Iminothiol/thiourea tautomeric equilibrium in thiourea lipids impacts DNA compaction by inducing a cationic nucleation for complex assembly

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 Abstract: Our research on lipidic vectors for transfection led us to develop thiourea lipids able to interact with DNA. Hence, we developed a series of lipopolythioureas based on the strong hydrogen bond donor ability of thiourea. More recently we have reported a branched hydroxylated bis-thiourea derivative with interesting transfecting properties. The last step of the syntheses involved a strong acidic condition, leading to an unstable product upon storage.Therefore we designed a new synthesis in mild acidic conditions. Though they exhibit the same mass, the lipids obtained in the two different conditions differ by their interaction with DNA. We therefore explored the physicochemical properties of these two lipids by different means that we describe in this article. In order to insure easier and reliable 13 C-NMR studies of the thiourea group we have designed the synthesis of the corresponding 13 C-labelled thiourea lipids. We have thus shown that when the lipid was submitted to mildly acidic medium; only the thiourea group was observed; while a thiourea/charged and/or uncharged iminothiol tautomeric equilibrium formed when the last step of the synthesis was submitted to low pH. NMR experiments showed that this tautomeric equilibrium could not form in polar solvents. However, UV experiments on the liposomal form of the lipopolythiourea showed the presence of the tautomers. Lipid/DNA interaction consequently differed according to the acidic treatment applied. Eventually, these results revealed that on this particular thiourea lipid, electrostatic interactions due to cationic thioureas are likely to be responsible for DNA compaction and that this tautomeric form of the thiourea could be stabilised by hydrogen bonds in a supramolecular assembly. Nevertheless, this does not reflect a general thiourea lipid/DNA interaction as other thiourea lipids that are able to compact DNA do not undergo an acidic treatment during the final stage of their synthesis.

Lipopolythiourea/DNA interaction: A biophysical study

“…As referred to DNA condensation via carbohydrate based hydrogen bonds [35], these data could support a multi-layer model in which a first LPT layer would interact rapidly with DNA through thiourea/phosphate interaction, then this primary construct would be stabilized by the addition of more LPT to enhance the hydrophobic forces probably via hydrogen bonds. This hypothesis is consistent with the disassembly of supramolecular constructs by the addition of salt or trifluoroethanol able to displace hydrogen bonds [32].…”

“…Interaction could be detected as low as 1 LPT/PO reaching a plateau at 4 LPT/PO and led to a DNA condensed state. The fact that the TU4OH family was more efficient to interact with DNA than the other lipids has also been investigated by 13 C NMR, and tends to indicate that the thiourea moieties in these lipids are in a tautomeric thiourea/iminothiol form which might explain the strong difference of interaction obtained with these lipids [32]. The mixture of both ionic and hydrogen bonds would be a key to improve gene transfection as also indicated in a recent work where the authors added thiourea functions to their positively charged cyclodextrine based vectors [33,34].…”

Virtual screening identifies broad-spectrum β-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases

“…Of these thirty-one compounds, 24, 40, 46, 52 and 54, showed an inhibition activity higher than 70% and were all originally identified as potential ligands of a MBL. All the last present a thiol, a thiosemicarbazide or thiosemicarbazone moiety able to undergo an iminothiol/thiourea tautomerization and to generate a thiol group 30 , 31 . It must be noted that the p K a of sulfhydryl groups in MBL decreases by about two orders of magnitude, due to the zinc presence, leading to deprotonation and strong coordination of the metal ions 32 , 33 .…”

Lipothioureas as Lipids for Gene Transfection: A Review

“…Deprotection led to compounds of similar molecular mass, but with either an iminothiol or a thiourea function depending on the strength of the acid used (strong hydrochloric acid or weak acetic acid). The presence in liposomes of the iminothiol which can only form and remain stable in drastic conditions could only be explained by the favoured hydrogen bonds of the lipidic auto-assembly [36]. …”