Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates

Article abstract of DOI:10.1016/j.bmcl.2009.12.007

Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (k_i) ranging from 0.3 × 10⁵ M^-1 min^-1 to 10.4 × 10⁵ M^-1 min^-1. When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE's (? 1 × 10⁷ M^-1 min^-1) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling.

Full text of DOI:10.1016/j.bmcl.2009.12.007

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1194–1197
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates
Lilu Guo a,d, Alirica I. Suarez c, Michael R. Braden b, John M. Gerdes b, Charles M. Thompson b,d,
*
a Department of Chemistry, The University of Montana, Missoula, MT 59812, United States
b Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, United States
c Facultad de Farmacia, Universidad Central de Venezuela, Caracas VZ, United States
d ATERIS Technologies, 901 N Orange Street, Missoula MT 59802, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group
and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholines-
terase. The inhibitors showed bimolecular inhibition constants (ki) ranging from 0.3 ꢀ 105 Mꢁ1 minꢁ1 to
10.4 ꢀ 105 Mꢁ1 minꢁ1. When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the
corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chro-
mophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMo-
AChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups,
showed excellent inhibitory potency against both AChE’s (ffi 1 ꢀ 107 Mꢁ1 minꢁ1) indicating, in general,
that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dab-
syl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic
analysis, and visualized using molecular modeling.
Received 24 August 2009
Revised 28 November 2009
Accepted 2 December 2009
Available online 4 December 2009
Keywords:
Fluorophosphonate
Acetylcholinesterase
Chromophore
Inhibition
Modeling
Ó 2009 Elsevier Ltd. All rights reserved.
Acetylcholinesterase (AChE), the enzyme responsible for hydro-
lysis of the neurotransmitter acetylcholine (ACh) contains three
key motifs: an active-site (A-site), a peripheral anionic site (P-site),
and a long narrow hydrophobic gorge connecting the A- and P-
sites.1 The A-site contains a catalytically reactive serine located
20 Å from the protein surface.2–4 The P-site is located at the protein
perimeter and transiently attracts cation substrates prior to trans-
location to the A-site.5 The gorge is lined with hydrophobic resi-
dues to facilitate passage of substrate and other molecules. The
A-site is the principal target for non-covalent inhibitors such as ta-
crine and acridine, and covalent modifiers such as organophos-
phates, carbamates and sulfonyl halides.6–11
The primary objective of this study was to select and evaluate
AChE inhibitors (Fig. 1) that anchor chromophores (C) in the gorge
but near to the protein surface. Such structures could serve as re-
porter groups for protein structure changes without interrupting
the favorable ACh to P-site interaction or as visualization tools in
proteomics. To seat and locate the proper chromophore probe, a
reactive phosphonate group bearing a fluorine as leaving group
(LG) was used to attach the molecule to the active site serine
(Fig. 1). To more precisely place chromophore groups in the gorge
just below the P-site entrance, a panel of chromophore-linked OPs
that vary in the chromophore structure were designed with an
interatomic distance of slightly less than 20 Å. Owing to slight
differences in the chromophore attachment and size, structures
were prepared with three- and four-carbon bond linker groups
(one methylene spacer variance) to maintain net interatomic
distances less than or equal to 20 Å. Chromophores were linked
via a triazole to reactive OP groups using click chemistry12
(Fig. 1).13–15
Variation in the chromophore group is important because dif-
ferences in size and chemical properties can play a role in protein
binding. For example, the propargyl amide of Lissamine has an IC50
of 50 nM with recombinant mouse AChE whereas other chromo-
phore amides are poor inhibitors.16 In this study, chromophore-
linked reactive phosphonates were synthesized, inhibition of two
AChE’s determined, the contribution of the chromophore assessed,
and an optimized inhibitor used to covalently modify AChE.
Thefluorophosphonate chromophore (FP-C)structureswere pre-
pared as shown (Scheme 1). Seven amine-reactive chromophores
(sulfonyl-Cl or activated carboxylic ester/NHS) were reacted with
propargyl amine to afford the propargyl amides 1a–g.17 O,O-Di-
methyl-(3-bromo propyl)phosphonate 318 and O,O-dimethyl-(4-
O
O
N
N
P
+
C
N3
RO
LG
P
N
RO
LG
C
C
= chromophore
LG = leaving group
* Corresponding author.
E-mail address: charles.thompson@umontana.edu (C.M. Thompson).
Figure 1. Proposed design for chromophore-linked OPs using click reaction.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.007

