The Synthesis of Blood Group Antigenic A Trisaccharide and Its Biotinylated Derivative

Blood group antigenic A trisaccharide represents the terminal residue of all A blood group antigens and plays a key role in blood cell recognition and blood group compatibility. Herein, we describe the synthesis of the spacered A trisaccharide by means of an assembly scheme that employs in its most complex step the recently proposed glycosyl donor of the 2-azido-2-deoxy-selenogalactoside type, bearing stereocontrolling 3-O-benzoyl and 4,6-O-(di-tert-butylsilylene)-protecting groups. Its application provided efficient and stereoselective formation of the required α-glycosylation product, which was then deprotected and subjected to spacer biotinylation to give both target products, which are in demand for biochemical studies.

Two coexisting pseudo-mirror heteromolecular telomeric G-quadruplexes in opposite loop progressions differentially recognized by a low equivalent of Thioflavin T

The final 3′-terminal residue of the telomeric DNA G-overhang is inherently less precise. Here, we describe how alteration of the last 3′-terminal base affects the mutual recognition between two different G-rich oligomers of human telomeric DNA in the formation of heteromolecular G-quadruplexes (hetero-GQs). Associations between three- and single-repeat fragments of human telomeric DNA, target d(GGGTTAGGGTTAGGG) and probe d(TAGGGT), in Na+ solution yield two coexisting forms of (3 + 1) hybrid hetero-GQs: the kinetically favourable LLP-form (left loop progression) and the thermodynamically controlled RLP-form (right loop progression). However, only the adoption of a single LLP-form has been previously reported between the same probe d(TAGGGT) and a target variant d(GGGTTAGGGTTAGGGT) having one extra 3′-end thymine. Moreover, the flanking base alterations of short G-rich probe variants also significantly affect the loop progressions of hetero-GQs. Although seemingly two pseudo-mirror counter partners, the RLP-form exhibits a preference over the LLP-form to be recognized by a low equivalent of fluorescence dye thioflavin T (ThT). To a greater extent, ThT preferentially binds to RLP hetero-GQ than with the corresponding telomeric DNA duplex context or several other representative unimolecular GQs.

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

<p>The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from <i>Akkermansia (Sp). muciniphila</i> cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This <a>provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being <i>de facto</i> O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite. </a>Here we use methods that combine collision- and electron-based fragmentation to characterize the number of <i>O-</i>glycosites that are present in <i>O-</i>glycopeptides derived from OpeRATOR digestion of four known <i>O-</i>glycoproteins. Our data show that over 50% of <i>O-</i>glycopeptides generated from combined digestion using OpeRATOR and trypsin contain multiple <i>O-</i>glycosites, indicating that collision-based fragmentation alone is not sufficient. Electron-based dissociation methods are necessary to capture the <i>O-</i>glycopeptide diversity present in OpeRATOR digestions. </p>

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

<p>The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from <i>Akkermansia (Sp). muciniphila</i> cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This <a>provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being <i>de facto</i> O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite. </a>Here we use methods that combine collision- and electron-based fragmentation to characterize the number of <i>O-</i>glycosites that are present in <i>O-</i>glycopeptides derived from OpeRATOR digestion of four known <i>O-</i>glycoproteins. Our data show that over 50% of <i>O-</i>glycopeptides generated from combined digestion using OpeRATOR and trypsin contain multiple <i>O-</i>glycosites, indicating that collision-based fragmentation alone is not sufficient. Electron-based dissociation methods are necessary to capture the <i>O-</i>glycopeptide diversity present in OpeRATOR digestions. </p>

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

<p>The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from <i>Akkermansia (Sp). muciniphila</i> cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This <a>provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being <i>de facto</i> O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite. </a>Here we use methods that combine collision- and electron-based fragmentation to characterize the number of <i>O-</i>glycosites that are present in <i>O-</i>glycopeptides derived from OpeRATOR digestion of four known <i>O-</i>glycoproteins. Our data show that over 50% of <i>O-</i>glycopeptides generated from combined digestion using OpeRATOR and trypsin contain multiple <i>O-</i>glycosites, indicating that collision-based fragmentation alone is not sufficient. Electron-based dissociation methods are necessary to capture the <i>O-</i>glycopeptide diversity present in OpeRATOR digestions. </p>