Exploring the Chemistry of the Mechanical Bond: Synthesis of a [2]Rotaxane through Multicomponent Reactions

The synthesis of a [2]rotaxane through three- or five-component coupling reactions has been adapted to an organic chemistry experiment for upper-division students. The experimental procedure addresses the search for the most favorable reaction conditions for the synthesis of the interlocked compound, which is obtained in a yield of up to 71%. Moreover, the interlocked nature of the rotaxane is proven by NMR spectroscopy. The content of the sessions has been designed on the basis of a proactive methodology whereby upper-division undergraduate students have a dynamic role. The laboratory experience not only introduces students to the chemistry of the mechanical bond but also reinforces their previous knowledge of basic organic laboratory procedures and their skills with structural elucidation techniques such as NMR and FT-IR spectroscopies. The experiment has been designed in such a customizable way that both experimental procedures and laboratory material can be adapted to a wide range of undergraduate course curricula.


Aims
-To introduce students to the usual methods for the synthesis of rotaxanes.
-To highlight the key role of the template effect in the synthesis of interlocked compounds.
-To synthesize a [2]rotaxane through a five or three-components clipping reaction.
-To reinforce key organic chemistry techniques and learn new ones.
-To consolidate previously acquired principles of FT-IR and NMR spectroscopy and apply them to the structural elucidation of an interlocked compound.

Learning Outcomes
At the end of these sessions, you should be able to: -Extract relevant information from the bibliography given by the instructor.
-Perform chemical reactions under high dilution conditions.
-Compare, discuss, and interpret a set of results obtained under different reaction conditions on the basis of the provided background.
-Check the purity of organic compounds by thin layer chromatography (TLC).
Mechanically interlocked molecules (MIMs) are a type of compounds constituted by at least two submolecular components. 1,2These components are not covalently linked, but they are topologically interlocked one to each other.This particular link between the subcomponents of these molecules is known as the mechanical bond. 3 order to form a mechanical bond, it is necessary to arrange the different components in an orthogonal geometry.For this purpose, different methods for the assembly of MIMs have been developed over the years.The assembly employing transition metals as templates allows this union to be established by coordination of both components with a metal ion.Through the hydrophobic effect, several components can be joined by forming a hydrophobic cavity.The host-guest interactions between π-donors and acceptors allow the interconnection by the attraction of the components.Another particularly useful approach for obtaining MIMs is the establishment of hydrogen bond interactions between both components. 4,5,6,7,8drogen bonds are characterized by their directionality and ability to act cooperatively, forming associations between donor and acceptor groups (Figure S1).In this type of bond, the donor group, the hydrogen atom, and the acceptor one (usually a heteroatom) are aligned.These bonds have a high dipolar character.Within the wide range of synthetic MIMs, two families must be highlighted due to their versatility and their extraordinary properties: catenanes and rotaxanes (Figure S2).
Catenanes are molecules in which at least two macrocycles are interlocked, mimicking the chain links. 11In the case of rotaxanes, the two components are of different type, one linear and one cyclic.The simplest example of a rotaxane could be considered as a dumbbell, a linear component with bulky groups at the ends, surrounded by a cyclic one.
The bulky groups are known as stoppers and their purpose is to prevent the dethreading of the components. 12One would think that, to achieve the synthesis of a rotaxane, it is necessary to know how to sew molecules.The linear component, better known as axis or thread, should be introduced through the cyclic component or macrocycle.The first rotaxane was synthesized by Harrison and Harrison more than five decades ago via a statistical approach (Figure S3a). 13This type of protocol is characterized by an absence of interactions between the precursors to allow their efficient orientation, affording the interlocked species in very low yields.Later, Professor Schill used a covalent-bond-directed synthesis to obtain another rotaxane (Figure S3b).Although the product was obtained in a higher yield, the synthetic route involved a high number of reaction steps. 14
Since the publication of these early examples, the synthesis of rotaxanes has undergone a remarkable development characterized by the variety of available methods and a significant increase in reaction yields.The most advantageous synthetic methods employ a template that appropriately orients the precursors in space.These methods involve the prior formation of a supramolecular complex stabilized by non-covalent interactions. 15Subsequent covalent modifications prevent the dissociation of these components.
Template-based methods for the obtention of rotaxanes can be classified into five main types (Figure S4).The capping methodology (Figure S4a), which involves the prior formation of a pseudorotaxane and its subsequent capping.In the snapping methodology (Figure S4b), once a semirotaxane has been formed, the capping of the non-stopper end Supporting Information S5 is accomplished.In the slipping methodology (Figure S4c), the cyclic component is threaded onto a thread having stoppers under high temperature conditions.The clipping methodology (Figure S4d) involves the cyclisation of a ligand around a thread having stoppers.In the metal-active template method (Figure S4e), the metal plays a dual role, orienting the ligands in the appropriate geometry and catalyzing the formation of the covalent bond that captures the interlocked species.Rotaxanes are the most interesting type of MIMs because of their greater variety of motions and large number of applications. 12 Nature, MIMs play a fundamental role in many processes occurring within the organisms.The establishment of mechanical bonds is critical for the development of various biological processes, such as mitochondrial scission, selective ion transport or DNA replication, which proceed through mechanically interlocked intermediates. 1,16,17,18,19Nature is always ahead of science.Whatever a scientist thinks of designing, Nature did it previously.Therefore, Nature is a great source of inspiration, in particular a wide number of biological systems.There is a duality that may seem contradictory.In order to understand Nature, it is necessary the progress in science and, for the improvement in research, it is essential to interpret Nature.Thus, chemists have dedicated a lot of effort to understand the chemistry of the mechanical bond, thus overcoming the inherent difficulty in achieving the adequate spatial arrangement of the different components.

