Chemoenzymatic Synthesis of Optically Active Alcohols Possessing 1,2,3,4-Tetrahydroquinoline Moiety Employing Lipases or Variants of the Acyltransferase from Mycobacterium smegmatis †

: The enzymatic kinetic resolution (EKR) of racemic alcohols or esters is a broadly recognized methodology for the preparation of these compounds in optically active form. Although EKR approaches have been developed for the enantioselective transesteriﬁcation of a vast number of secondary alcohols or hydrolysis of their respective esters, to date, there is no report of bio-or chemo-catalytic asymmetric synthesis of non-racemic alcohols possessing 1,2,3,4-tetrahydroquinoline moiety, which are valuable building blocks for the pharmaceutical industry. In this work, the kinetic resolution of a set of racemic 1,2,3,4-tetrahydroquinoline-propan-2-ols was successfully carried out in neat organic solvents (in the case of CAL-B and BCL) or in water (in the case of MsAcT single variants) using immobilized lipases from Candida antarctica type B (CAL-B) and Burkholderia cepacia (BCL) or engineered acyltransferase variants from Mycobacterium smegmatis (MsAcT) as the biocatalysts and vinyl acetate as irreversible acyl donor, yielding enantiomerically enriched ( S )-alcohols and the corresponding ( R )-acetates with E -values up to 328 and excellent optical purities (>99% ee). In general, higher ee-values were observed in the reactions catalyzed by lipases; however, the rates of the reactions were signiﬁcantly better in the case of MsAcT-catalyzed enantioselective transesteriﬁcations. Interestingly, we have experimentally proved that enantiomerically enriched 1-(7-nitro-3,4-dihydroquinolin-1(2 H )-yl)propan-2-ol undergoes spontaneous ampliﬁcation of optical purity under achiral chromatographic conditions.


Introduction
The implementation of simple, cost-efficient, and environmentally benign synthetic methods that allow the preparation of chiral compounds in high chemical and optical purities is of paramount interest to academia and industry.In this regard, biocatalytic processes which rely on biodegradable and relatively inexpensive enzymes, which can be easily produced in significant amounts by microbial fermentation, are of interest to possibly satisfy the economic and sustainable demand [1][2][3][4][5].The great attractiveness of enzymes in synthetic organic chemistry mainly stems from their ability to catalyze the transformation of a broad scope of xenobiotic substrates with excellent regio-, chemo-and enantio-selectivity [6].These particular features enable the synthesis of chiral products Catalysts 2022, 12, 1610 2 of 23 with excellent optical purities by following shortened synthetic routes and generating less waste to be recycled, which all together constitute a strong driving force in a vast number of industrial applications [7,8], including the manufacturing of pharmaceuticals [9][10][11][12][13][14][15], agrochemicals [16,17] and cosmetic ingredients [18].
The most popular enzymes used in biotechnological applications worldwide are lipases (EC 3.1.1.3,triacylglycerol ester hydrolases), which global market was estimated at 425 mln USD in 2018 with a Compound Annual Growth Rate (CAGR) of 6-8% [19,20].Lipases outperform enzymes from other classes mainly because they exhibit remarkable catalytic activity and high operational stability under non-physiological conditions, including nearly anhydrous organic solvents [21][22][23][24][25][26].Moreover, an increased preference for the use of lipases in industry, and especially in the production of pharmaceuticals and their building blocks [27][28][29][30], is due to the fact that the reactions catalyzed by these enzymes do not require elevated temperatures, high pressures, costly and/or difficult-to-regenerate cofactors, anhydrous and oxygen-free conditions, special handling techniques or sophisticated laboratory equipment.Furthermore, the relatively low price and wide commercial availability of lipase preparations, either in a native or immobilized form, is also a key advantage for the development of enzyme-based biotechnologies.
On the other hand, the acyltransferase from Mycobacterium smegmatis (MsAcT) is a recently identified alternative catalyst with extraordinary catalytic activity for a wide array of reactions, including (trans)esterification, (trans)amidation, and perhydrolysis [89][90][91].Amazingly, a promiscuous MsAcT offers efficient acyl transfer between various esters (i.e., ethyl acetate, benzyl acetate, vinyl acetate, etc.) and a broad range of alcohols in water as an environmentally benign solvent [92][93][94][95][96].Such outstanding ability to perform transesterification in an aqueous solution is relatively uncommon since water usually promotes hydrolysis over synthesis, thus leading to the unfavorable equilibrium of the synthesis of the ester.Strikingly, the unique advantage of catalyzing (trans)esterification of alcohols in aqueous media can reduce the usage of toxic organic solvents and thus lower waste generation.Because of this reason, MsAcT has also attracted attention for its ability to catalyze selective transacylation of racemic alcohols [97,98] performed under kinetically-controlled conditions.
Interestingly, although lipases and MsAcT have proven beneficial in enantioselective C-O functional group chemistry of racemic substrates bearing hydroxyl or acyl moiety, their application in asymmetric biocatalytic transformations of alcohols/esters substituted with 1,2,3,4-tetrahydroquinoline is still unexplored.Moreover, since chiral tetrahydroquinolinebased derivatives serve as essential building blocks for various natural products [99,100], pharmaceuticals [101], and promising drug candidates [102][103][104] (Figure 1), the elaboration of novel synthetic routes toward obtaining these compounds in enantiomerically pure form is highly required.Therefore, as a part of our ongoing research program, which is focused on biocatalytic preparation of optically active heteroaromatic alcohols, and with the aim to widen the biosynthetic repertoire of both titled enzymes towards racemic substrates, an efficient chemoenzymatic synthesis of enantiomerically enriched 1-(hydroxypropyl)-1,2,3,4-tetrahydroquinolines is reported for the first time.

