Received 14th February 2013,Accepted 19th March 2013
DOI: 10.1039/c3ob40324f
www.rsc.org/obc
Chemical approaches for detection and destructionof nerve agents†
Dariush Ajami and Julius Rebek, Jr.*
Since the introduction of organophosphorus (OP) compounds as nerve agents and pesticides, methods
of dealing with their toxicity to humans have been intensely researched. There are studies on sensing,
pretreatments, prophylactics, antidotes and therapies. There is some overlap in all of these endeavors
because they have to deal with the reactivity of the phosphorus atom in various contexts. The contexts
range from large spaces, the thinly spread vapors in the air, to very small spaces in the active sites of
enzymes – acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) – that have reacted with the OP
agent.
Our entry into this field was inspired by a publication ofSwager1 a decade ago. He observed that an intensely fluor-escent compound could be generated by the action of an OPon a carefully crafted pyridine structure. We will use OP gener-ally to include both live nerve agents and the typical mimicssuch as diethyl chlorophosphate (DCP) or diisopropyl fluoro-phosphates (DFP) that are used with caution in the laboratory.The reaction observed by Swager was a cyclization reaction(Fig. 1) that led to an extensively conjugated rigid molecule,triggered by the OP; that is, the reaction created a fluorescentdye and was a useful as a sensor for OP’s.
We had observed a related cyclization reaction, some twentyyears ago2 (Fig. 2); this reaction did not create a conjugated,fluorescent molecule, but it suggested how it could be used toturn on a fluorescent signal. The reaction occurred when therigid amino ester shown was prepared from the correspondingalcohol. Mere evaporation of a solution of the amino esterquickly converted it to a quaternary ammonium salt. The reac-tion was improbably rapid: the tertiary amine is not a greatnucleophile and the carboxylate is a poor leaving group. More-over, in the case shown, it is clear from the geometry of themolecule’s framework (made from Kemp’s3 triacid) that boththe nucleophile (N: atom) and the leaving group (−OAc) are onthe same side (above) the plane defined by the 3 carbons(green spheres). In other words, nucleophile, carbon andleaving group cannot achieve a rectilinear (180°) arrangementexpected for an intramolecular version of an SN2 reaction – atleast not without some considerable contortions of the mole-cular skeleton. Accordingly, the fast reaction of this moleculeis driven by the release of strain.
This reaction is a version of the Menschutkin reaction, andhas been studied for nearly 100 years. It usually requires veryforcing conditions: neat liquids at 200 degrees,4 or in solventsin sealed tubes5 are typically used to get a tertiary amine toreact with a simple methyl ester (Fig. 3). We also hadexperience with this reaction in the very close quarters of acavitand – about which, more later.
Fig. 1 Intramolecular reaction subsequent to phosphylation of an alcohol gen-erates a rigid fluorescent heterocycle.
Fig. 2 Relief of strain drives a cyclization reaction with an unlikely geometry.
Fig. 3 Menschutkin reactions between poor nucleophiles and electrophilesrequire forcing conditions.
†Dedicated to Andrew Hamilton on the occasion of his 60th birthday.
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037,
USA. E-mail: [email protected]; Fax: +1 858-784-2876; Tel: +1 858-784-2250
The reactivity of the Kemp’s skeleton suggested to us ameans of attaching any fluorescent dye to expand the responseof the sensor. There are many such dyes with a wide range ofwavelengths (extending even to the visible and infrared) avail-able to choose from. In such a dye, the fluorophore absorbsphotons of one wavelength that convert it to an excited state,then emits (fluoresces) photons at a longer wavelength. It wasalready known that when a dye is excited near a basic amine,photo-electron transfer from the amine to the excited state ofthe dye takes place. This quenches the system and causes thefluorescence of the dye be turned off (Fig. 4). If the lone pair ofthe amine is not available, the fluorescence resumes.
We adapted the amino ester cyclization and its triggering offluorescence for use in sensing OP’s. Typical organophos-phates are much weaker bases than the carboxylate, andtherefore better leaving groups. On reaction with the alcohol,the OP gives a phosphate ester that should cyclize rapidly andindeed, this proved to be the case. We prepared a series offluorophores such as pyrene and coronene and attached themat different distances from the amine, which established thatthe closer, the better.6 The sensors worked quite well andrapidly. For example, Fig. 5 shows a piece of filter paper thathad been soaked in a dilute chloroform solution of the sensor,then dried. It was then used to cover a vial with PFP for5 seconds, and the enhanced fluorescence is clearly visible.The enhancement with pyrene was only several fold (see below)
but with the known coumarin dye attached to the trigger, wewere able to show some 50-fold enhancement of fluorescence.
