The synthesis and kinetic evaluation of aryl α-aminophosphonates as novel inhibitors of T. cruzi trans-sialidase

Article abstract of DOI:10.1016/j.ejmech.2018.08.089

The trans-sialidase protein expressed by Trypanosoma cruzi is an important enzyme in the life cycle of this human pathogenic parasite and is considered a promising target for the development of new drug treatments against Chagas' disease. Here we describe

Full text of DOI:10.1016/j.ejmech.2018.08.089

Accepted Manuscript
The synthesis and kinetic evaluation of aryl α-aminophosphonates as novel inhibitors
of T. cruzi trans-sialidase
Zexin Chen, Patricia Marcé, Ricardo Resende, Pedro M. Alzari, A. Carlos Frasch,
Jean M.H. van den Elsen, Susan J. Crennell, Andrew G. Watts
PII:
S0223-5234(18)30762-1
10.1016/j.ejmech.2018.08.089
EJMECH 10700
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 June 2018
Revised Date: 21 August 2018
Accepted Date: 29 August 2018
Please cite this article as: Z. Chen, P. Marcé, R. Resende, P.M. Alzari, A.C. Frasch, J.M.H. van den
Elsen, S.J. Crennell, A.G. Watts, The synthesis and kinetic evaluation of aryl α-aminophosphonates
as novel inhibitors of T. cruzi trans-sialidase, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.08.089.
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ACCEPTED MANUSCRIPT
The synthesis and kinetic evaluation of aryl α-aminophosphonates
as novel inhibitors of T. cruzi trans-sialidase
Zexin Chen1, Patricia Marcé2, Ricardo Resende2, Pedro M. Alzari3, A. Carlos Frasch4, Jean
M.H. van den Elsen1, Susan J. Crennell1, Andrew G. Watts2*
1 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
2 Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
3 Institut Pasteur, Unité de Microbiologie Structurale, CNRS URA 3528 & Université Paris
Diderot, 25 rue de Dr. Roux, 75724, Paris, France.
4 Instituto de Investigaciones Biotecnológicas, Universidad de General San Martin, Casilla de
Correo 30, 1650 San Martin, Argentina.
* Corresponding author
Email: a.watts@bath.ac.uk
Phone: +44 (0)1225 386788
Abbreviations:
TcTS, Trypanosoma cruzi trans-sialidase; CAZy, Carbohydrate-Active EnZymes database;
DANA, N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid; GH, glycoside hydrolase; MuNANA,
2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid;
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Abstract
The trans-sialidase protein expressed by Trypanosoma cruzi is an important enzyme in the life cycle
of this human pathogenic parasite and is considered a promising target for the development of new
drug treatments against Chagas’ disease. Here we describe α-amino phosphonates as a novel class
of inhibitor of T. cruzi trans-sialidase. Molecular modelling studies were initially used to predict the
active-site binding affinities for a series of amino phosphonates, which were subsequently
synthesised and their IC50s determined in vitro. The measured inhibitory activities show some
correlation with the predictions from molecular modelling, with 1-napthyl derivatives found to be
the most potent inhibitors having IC50s in the low micromolar range. Interestingly, kinetic analysis of
the mode of inhibition demonstrated that the α-aminophosphonates tested here operate in a non-
competitive manner.
Keywords
Trans-sialidase, Trypanosoma cruzi, α-aminophosphonates, inhibitors, non-competitive
1. Introduction
Trans-sialidase (TcTS) is a crucial enzyme in the life cycle of the protozoan parasite
Trypanosoma cruzi, the causative agent of Chagas’ disease [1 - 3]. T. cruzi is unable to synthesise
sialic acid de novo, so TcTS functions to sequester sialic acid from host cell surfaces and transfer it to
mucins on the surface of the parasite [4, 5]. The process of surface sialylation plays an important
role in the pathogenicity of T. cruzi, by facilitating its invasion into host cells and helping to mask
itself from the host immune system [6 - 9]. For these reasons, TcTs is considered a promising
drugable target, with considerable effort being put into the discovery of trans-sialidase inhibitors as
potential therapeutics for the treatment of Chagas’ disease [10 - 11].
The 3-D structure and enzymatic mechanism of TcTS have been studied extensively. TcTS
belongs to the glycoside hydrolase family GH33 (CaZY) [12], which contains most of the prokaryotic
and some eukaryotic sialidases. Despite its inclusion in a glycoside hydrolase family, the
predominant function of TcTS is trans-glycosidation, where α-2,3-linked sialic acid residues are
preferentially transferred between galactose residues on glycoconjugates [13 - 15]. This trans-
glycosidation reaction has been demonstrated to operate through a double-displacement
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mechanism utilising a tyrosine residue as the catalytic nucleophile and displays classical ping-pong
reaction kinetics [16 - 18].
The catalytic domain of TcTS comprises a six-bladed beta propeller topology, also observed
with viral, bacterial and human sialidases [19]. Similarly, the active site of TcTS is highly conserved
amongst members of the sialidase super family (GH33, 34 and 83) comprising eight strictly
conserved amino acids which span three distinct binding motifs, the sialic acid, carboxylate and
lactose binding sites (Fig. 1a) [17, 19]. Studies into the design of inhibitors of TcTS have investigated
derivatives targeted towards all three of these binding motifs, either individually, or in combination
[10]. Compounds based on derivatives of sialic acid have been found generally to be very poor
inhibitors of TcTS. DANA (1) (Fig. 1b) is a low micromolar inhibitor of influenza neuraminidases,
however was found to be only a weak inhibitor of TcTS (Ki = 12.3 mM) [20]. Compounds binding to
the lactose site have shown more promise as inhibitors, such as the pentasaccharide
Galp(β1→2)[Galp(β1→3)]Galp(β1→6) [Galf(β1→4)]GlcNAc-4)]GlcNAcol with an IC50 of 0.61 mM
towards TcTS [21]. Also, the reduced disaccharide lactitol (2) (Fig. 1b) was shown to inhibit TcTS with
a KM = 0.26 mM and was also shown to reduce infection of mammalian cells [22]. Compounds
incorporating a phosphonate group such as 3 (IC50 = 5 mM) [23] and 4 (Ki = 7.3 mM) [24] have also
been investigated in attempts to improve affinity to the carboxylate binding Arg triad, but have been
found to be only weak inhibitors. Alternatively, the most potent inhibitors of TcTS found to date are
not carbohydrate based at all, but instead contain multiple aromatic rings such as the anthraquinone
(5) [25] and chalcone (6) [26] with Ki = 0.89 µM and IC50 = 0.9 µM, respectively (Fig. 1b).
Interestingly, compounds 4 and 5, which contain aromatic rings, were both found to inhibit TcTS in a
non-competitive manner.
Here we describe the design, synthesis and kinetic evaluation of a series of aryl α-
aminophosphonates as inhibitors of TcTS. These compounds were designed to target all three
binding motifs of the trans-sialidase (sialic acid, carboxylate and lactose), but incorporate substituted
aromatic residues to occupy the carbohydrate binding regions, which simplifies their chemical
synthesis significantly.
