Lipase-Catalyzed Kinetic Resolution of Dimethyl and Dibutyl 1-Butyryloxy-1-Carboxymethylphosphonates

: The main objective of this study is the enantioselective synthesis of carboxyhydroxyphosphonates by lipase-catalyzed reactions. For this purpose, racemic dimethyl and dibutyl 1-butyryloxy-1-carboxymethylphosphonates were synthesized and hydrolyzed, using a wide spectrum of commer-cially available lipases from different sources (e.g., fungi and bacteria). The best hydrolysis results of dimethyl 1-butyryloxy-1-carboxymethylphosphonate were obtained with the use of lipases from Candida rugosa , Candida antarctica , and Aspergillus niger , leading to optically active dimethyl 1-carboxy-1-hydroxymethylphosphonate (58%–98% enantiomeric excess) with high enantiomeric ratio (reaching up to 126). However, in the case of hydrolysis of dibutyl 1-butyryloxy-1-carboxymethylphosphonate, the best results were obtained by lipases from Burkholderia cepacia and Termomyces lanuginosus , leading to optically active dibutyl 1-carboxy-1-hydroxymethylphosphonate (66%–68% enantiomeric excess) with moderate enantiomeric ratio (reaching up to 8.6). The absolute conﬁguration of the products after biotransformation was also determined. In most cases, lipases hydrolyzed ( R ) enantiomers of both compounds.


Introduction
Enantioselective biocatalysis has long been used as an alternative to traditional methods for obtaining pure chemical isomers [1]. It has been used in many industrial fields; in particular, the use of whole-cell biocatalysts and enzymes has become common in producing enantiomeric active drugs [2][3][4][5]. This is due to the associated highly regioselective and enantioselective reactions, carried out mainly in water under mild conditions [6]. Biotransformations are applied to obtain enantiopure compounds by enantioselective reactions, such as deracemization, desymmetrization, or asymmetric synthesis [7,8]. Hydroxyphosphonates are among the countless different classes of compounds obtained by enantioselective biocatalysis [9,10]. These compounds have a wide range of biological properties, serving as antibacterial, antiviral, and anticancer agents, as well as enzyme inhibitors or pesticides; they can also be used as precursors of other biologically active compounds [11][12][13][14]. Their bioavailability depends on their three-dimensional structure; therefore, obtaining them as enantiopure isomers with a specific absolute configuration is crucial for therapeutic effectiveness and drug safety [15].
The aim of this study was to synthesize two-as yet unexploited-1-carboxy-1hydroxymethylphosphonates, obtaining them with good enantiomeric excesses. For this The aim of this study was to synthesize two-as yet unexploited-1-carboxy-1-hydroxymethylphosphonates, obtaining them with good enantiomeric excesses. For this purpose, biocatalytic hydrolysis by lipases was carried out. Despite the fact that the biological activity of carboxyhydroxyphosphonates is unknown, pure enantiomers of these compounds may be used as chiral auxiliaries, useful in 31 P NMR spectroscopy to determine the enantiomeric purity and absolute configuration of different classes of compounds [18]. Moreover, they can be used as precursors of aminophosphonates [12], due to the fact that many of aminophosphonic acids are biologically active [19]. Dimethyl 1carboxy-1-hydroxymethylphosphonate can be transformed to its (4-nitrophenyl)methyl ester, which can be used in the synthesis of 2-substitued-3-carboxy carbapenem antibiotics [20].

Enzymatic Hydrolysis
Compounds 2 were hydrolyzed by different lipases, in order to obtain optically pure hydroxyphosphonates 1 (Scheme 2 and Figure 1). It was only possible to obtain compound 1a with an ee > 98% when using a lipase from Candida rugosa (Table 1). Satisfying results were obtained during the hydrolysis of butyryloxyphosphonate 2a using Candida antarctica and Aspergillus niger lipases (enantioselectivities of 6.3 and 5.4, respectively). Hydroxyphosphonate 1b was obtained with moderate enantioselectivity when using Burkholderia cepacia and Termomyces lanuginosus lipases (enantioselectivities of 8.6 and 6.8, respectively; Table 2). As can be observed, only Aspergillus niger lipase hydrolyzed fairly well both substrates, and only compound 2b was hydrolyzed by all tested enzymes. Scheme 2. Biocatalytic hydrolysis of compounds 2. Scheme 1. Synthesis of 1-carboxy-1-hydroxymethylphosphonates 1 and 1-butyryloxy-1-carboxymethylphosphonates 2.

