METHOD FOR PREPARING ALPHA-BRANCHED BETA'-HYDROXY CARBONYL COMPOUNDS BY ENZYMATIC-CATALYZED REDUCTIVE ALDOL REACTION
20250250593 · 2025-08-07
Inventors
- Tobias Jürgen Erb (Marburg, DE)
- Matthias Tinzl (Marburg, DE)
- Gabriele Stoffel (Marburg, DE)
- Patrick Gerlinger (Marburg, DE)
Cpc classification
C12Y103/01085
CHEMISTRY; METALLURGY
C12P7/40
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a method for preparing -branched -hydroxy carbonyl compounds through enzymatic-catalyzed reductive aldol reaction by reacting ,-unsaturated carbonyl donors with carbonyl acceptors in the presence of a polypeptide capable of catalyzing reductive aldol reactions and a cofactor, wherein the polypeptide is an enoyl-CoA carboxylase/reductase (Ecr). The replacement of the native CO.sub.2 electrophile in enoyl-CoA carboxylases/reductases (Ecrs) by different carbonyl acceptors advantageously creates a new-to-nature biocatalytic route towards -branched -hydroxy carbonyl compounds.
Claims
1. A method for preparing an -branched -hydroxy carbonyl compound by reductive aldol reaction, wherein the method comprises the steps: a) providing an ,-unsaturated carbonyl donor, wherein the ,-unsaturated carbonyl donor is a coenzyme A thioester of an ,-unsaturated carboxylic acid; b) providing a carbonyl acceptor, wherein the carbonyl acceptor is an aldehyde; c) performing an enzymatic-catalyzed reductive aldol reaction with the ,-unsaturated carbonyl donor and the carbonyl acceptor by using a polypeptide capable of catalyzing reductive aldol reactions in the presence of a cofactor, wherein the polypeptide is an enoyl-CoA carboxylase/reductase.
2. The method according to claim 1, wherein the polypeptide is a crotonyl-CoA carboxylase/reductase.
3. The method according to claim 1, wherein the polypeptide comprises at least 95% sequence identity to amino acid sequence SEQ ID NO: 1 or SEQ ID NO:2.
4. The method according to claim 1, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 29.
5. The method according to claim 1, wherein the polypeptide is a wild type crotonyl-CoA carboxylase/reductase.
6. The method according to claim 1, wherein the polypeptide is a crotonyl-CoA carboxylase/reductase obtained from Kitasatospora setae or Caulobacter crescentus.
7. The method according to claim 1, wherein the cofactor is NADPH.
8. The method according to claim 1, wherein the ,-unsaturated carboxylic acid has the general formula (I): ##STR00021## wherein R.sup.1 is selected from hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl.
9. The method according to claim 8, wherein R.sup.1 represents H, cyclo-C.sub.3H.sub.5, cyclo-C.sub.4H.sub.7, cyclo-C.sub.5H.sub.9, cyclo-CH.sub.11, cyclo-C.sub.7H.sub.13, cyclo-C.sub.8H.sub.15, -Ph, CH.sub.2-Ph, C.sub.2H.sub.4Ph, CPh.sub.3, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, CH(CH.sub.3).sub.2, C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)C.sub.2H.sub.5, C(CH.sub.3).sub.3, C.sub.5H.sub.11, CH(CH.sub.3)C.sub.3H.sub.7, CH.sub.2CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)CH(CH.sub.3).sub.2, C(CH.sub.3).sub.2C.sub.2H.sub.5, CH.sub.2C(CH.sub.3).sub.3, CH(C.sub.2H.sub.5).sub.2, C.sub.2H.sub.4CH(CH.sub.3).sub.2, CH.sub.13, C.sub.3H.sub.6CH(CH.sub.3).sub.2, C.sub.2H.sub.4CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3)C.sub.3H.sub.7, CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2, C.sub.7H.sub.15, C.sub.8H.sub.17, C(CH.sub.3).sub.2C.sub.3H.sub.7, CH(CH.sub.3)CH(CH.sub.3)C.sub.2H.sub.5, CH.sub.2CH(CH.sub.3)CH(CH.sub.3).sub.2, CH.sub.2C(CH.sub.3).sub.2C.sub.2H.sub.5, CH(CH.sub.3)C(CH.sub.3).sub.3, C(CH.sub.3).sub.2CH(CH.sub.3).sub.2, C.sub.2H.sub.4C(CH.sub.3).sub.3, CHCH.sub.2, CH.sub.2CHCH.sub.2, C(CH.sub.3)CH.sub.2, CHCHCH.sub.3, C.sub.2H.sub.4CHCH.sub.2, CH.sub.2CHCHCH.sub.3, CHCHC.sub.2H.sub.5, CHC(CH.sub.3).sub.2, CH.sub.2C(CH.sub.3)CH.sub.2, CH(CH.sub.3)CHCH, C(CH.sub.3)CHCH.sub.3, CHCHCHCH.sub.2, C.sub.3H.sub.6CHCH.sub.2, C.sub.2H.sub.4CHCHCH.sub.3, CH.sub.2CHCHC.sub.2H.sub.5, CHCHC.sub.3H.sub.7, CH.sub.2CHCHCHCH.sub.2, CHCHCHCHCH.sub.3, C.sub.2H.sub.4CHCHCH.sub.3, CH.sub.2CHCHC.sub.2H.sub.5, CH.sub.2CHCHCHCH.sub.2, CHCHCHCHCH.sub.3, CHCHCH.sub.2CHCH.sub.2, C(CH.sub.3)CHCHCH.sub.2, CHC(CH.sub.3)CHCH.sub.2, CHCHC(CH.sub.3)CH.sub.2, CH.sub