In the Laboratory
W
Synthesis of Chemiluminescent Esters: A Combinatorial Synthesis Experiment for Organic Chemistry Students Robert Duarte Department of Math, Science and Technology, Farquhar School of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, FL 33314 Janne T. Nielsen and Veljko Dragojlovic* Oceanographic Center, Nova Southeastern University, Dania, FL 33004-3078; *
[email protected] Introduction Combinatorial synthesis is a name for a group of techniques whose goal is to synthesize a large number of structurally diverse compounds, called a library, in a short period of time (1). Several publications in this Journal described combinatorial synthesis laboratory exercises suitable for undergraduate students (2–4), including an experiment on synthesis of esters (5). Synthesis of esters and examination of their properties are important elements of both the lecture and the laboratory components of an organic chemistry course. We developed a laboratory exercise that integrates concepts traditionally included in an organic chemistry course with those of combinatorial synthesis.
Combinatorial Synthesis Methods Two common combinatorial synthesis methods are parallel combinatorial synthesis and mix-and-split combinatorial synthesis. In parallel combinatorial synthesis each compound is made separately. Thus, if one wants to prepare a library of compounds that are made by joining two building blocks, A and B, and there are six different A and three different B components, a total of 18 reactions are required to synthesize all the possible compounds. Mix-and-split combinatorial synthesis involves the preparation of mixtures of compounds rather than individual compounds. The result is a considerable reduction in time and materials as fewer reactions are done. Thus for the same library of 18 compounds only nine reactions are required. All six of the A compounds are mixed together, the mixture is split into three portions and each portion is reacted with one of the individual B compounds (B1, B2, or B3) to give three mixtures of products. The same is repeated in a reverse fashion. The B compounds are mixed together and split into six portions. Each portion is reacted with one of the A compounds to give six mixtures of products. Thus, a total of nine reactions are performed and, as a result, nine mixtures are obtained. Once a library of compounds is synthesized, the compounds are tested for the desired activity. Since parallel synthesis yields each individual compound, tests directly reveal the active compounds. The mix-and-split method results in mixtures of compounds. Thus, a positive test means that one or more of the compounds in the mixture have the desired activity. To identify the active compound, one has to perform deconvolution. Chemiluminescence Some aromatic oxalate esters exhibit chemiluminescence when treated with hydrogen peroxide in the presence of a suitable acceptor molecule (Scheme I). Reaction between such 1010
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an ester, hydrogen peroxide, and the acceptor results in a fragmentation reaction. The reaction mechanism is complex and includes multiple competing fragmentation reactions (6, 7). In the course of fragmentation, energy is transferred to the acceptor molecule, thus promoting one of its electrons into an excited state. The acceptor returns to the ground state by releasing a quantum of visible light. An article on chemiluminescence as a lecture demonstration appeared in this Journal in 1934 (8). Since then there has been a steady stream of publications that describe “substances that emit cold light upon proper oxidation” (9) including unusual ones such as oxidation of legume extracts (10) or oxidation of urine with a mixture of peroxide and blood (9). Some of the commonly used chemiluminescent compounds are luminol (8, 9, 11–16), various derivatives of oxalic acid (17–20), 1,2-dioxetanes (21, 22), lucigenin (23), tris(2,2′-bipyridyl)ruthenium(II) (24, 25), violanthrone (26), and lophine (27). Some of the acceptors used in chemiluminescence experiments are rubrene, 9,10-diphenylanthracene, 9,10-bis(phenylethynyl)anthracene, rhodamine B, and fluorescein (15, 17, 18, 23). Rubrene, 9,10-bis(phenylethynyl)anthracene, and 9,10-diphenylanthracene are relatively expensive. 9,10-Diphenylanthracene can be synthesized by the students in a separate experiment (28). Rhodamine B is a cancer suspect agent. Fluorescein is inexpensive, but in a chemiluminescence reaction only gives a weak emission of light. We found that a number of inexpensive acceptors, including anthracene (29) and chlorophyll, are suitable for a chemiluminescence experiment.
O
O R1
C
OR2
+
HO
OH
O
C
R1
C O
O
R1
acceptor decomposition + acceptor* products
acceptor
+
light
Scheme I. General reaction to observe chemiluminescence from an ester.
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In the Laboratory
Experimental Overview
Discussion
In this exercise, students prepared a library of esters made in series of reactions between an acyl chloride and a phenol, tested the compounds for chemiluminescence, and, finally, identified a general structure of a chemiluminescent ester by comparing structural properties of active and inactive esters.1 This combinatorial synthesis exercise can be run as either a one or a two-day experiment. A detailed experimental procedure is provided in the Supplemental Material.W We followed a modified procedure of Bell and coworkers (30). Hazards Acetone and ethyl acetate are flammable. Acyl chlorides should be handled in a fume hood. They are toxic and hydrolyze in air to produce HCl. Phenols cause skin burns. Some of the phenols are toxic. Students should wear gloves and safety glasses at all times. They should be instructed not to touch faucets, door knobs, et cetera with gloves on their hands. Students must be well supervised. In our labs, each laboratory section is limited to 16 students and is supervised by both a professor and a teaching assistant.
