 C; H2 and H8 are both either 1 H, 2 H, or 3 H when R 1 is isotopically labeled adenine; H8 is either 1 H, 2 H, or 3 H when R 1 is isotopically labeled guanine; N3 and N9 are both either 14 N or 15 N, when R 1 is isotopically labeled adenine; N2, N3 and N9 are all either 14 N or 15 N, when R 1 is isotopically labeled guanine; Nl and N6 are either 14 N or 15 N when R 1 is isotopically labeled adenine; Nl is either 14 N or 15 N when R 1 isotop
  • a methods of isotopically labeling a purine base in a ribonucleoside triphosphate with 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O or 18 O is provided.
  • the methods includes combining enzymes from the pentose phosphate pathway and the denovo purine synthesis pathway with glucose, glutamine, serine, ammonia and carbon dioxide precursors and folate, aspartate, glutamine, ATP and NADPH co-factors where at least one of the precursors is isotopically labeled.
  • ribonucleoside triphosphates isotopically labeled at least in the purine base may be converted to other ribonucleoside phosphates, ribonucleoside, deoxyribonucleoside phosphates and deoxyribonucleosides by methods known to those of skill in the art.
  • a method of synthesizing 3 C-10-formyl-tetrahydrofolate is
  • the method includes combining tetrahydrofolate, ; serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD).
  • a method of for immobilizing recombinant enzymes of the pentose phosphate pathway and the denovo purine synthesis pathway with fusion affinity domains attached to a metal chelate resin is provided.
  • Figure 1 illustrates substrates and products of the biosynthesis for: (a) ATP (b) GTP and the metabolic origin of purine atoms for: (c) adenine, (d) guanine.
  • FIG. 2 illustrates a scheme for the enzymatic synthesis of purine nucleotides.
  • Conversion of Glucose to PRPP Glucose and ATP are converted to glucose-6-phosphate (G6P) by the action of glucokinase (glk) with the production of ADP.
  • G6P and NADP + are converted to 6-phosphogluconate (6PG) by glucose-6-phosphate dehydrogenase (zwf) with the production of NADPH.
  • 6PG and NADP + are converted to ribulose-5-phosphate (Ru5P) by the action of 6-phosphogluconate dehydrogenase (gndA) with the production of NADPH.
  • Phosphoriboisomerase interconverts Ru5P and ribose-5-phosphate (R5P).
  • R5P and ATP are converted to phosphoribose-pyrophosphate (PRPP) by the action of PRPP synthase (prsA) with the production of AMP.
  • PRPP phosphoribose-pyrophosphate
  • PRPP phosphoribose-pyrophosphate
  • prsA PRPP synthase
  • PRA glycine and ATP are converted to phosphoribosyl-glycinamide (GAR) by the action of phosphoribosylamine-glycine ligase (purD) with the production of ADP.
  • GAR and 10- formyl-tetrahydrofolate are converted to phosphoribosyl-formylglycinamide (FGAR) by the action of phosphoribosyl-glycine amide formyltransferase (purN) with the production of tetrahydrofolate (THF).
  • FGAR, glutamine and ATP are converted to phosphoribosyl- formylglycinamidine (FGAM) by the action of FGAM synthase (purL).
  • FGAM and ATP are converted to phosphoribosyl-aminoimidazole (AIR) by phosphoribosyl-formyl glycinamidine cyclo-ligase (purM) with the production of ADP.
  • AIR, CO 2 and ATP (47) are converted to phosphoribosyl-amino-carboxy-imidazole (CAIR) by the action of phosphoribosyl-amino-imidazole carboxylase (purE, purK) with the production of ADP.
  • CAIR, aspartate and ATP are converted to phosphoribosyl-amino- succinocarboxamide-imidazole (SAICAR) by phosphoriobsyl-aminoimidazole-succino- carboxamide synthase ipurC) with the production of ADP.
  • SAICAR is converted to phosphoribosyl-amino-imidazole carboxamide (AICAR) by adenylosuccinate lyase (purB) with the production of fumarate.
  • AICAR and 10-formyl-THF are converted to phosphoribosyl-formylamido-imidazole carboxamide (FAICAR) by the action of phophoribosylamino-imidazole-carboxamide formyltransferase (purH) with the production of THF.
  • FAICAR is converted to inosine monophosphate (IMP) by IMP cyclohydrolase (purH) with the production of H 2 O.
  • IMP and GTP are converted to adenylosuccinate (AdS) by adenylosuccinate synthase (purA) with the production of GDP.
  • AdS is converted to AMP by the action of purB with the production of fumarate.
  • IMP and NAD + are converted to xanthosine monophosphate (XMP) by the action of IMP dehydrogenase (guaB) with the production of NADH.
