Chemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras.

Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.

Behavior and Fos activation reveal that male and female rats differentially assess affective valence during CTA learning and expression.

Females are more affected by psychiatric illnesses including eating disorders, depression, and post-traumatic stress disorder than males. However, the neural mechanisms mediating these sex differences are poorly understood. Animal models can be useful in exploring such neural mechanisms. Conditioned taste aversion (CTA) is a behavioral task that assesses how animals process the competition between associated reinforcing and aversive stimuli in subsequent task performance, a process critical to healthy behavior in many domains. The purpose of the present study was to identify sex differences in this behavior and associated neural responses. We hypothesized that females would value the rewarding stimulus (Boost®) relative to the aversive stimulus (LiCl) more than males in performing CTA. We evaluated behavior (Boost® intake, LiCl-induced behaviors, ultrasonic vocalizations (USVs), CTA performance) and Fos activation in relevant brain regions after the acute stimuli [acute Boost® (AB), acute LiCl (AL)] and the context-only task control (COT), Boost® only task (BOT) and Boost®-LiCl task (BLT). Acutely, females drank more Boost® than males but showed similar aversive behaviors after LiCl. Females and males performed CTA similarly. Both sexes produced 55 kHz USVs anticipating BOT and inhibited these calls in the BLT. However, more females emitted both 22 kHz and 55 kHz USVs in the BLT than males: the latter correlated with less CTA. Estrous cycle stage also influenced 55 kHz USVs. Fos responses were similar in males and females after AB or AL. Females engaged the gustatory cortex and ventral tegmental area (VTA) more than males during the BOT and males engaged the amygdala more than females in both the BOT and BLT. Network analysis of correlated Fos responses across brain regions identified two unique networks characterizing the BOT and BLT, in both of which the VTA played a central role. In situ hybridization with RNAscope identified a population of D1-receptor expressing cells in the CeA that responded to Boost® and D2 receptor-expressing cells that responded to LiCl. The present study suggests that males and females differentially process the affective valence of a stimulus to produce the same goal-directed behavior.

Deciphering the substrate recognition mechanisms of the heparan sulfate 3-O-sulfotransferase-3.

The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.

Structural and substrate specificity analysis of 3-O-sulfotransferase isoform 5 to synthesize heparan sulfate.

Heparan sulfate 3-O-sulfotransferase (3-OST) transfers a sulfo group to the 3-OH position of a glucosamine saccharide unit to form 3-O-sulfated heparan sulfate. 3-O-sulfation is known to be critically important for bestowing anticoagulant activity and other biological functions of heparan sulfate. Here, we report two ternary crystal structures of 3-OST-5 with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates. We also used 3-OST-5 to synthesize six 3-O-sulfated 8-mers. Results from the structural analysis of the six 3-O-sulfated 8-mers revealed the substrate specificity of 3-OST-5. The enzyme prefers to sulfate a 6-O-sulfo glucosamine saccharide that is surrounded by glucuronic acid over a 6-O-sulfo glucosamine saccharide that is surrounded by 2-O-sulfated iduronic acid. 3-OST-5 modified 8-mers display a broad range of anti-factor Xa activity, depending on the structure of the 8-mer. We also discovered that the substrate specificity of 3-OST-5 is not governed solely by the side chains from amino acid residues in the active site. The conformational flexibility of the 2-O-sulfated iduronic acid in the saccharide substrates also contributes to the substrate specificity. These findings advance our understanding for how to control the biosynthesis of 3-O-sulfated heparan sulfate with desired biological activities.

Palladium Catalyzed Arylation and Benzylation of Nitroarenes Using Aryl Sulfonates and Benzyl Acetates.

Pd-catalyzed arylation or benzylation of nitroazoles using aryl sulfonates or benzyl acetates is described. Electronically varied aryl tosylates and mesylates, as well as benzyl acetates, afford the arylated and benzylated products. Arylation of nitrobenzene is also reported. The relative rate for the arylation of halides is greater than that of tosylates using the reported reaction parameters. These studies enhance the scope of electrophiles for nitroarene arylations and benzylations, which was hitherto limited to the use of halide electrophiles.