Introduction of Chiral Centers to α- and/or β-Positions of Carbonyl Groups by Biocatalytic Asymmetric Reduction of α,β-Unsaturated Carbonyl Compounds

Abstract

Biocatalytic asymmetric reductions of acyclic and cyclic α,β-unsaturated carbonyl compounds are favorable protocols for introduction of chiral centers to α- and/or β-positions of the carbonyl groups. Representative biocatalytic reductions of electron deficient olefins are compiled from a synthetic point of view according to compound types from the papers in 2012 to early 2022. Applications to syntheses of some enantiomericaly enriched perfumery ingredients are presented to show the feasibility of the biocatalytic reductions.

Keywords

asymmetric conjugate reduction α,β-unsaturated carbonyl compound biocatalytic reduction ene reductase perfumery ingredient

Introduction of chiral centers to organic molecules is an important task, especially for total syntheses of physiologically active compounds. Carbonyl groups have been utilized to introduce chiral centers at the α- or β-position by asymmetric nucleophilic reactions of enolates,¹ asymmetric protonation^2,3 or asymmetric conjugate additions to α,β-unsaturated carbonyl compounds (Figure 1).^4,5 Catalytic asymmetric hydrogenation of a double bond employing chiral organometallic catalysts has an alternative advantage, especially in the reduction of α,β-unsaturated carbonyl compounds, which play a major role not only on a laboratory scale but also on a process scale to introduce up to two stereogenic centers in a single step.⁶ Compared to other asymmetric inductions, the catalytic reductions do not require any extra reagents other than catalysts, which is superior in atom economy, benign and sustainable, with less waste. A simple synthetic operation is an extra advantage.

Figure 1.

Introduction of chiral centers at α- and/or β-positions of carbonyl groups.

In addition to the catalytic asymmetric hydrogenation to introduce chiral centers at the α- and/or β-positions of carbonyl groups, biocatalytic asymmetric reductions of α,β-unsaturated carbonyl compounds have attracted more and more attention employing enzymes or whole cells, which proceed in aqueous solvents under neutral conditions at room temperature under atmospheric pressure. The reaction condition is benign and environmentally friendly. An additional advantage is that there is no contamination of heavy metals in the products and wastes, which is different from catalytic hydrogenations. Chemoselectivity of the reduction is high, because the reaction proceeds only with electron-deficient olefins. Isolated double bonds, carbonyl, formyl, nitro, and cyano groups, and benzyl ethers are left intact under the reaction conditions, which could be reduced by catalytic hydrogenations. Although selective reduction of a double bond of an α,β-unsaturated carbonyl compound in the presence of an isolated double bond could be carried out chemoselectively by electron transfer reduction with alkaline metals, the reaction is not suitable for chiral induction.

These characteristic features of biocatalytic reductions are favorable for syntheses of physiologically active compounds including not only flavor and fragrance substances,⁷ but also pharmaceutical ingredients.⁸

The quality of perfumery ingredients highly depends on the absolute stereochemistries, their enantiomeric purities, and on the character of very small amounts of contaminants. The benign nature of biocatalytic reduction might be suitable to apply the flavor substances to human bodies. Some representative flavor and fragrance substances having chiral centers at α- or β-positions of carbonyl groups are highlighted in Figure 2.

Figure 2.

Some representative flavor ingredients having chiral centers at α- and/or β-positions of carbonyl groups and their related derivatives.

A great deal of research data has been accumulated on the biocatalytic asymmetric reduction of electron-deficient olefins,^9–14 and during preparation of this manuscript, a comprehensive review on the topic by Hollmann, Opperman and Paul has appeared.¹³ Synthetic organic chemists are familiar with reagent-based reductions, in spite of the big advantage of biocatalytic reduction. In this regard, the present review deals with developments in enantioselective biocatalytic reduction of electron deficient olefins to emphasize the synthetic utility of biocatalytic reduction, in which more specific examples of bioreductions and applications to the syntheses of some perfumery ingredients and physiologically active small molecules are listed to exemplify the further feasibility of bioreduction in organic synthesis.

Among recent progress in biocatalytic asymmetric reductions, old yellow enzymes (OYE: NADPH dehydrogenase, EC 1.6.99.1) have been studied to reduce electron-deficient olefins, such as α,β-unsaturated carbonyl compounds. A variety of OEYs are found in a variety of plants such as tomatoes and microorganisms, which are relatively stable and available on a large scale. Their structures are well established by X-ray crystallography.^15,16 With the development of genetic engineering,¹⁷ variants by genetic modification and homologues by cloning have been created to elucidate the reaction mechanism, to expand the scope of applications, and to improve the reaction conditions.

The mechanism of the reduction by OYE is considered as shown in Figure 3.^9,18–20 The substrate taken up in the enzyme is reduced in trans-fashion by 1,4-addition of the hydride by the flavin residue present in the protein, which is in contrast to the cis-type addition in the catalytic reduction in the presence of metal catalyst under a hydrogen atmosphere. The oxidized flavin residue is reduced by a cofactor NADPH, which starts the reduction cycle again. The resulting NADP⁺ is reduced back to NADPH by a combination of glucose and glucose dehydrogenase to turn the catalytic cycle. A combination of glucose-6-phosphate and glucose-6-phosphate dehydrogenase, formate and formate dehydrogenase, 2-propanol and alcohol dehydrogenase, or phosphite and phosphite dehydrogenase can also be used in this cofactor recycling process.

Figure 3.

Representative OYE catalyzed reduction of electron deficient olefins by NADPH recycling with glucose. EWG = electron-withdrawing group. GDH = Glucose dehydrogenase.

Biocatalytic Reductions of Cyclic α,b-Unsaturated Ketones

Cyclic ketones having chiral centers at α- and/or β-positions of ketones are important chiral building blocks for total syntheses of natural products. Some representative results to introduce chiral centers by bioreductions at β-positions of β-substituted cyclic enones are shown in entries 1∼8, Table 1. In entry 1, whole cells of E. coli BL21(DE3)(pOYE-pET3b) were employed to reduce 3-methylcyclohexenone in 100% conversion with 96% enantioselectivity (ee) without adding NADP⁺ and GDH. Cyclohexenones having larger substituents also resulted in high enantioselectivity. but in lower yield. β-Cyclodextrin was added to promote solubility of the substrate in aqueous media. In entry 2, glucose was added to maintain the strain. In entry 3, OYE homologue, YqjM, enabled the reduction of cyclohexenone having a longer substituent. In entries 4 and 5, both enantiomers were obtained by changing ene reductases, and also in 6 and 7 by changing YqjM mutants. (R)-(–)-Muscone (2) is a precious perfumery component, as well as a medicinal ingredient to activate the cardiovascular system. Entry 8 shows that the ene reductase from S. salmonicolor TPU 2001 furnished unnatural (S)-( + )-muscone in 95% yield and 100% ee without adding glucose and GDH.²⁵ The catalyst was specific for the reduction of (E)-3-methyl-2-cyclopentadecenone, but not active for the (Z)-enone, along with enones of smaller ring size. Development of a new catalytic system to provide the precious natural (R)-(–)-muscone (2) is expected. The synthetic pathway leading to (E)-enone is shown in Figure 4, which involves intramolecular cyclization in vapor phase in the process scale.⁴² The enzyme was compatible for reduction of either (E)-2-cyclododecenone or (E)-2-cyclopentadecenone.

Figure 4.

Synthesis of unnatural (S)-( + )-muscone (16).⁴².

