Fatiha Benyahya

Isabelle Nadaud

Ludovic Georges

Alain Loussert

Isabelle Lhommet

Hélène Rimbert

Philippe Leroy

Pierre Sourdille

Have a break: how to decipher Spo11.2 role in Triticum aestivum?

Spo11.2 protein is part of the Spo11 complex that initiates meiotic recombination in plant (1). Spo11.2 role is well-documentated in diploid species but remains unknown in polyploid species (3). The objectives of this study were to decipher the Spo11.2 protein's role in the hexaploid bread wheat (Triticum aestivum L., AABBDD; 2n = 6X=42) using simple, double and triple mutants. We compared them between each other at different meiotic scales: phenotype, pollen integrity and meiosis progression (Part 1); homologous recombination (Part 2); genomic and transcriptomic sequences (Part 3). Have a break: how to decipher Spo11.2 role inTriticum aestivum? Triticum aestivum Spo11.2 has the same role in recombination as in diploid species. The abd -/-mutants were sterile, present meiosis anomalies leading to synapse failure while sterile bd -/- mutants conserved again the capacity to form some chiasmata. However the loss of Ta Spo11.2-A function could constitute the first step to the wheat genome fractionation (4). Spo11.2 in hexaploid Triticum aestivum is essential to successfully meiotic recombination as in diploids species. Mutants bd -/-and abd -/-show anomalies of meiosis with appariement defaults in metaphase I (presence of singletons) and polyades formation.

The percentage of pollen viability is very significantly reduced in Spo11.2 bd -/-(70%) and abd -/- mutants (88%) Mutant Spo11.2 abd -/-is sterile.

How takes place meiosis?

Are mutants fertiles?

Fatiha

  Benyahya 1,2 , Isabelle Nadaud 1 , Ludovic Georges 1 , Alain Loussert 1 , Isabelle Lhommet 1 , Hélène Rimbert 1 , Philippe Leroy 1 , Pierre Sourdille 1 1 UMR INRA / UCA 1095 Unité GDEC, 5 chemin de beaulieu 63000 Clermont-Ferrand, France 2 UCA 24 avenue des Landais 63170 Aubière, France 3 CRBC Plate-forme CLIC faculté de médecine, 28 place Henri Dunant 63000 Clermont-Ferrand, France Acknowlegments: Véronique Lesage 1 for providing Renan seeds irradiated and Caroline Vachias 3 for help to acquire confocal LSM 800 images. EMBO Meiosis Workshop 25 th -29 th August 2019 La Rochelle

  of meiosis atlas ABD +/+ vs mutants Spo11.2 bd -/-and abd -/-1.B. Viability of pollen (%)

Part 1

The deletion of 2 nucleotides and the presence of splicing site in Ta Spo11.2-A copy lead to an impaired protein.

Part 3

Why genome A copy of Spo11.2 remains active? The number of chiasmata is strongly reduced (X8) for bd -/-mutants.

The copy A conserved a slight activity while abd mutants cannot pair.

Can homologous chromosomes recognize each other?

ASY1

Protein of lateral axe

How recombination occurs?

ABD +/+ : 727 focis were counted on average (more or less 193) bd -/-and abd -/-mutants: the number of DMC1 focis is strongly reduced by a factor of 4 and 10 respectively. T-Student NS: non significant (1) ABD +/+ p<0,0005

(2) bd -/-p<0,05

T-Student

(1) ABD +/+ p<0,005

(2) ABD +/+ p<0,05

(3) bd -/-p<0,05


Olivier Abodja

Nadia Touati

Mathieu Morel

Sergii Rudiuk

Damien Baigl
email: damien.baigl@ens.psl.eu

ATP/azobenzene-guanidinium self-assembly into fluorescent and multi-stimuli responsive supramolecular aggregates

come

Introduction

There is a general interest in developing stimuli-responsive systems due to their ability to modulate their functions in response to endogenous signals or external stimulations [START_REF] Wei | Stimuli-Responsive Polymers and Their Applications[END_REF][START_REF] Mendes | Stimuli-Responsive Surfaces for Bio-Applications[END_REF][START_REF] Blanco-Gómez | Controlled Binding of Organic Guests by Stimuli-Responsive Macrocycles[END_REF] . A widely used strategy to convert a passive system into a responsive one, consists in tethering a stimuli-responsive group, such as a photochrome, a pH-sensitive group or a redox species [START_REF] Volarić | Molecular Photoswitches in Aqueous Environments[END_REF][START_REF] Szymański | Reversible Photocontrol of Biological Systems by the Incorporation of Molecular Photoswitches[END_REF][START_REF] Yusran | Electroactive Covalent Organic Frameworks : Design , Synthesis , and Applications[END_REF][START_REF] Sun | Photoresponsive Covalent Organic Frameworks with Diarylethene Switch for Tunable Singlet Oxygen Generation[END_REF][START_REF] Bapat | Redox-Responsive Dynamic-Covalent Assemblies: Stars and Miktoarm Stars[END_REF] through adequate covalent modification. With the repertoire of known chemical reactions and coupling methods, this powerful approach can lead to virtually any user-defined system designed to respond to a desired stimulus. However, the necessity of covalently modifying the entity to be stimulated can alter its properties. It also requires to adjust the modification conditions for each system, making the approach case-specific. Stimulus-responsive selfassembly of molecular bricks through non-covalent interactions appears as an interesting alternative as it opens the possibility of dynamic actuation of inherently passive molecules without needing to modify them. This approach has already been demonstrated relevant for a large number of applications ranging from chemical sensors to drug-delivery systems [START_REF] Wang | Adaptive Soft Molecular Self-Assemblies[END_REF] . The resulting stimuli-responsive self-assembled systems are of particular interest when involving biological molecules to dynamically control their functions [START_REF] Lu | DNA Assembly-Based Stimuli-Responsive Systems[END_REF][START_REF] Shigemitsu | Supramolecular Assemblies Responsive to Biomolecules toward Biological Applications[END_REF][START_REF] Wang | Controlling the Self-Assembly of Biomolecules into Functional Nanomaterials through Internal Interactions and External Stimulations: A Review[END_REF][START_REF] Uhlenheuer | Combining Supramolecular Chemistry with Biology[END_REF] . Among biologically active molecules, nucleotides and analogues appear as particularly interesting targets for such a purpose [START_REF] Pu | Nucleosides, and Nucleotides: Versatile Biomolecules for Generating Functional Nanomaterials[END_REF] . For instance, adenosine 5'-triphosphate (ATP) is the universal energetic and ubiquitous molecule driving biological processes such as cellular division and whose concentration can correlate with pathological conditions, for instance in Parkinson's disease [START_REF] Zingman | ATP-Sensitive Potassium Channels: Metabolic Sensing and Cardioprotection[END_REF] .

