L-Histidyl-L-serine 3.7-hydrate: water channels in the crystal structure of a polar dipeptide

Dipeptides may form nanotubular structures with pore diameters in the range 3.2-10 Å. These compounds normally contain at least one and usually two hydro­phobic residues, but L-His-L-Ser hydrate, C₉H₁₄N₄O₄·3.7H₂O, with two hydro­philic residues, forms large polar channels filled with ordered as well as disordered water mol­ecules.

S1. Comment

The structure of L-His-L-Ser was investigated by Padiyar (1998) as a zwitterionic hydrate, (I), after having first been studied in a 1:1 complex with Gly-L-Glu by Suresh & Vijayan (1985), (II). In the process of elucidating the ability of small peptides to form nanotubular crystal structures (Görbitz, 2007), the former structure appeared to be of considerable interest becuase of its high water content (presented as a trihydrate), but it was difficult to get a clear picture of the actual water structure as no atomic coordinates were available in the Cambridge Structural Database (CSD) (CSD refcode WADXIJ; CSD, Version 5.31 of November 2009; Allen, 2002). A low-temperature reinvestigation of the uncomplexed dipeptide, (I), has thus been carried out.

The crystal structure of (I) is shown in Fig. 1(a). Bond lengths and bond angles are normal. The main chain is fairly extended, as reflected by the torsion angles listed in Table 1, and quite similar to the conformation of L-His-L-Ser in (II) (Fig. 1b), as also reported by Padiyar (1998). The L-His side- chain conformation, with N1—C1—C2—C3 = gauche- and C1—C2—C3—N2 = gauche- coincides with the conformation taken by three out of six N-terminal His-residues in dipeptide structures in the CSD (Allen, 2002) and effectively separates the imidazole moiety from the rest of the molecule, thus avoiding potential steric conflict between hydrogen-bond donors and acceptors interacting with the L-His side chain and functional groups in the backbone, respectively. In (II) C1—C2—C3—N2 is gauche+ (77.8°), giving a slightly different appearance. The L-Ser conformation, with N4—C7—C8—O2 = gauche+ and C7—C8—O2—H2 = gauche-, recurs in (II) and has also been observed for six out of 12 C-terminal Ser residues in other dipeptides, often with a corystallized water molecule as acceptor for the H atom of the hydroxyl group as seen for (I) in Fig 1(a).

In addition to the ordered water molecules 1 and 2 (Fig. 1a), which were also identified by Padiyar (1998), coordinates were refined for seven low-occupancy water sites (see Experimental). All are arranged along conspicuous water channels running parallel to the a axis, highlighted by ellipse 1 in Fig. 2(a), where they provide acceptors for the side-chain >N—H donor of L-His as well as one of the H atoms of water molecule 1. These disordered water molecules define the solvent channel highlighted in ellipse 2 with a volume of 77.1 ų per unit cell (calculated by PLATON; Spek, 2009) and an average cross section of 16.0 Ų. The electron count within the channel, 27.3, corresponds to 3.41 water molecules per channel or 1.71 water molecules per peptide molecule, which fits nicely with the sum of refined occupancies for the seven disordered water molecules, 1.70. Together with water molecules 1 and 2 the complete solvent system, ellipse 3 in Fig. 2(a), has a total volume of 175.2 ų with average cross section 36.4 Ų.

