Chemical amplification with encapsulated reagents Jian Chen*, Steffi Ko¨rner*, Stephen L. Craig†, Shirley Lin*, Dmitry M. Rudkevich‡, and Julius Rebek, Jr.*§ *The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; †Duke University, Dept. of Chemistry, Box 90346, Durham, NC 27708-0346; and ‡University of Texas at Arlington, Department of Chemistry and Biochemistry, Box 19065, 502 Yates Street, Arlington, TX 76019-0065 Contributed by Julius Rebek, Jr., December 28, 2001
E
ncapsulation complexes are reversibly formed assemblies in which small molecular guests are temporarily surrounded by larger molecular hosts (1). The complexes are held together by weak intermolecular forces such as hydrogen bonds, van der Waals interactions or metal-ligand binding (2–5). Their lifetimes vary from milliseconds to days, a range that makes them useful as nanometric reaction chambers (6), as means to stabilize reagents (7), and as spaces where new forms of stereochemistry can emerge (8, 9). When encapsulated, guests are unreactive to reagents in solution. The capsule provides a barrier between reagents that are either free in solution where they exhibit normal reactivity, or encapsulated where they are unreactive. Guest exchange between the environments becomes a means of regulating reactivity. Experimental Procedures General. 1H NMR and 13C NMR spectra were recorded on a Bruker DRX-600 spectrometer. Chemical shifts were measured relative to residual nondeuterated solvent resonances. Both p-[N-(p-ethylbenzene)]toluamide and p-[N-(p-ethylbenzene)]ethylbenzamide were synthesized in accord with the literature protocols (7). 1H NMR Spectroscopy. For a typical NMR experiment, a 5-mm NMR tube was charged with a solution of 1,3-dicyclohexylcarbodiimide (DCC, 0.227 mg, 0.0011 mmol), cavitand 1 (3.71 mg, 0.0022 mmol), toluic acid (0.15 mg, 0.0011 mmol), and 4-ethyl-phenylamine (0.666 mg, 0.0055 mmol) in deuterated mesitylene-d12 (0.55 ml) at 295 K. 1H NMR spectra were taken at time intervals indicated on the figures, and good reproducibility was observed. Simulations of the kinetic behavior of the DCC兾capsule system were performed using the CHEMICAL KINETICS SIMULATOR program (developed at IBM Almaden, San Jose, CA) with the reactions and rate constants shown in Table 1.
Kinetic Studies by
Results and Discussion The dimeric capsule 1 (Fig. 1) is held together by eight bifurcated hydrogen bonds in organic solvents that do not compete well for hydrogen bond donors and acceptors. Encapsulation of appropriate guests occurs readily in those solvents that do not fit well inside the capsule. Specifically, deuterated mesitylene is appropriate for most guests and provides a convenient solvent for NMR spectroscopy of the encapsulation complexes. The capsule distinguishes shape and size in the molecular recognition of potential guests (10, 11). For example, trans stilbene 2 is encapsulated but cis stilbene 3 is not (Eq. 1). With rigid guests, length www.pnas.org兾cgi兾doi兾10.1073兾pnas.052706499
Table 1. Reactions and rate constants used for simulations of the kinetic of the DCC兾capsule system Reaction DCC ⫹ acid ⫽ I1 I1 ⫹ amine ⫽ amide ⫹ DCU I1 ⫹ acid ⫽ anhydride ⫹ DCU anhydride ⫹ amine ⫽ amide EDCC ⫽ DCC ⫹ EC EDCC ⫹ amide ⫽ Eamide ⫹ DCC EDCC ⫹ DCU ⫽ EDCU ⫹ DCC EDCU ⫹ amide ⫽ Eamide ⫹ DCU
kforward, liters䡠mol⫺1䡠s⫺1
kreverse, liters䡠mol⫺1䡠s⫺1
0.0025 0.015 0.017 0.012 10⫺7 0.01 0.01 10⫺6
0 0 0 0 0.1 0.002 0.002 10⫺6
E, encapsulated species; I1, intermediate; EC, ‘‘empty capsule’’ intermediate; number of molecules ⫽ 1 ⫻ 106; number of events ⫽ 8 ⫻ 105. Starting concentrations for the simulations were: [EDCC], 2 mM; [acid], 2 mM; [amine], 10 mM; other species were not present. For Fig. 3a, the one-product simulation was conducted without amide displacing DCC; the three-product simulation was carried out with another product formed in the reaction, which displaced DCC with the same kforward and kreverse values as for amide and DCU. For Fig. 3b, the starting concentration of amide was 0.5 mM.