Results and Discussion
Herein, we report a straightforward chemoenzymatic method for the preparation of optically active alcohols possessing 1,2,3,4-tetrahydroquinoline moiety (S)-(+)-and (R)-(-)-2a-c (Scheme 1).In order to obtain the desired products, two alternative biocatalytic routes, relying on enantioselective transesterification of racemic 1-(3,4-dihydroquinolin-1(2H)-yl)propan-2-ols rac-2a-c were performed under kinetically-controlled conditions.In this context, one of the approaches focused on lipase-catalyzed KR of rac-2a-c carried out in neat organic solvents.In contrast, the second attempt was realized by employing engineered variants of the acyltransferase from Mycobacterium smegmatis (MsAcT) in a waterabundant medium.Scheme 1. Enzyme-catalyzed kinetic resolution of racemic alcohols possessing 1,2,3,4-tetrahydroquinoline moiety rac-2a-c using lipases or acyltransferases as the biocatalysts.Therefore, as a part of our ongoing research program, which is focused on biocatalytic preparation of optically active heteroaromatic alcohols, and with the aim to widen the biosynthetic repertoire of both titled enzymes towards racemic substrates, an efficient chemoenzymatic synthesis of enantiomerically enriched 1-(hydroxypropyl)-1,2,3,4tetrahydroquinolines is reported for the first time.
Catalysts 2022, 12, x FOR PEER REVIEW 3 of 23 elaboration of novel synthetic routes toward obtaining these compounds in enantiomerically pure form is highly required.Therefore, as a part of our ongoing research program, which is focused on biocatalytic preparation of optically active heteroaromatic alcohols, and with the aim to widen the biosynthetic repertoire of both titled enzymes towards racemic substrates, an efficient chemoenzymatic synthesis of enantiomerically enriched 1-(hydroxypropyl)-1,2,3,4-tetrahydroquinolines is reported for the first time.
In the first two steps of the synthesis, the required racemic alcohols rac-2a-c and their respective esters rac-3a-c (used as analytical standards for monitoring the reactions' progress and establishment of enantiomeric excess of KR products) were prepared by using standard procedures.In this regard, a regioselective oxirane-ring opening of racemic propylene oxide with the respective 1,2,3,4-tetrahydroquinoline (1a-b) was performed in the presence of sodium bicarbonate (NaHCO 3 ) as a base in anhydrous ethanol (EtOH) at 100 • C for 72 h, leading to rac-2a-b in the 49-82% yield range.In the case of the nitroderivative 1c, extended reaction time (168 h), as well as increased amounts of propylene oxide (8 equiv) and NaHCO 3 (4.5 equiv), were necessary to obtain rac-2c in 83% yield.Due to the low boiling temperature of propylene oxide, all the reactions were performed in a high-pressure glass bottle.In turn, N,N-dimethylpyridin-4-amine (DMAP)-catalyzed esterification of rac-2a-c with an excess of acetyl chloride in the presence of triethylamine (Et 3 N) as a base in dry dichloromethane (CH 2 Cl 2 ) furnished racemic acetates rac-3a-c in an acceptable 36-52% yield range, respectively.The conditions for the lipase-catalyzed transesterification of racemic 1-(3,4-dihydroquinolin-1(2H)-yl)propan-2-ols rac-2a-c were optimized according to the conventional stepwise method reported previously [105].Shortly, the effects of lipase, co-solvent, temperature, and time-course of the enzymatic acetylation of alcohols were evaluated on the KR outcome.
Preliminary enzyme-catalyzed KR studies were performed toward a model racemic substrate rac-2a by using a set of commercially available lipase preparations originating from fungi, bacteria, or plants.The respective biocatalytic system for the resolution of rac-2a consisted of the appropriate biocatalyst suspended in a mixture of vinyl acetate as an irreversible acyl donor and methyl tert-butyl ether (MTBE) as a standard solvent at 40   In the first two steps of the synthesis, the required racemic alcohols rac-2a-c and their respective esters rac-3a-c (used as analytical standards for monitoring the reactions' progress and establishment of enantiomeric excess of KR products) were prepared by using standard procedures.In this regard, a regioselective oxirane-ring opening of racemic propylene oxide with the respective 1,2,3,4-tetrahydroquinoline (1a-b) was performed in the presence of sodium bicarbonate (NaHCO3) as a base in anhydrous ethanol (EtOH) at 100 °C for 72 h, leading to rac-2a-b in the 49-82% yield range.In the case of the nitro-derivative 1c, extended reaction time (168 h), as well as increased amounts of propylene oxide (8 equiv) and NaHCO3 (4.5 equiv), were necessary to obtain rac-2c in 83% yield.Due to the low boiling temperature of propylene oxide, all the reactions were performed in a high-pressure glass bottle.In turn, N,N-dimethylpyridin-4-amine (DMAP)catalyzed esterification of rac-2a-c with an excess of acetyl chloride in the presence of triethylamine (Et3N) as a base in dry dichloromethane (CH2Cl2) furnished racemic acetates rac-3a-c in an acceptable 36-52% yield range, respectively.
According to rational rules of optimizing lipase-catalyzed kinetic resolutions, we then investigated the proper choice of organic solvent.The selection of the appropriate reaction medium is a critical factor for biocatalysis [107], as solvent variation in many cases of lipase-catalyzed KR is known to influence the reaction rate and the equilibrium position [108,109], as well as enantioselectivity [110][111][112][113][114], regioselectivity [115], enantiotopic (prochiral) selectivity [116], and thermal stability [117] of the enzymes.Noteworthy, in rare cases, the organic solvent can also reverse stereo-preference of the lipases [118].
With the aim to obtain both enantiomeric forms of benchmark alcohol rac-2a with the highest possible enantiomeric enrichment, acetylations of rac-2a with vinyl acetate were performed in various organic solvents of varying polarity (log P from 0.20 to 2.52) in the presence of the most enantioselective enzymes, either Chirazyme L-2, C-3 or Amano PS-IM, at 40 • C for 24 h, respectively (Table 2).Moreover, since in the course of transesterification reactions with vinyl acetate, a highly volatile and toxic acetaldehyde is generated as a by-product, we decided to reduce the amounts of acyl donor from >80 equiv to 5 equiv with respect to rac-2a.Decreasing vinyl acetate is also reasonable since in situ forming acetaldehyde deactivates enzymes due to the formation of stable Schiff base-type adducts with amino groups of the side chains belonging to peripheral lysine residues [119].Furthermore, to minimize the costs of the overall enzymatic process, the reduction of biocatalyst loading (from 100% to 20% w/w with respect to rac-2a) was investigated under optimal EKR conditions.
The results presented in Table 2 show that enzyme selectivity (E > 100) was sufficient for practical use in most of the tested organic solvents except acetone for the reaction catalyzed by Amano PS-IM, or acetone, ethyl acetate (EtOAc), 2-methyltetrahydrofuran (2-MeTHF), and toluene (PhCH 3 ) in the case of Chirazyme L-2, C-3.Among the solvents tested, MTBE gave the best results regarding enantioselectivity and reaction rate and, thus, was considered a suitable solvent for further optimization studies.In the case of Chirazyme L-2, C-3-catalyzed KR of rac-2a, it was observed that the effective resolution also occurred in tert-amyl alcohol (2-methyl-2-butanol) (E = 175), which resulted in the isolation of (S)-(+)-2a (94% ee) and (R)-(+)-3a (96% ee) with 50% conv.Interestingly, when comparing KR reactions conducted with both lipases suspended in PhCH 3 , one can see that E-values were radically different (Table 2, entry 7 vs. entry 14).Changing the reaction's stoichiometry by using less vinyl acetate significantly altered the reaction rates, leading to lower conversions than in the previous step.On the other hand, thanks to this change, the enantioselectivity of the reactions carried out in MTBE and other studied solvents improved to achieve E-value up to 307.This time, since the conversions did not exceed 50%, alcohol (S)-(+)-2a was isolated with 5-94% ee.Conversely, acetate (R)-(+)-3a was obtained with up to >99% ee. also occurred in tert-amyl alcohol (2-methyl-2-butanol) (E = 175), which resulted in the isolation of (S)-(+)-2a (94% ee) and (R)-(+)-3a (96% ee) with 50% conv.Interestingly, when comparing KR reactions conducted with both lipases suspended in PhCH3, one can see that E-values were radically different (Table 2, entry 7 vs. entry 14).Changing the reaction's stoichiometry by using less vinyl acetate significantly altered the reaction rates, leading to lower conversions than in the previous step.On the other hand, thanks to this change, the enantioselectivity of the reactions carried out in MTBE and other studied solvents improved to achieve E-value up to 307.This time, since the conversions did not exceed 50%, alcohol (S)-(+)-2a was isolated with 5-94% ee.Conversely, acetate (R)-(+)-3a was obtained with up to >99% ee.>99 257 a Conditions: rac-2a 25 mg, lipase 5 mg, organic solvent 2 mL, vinyl acetate 56 mg (60 μL, 5 equiv), 24 h at 40 °C, 800 rpm (magnetic stirrer); b Logarithm of the partition coefficient of a given solvent between n-octanol and water according to ChemBioDraw Ultra 13.0 software indications; c Based on GC, the % conversion was calculated from the enantiomeric excess of the slower-reacting (S)alcohol (ees) and the formed (R)-acetate (eep) according to the formula conv.= ees/(ees + eep); d Determined by chiral HPLC analysis by using a Chiralcel OJ-H column; e Calculated according to Chen et al. [106], using the equation: To achieve a good conversion in a reasonable time span, we further tested the influence of temperature on Amano PS-IM-catalyzed KR of rac-2a with 5 equiv of vinyl acetate at temperatures ranging from 40 °C to 60 °C (Table 3).Since decreasing amounts of vinyl acetate lowered the reaction rates, all the acetylations of rac-2a were carried out for 72 h.These experiments revealed that elevated temperatures did not induce a notable acceleration of the reaction rate, but the enantioselectivity decreased.Since we could not observe any advantages of heating KR of rac-2a at higher temperatures in terms of reaction rates and enantioselectivity, the subsequent optimization attempts were performed at 40 °C.In turn, intensifying the CAL-B-catalyzed reactions by using elevated temperatures was not investigated since this lipase allowed the obtaining of very high conversions after relatively short reaction times ( a Conditions: rac-2a 25 mg, lipase 5 mg, organic solvent 2 mL, vinyl acetate 56 mg (60 µL, 5 equiv), 24 h at 40 • C, 800 rpm (magnetic stirrer); b Logarithm of the partition coefficient of a given solvent between n-octanol and water according to ChemBioDraw Ultra 13.0 software indications; c Based on GC, the % conversion was calculated from the enantiomeric excess of the slower-reacting (S)-alcohol (ee s ) and the formed (R)-acetate (ee p ) according to the formula conv.= ee s /(ee s + ee p ); d Determined by chiral HPLC analysis by using a Chiralcel OJ-H column; e Calculated according to Chen et al. [106], using the equation: To achieve a good conversion in a reasonable time span, we further tested the influence of temperature on Amano PS-IM-catalyzed KR of rac-2a with 5 equiv of vinyl acetate at temperatures ranging from 40 • C to 60 • C (Table 3).Since decreasing amounts of vinyl acetate lowered the reaction rates, all the acetylations of rac-2a were carried out for 72 h.These experiments revealed that elevated temperatures did not induce a notable acceleration of the reaction rate, but the enantioselectivity decreased.Since we could not observe any advantages of heating KR of rac-2a at higher temperatures in terms of reaction rates and enantioselectivity, the subsequent optimization attempts were performed at 40 • C. In turn, intensifying the CAL-B-catalyzed reactions by using elevated temperatures was not investigated since this lipase allowed the obtaining of very high conversions after relatively short reaction times (Table 2).
Kinetic control of enzymatic reactions is crucial for the outcome of the enantiomers' resolution processes in terms of optical purity and yield of the products [6,120].In the case of lipase-catalyzed KR, the product of the faster-reacting enantiomer is obtained with higher % ee-values when the reaction is arrested at <50% conv.On the other hand, a slowerreacting enantiomer is received with higher ee-values when KR of the racemic mixture is terminated at >50% conv.Depending on the time course of lipase-catalyzed KR, the yield of the products can vary significantly according to substrate conversion.Therefore, it is fundamental to select for KR process the most enantioselective enzyme (E >> 500), which would allow for the obtaining of both non-racemic products in a yield close to the maximum theoretical value (50%) and with excellent enantiomeric purity (>99% ee).Kinetic control of enzymatic reactions is crucial for the outcome of the enan resolution processes in terms of optical purity and yield of the products [6,120].In of lipase-catalyzed KR, the product of the faster-reacting enantiomer is obtain higher % ee-values when the reaction is arrested at <50% conv.On the other slower-reacting enantiomer is received with higher ee-values when KR of the mixture is terminated at >50% conv.Depending on the time course of lipase-cataly the yield of the products can vary significantly according to substrate conversion fore, it is fundamental to select for KR process the most enantioselective enzym 500), which would allow for the obtaining of both non-racemic products in a yie to the maximum theoretical value (50%) and with excellent enantiomeric purity (> Considering the above-mentioned facts, our next objective was to establish propriate time/conversion after which enzymatic reaction could afford KR prod (+)-2a and (R)-(+)-3a with the highest possible enantiomeric excesses (Table 4).[106], using the equation: Considering the above-mentioned facts, our next objective was to establish the appropriate time/conversion after which enzymatic reaction could afford KR products (S)-(+)-2a and (R)-(+)-3a with the highest possible enantiomeric excesses (Table 4).Conditions: rac-2a 25 mg, Amano PS-IM 5 mg, MTBE 2 mL, vinyl acetate 56 mg (60 μL, 5 equiv), 72 h at 40-60 °C, 800 rpm (magnetic stirrer); b Based on GC, the % conversion was calculated from the enantiomeric excess of the slower-reacting (S)-alcohol (ees) and the formed (R)-acetate (eep) according to the formula conv.= ees/(ees + eep); c Determined by chiral HPLC analysis by using a Chiralcel OJ-H column; d Calculated according to Chen et al. [106], using the equation: Kinetic control of enzymatic reactions is crucial for the outcome of the enantiomers' resolution processes in terms of optical purity and yield of the products [6,120].In the case of lipase-catalyzed KR, the product of the faster-reacting enantiomer is obtained with higher % ee-values when the reaction is arrested at <50% conv.On the other hand, a slower-reacting enantiomer is received with higher ee-values when KR of the racemic mixture is terminated at >50% conv.Depending on the time course of lipase-catalyzed KR, the yield of the products can vary significantly according to substrate conversion.Therefore, it is fundamental to select for KR process the most enantioselective enzyme (E >> 500), which would allow for the obtaining of both non-racemic products in a yield close to the maximum theoretical value (50%) and with excellent enantiomeric purity (>99% ee).
Considering the above-mentioned facts, our next objective was to establish the appropriate time/conversion after which enzymatic reaction could afford KR products (S)-(+)-2a and (R)-(+)-3a with the highest possible enantiomeric excesses (Table 4).Consequently, Novozym 435 and Amano PS-IM were arbitrarily selected for kinetic studies on KR of rac-2a.Replacing Chirazyme L-2, C-3 with Novozym 435 was found crucial for further experiments since Chirazyme L-2, C-3 is no longer available at chemical suppliers.Moreover, the performed change among the CAL-B biocatalysts was dictated by the fact that Novozym 435 was significantly more active toward the model racemic substrate rac-2a than Chirazyme L-2, C-3.All the acetylations of rac-2a were carried out by using 20% w/w of lipase preparation with respect to rac-2a in the presence of 5 equiv of vinyl acetate in MTBE at 40 • C and terminated after specific time intervals optimal/tailored for each biocatalyst.From the results collected in Table 4, it was clear that in order to obtain enantiomerically pure alcohol (S)-(+)-2a (>99% ee), the reaction catalyzed by Novozym 435 had to be stopped after 18 h when 57% conv.was achieved.In the case of Amano PS-IM-catalyzed KR of rac-2a, termination of the process after 24 h led to enantiomerically pure acetate (R)-(+)-3a (>99% ee) with 31% conv.It is evident that further elongation of the reaction time deteriorated the enantiomeric purity of (R)-(+)-3a.Moreover, Amano PS-IM-catalyzed acetylation of rac-2a was impossible to run with higher conversions than 50%, even after extending the time to 120 h.
For the purpose of examining the synthetic potential of the present approach, a preparative-scale lipase-catalyzed KR of rac-2a was conducted under optimal reaction conditions by using either Novozym 435 or Amano PS-IM as a biocatalyst (Table 5).The performed experiments revealed that Novozym 435-catalyzed KR of rac-2a afforded (S)-(+)-2a in 39% yield and with 98% ee.On the other hand, the reaction catalyzed by Amano PS-IM allowed the obtaining of optically active acetate (R)-(+)-3a in 25% yield and 99% ee.substrate rac-2a than Chirazyme L-2, C-3.All the acetylations of rac-2a were carried out by using 20% w/w of lipase preparation with respect to rac-2a in the presence of 5 equiv of vinyl acetate in MTBE at 40 °C and terminated after specific time intervals optimal/tailored for each biocatalyst.From the results collected in Table 4, it was clear that in order to obtain enantiomerically pure alcohol (S)-(+)-2a (>99% ee), the reaction catalyzed by Novozym 435 had to be stopped after 18 h when 57% conv.was achieved.In the case of Amano PS-IM-catalyzed KR of rac-2a, termination of the process after 24 h led to enantiomerically pure acetate (R)-(+)-3a (>99% ee) with 31% conv.It is evident that further elongation of the reaction time deteriorated the enantiomeric purity of (R)-(+)-3a.Moreover, Amano PS-IM-catalyzed acetylation of rac-2a was impossible to run with higher conversions than 50%, even after extending the time to 120 h.
For the purpose of examining the synthetic potential of the present approach, a preparative-scale lipase-catalyzed KR of rac-2a was conducted under optimal reaction conditions by using either Novozym 435 or Amano PS-IM as a biocatalyst (Table 5).The performed experiments revealed that Novozym 435-catalyzed KR of rac-2a afforded (S)-(+)-2a in 39% yield and with 98% ee.On the other hand, the reaction catalyzed by Amano PS-IM allowed the obtaining of optically active acetate (R)-(+)-3a in 25% yield and 99% ee.Moreover, to study the effect of diverse substituents on the benzene ring of the 1,2,3,4-tetrahydroquinoline unit, two other derivatives rac-2b-c were subjected to preparative-scale enzymatic KR.The obtained results show that both examined lipases were less enantioselective toward substituted derivatives rac-2b-c (E = 21-140) than when tested along with rac-2a.Moreover, the racemic substrate bearing strong electron-withdrawing nitro group rac-2c turned out to be more challenging for lipases in terms of selectivity in enantiomeric discrimination than the one possessing electron-donating methyl group rac-2b.In the best reaction scenario, optically pure alcohol (S)-(+)-2c (>99% ee) was isolated  C for 18-24 h, 800 rpm (magnetic stirrer); b Based on GC, the % conversion was calculated from the enantiomeric excess of the slower-reacting (S)-alcohol (ee s ) and the formed (R)-acetate (ee p ) according to the formula conv.= ee s /(ee s + ee p ); c Determined by chiral HPLC analysis by using a Chiralcel OJ-H or Chiralcel OD-H column, respectively; d Isolated yield after column chromatography; e Calculated according to Chen et al. [106], using the equation: Moreover, to study the effect of diverse substituents on the benzene ring of the 1,2,3,4tetrahydroquinoline unit, two other derivatives rac-2b-c were subjected to preparativescale enzymatic KR.The obtained results show that both examined lipases were less enantioselective toward substituted derivatives rac-2b-c (E = 21-140) than when tested along with rac-2a.Moreover, the racemic substrate bearing strong electron-withdrawing nitro group rac-2c turned out to be more challenging for lipases in terms of selectivity in enantiomeric discrimination than the one possessing electron-donating methyl group rac-2b.In the best reaction scenario, optically pure alcohol (S)-(+)-2c (>99% ee) was isolated from the reaction catalyzed by Novozym 435 in 35% isolated yield.Noteworthy, lipase-catalyzed KR employing both the racemic substrates rac-2b and rac-2c proceeded with higher reaction rates when compared to rac-2a, thus leading to 60-62% conv.when Novozym 435 was applied.The nitro-derivative rac-2c was also faster transformed by Amano PS-IM, leading to 46% conv.after 24 h.Since the nitro group present in rac-2c boosted the reaction rates, a drop in enantiomeric excess of the formed acetate (R)-(+)-3c was observed.
The most interesting result was obtained in the case of (R)-(-)-2c, which has been isolated with a higher enantiomeric excess (99% ee) than the initial substrate (R)-(+)-3c (94% ee).As we were puzzled by such an outcome, the hydrolytic conditions have also been applied toward less enantiomerically enriched acetate (R)-(+)-3c (67% ee) obtained via biocatalytic resolution.According to chiral HPLC, this attempt resulted in the isolation of the corresponding alcohol (R)-(-)-2c with 72% ee, which unambiguously proved that enantiomeric enrichment occurs in the case of nitro-derivative.Such phenomenon might be attributed to the spontaneous amplification of optical purity under achiral chromatographic conditions, which have already been reported for various 'not-completely-racemic' chiral compounds [121][122][123][124].More in detail, the reason for the observed chiral recognition may lay in the ability of (R)-(-)-2c to act as a donor and an acceptor of hydrogen bonds and hence the intermolecular interactions between the enantiomers, leading to the formation of homochiral (R,R or S,S) and heterochiral (R,S)-associations.In turn, such dimeric species are able to interact selectively with active centers of the silica gel, as is often observed in the case of diastereoisomers.However, the experimental observations should be correlated with the thermodynamic analysis of the potential equilibrium between possible stereoisomeric aggregates to prove that the effect of 'self-disproportionation of enantiomer' (SDE) occurred in the case of non-racemic alcohol (R)-(-)-2c.Interestingly, when we additionally performed KR of rac-2c and analyzed the crude reaction mixture using HPLC, it turned out that the % ee-values of (S)-(+)-2c and (R)-(+)-3c were the same as for the reaction, which KR products were purified using SiO 2 -based column chromatography.These results revealed that the SDE phenomenon is absent in more complex mixtures containing corresponding alcoholester pairs, providing clear proof that optical purities of the KR products, and thus E-values calculated for enzymatic reactions, are not affected.

