Synthesis and 31P chemical shift identification of tripeptide active site models that represent human serum acetylcholinesterase covalently modified at serine by certain organophosphates.

Article abstract of DOI:10.1021/tx960097w

Most organophosphorus (OP) insecticides impart their toxic action via inhibition of cholinesterases by reacting at an essential serine hydroxyl group. The inhibition process is dependent upon the reactivity, stereochemistry, leaving group, and the mechani

Full text of DOI:10.1021/tx960097w

Chem. Res. Toxicol. 1996, 9, 1325-1332
1325
Syn th esis a n d 31P Ch em ica l Sh ift Id en tifica tion of
Tr ip ep tid e Active Site Mod els Th a t Rep r esen t Hu m a n
Ser u m Acetylch olin ester a se Cova len tly Mod ified a t
Ser in e by Cer ta in Or ga n op h osp h a tes
Charles M. Thompson,* Alirica I. Suarez, and Oscar P. Rodriguez
Department of Chemistry, University of Montana, Missoula, Montana 59812
Received J une 14, 1996X
Most organophosphorus (OP) insecticides impart their toxic action via inhibition of cho-
linesterases by reacting at an essential serine hydroxyl group. The inhibition process is
dependent upon the reactivity, stereochemistry, leaving group, and the mechanism of
phosphorylation and/or reactivation (or aging) inherent to the OP compound under consider-
ation. Because a wide array of phosphorylated structures are possible following inhibition by
an OP, a simple model system was sought to investigate the mechanistic details of these and
related reactions. In the present study, the tripeptide N-CBZ-Glu-Ser(OH)-Ala-OEt (chosen
as a truncated form of human serum cholinesterase) was chemically modified at the serine
hydroxyl group by various O-methyl phosphate groups and the 31P NMR chemical shift recorded.
Six tripeptides, representing (a) phosphorylation by dimethyl phosphorothionates (N-CBZ-
Glu-Ser[O-P(S)(OMe)2]Ala-OEt; 5), (b) phosphorylation by dimethyl phosphates (N-CBZ-Glu-
Ser[O-P(O)(OMe)2]Ala-OEt; 6), (c) phosphorylation by O,S-dimethyl phosphorothiolates (N-
CBZ-Glu-Ser[O-P(O)(OMe)(SMe)]Ala-OEt; 7), (d) aging following inhibition by dimethyl
phosphorothionates (N-CBZ-Glu-Ser[O-P(O)(OMe)(S-)]Ala-OEt; 8), (e) aging following inhibition
by dimethyl phosphates (N-CBZ-Glu-Ser[O-P(O)(OMe)(O-)]Ala-OEt; 9), and (f) phosphorylation
by (R/S)PSc-isomalathion stereoisomers (N-CBZ-Glu-Ser[O-P(O)(OMe)(SCH(CO2CO2Et)CH2-
CO2Et)]Ala-OEt; 10) have been synthesized. Tripeptides 5 and 6 were prepared via preliminary
formation of an intermediate tripeptide phosphite followed by direct conversion to 5 using S8
or to 6 with m-CPBA, respectively. Tripeptides 8 and 9 were prepared by dealkylation of 5
and 6, respectively. Tripeptides 7 and 10 were prepared by reaction of 8 with dimethyl sulfate
and (R)- or (S)-diethyl (trifluoromethanesulfonyl)malate, respectively.
restoration of cholinesterase activity is accompanied by
scission of the serine-phosphate linkage. OP-inhibited
cholinesterase may also undergo “aging” or cleavage of
a phosphate-ester bond (P-O-R) or “nonreactivation”
pathways (eq 1). Whereas an aging mechanism results
from cleavage of a phosphorus ester (P-O-R) bond to
give a phosphate anion (5), the non-reactivation mech-
anism(s) are less well characterized and possibly due to
changes in the protein structure that lead to an irrevers-
ibly inactivated enzyme. Although the aging mechanism
is responsible for the inactivation of neuropathy target
esterase leading to OP-induced delayed neuropathy (6),
cholinesterase aging and nonreactivation mechanisms
both lead to sustained acetylcholine levels that may be
mistaken for simple inhibition. Since the ill health
effects and treatment of aging and nonreactivation of
cholinesterase differ from the treatment of straight-
forward inhibition, recognition of the correct mechanism
is important for clinical treatment. Moreover, the rate
and extent to which a particular OP agent causes an
irreversible, postinhibitory reaction to occur likely cor-
relate with its delayed toxic action.
In tr od u ction
Most organophosphate (OP)1 insecticides are toxic to
target and nontarget organisms by virtue of covalent
phosphorylation of cholinesterase(s) at an essential serine
residue. Once modified at this residue by the OP,
cholinesterase is unable to hydrolyze essential neuro-
transmitters leading to neurochemical imbalances (1).
The mechanism of cholinesterase inactivation by OP
agents occurs with the ejection of a leaving group Z
synchronous with formation of a phosphoserine linkage
(eq 1). To be a potent inhibitor of cholinesterase, an OP
compound usually contains a phosphoryl π-bond (PdO)
and Z must be a good leaving group (2-4).
Several postinhibitory outcomes are also possible for
OP-inhibited cholinesterase including reactivation (eq 1),
which leads to restoration of all or part of the cholinest-
erase activity following hydrolysis with water (as OH-)
or certain oxime antidotes (e.g., 2-pyridine aldoxime
methiodide; 2-PAM). It is generally accepted that the
* Corresponding author: Prof. Charles M. Thompson, Department
of Chemistry, University of Montana, Missoula, MT 59812;
Telephone/FAX: (406) 243-4643; e-mail: cmthomp@selway.umt.edu.