Water channels have previously been found in the structures of highly hydrated dipeptide species like L-Val-L-Ser trihydrate (Johansen et al., 2005), L-His-L-Asp trihydrate (Cheng et al., 2005) and in particular L-Leu-L-Ile 2.5 hydrate (Görbitz & Rise, 2008) and L-Leu-L-Ala tetrahydrate (Fig. 2b) (Görbitz, 1997). All these channels have completely ordered water structures. The same applies to those members of the Phe-Phe class of nanotubular dipeptides which have channels small enough to be spanned by individual water molecules, e.g. L-Leu-L-Leu 0.87 hydrate and L-Leu-L-Phe 0.86 hydrate (Görbitz, 2001). In contrast, the only slightly larger channels of L-Phe-L-Leu 1.26 hydrate (Fig. 2c) (Görbitz, 2001), which are comparable in size (average cross section 19.4 Ų) to the central channel in ellipse 2 in Fig. 2(a) for (I), contain disordered water molecules. These two structures together with the hexagonal structure of L-Phe-L-Phe 2.47 hydrate (Görbitz, 2001) are unique among dipeptides in having channels with disordered and presumably movable water molecules (Febles et al., 2006).

Considering the crystal packing arrangement of dipeptides, compounds with at least one hydrophobic residue frequently form distinct layers (Görbitz, 2010). When both residues are polar or charged, however, layers are usually less obvious or even absent. The crystal packing of (I) can nevertheless conveniently be regarded as being composed of layers, with each layer being in turn being constructed from two individual sheets (Fig. 2a). Interactions between layers are very weak, essentially being confined to interactions involving the disordered water molecules at the centre of the solvent channel, as the long >C5—H51···O3 contact included in Fig. 2(a) is not very significant (Wood et al., 2009). This would explain the fragile nature of the crystals grown.

The hydrogen-bonding pattern within a sheet in the structure of (I) may be compared with patterns observed in other dipeptide structures. In a recent survey (Görbitz, 2010) it was found that two or even three head-to-tail hydrogen-bonded chains, involving the N-terminal amino groups and C-terminal carboxylate groups, co-exist in more than two thirds of all crystal structures. In most of them two such chains generate hydrogen-bonded sheets that can be classified into four basic patterns called S4, T4, S5 and T5, where the initial capital letter denotes the type of symmetry involved in moving from one molecule in the chain to the next (T = translation, S = screw axis) and the number describes the hydrogen bonding of the amide N—H group [4 = C(4) chain, 5 = C(5) chain; for graph-set theory, see Etter et al., 1990]. A model T4 pattern is depicted in Fig. 3(a) showing hydrogen-bonded tapes incorporating the amide C(4) chains as well as C^α—H···O interactions. For each of the four basic patterns the separation between tapes, called d for the T4 pattern in Fig. 3(a), must be small enough for amino groups and carboxylate groups to form the direct hydrogen bonds that define the two C(8) chains. Occasionally, d is too large for this to be possible, and one or two C(8) chains may be lost compared to the parent pattern (Görbitz, 2010). In the pattern code an asterisk is used to denote such a missing chain; S4* accordingly means a pattern that is derived from the regular S4 pattern, but with only one remaining C(8) chain. Fig. 3(b) shows that the pattern of (I) can be classified as T4**, with the charged termini being bridged by the solvent water molecules and the functional groups of the L-Ser and L-His side chains. The only other known example of a T4** structure, Gly-L-Tyr dihydrate (Cotrait & Bideau, 1974), is shown in Fig. 3(c). Despite its overall apparently quite different crystalline arrangement, the solvent water molecules and side-chain hydroxyl groups play roles remarkably similar to [the ones] they do in the structure of (I).

Related literature

For related literature, see: Allen (2002); Cheng et al. (2005); Cotrait & Bideau (1974); Etter et al. (1990); Febles et al. (2006); Görbitz (1997, 2001, 2007, 2010); Görbitz & Rise (2008); Johansen et al. (2005); Padiyar (1998); Spek (2009); Suresh & Vijayan (1985); Wood et al. (2009).

S2. Experimental

Fragile, needle-shaped crystals were grown by vapour diffusion of acetonitrile into 30 µl of an aqueous solution containing about 1 mg of the peptide.

