Synthesis and characterization of photolabile choline precursors as reversible inhibitors of cholinesterase: Release of choline in the microsecond time range

Article abstract of DOI:10.1021/jo951190c

Three o-nitrobenzyl ether derivatives of choline (compounds A-C) were synthesized as photolabile cholinesterase inhibitors in order to study the mechanism of choline release from the enzyme active site. The key step of the synthesis was a simple and efficient Lewis acid-catalyzed opening of dioxolane rings derived from o-nitrobenzaldehyde and o-nitroacetophenone. Laser flash photolyses of compounds A-C were analyzed by UV spectroscopy, HPLC, and an enzymatic assay for choline. The quantum yields of photoconversion were determined, and the kinetics of choline release were analyzed by studying the decay of the transient aci-nitro intermediate at 405 nm.The observed rates varied considerably in function of both pH and the substituent at the α-benzylic position. Furthermore, we demonstrated that all three compounds possessed reversible inhibitory properties on both purified Torpedo acetylcholinesterase and purified human plasma butyrylcholinesterase. Compound A, O-[1-(2-nitrophenyl)ethyl]choline iodide, which displayed the highest quantum yield (0.27) and the most rapid photolysis rate (6.8 x 10⁴ s^-1 at pH 6.5), represents therefore an interesting tool for the study of the fast process of choline release from cholinesterases by time-resolved Laue crystallography.

Full text of DOI:10.1021/jo951190c

J . Org. Chem. 1996, 61, 185-191
185
Syn th esis a n d Ch a r a cter iza tion of P h otola bile Ch olin e P r ecu r sor s
a s Rever sible In h ibitor s of Ch olin ester a ses: Relea se of Ch olin e in
th e Micr osecon d Tim e Ra n ge
Ling Peng and Maurice Goeldner*
Laboratoire de Chimie Bio-organique, URA 1386 CNRS-Faculte´ de Pharmacie,
Universite´ Louis Pasteur Strasbourg, BP 24, 67401 Illkirch Cedex, France
Received J uly 3, 1995 (Revised Manuscript Received October 5, 1995X)
Three o-nitrobenzyl ether derivatives of choline (compounds A-C) were synthesized as photolabile
cholinesterase inhibitors in order to study the mechanism of choline release from the enzyme active
site. The key step of the synthesis was a simple and efficient Lewis acid-catalyzed opening of
dioxolane rings derived from o-nitrobenzaldehyde and o-nitroacetophenone. Laser flash photolyses
of compounds A-C were analyzed by UV spectroscopy, HPLC, and an enzymatic assay for choline.
The quantum yields of photoconversion were determined, and the kinetics of choline release were
analyzed by studying the decay of the transient aci-nitro intermediate at 405 nm. The observed
rates varied considerably in function of both pH and the substituent at the R-benzylic position.
Furthermore, we demonstrated that all three compounds possessed reversible inhibitory properties
on both purified Torpedo acetylcholinesterase and purified human plasma butyrylcholinesterase.
Compound A, O-[1-(2-nitrophenyl)ethyl]choline iodide, which displayed the highest quantum yield
(0.27) and the most rapid photolysis rate (6.8 × 104 s-1 at pH 6.5), represents therefore an interesting
tool for the study of the fast process of choline release from cholinesterases by time-resolved Laue
crystallography.
In tr od u ction
P3-ester of ATP (“caged” ATP)10 was the first example of
a thorough photochemical study, demonstrating that the
decay of an “aci-nitro” intermediate could be correlated
to the formation of ATP. Similar photochemical frag-
mentation mechanisms have also been proposed for the
photolysis of o-nitrobenzyl derivatives, such as ethers,11
carbamates,12 carboxylic esters,13 and amines.14 These
photolabile compounds have become interesting tools for
the investigation of several fast biological processes.1
In the present paper, we describe the synthesis and
characterization of three o-nitrobenzyl ethers of choline
(Scheme 1, compounds A-C) as photolabile choline
precursors which are reversible inhibitors of cholin-
esterases. These molecules differ from the classical
photolabile “caged” compounds in their capability to
interact with the enzymes, prior to photoactivation. In
particular, probe A seems best adapted for further
photoregulatory investigation of choline release from
cholinesterases.
Photolabile compounds can provide control of temporal
and spatial release of biologically interesting molecules
by rapid photolysis and are thus important tools for the
time-resolved study of fast biological processes.1 Acetyl-
cholinesterase (AChE) and butyrylcholinesterase (BuChE)
are fast-acting enzymes which catalyze the hydrolysis of
the neurotransmitter acetylcholine.2 AChE operates at
a nearly diffusion-limited rate3 and possesses a turnover
of 20 000 per second.4 Although the 3-D structures of
both AChE5 and AChE-inhibitor complexes6 as well as
the deduced model structure of BuChE7 were described,
it is still unclear how choline is rapidly released from the
active site of the enzyme in order to maintain its high
turnover. To address this problem, we conceived photo-
labile precursors of choline, as inhibitors of cholin-
esterases, to investigate the process of rapid choline
release from cholinesterases by future time-resolved
crystallographic studies.8
(8) (a) Schlichting, I.; Almo, S. C.; Rapp, G.; Wilson, K.; Petratos,
K.; Lentfer, A.; Wittinghofer, A.; Kabsch, W.; Pai, E. F.; Petsko, G. A.;
Goody, R. S. Nature 1990, 345, 309-315. (b) Pai, E. M. Curr. Biol.
1992, 2, 821-827. (c) Time-resolved macromolecular crystallography.
Philos. Trans. R. Soc. London A 1992, 340, 167-334.
(9) Pillai, V. N. R. Synthesis 1980, 1, 1-26.
o-Nitrobenzyl groups have been used as photolabile
protecting groups9 to mask or “cage” biological molecules.1
The photochemical release of ATP from the o-nitrobenzyl
X Abstract published in Advance ACS Abstracts, December 15, 1995.
(1) (a) Corrie, J . E. T.; Trentham, D. R. in Bioorganic Photochem-
istry; Morrison, H., Ed.; Wiley: New York, 1993; Vol. 2, pp 243-305.
