Penicillin G acylase-mediated kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and their esters

Article abstract of DOI:10.1016/j.molcatb.2016.05.011

Extensive studies on the penicillin G acylase-mediated kinetic resolution of N-acylated 1-aminoalkylphosphonic and 1-aminoalkylphosphinic acids as well as their esters were carried out to recognise the relationships between the substrate structure, reacti

Full text of DOI:10.1016/j.molcatb.2016.05.011

Accepted Manuscript
Title: Penicillin G acylase-mediated kinetic resolution of
racemic 1-(N-acylamino)alkylphosphonic and
1-(N-acylamino)alkylphosphinic acids and their esters
´
Author: Katarzyna Zielinska Roman Mazurkiewicz Katarzyna
´
˛
Szymanska Andrzej Jarzebski Sylwia Magiera Karol Erfurt
PII:
S1381-1177(16)30088-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.05.011
MOLCAB 3372
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
12-11-2015
4-5-2016
23-5-2016
´
Please cite this article as: Katarzyna Zielinska, Roman Mazurkiewicz, Katarzyna
´
˛
Szymanska, Andrzej Jarzebski, Sylwia Magiera, Karol Erfurt, Penicillin G acylase-
mediated kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic and 1-(N-
acylamino)alkylphosphinic acids and their esters, Journal of Molecular Catalysis B:
Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.05.011
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Penicillin G acylase-mediated kinetic resolution of racemic 1-(N-
acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and
their esters
Katarzyna Zielińskaa, Roman Mazurkiewicza*, Katarzyna Szymańskab, Andrzej Jarzębskib,
Sylwia Magierac, Karol Erfurtd
aDepartment of Organic and Bioorganic Chemistry and Biotechnology, Faculty of Chemistry,
Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland
bDepartment of Chemical and Process Engineering, Faculty of Chemistry,
Silesian University of Technology, Strzody 7, 44-100 Gliwice, Poland
cDepartment of Inorganic, Analytical Chemistry and Electrochemistry, Faculty of Chemistry,
Silesian University of Technology, Krzywoustego 6, 44-100 Gliwice, Poland
dDepartment of Chemical Organic Technology and Petrochemistry, Faculty of Chemistry,
Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland
1

Graphical abstract
Highlights
 The penicillin G acylase-mediated hydrolysis of 1-(N-acylamino)alkylphosphonic acids
and 1-(N-acylamino)alkylphosphinic acids, as well as their dimethyl esters proved to be a
highly effective method of their kinetic resolution for 13 of 16 tested substrates.
 The initial hydrolysis rates of 1-(N-acylamino)alkylphosphonic acids and their esters
rapidly decrease with the increasing steric effect of the substituent at the -position.
 Bulky substituents at phosphorus hinder the enzymatic hydrolysis to a much lesser degree.
 Penicillin G acylase exhibited stereochemical preference for the (R)-substrate.
Abstract
Extensive studies on the penicillin G acylase-mediated kinetic resolution of N-acylated
1-aminoalkylphosphonic and 1-aminoalkylphosphinic acids as well as their esters were
carried out to recognise the relationships between the substrate structure, reaction conditions,
and the enzymatic hydrolytic deacylation efficiency and stereoselectivity. Reactivity of 1-(N-
acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and their esters in the
penicillin G acylase-mediated hydrolytic deacylation reaction depends strongly on the kind of
their N-acyl group, with high preference to the hydrolytic splitting of the N-phenylacetyl
moiety. The initial hydrolysis rates of 1-(N-phenylacetylamino)alkylphosphonic acid dimethyl
esters 2 are mostly distinctly lower in comparison with the corresponding free acids 3 and
rapidly decrease with the increasing steric effect of the substituent at the -position. In
contrary to the substituents at the -carbon, bulky substituents at the phosphorus hinder the
enzymatic hydrolysis to a much lesser degree. The penicillin G acylase-mediated
stereospecific hydrolysis of N-acyl group of both racemic 1-(N-acylamino)alkylphosphonic
acids 3 and their dimethyl esters 2 proved to be, in most cases, a highly effective method for
2

the kinetic resolution of these compounds. High enzyme enantioselectivity E-values
exceeding 100, or synthetically useful E-values exceeding 20 (in two cases) were obtained for
the N-acylated phosphonic acid analogues of alanine, phenylalanine, valine, leucine, and
asparagine, as well as for their dimethyl esters, with the exception of the dimethyl ester of
phosphonic analogue of valine 2e, that E-value was low (E = 1.2). Also for the N-acylated H-
phosphinic acid analogues of alanine, as well as phenylphosphinic acid analogue of alanine,
high enzyme enantioselectivity values exceeding 100 were obtained. In contrary, E-values for
both diastereomers of ethyl ester of phenylphosphinic analogue of alanine 2k were low (E = 7
and 13). For the all accomplished assignments penicillin G acylase exhibited stereochemical
preference for the (R)-substrate.
Keywords:
Penicillin G acylase
Enantioselective kinetic resolution
Hydrolytic deacylation
1-(N-acylamino)alkylphosphonic acids
1-(N-acylamino)alkylphosphinic acids
1. Introduction
Chiral, non-racemic -aminoalkylphosphonic and -aminoalkylphosphinic acids, as
structural analogues and mimetics of natural -amino acids, exhibit inhibitory activity against
a plethora of different enzymes, resulting in their diversified biological activity, with a wide
range of application, mainly as pharmaceuticals and agrochemicals [1-6]. Due to that, they
attract significant attention of chemists and biochemist for many years. The biological
activities of these compounds are strongly dependent on their configuration at the stereogenic
-carbon [3,4,6]. Several groups of methods for the asymmetric synthesis of -
aminoalkylphosphonic and -aminoalkylphosphinic acids derivatives have been developed
[2,7-10]; however, due to disadvantages most of them (complicated and laborious procedures,
expensive chiral catalysts) the enzymatic kinetic resolution of racemic mixtures of these
compounds has become an interesting alternative [2].
Hydrolase-mediated kinetic resolution of racemic -amino acid derivatives is a well
established method for producing optically pure products, with a large number of practical
applications [11-13]. In contrast to -amino acids, literature data on hydrolases-mediated
3

kinetic resolution of -aminoalkylphosphonic and -aminoalkylphosphinic acids derivatives
are limited to a few papers. The resolution of racemic 1-(N-acylamino)alkylphosphonic acids
using porcine kidney acylase described by Telegdi et al. [14,15] was not confirmed by other
authors in later experiments [16]. Solodenko et al. [4, 17-19] applied penicillin acylase from
E. coli for stereoselective hydrolytic kinetic resolution of a few relatively simple N-acylated
1-aminoalkylphosphonic acids, 1-(N-phenylacetylamino)ethylphosphonous acid, as well as
dimethyl and diisopropyl esters of 1-(N-phenylacetylamino)ethylphosphonic acid.
Zimmermann et al. described in the US Patent [20] and other analogical patents the kinetic
resolution of phosphonic analogues of a few N-acylated -amino acids using lyophilised,
carrier–bound penicillin G acylase (Boehringer Mannheim GmBH).
Recently, we have developed a new, effective two-stage method for the transformation
of N-acyl-α-amino acids into their phosphonic or phosphinic analogues via the
electrochemical decarboxylative α-methoxylation of N-acyl-α-amino acids to N-(1-
methoxyalkyl)amides and the Michaelis-Arbuzov-type amidoalkylation of the proper
phosphorus nucleophile with the latter compounds [21-24]. The method enables easy access
to a variety of N-acylated -aminoalkylphosphonic and -aminoalkylphosphinic acid esters of
diversified structure, including the most interesting group of these compounds with the
substituents at the -position identical with those ones characteristic for natural, -amino
acids, both proteinogenic and unproteinogenic. The main drawback of this method is the
racemization of N-acyl-α-amino acid already on the step of decarboxylative α-methoxylation
of N-acyl-1-amino acid. With a large library of structurally diversified racemic -
aminoalkylphosphonic and -aminoalkylphosphinic acid derivatives in hand, we performed
an extensive study on the penicillin G acylase-mediated kinetic resolution of N-acylated -
aminoalkylphosphonic and -aminoalkylphosphinic acids as well as their esters to recognise
the relationships between the substrate structure, reaction conditions, and the enzymatic
hydrolytic deacylation efficiency and stereoselectivity.
2. Experimental
2.1. General
Melting points were determined using capillary tubes in Stiriling SMP 3 apparatus and
were uncorrected. IR-spectra were measured on a Nicolett 6700 FT-IR spectrophotometer
1
13
(ATR method). H and C NMR spectra were recorded on Varian instruments at operating
4

