Introducing Dynamic Combinatorial Chemistry: Probing the Substrate

In the Laboratory

Introducing Dynamic Combinatorial Chemistry: Probing the Substrate Selectivity of Acetylcholinesterase € m* Marcus Angelin, Rikard Larsson, Pornrapee Vongvilai, and Olof Ramstro Department of Chemistry, The Royal Institute of Technology, Teknikringen 30, S-10044, Stockholm, Sweden *[email protected]

Combinatorial chemistry is an effective approach to obtain a vast number of molecules in a limited time period. It is an important tool in many scientific fields, with the drug discovery industry possibly being the most widespread example (1). Even though compartmentalization and solid-phase synthesis protocols have made the process more streamlined and efficient, the compounds are still generally prepared and analyzed individually. This requires a lot of work and is only feasible due to the constant improvement of automated, high-throughput techniques. In an ideal system, the target molecule directly selects the best substrate analogue or inhibitor from the library pool. If the library could then change its composition and adapt to the selection pressure provided by the target, an amplification of the best candidates would be possible. Dynamic combinatorial chemistry (DCC) is a recent approach that is founded on these principles (Figure 1, blue box) (2, 3). The dynamics is created by using reversible chemical transformations. A number of reactants combine under thermodynamic control, forming a library under dynamic equilibrium: the dynamic combinatorial library (DCL). The target molecule, such as an enzyme or a receptor protein, is then allowed to interact with the library, thereby affecting its thermodynamic properties. For example, if one or more components favorably interact with the target, they would be brought to a lower energy state. Subsequently, the DCL would respond by producing more of these “selected” components at the expense of the other components. There are, however, situations where thermodynamic screening is difficult to apply. One example is when the amplified library member is not stable enough without the target, thereby making it hard to isolate and characterize. Also, thermodynamic screening procedures often require stoichiometric amounts of target to get notable amplification effects. This can be problematic when working with rare pharmaceutical targets, available only in small quantities. A way around these problems is to

couple the selection to a kinetically controlled secondary reaction. In this case, only a catalytic amount of target molecule is needed. It may also result in a stable molecule, making isolation and characterization more straightforward. This concept is termed dynamic combinatorial resolution (DCR) and has been described in the literature (Figure 1, blue and red box) (4-6). In this laboratory experiment, students are challenged to use DCC in general and DCR in particular to probe the substrate selectivity of acetylcholinesterase (AChE). During the exercise, students prepare a small DCL of thiols and thioesters. Then AChE is added, and the selection process is monitored through 1H NMR analysis. The experiment is based on a previous research project (4, 5) that has been adapted as a laboratory experiment for undergraduate students. It also represents an authentic and fun way to introduce DCC into the curriculum. The System The DCL is based on reversible transthioesterification (Figure 2). This is a biologically fundamental process involved in, for example, the production of acetyl-coenzyme A (acetylCoA) in the Krebs cycle. It is a rapid and robust reaction that works well in water. This is rare for most organic transformations but a desirable feature in the pharmaceutical industry. The selector, AChE, is a serine hydrolase and has a central role in the nervous system. It works by terminating nerve impulses at cholinergic synapses. This is accomplished by hydrolyzing the neurotransmitter acetylcholine (ACh) (Figure 3). AChE is important in the pharmaceutical industry because it serves as a target for various conditions, including Alzheimer's disease (7). AChE is a well-known enzyme, with a high specific activity approaching the diffusion-controlled limit. The active site has been the subject of several studies (8-11) and is composed of two subsites: an esteratic subsite that contain the catalytic triad

Figure 1. The principles of stoichiometric DCC (blue box) and DCR (blue and red box).

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Vol. 87 No. 11 November 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100400v Published on Web 09/10/2010

In the Laboratory

and an anionic subsite where aromatic residues interact with the quaternary ammonium ion through cation-π interactions. In this experiment, a small DCL was designed to investigate the sensitivity of AChE to substitution on the acyl side. Three thioesters with substitution ranging from methyl to phenyl were combined with thiocholine in deuterated buffer (pD 8) to form a DCL of eight compounds (Figure 4). Experimental Procedure Part 1: Preparations Noncommercial starting materials and buffer and enzyme solutions were prepared by the instructor prior to the experiment (details in the supporting information). Part 2: The Experiment1 After an introduction, students were divided into groups of two. A 0.1 M solution of thiocholine in D2O was prepared and mixed with 50 mol% phosphine.2 Next, 0.1 M solutions of each thioester were prepared in buffer solution. Sixty microliters of

each solution was added to the NMR tube and diluted to 600 μL with D2O. This initiated the transthioesterification reactions and subsequent establishment of the dynamic library. During the equilibration time (around 2 h), the students worked on the questions in the student handout (see supporting information). Most importantly, students analyzed the instructor-provided 1 H NMR spectra of the individual transesterification reactions. This was necessary to analyze and identify all components in the dynamic library. A short lunch break also occurred during this time. After the break, a 1H NMR spectrum was recorded of the established equilibrium. Then, the enzyme solution (20 μL) was added, and the selection process was followed by 1H NMR analysis (immediately and every 30-45 min for 150 min). During this time, students analyzed the completed NMR spectra of the library mixture and, subsequently, could identify which substance or substances were consumed, formed, and amplified during the enzymatic selection step. Hazards Preparation of the library mixture was performed in a fume hood and students wore standard protective clothing and safety goggles. No solvents were used except deuterated water. The sulfur-containing molecules produced little or no smell due to low volatility. Detailed information about all chemicals is provided in supporting information. Results and Discussion

Figure 2. Schematic of the reversible transthioesterification reaction.

