FK228 from Burkholderia thailandensis MSMB43

Xiang-Yang Liu; Cheng Wang; Yi-Qiang Cheng

doi:10.1107/S160053681203601X

. 2012 Aug 23;68(Pt 9):o2757–o2758. doi: 10.1107/S160053681203601X

FK228 from Burkholderia thailandensis MSMB43

Xiang-Yang Liu ^a, Cheng Wang ^a,^*, Yi-Qiang Cheng ^a,^*

PMCID: PMC3435793 PMID: 22969639

Abstract

FK228 [systematic name: (1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-di(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetrazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone], C₂₄H₃₆N₄O₆S₂, also known as FR901228, depsipeptide, NSC 630176, romidepsin, and marketed as Istodax by Celgene Corporation, is crystallized from ethyl acetate in P2₁ as compared to the absolute configuration of FK228, first crystallized from methanol in P2₁2₁2₁ [Shigematsu et al. (1994 ▶). J. Antibiot. 47, 311–314]. A slight difference is observed between the absolute configuration of FK228 and the present structure. The molecular structure is stabilized by intramolecular N—H⋯O hydrogen bonds. In the crystal, molecules are linked via N—H⋯O hydrogen bonds.

Related literature

For diverse natural products, see: Nguyen et al. (2008 ▶); Knappe et al. (2008 ▶); Seyedsayamdost et al. (2010 ▶); Biggins et al. (2011 ▶); Klausmeyer et al. (2011 ▶); Wang et al. (2011 ▶, 2012 ▶). For large-scale genome sequencing, see: Yu et al. (2006 ▶); Mukhopadhyay et al. (2010 ▶); Zhuo et al. (2012 ▶). For the initial discovery of FK228 from Chromobacterium violaceum No. 968 and its crystal structure report, see: Shigematsu et al. (1994 ▶); Ueda, Nakajima, Hori, Fujita et al. (1994 ▶). For the biological activities and mode of action of FK228, see: Furumai et al. (2002 ▶); Ueda, Manda et al. (1994 ▶); Ueda, Nakajima, Hori, Goto & Okuhara (1994 ▶). For biosynthetic studies of FK228, see: Cheng et al. (2007 ▶); Potharla et al. (2011 ▶); Wesener et al. (2011 ▶). For clinical application of FK228, see: Robey et al. (2011 ▶); StatBite (2010 ▶).

Experimental

Crystal data

C₂₄H₃₆N₄O₆S₂
M _r = 540.69
Monoclinic,
a = 9.1085 (2) Å
b = 16.2431 (4) Å
c = 9.4192 (2) Å
β = 92.096 (1)°
V = 1392.64 (5) Å³
Z = 2
Cu Kα radiation
μ = 2.10 mm⁻¹
T = 100 K
0.26 × 0.14 × 0.07 mm

Data collection

Bruker SMART APEXII area-detector diffractometer
Absorption correction: analytical (SADABS; Sheldrick, 2003 ▶) T _min = 0.611, T _max = 0.867
22577 measured reflections
4918 independent reflections
4859 reflections with I > 2σ(I)
R _int = 0.025

Refinement

R[F ² > 2σ(F ²)] = 0.025
wR(F ²) = 0.065
S = 1.02
4918 reflections
343 parameters
1 restraint
H atoms treated by a mixture of independent and constrained refinement
Δρ_max = 0.29 e Å⁻³
Δρ_min = −0.15 e Å⁻³
Absolute structure: Flack (1983 ▶), 2188 Friedel pairs
Flack parameter: 0.022 (9)

Data collection: APEX2 (Bruker, 2007 ▶); cell refinement: SAINT (Bruker, 2007 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL and OLEX2 (Dolomanov et al., 2009 ▶); software used to prepare material for publication: SHELXTL.

