Hydroxy oximes as organophosphorus nerve agent sensors

Article abstract of DOI:10.1002/anie.200902820

Find and destroy: A series of oximes were constructed to simultaneously detect and detoxify organophosphorus-based nerve agents. They function as optical sensors employing the oxime reactivity and incorporating a β-hydroxyl group to undergo an intramolecu

Full text of DOI:10.1002/anie.200902820

Communications
DOI: 10.1002/anie.200902820
Nerve Agent Sensors
Hydroxy Oximes as Organophosphorus Nerve Agent Sensors**
Trevor J. Dale and Julius Rebek, Jr.*
The G-series of nerve agents including Sarin, Soman, and
Tabun are inhibitors of serine proteases and their primary
toxicity results from the inhibition of acetylcholinesterase.
They can be lethal within minutes if inhaled as the esterifi-
cation of the active site serine residue by the OP agent
renders the enzyme inoperative. This results in a buildup of
acetylcholine in the cholinergic synapses that paralyzes the
oxime is not without liabilities—the resultant esters with
OP agents are extremely powerful inhibitors of the relevant
enzymes and their destruction is paramount in any detection
[
9,10]
scheme.
To overcome some of these liabilities, we
returned to a neighboring group tactic by incorporating a b-
hydroxy function for cyclization and displacement of the
initially formed (and likely lethal) OP ester (Scheme 1c). The
resulting isoxazole becomes the source of a fluorescent signal
that reports the presence and destruction of the OP agent.
Oximes possess significantly enhanced reactivity attrib-
[1]
central nervous system. Recent reports describe detection
[
2]
methods for these nerve agents using enzymes, electro-
[3]
[4–6]
chemistry, or luminescence,
but limitations of these
[
11]
systems include low sensitivity, operational complexity, and
non-portability. The ease of production and extreme toxicity
of these organophosphorus (OP) nerve agents underscores
the need to rapidly detect and, ideally, neutralize these
odorless and colorless chemicals. We report here progress
towards this goal.
In previous work, a series of sensors was described in
which the reaction of a primary alcohol with an OP toxin
initiates an intramolecular
uted primarily to the a-effect. Nucleophiles such as oximes
containing heteroatoms adjacent to the nucleophilic center
have greater reactivity than what is predicted from their
basicity. For example, oximes and phenols are oxygen
nucleophiles of similar pK , but oximes react ca. 100 times
a
[12]
faster with phosphorus-based electrophiles. They have also
been shown to react much faster with phosphorus electro-
[
7]
philes than do hydrazones, another a-effect function.
cyclization to generate a
fluorescence
(
response
Scheme 1a). While this
system had the advantage
of compatibility with
[
6]
a
range of fluorophores, its
response time to ppm
vapor concentrations was
slow (seconds). To enhance
the detection rate, the alco-
hol was replaced with a
more nucleophilic oxime
group. This function is
known as an excellent
reagent for OP agents with
[
5,7]
rapid detection abilities,
and is even the basis for
antidotes presently used
[6]
Scheme 1. a) A previously reported fluorescence detection scheme and b) the optimized sensor with this
against OP poisoning (e.g. scaffold, 1a. c) The oxime-based detection sequence: reaction of an unsaturated b-hydroxy oxime with an OP
2
-pyridinealdoxime
(2- nerve agent is followed by cyclization of the OP ester to the isoxazole in the presence of a base. d) Currently
PAM), Scheme 1d). The
[8]
employed OP agent poisoning antidote, 2-pyridinealdoxime methiodide (2-PAM).
[
*] Dr. T. J. Dale, Prof. Dr. J. Rebek, Jr.
The Skaggs Institute for Chemical Biology & Department of
Chemistry, The Scripps Research Institute
Installing aromatic cores between the oxime and hydroxy
groups served to increase the desired enol tautomer as well as
enhance the practicality of the materials by shifting the
absorption of the chromophore to longer wavelengths. Four
oxime-based sensors, 1–4, were synthesized with the aromatic
cores naphthalene, pyrene, coumarin, and pyridine
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2876
E-mail: jrebek@scripps.edu
Homepage: http://www.scripps.edu/rebek/
(
Scheme 2). These were all synthesized as previously de-
[
**] We thank the Skaggs Institute for Chemical Biology for financial
[13]
support. T.J.D. is a Skaggs postdoctoral fellow.
scribed from the corresponding salicylaldehyde derivatives.
