Chemical Deuteration at the European Spallation Source, part of thebroader Deuteration Network (DEUNET)
Anna Leung, Oliver Bogojevic, Hanna-Wacklin-Knecht,2 Zoë Fisher,3 Arno HiessScientific Activities Division, European Spallation Source ERIC, 22100 Lund, Sweden; 2Division of Physical
Chemistry, Chemistry Department, Lund University, 22363 Lund, Sweden; 3Molecular Cell Biology Unit & Lund
Protein Production Platform (LP3), Biology Department, Lund University, 22362 Lund, Sweden.

The chemical deuteration laboratory is one of the three pillars of the European Spallation
Source (ESS) Deuteration and Macromolecular Crystallisation (DEMAX) platform for users of
the ESS neutron instruments. DEMAX will begin responding to service requests in early 2019
in order to be well-prepared for the installation and commissioning of the ESS instruments
and subsequent user program. The chemical deuteration laboratory will offer, in the firstinstance, saturated fatty acids, fatty alcohols, alkyl halides, oleic acid and a limited range of
surfactants and glycero- and phospholipids.
The ESS chemical deuteration laboratory is using known chemical deuteration
methodologies to produce deuterated molecules, as well as establishing new biochemicalmethods to enable a broader suite of molecules to be synthesised with greater efficiency.
Some of the results of both the chemical and the biochemical syntheses at ESS will bepresented.
The ESS DEMAX platform operates within the broader Deuteration Network (DEUNET), acollaboration between several laboratories with expertise in deuteration techniques,
including the deuteration laboratories at J-PARC Centre/CROSS. This collaboration is initially
funded by SINE2020, a European project funded by the Horizon 2020 program. Some recent
results from the European partner laboratories will also be presented.

Recent research progress in heterogeneous platinum group metalcatalyzed H–D exchange reactions
Tsuyoshi Yamada, Yoshinari Sawama, Hironao Sajiki
Laboratory of Organic Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, JapanThe utility of deuterium (D)-labeled compounds has been expanding in various fields in thepast few decades, such as elucidation of reaction mechanisms, deuterated semiconductor
devices, and heavy drugs and so on. Therefore, the development of easy and efficient D
incorporation method to various substrates is quite desirable.
The hydrogen(H)–D exchange reactions based on the C–H activation is a strong andstraightforward tool to introduce deuterium atoms into target compounds. We havedeveloped platinum group metal on carbon-catalyzed H–D exchange methods of various
aromatic and aliphatic compounds using D2O as a deuterium source in the presence ofhydrogen gas (H2) as an activator of the metal catalyst. Recently, we have also developed
alternative deuterium labeling methods using in situ generated H2 based on the platinum (Pt)catalyzed dehydrogenation of 2-propanol. Since Pt/C could be effectively activated by quitesmall amount of in situ generated H2, the H–D exchange of acrylic and methacrylic acidderivatives also smoothly proceeded without the reduction of alkene functionalities.
Furthermore, Pt or iridium on carbon-catalyzed deuteration of aromatic rings in D2O and 2propanol-mixed solvent at room temperature have also been accomplished.

Chemical Deuteration of Small Organic Molecules and its
Application to Neutron Studies
Kazuhiro Akutsu,1 Tsukasa Miyazaki,1 Taiki Tominaga,1 Kwihwan Park,2 Yoshinari Sawama,2and Hironao Sajiki2
1Research Center for Neutron Science and Technology, Comprehensive Research Organization for Science andSociety (CROSS), 162-1 Shirakata, Tokai, Ibaraki 319-1106, Japan.; 2Laboratory of Organic Chemistry, Gifu
Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan.