Acyltransfarase-Catalyzed KR of Racemic 1,2,3,4-Tetrahydroquinoline-Based Alcohols rac-2a-c Using Vinyl Acetate in Water
It is well-known fact that wild-type acyltransferase from Mycobacterium smegmatis (wt-MsAcT) displays poor enantioselectivity toward secondary alcohols (E < 20), and low catalytic activity, especially for aromatic substrates, thus catalyzing acyl transfer with unsatisfactory rates and substrate conversions [97].Recently, to overcome these drawbacks, novel variants of MsAcT have been developed by means of site-directed mutagenesis [98].The best variants possessed a less bulky amino acid instead of phenylalanine in the active site in positions (F150, F154, F174) [98,125].Especially single and double variants, having in these positions an alanine or valine, offered exquisite control in enantioselective acyl transfer (up to E > 200) between ethyl acetate or vinyl acetate and various racemic alcohols (i.e., phenyl or aliphatic secondary alkanols and alkynols at varying sizes).
Taking this advantage into account, in the next step of the biocatalytic studies, we have investigated MsAcT-catalyzed KR of rac-2a-c using the library of made-in-home MsAcT variants in an aqueous 200 mM potassium phosphate buffer (pH 7.5) supplemented with vinyl acetate (10%, v/v, 1 M final conc.) as acyl donor (Table 6).
In the case of rac-2c, all the examined MsAcT variants displayed low-to-moderate enantioselectivity (E = 1-27), showing that enantiomers of racemic substrate possessing the nitro group in the benzene ring were difficult to distinguish.For the most enantioselective F154A variant, a conversion of 22% was observed, while the (R)-enantiomer of the corresponding acetate (R)-(+)-3c was formed with 91% ee and an E-value of 27.In turn, F150A/F154A variant turned out to be significantly more active toward rac-2c than F154A.In this case, 58% of alcohol rac-2c was converted to the (R)-configured acetate (R)-(+)-3c with 55% ee resulting in an E-value of 8. Further experiments with novel Ms-AcT variants are needed to achieve more enantioselective biotransformations of racemic 1-(3,4-dihydroquinolin-1(2H)-yl)propan-2-ols rac-2a-c.Table 6.Screening conditions for MsAcT-catalyzed KR of rac-2a-c using vinyl acetate in an aqueous buffer.

