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

Summary

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.

Abstract

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).

Introduction

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 as a buil....

Protocol

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 E. coli.
   1. Streak Top10 E. coli transformed with pBAD-TreT expression vector on a lysogeny broth (LB) agar plate containin....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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, monobasic	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, Sigma Aldrich, Santa Cruz Biotechnology, American Radiolabeled Chemicals		Examples of vendors that offer numerous glucose analogues
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