The first three cores were of particular interest as they are
fluorescent species, thus expanding the application of any
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200902820.
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Chemie
observed with the alcohol-based sensor 1a. The
electronic difference between the oximes and the
cyclized arylisoxazoles observed from these opti-
cal measurements confirm that the oxime cycliza-
tion is a plausible construct for a practical OP
nerve agent sensor.
The detection reaction rates for oximes 1–4
were determined and compared with the previ-
ously developed sensor 1a. In all cases, kinetic
analysis revealed that initial nucleophilic reaction
with an electrophile was rate-limiting, preceding a
fast intramolecular cyclization. This result is key
to the function, as a slow secondary reaction
would limit the sensitivity of the oxime sensors for
OP agents. Using TsCl as a generic electrophile, a
rate increase of some four to five orders of
Scheme 2. Four ortho-hydroxy oxime targets, 1–4, to be used as OP nerve agent
sensors and their corresponding arylisoxazoles 5–8.
formed chromogenic sensors to be used as fluorogenic sensors
as well. Initially, oximes 1–4 were reacted with p-toluenesul-
fonyl chloride (TsCl) in the presence of an amine base to
magnitude was observed with the oximes compared to the
alcohol 1a (Table 2).
[
13]
generate the desired arylisoxazoles 5–8 (Scheme 1). As a
more representative test of their utility as sensors, the oximes
were found to cyclize upon reaction with diethylchlorophos-
phate (DCP), a reactive nerve gas mimic, under the same
conditions.
The cyclization to the isoxazoles (5–8) produced meas-
urable shifts in the absorption and emission properties, as well
as altered the magnitude of both the extinction coefficients
and the fluorescence intensities (see Table 1, Figure 1, and
Figures S1–S3 in the Supporting Information). Cyclization of
the naphthyl-based sensor 1 produced the greatest fluores-
cence enhancement of 62-fold—three times the enhancement
Table 2: Reaction rate comparison between the oxime sensors 1–4 and
the previously reported sensor 1a.
[a]
ꢀ
1
Sensor
Relative rate
kobs [min
]
t [min]
1
=
2
4
2
3
1
132000
31000
14000
10000
1.0
25.1ꢁ0.1
5.9ꢁ0.1
2.6ꢁ0.2
1.9ꢁ0.1
0.028
0.12
0.27
0.36
3650
1
a
0.00019ꢁ0.00001
[
a] Conditions: [sensor]=0.07 mm, [TsCl]=3 mm, [iPr NEt]=10 mm in
2
CH CN. Experimental errors were calculated from repeated measure-
3
ments.
An additional advantage of the b-hydroxy oxime system is
access to more practical water-soluble nerve agent detectors.
Many of the G-series of nerve agents contain a relatively inert
PꢀF bond and do not readily decompose under ambient
aqueous conditions, a property that makes the agents such
insidious chemical weapons. The hydrophobicity of the
trimethylcyclohexyl scaffold renders the sensors described
in Scheme 1a insoluble in water. The oxime-based com-
pounds, however, can be easily solubilized for aqueous
conditions with various cores due to the simple, modular
design.
Sensor development under aqueous conditions is ham-
pered by the instability of suitable chloride containing
electrophiles (DCP is hydrolyzed in water buffered to pH 7
within a few minutes) and requires the use of more toxic nerve
Figure 1. Absorbance (thin lines) and emission (thick lines) spectra for
oxime 1 and isoxazole 5. lexc =310 nm; concentration=3ꢀ10 m in
ꢀ5
CH Cl .
2
2
Table 1: Summary of optical data measured for the cyclizations of oximes 1–4 to arylisoxazoles 5–8 (all spectra were obtained on purified compounds).
Absorption Fluorescence
lmax (isox)
[
a]
[c]
Cyclization
lmax (oxime)
Dlmax
eisox/eoxime
lmax (oxime)
lmax (isox)
Dlmax
Flisox/Floxime
1
2
3
4
!5
!6
!7
!8
354 nm
423 nm
286 nm
325 nm
326 nm
399 nm
300 nm
ꢀ28
ꢀ24
14
0.5
1.8
4.2
392 nm
440 nm
430 nm
N/A
361 nm
415 nm
435 nm
N/A
ꢀ31
ꢀ25
5
62
46
9
[
b]
<280 nm
>45
1.4
N/A
N/A
[a] The ratio of extinction coefficients was calculated at lmax for each cyclization. [b] Compound 8 does not have a lmax >280 nm; therefore the
extinction coefficient at 300 nm was used. [c] The maximum fluorescence ratio at any single wavelength is tabulated.