.2CHC(CH.sub.3).sub.2, C(CH.sub.3)C(CH.sub.3).sub.2, C.sub.2H.sub.4CHCH.sub.2, CHCHC.sub.2H.sub.5, CHC(CH.sub.3).sub.2, CH.sub.2CHCHCH.sub.3, CHCHCHCH.sub.2, C.sub.3H.sub.6CHCH.sub.2, CHCHC.sub.3H.sub.7, C.sub.4H.sub.8CHCH.sub.2, CHCHC.sub.4H.sub.9, C.sub.3H.sub.6CHCHCH.sub.3, CH.sub.2CHCHC.sub.3H.sub.7, C.sub.2H.sub.4CHCHC.sub.2H.sub.5, CH.sub.2C(CH.sub.3)C(CH.sub.3).sub.2, C.sub.2H.sub.4CHC(CH.sub.3).sub.2, CH.sub.2CCH, CCH, CCCH.sub.3, C.sub.2H.sub.4CCH, CCC.sub.2H.sub.5, CH.sub.2CCCH.sub.3, CCCHCH.sub.2, CHCHCCH, CCCCH, C.sub.3H.sub.6CCH, CCC.sub.3H.sub.7, C.sub.2H.sub.4CCCH.sub.3, CH.sub.2CCC.sub.2H.sub.5, CH.sub.2CCCHCH.sub.2, CH.sub.2CHCHCCH, CH.sub.2CCCCH, CCCHCHCH.sub.3, CHCHCCCH.sub.3, CCCCCH.sub.3, CCCH.sub.2CHCH.sub.2, CHCHCH.sub.2CCH, CCCH.sub.2CCH, C(CH.sub.3)CHCHCH.sub.2, CHC(CH.sub.3)CHCH.sub.2, CHCHC(CH.sub.3)CH.sub.2, C(CH.sub.3)CHCCH, CHC(CH.sub.3)CCH, CCC(CH.sub.3)CH.sub.2, C.sub.4H.sub.8CCH, CCC.sub.4H.sub.9, C.sub.3H.sub.6CCCH.sub.3, CH.sub.2CCC.sub.3H.sub.7, C.sub.2H.sub.4Ph, CHCH-Ph, CC-Ph, CH.sub.2NH.sub.2, CH.sub.2OH, CH.sub.2SH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, C.sub.6H.sub.4OCH.sub.3, C.sub.6H.sub.4OH, CH.sub.2CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OH, CH.sub.2OCH.sub.3, CH.sub.2C.sub.6H.sub.4OCH.sub.3, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2R.sup.2, CH.sub.2CH.sub.2R.sup.2, or CH.sub.2CH.sub.2CH.sub.2R.sup.2; and R.sup.2 represents NH.sub.2, OH, SH, F, Cl, Br, I, CN, N.sub.3, OCN, NCO, SCN, or NCS.
10. The method according to claim 1, wherein the carbonyl acceptor has the general formula (II): ##STR00022## wherein R.sup.3 represents H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, CH(CH.sub.3).sub.2, C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)C.sub.2H.sub.5, C(CH.sub.3).sub.3, C.sub.5H.sub.11, CH(CH.sub.3)C.sub.3H.sub.7, CH.sub.2CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)CH(CH.sub.3).sub.2, C(CH.sub.3).sub.2C.sub.2H.sub.5, CH.sub.2C(CH.sub.3).sub.3, CH(C.sub.2H.sub.5).sub.2, C.sub.2H.sub.4CH(CH.sub.3).sub.2, C.sub.6H.sub.13, C.sub.3H.sub.6CH(CH.sub.3).sub.2, C.sub.2H.sub.4CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3)C.sub.3H.sub.7, CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2, -Ph, or CH.sub.2-Ph.
11. The method according to claim 1, wherein the carbonyl acceptor is selected from the group comprising or consisting of formaldehyde, acetaldehyde and propionaldehyde.
12. The method according to claim 1, wherein the ,-unsaturated carboxylic acid is selected from the group comprising or consisting of crotonic acid (trans-2-butenoic acid), trans-cinnamic acid, 5-chloro-2-pentenoic acid, trans-2-hexenoic acid, 5-methyl-2-hexenoic acid, trans-2-penten-4-ynoic acid and penta-2,4-dienoic acid.
13. The method according to claim 1, wherein the enzyme-catalyzed reductive aldol reaction is performed in the presence of an acyl-CoA oxidase.
14. The method according to claim 1, further comprising the following step: d) performing a hydrolysis reaction with the coenzyme A thioester of the -branched -hydroxy carbonyl compound under basic conditions or by using a thioesterase.
15. The method according to claim 1, wherein the -branched -hydroxy carbonyl compound has the general formula (IIIa) and the -branched -hydroxy acyl-CoA has the general formula (IIIb): ##STR00023## and R.sup.1, R.sup.2, and R.sup.3 have the same meanings as defined in any one of the claims 8-10.
Description
DESCRIPTION OF THE FIGURES
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[0544]
[0545]
[0546]
[0547]
[0548]
[0549]
[0550]
[0551]
[0552] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
EXAMPLES
Materials and Methods
Chemicals
[0553] Crotonic Anhydride, formaldehyde solution 37%, acetaldehyde, hexanoic acid, 5-methylhexanoic acid, 4-pentynoic acid, trans-cinnamic acid and 5-chloropentanoic acid were purchased from Sigma Aldrich AG. Coenzyme A trilithium salt and DNAse I were purchased from Roche Diagnostics and NADPH Na.sub.4 (98%) from Carl Roth GmbH. Solvents and salts were all analytical grade or better. Crotonyl-CoA was synthesized according to Peter, D. M., Vgeli, B., Cortina, N. S. & Erb, T. J. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters. Molecules 21, 517 (2016).