frequencies of 400 or 600 and 100 or 150 MHz, respectively, using TMS as resonance shift
31
standard. P NMR spectra were recorded on a Varian instrument at operating frequency of
161.8 MHz with 80% orthophosphoric acid as an external resonance shift standard. All
chemical shifts (δ) are reported in ppm and coupling constants (J) are in Hz. HPLC analyses
were carried out using a LaChrom ULTRA HPLC system (Merck Hitachi, Germany),
including a binary pump (Model L-2 2160U), diode array detector (Model L-2455U),
autosampler (Model L-2200), a temperature–controled column compartment (Model L-
2350U), and a degasser module. The data were evaluated by EZ Chrom Elite System
Manager. High-resolution mass spectrometry analyses were performed on a Waters Xevo G2
Q-TOF mass spectrometer (Waters Corporation) equipped with an ESI source operating in
positive ion modes. Full-scan MS data were collected from 100 to 1000 Da in a positive ion
mode with a scan time of 0.1 s. Column chromatography was carried out using Merck 60
silica gel (70-230 mesh). Routine monitoring of reactions was done with the use of thin layer
chromatography, using TLC plates coated with silica gel (Merck 60 F254). Ion-exchange
chromatography was carried out using DOWEX 50W X8 in the H+ form. Optical rotations
were measured on a JASCO V-650 polarimeter.
2.2. Chemicals and enzyme
All used -amino acids, N-Cbz-DL-alanine, N-Cbz-O-t-Bu-DL-serine, acetyl chloride,
phenylacetyl
chloride,
N,N-diisopropylethylamine,
ammonium
phosphinate,
hexamethyldisilazane and 3-(1-piperidino)propyl functionalized silica gel (SiO2-Pip, 200-400
mesh, 1.1 mmol/g) were purchased from Sigma-Aldrich. Penicillin G acylase from
Escherichia coli (20.34 U/mg) was purchased from CBC Biotech S.r.l. (Italy).
The specific activity of the penicillin G acylase was determined with penicillin G as a
substrate according to the PDAB method [25].
2.3. Substrate synthesis
2.3.1. N-Phenylacetylation of -amino acids
To a solution of amino acid (20 mmol) in 2M NaOH (20 mL), phenylacetyl chloride
(2.9 mL, 3.4 g, 22 mmol) was added. The reaction mixture was stirred at room temperature
for 2 hours and then acidified by the addition of 2M HCl. The crude product was filtered off,
washed with distilled water and recrystallized from ethanol.
5

2.3.2. Bis-(trimethylsilyl) phosphonite
The synthesis was performed following the procedure described by Olszewski et al.
[26] and Boyd et al. [27]. Ammonium phosphinate (3.32g, 40 mmol) and
hexamethyldisilazane (8.8 mL, 6.78 g, 42 mmol) were heated at 110°C for 2 hours under
argon in a three-necked flask with a septum and a condenser. The reaction mixture was cooled
to room temperature, dichloromethane (12.5 mL) was added, and the mixture was stirred for
another hour. The solution of the crude product in CH2Cl2 was used for further reactions
without purification.
2.3.3. Racemic dimethyl 1-(N-acylamino)alkylphosponates 2 (Procedure A)
To an undivided cylindrical glass electrolyser (85 mL) with a thermostatic jacket, a
magnetic stirrer, a cylindrical Pt mesh anode (47 cm2), and a similar cathode (44 cm2),
arranged concentrically each to other at a distance of 2.5 ± 0.5 mm, methanol (30 mL), N-
acyl--amino acid 1 (3.0 mmol), and SiO2-Pip (200 mg, 0.22 mmol) were added. Electrolysis
was carried out with stirring at a current density of 0.3 A/dm2 at 10oC until 3-3.75 F/mol
charge was passed. SiO2-Pip was filtered off and methanol was evaporated under reduced
pressure to obtain the corresponding N-(1-methoxyalkyl)amide. The crude N-(1-
methoxyalkyl)amide and triphenylphosphonium tetrafuluoroborate (2.0 mmol) were dissolved
in CH2Cl2 (2 mL) and the solution was left at room temperature for 0.5 h. After evaporation
of the solvent, the corresponding crude 1-(N-acylamino)alkyltriphenylphosphonium
tetrafluoroborate was obtained. A solution of the phosphonium salt (2 mmol) in CH2Cl2 (6
mL), triphenylmethylphosphonium iodide (0.5 mmol), diisoprophylethylamine (0.03 mL, 0.2
mmol) and trialkylphosphite (3 mmol) or diethoxy phenyl-phosphonite (3 mmol) were placed
o
in a glass vial sealed with a screw-cap. The mixture was kept at 60 C for the time given in
Table 1. On the reaction completion, the solvent was evaporated under reduced pressure and
the residue was extracted with toluene (4 x 2 mL). After evaporation of toluene, the crude
product was purified by column chromatography (silica gel, CH2Cl2/MeOH, 20:1 v/v).
2.3.3.1 1-(N-Acetylamino)ethylphosphonic acid dimethyl ester 2a [28]
31P NMR (CDCl3): δ = 30.9;
1H NMR (CDCl3): δ= 1.35 (dd, J1 = 7.6, J2 = 16.8 Hz, 3H, CH3), 2.02 (s, 3H, CH3CO), 3.76
(d, J = 10.4 Hz, 3H, OMe), 3.78 (d, J = 10.4 Hz, 3H, OMe), 4.54 (ddq, J1 = 7.2 Hz, J2 = 9.6
Hz, J3 = 16.4 Hz, 1H, CH), 6.22 (d, J = 8.0 Hz, 1H, NH).
2.3.3.2 1-(N-Benzyloxycarbonylamino)ethylphosphonic acid dimethyl ester 2b [29,30]
31P NMR (CDCl3): δ = 28.5;
6

1H NMR (CDCl3): δ= 1.40 (dd, J1 = 7.2, J2 = 16.8 Hz, 3H, CH3), 3.74 (d, J = 11.2 Hz, 3H,
OMe), 3.75 (d, J = 10.4 Hz, 3H, OMe), 4.18 (m, 1H, CH), 4.95 (d, J = 8.0 Hz, 1H, NH), 5.12
(s, 2H, OCH2Ph), 7.25-7.36 (m, 5H, Ph).
2.3.3.3 1-(N-Phenylacetylamino)ethylphosphonic acid dimethyl ester 2c [31]
31P NMR (CDCl3): δ = 28.21;
1H NMR (CDCl3): δ = 1.30 (dd, J1 = 7.6 Hz, J2 = 17.2 Hz, 3H, CH3), 3.55 (s, 2H, CH2Ph),
3.68 (d, J = 10.4 Hz, 3H, OMe), 3.60 (d, J = 10.4 Hz, 3H, OMe), 4.53 (ddq, J1 = 7.6 Hz, J2 =
10.0 Hz, J3 = 15.2 Hz, 1H, CH), 6.18 (d, J = 9.6 Hz, NH), 7.24-7.32 (m, 5H, Ph).
2.3.3.4 1-(N-Phenylacetylamino)-2-phenylethylphosphonic acid dimethyl ester 2d
31P NMR (CDCl3): δ = 26.2;
1H NMR (CDCl3): δ = 2.78 (ddd, J1 = J2 = 10.0 Hz, J3 = 14.8 Hz, 1H, CHCH2Ph), 3.17 (ddd,
J1 = 4.8 Hz, J2 = 8.4 Hz, J3 = 14.4 Hz, 1H, CHCH2Ph), 3.45 (s, 2H, CH2Ph), 3.64 (d, J = 10.6
Hz, 3H, OMe), 3.74 (d, J = 10.6 Hz, 3H, OMe), 4.78 (dddd, J1 = 4.0 Hz, J2 = 9.6 Hz, J3 =
10.4 Hz, J4 = 15.2 Hz, 1H, CH), 5.81 (d, J = 9.6 Hz, NH), 6.94-7.82 (m, 10H, Ph);
13C NMR (CDCl3): δ = 35.3 (d, J = 3.2 Hz) 43.4, 45.7 (d, J = 154.8 Hz), 53.0 (d, J = 6.6 Hz),
53.2 (d, J = 7.1 Hz), 126.8, 127.2, 128.4, 128.9, 129.2, 132.1, 134.3, 136.2, 170.3 (d, J = 4.4
Hz);
IR (ATR) ν (cm−1): 3252, 2956, 1672, 1541, 1228, 1180, 1018, 838, 734, 697;
HRMS(ESI): calc. for C18H23NO4P [M + H]+ m/z 348.1365, found m/z 348.1366.
2.3.3.5 1-(N-Phenylacetylamino)-2-methylpropylphosphonic acid dimethyl ester 2e
31P NMR (CDCl3): δ = 26.6;
1H NMR (CDCl3): δ = 0.85 (d, J= 6.9 Hz, 3H, CH(CH3)2), 0.93 (dd, J1 = 1.3, J2 = 6.8 Hz, 3H,
CH(CH3)2), 2.06-2.22 (m, 1H, CH(CH3)2), 3.62 (d, J = 10.6 Hz, 3H, OMe), 3.63 (s, 2H,
CH2Ph), 3.73 (d, J = 10.7 Hz, 3H, OMe), 4.38 (ddd, J1 = 4.4 Hz, J2 = 10.4 Hz, J3 = 17.9 Hz,
1H, CH), 5.66 (d, J = 9.7 Hz, NH), 7.25-7.40 (m, 10H, Ph);
13C NMR (CDCl3): δ = 17.7 (d, J = 4.6 Hz), 20.3 (d, J = 12.5 Hz), 28.8 (d, J = 3.5 Hz), 43.7,
49.6 (d, J = 152.2 Hz), 52.7 (d, J = 7.5 Hz), 52.8 (d, J = 6.7 Hz), 127.4, 129.0, 129.2, 134.6,
170.7 (d, J = 5.1 Hz);
IR (ATR) ν (cm−1): 3261, 1665, 1541, 1244, 1041, 1021, 828, 746, 711, 697;
HRMS(ESI): calc. for C14H23NO4P [M + H]+ m/z 300.1365, found m/z 300.1354.
2.3.3.6 1-(N-Phenylacetylamino)-3-methylbutylphosphonic acid dimethyl ester 2f
31P NMR (CDCl3): δ = 27.6;
7

