The glycolipid flocculosin-A from the fungus Anthracocystis flocculosa, or how to deal with cotton-wool-like crystals

This article is part of a special issue on current research in crystal growth and related characterization

Flocculosin A is a strong antifungal metabolite secreted by the Basidiomycota fungus Anthracocystis flocculosa (formerly Pseudozyma flocculosa). It crystallizes in the form of long, soft cotton-wool-like needles, usually observed together with the mycelium in liquid culture conditions. Many crystallization tests were carried out. Among them, only a very slow cooling technique was able to yield suitable crystals, which were needle-like with a maximum thickness of 5 µm and several millimetres long. These crystals permitted us to record single-crystal X-ray diffraction data and solve the crystal structure as well as to determine the absolute configurations of the three chiral centres present in the two lipid chains.

1. Introduction

Anthracocystis flocculosa is a basidiomycetous fungal yeast with powerful antagonistic activity against powdery mildews (Avis & Bélanger, 2002). For many years, this inhibitory activity had been believed to be mainly due to the production of a single glycolipid, flocculosin, whose 3D structure has been the subject of numerous investigations (Cheng et al., 2003; Mimee et al., 2005). Although a recent study showed that flocculosin is not associated to a biocontrol activity (Santhanam et al., 2021), this compound and analogs are still an attractive research subject due to their broad spectrum of antimicrobial properties (Mimee et al., 2005; Mimee et al., 2009). Considerable interest has developed in flocculosin derivatives, not only for their strong fungicidal activities, but also for evaluation as future economic sources of ω-hydroxy fatty acids (Becker et al., 2020) and surfactants molecules (Paulino et al., 2017). It is interesting to note that many other strains of basidiomycete yeast produce cellobiose lipids, including ustilagic acid analogues (Kulakovskaya et al., 2005), which can also be exploited as a source of new active molecules.

In order to fully understand the structure–activity relationships of flocculosins, we performed a study of isolation and structure elucidation of the leading compound, flocculosin-A, whose structural scaffold consists of a cellobiose disaccharide with different degrees of acetyl­ation, linked to a hy­droxy-palmitoic acid and a hy­droxy-caproic acid via glycosidic bonds. However, the structure of flocculosin-A poses a serious challenge because the product always crystallizes as very long, curved, soft thin needles, less than 5 µm thick and of poor crystallinity.

2. Experimental

2.1. Materials

HPLC-grade solvents and reagents were purchased from Sigma-Aldrich (Quentin-Fallavier, France). Vacuum-liquid chromatography (VLC) was performed using C18 (50–70 mesh) resin obtained from Sigma–Aldrich. Column chromatography was conducted on Merck silica gel (70–230 mesh). Thin layer chromatography was performed on glass pre-coated silica gel 60 F254 plates (Merck, Darmstadt, Germany).

Product characterizations: NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for ¹H and 100 MHz for ¹³C). High-resolution mass spectra were obtained on a Thermo Instruments ESI-MS system connected to a Thermo Instruments HPLC system.

2.2. Isolation and purification of flocculosin-A

The fungus Anthracocystis flocculosa was isolated on carnation leaves grown in INRAE greenhouses (Ponchet, 1988). It was cultured on glucose asparagine medium (Picard et al., 2000). Ten (1 L) Erlenmeyer flasks containing 200 ml of medium were inoculated with 500 µL of a suspension of yeast-like bodies (10⁶ propagules per ml). The cultures were maintained under constant agitation (80 rpm) in the dark for four days.

The fully fermented cultures were filtered to separate the culture filtrate from the mycelium.

Two litres of the culture medium were applied to C18 VLC (10 × 10 cm) and eluted with successive CH₃OH/H₂O mixtures (25:75, 50:50, 80:20, 100:0, each ∼0.8 L) followed by 0.8 L of a CH₂Cl₂/CH₃OH mixture (50:50) to yield five fractions (M1–M5). Fraction M3 (4.5 g) was pure flocculosin-A (compound 1). The other fractions contain minor compounds or isomers. For example, fraction M2 (223 mg), purified by silica gel column chromatography (1.5 × 60 cm), and eluted with three ternary mixtures CHCl₃/EtOH/H₂O (7:1:0.1, 6:1:0.1, 5:1:0.1) yielded besides additional flocculosin-A (71 mg), two other derivatives named flocculosin-B (25 mg) and flocculosin-C (18 mg). All in all, seven parent derivatives bearing the same cellobiose moiety, either intermediates or precursors with various substitutions were isolated in addition to the major compound flocculosin-A (named flocculosin-B to -H). They will be described elsewhere.

Flocculosin-A was obtained as an amorphous powder. Molecular formula: C₄₀H₇₀O₁₉. High-resolution electron spray ionization mass spectrometry = 853.4474 [M−H]⁻ (calcd for C₄₀H₆₉O₁₉⁻ 853.4439) = 855.4577 [M+H]⁺ (calcd for C₄₀H₇₁O₁₉⁺ 855.4584).

2.3. Crystallization of flocculosin-A

Solvents were first distilled, filtered through fritted glass No. 5, and stored in a cold place (4°C).

