Biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and the determination of the absolute configuration of all isomers.

Bioorganic Chemistry 61 (2015) 28–35

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Biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and the determination of the absolute configuration of all isomers Paulina Majewska ⇑ ´ skiego 27, 50-370 Wrocław, Poland Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzez_ e Wyspian

a r t i c l e

i n f o

Article history: Received 27 February 2015 Available online 21 May 2015 Keywords: Biocatalysis Hydroxyphosphonates Lipolytic activity

a b s t r a c t 2-Hydroxy-2-(ethoxyphenylphosphinyl)acetic acid, a new type of organophosphorus compound possessing two stereogenic centers, was investigated. Racemic 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid was synthesized and hydrolyzed using four bacterial species as biocatalysts. In all cases the reaction was more or less stereoselective and isomers bearing a phosphorus atom with an (SP)-configuration were hydrolyzed preferentially. The observed 1H and 31P NMR chemical shifts of Mosher esters of 2-hydro xy-2-(ethoxyphenylphosphinyl)acetic acid were correlated with the configurations of both stereogenic centers of all four stereoisomers. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Stereoselective biocatalytic processes employing whole cells of microorganisms and isolated enzymes are becoming increasingly widely used in the preparation of chiral compounds. Among several classes of enzymes involved in enantioselective biotransformations lipases are the most commonly used, because of their wide tolerance toward synthetic substrates and substantial stereoselectivity of catalyzed reactions. One of the most difficult applications in biocatalysis is the separation of enantiomers. Because the biological activity of many classes of compounds is often inherent in their stereochemistry, it is very important to obtain pure enantiomers. Kinetic resolution of enantiomers using microorganisms or enzymes is one of the ways in which separation is enabled and made relatively easy and cost effective [1,2]. Among the methods for the determination of the absolute configuration of the various classes of compounds, a significant role is played by Nuclear Magnetic Resonance (NMR) spectroscopy (although the most common methods are still based on measurements and interpretation of X-ray diffraction spectra or Circular Dichroism). The NMR analysis is available in almost every bigger research center. Also, a small quantity of studied compound (in many cases it can even be recovered) and the ease of sample preparation and speed of the analysis make this method of structure elucidation an important one [3]. Additionally, the NMR method can determine the absolute configuration of both solids and liquid (which is impossible using crystallography) and test compounds do not need to be 100% optically pure. But the NMR method cannot ⇑ Fax: +48 713202427. E-mail address: [email protected] http://dx.doi.org/10.1016/j.bioorg.2015.05.006 0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

distinguish enantiomers located in an achiral environment which is usually deuterated solvent, because the signals of enantiomeric nuclei have identical chemical shifts. To distinguish between them the studied compound needs to be transformed into diastereomeric mixture. The first of the two most popular methods is to create a derivative with a chiral derivatizing agent (CDA), by creating a covalent bond and the introduction of an additional stereogenic center [3]. The second method is based on an analysis using chiral solvating agents (CSA), which form complexes with the analysed compound by weak interactions (e.g., hydrogen bonds, Van der Waals forces) [4]. Chiral hydroxyphosphonates are important precursors for the synthesis of a variety of important organophosphates [5–7]. Many of the known hydroxyphosphonates not yet been tested for their biological properties, however, are known to have potential antiviral [8], antibacterial [9] and antitumor properties [5]. Although hydroxyphosphonoacetic acid is a known compound having an application as a corrosion inhibitor [10–12], its derivatives are not well-studied compounds. However, due to their structure they are of great interest because they can find potential use as chiral auxiliaries in NMR methods for the determination of the enantiomeric purity and absolute configuration of various classes of compounds (data not yet published). Chiral auxiliaries may only be used if they are pure enantiomers [4]. Therefore, it is important to find a way to obtain these compounds in enantiomerically pure form. The purpose of this study was to obtain, biocatalytically, 2-hy droxy-2-(ethoxyphenylphosphinyl)acetic acid 1 with good enantiomeric excess in the most simple and effective way by whole-cell hydrolysis of 2-butyryloxy-2-(ethoxyphenylphosphiny l)acetic acid 2.