Table 1.

Biocatalytic Reductions of Cyclic α,β-Unsaturated Ketones.

Entry	Enzyme	Reaction conditions	Yields	Ref.
1	Whole cells of E. coli BL21(DE3)(pOYE-pET3b)	Ampicillin, IPTG, β-cyclodextrin	100% conv., 96% ee	21
2	OYE from Candida sake DBVPG6162	pH 7.0, glucose, EtOH	75%, 98% ee	22
3	YqjM mutant C26D/I69T	NADP⁺, GDH, glucose	92%, 99% ee	23
4	YqjM C26G		100% conv., 96% ee	23
5	OPR1	DMSO or EtOH, GDH, NADH, pH 7.5	99% conv. 99% ee	24
6	YqjM wild type	NADP⁺, GDH, glucose	100% conv., 93% ee	23
7	YqjM C26G		100% conv., 96% ee	23
8	Reductase from Sporidiobolus salmonicolor TPU 2001	pH 7.0, DMSO, NADPH	95%, 100%ee	25
9	Whole cells of E. coli BL21(DE3)(pOYE-pET3b)	Ampicillin, IPTG, β-cyclodextrin	100%, 94% ee	21
10	90 kDa reductase from Nicotiana tabacum	NADPH, pH 8.0, 2-mercaptoethanol, dithiothreitol	95% conv. 99% ee	26
11	YqjM	Rose bengal, TEOA, CaCl₂, white LED, pH 7.5	84%, 99% ee	27
12	44 kDa reductase from N. tabacum,	NADPH, pH 8.0, 2-mercaptoethanol, dithiothreitol	80% conv., >99% ee	26
13	Whole cells of E. coli BL21(DE3)(pOYE-pET3b)	Ampicillin, IPTG, β-cyclodextrin	100% conv. 96% ee	21
14	74 kDa reductase from N. tabacum	NADPH, pH 8.0, 2-mercaptoethanol, dithiothreitol	99% conv., 97% ee	26
15	Purified ClER	GDH, glucose, NADP⁺, pH 6.0	>99% conv., 93% ee	28
16	OPR1	NAD⁺, GDH, glucose	69%, 72% ee	29
17	Purified OYE 2.6 wild type	pH 7.0, NADP⁺, glucose, GDH	>99%, >98% ee	30
18	OYE1	NADH, tris-HCl buffer	69% conv., 94% ee	31
19	XenA	DMSO or EtOH, NADH, pH 7.5	99%, 99% ee	24
20	OPR1	NADP⁺, G6PDH, glucose-6-phosphate,	95%, 91% ee	29
21	Crude ClER	GDH, glucose, NADP⁺, pH 6.0	>99% conv., 96% ee	28
22	Recombinant whole cells from Saccharomyces cerevisiae BY4741ΔOye2	Glucose, pH 7.0, EtOH	89%, 80% ee	22
23	Progesterone 5β-reductase (At5β-24StR) from Arabidopsis thaliana	GDH, glucose, NADPH, pH 7.5	99%, 88% ee	32
24	NCR	Degassed tris-HCl buffer pH 7.0, under argon	98%, 88% ee	33
25	YqjM	FMN, methylviologen, CdSe quantum dots, TEOA, Xe lamp (410-520 nm)	No yield, 64% ee	34
26	TsOYE	Au-TiO₂, FMN, Xe-lamp (200 W), 50 °C, pH 7.0	66% conv. 86% ee	35
27	Purified ClER	GDH, glucose, NADP⁺, pH 6.0	99% conv., 93% de	28
28	LacER from Lactobacillus casei	GDH, glucose, NADP⁺, pH 7.0, DMSO	99%, 98% de	36
29	NostocER1 from Nostoc sp. PCC 7120	pH 7.0, NADH	99%, 99% de	37
30	Nostoc sp. PCC 7120	NADH, FDH, NaHCO₂, [BMIM][PF₆] 10% (v/v) or resin XAD4 (AR), pH 6.3	>96%, >97% de	38
31	Purified ClER	GDH, glucose, NADP⁺, pH 6.0	99% conv., 99% de	28
32	Progesterone 5β-reductase	NADP⁺, glucose-6-phosphate, G6PDH	100%	39 –41

Ampicillin, aminobenzylpenicillin; ClER = OYE homologue from Clavispora lusitaniae; FDH, formate dehydrogenase; FMN, flavin mononucleotide; GDH, glucose dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; IPTG, isopropylthio-β-D-galactoside; NCR, nicotinamide-dependent cyclohexenone rductase; OPR, OYE homologue from Lycopersicon esculentum (tomato); OYE 2.6, OYE homologue from Pichia stipitis; TEOA, triethanolamine; TsOYE, OYE homologue from Thermus scotoductus; XenA, Oxidized OYE from Pseudomonas putida; YqjM, OYE homologue from Bacillus subtili.

Some examples of asymmetric bioreductions of α-substituted cyclic enones to introduce chiral centers at α-positions are presented in entries 9∼19. The same reaction condition in entry 1 was applicable to the reduction of 2-methylcyclohexenone in entry 9, with the same efficiency. The same enone was reduced by 90 kDa reductase from Nicotiana tabacum plant culture in entry 10, while the opposite enantiomer was obtained in entry 12 by 44 kDa reductase from the same plant. In entry 11, the bioreduction was accomplished without addition of cofactor, NADH, in which photo-excited rose bengal reduced the oxidized OYE to turn the catalytic cycle. The oxidized rose bengal was driven back to rose bengal by triethanolamine (TEOA). The process is clean, cost effective and favorable for large scale preparation. In entry 13, the same reaction conditions to reduce 3-methylcyclohexeneone in entry 1 was applied to the reduction of 2-methylcyclohexenone. The same (R)-2-methylcyclohexanone was obtained from 2-methylenecyclohexanone in entry 14. Similarly, 2-methylcyclopentenone was reduced by ClER and OPR1 in entries 14 and 15. The Baylis − Hillman substrate was reduced by OYE 2.6 in entry 16. Among various mutants investigated, the wild type mutant exhibited the best performance. In entry 18 and 19, the bioreductions were carried out without employing the GDH recycling system, in which the reduction of entry 18 was scaled up to 400 mg of the substrate.

Bioreduction of ketoisophorone (oxophorone) (17) has been the most widely investigated, as shown in entries 20∼26 as a model reaction to test new reduction conditions. The product, levodione (18), has been employed for the synthesis of carotenoids as a key starting material.^43–45 In entries 20 and 21, OPR1 and ClER were applicable not only to the bioreduction of cyclic enones (entries 5, 15 and 16), but also ketoisophorone. Entry 22 shows cofactor free bioreduction using whole cells of recombinant S. cerevisiae BY4741ΔOye2. The cofactor free process is cost effective and might be useful for large-scale reduction. In entry 23, an alternative nicotinamide-independent bioreductase, progesterone 5β-reductase, was employed. In entry 24, an equivalent amount of N-Boc-pyrrolidinone was used as a hydrogen donor under anaerobic conditions to prevent generation of H₂O₂, which may cause epoxidation of the double bond of the substrate. N-Boc-pyrrolidinone was favorable, because the co-product, 3-hydroxypyrole, may not inhibit the activity of the catalyst. In entry 25, CdSe quantum dots, methylviologen and triethanolamine (TEOA) were used to reduce cofactor flavin mononucleotide (FMN) under visible light irradiation, which was followed by reduction of ketoisophorone with YqjM. Although a variety of attempts have been reported on the bioreduction of ketoisophorone, only (R)-levodione was obtained by ene reductase reduction. In entry 26, the bioreduction was carried out without adding NADP⁺, in which ene reductase was regenerated via a photocatalytic oxidation cycle of water employing titanium dioxide based photocatalyst. FMN plays a dual role as an enzyme prosthetic group and a redox mediator delivering electrons from the photocatalyst to the enzyme. Medium enantioselectivity might be the result of asymmetric hydrogenation by H₂ generated by the oxidation of water.

