Received 3 February 2011
Acidic and anionic forms of 1,3-cyclic dihydroxyacetone phosphate (cDHAP) dimethyl acetal
The six-membered cyclic phosphate diester, 5,5-dimethoxy-2-hydroxy-1,3,2-dioxaphosphorinan-2-one, C5H11O6P or (MeO)2cDHAP, which is the dimethyl acetal of cyclic dihydroxyacetone phosphate (cDHAP), has been obtained in the form of two new cyclohexylammonium (cha) salts, cyclohexylammonium 5,5-dimethoxy-2-oxo-1,3,2-dioxaphosphorinan-2-olate monohydrate, (cha)[(MeO)2cDHAP]·H2O or C6H14N+·C5H10O6P-·H2O, and cyclohexylammonium 5,5-dimethoxy-2-oxo-1,3,2-dioxaphosphorinan-2-olate, (cha)[(MeO)2cDHAP] or C6H14N+·C5H10O6P-, as well as in the form of the anhydrous free acid, (MeO)2cDHAP. It is shown that protonation of the cyclic phosphate group influences the chair conformation of the P/O/C/C/C/O 1,3,2-dioxaphosphorinane ring, and that differences in the ring conformation correlate with different deformations observed in the ionized and protonated phosphate groups. The ring is more evenly puckered in the anions, in contrast with the flattening observed in the structure of the free acid.
Six-membered cyclic phosphate esters are constituents of a number of biologically important molecules, such as 3':5'-cyclic nucleotides, e.g. cAMP. Dihydroxyacetone phosphate (DHAP), the linear form of cDHAP, is one of the most important biochemical intermediates and of great importance for all living cells [for a review, see Slepokura & Lis (2010)]. The cyclic form, cDHAP, has aroused interest recently as a new molecule of biological importance (Goswami & Adak, 2002). Occurring in living organisms, small cyclic phosphates of cDHAP-like structure began to attract attention when their biological activity as signalling molecules was suggested (Shinitzky et al., 2000). Cyclic glycerophosphates can be formed by enzymatic degradation of phospholipids, e.g. 1,3-cyclic glycerophosphate is naturally formed by the action of phospholipase C on phosphatidyl glycerol.
Only five of more than 160 hits for cyclic phosphates with six-membered rings deposited in the Cambridge Structural Database (CSD, Version 5.32; Allen, 2002) bear the H atom at the exocyclic O atom. We will discuss just three of the reported protonated cyclic phosphates [CSD refcodes ETPHOS (Gerlt et al., 1980), KADPUA (Johnson et al., 1989) and SEZRUL (Samas et al., 2007)], because the remaining two have high R factors and low bond precision, and the positions of the H atoms were not determined.
Previously, we have reported the synthesis and structural investigations of nine different salts of (MeO)2cDHAP with both organic and inorganic cations [(5a)-(5e) and (5e')-(5h); Slepokura, 2008], along with its phenyl derivative, (MeO)2cDHAP(Ph) [(4); Slepokura & Lis, 2004b]. Among these, the structures of two cyclohexylammonium (cha) salts and the acid in the form of an oxonium salt were presented: (cha)[(MeO)2cDHAP]·3H2O, (5a), (cha)[(MeO)2cDHAP]·H2O, (5b), and (H5O2)[(MeO)2cDHAP], (5c).
The present paper concerns the synthesis and crystal structure of 5,5-dimethoxy-2-hydroxy-1,3,2-dioxaphosphorinan-2-one, the dimethyl acetal of cyclic dihydroxyacetone phosphate, (MeO)2cDHAP, in the form of two crystalline cha salts, (cha)[(MeO)2cDHAP]·H2O, (5i) [polymorphous form of (5b)], and anhydrous (cha)[(MeO)2cDHAP], (5j), as well as in the form of the free acid, (MeO)2cDHAP, (5k).