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[
]
agent mimics. Diisopropylfluorophosphate (DFP) * mimics
the reactivity of G-series agents more accurately than
chloride based electrophiles and it is not hydrolyzed by
water near pH 7 at an appreciable rate, allowing for aqueous
testing. We studied the reaction rates between DFP and the
four sensors, 1–4, in aqueous solution buffered to pH 7.5 with
[2] M. Pohanka, P. Dobes, L. Drtinova, K. Kuca, Electroanalysis
2
2
009, 21, 1177 – 1182; b) J. P. Walker, S. A. Asher, Anal. Chem.
005, 77, 1596 – 1600; c) X. Ji, J. Zheng, J. Xu, V. K. Rastogi, T.-c.
Cheng, J. J. DeFrank, R. M. Leblanc, J. Phys. Chem. B 2005, 109,
793 – 3799; d) A. L. Simonian, J. K. Grimsley, A. W. Flounders,
3
J. S. Schoeniger, T. C. Cheng, J. J. DeFrank, J. R. Wild, Anal.
Chim. Acta 2001, 442, 15 – 23; e) A. Mulchandani, P. Mulchan-
dani, I. Kaneva, W. Chen, Anal. Chem. 1998, 70, 4140 – 4145.
[3] G. Liu, J. Wang, R. Barry, C. Petersen, C. Timchalk, P. L.
Gassman, Y. Lin, Chem. Eur. J. 2008, 14, 9951 – 9959; b) I. Oh,
R. I. Masel, Electrochem. Solid-State Lett. 2007, 10, J19 – J22;
c) G. D. Liu, Y. H. Lin, Anal. Chem. 2006, 78, 835 – 843.
4] J. M. Rathfon, Z. M. Al-Badri, R. Shunmugam, S. M. Berry, S.
Pabba, R. S. Keynton, R. W. Cohn, G. N. Tew, Adv. Funct. Mater.
2009, 19, 689 – 695; b) R. Shunmugam, G. N. Tew, Chem. Eur. J.
2008, 14, 5409 – 5412; c) H. S. Hewage, K. J. Wallace, E. V.
Anslyn, Chem. Commun. 2007, 3909 – 3911; d) D. Knapton, M.
Burnworth, S. J. Rowan, C. Weder, Angew. Chem. 2006, 118,
1
0 mm HEPES buffer. Sensors 1, 3, and 4 reacted with DFP
with similar rates, but pyrene-based sensor 2 was insoluble
under these reaction conditions. The previously described
trimethylcyclohexyl system was completely insoluble and
direct comparisons could not be made. Instead, the rates were
compared to the reaction of DFP with 2-pyridinealdoxime
[
[10,14]
methiodide (2-PAM, Scheme 1d),
a known oxime based
antidote for OP poisoning, under identical conditions. The
three sensors measured were all found to react faster with
DFP in solution than 2-PAM (Table 3).
5
957 – 5961; Angew. Chem. Int. Ed. 2006, 45, 5825 – 5829; e) S.
Bencic-Nagale, T. Sternfeld, D. R. Walt, J. Am. Chem. Soc. 2006,
28, 5041 – 5048; f) E. R. Menzel, L. W. Menzel, J. R. Schwierk-
1
Table 3: Relative reaction rate comparison for the oxime sensors with
[
a]
ing, Talanta 2005, 67, 383 – 387; g) S.-w. Zhang, T. M. Swager, J.
Am. Chem. Soc. 2003, 125, 3420 – 3421; h) K. A. Van Houten,
D. C. Heath, R. S. Pilato, J. Am. Chem. Soc. 1998, 120, 12359 –
12360.
DFP in aqueous solution.
ꢀ1
Compound
Relative rate
kobs [min
]
t [min]
=
1
2
1
1.8
N/A
1.9
1.4
1.0
0.0095ꢁ0.0005
73
N/A
69
92
133
[
b]
2
3
4
2
N/A
[5] K. J. Wallace, R. I. Fagbemi, F. J. Folmer-Andersen, J. Morey,
V. M. Lynth, E. V. Anslyn, Chem. Commun. 2006, 3886 – 3888.
[6] T. J. Dale, J. Rebek, Jr., J. Am. Chem. Soc. 2006, 128, 4500 – 4501.
