Mathieu Cousy
email: mathieu.cousy@enac.fr

Vincent Peyruqueou

Christophe Pierre

Cyrille Priou

Dong Bach Vo

Jérémie Garcia

Mihaela Mitici
email: m.a.mitici@uu.nl

Simon Van Oosterom

Alexei Sharpanskykh
email: a.sharpanskykh@tudelft.nl

Malte Von Der Burg

Paul Roling

Elisa Spiller

Samuele Gottofredi

Paola Lanzi
email: paola.lanzi@dblue.it

AEON: TOWARD A CONCEPT OF OPERATION AND TOOLS FOR SUPPORTING ENGINE-OFF NAVIGATION FOR GROUND OPERATIONS

Keywords: Engine-Off Taxiing, Human-Machine-Interaction, Multi-Agent System, Human Performance, Safety, liability

The SESAR Advanced Engine-off Navigation project (AEON) aims at exploring the reduction of ground operations environmental impact based on the use of three classes of greener taxiing solutions: singleengine taxiing solutions, hybrid towing taxiing solutions and electric engine solutions. This approach requires a novel concept of operation and new support tools for sustainable airport ground operations to cope with the additional vehicles on the ground to tow aircraft, discrepancies between aircraft in terms of ground speed according to the taxiing technique and the management of the fleet of towing vehicles. In this paper we first describe the motivation and the context of the project. We then introduce the AEON concept of operation that includes a new role responsible for the supervision of towing vehicles. We also articulate the architecture of our solution and describe three inter-operating support tools: (1) an optimization tool to estimate the number of necessary tugs and assign them to the aircraft before the operations; (2) a multiagent path planning system providing real-time, conflict-free routes including speed profiles to maximize capacity as well as fuel efficiency. (3) Human-Machine Interfaces for supporting Air Traffic Controllers and the Tug Fleet Manager to control and supervise the whole traffic. Then, we present the validation activities including an evaluation with Air Traffic Controllers using realistic real-time simulations on the Paris Charles de Gaulle airport. Finally, we conclude with a discussion on how this concept could impact on a number of key performance indicators (KPA), such as human performance, safety, capacity, environmental impact and proactive liability allocation in case of an accident.

INTRODUCTION

Aircraft jet engines are made for flying, not taxiing. Using main engines for ground operations is inefficient and taxiing phase can represent from 10 to 30% of flight time in Europe [START_REF] Deonandan | Evaluation of strategies for reducing taxi-out emissions at airports[END_REF]. Regarding fuel consumption, Stettler et al. estimated that taxiing to burn 36% of the fuel on a standard Landing and Take Off cycle [START_REF] Stettler | Air quality and public health impacts of UK airports. Part I: Emissions[END_REF]. They also estimated that the taxi phase accounts for 12% of NOx, 89% of CO and 91% of HC emissions.

Several alternatives are being developed to reduce fuel consumption and noxious emissions during ground operations [START_REF] Lukic | State of the art of electric taxiing systems[END_REF]. They can be grouped in two categories: autonomous techniques in which the aircraft uses its own equipment such as electric engines in the rear or landing gear; and non-autonomous technique in which external equipment is used such as specifically designed tugs coupled to the aircraft and controlled by the pilot. While each of these solutions can help saving fuel, they all have advantages and drawbacks. Autonomous electric taxiing aircraft have lower dynamic performances (speed and acceleration), nonautonomous solutions add new vehicles to be managed on the taxiways (empty tugs). However, all these techniques share the need for engines warmup and start management for departing aircraft. For this reason, Single Engine Taxiing (SET) is used mainly for taxi-in of arriving aircraft and almost never for departures [START_REF]Single-Engine Taxi-out Evaluations[END_REF].

In this paper, we present our work during the SESAR AEON project to explore a new concept of operation and dedicated supporting tools for enabling the introduction of engine-off taxiing techniques. More specifically, we worked in close collaboration with multiple stakeholders from two major airports in Europe, Paris CDG (LFPG, France)and Amsterdam Schiphol (EHAM, Netherlands), including ATC officers, pilots, airlines, airport management, and ground handlers to explore and refine our concept and associated tools. We iteratively designed and assessed interconnected solutions to enable an optimized allocation of a fleet of tugs to aircraft, centralized routing providing speed profiles to avoid conflicts, dedicated HMI for Air Traffic Controllers as well as a new role, the Tug Fleet Manager. We describe the main aspects of our concept of operation and the overall architecture of the solution before presenting each component in more details. We then describe the experimental platform we created to perform real-time evaluations of our prototypes on 12x30 minutes long exercises simulating a normal day of traffic at Paris-CDG airport. We conclude with a discussion on how this concept could impact on several key performance indicators (KPA), such as human performance, safety, capacity, environmental impact and proactive liability allocation in case of an accident, and how it could be integrated with existing tools used to manage airport operations such as the Airport Collaborative Decision Making (A-CDM) tool.

RELATED WORK

Introducing engine-off taxiing techniques might have a strong impact on the airport capacity and the work of airports' operators due to specific path planning activities and tugs supervision [START_REF] Baaren | Design of a zero emission aircraft towing system[END_REF].

Electric taxiing and airport ground operations have been modelled extensively in the past years using operations research methods. Prior work modelled the use of a fleet of towing vehicles for parts of the taxiing phases including the optimal allocation of vehicles to aircraft [START_REF] Baaren | Design of a zero emission aircraft towing system[END_REF][START_REF] Soltani | An eco-friendly aircraft taxiing approach with collision and conflict avoidance[END_REF], battery charging of vehicles [START_REF] Hiermann | The Electric Fleet Size and Mix Vehicle Routing Problem with Time Windows and Recharging Stations[END_REF], or the sizing of the fleet [START_REF] Han | Optimal scheduling of airport ferry vehicles based on capacity network[END_REF]. Most work focus on fleet management at a strategic level but dedicated optimization models for the management of a fleet of electric towing vehicles are not available for the tactical phase when disruptions impact the usage of the towing vehicles for arrival/departure flights.

Several path algorithms have been introduced over the years using multi-agent systems (MAS) [60]. The success of these algorithms is highly dependent on the environment and constraints that are imposed [START_REF] Durfee | Scaling Up Agent Coordination Strategies Coordination Strategies[END_REF]. Centralized global optimization methods suffer from scalability and computational complexity issues, as the state space and time grow exponentially with the increase of the number of aircraft [START_REF] Sharon | Conflict-based search for optimal multiagent pathfinding[END_REF][START_REF] Wang | MAPP: A scalable multi-agent path planning algorithm with tractability and completeness guarantees[END_REF]. Multi Agent Path Finding (MAPF) and Multi Agent Path Planning (MAPP) [START_REF] Erdem | A general formal framework for pathfinding problems with multiple agents[END_REF][START_REF] Honig | Multi-agent path finding with kinematic constraints[END_REF][START_REF] Ma | Searching with consistent prioritization for multi-agent path finding[END_REF] is a class of decentralized, multiagent planning and scheduling techniques that can address this issue as they scale polynomially and allow for high flexibility and robustness of plans that can be generated and modified in real time. Such techniques would also allow for more efficient, multiagent, anticipatory identification and resolution of conflicts in polynomial time. Effective conflict identification and resolution mechanisms are essential for safety and allow for efficient and smooth traffic flow with the minimum number of stops. Considering a control of the speed profiles of aircraft also enable to eliminate strops and reduce fuel consumption [START_REF] Van Arem | The Impact of Cooperative Adaptive Cruise Control on Traffic-Flow Characteristics[END_REF].

To support Air Traffic Controllers (ATCOs), Advanced-Surface Management Ground Control Systems (A-SMGCS) routing service must provide path planning decision support [START_REF] Lane | EUROCONTROL Specification for A-SMGCS Services[END_REF]. Prior work investigated interaction to help ATCOs validating recommended routes or editing them [START_REF] Chua | Initial assessment of the impact of modern taxiing techniques on airport ground control[END_REF] but did not considered having a variety of taxiing techniques with different speeds capabilities. Using speed profiles have been investigated specific displays to guide pilots following speed targets [START_REF] Bernatzky | Development and evaluation of a speed guidance interface for trajectory-based dispatch towing[END_REF]. To build an effective distributed system, both human and automated agents such as path planning algorithms will require various inputs to operate and dedicated mechanisms to share their solutions or alternatives to other agents. The Human-Automation Teaming [START_REF] Battiste | Human-Automation Teaming : Lessons Learned and Future Directions[END_REF] will require providing dedicated Situation Awareness [START_REF] Endsley | From Here to Autonomy: Lessons Learned From Human-Automation Research[END_REF][START_REF] Salmon | Distributed situation awareness: Theory, measurement and application to teamwork[END_REF] elements that are required by each stakeholder, including algorithms, to operate efficiently.

Our goal is to build upon research operations, multi-agent path planning and Human-Automation teaming work to explore a new concept of operation with adequate supporting tools enabling the use of engine off navigation techniques on ground.

AEON REQUIREMENTS, SOLUTIONS AND HYPOTHESES

We started by conducting several workshops and interviews with professional stakeholders to better understand the current contexts, the challenges and to identify needs to inform our concept of operation. Based on the results, we elicited requirements covering three aspects of the ground operations, airport management; air traffic control and piloting as detailed in Table 1.

Overview of the solutions

The AEON concept of operations introduces solutions for using green taxiing techniques at strategic and tactical levels. The overall framework of our solution is presented in Figure 1.

In the strategic phase, i.e., up to 1 hour before the flight is scheduled, we integrate with the airportcollaborative decision making (A-CDM) platform to support discussion and negotiation of the desired taxi technique for the aircraft between the stakeholders. This choice will have an impact on the taxi speed and hence on the flight's in-block or off-block time. In addition, it also has an impact on the organization of ground handling activities (the tugs, marshallers... need to be available). Given these choices and tug availability, a first set of support algorithm computes an initial tug allocation plan. In the tactical phase, i.e., during the operations, we introduce a multiagent system suggesting optimal routes for tugs and aircraft. The multi agent system uses flight plan and surveillance data to compute routes and speed profiles to avoid conflicts as well as stop and go at intersections. The path and speed information can be distributed to pilots and tug drivers via the Electronic Flight Bag. We also designed a radar image providing additional situation awareness elements to the ATCOs about the taxiing techniques and supporting the interaction with the optimal route suggestion algorithm. Finally, we designed a moving map for tug drivers and pilots to use the path and speed information.