X Abstract published in Advance ACS Abstracts, November 1, 1996.
1 Abbreviations: OP, organophosphate; 2-PAM, 2-pyridine aldoxime
methiodide; TMS, tetramethylsilane; PMA, phosphomolybdic acid;
DBQ, dibromoquinone 4-chloroimide; CBZ, carbobenzyloxy; m-CPBA,
m-chloroperoxybenzoic acid; PEX, potassium ethyl xanthate; TEA,
triethylamine; DCC, dicyclohexylcarbodiimide; DIPC, diisopropylcar-
bodiimide; ((iPr)2N)2P-OMe, bis-N,N-(diisopropylamino)methoxyphos-
phine; ((nPr)2N)P(OMe)2, N,N-(di-n-propylamino)bismethoxyphosphine;
DMS, dimethyl sulfate.
Most postinhibitory mechanisms and their toxicological
consequences have been less studied than OP inhibition
mechanisms, but the primary chemical alterations caused
to the active site peptide structure must necessarily
preface protein structural changes. The initial alter-
ations at the active site may involve localized changes
to peptide conformation, charge repulsion (e.g., with
S0893-228x(96)00097-5 CCC: $12.00 © 1996 American Chemical Society

1326 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Thompson et al.
(1)
Sch em e 1. P h osp h or oth ion a tes, Oxon s, a n d
P h osp h or oth iola tes a n d Th eir Mech a n ism of
Ch olin ester a se In h ibition
Sch em e 2. Exp a n sion of th e Active Site Region of
Cer ta in Ch olin ester a ses Th a t Ad join th e Ca ta lytic
Ser in e Resid u e
From this introduction, it is evident that a number of
OP-modified cholinesterase species are possible even
when a limited class of common OP inhibitors is consid-
ered. Therefore, determination of an individual phos-
phorylation mechanism is relatively complex and further
complicated by the usual difficulties associated with the
analysis of high molecular weight proteins. As such,
more simplified model systems would help to decipher
inhibition and reactivation pathways.
The local peptide sequence neighboring the essential
serine residue at the active site of certain cholinesterases
(e.g., Torpedo californica AChE and human serum but-
yrylcholinesterase) and select serine proteases show
homology as a Glu-Ser-Ala tripeptide (Scheme 2) (1).
Since human serum cholinesterase is a likely primary
target in mammalian OP poisoning episodes (4, 13, 14),
the Glu-Ser-Ala tripeptide sequence should be an excel-
lent template and starting point to study mechanisms of
cholinesterase inhibition and recovery. OP-modified Glu-
Ser-Ala sequences could be used to evaluate local changes
in peptide structure and determine inhibition and postin-
hibitory mechanisms that would not be possible with
intact protein. In addition to their utility as mechanistic
probes of cholinesterase intoxication by certain OP
agents, modified Glu-Ser-Ala tripeptides could also serve
as spectral and biochemical markers, as mechanistic
probes, and as potential conjugates for immunochemical
assays.
With this preliminary aim, we first undertook the
synthesis of select Glu-Ser-Ala tripeptides that have been
modified at the serine hydroxyl by phosphorus-containing
groups. Six phosphorylated tripeptides 5-10 were se-
lected to mimic covalently modified cholinesterase result-
ing from inhibition by various dimethyl phosphates and
phosphorothionates (Chart 1). Tripeptides 5, 6, and 7
represent inhibition of cholinesterase by O,O-dimethyl
phosphorothionates 1 (e.g., parathion methyl, malathion),
O,O-dimethyl phosphates 2 (e.g., paraoxon, malaoxon),
and O,S-dimethyl phosphorothiolates 3 (e.g., isoparathion
methyl, isomalathion), respectively. Tripeptides 8 and
9 depict postinhibitory reactions that occur following
reaction of cholinesterase with an O,O-dimethyl phos-
aging), or steric repulsion imposed by the phosphoryl
group, all or any of which may cause disruption in the
orientation of the catalytic residues. Although the cho-
linergic system is likely restored through protein syn-
thesis, the toxicological role of the irreversibly OP-
inhibited cholinesterase remains largely unknown. In
light of the numerous delayed neuropathic maladies that
cannot be explained by simple cholinesterase inhibition
by OP agents, an investigation of postinhibitory mech-
anisms would be beneficial, particularly studies that
identify OP agents that lead to irreversible inhibition.
A number of commercial OP insecticides are O,O-
dimethyl phosphorothionates (MeO)2P(S)Z (1), a com-
pound class that includes malathion (1; Z ) -SCH(CO2-
Et)CH2CO2Et) and parathion methyl (1; Z ) -OPh-4-
NO2). Prior to reaction with cholinesterase, most
phosphorothionates must be transformed from the rela-
tively unreactive phosphorothionate form (1; PdS) into
the more reactive oxon form (2; PdO) (Scheme 1) (7).
Oxons react with cholinesterases to afford O,O-dimethyl
phosphorylated enzymes via ejection of the leaving group
Z to give O,O-dimethyl phosphate inhibited ChE repre-
senting the major mechanism of action of phospho-
rothionate insecticides. Alternatively, O,O-dimethyl phos-
phorothionates 1 may isomerize to form phosphorothiolates
(3) (Scheme 1), a process that occurs during the manu-
facture, storage, or environmental lifetime of the OP (8-
10). Because a PdO bond is formed during the isomer-
ization, phosphorothiolates (3) like oxons are more potent
anticholinesterase agents than thionates (11). Phospho-
rothiolates differ from oxons in their reaction with
cholinesterases in that an O,S-dimethyl phosphorylated
product is likely to result (Scheme 1) (12) although
ejection of the thiomethyl (MeS-) rather than the Z group
is a possible alternative mechanism (not shown). The
inhibition of cholinesterase by the parent phospho-
rothionate (1) is also shown in Scheme 1 but is a poor
reaction.

