The self-assembling zwitterionic form of L-phenyl­alanine at neutral pH

The title zwitterion (2S)-2-aza­niumyl-1-hy­droxy-3-phenyl­pro­pan-1-olate, C₉H₁₁NO₂, also known as L-phenyl­alanine, was characterized using synchrotron X-rays. It crystallized in the monoclinic space group P2₁ with four mol­ecules in the asymmetric unit. The 0.62 Å resolution structure is assumed to be closely related to the fibrillar form of phenylalanine, as observed by electron microscopy and electron diffraction. The structure exists in a zwitterionic form in which π–π stacking and hydrogen-bonding inter­actions are believed to form the basis of the self-assembling properties.

S1. Introduction

L-Phenyl­alanine is an essential aromatic and hydro­phobic amino acid and is one of the 20 amino acids found in proteins. X-ray diffraction studies by Gurskaya & Vainshtein (1963) started with the acidic form of L-phenyl­alanine. These authors noted an orthorhombic cell with space group P2₁2₁2₁ and unit-cell dimensions a = 27.68 Å, b = 6.98 Å and c = 5.34 Å. Their model was derived from a three-dimensional Patterson analysis and described a layered structure in which the groups of hydrogen-bonded phenyl­alanine molecules are stabilized by van der Waals forces. Some time later, Al-Karaghouli & Koetzle (1975) conducted a single-crystal neutron study of the same molecule, allowing precise determination of the H-atom positions and the hydrogen-bonding network. The space group and unit-cell dimensions were consistent with the X-ray results (a = 27.76 Å, b = 7.07 Å and c = 5.38 Å). The structure formed a layered system held together by hydrogen-bonded chloride ions with each chloride ion linking four molecules. The inter-layer inter­actions were also assumed to be weak van der Waals forces, confirming the work of Gurskaya & Vainshtein (1963). Powder diffraction studies have also been carried out: Khawas (1970) crystallized L-phenyl­alanine from aqueous solution but had difficulties growing large L-phenyl­alanine single crystals. Their samples were nonetheless highly crystalline and X-ray powder diffraction experiments were carried out yielding a unit cell of a = 13.13 Å, b = 6.59 Å, c = 10.348 Å and β = 104.38°, space group P2₁.

L-Phenyl­alanine is inter­esting for its importance in human health and implications in conditions such as lethargy, liver damage and phenyl­ketonuria (PKU). PKU is a good example of the consequences of an excess of L-phenyl­alanine in the brain. High concentrations of phenyl­alanine are extremely harmful especially during early infancy and, if not diagnosed and treated immediately (with a phenyl­alanine-reduced diet), result in profound and permanent mental retardation, epilespy and microcephaly (Martynyuk et al., 2005).

Recently, L-phenyl­alanine has attracted inter­est for its possible link to self-assembly in amyloid type systems. Studies on the islet amyloid polypeptide (IAPP) (Tenidis et al. (2000)), known to be associated with Type II diabetes, have shown that phenyl­alanine plays a crucial role in the formation of amyloid fibrils. Azriel et al. (2001) have argued that there is `experimental support for the key role of the phenyl­alanine residue in self-assembly associated with amyloid formation`.

Over the past decade, there has been extensive research into the assembly properties of a wide range of peptide systems and their ability to form nanotubes are of potential commercial significance. More recently, a number of groups have reported the assembly properties of simpler systems, such as di­phenyl­alanine, and also a number of amino acid derivative systems. For example, Görbitz (2001) observed nanotube formation from di­phenyl­alanine, and Gazit and co-workers (Adler-Abramovich et al., 2006; Tamamis et al., 2009)) have subsequently studied the physical and thermal properties of these nanotubes. Adler-Abramovich et al. (2012) has shown that L-phenyl­alanine, like di­phenyl­alanine, has remarkable properties of self-assembly from aqueous solution, forming stable nanofilaments that have been observed by electron microscopy. Hence, this relatively simple molecule appears to have complex behaviour that may be of relevance for a number of the biomedical issues.

S2. Experimental

S2.1. Synthesis and crystallization

L-Phenyl­alanine was purchased from Sigma–Aldrich. The best crystals were obtained in crystallization screenings using drops of 20 ml containing 10 mg ml^-1 of L-phenyl­alanine in 15% polyethyl­ene glycol (PEG) 4 K, 15% propan-2-ol and 0.05 M NaCl. Crystals formed over a period of one week. There were then cryocooled straight from the drop without the addition of cryoprote­ctant.

X-ray diffraction data were collected on ID23-1 at the ESRF to a resolution of 0.62 Å.

