Divergent Behavior of Glycosylated Threonine and Serine Derivatives in Solid Phase Peptide Synthesis

Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing conditions, glycosylated threonine analogs undergo β-elimination, rather than epimerization. Mechanistic studies and molecular modeling were used to understand the origin of the differences in reactivity

Competition between Serum IgG, IgM, and IgA Anti-Glycan Antibodies

<div><p>Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies.</p></div

Dependence of inhibition on carbohydrate antigen structure.

<p>Measured IgG signals to Forssman disaccharide and tetrasaccharide (A) and four blood group A antigens (B) in the absence of added IgM (0 μg/mL) or in the presence of varying amounts of IgM (50–400 μg/mL).</p

Competition between serum IgG, IgA, and IgM anti-glycan antibodies.

<p>(A) Addition of IgM and IgA to IgG. Polyclonal IgG isolated from serum was first profiled on the array alone. Separately, IgG was premixed with varying amounts of IgM or IgA and then profiled on the array. For each array component, the change in IgG signal in the presence of IgM or IgA was determined. The box plots depict the range of IgG changes (on a log base 2 scale) measured on the array upon addition of 4 serum equivalents of IgM or IgA. The line in the middle of the box is the median, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. (B) Addition of IgG and IgA to IgM. An analogous protocol as above was used to evaluate effects of IgG and IgA on IgM signals. (C) Addition of IgM and IgG to IgA. An analogous protocol as above was used to evaluate effects of IgG and IgM on IgA signals. The box plots demonstrate significant decreases in IgG and IgA signals in the presence of IgM for the vast majority of array components.</p

Changes in serum IgG and IgM signals in a pooled serum sample in the presence of varying amounts of added IgG, IgA, or IgM.

<p>(A) Changes in IgG signals upon addition of IgM. (B) Changes in IgG signals upon addition of IgA. (C) Changes in IgM signals upon addition of IgG. (D) Changes in IgM signals upon addition of IgA. IgG and IgM signals in a pooled serum sample were first profiled on the array alone. Separately, the serum sample was pre-mixed with varying amounts of IgG, IgA, or IgM and then profiled on the array. The box plots depict the range of IgG or IgM signals (on a log base 2 scale) alone or in the presence of varying amounts of IgG, IgA, or IgM. The line in the middle of the box is the median signal, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. The only significant decreases observed are for IgG signals upon addition of IgM.</p

Enhanced Epimerization of Glycosylated Amino Acids During Solid-Phase Peptide Synthesis

Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare <i>O</i>-linked glycopeptides result in substantial amounts of epimerization at the α position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to nonglycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens