SLIM: A Short‐Linked, Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

Abstract

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules,p referably acquired under native conditions.I nt his regard, pulsed dipolar EPR spectroscopy( PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However,t he currently available spin labels have diverse deficiencies for in-cell applications,f or example,l ow radical stability or long bioconjugation linkers.Inthis work, asynthesis strategy is introduced for the derivatization of trityl radicals with am aleimide-functionalized methylene group.T he resulting trityl spin label, called SLIM, yields narrowd istance distributions,e nables highly sensitive distance measurements down to concentrations of 90 nm,a nd shows high stability against reduction. Using this label, the guanine-nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O( YopO) is shown to changei ts conformation within eukaryotic cells.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Ox‐SLIM: Synthesis of and Site‐Specific Labelling with a Highly Hydrophilic Trityl Spin Label

“…Next, the carboxylation of trityl alcohol 21 was carried out by deprotonation with sBuLi in TMEDA/n-hexane using gaseous CO 2 as electrophile. Other frequently applied carboxylation reagents such as Boc 2 O [16,24,40] more likely contain traces of water,s everelyd iminishing the yield of the dicarboxylated product. The obtained dicarboxylic acid was then converted into the corresponding tert-butyl diester 22 using O-tert-butyldiisopropylisourea, [42] since classical Steglichc onditions [43] (DCC, DMAP)o rthec ombination Boc 2 O/DMAP [44] yielded only traces of product.…”

“…Initial studies with 3C [18] provedt he suitability of trityl radicals for this purpose in general,t hought he bioconjugation proceeded with low efficiencya nd inseparable aggregates. Trityl spin labels with functionalg roups forb ioconjugation based on maleimides (4C, 5C, 6C) [22][23][24] coped with this challenge by providing highly selective and efficient linkaget oc ysteines. While 2C-5C are simple esters/ amides of 1C,t he benzylic CH 2 -linker used to construct the socalled SLIM-trityl 6C was showntoimprovethe stability towards intracellular reduction by shifting its redox potentials.C oncomitantly, as hort linkeri si ntroduced giving rise to narrow distance distributions.…”

“…While 2C-5C are simple esters/ amides of 1C,t he benzylic CH 2 -linker used to construct the socalled SLIM-trityl 6C was showntoimprovethe stability towards intracellular reduction by shifting its redox potentials.C oncomitantly, as hort linkeri si ntroduced giving rise to narrow distance distributions. [24] Another inherentd rawback caused by the lipophilic core of these first generation trityl spin labels is aggregation with themselves [25,26] and hydrophobic interactions with proteins. [18,19,22,23] This complicates the spin labelling of proteins and can adversely influence the PDS-derived dis-tance distributions.…”

Excitation Energy Transfer and Exchange‐Mediated Quartet State Formation in Porphyrin‐Trityl Systems

“…Porphyrins were chosen as the chromophores since they are highly photostable, [24] exhibit characteristicE PR signatures, [25] and their central metal can easily be modified, enabling av ariation of the excited state energetics while avoiding major changes to the molecular structure. At etrathiaryl trityl radical, derivatives of which are commonly employeda ss pin labels, [26][27][28][29] was attached to the porphyrin via ap henyl linker and is used hereast he stable radicalm oiety.C ompared to more commonly employed stable radicals, such as nitroxides, trityl radicals are characterised by a very narrow EPR line and slow spin-lattice relaxation, [30] which might be particularly interesting for spin information storage applications. In addition, the chosen molecular design and synthetic approacha llows as ystematic modification of the individual buildingb locks of this photogenerated three-spin system.…”

A Two‐Armed Probe for In‐Cell DEER Measurements on Proteins**

“…The resistance of nitroxides towards reductionc an be increased through modification of the nitroxide-containingr ing, usually by attaching substituents. [18,19] Other approaches replaced the nitroxide by other radicalt ypes, such as the trityl radical; [20,21] see also the review by Bonucci et al [22] Here we focus on one such alternative, the Gd III ion, with S = 7/2, which has ab etter stability than the standard nitroxides and, especially at high magnetic field, provides high sensitivity and therefore is ag ood candidate for in-cell DEER measurements. [23][24][25] AD OTA( 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid) based Gd III complex, functionalizedwith amaleimide group,w as successfully used for in-cell DEER on ap rotein, althoughawide distance distribution was found, due to the flexibility of the linker.…”