• i j The present invention relates to separating individual compounds from complex chemical mixtures. In particular, the invention relates the separation of optically-active organic molecules. 5 Separating drug stereoisomers is of considerable importance, especially in the field of pharmaceuticals. Generally, only one of the stereoisomers in a particular pharmaceutical is responsible for the desirable pharmaceutical effect. A classic example is thalidσmide. When first developed, the drug was

10 administered as a mixture of stereoisomers. Only one of the stereoisomers had the desired pharmacological effect. As is well known today, the other stereoisomer not only had no beneficial pharmacological effect, but was responsible for severe birth defects in children of mothers taking the drug.

15 Presently, when a race ic mixture is proposed for use as a drug in the United States, the Federal Drug Administration (FDA) requires proof, which can be time consuming and expensive, that a non-pharmaceutically effective stereoisomer is harmless. Alternatively, the FDA requires use of only the pure stereoisomer

20 having the pharmacological effect.

Obtaining only the stereoisomer having beneficial pharmaceutical effects requires either synthesis of only the effective stereoisomer or separating the effective stereoisomer from a racemic mixture including the effective and non-effective

25 stereoisomers.

In accordance v/ith the present invention there is provided a method of separating a compound from a solution comprising

> passing the solution through crystals of biological acromolecules capable of selectively binding the compound. In

30 a further aspect of the present invention, the three-dimensional structure of the compound is determined while bound to the

crystals. In another aspect of the present invention, the crystals used in the separation are cross-linked.

Single crystals of proteins and other biological macromolecules have large solvent contents. Matthews, J. Mol. Biol. 33 : 491-497 (1968) . Typically, 20-80% of the volume of these crystals is occupied by mobile solvent channels and cavities. The crystals exist as an ordered matrix of the macromolecule with open channels of solvents that run through the crystals. Bugg, J. Crystal Growth 76 : 535-544 (1986) . Useful crystals of biological macromolecules include a wide variety of chemical materials ranging from globular proteins to highly charged nucleic acids. Exemplary crystals include human purine nucleoside phosphorylase (PNP) and influenza neuraminidase. Preferably, the crystals have a size of about 10- 600 μ , most preferably 300-600 μ . The solvent content of the macromolecular crystals is preferably in the range of 30-90% of the crystal volume. The molecular weight of the macromolecular crystals would generally be in excess of 5,000 daltons. The solvent channels through the crystals have a minimum width sufficiently large enough to accommodate the materials of interest. Preferably, the minimum width of the solvent channels is about 12 A. The amount of crystals used in separation varies greatly and depends on the amount of solution that is to pass therethrough. For example, as little as one milligram can be used for structural analysis, while columns used to handle large volumes of solution would typically hold up to 10 grams.

The crystals can be arranged in various ways in accordance with the present invention. For example, the crystals can be packed into a column or simply placed in a dish. Solutions can then be poured through the column and either gravity or vacuum used to pull the solution through the crystals. If the crystals are in a dish, the solution is simply mixed with the crystals, and the solution is then filtered off under gravity or vacuum, or individual crystals are removed from solution using a syringe or pipette.

The crystals permit separation of numerous compounds from solutions, the requirement being that the compound have a binding

affinity for the biological macromolecule that is crystallized, and be sufficiently small to diffuse through the channels in the crystalline matrix. For example, crystals of the enzyme human purine nucleoside phosphorylase (PNP) selectively binr* to substrates for the enzyme, substrate analogues, and enzyme inhibitors. The enzyme also separates the stereoisomers of 9- l-(3-chlorophenyl) -2-carfaoxyet yi ] -9-deazaguanine by selectively binding to only the S-isomer. Examples of other optically active compounds that bind selectively to the enzyme include formycin b and 5 '-iodo-9-deazainosine.

Removal of the separated compound from the crystalline matrix is performed using appropriate eiuants. The specific eiuants depend on the type of binding between the crystals and separated compound and -will be readily apparent to the skilled artisan. For example, che eiuant can contain a substrate, sucstrate analog, cr ether inhibitor that competes for the protein binding site, thus releasing the compound of interest. Exemplary eiuants for crystals of PNP include, for example, aqueous solutions containing acylovir diphosphate, in the millimolar to icromolar range. Other suitable eiuants will be readily determinable to one of ordinary skill in the art.