Dihydrocarvone (1) is a very versatile chiral building block for natural product syntheses, because it has a six-membered ring, two chiral centers and two functional groups for further transformations.⁴⁶ Chemo- and diastereoselective bioreductions of the enone moiety of carvone are listed in entries 27∼32, in which both enantiomers were obtained. Although selective reduction of an enone moiety in the presence of either a non-conjugated double bond or ketone is usually carried out by an electron transfer process to avoid over-reduction of such substituents, stereoselectivity of the process is not always satisfactory. Compared to such a process, purified ClE was active in reducing both (R)- and (S)-carvones to corresponding dihydrocarvones in entries 27 and 31, with high efficiency. LacER from Lactobacillus casei in entry 28 and NostocER1 from the cyanobacterium, Nostoc sp. PCC 7120 in entry 29 provided (2R,5R)-dihydrocarvone (1), efficiently. Biphasic bioreduction in the presence of hydrophobic ionic liquid, [BMIM][PF₆], suppressed a host cell-mediated epimerization of (2R,5R)-dihydrocarvone (1) in entry 30 and enabled large scale bioreduction on a liter scale. No further reduction of the carbonyl group occurred. Heterogeneous reduction on resin XAD4 (AR) was also effective, resulting in a shorter time scale. Progesterone was reduced stereoselectively in entry 32.

Biocatalytic Reduction of Acyclic α,b-Unsaturated Ketones

In entry 1, Table 2, bioreduction by microorganism, S. cerevisiae, was carried out by adsorbing the substrate in filter paper. On the other hand, the opposite enantiomer was obtained by the cascade reaction in entry 1, Table 2, which was applied to the total synthesis of (R)-tropinal® (6) in Figure 5. Acyclic Baylis-Hillman product was reduced by YqjM variant in entry 2. Bioreduction of doubly activated double bonds in entries 3 and 4 proceeded enantioselectively to afford both enantiomers according to the geometry of the double bonds of the substrates. Phosphonate group was also effective as a double activating group in entry 5. Enantiomerically pure α-haloketone is difficult to prepare owing to higher acidity of the α-proton. In entry 6, α-bromoketone was obtained, in which enantioselectivity could not be determined due to isomerization. However, enantioselectivity was maintained in the case that the present process was combined to cascade process, in which subsequent reduction of the carbonyl group in one pot operation led to bromohydrin in entry 4, Table 2.

Figure 5.

Synthesis of (R)-tropional (6), a flavor of lily of the valley.⁴⁷.

Table 2.

Biocatalytic Reductions of Acyclic α,β-Unsaturated Ketones.

Entry	Enzyme	Reaction conditions	Yields	Ref
1	S. cerevisiae type II	H₂O The substrate was adsorbed in filter paper.	73%, 75% ee	47
2	YqjM C26N/I69A variant	pH 7.0, GDH, glucose, NADP⁺	95% conv., 93% ee	48
3	OYE1	DMSO or EtOH, NADH, pH 7.5,	99% conv., 92% ee	24
4	OYE1	DMSO or EtOH, NADH, pH 7.5	99% conv., 76% ee	24
5	OYE3,	EtOH, pH 7.5, formate dehydrogenase, NADP⁺, NH₄ ⁺ HCO₂^–	R = Me or Et, 99% 99% ee	49
6	OYE1 from S. pastorianu OYE2∼3 from S. cerevisiae	GDH, glucose, NADP⁺ 1% DMSO, pH 6.0	>99% conv. ee unknown	50

Biocatalytic Reductions of α,b-Unsaturated Aldehydes

Some aldehydes of low molecular weight are versatile fragrance components due to their volatility. Introduction of chiral centers at α- or β-position of a formyl group is not well exploited because of inherent sensitive nature of a formyl group toward various chemical transformations. An asymmetric catalytic hydrogenation is not feasible due to the possibility of reduction of the formyl group into primary alcohol. Citronellal (4) in Figure 2 is a very important perfumery ingredient and a useful synthon to install a chiral secondary methyl group distal to a functional group in natural product syntheses. Reduction of citral with PhENR provided natural citronellal (4) in high enantioselectivity albeit in low yield in entry 1, Table 3. On the other hand, its antipode was obtained in high yield with high enantioselectivity in entry 2 by progesterone reductase. In entry 3, isoenzyme OPR obtained from tomato was also effective for the reduction. The α,β-unsaturated aldehydes having a substituent at α-position were successfully reduced in entries 5∼8. In entry 5, by employing [Ru(bpz)₂(Clbpy)]Cl₂ as a photosensitizer and methyl viologen dichloride ([MV²⁺]Cl₂) as an electron transfer mediator, light-driven biocatalytic reduction was carried out to realize regeneration of costly redox coenzymes in 100 turnover frequency with 65% yield and 85% ee, which might be useful for process scale reduction. Electron rich unsaturated aldehyde was reduced by OEY 3 in gram scale in entry 6 by impregnation of the aldehyde on XAD 1180 resin with Et₂O. Subsequent oxidation of the formyl group led to a key precursor of anti-diabetic drugs of the PPAR-α/g agonists such as tesaglitazar. Two olfactory principles of the lily-of-the-valley, lilial® (5) and tropional® (6), were obtained in entries 7 and 8. Slow reaction rate in buffer increased by carrying out the bioreduction in a biphasic system.

Table 3.

Biocatalytic Reductions of α,β-Unsaturated Aldehydes.

Entry	Product	Enzyme	Reaction conditions	Yields	Ref.
1		FMN-binding proein from Pyrococcus horikoshii (PhENR)	Tris-HCl buffer, NADH or NADPH	19% conv., 96% ee	16
2		Progesterone 5β-reductase (At5β-StR) from A. thaliana	pH 7.5, NADPH	99% conv., 99% ee	32
3		OPR1	NADH or NADPH	99% conv. 95% ee	29
4		NCR	Degassed Tris-HCl buffer pH 7.0 (1 eq)	99% conv., 99% ee	33
5		PETNR₃₄₂C	[Ru(bpz)₂(Clbpy)]Cl₂, [MV²⁺]Cl₂, pH 8.0, NADP⁺, G6PDH, O₂ free, glucose-6-phosphate, pH 8.0, n-octanol, isooctane, hν 150 W	65%, 85% ee	51
6		OYE3	XAD 1180 resin, Et₂O Evaporation GDH, NADP⁺, pH 7.0	100%, 99% ee	52
7	(S)-Lilial® (5)	OYE3	pH 7.5, t-BuOMe	32% conv., >95% ee	53
8		OYE2	pH 7.5, t-BuOMe	99%, >95% ee	53

Biocatalytic Reductions of α,b-Unsaturated Acids, Esters and Lactones

Unsaturated acids and esters, especially those having extra electron- withdrawing groups (EWG), are suitable for bioreduction, in which the EWG groups make the reduction easier by both lowering the barrier and stabilizing the product so shifting the equilibrium compared to substrates with electron donating groups (Table 4).