1H NMR (CDCl3): δ = 0.88 (d, J = 6.0 Hz, 6H, CH(CH3)2), 1.45-1.60 (m, 3H, CH2CH(CH3)2),
3.59 (s, 2H, CH2Ph), 3.63 (d, J = 10.8 Hz, 3H, OMe), 3.73 (d, J = 10.4 Hz, 3H, OMe), 4.47-
4.59 (m, 1H, CH), 5.56 (d, J = 10.0 Hz, 1H, NH), 7.23-7.53 (m, 5H, Ph);
13C NMR (CDCl3): δ = 21.2, 23.2, 24.5 (d, J = 13.3 Hz), 38.1 (d, J = 1.7 Hz), 43.2 (d, J =
154.6 Hz), 43.7, 52.9 (d, J = 6.4 Hz), 53.0 (d, J = 7.1 Hz), 127.4, 128.9, 129.2, 134.5, 170.4
(d, J = 4.2 Hz);
IR (ATR) ν (cm−1): 3255, 2956, 1655, 1541, 1229, 1031, 831, 725;
HRMS(ESI): calc. for C15H25NO4P [M + H]+ m/z 314.1521, found m/z 314.1523.
2.3.3.7 1-(N-Phenylacetylamino)-2-carbamoylethylphosphonic acid dimethyl ester 2g
31P NMR (DMSO-d6): δ = 27.1;
1H NMR (DMSO-d6): δ = 2.33-2.42 (m, 1H, CH2CH), 2.45-2.55 (m, 1H, CH2CH), 3.42 (d, J
= 14.0 Hz, 1H, CH2Ph), 3.46 (d, J = 14.0 Hz, 1H, CH2Ph), 3.56 (d, J = 10.0 Hz, 3H, OMe),
3.60 (d, J = 10.4 Hz, 3H, OMe), 4.65-4.75 (m, 1H, CH), 6.91 (s, 2H, NH2), 7.19-7.29 (m, 5H,
Ph), 8.36 (d, J = 9.2 Hz, 1H, NH);
13C NMR (DMSO-d6): δ = 35.5 (d, J = 4 Hz), 42.2 (d, J = 157.2 Hz), 42.3, 53.0 (d, J = 6.4
Hz), 53.3 (d, J = 6.8 Hz), 126.7, 128.5, 129.3, 136.6, 169.8 (d, J = 2 Hz), 170.6 (d, J = 15.1
Hz);
IR (ATR) ν (cm−1): 3404, 1671, 1548, 1224, 1030;
HRMS(ESI): calc. for C13H20N2O5P [M + H]+ m/z 315.1110, found m/z 315.1101.
2.3.3.8 1-(N-Benzyloxycarbonylamino)-2-tert-butoxyethylphosphonic acid dimethyl ester 2h
31P NMR (CDCl3): δ = 26.1;
1H NMR (CDCl3): δ =1.18 (s, 9H, t-Bu), 3.54 (dd, J1 = 4.0 J2 = 9.6 Hz, 1H, CH2), 3.61 (dd, J1
= 3.6 Hz, J2 = 9.2 Hz, 1H, CH2), 3.75 (d, J = 10.8 Hz, 3H, OMe), 3.76 (d, J = 10.8 Hz, 3H,
OMe), 4.26 (m, 1H, CH), 5.13 (s, 2H, OCH2Ph), 5.28 (d, J = 10.4 Hz, 1H, NH), 7.31-7.37 (m,
5H, Ph);
13C NMR (CDCl3): δ = 27.4, 48.3 (d, J = 155.6 Hz), 52.7 (d, J = 6.1 Hz), 53.3 (d, J = 6.1 Hz),
60.6, 67.2, 73.7, 128.1, 128.2, 128.5, 136.2, 153.5 (d, J = 5.1 Hz),
IR (ATR) ν (cm−1): 3225, 2971, 1705, 1552, 1276, 1223, 1033, 904, 776, 696;
HRMS(ESI): calc. for C16H27NO6P [M + H]+ m/z 360.1576, found m/z 360.1578.
2.3.3.9 N-Phenylacetylpyrrolidine-2-phosphonic acid dimethyl ester 2j
31P NMR (CDCl3): δ = 22.5;
8

1H NMR (CDCl3): δ = 1.84-2.34 (m, 4H, (CH2)2), 3.50-3.61 (m, 2H, NCH2), 3.69 (d, J = 10.8
Hz, 3H, OMe), 3.79 (d, J = 10.4 Hz, 3H, OMe), 3.70 (s, 2H, CH2Ph), 5.60-5.66 (m, 1H, CH),
7.21-7.35 (m, 5H, Ph);
13C NMR (CDCl3): δ = 24.7, 26.3, 42.1, 47.3, 52.1 (d, J = 158.2 Hz), 52.8 (d, J = 6.4 Hz),
52.9 (d, J = 7.7 Hz), 126.8, 128.6, 128.9, 134.5, 169.9 (d, J = 2 Hz);
IR (ATR) ν (cm−1): 3471, 2955, 1640, 1405, 1231, 1023, 828, 722;
HRMS(ESI): calc. for C14H21NO4P [M + H]+ m/z 298.1208, found m/z 298.1209.
2.3.3.10 1-(N-Phenylacetylamino)ethyl(phenyl)phosphinic acid ethyl ester 2k (a mixture of
diastereomers in a ratio of 0.55:0.45)
31P NMR (CDCl3): δ = 36.8 (major), 36.3 (minor);
1H NMR (CDCl3): δ = 1.18 (dd, J1 = 7.8 Hz, J2 = 15.8 Hz, 3H, -CH3, minor), 1.19 (t, J = 7.1
Hz, 3H, OCH2CH3, minor), 1.27 (t, J = 7.1 Hz, 3H, OCH2CH3, major), 1.39 (dd, J1 = 7.3 Hz,
J2 = 14.6 Hz, 3H, -CH3, major), 3.37 (s, 2H, CH2Ph, major), 3.59 (s, 2H, CH2Ph, minor),
3.77-4.22 (m, 2H, OCH2CH3), 4.48-4.62 (m, 1H, CH), 4.62-4.81 (m, 1H, CH), 6.16 (d, J = 9.7
Hz, 1H, NH, major), 6.24 (d, J = 8.1 Hz, NH, minor), 6.90-7.87 (m, 5H, Ph);
13C NMR (CDCl3): δ = 14.4, 15.0 (d, J = 3.2 Hz), 16.4 (d, J = 6.2 Hz ), 42.9 (d, J = 113.8
Hz), 43.4, 43.8, 43.9 (d, J = 108.5 Hz), 61.4 (d, J = 6.8 Hz), 61.41 (d, J = 6.7 Hz), 127.2,
127.4, 128.4, 128.5, 128.6, 128.76, 128.8, 128.9, 129.1, 129.3, 132.0 (d, J = 15.2 Hz), 132.1
(d, J = 15.9 Hz), 132.6 (d, J = 3.0 Hz), 132.8 (d, J = 3.2 Hz), 134.4, 134.7, 170.0 (d, J = 5.4
Hz), 170.2 (d, J = 5.3 Hz);
IR (ATR) ν (cm−1): 3244, 1668, 1545, 1209, 1122, 1024, 953, 733, 702, 690;
HRMS(ESI): calc. for C18H23NO3P [M + H]+ m/z 332.1415, found m/z 332.1416.
2.3.4. Racemic
1-(N-acylamino)alkylphosponic
acids
3
and
1-
(phenylacetylamino)ethyl(phenyl)phosphinic acid 4d (Procedure B)
A mixture of dimethyl 1-(N-acylamino)alkylphosponate 2 or ethyl 1-(N-
phenylacetyl)ethyl(phenyl)phosphinate 2k (0.2 mmol), aqueous HCl (1M, 2 mL), and acetone
(1 mL; only in the case of compounds 2d-f) was heated under reflux for 2 hours (for
compounds 2a-c, 2e and 2g-j) or for 4 hours (for compounds 2d and 2f). After evaporation of
the solvent under reduced pressure, the residue was recrystallized by dissolution in ethanol
(96%) and precipitation with diethyl ether.
2.3.4.1 1-(N-Acetylamino)ethylphosphonic acid 3a [32]
31P NMR (D2O): δ = 21.7;
1H NMR (D2O): δ = 1.31 (dd, J1 = 6.8 Hz, J2 = 16.0 Hz, 3H, CH3), 2.01 (d, J = 1.2 Hz, 3H,
CH3CO), 4.21 (dq, J1 = 7.2 Hz, J2 = 14.8 Hz, 1H, CH).
9

