Enantioselective desymmetrisation of citric acid catalysed by the substratetolerant petrobactin biosynthetic enzyme AsbA

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1 This journal is The Royal Society of Chemistry 9 Supporting information Enantioselective desymmetrisation of citric acid catalysed by the substratetolerant petrobactin biosynthetic enzyme AsbA Daniel Oves-Costales, Lijiang Song and Gregory L. Challis * Materials, methods and procedures. Synthesis of racemic N 8 -citryl-spermidine The synthesis of unlabelled N 8 -citryl-spermidine was accomplished using the methodology previously described by us.. Cloning and overexpression of asba in Escherichia coli. The cloning, overproduction and purification of AsbA was carried out using the methodology previously described by us.. Incubation of [, - C ]acetic acid, coenzyme A, ATP, oxaloacetic acid, spermidine and Mg + with acetyl-coa synthetase, si-citrate synthase and purified recombinant His 6 -AsbA The experiment was carried out in a sequential manner, starting with the synthesis of S-[, - C ]citric acid. Thus, in a total volume of ml, [, - C ]acetic acid (.5 mmol), CoASH (.75 mmol), ATP (. mmol), MgCl (.5 mmol), Tris-HCl (ph 8.,. mmol) and Acetyl-CoA synthetase (.6 units) were incubated at C for minutes. The mixture was centrifuged ( rpm, minutes) and oxaloacetic acid (.5 mmol) and si-citrate synthase (44 units) were added to the supernatant. The resulting mixture was incubated for minutes at C, followed by centrifugation ( rpm, minutes). To the supernatant spermidine (.5 mmol), ATP (. mmol), Tris-HCl (ph 8.,.4 mmol), His 6 -AsbA (. µmol) were added and the total volume was made up to ml with distilled water. The solution was incubated at C for 5 hours. The sample was centrifugated ( rpm, minutes), the supernatant was passed through a.45 micron filter, diluted 4-fold with water, and partially-purified by reverse phase HPLC (Agilent- Zorbax XDB-C8 column, X mm, 5 micron, retention time ~6 min] detecting absorbance at nm using the elution profile in table. Table Time (min) Water (.% TFA) MeCN (.% TFA) Flow (ml / min) The collected fractions were analyzed by ESI-MS and those containing the compound with m/z. were freeze-dried. The semi-purified C-labeled N 8 -citryl-spermidine thus obtained was further purified by reverse phase HPLC (Phenomenex Synergi fusion-rb 8, 5 x mm, 4 micron, retention time ~ min) detecting absorbance at nm using the elution profile in table. S

2 This journal is The Royal Society of Chemistry 9 Table Time (min) Water (.% TFA) MeCN (.% TFA) Flow (ml / min) The collected fractions were analyzed by ESI-MS and those containing the compound with m/z. were freeze-dried. The purified C-labeled N 8 -citryl-spermidine obtained was analyzed by ESI-TOF-MS (Bruker MicroTof) and NMR spectroscopy (D O + drop of CD CN for calibration purposes; H, C, COSY, HSQC and HMBC; Bruker Avance 7 spectrometer equipped with a TCI cryoprobe). Its identity was confirmed by comparison of the NMR data with that of unlabeled N 8 -citryl-spermidine. Analysis of the HMBC and HSQC spectra indicated that a and not b, was the product of the AsbA-catalyzed reaction. To further confirm this conclusion, a 7 MHz C-NMR spectrum of a mixture of a mixture of the C-labeled N 8 -citryl-spermidine and chemically-synthesised racemic N 8 -citryl-spermidine was recorded. This spectrum showed unequivocally that compound a was the product of the AsbA-catalyzed reaction..4 Incubation of His 6 -AsbA with citric acid and spermidine analogues. mm citric acid, 4 mm ATP, 7.5 mm MgCl, mm Tris-HCl (ph 8.),.85 μm His 6 -AsbA (after Ni-NTA purification) and mm amine (,-propanediamine,,4-butanediamine,,5- pentanediamine,,7-heptanediamine,,8-octanediamine, nor-spermidine,,4-dihydroxybenzoylspermidine,) in a final volume of 5 μl were incubated for 9 minutes at 7 C. The reactions were initiated by addition of the enzyme and were stopped by addition of 75 μl of a 5% trichlororoacetic acid solution. Reaction mixtures were passed through a.45 micorn filter prior to analysis. The corresponding controls were carried out in the same way using denatured His 6 -AsbB (heated at C for minutes prior to addition to the incubation mixture). LC-MS analysis of the reaction mixtures were carried out using a reverse phase column (Agilent Eclipse XDB-C8, 5 X 4.6 mm, 5 micron) connected to an Agilent HPLC instrument. The outflow was routed via a splitter (% to mass spectrometer, 9% to waste) to a Bruker HCT+ spectrometer fitted with an electrospray source operating in positive ion mode. The eluting profile shown in table was used. Table Time (min) Water (.% TFA) MeCN (.% TFA) Flow (ml / min) Scaled-up incubation of His 6 -AsbA with citric acid and,5-pentanediamine The enzymatic incubation using citric acid and,5-pentanediamine was scaled-up (total volume of ml) and the corresponding product was partially-purified using reverse phase HPLC (Agilent S

