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

Abstract

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 commercially 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.

1. 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].

Similar compounds, diethyl 1-carboxy-1-hydroxymethylphosphonate and its ester were previously synthesized, and enantioselective kinetic resolution of diethyl 1-carboxy-1-hydroxyphosphonate was investigated [16]. Dimethyl, dibutyl and diisopropyl 1-carboxy-1-hydroxymethylphosphonates were also previously synthesized as intermediates of the synthesis to obtain dioxolanone-substituted dialkyl phosphonates, and their spectroscopic data were not determined [17].

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 ³¹P 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 1-carboxy-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].

2. Results

2.1. Synthesis of Enzymatic Hydrolysis Substrates

Racemic dimethyl 1-carboxy-1-hydroxymethylphosphonate 1a and dibutyl 1-carboxy-1-hydroxymethylphosphonate 1b were obtained by the addition of dimethylphosphite or dibutylphosphite to glyoxylic acid with isolated yield reaching up to 98.1% for compound 1a and 82.3% for compound 1b. Afterwards, dimethyl 1-butyryloxy-1-carboxymethylphosphonate 2a and dibutyl 1-butyryloxy-1-carboxymethylphosphonate 2b were obtained by simple acylation of compounds 1 with butyryl chloride (see Scheme 1) with moderate yield reaching up 26.4% and 32.8% for compounds 2a and 2b, respectively.

2.2. 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. Lipase-catalyzed hydrolysis of dimethyl 1-butyryloxy-1-carboxymethylphosphonate 2a.

Lipase	Time [h]	Conversion [%]	ee of Product (R)	ee of Substrate (S)	E
CRL	168	12	>98	25	126
CAL	24	24	68	20	6.3
ANL	168	28	58	36	5.4
PCL	168	<10	-	-	-
PFL	168	<10	-	-	-
BCL	168	<10	-	-	-
MRL	168	<10	-	-	-
RNL	168	<10	-	-	-
MJL	168	<10	-	-	-
RML	168	<10	-	-	-
PPL	168	<10	-	-	-
N435	168	<10	-	-	-
ROL	168	<10	-	-	-
MCL	168	<10	-	-	-

). 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. Lipase-catalyzed hydrolysis of dibutyl 1-butyryloxy-1-carboxymethylphosphonate 2b.

Lipase	Time (h)	Conversion (%)	ee of Product (R)	ee of Substrate (S)	E
BCL	168	52	68	50	8.6
TLL	168	68	66	36	6.8
PCL	168	30	57	20	4.4
PFL	168	48	58	10	4.1
ANL	48	58	40	41	3.4
MRL	144	48	17	29	2.8
RNL	168	25	32	5	2.0
MJL	168	31	37	<5	-
CRL	20	30	40	<5	-
RSL	168	34	21 1	<5	-
RML	168	14	9 1	<5	-
PPL	96	43	<5	<5	-
N435	168	17	<5	<5	-
ROL	168	16	<5	<5	-
MCL	168	12	<5	<5	-

¹ Reversed configuration.

). As can be observed, only Aspergillus niger lipase hydrolyzed fairly well both substrates, and only compound 2b was hydrolyzed by all tested enzymes.

2.3. Optical Rotation of Compounds 1 and 2

After biotransformation and separation of the obtained products, 36 mg of compound (R)-1b (ee = 68%), 50 mg of compound (S)-2b (ee = 50%), 204 mg of compound (R)-1a (ee = 58%), and 104 mg of compound (S)-2a (ee = 36%) were obtained and their optical rotation was measured. The optical rotation for compound (R)-1b (ee = 68%) was [α]_D = +8.7 (c 2.0, CHCl₃, 22 °C); for compound (S)-2b (ee = 50%), it was [α]_D = −14.5 (c 2.0, CHCl₃, 22 °C); that for (R)-1a (ee = 58%) was [α]_D = +8.0 (c 2.0, CHCl₃, 22 °C); and for compound (S)-2a (ee = 36%) it was [α]_D = −2.8 (c 2.0, CHCl₃, 22 °C).

2.4. 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. ³¹P 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.

¹H NMR chemical shifts of the -CH₃ groups (OCH₂CH₂CH₂CH₃) of compound 3 were assigned as follows: (R,R), 0.82 ppm and 0.87 ppm; (S,R), 0.86 ppm and 0.90 ppm. In this case, it can be seen that the signals resulting from hydrogen atoms from -CH₃ groups of the isomer possessing (R)-configuration at α-carbon atom were upfield, compared to the same groups of the (S)-isomer (Figure 3).