2.3.4.2 1-(N-Phenylacetylamino)ethylphosphonic acid 3b [4]
31P NMR (D2O): δ = 23.9;
1H NMR (D2O): δ = 1.33 (dd, J1 = 7.6 Hz, J2 = 16.4 Hz, 3H, CH3), 3.64 (s, 2H, CH2Ph), 4.22
(dq, J1 = 7.6 Hz, J2 = 14.8 Hz, 1H, CH), 7.32-7.44 (m, 5H, Ph).
2.3.4.3 1-(N-Phenylacetylamino)-2-phenylethylphosphonic acid 3c [4]
31P NMR (D2O): δ = 19.9;
1H NMR (D2O): δ = 2.78 (ddd, J1 = 7.6 Hz, J2 = 12.4 Hz, J3 = 14.0 Hz, 1H, CHCH2Ph), 3.23-
3.29 (m, 1H, CHCH2Ph), 3.41 (d, J = 14.8 Hz, 1H, CH2Ph), 3.48 (d, J = 14.8 Hz, 1H, CH2Ph),
4.43 (ddd, J1 = 2.8 Hz, J2 = 12.4 Hz, J3 = 16.4 Hz, 1H, CH), 7.21-7.32 (m, 10H, Ph).
2.3.4.4 1-(N-Phenylacetylamino)-2-methylpropylphosphonic acid 3d [20]
31P NMR (D2O): δ = 22.3;
1H NMR (D2O): δ = 0.92 (d, J = 2.9 Hz, 3H, CH(CH3)2), 0.94 (d, J = 3.2 Hz, 3H, CH(CH3)2),
2.07-2.25 (m, 3H, CH(CH3)2, ), 3.65 (d, J = 14.8 Hz, 1H, CH2Ph), 3.73 (d, J = 14.8 Hz, 1H,
CH2Ph), 4.06 (dd, J1 = 5.6 Hz, J2 = 17.6 Hz, 1H, CH), 7.30-7.76 (m, 5H, Ph);
13C NMR (D2O): δ = 18.4 (d, J = 6.1 Hz) 20.9 (d, J = 11.0 Hz), 29.6 (d, J = 3.1 Hz), 43.3,
53.5 (d, J = 150.2 Hz), 128.2, 129.8, 130.1, 136.0, 175.4 (d, J = 5.7 Hz);
IR (ATR) ν (cm−1): 3415, 2969, 2285, 1530, 1181, 981, 935, 709;
HRMS(ESI): calc. for C12H19NO4P [M + H]+ m/z 272.1052, found m/z 272.1052.
2.3.4.5 1-(N-Phenylacetylamino)-3-methylbutylphosphonic acid 3e [4]
31P NMR (D2O): δ = 22.9;
1H NMR (D2O): δ = 0.78 (d, J = 6.0 Hz, 3H, CH(CH3)2), 0.87 (d, J = 6.0 Hz, 3H, CH(CH3)2),
1.47-1.67 (m, 3H, CH2CH(CH3)2 + CH2CH(CH3)2), 3.59 (d, J = 14.8 Hz, 1H, CH2Ph), 3.67
(d, J = 14.4 Hz, 1H, CH2Ph), 4.23 (ddd, J1 = 2.8 Hz, J2= 12.4 Hz, J3 = 15.6 Hz, 1H, CH),
7.29-7.41 (m, 5H, Ph).
2.3.4.6 1-(N-Phenylacetylamino)-2-carbamoylethylphosphonic acid 3f
31P NMR (D2O): δ = 18.1;
1H NMR (D2O): δ = 2.55-2.63 (m, 1H, CH2), 2.84-2.91 (m, 1H, CH2), 3.60 (d, J = 14.8 Hz),
3.65 (d, J = 14.8 Hz), 4.45-4.56 (m, 1H, CH), 7.29-7.42 (m, 5H, Ph);
13C NMR (D2O): δ = 35.4 (d, J = 3.8 Hz), 42.2, 44.9 (d, J = 153.3 Hz), 127.2, 128.8, 129.0 (d,
J = 3.0 Hz), 134.8, 173.9 (d, J = 4.6 Hz), 174.9;
IR (ATR) ν (cm−1): 3263, 2954, 1732, 1656, 1542, 1226, 1165, 1000, 695;
HRMS(ESI): calc. for C11H16N2O5P [M + H]+ m/z 287.0797, found m/z 287.0799.
2.3.4.7 N-Phenylacetylpyrrolidine-2-phosphonic acid 3h
31P NMR (D2O): δ = 23.2;
10

1H NMR (D2O): δ = 1.89-2.24 (m, 4H, (CH2)2), 3.59-3.78 (m, 2H, NCH2), 3.81-3.85 (m, 2H,
CH2Ph), 4.24-4.33 (m, 1H, CH), 7.24-7.42 (m, 5H, Ph);
13C NMR (DMSO-d6): δ = 27.2, 29.3, 31.1, 32.5, 45.6, 46.2, 51.0, 52.6, 59.6 (d, J = 148.6
Hz), 60.0 (d, J = 153.9 Hz), 131.3, 131.6, 133.2, 133.4, 134.5, 134.6, 140.4, 141.9, 174.4,
175.6;
IR (ATR) ν (cm−1): 1608, 1451, 1178, 998, 772;
HRMS(ESI): calc. for C12H17NO4P [M + H]+ m/z 270.0895, found m/z 270.0892.
2.3.4.8 1-(N-Phenylacetylamino)ethyl(phenyl)phosphinic acid 4d
31P NMR (D2O): δ = 37.7;
1H NMR (D2O): δ = 1.37 (dd, J1 = 7.4 Hz, J2 = 14.9 Hz, 3H, CH3), 3.40 (s, 1H, CH2Ph), 4.38
(dq, J1 = 7.2 Hz, J2 = 9.6 Hz, 1H, CH), 6.98-7.87 (m, 10H, Ph);
13C NMR (D2O): δ= 12.7, 42.0, 44.4 (d, J = 111.4 Hz), 127.1, 128.5, 128.7, 128.9, 131.4,
131.5, 132.7, 134.5, 172.8 (d, J = 4.6 Hz);
IR (ATR) ν (cm−1): 3233, 3057, 1634, 1544, 1167, 950, 690;
HRMS(ESI): calc. for C16H19NO3P [M+H]+ m/z 304.1103, found m/z 304.1102.
2.3.4.9 1-(N-Benzyloxycarbonylamino)-2-hydroxyethylphosphonic acid dimethyl ester 2i
31P NMR (D2O): δ = 26.4;
1H NMR (D2O): δ = 3.72-3.80 (m, 1H, CH2OH), 3.75 (d, J = 12.0 Hz, 6H, OMe), 3.85-3.90
(m, 1H, CH2OH), 4.22-4.29 (m, 1H, CH), 5.12 (d, J = 12.0 Hz, 1H, PhCH2O), 5.19 (d, J =
12.0 Hz, 1H, PhCH2O), 7.37-7.68 (m, 5H, Ph);
13C NMR (D2O): δ = 26.4, 49.7 (d, J = 152.5 Hz), 53.8 (d, J = 7.6 Hz), 53.8 (d, J = 7.1 Hz),
67.3, 127.6, 128.4, 128.7, 136.2, 157.8;
IR (ATR) ν (cm−1): 3283, 2956, 1698, 1533, 1215, 1024, 696;
HRMS(ESI): calc. for C12H19NO6P [M + H]+ m/z 304.0949, found m/z 304.0950.
2.3.5. Racemic 1-(N-acylamino)alkyl-H-phosphinic acids 4a-c (Procedure C)
Syntheses of crude 1-(N-acylamino)alkyltriphenylphosphonium tetrafluoroborates
from the corresponding N-acylaminoacids 1 were carried out as described in procedure A. A
solution of the phosphonium salt (2 mmol) in CH2Cl2 (6 mL), triphenylmethylphosphonium
iodide (0.020 g, 0.5 mmol), diisoprophylethylamine (0.03 mL, 0.2 mmol) and
bis(trimethylsilyl)phosphonite (3 mmol) was placed in a glass vial sealed with a screw-cap.
The mixture was kept at room temperature for a given period of time (Table 1). Then
methanol (2 mL) was added and the mixture was left to stay overnight at room temperature.
After filtration, the solvent was evaporated under reduced pressure and the remaining was
11