is particularly important: the anilide 4a is encapsulated (Eq. 2), but the slightly longer 4b is not. Functional groups also play a role: dicyclohexylcarbodiimide (5, DCC) is readily encapsulated within 1 but its hydration product, dicyclohexylurea (6, DCU), is an even better guest. The urea functionality is able to interact with both the donors and acceptors in the seam of the capsule’s hydrogen bonds. The relative affinity of the capsule for the guests is DCU ⬃ 4a ⬎ DCC. The discrimination of subtle structural differences coupled with the compartmentalization of reagents gives rise to nonlinear kinetics and autocatalytic behavior in reactions involving encapsulated DCC (12). Specifically, the reaction of p-toluic acid (7a) and p-ethyl-aniline (8) with encapsulated DCC in deuterated mesitylene shows a distinctly sigmoidal reaction profile (Fig. 2). At millimolar concentrations of capsule and DCC, the equilibrium concentration of free DCC is too low to detect by NMR experiments. Nonetheless, the reaction proceeds slowly through whatever concentration of DCC is available in solution. The products are DCU 6, the anilide 4a and a side product (13, 14) N-acylurea (not shown). The capsule gradually fills with DCU and anilide. The provenance of this kinetic behavior was established through the following control experiments. First, direct competition between the two acids 7a and 7b with the aniline and DCC in the same solvent were undertaken; all reactants were free in solution. The anilides 4a and 4b are formed in comparable yields and at nearly the same rates. This is expected from the insignificant differences in their remote, para substituents. Abbreviations: DCC, dicyclohexylcarbodiimide; DCU, dicyclohexylurea. §To
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PNAS 兩 March 5, 2002 兩 vol. 99 兩 no. 5 兩 2593–2596
CHEMISTRY
Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting selfregulation is sensitive to the highly selective molecular recognition properties of the capsule.
[2]
Fig. 1. A cylindrical capsule held together by eight bifurcated hydrogen bonds; line drawing, ball and stick molecular model and cartoon representation used elsewhere in this publication.
Second, the initial rates of product (anilide or DCU) formation were determined separately for the two acids in the presence of a stoichiometric amount of the capsule 1. Only the initial rates (up to ⬇25% conversion of the reaction) were followed to reduce curvature from the reaction profiles. These rates are quite different [Fig. 3a, open triangles (7b) and open squares (7a)]. When the DCC is encapsulated, the shorter acid generates products several times faster than the longer one. Third, the reactions were performed in the presence of the respective products—the anilides 4a and 4b. The shorter anilide accelerates the rate of its formation (Fig. 3a, solid squares), the longer amide does not (Fig. 3a, solid triangles). Fourth, the rate acceleration for the formation of the shorter anilide 4a was shown to be a function of the concentration of added 4a at the onset of the reaction. Fig. 3b shows these effects (added [4a] ⫽ 0.0–0.8 mM). This behavior results from feedback loops in a self-regulating reaction cycle (Fig. 4). The urea 6 and the shorter anilide 4a are both good guests for the host 1. Once formed, each of these products displaces the DCC from the capsule into the bulk solvent where it can react with the acid. A single molecule of DCC reacts to yield one molecule each of the urea and anilide, which then displace additional DCC, leading to more urea and anilide and resulting in chain reaction kinetics. The capsule does not influence the reaction between acid and DCC; it only limits the rate at which reagents encounter each other, leading to kinetics that accelerate with the increased concentration of free, ‘‘reactive’’ DCC. The sigmoidal shape in the plot of [DCC]encapsulated versus time depends on two conditions: (i) displacement of DCC due to the formation of at least two products which are better guests for the capsule by at least an order of magnitude (relative affinity of the capsule for product versus DCC ⱖ 50); (ii) the initial release of DCC and its subsequent displacement by better guests must be slower than the coupling reaction. A simulation of the rate of DCC release in these various situations is shown in Fig. 5. Fig. 5a compares reaction profiles for systems in which one product, two products, and three products can displace the encapsulated reagent. ‘‘Autocatalysis,’’ as evidenced by sigmoidicity in the rate profile, does not occur when only one good guest displaces DCC.
