Symposium on Teaching Crystallography ..
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Teaching Biochemists and Pharmacologists How To Use Crystallographic Data William L. Duax Medical Foundation of Buffalo, Inc., 73 High Street, Buffalo, NY 14203 The three-dimensional structure of most biologically active molecules olavs a orimarv role in eovernine their interproactions and ac'tivkies.'~-ra~crystal~~~ra~hic~tudies vide the most reliable data concerning ground state structure. T h e Cambridge Structural Database [ I ) now contains complete biblioera~hic.chemical. and numerical details for some 55,000 organic carbon crystal structures. The Protein Data Bank (2) is an international repository of crystal structure studies of hundreds of proteins, tRNA's, polynucleotides, and polysaccharides. By combining solid state data with physical chemical data on structure in solution and with molecular energy calculations a reasonable picture of dynamic properties of hormones, carcinogens, antibiotics, and proteins can be constructed. Bv combining this information with biochemical. pharmacological, and physiological data, a better understandine of the molecular mechanisms of hiosvnthesis. metabolism, membrane support, receptor binding, and nuclear interaction can be achieved. The demand for trained crystallographers in such fields as genetic engineering, rational drug design, and solid state and materials research is generating a shortage of educators and research crystallographers in the academic community a t a time when the need for more effective teaching of basic crystallography and the effective use of crystallographic data are most needed. A course in crystallography designed for biochemists and pharmacologists should address the following questions:
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How does one get the information one wants from a crystal strueture publication? How does one compare structures derived from X-ray analysis, molecular mechanics calculations,and solution spectroscopy? How is a molecular structure observed in a crystal related to the structure in solution or in the biological setting? Can analysis of structuraldataon a class of compounds be used to predict biological activity of new analogues? How can molecular conformation be analyzed qualitatively and quantitatively? Haw does one model substrate-protein interactions, and how can model studies be related to biological response and drug design?
C. Bond Lengths and Angles D. ConformationalAnalysis, Torsion Angles E. Using the Cambridge Structural Database F. Use of X-ray Data in Solution Spectra Interpretation G. Molecular Mechanics Empirical Energy Calculations H. Structural Representation and Computer Graphics In. Application A. Empirical Drug Design 8. Protein Substrate Interaction
Crystal Composltlon Students are introduced to lattice and crystal systems by requiring them to identify two-dimensional repeat patterns in wallpaper and tile samples and are asked to construct one or more unit cells from paper or cardboard. The way in which symmetry operators (rotation, translation, inversion) relate subunits (usually molecules) within a unit cell is demonstrated (Fig. 1).The fact that molecules related by crystal symmetry are identical and have identical crystalline environments is stressed. Examples of crystals containing two or more molecules in the crystallographic asymmetric unit are also presented. These molecules do not have identical environments and may or may not have similar shapes. In this connection it is instructive to contrast the crystal structures of cyclo(hexaglycy1) (3)and cholesterol (4). Crystals of cyclo(hexaglycyl) contain five crystallographically independent molecules that have significantly different shapes (Fig. 2). Crystals of cholesterol contain eight crystallographically distinct molecules (Fig. 3) that have very similar overall shapes. The cholesterol molecules fall into two groups differing only by a subtle conformational twist in the side chain. Althoueh there is oseudosvmmetrv between some of them. they & crystallo&aphicaliy and structurally distinct. I t is also instructive to provide examples of polymorphism in which more than one crystal form is generated by different modes of association of a single molecule. Useful examples of polymorphism include the steroid estrone (5)and the peptide enkephalin (6-8).Full X-rav analvsis reveals that the four mole&les of estrone observed in th;ee polymorphic
The following is a description of a one-semester course designed to teach first-year graduate students of pharmacology, medicinal chemistry, and biochemistry how to extract, interpret, evaluate, and use the information provided by an X-ray crystallographic crystal structure determination. Course Outline The following outline has been followed in presenting the course: I. Crystal Composition A. Lattice and Crystal Systems B. Space Groups and Symmetry C. Crystal Packing and Multiple Asymmetric Units 11. Molecular Geometry A. Point Groups and Molecular Symmetry B. Isomerism and Chirality 502
Journal of Chemical Education
Figure 1. Illustration of (a) e single molecule of glucose in a unit cell and (b) the packing of 12 molecules in and around the unit cell.