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a)
OH
CH2OH
HO
OH
OH
HO
HO
HO
O
O
O
CO2H
HO
OH
OH
HO
AcHN
P
OEt
O
AcNH
OH
OH
HOH2C
HO
3
( ) IC50 = 5 mM
1
(2) KM = 0.26 mM
( ) Ki = 12.3 mM
O
O
O
O
H
HO
OH
OH
O
OH
OH
O
S
Cl
Me
N
H
-2O3PO
N
OH
OH
O
6
( ) IC50 = 0.9 µM
(5) Ki = 0.89 µM
(4) Ki = 7.3 mM
b)
Fig. 1. a) Active site of TcTS with sialyl lactose bound (PDB; 1S0I) showing the strictly conserved amino acids
that define the sialic acid, carboxylate and lactose binding regions. b) Structures of known inhibitors of TcTS
and their reported inhibitory activities.
2. Results and Discussion
2.1. Molecular docking studies
Molecular docking simulations were performed using AutoDock Vina [27] to
investigate potential binding modes of a series of aryl α-aminophosphonates in the active
site of TcTS and to predict the impact of substitutions to the aromatic rings of groups X- and
Y-, designed to bind to the sialic acid and lactose binding sites, respectively (Table 1). The
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crystallised structure of TcTS chosen to perform the docking studies on was 1S0I (PDB code)
[17] which is a complex (soaked) of trans-sialidase with the substrate α-(2,3)-sialyl lactose. It
was considered important to use a structure representing the Michaelis complex, where
residue Tyr119 has rotated into the ‘closed’ binding conformation, in order to evaluate
potential π-π stacking interactions in the lactose binding site. Additionally, as the α-carbon
of the proposed aminophosphonates is chiral, docking studies were performed on both the
(R)- and (S)- isomers of each derivative to evaluate the preferred stereochemistry.
Table 1.
Predicted binding energies (kcal/mol) of the (R)- and (S)- isomers of α-aminophosphonates 7a-7k using
AutoDock Vina [27].
Predicted affinity
(kcal/mol)
Compound
X
Y
(R)-isomer (S)-isomer
phenyl
-4.5
-8.6
-6.8
-4.8
-4.6
-8.9
-8.8
-7.5
-9.6
-9.8
-8.2
-3.8
-7.0
-5.6
-3.6
-3.7
-7.4
-7.7
-6.6
-8.1
-8.0
-7.6
7a
7b
7c
7d
7e
7f
1-napthyl
2-napthyl
2-biaryl
3-biaryl
phenyl
4-methoxy phenyl
4-chloro phenyl
7g
7h
7i
4-trifluoromethyl phenyl
4-N-acetamido phenyl
4-nitro phenyl
1-napthyl
7j
3,5-di-trifluoromethyl phenyl
7k
Initially, X was fixed as a phenyl group (sterically conservative in comparison to sialic
acid) and a series of Y = aryl derivatives screened for their predicted binding in the lactose
site. The phenyl, naphthyl and bi-aryl Y groups were chosen to investigate the importance
towards binding of number of aromatic groups, planarity and regiochemistry of attachment.
For all derivatives at Y modelled (7a-7e), docking studies predict the (R)-isomer to form
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slightly more favourable binding interactions with TcTS (Table 1). The 1-napthyl derivative
(R)-7b was predicted to bind most tightly (-8.6 kcal/mol), indicating that a planar group
consisting of two aromatic residues is preferred. The predicted binding position of (R)-7b is
presented in Fig. 2a, showing the phosphonate group occupying the carboxylate binding
pocket (Arg triad), while the phenyl group (X) occupies the sialic acid binding site and the
napthyl group (Y) occupies the lactose binding site, capable of forming π-stacking
interactions between Tyr119 and Trp312.
Based on the initial docking results, Y was then fixed as a 1-napthyl group and the X
position screened using a series of modified phenyl derivatives for predicted binding to the
sialic acid site. Again, the predicted binding energies for all modelled derivatives 7a-7f
slightly favoured the (R)- isomer. Encouragingly, the 4-N modified phenyl derivatives (R)-7d
and (R)-7e, which more resemble 4-N-acetyl neuraminic acid, were predicted to bind with
the highest affinities of -9.6 and -9.8 kcal/mol, respectively (Table 1). An overlay of the
predicted binding position of compounds (R)-7a-7f is shown in Fig. 2b. It can be seen that all
derivatives, except for 7h (Fig. 2b: green sticks), are predicted to bind in a very similar
position, with 7h predicted to have the poorest binding affinity.
7f
7g
7h
7i
7j
7k
a)
b)
Fig. 2. a) Predicted binding position of (R)-isomer of compound 7b (grey sticks) in the TcTS active site (side-
chains in blue sticks). b) Overlay of predicted binding positions for (R)-isomer of compounds 7f-7k in TcTS.
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2.2. Chemistry
Each of the α-aminophosphonates modelled in the docking experiments were then
synthesised chemically to facilitate their biochemical evaluation as inhibitors of TcTS. One of
the most convenient methods for the synthesis of α-aminophosphonates is reported by
Naydenova et al. [28] which uses a one-pot, solvent-free modification of the original
Kabachnik-Fields reaction [29, 30]. Here, the relevant X-benzaldehyde and Y-amine
derivatives were heated by microwave irradiation in dimethylphosphite to generate the
respective α-aminophosphonate dimethyl esters 8a-8k (Scheme 1). Treatment of the
protected phosphonates 8a-8k with trimethylsilyl bromide then gave the target compounds
7a-7k, which were isolated as racemates in generally good to excellent yields over the two
steps (Table 2). Considerable attempts were made to isolate the products 8a-8k as pure
enantiomers using methods such as co-crystallisation with chiral resolving agents and chiral
HPLC. As none of these methods were successful in providing pure enantiomers (or even
any enantiomeric enrichment), the kinetic evaluations were performed on the racemates.
O
P
O
P
O
MeO
OMe
HO
OH
a
b
Y
+
Y
Y
H2N
X
X
N
X
N
H
H
H
H
8 a-k
7a-k
Scheme 1. Reagents and conditions: (a) Microwave (90W, time specified in Table 2), dimethylphosphite (10
equiv. w/v). (b) TMSBr (equiv. specified in Table 2), CH2Cl2/DMF, 0°C, 30 minutes. X and Y are defined in Table
1.
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Table 2
Specific reaction conditions and product yields for the synthesis of compounds 7a-7k.
Time
(min)
Yield of 8
TMSBr
(equiv.)
