4C1 Chair Research Articles

ChemInform Abstract: A Double Diastereoselective Michael‐Type Addition as an Entry to Conformationally Restricted Tn Antigen Mimics.

A totally stereocontrolled C-Michael addition of serine-equivalent C-nucleophiles to tri-O-benzyl-2-nitro-d-galactal was used as the key step to synthesize several pyrano[3,2-b]pyrrole structures. These scaffolds could be regarded as conformationally restricted Tn antigen mimics, as we have demonstrated by biological assays. The pyranose rings retain their 4C1 chair conformation, as shown by molecular modeling and NMR spectroscopy. The expected bioactivity was established by a competition-tailored enzyme-linked lectin assay using both soybean and Vicia villosa agglutinins as model lectins. The facile described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational features and bioactivity demonstrate the prepared glycomimics to be promising candidates for further exploitation of this scaffold to give glycans for lectin blocking and vaccination.

Structures of the Toxoplasma gliding motility adhesin.

Micronemal protein 2 (MIC2) is the key adhesin that supports gliding motility and host cell invasion by Toxoplasma gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1-6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2-M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal structures of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual (1)C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state.

Conformational interchange of a carbohydrate by mechanical compression at the air–water interface

Methyl xylopyranoside containing three 4-(pyrene-1-yl)benzoyl groups (PyXy) undergoes conformational interchange within a Langmuir monolayer upon mechanical compression. This xylose-type molecular machine PyXy was immobilized within two different matrix lipids, methyl stearate and methyl 2,3,4-tri-O-stearoyl-β-D-xylopyranoside, which respectively form rigid and soft monolayers. Structural properties of the monolayer were characterized by assessing the compressibility, compression modulus, and ideal limiting molecular area of PyXy, all of which were estimated from the π-A isotherm measurements. Only the rigid monolayer exhibited a transition to the condensed phase with a limiting molecular area of PyXy smaller than that of the cross-sectional area of the xylopyranose ring in its C1 chair conformation. This suggests conformational interchange of PyXy from the most stable (4)C1 (C1) form to the metastable (1)C4 (1C) form. Surface-reflective fluorescence spectroscopy of the monolayer was applied to detect excimer emission resulting from the face-to-face dimerization of pyrenes attached at the O-2 and O-4 positions of xylose. Fluorescence intensity of the excimer increased abruptly in the condensed region only when the rigid monolayer was applied. These results indicate that the rigidity of the matrix monolayer is a critical aspect of the precise manipulation of molecular machines at interfaces. Consequently, this study demonstrates that including a molecular machine into a rigid lipid matrix is a promising means for the preparation of a novel nanoassembly with dynamic functionalities variable depending on a mechanical stimulus.

Microsecond kinetics in model single- and double-stranded amylose polymers.

Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.

Facile Synthesis of the Naturally Cytotoxic Triterpenoid Saponin Patrinia-Glycoside B-II and Its Conformer

The first chemical synthesis of the natural triterpenoid saponin Patrinia-glycoside B-II, namely oleanolic acid 3-O-α-l-rhamnopyranosyl-(1→2)-[β-d-gluco-pyranosyl-(1→3)]-α-l-arabinopyranoside, has been accomplished in a linear 11-step sequence 11 with 9.4% overall yield. The abnormal 1C4 conformation of the arabinose residue was found to occur via conformational fluctuation during preparation of the intermediates. Molecular mechanism and quantum chemistry calculations showed that Patrinia-glycoside B-II and its conformer 1 cannot interconvert under normal conditions. Preliminary structure-activity relationships studies indicated that the 4C1 chair conformation of the arabinose residue in the unique α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl disaccharide moiety is one of the chief positive factors responsible for its cytotoxic activity against tumors.

A Double Diastereoselective Michael-Type Addition as an Entry to Conformationally Restricted Tn Antigen Mimics

A totally stereocontrolled C-Michael addition of serine-equivalent C-nucleophiles to tri-O-benzyl-2-nitro-d-galactal was used as the key step to synthesize several pyrano[3,2-b]pyrrole structures. These scaffolds could be regarded as conformationally restricted Tn antigen mimics, as we have demonstrated by biological assays. The pyranose rings retain their (4)C1 chair conformation, as shown by molecular modeling and NMR spectroscopy. The expected bioactivity was established by a competition-tailored enzyme-linked lectin assay using both soybean and Vicia villosa agglutinins as model lectins. The facile described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational features and bioactivity demonstrate the prepared glycomimics to be promising candidates for further exploitation of this scaffold to give glycans for lectin blocking and vaccination.