S2.2. Refinement

Crystal data, data collection and structure refinement details are summarized in Table 1. H atoms were positioned geometrically and refined as riding, with C—H = 0.95 (aromatic), 0.99 (methyl­ene) or 1.0 Å (NCR₂) and N—H = 0.91 Å (tertiary amine), and with U_iso(H) = 1.5U_eq(N) for amine H and 1.2U_eq(C) for all others. Nine non-H atoms (C1A, N1A, C2A, C1C, C2C, O1C, C1D, N1D and C2D) were refined with isotropic displacement parameters to avoid nonpositive definites (NPDs).

S3. Results and discussion

As part of a set of X-ray diffraction experiments designed to characterize this phenyl­alanine nanotube structure, a new crystalline form of zwitterionic L-phenyl­alanine [systematic name: (2S)-2-aza­niumyl-1-hy­droxy-3-phenyl­propan-1-olate] has been identified (Fig. 1 and Table 2). Extensive macroscopic characterization work on phenyl­alanine has led to a full structural characterization of this new crystalline form. It should be noted that although this structure was indexed as a primitive unit cell, it exhibits a pseudo-B-face-centring geometry, as can be seen in Fig. 2. A layered structure stabilized by alternating hydro­phobic (aromatic environment) and hydro­philic inter­actions is observed in the crystal packing. Each layer is composed of two rows of phenyl­alanine molecules held together by hydrogen bonds, whereas the inter­layer inter­actions between the hydro­phobic rings are thought to be related to π–π stacking (Figs. 3 and 5). Previous studies on aromatic inter­actions between phenyl rings implicated π–π stacking inter­actions as playing a critical role in self-assembly (Gillard et al., 1997). Olsztynska et al. (2006) suggested that the presence of hydro­phobic inter­actions in L-phenyl­alanine dissolved in water leads to the aggregation of the molecules via π–π stacking. Our structure adopts laterally displaced rings involving residues B and D, and A and C. Furthermore, edge-to-face ring inter­actions (Jennings et al., 2001) are noted between layers, with the rings forming an angle of approximately 45° with respect to each other. Both L-phenyl­alanine–L-phenyl­alanine malonate (Görbitz & Etter, 1992) and L-phenyl­alanine hydro­chloride (Al-Karaghouli & Koetzle, 1975) adopt a similar structure to the one published here, with a combination of an edge-to-face and parallel-displaced conformations. However, in both these studies the rings are all in register, as opposed to the four different conformations adopted by the molecules presented here. To the best of our knowledge, this is the only L-phenyl­alanine structure showing four distinct conformations of the amino acid. The existence of two types of π-stacking inter­actions within this structure is also consistent with the structure of the amyloid forming peptide KFFEAAAKKFFE solved by Makin et al. (2005). This configuration corresponds to an energically favoured stacking arrangement (McGaughey et al., 1998).

Within the individual asymmetric units, hydrogen bonding occurs between the carboxyl­ate and amine groups (Fig. 4) forming a two-dimensional hydrogen-bond network parallel with the ac plane. Each molecule takes part in four inter­molecular N—H···O hydrogen bonds (Table 2), three of which are illustrated in Fig. 4. The fourth N—H···O inter­action is not visible in Fig. 4 since it occurs between residues in neighbouring layers of the lattice. Collectively, these hydrogen bonds, within and between layers, in combination with the π–π stacking geometry described above, form a strong set of inter­action that hold this structure together and that are likely to be significant in the self-assembly of phenyl­alanine fibrils (Figs. 3 and 5). Our observations in this structure that the phenyl­alanine rings are located in the hydro­phobic region of the structure can be related to the observations of Chelli et al. (2002) that π-stacking inter­actions between the rings are more frequent in the hydro­phobic core of the protein.

As seen in Fig. 6, L-phenyl­alanine is found by light microscopy to form fibrous structures, reflecting its fundamental tendency to self-assemble into fibrils.

The relationship between this water-free structure and the filamentous structure as seen by fibre diffraction and electron microscopy is part of an ongoing study that will be published elsewhere (Mossou et al., 2014). It appears likely that the π–π stacking between neighbouring phenyl rings is the basis of the unique specific-aggregation properties that lead to the formation of the nanotube structures, with the short axis of the unit cell corresponding to the fibre stacking axis of the fibrils.

Related literature

For related literature, see: Adler-Abramovich, Reches, Sedman, Allen, Tendler & Gazit (2006); Adler-Abramovich, Vaks, Carny, Trudler, Frenkel & Gazit (2012); Al-Karaghouli & Koetzle (1975); Azriel & Gazit (2001); Chelli et al. (2002); Gillard et al. (1997); Görbitz (2001); Görbitz & Etter (1992); Gurskaya & Vainshtein (1963); Jennings et al. (2001); Khawas (1970); Makin et al. (2005); Martynyuk et al. (2005); McGaughey et al. (1998); Olsztynska et al. (2006); Tamamis et al. (2009); Tenidis et al. (2000).

