Nucleotide–amino acid inter­actions in the L-His–IMP·MeOH·H₂O complex

In the crystal structure of the methanol-solvated mono­hydrated complex of L-histidine (His) with inosine 5′-monophosphate (IMP), namely L-histidinium inosine-5′-phosphate methanol solvate monohydrate, C₆H₁₀N₃O₂⁺·C₁₀H₁₂N₄O₈P⁻·CH₃OH·H₂O, most of the inter­actions between IMP anions (anti/C3′-endo/gauche–gauche conformers) are realized between the riboses and hypoxanthine bases in a trans sugar-edge/sugar-edge geometry, and between the phosphate groups. The base Watson–Crick edge is involved in additional methanol-mediated IMP...MeOH...IMP contacts. Specific and nonspecific nucleotide–amino acid (IMP...His) inter­actions engage the Hoogsteen edges of the base and phosphate group, respectively. Additional stabilization of His...IMP contacts is provided by π–π stacking between the imidazolium ring of His and the hypoxanthine base of IMP. The results may indicate the possible recognition mechanism between His and IMP.

S1. Comment

Specific protein–nucleotide/nucleic acid interactions are realised mainly via direct hydrogen bonds between the nucleotide base and amino acid side-chain, hydrophobic interactions, and π–π stacking between the base and aromatic side-chain (Rice & Correll, 2008). For base contacts, significant amino acid–base type correlations have been revealed, with the most obvious being the arginine–guanine pair (Luscombe et al., 2001; Hoffman et al., 2004). Inspection of DNA–protein complexes has also shown a histidine–guanine preference (Luscombe et al., 2001). In protein–DNA complexes, the main specificity is ascribed to the major rather than the minor groove. In protein–RNA, as well as in protein–nucleotide recognition, the different edges of a base may play a similar role in protein binding. According to the descriptive nomenclature based on geometry introduced by Leontis & Westhof (2001) for classification of non-Watson–Crick RNA base pairing, three distinct edges in the nucleotide base may be distinguished: the Watson–Crick edge, the Hoogsteen edge (equivalent to the B-DNA major groove and RNA A-type helix deep groove), and the sugar edge (which includes the 2'-OH group and is equivalent to the B-DNA minor and RNA shallow groove).

We believe that the high-resolution crystal structures of amino acid–nucleotide complexes could be a reliable and informative tool in the investigation of protein–nucleic acid interactions and their manner of recognition. We have therefore undertaken the synthesis and structural analysis of the title L-His–IMP.MeOH.H₂O complex, (I), presented here, which reveals both the specific and the nonspecific interactions of L-histidine (His) with inosine 5'-monophosphate (IMP), commonly found in tRNA nucleotides. The conformation and binding mode of IMP are compared with those found in three other amino acid–IMP complexes deposited in the Cambridge Structural Database (CSD; Allen, 2002) and in high-resolution protein–IMP complexes deposited in the Protein Data Bank (PDB; Berman et al., 2000). The three isomorphous complexes of IMP with L-Ser (CSD refcode ZUWQEN; Mukhopadhyay et al., 1995), L-Glu (QUSMIA; Bhattacharya et al., 2000) and L-Gln (LIRQUY; Bera et al., 1999), along with two complexes of N⁷-methylguanosine 5'-monophosphate (m⁷GMP) with L-Phe and Trp–Glu (DUMJEA10 and SEKXIP10, respectively; Ishida et al., 1988, 1991) are the only examples of amino acid/oligopeptide–nucleotide complexes reported so far (not counting five coordination compounds containing cobalt or platinum cations interfering with the amino acid–nucleotide interactions). Unfortunately, the quality of the data for the previously reported complexes is not satisfactory. In addition, in the highly hydrated IMP–Ser/Glu/Gln complexes, no direct specific nucleobase–amino acid functional site interactions can be observed.

The asymmetric unit of (I) consists of an IMP monoanion, a His cation and two solvent molecules (methanol and water), as shown in Fig. 1. The nucleotide adopts an anti conformation, typical for both free and amino acid-complexed IMP, about the N-glycosidic C1'—N9 bond (χ_CN O4'—C1'—N9—C4 close to a common value of 140°; Table 1), and also a typical gauche–gauche conformation about the C4'—C5' bond. The relative orientation of the hypoxanthine (Hyp) base and the ester atom O5' is stabilized by an intramolecular C8—H8···O5' hydrogen bond (Table 2), shown as a dotted line in Fig. 1.

Interestingly, while the high-resolution crystal structures of protein-bound IMP [RNase A–IMP complex, PDB ID 1Z6D (Hatzopoulos et al., 2005), and NTPase–IMP complex, 2DVN (Lokanath et al., 2008)] reveal a slight preference for anti over syn conformations about the glycosidic bond, the same structures show that the IMP molecules, upon binding to the protein active site, adopt diverse conformations about the C4'—C5' bond, i.e. gauche–trans, trans–gauche and gauche–gauche.