extracted with toluene (4 x 2 mL). The residue from extraction was subjected to ion-exchange
chromatography using DOWEX 50W X8 in the H+ form, and water as elution agent to isolate
the product.
2.3.5.1 1-(N-Benzyloxycarbonylamino)ethylphosphinic acid 4a [33]
31P NMR (D2O): δ =28.1;
1H NMR (D2O): δ = 1.28 (dd, J1 = 7.6 Hz, J2 = 16.4 Hz, 3H, CH3), 3.78 (dq, J1 = 7.2 Hz, J2 =
15.2 Hz, 1H, CH), 5.13 (s, 2H, CH2Ph), 6.87 (d, J = 543.6 Hz, 1H, PH), 7.37-7.46 (m, 5H,
Ph).
2.3.5.2 1-(N-Phenylacetylamino)ethylphosphinic acid 4b [31]
31P NMR (D2O): δ = 26.9;
1H NMR (D2O): δ = 1.25 (dd, J1 = 7.6 Hz, J2 = 15.6 Hz, 3H, CH3), 3.64 (s, 2H, CH2Ph), 3.96
(dq, J1 = 7.2 Hz, J2 = 14.8 Hz, 1H, CH), 6.81 (d, J = 530 Hz, 1H, PH), 7.31-7.42 (m, 5H, Ph).
2.3.5.3 1-(N-Benzyloxycarbonylamino)-2-tert-butoxyethylphosphinic acid 4c
31P NMR (D2O): δ = 23.3;
1H NMR (D2O): δ = 1.16 (s, 9H, t-Bu), 3.52-3.65 (m, 1H, CH), 3.62-3.78 (m, 2H, CH2-O-t-
Bu), 5.18-5.87 (m, 2H, OCH2Ph), 7.35-7.64 (m, 5H, Ph);
13C NMR (D2O): δ = 27.2, 51.2 (d, J = 103.5 Hz), 59.9, 67.4, 73.9, 128.5, 132.0, 132.1, 136.0,
156.5;
IR (ATR) ν (cm−1): 2380, 1697, 1525, 1365, 1123, 962, 794, 696;
HRMS(ESI): calc. for C14H23NO5P [M + H]+ m/z 316.1313, found m/z 316.1308.
2.4. Enzymatic reactions
Racemic substrates 2a-k, 4a-d or 3a-i (1 mmol) were dissolved in 0.2 M pH 7.0
phosphate buffer (20 mL) and the temperature of the mixture was adjusted to 25oC. The pH
was adjusted to 7.0 by the addition of 1 M aqueous NaOH as necessary. A solution of the
proper amount of penicillin G acylase (see Table 1) in phosphate buffer (0.45 mL) was then
added and the resulting mixture was stirred at 25oC for the time given in Table 1.
2.4.1. Kinetic experiments
In the case of kinetic experiments, the progress of the substrate conversion was
determined by extracting a 2 mL of aliquot of the reaction mixture, acidification to pH 3.5
with 0.1M to aqueous HCl (in the case of free acids 3 or 4) or with 0.2 M pH 3.5 acetate
buffer in the case of esters 2. After evaporation of water under reduced pressure, a precisely
measured amount of a solution of pivalic acid in D2O was added as the internal standard
(about 0.5 mg of the pure compound), the residue was dissolved in D2O, and the amount of
substrate and hydrolysis product was determined by 1H NMR.
12

2.4.2. Preparative experiments
2.4.2.1.Separation of deacylation products 5a-h and remaining N-acylated esters 2a-k
The reaction mixture was acidified with 0.2 M pH 3.5 acetate buffer to pH = 4.0 and water
was evaporated under reduced pressure. The residue was extracted with CH2Cl2 (4 x 3 mL),
the extract was dried with MgSO4, the solvent was evaporated under reduced pressure, and
the residue was separated by column chromatography (silica gel, CH2Cl2/MeOH 20:1).
2.4.2.1.1 (R)-1-Aminoethylphosphonic acid dimethyl ester 5a [34]
31P NMR (CDCl3): δ = 31.1;
1H NMR (CDCl3): δ = 1.45 (dd, J1 = 7.2 Hz, J2 = 18.0 Hz, 3H, CH3), 3.21 (dq, J1 = 7.2 Hz, J2
= 9.6 Hz, 1H, CH), 3.87 (d, J = 10.8 Hz, 3H, OMe), 3.89 (d, J = 10.4 Hz, 3H, OMe).
2.4.2.1.2 (R)-1-Amino-2-phenylethylphosphonic acid dimethyl ester 5b [35]
31P NMR (CDCl3): δ = 30.1;
1H NMR (CDCl3): δ = 3.19 (ddd, J1 = 4.0 Hz, J2 = 7.6 Hz, J3 = 14.0 Hz, 1H, CHCH2), 3.34
(ddd, J1 = 3.6 Hz, J2 = 11.2 Hz, J3 = 14.8 Hz, 1H, CHCH2), 3.74 (ddd, J1 = 3.6 Hz, J2 = 8.4
Hz, J3 = 14.0 Hz, 1H, CH), 3.78 (d, J = 10.4 Hz, 3H, OMe), 3.80 (d, J = 10.4 Hz, 3H, OMe),
7.33-7.19 (m, 5H, Ph);
HRMS(ESI): calc. for C10H17NO3P [M + H]+ m/z 230.0946, found m/z 230.0947.
2.4.2.1.3 (R)-1-Amino-2-methylpropylphosphonic acid dimethyl ester 5c
31P NMR (CDCl3): δ = 31.0;
1H NMR (CDCl3): δ = 1.02 (d, J = 6.4 Hz, 3H, CH(CH3)2), 1.05 (d, J = 7.2 Hz, 3H,
CH(CH3)2), 2.08-2.15 (m, 1H, CH(CH3)2), 2.94 (dd, J1 = 4.0 Hz, J2 = 14.4 Hz, 1H, CH), 3.79
(d, J = 10.4 Hz, 3H, OMe), 3.78 (d, J = 10.4 Hz, 3H, OMe);
13C NMR (CDCl3): δ = 17.3 (d, J = 4.6 Hz), 20.5 (d, J = 12.9 Hz) 29.2 (d, J = 3.5 Hz), 52.7
(d, J = 6.8 Hz), 52.8 (d, J = 7.6 Hz), 53.8 (146.4 Hz);
IR (ATR) ν (cm−1): 3392, 2958, 1563, 1465, 1373, 1221, 1027, 827, 699;
HRMS(ESI): calc. for C6H17NO3P [M + H]+ m/z 182.0946, found m/z 182.0945.
2.4.2.1.4 (R)-1-Amino-3-methylbutylphosphonic acid dimethyl ester 5d [34]
31P NMR (CDCl3): δ = 29.2;
1H NMR (CDCl3): δ = 0.94 (d, J = 6.4 Hz, 3H, CH(CH3)2), 0.98 (d, J = 6.4 Hz, 3H,
CH(CH3)2), 1.64-1.82 (m, 3H, CH2CH(CH3)2 + CH(CH3)2), 3.64 (ddd, J1 = 4.0 Hz, J2 = 10.0
Hz, J3 = 14.4 Hz, 1H, CH), 3.87 (d, J = 10.8 Hz, 3H, OMe), 3.89 (d, J = 10.8 Hz, 3H, OMe).
2.4.2.1.5 (R)-1-Amino-2-carbamoylethylphosphonic acid dimethyl ester 5e
31P NMR (CD3CN): δ = 29.9;
13