Moreover, ATP bears negatively charged phosphates as well as an aromatic π-conjugated adenine ring allowing, multiple electrostatic, π-π and hydrogen bonding interactions, thus making it prone to be engaged in supramolecular self-assembled systems [START_REF] Deng | ATP-Responsive and ATP-Fueled Self-Assembling Systems and Materials[END_REF][START_REF] Mishra | ATP-Driven Synthetic Supramolecular Assemblies: From ATP as a Template to Fuel[END_REF] . Several stimuliresponsive ATP-based supramolecular systems have thus been developed but for only a few specific stimuli. One strategy consisted in exploiting ATP as an enzyme substrate to build enzyme-responsive assemblies. This was achieved in particular using phosphatase to control the fluorescence or chirality of the assemblies [START_REF] Li | Adenosine Triphosphate Templated Self-Assembly of Cationic Porphyrin into Chiral Double Superhelices and Enzyme-Mediated Disassembly[END_REF][START_REF] Wang | An Enzyme-Responsive Polymeric Superamphiphile[END_REF][START_REF] Mishra | Biomimetic Temporal Self-Assembly via Fuel-Driven Controlled Supramolecular Polymerization[END_REF][START_REF] Kumar | A Dynamic Supramolecular Polymer with Stimuli-Responsive Handedness for in Situ Probing of Enzymatic ATP Hydrolysis[END_REF] . Another approach explored the chelating properties of ATP with metals to develop ion-responsive supramolecular structures but these investigations were limited to small-sized complexes [START_REF] Sigel | Nucleoside 5′-Triphosphates: Self-Association, Acid-Base, and Metal Ion-Binding Properties in Solution[END_REF][START_REF] Yao | Colorimetric Detection of Copper Ions Based on a Supramolecular Complex of Water-Soluble Polythiophene and ATP[END_REF][START_REF] Yeung | Phosphate Derivative-Induced Supramolecular Assembly and NIR-Emissive Behaviour of Alkynylplatinum(Ii) Terpyridine Complexes for Real-Time Monitoring of Enzymatic Activities[END_REF] . Besides enzymes and metals, light constitutes a highly desired trigger to control self-assemblies due to its precise spatiotemporal control, tunability (wavelength, power) and remote character [START_REF] Yagai | Recent Advances in Photoresponsive Supramolecular Self-Assemblies[END_REF] . Light-responsive ATP-based systems to photochemically control the release of this nucleotide have been described but this involved photocleavable bounds, therefore precluding any photo-reversible response [START_REF] Jeschik | Photocaged and Mixed Photocaged Bioreversible-Protected ATP Derivatives as Tools for the Controlled Release of ATP[END_REF] . To our knowledge, photoreversible supramolecular assemblies involving unmodified ATP have yet to be demonstrated. To reach such a goal, azobenzene-containing molecules constitute promising co-assembly partners as they have been successfully exploited in photocontrolled supramolecular assemblies for their robustness and for a rapid and reversible trans-cis photoisomerization [START_REF] Schultz | Mechanism and Dynamics of Azobenzene Photoisomerization[END_REF] . Cationic derivatives of azobenzene can be used in particular to electrostatically interact with negatively charged mono-or polynucleotides, a process that can be rendered photodependent through the polarity and/or geometric changes upon azobenzene trans/cis isomerization [START_REF] Lubbe | Recent Developments in Reversible Photoregulation of Oligonucleotide Structure and Function[END_REF] . This was used for instance to photocontrol phenomena such as DNA compaction [START_REF] Estévez-Torres | DNA Compaction: Fundamentals and Applications[END_REF][START_REF] Rudiuk | Enhancement of DNA Compaction by Negatively Charged Nanoparticles. Application to Reversible Photocontrol of DNA Higher-Order Structure[END_REF][START_REF] Diguet | Photosensitive Surfactants with Various Hydrophobic Tail Lengths for the Photocontrol of Genomic DNA Conformation with Improved Efficiency[END_REF][START_REF] Sollogoub | Photocontrol of Single-Chain DNA Conformation in Cell-Mimicking Microcompartments[END_REF][START_REF] Venancio-Marques | Photosensitive Polyamines for High-Performance Photocontrol of DNA Higher-Order Structure[END_REF][START_REF] Zakrevskyy | DNA Compaction by Azobenzene-Containing Surfactant[END_REF][START_REF] Schimka | [END_REF] , co-acervation 36 , DNA intercalation [START_REF] Bergen | Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature[END_REF][START_REF] Dohno | Photoswitchable Molecular Glue for DNA[END_REF] or G-quadruplex formation [START_REF] Wang | Conformational Switching of G-Quadruplex DNA by Photoregulation[END_REF] . With the objective to build extended photoreversible ATP assemblies in water, and thus limit the possibility of precipitation, we avoided classical azobenzene cationic surfactants [START_REF] Rudiuk | Light-Regulated MRNA Condensation by a Photosensitive Surfactant Works as a Series Photoswitch of Translation Activity in the Presence of Small RNAs[END_REF][START_REF] Venancio-Marques | Modification-Free Photocontrol of β -Lactam Conversion with Spatiotemporal Resolution[END_REF][START_REF] Anyfantakis | Dynamic Photocontrol of the Coffee-Ring Effect with Optically Tunable Particle Stickiness[END_REF] and opted instead for a hydrophilic azobenzene partner, choosing guanidium as the cationic group for its strong affinity for nucleotide phosphate groups, including those of ATP [START_REF] Noguchi | Nonlinear Fluorescence Response Driven by ATP-Induced Self-Assembly of Guanidinium-Tethered Tetraphenylethene[END_REF][START_REF] Roy | Nucleotide Sensing with a Perylene-Based Molecular Receptor via Amplified Fluorescence Quenching[END_REF][START_REF] Okuro | Boronic Acid-Appended Molecular Glues for ATP-Responsive Activity Modulation of Enzymes[END_REF] . We thus used AzoDiGua, a symmetric diguanidinium azobenzene derivative and photodependent DNA intercalator [START_REF] Bergen | Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature[END_REF] which was shown to co-assemble with guanosine monophosphate into photoswitchable and extended fluorescent crystals [START_REF] Zhou | Photoswitchable Fluorescent Crystals Obtained by the Photoreversible Coassembly of a Nucleobase and an Azobenzene Intercalator[END_REF] . In this work we coassembled AzoDiGua with ATP and observed the spontaneous formation in water of the first photo-reversible micrometer-sized supramolecular assemblies containing ATP. The self-assembled structures scattered light and exhibited fluorescence only in their assembled state, resulting in a reversible photoswitching of both turbidity and fluorescence. We also exploited the presence of ATP in the supramolecular structures to dynamically control their self-assembly by other stimuli, including metal ions and alkaline phosphatase (ALP) enzyme. With a combination of methods including optical microscopy, fluorescence spectroscopy, and electronic paramagnetic resonance (EPR), we characterized the reversibility, highly dynamic character and the switchable optical properties of these multi-stimuli responsive ATP/azobenzene-guanidinium assemblies of a new kind.