The AEON concept also introduces a new role at the airport, the tug fleet manager (TFM), which is envisioned to be responsible for operating and maintaining the tugs. Throughout the day, the TFM ensures the best availability of the vehicle fleet by assigning tugs to arriving/departing aircraft, by monitoring their status (the state-of-charge of the battery, the availability of the tug to perform a towing task) and by handling operational delays of arriving/departing aircraft. The TFM will oversee the towing vehicles being on time to pick-up arriving/departing aircraft and tow these aircraft to the gates/runways. The TFM is also expected to assign towing vehicles to the specific aircraft. These assignments are supported by the dedicated algorithms for allocating vehicles to aircraft and for identifying efficient routes to follow. Once a towing mission is assigned, it becomes the joint responsibility of the TFM and ATCO to reach the aircraft to be towed on time for smooth operations.

Hypotheses

Concerning the non-autonomous taxiing technique, it has been considered that the tugs would only move on the taxiways network, even when not towing any aircraft. The use of service roads would have been too complex since they are also used by a wide variety of other vehicles and the route planning would have impeded. In addition, the aircraft loading and unloading processes were only possible at dedicated areas, namely the deicing pads that are close to the runway entry and exit (respectively for departure and arrival aircraft). Finally, no assumptions were made concerning the economic model for the non-autonomous taxiing technique (whether the service would be provided by the airport, the ground handler, or the airline). Actually, this choice has no impact on the operations that are proposed and evaluated in AEON project.

In the setup put in place for the concept validation, the ground ATCO and TFM were in separated rooms (tower and apron ATCOs were simulated). Both had a radar image view of the airport and the TFM had a dedicated HMI for tugs allocations. The ATCO could talk to the aircraft' pilots directly over radio on a given frequency and the TFM talked to tug drivers only on another frequency. Finally, 3 people acted as pseudo pilots and tug drivers, responding to ATCO and TFM requests and manipulating the simulation engine accordingly.

INTER-OPERATING SUPPORTING TOOLS

In this section we describe the tools that were developed during the project that include an allocation tool for optimizing the fleet of vehicles operations, a multi-agent path planning tools providing conflict free routes and speed targets for aircrafts, and interfaces and interactions for ground controllers and the tug fleet manager to carry out their tasks with the support of the algorithms.

Allocation of tugs to aircrafts

During the strategic planning phase, the AEON fleet management algorithm helps estimate the adequate number of tugs to operate a given airport in a given period, considering its specific traffic. Taking into account the arrival and departure sequences, plus the operational constraints of the tugs fleet, the AEON fleet management algorithm sizes the fleet of tugs needed and at the tactical level can reallocate the fleet depending on operational events.

Algorithm inputs

The tug scheduling algorithm uses three inputs:

• The airport road networks, including their types and the time it takes to traverse the airport using the different networks. • The flight schedule during a day of operation to estimate the drop-off time and corresponding energy based on a single-agent version of the path planning algorithm detailed in Section 4.2. • A list of tugs present at the airport that has to start and end their day of operations at the depot and with a full battery. Charging the battery can be done at a station for a minimum time of 𝑡 ! "#$ = 1 hour.

Allocation algorithm

From those input parameters, the tug fleet management algorithm creates a tug schedule. This schedule contains: which aircraft are to be towed, which tug tows which aircraft, and when (and where) the tugs are going to recharge. The algorithm aims to maximize the environmental impact of the tugs and uses a greedy search approach to determine which flights are towed. We first compute an initial schedule and later use a rolling horizon approach to adapt the tugs schedule to events throughout the day. The algorithm is run for each weight class of the aircraft (medium/heavy/ultra-heavy). The algorithm initializes the fleet at the start of the day at the depot with a full battery. To allocate the tugs, the algorithm iterates in time. During each iteration, the next available tug is assigned to a flight, or is sent to the depot to recharge for the end of the day. The flight which is assigned to the tug is the flight 𝑎 & from a subset 𝐴 % ⊂ 𝐴 & with the highest marginal emission savings. The set 𝐴 % confirms to (1) its flights have not been assigned to tugs (yet), ( 2) the flights from it can be towed by the tug without running into battery life problems, (3) the tug can tow any flight from 𝐴 % and still be at the depot with a full battery by the end of the day, (4) the flight with the earliest arrival/departure time which conforms to these three conditions is in 𝐴 % , and (5) no pair of flights from 𝐴 % can be towed consecutively by this tug. Finally, if after a number of iterations all tugs are at the depot by the end of the day, the algorithm terminates the loop and post-processes the charging schedule using the algorithm from van der Klauw et al. [START_REF] Van Der Klauw | Scheduling of electricity storage for peak shaving with minimal device wear[END_REF].

Rolling horizon approach for unplanned events

To cope with a number of events that may occur such as flight delays, tug breakdowns, or changes to the schedule because of manual preferences by the tug fleet manager. A rolling horizon approach is used to adapt the schedule. When an event occurs, the input information is updated and we reuse the allocation algorithm to re-optimize the tug schedule for the remainder of the day.

In the case that the TFM assigns a flight to a specific tug, this is considered as a fixed point in the day for this tug and the algorithm ensures that this flight is always in the tug schedule. In case a flight is manually unassigned from a tug, it is always left out of the set 𝐴 % for this tug. Figure 2 illustrates an initial schedule and an example of the rolling horizon approach. A fleet of five tugs for aircraft class medium tows flights from 6 AM to 6 AM (the next day). At 2 PM, tug 5 breaks down, and the schedule is re-optimized for the remaining tugs. At 4 PM tug 5 is repaired and enters service again, and serves the remainder of the day.

Path Planning

The hybrid multi-agent system (MAS) computes conflict-free paths and monitors the subsequent plan execution of all aircraft and tugs moving over the airport surface concurrently. It comprises a distributedhierarchical structure of both centralized and distributed agents as illustrated in Figure 3. The centralized Routing Agent computes conflict-free routes for all vehicles that are scheduled to be taxiing within a predefined planning window and is repeated at least once per replanning period. To compute paths, the airport layout is represented as graph 𝐺 = (𝑉, 𝐸) with nodes 𝑉 as junction points or end points such as gates, and edges 𝐸 as center lines of taxiways and service roads. We use motion planning to account for vehicle kinematics in planning since heavy vehicles such as aircraft entail finite acceleration and deceleration. To ensure conflict-free paths, we deploy a two-level search based on Priority-Based Search (PBS) [START_REF] Ma | Searching with consistent prioritization for multi-agent path finding[END_REF] with an augmented version of the Safe Interval Path Planning (SIPP) algorithm [START_REF] Phillips | SIPP: Safe interval path planning for dynamic environments[END_REF], which will be outlined in the next sections. Once a route is cleared by ATC, the Localized Agents, in their current form conceptualized to be positioned at every junction, monitor the execution of the cleared route, and trigger central replanning if the deviation exceeds time thresholds.

The MAS can be utilized by the fleet allocation algorithm (4.1) to obtain estimates for time and distance of paths. Furthermore, the MAS interacts with the human-machine interface to facilitate co-design of the vehicle routes. For that, the Routing Agent takes constraints of the ATCOs into account during path planning and poses route suggestions to them. Additionally, the vehicle-specific objective functions can be adjusted to influence the path planning. These are currently based on a linear combination of taxi time and distance but may later be extended with other cost-parts. Besides that, the estimated arrival time and remaining distance per vehicle as well as the deviations during plan execution computed by the Localized Agents can be visualized in the HMI to keep stakeholders up to date.

Single Agent Motion Planning based on SIPP

In the low-level search, we plan separately for each vehicle using SIPP. In contrast to classical path finding algorithms such as Dijkstra or A*, SIPP uses time intervals to specify the time dimension in its state definition. This reduces the number of possible states and makes SIPP efficient to use for motion planning with vehicle kinematics. We adapted the successor-generation of SIPP to account for constraints, vehiclespecific kinematics, and an activity sequence. The latter is used to include the various possible taxiing phases in the same planning, modelled as one of three activities: go-to activities such as regular taxiing have both a path and speed degree of freedom. Follow activities such as pushback operations have a predefined path associated to it, so that speed is the only remaining degree of freedom for these route segments. Wait activities such as coupling to or decoupling from a tug are fixed time periods at a set of given locations (nodes) that have to be accounted for in planning. During the search, constraints and ATCO preferences for taxiway-directionality are taken into account. While constraints limit the possible set of edges and are used to represent blocked taxiway-segments such as a crossing of an active runway, ATCO preferences influence the chosen path by assigning weights to the cost of traversing an edge. The possible motions along an edge are bound by the type-specific vehicle kinematic values for maximal speed, speed limit in turns, and fixed acceleration/deceleration rates.

Multi-Agent coordination using PBS

To compute collision-free paths for all vehicles, we utilize the Priority-Based Search algorithm. Using a depth-first search, PBS creates a binary search tree that establishes a priority order between conflicting vehicles. The vehicle dimensions are converted into circular shapes and used to compute unsafe time intervals of the vehicle motion over each edge. This set of edges includes their path as well as the swiped adjacent edges. Two vehicles are in conflict if their unsafe intervals on any edge overlap. PBS then creates two child nodes, giving priority to one of the two conflicting vehicles in each. SIPP is called again to account for the newly set unsafe intervals the prioritized vehicle while determining a new path for the deprioritized one. PBS continues the conflict detection and resolution with the child node that has the lowest overall sumof-cost of all paths. The path planning terminates once all conflicts have been detected and resolved successfully.