3 This journal is The Royal Society of Chemistry 9 Zorbax XDB-C8 column, 5 X mm, 5 micron), detecting absorbance at nm and with the elution profile shown in table 4 (retention time. min) Table 4 Time (min) Water (.% TFA) MeCN (.% TFA) Flow (ml / min) The identity of compound was confirmed as 8 by ESI-TOF-MS (Bruker MicroTof, calculated for C H N O ; found 77.44) and ESI-MS/MS (Bruker HCT+)..4. Scaled-up incubation of His 6 -AsbA with citric acid and,7-heptanediamine The enzymatic incubation using citric acid and,7-pentanediamine was scaled-up (total volume of ml) and the corresponding product was partially purified using reverse phase HPLC (Agilent Zorbax XDB-C8 column, 5 X mm, 5 micron), detecting absorbance at nm and with the elution profile shown in table 5 (retention time 8.5 min). Table 5 Time (min) Water (.% TFA) MeCN (.% TFA) Flow (ml / min) The identity of the compound was confirmed as 7 by ESI-TOF-MS (calculated for C H 5 N O ; found 5.7) and ESI-MS/MS..4. Scaled-up incubation of His 6 -AsbA with citric acid and,8-heptanediamine The enzymatic incubation using citric acid and,7-pentanediamine was scaled-up (total volume of ml) and the corresponding product was partially purified using reverse phase HPLC (Agilent Zorbax XDB-C8 column, 5 X mm, 5 micron), detecting absorbance at nm and with the elution profile shown in table 5 (above, retention time 9.8 min). The identity of the compound was confirmed as 6 by ESI-TOF-MS (calculated for C 4 H 7 N O ; found 9.867) and ESI- MS/MS..4.4 Scaled-up incubation of His 6 -AsbA with citric acid and norspermidine The enzymatic incubation using citric acid and norspermidine was scaled-up (total volume of ml) and the corresponding product was partially purified using reverse phase HPLC (Agilent Zorbax XDB-C8 column, 5 X mm, 5 micron), detecting absorbance at nm and with the elution profile shown in table 5 (above, retention time min). The identity of the compound was confirmed as 5 by ESI-TOF-MS (calculated for C H 4 N O ; found 6.645) and ESI- MS/MS. S