Results and discussion

Preparation of ATP/AzoDiGua supramolecular aggregates in solution Figure 1 shows the main components and concept of this work. ATP (Figure 1A, left) was used as a biologically active brick to be self-assembled. To ensure the formation of extended and light-responsive assembly, ATP was co-assembled with the photosensitive DNA intercalator AzoDiGua [START_REF] Bergen | Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature[END_REF] which contains two guanidinium groups with a strong affinity for phosphates and an azobenzene group enabling both π-π interaction and photoswitchability (Figure 1A, right). We hypothesized that mixing ion-and enzyme-sensitive ATP with photosensitive AzoDiGua would lead to the spontaneous formation of multi-stimuli responsive assemblies in water at room temperature (Figure 1B). The direct equimolar mixture of the two components ([ATP] = [AzoDiGua] = 1 mM) at physiological pH (7.4) and room temperature led to the immediate formation of a turbid solution. Optical microscopy observations revealed the presence of micrometer-sized entities that progressively aggregated with each other over time (Figures 1C, S1, Movie S1). After 10 min, the aggregates reached an average size of around 20 µm in average. Assemblies of similar size and morphology were recently reported when ATP was mixed with oppositely charged ammonium-or guanidinium-rich polycations, highlighting the interest of exploiting electrostatic interactions but the resulting structures were not responsive [START_REF] Cakmak | Prebiotically-Relevant Low Polyion Multivalency Can Improve Functionality of Membraneless Compartments[END_REF] . To evaluate the role of the phosphate groups in the ATP-AzoDiGua selfassembly process, we performed the same experiments using ATP analogs, and especially the monophosphate (adenosine monophosphate, AMP) and the diphosphate (adenosine diphosphate, ADP) derivatives (Figure S2). After the same self-assembly time (10 min), a markedly different behaviour was observed between AMP, ADP and ATP. With AMP, the solution remained transparent and no aggregate could be detected by optical microscopy (Figure S2A), in agreement with results previously obtained after a thermal annealing treatment [START_REF] Zhou | Photoswitchable Fluorescent Crystals Obtained by the Photoreversible Coassembly of a Nucleobase and an Azobenzene Intercalator[END_REF] . In contrast, both ADP and ATP led to the formation of supramolecular aggregates but those obtained with ADP were of much smaller size (8 ± 1 µm) and led to a less turbid solution (Figure S2B) than that with ATP (Figure S2C). This evolution not only emphasizes the crucial role of the phosphate groups for the supramolecular self-assembly to occur but provides also a way to tune the aggregate characteristics by the number of phosphate per AzoDiGua nucleotide partner. When using ATP at an acidic pH (2.0 instead of 7.4), the ATP/AzoDiGua mixture solution remained transparent and no extended supramolecular structures could be detected by optical microscopy (Figure S3), which was attributed to a lower ionization degree of ATP (-2 ionization state at acidic pH), its possible hydrolysis to ADP at acidic pH or a combination of both effects [START_REF] Deng | ATP-Responsive and ATP-Fueled Self-Assembling Systems and Materials[END_REF] . All these results show the important role of the electrostatic interactions between the negatively charged phosphate groups of ATP and the cationic guanidinium moieties of AzoDiGua. To have further insights into the molecular interactions between AzoDiGua and ATP at a low concentration where microscopic aggregation did not occur, a spectroscopic titration of AzoDiGua by ATP was performed. We observed a hypochromic effect on the π-π * transition of AzoDiGua at 360 nm with increasing amounts of ATP (Figure S4). This effect is in agreement with the establishment of π-π interactions between ATP and AzoDiGua, which are probably involved in the formation of the supramolecular aggregates at higher ATP and AzoDiGua concentrations. ATP is thus shown as a new binding partner to AzoDiGua, a DNA intercalant known so far for its propensity to form π-π and electrsotatic interactions within double-stranded DNA [START_REF] Bergen | Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature[END_REF] or with guanosine monophosphate nucleotide [START_REF] Zhou | Photoswitchable Fluorescent Crystals Obtained by the Photoreversible Coassembly of a Nucleobase and an Azobenzene Intercalator[END_REF] . In summary, ATP and AzoDiGua spontaneously self-assembled at room temperature and physiological pH into supramolecular aggregates through combined effects of electrostatic interactions and π-π stacking. These aggregates reached a size of a few micrometers in only a few minutes. To characterize their properties, including their reversibility and dynamic character, we fixed their composition ([ATP] = [AzoDiGua] = 1 mM) and assembly time to 10 min before studying their fluorescence and response to the application of different physical, chemical and biological stimuli.

Fluorescence of the supramolecular assembly in solution

The presence of the AzoDiGua in the aggregates led us to explore whether fluorescent properties could be acquired by these assemblies through the aggregation-induced fluorescence mechanism of azobenzene 48,49,50,51,52,53. Using fluorescence spectroscopy, we found that the ATP/AzoDiGua aggregates displayed a broad fluorescence excitation/emission with maxima at 400 nm and 650 nm for the excitation and emission, respectively (Figures 2A,S5). Notably, neither ATP nor AzoDiGua alone was significantly absorbing for a fluorescence emission at 650 nm (Figure S5) nor fluorescent when excited at 400 nm (Figure 2A, S6). Moreover, the same system prepared with ADP or AMP, instead of ATP, displayed a weak fluorescence for ADP/AzoDiGua where small aggregates formed, and no detectable signal for AMP/AzoDiGua where no assembly was observed (Figure S7). All these results show that the self-assembly between ATP and AzoDiGua was instrumental in the emergence of the fluorescence from the ATP/AzoDiGua suspension. Fluorescence microscopy (Figure 2B) evidenced that the fluorescence emission was mainly localized at the position of the micrometer-sized ATP/AzoDiGua aggregates (Figure 1C), which was thus attributed to the aggregation-induced fluorescence of azobenzene within the supramolecular aggregates. Therefore, ATP and AzoDiGua self-assembled to form fluorescent aggregates while their individual components, AzoDiGua and ATP alone, were not intrinsically fluorescent in solution. We then studied how different stimuli could dynamically control the assembly and therefore affect its aggregationinduced fluorescence properties.

Photoresponse of ATP/AzoDiGua assembly

Since the aggregates resulted from the co-assembly of AzoDiGua with ATP, we explored the challenge to realize the first photoreversible ATP-based supramolecular assemblies. Indeed, pure AzoDiGua is able to reversibly photoisomerize between trans and cis upon UV and blue light irradiation, respectively (Figure 3A) [START_REF] Bergen | Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature[END_REF] . To characterize the photoresponse of the supramolecular aggregates, we first prepared the ATP/AzoDiGua aggregates in the regular conditions ([ATP] = 1 mM, [AzoDiGua] = 1 mM, 10 min incubation at room temperature) using pure trans-AzoDiGua as a starting material and exposed them to successive irradiations by UV (395/25 nm, 26 mW•cm -2 ) for 3 min and blue (480/30 nm, 40 mW•cm -2 ) for 3 min. The first UV application resulted in the progressive disassembly of the supramolecular aggregates until their complete disappearance in less than 1 min (Movie S2, Figure 3B middle). The subsequent blue irradiation led to the recovery of the assembly with a similar kinetics (Movie S2, Figure 3B right). This was accompanied by a photocontrol of the optical properties of the ATP/AzoDiGua suspension. The first UV irradiation led to the loss of the suspension turbidity due to the vanishing of the light-scattering entities and appearance of the characteristic color of the cis-AzoDiGua isomer enriching the solution (Figure 3B middle, Figure S8). Similarly, the subsequent exposure to blue irradiation induced a change of the optical aspect of the suspension which became turbid again, due to the reappearance of the micrometer-sized ATP/AzoDiGua aggregates (Figure 3B right). Note that, compared to the initial suspension composed of pure trans-AzoDiGua, the suspension after UV and blue irradiation was less turbid and the aggregates in smaller number and with a smaller size due to an incomplete trans-isomer enrichment upon blue irradiation in the photostationary state [START_REF] Bergen | Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature[END_REF] . This successive disassembly/reassembly of the ATP/AzoDiGua aggregates upon UV/blue irradiation was then successfully repeated for five complete consecutive cycles (Figure S9). It resulted in a dynamic and reversible photocontrol of the suspension fluorescence correlating with the assembly state, i.e., high intensity for the blue-induced assembly and low intensity after UV-induced disassembly, respectively, with only small fatigue appearing from the third cycle (Figure 3C).

Again, the fluorescence after the first UV/blue cycle was less than in the initial solution (Figure S10) due to a lower fraction of trans-AzoDiGua in the photostationary state compared to the pure trans solution before irradiation. By exploiting the co-assembly between ATP and AzoDiGua, we could achieve both photoswitchable and aggregation-induced fluorescence of ATP/AzoDiGua aggregates leading to, the best of our knowledge, the first photoswitchable micro-meter sized ATP-based assembly.