Table 4.

Biocatalytic Reductions of α,β-Unsaturated Acids, Esters and Lactones.

Entry	Enzyme	Reaction conditions	Yields	Ref.
1	OPR 1-wild type	pH 7.5, NADH	96%, 99% ee	54
2	XenA	pH 7.5, GDH, glucose, NADP⁺	94%, 99% ee	55
3	OYE3	DMSO, pH 7.0, glucose, NADP⁺, GDH	R = Me, 100%, 97% ee R = (CH₂)₂CO₂Me, 100%, 94% ee	56
4	OYE1-W116L	pH 6.0, glucose, NADP⁺, GDH	90∼100% conv. Up to 99%ee	57
5	OYE1-W116A		85∼100% conv. Up to 94%ee	57
6	YqjM wt variant		47% conv., 90% ee	48
7	YqjM C26N/I69A variant		44% conv., 88% ee	48
8	OYE3	NADPH	98% conv., 96% ee	58
9	OYE3	NADH	99% conv., 99% ee	58
10	YqjM wt variant	KPi, pH 6.5	55% conv., 99% ee	58
11	OYE3	DMF, pH 7.0, glucose, NADP⁺, GDH	100%, 97% ee	59
12	OYE3	DMSO, pH 7.5, glucose, GDH, NADH⁺	R = SPh, 66%, 96% ee R = CH₂Br, 54%, 99% ee	60
13	OYE 2	IPTG, glucose, K₂HPO₄, ampicillin, vitamin, pH 6.8, air	92%, 96% ee	61
14	Yqj M		69%, 76% ee	61

Regio- and asymmetric reduction of (E)-β-cyanoacrylic acid is depicted in entry 1, in which the presence of γ,d-unsaturation was vital for substrate acceptance. The (Z)-double bond isomer was not reduced. Catalytic hydrogenation of the product led to the γ²-aminoester (S)-pregabalin (25), an analogue of the neurotransmitter GABA (Figure 6).

Figure 6.

Synthesis of (S)-pregabalin (25).⁵⁴.

In entry 2, by employing an oxidized OYE, XenA, the unsaturated acid, led to (R)-naproxen, an antipode of the anti-inflammatory drug, (S)-naproxen. Development to afford the pharmacologically important (S)-isomer is expected.

Electron deficient (E)-β-cycanoacrylate was reduced by OYE3 in entry 3. Bioreduction of the corresponding (Z)-isomer was less efficient. The present protocol was applied to the synthesis of a GABA analogue (26), after hydride reduction and protection, in one pot (Figure 7).

Figure 7.

Synthesis of γ²-aminoester (26).⁵⁶.

When Trp in position 116 in OYE1 was replaced by Leu, the reduction of β-cycanoacrylate provided (R)-3-cyano-3-phenylpropanoate in entry 4; replacement with Ala led to the (S)-enantiomer in entry 5. Similarly, both enantiomers were obtained from Baylis-Hillman product by two alternative YqjM variants in entries 6 and 7. In entries 8 and 9, (Z)- and (E)-2-methylsuccinate furnished by the same enzyme (R)- and (S)-enantiomers, respectively. The isolated double bond was stable under the same reaction conditions in entry 10. It is worthy of note that both (Z)- and (E)-α-bromocrotonate were reduced by OYE3 quite efficiently in entry 11 to provide (S)-2-bromobutanoate, which is difficult to prepare by other means, such as catalytic hydrogenation, owing to the labile nature of the C–Br bond toward hydrogenation, along with easy racemization. α-Chlorocrotonate was also reduced by the same enzyme to afford the (S)-enantiomer highly efficiently. In spite of the absence of an electron-withdrawing group, β-substituted butenolides were reduced in entry 12. Both enantiomers of β-methyl-δ-valerolactone were prepared in entries 13 and 14, which is superior than transition metal catalyzed asymmetric reduction. Scale-up synthesis of the (R)-enantiomer by OYE2 in entry 13 was accomplished in a 1.3 L batch reactor to result in a final concentration of 1.0 g L^–1.

Cascade Reductive Biotransformations

Synthesis by multiple biotransformations in a one pot operation is very favorable in terms of pot economy.⁶² Several efforts have been reported by either combining two different enzymes, or a chemical reaction and enzyme.^63–65

Entry 1 in Table 5 shows that reduction of an acetoxy-enone provided (R)-α-methylketone in 63% and 96% ee in a 5 mmol scale. The reduction was carried out by adsorption of the substrate on filter paper. The product was applied to the synthesis of (R)-tropinal® (6), a flavor of lily of the valley, which is an important fragrance ingredient (Figure 5). Haloform reaction of the product, followed by treatment with chloroformate, led to a mixed anhydride, which was reduced to primary alcohol, and subsequent TEMPO oxidation furnished (R)-tropinal® (6) in 49% overall yield and 94% ee. The authors proposed that the initial reaction proceeded via S_N2’ elimination of the acetoxy group by the attack of the hydride from flavin mononucleotide to provide α-methylene ketone, which was reduced to give (R)-methylketone. It is interesting to note that the opposite (S)-enantiomer was obtained under the same enzymatic reaction conditions in entry 1, Table 2, when the substrate without the acetoxy group was employed.

Table 5.

Cascade Reactions Involving Biocatalytic Reduction.

Entry	Enzyme	Reaction conditions	Yields	Ref.
1	S. cerevisiae type II	H₂O The substrate was adsorbed on filter paper.	63%, 96% ee	47
2	1. YqjM, GDH 2. Prelog-ADH	glucose, NADP⁺, pH 7.0	75%, >99% ee	66
3	1. YqjM, glucose GDH, 2. ADH_LK	NADP⁺, pH 7.0	85%, >99% ee	66
4	OYE3 from Saccharomyces cerevisiae, ADH EVO030 (6:1)	DMSO, GDH, glucose, NAD⁺, NADP⁺, pH 7.0	99% conv. 99% syn	50
5	Mutant of Candida macedoniensis old yellow enzyme (CmOYE)	DMSO, GDH, glucose, NAD⁺, NADP⁺, pH 7.0	90% ee unknown	67
6	NtDBR, MMR	GDH, glucose, NADP⁺, pH 7.0,	79%, de unknown	68
7	NtDBR, MNMR	GDH, glucose, NADP⁺, pH 7.0,	90%, de unknown	68
8	Recombinant S. cerevisiae BY4741ΔOye2	pH 7.0, EtOH	100%, 98% ee	22
9	Baker's yeast	D-glucose, EtOH	49%, 97% ee	69
10	1. OYE3 2. (S)-ω-transaminase ATA-113	1. DMSO, GDH, glucose, pH 7.0, NADP⁺ 2. i-Pr₂N, PLP	>99% conv. 96% ee	70
11	1. OYE3 2. (R)-ω-transaminase ATA-251	1. DMSO, GDH, glucose, pH 7.0, NADP⁺ 2. i-Pr₂N, PLP	>99% conv. 96% ee	70
12	1. OYE1 2. (S)-ω-transaminase ATA-237	1. DMSO, GDH, glucose, pH 7.0, NADP⁺ 2. i-Pr₂N, PLP	78%, >99% ee	70
13	CgrAlcOx, OYE2, horseradish peroxidase, catalase	glucose, NADP⁺, pH 8.0, 1% acetone	95% conv., 99% ee	71
14	CgrAlcOx, GluER, BsGDH	glucose, NADP⁺, pH 8.0, 1% acetone	95% conv., 96% ee	71
15	Recombinant ene reductase from Gluconobacter oxydans, recombinant aldehyde dehydrogenase from Pseudomonas putida	citric acid-NaOH buffer, NADPH,	97% conv., 99% ee	72
16	OYE2, aldehyde dehydrogenase	DMSO or EtOH, NADH, pH 7.0	98%, 99% ee	73
17	1. BOU730 cells containing P450-BM3 mutant 2. cells containing YqjM (R)-selective mutant	GDH, glucose, NADP⁺	85%, 99% ee	74
18	1. Rh₂(OPiv)₄ 2. OPR1 from Lycopersicum esculentum	1. CH₂Cl₂, −78 °C, evaporation 2. GDH, glucose, NADP⁺, pH 7.5, DMSO	54%, 99% ee TON 440	75