Neither the Swager system nor ours incorporate any mole-cular recognition – any acid chloride, acylating agent or evensulfonating agent would yield the same result – so they are notspecific to OP’s. However, acid chlorides and sulfonyl halidesare also unhealthy things to have in the breathable atmos-phere, so their sensing and destruction are not without merit.
As a practical application, a French team at Grenoble CEA,has used the concept and scaffold of this molecular trigger tobuild a electronic device for sensing OP nerve agents.7 Theirsystem involves a silicon nanowire between two electrodes,and their wire is coated with covalently linked tertiary amine(Fig. 6). On exposure to the agent mimic, cyclization takesplace and creates a positive charge of the ammonium group.The change in charge creates a change in resistance and asignal. In short, the intra-molecular reaction, what is known inphysical organic chemistry as neighboring group participation,can be applied to sense nerve agents.8
As can be seen on the time axis, the full response of thisdevice is on the order of minutes, and this is due to the slowstep of the process: the reaction of the alcohol with the OP.This reaction is aided by general base catalysis that is revealedin the X-ray structure of the amino alcohol, produced by theFrench team. The strong hydrogen bond between amine baseand hydroxyl group accounts for some of the background
Fig. 5 Left: rapid response of a pyrene derivative to DFP vapor. Right: large enhancements of fluorescence with a coumarin derived sensor.
Fig. 4 Quenching of fluorescence by intramolecular photoelectron transfer by an amine. When the amine is converted to a quaternary ammonium group, theexcited dye will fluoresce.
fluorescence in the resting state of our system. In retrospect,the amine’s electrons for PET quenching in molecules of Fig. 5are already less available because they are tied up in the hydro-gen bond. Ameliorative measures were called for in the formof more reactive, faster nucleophiles, and we turned to thosesystems that had been studied with organophosphates fordecades; namely, oximes.
The oxime functional group has a pKa in the range of 9 to10 in aqueous solution, and requires deprotonation to be reac-tive, and typically only a few percent is in the active form atphysiological pH. There are good correlations by Cashman9
and others on the inverse relationship between acidity ofoximes and their reactivity to the enzymes disabled by OP’s.Our starting point was the classical compound, pralidoxime(PAM, Fig. 7) and it congeners that are known and are actuallyapproved for human use as reactivators of the OP-conjugatedAChE. Specifically, they show excellent reactivity with the
disabled enzyme. However, they are not particularly good asprophylactics or pretreatments, one of the problems being thatwhen the reaction with the OP occurs, the PAM–OP ester isstill capable of phosphylating the enzyme. Essentially, it reactsreversibly with the enzyme, as indicated by k+ and k− in theequation of Fig. 8.
Whatever else its faults are, including the inability to accessthe central nervous system, we intended to correct the reversi-bility problem of PAM-like compounds by engineering, onceagain, neighboring group participation into the molecule. Weincorporated a hydroxyl group ortho to the nucleophilic oxime.On reaction with the OP, the oxime’s OP ester can act as aleaving group, and the hydroxyl group can act as an internalnucleophile (Fig. 9). Cyclization to the benzisoxazole occurswith ejection of the harmless OP fragment.
It was reasonable to expect that the heterocyclic productswould have different absorption and emission spectra than thestarting materials, and that the systems could also be used asgood fluorescent sensors.10 That proved to be the case, as thedifferent photochemical signatures show in Fig. 9. We pre-pared a number of aromatic hydroxy oximes, and showed thatthey reacted with OP mimics and surrogates much faster thanthe simple alcohols to give the aromatic isoxazoles.11
When these results were published, we were contacted byDrs Fry and Creasy, researchers at the Edgewood Arsenal whooffered to test our molecules on live nerve agents. They estab-lished that these hydroxy oximes react rapidly with sarin andthe like to give the isoxazoles.12 The new fluorescence appearsonly after the nerve agent has been destroyed so the molecules
Fig. 6 Structure and function of a silicon nanowire device coated with an amino alcohol. Cyclization alters the resistance of the device.
Fig. 7 Oximes used for reactivation of OP-conjugated cholinesterases.