Computing details

Data collection: MxCuBE (Gabadinho et al., 2010); cell refinement: XDS (Kabsch, 1993); data reduction: XDS (Kabsch, 1993); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL2013 (Sheldrick, 2008); molecular graphics: PLATON (Spek, 2003, 2009); software used to prepare material for publication: publCIF (Westrip, 2010).

Figures

	Fig. 1. The contents of the asymmetric unit with residues A to D from the zwitterionic L-phenylalanine structure. Displacement ellipsoids are drawn at the 50% probability level.
	Fig. 2. The monoclinic unit cell of L-phenylalanine.
	Fig. 3. Diagram showing the layers of phenylalanine residues.
	Fig. 4. The two-dimensional hydrogen-bond network in the L-phenylalanine structure.
	Fig. 5. Diagram showing the interactions holding the L-phenylalanine structure together. The π-stacking interactions between the rings within a layer are dominated by so-called nearly parallel-displaced interactions, while the interactions between them are based on edge-to-face interactions between the rings. Hydrogen bonds between the carboxylate and amine group contribute to the stacking.
	Fig. 6. Optical microscopy image showing filaments formed from L-phenylalanine.

(I)

Crystal data

C9H11NO2	F(000) = 704
Mr = 165.19	Dx = 1.349 Mg m^−3
Monoclinic, P21	Synchrotron radiation, λ = 0.61995 Å
a = 6.0010 (5) Å	Cell parameters from 841 reflections
b = 30.8020 (17) Å	θ = 1–20°
c = 8.7980 (4) Å	µ = 0.10 mm^−1
β = 90.120 (4)°	T = 100 K
V = 1626.24 (17) Å^3	Polygon, colourless
Z = 8	0.20 × 0.10 × 0.07 mm

Data collection

MD2M mini diffractometer	Rint = 0.064
Radiation source: synchrotron	θmax = 30.5°, θmin = 2.0°
profile from θ /2θ scans	h = −9→9
45788 measured reflections	k = −47→47
12740 independent reflections	l = −13→12
10932 reflections with I > 2σ(I)

Refinement

Refinement on F^2	Hydrogen site location: inferred from neighbouring sites
Least-squares matrix: full	H-atom parameters constrained
R[F^2 > 2σ(F^2)] = 0.064	w = 1/[σ^2(Fo^2) + (0.0808P)^2 + 1.0335P] where P = (Fo^2 + 2Fc^2)/3
wR(F^2) = 0.170	(Δ/σ)max < 0.001
S = 1.15	Δρmax = 0.91 e Å^−3
12740 reflections	Δρmin = −0.75 e Å^−3
389 parameters	Absolute structure: Flack x determined using 4805 quotients [(I+)-(I-)]/[(I+)+(I-)] (Parsons and Flack (2004), Acta Cryst. A60, s61).
1 restraint	Absolute structure parameter: 0.0 (4)

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.

