Open Call for Chemical and Biodeuteration Proposals at ESS

The Deuteration and Macromolecular Crystallisation (DEMAX) platform at the ESS is issuing a pilot call for chemical and biological deuteration proposals.

Deadline for proposal submission: 25th March 2022

Proposers notified of acceptance: May 2022

Molecules/support delivered: from September 2022

Submit proposals here: https://useroffice.ess.eu

More information here: https://europeanspallationsource.se/article/2022/01/10/pilot-call-chemical-and-biodeuteration-support-demax-platform

Questions? Get in touch! demax@ess.eu

Deuteration and Macromolecular Crystallisation (DEMAX) at ESS

The ESS DEMAX platform produces deuterated materials such as biomass, proteins, lipids and other small molecules for neutron techniques such as small angle scattering, reflectometry, protein crystallography, spectroscopy, and powder diffraction. DEMAX also supports crystallisation optimisation for large protein crystal growth (protiated and/or perdeuterated) for crystallography.

Scientists can submit proposals for deuterated materials for use in their neutron experiments at other facilities. Access is based upon peer review of proposals and scientific merit and is currently not limited to ESS member countries. The next call for proposals will be advertised at https://deuteration.net. For more information email demax@ess.eu.

DEMAX Publications:

  • F. Kozielski, C. Sele, V. O. Talibov, J. Lou, D. Dong, Q. Wang, X. Shi, M. Nyblom, A. Rogstam, T. Krojer, Z. Fisher, W. Knecht “Identification of fragment binding to SARS-CoV-2 nsp10 reveals ligand binding sites in conserved interfaces between nsp10 and nsp14/16” RSC Chemical Biology (2021) https://doi.org/10.1039/d1cb00135c
  • V. Kelpsas, A. Leung, C. von Wachenfeldt “Evolving Escherichia coli Host Strains for Efficient Deuterium Labeling of Recombinant Proteins Using Sodium Pyruvate- d3” International Journal of Molecular Science 22, 9678 (2021) https://doi.org/10.3390/ijms22189678
  • S. Aggarwal, C. von Wachenfeldt, S.Z. Fisher, E. Oksanen “A protocol for production of perdeuterated OmpF porin for neutron crystallography” Protein Expression & Purification 188, 105954 (2021) https://doi.org/10.1016/j.pep.2021.105954
  • A.U. Mushtaq, J.Ådén, L.A. Clifton, H.Wacklin-Knecht, M. Campana, A.P. G. Dingeldein, C.Persson, T.Sparrman and G. Gröbner, “Neutron reflectometry and NMR spectroscopy of full length Bcl-2 protein reveal its membrane localization and conformation”, Communications Biology  4,507 (2021). https://doi.org/10.1038/s42003-021-02032-1
  • S. Jamali, V. V. Mkhitaryan, H. Malissa, A. Nahlawi, H. Popli, T. Grünbaum, S. Bange, S. Milster, D. M. Stoltzfus, A. E. Leung , T. A. Darwish, P. L. Burn, .J. M. Lupton, C. Boehme “Floquet spin states in OLEDs “ Nature Communications 12, 465 (2021) https://doi.org/10.1038/s41467-020-20148-6
  • J. Larsson, A.E. Leung, C Lang, B. Wu, M. Wahlgren, T. Nylander, S. Ulvenlund, A. Sanchez-Fernandez “Tail unsaturation tailors the thermodynamics and rheology of a self-assembled sugar-based surfactant” Journal of Colloid and Interface Science 585, 178-183 (2021) https://doi.org/10.1016/j.jcis.2020.11.063
  • J. Larsson, A. Sanchez-Fernandez, A.E. Leung, R. Schweins, B. Wu, T. Nylander, S. Ulvenlund, M. Wahlgren “Molecular structure of maltoside surfactants controls micelle formation and rhelogical behaviour” Journal of Colloid and Interface Science 581, 895-904 (2021) https://doi.org/10.1016/j.jcis.2020.08.116
