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Novel Chemical Probes for Tuberculosis Diagnosis and Treatment

Final Report Summary - GLYCOTUP (Novel Chemical Probes for Tuberculosis Diagnosis and Treatment)

Tuberculosis (TB) is a common and deadly infectious disease caused by mycobacteria, mainly Mycobacterium tuberculosis (Mtb). In the developed world, the advent of powerful antibiotics and a concerted inoculation program saw the near extinction of TB. However, the rise of drug-resistant strains, HIV infections, and the neglect of TB control programs have increased the urgency for an alternative treatment and diagnosis. It has been identified a sugar types for their varying potential utility in Mtb-specific labelling. We initially focused on trehalose (D-Glc(α1,1)α-D-Glc, Tre) as one of the key sugar types for Mtb-specific labelling. Mammalian cells do not utilize trehalose and the highly impermeable nature of trehalose to most cell types has led to its use as an inert blood component surrogate. However, in Mtb trehalose plays a striking and remarkable role as a mycolic acid carrier. We proposed a program targeted at the development of innovative methodologies for the preparation of fluorosugars and glycolipid analogues that will allow direct imaging and treatment of TB. As originally planned in the project, the first part of the work was the design and synthesis of 2-deoxy-2-fluoro-trehalose derivatives and their use as imaging agents (alternative TB diagnosis agents: 18F PET and fluorescence imaging). The second part will focused on the mechanistic trapping of Mtb-associated carbohydrate-processing enzymes and the developtment of general protein engineering methods for this purpose. A brief summary of the major accomplishments is listed below:

1. Development of a dehydrative glycosylation protocol for the preparation of symmetrical 2,2'-dideoxy-2,2'-dihalo-α,α-trehalose analogues from readily available precursors.
2. Expansion of the above protocol for the preparation of unsymmetrical 2-deoxy-2-halo-α,α-trehalose analogues
3. Development of an acid-catalyzed addition of FDG to glycals from readily available precursors that allow for 18F incorporation.
4. Confirmation that unsymmetrical 2,2'-dideoxy-2-fluoro-trehalose and related derivatives are efficiently processed by M. tuberculosis mycolyltransesterase enzyme ag85C in vitro.
5. Confirmation that M. tuberculosis culture in vitro takes up decent amounts of single label 14C-labeled trehalose and derivatives.
6. Confirmation that M. tuberculosis can be selectively labelled within macrophages with a fluorescein-containing Tre probe.
7. Synthesis of carbohydrates with different configuration (gluco, galacto, manno), C-2 group (F, NHAc) and anomeric linkage (S, Se) for OtsA-mediated preparation of unsymmetrical 2-deoxy-2-substituted-α,α-trehalose and also for the development of protein engineering methods for studing the mechanistic trapping of Mtb-associated carbohydrate-processing enzymes (antigen 85 proteins).
8. Development of a combined chemical tagging method followed by Endo-A catalyzed elongation allows access to homogeneous, elaborated glycoproteins. A survey of different linkages and sugars (including F-sugars) demonstrated not only that unnatural linkages can be tolerated but they can provide insight into the scope of Endo-A transglycosylation activity.
9. Exploring new methods for the preparation of dehydroalanine as a handle for the development of Mtb-associated protein modification methods.
10. Synthesis of SeS-linked homogeneous glycoproteins as a privilege tag for X-ray analysis of Mtb-associated proteins.
11. Development of a chemical site-selective method for the incorporation of [18F]fluorine into proteins.

Contact details:

Prof. Benjamin G. Davis and Dr. Omar Boutureira

Chemistry Research Laboratory
Department of Chemistry, University of Oxford, 12 Mansfield Road,
Oxford OX1 3TA, UK

Ben.Davis@chem.ox.ac.uk
Omar.Boutureira@chem.ox.ac.uk