Koji KANO and Hiroaki KITAGISHI (Doshisha University, Kyoto, Japan)

1. Cyclodextrin Dimers as Simple Myoglobin Models in Aqueous Solution Koji KANO and Hiroaki KITAGISHI (Doshisha University, Kyoto, Japan)

2. Carrier of Diatomic Molecules

3. His 64 (distal His) His 93 (proximal His) • Myoglobin (Mb) is an oxygen-storage hemoprotein. • Oxygen bound to Mb is stabilized by two His residues. • Heme center is surrounded by a hydrophobic wall of the protein.

4. A picket-fence Por prepared by Prof. Collman’s group Collman, J. P.; Boulatov, R.; Sunderland, C. J.; Fu. L. Chem. Rev.2004, 104, 561-588. Jameson, G. B.; Rodley, G. A.; Robinson, W. T.; Gagne, R. R.; Reed, C. A.; Collman, J. P. Inorg. Chem. 1978, 17, 850-857.

6. Many artificial dioxygen receptors have been prepared. • These model compounds can bind dioxygen only in absolute organic solvents such as toluene. • Dioxygen adducts are easily autoxidized in the presence of a trace amount of water.

7. Dendrimers as Mb models Zingg, A.; Felber, B.; Gramlich, V.; Fu, L.; Collman, J. P.; Diederich, F. Helv. Chim. Acta2002, 85, 333-351. Jiang, D.-L.; Aida, T. Chem. Commun. 1996, 1523-1524.

8. Why is modeling of the Mb or Hb functions so difficult? • Difficulty in preparation of five coordinate Fe(II)Por

9. Why is modeling of the Mb or Hb functions so difficult? • Easy oxidative dimerization of O2-Fe(II)Por yielding a m-oxo-dimer of Fe(III)Por

10. Why is modeling of the Mb or Hb functions so difficult? • Direct oxidation of Fe(II)Por to Fe(III)Por with O2

11. Why is modeling of the Mb or Hb functions so difficult? • Water-promoted autoxidation of O2-Fe(II)Por

12. The main reason why modeling in aqueous solution is so difficult.

13. pKa 4.8 pKa 0.4 Chem. Lett. 1996, 925-926. J. Am. Chem. Soc. 2002, 124, 9937-9944.

14. Inorg. Chem. 2006, 45, 4448-4460. J. Am. Chem. Soc. 2008, 130, 8006-8015.

15. Py2CD Py3CD Synthetic route of Py2CD and Py3CD

16. Experimental procedures for examining O2 and CO binding of Fe(II)PCD in an aqueous solution at pH 7.0.

17. UV-vis spectra of Fe(II)PCD, O2-Fe(II)PCD and CO-Fe(II)PCD in phosphate buffer at pH 7.0 and 3 oC.

18. O2 affinity at 25 oC and pH 7.0 P1/2: hemoCD17 Torr Fe(II)PCD 176Torr P1/2: Mb (sperm whale) 0.29 Torr Hb (human R) 0.17 Hb (human T) 26 Model systems in organic solvents 0.1 ~ 2150

19. CO affinity at 25 oC and pH 7.0 P1/2: hemoCD1.5 x 10-5 Torr Fe(II)PCD 0.016 Torr P1/2: Mb (sperm whale) 0.02 Torr Hb (human R) 0.013

20. A conformation of hemoCD is similar to that of leghemoglobin. A conformation of Fe(II)PCD is similar to that of Mb or Hb.

21. Cage Effects The Fe center of FeTPPS is completely capped with the two CD cavities. O2 as well as CO released from the Fe(II) center cannot slip out of the cleft of CDcapsule because of its hydrophobic nature. Released O2 or CO rebinds to the Fe(II) center. Fe(II)PCD

22. Picket-fence porphyrin

23. Oxy-hemoCD

24. Reductive nitrosylation of Fe(III)PCD and oxidation of (NO)Fe(II)PCD

25. Nitric Oxide NO is biosynthesized from arginine and dioxygen by nitric oxide synthases (NOS). NO causes relaxation of smooth muscle to control blood pressure. NO stimulates the soluble guanylate cyclase leading to subsequent formation of cyclic GMP. Macrophages generate NO to kill antigen. etc.

26. Nitrosylation of Fe(II)PCD lmax 420 nm

27. Reductive nitrosylation of Fe(III)P(TMe-b-CD)2 complex lmax 401 nm

28. No reductive nitrosylation occurs in the absence of cyclodextrin.

29. lmax = 420 nm lmax = 401 nm No reductive nitrosylation

30. This mechanism has been well established. Oxy-Mb regulates NO in biological system. The mechanism has not been clarified. NO inhibits the activities of proteins such as cyt P450, cyt c oxidase, nitrile hydrase, and catalase.

31. (NO)Fe(II)PCD is gradually oxidized to Fe(III)PCD and NO3- in an aerobic aqueous solution at pH 7.0, • (NO)Fe(II)P(TMe-b-CD)2 is not oxidized at all.

32. In the case without cyclodextrin • In the absence of CD, reductive nitrosylation cannot be applied. • (NO)Fe(II)TPPS can be prepared from nitrosylation of Fe(II)TPPS in a glove box. • (NO)Fe(II)TPPS is very unstable in an aerobic aqueous solution. Ring-opening reaction of FeTPPS may occur.

33. t1/2 = 6.3 h t1/2 = ∞ No oxidation occurs. Decomposition of the porphyrin ring occurs.

34. kmax : the maximum reaction rate constant for autoxidation of (NO)Fe(II)PCD kmax = 5.1 x 10-5 s-1 koff : the reaction rate constant for the dissociation of NO from (NO)Fe(II)PCD koff = 5.6 x 10-5 s-1

35. Mechanism for oxidation of (NO)Fe(II)PCD with dioxygen Rate-determining step

36. A large activation enthalpy change reflects the endothermic dissociation of the NO-Fe(II) bond. • Since activation entropy change is almost zero, no bimolecular reaction participates in the rate-determining step. Eyring plot for autoxidation of (NO)Fe(II)PCD. The thermodynamic parameters support the reaction mechanism proposed herein. DH‡ = 98.9 kJ mol-1 DS‡ = 0.17 J mol-1K-1

37. Oxidation of (NO)Mn(II)PCD by O2

38. Autoxidation of (NO)Mn(II)PCD (NO)Mn(II) t1/2 ~ 35 h Mn(III) Zero-order kinetics was observed for the aut-oxidation of (NO)Mn(II)PCD.

39. very slow If the equilibrium A ⇌ B exclusively shifts to A and a very small amount of B existing in the system relatively rapidly reacts to yield a final product, such a reaction obeys zero-order kinetics.

40. The rate of autoxidation of Mn(II)PCD is much faster than that of (NO)Mn(II)PCD.

41. Autoxidation of (NO)Mn(II)P(TMe-b-CD)2 (NO)Mn(II) Mn(III) Mn(II)

42. Mechanism for Oxidation of (NO)Mn(II)PCD with Dioxygen

43. Mechanism for oxidation of (NO)Fe(II)PCD with dioxygen

44. Summary • HemoCD and Fe(II)PCD are good carriers of simple diatomic molecules such as O2, CO, and NO in aqueous solution. • HemoCD shows the extremely high CO affinity that might be used for medicinal purposes. • Fe(II)PCD is a good model for studying interactions of NO with hemoproteins. • The mechanism for oxidation of (NO)Fe(II)Por by O2 was clarified in the present study.