¹H NMR chemical shifts of -OCH₃ groups of compound 3 were assigned as follows: (R,R), 3.68; (S,R), 3.55 ppm. In this case, it can be seen that the signals resulting from hydrogen atoms of the (S)-configuration isomer at the α-carbon atom were upfield, compared with the (R)-isomer (Figure 4).

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.

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 [α]_D = +3.5 (c 0.4, H₂O, 22 °C), while that for compound (R)-4b (ee = 32%) was [α]_D = +5.0 (c 0.4, H₂O, 22 °C). After comparison of optical rotation of 1-carboxy-1-methylphosphonic acids 4 obtained after hydrolysis of compounds 1a and 1b, the absolute configuration of compound 1a was determined to be the same as that of compound (R)-1b.

¹H, ³¹P, ¹³C, ¹H-¹H COSY, ¹H-¹³C HMQC and ¹H-¹³C HMBC spectra of compound 1a–4 are shown in Supplementary Materials.

3. 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-1-hydroxyphosphonate 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.

4. Materials and Methods

4.1. General Informations

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), POCh (Gliwice, Poland), or BIOCORP (Warsaw, Poland), and were used without further purification.

NMR (nuclear magnetic resonance) spectra were measured using a Bruker Avance™ 600 (Bruker, Karlsruhe, Germany) at 600.58 MHz for ¹H, 243.12 MHz for ³¹P, and 151.02 MHz for ¹³C; or on a Jeol ECZ 400S (Jeol Ltd., Tokyo, Japan) at 399.96 MHz for ¹H, 161.92 MHz for ³¹P, and 100.6 MHz for ¹³C, in CDCl₃ (99.8% of D, containing 0.03% v/v TMS) or in D₂O (99.8% of D, containing 0.75% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid). ¹H NMR were referenced to the internal standards TMS (δ = 0.00) or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (δ = 0.00), ¹³C NMR spectra to the central line of CHCl₃ (δ = 77.23), and 85% phosphoric acid in H₂O for ³¹P NMR spectra of synthesized compounds was used as external reference (δ = 0.00). Chemical shifts (δ) are reported in ppm, in relation to standards. The biotransformation products were analyzed by ³¹P NMR, with quinine used as a chiral solvating a(CSA) and without the addition of an external standard for more readable spectra. The assignment of signals in ¹H and ¹³C NMR was confirmed using ¹H-¹H COSY, ¹H-¹³C HMQC, and ¹H-¹³C HMBC spectra.

The optical rotation was measured in CHCl₃, using a polAAr-31 polarimeter at 578 nm (Optical Activity Ltd., Cambridgeshire, UK).

MS spectra were obtained using a high-resolution mass spectrometer with time-of-flight analyzer (TOFMS) from LCT PremierTM XE (Waters, Milford, MA, USA).

The synthesized compounds were purified by a medium-pressure liquid chromatography system: Combi Flash^® Rf 150 (Teledyne ISCO, Lincoln, NE, USA) on reversed phase column PuriFlash C18-HP, 15 μm, 120 g (Interchim, Montluçon, France) or by high-pressure liquid chromatography: ACCQ Prep HP125 UV–Vis (Teledyne ISCO, Lincoln, NE, USA) on reversed phase column Reprospher 100 C18, 5 μm, length: 250 mm, ID: 20 mm (Dr. Maish, Ammerbuch-Entringen, Germany).

The general procedure of purification using MPLC was as follows: 3 min of isocratic flow of pure water, 37 min from 0% to 100% of acetonitrile in water, 10 min of isocratic flow of pure acetonitrile; flow 10 mL/min, death time 3 min, R_f1a = 5 min, (compound 1a) R_f2a = 18 min (compound 2a), R_f1b = 26 min (compound 1b), and R_f2b = 30 min (compound 2b).

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).

4.2. 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.

Compound 1a:

Yield: 3.61 g, 98.1%

MS(TOF MS ES-) Calcd for C4H8O6P [M − H]⁻ 183.0058; found: 183.0064.

³¹P NMR δ(ppm): 19.78; ¹H NMR: δ(ppm) 3.91 (d, J = 11.0 Hz, 3H, OCH₃), 3.92 (d, J = 10.7 Hz, 3H, OCH₃), 4.62 (d, J = 16.3 Hz, 1H, PCH); ¹³C NMR δ(ppm): 54.71 (d, J = 6.9 Hz, 1C, OCH₃), 55.28 (d, J = 6.9 Hz, 1C, OCH₃), 68.48 (d, J = 157.7 Hz, 1C, PC), 170.36 (1C, COOH).

