Chemistry - A European Journal
10.1002/chem.201701388
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line HPLC measurements. Additional benchmarking involved
repeat biotransformations conducted over several weeks and
this established the excellent reproducibility and robustness of
this novel analytical approach. In conclusion, we believe that
additional optimisation and configuration of the UVRR
instrument set up will make this approach amenable to
miniaturization and in situ point-and-shoot analyses,[
thus
enhancing the potential for wider application. The method could
also be developed as a high throughput screening technique for
enzyme activity, including the monitoring of cascade
biotransformations, as well as for investigating enzyme inhibitors.
Acknowledgements
CW and HF are grateful to BBSRC for their PhD studentships.
HF wishes to thank Jonathan Latham for NHase insights. YX
thanks the Cancer Research UK for funding (including an
Experimental Cancer Medicine Centre award). AJC would like to
acknowledge BBSRC for funding (BB/M028631/1). NJT, JM and
RG are also indebted to BBSRC and GSK for financial funding
Figure 5. An MCR-ALS model was applied to the UVRR data where it
successfully deconvolved spectra into its pure components for
biotransformation 4: a) hypoxanthine b) xanthine and c) uric acid. d) Shows
the reaction dynamics from real-time UVRR measurements (denoted by
outlined symbols) and off-line HPLC data (denoted by solid symbols) as a
function of time for the conversion of hypoxanthine to xanthine to uric acid.
UVRR spectra were obtained for 20 s with baseline correction, normalisation
and smoothing applied (see SI ‘data processing’ for full details). Data shown
from replicate 1.
(
grant BB/K00199X/1). NJT thanks the Royal Society for a
Wolfson Research Merit Award.
Conflict of interest
The authors declare no conflict of interest
highly similar spectra between the three analytes. Noticeably,
2
the R value of xanthine was lower (biotransformation 4) than
previous – this is due to the low overall concentration of xanthine
Keywords: UV resonance Raman (UVRR) • on-line • reaction
monitoring • biotransformation • quantification
(
<8%) throughout the reaction. This is further supported by the
proposed mechanism of XO (based on xanthine dehydrogenase,
XDH, from Rhodobacter capsulatus) whereby hypoxanthine
binds to the active site and is converted to xanthine by oxidation
at the C-2 position. Xanthine is then released, before binding in
a different orientation to present the C-8 for oxidation to give uric
acid.[ This means that the concentration of the intermediate
remains low throughout.
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conversion of nitrile containing compounds into their
corresponding carboxylic acid: either in a single step (nitrilase)
or a two-step process (nitrile hydratase and amidase) (see SI
Figure S12a). Fluorometric and colorimetric assays have
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further demonstrate the general utility of the UVRR approach for
enzyme reaction monitoring.
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In this study we have developed a label-free, rapid, on-line
screening method to monitor biological and industrially relevant
biotransformations based on UVRR spectroscopy. To
demonstrate the general utility of this approach, multiple
substrates and enzyme systems were investigated which
included single, multiple and cascade enzyme systems. UVRR
spectra acquisitions were rapid (20 s per measurement) and
when combined with MCR-ALS produced substrate(s) and
product(s) concentrations that were wholly in agreement with off-
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