In the Laboratory

Supramolecular Inclusion in Cyclodextrins: A Pictorial Spectroscopic Demonstration Basudeb Haldar, Arabinda Mallick, and Nitin Chattopadhyay* Department of Chemistry, Jadavpur University, Calcutta 700 032, India; *[email protected]

Supramolecular chemistry is a hot topic in contemporary chemical research. It deals with highly organized structures involving two or more individual molecules or parts of large macromolecules held together by intermolecular forces. Supramolecular systems are becoming increasingly important in various areas of chemistry such as control and catalysis of chemical reactions, organic synthesis, molecular recognition, design of materials for molecular scale electronics, chemical separations, modulation of solution properties of some hydrophobically tailored polymers, and so forth. Considering the enhanced efficacy of various drugs encapsulated in different systems for a targeted delivery, encapsulation of probes within cyclodextrin cavities is receiving immense attention in the laboratory and research fields. Inclusion complexes involving organic molecules and cyclodextrins of proper cavity dimensions constitute a major part of the present day supramolecules. The spectroscopic parameters of an organic fluorophore often change dramatically upon inclusion in cyclodextrins­—allowing one to monitor the inclusion phenomenon through this change. In the experiment described here, the concept of inclusion phenomenon is illustrated following the changes in the absorption and fluorescence spectra of a probe upon formation of supramolecular entities. The most evident and pictorial part comes from the fluorescence. The photophysics of the fluorophore involves a specific photophysical process, namely, intramolecular excimer formation. Background and Overview Excimer (an excited dimer) formation is a bimolecular photophysical process where a complex is formed between an electronically excited species, M*, and another similar groundstate molecule, M. Generally the complex is stabilized by some charge-transfer interaction. The M*M complex is an electronically excited metastable species that possesses observable properties distinct from those of M* (1). According to the definition of J. B. Birks (2), the two fluorophores must be sufficiently far apart when light is absorbed, so that the excitation is localized on one of them. This excited fluorophore, often referred to as “locally excited” fluorophore, gives rise to the “monomer” emission. The diffusive encounter between the fluorophoric probes results in the observation of excimer emission. The excimer generates an emission band that is entirely distinct from the emission of its parent monomer molecule. Excimer emmission is broad and unstructured. It is often observed from planar aromatic molecules, such as naphthalene, pyrene, and so forth, when the two molecules are in a nearly parallel-stacked configuration (3, 4). The fluorescence spectroscopy applied to the excimer formation with pyrene or its derivatives has been proved to be useful for assigning the association behavior of the hydrophobically modified polymers having fluorophore labels (4, 5). These polymers have hydrophobic groups as attachments either at the two ends or along the various parts of the backbone of a water-soluble homopolymer. Thus, the modified polymers are amphiphilic in nature and show unique solution properties in

aqueous medium. The hydrophobic attachments lead to the association among themselves and modify the rheological properties of the aqueous solution significantly compared to their conjugate homopolymer (6, 7). They serve as simple models for studying the intramolecular associative interactions at the molecular level. It is possible to control the self-association behavior of the hydrophobically end-capped polymers and hence their rheological properties simply by the addition of cyclodextrins. The control, at its molecular level, can be monitored from the relative yield of the excimer to the monomer emission. Cyclodextrins (CDs) are cyclic oligosachharides that have a hydrophobic cavity with a hydrophilic exterior due to the positioning of the primary and secondary hydroxyl groups at the rims (8–10). CDs composed of six, seven, or eight glucopyranose units are known as α-, β-, and γ-CD, respectively. They are commercially available cagelike organic molecules, commonly known as “molecular buckets”, with the accessible internal cavities for various organic guest molecules to form host–guest inclusion complexes. The inclusion of the guest molecules in the CD cavity is driven by hydrophobic effect, van der Waals interaction, and entropy effect of displacement of water molecules from the cavity. These complexes fit the definition of supramolecules, since they do not involve covalent bonding between the guest and the host. A generalized structure of α-cyclodextrin (containing six sugar units) and a comparative illustration of the three CDs are shown in Scheme I. OH O HO OH

O HO

OH

O OH

HO O

OH HO

OH HO

HO O OH

O

OH

HO OH O

OH

HO

13.7 Å

15.3 Å

16.9 Å

5.7 Å

7.8 Å

9.5 Å

7.9 Å

B-cyclodextrin

C-cyclodextrin

H-cyclodextrin

Scheme I. Structure of α-cyclodextrin and dimensions of α-, β-, and γ-cyclodextrins.

