1. Yun Hee Jang, Mario Blanco, Siddharth Dasgupta, William A. Goddard, III MSC, Beckman Institute, Caltech David A. Keire, John E. Shively The Beckman Research Institute of the City of Hope

2. Antibody Tumor cell targeting 90  -emitting (2.25 MeV, t 1/2 =64h) Chelating ligand (DOTA) Therapy: 90 Y 3+ (64h) Diagnosis: 111 In 3+ (2.8d) 64 Cu 2+ (12.8h) MRI contrast agent: Gd 3+ D. Parker, Chem. Soc. Rev. 19, 271 (1990)

3. Rapid complexation Kinetic inertness at pH 2~8 w.r.t. acid- promoted dissociation DTPA (log K=22.1) DOTA (log K=24.8) Not inert leading to bone-marrow toxicity <0.5% dissociated over 18 days in serum (pH 7.4, 37 o C): inert x1600 slower than Y-DTPA formation Lewis, Raubitschek and Shively, Bioconjugate Chem. 5, 565 (1994) Thermodynamic stability

4. Type I: labile + Y 3+ Type II: stable/inert or fast slow Y 3+ + H 2 (DOTA) 2- Y 3+ E.T. Clark and A.E. Martell, Inorg.Chim.Acta 190, 27 (1991) X.Y. Wang, et al. Inorg.Chem. 31, 1095 (1992)

5. Calculate structure/energy change occurring during complex-formation Design new chelating agent and predict its energetics/kinetics B3LYP/LACVP* // HF/LACVP* (6-31g* for C/H/O/N; Hay-Wadt ECP for Y) Jaguar 3.5 (Schrodinger Inc.) Vibration analysis  ZPE / thermodynamic quantity  Gibb’s free energy Continuum solvation calculation by solving Poisson-Boltzmann equation Identify the rate-determining step: Deprotonation or conformation change?

6. Y 3+ moves into the cage spontaneously with deprotonation. RMS deviation between ring conformations < 0.5 Å. Deprotonation is the rate-determining step. Y 3+ moves into the cage spontaneously with deprotonation. RMS deviation between ring conformations < 0.5 Å. Deprotonation is the rate-determining step. -H + YH 2 (DOTA) + YH(DOTA)Y(DOTA) - Y 3+ outside the cagethe same as x-ray structure of final complex

7. Direct attack of outside base on the ring proton? No room for it. top viewside viewbottom view

8. Conformation change to the one favorable to attack? Too high cost, especially, for YH(DOTA) Conformation change to the one favorable to attack? Too high cost, especially, for YH(DOTA) YH 2 (DOTA) + 16.6* (12.1)** kcal/mol 42.7* (34.5)** kcal/mol YH(DOTA) 21.6* (24.6)** kcal/mol * 1.807 Å for r(Y) ** 1.673 Å for r(Y) in solvation calculation 4-coordinate2-coordinate3-coordinate

9. Proton transfer is easier than conformation change. Calculated activation free energy is in agreement with experimental value. Proton transfer is easier than conformation change. Calculated activation free energy is in agreement with experimental value. Proton transfer from ring NH to COO (more accessible to outside base)? reactant (NH...COO)TS (N..H..COO)product (N...COOH) ***experimental  G for Eu,Gd,Ce,Ca-complexes (Inorg.Chem. 32, 4193 (1993))

10. Structural change leading to more stable TS: 6-membered ring of DO3A1Pr rather than 5-membered ring of DOTA Structural change leading to more stable TS: 6-membered ring of DO3A1Pr rather than 5-membered ring of DOTA DO3A1Pr (Pr=propionate) TS (DO3A1Pr) TS (DOTA)

11. Protonation at propionate site is more stable.  6-membered ring TS H pr (DO3A1Pr): 0.0 kcal/mol H ac (DO3A1Pr): 7.8 kcal/mol

12. Deprotonation from ring nitrogen is the rate-determining step. Deprotonation occurs by proton transfer from ring nitrogen to carboxylate. Adding CH 2 to one carboxylate arm can improve the incorporation rate. Explicitly-coordinated water molecules How many water molecules? Effect on structure/energetics Introduction of amide linkage

13. Caltech William A. Goddard, III Siddharth Dasgupta Mario Blanco Daniel Mainz Sungu Hwang City of Hope John E. Shively David Keire Supported by NSF