Flocculosin-A is sparingly soluble in water, moderately in hy­droxy­lated solvents, and very soluble at high temperatures in lipophilic solvents such as chloro­form, acetone, etc. All attempts to get crystals by temperature variations in all of these solvents, and mixtures thereof, led invariably to cotton-wool-like fibres, unsuitable for diffraction studies.

Obtaining fibres is usually the result of a large difference in crystal growth along three perpendicular directions. Flocculosin-A is in this case with a lateral/longitudinal growth ratio of 1:1000. The result is fibres several millimetres long and only a micron in diameter. To solve this problem and improve this ratio, we adopted a method of cyclic temperature variation between partial crystal dissolution (50°C) and complete crystallization (−10°C) over a slow time variation in a suitable solvent, compound TFT (tri­fluoro-toluene, CF₃-C₆H₅) is considered to be a substitute for chloro­form or di­chloro­methane. Due to the thermal stability of flocculosin-A, the procedure that led to exploitable crystals was as follows.

A solution of flocculosin-A (10 mg in 1 ml of TFT) in a small glass vials (2 mL) was heated to 100°C until complete dissolution occurred. The vials were isolated from the exterior with silicon stoppers and placed in a dewar filled with 200 ml of hot (100°C) NaCl brine, acting as a temperature buffer. The dewar was then enclosed in a large polystyrene box also containing a large volume of hot brine. The closed box was allowed to cool down at room temperature (the internal temperature was followed by an external thermocouple reader fixed to the box). The temperature equilibrium was achieved within a full day. The box was then placed in a freezer and cooled down to −10°C for half a day. The crystallization of flocculosin-A was checked at this stage under a microscope equipped with a polarizer. This completed the first temperature cycle.

Afterwards, two more temperature cycles were made, i.e. for each, heating the box up to 50°C to partially dissolve the needles, and cooling it again at room temperature, then down to −10°C. It was expected that the dissolution/recrystallization of the needles will be mostly along the fastest growing axis than along the slowest, and that crystal growth will favour increasing the thickness of the needles after recrystallization, because of the physical limitation of the needle's elongation in the small cylinder of crystallization. Each cycle was completed over one or two days.

At the end of the third cycle, needles of flocculosin-A were harvested, dispersed on a glass plate under a microscope, with a few drops of perfluoro-polyether Mw4500 (Alfa-Aesar, Karlsruhe, Germany) over them [Fig. 1(a)]. Several selected needles, 3 to 5 µm thick, the maximum we observed, were selected, cut at about 200 µm in length and fished out with a Hampton cryo-loop, being careful to avoid any twisting or bending of the crystal due to the surface tension of the drop [Fig. 1(b)]. They were immediately frozen in liquid nitro­gen.

Figure 1 (a) Crystals of flocculosin-A. (b) A 3 × 3 × 200 µm crystal mounted on a cryo-loop.

2.4. X-ray data collection

X-ray diffraction data were recorded at synchrotron SOLEIL (St Aubin, France) on the micro-focus beamline PROXIMA 2A (Duran et al., 2013). The X-ray wavelength was set at λ = 0.872 Å. The quality of diffraction differs greatly along the needle axis. A good position was selected from a small number of X-ray diffraction frames, showing an acceptable diffraction quality that can be characterized by a good mosaicity and no elongation of Bragg spots. The X-ray diffraction data were recorded in the standard rotation method using a DECTRIS EIGER X 9M pixel detector. The data were processed with the XDS software (Kabsch, 2010), and formatted for the SHELX suite of programs (Sheldrick, 2008).

3. Results and discussion

The crystal structure of flocculosin-A was solved by direct methods (SHELXS) and refined (SHELXL) with individual isotropic, then anisotropic thermal factors for the O atoms. H atoms were introduced at theoretical positions but were not refined. Crystal data and refinement statistics are reported in Table 1. In the final refined structure, the carboxyl­ate group of the palmitoic chain was found disordered and refined into two positions, roughly perpendicular to each other (Fig. 2). As flocculosin-A was initially isolated from an ethano­lic extract, the crystal structure interestingly retains one water (ordered) and one (disordered and partially occupied) molecule of ethanol in its crystal packing.

Table 1
Experimental details

Crystal data
Chemical formula	C40H70O19·C2.64H5.96O1.66
Mr	894.97
Crystal system, space group	Orthorhombic, P21212
Temperature (K)	103
a, b, c (Å)	16.72 (1), 54.08 (1), 5.49 (1)
V (Å^3)	4964 (10)
Z	4
Synchrotron, λ (Å)	0.872
μ (mm^−1)	0.09
Crystal size (µm)	3 × 3 × 200
Data collection
No. of measured, independent and observed [I > 2σ(I)] reflections	3646, 3646, 2986
θmax (°)	29.9
(sin θ/λ)max (Å^−1)	0.571
Refinement
R[F^2 > 2σ(F^2)], wR(F^2), S	0.083, 0.204, 1.54
No. of reflections	3646
No. of parameters	668
No. of restraints	13
H-atom treatment	H atoms treated by a mixture of independent and constrained refinement
(Δ/σ)max	0.845
Δρmax, Δρmin (e Å^−3)	0.36, −0.32
Computer program: SHELXL2018/3 (Sheldrick, 2018).