The nucleotide ribose ring in (I) is puckered in an envelope C3'-endo (³E) manner, as confirmed by the pseudorotation parameters (Rao et al., 1981) P = 13.9 (5)° and τ_m = 33.7 (3)° (reference bond C2'—C3'), as well as by the Cremer & Pople (1975) puckering parameters q₂ = 0.328 (6) Å and ϕ₂ = 283 (1)°. The envelope ³E conformation is one of two typically observed in free IMP and other free and protein-bound nucleotides (Allen, 2002; Moodie & Thornton, 1993), but was not observed in the previously reported amino acid–nucleotide (both IMP and m⁷GMP) complexes, in which C2'-endo (²E) puckering is the most common. Similarly, the ribose in protein-bound IMP does not reveal any conformational preference and is found in various puckering conformations, i.e. C2'-endo (²E), C2'-exo (E₂), C1'-endo (¹E), C3'-endo (³E) and C4'-exo (E₄).

As seen from the β torsion angle (P1—O5'—C5'—C4'; Table 1), the phosphate group of the inosine nucleotide of (I) is oriented in an antiperiplanar manner relative to the ribose ring, which is also a characteristic of amino acid-complexed IMP, but not the only preferred conformation for protein-bound IMP and other nucleotides (Moodie & Thornton, 1993; Berman et al., 2000). The orientation of terminal phosphate O atoms relative to atom C5', with the two O atoms (O8 and O9) being in ±sc psoitions and the third (hydroxyl O7) in an ap position, is typical for organic phosphate monoesters. However, the location of the H atom in the phosphate group at the antiperiplanar O atom is not often observed in monoanionic phosphate esters. The geometry of the monoionized IMP phosphate group reveals a significant deformation from the ideal tetrahedral shape (Table 1), with the O7—P1—O5' angle, formed by the ester and hydroxyl O_ap atom, being the smallest of all the phosphate angles (\sim 100°) and the remaining O—P—O angles being in the range 107–114°. The value of the O7—P1—O5' angle correlates with the P1—O5' and P1—O7 bond lengths, being significantly longer than the other P—O bonds (Table 1).

The His cation of (I), with the carboxyl group deprotonated and both amino atom N11 and imidazole atom N16 (N^ε) protonated, reveals a perpendicular orientation of the imidazolium ring and C^α12—C^β13—C^γ14 fragment, which is reflected in the interplanar angle of 86.8 (4)° and in the χ² (C^α12—C^β13—C^γ14—N^δ18/C^δ15) torsion angles, which are close to ∓90° (Table 1). These, in combination with a χ¹ (N11—C^α12—C^β13—C^γ14) angle close to 65°, indicate the location of the protonated atoms N11 and N18 (N^δ) on the same side of the His cation, as shown in Fig. 1, which plays a role in the His···IMP interactions, as will be discussed later.

Among all the intermolecular interactions observed in the crystal structure of (I), three general types of direct contacts may be distinguished: (a) His···His interactions between adjacent His cations, (b) IMP···IMP interactions between nucleotide anions, and (c) His···IMP interactions, crucial to the amino acid–nucleotide recognition process. In addition, indirect solvent-mediated hydrogen bonds are present.

Both His···His and IMP···IMP interactions arrange adjacent His cations or IMP^- anions (related by a 2₁ axis and a direct b-axis translation) into infinite helices running down the b axis, as shown in Fig. 2. Within the His helix, the ammonium and carboxylate groups of adjacent His cations interact with each other via N11—H11C···O12^viii hydrogen bonds of a salt-bridge type (Table 2). As a result, the His functional groups (protonated imidazole rings) are exposed outside the helix, which enables the specific amino acid–nucleotide interactions. The structure of the His helix is additionally stabilized by interactions between adjacent cations related by a direct b-axis translation (Fig. 2a, Table 2): unusually short C—H···O hydrogen bonds [C^α12—H12···O12ⁱ; symmetry code: (i) x, y + 1, z] and C—H···π interactions [C^β13—H13B···Cg1ⁱ (Cg1 is the centroid of the protonated imidazole ring of the His cation), with a perpendicular H13B···ringⁱ distance of 2.70 Å, an H13B···Cgⁱ distance of 2.97 Å and a C13—H13B···Cg1ⁱ angle of 146°].

Similarly, in the IMP helix, a direct b-axis translation and strong phosphate···phosphate O7—H7···O9ⁱ hydrogen bonds, along with close sugar···sugar C—H···O contacts and possible base···base C═O···π interactions [C6═O6···Cg2ⁱ (Cg2 is the centroid of the N1–C6 ring), with a perpendicular O6···ringⁱ distance of 3.289 Å, an O6···Cg2ⁱ distance of 3.488 (5) Å and a C6═O6···Cg2ⁱ angle of 101.0 (4)°] generate infinite chains of IMP anions running down the b axis. The mutual orientation and interactions between two parallel chains related by the action of a 2₁ screw axis result in the helices having the IMP anions arranged in a trans manner and pointing their sugar edges towards each other, as shown in Fig. 2(b). Therefore, most of the intrahelical IMP···IMP interactions (beside those between the phosphate groups) are realised between the riboses and the hypoxanthine (Hyp) bases in a trans sugar edge/sugar edge geometry (Leontis & Westhof, 2001). These are sugar···base [O2'—H2'···N3(Hyp)ⁱⁱ and C2(Hyp)—H2···O3'^ix; symmetry codes: Fill in from table] and sugar···sugar close C—H···O contacts which also involve the ribose atom O3' (in polynucleotides this atom constitutes the nucleic acid backbone and is thus not considered as a sugar-edge atom). The Watson–Crick edges are engaged in methanol-mediated IMP···MeOH···IMP interactions involving the hypoxanthine atoms N1 and O6: N1—H1···O31—H31···O6^v (Table 2). In the previously reported IMP complexes, the IMP···IMP contacts, seeming to be genuinely predominant, were realised mainly via phosphate···ribose interactions involving both atoms O2' and O3', and via N1(Hyp)···phosphate contacts, which are additionally possible in Ser–IMP and Gln–IMP, but not in Glu–IMP.