(b) Hess, G. P. Biochemistry 1993, 32, 989-1000.
(2) (a) Barnard, E. A. in The Peripheral Nervous System; Hubbard,
J . L., Ed.; Plenum: New York, 1974; pp 201-224. (b) Chatonnet, A.;
Lockridge, O. Biochem. J . 1989, 260, 625-634.
(3) (a) Hasinoff, B. B. Biochim. Biophys. Acta 1982, 704, 52-58. (b)
Bazelyansky, M.; Robey, E.; Kirsch, J . F. Biochemistry 1986, 25, 125-
130.
(10) Walker, J . W.; Reid, G. P.; McCray, J . A.; Trentham, D. R. J .
Am. Chem. Soc. 1988, 110, 7170-7177.
(11) (a) Walker, J . W.; Martin, H.; Schmitt, F. R.; Barsotti, R. J .
Biochemistry 1993, 32, 1338-1345. (b) Corrie, J . E. T. J . Chem. Soc.,
Perkin Trans. 1 1993, 2161-2166.
(12) (a) Walker, J . W.; McCray, J . A.; Hess, G. P. Biochemistry 1986,
25, 1799-1805. (b) Milburn, T.; Matsubara, N.; Billington, A. P.;
Udgaonkar, J . B.; Walker, J . W.; Carpenter, B. K.; Webb, W. W.;
Marque, J .; Denk, W.; McCray, J . A.; Hess, G. P. Biochemistry 1989,
28, 49-55.
(13) (a) Wilcox, M.; Viola, R. W.; J ohnson, K. W.; Billington, A. P.;
Carpenter, B. K.; McCray, J . A.; Guzikowski, A. P.; Hess G. P. J . Org.
Chem. 1990, 55, 1585-1589. (b) Wieboldt, R.; Gee, K. R.; Niu, L.;
Ramesh, D.; Carpenter, B. K.; Hess, G. P. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 8752-8756. (c) Gee, K. R.; Wiedoldt, R.; Hess, G. P. J . Am.
Chem. Soc. 1994, 116, 8366-8367.
(14) (a) Muralidharan, S.; Maher, G. M.; Boyle, W. A.; Nerbonne, J .
M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5199-5203. (b) Wieboldt,
R.; Ramesh, D.; Carpenter, B. K.; Hess, G. P. Biochemistry 1994, 33,
1526-1533.
(4) (a) Rosenberry, T. L. Adv. Enzymol. 1975, 43, 103-208. (b)
Quinn, D. M. Chem. Rev. 1987, 87, 955-979.
(5) Sussman, J . L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.;
Toker, L.; Silman, I. Science 1991, 253, 872-879.
(6) Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner,
M.; Hirth, C.; Axelsen, P. H.; Silman, I.; Sussman, J . L. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 9031-9035.
(7) Harel, M.; Sussman, J . L.; Krejci, E.; Bon, S.; Chanal, P.;
Massoulie´, J .; Silman, I. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 10827-
10831.
0022-3263/96/1961-0185$12.00/0 © 1996 American Chemical Society

186 J . Org. Chem., Vol. 61, No. 1, 1996
Peng and Goeldner
Sch em e 1. Syn th esis of 2-Nitr oben zyl Eth er s of Ch olin e, Com p ou n d s A-Ca
a
Reagents and conditions: (i) NaBH3CN, TiCl4, CH3CN, rt, 2 h; (ii) MsCl, TEA, ether, rt, 1 h; (iii) NaI, acetone, reflux, 2 h; (iv) NMe3,
toluene, rt, 24 h; (v) TMSCN, ZnI2, rt, 15 min; (vi) (a) BH3‚THF, rt, 4 h, (b) 6 N HCl, (c) 4 N NaOH; (vii) di-tert-butyl dicarbonate, TEA,
THF, rt, 45 min; (viii) (a) CF3COOH, CH2Cl2, rt, 10 min, (b) CSCl2, NaHCO3, CH2Cl2/H2O, rt, 10 min.
Sch em e 2. P r op osed Mech a n ism of th e P h otoch em ica l Rea ction of 2-Nitr oben zyl Eth er s of Ch olin e
Resu lts
Ta ble 1. Sp ectr a l P r op er ties, HP LC Ch a r a cter iza tion ,
a n d Qu a n tu m Yield s of com p ou n d s A-C
Syn th esis of 2-Nitr oben zyl Ch olin e Der iva tives
A-C. The synthetic routes to the three photolabile
choline derivatives (compounds A-C) are outlined in
Scheme 1. The introduction of the choline moiety used
a common strategy for all three compounds, namely the
opening of the cyclic acetal 1a and ketal 1b.
Treatment of ketal 1a with sodium cyanoborohydride,
in the presence of TiCl4,15 led to the opening of the
dioxolane ring and yielded ether 2a which was subse-
quently mesylated (3a ) and iodinated to give 4a . Reac-
tion of 4a with trimethylamine in dry toluene led to the
final product A. The use of dry toluene as solvent
facilitated the final isolation and purification of quater-
nary ammonium compounds as they precipitated from
the reaction medium. Starting from acetal 1a , compound
B was prepared in the same way.
Acetal 1b was treated with trimethylsilyl cyanide in
the presence of ZnI216 to give nitrile 5, which was further
reduced with BH3‚THF to the corresponding amine 6 and
protected as the tert-butyl carbamate 7. This compound
was subsequently mesylated (8) and transformed to the
corresponding iodide 9. The removal of the Boc protect-
ing group in 9 by trifluoroacetic acid, followed by treat-
ment with thiophosgene, gave the isothiocyanate deriva-
tive 10. The final compound C was obtained by a SN2-
substitution of the iodine in 10 with trimethylamine in
toluene.
a
c
λmax
,
nm
ꢀ,a M-1 cm-1
tR,b min
Φ
A
B
C
261
261
260
5300
5000
4300
11.1
9.3
16.3
0.27d
0.19e
0.26d
a
Absorption properties were determined in 0.1 M phosphate
b
buffer, pH 7.2, at 20 °C. The HPLC retention time (tR) of each
compound on a reversed phase column using acetonitrile (40%)
and a solution of 5 mM sodium dodecylsulfate and 5 mM sodium
sulfate at pH 2.00 (60%). c Quantum yields (Φ). Determined
d
relative to 1-(2-nitrophenyl)ethyl carbamylcholine. e Determined
relative to compound C as a reference.
pounds A-C showed a maximum around 260 nm (Table
1), which is characteristic of the 2-nitrobenzyl moiety.