Figure 2 Mercury (Macrae et al., 2006; 2020) view of the flocculosin-A packing along the shortest axis. The two palmitoic and caproic chains are along the c axis, leading to a strongly dissymmetric cell. Water and ethanol solvate molecules have been removed for clarity.

The complete structure of flocculosin-A is built around an acetyl­ated cellobiose moiety containing two lipid chains (Fig. 3). The characteristic of these two chains is a hy­droxy­lation whose absolute configuration was unknown. Due to the known absolute configuration of the cellobiose moiety [formerly β-D-glucopyran­osyl(1→4) D-glucopyran­ose], the absolute configuration of the three chiral centres of the lipidic chains–two in the palmitoic chain and one in the caproic chain–we can now confidently extrapolate from that of the glucose moieties, as shown in Fig. 3.

Figure 3 The chemical formula of flocculosin-A (top) and a pyMOL (Schrödinger & DeLano, 2020) view (bottom) of the molecule with H atoms in theoretical positions (not refined).

4. Conclusion

By adopting a special protocol of crystallization employing slow cyclic temperature variations, we were able to improve the quality and size of the crystalline fibres of flocculosin-A. Its structure was solved by X-ray diffraction, which makes it possible to determine by comparison the absolute configuration of the asymmetric C atoms of the lipid chains attached to the cellobiose moiety of the molecule. The evaluation of the anti-fungal properties of flocculosin-A and its analogs are underway.

Supporting information

Computing details

(floc)

Crystal data

C40H70O19·C2.64H5.96O1.66	F(000) = 1848
Mr = 894.97	Dx = 1.144 Mg m^−3
Orthorhombic, P21212	Synchrotron radiation, λ = 0.872 Å
a = 16.72 (1) Å	Cell parameters from 3646 reflections
b = 54.08 (1) Å	θ = 1.0–29.0°
c = 5.49 (1) Å	µ = 0.09 mm^−1
V = 4964 (10) Å^3	T = 103 K
Z = 4	Needle, colourless

Data collection

Pixel detector diffractometer	3646 independent reflections
Radiation source: SOLEIL	2986 reflections with I > 2σ(I)
screenless rotation scans	θmax = 29.9°, θmin = 0.9°
3646 measured reflections

Refinement

Refinement on F^2	Hydrogen site location: inferred from neighbouring sites
Least-squares matrix: full	H atoms treated by a mixture of independent and constrained refinement
R[F^2 > 2σ(F^2)] = 0.084	w = 1/[σ^2(Fo^2) + (0.1P)^2] where P = (Fo^2 + 2Fc^2)/3
wR(F^2) = 0.204	(Δ/σ)max = 0.845
S = 1.54	Δρmax = 0.36 e Å^−3
3646 reflections	Δρmin = −0.32 e Å^−3
668 parameters	Absolute structure: No quotients, so Flack parameter determined by classical intensity fit
13 restraints	Absolute structure parameter: −2 (2)

Special details

Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Ų)