Synthesis of Reference Compounds (2S,3S)-2-Ethyl-3-hydroxybutanoic acid and (2S,3R)-2-Ethyl-3-hydroxybutanoic acid
[0554] All non-aqueous reactions were carried out using flame-dried glassware under argon atmosphere. All solvents were distilled by rotary evaporation. Solvents for non-aqueous reactions were dried as follows prior to use: Tetrahydrofuran (THF) was dried with potassium hydroxide and subsequently distilled from Solvona. Methylene chloride, diisopropylamine and diisopropylethylamine were distilled from calcium hydride. All other commercially obtained reagents were used as received.
[0555] Reactions were monitored by thin layer chromatography (TLC) using Merck Silica Gel 60 F.sub.254-plates and visualised by fluorescence quenching under UV-light. In addition, TLC-plates were stained using a potassium permanganate or cerium sulfate/phosphomolybdic acid stain. Chromatographic purification of products was performed on Merck Silica Gel 60 (230-400 mesh) unless otherwise noted using a forced flow of eluents. Concentration in vacuo was performed by rotary evaporation at 40 C. and appropriate pressure and by exposing to fine vacuum at room temperature if necessary.
[0556] NMR spectra were recorded on a Bruker AV 300 MHz, AV III 500 MHz, AV III HD 500 MHz spectrometer at room temperature. Chemical shifts are reported in ppm with the solvent resonance as internal standard. Data are reported as follows: s=singlet, bs=broad singlet, d=doublet, t=triplet, q=quartet, quint=quintet, sext=sextet, m=multiplet. Diastereomers are featured with a/b, rotamers with */**.
[0557] Mass spectra were recorded by the mass service department of the Philipps-Universitt Marburg. HR-EI, ESI or APCI mass spectra were acquired with an LTQ-FT mass spectrometer (Thermo Fischer Scientific). The resolution was set to 100 000.
[0558] IR spectra were recorded on a Bruker IFS 200 spectrometer. The absorption bands are given in wave numbers (cm.sup.1), intensities are reported as follows: s=strong, m=medium, w=weak, br=broad band.
[0559] Optical rotations were determined at 20 C. for Na-D wavelength (589 nm) with a Krss P8000-T polarimeter.
(S)-4-Benzyl-3-butyryloxazolidin-2-one
##STR00016##
[0560] To a solution of (S)-oxazolidinone (2.00 g, 11.3 mmol, 1.00 eq.) in tetrahydrofuran (25 mL) at 78 C., under an atmosphere of argon was added a solution of n-butyllithium in hexane (2.5 M, 4.74 mL, 11.9 mmol, 1.05 eq.) dropwise over 10 min. The mixture was stirred for 15 min and butyryl chloride (1.28 mL, 12.4 mL, 1.10 eq.) was added dropwise over 10 min. After 3 h at 78 C. a solution of sat. ammonium chloride solution was added. The mixture was extracted with ethyl acetate (3) and the combined extracts were dried (magnesium sulfate). After removal of the solvent in vacuo the residue was purified by flash chromatography (n-pentane/ethyl acetate 5:1) to yield the oxazolidinone product (2.50 g, 10.1 mmol, 90%) as colourless oil. R.sub.f=0.80 (n-pentane/ethyl acetate 1:1); .sup.1H-NMR: (300 MHz, CDCl.sub.3): =7.37-7.20 (m, 5H, Ph-CH), 4.43 (ddd, 1H, .sup.3J=13.4, 7.0, 3.5 Hz, NCH), 4.24-4.14 (m, 2H, Ph-CH.sub.2), 3.31 (dd, 1H, .sup.2J=13.4 Hz, .sup.3J=3.5 Hz, OCH.sub.2), 3.04-2.72 (m, 3H, OCH.sub.2, CH.sub.2CH.sub.2CH.sub.3), 1.74 (sext, 2H, CH.sub.2CH.sub.2CH.sub.3), 1.02 (t, 3H, .sup.3J=7.4 Hz, CH.sub.3) ppm. The analytical data is in accordance to literature..sup.[1]
(S)-4-Benzyl-3-((2S,3R)-2-ethyl-3-hydroxybutanoyl)oxazolidin-2-one
##STR00017##
