Description:DESCRIPTION

FIELD OF INVENTION
The present invention relates to the development of a process of use of Baliospermum montanum hydroxynitrile lyase (BmHNL) in the stereoselective synthesis of optically pure ꞵ-nitroalcohols. More particularly the present invention includes the use of BmHNL in the stereoselective synthesis of ꞵ-nitroalcohols where one or more chiral centers are present.
BACKGROUND OF THE INVENTION

Optically pure β-nitroalcohols are important chiral synthons for the synthesis of various enantiopure drugs, biologically active molecules and natural products[1-3]. Henry reaction or nitroaldol reaction is an important transformation that involves a carbon-carbon bond formation, coupling between a nucleophilic nitroalkane with an electrophilic aldehyde (or ketone) to form β-nitroalcohol. In organic chemistry, several metals and non-metal-based catalysts have been reported in the asymmetric synthesis of β-nitroalcohols, however by virtue of their chemical nature they do not serve as green and ecofriendly catalysts. Due to their environment friendly nature and high selectivity, enzyme catalysts have lured pharma industries in recent years.
The existing biocatalytic routes for the synthesis of enantiopure β-nitroalcohols are kinetic resolution, retro-Henry reaction, dynamic kinetic resolution, asymmetric reduction, and asymmetric Henry reaction. The latter one represents the most important biocatalytic route to synthesize chiral β-nitroalcohols and uses HNLs as catalysts. In addition to their natural enantioselective hydrocyanation activity, a few HNLs catalyze promiscuous stereoselective Henry reaction. Two (S)-selective HNLs from Hevea brasiliensis (HbHNL) and BmHNL and three (R)-selective HNLs from Arabidopsis thaliana (AtHNL), Granulicella tundricola (GtHNL) and Acidobacterium capsulatum (AcHNL) are reported to catalyze the stereoselective nitroaldol reaction. Until recently, HbHNL is the only (S)-selective HNL reported to catalyze promiscuous Henry reaction in one step starting from the achiral aldehyde. It catalyzes the carboligation reaction between aromatic aldehydes and both nitromethane and nitroethane and produced various β-nitroalcohols with good % ee and % de. Baliospermum montanum HNL (BmHNL), earlier reported for cyanohydrin synthesis was recently combined in a cascade along with Horse liver alcohol dehydrogenase, i.e., HLADH-BmHNL to synthesize ten different aromatic (S)-β-nitroalcohols from their corresponding primary alcohols. Despite the use of two enzymes, yield of the products did not significantly improve. Overall, HNL catalyzed enantioselective and diastereoselective synthesis of chiral β-nitroalcohols suffer with (i) less number of available biocatalysts (HNLs), (ii) poor substrate scope and (iii) long reaction time. The current invention addresses these challenges using BmHNL catalyzed stereoselective Henry and retro-Henry reaction.
HbHNL is the only (S)-selective HNL tested so far towards the enantioselective synthesis of (S)-β-nitroalcohols by Henry reaction, using nitromethane as the co-substrate. The biocatlytic reaction was carried out in citrate phosphate buffer (CPB) pH 5.5 using t-butyl methyl ether (TBME) as the organic solvent, incubated for 48 h. Among fourteen aromatic and aliphatic aldehydes used in this study, twelve of them produced corresponding (S)-β-nitroalcohols with 13 – 57% conversion and 64-99% ee (Scheme 1)

Scheme 1: HbHNL-catalyzed enantioselective nitroaldol reaction.
Additionally, HbHNL was also tested for its diastereoselective synthesis of β-nitroalcohol (Scheme 2). It catalysed the Henry reaction between benzaldehyde and nitroethane in a biphasic reaction system, where TBME was used as the organic solvent acquiring 50% of the reaction mixture. Phosphate buffer pH 7 was used as the reaction buffer and the reaction was incubated up to 48 h. HbHNL preferentially synthesized the anti (1S,2R) isomer of 2-nitro-1-phenylpropanol (2-NPP) as the major product with 67% isolated yield with 95% ee and 80% de.

Scheme 2: HbHNL catalyzed diastereoselective nitroaldol reaction.
The HNL catalyzed Henry reaction for the synthesis of enantiopure (S)-β-nitroalcohols is restricted to only one enzyme, i.e., HbHNL. The HbHNL catalyzed synthesis uses a high enzyme: substrate ratio, where 8.99 U of enzyme was used per µmol of the substrate, and it takes a long reaction time of 48 h. The yields of the chiral β-nitroalcohols were limited to 13-57% only. An acyl-peptide releasing enzyme from Sulfolobus tokodaii (ST0779) is known to catalyze enantioselective synthesis of β-nitroalcohols, however, the catalyst does not show uniform enantiopreference; rather, its enantioselectivity varies with the electronic effects of the substituents on the benzaldehyde ring.
Similarly, the HNL catalyzed diastereoselective β-nitroalcohol synthesis is confined to one biocatalyst i.e., HbHNL to obtain the (1S,2R)-stereo isomers. The HbHNL catalyzed synthesis of (1S,2R)-2-NPP uses 4495 U of enzyme for every mmole of benzaldehyde and the reaction time is 48 h long. Moreover, HbHNL catalyzed diastereoselective β-nitroalcohol synthesis was tested with only one substrate benzaldehyde. Although three nucleophiles other than nitromethane were studied in the HbHNL catalyzed diastereoselective Henry reaction, in case of 2-nitropropane it produced enantioselective product while no stereoselectivity and conversion were found with phenylnitromethane. Hence its diastereoselectivity is also limited to one nucleophile, nitroethane.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a process for the production of optically active ꞵ-nitroalcohols using BmHNL.
Another objective of this invention is to demonstrate the use of BmHNL as a catalyst in the stereoselective C−C bond formation reaction and synthesize chiral ꞵ-nitroalcohols having one or more chiral centers.
Another objective of this invention is to find the use of BmHNL in stereoselective Henry or retro-Henry reaction, where BmHNL exhibits large substrate scope towards Henry reaction, accept a broad range of aldehydes and nitroalkanes, while a wide range of racemic ꞵ-nitroalcohols are used as substrates in the retro-Henry reaction.
Yet another objective of this invention is to determine the stability of BmHNL in the biocatalytic reaction conditions, and its reusability in the synthesis of chiral ꞵ-nitroalcohol.
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.
The features and advantages of the present proposed system will become more apparent from the following detailed description along with the reference numerals
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 is 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.
The features and advantages of the present invention will become more apparent from the following detailed description along with the accompanying figures, which forms a part of this application and in which:
Fig 1 Summary of optimization of biocatalytic parameters of BmHNL catalyzed retro-Henry reaction using racemic β-nitroalcohols as substrates in accordance to the present invention;
Fig 2 Summary of optimization of biocatalytic parameters of BmHNL catalyzed nitroaldol reaction using benzaldehyde and nitroethane in accordance to the present invention;
Fig 3 Determining the tolerable substrate concentration towards the diastereoselective synthesis of 2-NPP using BmHNL as the biocatalyst in accordance to the present invention;
Fig 4 Summary of optimization of biocatalytic parameters of BmHNL catalysed nitroaldol reaction using benzaldehyde and 1-nitropropane as the substrates in accordance to the present invention;
Fig 5 Michaelis-Menten plot for determination of kinetic rate parameters of wt BmHNL catalysed synthesis of (1S,2R)-2-NPP in accordance to the present invention;
Fig 6 A comparison of BmHNL’s cyanogenesis vs. retro-nitroaldolase activity by spectrophotometric cleavage assay in accordance to the present invention;
Fig 7 Depicts the high stability in the reaction condition in accordance to the present invention;
Fig 8 Percentage of remaining biocatalytic activity of BmHNL in the enzyme recycling experiment in accordance to the present invention;
DETAILED DESCRIPTION OF THE INVENTION

The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.
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 a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.
