Karla S Vera
email: vera.karla@ustp.edu.ph

Eddie Caingles

Jeffrey Bergonia

B Ken

Israel A Balangao

Baguhin

STRENGTH PROPERTIES OF CHEMICALLY STABILIZED ROAD SUBBASE MATERIALS WITH LIME SLUDGE AND FLY ASH

Keywords: chemical stabilization, cement, lime sludge, fly ash, sub base

Current chemical stabilizing practices involve using cement, lime, bitumen, and other chemical stabilizers that are expensive because of their reliance on the use of manufactured chemicals. The purpose of this research is to assess the geotechnical properties of subbase materials when the industrial waste fly ash (FA) and lime sludge (LS) are used as stabilizers for road subbase coarse materials. This quantitative research considered five soil samples that were treated with FA contents of 10%, 20%, 30%, 40%, and 50% in addition to the constant LS and cement contents of 10% and 1% respectively. The untreated and treated soil samples were tested to identify the physical and index properties of subbase materials, compaction behavior, and California Bearing Ratio (CBR) of the samples. Results showed that the soil strength (CBR) tended to increase when FA content is increased. Moreover, the highest value of soil strength is obtained when the soil is mixed with 50% FA, 10% LS, and 1% cement contents. Therefore, it was concluded that the admixture of FA, LS, and Cement is a potential chemical stabilizer for road subbase coarse materials. Moreover, further study on increasing the FA content beyond 50% to the admixture is recommended.

INTRODUCTION

Civil engineering structures are founded on or on the surface of the earth. Soil serves as the ultimate foundation for all structures, thus, its geotechnical properties will greatly contribute to the stability of these structures. In designing a pavement, success does not solely depend on the quality of the top layer, such as the concrete pavement or asphalt, but also on the underneath layers like base, subbase, and subgrade [START_REF] Coban | The use of lime sludge for soil stabilization[END_REF]. Subbases/bases are usually constructed materials out of smaller rocks of various shapes and fragments that often have holes or gaps and can be compacted to create a hard surface. They act as structural layers that help in spreading the wheel load before it transmits to the subgrade thus, the subgrade layer will not be over-stressed [START_REF] Rajesh | Soil stabilization using industrial waste and lime[END_REF]. Improving the geotechnical properties of these pavement layers, through stabilization, is significantly important to save resourceful construction materials. Hence, well-stabilized soil is vital for the appropriate purpose and proper function of the structures. Soil stabilization is the treatment of soils to enable their strength and durability to be improved such that they become totally suitable for construction beyond their original classification [START_REF] Alhassan | Effect of Rice Husk Ash on Cement Stabilized Laterite[END_REF]. It improves soil properties and transforms unsuitable soil into the required condition. More so, it increases bearing capacity or reduces settlement, water permeability, or risk of liquefaction [START_REF] Zorluer | The usability of industrial wastes on soil stabilization[END_REF]. The stabilization process is divided into two broad fields namely; mechanical and chemical stabilization. Mechanical stabilization requires compaction, aggregate mixing, gradient improvement, and asphalt cement extension. Chemical stabilization requires the mixing of chemicals in the soil like cement, lime, asphalt, or fly ash. The most common chemicals adopted for soil treatment are cement and lime [START_REF] James | Industrial wastes as auxiliary additives to cement/lime stabilization of soils[END_REF]. However, the construction cost of soil stabilization using the latter chemicals is remained financially high because of the over-dependence on the use of manufactured additives that strengthen the soil. Various engineering studies have revealed the possible replacement of cement and lime in soil stabilization by exploring the effectiveness of using industrial wastes as stabilizers. These include by-products from paper milling and sugar milling companies known as lime sludge [START_REF] Chandak | Effect of lime sludge on strength and compaction of soil[END_REF][START_REF] Nagaraju | An experimental study on paper mill lime sludge as stabilizing agent[END_REF][START_REF] Daleon | Effects of Sugar Mill Lime Sludge Treatment on the CBR and UCS of Expansive Soil in Bukidnon[END_REF][START_REF] Suthar | Bearing ratio and leachate analysis of pond ash stabilized with lime and lime sludge[END_REF] and hypo sludge [START_REF] Usha | Soil stabilization for pavement using lime and hyposludge[END_REF], by-products from rice milling companies known as rice husk ash [START_REF] Roy | Soil stabilization using rice husk ash and cement[END_REF], from coal-fired thermal power plants called fly ash [START_REF] Simatupang | The mechanical properties of fly-ash-stabilized sands[END_REF][START_REF] Turan | Use of class C fly ash for stabilization of fine-grained soils[END_REF][START_REF] Unsever | Stabilization of clay soils using fly ash[END_REF][START_REF] Sharma | Effect of fly ash, construction demolition waste and lime on geotechnical characteristics of a clayey soil: a May-June comparative study[END_REF][START_REF] Dahale | Effect OF flyash and lime on stabilization of expansive soil[END_REF] among others. The municipality of Villanueva in Northern Mindanao, Philippines has a coal-fired power plant. The fly ash generated by the plant is currently utilized by the host town as an admixture to cement to manufacture construction materials such as concrete hollow blocks (CHBs), bricks, and pavers. However, the mechanical and environmental properties of these construction materials were not determined. With the continuous generation of fly ash at the plant, the best option for reducing it is to utilize it for construction purposes such as those of other agro-industrial wastes [START_REF] Chandak | Effect of lime sludge on strength and compaction of soil[END_REF][START_REF] Nagaraju | An experimental study on paper mill lime sludge as stabilizing agent[END_REF][START_REF] Daleon | Effects of Sugar Mill Lime Sludge Treatment on the CBR and UCS of Expansive Soil in Bukidnon[END_REF][START_REF] Suthar | Bearing ratio and leachate analysis of pond ash stabilized with lime and lime sludge[END_REF][START_REF] Usha | Soil stabilization for pavement using lime and hyposludge[END_REF][START_REF] Roy | Soil stabilization using rice husk ash and cement[END_REF][START_REF] Simatupang | The mechanical properties of fly-ash-stabilized sands[END_REF][START_REF] Turan | Use of class C fly ash for stabilization of fine-grained soils[END_REF][START_REF] Unsever | Stabilization of clay soils using fly ash[END_REF][START_REF] Sharma | Effect of fly ash, construction demolition waste and lime on geotechnical characteristics of a clayey soil: a May-June comparative study[END_REF][START_REF] Dahale | Effect OF flyash and lime on stabilization of expansive soil[END_REF][START_REF] Balangao | DESIGN AND DEVELOPMENT OF A MANUALLY-OPERATED HYDRAULIC HOT-PRESSING MACHINE FOR AGRICULTURAL WASTES-BASED PARTICLEBOARD APPLICATION[END_REF][START_REF] Jabile | UTILIZATION OF SAWDUST AND RICE HUSK FOR PARTICLE BOARD APPLICATION[END_REF][START_REF] Jabile | Assessing the suitability of fly ash and rice husk ash from Misamis Oriental, Philippines in producing bricks for pedestrian and light traffic applications[END_REF][START_REF] Baguhin | Investigation on Load-Bearing Concrete Hollow Block Reinforced with Coconut Coir Fiber[END_REF]. Consisting of predominantly sphericallyshaped particles [START_REF] Balangao | Production of Autoclaved Aerated Concretes with Fly Ash from a Coal Power Plant in Misamis Oriental, Philippines[END_REF][START_REF] Khairul Nizar | Study on physical and chemical properties of fly ash from different area in Malaysia[END_REF][START_REF] Bada | Evaluation and treatment of coal fly ash for adsorption application[END_REF], fly ash contains heavy metals [START_REF] Xu | Status of trace element emission in a coal combustion process: a review[END_REF][START_REF] Levandowski | Chemical and petrographical characterization of feed coal, fly ash and bottom ash from the Figueira Power Plant, Paraná, Brazil[END_REF] which make it hazardous. Therefore, there should be no environmental problems with any construction materials with fly ash in order that they are safe to use. One possible way to immobilize the heavy metals in the fly ash prior to its utilization is the addition of other pozzolanic and cementitious resources into the mix. The idea of coming up with a study of adding lime sludge with the fly ash to further stabilize soil was considered when [START_REF] Daleon | Effects of Sugar Mill Lime Sludge Treatment on the CBR and UCS of Expansive Soil in Bukidnon[END_REF] was able to utilize it in the stabilization of clay. The said lime sludge is produced by a sugar milling plant in Bukidnon, Philippines, and is currently not utilized and properly disposed of, thereby, piling it in an open dump. According to the study, major proportions of lime sludge such as silicon dioxide (SiO2), aluminum oxide (Al2O3), and calcium oxide (CaO) were the same as major components found in the Portland cement. Results of their study revealed that the addition of 10% by weight of lime sludge increases the California Bearing Ratio (CBR) and Unconfined Compressive Strength (UCS) of the said clay as subgrade soil. The combination of fly ash and lime sludge to replace cement and lime to stabilize the base/subbase layer of road pavement is promising; just like any other resource combinations May-June processed for a specific application [9-10, 17-19, 26-28]. The process would be economic-and environment-friendly promoting a reduction of the time and costs of production especially for cement and lime and preventing harmful gases that are to be emitted from the process of manufacturing them. It also saves natural land resources [START_REF] Xue | Utilization of municipal solid waste incineration ash in stone mastic asphalt mixture: Pavement performance and environmental impact[END_REF] which are to be used in manufacturing them prior to their use in construction. It also promotes sustainability in production since it actually utilizes wastes that are generated daily from their sources. This work is intended to evaluate the potential application of fly ash and lime sludge as stabilizing agents to improve the properties of a road subbase material. Basically, this study focused on determining the mixture of soil and lime sludgefly ash-cement with respect to its curing period that has attained the highest value of California Bearing Ratio (CBR) of soil intended as a subbase material. The untreated and treated soil samples were identified as the input parameters. The process was mainly laboratory testing of the untreated and treated soil samples. Lastly, for the expected output, the untreated samples acted as the control group which becomes the basis for evaluating the results if there are significant improvements in the geotechnical properties of soil as it is being treated with lime sludge (LS), fly ash (FA), and Ordinary Portland Cement (OPC).