Yield of 7
Compound
(%)
(%)
7a
7b
7c
7d
7e
7f
5
5
5
7
5
7
5
7
8
6
7
75
85
70
84
76
66
87
55
72
72
84
5
3
94
82
94
86
74
49
79
64
94
59
40
5
7.5
4.5
5
7g
7h
7i
4.5
5
2.5
4.5
3.5
7j
7k
2.3 Trans-sialidase inhibition studies
The inhibitory activities of the newly synthesised α-aminophosphonates 7a-7k were
then evaluated by determining their IC50 values towards TcTS following methods described
previously [31]. The assay employed uses 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic
acid (MuNANA) as the substrate to measure the hydrolytic activity of TcTS by detection of
the fluorescent product of hydrolysis, 4-methylumbelliferone [19]. The IC50s determined for
compounds 7a-7k are presented in Table 3.
For compounds 7a-7e, where the Y group was screened with the X group fixed as
phenyl, some broad agreement was observed between the predicted binding energies and
the IC50 values measured. The two napthyl derivatives 7b and 7c were predicted to bind
most favourably with TcTS (-8.6 and -6.8 kcal/mol, respectively) for this set of compounds
and both were indeed found to have the two lowest IC50s (0.47 and 0.51 mM, respectively).
However, docking studies predicted essentially no difference between the binding energies
of the two bi-aryl derivatives 7d and 7e, while the inhibition studies demonstrate a 10-fold
difference between the observed IC50s of 0.63 mM and 5.58 mM, respectively.
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Table 3
Inhibitory values determined for compounds 7a-7k against TcTS.
IC50
Std. error
(mM)
Ki
(mM)
Mode of
inhibition
Compound
(mM)a
>7.5 b
0.47
0.51
0.63
5.58
0.27
0.71
0.29
0.23
0.21
2.35
>20
7a
7b
7c
7d
7e
7f
±0.09
±0.09
±0.04
±2.37
±0.03
±0.05
±0.01
±0.03
±0.04
±0.83
0.43
Non-competitive
7g
7h
7i
0.19
0.14
Non-competitive
Non-competitive
7j
7k
a
Data are expressed as the mean average of three (n=3) independent
b
experiments. Saturating concentrations could not be reached owing
to solubility.
For the second set of compounds 7f-7k, where Y was fixed as a 1-napthyl group and
derivatives screened for X, there was again mixed agreement between predicted binding
energies and the observed IC50s. In general, compounds substituted at the 4-position of the
pheny group (7f-7j) were all found to have comparable IC50 values (0.21 – 0.71 mM), while
the 3,5-modified derivative 7k was found to be a significantly poorer inhibitor with an IC50 of
2.35 mM. Notably, the 4-trifluoromethyl derivative 7h was predicted to be the poorest
binder in docking studies, but its IC50 = 0.29 mM was found to be almost equivalent to the
best inhibitors.
To determine whether α-aminophosphonates display any selectivity for inhibition of
TcTS over other family GH33 sialidases (which includes the human sialidases), we
determined the IC50s of the most potent inhibitor, compound 7j, against the two bacterial
sialidases Clostridium perfringens neuraminidase (CpNA) and Staphylococcus pneumoniae
neuraminidase A (SpNA) (Supplementary Data). Compound 7j was found to be a poor
inhibitor of both enzymes, with IC50s of 2.39 mM and 1.41 mM against SpNA and CpNA,
respectively. As such, 7j displays 7 to 12 fold better inhibition against TcTS (IC50 0.21 mM)
than the bacterial sialidases suggesting that α-aminophosphonates possess some degree of
selectivity for trans-sialidase.
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In summary, the IC50 values determined here suggest that α-aminophosphonates are
effective inhibitors of TcTS and possess some selectivity for this enzyme over other family
GH33 sialidases. Furthermore, derivatives consisting of Y groups containing at least two
aromatic residues in a planar conformation and X groups modified at the C-4 position,
possess the most potent inhibitory activities towards trans-sialidase.
2.4 Mode of inhibition studies
Having demonstrated that α-aminophosphonates, as a class, are effective inhibitors of TcTS,
the mode of inhibition of the parent 1-napthyl compound 7b and the two most potent
compounds 7i and 7j were then investigated. Lineweaver-Burk plots were produced by
plotting residual enzyme activity against substrate concentration for a series of inhibitor
concentrations. The regression lines for each inhibitor concentration converge on the X-axis
demonstrating that all three compounds display non-competitive inhibition kinetics towards
TcTS (Fig. 3). The Ki values for 7b, 7i and 7j were determined to be 430 µM, 190 µM and 140
µM, respectively (Table 3). It is interesting to note that a number of inhibitors containing
multiple aromatic rings, including 5, have been shown previously by other groups to display
non-competitive inhibition kinetics towards TcTS [25].
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b)
0.25
a)
0.20
0.15
0.10
0.05
0.20
0.15
0.10
0.05
-0.01
0.00
0.01
0.02
-0.01
0.00
0.01
0.02
1/[S] (mM-1)
1/[S] (mM-1)
0.25
0.20
0.15
0.10
0.05
c)
-0.01
0.00
0.01
0.02
1/[S] (mM-1
)
Fig. 3. Plots of Lineweaver-Burk kinetics performed using [MuNANA] = 2.0, 1.0, 0.5, 0.2, 0.1, 0.05 and 0.01 mM
for: a) Compound 7b at 0 (ò), 0.05 (¢), 0.10 (Å), 0.20 (Ç) and 0.40 (ø) mM. b) Compound 7i at 0 (ò), 0.02
(¢), 0.05 (Å), 0.10 (Ç) and 0.15 (ø) mM. c) Compound 7j at 0 (ò), 0.05 (¢), 0.10 (Å) and 0.20 (Ç) mM.
3. Conclusions
We have successfully synthesised a series of eleven aryl α-aminophosphonates in
two chemical steps in good to excellent overall yields. Several of these derivatives were
demonstrated to inhibit TcTS in the low micromolar range, comparable to some of the most
potent carbohydrate-based inhibitors of this enzyme. Furthermore, we have shown that
these compounds display non-competitive inhibition kinetics towards TcTS, consistent with
several other compounds containing multiple aromatic residues. Together, these results
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demonstrate that α-aminophosphonates are a promising class of compounds for further
development towards more potent inhibitors of T. cruzi trans-sialidase.
4. Experimental protocols
4.1. General methods and materials
Chemical reagents were purchased from Sigma-Aldrich unless specifically stated.
Anhydrous solvents were purchased from Sigma-Aldrich and used without further
purification. Purified Clostridium perfringens neuraminidase (CpNA) was purchased from
Sigma-Aldrich. All other solvents were purchased from Fisher Scientific. Analytical thin layer
chromatography (TLC) was carried out on Merck aluminium backed TLC plates silica gel 60
F254 (0.25 mm thickness), viewed using UV light of wavelength 254 nm or stained with
potassium permanganate solution or para-anisaldehyde stain. Silica gel chromatography
was performed on silica gel 60 Å (200-400 mesh) from Sigma-Aldrich. Preparative reverse
phase chromatography (C-18) was performed using a VersaFlash hand held column (23 x
110 mm) from Supelco. Melting points were obtained using a Thermo Fisher IA9000 digital
1
melting point apparatus. H, 13C and 31P NMR spectra were recorded using Bruker Advance
1
III 400 MHz spectrometer with acquisition frequencies of 400 for H; 101 MHz for 13C; and
162 MHz for 31P. Deuterated solvents were purchased from Cambridge Isotope
Laboratories. The NMR chemical shifts δ were recorded in parts per million (ppm) with
reference to tetramethylsilane for 1H and 13C and phosphoric acid for 31P NMR spectroscopy.
