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

Article abstract of DOI:10.1021/ja9050235

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

Full text of DOI:10.1021/ja9050235

Published on Web 11/10/2009
Alternating Pattern of Stereochemistry in the Nonactin
Macrocycle Is Required for Antibacterial Activity and Efficient
Ion Binding
Brian R. Kusche,† Adrienne E. Smith,† Michele A. McGuirl,‡ and Nigel D. Priestley*,†
Department of Chemistry and Biochemistry and DiVision of Biological Sciences, UniVersity of
Montana, 32 Campus DriVe, Missoula, Montana 59812-1656
Received June 18, 2009; E-mail: nigel.priestley@umontana.edu
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 ob-
tain the Ka, ∆G, ∆H, and ∆S of formation for the K+, Na+, and NH4 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.
Introduction
Nonactin, 1 (Figure 1), is a member of the macrotetrolides,1-4
a class of ionophore antibiotic made by Streptomyces griseus
ETH A7796. Nonactin has antibacterial and anticancer proper-
ties2 and has also been shown to be an inhibitor of efflux pumps
in multidrug resistant cancers.5 As nonactin shows selectivity
for binding NH4+ ions, it has found widespread use in ammonia-
selective electrodes.6 The antibacterial effects of nonactin depend
+
upon its ability to form stable complexes with K+, Na+, or NH4
ions and to support the passive diffusion of these ions across
cell membranes.
The structure of nonactin is highly unusual for a natural
product.4 Nonactin is a cyclic tetraester assembled from nonactic
acid. Both (+)-nonactic acid and (-)-nonactic acid are needed
to construct the macrocycle, and they are joined head-to-tail in
† Department of Chemistry and Biochemistry.
‡ Division of Biological Sciences.
(1) Corbaz, R.; Ettinger, L.; Gaumann, E.; Keller-Schlierlein, W.; Kra-
dolfer, F.; Kyburz, E.; Neipp, L.; Prelog, V.; Zahner, H. HelV. Chim.
Acta 1955, 38, 1445.
Figure 1. Structures of nonactin and the naturally occurring macrotetrolides.
(2) Meyers, E.; Pansy, F. E.; Perlman, D.; Smith, D. A.; Weisenborn,
F. L. J. Antibiot. 1965, 18, 128.
(3) Gerlach, H.; Hutter, R.; Keller-Schlierlein, W.; Seibl, J.; Zahner, H.
HelV. Chim. Acta 1967, 50, 1782–1793.
an alternating (+)-(-)-(+)-(-) pattern. The overall structure
has S4 symmetry in both the free and complexed forms and is
achiral even though it is assembled from chiral precursors. Early
crystallography studies of nonactin7 and its potassium complex8
(4) Keller-Schierlein, W.; Gerlach, H. Fortschr. Chem. Org. Naturst. 1968,
26, 161–189.
(5) Borrel, M. N.; Pereira, E.; Fiallo, M.; Garnier-Suillerot, A. Eur.
J. Biochem. 1994, 223, 125–133.
(6) Karakus, E.; Pekyardimci, S.; Kilic, E. Artif. Cells, Blood Substitutes,
Biotechnol. 2006, 34, 523–534.
(7) Dobler, M. HelV. Chim. Acta 1972, 55, 1371–1384.
9
10.1021/ja9050235 CCC: $40.75  2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 17155–17165 17155

A R T I C L E S
Kusche et al.
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.
delineated the major structural changes on ion binding. Nonactin
assumes a flat doughnut-like structure in the absence of an ion
that is approximately 17 × 17 × 8.5 Å3 (Figure 2). During K+
binding there is a stepwise association of the carbonyl and
tetrahydrofuran oxygen atoms of nonactin with the ion, together
with the stripping of the water of hydration, that results in a
more spherical complex approximately 15 × 15 × 12 Å3 in
size. The eight coordinating oxygen atoms form a cubic
geometry that completely shields the ion from solvent. Although
the structural reorganization that occurs on ion binding appears
substantial, the great majority of the change is accounted for
by changes in the O16-C1-C2-C3 and C1-C2-C3-C4
torsion angles in each monomer (Figure 2) which account for
67% of the total torsion angle changes. Further studies of
nonactin, including the use of NMR9,10 and Raman spectros-
copy,11 together with molecular dynamics simulation,12 sub-
stantially confirmed the binding model.
As the structure of nonactin is unique, so is the biosynthesis
of the compound. Nonactin is a polyketide and is assembled
from acetate, propionate, and succinate. Much is known about
the precursors of nonactin biosynthesis from labeling experi-
ments,13-15 the genetics of nonactin biosynthesis from analysis
of the nonactin biosynthesis gene cluster,16,17 and the enzymol-
ogy of nonactin biosynthesis.18-20 These data show that, after
the formation of an early, achiral intermediate, the biosynthesis
pathway for nonactin branches into two mirror image pathways
for the generation of (+)- and (-)-nonactic acid. The latter
precursors are then stereoselectively assembled into nonactin.
Emerging evidence suggests that the two pathways arose from
gene duplication followed by divergent evolution. The central
question raised, therefore, by both the structure and the
biosynthesis of nonactin concerns the benefit, if any, to the
producing organism in generating the natural, achiral diastere-
oisomer and for maintaining two independent pathways for the
generation of nonactic acid.
To better understand the nonactin structure and biosynthesis
we prepared two alternate diastereoisomers of nonactin, one
prepared solely from (+)-nonactic acid and one prepared solely
from (-)-nonactic acid, referred to herein as ‘all-(+)-nonactin’
and ‘all-(-)-nonactin’ respectively. These diastereoisomers are
of interest as their stereochemical arrangement mimics that of
valinomycin, another naturally occurring potassium ionophore.21
In all-(-)-nonactin the (2R,3R,5S,8S) pattern of (-)-nonactate
is replicated four times around the macrocycle. Taking nonactate
as the monomer, nonactin might be described as a syndiotactic
polymer. In valinomycin the (R,S,S,S) pattern of D-valine-D-
hydroxyvalerate-D-valine-L-lactate is replicated three times
around the macrocycle. With the D-valine-D-hydroxyvalerate-
D-valine-L-lactate structure as the repeating monomer of vali-
nomycin, valinomycin and both all-(-)- and all-(+)-nonactin
might be described as isotactic polymers. Our evaluation of the
non-natural diastereoisomers with respect to their antibiotic
activity, the thermodynamics of ion binding, and their ability
to partition ions into organic liquids and through bulk organic
phase models of membranes confirm that the producing organ-
ism is required to use both enantiomers of nonactic acid in
nonactin formation to achieve biological activity.
(8) Kilbourne, B. T.; Dunitz, J. D.; Pioda, L. A. R.; Simon, W. J. Mol.
Biol. 1967, 30, 559–563.
(9) Saito, H.; Tabeta, R.; Yokoi, M. Magn. Reson. Chem. 1988, 26, 775–
786.
(10) Tabeta, R.; Saito, H. Biochemistry 1985, 24, 7696–7702.
(11) Phillies, G. D. J.; Asher, I. M.; Stanley, H. E. Biopolymers 1975, 14,
2311–2327.
(12) Marrone, T. J.; Mertz, K. M. J. Am. Chem. Soc. 1992, 114, 7542–
7549.
Results
(13) Ashworth, D. M.; Clark, C. A.; Robinson, J. A. J. Chem. Soc., Perkin
Trans. 1 1989, 1461–1467.
Synthesis. Nonactic acid has been the target of many synthetic
studies as it is a relatively small yet stereochemically complex
molecule.22,23 Fewer total syntheses of nonactin, on the other
hand, have been reported as it is, at least in principle,
(14) Spavold, Z. M.; Robinson, J. A. J. Chem. Soc., Chem. Commun. 1988,
4–6.
(15) Nelson, M. E.; Priestley, N. D. J. Am. Chem. Soc. 2002, 124, 2894–
2902.
(16) Kwon, H.-J.; Smith, W. C.; Xiang, L.; Shen, B. J. Am. Chem. Soc.
2001, 123, 3385–3386.
(17) Walczak, R. J.; Woo, A. J.; Strohl, W. R.; Priestley, N. D. FEMS
Letts. 2000, 183, 171–175.
(20) Woo, A. J.; Strohl, W. R.; Priestley, N. D. Antimicrob. Agents
Chemother. 1999, 43, 1662–1668.
