5-Methyl-6-aza-2′-deoxy­isocytidine

In the title compound, 3-amino-2-(2-deoxy-β-D-erythro-pento­furan­osyl)-6-methyl-1,2,4-triazin-5(2H)-one, C₉H₁₄N₄O₄, the conformation of the N-glycosidic bond is high-anti and the 2-deoxy­ribo­furan­osyl moiety adopts a North sugar pucker (²T₃). The orientation of the exocyclic C—C bond between the –CH₂OH group and the five-membered ring is ap (gauche, trans). The crystal packing is such that the nucleobases lie parallel to the ac plane; the planes are connected via hydrogen bonds involving the five-membered ring.

S1. Comment

Transposition of the amino and oxo groups of 2'-deoxycytidine results in 2'-deoxyisocytidine. This reversal of the substituent pattern of the nucleobase results in a change of the hydrogen-bond donor–acceptor motif. As a result, parallel-stranded (ps) DNA will be formed via reverse Watson–Crick base pairs between 2'-deoxyisocytidine and 2'-deoxyguanosine (Seela & He, 2000). Such ps DNAs have been also found for oligonucleotide duplexes incorporating 2'-deoxy-5- methylisocytidine by base pairing with 2'-deoxyguanosine, or 2'-deoxisoguanosine by base pairing with 2'-deoxycytidine (Sugiyama et al., 1996; Seela, He & Wei, 1999). However, because of the low acid stability of 2'-deoxyisocytidine and its 5-methyl derivative, oligonucleotides containing these two compounds show substantial degradation at the N-glycosidic bond. This degradation occurs in solution as well as during MALDI-TOF spectrometric analysis (positive mode, matrix: 3-hydroxy-picolinic acid). To overcome this problem, 6-aza-2'-deoxy-5-methylisocytidine, (I), which is much more acid stable, was used as a replacement. Oligonucleotides containing (I) can form parallel DNA duplexes with slightly lower thermal stability than those containing 2'-deoxy-5-methylisocytidine, (II), (Seela & He, 2003); acidic degradation was not observed under the conditions described above. As it is expected that the additional N atom of (I) causes stereochemical changes to the sugar moiety, the X-ray structure of (I) was determined and compared with that of (II).

The maximum deviation from the least-squares plane of the nucleobase of (I) is ±0.016 Å [N1 = 0.021, C2 = −0.024, N3 = 0.007, C4 = 0.012, C5 = −0.015 and N6 = 0.000 (1) Å]. The maximum deviation of the pyrimidine ring of (II) is 0.043 Å. Selected bond lengths of the base residue are summarized in Table 1. The C(5)═N(6) bond of the base of (I) is 0.059 Å shorter than the C(5)═C(6) bond of (II). This shortening is similar to that observed between 6-azacytidine and cytidine (0.056 Å; Singh & Hodgson, 1974a). The glycosidic bond length of I [N1—C1'] is 1.479 (2) Å, which is approximately the same as that of (II) (1.478 (5) Å; ?? Seela, He, et al., 2000).

Compound (I) adopts a different conformation around the glycosidic bond than (II). While (II) shows a syn conformation (χ_CN = 58.2°), that of (I) is high anti with χ_CN (O4'—C1'—N1—C2) = −103.4°. This value is in accordance with the observation that the favored conformation of ortho azanucleosides with an N atom next to the glycosidic bond would have a χ_CN not far from −90°, because of the Coulomb repulsion between non-bonding electron pairs of O4' and the N atom next to the glycosidic bond (N-8 for azapurine nucleosides, and N-6 for azapyrimidine nucleosides). For 6-azapyrimidine ribonucleosides, the χ_CN of 6-azacytidine and 6-azauridine was reported to be −80° and −93°, respectively (Singh & Hodgson, 1974b; Schwalbe & Saenger, 1973). The glycosidic torsion angle of corresponding 2'-deoxyribonucleosides is also in the high-anti range (χ_CN of 6-aza-2'-deoxythymidine is −86.6°; Banerjee & Saenger, 1978). For 8-azapurine nucleosides, the χ_CN of 8-azaadenosine amounts to −77° (Singh & Hodgson, 1974c) and that of 8-aza-1,3-dideaza-2'-deoxyadenosine to −77.1 ° (Seela et al., 2001). For 8-aza-7-deaza-2'-deoxyadenosine, χ_CN moves towards the anti range (−106.3°; Seela, Zuluf et al., 1999). 8-Aza-7-deaza-7-iodo-2'-deoxyadenosine shows a smaller value (χ_CN = −73.2°), similar to that of the corresponding 7-bromo compound (χ_CN = −74.1°), which also displays a high-anti conformation (Seela, Zuluf et al., 2000).