The invention relates to the development of a process where Baliospermum montanum hydroxynitrile lyase (BmHNL) has been used in the preparation of optically pure ꞵ-nitroalcohols by enantioselective synthesis and cleavage. It also includes BmHNL’s catalytic potential in the diastereoselective synthesis of ꞵ-nitroalcohols where more than one chiral center is present. To date, HbHNL is the only HNL reported to show this nitroaldol activity. HbHNL was tested with 14 aldehyde substrates for enantioselective synthesis where it showed 13-57% conversion with 64-99% ee of (S)-ꞵ-nitroalcohols. For the diastereoselective synthesis, only benzaldehyde was tested and it showed 67% yield with 95% ee and 80% de of the (1S,2R)- 2-nitro-1-phenylpropanol (NPP). In the present study, BmHN tested with twenty three aldehydes for its enantioselective synthesis of (S)-ꞵ-nitroalcohols showed up to >99% conversion and 99% ee. BmHNL on enantioselective cleavage of twenty two racemic ꞵ-nitroalcohols produced the enantiocomplementary products, i.e., (R)-ꞵ-nitroalcohols with up to 49.9% conversion and up to >99% ee. BmHNL was studied in the diastereoselective synthesis of nitroaldol products using twenty different aldehydes and two bulky nitroalkanes (nitroethane and 1-nitropropane) that resulted in the production of (1S,2R) isomers of thirty three different ꞵ-nitroalcohols with conversion, % ee, and % de up to >99%. This data showed BmHNL’s broad substrate scope, nucleophile promiscuity, and high stereoselectivity in nitroaldol synthesis, which is not found in case of HbHNL. BmHNL was found to stable for up to 2 months with a loss of only 14% of its activity over this time. This enzyme was tested for its recyclability where it could retain 82% of its activity after a consecutive 20 cycles of reaction, leading to a TTN of 37,170 compared to only 267 for HbHNL towards the same reaction. By recycling the enzyme, gram-scale synthesis of (1S,2R)-NPP was carried out starting from benzaldehyde and nitroethane with improved yield and stereoselectivity compared to HbHNL catalyzed reaction. This enzyme was also found to synthesize the (1S,2S)- syn stereoisomers of ꞵ-nitroalcohols when reaction time was extended while a shorter reaction time produced the (1S,2R)- anti stereoisomers.
To explore BmHNL’s applicability in the enantioselective Henry reaction a diverse set of 23 substrates including both aromatic and heteroaromatic aldehydes (Table 1) were investigated for the synthesis of enantiopure (S)-β-nitroalcohols (Scheme 3). Eleven of them were tested for the first time by HNL biocatalysis. The reactions were carried out using our previously optimized reaction conditions, where BmHNL was used in a cascade reaction. BmHNL produced high % conversion and enantiomeric excess for all the tested substrates in contrast to previously reported enzymes and methods. The aldehydes contained substitutions at the ortho-, meta- and para-positions of the phenyl ring. Substrates with more than one substitution were also included. The nature of substituents on the aldehyde were of both electron donating and electron withdrawing. Irrespective of the electronic nature and position of the substitution on the phenyl ring, high % conversion and ee of corresponding (S)-β-nitroalcohols were obtained for most of the aldehydes used (Table 1). Among the substrates investigated, nitro-substituted meta- and para-benzaldehydes displayed the highest % conversion of >99. In case of di-substituted benzaldehydes, 2,6-dichloro, 2,5-dimethoxy, 3,5-dichloro, and 2,4-dimethoxy benzaldehydes 96.4, 87.9, 75.8, and 69.2% conversion respectively were obtained with excellent enantioselectvity ranging from 97.5 to > 99%. Three bulky aldehydes (S. No. 9, 18, and 19, Table 1) could not be converted efficiently to corresponding (S)-β-nitroalcohols, the % conversion was between 22 to 53 only, however they showed good enantioselectivity of 86 to >99%. Another two bulky aldehydes, 1 or 2-napthaldehyde had a slight higher % conversion of 62 to 68% with 98.6% ee. Another two longer chain aldehydes, trans-cinnamaldehyde and 3-phenylpropionaldehyde showed almost 70% conversion, but the later showed poor % ee.

Scheme 3: BmHNL catalyzed Henry reaction in the enantioselective synthesis of (S)-β-nitroalcohols
Table 1: BmHNL-catalyzed Henry reactions between different aromatic aldehydes and nitromethane.
S. No. R time (h) % conversion % ee
1. 2,6-diClC6H3 24 96.4 >99
2. 3,5-diClC6H3 32 75.8 98.6
3. 3-OHC6H4 8 73.4 >99
4. 2,5-diOMeC6H3 24 87.9 97.4
5. 2,4-diOMeC6H3 16 69.2 >99
6. 4-OMeC6H4 8 61.8 93.6
7. 3-OMeC6H4 8 81 94.3
8. 2-OMeC6H4 8 88.9 86.2
9. 4-PhC6H4 24 43.7 >99
10. 4-BrC6H4 24 81.4 97.8
11. 4-MeC6H4 8 75 87.4
12. 3-MeC6H4 8 72.7 92
13. 2-MeC6H4 8 63.5 81.5
14. *4-ClC6H4 24 77.2 94.9
15. 3-ClC6H4 8 68 93
16. 4-NO2C6H4 8 >99 92.7
17. 3-NO2C6H4 16 >99 90.4
18. 3-PhCH2OC6H4 24 22.1 95.2
19. 3-PhOC6H4 40 53.3 86.5
20. 1-Naphthyl 16 62.4 98.6
21. 2-Naphthyl 24 68.3 98.6
22. *PhCH=CH 32 67.4 98
23. PhCH2CH2 24 70.5 56.4
* Indicates the final substrate concentration was 10 mM.
The existing biocatalytic synthesis of enantiopure (S)-β-nitroalcohols uses two main approaches. They are (a) kinetic resolution or retro-Henry reaction by (i) wt-AtHNL, (ii) celite AtHNL, (iii) lipase PS-IM starting from racemic ꞵ-nitroalcohol, and (b) nitroaldol reaction using (i) HbHNL using aldehydes as substrates, (ii) HLADH-BmHNL cascade starting from achiral aromatic alcohols. We observed that twelve among the 23 substrates have been studied by one or more of the above approaches. The kinetic resolution approach had a maximum conversion of ~50%, while HbHNL was reported for giving only 13% conversion with 66 % ee in case of 3-phenylpropionaldehyde. In contrary our BmHNL catalyzed nitroaldol reaction produced excellent conversion of 62 to >99% and 81 to >99% ee for twenty of the 23 substrates in their corresponding (S)-β-nitroalcohol synthesis (Table 1).
Experimental: A typical BmHNL catalysed enantioselective nitroaldol reaction consisted of 475 μL of TBME, 25 mM aldehyde, 30 U of BmHNL, 100 mM CPB pH 5.5, 1.5 M nitromethane, and 50 mg/mL sorbitol. The total volume of the reaction mixture was made up to 1 mL by adding the remaining volume of autoclaved double distilled water. This reaction mixture was incubated in a thermomixer at 30 °C at 1200 rpm.
2. Reaction condition optimization for BmHNL catalyzed retro-Henry reaction
As mentioned before HbHNL is the only (S)-selective HNL reported to synthesize enantiopure (S)-β-nitroalcohols using Henry reaction. Its application in retro-nitroaldol reaction is not explored yet. A HNL catalyzed retro-nitroaldol reaction often produces a chiral β-nitroalcohols having opposite stereoselectivity of the HNL. This inspired us to explore the retro-nitroaldol activity of BmHNL to prepare various (R)-β-nitroalcohols. For this purpose, 2,6-dichloro NPE, which showed excellent % conversion and % ee (96.4 % conv. with >99 % ee) in BmHNL catalyzed enantoselective synthesis of (S)-β-nitroalcohols, was chosen as the model substrate. As it has been observed that HNL catalyzed cleavage reactions are fast and efficient compared to synthesis reactions, so initially cleavage reaction was carried out using low enzyme content (12.5 U) compared to the enzyme amount used in the synthesis reaction (30 U). Unfortunately, the reaction showed negligible cleavage activity. So, to enhance the cleavage activity the enzyme-to-substrate ratio was increased 13 times, which improved the % conversion to 32.8 % with 55.4 % ee after 24 h. To improve the results further substrate concentration was decreased to 3 mM, while enzyme concentration was also doubled. 40 U of BmHNL with 3 mM enzyme showed 36.7% conversion with 63% ee. Increasing enzyme quantity to 80 U also did not improve the % conversion and % ee significantly. Hence, in the subsequent optimization, a change in substrate was explored to gain higher selectivity. Three other substrates, 3-methoxy NPE or 3 methyl NPE or 3,5-dimethoxy NPE, which showed good % conversion and % ee in the enantioselective synthesis were tested here. Except for 3,5-dichloro NPE, the other two substrates showed >99 % ee within 16 h. Hence, 40 U enzyme for every 3 mM substrate was considered as the optimized reaction condition to prepare different (R)-β-nitroalcohols from their corresponding racemic β-nitroalcohols.
