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Acta Crystallogr C. 2009 October 15; 65(Pt 10): o521–o524.
Published online 2009 September 19. doi:  10.1107/S0108270109033083
PMCID: PMC2816929

Pseudomerohedrally twinned monoclinic structure of unfolded ‘free’ nona­ctin: comparative analysis of its large conformational change upon encapsulation of alkali metal ions


The title compound, C40H64O12, crystallizes in a pseudo­merohedrally twinned primitive monoclinic cell with similar contributions of the two twin components. There are two symmetry-independent half-mol­ecules of nona­ctin in the asymmetric unit. Each mol­ecule has a pseudo-S 4 symmetry and resides on a crystallographic twofold axis; the axes pass through the mol­ecular center of mass and are perpendicular to the plane of the macrocycle. The literature description of the room-temperature structure of nona­ctin as an order–disorder structure in an ortho­rhom­bic unit cell is corrected. We report a low-temperature high-precision ordered structure of ‘free’ nona­ctin that allowed for the first time precise determination of its bond distances and angles. It possesses an unfolded and more planar geometry than its complexes with encapsulated Na+, K+, Cs+, Ca2+ or NH4 + cations that exhibit more isometric overall conformations.


Nonactin, (I), is a naturally occurring optically inactive macrotetrolide anti­biotic whose activity includes the ability to transport cations across biological and artificial membranes. New anti­biotics are urgently needed to combat persistent, emerging and re-emerging infectious diseases, hospital-acquired resistance, and bioterrorism agents. Currently, there is a renaissance of drug discovery from natural products. Exploration of microbial diversity in underexplored environments offers renewed hope for the discovery of novel anti­biotics, anti­cancer agents, and other drugs from nature. In a pilot effort to explore the Great Lakes as an untapped freshwater environment for microbial resources from which new chemical entities may be obtained as anti­biotics, dozens of actinomycete isolates were obtained from Lake Michigan sediment samples and found to produce chemical extracts with anti­microbial activities. In particular, a Streptomyces sp. isolate was found to produce a significant amount of what was proven later to be nona­ctin anhydride.

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Nonactin has been isolated previously from numerous bacterial sources as an anti­biotic and anti­cancer agent (Corbaz et al., 1955 [triangle]; Solov’eva, 1973 [triangle]; Wallhaeusser et al., 1964 [triangle]). This macrotetrolide ionophore consists of the (+)(−)(+)(−)-ester linkage of the enanti­omeric nona­ctic acid building blocks. However, the elucidation of the solid-state structure of nona­ctin has proven difficult. Dunitz (1964 [triangle]) determined that impure crystals (described as ‘not highly purified’) of nona­ctin gave diffraction patterns with para-ortho­rhom­bic symmetry. Dornberger-Schiff (1966 [triangle]) noted that the structure may be of the type ‘order–disorder’. The orthorhombic unit-cell dimensions were determined by Dominguez et al. (1962 [triangle]) as a = 47.6 Å, b = 31.5 Å and c = 5.70 Å, and Dobler (1972 [triangle]) reported the room-temperature order–disorder structure of nona­ctin in this cell, but noted that the superposition structure corresponds to the ortho­rhom­bic space group Pbam with a unit cell of a/2, b/2, c. Although Dobler reported the atomic parameters, he commented that the large magnitude of the standard deviations on bond distances and angles did not allow for a detailed discussion of the mol­ecular geometry of nona­ctin. Dobler observed diffuse streaks in the diffraction pattern of crystals of nona­ctin; we also recorded streaking in the diffraction pattern of our crystal.

Herein we report mol­ecular parameters of nona­ctin established with high precision. Our low-temperature (100 K) structural investigation of a colorless unknown (subsequently proven to be nona­ctin) proceeded as follows. The initial indexing of the unit cell suggested a C-centered ortho­rhom­bic lattice consistent with the reported dimensions (a = 31.147 Å, b = 47.166 Å, c = 5.569 Å and V = 8180.55 Å3), which is frequently a sign of trouble. The program CELL_NOW (Sheldrick, 2009 [triangle]) was used to index the reflections and the crystal appeared to be single. A full sphere of data was collected. The program XPREP (Sheldrick, 2008 [triangle]) suggested the space group Cmma, which is encountered in the Cambridge Structural Database (CSD; Allen, 2002 [triangle]) only 36 times. Not surprisingly, the structure could not be solved in this space group and a monoclinic unit cell was selected instead. Systematic absences were consistent with the space groups P2/n and Pn and the E statistics were indiscriminate as to the centrosymmetry. Although the structure was successfully solved in P2/n, the refinement stalled, with an R factor of ~22%. Scrutiny of the data revealed many F obs values much higher than the corresponding F calc values, a likely indication of twinning. The program PLATON (Spek, 2009 [triangle]) was then used to analyze the data, and indeed pseudomerohedral twinning was detected. The suggested transformation matrix (00An external file that holds a picture, illustration, etc.
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Object name is c-65-0o521-efi1.jpg00) corresponds to inter­change of the a and c axes that have very similar lengths. The suggested twin law was incorporated into the instruction file with a TWIN/BASF combination for the program XL (Sheldrick, 2009 [triangle]) and the batch scale factor refined to indicate a 45.08 (11)% contribution of the minor twin component. The resulting refinement converged to an R factor of 4.30%. Fig. 1 [triangle] shows the relationship between the primitive monoclinic unit cell and the apparent incorrect C-centered ortho­rhom­bic unit cell. The monoclinic unit cell can be converted into the C-centered ortho­rhom­bic cell with a transformation matrix (101, An external file that holds a picture, illustration, etc.
Object name is c-65-0o521-efi1.jpg01, 0An external file that holds a picture, illustration, etc.
Object name is c-65-0o521-efi1.jpg0). A recent related example of pseudomerohedral twinning of cyclo­penta­deca­none was reported by Noe et al. (2008 [triangle]).

Figure 1
Relationship between the correct monoclinic unit cell under P2/n symmetry and the apparent C-centered ortho­rhom­bic unit cell. The unique twofold symmetry axis is perpendicular to the plane of the page.

Nonactin (Fig. 2 [triangle]) crystallizes in a centrosymmetric lattice of P2/n symmetry, with two symmetry-independent half-mol­ecules in the asymmetric unit (four mol­ecules in the unit cell, Z = 4). Each mol­ecule occupies a crystallographic twofold axis. The two independent mol­ecules have virtually identical conformations with an approximate S 4 symmetry. The twofold and pseudo-S 4 axes pass through the mol­ecular center of mass and are perpendicular to the plane of the macrocycle. The mol­ecules form stacks in the [010] direction. There is a solvent-accessible void in the center of the macrocycle with an approximate volume of 69 Å3 (PLATON). The dimensions of this mol­ecular cavity can be described with the centroid–centroid distances between the tetra­hydro­furan rings related by the twofold axes. These dimensions are 7.688 (2) × 7.544 (2) Å in the O1 mol­ecule and 7.563 (3) × 7.593 (2) Å in the O1A mol­ecule. These continuous voids form channels in the crystal along the b axis (Fig. 3 [triangle]).

Figure 2
(a) The mol­ecular structure of nona­ctin, with displacement ellipsoids shown at the 40% probability level and all H atoms omitted. (b) Sideways view of the two mol­ecules along the crystallographic a axis. All atoms are shown ...
Figure 3
A packing diagram of nona­ctin, viewed along the b axis. The voids in the center of the macrocycle form continuous channels in the lattice. All atoms are shown with 40% probability ellipsoids, but the O-atom ellipsoids are drawn with octant shading. ...

The conformation of ‘free’ nona­ctin was compared with that observed in the previously reported nona­ctin complexes with Na+ (Dobler & Phizackerley, 1974 [triangle]), K+ (Kilbourn et al., 1967 [triangle]), Cs+ (Sakamaki et al., 1977 [triangle]), Ca2+ (Vishwanath et al., 1983 [triangle]) and NH4 + (Neupert-Laves & Dobler, 1976 [triangle]) using a new subroutine WBOX developed specifically for this project in the program OLEX2 (Dolomanov et al., 2009 [triangle]) to compute the dimensions of the smallest parallelepiped superscribing each of the nona­ctin complexes (Fig. 4 [triangle]). The obtained dimensions (Table 1 [triangle]) clearly show that ‘free’ nona­ctin per se is unfolded and relatively square-planar, but becomes globular when encapsulating a cation, a fact noted by others and now supported quanti­tatively. For example, the dimensions of the parallelepiped superscribing nona­ctin are ~9 × 17 × 17 Å, whereas those for the parallelepiped encompassing nona­ctin coordinated to a Ca2+ cation are ~12 × 14 × 14 Å. In the four metal cationic complexes of nona­ctin, the macrocycle coordinates to the metal with four carbonyl and four tetra­hydro­furan O atoms in a distorted cubic arrangement. In the cases of the smaller Na+ and Ca2+ cations (hard Lewis bases), the metal–oxygen distances to the carbonyl O atoms are shorter, whereas in the cases of the larger Cs+ and K+ cations (softer Lewis bases), the distances to the tetrahydrofuran O atoms are shorter. It is noteworthy that encapsulation of larger Cs+ and K+ cations produces more spheroidal and compact structures – the ‘encompassing’ box sizes for the corresponding complexes are smaller than those of the complexes involving Na+ and Ca2+ (Table 1 [triangle]). This is likely due to the better fit between the sizes of the larger cations and the inter­nal nona­ctin cavity. In the case of the ammonium salt, the four H atoms of the cation form hydrogen-bonding inter­actions with four tetra­hydro­furan O atoms. Thus, the conformational changes of nona­ctin are, as expected, dependent on the size and nature of the encapsulated cation.