Compound 1b:

Yield: 4.41 g, 82.3%

MS(TOF MS ES+) Calcd for C10H22O6P [M + H]⁺ 269.1154; found: 269.1192, calcd for C10H21O6PNa [M + Na]⁺ 291.0974; found: 291.0971.

³¹P NMR δ(ppm): 17.27; ¹H NMR: δ(ppm) 0.93 (t, J = 7.4 Hz, 6H, OCH₂CH₂CH₂CH₃), 1.38–1.45 (m, 4H, OCH₂CH₂CH₂CH₃), 1.65–1.71 (m, 4H, OCH₂CH₂CH₂CH₃), 4.15–4.23 (m, 4H, OCH₂CH₂CH₂CH₃), 4.61 (d, J = 16.8 Hz, 1H, PCH); ¹³C NMR δ(ppm): 13.67 (2C, OCH₂CH₂CH₂CH₃), 18.71 (2C, OCH₂CH₂CH₂CH₃), 32.53 (d, J = 5.0 Hz, 1C OCH₂CH₂CH₂CH₃), 32.56 (d, J = 5.0 Hz, 1C OCH₂CH₂CH₂CH₃), 68.12 (d, J = 7.2 Hz, 1C, OCH₂CH₂CH₂CH₃), 68.44 (d, J = 7.2 Hz, 1C, OCH₂CH₂CH₂CH₃), 68.55 (d, J = 156.2 Hz, 1C, PC), 170.64 (1C, COOH).

4.3. 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.

Compound 2a:

Yield: 1.34 g, 26.4%

MS(TOF MS ES-) Calcd for C8H16O7P [M − H]⁻ 253.0477; found: 253.0478.

³¹P NMR δ(ppm): 17.25; ¹H NMR: δ(ppm) 0.98 (t, J = 7.4 Hz, 3H, OCOCH₂CH₂CH₃), 1.67–1.74 (m, 2H, OCOCH₂CH₂CH₃), 2.41–2.51 (m, 2H, OCOCH₂CH₂CH₃), 3.88 (d, J = 6.1 Hz, 3H, OCH₃), 3.90 (d, J = 6.1 Hz, 3H, OCH₃), 5.57 (d, J = 17.8 Hz, 1H, PCH); ¹³C NMR δ(ppm): 13.61 (1C, OCOCH₂CH₂CH₃), 18.41 (1C, OCOCH₂CH₂CH₃), 35.62 (1C, OCOCH₂CH₂CH₃), 54.89 (t, J = 6.9 Hz, 2C, OCH₃), 67.65 (d, J = 162.4 Hz, 1C, PC), 166.20 (1C, OCOCH₂CH₂CH₃), 172.18 (d, J = 10.2 Hz, 1C, COOH).

Compound 2b:

Yield: 2.22 g, 32.8%

MS(TOF MS ES+) Calcd for C14H28O7P [M + H]⁺ 339.1573; found: 339.1572.

³¹P NMR δ(ppm): 14.45; ¹H NMR: δ(ppm) 0.93 (t, J = 7.4 Hz, 6H, OCH₂CH₂CH₂CH₃), 0.98 (t, J = 7.4 Hz, 3H, OCOCH₂CH₂CH₃), 1.37–1.45 (m, 4H, OCH₂CH₂CH₂CH₃), 1.62–1.74 (m, 6H, OCOCH₂CH₂CH₃, OCH₂CH₂CH₂CH₃), 2.37–2.51 (m, 2H, OCOCH₂CH₂CH₃), 4.14–4.24 (m, 4H, OCH₂CH₂CH₂CH₃), 5.54 (d, J = 17.8 Hz, 1H, PCH); ¹³C NMR δ(ppm): 13.69 (3C, OCOCH₂CH₂CH₃, OCH₂CH₂CH₂CH₃), 18.44 (1C, OCOCH₂CH₂CH₃), 18.73 (1C, OCH₂CH₂CH₂CH₃), 18.74 (1C, OCH₂CH₂CH₂CH₃), 32.50 (d, J = 2.9 Hz, 1C, OCH₂CH₂CH₂CH₃), 32.54 (d, J = 2.9 Hz, 1C, OCH₂CH₂CH₂CH₃), 35.70 (1C, OCOCH₂CH₂CH₃), 68.40 (d, J = 6.7 Hz, 1C, OCH₂CH₂CH₂CH₃), 68.49 (d, J = 6.4 Hz, 1C, OCH₂CH₂CH₂CH₃), 67.94 (d, J = 148.1 Hz, 1C, PC), 166.55 (1C, OCOCH₂CH₂CH₃), 172.17 (d, J = 10.2 Hz, 1C, COOH).

