Practical 1: Cyclodextrin Host-Guest Chemistry

Introduction

Cyclodextrins (CD) are a class of cyclic oligosacchariders that have a hydrophobic molecule-sized cavity (Del Valle, 2004). The most common CDs are toroidally (‘trash

-can’) shaped polysaccharides composed of 6, 7, or 8 D-glucopyranoside units joined by a 1,4-glycosidic link; and are referred to as α-, β- and γ-cyclodextrin, respectively (Figure 1). The ring size increases from α – 0.49nm, β – 0.62nm and γ – 0.8nm. Smaller and larger ring structures are also known and used.

The shape of the CD is often represented as a funnel with an upper wider rim and lower narrower rim. The upper rim consists of the secondary hydroxyl groups and the lower with primary hydroxyl groups. The larger number of hydrophilic hydroxyl groups at the rim of the funnel and the hydrophobic cavity make CDs a useful host for hydrophobic guest molecules.

CDs can therefore act as hosts to guest molecules that can fit into the cavity and prefer a hydrophobic environment. Febreeze®, a brand of household odor eliminators, is based on the inclusion of odor molecules in a CD cavity. Also, solubility of drugs can be enhanced by inclusion in CDs. CDs have also been used in separation science applications. Capillary electrophoresis, liquid chromatography, and gas chromatography employ the CD inclusion phenomenon to separate structurally similar molecules, even chiral isomers! These applications have spurred the development of many CD derivatives by replacing the hydroxyls on the exterior of the CD with a variety of charged and uncharged groups.

Figure 1: Schematic representations of (a) the general chemical structure and (b) the tridimensional structure of cyclodextrins, and (c) chemical structure and dimensions for α-, β- and γ-cyclodextrin (n = 6, 7 and 8, respectively). Image from (Crini et al, 2018).

Aim

This practical examines host-guest interactions between a small molecule (8-anilino-1- naphthalenesulfonate (ANS, Figure 2a) and Methyl-β-Cyclodextrin (ME β-CD) (Figure 2b). ME β-CD is a derivative of β-CD which has been modified to give a more hydrophobic cavity. ANS displays increased fluorescence intensity in a hydrophobic (non-polar) environment compared to in a polar solvent such as water. Therefore, when ANS associates with a β-CD molecule, an enhancement in fluorescence can be seen. This binding interaction between the two molecules will be examined in this lab session using fluorescence spectroscopy. An estimate of the binding constants of the complexes will also be examined.

Figure 2. a) Left – 8-Anilinonaphthalene-1- sulfonic acid (ANS). b) Right Structure of the Methyl-β-cyclodextrins as will be used in this laboratory exercise. For ME b-CD: R= -H or -CH3

Precautions

  • 2-Butanol is an irritant and may cause drowsiness. Avoid breathing fume.
  • Dispose all samples in designated waste container.

Experimental Method

Instrument:

You will use the Cary Eclipse Fluorescence Spectrophotometer by Agilent Technologies for measurement of fluorescence.

  1. Turn on the Spectrophotometer for at least 5 minutes before use. The power switch is on bottom right corner.
  2. Log in to the associated laptop (username: cbms-teaching; pw: 200300).
  3. Open the Cary Eclipse Scan Application by clicking on the Scan Icon on    the left of the Task Bar (bottom left corner of the screen).

These steps will be done in advance for you by the lab technician.

 PART I: Fluorescence enhancement of ANS by different solvents                                                                                                                                        

Solutions: You will be provided with the following stock solutions and samples.

  • 400µM ANS
    • 1 X Phosphate Buffer Saline (PBS) pH7
    • Absolute methanol
    • Absolute ethanol
    • 2-butanol

Method:

  1. Into clean 2 ml microcentrifuge tubes, mix 100 μl of ANS solution with 1.9 ml of different solvents (PBS, methanol, ethanol and 2-butanol). Label your 4 microcentrifuge tubes properly.
  2. Incubate for 5 mins in ultrasonicator bath. Now your samples are ready for ANS

fluorescence measurement with Cary Eclipse Spectrophotometer.

  • In the Cary Eclipse Scan Application, click on Setup to input your parameters to obtain the fluorescence emission scan measurements as follows:
    • Cary tab:
      • Excitation: 340 nm
      • Start: 400nm
      • Stop: 600nm
      • Excitation slit nm = 5
      • Emission slit nm = 5
      • Scan control: medium
    • Options tab:
      • Y minimum: 0.0
      • Y maximum: 400.0
      • Check the Smoothing box
    • Reports tab:
      • Under Peaks: Tick Maximum Peak and Untick All peaks
      • Change Threshold to 10
    • Leave the rest of the parameters as default.
  • You will be provided with a blank fluorimeter cuvette which contains 2mL of the

blank solvent – water.