The other major conformational parameter of interest is the pucker of the deoxyribofuanosyl moiety. The maximum amplitude of puckering (Ψ_m) of (I) is 26.3 (3)°, which is significantly smaller than the average value of 38.6±3° (Saenger, 1984). The phase angle of pseudorotation (P) of (I) is 344.6°, which corresponds to a ²T₃ sugar pucker. Therefore, the additional N atom of the nucleobase of (I) causes the sugar moiety to adopt an N-type instead of the S-type sugar pucker preferred by most 2'-deoxy-β-D-ribofuranosyl nucleosides. The N(3'-endo) \rightleftharpoons S (2'-endo) equilibrium of the sugar moiety is controlled by various gauche effects. It is known that the N-type conformer population increases linearly with the electronegativity of the 2'-substituent (Guschlbauer & Jankowski, 1980). It was also reported from a measurement performed in solution that the higher the electron-withdrawing effect of the 7-substituents of 7-deaza-2'-deoxyadenosines, the more the N \rightleftharpoons S equilibrium of the sugar moieties is biased towards the N conformation (Rosemeyer et al., 1997). In the crystalline state, the 8-aza-7-deaza-2'-deoxyadenosine adopts an S-type sugar puckering (P = 182.2°, ²T₃; Seela, Zulauf et al., 1999), while 8-aza-7-deaza-7-iodo-2'-deoxyadenosine (P = 309.4°, ¹E) and 8-aza-7-deaza-7-bromo-2'-deoxyadenosine (P = 310.9°, ¹E) adopt a sugar pucker that is close to N-type (Seela, Zulauf et al., 2000). For comparison, 7-halogenated 8-aza-7-deaza-2'-deoxyguanosines also exhibit an N-type sugar pucker (Seela, Becher et al., 1999).

The conformation about the C5'—C4' bond of (I) is ap (gauche,trans) with a torsion angle γ (O5'—C5'—C4'—C3') of 179.5°. Thus, atom O5' is located away from the sugar ring. The ap conformation means that the nucleobase and the CH₂OH group undergo a disrotatory motion so that the Coulomb repulsion between N6 (pyrimidine numbering) and O5', as well as O4', is minimized.

Intermolecular hydrogen bonds generate a three-dimensional network and provide additional crystal stabilization (Table 2). The crystal packing shows distinct formation of layers (Fig. 2). All nucleobases show a parallel orientation to the xz plane. Two types of hydrogen bonds are observed within the planes: type (i) (numbering in same order as Table 2) connects the H atom of the amino group of one molecule with the O atom of the 3'-hydroxyl group of another molecule, and type (iii) connects atom N3 with the H atom of the 3'-hydroxyl group. The sugar rings are approximately perpendicular to the nucleobase plane. Neighbouring planes are connected via a hydrogen bond from the second H atom of the amino group to the O atom of the 5'-hydroxyl group (ii) and another hydrogen bond from the H atom of the 5'-hydroxyl group to the N atom of the amino group (iv).

S2. Experimental

Compound (I) was synthesized as described by Seela & He (2003) and crystallized from acetone/MeOH (8:2).

S3. Refinement

In the absence of suitable anomalous scattering, Friedel equivalents could not be used to determine the absolute structure. Refinement of the Flack parameter (Flack, 1983) led to inconclusive values (Flack & Bernardinelli, 2000) for this parameter [−0.3 (0)]. Therefore, Friedel equivalents [20] were merged before the final refinement. The known configuration of the parent molecule was used to define the enantiomer employed in the refined model. All H atoms were initially found in a difference Fourier synthesis. In order to maximize the data–parameter ratio, H atoms bonded to C atoms were placed in geometrically idealized positions (C—H=0.93–0.98 Å) and constrained to ride on their parent atoms. The coordinates of other H atoms were refined freely, starting from difference map positions. An overall isotropic displacement parameter was refined for all H atoms.

Computing details

Data collection: XSCANS (Siemens, 1996); cell refinement: XSCANS; data reduction: SHELXTL (Sheldrick, 1997b); program(s) used to solve structure: SHELXS97 (Sheldrick, 1990); program(s) used to refine structure: SHELXL97 (Sheldrick, 1997a); molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL.

