J. Am. Chem. Soc. 1991, 113, 7433-7434
`
`7433
`
`most reasonably attributed to a rotational isotope effect, 11 is
`consistent with concert. For the isotope effect to be consistent
`with the stereospecific biradical pathway, closure to the five(cid:173)
`membcrcd 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 l,J.shift would appe<1r to be concerted.
`
`Acknowledgment. \Ve 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,
`
`(l I) Dai, S. H.; Dolbier, J. J. Am. Chem. Soc. 1910, 92, 1774; 1972, 9./,
`3946. Crawford, R. J,; Chang, M. H. Tetrohtdron 1981, J8, 837. Gajewski,
`J, J.; Benner, C. W.;Stahly, B. N.; Hall, R. F.; Sato, R. I. Tttrahtdron 1981,
`38, 853.
`
`Atomic Structure of the Rapamycln Human
`Immunophllin FKBP-12 Complex
`
`Gregory D. Van Duyne,1 Robert F. Standaert,•
`Stuart L. Schreiber,•·! and Jon Clardy•·t
`
`Departn1e11t of Chemistry, Cornell University
`Ithaca, New York 14853
`Departn1ent of Chenrlstry, Harvard University
`Cambridge, Massachusetts 02 I 38
`Received June 3. I 99 I
`
`Complexes of immunophilins with immunosuppressive drugs
`interfere with a varietl of signal transduction pathways in the
`cytoplasm of the cell. 1- Rapamycin' (1) is a high affinity ligand
`(K, ~ 0.2 nM) 2 to the immunophilin FKBP-12,_, and appears
`to be a general and potent antiproliferative agent. 1 The p!eiotropic
`actions or 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.'
`Herein we report the three-dimensional structure of the complex
`of human FKBP-12 and rapamycin, detennined lo 1.7-A resolution
`
`t Cornell University.
`I Harvard University.
`(I) Schreiber, S. L. Scltnce 1991, 251, 283.
`(2) Bierer, 8. E.; Malila, P. S.; Standaert, R. F.; Hcrzcnbcrg, L. A.;
`Burakoff, S. J.; Crabtree, G.; Schreiber, S. L. Proc. Noll, Acad, Sci. U.S.A.
`1990, 87, 9231-92)5.
`(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. Chtm. 1980, 58, 519.
`(5} The family of proteins that bind to FK506 and rapamycin have been
`colloclivcly named FKBP's (FK506 binding proteins), with a suffix designating
`the approximate molecular weight. Human FKBP·l2, the most abundant
`FKDP found in the cytoplasm, ls a 12 lcD protein oC 107 amino acids 1ha1 blnds
`FK506 and rapamycin and catalyzes the els-trans lsomcrization of pcpti·
`dyl-prolyl amide bonds In peptide substrates.
`(6) Harding, ?>.f. W.; Oalat, A.; Uehling, D, E.; Schreiber, S. L. Naturt
`1989, U/, 758.
`(7) Sickicrka, J. J.; Hung, S. H. Y.; Poe. M.; Lin, C. S.; Sigal, N. H.
`Nallut 1989, J.//, 755.
`(8) Koltin, Y.; Faucette, L.; ~ergsma, D. J.; Levy, ~f. A.; Caffcrkcy, R.;
`Koser, P. L.; Johnson, R, K.; L1v1, G. P. Mo/. Ct/I. Biol. 1991, //, 1718.
`
`Figure t. (a, top) A slereoview of 1he a·carbon tracing of FKBP-12 and
`rapamycin. The N· and C·terminal a·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.
`
`1 • Rapamydl'l
`2. FKSOS
`As shown in Figure I, the protein component of the FKBP·
`12/rapamycin complex forms a five·stranded antiparallel ~·sheet
`
`(9) Crystals of the FKBP-12/rapamycln 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 P2 12121 with a= 45.42 A, b = 49.16 A, c
`"'54.74 A, and one molecule in the asymmetric unit. Data were measured
`using a SanDiego Multiwirc Systems Mark 11 dctoctor and a rotating anode
`source to l.7·A resolution. A total of 81 484 reflections were measured (12991
`unique, 93% complete, R,yra • 0.056, 10633 with F~ 3a) from two crystals.
