Description:4. DETAILED DESCRIPTION

TECHNICAL FIELD OF THE INVENTION
The invention relates to the discovery of hydroxynitrile lyase variants and their use in diastereoselective synthesis of ꞵ-nitroalcohols. More specifically, the present invention relates to on variants of a (R)-selective α/β hydrolase fold hydroxynitrile lyase and their use in diastereoselective synthesis of ꞵ-nitroalcohols.

BACKGROUND OF THE INVENTION

Chiral -nitroalcohols with more than one chiral center are key building blocks that find application in the synthesis of pharmaceuticals, and bioactive molecules .

Pseudomonas flourescens lipase catalyzed kinetic resolution (KR) of three aromatic ꞵ-nitroalcohols each having two chiral centers has produced the (R)-β-nitroalcohols, where R= 4-ClPh and 4-COOMePh (Scheme 1). However, the chirality of the second chiral carbon with –NO2 was not specified in the case of both of these substrates. KR of the third substrate 2-hydroxy-3-nitro-3-phenyl-1-benzyl-oxypropane has resulted in the synthesis of the (R,S)-diastereomer in 85% ee, 50% yield, and the (S,R)-diastereomer of the 2-acetoxy-3-nitro-3-phenyl-1-benzyl-oxypropane in 82% ee, 45% yield. While the yield is not high, the % ee of the products obtained by this method is not more than 85. Further, this process requires additional steps of synthesis of its starting substrate racemic β-nitroalcohol.

Scheme 1: Pseudomonas flourescens lipase catalyzed KR in the synthesis of ꞵ-nitroalcohol diastereomers.

In the KR based stereoselective transacetylation of racemic trans-2 nitrocyclohexanol using vinyl acetate as an acylating agent, Pseudomonas flourescens and Candida antarctica lipase B (CALB) have produced 49-50% conversion and >98% ee of each of the stereoisomers (Scheme 2a). Preparative scale transacetylation by Pseudomonas flourescens has produced (1R,2R)-trans-2-nitrocyclohexyl acetate in 99% ee, 49% yield, and (1S,2S)-trans-2-nitrocyclohexanol in 99% ee, 48% yield. In the transesterification of racemic cis-2-nitrocyclohexanol in the presence of vinyl acetate four enzymes, Candida cylindracea C1, Pseudomonas stutzeri, Alcaligenes spp. and Pseudomonas flourescens showed E >400, with a conversion range of 47-81% and up to >98% ee of the product (Scheme 2b). The cis-2-nitrocyclohexanol on preparative scale transesterification produced (1R,2S)-cis-2-nitrocyclohexyl acetate and (1S,2R)-cis-2-nitrocyclohexanol in 99% ee, however, the cis-acetate was decomposed during silica gel based separation. For the hydrolysis of racemic trans-2-nitrocyclohexyl acetate, Candida cylindracea C1, Alcaligenes spp., Pseudomonas cepacia P1, Pseudomonas stutzeri, and Pseudomonas cepacia P2 have shown high enantioselectivity (Scheme 2c). The hydrolysis based KR by these enzymes has produced (1S,2S)-trans-2-nitrocyclohexyl acetate in 88-99% ee, 47-53% conversion, and (1R,2R)-trans-2-nitrocyclohexanol in 91-99% ee. Substrates for the kinetic resolution, i.e., racemic trans β-nitroalcohols and their esters, requires additional chemical steps for their synthesis, and hence is not an economical route. In addition to the restricted yield of a single diastereomer the kinetic resolution reported in both Scheme 1 and 2 suffers from long reaction time (up to 60 h), and limited substrate scope.

() (1R,2R) (1S,2S)

() (1R,2S) (1S,2R)

() (1S,2S) (1R,2R)
Scheme 2: KR of racemic 2-nitrocyclohexanol and 2-nitrocyclohexyl acetate via transesterification and hydrolysis in the synthesis of ꞵ-nitroalcohol diastereomers.

The one-pot two-step dynamic kinetic resolution (DKR) of 2-methyl-2-nitrocyclohexanol using 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) catalysed intramolecular nitroaldol (Henry) reaction followed by immobilized CALB catalyzed KR has produced the (1R,2S)-cis-2-methyl-2-nitrocyclohexyl acetate and (1R,2R)-trans-2-methyl-2-nitrocyclohexyl acetate in 97 and 96% ee with 18 and 53% conversion respectively. While the acetates had 49.3% de (trans), corresponding alcohols had 21.4% de (trans) (Scheme 3). This is the only DKR method known for diastereoselective synthesis of β-nitroalcohol. It used only one substrate and involved a chemo-enzymatic process.

