Synthesis, lipase catalyzed kinetic resolution, and determination of the absolute configuration of enantiomers of the Morita-Baylis-Hillman adduct 3-hydroxy-2-methylenebutanenitrile

3( RS )-Hydroxy-2-methylenebutanenitrile was synthesized from acetaldehyde and acrylonitrile with DABCO as a catalyst. Optimization of the reaction conditions was conducted because some literature procedures were not reproducible. Asymmetric transesterification of the Morita-Baylis-Hillman adduct was carried out using ten lipases. The hydroxyl-nitrile was also esterified chemically and subjected to enzymatic hydrolysis with the same set of enzymes. The best results were obtained with lipase from Candida antarctica and Novozyme 435. The absolute configuration of an optically pure hydroxyl-nitrile was determined by Mosher’s method


Introduction
The Morita-Baylis-Hillman (MBH) reaction is a useful tool for the synthesis of multifunctionalized compounds from electrophiles and activated alkenes (Scheme 1).Scheme 1.General scheme of the MBH reaction.
7][8] Biocatalysts [9][10][11] have also been employed as chiral auxiliaries in MBH reactions, but in general they are less effective in terms of both substrate conversion and stereoselectivity.3][14][15] We have focused recently on the possibility of using MBH adducts in the synthesis of novel δ-lactones with potential olfactory properties.We chose 3-(RS)-hydroxy-2methylenebutanenitrile 1(RS) as a model substrate for further studies.][18] Most studies of this sort have been carried out using ring-substituted benzaldehydes.
β-Hydroxy-nitriles are less commonly known to possess interesting aroma profiles but there is some evidence of their usefulness as fragrance agents. 19The synthesis of Morita-Baylis-Hillman adducts in the absence of chiral auxiliaries results in stereomeric mixtures of products.1][22] The use of enzymes for the kinetic resolution of secondary carbinols is well documented, 23,24 and has proved to be economically and environmentally advantageous.Previous studies regarding biocatalytic resolution of enantiomers of 3(RS)-hydroxy-2methylenebutanenitrile 1(RS) were focused on the optimization of transesterification reaction conditions using lipase from Pseudomonas cepacia. 25The optimized reaction conditions resulted in a moderate conversion of MBH adduct (27%) with excellent ee (98%).A major disadvantage of this procedure is the very long reaction time (474 h), which makes it unpractical.Rotation of the plane of polarized light of the acetylated product was described as levorotary but the specific rotatory power was not determined.Adduct 1(RS) was also used to study nitrile hydrolase activity of Rhodococcus erythropolis SET1. 26The yield of recovered nitrile and reaction time were satisfactory (42% and 48 h respectively); ee was moderate (76.5%).The configuration of the stereogenic secondary carbinol center was assigned only tentatively (R), and the specific rotatory power also was not determined.
The aim of studies presented herein was to optimize the synthesis and biocatalytic kinetic resolution of racemic aliphatic MBH adduct to give optically pure model β-hydroxy-nitriles and their acetates.In addition, we aimed to determine the absolute configuration of the 3-hydroxy-2-methylenebutanenitrile.

Results and Discussion
3(RS)-Hydroxy-2-methylenebutanenitrile 1(RS) was prepared by the Morita-Baylis-Hillman reaction of acetaldehyde and acrylonitrile using Optimization of the reaction conditions was carried out in a solvent-free environment (Table 1), and in THF, 1,4-dioxane, and their water mixtures (Table 2).An accelerated rate and increased yields in the Morita-Baylis-Hillman reaction of acetaldehyde and acrylonitrile was observed, as in previous reports regarding aromatic aldehydes and activated alkenes. 18,27In general, a stoichiometric amount of a base is usually used for MBH transformations.In this report we show that an increase in the molar ratio of DABCO results in lower product yields (entries 1-4).Increasing the molar ratio of acetaldehyde is more favorable than of acrylonitrile, and results in higher yields and fewer side products (entries 5 and 6).Optimization of the reaction conditions was also carried out in THF, 1,4-dioxane and their water mixtures using equimolar amounts of both substrates, and 20 mol% DABCO.Yields of MBH transformations in pure solvents were moderate to poor (entries 1 and 4), but in their water mixtures reactions yields were excellent.Similar outcomes were obtained in the coupling of acetaldehyde and methyl acrylate in organic solvent-water systems. 28ble 2. Optimization of MBH reaction of acetaldehyde and acrylonitrile carried out in THF, 1,4-dioxane, and their mixtures with water a Molar ratio.b Volume ratio.c Isolated yield.
The first attempt at kinetic resolution of 1(RS) was based on enzymatic transesterification (Scheme 3) under anhydrous conditions with vinyl acetate as an acyl donor.We chose ten lipases for the initial screening (Table 3).Entry numbers in Table 3 identify the enzymes used in Tables 4 and 5.As a result of these biotransformations we were able to obtain acetate 2(S) in excellent enantiomeric excess, and moderate to very good yield (Table 4, entries 1-3 and 8).Novozyme 435 and lipase A from Candida antarctica transformed S-alcohol most rapidly (Table 4 entries 1 and 8; 25% and 39% of substrate conversion after 1 h).PS Amano lipase from Pseudomonas cepacia and immobilized lipozyme from Mucor miehei required more time to achieve higher conversion, and in these cases there was little or no effect on enantioselectivity.
The second strategy for obtaining pure enantiomers of 3-hydroxy-2-methylenebutanenitrile was enzymatic hydrolysis of 3(RS)-acetoxy-2-methylenebutanenitrile 2(RS), which was synthesized from 1(RS) using acetyl chloride and pyridine in anhydrous dichloromethane (Scheme 4).Both compounds were found to be not useful as fragrances: alcohol 1(RS) is odorless, and acetate 2(RS) causes irritation to the nasal mucosa.Biotransformations were carried out using the same set of enzymes at room temperature with shaking (150 rpm) in a biphasic system consisting of a phosphate buffer (pH 7) and i-Pr 2 O (Scheme 4).The three tested enzymes gave acceptable results (Table 5, entries 1,2 and 8).It can be seen that enzymatic hydrolysis results in higher conversions than transesterification reactions, but at the expense of enantioselectivity.The (S)-acetate is consumed considerably faster than the R-isomer, but the hydrolysis in a biphasic system is a slower process, and in this case it requires 24 h to obtain excellent ees of an acetate (>99%).Conversions above 50% indicate that both isomers of acetate 2 are being consumed.Careful monitoring of the hydrolysis progress is required to obtain optically pure 1(S).The last phase of this study was determination of the absolute configuration of enantiomers of the Morita-Baylis-Hillman adduct 1.We chose the Mosher ester method. 29Optically pure acetate obtained via enzymatic hydrolysis (intact substrate, Table 5 entry 2, 24 h) was hydrolyzed chemically in a biphasic system.Subsequent esterification with both enantiomers of Mosher's acid chloride in anhydrous conditions gave two esters 3(R,R) and 3(S,R) (Scheme 5).
O N

