1-Deoxy-Erythrulose by Cell-Free Extracts of Bacteria And

Agric. Biol. Chem., 48 (6), 1643-1645, 1984 1643

Note preparation of enzyme solutions, gas chromatography, and physical and chemical analyses were described in the previous paper.X) PCand TLCwere performed with one of Formation of 1-Deoxy-D-fructose, the following solvent systems (by vol.): (A) isobutanol- 1 -Deoxy-D-sorbose, pyridine-acetate-water (12 : 6 : 1 : 4); (B) ethyl acetate- pyridine-water (30 : ll : 6); (C) ethyl acetate-pyridine- 1 -Deoxy-D-tagatose and water (40 : ll : 6); (D) ethyl acetate-acetate-water (3 : 3 : 1-Deoxy-erythrulose by 1); (E) ethyl acetate-petroleum ether (3: 1); (F) ethyl Cell-free Extracts of Bacteria acetate-benzene (1 : 9). WhenD-erythrose (1 g) was incubated with methyla- and Actinomycetes cetoin (4g) in the presence of a partially purified enzyme preparation of the mutant BG2532,a newly formed spot Akira Yokota and Ken-ichi Sasajima was detected on PC CR/0.29, solvent C). The product (!) was purified and isolated by a similar method to that Institute for Fermentation, described previously1* as crystals (678 mg), mp 77 ~78°C, Osaka, 1 7-85, Juso-honmachi 2-chome, [a]D -102° (c=l, water). PMR (90 MHz, D2O) 3: 1.57, Yodogawa-ku, Osaka 532, Japan 1.62, 1.67 and 2.50 (total 3H, 4 singletsinthe ratio 4 : 9 : 78 Received November 14, 1983 : 9, C-CH3), 3.70-4.40 (5H, m). Anal. Found: C, 43.67; H, 7.38. Calcd. for C6H12O5: C, 43.90; H, 7.37%. PC; Rf

In a previous paper,1} we described that cell-free extracts Table I. Comparison of Physical Data of of various microorganisms catalyzed the syntheses of 1- Compounds 1, 2 and 3 with Those deoxy-D-r/zra?-pentulose and l -deoxy-L-r/jreo-pentulose of Known1-Deoxy-hexuloses by an acyloin-type condensation reaction between pyruvate and D-glyceraldehyde or L-glyceraldehyde. The Compound mp (°C) [a]D (°) Ref. following investigation revealed that, with a similar method using pyruvate and D-erythrose, D-threose or 1-Deoxy-hexulose1 77-78 -102 glycolaldehyde, 1-deoxy-D-fructose (1), 1-deoxy-D-sor- 1-Deoxy-hexulose2 133-134 +48 bose (2), 1-deoxy-D-tagatose (3) or 1-deoxy-erythrulose 1-Deoxy-hexulose3 123-124 -ll.4 (3,4-dihydroxy-2-butanone) (4) was also synthesized 1-Deoxy-D-fructose 77-78 -90, -85, 2-7 by cell-free extracts of many strains of bacteria and actinomycetes. -82, -81 1 -Deoxy-L-fructose - + 85 3 The present paper deals with the enzymatic formation of 1 -Deoxy-D-sorbose - - 1 2 1-deoxy-ketoses 1, 2, 3 and 4, by using a partially purified 1-Deoxy-L-sorbose 149- 151 -51 ll enzymepreparation of a transketolase mutant of Bacillus 1-Deoxy-D-tagatose 130, -14 8, 9 pumilus IFO 12089, BG2532, and also with the distri- 121-123 bution of the enzyme activities in bacteria and 1-Deoxy-D-psicose 97-98 +1.5, -0.1 10, ll actinomycetes. 1-Deoxy-L-psicose Syrup -1.0, -0.25 2, 4 Microorganisms, culture media, cultural conditions,

CH3 CH3 CH3 CH3 C=0 C=N-NH-C6H5 C=0 C=0 HOCH C=N-NH-C6H5 HCOH HOCH HCOH HCOH HOCH HOCH HCOH HCOH HCOH HCOH CH2OH CH2OH CH2OH CH2OH

1 la 2 3

9H3 CH3 CH3 C=O C=N-NH-C6H3 (NO2) 2 V=0 HCOH H9OH H9OCOC6H5 CH2OH CH2OH CH2OCOC6H5

4 4a 4b

Fig. 1. Chemical Structures of 1-Deoxy-ketoses and Their Derivatives. Abbreviations: TPP, thiamine pyrophosphate; PC, paper chromatography; TLC, thin-layer chromatography. 1644 A. Yokota and K. Sasajima