Conformations of the iduronate ring in short heparin fragments described by time-averaged distance restrained molecular dynamics

The polyconformational behavior of L-iduronic acid (L-IdoA2S) in heparin derivatives has been previously analyzed in terms of intra-ring proton-proton vicinal coupling constants ((3)JHH) through mathematical fit of experimental and theoretical values (Ferro DR, Provasoli A, Ragazzi M, Casu B, Torri G, Bossennec V, Perly B, Sinay P, Petitou M, Choay J. 1990. Conformer Populations of L-Iduronic Acid Residues in Glycosaminoglycan Sequences. Carbohydr Res. 195:157-167; Muñoz-García JC, López-Prados J, Angulo J, Díaz-Contreras I, Reichardt N, de Paz JL, Martín-Lomas M, Nieto PM. 2012. Effect of the substituents of the neighboring ring in the conformational equilibrium of iduronate in heparin-like trisaccharides. Chemistry. 18:16319-16331.). However, this methodology is subjected to the experimental uncertainties of the J-coupling measurements, the force field deviations and the goodness of the least-squares fit. In the present work, we have used time-averaged distance restrained molecular dynamics (tar-MD) to largely reduce these errors, which enables accurate quantification of the population of conformers, or puckers, of the L-IdoA2S residue, in a set of eight heparin-like trisaccharides following the general sequence d-glucosamine (GlcN)-IdoA-GlcN, directly from the time evolution of the puckering coordinates θ and . Thus, by carrying out tar-MD simulations in explicit water, with the exclusive nuclear overhauser enhancement (NOE)-derived distance H2-H5 of the L-IdoA2S (2)SO conformer as the unique imposed constraint, we have been able to accurately and easily determine the different extents to which the iduronate ring populates the polar ((1)C4 chair) and equatorial ((2)SO skew-boat) areas of the puckering sphere depending on the sulfation pattern of the flanking GlcN residues, under low temperature conditions (278 K). The results indicate that 6-O-sulfation at the reducing-end GlcN residue facilitates the (1)C4 to (2)SO transitions of the iduronate ring by augmenting the flexibility of the C2-C3 torsion, driving the conformational equilibrium toward a majority of equatorial conformers.

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)4, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with Ki values of 2.01 × 10(-5) and 1.84 × 10(-6) m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔHr°), which is similar to those obtained for 2 and (GlcNAc)4. However, the entropy loss (-TΔSr°) for the binding of 3 was smaller than those of 2 and (GlcNAc)4. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the Kd value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites -4 to -1 and the moranoline moiety adopts an undistorted (4)C1 chair conformation almost overlapping with the -1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835-838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.

Chair interconversion and reactivity of mannuronic acid esters

Mannopyranosyluronic acids display a very unusual conformation behavior in that they often prefer to adopt a (1)C4 chair conformation. They are endowed with a strikingly high reactivity when used in a glycosylation reaction as a glycosyl donor. To investigate the unusual conformational behavior a series of mannuronic acid ester derivatives, comprising anomeric triflate species and O-methyl glycosides, was examined by dynamic NMR experiments, through lineshape analysis of (1)H and (19)F NMR spectra at various temperatures from -80 °C to 0 °C. Exchange rates between (4)C1 and (1)C4 chair conformations were found to depend on the electronic properties and the size of the C2 substituent (F, N3 or OBn) and the aglycon, with higher exchange rates for the glycosyl triflates and smaller C2 substituents. Low temperature (19)F exchange spectroscopy experiments showed that the covalently bound anomeric triflates did not exchange with free triflate species present in the reaction mixture. To relate the conformational behavior of the intermediate triflates to their reactivity in a glycosylation reaction, their relative reactivity was determined via competition reactions monitored by (1)H NMR spectroscopy at low temperature. The 2-O-benzyl ether compound was found to be most reactive whereas the 2-fluoro compound - the most flexible of the studied compounds - was least reactive. Whereas the ring-flip of the mannuronic acids is important for the enhanced reactivity of the donors, the rate of the ring-flip has little influence on the relative reactivity.

