Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

Abstract

Ion channel-forming peptides and proteins offer tremendous opportunities for fundamental and applied studies of function on individual molecules. An ongoing challenge in ion channel research is the lack of simple and accessible synthetic methods to engineer pores with tailored chemical and physical properties. This paper describes a practical synthetic route to rapidly generate C-terminally modified derivatives of gramicidin A (gA), an ion channel-forming peptide, through the use of two chemically reactive gA-based building blocks. These amine- and azide-containing building blocks can react readily with typical substrates for amidation and 1,3-dipolar cycloaddition ("click") reactions to present molecules with desired structure and functionality near the opening of a gA pore. These derivatives of gA are stable under typical aqueous conditions for ion channel recordings and retain characteristic single ion channel conductance properties in planar lipid bilayers. Additionally, the synthetic methods described here afford useful quantities of these gA derivatives in good purity and yield with minimal purification. We demonstrate that derivatives of gA can be used for studying, in situ, a change in conductance through a channel upon performing a "click" reaction on an azide moiety attached to the gA pore. We also demonstrate that these gA-based building blocks can be used to construct sensors for the recognition of specific protein-ligand binding interactions in solution. This widely accessible, enabling synthetic methodology represents a powerful new tool to study relationships between chemical structure and function on the single molecule level.

Reactive derivatives of gramicidin enable light- and ion-modulated ion channels

“…We, therefore, used methods to esterify gA (1) to generate gA-derivatives 2-4 ( Figure 2). [1][2][3] The relatively poor chemical reactivity of the C-terminal alcohol on native gA and the instability of esters and carbamates, however, has been a limiting factor towards constructing tailored sensors based on gA. We, therefore, recently reported a practical synthesis of three gA-based synthetic building blocks-desethanolamine gA (5), gramicidamine (6), and gramicidazide (7)-in large quantities from commercially available (and relatively inexpensive) gA (1). [1] These building blocks make it possible to rapidly synthesize a wide range of stable C-terminal derivatives of gA. Figure 2 shows a general synthetic scheme for the synthesis of 5-7 from gA (1).…”

“…[1][2][3] Synthesis of 18-crown-6 gA derivative 8 (gA-18c6). We dissolved 4 mg (2.2 µmol) of desethanolamine gramicidin A 5 in 0.3 mL of anhydrous tetrahydrofuran (THF).…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] An ion channel assembly can conduct >10 3 ions per millisecond from a single channel opening event, [14] making it possible to detect the ion conducting functional properties of a single supramolecular structure. Developing analytical assays using ion channels, therefore, offers the potential advantage of high sensitivity with, in principle, single molecule detection limit due to the amplification characteristics of an ion pore.…”

“…In order to realize such sensitive assays, several groups have reported methods to engineer tailored ion channel proteins and peptides that respond to specific external stimuli. [1][2][3][15][16][17][18][19] The paper presented here will explore the conductance properties of synthetically modified derivatives of gramicidin A (gA), a natural ion channel-forming peptide. [20][21][22][23][24] We will describe some basic design criteria for development of sensors based on these semi-synthetic derivatives of gA and present early examples of the engineering of ion channel-based sensors for detection of chemical analytes in solution as well for the controlled and reversible modulation of ion conductance across membranes using specific wavelengths of light (i.e., lightmodulated ion channels).…”

“…[1][2][3][15][16][17][18][19] Here, we demonstrate how changes in charge on chemical groups presented near the opening of a gramicidin pore can be used to sense specific chemical analytes in solution. We also present how changes in charge of functional groups attached to the C-terminus of gA can be used indirectly for the detection of different wavelengths of light.…”