P. Majewska / Bioorganic Chemistry 61 (2015) 28–35

2. Materials and methods All materials were purchased from Sigma Aldrich (St. Louis, Missouri, United States), POCh (Gliwice, Poland) or BIOCORP (Warszawa, Poland) and were used without further purification. Bacillus subtilis and Serratia liquefaciens were derived from our own collection and were identified by the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Escherichia coli and Pseudomonas fluorescens were purchased from DSMZ. NMR (Nuclear Magnetic Resonance) spectra were measured on a Bruker Avance™ 600 at 600.58 MHz for 1H; 243.12 MHz for 31P and 151.02 MHz for 13C in CDCl3 (99.8% of atom D, containing 0.03% v/v TMS) or on a Bruker Avance™ DRX 300 instrument operating at 300.13 MHz for 1H; 121.50 MHz for 31P and 75.45 MHz for 13 C in CDCl3 (99.8% of atom D, containing 0.03% v/v TMS). Chemical shifts (d) are reported in ppm and coupling constants (J) are given in Hz. 1H NMR are referenced to the internal standard TMS (d = 0.00) and 13C NMR spectra to the central line of CHCl3 (d = 77.23).

29

The amount of 3.40 g (20 mmol) of ethyl phenylphosphinate was mixed with 1.84 g (20 mmol) of glyoxylic acid monohydrate and 2.79 mL (20 mmol) triethylamine. The resulting solution was stirred for 2 h at room temperature. Thereafter, the reaction mixture was dissolved in 100 mL of chloroform, placed in an ice bath and 2.07 mL (20 mmol) of butyryl chloride was slowly added dropwise. After completion of the reaction, which lasted for two days as monitored by TLC, the resulting solution was extracted with 100 mL of distilled water, the organic phase was dried by magnesium sulfate anhydrous, evaporated and the product was purified by column chromatography (Merck Silica Gel 60; 63-230 mesh) using dichloromethane:isopropanol at a ratio of 100:5 as eluent. After purification, 3.89 g (61.9% yield) of pure 2-butyryloxy-2-(e thoxyphenylphosphinyl)acetic acid 2 was obtained. MS (TOF MS ES+) Calcd for C14H20O6P [M + H]+ 315.3; found: 315.1; Calcd for C14H19O6PNa [M + Na]+ 337.2; found: 337.1.

2.2.1. (RP,S), (SP,R) 1

H NMR (CDCl3, d, ppm): 0.90 (t, J = 7.4 Hz, 3H, CH2CH2CH3), 1.37

(t, J = 7.0 Hz, 3H, POCH2CH3), 1.56–1.67 (m, 2H, CH2CH2CH3), 2.1. Synthesis of compound 1 Compound 1 was synthesized according to the method described below [13]: The amount of 3.40 g (20 mmol) of ethyl phenylphosphinate was mixed with 1.84 g (20 mmol) of glyoxylic acid monohydrate and 2.79 mL (20 mmol) triethylamine. The resulting solution was stirred for 2 h at room temperature. Thereafter, the reaction mixture was dissolved in 10 ml of distilled water and triethylamine was removed from the solution by ion exchange chromatography (DowexÒ 50W X8 50-100 mesh). After purification, 3.39 g (69.5% yield) of pure 2hydroxy-2-(ethoxyphenylphosphinyl)acetic 1, was obtained. MS (TOF MS ES+) Calcd for C10H14O5P [M + H]+ 245.2; found: 245.1; Calcd for C10H13O5PNa [M + Na]+ 267.2; found: 267.0. 2.1.1. (RP,S), (SP,R) 1

H NMR (CDCl3, d, ppm): 1.28 (t, J = 7.1 Hz, 3H, POCH2CH3),

4.07–4.25 (m, 2H, POCH2CH3), 4.73 (d, J = 10.9 Hz, 1H, PCH), 7.42–7.85 (m, 5H, PC6H5). 31 P NMR (CDCl3, d, ppm): 35.41. 13

C NMR (CDCl3, d, ppm): 16.41 (d, J = 5.7 Hz, 1C, POCH2CH3),

63.31 (d, J = 6.9 Hz, 1C, POCH2CH3), 71.01 (d, J = 105.2 Hz, 1C, PCH), 127.19 (d, J = 134.1 Hz, 1C, PC6H5), 128.59 (d, J = 13.2 Hz, 2C, PC6H5), 132.65 (d, J = 9.9 Hz, 2C, PC6H5), 133.28 (1C, PC6H5),