  • T. Cleveland IV, E. Blick, S. Krueger, A. Leung, T. Darwish, P. Butler “Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering” IUCrJ, 8 (2021) doi.org/10.1107/S2052252520013974
  • R. Delhom, A. Nelson, V. Laux, M. Haertlein, W. Knecht, G. Fragneto, H.P. Wacklin-Knecht, “The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry”, Nanomaterials 10, 2439 (2020) https://doi.org/10.3390/nano10122439
  • J.M.O. Rodriguez, E. Krupinska, H. Wacklin-Knecht, W. Knecht, Preparation of human dihydroorotate dehydrogenase for interaction studies with lipid bilayers, Nucleosides Nucleotides & Nucleic Acids 1-14 (2020) https://doi.org/10.1080/15257770.2019.1708100
  •  A. Luchini, R. Delhom, V. Cristiglio, W. Knecht, H. Wacklin-Knecht, G. Fragneto, Effect of ergosterol on the interlamellar spacing of deuterated yeast phospholipid multilayers, Chemistry and Physics of Lipids, 227, 104873 (2020) https://doi.org/10.1016/j.chemphyslip.2020.104873
  • A. Rogstam, M. Nyblom, S.Christensen, C. Sele, V.O. Talibov, T. Lindvall, A.A. Rasmussen, I. André, S.Z. Fisher, W. Knecht, F. Kozielski, “Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2” International Journal of Molecular Sciences 21, 7375 (2020) https://doi.org/10.3390/ijms21197375
  • K. Koruza, A.B. Murray, B.P. Mahon, J.B. Hopkins, W. Knecht, R. McKenna, S.Z. Fisher, “Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX” International Journal of Molecular Sciences 21, 5277 (2020) https://doi.org/10.3390/ijms21155277
  • M.T.B. Clabbers, S.Z. Fisher, M. Coinçon, X. Zou, H. Xu, “Visualizing drug binding interactions using microcrystal electron diffraction” Communications Biology  3, 417 (2020) https://doi.org/10.1038/s42003-020-01155-1
  • M. Budayova-Spano, K. Koruza, S.Z. Fisher, “Large crystal growth for neutron protein crystallography” Methods in Enzymology 634, 21-46 (2020) https://doi.org/10.1016/bs.mie.2019.11.015
  • O. Bogojevic, A.E. Leung, “Enzyme-assisted synthesis of high-purity, chain-deuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine” ACS Omega 5, 22395-22401 (2020) https://doi.org/10.1021/acsomega.0c0282320
  • A. Sanchez-Fernandez, A.E. Leung, E.G. Kelley, A.J. Jackson “Complex by design: Hydrotrope-induced micellar growth in deep eutectic solvents” Journal of Colloid and Interface Science 581, 292-298 (2020) https://doi.org/10.1016/j.jcis.2020.07.077
  • A. Sanchez-Fernandez, C. Diehl, J.E. Houston, A. Leung, J.P. Tellam, S.E. Rogers, S. Prevost, S. Ulvenlund, H. Sjögren, M. Wahlgren “An integrative toolbox to unlock the structure and dynamics of protein-surfactant complexes” Nanoscale Advances 2, 4011-4023 (2020) doi.org/10.1039/D0NA00194E
  • K. Koruza, B. Lafumat, M. Nyblom, B.P. Mahon, R. McKenna, S.Z. Fisher, ”Structural comparison of protiated, H/D exchanged, and deuterated human carbonic anhydrase IX” Acta Crystallographica D 75, 895-903 (2019) https://doi.org/10.1107/S2059798319010027
  • K., Koruza, B.P. Mahon, M.P. Blakeley, A. Ostermann, T. Schrader, W. Knecht, R. McKenna, S.Z. Fisher, “Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative” Journal of Structural Biology 205, 147-154 (2019) https://doi.org/10.1016/j.jsb.2018.12.009 
  • A. Raasakka, S. Ruskamo, R. Barker, O.C. Krokengen, G.H. Vatne, C.K. Kristiansen, E.I. Hallin, M.W.A. Skoda, U. Bergmann, H. Wacklin-Knecht, N.C. Jones, S.V. Hoffmann, P. Kursula, ”Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail”, PLoS One 14.  24 (2019) https://doi.org/10.1371/journal.pone.0216833 