Path co-design, clearing and replanning

ATCOs can co-design the routes by specifying certain locations that must be passed. These are added as via-nodes to the activity sequence of a vehicle. Once satisfied with the computed path, ATCOs can clear the vehicle route in the HMI. During replanning, only the speed profile over a cleared path will be adapted to resolve a conflict between two vehicles. The updated speed profiles are reported back to the HMI for further use as outlined in the next section.

User Interfaces and Interaction

We designed interfaces and interaction to support three types of users with their tasks: the new role of TFM that must supervise the tug fleet allocation plan and update it according to operational events; the ground ATCOs that must control aircraft with various taxiing techniques and additional towing vehicles that will have to attach and detach from aircraft; and the pilots that will have to follow speed targets and to turn on their engines at the most appropriate time. The envisioned position of the TFM is composed of a supervision HMI to monitor on the tugs' status and their allocation as well as a radar image which allows the spatial monitoring of all the vehicles on the apron and the taxiways, similarly to the tool used by ground ATCOs.

Radar image

We designed a radar image presented in Figure 5 right for both ATCOs and TFM. It provides situation awareness on aircraft's status, trajectory, and traffic on the airport taxiways. It also provides several vehicles' characteristics and status information that are either textual or iconographic, and suggests paths which are computed depending on the taxi technology used by each aircraft. The flights are represented by an aircraft icon and tugs are depicted with a triangle shape surrounded by a green circle. To facilitate the identification of each tug and aircraft on the airport taxiways, information such as callsign, aircraft type, bound type, origin, and destination locations are provided on each vehicle tag. While inbound flights' tag is colored in blue, outbound flights' is colored in red and tugs' tag is colored in green. These colors were inspired by the current radar color coding used at Paris-CDG airport. For each aircraft, the Taxi Mode of Operation (TMO) is presented as follows:

• Aircraft using all their engines are represented by 2 white semi-circles on both sides of the aircraft icon.

For single SET, only a single semi-circle is visible indicating that half of the engines are off. • Autonomous taxiing with electric engine is represented by highlighting in purple one of the 2 semicircles at the front and rear of the aircraft icon. When the aircraft uses the eTaxi or Wheeltug technologies, the semi-circle at the front or the back is highlighted, respectively; • Non autonomous taxiing, which necessitates aircraft to be tugged, is represented by a green dashed circle around the aircraft icon.

To help the ATCO to visualize the optimized routes computed by algorithms, we display the corresponding route when a flight or a tug is selected. This is meant to help ATCOs identifying possible conflicts with the vehicles on other tracks. The route suggestions use the same colors than for TMO: white for classic and SET, purple for electric and green for towing. In addition, information such as suggested coupling positions for an inbound flights or decoupling position for outbound flights are displayed. These suggestions are computed by the path planning algorithm and can be refined by the TFM. The final decision remains to the ATCO who can change a suggested route if needed before validation and clearance.

Moving map

The moving map tool, illustrated in Figure 5 left, displays information on routes, speeds and traffic awareness. It can be used inside the cockpit. The map centered on the aircraft helps pilots following the ATCO's cleared taxi route and the target taxiing speed as well as monitoring their TMO or other aircraft on the taxiways. The speed monitor instrument was designed according to Bernatzky et al. [START_REF] Bernatzky | Development and evaluation of a speed guidance interface for trajectory-based dispatch towing[END_REF] recommendations on speed guidance interface for trajectory based-dispatch towing. It allows pilot to optimize their speed according to the optimal traffic configuration computed in real time by the planning algorithms, to mitigate untimely stops, gas emission and delays and to fluidify the entire airport traffic. The moving map also provides the coupling and decoupling locations and assists pilots with the engines' warmup procedure. On the cleared route, an orange segment informs the pilot where (and when) the engine should be started. This is supplemented with a starting timer which is displayed on the moving map dashboard at the relevant time. The length of the segment, and therefore the duration, is computed according to the waiting time for the runway access.

VALIDATION METHODOLOGY

The AEON solution was validated using an iterative, participative, and multi-layered assessment approach that involved the Consortium and its stakeholders since the earliest phases of the project.

A three-step iterative process was adopted, which included three validation sessions: the initial validation session carried out with selected aviation stakeholders, the intermediate validation session in which the concept was discussed with personnel from Schiphol Amsterdam Airport and Paris Charles De Gaulle Airport, and the final validation session in which the AEON concept and tools were tested in real-time simulation (RTS) by 6 pairs of ATCOs as visible in Figure 6. During this overall process, the project explored the impact of the proposed solution on several interconnected Key Performance Areas (KPAs), namely: 1) Human Performance, 2) Liability, 3) Safety, 4) Cost-Benefit, 5) Capacity, 6) Efficiency, and 6) Environment.

Each KPA was than further detailed in specific objectives and criteria.

VALIDATION RESULTS

The several validations activities allowed us to improve the concept of operation and the supporting tools.

In the final validation, we were able to evaluate most of the tools altogether in a realistic scenario except the algorithms that were not integrated in the platform used for the real time simulation, although their logic were evaluated during debriefings with the involved ATCOs. We detail and discuss the results according to some of the KPI of the project.

Safety

One of the important identified safety issues was that pilots, air traffic controllers or other actors involved in ground operations may consider a taxiing aircraft-tug combination to be a lower priority towed aircraft. This would lead to false expectations about the behavior of this actor, which could result into reduced safety margins. One more important safety hazard stems from the fact that a tug connected in front of an aircraft is not visible from the aircraft's cockpit. This lack of visibility could result in unintended runway incursion, when the aircraft would stop too late at the runway holding point and the tug would appear at the protected area of the runway. Furthermore, it was evident from the preliminary safety analysis that operations at tug coupling/de-coupling locations needs to be studied in more detail, as they could present risks to both people (tug drivers) and equipment. These risks need to be quantified in a follow-up study. Also the location of such points on the airport surface should be considered from the safety point of view.

Human Performance

Several considerations arose from the results produced in the final evaluation. Although all the roles implied with taxiing operations appeared to be considered, the description of the tug fleet manager (TFM) need to be further detailed and improved to ensure the support of Ground ATCOs. Ground ATCO's performance appeared to benefit from situation awareness concerning the TFM activity and familiarity with the designed operations. The exchange of communication between these two roles was not adequate and caused ATCOs to manage the tug vehicles even though it was not her/his responsibility. In this light, the TFM and Ground ATCO shall be considered as a team. Two additional roles concerned by the AEON operational concept are the pilot and ground handling (GH) companies, since the e-taxi and tug vehicles usage will modify pushback and taxiing operations. Pilots will require new skills to take-over tug drivers when driving e-taxi-equipped aircraft. Similarly, tug drivers will need new competencies to operate the tug vehicles and manage the coupling and uncoupling of the aircraft. Staffing levels for Ground ATCOs will increase to accommodate the higher demand for TFM. The same will happen for GH companies which will need to increase their staffing levels to provide more tug drivers, but on the other hand, will see a decrease in demand for towing vehicle drivers.

Cost-Benefits

The cost benefits analysis shows that for 13 larger European airports there is a reasonable business case to implement towing, assuming each towing vehicle needs to offset an average of 1000 kg of fuel on a peak day. Further analysis for Amsterdam Airport Schiphol (AMS) and Paris CDG shows that towing all heavy and medium sized aircraft vehicles would most probably make sense on a 2018 year basis. For smaller, regional aircraft, utilization would be relatively low, limiting the business case if all aircraft where towed. It would make sense to only operate a few of these sized vehicles. For eTaxi without any engines on, the business model shows net savings if installed on all aircraft. If we would need at least 1000kg of fuel saved per day to offset the installations costs, only around 10% of the fleet would be equipped. Sensitivity analysis shows that the weight added must also be very limited, which could be a challenge. The business model thus seems a lot more limited than that for towing, especially as towing aircraft equipped with eTaxi would completely negate the benefit of the eTaxi system, as the main benefits in terms of on ground fuel consumption is largely at the same large airports as towing would be deployed. For towing the most important unknown is the operating cost of a towing vehicle, which can only be quantified by the manufacturer and the operator. To limit the operational costs, it would be beneficial to make the towing vehicles autonomous and not require a driver on board.

Liability

The liability assessment shows how developing and deploying new technological solutions within wellconsolidated procedures may have a relevant impact on the responsibility regimes of the operators involved. The analysis of the AEON solution reveals how the TFM, as a connection between strategic and tactical operations, needs to have an adequate understanding of the work of ATCOs, despite playing different roles. Otherwise, the liability risk exposure of ANSPs and their operators would increase until becoming unbearable. The linear design of the communication flow and the consistent deployment of the related means represent an essential precondition for minimizing risks related to carelessness due to poor situational awareness. Eventually, innovation of roles and procedures should be coupled with a dedicated review of internal policies, rescoping general and specific licenses according to the new skills required.

CONCLUSION AND DISCUSSION

We described the work carried on during the SESAR AEON project to explore a new concept of operation and dedicated supporting tools for enabling the introduction of engine-off taxiing techniques. We described the initially envisioned concept of operation and the set of supporting tools enabling it. These tools include an optimized allocation of a fleet of tugs to aircraft, centralized routing providing speed profiles to avoid conflicts and dedicated HMI for Air Traffic Controllers as well as a new role, the Tug Fleet Manager. We described the main aspects of our concept of operation and the overall architecture of the solution before presenting each component in more details. We described our validation activities and detailed the main results regarding safety, human performance, liability and cost benefit KPI.

The introduction of the TFM role posed specific challenges regarding the collaboration with the ground ATCOs and the liability aspects. Further research is required to offer adequate tools and define precisely the responsibilities and training of the TFM. Regarding the allocation algorithm, we were not able to integrate it in the validation platform and assess the quality of the allocation for realistic operations. However, based on the feedback from airport managers, we will explore its use as a decision support tool to identify best charging location or runs simulations on coming days to find the best day for planning maintenance on tugs. Regarding the path planning algorithms, we use a simpler version on our simulation because the computing time were too high to make it usable by ATCOs. We are currently improving the performances to be able to integrate it. The path planning tool currently respects the routing used by air traffic controllers. This make the results in line with ATCOs' current expectations but possibly less optimal. We are interested in running several simulation with different level of constraints and its impact on performances.