High resolution mass spectrometry was performed using a BrukerMicrOTOF electrospray
ionisation mass spectrometer.
4.2 Molecular Docking
A structure of TcTS obtained by X-ray diffraction with the ligand α(2,3)-sialyl lactose
bound (PDB code: 1S0I) was used for all docking studies. A model of the structure was
prepared using AutoDock tools, which involved removing the bound ligand, mutating
residue A59D, adding all hydrogens to the model, setting the lactose binding site residues
Y119 and W312 to be flexible, and limiting the region of docking to the catalytic site. 3-D
models of α-phosphonate ligands were built using Phenix eLBOW [32] or CCP4 JLigand [33].
Molecular docking experiments were run using AutoDock Vina [27] to screen for potential
binding positions and to calculate the predicted binding energy as the docking score.
4.3 General procedure for the synthesis of protected α-aminophosphonates 8a-8k.
A mixture of the aldehyde (4.7 mmol, 1 eq.) and the amine (4.7 mmol, 1 eq.) were
dissolved in dimethylphosphite (10 equiv. w/v) and the resultant solution subjected to
microwave heating (90 W) for the specified time (Table 2). The reaction mixture was then
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diluted with dichloromethane, and the organic phase washed sequentially with saturated
sodium bicarbonate solution and brine. The organic phases were then combined and dried
over anhydrous magnesium sulphate, filtered and the filtrate concentrated. The resultant
residue was then purified by column chromatography (35% EtOAc/petroleum ether) to give
the protected
α
-aminophosphonates 8a-8k.
4.3.1 (±) Dimethyl-[
α
-(phenyl)(phenylamino)methyl] phosphonate (8a)
The title compound was prepared from benzaldehyde and aniline. White solid; yield 75%; 1H
NMR (400 MHz, CDCl3): δ 7.50 – 7.40 (m, 2H, Ar), 7.35 – 7.27 (m, 2H, Ar), 7.27 – 7.18 (m, 1H,
Ar), 7.12 – 7.01 (m, 2H, Ar), 6.66 (dd, J = 7.3, 1.1 Hz, 1H, Ar), 6.61 – 6.52 (m, 2H, Ar), 4.81-
4.70 (m, 2H, NH, P-CH ), 3.71 (d, J = 10.6 Hz, 3H, OMe), 3.43 (d, J = 10.6 Hz, 3H, OMe).13C
NMR (100.6 MHz, CDCl3): δ 146.16, 136.12, 129.13, 128.65, 127.98, 127.76, 118.48, 113.85,
55.68 (d, J = 151.3 Hz), 53.75, 53.69. 31P NMR (161.93 MHz, CDCl3): δ 24.96. HRMS (ESI):
Calculated for C15H18NO3PNa (M+Na)+: m/z= 314.0922, found m/z = 314.0907.
4.3.2 (±) Dimethyl-[α-(phenyl)(napthalen-1-ylamino)methyl] phosphonate (8b)
The title compound was prepared from benzaldehyde and 1-aminonapthalene. White solid;
yield 85%; 1H NMR (400 MHz, CDCl3): δ 8.06-8.03 (m, 1H, Ar), 7.79- 7.75 (m, 1H, Ar), 7.56 –
7.45 (m, 4H, Ar), 7.38 – 7.19 (m, 5H, Ar), 6.49 – 6.37 (m, 1H, Ar), 5.49 (s, 1H, NH), 5.01 (d, J =
19.8 Hz, 1H, P-CH), 3.80 (d, J = 10.6 Hz, 3H, OMe), 3.53 (d, J = 10.6 Hz, 3H, OMe). 13C NMR
(101 MHz, CDCl3) δ 141.05, 135.25, 134.20, 128.72, 128.62, 128.07, 127.58, 126.13, 125.85,
125.11, 123.88, 119.97, 118.69, 106.54, 55.87 (d, J = 151.0 Hz), 53.85, 53.81. 31P NMR (162
MHz, CDCl3): δ 24.94. HRMS (ESI) calculated for C19H21NO3P [M+H]+: 342.1259. Found:
342.1252.
4.3.3 (±) Dimethyl-[α-(phenyl)(napthalen-2-ylamino)methyl] phosphonate (8c)
The title compound was prepared from benzaldehyde and 2-aminonapthalene. White solid;
1
yield 70%; H NMR (400 MHz, CDCl3): δ 8.03-8.01 (m, 1H, Ar), 7.78-7.76 (m, 1H, Ar), 7.53-
7.45 (m, 4H, Ar), 7.35-7.22 (m, 4H, Ar), 7.12-7.14 (m, 1H, Ar), 6.42-6.40 (m, 1H, Ar), 5.46 (s,
1H, NH), 4.97 (d, J = 21.5 Hz, 1H, P-CH), 3.78 (d, J = 10.8 Hz, 3H, OMe), 3.52 (d, J = 10.4, 3H,
OMe). 13C NMR (101 MHz, CDCl3) δ 141.12, 140.97, 135.24, 135.21, 134.20, 128.79, 128.76,
128.66, 128.14, 128.12, 127.63, 127.57, 126.18, 125.91, 125.75, 125.16, 123.87, 120.02,
118.72, 106.55, 56.33 (d, J = 149.6 Hz), 54.03, 53.96, 53.89. 31P NMR (162 MHz, CDCl3): δ
24.90. HRMS (ESI) calculated for C19H21NO3P [M+H]+: 342.1259. Found: 342.1250.
4.3.4 (±) Dimethyl-[α-(phenyl)([1,1'-biphenyl]-2-ylamino)methyl] phosphonate (8d)
The title compound was prepared from benzaldehyde and 1,1’-biphenyl-2-amine. White
solid; yield 76%; 1H NMR (400 MHz, CDCl3): δ 7.52 – 7.42 (m, 4H, Ar), 7.42 – 7.35 (m, 3H, Ar),
7.35 – 7.29 (m, 2H, Ar), 7.28 – 7.24 (m, 1H, Ar), 7.13 – 7.05 (m, 2H, Ar), 6.80 – 6.75 (m, 1H,
Ar), 6.55 – 6.50 (m, 1H, Ar), 4.97 (s, 1H, NH), 4.78 (d, J = 24.2 Hz, 1H, P-CH), 3.62 (d, J = 10.6
Hz, 3H, OMe), 3.47 (d, J = 10.6 Hz, 3H, OMe). 13C NMR (100.6 MHz, CDCl3): δ 143.14, 138.88,
13

ACCEPTED MANUSCRIPT
135.61, 130.25, 129.18, 128.92, 128.78, 128.62, 127.96, 127.62, 127.50, 118.28, 111.89,
56.13 (d, J = 150.8 Hz), 53.69, 53.52. 31P NMR (162 MHz, CDCl3): δ 24.51. HRMS (ESI)
calculated for C21H22NO3PNa [M+Na]+: 390.1235. Found: 390.1247.