[0561] To a solution of the oxazolidinone (0.50 g, 2.02 mmol, 1.00 eq.) in methylene chloride (10 mL) at 0 C., under an atmosphere of argon was added titanium tetrachloride (0.24 mL, 2.22 mmol, 1.10 eq.) dropwise. After 15 min diisopropylethylamine (0.53 mL, 3.03 mmol, 1.50 eq.) was added over a period of 5 min, and the mixture was stirred for 45 min. N-methylpyrrolidone (0.21 mL, 2.22 mmol, 1.10 eq.) was added and after 10 min acetaldehyde (0.23 mL, 4.04 mmol, 2.00 eq.). After 2 h half sat. ammonium chloride solution was added and the mixture extracted with methylene chloride (3). The combined extracts were dried (magnesium sulfate) and the solvent was removed in vacuo. The residue was purified by flash chromatography (n-pentane/ethyl acetate 5:1 to 1:1) to give the hydroxy oxazolidinone product (0.49 g, 1.72 mmol, 85%) as yellow oil. R.sub.f=0.50 (n-pentane/ethyl acetate 1:1); .sup.1H-NMR: (500 MHz, CDCl.sub.3): =7.37-7.22 (m, 5H, Ph-CH), 4.74 (ddd, 1H, .sup.3J=13.3, 7.0, 3.5 Hz, NCH), 4.22-4.13 (m, 2H, Ph-CH.sub.2), 4.13-4.06 (m, 1H, CHOH), 3.54 (dt, 1H, .sup.3J=9.4, 4.5 Hz, CHCH.sub.2), 3.37 (dd, 1H, .sup.2J=13.3, .sup.3J=3.2 Hz, OCH.sub.2), 2.71 (dd, 1H, .sup.2J=13.3, .sup.3J=10.0 Hz, OCH.sub.2), 2.46 (bs, OH), 1.96-1.81 (m, 1H, CH.sub.2CH.sub.3), 1.77-1.64 (m, 1H, CH.sub.2CH.sub.3), 1.23 (d, 3H, .sup.3J=6.4 Hz, CHCH.sub.3), 0.91 (t, 3H, .sup.3J=7.5 Hz, CH.sub.2CH.sub.3) ppm; .sup.13C-NMR: (125 MHz, CDCl.sub.3): =175.5 (NCO), 153.9 (CO.sub.2), 135.2 (Ph-Cq), 129.3 (2Ph-CH.sub.2), 129.0 (2Ph-CH.sub.2), 127.4 (Ph-CH), 68.6 (CHOH), 66.3 (Ph-CH.sub.2), 55.6 (NCH), 50.1 (CHCH.sub.2), 38.1 (OCH.sub.2), 20.7 (CH.sub.2CH.sub.3), 19.5 (CHCH.sub.3), 11.9 (CH.sub.2CH.sub.3) ppm; FT-IR: u.sub.max(film): 3482 (w), 3029 (w), 2969 (w), 2932 (w), 2877 (w), 1774 (s), 1691 (m), 1604 (w), 1455 (w), 1384 (m), 1350 (w), 1288 (w), 1272 (w), 1208 (s), 1151 (w), 1114 (m), 1077 (w), 1050 (w), 1017 (w), 983 (w), 948 (w), 910 (w), 822 (w), 763 (w), 746 (w), 702 (m), 593 (w), 572 (w), 506 (w) cm.sup.1.
(2S,3R)-2-Ethyl-3-hydroxybutanoic acid
##STR00018##
[0562] To a solution of the oxazolidinone (0.23 g, 0.79 mmol, 1.00 eq.) in a 4:1 mixture of tetrahydrofuran/water (5 mL) at 0 C. was added a solution of hydrogen peroxide (30% in water, 0.21 mL, 2.05 mmol, 2.60 eq.) and lithium hydroxide monohydrate (66 mg, 1.58 mmol, 2.00 eq.). The mixture was stirred overnight reaching room temperature. After careful addition of a sat. solution of sodium thiosulfate at 0 C., the mixture was extracted with ethyl acetate (3). The mixture was extracted with dichloromethane (3) and the pH value of the aqueous layer was adjusted (pH=2-3) with hydrochloric acid (6 M) and extracted with ethyl acetate (4). The combined extracts were dried (sodium sulfate), filtered and the volatiles removed in vacuo. The residue was purified by flash chromatography (ethyl acetate) to yield the product (50 mg, 0.38 mmol, 48%) as clear oil. R.sub.f=0.15 (ethyl acetate); .sup.1H-NMR: (500 MHz, CDCl.sub.3): =4.07 (t, 1H, .sup.3J=5.4 Hz, CHOH), 2.44-2.39 (m, 1H, CHCH.sub.2), 1.77-1.71 (m, 1H, CH.sub.2CH.sub.3), 1.70-1.61 (m, 1H, CH.sub.2CH.sub.3), 1.25 (d, 3H, .sup.3J=6.3 Hz, CHCH.sub.3), 1.00 (t, 3H, 3J=7.4 Hz, CH.sub.2CH.sub.3) ppm. (OH and CO.sub.2H were not detectable due to rapid exchange with the solvent); .sup.13C-NMR: (125 MHz, CDCl.sub.3): 179.7 (CO.sub.2H), 68.0 (CHOH), 53.5 (CHCH.sub.2), 20.3 (CH.sub.2CH.sub.3), 20.1 (CHCH.sub.3), 12.2 (CH.sub.2CH.sub.3) ppm; FT-IR: u.sub.max(film): 3348 (w), 2969 (m), 2933 (w), 2880 (w), 1704 (s), 1461 (w), 1409 (w), 1382 (w), 1263 (w), 1201 (m), 1152 (w), 1086 (m), 1051 (w), 1021 (w), 954 (w), 910 (w), 886 (w), 782 (w), 732 (m), 662 (w), 521 (w), 485 (w) cm.sup.1; HRMS (ESI): m/z calcd. for C.sub.6H.sub.12O.sub.3Na [M+Na].sup.+ 155.0684. found 155.0679; [].sup.20.sub.D: 5.6 (c=0.5, chloroform).