Finally, the nucleotide Hoogsteen edge, commonly used in protein–nucleic acid recognition, is also involved in amino acid–nucleotide (His···IMP) interactions in (I). As shown in Figs. 1 and 3, the histidine side chain–Hyp base recognition is realised via specific hydrogen bonds utilizing both Hyp atoms N7 and O6, i.e. N^ε16—H16···N7(Hyp) and C^δ15—H15···O6(Hyp) (Table 2). In DNA–protein complexes, histidine has been shown to prefer interactions with guanine (Luscombe et al., 2001), which is a 2-amino derivative of hypoxanthine and thus has the same Hoogsteen edge crucial for protien–DNA recognition. Direct amino acid side-chain–Hyp interactions were not observed in the other IMP–amino acid complexes, but they may be analysed in macromolecular protein–IMP complexes. Hyp atom N7 in one of the IMP molecules in the complexes RNase A–IMP (Hatzopoulos et al., 2005) and NTPase–IMP (Lokanath et al., 2008) is involved in such hydrogen bonding to O^γ(Thr) and N^η2(Arg), respectively. In the NTPase–IMP complex, the Hyp bases interact via O6···N^ζ(Lys), O6···N^ε(His) and O6···N^η1(Arg) contacts. In protein–IMP complexes, N1(Hyp)···amino acid side-chain interactions are also commonly observed. However, in the L-His–IMP complex presented here, this atom is involved in IMP···MeOH···IMP interactions, and therefore does not bind His at all.

The nucleotide anionic phosphate group in the L-His–IMP complex, (I), accepts four additional nonspecific N/C—H···O hydrogen bonds from three different His cations, two of which are bound in a monodentate manner (via the NH₃⁺ group or imidazolium ring) and the third in a bidentate manner via both ammonium atom N11 and imidazolium atom N18 (N^δ) (Fig. 3). The latter is facilitated by the specific His conformation with both atoms N11 and N18 located on the same side, as discussed above. Phosphate···His hydrogen bonds are also observed in the RNase–IMP complex, where the phosphate group of one of the three bound IMP molecules interacts with atoms N^δ and N^ε of two different His residues from the catalytic triad.

In the current L-His–IMP complex, the nucleotide ribose atom O3' is involved in water-mediated IMP···H₂O···His interactions: O3'—H3'···O21—H21A···O11ⁱⁱⁱ and O3'—H3'···O21—H21B···O11^iv [symmetry codes: From table]. Additional stabilization of the His···IMP contacts is provided by π–π stacking of the cation···π type, formed between the centroid Cg1 of the protonated imidazole ring of the His cation and the Hyp base of the IMP anion [centroid-to-centroid distance of 3.693 (4) Å], as shown in Fig. 4. A close P1—O8···Cg1^$ contact, which can be classified as a lone pair···π interaction, is also observed, with a perpendicular O8···ring^$ distance of 3.24 Å, an O8···Cg1^$ distance of 3.567 (4) Å and a P1—O8···Cg1^$ angle of 105.1 (2)° [symmetry code: ($) -x + 1, y - 1/2, -z + 1] (Fig. 4).

Related literature

For related literature, see: Allen (2002); Bera et al. (1999); Berman et al. (2000); Bhattacharya et al. (2000); Cremer & Pople (1975); Hatzopoulos et al. (2005); Hoffman et al. (2004); Ishida et al. (1988, 1991); Leontis & Westhof (2001); Lokanath et al. (2008); Luscombe et al. (2001); Moodie & Thornton (1993); Mukhopadhyay et al. (1995); Rao et al. (1981); Rice & Correll (2008).

S2. Experimental

A MeOH/water solution (Solvent ratio?) containing a 1:1 or 1:2 molar ratio of inosine 5'-phosphate disodium salt (IMPNa₂; Sigma–Aldrich) and L-histidine hydrochloride (L-His.HCl; Chemapol) was heated at 333 K for 15 min. Slow evaporation of the resulting solution at room temperature gave small needle-shaped, mostly twinned, crystals of L-His–IMP.MeOH.H₂O, (I).

S3. Refinement

All H atoms were found in difference Fourier maps. In the final refinement cycles, all except for water-bonded H atoms were positioned geometrically and treated as riding atoms, with C—H = 0.95–1.00 Å, N—H = 0.88–0.91 Å and O—H = 0.84 Å, and with U_iso(H) = 1.2U_eq(C,Nsp²) or 1.5U_eq(O,Nsp³). Water H atoms were refined with U_iso(H) = 1.5U_eq(O).

Computing details

Data collection: CrysAlis CCD (Oxford Diffraction, 2007); cell refinement: CrysAlis RED (Oxford Diffraction, 2007); data reduction: CrysAlis RED (Oxford Diffraction, 2007); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: XP in SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97 (Sheldrick, 2008).