  • Place your blank sample cuvette into the fluorimeter i.e. containing 2ml of water into the fluorimeter. This is your blank “Zero”. **Note, if using quartz cuvettes, be careful when handling as they are very expensive and can easily be broken or chipped. Avoid touching the face of the cuvette with your fingers.
  • Carefully remove the water blank cuvette out of the holder and put it back into the correct slot in the cuvette holder rack. Do not
  • In the rack, there are 4 cuvettes. Please select the correct cuvette to use. Decant 2mL

of your ANS in PBS solution into the PBS Sample cuvette. Carefully place it in the fluorimeter. Change the excitation and emission slit values to 10 and 10. Hit Start

– optional name the sample as ‘PBS’ in window after you hit start.

  • The software will record the wavelength for the maximum peak position and the value of the maximum fluorescence intensity. You can find these numbers in the Report on the right of the screen. You will need these values for your report.
  • Take your ANS Sample cuvette out. Empty your ANS sample mixture into the provided Liquid Waste bottle. Place the empty Sample cuvette back into its designated slot in the cuvette holder rack.
  • Using the same settings, repeat Step 7-9 for the three other ANS-solvent solutions you have prepared. Change the excitation and emission slit values to 5 and 5 for all remaining samples. Run the emission scan. Again, the wavelength for the maximum peak position and the value of the maximum fluorescence intensity for the ANS-

solvent solutions will be reported in the software. You will need these values for your report. A printout of fluorescence emission graphs and associated reports should be done once you have finished collecting your data.

 PART II: Binding constant of ANS:ME-β-CD                                                                                                                                        

Solutions: You will be provided with the following stock solutions:

  • 1 X Phosphate Buffer Saline (PBS) pH7
  • 400μM ANS
  • 20mM ME-b-CD in PBS

Method:

  1. Add 500 μl of 400μM ANS to 8 x clean 2ml microcentrifuge tubes.
  2. Add X μL of 20mM ME β-CD and Y μL of PBS according to the table below to increase the concentration of ME β-CD from 0 mM to 15 mM. The total volume should always be 2mL. **Have this table checked by a demonstrator BEFORE making your samples for the experiment.
      Sample[ME β- CD]X mL of ME β-CD (20mM)  mL of ANS (400μM)Y mL of PBS  Fluorescence intensity (F)      F-F0
10 mM05001500= F0 
23 mM 500   
35 mM 500   
47 mM 500   
59 mM 500   
611 mM 500   
713 mM 500   
815 mM 500   
  • Incubate each sample for 5 mins in the bath ultrasonicator.
  • In the Cary Eclipse Scan Application, click on Setup to input your parameters for the fluorescence measurement as below:
    • Cary tab:
      • Excitation: 340 nm
      • Start: 400nm
      • Stop: 600nm
      • Excitation slit nm = 2.5
      • Emission slit nm = 5
      • Scan Control: medium
  • Options tab:
    • Y minimum: 0.0
    • Y maximum: 500.0
    • Check the Smoothing box
    • Reports tab:
      • Under Peaks: Tick Maximum Peak and Untick All peaks
      • Change Threshold to 0.2
    • Leave the rest of the parameters as default.
  • Add 2ml of 1 X PBS into the fluorimeter cuvette. Use this as your blank (Zero).
  • Carefully remove/pipette the PBS out of cuvette after collecting the scan
  • Add 2mL of your first sample – Sample 1 – into the cuvette. Hit Start to measure.
  • Write down the maximum fluorescence intensity (F) into the table.
  • Pipette your Sample 1 back into its original tube.
  • Repeat Step 7-9 with remaining Samples 2 to 8. You are going up in concentration of the cyclodextrin so there is no need to wash the cuvette in between each sample. However, be sure to remove as much liquid out of the cuvette as possible before adding your next sample.
  • You can switch between viewing all graphs and individual graph by choosing either

All graph icon   or Focused graph icon  on the software program.

  1. Create a Benesi-Hildebrand Plot by plotting data as described in Part III. Using this graph, estimate the binding constant (Ka). It is important to record the Fluorescence intensity ‘F’ of the peak. Add these values to the table for each sample.

 Part III: Determine the binding constants using fluorescence spectrometry                                                                                                                                        

If the fluorescence of ANS changes upon inclusion in the CD cavity then the degree of the fluorescence change is related to the degree of binding. One can use this information

to then determine the equilibrium constant (Ka) for the complexation process. For a 1:1 ANS:CD complex, the association constant Ka can be defined as:

⇔  

ANS + CD 𝐾𝑎

ANS:CD                                   [Eqn 1]

𝑎  

𝐾   = [𝐴𝑁𝑆:𝐶𝐷]

[𝐴𝑁𝑆][𝐶𝐷]

[Eqn 2]

For a 1:1 complex, the equilibrium concentrations of ANS and CD are determined by by subtracting the concentration that has complexed [ANS:CD] from the initial concentrations ([ANS]0 and [CD]0):

[𝐴𝑁𝑆] = [𝐴𝑁𝑆]0 − [𝐴𝑁𝑆: 𝐶𝐷]                                    [Eqn 3]

