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A chemo-enzymatic cascade for the one-pot synthesis of 1-deoxy-d -xylulose 5-phosphate and 1-deoxy-d -xylulose
Tetrahedron Letters 53 (2012) 4809–4812
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
A chemo-enzymatic cascade for the one-pot synthesis of 1-deoxy-D-xylulose 5-phosphate and 1-deoxy-D-xylulose Shao-Bo Dai , Juan Liao , Jie Tian, Heng Li, Wen-Yun Gao ⇑ Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education) and College of Life Sciences, Northwest University, 229 North Taibai Road, Xi’an, Shaanxi 710069, PR China
a r t i c l e
i n f o
Article history: Received 20 February 2012 Revised 25 May 2012 Accepted 12 June 2012 Available online 18 June 2012 Keywords: 1-Deoxy-D-xylulose 5-phosphate 1-Deoxy-D-xylulose Chemo-enzymatic cascade Methylerythritol phosphate pathway
a b s t r a c t A chemo-enzymatic cascade for the one-pot preparation of 1-deoxy-D-xylulose 5-phosphate (DXP) and 1-deoxy-D-xylulose (DX) from stable, cheap, and easily available starting material R-glycidol is reported. The epoxide ring of R-glycidol was opened with phosphate to generate L-glycerol 3-phosphate, which was subsequently converted into the target molecules by combination of multi-enzymatic reactions in the same flask with purified overall yields of 27.6% (DXP) and 33% (DX), respectively. This approach represents the first one-pot chemo-enzymatic synthesis of these two biologically important compounds. Ó 2012 Elsevier Ltd. All rights reserved.
1-Deoxy-D-xylulose 5-phosphate (DXP, 1) is one of the key intermediates of the newly discovered 2-methyl-D-erythritol 4phosphate (MEP, 2) pathway for isoprenoid biosynthesis in the chloroplast of phototrophic organisms and in certain bacteria including human pathogens such as the malarial parasite Plasmodium falciparum (Scheme 1).1,2 Also, it is a precursor for the biosynthesis of vitamins B1 and B6 in some bacteria.3–5 Compound 1 is biosynthesized via the condensation of pyruvate 3 and D-glyceraldehyde 3-phosphate (D-GAP, 4) catalyzed by DXP synthase (DXS), a thiamine pyrophosphate (ThPP) dependent enzyme.6–8 Furthermore, 1 is converted into 2 in a reaction catalyzed by DXP reductoisomerase (DXR) in the presence of NADPH and a divalent cation through a retro-aldol/aldol mechanism.9–11 All enzymes of MEP pathway are promising targets for screening new antibiotics, especially DXR.12,13 Thus, there is an increasing demand for large-scale preparation of 1, the natural substrate of DXR. 1-Deoxy-D-xylulose (DX, 5), the dephosphorylated form of 1, is not a direct intermediate of the pathway, but it can be phosphorylated by D-xylulokinase (DXK) in plants and microbes to form 1 and then effectively incorporated into final terpenoids by organisms,14 so it is regarded as a potential intermediate in the route. Many chemical and enzymatic methods have been established up to date to obtain 1 and 5 due to their biological importance.15 Often, the chemical routes for these two compounds are not practical because they are normally multi-step procedures with low to ⇑ Corresponding author. Tel.: +86 29 88303446x852; fax: +86 29 88303572.
E-mail address: [email protected] (W.-Y. Gao). These two authors equally contributed to this work.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2012.06.062
medium overall yields. Although the enzymatic routes are always achieved in one-pot processes with acceptable yields, either too many enzymes or unstable sugar phosphates have to be involved. Two enzymatic methods have been set up by this group which are not very convenient because the triose phosphates used as starting materials are either unstable or too expensive.16,17 Combination of these two strategies to establish a ‘one-pot’, chemo-enzymatic synthesis of the two compounds has not been explored so far. Recently, Hecquet and co-workers developed a chemo-enzymatic method to synthesize dihydroxyacetone phosphate (DHAP, 6) from inexpensive, stable, and commercially available starting material rac-glycidol (rac-7), which underwent a chemical opening of the epoxide ring of rac-7 and then enzymatic oxidation to produce 6 with an overall yield of about 28% (If R-7 was used, the yield supposed to be 55%).18 We thought that the in situ generated 6 would be a possible source for the preparation of 1 and 5. Considering that L-glycerophosphate oxidase (L-GPO) exclusively and stereoselectively catalyzes the secondary alcohol oxidation of 19 L-glycerol 3-phosphate (L-G-3-P, 8), we replaced rac-7 with R-7 to make the process more effective. The sequence we used comprised a separated chemical epoxide ring opening followed by a multi-enzymatic conversion in the same flask and downstream purification. The whole event can be accomplished within 24 h (Scheme 2). The epoxide ring opening of R-7 with phosphate followed the Hecquet’s process with minor modification.18 After that, the reaction tube was cooled down to room temperature and the pH value was adjusted to 6.8 (an optimal value, see below) in order that the enzymes of next step could enjoy maximal catalytic activation.