BsGDH, glucose dehydrogenase from Bacillus subtilis; GluER, OYE from Gluconobacter oxydans; MNMR, menthone:( + )-neomenthol reductase from Mentha piperita; MMR, ( − )-menthone:( − )-menthol reductase from Mentha piperita; NtDBR, ene reductase from Nicotiana tabacum; PLP, pyridoxal-5’-phosphate.

In entries 2 and 3, after confirming the reduction of enone by the ene reductase, alcohol dehydrogenase was added sequentially in one pot to reduce the carbonyl group diastereoselectively, in which alcohols having two chiral centers were obtained with high enantioselectivity. OYE3 and alcohol dehydrogenase (ADH) EVO030 were added in one portion at the same time in entry 4 to provide syn-bromohydrin efficiently. However, when the two enzymes were added sequentially, the diastereoselectivity was lowered due to isomerization of the intermediary α-bromoketone. The resulting syn-bromohydrin was transformed to the tetrahydrofuran (28), the most pleasant roasted meat flavor, via enzymatic hydrolysis of the benzoate by lipase in a one pot operation, followed by cyclization and S_N2 substitution (Figure 8).

Figure 8.

Synthesis of the most pleasant roasted meat flavor (28).⁵⁰.

(4R,6R)-Actinol (29) is an important doubly chiral building block for the syntheses of carotenoids such as xanthoxin, zeaxathin, and related compounds.⁷⁶ Reduction of ketoisophorone (17) by the mutant of Candida macedoniensis old yellow enzyme (CmOYE) in entry 5 afforded (4R,6R)-actinol (29) in 90% yield, in which the chemical yield was improved compared with the two-enzyme system employing CmOYE and Corynebacterium aquaticum (6R)-levodione reductase (LVR). Menthols are known to be important precursors in the production of several pharmaceutical and commodity chemicals, such as fragrances, perfumes and flavors. Sequential diastereroselective reduction of pulegone (31) was carried out by combining ene reductase from Nicotiana tabacum (NtDBR) and ( − )-menthone: ( − )-menthol reductase (MMR) from Mentha piperita or menthone: ( + )-neomenthol reductase from Mentha piperita (MNMR), which led to pure ( − )-menthol (9) in entry 6 or ( + )-neomenthol (32) in entry 7, respectively. In entry 8, the double bond of the α,β-unsaturated aldehyde was reduced with whole cells involving recombinant BY4741ΔOye2 from S. cerevisiae, in which endogeneous carbonyl reductase subsequently reduced the formyl group. Baker's yeast was also effective to reduce sequentially the unsaturated (E)-aldehyde synthesized from 1,3-diisopropenylbenzene in entry 9 to give the alcohol in 97% ee, which was oxidized by pyridinium chlorochromate to furnish florhydral® (7), a marine odorant floral note (Figure 9), while an opposite enantiomer was obtained from (Z)-unsaturated enal.

Figure 9.

Synthesis of florhydral® (7).⁶⁹.

Transamination was combined with enone reduction in entries 10∼12. Two diastereomers were obtained independently by employing (S)-ω- or (R)-ω-transaminase in entries 10 and 11. Sequential addition of two enzymes was favorable for good conversion in entries 10 and 11, whereas mixing the two enzymes at one time was also effective in entry 12. In entry 13, a one-pot bienzymatic cascade from inexpensive geraniol (33) was carried out by combining a copper radical oxidase (CgrAlcOx) and OYE2 to afford (R)-citronellal (4) in 95% conversion with 96% ee. Alternatively, in entry 14, combination with GluER provided (S)-citronellal (24) in 95% conversion with 99% ee. In entry 15, the resulting intermediary (S)-citronellal (24), after the ene reduction, was oxidized to (S)-citronellic acid (32) with the co-existing aldehyde dehydrogenase in a one pot operation, in which NADPH was regenerated with aldehyde dehydrogenase to recycle the ene reductase. The process is called a hydrogen-borrowing cascade since it does not require a hydride source such as glucose in combination with glucose dehydrogenase, which makes the process cost effective. The oxidation process was effective also by separate oxidation of citronellal (24) to citronellic acid (32) with comparably high efficiency. Similar one pot two-enzyme reduction-oxidation systems have been reported without adding a hydride source (entry 16). The process was also applicable to other α-substituted aldehydes. Combination of allylic C-H oxidation, followed by ene reduction was realized in entry 17. Cyclohexenecarboxylate was treated with BOU730 cells containing P450-BM3 mutant to promote allylic C–H oxidation, and subsequent allyl alcohol oxidation led to cyclohex-2-enone. Addition of BOU730 cells containing YgjM (R)-selective mutant in one pot enabled enone reduction in high yield and high enantioselectivity to provide β-methoxycarbonylcyclohexanone. A combinationof chemical reaction and enzymatic reaction was reported in entry 18. A Rh-catalyzed diazocoupling reaction led to the (E)-succinate derivative, which was subjected to ene reduction without isolation after evaporation of dichloromethane to realize turnover number (TON) 440. The side product in diazocoupling, (Z)-alkene, was not reduced.

Conclusions

The introduction of chiral centers into the α− and/or β−positions of carbonyl compounds is an important task in organic synthesis, and a variety of methods are known, in which asymmetric alkylation or protonation at the α-position of the carbonyl group, catalytic hydrogenation, and conjugate addition to the β-position of an α,β-unsaturated carbonyl compound has been well investigated. Among them, the reduction of α,β-unsaturated carbonyl compounds using asymmetric organometallic catalysts has been widely studied and achieved in recent years, because the reaction proceeds cleanly and atom economically, does not require extra reagents, and produces less waste, and the operation is simple compared with other methods, which is a promising protocol for process scale asymmetric synthesis. In addition to the introduction of chiral centers by catalytic asymmetric hydrogenation, the biocatalytic asymmetric reduction of α,β-unsaturated carbonyl compounds is an alternative method that should be focused on.

In this review, the asymmetric biocatalytic reduction of α,β-unsaturated carbonyl compounds is summarized according to compound type from the viewpoint of synthetic organic chemistry. The progress in biocatalytic asymmetric reduction has been remarkable in recent years, and a great deal of research data have been accumulated. In particular, much effort has been devoted to the asymmetric reduction of electron-deficient olefins employing OYEs, and many remarkable studies have been reported, including the development of reaction conditions and the preparation of OYE variants by genetic engineering. Compared to the asymmetric catalytic hydrogenation with a chiral organometallic catalyst, only the electron-deficient olefins are reduced chemoselectively, in which isolated double bonds, formyl groups, ketones, benzyl ethers, etc remained intact under the reaction conditions. The reaction proceeds at ambient temperature under atmospheric pressure in aqueous solvent. There is no contamination of heavy metal in either the product or waste. These characteristic benign features are suitable for preparation of chiral building blocks as a starting material for natural products, syntheses of chiral fragrance compounds of low molecular weight, and for transformations of compounds with multifunctional groups, especially in the final stage of syntheses of pharmaceuticals. At the same time, various variants of OYEs have been prepared by genetic engineering and used either as purified enzymes or in whole cells, which led to the expansion of substrate scope and improvement of the reaction conditions. Development in the reduction using OYEs has made improvements in co-factor regeneration in various ways.