4.3.5 (±) Dimethyl-[α-(phenyl)([1,1'-biphenyl]-3-ylamino)methyl] phosphonate (8e)
The title compound was prepared from benzaldehyde and 1,1’-biphenyl-3-amino. White
solid; yield 84%; 1H NMR (400 MHz, CDCl3): δ 7.49 – 7.42 (m, 2H, Ar), 7.42 – 7.37 (m, 2H, Ar),
7.36 – 7.27 (m, 4H, Ar), 7.26 – 7.20 (m, 2H, Ar), 7.11 (t, J = 7.8 Hz, 1H, Ar), 6.87 (dd, J = 7.6,
1.7 Hz, 1H, Ar), 6.79 (t, J = 2.0 Hz, 1H, Ar), 6.52 (dd, J = 8.2, 2.4 Hz, 1H, Ar), 5.22 (s, 1H, NH),
4.75 (br d, J = 25.0 Hz, 1H, P-CH), 3.74 (d, J = 10.7 Hz, 3H, OMe), 3.43 (d, J = 10.6 Hz, 3H,
OMe). 13C NMR (101 MHz, CDCl3) δ 146.42, 143.45, 142.02, 135.56, 129.49, 128.70, 128.52,
128.03, 127.79, 127.73, 127.11, 126.96, 117.57, 112.81, 112.67, 55.71 (d, J = 151.4 Hz),
53.76, 53.69. 31P NMR (162 MHz, CDCl3): δ 24.88. HRMS (ESI) calculated for C21H23NO3P
[M+H]+: 368.1416. Found: 368.1416.
4.3.6 (±) Dimethyl-[α-(4-methoxyphenyl)(napthalen-1-ylamino)methyl] phosphonate (8f)
The title compound was prepared from 4-methoxybenzaldehyde and 1-aminonapthalene.
1
White solid; yield 66%; H NMR (400 MHz, CDCl3) δ 8.03-8.01 (m, 1H, Ar), 7.80-7.77 (m, 1H,
Ar), 7.53-7.42 (m, 4H, Ar), 7.26-7.23 (m, 2H, Ar), 7.20-7.16 (m ,1H, Ar), 6.88 – 6.82 (m, 2H,
Ar), 5.90 (br s, 1H, NH), 4.93 (d, J = 24.6 Hz, 1H, P-CH), 3.80 (s, 3H, Ar-OMe), 3.79 (d, J = 10.5
Hz, 3H, OMe), 3.55 (d, J = 10.5 Hz, 3H, OMe). 13C NMR (101 MHz, CDCl3) δ 157.3, 144.4,
134.21, 128.73, 128.68, 128.59, 126.12, 125.80, 125.06, 123.93, 119.96, 118.65, 114.21,
114.18, 106.62, 55.51 (d, J = 150.3 Hz), 53.80, 53.69, 34.52. 31P NMR (162 MHz, CDCl3): δ
25.12. HRMS (ESI) Calculated for C20H23NO4P [M+H]+: 372.1364. Found 372.1359.
4.3.7 (±) Dimethyl-[α-(4-chlorophenyl)(napthalen-1-ylamino)methyl] phosphonate (8g)
The title compound was prepared from 4-chlorobenzaldehyde and 1-aminonapthalene.
1
White solid; yield 87%; H-NMR (400 MHz, CDCl3): δ 8.02 - 7.99 (m, 1H, Ar), 7.81 - 7.77 (m,
1H, Ar), 7.55 - 7.44 (m, 4H, Ar), 7.33 - 7.30 (m, 2H, Ar), 7.27 – 7.24 (m, 1H, Ar), 7.19-7.15 (m,
1H, Ar), 6.38 – 6.35 (m, 1H, Ar), 5.42 (s, 1H, NH), 4.98 (d, J = 24 Hz, 1H, P-CH), 3.82 (d, J =
10.8 Hz, 3H, OMe), 3.62 (d, J = 10.8 Hz, 3H, OMe). 13C NMR (101 MHz, CDCl3): δ 140.83,
140.69, 134.21, 133.93, 133.89, 128.96, 128.93, 128.85, 128.68, 126.04, 125.96, 125.84,
123.85, 119.87, 119.02, 106.59, 56.09 (d, J = 150.3 Hz, P-CH), 54.59, 54.08. 31P NMR (162
MHz, CDCl3): δ 24.10. HRMS (ESI) Calculated for C19H19ClNNaO3P [M+Na]+: 398.0688. Found
398.0674.
4.3.8 (±) Dimethyl-[α-(4-(trifluoromethyl)phenyl)(napthalen-1-ylamino)methyl] phosphonate
(8h)
The title compound was prepared from 4-trifluoromethylbenzaldehyde and 1-
1
aminonapthalene. White solid; yield 56%; H NMR (400 MHz, CDCl3): δ 8.01 (dd, J = 7.7, 1.7
Hz, 1H, Ar), 7.79 (dd, J = 7.5, 2.0 Hz, 1H, Ar), 7.70 – 7.44 (m, 6H, Ar), 7.26 (d, J = 8.2 Hz, 1H,
14

ACCEPTED MANUSCRIPT
Ar), 7.15 (dd, J = 8.2, 7.6 Hz, 1H, Ar), 6.42 – 6.18 (m, 1H, Ar), 5.45 (s, 1H, NH), 5.02 (d, J =
24.4 Hz, 1H, P-CH), 3.81 (d, J = 10.7 Hz, 3H, OMe), 3.61 (d, J = 10.7 Hz, 3H, OMe). 13C NMR
(101 MHz, CDCl3): δ 141.85, 140.66, 140.16, 139.28, 135.37, 132.46, 128.88, 128.48, 127.92,
126.43, 126.08, 125.65, 125.27, 124.36, 122.36, 120.07, 117.04, 58.31 (d, J = 153 Hz, P-CH),
53.97, 53.67. 31P NMR (162 MHz, CDCl3): δ 23.95. HRMS (ESI) Calculated for C20H19F3NNaO3P
[M+Na]+: 432.0952. Found 432.0942.
