`
`7433
`
`most reasonably attributed to a rotational isotope effect, I I is
`consistent with concert. For the isotope effect to be consistent
`with the stereospecific biradical pathway, closure to the five(cid:173)
`membered ring must be rate-determining. However, it is hard
`to reconcile the demand of slow closure to a five-membered ring
`relative to closure to a three-membered ring with the demand of
`rapid closure to a five-membered ring relative to bond rotation
`in the diradical. Thus, the 1,3-shift would appear to be concerted.
`
`Admowledgment. We thank the National Science Foundation
`for financial support, Prof. Christopher Samuel (U. Warwick),
`who provided the initial NMR data and spectral analysis of the
`product epoxide to warrant pursuit of this work, and Prof. Kevin
`Gilbert for his continued interest in this problem.
`
`Supplementary Material Available: Experimental details for
`the synthesis of 1 and NMR spectra of the methylene protons of
`the undeuterated epoxide, the deuterated epoxide from the three
`different reaction times, and simulations of mixtures (9 pages).
`Ordering information is given on any current masthead page.
`
`(11) Dai, S. H.: Dolbicr, J. J. Am. Chern. Soc. 1970, 92, 1774: 1971,94,
`3946. Crawford, R. J.; Chang, M. H. Tetrahedron 1982, 38,837. Gajewski,
`J. J.; Benner, C. W.; Stahly, B. N.; Hall, R. F.; Sato, R. I. Tetrahedron 1982,
`38.853.
`
`Atomic Structure of the Rapamycin Human
`Immunophilin FKBP-12 Complex
`
`Gregory D. Van Duyne,t Robert F. Standaert,l
`Stuart L. Schreiber, •. 1 and Jon Clardy··t
`
`Department of Chemistry, Cornell University
`Ithaca, New York 14853
`Department of Chemistry, Harvard University
`Cambridge, Massachusetts 02138
`Received June 3, 1991
`
`Complexes of immunophilins with immunosuppressive drugs
`interfere with a variety of signal transduction pathways in the
`cytoplasm of the cell. 1-3 Rapamycin4 (1) is a high affinity ligand
`(Kd = 0.2 nM)2 to the immunophilin FKBP-125-7 and appears
`to be a general and potent antiproliferative agent. 1 The pleiotropic
`actions of rapamycin on growth factor receptor signaling pathways
`have elevated this compound to a high status as a probe of sig·
`naling mechanisms. Although the precise details have yet to be
`elucidated, the complex of human FKBP-12 and rapamycin has
`been shown by genetic methods to function as the inhibitory agent. 8
`Herein we report the three-dimensional structure of the complex
`of human FKBP-12 and rapamycin, determined to 1.7-A resolution
`
`t Cornell University.
`I Harvard University.
`(I) Schreiber, S. L. Science 1991, 25J, 283.
`(2) Bierer, B. E.; MatHa, P. S.; Standaert, R. F.; Herzenberg, L. A.;
`Burakoff, S. 1.; Crabtree. G.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A.
`1990.87.9231-9235.
`(3) Bierer, B. E.; Somers, P. K.; Wandless, T. J.; Burakoff, S. J.; Schreiber,
`S. L. Science 1990, 250, 556-559.
`(4) Findlay, J. A.; Radics, L. Can. J. Chem. 1980,58,579.
`(5) The family of proteins that bind to FK506 and rapamycin have been
`collcctively named FKBP's (FKS06 binding proteins), with a suffix designating
`the approximate molecular weight. Human FKBP-12, the most abundant
`FKBP found in the cytoplasm. is a 12 kD protein of 107 amino acids that binds
`FK506 and rapamycin and catalyzes the cis-trans isomerization of pepti.
`dyl-prolyl amide bonds in peptide substrates.
`(6) Harding, M. W.: Galat, A.; Uehling, D. E.; Schreiber, S. L. Nature
`1989, 34J, 758.
`(7) Siekierka, J. J.; Hung, S. H. Y.; Poe, M.; Lin, C. S.; Sigal, N. H.
`Natule 1989, 34J, 755.
`(8) Kollin, Y.; Faucette, L.; Bergsma, D. J.; Levy, M. A.: Cafferkey, R.;
`Koser, P. L.: Johnson, R. K.; Livi, G. P. Mol. Cell. Bioi. 1991, II, 1718.
`
`~)~
`~.
