Crystallization of (20R) and (20S) analogs of 2-methylene-19-nor-24-dimethyl-1α,25-dihydroxyvitamin D3
United States Patent 8884039
Disclosed are methods of purifying (20R) and (20S) analogs of 2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 to obtain the (20R) and (20S) analogs in crystalline form. The method includes the steps of preparing a solvent of either diethyl ether or a mixture of 2-propanol and hexane, dissolving a product containing the (20R) and (20S) analog to be purified in the solvent, cooling the solvent and dissolved product below ambient temperature for a sufficient amount of time to form a precipitate of crystals, and recovering the crystals.
Inventors:
Deluca, Hector F. (Deerfield, WI, US)
Flores, Agnieszka (Madison, WI, US)
Thoden, James B. (Madison, WI, US)
Holden, Hazel M. (Fitchburg, WI, US)
Application Number:
Publication Date:
11/11/2014
Filing Date:
03/14/2013
Export Citation:
Wisconsin Alumni Research Foundation (Madison, WI, US)
Primary Class:
International Classes:
C07C401/00
Field of Search:
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US Patent References:
8664206DeLuca et al.514/1678604009DeLuca et al.514/167DeLuca et al.5843928DeLuca et al.5536713DeLuca et al.5086191DeLuca et al.
Other References:
Andrews et al., “A Direct, Regio- and Stereoselective 1Alpha-Hydroxylation of (5E)-Calciferol Derivatives”, Journal of Organic Chemistry, 35-1637.
Baggiolini et al., “Stereocontrolled Total Synthesis of 1[alpha],25-Dihydroxycholecaliferol and 1[alpha],25-Dihydroxyergocalciferol”, Journal of Organic Chemistry, 98-3108.
Calverley et al., “A Biologically Active Vitamin D Metabolite Analogue”, Tetrahedron, ): .
Choudhry et al., “Synthesis of a Biologically Active Vitamin-D2 Metabolite”, Journal of Organic Chemistry, 6-1500.
Lythgoe et al., “Calciferol and its Relatives. Part 22. A Direct Total Synthesis of Vitamin D2 and Vitamin D3”, J. Chem. Soc. Perkin I, 5.
Lythgoe, “Synthetic Approaches to Vitamin D and its Relatives”, Chem. Soc. Rev., 9-475.
Paaren et al., “Direct C-1 Hydroxylation of Vitamin D Compounds: Convenient Preparation of 1alpha-Hydroxyvitamin D3,1alpha-Dihydroxyvitamin D3 and 1alpha,Hydroxyvitamin D2”, Proc. Natl. Acad. Sci. USA, ): .
Paaren et al., “Direct C-1 Hydroxylation of Vitamin D3 and Related Compounds”, J. Org. Chem., 53-3258.
Perlman et al., “Novel Synthesis of 19-Nor-Vitamin D Compounds”, Tetrahedron Letters, 63-7666.
Nerinckx et al., “An Improved Synthesis of 1Alpha-Hydroxy Vitamin D3”, Tetrahedron, ): .
Sardina et al., “Studies on the Synthesis of Side-Chain Hydroxylated Metabolites of Vitamin D. 2. Stereocontrolled Synthesis of 25-Hydroxyvitamin D2”, Journal of Organic Chemistry, 64-1269.
Sicinski et al., “New 1alpha,25-Dihydroxy-19-norvitamin D3 Compounds of High Biological Activity: Synthesis and Biological Evaluation of 2-Hydroxymethyl, 2-Methyl, and 2-Methylene Analogs”, J. Med. Chem., 62-4674.
Sheldrick, “Phase Annealing in SHELX-90: Direct Methods for Larger Structures”, Acta Cryst., 1990, A46: 467-473.
Toh et al., “Studies on a Convergent Route to Side-Chain Analogues of Vitamin D:25-Hydroxy-23-oxavitamin D3”, Journal of Organic Chemistry, 14-1417.
Vanmaele et al., “A Stereocontrolled Partial Synthesis of 1Alpha-Hydroxy Vitamin D3”, Tetrahedron Letters, ): 995-998.
Vanmaele et al., “1Alpha-Hydroxy Previtamin D3 and its Selective Formation From 1-Keto Previtamin D3”, Tetrahedron, ): .
Vanmaele et al., “An Efficient Synthesis of 1Alpha-25-Dihydroxy Vitamin D3”, Tetrahedron, ): 141-144.
Flores et al., “A 20S Combined with a 22R Configuration Markedly Increases both in Vivo and in Vitro Biological Activity of 1[alpha],25-Dihydroxy-22-methyl-2-methylene-19-norvitamin D3”, Journal of Medical Chemistry, 52-4366.
International Search Report and Written Opinion, PCT International Application No. PCT/US, mailed Aug. 13, 2013.
Primary Examiner:
Qazi, Sabiha N.
Attorney, Agent or Firm:
Andrus Intellectual Property Law, LLP
Parent Case Data:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONSThe present application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/652,965, filed on May 30, 2012, the content of which is incorporated herein by reference in its entirety.
A compound having the formula
in crystalline form, wherein the methyl group attached to carbon 20 may be in its R or S orientation.
(20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 in crystalline form.
A crystalline form of (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 having molecular packing arrangement defined by space group P2(1) and unit cell dimensions a=7.57 ? b=14.79 ? c=14.48 ? α=90°, β=102.2° and γ=90°.
A three dimensional structure for (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 as defined by the molecular packing arrangement set forth in claim 3.