0.54 (solvent D). It reduced alkaline silver nitrate and 43.95; H, 7.40. Calcd. for C6H12O5; C, 43.90; H. 7.37%. alkaline triphenyltetrazolium chloride, and gave a brick PC; Rf0.27 (solvent C). TLC; Rf0.35 (solvent C). Minor red color with vanillin-perchloric acid reagent. TLC; Rf product (21mg), mp 123~124°C, [a]D -ll.4° (c=0.5, 0.45 (solvent A), 0.37 (solvent C). From these data, 1 was water). PMR(D2O) 5: 1.66 and 2.54 (total 3H, two singlets assumed to be a 1-deoxy-hexulose. By comparison of the in the ratio 97:3, C-CH3), 3.63-4.33 (m, 5H). Anal. physical data of 1 with those of known 1-deoxy- Found: C, 43.85; H, 7.39. Calcd. for C6H12O5; C, 43.90; hexuloses2~12) (Table I), 1 was deduced to be 1-deoxy-D- H, 7.37%. PC; Rf0.32 (solvent B). TLC; tf/0.38 (solvent fructose. Preparation of the phenylosazone derivative (la) C). Both compounds reduced alkaline silver nitrate and confirmed the chemical structure of 1. Crystalline la alkaline triphenyltetrazolium chloride, and 2 gave a (22mg from lOOmg of 1), mp 137~138°C, [a]D -25.0° brick red and 3 a reddish brown color with vanillin-per- (c= 1, pyridine). Anal Found: C, 63.19; H, 6.28; N, 16.01. chloric acid. From these data, the two products, 2 and 3, Calcd. for C15H22N4O3; C, 63.14; H, 6.48; N, 16.36%. were also assumed to be 1-deoxy-hexuloses. By compari- TLC; Rf0.56 and 0.1-6 (solvent E). (lit. mp 137~140°C son of their physical data with those of known 1-deoxy- and [a]5780 -28° (c= 1.3, pyridine),4) mp 136- 139°C and hexuloses2~12) (Table I), they were determined to be 1- [a]D -18.5° (c=0.6, pyridine),6) TLC; Rf0.44 and 0.10 deoxy-D-sorbose (2) and 1-deoxy-D-tagatose (3), respec- (solvent E)6).) tively. WhenD-threose (0.5 g, prepared from D-galactose by the When glycolaldehyde (1 g) was incubated with methyl- method described by Perlin and Brice13)) was incubated acetoin (4 g) in the presence of a partially purified enzyme with methylacetoin (2g) in the presence of a partially solution of the mutant BG2532, a newly formed spot (PC, purified enzyme solution of the mutant BG2532, a major i?/0.78, solvent C) was detected. The product was purified compound (PC, Rf 0.27, solvent C) (2) and a minor and isolated as crystals (4, 817mg), mp 24~25°C, [a]D compound (PC, Rf0.32, solvent C) (3) were formed. Each +0.3° (c=4, H2O). PMR (90MHz, D2O) 6: 2.46 (3H, s, product was purified and isolated as crystals from the C-CH3), 4.12 (2H, d, /=5.0Hz), 4.62 (1H, t, /=4.5Hz). reaction mixture. Major product (123mg), mp 133- PC; it/0.78 (solvent Q* It reduced alkaline silver nitrate 134°C, [a]D +48° (c=l, water). PMR (D2O) S: 1.66 and and alkaline triphenyltetrazolium chloride, and gave a 2.52 (total 3H, two singlets in the ratio 93:7, C-CH3), light brown color with vanillin-perchloric acid reagent. 3.40-3.63 (m, 1H), 3.66-4.10 (m, 4H). Anal. Found: C, Fromthese data, reaction product 4 was determined to be

Table II. Formation of 1-Deoxy-d-fructose and 1-Deoxy-erythrulose by Various Microorganisms The composition of the reaction mixture was similar to that described in the previous paper.1 Formation of . ..Microorganism. Strain _.__No. .. Medium,. Proteinf , .. , 1 TA 6 IFO (mg/ml) 1 -Deoxy-D- 1 -Deoxy- fructosefl erythmlose6

Acetobacter aceti 328 1 E 1.6 -c - Acetobacter aceti 328 1 E pptd + + Alcaligenes faecalis 1 3 1 1 1 B 5.6 + + Arthrobacter globiformis 12 1 37 B 2.2 + + Bacillus pumilus 1 2089 A 2. 8 + + Bacillus pumilus BG2532 A 3.3 + + Bacillus subtilis 1 3719 A 2.3 + + Escherichia coli 13168 B 3. 1 + + Klebsiella pneumoniae 33 1 7 B 8.4 + + Micrococcus luteus 3333 C 1.4 + + Pseudomonas aeruginosa 1 2689 B 7.4 + + Actinoplanes missouriensis 1 3243 F 2.2 + + Nocardia erythropolis 1 2682 F 2.2 + + The reaction mixture contained sodium pyruvate and D-erythrose as substrates. The reaction mixture contained sodium pyruvate and glycolaldehyde as substrates. The signs plus and minus show the results obtained by PC with solvent C and with vanillin-perchloric acid as spray reagent. The insoluble fraction of the disrupted cells, which was resuspended in the original volume of buffer A, was used. Enzymatic Formation of 1-Deoxy-ketoses 1645