Figures

	Fig. 1. The structures and atom-numbering schemes for the His cation, IMP anion and solvent molecules joined by hydrogen bonds (dashed lines) in the asymmetric unit of (I). The intramolecular C8—H8···O5' contact, stabilizing the IMP structure, is shown with a dotted line [Currently a dashed line - please revise plot]. Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radii.
	Fig. 2. The arrangement of (a) the histidinium cations and (b) the IMP anions within the separate helices running down the b axis. Intermolecular (a) His···His and (b) IMP···IMP interactions are shown as dashed lines, C—H···π interactions with dashed open lines and intramolecular C8—H8···O5' interactions with dotted lines. Symmetry codes are given in Table 2.
	Fig. 3. The specific and nonspecific His···IMP hydrogen bonds (dashed lines) observed in (I). Close P—O···π(His) and intramolecular C8—H8···O5' contacts are shown with dashed open and dotted lines, respectively. Symmetry codes are given in Table 2.
	Fig. 4. L-His cations and IMP anions interacting via π–π stacking and close P—O···π(His) contacts (dashed open lines). His···His C—H···π interactions (dashed open lines), and inter- and intramolecular hydrogen bonds (dashed and dotted lines, respectively) are also shown. Symmetry codes are given in Table 2. [Additionally, symmetry code: ($) -x + 1, y - 1/2, -z + 1.]

L-Histdinium inosine-5'-phosphate methanol solvate monohydrate

Crystal data

C6H10N3O2^+·C10H12N4O8P^−·CH4O·H2O	F(000) = 580
Mr = 553.43	Dx = 1.618 Mg m^−3
Monoclinic, P21	Mo Kα radiation, λ = 0.71073 Å
Hall symbol: P 2yb	Cell parameters from 3325 reflections
a = 14.195 (6) Å	θ = 4.2–42.0°
b = 4.816 (3) Å	µ = 0.20 mm^−1
c = 17.029 (6) Å	T = 110 K
β = 102.58 (3)°	Needle, colourless
V = 1136.2 (9) Å^3	0.24 × 0.02 × 0.01 mm
Z = 2

Data collection

Oxford Xcalibur PX κ-geometry diffractometer with CCD Onyx camera	1891 reflections with I > 2σ(I)
Radiation source: fine-focus sealed tube	Rint = 0.136
Graphite monochromator	θmax = 26.0°, θmin = 4.2°
ω and ϕ scans	h = −16→17
13113 measured reflections	k = −5→5
4289 independent reflections	l = −21→19

Refinement

Refinement on F^2	Secondary atom site location: difference Fourier map
Least-squares matrix: full	Hydrogen site location: difference Fourier map
R[F^2 > 2σ(F^2)] = 0.052	H atoms treated by a mixture of independent and constrained refinement
wR(F^2) = 0.070	w = 1/[σ^2(Fo^2) + (0.P)^2] where P = (Fo^2 + 2Fc^2)/3
S = 0.77	(Δ/σ)max < 0.001
4289 reflections	Δρmax = 0.34 e Å^−3
346 parameters	Δρmin = −0.38 e Å^−3
1 restraint	Absolute structure: Flack (1983), with 1785 Friedel pairs
Primary atom site location: structure-invariant direct methods	Absolute structure parameter: −0.04 (18)

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.

Refinement. Refinement of F² against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F², conventional R-factors R are based on F, with F set to zero for negative F². The threshold expression of F² > σ(F²) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F² 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 (Ų)