Scheme 3: One-pot DKR with repeated interconversion and lipase based KR

HNL catalysed diastereoselective synthesis is a direct approach to synthesize β-nitroalcohol diastereomers. Hevea brasiliensis HNL (HbHNL) catalyzed nitroethane addition to benzaldehyde has produced a mixture of diastereomers of 2-nitro-1-phenylpropanol (NPP) in 67% yield, wherein the anti stereoisomer (1S,2R)- was found in 80% de with 95% ee (Scheme 4). HbHNL catalyzed diastereoselective synthesis has reported the use of only one substrate and hence is limited by poor substrate scope (Scheme 4) along with a 48 h long reaction time. Further the enzyme loading was also very high 4000 U/mmol of substrate
Scheme 4: HbHNL catalyzed diastereoselective Henry reaction

Two (R)-selective bacterial hydroxynitrile lyases, Acidobacterium capsulatum (AcHNL) and Granulicella tundricula (GtHNL) and their mutants were tested for the diastereoselective synthesis of (1R,2S)-NPP using benzaldehyde and nitroethane. Wild type (WT) AcHNL produced highest activity of 77% conversion, 73% ee and 6% de (Scheme 5). To improve the stereoselectivity, mutation was introduced into some specific positions of the amino acid chain in both the enzymes. The mutants of these metalloenzymes could not show much improvement in terms of stereoselectivity or % conversion. In the case of GtHNL (A40R, and A40H/A42T/Q110H) and AcHNL (A40H, A40R, and A40H/A42T/Q110H) variants, some produced anti product, i.e., (1R, 2S)-NPP, and some gave the syn, i.e. (1R,2R)-NPP. No mutant gave high conversion, % ee and % de of a β-nitroalcohol diastereomer. While GtHNL-A40H/A42T/Q110H gave maximum 78% conversion, it showed only 69% ee of (1R,2S)-NPP with −27% of de, i.e., syn major. A maximum 89% ee was found in the case of AcHNL-A40H/A42T/Q110H, but the conversion was only 21% and 24% de of anti major. Similarly, a maximum of 54% de, anti major was found in the case of AcHNL-A40H, but the % conversion was only 16.4, and 85.7% ee of (1R,2S)-NPP. Further, AcHNL and GtHNL variants catalyzed diastereoselective synthesis has been tested with only one substrate benzaldehyde using only one nucleophile, nitroethane.

Scheme 5: GtHNL and AcHNL muteins catalyzed diastereoselective Henry reaction

The halohydrin dehalogenase (HheC) catalyzed regio and enantioselective ring opening of epoxide by nitrite was tested with 2,3-epoxyheptane, which showed high regioselectivity in the nitrite attack to the sterically less crowded position to produce (2R,3R)-2-nitroheptan-3-ol in >99% ee and 42% yield (Scheme 6) [7]. The epoxide ring opening by nitrite is an example of kinetic resolution. It is limited by poor yield, requires additional step of substrate preparation, and has not been tested with more than one substrate.

Scheme 6: Halohydrin dehalogenase catalyzed ring opening by nitrite in the synthesis of β-nitroalcohol diastereomer.

This invention relates to the preparation of (R)-selective Arabidopsis thaliana hydroxynitrile lyase variants with improved activity towards stereoselective Henry reaction that allows the production of chiral -nitroalcohols having two chiral centers. It further includes the preparation of a range of different -nitroalcohol distereomers from their corresponding carbonyl substrates.

OBJECTIVE OF THE INVENTION

It is an object of the present invention to provide an improved hydroxynitrile lyase by substituting different amino acids in the amino acid sequence of a WT enzyme, which can catalyze the diastereoselective synthesis of ꞵ-nitroalcohols via the Henry reaction.

This new improved hydroxynitrile lyase should carry out promiscuous nitroaldol reaction efficiently with different nitroalkanes.

This mutated biocatalyst should also have a broad substrate range, allowing it to accept broad range of aromatic aldehydes with structural diversity.