`lIe~'"
`~ /}.;.~
`Phe~ ~6
`
`Figure 1. (a, top) A stereoview of the a-carbon tracing of FKBp·12 and
`rapamycin. The N- and C-terminal ",-carbons are labeled. (b, bottom)
`A stereodrawing of the binding pocket showing all of the bound rapa(cid:173)
`mycin molecule and selected FKBP·12 residues.
`
`by X-ray crystallographic techniques.9 This structure provides
`a framework to interpret the effects of structural perturbation of
`either rapamycin or human FKBP-12 on signal transduction
`pathways.
`
`2 . FKS06
`1 • Rapamycin
`As shown in Figure I, the protein component of the FKBP-
`12/rapamycin complex forms a five-stranded anti parallel p-sheet
`
`(9) Crystals of the FKBP-12/rapamycin complex were grown from solu(cid:173)
`tions containing 10 mg/mL of protein complex, 300 mM ammonium sulfate,
`and 100 mM phosphate at pH 6.0 using the hanging drop' method at room
`temperature. The space group is P212121 with 0= 45.42 A, b = 49.16 A, c
`= 54.74 A, and one molecule in the asymmetric unit. Data were measured
`using a SanDiego Multiwire Systems Mark II detector and a rotating anode
`source to 1.7-A resolution. A total of81484 reflections were measured (12991
`unique, 93% complete, RI)'"' .0.056, 10633 with F <!: 311) from two crystals.
`The structure was sOlvCCl using the molecular replacement method with a
`search model composed of the protein comroonent of the FKBP-12/FK506
`complex and the MERLOT program system. 0 The structure was refined with
`X-PLOR II using least·squares minimization by conjugate gradients where the
`stereochemical restraints used in ligand refinement were restricted to terms
`for bond lengths, bond angles, and improper dihedral angles (for planar sp2
`carbons and chiral centers). The conformation of bound rapamycin was
`determined unambiguously from well-defined electron density in 2F.-F, maps.
`The R factor for the current model, including FKBP-12, rapamycin, and 85
`water molecules, is 0.165. All main chain atoms, all buried side-chain atoms,
`and all ligand atoms are well·defined in the final 2Fo - F, electron density
`map. The root-mean-square deviations of bond lengths and bond angles from
`their ideal values are 0.01 A and 2.8°, respectively.
`(10) Filzgerald, P. M. D. I. AppJ. CryS/aliogr. 1988,21,273.
`(11) Branger, A. T. X-PLOR Manual, Version 2.1: Yale University, New
`Haven, 1990.
`
`0002-7863/91/ I 5 13-7433$02,50/0
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`© 1991 American Chemical Society
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`J. Am. Chem. Soc. 1991, 113, 7434-7435
`
`wrapping with a right-handed twist around a short a-helix-the
`same folding topology found in the complex of FKBP-12 with
`FK50612 and in uncomplexed FKBP-12. 1l•14 The root-mean(cid:173)
`square (rms) deviations of a-carbons, backbone atoms, and all
`protein atoms between FKBP-12 complexed with rapamycin and
`FKBP-12 complexed with FK506 are 0.67, 0.67, and 1.51 A,
`respectively. Only one region, involving residues 31-34 in the loop
`between strands 4 and 5 of the !:i-sheet structure, adopts a different
`main chain conformation. These residues are not involved in
`protein-ligand interactions, but may be important recognition
`features of the complex.
`Rapamycin binds in a cavity between the !:i-sheet and a-helix
`with the pipecolinyl ring deeply buried in the protein (Figure la).
`The protein-ligand interface involves atoms from the pyranose
`ring through the C28 hydroxyl, with the remainder, including the
`C17-C22 triene, exposed. The CI ester, the pipecolinyl ring, the
`C8 and C9 carbonyls, and the pyranose ring adopt a conformation
`that is superimposable with the same groups in the FKBP-12/
`FK506 complex;,!2 Three hydrogen bonds between this region and
`FKBP-12 (lie-56 NH to C I carbonyl, Tyr-82 hydroxyl to C8
`carbonyl, and Asp-37 carboxylate to CIO hydroxyl) and a C9
`carbonyl binding pocket involving C-H .. ·O interactions with E(cid:173)
`hydrogens from Tyr-26, Phe-36, and Phe-99 are also identical with
`those found in the complex with FK506, thus confirming the
`identical binding roles of the common structural elements lS in the
`two immunosuppressant ligands.
