Research Terms
This strategy enables the diversity-oriented synthesis of various glycosphingolipids (GSLs). GSLs are a family of glycolipids with a sphingoid or a ceramide as the hydrophobic moiety linked to the downstream end of a carbohydrate chain through a glycosidic bond. GSLs are an essential component of the cell membrane and play a key role in many biological and pathological processes. Aberrant GSL expression and metabolism are associated with diseases such as cancer, diabetes, sclerosis, bacterial and viral infections, and Alzheimer’s disease, so exploring GSLs and their derivatives is popular in medical research. For example, neuroblastoma tumors surround themselves with orders of magnitude higher concentrations of GSLs, providing a target for treatment with monoclonal antibodies. However, the isolation of GSLs from nature is challenging, and it is only possible to obtain them in minor quantities. Chemical or enzymatic synthesis of GSLs is a promising alternative pathway to GSLs. However, these strategies suffer from low yields, high economic cost, poor solubility in water solutions, compatibility problems, and only provide individual structures one by one.
Researchers at the University of Florida have developed a strategy for high-yield synthesis of glycosphingolipids. This strategy combines chemical transformations and stepwise enzymatic elongations, rapidly producing diverse natural and functionalized glycolipids. These functional products can help achieve breakthroughs in research targeting specific cancers and degenerative diseases.
Diversity-oriented synthesis of glycosphingolipids important for diagnostic and biological applications, combining chemical and enzymatic transformations
This three-stage series of chemoenzymatic reactions efficiently synthesizes glycosphingolipids (GSLs). The first step involves the synthesis of a common GSL precursor by binding a protected mono- or disaccharide donor to a short lipid head via glycosylation and deprotection of the donor. The second step assembles a glycan in aqueous media through enzymatic elongation of the sugar chain. The final step comprises chemoselective reactions to construct the ceramide moiety on site. These reactions include metathesis to install the main lipid chain and azide reduction and acylation to install the N-linked lipid chain. All syntheses start or go through the same intermediates, making the process simple and reproducible. Furthermore, the synthesized GSLs may incorporate different lipids or functional groups, enabling a wide range of resulting GSL derivatives that may have valuable applications in drug development and medical and biological research.
This liquid chromatography, tandem mass spectrometry (LC-MS/MS) system utilizes an extensive mass spectrometry database and a two-step searching procedure to identify glycolipids and glycosphingolipids. Glycolipids play an important role in various physiological and pathological processes. More than 80 percent of glycan cells in the central nervous system are glycolipids related to many diseases such as cancer, Alzheimer’s disease, and depression. Identifying glycolipids in experimental samples remains challenging because of their unique chemical and physical properties. Available commercial software designed to identify lipids is not well suited for glycolipid analysis, especially for analysis of unknown glycolipids, since many glycolipids are absent from mass spectrometry libraries.
Researchers at the University of Florida have developed a tandem mass spectrometry system employing a more extensive library/database and a two-step process to search and identify specific glycolipids. The system analyzes the glycolipids and glycosphingolipids in bodily fluids and tissues to help identify biomarkers useful for diagnosis and therapy of diseases such as cancer, Alzheimer’s, or depression.
Improved mass spectrometry database and searching software that better identifies glycolipids and glycosphingolipids in tissue samples in order to develop biomarkers and therapeutic tools
The liquid chromatography, tandem mass spectrometry (LC-MS/MS) software applies a two-step process to analyze specific glycolipids, including glycosphingolipids, using an extensive database. The first step identifies the glycan moiety, and the second step identifies the lipid moiety. The mass spectrometry database will provide the tandem spectra to identify various glycolipids. Once the system obtains the MS/MS spectra of a sample, analysis using the database and new searching procedure identifies the glycolipid composition in terms of both glycan forms and lipid forms. The analytical system identifies glycolipids more efficiently than available commercial software designed for identifying lipids.
