Novel selective and irreversible mosquito acetylcholinesterase inhibitors for controlling malaria and other mosquito-borne diseases.

We reported previously that insect acetylcholinesterases (AChEs) could be selectively and irreversibly inhibited by methanethiosulfonates presumably through conjugation to an insect-specific cysteine in these enzymes. However, no direct proof for the conjugation has been published to date, and doubts remain about whether such cysteine-targeting inhibitors have desirable kinetic properties for insecticide use. Here we report mass spectrometric proof of the conjugation and new chemicals that irreversibly inhibited African malaria mosquito AChE with bimolecular inhibition rate constants (k(inact)/K(I)) of 3,604-458,597 M(-1)sec(-1) but spared human AChE. In comparison, the insecticide paraoxon irreversibly inhibited mosquito and human AChEs with k(inact)/K(I) values of 1,915 and 1,507 M(-1)sec(-1), respectively, under the same assay conditions. These results further support our hypothesis that the insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides with reduced insecticide resistance and low toxicity to mammals, fish, and birds for the control of mosquito-borne diseases.

Malaria is a n class="Species">human disease caused by a protozoan parasite transmitted by mosquitoes1 and has been a grave concern in human populations for ≥50,000 years23. According to the World Malaria Report 20114, approximately 3.3 billion people are at risk for contracting malaria, and an estimated 216 million cases led to nearly 655,000 deaths in 2010. Insecticides are a proven approach to controlling the disease. However, current insecticides are hampered by their toxicity to humans5 and insecticide resistance6. There is an urgent need for novel insecticides to control the mosquitoes that transmit malaria and other mosquito-borne diseases such as West Nile virus infection7 with reduced insecticide resistance and low toxicity to other species.

AChE is a hydrolase vital to the regulation of the neurotransmitter n class="Chemical">acetylcholine in mammals, fish, birds, and insects8910. Its catalytic serine hydrolyzes acetylcholine at the bottom of its active site (Fig. 1). Current anticholinesterase insecticides work through covalent modification of this serine, thus disabling its catalytic function and incapacitating insects. However, because this serine is also ubiquitous in the AChEs of mammals and other species (Fig. 1), anticholinesterase insecticides are toxic to mammals, fish, and birds.

Figure 1

New chemicals that target an insect-specific cysteine of AP-AChEs.

Upper left panel: cross-section of the AP-AChE and AChE active sites showing the locations of the insect-specific cysteine and the corresponding residue in non-insect species; upper right panel: chemical structures of PMn, PYn, PYS18, and PMS20; middle panel: two-step quiescent affinity labeling mechanism for PMn and PYn and definition of kinetic parameters; lower panel: syntheses of PMn, PYn, PYS18, and PMS20. DCM: CH2Cl2; DMF: N,N′-dimethylformamide; NBS: N-bromosuccinimide; TFA: CF3CO2; THF: tetrahydrofuran; : reflux.

Interestingly, disease-transmitting mosquitoes such as the African malaria mosquito (n class="Species">Anopheles gambiae sensu stricto) and the common house mosquito (Culex pipiens L.) have two AChEs—Ace-paralogous AChE (AP-AChE) and Ace-orthologous AChE—both of which have an insect-specific cysteine residue near its active site11. Responsible for cholinergic functions121314, AP-AChE has an insect-specific cysteine located at the rim of its active site11. This cysteine has mutated to phenylalanine in the corresponding enzymes of mammals, fish, and birds11 (Fig. 1). Because the AChEs of non-insect species lack this cysteine, cysteine-targeting AP-AChE inhibitors would have reduced off-species toxicity. Furthermore, these inhibitors have never been used as insecticides and therefore would have a lower propensity for inducing insecticide resistance relative to current anticholinesterase insecticides11.

Following this reasoning, we developed n class="Chemical">methanethiosulfonate derivatives that selectively and irreversibly inhibit insect AP-AChEs presumably through conjugation to the insect-specific cysteine1215. However, no direct proof of the conjugation of a sulfhydryl agent to the insect-specific cysteine in AP-AChE has been presented to date, and doubts have remained about whether the insect-specific cysteine is accessible for conjugation and whether cysteine-targeting AP-AChE inhibitors can be developed with kinetic properties comparable to those of insecticides. Here we report our redesigned inhibitors of An. gambiae sensu stricto AP-AChE (agAP-AChE) and direct proof for the conjugation of the new inhibitor to Cys286, the insect-specific cysteine in agAP-AChE. We also report kinetic data showing that the new inhibitors are superior to the insecticide paraoxon and discuss the feasibility of targeting the insect-specific cysteine to develop effective and environmentally safe insecticides.

Results

Design of agAP-AChE inhibitors

Encouraged by reports that small-molecule–conjugated fragments of cholinesterases can n class="Chemical">be detected using liquid chromatography mass spectrometry16171819202122, we wanted to perform a mass spectrometric study of recombinant agAP-AChE23 that was treated with our cysteine-targeting inhibitor before protein digestion to obtain direct proof of the conjugation of the sulfhydryl agent to Cys286. Our previously reported methanethiosulfonates form a disulfide bond with the insect-specific cysteine1215, and the methanethiosulfonate adducts are unstable in the presence of a disulfide-bond–cleavage agent during the digestion process. For this reason, we set out to develop maleimide-containing inhibitors that form a carbon-sulfur bond to Cys286, thereby their adducts are stable during the digestion process. We also sought to compare the kinetic properties of the new inhibitors with those of anticholinesterase insecticides.

As revealed by an n class="Chemical">agAP-AChE model refined using multiple molecular dynamics simulations (Protein Data Bank ID: 2AZG)24, Cys286 is stabilized by aromatic residues via sulfur-aromatic interactions25. To react with Cys286, the cysteine-targeting inhibitor must have adequate affinity for the active site to accumulate a local concentration around Cys286 high enough to offset the sulfur-aromatic interaction. The inhibitor should also have adequate flexibility to satisfy the directional requirement for covalent bond formation.

