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.

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.
<ol>
	<li>Prepare a 3 mL overnight culture of TreT-expressing E. coli.
	<ol>
		<li>Streak Top10 E. coli transformed with pBAD-TreT expression vector on a lysogeny broth (LB) agar plate containing 100 &#181;g/mL ampicillin.</li>
		<li>Incubate the plate at 37 &#176;C for approximately 48 hours.</li>
		<li>Pick a single colony from the plate and inoculate 3 mL of LB liquid medium containing 100 &#181;g/mL ampicillin in a culture tube.</li>
		<li>Place the tube in a shaking incubator at 37 &#176;C x 175 rpm overnight.</li>
	</ol>
	</li>
	<li>Induce protein expression in TreT-expressing E. coli.
	<ol>
		<li>Add 750 mL Terrific Broth supplemented with 100 &#956;g/mL ampicillin to a 2,800 mL Fernbach culture flask. Transfer 1 mL broth from the flask to a cuvette for later use as a blank.</li>
		<li>Add the 3 mL overnight culture generated in step 1.1.4 to the culture flask, then place the flask in an incubator and shake at 37 &#176;C x 200 rpm. Periodically check the absorbance of the culture at 600 nm versus the blank collected in step 1.2.1.</li>
		<li>Once the absorbance at 600 nm reaches between 0.5-1.0, induce TreT expression by adding 750 &#181;L of 1 M arabinose solution (1 mM final concentration) to the culture. Return the flask to the incubator and shake overnight at 37 &#176;C x 200 rpm.</li>
	</ol>
	</li>
	<li>Pellet and lyse the TreT-expressing E. coli cells.
	<ol>
		<li>Transfer the culture to a polypropylene bottle and centrifuge for 15 min at 4000 x g at 4 &#176;C.</li>
		<li>Discard the supernatant and re-suspend the pellet in 15 mL of phosphate-buffered saline (PBS).</li>
		<li>Transfer the cell suspension to a 50 mL conical tube and centrifuge for 15 min at 4,000 x g at 4 &#176;C. Discard the supernatant and either proceed to cell lysis (step 1.3.4) or store the pellet indefinitely at -80 &#176;C.</li>
		<li>Dissolve 1 protease inhibitor mini tablet in 20 mL of wash buffer (50 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 8.0) in a 50 mL conical tube.</li>
		<li>Transfer the protease inhibitor-containing wash buffer to the conical tube containing the pellet. Vortex until the pellet is re-suspended.</li>
		<li>Transfer re-suspended cells to a 100 mL beaker and lyse the cells by sonication (pulse sequence of 45 seconds on, 45 seconds off with a run time of 2 min and 15 seconds at an amplitude of 75 percent).</li>
		<li>Transfer the lysate to a 50 mL metal conical tube and centrifuge for 60 min at 15,000 x g at 4 &#176;C.</li>
		<li>Clarify the lysate by passage through a 0.2-0.45 &#956;m syringe filter into a 50 mL conical tube. 
		NOTE: The typical concentration of lysate obtained is 100 mg/mL.</li>
	</ol>
	</li>
	<li>Purify TreT from E. coli cell lysate using fast protein liquid chromatography (FPLC).
	<ol>
		<li>Set up the FPLC with a nickel affinity column (5 mL bed volume). Wash the column with 10 mL of deionized water or until the column is clean of any contaminants. Equilibrate the column using 20 mL of wash buffer (50 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 8.0) at a flow rate of 1 mL/min.</li>
		<li>Load the lysate (20 mL) obtained from step 1.3.8 onto the column and elute untagged proteins with wash buffer at a flow rate of 1 mL/min until the absorbance reaches background levels (typically 80-100 mL of wash buffer are required).</li>
		<li>Elute His-tagged TreT by using a linear gradient of elution buffer (50 mM NaH2PO4, 500 mM NaCl, 250 mM imidazole, pH 8.0) from 1-100% over 60 min at a flow rate of 1 mL/min. Collect 4 mL fractions until TreT has eluted and the absorbance reaches baseline level. 
		&#8203;NOTE: Typically, 60 mL of elution buffer are required to elute the protein, and the protein elutes in the 60-100% elution buffer range. Approximately 10-15 mL of pure TreT in elution buffer are obtained.</li>
		<li>Determine the concentration of TreT by measuring absorbance at 280 nm against an elution buffer blank.</li>
	</ol>
	</li>
	<li>Exchange TreT into tris(hydroxymethyl)aminomethane (Tris) buffer by dialysis.
	<ol>
		<li>After preparing the dialysis tubing according to the manufacturer&#39;s instructions, prime it by rinsing with deionized water and then Tris buffer (50 mM Tris, 300 mM NaCl, pH 8.0).</li>
		<li>Load the TreT sample into the dialysis tubing using a syringe and blunt needle. Dialyze overnight against 2 L of Tris buffer.</li>
		<li>Determine the concentration of TreT by measuring absorbance at 280 nm against a blank collected from the dialysis washing.</li>
		<li>Transfer the TreT solution to a 50 mL conical tube and proceed to trehalose analogue synthesis (step 2) or store the enzyme at 4 &#176;C. 
		&#8203;NOTE: TreT is a thermostable protein. The TreT was stored in Tris buffer at 4 &#176;C for several months without observing significant loss of activity.</li>
	</ol>
	</li>
</ol>
2. One-step Synthesis of Trehalose Analogues Using TreT Enzyme
NOTE: The protocol below describes a reaction scale based on 4 mL volume, which can deliver approximately 15-30 mg of trehalose analogue depending on reaction efficiency and molecular weight of the product. The reaction components can be scaled to obtain more or less trehalose analogue if desired.
<ol>