A method of purifying (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3, comprising the steps of: (a) preparing a solvent comp (b) dissolving a product containing (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 to be puri (c) cooling said solvent and dissolved product below ambient temperature for a sufficient amount of time to form a precipitate of (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3
and (d) separating the (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 crystals from the solvent.
The method of claim 5 including the further step of allowing said solvent and dissolved product to cool to ambient temperature prior to cooling below ambient temperature.
The method of claim 5 wherein said solvent comprises 100% diethyl ether, by volume.
The method of claim 5 wherein the step of separating comprises filtering the solvent and precipitate to obtain the crystals.
The method of claim 5 including a further step (e) comprising repeating steps (a) through (d) using the recovered crystals from step (d) as the product of step (b).
(20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 in crystalline form.
A crystalline form of (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 having molecular packing arrangement defined by space group C2 and unit cell dimensions a=27.33 ? b=6.68 ? c=19.22 ? α=90°, β=113.57° and γ=90°.
A three dimensional structure for (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 as defined by the molecular packing arrangement set forth in claim 11.
A method of purifying (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3, comprising the steps of: (a) preparing a solve (b) adding a product containing (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 to be purified to said hexane to form a suspension of the p (c) adding 2-propanol dropwise to the suspension to form a mixture of the product in the hexane and 2- (d) heating the mixture to dissolve the product containing (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 to be puri (e) cooling said mixture and dissolved product below ambient temperature for a sufficient amount of time to form a precipitate of (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3
and (f) separating the (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 crystals from the mixture.
The method of claim 13 including the further step of allowing said mixture and dissolved product to cool to ambient temperature prior to cooling below ambient temperature.
The method of claim 13 wherein the step of separating comprises filtering the mixture and precipitate to obtain the crystals.
The method of claim 13 including a further step (g) comprising repeating steps (a) through (f) using the recovered crystals from step (f) as the product of step (b).
The method of claim 13 wherein said mixture comprises about 15% 2-propanol and about 85% hexane, by volume.
Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under DK047814 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUNDThe field of the present invention relates to purification of organic compounds, and more particularly to the purification of (20S) and (20R) analogs of 2-Methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 (referred to herein as “MET-1” and “MET-2” respectively) by preparing them in crystalline form.Purification of organic compounds, especially those designated for pharmaceutical use, is of considerable importance for chemists synthesizing such compounds. Preparation of the compound usually requires many synthetic steps and, therefore, the final product can be contaminated not only with side-products derived from the last synthetic step of the procedure but also with compounds that were formed in previous steps. Even chromatographic purification, which is a very efficient but relatively time-consuming process, does not usually provide compounds which are sufficiently pure to be used as drugs.Depending on the method used to synthesize 1α-hydroxyvitamin D compounds, different minor undesirable compounds can accompany the final product. Thus, for example, if direct C-1 hydroxylation of the 5,6-trans geometric isomer of vitamin D is performed, followed by SeO2/NMO oxidation and photochemical irradiation, (see Andrews et al., J. Org. Chem. 51, ); Calverley et al., Tetrahedron 43, ); Choudry et al., J. Org. Chem. 58, )), the final 1α-hydroxyvitamin D product can be contaminated with 1β-hydroxy—as well as 5,6-trans isomers. If the method consists of C-1 allylic oxidation of the 4-phenyl-1,2,4-triazoline-3,5-dione adduct of the pre-vitamin D compound, followed by cycloreversion of the modified adduct under basic conditions, (see Nevinckx et al., Tetrahedron 47, ); Vanmaele et al., Tetrahedron 41, 141 (1985) and 40, ); Vanmaele et al., Tetrahedron Lett. 23. 995 (1982)), one can expect that the desired 1α-hydroxyvitamin can be contaminated with the pre-vitamin 5(10), 6,8-triene and 1β-hydroxy isomer. One of the most useful C-1 hydroxylation methods, of very broad scope and numerous applications, is the experimentally simple procedure elaborated by Paaren et al., J. Org. Chem. 45, ) and Proc. Natl. Acad. Sci. U.S.A. 75, ). This method consists of allylic oxidation of 3,5-cyclovitamin D derivatives, readily obtained from the buffered solvolysis of vitamin D tosylates, with SeO2/t-BuOOH and subsequent acid-catalyzed cycloreversion to the desired 1α-hydroxy compounds. Taking into account this synthetic path, it is reasonable to assume that the final product can be contaminated with 1α-hydroxy epimer, 5,6-trans isomer and the previtamin D form. 1α-hydroxyvitamin D4 is another undesirable contaminant found in 1α-hydroxyvitamin D compounds synthesized from vitamin D2 or from ergosterol. 1α-hydroxyvitamin D4 results from C-1 oxidation of vitamin D4, which in turn is derived from contamination of the commercial ergosterol material. Typically, the final product may contain up to about 1.5% by weight 1α-hydroxyvitamin D4. Thus, a purification technique that would eliminate or substantially reduce the amount of 1α-hydroxyvitamin D4 in the final product to less than about 0.1-0.2% would be highly desirable.The vitamin D conjugated triene system is not only heat- and light-sensitive but it is also prone to oxidation, leading to the complex mixture of very polar compounds. Oxidation usually happens when a vitamin D compound has been stored for a prolonged time. Other types of processes that can lead to a partial decomposition of vitamin D compounds consist of some water-elimination reactions. The driving force for these reactions is the allylic (1α-) and homoallylic (3β-) position of the hydroxy groups. The presence of such above-mentioned oxidation and elimination products can be easily detected by thin-layer chromatography.Usually, all 1α-hydroxylatation procedures require at least one chromatographic purification. However, even chromatographically purified 1α-hydroxyvitamin D compounds, although showing consistent spectroscopic data that suggests homogeneity, do not meet the purity criteria required for therapeutic agents that can be orally, parenterally or transdermally administered. Therefore, it is evident that a suitable method of purification of the 1α-hydroxylated vitamin D compounds MET-1 and MET-2 is required.SUMMARYDisclosed are methods of purifying MET-1 and MET-2 by means of crystallization to obtain MET-1 and MET-2 in crystalline form. The solvent plays an important role in the crystallization process, and is typically an individual liquid substance or a suitable mixture of different liquids. For crystallizing MET-1 and MET-2, the most appropriate solvent and/or solvent system is characterized by the following factors:
(1)(2)(3) significant dependence of solubility properties with regard to temperature (condition necessary for providing satisfactory crystallization yield); and(4) relatively low cost.