	x	y	z	Uiso*/Ueq	Occ. (<1)
C1A	0.4861 (3)	0.28263 (9)	−0.5511 (10)	0.0394 (13)
H1A	0.455350	0.285644	−0.397674	0.032 (12)*
C2A	0.4301 (3)	0.28209 (10)	−0.7631 (11)	0.0460 (14)
H2A	0.460141	0.281877	−0.920622	0.024 (12)*
O2A	0.3808 (2)	0.30430 (7)	−0.7426 (8)	0.0588 (12)
C3A	0.3737 (3)	0.26001 (10)	−0.7446 (12)	0.0503 (15)
H3A	0.334608	0.262924	−0.609799	0.041 (14)*
O3A	0.3302 (2)	0.25648 (7)	−0.9727 (8)	0.0551 (11)
C4A	0.4176 (3)	0.23576 (10)	−0.6994 (12)	0.0486 (15)
H4A	0.448430	0.230834	−0.847687	0.037 (14)*
O4A	0.3621 (2)	0.21699 (7)	−0.6365 (9)	0.0632 (12)
H4AO	0.332028	0.214142	−0.755751	0.062*
C5A	0.4735 (3)	0.23907 (9)	−0.4854 (11)	0.0451 (14)
H5A	0.440980	0.243247	−0.338198	0.009 (9)*
O5A	0.52653 (19)	0.25937 (6)	−0.5358 (7)	0.0441 (10)
C6A	0.5253 (4)	0.21701 (13)	−0.4268 (16)	0.074 (2)
H6A1	0.492520	0.201777	−0.431080	0.08 (2)*
H6A2	0.567926	0.215424	−0.551156	0.09 (2)*
O6A	0.5599 (3)	0.21959 (10)	−0.1971 (11)	0.0885 (16)
H6A	0.588945	0.232259	−0.195066	0.085*
O4B	0.5425 (2)	0.30081 (7)	−0.5809 (7)	0.0481 (10)
O1B	0.7224 (2)	0.35177 (7)	0.0788 (8)	0.0510 (11)
C1B	0.6985 (3)	0.33966 (10)	−0.1381 (11)	0.0456 (15)
H1B	0.739592	0.341832	−0.268790	0.024 (11)*
C2B	0.6883 (3)	0.31288 (9)	−0.0760 (11)	0.0459 (15)
H2B	0.652945	0.311860	0.070479	0.059 (17)*
O2B	0.7630 (3)	0.30160 (8)	−0.0149 (9)	0.0673 (12)
H2BO	0.800364	0.311603	−0.042143	0.068*
C3B	0.6479 (4)	0.29925 (10)	−0.2791 (13)	0.0569 (17)
H3B	0.686036	0.297956	−0.418713	0.051*
O3B	0.6332 (4)	0.27465 (8)	−0.1852 (11)	0.0927 (17)
H3BO	0.591803	0.268822	−0.250045	0.088*
C4B	0.5744 (3)	0.31198 (10)	−0.3649 (12)	0.0483 (15)
H4B	0.533126	0.311533	−0.233291	0.023 (12)*
C5B	0.5944 (3)	0.33867 (10)	−0.4245 (12)	0.0490 (16)
H5B	0.635754	0.339321	−0.556051	0.021 (11)*
O5B	0.6243 (2)	0.35023 (6)	−0.2092 (8)	0.0536 (11)
C6B	0.5195 (4)	0.35329 (12)	−0.5032 (17)	0.080 (2)
H6B1	0.487680	0.343723	−0.622681	0.077*
H6B2	0.485312	0.357092	−0.360737	0.077*
O6B	0.5505 (4)	0.37630 (10)	−0.6146 (14)	0.106 (2)
C1	0.7496 (4)	0.37675 (11)	0.0424 (12)	0.0564 (17)
H1C	0.706774	0.386820	−0.033011	0.051*
H1D	0.796775	0.376953	−0.066496	0.028 (13)*
C2	0.7710 (3)	0.38692 (10)	0.2865 (12)	0.0484 (15)
H2	0.722228	0.386123	0.391475	0.016 (10)*
O2	0.8309 (3)	0.37186 (8)	0.3961 (10)	0.0827 (15)
H2O	0.845725	0.378306	0.527863	0.078*
C3	0.7974 (4)	0.41367 (10)	0.2735 (12)	0.0550 (16)
H3C	0.758244	0.423033	0.174370	0.054 (14)*
H3D	0.849671	0.414510	0.189436	0.023 (12)*
C4	0.8049 (4)	0.42594 (10)	0.5207 (12)	0.0567 (16)
H4C	0.844624	0.416754	0.619285	0.090*
H4D	0.752822	0.424969	0.605812	0.090*
C5	0.8306 (4)	0.45304 (11)	0.5043 (13)	0.0629 (18)
H5C	0.880230	0.454172	0.406737	0.049 (15)*
H5D	0.788526	0.462535	0.418350	0.059 (18)*
C6	0.8451 (4)	0.46467 (10)	0.7533 (12)	0.0540 (16)
H6C	0.795834	0.463135	0.852369	0.062 (19)*
H6D	0.888086	0.455440	0.837429	0.048 (15)*
C7	0.8681 (4)	0.49144 (12)	0.7381 (14)	0.0677 (19)
H7C	0.825537	0.500662	0.651958	0.065*
H7D	0.917912	0.492984	0.641560	0.062*
C8	0.8812 (4)	0.50297 (11)	0.9884 (12)	0.0585 (17)
H8C	0.922886	0.493453	1.075794	0.033 (13)*
H8D	0.831047	0.501694	1.083419	0.021 (11)*
C9	0.9070 (4)	0.53052 (10)	0.9759 (13)	0.0565 (17)
H9C	0.955818	0.531898	0.874936	0.020 (11)*
H9D	0.864206	0.540178	0.895248	0.067 (19)*