Experimental: BmHNL catalyzed retro-Henry reaction involved the following set of experiments.
First optimization of enzyme to substrate ratio: A typical reaction consisted of TBME 480 µL, 12.5 U BmHNL, 100 mM CPB pH 5.5 (100 µL, 1 M), 50 mg/mL Sorbitol (63 µL, 0.8 g/mL), and 20 mM 2,6-dichloro NPE (20 µL, 1 M), incubated at 30° C, 1200 rpm. Here enzyme to substrate ratio was 12.5 U: 20 mM. Racemic substrate stock was prepared using TBME as the solvent. Total reaction volume was made up to 1 mL by adding remaining volume of double distilled water.
Second optimization of enzyme to substrate ratio: A typical reaction consisted of TBME 480 µL, 40 U BmHNL, 100 mM CPB pH 5.5 (100 µL, 1 M), 50 mg/mL Sorbitol (63 µL, 0.8 g/mL), and 5 mM 2,6-dichloro NPE (20 µL, 0.25 M), incubated at 30° C, 1200 rpm. Here enzyme to substrate ratio was 40 U: 5 mM. Racemic substrate stock was prepared using TBME as the solvent. Total reaction volume was made up to 1 mL by adding remaining volume of double distilled water.
Third optimization of enzyme to substrate ratio: A typical reaction consisted of TBME 488 µL, 40/80 U BmHNL, 100 mM CPB pH 5.5 (100 µL, 1 M), 50 mg/mL Sorbitol (63 µL, 0.8 g/mL), and 3 mM 2,6-dichloro NPE (12 µL, 0.25 M), incubated at 30° C, 1200 rpm. Here enzyme to substrate ratio was 40/80 U: 3 mM. Racemic substrate stock was prepared using TBME as the solvent. Total reaction volume was made up to 1 mL by adding remaining volume of double distilled water.
Optimization using different substrates: A typical reaction consisted of TBME 488 µL, 40 U BmHNL, 100 mM CPB pH 5.5 (100 µL, 1 M), 50 mg/mL Sorbitol (63 µL, 0.8 g/mL), and 3 mM different racemic substrate, i.e., 3-methoxy NPE or 3 methyl NPE or 3,5-dimethoxy NPE (12 µL, 0.25 M), incubated at 30° C, 1200 rpm. Racemic substrate stock was prepared using TBME as the solvent. Total reaction volume was made up to 1 mL by adding remaining volume of double distilled water.
3. BmHNL catalysed enantioselective C−C bond cleavage in the production of different (R)‐β‐nitroalcohols
To explore the substrate scope of this BmHNL catalyzed retro-Henry reaction, 22 racemic β-nitroalcohols, whose corresponding aldehydes were used in Table 1, were used in the preparation of corresponding (R)-β-nitroalcohols (Scheme 4, Table 2). Surprisingly, among the twenty two substrates studied, nineteen of them could undergo retro-nitroaldol reaction with high enantioselectivity of 72 to >99% ee and 32-50% conversion except, 2-Me-NPE. This depicts the high stereoselectivity and broad substrate scope of BmHNL towards retro-nitroaldol reaction. This is the first report where a single enzyme is used to prepare a large number of (R)‐β‐nitroalcohols. The earlier report of AtHNL catalyzed nitroaldol reaction in the synthesis of (R)‐β‐nitroalcohols included 16 substrates, but the yield was in the range of 0 to 34%. Another study of AcHNL, GtHNL and their engineering was reported in the synthesis of (R)‐β‐nitroalcohols, however a maximum of three aldehydes were converted to their corresponding nitroaldol products. Our study revealed that irrespective of the position of the substitution at the phenyl ring of the NPE, a dozen of substrates resulted in the >90% ee of corresponding products in the retro-nitroaldol reaction. It was difficult to draw a correlation between the electronic nature of the substituents on the phenyl ring of a NPE and the stereoselectivity or conversion of the (R)‐β‐nitroalcohols obtained. It was noticed that even the racemic β‐nitroalcohols with bulky groups i.e., 1-naphthyl and 2-naphthyl could undergo retro-nitroaldol reaction with 32.1 and 44.7% conversion respectively and >99% ee.

Scheme 4: BmHNL catalyzed retro-Henry reaction in the preparation of (R)-β-nitroalcohols
Table 2: BmHNL catalyzed retro-Henry reaction in the preparation of different (R)-β-nitroalcohols
S. No. R Reaction time (h) % conversion % ee
1 2,4-diOMeC6H3 8 49.9 >99
2 4-OMeC6H4 10 49.5 >99
3 3-MeC6H4 16 48.5 >99
4 2-Naphthyl 40 44.7 >99
5 3-OHC6H4 8 41.2 >99
6 3-OMeC6H4 8 37.1 >99
7 1-Naphthyl 24 32.1 >99
8 4-MeC6H4 16 42.4 98.3
9 4-BrC6H4 40 47 94.6
10 2,5-diOMeC6H3 48 39.3 93.4
11 2-MeC6H4 8 17.3 92.7
12 4-PhC6H4 48 44.1 90.3
13 3-ClC6H4 48 38 89.1
14 2-OMeC6H4 24 35.3 86.6
15 3-PhCH2OC6H4 48 32.2 79.9
16 3-PhOC6H4 64 45.7 74.6
17 4-NO2C6H4 40 31.9 73.5
18 3-NO2C6H4 48 42.8 71.9
19 2,6-diClC6H3 24 35.7 71.9
20 4-ClC6H4 16 29.9 59
21 3,5-diClC6H3 24 44.1 10.3
22 3-PhCH2CH2 24 50.6 2.9
Experimental: The following reaction conditions were used to carry out BmHNL catalyzed retro-nitroaldol reaction. The reaction mixture contained 488 μL TBME, 3 mM racemic β-nitroalcohol (12 µL, 0.25 M), 40 U of BmHNL, 100 mM CPB pH 5.5 (100 µL, 1 M), and 50 mg/mL (63 µL, 0.8 g/mL) sorbitol. The total volume of the reaction mixture was made up to 1 mL by adding the remaining volume of autoclaved double distilled water. This reaction mixture was incubated in a thermomixer at 30 °C at 1200 rpm.
4. Promiscuous diastereoselective nitroaldol reaction to synthesize 2-nitro-1-phenyl propanol (2-NPP)
HNLs are a group of versatile biocatalysts that are well known for their reversible cyanogenesis activity and are used for the synthesis of enantiopure cyanohydrins from aldehydes or ketone and HCN. These excellent biocatalysts are also capable of catalyzing the carboligation reaction between carbonyl substrates and nitroalkanes to produce ꞵ-nitro alcohols which possess great synthetic value. Though this biocatalysis approach holds great synthetic applicability but till date only one HNL is reported to catalyze the diastereoselective synthesis of (S)-ꞵ-nitro alcohol. HNL from Hevea brasiliensis or HbHNL preferentially produced (1S,2R)-2-NPP from benzaldehyde and nitroethane and shows 67% yield with 95% ee and 80% de. After finding the promiscuous nitroaldol activity of BmHNL that it can enantioselectively add nitromethane to a range of aromatic aldehydes, the nitroaldol reaction between benzaldehyde and nitroethane was tested with its crude form. BmHNL showed 60% conversion with 97% ee and 65% de and it also preferentially synthesized (1S,2R)-2-NPP, the same stereoisomer produced by HbHNL.
Experimental: The reaction mixture contained 600 µL of n-butyl acetate, 50 mM reaction buffer (42 µL, 1.5 M), 15 mg of crude wild type BmHNL, 1 M nitroethane (91.8 µL), and 20 mM benzaldehyde solution (25 µL, 1 M). Final reaction volume was kept 1.25 mL by adding remaining amount of double-distilled water. Benzaldehyde stock was prepared in n-butyl acetate.