MATERIALS AND METHODS

Selection of Site for Soil Sampling

Locally available sources of subbase course soils were randomly picked as suggested by the Department of Public Works and Highways (DPWH) of the Philippines. There were no particular parameters set on the selection of soil samples for this study because most of the time in the actual construction, the material used for this pavement layer is being blended and modified to meet DPWH standards.

Collection and Preparation of Soil Sample

In this stage, a soil sample was hauled from the site and placed in a suitable container to preserve moisture. As to the curing process, for the CBR test, the soil sample was soaked in a curing tank with water for 4 days before being penetrated by the CBR machine. The amount of water for the CBR test was computed using a formula based on the volume-density relationship as given in Equation 1. The hygroscopic moisture content (HMC) is the in-situ moisture content of the soil which was obtained by ovendrying the soil samples for 24 hours at 100+10 o C. The OMC is the moisture content at which the maximum dry density is obtained in the compaction test. Eq. 1

Collection and Preparation of Lime Sludge, Fly Ash, and Cement

Lime sludge was collected from a sugar milling company in Bukidnon, Philippines. The coal fly ash was taken from a coal power plant in Villanueva, Misamis Oriental, Philippines. Both the lime sludge and fly ash were air-dried to be easily pounded to finer grains. Sieving of the raw materials using sieve no. 200 was done. More so, the loss on ignition (LOI) test was performed before these materials were mixed with the cement and soil to ensure that the grain particles of lime sludge and fly ash are as fine as the cement. Type 1 Portland cement available in the market was used. The cement was sieved using no. 200 before mixing it with soil, lime sludge, fly ash, and water. The soil samples were thoroughly mixed until a uniform color is observed.

Preparation of Blended Sample

The variation of the mixtures was based on the minimum standard set by the DPWH and the best percentage suggested by previous studies. The variation of mixes is presented in Table 1. As stipulated in the DPWH Blue Book (2013), the amount of cement to be added to the soil aggregate shall be from 6 -10 mass percent of the dry soil. Hence, in this study, the maximum amount of cement added was within 1% for the sets of the mixture. The percentage addition of fly ash ranged from 10% to 50% by soil dry weight with an interval of 10%. Meanwhile, for the lime sludge, the percentage addition by soil dry weight was 10% to sets of the mixture. After attaining the required amount of soil aggregate, lime sludge, fly ash, and OPC in each soil sample, it was then tested for compaction and CBR to evaluate its properties as a subbase material. Similar curing procedures were adopted for the treated soil. For the CBR test, the amount of water was computed using a formula using the volume-density relationship as shown in Equation 2. Meanwhile, Equations 3 -5 were used to determine the weight of stabilizers in each soil sample, respectively.

May-June 𝑥 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑂𝑃𝐶) Eq. 5

RESULTS AND DISCUSSION

Physical and Index Properties of the Subbase Material

The sieve analysis revealed that the soil sample consisted of 0% gravel or soil particles with more than 2mm size, 91% sand or soil particles with sizes ranging from 2mm to 0.075mm, and 9% silt and clay or soil particles with less than 0.075mm size. This means the soil samples contain dominantly sand with very little clay. Moreover, the grading criteria passed the DPWH requirement for subbase material.

Based on the Atterberg limit values, the liquid limit and plastic limit of the soil were 26% and 0%, respectively. This indicates that soil exhibited non-plasticity properties. Nonplastic soil is a non-cohesive soil that does not bond or stick together due to a lack of cohesion. The soil was found to be under the A-1-a subgroup considering the soil classification system of the American Association of State Highways and Transportation Officials (AASHTO), Further, the soil was classified as silty sands or sand silt mixture according to Unified Soil Classification System (USCS). In general, the material passed the DPWH requirements for Item 200 -Aggregate Subbase Course based on its physical and index property values.

CBR Values of untreated and treated Subbase course

Table 5 shows the CBR values of the mixture. Among the mixtures added with lime sludge, OPC, and FA, the addition of 50% FA gave the highest CBR value at 287%, while the lowest CBR value of 31% was observed from the mixture added with 10% fly ash. Coban (2017) observed similar results in his study on the use of lime sludge as a soil stabilizer. He said that this is because the lower lime sludge content did not respond as effectively in flocculation, the CBR value barely changed, and higher lime sludge contents were required to achieve this. Moreover, soaked CBR value amplified along with the increased amount of fly ash from 10% to 50% obtaining the highest CBR value at 50% fly ash addition equal to 287%. This increase in CBR was a result of the pozzolan reaction between alumina and silica of cement, lime sludge, and fly ash with water. According to Cherian and Arnepalli [START_REF] Cherian | A critical appraisal of the role of clay mineralogy in lime stabilization[END_REF], pozzolan reactions started when the hydroxyl ion increased from the lime leading to a pH rise in the soil's water, with which the silicate and the aluminum sheets may start to dissolve. An increase in the CBR values of soils treated with lime sludge was also observed by Daleon and Lorenzo [START_REF] Daleon | Effects of Sugar Mill Lime Sludge Treatment on the CBR and UCS of Expansive Soil in Bukidnon[END_REF] in their study on the treatment of clay soil with sugar-mill lime sludge. They explained that, as silica and/or alumina are released, they can be combined with calcium to form hydrates of calcium silicate and/or calcium aluminum, which can be used to cement the soils together. After reaching its peak value, the CBR value decreased as the amount of lime sludge was further increased to 16%. This may be attributed to carbonation reactions which happen due to the presence of excess lime that reduces the bearing capacity of the soil.

May-June

CONCLUSION

Based on the laboratory results, the material has passed the specifications stipulated in the DPWH Blue Book for Item 200. For the treated mixture, the CBR values increase as the percentage addition of fly also increases. It is then concluded that the OPC, LS, and FA can be used as stabilizing agents to improve the California Bearing Ratio of the subbase coarse material.

ACKNOWLEDGEMENT

Table 1 . Variation of Mixtures Soil Samples Aggregate Base/Subbase Course (% OPC + %LS + %OPC)

 1

	A	100% Soil	1%OPC + 10%LS
	B	100% Soil	1%OPC + 10%LS + 10% FA
	C	100% Soil	1%OPC + 10%LS + 20% FA
	D	100% Soil	1%OPC + 10%LS + 30% FA
	E	100% Soil	1%OPC + 10%LS + 40% FA
	F	100% Soil	1%OPC + 10%LS + 50% FA

Table Summary of Physical and Index Properties of the

 Summary

		3.2
	Subbase Material
	Property	Quantity
	Gravel, %	0.00
	Sand, %	91.00
	Clay, %	9.00
	Liquid Limit, %	26.00
	Plastic Limit, %	Non-Plastic
	Plasticity Index, %	Non-Plastic
	AASHTO Classification	A-1-a
	USCS Classification	SM

. Compaction and California Bearing Ratio Tests

  The soil sample was subjected to a compaction test based on modified proctor compaction test method to obtain the Maximum Dry Density (MDD) and Optimum Moisture Content (OMC). The MDD and OMC values were displayed in Table3. The CBR value of 103.9% surpassed the requirement of 30% CBR soaked value of the DPWH for Item 200 -Aggregate Subbase Course.

Table 3 . Summary of Compaction Test and CBR Test Results of the untreated subbase course

 3

	Mixture ID	Tests	Result
	Subbase Materials	Maximum Dry Density (Kg/cu.m.)	2020
		Optimum Moisture Content (%)	8.6
		California Bearing Ratio (%)	103.9
	3.3. Compaction behaviour of treated subbase course
	The compaction results of the treated soil samples were
	shown in

Table 4 .

 4 The treated soil sample A obtained the highest MDD of 1910 kg/ m 3 with OMC of 12.2% while soil sample F had the lowest MDD of 1748 kg/ m 3 with OMC of 16.0%. Further, as the fly ash was added to the mixtures, the MDD values decreased. The incorporation of 1% cement into the mix resulted in greater density, resulting in the flocs repositioning, hence showing denser compacts.