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OH O
OH 5 OH DXK
O COOO
3
NADPH NADP+ HO
OH
CO2 O
+ DXS
OH
OPO32-
H OH
OPO32-
OPO32DXR
terpenoids
OH OH 2
1
4 Scheme 1. MEP pathway for terpenoid biosynthesis.
O R-7
HO NaH2PO4
OPO3H2 OH + OH
OH
9 OPO3H2
HO
one step, one-pot
1
AK
5
8 H2O
DXS ThPP Mg2+ 3
1/2 O2 L-GPO H2O2 catalase O HO
OPO3H2
TIM
4
6 Scheme 2. Two-step one-pot reaction leading to DXP and then to DX from Rglycidol.
Subsequently, compound 3, ThPP, Mg2+, and the enzymes L-GPO, catalase, triosephosphate isomerase (TIM), and DXS were added into the mixture and incubation was performed at 37 °C for 6 h. Formation of DXP was monitored by paper chromatography. In the enzymatic reaction, 8 formed from R-7 was aerobically oxidized to 6 with the concomitant reduction of O2 to H2O2 that was poisonous to the enzymes and was thus decomposed back to O2 by catalase. The product O2 could further support the oxygenation for L-GPO.19 We found that an extra O2 supply could not enhance the DXP production. In the following reactions, 6 was isomerized by TIM to 4 which was subsequently transformed into 1 by DXS in the presence of ThPP, 3 and Mg2+. Although the equilibrium between the two triose phosphates largely favors 6 side with a 6 to 4 ratio being about 96 to 4 as reported,20 the consumption of 4 by DXS made the reaction directionally move toward 4 and the isomerization could smoothly carry out. Furthermore, the combined enzymatic conversion of 6 to the final product could not only prevent its inhibition of L-GPO and thus ensured effective conversion of L-G-3-P 8, but also suppresses nearly any possible decomposition of 6, especially hydrolysis, because of the relatively low concentration of 6.19 The multi-enzymatic reaction was preferably run under a slightly acidic condition (pH 6.8) to make sure that 6 was maximally stable and to maintain an excellent conversion rate. To maximize the production of 1, we optimized the reaction by changing pH value, reaction temperature, amounts of L-GPO and catalase, phosphate source, and the molar ratio of R-7 to phosphate. The other parameters such as the amounts of TIM, DXS and so on have already been evaluated in the previous work.16 Production of 1 was monitored in accordance with the published method by extending the reaction to formation of 2 in the presence of DXR and NADPH21 and each experiment was repeated three times to avoid accidental error. All the results were depicted in Figure 1. As shown in Panel A, three different temperatures were tested and the results displayed that 37 °C was the best for the activities of all four enzymes. When the temperature reached 45 °C, barely no DXP was obtained, indicating that L-GPO or/and
catalase were very sensitive to relatively high temperature because we have shown that TIM and DXS still exhibited catalytic activity even at 50 °C.16 To simplify the experiment, the pH effect on the four enzymes was considered as a single factor and tested together, which was shown in Panel B. As described previously,16 the optimum pH for TIM and DXS in preparation of 1 was 7.5. But when they were combined with L-GPO and catalase, reaction performed at pH 7.5 gave lower yield of 1 than that performed at pH 6.8. So we finally chose pH 6.8 for the multi-enzymatic reaction. L-GPO and catalase both were critical for the formation of the intermediate 6. Panel C showed that with increase of L-GPO, the concentration of 1 first increased then decreased and best 1 yield was achieved when 1.0 unit of L-GPO was utilized for every 25 lmol of R-7. Panel D displayed the influence of catalase on the yield of 1, from which one could find that when catalase reached 45 units for 25 lmol of R-7, the maximum formation of 1 was acquired. We observed that, just like L-GPO, more catalase conversely decreased the production of 1 and the reason remained ambiguous. In addition, we appraised the influence of the phosphate salts and the molar ratio of R-7 to the phosphate on the formation of 1. Four phosphate salts were tested and the results (Panel E) showed when K2HPO4 or Na2HPO4 was used, higher 1 concentration was detected, whereas less 1 was produced when dihydrogen phosphates (KH2PO4 or NaH2PO4) were employed. It is obvious that the hydrogen phosphates are stronger base and thus much better nucleophiles than dihydrogen phosphates, so we selected Na2HPO4 to open the epoxide ring of R-7. Further on, we measured the impact of molar ratio of R-7 to the phosphate on 1 yield and the data (Panel F) indicated that both molar ratios of 2:1 and 1:1 led to good yields of 1 with the former one being slightly higher. We eventually determined 1:1 as the R-7 to phosphate ratio. Combining the optimized factors, a preparatory reaction was conducted accordingly. After reaction, the concentration of 1 was measured photometrically by extending the reaction to formation of MEP 2 in the presence of DXR and NADPH21 and the result showed that the yield of compound 1 was about 40%. Subsequent purification using the ion-exchange protocol16 afforded 1 in little more than 12% yield with purity higher than 95% as shown by its 1 H NMR spectrum. The left part of 1 was contaminated by L-glycerol 2-phosphate (9, Scheme 1) that was a byproduct of the epoxide ring opening of R-7 and could not be oxidized by L-GPO. Repeated ion-exchange purification would largely remove 9 from 1, but decomposition of 1 during re-chromatographic procedure could also be enormous. To improve the production of 1, we tried to isolate the compound with silica gel column chromatograph and found that the solvent system i-propanol/concd NH4OH–H2O (6:3:1) reported by Keller22 resulted in good separation of compounds 1 and 9 (Rf values for 1 and 9 were 0.33 and 0.25, respectively). Silica gel column chromatography using the selected solvent system followed by ion-exchange on a Dowex 50w 8 resin (H-form) column gave the target 1 as free acid in a yield around 28%. To get compound 5, we treated the reaction mixture with alkaline phosphatase and 1 was thus converted into 5 (Scheme 1).