The Assignment of the Absolute Configuration of Enantiomeric Products 2a-c and 3a-c
To the best of our knowledge, no data are available for the absolute configurations of chiral alcohols 2a-c or their acetates 3a-c.Therefore, the absolute configurations of the KR products were determined by comparison of the peak elution order registered with chiral HPLC using optically active chemical standards, (R)-(-)-2a (>99% ee), (R)-(-)-2b (59% ee), and (R)-(-)-2c (>99% ee), synthesized from commercial (R)-(+)-propylene oxide (>99% ee).According to our investigation, the slower-reacting alcohols 2a-c and their acetates 3a-c have the (S)-(+)-and (R)-(+)-configurations, respectively.This assignment agrees well with the Kazlauskas-rule postulated for lipases [126].In this context, all tested MsAcT variants also showed a clear stereo-preference for the (R)-enantiomer of the employed racemic substrates rac-2a-c.Moreover, as a proof-of-principle, the absolute configuration of the synthesized nitro-alcohol (S)-(+)-2c (>99% ee) was unambiguously confirmed by determining the crystal structure from single crystals using X-ray diffraction (XRD) analysis (Figure 2).For details concerning the crystal growth conditions and routine crystal structure determination using single-crystal XRD analysis, please see Experimental Section 3.11 and Supporting Information.also showed a clear stereo-preference for the (R)-enantiomer of the employed racemic substrates rac-2a-c.Moreover, as a proof-of-principle, the absolute configuration of the synthesized nitro-alcohol (S)-(+)-2c (>99% ee) was unambiguously confirmed by determining the crystal structure from single crystals using X-ray diffraction (XRD) analysis (Figure 2).For details concerning the crystal growth conditions and routine crystal structure determination using single-crystal XRD analysis, please see Experimental Section 3.11 and Supporting Information.The following crystal structure has been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC-2217269.Ellipsoids are set to 50% probability.Nitrogen atoms are presented in blue, oxygen atoms in maroon, carbon atoms in black, whereas hydrogen atoms in gray.
Analytical scale lipase-catalyzed reactions were performed in thermo-stated glass vials (V = 4 mL) placed in Chemglass CG-1991-04 GOD Anodized Aluminum Reaction Block, 48 Position, 19 mm Hole Depth, For Circular Top Hot Plate Stirrer.
Melting point (mp) ranges, uncorrected, were determined with a commercial apparatus (Thomas-Hoover "UNI-MELT" capillary melting point apparatus (Philadelphia, PA, USA)) on samples contained in rotating capillary glass tubes open on one side (1.35 mm inner diam.and 80 mm length).
Analytical thin-layer chromatography was carried on TLC aluminum plates with silica gel Kieselgel 60 F254 from Merck (Darmstadt, Germany) (0.2 mm thickness film containing a fluorescence indicator green 254 nm (F254) using UV light as a visualizing agent.

