Deuterated carbohydrate probes as ‘label-free’ substrates for probing nutrient uptake in mycobacteria by nuclear reaction analysis† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc09588j Click here for additional data file.

Deuterated sugars that are transported into mycobacteria can be detected using ion beam (nuclear reaction) analysis.


General Methods and Materials
All chemicals were used as supplied unless otherwise stated. Ruthenium on carbon

Physical and Analytical Methods
NMR spectroscopy ( 1 H) measurements were conducted on either Bruker DPX-300 or DPX-400 spectrometers using deuterium oxide as a solvent. All chemical shifts are reported in ppm (δ) relative to the residual solvent. All spectra were acquired using a zg30 pulse sequence with a minimum of 16 scans. Mass spectral analyses were recorded on an Agilent 6130B single Quad spectrometer using electrospray ionisation (ESI) in positive mode on samples prepared in methanol. Lyophilisation was conducted on a Heto lyolab 300 freeze dryer. Optical density measurements were conducted at a wavelength of 600 nm on a Shimadzu UVmini-1240 UV visible spectrophotometer. The ion beam accelerator is a National Electrostatics Corporation (USA) 5SDH Pelletron accelerator, having a target station equipped with a liquid nitrogen cooling system, based at the University of Durham, UK.

Typical Procedure for the Ru/C-Catalysed H-D Exchange of Sugars
Trehalose (1.5 mmol, 0.51 g) was dissolved into 2 mL of D 2 O to which Ru/C was added (10 mol % relative to the substrate). The solution was stirred in a butyl crimp sealed reaction vessel, and degassed with hydrogen, after which a balloon filled with hydrogen gas was fitted to the vessel, to maintain a pressure of ~ 1 bar. The vessel was subsequently heated to 80 °C for 72 hours. After this time, the mixture was cooled to room temperature and the solid catalyst removed using a syringe membrane filter (Sartorius Stedim, Minisart ® , 0.2 µm). The filtrate was then lyophilised to yield a white solid. This same procedure was followed for the deuteration of monosaccharides but with 5 mol % of 5 % Ru/C and heated at 80 o C for 24 hours.

Evaluation of deuterium content
The incorporation of deuterium was demonstrated by using ESI mass spectrometry and the deuterium content was quantified by 1 H NMR by integration of the protons on the methyl protons of the methoxy group, relative to the residual, non-exchanged ring-protons. In the case of the disaccharide carbohydrate: trehalose, protons at the C1 (anomeric) position were utilised instead of the methyl protons.
Calculation for average deuteration of carbohydrate, Sawama et al [1] ) Proton positions H 2-4 , H 6 are able to undergo deuteration (Compound data (2)) due to their proximity to the hydroxyl group on the adjacent carbon. Therefore the total protons of non-deuterated Methyl-β-D-galactopyranoside at these positions is equal to five.
It should be noted here that this calculation provides an average of all the possible deuteration combinations and that for any given degree of deuteration per sugar (e.g. 2 per molecule) there exist many possible structures and this leads to increased complexity in the NMR spectra. This does not prevent the analysis since the overall % deuteration is required here for the NRA.
In order to convert the relative mols of carbohydrate taken up by Mycobacterium smegmatis, the NRA signal was divided by the average number of deuterium atoms per sugar to give the relative mol carbohydrate.

Nuclear Reaction Analysis
Lyophilised samples were pressed into pellets on aluminium foil using a press to prepare specimens that were homogenous and thick compared to the range (~4 microns) of the 0.7 MeV 3 He + ion beam. The choice of beam energy coincident with a broad maximum in the nuclear reaction cross-section; therefore enables the most sensitive detection of deuterons. A defined charge of 3 He + ions, typically 4 mC, was directed onto the sample at normal incidence and backscattered ions and reaction products were detected using a 1.5 mm thick Canberra PIPS detector with nominal resolution of 19 keV. Data analysis was carried out using the Surrey University Datafurnace programme, to provide a quantitative simulation of the fast proton spectrum under the instrument settings which we have established previously. Due to the relatively low count rate for the data obtained, and the homogenous nature of the samples, fitting to complex depth profiles was neither practical nor necessary. Instead the relative yield of fast protons was analysed to determine the relative content of 2 H-carbohydrate, since under the constant experimental conditions, these factors are directly proportional to one another. Example data and fitting is shown in Figure S16