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

	x	y	z	Uiso*/Ueq
O2A	0.5490 (3)	0.02363 (6)	0.7803 (2)	0.0063 (3)
O1A	0.2463 (3)	0.01989 (7)	0.9286 (2)	0.0081 (3)
C1A	0.4529 (4)	0.01845 (7)	0.9055 (3)	0.0034 (4)*
N1A	0.5090 (3)	0.03736 (6)	1.1741 (2)	0.0049 (3)*
H11A	0.5181	0.0658	1.1463	0.007*
H12A	0.5929	0.0329	1.2590	0.007*
H13A	0.3645	0.0305	1.1939	0.007*
C2A	0.5939 (4)	0.00934 (7)	1.0478 (3)	0.0038 (4)*
H21A	0.7538	0.0163	1.0271	0.005*
C3A	0.5733 (4)	−0.03755 (8)	1.1029 (3)	0.0083 (4)
H31A	0.6520	−0.0401	1.2015	0.010*
H32A	0.4138	−0.0438	1.1214	0.010*
C4A	0.6634 (4)	−0.07162 (8)	0.9970 (3)	0.0077 (4)
C5A	0.5310 (5)	−0.08962 (9)	0.8829 (3)	0.0135 (5)
H51A	0.3845	−0.0788	0.8668	0.016*
C6A	0.6109 (7)	−0.12319 (10)	0.7923 (4)	0.0215 (7)
H61A	0.5191	−0.1350	0.7144	0.026*
C7A	0.8230 (7)	−0.13945 (10)	0.8148 (4)	0.0232 (7)
H71A	0.8761	−0.1629	0.7547	0.028*
C8A	0.9577 (6)	−0.12114 (11)	0.9263 (5)	0.0219 (7)
H81A	1.1042	−0.1319	0.9421	0.026*
C9A	0.8794 (5)	−0.08719 (9)	1.0148 (4)	0.0138 (5)
H91A	0.9746	−0.0744	1.0885	0.017*
O2B	0.5510 (3)	0.11439 (6)	0.9990 (2)	0.0069 (3)
O1B	0.2387 (3)	0.11734 (7)	0.8591 (2)	0.0088 (3)
N1B	0.5026 (3)	0.10167 (7)	0.6082 (2)	0.0043 (3)
H11B	0.5811	0.1075	0.5222	0.006*
H12B	0.3548	0.1058	0.5901	0.006*
H13B	0.5265	0.0736	0.6366	0.006*
C2B	0.5773 (4)	0.13141 (8)	0.7326 (3)	0.0038 (4)
H21B	0.7402	0.1273	0.7519	0.005*
C3B	0.5340 (4)	0.17805 (8)	0.6786 (3)	0.0072 (4)
H31B	0.6239	0.1835	0.5863	0.009*
H32B	0.3750	0.1808	0.6498	0.009*
C4B	0.5883 (4)	0.21249 (8)	0.7953 (3)	0.0065 (4)
C1B	0.4455 (4)	0.12022 (8)	0.8765 (3)	0.0040 (4)
C5B	0.4241 (4)	0.24153 (9)	0.8420 (3)	0.0103 (5)
H51B	0.2777	0.2388	0.8018	0.012*
C9B	0.8012 (5)	0.21697 (9)	0.8562 (3)	0.0108 (5)
H91B	0.9159	0.1976	0.8259	0.013*
C7B	0.6817 (5)	0.27839 (9)	1.0067 (3)	0.0127 (5)
H71B	0.7137	0.3005	1.0788	0.015*
C8B	0.8480 (5)	0.24964 (9)	0.9611 (3)	0.0120 (5)
H81B	0.9941	0.2523	1.0019	0.014*
C6B	0.4693 (5)	0.27442 (10)	0.9461 (4)	0.0150 (5)