L. Guo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1194–1197
1195
Table 1
Inhibition rates constants (ki) for 6, 7, 10a–g and 11a–g against rMAChE and EEAChE
H2
C
H2
C
i
+
Cl
H2N
N
H
C
C
kia (Mꢁ1 minꢁ1
)
kia (Mꢁ1 minꢁ1
)
90%
Compound
1a-1g
rMAChE
EEAChE
6
7
1.3 0.1 ꢀ 107
1.0 0.1 ꢀ 106
1.0 0.1 ꢀ 106
1.9 0.5 ꢀ 105
7.5 1.0 ꢀ 105
6.5 1.1 ꢀ 104
4.0 0.6 ꢀ 105
ND
7.7 0.1 ꢀ 106
O
OCH3
O
OCH3
O
F
OCH3
0.96 0.08 ꢀ 106
1.3 0.1 ꢀ 105
6.2 0.1 ꢀ 104
3.0 0.3 ꢀ 105
1.1 0.1 ꢀ 105
1.8 0.3 ꢀ 105
ND
iii, iv
ii
72% H3CO
P
P
P
10a
11a
10b
11b
10c
11c
10d
11d
10e
11e
10f
H3CO
(CH2)nBr
(CH2)nN3 45%
(CH2)nN3
2: n = 3
3: n = 4
4: n = 3
5: n = 4
6: n=3
7: n=4
O
P
N
N
2.1 0.7 ꢀ 105
1.4 0.2 ꢀ 105
6.5 0.6 ꢀ 105
3.5 0.6 ꢀ 104
ꢂ1 ꢀ 104
2.5 0.4 ꢀ 105
3.3 0.8 ꢀ 104
1.8 0.1 ꢀ 104
2.7 1.0 ꢀ 104
ꢂ1 ꢀ 104
H
N
C
1a-1g
+
4 or 5
v
N
H3CO
(CH2)n
80%
X
8a-8g: X = CH3O; n = 3
9a-9g: X = CH3O; n = 4
11a-11g: X = F; n = 4
ꢂ1 ꢀ 104
ꢂ1 ꢀ 104
iii, iv
50%
iii, iv 50%
11f
ꢂ1 ꢀ 104
ꢂ1 ꢀ 104
10a-10g: X = F; n = 3
10g
11g
Paraoxon
ꢂ1 ꢀ 104
ꢂ1 ꢀ 104
4.0 ꢀ 105
3.0 ꢀ 105
C
= chromophore in compound 1, 8, 9, 10 and 11
ND = not determined, poor solubility.
a
All ki values (means SEM) were determined from P3 different experiments.
O
S O
O
S O
O
N
S
N
NMe2
O
mined in a concentration-dependent manner21 using 1/[i] = (
ln
and 11f) and lissamine-based FPs (10g/11g). Five inhibitor concen-
trations causing a range of 20%–90% inhibition were used along
with a control to determine ki and the resulting linear plot (1/
Dt/
NMe2
D
c
)kiꢁ(1/KD) for all compounds except the Texas Red-FPs (10f
a: C = dansyl
b: C = dabsyl
c: C = pyrene
NEt2
C
O
C
O
OMe
O
[i] ꢂ
Dt/D
lnv) was used in cases when R2 > 0.98 (n P 3). For the
Texas Red-linked FPs (10f and 11f) and lissamine-based FPs
(10g/11g) less than 50% inhibition was obtained. As a result of this
poor inhibition, the resultant plots afforded R2 P 0.80 and other
estimated ki(s) 6 104 Mꢁ1 minꢁ1 were obtained.
O
O
O
d: C = 7-diethylamino
coumarin
e: C = 7-methoxy
coumarin
-O3S
-O3S
O
Paraoxon (EtO)2P(O)OPhNO2 was used as a reference inhibitor
O
with
a
ki of 4.0 ꢀ 105 Mꢁ1 minꢁ1 for rMAChE22,23 and 3.0 ꢀ
S
S
105 Mꢁ1 minꢁ1 for EEAChE,24 which are in excellent agreement
with literature values.
O
O
The ki values for the control compounds 6 and 7 were approxi-
mately 106–107 Mꢁ1 minꢁ1 or 10–100-fold stronger than paraoxon.
The propyl analog 6 was 10-fold more potent inhibitor than 7 to-
ward both AChE’s.
N+
O
N
O
NEt2
+Et2N
g: C = lissamine
f: C = Tex as Red
The twelve chromophore-linked FPs (10a–g and 11a–g) were
all found to be good inhibitors of both rMAChE and EEAChE and
comparable to paraoxon except 11c. The dansyl- and dabsyl-linked
FPs, 10a and 10b, were the most potent inhibitors in the series and