The overall structures of the (MeO)2cDHAP anions in compounds (5i) and (5j) bear great similarities with each other and with the previously reported anions in (5a)-(5h) (Slepokura, 2008). The six-membered 1,3,2-dioxaphosphorinane ring adopts a chair (C) conformation only slightly distorted towards an envelope (E), which is reflected in the values of the dihedral angles between the least-squares plane through the four central atoms of the ring (O1/O3/C1/C3) and the O1/P/O3 and C1/C2/C3 planes (1 and 2), as well as in the Cremer-Pople puckering parameters (Cremer & Pople, 1975; see Table 1). The values of |2 - 1| for (5a), (5b), (5i) and (5j) clearly show that the flattening of the dioxaphosphorinane ring at the P atom is negligible in the (MeO)2cDHAP anions and that the conformation of the rings is close to an ideal chair.
Selected geometric parameters for (5i)-(5k) are given in Table 2. The deformation of the phosphate group from the ideal tetrahedral shape, which was observed previously in (5a)-(5h), is also observed in (5i) and (5j). The deformation of the ionized cyclic phosphate is seen particularly in the endocyclic O1-P1-O3 and exocyclic O4-P1-O5 bond angles, which are, respectively, the smallest [101.73 (11)° on average for (5a)-(5j)] and the largest [119.24 (12)° on average]. The values of the endo- and exocyclic O-P-O angles correlate with the respective P-O bond lengths. In all the known (MeO)2cDHAP anions, the P-Oendo bonds are all approximately 1.60 Å and more than 0.1 Å longer than the P-Oexo bonds.
A completely different deformation is present in the protonated phosphate group of the (MeO)2cDHAP molecule in (5k). The hydroxy group in (5k) adopts an axial position similar to the previously reported structures ETPHOS, KADPUA and SEZRUL. As can be seen by the P-Oendo, P-O(H) and P=O distances (Table 2), the protonation of the phosphate group affects to a larger extent the length of the P-Oendo bonds (becoming, on average, 0.03 Å shorter than in the anion) than that of the exocyclic equatorially oriented P1=O5 bond [becoming formally double, but only slightly shortened compared with the P-Oexo bonds in the (MeO)2cDHAP anions]. Within the O-P-O angles in (5k), the exocyclic O4-P1-O5 angle is the largest [115.13 (6)°], although none of them is distinctly smaller than the others. Instead, in the protonated phosphate group, three lower values for the angles involving endocyclic O atoms and three higher values for the angles involving P=O bonds are observed. It may be noted that the geometry of the phosphate group in (5k) is similar to that observed in the phenyl derivative, (4). These differences in the deformations observed in the ionized and protonated phosphate groups are accompanied by different distortions of the P/O/C/C/C/O rings. In contrast with the almost ideal chair conformation in the (MeO)2cDHAP anions of (5a)-(5j), the ring in acidic (5k) is significantly flattened at the P atom (see Table 1), which is comparable with the conformations observed in phenyl derivative (4) and the acidic cyclic phosphates ETPHOS and SEZRUL.
It has been shown that the acetal group in the analogous linear compounds, different salts of (MeO)2DHAP and (MeO)2DHAP(Ph) [(6a)-(6e); Slepokura & Lis, 2006], and in the unphosphorylated species (MeO)2DHA [(3); Slepokura & Lis, 2004a] seems to be very rigid, and its conformation is independent of phosphorylation, the ionization state of the inserted phosphate group, or additional substitution. It is likely that such a conformation is determined and stabilized by the generalized anomeric effect. The acetal group in the cyclic compounds (5i)-(5k), as in (4) and (5a)-(5h), reveals some common features with linear (3) and (6a)-(6e) (see scheme above): the relevant C4-O21-C2-O22 and C5-O22-C2-O21 torsion angles show a synclinal orientation of the methyl groups (C4 and C5) in relation to the acetal atoms O22 and O21. Similarly, as was observed in the structures of (3), (4), (5a)-(5h) and (6a)-(6e), two of the angles with their vertex on acetal atom C2 are much smaller than the others (Table 2).
The cations and anions in (5i) and (5j) are arranged in a way that leads to the aggregation of their hydrophilic and hydrophobic groups into distinct regions in the crystals. The packing schemes of (5i) and (5j) are dominated by N+-HO- hydrogen bonds, but in the hydrated salt (5i), as in (5a) and (5b) (Slepokura, 2008), an additional important role is played by contacts of the N+-HOW and OW-HO- type (OW is the water O atom). Geometric parameters of hydrogen bonds and close contacts are given in Table 3.