Fig. 8 Pathways of reaction between OPs, enzyme and oximes.
are both potential pretreatments and sensors. The specialinteraction of the ortho hydroxyl with the oxime extends toexcited state of the compounds. Our collaborators in Athensshowed that photoexcitation leads to intramolecular protontransfer from the phenol to the quinoid tautomer.13
An unexpected dividend of this research came when ourcollaborators, Prof. Taylor and Dr Radic at the UCSD School ofPharmacy tested some of the hydroxy oximes as reactivatorsfor OP-disabled enzymes. While none of the new compoundswere able to reactivate the AChE conjugates faster than PAM,one of the compounds showed good activity – some 5–10 timesbetter than PAM – on the butyryl enzyme (hBuChE) disabledby paraoxon, cyclosarin or VX (Fig. 10).14 The butyryl enzymehas been under development for many years as a prophylacticfor nerve agent exposure. Because of its very large size (morethan 300 000 D) and because of considerable expense (it isextracted from human blood) and because it acts a stoichio-metric scavenger, the number of people that can be injectedfor pretreatment is limited. However, as hBuChE can be regen-erated through the reactivator, it could become a catalytic,rather than the stoichiometric agent. As such, it would be
accessible to a much larger population as a prophylactic. It hasalready shown efficacy in protection of animals from live nerveagents.
What causes the unusual behavior of this reactivator? Thereare three features: the first is that the positive charge, unlikethe other reactivators, is not in the pyridium ring, rather thetrimethyl ammonium group is exocyclic to the aromaticscaffold. Secondly, because of this bulky ammonium grouportho to the oxime, the oxime itself must be twisted out of theplane of the aromatic, i.e. the nucleophilic oxygen is directedout of the usual plane (Fig. 10). Finally, the hydroxyl groupnext to the oxime allows the initial reaction product of reactiv-ation to cyclize quickly to the corresponding benzisoxazole.We are now attempting to further improve the activity of thisnew lead compound by applying medicinal chemistry toimprove its bioavailability, pharmacokinetics and potency.
As described above, the oxime is used mostly in the contextof reactivation of the disabled enzymes but its role is beingexpanded. Shown in Fig. 11 are the efficacies of a series ofoximes with a nerve agent mimic relative to the standard2PAM.15 It is clear that the ortho hydroxyl enhances reactivity 5to 10-fold with aldoximes and amidoximes. We are pleasedthat the hydroxy oxime has also been incorporated into OPdestroying molecules devised in other laboratories.16–18
As mentioned above, it is desirable and even necessary tohave the effective nucleophiles show selectivity for the targetnerve agents rather than any biological electrophile such as athioester or even the ubiquitous ATP. Fortunately, the nerveagents have side chains that were designed to fit into thebinding pocket of their own targets, the cholinesteraseenzymes. These side chains (Fig. 12) are the recognitionelements and have affinity for other naturally-occurring com-pounds like cyclodextrins as recently exploited by Kubik.19 Theagents also disable other serine hydrolase enzymes20 andcause collateral neuropathic damage.
Recognition studies have seen the decline of simple macro-cycles and the emergence of cleft-like structures21 with conver-gent functional groups22 that are particularly effective in
Fig. 9 Cyclization of a hydroxyl oxime triggered by OPs. The product benzisox-azole has different absorption and emission spectra than the parent hydroxyloxime.
Fig. 10 Structure and reactivity of a BuChE reactivator.
sequestering heterocyclic23 compounds. But the mostadvanced synthetic models of enzymes and receptors are cavi-tands bearing inwardly-directed functions: they fold aroundtheir targets, isolate them from the bulk medium, place themin hydrophobic environments and present them with reactivegroups. The side chain structures are shown in the figure.They are either hydrophobic, such as the cyclohexyl of cyclo-sarin, or they resemble the alkyl ammonium group of theenzyme’s substrate acetylcholine.
We drew on our experience with the cavitands to applythem to recognize these side chains. We had earlier engineereddeep cavitands and we were able to stabilize the vase-likecontainer structure of the several conformations these receptormolecules can take by introducing hydrogen bonds along theupper rim. This shape was apparent from the NMR spectra oftheir complexes in solution, and also by the X-ray structure
solved by our collaborators in the solid state (Fig. 12).24 More-over, we had limited experience with a cavitand bearing fourcarboxylates along the upper rim. These negative chargesimpart water solubility at high (>8) pH, and they attract tri-methyl ammonium groups (as in choline) as guests.25
In addition, we had experience in functionalizing the upperrim with the various reactive groups. Shown in Fig. 13 are anintroverted carboxylic acid and its methylester, again, derivedfrom the Kemp’s scaffold. In this context, an inwardly-directedmethyl ester of a cavitand reacts very rapidly with tertiaryamines, such as quinuclidine that fit inside the cavitand.26
The cavitand folds around the amine and presents the methylester in an ideal arrangement for the Menschutkin reaction atvery close quarters. The cavitand reaction is some 100 000times faster than the comparable bimolecular version. Onmere mixing of the ester with the base quinuclidine at room
Fig. 11 Relative reaction rates of oximes with an OP (from ref. 15); the ortho hydroxyl enhances the nucleophilicity.
