Enzyme Catalysis for the Production of Perdeuterated Complex Molecules Established at ESS DEULAB

The chemical deuteration laboratory at ESS (DEULAB) is working to establish innovative methods of producing complex perdeuterated molecules with exciting applications in neutron scattering.

The first method focuses on the production of perdeuterated chiral molecules. Chiral molecules exist in two different forms (enantiomers) which are mirror images of each other but otherwise identical. The synthesis of a chiral molecule as a single enantiomer, without its mirror image, is challenging, and the inclusion of isotopic labelling imposes even more requirements upon equipment, methods and synthetic design. This is one reason why the number of perdeuterated chiral molecules available to the neutron scattering community is so limited.

Our method uses enzyme catalysts, which, due to the intricate design of their active site, often have full specificity for one enantiomer only, and can transform relatively economical, achiral precursors into valuable chiral molecules. Using deuterated analogues of  the precursors allows us to use this process to produce deuterium-labelled chiral molecules. Our first target molecule was lactic acid, which exists as both D-lactic acid, and L‑lactic acid (Figure 1). Lactic acid is best known as the molecule produced in the body during exercise, but it is also of interest in the fields of material science and biomedical technology as the monomer for poly(lactic acid) polymers which are renewable, biodegradable and biocompatible.

D and L lactic acid

Figure 1. The two enantiomers of lactic acid.

The lactate dehydrogenase enzyme is responsible for the production of lactic acid from pyruvate during normal metabolism and exercise, and normally it exists in only the L-form producing only L-lactic acid. However, D‑lactate dehydrogenase, which produces and acts exclusively on the other enantiomer D-lactic acid, has also been expressed recombinantly in bacterial hosts. We first used D‑lactate dehydrogenase to produce perdeuterated D-lactic acid-d4. In addition to the enzyme, we used two deuterated reagents, sodium pyruvate-d3, which we can easily synthesise in our lab and sodium formate-d1, which is commercially available. The other components of the catalytic system, including a buffer, a coenzyme, an acid and water as solvent – were all standard, unlabelled reagents. We isolated the product as D-lactic acid-d4 and analysed it using mass spectrometry to determine the level of deuterium incorporation (Figure 2). Pleasingly, we could see that the molecular weight of the product was four atomic mass units higher than for unlabelled lactic acid (one atomic mass unit per deuterium atom).

LA-d4 MS

Figure 2. Top: mass spectrum of unlabelled lactic acid (89.0 amu). Bottom: mass spectrum of D-lactic acid-d4; shifted by amu (93.0 amu).

This process is equally applicable to producing perdeuterated L-lactic acid-d4, by using L-lactate dehydrogenase as the catalyst. Both samples will be used by our Deuteration Network (DEUNET) partners at the Jülich Research Centre (FZJ) in Germany to synthesise tailored poly(lactic acid) samples, which will be analysed by neutron scattering to correlate the arrangement of the chiral monomers with the structure and physical properties of the polymers.

The number of similar enzymes which are commercially available, and the substrates they accept, suggests that this method will have broad applicability in the synthesis of chiral deuterated molecules, and so, at ESS, we are developing this enzyme-catalysis to produce more novel perdeuterated chiral molecules which are presently unavailable.

Deuteration for Neutron Scattering – DEUNET Workshop: Thanks for an Insightful and Inspiring Meeting!

From Monday 15th-Wednesday 17th May, the Deuteration for Neutron Scattering – DEUNET Workshop was held in Oxford, England, organised jointly by the STFC Deuteration Facility and the European Deuteration Network (DEUNET).

Prominent researchers working in the fields of lipids and membranes, energy materials, surfactants, polymers, protein structure and function, colloids, and other small molecules, presented their research which has exploited the power of the tandem techniques of deuterium-labelling and neutron scattering. The programme demonstrated the impact that deuterium labelling has on neutron science, and the ways in which this leads to high-impact science. Each of the facilities involved in the European Deuteration Network presented the current capabilities of their laboratories, in addition to projects and development being undertaken. It was very enjoyable to host delegates from other facilities around the world who work in the field of deuteration, such as J-PARC (Japan) and ORNL (USA), and to hear about the work undertaken in their laboratories.

There was a great deal of opportunity for delegates to interact outside of the presentations, during discussion sessions, a poster session, and within meal and coffee breaks. An advisory board was appointed for the DEUNET, comprised of Trevor Forsyth (ILL), Jian Lu (University of Manchester), Thomas Hellweg (Bielefeld University) and Peter Holden (ANSTO). We trust that they will succeed in providing the network with invaluable advice and insight.

We hope to hold the next STFC – DEUNET Deuteration for Neutron Scattering workshop in late 2018. Further information will be provided at Deuteration.net as it becomes available.

Thanks to all those involved in the workshop for a productive and inspiring meeting!

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The delegates of the Deuteration for Neutron Scattering – DEUNET Workshop held in Oxford, 15-17th May 2017.