Interestingly, hexane, so frequently used for crystallization purposes, was found less suitable as the sole solvent for crystallization of MET-1 and MET-2. However, it was found that diethyl ether (Et2O) was most useful for the crystallization of MET-1, and a mixture of 2-propanol and hexane, was most useful for the crystallization of MET-2. In particular, it was determined that a mixture of about 10% to about 20% 2-propanol with about 90% to about 80% hexane by volume, (preferably 15% 2-propanol with about 85% hexane by volume) performed well. The diethyl ether solvent and the 2-propanol/hexane solvent mixture both were easy to remove by evaporation or other well-known methods. In all cases the crystallization process occurred easily and efficiently. The precipitated crystals were sufficiently large to assure their recovery by filtration or other means, and thus were suitable for x-ray analysis.Accordingly, disclosed is a compound having the formula:
in crystalline form, where the wavy line at carbon 20 indicates the methyl group attached to carbon 20 may be in its R or S orientation. More specifically, disclosed are (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 in crystalline form (otherwise referred to as “MET-1”), and (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 in crystalline form, (otherwise referred to as “MET-2”). BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 is an illustration of the three dimensional molecular structure for MET-1 as defined by the atomic positional parameters discovered and set forth herein.FIG. 2 is an illustration of the three dimensional molecular structure for MET-2 as defined by the atomic positional parameters discovered and set forth herein.DETAILED DESCRIPTIONDisclosed herein is (20S)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 (MET-1) in crystalline form, which is a pharmacologically important compound characterized by the formula I shown below: Also disclosed herein is (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 (MET-2) in crystalline form, which also is a pharmacologically important compound characterized by the formula II shown below: Also disclosed herein are methods of purifying MET-1 and MET-2. The purification technique typically involves obtaining the MET-1 and MET-2 products in crystalline form by utilizing a crystallization procedure wherein the material to be purified is dissolved using as the solvent either diethyl ether (Et2O) as the sole solvent to obtain MET-1, or a mixture comprised of 2-propanol and hexane to obtain MET-2. In particular, it was determined that a mixture of about 10% to about 20% 2-propanol (v/v) with about 90% to about 80% hexane (v/v) performed well. Preferably the mixture comprises about 15% 2-propanol (v/v) and about 85% hexane (v/v). Thereafter, the solvent can be removed by evaporation, with or without vacuum, or other means as is well-known in the art. Alternatively, the resultant crystals may be filtered from the mother liquor. The technique can be used to purify a wide range of final products containing MET-1 and MET-2 obtained from any known synthesis thereof, and in varying concentrations, which may range from microgram amounts to kilogram amounts. As is well known to those skilled in this art, the amount of solvent utilized should be modulated according to the amount of MET-1 and MET-2 to be purified.EXAMPLESThe following examples are illustrative and should not be interpreted as limiting the claimed subject matter.The usefulness and advantages of the present crystallization procedure is shown in the following specific Examples. After crystallization, the precipitated material was observed under a microscope to confirm its crystalline form. Yields of crystals were relatively high and the obtained crystals showed a relatively sharp melting point of 99° C. (MET-1) and 154° C. (MET-2).The described crystallization process of the synthetic MET-1 and MET-2 products represents a valuable purification method, which can remove most side products derived from the synthetic path. Such impurity may result from contamination of starting raw materials. The crystallization process occurred easily and efficiently. The precipitated crystals were sufficiently large to assure their recovery by filtration, or other means, and thus were suitable for x-ray analysis.Example 1Crystallization of (20S)-2-methylene-19-nor-22-dimethyl-1α,25 dihydroxyvitamin D3 (MET-1)Crystallization from Diethyl Ether.