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four independent molecules are observed in a horseshoe shaned conformation (Fig. 4). It becomes clear that there may be many ways in which the same molecule can aggregate to form crystals. While multiple crystal forms and crystal forms with more than one molecule in the repeat unit provide information on the flexibility of molecules, flexibility per se is not a prerequisite for polymorphism.
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Crystallographic Parameters and Thelr Accuracy
Although the theory of diffraction physics and of crystal structure determination and refinement are not a part of the course, it is important to outline precisely what information is contained in a published structure and to discuss the criterion for evaluating the accuracy of a determination (9).The methods used to find atomic coordinates yield approximate positions, and the rough positional parameters are refined by least-squares techniques. The accuracy of a crystal structure determination is governed by the size and quality of the crystal, the method used for data collection, and the means by which the atomic coordinates of the structure have been refined. The accuracy of a given structure report can be judged on the basis of (1)the method of data collection (film or diffractomer), (2) the reliability factor, R, (3) the ratio between the number of data used for refinement and the number of atomic positional and thermal parameters which were refined, (4) whether anisotropic thermal motion was considered during structure refinement, and (5) whether hydrogen atoms could be located. Intensity data measured by a diffractometer are normally more accurate than data recorded on photographic film. The R factor is a numerical measure of the agreement between the magnitudes of observed intensities, IFol, of the diffraction data and the magnitudes of the calculated intensities, IF
Figure2. Superpositionstereoviewsoffivedifferentmnformationsofcycio(hexagiycyi)found inasinglecrystalform. The conformers fail into two major confwmtionai groupings, me members of one group are further subdivided by variation in the orientallon of carbonyl axygens at either end of the molecule.
Figure 3. Cholesterol aggregates found in the anhydrous forms of cholesterol.
Figure 4. Two stable conformations of the pntapeptide [Leuslenkepheiin found repeatedly in the solid state.
Small values of R indicate good agreement between the observed and calculated data and are consistent with a reliable structure determination. An R factor less than 5-6% denotes a high-precision analysis. If the R factor is in the range 1020%. the overall molecular conformation is nrobahlv correct. but some of the experimental data may be &accurate, or the structure could be further refined. Although crystallographers use the R factor as a convenient barometer of the accuracy of a determination, other factors should he carefully considered. The intensities of the X-ray reflections are a function of the time-averaged electron density. Consequently, they depend on thethermal motion of the atoms as well as the mean atomic positions. Thermal motion mav be described hv"~~~ either an isotrooic (spherical) or anisotropic (ellipsoidal) model. In general, the mean atomic positions and the geometrical parameters calculated from them are more accurate if the more sophisticated anisotropic model has been used for the thermal motion ~~
forms exhibit only subtle differences in overall conformation and hydrogen bonding. In two crystal forms of [Leuslenkephalin, six crystallographically independent molecules take up extended conformations, whereas, in a third crystal form,
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eg C-C in C ~ C ~ mean mad. d(s)
O ~ Q X ~ ~ Q :
1.q. uq. Nobs 1.535 1535 0.016 1.525 1545 2814
Endocyclic torsion angles redundantly define the conformations of rings (14). Clearly, if all hond lengths and angles are held constant and closure is required, two endocyclic torsion angles having only one common bond will fully define the conformation of a six-membered ring. Consequently, a two-parameter definition of the conformation of a ring would he a desirable improvement over the six-torsion-angle definition. It has been generally recognized that the symmetry in a ring provides a qualitative means of defining the ring's conformation. A thorough analysis of symmetry in rings of various sizes has been described by Hendrickson (15) and Pitzer and Donath (16). The most widely used quantitative descriptors of ring conformation other than torsion angles are the pseudo-rotation parameters (A and 6) defined by Altona, Geise, and Romers (17). The relationship between molecular shape and the location of rotational and mirror-plane symmetry elements is present in Figure 6. A quantitative evaluation of the conformations of rings of any size from which comparative analysis
