Chemoenzymatic Synthesis of Trehalose Analogues: Rapid Access to Chemical Probes for Investigating Mycobacteria

Abstract

Trehalose analogues are emerging as valuable tools for investigating Mycobacterium tuberculosis, but progress in this area is slow due to the difficulty in synthesizing these compounds. Here, we report a chemoenzymatic synthesis of trehalose analogues that employs the heat-stable enzyme trehalose synthase (TreT) from the hyperthermophile Thermoproteus tenax. By using TreT, various trehalose analogues were prepared quickly (1 h) in high yield (up to >99 % by HPLC) in a single step from readily available glucose analogues. To demonstrate the utility of this method in mycobacteria research, we performed a simple "one-pot metabolic labeling" experiment that accomplished probe synthesis, metabolic labeling, and imaging of M. smegmatis in a single day with only TreT and commercially available materials.

Tailoring trehalose for biomedical and biotechnological applications

“…Thus, this work established a robust method for the rapid and convenient synthesis of fluorine-modified trehalose analogues, confirmed that the analogues mimic the structure of native trehalose, and demonstrated that they could bind to mycobacteria through a non-mammalian trehalose recycling pathway. Together, the pioneering work of the Barry and Davis groups, 52,115 combined with our contributions, 82,83 should help to pave the way for continued development of trehalose-based PET imaging probes for tuberculosis diagnostics and other applications.…”

“…In our initial report in 2014, we showed that this enzyme could indeed convert a wide variety of glucose analogues to trehalose analogues in high yield (up to 99% yield as determined by HPLC, Figure 7). 82 The method has now been applied to the semi-preparative synthesis of numerous analogues bearing a variety of modifications (e.g., fluoro-, deoxy-, azido-, thio-, stereochemical, and isotopic modifications), with typical isolated yields of 60–80%. 82–84 The rapid and facile procedures we have developed for TreT-catalyzed synthesis and product purification have facilitated access to novel trehalose analogues for mycobacteria research, as discussed in more detail later.…”

“…82 The method has now been applied to the semi-preparative synthesis of numerous analogues bearing a variety of modifications (e.g., fluoro-, deoxy-, azido-, thio-, stereochemical, and isotopic modifications), with typical isolated yields of 60–80%. 82–84 The rapid and facile procedures we have developed for TreT-catalyzed synthesis and product purification have facilitated access to novel trehalose analogues for mycobacteria research, as discussed in more detail later. As mentioned above, the cost of UDP-glucose is a limitation, plus T. tenax TreT showed poor activity on glucose analogues bearing 4-position modifications.…”

“…Certainly, the chemoenzymatic approaches to trehalose analogue synthesis discussed above could be applied to the production of 14 C-trehalose. For example, the unidirectional TreT enzyme from T. tenax 81,82 could provide a rapid and high-yielding synthesis of 14 C-trehalose from relatively inexpensive 14 C-glucose, without the need for cell culture or laborious purification steps.…”

“…82 The TreT enzyme was capable of generating 3- and 6-TreAz from the corresponding commercially available azido glucoses in a single step in 95 and 99% yield (HPLC), respectively. In addition, a purification protocol was developed that utilizes spin dialysis and ion exchange to deliver pure products quickly and in aqueous solution.…”