Synthesis of Phosphorylated Tripeptides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1327
Ch a r t 1. P h osp h or yla ted Tr ip ep tid es
Rep r esen tin g Ch olin ester a se Mod ified by
Or ga n op h osp h a tes
modified Glu-Ser-Ala tripeptides are likely to match
fragments obtained via denaturation or digestion of intact
protein.
Exp er im en ta l Section
Melting points were determined on a Mel-Temp melting point
apparatus and are uncorrected. 1H, 13C, and 31P were taken at
400, 100.6, and 161.9 MHz, respectively. 1H and 13C NMR
spectra were taken in D2O or CDCl3 using 0.05% dioxane or
tetramethylsilane (TMS) as internal standards, respectively. 31
P
NMR spectra were taken in the indicated deuterated solvents
using 85% phosphoric acid as an external standard.
Analytical thin-layer chromatography (TLC) was conducted
on E. Merck aluminum-backed, 0.2 mm silica gel 60F254 plates.
Visualization was accomplished with an ultraviolet (UV) lamp
and/or ninhydrin, phosphomolybdic acid (PMA), dibromoquinone
4-chloroimide (DBQ), or ammonium molybdate staining re-
agents. All solvents and reagents were purified by standard
literature methods. Air- or moisture-sensitive reactions were
conducted under an argon atmosphere by utilizing standard
techniques. Flash chromatography was done on Kieselgel 60,
230-400 mesh silica gel in the solvent(s) indicated. Com-
mercially available reagents, amino acids (only L-amino acids
used in this study), and solvents were purified when necessary
by standard literature methods. Elemental analyses were
performed by Midwest Microlab Ltd., Indianapolis, IN. Optical
rotations were recorded (Na lamp) at room temperature in the
specified solvents using a Perkin Elmer Model 241 polarimeter.
Ca u tion : The OP compounds used in this study are hazard-
ous and should be handled in a well-ventilated hood by trained
personnel. These materials may be destroyed by stirring with 1
N NaOH overnight.
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser y-
la la n in e Eth yl Ester (4). Step 1. To a 100 mL, two-neck flask
fitted with a stir bar, septum, and gas inlet was added a solution
of CBZ-serylalanine ethyl ester (11) (1 g, 2.9 mmol) in 20 mL of
absolute ethanol followed by 10% Pd/C (25 mg). The flask was
degassed while stirred (using a water aspirator) and purged with
argon thrice, degassed again, and finally reacted with H2 via a
balloon. The reaction was maintained at ambient balloon H2
pressure for 3-4 h, replacing the H2 in the balloon at 1 h
intervals. When the reaction showed no starting material
remained, the reaction mixture was evacuated of hydrogen,
flushed with nitrogen, and filtered through a 0.5 cm pad of
Celite, and the solvent evaporated to afford serylalanine ethyl
ester as a light yellow semisolid. This material was used
directly in the next step but recrystallization (small scale) from
absolute ethanol-diethyl ether afforded white crystals for
spectral characterization. Yield ) 72%; mp ) 71-73 °C. 1H
NMR (CDCl3): δ 1.25 (3 H, t), 1.47 (3 H, t), 2.0-2.3 (3 H, br s),
3.43 (1 H, t), 3.66-3.72 (2 H, q), 3.76-3.82 (1 H, q), 4.12-4.21
(2 H, q), 4.46-4.56 (1 H, m), 7.76 (1 H, d).
phorothionate 1 and O,O-dimethyl phosphate 2 (oxon),
respectively. Tripeptide 10 is one of two model structures
representing the inhibition of cholinesterase by the
stereoisomers of isomalathion (3; -SCH(CO2Et)CH2CO2-
Et). Based on comparative kinetic analyses, we previ-
ously found that the stereoisomers of isomalathion inhibit
rat brain acetylcholinesterase via two different mecha-
nisms, namely, ejection of the -SCH(CO2Et)CH2CO2Et or
the -SCH3 group (eq 2), the latter leading to irreversibly
(2)
Step 2. Serylalanine ethyl ester (1 g, 4.23 mmol) and DCC
(0.86 g, 4.23 mmol) were dissolved in 30 mL of freshly distilled
CH2Cl2 and chilled to 0 °C. CBZ-glutamic acid 5-methyl ester
(1.18 g, 4.23 mmol) was added, and the reaction mixture was
stirred under argon for 4 h at 0 °C. Upon completion (as
determined by TLC), the mixture was filtered through a fritted
funnel containing a 1 cm layer of Celite, and the resulting
solution was carefully neutralized to pH 8 by the addition of
5% sodium bicarbonate. The organic layer was separated and
dried over anhydrous magnesium sulfate, the solvent was
evaporated in vacuo, and the crude mixture was chromato-
graphed on silica gel using a 3:1 mixture EtOAc/petroleum ether
to afford 4 as a white powder. Yield ) 56%; mp ) 120-121 °C;
inhibited enzyme (12, 15-17). The supposition that dual
mechanisms were operative was based upon the ready
reactivation of acetylcholinesterase following inhibition
by either of the (RP)-isomalathion stereoisomers yet
virtual nonreactivation following inhibition by either of
the (SP)-isomalathion stereoisomers (17). To confirm this
mechanistic anomaly, two distinct tripeptide models
representing the ejection of the -S(CHCO2Et)CH2CO2Et
group (tripeptide 7) and ejection of the -SCH3 (tripeptide
10) are needed.
