Enzymatic Synthesis of Disialyllacto-N-Tetraose (DSLNT) and Related Human Milk Oligosaccharides Reveals Broad Siglec Recognition to the Atypical Neu5Acα2-6GlcNAc Motif.

Sialic acids (Sias) are ubiquitously expressed on all types of glycans, typically as terminating residues. They usually link to galactose, N-acetylgalactosamine, or other Sia residues, forming ligands of many glycan-binding proteins. An atypical linkage to the C6 of N-acetylglucosamine (GlcNAc) has been identified in human milk oligosaccharides (HMOs, e.g., DSLNT) and tumor-associated glycoconjugates. Herein, we achieved the systematic synthesis of these HMOs in an enzymatic modular manner. The synthetic strategy relies on a novel activity of ST6GalNAc6 for efficient construction of the Neu5Acα2-6GlcNAc linkage, and another 12 specific enzyme modules for sequential HMO assembly. The structures enabled comprehensive exploration into their structure-function relationships using glycan microarray, revealing broad yet distinct recognitions by Siglecs to the atypical Neu5Acα2-6GlcNAc motif. The work provides tools and new insights for functional study and potential applications of Siglecs and HMOs.

Divergent synthesis of amino acid-linked O-GalNAc glycan core structures.

O-GalNAc glycans, also known as mucin-type O-glycans, are primary constituents of mucins on various mucosal sites of the body and also ubiquitously expressed on cell surface and secreted proteins. They have crucial roles in a wide range of physiological and pathological processes, including tumor growth and progression. In addition, altered expression of O-GalNAc glycans is frequently observed during different disease states. Research dedicated to unraveling the structure-function relationships of O-GalNAc glycans has led to the discovery of disease biomarkers and diagnostic tools and the development of O-glycopeptide-based cancer vaccines. Many of these efforts require amino acid-linked O-GalNAc core structures as building blocks to assemble complex O-glycans and glycopeptides. There are eight core structures (cores one to eight), from which all mucin-type O-glycans are derived. In this protocol, we describe the first divergent synthesis of all eight cores from a versatile precursor in practical scales. The protocol involves (i) chemical synthesis of the orthogonally protected precursor (3 days) from commercially available materials, (ii) chemical synthesis of five unique glycosyl donors (1-2 days for each donor) and (iii) selective deprotection of the precursor and assembly of the eight cores (2-4 days for each core). The procedure can be adopted to prepare O-GalNAc cores linked to serine, threonine and tyrosine, which can then be utilized directly for solid-phase glycopeptide synthesis or chemoenzymatic synthesis of complex O-glycans. The procedure empowers researchers with fundamental organic chemistry skills to prepare gram scales of any desired O-GalNAc core(s) or all eight cores concurrently.

Design and application of near-infrared fluorophore based on a novel thiazolidinedione-functionalized dicyanoisophorone.

A novel dicyanoisophorone (DCI)-based NIR fluorophore employing 2, 4-thiazolidinediones as the modification site was designed for fluorescence imaging. The fluorophore was assessed as a switchable reporter for H2O2 and the probe exhibited lysosomes-targeted, a large turn-on fluorescence signal at 720 nm with a large stokes shift (150 nm) and can be used in biological systems. The ability of the novel fluorophore to emit NIR fluorescence through a "turn-on" activation mechanism makes it a promising fluorophore for in vivo imaging applications. The strategy of introducing the thiazolidinediones with the easy modification site into the fluorophore has a good application prospect to expand the application of the NIR fluorophore.

Establishment of a Customizable Fluorescent Probe Platform for the Organelle-Targeted Bioactive Species Detection.

A customizable fluorescent probe platform that can be used to detect various bioactive analytes offers significant potential for engineering a wide range of bioprobes with diverse sensing and imaging functions. Here, we show a facile and innovative strategy for introducing cis-amino-proline as a carrier scaffold, which is appended with three specific functional groups: a target group, a water-soluble group, and fluorophores with triggers. The potency of the designed strategy could be customized to generate variable multifunctional fluorescent probes for detecting bioactive species of interest, including reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive sulfur species (RSS), ROS/RSS, and even enzymes. We designed and synthesized five representative water-soluble and organelle-targeted compounds, PMB, PMN, PMD, PRB, and PME, with emission wavelengths of these fluorescent probes varying from blue to red (465, 480, 535, 550, 565, and 640 nm). This strategy could be exemplified by its application to develop a mitochondria-/lysosome-targeting multifunctional fluorescent probe capable of imaging bioactive species of interest in live cells and nude mice.

AIEgens Conjugation Improves the Photothermal Efficacy and Near-Infrared Imaging of Heptamethine Cyanine IR-780.

Near-infrared (NIR) fluorescent probes can deeply penetrate through tissues with little damage. To facilitate image-guided theranostics, researchers usually apply a desired amount of photosensitizers to achieve effective photothermal responses. However, these probes could easily suffer from low photostability and aggregated-caused quenching effect in high concentrations. In this paper, the rational incorporation of an aggregated-induced emission (AIE) unit into the structure of heptamethine cyanine IR-780 is reported. Using tetraphenylethene (TPE) as an AIE core, we synthesize three TPE-modified IR-780 probes (IR-780 AIEgens) via different linkages. The IR-780 derivatives all show enhanced AIE features, in which the probe with an ether linkage (IR780-O-TPE) is superior in rapid cell uptake, high targeting capacity, and good photostability. Moreover, IR780-O-TPE exhibits the strongest cytotoxicity to HeLa cells (IC50 = 3.3 µM). The three IR-780 derivatives displayed a photothermal response in a concentration-dependent manner, in which IR-780 AIEgens are more cytotoxic than IR-780, with IC50 of 0.3 µM under 808 nm laser irradiation. In tumor-bearing mice, the optimal probe IR780-O-TPE also showed a more effective photothermal response than IR-780. By illustrating the relationship between aggregation state with photophysical properties, cell imaging, and cytotoxicity, this work is helpful in modulating NIR-based photosensitizers into AIE features for efficient image-guided theranostics.

A highly selective and sensitive fluorescent probe for simultaneously distinguishing and sequentially detecting H2S and various thiol species in solution and in live cells.

A novel dual-channel fluorescent probe (NCR) based on differences in reactivity among H2S, Cys/Hcy, and GSH was rationally designed for simultaneously distinguishing and sequentially sensing H2S, Cys/Hcy, and GSH using two emission channels, which also demonstrated that NCR can be used for targeting mitochondria in mammalian cells.