In spite of the abundance of research work on the biocatalytic reduction of α,β-unsaturated carbonyl compounds, there still remains some issues to be considered. Owing to the higher substrate specificity of the enzymes, diversity of substrates is limited. Only one enantiomer can be obtained by a specific enzyme, and only one enantiomer can be accepted by an enzyme. Substrates of higher molecular weight having more complex structures are difficult to be reduced. Asymmetric biocatalytic reduction of a tetra-substituted olefin is rare introducing two chiral centers at α− and β−positions of a carbonyl group at the same time.⁷⁷ In a process scale reaction, recycling of the catalyst system and improvement of the turnover number are desired. In order to stabilize unstable enzymes and make them reusable, immobilization on solid or liquid support is suggested, which might be useful also for flow synthesis. Even though the reduction by enzymes is useful in organic synthesis, synthetic organic chemists are not used to isolate or bioengineer enzymes on a preparative scale. Utility of these enzymes like stock reagents is expected. How to introduce efficiently chiral centers into the α- and/or β-positions of carbonyl compounds is a long-lasting topic in synthetic organic chemistry. It is a desired challenge to improve these issues. Since biocatalytic reactions are basically sustainable and atom economical, their potential is high. Further progress in this area is expected.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

ORCID iD

Hisahiro Hagiwara

Trial Registration

Not applicable, because this article does not contain any clinical trials.

References

Cano

Zakarian

McGlacken

. Direct asymmetric alkylation of ketones: still unconquered. Angew Chem Int Ed Engl. 2017;56(32):9278‐9290. doi:10.1002/anie.201703079 and earlier references cited therein.

Wang

Goh

CMT

Xiao

Tan

C-H

. Enantioselective protonation catalyzed by chiral Brönsted bases. J Synth Org Chem Japan. 2013;71(11):1145‐1151. doi:10.5059/yukigoseikyokaishi.71.114.

Phelan

Ellman

. Conjugate addition–enantioselective protonation reactions. Beilstein J Org Chem 2016;12:1203‐1228. doi:10.3762/bjoc.12.116 and earlier references cited therein.

Schmid

Drissi-Amraoui

Crévisy

Baslé

Mauduit

. Copper-catalyzed asymmetric conjugate addition of organometallic reagents to extended Michael acceptors. Beilstein J Org Chem. 2015;11:2418‐2434. doi:10.3762/bjoc.11.263.

Shockley

Holder

Stoltz

. Palladium-catalyzed asymmetric conjugate addition of arylboronic acids to α,β-unsaturated cyclic electrophiles. Org Process Res Dev. 2015;19(8):974‐981. doi:10.1021/acs.oprd.5b00169 and earlier references cited therein.

Hagiwara

. Chemo- and enantioselective catalytic hydrogenation of((((-unsaturated ketones and aldehydes as a tool to introduce chiral centers at (- or (-positions of ketones. Nat Prod Commun. 2018;13(4):489‐499. doi:10.1177/1934578X1801300427.

Schempp

Drummond

Buchhaupt

Schrader

. Microbial cell factories for the production of terpenoid flavor and fragrance compounds. J Agric Food Chem. 2018;66(10):2247‐2258. doi:10.1021/acs.jafc.7b00473.

Huisman

Collier

. On the development of new biocatalytic processes for practical pharmaceutical synthesis. Curr Opin Chem Biol. 2013;17(2):284‐292. doi:10.1016/j.cbpa.2013.01.017.

Some representative reviews, [a] Winkler

Tasnádi

Clay

Hall

Faber

. Asymmetric bioreduction of activated alkenes to industrially relevant optically active compounds. J Biotechnol. 2012;162(2):381‐389. doi:10.1016/j.jbiotec.2012.03.023.

10.

Toogood

Scrutton

. New developments in ‘ene’-reductase catalysed biological hydrogenations. Curr Opin Chem Biol. 2014;19(1):107‐115. doi:10.1016/j.cbpa.2014.01.019.

11.

Domínguez

Schell

Bisagni

Kalthoff

. Reduction of activate carbon-carbon double bonds using highly active and enantioselective double bond reductases. Johnson Matthey Technol. 2016;60(4):243‐249. doi:10.1595/205651316X692572.

12.

Winkler

Faber

Hall

. Biocatalytic reduction of activated C=C-bonds and beyond: emerging trends. Curr Opin Chem Biol. 2018;43(1):97‐105. doi:10.1016/j.cbpa.2017.12.003.

13.

Hollmann

Opperman

Paul

. Biocatalytic reduction reactions from a chemist’s perspective. Angew Chem Int Ed Engl. 2021;60(11):5644‐5665. doi:10.1002/anie.202001876 and earlier references cited in these references.

14.

Lauterbach

Lenz

Vincent

. H₂-driven cofactor regeneration with NAD(P)⁺-reducing hydrogenases. FEBS J. 2013;280(13):3058‐3068. doi:10.1111/febs.12245.

15.

For example, Kitzing

Fitzpatrick

Wilken

, et al. The 1.3 Å crystal structure of the flavoprotein YqjM reveals a novel class of old yellow enzymes. J Biol Chem. 2005;280(30):27904‐27913. doi: 10.1074/jbc.M502587200.

16.

Steinkellner

Gruber

Pavkov-Keller

, et al. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations. Nature Commun. 2014;5:4150. doi:10.1038/ncomms5150.

17.

Amato

Stewart

. Applications of protein engineering to members of the old yellow enzyme family. Biotechnol Adv. 2015;33(5):624‐631. doi:10.1016/j.biotechadv.2015.04.011.

18.

Hall

Stueckler

Hauer

, et al. Asymmetric bioreduction of activated C=C bonds using Zymomonas mobilis NCR enoate reductase and old yellow enzymes OYE 1–3 from yeasts. Eur J Org Chem. 2008;2008(9):1511‐1516. doi:10.1002/ejoc.200701208.

19.

Pang

Hay

Scrutton

Sutcliffe

. Deep tunneling dominates the biologically important hydride transfer reaction from NADH to FMN in morphinone reductase. J Am Chem Soc. 2008;130(22):7092‐7097. doi 10.1021/ja800471f.

20.

Lonsdale

Reetz

. Reduction of α,β-unsaturated ketones by old yellow enzymes: mechanistic insights from quantum mechanics/molecular mechanics calculations. J Am Chem Soc. 2015;137(46):14733‐14742. doi: 10.1021/jacs.5b08687.

21.

Swiderska

Stewart

. Stereoselective enone reductions by Saccharomyces carlsbergensis old yellow enzyme. J Mol Catal B. 2006;42(1):52‐54. doi:10.1016/j.molcatb.2006.06.023

22.

Romano

Contente

Molinari

, et al. Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes. [b] Romano, D, Contente, ML, Molinari, F. Microb Cell Fact. 2014;13(4):60‐68. doi:10.1186/1475-2859-13-60

23.