Table 4 .

 4 MDD and OMC of treated samples

	Soil Samples	Aggregate Base/Subbase Course	(% OPC + %LSA+ %FA)	MDD (Kg/m 3 )	OMC (%)
	A	100% Soil	1%OPC +10%LS	1910	12.2
			1%OPC
	B	100% Soil	+10%LS +
			10% FA	1869	12.0
			1%OPC
	C	100% Soil	+10%LS +
			20% FA	1854	14.4
			1%OPC
	D	100% Soil	+10%LS +
			30% FA	1902	13.9
			1%OPC
	E	100% Soil	+10%LS +
			40% FA	1824	13.6
			1%OPC
	F	100% Soil	+10%LS +
			50% FA	1748	16.0

Table 5 . CBR values of the mixtures Soil Samples

 5

		Aggregate Base/Subbase Course	(% OPC + %LS+ %OPC)	CBR Value (%)
	A	100% Soil	1%OPC +10%LS	62
			1%OPC
	B	100% Soil	+10%LS +	31
			10% FA
			1%OPC
	C	100% Soil	+10%LS +	122
			20% FA
			1%OPC
	D	100% Soil	+10%LS +	170
			30% FA
			1%OPC
	E	100% Soil	+10%LS +	178
			40% FA
			1%OPC
	F	100% Soil	+10%LS +	287
			50% FA
	It could also be observed that the CBR value of 31% obtained
	by the soil sample added with 1% has already passed the
	minimum requirement of 30% soaked CBR Value for Item
	200 -Aggregate Subbase Course as stipulated in the DPWH
	Blue Book which means that this mixture could already be
	considered for adaptation depending the on the pavement
	design parameters.

The authors would like to thank the University of Science and Technology of Southern Philippines in Cagayan de Oro City, Philippines for funding this research study.


Yining Jiang

Suvasthika Indrajith

Ariel Francis

Perez Mellor

Thomas Bürgi

Marc Lecouvey

Carine Clavaguéra

Enrico Bodo

Chantal Houée-Levin

Ariel Francis Perez Mellor

Estelle Loire

Giel Berden

Jos Oomens

Debora Scuderi

la diffusion de documents scientifiques

Keywords: One-electron oxidation, -radiolysis, IRMPD spectroscopy, tandem mass spectrometry, cyclic peptides

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

they are crucial signaling agents in various biological processes, essential for sustaining the life of cells, their uncontrolled production is generally considered to be an important factor in the pathophysiology of various states, including ischemia, atherosclerosis, heart failure and cancer [START_REF] Grune | Decreased Proteolysis Caused by Protein Aggregates, Inclusion Bodies, Plaques, Lipofuscin, Ceroid, and "aggresomes" during Oxidative Stress, Aging, and Disease[END_REF] .

ROS are produced in living cells by Fenton reaction [START_REF] Halliwell | Oxygen-Derived Species: Their Relation to Human Disease and Environmental Stress[END_REF] and/or enzymes such as NADPH oxidase.

An imbalance of the equilibrium between the free radical formed during the oxidation process and the antioxidant defense mechanisms leads to oxidative stress and a variety of critical biological molecules, including DNA and cellular proteins, are subjected to oxidative damage 3 . • OH, which is the most reactive oxygen species, is also generated by the radiolysis of water molecules according to the so-called indirect effect of ionizing radiation, involving hydrogen peroxide [START_REF] Wojcik | DNA Interstrand Crosslinks Are Induced in Cells Prelabelled with 5-Bromo-2′-Deoxyuridine and Exposed to UVC Radiation[END_REF] .

Gamma radiolysis can in this way mimic oxidative stress in cells.

Almost all peptide residues can be oxidized by OH radicals. Most of these oxidized residues are toxic. Two major exceptions are the sulfur containing residues (cysteine and methionine), which not only are very easily oxidized but can also be repaired by cellular machineries (thiol-disulfide cycles for the former and methionine sulfoxide reductases for the latter). Methionine (Met) residues are often considered as endogenous antioxidants that can detoxify the cell and protect crucial regions of proteins [START_REF] Levine | Methionine Residues May Protect Proteins from Critical Oxidative Damage[END_REF][START_REF] Nauser | Calmodulin Methionine Residues Are Targets for One-Electron Oxidation by Hydroxyl Radicals: Formation of S∴N Three-Electron Bonded Radical Complexes[END_REF] . Other studies have also shown pro-oxidant properties thanks to intramolecular long-range electron transfer at the free radical level [START_REF] Mozziconacci | Methionine Residue Acts as a Prooxidant in the •OH-Induced Oxidation of Enkephalins[END_REF] . The condition under which Met can provide its antioxidant action is that the oxidation should produce methionine sulfoxide (MetSO) but no other species containing -SO2 or -SO3.

In this context, considerable efforts have been devoted to developing analytical strategies to detect oxidized Met residues (see [START_REF] Maitre | Applications of Infrared Multiple Photon Dissociation (IRMPD) to the Detection of Posttranslational Modifications[END_REF] and references therein) and among them tandem mass spectrometry (MS/MS) is largely used, especially in combination with the electrospray ionization technique (ESI). [START_REF] Whitehouse | Electrospray Interface for Liquid Chromatographs and Mass Spectrometers[END_REF] . Tandem mass spectrometry is a highly and efficient analytical tool, whose potential was impeded by the limited structural information which it can provide [START_REF] Bridgewater | The Effect of Histidine Oxidation on the Dissociation Patterns of Peptide Ions[END_REF] . Tandem mass spectrometry combined with IRMPD spectroscopy has become a powerful tool for the characterization of molecular ion structures in the gas phase, allowing the localization of the post-translational modifications and giving information on their nature [START_REF] Maitre | Applications of Infrared Multiple Photon Dissociation (IRMPD) to the Detection of Posttranslational Modifications[END_REF] .

IRMPD spectroscopy experiments need to be performed with free electrons lasers (FEL) at the FELIX (Free Electron Laser for Infrared eXperiment) laboratory in the Netherlands [START_REF] Oomens | Gas-Phase Infrared Photodissociation Spectroscopy of Cationic Polyaromatic Hydrocarbons[END_REF] or at the CLIO (Centre Laser Infrarouge d'Orsay) facility in France [START_REF] Lemaire | Gas Phase Infrared Spectroscopy of Selectively Prepared Ions[END_REF] , which are characterized by a high power and IR tunability.

In previous studies [START_REF] Ignasiak | Oxidation of Methionine-Containing Peptides by OH Radicals: Is Sulfoxide the Only Product? Study by Mass Spectrometry and IRMPD Spectroscopy[END_REF] performed at the CLIO facility in Orsay, we have investigated the oxidation products formed from linear dipeptides containing methionine by tandem mass spectrometry and IRMPD spectroscopy. The sulfoxide signature has been recognized near 1000 cm -1 in the IRMPD spectrum of the oxidation products of dipeptides such as Val-Met, Tyr-Met and Met-Met.

In Lys-Met, we were able to unravel the mechanism involved in the formation of the sulfoxide [START_REF] Ignasiak | Characterization by Mass Spectrometry and IRMPD Spectroscopy of the Sulfoxide Group in Oxidized Methionine and Related Compounds[END_REF] .

In the dipeptides Trp-Met and Met-Trp we were able to show the formation of a sulfoxide on both peptides and on the latter one we shown evidence of the oxidation occurring also on the aromatic ring of Trp [START_REF] Scuderi | Tandem Mass Spectrometry and Infrared Spectroscopy as a Tool to Identify Peptide Oxidized Residues[END_REF] .

In the present work, our aim is to shed light on the mechanism of these damaging events, by investigating in more detail the role of the neighboring groups on the final products of the oxidation of model cyclic dipeptides containing methionine (Figure 1). We further aim to understand the relationship between the 2-centre 3-electron free radical formed in the first step of the oxidation process and the final compound. Few studies have attempted to obtain a complete picture of Met oxidation, i.e., from the free radicals to the final products [START_REF] Bergès | Toward an Understanding of the Oxidation Process of Methionine Enkephalin: A Combined Electrochemistry, Quantum Chemistry and Quantum Chemical Topology Analysis[END_REF] . The chemical properties of these free radicals and especially the relationship between their structure and the final products are not yet completely known.