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In the Laboratory

M*

E* monomer emission

Absorbance

CH2O(CH2CH2O)nCH2

excimer emission

290

M

associated pair

Scheme II. Structure and photophysics of PYPY [pyrene end-capped poly(ethylene oxide)]. (Reproduced with permission from ref 11.)

From this experiment, one can learn how the formation of excimer from the intramolecular association of hydrophobic end-groups of a representative hydrophobically modified polymer, pyrene end-capped poly(ethylene oxide) (PYPY, Scheme II), can be controlled by supramolecular inclusion with different CDs with varying cavity dimensions. The PYPY molecular system contains pyrene units, at both ends of the chain, capable of formation of the excimer. Experimental Procedure The overall experiment is simple and the observations are neat. The experimental procedure is given in the online supplement. Hazards Exposure to direct UV light is a potential hazard. UV light can cause permanent damage to the eye if it falls directly onto it. Prolonged exposure to UV light can also cause skin damage. The experimenter should wear UV protective glasses or a UV face shield. Cyclodextrins are chemically safe and all toxicity tests have shown that if administered orally they are harmless. The polymer (PYPY) may be harmful if swallowed or if absorbed through the skin. It is also a respiratory and eye irritant. Results Pyrene produces excimer emission only when the concentration of the solution becomes millimolar or higher and formation of its excimer is explained to be diffusion controlled (3). For PYPY, however, the excimer emission is prominent even at micromolar concentration of the solution confirming

430

320

350

380

Wavelength / nm

410

Figure 1. Absorption spectra of PYPY in aqueous γ-CD solutions. The γ-CD concentration increases along the direction of the arrows from 0 to 7.36 mM. (Reproduced with permission from ref 11.)

that the excimer formation, here, is principally intramolecular in nature. In dilute aqueous solution, even in the ground state, some of the PYPY molecules form intramolecular associated pair using the two pyrene end-group moieties (Scheme II). Aqueous solutions of PYPY show the characteristic absorption bands of the pyrenyl end-groups between 300 and 390 nm. Upon excitation, the polymer gives a structured fluorescence corresponding to pyrene monomer in the range 350–450 nm and a broad and unstructured excimer emission with maximum at ~480 nm. Interaction with Different Cyclodextrins Effect on the Absorption Spectrum The absorption spectrum of an aqueous solution of PYPY remains unchanged with the addition of α-CD indicating that the fluorophore does not form an inclusion complex with this CD. Addition of β-CD, however, shows a little change in the absorption spectrum. A careful observation shows two isosbestic points at 330 nm and 349 nm indicating an interaction of the polymer and β-CD. This is interpreted in terms of inclusion of the pyrenyl end-groups within the β-CD cavity. Addition of γ-CD to the aqueous solution of PYPY, however, shows a prominent modification in the absorption spectrum compared to that observed in the presence of β-CD. In contrast to the two isosbestic points as observed in β-CD solution, three isosbestic points (at 330, 337, and 349 nm) are observed in the γ-CD environment. The marked difference in the extent of modification in the absorption spectrum with an additional isosbestic point in γ-CD solution of PYPY indicates that the mode of inclusion in γ-CD is different from that in β-CD solution (12). The change in the absorption spectrum of PYPY in the presence of varying concentrations of γ-CD is presented in Figure 1.

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In the Laboratory

Fluorescence Intensity

B-CD

i-v

350

400

450

500

550

600

Wavelength / nm

Fluorescence Intensity

C-CD

x i i x

any appreciable interaction of the probe with α-CD and hence negates the possibility of formation of an inclusion complex between the two. With an increase in the β-CD concentration, the excimer band at 480 nm decreases in intensity with a concomitant increase in the intensity of the monomer emission. An isoemissive point develops at 440 nm. The absorption spectra indicated a change in the microenvironment around the fluorophore and suggested the inclusion of PYPY into the β-CD cavity. The observations from the fluorescence measurements reinforce this phenomenon on the photophysics of PYPY. That the formation of the excimer is restricted upon the addition of β-CD to the aqueous solution of PYPY is evident from the fluorescence spectra. A rather different and interesting observation is obtained with the addition of γ-CD in the aqueous solution of PYPY; the excimer emission is found to increase at the cost of the monomer emission, again through an isoemissive point at 440 nm. The fluorometric observation with PYPY in γ-CD is qualitatively opposite of what is observed with the fluorophore in β-CD solution. Both the absorption and the fluorescence spectral study support that the mode of inclusion of the fluorophore in the γ-CD cavity is different from that with the β-CD. Discussion