Enzymatic Hydrolysis
Compounds 2 were hydrolyzed by different lipases, in order to obtain optically pure hydroxyphosphonates 1 (Scheme 2 and Figure 1). It was only possible to obtain compound 1a with an ee > 98% when using a lipase from Candida rugosa (Table 1). Satisfying results were obtained during the hydrolysis of butyryloxyphosphonate 2a using Candida antarctica and Aspergillus niger lipases (enantioselectivities of 6.3 and 5.4, respectively). Hydroxyphosphonate 1b was obtained with moderate enantioselectivity when using Burkholderia cepacia and Termomyces lanuginosus lipases (enantioselectivities of 8.6 and 6.8, respectively; Table 2). As can be observed, only Aspergillus niger lipase hydrolyzed fairly well both substrates, and only compound 2b was hydrolyzed by all tested enzymes.
with an ee > 98% when using a lipase from Candida rugosa (Table 1). Satisfying results were obtained during the hydrolysis of butyryloxyphosphonate 2a using Candida antarctica and Aspergillus niger lipases (enantioselectivities of 6.3 and 5.4, respectively). Hydroxyphosphonate 1b was obtained with moderate enantioselectivity when using Burkholderia cepacia and Termomyces lanuginosus lipases (enantioselectivities of 8.6 and 6.8, respectively;

Determination of the Absolute Configuration
The Mosher method based on the NMR technique is one of the most commonly used methods for the determination of absolute configuration when obtaining a pure isomer is impossible [21]. In this case, double derivatization was used. Dibutyl 1-carboxy-1-hydroxymethylphosphonate 1b, in a 1.0:1.9 molar ratio of isomers (S):(R) (ee = 32%), obtained after biotransformation of 2b by Burkholderia cepacia lipase, was acylated by (S)-(+)-MTPA-Cl, resulting in a mixture of (S,R):(R,R) isomers of Mosher ester 3 with molar ratio of 1.0:1.7. 31 P NMR chemical shifts of Mosher ester 3 were assigned as follows: (S,R), 13.10 ppm; (R,R), 12.84 ppm. The signals resulting from the phosphorus atom of the isomer possessing (R)-configuration at α-carbon atom were upfield, compared to the (S)-isomer, as can be seen from Figure 2.

Determination of the Absolute Configuration
The Mosher method based on the NMR technique is one of the most commonly used methods for the determination of absolute configuration when obtaining a pure isomer is impossible [21]. In this case, double derivatization was used. Dibutyl 1-carboxy-1-hydroxymethylphosphonate 1b, in a 1.0:1.9 molar ratio of isomers (S):(R) (ee = 32%), obtained after biotransformation of 2b by Burkholderia cepacia lipase, was acylated by (S)-(+)-MTPA-Cl, resulting in a mixture of (S,R):(R,R) isomers of Mosher ester 3 with molar ratio of 1.0:1.7. 31 P NMR chemical shifts of Mosher ester 3 were assigned as follows: (S,R), 13.10 ppm; (R,R), 12.84 ppm. The signals resulting from the phosphorus atom of the isomer possessing (R)-configuration at α-carbon atom were upfield, compared to the (S)-isomer, as can be seen from Figure 2.

