Substituted phosphonic analogues of phenylglycine as inhibitors of phenylalanine ammonia lyase from potatoes

Article abstract of DOI:10.1016/j.biochi.2018.06.005

A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato L-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (

Full text of DOI:10.1016/j.biochi.2018.06.005

Biochimie 151 (2018) 119e127
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Research paper
Substituted phosphonic analogues of phenylglycine as inhibitors of
phenylalanine ammonia lyase from potatoes
a
a
b
b
ꢀ
Weronika Wanat , Michał Talma , Jozef Hurek , Małgorzata Pawełczak ,
,
*
Paweł Kafarski a
a
_
ꢀ
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeze Wyspianskiego 27, 50-370, Wrocław,
Poland
b Department of Chemistry, University of Opole, ul. Oleska 48, 45-052, Opole, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 February 2018
Accepted 6 June 2018
A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been
synthesized and evaluated for their inhibitory activity towards potato L-phenylalanine ammonia lyase.
Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of
their binding performed using molecular modeling have shown that they might be bound either in active
site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby
the to the entrance channel for substrate into active site.
Keywords:
PAL inhibitors
Aminophosphonates
Molecular modeling
Structure-activity relationship
© 2018 Published by Elsevier B.V.
1. Introduction
Enzyme inhibitors are used as tools for studying mechanisms of
enzymatic catalysis and as compounds for treating certain physi-
Phenylalanine ammonia lyase (PAL) catalyzes non-oxidative
transformation of -phenylalanine into trans-cinnamic acid and
ologic disorders. Phosphonic analogues of amino acids are well-
recognized class of inhibitors of enzymes of variable activities
and thus are believed to be valuable potential dugs and pesticides
[16e18]. Such analogues of aromatic amino acids also rank amongst
the most interesting inhibitors of phenylalanine ammonia lyase
[19e23], with a strained analogue of substrate - 2-aminoindane-2-
phosphonic acid (compound 1 in Scheme 1B) being the most potent
[24e26] and with analogues of phenylglycine (compound 2,
Scheme 1B) being the simplest ones with micromolar inhibitory
constants (value of which depends on enzyme source) [27].
Therefore, we have synthesized a series of analogues of phenyl-
glycine bearing fluorine atoms in phenyl ring. For comparison, a
series of variously substituted phenylglycine analogues was also
obtained. All the compounds (see Table 1) were evaluated for their
potency to inhibit activity of phenylalanine ammonia lyase isolated
from potatoes.
Fluorination has become recently an increasingly popular
strategy in medicinal chemistry and protein biochemistry.
Replacement of phenylalanine, tyrosine or tryptophan by their
fluorinated analogues has been recently described as a mean to
study amino acid interactions within the protein fold [28,40],
whereas the judicious introduction of fluorine into an inhibitor
molecule can productively influence conformation, pKa, intrinsic
L
ammonia (Scheme 1A). This reaction is a first step for the chan-
neling of carbon from primary metabolism into phenylpropanoid
secondary metabolism in plants [1e3]. Since phenylpropanoids
play a vital role in development and response to environmental
stimuli this enzyme have been intensively studied with respect to:
its role in plants, catabolic function in fungi, its mechanism of ac-
tion, and three-dimensional structure [4e7]. It was also considered
as a possible target in the search for new compounds of herbicidal
activity, however, phenylopropanoids are so important to plants
that blocking the activity of phenylalanine ammonia lyase launches
their biosynthesis by alternative pathway [8e10]. Additionally, PAL
natural ability to break down L-phenylalanine makes it a reliable
treatment for the genetic condition phenylketonuria, an inherited
disorder that increases the levels of phenylalanine in the blood
[11,12]. Finally, reversed reaction catalyzed by PAL has been suc-
cessfully utilized for the synthesis of structurally diverse analogues
of phenylalanine, useful building blocks in medicinal chemistry
[13e15].
* Corresponding author.
E-mail address: pawel.kafarski@pwr.edu.pl (P. Kafarski).
https://doi.org/10.1016/j.biochi.2018.06.005
0300-9084/© 2018 Published by Elsevier B.V.

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W. Wanat et al. / Biochimie 151 (2018) 119e127
Scheme 1. Reaction catalyzed by L-phenylalanine ammonia lyase (A) and structures of the chosen inhibitors of the enzyme (B).
potency, membrane permeability, metabolic pathways, and phar-
macokinetic properties of new drug candidate [29e33]. Although a
C-F bond (1.34 Å for an sp3 carbon) is about 20% longer than a C-H
bond (1.09 Å), fluorination has been shown as being well tolerated
by a variety of proteins without introducing much steric pertur-
bation to the parent structure.
with bromine, chlorine and methyl group being the most suitable
(compounds 5, 6 and 20). This suggests that steric effects are
playing the most important role here. Analogues substituted with
nitrile moiety appeared to be completely inactive showing that
there is a certain limit of para-substitution.
Moreover, the introduction of fluorine as an additional substit-
uent in position 3 usually resulted in enhanced inhibitory activity
(see compounds 16 and 17), an effect, which is in opposition to
introduction of this atom in position 2 of aromatic ring (compounds
14 and 21). Summing up, introduction of one fluorine atom into
phenyl ring of phosphonic analogues did not affect significantly
activity of these compounds against PAL.
Thus, despite of descriptive analysis, there is no possibility to
build-up the simple structure-activity relationship for phosphonic
analogues of phenylalanine also because modeling studies (see next
paragraph) indicate that they might be bound in two neighboring
binding sites of the enzyme e either in the active site (subunit A) or
in site, which has no physiological meaning located nearby to the
entrance channel of substrate phenylalanine (subunit B).
2. Results and discussion
2.1. Synthesis
Synthesis of the series of racemic ring-substituted 1-
aminobenzylphosphonates, analogues of phenylglycine, was per-
formed by applying three-component amidoalkylation reaction
designed by Oleksyszyn and Soroka [34e36]. This procedure is
simple and straightforward and provided the desired compounds
in satisfactory yields.
2.2. Inhibitory activity
Inhibitory potency of the obtained compounds was evaluated
using phenylalanine ammonia lyase isolated from potatoes (Sola-
num tuberosum L.) and compared with literature data for buck-
wheat (Fagopyrum esculentum) enzyme [27]. Results presented in
Table 1 indicate that, with exception of compounds 3, 15, 25 and 26,
which are inactive, all analogues of phenylglycine appeared to be
weak or moderate inhibitors of PAL. Generally, analogues of phe-
nylglycine exhibit inhibitory activity towards buckwheat enzyme
higher than towards potato PAL. They are also equipotent with or
even more active than aminobenzylphosphonic acid 2, a formal
analogue of phenylglycine, against parsley (Petroselinum crispum)
enzyme [21,23]. Additionally, the most active compounds: 5, 6, 17,
18 and 20 were significantly less active than cyclic derivative 29
(Scheme 1B) towards buckwheat enzyme [22].