	<li>Add glucose analogue (0.080 mmol, mass will depend on the molecular weight), UDP-glucose (0.160 mmol, 97.6 mg), and MgCl2 (0.080 mmol, 16.3 mg) to a 15 mL conical tube. The final concentrations of these components will be 20 mM, 40 mM, and 20 mM, respectively.</li>
	<li>Add TreT in Tris buffer (obtained from step 1.5.4) and, if necessary, an appropriate volume of Tris buffer (50 mM Tris, 300 mM NaCl, pH 8.0) to achieve a final enzyme concentration of 300 &#956;g/mL and a final volume of 4 mL. Pipet the mixture up and down gently or invert the tube to dissolve the solids.</li>
	<li>Incubate the reaction at 70 &#176;C with shaking at 300 rpm for 1 hour, then place the tube on ice to cool.</li>
</ol>
3. Purification of Trehalose Analogues from Crude Enzymatic Reaction Mixture
<ol>
	<li>Pre-rinse a centrifugal filter unit (nominal molecular weight limit (NWML) 10 kDa) to remove trace glycerol in the membrane by adding 3 mL of deionized water to the centrifugal filter unit and centrifuging at 3,000 x g until all liquid passes through the filter into the tube (approximately 20 min). Repeat two additional times. Complete this step immediately prior to or during the reaction (step 2.3).</li>
	<li>After cooling the enzymatic reaction mixture (obtained from step 2.3), transfer it to the pre-rinsed centrifugal filter unit. Rinse the reaction tube with 1 mL of deionized water and transfer to the centrifugal filter unit. Repeat rinsing of the reaction tube for maximum recovery of product.</li>
	<li>Centrifuge the centrifugal filter unit at 3,000 x g until all liquid passes through the filter into the tube (approximately 20 min). Rinse the upper chamber of the centrifugal filter unit with 3 mL of deionized water and centrifuge at 3,000 x g until all liquid passes through the filter into the tube (approximately 20 min). Repeat rinsing for maximum recovery of product.</li>
	<li>Discard the upper chamber of the centrifugal filter unit. Add mixed-bed ion-exchange resin (3 g) to the filtrate at the bottom of the tube (typical filtrate volume is 8-15 mL depending on the number of rinses). Stir at room temperature for 1 hour with a magnetic stir bar at a speed sufficient to keep the resin beads suspended in the solution.</li>
	<li>Decant the supernatant and filter it to remove the resin. Add 5 mL of deionized water to rinse the remaining resin. Decant the supernatant and filter it, combining it with the product solution from the first decantation. Repeat rinsing of the resin for maximum recovery of product.</li>
	<li>Analyze the reaction by thin-layer chromatography (TLC) or HPLC to determine whether complete conversion of the glucose analogue starting material to the trehalose analogue product was achieved. See step 4.1 for TLC analysis and step 4.2 for HPLC analysis.</li>
	<li>Remove water by lyophilization or rotary evaporation to give the dried product. If no unreacted glucose analogue was observed during TLC or HPLC analysis, purification by chromatography is unnecessary. Weigh the product to obtain the reaction yield and perform nuclear magnetic resonance (NMR) spectroscopic analysis (step 4.3) to confirm product structure and purity.</li>
	<li>If unreacted glucose analogue was observed during TLC analysis, separate it from the trehalose analogue using a size exclusion column.
	<ol>
		<li>Prepare a 1 x 100 cm column containing deionized water-saturated, extra-fine P2 polyacrylamide bead size exclusion media according to the manufacturer&#39;s instructions. 
		&#8203;NOTE: The size exclusion column can be reused after washing with deionized water.</li>
		<li>Re-dissolve the dried enzymatic reaction product (obtained from step 3.7) in 0.5 mL of deionized water. Apply the product solution to the size exclusion column manually or by using a column flow adapter. Rinse the vial that contained the crude product with another 0.5 mL deionized water, and load it into the size exclusion column.</li>
		<li>Elute the product with deionized water by gravity flow and collect fractions of approximately 2 mL volume.</li>
		<li>Analyze the fractions by TLC (step 4.1). Pool the fractions containing pure trehalose analogue.</li>
		<li>Remove water by lyophilization or rotary evaporation to give the dried product. Weigh the product to obtain the reaction yield and proceed to NMR analysis (see step 4.3).</li>
	</ol>
	</li>
</ol>
4. Analysis of Trehalose Analogue Products
<ol>
	<li>Perform thin-layer chromatography (TLC) analysis of TreT reaction. 
	NOTE: This procedure can also be used to analyze size exclusion column fractions. It may be necessary to concentrate the reaction mixture or column fractions prior to TLC analysis to observe compound staining on the TLC plate.
	<ol>
		<li>Mark lanes on the TLC plate surface with a pencil and apply analyte(s) and relevant standard(s) to the appropriate lanes, including the glucose analogue standard, the trehalose analogue standard (if available), the reaction mixture (or fractions collected from size exclusion column purification), and a co-spot. After applying each sample to the TLC plate, allow the plate to dry. 
		NOTE: For reaction analysis, typically 2 &#181;L of sample is applied to the TLC plate.</li>
		<li>Develop the TLC plate using n-butanol/ethanol/deionized water (5:3:2).</li>