[𝐶𝐷] = [𝐶𝐷]0 − [𝐴𝑁𝑆: 𝐶𝐷]                                          [Eqn 4]

However, because the [CD]0 >> [Complex], we can simplify [Eqn 4] to:

[𝐶𝐷] = [𝐶𝐷]0                                                                      [Eqn 5]

Substitution of [Eqn 3] and [Eqn 5] into the equilibrium expression [Eqn 2] yields:

1  

𝐾 =               [𝐴𝑁𝑆:𝐶𝐷]

([𝐴𝑁𝑆]0,[𝐴𝑁𝑆:𝐶𝐷])([𝐶𝐷]0)

[Eqn 6]

The next equation is important because it links the equilibrium concentrations of the species involved to fluorescence intensity, which you have measured. The observed fluorescence intensity of ANS in the cyclodextrin solutions becomes a weighted average of the intensity from the free and the complexed ANS, as follows:

[𝐴𝑁𝑆:𝐶𝐷]  =  𝐹,𝐹0

[Eqn 7]

[𝐴𝑁𝑆]0              𝐹#,𝐹0

F0 is the measured fluorescence intensity in the absence of CD, where F is the measured fluorescence intensity in the presence of CD, and 𝐹 is the fluorescence intensity of ANS completely complexed with the CD (that is, no major increase in fluorescence can be seen with increased concentration of CD). In other words, the fraction of the total

possible intensity change 𝐹 − 𝐹//𝐹0 − 𝐹/ is proportional to the fraction of ANS

complexed [ANS:CD]/[ANS]0. Substitution of [Eqn 7] into [Eqn 6], followed by rearrangement, yields:

*

(𝐹-𝐹0)

=         *

𝐾𝑎(𝐹#-𝐹0)

𝑥     *

[𝐶𝐷]0

+      *

(𝐹#-𝐹0)

Eqn 8

Note, Eqn 8 is in the form of y = mx + b

If we then plot

*

(𝐹-𝐹0)  versus

*

[𝐶𝐷]0, the value of Ka can be obtained by dividing the

intercept by the slope.

This equation is only true for 1:1 complexes which need to be proven before it can be used to determine Ka. It may be possible that the host and guest form something other than a 1:1 complex. The above method is referred to as creating a Benesi-Hildebrand. It may be possible that the host and guest form something other than a 1:1 complex.

Report

Data Presentation and Analysis: for your results section

  1. From the date collected in Experimental Part I, comment on the effect of the different solvents on the fluorescence emission properties of ANS. Is there a shift in the maximum wavelength value of the emission peak? Why? Compare this also to the fluorescence emission of ANS in the most concentrated CD solution (from Part II). Can you make a comment about the polarity of the interior of the CD cavity?
  • Using the results from Part II, complete the table that includes the fluorescence intensity at one wavelength (preferably at the peak of the most intense sample) for each [CD]. Determine FF0 for each [CD].
  • Create a Benesi-Hildebrand Plot. To do this, plot the data of 1/(FF0) (y-axis) vs. 1/[CD]0 (x-axis). Fit the data to [Eqn 8]. The fitting parameters of the data to a straight-line equation (y = mx + b) should allow you to calculate Ka. Take care with units, concentrations will need to be M when reporting the Ka value.
  • When writing your report, present YOUR data as obtained during the laboratory session. If needed, a ‘good’ data set for Part I and II may also be made available on ilearn. Use the ‘good’ date that is given on ilearn to create the Benesi- Hildebrand Plot. Contrast these results with YOUR experimental results. If appropriate, comment on what went wrong? What could be done better next time?

Questions to consider in your report: The answers to the questions below are designed to make you think about different aspects of your experiment and should be incorporated into the results or discussion section of report, not answered separately.

  • How will the different solvents affect ANS? Explain the differences. (Part I)
    • Discuss why the complexion between ANS: ME β-CD is a 1:1 stoichiometry?
    • With your estimated binding constant, compare your value with other well- known complexes from the literature (e.g. Biotin/Streptavidin). Is this a strong or weak interaction?
    • Describe some other methods/plots that could have been used to determine the stoichiometry and/or binding constants.

References

Crini G., Fourmentin S., Fenyvesi É., Torri G., Fourmentin M., Morin-Crini N. (2018) Fundamentals and Applications of Cyclodextrins. In: Fourmentin S., Crini G., Lichtfouse E. (eds) Cyclodextrin Fundamentals, Reactivity and Analysis. Environmental Chemistry for a Sustainable World, vol 16. Springer, Cham

Del Valle, E. M. M. (2004). Cyclodextrins and their uses: a review. Process Biochemistry 39(9): 1033- 1046.

Peña, A. M., F. Salanas, et al. (1993). Absorptiometric and spectrofluorimetric study of the inclusion complexes of 2-naphthyloxyacetic acid and 1-naphthylacetic acid with β-cyclodextrin in aqueous solution. Journal of inclusion phenomena and molecular recognition in chemistry, Kluwer Academic Publishers. 15: 131-143.

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