2.3. Docking of most potent inhibitors to Petroselinum crispum PAL
Docking studies might contribute to understanding of mode of
binding of phenylglycine analogues by the enzyme and to identi-
fication of amino acids vital for this process. Quite surprisingly,
docking studies on parsley enzyme indicated that analogues of
phenylalanine could be bound in two subunits, namely in the active
site (subunit A marked as green in Fig. 1) and, unexpectedly, in
adjoining deep site located in subunit B (marked in red in Fig. 1).
The second site is located nearby the to the channel, which forms
an entrance for substrate into active site in unit A. Modeling pre-
dicts binding of compounds: 5, 6 and 10 (only S isomers), and both
isomers of compounds: 7, 11, 20 and 22 in this site, whereas
remaining ones are bound, as expected, in subunit A. It is worth to
mention, that in the case of aminophosphonic acids R-isomers are
mimicking S-amino acids. From modeling it is clearly seen that
Arg354 plays a major role for binding in site A forming strong
electrostatic interactions with phosphonic moiety, while in site B
similar role play Lys 336 and Lys345.
It is worth to note that total replacement of hydrogen atoms in
phenylglycine analogue 2 leading to perfluorinated compound 3
(Scheme 1B) resulted in total abolishment of inhibitory activity.
When analyzing single substitution of phenyl ring it is seen that
substitution of parent compound 2 in para-position if of choice

W. Wanat et al. / Biochimie 151 (2018) 119e127
121
binding sites or may reflect the differences between parsley and
potato enzymes. The process of movement of substrates and in-
hibitors along entrance channel has not been, however, analyzed in
he literature yet. This speculation is to some extend supported by
recent finding that some inhibitors of phenylalanine ammonia
lyase act as slow-binding ones [23].
Table 1
Structures and in vitro inhibitory potency of phosphonic analogues of phenylglycine
towards potato PAL.
Compound Structure
IC50
[
m
M] [S-Phe] ¼ 1 mM Ki [
mM]
2
all-H
6.5 [21]
For comparison, we have constructed models of pharmaco-
phores for these two binding sites (Fig. 2). These models are quite
similar to each other and thus, only small differences decide, which
site (A or B) is occupied by inhibitor.
As seen from Fig. 3 both enantiomers of compound 17 are
effectively bound to the site A of the enzyme, although in visibly
different manner. This is, more or less, typical situation for ana-
logues of phenylglycine studied in this work. It is also worth to
mention that R-phenylalanine acts as moderate inhibitor of the
enzyme [37], while S-isomer is its substrate.
Identified interactions of R-3-fluoro-4-chlorobenzylphosphonic
acid with the active site of the enzyme, shown in Fig. 4, are rela-
tively simple. The Arg 354 electrostatically stabilizes the two oxide
atoms of phosphonic acid group when its third oxygen atom in-
teracts with Phe 400 (blue arrow) and Tyr351. Phe116 interacts
41.5 [23]
3
4
perfluoro
4-F
naa
57.47 2.31
11.86 0.86
10.25 1.24
19.33 1.02
c
1.8 [27]
5
6
4-Cl
4-Br
1.85 0,23
0.21 [27]
4.65 0,48
0.29 [27]
10.08 3.43
ndb
na [27]
nd
na [27]
nd
7
8
4-I
2-F
106.87 6.13
628.76 58.24
9
2-Cl
2-Br
115.60 6.14
135.09 5.96
10
1.0 [27]
11
12
3-F
3-Cl
81.86 5.82
16.86 1.31
5.78 2.34
1.75 0.40
1.0 [27]
34.21 1.70
10.91 1.62
13
14
15
16
17
18
19
20
2-F,4-F
98,68 6,10
76.48 6.61
na
46.51 5.15
10.94 0.96
8.22 0.59
476.47 16.35
11,46 1,58
with phenyl ring of the inhibitor 17 (yellow arrow) by pep inter-
2-F,4-Cl
2-Cl,4-Cl
3-F,4-F
3-F,4-Cl
3-Cl,4-F
2,4,6-F,F,F
4-CH3
action. This interaction, however, is not of vital for inhibitory action
since any visible dependence of the inhibitory potential of the full
set of inhibitors bound in site A on electron density in their phenyl
ring is seen (data not shown).
4.72 1.05
3.14 0.76
1.65 0.48
nd
6,00 1,35
0.25 [27]
6.53 1.89
nd 8.3 [27]
nd
21
22
23
24
25
26
27
28
29
2-F,4-CH3
4-CF3
2-F,4-OCHF2
3-F,5-CF3
4-CN
2-F,4-CN
4-OCH3
4-NH2
101.78 3.55
130.11 6,65
118.02 3.43
181.79 7.91
na
2.4. Conclusions
nd
As seen, from this work and literature data, phosphonic acid
analogues of phenylglycine appear to be, in most cases, moderate
inhibitors of phenylalanine ammonia lyase, with inhibitory po-
tential being strongly dependent on the source of the enzyme.
Molecular modeling done for parsley enzyme suggest that these
inhibitors can be bound in two pockets being in vicinity to each
other and interconnected by a channel directing substrate into
active site cavity. Therefore, we also modeled binding of the most
na
1038.60 41.28
348.83 11.95
nd
nd
0.047 [22]
cyclobutyl derivative
a
na ¼ not active at concentration of 1 mM.
nd ¼ not determined.
b
c
Data in references 18 and 20 consider parsley enzyme, whereas data in refer-
ences 19 and 24 buckwheat enzyme.
effective
inhibitor
of
buckwheat
enzyme
-
1-
aminobenzocyclobutene-1-phosphonic acid (29) [22]. Modeling
predicts that this compound might be effectively bound in four
different sites of the enzyme (Fig. 5). This indicates that the
determination of binding of phenylglycine analogues require
crystallographic studies to be done in order to better understand
their action on PAL and to design more effective inhibitors.
Quite interestingly modeling predicts quite effective binding of
all the studied compounds with preferable binding of two isomers
of compound 1. However, as seen from Table 1 this compound is
inactive. We speculate that the lack of activity might result from
restricted ability of this molecule to reach any of the possible
Fig. 1. Binding of the studied analogues by two subunits of parsley PAL. Active site is located in subunit A marked by green tertiary structure, whereas non-physiologic site B is
located in subunit B marked in red Mode of binding in these two sites is also shown.

122
W. Wanat et al. / Biochimie 151 (2018) 119e127
Fig. 2. Pharmacophore models for binding sites A (left panel) and B (right panel). The colors denote regions where: acceptors of hydrogen bonds are present (green), hydrophobic
interactions resulting from he presence of aromatic ring are seen (orange), other hydrophobic interactions appear (light blue), and positively (red) and negatively (dark blue)
charged fragments of inhibitor appear.
Fig. 3. Modes of binding of R- (left panel) and S- (right panel) enantiomers of 3-fluoro-4-chlorobenzylphosphonic acid (compound 17) by parsley PAL.