In this paper, we report full experimental procedures
for the synthesis of the native Glu-Ser-Ala tripeptide, OP-
modified tripeptides 5-10, and a brief comparative 31P
chemical shift analysis of the phosphorylated tripeptides.
As precise representations of the cholinesterase primary
structure, the spectral features of the proposed OP-
[R]23 ) -15.66° (c 1.10, CH2Cl2). 1H NMR (CDCl3): δ 1.20 (3
D
H, t, J ) 7.1 Hz), 1.35 (3 H, d, J ) 7.2 Hz), 1.71 (1 H, dt, J )
7.1, 6.9 Hz), 1.90-2.11 (1 H, dt, J ) 7.6, 6.2 Hz), 2.15 (2 H, t, J
) 7.6 Hz), 2.38-2.42 (2 H, m), 3.61 (3 H, s), 3.72 (1 H, q, J )
7.8 Hz), 4.12 (1 H, q, J ) 7.6 Hz), 4.35 (1 H, m), 4.50 (2 H, m),
5.06 (2 H, m), 6.21 (1 H, d, J ) 7.6 Hz), 7.29 (5 H, m), 7.43 (1 H,
d). 13C NMR (CDCl3): δ 14.1, 17.6, 23.3, 27.9, 30.0, 33.7, 42.3,

1328 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Thompson et al.
48.4, 51.8, 54.4, 61.6, 62.7, 67.0, 127.9, 128.1, 128.4, 136.0, 156.4,
170.0, 171.9, 172.8, 173.7. Anal. Calcd for C22H31N3O9: C,
54.87; H, 6.49; N, 8.72. Found: C, 55.10; H, 6.55; N, 8.63.
H, s), 3.74 (3 H, dd, J ) 12.7 Hz), 4.15 (2 H, q, J ) 7.1 Hz), 4.26
(2 H, m), 4.46 (2 H, m), 4.79 (1 H, m), 5.08 (2 H, s), 6.02 (1 H,
m), 7.24 (1 H, s), 7.31 (5 H, m). 13C NMR (CDCl3): δ 14.1, 17.8,
24.9, 30.1, 30.2, 33.9, 48.5, 51.9, 53.0, 53.1, 53.2, 54.8, 54.9, 61.4,
67.1, 127.8, 127.9, 128.1, 128.5, 136.1, 167.5, 167.6, 171.0, 172.1.
31P NMR: δ 33.2 and 33.6. Anal. Calcd for C24H36N3PO11S:
C, 47.60; H, 5.99; N, 6.93. Found: C, 47.40; H, 6.14; N, 7.06.
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser yl-
O-[O,O-d im eth ylth iop h osp h or yl]a la n in e Eth yl Ester (5).
Tripeptide 4 (0.3 g, 0.58 mmol) was dissolved in 50 mL of freshly
distilled CH2Cl2 and chilled to 0 °C. Tetrazole (0.14 g, 2 mmol)
was added, and the reaction was stirred for 0.5 h, whereupon
dimethoxy N,N-di-n-propylaminophosphite (0.15 g, 0.76 mmol)
was added and the ice bath removed. Stirring was continued
for 0.5 h or until TLC showed consumption of the starting
material. The reaction mixture was concentrated in vacuo, and
the residue was suspended in 25 mL of dry toluene. Freshly
recrystallized elemental sulfur (700 mg) was added, and the
reaction was stirred under an argon atmosphere for 48 h at room
temperature. The mixture was diluted with 30 mL of ether,
the excess sulfur filtered through a pad of Celite, the solvent
evaporated in vacuo, and the crude product purified by flash
chromatography using ether to afford 5 as a white powder. Yield
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser yl-
O-[h yd r ogen -O-m eth ylth iop h osp h or yl]a la n in e Eth yl Es-
ter (8, Sa lt; 13, F r ee Acid ). A solution of tripeptide 5 (0.2 g,
0.315 mmol) and potassium ethyl xanthate (0.096 g, 0.6 mmol)
in 25 mL of dry acetone was heated to reflux under argon for 4
h. The solution was cooled to 25 °C and citric acid (0.06 g, 43
mmol) was added. The mixture was stirred for 2 h and filtered,
and the solvent removed in vacuo to afford a solid material. The
product was purified by flash chromatography using 9:1 CHCl3/
MeOH to give a 2:1 diastereomeric mixture of 13. Yield ) 80%;
mp ) 144-145 °C; [R]23 ) -9.62 (c 0.66, CHCl3). 1H NMR
D
(CDCl3): δ 1.23 (3 H, t, J ) 6.0 Hz), 1.39 (3 H, d, J ) 6.9 Hz),
2.0-2.4 (2 H, m), 2.82-2.86 (3 H, m), 3.50 (3 H, d, J ) 16.5
Hz), 3.57 (3 H, s), 4.14 (2 H, q, J ) 7.0 Hz), 4.31-4.60 (4 H, m),
5.06-5.14 (3 H, m), 7.27 (5 H, m). 13C NMR (CDCl3): δ 14.0,
17.5, 29.7, 30.0, 48.7, 51.9, 53.6, 53.7, 54.4, 61.6, 67.5, 128.0,
128.1, 128.5, 135.9, 156.7, 172.4, 172.5, 176.3. 31P NMR
(CDCl3): δ 57.6 and 59.5 in a 2:1 ratio, respectively. Anal. Calcd
for C23H34PN3SO10‚H2O: C, 45.32; H, 5.95; N, 6.89. Found: C,
45.15; H, 5.52; N, 6.79.