  • XMP, glutamine and ATP are converted to GMP by the action of GMP synthase (guaA) with the production of AMP and glutamate.
  • guaA GMP synthase
  • AMP and ATP are converted to ADP by the action of adenylate kinase ⁇ pis A).
  • GMP and ATP are converted to GDP by the action of guanylate kinase (spoR) with the production of ADP.
  • NDP' s and creatine phosphate are converted to NTP' s by the action of creatine phosphokinase (ckmT) with the production of creatine.
  • Nicotinamide Adenine Dinucleotide Regeneration NAD(P)H, ⁇ -ketoglutarate ( ⁇ - KG) and NH 3 are converted to NAD(P) + by the action of glutamate dehydrogenase (gdhA) with the production of glutamate.
  • Glutamine Recycling Glutamate, ATP and NH 3 are converted to glutamine by the action of glutamine synthase (glnA) with the production of ADP. Aspartate Recycling.
  • Fumarate and NH 3 are converted to aspartate by the action of aspartate ammonia-lyase (aspA). Folate Regeneration. THF and serine are converted to 5,10-CH 2 -THF by the action of glycine hydroxymethyl-transferase (glyA) with the production of glycine. 5,10-CH 2 -THF and NADP + is converted to 5,10-CH-THF by the action of methylene-THF dehydrogenase (folD) with the production of NADPH. 5,10-CH-THF is converted to 10-CHO (formyl) THF by the action of methenyl-THF cyclohydrolase (folD) with the production of H 2 O.
  • Figure 3 illustrates the pathway engineered synthesis scheme for: (a) ATP and (b) GTP. Cofactors are added in catalytic amounts, and intermediates that are generated in situ are shown in parentheses.
  • Figure 4 illustrates HPLC chromatograms which show the time course of the U- 3 C GTP-forming reaction. The early-eluting peaks are from the NAD(P) + and THF cofactors.
  • Buffer includes, but is not limited to, 2-amino-2-methyl-l,3-propanediol, 2-amino- 2-methyl-l-propanol, L- (+) -tartaric acid, D-(-)-tartaric acid, ACES, ADA, acetic acid, ammonium acetate, ammonium bicarbonate, ammonium citrate, ammonium formate, ammonium oxalate, ammonium phosphate, ammonium phosphate, ammonium sodium phosphate, ammonium sulfate, ammonium tartrate, BES, BICINE, BIS-TRIS, bicarbonate, boric acid, CAPS, CHES, calcium acetate, calcium carbonate, calcium citrate, citrate, citric acid, diethanolamine, EPP, ethylenediaminetetraacetic acid disodium salt, formic acid solution, Gly-Gly-Gly, Gly-Gly, glycine, HEPES, imidazo
  • Salt refers to a salt of a purine nucleotide isotopically labeled in the purine base, which possesses the desired activity of the parent compound.
  • Such salts include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane- disulfonic acid, 2-hydroxyethanesulfonic acid,
  • a alkali metal ion e.g. , sodium or potassium
  • an alkaline earth ion e.g., calcium or magnesium
  • an aluminum ion or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N- methylglucamine, morpholine, piperidine, dimethylamine, diethylamine and the like.
  • salts of amino acids such as arginates and the like, and salts of organic acids like glucurmic or galactunoric acids and the like.
  • the present invention provides purine nucleotides isotopically labeled in the purine base, methods of making thereof and uses thereof, for example, in biochemical and biophysical studies of nucleic acid structure and function. Also provided is a method of synthesizing 13 C- 10-formyl-tetrahydrofolate.
  • Z is hydrogen, -PO 3 , - P(O)O-P ⁇ 3 or -P(O)O-P(O)O-P ⁇ 3 wherein the oxygen atoms in the phosphate groups are independently 16 O, 17 O or 18 O;
  • 02', 03', 04', 05', and 06 are independently 16 O, 17 O, or 18 O; Cl', C2 ⁇ C3 ⁇ C4', C5', are independently 11 C, 12 C, 13 C or 14 C; C2 and C8 are both either 11 C, 12 C, 13 C or 14 C; H2 and H8 are both either 1 H, 2 H, or 3 H when R 1 is isotopically labeled adenine; H8 is either 1 H, 2 H, or 3 H when R 1 is isotopically labeled guanine; N3 and N9 are both either 14 N or 15 N, when R 1 is isotopically labeled adenine; N2, N3 and N9 are all either 14 N or 15 N, when R 1 is isotopically labeled guanine; Nl and N6 are either 14 N or 15 N when R 1 is isotopically labeled adenine; Nl is either 14 N or 15 N when R 1 is isotopically labeled guanine
  • R is isotopically labeled adenine
  • R 2 is O2'H and Z is hydrogen.