Methyl (2S,3S)-2-ethyl-3-hydroxybutanoate
##STR00019##
[0563] To a solution of the diisopropylamine (6.19 mL, 44 mmol, 2.60 eq.) in tetrahydrofuran (30 mL) at 0 C. under an atmosphere of argon was added a solution of n-butyllithium in hexane (2.5 M, 16.9 mL, 42.3 mmol, 2.50 eq.) dropwise. After 30 min the solution was cooled to 78 C. and a solution of hydroxy ester (1.87 mL, 16.9 mmol, 1.00 eq.) in tetrahydrofuran (8 mL) was added over a period of 20 min. The solution was stirred for 4 h reaching 30 C. and after recooling to 78 C. a solution of ethyl iodide (5.04 mL, 62.6 mmol, 3.70 eq.) in tetrahydrofuran (7 mL) was added over a period of 20 min. The solution was stirred overnight reaching room temperature. A sat. solution of ammonium chloride was added and the mixture extracted with diethyl ether (3). The combined extracts were dried (sodium sulfate), filtered and the volatiles carefully removed in vacuo. The residue was purified by flash chromatography (n-pentane/diethyl ether 3:1 to 1:1) to yield the ester product (2.40 g, 16.4 mmol, 97%) as pale-yellow liquid. R.sub.f=0.60 (n-pentane/ethyl acetate 1:1); .sup.1H-NMR: (300 MHz, CDCl.sub.3): =3.92 (quint, 1H, .sup.3J=6.3 Hz, CHOH), 3.72 (s, 3H, CO.sub.2CH.sub.3), 2.33 (dt, 1H, .sup.3J=8.0, 6.3 Hz, CHCH.sub.2), 1.75-1.61 (m, 2H, CH.sub.2CH.sub.3), 1.22 (d, 3H, .sup.3J=6.4 Hz, CHCH.sub.3), 0.92 (t, 3H, .sup.3J=7.5 Hz, CH.sub.2CH.sub.3) ppm. (OH was not detectable due to rapid exchange with the solvent). The analytical data is in accordance with literature..sup.[2]
(2S,3S)-2-Ethyl-3-hydroxybutanoic acid
##STR00020##
[0564] To a solution of the ester (400 mg, 2.74 mmol, 1.00 eq.) in a 5:1 mixture of water/tetrahydrofuran (9 mL) at room temperature was added potassium hydroxide (1.53 g, 27.4 mmol, 10.0 eq.) in one portion. After stirring overnight water was added and the pH-value was adjusted (pH=3) with hydrochloric acid (1 M). The mixture was extracted with ethyl acetate (3), the combined extracts dried (magnesium sulfate), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (n-pentane/ethyl acetate 1:1) to yield the acid product (300 mg, 2.27 mmol, 83%) as pale-yellow oil. R.sub.f=0.15 (ethyl acetate); .sup.1H-NMR: (500 MHz, CDCl.sub.3): =3.98 (t, 1H, .sup.3J=6.3 Hz, CHOH), 2.33 (dt, 1H, .sup.3J=8.0, 6.3 Hz, CHCH.sub.2), 1.77-1.65 (m, 2H, CH.sub.2CH.sub.3), 1.28 (d, 3H, .sup.3J=6.3 Hz, CHCH.sub.3), 0.99 (t, 3H, .sup.3J=7.5 Hz, CH.sub.2CH.sub.3) ppm. (OH was not detectable due to rapid exchange with the solvent); .sup.13C-NMR: (125 MHz, CDCl.sub.3): 179.8 (CO.sub.2H), 68.0 (CHOH), 54.2 (CHCH.sub.2), 22.4 (CH.sub.2CH.sub.3), 21.4 (CHCH.sub.3), 11.6 (CH.sub.2CH.sub.3) ppm; FT-IR: u.sub.max(film): 3366 (w), 2969 (m), 2937 (w), 2880 (w), 1704 (s), 1461 (w), 1383 (w), 1273 (w), 1201 (m), 1147 (w), 1116 (m), 1090 (w), 1058 (w), 1018 (w), 953 (w), 884 (w), 841 (w), 782 (w), 657 (w), 514 (w) cm.sup.1; HRMS (ESI): m/z calcd. for C.sub.6H.sub.12O.sub.3Na [M+Na].sup.+ 155.0684. found 155.0676; [].sup.20.sub.D: 4.0 (c=0.5, chloroform).
Cloning and Mutagenesis
[0565] Acyl-CoA ligases RevS and At4CL4 were identified by literature research. Codon optimized genes containing either N- or C-terminal 6 His-tags for RevS and At4CL4, respectively were synthesized and cloned into pET-16b and pET-21a expression vectors by Baseclear.
[0566] The KsCcr gene was provided by the JGI. Enzyme variants KsCcrl167A, M356A and M365V variants were generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA). Employed primer pairs are reported in Table 6.
TABLE-US-00018 TABLE6 PrimerpairsemployedforthegenerationofKsCcrvariants. ForwardPrimer SEQ ReversePrimer SEQ Mutation 5.fwdarw.3 IDNO: 5.fwdarw.3 IDNO: I176A GACCCAGAGCAGCG SEQIDNO: GTTTCGAAGCC SEQIDNO: CGCCTGGGGCTTC 28 CCAGGCGCGCT 32 GAAAC GCTCTGGGTC M356A CAACCGTTACCTGTG SEQIDNO: GATACGTTTCA SEQIDNO: GGCGTCCCTGAAAC 33 GGGACGCCCAC 34 GTATC AGGTAACGGTT M356V CCGTTACCTGTGGGT SEQIDNO: GTTTCAGGGAC SEQIDNO: GTCCCTGAAAC 35 ACCCACAGGTA 36
Gene Expression and Protein Purification
[0567] His-tagged KsCcr and its variants were expressed in E. coli BL21 (DE3). Cells in terrific broth were grown at 37 C. to an OD.sub.600=0.8-1.0 upon which expression for 12-16 h at 23 C. was induced by the addition of 500 M IPTG (Isopropyl-D--thiogalactopyranoside). Cells were harvested for 15 min at 7500 g at 4 C. then resuspended in 2 mL of buffer A (50 mM Tris, 500 mM NaCl, 1 M L-Proline, pH=7.5) per gram of pellet. The suspension was treated with 10 mg/mL of DNAse I, 5 mM MgCl.sub.2, 10 g/ml of lysozyme and incubated on ice for 20 min upon which cells were lysed using a sonicator. The lysate was clarified at 45000 g at 4 C. for 40 min and then loaded onto a pre-equilibrated 1 mL HisTrap FF column and washed with 15% buffer B (50 mM Tris, 500 mM NaCl, 1 M L-Proline, 500 mM imidazole, pH=7.5) for 20-30 column volumes until the UV 280 nm reached the baseline level. The protein was eluted by applying 100% buffer B, collected then pooled and desalted into 12.5 mM Tris, 125 mM NaCl, 1 M L-Proline pH=7.5. The addition of L-Proline to each buffer increased the yields for the protein purification by making the protein more soluble as previously reported. The protein was flash frozen in N2 (I) and stored at 80 C. if not immediately used for assays.