Accordingly, we designed PMn and PYn (Fig. 1) as prototypic cysteine-targeting n class="Chemical">agAP-AChE inhibitors that were expected to follow the two-step quiescent affinity labeling mechanism26 as depicted in Scheme 1 (Fig. 1). Specifically, these compounds were designed to react with Cys286 only after they reversibly bind in the vicinity of Cys286 with adequate affinity to impart target enzyme selectivity. The PMn series was inspired by a report that methylpyridinium binds well at the AChE active site27. The PYn series was designed purposely to have reduced affinity for the active site to investigate the effect of the inhibitor affinity on the inhibitor reactivity toward Cys286. The use of long alkylene chains in the prototypes was based on the chain-length–activity relationship of our reported irreversible AP-AChE inhibitors1215 and supported by 100 10-ns-long molecular dynamics simulations (each with unique initial velocities and a 1.0-fs time step) of agAP-AChE in reversible complex with PM20 using an explicit water model282930. These simulations predicted that PM20 was capable of spanning the active site of agAP-AChE with its pyridinium group forming cation-pi interactions with Trp84, Tyr121, Tyr130, and Tyr328 and with its maleimide alkene carbon atom located 3.6 Å away from the sulfur atom of Cys286 (Fig. 2). To estimate the binding affinity of PYn and PMn, we also designed PYS18 and PMS20 whose maleimide is replaced with succinimide that cannot react with cysteine but is sterically almost identical to maleimide (Fig. 1). Notably, we made and tested PMn and PYn with n ranging from 10 to 22, but we report herein the representatives with n in the range of 16–20.

Figure 2

Close-up view of agAP-AChE in reversible complex with PM20 predicted by microsecond molecular dynamics simulations.

The nitrogen, oxygen, and sulfur atoms are in blue, red, and green, respectively. The carbon atoms in agAP-AChE and PM20 are in tangerine and yellow, respectively. The mesh depicts the portion of PM20 that is inserted in the active site of agAP-AChE. The simulation protocol is provided in the Supplementary Information.

Synthesis of agAP-AChE inhibitors

PMn and PYn were readily prepared in excellent yields following Scheme 2 (Fig. 1). The protected maleimide31 was attn class="Gene">ached to an ω-substituted alkylene chain through reaction with Br(CH2)nOH. The hydroxyl group of the resulting intermediate was then substituted with bromide and subsequently converted to pyridinium or piperidine. Deprotection of the corresponding intermediate yielded PMn or PYn. Reduction of PY18 and PM20 gave PYS18 and PMS20, respectively (Fig. 1).

Selective and irreversible inhibition of agAP-AChE

To determine whether PMn and PYn irreversibly inhin class="Chemical">bit recombinant agAP-AChE23, we performed time-course experiments of AChE inhibition and found that PMn and PYn irreversibly inhibited recombinant agAP-AChE but not recombinant human AChE (hAChE). As shown in Figure 3, the irreversible inhibition is indicated by progressive inhibition over time, whereas reversible inhibition is evident from constant inhibition over time. As expected, the control inhibitor paraoxon irreversibly inhibited both enzymes, whereas PMS20 and PYS18 irreversibly inhibited neither (Fig. 3). The irreversible inhibition was then confirmed with dilution experiments32 (Fig. 3), which determine the inhibitor dissociation from the enzyme upon dilution of the complex through measuring the reduction of the enzyme inhibition upon the dilution. If the complexation is irreversible, no complex dissociation occurs upon the dilution, and hence the percentage of enzyme inhibition remains constant after the dilution. Figure 3 demonstrates clearly that PY18 and PM20 irreversibly inhibited agAP-AChE but not hAChE.

Figure 3

Time-course and dilution experiments for the inhibition of agAP-AChE and hAChE by PY18, PM20, PMS20, paraoxon, and NEM.

% enzyme activity: the enzyme activity compared to that without inhibitor treatment. Before the 10-fold dilution, the concentrations of PY18, PM20, PMS20, paraoxon, and NEM were 0.1, 0.001, 1.67, 0.2, and 100 μM for the agAP-AChE inhibition assays and 6.67, 0.5, 0.833, 0.2, and 100 μM for the hAChE inhibition assays, respectively.

Proof of the Cys286 conjugation in agAP-AChE

To prove that the observed selective and irreversin class="Chemical">ble inhibition of agAP-AChE by PM20 is due to the conjugation of the inhibitor to Cys286, we performed nano-flow liquid-chromatography electrospray ionization tandem mass spectrometry (nanoLC–ESI-MS/MS) analysis33 on recombinant agAP-AChE23 that was treated with PM20 followed by protein digestion. We identified a doubly-charged ion from a high-resolution Orbitrap survey scan with the monoisotopic mass of 1082.6122 (Fig. 4). The mass of this ion was within 0.2 parts per million (ppm) of the calculated mass over charge ratios (m/z) of 1082.6120 (82.6%) and 1083.1137 (100.0%) for [GICEFPFVPVVDGAFL+H]+2, a fragment of agAP-AChE. C is Cys286 conjugated with PM20 via Michael addition34, and two formal charges reside on the PM20 pyridinium ring and the N-terminus.

Figure 4

Mass spectrometric proof of the conjugation of PM20 to Cys286 in agAP-AChE.

Upper panel: high-resolution Fourier transform mass spectrometry survey scan of the [M+H]+2 ion (m/z of 1082.6122) corresponding to the PM20-labeled fragment of agAP-AChE with calculated (cal.) m/z of 1082.6120. Middle panel: supporting tandem mass spectrum showing the observed b- and y-type ions resulting from fragmentations of the [M+H]2+ ion. Lower panel: mechanisms for the oxazolone formation and b-ion cyclization.

The conjugated peptide identification was then confirmed with the tandem mass spectrometry showing the b- and y-type ions35 resulting from collision-induced fragmentations of [GICEFPFVPVVDGAFL+H]+2 in the linear ion trap component of the spectrometer. It has n class="Chemical">been reported that the bn ions, which are generated by collision and display cleavage at the peptide bond of the nth residue counting from the N-terminus, exist in a linear peptidic form with a C-terminal oxazolone363738394041, in a cyclic peptidic form resulting from the reaction between the N-terminus and the oxazolone group41424344454647 (Fig. 4), or as a mixture of the two40. Smaller b ions (n = 2 or 3) reportedly adopt the linear form exclusively, whereas b8 ion adopts exclusively the cyclic form40. The masses of both linear and cyclic forms are one hydrogen atom less than that of the corresponding linear peptide without the oxazolone group. In this study, we were interested in the masses of the bn ions rather than their structures and therefore calculated the bn (n > 3) ion masses using the cyclic form. The calculated b-ion m/z values of cyclo(GICE)+, cyclo(GICEF)+, cyclo(GICEFP)+, cyclo(GICEFPF)+, cyclo(GICEFPFV)+, cyclo(GICEFPFVPV)+, cyclo(GICEFPFVPVV)+, and cyclo(GICEFPFVPVVD)+ were 857.52, 1004.59, 1101.64, 1248.71, 1347.78, 1543.90, and 1643.97, respectively. These values corresponded well with the observed b ions (Fig. 4). For the yn ions that have breakage at the peptide bond of the nth residue counting from the C-terminus, the calculated y ion m/z values of [P+H]+ and [P+H]+ were 817.45 and 1160.64, respectively, which were in excellent agreement with the observed y ions (Fig. 4). The conjugated peptide identification was also confirmed with the tandem mass spectra showing the same b- and y-type ions resulting from collision-induced fragmentations of [GICEFPFVPVVDGAFL+H]+2 and the PM20-conjuated synthetic peptide of the same sequence (data not shown).