2(R)
OH N All relevant chemical shifts of Mosher's esters are shown in Table 6.Methyl and hydrogens of the (S)-ester lie downfield with respect to peaks of the (R)-ester, and methylene hydrogens the opposite.This shielding/deshielding pattern (Figure 1) indicates that initial alcohol has the R configuration.

Conclusion
We report above the strategy of the synthesis of the multifunctional building block 3-hydroxy-2-methylenebutanenitrile 1, its kinetic resolution into optically pure isomers, and determination of the absolute configuration of the isomers of the title compound.Solvent-free Morita-Baylis-Hillman reaction has proven to give the best results (Table 1 entry 1).Two strategies of kinetic resolution allowed us to obtain the (S)-acetate with excellent ee (97% enzymatic transesterification -Table 4 entry 1), and optically pure (S)-alcohol (>99% -Table 5 entry 1) and (R)-acetate (>99% -Table 5 entries 1,2 and 8).The absolute configuration of the enzymatic hydrolysis product (Scheme 5) was determined by Mosher's method; the shielding/deshielding pattern indicates that the initial alcohol has the R configuration.

Experimental Section
General.All reagents obtained from commercial suppliers (Sigma-Aldrich, Poznań, Poland; Merck, Warszawa, Poland; Avantor, Gliwice, Poland) were of analytical grade and were used without further purification.The course of all reactions, composition of products, and their purities were monitored by gas chromatography (GC).Analytical GC was performed on Agilent 7890A apparatus with flame ionization detector with nitrogen as make up gas.Analyses were carried out on Thermo TR-5 column [(5% phenyl)-methylpolysiloxane] 30 m×0.32 mm ID×1.0 µm film using helium as carrier gas at a flow rate of 3.8462 mL/min in a split ratio of 1:100 and the following programme: (a) 40 °C for 5 min; (b) rate of 10.0 °C/min from 40 to 190 °C and hold for 5 min.Chiral gas chromatography was carried out with Varian CP Chirasil-Dex CB column, 25 m x 0.25 mm ID x 0.25 μm film.Analyses were carried out using helium as carrier gas, flow 0.5 mL/min, split ratio 35:1 with programme: (a) 120 °C for 10 min.Injector and detector were held at 250 °C.Injector and detector were held at 250 °C.High resolution mass spectra (HRMS) were recorded using electron spray ionization (ESI) technique on Waters LCT Premier XE mass spectrometer.The middle-infrared spectra (4000-400 cm -1 ) were collected on a Fourier transform, Bruker VERTEX 70 V vacuum spectrometer equipped with an air-cooled DTGS detector (Ettlingen, Germany).The liquid samples were placed on the diamond crystal of the ATR accessory.The spectral data were recorded at a resolution of 2 cm -1 with 64 scans collection and further elaborated using Bruker OPUS software. 1H and 13 C NMR spectra were recorded in CDCl 3 with TMS as an internal standard on Bruker Avance TM DRX 600 instrument.Chemical shifts (δ) are reported in ppm and coupling constants (J) are given in Hz.Optical rotation measurements were carried out on a Krüss P3000 automatic polarimeter.

Scheme 5 .
Scheme 5. Synthesis of Mosher's esters from the enzymatic hydrolysis product.

Table 1 .
Optimization of MBH reaction of acetaldehyde and acrylonitrile under solvent-free conditions a Molar ratio.b Isolated yield.

Table 3 .
Biocatalysts selected for the screening procedure