Computational Studies of Protonated β-d-Galactose and Its Hydrated Complex: Structures, Interactions, Proton Transfer Dynamics, and Spectroscopy

We present an exploration of proton transfer dynamics in a monosaccharide, based upon ab initio molecular dynamic (AIMD) simulations, conducted "on-the-fly", in β-d-galactose-H(+) (βGal-H(+)) and its singly hydrated complex, βGal-H(+)-H2O. Prior structural calculations identify O6 as the preferred protonation site for O-methyl α-d-galactopyranoside, but the β-anomeric configuration favors the inversion of the pyranose ring from the (4)C1 chair configuration, to (1)C4, and the formation of proton bridges to the (axial) O1 and O3 sites. In the hydrated complex, however, the proton bonds to the water molecule inserted between the O6 and Ow sites, and the ring retains its original (4)C1 conformation, supported by a circular network of co-operatively linked hydrogen bonds. Two distinct proton transfer processes, operating over a time scale of 10 ps, have been identified in βGal-H(+) at 500 K. One of them leads to chemical reaction and the formation of an oxacarbenium ion (accompanied by the loss of an H2O molecule). In the hydrated complex, βGal-H(+)-H2O, this reaction is suppressed, and the proton transfer, which involves multiple jumps between the sugar and the H2O, creates an H3O(+) ion, relevant, perhaps, to the reactivity of protonated sugars both in the gas and condensed phases. Anticipating future spectroscopic investigations, the vibrational spectra of βGal-H(+) and βGal-H(+)-H2O have also been computed through AIMD simulations conducted at average temperatures of 300 and 40 K and also through vibrational self-consistent field (VSCF) calculations at 0 K.

Theoretical investigation of reaction mechanisms of alkaline hydrolysis of 2,3,6-trinitro-β-d-glucopyranose as a monomer of nitrocellulose

The 2,3,6-trinitro-β-d-glucopyranose as a monomer of nitrocellulose in the 4C1 chair conformation was selected for the alkaline hydrolysis of nitrocellulose within SN2 framework in the gas phase and in the bulk water solution. Both the direct and angular attacks of OH− in the hydrolysis reactions were considered. Geometries were optimized at the B3LYP/6-311G(d,p) level both in the gas phase and in bulk water solution. Effect of bulk water solution was modeled using the PCM approach. Nature of potential energy surfaces of the local minima and transition states was ascertained through harmonic vibrational frequency analysis. Intrinsic reaction coordinate calculations were also performed to validate the computed transition state structures. Effect of electron correlation on computed energies was considered at the MP2/cc-pVTZ//B3LYP/6-311G(d,p) level. It was found that the angular attack of OH− in the hydrolysis reaction will require significantly larger activation energy than the direct attack. Computed transition states correspond to the structure where the presence of hydrogen bonds between the OH− and various sites of nitrocellulose was the necessary stabilizing factor. The ring breaking through the C–O ring bond was not found to be the first step in the alkaline hydrolysis reactions. It was predicted that alkaline hydrolysis would be driven by the addition–elimination (substitution) reaction starting at the C3 site and will progress in the C2 → C6 direction. Entropy of system in water solution will have profound effect on alkaline hydrolysis reaction of nitrocellulose.

Is N-acetyl-D-glucosamine a rigid 4C1 chair?

Understanding microsecond-timescale dynamics is crucial to establish three-dimensional (3D) structure–activity relationships in sugars but has been intractable to experiments and simulations. As a consequence, whether arguably the most important chemical scaffold in glycobiology, N-acetyl-d-glucosamine (GlcNAc), deviates from a rigid 4C1 chair is unknown. Here, conformer populations and exchange kinetics were quantified from the longest aqueous carbohydrate simulations to date (0.2 ms total) of GlcNAc, four derivatives from heparan sulfate and their methylglycosides. Unmodified GlcNAc took 3–5 μs to reach a conformational equilibrium, which comprised a metastable 4C1 chair that underwent 4C1 ↔ 1C4 transitions at a predicted forward rate of 0.8 μs−1 with an average 1C4-chair lifetime of 3 ns. These predictions agree with high-resolution crystallography and nuclear magnetic resonance but not with the hypothesis that GlcNAc is a rigid 4C1 chair, concluded from previous experimental analyses and non-aqueous modeling. The methylglycoside was calculated to have a slower forward rate (0.3 μs−1) and a more stable 4C1 conformer (0.2 kcal mol−1), suggesting that pivotal 3D intermediates (particularly 2SO, 1S5 and B2,5) increased in energy, and water was implicated as a major cause. Sulfonation (N-, 3-O and 6-O) significantly augmented this effect by blocking pseudorotation, but did not alter the rotational preferences of hydroyxl or hydroxymethyl groups. We therefore propose that GlcNAc undergoes puckering exchange that is dependent on polymerization and sulfo substituents. Our analyses, and 3D model of the equilibrium GlcNAc conformer in water, can be used as dictionary data and present new opportunities to rationally modify puckering and carbohydrate bioactivity, with diverse applications from improving crop yields to disease amelioration.