O Cl Cl O O O Cl Cl Cl
Cl O
O OH
OH
OH Cl
H3CO
OCH3 OCH3
CH3
Cl
OH
OH H 3C
Cl
OH NO2
CH3
CH3 NO2
NO2 OH
OH F
F
F Figure 1. Building blocks for the combinatorial synthesis.
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One-Day versus Two-Day Experiment We examined the feasibility of both a parallel combinatorial synthesis experiment as well as a mix-and-split combinatorial synthesis experiment by assigning the experiments to different laboratory sections of second-semester organic chemistry students. Building blocks for combinatorial synthesis were acyl chlorides and phenols (Figure 1). One- and two-day experiments, performed during threehour laboratory sessions, were explored for both parallel and mix-and-split combinatorial syntheses. In the two-day parallel combinatorial synthesis experiment, the first day was devoted to synthesis of esters of dicarboxylic acids, and the second day to synthesis of aromatic oxalates. The one-day experiment consisted only of synthesis of aromatic oxalates. In the two-day mix-and-split experiment, the first day was devoted to synthesis of mixtures of various aromatic and aliphatic esters of dicarboxylic acids (oxalic, adipic, and phthalic) and the second day to synthesis of mixtures of aromatic esters. The one-day experiment consisted only of the synthesis of mixtures of aromatic esters. After trying the various versions on different laboratory sections of our organic chemistry students, we settled on a one-day experiment, concluding with the students investigating the effect of the nature of the acceptor on a chemiluminescence reaction. Parallel Combinatorial Synthesis In parallel combinatorial synthesis each individual ester was prepared and tested for activity. In this experiment students prepared a library of 9 to 12 esters. Each student prepared one or two esters and tested them for chemiluminescence. The esters that were expected to be active were assigned to more than one student. Results from the entire class were combined and posted on the board. The parallel combinatorial synthesis of chemiluminescent esters experiment was relatively short and a student could prepare 2–3 esters in the course of a three-hour laboratory session or could repeat an unsuccessful preparation. The parallel combinatorial synthesis experiment worked well. However, preparation of the esters was not trivial. The students had to be careful and had to have reasonably good technical skills. The experiment was done by our second-semester organic chemistry students who were familiar with reactions executed under anhydrous conditions, use of syringes to measure and deliver reagents, and precautions when dealing with corrosive compounds. Still we found that the experiment was challenging for some of the students. Thus, it was necessary to assign preparation of the active esters to more than one student. One of the major problems with this experiment was cross contamination of the reagents. To minimize this we color coded the reagents and syringes. Phenols with electron-withdrawing substituents were expected to give positive reactions. Good results were obtained with oxalate esters of 4-nitrophenol, 2,4,6-trichlorophenol, and 2,3,6-trifluorophenol with rubrene as an acceptor. Although 2,4-dinitrophenol gave the oxalate ester that exhibited the most intense chemiluminescence, 2,4-dinitrophenol had to be dried before the reaction and its oxalate ester appeared to be less stable compared to other oxalate esters. Thus, some students had difficulty getting good results
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with it. There were few problems with 4-nitrophenol compared to 2,4-dinitrophenol. Some of the chemiluminescent esters emitted low-intensity light over a period of time. If viewing conditions are not optimal such emission may not be noticed by the students. Addition of a catalyst (imidazole or sodium salicylate) resulted in a brief and more intense flash of light (20). Thus, in a parallel combinatorial synthesis experiment, a catalyst could be added to identify all of the active compounds. We do not recommend use of such catalysts in a mix-and-split combinatorial synthesis experiment, as the results would be too difficult to interpret. A simple experiment might include p-cresol (a phenol with a moderate electron-donating substituent), 3,4,5-trimethoxyphenol (a phenol with three strong electron-donating substituents), and 4-nitrophenol (a phenol with a strong electron-withdrawing substituent). A more complex experiment might include a number of phenols (e.g., p-cresol, 4nitrophenol, 2,4-dinitrophenol, 2,4,6-trichlorophenol, 2,3,6-trifluorophenol, and 3,4,5-trimethoxyphenol) with the goal of identifying differences in the activities of the resulting oxalate esters. This may be a good time to discuss with
OH
OH
O Cl Cl
+
+
O CH3
NO2
CH3
O O O O H3C
+ NO2
O O O O H3C
+ NO2
O O O O O2N
Scheme II. Example of mixed ester products in the mix-and-split method.