) 81%; mp ) 118-119 °C; [R]23 ) -13.9 (c 0.24, CHCl3). 1H
D
NMR (CDCl3): δ 1.23 (3 H, t, J ) 7.1 Hz), 1.35 (3 H, d, J ) 7.2
Hz), 1.99-2.03 (1 H, m), 2.14-2.21 (1 H, m), 2.43-2.50 (2 H,
m), 3.64 (3 H, s), 3.66 (3 H, d, J ) 14.2 Hz), 3.71 (3 H, d, J )
14.2 Hz), 4.12-4.20 (1 H, m), 4.43-4.50 (1 H, m), 4.72-4.76 (1
H, m), 5.08 (2 H, s), 6.02 (1 H, d, J ) 7.6 Hz), 7.28-7.31 (5 H,
m). 13C NMR (CDCl3): δ 14.2, 18.0, 27.3, 30.4, 48.6, 52.1, 53.0,
53.1, 54.9, 55.0, 55.2, 61.5, 67.2, 119.3, 127.7, 127.8, 127.9, 128.2,
128.4, 128.5, 135.8, 167.4, 171.2, 172.1. 31P NMR (CDCl3): δ
72.8. Anal. Calcd for C24H36N3PO11S: C, 47.60; H, 5.99; N, 6.94.
Found: C, 47.75; H, 6.00; N, 6.84.
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser yl-
O-[O-sod io,O-m eth ylp h osp h or yl]a la n in e Eth yl Ester (9).
A solution of O,O-dimethylphosphoryl tripeptide 6 (0.2 g, 0.34
mmol) and sodium iodide (0.05 g, 0.34 mmol) in dry butanone
(5 mL) was heated to reflux for 1 h. The solution was allowed
to cool to room temperature, the solvent evaporated, and the
residue purified by column chromatography using 9:1 CH2Cl2/
MeOH to give 9 as the sodium salt. Yield ) 92%; mp ) 112 °C;
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser yl-
O-[O,O-d im eth ylp h osp h or yl]a la n in e Eth yl Ester (6). To
a stirred, room temperature solution of tripeptide 4 (0.25 g, 0.5
mmol) in 25 mL of dry CH2Cl2 were added tetrazole (0.07 g, 1
mmol) and N,N-di-n-propylbismethoxyphosphine (0.15 g, 0.77
mmol). The phosphitylation step was stirred for 1 h, and
m-CPBA (0.25 g, 1.45 mmol) was added. Stirring was continued
approximately 1 h or until TLC indicated that the oxidation step
(conversion to phosphoryl) was complete. The reaction was
terminated by the addition of 25 mL of ether and 25 mL of 5%
NaOH, and after 0.5 h of stirring, the organic layer was
separated, washed with 5% NaHCO3 and brine, and dried with
Na2SO4. The solution was filtered and evaporated in vacuo to
yield 6 as a yellow oil. Purification by flash chromatography
using 3:1 EtOAc/petroleum ether gave 6 as a white power. Yield
[R]23 ) -2.06 (c 0.8, H2O). 1H NMR (CHCl3): δ 1.18-1.20 (3
D
H, t, J ) 7.0 Hz), 1.37 (3 H, d), 1.8-2.5 (4 H, m), 3.52-3.59 (6
H, m), 4.13 (3 H, q, J ) 7.0 Hz), 4.21-4.52 (4 H, m), 4.81-5.22
(3 H, m), 6.2 (1 H, br), 7.26 (5 H, s), 7.6 (1 H, br s), 7.8 (1 H, br
s). 13C NMR (90% CDCl3/10% DMSO-d6): δ 14.0, 17.5, 17.8,
30.1, 30.9, 48.4, 48.5, 52.9, 53.0, 53.1, 54.8, 54.9, 61.4, 67.0,
127.8, 127.9, 128.1, 128.4, 136.10, 167.5, 167.6, 171.7, 172.1. 31
P
NMR (CDCl3): δ 4.3. Anal. Calcd for C23H33N3PO12Na: C,
46.23; H, 5.57; N, 7.03. Found: C, 45.95; H, 5.72; N, 6.88.
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser yl-
O-[O-m eth yl-S-d ieth ylsu ccin ylp h osp h or yl]a la n in e Eth yl
Ester (Dia ster eom er s; 10). To 20 mL of dry THF in a flask
fitted with a reflux condenser and nitrogen inlet tube were
added tripeptide 5 (0.3 g, 0.47 mmol) and PEX (0.144 g, 0.5
mmol). The mixture was brought to reflux for 4 h, and then
cooled to 0 °C whereupon (S)-diethyl (trifluoromethanesulfonyl)-
succinate (0.156 g, 0.47 mmol) was added as a 10 mL solution
in THF and stirred for 1 h at 0 °C. Water (20 mL) was added
and the mixture extracted with ether (3 × 25 mL). The ether
extracts were combined, washed with brine, separated, and
dried over sodium sulfate. Flash chromatography using 100%
ethyl acetate affords the pure product as a light yellow wax.
Recrystallization from methylene chloride-petroleum ether
afforded white crystals of 10. Yield ) 65%; mp ) 68-70 °C;
) 52%; mp ) 104-105 °C; [R]23 ) -19.50 (c 0.5, CHCl3). 1H
D
NMR (CDCl3): δ 1.24 (3 H, t, J ) 7.1 Hz), 1.38 (3 H, d, J ) 7.2
Hz), 1.51 (3 H, dt, J ) 7.1 Hz), 2.21-2.25 (3 H, dt, J ) 7.1 Hz),
2.39-2.54 (2 H, m), 3.65 (3 H, s), 3.74 (3 H, d, J ) 11.2 Hz),
3.79 (3 H, d, J ) 11.2 Hz), 4.15-4.16 (1 H, q, J ) 7.1 Hz), 4.26-
4.34 (1 H, m), 5.07-5.09 (2 H, dd, J ) 12 Hz), 6.15 (1 H, d, J )
6.6 Hz), 7.35 (5 H, m), 7.72-7.78 (1 H, d, J ) 7.9 Hz). 13C NMR
(CDCl3): δ 14.5, 18.2, 25.3, 25.9, 30.6, 34.3, 48.8, 49.7, 52.3, 53.6,
55.1, 55.2, 55.3, 61.8, 67.5, 128.4, 128.5, 136.5, 158.2, 168.0,
172.0, 172.5, 173.3. 31P NMR (CDCl3): δ 2.2. Anal. Calcd for
C24H36N3O12P: C, 48.90; H, 6.15; N, 7.12. Found: C, 49.07; H,
6.30; N, 7.06.