Of eight cysteine residues in our recomn class="Chemical">binant agAP-AChE23, the nanoLC–ESI-MS/MS analysis showed that only Cys286 was conjugated with PM20 and that others were identified as carboxamidomethyl cysteine due to reduction of the three intramolecular and one intermolecular disulfide bonds of the recombinant enzyme and subsequent alkylation of the resulting thiols during the enzyme digestion process. It is known that only Cys, Lys, and His react with maleimides4849. The agAP-AChE model24 shows that the active-site and peripheral-site regions contain only one His residue (His439) and no Lys residue. The nanoLC–ESI-MS/MS analysis did not identify any His439-containing fragment carrying the PM20 adduct. These results provide direct proof for the conjugation of PM20 to the insect-specific cysteine in agAP-AChE.

Kinetic studies of AP-AChE inhibitors

After confirming the mechanism for the selective and irreversible inhin class="Chemical">bition of agAP-AChE by PMn, we determined bimolecular inhibition rate constants (inact/KI or ki) and pseudo-unimolecular inhibition rate constants (inact or k2) of our irreversible inhibitors and the control inhibitor paraoxon (Fig. 5 and Table 1), using nonlinear regression fitting analysis50 according to Equations 1 and 2 (Fig. 1). We found that the bimolecular inhibition rate constants (inact/KI or ki) for inhibiting agAP-AChE and hAChE by paraoxon were 1,915 and 1,507 M−1sec−1, respectively, at high paraoxon concentrations of 333–2000 nM with incubation times of 15 minutes or less (Fig. 5 and Table 1). These values changed to 9,862 and 10,013 M−1sec−1, respectively, at low paraoxon concentrations of 17–100 nM with incubation times of 75 minutes or less. These results are consistent with reports that the kinetics of AChE inhibition by paraoxon is concentration dependent515253. Our bimolecular inhibition rate constants measured at low paraoxon concentrations are comparable to those reported for agAP-AChE23 and mammalian AChEs5152535455.

Figure 5

Progressive AChE inhibition as a function of time and inhibitor concentration indicating the two-step quiescent affinity labeling mechanism for the test inhibitors.

% enzyme activity: the enzyme activity compared to that without inhibitor treatment. Left: linear plots of natural log of % AChE activity versus time in second at different inhibitor concentrations; right: nonlinear plots of the observed inhibition rate in 1/second versus inhibitor concentration in nanomolar.

Table 1

Irreversible and Reversible Inhibition of agAP-AChE and hAChE

Irreversible Inhibition
Inhibitor	Incubation (min)	kinact (hr−1)a	KI (μM)b	kinact/KI (M−1s−1)c
	agAP-AChE
PY16 (100–600 nM)	≤15	15.4 ± 1.5	1.19 ± 0.13	3604 ± 45
PY18 (100–600 nM)	≤15	16.7 ± 0.4	0.58 ± 0.04	8059 ± 743
PM18 (4–26 nM)	≤15	10.4 ± 0.9	0.013 ± 0.001	230155 ± 26492
PM20 (3–18 nM)	≤15	56.3 ± 22.4	0.034 ± 0.017	458597 ± 111134
Paraoxon (333–2000 nM)	≤15	25.6 ± 5.6	3.71 ± 1.24	1915 ± 336
Paraoxon (17–100 nM)	≤75	10.3 ± 0.4	0.289 ± 0.009	9862 ± 96
	hAChE
Paraoxon (333–2000 nM)	≤15	32.7 ± 2.7	6.03 ± 0.49	1507 ± 2
Paraoxon (17–100 nM)	≤75	5.8 ± 1.2	0.161 ± 0.048	10,013 ± 721

akinact: pseudo-unimolecular inhibition rate constant; bKI: the inhibitor concentration that yields a rate of inactivation that is equal to a half of kinact; ckinact/KI: bimolecular inhibition rate constant; dKi: equilibrium dissociation constant.

Because we were interested in knowing how quickly our compounds inhin class="Chemical">bited agAP-AChE and the inhibitory potency of the redesigned inhibitors relative to that of paraoxon, we measured the kinetic data of PMn and PYn over time courses of ≤15 minutes. In this context, we found that PMn and PYn irreversibly inhibited agAP-AChE with pseudo-unimolecular inhibition rate constants of 10–56 hr−1 and bimolecular inhibition rate constants of 3,013–458,597 M−1sec−1 (Table 1 and Fig. 5). We also found that PMn and PYn reversibly inhibited hAChE with equilibrium dissociation constants (Ki) of 0.12–20.37 μM (Table 1 and Fig. 6) and that PYS18 and PMS20 reversibly inhibited insect and human enzymes with Ki values of 26.18 and 0.42 μM, respectively (Table 1 and Fig. 6).

Figure 6

Determination of equilibrium dissociation constants (Ki).

Left: reciprocal hydrolysis rate (1/v in second per optical density) was plotted against reciprocal substrate concentration (1/[ATCh] in 1/mM) in the absence and presence of an inhibitor at varying concentrations; right: the slope of the double reciprocal plot was plotted against inhibitor concentration ([I] in μM). Ki was obtained from the negative x intercept of the slope replot.

In addition, as shown in Figure 5, the observed inhin class="Chemical">bition rate constants (obs) of PMn and PYn were nonlinear functions of inhibitor concentration ([I]), indicating that the designed inhibitors indeed followed the two-step quiescent affinity labeling mechanism rather than the one-step nonspecific affinity labeling mechanism characterized by a linear relationship between obs and [I]26.