Bougioukou

Kille

Taglieber

Reetz

. Directed evolution of an enantioselective enoate-reductase: testing the utility of iterative saturation mutagenesis. Adv Synth Catal. 2009;351(18):3287‐3305. doi:10.1002/adsc.200900644.

24.

Turrini

Cioc

van der Niet , et al. Biocatalytic access to nonracemic γ-oxo esters via stereoselective reduction using ene-reductases. Green Chem. 2017;19(2):511‐518. doi:10.1039/c6gc02493a.

25.

Yamamoto

Oku

Ina

, et al. Purification and characterization of an enone reductase from Sporidiobolus salmonicolor TPU 2001 reacting with large monocyclic enones. ChemCatChem. 2017;9(19):3697‐3704. doi:10.1002/cctc.201700244.

26.

. Hirata

Shimoda

Gondai

. Asymmetric hydrogenation of the C-C double bond of enones with the reductases from Nicotiana tabacum. Chem Lett. 2000;29(8):850‐851. doi:10.1246/cl.2000.850

27.

Lee

Choi

Pesic , et al. Cofactor-free, direct photoactivation of enoate reductases for the asymmetric reduction of C=C bonds. Angew Chem Int Ed Engl. 2017;56(30):8681‐8685. doi:10.1002/anie.201702461

28.

H-L

Lin

G-Q

J-H

. An ene reductase from Clavispora lusitaniae for asymmetric reduction of activated alkenes. Enzyme Microb Technol. 2014;56(1):40‐45. doi:10.1016/j.enzmictec.2013.12.016.

29.

Hall

Stueckler

Kroutil

Macheroux

Faber

. Asymmetric bioreduction of activated alkenes using cloned 12-oxophytodienoate reductase isoenzymes OPR-1 and OPR-3 from Lycopersicon esculentum (tomato): a striking change of stereoselectivity. Angew Chem Int Ed Engl. 2007;46(21):3934‐3937. doi:10.1002/anie.200605168.

30.

Walton

Sullivan

Patterson-Orazem

Stewart

. Residues controlling facial selectivity in an alkene reductase and semi-rational alterations to create stereocomplementary variants. ACS Catal. 2014;4(7):2307‐2318. doi:10.1021/cs500429k.

31.

Köcninger

Baraibar

Mügge , et al. Recombinant cyanobacteria for the asymmetric reduction of C=C bonds fueled by the biocatalytic oxidation of water. Angew Chem Int Ed Engl. 2016;55(18):5582‐5585. doi:10.1002/anie.201601200.

32.

Durchschein

Wallner

Macheroux

, et al. Nicotinamide-dependent ene reductases as alternative biocatalysts for the reduction of activated alkenes. Eur J Org Chem. 2012;2012(26):4963‐4968. doi:10.1002/ejoc.201200776.

33.

Winkler

Clay

Entner

Plank

Faber

. NAD(P)H-independent asymmetric C=C bond reduction catalyzed by ene reductases by using artificial co-substrates as the hydrogen donor. Chem Eur J. 2014;20(5):1403‐1409. doi:10.1002/chem.201303897.

34.

Burai

Panay

Zhu

Lian

Lutz

. Light-driven, quantum dot-mediated regeneration of FMN to drive reduction of ketoisophorone by Old Yellow Enzyme. ACS Catal. 2012;2(4):667‐670. doi:10.1021/cs300085h.

35.

Mifsud

Gargiulo

Iborra

Arends

IWCE

Hollmann

Corma

. Photobiocatalytic chemistry of oxidoreductases using water as the electron donor. Nature Chem. 2014;5:3145‐3150. doi:10.1038/ncomms4145.

36.

Gao

Ren

Zhu

. Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei. Enzyme Microb Technol. 2012;51(1):26‐34. doi:10.1016/j.enzmictec.2012.03.009.

37.

Castiglione

Weuster-Botz

. Comparative characterization of novel ene-reductases from cyanobacteria. Biotechnol Bioeng. 2013;110(5):1293‐1301. doi:10.1002/bit.24817.

38.

Castiglione

Polte

Leupold

Meo

Weuster-Botz

. Asymmetric whole-cell bioreduction of (R)-carvone by recombinant Escherichia coli with in situ substrate supply and product removal. Bioch. Eng J. 2017;117(1):102‐111. doi:10.1016/j.bej.2016.10.002.

39.

Bauer

Rudolph

Müller-Uri

Kreis

. Vein Patterning 1-encoded progesterone 5b-reductase: activity-guided improvement of catalytic efficiency. Phytochemistry. 2012;77:53‐59. doi:10.1016/j.phytochem.2012.01.022.

40.

Bauer

Munkert

Brydziun

, et al. Highly conserved progesterone 5(-reductase genes (P5bR) from 5(-cardenolide-free and 5(-cardenolide-producing angiosperms. Phytochemistry. 2010;71(13):1495‐1505. doi:10.1016/j.phytochem.2010.06.004.

41.

Herl

Fischer

Mü¨ller-Uri

Kreis

. Molecular cloning and heterologous expression of progesterone 5(-reductase from Digitalis lanata Ehrh. Phytochemistry. 2006;67(3):225‐231. doi:10.1016/j.phytochem.2005.11.013.

42.

Hagiwara

Adachi

Doya

Hoshi

Suzuki

. Expedient synthesis of E-3-methylcyclopentadec-2-en-1-one, an important precursor for (R)-(–)-muscone. Flavour Fragr J. 2012;27(1):54‐59. doi:10.1002/ffj.2080.

43.

Some representative recent works on carotenoid synthesis from levodione. Ernst

. Recent advances in industrial carotenoid synthesis. Pure Appl Chem. 2002;74(8):1369‐1382.

44.

Yamano

Eno

Hikita

Kurimoto

Wada

. Stereo controlled first total syntheses of salmoxanthin and deepoxysalmoxanthin. Curr Org Synth. 2015;12(2):180‐188. doi:10.2174/1570179411999141106102507.

45.

Rivas

Alvarez

de Lera

. Stereocontrolled synthesis and configurational assignment of (R)-all-trans-11,12-dihydro-3-hydroxyretinol. Tetrahedron Lett. 2019;60(35):150972. doi:10.1016/j.tetlet.2019.150972.

46.

Hagiwara

. Recent advances of total syntheses of diterpenoids starting from carvone. Nat Prod Commun. 2016;11(9):1381‐1402. doi:10.1177/1934578X1601100947.

47.

Paula

BRS

Zampieri

Rodrigues

JAR

Morana

PJS

. Bioreduction of (-acetoxymethyleEnones: proposal for an S_N2’ mechanism catalyzed by enereductase. Adv Synth Catal. 2016;358(22):3555‐3571. doi:10.1002/adsc.201600601.

48.

Rüthlein

Classen

Dobnikar

Schclzel

Pietruszka

. Finding the selectivity switch–A rational approach towards stereocomplementary variants of the Ene reductase YqjM. Adv Synth Catal. 2015;357(8):1775‐1786. doi:10.1002/adsc.201500149.

49.

Janicki

Kiełbasiński

Turrini

Faber

Hall

. Asymmetric bioreduction of (-activated vinylphosphonate derivatives using ene-reductases. Adv Synth Catal. 2017;359(23):4190‐4196. doi:10.1002/adsc.201700716.

50.

Brenna

Crotti

Gatti

Marinoni

Monti

Quaiato

. Exploitation of a multienzymatic stereoselective cascade process in the synthesis of 2-methyl-3-substituted tetrahydrofuran precursors. J Org Chem. 2017;82(4):2114‐2122. doi:10.1021/acs.joc.6b02927.