During laser flash photolysis of A, a decrease in absorp-
tion at 260 nm was observed concomitant with an
increase at 225 nm and the appearance of a new peak at
310 nm (Figure 1a), corresponding to the formation of
the aromatic nitroso byproduct (Scheme 2). The observed
isobestic points at 275, 248, and 210 nm are consistent
with a unique photodecomposition process. This was
further confirmed by HPLC analysis using an ion-pair
partitioning technique on a reversed phase column
(Figure 1b). To avoid the positively charged molecules
A-C being immediately eluted in the void volume, we
used the negatively charged dodecyl sulfate as a coun-
terion which conferred to these probes a reasonable
retention time (Table 1). Figure 1b shows an HPLC
analysis during the photochemical decomposition of
compound A. The photolytic byproduct, 2-nitroso-
acetophenone, eluted earlier (retention time of 6.1 min)
than the parent compound (retention time of 9.0 min).
The decay of compound A was concomitant with the
P h otoch em ica l Relea se of Ch olin e fr om Com -
p ou n d s A-C. The UV-vis absorption spectra of com-
(15) Adam, G.; Seebach, D. Synthesis 1988, 5, 373-375.
(16) Kirchmeyer, S.; Mertens, A.; Arvanaghi, M.; Olah, G. A.
Synthesis 1983, 6, 498-500.

Release of Choline in the Microsecond Time Range
J . Org. Chem., Vol. 61, No. 1, 1996 187
F igu r e 1. Laser flash photolysis of compound A was analyzed by (a) UV spectroscopy, (b) HPLC analysis, and (c) an enzymatic
assay specific for choline. A solution of 1 mM compound A in 100 mM phosphate buffer at pH 6.5 was exposed to 351 nm laser
flashes. (a) UV spectral recording of the photolysis. The lowest trace at 310 and 225 nm and the highest trace at 260 nm correspond
to the starting material. b) Aliquots of irradiated sample (100 µL) were analyzed by HPLC with acetonitrile (45%) and a solution
containing 5 mM sodium dodecylsulfate, 5 mM sodium sulfate at pH 2.00 (55%). Compound A has an HPLC retention time of 9.0
min, and the appearing peak at 6.1 min corresponds to the photolysis byproduct, 2-nitrosoacetophenone. (c) The photoreleased
choline was quantified by an enzymatic assay17 using 50 µL aliquots of samples (see Experimental Section).
formation of the corresponding aromatic nitroso product,
implying a stoichiometric photoconversion with no other
UV-absorbing byproducts being observed during this
photolytic reaction.
transients have been observed with a variety of 2-nitro-
benzyl compounds and were attributed to the aci-nitro
species1 (Scheme 2). Most importantly, the decay of this
species can be used as a kinetic measure of release of
the corresponding product.10 For compound A, the decay
of the aci-nitro transient is a first-order reaction (Figure
2b), while a two-component decay was observed for
compounds B and C (data not shown). Furthermore, the
decay rate of the transient was first order in [H+] (Figure
2c and Table 2) and was sensitive to substitution at the
R-benzylic position (Table 2). The aci-nitro signal for
compound A decayed about 50 times faster than com-
pound C and approximately 800 times faster than
compounds B at pH 6.5, 20 °C (Table 2). The fastest
photolysis rate (6.8 × 104 s-1) was observed with com-
pound A at pH 6.5, a rate which is comparable with the
turnover rate of acetylcholinesterase.
Biologica l P r op er ties of Com p ou n d s A-C. Both
AChE and BuChE display high affinity for quaternary
ammonium ligands through specific interaction with
aromatic residues at the active sites of the enzymes.4,6,18
All three compounds A-C showed reversible inhibitory
properties on both purified Torpedo acetylcholinesterase
(mixed type of inhibition) and purified human plasma
butyrylcholinesterase (competitive inhibition) with inhi-
bition constants in the 10-5-10-6 M range (Table 3).
AChE and BuChE remained fully stable when exposed
to 351-nm laser flashes. The photolytic byproduct from
compound A, 2-nitrosoacetophenone, which could be
scavenged by dithiothreitol or glutathione as previously
described,1 had no toxic effects on the activity of cho-
linesterases, even at 1 mM concentration (data not
shown).
In this study, the photoproduct of primary interest was
choline. After photolysis of compounds A-C, the re-
leased choline was detected and quantified with a specific
enzymatic assay17 (Figure 1c). The amount of choline
formed increased with increasing exposure to light and
coincided with the disappearance of the parent compound
A (Figure 1). This result also indicated a stoichiometric
conversion of choline from its precursor. Similar results
were also obtained for compounds B and C (data not
shown).
Qu a n tu m Yield s for P h otocon ver sion . The quan-
tum yields for the photoconversion of compounds A-C
were determined by comparison with the photolysis of
1-(2-nitrophenyl)ethyl carbamylcholine (Φ ) 0.25).12 The
extent of the photolytic conversion in equimolar mixtures
of choline derivatives and the reference compound was
analyzed on HPLC (see Experimental Section). Com-
pounds A and C photolyzed as efficiently as 1-(2-nitro-
phenyl)ethyl carbamylcholine and possessed quantum
yields of 0.27 and 0.26, respectively (Table 1). However,
1-(2-nitrophenyl)ethyl carbamylcholine could not be used
for the determination of the quantum yield of compound
B because of their similar HPLC retention time, and we
therefore used compound C as an internal reference. As
compound B photolyzed 1.25-fold less efficiently than
compound C, a quantum yield of 0.19 was deduced for
compound B.
La ser F la sh Kin etic Stu d ies. The kinetics of the
photolysis of compounds A-C was analyzed by monitor-
ing the formation and decay of the presumed aci-nitro
intermediate. Laser flash photolysis of A-C induced a
rapid increase in absorbance around 400 nm, followed
by an exponential decay to the initial level (Figure 2).