`Two additional hydrogen bonds are involved in rapamycin
`binding to FKBP-12 (Figure 1 b). The first is from Glu-54 main
`chain carbonyl to C28 hydroxyl, which along with the lie-56 NH
`to C 1 carbonyl-hydrogen bond may mimic the interaction of the
`dipeptide portion of a natural substrate with FKBP-12. It has
`been noted that the pyranose-pipecolinyl region also mimics a
`dipeptide,16 making rapamycin, like FK506, a possible example
`of an extended peptide mimic. This hydrogen bond is analogous
`to the one from Glu-54 main chain carbonyl to C24 hydroxyl found
`in the FKBP-12/FK506 complex. 12 The second hydrogen bond
`is from GIn-53 main chain carbonyl to the C40 hydroxyl. In the
`rapamycin complex the cyclohexyl group (C3S-C42) is bound
`to the protein through this hydrogen bond, while the FK506
`complex has no such cyclohexyl-protein interaction. FKS06's (2)
`C27-C28 double bond restricts the orientations of the cyclohexane
`while in rapamycin (I) the cyc\ohexyl ring can swing about the
`C35-C36 bond to form a GIn-53 carbonyl to C40 hydroxyl hy(cid:173)
`drogen bond.
`The conformation of bound rapamycin is virtually identical with
`that seen in the free, crystalline state,4 with an rms difference of
`0.49 A. Unlike FKS06. which undergoes a cis to trans isomer(cid:173)
`ization of the amide bond accompanied by a dramatic change in
`overall conformation on binding to FKBP-12,12 rapamycin pos(cid:173)
`sesses a high degree of structural preorganization for binding. This
`preorganization, along with the anchoring of the cyclohexyl group,
`may explain the twofold higher affinity (Kd = 0.2 nM) of rapa(cid:173)
`mycin for FKBP-12 compared to FKS06 (Kd = 0.4 nM).2
`The view of the FKBP-12/rapamycin complex as the biological
`effector in immunosuppressive function requires a focus on the
`complex as a whole-in particular the exposed regions of bound
`rapamycin and the FKBP-12 loops flanking the binding site. The
`likely role of FKBP-12 and other FKBP's in the disruption of
`signal transduction in T-cells is to present rapamycin (or FKS06)
`to as yet unknown biological acceptors, or partner proteins. The
`FKBP-12/rapamycin complex described may be best viewed in
`this context as the ligand, now known at atomic resolution, to a
`partner protein involved in cytoplasmic signal transduction.
`
`(12) Van Duyne. G. D.; Standacrt. R. F.; Karplus, P. A.; Schreiber. S. L.;
`Clardy, 1. C. Science 1991, 252, 839.
`(13) Michnick, S. W.; Rosen, M. K.; Wandlcss, T. J.; Karplus, M.;
`Schreiber, S. L. Science 1991, 252, 836.
`(14) Moore, J. M.; Peattie, D. A.; Fitzgibbon, M. J.; Thomson. J. A.
`Natute 1991, 351, 248.
`(I S) Wandless, T. J.; Michnick, S. W.; Rosen, M. K.; Karplus, M.;
`Schreiber, S. L. J. Am. Chem. Soc. 1991, 113,2339.
`(16) Albers, M, W,; Walsh, C. T.; Schreiber, S. L. J. Org. Chem. 1990.
`55,4984.
`
`Acknowledgment. We thank T. J. Stout and P. A. Karplus for
`technical assistance and WaIt Cullen and Jack Oscarson of the
`National Products Discovery and Fermentation Department at
`Pfizer, Inc. for supplying the rapamycin used in this study.
`Support from the National Cancer Institute (CA-24487, J.C.)
`and the National Institute of General Medical Sciences (GM-
`38627, S.L.S.) is gratefully acknowledged. The area detector
`facility was supported by NSF Grant DIR-8820910. Coordinates
`of the bound rapamycin molecule are available from the authors.
`The complete refined coordinates of the FKBP-12/rapamycin
`complex will be deposited in the Brookhaven Protein Databank.