(20 S2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 (MET-1) (14.3 mg), was dissolved in boiling diethyl ether (3 mL) and left at room temperature for about 1 hour, then it was kept in a refrigerator for about 48 hours. The precipitated crystals were filtered off, washed with a small volume of a cold (0° C.) diethyl ether, and dried to give crystalline material.Experimental.A colorless prism-shaped crystal of dimensions 0.25×0.34×0.55 mm was selected for structural analysis. Intensity data were collected using a Bruker AXS Platinum 135 CCD detector controlled with the PROTEUM software suite (Bruker AXS Inc., Madison, Wis.). The x-ray source was CuK radiation (1.54178 ?) from a Rigaku RU200 x-ray generator equipped with Montel optics, operated at 50 kV and 90 mA. The x-ray data were processed with SAINT version 7.06A (Broker AXS Inc.) and internally scaled with SADABS version 2005/1 (Broker AXS Inc.). The sample was mounted on a glass fiber using vacuum grease and cooled to 100 K. The intensity data were measured as a series of phi and omega oscillation frames each of 1° for 10-25 sec/frame. The detector was operated in
mode and was positioned 4.5 cm from the sample. Cell parameters were determined from a non-linear least squares fit of 3265 peaks in the range of 4.0&theta&55°. The data were merged to form a set of 4624 independent data with R(int)=0.0287.The monoclinic space group P2(1) was determined by systematic absences and statistical tests and verified by subsequent refinement. The structure was solved by direct methods and refined by full-matrix least-squares methods on F2, (a) G. M. Sheldrick (1994), SHELXTL Version 5 Reference Manual, Broker AXS Inc.; (b) International Tables for Crystallography, Vol. C, Kluwer: Boston (1995). Hydrogen atom positions were determined from difference peaks and ultimately refined by a riding model with idealized geometry. Non-hydrogen atoms were refined with anisotropic displacement parameters. In addition to the molecule of MET-1, one molecule of diethyl ether was present in the asymmetric unit of the crystalline lattice. A total of 334 parameters were refined against 1 restraint and 4624 data to give wR2=0.0995 and S=1.040 for weights of w=1/[s2(F2) (0.0675P)2], where P=[Fo2+2Fc2]/3. The final R(F) was 0.0332 for the 4624 observed data. The largest shift/s.u. was 0.001 in the final refinement cycle and the final difference map had maxima and minima of 0.238 and -0.201 e/?3, respectively. The absolute structure was determined by refinement of the Flack parameter, H. D. Flack, Acta Cryst. A, vol. 39, 876-881 (1983).The three dimensional structure of MET-1 as defined by the following physical data and atomic positional parameters described and calculated herein (Tables 1-8) is illustrated in FIG. 1.Example 2Crystallization of (20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 (MET-2)Crystallization from 2-propanol/hexane.(20R)-2-methylene-19-nor-22-dimethyl-1α,25-dihydroxyvitamin D3 (22.7 mg), was suspended in hexane (4 mL) and then 2-propanol was added dropwise to the suspension. The mixture was heated in a water bath to dissolve the vitamin, then was left at room temperature for about 1 hour, and finally was kept in a refrigerator for about 48 hours. The precipitated crystals were filtered off, washed with a small volume of a cold (0° C.) 2-propanol/hexane (3:1) mixture, and dried to give crystalline material. It should be noted that an excess of 2-propanol should be avoided to get the point of saturation, i.e. only about 1 mole or less of 2-propanol should be added.Experimental.A colorless prism-shaped crystal of dimensions 0.36×0.17×0.03 mm was selected for structural analysis. Intensity data were collected using a Bruker AXS Platinum 135 CCD detector controlled with the PROTEUM software suite (Bruker AXS Inc., Madison, Wis.). The x-ray source was CuK radiation (1.54178 ?) from a Rigaku RU200 x-ray generator equipped with Montel optics, operated at 50 kV and 90 mA. The x-ray data were processed with SAINT version 7.06A (Bruker AXS Inc.) and internally scaled with SADABS version 2005/1 (Bruker AXS Inc.). The sample was mounted in a quartz capillary tube and data collected at 298 K. The intensity data were measured as a series of phi and omega oscillation frames each of 1° for 20-40 sec/frame. The detector was operated in