ATP/AzoDiGua assembly response to metal ions

To increase the stimulus-responsivity of the photoswitchable aggregates, we exploited the chelating properties of ATP to study the possibility to control this assembly with ions. Knowing the strong affinity of ATP with divalent metal ions [START_REF] Sigel | Nucleoside 5′-Triphosphates: Self-Association, Acid-Base, and Metal Ion-Binding Properties in Solution[END_REF][START_REF] Wilson | Chelation of Divalent Cations by ATP, Studied by Titration Calorimetry[END_REF] , our hypothesis was that the addition of such cations would lead to the disassembly of the aggregates and the decrease of their fluorescence (Figure 4A). To characterize this effect, we started from the preformed ATP/AzoDiGua aggregates and added different metal cations (M n+ ) to ensure a final molar ratio [M n+ ]/[ATP] = 2. Interestingly, it was found that the turbidity of the solution immediately disappeared for the tested divalent cations (Mg 2+ , Ca 2+ , and Cu 2+ ) but remained for the monovalent ones (Na + , K + ) (Figure 4B), showing that the ion-triggered disassembly was valence-selective. The color due to the presence of trans-AzoDiGua (Figure S8) remained upon ion-triggered disassembly as the response was mediated by ATP chelation and not by a change of AzoDiGua isomerization state. The mechanism of the metal-mediated disassembly of the aggregates was then further investigated in the case of Cu 2+ using EPR spectroscopy, keeping all concentrations identical to that used for the assembly/disassembly experiments ([ATP] = 1 mM ; [AzoDiGua] = 1 mM ; [Cu 2+ ] = 2 mM). The EPR signal of pure Cu 2+ (2 mM) shifted to lower fields with appearance of a marked shouldering when it was mixed with ATP (1 mM) while ATP alone had no EPR signature for the same field range (Figure S11A). These effects, in agreement with previous reports [START_REF] Wang | A Cu(II)-ATP Complex Efficiently Catalyses Enantioselective Diels-Alder Reactions[END_REF] , are attributed to the interaction between Cu 2+ and ATP upon chelation [START_REF] Hoffmann | Copper(II) Ions Interactions in the Systems with Triamines and ATP. Potentiometric and Spectroscopic Studies[END_REF] . On the contrary, the EPR signal of pure Cu 2+ did not evolve upon addition of AzoDiGua (1 mM) showing the absence of interaction between these two cationic species (Figure S11B). Interestingly, AzoDiGua/ATP aggregates did not have any EPR signal but, after addition of Cu 2+ , a clear paramagnetic signal appeared and displayed the same features as the one of Cu 2+ /ATP mixture (Figure 4C). This new signal was thus attributed to the coordination of Cu 2+ with ATP and shows that the disassembly of ATP/AzoDiGua aggregates upon Cu 2+ addition is due to the competitive complexation between ATP and Cu 2+ . Optical microscopy observations of the ATP/AzoDiGua aggregates ([ATP] = 1 mM ; [AzoDiGua] = 1 mM) exposed to increasing amounts of Cu 2+ evidenced a gradual disassembly with an increase in [Cu 2+ ] and showed that an excess of Cu 2+ (2 mM) was necessary to ensure full and permanent disassembly (Figure S12). This ion-stimulated disassembly was then used to control the fluorescence of ATP/AzoDiGua co-assembly (Figure 4D, S13), showing either a way to chemically adjust the fluorescence of the system or an original method to titrate the presence of divalent cations (here, Cu 2+ ) by fluorescence. After disassembly for a ratio [Cu 2+ ]/[ATP]=2, further addition of ATP (3 mM) led to the re-assembly of ATP/AzoDiGua aggregates and appearance of suspension turbidity (Figure S14), emphasizing the reversibility of the supramolecular aggregates and the role of free ATP for the assembly to occur. By exploiting the chelating properties of ATP and its strong and specific affinity for divalent cations, we demonstrated that ATP-AzoDiGua aggregates were cation-responsive and valence-selective, offering the possibility not only to dynamically control both their assembly/disassembly behaviour and their fluorescence through a chemical stimulus but also to envision the principle of a divalent-specific ionic sensor.

Enzymatic response of ATP/AzoDiGua assembly

We finally explored the ability to use an enzyme as a stimulus. Alkaline phosphatase (ALP) was chosen as an enzymatic cleavage agent for its ability to catalytically hydrolyze ATP into adenosine and three equivalents of monophosphate groups in very mild conditions. We exploited this property and used ALP as a trigger to dynamically control ATP/AzoDiGua coassembly. Adding ALP to the preformed ATP/AzoDiGua aggregates ([ATP] = 1 mM, [AzoDiGua] = 1 mM) led to their complete disassembly in 10 min (Figure S15). This disassembly was accompanied by a loss of turbidity of the ATP/AzoDiGua solution after adding the enzyme to the mixture (Figure 5A). When the same experiment was performed with a previously denaturated ALP (treatment at 90 °C), the suspension turbidity remained and no disassembly was observed (Figure S16), confirming that ALP-triggered disassembly was due to ATP hydrolysis by the active enzyme. This enzyme-mediated disassembly was accompanied by a gradual decrease of suspension fluorescence down to a minimal level corresponding to that of free AzoDiGua after 10 min of reaction (Figure 5B), confirming the complete disassembly and release of free AzoDiGua. These results thus demonstrated how both aggregation-induced fluorescence of azobenzene and the characteristics of a self-assembled structure can be dynamically controlled through the action of an active enzyme having as a substrate one of two co-assembled partners.

Conclusion

Adenosine 5'-triphosphate (ATP) is involved in many biological and biotechnological processes, making its dynamic actuation, and especially by light, a valuable challenge for a broad range of applications of fundamental and/or practical interest. Instead of the conventional approach involving a covalent modification to render it stimulus-responsive, we have described an original strategy where ATP is associated with a photosensitive nucleotide binder called AzoDiGua to form multi-stimuli responsive supramolecular structures through non-covalent interactions. AzoDiGua is a water-soluble azobenzene molecule with two guaninidium groups as its extremity and known as a photosensitive DNA intercalator. Here, we exploited its phototunable capacity to interact with nucleotides through π-π and electrostatic interaction to build a new kind of photoresponsive co-assembly using ATP as a co-assembly partner. We showed in particular that simply mixing AzoDiGua and ATP led to the spontaneous assembly of the two partners into micrometer-sized aggregates in a few minutes in water. The interest of such an approach is multifold. First, the approach allows to dynamically control the assembly/release of ATP by light without having to modify it, and in mild conditions (physiological pH, room temperature, ATP concentrations similar to intracellular one), making the approach particularly robust, prone to generalization to other targets and opening interesting perspectives for in situ non-invasive actuation. Second, the non-covalent assembly allows to maintain the stimulus-responsiveness of the individual bricks in the supramolecular structure.

For instance, AzoDiGua remains photoswitchable and enables photoreversible assembly/disassembly of the aggregates by short UV/blue irradiation pulses for several consecutive cycles. Similarly, intrinsic chelation properties and enzymatic reactivity of ATP could be exploited to dynamically trigger fast disassembly of the structures by the action of metal cations or enzymes. Third, fluorescence appeared as an emerging property arising from the self-assembly itself. Indeed, although ATP and AzoDiGua have only very weak fluorescent properties when they are individually dispersed in solution, their co-assembly lead to significantly fluorescent superstructures. Such aggregation-induced fluorescence of azobenzene is a well-known phenomenon but is in many case obtained with pure azobenzene compounds loosing most of their phoswitchability in the aggregated state. Here, the coassembly with ATP allowed us not only to combine both aggregation-induced fluorescence and photoswitchability in the same system, but also to realize multi-stimuli responsive fluorecence.

For instance, the fluorescence of the ATP/AzoDiGua supramolecular aggregates was successfully and dynamically controlled by various physical (light), chemical (metal cations) and biological (enzyme) stimuli. This not only further evidenced the dynamic control over the self-assembly achieved by these different stimuli but also provides opportunities for the development of sensing strategies for detecting ATP, analyzing the presence of metal cations with valence-selectivity, or assessing enzymatic activity. Demonstrated here with two particular bricks, ATP and AzoDiGua, we think this co-assembly strategy could be explored with other nucleotide or DNA/RNA targets and/or other stimuli-responsive DNA intercalators to build highly dynamic and multifunctional supramolecular structures in a particularly robust, userfriendly and multi-purpose way.

Figure 1 .

 1 Figure 1. Spontaneous assembly of an azobenzene diguanidinium nucleotide binder

Figure 2 .

 2 Figure 2. ATP/AzoDiGua aggregates are fluorescent upon assembly of non-fluorescent

Figure 3 .

 3 Figure 3. Photoreversible assembly of fluorescent ATP/AzoDiGua aggregates. A)

Figure 4 .

 4 Figure 4. ATP/AzoDiGua aggregates are cation-responsive and valence-selective. A)

Figure 5 .