1H NMR (CD3CN): δ = 2.28 (ddd, J1 = 8.2 Hz, J2 = 10.9 Hz, J3 = 18.7 Hz, 1H, CHCH2), 2.51
(ddd, J1 = 3.2 Hz, J2 = 8.6 Hz, J3 = 15.2 Hz, 1H, CHCH2), 3.44 (ddd, J1 = 3.6 Hz, J2 = 10.9
Hz, J3 = 14.1 Hz, 1H, CH), 3.71 (d, J = 10.4 Hz, 3H, OMe), 3.74 (d, J = 10.4 Hz, 3H, OMe);
13C NMR (CD3CN): δ = 38.5, 46.6 (d, J = 153.7 Hz), 54.1 (d, J = 6.9 Hz), 54.3 (d, J = 7.0
Hz), 174.0 (d, J = 3.7 Hz);
IR (ATR) ν (cm−1): 3342, 1668, 1555, 1408, 1203, 1042, 780;
HRMS(ESI): calc. for C5H14N2O4P [M + H]+ m/z 197.0691, found m/z 197.0690.
2.4.2.1.6 (R)-1-Aminoethyl(phenyl)phosphinic acid ethyl ester 5i (a mixture of diastereomers
in a ratio of 0.63 : 0.37) [36]
31P NMR (CDCl3): δ = 48.0 (minor), 48.9 (major);
1H NMR (CDCl3): δ = 1.16 (dd, J1 = 7.2 Hz, J2 = 17.2 Hz, 3H, -CH3, major ), 1.307 (dd, J1 =
7.2 Hz, J2 = 16.4 Hz, 3H,-CH3, minor), 1.309 (t, J = 6.8 Hz, 3H, OCH2CH3, major ), 1.312
(t, J = 7.2 Hz, 3H, OCH2CH3, minor), 3.18-3.26 (m, 1H, CH), 3.87-3.97 and 4.07-4.19 (m,
2H, OCH2CH3), 7.18-7.82 (m, 6H, NH, Ph).
2.4.2.2.Separation of deacylation products 6a-f or 7a-c and remaining N-acylated acids 3a-h
or 4a-d
The reaction mixture was acidified with 0.1 M aqueous HCl to pH 3.5 and water was
evaporated under reduced pressure. The residue was dissolved in water (2 mL) and passed
through a DOWEX 50W X8 (H+ form) column (50 mL), which was rinsed with water. The
first fractions contained remaining free acids 3a-h or 4a-d, whereas further, ninhydrin-
positive fractions contained deacylation products 5a-i.
2.4.2.2.1 (R)-1-Aminoethylphosphonic acid 6a [37,38]
31P NMR (D2O): δ = 14.9;
1H NMR (D2O): δ = 1.45 (dd, J1 = 6.8 Hz, J2 = 15.6 Hz, 3H, CH3), 3.44 (m, 1H, CH).
2.4.2.2.2 (R)-1-Amino-2-phenylethylphosphonic acid 6b [4,6,37]
31P NMR (D2O): δ = 11.7;
1H NMR (D2O): δ = 2.63-2.88 (m, 1H, CH2Ph), 3.21-3.29 (m, 1H, CH2Ph), 3.57-3.64 (m, 1H,
CH) 7.22-7.42 (m, 5H, Ph).
2.4.2.2.3 (R)-1-Amino-2-methylpropylphosphonic acid 6c [6,37,38]
31P NMR (D2O): δ = 13.9;
1H NMR (D2O): δ = 1.11 (d, J = 6.8 Hz, 3H, CH(CH3)2), 1.14 (d, J = 7.2 Hz, 3H, CH(CH3)2),
2.23-2.32 (m, 1H, CH(CH3)2), 3.30 (dd, J1 = 6.0 Hz, J2 = 14.4 Hz, 1H, CH).
2.4.2.2.4 (R)-1-Amino-3-methylbutylphosphonic acid 6d [4,37]
31P NMR (D2O): δ = 14.4;
14

1H NMR (D2O): δ = 0.94 (d, J = 6.0 Hz, 3H, CH(CH3)2), 0.98 (d, J = 5.6 Hz, 3H, CH(CH3)2),
1.67-1.70 (m, 3H, CH2CH(CH3)2 + CH2CH(CH3)2), 3.13-3.18 (m, 1 H, CH).
2.4.2.2.5 (R)-1-Amino-2-carbamoylethylphosphonic acid 6e [39]
31P NMR (D2O): δ =11.5;
1H NMR (D2O): δ = 2.69-2.73 (m, 1H, CHCH2), 2.91-2.96 (m, 1H, CHCH2), 3.63-3.67 (m,
1H, CH).
2.4.2.2.6 (R)-1-Aminoethyl-H-phosphinic acid 7a [40]
31P NMR (D2O): δ = 21.4;
1H NMR (D2O): δ = 1.41 (dd, J1 = 7.2 Hz, J2 = 15.6 Hz, 3H, CH3), 3.24 (dq, J1 = 7.2 Hz, J2 =
9.6 Hz 1H, CH), 6.97 (d, J = 531.6 Hz, 1H, PH).
2.4.2.2.7 (R)-1-Aminoethyl(phenyl)phosphinic acid 7c [41]
31P NMR (D2O): δ = 27.3;
1H NMR (D2O): δ = 1.38 (dd, J1 = 7.2 Hz, J2 = 14.0Hz, 3H, CH3), 3.54 (dq, J1 = 7.2 Hz, J2 =
9.6 Hz, 1H, CH), 7.41-7.80 (m, 5H, Ph).
2.5. Enantiomeric excess determination
2.5.1. NMR methods
The enantiomeric excesses of unreacted N-acylated acids 3a-h and 4a-c (ees) were
determined by using 31P NMR spectroscopy in the presence of quinine as the chiral
discriminating agent. The analysed sample (5 mg) and quinine (10-25 mg) were dissolved in a
mixture of CDCl3 (0.55 mL) and CD3OD (0.1 mL) and 31P NMR spectrum was measured.
The enantiomeric excess was determined based on the integration of phosphorus signals of
both enantiomers.
The enantiomeric excesses of unreacted N-acylated esters 2b-g, 2k, as well as
ethyl(phenyl)phosphinic acid 4d, was determined by 1H NMR spectroscopy in the presence of
quinine. The analysed sample (5 mg ) and quinine (10-25 mg) ware dissolved in CDCl3 (0.65
1
mL) and H NMR spectrum was measured. The enantiomeric excesses of esters 2a-g were
determined based on the integration of OMe group signals of both enantiomers. In the case of
ethyl(phenyl)phosphinic acid derivatives 2k and 4d, signals of CH2 protons of benzyl group
and signals of -Me group were integrated, respectively
In the all cases, the satisfactory resolution of enantiomers signals was verified by
adding 1-2 mg of the corresponding racemate to the analysed sample and repeating the
measurement.
2.5.1. Chiral HPLC
15

For most N-acylated acids 3a-f and 4a and 4d as well as esters 2c, 2e and 2f,
enantiomeric excesses were also successfully determined by means of chiral HPLC. The
chromatographic separation of enantiomers of acids 3a-f and 4a and 4d was performed on a
CHIRALPAK QN-AN (150x4.6 mm, 5 m) column (Daicel/Chiral Technologies Illkirch,
France)
maintained
at
25oC.
The
mobile
phase
was
composed
of
MeOH/CH3CO2H/ammonium acetate (98:2:0.5; v/v/w) using an isocratic elution with a flow
rate set at 0.8 mL/min. The detector wavelength was set at 232 nm and injection volume was
20 L. Chromatographic separation of enantiomers of esters 2c, 2e and 2f was achieved on a
CHIRALCEL OD-RH (150x4.6 mm, 5 m) column (Daicel/Chiral Technologies Illkirch,
France). Mobile phase consisted of a mixture of hexane/isopropanol (92:8; v/v for ester 2c
and 2e, and 97:3; v/v for ester 2f) at 1.0 mL/min flow rate. The column temperature was
maintained at 25oC, the injection volume was 20 L, the detection was performed at 222 nm.
3. Results and discussion
The N-acylated -aminoalkylphosphonic and -aminoalkylphosphinic acids esters
used in this work were prepared as racemic mixtures using the method described in our
previous papers [21-24] (Table 1). Their hydrolysis in 2 M hydrochloric acid to N-acylated
acids was satisfactorily performed according to a well-known method [42] (Scheme 1, Table
1). Only in the case of ester 2h, the product of deprotection of hydroxymethyl group with
untouched dimethoxyphosphonyl group 2i was obtained instead of the expected product 3g.
The obtained N-acylated acids were isolated by cation-exchange chromatography and purified
by recrystallization. The structures of the obtained compounds were confirmed by their
spectroscopic (1H-, 13C-, 31P NMR, IR) and HRMS data (see Experimental).
Enzymatic deacylation of N-acylated -aminoalkylphosphonic and -aminoalkylphosphinic
acids as well as their esters was carried out in a water solution of phosphate buffer (pH 7.0) at
25oC, using various reaction time and amount of enzyme depending on the specific reactivity
of substrate. The optimal reaction conditions for every specific substrate, assuring a racemic
substrate conversion close to 50%, were determined in a series of preliminary experiments, in
which initial hydrolysis rates were measured (Table 2). The concentrations of the reaction
1
product and unconverted substrate in reaction mixtures were determined by means of H
NMR spectroscopy using pivalic acid as the internal standard.
16