3. Experimental section
3.1. Materials and methods
All chemicals were purchased from commercial Polish suppliers
(Sigma Aldrich, Trimen Chemicals and POCh), and used without
further purification. 1H NMR spectra were recorded on a Bruker
Avance II Ultrashield Plus or on a Bruker Avance III 500 MHz
(Bruker, Rheinstetten, Germany) spectrometersv c/operating at
600.58 MHz and 400 MHz, 19F NMR at 565.00 MHz and 376 MHz,
13C NMR and 31P NMR{1H} (100.61MHz/151 MHz and 162.01MHz/
243 MHz), respectively. Samples of the products were diluted in
D2O (99.8% at % D) with addition of D2SO4 or NaOD in cases of poor
solubility. Deuterated solvents were supplied by ARMAR AG (Dot-
tingen, Switzerland). Chemical shifts are reported relative to in-
ternal TMS (1H NMR), CFCl3 19F NMR) and 85% H3PO4 31P NMR)
( (
standards and are given in parts per million (ppm), while coupling
constant are reported in Herz. Multiplicities are shown as the
Fig. 4. Interactions of R-3-fluoro-4-chlorobenzylphosphonic acid with active site of
parsley PAL.

W. Wanat et al. / Biochimie 151 (2018) 119e127
123
Fig. 5. Multisite binding of two enantiomers of 1-aminobenzocyclobutene-1-phosphonic acid (29) by parsley PAL, as predicted by molecular modeling.
abbreviations: s (singlet), bs (broad singlet), d (doublet), t (triplet),
m (multiplet). Mass spectra were recorded at the Faculty of
Chemistry, Wroclaw University of Science and Technology by using
a Waters LCT Premier XE mass spectrometer (electrospray ioniza-
tion, ESI) (Waters, Milford, MA, USA). Melting points were deter-
mined on an SRS Melting Point Apparatus OptiMelt MPA 100
(Stanford Research Systems, Sunnyvale, CA, USA) and reported
uncorrected.
3.2.2. Amino-[(4-fluorophenyl)methyl]phosphonic acid (4)
White solid, m p. 293 ꢀC (lit [27]. m p. 273e275 ꢀC); yield: 55%;
1H NMR (600 MHz, D2O)
, ppm: 4.27 (d, J ¼ 15.5 Hz, 1H, CHP), 4.71
(bs, 1H, NH), 7.11 (t, J ¼ 8.8 Hz, 2H, 2xCHar), 7.41 (dd, J ¼ 7.9, 4.5 Hz,
2H 2xCHar); 13C NMR (101 MHz, D2O)
d
d
, ppm: 163.50 (d, J ¼ 2.3 Hz,
Car), 161.88 (d, J ¼ 2.1 Hz, Car), 129.82 (dd, J ¼ 8.6, 5.1 Hz, Car),
128.84e128.71 (m, Car), 115.85 (d, J ¼ 22.0 Hz, 2xCar), 52.81 (d,
J ¼ 139.5 Hz, CHP); 19F NMR (565 MHz, D2O)
d
, ppm: ꢁ113.54 (d,
6J ¼ 3.4 Hz, 1F, FP); 31P NMR (162 MHz, D2O)
d
ppm: 9.75 (d,
6J ¼ 3.3 Hz, 1P, PF); HRMS (ESI-MS) m/z [MH]þ calcd. for
3.2. Synthesis of aminobenzylphosphonic acids e general procedure
C7H9FNO3P: 206.0382, found: 206.0361.
Synthetic procedure was based on this elaborated earlier by
Oleksyszyn and Soroka [34e36]. Thus, acetamide 1.18 g (0.02 mol)
was dissolved in glacial acetic acid (4 mL, 4.20 g, 0.07 mol). The
solution was cooled in an ice-bath at 0 ꢀC and acetyl chloride
(0.71 mL, 0.78 g, 0.01 mol) was added observing the formation of a
crystalline by-product. After a few minutes appropriate aldehyde
(0.01 mol) was added, and the mixture was kept in ice-bath for
30 min and then left for 1 day at room temperature with constant
stirring. Then the mixture was cooled again to 0 ꢀC and phosphorus
trichloride was added dropwise (0.87 mL, 1.37 g, 0.01 mol). After
stirring for 30 min the mixture was allowed to warm to room
temperature, and finally heated for 1e1.5 h at 75e80 ꢀC. Evapora-
tion of the volatile components of the reaction mixture resulted in
an oily product, which was refluxed for 8 h in concentrated hy-
drochloric acid (50 mL). After the removal of volatile components of
the reaction mixture oily product was dissolved in ethanol (10 mL)
and left for crystallization. Resulting aminobenzylphosphonic acid
was then recrystallized from ethanol or ethanol-water.
3.2.3. Amino-[(4-chlorophenyl)methyl]phosphonic acid (5)
White solid, m p. 270 ꢀC (lit [27]. m p. 265e268 ꢀC); yield: 66%;
1H NMR (600 MHz, D2O)
d
, ppm: 3.77 (d, J ¼ 17.0 Hz, CHP), 5.06 (bs,
1H, NH), 6.54 (dd, J ¼ 8.6 Hz, 1.9 Hz, 2H, 2xCHar), 6.59 (d, J ¼ 8.6 Hz,
2H, 2xCHar); 13C NMR (151 MHz, D2O)
d
, ppm: 134.17 (d, J ¼ 2.8 Hz,
Car), 131.53 (d, J ¼ 4.7 Hz, Car), 129.24 (d, J ¼ 5.0 Hz, 2xCar), 128.98 (d,
J ¼ 1.7 Hz, 2xCar), 52.90 (d, J ¼ 138.3 Hz, CHP); 31P NMR (162 MHz,
D2O) d
, ppm: 11.72 (s, 1P); HRMS (ESI-MS) m/z [MH]- calcd. for
C7H9ClNO3P: 219.9930, found: 219.9919.
3.2.4. Amino-[(4-bromophenyl)methyl]phosphonic acid (6)
White solid, m p. 290 ꢀC (lit [27]. m p. 290e296 ꢀC); yield: 67%;
1H NMR (600 MHz, D2O)
d
, ppm: 4.28 (d, J ¼ 16.0 Hz, 1H, CHP), 4.68
(bs, 1H, NH), 7.23 (dd, J ¼ 8.5,1.8 Hz, 2H, CHar), 7.49 (d, J ¼ 8.5 Hz, 2H,
CHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm: 141.10 (s, Car),
130.84 (d, J ¼ 2.0 Hz, 2xCar), 129.48 (d, J ¼ 4.9 Hz, 2xCar), 119.29 (dd,
J ¼ 3.2,0.7 Hz, Car), 55.10 (d, J ¼ 130.1 Hz, CHP); 31P NMR (162 MHz,
D2O) d
, ppm: 10.80 (s, 1P); HRMS (ESI-MS) m/z [MH]- calcd. for
C7H9BrNO3P: 263.9425, found: 263.9417.
