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

catalysts

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

Paulina Majewska

Laboratory of Biotechnology, Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeze˙ Wyspia´nskiego27, 50-370 Wrocław, Poland; [email protected]; Tel.: +48-71-320-4614

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 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. Keywords: biocatalysis; hydroxyphosphonates; lipolytic activity; determination of absolute configu- Citation: Majewska, P. Lipase- ration; enantiomers Catalyzed Kinetic Resolution of Dimethyl and Dibutyl 1-Butyryloxy-1- carboxymethylphosphonates. Catalysts 2021, 11, 956. https:// 1. Introduction doi.org/10.3390/catal11080956 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; Academic Editor: Takeshi Sugai in particular, the use of whole-cell biocatalysts and enzymes has become common in producing enantiomeric active drugs [2–5]. This is due to the associated highly regioselective Received: 15 July 2021 and enantioselective reactions, carried out mainly in water under mild conditions [6]. Accepted: 9 August 2021 Published: 10 August 2021 Biotransformations are applied to obtain enantiopure compounds by enantioselective reactions, such as deracemization, desymmetrization, or asymmetric synthesis [7,8]. Hy-

Publisher’s Note: MDPI stays neutral droxyphosphonates are among the countless different classes of compounds obtained by with regard to jurisdictional claims in enantioselective biocatalysis [9,10]. These compounds have a wide range of biological published maps and institutional affil- properties, serving as antibacterial, antiviral, and anticancer agents, as well as enzyme iations. inhibitors or pesticides; they can also be used as precursors of other biologically active compounds [11–14]. 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 Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. were previously synthesized, and enantioselective kinetic resolution of diethyl 1-carboxy-1- This article is an open access article hydroxyphosphonate was investigated [16]. Dimethyl, dibutyl and diisopropyl 1-carboxy- distributed under the terms and 1-hydroxymethylphosphonates were also previously synthesized as intermediates of the conditions of the Creative Commons synthesis to obtain dioxolanone-substituted dialkyl phosphonates, and their spectroscopic Attribution (CC BY) license (https:// data were not determined [17]. creativecommons.org/licenses/by/ The aim of this study was to synthesize two—as yet unexploited—1-carboxy-1- 4.0/). hydroxymethylphosphonates, obtaining them with good enantiomeric excesses. For this

Catalysts 2021, 11, 956. https://doi.org/10.3390/catal11080956 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, x FOR PEER REVIEW 2 of 11

The aim of this study was to synthesize two—as yet unexploited—1-carboxy-1-hy- Catalysts 2021, 11, 956 droxymethylphosphonates, obtaining them with good enantiomeric excesses. For2 ofthis 10 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 deter- minepurpose, the biocatalyticenantiomeric hydrolysis purity and by lipasesabsolute was configuration carried out. of Despite different the factclasses that of the com- bio- poundslogical activity [18]. Moreover, of carboxyhydroxyphosphonates they can be used as precursors is unknown, of aminophosphonates pure enantiomers [12], of these due compounds may be used as chiral auxiliaries, useful in 31P NMR spectroscopy to determine to the fact that many of aminophosphonic acids are biologically active [19]. Dimethyl 1- the enantiomeric purity and absolute conﬁguration of different classes of compounds [18]. carboxy-1-hydroxymethylphosphonate can be transformed to its (4-nitrophenyl)methyl Moreover, they can be used as precursors of aminophosphonates [12], due to the fact ester, which can be used in the synthesis of 2-substitued-3-carboxy carbapenem antibiotics that many of aminophosphonic acids are biologically active [19]. Dimethyl 1-carboxy-1- [20]. 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 2.1.2. Results Synthesis of Enzymatic Hydrolysis Substrates 2.1. SynthesisRacemic ofdimethyl Enzymatic 1-carboxy- Hydrolysis1-hydroxymethylphosphonate Substrates 1a and dibutyl 1-carboxy-1-hydroxymethylphosphonateRacemic dimethyl 1-carboxy-1-hydroxymethylphosphonate 1b were obtained by the1a and addition dibutyl 1-carboxy-of dime- thylphosphite1-hydroxymethylphosphonate or dibutylphosphite1b were to glyoxylic obtained byacid the with addition isolated of dimethylphosphiteyield reaching up orto 98.1%dibutylphosphite for compound to glyoxylic 1a and 82.3% acid with for compound isolated yield 1b reaching. Afterwards, up to dimethyl 98.1% for 1-butyryloxy- compound 1a 1-carboxymethylphosphonateand 82.3% for compound 1b. Afterwards, 2a and dibutyl dimethyl 1-butyryloxy-1-carboxymethylphosphonate 1-butyryloxy-1-carboxymethylphosphonate 2b2a andwere dibutyl obtained 1-butyryloxy-1-carboxymethylphosphonate by simple acylation of compounds 1 with2b butyrylwere obtained chloride by (see simple Scheme acy- 1)lation with of moderate compounds yield1 with reaching butyryl up chloride26.4% and (see 32.8% Scheme for1 )compounds with moderate 2a and yield 2b reaching, respectively.up 26.4% and 32.8% for compounds 2a and 2b, respectively.

