Alternating pattern of stereochemistry in the nonactin macrocycle is required for antibacterial activity and efficient ion binding

Abstract

Nonactin is a polyketide antibiotic produced by Streptomyces griseus ETH A7796 and is an ionophore that is selective for K(+) ions. It is a cyclic tetraester generated from two monomers of (+)-nonactic acid and two of (-)-nonactic acid, arranged (+)-(-)-(+)-(-) so that nonactin has S4 symmetry and is achiral. To understand why achiral nonactin is the naturally generated diastereoisomer, we generated two alternate diastereoisomers of nonactin, one prepared solely from (+)-nonactic acid and one prepared solely from (-)-nonactic acid, referred to here as 'all-(+)-nonactin' and 'all-(-)-nonactin', respectively. Both non-natural diastereoisomers were 500-fold less active against gram positive organisms than nonactin confirming that the natural stereochemistry is necessary for biological activity. We used isothermal calorimetry to obtain the K(a), DeltaG, DeltaH, and DeltaS of formation for the K(+), Na(+), and NH(4)(+) complexes of nonactin and all-(-)-nonactin; the natural diastereoisomer bound K(+) 880-fold better than all-(-)-nonactin. A picrate partitioning assay confirmed that all-(-)-nonactin, unlike nonactin, could not partition K(+) ions into organic solvent. To complement the thermodynamic data we used a simple model system to show that K(+) transport was facilitated by nonactin but not by all-(-)-nonactin. Modeling of the K(+) complexes of nonactin and all-(-)-nonactin suggested that poor steric interactions in the latter complex precluded tight binding to K(+). Overall, the data show that both enantiomers of nonactic acid are needed for the formation of a nonactin diastereoisomer that can act as an ionophore and has antibacterial activity.

Figure 1

Structures of nonactin and the naturally occurring macrotetrolides.

Figure 2

Structure of nonactin and the nonactin-K⁺ complex. Coordinates for the structures were taken from crystallography data. The left and center structures are those of nonactin without a coordinated metal ion. The main macrocycle ring of nonactin has been emphasized and color coded in the central structure to show the changes in torsion angle between nonactin and the nonactin-K⁺ complex – the color map runs from red (small change) to yellow (extensive change). The right structure is that of the nonactin-K⁺ complex with the ion omitted for clarity.

Figure 3

Determination of ionophore-K⁺ partition into an immiscible organic phase as a thermodynamic measure of ionophore ability. Differing amounts of ionophore were partitioned between equal volumes of CHCl₃ and an aqueous solution of potassium picrate solution. The amount of ionophore-K⁺ complex in the CHCl₃ phase was quantified by measuring the concentration of the picrate counter ion via its absorbance at 357 nm. This assay is a modification of the partition assay used to quantify tetranactin developed by Suzuki et al. Inset picture: the picrate anion is quite visible in the lower, CHCl₃ layer when nonactin is used as the ionophore (A) and not present when all-(−)-nonactin is used (B).

Figure 4

Determination of ionophore-K⁺ transport rates between two aqueous phases through an intermediate bulk organic solvent as a kinetic measure of ionophore ability and a simple model of passive membrane diffusion. The transport rate of K⁺ from the one aqueous phase to the other was measured by following the absorbance of the ‘receiving’ phase at 357 nm, that is, by measuring co-transport of the picrate anion. Fit lines were calculated by non-linear least squares regression to a simple rate equation modeling a first order approach to an equilibrium, A₃₅₇(t) = k₁.(1 − exp[−k₂t]). ● Nonactin as the ionophore; k₁ = 1.8, k₂ = 0.0009. ○ All-(−)-nonactin as the ionophore; k₁ = 1.9, k₂ = 0.100.

Figure 5

Representation of the nonactin and all-(−)-nonactin structures bound to potassium. 1, Distribution of K⁺-carbonyl oxygen distances for nonactin (blue) and all-(−)-nonactin (red) obtained from molecular dynamics simulation. 2, Representations of the poor steric interactions (A) that are avoided in all-(−)-nonactin by distorting the complex to a less favorable conformation for binding or that are absent (B) in the natural diastereoisomer, nonactin. For nonactin, H-3 and H-6 face inwards towards the cation; for all-(−)-nonactin H-3 and H-6 face outwards toward solvent. 3, Distribution of K⁺-tetrahydrofuran oxygen distances for nonactin (blue) and all-(−)-nonactin (red) obtained from molecular dynamics simulations.

Scheme 1

Synthesis of orthogonally protected nonactic acid monomers and dimers.

Scheme 2

Completion of the synthesis of all-(−)-nonactin.