H61B	0.3552	0.2941	0.9754	0.018*
O1C	0.7424 (3)	0.01836 (6)	0.4276 (2)	0.0087 (3)*
O2C	1.0545 (3)	0.02108 (6)	0.2877 (2)	0.0068 (3)
N1C	1.0008 (3)	0.03647 (7)	0.6786 (2)	0.0047 (3)
H11C	1.0162	0.0646	0.6484	0.007*
H12C	1.0814	0.0320	0.7648	0.007*
H13C	0.8545	0.0308	0.6971	0.007*
C2C	1.0835 (4)	0.00685 (7)	0.5555 (3)	0.0032 (4)*
H21C	1.2452	0.0125	0.5368	0.004*
C4C	1.1204 (4)	−0.07435 (8)	0.5024 (3)	0.0070 (4)
C9C	1.3395 (4)	−0.07789 (9)	0.4487 (3)	0.0114 (5)
H91C	1.4494	−0.0577	0.4807	0.014*
C3C	1.0525 (4)	−0.03957 (8)	0.6140 (3)	0.0074 (4)
H31C	1.1411	−0.0432	0.7083	0.014 (10)*
H32C	0.8938	−0.0438	0.6406	0.017*
C5C	0.9634 (5)	−0.10435 (9)	0.4522 (4)	0.0125 (5)
H51C	0.8140	−0.1021	0.4869	0.015*
C6C	1.0202 (5)	−0.13751 (10)	0.3527 (4)	0.0169 (6)
H61C	0.9102	−0.1576	0.3197	0.020*
C8C	1.3957 (5)	−0.11105 (10)	0.3487 (4)	0.0148 (5)
H81C	1.5442	−0.1131	0.3121	0.018*
C1C	0.9501 (4)	0.01616 (7)	0.4101 (3)	0.0036 (4)*
C7C	1.2387 (5)	−0.14121 (9)	0.3013 (4)	0.0157 (5)
H71C	1.2797	−0.1641	0.2346	0.019*
O1D	0.7469 (3)	0.11726 (7)	0.3557 (2)	0.0082 (3)
O2D	1.0503 (3)	0.11366 (6)	0.5056 (2)	0.0062 (3)
C3D	1.0828 (4)	0.17255 (8)	0.1799 (3)	0.0075 (4)
H31D	1.1711	0.1749	0.0853	0.009*
H32D	0.9258	0.1790	0.1538	0.009*
C2D	1.0964 (4)	0.12565 (7)	0.2371 (3)	0.0040 (4)*
H21D	1.2550	0.1179	0.2593	0.005*
N1D	1.0083 (3)	0.09752 (6)	0.1121 (3)	0.0043 (3)*
H11D	1.0965	0.1003	0.0287	0.006*
H12D	0.8669	0.1058	0.0885	0.006*
H13D	1.0077	0.0693	0.1432	0.006*
C4D	1.1644 (4)	0.20645 (8)	0.2902 (3)	0.0065 (4)
C5D	1.0215 (5)	0.22383 (9)	0.3992 (3)	0.0115 (5)
H51D	0.8748	0.2126	0.4084	0.014*
C9D	1.3814 (4)	0.22282 (9)	0.2807 (4)	0.0115 (5)
H91D	1.4830	0.2108	0.2095	0.014*
C8D	1.4493 (5)	0.25671 (10)	0.3754 (4)	0.0147 (5)
H81D	1.5964	0.2679	0.3673	0.018*
C6D	1.0893 (5)	0.25721 (10)	0.4944 (4)	0.0157 (5)
H61D	0.9902	0.2685	0.5686	0.019*
C1D	0.9534 (4)	0.11820 (7)	0.3788 (3)	0.0036 (4)*
C7D	1.3045 (5)	0.27409 (9)	0.4805 (4)	0.0148 (5)
H71D	1.3509	0.2975	0.5434	0.018*