		<li>Dry the developed TLC plate, then dip it in 5% H2SO4 in ethanol (sugar stain) and heat on a hot plate on high setting until sugar-containing spots can be visualized (typically 5 min).</li>
	</ol>
	</li>
	<li>Perform HPLC analysis of TreT reaction mixtures using any HPLC system capable of separating and detecting carbohydrates. This protocol involves carbohydrate separation using an aminopropyl HPLC column and detection using refractive index.
	<ol>
		<li>Attach aminopropyl column (4.6 x 250 mm) containing a pre-column guard to the HPLC.</li>
		<li>Equilibrate aminopropyl column with 80% acetonitrile in deionized water at a flow rate of 0.4 mL/min.</li>
		<li>Load the solution of reaction product (or standard) onto the aminopropyl column.</li>
		<li>Elute the product (or standard) with 80% acetonitrile in deionized water at a flow rate of 0.4 mL/min and a column temperature of 50 &#176;C. Typically, the run time used is 40 min. 
		NOTE: Both the glucose analogue starting material and the trehalose analogue product can be detected by refractive index, although other methods such as evaporative light scattering detection (ELSD) could be used. Using the conditions described, glucose analogues typically elute between 10-15 min and trehalose analogues elute between 15-25 min.</li>
	</ol>
	</li>
	<li>NMR analysis of purified trehalose analogues.
	<ol>
		<li>Dissolve purified trehalose analogue in D2O (700 &#956;L) and transfer the solution to an NMR tube.</li>
		<li>Acquire 1H and 13C NMR spectra according to appropriate NMR facility protocols.</li>
	</ol>
	</li>
</ol>
5. Application of TreT-synthesized Trehalose Analogues to the Detection of Mycobacteria
<ol>
	<li>Synthesize, purify, and administer 6-TreAz to M. smegmatis (Msmeg).
	<ol>
		<li>Add 6-azido-6-deoxy glucopyranose (6-GlcAz, 0.020 mmol, 4.1 mg), UDP-glucose (0.040 mmol, 24.4 mg), and MgCl2 (0.020 mmol, 4.1 mg) to a 15 mL conical tube.</li>
		<li>Add TreT in Tris buffer (obtained from step 1.5.4) to achieve a final enzyme concentration of 300 &#956;g/mL and a final volume of 1 mL. Pipet the mixture up and down gently or invert the tube to dissolve the solids.</li>
		<li>Incubate the reaction at 70 &#176;C with shaking for 15 min.</li>
		<li>Dilute the enzymatic reaction mixture with 3 mL of deionized water and transfer it to a pre-washed centrifugal filter unit (NMWL 10 kDa). Centrifuge the filter unit at 3,000 x g until most of the liquid passes through the filter into the tube, approximately 10 min.</li>
		<li>Discard the upper chamber of the centrifugal filter unit. Add mixed-bed ion-exchange resin (0.75 g) to the tube and stir/shake at room temperature for 25 min. Decant the supernatant and filter it to remove the resin. 
		&#8203;NOTE: Steps 5.1.1-5.1.5 provide an aqueous solution of 6-azido-trehalose (6-TreAz) at approximately 5 mM concentration in less than 1 hour. The 5 mM concentration is estimated based on the quantitative conversion of substrate to product and the dilution that takes places during the purification steps, assuming minimal loss of product during these steps. The solution can be sterile-filtered before addition to a biological sample if desired.</li>
		<li>Add the appropriate volume of 6-TreAz product solution to a log-phase culture of M. smegmatis (Msmeg), typically to achieve a final culture volume of 100-1,000 &#956;L and a final 6-TreAz concentration of ~25 &#956;M. Incubate the cells at 37 &#176;C for the desired amount of time, typically 60 min.</li>
	</ol>
	</li>
	<li>Perform click chemistry to conjugate a fluorophore to azide-labeled cells. In this protocol, use Cu-catalyzed azide-alkyne cycloaddition (CuAAC) to deliver a fluorophore to cell-surface azides in Msmeg.
	<ol>
		<li>Centrifuge the cells at 3,900 x g for 5 min, and then wash the cells with PBS containing 0.5% bovine serum albumin. Repeat two times.</li>
		<li>Re-suspend the pelleted cells in 4% para-formaldehyde in PBS to fix them. After incubating for 10 min, repeat step 5.2.1 to wash the cells.</li>
		<li>Re-suspend pelleted cells in 138 &#956;L PBS.</li>
		<li>Add 3 &#956;L of a 1 mM stock solution of alkyne-carboxyrhodamine 110 (Alkyne-488) in DMSO.</li>
		<li>Add 3 &#956;L of a freshly prepared 60 mM stock solution of sodium ascorbate in deionized water.</li>
		<li>Add 3 &#956;L of a 6.4 mM stock solution of Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) in tert-butanol/dimethylsulfoxide (DMSO) 4:1.</li>
		<li>Add 3 &#956;L of a 50 mM stock solution of CuSO4 in deionized water.</li>
		<li>Pipet the cell suspension up and down, then incubate in the dark at room temperature for 30 min.</li>
		<li>Repeat step 5.2.1 to wash the cells. Re-suspend the cells in 150 &#956;L PBS.</li>
	</ol>
	</li>
	<li>Perform cellular fluorescence analysis. In this protocol, use fluorescence microscopy to visualize cellular fluorescence of labeled Msmeg.
	<ol>
		<li>Add 10 &#956;L of bacterial cells suspended in PBS to a microscope slide and lightly spread the liquid into a thin layer using the edge of a coverslip. Allow to air dry in the dark.</li>
		<li>Add 10 &#956;L of mounting medium over the dried sample, then place cover slips over the sample and apply adhesive (e.g., nail polish) to immobilize.</li>
		<li>Image the slides using a fluorescence microscope at 100X magnification.</li>
	</ol>
	</li>
</ol>