(18) Cox, J. E.; Priestley, N. D. J. Am. Chem. Soc. 2005, 127, 7976–7977.
(19) Kwon, H.-J.; Smith, W. C.; Scharon, J.; Hwang, S. H.; Kurth, M. J.;
Shen, B. Science 2002, 297, 1327–1330.
(21) MacDonald, J. C. Can. J. Microbiol. 1969, 15, 236–8.
(22) Arco, M. J.; Trammell, M. H.; White, J. D. J. Org. Chem. 1976, 43,
479–482.
9
17156 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Alternating Stereochemistry in the Nonactin Macrocycle
A R T I C L E S
straightforward to generate from nonactic acid.24,25 The practical
challenges in nonactin synthesis, however, lie in the need to
prepare both enantiomers of nonactic acid and in the final
reaction, the formation of a 32-membered macrocycle from a
linear precursor, challenges for which excellent solutions are
available.24,25 We were fortunate that syntheses of non-natural
diastereoisomers of nonactin could be patterned directly from
known routes leaving the production of both enantiomers of
nonactic acid as the main technological hurdle. As nonactin is
readily available to us on a large scale we decided to use the
natural product as a starting point rather than generate nonactic
acid de novo. Methanolysis of nonactin13,26 is a reliable
procedure, and we have used this in the past to generate tens of
grams of methyl nonactate, 8, albeit as the racemate. Resolution
of methyl nonactate was accomplished on a large scale using a
Rhodococcus-mediated oxidation;27 Rhodococcus cultures are
effective in stereoselectively oxidizing methyl (-)-nonactate to
methyl (-)-8-ketononactate leaving unreacted methyl (+)-
nonactate, (+)-8. After biotransformation, separation of methyl
(+)-nonactate and methyl (-)-8-ketononactate by flash chro-
matography is undemanding. Although (-)-8 can be obtained
through reduction of methyl (-)-8-ketononactate in an anaerobic
Rhodococcus culture,27 we chose to isolate (-)-nonactic acid
directly from a culture of S. griseus ∆nonD, a mutant strain of
the nonactin producing organism wherein the chromosomal copy
of the cocaine esterase-like nonactin biosynthesis gene nonD
had been disrupted. Overall, by degrading the natural product
and by exploiting a mutant strain of the nonactin producing
organism, we had access to many grams of both enantiomers
of 8.
Scheme 1. Synthesis of Orthogonally Protected Nonactic Acid
Monomers and Dimers
Scheme 2. Completion of the Synthesis of All-(-)-nonactin
The conversion of 8 into nonactin was predominantly an
exercise in manipulating protecting groups. Thus, protection of
the C-8 alcohol of 8 was achieved using TBSCl prior to
saponification to generate the TBS-protected acid 9 (Scheme
1). Similarly, saponification of 8 prior to conversion to the
corresponding benzyl ester lead to 11. Compounds 9 and 11
were the sole orthogonally protected nonactic acid monomers
needed to complete the synthesis of all-(-)-nonactin, (-)-17.
Coupling of 9 and 11 to afford the dimer 12 was accomplished
using DCC (Scheme 1). Compound 12 was then deprotected
by removing the silyl ether to afford benzyl protected alcohol
14 and by hydrogenolysis to afford protected acid 13. Following
the guide of Fleming, 13 and 14 could be coupled to give a
protected linear tetramer 15 using 2,4,6-trichlorobenzoyl chloride
(Scheme 2).24 Removal of the benzyl ester (hydrogenolysis) and
the silyl ether (acidification) protecting groups from 15 was
straightforward leading to the linear tetramer 16. At this point
in the synthesis we had some concern that the macrocyclization
reaction to form all-(-)-nonactin would prove problematic. It
might be argued that the alternating (+)-(-)-(+)-(-) stereo-
chemical pattern of nonactin may influence the manner in which
a linear, tetrameric precursor might fold and therefore affect
(23) Bartlett, P. A. M.; James, D.; Ottow, E. J. Am. Chem. Soc. 1984, 106,
5304–11.
(24) Fleming, I. G.; Sunil, K. J. Chem. Soc., Perkin Trans. 1 1998, 17,
2733–2748.
the cyclization reaction. The Yamaguchi coupling24,28 to form
all-(-)-nonactin from (-)-16, however, was quite straightfor-
ward. The simplicity of the cyclization reaction suggests that
(25) Gerlach, H.; Konrad, O.; Thalmann, A.; Servi, S. HelV. Chim. Acta
1975, 58, 2036–43.
(26) Dinges, J. M.; Bessette, B. A.; Cox, J. E.; Redder, C. R.; Priestley,
N. D. Biotechnol. Prog. 2006, 22, 1354–1357.
(27) Nikodinovic, J.; Dinges, J. M.; Bergmeier, S. C.; McMills, M. C.;
Wright, D. L.; Priestley, N. D. Org. Lett. 2006, 8, 443–445.
(28) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, T. Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17157

A R T I C L E S
Kusche et al.
Table 1. Antimicrobial Activity of Nonactin, All-(+)-nonactin and
All-(-)-nonactin against a Range of Microorganisms
surprisingly clear-cut. The latter compounds showed no sig-
nificant antibiotic activity at concentrations up to their solubility
limits.
minimum inhibitory concentration/µM
organism
nonactin all-(-)-nonactinb all-(+)-nonactinb
Ion Binding - Microcalorimetry. Having shown that all-(-)-
nonactin and all-(+)-nonactin have no significant antibiotic
activity, we needed to understand why. Nonactin acts as an
antibiotic, as it is able to form stable complexes with alkali metal
Bacillus anthracis
Bacillus cereus
Bacillus subtilis
Enterococcus faecalis
E. faecalis - Vancomycin resistant
Staphylococcus aureus
S. aureus - methicillin resistant
Escherichia coli
2
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
4
1
2
2
2
2
+
and NH4 ions catalyzing their passive diffusion down the
electrochemical gradients that exist across bacterial cell mem-
branes. The first question that we chose to address concerned,
therefore, the ability of non-natural diastereoisomers to form
complexes with metal ions. Microcalorimetry was the technique
of choice, as we would be able to extract much thermodynamic
detail concerning ion binding. We chose to conduct these
experiments in methanol. First, the solubility of nonactin in
water is so low that conducting these experiments on aqueous
samples was not practical.29 Second, the comparison of binding
data across the diastereoisomer series would be sufficient. The
thermodynamic constants for metal ion binding were determined
from titrations of KNCS, NaNCS, or NH4NCS into a solution
of the appropriate nonactin diastereoisomer. The heat change
observed during the titration series was used to compute
association constants (Ka) and ∆H of binding from which ∆G
and ∆S could then be derived (Table 2). We chose to use only
all-(-)-nonactin to test the binding of non-natural diastereoi-
somers. This choice was reasonable, as there can be no
stereoselectivity involved in the binding reactions to achiral ions.
Good data were obtained for nonactin with each of the ions
tested which was in stark contrast to the situation for all-(-)-
nonactin. In the latter case we were able to obtain reliable data
only for K+ and Na+ binding. In the case of NH4+, the signal
>1000a
>1000a
>1000a
Pseudomonas aeruginosa
Candida glabrata
a MIC determinations are limited by the low solubility of nonactin.
b Solubility of all-(+)-nonactin and all-(-)-nonactin is lower than that of
the natural product.
the ease of folding plays no role in the observed stereochemistry
of the natural product, nonactin. After generating all-(-)-
nonactin the entire synthetic sequence was repeated to generate
all-(+)-nonactin with similar results. Comparison of the 1H and
13C NMR spectra of the linear (+)-(-)-(+)-(-)-tetramer and
nonactin, as demonstrated by Fleming,24 revealed a significant
simplification in the resonances assigned to the methyl groups
at C-2 and C-8 of nonactic acid when the linear tetramer is
converted into a cyclic form. The eight resolved doublets in
the linear tetramer collapse to two doublets showing that in
nonactin there is S4 symmetry and that the four C-2 methyl
groups and the four C-8 methyl groups are degenerate. Similar
1
changes in the H and 13C NMR spectra were observed upon
formation of both all-(-)-nonactin and all-(+)-nonactin. These
data show that there is C4 symmetry, and degeneracy of the
methyl groups, in the non-natural diastereoisomers of nonactin.