350

400

450

500

550

600

Wavelength / nm

Fluorescence Intensity

H-CD

i x x i

0

350

400

450

500

550

600

Wavelength / nm Figure 2. Fluorescence spectra of PYPY in aqueous α-CD (0–4.2 mM), β-CD (0–1.1 mM), and γ-CD (0–9.5 mM) (λex = 330 nm). Roman numerals increase with concentration. (Reproduced with permission from ref 11.)

Effect on the Fluorescence Spectrum The differential effect of addition of the three CDs on the fluorescence spectra is much more prominent and informative than the absorption spectra. Figure 2 presents the variation of the fluorescence spectra of PYPY in the presence of varying concentrations of the three CDs. As we see, addition of α-CD to the aqueous solution of PYPY does not affect either the fluorescence intensities or the spectral pattern. This observation, coupled with the lack of change of absorption spectra, rules out

The differential effect in the spectroscopic observations of PYPY in the presence of the three cyclodextrins can be rationalized considering the different modes of inclusion of the fluorophore in the CDs (13, 14). For the optimized geometry of the fluorophore molecule the diameter of the pyrene unit has been calculated to be 7.25 Å. With a glance at Scheme I, one realizes that even one pyrene end-group of the fluorophore is too large to be encapsulated within the α-CD cavity. This justifies the lack of effect of addition of α-CD on the photophysics of the probe in terms of the spectroscopic observables. The cavity dimension of β-CD, however, is large enough to encapsulate a single pyrene unit from the end-capped polymer leaving the other one in the bulk aqueous phase. This forbids formation of the excimer because of the walling effect of the β-CD and leads to a decrease in the excimer emission with a concomitant increase in the monomer emission. Enhanced excimer emission relative to the monomer emission of PYPY in the presence of γ-CD can be rationalized by considering the inclusion of both the pyrene units of a single polymer molecule within the same γ-CD cavity. A similar observation with the free pyrene molecular system suggests that the poly(ethylene oxide) chain does not affect the inclusion behavior significantly (15, 16). Rather the polymer having pyrene at both ends of the water-soluble polymer chain leads to the formation of the intramolecular excimer and hence, allows one to perform the experiment at a very low probe concentration. In γ-CD solutions of PYPY the total fluorescence comprises emissions from both the monomer and excimer. Monomer emission comes from a section of the fluorophore molecules residing in the bulk water phase as well as from the molecules embedded within the CD cavity that do not have the proper conformation to form the excimer where a single pyrene moiety is included within γ-CD, similar to the case of β-CD. When the concentration of γ-CD is

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In the Laboratory

Literature Cited

HO

OH

HO

OH

OH

OH

OH

Scheme III. Schematic representation of inclusion of PYPY in α-, β-, and γ-cyclodextrin, respectively.

much higher compared to that of the fluorophore (i.e., [PYPY] ≈ 10‒6 mol dm‒3 and [γ-CD] ≈ 10‒3 mol dm‒3) the concentration of the free molecules residing in the bulk water can easily be neglected and the photophysical behavior can be considered primarily to correspond to the embedded fluorophores. The variation in the spectroscopic observations to the differential mode of inclusion of the polymer end-groups in the three CDs can be expressed as shown in Scheme III. Thus, with a proper choice of the cyclodextrin, it is possible to control the association behavior and hence thickening properties of the hydrophobically modified water-soluble polymers dramatically. Conclusions This lab experiment allows the students (i) to deal simultaneously with the normal and the excimer fluorescences; (ii) to realize the formation of the various types of probe–CD supramolecular inclusion complexes depending on the relative size of the probe and the CD cavities; (iii) to realize the effect on its photophysics of formation of a probe–CD supramolecular complex for the water-soluble polymeric fluorophore; and (iv) to know how the inclusion phenomenon can be externally controlled through a choice of a particular cyclodextrin. Acknowledgments Financial support from the D.S.T. and C.S.I.R., Government of India, is gratefully acknowledged.

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