General Procedure for the Synthesis of Racemic Esters rac-3a-c
To a solution of the respective racemic alcohol rac-2a-c (1.00 mmol) in dry CH 2 Cl 2 (10 mL), Et 3 N (152 mg, 1.50 mmol, 209 µL) and DMAP (10 mg) were added.The mixture was cooled to 0-5 • C in an ice bath.Next, acetyl chloride (118 mg, 1.50 mmol, 106 µL for rac-2a-b or 157 mg, 2.00 mmol, 142 µL for rac-2c) was dissolved in dry CH 2 Cl 2 (5 mL) and added dropwise to the reaction mixture by using a syringe.Afterward, the cooling bath was removed, and the resulting mixture was stirred at 25 • C for 24 h for rac-2a-b or 72 h for rac-2c.The crude mixture was diluted with CH 2 Cl 2 (20 mL), subsequently quenched with H 2 O (40 mL), and the water phase was extracted with CH 2 Cl 2 (3 × 20 mL).The combined organic layer was washed with a saturated aqueous solution of NaHCO 3 (80 mL) and brine (80 mL) and dried over anhydrous MgSO 4 .After evaporation of the residuals of solvent under reduced pressure, the crude product was purified by column chromatography on silica gel, using a gradient of n-hexane/EtOAc (70:30 v/v) mixture as eluent, thus yielding desired racemic ester rac-3a-c.

