Competition expt between 18-crown-6 and 1,3-xylyl-18crown-5 with t-BuNH3ClO4

Weak complex formation Strong complex formation

- 1eq of guest addition makes ppt in NMR tube, cannot proceed further titration

Prerequisite: Complex chemical shifts of both known and unknown and Kassoc of weak/reference complex should be known to use this method.

Ref: Rehoudt et.al J.Tetrahedron Lett, 1976, 1375-1378

Competition experiments by NMR binding titration:

Reference cell with pure solvent, sample cell with a solution of one of the host-guest partner(host). On addition of guest solution a heat effect occurs that is counter-regulated by the cell feed back current to maintain ΔT at zero. Output raw data consist in a number of heat pulses that decrease in magnitude following the progressive saturation of the host binding site by the incremental addition of guest species. Ref: Freyer, MW; Lewis, EA. Methods Cell Biol. 2008, 84, 79-113.

Isothermal titration calorimetry:

Non-linear regression is used to determine to obtain best values for fitting parameters(K, ΔH and n)

Theory: ( Teach on board)

For 1:1 binding isotherm:

Xr =[X]tot/[M]tot1/r =K*[M]tot

[MX]= 1/2 {(Mt+Xt+1/K)±√ (Mt+Xt+1/K)2-4MtXt }

Ref: Wiseman et.al, Analy.Biochem. 1989, 179,131-137.

The heat change is then simply calculated by integrating the heater powerover the time (sec) of the measurement.

The raw signal in the power compensationcalorimeter is the power (m cal/sec or m J/sec) applied to the control heater that is required to keep the calorimeter cell from changing temperature as a function oftime.

Two ligand molecules bind to receptor molecule.

1. Where enthalpy change and binding affinity for both ligands are close enough to be thermodynamically indistinguishable

2. Where binding site with higher affinityIs accompanied by a more exothermic enthalpy change

3. Where the binding affinity site demonstrate a less exothermic enthalpy change than the lower

binding affinity.

Strong complexes: low macro molecule concentration is enough to produce heat changes; weak complex(<104) need high macromolecule concentration- This play role in curvature.(enough heat to be detected and quantified)Thermogram with acceptable curvature for non-linear regression analysis required to obtainAccurate K value

Tuning C value C=[HOST]* K value Xr =[X]tot/[M]tot

Dilution effects for all solute species is common between macromolecule and ligand solutionAnd these can be calculated and

Heat of dilution for ligand itself must be measure in blank experiment. Ligand solution is titrated into buffer in the sample cell.

The heat of dilution of the macromolecule should also be measured in another blank experiment. This can be done by injecting buffer from the syringe into macromolecule solution in the sample cell.

Another blank experiment: buffer into buffer

Sample solutions should be degassed prior to filling the cell and injection syringe since The generation of bubbles in the sample solutions will generate spurious heat signals in ITC.

http://www-ibmc.u-strasbg.fr/arn/plateformes/microcal/ITC%20-%20Derived%20Binding%20Constants.pdf

Campoy, AV; Freire ,E. Nature Protocols 2006, 1, 187-191.

References: for slide 6 and 7.