  • R is isotopically labeled adenine
  • R 2 is O2 ⁇ and Z is - PO 3 .
  • R is isotopically labeled adenine
  • R 2 is O2'H and Z is -
  • R is isotopically labeled adenine
  • R 2 is O2'H and Z is -P(O)O-P(O)O-P ⁇ 3 .
  • R is isotopically labeled adenine
  • R 2 is H2" and Z is hydrogen.
  • R 1 is isotopically labeled adenine
  • R 2 is H2" and Z is -PO3.
  • R isotopically labeled adenine
  • R 2 is H2" and Z is -P(O)O-P ⁇ 3 .
  • R isotopically labeled adenine, R 2 is H2" and Z is -P(O)O-P(O)O-PO 3 .
  • R 1 is 2,8- 2 H-2,4,5,6,8- 13 C-l,3,6,9- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-l,6,7- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-l,6- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-3,7,9- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-3,9- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-7- 15 N- adenine, 2,8- 2 H-2,4,5,6,8- 13 C-adenine, 2,8- 2 H-2,4,5,8- 13 C-l,3,6,7,9- 15 N-adenine, 2,8- 2 H- 2,4,5,8- 13 C-l,3,6,9- 15 N-adenine, 2,8- 2 H- 2,4,5,8- 13 C-l,3,6,9
  • R 1 is isotopically labeled guanine
  • R 2 is O2'H and Z is hydrogen.
  • R 1 is isotopically labeled guanine
  • R 2 is O2 ⁇ and Z is - PO 3 .
  • R is isotopically labeled guanine
  • R 2 is O2'H and Z is - P(O)O-PO 3 .
  • R is isotopically labeled guanine, R 2 is O2'H and Z is -P(O)O-P(O)O-P ⁇ 3 .
  • R 1 is isotopically labeled guanine
  • R 2 is H2" and Z is hydrogen.
  • R is isotopically labeled guanine
  • R 2 is H2" and Z is -PO 3
  • R is isotopically labeled guanine
  • R 2 is H2' ' and Z is -P(O)O-P ⁇ 3
  • R is isotopically labeled guanine
  • R 2 is H2" and Z is -P(O)O-P(O)O-PO 3 .
  • R 1 is 8- 2 H-2,4,5,6,8- 13 C-l,2,3,9- 15 N-guanine, 8- 2 H- 2,4,5,6,8- 13 C-l,7- 15 N-guanine, 8- 2 H-2,4,5,6,8- 13 C-l- 15 N-guanine, 8- 2 H-2,4,5,6,8- 13 C-2,3,7,9- 15 N-guanine, 8- 2 H-2,4,5,6,8- 13 C-2,3,9- 15 N-guanine, 8- 2 H-2,4,5,6,8- 13 C-7- 15 N-guanine, 8- 2 H- 2,4,5,6,8- 13 C-guanine, 8- 2 H-2,4,5,8- 13 C-l,2,3,7,9- 15 N-guanine, 8- 2 H-2,4,5,8- 13 C-l,2,3,9- 15 N-guanine, 8- 2 H-2,4,5,8- 13 C-l,2,3,9- 15 N-guanine, 8- 2 H
  • Figure 1 illustrates substrates and products of the biosynthesis for: (a) ATP (b) GTP and the metabolic origin of purine atoms for: (c) adenine, (d) guanine.
  • Purine nucleotides isotopically labeled in the purine base may exist in several tautomeric forms and mixtures thereof. Purine nucleotides isotopically labeled in the purine base may exist in unsolvated forms as well as solvated forms, including hydrated forms or as N-oxides. In general, hydrated, solvated forms and N-oxides are within the scope of the present invention. Certain purine nucleotides isotopically labeled in the purine base may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • a method of isotopically labeling a purine base in a ribonucleoside triphosphate with 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O or 18 O comprising combining enzymes from the pentose phosphate pathway and the denovo purine synthesis pathway with glucose, glutamine, serine, ammonia and carbon dioxide precursors and folate, aspartate, glutamine, ATP and NADPH co-factors wherein at least one of the precursors is isotopically labeled.
  • FIG. 2 illustrates a scheme for the enzymatic synthesis of purine nucleotides.
  • Glucose and ATP are converted to glucose-6-phosphate (G6P) by the action of glucokinase (glk) with the production of ADP.
  • G6P and NADP + are converted to 6-phosphogluconate (6PG) by glucose-6-phosphate dehydrogenase (zwj) with the production of NADPH.
  • 6PG and NADP + are converted to ribulose-5-phosphate (Ru5P) by the action of 6- phosphogluconate dehydrogenase (gndA) with the production of NADPH.