Deuteration for Neutron scattering – DEUNET workshop, Oxford 15-17 May 2017

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We are pleased to announce the Deuteration for Neutron Scattering – DEUNET workshop, organised jointly by the STFC Deuteration facility and the DEUNET European Deuteration Network which will be held at the Oxford Spires Hotel, Oxford, UK 15-17th of May 2017.

Deuteration benefits neutron scattering investigations of many types of soft and biological material ranging from macromolecular structures to energy material function. The workshop aims to demonstrate the impact of deuterium-labelling in neutron science, and to establish applications in which this technique will play a key role in generating the greatest future scientific potential. The workshop will consist of a series of scientific presentations on the state-of-the art applications of neutron scattering and deuterium labelling and updates on the current capabilities of deuteration facilities in Europe and around the world. Extensive interactions between the delegates will be facilitate by dedicated discussion sessions in addition to a poster session.

Detailed information, including a preliminary program and list of confirmed speakers can be found at: https://indico.esss.lu.se/event/756/

Registration is now open and limited to 60 participants. Abstracts for posters are welcome until the 2nd April 2017.

We hope to see you in Oxford!

The organising committee:

Peixun Li, Marek Jura, John Webster STFC
Giovanna Fragneto ILL
Jürgen Allgaier FZJ
Hanna Wacklin ESS

Deuteration User Survey

 

Within the SINE2020 project we have setup an European Deuteration Network (DEUNET) that aims to increase the availability and accessibility of complex deuterated molecules to the European neutron scattering community. The existing capabilities of the laboratories at ISIS, ILL and FZJ which currently produce deuterated materials for neutron scattering will be complemented by an additional laboratory at ESS. Furthermore, the organisation of the facilities into a network leverages the unique specialisations of each of the laboratories.

Under this project we have prepared a short user survey to help us to get a better overview of your demands for deuterated materials. We hope you will participate in this survey.

Thank you for your help,

WP5: Chemical Deuteration Meeting Held at ILL, Grenoble

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Attendees (left-right): Peixun Li (ISIS), John Webster (ISIS), Kun Ma (ISIS), Marek Jura (ISIS), Jürgen Allgaier (FZJ), Hanna Wacklin (ESS), Robin Delhom (ILL), Giovanna Fragneto (ILL), Andreas Raba (FZJ), Anna Leung (ESS), Rachel Morrison (ILL).

January, 2017: A two-day SINE2020 Work Package 5: Chemical Deuteration meeting was recently hosted by the Institut Laue-Langevin (ILL) in Grenoble, France. The event was well-attended, with all of those from ISIS, ILL, FZJ and ESS involved in the Chemical Deuteration project joining the meeting.

Reports from each of the facilities, detailing progress made towards their projects and deliverable objectives, opened the meeting on 18th January. More information about the projects being undertaken by the Deuteration Network can be found here. Kun Ma, who recently joined the ISIS Deuteration Facility, was welcomed to the network.

Subsequent sessions were allocated to the discussion of a collaborative User Workshop for chemical deuteration, to be hosted by ISIS in Oxford, UK in May 2017; methods to survey the requirements of the neutron scattering community for deuterated chemicals; and strategies to ensure the sustainability of the Deuteration Network into the future.

Thank you to Rachel Morrison for orchestrating a very successful meeting, and for all members of the network for their attendance and enthusiastic contribution!

 

First Deuterated Molecule Produced at the Chemical Deuteration Lab, ESS

The chemical deuteration laboratory at ESS recently produced its first chemically deuterated molecule, sodium pyruvate-d3. It was produced by reacting pyruvic acid with deuterium oxide (D2O) and sodium bicarbonate:

pyruvic-acid-deuteration

Sodium pyruvate-d3 was analysed by nuclear magnetic resonance (NMR) spectroscopy to determine the identity and purity of the molecule, and to quantify the deuteration at each carbon atom. From the 13C NMR spectrum, it was observed that the sample was highly deuterated, and that the integrity of the sample was maintained.

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13C NMR spectrum showing a pattern indicative of deuteration at a carbon atom (25 ppm).

The high deuteration level was confirmed by mass spectrometry, with a peak observed at the value expected (90.0 atomic mass units).

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Mass spectrum showing the a signal at the expected atomic mass for pyruvate-d3 (90.0 amu), shifted by three atomic mass units from pyruvate (87.0 amu), which is consistent with the exchange of three 1H atoms for three 2H atoms.

The pyruvate ion (CH3COCOO) plays an important biochemical role, providing energy to cell via a series of chemical reactions known as the Krebs cycle. It is also a substrate for the lactate dehydrogenase enzyme, from which lactic acid is produced during normal metabolism and exercise. For the chemical deuteration laboratory, sodium pyruvate-d3 will serve as a precursor to deuterated lactic acid-d4. Lactic acid-d4 is a chiral molecule and so exists in two forms, D-lactic acid-d4 and L-lactic acid-d4; the use of an enzyme to produce lactic acid-d4 from sodium pyruvate-d3 will allow us to produce one or the other, instead of a mixture of both which would have to be separated.