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A. 107, 21761-21766 (2010).</li></ol>

The authors have nothing to disclose.

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 monosubstituted trehalose analogues 9, 13, 14. Alternative synthetic approaches are needed to rapidly and efficiently produce various types of trehalose analogues that may have value in the aforementioned areas. Biocatalytic approaches that exploit trehalose-synthesizing enzymes from nature hold the most promise. Although several trehalose-synthesizing pathways exist in nature, we consider TreT from T. tenax18 to be the ideal enzyme for trehalose analogue synthesis for several reasons. 
TreT from T. tenax is thermostable, allowing reactions to be heated for improved speed and for prevention of microbial contamination in large-scale production settings. TreT from T. tenax is a unidirectional enzyme that is not capable of degrading trehalose. TreT from T. tenax is amenable to expression and purification from E. coli, and its shipment and storage is facile. The TreT reaction is a single step and it involves the simple substrates glucose and UDP-glucose. A large number of structurally/functionally diverse glucose analogues are available from numerous commercial vendors. UDP-glucose is a relatively inexpensive glucose donor, especially in comparison to the TreP donor &#946;-D-glucose 1-phosphate. TreT from T. tenax has high promiscuity for the glucose substrate structure, so various trehalose analogues are accessible. Product purification from the TreT reaction mixture is fast and straightforward.
Herein, we reported a detailed optimized protocol for trehalose analogue synthesis that capitalizes on the above positive attributes of the TreT enzyme. The TreT process can be used to access a variety of pure trehalose analogues on a semi-preparative scale (10-100 mg), though additional scale-up should be possible if desired. Numerous types of analogues were accessed in our prior and present work, including azido-, deoxy-, fluoro-, and thio-trehaloses, as well as trehalose stereoisomers, but other modifications will also be possible (e.g., stable isotope-labeled and radiolabeled trehaloses are of particular interest). The synthesis and purification steps of the TreT process are operationally simple and can easily be carried out by personnel who are not trained in synthetic chemistry. One of the most attractive features of the TreT process is that both the synthesis and purification steps can be executed in a very short period of time, ranging from 1-4 hours depending on the number of wash steps desired (note: if size exclusion column purification is needed to separate the trehalose analogue product from unreacted glucose analogue, this increases the total time to about 2 days, which is still substantially faster than existing methods for trehalose analogue synthesis).
Although the TreT process is the most efficient enzymatic route to accessing trehalose analogues reported yet, it has some drawbacks that provide opportunity for future improvement. First, although UDP-glucose is relatively inexpensive compared to other sugar donors, the efficiency of the TreT process could be further improved by coupling it with an enzymatic synthesis of UDP-glucose from a cheaper source, e.g., &#945;-D-glucose 1-phosphate, or by identifying an inexpensive surrogate for UDP-glucose. A second potential limitation of this method is that the reaction scope is ultimately governed by the substrate tolerance of TreT. While the tolerance of wild-type TreT is quite high (see Table 1), some analogues cannot be synthesized using this method (e.g., 4-position-modified analogues). Therefore, engineered versions of TreT with increased substrate tolerance will be valuable. Third, while many TreT-catalyzed reactions proceed with quantitative conversion, lower conversion necessitates a chromatographic purification step. In this protocol, we focused on using a relatively slow size exclusion process since it is inexpensive, does not require any specialized equipment, and can be performed solely with water elution. Alternatively, HPLC purification employing a preparative-scale aminopropyl column could be used as a faster method for product purification. Despite some of these limitations, the TreT process provides a powerful platform for the rapid and efficient preparation of trehalose analogues, and should expedite their evaluation and application in the bio(techno)logical and biomedical fields. Future improvements to the TreT process, discussed above, will further strengthen research and applications related to trehalose and its derivatives.
Trehalose analogues have potential value in various areas. As mentioned in the introduction, natural trehalose is used for biopreservation and food sweetening applications, so trehalose analogues with properties such as degradation resistance may be attractive. In addition, a variety of stable isotope-labeled and radiolabeled versions of natural trehalose are commercially available for research purposes, and the TreT process can certainly provide fast and efficient access to these molecules. Trehalose analogues are also of interest for targeting pathogens with probes and inhibitors since trehalose is absent from mammals. One application in this area that is of particular interest is the detection of mycobacteria. M. tuberculosis, the causative agent of tuberculosis, which kills 1.5 million people per year, contains unique trehalose-processing pathways that are absent from mammals. For example, the trehalose-recycling transporter and downstream enzymes that incorporate trehalose into outer membrane-resident glycolipids are not present in mammals 22. Targeting this mycobacteria-specific machinery with detectable trehalose analogues represents an attractive approach to in vivo imaging of M. tuberculosis infection in model systems and possibly human patients. Indeed, the above examples of FITC-keto-trehalose and TreAz (Figure 1B) have paved the way for such developments 8,9. To facilitate access of these tools to the research community, we reported herein a protocol for rapidly synthesizing and using 6-TreAz to image mycobacteria in combination with click chemistry.
This protocol can be adapted for the development of other detectable trehalose analogues that may be useful for tuberculosis diagnostic applications. For instance, Barry and Davis first proposed the possible use of 18F-fluoro-trehalose as a positron emission tomography (PET) probe for in vivo imaging of mycobacterial infection 8. The TreT process may be ideal for realizing this goal. TreT is capable of rapidly and quantitatively generating 2-fluoro-trehalose from 2-fluoro-glucose (see Table 1), which is critical because 18F-2-fluoro-glucose is short-lived (18F half-life = 110 min), necessitating extremely fast chemistry. Furthermore, 18F-2-fluoro-glucose is readily available because it is already used in the clinic for PET imaging of tumors. Use of the described TreT process for the development of 18F-fluoro-trehalose and other trehalose analogues for various applications is currently underway in our laboratories.

