Structure Dependence of Pyridine and Benzene Derivatives on Interactions with Model Membranes

Abstract

Pyridine-based small-molecule drugs, vitamins, and cofactors are vital for many cellular processes, but little is known about their interactions with membrane interfaces. These specific membrane interactions of these small molecules or ions can assist in diffusion across membranes or reach a membrane-bound target. This study explores how minor differences in small molecules (isoniazid, benzhydrazide, isonicotinamide, nicotinamide, picolinamide, and benzamide) can affect their interactions with model membranes. Langmuir monolayer studies of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE), in the presence of the molecules listed, show that isoniazid and isonicotinamide affect the DPPE monolayer at lower concentrations than the DPPC monolayer, demonstrating a preference for one phospholipid over the other. The Langmuir monolayer studies also suggest that nitrogen content and stereochemistry of the small molecule can affect the phospholipid monolayers differently. To determine the molecular interactions of the simple N-containing aromatic pyridines with a membrane-like interface, ¹H one-dimensional NMR and ¹H-¹H two-dimensional NMR techniques were utilized to obtain information about the position and orientation of the molecules of interest within aerosol-OT (AOT) reverse micelles. These studies show that all six of the molecules reside near the AOT sulfonate headgroups and ester linkages in similar positions, but nicotinamide and picolinamide tilt at the water-AOT interface to varying degrees. Combined, these studies demonstrate that small structural changes of small N-containing molecules can affect their specific interactions with membrane-like interfaces and specificity toward different membrane components.

Figure 1.

Structures of isoniazid (INH), benzhydrazide (BHZ), isonicotinamide (iNIC), benzamide (BA), nicotinamide (NIC), and picolinamide (PIC), with protons labeled for ¹H NMR peak labeling. The protons in the ¹H NMR spectra has H_a as the most downfield ¹H NMR peak, Hb the next one, etc. See Figure S1 for enlargement.

Figure 2.

The Langmuir monolayer (A) shows how a molecule can affect a phospholipid interface by penetrating and condensing (red arrows) or spreading (blue arrows) the phospholipids during a compression isotherm. The schematic depiction of a RM (B) outlines the area in which a molecule may reside, such as the bulk water (a), interfacial Stern layer (b), AOT tail region (c), and isooctane (d). The black oval demonstrates how a molecule can have varying depths within the RM interface along with different orientations. Figure adapted from Peters et al.

Figure 3.

Compression isotherms of DPPC (left column) or DPPE (right column) in the presence of INH (A and B) or BHZ (C and D). The solid black curves correspond to the control films without any hydrazide present. The other curves correspond to 10mM hydrazide (red dashed line), 1 mM (blue dotted line), and 0.1 mM (green dashed and dotted line) present in the 20 mM sodium phosphate buffered subphase (pH 7.4). Each curve is an average of at least 3 trials with standard deviations. The R group for each phospholipid includes the phosphate, glycerol, and fully saturated C₁₆ tails. See Figures S4 and S5 for enlarged versions.

Figure 4:

The resulting surface pressure compression isotherms of DPPC (left column) and DPPE (right column) in the presence of BA (A and B), PIC, (C and D), NIC (E and F), iNIC (G and H) at concentrations of 0 mM (black solid line), 0.1 mM (green dashed and dotted line), 1.0 mM (blue dotted line), and 10 mM (red dashed line) in the 20 mM sodium phosphate buffered subphase (pH 7.4). Each curve is an average of at least 3 trials with standard deviations. The R group for each phospholipid includes the phosphate, glycerol, and fully saturated Ci6 tails. See Figures S7-S10 for enlarged versions.

Figure 5.

1D ¹H NMR spectra obtained using a 400 MHz Varian NMR of INH, iNIC, BHZ, BA, NIC, and PIC in D₂O and varying sizes of RMs (w₀) given on the left of each stack of spectra. See Figure 1 for labeled structures corresponding to peak labels.

Figure 6:

¹H-¹H 2D ROESY NMR spectra acquired using a 400 MHz Inova NMR of INH (A1-A3) and iNIC (B1-B3) using 200 ms, 100 ms, and 0 ms mixing time (1–3) and a relaxation delay of 1.5 s. The diagonal is indicated by the diagonal line. The lines also highlight any off diagonal cross peaks.

Figure 7:

Pictorial representation of the placement of INH, iNIC, BHZ, BA, PIC, and NIC within the RM as determined through ID and 2D ¹H NMR studies. It is important to note that this system is highly dynamic and therefore these are average positions/orientation within the AOT interface based on the ID and 2D NMR data presented in this study.