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
O2A	0.0057 (7)	0.0107 (8)	0.0026 (8)	0.0018 (6)	0.0002 (6)	0.0016 (6)
O1A	0.0020 (7)	0.0187 (9)	0.0035 (8)	−0.0010 (6)	−0.0015 (6)	0.0018 (7)
C3A	0.0111 (10)	0.0099 (10)	0.0041 (11)	0.0028 (8)	0.0001 (8)	0.0020 (8)
C4A	0.0094 (10)	0.0067 (9)	0.0068 (12)	0.0008 (8)	−0.0004 (8)	0.0024 (8)
C5A	0.0174 (12)	0.0118 (11)	0.0112 (13)	0.0000 (9)	−0.0082 (10)	0.0018 (9)
C6A	0.0405 (19)	0.0116 (12)	0.0124 (15)	−0.0031 (12)	−0.0041 (13)	−0.0009 (10)
C7A	0.040 (2)	0.0094 (12)	0.0206 (18)	0.0034 (12)	0.0120 (14)	−0.0010 (11)
C8A	0.0177 (14)	0.0130 (12)	0.035 (2)	0.0065 (11)	0.0084 (13)	0.0013 (12)
C9A	0.0094 (11)	0.0111 (11)	0.0210 (15)	0.0030 (9)	−0.0036 (10)	0.0005 (10)
O2B	0.0061 (7)	0.0119 (8)	0.0028 (8)	−0.0003 (6)	−0.0023 (6)	0.0014 (6)
O1B	0.0032 (7)	0.0203 (9)	0.0028 (9)	0.0004 (6)	0.0007 (6)	0.0031 (7)
N1B	0.0022 (7)	0.0085 (9)	0.0023 (9)	0.0002 (6)	0.0008 (6)	−0.0003 (6)
C2B	0.0016 (8)	0.0086 (9)	0.0010 (10)	0.0002 (7)	0.0003 (7)	−0.0003 (7)
C3B	0.0107 (10)	0.0082 (10)	0.0027 (11)	−0.0002 (8)	0.0002 (8)	0.0027 (7)
C4B	0.0074 (9)	0.0075 (9)	0.0045 (11)	−0.0018 (8)	−0.0005 (8)	0.0019 (7)
C1B	0.0033 (8)	0.0083 (9)	0.0005 (10)	0.0007 (7)	0.0006 (7)	0.0005 (7)
C5B	0.0070 (10)	0.0117 (11)	0.0123 (13)	0.0002 (8)	0.0006 (8)	−0.0005 (9)
C9B	0.0086 (10)	0.0102 (11)	0.0134 (13)	−0.0006 (8)	−0.0015 (9)	0.0021 (9)
C7B	0.0175 (13)	0.0089 (10)	0.0117 (13)	−0.0069 (9)	0.0022 (10)	0.0006 (9)
C8B	0.0113 (11)	0.0131 (11)	0.0115 (13)	−0.0051 (9)	−0.0040 (9)	0.0037 (9)
C6B	0.0162 (12)	0.0117 (11)	0.0171 (15)	−0.0004 (10)	0.0048 (10)	−0.0036 (10)
O2C	0.0074 (7)	0.0114 (8)	0.0018 (8)	0.0014 (6)	0.0004 (6)	0.0023 (6)
N1C	0.0028 (8)	0.0089 (9)	0.0025 (9)	0.0000 (6)	−0.0020 (6)	−0.0001 (7)
C4C	0.0096 (10)	0.0068 (10)	0.0045 (11)	0.0016 (8)	0.0008 (8)	0.0016 (7)
C9C	0.0086 (10)	0.0122 (11)	0.0133 (13)	0.0005 (8)	0.0004 (9)	−0.0001 (9)
C3C	0.0101 (10)	0.0087 (10)	0.0033 (11)	0.0015 (8)	−0.0002 (8)	0.0020 (8)
C5C	0.0108 (11)	0.0134 (11)	0.0132 (14)	−0.0007 (9)	0.0029 (9)	−0.0008 (9)
C6C	0.0198 (13)	0.0136 (12)	0.0173 (16)	−0.0036 (10)	0.0016 (11)	−0.0042 (10)
C8C	0.0126 (12)	0.0148 (12)	0.0170 (15)	0.0057 (9)	0.0050 (10)	0.0007 (10)
C7C	0.0213 (14)	0.0108 (11)	0.0150 (15)	0.0031 (10)	0.0017 (11)	−0.0027 (10)
O1D	0.0030 (7)	0.0203 (9)	0.0015 (8)	0.0026 (6)	0.0009 (6)	0.0023 (6)
O2D	0.0061 (7)	0.0103 (8)	0.0022 (8)	0.0003 (6)	−0.0017 (6)	0.0012 (6)
C3D	0.0108 (10)	0.0074 (9)	0.0042 (11)	0.0004 (8)	0.0016 (8)	0.0032 (7)
C4D	0.0068 (9)	0.0062 (9)	0.0065 (11)	0.0006 (7)	0.0007 (8)	0.0029 (7)
C5D	0.0102 (11)	0.0107 (11)	0.0135 (14)	−0.0024 (8)	0.0058 (9)	−0.0016 (9)
C9D	0.0069 (10)	0.0122 (11)	0.0153 (14)	−0.0008 (8)	0.0031 (9)	0.0034 (9)
C8D	0.0097 (11)	0.0138 (12)	0.0205 (15)	−0.0036 (9)	−0.0028 (10)	0.0036 (10)
C6D	0.0204 (13)	0.0136 (12)	0.0132 (15)	0.0000 (10)	0.0038 (11)	−0.0034 (10)
C7D	0.0192 (13)	0.0098 (11)	0.0152 (14)	−0.0029 (10)	−0.0044 (11)	0.0009 (9)