[0568] The same procedure was employed for the expression and purification of CcCcr.sub.PAG with a modified buffer composition. Buffer A contained 50 mM Tris HCl pH=7.9, 500 mM NaCl, buffer B contained 50 mM Tris HCl pH=7.9, 500 mM NaCl and the desalting buffer contained 25 mM Tris HCl pH=7.9, 125 mM NaCl, 30% glycerol.
[0569] Acyl-CoA oxidase Acx4 was expressed in E. coli BL21-AI cells (Invitrogen). Cells were grown at 37 C. to an OD.sub.600=1 and the expression was induced by the addition of 500 M IPTG and L-arabinose (0.02% w/v). Cells were harvested after an additional 6 hours of incubation at 30 C. The purification procedure followed the same protocol as for KsCcr with buffer A containing 50 mM HEPES pH=7.6, 500 mM NaCl, buffer B containing 50 mM HEPES pH=7.6, 500 mM NaCl, 500 mM imidazole and desalting buffer containing 25 mM TrisHCl pH=7.9, 150 mM NaCl, 30% glycerol. Additionally, the concentration of FAD in Acx4 batches was calculated using a standard curve for absorption at 450 nm (.sub.FAD 450 nm=11.3 mM.sup.1 cm.sup.1) and reconstituted to equimolar concentrations of protein and cofactor.
Chemical Synthesis of (2S,3S)-2-EHB-CoA and (2S,3R)-2-EHB-CoA
[0570] Enoyl-CoAs where synthesized using the mixed anhydride method adapted from a known protocol. The unsaturated acid (63 mol) and triethylamine (70 mol) were dissolved in CH.sub.2Cl.sub.2 (2 ml) and stirred at 23 C. for 30 min. The reaction was cooled to 4 C. and ethylchloroformate (70 mol) was added. After 2 h the solvent was evaporated and a solution of CoA-trilithium salt (31 mol) in 0.4 M KHCO.sub.3 (2 ml) was added. The reaction procedure was monitored by mixing 5 l of reaction mixture with 35 l of an aqueous 5,5-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) solution. Upon completion the reaction was acidified to pH=3-4 with formic acid, diluted to 35 ml with H.sub.2O and lyophilized. The product was resuspended in H.sub.2O and purified by reverse phase HPLC over a Gemini 10 m NX-C18 110 , 10021.2 mm, AXIA packed column (Phenomenex) using a gradient from 5% to 65% methanol with 25 mM ammonium formate pH=8.1 as the aqueous phase. Fractions containing the product were pooled and lyophilized. Lyophilized fractions were analyzed by HPLC over an Agilent Eclipse Plus C18, 3.5 m 100 , 1002.1 mm, 4.6100 mm column using 5% to 30% acetonitrile gradient over 7.5 min with 25 mM ammonium formate pH=8.1 as the aqueous phase. The fractions containing the expected product were pooled, lyophilized and stored at 20 C. if not used.
Enzymatic Synthesis of Enoyl-CoAs for the Substrate Promiscuity Screen
[0571] Synthesis was carried out using an adopted, one-pot enzymatic synthesis setup. The final assay volume was 6 ml and contained 100 mM HEPES pH=7.5, 20 mM MgCl.sub.2, 100 mM KHCO.sub.3, 4.35 mM (20 mg) CoA, 17.38 mM ATP and 20.86 mM of the corresponding carboxylic acid. Despite their low solubility in water, cinnamic acid and 5-methylhexanoic acid could be added to the assay directly while still enabling full CoA depletion.
[0572] Upon equilibration of the assay mixture at 30 C., the enzymes were added to a final concentration of 5 M ligase (At4CL4 for the mixture containing cinnamic acid, RevS for all other setups) and 3 M Acx4. The reactions were incubated for 2 h 30 C. at 200 rpm, while monitoring CoA consumption using Ellman's reagent. Upon completion, the reaction was quenched with a final concentration of 10% (v/v) formic acid, centrifuged for 10 min at 5000 g, filtered through a 0.2 m syringe filter and flash frozen in N2 (I) and stored at 80 C. if not immediately subjected to HPLC-MS purification.
[0573] All enoyl-CoAs were purified via reverse phase LC/MS using a Gemini 10 m NX-C18 110 , 10021.2 mm, AXIA packed column (Phenomenex). Using 50 mM NH.sub.4HCO.sub.2 pH 8.2 as aqueous phase, the column was equilibrated after injection for 2 min with 5% MeOH, followed by a gradient from 5% to 40% MeOH in 19 min, a 2 min washing step at 95% MeOH and a re-equilibration step of 3 min at 5% MeOH. The flow rate was kept constant at 25 ml min.sup.1. Fractions containing the product were pooled, flash frozen in liquid N2, lyophilized and stored at 20 C. until use. The concentration was determined via UV/Vis at 260 nm assuming an extinction coefficient of 22.4 mM.sup.1 cm.sup.1. For cinnamoyl-CoA .sub.cinnamoyl-CoA 308 nm=16.6 mM.sup.1 cm.sup.1 was employed.