	x	y	z	Uiso*/Ueq
P1	0.34947 (11)	−0.1216 (4)	0.57602 (8)	0.0210 (4)
O7	0.4318 (2)	0.0994 (7)	0.5780 (2)	0.0198 (10)
H7	0.4079	0.2597	0.5744	0.030*
O8	0.2523 (2)	−0.0336 (7)	0.52848 (17)	0.0224 (10)
O9	0.3854 (2)	−0.3940 (7)	0.54845 (17)	0.0192 (10)
O2'	0.3828 (3)	−0.2706 (8)	0.9686 (2)	0.0241 (10)
H2'	0.3580	−0.1599	0.9964	0.036*
O3'	0.2451 (2)	−0.0022 (9)	0.85687 (18)	0.0237 (10)
H3'	0.2006	0.0203	0.8159	0.035*
O4'	0.4219 (2)	−0.4579 (8)	0.80445 (17)	0.0178 (9)
O5'	0.3492 (2)	−0.1397 (8)	0.66918 (16)	0.0202 (9)
O6	0.8424 (3)	0.2979 (9)	0.8626 (2)	0.0301 (12)
N1	0.8292 (3)	−0.0404 (10)	0.9557 (2)	0.0266 (13)
H1	0.8885	−0.0042	0.9821	0.032*
N3	0.6906 (3)	−0.3145 (10)	0.9549 (2)	0.0202 (12)
N7	0.6313 (3)	0.1524 (9)	0.7875 (2)	0.0169 (12)
N9	0.5579 (3)	−0.1687 (10)	0.8467 (2)	0.0189 (12)
C1'	0.4792 (4)	−0.3208 (11)	0.8723 (3)	0.0162 (14)
H1'	0.5073	−0.4610	0.9142	0.019*
C2'	0.4135 (3)	−0.1213 (13)	0.9062 (3)	0.0166 (13)
H2'A	0.4467	0.0568	0.9258	0.020*
C3'	0.3304 (3)	−0.0793 (11)	0.8330 (3)	0.0158 (14)
H3'A	0.3484	0.0700	0.7981	0.019*
C4'	0.3243 (3)	−0.3533 (11)	0.7889 (3)	0.0155 (14)
H4'	0.2827	−0.4844	0.8118	0.019*
C5'	0.2887 (4)	−0.3376 (11)	0.6986 (2)	0.0183 (14)
H5'A	0.2931	−0.5220	0.6741	0.022*
H5'B	0.2205	−0.2762	0.6850	0.022*
C2	0.7815 (4)	−0.2420 (13)	0.9852 (3)	0.0288 (17)
H2	0.8152	−0.3400	1.0312	0.035*
C4	0.6511 (4)	−0.1554 (14)	0.8900 (3)	0.0197 (14)
C5	0.6962 (4)	0.0430 (13)	0.8540 (3)	0.0178 (14)
C6	0.7931 (4)	0.1173 (13)	0.8866 (3)	0.0230 (16)
C8	0.5486 (4)	0.0194 (11)	0.7861 (3)	0.0203 (14)
H8	0.4904	0.0521	0.7476	0.024*
O11	1.0199 (3)	0.7180 (8)	0.68428 (19)	0.0278 (11)
O12	0.9791 (2)	0.5547 (8)	0.55834 (18)	0.0208 (9)
N11	0.8602 (3)	0.9621 (9)	0.50218 (19)	0.0198 (12)
H11A	0.8286	1.1240	0.4865	0.030*
H11B	0.8166	0.8211	0.4979	0.030*
H11C	0.9023	0.9262	0.4701	0.030*
N16	0.6764 (3)	0.5694 (10)	0.6901 (2)	0.0203 (12)
H16	0.6560	0.4380	0.7183	0.024*
N18	0.6788 (3)	0.8814 (10)	0.5999 (2)	0.0180 (11)
H18	0.6599	0.9903	0.5579	0.022*
C11	0.9757 (4)	0.7274 (13)	0.6119 (3)	0.0231 (15)
C12	0.9142 (3)	0.9865 (12)	0.5875 (2)	0.0171 (14)
H12	0.9610	1.1421	0.5882	0.021*
C13	0.8483 (4)	1.0789 (12)	0.6422 (3)	0.0186 (14)
H13A	0.8889	1.1143	0.6964	0.022*
H13B	0.8184	1.2578	0.6217	0.022*
C14	0.7699 (4)	0.8851 (13)	0.6511 (3)	0.0194 (13)
C15	0.7671 (4)	0.6853 (12)	0.7074 (3)	0.0202 (15)
H15	0.8186	0.6354	0.7508	0.024*
C17	0.6248 (4)	0.6869 (11)	0.6244 (3)	0.0149 (13)
H17	0.5603	0.6405	0.5991	0.018*
O21	0.1199 (4)	0.2157 (10)	0.7288 (3)	0.0437 (15)
H21A	0.082 (5)	0.095 (15)	0.713 (4)	0.066*
H21B	0.111 (5)	0.359 (16)	0.716 (4)	0.066*
O31	0.9968 (3)	0.0716 (11)	1.0600 (2)	0.0416 (13)
H31	1.0388	−0.0497	1.0769	0.062*
C32	0.9766 (5)	0.227 (2)	1.1282 (4)	0.083 (3)
H32A	1.0102	0.4064	1.1324	0.100*
H32B	0.9993	0.1212	1.1778	0.100*
H32C	0.9070	0.2588	1.1201	0.100*