O O Cl O O O O O O O R P R P O NEt3 R P O O OH O H + OH O OH R O CH Cl R H R OH 3 O

1: a R = CH3 2: a R = CH3 b R = C4H9 b R = C4H9 Scheme 1.1. SynthesisSynthesis ofof 1-carboxy-1-hydroxymethylphosphonates1-carboxy-1-hydroxymethylphosphonates 11 andand 1-butyryloxy-1-carboxy- 1-butyryloxy-1-carbox- ymethylphosphonatesmethylphosphonates 2 .2.

2.2. Enzymatic Enzymatic Hydrolysis Compounds 22 werewere hydrolyzed hydrolyzed by by different different lipases, lipases, in order in order to obtain to obtain optically optically pure hydroxyphosphonatespure hydroxyphosphonates 1 (Scheme1 (Scheme 2 and Figure2 and 1). Figure It was1). only It was possible only to possible obtain compound to obtain 1a Candida rugosa 1acompound with an ee with> 98% an when ee > 98%using when a lipase using from a lipase Candida from rugosa (Table 1).(Table Satisfying1). Satisfying results results were obtained during the hydrolysis of butyryloxyphosphonate 2a using Candida were obtained during the hydrolysis of butyryloxyphosphonate 2a using Candida antarc- antarctica and Aspergillus niger lipases (enantioselectivities of 6.3 and 5.4, respectively). tica and Aspergillus niger lipases (enantioselectivities of 6.3 and 5.4, respectively). Hydrox- Hydroxyphosphonate 1b was obtained with moderate enantioselectivity when using yphosphonate 1b was obtained with moderate enantioselectivity when using Burkholderia Burkholderia cepacia and Termomyces lanuginosus lipases (enantioselectivities of 8.6 and 6.8, cepacia and Termomyces lanuginosus lipases (enantioselectivities of 8.6 and 6.8, respectively; respectively; Table2). As can be observed, only Aspergillus niger lipase hydrolyzed fairly Table 2). As can be observed, only Aspergillus niger lipase hydrolyzed fairly well both sub- well both substrates, and only compound 2b was hydrolyzed by all tested enzymes. strates, and only compound 2b was hydrolyzed by all tested enzymes. 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 - - - SchemePFL 2. Biocatalytic 168 hydrolysis of <10 compounds 2. - - - 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 - - - Catalysts 2021, 11, x FOR PEER REVIEW 2 of 11

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 dime- thylphosphite 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.

O O Cl O O O O O O O R P R P O NEt3 R P O O OH O H + OH O OH R O CH Cl R H R OH 3 O

1: a R = CH3 2: a R = CH3 b R = C4H9 b R = C4H9 Scheme 1. Synthesis of 1-carboxy-1-hydroxymethylphosphonates 1 and 1-butyryloxy-1-carboxymethylphosphonates 2.