The short-lived transient was detected with a delay of
0.2 µs after laser flash photolysis of A and showed a
spectral maximum around 405 nm (Figure 2a). Similar
Discu ssion
The goal of this work was to synthesize and character-
ize photolabile precursors of choline to be used as
cholinesterases inhibitors allowing a fast and efficient
photorelease of choline. Such photolabile inhibitors
should fulfill a number of criteria: they should show
(17) Takayama, M.; Itoh, S.; Nagasaki, T.; Tanimizu, I. Clin.
Chimica Acta, 1977, 79, 93-98.
(18) Dougherty, D. A.; Stauffer, D. A. Science 1990, 250, 1558-1560.

188 J . Org. Chem., Vol. 61, No. 1, 1996
Peng and Goeldner
Ta ble 3. In h ibition Con sta n ts of Com p ou n d s A-C on
ACh E a n d Bu Ch E
AChE (M)
BuChE (M)
A
B
C
1.30 × 10-5
1.00 × 10-5
3.73 × 10-6
1.11 × 10-5
1.94 × 10-5
3.13 × 10-6
the synthesis of 2-nitrobenzyl ether derivatives,11 none
of them seemed suitable for the synthesis of the com-
pounds A-C. The basic reaction conditions of William-
son ether synthesis gave mainly tarry mixtures, while
the silver(I) oxide catalyzed condensations19 proved un-
successful for the 2-nitrobenzyl ether series (data not
shown). The syntheses of compounds A-C (Scheme 1)
in the present work took advantage of the possibility of
using cyclic ketals and acetals as precursors of the choline
moiety. Noticeably, the reductive and nucleophilic open-
ings of the dioxolane rings were achieved in near-
quantitative yield in the presence of Lewis acids (ZnI2
and TiCl4).
Previous work1,9,11 has shown that the 2-nitrobenzyl
ethers can be readily and efficiently photodecomposed.
Photorelease of choline from compounds A-C was in-
vestigated and analyzed by UV spectra, HPLC, and an
enzymatic assay for choline (Figure 1). The kinetics of
choline release was analyzed by studying the decay of
the transient aci-nitro intermediate (Scheme 2). The
observed decay rates were very different for the three
compounds A-C (Table 2), showing a fast exponential
decay for compound A but a slower biphasic process20 for
both compounds B and C. The strong dependence of the
decay rate on the substituent at the R-benzylic position,
although well-documented in analogous series,11-13 is in
our case remarkable by its extent; i.e., 4 orders of
magnitude difference were found between the decay rates
observed when R ) CH3 (A) and R ) H (B). Further-
more, the observed first order pH-dependence of the
decay rates (compound A, Figure 2c) is in agreement with
the proposed mechanism (Scheme 2). Compound A
shows excellent kinetic properties (k ) 6.8 × 104 s-1 at
pH 6.5, 20 °C) for photolytic release of choline, one of the
fastest photolysis reactions described with o-nitrobenzyl
derivatives. This photolytic release of choline from probe
A in the microsecond time range (t1/2 ≈ 10 µs) is even
faster than the enzymatic choline release from acetyl-
cholinesterase (≈50 µs).4 Finally, the observed quantum
yields for the three probes A, B and C, respectively, 0.27,
0.19 and 0.26 (Table 1), are sufficient to ensure an
efficient photorelease of choline.
F igu r e 2. Kinetic analysis on the laser flash photolysis of
compound A. A solution of 1 mM compound A in 0.1 M
phosphate buffer was exposed to a single 351 nm laser pulse
at 20 °C. (a) The UV spectrum of a transient was observed by
recording the spectral change before and after (0.2 µs delay)
laser flash photolysis of compound A. (b) Kinetic record at 405
nm after a single laser flash photolysis of compound A at pH
6.5. Arrow indicates the begining of the laser flash. The
transient was formed immediately and followed by an expo-
nential decay. (c) Rate constants for the decay of the transient
intermediate in the photolysis of compound A as a function of
pH.
Ta ble 2. Ha lf-Life (t1/2) of th e a ci-Nitr o Tr a n sien ts fr om
Com p ou n d s A-C a s a F u n ction of p H
All three compounds are reversible inhibitors of both
AChE and BuChE. The inhibition constants are in the
µM range (Table 3) and are expected reasonable values
for aromatic molecules carrying a quaternary ammonium
group.4 Clearly, probe C did not react covalently with
the catalytic serine as anticipated, suggesting an un-
favorable positioning of the isothiocyanato moiety within
the active site. Furthermore, the photolytic byproduct
of compound A, 2-nitrosoacetophenone, has no toxic effect
on the enzymes even at concentrations up to 1 mM.
AChE is a fast-acting serine protease which terminates
signals at cholinergic synapses.21 Despite the report on
t1/2, pH 6.5a
t1/2, pH 7.2a
t1/2, pH 8.1a
A
B
C
10.2 µs
110 µs
525 µs
82.3 msb
0.56 msb
521 msb
1.11 msb
2.42 sb
1.84 msb
a
Spectral transients observed in flash photolysis were analyzed
b
in 0.1 M phosphate buffer at 20 °C. Value corresponding to the
slower phase of the decay rate were presented.
inhibitory effect on the enzymes prior to photolysis; they
should photorelease choline efficiently and rapidly; and
the byproducts of photolysis should be chemically inert,
biologically inactive, and nontoxic for the enzymes.
Three photolabile precursors of choline, A-C, were
synthesized. Each contains a 2-nitrobenzyl moiety at-
tached via an ether linkage to the oxygen of choline.
Although several methods had previously been used for
(19) Fukase, K.; Tanaka, H.; Torii, S.; Kusumoto, S. Tetrahedron
Lett. 1990, 31, 389-392.
(20) Biphasic decays have already been reported for ortho-nitroben-
zyl ethers (ref 11b).
(21) Barnard, E. A. In The Peripheral Nervous System; Hubbard, J .
I., Ed.; Plenum: New York, 1974, pp 201-224.