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
P1	0.0213 (9)	0.0203 (10)	0.0191 (8)	−0.0036 (9)	−0.0002 (7)	0.0009 (7)
O7	0.018 (2)	0.020 (3)	0.021 (2)	−0.001 (2)	0.0034 (17)	0.0022 (19)
O8	0.014 (2)	0.022 (3)	0.0262 (19)	0.0054 (18)	−0.0048 (16)	−0.0005 (18)
O9	0.029 (2)	0.017 (3)	0.0127 (18)	0.005 (2)	0.0055 (17)	0.0007 (16)
O2'	0.025 (2)	0.033 (3)	0.014 (2)	0.005 (2)	0.0043 (17)	0.0014 (19)
O3'	0.014 (2)	0.033 (3)	0.0195 (18)	0.007 (2)	−0.0059 (16)	0.0011 (19)
O4'	0.013 (2)	0.021 (3)	0.019 (2)	0.0036 (19)	0.0018 (16)	−0.0037 (18)
O5'	0.023 (2)	0.021 (3)	0.0150 (18)	−0.006 (2)	0.0009 (16)	−0.0042 (19)
O6	0.025 (3)	0.031 (3)	0.032 (2)	−0.010 (2)	0.0037 (19)	0.0034 (19)
N1	0.018 (3)	0.034 (4)	0.024 (2)	−0.004 (3)	−0.002 (2)	0.009 (2)
N3	0.024 (3)	0.022 (3)	0.013 (2)	−0.003 (3)	0.001 (2)	0.005 (2)
N7	0.021 (3)	0.014 (3)	0.015 (2)	0.002 (2)	0.004 (2)	−0.001 (2)
N9	0.009 (3)	0.030 (4)	0.016 (2)	0.000 (2)	0.000 (2)	−0.004 (2)
C1'	0.016 (3)	0.016 (4)	0.017 (3)	−0.007 (3)	0.005 (3)	0.009 (3)
C2'	0.010 (3)	0.017 (3)	0.022 (3)	−0.003 (3)	0.000 (2)	0.005 (3)
C3'	0.012 (3)	0.011 (4)	0.021 (3)	0.001 (3)	−0.003 (2)	−0.003 (2)
C4'	0.014 (3)	0.020 (4)	0.015 (3)	0.001 (3)	0.007 (2)	0.002 (2)
C5'	0.021 (3)	0.022 (4)	0.012 (3)	−0.008 (3)	0.004 (2)	−0.001 (2)
C2	0.021 (4)	0.035 (5)	0.031 (4)	0.003 (3)	0.006 (3)	0.014 (3)
C4	0.020 (3)	0.025 (4)	0.017 (3)	−0.002 (3)	0.011 (3)	−0.004 (3)
C5	0.017 (3)	0.020 (4)	0.015 (3)	−0.003 (3)	0.002 (3)	−0.005 (3)
C6	0.019 (4)	0.030 (5)	0.020 (3)	0.007 (3)	0.003 (3)	−0.006 (3)
C8	0.027 (4)	0.020 (4)	0.015 (3)	0.005 (3)	0.006 (3)	0.005 (3)
O11	0.031 (3)	0.028 (3)	0.020 (2)	0.004 (2)	−0.0038 (19)	−0.001 (2)
O12	0.015 (2)	0.017 (3)	0.030 (2)	0.001 (2)	0.0028 (17)	0.006 (2)
N11	0.018 (3)	0.018 (3)	0.027 (3)	0.009 (2)	0.012 (2)	0.005 (2)
N16	0.024 (3)	0.014 (3)	0.026 (3)	0.000 (3)	0.012 (2)	−0.003 (2)
N18	0.015 (3)	0.022 (3)	0.015 (2)	−0.002 (3)	0.000 (2)	0.000 (2)
C11	0.025 (4)	0.019 (4)	0.026 (4)	−0.015 (3)	0.007 (3)	0.002 (3)
C12	0.006 (3)	0.031 (4)	0.014 (3)	0.001 (3)	0.002 (2)	0.004 (3)
C13	0.018 (3)	0.015 (4)	0.023 (3)	0.003 (3)	0.004 (2)	−0.004 (3)
C14	0.024 (4)	0.021 (4)	0.015 (3)	0.002 (3)	0.009 (3)	−0.001 (3)
C15	0.012 (3)	0.030 (4)	0.016 (3)	0.005 (3)	−0.003 (2)	−0.005 (3)
C17	0.019 (3)	0.009 (4)	0.020 (3)	−0.003 (3)	0.011 (3)	−0.002 (3)
O21	0.039 (3)	0.026 (4)	0.056 (3)	0.011 (3)	−0.012 (2)	0.005 (3)
O31	0.021 (3)	0.055 (4)	0.042 (2)	0.015 (2)	−0.009 (2)	−0.008 (2)
C32	0.057 (6)	0.111 (9)	0.083 (6)	0.014 (6)	0.018 (5)	0.036 (6)

Geometric parameters (Å, º)