3.2.1. Amino-[(2,3,4,5,6-pentafluorophenyl)methyl]phosphonic acid
(3)
White solid, m p. 247 ꢀC; yield: 40%; 1H NMR (600 MHz,
3.2.5. Amino-[(4-iodophenyl)methyl]phosphonic acid (7)
D2O þ D2SO4)
d
, ppm: 3.94 (d, J ¼ 18.4 Hz, 1H, CHP), 5.06 (bs, 1H,
White solid, m p. 280 ꢀC, yield: 63%; 1H NMR (600 MHz,
NH); 13C NMR [19F] (101 MHz, D2O þ NaOD)
d
, ppm: 145.37 (s, Car),
D2O þ NaOD) , ppm: 3.61 (d, J ¼ 15.9 Hz, 1H, CHP), 4.67 (bs, 1H,
d
144.45 (s, Car), 139.92 (s, Car), 137.89 (s, Car), 136.95 (s, Car), 115.66 (s,
NH), 7.02 (dd, J ¼ 8.3,1.7 Hz, 2H, 2xCHar), 7.56 (d, J ¼ 8.3 Hz, 2H,
Car), 48.17 (d, J ¼ 131.0 Hz, CHP); 19F NMR (376 MHz, D2O þ D2SO4)
2xCHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm: 141.91 (d,
J ¼ 2.6 Hz, Car), 136.86 (d, J ¼ 2.1 Hz, 2xCar), 129.71 (d, J ¼ 4.9 Hz,
d
, ppm: ꢁ161.90 (qd, J ¼ 8.3, 3.6 Hz, 2F), ꢁ152.73 (tdd, J ¼ 21.1, 4.9,
2.4 Hz, 1F), ꢁ141.95 (dd, J ¼ 12.3, 8.2, 5.0 Hz, 2F); 31P NMR
2xCar), 90.65 (d, J ¼ 3.5 Hz, Car), 55.17 (d, J ¼ 129.9 Hz, CHP); 31P
(162 MHz, D2O þ D2SO4)
d, ppm: 8.36e8.23 (m); HRMS (ESI-MS)
NMR (162 MHz, D2O þ NaOD)
d ppm: 17.85 (s, 1P); HRMS (ESI-MS)
m/z [MH]þ calcd. for C7H5F5NO3P: 278.0005, Found: 278.0010.
m/z [MH]þ calcd. for C7H9INO3P: 313.9443, found: 313.9448.

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W. Wanat et al. / Biochimie 151 (2018) 119e127
3.2.6. Amino-[(2-fluorophenyl)methyl]phosphonic acid (8)
3.2.12. Amino-[(4-chloro-2-fluorophenyl)methyl]phosphonic acid
(14)
White solid, m p. 253 ꢀC (lit [27]. m p. 257e260 ꢀC); yield: 95%; 1H
NMR (600 MHz, D2O)
d
, ppm: 4.30 (d, J ¼ 17.5 Hz, 1H, CHP), 4.95 (bs,
White solid, m p. 263 ꢀC; yield: 71%; 1H NMR (600 MHz, D2O)
1H, NH), 6.61 (t, J ¼ 9.6, 1H, CHar), 6.68 (t, J ¼ 7.6, 1H, CHar), 6.87 (dt,
d
ppm: 4.65 (d, J ¼ 16.7 Hz, 1H, CHP), 4.71 (bs, 1H, NH), 7.27 (d,
J ¼ 13.9,7.5 Hz, 2H, 2xCHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm:
J ¼ 8.9 Hz, 2H, 2xCHar), 7.46 (dt, J ¼ 8.4,4.3 Hz, 1H, CHar); 13C NMR
161.33 (d, J ¼ 5.8 Hz, Car),158.91 (d, J ¼ 5.8 Hz, Car),129.17e128.79 (m,
Car), 127.89 (dd, J ¼ 8.4,2.4 Hz, Car), 124.04 (dd, J ¼ 3.2,2.3 Hz, Car),
115.11 (dd, J ¼ 22.9,1.8 Hz,Car), 48.38 (dd, J ¼ 132.0,1.6 Hz, CHP); 19F
(101 MHz, D2O þ NaOD) , ppm: 159.85 (dd, J ¼ 246.6,5.7 Hz, Car),
d
131.88 (dd, J ¼ 10.7,2.9 Hz, Car),129.83 (dd, J ¼ 4.8,4.3 Hz, Car),127.81
(d, J ¼ 14.2 Hz, Car), 124.26 (t, J ¼ 3.0 Hz, Car), 115.67 (dd,
J ¼ 26.8,1.7 Hz, Car), 48.06 (d, J ¼ 131.5 Hz, CHP); 19F NMR (376 MHz,
NMR (376 MHz, D2O)
(162 MHz, D2O)
d
, ppm: ꢁ117.14 to ꢁ117.26 (m); 31P NMR
d
ppm: 12.26 (s, 1P); HRMS (ESI-MS) m/z [MH]þ
D2O)
d
, ppm: ꢁ114.39 to ꢁ114.51 (m, 1F); 31P NMR (162 MHz, D2O)
calcd. for C7H9FNO3P: 206.0382, found: 206.0377.
d
, ppm: 9.64 (d, 7J ¼ 3.5 Hz, 1P); HRMS (ESI-MS) m/z [MH]þ calcd.
for C7H8ClFNO3P: 239.9993, found: 239.9990.
3.2.7. Amino-[(2-chlorophenyl)methyl]phosphonic acid (9)
White solid, m p. 244 ꢀC (lit [27]. m p. 247e249 ꢀC); yield: 38%;
3.2.13. Amino-[(2,4-dichlorophenyl)methyl]phosphonic acid (15)
1H NMR (600 MHz, D2O)
d, ppm: 4.71 (bs, 1H, NH), 4.95 (d,
White solid, m p. 245 ꢀC; yield: 72%; 1H NMR (400 MHz,
J ¼ 16.5 Hz, 1H, CHP), 7.37e7.30 (m, 2H, 2xCHar), 7.46 (d, J ¼ 7.4 Hz,
D2O þ D2SO4) , ppm: 4.13 (d, J ¼ 17.4 Hz, 1H, CHP), 5.10 (bs, 1H,
d
1H, CHar), 7.55 (dt, J ¼ 7.6, 1.9 Hz, 1H, CHar); 13C NMR (101 MHz,
NH), 6.49 (dd, J ¼ 59.1,8.5,2.1 Hz, 2H, 2xCHar), 6.59 (dd, J ¼ 2.1 Hz,
D2O þ NaOD) , ppm: 139.87 (d, J ¼ 0.8 Hz, Car), 133.23 (d, J ¼ 7.4 Hz,
d
1.0 Hz, 1H, CHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm: 138.91
Car), 129.06 (d, J ¼ 1.6 Hz, Car), 128.68 (d, J ¼ 3.7 Hz, Car), 127.64 (d,
J ¼ 2.3 Hz, Car), 126.90 (d, J ¼ 2.2 Hz,Car), 51.01 (d, J ¼ 131.3 Hz, CHP);
(d, J ¼ 0.9 Hz, Car), 133.88 (d, J ¼ 7.4 Hz, Car), 131.72 (d, J ¼ 2.8 Hz,
Car), 129.66 (d, J ¼ 3.7 Hz, Car), 128.52 (d, J ¼ 1.6 Hz, Car), 127.00 (d,
J ¼ 2.3 Hz, Car), 50.68 (d, J ¼ 131.0 Hz, CHP); 31P NMR (162 MHz,
31P NMR (162 MHz, D2O)
d ppm: 9.56 (s, 1P); HRMS (ESI-MS) m/z
[MH]þ calcd. for C7H9ClNO3P: 222.0087, found: 220.0088.