2.31–2.46 (m, 2H, CH2CH2CH3), 4.22–4.38 (m, 2H, POCH2CH3), 5.63 (d, J = 10.4 Hz, 1H, PCH), 7.48–7.91 (m, 5H, PC6H5). 31 P NMR (CDCl3, d, ppm): 33.21. 13

C NMR (CDCl3, d, ppm): 13.40 (1C, CH2CH2CH3), 16.38

(d, J = 5.3 Hz, 1C, POCH2CH3), 18.15 (1C, CH2CH2CH3), 35.50 (1C, CH2CH2CH3), 63.52 (d, J = 6.1 Hz, 1C, POCH2CH3), 70.29 (d, J = 106.6 Hz, 1C, PCH), 127.16 (d, J = 142.0 Hz, 1C, PC6H5), 128.65 (d, J = 13.5 Hz, 2C, PC6H5), 132.45 (d, J = 9.8 Hz, 2C, PC6H5), 133.46 (1C, PC6H5), 166.08 (1C, COCH2CH2CH3), 171.69 (1C, COOH).

2.2.2. (RP,R), (SP,S) 1

H NMR (CDCl3, d, ppm): 0.79 (t, J = 7.4 Hz, 3H, CH2CH2CH3), 1.37

(t, J = 7.0 Hz, 3H, POCH2CH3), 1.40–1.51 (m, 2H, CH2CH2CH3), 2.11– 2.29 (m, 2H, CH2CH2CH3), 4.17–4.41 (m, 2H, POCH2CH3), 5.79 (d, J = 13.6 Hz, 1H, PCH), 7.48–7.91 (m, 5H, PC6H5). 31 P NMR (CDCl3, d, ppm): 35.19. 13

C NMR (CDCl3, d, ppm): 13.47 (1C, CH2CH2CH3), 16.38

(d, J = 5.3 Hz, 1C, POCH2CH3), 18.06 (1C, CH2CH2CH3), 35.46 (1C, CH2CH2CH3), 63.52 (d, J = 6.1 Hz, 1C, POCH2CH3), 69.96 (d, J = 109.9 Hz, 1C, PCH), 126.76 (d, J = 141.4 Hz, 1C, PC6H5), 128.62 (d, J = 13.5 Hz, 2C, PC6H5), 132.45 (d, J = 9.8 Hz, 2C, PC6H5), 133.46

170.73 (1C, COOH).

(1C, PC6H5), 165.83 (1C, COCH2CH2CH3), 171.81 (1C, COOH).

2.1.2. (RP,R), (SP,S)

2.3. Synthesis of compound 3

1

H NMR (CDCl3, d, ppm): 1.30 (t, J = 7.1 Hz, 3H, POCH2CH3),

4.07–4.25 (m, 2H, POCH2CH3), 4.78 (d, J = 13.1 Hz, 1H, PCH), 7.42–7.85 (m, 5H, PC6H5). 31 P NMR (CDCl3, d, ppm): 36.42. 13


63.12 (d, J = 7.1 Hz, 1C, POCH2CH3), 70.54 (d, J = 106.5 Hz, 1C, PCH), 126.87 (d, J = 134.1 Hz, 1C, PC6H5), 128.62 (d, J = 13.3 Hz, 2C, PC6H5), 132.54 (d, J = 9.9 Hz, 2C, PC6H5), 133.28 (1C, PC6H5), 170.80 (1C, COOH). 2.2. Synthesis of compound 2 Compound 2 was synthesized in two step reaction from ethylphenylphosphinate without purification of intermediate 1 according to a modified method described in the literature [14]:

Compound 1 of various stereomeric compositions was acylated by (S)-(+)MTPA-Cl according to the literature [15]: Dry pyridine (300 lL) was added to dry bottle with septum using a syringe with a needle. Then, also using a syringe, 0.14 mmol (26 lL) of (S)-(+)MTPA-Cl was added. Then, 0.10 mmol (24 mg) of compound 1 dissolved in the dry dichloromethane (300 lL) was added. The mixture was left for 24 h at room temperature. An excess of 3-dimethylamino-1-propylamine (0.20 mmol, 24 lL) was then added and after 5 min at room temperature, the mixture was diluted with diethyl ether (10 mL), washed with cold dilute HCl (10 mL) and water (10 mL), and dried over anhydrous magnesium sulfate. After filtration of the drying agent, ether was evaporated and compound 3 was purified by column chromatography (Merck Silica Gel 60; 63-230 mesh) using ethyl acetate/n-hexane/2-propanol at a ratio of 10:10:1 as eluent.

