Synthesis and Self-Assembly of the "Tennis Ball" Dimer and Subsequent Encapsulation of Methane. An Advanced Organic Chemistry Laboratory Experiment

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1 Synthesis and Self-Assembly of the "Tennis Ball" Dimer and Subsequent Encapsulation of Methane An Advanced rganic Chemistry Laboratory Experiment Lab documentation Written material for use by students Abstract While important to the biological and materials sciences, noncovalent interactions, self-folding, and self-assembly often receive little discussion in the undergraduate chemistry curriculum. The synthesis and MR characterization of a molecular Tennis Ball in an advanced undergraduate organic chemistry laboratory is a simple and effective way to introduce the relevance of these concepts. In appropriate solvents, 1 dimerizes through a seam of 8 hydrogen bonds with encapsulation of a guest molecule and symmetry reminiscent of a tennis ball. The entire experiment can be completed in three lab periods, however large scale synthetic preparation of 1 by a teaching assistant would reduce the laboratory to a single lab period for MR studies. Introduction While covalent bonds determine the primary structure of molecules, it is noncovalent interactions that control the supramolecular organization of molecules. 1 A wide variety of noncovalent interactions (electrostatic interactions, van der Waals forces, hydrogen bonding, metal-ligand interactions, the hydrophobic effect, cation-ϖ forces, and aromatic-aromatic forces) play important roles in biological chemistry, materials science, and information technology. The dynamic and reversible nature of these forces allows for the organization of simple molecules into more complicated systems. ften, this organization gives rise to functions not present in the simple, unorganized state. The self-organizing processes that take place are grouped into the categories of self-folding (organization within a single molecule) and self-assembly (organization of more than one molecule). In this experiment a simple organic molecule is synthesized and its self-assembling behavior is studied using MR. Supplementary Information 1

2 Background Compound 1 is a curved molecule that displays self-complementary hydrogen bonding sites. In solvents that effectively compete for hydrogen bonds, 1 exists as a free monomer. In noncompetitive solvents and in the presence of a suitable guest molecule, 1 self-associates through hydrogen bonds, forming a dimeric structure that encapsulates the smaller guest molecule within the cavity. The symmetry of this complex has led to it being dubbed the tennis ball (Figure 1). 2,3 Hydrogen bonding interactions between monomers drive assembly of the complex. 2 H H 1 Figure 1. Presence of guest drives dimeration of 1 to form the tennis ball. enyl groups on the right-hand monomer have been omitted for clarity. The assembling behavior of this molecule in solution can be studied using two characteristic features in the 1 H-MR spectrum: 1) The chemical shift of protons involved in hydrogen bonds typically shift downfield due to a decrease in electron density near the nucleus. Moreover, the formation of a hydrogen bond in a discrete and kinetically stable species results in a strong and concentration independent change in the chemical shift to lower field. 2) Protons belonging to the encapsulated guest molecule experience a shielding effect from the adjacent aromatic walls of the capsule. In the case of kinetically stable complexes this results in upfield shifted signals that can only arise from encapsulated guest molecules. Supplementary Information 2

3 The MR studies that make up this experiment will show compound 1 in its monomeric state, self-assembled while encapsulating a solvent molecule, and self-assembled while encapsulating a small guest molecule (methane). Experimental Procedure Part A - Synthesis 2 + H 2 H 2 H +, C 6 H 6 2 H 2 H H 2 2 H H 1) KH, DMS 2) BrH 2 C CH 2 Br H H BrH 2 C CH 2 Br 1 Reagents Per student, this laboratory requires benzil (2.0 g); urea (1.14 g); toluene (25 ml); trifluoroacetic acid (TFA, 1 ml); dichloromethane (100 ml); methanol (50 ml); dimethylsulfoxide (DMS, 20 ml); potassium hydroxide (KH, 85%, 1.04 g); 1,2,4,5-tetrakis(bromomethyl)benzene (0.18 g); magnesium sulfate (~5 g). Equipment The following equipment is required: Pasteur pipets, spatulas, stir bars, magnetic stirrer, heating mantle, 50 and 100 ml round-bottom-flasks, Dean-Stark trap, condenser, drying tube, filter flask, Büchner funnel, filter paper, mortar and pestle, Erlenmeyer flasks, rotary evaporator. Hazards Warning! Trifluoroacetic acid, potassium hydroxide, and dichloromethane cause severe chemical burns. Chloroform-d, dichloromethane, methanol, toluene, are trifluoroacetic acid are toxic substances and Supplementary Information 3

