New Deuterated Phospholipids with Unsaturated and Branched Alkyl Chains from the National Deuteration Facility

 
Authors

Tamim Darwish, Nageshwar Rao Yepuri, Greta Moraes, Marie Gillon, Peter Holden and Michael James

The surface of a biological cell is of great interest because it mediates interactions and exchanges between the cell and its surroundings.

Phospholipids bearing different saturated and unsaturated fatty acid chains are major components of biological membranes and have been the focus of vast numbers of scientific studies in fields such as molecular biology, biochemistry, chemistry, biophysics and pharmacology.

There exists substantial demand for isotopically labelled lipids for many studies. Deuteration is an essential prerequisite in many 2H NMR (1), mass spectroscopy (2), and neutron-scattering (3) studies. 

While the use of commercially available deuterated phospholipids with saturated acyl chains has been prevalent in neutron scattering studies, investigations of highly biologically relevant biomimetic cellular membranes have been lacking due to the non-availability of deuterated lipids and fatty acids with unsaturated (e.g., oleyl) and branched (e.g., phytanyl) chains.  The double bond in 1,2-dioleoyl-sn-glycero-3-phosphocholine ( DOPC) produces a kink in the alkane chain, which disrupts the lipid packing.

This disruption creates extra free space within the bilayer model which allows additional flexibility in the adjacent chains and makes the model resembles the structure of cell membrane in real life.

Figure 1. Phospholipid bilayer membrane, indicating hydrophilic headgroups and hydrophobic core, composed of lipid tails.

1,2-diphytanoyl-sn-glycero-3-phosphocholine ( DPhPC), a phytanyl-based lipid, is found in the cellular membranes of extremophile bacteria such as archaebacteria, which can survive in harsh environments, and is stable over a wide range of temperatures. The key reasons behind the utility of phytanyl-based lipids are a combination of their excellent chemical and mechanical stability and their capacity to form highly electrically insulating bilayer membranes. 

It is this last feature that has for decades allowed the effective study of pore-generating proteins using patch-clamping, electrical impedance spectroscopy and other electrochemical methods.  In nuclear magnetic resonance spectroscopy studies, 2H-NMR has proven to be a very effective probe for the elucidation of lipid structure and motion in model and biological membranes. However, relatively few research groups have employed this technique due to the difficulty in the synthesis of suitably 2H–labelled lipids.

With this background in mind, it becomes evident that there is a significant need at our National Deuteration Facility (NDF) to develop a convenient synthesis of deuterated DOPC and DPhPC.  In the last couple of years the NDF has been successful in producing gram-quantities of oleic acid-d32 by constructing the molecule from deuterated precursors, using multi-step organic synthesis reactions.

This has allowed us now to produce scalable quantities of DOPC-d64 by a single step reaction by using our oleic acid-d32 and a commercially available phosphocholine adduct.  Moreover, we have developed the capability to produce gram-quantities of specifically deuterated DOPC-d9 where deuteration is selectively incorporated at its hydrophilic choline head (Figure 2).
 

Figure 2. Deuterated DOPC at a) the hydrophobic alkyl chains (DOPC-d64), b) the hydrophilic choline head (DOPC-d9).

Figure 3. 1H NMR spectra (400 MHz, CDCl3) of top: commercially protonated DOPC (Avanti lipids), bottom: DOPC-d64  (NDF) which shows depletion of protons signals from the alkyl chains (region of low frequency (0-3 ppm)).

In addition, the chemical deuteration team at NDF has recently developed a method for stereoselective synthesis of fully deuterated Phytanic acid-d39 and its phospholipid derivative 1,2-diphytanoyl-sn-glycero-3-phosphocholine-d78. DPhPC in its protonated form was recently used to form tethered model membranes for neutron reflectometry studies, and was effective at revealing the membrane structure against a solution interface comprised of heavy water (D2O)(4). 

Membranes formed using protonated DPhPC however would not be an effective platform for studies seeking to investigate the location and structure of integral and trans-membrane proteins, as both lipid and protein have similar neutron scattering length densities.  In these instances, tail-deuterated DPhPC-d78 would be essential to support such studies, and the NDF is now able to produce these molecules which have been commercially unavailable before in their deuterated forms.

Figure 4. Deuterated a) DPhPC-d78 and b) deuterated phytanic acid-d39 produced at NDF.

References:

1. For example see: Gehman, J. D., Sani, M. A and Separovic, F. (2011), Solid-state NMR of membrane-acting antimicrobial peptides. In Biomolecular NMR Spectroscopy, eds. A. Dingley and S. Pascal, IOS Press, Amsterdam, The Netherlands, Chapter 8, pp 137-161.
2. For example see: W K Rohwedder, W. K. (1985), Mass spectrometry of lipids labeled with stable isotopes, Prog. Lipid Res. 24, 1-18.
3. For example see: Darwish, T. A., Smith, A. R. G., Gentle, I. R., Burn, P. L., Luks, E., Moraes, G., Gillon, M., Holden, P. J., and James, M. (2012), Deuteration of conjugated aromatic heterocycles for morphological studies of organic light emitting devices, Tetrahedron Letters 53, 931–935.
4. Duncan J. McGillivray, Gintaras Valincius, David J. Vanderah, Wilma Febo-Ayala, John T. Woodward, Frank Heinrich, John J. Kasianowicz and Mathias Lösche, 2007, Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes, Biointerphases 2, 21-33.