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). 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). Hydrox- yphosphonate 1b was obtained with moderate enantioselectivity when using Burkholderia

Catalysts 2021, 11, 956 cepacia and Termomyces lanuginosus lipases (enantioselectivities of 8.6 and 6.8, respectively;3 of 10 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.

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Scheme 2. Biocatalytic hydrolysis of compounds 2.

Figure 1. 31P31 NMRP NMR spectrum spectrum of products of products after afterbiotrans biotransformationformation of dibutyl of dibutyl 1-butyryloxy-1-carbox- 1-butyryloxy-1- carboxymethylphosphonateymethylphosphonate by Amano by Amano Lipase Lipase PS from PS Burkholderia from Burkholderia cepacia cepacia after 168after h. 168 h.

TableTable 2.1. Lipase-catalyzedLipase-catalyzed hydrolysishydrolysis ofof dibutyldimethyl 1-butyryloxy-1-carboxymethylphosphonate 1-butyryloxy-1-carboxymethylphosphonate2b .2a. ee of Product ee of Substrate LipaseLipase Time Time [h] (h) Conversion Conversion (%) [%] ee of Product (R) ee of Substrate (S)EE BCL 168 52 68(R) (S 50) 8.6 CRLTLL 168 168 68 12 66 >98 25 36 126 6.8 CALPCL 24 168 30 24 57 68 20 20 6.3 4.4 ANLPFL 168 168 48 28 58 58 36 10 5.4 4.1 ANL 48 58 40 41 3.4 PCLMRL 168 144 <10 48 17 - - 29 - 2.8 PFLRNL 168 168 <10 25 32 - - 5 - 2.0 BCLMJL 168 168 <10 31 37 - - <5 - - MRLCRL 168 20 <10 30 40 - - <5 - - RSL 168 34 1 <5 - RNL 168 <10 21 - - - RML 168 14 9 1 <5 - MJLPPL 168 96 <10 43 <5 - - <5 - - RMLN435 168 168 <10 17 <5 - - <5 - - PPLROL 168 168 <10 16 <5 - - <5 - - N435MCL 168 168 <10 12 <5 - - <5 - - 1 ReversedROL conﬁguration.168 <10 - - - MCL 168 <10 - - - 2.3. Optical Rotation of Compounds 1 and 2

TableAfter 2. Lipase-catalyzed biotransformation hydrolysis and separationof dibutyl 1-butyryloxy-1-carboxymethylphosphonate of the obtained products, 36 mg of compound 2b. (R)-1b (ee = 68%), 50 mg of compound (S)-2b (ee = 50%), 204 mg of compound (R)-1a ee of Product ee of Substrate (eeLipase = 58%), andTime 104 mg(h) ofConversion compound (S(%))-2a (ee = 36%) were obtained and their opticalE rotation was measured. The optical rotation for compound(R) (R)-1b (ee = 68%)(S) was [α]D = +8.7 ◦ (c 2.0,BCL CHCl 3, 22168C); for compound 52 (S)-2b (ee = 50%), 68 it was [α]D = 50− 14.5 (c 2.0, CHCl 8.6 3, 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 -

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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.

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)- Catalysts 2021, 11, 956 1a (ee = 58%), and 104 mg of compound (S)-2a (ee = 36%) were obtained and their optical4 of 10 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 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).◦ (S)-2a (ee = 36%) it was [α]D = −2.8 (c 2.0, CHCl3, 22 C).