Figure 5. Thedistribution of 2814 G C bond lengths observed in X-raystudies of ~rystalscontaining cyclohexsne. during structure refinement. Since hydrogen atoms have only a single electron, they scatter X-rays very weakly, and they can be observed experimentally only if the data are of good quality. Finally, in the absence of systematic errors in data collection or refinement, the greater the number of observed data relative to the number of independent atoms, the better the atomic resolution will be. Molecular Geometry Introduction to Stereochemistry (10) provides a suitable review of point groups and molecular symmetry and numerous useful exercises. Each student should obtain a simple model building kit' in order to be able to construct conform~tionalisomms and enantiomeric pairs of molecules. Recause different types of modeling equipment have different strengths and weaknesses, a variety of iolecular representation (both physical and computer graphic) are useful for instructional purposes. The Cambridge Structural Database (CSD) has reliable information ahout a wide ranee of molecular fragments. Bv retrieving all of the enamplecof a given fragmeGt from thk database and examinine the ranees of observed bond lenpth. valence angle, and torsion anae, a student may gai;an appreciation for the fact that the magnitudes of hond lengths and angles are generally very constant and impervious to intermolecular interaction. There are many reviews describing the use of the CSD to determine mean geometries and the ranges of hond lengths and angles commonly encountered in organic and bioorganic molecules (11). For example, on the basis of 2814 observations, the C-C bond lenpth in cvclohexane has a mean value of 1.535 A with a staidard diviation uf 0.016 A. The observed distribution is Gaussian in nature and half of the observations fall between 1.525 and 1.545 (Fig. 5).
PLANAR
CHAIR
BOAT
SOFA
Figure 6. The shapes and symmetry elements (mirrorsand twofold rotations) Present in the most commonly observed conformations of six-membered rings.
Molecular Conformailon Molecular conformation is most conveniently and precisely characterized by torsion angles. An excellent discussion of the torsion angle concept in conformational analysis appears in an article by Bucourt (12). The relationship between the torsion-angle magnitudes and the observed conformation of rings, hydrocarbon chains, peptides, proteins, and nucleic acids should be illustrated with molecular models and computer graphic illustrations (13).
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Cochranes of Oxford Limited. Leafield. Oxford. England, make an inexpensive kit with a useful 50-page booklet. 504
Journal of Chemical Education
Figure 7. Steroidal cyclabxanone rings in half-chair (a) and sofa conformations fb).
can be easilv made is obtainable from a mathematical combination of torsion angles. This analysis is based upon consideration of ao~roximatesvmmetrv ~ossessedbv most rines (13). stable-conformers of cyclohexene rings lie in a range
between the sofa and half-chair conformation illustrated in Figure 7. The conformation of most cyclohexene rings for which X-ravanalvsis is available can be auantitativelv evaluated using symmetry analysis, and their position jn the continuum between the sofa and half-chair conformations identified (18). The nomenclature for defining peptide torsion angles is well established (19). It is instructive to illustrate the relationship between the Ramachandran plots of peptide torsion angles (& and +) and the simplest polypeptide conformations (p sheets, a helix, 3-10 helices, etc.) (Fig. 8). Single-crystal X-ray analysis of double-stranded DNA molecules of predetermined sequence provides valuable information on the shape and flexibility of DNA (Fig. 9, (20)). In addition, X-ray determinations have been completed on complexes of double strands of helical DNA and intercalating drugs (21) and a DNA restriction enzyme (Fig. 10, (22)). These studies demonstrate the flexibility of the DNA molecule and how it responds to interaction with small and macromolecules.
