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Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Published: February 17th, 2017



1Department of Chemistry and Biochemistry, Central Michigan University, 2Department of Chemistry, University of Southern Maine

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a "green" biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose is a symmetrical non-reducing disaccharide consisting of two glucose moieties that are joined by a 1,1-α,α-glycosidic bond (Figure 1A). While trehalose is absent from humans and other mammals, it is found commonly in bacteria, fungi, plants, and invertebrates 1. The primary role of trehalose in most organisms is to protect against environmental stresses, such as desiccation 1. In addition, some human pathogens require trehalose for virulence, including the tuberculosis-causing Mycobacterium tuberculosis, which utilizes trehalose as a mediator of cell envelope biosynthesis and....

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1. Expression and Purification of TreT from Top10 E. coli

NOTE: Please contact the authors to request the TreT-expressing E. coli strain (pBAD TreT plasmid, containing the T. tenax tret gene under the control of the AraC protein, transformed into Top10 E. coli 19) and the accompanying material transfer agreement. The following protocol typically gives a protein yield of approximately 4 mg/L.

  1. Prepare a 3 mL overnight culture of TreT-expressing .......

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T. tenax TreT was obtained from E. coli in a yield of approximately 4 mg/L using standard protein expression and purification techniques. A single nickel affinity chromatography step was sufficient to purify TreT from E. coli lysate (a representative FPLC trace is shown in Figure 4). As established in our initial publication on the TreT synthesis process, recombinant T. tenax TreT is capable of converting a broad of variety glucose anal.......

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Trehalose analogues have the potential to impact various fields, from preservation of food and pharmaceuticals to diagnosis and treatment of microbial infections 6. Existing multistep chemical synthesis methods are useful for producing complex trehalose analogues with multiple sites of modification (e.g., naturally occurring complex mycobacterial glycolipids). However, these methods are invariably lengthy and inefficient, even when applied to the synthesis of comparatively simple monosubs.......

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This work was funded by a grant from the National Institutes of Health (R15 AI117670) to B.M.S and P.J.W, as well as a Cottrell College Scholar Award from the Research Corporation (20185) to P.J.W. L.M.M. was supported by a Provost's Fellowship from CMU.


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Name Company Catalog Number Comments
LB agar Research Products International L24021
Ampicillin sodium salt Sigma Aldrich A9518
Luria broth Research Products International L24045
Terrific Broth Research Products International T15050
L-(+)-Arabinose Sigma Aldrich A3256
Phosphate-buffered Saline GE Healthcare SH30256
Imidazole Sigma Aldrich I5513
Sodium chloride BDH BDH9286
Sodium phosphate, Fisher Scientific S374
Syringe filter, 0.45 µm Fisher Scientific 09719D
Protease Inhibitor mini-tablets, EDTA-free Thermo Scientific 88666
HisTrap HP nickel affinity column, 5 mL GE Healthcare 17-5248-02
TRIS base ultrapure Research Products International T60040
Dialysis tubing, MWCO 12–14,000 Fisher Scientific 21-152-16
Glucose analogues CarboSynth, Examples of vendors that offer numerous glucose analogues
Sigma Aldrich,
Santa Cruz Biotechnology, American Radiolabeled Chemicals
6-Azido-6-deoxy glucopyranose (6-GlcAz) CarboSynth MA02620
UDP-Glucose abcam Biochemicals ab120384
Magnesium chloride hexahydrate  Fisher Scientific M33
Amicon Ultra-15 centrifugal filter unit EMD Millipore UFC901008
Bio-Rex RG 501-X8 mixed-bed ion-exchange resin Bio-Rad 444-9999
Extra-Fine Bio-Gel P2 media Bio-Rad 150-4118
Glass-backed silica gel thin-layer chromatography plates EMD Millipore 1056280001
n-Butanol Fisher Scientific A399
Ethanol Fisher Scientific S25310A
Sulfuric acid Fisher Scientific A300
Acetonitrile EMD Millipore AX0145
Deuterium oxide, 99.8% Acros Organics 351430075
Aminopropyl HPLC column Sigma Aldrich 58338
Bovine serum albumin Sigma Aldrich 5470
Para-formaldehyde Ted Pella 18505
Alkyne-488 Sigma Aldrich 761621
Sodium ascorbate Sigma Aldrich A7631
Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) Click Chemistry Tools 1061
tert-Butanol Sigma Aldrich 360538
Dimethylsulfoxide Sigma Aldrich W387520
Copper(II) sulfate Sigma Aldrich C1297
Fluoromount-G mounting medium Southern Biotechnology 10001

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