Screening Conditions for MsAcT-Catalyzed KR of rac-2a-c Using Vinyl Acetate in Water
The EKR reaction was initiated by the addition of substrate rac-2a-c (50 µL) from a 0.5 M stock solution prepared by dissolving rac-2a (95.6 mg) or rac-2b (102.6 mg) or rac-2c (118.1 mg) in vinyl acetate (1 mL), and the lyophilized cell-free extract (CFE) of the respective MsAcT (5 µL) from a stock solution prepared by suspending CFE (25 mg) in 200 mM KPi buffer (pH 7.5, 0.5 mL).In general, biotransformations were conducted in 0.5 mL final volume composed of 200 mM KPi buffer (pH 7.5)/vinyl acetate (90:10, v/v) in glass vials (V = 1.5 mL) at 30 • C using a laboratory shaker (500 rpm) for 5 h.After this time, each reaction was stopped by extracting the content of the vial with MTBE (3 × 0.5 mL).The combined organic phase was dried over anhydrous MgSO 4 , the filtrate was additionally centrifuged (5 min, 6000 rpm), and only then was the supernatant was transferred into separate HPLC vials and concentrated under vacuum.The oil residue in one of the vials was used to determine % conv.by using GC analysis, and the other oil residue was redissolved in an HPLC-grade mixture of n-hexane/2-PrOH (1.5 mL; 3:1, v/v), passed through a short pad of silica gel loaded into a Pasteur pipette with cotton wool (in order to remove residuals of the cell components), and analyzed directly by HPLC on a chiral stationary phase to establish enantiomeric excesses of optically active alcohol (S)-(+)-2a-c and acetate (R)-(+)-3a-c.Blank reactions were prepared with buffer (5 µL, KPi 200 mM, pH 7.5) instead of MsAcT enzyme solution.For details, see Table 6  A single crystal of sufficient quality for X-ray diffraction (XRD) analysis was prepared using solvent evaporation methodology.In this regard, (S)-(+)-2c (15 mg, >99% ee) was dissolved in EtOAc (1 mL) and transferred into an open-neck glass vial (V = 4 mL).The vessel was sealed with Parafilm M (Sigma Aldrich) with a small hole punctured to enable very slow evaporation of the solvent and stored at room temperature.After two weeks, single crystals of morphology suitable to perform reliable XRD analysis were grown.