Molecular Flexlblllty and Relative Potential Energy
Figure 8. A Ramachmdran plot Illushatingthe regions of lhe plot In which the # and J. values correspond to ma Ideal forms of &sheet and a-helical tertiary structure.
Flgue 9. A wmparlson of lhe crystallographically obsened conformation of (a) lhree A-hellcal tetramers wnh a wmmon helical axls, (b) the Bhellcal dodecamer CGCQAATTBrCOCG and (c) three Z-helical teharners with a Wmmon helical axls.
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It is important to address the question of the extent to which the factors governing aggregation of molecules in the solid state alters their shape and how these factors may be related to the forces acting upon molecules in solution and in the biological milieu. These questions can be addressed by comparing the results of X-ray crystallographic determinations with information on molecular geometry and conformation from theoretical calculations and other physical measurements made in the gas phase, in solids, and in solution. I t is particularly useful and interesting to contrast the results of molecular mechanics empirical energy calculations with the results of X-ray studies (23,24). Molecular mechanics calculations provide useful information concerning variation in potential energy as a function of variation in molecular structure. Several molecular mechanics programs are available from the quantum chemical program exchange (QCPE)or as part of computer graphics software systems that are widely distributed in the academic community. The accuracy Number 6
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with which a given program successfully identifies the global minimum energy conformations and the height and shape of barriers to conformational transition is difficult for the average user of these programs to estimate. Consequently, i t is valuable for the novice to use more than one program if ~ossible.and it is essential that the user understand the kariahleb and the relative contrihution that each makes to the overall calculated enerev. -- This requires knowledge of what goes into the program, how it works, and careful examination of the details of the output. Molecular mechanics programs calculate the potential enerev of a molecule as a summation of the individual energies associated with bond stretching and compression, bond-anele heudine, bond torsion, nonbonding interactions, charge, &d dipolekoment energies. In most cases the individual contributors to the total energy have a corresponding structural feature that can he derived directly from X-ray data. I t is unwise to assume that a molecular mechanics program has been adequately parameterized to deal with all of the structural features in a molecule of interest. If possible, one can assemhle a data hase of crystal structure determinations of molecules, or molecular fragments closely related to those of interest. Thisdata base should be examined carefully in order to detect any patterns that can be useful in evaluating the relative population of conformational isomers, the relative flexibility of those isomers, and evidence of intermolecular h e . hydrogen . . bonding) or intramolecular (substitution effect) on conformatinn. Representative structures in the data haseshould be subjected toenergy minimization. Computer output for the calculation on one or more
structures should be examined carefully. What values for individual parameters is the program attempting to impose upon the refined structure? How successful is the program in achievine a structure havine these ideal or averaee parameters? ~ 6 a are t the magnitLdes of the contrihu~o&to the total energy of the molecule? How does the geometry of the redefined structures compare with that of the crystallographically determined structure? I t is particularly instructive to use as a model a molecule for which two or more significantly different conformations have been observed cr~stallographically.If there is excellent agreement between the crystallographically observed structures and those minimized, you are in luck. If the differences between the observed and calculated structures are randomly distributed, i t may be difficult to determine to what extent the disparity is due to crystal packing forces or to inaccurate parameterization of the program. If systematic differences are observed, it should he possible to identify their source. The phvsical significance of eeometric differences between crvsialiographically ohserveb and energy-minimized structu;es should be examined using molecular graphical superposition of the observed and minimized structures (Fig. 11). Students should be challenged to account for the source of differences. If students conclude that crystal packing forces distort the molecule, they should he asked to substantiate their conclusion identifying unusually close contacts, directional forces, etc. If they question the accuracy of the calculations, they should offer specific support for their criticism.