Effect of inhibitor affinity on inhibitor reactivity

To evaluate the prospect of using cysteine-targeting inhin class="Chemical">bitors as insecticides with consideration to their potential reactions with off-target cysteines, we studied the effect of inhibitor affinity on inhibitor reactivity toward the cysteine. As shown in Figure 7, treating agAP-AChE with 50 μM N-ethylmaleimide (NEM) immediately resulted in 36 ± 2% inhibition of the enzyme activity, and extending the treatment for 30 minutes slightly increased the inhibition to 40 ± 1%, whereas treating agAP-AChE with a combination of 50 μM NEM and 6.7 nM PM20 for 30 minutes decreased the enzyme activity by 94 ± 3%. In addition, Figure 3 shows that, when agAP-AChE was incubated with 100 μM NEM for 30 minutes, 45 ± 2% of the enzyme activity was inhibited; when the incubation solution was diluted by 10-fold, the enzyme inhibition was reduced to 14 ± 4%. These results demonstrate the inability of NEM to irreversibly inhibit agAP-AChE. However, the inability of NEM to irreversibly inhibit agAP-AChE does not imply that NEM cannot react with Cys286, because NEM lacks a long chain that can physically block the active site. Nevertheless, when agAP-AChE was incubated with 50 μM NEM, only 40 ± 1% of the enzyme activity was inhibited; when the incubation solution was then treated with 6.7 nM PM20 for 30 minutes, 96 ± 4% of the enzyme activity was inhibited (Fig. 7). These results indicate that 50 μM NEM cannot react with Cys286. Otherwise, Cys286 would be blocked by NEM and the subsequent 30-minute treatment with 6.7 nM PM20 would not lead to the 96 ± 4% enzyme inhibition (Fig. 7). We also found that 1-ethylpyrrolidine-2,5-dione, a close analog of NEM, did not irreversibly or reversibly inhibit agAP-AChE and hAChE at inhibitor concentrations up to 10 mM (data not shown), indicating that NEM has much lower affinity for agAP-AChE than PMn or PYn. These studies reveal the correlation between the low affinity of NEM for agAP-AChE and its inability to react with Cys286. More importantly, these studies demonstrate that the markedly increased reactivity of the maleimide in PM20 toward Cys286 is due to the affinity gained from attaching maleimide to N-alkylpyridinium.

Figure 7

Effects of 50 μM NEM and 6.7 nM PM20 on agAP-AChE and hAChE activities.

% enzyme activity: the enzyme activity compared to that without inhibitor treatment.

Discussion

The present studies show that PMn and PYn can selectively and irreversibly inhin class="Chemical">bit agAP-AChE. The mechanism for the irreversible inhibition is now known due to the conjugation of the inhibitor to Cys286, the insect-specific cysteine in agAP-AChE, according to the nanoLC–ESI-MS/MS analysis. The mass spectroscopic data are consistent with our previous reports1215 that the irreversible inhibition of insect AChEs by a methanethiosulfonate could be partially restored by β-mercaptoethanol that cleaves in theory the disulfide bonds in the enzymes (leading to enzyme inhibition) and the disulfide bond of the methanethiosulfonate to Cys286 in agAP-AChE or Cys289 in greenbug AP-AChE (leading to enzyme reactivation). Given the experimental data of our previous and present studies, we conclude that the insect-specific cysteine in AP-AChE is accessible for conjugation with high-affinity and flexible sulfhydryl agents and that such conjugation can lead to the selective and irreversible inhibition of AP-AChEs.

Furthermore, the present work demonstrates the correlation between the reactivity of the n class="Chemical">cysteine-targeting inhibitors and their binding affinity. Because the bimolecular inhibition rate constant (inact/KI) of PM20 is 239-fold higher than that of paraoxon (Table 1), structural modification of PM20 to change its Ki for agAP-AChE from the present 400 nM to 4 nM can, in theory, yield a new inhibitor with inact/KI equivalent to that of paraoxon but with an electrophile that can be ~24,000-fold less reactive than maleimide. Given the report that the AChE inhibitor Ki can be improved to 33 fM56, the expectation that the Ki of PM20 can be improved to 4 nM is conservative. The replacement of maleimide with such a weak electrophile would prevent the new inhibitor from reacting with off-target cysteines. For that reasons, we believe that the insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides with reduced insecticide resistance and low toxicity to mammals, fish, and birds for the control of malaria and other mosquito-borne diseases.

Methods

Materials

Acetylthiocholine (n class="Chemical">ATCh) and Triton X-100 were purchased from ACROS (Morris Plains, NJ), and 5,5′-dithiobis-2-nitrobenzoate (DTNB) was ordered from Sigma-Aldrich (St. Louis, MO). Recombinant agAP-AChE with a specific activity of ≥461 U/mg (36.67 ng/μL) in the presence of 5% glycerol was made according to a published protocol23. Recombinant hAChE with a specific activity of ≥1500 U/mg was purchased as a lyophilized powder from Sigma-Aldrich (catalog number: C1682) and dissolved in 50 mM phosphate buffer (pH 8.0) containing 0.1% Triton X-100 as a stock solution. Peptide GICEFPFVPVVDGAFL was purchased from GenScript (Piscataway, NJ).

General description of chemical synthesis

1H NMR (400 MHz) and n class="Chemical">13C NMR (100 MHz) spectra were recorded on a Mercury 400 spectrometer from Varian (Palo Alto, CA). Chemical shifts are reported in ppm using the solvent peak as an internal standard. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, and m = multiplet), coupling constant, and integration. Low-resolution mass spectra (LRMS) were recorded using either a Hewlett Packard 5973 Mass Spectrometer with SIS Direct Insertion Probe (Palo Alto, CA) or a Waters ZQ 2000 Mass Spectrometer (Milford, MA). High-resolution mass spectra (HRMS) were obtained on a Bruker BioTOF II ESI. IR spectra were obtained on a ThermoNicolet Avatar 370 FT-IR (Waltham, MA) using a KBr pellet. A Biotage SP-1 (Charlotte, NC) was used for medium pressure liquid chromatography (MPLC) purification using silica gel as the packing material. 16-Bromo-1-hexadecanol (1a) was purchased from Astatech, Inc. (Bristol, PA), octadecanedioic acid and eicosanedioic acid from TCI America (Portland, OR), respectively, and used as received. Anhydrous tetrahydrofuran (THF), LiAlH4, and 48% hydrobromic acid were purchased from Sigma-Aldrich (St. Louis, MO). N-Bromosuccinimide (NBS) was purchased from Fisher Scientific (Pittsburgh, PA), and recrystallized from water before use.