[N-(Ben zyloxyca r bon yl)glu ta m yl-γ-m eth yl ester ]ser yl-
O-[O,S-d im eth ylp h osp h or yl]a la n in e Eth yl Ester (7). To
a 100 mL flask, fitted with a condenser, argon inlet/outlet tube,
and a stir bar was added 5 (0.2 g, 0.31 mmol) as a solution in
40 mL of anhydrous acetone. Potassium ethyl xanthate (PEX)
(0.096 g, 0.6 mmol) was added and the solution heated to reflux
for 5 h. The reaction was returned to room temperature,
dimethyl sulfate (0.6 mmol) was added, and the reaction was
brought to reflux for 1 h. The solution was again returned to
room temperature, and the solvent was evaporated in vacuo to
afford the crude product as a semisolid, which was purified using
flash chromatography (100% ethyl acetate) to give 7 as a
diastereomeric mixture of products in a near 1:1 ratio. Yield )
56%; mp ) 61-62 °C; [R]23D ) -17.80 (c 0.13, CHCl3). 1H NMR
(CDCl3): δ 1.23 (3 H, t, J ) 7.1 Hz), 1.36 (3 H, d, J ) 7.2 Hz),
2.01 (2 H, m), 2.25 (3 H, d, J ) 15.3 Hz), 2.44 (2 H, m), 3.63 (3
[R]23 ) +20.31 (0.6, CH2Cl2). 1H NMR (CDCl3): δ 1.01 (3 H,
D
d, J ) 4 Hz), 1.21 (3 H, m), 1.35 (3 H, m), 2.02 (2 H, m), 2.23 (2
H, m), 2.62 (3 H, m), 2.82-3.08 (3 H, m), 3.4 (1 H, br s), 3.63 (3
H, s), 3.81 (3 H, d). 4.15 (5 H, m), 4.23 (3 H, m), 4.6-4.8 (4 H,
m), 5.1 (2 H, m), 5.8 (1 H, br s), 7.25 (5 H, s). 13C NMR
(CDCl3): δ 14.05, 14.1, 17.6, 23.4, 27.4, 30.1, 36.1, 36.3, 42.2,
48.4, 51.8, 53.1, 53.2, 54.6, 54.7, 61.4, 66.7,121.4, 127.8, 127.85,
128.0, 128.4, 136.0,167.5, 169.9, 170.0, 170.2, 171.8, 172.1,
173.55. 31P NMR (CDCl3): δ 35.7 and 35.8. Anal. Calcd for
C31H46N3O15PS: C, 48.75; H, 6.07; N, 5.50. Found: C, 48.70;
H, 6.41; N, 5.90.
N-(Ben zyloxyca r b on yl)ser yla la n in e E t h yl E st er (11)
(18). CBZ-serine (3.87 g, 16.2 mmol) and dicyclohexylcarbodi-
imide (DCC) (3.34 g, 16.2 mmol) were dissolved in 30 mL of CH2-

Synthesis of Phosphorylated Tripeptides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1329
Sch em e 3. Syn th esis of Tr ip ep tid e Ta r get a n d Mod el Rea ction of Dip ep tid e w ith P h osp h ityla tin g Agen ts
Cl2 and chilled to 0 °C. Alanine ethyl ester (16.1 mmol; formed
in situ from equimolar reaction between alanine ethyl ester
hydrochloride and triethylamine in THF) was added as a
solution in 2 mL of CH2Cl2 and the reaction stirred for 4-5 h
at 0 °C. The reaction was monitored by TLC for loss of starting
material and then filtered through a 1 cm pad of Celite, and
the solvent was removed in vacuo to give the crude product as
a sticky yellow oil. Purification by flash chromatography using
100% ether afforded white crystals. Yield ) 50%; mp ) 103-
3. Alanine ethyl ester hydrochloride was converted to
its free base using triethylamine (TEA) in THF and
immediately coupled to N-CBZ-serine with dicyclohexyl-
carbodiimide (DCC) to form N-CBZ-serylalanine ethyl
ester (11) in 50% yield. The CBZ group was removed by
hydrogenolysis (10% Pd/H2) to afford serylalanine ethyl
ester as the primary amine. Because serylalanine ethyl
ester undergoes intramolecular cyclization at room tem-
perature, it was immediately reacted with DCC and
N-CBZ-glutamic acid 5-methyl ester to form tripeptide
4 in 56% yield for the two steps. The use of 1,3-
diisopropylcarbodiimide (DIPC) as coupling reagent for
formation of 4 and 11 gave slightly lower yields but with
a noticeable reduction of side products.