Crystal Structure Determination of (S)-(+)-2c
A suitable crystal of (S)-(+)-2c with dimensions 0.83 × 0.50 × 0.17 mm 3 was selected and mounted on an Oxford Diffraction κ-CCD Gemini A Ultra diffractometer.The crystal was measured with mirror mono-chromated CuKα radiation at room temperature.Data collection, data reduction, scaling, and absorption correction were performed using CrysAl-isPro 1.171.42.72a [127].The final completeness is 100.00%out to 67.002 in Θ. Empirical absorption correction using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm, was applied.The structure was solved with the ShelXT 2018/2 [128] solution program using dual methods and Olex2 1.5 [129] as the graphical interface.The model was refined with ShelXL 2019/2 [130] using full matrix least squares minimization on F 2 .All non-hydrogen atoms were refined aniso-tropically.Hydrogen atom positions were calculated geometrically and refined using the riding model, except hydrogen atom from the hydroxy group, which was picked from the electronic differential map and refined freely.The Flack parameter was refined to 0.03 (9).A slightly increased error on this parameter is caused by a disorder observed in the crystal structure of (S)-(+)-2c with two conformations of a six-membered aza ring.For more detail, see Supporting Information.An absolute configuration (S) for the molecule was successfully determined using anomalous dispersion effects.The Flack parameter calculated from selected 849 quotients (Parsons' method) equals 0.04 (10) [131].Further analysis of the absolute structure was performed using likelihood methods with PLATON [132] using Bijvoet pairs to obtain the Hooft parameter [133].A total of 915 Bijvoet pairs (coverage of 0.99) were included in the calculations.The resulting value of the Hooft parameter was 0.05 (8), with a P3 probability for an inverted structure smaller than 0.1 × 10 −27 .CCDC 2217269 contains the supplementary crystallographic data for compound (S)-(+)-2c.This can be obtained free of charge on application to CDC, 12 Union Road, Cambridge CB21EZ, UK (Fax: (+44)1223-336-033; email: deposit@ccdc.cam.ac.uk).