The laboratory experiment was tested on a small class of second-year undergraduate chemistry students (4 students divided into 2 subgroups). A full day3 (9 a.m.-5 p.m. including a lunch break) was dedicated to the experiment. After a general introduction and discussion about the experiment and the

Figure 3. Hydrolysis of neurotransmitter ACh by the enzyme AChE to form choline and acetic acid.

Figure 4. The DCL used for determination of AChE substrate selectivity.

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irreversible enzyme-catalyzed hydrolysis, the library responds by producing more at the expense of 3-(acetylthio)propionic acid. This goes on until both components are completely consumed, displaying an amplification effect and a clear example of DCR.6 After the experimental part was finished, approximately 30 min remained for discussion of the results and the provided questions. The students did not have time to complete everything during the laboratory session, so the final touches were made outside of the laboratory and the reports (i.e., answers to the questions) were handed in a few days after the actual experiment. Conclusions

Figure 5. Selection process of AChE on the dynamic combinatorial library.

principles upon which it is based, subgroups (2 students in each) were formed and the experimental part started. The students calculated stoichiometry, prepared solutions, and mixed the components in the NMR tube, initiating the library formation. The weighing took additional time due to the inexperience of working with chemicals in small quantities. In total, this part took approximately 2 h, leaving the groups with 1-1.5 h to work with the instructor-provided NMR spectra of the individual thioester exchange (and other questions in the student handout). The students who were less experienced with NMR analysis required help from the instructor to interpret the NMR spectra.4 After the lunch break (0.5-1 h), the library had reached equilibrium and a 1H NMR was recorded to visualize its composition (Figure 5A displays parts of the spectrum5). The enzyme solution was added and the experiment was followed by 1H NMR analysis (immediately and every 30-45 min for the next 2.5 h; Figure 5). During the time intervals, the students analyzed their recorded NMR spectra and translated the information from the instructor-provided spectra. They observed that acetylthiocholine was hydrolyzed quickly after enzyme addition. This was logical as it was the substance most similar to acetylcholine, the natural substrate. Consequently, a new peak corresponding to acetic acid was observed. With time, 3-(acetylthio)propionic acid was also consumed. The students realized that this could not be due to enzyme hydrolysis. Both because 3-(acetylthio)propionic acid does not contain a positively charged ammonium ion, known to be required for acetylcholinesterase hydrolysis, and that there were no new peaks from potential hydrolysis products. Instead, this effect could be explained by the dynamic properties of the system. As acetylthiocholine is removed from the equilibrium through 1250


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Overall, the students were positive and satisfied with the laboratory experiment. They thought it was nice to be able to learn about this new concept and liked that it combined organic chemistry and biochemistry. They were also happy to use complex instruments, such as the NMR, first hand and not only analyzing spectra from a book or printed by an instructor. The questions were appreciated and forced the students to think about basic and important concepts, such as the cation-π interaction and new ways to think about the reversibility concept. This experiment is best suited for small groups of students (maximum 6-8 subgroups and represents an effective way to introduce DCR to chemistry students. The concept is presented through a fun experiment, in a realistic laboratory setting, using a target molecule of great biological significance. Notes 1. A detailed experimental description is provided in the supporting information. 2. Specifically, the phosphine used was the water-soluble triphenylphosphine-3,30 ,300 -trisulfonic acid trisodium salt. It was necessary to reduce the small quantities of disulfide present in the thiocholine starting material and formed during the experiment. 3. Suggestions to adapt the experiment to shorter laboratory periods are available in the in the supporting information. 4. Students could also work with the instructor-provided NMR spectra outside of the laboratory, prior to the actual experiment. 5. For a full picture of the library with peak assignment, see supporting information. 6. It was also possible to see other small effects on the library composition during the enzyme-catalyzed hydrolysis. These, however, were more difficult to see and of less pedagogical importance.

Literature Cited 1. Terett, N. K. Combinatorial Chemistry; Oxford Univ. Press: Oxford, 1998. 2. Ramstr€om, O.; Lehn, J.-M. Nat. Rev. Drug Discovery 2002, 1, 26–36. 3. Corbett, P. T.; Leclaire, J.; Vial, L.; West, K. R.; Wietor, J.-L.; Sanders, J. K. M.; Otto, S. Chem. Rev. 2006, 106, 3652– 3711. 4. Larsson, R.; Pei, Z.; Ramstr€om, O. Angew. Chem., Int. Ed. 2004, 43, 3716–3718. 5. Larsson, R.; Ramstr€om, O. Eur. J. Org. Chem. 2006, 285–291.


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6. Vongvilai, P.; Angelin, M.; Larsson, R.; Ramstr€om, O. Angew. Chem., Int. Ed. 2007, 46, 948–950. 7. Jakob-Roetne, R.; Jacobsen, H. Angew. Chem., Int. Ed. 2009, 48, 3030–3059. 8. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253, 872–879. 9. Ordentlich, A.; Barak, D.; Kronman, C.; Flashmert, Y.; Leitner, M.; Segall, Y.; Ariel, N.; Cohen, S.; Velan, B.; Shafferman, A. J. Biol. Chem. 1993, 268, 17083–17095.


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10. Harel, M.; Schalk, I.; Ehret-Sabtier, L.; Bouet, F.; Goeldner, M.; Hirth, C.; Axelsen, P. H.; Sussman, J. L. Proc. Natl. Acad. Sci. 1993, 90, 9031–9035. 11. Radic, Z.; Duran, R.; Vellom, D. C.; Li, Y.; Cervenansky, C.; Taylor, P. J. Biol. Chem. 1994, 269, 11233–11239.

Supporting Information Available Student handout; experimental description; hazard section; NMR spectra. This material is available via the Internet at http://pubs.acs.org.


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Introducing Dynamic Combinatorial Chemistry: Probing the Substrate

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