SUMMARY OF THE INVENTION

The following summary is provided to facilitate a clear understanding of the new features in the disclosed embodiment and it is not intended to be a full, detailed description. A detailed description of all the aspects of the disclosed invention can be understood by reviewing the full specification, the drawing and the claims and the abstract, as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

The manner in which the present invention is formulated is given a more particular description below, briefly summarized above, may be had by reference to the components, some of which are illustrated in the appended drawing It is to be noted; however, that the appended drawing illustrates only typical embodiments of this invention and are therefore should not be considered limiting of its scope, for the system may admit to other equally effective embodiments.

Fig 1: Mutant screening towards the diastereoselective synthesis of 2-NPP by AtHNL wt and mutants.
Fig 2 Summary of optimization of biocatalytic parameters of Y14C catalyzed nitroaldol reaction using benzaldehyde and nitroethane as the substrate.
Fig 3: Mutant screening towards the diastereoselective synthesis of 2-NPB by AtHNL wt and mutants.
Fig 4: Summary of optimization of biocatalytic parameters of Y14A catalyzed nitroaldol reaction using benzaldehyde and 1-nitropropane as the substrate.
Fig: 5. Initial kinetic rate parameter determination of A. WT AtHNL and B. Y14C mutein towards the synthesis of (1R,2S)-2-NPP.
Fig: 6. Comparison of cyanogenesis and nitroaldolase activity of wt AtHNL, Y14C, and Y14A.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are constructed such that it provides a complete and thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.

Throughout this specification, various indications have been given as to preferred and alternative embodiments of the invention. It should be understood that it is the appended claims, including all equivalents, which are intended to define the spirit and scope of this invention.

This invention relates to the preparation of (R)-selective Arabidopsis thaliana hydroxynitrile lyase variants with improved activity towards diastereoselective Henry reaction that allows the production of -nitroalcohols having two chiral centers. It further includes the preparation of a range of different chiral -nitroalcohols from their corresponding carbonyl substrates.

The present invention uses two (R)-selective HNL mutein for the direct addition of nitroalkane into a carbonyl substrate in the diastereoselective synthesis of β-nitroalcohols (Scheme 7). The engineered enzymes showed high % conversion and stereoselectivity in the addition of nitroalkane nucleophiles into the carbonyl center and produced β-nitroalcohols with two chiral centers. This process is exemplified using a number of carbonyl substrates especially aldehydes, including molecules that could synthesize products of pharmaceutical significance. The current process also used more than one nucleophile, especially nitroalkane in the stereoselective addition to a carbonyl center and produced β-nitroalcohol diastereomers. Unlike the existing HNLs, the enzymes reported here have shown high selectivity with respect to both the substrates, i.e., aldehyde, and nitroalkane. The muteins also showed high conversion, high % ee, and high % de, i.e., up to >99%, while the major diastereomer was found to be of (1R,2S) configuration. The present invention also describes the discovery of two highly stereoselective enzymes with single mutation each in their amino acid sequence compared to the WT enzyme.

