Biocatalytic Enantioselective Oxidation of Sec‐Allylic Alcohols with Flavin‐Dependent Oxidases

Abstract The oxidation of allylic alcohols is challenging to perform in a chemo‐ as well as stereo‐selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin‐dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from Arabidopsis thaliana (AtBBE15) and the 5‐(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of sec‐allylic alcohols. While primary and benzylic alcohols as well as certain sugars are well known to be transformed by flavin‐dependent oxidases, sec‐allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E‐value of up to >200. For instance HMFO V465S/T oxidized the (S)‐enantiomer of (E)‐oct‐3‐en‐2‐ol (1 a) and (E)‐4‐phenylbut‐3‐en‐2‐ol with E>200 giving the remaining (R)‐alcohol with ee>99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e. g. dimethyl sulfoxide).


Enzymes and chemicals
Reagents and organic solvents were obtained from commercial suppliers in reagent grade quality and used without further purification, unless otherwise stated. The 5-hydroxymethylfurfural oxidase variants (HMFO V465S, V465T, W466H and W466H/V465T) as well as wild type were employed as a purified enzyme solution, which was prepared as previously reported. [1] HMFO V367R/W466F was purchased form GECCO (Groningen, Netherlands). Variants of the berberine bridge enzyme analogue from Arabidopsis thaliana (AtBBE-like15 L182V/L178V/I184V and L182V/I409V) were employed as a purified enzyme solution. Catalase from Micrococcus lysodeikticus (170000 U/mL) was purchased from Sigma-Aldrich.

Synthesis of allylic alcohols from their corresponding ketones
For procedure see main paper. The yields after the purification are reported in Table S1.

390) and HMFO V465T/W466H (pEG 395)
For the different variants of HMFO, the same expression and purification method was used as mentioned in the main manuscript.
His-Tagged HMFO was purified by immobilized Ni-affinity chromatography (5 mL HisTrap FF column, GE Healthcare) applying a 5 to 500 mM gradient of imidazole. Collected fractions were analyzed by SDS-PAGE (see Figure S1). Fractions containing HMFO were pooled, concentrated by ultrafiltration and desalted.

Cosolvent study with substrate 4a by using HMFO variants (1.4 µM)
Substrate 4a was chosen for the cosolvent study by using four different variants of HMFO including V465S, V465T, W466H and V465T/W466H. DMSO, isooctane, n-heptane and glycerol were chosen as cosolvents and different ratio starting from 5% v/v to 50% v/v of these cosolvents was tested in the oxidation reaction.

i) HMFO V465S
Results from testing V465S, revealed that better conversion levels were achieved by using glycerol as cosolvent (Table S4, Figure S2). By using 20% v/v of glycerol 29% conversion (entry 3) was observed but by increasing the cosolvent ratio to 50%, conversion was dropped to 16% (entry 5). In case of water immiscible cosolvent no big different in term of conversion levels in the presence of different ratio of cosolvent was observed.

ii) HMFO V465T
Results from testing V465T revealed that better conversions were achieved by using glycerol as cosolvent (Table S5, Figure S3).  Testing different cosolvents with HMFO V465T DMSO Glycerol Isooctane n-Heptane

iii) HMFO W466H
Results from testing W466H with different cosolvents are shown in Table S6 and Figure S4. As results reveal this mutation considerably reduced the activity of the enzyme since very poor conversions were observed by using this variant at different conditions.  Figure S4. Cosolvent study with substrate 4a by using HMFO W466H

iv) HMFO V465T/W466H
Results from oxidation of 4a with V465T/W466H in the presence of various cosolvents revealed that better conversions were achieved by using glycerol as cosolvent (Table S7, Figure S5). By using 30% v/v of glycerol, conversion was reached to 25.1% (entry 3). n-Heptane was not accepted by the enzyme, since no conversion was observed in the presence of these cosolvents. Testing different cosolvents with HMFO W466H

Investigating the oxygen pressure effect on the oxidation of various substrates by using two different variants of HMFO
Substrate screening in the oxidation step was done by using two variants of HMFO including V465S and V465T in the presence of 10% v/v of DMSO and glycerol as cosolvent. The reactions were performed at two different conditions (with and without oxygen pressure) in parallel. Results revealed that in most of the cases, by using V465T variants higher conversion was achieved. It is worth to mention that in case of α-ionol (6a) very low conversion level (˂3%) was observed in all tested conditions. In general by using DMSO as cosolvent low conversions were obtained. In the same line as the other results, higher conversions were obtained by using glycerol as cosolvent. Results are shown in Table   S8. Testing different cosolvents with HMFO V465T/W466H  In the oxidation of substrate 1a, by applying oxygen pressure the conversion level dropped. In the oxidation of substrates 2a, 3a and 4a (aromatic substrates) using HMFO V465T and V465S variants, the oxygen did not have a high impact on the conversion level. In the oxidation of substrate 5a, with using different variants by applying oxygen pressure, especially in case of glycerol, an improvement in terms of conversion was observed.

DMSO Glycerol Isooctane
The results from oxidation of substrates 3a-5a with different HMFO variants as well as wild type without using any cosolvent in the presence of air, 2 and 4 bar oxygen pressure are shown in Table S9.      11.8 (S), 14.3 (R) [14] 24.0

GC with a achiral phase:
For substrate 1a, GC equipped with chiral column was used. Chiral GC measurements were performed on an Agilent Technologies 7890 A GC system equipped with a FID-detector and a 7683B injector in combination with a 7683 Series Autosampler and using a Chirasil ChiralDexCB column (25m x 320μm x 0.25μm) and H 2 as carrier gas.    14.6 (S), 15.0 (R) [15] 13.9 [a] Injector temperature: 250 °C;    Figure S20. 1 H NMR of 5b purified from upscaling in CDCl3 Figure S21. 13