4.4. Enzyme Source

ANL, Amano lipase A from Aspergillus niger (Sigma-Aldrich); CAL, Lipase A from Candida antarctica (Fluka, Buchs, Switzerland); CRL, Candida rugosa lipase (Sigma-Aldrich); PFL, Amano lipase AK, from Pseudomonas fluorescens (Sigma-Aldrich); MCL, Mucor circinelloides lipase (gift from Lodz University of Technology, Lodz, Poland); PCL, Amano Lipase G, from Penicillium camemberti (Sigma-Aldrich); PPL, Lipase Type II, Crude from Porcine Pancreas (Sigma-Aldrich); TLL, Lipase from Termomyces lanuginosus (Fluka); BCL, Amano Lipase PS from Burkholderia cepacia (Sigma-Aldrich); MRL, Mucor Racemosus lipase (gift from Lodz University of Technology); RNL, Lipase from Rhizopus niveus (Sigma-Aldrich); MJL, Amano Lipase M from Mucor javanicus (Sigma-Aldrich); RSL, Lipase from Rhizopus sp. (SERVA, Heidelberg, Germany); RML, Lipase from Rhizomucoer miehei produced in Aspergillus oryzae (Sigma-Aldrich); N435, Novozym^® 435, immobilised lipase from Candida antarctica B (Novozymes, Bagsværd, Denmark); ROL, Lipase from Rhizopus oryzae (Sigma-Aldrich).

4.5. 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 ³¹P 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.

4.6. Enantioselectivity Assignment

The mixtures of biotransformation products (alcohol and unreacted ester) were analyzed by ³¹P NMR spectroscopy using quinine as a chiral solvating agent. The degree of enantiomeric excess was expressed as a percentage (%), and is defined as:

$$\mathrm{ee}=\frac{P_1-P_2}{P_1+P_2}\times 100\%$$

(1)

where P₁ and P₂ 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]:

$$\mathrm{E}=\frac{\ln \left[{\mathrm{ee}}_p\frac{\left(1-{\mathrm{ee}}_s\right)}{\left({\mathrm{ee}}_s+{\mathrm{ee}}_p\right)}\right]}{\ln \left[{\mathrm{ee}}_p\frac{\left(1+{\mathrm{ee}}_s\right)}{\left({\mathrm{ee}}_s+{\mathrm{ee}}_p\right)}\right]}$$

(2)

where ee_p is the enantiomeric excess of product and ee_s is the enantiomeric excess of substrate.

4.7. Procedure of Determination of Optical Rotation and Determination of the Absolute Configuration

4.7.1. Preparation of Non-Equimolar Mixture of Enantiomers of Compounds 1 and 2 for Determination of Optical Rotation and of the Absolute Configuration

Butyryloxyphosphonates 2 were partially hydrolyzed by Amano Lipase PS from Burkholderia cepacia (compound 2b) or by Aspergillus niger lipase (compound 2a), according to the general procedure of enzymatic hydrolysis. Unreacted butyryloxyphosphonates 2 were separated from hydroxyphosphonates 1 by MPLC. Separated compounds were analyzed by NMR with quinine as a chiral solvating compound and optical rotation was measured.

4.7.2. Synthesis of Dibutyl 1-Carboxy-1-(3,3,3-Trifluoro-2-Methoxy-2-Phenylpropanoxy)Methylphosphonate (compound 3)

The non-equimolar mixture of enantiomers of compound 1b obtained according to the procedure described in Section 4.7.1, was acylated by (S)-(+)MTPA-Cl, according to the literature [17]. Dry pyridine (300 μL) was added to a dry bottle with septum using a syringe with a needle. Then, also using a syringe, (S)-(+)MTPA-Cl (50 μL, 0.27 mmol) was added. After that, compound 1b (36 mg, 0.15 mmol) dissolved in dry chloroform (300 μL) was added. The mixture was left for 24 h at room temperature. An excess of 3-dimethylamino-1-propylamine (50 μL, 0.40 mmol) was then added and, after 5 min at room temperature, the mixture was diluted with chloroform (10 mL), washed with cold dilute HCl (10 mL) and water (10 mL), and dried over anhydrous magnesium sulphate. After filtration of the drying agent, the ether was evaporated and compound 3 was purified by HPLC.

MS (TOF MS ES-) Calcd for C20H27O8PF3 [M − H]⁻ 483.1396; found: 483.1396.