S3. Refinement

Peptide H atoms were positioned with idealized geometry and fixed C/N—H distances for NH₃, NH, CH₂, CH (methine) and CH (sp²) at 0.91, 0.88, 0.99, 1.00 and 0.95 Å, respectively. Free rotation was permitted for the amino group. Restraints were imposed on the O—H distances of L-Ser and the ordered water molecules 1 and 2 (DFIX 0.85 0.01) and on the H···H distances of water molecules (DFIX 1.35 0.02) to give O—H bond lengths in the range 0.84–0.85 Å and H—O—H angles in the range 105–109°.

In the standard refinement of the structure of (I), electron density from disordered solvent molecules within the channels was modelled by seven O atoms, three with occupancies in the range 0.42 (3)–0.457 (13) that were refined isotropically and four with occupancies in the range 0.042 (10)–0.159 (19) that were assigned a fixed isotropic temperature factor of 0.06.

Structure refinement (not tabulated, downloadable cif) has also been carried out with a modified. hkl file from which the contribution from disordered solvent had been eliminated by the SQUEEZE routine of the PLATON program (Spek, 2009). This procedure gave R = 0.045, wR(F²) = 0.112, and reductions in the standard uncertainties of calculated geometric parameters, amounting to 0.001 Å for bond lengths and 0.1° for angles and torsion angles, but no significant changes to the geometric parameters themselves.

Computing details

Data collection: APEX2 (Bruker, 2007); cell refinement: SAINT-Plus (Bruker, 2007); data reduction: SAINT-Plus (Bruker, 2007); program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to refine structure: SHELXTL (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL (Sheldrick, 2008).

Figures

	Fig. 1. (a) The asymmetric unit of (I) with atom numbering indicated. Displacement ellipsoids (or spheres for O atoms in water molecules with low occupancy) are drawn at the 50% probability level; H atoms are spheres of arbitrary size. (b) Ball-and-stick model of L-His-L-Ser as it appears in the complex with Gly-L-Glu (Suresh & Vijayan, 1985). The CSD entry (refcode DIYZOA; Allen, 2002) does not contain coordinates for H, so H atoms were introduced in idealized positions with the hydroxyl group pointing in the direction of the closest acceptor atom.
	Fig. 2. (a) Unit cell and molecular packing of (I) viewed along the a axis. A hydrogen-bonded sheet, encompassing ordered water molecules and peptide molecules in the ab plane, is highlighted in blue. Notably, the L-Ser hydroxyl group, with O atom coloured in violet, is located outside the sheet and in fact constitutes an essential part of the neighbouring sheet. The two sheets connected this way are said to form a layer. A wavelike interface between two such layers has been indicated by a dashed line, and it is crossed only by the idicated [indicated?], very weak C5—H51···O3 contacts (H···O = 2.62 Å, C—H···O = 120°). The ellipses in grey show water-filled channels running through the crystal parallel to the a axis. In 1 the positions of all water molecules have been included, with low-occupancy water positions coloured in orange, while in 2 only the ordered water molecules (with OW1 and OW2) have been retained, leaving an empty column shown in orange (three more examples included). In 3 all water molecules have been removed, giving a much larger channel. (b) Water channels in the structure of L-Leu-L-Ala tetrahydrate (Görbitz, 1997) comparable in size to the channels of (I). Out of the four water molecules in the asymmetric unit, one (indicated by arrows) is tightly associated with the peptide molecules, while the other three form the bulk of the channel. To the right a column is illustrated in the same way as column 3 in (a). (c) A hydrophilic channel without ordered water molecules running through the crystal structure of L-Phe-L-Leu (Görbitz, 2001). All illustrations are on approximately the same scale and were prepared by Mercury (Macrae et al., 2008) with application of a 1.2 Å probe radius and 0.5 Å grid spacing for calculation of voids.
	Fig. 3. (a) A standard T4 hydrogen-bonding pattern found in the crystal structures of dipeptides (Görbitz, 2010). The two head-to-tail chains are coloured in red; the C(4) chain involving the amide N—H···O and two additional C^α—H···O interactions form one-dimensional hydrogen-bonded tapes coloured in blue. The separation between tapes is called d. (b) Hydrogen bonding in an individual sheet of (I) (highlighted in blue in Fig. 2a). The O atoms of the L-Ser hydroxyl groups coming from the adjacent layer have, as in Fig. 2(a), been coloured in violet. L-His H atoms on C^β have been omitted. (c) Hydrogen bonding in a sheet in the structure of Gly-L-Tyr dihydrate (Cotrait & Bideau, 1974). The L-Tyr side chains have been hidden, except the hydroxyl groups of dipeptide molecules in the adjacent layer. Hydrogen-bonded tapes have been coloured in blue in (b) and (c) in the same way as in (a).

