Cyclo(-l-prolyl-l-valinyl-) from Burkholderia thailandensis MSMB43

The title compound [systematic name: (3S,8aS)-3-isopropylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione], C10H16N2O2,, is a newly isolated cyclic dipeptide from Burkholderia thailandensis MSMB43. There are two independent molecules in the asymmetric unit. Two C atoms and their attached H atoms in the five-membered ring of one of the molecules are disordered over two sets of sites in a 0.715 (5):0.285 (5) ratio. The two independent molecules have the same configuration and the absolute configurations of the chiral centers were determined based on the observation of anomalous dispersion. In the crystal, two types of N—H⋯O hydrogen bonds link pairs of independent molecules.

The title compound [systematic name: (3S,8aS)-3-isopropylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione], C 10 H 16 N 2 O 2 ,, is a newly isolated cyclic dipeptide from Burkholderia thailandensis MSMB43. There are two independent molecules in the asymmetric unit. Two C atoms and their attached H atoms in the five-membered ring of one of the molecules are disordered over two sets of sites in a 0.715 (5):0.285 (5) ratio. The two independent molecules have the same configuration and the absolute configurations of the chiral centers were determined based on the observation of anomalous dispersion. In the crystal, two types of N-HÁ Á ÁO hydrogen bonds link pairs of independent molecules.

Experimental
The title compound is a cyclic dipeptide of L-proline and L-valine. The structural skeleton is fused by a five-membered pyrrolidine ring and a six-membered piperazine ring. The pyrrolidine ring adopts an envelope configuration and the piperazine ring has a boat configuration. These two rings are located on nearly the same plane and the dihedral angles of these two least-squares planes are 18.2 (1)° for the non-disordered molecule, and 30.6 (1)° for the major component of the disordered molecule. There are two independent molecules in the asymmetric unit of the crystal. Atoms C3A and C4A of one of the molecules are disordered over two positions with a major component contribution of 71.5 (5)%. The two molecules have the same configuration and the absolute configurations of C2, C2A, C7 and C7A are S based on the results of anomalous dispersion. There are two intermolecular hydrogen bonds present between two independent molecules in the different asymmetric unit and connect them to form a pair of molecules (Table 1, Fig. 1 and Fig. 2).

Experimental
Isolation of the title compound Bacterial strain and culture conditions B. thailandensis strain MSMB43 was obtained from the US Centers for Disease Control (CDC) and was routinely activated on LB agar containing 50 mg ml -1 of apramycin (Am 50 ) 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 Am 50 , and the culture were growing at 37°C for 24 h as seed culture. For fed-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 M11 medium (10.0 g/L dextrose, 2.0 g/L pancreatic digest of casein, 1.0 g/L yeast extract, 1.0 g/L beef extract; pH 7.0). Fermentation was proceeded at 37°C, 300 rpm for 72 h, during which the pH was automatically adjusted by the fermentor with 1 N HCl or 1 N NaOH. Three liters of 10X M11 was fed to the fermentor from 24 h to 48 h at a flow rate of 0.125 ml/min.

Recovery of the crude extract
Bacterial cells and debris were removed by centrifugation of broth at 6,000 g for 15 min. Supernatant was applied to a 2-L column (Φ 8.0 x 40 cm) packed with a 50/50 mixture of Diaion HP-20 resin (Sigma-Aldrich, USA) and Amberlite supplementary materials XAD16 resin (Sigma-Aldrich) to allow absorption to occur. The resins were subsequently dried and extracted repeatedly with ethyl acetate. Organic extractions were pooled and dried in a rotary evaporator to yield a crude extract.

Crystallization
The purified title compound was dissolved in ethyl acetate and the crystals were obtained after a slow evaporation of the solvent at room temperature for 5 days.

Refinement
All hydrogen atoms attached to the carbon atoms were placed in geometrically idealized positions (C-H = 0.98, 0.99 and 1.00 Å on the primary, secondary and tertiary aliphatic C atoms 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. The hydrogen atoms attached to N were located in the difference map and refined independently with restraints and constraints. The H atoms on the N were constrained to have the distances of 0.88 Å and the U iso value were assigned as equal to 1.2 times the U eq of the attached atoms. program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to refine structure: SHELXTL (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008) and OLEX2 (Dolomanov et al., 2009); software used to prepare material for publication: SHELXTL (Sheldrick, 2008) and OLEX2 (Dolomanov et al., 2009   Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
x y z U iso */U eq Occ. (