+
derived from the heat of dissolution of NH4 in methanol
+
completely swamped out any signal due to NH4 binding that
may, or may not, have been present. The negative entropy
changes on ion binding to nonactin (Table 2) show that entropy
loss from organization of the ionophore around the ion is greater
than the entropy gain from the ion being stripped of solvent.
The large and favorable enthalpy changes upon K+-nonactin
formation derive from the coordination of the carbonyl and
tetrahydrofuran oxygen atoms to the ion. The coordination effect
is less favorable for Na+ than K+, as the cavity in nonactin is
a better fit for K+. In the crystal structure of the nonactin-K+
complex, the K-O distance is close to the sum of the ionic
radii of K+ and O. The better fit for K+ to nonactin is reflected
in the association constant for the nonactin-K+ complex which
is approximately 200-fold larger than that of the nonactin-Na+
complex. The K+-all-(-)-nonactin complex, however, is ap-
proximately 900-fold less stable than the K+-nonactin complex.
The low enthalpy change on formation of the K+-all-(-)-
nonactin complex suggests that only one to two oxygen atoms
likely coordinate to the K+. When combined with the low,
positive entropy change on K+-all-(-)-nonactin complex forma-
tion, the data suggest a lack of a sequential binding pathway
and that all-(-)-nonactin, unlike nonactin, cannot easily fold
around the ion to allow for further coordination and the
formation of a stable complex. The enthalpy change of Na+
binding to all-(-)-nonactin suggests that multiple coordinate
bonds are formed to the Na+ although the association constant
is still low. While the solution structure of all-(-)-nonactin
remains to be determined, it is quite clear from the data that
We did two further experiments in addition to the spectro-
scopic and spectrometric characterization to confirm that we
had prepared all-(-)-nonactin and all-(+)-nonactin. First, we
subjected small samples of all-(-)-nonactin and all-(+)-nonactin
to methanolysis to generate samples of methyl nonactate. The
stereochemical purity of the methyl nonactate was confirmed
by GC analysis using a chiral stationary phase. In each case
the GC analysis demonstrated that only one enantiomer of 8,
and also the expected enantiomer, was present after degradation
thereby confirming that no inadvertent scrambling of synthetic
samples had occurred during the synthesis. Second, conductivity
measurements of methanol solutions of all-(-)-nonactin and all-
(+)-nonactin confirmed that the synthetic samples did not
contain any significant cations, the presence of which might
confound later analysis.
Biological Activity. We measured minimal inhibitory con-
centrations (MIC) for nonactin, all-(-)-nonactin, and all-(+)-
nonactin against a range of Gram positive and Gram negative
bacteria (Table 1). As expected, nonactin showed significant
potency against Gram positive organisms, including drug
resistant forms of the pathogens Staphylococcus aureus and
Enterococcus faecalis. Also, as expected, nonactin showed no
activity against Gram negative organisms or against the
pathogenic fungus Candida glabrata. In the latter cases, true
MIC values could not be obtained as the solubility of nonactin
became limiting. Regardless, it is reasonable that compounds
that show MIC values in excess of 1 mM are classified as
‘inactive’ as such levels of potency have no practical application.
The results for all-(-)-nonactin and all-(+)-nonactin were
(29) Izatt, R. M.; Bradshaw, J. S.; Nielsen, S. A.; Lamb, J. D.; Christensen,
J. J.; Sen, D. Chem. ReV. 1985, 85, 271–339.
9
17158 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Alternating Stereochemistry in the Nonactin Macrocycle
A R T I C L E S
Table 2. Isothermal Calorimetry Data for the Formation of Ionophore-Ion Complexes in Methanol at 298 Ka
nonactin
all-(-)-nonactin
K+
Na+
NH4+
K+
Na+
∆G/kcal·mol-1
∆H/kcal·mol-1
∆S/cal·mol-1 ·K-1
Ka/dm3 ·mol-1
-6.24 ( 0.02
-12.9 ( 0.1
-3.08 ( 0.01
-6.3 ( 0.1
-10.8 ( 0.3
184 ( 4
-6.40 ( 0.01
-17.8 ( 0.7
-38 ( 2
-2.24 ( 0.05
-2.1 ( 0.1
0.45 ( 0.40
44 ( 4
-2.20 ( 0.03
-13.5 ( 0.5
-38 ( 2
-22.4 ( 0.3
(38.6 ( 1.3) × 103
(51.9 ( 0.4) × 103
42 ( 2
a
+
The interaction between NH4 and all-(-)-nonactin could not be determined.
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 counterion via its absorbance at 357 nm. This assay is a modification of
the partition assay used to quantify tetranactin developed by Suzuki et al.30 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).
the non-natural diastereoisomers of nonactin cannot effectively
bind to monovalent cations and that the poor binding is directly
related to their lack of biological activity.
methanol as a solvent, the partition experiment provides an
alternative approach wherein the effects of water can be
evaluated. Typical binding curves were obtained for both
nonactin and all-(-)-nonactin. For equal concentrations of
nonactin and all-(-)-nonactin, the amount of picrate present in
the organic layer was minimally 100-fold less for all-(-)-
nonactin than for nonactin. The data match well with the relative
association constants for the two complexes that we obtained
in methanol solution. The difference in ion binding and
subsequent partitioning is demonstrated in the inset photograph
in Figure 3 where two vials are identically set up with the
exception that one contains nonactin (3A) and one contains all-
(-)-nonactin (3B). The lack of picrate color in the lower CHCl3
phase is quite evident when all-(-)-nonactin is the ionophore.
Partitioning Assay. We regularly use a partitioning assay to
quantify the amount of nonactin that is formed in fermentative
cultures of S. griseus.30 The assay relies upon the ability of the
nonactin-K+ complex to partition into the organic phase of an
organic-aqueous biphasic system. As the complex is formed
and moves into the organic phase, a counterion must follow to
maintain electric neutrality. Commonly, the counterion used is
picrate which imparts a distinct yellow color to the organic layer
(Figure 3A). Conventionally, the assay is used to quantify
nonactin concentrations, as the amount of picrate found in the
organic layer, quantified through UV spectroscopy, is propor-
tional to the amount of nonactin present. We adapted the assay
to obtain a standard curve for both nonactin and all-(-)-nonactin
(Figure 3). As our microcalorimetry data were obtained using
Transport Assay. At this point we have demonstrated that
the lack of biological activity of the non-natural diastereoisomers
of nonactin is most likely due to their inability to form
complexes with monovalent cations. The data we have, however,
are purely thermodynamic even though the data are consistent
(30) Suzuki, K.; Nawata, Y.; Ando, K. J. Antibiot. 1971, 24, 675.
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17159

A R T I C L E S
Kusche et al.
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 cotransport of the picrate anion. Fit lines were calculated by nonlinear
least-squares regression to a simple rate equation modeling a first-order approach to an equilibrium, A357(t) ) k1(1 - exp[-k2t]). (b) Nonactin as the
ionophore; k1 ) 1.8, k2 ) 0.0009. (O) All-(-)-nonactin as the ionophore; k1 ) 1.9, k2 ) 0.100.
in both methanol and water-CHCl3. To round out our data we
sought to observe kinetic differences in the transport of
monovalent ions by nonactin and its non-natural diastereoiso-
mers. There is a substantial body of literature on the kinetics
of nonactin-mediated ion transport through black lipid mem-
branes, the latter being used as a surrogate for cell membranes.31,32
For our purpose, however, there is a simpler experimental
system that can mimic a cell membrane and ion transport. It
has been demonstrated that ionophores can mediate the move-
ment of K+ between two aqueous phases that are separated by
a common organic layer.33-35 The K+-nonactin complex that
forms in one aqueous phase can partition into the organic layer.
Since the bulk organic solvent is being mechanically stirred,
there is a uniform concentration of K+-nonactin complex that
can then partition into the second aqueous layer and dissociate.
The free nonactin can move back through the organic phase to
form further K+-nonactin complex in the first aqueous phase.
Overall, the process results in the bulk transport of K+ from
one aqueous phase to the other if they have different initial K+
concentrations. The transport process can be followed by
monitoring cotransport of picrate as the counterion. We used
this approach to measure the relative rates of K+ transport that
could be mediated by nonactin and all-(-)-nonactin (Figure 4).
The rate of K+ transport between the two aqueous phases is
dependent upon the K+ concentration difference between them.