30


2.3.1. (SP,S,R) 1

2.3.3. (RP,S,R) 1

H NMR (CDCl3, d, ppm): 1.27 (t, J = 7.1 Hz, 3H, POCH2CH3), 3.46


(s, 3H, OCH3), 4.01–4.31 (m, 2H, POCH2CH3), 5.79 (d, J = 14.3 Hz,


1H, PCH), 7.30–7.81 (m, 5H, PC6H5). 31 P NMR (CDCl3, d, ppm): 30.73.


13

13



55.61 (1C, OCH3), 62.87–63.92 (1C, POCH2CH3), 72.28 (d,

55.61 (1C, OCH3), 62.87–63.92 (1C, POCH2CH3), 72.28 (d,

J = 106.4 Hz, 1C, PCH), 84.60–85.62 (1C, C(CF3)), 123.56 (d,

J = 106.4 Hz, 1C, PCH), 84.60–85.62 (1C, C(CF3)), 123.10 (d,

J = 287.3, 1C, CF3), 127.41–129.97 (6C, PC6H5), 131.20–133.98

J = 289.2, 1C, CF3), 127.41–129.97 (6C, PC6H5), 131.20–133.98

(C(C6H5)), 163.70–165.90 (2C, COOH, C@O).

(C(C6H5)), 163.70–165.90 (2C, COOH, C@O).

2.3.2. (RP,R,R) 1

2.3.4. (SP,R,R)


1




1H, PCH), 7.30–7.81 (m, 5H, PC6H5). 31 P NMR (CDCl3, d, ppm): 30.68. 13



13


55.94 (1C, OCH3), 62.87–63.92 (1C, POCH2CH3), 71.96 (d,

56.05 (1C, OCH3), 62.87–63.92 (1C, POCH2CH3), 72.39 (d,

J = 105.5 Hz, 1C, PCH), 84.60–85.62 (1C, C(CF3)), 123.56 (d,

J = 105.5 Hz, 1C, PCH), 84.60–85.62 (1C, C(CF3)), 123.10 (d,

J = 287.3, 1C, CF3), 127.41–129.97 (6C, PC6H5), 131.20–133.98

J = 289.2, 1C, CF3), 127.41–129.97 (6C, PC6H5), 131.20–133.98

(C(C6H5)), 163.70–165.90 (2C, COOH, C@O).

O

O P

O H

O

+

OH

(C(C6H5)), 163.70–165.90 (2C, COOH, C@O).

NEt3 O

H

O P

O

2.4. Microorganisms, growth and whole cell biotransformation conditions

OH OH

Microorganisms were cultivated in a nutrient broth. Cell cultures before biotransformations were grown in a medium tested previously for stimulating vigorous growth and lipolytic activity [16]. One liter of the medium contained 10 g soluble starch, 1 g yeast extract, 5 g (NH4)2SO4, 2 g K2HPO4, and 100 lL of tributyrin and 1 L distilled water. In one case (when B. subtilis was used as biocatalyst), nutrient broth was also tested as a medium for growing bacteria strain. The microorganisms were incubated at 26 °C with shaking at 150 rpm for 24 h. Subsequently, the cells were centrifuged at 1449g for 10 min. Biotransformations were performed in 100 mL solution of 0.017 M phosphate buffer, pH 7.0, and 50 lL of substrate with (150 rpm shaking; room temperature). Then the biomass was centrifuged at 1449g, the supernatant evaporated and the products of biotransformation were extracted three times from solid residue with acetonitrile. Then, the organic solvent was evaporated and the products were analyzed via 31P NMR spectroscopy

1 Cl

CH3Cl O

O

O P

O OH O O 2

Scheme 1. Synthesis of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and 2butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2.