We are currently pursuing the analysis of the results from our validation activities to investigate the impact on capacity and environmental impact of our solution. Based on these additional results, we will articulate an updated version of the concept of operation. Future efforts will consider the use of autonomous vehicles for towing aircraft and improvements on the allocation, planning and HMI tools based on our validation results.

Figure 1 :

 1 Figure 1: AEON concept image highlighting the actors, technologies, and operational phases taken into account.

Figure 2 :

 2 Figure 2: Right: Tug schedule at the start of the day of operations. Left: Tug schedule after 4 PM, tug 5 has been repaired and remains in service.

Figure 3 :

 3 Figure 3: Multi-agent system and interaction with HMI and fleet management algorithm.

Figure 4 :

 4 Figure 4: Supervision HMI with arriving and departing aircraft on the top and the tugs underneath with allocations.Tug Fleet Management HMIThe supervision HMI present a synoptic view of the status of fleet of tugs and their planned allocation to departing and arriving aircraft as presented in Figure4. The HMI presents the data through a horizontal timeline with two main panels. The top panel provides inbound and outbound flights schedule information and their expected TMO for each runway. The top area uses horizontal ribbons to present the planned departures and arrivals for each runway. All the departures and arrival items also display the taxi mode of the aircraft and in particular any tug request. The bottom panel displays allocations for each tug available to the operator. For each tug, the callsign and the battery status are displayed. Each tug is associated to a line which represents it schedule throughout the day. On each line, aircraft and tugs allocations, expected journeys and tug unavailability duration are shown. Although most of the allocations are computed and set by AEON algorithms using data from the A-CDM, the operator can made change via the interface to amend or cancel allocations.

Figure 5 :

 5 Figure 5: Left: Portion of the radar image representing aircraft, their taxiing techniques and the towing vehicles. Right: Moving map representing the route, a speed indicator and indications for engine start-up. engines

Figure 6 :

 6 Figure 6: Experimental setup of the final assessment. Left: ATCO with the radar HMI and a realistic Paris-CDG tower simulation. Center: TFM with the supervision HMI and the radar HMI. Right: Pseudo pilots positions.

Table 1 : Elicited requirements from field work

 1

	Ground operation	Requirements
	aspects
	Airport management	• Defining and sharing the taxiing technique choices for all scheduled aircraft
	and ground handling	• Supporting the allocation of the tugs to aircrafts and their reallocation
	Air Traffic Control	• Enhancing situation awareness of the taxiing technique for each aircraft.
		• Supporting routing decision with conflict free routes
	Piloting	• Providing speed profiles to avoid stops during taxiing phases
		• Providing remaining taxi time to anticipate engines start-up

ACKNOWLEDGMENTS

This work has received funding from the SESAR Joint Undertaking under grant agreement No 892869 under European Union's Horizon 2020 research and innovation programme. We are grateful to all advisory board members and participants that provided useful feedback on comments on our work and helped shaping the current concept of operation.


Yuanyuan He

Arnaud Buch

Cyril Szopa

Amy Williams

Caroline Freissinet

Melissa Guzman

Maëva Millan

David Coscia

Jean-Yves Bonnet

Michel Cabane

The application of TMAH thermochemolysis on the detection of nucleosides: applications for the SAM and MOMA instruments

Keywords: Nucleosides, TMAH thermochemolysis, Py-GC/MS, Mars, SAM Conceptualization, Formal analysis, Investigation, Methodology, Writingoriginal draft Conceptualization, Methodology, Formal analysis, Supervision. Cyril Szopa: Conceptualization, Methodology, Formal analysis, Supervision. Caroline Freissinet: Conceptualization, Methodology, Formal analysis, Supervision. Amy

Tetramethylammonium hydroxide (TMAH) thermochemolysis has been utilized by the Curiosity rover's Sample Analysis at Mars (SAM) instrument and will be utilized by the ExoMars 2022's Mars Organic Analyzer (MOMA) instrument. TMAH thermochemolysis is one of the main techniques that enables the detection of bioindicators when it is combined with pyrolysis-gas chromatography and mass spectrometry (Py-GC/MS) at the surface of Mars. This study identifies the thermochemolysis products of targeted nucleosides using flash pyrolysis and a SAM-like ramp pyrolysis. Results indicate that the methylated nucleosides can be detected when nucleosides are submitted to TMAH thermochemolysis, which demonstrates that the TMAH thermochemolysis protocol does not result in the decomposition of the nucleosides. To detect the whole structure of each nucleoside, low thermochemolysis temperature is required. 200 °C is the optimal temperature. Methylated nucleobases are the main products of the tested nucleosides when the thermochemolysis temperature is higher than 300 °C. Furfuryl methyl ether, one of the degradation products of these nucleosides, were also identified from 200 to 600 °C in this study. These experiments will establish a reference database for interpretation of the data acquired by the SAM and MOMA experiments during operations on Mars.

Introduction

The Sample Analysis on Mars (SAM) instrument is one of ten instruments on the Curiosity rover designed to assess habitability on Mars and search for organic and inorganic molecules. The Mars Organic Molecule Analyzer (MOMA) instrument onboard the ExoMars 2022 rover has a main goal of looking for biochemical precursors at the Mars surface. Some known chemical biosignatures and bioindicator could be organic compounds such as fatty acids [START_REF] Williams | Recovery of fatty acids from mineralogic Mars analogs by TMAH thermochemolysis for the Sample Analysis at Mars wet chemistry experiment on the Curiosity Rover[END_REF], amino acids and carboxylic acids [START_REF] Stalport | The influence of mineralogy on recovering organic acids from Mars analogue materials using the one-pot derivatization experiment on the Sample Analysis at Mars (SAM) instrument suite[END_REF], pigments [START_REF] Simoneit | Molecular indicators (biomarkers) of past life[END_REF], nucleobases [START_REF] Simoneit | Molecular indicators (biomarkers) of past life[END_REF], sugars/polysaccharides [START_REF] Simoneit | Molecular indicators (biomarkers) of past life[END_REF], isoprenoids, and steriods [START_REF] Simoneit | Biomarkers as tracers for life on early Earth and Mars[END_REF], etc. Among these chemical products, nucleosides, nucleotides, and nucleobases are essential components of life on Earth. They constitute essential building blocks of nucleic acids, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), and are involved in the regulation and modulation of various physiological process in the human body [5,[START_REF] Jacobson | Purine and pyrimidine (P2) receptors as drug targets[END_REF]. DNA, as an information carrier, stores an organism's genetic information with unique orders of four nucleobases (A, T, C, G). This method storing genetic information has proved robust across billion-year long timescales on Earth. Some studies have suggested that DNA could be placed in extremely cold regions on Earth or even on Mars to enable millennium-long preservation [START_REF] Dong | DNA storage : research landscape and future prospects[END_REF]. The first life on Earth likely used RNA, which is made up of sequences of four different nucleotides, the latter of which can be formed through a chemical reaction source. RNA polymers may have emerged very quickly after the deposition of meteorites containing nucleobases (less than a few years), and the synthesis of nucleotides and their polymerization into RNA may occur over one to a few wet-dry cycles of warm little ponds [START_REF] Pearce | Origin of the RNA world: The fate of nucleobases in warm little ponds[END_REF]. However, biological RNA and DNA are universal, unique, and very conservative. The role of RNA in the origin of life is well established, and how RNA emerged on the early Earth is one of the first steps in understanding the origin of life [START_REF] Biscans | Exploring the emergence of RNA nucleosides and nucleotides on the early earth[END_REF]. This means that the emergence of nucleic acids is central to many theories for the origins of life, and that opens questions including the original source and structure of the building blocks of these molecules, such as nucleobases, sugars, nucleosides and nucleotides [START_REF] Fialho | Glycosylation of a model proto-RNA nucleobase with non-ribose sugars: Implications for the prebiotic synthesis of nucleosides[END_REF]. Therefore, the formation and detection of nucleosides and nucleotide abiogenesis has been widely studied [START_REF] Pearce | Origin of the RNA world: The fate of nucleobases in warm little ponds[END_REF][START_REF] Menor-Salván | From the Dawn of Organic Chemistry to Astrobiology: Urea as a Foundational Component in the Origin of Nucleobases and Nucleotides[END_REF][START_REF] Basiuk | Pyrolysis of simple amino acids and nucleobases: survivability limits and Implications for extraterrestrial dielivery[END_REF].

The structure of nucleobases, nucleosides, and nucleotides are well known today.