Safety and Hazards
Chemistry laboratories are potentially dangerous places and therefore must be treated with respect.The following rules are for the safety of you, other students and members of staff and should be always followed.
-You should wear appropriate personal protective equipment, such as disposable gloves, goggles, closed shoes and a lab coat.
-The procedures must be performed in a fume hood or similarly ventilated workspace.
-Make sure that all glassware containing chemicals is properly labelled.
-Liquid and solid waste must be disposed into sealed and appropriately labeled containers.
-The rinsed syringe should be disposed into a dedicated, appropriately labelled disposal container.The rinsed needle can be reused after being dried in an oven.
-Report all accidents or near accidents to the instructor.
-Do not remove samples or chemicals from the laboratory.
Safety information for all reagents is available via the appropriate Safety Data Sheet (SDS).The Globally Harmonised System (GHS) is a single worldwide system for classifying and communicating the hazardous properties of industrial and consumer chemicals.

General Experimental Information
All commercially available compounds were purchased from Merck, Acros Organics, or Alfa-Aesar Chemical Co. and used without purification.N 1 ,N 1 ,N 4 ,N 4 -Tetrabutylfumaramide (2) was prepared from fumaroyl dichloride and dibutylamine. 20e pre-formed U-shaped component, N 1 ,N 3 -bis[4-(aminomethyl)benzyl]isophthalamide (5), was generated in situ by the acidic deprotection of the corresponding di-Boc derivative, 21 following previously described procedures.HPLC grade solvents (Scharlab) were nitrogen-saturated as well dried and deoxygenated using an Innovative Technology Inc. Pure-Solv 400 Solvent Purification System.Deionized water was used in the preparation of all aqueous solutions.Brine refers to a saturated aqueous solution of sodium chloride.
TLC are performed on precoated silica gel on aluminum cards (0.25 mm thick, with 254 nm fluorescent indicator, Fluka) and observed under UV light (254 or 365 nm).
The additions are made with kd Scientific motor-driven syringe pumps (model number 101) and stainless steel 304 syringe needle, noncoring point (30 cm) from Sigma-Aldrich.
Under reduced pressure refers to the use of a Büchi Rotavapor R-3000 or a Heidolph Hei-Vap Value G3 apparatus with a Vacuumbrand CVC 3000 vacuum pump equipped with a water bath for aiding to remove the solvent.
Melting point (m.p.) is determined on a Kofler hot-plate melting point apparatus and is uncorrected.
Infrared spectroscopy is performed using a PerkinElmer Spectrum 65, FT-IR Spectrometer (ATR) in the range 4000-600 cm -1 .The intensities of the absorption bands are indicated as vs (very strong), s (strong), m (middle) and w (weak).