Scheme 7: Diastereoselective synthesis of ꞵ-nitroalcohols via Henry reaction by using AtHNL mutants as the catalyst.
AtHNL variant library screening towards promiscuous nitroaldol reaction to synthesize 2-nitro-1-phenyl propanol (2-NPP)
WT AtHNL catalyses the addition of nitromethane to benzaldehyde by forming a CC bond and synthesizes (R)-2-nitro-1-phenylethanol (NPE) but it failed to show such stereoselectivity when it was tested with nitroethane as a nucleophile in the asymmetric Henry reaction. So, to find an efficient mutant that can catalyse this promiscuous reaction, two site saturation mutant libraries of AtHNL created at Y14 and F179 position were screened using crude cell lysates. Four mutants (Y14C, Y14E, Y14G, and Y14H) of the Y14 library shown stereoselective synthesis of 2-NPP. These mutants selectively synthesized the (1R,2S) isomer of the product in 96, 53, 36.6, and 54.5% ee respectively (Fig 1). Except Y14C where 58% conversion was found, other variants did not show significant increase in the conversion compared to the WT enzyme. From the F179 series, only one variant, F179L showed 46.3% enantiomeric excess with 28% conversion. This study shows that the replacement of cysteine in place of tyrosine in the 14 position played a very crucial role in gaining the new catalytic activity by this enzyme. HbHNL, which catalyzed this same promiscuous reaction for the synthesis of (1S,2R)-2-NPP, also possesses cysteine and leucine at its 14 and 179 positions repectively.
Optimization of reaction conditions towards the stereoselective synthesis of (1R,2S)-2-NPP using AtHNL-Y14C as the catalyst
A thorough optimization of reaction conditions was carried out to maximize the production of (1R,2S)-2-NPP. Five different reaction parameters were systematically varied to attain the optimum biocatalytic reaction conditions for this reaction. After optimizing these parameters, the desired product was synthesized in 81% conversion with 97% ee and 95% de. We began the optimization experiment by testing the reaction in two different buffers potassium phosphate buffer (KPB) and citrate phosphate buffer (CPB) varying their pH from 5.5 to 7. Previous studies of HNL catalysed synthesis of 2-NPP also used KPB as buffer. While in the case of HbHNL, KPB pH 7.0 was used, [5] KPB pH 6.0 was the choice in AcHNL and GtHNL catalysed biocatalysis.[6] With increasing pH, the % ee of the (1R,2S) anti-isomer decreased both in case of KPB and CPB. At CPB pH 5.5, Y14C mutant showed 58.5% conversion with 95.3% ee and this buffer was chosen for further studies. HNL biocatalysis is commonly done in a biphasic reaction system, which consists of both nonpolar organic solvent and an aqueous buffer. Use of a biphasic system not only improves the substrate solubility but also helps to avoid substrate inhibition on enzymes. Previously tert-butyl methyl ether (TBME) was used as the organic solvent in the synthesis of 2-NPP using HbHNL[5], AcHNL, and GtHNL mutants.[6] In the WT AtHNL catalysed enantioselective synthesis of 2-nitro-1-phenylethanol, n-butylacetate (NBA) was found to be the best organic solvent.[8] In this optimization study, among the five solvents tested TBME gave the best result in terms of % conversion and % ee towards the synthesis of 2-NPP and showed 17% more conversion compared to n-butyl acetate. Nitroethane functions as the co-substrate in this diastereoselective biocatalysis along with the substrate benzaldehyde and its concentration was optimized in the subsequent experiment. AcHNL and GtHNL catalysed diastereoselective synthesis used 1 M nitroethane. [6] In this study, the Y14C variant showed increased activity and stereoselectivity with the increasing nitroethane concentration up to 1.75 M and decreased slightly in 2 M nitroethane. The nitroethane concentration optimisation study resulted in 78% conversion with 95.4% ee and 94% de of (1R,2S)-2-NPP. Optimization of the concentration of CPB in the diastereoselective synthesis was done using a range of buffer concentrations from 50 to 250 mM. Although Y14C variant showed a slight decrease in % conversion with the increasing buffer concentration, % ee increased in a reversible manner. With 100 mM buffer the highest, 97.6% enantiomeric excess of (1R,2S)-2-NPP was achieved with 77.8% conversion. To improve the product conversion further enzyme amount was optimized. By increasing the enzyme amount an increase in the activity was observed up to 15 mg and after that, the % conversion and % ee remained the same. (Fig: 2)
Substrate scope of AtHNL-Y14C in the diastereoselective synthesis of anti-isomer of different β-nitroalcohols using nitroethane as a nucleophile
Using the optimized biocatalytic reaction conditions, several aromatic aldehydes having substituents at different positions in the aromatic ring were converted into their corresponding (1R,2S) anti-diastereomer of β-nitroalcohols. This versatile substrate set contained both electron-donating and withdrawing groups, single and double substitutions in the aromatic ring, and substitutions were present at the ortho, meta, and para positions of the aromatic ring in different combinations (Table 1). When benzaldehyde was used as the substrate, the Y14C has produced its (1R,2S) stereoisomer of the β-nitroalcohol with 97.2% conversion, >99% ee and 98% de. Two other (R)-selective HNLs, GtHNL and AcHNL have been reported in the synthesis of this same stereoisomer from benzaldehyde.[6] WT AcHNL showed very poor diastereomeric excess (6%) with 73% ee and 77% conversion to (1R,2S)-NPP. Even after engineering, the AcHNL A40H gave only 27% de, 88% ee of the product, with a decreased conversion of 66%. WT HbHNL was reported to show such promiscuous reaction, however it produced (1S,2R)-NPP with 67% conversion, 95% ee, and 80% de.[5] Among the twenty aldehydes investigated by us in the Y14C catalyzed biocatalysis (Table 1), except benzaldehyde none of the others were studied earlier by any HNL towards Henry reaction based diastereoselective synthesis of β-nitroalcohols. Biocatalytic synthesis of eighteen out of the nineteen (1R,2S)- isomers of β-nitroalcohols are reported for the first time, while there exist only a single biocatalytic approach, i.e., Pseudomonas flourescens lipase catalyzed KR to produce 1-(4-chlorophenyl)-2-nitropropanol in 77% conversion. Unfortunately, the stereoselectivity of this KR product was not specifically mentioned.
Except 4-methoxybenzaldehyde (Table 1), Y14C catalyzed the conversion of all the tested aldehydes to their corresponding diastereoselective β-nitroalcohol products. Irrespective of the presence of electron donating or withdrawing group in the aldehyde, Y14C has displayed excellent stereoselectivity. For example, electron donating group substituted aldehydes like 3-methoxy or 3-methylbenzaldehyde showed 74 and 73% conversion with 98.2% and 98.6% ee of their (1R,2S)- isomers respectively. Similarly 3-chlorobenzaldehyde possessing electron withdrawing group (negative inductive effect) also showed very good conversion of 93% with >99% ee of its (1R,2S)- isomer. The position of substitution in the aromatic ring also did not obstruct the stereoselectivity of the product. More than 70% conversion was found with eleven aldehydes, with another six, moderate to good conversion was observed while 92-99% ee of the (1R,2S)- isomers of β-nitroalcohols were obtained with fifteen aromatic aldehydes. We also checked whether Y14C can accommodate aldehydes having bulky substitutions or double substitution in their aromatic ring and found that bulky substitutions hamper the % conversion significantly. For example, 1-napthaldehyde, 3-benzyloxybenzaldehyde and 2,5-dimethoxybenzaldehyde showed only 54, 40, and 18.5% conversion respectively. Increase in the number of carbons in the alkyl chain of the aldehyde affected the catalytic Henry reaction activity of Y14C severely. 3-Phenylpropionaldehyde, which has two more carbons in the alkyl chain showed only 5% conversion of the product with only 41% ee of its (1R,2S)- isomer. (Table 1)
AtHNL variant library screening towards promiscuous nitroaldol reaction to synthesize 2-nitro-1-phenyl butanol (2-NPB)