Fig. 12 Side chains of common nerve agents and the chemical formula and X-ray structure of a deep cavitand.
Fig. 13 A cavitand with an inwardly-directed carboxylic acid and the cartoon of its methylester with quinuclidine in the cavity. The Menschutkin reaction occursvery rapidly since the reagents are properly aligned.
temperature, an extraordinary methyl transfer reaction tookplace that was several orders of magnitude faster than the cor-responding reaction in bulk solution.
The recognition properties and the reactivity of these cavi-tand functions augurs well for their use in recognizing andbinding the side chains of typical nerve agents. We prepared aseries of these with neutral, water-soluble side chains, includ-ing the 4 tetraethylene glycol side chains,27 and the 4 glucoseside chains (Fig. 14). For the latter we “clicked” the carbo-hydrates on by way of the copper-catalyzed azide/acetylenecycloaddition reaction.28 In addition, 4 ammonium groupswere added as another example of a cavitand that was solubleat physiological pH. As shown by the NMR spectra, this
ammonium cavitand bound cyclohexanol, the pinacolonederived alcohol derivative and, much more weakly, the isopro-pyl group of the agent sarin.29 We also tested the carbohydratederivative in human plasma and found that it operates quitewell to bind a hydrophobic guest in this unusual medium, asshown in the figure.30 Apparently, the proteins and lipidmaterials in the plasma cannot compete with intended guestsfor this water-soluble cavitand. The cavitand also functions inhuman urine.31
Our current efforts are directed at functionalizing the water-soluble versions of the cavitands, and these have proved to bequite challenging. The water-soluble cavitands tested showedlow toxicity toward cell lines and crossed artificial membranes;
Fig. 14 Structures of ammonium and glucosyl cavitands. The spectra show binding of the OP side chains in water (D2O) and biological fluids.
Fig. 15 Formula of a water-soluble cavitand with a functional acetal wall and models of its hydroxy oxime derivative shown with cyclosarin inside.
these will be developed further by functionalizing the upperrims with groups known to react with nerve agents.
The intent is to install an effective nucleophile onto theupper rim of a water-soluble cavitand. One target structure isshown in Fig. 15 where the acetal provides a site of functional-ization,32 a hydroxy-oxime is the nucleophile and the tetraethyleneglycol “feet” of the cavitand provide pH-independentwater solubility. This is expected to provide a reactive, albeitstoichiometric, agent that is selective for the OP side chains asshown for cyclosarin.
Ideally, a prophylactic that acts as a catalytic scavenger ofOP’s is desired. While progress is being made on modifying anenzyme, paraoxonase for this purpose,33 a smaller moleculesolution to the pretreatment problem would be accessible to alarger population. We are working to achieve this goal withcavitands.
While it has been an honor to write this for Andrew Hamil-ton, and a pleasure to use the casual tone of a perspective, thepresent work is not a safe substitute for a comprehensive treat-ment of the subject. Fortunately, there is a timely and excellentreview to which interested readers are referred.34
We are grateful to the United States Department of Defense,Defense Threat Reduction Agency and the National Institutesof Health (1R21 NS072086) for financial support. We thankour co-workers and collaborators whose names appear in thecited references.
Notes and references
1 S.-W. Zhang and T. Swager, J. Am. Chem. Soc., 2003, 125,3420.
2 P. Ballester, B. M. Tadayoni, N. Branda and J. Rebek Jr.,J. Am. Chem. Soc., 1990, 112, 3685.
3 D. S. Kemp and K. S. Petrakis, J. Org. Chem., 1981, 46, 5140.4 E. L. Eliel and R. P. Anderson, J. Am. Chem. Soc., 1952, 74,
547.5 M. S. Newman and H. A. Lloyd, J. Am. Chem. Soc., 1952, 74,
2672.6 T. J. Dale and J. Rebek Jr., J. Am. Chem. Soc., 2006, 128,
4500.7 S. Clavaguera, A. Carella, L. Caillier, C. Celle, J. Pecaut,
S. Lenfant, D. Vuillaume and J.-P. Simonato, Angew. Chem.,Int. Ed., 2010, 49, 4063.
8 For other mechanism-based approaches, see: K. Chulvi,P. Gavina, A. M. Costero, S. Gil, M. Parra, R. Gotor, S. Royo,R. Martınez-Manez, F. Sancenon and J.-L. Vivancosade,Chem. Commun., 2012, 48, 10105–10107; R. Gotor,A. M. Costero, S. Gil, M. Parra, R. Martınez-Manez andF. Sancenon, Chem.–Eur. J., 2011, 17, 11994.
