Chemoenzymatic method to enantiopure sulphur heterocyclic β -hydroxy nitriles

Sulphur heterocyclic β -hydroxy nitriles are prepared from the suitable aldehyde and (trimethylsilyl)acetonitrile in the presence of lithium acetate at good yields. Burkholderia cepacia lipase allowed the formation of the both enantiomers of β -hydroxy nitriles in highly enantiopure forms (ee 99%) through acylation of a racemic mixture and alcoholysis of the acylated enantiomer in tert -butyl methyl ether.


Introduction
The enantiomers of β-hydroxy nitriles possess high potential as intermediates in organic synthesis and medicinal chemistry.This is due to the easy transformation of the cyano group into other functionalities, leading to the formation of β-hydroxy acids, their derivatives and γ-amino alcohols.As a result, synthetic methods to access both racemic and enantiopure β-hydroxy nitriles are continuously improved.
Anti-depressants with a 3-amino-1-arylpropan-1-ol structure are widely used for the treatment of psychiatric and metabolic disorders.Chiral β-hydroxy nitriles are convenient intermediates for their synthesis.When a β-hydroxy nitrile motif is attached to a heteroaromatic scaffold a broad range of potential biologically active compounds can be prepared.An illustrative example is (S)-duloxetine, 1 a (S)-3-amino-1-(2-thienyl)propan-1-ol-based serotoninnorepinephrine reuptake inhibitor for treating diabetic neuropathy, depression and anxiety. 2 In this context the need for reliable methods to access the enantiomers of new sulphur containing βhydroxy nitriles is understandable.Herein we describe the chemoenzymatic syntheses of the both enantiomers of new sulphur heterocycles 1(b-f) (Scheme 1).Compound 1a in enantiopure forms is known and serves as a reference compound for our work. 11c is used as a model substrate for optimization.Lipase-catalyzed asymmetric acylation is the enzymatic step used for enantioseparation while lipase-catalyzed alcoholysis is used to transform the acylated enantiomers (R)-2(a-e) back into the corresponding β-hydroxy nitrile enantiomers under mild conditions.The study is continuation to the previous works in the group with sulphur heterocyclic aldehydes as starting materials where dynamic kinetic resolution (DKR) methods by lipases have been developed in the formation of α-hydroxy nitriles (cyanohydrins).3a,b Early biocatalytic attempts to access enantiopure β-hydroxy nitriles have been based on the baker's yeast reduction of the corresponding β-keto nitriles. 4The first lipase-catalyzed access consisted of the hydrolysis of the esters of β-hydroxy nitriles.5a,b Kamal's group has used lipasecatalyzed transesterification for the kinetic resolution of 3-hydroxy-3-phenylpropanenitriles 6a,b and 4-aryloxy-3-hydroxybutanenitriles. 7Bäckvall's group combined lipase catalysis with Rucomplex catalysis and developed DKR to prepare enantiopure β-hydroxy nitriles.8a,b Previously, also racemic β-hydroxy nitriles have been enantioselectively hydrolyzed into the corresponding enantiopure acids and amides with satisfactory enantioselectivity using microbial whole cells containing nitrile hydratase/amidase activity. 9