4.3.9 (±) Dimethyl-[α-(4-N-acetamidophenyl)(napthalen-1-ylamino)methyl] phosphonate (8i)
The title compound was prepared from 4-N-acetamidobenzaldehyde and 1-
aminonapthalene. White solid; yield 72%; 1H NMR (400MHz, CDCl3): δ 8.00 (d, J = 6.4Hz, 1H,
Ar), 7.83-7.71 (m, 1H, Ar), 7.53 - 7.71 (m, 6H, Ar), 7.25 (d, J = 8.0 Hz, 1H, Ar), 7.18 - 7.14 (m,
1H, Ar), 6.41 (d, J = 6.0 Hz, 1H, Ar), 5.39 (s, 1H, NH), 4.97(d, J = 20 Hz, 1H, P-CH), 3.81 (d, J =
10.4Hz, 3H, OMe), 3.57 (d, J = 9.6Hz, 3H, OMe), 2.15(s, 3H, NAc). 13C NMR (101 MHz, CDCl3):
δ 163.63, 141.15, 141.06, 138.28, 134.37, 130.65, 128.85, 128.38, 128.07, 126.33, 126.07,
125.37, 124.06, 123.33, 120.05, 119.04, 55.55 (d, J = 151 Hz, P-CH), 54.23, 54.07, 24.63. 31P-
NMR (162 MHz, CDCl3): δ 25.57. HRMS (ESI) Calculated for C21H23N2NaO4P [M+H]+:
421.1293. Found 421.1269.
4.3.10 (±) Dimethyl-[α-(4-nitrophenyl)(napthalen-1-ylamino)methyl] phosphonate (8j)
The title compound was prepared from 4-nitrobenzaldehyde and 1-aminonapthalene.
Yellow solid; yield 72%; 1H NMR (400 MHz, CDCl3): δ 8.24 – 8.10 (m, 2H, Ar), 8.05 – 7.92 (m,
1H, Ar), 7.79 (dd, J = 7.6, 1.7 Hz, 1H, Ar), 7.74 – 7.64 (m, 2H, Ar), 7.59 – 7.44 (m, 2H, Ar), 7.30
– 7.20 (m, 1H, Ar), 7.18 – 7.09 (m, 1H, Ar), 6.35 – 6.16 (m, 1H, Ar), 5.45 (s, 1H, NH), 5.06 (d, J
= 24.8 Hz, 1H, P-CH), 3.82 (d, J = 10.8 Hz, 3H, OMe), 3.66 (d, J = 10.8 Hz, 3H, OMe). 13C NMR
(101 MHz, CDCl3): δ 143.63, 143.28, 140.06, 134.32, 128.77, 128.43, 128.38, 126.16, 125.91,
125.49, 123.92, 123.88, 123.84, 119.75, 119.53, 106.60, 55.41 (d, J = 151 Hz, P-CH) 54.21,
53.93. 31P NMR (162 MHz, CDCl3): δ 23.26. HRMS (ESI) Calculated for C19H20N2O5P [M+H]+:
387.1109. Found 387.1099.
4.3.11
(±)
Dimethyl-[α-(3,5-bis(trifluoromethyl)phenyl)(napthalen-1-ylamino)methyl]
phosphonate (8k)
The title compound was prepared from 3,5-bis(trifluoromethyl)benzaldehyde and 1-
1
aminonapthalene. White solid; yield 84%. H NMR (400 MHz, CDCl3): δ 8.12 - 8.04 (m, 3H,
Ar), 7.83 (d, J = 8.2 Hz, 2H, Ar), 7.58-7.50 (m, 2H, Ar), 7.32 (d, J = 8.4 Hz, 1H, Ar), 7.21 (m, 1H,
Ar), 6.29 (d, J = 5.6 Hz, 1H, Ar), 5.48 (s, 1H, NH), 5.08 (d, J = 24.4 Hz, 1H, P-CH), 3.85 (d, J =
10.8 Hz, 3H, OMe), 3.69 (d, J = 10.8 Hz, 3H, OMe). 13C NMR (101 MHz, CDCl3): δ 142.65,
140.15, 140.06, 137.18, 133.27, 131.56, 128.95, 127.87, 126.23, 126.17, 125.37, 125.07,
124.76, 122.36, 121.83, 120.05, 116.04, 56.35 (d, J = 153.3 Hz, P-CH), 54.07, 53.97. 31P NMR
(162 MHz, CDCl3): δ 21.06. HRMS (ESI) Calculated for C21H19F6NO3P [M+H]+: 478.1006. Found
478.1003.
15

ACCEPTED MANUSCRIPT
4.4 General procedure for the synthesis of
α
-aminophosphonates 7a-7k.
TMSBr (equiv. specified in Table 3) was added dropwise to a solution of the dimethyl α-
aminophosphonate (100 mg, 1.0 equiv.) in anhydrous CH2Cl2/DMF (2:1, 3 mL) at 0°C and the
mixture held at this temperature for 30 minutes. The solution was then allowed to warm to
room temperature and concentrated in vacuo. The resultant residue was then diluted with
water (2.5 mL), neutralized (aq. 1M NaOH) and purified by C-18 reverse phase
chromatography (20-40 % MeOH/H2O) to give the title phosphonic acid.
4.4.1 (±) α-(phenyl)(phenylamino)methyl phosphonic acid (7a)
1
The title compound was prepared from 8a. White solid; yield 94%. H NMR (400 MHz,
CD3OD): δ 7.54 (d, J = 8.0 Hz, 2H, Ar), 7.25 - 7.20 (m, 2H, Ar), 7.12 - 7.09 (m, 1H, Ar), 7.03 -
13
6.89 (m, 2H, Ar), 6.60 - 6.44 (m, 3H, Ar), 4.49 (d, J = 21.6 Hz, 1H, P-CH). C NMR (101 MHz,
CD3OD): δ 150.25, 144.04, 129.62, 129.15, 128.62, 126.46, 117.34, 114.74, 60.64 (d, J = 130
Hz). 31P NMR (162 MHz, CD3OD): δ 16.46. HRMS (ESI): Calculated for C13H13NO3P [M-H]-:
262.0633. Found 262.0633.
4.4.2 (±) α-(phenyl)(napthalen-1-ylamino)methyl phosphonic acid (7b)
1
The title compound was prepared from 8b. White solid; yield 82%. H NMR (400 MHz,
CD3OD): δ 8.32 (dd, J = 7.9, 1.5 Hz, 1H, Ar), 7.76 – 7.69 (m, 1H, Ar), 7.59 (dt, J = 8.0, 1.7 Hz,
2H, Ar), 7.52 – 7.35 (m, 2H, Ar), 7.24 (dd, J = 8.4, 6.9 Hz, 2H, Ar), 7.19 – 7.01 (m, 3H, Ar), 6.32
(dd, J = 6.6, 2.2 Hz, 1H, Ar), 4.64 (d, J = 21.3 Hz, 1H, P-CH). 13C NMR (101 MHz, CD3OD): δ
145.19, 143.55, 135.87, 129.13, 129.07, 128.60, 127.42, 126.46, 126.24, 125.27, 124.96,
122.25, 116.77, 106.31, 60.57 (d, J = 131 Hz). 31P NMR (162 MHz, CD3OD): δ 15.74. HRMS
(ESI) calculated for C17H15NO3P [M-H]-: 312.0790. Found: 312.0796.