  • Phosphoriboisomerase interconverts Ru5P and ribose-5-phosphate (R5P).
  • R5P and ATP are converted to phosphoribose-pyrophosphate (PRPP) by the action of PRPP synthase (prsA) with the production of AMP.
  • PRPP and glutamine are converted to phosphoribose-amine (PRA) by the action of amidophosphoribosyl-transferase (purF) with the production of glutamine.
  • PRA glycine and ATP are converted to phosphoribosyl- glycinamide (GAR) by the action of phosphoribosylamine-glycine ligase (purD) with the production of ADP.
  • GAR and 10-formyl-tetrahydrofolate are converted to phosphoribosyl- formylglycinamide (FGAR) by the action of phosphoribosyl-glycine amide formyltransferase (purN) with the production of tetrahydro folate (THF).
  • FGAR, glutamine and ATP are converted to phosphoribosyl-formylglycinamidine (FGAM) by the action of FGAM synthase (purL).
  • FGAM and ATP are converted to phosphoribosyl-aminoimidazole (AIR) by phosphoribosyl-formyl glycinamidine cyclo-ligase (purM) with the production of ADP.
  • AIR, CO 2 and ATP (47) are converted to phosphoribosyl-amino-carboxy- imidazole (CAIR) by the action of phosphoribosyl-aminoimidazole carboxylase (purE, purK) with the production of ADP.
  • CAIR, aspartate and ATP are converted to phosphoribosyl-amino-succinocarboxamide-imidazole (SAICAR) by phosphoriobsyl-aminoimidazole-succino-carboxamide synthase (purC) with the production of ADP.
  • SAICAR is converted to phosphoribosyl-amino-imidazole carboxamide (AICAR) by adenylosuccinate lyase (purB) with the production of fumarate.
  • AICAR and 10-formyl-THF are converted to phosphoribosyl-formylamido-imidazole carboxamide (FAICAR) by the action of phophoribosylamino-imidazole-carboxamide formyltransferase ⁇ purH) with the production of THF.
  • FAICAR is converted to inosine monophosphate (IMP) by IMP cyclohydrolase (purH) with the production of H 2 O.
  • IMP and GTP are converted to adenylosuccinate (AdS) by adenylosuccinate synthase (purA) with the production of GDP.
  • AdS is converted to AMP by the action of purB with the production of fumarate.
  • IMP and NAD + are converted to xanthosine monophosphate (XMP) by the action of IMP dehydrogenase (guaB) with the production of NADH.
  • XMP, glutamine and ATP are converted to GMP by the action of GMP synthase (guaA) with the production of AMP and glutamate.
  • AMP and ATP are converted to ADP by the action of adenylate kinase (plsA).
  • GMP and ATP are converted to GDP by the action of guanylate kinase (spoR) with the production of ADP.
  • NDP' s and creatine phosphate are converted to NTP' s by the action of creatine phosphokinase (ckmT) with the production of creatine. Nicotinamide Adenine Dinucleotide Regeneration.
  • NAD(P)H, ⁇ -ketoglutarate ( ⁇ -KG) and NH 3 are converted to NAD(P) + by the action of glutamate dehydrogenase (gdhA) with the production of glutamate.
  • Glutamate, ATP and NH 3 are converted to glutamine by the action of glutamine synthase (glnA) with the production of ADP.
  • Fumarate and NH 3 are converted to aspartate by the action of aspartate ammonia-lyase (aspA).
  • THF and serine are converted to 5, 10-CH 2 - THF by the action of glycine hydroxymethyl-transferase (glyA) with the production of glycine.
  • 5,10-CH 2 -THF and NADP + is converted to 5,10-CH-THF by the action of methylene-THF dehydrogenase (folD) with the production of NADPH.
  • 5,10-CH-THF is converted to 10-CHO (formyl) THF by the action of methenyl-THF cyclohydrolase (folD).
  • the enzymes are recombinant enzymes. In other embodiments, the enzymes are attached to a resin. In still other embodiments, the enzymes are attached to the resin with a covalent bond. In still other embodiments, the covalent bond is formed between amine, carboxylate or sulfhydryl groups on the enzymes and the resin. In still other embodiments, the resin is functionalized sepharose, agarose, polyacrylamide or polystyrene. In some embodiments, the enzymes are attached to the resm non-covalently.
  • the enzymes are attached to the resin with fusion affinity domains
  • the fusion affinity domains are hexahistidine, glutathione-S-transferase, maltose binding protein, S peptide, TAP, chitin binding domain, FLAG, streptavidin binding peptide or MAT.
  • the enzymes, the precursors and co-factors are combined in a buffer which includes magnesium chloride and dithiothreitol.
  • the buffer includes sodium chloride and/or potassium chloride.