The relatively large channels present in the crystals of biological macromolecules create a high solvent content, which permits various types of compounds to be diffused through the crystals. The crystals also possess inherent binding activities, e.g., enzyme crystals bind substrates and substrate analogues for the enzyme. When solutions containing the compound are passed through the crystals, the compound -will bind to the crystals and be retained in the crystal lattice 'while the remainder of the solution passes through and cut of the crystals. Therefore, the crystals can be used to separate individual compounds from complex chemical mixtures. For example, enzyme crystals can separate substrates for the enzyme that 'would not be degraded by being coupled to the enzyme crystals as well as being able to separate enzyme-substrate analogues. Such separation procedures according to the present invention are useful, e.g., in purifying

drugs (such as substrate analogues, ..nich block enzyme-suostrate bmcmg; and separating stereoisomers from racemic mixtures.

In a preferred emcodiment, the crystallograpny techniques are used to determine the three-dimensional structure of the separated compounds while bound in the crystal matrix. In this embodiment the crystals are then characterized using standing X- ray or neutron diffraction techniques to determine the structure of the protein complex (T. . Blundell and L. N. Johnson, Protein CrystallograDhy, Academic Press, New /ork, 1976, pp. 333-336). Because the separated compound is bound in an ordered crystal matrix, it is also oriented in an ordered crystalline array. By first determining the three-dimensional structure of the biological macromolecule crystal matrix, then the approximate pnases that are required to solve the unknown structure will also be -mown. The unknown structure can then ce determined by calculating a difference Fourier map. The map ould be calculated by a Fourier series using the pnase angles oDtained from the structure of the uncomplexed protein, and Fourier coefficients that are the difference between the structure factors for the protein complex and the structure factors for the uncomplexed protein. The structure of the unknown molecule might then be obtained with higher precision by refining the structure of the protein complex, using X-ray diffraction data collected from crystals of the complex. Useful crystallography techniques include X-ray diffraction or neutron diffraction, the procedures of vhicn will be readily apparent to the skilled artisan, such as disclosed in T. L. Blundell and L. N. Johnson, Protein Crystallography, Academic Press, New York, 1976, pp. 333-336, Ealick et al., Proc Nat Acaα Sci USA 88 : 11540-11544 (1991) , COOK, et al. , J Biol Chem 256 : -079-4080 ^980) , Cook, et al . , J Biol Chem 260 : 12968-12969 (1985) , and Ealick, et al., Annals New ork Academy Sci. 451: 311-312 (1986). The three-dimensional structure obtained m this -anner s useful, e.g., in confirming the presence of only a desired stereoisomer and _ designing pharmaceuticals. Once the three-dimensional structure of a compound s known, then drugs can be designed smg known methodologies as disclosed in Goodford, J Med Chem 27: 557-564

(1984) , Blundell, et al., Nature 304 : 2723-275 ((1983) , Johanson, et al., J. Biol. Chem. 260 : 1465-1474 (1985) , and Wilton, et al., Biotechnology, June 1984, pp. 511-519, Ealick et al., Proc. Nat. Acad. Sci. USA 88 : 11540-11544 (1991) , the disclosures of which are incorporated herein by reference. The general steps that are followed in this process are depicted in the flow chart shown in Figure 2 of Ealick et al., ibid. The structure of the complex shows the mechanism whereby the unknown compound binds to the protein. This structural information is then used to model new chemical compounds that are expected to bind even more tightly to the protein. These new compounds would then be synthesized, bound to the protein in the crystals, and characterized by crystallographic methods. Using this iterative approach, which is referred to as structure-based drug design as disclosed in Ealick et al., ibid . , new chemical compounds that bind tightly to the protein and thereby block sites required for normal biological functions can be developed.

In another preferred embodiment, the crystals are cross- linked before the separation procedure. Cross-linking of the crystals can be performed by known procedures for cross-linking protein crystals, such as by suspending the crystals in glutaraldehyde as disclosed in Quiocho, et al., Proc. N.A.S. 52 : 833- 839 (1964) , Korn, et al., J. Mol. Biol. 65 : 525-529 (1972) , Richards, et al., J. Mol. Biol. 37 : 231-233 (1968) , and Matthews, et al. J. Mol. Biol. 82 : 513-526 (1974) , the disclosures of which are incorporated herein by reference.

In order to more particularly describe the present invention, the following non-limiting examples are provided. All parts and percentages reported in the examples are by weight unless indicated otherwise.

EXAMPLE 1

In this example crystals of human PNP are used to separate the human-PNP substrate guanine from solution. Crystals of PNP are grown as disclosed in the aforesaid Cook, et al., J. Biol. Chem. 256 : 4079-4080 (1980) .

The crystals are then transferred to a solution that has the same chemical composition as the solution that was used to crystallize PNP, but has a new concentration of ammonium sulfate that is 5%-15% higher in ammonium sulfate concentration than the original crystallizing solution. This higher concentration of ammonium sulfate prevents the crystals from dissolving. This stabilizing solution is often referred to as an artificial mother liquor. Guanine is added to this solution, which is then allowed to diffuse into the crystals of PNP and guanine. The time required for fusion is 1-24 hours, after which the crystals will exist as a complex of PNP and guanine. These crystals can then be studied by X-ray or neutron diffraction methods in order to study their structure.