Supplementary Material

Crystal structure: contains datablock(s) global, I. DOI: 10.1107/S160053681203601X/jj2148sup1.cif

e-68-o2757-sup1.cif^{(25.6KB, cif)}

Structure factors: contains datablock(s) I. DOI: 10.1107/S160053681203601X/jj2148Isup2.hkl

e-68-o2757-Isup2.hkl^{(240.9KB, hkl)}

Supplementary material file. DOI: 10.1107/S160053681203601X/jj2148Isup3.cdx

Additional supplementary materials: crystallographic information; 3D view; checkCIF report

Table 1. Hydrogen-bond geometry (Å, °).

D—H⋯A	D—H	H⋯A	D⋯A	D—H⋯A
N1—H3⋯O5	0.84 (2)	2.27 (2)	3.0123 (18)	147.1 (17)
N2—H4⋯O6	0.82 (2)	2.05 (2)	2.7867 (18)	149.1 (18)
N3—H16⋯O3ⁱ	0.87 (2)	2.18 (2)	3.0449 (17)	175.9 (18)

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Symmetry code: (i) Inline graphic .

Acknowledgments

Support for this work was obtained from a Research Growth Initiative Award from the University of Wisconsin–Milwaukee and NIH/NCI grant R01 CA 152212 (both to YQC). The authors thank Lara C. Spencer and Ilia A. Guzei (University of Wisconsin–Madison, Department of Chemistry, Crystallography Facility) for collecting the crystallographic data.

supplementary crystallographic information

Comment

Diverse natural products, including thailandamides (Nguyen et al., 2008), capistruin (Knappe et al., 2008), bactobolin A—D (Seyedsayamdost et al., 2010), burkholdacs A—B (Biggins et al., 2011), spiruchostatin C (Klausmeyer et al., 2011), and thailandepsins (Wang et al., 2011, 2012), were discovered recently from Brukholderia thailandensis E264. In conjunction with large-scale genome sequencing (Yu et al., 2006; Mukhopadhyay et al., 2010; Zhuo et al., 2012), the Burkholderia thailandensis species have drawn much attention due to their capability to synthesize novel compounds with antibacterial, antitumor and antiviral activities.

While exploring novel natural products from the culture broth of B. thailandensis MSMB43, the title compound FK228 was isolated in large quantity (~100 mg/L). The physicochemical properties (UV spectrum, IR spectrum, MS and NMR) of the FK228 from B. thailandensis MSMB43 are identical to those of the FK228 isolated in the early 1990s from Chromobacterium violaceum No. 968 (Ueda, Nakajima, Hori, Fujita et al., 1994; Shigematsu et al., 1994). The crystal structure of the newly isolated FK228, herein, is slightly different than that reported by (Ueda, Manda et al., 1994), due to different crystallization conditions employed.

FK228 is bicyclic depsipeptide and consists of five building blocks, D-cysteine (D-Cys), D-valine (D-Val), 4-amino-3-hydroxy-5-methylheptanoic acid (Ahhp, derived from an isoleucine and an acetate unit), L-valine (L-Val), Z-dehydrobutyrine(Z-Dhb) and 3-hydroxy-7-mercapto-4-heptenoic acid (Acyl, derived from a cysteine and two acetate units). The primary structure of FK228 is (L)Val-(Z)Dhb-(D)Cys-Ahhp-(D)Val. X-ray crystallographic analysis indicates that the skeleton of FK228 is a [8.7.6] 23-membered ring adopting an uncommon cage-shape including a 16-membered macrocyclic lactone and a 15-membered ring containing a signature disulfide bond.

