Preparation of SNS Cobalt(II) Pincer Model Complexes of Liver Alcohol Dehydrogenase

Summary

The preparation of SNS pincer cobalt(II) model complexes of liver alcohol dehydrogenase is presented here. The complexes can be prepared by reacting the ligand precursor with CoCl₂·6H₂O and can then be recrystallized by allowing diethyl ether to slowly diffuse into an acetonitrile solution that contains the cobalt complex.

Abstract

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each possessing two sulfur and one nitrogen donor atom functionalities (SNS) and based on bis-imidazole or bis-triazole compounds) are metallated with CoCl₂·6H₂O to afford tridentate SNS pincer cobalt(II) complexes. Preparation of the cobalt(II) model complexes for liver alcohol dehydrogenase is facile. Based on a quick color change upon adding the CoCl₂·6H₂O to acetonitrile solution that contains the ligand precursor, the complex forms rapidly. Formation of the metal complex is complete after allowing the solution to reflux overnight. These cobalt(II) complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH). The complexes are characterized using single crystal X-ray diffraction, electrospray mass spectrometry, ultra-violet visible spectroscopy, and elemental analysis. To accurately determine the structure of the complex, its single crystal structure must be determined. Single crystals of the complexes that are suitable for X-ray diffraction are then grown via slow vapor diffusion of diethyl ether into an acetonitrile solution that contains the cobalt(II) complex. For high quality crystals, recrystallization typically takes place over a 1 week period, or longer. The method can be applied to the preparation of other model coordination complexes and can be used in undergraduate teaching laboratories. Finally, it is believed that others may find this recrystallization method to obtain single crystals beneficial to their research.

Introduction

The purpose of the presented method is to prepare small-molecule analogs of LADH to further understand the catalytic activity of metalloenzymes. LADH is a dimeric enzyme that contains a cofactor-binding domain and zinc(II) metal-containing catalytic domain¹. LADH, in the presence of co-factor NADH, can reduce ketones and aldehydes to their respective alcohol derivatives². In the presence of NAD⁺, LADH can perform reverse catalysis of oxidation of alcohols to ketones and aldehydes². The crystal structure of LADH’s active site shows that its zinc(II) metal center is bound to one nit....

Protocol

1. Synthesis of chloro-(n³-S,S,N)-[2,6-bis(N-isopropyl-N’-methyleneimidazole-2-thione)pyridine]cobalt(II)tetrachlorocobaltate [4]

1. To prepare complex 4, add 0.121 g (3.12 x 10^-4 mol) of 2,6-bis(N-isopropyl-N’-methyleneimidazole-2-thione)pyridine (C₁₉H₂₅N₅S₂)⁶ to 15 mL of acetonitrile in a 100 mL round bottom flask. Next, to this solution, add 0.0851 g (3.58 x 10^-4 mol) of cobalt chl.......

Discussion

The preparation of complexes 4 and 5 is facile. The key step is to add the solid CoCl₂·6H₂O to an acetonitrile solution that contains the respective ligand precursor. The solution turns dark green within seconds after the addition of CoCl₂·6H₂O to form complex 4. The solution turns bright blue after the addition of CoCl₂·6H₂O to form complex 5. To ensure complete reaction, t.......

Materials

Name	Company	Catalog Number	Comments
100 mL Round Bottomed Flask	Chem Glass	CG150691	100mL Single Neck Round Bottomed Flask, 19/22 Outer Joint
Acetonitrile	Fisher	HB9823-4	HPLC Grade
Chiller for roto-vap	Lauda	L000638	Alpha RA 8
Cobalt Chloride hexahydrate	Acros Organics	AC423571000	Acros Organics
Diethyl Ether	Fisher	E-138-1	Diethyl Ether Anhydorus
graduated cylinder	Fisher	S63456	25 mL graduated cylinder
hotplate	Fisher	11-100-49SH	Isotemp Basic Stirring Hotplate
jars	Fisher	05-719-481	250 mL jars
Ligand	-----	-----	Synthezied previously by Professor Miecznikowski
medium cotton balls	Fisher	22-456-80	medium cotton balls
one dram vials	Fisher	03-339	one dram vials with TFE Lined Cap
pipet	Fisher	13-678-20B	5.75 inch pipets
pipet bulbs	Fisher	03-448-21	Fisher Brand Latex Bulb for pipet
recrystallizing dish for sand bath	Fisher	08-741 D	325 mL recrystallizing dish for sand bath
reflux condensor	Chem Glass	CG-1218-A-22	Condenser with 19/22 inner joint
Rotovap	Heidolph Collegiate	36000090	Brinkmann; Heidolph Collegiate Rotary Evaporator with Heidolph WB eco bath Heidolph Rotary Evaporator
sea sand for sandbath	Acros Organics	612355000	washed sea sand for sand bath
Stir bar	Fisher	07-910-23	Egg-Shaped Magnetic Stir Bar
Vacum grease	Fisher	14-635-5D	Dow Corning High Vacuum Grease
vacuum pump for rotovap	Heidolph Collegiate	36302830	Heidolph Rotovac Valve Control