5. Optimization of reaction conditions towards the stereoselective synthesis of (1S,2R)-2-NPP using BmHNL as the catalyst
Several important reaction parameters were optimized to improve the production of (1S,2R)-2-NPP with respect to % conversion, enantio- and diastereoselectivity. For the synthesis of this same isomer using HbHNL, phosphate buffer (KPB) pH 7 was used, whereas AcHNL and GtHNL mutein preferred KPB pH 6 to synthesize the opposite anti-isomer. We used KPB and CPB, varying their pH from 5.5 to 7. At pH 5.5, BmHNL showed 56.7% conversion with 97.4% ee and its % de improved to 72%. To find a suitable organic solvent for this biphasic reaction system five solvents were tested n-butylacetate (NBA), tert-butyl methyl ether (TBME), diethyl ether (DEE), toluene and di-isopropyl ether (DIPE). Among these five, TBME gave the best result in terms of % conversion and % ee and showed a 16% improvement in % conversion compared to NBA. The same solvent was also found to be suitable for HbHNL-catalysed diastereoselective synthesis of 2-NPP. Co-substrate nitroethane concentration was optimized in the next experiment. Previously HbHNL used 10 times more nitroethane than benzaldehyde, here nitroethane concentration was varied from 0.75 M to 2 M against 25 mM benzaldehyde substrate. Catalytic activity with respect to % conversion and enantioselectivity of BmHNL increased with the increasing nitroethane concentration up to 1.75 M and slightly decreased further in 2 M nitroethane. Use of 1.75 M nitroethane improved % ee and % de to >99% and 77.4% respectively from 97% and 69%. CPB was found to be the best reaction buffer to catalyse this experiment. Its concentration was optimized in the subsequent experiment. With increasing buffer concentration there was a slight improvement in % de with constant % conversion. BmHNL showed 80% conversion in this optimized condition with >99% ee and 81.4% de. In the previous reports, for the synthesis of 2-NPP by HNLs, only purified proteins were used. Since our study has been carried out by crude enzyme, the amount of enzyme has to be optimized. Thus, enzyme amounts of 5, 10, 15, 20, and 25 mg were tested in this experiment. With an increase in enzyme amount, crude lysate of wt BmHNL showed an increase in the activity up to 10 mg and beyond that, the % conversion and % ee remained the same. (Figure 2)
Experimental: To obtain higher % conversion, ee and de in the BmHNL catalyzed diastereoselective nitroaldol reaction using benzaldehyde and nitroethane, various reaction conditions like buffer type, buffer pH, buffer concentration, organic solvent, nitroethane (co-substrate) concentration, and enzyme content were optimized.
Buffer and pH optimization: In this experiment, two buffers KPB (potassium phosphate buffer) and CPB were tested varying its pH from 5.5 to 7. Reaction mixture contained 600 µL NBA, 50 mM reaction buffer (42 µL, 1.5 M), 15 mg of crude wild type BmHNL, 1 M nitroethane (91.8 µL), and 20 mM benzaldehyde solution (25 µL, 1 M). Final reaction volume was kept 1.25 mL by adding remaining amount of double-distilled water. Benzaldehyde stock was prepared in NBA.
Organic solvent optimization: Five organic solvents used in this study were NBA, TBME, DEE, toluene and DIPE. The reaction mixture composed of 600 µL of organic solvent, 50 mM CPB pH 5.5 (42 µL, 1.5 M), 15 mg of crude wild type BmHNL, 1 M nitroethane (91.8 µL), and 20 mM benzaldehyde solution (25 µL, 1 M). Final reaction volume was kept 1.25 mL by adding the remaining amount of double-distilled water. Benzaldehyde stock was prepared in different corresponding organic solvents.
Nitroethane concentration optimization: Co-substrate concentration is an important reaction parameter in HNL catalyzed Henry reaction. Six different concentrations of nitroethane were used to improve the % conversion and stereoselectivity of the BmHNL. The reaction mixture consisted of 600 µL TBME, 50 mM CPB pH 5.5 (42 µL, 1.5 M), 15 mg of crude wild type BmHNL, 0.75 M (69 µL)/ 1 M (92 µL)/ 1.25 M (114.7 µL)/ 1.5 M (138 µL)/ 1.75 M (161 µL)/ 2 M (184 µL) nitroethane, and 20 mM benzaldehyde solution (25 µL, 1 M). Final reaction volume was kept 1.25 mL by adding the remaining amount of double-distilled water. Benzaldehyde stock was prepared in TBME.
CPB concentration optimization: The concentration of CPB was optimized in this experiment. Buffer concentration was varied from 50 to 250 mM. The reaction mixture used 600 µL TBME, 50 mM CPB pH 5.5 (42 µL, 1.5 M)/ 100 mM (83 µL, 1.5 M)/ 150 mM (125 µL, 1.5 M)/ 200 mM (167 µL, 1.5 M)/ 250 mM (208 µL, 1.5 M), 1.75 M (161 µL) nitroethane, 20 mM benzaldehyde solution (25 µL, 1 M), and 15 mg of crude wild type BmHNL. Final reaction volume was maintained to 1.25 mL by adding the remaining amount of double-distilled water. Benzaldehyde stock was prepared in TBME.
Enzyme content optimization: To optimize the amount of enzyme required to increase the yield and enantiopurity of the product, six different enzyme concentrations of enzymes were taken into consideration starting from 5 to 25 mg of enzyme. The reaction mixture composed of 600 µL TBME, CPB pH 5.5 100 mM (83 µL, 1.5 M), 5/ 10/15/20/25 mg of crude wild type BmHNL, 1.75 M (161 µL) nitroethane, 20 mM benzaldehyde solution (25 µL, 1 M). Final reaction volume was kept 1.25 mL by adding the remaining amount of double-distilled water. Benzaldehyde stock was prepared in TBME.
6. Determining the tolerable substrate loading and TTN calculation
To determine the maximum substrate loading in the BmHNL catalyzed promiscuous reaction, benzaldehyde concentration was varied from 20 to 130 mM. With the increasing substrate concentration, % conversion and % ee remained constant up to 70 mM and later % conversion decreased gradually. The total turnover number (TTN) was calculated by considering the product formation at 70 mM substrate concentration and it was determined to be 19900 [Figure 3]. A comparison of this biotransformation with wt HbHNL, the only HNL reported to catalyse this reaction, showed a TTN of 267 which is almost 74.5 times less compared to our result.
Experimental: The reaction condition was similar to the previous experiment, except in the case of higher substrate concentrations, the volume of TBME was adjusted in such a way that the total volume of TBME and substrate stock solution remained 625 µL. TTN calculated using the formula: Total turnover number=(µmol of product formed)/(µmol of enzyme used)
7. Purified wt BmHNL catalysed stereoselective synthesis of (1S,2R) or anti-isomer of different β-nitroalcohols using nitroethane as the co-substrate
Using the optimized biocatalytic reaction conditions, several aromatic aldehydes having substituents at different positions on the phenyl ring were converted into their corresponding anti diastereomers of β-nitroalcohols (Scheme 5). 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 3). To date there is only one (S) selective HNL, i.e., HbHNL reported to carry out such promiscuous reaction to produce (1S,2R)-NPP. HbHNL showed 67% yield and 95% ee with 80% de. Two other (R)-selective HNLs GtHNL and AcHNL were reported to synthesize the opposite stereoisomer, i.e., (1R,2S)-NPP from benzaldehyde. Wild-type AcHNL showed very poor diastereomeric excess (6%) with 73% ee and 77% conversion to (1R,2S)-NPP. This poor diastereomeric excess and % ee was improved to 27% and 88% respectively when AcHNL-A40H was used, however % conversion decreased to 66%. Here wt BmHNL showed up to >99% conversion with >99% ee and 92.4% diastereomeric excess towards the synthesis of different (1S,2R)-NPP derivatives from their corresponding aromatic aldehydes. Among the twenty substrates used here (Table 3), except benzaldehyde none of them were studied earlier by HNL catalysed Henry reaction to produce their anti isoforms starting from their corresponding aldehydes. The only other biocatalytic approach known so far is Pseudomonas flourescens lipase catalysed kinetic resolution, where three racemic β-nitroalcohols having two chiral centres were used. However the yield were limited to ~50% and the stereoselectivity of the product was not clear.