C10	0.9232 (4)	0.54148 (11)	1.2226 (13)	0.0594 (17)
H10A	0.969037	0.532742	1.297733	0.062 (19)*
H10B	0.875949	0.538889	1.328139	0.025 (11)*
C11	0.9419 (4)	0.56919 (10)	1.2102 (13)	0.0599 (18)
H11C	0.990436	0.571576	1.109447	0.053 (16)*
H11D	0.897124	0.577679	1.127050	0.069 (19)*
C12	0.9552 (4)	0.58131 (10)	1.4548 (14)	0.0627 (18)
H12C	0.910859	0.576705	1.565362	0.052 (16)*
H12D	1.005497	0.574935	1.526262	0.07 (2)*
C13	0.9599 (4)	0.60913 (11)	1.4390 (12)	0.0606 (17)
H13C	0.908661	0.615446	1.373661	0.043 (15)*
H13D	1.002474	0.613613	1.321543	0.07 (2)*
C14	0.9772 (4)	0.62215 (10)	1.6824 (12)	0.0522 (16)
H14	0.937831	0.615930	1.805130	0.08 (2)*
O14	1.0534 (3)	0.61623 (8)	1.7641 (10)	0.0728 (14)
H14O	1.081062	0.629161	1.773207	0.074*
C15	0.9658 (5)	0.64943 (12)	1.6581 (14)	0.0679 (19)
H15C	0.913083	0.652344	1.580945	0.087*
H15D	1.007038	0.655768	1.544836	0.087*
C16A	0.9695 (13)	0.6647 (3)	1.889 (4)	0.050 (7)	0.5
O16A	1.0163 (10)	0.6585 (2)	2.048 (3)	0.083 (3)	0.5
O16B	0.9341 (8)	0.68435 (19)	1.890 (2)	0.098 (4)	0.5
H16B	0.940700	0.691337	2.025492	0.098*	0.5
C16B	0.9911 (16)	0.6623 (8)	1.882 (5)	0.089 (12)	0.5
O16C	1.0579 (11)	0.6705 (3)	1.902 (4)	0.135 (5)	0.5
O16D	0.9517 (10)	0.6594 (4)	2.064 (4)	0.116 (5)	0.5
H16D	0.980294	0.662189	2.186939	0.112*	0.5
C1'	0.3648 (12)	0.4395 (4)	−1.635 (4)	0.282 (11)
H1A'	0.348754	0.453423	−1.738810	0.250*
H1B'	0.407740	0.444816	−1.525476	0.250*
H1C'	0.384009	0.425912	−1.737778	0.250*
C2'	0.2945 (9)	0.4310 (3)	−1.488 (4)	0.209 (8)
H2A'	0.250912	0.425973	−1.600293	0.153*
H2B'	0.274640	0.444875	−1.388015	0.154*
C3'	0.3146 (9)	0.4096 (3)	−1.325 (3)	0.239 (9)
H3A'	0.352022	0.398504	−1.411844	0.151*
H3B'	0.342864	0.416034	−1.179443	0.155*
C4'	0.2449 (7)	0.3947 (2)	−1.241 (3)	0.149 (5)
H4A'	0.203180	0.406632	−1.190270	0.156*
H4B'	0.223882	0.386019	−1.386394	0.159*
C5'	0.2511 (8)	0.37759 (19)	−1.060 (3)	0.136 (4)
H5A'	0.196080	0.373383	−1.008558	0.23 (7)*
H5B'	0.277277	0.385785	−0.920238	0.21 (7)*
C6'	0.2919 (7)	0.35505 (19)	−1.101 (2)	0.115 (3)
HA6'	0.346031	0.359305	−1.166362	0.12 (4)*
O6'	0.2495 (5)	0.34202 (17)	−1.2926 (19)	0.155 (3)
H6'	0.275659	0.329431	−1.333650	0.153*
C7'	0.3048 (5)	0.33749 (16)	−0.8800 (18)	0.094 (3)
H7A'	0.328999	0.347002	−0.744741	0.084*
H7B'	0.252120	0.331348	−0.823913	0.086*
C8'	0.3575 (4)	0.31577 (13)	−0.9377 (15)	0.0651 (18)
O8'	0.3758 (4)	0.30956 (10)	−1.1437 (13)	0.0969 (17)
C1"	0.5125 (8)	0.3857 (3)	−0.785 (3)	0.126 (4)
O1"	0.4565 (7)	0.3763 (2)	−0.885 (3)	0.213 (6)
C2"	0.5446 (8)	0.4046 (3)	−0.905 (3)	0.178 (6)
H2A"	0.589694	0.411280	−0.810973	0.158*
H2B"	0.563623	0.398996	−1.064002	0.152*
H2C"	0.504272	0.417578	−0.926913	0.158*
O1#	0.2229 (3)	0.27563 (8)	−0.8156 (10)	0.0708 (13)
C1#	0.2545 (4)	0.26365 (11)	−0.9811 (13)	0.0549 (16)
C2#	0.2145 (4)	0.25732 (14)	−1.2169 (13)	0.0695 (19)
H2A#	0.251311	0.247752	−1.318783	0.065*
H2B#	0.166361	0.247504	−1.184504	0.077*
H2C#	0.199506	0.272575	−1.301922	0.074*
OW	0.3539 (3)	0.17811 (8)	−0.3270 (9)	0.0743 (13)
C1W_1	0.615 (4)	0.4675 (7)	1.823 (9)	0.17 (3)	0.3
C2W_1	0.627 (3)	0.4827 (13)	1.599 (9)	0.15 (3)	0.3
O1W_1	0.609 (3)	0.4815 (9)	2.042 (8)	0.19 (3)	0.3
C1X_1	0.606 (4)	0.4900 (13)	1.76 (2)	0.21 (4)	0.3
C2X_1	0.581 (4)	0.4835 (13)	1.517 (18)	0.21 (3)	0.3
O1X_1	0.591 (3)	0.4716 (9)	1.931 (15)	0.167 (17)	0.3