Isotopic labelling of organic molecules with the stable and non-radioactive isotope,
deuterium (2H), using chemical processes is effective to enhance contrast and reduce
incoherent background of the organic samples in neutron scattering experiments. The Japan
Proton Accelerator Research Complex (J-PARC), Comprehensive Research Organisation for
Science and Society (CROSS), Japanese Atomic Energy Agency (JAEA), and National Institutes
for Quantum and Radiological Science and Technology (QST) have cooperated to promote thedeuteration activities at J-PARC. Recently, Parr Reactor system which is used in metalcatalysed H/D exchange reactions and liquid chromatography–mass spectrometry (LC-MS)
were installed and their test operation has been performed for the chemical deuteration
research.
We have not only developed a simple and effective method for the deuteration of smallhydrocarbon molecules, ionic liquids and monomers, on gram scale starting from their
protonated versions but also we have installed and developed the methods of deuterationlevel analysis using LC-MS and elemental analysis techniques. In this presentation, we willshow the summary of deuteration works and the results of practical application of thedeuterated compounds to neutron studies.

Biological deuteration and support for macromolecular
crystallization at the European Spallation SourceZoë Fisher1,2, Katarina Koruza2, Wolfgang Knecht2,Hanna-Wacklin-Knecht1, Anna Leung1,Arno Hiess1
1

Scientific Activities Division, European Spallation Source ERIC, 22100 Lund, Sweden; 2Molecular Cell Biology
Unit & Lund Protein Production Platform (LP3), Biology Department, Lund University, 22362 Lund, Sweden;

DEMAX is the Deuteration and Macromolecular Crystallography support lab for soft matterand life science users of the European Spallation Source (ESS). DEMAX has three support pillars
and will be available to all users of ESS instruments. This presentation will focus on support
for biological deuteration and large protein crystal growth. For these biological support
functions we have established a close partnership with Lund University’s Lund Protein
Production Platform (LP3), a cross-faculty support lab for the production and crystallization ofproteins, both unlabeled and labeled (13C, 15N, 2H) using bacteria, insect cells, and yeast.
DEMAX and LP3 are co-located in the Biology Department of LU. DEMAX will offer deuterated
biomass production of yeast and bacteria and are also growing algae in heavy water to be
used for making rich deuterated media. The LP3 and DEMAX share a state-of-the-art
crystallization laboratory, fully equipped with a variety of crystallization robots and set-ups foreverything from small volume high-throughput screening to fine-screening and large volume
crystal optimization. We have access to a Mosquito with LCP, Opticrys, Oryx8, Dragonfly andTECAN screen formulators, and crystallization plate storage and UV imaging. The second halfof the talk will feature a science highlight from a recent research project.

Protein Deuteration for Neutron Scattering
Satoru Fujiwara
Tokai Quantum Beam Science Center, Quantum Beam Science Research Directorate, National Institutes for
Quantum and Radiological Science and Technology, 2-4 Shirakata, Tokai, Ibaraki, 319-1106, Japan.

Neutrons provide unique tools to investigate structures and dynamics of biologicalmacromolecules. The information available with the neutron techniques includes, for example,
the positions of the hydrogen atoms by neutron crystallography, the in situ structural
information by small-angle neutron scattering (SANS), and the dynamics of the molecules byquasielastic neutron scattering (QENS). Protein deuteration makes significant contributions tousefulness of these techniques applied to the protein studies. In neutron crystallography, theproblem of cancellation of the density due to the negative density of hydrogens can beavoided, and the background mainly due to the incoherent scattering from hydrogens isreduced. In SANS, the technique of selected deuteration is applied to obtain the structural
information on the selected component within the complex. The QENS measurements on themacromolecular complex consisting of the deuterated components and the usual protonatedcomponent provide the information on the dynamics of the protonated component within thecomplex. Perdeuteration of the proteins is required for neutron crystallography and QENS,
while for SANS, the proteins deuterated to various degrees can be utilized. This is because thescattering length density of D2O solvent matches to that of the proteins with the deuterationlevel at about 80%. There are also various SANS techniques using the proteins deuterated tovarious degrees. It is thus useful to control the degrees of deuteration in producing thedeuterated proteins.
Based on the technique of protein deuteration using algal peptone, we have developed atechnique of producing the proteins at various deuteration levels. In this talk, I will describe asimple technique to produce such deuterated proteins in detail.