2.4. Determination of th thee Absolute ConfigurationConfiguration The Mosher Mosher method method based based on on the the NMR NMR technique technique is one is oneof the of most the most commonly commonly used usedmethods methods for the for determination the determination of absolute of absolute configuration configuration when obtaining when obtaining a pure isomer a pure is isomerimpossible is impossible [21]. In this [21 ].case, In this double case, doublederivatization derivatization was used. was used.Dibutyl Dibutyl 1-carboxy-1-hy- 1-carboxy- 1-hydroxymethylphosphonatedroxymethylphosphonate 1b, in1b, a in1.0:1.9 a 1.0:1.9 molar molar ratio ratio of isomers of isomers (S):( (RS)):( (eeR) = (ee 32%), = 32%), ob- obtainedtained after after biotransformation biotransformation of 2b of 2bby byBurkholderiaBurkholderia cepacia cepacia lipase,lipase, was was acylated acylated by ( byS)-(+)- (S)- (+)-MTPA-Cl,MTPA-Cl, resulting resulting in a inmixture a mixture of (S of,R ():(S,RR,):(R)R isomers,R) isomers of Mosher of Mosher ester ester 3 with3 withmolar molar ratio ratioof 1.0:1.7. of 1.0:1.7. 31P NMR31P NMRchemical chemical shifts shiftsof Mosher of Mosher ester 3 ester were3 assignedwere assigned as follows: as follows: (S,R), (13.10S,R), 13.10ppm; ppm;(R,R), ( R12.84,R), 12.84 ppm. ppm. The Thesignals signals resulting resulting from from the the phosphorus phosphorus atom atom of of the the isomer possessing ((RR)-configuration)-configuration at atα α-carbon-carbon atom atom were were upfield, upfield, compared compared to to the the (S )-isomer,(S)-isomer, as canas can be be seen seen from from Figure Figure2. 2.

Figure 2. Anisotropic effect of phenyl group on phosphorus atom.atom.

1 1HH NMR NMR chemical chemical shifts shifts of of the the -CH -CH3 groups3 groups (OCH (OCH2CH2CH2CH22CHCH23C) ofH 3compound) of compound 3 were3 wereassigned assigned as follows: as follows: (R,R), ( R0.82,R), ppm 0.82 ppmand 0.87 and ppm; 0.87 ppm; (S,R), (S 0.86,R), 0.86ppm ppm and and0.90 0.90ppm. ppm. In this In thiscase, case, it can it canbe seen be seen that that the thesignals signals resulting resulting from from hydrogen hydrogen atoms atoms from from -CH -CH3 groups3 groups of ofthe the isomer isomer possessing possessing (R ()-configurationR)-conﬁguration at at αα-carbon-carbon atom atom were were upfield, upﬁeld, compared compared to thethe same groups of thethe ((S)-isomer)-isomer (Figure(Figure3 3).).

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Figure 3. Anisotropic effect of phenyl group onon hydrogenhydrogen atomsatoms ofof butylbutyl groups.groups.

1 1H NMR chemical shifts of -OCH3 groups of compound 3 were assigned as follows: ((RR,,RR),), 3.68;3.68; ((SS,R,R),), 3.55 3.55 ppm. ppm. In In this this case, case, it it can can be be seen seen that that the the signals signals resulting resulting from from hydro- hy- α gendrogen atoms atoms of the of (theS)-conﬁguration (S)-configuration isomer isomer at the at the-carbon α-carbon atom atom were were upﬁeld, upfield, compared com- withpared the with (R)-isomer the (R)-isomer (Figure (Figure4). 4).

FigureFigure 4.4. Anisotropic effecteffect ofof P=OP=O group on hydrogen atomsatoms ofof methoxymethoxy groups.groups.

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.

Scheme 3. Acidic hydrolysis of hydroxyphosphonates 1.

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.

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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).

Catalysts 2021, 11, 956 5 of 10 Figure 4. Anisotropic effect of P=O group on hydrogen atoms of methoxy groups.