D2O þ D2SO4)
d
, ppm: 10 l9 (s, 1P); HRMS (ESI-MS) m/z [MH]þ
calcd. for C7H8Cl2NO3P: 255.9697, found: 255.9706.
3.2.8. Amino-[(2-bromophenyl)methyl] phosphonic acid (10)
White solid, m p. 234 ꢀC (lit [27]. m p. 246e248 ꢀC); yield: 38%; 1H
NMR (400 MHz, D2O)
d
, ppm: 4.66 (bs 1H, NH), 4.91 (d, J ¼ 16.4 Hz,
3.2.14. Amino-[(3,4-difluorophenyl)methyl]phosphonic acid (16)
White solid, m p. 294 ꢀC (lit [27]. m p. 286e289 ꢀC); yield: 78.5%;
1H, CHP), 7.16e7.23 (m,1H, CHar), 7.35 (t, J ¼ 7.6 Hz,1H, CHar), 7.52 (dt,
J ¼ 7.9 Hz, 1.7 Hz, 1H, CHar), 7.60 (d, J ¼ 8.1 Hz, 1H, CHar); 13C NMR
1H NMR (400 MHz, D2O)
d
, ppm: 4.40 (d, J ¼ 15.7 Hz, 1H, CHP), 4.83
(bs, 1H, NH), 7.38e7.23 (m, 2H, 2xCHar), 7.42 (ddt, J ¼ 11.5,7.5,1.9 Hz,
1H, CHar); 13C NMR (101 MHz, D2O)
, ppm: 155.76 (s, Car), 155.15 (s,
(101 MHz, D2O þ NaOD)
d, ppm: 148.63 (s, Car), 141.65 (s, Car), 132.40
(d, J ¼ 1.4 Hz, Car),128.82 (d, J ¼ 3.7 Hz, Car),127.79 (dd, J ¼ 44.6,2.2 Hz,
d
Car), 124.32 (d, J ¼ 8.0 Hz, Car), 53.83 (d, J ¼ 131.2 Hz, CHP); 31P NMR
Car), 129.81 (s, Car), 124.50 (s, Car), 117.81 (d, J ¼ 17.6 Hz, Car), 116.90
(162 MHz D2O) d
, ppm: 10.16 (s,1P); HRMS (ESI-MS) m/z [MH]- calcd.
(d, J ¼ 15.2 Hz, Car), 52.38 (d, J ¼ 140.2 Hz, CHP); 19F NMR (565 MHz,
for C7H9BrNO3P: 263.9425, found: 263.9431.
D2O)
d
, ppm: ꢁ137.34 (d, J ¼ 21.4 Hz, 1F), 138.15 (dd, J ¼ 21.4 Hz,
4.0 Hz, 1F); 31P NMR (162 MHz, D2O)
d
, ppm: 10.53 (d, J ¼ 2.7 Hz,
3.2.9. Amino-[(3-fluorophenyl)methyl]phosphonic acid (11)
1P); HRMS (ESI-MS) m/z [MH]- calcd. for C7H8F2NO3P: 222.0132,
White solid, m p. 325 ꢀC (lit [27]. m p. 273e275 ꢀC); yield: 76%;
found: 222.0131.
1H NMR (400 MHz, D2O)
d, ppm: 4.40 (d, J ¼ 16.0 Hz, 1H, CHP), 4.71
(br s, 1H, NH), 7.09e7.15 (m, 1H, CHar), 7.15e7.23 (m, 2H, 2xCHar)
3.2.15. Amino-[(4-chloro-3-fluorophenyl)methyl]phosphonic acid
(17)
7.40 (td, J ¼ 8.1,6.0 Hz, 1H, CHar); 13C NMR (101 MHz, D2O)
d, ppm:
163.70 (s, Car), 135.16 (s, Car), 130.83 (d, J ¼ 8.2 Hz, Car), 123.64 (d,
J ¼ 3.7 Hz, Car), 115.68 (d,J ¼ 20.1 Hz, Car), 114.64 (dd, J ¼ 23.0,4.7 Hz,
White solid, m p. 251 ꢀC; yield: 52.5%; 1H NMR (600 MHz, D2O)
d
, ppm: 4.39 (d, J ¼ 16.1 Hz, 1H, CHP), 4.71 (bs, 1H, NH), 7.19 (d,
Car), 53.07 (d, J ¼ 138.5 Hz, CHP); 19F NMR (376 MHz, D2O)
d,
J ¼ 8.3 Hz, 1H, CHar), 7.30 (dt, J ¼ 10.2,1.9 Hz, 1H, CHar), 7.50 (t,
ppm: ꢁ112.58 to ꢁ112.68 (m, 1F); 31P NMR (243 MHz, D2O)
d, ppm:
J ¼ 8.0 Hz, 1H, CHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm:
10.84 (s, 1P); HRMS (ESI-MS) m/z [MH]þ calcd. for C7H9FNO3P:
157.28 (dd, J ¼ 244.2,2.2 Hz, Car), 143.53 (dd, J ¼ 6.6,2.6 Hz, Car),
129.68(d, J ¼ 2.0 Hz, Car), 124.25 (dd, J ¼ 4.9,3.4 Hz, Car), 117.37 (dd,
J ¼ 17.6,3.1 Hz, Car), 115.48 (dd, J ¼ 21.5,4.8 Hz, Car), 54.95 (dd,
206.0382, found: 206.0377.
3.2.10. Amino-[(3-chlorophenyl)methyl]phosphonic acid (12)
J ¼ 129.4,1.2 Hz, CHP); 19F NMR (376 MHz, D2O)
d, ppm: 114.99 (t,
White solid, m p. 260 ꢀC (lit [27]. m p. 271e274 ꢀC); yield: 41%;
5J ¼ 9.0 Hz, 1F); 31P NMR (162 MHz, D2O)
d,
ppm: 9.89 (d,
1H NMR (600 MHz, D2O)
d, ppm: 3.67 (d, J ¼ 15.9 Hz, 1H, CHP), 4.72
5J ¼ 1.6 Hz, 1P); HRMS (ESI-MS) m/z [MH]þ calcd. for C7H8ClFNO3P:
(bs, 1H, NH), 7.13e7.22 (m, 3H, 3xCHar), 7.29 (d, J ¼ 1.5 Hz, 1H, CHar);
239.9993, found: 240.0314.