  • the buffer is sodium phosphate, potassium phosphate, Tris-HCl, MOPS, or HEPES.
  • the buffer includes sodium chloride and/or potassium chloride.
  • the enzymes, the precursors and the co-factors are combined at a temperature between about O 0 C and about 100 0 C. In other embodiments, the enzymes, the precursors and the co-factors are combined at a temperature between about 0 0 C and about 55 0 C. In still other embodiments, the enzymes, the precursors and the co-factors are combined at a temperature between about 15 0 C and about 45 0 C. In still other embodiments, the enzymes, the precursors and the co-factors are combined at a temperature between about 2O 0 C and about 4O 0 C. In still other embodiments, the enzymes, the precursors and the co- factors are combined at a temperature between about 25 0 C and about 4O 0 C. In still other embodiments, the enzymes, the precursors and the co-factors are combined at a temperature of about 37 0 C.
  • the recombinant enzymes, the precursors and the co-factors are combined at a pH between about 5.0 and about 9.0. In other embodiments, the recombinant enzymes, the precursors and the co-factors are combined at a pH between about 6.0 and about 8.0. In still other embodiments, the recombinant enzymes, the precursors and the co-factors are combined at a pH between about 6.5 and about 8.0. In still other embodiments, the recombinant enzymes, the precursors and the co-factors are combined at a pH of about 7.5.
  • the enzymes, the precursors and the co-factors are combined at a temperature between about 25°C and about 40 0 C and at a pH between about 6.5 and about 8.0. In other embodiments, the enzymes, the precursors and the co-factors are combined at a temperature of about 37 0 C and a pH of about 7.5.
  • the enzyme concentration is between about 0.1 mg/ml and about 10 mg/ml. I other embodiments, the enzyme concentration is between about 0.2 mg/ml and about 4 mg/ml. In still other embodiments, the enzyme concentration is about 0.2 mg/ml.
  • the enzymes, the precursors and the co-factors are combined at a temperature between about 25°C and about 40 0 C, at a pH between about 6.5 and about 8.0 and the enzyme concentration is between about 0.1 mg/ml and about 0.2 mg/ml.
  • the precursor concentration is between about 1 mM and 100 mM. In other embodiments, the precursor concentration is between about 5 mM and 50 mM. In still other embodiments, the precursor concentration is between about 10 mM and 20 mM.
  • the enzymes, the precursors and the co-factors are combined at a temperature between about 25°C and about 40 0 C, at a pH between about 6.5 and about 8.0, the enzyme concentration is between about 10 mg/ml and about 20 mg/ml and the precursor concentration is between about 10 mM and 20 mM.
  • the co-factor concentration is between about 0.0001 mM and 0.01 mM. In other embodiments, the co-factor concentration is between about 0.01 mM and 0.1 mM.
  • the enzymes, the precursors and the co-factors are combined at a temperature between about 25 0 C and about 4O 0 C, at a pH between about 6.5 and about 8.0, the enzyme concentration is between about 10 mg/ml and about 20 mg/ml, the precursor concentration is between about 10 mM and 20 Mm and the co- factor concentration is between about 0.01 mM and 0.1 mM.
  • the enzymes, the precursors and the co-factors are combined at a temperature of about 37°C, a pH of about 7.5, the enzyme concentration is about 0.2 mg/ml, precursor concentration is about 10 mM and the co-factor concentration is between about 0.1 mM.
  • the enzymes from the pentose phosphate pathway include glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluonate dehydrogenase, phosphoriboisomerase and phosphoribose-pyrophosphate synthase.
  • the enzymes from the denovo purine synthesis pathway include amidophosphoribosyl- transferase, phosphoribosylamine-glycine ligase, phosphoribosylamine-glycine amide formyltransferase, phosphoribosylamine-formylglycinamide, phosphoribosylamine- formylglycinamidine cyclo-ligase, phosphoribosyl-amino-imidazole carboxylase, phosphoriobsyl-aminoimidazole-succino-carboxamide synthase, adenylosuccinate lyase, phophoribosylamino-imidazole-carboxamide formyltransferase, IMP cyclohydrolase and (adenylosuccinate synthase) and/or (IMP dehydrogenase and GMP synthase), adenylate kinas
  • the isotopically labeled purine bases made by the methods described above are 2,8- 2 H-2,4,5,6,8- 13 C-l,3,6,7,9- 15 N-adenine, 2,8- 2 H-l,3,6,7,9- 15 N-adenine, 2,4,5,6,8- 13 C-l,3,6,7,9- 15 N-adenine, 2,4,5,6,8- 13 C-adenine, l,3,6,7,9- 15 N-adenine, l,6,7- 15 N-adenine, 7- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-l,3,6,9- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-l,6,7- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-l,6- 15 N-adenine, 2,8- 2 H-2,4,5,6,8- 13 C-l,6- 15 N-adenine, 2,8-
  • the ribonucleoside triphosphate is converted to a ribonucleoside diphosphate. In other embodiments, the ribonucleoside triphosphate is converted to a ribonucleoside diphosphate by pyruvate kinase in the presence of pyruvate. In still other embodiments, the ribonucleoside diphosphate is converted to a ribonucleoside monophosphate. In still other embodiments, the ribonucleoside diphosphate is converted to a ribonucleoside monophosphate by adenylate kinase or guanylate kinase.
