Comment top
In 2006, Clardy and colleagues isolated phaeosphaeride A from an endophytic fungus FA39 (Maloney et al., 2006). Phaeosphaeride A turned out to be an inhibitor of signal transduction and an activator of transcription 3 (STAT3)-dependent signaling. It was reported to selectively inhibit STAT3/DNA binding with an IC50 of 0.61 mM and to exhibit promising cell growth inhibition in STAT3-dependent U266 multiple myeloma cells with an IC50 of 6.7 µM.
While the relative stereochemistry of phaeosphaeride A was deduced on the basis of NOE experiments, its absolute configuration remained undetermined (Maloney et al., 2006). Moreover, the attempts of total synthesis of phaeosphaeride A showed considerable differences in 1H and 13C NMR data between the synthetic and natural phaeosphaeride A (Kobayashi et al., 2011; Chatzimpaloglou et al., 2012, 2014). In 2015, Kobayashi and colleagues established the relative and absolute configurations of natural phaeosphaeride A by completing the first total synthesis of ent-phaeosphaeride A (Kobayashi et al., 2015).
Our research group isolated phaeosphaeride A from a fungal strain belonging to the genus Phoma. Phaeosphaeride A was obtained as an optically active (-108.33 (c 0.06, CH2Cl2)) yellow glass. 1H and 13C NMR data as well as mass spectra of our phaeosphaeride A match with the data reported for Clardy's natural phaeosphaeride A (Maloney et al., 2006). Optical rotation of Clardy's product (-93.6 (c 2.0, CH2Cl2)) and our phaeosphaeride A have the same sign. In this work we describe the crystal structure of natural phaeosphaeride A.
Experimental top
NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer in DMSO-d6. The same solvent was used as an internal standard. High-resolution mass spectra (HRMS) were recorded on a LTQ Orbitrap Velos spectrometer. Optical rotations were determined on an Optical Activity AA-55 polarimeter using a 20 cm cell with a Na 589 nm filter.
Phaeosphaeride A was isolated from solid culture of the fungus Phoma sp. N 19. The microorganism was obtained from leaves of Cirsium arvense (L.) Scop. and deposited in the culture collection of the All-Russian Institute of Plant Protection (Saint-Petersburg, Russian Federation). The metabolite was purified from the fungal extract with a combination of preparative column chromatography and TLC on silica gel to give phaeosphaeride A as a yellow precipitate. (-108.33 (c 0.06, CH2Cl2); 1H NMR (400 MHz, DMSO-d6) δ 5.44 (d, J = 5.8 Hz, 1H), 4.97 (s, 2H), 4.92 (s, 1H), 4.07 (d, J = 11.3 Hz, 1H), 3.86 (d, J = 5.8 Hz, 1H), 3.79 (s, 3H), 1.82 (m, 1H), 1.58-1.22 (m, 7H), 1.19 (s, 3H), 0.86 (t, J = 6.4 Hz, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 166.53, 155.30, 137.12, 104.80, 90.80, 86.25, 70.96, 64.36, 63.76, 30.90, 27.60, 26.11, 21.96, 20.40, 13.85; HRMS [M + H]+ calcd for C15H24NO5 298.16490, found 298.16493. Recrystallization from heptane yielded yellow crystals (-116.66; -108.33 (c 0.06, CH2Cl2).
Refinement top
Crystal data, data collection and structure refinement details are summarized in Table 1.
H atoms bonded to C atoms were included in calculated positions and refined using a riding model, with Uiso(H) set to 1.2Ueq(C) and C–H = 0.97 Å for CH2 groups, Uiso(H) set to 1.5Ueq(N) and C–H = 0.96 Å for CH3 groups and Uiso(H) set to 1.2Ueq(N) and C–H = 0.93 Å for CH groups. All H atoms bonded to O atoms were located in a difference Fourier map and were refined with distance restraints and constrained displacement parameters OH 0.82 Å and Uiso(H) set to 1.2Ueq(O). The large thermal ellipsoid on C13 is characteristic for the distal end of long alkyl chains.
The structure of phaeosphaeride A (Fig.1) was refined as rotational twin [by a two-fold rotation about (001)] with twin fractions of 0.718(6) and 0.282(6). The 'HKLF 5' format file for the final refinement was generated by the TwinRotMat facility in Platon (Spek, 2009). The ratio (Fc2-Fo2)/esd for the reflections with the highest error in final refinement model (as a rotational twin) has lower residuals than in the initial solution. We have used Bayesian Statistics for verifying absolute structure. Supplementary crystallographic data for this paper have been deposited at Cambridge Crystallographic Data Centre (CCDC 1412515) and can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.
Geometry top
The asymmetric unit contains two independent molecules of phaeosphaeride A (I and II) (fig. 1). Each molecule of phaeosphaeride A (numbering of atoms of phaeosphaeride A is given according to Clardy (Maloney et al., 2006)) contains two primary sections; an alkyl chain consisting of C(13)—C(12)—C(11)—C(10)—C(9) atoms and a cyclic system consisting of five and six membered rings with adjacent atoms.