Deuterated PEG Oligomers at JCNS

Polyethylene glycol (PEG) is one of the most widely used polymers. There is a large number of procedures to synthesize PEG and PEG containing materials in the hydrogenous version but also deuterium labelled. The situation is different for oligomeric PEG, having a molecular weight range of about 200 to 1,000 g/mol. These compounds play an important role in pharmaceutical and cosmetic products, in functional fluids like lubricants or in polymer products. In fundamental research oligomeric PEGs are used as building blocks in synthetic chemistry, as solvent but also in many biological applications. Oligomeric PEG is produced industrially in large quantities. So far, procedures for its synthesis in small quantities using ordinary lab equipment do not exist. Consequently, deuterated PEG oligomers are not available.

At the deuteration lab at JCNS we have established a procedure which now allows the synthesis of these compounds in the lab scale. The process is based on the oligomerization of commercially available deuterated ethylene oxide (d-EO) using deuterated and partially potassium metalated ethylene glycol as initiator (see Figure 1). The reaction can be carried out in a steel reactor at a moderately elevated pressure in the batch mode.

Figure 1. Synthesis of deuterium labelled PEG oligomers using partially potassium metalated ethylene glycol-d4 and ethyleneoxide-d4 as educts.

The key step of this process is the design of an initiator, which solubilizes in organic solvents. Usually, the transformation of a diol like ethylene glycol to its alkali alcoholate yields highly insoluble products already at low metalation degrees. The same holds for KOH, which is used in industrial high temperature processes for PEG production. This leads to an inhomogeneous initiation process in the ethoxylation step and a broad MW distribution of the product. For this reason, only about 4% of the OH-groups in d-ethylene glycol were replaced by OK-groups with the help of potassium metal and solubilized in 1,4-dioxane. The reaction with d-EO occurred at 100 °C. This procedure was used to synthesize a series of d-PEGs in the molecular weight range between 200 and 600 g/mol. A SEC trace of the product d-PEG 400 is shown exemplary in Figure 2 together with its commercial equivalent. In this case the deuterated variant shows even a slightly narrower MW distribution than the commercial material.

Figure 2. SEC traces of deuterated PEG 400 (black) and the commercial hydrogenous equivalent (red).

As a result of the normal work-up procedure the hydroxyl groups exist in the OH-version. With the help of D2O, they can be converted to OD. In Figure 3 the 2H-NMR spectrum of deuterated PEG 400 is shown. In pyridine as solvent the OD-end groups appear at 6.0 ppm, the methylene units next to the OD-functionalities are located at 3.85ppm and all residual methylene groups at 3.5 ppm. Using mixtures of h-EO and d-EO, partially deuterated PEGs are accessible. This can be useful for example if a lower scattering length density of the PEG is needed for contrast matching with D2O.

Figure 3. 2H-NMR spectrum of deuterated PEG 400 in dry h-pyridine.

ESS DEMAX lab priority access for COVID-19 related proposals

The Deuteration & Macromolecular Crystallisation (DEMAX) platform at ESS is offering prioritised access to laboratory services for scientists and researchers working on COVID-19-related research projects.

DEMAX, which is currently running it second pilot proposal round, is able to provide expertise, advice and limited materials to support research aimed at the critical need to gain a better understanding of COVID-19.

ESS will evaluate prioritised access to the laboratory services for COVID-19-related proposals on a continuous basis.

Find out more: DEMAX COVID-19 support

Figure 1: Ribbon diagram of methyltransferase in complex with activator protein of SARS-CoV2 (PDB ID 6w4h). 

The first molecules requested from the ESS DEMAX team have been delivered and utilised in a successful neutron experiment

blog post B-hdm-d31ILL_HDM_d[1]

Beta-hexadecyl maltoside-d31, the surfactant produced at the ESS chemical deuteration lab; and the SANS data it contributed to (collected on the D11 instrument at the ILL).