4 should be handled carefully. Tetrabromodurene is both corrosive and a lachrymator. Dimethylsulfoxide facilitates the absorption of other chemicals through the skin. Toluene and methanol are flammable and should be kept a way from open flames. All chemicals should be handled in a fume hood while wearing gloves, safety glasses, and a lab coat. Waste Disposal All reagents can be disposed in an organic waste container, except KH which should be placed in a solid or aqueous waste, aqueous filtrate from synthesis of tennis ball monomer, and chloroform which may require disposal in a halogenated waste. Be sure to keep trifluoroacetic acid and other strong acids segregated from strong bases such as KH. Procedure Day 1 Diphenylglycoluril. Combine benzil (2.00 g), urea (1.14 g), toluene (15 ml) and trifluoroacetic acid (1 ml) in a single neck 50 ml round bottom flask fitted with a magnetic stir bar, a Dean-Stark trap, a reflux condenser, and a drying tube. (Caution! Trifluoroacetic acid causes severe chemical burns and can leave significant scarring!) Using a heating mantle and a magnetic stirring plate bring the mixture to reflux for hours. After cooling to room temperature filter the mixture and wash the filter cake with methanol (50 ml) and dichloromethane (50 ml) to remove soluble impurities. This gives pure diphenylglycoluril as a white powder (2.56 g, 91%). The solid can be stored in air until it is used in the next step. Procedure Day 2 Tennis ball monomer (1). Grind some technical grade (85%) KH using a mortar and pestle. (Caution! Finely ground KH causes severe chemical burns!) Add the freshly ground KH (1.04 g) to a suspension of diphenylglycoluril (2.32 g) and dimethylsulfoxide (DMS, 20 ml) in a 50 ml round bottom flask. Fit the flask with a condenser and heat it rapidly to 120 C using a preheated heating mantle. After 5 minutes add 1,2,4,5-tetrakis(bromomethyl)benzene (0.18 g) as a solid and continue heating for 45 minutes. Cool the reaction to room temperature and pour it into an Erlenmeyer flask containing water (300 ml). Collect the white precipitate by vacuum filtration on a Büchner funnel. Re-suspend the residue in an additional 100 ml of water, stir for 5 minutes, and filter. (The filtration may take up to one hour). To Supplementary Information 4

5 extract the product from the solid, place the solid filter cake in a single neck 100 ml flask, add dichloromethane (50 ml) and fit the flask with a condenser. Use a heating mantle to heat the mixture briefly to reflux, cool it to room temperature, and filter it again, washing with more dichloromethane (50 ml). Collect the combined organic filtrates, dry them over magnesium sulfate, and concentrate the resulting solution to dryness on a rotary evaporator to give the crude tennis ball monomer (1) as a pale yellow powder (40-75 mg). This material can be used for the following MR studies without further purification. Supplementary Information 5

6 Experimental Procedure Part B MR Studies Reagents and Equipment Per student, this laboratory requires compound 1 (~15 mg), CDCl 3 (2 ml), DMS-d 6 (1 ml), and three MR tubes. Procedure Three samples are prepared for 1 H MR analysis (see Table). Sample A contains 4 6 mg of 1 in 0.6 ml DMS-d 6. Sample B contains 4-6 mg of 1 in 0.6 ml CDCl 3. Sample C is prepared by bubbling in-house natural gas (methane) slowly into a solution prepared similarly to B for 5 minutes. (Caution! Methane is a flammable gas. Be sure to remove all possible sources of ignition before use!) Collect the MR spectrum of each sample, scanning from 2 to 11 ppm. Analysis of MR results A complete analysis the MR spectra is confounded by the presence of impurities and complicated aromatic and aliphatic regions. However, the analysis is simplified by the presence of key signals for hydrogen bonding protons and encapsulated guests in the unobscured downfield and upfield windows, respectively. Record the shift of signals from 8.2 to 10.0 ppm and from 1 to 1 ppm for each sample. Representative MR data for samples A-C is shown in the Table (see below). Table: Representative chemical shifts (ppm) of selected signals for samples of 1. Sample H CH 2 guest (free, bound) A (DMS-d 6 ) , 4.71 /A B (CDCl 3 ) , 3.95 /A C (CDCl 3 + CH 4 ) 9.23, , , 0.91 Supplementary Information 6

7 Questions to address in your lab report: 1) The synthesis of 1 is hampered by the formation of one major isomeric side product during the alkylation of 1,2,4,5-tetrakis(bromomethyl)benzene. What is this side product? 2) From the MR evidence that you have collected, what is the nature of the assembled state of 1 in each of samples A, B, and C? 3) What behavior emerges from the self-assembly of 1 that is not present in the unassembled state? 4) Predict which other guest molecules may be appropriate for encapsulation by 1 and rationalize your choices. 5) The self-folding and self-assembly of proteins is a problem of great interest to biochemical researchers. Based on what you know about the different amino acid structures, tabulate the major noncovalent interactions and the amino acids that would be associated with each type of interaction. References 1) Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspectives; Wiley & Sons: ew York, ) Wyler, R.; de Mendoza, J.; Rebek, J., Jr. Angew. Chem. Int. Ed. Engl. 1993, 32, ) Branda,.; Wyler, R.; Rebek, J., Jr. Science 1994, 263, Supplementary Information 7