Furthermore, as K+ is transported, the concentration difference
decreases; the process can be conveniently modeled by a first-
order approach to an equilibrium. In this system, with all else
other than the identity of the ionophore being equal, nonactin
can support transport rates that are minimally 100-fold higher
than those mediated by all-(-)-nonactin. It is noteworthy that
the K+ transport rate with all-(-)-nonactin is not zero. Although
the transport rate with all-(-)-nonactin is much lower than that
with nonactin, the end point, the equilibrium distribution of K+
and picrate, is the same. The difference between the equilibrium
values of A357 of 1.8 and 1.9 (Figure 4) is a result of the large
extrapolation needed to calculate the equilibrium value when
the K+ transport rates are low in the case of all-(-)-nonactin.
Molecular Modeling. To gain insight into the structural
differences between nonactin (1) and all-(-)-nonactin (17) in
solution we used molecular dynamics to simulate their potassium
complexes using an explicit water solvent model. Our work was
guided by molecular dynamics studies of nonactin by Merz12
and by our own previous studies of macrotetrolide selectivity.36
Nonactin retains a tight, approximately cubic coordination to
the K+ ion throughout the simulation. The K+-carbonyl oxygen
distances (Figure 5) are stable (2.90 ( 0.30 Å) as are the K+-
tetrahydrofuran oxygen distances (3.60 ( 0.42 Å) for nonactin.
The all-(-)-nonactin K+ complex has a much less rigid structure.
The K+-carbonyl oxygen distances (Figure 5) (3.73 ( 0.86 Å)
and the K+-tetrahydrofuran oxygen distances (3.50 ( 0.98 Å)
show much more variation than similar distances in nonactin.
Significantly, in the nonactin-K+ complex the hydrogen atoms
at C3 and C6 of each monomer are on the inside of the structure
‘facing’ the cation. In all-(-)-nonactin only the H-3 and H-6
(31) van Dijk, C.; de Levie, R. Biophys. J. 1985, 48, 125–136.
(32) Hall, J. E.; Latorre, R. Biophys. J. 1976, 16, 99–103.
(33) Burger, H. M.; Seebach, D. HelV. Chim. Acta 2004, 76, 2570–2580.
(34) Shinji, T.; Seno, M. J. Phys. Chem. A 1994, 1994, 1682–1688.
(35) Tajima, I.; Okada, M.; Sumitomo, H. J. Am. Chem. Soc. 1981, 103,
4096–4100.
(36) Lee, J. W.; Priestley, N. D. Bioorg. Med. Chem. Lett. 1998, 1725–
1728.
9
17160 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Alternating Stereochemistry in the Nonactin Macrocycle
A R T I C L E S
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 (B) that are absent in the natural diastereoisomer, nonactin. For
nonactin, H-3 and H-6 face inward toward the cation; for all-(-)-nonactin, H-3 and H-6 face outward toward solvent. 3, Distribution of K+-tetrahydrofuran
oxygen distances for nonactin (blue) and all-(-)-nonactin (red) obtained from molecular dynamics simulations.
hydrogen atoms of two nonadjacent monomer units are inside
the structure; the two other H-3 and H-6 sets face out toward
the solvent. The structure becomes distorted so that poor steric
interactions between a tetrahydrofuran ring and a carbonyl group
on the other edge of the closing macrocycle are avoided. The
steric interaction prevents the macrocycle from closing to give
sufficiently close contacts between the coordinating oxygen
atoms and the central cation. Only if the stereochemistry of the
monomer units alternates around the macrocycle, as is found
in nonactin, can a conformation be accessed where all the H-3
and H-6 atoms are on the inside face of the macrocycle allowing
for a tight complex to be formed.
and proceeded in high yield. Formation of the non-natural
nonactin diastereoisomers was not noticeably slower than
formation of nonactin. These data strongly suggest that the
folding of precursor species plays no significant part in the
observed stereochemistry of the natural product.
If the natural diastereoisomer of nonactin was more active
than nonactin assembled from homochiral nonactic acid, how
much more active would it be? We were surprised that the
differences in biological activity of nonactin and its non-natural
diastereoisomers were so clear (Table 1), as nonactin was
minimally 100-fold more active. Should S. griseus derive a
benefit from the antibiotic activity of nonactin, then the large
difference in activity between the natural and non-natural
diastereoisomers can readily account for the evolution, and
maintenance, of parallel biosynthesis pathways.
Discussion
The stereochemistry of nonactin is unique among natural
products in that the producing organism must generate both
enantiomers of nonactic acid. It is clear from analysis of the
nonactin biosynthesis gene cluster that there are separate,
enantiocomplementary pathways to each enantiomer of nonactic
acid and that there are parallel sets of biosynthesis enzymes.
The stereochemical course of nonactin biosynthesis is complex,
and this complexity has been maintained even against natural
genetic drift. There is most likely a benefit to the producing
organism in generating the achiral diastereoisomer of nonactin
that outweighs the cost of running parallel biosynthesis path-
ways. At the outset we had two hypotheses. First, it would be
quite conceivable that nonactin would be more active than all-
(+)-nonactin or all-(-)-nonactin; however, the relative activity
of the diastereoisomers was open to question. Second, we
thought that during the late stages of nonactin biosynthesis there
might be substantial differences in the manner in which (+)-
(-)-(+)-(-) and (-)-(-)-(-)-(-) linear tetramers of nonactic
acid could fold that might be reflected in the synthesis of the
natural diastereoisomer being the more efficient process. The
data that we obtained settle both of these questions.
The antibacterial activity of nonactin derives from its ability
to form stable complexes with K+, Na+, and NH4+. Since all-
(-)-nonactin and all-(+)-nonactin are not active against the set
of Gram negative bacteria we tested, it was a logical step to
determine if they were able to form ion complexes; the
microcalorimetry data were convincing that they could not to
any appreciable degree. The association constant, Ka, for the
K+-nonactin complex in methanol was found to be ap-
proximately 900-fold higher than that of the K+-all-(-)-nonactin
complex (Table 2). Similarly, nonactin allowed for approxi-
mately 100-fold greater partitioning of potassium picrate into
CHCl3 than all-(-)-nonactin (Figure 3). The data show a clear
thermodynamic advantage for the natural product.
While the antibacterial activity and thermodynamic data show
that the natural product is favored, we chose a simple transport
assay to obtain comparative kinetic data. Again, the natural
product was superior. Rates of K+ transport were over 100-
fold higher when nonactin was used as the ionophore than when
all-(-)-nonactin was used. No significant transport was observed
without ionophore being present, and it was noteworthy that
transport rates, although very low (Figure 4), were reliably
measured for all-(-)-nonactin. Overall, the binding of K+ to
all-(-)-nonactin is poor but sufficiently large so that transport
rates can be measured; however, the extent of the binding and
The relative efficiency by which the folding of linear
tetrameric species to form each of the nonactin diastereoisomers
became apparent during the macrolactonization reactions that
we used to form the latter. Each of the reactions was efficient
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17161

A R T I C L E S
Kusche et al.
Methyl (-)-Nonactate ((-)-8). A 4 day, 10 L fermentation of
Streptomyces griseus ∆nonD was clarified by centrifugation. The
clarified broth was acidified to pH 2 by the addition of conc. aq.
HCl. The broth was extracted twice with an equal volume of EtOAc.
The combined EtOAc extracts were dried over MgSO4, filtered,
and concentrated to yield a crude oil. (-)-Nonactic acid was isolated
from the oil by chromatography on silica gel, eluting with
EtOAc-hexanes-AcOH, as a colorless oil (3.1 g). The (-)-
nonactic acid was converted into (-)-8 by Fisher esterification and
was shown to be identical to an authentic standard (>98% ee by
GC18).
transport mediation are not sufficient to impart significant
biological activity.
We used molecular dynamics to give us some structural
insight into the structures of the all-(-)-nonactin and nonactin
K+ complexes. Only in nonactin, with its alternating stereo-
chemistry, can conformations where the H-3 and H-6 atoms of
each tetrahydrofuran ring face the center of the complex be
accessed. If two monomer units are inverted, as is the case with
all-(-)-nonactin, two tetrahydrofuran rings have poor steric
interactions with a carbonyl group (Figure 5) on the other edge
of the molecule, preventing tight closure around the cation. Such
modeling data allow us to account for the activity and affinity
differences between nonactin and our non-natural diastereoi-
somers.