Table 1 Biotransformations with whole cells of four bacterial species. Microorganism

Reaction time (days)

Escherichia coli Bacillus subtilis Pseudomonas fluorescens Serratia liquefaciens

3 4 6 6

Conversion (%)

ee of product (%)

ee of substrate (%)

E

(RP,R), (SP,S)

(RP,S), (SP,R)

(RP,R), (SP,S)

(RP,S), (SP,R)

(RP,R), (SP,S)

(RP,S), (SP,R)

(RP,R), (SP,S)

(RP,S), (SP,R)

50 49 37 40

51 47 36 51

3 32 22 40

1 19 6 18

11 22 9 3

15 32 18 8

1.2 2.4 1.7 2.4

1.1 1.9 1.3 1.6

Table 2 Biotransformations with Bacillus subtilis grown in two different culture media. Medium

Reaction time (days)

Nutrient broth Lipolytic medium

4 4

Conversion (%)

ee of product (%)

ee of substrate (%)

E

(RP,R), (SP,S)

(RP,S), (SP,R)

(RP,R), (SP,S)

(RP,S), (SP,R)

(RP,R), (SP,S)

(RP,S), (SP,R)

(RP,R), (SP,S)

(RP,S), (SP,R)

54 49

51 47

80 32

60 19

91 22

79 32

27.6 2.4

9.4 1.9


with and without addition of quinine used as a chiral discriminator.

3. Results and discussion

31

3.1.2. Microbiological methods The starting substrate 2 was hydrolyzed using whole cells of four bacterial species. As seen from Table 1, some stereoselectivity can be observed and isomers bearing phosphorus atom with an (SP)-configuration were hydrolyzed preferentially.

3.1. Resolution of stereoisomers 3.1.1. Physical methods A new type of organophosphorus compound 2-hydroxy-2-(e thoxyphenylphosphinyl)acetic acid 1 was synthesized by phospho-aldol reaction between ethyl phenylphosphinate and glyoxylic acid. Its acyloxy derivative was obtained by simple acylation of compound 1 with butyryl chloride (Scheme 1). Both compounds, 2-hydroxy-2-(ethoxyphenylphosphinyl)ace tic acid 1 and 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2, are mixtures of four stereoisomers–two pairs of enantiomers. The signal of the first one is situated on the left side on 31 P NMR spectra (higher ppm) corresponds to the pair of enantiomers of (RP,R), (SP,S) configurations; the second one–on the right side (lower ppm)–the pair of (RP,S), (SP,R) enantiomers. After purification of compounds 1 and 2 attempts were made to resolve the diastereoisomers using column chromatography by the application of various eluents or by application of medium-pressure chromatography system; however, both appeared to be unsuccessful. Only when silica gel column and mixture of ethyl acetate/n-hexane/2-propanol (4:7:1 v/v) as eluent was used was some enrichment of the isomers of compound 2 obtained. In one of the fractions small amount of compound 2 with a molar ratio of diastereomers 1:0.3 was found. The combined content of the next few fractions afforded a product with 1:0.7 molar ratio of diastereomers. Similar results were achieved when attempts were made to separate the diastereomers of compound 1 using the medium-pressure chromatography system with reverse-phase column in gradient of water and acetonitrile (10 min of pure water, 5 min from pure water to 5% of acetonitrile in water, 10 min of isocratic flow, 5 min from 5% to 10% of acetonitrile in water, 35 min of isocratic flow, followed by a 10 min increasing gradient to 100% of acetonitrile, then maintained for 15 min). The identified retention time was blurred, and the signal from compound 1 appeared after 45 min and reached up to 85 min. Subsequent fractions were collected into separate tubes and in one of them small amount of compound 1 with a molar ratio of diastereomers 0.2:1 was found. The content of few next tubes was mixed giving a molar ratio of diastereomers 0.5:1.

Ph OH

(a) O

O

(RP,R)

(b)

C OO H

C OO H

Ph OH

(SP,S)

H3CH2CO OH O

Fig. 3. 31P NMR of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 with molar ratio of diastereomers 0.5:1.

H3CH2CO H OCH2CH3 H

C OO H

Fig. 2. 1H NMR of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 with molar ratio of diastereomers 0.5:1 (signals from hydrogen atom from group PACH).

Ph H Ph H

(SP,R)

O

C OO H

OCH2CH3 OH

(RP,S)

Fig. 1. A Newman projections of isomers of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1: (a) one pair of enantiomers; (b) second pair of enantiomers.

Fig. 4. 1H NMR of 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 with molar ratio of diastereomers 1:0.7 (signals from hydrogen atom from group PACH).