31P NMR (162 MHz, D2O)
d ppm: 17.24 (s, 1P); HRMS (ESI-MS) m/z
[MH]- calcd. for C7H9ClNO3P: 219.9930, found: 219.9929.
3.2.16. Amino-[(3-chloro-4-fluorophenyl)methyl]phosphonic acid
(18)
3.2.11. Amino-[(2,4-difluorophenyl)methyl]phosphonic acid (13)
White solid, m p. 270 ꢀC; yield: 71%; 1H NMR (600 MHz, D2O)
d,
White solid, m p. 260 ꢀC; yield: 84%; 31P NMR (162 MHz,
ppm: 4.58 (d, J ¼ 16.6 Hz, 1H, CHP), 4.72 (bs, 1H, NH), 6.95e7.03 (m,
D2O þ D2SO4)
d
, ppm: 12.33; 1H NMR (400 MHz, D2O þ D2SO4)
d,
2H, 2xCHar), 7.50e7.54 (m, 2H, 2xCHar); 13C NMR (101 MHz,
ppm: 4.13 (d, J ¼ 16.8 Hz, 1H, CHP), 4.90 (bs, 1H, NH), 6.84 (t,
J ¼ 8.9 Hz, 1H, CHar), 6.93e6.96 (m, 1H, CHar), 7.13 (dt, J ¼ 6.8,2.2 Hz,
D2O þ NaOD) , ppm: 161.76 (dd, J ¼ 18.6,12.5,4.3 Hz, Car), 159.32
d
(dd, J ¼ 18.6,12.4,4.2 Hz, Car), 129.65 (dd, J ¼ 9.9,6.0,3.9 Hz, Car),
125.09 (dd, J ¼ 14.9,3.6,1.7 Hz, Car), 110.88 (dd, J ¼ 21.0,3.6,2.1 Hz,
Car), 103.18 (dd, J ¼ 27.3,25.4,1.8 Hz, Car), 47.82 (dd, J ¼ 132.9,1.2 Hz,
1H, CHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm: 156.30 (dd,
J ¼ 243.4,2.8 Hz, Car), 139.42 (dd, J ¼ 3.7,2.5 Hz, Car), 129.31 (d,
J ¼ 4.9 Hz, Car), 127.50 (dd, J ¼ 7.3,5.0 Hz, Car), 119.25 (dd,
J ¼ 17.6,2.3 Hz, Car), 115.82 (dd, J ¼ 20.9,2.0 Hz, Car), 54.64 (d,
CHP); 19F NMR (565 MHz, D2O)
d,
ppm: ꢁ109.15 (q,
J ¼ 9.0,5.6,3.7 Hz, 1F), ꢁ112.84 (q, J ¼ 8.8,4.5,3.4 Hz, 1F); 31P NMR
J ¼ 130.5 Hz, CHP); 19F NMR (565 MHz, D2O)
, ppm: ꢁ115.75 (dd,
d
(243 MHz, D2O)
d
, ppm: 9.12 (t, 4J ¼ 3.4 Hz, 1P); HRMS (ESI-MS) m/z
J ¼ 11.1,8.9,4.4 Hz, 1F); HRMS (ESI-MS) m/z [MH]þ calcd. for
[MH]- calcd. for C7H8F2NO3P: 222.0132, found: 222.0139.
C7H8ClFNO3P: 239.9993, found: 239.9994.

W. Wanat et al. / Biochimie 151 (2018) 119e127
125
3.2.17. Amino-[(2,4,6-trifluorophenyl)methyl] phosphonic acid (19)
White solid, m p. 248 ꢀC; yield: 48%; 1H NMR (400 MHz,
D2O þ NaOD)
d
, ppm: 3.76 (d, J ¼ 16.5 Hz, 1H, CHP), 4.67 (bs, 1H,
NH), 7.21 (dd, J ¼ 19.8,9.5 Hz, 2H, 2xCHar), 7.39 (s, 1H, CHar); 13C
D2O þ D2SO4)
d
, ppm: 4.52 (d, J ¼ 17.6 Hz, 1H, CHP), 6.85 (t,
NMR (101 MHz, D2O þ NaOD) , ppm: 161.93 (dd, J ¼ 244.1,2.4 Hz,
d
J ¼ 9.0 Hz, 3H, 3xCHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm:
Car), 146.30 (dd, J ¼ 7.6,2.3 Hz, Car), 130.84 (qdd, J ¼ 32.5,8.6,2.0 Hz,
162.42e161.89 (m, Car), 159.99e159.44 (m, 2xCar), 113.30 (tdd,
J ¼ 18.7,4.6,1.1 Hz, Car), 100.26 (t, J ¼ 27.7 Hz, 2xCar), 46.71 (d,
C
arCF3), 123.66 (qd, J ¼ 271.7,3.1 Hz, CarCF3), 120.28 (dq,
J ¼ 7.7,3.8 Hz, Car), 117.96 (dd, J ¼ 21.9,4.6,1.0 Hz, Car), 110.19 (dd,
J ¼ 132.4 Hz, CHP); 19F NMR (376 MHz D2O þ D2SO4)
d,
J ¼ 25.1,3.9,2.7 Hz, Car), 55.24 (dd, J ¼ 128.4,1.6 Hz, CHP); 19F NMR
ppm: ꢁ110.12 (td, J ¼ 8.5,3.2 Hz, 2F), ꢁ106.47 to ꢁ106.60 (m, 1F);
(565 MHz, D2O þ NaOD)
d,
ppm: ꢁ112.89 (t, J ¼ 9.5 Hz, 1F,
31P NMR (162 MHz D2O þ D2SO4)
d
, ppm: 8.72 (dd, 4,6J ¼ 7.5 Hz,
FCar), ꢁ62.11 (s, 3F, CF3); 31P NMR (162 MHz, D2O þ NaOD)
d, ppm:
3.6 Hz, 1P); HRMS (ESI-MS) m/z [MH]þ calcd. for C7H7F3NO3P:
16.97 (s, 1P); HRMS (ESI-MS) m/z [MH]þ calcd. for C8H8F4NO3P:
242.0194, found: 242.0191.
274.0256, found: 274.0264.
3.2.18. Amino-[(4-methylphenyl)methyl]phosphonic acid (20)
3.2.23. Amino-[(4-cyanophenyl)methyl]phosphonic acid (25)
White solid, m p. 280 ꢀC, (lit [27]. mp 254e256 ꢀC); yield: 84%;
White solid, m p. 295 ꢀC; yield: 84%; 1H NMR (600 MHz,
1H NMR (600 MHz, D2O)
d, ppm: 2.28 (s, 3H, CH3), 4.33 (d,
D2O þ D2SO4) , ppm: 3.14 (d, J ¼ 17.6 Hz, 1H, CHP), 5.30 (bs, 1H,
d
J ¼ 15.9 Hz, CHP), 4.70 (bs, 1H, NH), 7.23 (d, J ¼ 7.6 Hz, 2H, 2xCHar),
NH), 5.91 (dd, J ¼ 8.6,1.8 Hz, 2H, 2xCHar), 6.39 (d, J ¼ 8.2 Hz, 2H,
7.29 (dd, J ¼ 8.3,1.9 Hz, 2H, 2xCHar); 13C NMR (151 MHz, D2O)
d
,
2xCHar); 31P NMR (162 MHz, D2O þ D2SO4)
d, ppm: 5.30 (s, 1P);
ppm: 139.19 (d, J ¼ 2.4 Hz, Car), 129.51 (d, J ¼ 1.6 Hz, Car), 127.69 (d,
HRMS (ESI-MS) m/z [MH]- calcd. for C8H8N2O3P: 212.0351, found:
J ¼ 5.1 Hz. Car), 53.25 (d, J ¼ 139.8 Hz, CHP), 20.21 (s, 1C, CH3); 31P
212.0408.