Molecular Flexibility in Solid and Solution The relations hi^ between molecular conformation in solid and solution shouid he discussed. Molecules of limited flexibility can he expected to have nearly identical conformations in the solid state and in solution. Molecules having extensive conformational flexihilitv a ereater chal. provide . lenge. In general, such molecules exist in solution as an equilibrium mixture of different conformers that is solvent and temperature dependent. Consequently, spectral measurements made on flexihle molec~tlesmay he diffic~~lt to interpret unambiguously. The signals may represent an a\,erage structure or a mixtltre of conformers. Bv carefullv mani~ulatine nmditions. ir is -crvstallizntion . usually possible to obtain one or more crystal forms of the com~oundin which low-enerw conformers are stabilized bv favorable intermolecular int&actions. Some crystal form; may contain substantial amounts of solvent. Some forms will haie more than one molecule in the cryrtallopraphic repeat unit that have significantly different confmnations. In the latter case, is it ieasonabie to conclude that the potential Figure 10. Schematic backbone drawing of aoe subunit of (dimerid Eco RI energies of the two conformers are comparable? endonuclease and bath strands of me DNA in the complex. Some illustrative examples include: The potassium complex of valinomycin in which a flexible dodeeadepsipeptide takes up a conformation for which X-rav results and solution spectra are in complete agreement (25,26). The uncomplexed form of valinomycin for which spectral measurement in solution have been interpreted to reflect half a dozen conformations (27, 28) that differ from the one observed in the solid state (29). The structure of vitamin D for which spectral data indicate the Fiaure 11. Stereo-suosrwsition presence of molecules having A " .~~of molecules obtained hom X-rau data (solid line1 and molecular mechanics calculatton (dashed line) for a steroid ShuCtJle 16n-memyl-3.20d~ox0bpre~~n-17-yI poplonate). T3.t wbt e rings in two different chair condinerence m the orientation of the propionate may be aslociatsd with me inllutnm ol packing mtsranmns. The formations and a crystal form rignilicant dittersnce in me orientation ot me A rmg relssve to rest of me steroid a due to nsdequscies m containing both forms has been paramaterimtian of Me fused kn-3-ane A ring prepared (30,3I). ~
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upon crystallographic analysis for the action of calcium channel antagonists (4.3, suicide suhstrates for As-3-ketosteroid isomerase (44, 45), cardioactive drugs (463, thyrotropin-releasing hormone analogues (471, and other examples of particular interest to the student or instructor. If the student has an interest in a specific class of drugs or hor~ g w 12 e T l scenar o f o r me complexat on of potasslum ,on by valinomyem using rwo wysle.1 structures From 1eI1 mones, it may be possible to de10 imht. uncomplexed vahnomycm (Kt has oeen added), lntermedlale (modelled by c n a n g e s in torsfon angles), and sign his or her assignments complexed valinomycin. Kt Is depicted cross-hatched. oxygen striped, a n d nitrogen stipled. around those compounds when constructina molecular models, searching the Cambridge Structural Database, performing Structure and Blologlcal Action energy minimization calculations and reviewing the literaThe students should be challenged to speculate about the ture concerning the molecular basis for biological activity. relevance of structural information derived from gas phase, solid, and solution to the structures in the biological setting. In the case of molecules that are bound to receptors and enzymes in order to effect their biological response, does the protein-substrate interaction more nearly resemhle interactions found in solution or in the solid state? In this context it is useful to examine crystal structure studies of biologically active proteins, the inhibitors and cofactors of those proteins, and the complexes between the protein and the inhibitor and cofactors. Some useful examples include: The structure of methotreaste observed in single-crystal studies (32) d i f f e r s significantly from the structure of the inhihitor d i h y d r o f o l a t e reductase when it is hound in the a c t i v e site of the enzyme (33). When hound to the tryptophan repressor, tryptophan i t s e l f is a little distorted from the conformation observed in single nystals (34),but the protein undergoes a conformational change that is critical to its interaction with DNA (35). At the contact region between l y s o z y m e and an antibody to lysozyme (36),the surfaces of both proteins appear to he little changed from what was observed in the X-ray studies of the i s o l a t e d components (37-39).