32


B. subtilis appeared to carry out hydrolysis most effectively and with the best enantioselectivity (range 2.4 for (RP,R), (SP,S) pair of enantiomers and 1.9 for (RP,S), (SP,R) pair) compared with three other organisms. Also some enantioselectivity was observed when S. liquefaciens was used as a biocatalyst, but enantiomeric excess did not exceed 40%. In order to test the effect of the type of culture medium on the enantioselectivity of reaction, experiments were also carried out

Fig. 5. 31P NMR of 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 with molar ratio of diastereomers 1:0.7.

using B. subtilis strain in nutrient broth. As can be seen (Table 2) the application of this medium significantly improved the enantioselectivity. 3.2. Configurational assignments 3.2.1. NMR experiments The assignment of the absolute configuration started from the analysis of the three-dimensional structure of compounds 1 and 2 (Fig. 1). For this purpose the NMR experiments were performed. An early assumption was that the hydrogen atom from acid group (ACOOH) forms a hydrogen bond with the oxygen atom of the phosphoryl group (P = 0). In such a case, the phenyl group has an anisotropic effect on the hydrogen atom moving signals upfield in 1H NMR spectrum (Fig. 2). So, it might be concluded that the signals situated in the lower ppm region derived from a pair of (RP,S), (SP,R) enantiomers and those situated at higher ppm come from a pair of enantiomers of (RP,R), (SP,S) configurations. By comparison of 31P and 1H NMR spectra of 2-hydroxy-2-(e thoxyphenylphosphinyl)acetic acid 1 of diastereomeric ratio of 0.5:1 it can be deduced that signals in 31P NMR spectra (Fig. 3) located at lower ppm values are derived from the pair of (RP,S), (SP,R) enantiomers, whereas those located at higher ppm are from the pair of (RP,R), (SP,S) enantiomers. The same situation occurs in the case of esters (Figs. 4 and 5). However, to confirm the correctness of this assumption, NOESY measurements were made for the equimolar mixture of all isomers of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1

Fig. 6. The NOESY spectrum of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1.


and equimolar mixture of all isomers of 2-butyryloxy-2-(ethoxy phenylphosphinyl)acetic acid 2. The proximity of atoms derived from the hydrogen atoms from phenyl group relative to the hydrogen atom from the PACH group has been confirmed (Fig. 6), as the signal from (RP,S) and (SP,R) isomers is stronger than that deriving from the (RP,R) and (SP,S) isomers. As can be seen in Fig. 6 the interaction between the hydrogen atom derived from PACH group and the proton atoms derived from the ethyl group (AOACH2ACH3) is more pronounced for (RP,R) and (SP,S) isomers. Similar relations are also seen from the NOESY spectrum of compound 2 (Fig. 7). In addition, a stronger signal can be seen for (RP,S) and (SP,R) isomers in the case the correlation between the hydrogen atoms from the ethyl group (AOACH2ACH3) and the butyryloxy group (AOACOACH2ACH2ACH3). Similarly, stronger signals are visible in the case of (RP,R) and (SP,S) isomers for the correlation between hydrogen atoms from the phenyl group and butyryloxy moiety (AOACOACH2ACH2ACH3).

33

In order to confirm that the signals from the same pair of enantiomers are situated at the same side of the 31P NMR spectra of compounds 1 and 2, non-equimolar mixture of diastereomers of 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 (see Fig. 5) was hydrolyzed with 3 M HCl to give a non-equimolar mixture of diastereomers of 2-hydroxy-2-(ethoxyphenylphosphinyl)a cetic acid 1 with a similar ratio (Fig. 8). 3.2.2. Mosher esters One of the commonly used method for the determination of absolute configuration, when obtaining pure isomer is impossible is the Mosher method based on the NMR technique [15]. In this case double derivatization was used. 3.2.2.1. Synthesis of Mosher ester I. In the first experiment, a non-equimolar mixture of all isomers of 2-hydroxy-2-(ethoxyphe nylphosphinyl)acetic acid at a 3.4:0.7:0.4:1.6 molar ratio of isomers (SP,S):(RP,R):(RP,S):(SP,R), obtained by means of biocatalytic hydrolysis with B. subtilis which was acylated with (S)-(+)MTPA-Cl

Fig. 7. The NOESY spectrum of 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2.