L-Histidyl-L-serine 3.7-hydrate

Crystal data

C9H14N4O4·3.7H2O	F(000) = 660
Mr = 308.89	Dx = 1.450 Mg m^−3
Orthorhombic, P212121	Mo Kα radiation, λ = 0.71073 Å
Hall symbol: P 2ac 2ab	Cell parameters from 1250 reflections
a = 4.812 (3) Å	θ = 2.5–19.9°
b = 15.505 (8) Å	µ = 0.13 mm^−1
c = 18.958 (10) Å	T = 105 K
V = 1414.6 (13) Å^3	Needle, colourless
Z = 4	0.62 × 0.06 × 0.04 mm

Data collection

Bruker APEXII CCD diffractometer	1544 independent reflections
Radiation source: fine-focus sealed tube	1076 reflections with I > 2σ(I)
Graphite monochromator	Rint = 0.083
Detector resolution: 8.3 pixels mm^-1	θmax = 25.4°, θmin = 1.7°
Sets of exposures each taken over 0.5° ω rotation scans	h = −4→5
Absorption correction: multi-scan (SADABS; Bruker, 2007)	k = −18→18
Tmin = 0.836, Tmax = 0.996	l = −19→22
7345 measured reflections

Refinement

Refinement on F^2	Primary atom site location: structure-invariant direct methods
Least-squares matrix: full	Secondary atom site location: difference Fourier map
R[F^2 > 2σ(F^2)] = 0.048	Hydrogen site location: inferred from neighbouring sites
wR(F^2) = 0.131	H atoms treated by a mixture of independent and constrained refinement
S = 1.08	w = 1/[σ^2(Fo^2) + (0.0531P)^2 + 0.7553P] where P = (Fo^2 + 2Fc^2)/3
1544 reflections	(Δ/σ)max = 0.002
219 parameters	Δρmax = 0.27 e Å^−3
7 restraints	Δρmin = −0.26 e Å^−3

Special details

Experimental. Fragile needles.

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. Electron density from disordered solvent molecules within the channels was modelled by seven O atoms, three with occupancies in the range 0.42 (3) - 0.457 (13) that wererefined isotropically and four with occupancies in the range 0.042 (10) - 0.159 (19) that were assigned a fixed isotropic temperature factor of 0.06. Refinement of F² against ALL reflections.