Figure 4
Illustration of the smallest parallelepiped containing free nona­ctin (left) and nona­ctin coordinating a Cs+ cation. The box on the right is noticeably more isometric. All atoms are shown with their van der Waals spheres (see Table 1 ...
Table 1
Symmetry and size of selected nonactin complexes

The elusive structure of anhydrous ‘free’ nona­ctin has finally been unambiguously established. Nonactin crystallizes in a pseudomerohedrally twinned primitive monoclinic cell with two twin components of similar sizes.


Nonactin anhydride was isolated as an amorphous powder from the culture broth of Streptomyces sp. by ethyl acetate extraction, followed by silica-gel chromatography and reverse phase preparation high-pressure liquid chromatography. Its anti­microbial activities were monitored by agar diffusion assays during purification steps. A crystalline sample was isolated after being recrystallized three times from acetone.

Crystal data

  • C40H64O12
  • M r = 736.91
  • Monoclinic, An external file that holds a picture, illustration, etc.
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  • a = 28.252 (10) Å
  • b = 5.569 (2) Å
  • c = 28.270 (11) Å
  • β = 113.120 (19)°
  • V = 4090 (3) Å3
  • Z = 4
  • Cu Kα radiation
  • μ = 0.71 mm−1
  • T = 100 K
  • 0.55 × 0.20 × 0.12 mm

Data collection

  • Bruker SMART APEXII area-detector diffractometer
  • Absorption correction: multi-scan (SADABS; Bruker, 2009 [triangle]) T min = 0.694, T max = 0.918
  • 51667 measured reflections
  • 7460 independent reflections
  • 7285 reflections with I > 2σ(I)
  • R int = 0.024


  • R[F 2 > 2σ(F 2)] = 0.043
  • wR(F 2) = 0.110
  • S = 1.05
  • 7460 reflections
  • 478 parameters
  • H-atom parameters constrained
  • Δρmax = 0.55 e Å−3
  • Δρmin = −0.20 e Å−3

All H atoms were placed in geometrically idealized locations, with primary, secondary and tertiary C—H distances of 0.98, 0.99 and 1.00 Å, respectively. The H atoms were refined as riding, with isotropic displacement coefficients of U iso(H) = 1.5U eq(C) for methyl groups or 1.2U eq(C) otherwise.

Data collection: APEX2 (Bruker, 2009 [triangle]); cell refinement: SAINT-Plus (Bruker, 2009 [triangle]); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXTL (Sheldrick, 2008 [triangle]); program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL and OLEX2 (Dolomanov et al., 2009 [triangle]); software used to prepare material for publication: SHELXTL.

Supplementary Material

Crystal structure: contains datablocks global, I. DOI: 10.1107/S0108270109033083/sq3212sup1.cif

Structure factors: contains datablocks I. DOI: 10.1107/S0108270109033083/sq3212Isup2.hkl


The manuscript was prepared with beta test version 1.9.3 of the program publCIF (Westrip, 2009 [triangle]) and the programs FCF_filter, INSerter, and modiCIFer (Guzei, 2007 [triangle]). This work was supported in part by a grant from the US National Institute of Health (grant No. R03 AI073498 to YQC). We thank Lihua Jiang (UW–Milwaukee) for technical assistance in the assay-guided purification process. We are grateful to Professor Lawrence F. Dahl (UW–Madison) for fruitful discussions and to Dr A. I. Yanovsky (Pfizer) for insightful suggestions.


Supplementary data for this paper are available from the IUCr electronic archives (Reference: SQ3212). Services for accessing these data are described at the back of the journal.


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