slightly better inhibitors than paraoxon. The chromophore-linked
FPs were all weaker inhibitors (smaller ki) than the control com-
pounds 6/7 indicating that the anchored chromophores reduced
inhibition. For the dansyl-, dabsyl, methoxy- and diethylamino
coumarin linked FPs, the propyl-linked analogs ( 10a, 10b, 10d,
and 10e) were more potent than the corresponding butyl-linked
( 11a, 11b, 11d, and 11e). The ki values for 10/11a, 10/11b, 10/
11d, and 10/11e suggest that the smaller-sized chromophores
likely access the protein and modify the active site with either 3-
or 4-carbon linker length. However, the relatively poor ki values
for FP-Cs 10/11f indicates that the sterically demanding chromoph-
ores may exceed the P-site portal and hinder entry such that the
reactive P–F moiety cannot reach/covalently modify the A-site.
As such, the kinetic data does not represent an irreversible inhibi-
tor. Recently, we reported that lissamine propargyl amide 1g is a
reversible inhibitor of AChE and acting by an independent
mechanism.16
Scheme 1. Synthesis of a propyl- and butyl-tethered, chromophore-linked fluor-
ophosphonates. Reagents and conditions: (i) TEA, CH2Cl2; (ii) NaN3, CH3CN/H2O;
(iii) NaOH, 50–60 °C; H+; (iv) C3N3F3, CH2Cl2; (v) CuSO4, sodium ascorbate, t-BuOH/
H2O. All compounds were fully characterized.
bromo butyl)phosphonate 4 were converted to the azo-linked OPs 4
and 5. Chromophore-linked phosphonates 8a–g/9a–g were pre-
pared in excellent yield from alkynes (1a–g) and azides (4/5) using
Cu(I) catalysis.12 Phosphonates 8a–g/9a–g were converted to the
reactive fluorophosphonate products (10a–g/11a–g; FPCs) via
formation of the phosphorus monoacid followed by reaction with
cyanuric fluoride (C3N3F3).19,20 Conversion to the P–F bond was
monitored by 31P NMR. The phosphorus diesters P(OCH3)2,
(ꢂ34 ppm) shift 3 ppm upfield as the monoacid (ꢂ31 ppm). Follow-
ing conversion with cyanuric fluoride, the product shows character-
istic P–F coupling constant (JP–F = 1072 Hz).
The syntheses of control compounds, azopropyl- and azobutyl-
linked FPs 6 and 7, were also conducted to assess the anti-AChE
activity of the reactive phosphonate without a chromophore. The
same hydrolysis and fluorination reactions were applied to 4 and
5 to afford FPs 6 and 7.
To demonstrate that select chromophore-linked FPs became
covalently attached to the enzyme and result in a chromophore-
labeled enzyme, dabsyl-linked FP 10b was reacted with rMAChE
until complete inhibition. Excess inhibitor was removed from the
Chromophore-linked FPs 6,7, 10a–g, and 11a–g were evaluated
as irreversible inhibitors (Table 1). Rate constants (ki) were deter-