Figures

Fig. 1. A perspective view of (I). Displacement ellipsoids are shown at the 50% probability level, and H atoms are shown as spheres of arbitrary size. Fig. 2. The intermolecular hydrogen-bond network of the crystal packing, viewed perpendicular to the xy plane. Hydrogen bonds are indicated by dashed lines. H atoms not involved in hyddrogen bonding have been omitted.

(I)

Crystal data

C9H14N4O4	F(000) = 256
Mr = 242.24	Dx = 1.481 Mg m^−3
Monoclinic, P21	Mo Kα radiation, λ = 0.71073 Å
a = 8.682 (2) Å	Cell parameters from 39 reflections
b = 7.8835 (16) Å	θ = 5.3–20.6°
c = 8.998 (3) Å	µ = 0.12 mm^−1
β = 118.088 (18)°	T = 293 K
V = 543.3 (2) Å^3	Transparent needle, colourless
Z = 2	0.6 × 0.3 × 0.2 mm

Data collection

Bruker P4 diffractometer	Rint = 0.013
Radiation source: fine-focus sealed tube	θmax = 31.1°, θmin = 2.6°
Graphite monochromator	h = −12→1
2θ/ω scans	k = −1→11
2000 measured reflections	l = −11→13
1848 independent reflections	3 standard reflections every 97 reflections
1801 reflections with I > 2σ(I)	intensity decay: none

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)] = 0.036	w = 1/[σ^2(Fo^2) + (0.0769P)^2 + 0.0237P] where P = (Fo^2 + 2Fc^2)/3
wR(F^2) = 0.105	(Δ/σ)max < 0.001
S = 1.07	Δρmax = 0.28 e Å^−3
1848 reflections	Δρmin = −0.27 e Å^−3
169 parameters	Extinction correction: SHELXL97, Fc^*=kFc[1+0.001xFc^2λ^3/sin(2θ)]^-1/4
1 restraint	Extinction coefficient: 0.068 (12)
Primary atom site location: structure-invariant direct methods	Absolute structure: Flack H D (1983), Acta Cryst. A39, 876-881
Secondary atom site location: difference Fourier map	Absolute structure parameter: 0 (10)

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.

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
N1	0.25455 (13)	1.04157 (19)	0.51327 (13)	0.0291 (2)
C2	0.12052 (16)	1.0331 (2)	0.35471 (15)	0.0295 (3)
N2	0.15107 (17)	1.0263 (3)	0.22308 (15)	0.0407 (3)
H2A	0.057 (4)	1.029 (4)	0.126 (3)	0.0477 (18)*
H2B	0.248 (4)	1.033 (4)	0.232 (3)	0.0477 (18)*
N3	−0.04618 (15)	1.0321 (3)	0.32315 (16)	0.0389 (3)
C4	−0.08206 (17)	1.0302 (3)	0.45402 (19)	0.0371 (3)
O4	−0.23308 (15)	1.0281 (3)	0.43489 (19)	0.0610 (5)
C5	0.06730 (17)	1.0298 (3)	0.62485 (18)	0.0344 (3)
C6	0.0367 (2)	1.0213 (4)	0.7742 (2)	0.0520 (5)
H6A	0.1453	1.0007	0.8736	0.0477 (18)*
H6B	−0.0434	0.9309	0.7594	0.0477 (18)*
H6C	−0.0117	1.1269	0.7859	0.0477 (18)*
N6	0.22577 (15)	1.0367 (2)	0.64992 (14)	0.0337 (3)
C1'	0.44245 (14)	1.03544 (19)	0.56119 (14)	0.0252 (2)
H1'	0.4577	1.0573	0.4617	0.0477 (18)*
C2'	0.54904 (17)	1.16224 (19)	0.69809 (17)	0.0279 (2)
H2'1	0.4771	1.2567	0.6974	0.0477 (18)*
H2'2	0.6454	1.2056	0.6831	0.0477 (18)*
C3'	0.61549 (15)	1.06224 (18)	0.86083 (14)	0.0249 (2)
H3'	0.5344	1.0737	0.9077	0.0477 (18)*
O3'	0.78464 (14)	1.11949 (19)	0.97975 (12)	0.0379 (3)
H3'1	0.790 (4)	1.079 (4)	1.072 (3)	0.0477 (18)*
C4'	0.61588 (15)	0.87897 (18)	0.80461 (15)	0.0249 (2)
H4'	0.7354	0.8476	0.8313	0.0477 (18)*
O4'	0.50792 (14)	0.87332 (15)	0.62548 (12)	0.0306 (2)
C5'	0.5492 (2)	0.7544 (2)	0.88852 (18)	0.0319 (3)
H5'1	0.6221	0.7607	1.0096	0.0477 (18)*
H5'2	0.4315	0.7858	0.8635	0.0477 (18)*
O5'	0.54866 (16)	0.58482 (15)	0.83457 (16)	0.0350 (2)
H5'	0.455 (4)	0.569 (4)	0.772 (3)	0.0477 (18)*

