Biomolecules & Bioconjugates as Probes of Mechanism in Biology

These synthetic methods have allowed creation of previously unavailable or unnatural biomolecules to precisely test molecular, mechanistic hypotheses in biology.

Nature has accessed only a fraction of putative structure; unnatural variants can therefore uniquely answer questions that may elude traditional biochemical study of natural products. Our goal in developing Synthetic Protein Biology was to create probes that can move beyond the limited palette available in the late 1990s. Since then, we have used the methods described in the previous section to create molecules and higher-order bioconjugates (supra- and multi-molecular, particle[pub 106], virus-like[pub 179], cell-like[pub 120], modified viruses[pub 60] and cells[pub 172]). These chemistries have been logically coordinated in designed syntheses of proteins, peptides, nucleic acids, oligosaccharides, glyco-peptides, glyco-lipids, glyco-proteins and bioconjugates that have employed novel synthetic methods, routes and strategies including the development of novel biocatalytic methods, protecting-group-free (or minimized) chemical methods and dual chemo-enzymatic glycosylation strategies. We have built logical and systematically-varying probes that test hypotheses in biological mechanism and biomimicry in vitro, intracellularly and in vivo that have contributed to an approach now referred to, by some, as ‘Chemical Biology’.

A general strategy for mimicking different post-translational modifications in synthetic proteins[pub 49][pub 85][pub 97] has allowed the recapitulation of their functional effects, allowing precise appraisal of structure-activity relationships in highly relevant contexts such as glycobiology, immunology, inflammation and epigenetics. Many of our reactions are mutually compatible and so PTMs can be applied in multiple combinations[pub 85] to assess diverse biomolecule structure and function. Application of these methods to widely-occurring biological motifs (e.g., phosphorylation[pub 98][pub 197] or protein glycans[pub 85][pub 144]) has enabled new insight into their roles in biology. For example, the first multi-site-selective protein reactions were used to synthesize mimics of human inflammatory pathway molecules (e.g. PSGL-1 protein) that revealed the structural origin of function (this resided in both PTM sulfation & glycosylation).[pub 85] By paring down these structures to the bare-minimum needed for successful function, we have demonstrated e.g. that branching to allow correct presentation of ‘tip’ sugars is the only essential component needed by sugars on protein to interact with certain pathogens[pub 51] or antigen-presenting cells.[pub 179] Design and synthesis of unnatural oligosaccharide analogues from the HIV1-gp120 virus-coat protein and their attachment to protein carriers revealed the most potent sugar ligands of HIV-neutralizing antibody 2G12 and the structural basis of the associated immune response (with Wilson).[pub 144] Even synthetic cellular mimics have been constructed; we have assembled artificial chemical cells equipped with an autocatalytic carbohydrate-metabolism.[pub 120] These cells display successful symbiosis with living cells that has provoked new analyses in cellular science.

When used in alternative analytical, biophysical & high-throughput strategies, these probe molecules have been used as substrates, inhibitors and ligands to map & characterize the shapes, reactions and binding modes involved in protein-ligand interactions. These have focused particularly on carbohydrate–protein interactions; 3 example carbohydrate probe families illustrate this approach. Gas-phase spectroscopic approaches (with Simons) applied to probe molecules have elucidated the inherent conformational biases of glycoconjugates & oligosaccharides. By relating these structures to biological function, we have proposed structural hypotheses as to why some sugars (& not others) are conserved in biology. For example, direct detection of the possible chemical origins of endo- & exo-anomeric effects (as well as magnitude and modulation) in an unbiased form revealed a pivotal role for the C-2 substituent of sugars.[pub 150] Detailed & novel kinetic methods (e.g. through quantitative, high-throughput mass spectrometry) allow label-free enzyme assay. The elucidation of an unusual mechanism – the first demonstrated SNi in biology[pub 164] – relied upon new probes as well as isotopic and MS methods. This mechanism is likely to be relevant to many enzymes and is now guiding structure-based inhibitor design in academia and industry, including synergistic inhibitors that display true transition-state mimicry of glycosyltransferases (with Davies). Similarly, mechanism-based enzyme probes have been discovered and used by us to map pathologically-relevant TB sugar biology (with Barry III),[pub 155] thus creating a diagnostic method that is now being developed with the NIH (USA) and the Gates Foundation.

Prof Benjamin G. Davis
University of Oxford
Chemistry Research Laboratory
Mansfield Road
Oxford, OX1 3TA, UK
Phone: + 44 (0)1865 275652
Fax: + 44 (0)1865 275674