Polynucleotides are composed of pentose sugar moieties linked by 3',5'-phosphodiester bonds forming a sugar-phosphate backbone [START_REF] Davies | Conformations of nucleosides and nucleotides[END_REF][START_REF] Steenken | Purine Bases, Nucleosides, and Nucleotides: Aqueous Solution Redox Chemistry and Transformation Reactions of Their Radical Cations and e~ and OH Adducts[END_REF][START_REF] Altona | Conformational analysis of the sugar ring in nucleosides and nucleotides. Improved method for the interpretation of proton magnetic resonance coupling constants[END_REF][START_REF] Jeffrey | Hydrogen bonding in nucleosides and nucleotides[END_REF]. Nucleosides such as uridine, cytidine, adenosine and thymidine can be synthesized by simple two molecule reactions, i.e., from the reaction of ribose or 2'-deoxyribose with the canonical nucleobases (cytosine, uracil, adenine, guanine, and thymine) [START_REF] Saladino | Chemomimesis and molecular darwinism in action: From abiotic generation of nucleobases to nucleosides and RNA[END_REF]. The study of the formation of nucleosides from a one-pot reaction J o u r n a l P r e -p r o o f makes it possible to analyze the factors that might have played a role in the condensation of nucleotides into polymers, and which led to the evolution of extant nucleic acids [START_REF] Saladino | Proton irradiation: A key to the challenge of N-glycosidic bond formation in a prebiotic context[END_REF]. Abiotic phosphorylation of nucleosides is a key chemical step that may have provided nucleotides to the prebiotic world. Some minerals played a key role in that process. Borate can work in formamide to guide the reactivity of nucleosides under conditions where they are phosphorylated [START_REF] Furukawa | Abiotic regioselective phosphorylation of adenosine with borate in formamide[END_REF]. Adsorptions of K and Mg by goethite could also have played an important role in peptide synthesis and the formation of nucleosides [START_REF] Dubina | Potassium Ions are More Effective than Sodium Ions in Salt Induced J o u r n a l P r e -p r o o f Peptide Formation[END_REF][START_REF] Sheng | Comprehensive investigation of the energetics of pyrimidine nucleoside formation in a model prebiotic reaction[END_REF].Additionally, clays such as montmorillonite appear to play a protective role for nucleosides against ionizing radiation [START_REF] Aguilar-Ovando | Radiolysis of Nucleosides: Study of Sedimentary Microenvironment Models for the Protection of Bio-Organic Molecules on Early Earth[END_REF]. The survival of nucleosides exposed to a high radiation field in an aqueous solution and adsorbed on Na-montmorillonite indicates that the pyrimidine nucleosides were more resistant to heat and less resistant against ionizing radiation even when adsorbed in clay [START_REF] Aguilar-Ovando | Radiolysis of Nucleosides: Study of Sedimentary Microenvironment Models for the Protection of Bio-Organic Molecules on Early Earth[END_REF]. The purine-based nucleosides (adenosine and guanosine) showed a much higher radiolysis resistance than the pyrimidine-based nucleosides (cytidine and uridine) [START_REF] Cataldo | Radiolysis and radioracemization of RNA ribonucleosides: implications for the origins of life[END_REF]. Given these results, it is perhaps unsurprising that purine nucleobases are found in practically all carbonaceous chondrite meteorites while the pyrimidine nucleobases are absent or below the detection limits of current analytical techniques [START_REF] Cataldo | Radiolysis and radioracemization of RNA ribonucleosides: implications for the origins of life[END_REF].

Nucleobases have been found in meteorites (guanine, adenine, and uracil) with concentrations of 0.25 to 515 ppb [START_REF] Callahan | Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases[END_REF][START_REF] Pearce | Seeding the Pregenetic Earth: Meteoritic Abundances of Nucleobases and Potential Reaction Pathways[END_REF]. Derivatives of nucleobases (guanine> hypoxanthine) have also been identified in meteorites [START_REF] Callahan | Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases[END_REF][START_REF] Martins | Extraterrestrial nucleobases in the Murchison meteorite[END_REF]. In addition, purine nucleobases such as guanine and xanthine, and pyrimidine nucleobases such as cytosine, uracil and thymine have been detected in numerous meteorites previously, and the isotopic composition of nucleobases isolated from carbonaceous chondrites support their extraterrestrial origin [START_REF] Martins | Extraterrestrial nucleobases in the Murchison meteorite[END_REF][START_REF] Oba | Extraterrestrial hexamethylenetetramine in meteorites-a precursor of prebiotic chemistry in the inner solar system[END_REF][START_REF] Yasuhiro Oba | Identifying the wide diversity of extraterrestrial purine and pyrimidine nucleobases in carbonaceous meteorites[END_REF]. In addition, sugar phosphates are the backbone of the genetic molecules DNA and RNA.

Ribose is particularly essential as a building block of RNA, because it could have both stored information and catalyzed reactions in primitive life on Earth. Extraterrestrial sugars in the NWA 801 and Murchison meteorites are isotopically distinct from terrestrial sugars and provide clear evidence of an extraterrestrial origin for these sugars in primitive meteorites [START_REF] Furukawa | Extraterrestrial ribose and other sugars in primitive meteorites[END_REF]. The structure of nucleobases and nucleosides also influence their behavior. The perpendicular structure of nucleosides makes it difficult for the nucleoside to interact with the mineral structure and consequently, nucleosides have lower adsorption than corresponding nucleobases [START_REF] Villafañe-Barajas | Salinity Effects on the Adsorption of Nucleic Acid Compounds on Na-Montmorillonite: a Prebiotic Chemistry Experiment[END_REF]. This demonstrated that the ribose structure of nucleosides also played an important role compared to nucleobases. Above all, with the detection of extraterrestrial nucleobases and ribose (the main fragments of nucleosides), nucleosides are interesting target compounds for astrobiology even if there is less information about extraterrestrial nucleosides.

J o u r n a l P r e -p r o o f

Since TMAH thermochemolysis will be applied to search for organic compounds on Mars, this study focuses on the analysis of nucleosides by TMAH thermochemolysis.

Therefore, in this study, the application of TMAH thermochemolysis on six deoxynucleosides standards and mixtures pyrolyzed at different temperatures was studied. The temperature of the TMAH thermochemolysis reaction was optimized from 200 to 600°C for this set of nucleosides and all the products of these nucleosides following TMAH thermochemolysis were identified. Moreover, thermochemolysis of these nucleosides was also studied using a -slow‖ pyrolysis ramp (35 °C min -1 ) mimicking the pyrolytic conditions of the SAM experiment in order to assess its capability to detect nucleosides. From our knowledge, this is the first study of the behavior of nucleosides submitted to TMAH thermochemolysis under a SAM-like ramp pyrolysis (from 50 to 850 °C at a heating rate of 35 °C min -1 ) [START_REF] Leshin | Volatile, isotope, and organic analysis of martian fines with the Mars curiosity rover[END_REF].

Additionally, the thermochemolysis mechanisms of all nucleosides with TMAH were analyzed. This study provides important reference data that constrains the nucleosides or nucleoside derivatives that will be detectable using in-situ TMAH thermochemolysis as part of in situ analyses performed on Mars by the SAM and MOMA space experiments.

Experimental

Materials

In this study, nucleosides include deoxyadenosine (dA, Sigma, 99%, stored 2-8 °C), deoxyguanosine (dG, Sigma, 98-100%), thymidine (dT, Sigma, 99%), 2'-deoxyuridine (dU, Sigma, 99-100%), 2'-deoxycytidine (dC, Sigma, 99%, HPLC, stored -20 °C); deoxyinosine (dI, Sigma, 98%, stored -20 °C) were used. The structures of the selected nucleosides can be seen in Figure 1. All nucleosides were dissolved in deionized water. The concentration of each nucleoside and the concentration of their mixtures are shown in Table 1.

3 µl of TMAH (25 wt. % in methanol, Sigma) was applied in this study, which is always in excess volume and the amount of methyl functional group compared to the nucleosides. 0.2 µl of Naphthalene-D8 at a concentration of 0.005 mol•L -1 (Sigma-Aldrich, isotopic purity, 99 atom % D) was used as the internal standard (IS).

Py-GC/MS and methods

J o u r n a l P r e -p r o o f

The pyrolysis experiments were performed with an evolved gas analysis (EGA)/PY-3030D micro-oven pyrolyzer (Frontier Lab) installed on a Split/Splitless (SSL) injector of a

Trace GC Ultra gas chromatograph (ThermoScientific) coupled to a quadrupole mass spectrometer (ISQ LT, Thermo Scientific). Flash pyrolysis and SAM-like ramp pyrolysis were applied for the tests described in this article. For the flash pyrolysis, the pyrolysis temperatures were at 200, 300, 400, 500, and 600 °C. The nucleosides solution (in H 2 O, 0.5 µl of dA, dT, dU, dC, dI; 1 µl of dG, respectively) was put in a small stainless-steel cup (Frontier large volume cup), followed by the addition of 3 µl of TMAH over the nucleosides solution.

Then the stainless-steel cup containing a mixture of nucleosides and TMAH was quickly introduced into the oven already heated at the designated temperature. Helium (99.9999%) was used as the carrier gas to carry the volatile products from the pyrolyzer to the GC/MS.

The SAM-like ramp pyrolysis is the same as in previous work [START_REF] He | Influence of J o u r n a l P r e -p r o o f Calcium Perchlorate on the Search for Organics on Mars with Tetramethylammonium Hydroxide Thermochemolysis[END_REF][START_REF] He | Influence of Calcium Perchlorate on the Search for Martian Organic Compounds with MTBSTFA/DMF Derivatization[END_REF]. In this process, a cup containing the sample was heated using heart-cut EGA temperature zones from 50 °C to reach the final temperature (850 °C held for 1 min) at the SAM heating rate of 35 °C min -1 . The pyrolysis sequence being too long to proceed to a sharp injection of the analytes into the GC, liquid nitrogen was used to cool down the chromatographic column inlet where the volatiles produced by the samples condense and were trapped in a small volume. Once the pyrolysis achieved, the cryogenic cooling was stopped and all the products were then released and sent to the GC/MS via the helium flow. All experiments were repeated at least 5 times each.

The GC was equipped with a Supelco SLB-5MS Inferno column (30 m × 0.25 mm i.d.

× 0.25 μm film thickness). The temperature programming of the column starts at 40 °C and is held for 2 min, then ramped at a heating rate of 6 °C min -1 up to 130 °C and then heated again to 300 °C at a rate of 10 °C min -1 and maintained for 1 min. Helium was used as the carrier gas and the helium flow rate in the column was 1.2 mL min -1 . The split flow was 24 mL min -1 .

The temperature of the SSL injector was set at 280 °C. The ions were scanned between m/z 40 and m/z 500. The ionization energy of the electron ionization source was 70 eV.

Results

TMAH thermochemolysis for nucleosides

To determine the main reaction products of nucleosides with TMAH thermochemolysis, each nucleoside was submitted to flash pyrolysis conditions at 200 °C in the presence of TMAH. Figure 2 shows the chromatogram with the TMAH thermochemolysis J o u r n a l P r e -p r o o f products of nucleosides following a flash pyrolysis at 200 °C. We observe that the main thermochemolysis products are the methylated dA, dT, dU, and dC for all the nucleosides analyzed except for dG and dI. Though there are various methylated compounds of nucleobases as identified in our previous study [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF] , the methylated compounds with the highest intensity were selected as the possible target compounds that could allow the identification of nucleosides analyzed with TMAH thermochemolysis. Table 2 shows the main products of nucleosides reaction with TMAH thermochemolysis following flash pyrolysis at 200 °C. 200°C and could be the direct evidence of the presence of DNA molecule fragments. Figure 3 shows the mass spectrum of different methylated products from nucleosides following thermochemolysis with TMAH.