mode and was positioned 4.5 cm from the sample. Cell parameters were determined from a non-linear least squares fit of 9999 peaks in the range of 2.51&theta&58.69°. The data were merged to form a set of 3985 independent data with R(int)=0.0473.The monoclinic space group C2 was determined by systematic absences and statistical tests and verified by subsequent refinement. The structure was solved by direct methods and refined by full-matrix least-squares methods on F2, (a) G. M. Sheldrick (1994), SHELXTL Version 5 Reference Manual, Bruker AXS Inc.; (b) International Tables for Crystallography, Vol. C, Kluwer: Boston (1995). Hydrogen atom positions were determined from difference peaks and ultimately refined by a riding model with idealized geometry. Non-hydrogen atoms were refined with anisotropic displacement parameters. In addition to the molecule of MET-2, one molecule of isopropanol was present in the asymmetric unit of the crystalline lattice. A total of 325 parameters were refined against 1 restraint and 3985 data to give wR2=0.1600 and S=1.073 for weights of w=1/[s2(F2)+(0.1033P)2], where P=[Fo2+2Fc2]/3. The final R(F) was 0.0844 for the 3985 observed data. The largest shift/s.u. was 0.001 in the final refinement cycle and the final difference map had maxima and minima of 0.140 and -0.190 e/?3, respectively. The absolute structure was determined by refinement of the Flack parameter, H. D. Flack, Acta Cryst. A, vol. 39, 876-881 (1983).The three dimensional structure of MET-2 as defined by the following physical data and atomic positional parameters described and calculated herein (Tables 9-16) is illustrated in FIG. 2.TABLE 1Crystal data and structure refinement for MET-1.Empirical formulaC33H58O4Formula weight518.79Temperature100(1) KWavelength1.54178 ?Crystal system, space groupMonoclinic, P2(1)Unit cell dimensionsa = 7.5780(15)?α = 90°b = 14.792(3) ?β = 102.22(3)°c = 14.481(3) ?γ = 90°Volume) ?3Z2Calculated density1.086 Mg/m3Absorption coefficient0.532 mm-1F(000)576Crystal size0.25 × 0.34 × 0.55 mmTheta range for data collection3.12 to 63.06°Limiting indices-8 &= h &= 8, -17 &= k &= 16,0 &= l &= 16Reflections collected/unique [R(int) = 0.0287]Data/restraints/parametersGoodness-of-fit on F21.040Final R indices [I & 2□(I)]R1 = 0.0330, wR2 = 0.0955R indices (all data)R1 = 0.0332, wR2 = 0.0959Absolute structure parameter-0.01(15)Largest diff. peak and hole0.238 and -0.201 e/?3 TABLE 2Atomic coordinates (?2 × 104) and equivalent isotropicdisplacement parameters (?2 × 103) for MET-1 U(eq)is defined as one third of the trace of the orthogonalized Uij tensor.xyzU(eq)O(3)-1435(2)3847(1)11389(1)26(1)O(100)-1576(2)5643(1)10959(1)32(1)O(25)-4404(2)-6333(1)12194(1)27(1)O(1)3382(2)2361(1)11284(1)28(1)C(5)-1471(2)1831(1)11117(1)22(1)C(13)-3621(2)-1411(1)13186(1)22(1)C(6)-2465(2)1391(1)11638(1)23(1)C(20)-3217(2)-3144(1)13759(1)23(1)C(14)-2234(2)-617(1)13380(1)23(1)C(2)716(2)3237(1)10539(1)24(1)C(22)-2071(2)-3839(1)14466(1)27(1)C(7)-1882(2)618(1)12255(1)23(1)C(4)-2313(2)2542(1)10414(1)24(1)C(8)-2850(2)217(1)12818(1)24(1)C(23)-2980(2)-4791(1)14332(1)28(1)C(3)-1246(2)3423(1)10520(1)25(1)C(21)-5206(2)-3181(1)13826(1)30(1)C(24)-3359(2)-5161(1)13320(1)27(1)C(1)1560(2)2561(1)11290(1)23(1)C(10)502(2)1670(1)11152(1)22(1)C(15)-456(2)-1058(1)13319(1)26(1)C(12)-5392(2)-1054(1)13413(1)28(1)C(9)-4631(2)555(1)12982(1)29(1)C(17)-2546(2)-2145(1)13863(1)22(1)C(101)-280(3)6264(1)11455(1)40(1)C(29)1591(3)3619(1)9940(1)31(1)C(30)-125(2)-3978(1)14326(1)31(1)C(16)-580(2)-2012(1)13723(1)25(1)C(18)-3930(2)-1723(1)12152(1)24(1)C(11)-6056(2)-197(1)12851(1)29(1)C(25)-3972(3)-6149(1)13189(1)31(1)C(201)-3380(3)6000(1)10736(1)38(1)C(27)-2516(4)-6804(2)13656(2)65(1)C(28)-1941(3)-3553(1)15498(1)35(1)C(202)-4591(3)5306(2)10178(2)48(1)C(26)-5726(3)-6301(2)13518(1)50(1)C(102)1535(3)5832(2)11596(2)54(1) TABLE 