S.-B. Dai et al. / Tetrahedron Letters 53 (2012) 4809–4812
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Figure 1. Optimization of the chemo-enzymatic formation of DXP. Standard chemical process: 0.1 mmol R-7 and 0.1 mmol phosphate in 100 lL H2O were incubated at 100 °C for 5 h. After cooling down and pH adjustment, 25 lL was used for next step. The multi-enzymatic reaction mixture contained 120 mM Tris–HCl, 10 mM MgCl2, 2 mM ThPP, 2 mM mercaptoethanol, 2.8 mg sodium pyruvate, 1 unit L-GPO, 45 units catalase, 2 units TIM, 50 lg DXS in a volume of 100 lL. (A) Temperature dependency at pH 7.5; (B) pH dependency at 37 °C; (C) L-GPO dependency at 37 °C and pH 6.8; (D) catalase dependency at 37 °C and pH 6.8; (E) phosphate dependency; (F) R-7 to phosphate ratio dependency.
After single purification on silica gel, pure 5 was obtained with an overall yield of about 33%.23 Based on the above investigation, we conclude that this method provides an attractive one-pot, chemo-enzymatic procedure for the syntheses of 1 and 5 from cheap, stable, and easily available starting materials. Acknowledgments We gratefully acknowledge Prof. C. Dale Poulter, Chemistry Department of University of Utah, for providing the plasmid pFMH30 for the preparation of DXS. We also thank Prof. Z.-F. Wang and Prof. L.-J. Huang of this institute for assistance with the ESI-MS measurements. This work was supported by the National Science Foundation of China (NSFC) Grants 20772095 and 21172179 and Northwest University innovation funds for the graduate students (09YZZ59 and 10YYB02). Supplementary data Supplementary data (preparation of DXS and NMR spectra of DXP and DX) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.06.062. References and notes 1. Rohmer, M. In Comprehensive Natural Products II, Chemistry and Biology; Mander, L., Liu, H. W., Eds.; Elsevier, 2010; Vol. 1, pp 517–555. 2. Eisenreich, W.; Bacher, A.; Arigoni, D.; Rohdich, F. Cell. Mol. Life Sci. 2004, 61, 1401. 3. David, S.; Estramareix, B.; Fischer, J. C.; Thérisod, M. J. Chem. Soc., Perkin Trans. 1 1982, 2131. 4. Hill, R. E.; Sayer, B. G.; Spenser, I. D. J. Am. Chem. Soc. 1989, 111, 1916. 5. Cane, D. E.; Du, S.; Robinson, J. K.; Hsiung, Y.; Spenser, I. D. J. Am. Chem. Soc. 1999, 121, 7722. 6. Sprenger, G. A.; Schoerken, U.; Wiegert, T.; Grolle, S.; de Graaf, A. A.; Taylor, S. V.; Beggley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12857. 7. Lois, L. M.; Campos, N.; Rosa-Putra, S.; Danielsen, K.; Rohmer, M.; Boronat, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2105. 8. Lange, B. M.; Wildung, M. R.; McCaskill, D.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2100.