J o u r n a l P r e -p r o o f

In addition to the main methylated products of nucleosides with TMAH thermochemolysis, some byproducts of nucleobases are also identified following TMAH thermochemolysis at a flash pyrolysis of 200 °C, details of which can be found in Table S1-6 in the appendix.

Optimal temperature of nucleosides with TMAH thermochemolysis.

To determine the optimal temperature for TMAH thermochemolysis of nucleosides,

we performed thermochemolysis at different temperatures from 200 °C to 600 °C. Figure 4 shows the chromatograms of the compounds produced by TMAH thermochemolysis of each nucleoside at different temperatures. For a given nucleoside, the chromatograms obtained for the different thermochemolysis temperatures are quite comparable and the compounds detected are the same. However, the peak intensities of some main products varied with temperature. For example, the abundance of 2'-deoxy-N,N,O,O-tetramethyl-adenosine (RT = 33.1 min) decreased significantly with increasing thermochemolysis temperature. And in a more general way, intense peaks observed at high retention times at 200 °C tend to decrease in intensity whereas less retained species show higher intensities. This is often representative of an increase of the fragmentation of heavy species into smaller during the TMAH thermochemolysis reaction which is allowed at higher temperatures. This demonstrated that the bond between the ribose and the nucleobase is more easily broken at high temperature. Thus, the yield of methylated nucleobases increased when the temperature increased from 300 to 600 °C. For instance, the intensity of N,N,9-trimethyladenine at 600 °C is 2 times of that of 300 °C. Similarly, the yield of methylated ribose, 2methoxymethyl-furan, increased with the temperature increase, which also demonstrated that the high temperatures are more favorable to break the nucleosides into their respective the nucleobases and ribose. The methylated nucleobases from the nucleosides with TMAH thermochemolysis were detected at the highest tested temperature of 600 °C. Concerning the J o u r n a l P r e -p r o o f direct detection of nucleosides under their TMAH methylated form, it can be concluding that 200 °C is the optimal temperature because the nucleosides thermal degradation is limited at this temperature. On the other hand, the highest A i /A is value at 200 °C is very high maybe due to the lower temperature compared to the boiling temperature (218 °C) of the internal standard. However, the case may be different for nucleosides inside a solid matrix, where a higher temperature may be necessary to desorb the nucleotides from its inorganic matrix.

Analysis of a mixture of nucleosides

The methylated products of nucleosides and their target compounds at different temperatures have been analyzed. To determine whether there are coelutions among these nucleoside products, a mixture of the nucleosides, including dA, dU, dC, dT, and dI, has been analyzed by TMAH thermochemolysis between the temperatures of 200 to 600 °C (Figure 6).

dG was not mixed with the other five nucleosides, because according to our previous research, we found that dG can decompose into caffeine, which is one of the byproducts of hypoxanthine with TMAH thermochemolysis [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF]. The target peak of each nucleoside has been identified in the chromatogram. For example, the methylated compounds of dU and dT were separated completely, even if their structure differs only by one methyl group, with methylated uracil (peak 4) at a retention time of 20. J o u r n a l P r e -p r o o f

SAM-like ramp

We proceeded to TMAH thermochemolysis of nucleosides using a program of temperature of the pyrolyzer starting from 50°C then heating the sample at a 35°C.min -1 up to 850 °C (the volatiles was trapped in the column inlet) to simulate the way Mars samples are heated in the SAM instrument onboard the Curiosity rover. Figure 8 shows the products from the TMAH thermochemolysis of nucleosides under the SAM-like ramp of pyrolysis.

Compared with the chromatogram of nucleosides with TMAH thermochemolysis using a flash pyrolysis (Figure 2), it was found that the methylated compounds of each nucleoside and the byproducts from each nucleoside with TMAH thermochemolysis using a SAM-like ramp pyrolysis are the same.

Figure 9 shows the chromatogram of the mixtures of five kinds of nucleosides with TMAH thermochemolysis under a SAM-like ramp pyrolysis. As discussed previously, dG

was not mixed with the other five nucleosides because guanine can decompose into caffeine and influence the quantitation of hypoxanthine. Results showed that there is no overlap of the products among these five nucleosides with TMAH thermochemolysis. The target compounds of each nucleoside with TMAH thermochemolysis is shown in Figure 9. Figure 10 shows the abundance of the target compounds from each nucleoside with TMAH thermochemolysis.

When the peak intensities of the target compounds from each nucleoside are compared, the corresponding values of these products as detected by GC/MS decrease in the following manner: dA > dG > dT > dU > dI > dC. These results demonstrate that dA is the most likely to be identified among the tested nucleosides. The result is consistent with the conclusion in our previous work since adenine is the most detectable nucleobase in the context of the limits of detection of GC/MS instrumentation currently used for analysis of martian samples [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF].

Discussion

Methylated nucleobases and nucleosides

The main derivatized compounds of nucleosides are listed in Table 2. Most of the derivatized compounds are the methylated products of six nucleosides, including methylated nucleobases, methylated ribose derivatives, and methylated nucleosides (Figure 11). However, J o u r n a l P r e -p r o o f there are several different methylated products because of the different tautomeric forms of nucleobases.

For dA, there are four possible methylation sites: two are on the nucleobase site and the two others are on the glucose sites. The product of a full methylation of adenosine is 2'deoxy-N,N,O,O-tetramethyl-adenosine, mainly in N9H form. This demonstrates that the N9H form of adenosine is the most stable form. Both N9H and N3H forms of adenine, the degradation products of adenosine, were detected. The abundance of the N9H form of adenine is higher than the N3H form. This is because the N9H tautomeric form is the major form of adenine while the N3H tautomeric form is the minor form. Moreover, the N9H form of adenine was the most stable tautomer [START_REF] Guerra | Adenine tautomers: Relative stabilities, ionization energies, and mismatch with cytosine[END_REF][START_REF] Hanus | Correlated ab Initio Study of Nucleic Acid Bases and Their Tautomers in the Gas Phase, in a Microhydrated Environment, and in Aqueous Solution. Part 3. Adenine[END_REF]. N,9-dimethyl-adenine at low abundance was detected, which is also the methylated derivative of the N9H form of adenine. The relative abundance of trimethylated-adenine is much higher than the dimethylated adenine, because the stability of the trimethylated adenine is higher than the dimethylated one [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF].

Trimethyl-and tetramethyl-guanine are the main degradation products from dG with TMAH thermochemolysis. There are several tautomeric methylated products of guanine with the keto and enol forms being the most stable. Methylated 1,3,7-tri-methylxanthine (caffeine)

was detected from the TMAH thermochemolysis of dG. This could be caused by the deamination and oxidation of guanine to xanthine, followed by methylation by TMAH.

Methylated dC (2'-deoxy-N,N,O,O-tetramethyl-cytidine) and methylated cytidine are the main products of dC with TMAH thermochemolysis. There are four methylation sites on dC: two sites are on the nucleobase side and two sites are on the glucose side. The thermochemolysis temperature (from 200 to 500°C) had no effect on the number of functionalized derivatives that were detected from dC. However, at 600 °C, the intensity of methylated dC and N,N,N'-trimethyl-cytosine is the highest (excluding the effect of internal standard at 200 °C), which demonstrated that increasing temperature leads to the decomposition of dC. 600 °C will be the optimal temperature for the detection of dC.

Compared with other nucleosides tested in this study, the peak intensity of dC is the lowest, so it is more difficult to detect. This is consistent with the properties of cytosine with TMAH thermochemolysis [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF]. When the temperature of the TMAH thermochemolysis increased from 200 to 500 °C, methylated thymine and uracil were detected as the main products of dU and dT, respectively. This is caused by the thermal degradation of dU and dT, since the C-N sugar-base bond (293 kJ/mol) is one of the thermally weakest bonds in the DNA structure [START_REF] Alongi | Thermal degradation of DNA, an all-in-one natural intumescent flame retardant[END_REF]. The canonical forms of methylated thymine and uracil were predominantly detected because the stability of the canonical form is much higher than other forms, and the keto-enolic thermodynamic equilibrium shifted in favor of the formation of the ketone form of uracil and thymine, leading to the absence of the enolactic tautomeric forms of thymine and uracil [START_REF] Morsy | Fluorescence of Thymine Tautomers at Room Temperature in Aqueous Solutions[END_REF][START_REF] Shukla | Interaction of water molecules with cytosine tautomers: An excited-state quantum chemical investigation[END_REF][START_REF] Tsuchiya | Keto-enol tautomer of uracil and thymine[END_REF][START_REF] Brady | The electronic spectra of the pyrimidine bases uracil and thymine in a supersonic molecular beam[END_REF][START_REF] Rejnek | Correlated ab initio study of nucleic acid bases and their tautomers in the gas phase, in a microhydrated environment and in aqueous solution. Part 3. Adenine[END_REF]. This is similar to the result obtained from the study of nucleobases with TMAH thermochemolysis [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF]. The consistency of these results demonstrates that the TMAH thermochemolysis of nucleosides does not influence the additional structure of deoxyribose. When dT and dU were heated, ribose was released from the thymidine and uridine molecules, radicals of thymine and uracil were formed and they were methylated by TMAH. The stability of dT and dU was reduced at high temperature. Thus, methylated thymine and uracil were the main products of thymidine and uridine from TMAH thermochemolysis at 600 °C.

For dI, 1,7-dimethyl-hypoxanthine and methylated dI are the main products.

