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

Conformational Determinants of the Activity of Antiproliferative Factor Glycopeptide

The antiproliferative factor (APF) involved in interstitial cystitis is a glycosylated nonapeptide (TVPAAVVVA) containing a sialylated core 1 α-O-disaccharide linked to the N-terminal threonine. The chemical structure of APF was deduced using spectroscopic techniques and confirmed using total synthesis. The synthetic APF provided a platform to study amino acid modifications and their effect on APF activity, based on which a structure–activity relationship (SAR) for APF activity was previously proposed. However, this SAR model could not explain the change in activity associated with minor alterations in the peptide sequence. Presented is computational analysis of 14 APF derivatives to identify structural trends from which a more detailed SAR is obtained. The APF activity is found to be dictated by the close interplay between carbohydrate–peptide and peptide–peptide interactions. The former involves hydrogen bond and hydrophobic interactions, and the latter is dominated by hydrophobic interactions. The highly flexible hydrophobic peptide adopts collapsed conformations separated by low energy barriers. APF activity correlates with hydrophobic clustering associated with amino acids 4<b>A</b>, 6<b>V</b>, and 8<b>V</b>. Peptide conformations are highly sensitive to single point mutations, which explain the experimental trends. The presented SAR will act as a guide for lead optimization of more potent APF analogues of potential therapeutic utility

GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins

In 2011, a new type of protein <i>O</i>-glycosylation was discovered in which <i>N</i>-acetylgalactosamine is attached to the side chain of tyrosine (GalNAc-Tyr). While present on dozens of proteins, the biological roles of GalNAc-Tyr are unknown. To gain insight into this new type of modification, we synthesized a group of GalNAc-Tyr glycopeptides, constructed microarrays, and evaluated potential recognition of GalNAc-Tyr by a series of glycan-binding proteins. Through a series of >150 microarray experiments, we assessed binding properties of a variety of plant lectins, monoclonal antibodies, and endogenous lectins. VVL, HPA, and SBA were all found to bind tightly to GalNAc-Tyr, and several Tn binding antibodies and blood group A antibodies were found to cross-react with GalNAc-Tyr. Thus, detection of GalNAc-Tyr modified proteins is an important consideration when analyzing results from these reagents. Additionally, we evaluated potential recognition by two mammalian lectins, human (hMGL) and murine (mMGL-2) macrophage galactose type C-type lectins. Both hMGL and mMGL-2 bound tightly to GalNAc-Tyr determinants. The apparent <i>K</i>_d values (∼1–40 nM) were on par with some of the best known ligands for MGL, such as the Tn antigen. hMGL also bound the natural beta-amyloid peptide containing a GalNAc-Tyr epitope. STD NMR experiments provided structural insights into the molecular basis of recognition. Finally, GalNAc-Tyr was selectively captured by mMGL-2 positive dendritic cells. These results provide the first evidence that GalNAc-Tyr modified proteins and/or peptides may be ligands for hMGL and mMGL-2 and offer unique structures for the design of MGL targeting agents

1D NMR spectra of () T7 and () T7T8 imino regions at 200 μM strand concentration

<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p> Additional peaks that were not observed in 1–2 mM solutions are indicated with arrows

Changes in the chemical shift of the imino protons between the -MCT- and -MCT- modified ODN species and the native DD at 5°C () and at 25°C ()

<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p

NMR spectra of the imino H region of the -MCT modified oligos at various temperatures

<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p> The assignments are as follows: filled diamond T8, filled square T7, filled triangle G2, filled circle G4, star G10 and double dagger, G12

Changes in the chemical shift of select non-exchangeable protons between the -MCT- () and -MCT- () modified ODN species and the native DD at 25°C

<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p

Experimental and Chemoinformatics Study of Tautomerism in a Database of Commercially Available Screening Samples

We investigated how many cases of the same chemical sold as different products (at possibly different prices) occurred in a prototypical large aggregated database and simultaneously tested the tautomerism definitions in the chemoinformatics toolkit CACTVS. We applied the standard CACTVS tautomeric transforms plus a set of recently developed ring–chain transforms to the Aldrich Market Select (AMS) database of 6 million screening samples and building blocks. In 30 000 cases, two or more AMS products were found to be just different tautomeric forms of the same compound. We purchased and analyzed 166 such tautomer pairs and triplets by ¹H and ¹³C NMR to determine whether the CACTVS transforms accurately predicted what is the same “stuff in the bottle”. Essentially all prototropic transforms with examples in the AMS were confirmed. Some of the ring–chain transforms were found to be too “aggressive”, i.e. to equate structures with one another that were different compounds

Glycoamino Acid Analogues of the Thomsen–Friedenreich Tumor-Associated Carbohydrate Antigen: Synthesis and Evaluation of Novel Antiproliferative Factor Glycopeptides

Glycoamino acid analogues of the Thomsen–Friedenreich antigen disaccharide, where the 4′ and 4″ hydroxyl groups were substituted with fluorine or hydrogen, were synthesized and incorporated into the asialylated antiproliferative factor (<i>as</i>-APF), a biologically active form of APF, a glycopeptide found in the urine of patients with interstitial cystitis. Various strategies were employed to incorporate the fluorine atom at the 4-positions of either the galactose or <i>N</i>-acetylgalactosamine unit of the disaccharide antigen, based on stereochemistry and reactivity. These glycopeptides were evaluated in antiproliferative assays on both primary normal bladder epithelial cells and T24 bladder carcinoma cells. Unlike many previously published substitutions to APF, mono-4′-fluorination of the GalNAc residue did not affect the activity, whereas fluoro-derivatives of the galactose 4″-position or both 4′ and 4″ hydroxyls showed a reduced potency relative to the monosubstituted GalNAc derivative. A fourth compound where the 4″ position of galactose was deoxygenated showed a lower potency than the parent and monosubstituted compounds. These results suggest that specific substitutions in the sugar moieties in the APF can be tolerated, and the glycomimetic design of APF analogues can include fluorine in the GalNAc sugar of the disaccharide