1-(16-Hydroxyhexadecyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (2a)

To a stirred solution of 16-bromo-1-hexadecanol (1a, 0.32 g, 1.00 mmol) in DMF (10 mL) at room temperature was added 3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (0.16 g, 1.00 mmol) then followed by cesium carbonate (0.49 g, 1.50 mmol). The mixture was stirred at room temperature for 18 hours. Diluted with water (10 mL), extracted with methylene chloride (10 mL), dried (MgSO4), filtered, and then concentrated, further dried under high vacuum to give 0.41 g (100%) of 2a as a white powder, mp 84–86°C;1H NMR (400 MHz, CDCl3) δ 6.49 (s, 2H), 5.24 (s, 2H), 3.61 (t, J = 6.6 Hz, 2H), 3.43 (t, J = 7.4 Hz, 2H), 2.81 (s, 2H), 1.56–1.51 (m, 5H), and 1.31–1.22 (m, 24H); 13C NMR (100 MHz, CDCl3) δ 176.56, 136.76, 81.11, 63.28, 47.60, 39.27, 33.03, 29.85, 29.83, 29.75, 29.66, 29.34, 27.82, 26.91, and 25.96; IR (KBr) ν 3422, 3363, 2925, 2849, 1698, and 879 cm−1; LRMS (EI) m/z 338 (100%, [M-C4H4O]+); HRMS (ESI) m/z 338.2692 ([M-C4H4O+H]+, C20H36NO3+ requires 338.2695). Anal. calcd for C24H39NO4·0.5H2O: C, 69.53; H, 9.72; N, 3.38. Found: C, 69.67; H, 9.36; N, 3.38.

1-(18-Hydroxyoctadecyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (2b)

Compound 2b was syn class="Chemical">nthesized from 1b, which was prepared according to a published protocol57, in 96% yield in the same manner as the synthesis of 2a, mp 84–86°C; 1H NMR (400 MHz, CDCl3) δ 6.49 (s, 2H), 5.24 (s, 2H), 3.61 (t, J = 6.6 Hz, 2H), 3.43 (t, J = 7.4 Hz, 2H), 2.81 (s, 2H), 1.66 (brs, 1H), 1.56–1.48 (m, 4H), and 1.31–1.22 (m, 28H); 13C NMR (100 MHz, CDCl3) δ 176.57, 136.76, 81.11, 63.26, 47.59, 39.26, 33.03, 29.88, 29.83, 29.76, 29.68, 29.67, 29.35, 27.82, 26.91, and 25.97; IR (KBr) ν 3426, 2923, 2848, 1698, and 879 cm−1; LRMS (EI) m/z 365 (100%, [M-C4H4O]+); HRMS (ESI) m/z 388.2816 ([M-C4H4O+Na]+, C22H39NO3Na+ requires 388.2828). Anal. calcd for C26H43NO4·H2O: C, 69.14; H, 10.04; N, 3.10. Found: C, 70.88; H, 10.16; N, 3.10.

1-(20-Hydroxyeicosyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (2c)

Compound 2c was synthesized from 1c, which was prepared according to a pun class="Chemical">blished protocol57, in 100% yield in the same manner as the synthesis of 2a, mp 79–82°C;1H NMR (400 MHz, CDCl3) δ 6.50 (s, 2H), 5.25 (s, 2H), 3.63 (t, J = 6.6 Hz, 2H), 3.45 (t, J = 7.4 Hz, 2H), 2.82 (s, 2H), 1.57–1.52 (m, 5H), and 1.33–1.23 (m, 32H); 13C NMR (100 MHz, CDCl3) δ 176.56, 136.77, 81.12, 63.33, 47.61, 39.28, 33.04, 29.90, 29.85, 29.78, 29.70, 29.67, 29.36, 27.84, 26.92, and 25.97; IR (KBr) ν 3430, 2920, 2849, 1700, and 878 cm−1; LRMS (EI) m/z 393 (100%, [M-C4H4O]+); HRMS (ESI) m/z 484.3414 ([M+Na]+, C28H47NO4Na+ requires 484.3403). Anal. calcd for C28H47NO4·H2O: C, 70.11; H, 10.30; N, 2.92. Found: C, 70.73; H, 10.36; N, 2.71.

1-(16-Bromohexadecyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (3a)

NBS (89 mg, 0.50 mmol) was added into a stirred solution of 2a (0.20 g, 0.50 mmol) and n class="Chemical">triphenylphosphine (0.13 g, 0.50 mmol) in dry THF (10 mL) at room temperature. After 5 minutes, water (10 mL) was added, layers were separated, the aqueous layer was extracted with methylene chloride (2 × 10 mL), organic layers were combined, dried (MgSO4), filtered, and then purified by MPLC (silica gel, EtOAc:Hex = 1:4) to give 0.17 g (75%) of 3a as a white solid, mp 76–78°C; 1H NMR (400 MHz, CDCl3) δ 6.50 (s, 2H), 5.25 (s, 2H), 3.45 (t, J = 7.4 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 1.88–1.81 (m, 2H), 1.57–1.52 (m, 2H), 1.43–1.41 (m, 2H), and 1.39–1.23 (m, 24H); 13C NMR (100 MHz, CDCl3) δ 176.47, 136.73, 81.08, 47.58, 39.19, 34.29, 33.03, 29.83, 29.80, 29.73, 29.64, 29.31, 28.96, 28.38, 27.78, and 26.86; IR (KBr) ν 2919, 2848, 1704, 1400, and 876 cm−1; LRMS (EI) m/z 399 (100%, [M-C4H4O]+). Anal. calcd for C24H38BrNO3: C, 61.53; H, 8.18; N, 2.99. Found: C, 61.10; H, 8.46; N, 2.94.

1-(18-Bromooctadecyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (3b)

Compound 3b was synthesized from 2b (75% yield) in the same manner as the synthesis of 3a, mp 66−69°C;1H NMR (400 MHz, CDCl3) δ 6.51 (s, 2H), 5.26 (s, 2H), 3.45 (t, J = 7.4 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 2.82 (s, 2H), 1.86–1.81 (m, 2H), 1.43–1.39 (m, 2H), and 1.27–1.23 (m, 28H); 13C NMR (100 MHz, CDCl3) δ 176.53, 136.76, 81.12, 47.60, 39.26, 34.35, 33.07, 29.91, 29.86, 29.78, 29.70, 29.67, 29.35, 29.00, 28.41, 27.83, and 26.91; IR (KBr) ν 2919, 2848, 1704, 1401, and 876 cm−1; LRMS (EI) m/z 495 (2%, [M]+), 429 (100%, [M-C4H4O]+); HRMS m/z 427.2066 ([M-C4H4O]+, C22H38BrNO2+ requires 427.2086). Anal. calcd for C26H42BrNO3: C, 62.89; H, 8.53; N, 2.82. Found: C, 62.69; H, 8.90; N, 2.70.