Geometric parameters (Å, º)

O2A—C1A	1.255 (3)	O1C—C1C	1.258 (3)
O1A—C1A	1.258 (3)	O2C—C1C	1.256 (3)
C1A—C2A	1.535 (3)	N1C—C2C	1.501 (3)
N1A—C2A	1.498 (3)	N1C—H11C	0.9100
N1A—H11A	0.9100	N1C—H12C	0.9100
N1A—H12A	0.9100	N1C—H13C	0.9100
N1A—H13A	0.9100	C2C—C3C	1.531 (3)
C2A—C3A	1.528 (3)	C2C—C1C	1.534 (3)
C2A—H21A	1.0000	C2C—H21C	1.0000
C3A—C4A	1.505 (4)	C4C—C5C	1.391 (4)
C3A—H31A	0.9900	C4C—C9C	1.402 (4)
C3A—H32A	0.9900	C4C—C3C	1.510 (4)
C4A—C9A	1.390 (4)	C9C—C8C	1.390 (4)
C4A—C5A	1.394 (4)	C9C—H91C	0.9500
C5A—C6A	1.392 (5)	C3C—H31C	0.9900
C5A—H51A	0.9500	C3C—H32C	0.9900
C6A—C7A	1.381 (6)	C5C—C6C	1.389 (4)
C6A—H61A	0.9500	C5C—H51C	0.9500
C7A—C8A	1.390 (6)	C6C—C7C	1.393 (5)
C7A—H71A	0.9500	C6C—H61C	0.9500
C8A—C9A	1.386 (5)	C8C—C7C	1.387 (4)
C8A—H81A	0.9500	C8C—H81C	0.9500
C9A—H91A	0.9500	C7C—H71C	0.9500
O2B—C1B	1.262 (3)	O1D—C1D	1.256 (3)
O1B—C1B	1.253 (3)	O2D—C1D	1.264 (3)
N1B—C2B	1.496 (3)	C3D—C4D	1.506 (4)
N1B—H11B	0.9100	C3D—C2D	1.532 (3)
N1B—H12B	0.9100	C3D—H31D	0.9900
N1B—H13B	0.9100	C3D—H32D	0.9900
C2B—C1B	1.533 (3)	C2D—N1D	1.496 (3)
C2B—C3B	1.535 (3)	C2D—C1D	1.532 (3)
C2B—H21B	1.0000	C2D—H21D	1.0000
C3B—C4B	1.511 (4)	N1D—H11D	0.9100
C3B—H31B	0.9900	N1D—H12D	0.9100
C3B—H32B	0.9900	N1D—H13D	0.9100
C4B—C9B	1.392 (4)	C4D—C5D	1.395 (4)
C4B—C5B	1.393 (4)	C4D—C9D	1.399 (4)
C5B—C6B	1.392 (4)	C5D—C6D	1.387 (4)
C5B—H51B	0.9500	C5D—H51D	0.9500
C9B—C8B	1.394 (4)	C9D—C8D	1.396 (4)
C9B—H91B	0.9500	C9D—H91D	0.9500
C7B—C6B	1.386 (4)	C8D—C7D	1.379 (5)
C7B—C8B	1.394 (4)	C8D—H81D	0.9500
C7B—H71B	0.9500	C6D—C7D	1.398 (4)
C8B—H81B	0.9500	C6D—H61D	0.9500
C6B—H61B	0.9500	C7D—H71D	0.9500
O2A—C1A—O1A	126.3 (2)	C2C—N1C—H11C	109.5
O2A—C1A—C2A	119.1 (2)	C2C—N1C—H12C	109.5
O1A—C1A—C2A	114.6 (2)	H11C—N1C—H12C	109.5
C2A—N1A—H11A	109.5	C2C—N1C—H13C	109.5
C2A—N1A—H12A	109.5	H11C—N1C—H13C	109.5
H11A—N1A—H12A	109.5	H12C—N1C—H13C	109.5
C2A—N1A—H13A	109.5	N1C—C2C—C3C	106.5 (2)
H11A—N1A—H13A	109.5	N1C—C2C—C1C	108.36 (18)
H12A—N1A—H13A	109.5	C3C—C2C—C1C	113.04 (19)
N1A—C2A—C3A	106.3 (2)	N1C—C2C—H21C	109.6
N1A—C2A—C1A	108.17 (18)	C3C—C2C—H21C	109.6
C3A—C2A—C1A	112.74 (19)	C1C—C2C—H21C	109.6
N1A—C2A—H21A	109.8	C5C—C4C—C9C	118.5 (2)
C3A—C2A—H21A	109.8	C5C—C4C—C3C	119.6 (2)
C1A—C2A—H21A	109.8	C9C—C4C—C3C	121.9 (2)
C4A—C3A—C2A	115.7 (2)	C8C—C9C—C4C	120.0 (3)
C4A—C3A—H31A	108.4	C8C—C9C—H91C	120.0
C2A—C3A—H31A	108.4	C4C—C9C—H91C	120.0
C4A—C3A—H32A	108.4	C4C—C3C—C2C	114.2 (2)
C2A—C3A—H32A	108.4	C4C—C3C—H31C	108.7
H31A—C3A—H32A	107.4	C2C—C3C—H31C	108.7
C9A—C4A—C5A	118.2 (3)	C4C—C3C—H32C	108.7
C9A—C4A—C3A	120.5 (2)	C2C—C3C—H32C	108.7
C5A—C4A—C3A	121.2 (2)	H31C—C3C—H32C	107.6
C6A—C5A—C4A	120.8 (3)	C6C—C5C—C4C	121.4 (3)
C6A—C5A—H51A	119.6	C6C—C5C—H51C	119.3
C4A—C5A—H51A	119.6	C4C—C5C—H51C	119.3