P1—O5'	1.590 (3)	C2—H2	0.95
P1—O7	1.576 (3)	C4—C5	1.367 (7)
P1—O8	1.501 (3)	C5—C6	1.412 (7)
P1—O9	1.519 (4)	C8—H8	0.95
O7—H7	0.84	O11—C11	1.256 (5)
O2'—C2'	1.428 (5)	O12—C11	1.243 (6)
O2'—H2'	0.84	N11—C12	1.492 (5)
O3'—C3'	1.409 (5)	N11—H11A	0.91
O3'—H3'	0.84	N11—H11B	0.91
O4'—C1'	1.422 (5)	N11—H11C	0.91
O4'—C4'	1.443 (5)	N16—C17	1.323 (6)
O5'—C5'	1.445 (5)	N16—C15	1.375 (6)
O6—C6	1.240 (6)	N16—H16	0.88
N1—C2	1.343 (7)	N18—C17	1.334 (6)
N1—C6	1.401 (6)	N18—C14	1.393 (6)
N1—H1	0.88	N18—H18	0.88
N3—C2	1.328 (6)	C11—C12	1.528 (7)
N3—C4	1.362 (6)	C12—C13	1.523 (6)
N7—C8	1.333 (6)	C12—H12	1.00
N7—C5	1.398 (6)	C13—C14	1.486 (7)
N9—C8	1.357 (6)	C13—H13A	0.99
N9—C4	1.369 (6)	C13—H13B	0.99
N9—C1'	1.478 (6)	C14—C15	1.365 (7)
C1'—C2'	1.537 (7)	C15—H15	0.95
C1'—H1'	1.00	C17—H17	0.95
C2'—C3'	1.531 (6)	O21—H21A	0.80 (6)
C2'—H2'A	1.00	O21—H21B	0.73 (7)
C3'—C4'	1.511 (7)	O31—C32	1.462 (8)
C3'—H3'A	1.00	O31—H31	0.84
C4'—C5'	1.512 (6)	C32—H32A	0.98
C4'—H4'	1.00	C32—H32B	0.98
C5'—H5'A	0.99	C32—H32C	0.98
C5'—H5'B	0.99
O5'—P1—O7	100.3 (2)	C5—C4—N9	106.5 (5)
O5'—P1—O8	110.6 (2)	C4—C5—N7	109.7 (5)
O5'—P1—O9	109.6 (2)	C4—C5—C6	120.8 (5)
O7—P1—O8	114.3 (2)	N7—C5—C6	129.5 (5)
O7—P1—O9	107.0 (2)	O6—C6—N1	121.8 (5)
O8—P1—O9	114.1 (2)	O6—C6—C5	128.3 (5)
P1—O7—H7	109.5	N1—C6—C5	109.9 (5)
C2'—O2'—H2'	109.5	N7—C8—N9	111.6 (4)
C3'—O3'—H3'	109.5	N7—C8—H8	124.2
C1'—O4'—C4'	110.1 (4)	N9—C8—H8	124.2
C5'—O5'—P1	120.9 (3)	C12—N11—H11A	109.5
C2—N1—C6	125.7 (5)	C12—N11—H11B	109.5
C2—N1—H1	117.1	H11A—N11—H11B	109.5
C6—N1—H1	117.1	C12—N11—H11C	109.5
C2—N3—C4	111.1 (5)	H11A—N11—H11C	109.5
C8—N7—C5	104.8 (4)	H11B—N11—H11C	109.5
C8—N9—C4	107.5 (4)	C17—N16—C15	109.1 (5)
C8—N9—C1'	126.9 (4)	C17—N16—H16	125.4
C4—N9—C1'	124.6 (4)	C15—N16—H16	125.4
O4'—C1'—N9	109.0 (4)	C17—N18—C14	109.3 (5)
O4'—C1'—C2'	108.0 (4)	C17—N18—H18	125.4
N9—C1'—C2'	111.1 (4)	C14—N18—H18	125.4
O4'—C1'—H1'	109.6	O12—C11—O11	127.2 (6)
N9—C1'—H1'	109.6	O12—C11—C12	117.3 (4)
C2'—C1'—H1'	109.6	O11—C11—C12	115.5 (5)
O2'—C2'—C3'	111.5 (4)	N11—C12—C13	111.4 (4)
O2'—C2'—C1'	105.9 (4)	N11—C12—C11	110.0 (4)
C3'—C2'—C1'	101.5 (4)	C13—C12—C11	117.5 (4)
O2'—C2'—H2'A	112.4	N11—C12—H12	105.6
C3'—C2'—H2'A	112.4	C13—C12—H12	105.6
C1'—C2'—H2'A	112.4	C11—C12—H12	105.6
O3'—C3'—C4'	114.4 (4)	C14—C13—C12	116.9 (5)
O3'—C3'—C2'	111.0 (4)	C14—C13—H13A	108.1
C4'—C3'—C2'	103.9 (4)	C12—C13—H13A	108.1
O3'—C3'—H3'A	109.1	C14—C13—H13B	108.1
C4'—C3'—H3'A	109.1	C12—C13—H13B	108.1
C2'—C3'—H3'A	109.1	H13A—C13—H13B	107.3
O4'—C4'—C3'	105.3 (4)	C15—C14—N18	105.5 (5)
O4'—C4'—C5'	107.4 (4)	C15—C14—C13	130.6 (5)
C3'—C4'—C5'	115.6 (4)	N18—C14—C13	123.9 (5)
O4'—C4'—H4'	109.4	C14—C15—N16	107.7 (4)
C3'—C4'—H4'	109.4	C14—C15—H15	126.1
C5'—C4'—H4'	109.4	N16—C15—H15	126.1
O5'—C5'—C4'	107.1 (4)	N16—C17—N18	108.4 (5)
O5'—C5'—H5'A	110.3	N16—C17—H17	125.8
C4'—C5'—H5'A	110.3	N18—C17—H17	125.8
O5'—C5'—H5'B	110.3	H21A—O21—H21B	122 (8)
C4'—C5'—H5'B	110.3	C32—O31—H31	109.5
H5'A—C5'—H5'B	108.6	O31—C32—H32A	109.5
N3—C2—N1	124.9 (5)	O31—C32—H32B	109.5
N3—C2—H2	117.6	H32A—C32—H32B	109.5
N1—C2—H2	117.6	O31—C32—H32C	109.5
N3—C4—C5	127.4 (5)	H32A—C32—H32C	109.5
N3—C4—N9	126.1 (5)	H32B—C32—H32C	109.5
O7—P1—O5'—C5'	−175.2 (4)	C8—N9—C4—C5	0.0 (6)
O8—P1—O5'—C5'	63.8 (4)	C1'—N9—C4—C5	−169.1 (5)
O9—P1—O5'—C5'	−62.9 (4)	N3—C4—C5—N7	178.0 (5)
C4'—O4'—C1'—N9	−118.1 (5)	N9—C4—C5—N7	−0.6 (6)
C4'—O4'—C1'—C2'	2.8 (5)	N3—C4—C5—C6	−4.3 (9)
C8—N9—C1'—O4'	53.8 (7)	N9—C4—C5—C6	177.0 (5)
C4—N9—C1'—O4'	−139.2 (5)	C8—N7—C5—C4	0.9 (6)
C8—N9—C1'—C2'	−65.1 (6)	C8—N7—C5—C6	−176.5 (6)
C4—N9—C1'—C2'	101.9 (6)	C2—N1—C6—O6	−179.1 (6)
O4'—C1'—C2'—O2'	94.6 (4)	C2—N1—C6—C5	2.9 (8)
N9—C1'—C2'—O2'	−145.9 (4)	C4—C5—C6—O6	−177.4 (6)
O4'—C1'—C2'—C3'	−22.0 (5)	N7—C5—C6—O6	−0.3 (10)
N9—C1'—C2'—C3'	97.6 (5)	C4—C5—C6—N1	0.5 (8)
O2'—C2'—C3'—O3'	43.1 (6)	N7—C5—C6—N1	177.6 (5)
C1'—C2'—C3'—O3'	155.4 (4)	C5—N7—C8—N9	−0.9 (6)
O2'—C2'—C3'—C4'	−80.3 (5)	C4—N9—C8—N7	0.5 (6)
C1'—C2'—C3'—C4'	32.0 (5)	C1'—N9—C8—N7	169.4 (5)
C1'—O4'—C4'—C3'	18.2 (5)	O11—C11—C12—N11	−175.9 (4)
C1'—O4'—C4'—C5'	141.9 (4)	O12—C11—C12—N11	6.4 (7)
O3'—C3'—C4'—O4'	−152.8 (4)	O11—C11—C12—C13	−46.9 (6)
C2'—C3'—C4'—O4'	−31.6 (5)	O12—C11—C12—C13	135.3 (5)
O3'—C3'—C4'—C5'	88.8 (5)	N11—C12—C13—C14	64.8 (6)
C2'—C3'—C4'—C5'	−150.0 (4)	C11—C12—C13—C14	−63.4 (6)
P1—O5'—C5'—C4'	165.4 (3)	C17—N18—C14—C15	−0.4 (6)
O4'—C4'—C5'—O5'	−63.0 (5)	C17—N18—C14—C13	−179.2 (5)
C3'—C4'—C5'—O5'	54.2 (6)	C12—C13—C14—N18	−87.7 (7)
C4—N3—C2—N1	−0.6 (8)	C12—C13—C14—C15	93.8 (7)
C6—N1—C2—N3	−3.0 (9)	N18—C14—C15—N16	−0.6 (6)
C2—N3—C4—C5	4.3 (8)	C13—C14—C15—N16	178.1 (5)
C2—N3—C4—N9	−177.4 (6)	C17—N16—C15—C14	1.4 (6)
C8—N9—C4—N3	−178.6 (5)	C15—N16—C17—N18	−1.7 (6)
C1'—N9—C4—N3	12.3 (9)	C14—N18—C17—N16	1.3 (6)