1-(20-Bromoeicosyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (3c)

Compound 3c was synthesized from 2c (73% yield) in the same manner as the synthesis of 3a, mp 81–83°C; 1H NMR (400 MHz, CDCl3) δ 6.50 (s, 2H), 5.26 (s, 2H), 3.45 (t, J = 7.4 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.82 (s, 2H), 1.88–1.81 (m, 2H) 1.43–1.39 (m, 2H), and 1.27–1.23 (m, 32H); 13C NMR (100 MHz, CDCl3) δ 176.49, 136.81, 136.64, 81.25, 80.96, 47.83, 47.37, 39.25, 34.28, 33.06, 29.83, 29.76, 29.67, 29.34, 28.98, 28.40, 27.83, and 26.90; IR (KBr) ν 2913, 2844, 1704, 1401, and 870 cm−1; LRMS (EI) m/z 523 (6%, [M]+), and 455 (77%, [M-C4H4O]+). Anal. calcd for C28H46BrNO3: C, 64.11; H, 8.84; N, 2.67. Found: C, 64.10; H, 9.24; N, 2.59.

1-(16-(Piperidin-1-yl)hexadecyl)-1H-pyrrole-2,5-dione hydrochloride (PY16)

A solution of compound 3a (150 mg, 0.32 mmol) and piperidine (150 μL, 1.50 mmol) in n class="Chemical">acetonitrile (10 mL) and methylene chloride (5 mL) was stirred at room temperature overnight. The solvent was removed and the crude product was purified using MPLC (silica gel/DCM/MeOH) to give the intermediate as a white solid. The intermediate was treated with 0.5 mL of 3 M methanolic HCl and then evaporated to dryness. The residue was heated to reflux in anisole (3 mL) for 20 min. The solvent was removed, yielding pure PY16 as an off-white solid (128 mg, 93% yield), mp 118–120°C; 1H NMR (400 MHz, CDCl3) δ 10.92 (s, 1H), 6.63 (s, 2H), 3.49–3.42 (m, 2H), 3.42 (t, J = 7.2 Hz, 2H), 2.92–2.85 (m, 2H), 2.75–2.63 (m, 2H), 2.31–2.18 (m, 2H), 1.87–1.76 (m, 4H), 1.53–1.45 (m, 2H), 1.30–1.13 (m, 26H); 13C NMR (100 MHz, CDCl3) 171.09, 134.25, 57.78, 53.30, 38.09, 29.78, 29.73, 29.70, 29.64, 29.57, 29.29, 29.22, 28.72, 27.06, 26.91, 23.71, 22.74, and 22.23; IR (KBr) ν 3447, 2921, 2849, 1699 cm−1; LRMS (ESI) m/z 405.28 (100%, [M+H]+); HRMS (ESI) m/z 405.3469 ([M+H]+, C25H45N2O2+ requires 405.3481). Anal. calcd for C25H45ClN2O2: C, 65.40; H, 10.32; N, 6.10. Found: C, 65.69; H, 10.51; N, 6.07.

1-(18-(Piperidin-1-yl)octadecyl)-1H-pyrrole-2,5-dione hydrochloride (PY18)

Compound PY18 was synthesized from 3b (71% yield) in the same manner as the synthesis of PY16, mp 129−131°C; 1H NMR (400 MHz, CDCl3) δ 11.58 (s, 1H), 6.65 (s, 2H), 3.60–3.45 (m, 2H), 3.49 (t, J = 7.2 Hz, 2H), 2.95–2.85 (m, 2H), 2.70–2.55 (m, 2H), 2.40–2.25 (m, 2H), 1.95–1.80 (m, 8H), 1.60–1.50 (m, 2H), and 1.35–1.18 (m, 26H); 13C NMR (100 MHz, CDCl3) 171.16, 134.28, 58.07, 53.59, 38.17, 29.88, 29.85, 29.80, 29.78, 29.72, 29.64, 29.36, 29.31, 28.78, 27.19, 26.98, 23.80, 22.87, and 22.46; IR (KBr) ν 3445, 2918, 2850, and 1703 cm−1; LRMS (ESI) m/z 433.15 (100%, [M+H]+); HRMS (ESI) m/z 433.3788 ([M+H]+, C27H49N2O2+ requires 433.3794). Anal. calcd for C27H49ClN2O2·2.5H2O: C, 63.07; H, 10.59; N, 5.45. Found: C, 63.08; H, 10.51; N, 5.48.

1-(18-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)octadecyl)pyridinium bromide (PM18)

Compound 3b (50 mg, 0.10 mol) was dissolved in n class="Chemical">acetone (2 mL), followed by pyridine (81 μL, 1.00 mmol). The resulting mixture was refluxed for 17 hours. Acetone was evaporated in vacuo, the residue was dissolved in anisole (1.0 mL), then refluxed for 15 min. Cooled to room temperature, anisole was evaporated by blowing N2 to give 51 mg (100%) of PM18; mp 94–105°C; 1H NMR (400 MHz, CD3OD) δ 9.04 (d, J = 5.7 Hz, 2H), 8.61 (t, J = 7.8 Hz, 1H), 8.13 (t, J = 7.0 Hz, 2H), 6.80 (s, 2H), 4.66 (t, J = 7.5 Hz, 2H), 3.47 (t, J = 7.1 Hz, 2H), 2.05–2.01 (m, 2H), 1.57–1.53 (m, 2H), and 1.39–1.27 (m, 28H); 13C NMR (100 MHz, CD3OD) δ 171.40, 145.68, 144.78, 134.17, 128.33, 61.94, 37.36, 31.37, 29.59, 29.55, 29.48, 29.46, 29.43, 29.34, 29.03, 28.96, 28.33, 26.61, and 26.02; IR (KBr) ν 3055, 2918, 2849, 1703, and 696 cm−1; LRMS (ESI) m/z 427 (100%, [M]+); HRMS m/z 427.3324 ([M]+, C27H43N2O2+ requires 427.3325). Anal. calcd for C27H43BrN2O2·H2O: C, 61.70; H, 8.63; N, 5.33. Found: C, 61.85; H, 8.92; N, 5.27.