`The structure was so[vecl using the molecular rcpla«ment method with a
`search model composed of the protein comroncnt of the FKBP·12/FKS06
`complex and the MERLOT program system. 0 The structure was refined with
`X·PLOR 11 using least-squares minimization by conjugate gradients where the
`stcreochemiea\ restraints used in ligand refinement were restricted 10 terms
`for bond lengths, bond angles, and improper dihedral angles (for planar sp1
`carbons and chiral «nters). The conformation of bound rapamycln was
`dctcrmin«I unambiguously from wcll·dcnnod cloctron density In 2F0-Fc maps.
`The R fac1or for the current model, including FKBP-12, rapamycln, and 85
`water molecules, is 0.165. All main chain atoms, all buried side-chain atoms,
`and all ligand atoms arc wcll·dcfincd in the final 2F0 - F. e\C(tron density
`map. The root-mcan·square dcviatloni of bond lengths and bond angles from
`their ideal values arc 0.01 A and 2.8°, respectively.
`(10) Fitz8crald, P. M. D. J. Appl. Crystollogr. 1988, 21, 273.
`(11) BrOngcr, A. T. X·PLOR Manual, Vtrslon 2.1; Yale University, New
`Haven, 1990.
`
`0002·7863/91/1513·7433S02.SO/O © 1991 American Chemical Society
`Breckenridge Exhibit 1006
`Breckenridge v. Novartis AG
`
`

`
`7434
`
`J. Am. Chem. Soc. 1991, JJJ, 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
`FK506 12 and in uncomplexed FKBP-12."·14 The root-mean(cid:173)
`square (rms) deviations of a-carbons, backbone atoms, and all
`protein atoms between FKBP~I2 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 ,8-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 .B·sheet and a-helix
`with the pipecclinyl ring deeply buried in the protein (Figure la).
`The protein-ligand interface involves atoms from the pyranose
`ring through the C2S hydroxyl, with the remainder, including the
`CJ 7-C22 triene, exposed. The Cl ester, the pipecolinyl ring, the
`CS and C9 carbonyls, and the pyranose ring adopt a conformation
`that is superimposable with the same groups in the FKBP-12/
`FK506 complex. 12 Three hydrogen bonds between this region and
`FKBP-12 (lle-56 NH to CJ carbonyl, Tyr-S2 hydroxyl to CS
`carbonyl, and Asp.J7 carboxylate to CJO hydroxyl) and a C9
`carbonyl binding pocket involving C-tt ... o interactions with f(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 or the common structural elements15 in the
`two immunosuppressant ligands,
`Two additional hydrogen bonds are involved in rapamycin
`binding to FKBP-12 (Figure lb). The first is from Glu-54 main
`chain carbonyl to C28 hydroxyl, which along with the lle-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. 11 The second hydrogen bond
`is from Gln-53 main chain carbonyl to the C40 hydroxyl. In the
`rapamycin complex the cyclohexy! group (C35-C42) is bound
`to the protein through this hydrogen bond, while the FK506
`complex has no such cyclohexyl-protein interaction. FK506's (2)
`C27-C28 double bond restricts the orientations of the cyclohexane
`while in rapamycin (I) the cyclohexyl ring can swing about the
`CJ 5-C36 bond to form a Gln-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 FK506, 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 (K, = 0.2 nM) of rapa(cid:173)
`mycin for FKBP-12 compared to FK506 (K, = 0.4 nM).'
`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 FK506)
`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.; Standaert, R. F.; Karplus, P, A.; Schreiber, S, L.;
`Clardy, J, C. Sdtnrt 199t, 252, 839.
`(13) Mlchnick, S. W.; Rosen, M. K.; Wandless, T. J.; Karplus, M.;
`Schreiber, S. L. Scltnct 1991, 252, 836.
`(14) Moore, J.M.; Peattie, D. A.; Fitzgibbon, M. J.; Thomson, J. A.