For the five-membered ring C(1)—N(2)—C(3)—C(4)—C(5) of molecule I the Cremer-Pople (Cremer & Pople, 1975) parameters are Q=0.0714 Å, φ=197.99°, revealing a slightly distorted half-chair (2T1) conformation. Cremer-Pople parameters of Q=0.501 Å, θ=128.71°, φ=88.01° for the six-membered heterocycle of molecule I are consistent with a half-chair conformation (5H4). The geometric parameters of the six-membered rings for both molecules are similar, but the five-membered rings have different conformations. The five-membered ring in molecule II exhibits an envelope (1E) conformation.
Torsion angles O(1)—C(4)—C(5)—C(1) and C(3)—C(4)—C(5)—C(6) are -178.2(4)° and -177.1(4)° respectively, corresponding to co-planar conformation between the five and six-membered rings. The geometry of the heterocyclic ring system of the molecule (base of the half chair and the five-membered ring) is close to planar.
The exocyclic alkyl chain of I and the back of the half-chair lie approximately in the same plane. The deviation between the plane of alkyl atoms C(8)—C(9)—C(10)—C(11)—C(12)—C(13) and the back of the chair is 9.7(4)°.
The geometric parameters are similar for both molecules I and II forming the weak hydrogen-bonded dimer through O2—H2···O2A. But angles characterizing the methoxy groups N(2)—O(5)—C(16) are slightly different (109.9(3)° for I and 110.4(4)° for II). The difference between angles O(4)—C(1)—C(5) is much greater with values of 129.4(4)° and 123.2(4)° for I and II respectively.
The molecules form layered structures. Nearly planar sheets, parallel with the (001) plane, form primary layers of two-dimensional hydrogen-bonded networks with the hydroxyl moieties located on the interior of the sheets. The network (Fig. 2) is dependent primarily on four hydrogen bonds (Table 1) O2—H2···O2A, O3—H3···O4, O2A—H2A···O4, O3A—H3A···O4A (Desiraju et al., 1999; Arunan et al., 2011). In the (001) plane, two-dimensional hydrogen-bonded networks form a zig-zag pattern. The aliphatic butyl chains interdigitate with the butyl chains on the adjacent sheets.
Structure description top
In 2006, Clardy and colleagues isolated phaeosphaeride A from an endophytic fungus FA39 (Maloney et al., 2006). Phaeosphaeride A turned out to be an inhibitor of signal transduction and an activator of transcription 3 (STAT3)-dependent signaling. It was reported to selectively inhibit STAT3/DNA binding with an IC50 of 0.61 mM and to exhibit promising cell growth inhibition in STAT3-dependent U266 multiple myeloma cells with an IC50 of 6.7 µM.
While the relative stereochemistry of phaeosphaeride A was deduced on the basis of NOE experiments, its absolute configuration remained undetermined (Maloney et al., 2006). Moreover, the attempts of total synthesis of phaeosphaeride A showed considerable differences in 1H and 13C NMR data between the synthetic and natural phaeosphaeride A (Kobayashi et al., 2011; Chatzimpaloglou et al., 2012, 2014). In 2015, Kobayashi and colleagues established the relative and absolute configurations of natural phaeosphaeride A by completing the first total synthesis of ent-phaeosphaeride A (Kobayashi et al., 2015).
Our research group isolated phaeosphaeride A from a fungal strain belonging to the genus Phoma. Phaeosphaeride A was obtained as an optically active (-108.33 (c 0.06, CH2Cl2)) yellow glass. 1H and 13C NMR data as well as mass spectra of our phaeosphaeride A match with the data reported for Clardy's natural phaeosphaeride A (Maloney et al., 2006). Optical rotation of Clardy's product (-93.6 (c 2.0, CH2Cl2)) and our phaeosphaeride A have the same sign. In this work we describe the crystal structure of natural phaeosphaeride A.
NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer in DMSO-d6. The same solvent was used as an internal standard. High-resolution mass spectra (HRMS) were recorded on a LTQ Orbitrap Velos spectrometer. Optical rotations were determined on an Optical Activity AA-55 polarimeter using a 20 cm cell with a Na 589 nm filter.
Phaeosphaeride A was isolated from solid culture of the fungus Phoma sp. N 19. The microorganism was obtained from leaves of Cirsium arvense (L.) Scop. and deposited in the culture collection of the All-Russian Institute of Plant Protection (Saint-Petersburg, Russian Federation). The metabolite was purified from the fungal extract with a combination of preparative column chromatography and TLC on silica gel to give phaeosphaeride A as a yellow precipitate. (-108.33 (c 0.06, CH2Cl2); 1H NMR (400 MHz, DMSO-d6) δ 5.44 (d, J = 5.8 Hz, 1H), 4.97 (s, 2H), 4.92 (s, 1H), 4.07 (d, J = 11.3 Hz, 1H), 3.86 (d, J = 5.8 Hz, 1H), 3.79 (s, 3H), 1.82 (m, 1H), 1.58-1.22 (m, 7H), 1.19 (s, 3H), 0.86 (t, J = 6.4 Hz, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 166.53, 155.30, 137.12, 104.80, 90.80, 86.25, 70.96, 64.36, 63.76, 30.90, 27.60, 26.11, 21.96, 20.40, 13.85; HRMS [M + H]+ calcd for C15H24NO5 298.16490, found 298.16493. Recrystallization from heptane yielded yellow crystals (-116.66; -108.33 (c 0.06, CH2Cl2).