Among the twenty substrates used here, except 4-methoxy benzaldehyde, BmHNL was able to catalyse this reaction to synthesize the corresponding ꞵ-nitroalcohols with all other aldehydes. The number of functional substitutions attached to the aromatic ring or their chemical nature did not affect critically on the stereoselectivity of the enzyme. For example, methyl and methoxy substitutions which are electron donating and bromo and chloro substitutions that have electron withdrawing nature showed 75 to >99% conversion with 98.8 to >99% ee except for 4-methyl benzaldehyde, where 66% conversion was observed. Aromatic aldehydes having double substitutions in their aromatic ring also exhibited very high catalytic activity, for example, 2,3-dichloro, 2,3-dimethoxy, and 2,5-dimethoxy benzaldehyde showed >99, 89, and 94% conversion respectively with >99% ee in every case. However, BmHNL showed less catalytic activity when functional groups were introduced at the para position of the benzene ring. For example in case of 4-chloro benzaldehyde 75% conversion was found, while 2-chloro and 3-chloro benzaldehyde produced their corresponding ꞵ-nitroalcohols in >99% and 93% conversion respectively. A similar phenomenon was found in the case of 4-methyl and 4-methoxy benzaldehyde (Table 3), while 4-nitro is an exception. Bulky substrates like 1-napthaldehyde and 3-benzyloxy benzaldehyde, which possess a bicyclic aromatic ring and bulky benzyloxy substitution respectively were also converted to their corresponding chiral (1S,2R)-ꞵ-nitroalcohols and showed >99% conversion with >99% ee and 88.3% conversion with >99% ee respectively. An increase in the alkyl carbon chain length in the benzaldehyde impaired BmHNL’s catalytic activity and showed only 10% conversion with 95% ee in case of 3-phenyl propionaldehyde.

Scheme 5: BmHNL catalyzed diastereoselective Henry reaction in the synthesis of chiral β-nitroalcohols using nitroethane as co-substrate
Table 3: BmHNL catalysed synthesis of stereoenriched (1S,2R)-ꞵ-nitroalcohols from corresponding achiral aromatic aldehydes.
S. No. Substrate Time (h) Total % conversion % ee of (1S,2R) % ee of (1S,2S) Diastereomeric excess of anti product
1 1-napthaldehyde 8 >99 >99 >99 75.3
2 2-nitro benzaldehyde 8 >99 >99 93.1 55.2
3 2,3-dichloro benzaldehyde 8 >99 >99 >99 86.3
4 4-nitro benzaldehyde 8 >99 >99 >99 92.4
5 2-chloro benzaldehyde 8 >99 98.8 >99 85.0
6 2-bromo benzaldehyde 8 97.6 >99 >99 80.0
7 2,5-dimethoxy benzaldehyde 16 94.1 98.8 >99 49.4
8 3-chloro benzaldehyde 8 92.9 >99 >99 86.1
9 2,3-dimethoxy benzaldehyde 8 89.1 >99 >99 80.4
10 2-methoxy benzaldehyde 8 88.6 >99 >99 69.6
11 3-benzyloxy benzaldehyde 48 88.3 >99 >99 0.8
12 3-bromo benzaldehyde 8 86.1 >99 91.7 81.2
13 3-methoxy benzaldehyde 16 85.5 >99 >99 7.4
14 benzaldehyde 2 82.9 >99 >99 87.2
15 3-methyl benzaldehyde 16 82.5 >99 >99 2.9
16 2-methyl benzaldehyde 16 77.6 >99 >99 9.1
17 4-chloro benzaldehyde 8 75.1 >99 90.8 87.8
18 4-methyl benzaldehyde 24 66.2 >99 98.9 18.3
19 3-phenyl propionaldehyde 24 9.8 94.3 −4.9 21.8
20 4-methoxy benzaldehyde 24 no conversion

Experimental: Using the optimized reaction conditions twenty different aromatic aldehydes (Table 3) were converted to their corresponding β-nitroalcohols. A typical reaction contained 600 µL of TBME, 5 mM aldehyde (25 µL, 250 mM), 1.75 M nitroethane (161 µL), 100 mM CPB pH 5.5 (83 µL, 1.5M), and 1 mg of purified wild type BmHNL. Stock solutions of aldehydes were made in TBME. The total reaction volume was made up to 1.25 mL by adding the remaining volume of autoclaved double distilled water. 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 sulphate to remove any aqueous part, and then centrifuged at 15000g for 5 min at 4 °C. A 20 μL of this sample was analysed by HPLC using appropriate chiral column.
8. Promiscuous nitroaldol reaction to synthesize 2-nitro-1-phenyl butanol (2-NPB) by using 1-nitropropane
After the successful stereoselective synthesis of a series of nitrophenyl propanol derivatives using nitroethane as a nucleophile, the same experiment was planned with a bulkier nucleophile to test its catalytic promiscuity towards other nucleophilic co-substrate. 1-Nitropropane was chosen for this purpose and this is the first report, where different 2-nitro-1-phenyl butanol (NPB) derivatives were synthesized using any biocatalytic means. The optimized reaction condition obtained from the nitroethane study was employed here to produce enantiopure 2-NPB. Unlike the case of nitroethane, here crude BmHNL showed very poor activity with 1-nitropropane, so the reaction was repeated with purified enzyme, where a considerably better activity was observed. Purified BmHNL showed 36% conversion with >99% ee and 81.3% de towards the synthesis of (1S,2R)-2-NPB starting from benzaldehyde. To improve the activity further, a few reaction parameters were optimized afterward.
Experimental: A typical reaction contained 600 µL of TBME, 5 mM benzaldehyde (25 µL, 250 mM), 1 M nitropropane (156 µL), 100 mM CPB pH 5.5 (63 µL, 2 M), and 1 mg purified BmHNL. Substrate stock preparation and other reaction parameters were kept similar. Product extraction and HPLC were done according to the previously described method.
9. Optimization of reaction conditions towards the stereoselective synthesis of (1S,2R)-2-NPB using BmHNL as the catalyst
The amount of purified enzyme was optimized to further improve the % conversion of the (1S,2R) anti-isomer of 2-NPB by wt BmHNL. With increasing enzyme content, BmHNL showed improved % conversion but it failed to retain its stereoselectivity. For example use of 1 mg purified BmHNL produced 63% conversion with >99% ee and 70% de after 72 h, but with 5 mg of enzyme the % de decreased up to 10%. 1-Nitropropane concentration was optimized in the subsequent experiment, and was varied from 0.25 to 1.75 M. The % conversion increased with increasing 1-nitropropane concentration up to 1 M and then slightly decreased on further higher concentrations. BmHNL showed 39 % conversion with >99% ee and >99% de with 1 M 1-nitropropane after 24 h incubation (Figure 4).
Experimental: The following parameters were exclusively optimized towards the BmHNL catalyzed diastereoselective 1-nitropropane addition to benzaldehyde.
Optimization of reaction time and catalyst amount: To optimize the reaction time and catalyst amount different amount of BmHNL was used, incubated for longer time periods and aliquots were taken at different time intervals. Purified BmHNL, 1, 2.5, and 5 mg were used for this study. All other reaction conditions were the same as described previously.
Optimization of 1-nitropropane concentration: To optimize the 1-nitropropane concentration in the biocatalytic Henry reaction a range of 1-nitropropane concentrations was used from 0.25 - 1.75 M. The reaction condition was as follows: 600 L of TBME, 0.25 – 1.75 M 1-nitropropane, 1 mg BmHNL, 100 mM CPB pH 5.5 (63 L, 2 M), and 5 mM benzaldehyde (25 L, 250 mM). The reaction volume was made up to 1.25 mL using double distilled water. Substrate stock preparation and other reaction parameters were kept similar. Product extraction and HPLC were done according to the previously described method.