Release of Choline in the Microsecond Time Range
J . Org. Chem., Vol. 61, No. 1, 1996 189
the 3-D structure of AChE,5 the mechanism of the rapid
clearance of choline from the active site of the enzyme
remains unclear.22,23 This problem could be addressed
by time-resolved Laue crystallography at the atomic
level8 with suitable photolabile molecules that can release
choline rapidly and efficiently. Among the synthesized
photolabile inhibitors of cholinesterases, compound A is
the most promising candidate for such studies because
of its ability of rapid photorelease of choline with high
quantum yield.
(m, 1H), 7.62-7.69 (m, 1H), 7.76 (dd, 1H, J ) 7.9, 1.4 Hz),
7.89 (dd, 1H, J ) 8.1, 1.1 Hz).
2-[(2-Nitr oben zyl)oxy]eth a n ol (2b). According to the
general procedure I, acetal 1b (39 mg, 0.20 mmol) in CH3CN
(1 mL) was treated with titanium tetrachloride (0.25 µL, 0.22
mmol) and sodium cyanoborhydride (13.2 mg, 0.21 mmol).
Purification on silica gel with EtOAc/hexane 1:4 gave 2b (37
mg, 95%) as a colorless oil: 1H NMR (CDCl3) δ 2.02 (br s, 1H),
3.70-3.74 (m, 2H), 3.84 (br m, 2H), 4.96 (s, 2H), 7.43-7.50
(m, 1H), 7.63-7.71 (m, 1H), 7.78-8.00 (m, 1H), 8.07 (dd, 1H,
J ) 8.1, 1.2 Hz).
Meth a n esu lfon ic Acid 2-[(1-Nitr op h en yl)eth oxy]eth yl
Ester (3a ). According to the general procedure II, alcohol 2a
(0.46 g, 2.2 mmol) in ether (10 mL) was treated with triethyl-
amine (0.40 mL, 2.9 mmol) and methanesulfonyl chloride (0.19
mL, 2.4 mmol). Purification on silica gel with hexane/EtOAc
1:1 gave 3a (0.61 g, 97%) as a colorless oil: 1H NMR (CDCl3)
δ 1.54 (d, 3H, J ) 6.5 Hz), 3.10 (s, 3H), 3.47-3.67 (m, 2H),
4.28-4.39 (m, 2H), 5.07 (q, 1H, J ) 6.4 Hz), 7.40-7.48 (m,
1H), 7.63-7.71 (m, 1H), 7.76-7.80 (m, 1H), 8.06 (dd, 1H, J )
8.1, 1.1 Hz).
Meth a n esu lfon ic Acid 2-[(2-Nitr oben zyloxy)eth yl Es-
ter (3b). According to the general procedure II, alcohol 2b
(35 mg, 0.18 mmol) in ether (0.5 mL) was treated with
triethylamine (32 µL, 0.23 mmol) and methanesulfonyl chloride
(15 µL, 0.19 mmol). Purification on silica gel with hexane/
EtOAc 1:1 gave 3b (0.48 mg, 98%) as a colorless oil: 1H NMR
(CDCl3) 3.10 (s, 3H), 3.84-3.89 (m, 2H), 4.43-4.47 (m, 2H),
4.96 (s, 2H), 7.42-7.51 (m, 1H), 7.63-7.71 (m, 1H), 7.76-7.80
(m, 1H), 8.06 (dd, 1H, J ) 8.1, 1.2 Hz).
Exp er im en ta l Section
Melting points are uncorrected. Elemental analyses and
mass spectral data were obtained at the Faculte´ de Chimie,
Universite´ Louis Pasteur, Strasbourg. 1H NMR and 13C NMR
were run at 200 and 50 MHz, respectively. Chemical shifts
are given in parts per million (ppm) using the residue solvent
peaks as reference relative to TMS. A C18 column (3.9 × 300
mm) was used for HPLC analysis.
Acetylcholinesterase and butyrylcholinesterase were puri-
fied as described.24,25 2-Methyl-2-(2-nitrophenyl)-1,3-dioxolane
(1) and 2-(2-nitrophenyl)-1,3-dioxolane (2) were prepared from
2-nitroacetophenone and 2-nitrobenzylaldehyde, respectively.
Isothiocyanate derivative was purified on neutral aluminum
oxide 90 (70-230 mesh) by column chromatography; all other
compounds were purified by flash column chromatography on
silica gel (230-400 mesh).
1-[1-(2-Iod oeth oxy)eth yl]-2-n itr oben zen e (4a ). Accord-
ing to the general procedure III, 3a (0.71 g, 2.5 mmol) in
acetone (20 mL) was treated with sodium iodide (1.9 g, 12.5
mmol). Purification on silica gel with hexane/ether 4:1 gave
4a (0.78 g, 97%) as a colorless oil: 1H NMR (CDCl3) δ 1.55 (d,
3H, J ) 6.3 Hz), δ 3.24 (t, 2H, J ) 6.50 Hz), 3.47-3.67 (m,
2H), 5.09 (q, 1H, J ) 6.3 Hz), 7.39-7.48 (m, 1H), 7.64-7.71
(m, 1H), 7.84 (dd, 1H, J ) 7.9, J ) 1.4), 7.92 (dd, 1H, J ) 8.2,
1.2 Hz).
1-[(2-Iod oeth oxy)m eth yl]-2-n itr oben zen e (4b). Accord-
ing to the general procedure III, 3b (45 mg, 0.16 mmol) in
acetone (0.5 mL) was treated with sodium iodide (120 g, 0.8
mmol). Purification on silica gel with hexane/ether 4:1 gave
4b (50 mg, 96%) as a colorless oil: 1H NMR (CDCl3) δ 3.36 (t,
2H, J ) 6.50 Hz), 3.87 (t, 2H, J ) 6.50 Hz), 4.97 (s, 2H), 7.42-
7.50 (m, 1H), 7.64-7.71 (m, 1H), 7.87 (d, 1H, J ) 7.8 Hz), 8.06
(dd, 1H, J ) 8.1, 1.2 Hz).
Syn th eses. Gen er a l P r oced u r e I for th e Red u ctive
Op en in g of Keta l or Aceta l. To a solution of the ketal 1a
or acetal 1b (1 equiv) in CH3CN at 0 °C were added titanium
tetrachloride (1.3 equiv) and sodium cyanoborohydride (1.2
equiv). The resulting yellow suspension was stirred at room
temperature for 2 h and then neutralized with saturated
NaHCO3 solution before extraction of the product into CH2Cl2.