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the students how electron-withdrawing versus electron-donating substituents on the leaving group affect the rate of nucleophilic acyl substitution reactions, as well as the difference in reactivity between phenols and alcohols. Relative reactivities of carboxylic acids, acyl chlorides, and p-nitrophenyl esters in a nucleophilic acyl substitution reaction were discussed in a recent article in this Journal (31).
Mix-and-Split Combinatorial Synthesis Mix-and-split combinatorial synthesis involves the preparation of mixtures of esters rather than an individual ester. Since both reagents and reaction products are moisture sensitive, care was taken to minimize possible problems and hence the mixtures of phenols (p-cresol, 4-nitrophenol, and 2,4-dinitrophenol) and acyl chlorides (adipoyl chloride, oxalyl chloride, and phthaloyl chloride) were prepared for the students in advance. Each student was assigned a total of two series of reactions. One series of reactions (e.g., reaction of each phenol with the mixture of acyl chlorides) was done first, and the mixtures were tested for chemiluminescence and the results were briefly discussed. Then students were assigned to do the next series of reactions (reaction of each acyl chloride with the mixture of phenols). Doing one series of reactions followed by another helped students understand the principle of mix-and-split synthesis. Students prepared a total of 6 to 12 different mixtures of esters. In order to make sure that the class obtained a positive reaction when there should be one, preparation of certain mixtures of esters was assigned to more than one student. At the end of the laboratory session, all the results were written up on the board. The students were required to perform deconvolution on their own after the laboratory exercise (details are provided in the Supplemental MaterialW) and write a formal report for the experiment. It should be noted that in a mix-and-split method there is a possibility of the formation of mixed esters (Scheme II). This is a drawback of the method that we did not mention to the students as this would complicate deconvolution. If a goal of the laboratory exercise is to include comparison of the activity of individual mixed esters, students can prepare them by following a known procedure (32). Results of a detailed examination of chemiluminescence of mixed oxalate esters were reported by Orosz (33). Experiments with Nonphenolic Alcohols Alcohols other than phenols can be used in a parallel combinatorial synthesis experiment. Of course, the corresponding alkyl oxalate esters as well as phthaloyl and adipoyl esters are inactive in a chemiluminescence experiment. However, presence of an alcohol may present a problem in a mixand-split combinatorial synthesis experiment. Some of the alcohols are difficult to dry and to keep dry. Also, intrinsic reactivity of most aliphatic alcohols is higher compared to phenols, in particular phenols with electron withdrawing groups. Thus, if a student were to add an insufficient quantity of oxalyl chloride, it is the phenol that would not be converted into a diester. We abandoned a mix-and-split combinatorial synthesis experiment involving alcohols as it was experimentally difficult and only a few of the students were expected to obtain a positive result. Therefore, it was not rewarding enough for the students.
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In the Laboratory Table 1. Emissions of Oxalate Esters in the Presence of Hydrogen Peroxide with Rubrene As a Fluorophore Int e ns it y of Emis s ion
Es t e r
Durat ion/ min
bis (4-nit rophe ny l) ox alat e
s t rong
15–20
bis (p-cre s y l) ox alat e
no e mis s ion
---
bis (2,4-dinit rophe ny l) ox alat e
v e ry s t rong
10–15
bis (2,4,6-t richlorophe ny l) ox alat e
s t rong
15–20
bis (3,4,6-t rif luorophe ny l) ox alat e
s t rong
< 5a
bis (3,4,5-t rime t hox y phe ny l) ox alat e
no e mis s ion
---
a
B is (2,3,6-t rif luorophe ny l) ox alat e dis colore d s olut ions of acce pt ors ( m o s t l i k e l y o x i d a t i o n o f t he a c c e p t o r b y hy d r o g e n p e r o x i d e ) . Emis s ion of light did not las t long. A f t e r addit ion of s olut ion of acce pt or t o s uch a re act ion mix t ure , e mis s ion of light re s ume d.