The ESS DEMAX team held its first pilot call for scientific proposals earlier this year, in order to establish a system that runs smoothly in time for the first science to be performed on ESS instruments. The call offered a range of biologically and chemically deuterated materials as well as support for crystallisation. Nineteen proposals were received, requesting services across the three areas of expertise.

In June and July, the first deuterated molecules were delivered to proposers, and in July, one of these molecules was utilised in a SANS experiment on the D11 instrument at ILL. Johan Larsson, the principle investigator in the work, studies the self-assembly of sugar-based surfactants with applications in the formulations field, including shampoos and cosmetics. In order to tune the properties of the surfactants, he is investigating the use of combinations of sugar-based surfactants and other surfactant-like components. Using deuterated sugar-based surfactants allowed him to discriminate between the different components in the mixture.

Surfactants such as this one; lipids; monomers and other small molecules were requested from the ESS chemical deuteration during the first proposal call. Part of the significance of chemical deuteration is that it can be used to produce unnatural molecules, and we can introduce deuterium judiciously into the molecule. This unnatural surfactant molecule can be produced at ESS, with deuterium located at very specific locations – those which are required for the SANS experiment.

The ‘NET widens

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The DEUNET is growing! Our most recent meeting was held in Lund last week and included several new members. The Lund Protein Production Platform have joined the DEUNET, and biological deuteration and crystallisation at ESS has joined chemical deuteration in the network. Larodan AB also joined their first DEUNET meeting, and we were very fortunate to have Professor Hironao Sajiki from Gifu Pharmaceutical University, Japan, present some of his recent work in the field of deuteration chemistry.

The member labs provided updates and several possible collaborative projects were identified. We are, as always, grateful to the DEUNET advisory panel for travelling to the meeting and providing valuable advice. The DEUNET advisory panel consists of both deuteration facility members (Peter Holden from ANSTO, and Trevor Forsyth from ILL), and neutron users (Karen Edler, Jian Lu and Thomas Hellweg).

DEMAX’s first call for proposals a great succESS

The ESS DEMAX platform recently published its first call for proposals for deuteration and crystallisation support. Nineteen proposals were received, covering chemical deuteration, biological deuteration and crystallisation. The review process is underway and results will be communicated by the end of May. Thanks to all those neutron experimenters who submitted proposals!

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BrightnESS²: Enabling Further Collaboration Between Deuteration Partners

BrightnESS² is a three-year, EU-funded project within the European Commission’s Horizon 2020 Research and Innovation programme which focuses on the long-term sustainability of ESS and neutron scattering in Europe, and to further strengthen the network of facilities for research using neutrons. A total of 15 institutes and universities from Europe and South Africa are participants within the project.

As part of BrightnESS² the Deuteration Facility at the SFTC (UK) and the ESS Chemical Deuteration Laboratory (SE) are collaborating on the synthesis of structured deuterated phospholipids. At the moment, a limited number of deuterated, mixed acyl phospholipids (Figure 1) are available, and those that are available and prohibitively expensive for many neutron users and experiments.

 

general mixed acyl phospholipid.jpg
Mixed acyl phosphocholine lipids, where R1 and Rare different acyl tails.

The STFC Deuteration Laboratory has a wealth of experience in the synthesis of homo-acyl phospholipids and an active user community which continuously requests new phospholipid analogues for neutron experiments. The ESS Chemical Deuteration Laboratory is researching the application of enzymatic catalysis to the synthesis of deuterated molecules. Together, the facilities are working together to establish a new, modular method for the synthesis of tail-deuterated, mixed-acyl phospholipids, using enzymes to allow the regiospecific substitution of tails at the 1- and 2-positions. The modularity of the approach should mean that a large number of molecules can be accessed. The first target molecules are 1-palmitoyl-d31-2-oleoyl-d33-sn-3-phosphocholine (POPC-d64, Figure 2a) and 1-palmitoyl-d31-2-(9-oxononanoyl)-sn-3-phosphocholine (PoxnoPC-d31, Figure 2b).

POPC and PoxnoPC
2a) POPC-d64d31.