No abstract

The complexes [Fe(CO),{q6-RC6H4C(OX)(C,H,,)}] [X = SiMe,, R = H (2), m-Me(3), p-Me (4), or p-Ph (5); X = Et, R = H (6) orp-Me ( 7 ) ] have been prepared by the novel reaction of tricarbonyl(cyclo-octa-l,5-diene)iron (1) with aryl-lithium reagents in diethyl ether at low temperature, followed by alkylation of the adducts formed with SiMe,CI or Et,OBF,. The oxidation of (2) and (4) with [NH,], [Ce(NO,),] in ethanol solution at low temperature led to dissociation of the Fe(CO), moiety to give 9-aryl-9-trimethylsiloxybicyclo[4.2.1] non-2-ene (aryl = Ph or p-MeC,H,) in high yields. The new compounds were characterized by elemental analyses, i.r., 'H n.m.r., and mass spectra. In addition, the structure of complex (4) was confirmed unambiguously by X-ray diffraction analyses: triclinic, space group PT, with a = 9.689(3), b = 9.801 (2), c = 13.1 85(7) A, CY = 75.20(3), p = 68.1 9(3), y = 64.47(2)", Z = 2; R = 0.036 and R' = 0.026 for 3 166 reflections with I 3 30(/).In recent years, the preparation and characterization of olefinco-ordinated metal carbene complexes have been studied extensively in this laboratory.'--6 We discovered that different cyclopolyene-ligated carbonyliron compounds reacted with aryl-lithium reagents, followed by alkylation of the resulting acylmetalate intermediates with Et,OBF,, to give different products. For instance, reaction of tricarbonyl(cyc1ohexa-1,3diene)-and tricarbonyl(cyc1o-0ctatetraene)-iron gave a series of novel isomerized olefin-metakarbene c o m p l e x e ~, ~' ~ in which the cyclo-olefin ligand was bonded to the 'carbene' carbon atom to form diallyldicarbonyliron complexes without opening the ring, while in the case of tricarbonyl(cycloheptatriene)iron, ring opening occurred to generate novel ring-opened diallyl complexes.6 In an extension of our research, we have chosen tricarbonyl(cyc10-octa-1,5-diene)iron (1) as the starting material, where the olefin ligand is a non-conjugated cyclodiene, in order further to investigate the reaction of different cyclopolyene ligands and to reveal the reaction mechanism. Compound (1) adopts a quite interesting route to give novel (q6-arene)dicarbonyliron complexes which are difficult to prepare by other methods.

No abstract

Crystal habits can be used as indicators of conformational changes in their constituent proteins. As in the conversion of unliganded hemoglobin to the oxygenated form, the addition of a small hapten to a suspension of platy crystals of an unliganded Fab (NC6.8) results in the immediate disintegration of the plates and their replacement with prisms of the ligand-protein complex. Examination of the native and liganded forms by X-ray crystallography reveals that the space groups and protein structures are different. During complexation there are ligand-induced conformational changes both in the antigen combining site (local alterations) and in more distal portions of the molecule (allosteric changes). There is an extension of the light chain (10 A increase in length), a commensurate shortening of the heavy chain (by flexing), and a decrease in the "elbow bend" angle of 31 degrees (184 degrees to 153 degrees). Relative to the variable domains, the constant domain pair moves mainly as a unit in such a way that the carboxyl end of the heavy chain is displaced by 19 A. In an intact antibody this displacement may be relayed as a tug (by tensile forces) on the segment connecting the Fab to the Fc region, perhaps altering the orientations of the constituents responsible for such effector functions as complement activation.

The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use.