Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions.

Conservative mutation of transthyretin's surface residues can predispose an individual to familial amyloidosis by dramatically changing the energetics of misfolding. Senile systemic amyloidosis (SSA), however, cannot be explained in this fashion because wild-type (WT) transthyretin (TTR) misfolds and misassembles into amyloid. Since various modifications of the SH functionality of Cys10 have been reported in humans, we sought to understand the extent to which these modifications alter the stability and amyloidosis of WT TTR as a possible explanation for SSA.

A new strategy for the site-specific modification of proteins in vivo.

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli.

Mechanistic studies of beta-arylsulfotransferase IV.

Sulfotransferases are an important class of enzymes that catalyze the transfer of a sulfuryl group to a hydroxyl or amine moiety on various molecules including small-molecule drugs, steroids, hormones, carbohydrates, and proteins. They have been implicated in a number of disease states but remain poorly understood, complicating the design of specific, small-molecule inhibitors. A linear free-energy analysis in both the forward and reverse directions indicates that the transfer of a sulfuryl group to an aryl hydroxyl group catalyzed by beta-arylsulfotransferase IV likely proceeds by a dissociative (sulfotrioxide-like) mechanism.

Synthesis of sugar arrays in microtiter plate.

1,3-Dipolar cycloadditions between azides and alkynes were exploited to attach oligosaccharides to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface. Synthesis of sugar arrays was performed on a micromolar scale in situ in the microtiter plate. As a model study, the beta-galactosyllipid 5 was displayed on a 4-micromol scale.

Native state hydrogen exchange study of suppressor and pathogenic variants of transthyretin.

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease variants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallographic comparison of the disease-associated variants to the T119M interallelic trans-suppressor.

Saccharide display on microtiter plates.

New insight into the importance of carbohydrates in biological systems underscores the need for rapid synthetic and screening procedures for them. Development of an organic synthesis-compatible linker that would attach saccharides to microtiter plates was therefore undertaken to facilitate research in glycobiology. Galactosyllipids containing small, hydrophobic groups at the anomeric position were screened for noncovalent binding to microtiter plates.

Synthesis of alpha-galactosyl ceramide, a potent immunostimulatory agent.

Alpha-galactosyl ceramide has been identified to be a potent stimulatory agent for a novel immunological process, mediated by CD1 molecules. This paper describes a short and efficient synthesis of alpha-galactosyl ceramide. Starting from commercially available 2-deoxy galactose, a suitable sphingosine derivative was obtained in nine steps and 36% overall yield, which was subsequently glycosylated to give the target molecule.

Inhibition studies of porphobilinogen synthase from Escherichia coli differentiating between the two recognition sites.

Porphobilinogen synthase condenses two molecules of 5-aminolevulinate in an asymmetric way. This unusual transformation requires a selective recognition and differentiation between the substrates ending up in the A site or in the P site of porphobilinogen synthase. Studies of inhibitors based on the key intermediate first postulated by Jordan allowed differentiation of the two recognition sites.

Design of allele-specific protein methyltransferase inhibitors.

Protein arginine methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to arginine side chains in target proteins, regulate transcription, RNA processing, and receptor-mediated signaling. To specifically address the functional role of the individual members of this family, we took a "bump-and-hole" approach and designed a series of N(6)-substituted S-adenosylhomocysteine (SAH) analogues that are targeted toward a yeast protein methyltransferase RMT1. A point mutation was identified (E117G) in Rmt1 that renders the enzyme susceptible to selective inhibition by the SAH analogues.

Dihydroxylation of Polyenes Using Narasaka's Modification of the Upjohn Procedure.

Dihydroxylation of a variety of commercially available polyenes has been investigated using phenylboronic acid, N-methylmorpholine N-oxide (NMO), and osmium tetroxide in anhydrous solvent. The diastereoselectivity of multiple oxidation steps is in some cases affected by the in situ protection of the intermediate ene-diols as phenyboronic esters, affording polyols not available from the standard Upjohn dihydroxylation procedure. A convenient oxidative deprotection of the phenylboronic esters is also described.

Opening of thiiranes: preparation of orthogonal protected 2-thioglyceraldehyde.

Treatment of acrolein diethyl acetal sulfide 8 with methanesulfenyl bromide at low temperature results in an efficient thiirane ring opening to a halo disulfide 9. The bromine in this halo disulfide is easily substituted by silver acetate, sodium azide, sodium iodide, and silver nitrate. Treatment of 9 with tetrabutylammonium acetate yields a novel dehydrohalogenation product 12.

A simple method for the preparation of N-sulfonylsulfilimines from sulfides.

While excellent methods exist for the oxidation of sulfides to sulfoxides R1R2S-->R1R2SO, the azaversion of this atom transfer redox process, i.e., R1R2S-->R1R2S=N-SO2R3, has been less reliable.

Olefin epoxidation with bis(trimethylsilyl) peroxide catalyzed by inorganic oxorhenium derivatives. Controlled release of hydrogen peroxide.

The replacement of organometallic rhenium species (e.g., CH(3)ReO(3)) by less expensive and more readily available inorganic rhenium oxides (e.

Toward automated synthesis of oligosaccharides and glycoproteins.

The discovery of previously unknown functions associated with carbohydrates and the study of their structure-function relations are of current interest in carbohydrate chemistry and biology. Progress in this area is, however, hampered by the lack of convenient and effective tools for the synthesis and analysis of oligosaccharides and glycoconjugates. Development of automated synthesis of such materials is necessary to facilitate research in this field.

Euclidean shape-encoded combinatorial chemical libraries.

A method for the encoding of split/mix combinatorial chemical libraries based on Euclidean shapes is described. The shapes are fashioned from a polymeric matrix designed to swell in common organic solvents while retaining their unique forms, and exhibit good mechanical strength. The lightly crosslinked gel-type polymer was processed into an array of Euclidean forms that serve as encoding elements in the synthesis of combinatorial chemical libraries by using the split/pool methodology.

A soluble polymer-supported triflating reagent: a high-throughput synthetic approach to aryl and enol triflates.

[reaction: see text] The high-yielding synthesis and application of the first example of a polymer-supported reagent for the preparation of trifluoromethanesulfonates (triflates) is described. This new reagent efficiently triflates aryl alcohols and lithium enolates in high yield (>90%). A simple precipitation and filtration to remove the excess reagent and byproduct facilitate purification of the triflate products.