9 J. Kalisiak, E. C. Ralph, J. Zhang and J. R. Cashman, J. Med.Chem., 2011, 54, 3319.
10 T. J. Dale, A. Sather and J. Rebek Jr., Tetrahedron Lett., 2009,50, 6173.
11 T. J. Dale and J. Rebek Jr., Angew. Chem., Int. Ed., 2009, 48,7850.
12 W. Creasy and R. Fry, SAIC/Edgewood Chemical BiologicalCenter, personal communication.
13 I. S. K. Kerkines, I. D. Petsalakis, G. Theodorakopoulos andJ. Rebek Jr., J. Phys. Chem. A, 2011, 115, 834.
14 Z. Radić, T. Dale, Z. Kovarik, S. Berend, E. Garcia, L. Zhang,G. Amitai, C. Green, B. Radić, B. M. Duggan, D. Ajami,J. Rebek Jr. and P. Taylor, Biochem. J., 2013, 450, 231.
15 G. Saint-Andre, M. Kliachyna, S. Kodepelly, L. Louise-Leriche, E. Gillon, P.-Y. Renard, F. Nachon, R. Baati andA. Wagner, Tetrahedron, 2011, 67, 6352.
16 L. Louise-Leriche, E. Paunescu, G. Saint-Andre, R. Baati,A. Romieu, A. Wagner and P.-Y. Renard, Chem.–Eur. J.,2010, 16, 3510.
17 G. Mercey, T. Verdelet, G. Saint-Andre, E. Gillon,A. Wagner, R. Baati, L. Jean, F. Nachon and P.-Y. Renard,Chem. Commun., 2011, 47, 5295.
18 G. Mercey, T. Verdelet, J. Renou, M. Kliachyna, R. Baati,F. Nachon, L. Jean and P.-Y. Renard, Acc. Chem. Res., 2012,45, 756.
19 M. Zengerle, F. Brandhuber, C. Schneider, F. Worek,G. Reiter and S. Kubik, Beilstein J. Org. Chem., 2011, 7, 1543.
20 D. K. Nomura, J. L. Blankman, G. M. Simon, K. Fujioka,R. S. Issa, A. M. Ward, B. F. Cravatt and J. E. Casida, Nat.Chem. Biol., 2008, 4, 373.
21 T. K. Park, J. Schroeder and J. Rebek Jr., J. Am. Chem. Soc.,1991, 113, 5125.
22 A. Galán, J. de Mendoza, C. Toiron, M. Bruix,G. Deslongchamps and J. Rebek Jr., J. Am. Chem. Soc.,1991, 113, 9424.
23 K. S. Jeong, A. V. Muehldorf and J. Rebek Jr., J. Am. Chem.Soc., 1990, 112, 6144.
24 A. Shivanyuk, D. M. Rudkevich, K. Rissanen and J. RebekJr., Helv. Chim. Acta, 2000, 83, 1778.
25 F. Hof, L. Trembleau, E. C. Ullrich and J. Rebek Jr., Angew.Chem., Int. Ed., 2003, 42, 3150.
26 B. W. Purse, P. Ballester and J. Rebek Jr., J. Am. Chem. Soc.,2003, 125, 14682.
27 A. Lledo and J. Rebek Jr., Chem. Commun., 2010, 46, 8630.28 V. V. Rostovtsev, L. G. Green, V. V. Fokin and
K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596.29 S. Javor and J. Rebek Jr., J. Am. Chem. Soc., 2011, 133,
17473.30 D. A. Ryan and J. Rebek Jr., J. Am. Chem. Soc., 2011, 133,
19653.31 D. A. Ryan and J. Rebek Jr., Analyst, 2013, 138, 1008.32 M. Degardin, E. Busseron, D.-A. Kim, D. Ajami and
J. Rebek Jr., Chem. Commun., 2012, 48, 11850.33 M. Goldsmith, Y. Ashani, Y. Simo, M. Ben-David,
H. Leader, I. Silman, J. L. Sussman and D. S. Tawfik, Chem.Biol., 2012, 19, 456.
34 For an admirable review, see: K. Kim, O. G. Tsay, D. A.Atwood and D. G. Churchill, Chem. Rev., 2011, 111, 5345.