 5 Figure 5. Enzymatic control of the ATP/AzoDiGua assembly and fluorescence. A)

Acknowledgement

We thank Laurent Binet (IMRCP, Chimie ParisTech, Université PSL, CNRS) for EPR support and discussion; Hélène Bertrand (LBM, Sorbonne Université, ENS-PSL, CNRS) for experimental support in the fluorescence spectroscopy measurements.

Funding This work was supported by the European Research Council (ERC) (European Community's Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement No. 258782) and the French National Research Agency ANR through contracts DYOR (ANR-18-CE06-0019) and SCDiag (ANR-21-CE18-0051).

Conflict of interest

The authors declare no conflict of interest.


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Dr Vangelis Agouridas

Oleg Melnyk
email: oleg.melnyk@ibl.cnrs.fr

] R Desmet

Dr Pr A Tasiemski

An Iron-Catalyzed Protein Desulfurization Method Reminiscent of Aquatic Chemistry

Keywords: Iron, carbonate, protein, desulfurization, aquatic chemistry

One pillar of protein chemical synthesis based on the application of ligation chemistries to cysteine is the group of reactions enabling the selective desulfurization of cysteine residues into alanines. Modern desulfurization reactions use a phosphine as a sink for sulfur under activation conditions involving the generation of sulfur-centered radicals. Here we show that cysteine desulfurization by a phosphine can be effected efficiently by micromolar concentrations of iron under aerobic conditions in hydrogen carbonate buffer, that is using conditions that are reminiscent of ironcatalyzed oxidation phenomena occurring in natural waters. Therefore, our work shows that chemical processes taking place in aquatic systems can be adapted to a chemical reactor for triggering a complex chemoselective transformation at the protein level, while minimizing the resort to harmful chemicals.

Introduction

Protein chemical synthesis is now established as a powerful means for accessing proteins harboring any type of modifications at precise positions. Proteins are usually produced starting from shorter peptide segments assembled through the application of chemoselective peptide ligation methods. [1] Among these, those exploiting the reactivity of cysteine are intensively developed owing to the special reactivity of this thiol amino acid and its natural presence in protein sequences. [1a, 1c] In particular, the native chemical ligation (NCL) involving the reaction of peptide thioesters with cysteinyl (Cys) peptides has met a lot of success in the field for being highly efficient in water under mild conditions (Fig. 1a). [1a, 2] However, one limitation of NCL is the low frequency of Cys residues in proteins (~1.5%). [3] The scope of NCL has been significantly enlarged with the introduction of selective cysteine desulfurization methods (Fig. 1a). [4] When applied as a post-ligation treatment, these methods enable the production of peptide junctions at alanine (Ala) that are more frequent in proteins (8%) than peptide bonds to Cys. Since the pioneering works on Cys desulfurization, [4a] the concept has been extended to the production of almost all types of junctions, including the formation of isopeptidic bonds. [2, 4b] Although the importance of Cys desulfurization in the chemical production of peptides and proteins is clearly established, the methods that enable to perform this transformation in water on unprotected polypeptides can be counted on the fingers of one hand. [4b, 5] The first approaches exploited the ability of metals such as Pd/Al2O3 and Raney nickel to desulfurize alkylthiols. [4a] A significant advance in the field was the discovery that unprotected Cys peptides can be desulfurized by a water soluble phosphine in the presence of a radical initiator through the intermediacy of thiyl radicals (Fig. 1a). [6] A popular phosphine enabling peptide desulfurization is tris(2-carboxyethyl)phosphine (TCEP), which is classically used in combination with an excess of the water-soluble azo radical initiator VA-044 (Fig. 1b). The phosphoranyl radical intermediate obtained by reaction of the Cys thiyl radical with the phosphine undergoes a homolytic cleavage to produce an alanyl radical with the concomitant formation of a sulfenylphosphine. Finally, the alanyl radical evolves into an Ala peptide upon reaction with a hydrogen donor such as a thiol. This method is particularly popular and accounts for the majority of the desulfurization reactions performed today. [3] Conceptually, the majority of the Cys desulfurization methods designed afterwards also rely on the powerfulness of TCEP to combine with Cys thiyl radicals (Fig. 1b), but differ by the way the latter are generated using either chemical (boron compounds [7] ), photochemical (UV light irradiation, [8] light irradiation in the presence of ruthenium or iridium metal complexes, [9] ) or electrochemical [10] methods. The discovery of novel Cys desulfurization methods that do not resort on harmful additives, rare metals or complex experimental settings is desirable to enlarge the scope of the NCL/desulfurization synthetic strategy and make it easier to implement. As discussed above, a key step for enabling Cys desulfurization by a phosphine is the formation of thiyl radicals from Cys thiols. This transformation is a single-electron transfer process for which the Fe(II)/Fe(III) couple is particularly competent. [11] We show here that micromolar concentrations of iron in sodium hydrogen carbonate buffer under aerobic conditions are sufficient to effect the selective and efficient desulfurization of Cys thiols in proteins, including as a post ligation treatment in one-pot. An important lesson taken from this work is that a chemical process found in nature can be adapted to serve as a production tool for peptides or proteins of biological interest. By doing so, we succeeded in substituting harmful chemicals by others that are abundant and non-toxic. This work, by showing the efficacy of homogenous iron catalysis for promoting complex chemoselective transformations at the protein level, adds also a novel avenue to the flourishing area of iron-catalysis in organic chemistry. [12]

Results and Discussion

Screening of Peptide Desulfurization Parameters

The discovery that micromolar concentrations of iron can effect the selective and efficient desulfurization of cysteine residues in the presence of a phosphine came by serendipity, after having noticed that a peptide got spontaneously and quickly desulfurized after being solubilized in ~0.25 M sodium hydrogen carbonate in the presence of tris(2-carboxyethyl)phosphine (TCEP). Experiments conducted using sodium phosphate as the buffering system showed that peptide desulfurization can also proceed in such media, albeit with a rate 2.7-fold lower than those obtained in sodium hydrogen carbonate solution of same concentration (see Supplementary Information). Sodium hydrogen carbonate proved also superior to Good's buffers, further pointing out the peculiarity of this buffer system. The spontaneous desulfurization of thiols in the presence of TCEP was noticed as a side reaction by several authors, but was never considered of synthetic value for being low yielding and erratic. [13] The extent of desulfurization in our experiment in the absence of any apparent radical source was thus surprising and stimulated us to scrutinize some reaction parameters, before investigating in more depth how thiyl radicals can form in such conditions (Fig. 2). We started by looking at the dependence of the reaction to the type of thiol additive used as the hydrogen donor in the desulfurization process (Fig. 2b). Glutathione (GSH), a classical thiol used for peptide desulfurization in the presence of azo radical initiators, [14] gave the worst results. Sodium 3-mercapto-1-propanesulfonate (MPSNa) proved to be superior to the other alkyl thiols examined, followed by sodium mercapto-2ethanesulfonate (MESNa). Interestingly, varying MPSNa concentration revealed that its presence in the desulfurization mixture is beneficial to the reaction but only when used at 10-30 mM (Fig. 2c). In contrast, MPSNa was strongly inhibitory above 50 mM. Regarding the phosphine used in the desulfurization process, TCEP proved to be by far superior to tris(3hydroxypropyl)phosphine (THPP, Fig. 2d), although their pKa are very similar (TCEP pKa 7.7, [15] THPP pKa 7.2 [16] ). Fixing the MPSNa concentration to 30 mM and varying TCEP concentration from 10 to 200 mM revealed that TCEP concentration had to be at least 50 mM to achieve the highest conversions (see supplementary information). The desulfurization of peptide 1a into 2a was scaled up successfully to 810 mg scale (80% isolated). The reaction was performed in plasticware with passive diffusion of oxygen into the aqueous solution. * By-product corresponding to peptide PILKESVHGV-NH2. The proportion of this by-product is increasing if air is bubbled into the reaction mixture (see Supplementary Information). For precedents describing the formation of Ser from Cys during the deselenization of selenocysteine, see refs [18] .