4.4.3 (±) α-(phenyl)(napthalen-2-ylamino)methyl phosphonic acid (7c)
1
The title compound was prepared from 8c. White solid; yield 94%. H NMR (400 MHz,
CD3OD): δ 7.61-7.48 (m, 4H, Ar), 7.36-7.34 (m, 1H, Ar), 7.24-7.21 (m, 3H, Ar), 7.14-7.03 (m,
3H, Ar), 6.49 (s, 1H, Ar), 4.60 (d, J = 21.1 Hz, 1H, P-CH). 13C NMR (101 MHz, CD3OD): δ
145.28, 145.17, 138.54, 134.52, 129.08, 128.42, 127.85, 127.81, 127.56, 127.29, 127.13,
126.62, 125.85, 122.55, 118.87, 105.93, 57.43 (d, J = 130 Hz). 31P NMR (162 MHz, CD3OD): δ
15.95. HRMS (ESI) calculated for C17H15NO3P [M-H]-: 312.0790. Found: 312.0776.
4.4.4 (±) α-(phenyl)([1,1'-biphenyl]-2-ylamino)methyl phosphonic acid (7d)
1
The title compound was prepared from 8d. White solid; yield 86%. H NMR (400 MHz,
CD3OD): δ 7.65 – 7.57 (m, 2H, Ar), 7.57 – 7.46 (m, 4H, Ar), 7.40 – 7.30 (m, 1H, Ar), 7.26 (t, J =
7.6 Hz, 2H, Ar), 7.12 (td, J = 7.2, 1.5 Hz, 1H, Ar), 7.02 – 6.90 (m, 2H, Ar), 6.60 (td, J = 7.4, 1.1
16

ACCEPTED MANUSCRIPT
Hz, 1H, Ar), 6.50 (dd, J = 8.4, 1.0 Hz, 1H, Ar), 4.54 (d, J = 21.5 Hz, 1H, P-CH). 13C NMR (101
MHz, CD3OD) δ 150.62, 144.09, 143.43, 142.86, 130.06, 129.53, 129.23, 128.69, 127.86,
126.50, 116.19, 113.90, 113.38, 60.82 (d, J = 129.8 Hz). 31P NMR (162 MHz, CD3OD): δ 15.72.
HRMS (ESI) calculated for C19H17NO3P [M-H]-: 338.0946. Found: 338.0962.
4.4.5 (±) α-(phenyl)([1,1'-biphenyl]-3-ylamino)methyl phosphonic acid (7e)
1
The title compound was prepared from 8e. White solid; yield 74%. H NMR (400 MHz,
CD3OD) δ 7.62 – 7.57 (m, 2H, Ar), 7.47 – 7.39 (m, 2H, Ar), 7.39 – 7.20 (m, 5H, Ar), 7.18 – 7.01
(m, 2H, Ar), 6.85 – 6.73 (m, 2H, Ar), 6.57 (dd, J = 8.2, 2.3 Hz, 1H, Ar), 4.51 (d, J = 21.5 Hz, 1H,
P-CH). 13C NMR (101 MHz, CD3OD) δ 150.62, 144.09, 143.43, 142.86, 130.06, 129.53, 129.23,
128.69, 127.86, 126.50, 116.19, 113.90, 113.38, 60.82 (d, J = 129.8 Hz). 31P NMR (162 MHz,
CD3OD): δ 16.28. HRMS (ESI) calculated for C19H17NO3P [M-H]-: 338.0946. Found: 338.0960.
4.4.6 (±) α-(4-methoxyphenyl)(napthalen-1-ylamino)methyl phosphonic acid (7f)
1
The title compound was prepared from 8f. White solid; yield 49%. H NMR (400 MHz,
CD3OD) δ 8.22-8.2 (m, 1H, Ar), 7.76-7.74 (m, 2H, Ar), 7.53-7.44 (m, 3H, Ar), 7.36-7.34 (m, 2H,
Ar), 7.12-7.10 (m, 2H, Ar), 6.38-6.36 (m, 1H, Ar), 4.68 (d, J = 21.0 Hz, 1H, P-CH), 3.69 (s, 3H,
Ph-OMe). 13C NMR (101 MHz, CD3OD): δ 157.08, 143.50, 143.20, 133.92, 128.73, 128.67,
128.27, 126.74, 126.18, 125.06, 123.06, 120.64, 116.32, 113.40, 105.40, 58.14 (d, J = 130.0
Hz), 55.21. 31P NMR (162 MHz, CD3OD): δ 15.52. HRMS (ESI) calculated for C18H17NO4P [M-
H]-: 348.0906. Found: 348.0902.
4.4.7 (±) α-(4-chlorophenyl)(napthalen-1-ylamino)methyl phosphonic acid (7g)
1
The title compound was prepared from 8g. White solid; yield 79%. H-NMR (400 MHz,
CD3OD): δ 8.41 – 8.30 (m, 1H, Ar), 7.78 – 7.67 (m, 1H, Ar), 7.65 – 7.52 (m, 2H, Ar), 7.50 –
7.35 (m, 2H, Ar), 7.27 – 7.17 (m, 2H, Ar), 7.14 – 6.98 (m, 2H, Ar), 6.25 (dd, J = 7.5, 1.4 Hz, 1H,
Ar), 4.65 (d, J = 21.1 Hz, 1H, P-CH). 13C NMR (101 MHz, CD3OD): δ 156.76, 147.94, 146.89,
144.98, 144.01, 132.43, 129.74, 129.50, 129.15, 126.64, 126.52, 125.37, 122.91, 122.40,
116.59, 106.32, 60.78 (d, J = 130 Hz). 31P NMR (CD3OD, 162 MHz): δ 14.89. HRMS (ESI)
calculated for C17H14ClNO3P [M-H]-: 346.0400. Found: 346.0414.
4.4.8 (±) α-(4-(trifluoromethyl)phenyl)(napthalen-1-ylamino)methyl phosphonic acid (7h)
1
The title compound was prepared from 8h. White solid; yield 64%. H NMR (400 MHz,
CD3OD): δ 8.40 (d, J = 8.2 Hz, 1H, Ar), 7.78 (d, J = 8.0 Hz, 2H, Ar), 7.76 – 7.68 (m, 1H, Ar), 7.57
– 7.37 (m, 4H, Ar), 7.13 – 6.97 (m, 2H, Ar), 6.23 (dd, J = 7.1, 1.7 Hz, 1H, Ar), 4.74 (d, J = 21.2
Hz, 1H, P-CH). 13C NMR (101 MHz, CD3OD): δ 149.08, 145.05, 135.919, 129.43, 129.39,
129.17, 128.59, 128.30, 127.63, 127.40, 126.37, 125.38, 125.26, 125.26, 125.08, 124.94,
122.29, 106.15, 61.44 (d, J = 130 Hz). 31P NMR (162 MHz, CD3OD): δ 14.50. HRMS (ESI)
calculated for C18H14F3NO3P [M-H]-: 380.0663. Found: 380.0679.
