Martin A. Fascione

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

Protein labeling is a pivotal technique in molecular and cell biology. Strategies include derivatization of cysteine residues, labeling lysine or N-terminal amino groups with activated esters, periodate or PLP-mediated oxidation of the N-terminus for oxime ligation, and native-chemical ligation. Each of these methods has its own associated challenges: Selective labeling of a single cysteine residue frequently requires rounds of site-directed mutagenesis to introduce the labeling site and/or remove other cysteine residues, and selective labeling of the N-terminal amino group requires careful control of pH to ensure lysine residues are not also modified. Other modern methods for chemoselective labeling often require the introduction of specific recognition sequences or nonnatural amino acids into the protein to be labeled. In most cases, a substantial excess of the labeling reagent is necessary to ensure complete conversion to the product. We report a method for chemoselective N-terminal labeling of recombinant proteins in quantitative yield using depsipeptide substrates for the transpeptidase sortase A. The method does not require engineering of the protein sequence beyond that typically used in contemporary recombinant protein purification strategies. Unlike previous approaches, the method requires only a single N-terminal glycine residue in a sterically unhindered position, a minimal excess of the labeling reagent and substoichiometric quantities of transpeptidase. Sortase A (SrtA) catalyzes the reversible attachment of virulence factors to the cell walls of Gram positive bacteria by C-terminal modification of proteins at an LPXTG recognition sequence. The enzyme catalyzes the covalent attachment of the LPXT motif to a cysteine residue in the catalytic site to form a thioester intermediate. An N-terminal oligoglycine motif in the peptidoglycan can then react with this intermediate to covalently attach the substrate to the cell wall. SrtA has been exploited extensively for the C-terminal modification of proteins. 9] However, this method has certain constraints: the LPXTG sequence must be engineered into the protein and excess nucleophilic labeling reagent is required to push the equilibrium toward formation of product as the transpeptidase reaction is reversible (Scheme 1 a).

Do Glycosyl Sulfonium Ions Engage in Neighbouring-Group Participation? A Study of Oxathiane Glycosyl Donors and the Basis for their Stereoselectivity

Neighbouring-group participation has long been used to control the synthesis of 1,2-trans-glycosides. More recently there has been a growing interest in the development of similar strategies for the synthesis of 1,2-cis-glycosides, in particular the use of auxiliary groups that generate sulfonium ion intermediates. However, there has been some debate over the role of sulfonium ion intermediates in these reactions: do sulfonium ions actually engage in neighbouring-group participation, or are they a resting state of the system prior to reaction through an oxacarbenium ion intermediate? Herein, we describe the reactivities and stereoselectivities of a family of bicyclic thioglycosides in which an oxathiane ring is fused to the sugar to form a trans-decalin-like structure. A methyl sulfonium ion derived from one such glycosyl donor is so stable that it can be crystallised from ethanol, yet it reacts with complete stereoselectivity at high temperature. The importance of a ketal group in the oxathiane ring for maintaining this high stereoselectivity is investigated using a combination of experiment and ab initio calculations. The data are discussed in terms of S(N)1 and S(N)2 type mechanisms. Trends in stereoselectivity across a series of compounds are more consistent with selective addition to oxacarbenium ions rather than a shift between S(N)1 and S(N)2 mechanisms.

A Protein-Based Pentavalent Inhibitor of the Cholera Toxin B-Subunit

Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pm for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.

Mechanistic Studies on a Sulfoxide Transfer Reaction Mediated by Diphenyl Sulfoxide/Triflic Anhydride