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
C1A	0.035 (3)	0.045 (3)	0.039 (3)	−0.004 (3)	0.002 (3)	−0.002 (3)
C2A	0.046 (3)	0.050 (3)	0.042 (4)	−0.005 (3)	0.000 (3)	−0.002 (3)
O2A	0.064 (3)	0.057 (2)	0.055 (3)	0.008 (2)	−0.003 (2)	−0.004 (2)
C3A	0.041 (3)	0.056 (3)	0.053 (4)	−0.006 (3)	−0.004 (3)	0.001 (3)
O3A	0.049 (2)	0.065 (2)	0.051 (3)	−0.0033 (19)	−0.003 (2)	−0.007 (2)
C4A	0.045 (3)	0.050 (3)	0.050 (4)	0.001 (3)	0.005 (3)	−0.003 (3)
O4A	0.061 (3)	0.061 (2)	0.068 (3)	−0.013 (2)	−0.010 (2)	0.006 (2)
C5A	0.041 (3)	0.048 (3)	0.046 (4)	−0.006 (3)	0.000 (3)	0.002 (3)
O5A	0.0386 (19)	0.047 (2)	0.047 (2)	−0.0027 (17)	0.0051 (19)	−0.0014 (19)
C6A	0.054 (4)	0.067 (4)	0.100 (6)	0.001 (3)	−0.007 (5)	0.010 (4)
O6A	0.071 (3)	0.093 (4)	0.102 (5)	−0.003 (3)	−0.012 (3)	0.021 (3)
O4B	0.053 (2)	0.051 (2)	0.041 (2)	−0.0013 (19)	−0.0010 (19)	0.0007 (19)
O1B	0.053 (2)	0.048 (2)	0.052 (3)	−0.0035 (18)	−0.002 (2)	−0.005 (2)
C1B	0.043 (3)	0.052 (3)	0.042 (4)	0.000 (3)	−0.005 (3)	−0.003 (3)
C2B	0.047 (3)	0.046 (3)	0.044 (4)	−0.001 (3)	0.000 (3)	−0.001 (3)
O2B	0.068 (3)	0.067 (3)	0.067 (3)	0.004 (2)	−0.010 (3)	−0.011 (3)
C3B	0.064 (4)	0.049 (4)	0.058 (4)	−0.001 (3)	−0.014 (3)	−0.006 (3)
O3B	0.126 (4)	0.058 (3)	0.094 (4)	−0.002 (3)	−0.045 (4)	0.002 (3)
C4B	0.046 (3)	0.053 (3)	0.046 (4)	−0.001 (3)	0.005 (3)	0.003 (3)
C5B	0.049 (3)	0.045 (3)	0.053 (4)	−0.009 (3)	−0.010 (3)	−0.005 (3)
O5B	0.056 (2)	0.051 (2)	0.054 (3)	−0.0023 (19)	−0.008 (2)	−0.001 (2)
C6B	0.093 (5)	0.054 (4)	0.092 (6)	−0.005 (4)	−0.029 (5)	0.002 (4)
O6B	0.118 (5)	0.084 (4)	0.117 (5)	0.015 (3)	−0.020 (5)	−0.002 (4)
C1	0.060 (4)	0.059 (4)	0.050 (4)	−0.010 (3)	−0.001 (4)	0.000 (3)
C2	0.048 (3)	0.049 (3)	0.048 (4)	−0.002 (3)	−0.004 (3)	0.000 (3)
O2	0.092 (3)	0.074 (3)	0.083 (4)	−0.002 (3)	−0.028 (3)	−0.011 (3)
C3	0.062 (4)	0.049 (3)	0.054 (4)	−0.007 (3)	−0.006 (4)	−0.005 (3)
C4	0.066 (4)	0.048 (3)	0.056 (4)	−0.006 (3)	−0.008 (4)	−0.006 (3)
C5	0.069 (4)	0.057 (4)	0.063 (4)	−0.005 (3)	−0.007 (4)	−0.004 (3)
C6	0.063 (4)	0.048 (3)	0.052 (4)	−0.006 (3)	−0.003 (3)	0.002 (3)
C7	0.074 (4)	0.061 (4)	0.069 (5)	−0.004 (3)	−0.009 (4)	−0.004 (4)
C8	0.069 (4)	0.056 (4)	0.051 (4)	−0.004 (3)	0.002 (4)	−0.007 (3)
C9	0.067 (4)	0.048 (3)	0.055 (4)	−0.005 (3)	−0.005 (4)	0.000 (3)
C10	0.069 (4)	0.050 (3)	0.060 (4)	−0.008 (3)	−0.004 (4)	0.002 (3)
