Design and synthesis of cysteine-specific labels for photo-crosslinking studies

Chemical cross-linking mass-spectrometry (XL-MS) represents a powerful methodology to map ligand/biomacromolecule interactions, particularly where conventional methods such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (EM) are not feasible. In this manuscript, we describe the design and synthesis of two new photo-crosslinking reagents that can be used to specifically label free thiols through either maleimido or methanethiosulfonate groups and facilitate PXL-MS workflows. Both crosslinkers are based on light sensitive diazirines – precursors of highly reactive carbenes which offer additional advantages over alternative crosslinking groups such as benzophenones and aryl nitrenes given the controlled rapid and more indiscriminate reactivity.


Introduction
Chemical crosslinking-mass spectrometry (XL-MS) is an increasingly important approach to map biomacromolecule and small-molecule/biomacromolecule interactions. 1-4 Furthermore, XL-MS is uniquely placed to facilitate analysis of transient and dynamic interactions and/or conformational changes. Given that cross-linking "covalently traps" a protein or its complex, XL-MS has the potential to map changes in conformation and interactions with time. However, to do so requires reactive intermediates that react rapidly and indiscriminately with proximal functionality to ensure supramolecular connectivity is accurately captured upon cross-linking. 5 In this regard photoinduced cross-linking (PXL) 6 is advantageous in that light is used to trigger cross-linking and the suite of functionalities used for PXL tend to be more indiscriminate in their reactivity than traditional cross-linking reagents 7-9 such as NHS-esters which require nucleophilic residues to be present at an interface. We recently synthesized two cysteine specic diazirines as PXL reagents and outlined a workow for mapping protein interactions that exploits tag-and-transfer ( Fig. 1a and  b). 10 Using a cysteine selective methanethiosulfonate (MTS) or maleimido group to attach the reagents to the protein allows precise positioning of the crosslinking functionality within the protein. The photosensitive diazirines represent ideal functional groups for PXL-MS workows 5,11-17upon irradiation, the resultant carbenes insert rapidly into X-H bonds (including O-H, N-H, S-H and C-H bonds). 10 Diazirines have been shown to be advantageous over other common cross-linking groups (e.g. Fig. 1 Overview of tag-transfer PXL for mapping PPIs and diazirine based PXL reagents, (a) tag-transfer PXL workflow: a thiol-containing "bait" protein (green) is labelled with the reagent (here MTS-diazirine). After adding a partner protein (blue), irradiation with 365 nm UV light, reveals a carbene that can react with the partner protein. Reduction of the disulphide reveals a thiol which can be further labelled, (b) previously described diazirine based PXL reagents (c) diazirine based PXL reagents described in this study. benzophenone and phenyl azide) arising as a consequence of more rapid and indiscriminate reactivity leading to effective encoding of supramolecular connectivity. 18 However the synthesis of dizarines is more challenging, limiting their use.
In our prior work, we introduced two heterobifunctional reagents (MTS-diazirine 1 and MTS-TFMD 2) (Fig. 1b) bearing a methanethiosulphonate group to facilitate specic labelling of Cys residues on a "bait" protein, creating a cleavable disulde bond bearing a diazirine. 10 Following cross-linking with partner proteins, this disulde could be reduced leaving a thiol on the partner protein. In this work we describe the design and synthesis of two further reagents; (i) N-maleimido-diazirine 3 and (ii) MTS-alkynyldiazirine 4 (Fig. 1c). N-Maleimido-diazirine 3 can be used to label thiols on peptides or proteins via a conventional conjugate addition, used widely in protein labelling. 19 MTS-alkynyldiazirine 4 can be used to label thiols on peptides or proteins, yet bears an additional bio-orthogonal group (the alkyne) that could be exploited to introduce further functionality (e.g. uorophore, biotin) by "click" chemistry 20,21 to support chemical proteomics workows.