104 °C [lit. mp ) 100-101 °C]; [R]23 ) -26.80 (c 1.22, CH2-
D
Cl2). 1H NMR (CDCl3): δ 1.22 (3 H, t), 1.24 (3 H, d), 3.7 (2 H,
q), 4.0 (1 H, d), 4.1-4.2 (2 H, m), 4.3-4.35 (1 H, m), 4.5-4.55 (1
H, m), 5.1 (2 H, s), 7.3 (5 H, s). 13C NMR (CDCl3): δ 14.0, 17.6,
48.4, 55.5, 61.7, 63.0, 67.2, 128.0, 128.2, 128.3, 128.4, 136.0,
156.4, 170.5, 172.9. Anal. Calcd for C16H22N2O6: C, 56.78; H,
6.56; N, 8.28. Found: C, 56.65; H, 6.50; N, 8.16.
In model studies intended to optimize phosphitylation
conditions, we first attempted to phosphorylate dipeptide
11 using the commercially available diamidite reagent
bis-N,N-(diisopropylamino)methoxyphosphine [(iPr2N)2-
POMe], which, following sequential reaction with metha-
nol and oxidant, would provide the [dimethyl]phospho-
rylated dipeptide. Despite apparent clean reaction to
phosphitylated dipeptide (by TLC), the exchange of the
second amidite group with methanol was relatively slow
and poor yielding. Clean phosphitylation of the dipeptide
was achieved by use of (N,N-di-n-propylamino)bismethox-
yphosphine [(nPr)2NP(OMe)2] (22) and eliminated the
need for the second exchange reaction with methanol
(Scheme 3). Oxidation was accomplished with m-chlo-
roperoxybenzoic acid (m-CPBA; >95% purity) or magne-
sium monoperoxyphthalate (MMPP; 23) to give the
dimethylphosphorylated dipeptide 12 in 54% overall
yield.
N -(Be n zyloxyca r b on yl)se r yl-O-[O,O-d im e t h ylp h os-
p h or yl]a la n in e Eth yl Ester (12). To a stirred solution of
dipeptide 11 (0.17 g, 0.5 mmol) in 25 mL of dry CH2Cl2 were
added tetrazole (0.07 g, 1 mmol) and N,N-di-n-propylbis-
methoxyphosphine (0.15 g, 0.77 mmol). The phosphitylation
step was stirred for 1 h and m-CPBA (0.25 g, 1.45 mmol) was
added. Stirring was continued approximately 1 h or until TLC
indicated that the oxidation step (conversion to phosphoryl) was
complete. The reaction was terminated by addition of 25 mL
of ether and 25 mL of 5% NaOH, and after 0.5 h of stirring, the
organic layer was separated, washed with 5% NaHCO3 and
brine, and dried with Na2SO4. The solution was filtered and
evaporated in vacuo to yield 12 as a semisolid. Purification by
flash chromatography using EtOAc gave a white power. Yield
) 54%. 1H NMR (CDCl3): δ 1.29 (3 H, t, J ) 7.1 Hz), 1.41 (3
H, d, J ) 7.2 Hz), 3.74 (3 H, d, J ) 11.0 Hz), 3.79 (3 H, d, J )
11.1 Hz), 4.1-4.26 (4 H, m), 4.42-4.64 (4 H, m), 5.15 (2 H, s),
6.2 (1 H, brd, J ) 7.7 Hz), 7.25 (1 H, brd, J ) 6.9 Hz), 7.35 (5
H, s). 13C NMR (CDCl3): δ 14.1, 18.2, 48.4, 54.6, 54.7, 61.4,
67.1, 127.9, 128.0, 128.4, 135.8, 155.9, 167.7, 172.1. 31P NMR
(CDCl3): δ 1.8. Anal. Calcd for C18H27N2O9P: C, 48.43; H, 6.10;
N, 6.28. Found: C, 48.14; H, 5.96; N, 6.28.
Based on this success, we began to explore the syn-
thesis of the target phosphorylated tripeptides 5-10.
Using the identical phosphitylation conditions performed
using the dipeptide 11, tripeptide 4 was reacted sequen-
tially with (nPr)2N-P(OMe)2/tetrazole and m-CPBA to
afford the dimethylphosphorylated tripeptide 6 in 52%
yield (Scheme 4). The formation of 6 was monitored by
the appearance of a 31P chemical shift at δ ) 2.2. This
chemical shift correlates well with known dimethyl alkyl
phosphates (31P; δ ) -3.0 to 3.7) (24) and the model O,O-
dimethylphosphorylated dipeptide 12 (31P; δ ) 1.8).
Resu lts a n d Discu ssion
Our synthetic strategy to prepare phosphorylated
tripeptides 5-10 was partitioned into three stages: (a)
preparation of a protected Glu-Ser-Ala tripeptide se-
quence using traditional carbodiimide-based coupling, (b)
chemoselective phosphitylation of the serine hydroxyl
group by phosphoramidites (19-21), and (c) transforma-
tion of the resulting phosphitylated tripeptides into the
target phosphate and phosphorothioate moieties.
The formation of the “aged” phosphorylated tripeptide
9 was next undertaken. The “aged” tripeptide represents
loss of one of the phosphate methyl esters leading to the
phosphate monoester that likely exists as the monoanion
The native N-CBZ-Glu-Ser-Ala-OEt tripeptide (4; R )
CBZ, R1 ) OEt) (18) was prepared according to Scheme

1330 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Sch em e 4. Syn th esis of P h osp h or yla te Tr ip ep tid e Ta r gets 5, 6, a n d 9
Thompson et al.