In the past few decades, mechanistic investigations by pulse radiolysis have highlighted the complexity of solution-phase one-electron oxidation of methionine and polypeptides by OH radicals [START_REF] Bobrowski | Formation and Stability of Intramolecular Three-Electron SN, SS, and SO Bonds in One-Electron-Oxidized Simple Methionine Peptides. Pulse Radiolysis[END_REF][START_REF] Bobrowski | Stabilization of Sulfide Radical Cations through Complexation with the Peptide Bond: Mechanisms Relevant to Oxidation of Proteins Containing Multiple Methionine Residues[END_REF] . Several Met free radicals can be formed depending on the geometry of the peptide and the neighboring residues, in particular, 2-center 3-electron bonded free radicals (2c-3e) have been discovered in which the odd electron of sulfur makes a bond with any atom possessing a lone pair, as confirmed by quantum-chemical calculations [START_REF] Bergès | Toward an Understanding of the Oxidation Process of Methionine Enkephalin: A Combined Electrochemistry, Quantum Chemistry and Quantum Chemical Topology Analysis[END_REF][START_REF] Scuderi | Methionine One-Electron Oxidation: Coherent Contributions from Radiolysis, IRMPD Spectroscopy, DFT Calculations and Electrochemistry[END_REF][START_REF] Hug | Conformational Influence on the Type of Stabilization of Sulfur Radical Cations in Cyclic Peptides[END_REF][START_REF] Archirel | Radical Cations of the Monomer and van Der Waals Dimer of a Methionine Residue as Prototypes of (2 Center-3 Electron) SN and SS Bonds. Molecular Simulations of Their Absorption Spectra in Water[END_REF][START_REF] Pilmé | Topological Analyses of Time-Dependent Electronic Structures: Application to Electron-Transfers in Methionine Enkephalin[END_REF][START_REF] Bergès | OH Oxidation of Methionine in the Presence of Discrete Water Molecules: DFT, QTAIM and Valence Bond Analyses[END_REF] . The sulfur radical cations formed as transient species are stabilized due to interaction with the oxygen and nitrogen atoms of the peptide bond [START_REF] Hug | Conformational Influence on the Type of Stabilization of Sulfur Radical Cations in Cyclic Peptides[END_REF] . In this way the formation of a sulfoxide is possible. Cyclic peptides are an interesting class of molecules as models for the interior of (linear) proteins, where terminal groups are absent, like calmodulin [START_REF] Nauser | Calmodulin Methionine Residues Are Targets for One-Electron Oxidation by Hydroxyl Radicals: Formation of S∴N Three-Electron Bonded Radical Complexes[END_REF] , a protein with adjacent methionine residues. c(LMetDMet) serves as a better model for calmodulin than c(LMetLMet) because the methionine lateral chains are pointing in opposite directions due to the cis configuration of the peptide bond in cyclic peptides. Various previous studies have employed IRMPD spectroscopy to efficiently characterize the structures of cyclic peptides in the gas phase [START_REF] Shek | Fragmentations of Protonated Cyclic-Glycylglycine and Cyclic-Alanylalanine[END_REF][START_REF] Zou | Competition between Diketopiperazine and Oxazolone Formation in Water Loss Products from Protonated ArgGly and GlyArg[END_REF][START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-Diphenylalanine in the Gas Phase[END_REF][START_REF] Oomens | Water Loss from Protonated XxxSer and XxxThr Dipeptides Gives Oxazoline-Not Oxazolone-Product Ions[END_REF] .

METHODS

Products. The peptides were synthesized in the group of Prof. Marc Lecouvey at Sorbonne University Paris Nord and used as received. To synthesize the different peptidic targets, classical methodologies were used [START_REF] Cortes-Clerget | Novel Easily Recyclable Bifunctional Phosphonic Acid Carrying Tripeptides for the Stereoselective Michael Addition of Aldehydes with Nitroalkenes[END_REF][START_REF] Ueda | Facile Synthesis of Cyclic Dipeptides and Detection of Racemization[END_REF] . A detailed description of the synthesis is reported in Figure S1 and

S2.

Nitrous oxide with a global purity of 99.998% was purchased by ALPHA GAZ.

Gamma Irradiation. To perform γ-irradiations the panoramic 60 Co γ-source IL60PL Cis-Bio International (France) in the Paris-Saclay University (Orsay, France) has been employed. The dose rate was determined by Fricke dosimetry [START_REF] Spinks | An Introduction to Radiation Chemistry[END_REF] . Before the irradiation, performed at room temperature, the samples were purged under a N2O atmosphere for 30 min. The well-known method of scavengers [START_REF] Kadlcik | Reductive Modification of a Methionine Residue in the Amyloid-β Peptide[END_REF] allows a quantitative production of free radicals by stationary γ-radiolysis according to the following reactions. The chosen oxidant species was the hydroxyl radical

e -aq + N2O + H2O → • OH + OH -+ N2 (1)
The radiation chemical yield (G) is equal to 0.55 μmol J -1 (equations 1 and 2) [START_REF] Kadlcik | Reductive Modification of a Methionine Residue in the Amyloid-β Peptide[END_REF] . H atoms are also created in much lower yield (0.05 μmol J -1 ), leading to the desulfuration of methionine. The H2O2 yield is also lower (0.07 μmol J -1 ).

Infrared absorption spectroscopy.

The infrared absorption spectra of c(LMetLMet), c(LMetDMet) and c(GlyLMet) were investigated in different environments: pure powder, a KBr pellet containing the powder at ~1%, and a DMSO-d6 solution. We used Attenuated Total Reflectance (ATR) for pure powder measurements, while for KBr and DMSO-d6 solution, we measured the absorbance in transmission. For the latter, we use pure KBr pellets and pure solvent as a reference spectrum. In all cases, the resolution is set at 4 cm -1 , and 100 scans were collected.

MS operations and IRMPD spectroscopy.

IRMPD spectroscopy experiments were performed both at the FELIX Laboratory in Nijmegen (The Netherlands) [START_REF] Oepts | The Free-Electron-Laser User Facility FELIX[END_REF] and at the CLIO Facility in Orsay (France) [START_REF] Prazeres | Two-Colour Operation of a Free-Electron Laser and Applications in the Mid-Infrared[END_REF] . IRMPD experiments at the CLIO Facility have been performed in the 1000-3700 cm -1 spectral range. Infrared ion spectra were recorded using the 7 T hybrid FT-ICR mass spectrometer (APEX-Qe, Bruker) coupled to the CLIO beamline or to the OPO/OPA system (LaserVision). This experimental setup has been previously described in detail [START_REF] Bakker | Gas-Phase Structure of a π-Allyl-Palladium Complex: Efficient Infrared Spectroscopy in a 7 T Fourier Transform Mass Spectrometer[END_REF] . To perform the IRMPD experiments mass-selected ions were stored and irradiated for 1 to 4 seconds with the IR-FEL (corresponding to 25-100 pulses) or OPO/OPA (10-40 pulses) light in the frequency range of interest. At 44 MeV electron energy, the time-averaged CLIO laser power is between 0.5 and 1.2 W. The OPO/OPA power is between 0.1-0.2 W. The bandwidth at room temperature is about 10 cm -1 in the IRMPD spectra. We used a CO2 laser synchronized with the CLIO FEL or the OPO/OPA system for few tens of milliseconds at the beginning of the irradiation period to improve the ion fragmentation (Universal Laser System, 10 W, continuous wave operation, wavelength 10.6 µm).

Room temperature experiments in the fingerprint region, 600-2100 cm -1 , were also performed at the FELIX laboratory. The FEL was coupled to a Bruker AmaZon ETD quadrupole ion trap mass spectrometer, which was modified to have optical access to the ion trapping region as is described in detail elsewhere [START_REF] Martens | Infrared Ion Spectroscopy in a Modified Quadrupole Ion Trap Mass Spectrometer at the FELIX Free Electron Laser Laboratory[END_REF] . The IR frequency was calibrated using a grating spectrometer. Protonated ions were generated from solution by ESI, mass-to-charge isolated in the trap, and irradiated with a single infrared laser pulse from the FEL (40-100 mJ per pulse, bandwidth 0.4% of the IR frequency) to induce wavelength-dependent IRMPD. Precursor and fragment ion intensities were determined from 6 averaged mass spectra at each IR frequency.

The IRMPD spectra are obtained by recording the photofragmentation yield, R = -ln[Ip/(Ip+ΣIf)] as a function of the wavenumber of the IR radiation, with Ip and If the precursor and fragment ion intensity. The IR yield has been linearly corrected for the frequency-dependent variation of the laser pulse energy [START_REF] Berden | An Automatic Variable Laser Attenuator for IRMPD Spectroscopy and Analysis of Power-Dependence in Fragmentation Spectra[END_REF] .

Computational details. To evaluate the effect of the keto or enol form of the peptide bond on the IR spectra, we carried out a theoretical investigation on neutral c(GlyLMet). Spectral assignment of protonated cyclic dipeptides has been achieved by comparing experimental IRMPD spectra to calculated vibrational spectra.

The conformer sampling of the cyclic dipeptides has been carried out using the iterative metadynamics sampling and genetic crossover (iMTD-GC) method implemented in the Conformer Rotamer Ensemble Sampling Tool (CREST) program [START_REF] Pracht | Automated Exploration of the Low-Energy Chemical Space with Fast Quantum Chemical Methods[END_REF][START_REF]Conformer-Rotamer Ensemble Sampling Tool Based on the Xtb Semiempirical Extended Tight-Binding Program Package Crest[END_REF] . The sampling has included additional geometry optimization of the final conformer ensemble. During this workflow, the calculations of the conformer energies have been based on the semi-empirical extended tight-binding method (GFN2-xTB) [START_REF] Bannwarth | GFN2-XTB-An Accurate and Broadly Parametrized Self-Consistent Tight-Binding Quantum Chemical Method with Multipole Electrostatics and Density-Dependent Dispersion Contributions[END_REF] . Thus, an ensemble of conformers within 40 kJ/mol of the minimum energy conformer has been generated and a clustering algorithm using principal component analysis has been used to select the 100 most representative conformers.