(R)-2-((2R,5S)-5-((S)-2-(tert-Butyldimethylsiloxy)propyl)tet-
rahydrofuran-2-yl)propanoic Acid ((+)-9). Imidazole (1.3 g, 19.5
mmol) and TBSCl (1.5 g, 9.8 mmol) were added to a stirred solution
of (-)-methyl nonactate, (-)-8 (0.85 g, 3.9 mmol), in DMF (10
mL), and stirring continued at ambient temperature for 2 h. The
reaction was quenched by being poured into water (50 mL); the
resulting aqueous solution was then extracted with Et2O (3 × 50
mL). The organic phases were combined and concentrated to a crude
oil. Chromatography on silica gel eluting with EtOAc/hexanes (1:
9) gave an intermediate TBS-protected ester as a clear oil (1.24 g,
93%): [R]D25 +11 (c 0.90, CHCl3); δH (400 MHz, CDCl3) 0.03 (d,
J ) 2.2 Hz, 6H), 0.87 (s, 9H), 1.10 (d, J ) 7 Hz, 3H), 1.11 (d, J
) 5.9 Hz, 3H), 1.40-1.60 (m, 4H) 1.94 (m, 2H), 2.5 (m, 2H) 3.68
(s, 1H), and 3.84-4.00 (m, 3H); δC (100 MHz, CDCl3) -4.95,
-4.65, 13.44, 18.07, 24.61, 25.88, 28.71, 31.29, 45.53, 46.01, 51.52,
66.05, 76.16, 80.25, and 175.36. In a similar manner, TBS
protection of (+)-8 (0.26 g, 1.2 mmol) gave the intermediate
Summary
We have successfully prepared two non-natural diastereoi-
somers of nonactin, one assembled from (+)-nonactic acid and
one from (-)-nonactic acid. We demonstrated that the latter
diastereoisomers are significantly less potent as antibacterial
agents than the natural product, nonactin. As nonactin is an
ionophore, we determined the ability of our analogues to bind
to K+, Na+, and NH4+. The microcalorimetry data showed that
no significant binding to non-natural nonactin diastereoisomers
could be achieved. In the case of K+ it is likely that only one
to two carbonyl groups can complex with the ion but, unlike
nonactin, this first association does not lead to a stepwise
wrapping of the ionophore around the ion. To complement the
microcalorimetry data, we further demonstrated that all-(-)-
nonactin was ineffective at partitioning K+ into an organic
solvent and only poorly mediated K+ transport through a bulk
organic phase. Modeling data suggest that the alternating
stereochemistry of the monomer units in nonactin allows for
the THF rings to adopt equivalent conformations where the
macrocycle can close efficiently around the cation. Although
the stereochemistry of nonactin biosynthesis is complex, and
S. griseus has a unique system with enantiocomplementary
biosynthesis pathways, our activity, thermodynamic, and kinetic
data all show that there is a likely benefit to S. griseus in having
such a superbly complicated biosynthesis pathway.
25
protected ester (0.36 g, 90%): [R]D -26° (c 0.52, CHCl3). A
solution of intermediate ester (1.1 g, 3.2 mmol) in THF (13 mL),
MeOH (4.4 mL), and aqueous KOH (5 M, 4.4 mL) was stirred at
ambient temperature for 16 h. The reaction was then concentrated;
the resultant aqueous solution was further diluted with water (25
mL), acidified with 10% citric acid, and extracted with Et2O (4 ×
25 mL). The organic phases were combined and washed with water
and brine, dried, and then concentrated to give (+)-9 as a clear oil
(0.95 g, 93% yield): [R]D25 +28° (c 0.75, CHCl3). Analytical data
were equivalent to the known compound.24 In a similar manner,
deprotection of the antipode (0.30 g, 0.9 mmol) gave (-)-9 (0.28
g, 96%); HRMS (ESI) m/z calcd for C16H32NaO4Si: 339.1968; found
339.1963 ([M + Na]+, ∆ppm 1.7).
(2R)-2-((2R,5S)-5-((2S)-2-Hydroxypropyl)tetrahydrofuran-2-yl)-
propanoic Acid; Nonactic Acid ((-)-10). A solution of (-)-8 (0.85
g, 3.9 mmol, [R]D25 -31° (c 0.29, CHCl3)) in MeOH (7.5 mL) and
aqueous KOH (2 M, 15 mL) was stirred for 2 h at ambient
temperature. The reaction was concentrated and diluted with water
(75 mL), acidified with aqueous 1 M HCl, and finally extracted
with EtOAc (3 × 25 mL). The organic phases were combined,
dried, and concentrated to give (-)-nonactic acid (-)-10 as a
colorless oil (0.49 g, 62% yield): analytical data were equivalent
to the known compound.24 In a similar manner, (+)-8 (0.30 g, 1.39
Experimental Section
General Procedures. Solvents were obtained from Fisher
Scientific. All other chemicals, unless noted otherwise, were
obtained from the Aldrich Chemical Co. (Milwaukee, WI). The
solvents MeOH (over Mg(OMe)2), CH2Cl2 (over CaH2), diethyl
ether (over Na/K-benzophenone), and THF (over Na/K-benzophe-
none) were dried and distilled prior to use. MgSO4 refers exclusively
to anhydrous MgSO4. Solutions were concentrated by evaporation
in vacuo. All synthesis procedures, unless noted otherwise, were
carried out under a slight positive pressure of argon gas at ambient
temperature (21-25 °C). Column chromatography was performed
using Merck Silica Gel 60. NMR spectra were acquired at 400 MHz
(1H) and 100 MHz (13C), were referenced to the residual solvent,
and are reported as follows: chemical shift (δ/ppm), splitting pattern,
coupling constant (J/Hz), and intensity. Nonactin was obtained from
Promiliad Biopharma Inc. (Alberton, MT).
25
mmol, [R]D +30.6° (c 0.29, CHCl3)) was used to prepare (+)-
nonactic acid (+)-10 (0.25 g, 89%).
(R)-Benzyl 2-((2R, 5S)-5-((S)-2-Hydroypropyl)tetrahydrofuran-
2-yl)propanoate ((-)-11). A solution of (-)-nonactic acid, (-)-10,
(0.49 g, 2.4 mmol) in dry DMF (30 mL) was treated with tert-
BuOK and stirred at 60 °C for 30 min under an atmosphere of
argon. Benzyl bromide (0.62 g, 3.6 mmol) was added to the stirred
solution by syringe, and the reaction was maintained at 60 °C for
1.5 h. The reaction was quenched by being poured into water, and
the resulting aqueous solution was brought to pH 2 with 1 M HCl
and extracted with Et2O (4 × 20 mL). The organic layers were
combined, dried, and concentrated. The crude oil was purified by
chromatography on silica gel eluting with EtOAc/hexanes (1:1) to
Methyl (()-Nonactate ((()-8). Methyl (()-nonactate was
obtained through methanolysis of nonactin as described by Ash-
worth et al.,13 adapted for a large scale as described by Dinges et
al.26
25
give (-)-11 as a clear oil (0.51 g, 72%): [R]D -6.8° (c 1.10,
Methyl (+)-Nonactate ((+)-8). Methyl (+)-nonactate was
obtained using a Rhodococcus-mediated resolution of racemic
methyl nonactate in >98% ee as described by Nikodinovic et al.27
and was shown to be identical to an authentic standard.
CHCl3); δH (400 MHz, CDCl3) 1.14 (d, J ) 7.0 Hz, 3H), 1.17 (d,
J ) 6.2 Hz, 3H), 1.52-1.72 (m, 4H), 1.97 (m, 2H), 2.59 (m, 1H),
2.71 (s, 1H), 3.93-4.16 (m, 3H), 5.14 (dd, J ) 2.2 and 12.5 Hz,
2H), and 7.27-7.40 (m, 5H); δC (100 MHz, CDCl3) 13.39, 23.23,
9
17162 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Alternating Stereochemistry in the Nonactin Macrocycle
A R T I C L E S
28.60, 30.60, 42.97, 45.30; 65.08, 66.12, 76.99, 80.78, 128.00,
128.06, 128.39, 135.99, and 174.50. In a similar manner, esterifi-
cation of (+)-10 (0.21 g, 1.0 mmol) gave (+)-11 (0.256 g, 84%).