C11	0.072 (4)	0.048 (3)	0.060 (4)	−0.011 (3)	−0.008 (4)	0.000 (3)
C12	0.067 (4)	0.051 (4)	0.070 (5)	−0.004 (3)	0.001 (4)	−0.003 (3)
C13	0.075 (4)	0.057 (4)	0.049 (4)	−0.002 (3)	0.001 (4)	0.001 (3)
C14	0.051 (4)	0.054 (4)	0.052 (4)	−0.006 (3)	0.001 (3)	−0.003 (3)
O14	0.076 (3)	0.074 (3)	0.068 (3)	−0.004 (2)	0.001 (3)	−0.007 (3)
C15	0.083 (5)	0.064 (4)	0.057 (5)	0.000 (3)	0.002 (4)	−0.008 (4)
C16A	0.071 (12)	0.035 (9)	0.043 (16)	−0.012 (9)	0.018 (12)	−0.005 (8)
O16A	0.101 (12)	0.087 (8)	0.060 (9)	−0.001 (7)	0.001 (10)	0.014 (6)
O16B	0.138 (10)	0.070 (7)	0.085 (9)	0.032 (7)	−0.027 (8)	−0.026 (6)
C16B	0.055 (13)	0.16 (3)	0.05 (2)	−0.003 (15)	0.020 (15)	0.015 (19)
O16C	0.133 (13)	0.143 (13)	0.130 (15)	0.015 (11)	−0.016 (12)	−0.041 (12)
O16D	0.089 (11)	0.148 (14)	0.112 (16)	−0.032 (10)	0.010 (10)	−0.027 (12)
C1'	0.29 (3)	0.31 (3)	0.24 (2)	−0.03 (2)	0.00 (3)	0.06 (2)
C2'	0.204 (16)	0.169 (13)	0.25 (2)	0.013 (12)	0.000 (19)	0.038 (16)
C3'	0.31 (2)	0.204 (17)	0.204 (19)	0.022 (18)	0.03 (2)	0.027 (16)
C4'	0.139 (10)	0.142 (10)	0.165 (13)	−0.016 (8)	−0.030 (10)	0.029 (10)
C5'	0.151 (11)	0.103 (8)	0.155 (12)	0.046 (7)	−0.030 (10)	0.000 (9)
C6'	0.118 (8)	0.128 (8)	0.099 (8)	0.017 (7)	−0.012 (7)	0.009 (7)
O6'	0.158 (7)	0.157 (7)	0.149 (8)	0.024 (5)	−0.008 (7)	0.006 (6)
C7'	0.093 (6)	0.101 (6)	0.088 (6)	0.022 (5)	−0.015 (5)	0.002 (5)
C8'	0.069 (4)	0.072 (4)	0.055 (5)	−0.003 (4)	0.001 (4)	0.003 (4)
O8'	0.110 (4)	0.106 (4)	0.074 (4)	0.018 (3)	0.004 (4)	0.009 (3)
C1"	0.091 (7)	0.150 (10)	0.137 (11)	0.010 (7)	0.009 (8)	0.055 (9)
O1"	0.142 (7)	0.261 (12)	0.237 (14)	−0.017 (8)	−0.038 (10)	0.087 (11)
C2"	0.177 (13)	0.190 (13)	0.167 (14)	0.024 (11)	0.000 (13)	0.027 (13)
O1#	0.068 (3)	0.081 (3)	0.064 (3)	−0.002 (2)	−0.005 (3)	0.001 (3)
C1#	0.054 (4)	0.063 (4)	0.047 (4)	−0.003 (3)	0.006 (4)	0.004 (3)
C2#	0.058 (4)	0.093 (5)	0.057 (5)	−0.001 (3)	−0.004 (4)	0.014 (4)
OW	0.084 (3)	0.064 (2)	0.075 (3)	0.002 (2)	0.019 (3)	0.000 (2)
C1W_1	0.10 (3)	0.27 (8)	0.14 (5)	0.01 (4)	0.05 (3)	−0.07 (5)
C2W_1	0.05 (3)	0.17 (5)	0.24 (7)	0.02 (3)	−0.02 (4)	−0.02 (5)
O1W_1	0.10 (2)	0.16 (4)	0.30 (7)	−0.05 (2)	−0.02 (3)	−0.03 (4)
C1X_1	0.09 (4)	0.20 (6)	0.35 (11)	0.02 (4)	0.01 (6)	0.18 (7)
C2X_1	0.11 (4)	0.21 (6)	0.30 (8)	0.06 (4)	−0.03 (6)	0.03 (6)
O1X_1	0.13 (4)	0.13 (3)	0.25 (5)	−0.03 (2)	0.00 (4)	0.03 (3)