Results and Discussion
Chemical synthesis of racemates Basically, two main strategies are available for the preparation of β-hydroxy nitriles: one exploits the ring opening of epoxides with cyanides from various sources as nucleophiles and the other the addition of deprotonated nitriles to carbonyl compounds, most often to aldehydes. 10,11,12We chose the aldehyde route for the preparation of β-hydroxy nitriles rac-1(a-f) because various sulphur-containing heterocyclic aldehydes are readily commercially available.Nowadays, alkali acetonitriles as cyanide sources in condensation with aldehydes have been more and more often replaced by (trimethylsilyl)acetonitrile (TMSCH 2 CN) in order to get satisfactory yields of βhydroxy nitriles. 10,13Accordingly, we followed the previous procedure 13 and prepared rac-1(a-f) through the cyanomethylation of aldehydes with TMSCH 2 CN catalyzed by lithium acetate as a Lewis base.The isolated yields of the products were usually higher than 80% as described in the Experimental Section.As exceptions, rac-1d was obtained at 65% and rac-1f only at 41% chemical yields.
Acetate (rac-2(a-d)) and butanoate (rac-2e) esters were prepared from the corresponding anhydrides and rac-1(a-e) by standard procedure.).Taking also conversion and time into account it is clear that lipase PS-D is the most effective of the studied catalysts.Accordingly, the acylation tends to stop at 50% conversion when the reactive Renantiomer of the initially racemic mixture has reacted (entry 1).6a,b,7,10 Lipase PS-D was a natural choice for further studies, and it was expected to show R-enantioselectivity in accordance with the known R-selectivity for the acylation of rac-1a.
Next rac-1c was subjected to acylation with vinyl acetate varying the lipase PS-D content from 50 through 25 and 10 to 5 mg mL -1 in TBME.The enzyme content had not an effect on enantioselectivity (E»200 was achieved) whereas conversions reached after 15 minutes dropped from 50 through 42 and 27 to 14% with the given decreasing enzyme contents.In the presence of 25 mg mL -1 of lipase PS-D with 42% conversion after 15 minutes TBME was shown to be the best solvent as the reaction in DIPE (conversion 37%) and toluene (conversion 36%) took place slower.Moreover, minor solubility problems of rac-1c were linked to DIPE and toluene.
When an acyl donor R 1 CO 2 R 2 reacts with a racemic alcohol like rac-1c in the formation of an ester like (R)-2c (Scheme 1) R 2 OH is liberated.For a successful kinetic resolution, it is important that the acyl transfer is irreversible, i.e., that the enzymatic reaction of R 2 OH with (R)-2c is prevented.Finally, the most common irreversible acyl donors were screened for the acylation of rac-1c in TBME using 25 mg mL -1 of lipase PS-D (Table 2).It is worth to remember that 2,2,2-trifluoroethyl butanoate is not necessarily irreversible as an acyl donor although it is generally expected to be as the nucleophilic character of the liberated 2,2,2trifluoroethanol is low and it is not reactive when added as such to the resolution mixture.Thus, interesterification between an amino ester and 2,2,2-trifluoroethyl esters was previously shown to be highly effective in the presence of lipase PS and CAL-B preparations.14a,b In the present work, the acylation of rac-1c proceeds in a highly enantioselective manner with the tested acyl donors while reactivity is excellent only in the case of vinyl esters (entries 1-4).Vinyl acetate as an acyl donor is somewhat more favourable over the butanoate (entry 3 compared to 4).Enhanced substrate concentration somewhat lowers reactivity (entry 1 compared to 2).Table 2. Effect of acyl donors (0.2 M) for the acylation of rac-1c (0.1 M) in the presence of lipase PS-D (25 mg mL -1 ) in TBME

Entry
Acyl donor Time (h) The set of β-hydroxy nitriles rac-1(a-f) was subjected to kinetic resolution with lipase PS-D in TBME (Table 3).Attention was paid to the amount of the enzyme needed to have a feasible process.Lipase PS-D possesses excellent enantioselectivity for all thiophen-based β-hydroxy nitriles rac-1(a-e) (entries 1-6) while enantioselectivity is only moderate in the case of thiazolbased rac-1f (Table 3, entry 7).As in the case of rac-1c (Table 2, entry 4) enzymatic reactivity for rac-1e with vinyl butanoate was low compared to the reaction with vinyl acetate as an acyl donor (data not shown).We achieved somewhat lower enantioselectivity for the lipase PS-Dcatalyzed acylation of rac-1a in TBME (Table 3, entry 1) compared to the previous acylation in DIPE (ee>99% for both resolution products at 50% conversion). 1 Vinyl butanoate (0.2 M) was used as an acyl donor due to analytical problems in ee determination with the acetate of 1e.

Enzymatic alcoholysis
It is natural that lipases catalyze the acylation of an alcohol with R 1 CO 2 R 2 and the deacylation of the corresponding ester with an alcohol based on the same enantiopreference (R in the present case as shown in Schemes 1 and 2).We investigated lipase PS-D-mediated alcoholysis primarily aiming to deacylate the (R)-esters 2(a-e) obtained through the preparative scale asymmetric acylation under mild conditions as shown below.In addition, we wanted to know how enentioselective the alcoholysis is.Accordingly, each of rac-2(a-e) (0.1 M) was subjected to alcoholysis with 1-butanol (0.3 M) in TBME (Table 4).Alcohol content was kept as low as possible so that (R)-1(a-e) was still completely formed because reactivity decreased with increasing alcohol concentrations.Alcohols are reported as competitive inhibitors to lipases.15a,b The usual enzyme content used was 50 mg mL -1 so that the alcoholysis smoothly proceeded to 50% conversion with excellent enantioselectivity (E»200).In the case of rac-2e very slow reactions were observed even at high enzyme contents and even at elevated temperature (entries 5-7).

