Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.

The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.

Highly ionic conductive solid polymer electrolytes have been prepared by blending polyethylene oxide) (MW 600 000) andpoly(2-vinylpyridine) (MW 200 000) orpoly(4-vinylpyridine) (MW 50 000) and LiC104. All blends were prepared by the solution blending process. Several different blend compositions have been studied and optimum compositions required for preparing solid polymer electrolytes have been determined. The polyethylene oxide) (85% by weight)/poly(2-vinylpyridine) (15% by weightVLiClCh blend at an ethylene oxide/Li+ mole ratio of 10 exhibits an ionic conductivity value of 1.0 X 10-® S cm-' at 25 °C and is an elastomeric material with dimensional stability. Furthermore, this blend exhibits ionic conductivities >3.0 X 10-® S cm-1 at 25 °C over a wide salt concentration range. Several other blends prepared are also elastomeric materials with ionic conductivities ~5.0 X10-8 S cm-1, e.g. polyethylene oxide) (85 % by weight)/ poly(2-vinylpyridine) (15% by weight)/LiC104 at an ethylene oxide to Li+ mole ratio of 6 exhibits a value of 7.0 X 10-8 S cm-1 at 30 °C. Studies indicate that the LiC104 salt compatibilizes the polyethylene oxide) and the poly(2-vinylpyridine) by the simultaneous interaction of the Li+ with the oxygens of the PEO and nitrogen of the pyridyl units. Scanning electron microscopy (SEM) on the internal structure of the blends shows the presence of a two phase microstructure, most likely, stabilized by the emulsifying effect of LiCICh.

Supporting InformationExperimental procedures and characterization data ( 1 H and 13 C NMR, HRMS) for all compounds and their precursors.General Experimental Section. NMR spectra were recorded on an Oxford 300 MHz or 500 MHz NMR spectrometer running Varian VNMR software. Chemical shifts are reported in parts per million (ppm) with reference to internal solvent. Multiplicities are abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint), multiplet (m), and broad (br). High-resolution mass spectra (EI, MALDI and FAB) were provided by California Institute of Technology Mass Spectrometry Facility. Molecular mass calculations were performed with ChemDraw Ultra 9 (Cambridge Scientific). Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254 precoated plates (0.25 mm thickness) with a fluorescent indicator. Visualization was performed using UV and iodine stain. Flash column chromatography was performed using silica gel 60 (230-400 mesh)

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Poly(2,5,8,11,14,17,20,23-octaoxapentacosyI methacrylate)-block-poly(4-vinylpyridine) semicomb polymers were synthesized using anionic living polymerization techniques. The polymers are white, powdery materials and were characterized by 'H and '3C nuclear magnetic resonance spectroscopy and gel-permeation chromatography. The polymers display monomodal molecular weight distributions which are relatively narrow. The block copolymers show microphase separation as indicated by the presence of two glass transition temperatures (Tg), a soft (oxyethylene) phase Tg is observed between -60°C and -45°C and a hard (4-vinylpyridine) phase Tg is observed between 135 "C and 150°C. The soft oxyethylene phase has been doped with LiC10, to obtain ionic conductors with electrical conductivities around 5 .S . cm-' at 25 "C and the hard 4-vinylpyridine phase has been complexed with 7,7,8,8-tetracyano-1,4-quinodimethane (TCNQ, 2,5-cyclohexadiene-t,4-diylidenedimalonitrile) to obtain electronic conductivities around S 1 cm-' at 25 "C. The mixed (electronic and ionic) conductivities are intermediate between the ionic and electronic conductivities. Higher electronic conductivities ( = 10 -5 S . cm-' at 30°C) are obtained for the polycation TCNQ-/TCNQo complexes.

Poly(ethylene oxide) (MW 600,000)/poly(2vinylpyridine) (MW 200,000)/LiClO4 blends have been prepared by the solution blending process. The ionic conductivities of the blends containing lower weight fractions (15, 17.5, 20 and 22.5%) of poly (2vinylpyridine) initially increases as the salt content is increased, reaches a maximum at an ethylene oxide/Li+ mole ratio of 10 and decreases as the salt content is further increased. Blends, which have higher weight fractions of poly(2vinylpyridine) (25 and 35%) display different electric behavior, i.e., the ionic conductivity continously increased as the salt content is increased to an ethylene oxide/Li+ mole ratio of 2. Thermal, 7Li solidstate NMR and semiempirical MNDO molecular orbital studies indicate that this contrasting behavior may be explained by the structure and ratios of the solvates (mixed solvate or homosolvate) of LiClO4 present in the blends. © 1995 John Wiley & Sons, Inc.

New procedures for the synthesis of N -heterocyclic carbenes with with multiple fused rings have been developed utilizing a key ring-closing metathesis step. Rhodium complexes were obtained via the pentafluorophenyl carbene adducts. Solid-state structural behavior of the new carbene ligands were analyzed via X-ray crystallography.N-heterocyclic carbenes (NHCs), first isolated by Arduengo et al. in 1991, have become a well-studied and well-utilized class of ligands in the field of transition metal-catalyzed reactions. 1 Their proficiency in catalysis, variable electronic properties, and ease of translation into complex architectural structures have allowed for the development of unique reactivity and targeted selectivity. [2][3][4][5][6] In the past 20 years, NHCs have been applied as ligands in reactions such as palladium catalyzed cross-coupling, olefin metathesis, asymmetric hydrosilylation and conjugate addition. [7][8][9][10][11][12] Free NHCs have also shown great efficacy as organocatalysts in a variety of reactions. 13,14 Exploration of novel structural motifs has enabled the development of novel applications for NHCs. Serving as a positive feedback loop, as the synthetic and catalytic applications have grown, the array of structural motifs has grown in parallel (Figure 1), illustrating the dynamic interchange between structure and function. 1,15,16 In particular, fused NHC structures are of interest, as it has been shown computationally that the rotational lability of the carbene can greatly influence the behavior and dynamics of the NHC-bound metal complex. 17,18 In this study, the syntheses of fused carbenes 6 and 7 were designed to allow for control of stereochemistry at the backbone of the NHC as well as facile modification of the N-bound arene fragment. A three-carbon chain from the backbone of the NHC to the aryl was chosen to form the cis-fused as well as the trans-fused structures (Figure 2). Independent synthesis of each carbene precursor allowed for separate cis and trans routes, bypassing a need to separate the meso from the racemic forms. Synthesis of the racemate could be easily modified to render the enantioenriched mixtures of the trans species via resolution with inexpensive L-(+)-tartaric acid. 19 Hermann and Blechert have synthesized similar NHC structures. 16,20,21 Carbene 4 was appended to ruthenium and shown to have limited metathesis activity. 16 Unfortunately, the synthesis of 4 yielded a 3:1 mixture of the meso and racemic forms, and only the meso form was isolated for study. 16 The crystal structure of carbene 4, which contains a C 2 linkage, exhibited planarity of the N-bound arene with the heterocycle, thereby rendering the ruthenium species noncanonical with standard ruthenium-based catalysts.rhg@caltech.edu. Supporting Information Available. Crystallographic data for complexes 17 and 18, experimental procedures and NMR data for all new compounds are available. NIH Public Access Author ManuscriptOrganometallics. Author manuscript; available in PMC 2011 September 13. NIH-PA Au...