Going Native: Synthesis of Glycoproteins and Glycopeptides via Native Linkages To Study Glycan-Specific Roles in the Immune System

Glycosylation plays a myriad of roles in the immune system: Certain glycans can interact with specific immune receptors to kickstart a pro-inflammatory response, whereas other glycans can do precisely the opposite and ameliorate the immune response. Specific glycans and glycoforms can themselves become the targets of the adaptive immune system, leading to potent antiglycan responses that can lead to the killing of altered self- or pathogenic species. This hydra-like set of roles glycans play is of particular importance in cancer immunity, where it influences the anticancer immune response, likely playing pivotal roles in tumor survival or clearance. The complexity of carbohydrate biology requires synthetic access to glycoproteins and glycopeptides that harbor homogeneous glycans allowing the probing of these systems with high precision. One particular complicating factor in this is that these synthetic structures are required to be as close to the native structures as possible, as non-native linkages can themselves elicit immune responses. In this Review, we discuss examples and current strategies for the synthesis of natively linked single glycoforms of peptides and proteins that have enabled researchers to gain new insights into glycoimmunology, with a particular focus on the application of these reagents in cancer immunology.Bio-organic Synthesi

6-Deoxyhexoses froml-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology

In the search for alternative non‐metabolizable inducers in the l ‐rhamnose promoter system, the synthesis of fifteen 6‐deoxyhexoses from l ‐rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3‐acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6‐deoxy‐d ‐allose, 6‐deoxy‐d ‐gulose and 6‐deoxy‐l ‐talose. Highly crystalline 3,5‐benzylidene rhamnonolactone gives easy access to l ‐quinovose (6‐deoxy‐l ‐glucose), l ‐olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2‐deoxy‐2‐fluoro‐l ‐rhamnose and 2‐deoxy‐2‐fluoro‐l ‐quinovose. Biotechnology provides access to 6‐deoxy‐l ‐altrose and 1‐deoxy‐l ‐fructose

Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications

Synthesis of asparagine derivatives harboring a Lewis X type DC-SIGN ligand and evaluation of their impact on immunomodulation in multiple sclerosis

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post‐translational N‐glycosylation of Asn31 of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis‐type glycan structures in the N‐glycan of MOG with the DC‐SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (LeX)‐containing Fmoc‐SPPS‐compatible asparagine building block (SPPS=solid‐phase peptide synthesis), as well as asparagine building blocks containing two LeX‐derived oligosaccharides: LacNAc and Fucα1‐3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG31‐55) and analyzed with respect to their ability to bind to DC‐SIGN in different biological setups, as well as their ability to inhibit the citrullination‐induced aggregation of MOG31‐55. Finally, a cytokine secretion assay was carried out on human monocyte‐derived DCs, which showed the ability of the neoglycopeptide decorated with a single LeX to alter the balance of pro‐ and anti‐inflammatory cytokines, inducing a tolerogenic response.Bio-organic Synthesi

Conditionally controlling human TLR2 activity via Trans-Cyclooctene Caged Ligands

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.Bio-organic Synthesi

Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-dihydroxypyrrolidines: Rhamnulose iminosugar mimics inhibit α-glucosidase

The key step in the syntheses of highly substituted trans-3,4-dihydroxypyrrolidines is introduction of bromide by stereospecific and regiospecific Hanessian-Hullar reactions; benzylidene lactones of l-rhamnonolactone and 6-deoxy-l-gulonolactone allow introduction of N at C2 with inversion or retention of configuration. Initially a protecting group, the benzylidene acetal then provides a bromide at C5 to allow formation of the pyrrolidine ring. With silyl protecting groups, bromide was introduced at C5 with inversion of configuration whereas benzoyl protection gave a mixture of retention and inversion, indicative of neighbouring group participation in a Hanessian-Hullar reaction. Four stereoisomeric pyrrolidines - iminosugar mimics of α- and β-l-rhamnulose and α- and β-6-deoxy-d-sorbose were prepared. Only the α-l-rhamnulose mimic showed moderate inhibition of rhamnosidase but some were good inhibitors of α-glucosidases; none inhibited rhamnose isomerase and they had a small effect as synthetic inducers of the rhamnose catabolic operon in E. coli

Efficient synthesis and enzymatic extension of an N-GlcNAz asparagine building block

N-Azidoacetyl-D-glucosamine (GlcNAz) is a particularly useful tool in chemical biology as the azide is a metabolically stable yet accessible handle within biological systems. Herein, we report a practical synthesis of FmocAsn(N-Ac3GlcNAz)OH, a building block for solid phase peptide synthesis (SPPS). Protecting group manipulations are minimised by taking advantage of the inherent chemoselectivity of phosphine-mediated azide reduction, and the resulting glycosyl amine is employed directly in the opening of Fmoc protected aspartic anhydride. We show potential application of the building block by establishing it as a substrate for enzymatic glycan extension using sugar oxazolines of varying size and biological significance with several endo-β-N-acetylglucosaminidases (ENGases). The added steric bulk resulting from incorporation of the azide is shown to have no or a minor impact on the yield of enzymatic glycan extension.Bio-organic Synthesi

Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-dihydroxypyrrolidines: rhamnulose iminosugar mimics inhibit alpha-glucosidase

The key step in the syntheses of highly substituted trans-3,4-dihydroxypyrrolidines is introduction of bromide by stereospecific and regiospecific Hanessian-Hullar reactions; benzylidene lactones of l-rhamnonolactone and 6-deoxy-this should be small unnpercase d not l why can I not correct this-gulonolactone allow introduction of N at C2 with inversion or retention of configuration. Initially a protecting group, the benzylidene acetal then provides a bromide at C5 to allow formation of the pyrrolidine ring. With silyl protecting groups, bromide was introduced at C5 with inversion of configuration whereas benzoyl protection gave a mixture of retention and inversion, indicative of neighbouring group participation in a Hanessian-Hullar reaction. Four stereoisomeric pyrrolidines - iminosugar mimics of α- and β-l-rhamnulose and α- and β-6-deoxy-d-sorbose were prepared. Only the α-l-rhamnulose mimic showed moderate inhibition of rhamnosidase but some were good inhibitors of α-glucosidases; none inhibited rhamnose isomerase and they had a small effect as synthetic inducers of the rhamnose catabolic operon in E. coli