Preparation of (R)-and (S)-1(a-e)
Finally the preparative scale resolution of rac-1(a-e) was performed under optimized conditions (Scheme 1).In accordance with excellent enantioselectivities, the reactions were stopped at 50% conversion and the formed R-esters and unreacted S-alcohols were separated by column chromatography as described in the Experimental section.Compound rac-1f was not resolved as only moderate enantioselectivity was achieved (Table 3, entry 7).Lipase PS-D-catalyzed alcoholysis with 1-butanol in TBME afforded the corresponding R-alcohols.In general, both (R)and (S)-1(a-e) were obtained in highly enantiopure forms as shown by the ee values and the consistency of the specific rotations for the antipodes (Table 5).The specific rotation +33.4 (entry 1) for the (R)-1a (ee=99%) is in accordance with the literature value 1 -33.5 calculated to the S-alcohol at 30 o C under otherwise the same conditions.

Conclusions
Simple chemoenzymatic method for the preparation of the enantiomers of sulphur heteroaromatic β-hydroxy nitriles has been described starting from the corresponding aldehydes and using lipase PS-D catalysis as the source of enantiopurity.

Experimental Section
General Procedures.All solvents were of the highest analytical grade and were dried by standard methods when necessary.Aldehydes, (trimethylsilyl)acetonitrile, lithium acetate and vinyl acetate were purchased from Aldrich.Vinyl butanoate was from Fluka and isopropenyl acetate from Merck.2,2,2-Trifluoroethyl butanoate was prepared from butanoyl chloride and the alcohol.Lipase PS from Burkholderia cepacia (previously Pseudomonas cepacia) immobilized on diatomaceous earth (lipase PS-D) and on ceramic particles (lipase PS-C II) and lipase AK-C from Pseudomonas fluorescence were purchased from Amano Europe, England.7 The enzymatic reactions were performed at room temperature (23 o C) if not otherwise stated.

Preparation of rac-1(a-f)
Racemates 1(a-f) were all prepared according to the known method. 13The preparation of rac-1c is used to illustrate the general procedure.

Lipase-catalyzed acylation of racemic β-hydroxynitriles 1(a-f)
In a typical small-scale experiment, lipase was added to the solution of rac-1(a-f) (0.1 M) and an acyl donor (0.2 M) in an organic solvent.The reaction mixture was shaken at room temperature.The progress of the reaction and the ee values of the products were followed by taking samples (10 µL) at intervals and analyzing them by HP 1090 Liquid Chromatograph equipped with Daicel Chiralcel (0.46 X 25 cm) OD-H column for compounds 1(b-f).In the case of compound 1a the samples (100 µL) were analyzed with a HP 6850 Series GC System equipped with a Cyclosil-B (30 m X 0.32 mm X 0.25 µm) column.

Lipase-catalyzed preparative scale kinetic resolution of rac-1(a-e)
The general procedure is described for rac-1c as a model compound.Lipase PS-D (1.09 g) was added to the solution of rac-1c (1.00 g, 4.36 mmol) and vinyl acetate (0.75 g, 8.72 mmol, 0.80 mL) in TBME (43 mL), and the mixture was shaken at room temperature for 1 h.The reaction was stopped by filtering off the enzyme.The enzyme was washed twice with TBME (2×10 ml).After evaporation, the unreacted (S)-1c and the produced (R)-2c were separated on silica gel by elution with ethyl acetate/hexane (3/7).
In the case of rac-1e vinyl butanoate was used as an acyl donor due to analytical problems in achieving good enantioseparation for the acetate product with the chiral HPLC method.

Lipase-catalyzed deprotection of (R)-2(a-e) by alcoholysis
The general procedure is described for rac-2c as a model compound.Lipase PS-D (0.27 g) and 1-butanol (0.15 mL) were added to the solution of (R)-2c (0.15 g, 0.55 mmol) in TBME (5.50 mL), and the mixture was shaken at room temperature for 1 h (to reach the conversion higher than 95%).The reaction was stopped by filtering off the enzyme.The produced (R)-1c was purified on a short silica gel column by elution with ethyl acetate/hexane (3/7).Conversion for the alcoholysis of (R)-1a was obtained using dihexyl ether as an external standard and for (R)-1b using acetophenone as an external standard.1,4-dimethoxybenzene was used in all the other cases.