Methylated dI has two forms: 2'-deoxy-3-methyl-3',5'-di-O-methyl-inosine and 2'-deoxy-3methyl-3',5'-di-O-methyl-hypothanxine. This could be caused by the oxidation of hypoxanthine. An increasing thermochemolysis temperature leads to the degradation of dI, forming methylated hypothanxine and furfuryl methyl ether. Deoxyribose and ribose generated by dihydrogen abstraction are important intermediates in radiation damage [START_REF] Téoule | Radiation-induced DNA damage and its repair[END_REF]. It is known that the furanose ring is puckered rather than planar [START_REF] Denifl | Electron J o u r n a l P r e -p r o o f attachment to 5-chloro uracil[END_REF]. The furanose molecule has relatively high internal energy because of this twisted molecular structure, and there are no -electrons in the furanose molecule. Therefore, it is easier for furanose to be decomposed. Neutral C 5 H 6 O 2 was proven to be one of the important products of thermal decomposition of deoxyribose [START_REF] Ptasińska | Inelastic electron interaction (attachment/ionization) with deoxyribose[END_REF]. The products of the dissociative electron attachment cross sections for the three heaviest fragment anions are

The formation of furan derivatives

Conclusions

The products of nucleosides with TMAH thermochemolysis were studied under flash pyrolysis and a SAM-like ramp pyrolysis. We conclude that the methylated nucleobases are the main compounds of nucleosides with TMAH thermochemolysis. The optimal temperature for the detection of methylated nucleosides is 200 °C; for methylated nucleobases and ribose derivatives such as furfuryl methyl ether is 600 °C. The structure of the original DNA/RNA fragments influence the detection of the characteristic compounds. For example, the abundance of methylated nucleosides following TMAH thermochemolysis of the tested nucleosides can vary substantially. However, the optimal temperature can be different if the nucleoside is contained within some solid matrix. A higher thermochemolysis temperature can aid in the detection of nucleosides by enabling a higher efficiency of desorption.

Nucleobases are the main products of nucleosides with TMAH thermochemolysis and the J o u r n a l P r e -p r o o f limit of detection has been reported in our previous work [START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF]. Though some of the thermochemolysis products may not be detected, a thermochemolysis temperature of 600 °C still enables the detection of diagnostic products of nucleosides with TMAH thermochemolysis, which is consistent with thermochemolysis temperatures that will be applied on SAM and MOMA. These results demonstrate that the temperature of TMAH

Figure 3

Mass spectrum of the organic compounds from nucleosides with TMAH thermochemolysis.

The information of each molecule fragment has been listed in the supplementary material (Table S7). Figure 5 Evolution of the amount of the main products detected after TMAH thermochemolysis of nucleosides at different temperatures (A i /A IS is the ratio of the area of an organic chromatographic peak and the area of the internal standard chromatographic peak). The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8). The information of each molecule fragment has been listed in the supplementary material (Table S2). 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10 9 0,0 2,0x10 9 4,0x10

J o u r n a l P r e -p r o o f

J o u r n a l P r e -p r o o f

Figure 5 Evolution of the amount of the main products detected after TMAH thermochemolysis of nucleosides at different temperatures (A i /A IS is the ratio of the area of an organic chromatographic peak and the area of the internal standard chromatographic peak). The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8). J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

Following the thermochemolysis of

  dI at 200 °C, 1,7-dimethyl-hypoxanthine is the main byproduct with a retention time of 27.6 min. Under the same experimental condition, N, N, N'-trimethyl-cytosine is the main methylated compound produced from the dC thermochemolysis with a retention time of 27.4 min. 1,3-dimethyl-uracil is the main methylated compounds of dU with a retention time of 20.2 min. 1,3-dimethyl-thymine is the key product of dT with a retention time of 21.2 min. Isomers of tetramethyl-guanine are the main methylated products of dG following TMAH thermochemolysis and have retention times of 28.1 and 29.4 min, respectively. The production of isomers is due to the molecular structure of guanine which enables different possibilities for the replacement of polar labile hydrogens with methyl functional groups from TMAH, leading to the appearance of several peaks of methylated guanine[START_REF] He | Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment[END_REF]. N, N, 9-trimethyl-adenine or its isomer, N, N, 3-trimethyladenine, at retention times of 25.5 min and 26.8 min, respectively, are the main methylated products of dA with TMAH thermochemolysis. In addition, not only were methylated nucleobase derivatives from each nucleoside detected, but the characteristic peak of methylated ribose from the nucleoside structure was also identified at a retention time of 6.1 min. This compound was identified as 2-methoxymethyl-furan or furfuryl methyl ether and is the methylated product of ribose from the nucleoside molecule. In addition to thermal degradation products, the methylated nucleosides were also detected in our study, such as methylated dC at a retention time of 34.4 min, methylated dU (2'-deoxy-3-methyl-3,5'-di-Omethyl-uridine) at a retention time of 30.1 min, methylated dT (2'-deoxy-N,N,O-trimethylthymidine) at a retention time of 30.5 min, and methylated dA (2'-deoxy-N,N,O,Otetramethyl-adenosine) at a retention time of 33.1 min. The peak intensities of the methylated nucleosides are high, which demonstrates that methylated nucleosides are thermally stable at

Figure 5

 5 Figure5shows the distribution of the products detected after TMAH thermochemolysis of nucleosides at different temperatures. The target peak intensity of each methylated nucleoside with TMAH thermochemolysis is the highest after flash pyrolysis at 200 °C. For dA, dT, and dU, their methylated compounds were detected, as 2'-decoxy-N,N,O,O-tetramethyl-adenosine, 2'-decoxy-N,O,O-trimethyl-thymine, 2'-decoxy-3-methyl-3',5'-di-O-methyl-uridine, respectively. With increasing temperature, the abundances of methylated nuclesides decreased, while the abundances of methylated nucleobases increased.

  2 min and methylated thymine (peak 5) at a retention time of 21.3 min. In addition, some of the methylated nucleosides were detected directly, including 2'-deoxy-3-methyl-3',5'-di-O-methyl-uridine (peak 10), 2'-deoxy-N,O,Otrimethyl-thymidine (peak 11), and 2'-deoxy-N,O,O,Otetramethyl-adenosine (peak 12). The detected methylated nucleosides are more stable and their structures are simpler than the other tested nucleosides. Additionally, the mass fragments of the detected nucleosides are within the selected mass range (m/z 40-500) of our analysis.

Figure 7

 7 Figure7shows the abundance of methyl furfuryl produced from the TMAH thermochemolysis of nucleoside mixtures at different temperatures. The abundance of methyl furfuryl was relatively consistent from 200 to 500 °C. The abundance of the methylated furfuryl is the highest at 600 °C. This demonstrated that methyl furfuryl is eliminated from the main structure of the nucleosides and detected at 200 °C. With an increase in temperature, the methylated nucleosides are split into two parts: methylated furfuryl and methylated nucleobases. Therefore, the abundance of the methylated nucleosides mixtures decreased with the increase in thermochemolysis temperature, which shows a phenomenon similar to that of individual nucleotides.

2 '

 2 -deoxy-N,O,O-trimethyl-thymidine and 2'-deoxy-3-methyl-3',5'-di-O-methyluridine were two of the main methylated products detected from the thermochemolysis of thymidine and uridine with TMAH. Thymidine and uridine produce similar derivatized pyrimidine bases since the molecules differ only by the presence of an extra methyl group at carbon position 5 in thymidine. For thymidine and uridine, the N1 position is the only labile J o u r n a l P r e -p r o o f site of the nucleobase ring, and the active hydrogen of the secondary amine is replaced by a methyl functional group during TMAH thermochemolysis. In addition, the hydrogens of hydroxyl from deoxyribose were methylated by TMAH thermochemolysis. Mainly the canonical forms of methylated thymidine and uridine were detected in this study, because the canonical tautomer of thymine and uracil is thermodynamically more stable than keto, enol and keto-enol tautomerism[START_REF] Morsy | Fluorescence of Thymine Tautomers at Room Temperature in Aqueous Solutions[END_REF][START_REF] Shukla | Interaction of water molecules with cytosine tautomers: An excited-state quantum chemical investigation[END_REF][START_REF] Tsuchiya | Keto-enol tautomer of uracil and thymine[END_REF][START_REF] Brady | The electronic spectra of the pyrimidine bases uracil and thymine in a supersonic molecular beam[END_REF][START_REF] Rejnek | Correlated ab initio study of nucleic acid bases and their tautomers in the gas phase, in a microhydrated environment and in aqueous solution : Part 4. Uracil and thymine[END_REF]. Therefore, 2'-deoxy-N,O,O-trimethyl-thymidine and 2'deoxy-3-methyl-3',5'-di-O-methyl-uridine are the main methylated products from dT and dU with TMAH thermochemolysis at low temperature, respectively.

2 -

 2 (methoxymethyl)furan and 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran are also the main methylated products from the TMAH thermochemolysis of nucleosides. 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran is the methylated product of 2-(hydroxymethyl)-2,3-J o u r n a l P r e -p r o o f dihydrofuran-3-ol, which is the product formed from the degradation of the nucleoside. Furfuryl alcohol is the product of 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran under dehydration. 2-(methoxymethyl)furan is formed when the hydrogen from the hydroxyl group of the furfuryl alcohol is replaced by the methyl functional group of TMAH. The abundance of 2-(methoxymethyl)furan was higher than that of 3-methoxy-2-(methoxymethyl)-2,3dihydrofuran, which demonstrated that the structure of 2-(methoxymethyl)furan was more stable than 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran.

C 5 H 9 O 4 ,

 594 C 5 H 7 O 3 , and C 5 H 6 O 2[START_REF] Ptasińska | Inelastic electron interaction (attachment/ionization) with deoxyribose[END_REF]. This demonstrated that C 5 H 7 O 3 , and C 5 H 6 O 2 could be the main radicals of the deoxyribose component of nucleosides under thermochemolysis at temperatures higher than 200 °C. Therefore, 2-(methoxymethyl)furan and 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran are the main products from the degradation of the deoxyribose component of nucleosides.

  thermochemolysis affects the efficiency of the detection of DNA fragments. If there are indications of past or present life on Mars at the surface or near subsurface in the form of molecular fragments analogous to DNA, the experimental conditions of life detection instrumentation should be further optimized in the future.