10. Purified wt BmHNL catalysed stereoselective synthesis of (1S,2R) anti-isomer of different β-nitroalcohols using 1-nitropropane as the co-substrate
Fourteen aromatic aldehydes were taken for this study to synthesize their corresponding enantiopure (1S,2R) isomer of β-nitroalcohols using 1-nitropropane as the nucleophile (Scheme 6, Table 4). Among these substrates except 2-furaldehyde, all others were studied with nitroethane in Table 3. Aldehydes with nitro substitutions on the phenyl ring were preferred compared to other functional groups, for example 4-nitro and 2-nitro benzaldehyde showed 97% and 89% conversion with 88% and >99% ee respectively. Unlike nitroethane, in the case of 1-nitropropane, BmHNL exhibited a preference toward certain functional group and positions over others and this preference was in the order of ortho > meta > para. For example, except for 2-methoxy benzaldehyde, other ortho substituted aldehydes such as 2-bromo, and 2,3-dichloro benzaldehyde showed 89% and 79% conversion with 96% and 97% ee respectively. Aromatic aldehydes having meta substitutions like 3-chloro, 3-methoxy, 3-bromo, and 3-methyl benzaldehydes showed 31-62% conversion with 88 to >99% ee. The para substituted aldehydes exhibited very minimum activity, i.e., 4-chloro and 4-methyl benzaldehyde both showed only 28% conversion with 96% and >99% ee respectively. Extension in the alkyl chain in the aldehyde substrate massively reduced BmHNL’s efficiency to catalyse the reaction. In case of 3-phenyl propionaldehyde which possesses two extra carbons in the alkyl chain showed only 1% conversion. Such poor conversion in case of 3-phenyl propionaldehyde was also found with nitroethane as a co-substrate.

Scheme 6: BmHNL catalyzed diastereoselective Henry reaction in the synthesis of chiral β-nitroalcohols using 1-nitropropane as co-substrate
Table 4: BmHNL catalyzed synthesis of enantioenriched (1S,2R)-ꞵ-nitroalcohols from corresponding achiral aromatic aldehydes.
S. No. Substrate Time (h) Total % conversion % ee of (1S,2R) % ee of (1S,2S) Diastereomeric excess of anti product
1 4-nitro benzaldehyde 48 97 87.8 2.8 75.4
2 2,3-dichloro benzaldehyde 48 89.3 95.7 −82.5 91.6
3 2-nitro benzaldehyde 48 89.2 >99 >99 80.8
4 2-bromo benzaldehyde 48 78.8 97.4 >99 78.1
5 3-chloro benzaldehyde 48 62.3 88.2 68.3 89.3
6 3-methoxy benzaldehyde 48 52.8 >99 >99 63.3
7 2-furaldehyde 24 49.8 97.7 - 62.8
8 3-bromo benzaldehyde 48 45.5 >99 46.7 94.1
9 benzaldehyde 48 44.2 >99 >99 76
10 3-methyl benzaldehyde 48 30.6 >99 >99 58.5
11 4-chloro benzaldehyde 72 28.2 95.7 45.3 65.3
12 4-methyl benzaldehyde 48 27.9 >99 0 >99
13 2-methoxy benzaldehyde 48 24.7 95.2 - 58.9
14 3-phenyl propionaldehyde 48 trace - - -

Experimental: The optimized reaction conditions of diastereoselective synthesis of 2-nitro-1-phenylbutan-1-ol, were used with other aromatic aldehydes to synthesize their corresponding chiral β-nitroalcohols. A typical reaction contained 600 µL TBME, 5 mM aldehyde (25 µL, 250 mM), 1 M nitropropane (156 µL), 100 mM CPB pH 5.5 (63 µL, 2 M), and 1 mg purified BmHNL. Substrate stock preparation and other reaction parameters were kept similar. Product extraction and HPLC were done according to the previously described method.
11. Determination of the kinetic parameters of BmHNL towards the diastereoselective Henry reaction
The initial reaction rate of wt BmHNL was calculated using chiral HPLC towards the synthesis of (1S,2R)-2-NPP. The KM and Vmax of BmHNL towards the synthesis of (1S,2R)-2-NPP were found to be 10.3 mM and 0.46 µmol/min. The catalytic efficiency (kcat/ KM) was calculated to be 2.6 mM−1min−1 (Figure 5).
Experimental: The initial reaction rate of BmHNL towards the synthesis of (1S,2R)-NPP was assessed through normal phase chiral HPLC. After optimizing the time point, the minimum time where almost 10% conversion obtained, was selected. Biocatalysis was carried out with varying substrate concentrations. The reaction mixture contained 1.75 M nitroethane (161 µL), 1 to 25 mM benzaldehyde, 100 mM CPB pH 5.5 (63 µL, 2 M), and 0.5 mg purified BmHNL. Substrate stock preparation was done in TBME. Total volume of organic solvent i.e., TBME and stock substrate solution was kept 625 µL. Total reaction volume was made up to 1.25 mL by adding double distilled water. For each substrate concentration, biocatalysis was incubated for 10 mins. Product extraction and HPLC were done according to the previously described method.
12. Comparison of BmHNL’s cyanogenesis vs. retro-nitroaldolase activity
The impressive enantioselective, and diastereoselective synthetic potential of BmHNL suggests that the same active site is able to accommodate diverse chiral β-nitroalcohols. We here compared substrate preference of BmHNL towards the promiscuous products, i.e. β-nitroalcohols vs. mandelonitrile, a close to natural substrate. To study this phenomena, we used cleavage of the three different β-nitroalcohols of benzaldehyde and mandelonitrile. The cleavage product benzaldehyde in case of mandelonitrile and β-nitroalcohols, obtained was used to calculate the cyanogenesis and retro-nitroaldolase activity of BmHNL. The cyanogenesis activity was found to be 12 U/mg (Figure 6). However, the same enzyme displayed less activity when tested for retro-nitroaldol reaction using different ꞵ-nitroalcohols. Towards cleavage of 2-nitro-1-phenyl ethanol, 2-nitro-1-phenyl propanol, and 2-nitro-1-phenyl butanol it showed a specific activity of 1.41, 3.94, and 1.05 U/mg respectively. This data clearly demonstrates BmHNL’s preference towards 2-nitro-1-phenyl propanol. The same is observed during BmHNL’s nitroaldol synthesis when nitroethane was used as the nucleophile as compared to 1-nitropropane (Figure 6).
Experimental: Here we compared the retro-nitroaldolase activity of three ꞵ-nitroalcohols differing in their nitroalkane carbon chain length. This promiscuous nitroaldolase activity was also compared with its natural cyanogenesis activity. Racemic mandelonitrile, 2-nitro-1-phenyl ethanol, 2-nitro-1-phenyl propanol, and 2-nitro-1-phenyl butanol were used as the substrates for the cleavage assay. The detailed reaction condition contained 160 µL of 50 mM CPB pH 5.5, 20 µL of 67 mM substrate prepared in 5 mM CPB pH 3.15, and 20 µL of purified enzyme solution of 1 mg/mL concentration. The benzaldehyde released was measured in a spectrophotometer at 280 nm.
13. Determination of BmHNL’s stability in the reaction medium and recycling of the enzyme
As α/β hydrolase fold HNLs are known to be inhibited at higher aldehyde (especially benzaldehyde) concentration, we aimed to study the stability of BmHNL when it is exposed to a higher aldehyde concentration for long time. In this regard, BmHNL was incubated in a reaction medium carrying 70 mM of benzaldehyde for different time periods. We observed only 14% loss of initial activity after incubating the enzyme for two months, which confirms its high stability in the reaction condition (Figure 7). This data inspired us to recycle the enzyme for the synthesis of the (1S,2R)-2-NPP. The minimum time to get the maximum % conversion was found to be 7 h. The enzyme was recycled for 20 consecutive cycles afterward. Until ten cycles, BmHNL could maintain >95% of its initial activity. After ten cycles a slight but steady decrease in enzyme activity was observed. After twenty cycles BmHNL could retain up to 82% of its activity compared to the first cycle. Throughout these twenty cycles of enzyme recycling, it could maintain >99% ee towards the synthesis of (1S,2R)-2-NPP (Figure 8). In this recycling experiment sequentially, a total of 1.75 mmol of benzaldehyde was introduced and 72% of it was converted to product accounting to 1.26 mmol. The total amount of enzyme used in this experiment was only 1 mg (MW of BmHNL 29.5 kDa) and hence a total turnover number (TTN) was found to be 37,170.