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
O7—H7···O9^i	0.84	1.74	2.549 (5)	162
O2′—H2′···N3^ii	0.84	2.04	2.863 (6)	165
O3′—H3′···O21	0.84	1.91	2.707 (6)	157
O21—H21A···O11^iii	0.80 (6)	2.03 (7)	2.805 (6)	164 (7)
O21—H21B···O11^iv	0.73 (7)	2.15 (7)	2.824 (6)	153 (8)
O31—H31···O6^v	0.84	1.92	2.716 (5)	158
N1—H1···O31	0.88	1.84	2.695 (6)	165
N11—H11A···O8^vi	0.91	1.99	2.891 (5)	168
N11—H11B···O8^vii	0.91	1.97	2.856 (6)	164
N11—H11C···O12^viii	0.91	1.95	2.737 (5)	144
N16—H16···N7	0.88	1.89	2.767 (6)	171
N18—H18···O9^vi	0.88	1.87	2.716 (5)	161
C1′—H1′···O2′^ix	1.00	2.70	3.683 (6)	168
C3′—H3′A···O4′^i	1.00	2.49	3.340 (7)	142
C4′—H4′···O3′^x	1.00	2.70	3.597 (7)	150
C2—H2···O3′^ix	0.95	2.38	3.064 (7)	128
C8—H8···O5′	0.95	2.34	3.179 (6)	147
C12—H12···O12^i	1.00	2.08	2.964 (7)	146
C15—H15···O6	0.95	2.47	3.221 (7)	136
C17—H17···O9^i	0.95	2.45	3.385 (6)	168
C32—H32B···O11^v	0.98	2.47	3.183 (8)	130
C32—H32C···O3′^ii	0.98	2.55	3.467 (8)	156

Symmetry codes: (i) x, y+1, z; (ii) −x+1, y+1/2, −z+2; (iii) x−1, y−1, z; (iv) x−1, y, z; (v) −x+2, y−1/2, −z+2; (vi) −x+1, y+3/2, −z+1; (vii) −x+1, y+1/2, −z+1; (viii) −x+2, y+1/2, −z+1; (ix) −x+1, y−1/2, −z+2; (x) x, y−1, z.