Mathias Dezetter
email: mathias.dezetter@gmail.com

Jean François Le Galliard

Olivier Lourdais

Behavioural hydroregulation protects against acute effects of drought in a dry-skinned ectotherm

Keywords: behaviour, humidity gradient, hydroregulation, microclimate selection, water

During extreme climate events, behavioural thermoregulation may buffer ectotherms from thermal stress and overheating. However, heatwaves are also combined with dry spells and limited water availability, and how much individuals can behaviourally mitigate dehydration risks through microclimate selection remains largely unknown. Herein, we investigated the behavioural and physiological responses to changes in air and microhabitat humidity in a terrestrial ectotherm, the asp viper (Vipera aspis). We exposed individuals to a simulated heatwave together with water deprivation for 3 weeks, and manipulated air water vapour density (wet air vs. dry air) and microclimate (wet shelter vs. dry shelter) in a two-by-two factorial design. Dry air conditions led to substantial physiological dehydration and muscle wasting. Vipers exposed to dry air more often used a shelter that provided a moist microclimate, which reduced dehydration and muscle wasting at the individual level. These results provide the first experimental evidence that active behavioural hydroregulation can mitigate specific physiological stress responses caused by a dry spell in an ectotherm. Future studies investigating organismal responses to climate change should consider moisture gradient in the habitat and integrate both hydroregulation and thermoregulation behaviours.

Introduction

Climate warming poses a major extinction threat to biodiversity because rising local temperatures force many terrestrial organisms to shift their range, alter their phenology, or expend more energy to maintain homeostasis (Riddell et al., 2019;[START_REF] Urban | Accelerating extinction risk from climate change[END_REF]. These physiological and ecological effects will largely depend on potential buffering by microclimatic conditions [START_REF] Pincebourde | Fine-Scale Microclimatic Variation Can Shape the Responses of Organisms to Global Change in Both Natural and Urban Environments[END_REF][START_REF] Potter | Microclimatic challenges in global change biology[END_REF]. Notably, for some terrestrial organisms, vulnerability to global warming depends less on global climate trends than on microclimate conditions at small spatial scales, such as those generated by heterogeneous vegetation and soil layers, and on behavioural strategies allowing exploitation of these microclimate conditions [START_REF] Potter | Microclimatic challenges in global change biology[END_REF][START_REF] Riddell | Exposure to climate change drives stability or collapse of desert mammal and bird communities[END_REF]. Thus, to predict ecological consequences of climate change, one should better characterise how organisms respond to microclimatic heterogeneity [START_REF] Sears | The World Still Is Not Flat: Lessons Learned from Organismal Interactions with Environmental Heterogeneity in Terrestrial Environments[END_REF][START_REF] Woods | Extended phenotypes: buffers or amplifiers of climate change?[END_REF].

In the context of global warming, the risks of overheating are significantly reduced when an organism is capable of fast behavioural responses [START_REF] Seebacher | Physiological mechanisms of thermoregulation in reptiles: a review[END_REF][START_REF] Terrien | Behavioral thermoregulation in mammals: a review[END_REF]. Behavioural thermoregulation allows organisms, especially ectotherms, to exploit spatial variability in thermal conditions to maintain an optimal body temperature despite temporal fluctuations in operative temperature [START_REF] Angilletta | Thermal Adaptation[END_REF][START_REF] Fuller | How dryland mammals will respond to climate change: the effects of body size, heat load and a lack of food and water[END_REF].

For example, many terrestrial ectotherms typically live in a complex thermal landscape where they can change their posture and coloration, seek shade, use burrows or shift their daily activity patterns to control heat gain, exploit cold and warm microclimates and ultimately avoid overheating risks [START_REF] Black | Behavioral Thermoregulation in Lizards : Strategies for Achieving Preferred Temperature[END_REF][START_REF] Forget-Klein | Toads use the subsurface thermal gradient for temperature regulation underground[END_REF][START_REF] Moore | Under the weather?-The direct effects of climate warming on a threatened desert lizard are mediated by their activity phase and burrow system[END_REF].