Comparison of all spectra allowed for assignment of the absolute configuration of enantiomersComparison of compounds of all spectra 1b and allowed 2b (Figure for assignment 1). of the absolute configuration of enantiomersSynthesis of compoundsof dimethyl1b and 2b1-carboxy-1-(3,3,3-trifluoro-2-methoxy-2-phenylpro-(Figure1). panoxy)methylphosphonatesSynthesis of dimethyl 1-carboxy-1-(3,3,3-trifluoro-2-methoxy-2-phenylpropanoxy) was unsuccessful. For this reason, it was decided to deter- methylphosphonatesmine the absolute configuration was unsuccessful. of compound For this 1a reason,using a itdifferent was decided approach. to determine the absoluteAfter configuration biotransformation of compound and separation1a using of a obtained different products, approach. 46 mg of compound 1a (ee = After18%) and biotransformation 54 mg of compound and separation 1b (major enantiomer of obtained R products,, ee = 32%) 46 were mg obtained. of compound Both 1awere(ee hydrolyzed = 18%) and by 54 HCl, mg of and compound 1-carboxy-1-methylphosphonic1b (major enantiomer R acids, ee = 4 32%)(Scheme were 3) obtained. were ob- Bothtained were and hydrolyzed their optical by rotation HCl, and was 1-carboxy-1-methylphosphonic measured. The optical rotation acids for4 compound(Scheme3) 4a were (ee obtained= 18%) was and [α their]D = +3.5 optical (c 0.4, rotation H2O, 22 was °C), measured. while that The for opticalcompound rotation (R)-4b for (ee compound = 32%) was4a ◦ (ee[α]D = 18%)= +5.0 was (c [0.4,α]D H=2 +3.5O, 22 (c 0.4,°C). HAfter2O, 22 comparisonC), while thatof optical for compound rotation ( Rof)-4b 1-carboxy-1-(ee = 32%) ◦ wasmethylphosphonic [α]D = +5.0 (c 0.4,acids H 24O, obtained 22 C). after After hydr comparisonolysis of compounds of optical rotation 1a and 1b of, 1-carboxy-1- the absolute methylphosphonicconfiguration of compound acids 4 obtained 1a wasafter determined hydrolysis to be of compoundsthe same as 1athatand of 1bcompound, the absolute (R)- configuration1b. of compound 1a was determined to be the same as that of compound (R)-1b.

Scheme 3.3. Acidic hydrolysis ofof hydroxyphosphonateshydroxyphosphonates 11..

1 31 13 1 1 1 13 1 13 1H, 31P,P, 13C,C, 1H-H-1HH COSY, COSY, 1H-H-13C CHMQC HMQC and and 1H-H-13C HMBCC HMBC spectra spectra of compound of compound 1a– 1a4 are–4 are shown shown in Supplementary in Supplementary Materials. Materials. 3. Discussion 3. Discussion Obtaining optically pure hydroxyphosphonates has great potential; however, there Obtaining optically pure hydroxyphosphonates has great potential; however, there are no easy, well-trodden paths to achieving this goal. Lipolytic enzymes have long been 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 used for this purpose; however, the research conducted so far has indicated that the ap- appropriate enzyme should be selected for each hydroxyphosphonate ester. This was also propriate enzyme should be selected for each hydroxyphosphonate ester. This was also the case with the hydrolysis of butyryloxyphosphonates 2. Hydroxyphosphonate 1a was 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 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 Candida rugosa to hydrolyze dimethyl butyryloxyphosphonate 2a. To obtain optically ac- hydroxyphosphonate 1b, it is better to use Burkholderia cepacia lipase. tive 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 Catalysts 2021, 11, 956 6 of 10

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 (Gli- wice, 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 31 13 for P, and 100.6 MHz for C, in CDCl3 (99.8% of D, containing 0.03% v/v TMS) or in 1 D2O (99.8% of D, containing 0.75% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid). H NMR were referenced to the internal standards TMS (δ = 0.00) or 3-(trimethylsilyl)propionic- 13 2,2,3,3-d4 acid (δ = 0.00), C NMR spectra to the central line of CHCl3 (δ = 77.23), and 31 85% phosphoric acid in H2O for P 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, Rf1a = 5 min, (compound 1a) Rf2a = 18 min (compound 2a), Rf1b = 26 min (compound 1b), and Rf2b = 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, Rf3 = 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: Catalysts 2021, 11, 956 7 of 10