  • the ribonucleoside triphosphate is converted to a ribonucleoside. In other embodiments, the ribonucleoside triphosphate is converted to a ribonucleoside by a phosphatase, a calf intestinal phosphatase or a bacterial alkaline phosphatase.
  • the ribonucleoside triphosphate is converted to a ribonucleoside monophosphate.
  • the ribonucleoside triphosphate is converted to a ribonucleoside.
  • the ribonucleoside triphosphate is converted to a deoxyribonucleoside triphosphate. In other embodiments, the ribonucleoside triphosphate is converted to a deoxyribonucleoside triphosphate by ribonucleoside triphosphate reductase.
  • Also provided herein is a method of synthesizing 13 C-10-formyl-tetrahydrofolate.
  • the method includes combining tetrahydrofolate, , serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD).
  • a buffer which includes magnesium chloride and dithiothreitol.
  • the buffer includes sodium chloride and/or potassium chloride.
  • the buffer includes sodium chloride and/or potassium chloride.
  • serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD) are combined at a temperature between about O 0 C and about 55 0 C.
  • tetrahydrofolate, ? serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD) are combined at a temperature between
  • tetrahydrofolate, serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD) are combined at a temperature between about 2O 0 C and about 4O 0 C.
  • tetrahydrofolate 5 serine hydroxymethylase (glyA) and methylene- tetrahydrofolate dehydrogenase (folD) are combined at a temperature between about 25 0 C
  • tetrahydrofolate, ? serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (fo ⁇ D) are combined at a temperature of about 37 0 C.
  • tetrahydrofolate, serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD) are combined at a pH between about 6.0 and about 8.0.
  • tetrahydrofolate, serine hydroxymethylase (glyA) and methylene- tetrahydrofolate dehydrogenase (folD) are combined at a pH between about 6.5 and about 8.0.
  • tetrahydrofolate, ? serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (fo ⁇ D) are combined at a pH of about 7.5.
  • the hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (fo ⁇ D) are combined at a temperature between about 25 0 C and about 4O 0 C and at a pH between about 6.5 and about 8.0.
  • the hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (fo ⁇ D) concentration is between about 1 mg/ml and about 30 mg/ml.
  • the hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folO) concentration is between about 10 mg/ml and about 20 mg/ml.
  • tetrahydrofolate, ? serine hydroxymethylase (gfyA)and methylene-tetrahydrofolate dehydrogenase (folD) are combined at a temperature between about 25 0 C and about 4O 0 C, at a pH between about 6.5 and about 8.0 and the hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD) concentration is between about 10 mg/ml and about 20 mg/ml.
  • the isotopically labeled serine concentration is between about 1 mM and 100 mM. In other embodiments, the isotopically labeled serine concentration is between about 10 mM and 75 mM. In still other embodiments, the isotopically labeled serine concentration is between about 20 mM and 50 mM.
  • gly A and methylene-tetrahydrofolate dehydrogenase (folD) are combined at a temperature between about 25 0 C and about 4O 0 C, at a pH between about 6.5 and about 8.0 and the hydroxymethylase (glyA), methylene-tetrahydrofolate dehydrogenase (folD) concentration is between about 10 mg/ml and about 20 mg/ml and the isotopically labeled serine concentration is between about 20 mM and 50 mM.
  • the tetrahydrofolate concentration is between about 0.0001 mM and 10.0 mM. In other embodiments, the tetrahydrofolate concentration is between about 1 mM and 2 mM.
  • glyA and methylene-tetrahydrofolate dehydrogenase are combined at a temperature between about 25 0 C and about 4O 0 C, at a pH between about 6.5 and about 8.0 and the hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (fo ⁇ D) concentration is between about 10 mg/ml and about 20 mg/ml, the isotopically labeled serine concentration is between about 20 mM and 50 mM and the tetrahydrofolate concentration is between about 0.001 mM and 2.0 mM.