Having clarified the importance of the type and concentration of phosphine and thiol additives, we examined the dependency of the desulfurization process to sodium hydrogen carbonate concentration. The rate of the desulfurization process increased markedly in the concentration range 0.05-0.15 M and plateaued above ~0.24 M (Fig. 2e, see also Supplementary Information). At 0.24 M and 25 °C, the desulfurization reaction followed a second order rate law with k = 0.189  0.005 M-1 s-1 (mean  SEM) and was completed in about 6 h. We also noticed that reactors made of glass were detrimental to the reaction compared to reactors made of polypropylene, perhaps due to a critical component of the desulfurization process that disappears from the solution by adsorbing on glass, or to the inhibition of soluble iron catalyst by silicates liberated in solution (Fig. 2e). [17]

Reproducibility, Scale-up and Scope

The optimal conditions defined above for effecting the desulfurization of Cys (TCEP 50-100 mM, MPSNa 30 mM, NaHCO3 0.24-0.3 M, 25-37 °C, plastic reactor) proved highly reproducible whether the desulfurization of peptide 1a into 2a was performed in the presence of guanidine hydrochloride (Gdn•HCl, mean HPLC yield 91%, standard error 1.6% for n = 12) or not (mean HPLC yield 91%, standard error 2.2% for n = 9). Importantly, the method was useful on a preparative scale by providing the desulfurized peptide 2a in good yields following HPLC purification (Fig. 3a). The reaction could be scaled up from 30 to 810 mg scale with success without changing the above reaction parameters and by taking care of using plasticware only (Fig. 3b, 78-81% isolated, see Supplementary Information), showing the suitability of the method for peptide production at g scale. We further showed that the method works as well when a methionine (Met) residue is present in the peptide sequence as for peptide 1b (Fig. 3a, see also Supplementary Information), or when the Cys residue is substituted by a penicillamine one (Pen, peptide 1c in Fig. 3a). [14] In the latter case, the desulfurization yields a valine residue and by doing so shows its potential for accessing different peptide junctions. One interesting application of peptide desulfurization is the production of site-specifically deuterated peptides, which are useful tools in life sciences. [19] To probe if the method might enable peptide deuteration, the desulfurization of peptide 1a was performed in deuterated water (Fig. 4a). Doing so resulted in the clean formation of peptide 2a D featuring a deuterium atom in replacement of the Cys thiol and was validated at a preparative scale (5 mg, Fig. 4b).

Implication of Radicals and Homogeneous Metal

Catalysis in the Desulfurization Process Having identified the reagents and conditions enabling peptide desulfurization to proceed efficiently, we next probed if Cys thiol desulfurization proceeds through the intermediacy of free radical species as proposed in Fig. 1c. The introduction of sodium ascorbate in the reaction mixture, a powerful reductant of oxygen free radicals and thiyl radicals and thus a strong inhibitor of the reactions proceeding through such species, strongly inhibited the desulfurization of peptide 1a from the lowest concentration tested (0.1 mM, see Supplementary Information). This experiment shows that free radicals are likely involved in the desulfurization process. We therefore looked for the mechanism leading to their formation. Oxygen free radicals and thiyl radicals are notoriously implicated in the thiol autoxidation into disulfides, a process that has been thoroughly investigated in the past for its importance in organic synthesis, in chemical industry processes such as oil sweetening and biology. [20] Thiolates can spontaneously react with molecular oxygen to produce thiyl radicals and peroxide ion, i.e. RS -+ O2 RS . + O2 .-. However, the catalysis of this process by some metal salts is so efficient that when present, most of the oxidation proceeds via the metal-catalyzed pathway. [START_REF] Tarbell | Organic Sulfur Compounds[END_REF] For example, copper has been shown to be particularly powerful in catalyzing the oxidation of cysteine or related thiols in neutral water. [20] Model peptide 1a was again used to investigate the implication of metal-catalysis in the process (Fig. 5a). One established means for blocking metal-catalysis during the autoxidation of thiols into disulfides is to add a metal complexing agent such as cyanide ion. [20][START_REF] Tarbell | Organic Sulfur Compounds[END_REF] In our case, the addition of cyanide ion to the desulfurization mixture resulted in a dramatic decrease of the reaction rate from the lowest concentration tested (0.1 mM, Fig. 5b). This experiment tells us that a metal is likely implicated in the desulfurization process but gives no information on its nature, because of the capacity of cyanide ion to bind many metal species. We therefore looked at the effect of other metal chelators that are more selective for copper or iron. The addition of tripeptide Gly-His-Lys (GHK) to the desulfurization mixture, a peptide that binds strongly and selectively copper (II), [START_REF] Lau | [END_REF] had almost no effect on the rate of desulfurization (Fig. 5c). In contrast, the addition of desferrioxamine B, [23] a naturally-occurring hydroxamate-type siderophore that has a strong preference for Fe(III) species, blocked the desulfurization of peptide 1a when used above 5.7 µM (Fig. 5c). These data point toward the potential role of iron catalysis in the desulfurization process.

Although desferrioxamine B has a strong preference for Fe(III), it can also bind other metal species such as Al(III) or Ga(III), and to a much lesser extent Cu(II). [23] To definitely establish the nature of the metal implicated in the desulfurization process, we looked at the effect of adding various metal salts to a desulfurization reaction inhibited by desferrioxamine B (Fig. 6a). The inhibition of the desulfurization reaction by desferrioxamine B (5.7 µM) could be removed by adding as low as 5-10 µM of iron sulfate to the reaction mixture (Fig. 6b). In contrast copper sulfate proved to be a poor catalyst (Fig. 6c) and none of the other metals tested (CoSO4, NiSO4, MnSO4) enabled to restore the efficiency of the desulfurization process at its initial levels as does iron (Fig. 6d). Iron is known to be a common contaminant of chemicals and buffers. [24] The reagents and buffer used for the desulfurization process were analyzed separately by inductively coupled plasma mass spectrometry (ICP-MS) to determine their iron content (Fig. 6e). The main source of iron was found to be the peptide, the phosphine TCEP•HCl and the sodium hydroxide used for pH adjustments and to a lesser degree the thiol additive MPSNa. The iron content of a 0.24 M sodium hydrogen carbonate solution was below the detection limit. Summing the individual contributions yields a total iron content of 1.27  0.03 µM. The desulfurization mixture was also analyzed the same way to give an iron content of 1.45  0.68 µM, a value which is in agreement with the preceding one obtained by summing individual contributions. To sum-up at this stage, low micromolar concentrations of iron are sufficient to promote the desulfurization of Cys residues in the presence of TCEP and sodium hydrogen carbonate under extremely mild conditions (25-37 °C, pH 7).

Implication of molecular oxygen in the desulfurization process

Having established that micromolar levels of an iron salt are sufficient for catalyzing the peptide desulfurization reaction in sodium hydrogen carbonate buffer, we looked at the mechanism enabling the recycling of the catalyst. Indeed, in the mechanism proposed in Fig. 1c, iron (III) species convert thiolates into thiyl radicals and by doing so are concomitantly reduced into iron (II) species. The latter need to be oxidized back to iron (III) to make the process catalytic in iron. A good candidate for recycling iron (II) species is molecular oxygen.

Figure 7. Evidence for the involvement of molecular oxygen in the peptide desulfurization process. a An NCL reaction was performed under nitrogen atmosphere in sodium hydrogen carbonate buffer in the presence of MPSNa. Then, TCEP and FeSO4 were added and the reaction mixture splitted into two equal parts. One sample was kept under nitrogen, while the other was aerated. b LC-MS analysis of the crudes. c The yield of desulfurized peptide 6 over time was determined by HPLC (UV detection at 215 nm). The experiment was performed once.