3Bond lengths [?] for MET-1.O(3)—C(3)1.440(2)O(100)—C(101)1.423(2)O(100)—C(201)1.437(2)O(25)—C(25)1.435(2)O(1)—C(1)1.4140(19)C(5)—C(6)1.342(2)C(5)—C(10)1.504(2)C(5)—C(4)1.508(2)C(13)—C(18)1.537(2)C(13)—C(12)1.542(2)C(13)—C(14)1.561(2)C(13)—C(17)1.570(2)C(6)—C(7)1.461(2)C(20)—C(21)1.532(2)C(20)—C(17)1.559(2)C(20)—C(22)1.575(2)C(14)—C(8)1.497(2)C(14)—C(15)1.516(2)C(2)—C(29)1.325(2)C(2)—C(3)1.506(2)C(2)—C(1)1.516(2)C(22)—C(28)1.536(2)C(22)—C(30)1.544(2)C(22)—C(23)1.562(2)C(7)—C(8)1.345(2)C(4)—C(3)1.524(2)C(8)—C(9)1.505(2)C(23)—C(24)1.533(2)C(24)—C(25)1.532(2)C(1)—C(10)1.533(2)C(15)—C(16)1.539(2)C(12)—C(11)1.532(2)C(9)—C(11)1.534(2)C(17)—C(16)1.558(2)C(101)—C(102)1.491(3)C(25)—C(27)1.516(3)C(25)—C(26)1.521(3)C(201)—C(202)1.495(3) TABLE 4Bond angles [°] for MET-1.C(101)—O(100)—C(201)113.47(15)C(6)—C(5)—C(10)125.17(14)C(6)—C(5)—C(4)120.68(15)C(10)—C(5)—C(4)114.14(13)C(18)—C(13)—C(12)110.52(13)C(18)—C(13)—C(14)111.04(12)C(12)—C(13)—C(14)106.64(13)C(18)—C(13)—C(17)110.70(13)C(12)—C(13)—C(17)117.67(13)C(14)—C(13)—C(17)99.63(12)C(5)—C(6)—C(7)126.18(15)C(21)—C(20)—C(17)109.44(13)C(21)—C(20)—C(22)110.45(13)C(17)—C(20)—C(22)115.60(13)C(8)—C(14)—C(15)120.05(14)C(8)—C(14)—C(13)113.68(13)C(15)—C(14)—C(13)103.82(13)C(29)—C(2)—C(3)122.61(16)C(29)—C(2)—C(1)123.89(16)C(3)—C(2)—C(1)113.49(13)C(28)—C(22)—C(30)107.31(14)C(28)—C(22)—C(23)107.77(13)C(30)—C(22)—C(23)105.76(14)C(28)—C(22)—C(20)111.43(14)C(30)—C(22)—C(20)114.34(13)C(23)—C(22)—C(20)109.89(13)C(8)—C(7)—C(6)125.67(15)C(5)—C(4)—C(3)112.89(14)C(7)—C(8)—C(14)123.11(15)C(7)—C(8)—C(9)124.84(15)C(14)—C(8)—C(9)112.04(14)C(24)—C(23)—C(22)115.43(13)O(3)—C(3)—C(2)110.16(13)O(3)—C(3)—C(4)108.15(12)C(2)—C(3)—C(4)110.21(13)C(25)—C(24)—C(23)116.71(14)O(1)—C(1)—C(2)113.40(13)O(1)—C(1)—C(10)107.71(12)C(2)—C(1)—C(10)110.12(13)C(5)—C(10)—C(1)110.88(13)C(14)—C(15)—C(16)104.17(13)C(11)—C(12)—C(13)111.93(13)C(8)—C(9)—C(11)111.72(14)C(20)—C(17)—C(16)114.06(13)C(20)—C(17)—C(13)118.64(13)C(16)—C(17)—C(13)102.46(12)O(100)—C(101)—C(102)108.10(16)C(15)—C(16)—C(17)107.60(13)C(12)—C(11)—C(9)113.01(14)O(25)—C(25)—C(27)108.39(16)O(25)—C(25)—C(26)104.95(14)C(27)—C(25)—C(26)111.5(2)O(25)—C(25)—C(24)107.75(13)C(27)—C(25)—C(24)112.43(16)C(26)—C(25)—C(24)111.40(16)O(100)—C(201)—C(202)108.38(16) TABLE 5Anisotropic displacement parameters (?2 × 103) for MET-1.The anisotropic displacement factor exponent takes the form:-2π2[h2a * 2U11 + . . . + 2hka * b * U12.U11U22U33U23U13U12O(3)26(1)17(1)37(1)0(1)10(1)1(1)O(100)40(1)19(1)37(1)2(1)9(1)3(1)O(25)28(1)28(1)26(1)-2(1)6(1)-7(1)O(1)17(1)23(1)44(1)-6(1)8(1)-2(1)C(5)21(1)16(1)27(1)-4(1)4(1)-2(1)C(13)23(1)21(1)24(1)0(1)8(1)-2(1)C(6)19(1)18(1)32(1)-2(1)6(1)0(1)C(20)28(1)20(1)23(1)1(1)8(1)-5(1)C(14)25(1)20(1)25(1)-2(1)6(1)-3(1)C(2)27(1)17(1)29(1)-5(1)7(1)-4(1)C(22)33(1)22(1)24(1)3(1)4(1)-6(1)C(7)19(1)18(1)30(1)-2(1)6(1)-1(1)C(4)21(1)21(1)30(1)1(1)3(1)1(1)C(8)25(1)18(1)29(1)-3(1)6(1)-1(1)C(23)38(1)21(1)25(1)3(1)6(1)-5(1)C(3)29(1)20(1)28(1)4(1)7(1)2(1)C(21)29(1)23(1)39(1)2(1)11(1)-6(1)C(24)34(1)22(1)24(1)2(1)6(1)-4(1)C(1)20(1)22(1)29(1)-2(1)8(1)0(1)C(10)21(1)20(1)27(1)2(1)7(1)2(1)C(15)20(1)23(1)33(1)3(1)4(1)-3(1)C(12)29(1)24(1)34(1)1(1)14(1)-2(1)C(9)30(1)24(1)38(1)2(1)16(1)4(1)C(17)25(1)20(1)20(1)0(1)6(1)-4(1)C(101)65(1)25(1)29(1)-1(1)10(1)-9(1)C(29)33(1)24(1)39(1)3(1)13(1)-2(1)C(30)33(1)23(1)36(1)9(1)1(1)-1(1)C(16)22(1)21(1)29(1)2(1)1(1)-2(1)C(18)25(1)20(1)26(1)0(1)4(1)-1(1)C(11)25(1)28(1)39(1)3(1)16(1)3(1)C(25)42(1)23(1)25(1)2(1)1(1)-6(1)C(201)48(1)28(1)44(1)11(1)22(1)15(1)C(27)93(2)25(1)55(1)0(1)-32(1)8(1)C(28)49(1)29(1)26(1)2(1)4(1)-12(1)C(202)35(1)46(1)58(1)14(1)2(1)13(1)C(26)75(2)50(1)31(1)-7(1)23(1)-34(1)C(102)54(1)41(1)57(1)2(1)-10(1)-14(1) TABLE 6Hydrogen coordinates (?2 × 104) and isotropicdisplacement parameters (?2 × 103) for