51.

Peers

Toogood

Heyes

Mansell

Coeb

Scrutton

. Light-driven biocatalytic reduction of α,β- unsaturated compounds by ene reductases employing transition metal complexes as photosensitizers. Catal Sci Technol. 2016;6(1):169‐177. doi:10.1039/c5cy01642h.

52.

Bechtold

Brenna

Femmer

, et al. Biotechnological development of a practical synthesis of ethyl (S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP): over 100-fold productivity increase from yeast whole cells to recombinant isolated enzymes. Org Proc Res Devel. 2012;16(2):269‐276. doi:10.1021/op200085k.

53.

Stueckler

Mueller

Winkler

, et al. Asymmetric synthesis of Lilial® and Helional®. Dalton Trans. 2010;39(36):8472‐8476. doi:10.1039/c002971h.

54.

Winkler

Clay

Turrini

, et al.

Nitrile as activating group in the asymmetric bioreduction of (-cyanoacrylic acids catalyzed by ene-reductases.

Adv Synth Catal. 2014;356(8):1878‐1882. doi:10.1002/adsc.201301055.

55.

Waller

Toogood

Karuppiah

, et al. Structural insights into the ene-reductase synthesis of profens. Org Biomol Chem. 2017;15(20):4440‐4448. doi:10.1039/c7ob00163k.

56.

Brenna

Gatti

Manfredi

Monti

Parmeggiani

. Old Yellow Enzyme-mediated reduction of (-cyano-(((-unsaturated esters for the synthesis of chiral building blocks: stereochemical analysis of the reaction. Catal Sci Technol. 2013;3(4):1136‐1146. doi:10.1039/c3cy20804d.

57.

Brenna

Crotti

Gatti

, et al. Opposite enantioselectivity in the bioreduction of (Z)-(-aryl-(-cyanoacrylates mediated by the tryptophan 116 mutants of Old Yellow Enzyme 1: synthetic approach to (R)- and (S)-(-aryl-(-lactams. Adv Synth Catal. 2015;357(8):1849‐1860. doi:10.1002/adsc.201500206.

58.

Hall

Stueckler

Hauer

, et al. Asymmetric bioreduction of activated C=C bonds using Zymomonas mobilis NCR enoate reductase and Old Yellow Enzymes OYE 1–3 from yeasts. Eur J Org Chem. 2008;2008(9):1511‐1516. doi:10.1002/ejoc.200701208.

59.

Brenna

Gatti

Manfredi

Monti

Parmeggiani

. Enoate reductase-mediated preparation of methyl (S)-2-bromobutanoate, a useful key intermediate for the synthesis of chiral active pharmaceutical ingredients. Org Proc Res Dev. 2012;16(2):262‐268. doi:10.1021/op200086t.

60.

Turrini

Hall

Faber

. Enzymatic synthesis of optically active lactones via asymmetric bioreduction using ene-reductases from the Old Yellow Enzyme family. Adv Synth Catal. 2015;357(8):1861‐1871. doi:10.1002/adsc.201500094.

61.

Zhang

Schneiderman

Cai

Tai

Y-S

Fox

Zhang

. Optically active β-methyl-δ-valerolactone: biosynthesis and polymerization. ACS Sustainable Chem Eng. 2016;4(8):4396‐4402. doi:10.1021/acssuschemeng.6b00992.

62.

Hayashi

. Time and pot economy in total synthesis. Acc Chem Res. 2021;54(6):1385‐1398. doi:10.1021/acs.accounts.0c00803.

63.

For example Multi-step enzyme catalysis, ed. Garcia-Junceda

. Wiley-VCH, 2008.

64.

Ricca

Brucher

Schrittwieser

. Multi-enzymatic cascade reactions: overview and perspectives. Adv Synth Catal. 2011;353(13):2239‐2262. doi:10.1002/adsc.201100256.

65.

Mayer

Kroutil

Faber

. Enzyme-initiated domino (cascade) reactions. Chem Soc Rev. 2001;30(6):332‐339. doi:10.1039/B105493G.

66.

Classen

Korpak

Schölzel

Pietruszka

. Stereoselective enzyme cascades: an efficient synthesis of chiral γ-butyrolactones. ACS Catal. 2014;4(5):1321‐1331. doi:10.1016/B978-0-12-819820-9.00020-X.

67.

Horita

Kataoka

Kitamura

, et al. An engineered old Yellow Enzyme that enables efficient synthesis of (4R,6R)-actinol in a one-pot reduction system. ChemBioChem. 2015;16(3):440‐445. doi:10.1002/cbic.201402555.

68.

Toogood

Cheallaigh

Tait

, et al. Enzymatic menthol production: one-pot approach using engineered Escherichia coli. ACS Synth Biol. 2015;4(10):1112‐1123. doi:10.1021/acssynbio.5b00092.

69.

Abate

Brenna

Negri

Fuganti

Serra

. Biocatalysed synthesis of the enantiomers of the floral odorant Florhydral®. Tetrahedron: Asym. 2002;13(8):899‐904. doi:10.1016/S0957-4166(02)00202-1.

70.

Monti

Forchin

Crotti

, et al. Cascade coupling of ene-reductases and (-transaminases for the stereoselective synthesis of diastereomerically enriched amines. ChemCatChem. 2015;7(19):3106‐3109. doi:10.1002/cctc.201300764.

71.

Ribeaucourt

Höfler

Yemloul

, et al. Tunable production of (R)- or (S)-citronellal from geraniol via a bienzymatic cascade using a copper radical alcohol oxidase and Old Yellow Enzyme. ACS Catal. 2022;12(2):1111‐1116. 10.1021/acscatal.1c05334.

72.

Winkler

Gröger

Hummel

. Enantioselective rearrangement coupled with water addition: direct synthesis of enantiomerically pure saturated carboxylic acids from (((-unsaturated aldehydes. ChemCatChem. 2014;6(4):961‐964. doi:10.1002/cctc.201300764.

73.

Knaus

Mutti

Humphreys

Turner

Scrutton

. Systematic methodology for the development of biocatalytic hydrogen-borrowing cascades: application to the synthesis of chiral α-substituted carboxylic acids from (-substituted (((-unsaturated aldehydes. Org Biomol Chem. 2015;13(1):223‐233. doi:10.1039/C4OB02282C.

74.

Agudoa

Reetz

. Designer cells for stereocomplementary de novo enzymatic cascade reactions based on laboratory evolution. Chem Commun. 2013;49(93):10914‐10916. doi:10.1039/c3cc46229c.

75.

Wang

Bartlett

Denard

Hartwig

Zhao

. Combining Rh-catalyzed diazocoupling and enzymatic reduction to efficiently synthesize enantioenriched 2-substituted succinate derivatives. ACS Catal. 2017;7(4):2548‐2552. doi:10.1021/acscatal.7b00254.

76.

Wada

Yoshizumi

Noda

, et al. Production of a doubly chiral compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, by two-step enzymatic asymmetric reduction. Appl Environ Microbiol. 2003;69(2):933‐937. doi:10.1128/AEM.69.2.933-937.2003.

77.

Brenna

Gatti

Monti

Parmeggiani

Serra

. Stereochemical outcome of the biocatalysed reduction of activated tetrasubstituted olefins by old yellow enzymes 1–3. Adv Synth Catal. 2012;354(1):105‐112. 10.1002/adsc.201100504.