Trehalose is a symmetrical non-reducing disaccharide consisting of two glucose moieties that are joined by a 1,1-&#945;,&#945;-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 building block for the construction of immunomodulatory glycolipids 2.
<img alt="Figure 1" src="/files/ftp_upload/54485/54485fig1.jpg" /> 
Figure 1: Trehalose and trehalose analogues. (A) Structures of natural trehalose and an unnatural trehalose analogue, where X is a structural modification. (B) Examples of trehalose analogues reported in the literature that have potential applications in biopreservation and bioimaging.
Due to its unique structure and physiological functions, trehalose has drawn significant attention for use in bio(techno)logical and biomedical applications 3. The protective properties of trehalose observed in nature&#8212;e.g., its striking ability to help sustain life in &#34;resurrection&#34; plants that have undergone extreme dehydration 4&#8212;have spurred its extensive use in biopreservation applications. Trehalose has been used to preserve a wide array of biological samples, such as nucleic acids, proteins, cells, and tissues 3. For instance, trehalose is used as a stabilizing additive in a number of pharmaceuticals that are on the market, including several anti-cancer monoclonal antibodies 3. As well, trehalose is used as a sweetener in the food industry, and it is extensively used for product preservation in both the food and cosmetics industries. The adoption of trehalose for these types of commercial applications was initially limited by the inability to obtain bulk quantities of pure trehalose from natural sources or through synthesis. However, an efficient enzymatic process for the economical production of trehalose from starch has recently been developed, which has spurred its widespread commercial use 5.
Chemically modified derivatives of trehalose, referred to herein as trehalose analogues, have gained increasing attention for various applications (generic structure shown in Figure 1A; specific examples of trehalose analogues shown in Figure 1B) 6. For example, lacto-trehalose is a trehalose analogue with one of its glucose units replaced with galactose, thus its 4-position hydroxyl group has an inverted stereochemical configuration. Lacto-trehalose has the same stabilizing properties as trehalose but is resistant to degradation by intestinal enzymes, making it attractive as a non-caloric food additive 6, 7.
Our group&#39;s interest in trehalose analogues primarily relates to their value as mycobacteria-specific probes and inhibitors. The Barry and Davis groups developed a fluorescein-conjugated keto-trehalose analogue, named FITC-keto-trehalose, which was shown to metabolically label the cell wall of live M. tuberculosis, enabling its detection by fluorescence microscopy 8. The Bertozzi lab developed smaller azido-trehalose (TreAz) analogues that could metabolically label the cell wall and subsequently be detected using click chemistry and fluorescence analysis 9. These advances point to the possibility of using trehalose-based probes as diagnostic imaging agents for tuberculosis. Trehalose analogues have also been pursued as inhibitors of M. tuberculosis due to their potential to disrupt pathways in the bacterium that are essential for viability and virulence 10,11,12.
So far, the main obstacle to developing trehalose analogues for bio(techno)logical and biomedical applications is the lack of efficient synthetic methods. The two traditional routes to producing trehalose analogues rely on chemical synthesis (Figure 2). One route involves desymmetrization/modification of natural trehalose, while the other involves starting with properly functionalized monosaccharide building blocks and performing chemical glycosylation to forge the 1,1-&#945;,&#945;-glycosidic bond. These approaches, which have recently been discussed in review articles 13, 14, have proven useful for accomplishing multistep synthesis of small quantities of complex trehalose-containing natural products, such as sulfolipid-1 from M. tuberculosis 15. However, both approaches are generally inefficient, time-consuming, inaccessible to non-chemists, and, additionally, are not considered to be environmentally friendly. Thus, for synthesizing certain types of trehalose analogues, these strategies are not ideal.