MET-1.H(3A)-124643911136040H(25A)-3854-59811192041H(1A)403327631155941H(6A)-363615981160427H(20F)-3157-33391312028H(14A)-2129-4511404528H(7A)-7433831225827H(4A)-352926651049529H(4B)-23982310978029H(23A)-2206-52171473934H(23B)-4112-47611454234H(3B)-17353823998830H(21A)-5886-27551339245H(21B)-5311-30331445845H(21C)-5668-37791367145H(24A)-4282-47871293640H(24B)-2270-50941307540H(1B)150228091191028H(10F)64613861056927H(10G)98312621166927H(15A)554-7271369031H(15B)-316-10871266931H(12A)-6313-15181326733H(12B)-5200-9231408433H(9A)-505810431254435H(9B)-44647951361835H(17A)-2559-19571451126H(10A)-55764121206148H(10B)-29868171109448H(29A)9944020948537H(29B)28033486997537H(30A)485-44081478047H(30B)509-34121441147H(30C)-160-42011370047H(16A)271-20751432230H(16B)-299-24631329030H(18A)-4591-12671175035H(18B)-4607-22761207735H(18C)-2787-18201198335H(11A)-7122261304835H(11B)-6400-3491218635H(20A)-340965511037146H(20B)-378261391131346H(27A)-1429-66891343597H(27B)-2910-74121349897H(27C)-2288-67261432897H(28A)-1234-39881591053H(28B)-3130-35221562753H(28C)-1377-29701560053H(20C)-580455341002271H(20D)-456047641054671H(20E)-41875175960771H(26A)-6077-69241342575H(26B)-6654-59231316175H(26C)-5554-61541417775H(10C)243362411192781H(10D)179856881099381H(10E)1544528811958 TABLE 7Torsion angles [deg] for MET-1.C(10)—C(5)—C(6)—C(7)-6.9(3)C(4)—C(5)—C(6)—C(7)172.04(14)C(18)—C(13)—C(14)—C(8)62.64(17)C(12)—C(13)—C(14)—C(8)-57.81(17)C(17)—C(13)—C(14)—C(8)179.34(12)C(18)—C(13)—C(14)—C(15)-69.49(16)C(12)—C(13)—C(14)—C(15)170.05(13)C(17)—C(13)—C(14)—C(15)47.21(14)C(21)—C(20)—C(22)—C(28)-68.60(17)C(17)—C(20)—C(22)—C(28)56.32(18)C(21)—C(20)—C(22)—C(30)169.50(14)C(17)—C(20)—C(22)—C(30)-65.58(18)C(21)—C(20)—C(22)—C(23)50.77(17)C(17)—C(20)—C(22)—C(23)175.69(13)C(5)—C(6)—C(7)—C(8)175.69(16)C(6)—C(5)—C(4)—C(3)129.93(16)C(10)—C(5)—C(4)—C(3)-50.98(18)C(6)—C(7)—C(8)—C(14)175.28(14)C(6)—C(7)—C(8)—C(9)-6.4(3)C(15)—C(14)—C(8)—C(7)-0.9(2)C(13)—C(14)—C(8)—C(7)-124.65(17)C(15)—C(14)—C(8)—C(9)-179.41(15)C(13)—C(14)—C(8)—C(9)56.87(17)C(28)—C(22)—C(23)—C(24)175.53(15)C(30)—C(22)—C(23)—C(24)-69.94(18)C(20)—C(22)—C(23)—C(24)53.94(19)C(29)—C(2)—C(3)—O(3)-116.79(17)C(1)—C(2)—C(3)—O(3)64.38(16)C(29)—C(2)—C(3)—C(4)123.95(17)C(1)—C(2)—C(3)—C(4)-54.88(17)C(5)—C(4)—C(3)—O(3)-69.91(16)C(5)—C(4)—C(3)—C(2)50.56(17)C(22)—C(23)—C(24)—C(25)170.72(15)C(29)—C(2)—C(1)—O(1)-1.0(2)C(3)—C(2)—C(1)—O(1)177.82(12)C(29)—C(2)—C(1)—C(10)-121.76(17)C(3)—C(2)—C(1)—C(10)57.05(16)C(6)—C(5)—C(10)—C(1)-128.78(16)C(4)—C(5)—C(10)—C(1)52.18(17)O(1)—C(1)—C(10)—C(5)-178.02(11)C(2)—C(1)—C(10)—C(5)-53.90(16)C(8)—C(14)—C(15)—C(16)-163.10(13)C(13)—C(14)—C(15)—C(16)-34.78(16)C(18)—C(13)—C(12)—C(11)-65.07(17)C(14)—C(13)—C(12)—C(11)55.72(17)C(17)—C(13)—C(12)—C(11)166.45(14)C(7)—C(8)—C(9)—C(11)130.33(18)C(14)—C(8)—C(9)—C(11)-51.22(17)C(21)—C(20)—C(17)—C(16)-175.23(13)C(22)—C(20)—C(17)—C(16)59.33(17)C(21)—C(20)—C(17)—C(13)-54.36(17)C(22)—C(20)—C(17)—C(13)-179.80(12)C(18)—C(13)—C(17)—C(20)-50.06(18)C(12)—C(13)—C(17)—C(20)78.34(18)C(14)—C(13)—C(17)—C(20)-167.02(12)C(18)—C(13)—C(17)—C(16)76.56(15)C(12)—C(13)—C(17)—C(16)-155.04(14)C(14)—C(13)—C(17)—C(16)-40.40(14)C(201)—O(100)—C(101)—C(102)-177.01(16)C(14)—C(15)—C(16)—C(17)8.55(16)C(20)—C(17)—C(16)—C(15)150.05(13)C(13)—C(17)—C(16)—C(15)20.53(16)C(13)—C(12)—C(11)—C(9)-54.82(19)C(8)—C(9)—C(11)—C(12)50.78(18)C(23)—C(24)—C(25)—O(25)175.01(14)C(23)—C(24)—C(25)—C(27)-65.6(2)C(23)—C(24)—C(25)—C(26)60.4(2)C(101)—O(100)—C(201)—C(202)177.20(15) TABLE 8Observed and calculated structure factors for MET-1.hkl10Fo10Fc10shkl10Fo10Fc10shkl10Fo10Fc10shkl10Fo10Fc10shkl10Fo10Fc10s0-160150149212047948952150414413-913737141113913621-1605252122021721023150343614-911881873511757212-1607774132011812014150959515-91192181361113112423-160134137242025324532160767416-9136371115415721-1501061091520565110-17174721-6-8125231811212012-150444516201951993-3-16141391-5-811421412-821474913-1503436172079741-2-16175771-4-8124211-72114314524-1509194182050491-1-161554-3-8144431-621403810-140137148113014114210-16151501-2-8166641-521464911-1401816123027228131-1611671701-1-81103981-421595112-1409896133025224732-1614640-811491613-32144842953-140110106143055601-4-15111211321-811871892-22114614614-1402221153070681-3-15111410912-811601511-12132833331-13010410716301511452-2-1511059913-818880102127527942-1307472173072701-1-151636614-81203198312137237143-130102951830303310-151575315-81115116222118919524-1301231172