Determination of the Absolute Configuration
The Mosher method based on the NMR technique is one of the most commonly used methods for the determination of absolute configuration when obtaining a pure isomer is impossible [21]. In this case, double derivatization was used. Dibutyl 1-carboxy-1-hydroxymethylphosphonate 1b, in a 1.0:1.9 molar ratio of isomers (S):(R) (ee = 32%), obtained after biotransformation of 2b by Burkholderia cepacia lipase, was acylated by (S)-(+)-MTPA-Cl, resulting in a mixture of (S,R):(R,R) isomers of Mosher ester 3 with molar ratio of 1.0:1.7. 31 P NMR chemical shifts of Mosher ester 3 were assigned as follows: (S,R), 13.10 ppm; (R,R), 12.84 ppm. The signals resulting from the phosphorus atom of the isomer possessing (R)-configuration at α-carbon atom were upfield, compared to the (S)-isomer, as can be seen from Figure 2.    Comparison of all spectra allowed for assignment of the absolute configuration of enantiomers of compounds 1b and 2b (Figure 1).
After biotransformation and separation of obtained products, 46 mg of compound 1a (ee = 18%) and 54 mg of compound 1b (major enantiomer R, ee = 32%) were obtained. Both were hydrolyzed by HCl, and 1-carboxy-1-methylphosphonic acids 4 (Scheme 3) were obtained and their optical rotation was measured. The optical rotation for compound 4a (ee = 18%) was  Comparison of all spectra allowed for assignment of the absolute configuration of enantiomers of compounds 1b and 2b (Figure 1). Synthesis of dimethyl 1-carboxy-1-(3,3,3-trifluoro-2-methoxy-2-phenylpropanoxy)methylphosphonates was unsuccessful. For this reason, it was decided to determine the absolute configuration of compound 1a using a different approach.

Discussion
Obtaining optically pure hydroxyphosphonates has great potential; however, there are no easy, well-trodden paths to achieving this goal. Lipolytic enzymes have long been used for this purpose; however, the research conducted so far has indicated that the appropriate enzyme should be selected for each hydroxyphosphonate ester. This was also the case with the hydrolysis of butyryloxyphosphonates 2. Hydroxyphosphonate 1a was obtained with very good enantiomeric excess: >98% in the case of using the lipase from Candida rugosa to hydrolyze dimethyl butyryloxyphosphonate 2a. To obtain optically active hydroxyphosphonate 1b, it is better to use Burkholderia cepacia lipase.

Discussion
Obtaining optically pure hydroxyphosphonates has great potential; however, there are no easy, well-trodden paths to achieving this goal. Lipolytic enzymes have long been used for this purpose; however, the research conducted so far has indicated that the appropriate enzyme should be selected for each hydroxyphosphonate ester. This was also the case with the hydrolysis of butyryloxyphosphonates 2. Hydroxyphosphonate 1a was obtained with very good enantiomeric excess: >98% in the case of using the lipase from Candida rugosa to hydrolyze dimethyl butyryloxyphosphonate 2a. To obtain optically active hydroxyphosphonate 1b, it is better to use Burkholderia cepacia lipase.
In previous work [16], enantioselective kinetic resolution of diethyl 1-carboxy-1hydroxyphosphonate by lipases and whole-cell biocatalysts (bacteria and fungi) was investigated. The best results were obtained when Aspergillus niger lipase (enantiomeric ratio E = 15.5) and whole cells of Aspergillus parasiticus (E = 30.6) were used. Comparing the results described in the previous and in the present work it can be seen that lipases catalyze butyryloxycarboxyphosphonates with moderate enantioselectivity and only Aspergillus niger lipase hydrolyzes fairly well all three substrates. Lipases from this fungus, both in the form of a purified enzyme and a lipase produced during a biotransformation reaction using whole cells of the microorganism, have often been used for the preparation of optically active hydroxyphosphonates [9,16,22]. However, in order to obtain pure enantiomers of hydroxyphosphonates the catalyst should be selected individually for each compound.
One of the challenges posed in these studies was to determine the absolute configuration of all biotransformation products. In the case of hydroxyphosphonate 1b, it was possible to determine the configuration of the enantiomer formed after the biotransformation of butyryloxyphosphonate 2b using the Mosher method. However, in the case of hydroxyphosphonate 1a, it was not possible to obtain the corresponding Mosher ester. In this case, an indirect method was used. Hydroxyphosphonates 1a and 1b obtained after biotransformations were hydrolyzed to compound 4 for optical rotation comparison. This allowed for determination of the absolute configuration of hydroxyphosphonate 1a and confirmed that most of the enzymes hydrolyzed the (R)-enantiomer of butyryloxyphosphonates 2 with greater selectivity.
MS spectra were obtained using a high-resolution mass spectrometer with time-offlight analyzer (TOFMS) from LCT PremierTM XE (Waters, Milford, MA, USA).
The general procedure of purification using HPLC was as follows: 12 min of isocratic flow of pure water, 8 min from 0% to 30% of acetonitrile in water, 12 min of isocratic flow of 30% acetonitrile in water, 4 min from 30% to 40% of acetonitrile in water, 15 min of isocratic flow of 40% acetonitrile in water, 10 min from 40% to 60% of acetonitrile in water, 10 min from 60% to 100% of acetonitrile in water, 10 min of isocratic flow of pure acetonitrile; flow 10 mL/min, death time 5 min, R f3 = 60 min (compound 3).