(R)-Benzyl 2-((2R, 5S)-5-((S)-2-((R)-2-((2R,5S)-5-((S)-2-(tert-Bu-
tyldimethylsilyloxy)propyl) Tetrahydrofuran-2-yl)propanoyloxy)-
propyl)tetrahydrofuran-2-yl)propanoate ((+)-12). To a solution of
acid (+)-9 (0.55 g, 1.8 mmol) and alcohol (-)-11 (0.51 g, 1.8
mmol) in dry CH2Cl2 (10 mL) was added DCC (0.38 g, 1.8 mmol)
followed by DMAP (0.06 g, 0.5 mmol). The mixture was stirred
under argon for 16 h and then concentrated prior to being
resuspended in Et2O and filtered, and the filtrate was then
concentrated. The crude oil was purified by flash chromatography
on silica gel eluting with EtOAc/hexanes (1:9) to give (+)-12 as a
clear oil (0.64 g, 64%): [R]D25 +9.8° (c 1.00, CHCl3); δH (400 MHz,
CDCl3) 0.03 (s, 6H), 0.87 (s, 9H), 1.07 (d, J ) 7.0, 3H), 1.11 (d,
J ) 5.9 Hz, 3H), 1.12 (d, J ) 7.0 Hz, 3H), 1.21 (d, J ) 6.6 Hz,
3H), 1.35-1.83 (m, 8H), 1.84-2.03 (m, 4H), 2.5 (q, J ) 7.0 Hz,
1H), 2.6 (q, J ) 7.0 Hz, 1H), 3.82-4.06 (m, 5H), 4.96 (sextet, J
) 6.2 Hz, 1H), 5.12 (d, J ) 12.5 Hz, 1H), 5.16 (d, J ) 12.5 Hz,
1H), and 7.27-7.39 (m, 5H); δC (100 MHz, CDCl3) -4.86, -4.59,
13.15, 13.26, 18.04, 20.60, 24.63, 25.87, 28.33, 28.41, 31.34, 31.45,
42.42, 45.32, 45.40, 46.07, 66.08, 66.20, 69.18, 76.24, 76.60, 80.12,
80.25, 128.01, 128.44, 136.13, 174.30, and 174.62; IR (film-ATR):
2953, 2930, 2856, 1732, 1461, 1376, 1254, 1188, 1155, 1055, and
835 cm-1; HRMS (ESI) m/z calcd. for C33H55O7Si: 591.3717; found
591.3712 ([M + H]+, ∆ppm 0.9). In a similar manner, coupling of
(-)-9 (0.24 g, 0.8 mmol) and (+)-11 (0.23 g, 0.8 mmol) gave (-)-
([M + H]+, ∆ppm 2.3). In a similar manner, deprotection of (-)-
12 (0.14 g, 0.23 mmol) gave (+)-14 (0.083 g, 76%): [R]D25 +2.9°
(c 0.41, CHCl3).
(S)-Benzyl 2-((2R,5S)-5-((S)-2-((R)-2-((2R,5S)-5-((S)-2-(((R)-
2-(((2R,5S)-5-((S)-2-((R)-2-((2R,5S)-5-((S)-2-(tert-Butyldimeth-
yl silyloxy)propyl)tetrahydrofuran-2-yl)propanoyloxy)propyl)-
tetrahydrofuran-2-yl)propanoyloxy)propyl)tetrahydrofuran-2-
yl)propanoyloxy)propyl)tetrahydrofuran-2-yl)propanoate ((+)-
15). To a stirred solution of acid (+)-13 (0.16 g, 0.32 mmol),
alcohol (-)-14 (0.16 g, 0.34 mmol), and DMAP (0.18 g, 1.44
mmol) in dry CH2Cl2 under argon 2,4,6-trichlorobenzoyl chloride
(0.16 g, 0.64 mmol) was added by syringe. The reaction was
allowed to stir for 21 h and then concentrated to give a yellow
semisolid. The residue was dissolved in EtOAc, and the organic
phase was washed with 10% citric acid, sat. NaHCO3, and then
brine, dried, and then concentrated. The crude oil obtained was
purified by chromatography on silica gel eluting with EtOAc/
25
hexanes (1:3) to give (+)-15 as a clear oil (0.31 g, 99%): [R]D
+8.0° (c 0.75, CHCl3); δH (400 MHz, CDCl3) 0.03 (s, 6H), 0.87
(s, 9H), 1.07 (d, J ) 7.0 Hz, 9H), 1.11 (d, J ) 6.2 Hz, 3H), 1.12
(d, J ) 7.0 Hz, 3H), 1.18-1.24 (m, 9H), 1.38-2.02 (m, 24H),
2.40-2.60 (m, 4H), 3.80-4.06 (m, 8H), 4.96 (m, 3H), 5.12 (d, J
) 12.5 Hz, 1H), 5.16 (d, J ) 12.5 Hz, 1H), and 7.28-7.38 (m,
5H); δC (100 MHz, CDCl3) -4.86, -4.58, 12.92, 12.97, 13.14,
13.26, 20.57, 20.60, 20.63, 24.62, 25.87, 28.13, 28.34, 28.43, 31.31,
31.37, 31.40, 31.43, 42.37, 45.21, 45.26, 45.30, 45.42, 46.07, 66.07,
66.19, 69.21, 69.25, 69.28, 76.23, 76.44, 76.48, 76.57, 80.12, 80.22,
80.25, 127.98, 128.01, 128.44, 136.13, 174.20, 174.24, 174.27, and
174.64; IR (film-ATR): 2973, 2937, 2882, 1731, 1460, 1377, 1255,
1190, 1054, and 540 cm-1; HRMS (APCI) m/z calcd for
C53H90NO13Si: 976.6181; found 976.6158 ([M + NH4]+, ∆ppm 2.4).
In a similar manner, coupling of (-)-13 (0.06 g, 0.12 mmol) and
(+)-14 (0.06 g, 0.13 mmol) gave (-)-15 (0.12 g, 99%): [R]D25 -21°
(c 0.18, CHCl3); HRMS (ESI) m/z calcd for C53H86NaO13Si:
981.5735; found 981.5737 ([M + Na]+, ∆ppm 0.2).
25
12 (0.28 g, 62%): [R]D -22° (c 0.29, CHCl3).