To find an efficient mutant to catalyze the promiscuous Henry reaction between benzaldehyde and 1-nitropropane as a nucleophile in the synthesis of 2-NPB, Y14 and F179 mutant libraries were screened. Crude cell lysate of each variant was used as the catalyst to carry out this promiscuous Henry reaction. In the Y14 series, five mutants Y14A, Y14C, Y14L, Y14N, and Y14S showed improved activity compared to the WT AtHNL, while none was found from the F179 series. The % conversion to 2-nitro-1-phenyl butanol by Y14A, Y14C, Y14L, Y14N, and Y14S were found to be 6.2, 7.3, 10.9, 3.6, and 3.3% respectively compared to 0.7% by the WT enzyme. (Fig 3) As the % conversion by these mutants was very poor, their efficiency was further checked by their purified form.
Stereoselective synthesis of 1-nitro-3-phenyl butanol using purified AtHNL mutants
Five mutants that showed better performance than the WT AtHNL were purified and used in the stereoselective biocatalysis. With purified enzyme, the % conversion increased up to 8 times compared to the crude enzyme. All five AtHNL mutants showed stereopreference for the (1R,2S) isomer of the product with >99% ee and >99% de. However, the % conversion obtained by these variants was not so impressive. The Y14A, Y14C, Y14L, Y14N, and Y14S showed 37.1, 28.6, 31, 20, and 24% conversion respectively in the synthesis of 1-nitro-3-phenyl butanol. To gain higher % of conversion in the synthesis of (1R,2S)-2-NPB, Y14A was selected and reaction engineering was performed.
Optimization of reaction conditions towards the stereoselective synthesis of (1R,2S)-2-NPB using AtHNL-Y14A
To improve the % conversion to (1R,2S) isomer of 1-nitro-3-phenyl butanol by AtHNL-Y14A, the amount of purified enzyme and reaction time were optimized. With the increasing amount of enzyme up to 6 mg, Y14A showed enhanced % conversion, while a further increase in the enzyme content to 10 mg did not produce any significant increase in the % conversion. Using 6 mg of Y14A pure enzyme, the 72 h reaction showed 59% conversion, >99% ee and >99% de. In our previous experiments with nitroethane, we experienced a 9% increase in the % conversion when its concentration was optimized. Towards the NPB synthesis, we optimised the biocatalysis by varying five different concentrations, 0.25, 0.6, 1, 1.4, and 1.75 M of 1-nitropropane. The Y14A biocatalysis showed increased % conversion with increasing 1-nitropropane concentration up to 1.4 M, beyond that it didn’t increase further. It produced 48.4% conversion of (1R,2S)-2-NPB with >99% ee and >99% de after 24 h. (Fig 4)
Substrate scope of AtHNL-Y14A in the diastereoselective synthesis of anti-isomer of different β-nitroalcohols using 1-nitropropane as a nucleophile
To prove the broad substrate scope of AtHNL-Y14A towards the diastereoselective synthesis, fourteen different aromatic aldehydes were selected and studied to obtain their corresponding nitrophenyl butanol derivatives. (Table 2) Similar to Y14C mutein, Y14A also displayed excellent stereoselectivity in the synthesis of the desired product for most of the tested substrates, where up to 90% conversion, >99% ee and > 99% de was achieved. Only in case of five substrates poor (<30%) conversion was observed. Except for 4-nitrobenzaldehyde and 3-phenylpropionaldehyde, which showed very poor stereoselectivity and no activity respectively, all other substrates showed excellent stereoselectivity from 93 to >99% ee and up to 75% conversion. (Table-2)
Determination of the kinetic parameters of Y14C mutant towards the diastereoselective Henry reaction
The steady state kinetic parameters of Y14C towards the synthesis of (1R,2S) isomer of 2-NPP was carried out by measuring the initial velocities against different substrate concentrations, measured by a chiral HPLC. The KM and Vmax of the Y14C mutant were found to be 25.1 mM and 0.4 µmol/min respectively. The catalytic efficiency (kcat/KM) of this mutant was found to be 0.3 mM−1min−1, which is 259 times greater than that the WT enzyme. The WT AtHNL had a catalytic efficiency of only 0.00116 mM−1min−1, however without any stereoselectivity (Fig 5).
Comparison of cyanogenesis and nitroaldolase activity of wt AtHNL, Y14C, and Y14A
The two variants, Y14C and Y14A, which showed improved activity in the promiscuous stereoselective Henry reaction with nitroethane and 1-nitropropane respectively were used in their purified form to measure their cleavage activity and compared with their WT form. In the cyanogenesis of mandelonitrile, Y14C and Y14A showed a specific activity of 94.3 and 76 U/mg respectively as compared to 68 U/mg by the WT AtHNL. Similarly, three different racemic ꞵ-nitroalcohols, 2-NPE, 2-NPP, and 2-NPB were selected and their cleavage activity was measured by the three enzymes. Wild type AtHNL showed almost similar activity towards all the three substrates. The Y14C and Y14A showed almost 7 and 2.3 times more specific activity compared to WT AtHNL towards 2-NPP and 2-NPB respectively. However, both the mutants showed similar poor activity like WT AtHNL, when 2-NPE was used as the substrate. This conveys that in case of Y14C, the Henry product corresponding to nitroethane fits best in its active site, while with Y14A the corresponding Henry product from 1-nitropropane fits best, which eventually shows highest activity in the biocatalysis. This study clearly states that these two single mutations not only gained promiscuous Henry reaction activity for nitroethane and 1-nitropropane but also improved their natural cyanogenesis activity.
Gram scale synthesis of (1R,2S)-2-NPP by using Y14C crude enzyme as the catalyst
From the optimization studies it was evident that the mutant Y14C can be efficiently used in its crude form. We aimed to carry out a large-scale synthesis of the anti diastereoisomer, (1R,2S)-2-NPP using the Y14C crude cell lysates. The preparative scale synthesis included 100 mM benzaldehyde (1.051 mL) in 6.8 mL of 1.5 M CPB pH 5.5, 49.5 mL of TBME, 13 mL of nitroethane, 24 mL AtHNL-Y14C crude enzyme (50.8 mg/mL) as the catalyst and 6.7 mL of water. The reaction mixture was incubated at room temperature for 24 h. The product was extracted by ethyl acetate and column purified using hexane: ethyl acetate (95:5 v/v). The product was formed with 55% isolated yield. The % ee of this final product was 98% and the de was >99%. This is the first report where large-scale synthesis of (1R,2S)-2-NPP has been executed by any biocatalytic means. The only report till date to synthesize this molecule in an analytical scale was through AcHNL- A40H mutant enzyme, where after 24 h 66% product conversion was observed starting from 20 µmol of the substrate and 2.5 mg of pure enzyme. The % ee and % de was also very poor i.e., 88% and 27% respectively . This gave a TTN of 33, whereas our gram scale synthesis showed a TTN of 3,438 which is >100 times greater than the AcHNL mutant result.
Table-1. Y14C mutein catalyzed synthesis of enantioenriched (1R,2S)-ꞵ-nitroalcohols from corresponding achiral aromatic aldehydes using nitroethane as a nucleophile.
S. No. Substrate name Time (h) Total % conversion % Enantiomeric excess for RS product % Enantiomeric excess for RR product % Diastereomeric excess for anti product Isomeric Content for RS product Isomeric Content for RR product
1 2-nitrobenzaldehyde 24 >99 95.2 22.7 74.7 85.3 7.7
2 2,3-dichloro benzaldehyde 16 97.3 98.5 65.9 95.4 97.0 1.9
3 2-bromo benzaldehyde 24 >99 97.8 66.0 92.5 95.2 3.1
4 2-chloro benzaldehyde 16 97.5 98.6 61.0 88.7 93.7 4.6
5 3-chloro benzaldehyde 16 92.9 >99 0 >99 >99 0.0
6 4-nitro benzaldehyde 16 95.5 4.4 11.2 2.3 26.7 27.16
7 2-methoxy benzaldehyde 24 89.7 98.2 85.7 75.9 87.2 11.2
8 3-bromo benzaldehyde 16 83.6 >99 >99 98.4 98.9 0.8
9 Benzaldehyde 8 79.1 >99 0 >99 >99 0
10 3-methoxy benzaldehyde 24 73.9 98.2 82.8 97.7 98.0 1.1
11 3-methyl benzaldehyde 24 73.0 98.6 1.5 98.1 98.4 0.5
12 3-benzyloxy benzaldehyde 24 40.0 >99 88.0 96.8 98.2 1.5
13 2-methyl benzaldehyde 24 37.9 >99 70.4 91.7 95.4 3.5
14 4-chloro benzaldehyde 24 35.5 97.5 3.0 82.4 90.1 4.5
15 1-napthaldehyde 48 54.1 92.3 80.6 73.4 83.3 12.0
16 4-methyl benzaldehyde 24 27.4 96.9 -14.0 90.6 93.8 2.0
17 2,3-dimethoxy benzaldehyde 24 21.7 51.1 28.0 34.8 50.9 20.9
18 2,5-dimethoxy benzaldehyde 24 18.5 52.8 2.1 27.2 48.6 18.6
19 3-phenyl propionaldehyde 24 4.9 41.3 31.8 −12.0 31.1 36.9
20 4-methoxy benzaldehyde 24 No conversion