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

	x	y	z	Uiso*/Ueq	Occ. (<1)
O1	0.9796 (6)	0.6563 (2)	0.16814 (17)	0.0332 (8)
O2	0.4379 (7)	0.4741 (2)	0.27865 (18)	0.0344 (8)
H2	0.375 (10)	0.5225 (19)	0.292 (3)	0.041*
O3	0.2902 (7)	0.4618 (2)	0.09421 (18)	0.0409 (9)
O4	0.5593 (8)	0.3579 (2)	0.13843 (17)	0.0402 (9)
N1	0.7587 (8)	0.8135 (2)	0.18444 (19)	0.0299 (10)
H1A	0.6582	0.8627	0.1900	0.045*
H1B	0.9019	0.8235	0.1542	0.045*
H1C	0.8273	0.7964	0.2269	0.045*
N2	0.1726 (8)	0.8799 (3)	0.0967 (2)	0.0370 (10)
N3	0.3374 (9)	0.9818 (3)	0.0296 (3)	0.0459 (12)
H3	0.3550	1.0324	0.0090	0.055*
N4	0.5621 (8)	0.5898 (2)	0.16028 (19)	0.0300 (9)
H4	0.3824	0.5970	0.1540	0.036*
C1	0.5775 (9)	0.7448 (3)	0.1554 (2)	0.0283 (11)
H11	0.3997	0.7430	0.1827	0.034*
C2	0.5137 (10)	0.7619 (3)	0.0772 (2)	0.0300 (11)
H21	0.6856	0.7529	0.0494	0.036*
H22	0.3750	0.7192	0.0608	0.036*
C3	0.4067 (9)	0.8494 (3)	0.0626 (2)	0.0295 (11)
C4	0.1394 (11)	0.9602 (4)	0.0749 (3)	0.0480 (15)
H41	−0.0064	0.9973	0.0897	0.058*
C5	0.5102 (11)	0.9125 (3)	0.0199 (3)	0.0367 (12)
H51	0.6679	0.9092	−0.0102	0.044*
C6	0.7271 (9)	0.6586 (3)	0.1627 (2)	0.0283 (11)
C7	0.6726 (10)	0.5024 (3)	0.1677 (2)	0.0297 (11)
H71	0.8618	0.5012	0.1459	0.036*
C8	0.7035 (10)	0.4803 (3)	0.2460 (3)	0.0327 (11)
H81	0.8032	0.4248	0.2510	0.039*
H82	0.8146	0.5255	0.2697	0.039*
C9	0.4930 (11)	0.4365 (3)	0.1294 (2)	0.0353 (12)
O1W	1.0395 (8)	0.2793 (2)	0.17930 (17)	0.0389 (9)
H11W	1.185 (7)	0.290 (3)	0.156 (3)	0.058*
H12W	0.909 (7)	0.310 (3)	0.164 (3)	0.058*
O2W	0.2563 (10)	0.6191 (3)	0.3409 (3)	0.0730 (14)
H21W	0.396 (11)	0.637 (4)	0.364 (4)	0.109*
H22W	0.184 (14)	0.664 (3)	0.322 (4)	0.109*
O31W	0.526 (2)	0.2031 (6)	0.0608 (5)	0.058 (4)*	0.431 (13)
O32W	0.585 (3)	0.1381 (8)	0.0105 (6)	0.040 (5)*	0.42 (3)
O33W	0.537 (2)	0.3163 (6)	0.0025 (5)	0.057 (4)*	0.457 (13)
O41W	0.737 (9)	0.183 (3)	0.008 (2)	0.060*	0.112 (11)
O42W	0.77 (3)	0.131 (8)	0.029 (6)	0.060*	0.042 (10)
O43W	0.470 (11)	0.155 (2)	−0.0043 (18)	0.060*	0.159 (19)
O44W	0.291 (13)	0.195 (4)	0.014 (3)	0.060*	0.079 (9)