1196
L. Guo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1194–1197
Table 2
phosphonylated enzyme using a Spin-OUT Micro Column and the
protein fractions were analyzed by UV–vis. The dabsylated rMAChE
solution showed two absorbance maxima at 280 nm and 475 nm
accounting for the aromatic side chains rMAChE and the dabsyl
moiety, respectively. The molar extinction coefficient for the en-
zyme–FP-dabsyl complex was 3.1 ꢀ 104 Mꢁ1 cmꢁ1 in 0.1 M PBS
7.6 at 475 nm, which was determined from the absorption spec-
trum of the dabsyl-linked FP under the same conditions. The molar
extinction coefficient for the enzyme was experimentally derived
and calculated by protparam in ExPASy25 as 9.8 ꢀ 104 Mꢁ1 cmꢁ1
and 10.0 ꢀ 104 Mꢁ1 cmꢁ1, respectively. The amount of enzyme-
bound inhibitor was calculated from the absorption at 475 nm
and corrected for the absorption caused by inhibitor and then
determined from the absorption at 280 nm (Fig. 2). An estimate
of enzyme concentration from its absorption spectrum results in
a dabsyl-FP to enzyme stoichiometry of 1.45–1.55 ( 0.2). Analysis
of the FP-dabsyl enzyme complex by MALDI did not reveal modifi-
cation of sites in addition to the active site serine.
GOLDSCORE fitness scores of the top-ranked individuals for virtual docking simula-
tions of dansyl-10a, dabsyl-10b, and Texas Red-linked FP 10f to rMAChE
Compound
Chromophore
Fitness
10a
10b
10f
10f
Dansyl
Dabsyl
Texas red, ortho
Texas red, para
80.14
77.08
8.47
ꢁ9.24
To visualize the position and possible interactions between FP-C
ligands and AChE, molecular modeling was conducted. Dansyl-10a,
dabsyl-10b, and the ortho- and para-Texas Red-linked FP 10f were
virtually docked into rMAChE using the software GOLD26 and stan-
dard settings. A single monomer of rMAChE extracted from the
crystal structure (1maa)2 was employed. The top-ranked orienta-
tions of dansyl-FP 10a and dabsyl-FP 10b scored significantly high-
er in the GOLDSCORE best ranking list compared to either Texas
Red-linked FP 10f (Table 2).
Qualitatively, the top-ranked pose for the dabsyl-linked FP 10b
placed the reactive phosphoryl group of this ligand in a better dis-
tance and orientation to Ser203 than the dansyl-linked FP 10a. The
top-ranked pose for dabsyl-linked FP 10b was merged with the
structure of rMAChE, a covalent bond was created between the
phosphate group and Ser203, and the energy of the merged struc-
ture was minimized using AMBERFF07 forcefield and Tripos Sybyl
(St. Louis, MO). The energy minimized merge structure was then
subjected to a 10,000 ꢀ 1 fs step molecular dynamics simulation
using AMBERFF07 at 300 K. After 1000 steps, the structure with
the lowest potential energy was again energy minimized as above.
The orientation of dabsyl-linked FP 10b in rMAChE places the chro-
mophore near aromatic residues (e.g., Trp286; Fig. 3) lining the
upper gorge region near the P-site.
Figure 3. Visualizations of AChE (turquoise) inhibited by dabsyl-FP (magenta)
showing phosphonylated enzyme bound at Ser203. The chromophore is
positioned near P-site residues (e.g., Trp286). Only residues that are 4 Å from
dabsyl-FP and polar hydrogens shown. Views A and B are rotated 90 degrees about
the vertical axis.
a
>90% of its original activity and the inhibition mixture subse-
quently diluted 50-fold with buffer to halt further inhibition. The
enzyme activity was monitored over 2 h, however, no spontaneous
reactivation was observed. To assess oxime-induced reactivation,
2-pyridine aldoxime methiodide (2-PAM) was added after the dilu-
tion step and showed that rMAChE recovered 30–40% of the origi-
nal activity in 2 h. When 2-PAM is applied at >1 mM and incubated
with inhibited enzyme for 0.5 h, the amount of reactivation was
proportional to the reactivator concentration. Thus, rMAChE
undergoes covalent modification by 10b and moreover, that the
reaction occurs at the active site serine.
Spontaneous and oxime-induced reactivation27–29 of rMAChE
inhibited by 10b was conducted to show that the serine hydroxyl
group was covalently modified. Only inhibition at the serine
hydroxyl would afford a functional enzyme upon reactivation. To
assess spontaneous reactivation, rMAChE was inhibited by 10b to
In sum, a series of chromophore-linked FP compounds were
prepared as potent inhibitors against AChE showing that rMAChE
is more reactive than EEAChE. The anti-AChE kinetics revealed that
the chromophore, linker and the molecule length collectively affect
the ligand binding to the protein. The irreversible inhibition of
rMAChE was confirmed by the spectroscopic analysis and the inac-
tivated enzyme can be recovered to 30–40% by 2-PAM. Molecular
modeling was used to illustrate that small chromophore-linked
FPs fit the active gorge of AChE and the chromophore is positioned
near the gorge entrance at the P-site, which can be used to explore
the protein structural features and functionalities. FPs containing
large chromophores such as Texas Red and lissamine, showed dif-
ferent kinetics than the other FPs, which is likely caused by their
larger size or charge attraction to the P-site.
Acknowledgements
This research was supported by NIH UO1-ES016102 (CMT), NIH
R43 ES016392 (ATERIS) and NIH U44 NS058229 (ATERIS). Support
from the Core Laboratory for Neuromolecular Production (NIH
Figure 2. UV–vis of rMAChE inhibited by dabsyl-FP 10b.

L. Guo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1194–1197
1197
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P30-NS055022), Center for Structural and Functional Neuroscience
(NIH P20-RR015583) and Molecular Computational Core Facility is
appreciated.
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