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
N1	0.0193 (4)	0.0482 (7)	0.0176 (4)	−0.0011 (5)	0.0069 (3)	0.0017 (5)
C2	0.0226 (5)	0.0403 (7)	0.0207 (5)	−0.0034 (6)	0.0061 (4)	0.0029 (5)
N2	0.0269 (5)	0.0722 (10)	0.0201 (5)	−0.0034 (7)	0.0086 (4)	0.0003 (6)
N3	0.0208 (5)	0.0648 (9)	0.0247 (5)	−0.0021 (6)	0.0054 (4)	0.0069 (6)
C4	0.0207 (5)	0.0561 (9)	0.0314 (6)	0.0009 (6)	0.0096 (5)	0.0091 (7)
O4	0.0208 (5)	0.1140 (16)	0.0454 (7)	0.0009 (8)	0.0134 (5)	0.0167 (9)
C5	0.0226 (5)	0.0543 (8)	0.0263 (5)	0.0031 (6)	0.0115 (4)	0.0060 (6)
C6	0.0345 (7)	0.0941 (16)	0.0350 (7)	0.0016 (10)	0.0227 (6)	0.0050 (10)
N6	0.0226 (4)	0.0575 (8)	0.0213 (4)	−0.0007 (6)	0.0106 (4)	0.0025 (5)
C1'	0.0188 (4)	0.0376 (6)	0.0178 (4)	−0.0020 (5)	0.0074 (3)	0.0020 (4)
C2'	0.0234 (5)	0.0304 (6)	0.0241 (5)	−0.0026 (4)	0.0063 (4)	0.0032 (4)
C3'	0.0194 (4)	0.0345 (6)	0.0183 (4)	−0.0008 (4)	0.0068 (4)	−0.0014 (4)
O3'	0.0252 (4)	0.0560 (7)	0.0225 (4)	−0.0074 (5)	0.0028 (3)	−0.0037 (5)
C4'	0.0216 (5)	0.0316 (5)	0.0226 (5)	0.0038 (4)	0.0113 (4)	0.0039 (4)
O4'	0.0352 (5)	0.0339 (5)	0.0208 (4)	0.0021 (4)	0.0117 (3)	−0.0027 (4)
C5'	0.0348 (6)	0.0353 (6)	0.0302 (6)	0.0009 (5)	0.0193 (5)	0.0060 (5)
O5'	0.0299 (5)	0.0337 (5)	0.0377 (5)	−0.0003 (4)	0.0129 (4)	0.0035 (4)

Geometric parameters (Å, º)