Optimized Strategies for Perdeuteration and Crystallization of
Human Carbonic Anhydrase for Neutron Crystallography
Katarina Koruza 1, Benedicte Lafumat 2, Maria Gourdon2, Wolfgang Knecht1,2, Kazuhiro
Akutsu3, Motoyasu Adachi4, Satoru Fujiwara4 and Zoë Fisher1,51Biology Department, Lund University, Sölvegatan 35, Lund, 22362, Sweden
Lund Protein Production Platform, Lund University, Sölvegatan 35, Lund, 22362, Sweden
3Neutron Science and Technology Center, Comprehensive Research Organization for Science and Society(CROSS), 162-1 Shirakata, Tokai-mura, Naka-gun, Ibaraki 319-1106, Japan
4Quantum Beam Science Research Directorate, National Institutes for Quantum and Radiological Science andTechnology, Tokai, Ibaraki, 319‐1106, Japan
5
Scientific Activities Division, European Spallation Source ERIC, Box 176, Lund, 22100, Sweden
2Neutron protein crystallography benefits from using fully deuterated protein by improved
signal-to-noise ratio and reduced background from hydrogen incoherent scattering. In ourstudy we use carbonic anhydrase IX (CA IX) as a model for deuterated protein expression andcrystallisation. Full-length native CA IX is a multi-domain membrane protein that is insoluble
and difficult to express or crystallise. To prepare crystals we are using the soluble catalytic
domain of CA IX with 6 surface mutations - CA IX surface variant (CA IX SV). Expression media
and cell culture conditions were optimized to produce high yields of deuterated protein. TheCA IX SV was then structurally characterized by X-ray, tested for deuterium incorporation bymass spectrometry, temperature stability, and propensity to crystallize. Using unlabeled
carbon source and recycled heavy water, we were able to get 65–77% deuterium
incorporation, sufficient for most neutron-based techniques, and in a very cost-effective way.
Expression of protein and cell culture conditions will be further optimized by using rich algal
peptone media at J-PARC labs. The crystallization of CA IX SV, was enabled by the use of
microseed matrix screening (MMS), commercial screens, and refinement screens. Theseresults demonstrate potential of MMS in growth of large crystals for neutron studies andprovide the basis for developing strategies for macromolecular crystallization.

Methyl Configurations Observed by Neutron Crystallography in
Perdeuterated Proteins
Motoyasu Adachi
Tokai Quantum Beam Science Center, Quantum Beam Science Research Directorate, National Institutes for
Quantum and Radiological Science and Technology, 2-4 Shirakata, Tokai, Ibaraki, 319-1106, Japan.

More than 20 neutron crystal structures of perdeuterated proteins have been deposited in
PDB database by 2017. The deposited number of crystal structures is estimated to be only
20% of total crystal structures solved by protein neutron crystallography. Althoughperdeuteration costs much to get deuterated chemicals and heavy water or commerciallyavailable medium for bacterial cultivation, crystallization and so on, the perdeuteration inprotein crystallography gives the three advantages as follows; 1) no cancellation of contour
maps by negative scattering length of hydrogen atoms, 2) higher contour map levels of
deuterium than hydrogen atoms due to longer scattering length for structural analysis, and 3)
reduction of background noise and enhancement of reflection signals with better S/N ratio forexperimental measurement.
Here, I would like to focus on the methyl configuration in proteins, because the cancellationof contour maps should adversely affect to the observation for methyl groups that includehydrogen atoms. The cancellation will cause the position of hydrogen atoms to be ambiguous.
The perdeuteration is needed to replace hydrogen atoms with deuterium atoms in the methylgroups. Recently, two neutron diffraction data sets for T4 lysozyme have been collected atboth room and cryo temperatures using perdeuterated protein crystals to 2.1 and 1.7 Å
resolution, respectively. As a result in joint refinement using X-ray and neutron diffraction
data, many differences of the methyl configurations are observed between the two conditions.
It appears that the methyl configurations are sensitive depending on its environment. In
addition, differences from methyl configurations are not always explained by steric hindrance
between atoms. These observations may provide a new insight into the analyses for protein
dynamics and by quantum chemical calculations. In this meeting, I would like to discuss about
importance for direct observations of the methyl configuration in proteins.