EXAMPLE 2

In this example crystals of human PNP are used to separate an analog for a human PNP substrate from solution.

Crystals are prepared and transferred to a solution of artificial mother liquor as in Example 1. A solution of the same components used to crystalize the PNP containing 8-aminoguanine as the substrate analog is added to the crystal preparation. The substrate analog diffuses into the crystals and binds tightly to the active site of the enzyme to form a complex. The crystals of the complex can be used to separate and purify the substrate analog. The purified substrate analogue is then eluted from the crystals by pH adjustment. The procedure is repeated for substrate analogues formycin b, and 5 ' -iodo-9-deazainosine.

EXAMPLE 3 In this example crystals of human PNP are used to separate an inhibitor of human PNP from solution.

A solution containing the inhibitor of human PNP, acyclovir diphosphate, is added to the artificial mother liquor containing crystals of PNP as in Example 1. The inhibitors diffuse into the crystals and bind tightly to the active site of PNP to form a complex. The purified inhibitor is eluted from the crystals of the complex by pH adjustment. The procedure is repeated for the

inhibitors 9-benzyl-9-deazaguanine and 9-cyclohexylmethyl-9- deazaguanine.

EXAMPLE 4 In this example crystals of human PNP are used to separate an isomer from a racemic mixture.

A solution is formed containing a mixture (0.5 mg) in an equal molar concentration of stereoisomers (S)-9-[l-(3- chlorophenyl) -2-carboxyethyl]-9-deazaguanine and (R)-9-[l-(3- chlorophenyl) -2-carboxyethyl]-9-deazaguanine added to 1.0 mol of the artificial mother liquor of Example 1 containing 1-5 crystals of PNP. The crystals have maximum dimensions of .6 mm, with minimum dimensions of 0.1 mm. The mixture slowly dissolves in the artificial mother liquor and the individual steroisomers diffuse into the crystals. The preferred (S) steroisomer binds tightly to the enzyme, and the undesired steroisomer remains free in the solution of the artifical mother liquor. The desired steroisomer is isolated by harvesting the crystals, which bind only the desired steroisomer. In order to demonstrate that these crystals contain only the (S) isomer, X-ray diffraction experiments with the crystals of the resulting PNP complex are conducted using procedures described in the foregoing specification. These X-ray diffraction experiments demonstrate that essentially every molecule of PNP in the crystals contains a molecule of the purified (S) isomer bound to the active site of the enzyme. The X-ray diffraction data also demonstrate that no (R) isomer can be observed in the crystals. Thus, the crystals are used effectively to separate and purify the desired (S) isomer which is a potent inhibitor of the enzyme.

In order to separate the stereoisomer from the PNP crystals, the crystals of the complex are dissolved in 1 ml of an aqueous solution containing either 0.1 molar tris-acetate, or 0.05 molar potassium phosphate, pH 7.5. The stereoisomer is then separated from the enzyme by gel filtration using a Sephadex G-100 column (1.9x50 cm). Either 0.1 molar tris-acetate or 0.05 molar

potassium phosphate buffer, pH 7.5, is employed to equilibrate the column and elute the stereoisomer.

EXAMPLE 5 Crystals of PNP are used to separate large quantities of stereoisomers by the following procedure: Several grams (10-100 grams) of PNP are crystallized as in Example 1. These crystals are cross-linked with glutaraldehyde using procedures described in the aforesaid specification to form a material that is insoluble in a range of aqueous solutions. Exemplary solutions would contain tris-acetate or potassium phosphate buffers, at concentrations in the range of 0.01 molar - .5 molar, pH 5-8, and ammonium sulfate in the range of 0-65% saturated. These cross- linked crystals are poured into a chromatographic column, which is used to separate the stereoisomers. Either 0.1 molar tris- acetate buffer or 0.05 molar potassium phosphate buffer, pH 7.5, is employed as the solution to equilibrate the column and to elute compounds from the column. The stereoisomer mixture is dissolved in 1 ml of the solution, which is placed on the column. The stereoisomers are then consecutively eluted from the column using the buffer solution. The stereoisomer that is specific for the enzyme will be relatively tightly bound to the column, and will thus elute after the undesired stereoisomer is washed from the column. Alternatively, the stereoisomers can be washed from the column using a concentration gradient ranging from 0 to 0.01 molar 8-aminoguanine in 0.1 molar tris-acetate buffer or 0.05 molar potassium phosphate buffer, pH 7.5, to elute the stereoisomers from the column.