Yield: 3.61 g, 98.1% MS(TOF MS ES-) Calcd for C4H8O6P [M − H]− 183.0058; found: 183.0064. 31 1 P NMR δ(ppm): 19.78; H NMR: δ(ppm) 3.91 (d, J = 11.0 Hz, 3H, OCH3), 3.92 13 (d, J = 10.7 Hz, 3H, OCH3), 4.62 (d, J = 16.3 Hz, 1H, PCH); C 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. 31 1 P NMR δ(ppm): 17.27; H 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 13 (m, 4H, OCH2CH2CH2CH3), 4.61 (d, J = 16.8 Hz, 1H, PCH); C 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. 31 1 P NMR δ(ppm): 17.25; H 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, 13 3H, OCH3), 3.90 (d, J = 6.1 Hz, 3H, OCH3), 5.57 (d, J = 17.8 Hz, 1H, PCH); C 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. 31 1 P NMR δ(ppm): 14.45; H 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), 13 4.14–4.24 (m, 4H, OCH2CH2CH2CH3), 5.54 (d, J = 17.8 Hz, 1H, PCH); C 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). Catalysts 2021, 11, 956 8 of 10

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 circinel- loides 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 Can- dida 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:

P − P ee = 1 2 × 100% (1) P1 + P2

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]: (1−ees) ln eep (ees+eep) E = (2) (1+ees) ln eep (ees+eep)

where eep is the enantiomeric excess of product and ees 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 Catalysts 2021, 11, 956 9 of 10

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) 31 1 P NMR δ(ppm): 12.84; H 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 13 (m, 3H, PC6H5), 7.67–7.71 (m, 2H, PC6H5); C 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) 31 1 P NMR δ(ppm): 13.10; H 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 13 (m, 3H, PC6H5), 7.61–7.65 (m, 2H, PC6H5); C 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).

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/catal11080956/s1, Figures S1–S42: 1H, 31P, 13C, 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC spectra of compound 1a–4. Catalysts 2021, 11, 956 10 of 10

Funding: This research was funded by a subsidy from the Ministry of Education and Science for the Faculty of Chemistry of Wrocław University of Science and Technology (subsidy nr: 8211104160). Acknowledgments: The author would like to thank students at the Faculty of Chemistry—P. Sławenta and K. Wi˛ech—whohelped in the implementation of the initial research on biotransformation reactions. Conﬂicts of Interest: The author declares no conﬂict of interest.