To investigate the importance of oxygen in the desulfurization process, we compared the extent of desulfurization achieved using classical aerobic conditions or an inert nitrogen atmosphere. However, and although the greatest precautions were taken to remove oxygen from the buffer and reactants prior to transferring them to the nitrogen atmosphere and running the desulfurization reaction, only a modest decrease in desulfurization yield was observed compared to the control (see Supplementary Information). A convincing proof for the involvement of oxygen in the desulfurization process came from studies aimed at developing a one-pot NCL/iron-catalyzed desulfurization process. Indeed, we found that the NCL reaction proceeds well in sodium hydrogen carbonate buffer in the presence of MPSNa (30 mM). The NCL reaction between peptides 3 and 4 as depicted in Fig. 7a proceeds with a halfreaction time of 1.1 h (k = 1.70  0.05 10 -4 s -1 ) and is completed in about 6 hours. Logically in such conditions lacking TCEP, no desulfurization can occur during NCL. We reasoned that the desulfurization of the ligated peptide might be triggered subsequently in one-pot by adding TCEP (50 mM) and iron sulfate (10 µM). In the experiment shown in Fig. 7a, extensively degassed solutions of TCEP and iron sulfate were added to the ligated peptide mixture under nitrogen atmosphere and the reaction mixture was split in two equal parts. One sample was aerated while the other was kept under nitrogen. The data presented in Fig. 7b show that the desulfurization process proceeded with the aerated sample, while the extent of desulfurization for the sample kept under nitrogen was insignificant. While in the previous experiment performing the desulfurization reaction under nitrogen atmosphere led only to a limited decrease in extent of desulfurization, in this case we found a striking difference between aerated and non-aerated samples. We believe that for the latter experiment, the oxygen that was present in the buffer at the beginning of the ligation step was consumed during NCL for oxidizing thiols into disulfides, so that no oxygen was still present in the solution when TCEP and iron sulfate were added, explaining the absence of desulfurization for the sample kept under nitrogen.

This experiment shows unambiguously the critical role played by molecular oxygen in the desulfurization reaction. In all the experiments discussed so far including the scale-up study, the entry of molecular oxygen into the reaction mixture was ensured by passive diffusion through the liquid-air interface. In one experiment (peptide 1a, 30 mg scale), air was bubbled into the reaction mixture or not to see if forcing the entry of molecular oxygen into the aqueous solution has any positive impact on the outcome of the desulfurization reaction. We found that bubbling air into the reaction mixture diminished the conversion of peptide 1a into peptide 2a and promoted the formation of a by-product, i.e. peptide PILKESVHGV-NH2 (see Fig. 3 and Supplementary Information), compared to the control. The formation of a seryl residue from Cys one during peptide dechalcogenation has some precedents and is likely due to the interception of intermediate alanyl radicals by molecular oxygen. [18] Having established the implication of iron, hydrogen carbonate and oxygen in the studied reaction, we performed iron(II) speciation calculations to have an idea of the iron-carbonate species formed during desulfurization and propose a candidate for catalyst recycling through oxidation by molecular oxygen. [25] Iron(II) speciation calculations using the iron concentration determined by ICP show that Fe(CO3) is by far the major species formed under our experimental conditions (88%), followed by Fe(HCO3) + (9%) and [Fe(CO3)2] 2-(1%, see Supplementary Information). Of these three iron species, the latter is known to react ~10.000 times faster with molecular oxygen than the other iron-carbonate species. [26] Therefore and although [Fe(CO3)2] 2-represents a minor fraction of total iron(II), it likely plays a major role in the in situ production of iron(III) species. In accord with this hypothesis, the fraction of [Fe(CO3)2] 2-is predicted to increase 7-fold by varying NaHCO3 buffer concentration from 47.5 mM to 285 mM otherwise being identical, a value which is consistent with the ~10-fold decrease in the half-reaction time observed experimentally (see Supplementary Information).

One-Pot NCL/Iron-Catalyzed Desulfurization

Designing one-pot NCL/iron-catalyzed desulfurization methods is an important goal to achieve for enabling a reduction of poteconomy during chemical protein synthesis, and thus a significant gain in time and yield. [27] During the first attempts to perform an NCL/desulfurization process in one-pot, the addition of TCEP to the ligation mixture triggered the conversion of Cys residue into Ala as expected but at a rate significantly less than those measured for the desulfurization experiments performed on purified peptides. The only difference in reaction mixture composition between the one-pot NCL/desulfurization process and the desulfurization of a purified peptide is the additional presence in the former conditions of a thiol that is released from the thioester component, i.e. 3-mercaptopropionic acid (MPA) for the experiment depicted in Fig. 7a. Since MPA was less powerful than MPSNa for promoting the desulfurization process (see Fig. 2b), the MPA thioester 3 was replaced by an analogue derived from MPSNa in the one-pot NCL/desulfurization process. However, this change did not improve the rate of the desulfurization step, ruling out the possibility that the MPA released during ligation was responsible for the problem. In contrast, adding iron sulfate (10 µM) after completion of the ligation step along with TCEP restored the rate of the desulfurization process to expected values, suggesting that part of the iron catalyst is lost during NCL under anaerobic conditions. With the simple precaution of adding iron sulfate together with TCEP after the ligation step, the one-pot NCL/desulfurization process proved of practical value (Figs. 8,9).

The conditions for performing one-pot NCL/iron-catalyzed desulfurization processes being defined, we sought to exemplify the usefulness of the method by synthesizing polypeptides of biological significance. The first example shown in Fig. 8 describes the synthesis of dermaseptin B2, an antibacterial peptide produced by the frog Phyllomedusa bicolor. [28] Dermaseptin B2 produced through the one-pot NCL/ironcatalyzed desulfurization process proved indistinguishable by LC-MS from an authentic sample produced by solid phase peptide synthesis (SPPS). Both peptides exhibited also the same antibacterial activity against E. coli or S. aureus strains (Minimum Inhibitory Concentration of 1.25 µM and 20 µM respectively, n = 3).

The second example provided in Fig. 9 shows the chemical synthesis of ubiquitin protein. Depending on the peptide thioester used for the assembly, the product of the NCL step features one (12a) or two (12b) internal Cys residues. In both cases, native ubiquitin was successfully obtained by performing the iron-catalyzed desulfurization after ligation in one-pot (see Fig. 9b for the crude ubiquitin obtained from the desulfurization of 12a). Importantly, the circular dichroism spectrum of synthetic ubiquitin was similar to the one obtained from recombinant ubiquitin, showing that the synthetic protein was correctly folded (Fig. 8c).