9. Takahashi, S.; Kuzuyama, T.; Watanabe, W.; Seto, H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9879. 10. Wong, U.; Cox, R. J. Angew. Chem., Int. Ed. 2007, 46, 4926. 11. Munos, J. W.; Pu, X.; Mansoorabadi, S. O.; Kim, H. J.; Liu, H. J. Am. Chem. Soc. 2009, 131, 2048–2049. 12. Rohdich, F.; Bacher, A.; Eisenreich, W. Biochem. Soc. Trans. 2005, 33, 785. 13. Ershov, Y. V. Appl. Biochem. Microbiol. 2007, 43, 115. 14. Wungsintaweekul, J.; Herz, S.; Hecht, S.; Eisenreich, W.; Feicht, R.; Rohdich, F.; Bacher, A.; Zenk, M. H. Eur. J. Biochem. 2001, 268, 310. 15. Li, H.; Dai, S. B.; Gao, W. Y. Helv. Chim. Acta 2012, 95, 683. 16. Zhou, Y. F.; Cui, Z.; Li, H.; Tian, J.; Gao, W. Y. Bioorg. Chem. 2010, 38, 120. 17. Li, H.; Jie, T.; Hui, W.; Yang, S. Q.; Gao, W. Y. Helv. Chim. Acta 2010, 93, 1745. 18. Charmantray, F.; Dellis, P.; Samreth, S.; Hecquet, L. Tetrahedron Lett. 2006, 47, 3261. 19. Fessner, W. D.; Sinerius, G. Angew. Chem., Int. Ed. 1994, 33, 209. 20. Veech, R. L.; Raijman, L.; Dalziel, K.; Krebs, H. A. Biochem. J. 1969, 115, 837. 21. Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H. Tetrahedron Lett. 1998, 39, 7913. 22. Keller, R. K. J. Chromatogr. 1993, 645, 161. 23. (1) General one-pot, chemo-enzymatic procedure for the preparation of 1deoxy-D-xylulose 5-phosphate (1). Disodium hydrogen phosphate (71 mg, 0.5 mmol) was dissolved in 460 lL ddH2O in a 2 mL centrifugal tube with efficient stirring (pH 10). Upon dissolution of the salt, R-7 (39 mg, 0.5 mmol) was added and the resulting mixture was incubated in boiling water for 5 h. The reaction vessel was cooled down to room temperature and the pH was adjusted to 6.8 with 2 M hydrochloric acid. Then sodium pyruvate (55 mg, 0.5 mmol), thiamin pyrophosphate (2 mM), MgCl2 (10 mM), bmercaptoethanol (2 mM), L-GPO (20 units), catalase (900 units), TIM (40 units), and recombinant R. capsulatus DXS (1 mg) were added into the mixture. ddH2O was subsequently supplemented to a total volume of 2 mL and the mixture was incubated at 37 °C for 6 h. The reaction mixture thus obtained was evaporated under reduced pressure to remove water and the residue was extracted with methanol (2 mL 3). Subsequent removal of methanol under a gentle blow of N2 furnished an oily residue which was then isolated on a silica gel column using i-propanol/concd NH4OH–H2O (6:3:1 (v/v/v)). Fractions of 10 mL were collected and analyzed by TLC. The parts containing DXP were pooled and evaporated under reduced pressure to a small volume before 10 mL ddH2O was added. The mixture was then neutralized with 2 M HCl and reevaporated under reduced pressure to remove organic solvents. The residue aqueous solution was applied to a Dowex 50w 8 resin (H-form) column to remove NH4+. The acidic eluate was collected on ice and concentrated in vacuo. An oily yellowish DXP as free acid was obtained (29.7 mg, yield 27.6%) and identified by MS, 1H NMR and 31P NMR. ½a20 D 1.3° (c 4.1, H2O). ESI-MS (negative mode) m/z (%) 213 [MH] (100%). 1H NMR (400 MHz, D2O): d = 2.33 (3H, s, 1-H), 4.05 (2H, m, 5-H), 4.43 (1H, ddd, J = 2 Hz, 2 Hz, 1 Hz, 4-H), 4.54 (1H, d, J = 2 Hz, 3-H). 31P NMR (121.5 MHz, D2O): d = 1.21 (s). (2) General procedure for the preparation of 1-deoxy-D-xylulose (5). Upon completion of the reaction for DXP synthesis, the pH of the mixture was adjusted to 9.0 with 1 M NaOH and alkaline phosphatase (100 units) was added. After incubation at 37 °C for 4 h, the reaction mixture was evaporated under reduced pressure to
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remove water and the residue was extracted with methanol (2 mL 3). Subsequent removal of methanol under a gentle blow of N2 gave an oily residue which was then isolated on a silica gel column using an acetone– trichloromethane solvent system (1:1 to 5:1 (v/v)). Fractions of 10 mL were collected and analyzed by TLC. The parts containing DX were pooled and evaporated under reduced pressure to get rid of organic solvents. Oily DX (22 mg, yield 33%) was obtained and detected by ESI-MS and 1H NMR. ½a20 D
1.8° (c 1.7, MeOH). ESI-MS (positive mode) m/z (%) 157 [M+Na]+ (100%). 1H NMR (400 MHz, D2O): d 1.45, 1.49 (anomers), 2.31 (open chain) (total 3H, 3s, in the ratio about 1:1:4), 3.62–3.83 (m, 2H, OCH2), 4.15–4.43 (m, 2H, OCHCHO).24 (3) The yields of DX and DXP were calculated from R-7. 24. Kennedy, I. A.; Hemscheidt, T.; Britten, J. F.; Spenser, I. D. Can. J. Chem. 1995, 73, 1329.
