Organometallic Catalysis Catches up with Enzymatic in the Regeneration of NADH

Abstract

A rational design, based on a deep understanding of the reaction mechanism, led to the development of an iridium organometallic catalyst with activity comparable to that of enzymes in the chemical regeneration of NADH, using phosphite as a reducing agent. The innovative structural elements were individuated in replacing pyridine with pyrazine and adding a carbohydrazide moiety in the bidentate amidate supporting ligand. Resulting [Cp*Ir-(pyza-NH₂)-Cl] (pyza-NH₂ = κ²-pyrazinecarbohydrazide; 1) outperforms both the analogous complex with pyridine, [Cp*Ir-(pica-NH₂)-Cl] (pica-NH₂= κ²-pyridincarbohydrazide; 2), and that missing the -NH₂ moiety, [Cp*Ir-(pyza)-Cl] (pyza = κ²-pyrazineamidate; 3). A maximum TOF of 13,090 h^-1 was observed for 1 ([NAD⁺] = 6 mM, [cat] = 7.5 μM, pH 6.58 by phosphite buffer 0.4 M, 313 K), a value never reached for any organometallic catalyst and comparable with that of enzymes. ¹H diffusion NMR experiments indicate that 1 and 2 undergo dimerization in water through the ionization of the Ir-Cl bond and coordination of the carbohydrazide moiety to a second iridium center. This leads to [Cp*Ir-(pyza-NH₂)]₂X₂ (1 _D ) and [Cp*Ir-(pica-NH₂)]₂X₂ (2 _D ) that were isolated as PF₆ ^- salts by anion metathesis with NH₄PF₆ and fully characterized both in solution (multinuclear multidimensional NMR) and in the solid state (single-crystal X-ray diffractometry). Catalytic NADH regeneration experiments were carried out starting from stock solutions of 1-3 complexes in acetonitrile, where diffusion NMR experiments ensure the main presence of mononuclear catalytic precursors, in order to avoid complications due to dimerization. In-depth kinetic studies evidenced that catalyst 1 in combination with the H₂PO₃ ^- donor is superior to 2 and 3 in all aspects, facilitating the formation of the Ir-H intermediate and the tendency to donate the hydride to NAD⁺, at the same time inhibiting the detrimental accumulation of the off-cycle cat/NAD⁺ adduct. The introduction of the pyrazine moiety, much less σ-donating and more π-accepting than the pyridine one, is likely responsible for most of the increased activity and stability of 1 with respect to 2. Meanwhile, the dandling -NH₂ carbohydrazide moiety might further accelerate the process by providing a basic functionality close to the reactive coordination position and introducing some steric hindrance to hamper the formation of the off-cycle cat/NAD⁺ adduct.

Keywords: NADH regeneration; NMR; X-ray diffractometry; iridium catalysts; organometallic chemistry.

1

Most efficient Ir catalysts for NADH regeneration.

2

Reaction mechanism proposed for the regeneration of NADH mediated by [Cp*Ir^III(N,N)­X] catalysts.

3

Sketch of the chemical structures of the complexes investigated.

4

Synthetic procedure for 1, 2 and 1 _D , 2 _D .

5

(a) Section of the ¹H NOESY NMR spectrum showing a NOE contact between H₆ and H₁₀ protons (DMSO-d₆, 298 K). (b) Section of the ¹H NOESY NMR spectrum of 1 showing NOE contacts between the aromatic protons H₅ and H₆ (DMSO-d₆, 298 K). (c) Section of the ¹H,¹³C HSQC spectrum of 1 showing the direct scalar correlations of aromatic protons and carbons (DMSO-d₆, 298 K).

6

Dimerization reaction of complex 1 leading to 1 _D (top); ¹H NMR spectrum of a mixture of 1 and 1 _D in D₂O at 298 K.

7

A section of the ¹H,¹H EXSY NMR spectrum (D₂O, 298 K) showing the exchange cross peaks between resonances of 1 (black) and those of 1 _D (red).

8

Semilogarithmic plot of ln­(I/I ₀) versus G ² for 1 (green) and 1 _D (light green) in D₂O.

9

ORTEP drawing of cations 1 _D and 2 _D . Ellipsoid at 50% probability, hydrogen atoms, and counterions are omitted for clarity. Color code: Ir = orange, N = blue, O = red, C = gray.

10

(a) Kinetics of the growth of the absorption band of NADH at 340 nm. (b) TON vs t course obtained by means of UV–vis spectroscopy for 1 (green), 2 (gray), and 3 (purple) at [cat] = 7.5 μM, [NAD⁺] = 4 mM; phosphite buffer 0.4 M pH 6.58, 313 K.

11

(a) TOF vs [NAD⁺] plot for catalysts 1 and 1 _D ([cat] = 7.5 μM, phosphite buffer 0.4 M pH 6.58, 313 K). (b) TOF vs [NAD⁺] plot for catalysts 1 and 1 _D ([cat] = 7.5 μM; [HCOOK] = 0.125 M; phosphate buffer pH 7 0.1 M, 313 K).

12

(a) TOF vs [NAD⁺] plot for catalysts 1–3 ([cat] = 7.5 μM, phosphite buffer 0.4 M, pH 6.58, 313 K). (b) TOF vs [NAD⁺] plot for catalysts 1–3 ([cat] = 7.5 μM; [HCOOK] = 0.125 M; phosphate buffer pH 7 0.1 M, 313 K).

13

Time course of TON in the presence of phosphite or formate as hydride donors with 1. ([cat] = 7.5 μM; [HCOO–/H₂PO₃−] = 0.125 M; phosphate buffer, pH 6.58 M, 313 K).

14

¹H NMR spectra for the hydrogenation of NAD⁺ (gray) with phosphonic acid, catalyzed by 1, showing the regioselective formation of 1,4-NADH (purple) and 1,6-NADH (blue) ([NAD⁺] = 5 mM, [cat] = 100 μM, buffer phosphite 0.4 M, pH = 6.58, T = 313 K).