Isolation and Structural Characterization of Aldol Products by NMR
[0574] Synthesis, isolation and preparation of formaldehyde aldol product 2-HMB-CoA:15 mg of crotonyl-CoA, 16 mg of NADPH, were dissolved in 100 mM KH.sub.2PO.sub.4 pH=8, 100 mM formaldehyde and the reaction started by addition of 10 M KsCcr in a final volume of 3 mL and incubated at 30 C. for 20 min. The reaction was quenched by addition of 100 L 50% formic acid and centrifuged at 17000g to precipitate the protein. 2-HMB-CoA was purified by preparative RPLC/MS over a Gemini 10 m NX-C18 110 , 10021.2 mm, AXIA packed column (Phenomenex) using a methanol gradient from 5% to 30% over 10.5 min with 25 mM ammonium formate pH=8.1 as the aqueous phase. Fractions containing the product were pooled, lyophilized and stored at 20 C. Prior to NMR experiments, 4.7 mg of 2-HMB-CoA were dissolved in 600 L 50 mM KH.sub.2PO.sub.4 pH=7.0 and lyophilized. The sample was resuspended in 600 L of D20 resulting in a final concentration of 9 mM in 50 mM KD.sub.2PO.sub.4 pD=7.4 and measured at 25 C., following procedures described earlier.
[0575] Synthesis, isolation and preparation of acetaldehyde aldol product 2-EHB-CoA: 10 mg of crotonyl-CoA 80 mg of NADPH, were dissolved in 100 mM KH.sub.2PO.sub.4 pH=8 with 500 mM acetaldehyde and the reaction started by addition of 2 M KsCcr in a final volume of 2 mL and incubated at 30 C. for 30 min at which point Acx4, to a final concentration of 24 M, was added. At 110 min and 3 h reaction time another half equivalent of Acx4 was added to reach a final concentration of 48 M. The reaction was monitored by HPLC and upon full conversion to 2-EHB-CoA. The reaction was quenched by addition of 100 L 50% formic acid and lyophilized. The reaction mixture was still contaminated with crotonyl-CoA and was incubated with 4 mM NADPH in 100 mM KH.sub.2PO.sub.4 pH=8 with enoyl reductase Etr1p to reduce the remaining crotonyl-CoA to butyryl-CoA. Upon completion the reaction was quenched with 50% formic acid and centrifuged for 10 min at 17000 g at 4 C. to precipitate the protein. 2-EHB-CoA was purified by preparative RPHPLC/MS over a Gemini 10 m NX-C18 110 , 10021.2 mm, AXIA packed column (Phenomenex) using a methanol gradient from 5% to 30% over 10.5 min with 25 mM ammonium formate pH=8.1 as the aqueous phase. Fractions containing the product were pooled, lyophilized and stored at 20 C. Prior to NMR experiments, 0.38 mg of 2-EMB-CoA were dissolved in 600 L 50 mM KH.sub.2PO.sub.4 pH=7.0 and lyophilized. The sample was resuspended in 600 L of D.sub.2O and measured at 25 C., following procedures described earlier.
Quantification of Formaldehyde and Acetaldehyde
[0576] Formaldehyde stock solution concentration was determined by following the formation of phenylhydrazone at 368 nm (.sub.Phenylhydrazone,368 nm=6.1 mM.sup.1 cm.sup.1). Assays were performed on a Cary-60 UV/Vis spectrophotometer (Agilent) at 30 C. using quartz cuvettes (10 mm path length; Hellma) contained K.sub.2HPO.sub.4 pH=8.0 and 3 mM phenylhydrazine.
[0577] Acetaldehyde stock solution concentration was determined by its reduction to ethanol using alcohol dehydrogenase adhE from E. coli and measuring the depletion of NADPH at 340 nm (.sub.NADPH,340 nm=6.22 mM.sup.1 cm.sup.1). Assays were performed on a Cary-60 UV/Vis spectrophotometer (Agilent) at 30 C. using quartz cuvettes (10 mm path length; Hellma) and contained 100 mM Hepes pH=7.5, 300 M NADH and adhE.
Quantification of 2-HMB-CoA and 2-EHB-CoA
[0578] 2-HMB-CoA was quantified by incubation with thioesterase YciA from E.Coli and following the reaction of CoA with Ellmann's reagent (5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)). The reaction releases 2-nitro-5-thiobenzoate (TNB.sup.) that ionizes to TNB.sup.2. The latter compound was detected spectrophotometrically on a Cary-60 UV/Vis spectrophotometer (Agilent) at 30 C. using quartz cuvettes (10 mm path length; Hellma) at a wavelength of 412 nm with .sub.TNB2-,412 nm=14.15 mM.sup.1 cm.sup.1. For quantification of 2-EHB-CoA, the compound was incubated in 1 M NaOH for 15 min at 80 C. to hydrolyze the CoA-ester and the solution neutralized using HCl. The released CoA was quantified using Ellmann's reagent as described for 2-HMB-CoA.