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
A chemo-enzymatic cascade for the one-pot synthesis of 1-deoxy-D-xylulose 5-phosphate and 1-deoxy-D-xylulose Shao-Bo Dai , Juan Liao , Jie Tian, Heng Li, Wen-Yun Gao ⇑ Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education) and College of Life Sciences, Northwest University, 229 North Taibai Road, Xi’an, Shaanxi 710069, PR China
a r t i c l e
i n f o
Article history: Received 20 February 2012 Revised 25 May 2012 Accepted 12 June 2012 Available online 18 June 2012 Keywords: 1-Deoxy-D-xylulose 5-phosphate 1-Deoxy-D-xylulose Chemo-enzymatic cascade Methylerythritol phosphate pathway
a b s t r a c t A chemo-enzymatic cascade for the one-pot preparation of 1-deoxy-D-xylulose 5-phosphate (DXP) and 1-deoxy-D-xylulose (DX) from stable, cheap, and easily available starting material R-glycidol is reported. The epoxide ring of R-glycidol was opened with phosphate to generate L-glycerol 3-phosphate, which was subsequently converted into the target molecules by combination of multi-enzymatic reactions in the same flask with purified overall yields of 27.6% (DXP) and 33% (DX), respectively. This approach represents the first one-pot chemo-enzymatic synthesis of these two biologically important compounds. Ó 2012 Elsevier Ltd. All rights reserved.
1-Deoxy-D-xylulose 5-phosphate (DXP, 1) is one of the key intermediates of the newly discovered 2-methyl-D-erythritol 4phosphate (MEP, 2) pathway for isoprenoid biosynthesis in the chloroplast of phototrophic organisms and in certain bacteria including human pathogens such as the malarial parasite Plasmodium falciparum (Scheme 1).1,2 Also, it is a precursor for the biosynthesis of vitamins B1 and B6 in some bacteria.3–5 Compound 1 is biosynthesized via the condensation of pyruvate 3 and D-glyceraldehyde 3-phosphate (D-GAP, 4) catalyzed by DXP synthase (DXS), a thiamine pyrophosphate (ThPP) dependent enzyme.6–8 Furthermore, 1 is converted into 2 in a reaction catalyzed by DXP reductoisomerase (DXR) in the presence of NADPH and a divalent cation through a retro-aldol/aldol mechanism.9–11 All enzymes of MEP pathway are promising targets for screening new antibiotics, especially DXR.12,13 Thus, there is an increasing demand for large-scale preparation of 1, the natural substrate of DXR. 1-Deoxy-D-xylulose (DX, 5), the dephosphorylated form of 1, is not a direct intermediate of the pathway, but it can be phosphorylated by D-xylulokinase (DXK) in plants and microbes to form 1 and then effectively incorporated into final terpenoids by organisms,14 so it is regarded as a potential intermediate in the route. Many chemical and enzymatic methods have been established up to date to obtain 1 and 5 due to their biological importance.15 Often, the chemical routes for these two compounds are not practical because they are normally multi-step procedures with low to ⇑ Corresponding author. Tel.: +86 29 88303446x852; fax: +86 29 88303572.
E-mail address: [email protected] (W.-Y. Gao). These two authors equally contributed to this work.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2012.06.062
medium overall yields. Although the enzymatic routes are always achieved in one-pot processes with acceptable yields, either too many enzymes or unstable sugar phosphates have to be involved. Two enzymatic methods have been set up by this group which are not very convenient because the triose phosphates used as starting materials are either unstable or too expensive.16,17 Combination of these two strategies to establish a ‘one-pot’, chemo-enzymatic synthesis of the two compounds has not been explored so far. Recently, Hecquet and co-workers developed a chemo-enzymatic method to synthesize dihydroxyacetone phosphate (DHAP, 6) from inexpensive, stable, and commercially available starting material rac-glycidol (rac-7), which underwent a chemical opening of the epoxide ring of rac-7 and then enzymatic oxidation to produce 6 with an overall yield of about 28% (If R-7 was used, the yield supposed to be 55%).18 We thought that the in situ generated 6 would be a possible source for the preparation of 1 and 5. Considering that L-glycerophosphate oxidase (L-GPO) exclusively and stereoselectively catalyzes the secondary alcohol oxidation of 19 L-glycerol 3-phosphate (L-G-3-P, 8), we replaced rac-7 with R-7 to make the process more effective. The sequence we used comprised a separated chemical epoxide ring opening followed by a multi-enzymatic conversion in the same flask and downstream purification. The whole event can be accomplished within 24 h (Scheme 2). The epoxide ring opening of R-7 with phosphate followed the Hecquet’s process with minor modification.18 After that, the reaction tube was cooled down to room temperature and the pH value was adjusted to 6.8 (an optimal value, see below) in order that the enzymes of next step could enjoy maximal catalytic activation.