Methyl 6-azido-6-deoxy-α-D-galactoside

The structure of the title compound, C7H13N3O5, was solved using data from a multiple fragment crystal. The galactoside ring adopts a 4 C 1 chair conformation. In the crystal, the molecules are linked by strong O—H⋯O hydrogen bonds, which build linkages around the screw axis of the cell in a similar way to the iodo analogue. These C-5 and C-6 packing motifs expand to R 2 2(10), C 2 2(7) and C 2 2 2(8) motifs, as found in closely related compounds.

4-Deoxy-4-fluoro-β-D-glucopyranose

4-Deoxy-4-fluoro-β-D-glucopyranose, C(6)H(11)FO(5), (I), crystallizes from water at room temperature in a slightly distorted (4)C(1) chair conformation. The observed chair distortion differs from that observed in β-D-glucopyranose [Kouwijzer, van Eijck, Kooijman & Kroon (1995). Acta Cryst. B51, 209-220], (II), with the former skewed toward a B(C3,O5) (boat) conformer and the latter toward an (O5)TB(C2) (twist-boat) conformer, based on Cremer-Pople analysis. The exocyclic hydroxymethyl group conformations in (I) and (II) are similar; in both cases, the O-C-C-O torsion angle is ∼-60° (gg conformer). Intermolecular hydrogen bonding in the crystal structures of (I) and (II) is conserved in that identical patterns of donors and acceptors are observed for the exocyclic substituents and the ring O atom of each monosaccharide. Inspection of the crystal packing structures of (I) and (II) reveals an essentially identical packing configuration.

ChemInform Abstract: Structure of Lactitol (4-O-β-D-Galactopyranosyl-D-glucitol) Monohydrate: An Artificial Sweetener

C12H24O11.H2O, Mr = 362.4, orthorhombic, P2(1)2(1)2(1), a = 7.808 (2), b = 12.685 (2), c = 15.931 (3) A, V = 1577.9 (6) A3, Z = 4, Dx = 1.525 Mg m-3, lambda(Mo K alpha) = 0.71073 A, mu = 0.13 mm-1, F(000) = 776, T = 295 K, R = 0.031 for 1781 unique reflections with I greater than 2.5 omega (I). The galactopyranosyl ring has the 4C1 chair conformation and the conformation of the bent glucitol C-atom chain is MAA. The torsion angles characterizing the conformation of the glycosidic linkage are -86.3 (2) degrees [O(5)--C(1)--O(1)--C(14)] and 116.8 (2) degrees [C(1)--O(1)--C(14)--C(13)]. All hydroxyl groups act as donors in hydrogen bonds; three bonds are intramolecular. With the exception of O(1) of the glycosidic link which is not an acceptor and O(6) of the glucitol residue which is a double acceptor, all O atoms accept one hydrogen bond. The water molecule donates two and accepts one hydrogen bond.

Methyl β-allolactoside [methyl β-D-galactopyranosyl-(1→6)-β-D-glucopyranoside] monohydrate

Methyl beta-allolactoside [methyl beta-D-galactopyranosyl-(1-->6)-beta-D-glucopyranoside], (II), was crystallized from water as a monohydrate, C(13)H(24)O(11).H(2)O. The betaGalp and betaGlcp residues in (II) assume distorted (4)C(1) chair conformations, with the former more distorted than the latter. Linkage conformation is characterized by phi' (C2(Gal)-C1(Gal)-O1(Gal)-C6(Glc)), psi' (C1(Gal)-O1(Gal)-C6(Glc)-C5(Glc)) and omega (C4(Glc)-C5(Glc)-C6(Glc)-O1(Gal)) torsion angles of 172.9 (2), -117.9 (3) and -176.2 (2) degrees , respectively. The psi' and omega values differ significantly from those found in the crystal structure of beta-gentiobiose, (III) [Rohrer et al. (1980). Acta Cryst. B36, 650-654]. Structural comparisons of (II) with related disaccharides bound to a mutant beta-galactosidase reveal significant differences in hydroxymethyl conformation and in the degree of ring distortion of the betaGlcp residue. Structural comparisons of (II) with a DFT-optimized structure, (II(C)), suggest a link between hydrogen bonding, pyranosyl ring deformation and linkage conformation.