Results and discussion
We rst prepared an alkyl diazirine based reagent that could be used to label proteins via Michael addition of a thiol to a maleimide. This bioconjugation reaction is widely used 19 and trivial to perform for non-specialists whilst avoids the use of more hazardous and less chemoselective alkylating reagents e.g. ahalocarbonyl containing or iodoalkyl reagents. N-Maleimidodiazirine 3 was prepared in two steps (Fig. 2a) from 4hydroxybutan-2-one 5. One pot introduction of the diazirine group to give compound 6 22 was achieved via iodine mediated oxidation of the diaziridine that results from reaction of the ketone hydroxylamine-O-sulfonic acid in liquid ammonia. This was followed by Mitsunobu reaction with N-maleimide 7, to yield the labelling reagent in 43% over two steps.
This reagent could in principle be used to label thiols on proteins and exploited in PXL-MS (Fig. 2b). A difference in using this reagent in contrast to the tag-and-transfer reagents described previously is that following cross-linking, a permanent covalent link between bait and partner proteins will be generated, which may be advantageous under conditions where the former reagents are unstable (e.g. the reducing environment of the cell).
The second reagent has been designed to be used as a tagand-transfer diazirine, but with an additional biorthogonal alkyne. Consequently, the synthesis was longer, however gram quantities could be prepared. Beginning with ethyl acetoacetate 8, alkylation with 3-bromopropyne 9 and protection of the ketone 10 as the cyclic acetal 11, followed by reduction of the ester to alcohol 12 23 and removal of the acetal afforded the hydroxyl ketone substrate 13 for diazirine ring formation. As before, the reaction of the ketone with hydroxylamine-Osulfonic acid in liquid ammonia followed by oxidation with iodine gave diazirine 14. 24 Subsequent conversion of the alcohol 14 to an alkyl iodide 15 and reaction with sodium methanethiosulfonate afforded the nal tag-transfer MTSalkynyldiazirine 4 (Fig. 3).
This label could be used in tag and transfer PXL-MS applications in the same way as our previously described workow. 10 Labelling of bait protein followed by photocrosslinking and reduction, transfers onto a partner protein a thiol group, but also an alkyne. We envision this second cysteine-specic label as being of use for the enrichment of cross-linked proteins where multiple thiols are already present in the partner protein or other components of the sample under analyses; the  biorthogonal advantages of the alkyne should be readily exploited. Alternatively, the alkyne could be used to ligate e.g. uorophores for the construction of biosensors. 25

Conclusions
In summary, we have described the design and synthesis of two new diazirine based cross-linking reagents that can be used to specically label free thiols. These labels could be applied in structural analyses of protein-protein interactions using PXL-MS by conjugating them to bait peptides or proteins and subsequent crosslinking with partner proteins. In principle, such a strategy could readily be applied to other ligand/ biomacrolecule interactions as long as a thiol is present in the bait ligand.

General considerations
All solvents were purchased from Fisher Scientic and all reagents were purchased from Sigma-Aldrich or Fluorochem unless otherwise stated and used without further purication. Purication by column chromatography was carried out using silica gel. Analytical thin layer chromatography (TLC) was conducted using Merck 0.25 mm silica gel pre-coated aluminium plates with uorescent indicator active at UV245. 1 H and 13 CNMR spectra were acquired on Bruker Avance III HD 400 series spectrometer at 400 MHz for 1 H, and 100 MHz for 13 C. Chemical shis are expressed as parts per million using solvent as internal standard (CDCl 3 7.26 ppm in 1 H and 77.16 ppm in 13 C spectra) and coupling constants are expressed in Hz. The following abbreviations are used: s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet and br for broad.

2-(3-Methyl-3H-diazirin-3-yl)ethanol (6) 22
NH 3 (approx. 100 mL) was condensed, using a dry ice/acetone condenser, into a ask containing 4-hydroxy-2-butanone 5 (15.2 mL, 170 mmol) and cooled to À78 C. Aer reuxing (À30 C bath temperature) for 5 h, hydroxylamine-O-sulfonic acid (21.15 g, 187 mmol) dissolved in MeOH (150 mL) was added at À78 C and the reaction mixture was allowed to heat to room temperature overnight. The resulting mixture was ltered, the solid residue was washed with MeOH (2 Â 20 mL), and the ltrate was concentrated to about 100 mL. Triethylamine (26 mL, 187 mmol) was added to the resulting solution followed by iodine in several portions while cooling the reaction mixture in ice. Aer adding 29.2 g (115 mmol) of I 2 , the colour of iodine persisted, indicating the end of the reaction. The solvents were carefully removed from the reaction mixture (25 C and 120 mbar) and the residue was partitioned between Et 2 O (200 mL) and brine (200 mL) containing sat. aq. Na 2 S 2 O 3 (10 mL). The organic layer was separated and the aqueous layer was extracted with Et 2 O (2 Â 100 mL). The combined organic extracts were dried over Na 2 SO 4 and concentrated to give crude diazirine 6, which was puried by column chromatography (SiO 2 , pentane/ Et 2 O 1/1) to give 8.64 g (51%) of product as a colourless liquid.

Conflicts of interest
There are no conicts to declare.