Improved characterization of supramolecular assemblies by non conventional Isothermal Titration Calorimetry experiments E. Bertaut, D. Landy landy@univ-littoral.fr

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)

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 3) IBU 0.5mM 4) 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 = -3326  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, 2630-2641

Towards and optimal strategy (direct experiments) : general 1:1 case (Release+Titration)  (Titration+Titration) (Release+Titration) is more accurate than (titration+Titration) for Kf < 30 000M-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.

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 = -2524  372 cal.mol-1 Experiments which are not informative individually Becomes very accurate with global analysis

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

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 = 153 266  18 154 M-1 H 1:1 = - 8 235  130 cal.mol-1 K 2:1 = 127 122  3 253 M-1 H 2:1 = - 5 091  - 121 cal.mol-1

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, 2630-2641

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 = 10 083  784 M-1 HBSA-Napht-1-ol= - 5927  - 353 cal.mol-1 n= 5  1 site aspecific interactions

… 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

Improved characterization of supramolecular assemblies by non conventional Isothermal Titration Calorimetry experiments E. Bertaut, D. Landy landy@univ-littoral.fr