Geometric parameters (Å, º)

C1A—O4B	1.373 (6)	C6—C7	1.500 (8)
C1A—O5A	1.430 (6)	C7—C8	1.525 (10)
C1A—C2A	1.494 (8)	C8—C9	1.553 (8)
C2A—O2A	1.461 (7)	C9—C10	1.503 (10)
C2A—C3A	1.525 (8)	C10—C11	1.533 (8)
O2A—C8'	1.298 (9)	C11—C12	1.511 (10)
C3A—O3A	1.461 (7)	C12—C13	1.509 (8)
C3A—C4A	1.523 (8)	C13—C14	1.538 (9)
O3A—C1#	1.325 (8)	C14—O14	1.388 (7)
C4A—O4A	1.417 (7)	C14—C15	1.494 (9)
C4A—C5A	1.512 (9)	C15—C16B	1.47 (3)
C5A—O5A	1.438 (6)	C15—C16A	1.51 (2)
C5A—C6A	1.510 (9)	C16A—O16A	1.216 (10)
C6A—O6A	1.394 (10)	C16A—O16B	1.219 (10)
O4B—C4B	1.434 (7)	C16B—O16C	1.208 (11)
O1B—C1B	1.416 (7)	C16B—O16D	1.209 (11)
O1B—C1	1.439 (7)	C1'—C2'	1.500 (10)
C1B—O5B	1.422 (7)	C2'—C3'	1.503 (10)
C1B—C2B	1.497 (8)	C3'—C4'	1.486 (10)
C2B—O2B	1.429 (7)	C4'—C5'	1.363 (15)
C2B—C3B	1.498 (9)	C5'—C6'	1.414 (13)
C3B—O3B	1.447 (8)	C6'—O6'	1.451 (13)
C3B—C4B	1.484 (8)	C6'—C7'	1.556 (14)
C4B—C5B	1.518 (8)	C7'—C8'	1.502 (11)
C5B—O5B	1.427 (7)	C8'—O8'	1.219 (9)
C5B—C6B	1.543 (9)	C1"—O1"	1.201 (16)
C6B—O6B	1.481 (10)	C1"—C2"	1.330 (17)
O6B—C1"	1.239 (14)	O1#—C1#	1.234 (8)
C1—C2	1.492 (9)	C1#—C2#	1.497 (10)
C2—O2	1.425 (8)	C1W_1—O1W_1	1.422 (11)
C2—C3	1.514 (8)	C1W_1—C2W_1	1.497 (11)
C3—C4	1.517 (9)	C1X_1—O1X_1	1.38 (10)
C4—C5	1.529 (8)	C1X_1—C2X_1	1.46 (14)
C5—C6	1.525 (9)
O4B—C1A—O5A	108.2 (4)	C1—C2—C3	112.3 (5)
O4B—C1A—C2A	110.6 (4)	C2—C3—C4	113.6 (5)
O5A—C1A—C2A	108.9 (4)	C3—C4—C5	113.0 (5)
O2A—C2A—C1A	106.1 (4)	C6—C5—C4	112.8 (5)
O2A—C2A—C3A	106.9 (4)	C7—C6—C5	112.9 (5)
C1A—C2A—C3A	110.5 (5)	C6—C7—C8	112.4 (6)
C8'—O2A—C2A	119.9 (5)	C7—C8—C9	113.1 (5)
O3A—C3A—C4A	105.5 (5)	C10—C9—C8	112.9 (5)
O3A—C3A—C2A	110.7 (5)	C9—C10—C11	112.5 (5)
C4A—C3A—C2A	112.8 (5)	C12—C11—C10	114.5 (6)
C1#—O3A—C3A	117.8 (5)	C13—C12—C11	112.9 (6)
O4A—C4A—C5A	107.5 (5)	C12—C13—C14	114.6 (5)
O4A—C4A—C3A	110.0 (4)	O14—C14—C15	111.9 (5)
C5A—C4A—C3A	108.8 (5)	O14—C14—C13	110.3 (5)
O5A—C5A—C6A	106.9 (4)	C15—C14—C13	110.5 (6)
O5A—C5A—C4A	108.8 (4)	C16B—C15—C14	110.7 (19)
C6A—C5A—C4A	115.3 (5)	C14—C15—C16A	117.2 (8)
C1A—O5A—C5A	113.0 (4)	O16A—C16A—O16B	123.3 (16)
O6A—C6A—C5A	110.6 (6)	O16A—C16A—C15	118.5 (12)
C1A—O4B—C4B	117.3 (4)	O16B—C16A—C15	117.3 (14)
C1B—O1B—C1	114.0 (4)	O16C—C16B—O16D	118 (3)
O1B—C1B—O5B	106.9 (4)	O16C—C16B—C15	121 (2)
O1B—C1B—C2B	106.7 (5)	O16D—C16B—C15	118.3 (17)
O5B—C1B—C2B	110.6 (4)	C1'—C2'—C3'	112.6 (12)
O2B—C2B—C1B	111.5 (5)	C4'—C3'—C2'	115.1 (12)
O2B—C2B—C3B	111.0 (4)	C5'—C4'—C3'	122.1 (12)
C1B—C2B—C3B	111.0 (5)	C4'—C5'—C6'	120.5 (13)
O3B—C3B—C4B	113.5 (5)	C5'—C6'—O6'	107.3 (10)
O3B—C3B—C2B	105.3 (5)	C5'—C6'—C7'	118.0 (11)
C4B—C3B—C2B	112.4 (5)	O6'—C6'—C7'	109.6 (9)
O4B—C4B—C3B	112.0 (5)	C8'—C7'—C6'	113.2 (8)
O4B—C4B—C5B	107.7 (5)	O8'—C8'—O2A	123.9 (7)
C3B—C4B—C5B	109.1 (5)	O8'—C8'—C7'	124.0 (7)
O5B—C5B—C4B	108.4 (5)	O2A—C8'—C7'	112.1 (7)
O5B—C5B—C6B	106.9 (5)	O1"—C1"—O6B	124.9 (14)
C4B—C5B—C6B	111.6 (5)	O1"—C1"—C2"	114.4 (15)
C1B—O5B—C5B	110.9 (4)	O6B—C1"—C2"	118.8 (13)
O6B—C6B—C5B	105.1 (6)	O1#—C1#—O3A	122.5 (6)
C1"—O6B—C6B	118.5 (9)	O1#—C1#—C2#	124.4 (6)
O1B—C1—C2	107.3 (5)	O3A—C1#—C2#	112.9 (6)
O2—C2—C1	109.7 (5)	O1W_1—C1W_1—C2W_1	114.3 (12)
O2—C2—C3	111.2 (5)	O1X_1—C1X_1—C2X_1	113 (8)