<img alt="Figure 2" src="/files/ftp_upload/54485/54485fig2.jpg" /> 
Figure 2: Approaches to trehalose analogue synthesis. Chemical approaches to trehalose analogue synthesis, shown on the left, use multistep procedures that involve difficult protection/deprotection, desymmetrization, and/or glycosylation steps. Enzymatic synthesis, shown on the right, uses enzyme(s) to stereoselectively convert simple, unprotected substrates to trehalose analogues in aqueous solution. The enzymatic protocol reported herein uses a trehalose synthase (TreT) enzyme to convert glucose analogues and UDP-glucose into trehalose analogues in a single step. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
An efficient biocatalytic route to trehalose analogues would facilitate the production, evaluation, and application of this promising class of molecules. While the commercial enzymatic process for trehalose production5 is not adaptable to synthesizing analogues because it utilizes starch as a substrate, there are other biosynthetic pathways in nature that may be exploited for trehalose analogue synthesis. However, research in this area, which was recently reviewed 6, has been limited. One report used a method inspired by the Escherichia coli trehalose biosynthetic pathway to access a single fluoro-trehalose analogue from the corresponding fluoro-glucose. However, this approach requires a three-enzyme system that has limited efficiency and generality 8. Another approach that has been explored is to use trehalose phosphorylase (TreP) in the reverse direction, which in principle permits the one-step synthesis of trehalose analogues from glucose analogues and glucose-1-phosphate 6, 16, 17. Although this approach may have future promise, both inverting and retaining TrePs currently have drawbacks for analogue synthesis. For example, inverting TrePs have a prohibitively expensive donor molecule (&#946;-D-glucose 1-phosphate) and retaining TrePs have poor enzyme expression yields/stability and limited substrate promiscuity. Significant improvements (e.g., via enzyme engineering) will be needed before TreP-mediated analogue synthesis is practical.
At present, the most practical approach for the enzymatic synthesis of trehalose analogues is to use a trehalose synthase (TreT) enzyme, which converts glucose and uridine diphosphate (UDP)-glucose into trehalose in a single step 6. We recently reported the use of Thermoproteus tenax TreT&#8212;a thermostable and unidirectional enzyme 18&#8212;to synthesize trehalose analogues from glucose analogues and UDP-glucose (Figure 3) 19. This enzyme only operates in the synthetic direction and avoids the problem of trehalose degradation found in the TreP system. This one-step reaction could be completed in 1 hour, and a broad variety of trehalose analogues were accessed in high yield (up to &#62;99% as determined by high performance liquid chromatography (HPLC)) from readily available glucose analogue substrates (see Table 1 in the Representative Results section).
<img alt="Figure 3" src="/files/ftp_upload/54485/54485fig3.jpg" /> 
Figure 3: TreT-catalyzed one-step synthesis of trehalose analogues. The TreT enzyme from T. tenax can stereoselectively join readily available glucose analogues and UDP-glucose to form trehalose analogues in one step. R1-R4 = Variable structural modification, for example azido-, fluoro-, deoxy-, thio-, stereochemical, or isotopic label modifications; Y = variable heteroatom, for example oxygen or sulfur, or isotopically labeled heteroatom.
Here, we provide a detailed protocol for the TreT synthesis process, including expression and purification of TreT from E. coli, optimized TreT reaction conditions, and an improved purification method that is carried out entirely in the aqueous phase. This modified protocol enables the expedient and efficient synthesis and purification of diverse trehalose analogues on a semi-preparative scale (10-100 mg). We also demonstrate the use of this protocol for preparing and administering a trehalose-based probe to mycobacteria in less than 1 hour, which enabled the rapid fluorescence detection of mycobacterial cells.