17

ACCEPTED MANUSCRIPT
4.4.9 (±)
α
-(4-N-acetamidophenyl)(napthalen-1-ylamino)methyl phosphonic acid (7i)
1
The title compound was prepared from 8i. White solid; yield 94%. H NMR (400 MHz,
CD3OD): δ 8.34 (d, J = 8.4 Hz, 1H, Ar), 7.67 (d, J = 10.4 Hz, 1H, H-Ar), 7.54-7.51 (m, 2H, Ar),
7.41-7.33 (m, 4H, Ar), 7.04-6.95 (m, 2H, Ar), 6.26 (d, J = 7.6 Hz, 1H, Ar), 4.63 (d, J = 20.8 Hz,
1H, P-CH), 2.063 (s, 3H, Me). 13C NMR (101 MHz, CD3OD): δ 171.41, 145.16, 139.97, 137.05,
135.76, 129.19, 129.15, 129.04, 127.33, 126.17, 125.16, 122.17, 121.02, 116.70, 106.25,
60.77 (d, J = 129.6 Hz), 23.61. 31P NMR (162 MHz, CD3OD): δ 15.94. HRMS (ESI) calculated for
C19H18N2O4P [M-H]-: 369.1004. Found: 369.1016.
4.4.10 (±) α-(4-nitrophenyl)(napthalen-1-ylamino)methyl phosphonic acid (7j)
1
The title compound was prepared from 8j. Yellow solid; yield 59%. H NMR (400 MHz,
CD3OD): δ 8.40 (d, J = 8.3 Hz, 1H, Ar), 8.12 (d, J = 8.4 Hz, 2H, Ar), 7.82 (dd, J = 8.8, 2.0 Hz, 2H,
Ar), 7.72 (d, J = 8.0 Hz, 1H, Ar), 7.53 – 7.39 (m, 2H, Ar), 7.07 (d, J = 4.3 Hz, 2H, Ar), 6.21 (t, J =
4.3 Hz, 1H, Ar), 4.81 (d, J = 21.3 Hz, 1H, P-CH). 13C NMR (101 MHz, CD3OD): δ 153.26, 147.42,
147.39, 144.81, 144.69, 135.93, 129.74, 129.70, 129.25, 127.34, 126.56, 125.27, 123.91,
122.20, 117.49, 106.22, 61.68 (d, J = 130 Hz). 31P NMR (162 MHz, CD3OD): δ 14.02. HRMS
(ESI) calculated for C17H14N2O5P [M-H]-: 357.0640. Found: 357.0635.
4.4.11 (±)
α
-(3,5-bis(trifluoromethyl)phenyl)(napthalen-1-ylamino)methyl phosphonic acid
(7k)
1
The title compound was prepared from 8k. White solid; yield 40%. H NMR (400 MHz,
CD3OD): δ 8.39 (d, J = 8.2 Hz, 1H, Ar), 8.18 (s, 2H, Ar), 7.77 – 7.58 (m, 2H, Ar), 7.56 – 7.32 (m,
2H, Ar), 7.06 (d, J = 6.1 Hz, 2H, Ar), 6.14 (dd, J = 6.1, 2.7 Hz, 1H, Ar), 4.75 (d, J = 20.8 Hz, 1H,
P-CH).13C NMR (101 MHz, CD3OD): 148.35, 148.30, 144.79, 135.94, 131.82, 131.47, 131.15,
129.32, 129.20, 127.34, 126.75, 126.74, 126.52, 125.28, 124.09, 122.27, 122.07, 117.54,
105.93, 61.43 (d, J = 130 Hz). 31P NMR (162 MHz, CD3OD): δ 13.55. HRMS (ESI) calculated for
C19H13F6NO3P [M-H]-: 448.0537. Found: 448.0566.
4.5 Protein expression and purification
The plasmid pTrcHisA was used for expression of TcTS as described previously by Paris et al.
[31]. This plasmid encodes for recombinant TcTS truncated to only contain the catalytic and
lectin-like domains, with seven surface point mutations (N58F, S495K, V496G, E520K,
D593G, I597D and H599R). These mutation were introduced to promote crystallisation and
have been shown to have no impact on catalytic activity of TcTS [19]. Over expression was
performed using E. coli strain BL21 (DE3) chemically competent cells (NEB).
4.6 IC50 inhibition studies
The hydrolytic activity of TcTS was determined using a continuous fluorometric assay by
measuring the rate of release of 4-methylumbelliferone from the substrate 2’-(4-
18

ACCEPTED MANUSCRIPT
methylumbelliferyl)-α-D-N-acteylneuraminic acid (MuNANA) as described previously [19].
Fluorescence was measured on a FLUOstar Omega (BMG labtech) using excitation and
emission filters at 360 and 460 nm, respectively. All assays were run in 20mM Tris-HCl and
50mM NaCl at pH 7.5, containing 0.01% Triton X100 at 35°C. TcTS (50 µg/mL) was pre-
incubated with each compound over at least seven inhibitor concentrations (ranging from 1
µM to 15 mM) for 10 minutes at 35°C, then the assay initiated by the addition of MuNANA
(200 µM) to give a final reaction volume of 100 µL. The fluorescence was analysed using
MARs data analysis software (BMG labtech), where a linear region of the raw data curve was
selected and the slope calculated (RFU/min) which represents hydrolytic activity of the
enzyme. The IC50 values were determined as the concentration of inhibitor required to
reduce hydrolytic activity of TcTS by 50% compared to the control (no inhibitor). All assays
were repeated in three (n=3) independent experiments.
4.7 Mode of inhibition studies
The Ki determinations were performed by measuring the residual activity of TcTS in the
presence of several different concentrations of MuNANA (0–400 µM) at specified inhibitor
concentrations. Residual hydrolytic activity of TcTS was determined using the assay method
described above. Nonlinear fitting was performed by GraphPad Prism software and Km/Vmax
of different concentration of inhibitors was calculated. Further Ki and its standard error
were calculated using competitive, non-competitive, uncompetitive and mixed inhibition
models, the best fitting model were selected as the inhibition mode of the inhibitors.
Lineweaver-Burk plots were generated from the reciprocals of both substrate concentration
and activity at each inhibitor concentration to present the mode of inhibition.
Acknowledgments
We thank Prof Gary Taylor (St Andrews) for the generous gift of plasmid for SpNA
expression. RR was supported by a University Research Scholarship from the University of
Bath.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://doi.org/xxxxxxx
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Highlights
•
•
•
Aryl α-aminophosphonates are effective inhibitors of T. cruzi trans-sialidase
Inhibition of trans-sialidase is improved using substituted aryl α-aminophosphonates
Aryl α-aminophosphonates inhibit trans-sialidase in a non-competitive manner