N1—C2	1.3531 (15)	C1'—C2'	1.5164 (19)
N1—N6	1.3656 (15)	C1'—H1'	0.9800
N1—C1'	1.4789 (15)	C2'—C3'	1.5176 (18)
C2—N2	1.3310 (17)	C2'—H2'1	0.9700
C2—N3	1.3373 (16)	C2'—H2'2	0.9700
N2—H2A	0.87 (3)	C3'—O3'	1.4232 (15)
N2—H2B	0.81 (3)	C3'—C4'	1.5313 (19)
N3—C4	1.352 (2)	C3'—H3'	0.9800
C4—O4	1.2395 (17)	O3'—H3'1	0.87 (3)
C4—C5	1.472 (2)	C4'—O4'	1.4338 (16)
C5—N6	1.2863 (17)	C4'—C5'	1.5104 (18)
C5—C6	1.489 (2)	C4'—H4'	0.9800
C6—H6A	0.9600	C5'—O5'	1.421 (2)
C6—H6B	0.9600	C5'—H5'1	0.9700
C6—H6C	0.9600	C5'—H5'2	0.9700
C1'—O4'	1.4077 (18)	O5'—H5'	0.75 (3)
C2—N1—N6	121.12 (11)	C2'—C1'—H1'	109.4
C2—N1—C1'	126.07 (10)	C1'—C2'—C3'	104.52 (11)
N6—N1—C1'	112.39 (10)	C1'—C2'—H2'1	110.8
N2—C2—N3	117.39 (12)	C3'—C2'—H2'1	110.8
N2—C2—N1	120.50 (12)	C1'—C2'—H2'2	110.8
N3—C2—N1	122.12 (12)	C3'—C2'—H2'2	110.8
C2—N2—H2A	113.7 (16)	H2'1—C2'—H2'2	108.9
C2—N2—H2B	123 (2)	O3'—C3'—C2'	110.46 (11)
H2A—N2—H2B	123 (2)	O3'—C3'—C4'	112.68 (11)
C2—N3—C4	119.02 (12)	C2'—C3'—C4'	103.32 (9)
O4—C4—N3	122.78 (14)	O3'—C3'—H3'	110.1
O4—C4—C5	119.94 (14)	C2'—C3'—H3'	110.1
N3—C4—C5	117.27 (12)	C4'—C3'—H3'	110.1
N6—C5—C4	121.78 (13)	C3'—O3'—H3'1	100.1 (19)
N6—C5—C6	118.32 (13)	O4'—C4'—C5'	109.89 (11)
C4—C5—C6	119.90 (13)	O4'—C4'—C3'	107.38 (10)
C5—C6—H6A	109.5	C5'—C4'—C3'	112.81 (10)
C5—C6—H6B	109.5	O4'—C4'—H4'	108.9
H6A—C6—H6B	109.5	C5'—C4'—H4'	108.9
C5—C6—H6C	109.5	C3'—C4'—H4'	108.9
H6A—C6—H6C	109.5	C1'—O4'—C4'	110.95 (10)
H6B—C6—H6C	109.5	O5'—C5'—C4'	112.43 (11)
C5—N6—N1	118.51 (12)	O5'—C5'—H5'1	109.1
O4'—C1'—N1	109.03 (11)	C4'—C5'—H5'1	109.1
O4'—C1'—C2'	106.91 (10)	O5'—C5'—H5'2	109.1
N1—C1'—C2'	112.51 (11)	C4'—C5'—H5'2	109.1
O4'—C1'—H1'	109.4	H5'1—C5'—H5'2	107.9
N1—C1'—H1'	109.4	C5'—O5'—H5'	104 (2)
N6—N1—C2—N2	−175.38 (19)	N6—N1—C1'—O4'	69.21 (16)
C1'—N1—C2—N2	−3.4 (3)	C2—N1—C1'—C2'	138.20 (15)
N6—N1—C2—N3	5.0 (3)	N6—N1—C1'—C2'	−49.21 (17)
C1'—N1—C2—N3	176.99 (16)	O4'—C1'—C2'—C3'	−25.28 (12)
N2—C2—N3—C4	176.7 (2)	N1—C1'—C2'—C3'	94.38 (12)
N1—C2—N3—C4	−3.6 (3)	C1'—C2'—C3'—O3'	145.92 (11)
C2—N3—C4—O4	−179.5 (2)	C1'—C2'—C3'—C4'	25.17 (12)
C2—N3—C4—C5	0.1 (3)	O3'—C3'—C4'—O4'	−136.32 (10)
O4—C4—C5—N6	−178.1 (2)	C2'—C3'—C4'—O4'	−17.09 (12)
N3—C4—C5—N6	2.3 (3)	O3'—C3'—C4'—C5'	102.46 (13)
O4—C4—C5—C6	1.9 (3)	C2'—C3'—C4'—C5'	−138.31 (11)
N3—C4—C5—C6	−177.7 (2)	N1—C1'—O4'—C4'	−106.97 (11)
C4—C5—N6—N1	−1.1 (3)	C2'—C1'—O4'—C4'	14.91 (13)
C6—C5—N6—N1	178.90 (19)	C5'—C4'—O4'—C1'	124.60 (12)
C2—N1—N6—C5	−2.5 (3)	C3'—C4'—O4'—C1'	1.56 (13)
C1'—N1—N6—C5	−175.46 (16)	O4'—C4'—C5'—O5'	59.72 (15)
C2—N1—C1'—O4'	−103.38 (16)	C3'—C4'—C5'—O5'	179.50 (11)

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
N2—H2A···O3′^i	0.87 (3)	2.21 (3)	2.982 (2)	147 (2)
N2—H2B···O5′^ii	0.81 (3)	2.16 (3)	2.9243 (18)	159 (3)
O3′—H3′1···N3^iii	0.87 (3)	2.06 (3)	2.8117 (19)	145 (2)
O5′—H5′···O4^iv	0.75 (3)	1.98 (3)	2.707 (2)	166 (3)

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