Analysis of the initial reaction rates obtained (Table 2) showed, that the reactivity of a
substrate depends strongly on the kind of its N-acyl group. The ratio of the initial hydrolysis
rate of 1-(N-acetylamino)- and 1-(N-phenylacetylamino)ethylphosphonic acids was found to
be about 1 : 38000, whereas the same ratio for methyl esters of 1-(N-acetylamino)-, 1-(N-
benzyloxycarbonyamino)-, and 1-(N-phenylacetylamino)ethylphosphonic acids was about 1 :
95: 5100000, respectively. Therefore, our results confirmed that penicillin G acylase showed
a high preference in catalysing the hydrolysis of N-phenylacetyl moiety [4,18,19].
The initial hydrolysis rates of 1-(N-phenylacetylamino)akylphosphonic acid dimethyl
esters 2 in most cases are distinctly lower compared to the rates obtained for the
corresponding free acids 3. Also the initial hydrolysis rate of ethyl ester of
ethyl(phenyl)phosphinic acid 2k is about two times lower compared to the corresponding acid
4d. Only in the case of the 1-(N-phenylacetylamino)ethylaminophosphonic acid dimethyl
ester 2c, reactivity of ester is about three times higher than the reactivity of the parent acid 3b.
For a series of 1-(N-phenylacetylamino)alkylphosphonic acids 3b-f the initial
hydrolysis rate rapidly decreased with the increasing steric effect of the substituent at the -
position. Reactivity of the phosphonic acid analogue of alanine (3b, R2 = Me) was found to be
2-3 orders of magnitude higher compared to the reactivities of phosphonic analogues of
phenylalanine, valine, leucine and asparagine (3c-f; R2 = PhCH2, i-Pr, i-PrCH2, and
H2NCOCH2, respectively). The analogous, but even more distinct effect of the reduction of
reactivity by bulky substituents at the -position was seen in a series of dimethyl esters of N-
acylated -aminophosphonic acids 2c-g; the reactivity of the phosphonic analogue of alanine
(2c, R2 = Me) was, in this case, 4-6 orders of magnitude higher than the reactivity of the
corresponding phenylalanine, valine, leucine and asparagine, t-Bu-serine and serine (2d-i; R2
= PhCH2, i-Pr, i-PrCH2, H2NCOCH2, t-BuOCH2, and HOCH2, respectively).
Our efforts to hydrolyse phosphonic analogues of N-phenylacetylproline, both as the
free acid (3h) and as its methyl ester (2j), failed, even with high concentration of penicillin G
acylase and a prolonged reaction time. It seems, that the presence of proton at the amide
nitrogen of the acylamine moiety of substrate is crucial to ensure the catalytic activity of
penicillin G acylase in hydrolysis of N-acylated -aminophosphonic acids and their esters.
The presence of bulky phenyl group at the phosphorus atom in phenylphosphinic acid
analogue of 1-(N-phenylacetylamino)alanine 4d exerts unexpectedly slight reduction of its
reactivity in comparison with its H-phosphinic acid analogue 4b; moreover, only a slightly
lower reactivity was found for its diethyl ester 2k. It seems that in contrary to the substituents
17

at the -carbon, the bulky substituents at the phosphorus hinder the enzymatic hydrolysis to a
much lesser degree.
Enantiomeric excesses of unreacted N-acylated acids 3a-f, 4a-b, and 4d were
31
determined based on the integration of P NMR signals of both enantiomers of a substrate,
using quinine as the chiral discriminating agent (Fig.1a). In the case of unconverted N-
acylated esters 2b-g, and 2k, as well as ethyl(phenyl)phosphinic acid 4d, more satisfactory
1
resolutions were obtained for H NMR signals of OMe groups, CH2 protons of benzyl group
or -Me group, respectively (Fig. 1b). In the case of most unconverted N-acylated acids 3 and
4, as well as esters 2c, 2e and 2f enantiomeric excesses were also successfully determined by
chiral reversed-phase HPLC, using a CHIRALPAK QN-AX column (for acids), or by chiral
normal-phase chromatography, using a CHIRALPAK OD-RH column (for esters). The
1
obtained results were in a reasonable agreement with the data obtained using the 31P or H
NMR method (Table 2).
The enzyme enantioselectivity E was calculated as a function of extent of conversion c
and enantiomeric excess of the remaining substrate ees, measured at conversions close to 50%
(Table 2). E was defined as:
ln[(1c)(1eeS )]
ln[(1c)(1 eeS )]
E 
[12]
In the case of 1-(N-acetylamino)alkylphosphonic acids 3a-h highly effective kinetic
resolutions with the E-values exceeding 100 were obtained for phosphonic acid analogues of
alanine 3a and 3b, phenylalanine 3c, leucine 3e, and asparagine 3f; synthetically useful E-
value exceeding 20 was obtained also for the analogue of valine 3d (E = 22). Enzyme
enantioselectivities obtained for 1-(N-acylamino)alkylphosphonic acid methyl diesters 2 also
exceeded the value 100 for phosphonic analogues of alanine 2b and 2c, phenylalanine 2d, and
asparagine 2g; a satisfactory result was obtained also for the leucine analogue 2f (E = 66).
Only in the case of dimethyl ester of phosphonic analogue of valine 2e, the E-value was low
(E = 1.2), and lower that this one for the corresponding free acid 3d.
Also for the 1-N-acylated H-phosphinic acid analogues of alanine (4a-b), as well as
phenylphosphinic acid analogue of alanine 4d, high enzyme enantioselectivity values
exceeding 100 were determined. In contrary, the E-value was low for both diastereomers of
the ethyl ester of the phenylphosphinic analogue of alanine 2k (E = 7 and 13).
In the case of a few investigated compounds of extremely low reactivity (2a, 2h, 2i
and 4c) attempts of kinetic resolution were not undertaken.
18

The absolute configuration of hydrolysis products and unconverted substrates was
assigned by measurements of their optical rotation and the comparison with literature data.
For all the accomplished assignments, penicillin G acylase exhibited stereochemical
preference for the (R)-substrate. The same stereochemical preference of penicillin G acylase
was found by Zimmermann et al. [20] and Solodenko et al. [4,18] for phosphonic analogues
of 1-N-phenylacetylated alanine, phenylalanine, and leucine, as well as H-phosphinic
analogue of 1-N-phenylacetylalanine.
Conclusions
Reactivity of 1-(N-acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic
acids and their esters in the penicillin G acylase-mediated hydrolytic deacylation reaction
depends strongly on the kind of its N-acyl group; the obtained results confirm high preference
of penicillin G acylase to catalyse the hydrolysis of the N-phenylacetyl moiety.
The initial hydrolysis rates of 1-(N-phenylacetylamino)alkylphosphonic acid dimethyl
esters 2 are, in most cases, distinctly lower compared to the corresponding free acids 3. Only
in the case of 1-(N-phenylacetylamino)ethylaminophosphonic acid dimethyl ester 2c, the
reactivity of ester is about three times higher than the reactivity of the parent acid 3b.
The initial hydrolysis rates of 1-(N-acylamino)alkylphosphonic acids and their esters
rapidly decrease with the increasing steric effect of the substituent at the -position.
Reactivities of the phosphonic analogues of alanine, both free acid 3b and their dimethyl
esters 2c (R2 = Me), are 2-6 orders of magnitude higher compared to that of the corresponding
phosphonic analogues of phenylalanine, valine, leucine, asparagine, t-Bu-serine, and serine
(3c-f and 2d-i). It seems that in contrary to the substituents at the -carbon, bulky substituents
at phosphorus hinder the enzymatic hydrolysis to a much lesser degree. The efforts to
hydrolyse phosphonic analogues of N-phenylacetylproline, both as the free acid (3h) and as
its dimethyl ester (2j), failed.
The penicillin G acylase-mediated stereospecific hydrolysis of N-acyl group of
both,racemic 1-(N-acylamino)alkylphosphonic acids 3 and their dimethyl esters 2 proved to
be, in most cases, the highly effective method for the kinetic resolution of these compounds.
The enzyme enantioselectivity E-values exceeding 100 were obtained for the N-acylated
phosphonic acid analogues of alanine, phenylalanine, leucine, and asparagine, as well as for
their dimethyl esters, with the exception of the leucine diester. A synthetically useful E-value
exceeding 20 was obtained also for the phosphonic acid analogue of valine 3d and for the
19

dimethyl ester of phosphonic analogue of leucine 2f; only in the case of dimethyl ester of
phosphonic analogue of valine 2e the E-value was low (E = 1.2).
Also for the 1-N-acylated H-phosphinic acid analogues of alanine (4a-c), as well as
phenylphosphinic acid analogue of alanine 4d, high enzyme enantioselectivity values
exceeding 100 were determined. In contrary, the E-value was low for both diastereomers of
the ethyl ester of the phenylphosphinic analogue of alanine 2k (E = 7 and 13).
For all the accomplished assignments, penicillin G acylase exhibited stereochemical
preference for the (R)-substrate.
Acknowledgments
The financial support of the National Science Centre Poland (Grant No.
2012/07/N/ST5/00253) is gratefully acknowledged.
20

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21

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Obshch. Khim., 62 (1992) 1472-1480.
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22

41. I. Popoff, L. Huber, B. Block, P. Morton, R. Riordan, J. Org. Chem., 28 (1963) 2898-
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23

a)
b)
31
Figure 1. Enatiomeric excess determination using NMR methods: a). P NMR; left - (R)-1-
(N-phenylacetylamino)ethylphosphonic acid [(R)-3b] obtained by kinetic resolution (8 mg)
with quinine (22 mg) in CDCl3/CD3OD (0.6 mL/ 0.1 mL); right – racemic (R,S)-3b – the
1
same conditions. b). H NMR; left – (R)-1-(N-phenylacetylamino)ethylphosphonic acid
dimethyl ester [(R)-2c] (5 mg) with quinine (17 mg) in CDCl3; left – sample of (R)-2c
enriched with racemic (R,S)-2c (2 mg) – the same conditions.
24

Scheme 1. Syntheses of racemic 1-(N-acylamino)alkylphosphonic and 1-(N-
acylamino)alkylphosphinic acids and their esters.
25