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

rapid one-step enzymatic synthesis and all-aqueous purification of trehalose analogues

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 &#34;green&#34; 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).

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 analogues-many of which are commercially available-to the corresponding trehalose analogues with high efficiency 19. Table 1, which gives HPLC-determined reaction yields for a number of starting glucose analogues using our initially reported protocol, illustrates the scope of TreT&#39;s substrate promiscuity and its suitability for synthesis. Diverse structural modifications, including fluoro-, deoxy-, azido-, thio-, and stereochemical modifications at different positions around the sugar ring are well-tolerated by the wild-type enzyme, with the main exception being modifications at the 4-position.
<img alt="Figure 4" src="/files/ftp_upload/54485/54485fig4.jpg" /> 
Figure 4: Representative results for TreT purification by FPLC. Purification of recombinant T. tenax TreT from E. coli lysate was carried out using nickel affinity chromatography as described in steps 1.4.1-1.4.4. Blue, ultraviolet (UV) absorbance trace; light green, concentration of elution buffer; dark green, pressure. The peak corresponding to TreT is indicated with an arrow. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54485/54485table1.jpg" /> 
Table 1: Representative yields for the TreT reaction. HPLC-determined and isolated yields of the TreT reaction for several trehalose analogues. N.D., not detected. -- Indicates the reaction was not performed on a semi-preparative scale using the optimized protocol. *Indicates that a size exclusion chromatography step was required. Horizontal dividing lines separate the position of modification on the trehalose sugar ring. Table adapted from reference 19 with permission; updated to include isolated yields from this work. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/Table1.docx">Please click here to download this file.</a>
Herein, we report optimizations to our initial protocol that improve the overall efficiency and speed of the TreT synthesis process. While the original protocol used 10 mM glucose analogue and 40 mM UDP-glucose, it was determined that comparable conversion could be obtained using 20 mM glucose and 40 mM UDP-glucose, effectively doubling the amount of product generated per reaction and limiting the waste of UDP-glucose, which is relatively expensive. Using equimolar amounts of glucose analogue and UDP-glucose resulted in lower conversions. If desired, reaction times can also be shortened to less than the initially reported 60 min while still retaining comparable conversion for many analogues, which was demonstrated by the quantitative conversion of 6-GlcAz to 6-TreAz in only 15 min at 70 &#176;C (Figures 5 and 6).
The purification protocol reported herein is substantially improved, replacing the original enzyme precipitation/silica gel chromatography method with a non-chromatographic spin dialysis/ion exchange method. The rationale for this modification is that the TreT reaction mixture consists entirely of ionic species except for the neutral trehalose analogue product. Therefore, the mixture can be spin-dialyzed to remove enzyme and then treated with mixed-bed ion exchange resin to remove all of the ionic species, delivering purified trehalose analogue in aqueous solution in as little as 45 min. This all-aqueous purification method avoids environmentally detrimental organic solvents, time-consuming evaporation and filtration steps, and dry-load silica gel chromatography, which is a slow and cumbersome process, especially for non-chemists. For TreT reactions that exhibit quantitative conversion as determined using the described TLC or HPLC analyses, this purification method provides isolated yields of approximately 60-80% at the reported reaction scale, with the product loss likely due to binding of some sugar to the ion exchange resin (see Table 1 for representative isolated yields). In cases where unreacted glucose analogue remains, it can be separated from the desired trehalose analogue product using a polyacrylamide bead-based size exclusion column, eluting with water. If preferred, this could also be accomplished using preparative-scale HPLC with an aminopropyl column. Product purity can be assessed by TLC, HPLC, and/or NMR spectroscopic analysis. Figure 5 shows representative analytical data for 6-TreAz, which was synthesized via the described protocol. The TLC and HPLC data indicated quantitative conversion of 6-GlcAz substrate to 6-TreAz product for this reaction, and the isolated yield after the spin dialysis/ion exchange purification steps was 58%. 1H and 13C NMR spectroscopic analysis confirmed the structure of the 6-TreAz product, most notably including &#945;,&#945;-stereochemistry of the newly formed glycosidic bond, as well as its purity.
<img alt="Figure 5" src="/files/ftp_upload/54485/54485fig5.jpg" /> 
Figure 5: Representative analytical data for the TreT reaction. (A) TLC and (B) HPLC analysis of TreT-catalyzed conversion of 6-GlcAz to 6-TreAz. (C) 1H NMR and (D) 13C NMR spectra of the 6-TreAz product obtained after spin dialysis/ion exchange purification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Given the aforementioned value of trehalose analogues for the specific detection of M. tuberculosis, the TreT process should facilitate the development and use of trehalose-based probes for tuberculosis research and diagnostic applications. To demonstrate this type of application, we used the optimized TreT process to rapidly prepare, purify, and administer 6-TreAz&#8212;a trehalose-based click chemistry probe 9&#8212;to mycobacteria to enable fluorescence detection (workflow and representative data are shown in Figure 6). Briefly, TreT was used to convert commercial 6-GlcAz to 6-TreAz quantitatively in 15 min, and then the reaction mixture was subjected to the spin dialysis/ion exchange purification method, which took only 45 min (multiple rinse steps were omitted to increase speed). Thus, an aqueous stock solution of pure 6-TreAz of known concentration (~5 mM) was generated in only 1 hour. The 6-TreAz stock was immediately administered to a growing culture of the model bacterium Msmeg (final 6-TreAz concentration ~25 &#181;M) to accomplish azide labeling of the cell surface, which embedded a handle for click chemistry-mediated ligation 20,21 of a fluorescent probe. Subsequently, labeled cells were analyzed by fluorescence microscopy, which showed strong fluorescence for 6-TreAz-treated cells. As expected, control samples that were not treated with 6-TreAz or that were missing a functional trehalose transporter 22, which is required for 6-TreAz uptake and metabolic incorporation 9, showed no fluorescence.