Experimental: The stability of BmHNL was examined by measuring the loss of activity using mandelonitrile cleavage assay. Aqueous layer of the biocatalytic reaction was collected after incubation for a definite time and used as enzyme source to calculate the specific activity using mandelonitrile cleavage assay as describe in the experimental section of 12, above. Biocatalytic reaction mixture contained, 538 µL of TBME, 1.75 M nitroethane (161 µL), 70 mM benzaldehyde (87.5 µL, 1 M), 100 mM CPB pH 5.5 (63 µL, 2 M), and 1 mg BmHNL. The recyclability of the enzyme was tested using the same reaction condition but here in every 7 h, the organic layer was removed and a fresh 70 mM benzaldehyde, 1.75 M nitroethane, and 538 µL TBME was added to the aqueous reaction mixture.
14. Gram scale synthesis of (1S,2R)-2-NPP by recycling the BmHNL enzyme
From the experiment on recyclability of BmHNL, it was observed that the enzyme can be safely used up to 10 cycles while retaining its % conversion and stereoselectivity. We aimed to carry out a large-scale synthesis of the anti diastereoisomer, (1S,2R)-2-NPP using the recycling approach. We started with 153.5 U of BmHNL (12 mg, 12.79 U/mg), and in each cycle of 7 h, 1.05 mmol of benzaldehyde was added into the reaction medium. By this approach, 1.11 g of benzaldehyde was converted into 0.99 g of product. Among the reported approaches this method appeared as the most efficient process for catalyzing this promiscuous Henry reaction. Previously reported wt HbHNL consumed 4495 U of enzyme/mmol of the substrate and showed a TTN of 267. In contrary our method used only 14.6 U of enzyme/mmol of substrate and the TTN calculated based on the amount of product produced from the ten cycles was found to be 13,228. Hence BmHNL catalyzed diastereoselective nitroaldol reaction was found to use ~308 times less enzyme per mmol of substrate and demonstrated a 50 times higher TTN than the HbHNL catalyzed method.
Experimental: The biocatalytic reaction condition for this experiment was as follows. The reaction mixture had 12 mg purified BmHNL, 100 mM CPB pH 5.5 (1.2 mL, 1. 25 M), TBME 50% (7.4 mL), 70 mM benzaldehyde (108.7 µL), nitroethane 1.75 M (1.932 mL), and 2.24 mL of water. The reaction was incubated at 30° C and 1200 rpm. After every 7 h the organic layer of the reaction containing chiral product, TBME, nitroethane, and remaining benzaldehyde was collected and replaced with fresh benzaldehyde, TBME, and nitroethane. Likewise, the enzyme was recycled for 10 times.
15. BmHNL catalysed diastereoselective synthesis of the syn stereoisomers of (1S,2S) configuration
Among the nineteen aldehydes (Table 3, Scheme 5) that were converted into their corresponding (1S,2R) β-nitroalcohols, nine of them showed ≥20% isomeric content of (1S,2S) stereoisomer, when the reaction mixture was incubated beyond 24 h. These nine substrates were chosen to synthesize the syn, i.e., (1S,2S)-stereoisomeric products. The optimized reaction condition used for synthesis of (1S,2R) stereoisomers were extended in the synthesis (1S,2S) enantiomer, except carrying out the biotransformations for a longer time. Among the substrates used aromatic aldehydes having single methyl and methoxy substitution showed >60% isomeric content (IC) of the (1S,2S)-stereoisomer with % conversion ranging from 79-94 and % ee from 98 to >99. Benzaldehyde and 3-benzyloxy benzaldehyde showed 88% conversion with 94% and >99% ee of their (1S,2S)-stereoisomer respectively. These two substrates displayed 51-59% IC. The remaining three substrates 2-nitro benzaldehyde, 2,3-dimethoxy and 2,5-dimethoxy benzaldehyde showed poor % IC ranging from 36-41 (Table 5).
Table 5: BmHNL catalyzed synthesis of enantioenriched (1S,2S)-ꞵ-nitroalcohols from corresponding achiral aromatic aldehydes.
Substrate Time (h) Total % conversion % ee of (1S,2R) % ee of (1S,2S) de of syn product IC of (1S,2R) IC of (1S,2S)
2-nitro benzaldehyde 72 >99 >99 91.2 −24.4 62.2 36.2
3-benzyloxy benzaldehyde 72 87.8 >99 >99 2.7 48.4 51.2
3-methoxy benzaldehyde 48 94 >99 >99 24.2 37.9 62.1
benzaldehyde 72 87.4 87.7 94 10.1 35.9 59.1
2,5-dimethoxy benzaldehyde 72 93.7 99.5 >99 −20.9 60.3 39.6
2-methyl benzaldehyde 72 82.4 98.6 >99 21.8 38.8 60.9
4-methyl benzaldehyde 79 72 98.3 97.5 29.1 35.1 63.7
3-methyl benzaldehyde 48 85.3 >99 >99 27.2 36.4 63.6
2,3-dimethoxy benzaldehyde 72 93 >99 98 −16.7 58.3 41.2

Experimental: The reaction condition used for the synthesis of (1S,2R)-stereoisomers was employed here. A typical reaction contained 600 µL of TBME, 5 mM aldehyde (25 µL, 250 mM), 1.75 M nitroethane (161 µL), 100 mM CPB pH 5.5 (63 µL, 2 M), and 1 mg of purified BmHNL. Substrate stock preparation and other reaction parameters were kept similar. Product extraction and HPLC were done according to the previously described method.
16. Addition experiment details
a. Materials: Culture media and ampicillin were purchased from HiMedia laboratory Pvt. Ltd, India. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from BR-BIOCHEM Pvt. Ltd, India. Aldehydes, nitromethane, nitroethane, 1-nitropropane, and mandelonitrile were purchased from Sigma Aldrich, AVRA, SRL, and Alfa-Aesar. HPLC grade solvents were obtained from FINAR India. The chemicals purchased were used without purification. Silica gel 100-200 mesh used in column chromatography purifications was purchased from Molychem. 1H- and 13C-NMR spectra were recorded on a Bruker 500 MHz instrument with deuterated chloroform as solvents as specified. 1H-NMR data are given as chemical shift (ä ppm), multiplicity (s = singlet, brs= broad singlet, d = doublet, t = triplet, q = quartet, m =multiplet, dd = doublet of doublet, dt = doublet of triplet, ddd = doublet of doublet of doublet), coupling constant (Hz), and integration. 13C-NMR data are reported in terms of chemical shift relative to CDCl3 (77.16 ppm). VCX500 ultrasonic processor was used for lysing the cells by sonication. Enzyme assay was carried out in a UV/VIS Microplate & Cuvette spectrophotometer, Multiscan Go, Thermo Fisher Scientific. TLC silica gel 60 F254 (Merck) plates were used to monitor organic reactions using a UV-lamp for visualization.
b. Chromatography: Analytical high-performance liquid chromatography (HPLC) and chiral analysis were performed in an Agilent 1260 Infinity II system or LC-2080, Shimadzu system. Chiral HPLC was conducted using an Agilent 1260 Infinity II chiral HPLC system with isopropanol and hexanes as the mobile phase. Lux 5 µm Amylose-1, Lux 5 µm Cellulose-1, and Daicel CHIRALPAK IB columns were used to separate stereoisomers (4.6 x 20 mm, 5 μm). For calculating the % conversion of few substrates, Shimadzu LC-2080 HPLC was utilized using water and acetonitrile as the mobile phase, along with Agilent InfinityLab Poroshell 120 EC-C18 column (4.6 x 150 mm, 4 μm).
c. Cloning: BmHNL (LOCUS: AB505969) synthetic gene cloned into pUC57 was procured from Gene Script, USA, and sub-cloned into pCold1 plasmid at BamHI and SalI and transformed into E. coli BL21 DE3 cells.