In order to get a better energy estimation, the geometries of characteristic conformers of dipeptides and their oxidized forms were re-optimized at the B3LYP/6-311G(d) level of the theory. The IR absorption spectra were computed at the same level of theory using a Lorentzian convolution with a 20 cm -1 FWHM. To account for anharmonicity, the calculated harmonic frequencies were uniformly scaled by 0.96, which turned out to improve the agreement between calculated and measured absorptions. However, the frequencies associated with the sulfur atom were left unscaled, which complied with previous evidence [START_REF] Correia | Vibrational Signatures of Protonated, Phosphorylated Amino Acids in the Gas Phase[END_REF][START_REF] Paciotti | O-Sulfation Probed by IRMPD Spectroscopy[END_REF][START_REF] Nei | Infrared Multiple Photon Dissociation Action Spectroscopy of Deprotonated DNA Mononucleotides: Gas-Phase Conformations and Energetics[END_REF] . All DFT static calculations were performed with the Gaussian 16 package [START_REF] Frisch | Revision B.01[END_REF] . Furthermore, single-point energy calculations were computed for the lowest-energies conformers at the DLPNO-CCSD(T)/cc-pvTZ level of theory using the ORCA program package [START_REF] Neese | The ORCA Program System[END_REF] . These electronic energies were used in combination with DFT thermal corrections to compute relative Gibbs energies. Thus, the final products of oxidation are the same for c(LMetLMet)H + and c(LMetDMet)H + . In addition, there is no clear difference in the final yields of oxidation products. Stabilization of the radical cation >S• + by the formation of either (S∴S) + or (S∴N) +16 does not impact the nature of the final products.

RESULTS & DISCUSSION

MS and MS

IR absorption spectra.

In order to determine whether a single structure or a mixture of multiple structures from different families (for instance, keto-enol) are present in the powder and in the solution, we recorded the IR absorption spectra of pure powder, a KBr pellet containing the powder at ~1%, and in DMSO-d6 solution for the three cyclic peptides. We simulated the IR absorbance spectrum for the c(GlyMet) system (the simplest cyclic system) at the B3LYP/6-311++g(d,p)/PCM(DMSO) level of the theory. This spectrum (Figure S6) shows that there is a clear difference in the C=O stretch region between the keto and enol forms. The keto spectrum has a single band around 1662 cm -1 due to the two CO stretches, while the enols have a similar peak, with an additional band due to the formation of the enol group at 1710 cm -1 . The latter is a sign of the presence of the enolic form.

In c(GlyMet), the keto form is more stable than the enol form by 96 kJ/mol. The sign of the presence of the enol form is missing from the experimental IR absorption spectra of c(GlyMet) (Figure S7), which indicates that the system remains in keto form. The presence of keto form and absence of enol can also be confirmed in the c(LMetLMet) and c(LMetDMet) spectra (Figure S8) They are observed at 1317, 1278 cm -1 and 1218 cm -1 for c(LMetDMet)H + . An intense band at 1301 cm -1 with some satellite peaks to the red is observed in the spectrum of c(GlyMet)H + .

due
The high frequency region is characterized by features that are similar for the three cyclic dipeptides: a broad band observed from 2600 to 3200 cm -1 , an intense and a narrow peak is observed at 2941 cm -1 for c(LMetLMet)H + , whose counterpart is found at 2942 cm -1 for c(LMetDMet)H + . This intense band is not observed in the IRMPD spectrum of c(GlyMet)H + . In the region of the NH-stretch modes, intense peaks are also observed at 3423 and 3416 cm -1 for c(LMetLMet)H + and c(LMetDMet)H + , respectively. This band is red shifted at 3389 cm -1 for c(GlyMet)H + . In the (OH) region, c(LMetLMet)H + shows a weak band at 3585 cm -1 . Two weak bands are observed at 3494 and 3567 cm -1 for c(LMetDMet)H + and 3564 and 3598 cm -1 for c(GlyMet)H + . IRMPD spectra of the oxo-forms of cyclic peptides show some differences compared to those of the native peptides, especially in relative band intensities and in the fingerprint region. We have already shown that the sulfoxide group exhibits a specific band around 900-1000 cm -1 14 . (SO) has been observed at 869 cm -1 in the IRMPD spectrum of S4-9). The calculated structure for c(LMetLMet) and its oxo-form referred to as LL and LLO; c(LMetDMet) and its oxo-form referred to as LD and LDO; c(GlyMet) and its oxo-form referred to as GM and GMO. The tautomerization type of the structures (keto for ketone forms, enol for enol forms, iminol for iminol forms, cycl for cyclic structures, and open for open structures) and an energetic ranking number is also indicated in the structure nomenclature for the sake of clarity.

For the non-oxidized forms, two protonation sites can be considered: the amide oxygen or nitrogen atoms. For c(LMetLMet), c(LMetDMet) and c(GlyMet), the most stable structures correspond to protonation at the amide oxygen atom, as already observed for similar systems investigated by IRMPD spectroscopy [START_REF] Zou | Competition between Diketopiperazine and Oxazolone Formation in Water Loss Products from Protonated ArgGly and GlyArg[END_REF][START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-Diphenylalanine in the Gas Phase[END_REF] . The amide bond remains planar or quasi planar. The DKP structure is puckered in a flattened boat structure or puckered with a puckering angle from 20 to 50°. Structures characterized by a hydrogen bond between the protonated carbonyl group and the sulfur atom of the methionine side chain show the methionine side chain in the plane of the DKP ring, while in the structures without hydrogen bond the methionine side chain is close to 90° compared to the DKP ring.

Oxidation of methionine to methionine sulfoxide derivatives increases the proton affinity of the sulfur atom [START_REF] Lioe | Experimental and Theoretical Proton Affinities of Methionine, Methionine Sulfoxide and Their N-and C-Terminal Derivatives[END_REF][START_REF] Afonso | Proton Affinities of the Commonly Occuring L-Amino Acids by Using Electrospray Ionization-Ion Trap Mass Spectrometry[END_REF] .

As a consequence, the lowest-energy conformers of the oxidized cyclic peptides are in a keto form and are protonated on the sulfoxide group. Protonation on the amide oxygen leads to conformers at higher energy, depending on the hydrogen bond network stabilizing the structure. These conformers can be formed under our experimental conditions during the gamma radiolysis in solution or during the electrospray process used to generate the gas-phase ions depending on the internal energies of the ions [START_REF] Morsa | Dissociation Pathways of Benzylpyridinium "Thermometer" Ions Depend on the Activation Regime: An IRMPD Spectroscopy Study[END_REF] .

The vibrational bands in the IRMPD spectrum are sequentially labeled alphabetically to facilitate the comparison with the calculated ones. Relative Gibbs energies are provided at the DFT level, and also, in parenthesis, by incorporating DLPNO-CCSD(T) electronic energies in Figure 4 to 9.

Considering this last approximation and as no significant differences have been found between the two sets of relative Gibbs energies, only DFT energies are reported in the following sections. Tentative assignments of IRMPD spectral bands of c(LMetLMet)H + are detailed in Table 1.

We The amide bond remains planar. The DKP structure is slightly puckered in a flattened boat structure. LLOketo02 is only 2.1 kJ/mol higher in energy than LLOketo01. The DKP structure adopts a flattened boat structure. LLOketo03 is 3.9 kJ/mol higher in energy than LLOketo01 and characterized by a hydrogen bond between the amide NH group and the non-oxidized sulfur atom.

It shows a boat structure. Finally, LLOketo12 is at 33 kJ/mol, protonated on the carbonyl moiety, and stabilized by a hydrogen bond between the amide NpH group and the S=O group.

A tentative assignment of the experimental bands is reported in Table 2. In the fingerprint region, band A at 869 cm -1 is assigned to the S=O stretch combined with SCH and SOH bending character.

This band is diagnostic of the sulfoxide formation during the oxidation process and it is not observed in the IRMPD spectrum of the protonated non-oxidized dipeptide.

A very intense band is predicted in the calculated IR spectra of LLOketo02 and LLOketo12 at 2337 and 2530 cm -1 , respectively, which is due to stretching of the OH of the protonated S=O group. The band assignments of c(LMetDMet)H + are detailed in Table 3.

c(LMetDMet)H + and [c(LMetDMet)(O)H]

A comparison of the IRMPD spectrum of the oxo-form of c(LMetDMet)H with the simulated spectra of the lowest energy structures LDOketo01, LDOketo02, LDOketo04, LDOketo06 and LDOimin01 is displayed in Figure 7. LDOketo01 is the lowest-energy conformer and is protonated on the sulfoxide oxygen, while LDOketo02, LDOketo04 and LDOketo06 are protonated on the amide oxygen. Finally, LDOimin01 is protonated on the iminol oxygen. The most stable structure LDOketo01 presents a flattened boat form, while LDOketo02, is characterized by a planar structure. LDOketo04 shows a puckering boat structure LDOketo06 (+14.9 kJ/mol) shows a planar structure with one side chain pointing directly above the DKP ring.