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
O1	0.0270 (18)	0.0304 (19)	0.0424 (19)	−0.0020 (16)	−0.0004 (15)	−0.0028 (15)
O2	0.0339 (17)	0.0280 (19)	0.041 (2)	0.0001 (17)	0.0034 (16)	0.0028 (16)
O3	0.0413 (19)	0.037 (2)	0.044 (2)	−0.0066 (18)	−0.0137 (18)	0.0087 (17)
O4	0.049 (2)	0.030 (2)	0.042 (2)	−0.0009 (18)	−0.0088 (18)	0.0004 (16)
N1	0.031 (2)	0.028 (2)	0.030 (2)	0.0022 (19)	−0.0032 (18)	−0.0009 (17)
N2	0.033 (2)	0.038 (3)	0.040 (2)	0.004 (2)	−0.003 (2)	−0.005 (2)
N3	0.050 (3)	0.026 (3)	0.061 (3)	−0.001 (2)	−0.018 (3)	0.003 (2)
N4	0.0233 (18)	0.026 (2)	0.041 (2)	−0.0035 (18)	−0.0040 (19)	0.0070 (19)
C1	0.027 (2)	0.027 (3)	0.031 (3)	−0.003 (2)	−0.003 (2)	0.004 (2)
C2	0.036 (3)	0.027 (3)	0.027 (2)	0.002 (2)	−0.002 (2)	0.000 (2)
C3	0.031 (2)	0.029 (3)	0.029 (3)	−0.001 (2)	−0.005 (2)	−0.002 (2)
C4	0.038 (3)	0.045 (4)	0.061 (4)	0.006 (3)	−0.014 (3)	−0.014 (3)
C5	0.041 (3)	0.033 (3)	0.036 (3)	0.000 (3)	−0.008 (3)	0.001 (2)
C6	0.029 (3)	0.028 (3)	0.028 (2)	0.000 (2)	−0.001 (2)	0.001 (2)
C7	0.031 (2)	0.024 (3)	0.034 (3)	−0.002 (2)	−0.005 (2)	0.006 (2)
C8	0.030 (2)	0.031 (3)	0.037 (3)	0.000 (2)	−0.006 (2)	0.003 (2)
C9	0.040 (3)	0.037 (3)	0.029 (3)	−0.006 (3)	0.000 (2)	0.003 (2)
O1W	0.042 (2)	0.038 (2)	0.036 (2)	0.0024 (19)	0.0018 (18)	−0.0003 (16)
O2W	0.067 (3)	0.039 (2)	0.114 (4)	0.011 (2)	−0.014 (3)	−0.007 (3)

Geometric parameters (Å, º)