Under the different crystallization conditions presented, FK228 formed a different molecular arrangement than previously described (Fig. 1). The title crystal structure was obtained from ethyl acetate crystallizing in the P2₁ space group, while the provirus structure co-crystallized with one methanol in asymmetric unit in the P2₁2₁2₁ space group. The absolute configurations of all the chiral centers in each of these two structures are the same: C1(S), C3(S), c7 (S) and C22 (R). The geometric configuration of the double bonds in the Acyl and Dhb components are all determined as E and Z respectively. In comparing the these two structures, the configuration of these skeletons are the same. But there is a slight difference at the end of L-Val group. Under the different crystallization conditions, C18 and H19 had the opposite positions in these two structures because of the free rotation of the single bond (Fig. 2). Hydrogen bonds N2—H4···O6 and the weak intermolecular interactions N1—H3···O5 and N3—H16···O3 are observed in the title crystal structure. (Table 1, Fig. 3).

Experimental

Bacteria and culture medium

Burkholderia thailandensis strain MSMB43 (obtained from the US Centers for Disease Control, CDC) was routinely activated on LB agar containing 50 mg ml^-1 of apramycin (Am50) at 37°C for 1 to 2 days as a master plate. A single colony was then transferred into a 1 L flask containing 300 ml of LB medium and Am50, and the culture were growing at 37°C for 24 h as seed culture. For batch fermentation 250 ml of seed culture was transferred into a 20 L fermentor (BioFlo IV, New Brunswick Scientific Co., USA) containing 12 L of M8 medium (0.5% glucose, 0.5% peptone, 0.3% NaCl, 0.12% Na₂HPO₄, and 0.05% KH₂PO₄; pH 7), and fermented at 30°C for 96 hr under 20 L/min aeration and 200 rpm agitation; pH was maintained at 7.0 with 1 N NaCl or 1M HCl. For fed-batch fermentation, feeding of 3 L of 10X concentrated medium between 24 hr and 48 hr was performed on top of the batch fermentation.

Recovery of the crude extract

Bacteria cells were removed by centrifugation of cell broth at 3,800 rpm for 15 min. The supernatant was applied on a 2 L column (Φ 8.0 × 40 cm) packed with a mixture of Diaion HP-20 (Sigma-Aldrich, USA) resin and Amberlite XAD16 (Sigma-Aldrich, USA), which has been equilibrated in water at 5 ml/min. The resin was dried and then extracted with ethyl acetate for three times. The ethyl acetate extracts were combined and concentrated to dryness in vacuo at 35°C.

Isolation and purification of the title compound

The ethyl acetate extract was subjected to a two step isolation and purification protocol. Briefly, the ethyl acetate extract was mixed with 50 g silica gel (230–400 mesh, Whatman Purasil, USA). The mixture of silica gel was dried overnight and then applied to a 120 - g silica gel column (300 ml), which has been equilibrated with hexane. The column was eluted sequentially with 1 L hexane, 1 L hexane: ethyl acetate (3:1, v/v), 1 L hexane: ethyl acetate (1:1, v/v), 1 L ethyl acetate, 1 L ethyl acetate: acetone (1:1, v/v), and finally 2 L of acetone. The obtained fractions were applied on a flash chromatography equipped with a silica gel universal column (Yamazen Corporation, 23 ×123 mm, 16 g) connected to an injection column (Yamazen Corporation, 20 × 65 mm, 14 g). The column was eluted by a mixture of chloroform and acetone with an increasing of polarity according to the following concentrations of acetone, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 65%, and 100%. Fractions by 15% and 20% acetone were concentrated into dryness in vacuo and dissolved in acetonitrile. The acetonitrile solution was applied on a preparative HPLC system equipped with an Agilent Prep-C18 column (21.2 × 250 mm, 10 µm, PN 410910–102, USA). The mobile phase was composed of acetonitrile and water (purified by NANO pure Diamond Life Science ultrapure water system). After loading the sample, the column was eluted by a linear gradient aqueous acetonitrile from 40% to 55% within 0–25 min, FK228 was collected at 21–25 min. The flow rate was 8 ml/min. The UV spectrum was monitored at 210 nm. The FK228 solution was concentrated into dryness in vacuo on a rotary evaporator at 35 °C. The dried FK228 was dissolved in ethyl acetate and the crystals were obtained at room temperature after 5–7 days.