Sch em e 5. Syn th esis of P h osp h or yla ted Tr ip ep tid es 7, 8, 10, a n d 13
at physiologic pH (25). Several hydrolysis reactions were
attempted to selectively cleave one of the phosphorus
methyl esters in the presence of the alanine ethyl ester
and glutamate 5-methyl ester, but most conditions that
removed the P-OMe group also gave hydrolysis of either
or both of the carboxylic esters. Interestingly, selective
cleavage at the Glu 5-methyl carboxy ester was realized
using LiOH (1 M, room temperature, 48 h), TMS-I
(1 equiv; 50 °C, CHCl3), or bis-tributyltin oxide (1 equiv;
toluene, reflux). Hydrolysis using NaOH gave a low yield
of a mixture of ethyl and methyl carboxy ester hydrolysis
products. The removal of one P-OMe group was suc-
cessfully conducted in 80% yield by dealkylation using
NaI in acetone or butanone at reflux. The product was
isolated and characterized as the sodium salt in an effort
to better simulate ionization of the phosphoric acid at
physiologic pH. The “aged” tripeptide 9 shows a 31P NMR
chemical shift of 4.3 ppm approximately 2 ppm downfield
from the O,O-dimethylphosphorylated tripeptide 6.
Synthesis of the tripeptide phosphorothionate target
5 was envisioned to proceed by thionation (PdO f PdS)
of phosphate 6 with P4S10 or Lawesson’s reagent (26).
Unfortunately, reaction of 6 with P4S10 showed no
conversion to the thionate after 3 days. Reaction with
Lawesson’s reagent in either toluene or benzene at reflux
yielded only a poor yield of dehydroalanine elimination
side products preliminarily identified by a lack of a 31P
signal and the appearance of geminal alkene protons in
several other reagents, we eventually found that the
target phosphorothionate 5 (31P δ ) 72.2) could be
prepared in 81% yield by phosphitylation of 4 with
[(nPr)2NP(OMe)2] followed by reaction of the intermediate
phosphite with S8 in toluene (27) at room temperature
(Scheme 4). Substituting phosphorus pentasulfide (P4S10)
for S8
gave a 28% yield. The next phosphorylated
tripeptide we sought to prepare was a model that
represents reaction of cholinesterase with O,S-dimethyl
phosphorothiolates (e.g., isoparathion methyl, isoma-
lathion) (11, 15). We first intended to use (MeO)(MeS)-
PN(iPr)2, a novel phosphoramidate reagent that following
oxidation would give 7 directly. However, we were
unable to find a suitable preparation of this reagent.
Since thionate 5 should be amenable to the thionate-
thiolate isomerization used in the preparation of simple
phosphorothiolates (8, 11), we subjected 5 to dealkylation
with potassium ethyl xanthate (PEX) to form the thioic
acid salt 8. Realkylation with dimethyl sulfate (DMS)
formed the O,S-dimethylphosphorothiolated tripeptide 7
in 56% yield (Scheme 5). Because tripeptide 7 contains
an asymmetric phosphorus atom, diastereomers at phos-
phorus are formed giving 31P peaks at δ ) 33.2 and 33.6
in a near 1:1 ratio. It is likely that no stereoselectivity
was observed because the first step occurred by a non-
discriminate dealkylation of the two phosphorus methyl
esters leading to a near equal mixture of thioic acid
diastereomers that subsequently react with dimethyl
sulfate to give 7. Since we wished to obtain chemical
1
the H NMR. After attempting the thionation of 6 with

Synthesis of Phosphorylated Tripeptides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1331
different 31P chemical shifts were found between the
sulfur-containing and non-sulfur-containing phosphate
groups attached to the serine residue; for example,
thionate (PdS) 5 and phosphate 6 (PdO)-modified tri-
peptides differ by over 70 ppm in the 31P NMR. Even
the modest substitution of a methyl thiolester (MeS-P)
for methyl ester (MeO-P) caused a 30 ppm chemical shift
difference. Importantly, a 2 ppm chemical shift differ-
ence has been established between tripeptides 7 and 10,
which represents the two truncated forms of human
serum cholinesterase inhibition by the isomalathion
stereoisomers. Since formation of 10 precludes a mech-
anism to nonreactivation and possibly delayed toxicity,
the 2 ppm chemical shift difference may help identify the
rate and extent to which this insidious postinhibitory
reaction occurs. Although the 31P chemical shifts re-
ported in this work may not precisely correlate with
chemical shifts with intact protein, the relative chemical
shift differences should be conserved and provide useful
standards for the spectroscopic analysis of OP inhibition
mechanisms.
Ch a r t 2. Ster eoisom er s of Isom a la th ion
shifts for both stereoisomers, the nonselective procedure
worked to advantage.
The intermediate tripeptide phosphorothioic acid 8
formed enroute to the O,S-dimethylphosphorothiolated
tripeptide 7 represents the second “aged” target. Al-
though phosphorothioic acids have not been formally
postulated as “aged” intermediates, they may arise from
dealkylation of a thioalkyl ligand or other related process.
Because the ionization constant of a thioic acid is
expected to be smaller than that of a phosphoric acid, it
may exist, in part, as the protonated form. Surprisingly,
the simple neutralization of 8 using a variety of dilute
acids (HCl, HOAc, H3PO4, NaH2PO4, H2SO4, etc.) did not
give 13 and led mostly to decomposition. Phosphorothioic
acid tripeptide 13 was eventually isolated in 80% yield
from 8 following neutralization of the reaction mixture
with aqueous citric acid. The 31P NMR chemical shift
for this tripeptide showed a 1:1 ratio of two peaks at 57.6
and 59.5 ppm corresponding to diastereomers at phos-
phorus.
Ack n ow led gm en t. Financial support for this work
was provided by the National Institute of Environmental
Health Sciences (Grant ESO4434). We also wish to
thank the National Science Foundation for an instru-
mentation grant (CHE9302468) used to purchase of the
400 MHz NMR instrument used in this study.
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The last target represents cholinesterase inhibited by
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(17), we found that isomalathion stereoisomers (Chart
2) inhibit cholinesterase via two predominant mecha-
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