O1—C6	1.220 (5)	C1—C2	1.538 (6)
O2—C8	1.424 (6)	C1—H11	1.0000
O2—H2	0.85 (2)	C2—C3	1.478 (6)
O3—C9	1.246 (6)	C2—H21	0.9900
O4—C9	1.271 (6)	C2—H22	0.9900
N1—C1	1.482 (6)	C3—C5	1.365 (6)
N1—H1A	0.9100	C4—H41	0.9500
N1—H1B	0.9100	C5—H51	0.9500
N1—H1C	0.9100	C7—C9	1.522 (6)
N2—C4	1.321 (7)	C7—C8	1.530 (7)
N2—C3	1.382 (6)	C7—H71	1.0000
N3—C4	1.327 (7)	C8—H81	0.9900
N3—C5	1.370 (6)	C8—H82	0.9900
N3—H3	0.8800	O1W—H11W	0.84 (2)
N4—C6	1.331 (6)	O1W—H12W	0.845 (19)
N4—C7	1.463 (6)	O2W—H21W	0.85 (2)
N4—H4	0.8800	O2W—H22W	0.85 (2)
C1—C6	1.524 (6)
C8—O2—H2	113 (4)	N2—C3—C2	120.7 (4)
C1—N1—H1A	109.5	N2—C4—N3	110.7 (5)
C1—N1—H1B	109.5	N2—C4—H41	124.6
H1A—N1—H1B	109.5	N3—C4—H41	124.6
C1—N1—H1C	109.5	C3—C5—N3	105.1 (5)
H1A—N1—H1C	109.5	C3—C5—H51	127.4
H1B—N1—H1C	109.5	N3—C5—H51	127.4
C4—N2—C3	106.0 (4)	O1—C6—N4	125.0 (4)
C4—N3—C5	108.9 (5)	O1—C6—C1	120.3 (4)
C4—N3—H3	125.5	N4—C6—C1	114.7 (4)
C5—N3—H3	125.5	N4—C7—C9	111.7 (4)
C6—N4—C7	121.5 (4)	N4—C7—C8	109.6 (4)
C6—N4—H4	119.2	C9—C7—C8	111.6 (4)
C7—N4—H4	119.2	N4—C7—H71	107.9
N1—C1—C6	108.5 (4)	C9—C7—H71	107.9
N1—C1—C2	110.6 (4)	C8—C7—H71	107.9
C6—C1—C2	109.4 (4)	O2—C8—C7	110.5 (4)
N1—C1—H11	109.4	O2—C8—H81	109.6
C6—C1—H11	109.4	C7—C8—H81	109.6
C2—C1—H11	109.4	O2—C8—H82	109.6
C3—C2—C1	114.0 (4)	C7—C8—H82	109.6
C3—C2—H21	108.7	H81—C8—H82	108.1
C1—C2—H21	108.7	O3—C9—O4	124.8 (5)
C3—C2—H22	108.7	O3—C9—C7	119.3 (4)
C1—C2—H22	108.7	O4—C9—C7	115.9 (4)
H21—C2—H22	107.6	H11W—O1W—H12W	109 (3)
C5—C3—N2	109.3 (4)	H21W—O2W—H22W	105 (3)
C5—C3—C2	129.9 (4)
N1—C1—C6—N4	159.6 (4)	N2—C3—C5—N3	0.9 (5)
C1—C6—N4—C7	−179.0 (4)	C2—C3—C5—N3	−176.6 (4)
C6—N4—C7—C9	−152.1 (4)	C4—N3—C5—C3	−1.0 (5)
N4—C7—C9—O3	3.8 (6)	C7—N4—C6—O1	3.2 (7)
N1—C1—C2—C3	−51.8 (5)	N1—C1—C6—O1	−22.5 (6)
C1—C2—C3—N2	−56.2 (6)	C2—C1—C6—O1	98.3 (5)
C1—C2—C3—C5	121.1 (5)	C2—C1—C6—N4	−79.6 (5)
N4—C7—C8—O2	67.4 (5)	C6—N4—C7—C9	−152.1 (4)
C7—C8—O2—H2	−85 (4)	C6—N4—C7—C8	83.7 (5)
C6—C1—C2—C3	−171.3 (4)	C9—C7—C8—O2	−56.9 (5)
C4—N2—C3—C5	−0.6 (5)	N4—C7—C9—O3	3.8 (6)
C4—N2—C3—C2	177.2 (4)	C8—C7—C9—O3	126.8 (5)
C3—N2—C4—N3	−0.1 (5)	N4—C7—C9—O4	−173.8 (4)
C5—N3—C4—N2	0.7 (6)	C8—C7—C9—O4	−50.7 (6)

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
O2—H2···O2W	0.85 (2)	1.85 (2)	2.684 (6)	167 (5)
N1—H1A···O2^i	0.91	1.88	2.755 (5)	160
N1—H1B···N2^ii	0.91	1.91	2.792 (6)	163
N1—H1C···O1W^iii	0.91	1.91	2.810 (5)	171
N3—H3···O32W^iv	0.88	1.98	2.724 (11)	142
N4—H4···O1^v	0.88	2.16	2.991 (5)	157
C1—H11···O1^v	1.00	2.44	3.197 (6)	132
C4—H41···O2W^vi	0.95	2.60	3.499 (8)	159
C7—H71···O3^ii	1.00	2.36	3.342 (6)	166
O1W—H11W···O4^ii	0.84 (2)	2.11 (3)	2.888 (5)	154 (6)
O1W—H12W···O4	0.85 (2)	1.90 (2)	2.725 (5)	166 (5)
O2W—H21W···O31W^i	0.85 (2)	1.79 (6)	2.504 (11)	140 (8)
O2W—H22W···O1W^i	0.85 (2)	2.09 (4)	2.889 (6)	156 (8)

Symmetry codes: (i) −x+1, y+1/2, −z+1/2; (ii) x+1, y, z; (iii) −x+2, y+1/2, −z+1/2; (iv) x, y+1, z; (v) x−1, y, z; (vi) −x, y+1/2, −z+1/2.