  • tetrahydrofolate tetrahydrofolate
  • serine hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (fo ⁇ D) are combined at a temperature of about 37 0 C, a pH of about 7.5, the hydroxymethylase (glyA) and methylene-tetrahydrofolate dehydrogenase (folD) concentration is about 0.2 mg/ml, the isotopically labeled serine concentration is about 20 mM and the tetrahydrofolate concentration is between about 1.0 mM.
  • the fusion affinity domain is hexahistidine, glutathione-S- transferase, maltose binding protein, S peptide, TAP, chitin binding domain, FLAG, streptavidin binding peptide or MAT.
  • the metal chelate resin is nickel iminodiacetic acid resin.
  • the fusion affinity domain is hexahistidine and the resin is nickel iminodiacetic acid resin.
  • the isotopically labeled purine bases described herein can be used in general for biochemical and biophysical studies of nucleic acid structure and function. More specific uses include mass tagging where the introduction of a stable isotope produces a mass-shift in the mass spectrum which can provide different mass shifts, thus allowing for multiplexing which has potential uses for SNP detection, PCR analysis, genotyping. etc. Further, detection of RNA or DNA molecules can be facilitated for quantitative studies, and different labeling patterns.
  • the isotopically labeled purine bases described herein can be used for metabolic labeling to monitor the synthesis and degradation of DNA or RNA in cells, plants or animals and also in specific isotope labeling to facilitate NMR studies by providing unique spin systems
  • Example 1 Enzyme Cloning.
  • the genes for all recombinant E. coli enzymes for the enzymes described above were cloned from the E. coli K12 MRE600 genome, based on the reported gene sequences in Genbank using standard procedures. Each gene was amplified from genomic DNA using PCR with gene specific primers containing compatible restriction sites of either BamHI, Hindlll, EcoRI, or Xhol for cloning into pET22-HT (derived from pET22b, Novagen) encoding a N-terminal hexahistidine tag. E. coli strain DH5 ⁇ was used for cloning and plasmid maintenance. All constructs were verified by DNA sequencing. Rabbit muscle creatine phosphokinase was amplified from pET17b-RMCK, a gift from George Kenyon, University of Michigan. A single point mutation, Y397F was introduced to glutamine synthase to enhance specific activity.
  • Plasmids were transformed into E. coli BL21(DE3) cells for protein overexpression. Overnight 5 mL cultures in LB with 100 ⁇ g/mL ampicillin were diluted 100 fold and grown at 37°C with shaking at 280 rpm until the OD OOO was -0.6. Protein expression was induced by addition of 1 niM IPTG, followed by growth for an additional 4 hours. Cells were harvested by centrifugation at 10000 g for 30 minutes. All steps after cell growth were carried out at 4°C.
  • the cell pellets from 6 L of growth were gently resuspended in 250 mL of 50 mM Tris-HCl (pH 7.5), 250 mM NaCl and cell lysis was accomplished by sonication with 20 cycles of a 30 s pulse and 2 min rest at 75% power.
  • the lysates were clarified by centrifugation at 31000 g and the supernatants were loaded directly to a Ni-NTA (Qiagen) column equilibrated with lysis buffer plus 20 mM imidazole. The column was washed with 6 volumes of the same buffer, and protein was eluted with 250 mM imidazole.
  • Protein containing fractions were checked by SDS-PAGE and combined prior to dialysis against 50 mM potassium phosphate with 5 mM ⁇ -mercaptoethanol and 50% (v/v) glycerol. Dialyzed enzymes were stored at -20° C and were active for at least three months. Yields generally ranged from 10-150 mg of purified protein per liter of culture depending on the enzyme.
  • Enzymatic activity is reported in terms of units (U), where 1 U is the amount of enzyme required to convert 1 ⁇ mole/min of substrate into product and the specific activity is reported as U/mg. Enzymatic activities were determined only for guaB, guaA, purB, purA, purF and purD and for all enzymes in Figure 2 except folD and glyA by coupling the reaction to consumption of ATP which is monitored by the change in A 340 due to the action of lactate dehydrogenase on ADP and NADH. To assay organic pyrophosphatase, the phosphate detection method of Michelson was used. When substrates were not available, the amount of enzyme was estimated empirically, using an assumed specific activity of 1 U/mg. Table 1 shows the amount of each enzyme added in U or mg for each synthesis.
  • Stoichiometric substrates 212 mg (1.2 mmoles) glucose, 1 g. (18.8 mmoles) NH 4 Cl, 687 mg (4.7 mmoles) glutamine, 500 mg (4.7 mmoles) 13 C 3 -serine, 235 mg (2.35 mmoles) KHCO 3 .
  • Fuel reagents 2.12 g (9.4 mmoles) ⁇ -KG, 7.7 g. (23.5 mmoles) creatine phosphate.