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This is the case of the reaction producing longer guest 4b, where only one product, the urea 6, is a better guest capable of releasing DCC from the capsule; autocatalytic acceleration cannot take place. The sigmoidal profile does appear when two products compete with the reagent for the capsule and becomes more pronounced when three products can do so. Fig. 5b demonstrates the effect of adding a better guest at the beginning of the reaction. The sigmoidal profile is lost in this case because of the immediate displacement of DCC; there is no initial ‘‘slow’’ period in the kinetic data and DCC concentration appears to decrease linearly with time. We emphasize that the combination of molecular recognition and compartmentalization still have an effect on the kinetics in these latter systems. The rate profiles differ from those of the reactions in the absence of capsule, but the effect is not sufficiently strong to induce sigmoidal character to the kinetics. Perhaps a more useful measure of the chemical amplification is to look at the apparent pseudo-first order rate constant of the coupling reaction as a function of time. This rate constant, k⬘(t), is obtained as shown in Eq. 3 by taking the slope of the rate profiles and normalizing by the concentration of DCC at that point in the reaction. Under pseudo-first order conditions, k⬘(t) is constant for conventional systems. In the systems described here, however, k⬘(t) increases by as much as a factor of five over the course of the reaction. Fig. 6 shows the effective k⬘(t) that
Fig. 2. The reaction of encapsulated DCC (2 mM) with toluic acid (7a, 2 mM) p-ethyl-aniline (8, 10 mM) in deuterated mesitylene at 295 K. The graph shows the generation of encapsulated DCU as a function of time during the reaction course.
Chen et al.
accompanies the reaction profile in Fig. 2. Although the reaction conditions are not appropriate to obtain truly pseudo-first order measurements (there is not a large excess of either acid or amine relative to DCC), the analysis is nevertheless useful despite the approximations. For conventional systems operating under these reaction conditions, one would expect the pseudo-first order rate constant to decrease by ⬇20% over the course of the reaction as both acid and amine are consumed. The analysis in Fig. 6 shows
Fig. 4. A self-regulating reaction cycle: release of one molecule of DCC results in the formation of two molecules of product (urea and anilide), which are capable of displacing two molecules of DCC.
Chen et al.
the opposite trend, demonstrating the significance of the increase in k⬘(t), a characteristic of chemical amplification. d关DCC]t兾dt ⫽ k⬘(t)[DCC]t, k⬘(t) ⫽ (1兾[DCC]t) ⫻ d[DCC]t兾dt [3] CHEMISTRY
Fig. 3. (a) Initial rates of DCC release from the capsule during the reaction of Fig. 2. All reactions involve encapsulated DCC (2 mM) and p-ethyl-aniline (8, 10 mM) in deuterated mesitylene at 295 K; open triangles, p-ethyl benzoic acid (7b, 2 mM); solid triangles, (7b, 2 mM) and added anilide 4b (0.5 mM); open squares, p-toluic acid (7a, 2 mM); solid squares, (7a, 2 mM) and added anilide 4a (0.5 mM). (b) Response of the initial rates of product formation in the reaction of encapsulated DCC with p-toluic acid and p-ethyl-aniline under the conditions defined for a, as a function of added anilide 4a; open triangles, no added anilide; solid triangles, [4a] ⫽ 0.4 mM; open squares, [4a] ⫽ 0.5 mM; solid squares, [4a] ⫽ 0.8 mM.
Fig. 5. (a) Rate of DCC release as a function of the number of guests capable of displacing reagent; open circles, one product; open squares, two products; open triangles, three products. (b) Rate of DCC release without anilide present at the start of the reaction (open circles) and with 0.25 equivalents of anilide added (open squares). All simulations were carried out in CHEMICAL KINETICS SIMULATOR with the following conditions: [DCC]encapsulated ⫽ 2 mM; [acid] ⫽ 2 mM; [amine] ⫽ 10 mM; (initial rate of anilide formation)兾(initial rate of capsule release) ⫽ 30; K ⫽ [([DCC]encapsulated[product])兾[product]encapsulated[DCC])] ⫽ 50, where product ⫽ DCU or anilide; number of molecules ⫽ 1 ⫻ 106; number of events ⫽ 8 ⫻ 105.