Detailed reaction conditions (1.25 mL): 600 µL TBME, 5 mM different aldehyde substrate (25 µL, 250 mM), 1.75 M nitroethane (161 µL), 100 mM CPB pH 5.5 (83 µL, 1.5 M), 3 mg purified Y14C enzyme and the remaining volume of autoclaved double distilled water. Different aromatic aldehyde stock solutions were made in TBME. This reaction mixture was incubated at 30 °C and 1,200 rpm in a Thermo shaker. A 100 μL of an aliquot of the reaction was mixed with 200 μL of hexane: 2-propanol (90:10). The mixture was vortexed vigorously, followed by drying over anhydrous sodium sulfate to remove any aqueous part, and then centrifuged at 15000 g for 5 min at 4 °C. A 20 μL of this sample was taken and analyzed in HPLC using appropriate chiral column.% Conversion, % enantiomeric excess, % diastereomeric excess, and % isomeric content were calculated by using following formula –

Table 2: Y14A mutein catalyzed synthesis of enantioenriched (1R,2S)-ꞵ-nitroalcohols from corresponding achiral aromatic aldehydes using nitropropane as a nucleophile.
Sl. no. Substrate name Time (h) Total %
conversion Enantiomeric excess for RS product Enantiomeric excess for RR product Diastereomeric excess for anti product Isomeric Content for RS product Isomeric Content for RR product
1 4-nitrobenzaldehyde 72 90.3 1.7 -4.3 5.0 26.7 22.7
2 3-chlorobenzaldehyde 24 74.8 100 0 100 100 0
3 2-furaldehyde 24 63.1 96.9 - 67.6 82.5 -
4 2,3-dichlorobenzaldehyde 24 57.8 93.1 15.1 80.1 86.9 5.7
5 2-nitrobenzaldehyde 48 52.3 90.3 0 100.0 95.2 0
6 Benzaldehyde 24 51 100 0 100 100 0
7 2-bromobenzaldehyde 24 47.1 96.4 0 100.0 98.2 0
8 3-methoxybenzaldehyde 24 41.8 100 0 100 100 0
9 3-bromobenzaldehyde 24 37.3 98.0 0 100.0 99.0 0
10 2-methoxybenzaldehyde 24 15.2 96.1 - 79.9 88.2 -
11 4-methylbenzaldehyde 48 8.1 100 0 100 100 0
12 3-methylbenzaldehyde 24 7.8 100 0 100.0 100.0 0
13 4-chlorobenzaldehyde 48 6.0 100 13 30.9 65.5 19.5
14 3-phenylpropionaldehyde 48 trace - - - - -

