Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are (D2O)k = 0.20 in 200 mM HCl, (D2O)k = 0.81 in 50 mM HCl, and (D2O)k = 4.2 in pure water. The formyl-H KIEs are (D)k = 0.80 in 200 mM HCl, (D)k = 0.77 in 50 mM HCl, (D)k = 0.75 in pure water, (D)k = 0.88 in 50 mM NaOH, and (D)(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of (18)O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be (18)k = 0.9917 under neutral conditions, (18)k = 1.0290 (water nucleophile) or (18)k = 0.989 (hydroxide nucleophile) under alkaline conditions, and (18)(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented.
Carbohydrate
binding proteins (CBPs) are attractive targets in
medicine and biology. Multivalency, with several glycans binding to
several binding pockets in the CBP, is important for high-affinity
interactions. Herein, we describe a novel platform for design of multivalent
carbohydrate cluster ligands by directed evolution, in which serum-stable
2′-fluoro modified RNA (F-RNA) backbones evolve to present
the glycan in optimal clusters. We have validated this method by the
selection of oligomannose (Man9) glycan clusters from a
sequence pool of ∼1013 that bind to broadly neutralizing
HIV antibody 2G12 with 13 to 36 nM affinities.
The high mannose
patch (HMP) of the HIV envelope protein (Env)
is the structure most frequently targeted by broadly neutralizing
antibodies; therefore, many researchers have attempted to use mimics
of this region as a vaccine immunogen. In our previous efforts, vaccinating
rabbits with evolved HMP mimic glycopeptides containing Man9 resulted in an overall antibody response targeting the glycan core
and linker rather than the full glycan or Manα1→2Man
tips of Man9 glycans. A possible reason could be processing
of our immunogen by host serum mannosidases. We sought to test whether
more prolonged dosing could increase the antibody response to intact
glycans, possibly by increasing the availability of intact Man9 to germinal centers. Here, we describe a study investigating
the impact of immunization regimen on antibody response by testing
immunogen delivery through bolus, an exponential series of mini doses,
or a continuously infusing mini-osmotic pump. Our results indicate
that, with our glycopeptide immunogens, standard bolus immunization
elicited the strongest HIV Env-binding antibody response, even though
higher overall titers to the glycopeptide were elicited by the exponential
and pump regimens. Antibody selectivity for intact glycan was, if
anything, slightly better in the bolus-immunized animals.
The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, (13)k(obs) = 1.0312, (18)k(obs) = 0.997, and (34)k(obs) = 0.995; for neutral conditions, (13)k(obs) = 1.022, (18)k(obs) = 1.010, and (34)k(obs) = 0.996; and for alkaline conditions, (13)k(obs) = 1.0263, (18)k(obs) = 0.992, and (34)k(obs) = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure.
Oligomannose glycans are of interest as HIV vaccine components, but they are subject to mannosidase degradation in vivo. Herein, we report the synthesis of oligosaccharides containing a thio linkage at the nonreducing end. A thio-linked dimannose donor participates in highly stereoselective glycosylations to afford trimannose and tetramannose fragments. Saturation transfer difference nuclear magnetic resonance (STD NMR) studies show that these glycans are recognized by HIV antibody 2G12, and we confirm that the reducing terminal S-linkage confers complete stability against x. manihotis mannosidase.
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