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NMR spectra are recorded at 298 K using a Bruker Avance 400 instrument (400 MHz 6 Experimental Procedure for the five-component synthesis of [2]rotaxane Protocol: The amounts of each reagent and solvent used, as well as the addition time for each pair of students are specified in Table S1. 1. CHCl 3 (n -40 mL) is added to a round bottom flask (RBF) equipped with a stir bar.
2. The thread 2 (x equiv) and Et 3 N (z/2 equiv) were added to the RBF.
3. The RBF is sealed with a septum and the solution is stirred vigorously.
4. Two 25 mL syringes are prepared with the needles previously dried in the oven or alternatively, two dried dropping funnels.
5. The first syringe or dropping funnel is filled with a solution of isophthaloyl dichloride (3) (y equiv) in CHCl 3 (20 mL).
7. The two syringes are adapted to the motor-driven syringe pump and programmed for the assigned addition time according to table S1.When dropping funnels are used instead of syringes, the addition rate should be controlled manually.
8. After completion of the addition, the reaction mixture is stirred for additional 10 minutes.
9. The resulting suspension is filtered through a Celite pad.
11.The organic phase was then dried with anhydrous MgSO 4 .
12. The solution is filtered, and the solvent was removed under reduced pressure.
13.The resulting solid is placed into a filter plate funnel and washed with diethyl ether (3 x 15 mL) until all the unreacted thread 2 is extracted.To identify the product on TLC it is a common practice to run a side-by-side comparison of all material potentially present in the crude mixture.Make a TLC (SiO 2 , CHCl 3 /acetone 9:1) after each extraction with 5 mL of ether, comparing with a pure sample of fumaramide 2.
Product identification: 1. Measure the melting point of your product and compare it with that described in the literature.
2. Performs an IR-ATR spectrum of your product.
3. Prepare a sample for NMR analysis with 15 mg of your product in CDCl 3 .
4. With the help of your instructor, analyze the following one-dimensional spectra: 1 H, 13 C and DEPT-135 (or APT) and two-dimensional spectra: 1 H, 1 H-COSY,

Protocol:
The amounts of each reagent and solvent used, as well as the addition time for each pair of students are specified in Table S1.
1. CHCl 3 (n -20 mL) is added to a round bottom flask (RBF) equipped with a stir bar.
2. The thread 2 (x equiv), the pre-formed U-shaped 5 (y equiv) and Et 3 N (z equiv) were added to the RBF.
3. The RBF is sealed with a septum and the solution is stirred vigorously.4. One 25 mL syringe is prepared with the needle previously dried in the oven.
6.The syringe is adapted to the motor-driven syringe pump and programmed for the assigned addition time according to table S1. 7.After completion of the addition, the reaction mixture is stirred for additional 10 minutes.
11.The solution is filtered, and the solvent was removed under reduced pressure.
12. The resulting solid is placed into a filter plate funnel and washed with diethyl ether (3 x 15 mL) until all the unreacted thread 2 is extracted.To identify the product on TLC it is a common practice to run a side-by-side comparison of all material potentially present in the crude mixture.Make a TLC (SiO 2 , CHCl 3 /acetone 9:1)

S13
after each extraction with 5 mL of ether, comparing with a pure sample of fumaramide 2.

Product identification:
1. Measure the melting point of your product and compare it with that described in the literature.
2. Performs an IR-ATR spectrum of your product.
3. Prepare a sample for NMR analysis with 15 mg of your product and CDCl 3 .
Complete the following tables (S4 and S5) with appropriate values for the reagents to be used.Some information has been provided to guide you.

Table S1 .
Hazards of the chemical compounds employed in the laboratory experiment.
The Chemical Abstracts Service (CAS) Numbers and Globally Harmonised System (GHS) Hazards of the chemical compounds and solvents used in this work are listed below: 13 frequency, 101 MHz 13 C frequency).Chemical shifts are quoted in parts per million (ppm), referenced to residual chloroform (7.26 ppm for 1 H NMR, 77.00 ppm for13C NMR), as internal standard, whereas coupling constants, J, are quoted in Hz.Multiplicities are

Table S3 .
Different reaction conditions carried out by the students

Table S5 .
Data for the three-component synthesis of[2]rotaxane 1