Finally, LDOimin01 is in a slightly puckered boat structure.

LDOketo01 is stabilized by a hydrogen bond between the protonated sulfoxide group and the amide oxygen. Both LDOketo02 and LDOketo04 feature a shared proton between the sulfoxide and amide oxygen atoms. LDOketo06 has an amide nitrogen involved in a hydrogen bond with the sulfoxide group NpH.....O=S. No particular intra-molecular interaction is seen in LDOimin01.

LDOketo01 owns a prominent band centered at 1298 cm -1 that can be associated with S8), but the results prove that keto forms are generally at lower energy levels and were close enough to explain the experimental spectrum.

Detailed band positions along with a concise description of the assigned vibrational modes for c(GlyMet)H + are listed in Table 5.

The IRMPD spectrum of [c(GlyMet)(O)]H + is compared with the predicted spectra for the GMOketo01, GMOketo02 and GMOketo03 in S9).

A tentative assignment of IRMPD features of [c(GlyMet)(O)]H + is proposed in Table 6.

Diagnostic band for the formation of the sulfoxide is observed in the fingerprint region. The band A (881 cm -1 ) is associated with a combination of SOH bending, SO and CC stretching modes.

The intense bands predicted in GMOketo03 and GMOketo01 are assigned to NpH stretching (2678 cm -1 ) and OH stretching of the protonated S=O group (2763 cm -1 ), respectively.

CONCLUSIONS

We studied the one-electron oxidation of cyclic methionine dipeptides by OH free radicals produced by gamma radiolysis. The first step of oxidation, i.e. the formation of the methionine free radicals has been studied some years ago by pulse radiolysis [START_REF] Bobrowski | Formation and Stability of Intramolecular Three-Electron SN, SS, and SO Bonds in One-Electron-Oxidized Simple Methionine Peptides. Pulse Radiolysis[END_REF][START_REF] Bobrowski | Stabilization of Sulfide Radical Cations through Complexation with the Peptide Bond: Mechanisms Relevant to Oxidation of Proteins Containing Multiple Methionine Residues[END_REF] . The authors demonstrated that the c(LMetLMet) isomer led to (S∴S) 2c-3e free radicals due to the proximity of the two sulfur atoms, while the c(LMetDMet) isomer formed (S∴N) 2c-3e free radicals. Our aim was to elucidate the fate of these two free radicals, which is still unknown. Our studies also gave elements to predict the reactivity of these free radicals. The final products were mass-isolated in the gas phase. CID-MS 2 tandem mass spectrometry and IRMPD spectroscopy have been performed. DFT calculations have been performed to explore their chemical structures in the gas phase and for vibrational band assignment.

Oxidation of both c(MetMet) peptides induced the formation of one or two sulfoxide groups on each sulfur atom. Oxidation of c(GlyMet) led only to one sulfoxide group. No sulfone has been detected. In addition, loss of dihydrogen was observed leading to a C=C double bond in the methionine side chain, due to the attack of OH radicals to the carbon atom adjacent to sulfur (formation of the so-called -S free radical) [START_REF] Hiller | Mechanism of the Hydroxyl Radical Induced Oxidation of Methionine in Aqueous Solution[END_REF] . These results are similar to those observed for e.g.

Met-Val and Val-Met peptides [START_REF] Gregori | One-Electron Oxidation of Methionine-Containing Dipeptides of Reverse Sequence: Sulfur relative Gibbs free energy in kJ/mol, calculated by a combination of the electronic CCSD(T) energies at the DLPNO-CCSD(T)/cc-pvTZ level of theory with thermal correction (298 K) from the B3LYP/6-311g(d) calculations[END_REF] . They demonstrate that both 2c-3e free radicals have the same the product with a C=C double bond (i) by the loss of H2. In the second pathway, the thioether group of (a) is attacked by OH radical to form hydroxysulfuranyl radicals (b) which are highly unstable and decompose rapidly to S-centered radical cations (c). Three possible 2c-3e free radicals (f)-(h) can be formed through intramolecular or intermolecular stabilizations with a reactive site owing an electron lone pair. Hydration and disproportion of 2c-3e free radicals give rise to the formation of sulfoxide product c(MetMetSO) (j). The amount of sulfoxide formed in both c(LMetLMet) and c(LMetDMet) are quite similar and low (ca.5 %). The same amount of sulfoxide is observed for c(GlyMet). In addition, the similar yield of sulfoxide shows that the chirality of S-containing side chains in cyclic methionine dipeptides has no impact on the production of the final product.

IRMPD spectroscopy and DFT calculations allowed us to identify the chemical structures of the final products in the gas phase. In particular, we have shown that the cyclic dipeptides adopt a keto form in the gas phase, except for c(LMetDMet), where an iminol isomer can also be populated in our experimental conditions. IRMPD spectra are well reproduced by the calculated IR absorption spectra of the lowest energy isomers.

The non-oxidized dipeptides are protonated on the amide oxygen. Oxidation leads to the increase of the proton affinity of the sulfoxide group. In the oxidized cyclic dipeptides, the protonation site is the sulfoxide group. Structures with the protonation of the amide oxygen, at higher energy are also populated under our experimental conditions. IR spectra performed on the powders or on the solution showed that the dipeptides adopt a keto form of the DKP ring in the solid state or in solution before oxidation. Structures at higher energies are likely formed during the gamma radiolysis or are due to kinetic trapping in the electrospray source.

ASSOCIATED CONTENT

Supporting information

The supporting information is available free of charge via the internet at xxx.com A detailed description of the synthesis process of cyclic peptides; the CID-MS 2 fragmentation mass spectra of protonated cyclic dipeptides and their oxidation products with the assignments of the observed peaks; experimental and simulated infrared absorption spectra in different environments (pure powder, KBr powder, DMSO-d6 solution); and geometries of the B3LYP/6-311g(d)-optimized stable conformers of protonated cyclic dipeptides and their oxidized forms, along with their calculated spectra and detailed structural information.

versus Sulfoxide Characterized by IRMPD Spectroscopy and Static and Dynamics DFT Simulations. J. Phys. Chem. B 2017, 121 (9), 2083-2094. The corresponding structures are reported on the right together with the relative Gibbs free energy (∆G) at 298 K in kJ/mol, calculated at B3LYP/6-311g(d) level of theory. Another kind of relative Gibbs free energy in kJ/mol, calculated by a combination of the electronic CCSD(T) energies at the DLPNO-CCSD(T)/cc-pvTZ level of theory with thermal correction (298 K) from the B3LYP/6-311g(d) calculations, is provided in parentheses. The corresponding structures are reported on the right together with the relative Gibbs free energy (∆G) at 298 K in kJ/mol, calculated at B3LYP/6-311g(d) level of theory. Another kind of relative Gibbs free energy in kJ/mol, calculated by a combination of the electronic CCSD(T) energies at the DLPNO-CCSD(T)/cc-pvTZ level of theory with thermal correction (298 K) from the B3LYP/6-311g(d) calculations, is provided in parentheses.

Final

  Products of One-Electron Oxidation of Cyclic Dipeptides Containing Methionine Investigated by IRMPD Spectroscopy: Does the Free Radical Choose the Final Compound? Final products of One-Electron Oxidation of Cyclic Dipeptides containing Methionine investigated by IRMPD Spectroscopy: does the Free Radical choose the Final compound? INTRODUCTION Reactive oxygens species (ROS) have special function in the biology of living organisms. While

2 - 2 .