d. Expression, crude enzyme preparation and purification of wild type BmHNL and preparation of extracts: Expression and purification of BmHNL was carried out using methods as reported earlier. Wild type BmHNL plasmid transformed E.coli BL21(DE3) cells were inoculated in sterile Luria Bertani broth comprised of 100 µg/mL ampicillin and incubated at 37 °C, 180 rpm for 12 – 16 h to prepare primary culture. A 2% (v/v) primary culture inoculum was used for secondary culturing and incubated at 37 °C until O.D600 reaches 0.5 – 0.8. Then cells were subjected to a cold shock by incubating at 4 °C for 2 h, followed by protein expression using 1 mM IPTG, incubated at 18 °C, 180 rpm for 24 h. Cells were harvested by centrifuging at 8000 rpm for 12 minutes and the cell pellet was suspended in 20 mM KPB pH 7. Suspended cells were sonicated over ice and centrifuged at 10000 rpm for 45 min. This supernatant was further used as crude enzyme extract or after protein purification by Ni-NTA agarose affinity chromatography in biocatalysis. The sonicated supernatant was loaded into a Ni-NTA agarose column pre-equilibrated with twice its volume with binding buffer (20 mM imidazole, 300 mM sodium chloride, 20 mM KPB, pH 7). After a 45 min incubation in a shaker at 4 oC, the flow through was collected, the column was subsequently washed with three supernatant volumes of wash buffer [50 mM imidazole, 300 mM sodium chloride, 20 mM KPB, pH 7). Finally the column was eluted with one supernatant volume of elution buffer (500 mM imidazole, 300 mM sodium chloride, 20 mM KPB, pH 7). The eluted protein solution was dialyzed in 20 mM KPB pH 7 for three times, 3 h each. The protein obtained was concentrated and used for biocatalysis.
e. HNL assay: The reaction was performed in a 96 well microtiter plate. Each well of the plate consisted of 160 µL of 50 mM CPB pH 5.5, 20 µL of purified BmHNL (1 mg/mL), and 20 µL of 67 mM mandelonitrile solution prepared in 5 mM CPB pH 3.15. The activity was calculated using a molar extinction coefficient of benzaldehyde (1380 M−1 cm−1). One unit of HNL activity is defined as the amount of enzyme that produced 1 µmol of benzaldehyde from mandelonitrile per minute. All measurements were performed in triplicates. The control experiment had all the reaction components except the enzyme, which was replaced by its corresponding buffer.
f. Chemical synthesis of racemic β-nitroalcohol standards: Racemic β-nitroalcohols were synthesized by the addition of nitromethane, nitroethane, or 1-nitropropane to an aldehyde taken in 10:1 molar ratio in the presence of 2-50 mol% of Ba(OH)2 as a catalyst. For every mmol of the substrate, 3 mL of water was added as the reaction solvent. This reaction mixture was incubated at room temperature. After completion of the reaction, the product and unreacted substrate were extracted with ethyl acetate, dried over anhydrous sodium sulfate and concentrated in vacuum. The crude mixture was then purified by silica gel column chromatography using hexane and ethyl acetate as the mobile phase.
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 that are intended to define the spirit and scope of this invention.
SEQ ID NO: 1 (259 AA)
MVSAHFILIHTICHGAWLWYKLIPLLQSAGHNATAIDLVASGIDPRQLEQIGTWEQYSEPLFTLIESIPEGKKVILVGESGGGINIALAAEKYPEKVSALVFHNALMPDIDHSPAFVYKKFSEVFTDWKDSIFSNYTYGNDTVTAVELGDRTLAENIFSNSPIEDVELAKHLVRKGSFFEQDLDTLPNFTSEGYGSIRRVYVYGEEDQIFSRDFQLWQINNYKPDKVYCVPSADHKIQISKVNELAQILQEVANSASDL
SEQ ID NO: 2 (792 nucleotides)
Nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1
GGATCCATGGTGAGCGCTCATTTTATTCTGATACACACAATATGCCACGGAGCTTGGCTATGGTATAAGCTCATACCTTTGTTACAGTCAGCTGGGCACAATGCCACTGCAATTGACCTTGTAGCCAGTGGGATTGACCCAAGACAACTGGAACAAATTGGCACATGGGAACAATACTCAGAGCCATTATTTACTTTAATCGAATCAATCCCTGAAGGAAAAAAGGTTATACTTGTTGGAGAGAGCGGTGGAGGAATAAACATCGCCTTAGCTGCTGAAAAATATCCAGAGAAAGTTTCTGCCTTAGTTTTCCACAATGCATTGATGCCTGACATTGATCACAGTCCAGCTTTTGTTTATAAAAAGTTCAGTGAGGTATTTACTGACTGGAAGGACAGCATTTTTTCAAATTACACTTATGGAAATGACACTGTAACTGCAGTAGAATTGGGTGACAGGACTTTAGCGGAGAATATTTTTAGCAATTCGCCTATTGAGGATGTAGAACTGGCAAAGCATTTGGTAAGGAAGGGATCATTCTTTGAACAAGATTTAGATACACTCCCAAACTTCACCAGCGAAGGTTATGGATCAATTAGAAGAGTTTATGTGTATGGAGAGGAAGACCAAATATTTTCAAGGGACTTTCAACTTTGGCAAATAAATAATTATAAACCAGACAAGGTTTATTGTGTCCCCAGTGCAGATCATAAAATTCAGATTTCCAAAGTCAACGAATTAGCCCAAATTCTACAAGAAGTTGCAAATTCTGCAAGTGACTTGTGAGTCGAC
The bold regions represent nucleotides correspond to restriction enzymes BamHI (5’ end) and SalI (3’ end) respectively. This gene cloned into the pCold1 vector at BamHI and SalI.
, Claims:CLAIMS
I/We Claim
1. The method of preparing chiral β-nitroalcohols using BmHNL comprises of:
(i) a polypeptide having amino acid sequence as shown in SEQ ID NO: 1;
(ii) a hydroxynitrile lyase gene as shown in SEQ ID NO: 2 encoding the hydroxynitrile lyase of amino acid sequence of SEQ ID NO: 1;
(iii) a plasmid comprising the gene of SEQ ID NO: 2 contained in a vector;
(iv) a transformant obtained by introducing the said recombinant vector into a host;
(v) a culture obtained by culturing the said transformant; and
(vi) a hydroxynitrile lyase obtained from the said culture
2. BmHNL in one of the form as described in claim 1. (iv) to 1. (vi) has exhibited nitroaldolase, i.e., synthesis of β-nitroalcohol and retro-nitroaldolase activity, i.e., cleavage of β-nitroalcohols activity.
3. A method for stereoselective synthesis of optically active β-nitroalcohols comprising of
(i) a carbonyl compound and a nitroalkane in the presence of BmHNL 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 of R-CHO, where R= aromatic/ substituted aromatic/bicyclic/furan ring;
(iv) the nitroalkane used as the second substrate has the following formula RCH2NO2 where R= H/CH3/an alkyl chain of higher carbon;
(v) the BmHNL used is up to 15 unit per mmol of carbonyl substrate,
(vi) the said BmHNL catalyzed process as described that synthesized optically active β-nitroalcohols having one chiral center of the formula R1CH(OH)CH2NO2 where R1= aromatic /substituted aromatic/ PhCH=CH- /PhCH2CH2-; optically active β-nitroalcohols having two chiral centers of the formula R1CH(OH)CH(R2)NO2 where R1= aromatic/substituted aromatic and R2= CH3/an alkyl chain of higher carbon, where the absolute configuration of the two chiral centers are same or different.
4. A method for enantioselective preparation of optically active β-nitroalcohols comprising of
(i) a racemic β-nitroalcohol as a substrate in the presence of BmHNL in any form as described in claim 1. (iv) to 1. (vi),
(ii) the biphasic medium in the previous claim consists of CPB and TBME as an organic solvent
(iii) the racemic β-nitroalcohol used as a substrate and the optically pure β-nitroalcohol obtained have a formula of R1CH(OH)CH2NO2 where R= aromatic/substituted aromatic /PhCH2CH2-.
5. Recyclability or reuse of the BmHNL in one of the form as described in claim 1. (iv) to 1. (vi), in the process as described in claim 3 above for twenty cycles or more in the stereoselective synthesis of ꞵ-nitroalcohols, where the BmHNL has retained >80% of its initial activity.
6. The catalytic efficiency of BmHNL in one of the form as described in claim 1. (iv) to 1. (vi), used in the process as described in claim 3 in the diastereoselective synthesis of ꞵ-nitroalcohols was found to be very high including a Total Turnover Number 37,170.
7. The stability of BmHNL in one of the form as described in claim 1. (iv) to 1. (vi) after incubation with 70 mM benzaldehyde and organic solvent was found for at least two months, where 86% of initial enzymatic activity was retained.