synthesis and self-assembly of the tennis ball dimer...

Supplementary Information 1

Synthesis and Self-Assembly of the "Tennis Ball" Dimer and Subsequent Encapsulation of Methane

An Advanced Organic 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 NMR 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 NMR 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-- 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. Often, 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 NMR.


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.

NHN

N NHPhPh

O

O

NHN

HN NPhPh

O

O 1

2

Figure 1. Presence of guest drives dimeration of 1 to form the tennis ball. Phenyl groups on the right-handmonomer have been omitted for clarity.

The assembling behavior of this molecule in solution can be studied using two characteristic

features in the 1H-NMR 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.


The NMR 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

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 (DMSO, 20 mL);

potassium hydroxide (KOH, 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

NHHN

HN NH

O

O

O

OH2N NH2

O2 +

H+, C6H6

NHHN

HN NH

O

O

1) KOH, DMSO

2) CH2Br

CH2BrBrH2C

BrH2C

NHN

N NHPhPh

O

O

NHN

HN NPhPh

O

O 1

–2 H2O

2

2


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 KOH 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 KOH.

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 12-24 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%) KOH using a mortar and pestle.

(Caution! Finely ground KOH causes severe chemical burns!) Add the freshly ground KOH (1.04 g) to a

suspension of diphenylglycoluril (2.32 g) and dimethylsulfoxide (DMSO, 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


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 NMR studies without further

purification.


Experimental Procedure Part B – NMR Studies

Reagents and Equipment

Per student, this laboratory requires compound 1 (~15 mg), CDCl3 (2 mL), DMSO-d6 (1 mL), and

three NMR tubes.

Procedure

Three samples are prepared for 1H NMR analysis (see Table). Sample A contains 4–6 mg of 1 in

0.6 mL DMSO-d6. Sample B contains 4-6 mg of 1 in 0.6 mL CDCl3. 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

NMR spectrum of each sample, scanning from –2 to 11 ppm.

Analysis of NMR results

A complete analysis the NMR 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 NMR 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 ∂ NH ∂ CH2 ∂ guest (free, bound)

A (DMSO-d6) 8.22 3.95, 4.71 N/A

B (CDCl3) 9.23 4.68, 3.95 N/A

C (CDCl3 + CH4) 9.23, 9.27 4.68, 3.95 0.22, –0.91


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 NMR 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: New York, 1995.

2) Wyler, R.; de Mendoza, J.; Rebek, J., Jr. Angew. Chem. Int. Ed. Engl. 1993, 32, 1699-1701.

3) Branda, N.; Wyler, R.; Rebek, J., Jr. Science 1994, 263, 1267-1268.


Instructor’s Notes

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 NMR 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 DMSO (≤ 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 KOH 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.


6) To remove all DMSO 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. Only 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 NMR 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.


Representative answers to questions

1) Sample A is in the monomeric state with hydrogen bonding protons well solvated by DMSO. 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 (CDCl3). 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 NMR spectra.

NH

NN

NH

OO

HN

NN

HN

OO

Ph

Ph

Ph

PhNH

NN

NH

OO

HN

NN

HN

OO

Ph

Ph

Ph

Ph

= =

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. Organic 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:


Noncovalent interaction Amino acid residues involved

The hydrophobic effect all nonpolar residues

Hydrogen bonding backbone amides, residues containing heteroatoms

Electrostatic forces charged residues (Glu, Asp, His, Arg, Lys)

Aromatic-Aromatic forces aromatic residues (Phe, Trp, Tyr)

Cation-- interactions positive and aromatic residues (Lys, Arg, His, Phe, Trp,

Tyr)

Metal ligation Cys, Ser, His, Thr, Tyr, Asp, Glu


List of chemicals used in this experiment

Chemical CAS Registry

Number

Product

Numbera

Size Cost

Benzil 134-81-6 B515-1 100 g $12.30

Chloroform-d 865-49-6 17,593-5 100 g $23.80

Dichloromethane 75-09-2 44,34-8 4 L $63.50

1,2,4,5-

Tetrakis(bromomethyl)benzene

15442-91-8 23,661-6 5 g $58.30

Methane 74-82-8 29,547-7 57 L $114.30

Methanol 67-56-1 17,933-7 1 L $16.20

Methyl sulfoxide [DMSO] 67-68-5 47,230-1 1 L $44.00

Methyl sulfoxide-d6 [DMSO-d6] 2206-27-1 15,187-4 25 g $43.90

Potassium hydroxide 1310-58-3 22,147-3 500 g $17.30

Magnesium sulfate, anhydrous 7757-82-6 20,785-3 500 g $15.90

Toluene 108-88-3 17,941-8 1 L $16.40

Trifluoroacetic acid 76-05-1 T6,220-0 100 g $19.30

Urea 57-13-6 U270-9 100 g $12.90

aAll chemicals were purchased from the Aldrich Chemical Company (Milwaukee,

Wisconsin, 1-800-558-916, http://www.sigma-aldrich.com)

synthesis and self-assembly of the tennis ball dimer...

Documents