Kinetic Characterization
[0579] For reactions in which the full conversion to the aldol product was achieved assays were performed on a Cary-60 UV/Vis spectrophotometer (Agilent) at 30 C. using quartz cuvettes (1 mm, 3 mm or 10 mm path length; Hellma). Reactions were performed in 100 mM K.sub.2HPO.sub.4 pH=8.0. Kinetic parameters for one substrate were determined by varying its concentration while the others were kept constant at 10 times their K.sub.M value. Reaction procedure was monitored by following the oxidation of NADPH at 365 nm (.sub.NADPH,365 nm=3.33 mM.sup.1 cm.sup.1). For reactions where both butyryl-CoA and the aldol product where formed the steady state parameters were determined using a discontinuous assay. Reactions of 100 L volume were performed in 100 mM K.sub.2HPO.sub.4 pH=8.0 at 30 C. and each concentration was assayed in triplicates. Samples of 20 L reaction were quenched with 2 L of 50% formic acid and centrifuged for 10 min at 17000 g at 4 C. 10 L of this solution were diluted in 40 L of 500 mM Tris HCl pH=7 and incubated overnight at room temperature. Samples were analyzed using by HPLC over an Agilent Eclipse Plus C18, 3.5 m 100 , 1002.1 mm, 4.6100 mm column using 5% to 30% acetonitrile gradient over 7.5 min with 25 mM ammonium formate pH=8.1 as the aqueous phase.
HPLC Analysis of Reaction Products
[0580] Reactions for product analysis contained saturating amounts (at least 10 times the K.sub.M) of crotonyl-CoA, NADPH, 100 mM K.sub.2HPO.sub.4 pH=8.0, and varying amounts of formaldehyde in a final volume of 100 L. The reaction procedure was monitored by decrease in absorbance of NADPH at 365 nm, quenched with 10 L of 50% formic acid at completion and spinned at 17000 g for 10 min to precipitate the protein. The reaction was diluted 10 times into 5% methanol/Buffer 8.1 and analyzed by HPLC over an Agilent Eclipse Plus C18, 3.5 m 100 , 1002.1 mm, 4.6100 mm column using 5% to 30% acetonitrile gradient over 7.5 min with 25 mM ammonium formate pH=8.1 as the aqueous phase.
Substrate Promiscuity Screen
[0581] 100 L Assays contained 1 mM enoyl-CoA, 5 mM NADPH, 500 mM aldehyde (formaldehyde was employed at 75 mM), 5 M CcCcr.sub.PAG, 3 M Acx4 and 0.1 g/L catalase. Assays were performed in 100 mM K.sub.2HPO.sub.4 pH=8.0 at 30 C. and 10 L samples were taken at different time points and quenched with 1 L of 50% formic acid. The samples were spinned at 17000 g for 10 min at 4 C. to precipitate the protein and then analyzed by LC-MS.
LC-MS Analysis of CoA Esters
[0582] Determination of CoA esters was performed using a HRES-LC-MS. The chromatographic separation was performed on an Thermo Scientific Vanquish HPLC System using a Kinetex Evo C18 column (500.12 mm, 100 , 1.7 m, Phenomenexc) equipped with a 202.1 mm guard column of similar specificity at a constant eluent flow rate of 0.3 mL/min and a column temperature of 40 C. with eluent A being 50 mM ammonium formate pH=8.1 and eluent B being MeOH (Honeywell) The injection volume was 1 l. The elution profile consisted of the following steps and linear gradients: 0-1 min constant at 2.5% B; 1-6 min from 2.5 to 90% B; 6-8 min constant at 90% B; 8-8.1 min from 90 to 2.5% B; 8.1-10 min constant at 2.5% B. A Thermo Scientific ID-X Orbitrap mass spectrometer was used in positive mode with an electrospray ionization source and the following conditions: ESI spray voltage 3800 V, sheath gas at 60 arbitrary units, auxiliary gas at 15 arbitrary units, sweep gas at 2 arbitrary units, ion transfer tube temperature at 300 C. and vaporizer temperature at 300 C. Scheduled targeted collision induced dissociation was performed on the two suspect molecules according to the table, applying a precursor ion scan at a mass range between 800 and 900 m/z with a mass resolution of 120000 using the orbitrap mass analyzer after quadrupole pre-isolation. Data dependent detection of MS2 spectra was performed at a normalized collision energy of 30% and an activation Time of 10 ms with an automatic definition of the scan range and a mass resolution (MS2) of 120000 using the orbitrap mass analyzer. Putative structures for the CoAs were generated using knowledge on the biology of the system. For 2-HMB-CoA and 2-EHB-CoA The fragment masses were obtained by inspecting the data files using Xcalibur Qual Browser and Freestyle software. The experimental fragments were used to verify the proposed structure of the molecules by drawing fragment structures to explain the observed fragment mass. The structures of the fragments were drawn using Chemdraw. All precursor ion and fragment ion masses differed by less than 5 ppm compared to the theoretical mass.
Computer Simulations
[0583] The construction of the molecular models was based on the crystal structure of KsCcr (PDB code 6NA4). The complete tetramer structure was used for the simulations considering the reaction in one active site of a closed subunit (catalytically competent state). Minimization and classical MD simulations were performed with the software Amber18 using the CHARMM36/CMAP force field combined with Cgenff parameters for substrate cofactor and electrophile. Free energy calculations to obtain pKa shifts of His365 and Glu171 were performed with GROMACS2019. The reaction mechanism was explored by means of QM/MM calculations using the DFTB3 semiempirical method (3OB-3-1 set of parameters). The adaptive string method was used to find the minimum free energy path (MFEP) that connects reactants and products in a space of collective variables for subsequent PMF (potential of mean force) calculations. Details about parameters, simulation setup, MD simulations and free energy calculations are provided in the Supporting Information.