Enzymatic synthesis and 3-D structure of anti-proliferative acidic (MeGlcUA) xylotetrasaccharide

Anomerically free acidic xylo-oligosaccharides have shown interesting biological properties when tested against Gram-positive and Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori, sarcoma-180 and other tumors. We report here a structure–activity relationship study on the role of 4-O-methyl glucuronic acid (MeGlcUA) in regulating aggregation of β-polyxylosides of (9H-fluoren-9-yl)- methanol obtained via the action of Thermotoga neapolitana xylanase. Neutral compounds from mono- to penta-β-1,4 xylosides were obtained from this biocatalyzed reaction. In addition, acidic components among products, carrying an α-1,2 4-O-methyl glucuronic acid (MeGlcUA) were also isolated. An anti-proliferative test of these compounds on human epithelial EFO 27 ovarian cancer cells indicated that the presence of MeGlcUA modulates their biological activity, while its absence induces molecular aggregation. The three-dimensional structure of the most active MeGlcUA β-polyxyloside was investigated by resorting to NOESY experiments supported by dynamic force-field calculations with/without constraints. The 3D structure is characterized by all sugars possessing a 4C1 chair conformation. The MeGlcUA moiety, and the external and middle xyloses adopt a hairpin-shaped conformation, generating a non-planar arrangement of the molecule with the aromatic ring folding back toward the carbohydrate chain. Such a non-planar conformation may justify the lack of aggregation.

The Chitopentaose Complex of a Mutant Hen Egg-White Lysozyme Displays No Distortion of the –1 Sugar Away from a 4C1 Chair Conformation

Glycosidase inhibitors frequently reflect either the charge or the ‘flattened’ shape of the oxocarbenium-ion like transition state. Much of the impetus for such inhibitory strategies derives from historical studies on ligand binding to hen egg white lysozyme (HEWL); not least those suggesting that product complexes of the enzyme showed distortion of the pyranosides in the –1 subsite. Ironically, while distortion is undoubtedly a defining feature of glycosidases, product complexes themselves are rarely distorted. Here we show that the chitopentaose product complex of a mutant E35Q HEWL, solved at 1.8 Å resolution, is bound with all sugars in 4C1 conformation.

2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl azide

The CuI-catalysed 1,3-dipolar cyclo­addition of an azide and a terminal alkyne is becoming an increasingly popular tool for synthetic chemists. This is the most representative of the so-called `click reactions' and it is used to generate 1,4-di­substituted triazoles in high yield. During studies on such cyclo­addition reactions, a reduced reactivity of an α-glucosyl azide with respect to the corresponding β-anomer was observed. With the aim of understanding this phenomenon, the structure of the title compound, C14H19N3O9, has been determined at 140 K. The glucopyranosyl ring appears in a regular 4C1 chair conformation with all the substituents in equatorial positions, except for the anomeric azide group, which adopts an axial orientation. The observed bond lengths are consistent with a strong anomeric effect, which is reflected in a change in dipolar character and hence reduced reactivity of the α-glucosyl azide.

Phenyl 2,3-O-isopropylidene-1-thio-α-D-rhamnopyranoside

In the title compound, C15H20O4S, a dioxolane ring is fused to the pyran ring of the sugar which carries a thio­phenyl substituent on the anomeric C atom. The dioxolane ring adopts an envelope conformation and the pyran ring system a distorted 4 C 1 chair. The structure is stabilized by O—H⋯O hydrogen bonds, forming centrosymmetric dimers that generate an R 2 2(10) ring motif. Additional C—H⋯O inter­actions form an extended network. Two C atoms of the phenyl ring are disordered over two positions; the site occupancy factors are ca. 0.7 and 0.3.