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OH O
OH 5 OH DXK
O COOO
3
NADPH NADP+ HO
OH
CO2 O
+ DXS
OH
OPO32-
H OH
OPO32-
OPO32DXR
terpenoids
OH OH 2
1
4 Scheme 1. MEP pathway for terpenoid biosynthesis.
O R-7
HO NaH2PO4
OPO3H2 OH + OH
OH
9 OPO3H2
HO
one step, one-pot
1
AK
5
8 H2O
DXS ThPP Mg2+ 3
1/2 O2 L-GPO H2O2 catalase O HO
OPO3H2
TIM
4
6 Scheme 2. Two-step one-pot reaction leading to DXP and then to DX from Rglycidol.
Subsequently, compound 3, ThPP, Mg2+, and the enzymes L-GPO, catalase, triosephosphate isomerase (TIM), and DXS were added into the mixture and incubation was performed at 37 °C for 6 h. Formation of DXP was monitored by paper chromatography. In the enzymatic reaction, 8 formed from R-7 was aerobically oxidized to 6 with the concomitant reduction of O2 to H2O2 that was poisonous to the enzymes and was thus decomposed back to O2 by catalase. The product O2 could further support the oxygenation for L-GPO.19 We found that an extra O2 supply could not enhance the DXP production. In the following reactions, 6 was isomerized by TIM to 4 which was subsequently transformed into 1 by DXS in the presence of ThPP, 3 and Mg2+. Although the equilibrium between the two triose phosphates largely favors 6 side with a 6 to 4 ratio being about 96 to 4 as reported,20 the consumption of 4 by DXS made the reaction directionally move toward 4 and the isomerization could smoothly carry out. Furthermore, the combined enzymatic conversion of 6 to the final product could not only prevent its inhibition of L-GPO and thus ensured effective conversion of L-G-3-P 8, but also suppresses nearly any possible decomposition of 6, especially hydrolysis, because of the relatively low concentration of 6.19 The multi-enzymatic reaction was preferably run under a slightly acidic condition (pH 6.8) to make sure that 6 was maximally stable and to maintain an excellent conversion rate. To maximize the production of 1, we optimized the reaction by changing pH value, reaction temperature, amounts of L-GPO and catalase, phosphate source, and the molar ratio of R-7 to phosphate. The other parameters such as the amounts of TIM, DXS and so on have already been evaluated in the previous work.16 Production of 1 was monitored in accordance with the published method by extending the reaction to formation of 2 in the presence of DXR and NADPH21 and each experiment was repeated three times to avoid accidental error. All the results were depicted in Figure 1. As shown in Panel A, three different temperatures were tested and the results displayed that 37 °C was the best for the activities of all four enzymes. When the temperature reached 45 °C, barely no DXP was obtained, indicating that L-GPO or/and
catalase were very sensitive to relatively high temperature because we have shown that TIM and DXS still exhibited catalytic activity even at 50 °C.16 To simplify the experiment, the pH effect on the four enzymes was considered as a single factor and tested together, which was shown in Panel B. As described previously,16 the optimum pH for TIM and DXS in preparation of 1 was 7.5. But when they were combined with L-GPO and catalase, reaction performed at pH 7.5 gave lower yield of 1 than that performed at pH 6.8. So we finally chose pH 6.8 for the multi-enzymatic reaction. L-GPO and catalase both were critical for the formation of the intermediate 6. Panel C showed that with increase of L-GPO, the concentration of 1 first increased then decreased and best 1 yield was achieved when 1.0 unit of L-GPO was utilized for every 25 lmol of R-7. Panel D displayed the influence of catalase on the yield of 1, from which one could find that when catalase reached 45 units for 25 lmol of R-7, the maximum formation of 1 was acquired. We observed that, just like L-GPO, more catalase conversely decreased the production of 1 and the reason remained ambiguous. In addition, we appraised the influence of the phosphate salts and the molar ratio of R-7 to the phosphate on the formation of 1. Four phosphate salts were tested and the results (Panel E) showed when K2HPO4 or Na2HPO4 was used, higher 1 concentration was detected, whereas less 1 was produced when dihydrogen phosphates (KH2PO4 or NaH2PO4) were employed. It is obvious that the hydrogen phosphates are stronger base and thus much better nucleophiles than dihydrogen phosphates, so we selected Na2HPO4 to open the epoxide ring of R-7. Further on, we measured the impact of molar ratio of R-7 to the phosphate on 1 yield and the data (Panel F) indicated that both molar ratios of 2:1 and 1:1 led to good yields of 1 with the former one being slightly higher. We eventually determined 1:1 as the R-7 to phosphate ratio. Combining the optimized factors, a preparatory reaction was conducted accordingly. After reaction, the concentration of 1 was measured photometrically by extending the reaction to formation of MEP 2 in the presence of DXR and NADPH21 and the result showed that the yield of compound 1 was about 40%. Subsequent purification using the ion-exchange protocol16 afforded 1 in little more than 12% yield with purity higher than 95% as shown by its 1 H NMR spectrum. The left part of 1 was contaminated by L-glycerol 2-phosphate (9, Scheme 1) that was a byproduct of the epoxide ring opening of R-7 and could not be oxidized by L-GPO. Repeated ion-exchange purification would largely remove 9 from 1, but decomposition of 1 during re-chromatographic procedure could also be enormous. To improve the production of 1, we tried to isolate the compound with silica gel column chromatograph and found that the solvent system i-propanol/concd NH4OH–H2O (6:3:1) reported by Keller22 resulted in good separation of compounds 1 and 9 (Rf values for 1 and 9 were 0.33 and 0.25, respectively). Silica gel column chromatography using the selected solvent system followed by ion-exchange on a Dowex 50w 8 resin (H-form) column gave the target 1 as free acid in a yield around 28%. To get compound 5, we treated the reaction mixture with alkaline phosphatase and 1 was thus converted into 5 (Scheme 1).