Discussion

The desulfurization process is catalyzed by iron species, is fueled by molecular oxygen and proceeds through the intermediacy of free radical species. The reaction is sensitive to the nature and concentration of the thiol additive, of the phosphine, as well as of the buffer system. The desulfurization process is particularly powerful in sodium hydrogen carbonate buffer and its rate increases markedly with hydrogen carbonate ion concentration. Overall, the data presented in this report support the mechanism proposed in Fig. 1c. According to this mechanism, the formation of thiyl radicals consumes Fe(III) species that are concomitantly reduced to Fe(II) species. In our experiments, the concentration of iron species is three orders of magnitude less than the peptide concentration. In such conditions, Fe(II) species must be recycled to achieve useful desulfurization yields. Several studies established that the rate of oxidation of Fe(II) into Fe(III) by molecular oxygen is critically dependent on iron speciation. [29] In particular, King and coworkers showed that in natural waters containing more than 1 mM of carbonate ions, the ferrous carbonate complexes Fe(CO3), [Fe(CO3)(OH)] -and [Fe(CO3)2] 2-dominate the speciation of Fe(II). [26] Of these three ferrous carbonate complexes, Fe(CO3)2 2-is the most kinetically active species at neutral pH for reacting with molecular oxygen. The consequence is that [Fe(CO3)2] 2-plays a major role in the degradation of organic matter in many aquatic systems, albeit it is a minor iron species in natural water. Although the exact nature of the iron species involved in the desulfurization process remains to be established, the ease of Fe(II) recycling through the oxidation of ferrous carbonate complexes by molecular oxygen offers a simple explanation for the high dependence of the desulfurization process to sodium hydrogen carbonate buffer concentration. It provides also an explanation for the strong inhibitory effect observed at high thiol additive concentrations, by considering that thiolates bind iron(II) and thus are expected to compete for carbonate ion binding to iron (see supplementary information). [30] Conversely, the speciation calculations indicate that the fraction of Fe(II)-thiolate complex at a total thiol concentration of 30 mM decreases significantly by increasing NaHCO3 concentration (~2% of total iron at 0.3 M NaHCO3). We think that part of the iron(II)-catalyzed peptide desulfurization process reported here is likely reminiscent of the iron-catalyzed oxidation phenomena occurring naturally in aquatic systems. [26,31] Note that phosphate ions have also been shown to promote the oxidation of Fe(II) by molecular oxygen in neutral solutions. [32] Consistent with this knowledge, we found that peptide desulfurization proceeds also well in concentrated sodium phosphate buffer, albeit at a rate significantly well below that measured in sodium bicarbonate buffer. It is well known that iron is a common contaminant of chemical reagents. The trace amounts of adventitious iron present in the HPLC purified peptide and the different reagents used for the desulfurization process, i.e. the phosphine TCEP, sodium hydroxide and the thiol additive MPSNa, result in a total iron concentration of about 1 µM in the desulfurization mixture. Apparently, such a low concentration of iron species is sufficient to ensure an efficient, reproducible and scalable desulfurization process, with the conditions that the buffer providing the carbonate anion catalyst is used at a minimal concentration of 0.24 M.

Conclusion

A hallmark of the desulfurization method reported in this work is the capacity of micromolar levels of a homogeneous iron catalyst to effect a sophisticated chemoselective reaction at the protein level, as well as the simplicity of the reagents needed to effect such a transformation. Iron was found to be by far the most active metal for promoting the desulfurization of Cys residue, and its complexation by carbonate ion is likely critical for achieving high catalytic efficiencies. We showed that the NCL reaction proceeds at a useful rate in sodium hydrogen carbonate buffer in the presence of sodium 3-mercaptopropyl sulfonate, giving the possibility to perform one-pot NCL/desulfurization processes by adding TCEP and FeSO4 to the ligation mixture and aerating the solution. By doing so, one of the most popular method for chemical protein synthesis can be setup with ease by using only cheap and harmless reagents for generating thiyl radicals, i.e. iron, hydrogen carbonate ion and molecular oxygen from air, and by exploiting chemical processes occurring naturally in aquatic systems. The growth of the field of homogeneous iron catalysis has been primarily stimulated by the need for alternatives to noble metal catalysts for effecting organic reactions on small molecules. This work shows that homogeneous iron catalysis has also a great role to play in protein chemistry.

Figure 1 .

 1 Figure 1. The native chemical ligation/desulfurization strategy is central to the field of protein chemical synthesis by enabling the production of peptide bonds to alanine, a frequent amino acid residue found in proteins. a The native chemical ligation/desulfurization strategy utilizing the capacity of phosphines to combine with thiyl radicals is extremely popular. b Methods used so far for in situ generation of thiyl radicals. c In this work, thiyl radicals are formed by reaction of thiolates with Fe(III) carbonate species. The proposed mechanism is reminiscent of the iron-catalyzed oxidation of thiols by molecular oxygen and of iron carbonatecatalyzed oxidative processes occurring in natural waters.

Figure 2 .

 2 Figure 2. Screening of desulfurization reaction parameters. a Studied reaction. b Influence of the type of thiol additive used. c Influence of MPSNa concentration (MPSNa 0-100 mM). d Influence of the type of phosphine used. e Dependency to sodium hydrogen carbonate concentration. The yield of desulfurization over time was determined by HPLC (UV detection at 215 nm). Reactions were performed once.

Figure 3 .

 3 Figure 3. Use of the desulfurization process on preparative scale. a Desulfurization of Cys-or Pen-containing peptides 1a-c at 5 mg scale. bThe desulfurization of peptide 1a into 2a was scaled up successfully to 810 mg scale (80% isolated). The reaction was performed in plasticware with passive diffusion of oxygen into the aqueous solution. * By-product corresponding to peptide PILKESVHGV-NH2. The proportion of this by-product is increasing if air is bubbled into the reaction mixture (see Supplementary Information). For precedents describing the formation of Ser from Cys during the deselenization of selenocysteine, see refs[18] .

Figure 4 .

 4 Figure 4. Use of the desulfurization process for site-specific peptide deuteration. a Desulfurization of peptide 1a in D2O yields mono-deuterated product 2a D . b HPLC analysis of the desulfurization reaction performed in D2O at 5 mg scale.

Figure 5 .

 5 Figure 5. Evidence for the implication of metal catalysis in the desulfurization process. a Reaction used for investigating the implication of a metal in the desulfurization reaction. b Effect of cyanide ion on the desulfurization process. c Effet of Gly-His-Lys (GHK) tripeptide, a strong Cu(II) binder, on the desulfurization process. d Effet of desferrioxamine B, a siderophore that binds efficiently iron(III), on the desulfurization process. The yield of desulfurization over time was determined by HPLC (UV detection at 215 nm). Reactions were performed once .

Figure 6 .

 6 Figure 6. Effect of adding various metal salts to the desulfurization reaction inhibited by desferrioxamine B (5.7 µM). a Reaction used for identifying the metal implicated in catalysis. In panels b-f, the control experiment corresponds to a desulfurization reaction run without added desferrioxamine B and metal salt. b The inhibition of the desulfurization reaction by desferrioxamine B (5.7 µM) is restored by adding iron sulfate to the reaction mixture. c Effect of adding copper sulfate. d Besides FeSO4, none of the other metal salts tested (CuSO4, CoSO4, NiSO4, MnSO4) enabled to restore the efficiency of the desulfurization process as it is observed for the control experiment. The yield of desulfurization over time was determined by HPLC (UV detection at 215 nm). Reactions were performed once. g Fe content of aqueous solutions of peptide 1a, TCEP•HCl, NaHCO3, NaOH, MPSNa and of the desulfurization mixture (1 mM peptide) was determined by

Figure 8 .

 8 Figure 8. One-pot NCL/iron-catalyzed desulfurization process applied to the synthesis of antimicrobial peptide dermaseptin B2. a Principle of the synthesis of dermaseptin B2. b UPLC-MS analysis of the crude NCL/desulfurization process leading to dermaseptin B2 formation.

Figure 9 .

 9 Figure 9. One-pot NCL/iron-catalyzed desulfurization process applied to the synthesis of ubiquitin protein. a Principle of the synthesis of ubiquitin. b UPLC-MS analysis of the crude NCL/desulfurization process leading to ubiquitin formation from peptide thioester 10a (the structure of ubiquitin was generated using pdb entry 1D3Z). c Circular dichroism spectra of synthetic and recombinant ubiquitin (25 °C, 0.1 mg/mL, 10 mM sodium phosphate buffer, pH 7). The synthetic ubiquitin used for CD measurements was produced from peptide thioester 10a. The data represent the mean and standard error (95%), n = 6.

Acknowledgements

We acknowledge financial support from CNRS, University of Lille, Pasteur Institute of Lille and INSERM. We warmly thank G. A. Gamov for useful discussions regarding the use of KEV online.

Entry for the Table of Contents

Cysteine desulfurization by a phosphine can be effected efficiently under aerobic conditions in hydrogen carbonate buffer by micromolar concentrations of iron. The desulfurization mechanism involves likely iron(III) species produced through the oxidation of iron(II)-carbonate complexes by molecular oxygen and is reminiscent of iron-catalyzed oxidation phenomena occurring in natural waters.