(R)-2-((2R,5S)-5-((S)-2-((R)-2-((2R,5S)-5-((S)-2-(tert-Butyldimeth-
ylsilyloxy)propyl)tetrahydrofuran-2-yl)propanoyloxy)propyl)tet-
rahydrofuran-2-yl)propanoic Acid ((+)-13). A mixture of (+)-12
(0.31 g, 0.53 mmol) and 10% Pd/C (0.03 g) in THF (10 mL) was
stirred under 1 atm of hydrogen for 16 h. The mixture was filtered
through Celite, washing with EtOAc, and the filtrate and washings
25
were concentrated to give (+)-13 as a clear oil (0.25 g, 93%): [R]D
+20° (c 0.36, CHCl3); δH (400 MHz, CDCl3) 0.031 (s, 6H), 0.87
(s, 9H), 1.08 (d, J ) 7.0 Hz, 3H), 1.11 (d, J ) 6.2 Hz, 3H), 1.17
(d, J ) 7.3 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.38-2.10 (m,
12H), 2.38-2.57 (m, 2H), 3.84-4.02 (m, 5H), and 5.00 (sextet, J
) 6.2 Hz, 1H); δC (100 MHz, CDCl3) -4.86, -4.57, 13.21, 13.27,
18.04, 20.45, 24.61, 25.87, 28.38, 29.02, 31.12, 31.42, 42.22, 44.82,
45.41, 46.08, 66.23, 68.78, 76.32, 80.14, 174.37, and 177.62; IR
(film-ATR): 2956, 2934, 2857, 1732, 1711, 1054, 835, 774, and
540 cm-1; HRMS (ESI) m/z calcd for C26H49O7Si: 501.3248; found
501.3242 ([M + H]+, ∆ppm 1.2). In a similar manner, hydro-
genolysis of (-)-12 (0.13 g, 0.22 mmol) gave (-)-13 (0.10 g, 91%):
(S)-2-((2R,5S)-5-((S)-2-((R)-2-((2R,5S)-5-((S)-2-((R)-2-(2R,5S)-5-
((S)-2-((R)-2-((2R,5S)-5-(((S)-2-hydroxypropyl)tetrahydrofuran-2-
yl)propanoyloxy)propyl)tetrahydrofuran-2-yl)propanoyloxy)pro-
pyl)tetrahydrofuran-2-yl)propanoyloxy)propyl)tetrahydrofuran-2-
yl)propanoic acid ((+)-16). Ester (+)-15 (0.25 g, 0.26 mmol) was
stirred with 10% Pd/C (0.025 g) in THF (5 mL) under 1 atm of
hydrogen for 20 h. The reaction was filtered through Celite which
was washed with EtOAc. The combined filtrates were concentrated
to give the intermediate TBS-protected acid as a clear oil (0.23 g,
25
99%): [R]D +13° (c 0.73, CHCl3); δH (400 MHz, CDCl3) 0.03
25
[R]D -25° (c 0.42, CHCl3); HRMS (ESI) m/z calcd for
(s, 6H), 0.86 (s, 9H), 1.05-1.09 (m, 9H), 1.11 (d, J ) 6.2 Hz,
3H), 1.16 (d, J ) 7.0 Hz, 3H), 1.23 (m, 9H), 1.38-2.08 (m, 25H),
2.41-2.55 (m, 4H), 3.81-4.03 (m, 9H), and 4.90-5.06 (m, 3H);
δC (100 MHz, CDCl3) -4.85, -4.58, 12.99, 13.14, 13.17, 13.28,
18.05, 20.40, 20.64, 24.63, 25.87, 28.17, 28.34, 29.09, 31.09, 31.31,
31.38, 31.44, 42.23, 42.38, 42.45, 45.26, 45.31, 45.63, 46.07, 66.21,
68.79, 69.22, 69.30, 76.15, 76.27, 76.39, 80.12, 80.19, 80.25, 80.35,
174.23, and 174.32; ESI-MS m/z 891.2 ([M + Na]+). The
intermediate TBS-protected acid (0.17 g, 0.19 mmol) and p-TsOH
monohydrate (0.005 g, 0.13 mmol) were stirred in AcOH (1.3 mL)
and water (0.14 mL) for 1 h at ambient temperature. The reaction
was diluted with water (30 mL) and extracted with EtOAc (4 × 20
mL). The organic phases were combined, dried, and concentrated.
The crude oil obtained was purified by chromatography on silica
gel eluting with EtOAc/MeOH (98:2) followed by EtOAc/MeOH/
C26H49O7Si: 501.3248; found 501.3251 ([M + H]+, ∆ppm 0.6).
(R)-Benzyl 2-((2R,5S)-5-((S)-2-((R)-2-((2R,5S)-5-((S)-2-Hydrox-
ypropyl)tetrahydrofuran-2-yl)propanoyloxy)propyl)tetrahydrofuran-
2-yl)propanoate ((-)-14). A solution of dimer (+)-12 (0.32 g, 0.54
mmol) and p-TsOH monohydrate (0.02 g, 0.09 mmol) in acetic
acid (2.5 mL) and water (0.27 mL) was stirred at ambient
temperature for 30 min. The reaction was diluted with water (15
mL) and extracted with Et2O (3 × 30 mL). The organic phases
were combined and washed with aqueous sat. NaHCO3 and brine,
dried, and then concentrated. The crude oil obtained was purified
by chromatography on silica gel eluting with EtOAc/hexanes (1:1)
25
to give (-)-14 as a clear oil (0.16 g, 62%): [R]D -3.4° (c 0.26,
CHCl3); δH (400 MHz, CDCl3) 1.09 (d, J ) 7.3 Hz, H), 1.11 (d, J
) 7.3 Hz, 3H), 1.19 (d, J ) 6.2 Hz, 3H), 1.21 (d, J ) 6.2 Hz, 3H),
1.43-1.81 (m, 8H), 1.87-2.03 (m, 4H), 2.42-2.62 (m, 2H) 3.81,
4.16 (m, 5H), 4.98 (m, 1H), 5.12 (d, J ) 12.5 Hz, 1H), 5.16 (d, J
) 12.5 Hz, 1H), and 7.28-7.39 (m, 5H); δC (100 MHz, CDCl3)
13.27, 13.36, 20.54, 23.17, 28.42, 28.70, 30.59, 31.32, 42.38, 42.76,
45.42, 45.49, 65.15, 66.11, 69.41, 76.41, 80.29, 81.11, 128.03,
128.44, 136.12, 174.22, and 174.69; IR (ATR-film): 3403, 2973,
25
AcOH (95:4:1) to give (+)-16 as a clear oil (0.14 g, 95%): [R]D
+3.0° (c 0.86, CHCl3); δH (400 MHz, CDCl3) 1.03-1.26 (m, 24H),
1.44-2.05 (m, 24H), 2.38-2.55 (m, 4H), 3.80-4.18 (m, 9H), and
4.90-5.05 (m, 3H); δC (100 MHz, CDCl3) 13.03, 13.15, 13.18,
13.35, 20.39, 20.54, 20.59, 23.09, 28.20, 28.32, 28.67, 28.87, 30.55,
31.11, 31.28, 42.21, 42.32, 42.40, 42.77, 44.77, 45.31, 45.48, 45.59,
65.13, 68.92, 69.32, 69.40, 76.25, 76.37, 76.86, 76.90, 80.09, 80.30,
80.35, 81.10, 174.21, and 174.36; IR (ATR-film): 3406, 2974, 2936,
2936, 1730, 1457, 1378, 1188, 1165, 1087, 1056, and 540 cm-1
HRMS (ESI) m/z calcd for C27H41O7: 477.2852; found 477.2863
;
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17163

A R T I C L E S
Kusche et al.
1728, 1460, 1377, 1192, 1054, and 539 cm-1; HRMS (ESI) m/z
calcd. for C40H66NaO13: 777.4401; found 777.4389 ([M + Na]+,
∆ppm 1.5). In a similar manner, double-deprotection of (-)-15
(0.093 g, 0.13 mmol) gave (-)-16 (0.043 g, 60% overall for two
thiocyanate solution was titrated into the macrolide solution.
Controls were performed by titrating the metal thiocyanate solution
into the cell containing only methanol. The reference cell for all
experiments contained MeOH. For measurements with NaSCN an
initial 3.0 µL injection was made prior to the standard series of 29
× 10.0 µL injections to attenuate the first titration point. Similarly
for measurements with KSCN an initial 1.0 µL injection was
followed by an additional 29 × 3.0 µL injections for each titration.
Reliable ITC data were not forthcoming from measurements
involving all-(-)-nonactin with NaSCN and NH4SCN due to poor
25
steps): [R]D -6° (c 0.10, CHCl3); HRMS (ESI) m/z calcd for
C40H67O13: 755.4582; found 755.4595 ([M + H]+, ∆ppm 1.7).
All-(-)-nonactin ((-)-17). To a stirred mixture of the hydroxy-
acid tetramer (+)-16 (0.067 g, 0.089 mmol), DMAP (0.044 g, 0.36
mmol), and 4 Å powdered molecular sieves (0.9 g) in dry CH2Cl2
(31 mL) 2,4,6-trichlorobenzoyl chloride (0.031 g, 0.125 mmol) was
added by syringe. The reaction was allowed to stir for 20 h at
ambient temperature. The reaction was then filtered, and the filtrate
was washed with dilute HCl, sat. NaHCO3, and brine. The filtrate
was dried and concentrated to afford a crude oil which was purified
by chromatography on silica gel eluting with EtOAc/hexanes (1:3)
to give (-)-17 as a clear oil (0.045 g, 68%): [R]D25 -8.8° (c 0.28,
CHCl3); δH (400 MHz, CDCl3) 1.07 (d, J ) 7.0 Hz, 12H), 1.25 (d,
J ) 6.2 Hz, 12H), 1.45-1.85 (m, 16H), 1.88-2.04 (m, 8H),
2.36-2.50 (m, 4H), 3.80-4.00 (m, 8H), and 4.90-5.02 (m, 4H);
δC (100 MHz, CDCl3) 13.61, 20.65, 28.66, 31.37, 42.46, 45.97,
69.64, 76.52, 80.75, and 174.18; HRMS (ESI) m/z calcd for
C40H65O12: 737.4476; found 737.4475 ([M + H]+, ∆ppm 0.4). In
a similar manner, cyclization of (-)-16 (0.042 g, 0.56 mmol) gave
(+)-17 (0.034 g, 82%): [R]D25 +7.7° (c 0.36, CHCl3); HRMS (ESI)
m/z calcd. for C40H65O12: 737.4476; found 737.4481 ([M + H]+,
∆ppm 0.7).