34


resulted in a mixture of (SP,S,R):(RP,R,R):(RP,S,R):(SP,R,R) isomers of Mosher ester 3 with molar ratio of 1.0:0.6:0.5:0.9. 3.2.2.2. Synthesis of Mosher ester II. During second experiment also non-equimolar mixture of all isomers of 2-butyryloxy-2-(ethoxy phenylphosphinyl)acetic acid at a molar ratio of the isomers of (SP,S):(RP,R):(RP,S):(SP,R) being 0.5:1.1:0.8:0.6, obtained as unreacted substrate of biotransformation reaction was chemically hydrolyzed with 3 M HCl and then acylated with (S)-(+)MTPA-Cl resulting in a mixture of (SP,S,R):(RP,R,R):(RP,S,R):(SP,R,R) isomers of Mosher ester 3 of a molar ratio being 0.5:1.0:0.7:0.5. 3.2.2.3. Synthesis of Mosher ester III. For greater certainty, third experiment was performed. A mixture of a different diastereomeric ratio of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid was used (molar ratio of isomers (SP,S):(RP,R):(RP,S):(SP,R) equal 1.0:1.0:0.6:0.6, see Fig. 8). This mixture was then also acylated with (S)-(+)MTPA-Cl. During analysis of spectra after this experiment it was observed that upon acylation some enantiopreference and/or racemization occurs. The signals of (SP,S,R):(RP,R,R):(RP,S,R):(SP,R,R) isomers of Mosher ester 3 have a ratio of 0.9:1.0:0.7:0.8 and thus different from composition starting mixture. This problem, on the one hand, overshadows the experimental results obtained above; however, on the other hand, explains the intensity of the difference in the signals before and after acylation. To completely and unambiguously determine the absolute configuration, an additional experiment was performed. All spectra of Mosher esters 3 (I–III) were used to determine the absolute configuration of each isomer of compounds 1 and 2. After analysis of 31P NMR spectra of Mosher ester I and II it was observed that the signals coming from (SP,S,R) and (SP,R,R) isomers are bigger than those from (RP,R,R) and (RP,S,R) signals of Mosher ester I. Contrasting observations were made in the case of Mosher ester II. After deeper analysis of this spectra and spectrum of products after biotransformation by B. subtilis, it was possible to assign signals deriving from isomers (RP,R,R) and (RP,S,R) to signals in 31P NMR of compounds 1 and 2 after biotransformation. Combining the above with the information obtained after the synthesis of Mosher’s ester III, a determination of the absolute configurations of all isomers is possible. 31 P NMR chemical shifts of Mosher ester 3 were assigned as follows: (SP,S,R)–30.73 ppm; (RP,R,R)–30.68 ppm; (RP,S,R)–30.60 ppm; (SP,R,R)–30.58 ppm. As can be seen, the signals resulting from the phosphorus atom of isomers with an (R)-configuration at a-carbon atom are upfield (see Fig. 9), compared with the (S)-isomers coming from the same pair of enantiomers of compounds 1.

1

H NMR chemical shifts of methoxy group of compound 3 were assigned as follows: (SP,S,R)–3.46 ppm; (RP,R,R)–3.53 ppm; (RP,S,R)–3.43 ppm; (SP,R,R)–3.61 ppm. In this case, it can be seen that the signals resulting from hydrogen atoms from methoxy group of isomers of (S)-configuration at a-carbon atom are upfield, compared with (R)-isomers (Fig. 10). Comparison of all the spectra allowed the assignment of the absolute configuration of all isomers of compounds 1 and 2 (Fig. 11). As can be observed in Fig. 11, in the case of a non-equimolar mixture of isomers of compound 1 and compound 2 the phenomenon of chiral self-discrimination occurs. This situation is present in both cases: the mixture of product after biotransformation and the pure compounds after their resolution. Just a small enantiomeric excess of one isomer of compound 1 or 2 is enough to observe this phenomenon. Self-discrimination was confirmed by applying 31P NMR in the presence of quinine as a chiral solvating agent (Fig. 12). upfield relative to

H3CH2CO O P HOOC

H3CO O H

HOOC O P H3CH2C

CF3

H3CO O

O H

CF3

O

O

(RP,R,R) 3

(SP,S,R) 3

upfield relative to H3CO HOOC O O P H3CH2CO H O

O CH2CH3 H3CO O P O HOOC CF3 H

CF3

O (RP,S,R) 3

(SP,R,R) 3

Fig. 9. Anisotropic effect of phenyl group on phosphorus atom.

upfield relative to

H3CO O

HOOC O P H3CH2C

O H

H3CH2CO O P HOOC

CF3

O

H3CO O H

CF3

O

(RP,R,R) 3

(SP,S,R) 3

upfield relative to

H3CO HOOC O O P H3CH2CO H O

(RP,S,R) 3 Fig. 8. 31P NMR of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 after chemical hydrolysis of compound 2 with diastereomeric ratio 1:0.7.