The crystal structure of the monohydrated salt (5i) has a layered architecture (Fig. 2), similar to that observed in the other hydrated cha salts, (5a) and (5b). Each cha cation is directly linked by two charge-assisted N-HO hydrogen bonds to two adjacent (MeO)2cDHAP anions, resulting in centrosymmetric R42(8) motifs (Fig. 3) [see Bernstein et al. (1995) for graph-set notation]. The same cation is linked to two additional anions via water-mediated hydrogen bonds. Thus, another type of ring is formed, R66(16), this time also involving water molecules in addition to cha cations and (MeO)2cDHAP anions. Another R42(8) ring results from the centrosymmetric OW-HO- bonds between two water molecules and two anions. The sequence of these three rings generates double layers parallel to the (001) plane, as shown in Figs. 2 and 3. The same types of interaction, namely cationanion, cationH2Oanion and anionH2Oanion, were also observed in the polymorphous salt (5b). However, as shown in Fig. 3, the construction of the layers observed in (5b) is different: three unique rings generate the layer, R44(12) involving two cations and two anions, R54(12) involving one cation, two anions and two water molecules, and R64(12) involving two cations, two anions and two water molecules.
The arrangement of organic ions in the crystal structure of the anhydrous salt (5j) is different. Each cha cation is linked directly by three N+-HO- hydrogen bonds to three adjacent (MeO)2cDHAP anions. These interactions generate R43(10) rings forming ribbons with ladder-type hydrogen bonding along the a axis, as shown in Fig. 4.
The crystal packing of (5k) is determined by the strong centrosymmetric almost linear O-HO hydrogen bonds formed by the phosphate groups of two adjacent (MeO)2cDHAP molecules. In this way, centrosymmetric molecular dimers are formed (Fig. 5), giving rise to R22(8) rings. Adjacent dimers interact with each other via weak C-HO contacts, as shown in Fig. 5, resulting in ribbons along .
In conclusion, we have shown that protonation of the cyclic phosphate group influences the conformation of the 1,3,2-dioxaphosphorinane ring, causing its flattening, i.e. deformation from the ideal chair towards an envelope. In other words, the ring is more evenly puckered in compounds with an ionized phosphate group than in the acid molecule. We have also demonstrated that differences in the ring conformation correlate with different deformations observed in the ionized and protonated phosphate groups. Some common features for analogous protonated and esterified compounds have been revealed. In addition, we have shown that the arrangement of the ions in the crystal structures of four different (cha)[(MeO)2cDHAP] salts results in different crystal architectures, depending on water content, and gives rise to layers in hydrated (5a), (5b) and (5i) and to ribbons in anhydrous (5j).
| || Figure 1 |
Views of (5i) (top), (5j) (middle) and (5k) (bottom), showing the atom-numbering schemes and the symmetry-independent N+-HO- and O-HO- hydrogen bonds (dashed lines). Displacement ellipsoids are drawn at the 50% probability level.
| || Figure 2 |
The double layer parallel to the (001) plane in (5i), built up from (MeO)2cDHAP anions (solid lines), cha cations (thin lines) and water molecules joined by N-HO and O-HO hydrogen bonds and C-HO contacts (dashed lines). H atoms not involved in hydrogen bonding have been omitted for clarity. [Symmetry codes: (i) -x, -y, -z + 1; (iii) -x + 1, -y + 1, -z + 1.]
| || Figure 3 |
A comparison of the layer constructions observed in two polymorphous forms of (cha)[(MeO)2cDHAP]·H2O, viz. monoclinic (5b) (top; Slepokura, 2008) and triclinic (5i) (bottom; this work). Phosphate and ammonium groups represent the anions and cations, respectively. N+-HO-/OW and OW-HO- hydrogen bonds are shown as dashed lines (blue and red, respectively, in the electronic version of the paper).
| || Figure 4 |
The polymeric ribbons with ladder-type hydrogen bonding formed in (5j) by the (MeO)2cDHAP anions (solid lines) and cha cations (open lines) along the a axis. N+-HO- hydrogen bonds are shown as dashed lines. H atoms not involved in hydrogen bonding have been omitted for clarity. [Symmetry codes: (ii) x - 1, y, z; (v) x - , -y + , -z + 1.]
| || Figure 5 |
The ribbons in (5k) resulting from centrosymmetric molecular dimers [with R22(8) rings formed by the (MeO)2cDHAP molecules joined via strong O-HO hydrogen bonds; dashed lines (red in the electronic version of the paper)] linked through C-HO contacts (black dashed lines). H atoms not involved in hydrogen bonding have been omitted for clarity. [Symmetry codes: (iii) -x + 1, -y + 1, -z + 1; (x) -x + 2, -y + 1, -z.]