The organic layer was dried over MgSO4, the solvent was
evaporated with a rotary evaporator, and the crude product
was purified by flash chromatography.
Gen er a l P r oced u r e II for th e P r ep a r a tion of th e
Mesyla te Der iva tives 3a ,b a n d 8. To a solution of the
corresponding alcohol (2a ,b, 7) in ether were added triethyl-
amine (1.3 equiv) and methanesulfonyl chloride (1.1 equiv).
After 1 h at 25 °C, the organic solvent was removed and the
residue was purified by flash chromatography.
Gen er a l P r oced u r e III for th e P r ep a r a tion of th e
Iod id e Der iva tives 4a ,b a n d 9. Sodium iodide (5 equiv) was
added to a solution of the mesylate derivative (3a ,b and 8) in
acetone. The reaction mixture was refluxed for 2 h, and then
the organic solvent was removed and the residue was purified
by flash chromatography.
Gen er a l P r oced u r e IV for th e SN2 Su bstitu tion of 4a ,b
a n d 9 w ith Tr im eth yla m in e. The iodide derivative (4a , b
and 9) was treated with a solution of toluene saturated with
trimethylamine. The reaction mixture was stirred at 25 °C
for 24 h. The precipitate was collected by centrifugation,
washed five times with toluene, and dried in vacuo.
O-[1-(2-Nitr op h en yl)eth yl]ch olin e Iod id e (A). Accord-
ing to the general procedure IV, the iodide 4a (128 mg, 0.37
mmol) was treated with toluene (20 mL) saturated with
trimethylamine. Purification by washing the precipitate with
toluene gave compound A (135 mg, 90%) as a light yellow
1
solid: mp 138-139 °C; H NMR (CD3CN) δ 1.58 (d, 3H, J )
6.3 Hz), 3.25 (s, 9H), 3.53-3.85 (m, 4H), 5.11 (q, 1H, J ) 6.3
Hz), 7.53-7.63 (m, 1H), 7.74-7.81 (m, 2H), 7.98 (ddd, 1H, J
) 8.0, 0.7, 0.7 Hz); 13C NMR (CD3CN) δ 22.7, 54.3, 62.9, 66.0,
74.2, 124.6, 128.0, 129.0, 134.2, 138.2; MS (C13H21N2O3, FAB
positive) 253.2 g/mol. Anal. Calcd for C13H21N2O3I: C, 41.07;
H, 5.58; N, 7.37. Found: C, 41.26; H, 5.57; N, 7.40.
2-[1-(2-Nitr op h en yl)eth oxy]eth a n ol (2a ). According to
the general procedure I, ketal 1a (0.59 g, 2.8 mmol) in CH3CN
(14 mL) at 0 °C was treated with titanium tetrachloride (0.40
mL, 3.7 mmol) and sodium cyanoborohydride (0.21 g, 3.4
mmol). Purification on silica gel with hexane/EtOAc 4:1
yielded product 2a (0.57 g, 95%) as a colorless oil: 1H NMR
(CDCl3) δ 1.52 (d, 3H, J ) 6.3 Hz), 2.20 (br s, 1H), 3.31-3.50
(m, 2H), 3.71 (br m, 2H), 5.05 (q, 1H, J ) 6.3 Hz), 7.38-7.47
O-(2-Nitr oben zyl)ch olin e Iod id e (B). According to the
general procedure IV, the iodide 4b (128 mg, 0.37 mmol) was
treated with toluene (20 mL) saturated with trimethylamine.
Purification by washing the precipitate with toluene gave
compound B (135 mg, 90%) as a light yellow solid: mp 148-
1
150 °C; H NMR (CD3CN) δ 3.18 (s, 9H), 3.62-3.66 (m, 2H),
3.97-4.05 (m, 2H), 4.94 (s, 1H), 7.55-7.62 (m, 1H), 7.75-7.79
(m, 2H), 8.07 (d, 1H, J ) 8.0 Hz); MS (C12H19N2O3, FAB
positive) 239.2 g/mol. Anal. Calcd for C12H19N2O3I: C, 39.36;
H, 5.23; N, 7.65. Found: C, 39.56; H, 5.17; N, 7.40.
(2-Nit r op h en yl)[2-[(t r im et h ylsilyl)oxy]et h oxy]a cet o-
n itr ile (5). Solid zinc iodide (20 mg) and trimethylsilyl
chloride (0.18 mL, 1.4 mmol) were successively added to 1b
(0.25 g, 1.3 mmol). After 5 min of stirring at room tempera-
ture, the reaction mixture was purified by chromatography
with hexane/ether 1:1 to afford product 5 (0.30 g, 80%) as a
colorles oil: 1H NMR (CDCl3) δ 0.15 (s, 9H), 3.82-3.95 (m,
(22) Gilson, M. K.; Straatsma, T. P.; McCammon, J . A.; Ripoll, D.
R.; Faerman, C. H.; Axelsen, P. H.; Silman, I.; Sussman, J . L. Science
1994, 263, 1276-1278.
(23) Kronman, C.; Ordentlich, A.; Barak, D.; Velan, B.; Schafferman,
A. J . Biol. Chem. 1994, 269, 27819-27822.
(24) Ehret-Sabatier, L.; Schalk, I.; Goeldner, M.; Hirth, C. Eur. J .
Biochem. 1992, 203, 475-481.
(25) Lockridge, O., La Du, B. N. J . Biol. Chem. 1978, 253, 361-
366.

190 J . Org. Chem., Vol. 61, No. 1, 1996
Peng and Goeldner
4H), 6.12 (s, 1H), 7.58-7.66 (m, 1H), 7.72-7.80 (m, 1H), 8.00
(d, 1H, J ) 8.3 Hz), 8.14 (d, 1H, J ) 8.1 Hz); IR (CHCl3) 2338
cm-1 (vC≡N).