NMR (162 MHz, D2O) d, ppm: 11.25 (s, 1P); HRMS (ESI-MS) m/z
[MH]- calcd. for C8H12NO3P: 200.0447, found: 200.0475.
3.2.24. Amino-[(4-cyano-2-fluorophenyl)methyl]phosphonic acid
(26)
3.2.19. Amino-[(2-fluoro-4-methylphenyl)methyl] phosphonic acid
(21)
White solid, m p. 265 ꢀC; yield: 100%; 1H NMR (600 MHz,
D2O þ D2SO4)
d
ppm: 3.30 (d, J ¼ 18.1 Hz, 1H, CHP), 5.26 (br s, 1H,
White solid, m p. 287 ꢀC; yield: 53%; 1H NMR (400 MHz, D2O)
d,
NH), 5.89 (td, J ¼ 7.7,2.1 Hz, 1H, CHar), 6.07 (d, J ¼ 10.8 Hz, 1H, CHar),
ppm: 1.56 (s, 3H, CH3), 4.05 (d, J ¼ 17.2 Hz, 1H, CHP), 4.96 (bs, 1H,
6.15 (dd, J ¼ 8.2,1.3 Hz, 1H, CHar), 13C NMR (101 MHz, D2O þ NaOD)
NH), 6.23e6.28 (m, 2H, 2xCHar), 6.67 (dd, J ¼ 9.8, 5.6,2.3 Hz, 1H,
d
, ppm: 159.71 (dd, J ¼ 244.1,5.6 Hz, Car), 136.36 (dd, J ¼ 6.9,2.5 Hz,
CHar); 13C NMR (101 MHz, D2O þ NaOD)
d
, ppm: 160.80 (dd,
Car), 131.78 (dd, J ¼ 13.9,3.5 Hz, Car), 128.60 (t, J ¼ 4.1 Hz, Car), 124.34
(d, J ¼ 2.4 Hz, Car), 115.40 (d, J ¼ 23.7 Hz, Car), 48.60 (d, J ¼ 130.4 Hz,
J ¼ 240.5,2.7 Hz, Car), 138.79 (t, J ¼ 7.2 Hz, Car), 136.61 (dd,
J ¼ 2.5,2.0 Hz, Car), 128.24 (dd, J ¼ 8.4, 3.6 Hz, Car), 115.96 (d,
J ¼ 20.1 Hz, Car), 112.15 (dd, J ¼ 0.9,2.1 Hz, Car), 50.01 (d, J ¼ 132.3 Hz,
CHP); 19F NMR (376 MHz, D2O þ D2SO4)
d, ppm: ꢁ116.87 (dd,
J ¼ 10.9,7.2,3.9 Hz, 1F); 31P NMR (162 MHz, D2O þ D2SO4)
d, ppm:
CHP), 19.41 (s, CH3); 19F NMR (376 MHz, D2O)
d
, ppm: ꢁ114.08 (dd,
10.71 (t, 4J ¼ 4.2 Hz, 1P); HRMS (ESI-MS) m/z [MH]þ calcd. for
J ¼ 15.2,10.1,4.5 Hz, 1F); 31P NMR (162 MHz, D2O)
d, ppm: 13.12 (d,
C8H8FN2O3P: 231.0335, found: 231.0337.
4J ¼ 4.2 Hz, 1P); HRMS (ESI-MS) m/z [MH]- calcd. for C8H11FNO3P:
218.0382, found: 218.0380.
3.2.25. Amino-[(4-metoksyphenyl)methyl] phosphonic acid (27)
White solid, m p. 272 ꢀC; yield: 54%; 1H NMR (400 MHz, D2O)
d,
3.2.20. Amino-[(4-trifluoromethylphenyl)methyl]phosphonic acid
(22)
ppm: 3.15 (s, 3H, CH3O), 3.93 (d, J ¼ 16.7 Hz, 1H, CHP), 6.37 (d,
J ¼ 8.8 Hz, 2H, 2xCHar), 6.75 (dd, J ¼ 8.8, 1.9 Hz, 2H, 2xCHar); 13C
White solid, m p. 295 ꢀC (lit [27]. m p. 277e280 ꢀC); yield: 82%;
NMR (101 MHz, D2O þ NaOD) , ppm: 157.23 (d, J ¼ 2.5 Hz, Car),
d
1H NMR (400 MHz, D2O)
d
, ppm: 4.49 (d, J ¼ 16.3 Hz, 1H, CHP), 4.71
134.79 (d, J ¼ 2.4 Hz, Car), 129.12 (d, J ¼ 5.3 Hz, Car), 128.87 (d,
J ¼ 5.1 Hz, Car), 118.05 (d, J ¼ 1.6 Hz, Car), 113.54 (d, J ¼ 1.4 Hz, Car),
55.44 (s, 3H, OCH3), 54.84 (d, J ¼ 132.4 Hz, CHP); 31P NMR (162 MHz
(br s, 1H, NH), 7.56 (d, J ¼ 7.6 Hz, 2H, 2xCHar), 7.72 (d, J ¼ 7.6 Hz, 2H,
2xCHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm: 168.46 (s, Car),
146.53 (s, Car), 127.84 (d, J ¼ 4.7 Hz, Car), 127.45 (qd, J ¼ 31.9,2.7 Hz,
D2O) d
, ppm: 13.46 (s, 1P); HRMS (ESI-MS) m/z [MH]- calcd. for
2xCar), 124.76 (qd, J ¼ 3.7,2.2 Hz, Car), 124.58 (qd, J ¼ 271.0,1.0 Hz,
C8H12NO4P: 216.0426, found: 216.0416.