The cyclohexylammonium salt of (MeO)2cDHAP was obtained by isolation of an intermediate in the basic hydrolysis of the cyclic triester derivative, using a method described previously (Slepokura, 2008). Excess cyclohexylamine was removed by washing the crude product with diethyl ether. Concentration of the resulting mixture under vacuum and then under a nitrogen stream at room temperature gave two kinds of crystals, viz. small plates of (5j) and needles of the monohydrated monoclinic form described previously, (5b). Recrystallization of (5b) from water at room temperature resulted in large plates of its triclinic form, (5i).
Crystals of (MeO)2cDHAP, (5k), were grown from an acidic solution prepared as follows. The cyclohexylammonium salt of (MeO)2cDHAP (250 mg dissolved in a minimum quantity of water) was passed through an ion-exchange column (Dowex 50-H+). The acidic solution was stirred at 313 K for 3.5 h. Subsequent evaporation of the solvent under a nitrogen stream yielded large blocks of (5k).
All H atoms were found in difference Fourier maps. In the final refinement cycles, water H atoms in (5i) were refined with Uiso(H) = 1.5Ueq(O). All remaining H atoms were treated as riding atoms in geometrically optimized positions, with C-H = 0.98-1.00 Å, N-H = 0.91 Å and O-H = 0.84 Å, and with Uiso(H) = 1.2Ueq(C) for CH and CH2, or 1.5Ueq(C,N,O) for CH3, NH3 and OH.
For all compounds, data collection: CrysAlis CCD (Oxford Diffraction, 2009); cell refinement: CrysAlis RED (Oxford Diffraction, 2009); data reduction: CrysAlis RED; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: XP (Bruker, 1998); software used to prepare material for publication: SHELXL97 and PLATON (Spek, 2009).
Supplementary data for this paper are available from the IUCr electronic archives (Reference: MX3045 ). Services for accessing these data are described at the back of the journal.
Allen, F. H. (2002). Acta Cryst. B58, 380-388.
Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555-1573.
Bruker (1998). XP. Version 5.1. Bruker AXS Inc., Madison, Wisconsin, USA.
Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354-1358.
Flack, H. D. (1983). Acta Cryst. A39, 876-881.
Gerlt, J. A., Chodosh, D. F., Drews, R. E. & Adams, R. D. (1980). J. Org. Chem. 45, 1282-1286.
Goswami, S. & Adak, A. K. (2002). Tetrahedron Lett. 43, 503-505.
Johnson, O., Jones, D. W. & Edmundson, R. S. (1989). Acta Cryst. C45, 142-145.
Oxford Diffraction (2009). CrysAlis CCD and CrysAlis RED, in Xcalibur PX and Kuma KM-4 CCD software. Versions 184.108.40.206. Oxford Diffraction Ltd, Yarnton, Oxfordshire, England.
Samas, B., Groendyke, T., Blackburn, A. C. & Godrej, D. B. (2007). Acta Cryst. E63, o1276-o1278.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122.
Shinitzky, M., Haimovitz, R., Nemas, M., Cahana, N., Mamillapalli, R. & Seger, R. (2000). Eur. J. Biochem. 267, 2547-2554.
Slepokura, K. (2008). Carbohydr. Res. 343, 113-131.
Slepokura, K. & Lis, T. (2004a). Carbohydr. Res. 339, 1995-2007.
Slepokura, K. & Lis, T. (2004b). Acta Cryst. C60, o315-o317.
Slepokura, K. & Lis, T. (2006). Carbohydr. Res. 341, 507-524.
Slepokura, K. & Lis, T. (2010). Carbohydr. Res. 345, 512-529.
Spek, A. L. (2009). Acta Cryst. D65, 148-155.