1-deoxy-erythrulose (3,4-dihydroxy-2-butanone). Prepa- the members of the Chemical Research Laboratories of rations of dibenzoate (4a) and 2,4-dinitrophenylhydra- Takeda Chemical Industries for the physicochemical zone (4b) derivatives confirmed the chemical structure of measurements. 4. Crystalline 4a (462mg from 200mg of4), mp 86-87°C. PMR (90MHz, CDC13) d: 2.33 (3H, s, C-CH3), 4.86 (2H, REFERENCES dd), 5.63 (1H, q), 7.27-7.73 (6H, m, aromatic protons), 7.93~8.23 (4H, m, aromatic protons). Anal. Found: C, 69.ll; H, 5.41. Calcd. for C18H16O5: C, 69.22; H, 5.16%. 1) A. Yokota and K. Sasajima, Agric. Biol. Chem., 48, TLC; Rf 0.50 (solvent F). (lit. mp 87°C15) and 149 (1984). 2) K. James and S. J. Angyal, Aust. J. Chem., 25, 1967 84~85°C16).) Crystalline 4b (7mg from 150mg of 4), mp (1972). 116~117°C. Anal. Found: C, 42.65; H, 4.42; N, 18.41. Calcd. for C10H12N4O6: C, 42.23; H, 4.22; N, 19.71%. 3) S. J. Angyal, G. S. Bethell, D. E. Cowley and V. A. TLC; Rf0.29 (solvent E). (lit. mp 118°C15).) Pickless, Aust. J. Chem., 29, 1239 (1976). Compounds 1, 2 and 3 have been chemically synthe- 4) A. Ishizu, B. Lindberg and O. Theander, Carbo- sized.2 ~9>12) Compound4 has been chemically synthesized hydr. Res., 5, 329 (1967). as dibenzoate15 ~17) and isopropylidene18) derivatives. The 5) J. Thiem, D. Rasch and H. Paulsen, Chem. Ber., previous papers1>19) and this paper have shown that a 109, 3588 (1976). series of 1-deoxy-ketoses are formed by a partially purified 6) C. R. Haylock, L. D. Melton, K. N. SlessorandA. S. enzyme preparation of a transketolase mutant of B. Tracey, Carbohydr. Res., 16, 375 (1971). pumilus IFO 12089, BG2532. 7) W. L. Dills, Jr. and W. L. Meyer, Biochemistry, 15, 4506 (1976). The mechanism of the formation of these sugars may be the same as that already described in the previous 8) W. L. Dills, Jr. and T. R. Covey, Carbohydr. Res., paper.1} Acetaldehyde or acetone was detected by gas 89, 338 (1981). chromatograpy as a by-product using DL-acetoin or 9) M. L. Wolfromand R. B. Bennett, /. Org. Chem., 30, 1284 (1965). methylacetoinas a substrate. The results on the formation of 1-deoxy-hexuloses other 10) M. L. Wolfrom, A. Thompson and E. F. Evans, /. than 1-deoxy-D-tagatose, together with our previous re- Am. Chem. Soc, 67, 1793 (1945). sults on 1-deoxy-Mreopentuloses1* and l-deoxy-D-fl/rro- ll) J. Smejkal and J. Farkas, Collect. Czech. Chem. heptulose phosphate19) formation, show that the reaction Commun., 28, 1345 (1963). 12) F. J. L. Aparicio, M. G. Guillenand1. 1. Cubero,An. products of these acyloin-type condensations are 1-deoxy- Quim., 72, 938 (1976). ketoses with the trans configuration of hydroxyls at carbons 3 and 4. However, the formation of 1-deoxy-D- 13) A. S. Perlin and C. Brice, Can. J. Chem., 34, 541 (1956). tagatose which has the cis configuration at carbons 3 and 4 is an unexpected result. The reason for the formation of 14) M. L. Wolfrom, D. I. Weisblat, W. H. Zophy and S. this compoundas a sub-component in the enzymereaction W. Waisbrot, /. Am. Chem. Soc, 63, 201 (1941). 15) H. O. L. Fischer, E. Baer, H. Pollock and H. remainsnowobscure. Various strains of bacteria and actinomycetes had the Nidecker, Helv. Chim. Ada, 20, 1213 (1937). ability to catalyze the acyloin-type condensation between 16) A. Sakurai and M. Goto, Tetrahedron Lett., 25, 2941 pyruvate and D-erythrose or glycolaldehyde, to form 1- (1968). deoxy-D-fructose or 1-deoxy-erythrulose (Table II). 17) A. Sakurai and M. Goto, /. Biochem., 65, 755 (1969). 18) F. J. L. Aparicio, F. J. L. Herrera and M. V. Acknowledgments. Wewish to thank Dr. T. Iijima Fernandez, An. Quim., 74, 1561 (1978). and Dr. K. Imai of this Institute, for their encouragement 19) A. Yokota and K. Sasajima, Agric. Biol. Chem., and valuable discussions. Weare also grateful to Mrs. 47, 1545 (1983). Ihomi Nishiura for her able assistance in this work and to