1. Improved characterization of supramolecular assemblies
by non conventional
Isothermal Titration Calorimetry experiments
E. Bertaut, D. Landy

2. Classical and non conventional protocols and 8 competitive experiments
Classical versus non conventional experiments
Classical and non conventional protocols
Classical protocols
Competitive
Direct
0, 1, 2 or 3 constituents
among H, GA, GB GA GA
0, 1, 2 or 3 constituents
among H, GA, GB
H + GB H
8 direct experiments
and 8 competitive experiments
One single expression is necessary to describe the heat for all experiments :
Coupling of non conventional experiments can be very useful (Global analysis)

3. Illustrated by 1:1 β-cyclodextrin/Ibuprofen complex
Description of direct experiments
Illustrated by 1:1 β-cyclodextrin/Ibuprofen complex
4 aqueous solutions : 1) buffer alone :  2) βCD 5mM ) IBU 0.5mM ) βCD 5mM + IBU 4.6mM
(Free IBU = 0.5mM)
Titration
Reverse
Titration
Release
Dilution
Attenuated
Titration
Attenuated
Reverse
Titration
Annihilated
Release
Annihilated
Reverse
Titration
Kf = 9558  129 M-1
ΔH =  13 cal.mol-1
One heat model describes all experiments
High accuracy on K (uncertainty < 2%), equivalent to 8 titrations!
Is there any optimal strategy of experimental coupling ?
In general ? For specific cases ?
E. Bertaut, D. Landy (2014) Beilstein J. Org. Chem. 10,

4. Towards and optimal strategy (direct experiments) : general 1:1 case
(Release+Titration)  (Titration+Titration)
(Release+Titration) is more accurate than (titration+Titration) for Kf < M-1
No other coupling of two or more protocols is more accurate
than these approaches on a wide range of Kf for 1:1 complexes …
… but some experimental associations might be more efficient in specific cases.

5. Towards and optimal strategy (direct experiments) : Weak 1:1 complexes
For K < 200 M-1, titration may lead to (uncertainty on K) > K
Example : β-cyclodextrin/Phenol
Reverse Titration
Annihilated Release
Injection Number
Injection Number
Kf = 87  6 M-1
H =  372 cal.mol-1
Experiments which are not informative individually
Becomes very accurate with global analysis

6. Towards and optimal strategy (direct experiments) : two set of sites
High uncertainties are generally produced
when a variable number of aspecific sites has to be introduced
Example : Bovine Serum Albumine (BSA) / Ibuprofen
Titration leads to uncertainty on thermodynamic parameters > parameters
Titration
Attenuated Titration
Injection Number
Injection Number

7. Towards and optimal strategy (direct experiments) : sequential binding sites
In the case of sequential complexes, non significant values can be obtained with simple classical titrations
(depending on the respective stability and inclusion enthalpy)
Example : mixture of 1:1 + 2:1 complexes (CD/metallic system)
Titration leads to uncertainty on thermodynamic parameters > parameters
Titration
Attenuated Titration
Injection Number
Injection Number
K 1:1 =  M-1
H 1:1 =  130 cal.mol-1
K 2:1 =  M-1
H 2:1 =  cal.mol-1

8. HPβCD-Ibuprofen at 10°C :
Exhaustive description of competitive experiments
HPβCD-Ibuprofen at 10°C :
athermic 1:1 complex
Characterization by direct protocols is IMPOSSIBLE …
… but it becomes very accurate with competitive protocols!
Competitive
Titrations
Competitive
Releases
Double
Titration
Double
Reverse
Titration
Double
Release
Double
Dilution
Again, one model describes all experiments,
with a high level of accuracy :
Kf = 5477  146 M-1
ΔH = -3  12 cal.mol-1
E. Bertaut, D. Landy (2014) Beilstein J. Org. Chem. 10,

9. One application of competitive release experiment : solubility issues
Example : Bovine Serum Albumine (BSA) / Naphthol
Negligible heats for titration of BSA 0.04mM by Naphthol 0.2mM
Prealable Titration
Competitive release
Injection Number
Injection Number
Kf BSA-Napht-1-ol =  784 M-1
HBSA-Napht-1-ol=  cal.mol-1
n= 5  1 site
aspecific interactions

10. … Take-Home Messages …
The world of ITC protocols is wider than we could think!
Global Analyses are of great help
Non conventional experiments may improve ITC characterization
not only for complexes with very high stability but also for :
Weak to moderate complexes
Complexes with low enthalpy of formation
Coexistence of different binding sites
Coexistence of different stoichiometries
Solubility issues
For both host-guest chemistry and molecules of biological interest

11. Improved characterization of supramolecular assemblies
by non conventional
Isothermal Titration Calorimetry experiments
E. Bertaut, D. Landy