Crystallographic data are being used with increasing frequency to rationalize biological activity of hormones, drugs, and proteins, to develop empirical models for biological response, and t o contribute to drug design. Other instructive examples include the following: The crystal structure analysis of the complex of antiviral agents and the human cold virus provides a plausible explanation for the basis for the antiviral activity of the drug (40). Analysis of X-ray data on over s dozen ligands that bind to the benzodiazapine receptor have been used to identify features common to potent antagonists, develop a detailed model that accounts f o r key features found in ligands for the receptor, and p r o v i d e an explanation for the spectrum of responses elicited by receptor binding (41). The crystal structures of complexed and uncomplexed f o r m s of valinomycin have been used to model the mechanism o f ion capture that is consistent with biochemical and kinetic data (Fig. 12, (29)). The crystal structures of estrogenic agonists and antagonists have been combined with empirical energy calculations and pharmacological data to develop an empirical model for the molecular basis for estrogen receptor binding and hormonalresponse (42). Student Semlnars
To complete the course students should prepare a short paper and present a seminar in whichthey critically examine a model for biological action of a drug, hormone, or protein that is based at least in part on crystallographic data. A list of topics should be provided that includes examples where conflicting models have been proposed by different investigators. In addition to the numerous examples already cited above, the list of topics might include models based
Literature Clled 1. Allen. F. H.: Bellard. S.: Brice. M. D.: Cartanieht. B. A: Doubledav. A.: H i m . H.:
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535342. Kade, I.L.:Karic. J. Acto Cryat. IW3,16,969475. Shieh, H.-S.;Hoard,L.G.;Nordman,C.E. Acto Cryst. 1981,837,15361543. Bu8etta.B.; Courseills. M.; Hospital, M. Act. Cryst. 1973.829,29%313. Smith,G. D.:Griffm, J.F.Science 1978,199,121&1216. Karle. I. L.: Karle, J.:Msstropsolo.O.:Camerman,A.;Camerman, N.AeIoCryst. 1983, B39.615437.
8 G~iffin,J.F.;Langa,D.A.;Sm~h.G.D.;Blundell.T.L.;Tieklc,I.J.:Bedarkar,S.Pm. Nofl. A c d Sei. USA 1986.53.32723276. 9. Glusker,J. P.;Truehlmd. K. N. CryalolSfruefure Anoiyau. APrrmer;OxfordUniverrify: New York. 1985. lo. Mislow, K. Introduefion to Storeoch~miatry;BenjaminiCummins: Reading, MA, 1965. 11. Allen,F.H.:Kennard,O.;Tsyior,R.Acc.Chom.Ro8.1983,16.146153. 12. Bumurt, R. Topics Stereocham. 1974 8,159-224. 1s. Duax, W. L.: Weeks, C. M.: Rohrer, D. C. Topics Stor#ocham. 1974.9.283-293. 14. Dunitz, J. D . J Chem. Educ. 1970.47.488, 15. Hendriekson,J.B. J.Am. Chom.Soc. 1961,83,4537. 16. Pitzor. K. S . ; h a t h , W.E. J.Am. Chem.Soe. 1959,81,3213, 17. Altona,C.;Oeise,H. J.:Romers,C. Tetrahedron 1968.21.13. ; New York, 1975. 18. Duar. W. L.; Narbn, D. A. Atlas o/SLeraid S t r u r l u r ~Plenum: 19. IUPACXUB Cornmiasion on Biochemical Nomenelsture. Eur. J. Blochem. I970,17, >02-.,n>
20. Dickerson.R.E.;Drew,H.R.;Conner,B.N.;Wing,R.M.:Fratini,A.V.;Kapka,M.L. Science 1982,216,415485. 21. Wong,A. H.J.; Ughetto, G.: Quigiey, G. J.: Hako3hima.T.; uander M a d . G. A.: van Bourn, J. H.; Rich, A. Science 1984.225.1115-1121. Grahle, J.; 22. MeClarin, J. A,: Frederick, C. A : Wang. B:C; Greene, P.; Boyer, H. W.; Rasenherg. J . M. Science 1986.234. 15261541. 28. Duai, W. L.;Griffin. J. F.;Rohrer,D. C. J.Am. Chem. Sac. 1981,103,6705-6712. 21. Dusx, W. L. Worbhop an Colculofion of Crwtol Pocking on Non-Bonded Forces; Palycrysfal Bmk Service: Dayton. OH, 1984. 25. Pinkerbn,M.; Steinreuf. L. K.:Dawkins. P. Bioehem. Biophya. Rer. Commun. 1969.