C7A—C6A—C5A	120.4 (3)	C5C—C6C—C7C	119.8 (3)
C7A—C6A—H61A	119.8	C5C—C6C—H61C	120.1
C5A—C6A—H61A	119.8	C7C—C6C—H61C	120.1
C6A—C7A—C8A	119.2 (3)	C7C—C8C—C9C	121.1 (3)
C6A—C7A—H71A	120.4	C7C—C8C—H81C	119.4
C8A—C7A—H71A	120.4	C9C—C8C—H81C	119.4
C9A—C8A—C7A	120.3 (3)	O2C—C1C—O1C	126.5 (2)
C9A—C8A—H81A	119.8	O2C—C1C—C2C	118.5 (2)
C7A—C8A—H81A	119.8	O1C—C1C—C2C	115.1 (2)
C8A—C9A—C4A	121.0 (3)	C8C—C7C—C6C	119.2 (3)
C8A—C9A—H91A	119.5	C8C—C7C—H71C	120.4
C4A—C9A—H91A	119.5	C6C—C7C—H71C	120.4
C2B—N1B—H11B	109.5	C4D—C3D—C2D	115.1 (2)
C2B—N1B—H12B	109.5	C4D—C3D—H31D	108.5
H11B—N1B—H12B	109.5	C2D—C3D—H31D	108.5
C2B—N1B—H13B	109.5	C4D—C3D—H32D	108.5
H11B—N1B—H13B	109.5	C2D—C3D—H32D	108.5
H12B—N1B—H13B	109.5	H31D—C3D—H32D	107.5
N1B—C2B—C1B	108.17 (19)	N1D—C2D—C3D	106.61 (19)
N1B—C2B—C3B	107.22 (19)	N1D—C2D—C1D	108.26 (18)
C1B—C2B—C3B	112.3 (2)	C3D—C2D—C1D	112.3 (2)
N1B—C2B—H21B	109.7	N1D—C2D—H21D	109.9
C1B—C2B—H21B	109.7	C3D—C2D—H21D	109.9
C3B—C2B—H21B	109.7	C1D—C2D—H21D	109.9
C4B—C3B—C2B	114.2 (2)	C2D—N1D—H11D	109.5
C4B—C3B—H31B	108.7	C2D—N1D—H12D	109.5
C2B—C3B—H31B	108.7	H11D—N1D—H12D	109.5
C4B—C3B—H32B	108.7	C2D—N1D—H13D	109.5
C2B—C3B—H32B	108.7	H11D—N1D—H13D	109.5
H31B—C3B—H32B	107.6	H12D—N1D—H13D	109.5
C9B—C4B—C5B	118.2 (2)	C5D—C4D—C9D	118.4 (2)
C9B—C4B—C3B	121.8 (2)	C5D—C4D—C3D	120.6 (2)
C5B—C4B—C3B	120.0 (2)	C9D—C4D—C3D	120.9 (2)
O1B—C1B—O2B	126.1 (2)	C6D—C5D—C4D	121.4 (3)
O1B—C1B—C2B	115.3 (2)	C6D—C5D—H51D	119.3
O2B—C1B—C2B	118.6 (2)	C4D—C5D—H51D	119.3
C4B—C5B—C6B	121.6 (3)	C8D—C9D—C4D	120.3 (3)
C4B—C5B—H51B	119.2	C8D—C9D—H91D	119.8
C6B—C5B—H51B	119.2	C4D—C9D—H91D	119.8
C4B—C9B—C8B	120.7 (3)	C7D—C8D—C9D	120.5 (3)
C4B—C9B—H91B	119.7	C7D—C8D—H81D	119.8
C8B—C9B—H91B	119.7	C9D—C8D—H81D	119.8
C6B—C7B—C8B	119.4 (3)	C5D—C6D—C7D	119.5 (3)
C6B—C7B—H71B	120.3	C5D—C6D—H61D	120.2
C8B—C7B—H71B	120.3	C7D—C6D—H61D	120.2
C9B—C8B—C7B	120.4 (3)	O1D—C1D—O2D	126.3 (2)
C9B—C8B—H81B	119.8	O1D—C1D—C2D	115.2 (2)
C7B—C8B—H81B	119.8	O2D—C1D—C2D	118.5 (2)
C7B—C6B—C5B	119.7 (3)	C8D—C7D—C6D	119.9 (3)
C7B—C6B—H61B	120.2	C8D—C7D—H71D	120.1
C5B—C6B—H61B	120.2	C6D—C7D—H71D	120.1

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
N1A—H11A···O2B	0.91	1.99	2.840 (3)	155
N1A—H12A···O1C^i	0.91	1.79	2.695 (3)	174
N1A—H13A···O2C^ii	0.91	2.06	2.950 (3)	166
N1B—H11B···O1D	0.91	1.80	2.707 (3)	177
N1B—H12B···O2D^iii	0.91	1.99	2.883 (3)	168
N1B—H13B···O2A	0.91	2.00	2.854 (3)	156
N1C—H11C···O2D	0.91	1.98	2.839 (3)	158
N1C—H12C···O1A^iv	0.91	1.79	2.694 (3)	176
N1C—H13C···O2A	0.91	1.99	2.884 (3)	168
N1D—H11D···O1B^v	0.91	1.80	2.694 (3)	167
N1D—H12D···O2B^vi	0.91	2.07	2.963 (3)	168
N1D—H13D···O2C	0.91	1.98	2.829 (3)	156

Symmetry codes: (i) x, y, z+1; (ii) x−1, y, z+1; (iii) x−1, y, z; (iv) x+1, y, z; (v) x+1, y, z−1; (vi) x, y, z−1.