Takeshi Tsumuraya

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of α-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Concise synthesis of ciguatoxin ABC-ring fragments and surface plasmon resonance study of the interaction of their BSA conjugates with monoclonal antibodies.

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

Cyclotrigermanes. Synthesis and thermal decomposition

Abstract The reactions of dihalogenogermanes R 2 GeX 2 (X = halogen) with Mg and MgBr 2 in tetrahydrofuran produce the corresponding cyclotrigermanes (R 2 Ge) 3 , and cyclotetragermanes (R 2 Ge) 4 ; the preferred ring size of the products depends on the substituents at the germanium atom. In the thermolysis of hexamesitylcyclotrigermane ( 2 ), dimesitylgermylene ( 11 ) and tetramesityldigermene ( 8 ) are generated. While 11 can be trapped directly with 2,3-dimethyl-1,3-butadiene to give dimesitylger macyclopent-3-ene ( 9 ), 8 rearranges to mesityl(trimesityl)germylene ( 12 ), which leads to the formation of mesityl(trimesitylgermyl)germacyclopent-3-ene ( 10 ). Nevertheless, in the thermolysis of 2 with paraformaldehyde, thiobenzophenone, or phenylacetylene as trapping reagents the expected cyclo-adducts of tetramesityldigermene ( 8 ) are formed. Furthermore when hexamesitylcyclotrigermane ( 2 ) is reacted with acetylene in the presence of catalytic amounts of palladium complexes, a C-unsubstituted germole 21 is obtained in good y

Synthesis of germathiiranes

Thermolysis of 7,7-diphenyl-7-germanorbornadiene with adamantanethioneproduced germathiirane which reacted with ethanol to give germylmercaptan 3. Dimesitylgermylene induced photochemically reacted with adamantanethione to give a crystalline germathiirane 5 characterized by spectrum data. Digermathiirane 6 and digermaepiselenide 7 were also synthesized by the photolysis of hexamesitylcyclotrigermane with S8 and Se.

Diverse structural solutions to catalysis in a family of antibodies.

BACKGROUNDnSmall organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation.nnnRESULTSnThe structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ.nnnCONCLUSIONSnOur results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.

Reaction of germylene with thioketenes: Synthesis of alkylidenedigermathietanes

Abstract In the reaction of dimethyl- or diphenylgermylene with di-tert-butylthioketene alkylidenedigermathietanes were obtained, probably via alkylidenegermathiiranes. The exact structure of alkylidenedigermathietane 2b was confirmed by X-ray crystal analysis.

Germathiirane intermediate from the reaction of germylene with thioketones

Abstract Digermadithiolane was obtained from the reaction of dimethylgermylene and thioketones via germathiirane intermediate. In the gas phase reaction digermathietane was obtained, probably by the [2+2] cycloaddition of tetramethyldigermene with thioketone.

Reductive coupling reactions of dihalogenogermanes with magnesium and magnesium bromide: simple preparation of cyclotrigermanes and cyclotetragermanes

The reactions of various dihalogenogermanes with Mg and MgBr2 in tetrahydrofuran produce the corresponding cyclotrigermanes and cyclotetragermanes; the ring size of the products depends on the steric bulk of substituents on germanium.