<img alt="Figure 6" src="/files/ftp_upload/54485/54485fig6.jpg" /> 
Figure 6: Representative results for the rapid detection of mycobacteria using a TreT-synthesized trehalose analogue. (A) Workflow for the rapid synthesis, purification, and use of 6-TreAz for fluorescence detection of Msmeg. Steps 5.1.1-5.1.6 of the protocol were used to synthesize and purify 6-TreAz (giving a ~5 mM aqueous solution in 1 hour), then administer it to live Msmeg to accomplish metabolic labeling of the cell surface with azides. Next, as described in steps 5.2.1-5.2.9, click chemistry (CuAAC) was performed to react cell-surface azides with an alkyne-modified fluorophore, alkyne-488. (B) Fluorescence imaging of 6-TreAz-treated Msmeg containing a functional trehalose transporter (wild type and &#916;sugC::sugC) showed strong fluorescence, while the control samples of Msmeg left untreated or lacking the trehalose transporter (&#916;sugC) did not. Figure adapted from reference 19 with permission; updated with workflow and imaging data from the improved TreT protocol. Scale bars, 5 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues at JoVE.com

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

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 ...

rapid-one-step-enzymatic-synthesis-all-aqueous-purification-trehalose

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.3K Views.  Central Michigan University. 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.

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

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Chemistry

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioengineering

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and &#62;50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination&#160;experiments and one-pot glycosylation of SSEA-4.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products and notably to generate chiral carbon centers. The crucial first step consists in the selective double hydroboration of CO2 catalyzed by an iron hydride complex. The product obtained with this 4 e- reduction is a rare bis(boryl)acetal, compound 1, which is subjected in situ to three different reactions in a second step. The first reaction concerns a condensation reaction with (diisopropyl)phenylamine affording the corresponding imine 2. In the second and third reaction, intermediate 1 reacts with triazol-5-ylidene (Enders&#39; carbene) to afford compounds 3 or 4, depending on the reaction conditions. In both compounds, C-C bonds are formed, and chiral centers are generated from CO2 as the only source of carbon. Compound 4 exhibits two chiral centers obtained in a diastereoselective manner in a formose-type mechanism. We proved that the remaining boryl fragment plays a key role in this unprecedented stereocontrol. The interest of the method stands on the reactive and versatile nature of 1, giving rise to various complex molecules from a single intermediate. The complexity of a two-step method is compensated by the overall short reaction time (2 h for the larger reaction time), and mild reaction conditions (25 &#176;C to 80 &#176;C and 1 to 3 atm of CO2).

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations are conducted in a one-pot two-step procedure for the synthesis of complex molecules. The selective 4 e- reduction of CO2 with a hydroborane reductant affords a reactive and versatile bis(boryl)acetal intermediate which is subsequently involved in condensation reaction or carbene-mediated C-C coupling generation.

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products ...

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

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