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

N N

N N

COO-

C

-OOC

-OOC

Y3+

O

N

H

Antibody

Tumor cell

targeting

90

-emitting(2.25 MeV, t1/2=64h)

Chelating ligand (DOTA) • Therapy: 90Y3+(64h)• Diagnosis: 111In3+(2.8d) 64Cu2+(12.8h)• MRI contrast agent: Gd3+

D. Parker, Chem. Soc. Rev. 19, 271 (1990)

Rapid complexation

NH+ N

N +HN

O

O-

-OO

O-

O

O

O-

N

-OOC

-OOC

N N

COO-

COO-

COO-

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 tobone-marrow toxicity

<0.5% dissociated over 18 days in serum (pH 7.4, 37oC): inert

x1600 slower than Y-DTPA formation

Lewis, Raubitschek and Shively, Bioconjugate Chem. 5, 565 (1994)

Thermodynamic stability

Type I: labileType I: labile

+Y3+

Type II: stable/inertType II: stable/inert

orfast slow

Y3+ + H2(DOTA)2- Y3+ + H2(DOTA)2-

NH+ N

N +HN

O

O-

-OO

O-

O

O

O-

Y3+

NH+ N

N +HN

O

O-

-OO

O-

O

O

O-

Y3+

NH+ N

N N

O

O-

-OO

O-

O

O

O-

Y3+

N N

N N

O

O-

-OO

O-

O

O

O-

NH+ N

N +HN

O

O-

-OO

O-

O

O

O-

E.T. Clark and A.E. Martell, Inorg.Chim.Acta 190, 27 (1991)

X.Y. Wang, et al. Inorg.Chem. 31, 1095 (1992)

• 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?

• Y3+ moves into the cage spontaneously with deprotonation.• RMS deviation between ring conformations < 0.5 Å.• Deprotonation is the rate-determining step.

• Y3+ moves into the cage spontaneously with deprotonation.• RMS deviation between ring conformations < 0.5 Å.• Deprotonation is the rate-determining step.

-H+ -H+

YH2(DOTA)+ YH(DOTA) Y(DOTA)-

Y3+ outside the cage the same as x-ray structureof final complex

Direct attack of outside base on the ring proton? No room for it.Direct attack of outside base on the ring proton? No room for it.

top view side view bottom view

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)

YH2(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-coordinate 2-coordinate3-coordinate

• 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)?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))

Relative energy (kcal/mol) reactant TS Product expt'l ***

Gas-phase E 0 2.0 -25.2

Aqueous-phase 0 12.2 *,** -8.8* (-6.0)**Gactivation(aq) 8.4 *,** 8.1~9.3

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)

B3LYP//HF in aqueous phase (kcal/mol) DO3A1Pr DOTA

Energy barrier 4.5 12.2

Activation free energy 3.9 8.4

NH+ N

N +HN

O

O-

-OO

O

O-

-OO

one more CH2

Protonation at propionate site is more stable. 6-membered ring TS Protonation at propionate site is more stable. 6-membered ring TS

Hpr(DO3A1Pr): 0.0 kcal/mol Hac(DO3A1Pr): 7.8 kcal/mol

N N

N +HN

O

O-

-OO

O

O-

-OO

one more CH2

+HNN

NN

O

O-

O-O

O

-O

O-O

one more CH2

• Deprotonation from ring nitrogen is the rate-determining step.

• Deprotonation occurs by proton transfer from ring nitrogen to carboxylate.

• Adding CH2 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

NH+ N

N +HN

O

O-

-OO

O

O

O-

N

H

?

Caltech

William A. Goddard, III

Siddharth Dasgupta

Mario Blanco

Daniel Mainz

Sungu Hwang

City of Hope

John E. Shively

David Keire

Supported by

NSF