Sulfoxides are frequently used in organic synthesis as chiral auxiliaries and reagents to mediate a wide variety of chemical transformations. For example, diphenyl sulfoxide and triflic anhydride can be used to activate a wide range of glycosyl donors including hemiacetals, glycals and thioglycosides. In this way, an alcohol, enol or sulfide is converted into a good leaving group for subsequent reaction with an acceptor alcohol. However, reaction of diphenyl sulfoxide and triflic anhydride with oxathiane-based thioglycosides, and other oxathianes, leads to a different process in which the thioglycoside is oxidised to a sulfoxide. This unexpected oxidation reaction is very stereoselective and proceeds under anhydrous conditions in which the diphenyl sulfoxide acts both as oxidant and as the source of the oxygen atom. Isotopic labelling experiments support a reaction mechanism that involves the formation of oxodisulfonium (S-O-S) dication intermediates. These intermediates undergo oxygen-exchange reactions with other sulfoxides and also allow interconversion of axial and equatorial sulfoxides in oxathiane rings. The reversibility of the oxygen-exchange reaction suggests that the stereochemical outcome of the oxidation reaction may be under thermodynamic control, which potentially presents a novel strategy for the stereoselective synthesis of sulfoxides.

Stereoselective glycosylations using oxathiane spiroketal glycosyl donors

Novel oxathiane spiroketal donors have been synthesised and activated via an umpolung S-arylation strategy using 1,3,5-trimethoxybenzene and 1,3-dimethoxybenzene. The comparative reactivity of the resulting 2,4,6-trimethoxyphenyl (TMP)- and 2,4-dimethoxyphenyl (DMP)-oxathiane spiroketal sulfonium ions is discussed, and their α-stereoselectivity in glycosylation reactions is compared to the analogous TMP- and DMP-sulfonium ions derived from an oxathiane glycosyl donor bearing a methyl ketal group. The results show that the stereoselectivity of the oxathiane glycosyl donors is dependent on the structure of the ketal group and reactivity can be tuned by varying the substituent on the sulfonium ion.

Benzyne arylation of oxathiane glycosyl donors

Summary The arylation of bicyclic oxathiane glycosyl donors has been achieved using benzyne generated in situ from 1-aminobenzotriazole (1-ABT) and lead tetraacetate. Following sulfur arylation, glycosylation of acetate ions proceeded with high levels of stereoselectivity to afford α-glycosyl acetates in a ‘one-pot’ reaction, even in the presence of alternative acceptor alcohols.

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

Abstract A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

Mechanistic Investigations into the Application of Sulfoxides in Carbohydrate Synthesis

Abstract The utility of sulfoxides in a diverse range of transformations in the field of carbohydrate chemistry has seen rapid growth since the first introduction of a sulfoxide as a glycosyl donor in 1989. Sulfoxides have since developed into more than just anomeric leaving groups, and today have multiple roles in glycosylation reactions. These include as activators for thioglycosides, hemiacetals, and glycals, and as precursors to glycosyl triflates, which are essential for stereoselective β‐mannoside synthesis, and bicyclic sulfonium ions that facilitate the stereoselective synthesis of α‐glycosides. In this review we highlight the mechanistic investigations undertaken in this area, often outlining strategies employed to differentiate between multiple proposed reaction pathways, and how the conclusions of these investigations have and continue to inform upon the development of more efficient transformations in sulfoxide‐based carbohydrate synthesis.

Pyrrolysine Amber Stop Codon Suppression : Development and Applications

The pyrrolysine tRNA synthetase‐tRNA pair is probably one of the most promiscuous tRNA–synthetase pairs found in nature, capable of genetically encoding a plethora of noncanonical amino acids through stop codon reassignment. Proteins containing reactive handles, post‐translational modification mimics or both can be produced in practical quantities, allowing inter alia the probing of biological pathways, generating antibody–drug conjugates and enhancing protein function. This Minireview summarises the development of pyrrolysine amber stop‐codon suppression, presents some of the considerations required to utilise this technique to its greatest potential, and showcases the creative ways in which this technique has led to a better understanding of biological systems.

Molecular Recognition‐Mediated Transformation of Single‐Chain Polymer Nanoparticles into Crosslinked Polymer Films

Abstract We describe single‐chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition‐mediated crosslinking process. The SCNPs utilise molecular recognition with surface‐immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra‐ to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to “wrap” surfaces displaying molecular recognition motifs—which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs—within a layer of polymer film.