This composition of disparate compounds achieves a powerful immunostimulant, boosting the efficacy of a wide variety of vaccines. Vaccines are an invaluable public health measure, crucial for fighting pandemics, child mortality, and cancer. Estimates suggest childhood vaccinations have saved over 100 million lives in the last 50 years. Vaccines are also emerging as weapons against cancer thanks to their ability to stimulate the immune system to attack tumors. The core of a vaccine is the antigen, a derivative of an infectious agent the immune system learns to recognize. However, the vaccine or antigen alone typically does not attract enough attention from the immune system to create a lasting immune response or memory. To overcome this problem, adjuvants, compounds that boost vaccines to induce a strong immune response, are widely applied alongside vaccines to achieve lasting immune memory. But the utility of each adjuvant is often specific to a single vaccine.
Researchers at the University of Florida have developed a new type of vaccine adjuvants by covalently bonding two distinct immunostimulant compounds, monophosphoryl lipid A (MPLA) and either 2,4-dinitrophenyl (DNP) or rhamnose that triggers two different immune response pathways. The concerted effect of these two immunostimulants promises the conjugate adjuvants with robust efficacy and broad applicability to boost vaccines that prevent viral and bacterial infections and treat cancer.
Activates a strong immune response a vaccine to create immune memory to infectious diseases or initiate an immune attack against cancerous cells
Compounds that help vaccines provoke immune responses in humans, known as adjuvants, are of great interest for biomedical applications. Vaccines alone typically do not stimulate the immune system enough by themselves to create a strong and lasting immune response, so adjuvants are necessary to achieve long-term benefits. Cancer immunotherapies are another emerging technology relying on adjuvants to achieve a strong immune response to kill cancer cells.
Monophosphoryl lipid A (MPLA) and 2,4-dinitrophenyl (DNP) are two effective adjuvants. MPLA, being derived from bacteria, activates the antibacterial response pathway through its interaction with the toll-like receptor 4 (TLR4), a protein designed to recognize foreign antigens, including certain bacteria, e.g., Gram-negative bacteria. For its part, DNP can recruit its corresponding endogenous antibodies in the immune system, which in turn bind to the Fc receptors of immune cells to initiate a cytotoxic response to the incursion. The sugar rhamnose can also substitute for DNP to serve the same purpose, as it activates the same antibody-Fc receptor pathway while also providing the additional benefit of combatting tumor, making it a doubly effective ingredient in cancer immunotherapy. As a result, the covalent linkage of DNP or rhamnose with MPLA produces a multiplicative impact on the potency of the conjugate adjuvants.
This method for modifying extracellular vesicles combines enzymatic glycan engineering with chemoselective functionalization to unlock applications such as targeted drug delivery. Extracellular vesicles are lipid bilayer-enclosed nanoparticles secreted by cells, acting as important messengers and substance transporters between cells. However, they must be modified to carry special molecular markers and functional cargos for practical application. Although many different modification methods exist, such as introducing lipophilic molecules to the vesicle’s membrane, they are difficult to control and often inadvertently influence the membrane structure or other properties of extracellular vesicles. Glycans or carbohydrates are prevalent and exposed on the surface of extracellular vesicles, rendering them ideal facilitators for modifying extracellular vesicles.
Researchers at the University of Florida have developed a method using enzymatic reactions to modify the surface glycans of extracellular vesicles. By introducing functionalized sugar residues, they can label carbohydrates on the surface of extracellular vesicles and enable the attachment of various molecules, including affinity and fluorescent tags, ligands, antigens, antibodies, and drugs.
Functionalizing extracellular vesicles for biomedical applications such as immunotherapy and targeted drug delivery
The basic objective of extracellular vesicle engineering is attaching functional groups to the surface of the extracellular vesicles. These functional groups serve diverse purposes such as labeling vesicles with specific tags, ligands, and antibodies for targeted drug delivery or cancer immunotherapy. The glycans coating extracellular vesicles provides a straightforward method to functionalization via enzymatic glycan engineering. The glycans serve as the attachment point for functionalization owing to their abundance and excellent locations on the extracellular vesicle surface and their flexibility as the first link in the functional chain. Accordingly, an unnatural sugar residue with an azide is enzymatically attached to the natural sugar residue, facilitating further modification with various molecules for biological applications. The enzyme that enters this functionalization is a glycosyltransferase, which catalyzes the formation of covalent bonds between sugars, called glycosidic bonds. This enzyme proves especially wide-reaching in its ability to introduce useful unnatural sugar residues into the glycan chains.