General Procedure of Synthesis of Dimethyl and Dibutyl 1-Carboxy-1-Hydroxymethylphosphonates 1
The procedure of synthesis of compounds 1 was carried out according to the procedure for similar compounds described previously [16,23]. To diethyl or dibutyl phosphonate (20 mmol), glyoxylic acid monohydrate (1.84 g, 20 mmol) was added, followed by the addition of triethylamine (2.79 mL, 20 mmol). All compounds were stirred for 2 h at room temperature. The crude residue was dissolved in 10 mL distilled water and triethylamine was removed by ion exchange chromatography (Dowex ® 50WX8 50-100 mesh, Sigma Aldrich). The crude residue was purified by MPLC, giving the product as a colorless oily liquid.

General Procedure of Synthesis of Dimethyl and Dibutyl 1-Butyryloxy-1-Carboxymethylphosphonates 2
The procedure of synthesis of compounds 2 was carried out according to the procedure for similar compounds described previously [16,24]. To diethyl or dibutyl phosphonate (20 mmol), glyoxylic acid monohydrate (1.84 g, 20 mmol) was added, followed by the addition of triethylamine (2.79 mL, 20 mmol). All compounds were stirred for 2 h at room temperature. The reaction mixture was placed in an ice bath, it was all dissolved in 100 mL of chloroform, and 2.07 mL (20 mmol) of butyryl chloride was slowly added dropwise. After completion of the reaction-which was monitored by TLC-the resulting solution was extracted with 100 mL of distilled water, and the organic phase was dried by anhydrous magnesium sulphate and evaporated. The crude residue was purified by MPLC, giving the product as a colorless oily liquid.

Enzymatic Hydrolysis General Procedure
Lipase-catalyzed reactions were prepared according to a procedure described previously [22]. Reactions were carried out in a biphasic system (3.8 mL) consisting of 0.05 M phosphate buffer (pH 7.0, 3.0 mL) and a mixture of diisopropyl ether (0.2 mL) with hexane (0.6 mL). After addition of 0.2 mmol of substrate (51 mg or 68 mg respectively) and 100 mg of a suitable lipase, reactions were carried out at room temperature with shaking (150 rpm) and stopped after certain periods of time, when the conversion degree reached up to 50%, by the addition of 2 mL of acetone and the filtration of precipitated protein. The solvent was evaporated, and the residues were dissolved in 5 mL of distilled water. The obtained solutions were purified by ion exchange chromatography on a column filled with Dowex (200-400 mesh), with the water as eluent. Then, the organic solvent was evaporated, and the products were analyzed by means of 31 P NMR spectroscopy. In the case where there was no clear separation of signals derived from enantiomers, the analysis was repeated with quinine as a chiral solvating agent.

Enantioselectivity Assignment
The mixtures of biotransformation products (alcohol and unreacted ester) were analyzed by 31 P NMR spectroscopy using quinine as a chiral solvating agent. The degree of enantiomeric excess was expressed as a percentage (%), and is defined as: where P 1 and P 2 are the values of the area under the signals coming from the major and minor enantiomers of the product or substrate, respectively. The enantiomeric ratio (E) was computed from the following formula [25]: where ee p is the enantiomeric excess of product and ee s is the enantiomeric excess of substrate.