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Figure 1. Optimization of the chemo-enzymatic formation of DXP. Standard chemical process: 0.1 mmol R-7 and 0.1 mmol phosphate in 100 lL H2O were incubated at 100 °C for 5 h. After cooling down and pH adjustment, 25 lL was used for next step. The multi-enzymatic reaction mixture contained 120 mM Tris–HCl, 10 mM MgCl2, 2 mM ThPP, 2 mM mercaptoethanol, 2.8 mg sodium pyruvate, 1 unit L-GPO, 45 units catalase, 2 units TIM, 50 lg DXS in a volume of 100 lL. (A) Temperature dependency at pH 7.5; (B) pH dependency at 37 °C; (C) L-GPO dependency at 37 °C and pH 6.8; (D) catalase dependency at 37 °C and pH 6.8; (E) phosphate dependency; (F) R-7 to phosphate ratio dependency.
After single purification on silica gel, pure 5 was obtained with an overall yield of about 33%.23 Based on the above investigation, we conclude that this method provides an attractive one-pot, chemo-enzymatic procedure for the syntheses of 1 and 5 from cheap, stable, and easily available starting materials. Acknowledgments We gratefully acknowledge Prof. C. Dale Poulter, Chemistry Department of University of Utah, for providing the plasmid pFMH30 for the preparation of DXS. We also thank Prof. Z.-F. Wang and Prof. L.-J. Huang of this institute for assistance with the ESI-MS measurements. This work was supported by the National Science Foundation of China (NSFC) Grants 20772095 and 21172179 and Northwest University innovation funds for the graduate students (09YZZ59 and 10YYB02). Supplementary data Supplementary data (preparation of DXS and NMR spectra of DXP and DX) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.06.062. References and notes 1. Rohmer, M. In Comprehensive Natural Products II, Chemistry and Biology; Mander, L., Liu, H. W., Eds.; Elsevier, 2010; Vol. 1, pp 517–555. 2. Eisenreich, W.; Bacher, A.; Arigoni, D.; Rohdich, F. Cell. Mol. Life Sci. 2004, 61, 1401. 3. David, S.; Estramareix, B.; Fischer, J. C.; Thérisod, M. J. Chem. Soc., Perkin Trans. 1 1982, 2131. 4. Hill, R. E.; Sayer, B. G.; Spenser, I. D. J. Am. Chem. Soc. 1989, 111, 1916. 5. Cane, D. E.; Du, S.; Robinson, J. K.; Hsiung, Y.; Spenser, I. D. J. Am. Chem. Soc. 1999, 121, 7722. 6. Sprenger, G. A.; Schoerken, U.; Wiegert, T.; Grolle, S.; de Graaf, A. A.; Taylor, S. V.; Beggley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12857. 7. Lois, L. M.; Campos, N.; Rosa-Putra, S.; Danielsen, K.; Rohmer, M.; Boronat, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2105. 8. Lange, B. M.; Wildung, M. R.; McCaskill, D.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2100.