 22 CID spectra. The mass spectra (MS) of the samples containing non-irradiated dipeptides and dipeptides oxidized by OH radicals are reported in Figure The most intense peaks at m/z 263 and 189 in the MS of the non-irradiated c(MetMet) and c(GlyMet), respectively, are attributed to the protonated dipeptides. Singly (M+16) and doubly (M+32) oxidized products, m/z 279 and 295, were found for c(LMetLMet) and c(LMetDMet) (Figure 2.a and b), while only the single-oxidation product (M+16) at (m/z 205) was observed for c(GlyMet) (Figure 2.c). For c(LMetLMet) and c(LMetDMet), the product of dehydrogenation at m/z 261 was also observed after radiolysis, as already observed in the case of linear Val-Met and Met-Val dipeptides[START_REF] Lemaire | Gas Phase Infrared Spectroscopy of Selectively Prepared Ions[END_REF] . A peak at m/z 214 was observed in the spectrum of c(GlyMet), corresponding to instrumental contamination (n-butyl benzenesulfonamide, n-BBS).To characterize the final oxidation products, collision induced dissociation (CID-MS 2 ) of mass selected products was performed. The identification of the main fragments for each oxidation product is summarized in Tables S1 c(LMetLMet)H + , S2 c(LMetDMet)H + and S3 c(GlyMet)H + in the supporting information.The CID-MS 2 fragmentation mass spectra (FigureS3-5) of protonated dipeptides are characterized by the loss of sulfenic acid (-HSCH3) from the methionine side chain. In the case of c(LMetLMet)H + and c(LMetDMet)H + a peak at m/z 167 was observed as well, which is attributed to the loss of two sulfenic acid molecules.The MS 2 -CID fragmentation of electrosprayed mass selected oxidation products of c(LMetLMet)H + and c(LMetDMet)H + show the loss of one oxidized sulfenic acid molecule (-HSCH3+O) indicating that the oxidation can occur on the sulfur atom of both methionine side chains. No sulfone formation was observed. As expected, the CID-MS 2 fragmentation mass spectra of oxidized c(GlyMet)H + shows only the loss of one oxidized sulfenic acid molecule.

  to a single band in the C=O stretching region.IRMPD spectra. The IRMPD spectra of protonated c(LMetLMet), c(LMetDMet), c(GlyMet) and of their corresponding oxo-forms are reported in Figure3. Both spectra of non-oxidized c(MetMet)H + and of c(GlyMet)H + show similar features in the 800-3700 cm -1 energy range. For the protonated cyclic peptides, the  (CO) stretch region is characterized by two features (a sharp peak and a broad band) located at 1755-1665 cm -1 in the spectrum of c(LMetLMet)H + and at 1751-1676 and 1763-1639 cm -1 for c(LMetDMet)H + and c(GlyMet)H + . These bands are typical of an amide I vibrational mode in DKP rings[START_REF] Zou | Competition between Diketopiperazine and Oxazolone Formation in Water Loss Products from Protonated ArgGly and GlyArg[END_REF][START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-Diphenylalanine in the Gas Phase[END_REF] . The NH-bending range displays two bands at 1487 and 1436 cm -1 in the spectrum of c(LMetLMet)H + , whose counterpart is found at 1495 and 1435 cm -1 for c(LMetDMet)H + and 1470 and 1425 cm -1 for c(GlyMet)H + . The region of the  (OH) and aliphatic  (CH) bending modes shows two bands at 1315 and 1219 cm -1 for c(LMetLMet)H + .

  c[(LMetLMet)(O)H] + , whose counterpart is found at 871 cm -1 in the IRMPD spectrum of c[(LMetDMet)(O)H] + and 881 cm -1 in the IRMPD spectrum of c(GlyMet)(O)H + . Theoretical Results and Band Assignment. The nomenclature used for the molecules is shown in Figure 1. A subscript p is employed to indicate the peptide bond atoms on the protonated moiety. C, C, or C𝛾 indicates the carbon atom position relative to the peptide bond. Different forms of cyclic peptides have been extensively explored. Among them, the ketone forms show the lowest calculated energy (all structures explored with corresponding IR spectra and calculated energies are displayed in Figure S9-42 and Tables

c

  (LMetLMet)H + and [c(LMetLMet)(O)H] + . The experimental IRMPD spectrum of c(LMetLMet)H + and the simulated IR spectra for LLketo01, LLketo02 and LLketo04 geometries are reported in Figure4together with the corresponding calculated geometries. The calculated IR spectra for the three keto forms show the best agreement with the IRMPD one and allow all peaks observed to be assigned. LLketo01, LLketo02 and LLketo04 are all characterized by protonation on the amide oxygen. In LLketo01, the proton is involved in a hydrogen bond with the sulfur atom. This conformer is also stabilized by an NH…..S interaction. Its calculated IR spectrum shows good agreement with the IRMPD one for the major bands from B to I. LLketo02, which shows a more puckering boat structure, is 19.7 kJ/mol higher in energy compared to LLketo01 and allows for a better assignment of band A. Its structure is stabilized by two non-covalent interactions: an NH…..S interaction and a COH….S one. The position of band F is predicted slightly towards the red in LLketo02 and allows to justify the broad band observed in the IRMPD spectrum. Meanwhile, it is necessary to populate a third conformation LLketo04 found at 31.3 kJ/mol to justify band J in the hydrogen stretching region Kinetic trapping could explain the presence of thermodynamically disfavored species[START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-Diphenylalanine in the Gas Phase[END_REF][START_REF] Piccirillo | Probing the Competition among Different Coordination Motifs in Metal-Ciprofloxacin Complexes through IRMPD Spectroscopy and DFT Calculations[END_REF][START_REF] Nieuwjaer | IRMPD Spectroscopy and Quantum Chemistry Calculations on Mono-and Bi-Metallic Complexes of Acetylacetonate Ligands with Aluminum, Iron, and Ruthenium Ions[END_REF] . The population of different conformers LLketo01, LLketo02 and LLketo04 justify the broad bands C, D, E and F observed in the IRMPD spectrum of c(LMetLMet)H + .

6 .

 6 + . A comparison of the calculated IR spectra of the most stable geometries of c(LMetDMet)H + , LDketo01, LDketo02 and LDketo06, with the experimental IRMPD spectrum is shown in Figure As observed for c(LMetLMet)H + , all three structures are keto forms with protonation on the amide oxygen. A combination of the calculated spectra for these three structures can explain the ensemble of features observed in the IRMPD spectrum of c(LMetDMet)H + . The most stable structure LDketo01 features a proton shared between the amide oxygen and the sulfur atom, which stabilizes the structure. The DKP ring is in a slightly twisted boat structure. LDketo02 and LDketo06 are required to account for the presence of the bands L and M. The band predicted at 1682 cm -1 for LDketo06 is slightly blue-shifted compared to its counterparts in the other two structures, and the contribution of this band accounts for the broadening of band G in the IRMPD spectrum. Both LDketo02 and LDketo06 are stabilized by a non-covalent NpH…..S interaction LDketo02 possesses a quasi-planar structure while LDketo02 presents a flattened boat DKP ring.

  experimental band D. Bands H, I, J and K observed in the IRMPD spectrum of [c(LMetDMet)(O)H] + also find a counterpart in the calculated IR spectrum of LDOketo01. A contribution of LDOketo02 and LDOketo04 are required for explaining the presence of the intense band A observed in the IRMPD spectrum. The population of LDOketo06 and LDOimin01 can justify the presence of the weak bands M and N in the NH/OH stretching region. Band assignment for [c(LMetDMet)(O)H] + is reported in Table 4. In the fingerprint region, bands A and B are related to the diagnostic SO stretching mode. The prominent band A, centered at 871 cm -1 , is assigned to the stretching modes of SO and CC bonds, along with the bending of CH2 and CH3. The asymmetric band B at 1074 cm -1 is attributed to the SO stretching mode accompanied by the CH2 and CH3 bending modes. c(GlyMet)H + and [c(GlyMet)(O)H] + . A comparison of the IRMPD spectrum of c(GlyMet)H + with the calculated IR spectra of the lowest energies keto form GMketo01, GMketo02, GMketo03 and GMketo05 is shown in Figure 8. GMketo01 is the most stable conformer of c(GlyMet)H + and allows to justify most of the experimental features observed in the IRMPD spectrum: especially bands B-H and J-K. The calculated IR spectra of GMketo01, GMketo02 and GMketo03 are quite similar. GMketo02 and GMketo03 show better agreement with band A than GMketo01. GMketo02 and GMketo03 are at 8.2 and 10.9 kJ/mol higher in energy compared to GMketo01, respectively. It is still necessary to assume population of a fourth conformer GMketo05, lying at 19.6 kJ/mol higher in energy compared to GMketo01, to justify the bands I and L in the high frequency region. The protonation site is always on the amide oxygen for these structures. Other possible families have also been investigated (Figure S33-36, energies are listed in Table

Figure 9 .

 9 GMOketo01, conformer is the lowest in energy. It is protonated on the sulfoxide group. The protonation site of GMOketo02 and GMOketo03 is on the amide oxygen. GMOketo01 is stabilized by a hydrogen bond with the carbonyl group S=O-H…..O=C and it shows in a flattened DKP structure with the amide group mildly twisted by the S=OH…..O interaction. It provides a general match of most of the spectral features. However, in order to justify all the IR absorption peaks observed in the IRMPD spectrum, we should admit that the following also populate in our experimental conditions: GMOketo02 to justify the band M and GMOketo03 for a better description of the band N. GMOketo02 and GMOketo03 lie 22.3 and 42.2 kJ/mol higher than GMOketo01, respectively. GMOketo02 displays in a puckered boat structure, whereas the DKP ring structure of GMOketo03 shows a flat character. We have extensively searched the existence of other families in the gas phase (FigureS37-42, relative free energies are listed in Table

  fate, disproportionation plus hydration to give sulfoxide. A proposed mechanism for the oneelectron oxidation of cyclic methionine dipeptide c(MetMet) is displayed in Figure 10. When hydroxyl-induced oxidation occurs, the initial cyclic dipeptide c(MetMet) (a) undergoes two possible pathways: H-atom abstraction or addition of OH radical. They are in competition with each other. The first pathway leads to the formation of -S free radicals (d) and (e) by removing one hydrogen atom from the -S position of (a). Disproportionation of (d) and (e) leads to (a) and

Figure 1 Figure 1 .Figure 3 .Figure 4 .Figure 5 . 6 )

 113456 Figure 1

Figure 6 .Figure 7 .