Scheme 2. Kinetic resolution of 1-(N-acylamino)alkylphosphonic and 1-(N-
acylamino)alkylphosphinic acids and their esters.
26

Table 1. Transformation of N-acyl-α-amino acids 1 into N-acylated 1-aminoalkylphosphonic and 1-aminoalkylphosphinic acids esters 2 and free
acids 3 or 4
N-acyl-α-amino
acid
Phosphorus
Michaelis-Arbuzov reaction conditions and results
Free acids 3 or 4
nucleophile PR4R5(OR6)
Yield
[%]
97
No. R1
R2
R3
R4
R5
R6
Proc.
Time [h] No. Yield [%] m.p. [ºC]
No. R7
Proc.
B
m.p.[ºC]
181-182
Me
Me
Me
Me
PhCH2
i-Pr
i-PrCH2
H2NCOCH2
t-BuOCH2
-(CH2)3-
Me
Me
t-BuOCH2
Me
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Me
Me
Me
Me
Me
Me
Me
Me
Me
A
A
A
A
A
A
A
A
A
C
C
C
A
2
2
2
4
2
4
4
2
83
78
84
61
92
83
66
86
50
65-67
oil
OH
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2g
2h
2j
3a
PhCH2O
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2O
PhCH2
PhCH2O
PhCH2
PhCH2O
PhCH2
85-86
93-94
62-63
102-104
156-157
68-69
oil
OH
OH
OH
OH
OH
OH
OH
H
B
B
B
B
B
B
B
-
97
98
94
76
99
140-141
145-146
142-143
139-142
oil
-
oil
109-110
139-140
oil
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
1g
1h
1i
a
-
4
75
85
91
94
97
H
H
H
H
OTMS TMS
OTMS TMS
OTMS TMS
24
24
24
2
not isolated
not isolated
not isolated
1b
1c
1h
1c
H
H
Ph
H
H
Ph
-
-
B
OEt
Et
90
81-83
91-92
2k
aThe product of deprotection of CH2OH group 2i (R1 = PhCH2O, R2= CH2OH, R3= H, R4= R5= OMe) was obtained in a yield of 91% (oil) instead of the expected product 3g.
27

Table 2. Enzymatic hydrolysis of N-acylated 1-aminoalkylphosphonic and 1-aminoalkylphosphinic esters 2 and free N-acylated acids 3 or 4
Substrate 2, 3 or 4
Kinetic resolution results
eesb
x100
[%]
[]2D0 [o]
Yield [%]a
Substrate Product Substrate Product
Enzyme
U/mmol
Initial
(μmol/Umin)
hydrolysis
Product
No.
Product
config.
No.
R1
R2
R3
R4
OMe OMe
R5
R7
Time [h] Conversion
Ec
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-
2000
2000
500
4
2.86 ±0.13 x10-6
1.32 ±0.21 x10-4
2.71 ±0.24 x10-4
1.46 ±0.1 x101
5.01 ±0.70
504
216
144
0.5
1
0.15
0.43
0.46
0.49
-
-
-
-
-
2a
3a
2b
2c
3b
2d
3c
2e
3d
2f
3e
2g
3f
2h
2i
5a
6a
5a
5a
6a
5b
6b
5c
6c
5d
6d
5e
6e
5f
5g
5h
6f
7a
7a
7b
5i
-
-
OH
-
-
OH
-
OH
-
OH
-
OH
-
OH
-
-
48
46
42
37
49
39
48
45
31
45
49
42
-
35
41
45
41
42
48
33
41
34
40
41
45
-
+15.6d
+14.2f
+42.4h
+37.9j
+27.5l
+40.4n
+3.33r
-0.62t
+25.4v
+31.4z
+6.6ab
+7.9ad
-
-16.3e
-6.2g
-6.8i
-5.1k
-22.5m
-50.6p
-1.38s
+0.58u
-16.6w
-24.8aa
-2.2ac
-32.5ae
-
R
R
R
R
R
R
67(77)
87
99(99)
99
>100
>100
>100
>100
380
>100
1.2
22
66
>100
>100
660
-
PhCH2O
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2O
PhCH2O
PhCH2
PhCH2
PhCH2O
PhCH2
PhCH2O
PhCH2
PhCH2
OMe OMe
OMe OMe
Me
-
-
4
0.50
PhCH2
PhCH2
i-Pr
OMe OMe
500
500
2000
500
500
400
500
500
2000
2000
2000
2000
4
5.21 ±0.40 x10-4
4.31 ±0.67 x10-3
5.69 ±0.04 x10-5
7.16 ±0.82 x10-4
6.89 ±0.03x10-4
7.44 ±0.55 x10-3
6.83 ±0.07 x10-4
4.19 ±0.72 x10-3
5.12±1.44 x10-6
~5.30x10-6
72
0.46
84
-
-
47
0.52
99(99)
14(8)
75(73)
82(85)
99(99)
99
99(95)
-
-
OMe OMe
92
0.37
i-Pr
-
-
48
0.47
i-PrCH2
i-PrCH2
H2NCOCH2
H2NCOCH2
t-BuOCH2
HOCH2
-(CH2)3-
-(CH2)3-
Me
OMe OMe
72
0.48
-
-
23
0.49
R
R
OMe OMe
48
0.48
-
-
24
0.49
OMe OMe
OMe OMe
OMe OMe
336
288
216
190
24
0.17
0.16
no reaction
no reaction
0.46
-
-
-
-
-
-
-
-
no reaction
-
-
-
-
-
-
2j
-
-
OH
H
H
H
-
no reaction
-
-
-
-
3h
4a
4b
4c
2kaj
4d
H
H
H
H
H
OH
OH
OH
OEt
OH
-
-
-
Ph
-
1.70 ±0.09 x10-1
6.20 ±0.37
40
29
-
41
34
-
+47.5af
+71ah
-
-5.9ag
-6.7ai
-
R
R
-
85(87)
99
>100
>100
-
Me
t-BuOCH2
Me
4
1
0.49
2000
10
8
3.89±0.38 x10-5
336
2
0.25
-
2.00 ±0.16ak
0.45al/048am 49an
47ap
48
+72.0ar
+76.5at
-14.7as
-35.8aw
52al/68am 7al/13am
Me
Ph
4.28 ±0.37
2
0.47
19
79(85)
>100
7c
b
c
aIsolated yield after chromatography. Based on 31P- or 1H NMR in the presence of quinine; results based on chiral HPLC are in parenthesis. If ees value was determined
d
independently by HPLC and NMR method, the E value was calculated based on the data obtained by a chiral HPLC method. c 0.3, 1M NaOH, ec 0.2, 1M NaOH, Lit.[37]
[]20D -16.9º (R) c 1, 1M NaOH, fc 0.5, CH2Cl2, Lit. [29] []20D +14.4 º (S) c 1 CH2Cl2, gc 0.4, MeOH, c 0.4 MeOH, Lit. [4] []20D +44.5º (S) c 1, MeOH, c 0.5, MeOH, c
h
i
j
l
0.5, H2O, Lit.[18] []20D +38o (S), c 0.5, H2O, kc 0.5, H2O, Lit.[4] []20D -5.5o (R) c 1, H2O, Lit. [37] []20D -16.9o (R) c 1, 1M NaOH, c 0.9, CHCl3, mc 1.1, MeOH, Lit. [35]
r
t
u
[]20 +25.9 (S) c 0.9 CHCl3, nc 0.2, 1M NaOH, pc 0.4, 1M NaOH, Lit.[37] []20D -49.0º (R) c 0.25 1M NaOH, c 0.2, CHCl3, sc 0.2, MeOH, c 0.2, 1M NaOH, c 0.2 1M
D
NaOH, vc 0.6, CHCl3, wc 0.3, CHCl3, zc 0.5, 1M NaOH, aac 0.5 1M NaOH, Lit. [37] []20D -25.0º (R) c 1 1M NaOH, abc 0.5 CH3CN, acc 0.4 CH3CN, adc 0.4 1M NaOH, aec 0.3
H2O, Lit. [39 []20D -33.0º (R) c 1 H2O, afc 0.1 1M NaOH, Lit. [33] []20D+48.3º (R) c 1 Acetic Acid, agc 0.5 H2O, Lit.[18] []20D -6.8º (R) c 0.5 H2O, ahc 0.1 H2O, Lit.[18]
ai
aj
al
[]20D +74.0º (S) c 0.5 H2O, c 0.8 H2O, Lit.[18] []20D -6.8º (R) c 0.5 H2O, A mixture of diastereomers in a ratio of 0.55:0.45. akFor a mixture of diastereomers. Major
diastereomer. amMinor diastereomer. anA mixture of diastereomers in a ratio of 0.56:0.44. apA mixture of diastereomers in a ratio of 0.63:0.37.arc 2.2 CHCl3, asc 1.5 CHCl3, atc
0.6, 1M NaOH, awc 0.2 1M NaOH.
28