9. Takahashi, S.; Kuzuyama, T.; Watanabe, W.; Seto, H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9879. 10. Wong, U.; Cox, R. J. Angew. Chem., Int. Ed. 2007, 46, 4926. 11. Munos, J. W.; Pu, X.; Mansoorabadi, S. O.; Kim, H. J.; Liu, H. J. Am. Chem. Soc. 2009, 131, 2048–2049. 12. Rohdich, F.; Bacher, A.; Eisenreich, W. Biochem. Soc. Trans. 2005, 33, 785. 13. Ershov, Y. V. Appl. Biochem. Microbiol. 2007, 43, 115. 14. Wungsintaweekul, J.; Herz, S.; Hecht, S.; Eisenreich, W.; Feicht, R.; Rohdich, F.; Bacher, A.; Zenk, M. H. Eur. J. Biochem. 2001, 268, 310. 15. Li, H.; Dai, S. B.; Gao, W. Y. Helv. Chim. Acta 2012, 95, 683. 16. Zhou, Y. F.; Cui, Z.; Li, H.; Tian, J.; Gao, W. Y. Bioorg. Chem. 2010, 38, 120. 17. Li, H.; Jie, T.; Hui, W.; Yang, S. Q.; Gao, W. Y. Helv. Chim. Acta 2010, 93, 1745. 18. Charmantray, F.; Dellis, P.; Samreth, S.; Hecquet, L. Tetrahedron Lett. 2006, 47, 3261. 19. Fessner, W. D.; Sinerius, G. Angew. Chem., Int. Ed. 1994, 33, 209. 20. Veech, R. L.; Raijman, L.; Dalziel, K.; Krebs, H. A. Biochem. J. 1969, 115, 837. 21. Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H. Tetrahedron Lett. 1998, 39, 7913. 22. Keller, R. K. J. Chromatogr. 1993, 645, 161. 23. (1) General one-pot, chemo-enzymatic procedure for the preparation of 1deoxy-D-xylulose 5-phosphate (1). Disodium hydrogen phosphate (71 mg, 0.5 mmol) was dissolved in 460 lL ddH2O in a 2 mL centrifugal tube with efficient stirring (pH 10). Upon dissolution of the salt, R-7 (39 mg, 0.5 mmol) was added and the resulting mixture was incubated in boiling water for 5 h. The reaction vessel was cooled down to room temperature and the pH was adjusted to 6.8 with 2 M hydrochloric acid. Then sodium pyruvate (55 mg, 0.5 mmol), thiamin pyrophosphate (2 mM), MgCl2 (10 mM), bmercaptoethanol (2 mM), L-GPO (20 units), catalase (900 units), TIM (40 units), and recombinant R. capsulatus DXS (1 mg) were added into the mixture. ddH2O was subsequently supplemented to a total volume of 2 mL and the mixture was incubated at 37 °C for 6 h. The reaction mixture thus obtained was evaporated under reduced pressure to remove water and the residue was extracted with methanol (2 mL 3). Subsequent removal of methanol under a gentle blow of N2 furnished an oily residue which was then isolated on a silica gel column using i-propanol/concd NH4OH–H2O (6:3:1 (v/v/v)). Fractions of 10 mL were collected and analyzed by TLC. The parts containing DXP were pooled and evaporated under reduced pressure to a small volume before 10 mL ddH2O was added. The mixture was then neutralized with 2 M HCl and reevaporated under reduced pressure to remove organic solvents. The residue aqueous solution was applied to a Dowex 50w 8 resin (H-form) column to remove NH4+. The acidic eluate was collected on ice and concentrated in vacuo. An oily yellowish DXP as free acid was obtained (29.7 mg, yield 27.6%) and identified by MS, 1H NMR and 31P NMR. ½a20 D 1.3° (c 4.1, H2O). ESI-MS (negative mode) m/z (%) 213 [MH] (100%). 1H NMR (400 MHz, D2O): d = 2.33 (3H, s, 1-H), 4.05 (2H, m, 5-H), 4.43 (1H, ddd, J = 2 Hz, 2 Hz, 1 Hz, 4-H), 4.54 (1H, d, J = 2 Hz, 3-H). 31P NMR (121.5 MHz, D2O): d = 1.21 (s). (2) General procedure for the preparation of 1-deoxy-D-xylulose (5). Upon completion of the reaction for DXP synthesis, the pH of the mixture was adjusted to 9.0 with 1 M NaOH and alkaline phosphatase (100 units) was added. After incubation at 37 °C for 4 h, the reaction mixture was evaporated under reduced pressure to
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remove water and the residue was extracted with methanol (2 mL 3). Subsequent removal of methanol under a gentle blow of N2 gave an oily residue which was then isolated on a silica gel column using an acetone– trichloromethane solvent system (1:1 to 5:1 (v/v)). Fractions of 10 mL were collected and analyzed by TLC. The parts containing DX were pooled and evaporated under reduced pressure to get rid of organic solvents. Oily DX (22 mg, yield 33%) was obtained and detected by ESI-MS and 1H NMR. ½a20 D
1.8° (c 1.7, MeOH). ESI-MS (positive mode) m/z (%) 157 [M+Na]+ (100%). 1H NMR (400 MHz, D2O): d 1.45, 1.49 (anomers), 2.31 (open chain) (total 3H, 3s, in the ratio about 1:1:4), 3.62–3.83 (m, 2H, OCH2), 4.15–4.43 (m, 2H, OCHCHO).24 (3) The yields of DX and DXP were calculated from R-7. 24. Kennedy, I. A.; Hemscheidt, T.; Britten, J. F.; Spenser, I. D. Can. J. Chem. 1995, 73, 1329.