Chemiluminescence with Amides A number of amides of oxalic acid are also chemiluminescent, but were not suitable for this experiment. Amides are more difficult to prepare compared to esters. Reaction between an amine and an acyl chloride may be very vigorous and as a result some students ended up with a black sludge. When students carefully followed the experimental procedure, the resulting amides worked very well in a chemiluminescent reaction. However, chemiluminescent amides are very reactive and, upon addition of hydrogen peroxide in the presence of an acceptor, there is only a brief intense flash of light. Oxalyl chloride is also chemiluminescent and gives off a flash of light under the same conditions (18). Thus, if the preparation of an amide does not go to completion, unreacted oxalyl chloride will give a false positive reaction. There was
no such problem with esters as the activity of oxalyl chloride (a brief flash of light) was easily distinguished from that of an ester (an emission of light that lasts for minutes). Results The success rate in a mix-and-split combinatorial synthesis experiment was only ∼50%, compared to ∼80% in parallel combinatorial synthesis. A success meant that a student observed a chemiluminescence reaction when there should have been one. In a subsequent experiment, students examined the chemiluminescence reaction itself. When doing initial tests on oxalate esters, the students used rubrene as an acceptor (Table 1). In the final part of the exercise, students examined the effect of the acceptor on the efficiency of a chemiluminescence reaction (as determined by the intensity of emitted light) and the effect of the acceptor on the color of emitted light. We found that a number of inexpensive acceptors are suitable for this experiment (Table 2). Among them, freshly isolated chlorophyll gave the best results. However, not all oxalate esters gave a positive chemiluminescence reaction with all of the acceptors. Among the acceptors we used, rubrene gave a positive reaction with the largest number of esters (Figure 2). A reaction mixture can be heated in a water bath to increase the intensity of the emitted light (Figure 3). It was interesting for the students to compare colors of light emitted by anthracene and 9,10-diphenylanthracene (Figure 4). Since colors of the emitted light depend on the degree of conjugation in the acceptors, by comparing the colors, or even better the emission spectra, students can make conclusions about the degree of conjugation between the two phenyl substituents and the anthracene ring in 9,10diphenylanthracene.
Table 2. Behavior of Acceptors in a Reaction of Bis(2,4-dinitrophenyl) oxalate with Hydrogen Peroxide Acceptor
Color of Emitted Light
Intensitya
Duration
anthracene
blue
medium
5 min
xanthone
yellow–white
weak
2 min
1,10-phenanthroline
pale yellow
very weak
few seconds
chlorophyll
red
strong
5 min
stilbene
pale yellow
weak
2–5 min
fluorescein
yellow
weak
5 min
9,10-diphenylanthracene
blue
strong
15 min
rubrene
yellow–orange
very strong
10–15 min
2-naphthol
pale blue
weak
2 min
2-nitro-1-naphthol
yellow
weak
1 min
2,4-dinitro-1-naphthol
yellow–green
medium
few seconds
naphthalene
pale yellow
very weak
few seconds
benzil
pale yellow
very weak
few seconds
dibenzalacetone
pale green
weak
few seconds
methyl salicylate β-carotene
pale blue
weak
1 min
blue-green
weak
1 min
a
Strong—emission can be seen in a room with lights off (emergency light was on, external light coming in through windows was not blocked); medium—can be seen in a room, but best seen in a dark box; weak—can be seen in a dark box; very weak—can be seen in a dark box after eyes have adapted to darkness.
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Figure 3. Chemiluminescence of bis(2,4-dinitrophenyl) oxalate with hydrogen peroxide in presence of chlorophyll (A) at room temperature and (B) in boiling water. (This figure appears in color in the table of contents on page 916.)
Figure 2. Chemiluminescence of bis(4-nitrophenyl) oxalate, bis(2,4dinitrophenyl) oxalate, bis(3,4,6-trifluorophenyl) oxalate, and bis(2,4,6-trichlorophenyl) oxalate (from left to right) upon addition of H2O2 in presence of (A) chlorophyll, (B) rubrene, (C) anthracene, and (D) 9,10-diphenylanthracene. (Figure 2B appears in color in the table of contents on page 916.)
Figure 4. Chemiluminescence of bis(2,4-dinitrophenyl) oxalate with hydrogen peroxide in the presence of anthracene (left beaker) and 9,10-diphenylanthracene (right beaker) after (A) 10 seconds, (B) 30 seconds, (C) 1 minute, and (D) 10 minutes. (This figure appears in color in the table of contents on page 916.)
Conclusions
Note
This is a discovery-based experiment that introduces a number of complex concepts (chemiluminescence, combinatorial synthesis, SAR, intrinsic reactivity, UV–vis, fluorescence spectroscopy, and photochemistry). The instructor can decide which and how many concepts to include. Once the esters were successfully prepared, positive results were relatively easy to obtain and were easily distinguished from negative results. While students went through some difficult concepts and had some confusion about the experimental details, at the end, they liked the experiment, which included brightly colored, luminescent solutions.
1. A part of this work was presented on April 11, 2002 at the 223rd American Chemical Society Meeting in Orlando, FL.
Acknowledgments We thank our CHEM 3310 students for testing this experiment. W
Supplemental Material
A handout to students, details of preparation, additional photographs, and information for the instructors are available in this issue of JCE Online.
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