Investigating Substrate Analogues for Mycobacterial MenJ: Truncated and Partially Saturated Menaquinones

Abstract

Menaquinones (MKs) are essential for electron transport in prokaryotes, and importantly, partially saturated MKs represent a novel virulence factor. However, little is known regarding how the degree of saturation in the isoprenyl side chain influences conformation or quinone redox potential. MenJ is an enzyme that selectively reduces the second isoprene unit on MK-9 and is contextually essential for the survival of Mycobacterium tuberculosis in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target for pathogenic mycobacteria. Therefore, fundamental information about the properties of this system is important, and we synthesized the simplest MKs, unsaturated MK-1 and the saturated analogue, MK-1­(H2). Using two-dimensional nuclear magnetic resonance spectroscopy, we established that MK-1 and MK-1­(H2) adopted similar folded–extended conformations (i.e., the isoprenyl side chain folds upward) in each solvent examined but the folded–extended conformations differed slightly between organic solvents. Saturation of the isoprenyl side chain slightly altered the MK-1 analogue conformation in each solvent. We used molecular mechanics to illustrate the MK-1 analogue conformations. The measured quinone redox potentials of MK-1 and MK-1­(H2) differed between organic solvents (presumably due to differences in dielectric constants), and remarkably, an ∼20 mV semiquinone redox potential difference was observed between MK-1 and MK-1­(H2) in pyridine, acetonitrile, and dimethyl sulfoxide, demonstrating that the degree of saturation in the isoprenyl side chain of MK-1 influences the quinone redox potential. Finally, MK-1 and MK-1­(H2) interacted with Langmuir phospholipid monolayers and Aerosol-OT reverse micelle (RM) model membrane interfaces, where MK-1 adopted a slightly different folded conformation within the RM model membrane interface.

Interactions of Truncated Menaquinones in Lipid Monolayers and Bilayers

“…Langmuir monolayers are often used to examine the ability of a molecule to penetrate an interface, to disrupt packing, and to affect the elasticity of the monolayer [ 7 , 29 , 30 , 31 ]. We have previously reported compression isotherms of mixed films in terms of area per molecule for MK-1 and MK-2 [ 6 , 7 ]. The Langmuir monolayer data may be analyzed differently depending on the system of interest (hydrophilic vs. hydrophobic target molecule).…”

“…[ 1 , 2 , 3 ] MKs consist of a naphthoquinone headgroup and an isoprenoid side chain of varying length ( Figure 1 ) [ 4 , 5 ]. We have previously found that MK’s structure allows it to fold into different molecular shapes depending on environment and side chain length [ 6 , 7 ]. MKs must be membrane-associated to function in the ETS [ 1 , 8 ], and current knowledge regarding the interaction and conformation of MK homologues in phospholipid bilayers is limited and often conflicting [ 9 , 10 , 11 ].…”

Synthesis of Naphthoquinone Derivatives: Menaquinones, Lipoquinones and Other Vitamin K Derivatives

“…Recently, we synthesized fully and partially saturated MK-derivatives in an effort to understand their structural and electrochemical properties in model membranes [ 12 , 37 , 38 ]. For the synthesis of MK-2(II-H 2 ), the condensation of isophytol 22 and menadiol 2 was accomplished in 11% yield using MgF 2 -48, a Coman et al inspired catalyst ( Scheme 11 ).…”

“…Despite the polarity of the quinone group, these molecules are very hydrophobic, even though this property does vary with the length and the nature of the isoprene side group. We have recently found by synthesis of truncated MK-derivatives that only MK-1, MK-2, and MK-3, including derivatives with fully or partially saturated counterparts, are soluble in aqueous solution [ 12 , 37 , 38 ]. This means that assays with MK-4, even though it is known as an enzyme substrate in vivo , may not demonstrate enzyme activity even if the aqueous assay includes surfactants [ 39 ].…”

“…Contemplating the many synthetic strategies available, consideration of the structure and the application of the MK-derivatives may be important when choosing the appropriate method for synthesis. So far, our group has focused on the preparation of truncated MK-derivatives, where the syntheses are relatively short and direct [ 12 , 37 , 38 ]. Therein, we used Friedel-Crafts alkylation using BF 3 ∙OEt 2 as a Lewis acid catalyst.…”