The asymmetric unit contains two independent molecules of phaeosphaeride A (I and II) (fig. 1). Each molecule of phaeosphaeride A (numbering of atoms of phaeosphaeride A is given according to Clardy (Maloney et al., 2006)) contains two primary sections; an alkyl chain consisting of C(13)—C(12)—C(11)—C(10)—C(9) atoms and a cyclic system consisting of five and six membered rings with adjacent atoms.
For the five-membered ring C(1)—N(2)—C(3)—C(4)—C(5) of molecule I the Cremer-Pople (Cremer & Pople, 1975) parameters are Q=0.0714 Å, φ=197.99°, revealing a slightly distorted half-chair (2T1) conformation. Cremer-Pople parameters of Q=0.501 Å, θ=128.71°, φ=88.01° for the six-membered heterocycle of molecule I are consistent with a half-chair conformation (5H4). The geometric parameters of the six-membered rings for both molecules are similar, but the five-membered rings have different conformations. The five-membered ring in molecule II exhibits an envelope (1E) conformation.
Torsion angles O(1)—C(4)—C(5)—C(1) and C(3)—C(4)—C(5)—C(6) are -178.2(4)° and -177.1(4)° respectively, corresponding to co-planar conformation between the five and six-membered rings. The geometry of the heterocyclic ring system of the molecule (base of the half chair and the five-membered ring) is close to planar.
The exocyclic alkyl chain of I and the back of the half-chair lie approximately in the same plane. The deviation between the plane of alkyl atoms C(8)—C(9)—C(10)—C(11)—C(12)—C(13) and the back of the chair is 9.7(4)°.
The geometric parameters are similar for both molecules I and II forming the weak hydrogen-bonded dimer through O2—H2···O2A. But angles characterizing the methoxy groups N(2)—O(5)—C(16) are slightly different (109.9(3)° for I and 110.4(4)° for II). The difference between angles O(4)—C(1)—C(5) is much greater with values of 129.4(4)° and 123.2(4)° for I and II respectively.
The molecules form layered structures. Nearly planar sheets, parallel with the (001) plane, form primary layers of two-dimensional hydrogen-bonded networks with the hydroxyl moieties located on the interior of the sheets. The network (Fig. 2) is dependent primarily on four hydrogen bonds (Table 1) O2—H2···O2A, O3—H3···O4, O2A—H2A···O4, O3A—H3A···O4A (Desiraju et al., 1999; Arunan et al., 2011). In the (001) plane, two-dimensional hydrogen-bonded networks form a zig-zag pattern. The aliphatic butyl chains interdigitate with the butyl chains on the adjacent sheets.
For details of the extraction of natural phaeosphaeride A and a discussion of its biological activities, see: Maloney et al. (2006). For details of trials of the synthesis of natural phaeosphaeride A, see: Kobayashi et al. (2011); Chatzimpaloglou et al. (2012, 2014); Kobayashi et al. (2015). Ring-puckering parameters are as defined by Cremer & Pople (1975). Hydrogen bonding is described in detail by Desiraju & Steiner (1999) and by Arunan et al. (2011). The twin law was identified using TwinRotMat in PLATON (Spek, 2009). Criteria for absolute configuration determination are described by Flack (1983) and Parsons et al. (2013).
Refinement details top
Crystal data, data collection and structure refinement details are summarized in Table 1.
H atoms bonded to C atoms were included in calculated positions and refined using a riding model, with Uiso(H) set to 1.2Ueq(C) and C–H = 0.97 Å for CH2 groups, Uiso(H) set to 1.5Ueq(N) and C–H = 0.96 Å for CH3 groups and Uiso(H) set to 1.2Ueq(N) and C–H = 0.93 Å for CH groups. All H atoms bonded to O atoms were located in a difference Fourier map and were refined with distance restraints and constrained displacement parameters OH 0.82 Å and Uiso(H) set to 1.2Ueq(O). The large thermal ellipsoid on C13 is characteristic for the distal end of long alkyl chains.
The structure of phaeosphaeride A (Fig.1) was refined as rotational twin [by a two-fold rotation about (001)] with twin fractions of 0.718(6) and 0.282(6). The 'HKLF 5' format file for the final refinement was generated by the TwinRotMat facility in Platon (Spek, 2009). The ratio (Fc2-Fo2)/esd for the reflections with the highest error in final refinement model (as a rotational twin) has lower residuals than in the initial solution. We have used Bayesian Statistics for verifying absolute structure. Supplementary crystallographic data for this paper have been deposited at Cambridge Crystallographic Data Centre (CCDC 1412515) and can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.