 67 Figure 6

Figure 8 .

 8 Figure 8

Figure 9 .Figure 10 Figure 10 .

 91010 Figure 9

  have calculated the IR spectra of other possible families for c(LMetLMet)H + . Their structures and the IR spectra are reported in Figures S9-14. These structures can be ruled out on energetic LLOketo01 is the most stable conformer for [c(LMetLMet)(O)H] + , stabilized by a network of two hydrogen bonds. Protonation occurs on the oxygen of the sulfoxide group, as in LLOketo02 and LLOketo03. The protonated sulfoxide group is involved in a hydrogen bond with the carbonyl group S=O-H…..O=C . LLOketo01 is also stabilized by an NH…..S interaction.

	criteria.
	A comparison of the calculated IR spectra of the most stable geometries of [c(LMetLMet)(O)H] + ,
	LLOketo01, LLOketo02, LLOketo03 and LLOketo12, with the experimental IRMPD one is
	reported in Figure5.

Table 1

 1

		Experiment	Assignment	LLketo01 LLketo02	LLketo04
	A	870	νSC, δSCH, δCNH		853
	B	1219	δSCH, δCH2		1222	1187
	C	1315	νCN, νCp-O, δCH2, δCN(p)H	1323	1287	1306
	D	1436	δCH2, δCN(p)H, νNC	1433	1433	1445
	E	1487	νCpC,	1477	1462
			δCN(p)H, δCH2, δCpOH
	F	1665	νNpC, δCNpH, δCpOH	1653	1633	1672
	G	1755	νC=O, δCNH	1734	1746	1737
		Broad band at	νOH	2451	2533
		ca.2600
	H	2941	νCH2, νCH3	2938	2940	2928
			νNpH, νasCH2			2981
			νNpH	3053
			νNpH		3092
	I	3423	νNH	3444	3454	3443
	J	3585	νOH			3588

Table 1 .

 1 Experimental and calculated vibrational frequencies for the selected isomers c(LMetLMet)H + at B3LYP/6-311g(d) level of the theory, and corresponding band assignments.

Table 2

 2

		Experiment	Assignment	LLOketo01 LLOketo02 LLOketo03 LLOketo12
	A	869	νS=OH + , δSCH, νS=O	824			927
	B	1082	νCN, δSOH, νS=O	1071	1054		1121
	C	1181	δCOH, δCH2, δSOH			1206	1202
	D	1289	νCOH, δSOH, δCH2	1279	1263	1320	1322
	E	1427	δNH, δCH2, δCH3	1423	1405	1423	1429
	F	1592	δCNH, δCH2 νCN, νCO,		1619	1623
	G	1652	νC=O, δCNH	1644			1673
	H	1745	νC=O, δCNH	1729	1717	1730	1730
		-	ν(S)OH		2337		2530
	I	2938	νCH2, νCH3	2934	2928	2933	2933
		Broad band	νNH	3232		3232
		between 2900-3200 cm -1	ν(S)OH	3303
	J	3418	νNH	3431	3421
	K	3497	νNH, νNpH			3438	3450
	L	3587	νOH				3545
	M	3623	νOH			3596

Table 2 .

 2 Experimental

	and calculated vibrational frequencies for the selected isomers of
	[c(LMetLMet)(O)H] + at B3LYP/6-311g(d) level of the theory, and corresponding band
	assignments.

Table 3

 3

		Experiment	Assignment	LDketo01	LDketo02	LDketo06
	A	847	νSC, δCH2, δCNpH	804
	B	1218	νCpO, δCpOH, νCNp, δCH2		1167	1206
	C	1278	νCN, δCpOH, δCH2	1250	1276	1266
	D	1317	νCpO, νCC, δCN(p)H	1322	1311	1311
	E	1435	δscissorCH2,δCH3, δCN(p)H	1434	1430	1438
	F	1495	νCpCp, δCN(p)H, δCpOH,	1493		1471
			δCH2
	G	1676	νCNp, δCNpH, δCpOH	1638	1650	1682
	H	1751	νC=O, δCNH	1727	1732	1740
	I	2942	νCH, νCH2, νCH3, νNpH	2936	2934	2926
	J	3371	νNpH	3396
	K	3416	νNH	3431
	L	3494	νNH		3446	3447
	M	3567	νOH		3579	3602

Table 3 .

 3 Experimental and calculated vibrational frequencies for the selected isomers of c(LMetDMet)H + at B3LYP/6-311g(d) level of the theory, and corresponding band assignments.

Table 4

 4

		Experiment	Assignment	LDOketo	LDOketo	LDOketo	LDOketo06 LDOimin01
				01	02	04
	A	871	νS=OH + , νS=O, νCC, δCH2, δCH3	814	905	919
	B	1074	νS=O, δCH2, δCH3				999
	C	1179	δSCH,νCN(p), νCC		1162	1159		1120
	D	1281	δSOH, νCN(p),δCpOH, δCH2	1298	1263	1271	1243
	E	1329	νC(p)O, νCN(p), δCH2, δCH3		1322		1320
	F	1425	νCpO, νCN, δCN(p)H, δCH2			1412	1431	1428
	G	1465	νCpCp, δCN(p)H		1509	1503
	H	1591	νC(p)O, νCN(p), δCN(p)H	1650	1634	1645		1629/1669
	I	1776	νC=O, δCNH, νCNp	1734	1732	1739	1686/1724
	J	2933	νCH2, νCH3	2931	2924	2935	2935	2925
		Broad band	νNpH			3113
		between 2900 and 3300 cm -1	ν(S)OH	3196
	K	3412	νN(p)H	3420	3407			3420
	L	3486	νNH		3444	3454	3448
	M	3545	νOH				3559
	N	3579	ν(S)OH					3593

Table 4 .

 4 Experimental

	and calculated vibrational frequencies for the selected isomers of
	[c(LMetDMet)(O)H] + at B3LYP/6-311g(d) level of the theory, and corresponding band
	assignments.

Table 5 Experiment

 5

			Assignment GMketo01 GMketo02 GMketo03 GMketo05
	A	879	δSCH, δCNpH		866	870	824
	B	1209	νCNp δCpOH, νCpO,	1167	1172	1173	1184
	C	1301	δCH2, δCH3,	1283	1277	1278	1255
			νCpO νCC,
			νCN
	D	1470	δCN(p)H, δCH2	1433	1435	1432	1441/1496
			, δCH3, νCpCp
	E	1639	νCNp, δCNpH	1661	1648	1649	1682
	F	1763	νC=O, δCNH	1743	1747	1749	1735
		Broad band	νNpH, νCH	2845	2896	2879
		between 2600
		and 3200 cm -1
	G	2865	νCH2				2866
	H	2944	νCH2, νCH3				2934
	I	3389	νNpH				3383
	J	3416	νNH	3456	3465	3466	3441
	K	3564	νOH	3565	3557	3561
	L	3598	νOH				3597

Table 5 .

 5 Experimental and calculated vibrational frequencies for the selected isomers of c(GlyMet)H + at B3LYP/6-311g(d) level of the theory, and corresponding band assignments.

Table 6

 6

		Experiment	Assignment	GMOketo01 GMOketo02 GMOketo03
	A	881	δSOH, νS=OH + , νS=O, νCC	911	925	919
	B	1059	δSCH νS=O, νCN(p), νCC, νCpO,		1077
	C	1152	δSCH,δCpOH, δCH2			1108
	D	1212	δCH2 νCN(p), νCpO, δCOH,	1251	1190	1200
	E	1271	νCN(p), νCC, δCH2, δCH3	1310		1274
	F	1397	νCN(p), δCN(p)H	1384
	G	1446	νC(p)C(p),	1471	1436	1439
			δCN(p)H, δCH 2
	H	1584	νCpCp, νCNp, δCpOH		1574
	I	1657	νC(p)O, νCN(p), δCN(p)H	1637	1679
	J	1763	νC=O, δCNH	1750	1740	1738
		Broad band	νNpH			2678
		between 2600 and 2800 cm -1	ν(S)OH	2763
	K	2931	νCH2	2930	2879	2877
	L	3415	νNH	3432	3466	3452
	M	3578	νOH		3541
	N	3633	νOH			3606

Table 6 .

 6 Experimental

	and calculated vibrational frequencies for the selected isomers of
	[c(GlyMet(O))H] + at B3LYP/6-311g(d) level of the theory, and corresponding band
	assignments.

AUTHOR INFORMATION

Corresponding Author

*Debora Scuderi

Université Paris-Saclay, CNRS, Institut de Chimie Physique, UMR8000, 91405 Orsay, France. E-mail: debora.scuderi@universite-paris-saclay.fr; Phone: +33 1 69 15 75 74.

ACKNOWLEDGMENT