15.9, 5.9 Hz), 4.10-4.16 (m, 1H), 4.33 (dd, 1H, J ) 15.9, 3.4
Hz), 5.54 (dd, 1H, J ) 6.0, 3.4 Hz), 7.68-8.06 (m, 3H), 8.29
(d, 1H, J ) 8.1 Hz,); 13C NMR (CD3CN) δ 50.2, 54.6, 63.7, 66.2,
76.4, 125.6, 129.2, 130.4, 133.0, 134.8, 148.2, 153.1; IR (KBr)
2061, 2139 cm-1 (v S)C)N); MS (C14H20N3O3S, FAB positive)
310.1 g/mol. Anal. Calcd for C14H20N3O3SI: C, 38.45; H, 4.61;
N, 9.61. Found: C, 38.64; H, 4.83; N, 9.60.
La ser F la sh P h otolysis. An excimer laser was operated
at 351 nm (XeF) with a pulse energy of 100-150 mJ and a
pulse width of about 20 ns. A pulsed Xe high-pressure arc
was used as the monitoring light. A shutter disposed between
the lamp and the sample cell (pathlength ) 4 cm) was opened
shortly before the laser flash to avoid photolysis of the starting
compound by the Xe arc. The laser flash was perpendicular
to the probing light from the pulsed Xe lamp.
Tr a n sien t Sp ectr u m Detection . The detection system
allowed the capture of the transient spectrum (300-600 nm)
at a given time delay after excitation (time window g5 ns).
The analyzing light was split with a prism and was focused
with a lens on the entrance slit of polychromator of OMA
(optical multichannel analyzer). The transient spectrum was
captured by a diode array camera viewing a gateable micro-
channel plate image intensifier. The absorption spectrum was
observed after a laser shot by using the spectral distribution
of the monitoring light prior to the laser pulse as the reference
light intensity at each wavelength.
Kin etics Recor d s. The detection system allowed the
simultaneous capture of the kinetics at a given wavelength
by using a transient digitizer. The analyzing light through
the cuvette was gathered with a lens and focused on the
entrance slit of a monochromator. The signal was recorded
by a photomultiplier and fed into the transient digitizer. Rate
constants were calculated by least-squares fitting of decay
curves with single or dual exponential function, as appropriate.
HP LC An a lysis of th e P h otolysis of Com p ou n d s A-C.
A solution (4 mL) of 1 mM A in 100 mM phosphate buffer, pH
6.5 was exposed to 351-nm laser flashes. Aliquots of samples
(100 µL) were injected into a reversed phase column, which
was equilibrated with acetonitrile (45%) and a solution con-
taining 5 mM sodium dodecylsulfate, 5 mM sodium sulfate at
pH 2.00 (55%), at a flow rate of 1.0 mL/min. The compounds
were detected by UV absorption at 310 nm. Compound A has
a retention time of 9.0 min and the corresponding photolytic
byproduct, 2-nitrosoacetophenone, a retention time of 6.1 min.
Experiments with B and C were performed similarly.
Exten t of P h otolysis of Com p ou n d s A-C. Solutions of
equimolar mixtures of A-C and 1-(2-nitrophenyl)ethyl car-
bamylcholine (each 0.26 mM) in 20 mM sodium phosphate pH
7.0 were exposed for varying times (10 to 60 s) to 365-nm line
from a 1000 W xenon-mercury lamp. Aliquots of the irradi-
ated samples (40 µl) were analyzed by reversed phase HPLC
(40% acetonitrile, 60% of a solution containing 5 mM sodium
dodecylsulfate, 5 mM sodium sulfate, pH 2.00). The HPLC
retention time for compounds A-C and 1-(2-nitrophenyl)ethyl
carbamylcholine were 11.1, 9.3, 16.3, and 9.1 min, respectively.
En zym a tic Assa y17 for Ch olin e. A solution (4 mL) of 1
mM A in 100 mM phosphate buffer, pH 6.5 was subjected to
351-nm laser flashes. Aliquots of samples (50 µL) were taken
and added to a 950 µL solution containing 1.6 units of choline
oxidase, 4.3 units of peroxidase, 0.74 mM 4-aminoantipyrene,
0.34 mM CaCl2‚H2O, and 5.3 mM phenol in 50 mM Tris buffer,
pH 7.8. After 20 min at 25 °C, the solution turned to red and
its absorbance was measured at 505 nm. The corresponding
amount of choline (Figure 1c) was deduced from a standard
reference. Experiments with B and C were performed simi-
larly.
2-[2-Am in o-1-(2-n itr op h en yl)eth oxy]eth a n ol (6). To the
nitrile 5 (0.96 g, 3.3 mmol) was added dropwise a 1 M
BH3‚THF solution (40 mL) at 0 °C. After 4 h of stirring at 25
°C, 6 N HCl (40 mL) was added to the reaction mixture, giving
a white precipitate. After evaporation of the organic solvent
in vacuo, the aqueous phase was basified with 4 N NaOH to
pH 10. The product was extracted with EtOAc, and the
organic phase was dried over MgSO4 and reduced in vacuo.
The residue (0.68 g, 92%) was purified by flash chromatogra-
phy with dichloromethane/methanol/triethylamine 8:2:1 to give
the amine 6 as a colorless oil: 1H NMR (CDCl3) δ 2.55 (br s,
3H), 2.83 (dd, 1H, J ) 13.4, 8.3 Hz), 3.15 (dd, 1H, J ) 13.4,
2.8 Hz), 3.39-3.62 (m, 2H), 3.68-3.78 (m, 2H), 5.00 (dd, 1H,
J ) 8.3, 2.7 Hz), 7.40-7.49 (m, 1H), 7.62-7.70 (m, 1H), 7.78
(dd, 1H, J ) 7.9, 1.2 Hz), 7.97 (dd, 1H, J ) 8.1, 0.9 Hz).
