Chemical Science Supporting Knaus Mutti Supporting Information Systematic methodology for the...
Embed Size (px)
Transcript of Chemical Science Supporting Knaus Mutti Supporting Information Systematic methodology for the...
Systematic methodology for the development of biocatalytic hydrogen‐borrowing cascades: Application to the synthesis of chiral α‐substituted carboxylic acids from α‐ substituted α‐β unsaturated aldehydes.
Tanja Knaus,†a Francesco G. Mutti,†b Luke D. Humphreys,c Nicholas J. Turnerb and Nigel S. Scrutton*a
aManchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, Manchester, UK bManchester Institute of Biotechnology School of Chemistry, University of Manchester, Manchester, UK cGSK Medicines Research Centre, Stevenage, Herts, UK
Table of Contents
Source and cloning/expression conditions for recombinant proteins S2
Expression and purification of recombinant proteins in E. coli host cells S2
Chemical synthesis of substrates and reference compounds S4
Biocatalytic synthesis of reference compounds S11
Experiments performed for the conversion of α‐methyl‐trans‐cinnamaldehyde (1a) S12
Experiments performed for the conversion of α‐phenyl‐trans‐cinnamaldehyde (2a) S15
Experiments performed for the conversion of trans‐2‐methyl‐2‐pentenal (3a) S17
Experiments performed for the conversion of (Z)‐N‐(1‐(4‐chlorophenyl)‐3‐oxoprop‐1‐en‐2‐yl)acet‐amide (4a) S19
Experiments performed for the conversion of 2H‐chromene‐3‐carbaldehyde (5a) S22
Analytical methods and determination of absolute configuration S25
Chiral HPLC chromatograms S28
Chiral GC chromatograms S31
†T.K. and F.G.M. contributed equally.
*Corresponding author. Email: firstname.lastname@example.org
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is © The Royal Society of Chemistry 2014
Source and cloning/expression conditions for recombinant proteins
Table S1. Source and cloning/expression conditions for ene‐reductases and aldehyde dehydrogenases used in this study.
name source plasmid restr. sites E. coli strain
IPTG [mM] OD600 expression His6‐tag ref.
PETNR Enterobacter cloacae pET21a NdeI/NotI BL21‐DE3 Star 1 0.8‐1 25 °C/o.n. C‐term 1
TOYE Thermoanaerobacter pseudethanolicus pET21b NdeI/XhoI Arctic
Express RP 0.5 0.6 25 °C/o.n. C‐term 2
OYE2 Saccharomyces cerevisiae pET21b NdeI/XhoI BL21‐DE3 0.5 0.8‐1 25 °C/o.n. C‐term 3
OYE3 Saccharomyces cerevisiae pET21b NdeI/XhoI BL21‐DE3 0.5 0.8‐1 25 °C/o.n. C‐term 3
XenA Pseudomonas putida pET21a NdeI/NotI BL21‐DE3 0.5 0.6 25 °C/o.n. C‐term 4
XenB Pseudomonas fluorescens pET21a NdeI/NotI BL21‐DE3 0.5 0.6 25 °C/o.n. C‐term 4
LeOPR1 Lycopersicon esculentum pET21a NdeI/XhoI BL21‐DE3 1 0.6‐ 0.8 25 °C/o.n. C‐term
NerA Agrobacterium radiobacter pET21a NdeI/NotI BL21‐DE3 0.5 0.6 25 °C/o.n. C‐term 6
GluOx Gluconobacter oxydans pET28b NdeI/XhoI BL21‐DE3 0.5 0.8‐1 25 °C/o.n. N‐term 7
YqjM Bacillus subtilis pET21b NdeI/XhoI BL21‐DE3 1 0.8‐1 25 °C/o.n. no tag 8 MR Pseudomonas putida pET21a NdeI/NotI BL21‐DE3 1 0.8‐1 25 °C/o.n. no tag 9 Ald‐DH‐EC E. coli pET28b NdeI/XhoI BL21‐DE3 0.5 0.8‐1 25 °C/o.n. N‐term 10 Ald‐DH BOV Bos taurus pET28b NdeI/XhoI BL21‐DE3 0.5 0.5 25 °C/o.n N‐term 11
Ald‐DH‐HL Equus caballus pET‐SUMO ‐ BL21‐DE3 0.6 0.5 25 °C/o.n N‐term 12
Expression and purification of recombinant proteins in E. coli host cells
For recombinant expression 800 mL of LB medium supplemented with the appropriate antibiotic (100 µg/mL ampicillin for pET21a or pET21b) and 50 µg/mL kanamycin for pET28b and pET‐SUMO plasmids) were inoculated with 15 mL of an overnight culture harboring the desired vector with genes for the expression of the ERs and Ald‐DHs. Cells were grown at 37 °C until the desired OD600 was reached and expression of protein was induced by the addition of IPTG. Protein expression was carried out overnight and after harvesting of the cells (4 °C, 8 x 103 rpm, 10 min), the remaining cell pellet was resuspended in buffer and cells were disrupted using a French Press. For flavin‐containing proteins a small spatula of free FMN was added to the lysate before cell disruption. The cell debris was removed by centrifugation at 18 x 103 rpm for 30 min at 4 °C.