040924916221-151393916-812727132116717025-1304142114031931842-15172731-4-712682705421667110-1204342124039941043-15148461-3-711101091521636511-120767213402502453-5-14157621-2-71153142162112312022-1206763144087811-4-14166721-1-71391378572113013323-120444015401531541-3-14171030-711851712821565014-1207586164031381-2-141494511-713543364-831222015-1207974174069741-1-141636512-712362272-7311019516-12066611840121810-141343513-712112093-631747411-11088881150485511-14183881-3-614804515-53111711912-110105103125024122232-14131301-2-615985756-43125123533-110158162135016316523-1411082-1-611521461-33114312814-1105052145015816714-141262810-613033024-23119118925-110133130255089871-5-13112512821-615625336-13140641366-110182216501921933-4-131414112-61254234203129328050-100186194175043421-3-13176761-3-51126122313136437341-1006557185035371-2-13157521-2-515455123124224122-10016717110603823668-1-1311371441-1-51358351333127528753-100238244116045343680-131919710-51275275243113713514-100158152236031230941-13119319611-5158661531363915-1009690146023222212-13119920512-513736163113413526-1009292156015815513-13182851-3-4140638610731848111-90185185166017017524-13129271-2-411981852831454412-90171177176013813325-13123211-1-413673683-841373813-908687186057611-6-121403910-412292331-74111311824-9018517831702562254-5-121807411-415085005-64111611625-9012813022701131223-4-121889212-412422282-54110810916-905355137087951-3-12164661-3-311461273-441696010-8026225264701191191-2-1211701761-2-311891892-34140238461-8016816425701801841-1-1211511471-1-314034124-24119118522-8044411670252310-12114714610-312892812-14136536743-801551491770737311-121394011-31377373304122723344-80232231408026725362-12112512812-31240242214150450185-80120115218017016443-12193941-2-21142145124123622826-8088851280414114-12130341-1-21330333334131529441-70254225338015415015-121403510-21274279244123523022-7012012314802312312-6-111818411-21375372354124324213-70899615801181151-5-111666512-211861964641798010-60346368968087851-4-11136331-8-1123271741485011-60458436478056571-3-11110121-7-111071071841535712-6024623321901801853-2-1111131121-2-1144481-851757513-6031830972901711762-1-1111221221-1-112282402-75113113521-5047531390878810-11112011010-1199951-65121823822-50247222249018617821-11113813811-113393443-55113112013-50168164459012913012-11126826622-113643869-45110611710-4092291610690505513-11112611917-111511572-35112912121-4032431731100665714-1111631632-801603-251535612-404054104210016817125-11135361-70163701-15135735141-301411411310023724326-11131321-6015760105127427642-30269280241001541521-6-10183811-3012262363151576710-201151132510096901-5-1011661673-201595251383711-204854885610091931-4-1012172153-101526532535128827042-202172105111090901-3-1012142161001128131145126627321-104604711121101051032-2-1011691751101144014541655130330722-10239259631101571622-1-101136145120178751651465217-1012012624110505210-101287290130139339167519293110086931511013313011-101188180160110110218512328120018115826110192212-101145142170161651-8616165130017317043120444013-1019193180157561-7612730160010510614120798614-1011191092-81124271-6611121101700252015120797515-10167701-7111101071-5612432413800303416120656216-10142431-61163581-46177841110467471511301071072-6-9132351-51125231-361484451112102532582213077731-5-911111052-4113113015-2615525751531027526753130102951-4-9122251-3115705286-1611511462410406383741301251171-3-911231211-2114649106130930155101261272513041421-2-91981011-111230240216156053396102182233114021172-1-9128171011959412612432346710121126221401009620-91349341211132534363614456181026241314011310611-91969412113723864461178179102011511234140252112-9159511311248249356110010016611781843-31411192-7-924648130262711-7622926176161601-214149451-6-926262160288961-6621071031-77113131114186882-5-923026170277801-562991011-67180841214133301-4-92210206380225231-4622322291-571233226231411092-3-92100971-81215151-3622472416-4712752712414129291-2-921361421-7121521552-26238038810-3711091091-41511161132-1-921561571-6121061021-1622131873-2711481424-315111710910-92971071-51213313720621931794-1713933796-21511049911-921651641-41216316721622732535071189170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