GC Analyses. To a solution of (-)-17 (1 mg) in anhydrous
MeOH (1 mL) in a sealable four dram vial was added conc. H2SO4
(50 µL). The vial was sealed and heated in a J-KEM reflux-reaction
block (lower temperature, 65 °C; upper temperature 10 °C) for 48 h.
After cooling to room temperature the reaction was quenched by
the addition of water (0.25 mL), aq. NaOH (4 M, 0.25 mL), and
aq. sat. NaHCO3 (0.5 mL) followed, finally, by EtOAc (1.0 mL).
The resulting mixture was shaken and allowed to separate; the upper
EtOAc layer was analyzed by GC employing a chiral stationary
phase. A Shimadzu GC-17A GC with flame ionization detection
was used, fitted with a Supelco ꢀ-DEX 110 (30 m × 0.25 mm, df
0.25 µm) column. The chromatogram was obtained by injecting 1
µL of the sample solution with a gas flow rate set at 1.5 mL/min
and a split ratio of 30:1. The injector, oven, and detector were set
at 220, 135, and 300 °C, respectively. The retention times under
these conditions were as follows: methyl (-)-nonactate, 49.5 min;
methyl (+)-nonactate, 50.7 min. The sample of all-(-)-nonactin
contained >97% (-)-methyl nonactate.
Antibacterial/Antifungal Assays. Antibacterial and antifungal
potency was measured in 96-well plate-based microbroth dilution
assays. Test compounds were prepared as stock solutions in DMSO
(50 mM) and stored at -20 °C until used. Each compound was
diluted in a 2-fold dilution series, and a small sample (1 µL) of
each was added to wells in a test plate so that each column contained
the dilution series for one compound. An inoculum (∼ 1 × 105
cfu) of a test organism in culture media (100 µL) was added to
each well resulting in a dilution series running from 500 to 2 µM.
Where necessary the measurements were repeated at lower con-
centrations of the test compound. After an incubation period
determined from the strain-specific doubling time, Alamar blue (10
µL) was added and allowed to incubate; each well was scored for
dye reduction.37 The MIC value was taken as the lowest concentra-
tion of test compound that inhibits growth such that less than 1%
reduction of the blue resazurin (λmax 570 nm) component of the
Alamar blue to the pink resorufin (λmax 600 nm) was observed.
Isothermal Calorimetry Measurements and Analysis. All ex-
periments were performed using the VP-ITC system from Microcal,
LLC. Macrolides and salt solutions (NaSCN, KSCN, and NH4SCN)
were prepared in dry MeOH, and all samples were degassed with
helium before each experiment. The ITC cell and syringe temper-
atures were set at 25 °C, and for all experiments the metal
binding in the case of Na+ and both poor binding and a large heat
+
of dissolution in the case of NH4
.
Ionophore-Mediated Partitioning Assay. As a measure of
ion-ionophore binding we performed a partitioning assay described
originally for tetranactin by Suzuki et al.30 A set of two-phase
mixtures were set up by mixing CHCl3 (5 mL) and Tris buffer (5
mL, pH 8.0) containing Tris-HCl (100 mM), KCl (100 mM), and
potassium picrate (0.17 mM). All-(-)-nonactin was added to the
mixtures to obtain a range of ionophore concentrations from 0.1 to
0.01 mg/mL. Each test sample was thoroughly mixed, and then
the aqueous and organic layers were left to separate. Where a K+-
ionophore complex was formed, it was able to partition into the
organic phase, bringing along the colored picrate anion to maintain
neutrality. The concentration of the K+-ionophore complex in the
organic phase was estimated by measuring the concentration of
picrate, the latter measurement relying upon the absorbance of the
picrate anion at 357 nm (ε357
,
1450 dm3 · mol-1 · cm-1).
The experiment was repeated with nonactin for comparison. The
wavelength 357 nm is somewhat off the absorbance maximum for
the picrate anion; this value was deliberately chosen to attenuate
the large absorbance signal obtained at the picrate concentrations
needed for this experiment.
Bulk Transport through an Organic Phase As a Simple
Mimic of Kinetic Transport through a Membrane. To evaluate
the relative rate at which all-(-)-nonactin and nonactin may likely
allow for passive diffusion of ions across a membrane we adapted
a bulk organic phase model.35
The basic experiment consists of a system wherein two separate
aqueous samples are in contact with one immiscible organic sample
allowing for diffusion from one aqueous phase to the other via the
organic phase. A relatively simple apparatus was assembled using
a one-piece vacuum trap assembly (Chemglass, #CG-4510-03) with
an outer diameter of 35 mm and an inner tube diameter of 16 mm.
The lower part of the trap was filled with CHCl3 (35 mL) so that
the bottom of the inner trap tube was slightly below the surface
of the organic layer. An aqueous Tris buffer (10 mL, 100 mM, pH
8.0) was placed in the outer tube. A solution of potassium picrate
and KCl (2.5 mL, 0.25 mM potassium picrate, 100 mM KCl) in
Tris buffer (100 mM, pH 8.0) was placed in the inner tube. The
test ionophore, either nonactin or all-(-)-nonactin (0.5 mg in 1.0
mL of CHCl3), was added to the lower organic phase by syringe.
The organic phase was mechanically stirred at ∼200 rpm using a
magnetic stirrer bar. The stirring rate was carefully adjusted so that
no ‘spilling’ between the two aqueous phases occurred. The
transport of K+ from the inner tube to the outer tube, through the
organic phase, mediated by the ionophore, was followed by
measuring the concentration of the cotransported picrate anion via
its absorption at 357 nm.
Molecular Modeling. The nonactin structure was based on the
X-ray crystal data for the KNCS complex,8 and the all-(-)-nonactin
structure was derived from it by direct manipulation. To obtain
reasonable atomic charges we generated a low energy conformation
of a cyclic nonactate dimer and obtained a minimum energy
conformation at the HF/6-31G* level of theory using GAMESS
(2005R2).38 GAMESS was used again to calculate an electrostatic
potential grid which was used as input to the RESP charge
calculation of the AMBER-7 package.39 Only partial charges on
hydrogen atoms of methyl groups were averaged in the charge
fitting. Dynamics calculations were done using the AMBER-7 suite
(37) Davey, K. G.; Szekely, A.; Johnson, E. M.; Warnock, D. W. J. Antibiot.
Chemother. 1998, 42, 439–444.
9
17164 J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009

Alternating Stereochemistry in the Nonactin Macrocycle
A R T I C L E S
of programs39 together with the GAFF force field.40 Calculations
were performed on the K+ complexes, neutralized with one Cl-
ion together with 7920 water residues as an explicit solvent in a
periodic box of 46.6 × 47.7 × 49.0 Å3. A time step of 0.5 fs was
used without application of the SHAKE procedure. After 100 ps
of simulation at a fixed temperature (300 K) and pressure (1 bar)
to equilibrate the systems, a simulation for 20 ps further was done
to provide coordinate data for analysis.
(38) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon,
M. S.; Jensen, J. J.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su,
S.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. J. Comput. Chem.
1993, 14, 1347–1363.
(39) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E., III;
Wang, J.; Ross, W. S.; Simmerling, T. A.; Darden, T. A.; Merz, K. M.;
Stanton, R. V.; Cheng, A. L.; Vincent, J. J. Crowley, M.; Tsui, V.;
Gohlke, H.; Radmer, R. J.; Duan, Y.; Pitera, J.; Massova, I.; Seibel,
G. L.; Singh, P. K.; Weiner, P. K.; Kollman, P. A. AMBER 7 ed.;
University of California: San Francisco, 2002.
Acknowledgment. We thank Promiliad Biopharma Inc. for
supplying nonactin. This work was supported in part by NIH Grants
CA77347 and AI072158.
Supporting Information Available: 1H and 13C NMR spectra
and mass spectra for compounds 8 to 17. This material is
available free of charge via the Internet at http://pubs.acs.org.
(40) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A.
J. Comput. Chem. 2004, 25, 1157–1174.
JA9050235
9
J. AM. CHEM. SOC. VOL. 131, NO. 47, 2009 17165