References 1. Patel, R.N. Synthesis of chiral pharmaceutical intermediates by biocatalysis. Coord. Chem. Rev. 2008, 252, 659–701. [CrossRef] 2. Nestl, B.M.; Nebel, B.A.; Hauer, B. Recent progress in industrial biocatalysis. Curr. Opin. Chem. Biol. 2011, 15, 187–193. [CrossRef] [PubMed] 3. Nguyen, L.A.; He, H.; Pham-Huy, C. Chiral drugs: An overview. Int. J. Biomed. Sci. 2006, 2, 85–100. [PubMed] 4. Calcaterra, A.; D’Acquarica, I. The market of chiral drugs: Chiral switches versus de novo enantiomerically pure compounds. J. Pharm. Biomed. Anal. 2018, 147, 323–340. [CrossRef][PubMed] 5. Pollard, D.J.; Woodley, J.M. Biocatalysis for pharmaceutical intermediates: The future is now. Trends Biotechnol. 2007, 25, 66–73. [CrossRef] 6. Tao, J.; Xu, J.-H. Biocatalysis in development of green pharmaceutical processes. Curr. Opin. Chem. Biol. 2009, 13, 43–50. [CrossRef] [PubMed] 7. Kagan, H.B.; Gopalaiah, K. Early history of asymmetric synthesis: Who are the scientists who set up the basic principles and the first experiments? N. J. Chem. 2011, 35, 1933–1937. [CrossRef] 8. Schober, M.; Faber, K. Inverting hydrolases and their use in enantioconvergent biotransformations. Trends Biotechnol. 2013, 31, 468–478. [CrossRef][PubMed] 9. Kafarski, P.; Lejczak, B. Application of bacteria and fungi as biocatalysts for the preparation of optically active hydroxyphosphonates. J. Mol. Catal. B Enzym. 2004, 29, 99–104. [CrossRef] 10. Kolodiazhnyi, O.I. Asymmetric synthesis of hydroxyphosphonates. Tetrahedron Asymmetry 2005, 16, 3295–3340. [CrossRef] 11. Sobhani, S.; Tashrifi, Z. Synthesis of α-functionalized phosphonates from α-hydroxyphosphonates. Tetrahedron 2010, 66, 1429–1439. [CrossRef] 12. Rádai, Z. α-Hydroxyphosphonates as versatile starting materials. Phosphorus Sulfur Silicon Relat. Elem. 2019, 194, 425–437. [CrossRef] 13. Kolodiazhnyi, O.I. Chiral hydroxy phosphonates: Synthesis, configuration and biological properties. Russ. Chem. Rev. 2006, 75, 227–253. [CrossRef] 14.R ádai, Z.; Keglevich, G. Synthesis and Reactions of α-Hydroxyphosphonates. Molecules 2018, 23, 1493. [CrossRef] 15. Ogawa, J. Industrial microbial enzymes: Their discovery by screening and use in large-scale production of useful chemicals in Japan. Curr. Opin. Biotechnol. 2002, 13, 367–375. [CrossRef] 16. Majewska, P. Biocatalytic hydrolysis of diethyl 1-butyryloxy-1-carboxymethylphosphonate and determination of the absolute configuration of obtained products. J. Mol. Struct. 2021, 1225, 129084. [CrossRef] 17. Kaczybura, N.; Brückner, R. Tandem claisen condensation/transesterification between arylacetate enolates and arylmethylene- substituted 2,2-dimethyl-1,3-dioxolan-4-ones: An improved synthesis of Z-configured pulvinones. Synthesis 2007, 1, 118–130. [CrossRef] 18. Majewska, P. Hydroxyphosphinylacetic acid as a chiral auxiliary compound. Phosphorus Sulfur Silicon Relat. Elem. 2019, 194, 585–590. [CrossRef] 19. Kafarski, P.; Lejczak, B. Biological activity of aminophosphonic acids. Phosphorus Sulfur Silicon Relat. Elem. 1991, 63, 193–215. [CrossRef] 20. Feigelson, G.B.; Curran, W.V.; Ziegler, C.B. 4-Substituted Azetidinones as Precursors to 2-Substituted-3-Carboxy Carbapenem Antibiotics and a Method of Producing Them. U.S. Patent US5656753A, 12 August 1997. Available online: https://patents.google. com/patent/US5656753A/en (accessed on 15 June 2021). 21. Dale, J.A.; Mosher, H.S. Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and α-methoxy-α-trifluoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 1973, 95, 512–519. [CrossRef] 22. Majewska, P.; Serafin, M.; Klimek-Ochab, M.; Brzezińska-Rodak, M.; Zymańczyk-Duda,E.˙ Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester. Bioorg. Chem. 2016, 66, 21–26. [CrossRef][PubMed] 23. Caplan, N.A.; Pogson, C.I.; Hayes, D.J.; Blackburn, G.M. The synthesis of novel bisphosphonates as inhibitors of phosphoglycerate kinase (3-PGK). J. Chem. Soc. Perkin Trans. 2000, 1, 421–437. [CrossRef] 24. Majewska, P. Biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and the determination of the absolute configuration of all isomers. Bioorg. Chem. 2015, 61, 28–35. [CrossRef] 25. Straathof, A.J.J.; Jongejan, J.A. The enantiomeric ratio: Origin, determination and prediction. Enzym. Microb. Technol. 1997, 21, 559–571. [CrossRef]