Nucleic Acids in Chemistry and Biology

9.6.2.2 Thermodynamics of Daunomycin: DNA Interactions. The details of the energetics of

anthracycline–DNA interactions have been studied by several groups.^19 In most thermodynamic charac-
terizations, the initial step involves determination of the binding free energy. This is readily achieved by
measurement of the equilibrium association (or binding) constant and use of the standard Gibbs relationship,

(9.10)

where Kobsis the observed equilibrium constant, R the gas constant and Tthe temperature. Brad Chaires has
conducted equilibrium binding experiments and constructed binding isothermsthat span a 100-fold range
in free ligand concentration. Such essentially complete binding curves allow the free energy to be estimated
in a model-free manner by use ofWyman’s concept of median ligand activity. The free energy of ligation
( GX) is defined as the free energy to go from a state where no ligand is bound to a degree of saturation X

• .
  For the binding of daunomycin to calf-thymus DNA, the value of GXwas found to be 32.6 kJ mol^1 for
  the full ligation of a daunomycin binding site.
  The daunomycin–DNA binding affinity is dependent on bulk salt concentration, as is explained by the
  polyelectrolyte theories of Record and Manning (Section 9.3). Daunomycin carries one positive charge at
  neutral pH, which arises from protonation of the amine (pKa8.2) on the sugar moiety. Binding of cationic
  ligands such as daunomycin to DNA results in the entropically favourable release of condensed counter-ions
  from the DNA sugar–phosphate backbone. One practical consequence of polyelectrolyte theory is that it
  allows the observed binding free energy to be partitioned into two components, Gpeand GT, the
  polyelectrolyte and nonpolyelectrolyte contributions, respectively. The former is the free energy arising from
  counter-ion release. For daunomycin binding to DNA, Gpeis 4.2 kJ mol^1 at a reference salt concen-
  tration of 0.2 M NaCl. This is about 13% of the total binding free energy.
  Once the free energy for a binding interaction has been determined, the next step in a detailed thermo-
  dynamic characterization is to determine the enthalpic and entropic components of the free energy. These
  contributions are defined using the standard thermodynamic relationship,

(9.11)

where H and Sare the enthalpy and entropy changes accompanying binding. At 20°C, H for the
daunomycin–DNA interaction is 37.6 kJ mol^1. By use of Equation 9.11, T Sis then calculated as
5.0 kJ mol^1 , and hence at 20°C the value of Sis 17.1 J mol^1 K^1. The negative sign for Sindicates
that entropy decreases upon binding. This unfavourable entropy term is overcome by a large favourable
(negative) enthalpy that drives the binding. The binding enthalpy for daunomycin is strongly dependent on
temperature. This is a consequence of a binding-induced change in heat capacity( Cp) that accompanies
the interaction:

(9.12)

By measurement of the enthalpy as a function of temperature and calculation of the slope of the resulting
linear graph, the Cpcan be determined. For daunomycin binding to DNA, this value is 669 J mol^1 K^1.
Determination of heat capacity change is useful, since this parameter can yield information on changes in
hydrophobic interactions and removal of nonpolar solvent accessible surface area. It is an important, experi-
mentally measurable quantity that is now used to carry out detailed partitioning of free energy terms
(Section 9.6.4).
Studies of daunomycin and adriamycin binding to DNA have revealed important differences between
the two drugs and served to illustrate the value of thermodynamic characterisation in the context of rational
drug design. The structures of the two ligands bound to DNA are virtually superimposable and molecular









C

H

p T





GHTS

   GRTKln obs

352 Chapter 9

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