Figure captions:Figure 1

 1 Figure captions:

Figure 2

 2 Figure 2 Chromatograms of the chemical compounds detected after the thermochemolysis of nucleosides with TMAH at flash pyrolysis of 200 °C. The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8). dI peak 1: Furfuryl ether, RT=6.1 min; 2:Caffeine, RT=26.9min; 3: 1,7-dimethyl hypoxanthine and isomers, RT=27.5 and 28.6 min; 4: 2'deoxy-3-methyl-3',5'-di-O-methyl-inosine, RT=32.65 min. dC peak 1: Furfuryl ether, RT=6.1 min; 2: 4-Dimethylamino-1-methyl-2(1H)-pyrimidinone and isomers, RT=27.4 min; 3: 2'-deoxy-N,N,O,Otetramethyl-Cytidine, RT=34.4 min; dU peak 1: Furfuryl ether, RT=6.1 min; 2: 1,3-dimethyl-uracil; RT=20.2 min; 3: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-uridine and isomers. dT peak 1: Furfuryl ether, RT=6.1 min; 3: 1,3-Dimethyl-thymidine; RT= 21.5 min. dG peak 1: Furfuryl ether, RT=6.1 min; 2: Caffeine, RT=27.0 min; 3: trimethyl-guanine, RT=28.0 min; 4: Tetramethyl-guanine, RT=29.14 min; 5: Methyl guanosine and isomers, RT=34.5 min. dA Peak 1: Furfuryl ether, RT=6.1 min; 2: N,9dimethyl-9H-Purin-6-amine or N,9-dimethyl-adenine, RT=25.0 min; 3: N,N,9-Trimethyl-9H-purin-6amine or N,N,9-Trimethyl-adenine and isomers, RT=25.9 min; 4: N,N,3-Trimethyl-9H-purin-6-amine or N,N,3-Trimethyl-adenine and isomers, RT=26.8 min; 5: 2'-deoxy-N,N,O,O-tetramethyl-adenosine and isomers, RT=33.1 min.

Figure 4

 4 Figure 4Chromatograms of the chemical compounds detected after the TMAH thermochemolysis of nucleosides for the different flash pyrolysis temperatures studied. The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8). dA: 1: 2methoxymethyl-Furan; 2: 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran; 3: 2,3-diethyl-5-methyl-Pyrazine; 4: N,N,9-Trimethyl-adenine; 5: N,N,3-trimethyl-adenine; 6: 2'-deoxy-N,NO,O-tetramethyladenosine. dG: 1: 2-methoxymethyl-Furan; 2: Caffeine;3: Hexadecanoic acid methyl ester; 4 and 5:Tetramethyl-guanine and isomers. dT: 1 and 2: 2-methoxymethyl-Furan ;3: 1,3-Dimethyl-thymidine; 4: 2'-deoxy-N,N,O-trimethylthymidine. dU:1 and 2: 2-methoxymethyl-Furan and isomers; 3: 1,3dimethyl-uracil; 4: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-Uridine. dC:1 and 2: 2-methoxymethyl-Furan and isomers; 3: 4-Dimethylamino-1-methyl-2(1H)-pyrimidinone and isomers; 4: 2'-deoxy-N,N,O,O-tetramethyl-Cytidine. dI: 1 and 2: 2-methoxymethyl-Furan and isomers; 3 and 4: 1,7-Dimethyl-hypoxanthine and isomers.

Figure 6

 6 Figure[START_REF] Jacobson | Purine and pyrimidine (P2) receptors as drug targets[END_REF] Chromatograms of the chemical compounds detected after TMAH thermochemolysis of the mixture of nucleosides (10 nmol) at different temperatures. The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8, RT=15.3 min). Peak 1 : RT=6.1 min, Furfuryl methyl ether; Peak 2 : RT=10.8 min, 2'-deoxy-Cytidine?; Peak 3 : RT=13.0 min, 3-

Figure 7

 7 Figure 7Abundance of methyl furfuryl detected after TMAH thermochemolysis of nucleosides at different temperatures (A i /A IS is the ratio of the chromatographic peak area of 2-methoxymethyl-furan and the chromatographic peak area of the internal standard).

Figure 8

 8 Figure 8Chromatograms of the products of TMAH thermochemolysis of nucleosides under a SAMlike ramp pyrolysis. The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8).

Figure 9

 9 Figure 9 The chromatogram following a SAM-like ramp pyrolysis of the mixture of nucleosides. Peak 1: Furfuryl methyl ether; 2: 1,3-dimethyl-uracil; 3: 1,3-dimethyl-thymine; 4: trimethyl-cytosine; 5: N,N,9-Trimethyl-adenine; 6,8: 1,7-dimethyl-hypoxanthine; 7: N,N,3-Trimethyl-adenine; 9: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-Uridine; 10: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-Thymidine; 11: 2'deoxy-N,N,O,O-tetramethyl-adenosine; 12: 2'-deoxy-N,N,O,O-tetramethyl-cytidine. IS stands for internal standard (Naphthalene-D8).

Figure 10

 10 Figure 10The abundance of each methylated nucleoside detected by Py-GC/MS under a SAM-like ramp pyrolysis with TMAH thermochemolysis.

Figure 11 20 Figure 1 Figure 2 22 Figure 3

 112012223 Figure 11The TMAH thermochemolysis schemes of six nucleosides.J o u r n a l P r e -p r o o f

Figure 4

 4 Figure 4 Chromatograms of the chemical compounds detected after the TMAH thermochemolysis of nucleosides for the different flash pyrolysis temperatures studied. The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8). dA: 1: 2methoxymethyl-Furan; 2: 3-methoxy-2-(methoxymethyl)-2,3-dihydrofuran; 3: 2,3-diethyl-5methyl-Pyrazine; 4: N,N,9-Trimethyl-adenine; 5: N,N,3-trimethyl-adenine; 6: 2'-deoxy-N,NO,Otetramethyl-adenosine. dG: 1: 2-methoxymethyl-Furan; 2: Caffeine;3: Hexadecanoic acid methyl ester; 4 and 5:Tetramethyl-guanine and isomers. dT: 1 and 2: 2-methoxymethyl-Furan ;3: 1,3-Dimethylthymidine; 4: 2'-deoxy-N,N,O-trimethylthymidine. dU:1 and 2: 2-methoxymethyl-Furan and isomers; 3: 1,3-dimethyl-uracil; 4: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-Uridine. dC:1 and 2: 2methoxymethyl-Furan and isomers; 3: 4-Dimethylamino-1-methyl-2(1H)-pyrimidinone and isomers; 4: 2'-deoxy-N,N,O,O-tetramethyl-Cytidine. dI: 1 and 2: 2-methoxymethyl-Furan and isomers; 3 and 4: 1,7-Dimethyl-hypoxanthine and isomers. The peaks at the beginning of the chromatogram are the solvent peak.

Figure 6

 6 Figure 6 Chromatograms of the chemical compounds detected after TMAH thermochemolysis of the mixture of nucleosides (10 nmol) at different temperatures. The signal plotted is the total ion current of the MS detector. IS stands for internal standard (Naphthalene-D8, RT=15.3 min). Peak 1 : RT=6.1 min, Furfuryl methyl ether; Peak 2 : RT=10.8 min, 2'-deoxy-Cytidine?; Peak 3 : RT=13.0 min, 3-methyl-1,2cyclopentanedione?; Peak 4 : RT=20.1 min, 1,3-dimethyl-uracil ; Peak 5 : RT=21.2 min, 1,3-dimethyl-thymine; Peak 6: RT=25.4 min, N,N,9-trimethyl-adenine; peak 7,9: RT=27.4 and 28.6 min, 1,7-dimethyl-hypoxanthine; peak 8: RT=28.1 min, N,N,3-trimethyl-adenine; peak 10: RT=30.0 min, 2'-deoxy-3-methyl-3',5'-di-O-methyl-Uridine; peak 11: RT=30.44 min, 2'-deoxy-N,O,O-trimethyl-thymidine ; peak 12 : RT=33.0 min, 2'-deoxy-N,O,O,O-tetramethyl-Adenosine.

Figure 7 Figure 8

 78 Figure 7 Abundance of methyl furfuryl detected after TMAH thermochemolysis of nucleosides at different temperatures (A i /A IS is the ratio of the chromatographic peak area of 2-methoxymethyl-furan and the chromatographic peak area of the internal standard). J o u r n a l P r e -p r o o f

Figure 9 29 Figure 10 30 Figure 11

 929103011 Figure 9 The chromatogram following a SAM-like ramp pyrolysis of the mixture of nucleosides. Peak 1: Furfuryl methyl ether; 2: 1,3-dimethyl-uracil; 3: 1,3-dimethyl-thymine; 4: trimethyl-cytosine; 5: N,N,9-Trimethyl-adenine; 6,8: 1,7-dimethyl-hypoxanthine; 7: N,N,3-Trimethyl-adenine; 9: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-Uridine; 10: 2'-Deoxy-3-methyl-3',5'-di-O-methyl-Thymidine; 11: 2'deoxy-N,N,O,O-tetramethyl-adenosine; 12: 2'-deoxy-N,N,O,O-tetramethyl-cytidine. IS stands for internal standard (Naphthalene-D8).J o u r n a l P r e -p r o o f

  ,67,55,82,162,177,148,121 ,

  r n a l P r e -p r o o f

3 J

 3 o u r n a l P r e -p r o o f

Table 1

 1 Detailed information of nucleoside samples and solutions used in this study.

	J o u r n a l P r e -p r o o f
	Compounds	Molecular Weight (g mol -1 )	Amount (nmol)	Injection volume (µl)	Concentration (mol•L -1 )

Acknowledgement

This work was supported by CNES, focused on Sample Analysis at Mars of the Mars Science Laboratory mission, and Mars Organic Molecules Analyzer of the Exomars 2022 mission. Y.H. was supported by the CSC scholarship (201701810036). We thank Vincent Butin and Jamila El Bekri for their help in experimental setup.

Declaration of Competing Interest

No competing interests exist.