`
`Self-Assembling, Alkali-Metal-Complexing Nickel
`Salicylaldimine Complexes
`Otto F. Schall, Kerry Robinson,t Jerry L. Atwood,t and
`George W. Gokel*
`Departments of Chemistry. University of Miami
`Coral Gables. Florida 33124
`University of Alabama. University. Alabama 35487-0336
`Received May 23. 1991
`
`Nature often achieves biological function in large molecules
`that are shaped and ordered by various feeble forces such as
`hydrogen bonding, salt-bridge formation, 1T-stacking, etc. Wei
`and others2 have been interested in this phenomenon especially
`from the perspective of developing relatively small molecular hosts
`that can assemble, organize, and bind. This phenomenon has two
`manifestations that should be distinguished, however. On the one
`hand, there are those that self-assemble to bind with little
`structural change. 3 On the other, there are hosts such as car(cid:173)
`boxypeptidase A that undergo significant structural change
`) when a guest is bound. 4 A model in the
`("induced-fit system fl
`former category was devised by Reinhoudt et aI., who used a
`macrocyclic salen-polyether-U02 complexes to afford a binding
`site for urea. 5 We now report an unusual nickel salicylaldimine
`system that was thought6 to be in the former category but actually
`forms an unusual bimetallic molecular cage.
`3-Hydroxysalicylaldehyde was converted into a series of 3-
`alkoxy-N-methylsalicylaldimine derivatives as previously de(cid:173)
`scribed.6 The side arms in the 3-position included methyl (CH3,
`1), 2-methoxyethyl (CH2CH20CH), 2), and 2-(2-methoxyeth(cid:173)
`oxy)ethyl (CH2CH 20CH2CH20CH). 3). It is known that such
`aId imine systems react with nickel to form square-planar nickel(II)
`complexes of the NiL2 variety.7 These complexes may undergo
`tetrahedral-square planar equilibria if the system is sterically
`hindered. Some diamagnetic, square-planar complexes further
`associate by forming paramagnetic dimers.8 We isolated the
`complex 12Ni as previously reported.6 The combustion analysis
`and mass spectrum were compatible with the indicated stoi(cid:173)
`chiometry. Assessment of stoichiometry in such cases by vapor
`pressure osmometry (VPO) has been eschewed as the results do
`not always accord with those of cryoscopic studies (see supple(cid:173)
`mentary material).9 Our studies using VPO indicated that 12Ni
`
`t University of Alabama.
`(I) (a) Kim, M.; Gokel, G. W. J. Chem. Soc., Chern. Cornrnun. 1987,
`1686. (b) Medina, J. C.; Li, C.; Bott, S. G.; Atwood, J. L.; Gokel, G. W. J.
`Am. Chem. Soc. 1991, 113, 367. (c) Gokel, G. W.; Medina, J. C.; Li, S.
`Synletl, in press.
`.
`.
`(2) (a) Rebek, J., Jr. Top. Curro Chern. 1988, 149, 189. (b) Shmkal, S.;
`Miyazaki, K.; Manabe, O. J. Chern. Soc., Perkin Trans. 2 1987,449.
`(3) Hamilton, A. Adv. Supramoleculat Chern. 19ge, 1, 1.
`(4) (a) Hartsuck, J. A.; Lipscomb, W. N. Enzymes 1971, 3, 1.
`Quiocho, F. A.; Lipscomb, W. N. Adv. Protein Chem. 1971, 25, 1.
`Lipscomb, W. N. Proc. Natl. Acad. Sci. U.S.A. 1980,77. 3875.
`(5) Van Staveren, C. J.; van Erden. J.; van VeegeJ, C. J. M.; Harkema,
`S.; Reinhoudt, D, N. J. Am. Chern. Soc. 1988. 110,4994-5008.
`(6) Schepartz, A.; McDevitt, J. J. Am. Chern. Soc. 1989, Ill, 5976.
`(7) (a) Holm, R. H.; Everett, G. W., Jr.; Chakravarty, A. Prog. Inorg.
`Chem. 1966. 7. 83. (b) Holm. R. H.; O'Connor, M. J. Prog.lnotg, Chern.
`1971,14,241.
`(8) Holm, R. H, J, Am. Chem. Soc. 1961, 83. 4683.
`(9) (a) Holm, R. H.; Swaminathan, K.lnorg. Chem. 1962,1,599. (b)
`Holm, R. H.; Swaminathan, K. Inorg. Chem. 1963, 2, 181.
`
`(b)
`(c)
`
`0002-7863/91/1513-7434502.50/0
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`© 1991 American Chemical Society
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`NOVARTIS EXHIBIT 2093
`Par v. Novartis, IPR 2016-01479
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