`Naturt 1991, JJ/, 248.
`(15) Wandless, T. J.; ~fichnick, S. W.; Rosen, ~f. K.; Karplus, ~L;
`Schreiber, S. L. /. Am. Chtm. Soc. 1991, 11 J, 2339.
`(16) Albers, M. W.; Walsh, C. T.; Schreiber, S. L. /, Org. Chtm. 199(1,
`Jj, 4984.
`
`Acknowledgment. We thank T. J. Stout and P.A. Karplus for
`technical assistance and \Vall 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-244S7, J.C.)
`and the National Institute of General Medical Sciences (GM-
`3S627, S.L.S.) is gratefully acknowledged. The area detector
`facility was supported by NSF Grant DIR-SS209IO. 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/ Jerry L. Atwood,t and
`George \V. Gokct•
`Departn1e11ts of Chenifstry, Uniuersity of Miarni
`Coral Gables. Florida ]JI 24
`Universiry of Alabanta, University, Alaban1a 35487~0336
`Received May 2J, 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, 'lr-Stacking, etc. We 1
`and others2 have been interested in this phenomenon especially
`from the persp«:tive 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·
`boxypeptidase A that undergo significant structural change
`("induced-fit system") when a guest is bound. 4 A model in the
`former category was devised by Reinhoudt et al., 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 arn1s in the 3-position included methyl (CH3,
`I), 2-methoxyethyl (CH1CH20CH 3, 2), and 2-(2·methoxyeth(cid:173)
`oxy)ethyl (CH 1CH20CH 2CH,OCH,, 3). It is known that such
`aldimine systems react with nickel to form square-planar nickel(Il)
`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 din1ers.8
`\Ve isolated the
`complex I2Ni 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~
`mentary material).9 Our studies using VPO indicated that 12Ni
`
`t University of Alabama.
`(I) (a) Kim, f\.L; Gokcl, G. \V. J. Chtm, Soc., Chtm. Commun. 1987,
`1686. (b) ~fc-Oina, J.C.; Li, C.; Bott, S. G.; Atwood, J. L.; Gokel, G. W, J.
`Am. Chun, Soc, 1991, 11 J, 367. (c) Gokel, G. W,; ~fcdina, J. C.; Li, S.
`Synltll, in press.
`.
`,
`(2) (a) Rcbek, J,, Jr. Top. Curr. Chtm. 1988, 149, 189. (b) Shmka1, S.;
`Miyazaki, K.; ~fanabc, 0. J, Chtm. Soc., Perkin Tran.s. 2 1987, 449.
`(3) Hamilton, A. Adv. Supramoltcular Chem. 1990, /, L
`(4) (a) HarlSuck, J, A.; Lipscomb, W, N. Enzymes 1971, 3, 1.
`(b)
`Quiocho, F. A.; Lipscomb, W, N. Adv. Protein Chtm. 1971, 15, I. (c)
`Lipscomb, W. N. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3875.
`(5) Van Staveren, C, J,; van Erden, J.; van Vcegel, C. J, M.; Harkema,
`S.; Reinhoudt, D. N. /.Am. Chem. Soc. 1988, J JO, 4994-5008.
`(6)Schepartz, A.; McDevitt, J. J. Am. Chtm. Soc. 1989, Ill, 5976.
`(7) (a) Holm, R.H.; Everett, 0. W., Jr.; Chakravarty, A. Prog. Jnorg.
`Chem. 1966, 7, 83. (b) Holm, R.H.; O'Connor, ~f. J, Prog. lnorg. Chtm.
`t971, 14, 241.
`(8) Holm, R. H. /,Am. Chtm, Soc. 1961, 83, 4683.
`(9) (a) Holm, R.H.; Swaminathan, K. lnorg. Chem. 1962, /, S99. (b)
`Holm, R.H.; Swaminathan, K. lnorg, Chun. 1963, 2, 18!.
`
`0002-7863/91/1513-7434$02.50/0 © 1991 American Chemical Sccicty