[2-(2-Hyd r oxyeth oxy)-2-(2-n itr op h en yl)eth yl]ca r ba m -
ic Acid ter t-Bu tyl Ester (7). To a solution of the amine 6
(0.43 g, 1.9 mmol) in THF (8 mL) were added triethylamine
(0.27 mL, 1.9 mmol) and di-tert-butyl dicarbonate (0.41 g, 1.9
mmol). The reaction mixture was stirred at room temperature
for 45 min and purified by flash chromatography. Elution with
hexane/EtOAc 1:2 afforded 0.63 g (98%) of the product 7: 1H
NMR (CDCl3) δ 1.37 (s, 9H), 2.41 (br s, 1H), 3.38-3.53 (m,
4H), 3.72 (br s, 2H), 5.03 (dd, 1H, J ) 5.2, 5.0 Hz), 5.13 (br s,
1H), 7.41-7.49 (m, 1H), 7.60-7.69 (m, 1H), 7.76 (dd, 1H, J )
8.0, 1.5 Hz), 8.00 (dd, 1H, J ) 8.2, 1.0 Hz).
Met h a n esu lfon ic Acid 2-[2-[(ter t-Bu t oxyca r b on yl)-
a m in o]-1-(2-n itr op h en yl)eth oxy]eth yl Ester (8). Accord-
ing to the general procedure II, alcohol 7 (0.59 g, 1.8 mmol) in
ether (8 mL) was treated with triethylamine (0.30 mL, 2.2
mmol) and methanesulfonyl chloride (0.17 mL, 2.0 mmol).
Purification on silica gel with hexane/EtOAc 1:1 gave 8 (0.69
g, 95%) as a colorless oil: 1H NMR (CDCl3) δ 1.37 (s, 9H), 3.10
(s, 3H), 3.51-3.64 (m, 4H), 4.33-4.37 (m, 2H), 4.98 (br s, 1H),
5.01 (dd, 1H, J ) 6.0, 4.3 Hz), 7.43-7.52 (m, 1H), 7.63-7.75
(m, 2H), 7.98 (d, 1H, J ) 8.1 Hz).
[2-(2-Iodoeth oxy)-2-(2-n itr oph en yl)eth yl]car bam ic Acid
ter t-Bu tyl Ester (9). According to the general procedure III,
mesylate 8 (0.59 g, 1.45 mmol) in acetone (8 mL) was treated
with sodium iodide (1.1 g, 7.25 mmol). Purification on silica
gel with hexane/ether 1:1 gave 4b (0.62 g, 98%) as a colorless
oil: 1H NMR (CDCl3) δ 1.35 (s, 9H), 3.22 (t, 2H, J ) 6.1 Hz),
3.38-3.66 (m, 4H), 5.02 (br s, 1H), 5.06 (dd, 1H, J ) 10.2, 3.9
Hz), 7.44-7.52 (m, 1H), 7.63-7.70 (m, 1H), 7.76 (d, 1H, J )
7.9 Hz), 7.94 (d, 1H, J ) 8.1 Hz).
1-[1-(2-Iod oeth oxy)-2-isoth iocya n a toeth yl]-2-n itr oben -
zen e (10). To a solution of the iodide 9 (100 mg, 0.23 mmol)
in dichloromethane (1 mL) was added trifluoroacetic acid (0.25
mL). After 10 min of stirring at room temperature, the
reaction solution was concentrated in vacuo. The obtained
residue was dissolved in CH2Cl2/H2O 1:1 (2 mL) and treated
with sodium bicarbonate (0.15 g, 1.8 mmol) and thiophosgene26
(33 µL, 0.42 mmol). After extraction of the product with
CH2Cl2, the combined organic phase was dried over MgSO4
and evaporated in vacuo. Purification by flash chromatogra-
phy on Al2O3 with hexane/ether 4:1 gave the isothiocyanate
product 10 (60 mg, 69%) as a light yellow oil:27 1H NMR
(CDCl3) δ 3.26-3.33 (m, 2H), 3.36-3.79 (m, 2H), 3.80 (dd, 1H,
J ) 14.6, 6.5 Hz), 3.95 (dd, 1H, J ) 14.5, 3.3 Hz), 5.24 (dd,
1H, J ) 6.4, 3.3 Hz), 7.50-7.59 (m, 1H), 7.72-7.80 (m, 1H),
7.92 (dd, 1H, J ) 7.8, 1.4 Hz), 8.06 (dd, 1H, J ) 8.2, 1.2 Hz);
IR (CHCl3) 2112, 2212cm-1 (v S)C)N).
O -[r-(I s o t h i o c y a n a t o m e t h y le n e )-2-n i t r o b e n z y l]-
ch olin e Iod id e (C). According to the general procedure IV,
the iodide 10 (80 mg, 0.21 mmol) was treated with toluene
(15 mL) saturated with trimethylamine. Purification by
washing the precipitate with toluene gave compound B (82
mg, 92%) as a light yellow solid: mp 121-123 °C; 1H NMR
(CD3CN) δ 3.38 (s, 9H), 3.75-3.95 (m, 3H), 4.11 (dd, 1H, J )
Activity Assa y of ACh E a n d Bu Ch E. AChE and BuChE
activities were measured in 50 mM phosphate buffer, pH 7.2,
at 20 °C by Ellman's test28 using acetylthiocholine and
butyrylthiocholine as the respective substrates.
In h ibition of A-C on ACh E a n d Bu Ch E. Kinetic
measurements of enzyme were performed at variable substrate
concentrations in the absence and presence of different
(26) Thiophosgene was handled in a well-ventilated cupboard.
(27) Purification of compound 10 was achieved by rapid filtration
on an Al2O3 column.
(28) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, M.
R. Biochem. Pharmacol. 1961, 7, 88-95.

Release of Choline in the Microsecond Time Range
J . Org. Chem., Vol. 61, No. 1, 1996 191
concentrations of inhibitor to constitute the Lineweaver-Burk
double reciprocal plots. The apparent competitive inhibition
constants were calculated by the increase in slope of (1 + [I]/Ki)
in the presence of inhibitor.
Bassani, and C. Lineweaver for careful reading of the
manuscript. This work was supported by the Associa-
tion Franc¸aise contre les Myophathies, the Centre
National de la Recherche Scientifique, and l'Association
Franco-Israelienne pour la Recherche Scientifique et
Technologique.
Ack n ow led gm en t. We are grateful to Prof. J . Wirz,
University of Basel, for his kind hospitality and expert
advice for laser pulse photolysis. We thank Drs. D.
J O951190C