  • Catalytic cofactors 6.43 mg (0.012 mmoles) ATP, 6.6 mg (0.012 mmoles) GTP, 8.8 mg (0.012 mmoles) NADP + , 68 mg (0.12 mmoles) THF, 38 mg (0.24 mmoles) fumarate.
  • Substrates and cofactors were combined in 50 mM KH 2 PO 4 ZK 2 HPO 4 pH 7.6, 20 mM MgCl 2 , 20 mM DTT, 100 ⁇ g/mL ampicillin, 50 ⁇ g/mL kanamycin. Enzymes were added according to Table 1 for compound 1, giving a final volume of 120 mL. HPLC chromatograms of the reaction time course are included in Figure 4.
  • Substrates and cofactors were added to 50 mM KH 2 PO 4 ZK 2 HPO 4 pH 7.6, 20 mM MgCl 2 , 20 mM DTT, 100 ⁇ g/mL ampicillin, 50 ⁇ g/mL kanamycin. Enzymes were added according to the Table 1 for compound 2 giving a final volume of 120 mL.
  • Substrates and cofactors were added to 25 mM KH 2 PO 4 /K 2 HPO 4 pH 7.6, 10 mM MgCl 2 , 10 mM DTT, 50 ⁇ g/mL ampicillin, 25 ⁇ g/mL kanamycin and flushed thoroughly with argon. Enzymes were added according to the Table 1 for compound 3 to give a final volume of 100 mL. Argon was gently bubbled again and the glass flask was sealed. An equal amount of additional creatine phosphate was added during this reaction due to increased consumption of (d)ATP by glnA.
  • Catalytic cofactors 2.68 mg (0.005 mmoles) ATP, 26.8 mg (0.050 mmoles) dATP, 3.7 mg (0.005 mmoles) NADP + , 3.5 mg (0.005 mmoles) NAD + , 5mg (0.0085 mmoles) THF, 8 mg (0.05 mmoles) fumarate.
  • Substrates and cofactors were added to 25 mM KH 2 PO 4 /K 2 HPO 4 pH 7.6, 10 mM MgCl 2 , 10 mM DTT, 50 ⁇ g/mL ampicillin, 25 ⁇ g/mL kanamycin and flushed thoroughly with argon. Enzymes were added according to Table 1 for compound 4 to give a final volume of 100 mL. Argon was gently bubbled again and the glass flask was sealed.
  • HIV-2 TAR RNA (5 'GGCC AG AUUG AGCCUGGG AGCUCUCUGGCC3 ' ) was synthesized by in vitro transcription with T7 RNA polymerase using a mixture of unlabeled UTP and CTP from Sigma and 13 C 2,8 -ATP and U- 15 N-GTP.
  • RNA was synthesized in a 20 mL reaction under optimized conditions: 21 mM total NTP's (5.25 mM each), 40 mM Tris HCl (pH 8.1), 0.1 mM spermidine, 10 mM DTT, 28 mM MgCl 2 , 0.001% Triton X-IOO, 80 mg/mL polyethylene glycol (8000 MW), 300 nM each DNA strand (Invitrogen), and 0.65 mg/mL T7 RNA polymerase, incubated at 37°C for 4 hours.
  • RNA was purified on denaturing 20% polyacrylamide gels, electro-eluted and desalted, lyophilized and diluted in 10 mM sodium phosphate (pH 6.5), 150 mM NaCl, 10% D 2 O for recording NMR spectra.

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Abstract

La présente invention concerne des nucléotides de purine marqués de manière isotopique dans la base purique, leurs procédés de fabrication et leurs utilisations, par exemple, dans des études biochimiques et biophysiques de la structure et de la fonction d’acides nucléiques. L’invention concerne également un procédé de synthèse du 13C-10-formyl-tétrahydrofolate et un procédé d’immobilisation d’enzymes recombinantes de la voie des pentoses phosphates et de la voie de synthèse de purine de novo avec des domaines d’affinité de fusion attachés à une résine d’un chélate de métal.
PCT/US2009/053225 2008-08-08 2009-08-07 Nucléotides de purine marqués de manière isotopique dans la base purique, leur procédés de fabrication et leurs utilisations Ceased WO2010017526A1 (fr)

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CN108866028A (zh) * 2017-05-09 2018-11-23 中国科学院微生物研究所 一种氨基裂解酶突变体蛋白及其编码基因与应用

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CN108866028A (zh) * 2017-05-09 2018-11-23 中国科学院微生物研究所 一种氨基裂解酶突变体蛋白及其编码基因与应用
CN108866028B (zh) * 2017-05-09 2020-04-10 中国科学院微生物研究所 一种氨基裂解酶突变体蛋白及其编码基因与应用

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