CF3), 55.52 (d, J ¼ 128.6 Hz, CHP); 19F NMR (565 MHz, D2O)
d,
ppm: ꢁ62.47 (s. 3F, CF3); 31P NMR (243 MHz, D2O)
d, ppm: 10.53 (d,
3.2.26. Amino-[(4-aminophenyl)methyl]phosphonic acid (28)
7J ¼ 1.9 Hz); HRMS (ESI-MS) m/z [MH]- calcd. for C8H9F3NO3P:
White solid, m p. 350 ꢀC; yield: 52%, 1H NMR (400 MHz, D2O)
d,
254.0194, found: 254.0197.
ppm: 3.75 (d, J ¼ 17.1 Hz, 1H, CHP), 6.52 (d, J ¼ 8.4 Hz, 2H, 2xCHar),
6.62 (dd, J ¼ 8.5, 2.0 Hz, 2H, 2xCHar); 13C NMR (101 MHz,
3.2.21. Amino-[(4-difluorometoxy-2-fluorophenyl)methyl]
D2O þ NaOD) , ppm: 144.19 (d, J ¼ 2.3 Hz, Car), 133.10 (d, J ¼ 2.7 Hz,
d
phosphonic acid (23)
2xCar), 128.63 (d, J ¼ 5.2 Hz, Car), 116.10 (d, J ¼ 1.9 Hz, 2xCar), 54.92
Yellow solid, m p. 255 ꢀC; yield: 26%; 1H NMR (600 MHz, D2O)
d,
(d, J ¼ 133.2 Hz, CHP); 31P NMR (162 MHz D2O)
d, ppm: 11.16 (s, 1P);
ppm: 2.16 (s, 1H, CHF2), 4.56 (d, J ¼ 16.0 Hz, 1H, CHP), 4.71 (bs, 1H,
HRMS (ESI-MS) m/z [MH]- calcd. for C7H11N2O3P: 201.0429, found:
NH), 6.30 (dd, J ¼ 14.5,10.4,2.5 Hz, 2H, 2xCHar), 6,93 (dd,
201.0423.
J ¼ 8.7,1.9 Hz, 1H, CHar); 13C NMR (101 MHz, D2O þ NaOD)
d, ppm:
165.73 (dd, J ¼ 11.7,1.7 Hz, Car), 161.33 (dd, J ¼ 243.0,6.1 Hz, Car),
129.35 (dd, J ¼ 7.0,3.8 Hz, Car), 114.34 (t, J ¼ 1.8 Hz, CHF2), 112.65
(dd, J ¼ 15.2,2.3 Hz, 2xCar), 104.45 (dd, J ¼ 20.6,1.3 Hz, Car), 47.77
3.3. Isolation of PAL
Phenylalanine ammonia lyase was extracted from 1 mm thick
slices of potato tuber (Solanum tuberosum L.) basing on the pro-
cedure described by Havir & Hanson [38]. Fresh potatoes were
purchased from local grocery store. The washed slices were put in
the Petri dishes lined with filter paper Whatmann 2 soaked with
neomycin sulfate solution (50 mg/l) and exposed to with luminous
flux (5220 lumen) at 22 ꢀC for 22 h. The harvested slices were then
washed with distilled water, dried with paper towel and weighted.
(dd, J ¼ 135.3,1.0 Hz, CHP); 19F NMR (565 MHz, D2O)
d,
ppm: ꢁ166.58 (s, 2F, F2CH), ꢁ115.36 (dd, J ¼ 12.1,8.7,3.7 Hz,1F, CarF);
31P NMR (162 MHz, D2O)
d
, ppm: 12.41 (d, 4J ¼ 3.6 Hz, 1P).
3.2.22. Amino-[(3-fluoro-5-trifluoromethylphenyl)methyl]
phosphonic acid (24)
White solid, m p. 247 ꢀC; yield: 83%; 1H NMR (600 MHz,

126
W. Wanat et al. / Biochimie 151 (2018) 119e127
The prepared plant material (500 g) was homogenized by 1 min
(speed 8500 rpm) in 0.1 M borate buffer (pH ¼ 8.7) containing
mercaptoethanol (80 l/100 ml buffer) and filtered through flip
phenylalanine ammonia lyase (PAL) from Petroselinum crispum was
obtained from the RCSB Protein Data Bank e ID: 1W27 [39].
Structure was protonated by the pH of experimental method
(pH ¼ 8.7) and minimalized by the Smart minimizer algorithm with
CHARMm force field. The partial charges were calculated by the
Momany-Rone algorithm. The ligand and the water molecules were
removed from the structure of the enzyme. New ligands were
considered taken into account stereochemistry, protonated and
minimized with molecular dynamics calculations. Compounds
were minimized by Steepest descent and conjugate gradient algo-
rithms, then the heating step was provided. Target temperature
was set to 300 K and simulation time to 10 ps. Equilibration's step
time was set at 100 ps and the production step with NVT type to
1000 ps. The lowest potential energy structures were selected and
minimized by the DFTcalculations with the use of B3LYP functional.
The long minimization calculations were used to obtain partial
charges on aromatic ring of the compounds for the other studies.
Dockings in the active center of the enzyme were made by the
usage of the GOLD Algorithm (5.5 version, CCDC, Cambridge, United
Kingdom).
b
-
m
flops layer. The filtrate was brought to pH ¼ 5.5 by using 1 M acetic
acid followed by addition of protamine sulfate solution
(333 mg þ 1.33 ml 1 M acetate buffer, pH ¼ 5.0 and supplemented
with distilled water to 16.67 ml volume). The mixture was then
centrifuged for 10 min (7000 rpm/min), in 0 ꢀC. The sediment was
discarded and to supernatant ammonium sulfate was added to 27%
salting, stirring for 30 min at 4 ꢀC in the dark and left for 24 h. After
this time, the mixture was centrifuged again under this same
condition (10min/7000 rpm/0 ꢀC) and resulting supernatant
repeatedly salt out with ammonium sulfate (to 40% of saturation)
and centrifuged once more. The precipitate after second fraction-
ation was dissolved in borate buffer (final volume 105 ml). The
mixture was divided into aliquots (15 ml) and suspended in
ammonium sulfate (to 40% of saturation). Individual portions of the
enzyme were directly purified before kinetic tests on G-75 Sepha-
dex with using NGC Medium-Pressure Chromatography controlling
the process by UV-VIS (280 nm). Thus, 15 ml of PAL solution was
applied to the column and eluted with 0.1 M borate buffer
(pH ¼ 8.7) collecting 3 ml fractions. Active PAL fractions was
combined and used as enzyme source. The specific activity of the
PAL was ranged 2e8mU/mg.
Acknowledgements
This work was supported by 2017/26/M/ST5/00437 grant. The
Biovia Discovery Studio package was used under a Polish coun-
trywide license. The use of software resources (Biovia Discovery
Studio program package) of the Wrocław Center for Networking
and Supercomputing is also kindly acknowledged.
3.4. Kinetic assays
Kinetics of the PAL-catalyzed reaction of L-phenylalanine were
determined in 0.1 M borate buffer, pH ¼ 8.7 at 37 ꢀC by following
formation of trans-cinnamic acid at 290 nm using an enzyme of
3.75mU/mg specific activity. The determined extinction coefficient
References
for trans-cinnamic acid is 8940 Mꢁ1cmꢁ1
. The determined
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Vmax
V0
KM KM
1
ꢁ 1 ¼
þ
$
$½Iꢂ
(1)
½Sꢂ
½Sꢂ Ki
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