O CF3

CH2CH3 H3CO O

O P HOOC H

CF3

O

(SP,R,R) 3

Fig. 10. Anisotropic effect of phenyl group on hydrogen atoms of methoxy group.


35

product was enriched with isomers with even higher enantiomeric excess (up to 91%). Obtaining isomers with an absolute configuration of products of biotransformation dependent on the phosphorus atom is an unexpected result, because such compounds with two stereogenic centers have previously been obtained by biotransformation of the same or similar microorganisms, resulting in a high enantiomeric excess of isomers of (S) configuration on the a-carbon atom [16,17]. Acknowledgments

Fig. 11.

31

P NMR spectrum of products after biotransformation by B. subtilis.

This study was financed from the Project: ‘‘Biotransformations for pharmaceutical and cosmetics industry’’ No. POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund for the Innovative Economy. The author would like to thank students at the Department of Chemistry: J. Szyszkowiak, A. Tysler, K. Gołe˛biowski, M. Nicpon´, E. Krawiec, who helped in the implementation of the initial research on biotransformation reactions. References

Fig. 12. 31P NMR spectrum of products after biotransformation by B. subtilis– spectrum with using quinine as a CSA.

4. Conclusions The use of biotransformation of B. subtilis cells allowed the production of a mixture of isomers (SP,S) and (SP,R) of compound 1 with a very high enantiomeric excess-as high as 80%. Unreacted

[1] A. Liese, K. Seelbach, C. Wandrey, Industrial Biotransformations, VILEY-VCH, Weinheim, 2006. [2] K. Faber, Biotransformations in Organic Chemistry, 6th ed., Springer-Verlag, Berlin Heidelberg, 2011. _ [3] K.M. Błazewska, T. Gajda, Tetrahedron: Asymm. 20 (2009) 1337–1361. [4] T.J. Wenzel, C.D. Chisholm, Progr. Nucl. Mag. Reson. Spectrosc. 59 (2011) 1–63. [5] O.I. Kolodiazhnyi, Tetrahedron: Asymm. 16 (2005) 3295–3340. _ [6] D. Plazuk, J. Zakrzewski, A. Rybarczyk-Pirek, Tetrahedron: Asymm. 17 (2006) 1975–1978. [7] O.I. Kolodiazhnyi, Tetrahedron: Asymm. 23 (2012) 1–46. [8] W.C. Magee, D.H. Evans, Antivir. Res. 96 (2012) 169–180. [9] A.H. Kategaonkar, R.U. Pokalwar, S.S. Sonar, V.U. Gawali, Eur. J. Med. Chem. 45 (2010) 1128–1132. [10] B. Greaves, Corrosion Control in Aqueous Systems Using Cationic Polymers in Combination with Phosphonohydroxyacetic Acid, US Patent 4692317. [11] S.M. Zefferi, R.C. May, Corrosion Inhibition of Calcium Chloride Brine, US Patent 5292455. [12] X. Wang, Z. Liu, Y. Gao, Z. Liu, Asian J. Chem. 25 (2013) 6669–6672. [13] N.A. Caplan, Ch.I. Pogson, D.J. Hayes, G.M. Blackburn, J. Chem. Soc., Perkin Trans. 1 (2000) 421–437. [14] J. Szyszkowiak, P. Majewska, BioTechnologia 94 (2013) 425–431. [15] J.A. Dale, H.S. Mosher, J. Am. Chem. Soc. 95 (1973) 512–519. [16] P. Majewska, P. Kafarski, B. Lejczak, I. Bryndal, T. Lis, Tetrahedron: Asymm. 17 (2006) 2697–2701. [17] P. Majewska, B. Lejczak, P. Kafarski, Tetrahedron: Asymm. 17 (2006) 2870– 2875.

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