All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi . The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major , the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Gal p α1,3Gal f β and Gal p α1,3Gal f β1,3Man p α glycotopes as diagnostic biomarkers for L. major- caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica- caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies.

The photolytic properties of N-acyl-7-nitroindolines make these compounds attractive as photocleavable protecting groups and "caged" compounds for the light-induced release ("uncaging") of biologically active compounds and as acylating reagents under neutral conditions. However, the synthesis of N-acyl-7-nitroindolines usually requires multiple steps, and the direct acylation of 7-nitroindolines can be quite challenging. 7-Nitroindolines with other types of N-carbonyl-containing groups may also be photoreactive and could potentially be better accessible. Here we demonstrate the short and efficient synthesis of 5-bromo-7nitroindoline-S-thiocarbamates, a new class of photoreactive compounds, and the study of some of their photochemical and photophysical properties. Using 5-bromo-7-nitroindoline-S-ethylthiocarbamate as a model compound, we show that it can undergo one-photon and two-photon photolysis at 350 and 710 nm, respectively. Our experimental data and quantum chemistry calculations support a photolysis pathway that differs from photolysis pathways previously reported for N-acyl-7-nitroindolines. The photolysis with 350 nm light results in 5-bromo-7-nitrosoindoline, which is in equilibrium with its dimeric form(s), as supported by experiment and theory. This study expands the scope of photoreactive 7-nitroindoline derivatives and informs the development of novel photocleavable compounds.