Reaction conditions (1.25 mL): 600 µL TBME, 5 mM different aldehyde substrate (25 µL, 250 mM), 1.4 M nitropropane (156 µL), 100 mM CPB pH 5.5 (63 µL, 2 M), 6 mg purified Y14A and the remaining volume of autoclaved double distilled water. Substrate stock preparation and other reaction parameters were kept similar to that explained earlier. Product extraction and HPLC were done according to the previously described method. % Conversion, % enantiomeric excess, % diastereomeric excess, and % isomeric excess were calculated by using similar formula mentioned above. 
, C , Claims:5. CLAIMS
I/We Claim:
1. Preparation of β-nitroalcohols with two chiral centers using AtHNL mutein, Y14X or F179X, in any one of the following form, i.e., (iv) to (vi);
where (a) X is any amino acid other than that is present in the corresponding position in the wild type enzyme, and (b) AtHNL mutein is described in (i) to (iii) below,
(i) A polypeptide having amino acid sequence as shown in SEQ ID NO: 1, Fig 7.
(ii) A hydroxynitrile lyase gene as shown in SEQ ID NO: 2, Fig 8, encoding the hydroxynitrile lyase of amino acid sequence of claim 1.(i).
(iii) A plasmid comprising the gene of claim 1. (ii) contained in a vector.
(iv) A transformant obtained by introducing the recombinant vector of claim 1. (iii) into a host.
(v) A culture obtained by culturing the transformant of claim 1. (iv).
(vi) A hydroxynitrile lyase obtained from the culture of claim 1. (v).