Conclusions
Herein, we present our efforts to design novel alternative chemoenzymatic routes to access optically active alcohols possessing 1,2,3,4-tetrahydroquinoline moiety.Interestingly, the asymmetric synthesis of such optically active alcohols has not been reported before, either via chemo-catalytic or biocatalytic methods.At the same time, these synthons are potentially useful in the development of novel therapeutics.To fill this gap, enzymecatalyzed kinetic resolution of titled racemic compounds was accomplished using either commercially available lipases from Candida antarctica type B or Burkholderia cepacia or madein-home engineered variants of acyltransferase originating from Mycobacterium smegmatis (MsAcT).
In general, in terms of the optical purities of KR products, higher ee-values were observed in the reactions catalyzed by lipases.However, it seems interesting that MsAcT variants exhibited improved enantioselectivity toward methyl-substituted derivative rac-2b (E = 328) than the lipases (E = 140).Moreover, it has to be mentioned that MsAcT variants were significantly influenced by the electronic properties caused by the substituents present in the benzene ring of the 1,2,3,4-tetrahydroquinoline moiety.In this context, the nitro group in rac-2c displayed an even higher adverse effect on E-values than for the reactions catalyzed by lipases.Considering the above conclusions, it is clear that both methods can be used complementarily depending on the employed racemic substrate; however, the attempted MsAcT-catalyzed KR should be considered more environmentally benign since it generates less volatile and toxic organic solvents as wastes and accelerates the enantiomers resolutions significantly.Thus, we believe that examined variants of MsAcT are valuable additions to the biocatalysts library useful in the kinetic resolution of secondary alcohols and that this study will lead to more practical biocatalytic applications.

Table 1 .
Lipase screening for lipase-catalyzed KR of rac-2a with vinyl acetate in MTBE.

Table 1 .
Lipase screening for lipase-catalyzed KR of rac-2a with vinyl acetate in MTBE.

Table 2 .
Co-solvent screening for lipase-catalyzed KR of rac-2a with vinyl acetate.

Table 2 .
Co-solvent screening for lipase-catalyzed KR of rac-2a with vinyl acetate.

Table 3 .
Effect of the temperature on Amano PS-IM-catalyzed KR of rac-2a with vinyl acetate after 72 h.

Table 4 .
Time-course of lipase-catalyzed KR of rac-2a with vinyl acetate as acyl donor.

Table 4 .
Time-course of lipase-catalyzed KR of rac-2a with vinyl acetate as acyl donor.

Table 6 .
Screening conditions for MsAcT-catalyzed KR of rac-2a-c using vinyl acetate in an aqueou buffer.