Fig. 6. Apparent pseudo-first order rate constant for DCC conversion as a function of time. The apparent rate of the reaction increases as DCC is released from the molecular compartment 1 by the products of its hydrolysis; see text for details. The data are shown for the reaction of encapsulated DCC (2 mM) with toluic acid (7a, 2 mM) p-ethyl-aniline (8, 10 mM) in deuterated mesitylene at 295 K. Kinetics were monitored until conversion of DCC exceeded 95%. PNAS 兩 March 5, 2002 兩 vol. 99 兩 no. 5 兩 2595
Autocatalysis based on molecular recognition is a phenomenon generally associated with molecular replication: the product acts as a template on which the reactants are gathered. Although the present system has some phenomenological similarities to autocatalysis, there are important differences. First, templated autocatalysis selects specific molecular products from a sea of reactive possibilities based on their ability to reproduce (15–23). Traditional autocatalysis involves direct competition of individual molecules. In the present system, released reagents are equally likely (within the limits of their intrinsic reactivities) to react with any partners present in solution. The autocatalytic behavior, therefore, is viewed more correctly as an emergent property of the system as a whole, rather than a property of specific molecules within the system. We are not aware of any terms that describe this particular behavior because it requires compartmentalization. A more general term such as ‘‘chemical amplification’’ may better describe the kinetic effect: the significance is in the behavior itself, rather than the molecular components of the reaction. Second, product inhibition is generally a consequence of template-mediated replication. Al-
though it is not inevitable, we know of no cases in which the product of a templated reaction does not measurably compete for reaction sites. For the case at hand, no direct contact exists between reagents and products; the two species simply exchange residences, and the products do not compete for reaction sites. Nature has long recognized the inherent benefits of compartmentalization and it is widely believed to be an important, if not essential, characteristic of living systems. The behavior described here should be general to a variety of encapsulation complexes and reactions. The utility may lie in such abilities as providing a rapid dose of a specific molecule in response to a chemical signal (the molecule itself, for example). The results augur well for the design of synthetic systems with novel and increasingly complex behaviors.
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12. Chen, J., Korner, S., Craig, S. L., Rudkevich, D. M. & Rebek, J., Jr. (2001) Nature (London), in press. 13. De Tar, D. F. & Silverstein, R. (1966) J. Am. Chem. Soc. 88, 1013–1019. 14. De Tar, D. F. & Silverstein, R. (1966) J. Am. Chem. Soc. 88, 1020–1023. 15. Von Kiedrowski, G. (1986) Angew. Chem. Int. Ed. Engl. 25, 932–934. 16. Orgel, L. E. (1992) Nature (London) 358, 203–209. 17. Tjivikua, T., Ballester, P. & Rebek, J., Jr. (1990) J. Am. Chem. Soc. 112, 1249–1250. 18. Nowick, J. S., Feng, Q., Tjivikua, T., Ballester, P. & Rebek, J., Jr. (1991) J. Am. Chem. Soc. 113, 8831–8839. 19. Conn, M. M., Winter, E. A. & Rebek, J., Jr. (1994) Acc. Chem. Res. 27, 198–203. 20. Lee, D. H., Granja, J. R., Martinex, J. A., Severin, K. & Ghadiri, M. R. (1996) Nature (London) 382, 525–528. 21. Yao, S., Ghosh, I., Zutshi, R. & Chmielewski, J. (1997) J. Am. Chem. Soc. 119, 10559–10560. 22. Wang, B. & Sutherland, I. O. (1997) Chem. Commun. 1495–1496. 23. Robertson, A., Sinclair, A. J. & Philp, D. (2000) Chem. Soc. Rev. 29, 141–152.
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We are grateful to the Skaggs Research Institute and the National Aeronautics and Space Administration Astrobiology Institute for support of this research. The A. von Humboldt Foundation and Novartis are gratefully acknowledged for fellowships to S.K. The National Institutes of Health provided fellowships to S.L.C. and S.L. J.C. is a Skaggs Postdoctoral Fellow.
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