4 This journal is The Royal Society of Chemistry 9.5 Incubation of His 6 -AsbA with spermidine and citric acid analogues. mm spermidine, 4 mm ATP, 7.5 mm MgCl, mm Tris-HCl (ph 8.),.85 μm His 6 -AsbA (after Ni-NTA purification) and mm citric acid analogue (-ketoglutaric acid, -ketoglutaric acid, D- glutamic acid, L-glutamic acid, DL-isocitric acid, glutaric acid and tricarballylic acid) in a final volume of 5 μl were incubated for 9 minutes at 7 C. The reactions were initiated by addition of the enzyme and were stopped by addition of 75 μl of a 5% trichlororoacetic acid solution. Reaction mixtures were passed through a.45 micron filter prior to analysis. The corresponding controls were carried out in the same way using denatured His 6 -AsbB (heated at C for minutes prior to addition to the incubation mixture). LC-MS analyses of the reaction mixtures were carried as described in section.4 above..5. Scaled-up incubation of His 6 -AsbA with tricarballylic acid and spermidine The enzymatic incubation using tricarballylic acid and spermidine was scaled-up (total volume of ml) and the corresponding product was partially purified using reverse phase HPLC (Agilent Zorbax XDB-C8 column, 5 X mm, 5 micron), detecting absorbance at nm and using the elution profile shown in table 6 (retention time 7.9 min). Table 6 Time (min) Water (.% TFA) MeCN (.% TFA) Flow (ml / min) The identity of the compound as was confirmed by ESI-TOF-MS (calculated for C H 6 N O ; found 4.86) and ESI-MS/MS analyses..6 References. D. Oves-Costales, N. Kadi, M. J. Fogg, L. Song, K. S. Wilson and G. L. Challis, J. Am. Chem. Soc. 7, 9, Spectroscopic data H-NMR spectrum of C-labeled N 8 -citryl-spermidine a (page S5) C-NMR spectrum of C-labeled N 8 -citryl-spermidine a (page S6) COSY spectrum of C-labeled N 8 -citryl-spermidine a (page S7) HSQC spectrum of C-labeled N 8 -citryl-spermidine a (page S8) HMBC spectrum of C-labeled N 8 -citryl-spermidine a (page S9) ESI-MS/MS spectrum of N-citryl-pentane-,5-diamine 8 (page S) ESI-MS/MS spectrum of N-citryl-heptane-,7-diamine 7 (page S) ESI-MS/MS spectrum of N-citryl-octane-, 8-diamine 6 (page S) ESI-MS/MS spectrum of N -citryl-norspermidine 5 (page S) ESI-MS/MS spectrum of N 8 -tricarballyl-spermidine (page S4) S4

5 This journal is The Royal Society of Chemistry 9 H-NMR spectrum of C-labeled N 8 -citryl-spermidine a S5

6 This journal is The Royal Society of Chemistry 9 C-NMR spectrum of C-labeled N 8 -citryl-spermidine a S6

7 This journal is The Royal Society of Chemistry 9 COSY spectrum of C-labeled N 8 -citryl-spermidine a S7

8 This journal is The Royal Society of Chemistry 9 HSQC spectrum of C-labeled N 8 -citryl-spermidine a S8

9 This journal is The Royal Society of Chemistry 9 HMBC spectrum of C-labeled N 8 -citryl-spermidine a S9

10 This journal is The Royal Society of Chemistry 9 Intens. x 7 +MS(77.),.-.7min #(68-79) x 6 4 +MS(77.),.7-.74min #(94-6) 59.7 x MS(77.->.),.5-.94min #(44-57) x 5 +MS(77.->.), min #(7-8) m/z ESI-MS/MS spectrum of N-citryl-pentane-,5-diamine 8 S

11 This journal is The Royal Society of Chemistry 9 Intens. x 7 +MS(5.),.9-.7min #(57-9) 5.4 x MS(5.),.-.8min #(-7) x MS(5.->87.), min #(57-75) 87. x MS(5.->87.), min #(8-97) m/z ESI-MS/MS spectrum of N-citryl-heptane-,7-diamine 7 S

12 This journal is The Royal Society of Chemistry 9 Intens. x 8 +MS(9.), min #(97-9) 9.6 x MS(9.),.8-.95min #(-45).5. x MS(9.->.),.-.8min #(54-66) x MS(9.->.), min #(74-89) x MS4(9.->.->45.), min #(94-) x 4 +MS4(9.->.->45.), min #(8-7) m/z ESI-MS/MS spectrum of N-citryl-octane-, 8-diamine 6 S

13 This journal is The Royal Society of Chemistry 9 Intens. x 8. +MS(6.),.6-.79min #(84-6) x MS(6.),.-.8min #(-4) x MS(6.->.), min #(6-7) x 6 +MS(6.->.), min #(47-6) m/z ESI-MS/MS spectrum of N -citryl-norspermidine 5 S

14 This journal is The Royal Society of Chemistry 9 Intens. x 7 6 +MS(4.), min #(98-) 4. 4 x 7 +MS(4.),.-.8min #(-4) x MS(4.->46.),.6-.8min #(5-66) x 5 +MS(4.->46.), min #(75-8) m/z ESI-MS/MS spectrum of N 8 -tricarballyl-spermidine S4

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2010

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2010 Synthesis of substrates 2,2-[ 2 H 2 ]-Decanoyl-CoA (3) was synthesised using an extension of the method previously described by us. 1 Thus, diethyl malonate 7 (Scheme 1) was deprotonated and the resulting