Isomer (R,R)

³¹P NMR δ(ppm): 12.84; ¹H NMR: δ(ppm) 0.82 (t, J = 7.4 Hz, 3H, OCH₂CH₂CH₂CH₃), 0.87 (t, J = 7.4 Hz, 3H, OCH₂CH₂CH₂CH₃), 1.19–1.67 (m, 8H, OCH₂CH₂CH₂CH₃), 3.68 (s, 3H, OCH₃), 3.78-4.20 (m, 4H, OCH₂CH₂CH₂CH₃), 5.60 (d, J = 16.5 Hz, 1H, PCH), 7.37–7.43 (m, 3H, PC₆H₅), 7.67–7.71 (m, 2H, PC₆H₅); ¹³C NMR δ(ppm): 13.66 (1C, OCH₂CH₂CH₂CH₃), 13.69 (1C, OCH₂CH₂CH₂CH₃), 18.55 (1C, OCH₂CH₂CH₂CH₃), 18.67 (1C, OCH₂CH₂CH₂CH₃), 32.35 (d, J = 6.1 Hz, 1C OCH₂CH₂CH₂CH₃), 32.42 (d, J = 6.6 Hz, 1C OCH₂CH₂CH₂CH₃), 56.22 (1C, OCH₃), 68.71 (d, J = 6.9 Hz, 1C, OCH₂CH₂CH₂CH₃), 69.02 (d, J = 7.1 Hz, 1C, OCH₂CH₂CH₂CH₃), 69.56 (d, J = 145.6 Hz, 1C, PC), 84.59-85.35 (C(CF₃)), 123.22 (q, J = 288.9 Hz, CF₃), 127.58-132.06 (C(C₆H₅)), 163.88 (COO), 165.78 (d, J = 11.3 Hz, COOH).

Isomer (S,R)

³¹P NMR δ(ppm): 13.10; ¹H NMR: δ(ppm) 0.86 (t, J = 7.4 Hz, 3H, OCH₂CH₂CH₂CH₃), 0.90 (t, J = 7.4 Hz, 3H, OCH₂CH₂CH₂CH₃), 1.19–1.67 (m, 8H, OCH₂CH₂CH₂CH₃), 3.55 (s, 3H, OCH₃), 3.78–4.20 (m, 4H, OCH₂CH₂CH₂CH₃), 5.62 (d, J = 16.5 Hz, 1H, PCH), 7.37–7.43 (m, 3H, PC₆H₅), 7.61–7.65 (m, 2H, PC₆H₅); ¹³C NMR δ(ppm): 13.68 (1C, OCH₂CH₂CH₂CH₃), 13.70 (1C, OCH₂CH₂CH₂CH₃), 18.65 (1C, OCH₂CH₂CH₂CH₃), 18.72 (1C, OCH₂CH₂CH₂CH₃), 32.49 (d, J = 6.1 Hz, 1C OCH₂CH₂CH₂CH₃), 32.44 (d, J = 6.0 Hz, 1C OCH₂CH₂CH₂CH₃), 55.75 (1C, OCH₃), 68.66 (d, J = 7.0 Hz, 1C, OCH₂CH₂CH₂CH₃), 68.97 (d, J = 6.9 Hz, 1C, OCH₂CH₂CH₂CH₃), 69.49 (d, J = 145.1 Hz, 1C, PC), 84.59–85.35 (C(CF₃)), 123.19 (q, J = 288.9 Hz, CF₃), 127.58–132.06 (C(C₆H₅)), 164.74 (COO), 165.70 (d, J = 11.3 Hz, COOH).

4.7.3. Synthesis of 1-Carboxy-1-Methylphosphonic Acid 4

The non-equimolar mixture of enantiomers of compounds 1a and 1b was hydrolyzed by HCl. To compound 1 (1a, 46 mg, 0.25 mmol, ee = 18%; 1b, 54 mg, 0.20 mmol, ee = 32%), distilled water (10 mL) and 36% HCl (10 mL) were added. The hydrolysis reaction was carried out under reflux for 2 h. After cooling the mixture, the solvents were evaporated and compound 4 was purified by MPLC.

MS(TOF MS ES-) Calcd for C2H5O6P [M − H]⁻ 154.9745; found: 154.9750.

³¹P NMR δ(ppm): 14.25; ¹H NMR: δ(ppm) 4.63 (d, J = 18.2 Hz, 1H, PCH); ¹³C NMR δ(ppm): 71.89 (d, J = 147.0 Hz, 1C, PC), 175.99 (d, J = 1.8 Hz, 1C, COOH).