1-(20-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)eicosyl)pyridinium bromide (PM20)

PM20 was synthesized from 3c (99% yield) in the same manner as the synthesis of PM18; mp 104–112°C 1H NMR (400 MHz, CD3OD) δ 9.03 (d, J = 6.2Hz, 2H), 8.61 (t, J = 7.8 Hz, 1H), 8.13 (t, J = 6.9 Hz, 2H), 6.80 (s, 2H), 4.65 (t, J = 7.6 Hz, 2H), 3.47 (t, J = 7.1 Hz, 2H), 2.04–2.01 (m, 2H), 1.57–1.53 (m, 2H), and 1.39–1.27 (m, 32H); 13C NMR (100 MHz, CD3OD) δ 171.40, 145.68, 144.80, 134.16, 128.33, 61.94, 37.35, 31.35, 29.59, 29.55, 29.48, 29.46, 29.43, 29.33, 29.03, 28.95, 28.32, 26.60, and 26.02; IR (KBr) ν 3054, 2918, 2849, 1703, and 696 cm−1; LRMS (EI) m/z 455 (100%, [M]+); HRMS (ESI) m/z 455.3620 ([M]+, C29H47N2O2+ requires 455.3638). Anal. calcd for C29H47BrN2O2·0.8H2O: C, 63.33; H, 8.91; N, 5.09. Found: C, 63.29; H, 9.50; N, 4.98.

1-(18-(Piperidin-1-yl)octadecyl)pyrrolidine-2,5-dione hydrochloride (PYS18)

To a round bottom flask equipped with a n class="Chemical">hydrogen balloon was added PY18 (10 mg, 0.023 mmol), MeOH (2 mL) and 10% Pd-C (1 mg). The reaction was stirred at room temperature overnight and the catalyst was filtered. The filtrate was evaporated to dryness, leaving a pure product PYS18 as a white solid (10 mg, 100% yield), mp 112–114oC;1H NMR (400 MHz, CDCl3) δ 11.69 (s, 1H), 3.60–3.45 (s, 2H), 3.48 (t, J = 7.2 Hz, 2H), 2.91 (s, 2H), 2.70 (s, 4H), 2.63 (s, 2H), 2.33 (s, 2H), 1.95–1.80 (m, 6H), 1.60–1.52 (m, 2H), 1.40–1.20 (m, 28H); 13C NMR (100 MHz, CDCl3) δ 177.58, 58.03, 53.54, 39.16, 29.88, 29.86, 29.85, 29.80, 29.79, 29.71, 29.64, 29.40, 29.30, 28.41, 27.95, 27.19, 27.10, 23.77, 22.84, and 22.46; IR (KBr) ν 3434, 2918, 2849, and 1694 cm−1; LRMS (ESI) m/z 435.28 (100%, [M+H]+); HRMS (ESI) m/z 435.3931 ([M+H]+, C27H51N2O2+ requires 435.3951). Anal. calcd for C27H51ClN2O2·3H2O: C, 61.75; H, 10.94; N, 5.33. Found: C, 61.74; H, 10.25; N, 5.02.

1-(20-(2,5-Dioxopyrrolidin-1-yl)eicosyl)pyridin-1-ium 2,2,2-trifluoroacetate (PMS20)

To a stirred solution of PM20 (20 mg, 0.037 mmol) in n class="Chemical">MeOH (2 mL) was added triphenylphosphine (10.8 mg, 0.041 mmol), the resulting mixture was heated at 75°C for 4 hours. The crude product was purified by HPLC. HPLC conditions: Phenomenex Gemini 5 μ C18 4.6 × 250 mm column, flow rate 1.0 mL/min, solvent A: H2O + TFA (0.1%), solvent B: MeCN : H2O = 9 : 1 + TFA (0.1%); linear gradient from 80% A to 0% A over 20 minutes (tR = 18.40 minutes portion was collected) to give 6 mg (40%) of PMS20 as a waxy half-solid. 1H NMR (400 MHz, CD3OD) δ 8.99 (d, J = 5.7 Hz, 2H), 8.60 (t, J = 7.7 Hz, 1H), 8.12 (t, J = 7.0 Hz, 2H), 4.62 (t, J = 7.5 Hz, 2H), 3.45 (t, J = 7.4 Hz, 2H), 2.67 (s, 4H), 2.06–1.96 (m, 2H), 1.57–1.50 (m, 2H), and 1.40–1.24 (m, 32H); 13C NMR (100 MHz, CD3OD) δ 178.86, 145.67, 144.74, 128.31, 61.93, 38.39, 31.32, 29.58, 29.53, 29.50, 29.44, 29.42, 29.32, 29.09, 28.93, 27.87, 27.46, 26.71, and 26.00; IR (KBr) ν 2919, 2850, 1698, 1205, and 1136 cm−1; LRMS (EI) m/z 457 (36%, [M]+); HRMS (ESI) m/z 457.3772 ([M]+, C29H49N2O2+ requires 457.3794). Anal. calcd for C29H49N2O2+·CF3CO2−·CF3CO2H·0.3H2O: C, 57.43; H, 7.39; N, 4.06. Found: C, 57.49; H, 7.59; N, 4.35.

Determination of the AChE activity

AChE activity or hydron class="Chemical">lysis rate (v) in the absence or presence of an inhibitor at concentrations of ≤30 μM was determined using the Ellman assay58 through measuring the change in ultraviolet absorbance of an assay solution at 405 nm over a period of 2 minutes at 25°C with a SpectraMax Plus 384 Absorbance Microplate Reader from Molecular Devices (Sunnyvale, CA). Justification for the use of the Ellman assay to test sulfhydryl agents is provided in the Supplementary Information.

Determination of irreversible inhibition through dilution

In a typical dilution experiment with a 96-well plate, to 300 μL of 50 mM phosphate buffer (pH 8.0) was added 3 μL of 2 μg/mL agAP-AChE or 1 μg/mL hAChE, and then 5 μL of neat dimethyl sulfoxide (DMSO) or an inhibitor in neat DMSO freshly prepared at a concentration leading to ~50% enzyme inhibition using a 10 μL Eppendorf Research plus pipette. The resulting solution was mixed through 10 repetitions of uptake and expulsion of part of the solution using the pipette while moving the pipette tip around the bottom of the well. The solution was then incubated for 30 minutes at 25°C. At the end of incubation, a 50 μL Eppendorf Research plus pipette was used to transfer 28 μL of the solution to a well preloaded with 252 μL of 50 mM phosphate buffer (pH 8.0), and the resulting solution was mixed with the pipette. To the original and diluted enzyme solutions were added sequentially 10 μL of 2.5 mM DTNB and 10 μL of 30 mM ATCh. The resulting solution was quickly mixed with the pipette and measured immediately for v. Each dilution experiment was performed in triplicates. The AChE inhibition was determined by calculating the difference of v in the presence and absence of a test inhibitor. The irreversible AChE inhibition was determined by the null effect of the dilution on the AChE inhibition.