2. A mutant mentioned in the claim 1 in one of the form as described in claim 1. (iv) to 1. (vi) has exhibited nitroaldolase, i.e., synthesis of β-nitroalcohol or retro-nitroaldolase activity, i.e., cleavage of β-nitroalcohol or hydroxynitrile lyase activity, i.e., cleavage of a cyanohydrin.
3. A method for stereoselective synthesis of optically active β-nitroalcohols comprising of
(i) a carbonyl compound and a nitroalkane in the presence of a AtHNL mutants in any form as described in claim 1. (iv) to 1. (vi), in a biphasic medium
(ii) the biphasic medium in the previous claim consists of CPB and TBME as an organic solvent
(iii) the carbonyl compound used as a substrate has the formula R-CHO where R= aromatic and substituted aromatic/ PhCH2OPh/bicyclic/furan ring/PhCH2CH2.
(iv) The nitroalkane used as the second substrate has the formula RCH2NO2 where R= CH3/an alkyl chain of higher carbon.

4. A AtHNL mentioned in claim 1 catalyzed process as described in claim 3, that synthesized optically active β-nitroalcohols having two chiral centers of the formula R1CH(OH)CH(R2)NO2 where R1= aromatic and substituted aromatic/ PhCH2OPh/bicyclic/furan ring/PhCH2CH2 and R2= CH3/an alkyl chain of higher carbon, where the absolute configuration of the two chiral centers are same or different.
5. These mutated enzymes showed a TTN (Total turnover number) up to 3,438 towards the synthesis of 2-NPP.

6. DATE AND SIGNATURE

Dated this 27th April 2023

Signature

(Mr. Srinivas Maddipati)
IN/PA 3124
Agent for applicant.