[7] K. J. Wallace, J. Morey, V. M. Lynch, E. V. Anslyn, New J. Chem.
[
c]
0.010 ꢁ0.001
0.0075ꢁ0.0005
0.0052ꢁ0.0005
-PAM
2
005, 29, 1469 – 1474.
[a] Conditions: [oxime]=0.07 mm, [DFP]=3 mm, buffered at pH 7.5
[
8] J. Kassa, K. Kuca, L. Bartosova, G. Kunesova, Curr. Org. Chem.
with 10 mm HEPES buffer. Experimental errors were calculated from
repeated measurements. [b] Oxime 2 was insoluble to the reaction
conditions. [c] A slow background reaction was observed for oxime 3 in
the absence of DFP.
2
8
007, 11, 267 – 283; b) J. Kassa, J. Toxicol. Clin. Toxicol. 2002, 40,
03 – 816; c) J. Acharya, A. K. Gupta, D. K. Dubey, S. K. Raza,
Eur. J. Med. Chem. 2009, 44, 1335 – 1340; d) C. Luo, M. Tong, N.
Chilukuri, K. Brecht, D. M. Maxwell, A. Saxena, Biochemistry
2007, 46, 11771 – 11779.
[
9] Y. Ashani, A. K. Bhattacharjee, H. Leader, A. Saxena, B. P.
Doctor, Biochem. Pharmacol. 2003, 66, 191 – 202; b) C. Luo, A.
Saxena, M. Smith, G. Garcia, Z. Radic, P. Taylor, B. P. Doctor,
Biochemistry 1999, 38, 9937 – 9947; c) H. Leader, A. Vincze, B.
Manisterski, N. Rothschild, C. Dosoretz, Y. Ashani, Biochem.
Pharmacol. 1999, 58, 503 – 515; d) F. Hobbiger, D. G. OꢀSullivan,
P. W. Sadler, Nature 1958, 182, 1498 – 1499.
In summary, new nerve agent sensors were developed
based on the reaction of b-hydroxy oximes with OP agent
mimics. The initial reaction induces a cyclization to an
(
aryl)isoxazole and displacement of the formed oxime–OP
group gives sizable, easily-monitored optical changes. A series
of sensors built on aromatic cores was synthesized, including
more practical water-soluble ones. Compared to previously
reported sensors, considerable reaction rate enhancements
with OP mimics and greatly increased sensitivity were
achieved. Additionally, any nerve agent detected is detoxified
upon reaction, creating the potential construction of hybrid
materials—devices that not only detect the toxins but
simultaneously decompose them.
[
10] I. B. Wilson, S. Ginsburg, Biochim. Biophys. Acta 1955, 18, 168 –
70.
[11] Y. Ren, H. Yamataka, J. Org. Chem. 2007, 72, 5660 – 5667; b) G.
Klopman, K. Tsuda, J. B. Louis, R. E. Davis, Tetrahedron 1970,
1
26, 4549 – 4554; c) J. O. Edwards, R. G. Pearson, J. Am. Chem.
Soc. 1962, 84, 16 – 24; d) W. P. Jencks, J. Carriuolo, J. Am. Chem.
Soc. 1960, 82, 1778 – 1786.
[
[
12] F. Terrier, P. Rodriguez-Dafonte, E. Le Guevel, G. Moutiers,
Org. Biomol. Chem. 2006, 4, 4352 – 4363.
13] T. J. Dale, J. Rebek, Jr., Tetrahedron Lett. 2009, in press.
[14] J. H. Wills, A. M. Kunkel, R. V. Brown, G. E. Groblewski,
Science 1957, 125, 743 – 744.
[15] According to the Material Safety Data Sheet (MSDS) of DFP,
the oral lethal dose low (LDLO) or the lowest dose at which
Received: May 26, 2009
Revised: July 9, 2009
Published online: September 15, 2009
ꢀ
1
Keywords: arylisoxazoles · fluorescence · nerve agents · oximes ·
death may occur is 2.1 mgkg .
.
sensors
[
1] K. Tuovinen, Toxicology 2004, 196, 31 – 39; b) F. R. Sidell, J.
Borak, Ann. Emerg. Med. 1992, 21, 865 – 871.
[
*] Note: DFP is a highly toxic compound and tremendous care must be
taken when handling.
[
15]
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