Determination of kinact and KI

To each well containing 270 μL of 50 mM phosphate n class="Chemical">buffer (pH 8.0) and 5 μL of 1 μg/mL agAP-AChE or 0.5 μg/mL hAChE was added 5 μL of DMSO as a control or an inhibitor in neat DMSO freshly prepared at varying concentrations, using a 10 μL Eppendorf Research plus pipette. The resulting solution was mixed with the pipette. To the control solution was added 10 μL of 2.5 mM DTNB and 10 μL of 30 mM ATCh. The resulting solution was quickly mixed with the pipette and measured immediately for v0. Each of the inhibitor-containing solutions was incubated for various amounts of time at 25°C. At the end of the incubation, to the incubation solution was added 10 μL of 2.5 mM DTNB and 10 μL of 30 mM ATCh. The resulting solution was quickly mixed with the pipette and measured immediately for v. The kobs value was obtained from the slope of the line of ln(v/v) versus time according to Equation 1 in Figure 1 using GraphPad Prism Version 5.0d for Mac OS X from GraphPad Software (San Diego, CA). Linear fitting was used and the lines were forced to go through x = 0 and y = 0. Each of the kobs values used for the subsequent kinact and KI determination was an average of two independent experiments. The kinact and KI values were then computed from the curve of kobs versus the associated [I] according to Equation 2 in Figure 1 using Prism 4 with nonlinear fitting.

Determination of Ki

To each well was added sequentially 270 μL of 50 mM phosphate n class="Chemical">buffer (pH 8), 5 μL of 1 μg/mL agAP-AChE or 0.5 μg/mL of hAChE, 5 μL of DMSO or an inhibitor in neat DMSO freshly prepared at varying concentrations, 10 μL of 2.5 mM DTNB, and 10 μL of 0.9375, 1.25, 1.875, 3.75, 7.5, or 15 mM ATCh. The resulting solutions were mixed with a 1–10 μL multichannel pipette from Thermo Fisher Scientific and measured immediately for v. Ki was then obtained from 1/v, 1/[ATCh], and [I] using Prism 4 with the Lineweaver-Burk plot59.

Liquid chromatography tandem mass spectrometric detection of the PM20 adduct

To agAP-n class="Gene">AChE (100 ng) in 21 μL of 50 mM phosphate buffer (pH 8.0) was added 0.5 μL of 10 mM PM20 in neat DMSO and the solution was incubated at room temperature for 1 hour. After enzyme activity and its complete loss before and after the incubation were confirmed, the sample was added to 4x sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (200 mM Tris buffer, pH 6.8, 400 mM dithiothreitol, 8% SDS, and 40% glycerol) and heated to 100°C for 5 minutes. The sample was then loaded on a 4–20% gradient SDS-PAGE gel and run under 100 volts for 90 minutes. After the silver-stained SDS-PAGE gel band of the agAP-AChE was destained with potassium ferricyanide and sodium thiosulfate according to a published protocol60, the gel band containing agAP-AChE was cut into small pieces and treated with 50 mM tris(2-carboxyethyl)phosphine in 50 mM Tris buffer (pH 8.1) at 55°C for 40 minutes then alkylated with 40 mM iodoacetamide at room temperature for 40 minutes in the dark. The protein was digested in situ with 0.15 μg chymotrypsin from Roche (Indianapolis, IN) in 25 mM Tris buffer (pH 8.1) with 0.0002% Zwittergent 3–16 at room temperature overnight followed by peptide extraction with 2% trifluoroacetic acid and then with acetonitrile. The pooled extracts were concentrated to <5 μL on a SpeedVac spinning concentrator from Savant Instruments (Holbrook, NY) and then brought up in 0.2% trifluoroacetic acid for peptide identification using a ThermoFinnigan LTQ Orbitrap hybrid mass spectrometer from Thermo Fisher Scientific coupled to a nano-scale, two-dimensional high-performance liquid chromatography system from Eksigent. The solution of the digested peptides of agAP-AChE was loaded onto a 250-nL OPTI-PAK trap from Optimize Technologies (Oregon City, OR) custom packed with Michrom Magic C8 solid phase (Michrom Bioresources, Auburn, CA). Chromatography was performed using 0.2% formic acid in both the A solvent (98% water with 2% acetonitrile) and the B solvent (80% acetonitrile with 10% isopropanol and 10% water) and a 5% B to 50% B gradient over 35 minutes at 325 nL/min through a hand packed PicoFrit from New Objective (Woburn, MA) 75 μm × 100-mm column (Michrom Magic C18 3 μm). The LTQ Orbitrap tandem mass spectrometer was set to perform a Fourier transform full scan from m/z 350–1450 with resolution at 60,000 (400 m/z) followed by linear ion trap MS/MS scans on the top five ions. Dynamic exclusion was set to 1 and selected ions were placed on an exclusion list for 15 seconds. The lock-mass option was enabled for the Fourier transform full scans using the ambient air polydimethylcyclosiloxane ion with m/z 445.120024 or a common phthalate ion with m/z 391.284286 for real-time internal calibration. ChemDraw Ultra Version 12.0.3.1216 from CambridgeSoft (Cambridge, MA) was used to calculate the m/z values for the identified agAP-AChE fragments. Tandem mass spectra were extracted using BioWorks version 3.2 and analyzed using Sequest from ThermoFinnigan (San Jose, CA). Sequest was searched with a fragment ion mass tolerance of 0.60 and a parent ion tolerance of 25 ppm. Oxidation of methionine and iodoacetamide derivative of cysteine were specified as variable modifications in addition to the modification by the conjugation of PM20.

Author Contributions

Y.-P.P. conceived, designed, and supervised the project. Y.-P.P. and J.G.P. designed PYn, PMn, PYS18 and PMS20. Y.-P.P. performed the computational study. D.D. first syn class="Chemical">nthesized PYn; S.R. then synthesized PMn; J.G.P. subsequently developed the improved synthetic scheme for PYn, PMn, PYS18 and PMS20 shown in Figure 1. D.D. designed the experiment shown in Figure 7, established the kinetics study protocols, and performed the kinetics studies. B.J.M. and D.D. designed and performed the nanoLC-ESI-MS/MS study. Y.-P.P., D.D., J.G.P., S.R. and B.J.M. analyzed the data. H.J. made the recombinant agAP-AChE. Y.-P.P. wrote the paper; D.D., J.G.P., S.R. and B.J.M. drafted parts of the methods section; Y.-P.P., D.D., J.G.P., S.R. and B.J.M. contributed with revisions.