8 Instructor s otes Safety and Hazards Summary Chloroform-d, dichloromethane, methanol, toluene, are trifluoroacetic acid are toxic substances and should be handled with appropriate care. Dichloromethane, trifluoroacetic acid and potassium hydroxide can cause severe chemical burns. 1,2,4,5-tetrakis(bromomethyl)benzene is a lachrymator. Dimethylsulfoxide facilitates the absorption of other chemicals through the skin. Toluene and methanol are flammable and should be kept away from open flames. All chemicals should be handled in a fume hood only when wearing appropriate protective equipment that includes safety glasses, gloves, and a lab coat. All reactions should be carried out in a safe environment that includes proper ventilation and protection from chemical fumes. Tips for the instructor 1) If desired, both synthetic steps are easily performed by teaching assistants on a scale large enough to produce material for many students (>20x the scale reported above). This eliminates the synthetic component for the student, and reduces the total laboratory to a single day of MR sample preparation and studies as outlined below. 2) The high melting point and low solubility of diphenylglycoluril make it impractical to characterize this compound using melting point or thin layer chromatography. Its low solubility, however, is used to aid in its purification. After washing with methanol (to remove excess urea) and dichloromethane (to remove other organics) it can be safely assumed that the resulting white powder is pure product. 3) In the formation of diphenylglycouril (see Procedure 1), p-toluenesulfonic acid (0.5 g) can be used in place of the more corrosive trifluoroacetic acid, with a reduction in yield (approximately 50%). 4) Reagent grade DMS ( 1% water) can be used as solvent for procedure 2. Best results are obtained with a newly opened bottle. 5) In Procedure 2, the solution of diphenylglucouril and KH in may become quite viscous and may be difficult to stir. As the reaction is heated and 1,2,4,5-tetrakis(bromomethyl)benzene is added, the reaction should become a relatively homogeneous suspension. Supplementary Information 8

9 6) To remove all DMS from compound 1, wash the residue with as much water as time permits. The vacuum filtration that follows the heating period during the preparation of compound 1 can be slow, so fast filter paper (Whatman Qualitative Grade #1) should be used. Similarly, multiple extractions with dichloromethane will improve the yield of the desired product. 7) The unique solubility of the desired product 1 is exploited in its purification. nly the desired product and a structural isomer are soluble in dichloromethane. The product can thus be washed from the filter cake and collected in the filtrate. 8) Representative MR spectra for samples A, B, and C taken at 300 MHz are shown below. 9) If in-house methane is not available, lecture bottles of methane are available from Aldrich. Supplementary Information 9

10 Representative answers to questions 1) Sample A is in the monomeric state with hydrogen bonding protons well solvated by DMS. Sample B displays the downfield shift of hydrogen bonding protons indicative of assembly, but no signal for encapsulated guest is visible because the guest is deuterated solvent (CDCl 3 ). Sample C contains signals for both the solvent-filled capsule and the methane-filled capsule. The signals for free and encapsulated methane are clearly visible at ~0.2 and 0.9 ppm, respectively. 2) The formation of the tennis ball requires the alkylation on both sides of the durene spacer to occur in a cis manner, to give the so-called C-isomer. If alkylation occurs in a trans manner, the result is the S-isomer shown below. This isomer cannot form self-assembled capsules and so its signals are not present in the downfield and upfield windows of the MR spectra. H H H H = = H H H H 3) The monomeric state is not capable of binding small molecules (such as chloroform and methane), but this behavior emerges as a result of the self-assembly. 4) Based on the small size of the cavity, organic molecules somewhere between the size of methane and chloroform are appropriate choices. rganic molecules with nonpolar surfaces (to match the inner aromatic walls of the capsule) would be best. Dichloromethane and ethene are also appropriate guests for the capsule formed by 1. 5) A list of noncovalent forces (and the residues involved) that may aid protein folding and assembly is as follows: Supplementary Information 10

11 oncovalent interaction The hydrophobic effect Hydrogen bonding Electrostatic forces Aromatic-Aromatic forces Cation-ϖ interactions Amino acid residues involved all nonpolar residues backbone amides, residues containing heteroatoms charged residues (Glu, Asp, His, Arg, Lys) aromatic residues (e, Trp, Tyr) positive and aromatic residues (Lys, Arg, His, e, Trp, Tyr) Metal ligation Cys, Ser, His, Thr, Tyr, Asp, Glu Supplementary Information 11

12 Chemical List of chemicals used in this experiment CAS Registry umber Product Size Cost umber a Benzil B g $12.30 Chloroform-d , g $23.80 Dichloromethane , L $ ,2,4,5- Tetrakis(bromomethyl)benzene , g $58.30 Methane , L $ Methanol , L $16.20 Methyl sulfoxide [DMS] , L $44.00 Methyl sulfoxide-d 6 [DMS-d 6 ] , g $43.90 Potassium hydroxide , g $17.30 Magnesium sulfate, anhydrous , g $15.90 Toluene , L $16.40 Trifluoroacetic acid T6, g $19.30 Urea U g $12.90 a All chemicals were purchased from the Aldrich Chemical Company (Milwaukee, Wisconsin, , Supplementary Information 12