His6‐tagged proteins were resuspended in lysis buffer (50 mM KH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) prior to cell disruption and protein purification was performed by Ni‐NTA affinity chromatography using pre‐packed Ni‐ NTA HisTrap HP columns (GE Healthcare), previously equilibrated with lysis buffer. After loading of the filtered lysate, the column was washed with sufficient amounts of wash buffer (50 mM KH2PO4, 300 mM NaCl, 25 mM imidazole,pH 8.0), and bound protein was recovered with elution buffer (50 mM KH2PO4, 300 mM NaCl, 200 mM imidazole, pH 8.0).
Non‐tagged proteins (YqjM and MR) were purified by anion exchange chromatography using a HiPrepQ HP 16/10 column (GE Healthcare). The lysate was dissolved in start buffer (20 mM Tris/HCl, pH 8.0 buffer) and after cell disruption and centrifugation loaded onto the column. The elution of the proteins was performed with a gradient between start buffer and elution buffer (20 mM Tris/HCl, 1M NaCl, pH 8.0 buffer).
After SDS‐PA 50 mM K2H concentratio coefficient fo
Figure S1. SDS Plus Protein™ OYE2, lane 5: NerA, lane 1 Precision Plus
AGE, fraction PO4/KH2PO4 on was dete or the protein
S‐PAGE of puri ™ Unstained Sta OYE3, lane 6: 0: GluOx, lane s Protein™ Uns
ns containing buffer (pH
ermined at 2 n bound flavin
fied ene‐reduc andard, lane 2 XenA, lane 7: e 11: YqjM, l tained Standar
the desired 7.0) overnig 280 nm or f n (approx. 45
ctases. Lane 1: 2: PETNR, lane XenB, lane 8: ane 12: MR, rd.
proteins in a ght, and con for flavin co 50 nm/11,300
Biorad Precisio 3: TOYE, lane LeOPR1, lane lane 13: Bior
a sufficient p ncentrated u ontaining pro 0 M‐1 cm‐1).
on 4: 9: ad
Figure dehydr Protein lane 3:
purity were p using Centrip oteins using
S2. SDS‐P rogenases. La n™ Unstained : Ald‐DH‐EC, la
pooled and d preps (Millipo the wavele
AGE of pu ane 1: Biorad Standard, lane ne 4: Ald‐DH‐H
dialyzed again ore). The fin ngth/extincti
rified aldehy d Precision P e 2: Ald‐DH‐BO HL.
nst nal on
yde lus OV,
Chemical synthesis of substrates and reference compounds
1) Synthesis of (E)‐2,3‐diphenylacrylaldehyde (α‐phenylcinnamaldehyde) 2a
Synthesis of (E)‐methyl 2,3‐diphenylacrylate (α‐phenylcinnamic acid methyl ester)
(E)‐2,3‐diphenylacrylic acid (α‐phenylcinnamic acid) (1 g , 4.46 mmol) was dissolved in dried MeOH (30 mL). The reaction mixture was cooled down to 4 °C and a solution of thionylchloride (650 µL, 2 eq., 8.92 mmol) dissolved in dried MeOH (2 mL), prior cooled to 4 °C, was added dropwise into the reaction mixture. The solution was then refluxed overnight at 42 °C. The progress of the reaction was analyzed by TLC (Petroleum ether/EtOAc, 9:1, v v‐1, Rf ester product = 0.5; the carboxylic acid does not elute from the base). Methanol was evaporated, the residue was treated with sodium carbonate (20 mL, 5% w w‐1) and extracted with EtOAc (2 × 15 mL). The combined organic phase was dried with MgSO4, filtered and evaporated. The product was recovered as pure solid in 89% yield (784 mg, 3.99 mmol). 1H‐NMR (CDCl3, 400 MHz, δ (ppm), J (Hz): 3.72 (3H, CH3, s), 6.95 – 6.97 (2H, CH, m), 7.04 – 7.18 (5H, CH, m), 7.26 – 7.33 (3H, CH, m), 7.78 (1H, CH, s).
GC‐MS: (m/z, (rel. intensity)): 239 (100), 205 (4), 178 (5), 121 (10). GC‐MS was measured with a Varian CP3800 gas chromatograph connected to a Varian Saturn 2000 GC/MS/MS (Agilent DB‐Wax column).
Synthesis of (E)‐2,3‐diphenylp