27. 28. 29. 30. 31. 32. 33. 34.
99.2032-2039. Urry,D. W.; Kumsr.N.G.8iochemlalry 1974.13.1829-1831. Ouehinnikov, Yu. A,: lusnov. V. T. Tetrahedron L974.30.1871-18W. Duax, W. L.;Hauptman, H.: Weeks,C. M.:Norlon,D.A. Science 1972,175,911-914. Oksmurs. W. H.; Norman, A. W.: Wing, R. M. P m . Norl. Arod. Sci. USA 1974. 71, 4194.4197. Trink-ban; DBLuca, H. F.;Dahl, L. F. J. Olg. Chem. 1978.41,34763478. Surbn,P.A.;Cody,V.:Smith.G. D. J A m Chem.Soc. 1986.IOS.41554158. Bolin. J . T.: Filman, D. J.: M a t t h e w D. A,; Hamlin, R. C.: Kraut, J. J. Bid. Chsm. 1962,257,1365C-13662. Takigsws, T.: Ashida, T.; Sasads. Y.: Kekudo, M. Bull. Chem. Soe. Jopon 1966.39, ,"CO A""".
35. 2hang.R.-g.;doschimicak.A.;La~an.C.L.:Scheui~,B. W.;Otwinouski,Z.;Sigler.P. B. Nolure 1987,327,591597. 36. Amit, A. G.; Mariuzzs, R. A,; Phillips, S. E. V.: Polyjak, R. J. Science 1986.233.747767
37. Segal, D. M.; Psdlsn. E. A,; Cohen. 0. H.: Rudikoff, S.; Potter M.: Daviea, D. R. !%c. Nntl. Arnd Sti. l l S A 191C 71.429R4302. ~~~38. Po1jak.R. J.;Am~l,L.M.;Avey.H.P.;Chen,B.L.;Phi~aekerley.R.P.;Saul.P.Pmc. N d . Aced. Sci. USA 1973.70.3305-3310. 39. Blake.C.C.F.;Koenig,D.F.;Msir,G.A.:North,A. C.T.:Phillips,D.C.;Sanoa,V.R. Nature 1966. ZGS, 757-7-761. 40. Smith, T. J.; Kremer, M. J.; Luo. M.; Vriend. 0.: Arnold, E.; Kamer, G.; Rassman, M. G.; MeKinlay, M. A,: Diana, G. D.: Otto, M. J. Science 1986.233,12%-1288. 41. Cddinrr P. W.:. Mair. A. K. S. MolsculorPharmocol. 1985.25. 176184. -~ 42 n.,...w ~..c..rn..~F J . ~ t . ~ ~ . o n . . . : h ~I Wm. Y ~ . . : ~ 180 43. h n b , D A.:Tri#dc, D. J. Molrr~lorPhormorr . 19RS,d7.541-!I*. 44 CIIIPII. H I.. C;.u~ker,.I P . CUYCTV.D F ,tktz010, F H K h n w (. H. J. Am Cnrm Soc 1978. IM.4282 4289 ~
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