Given this, the most efficient thermoregulators should be better capable of maintaining homeostasis in a warming world [START_REF] Huey | Predicting organismal vulnerability to climate warming: Roles of behaviour, physiology and adaptation[END_REF][START_REF] Kearney | The potential for behavioral thermoregulation to buffer "cold-blooded" animals against climate warming[END_REF]). Yet, climate change does not only challenge body temperature regulation but also puts a strong pressure on osmoregulation (i.e., the homeostatic maintenance of salt and body fluids balance) in terrestrial organisms (Riddell et al., 2019;[START_REF] Rozen-Rechels | When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms[END_REF]. For example, avoidance of overheating in desert endotherms involves thermoregulatory costs from evaporative cooling, which implies that birds and mammals must actively seek or save water to maintain an optimal body temperature in a warmer climate [START_REF] Mitchell | Revisiting concepts of thermal physiology: Predicting responses of mammals to climate change[END_REF]Riddell et al., 2019).

In many terrestrial ectotherms, behavioural trade-offs between thermoregulation and osmoregulation exist because evaporative water loss increases with body temperature and in the microclimate conditions relevant for basking (e.g., Dupoué et al., 2015b;[START_REF] Riddell | Trade-offs between water loss and gas exchange influence habitat suitability of a woodland salamander[END_REF]. In addition, climate warming often involves concurrent changes in temperature, rainfall and moisture, especially during extreme climate events such as summer hot and dry spells that increase water loss risks [START_REF] Trenberth | Global warming and changes in drought[END_REF][START_REF] Ummenhofer | Extreme weather and climate events with ecological relevance: A review[END_REF].

Organisms should partially buffer the water constraints of climate change by means of behavioural hydroregulation, defined as the range of behaviours that control water gains and water losses and help individuals maintain an optimal hydration state for their activity and functional capacities [START_REF] Pirtle | Hydroregulation ; A Neglected Behavioral Response of Lizards to Climate Change ?[END_REF][START_REF] Rozen-Rechels | When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms[END_REF]. For example, reptiles living under water stress can maintain their water balance by reducing their activity [START_REF] Davis | Seasonal Patterns of Body Condition, Hydration State, and Activity of Gila Monsters (Heloderma suspectum) at a Sonoran Desert Site[END_REF]Kearney et al., 2018) or by selecting cool [START_REF] Ladyman | The influence of dehydration on the thermal preferences of the Western tiger snake, Notechis scutatus[END_REF][START_REF] Pintor | Hydroregulation in a tropical dry-skinned ectotherm[END_REF] or wet microclimates (Dupoué et al., 2015b;[START_REF] Guillon | Microclimate preferences correlate with contrasted evaporative water loss in parapatric vipers at their contact zone[END_REF][START_REF] Lourdais | Hydric "Costs" of Reproduction: Pregnancy Increases Evaporative Water Loss in the Snake Vipera aspis[END_REF].

A wide range of hydroregulation behaviours have been observed in the wild, from moisture harvest in desert beetles, free-standing water seeking in large mammals to soil moisture preferences in frogs [START_REF] Rozen-Rechels | When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms[END_REF][START_REF] Wolcott | Role of Behavior in Meeting Osmotic Challenges[END_REF]. Despite that, our knowledge of the buffering capacity of these behavioural mechanisms still remains limited [START_REF] Pirtle | Hydroregulation ; A Neglected Behavioral Response of Lizards to Climate Change ?[END_REF][START_REF] Rozen-Rechels | Water restriction induces behavioral fight but impairs thermoregulation in a dry-skinned ectotherm[END_REF]. Behavioural hydroregulation is most obvious in wet-skinned amphibians [START_REF] Greenberg | Hydrothermal physiology and climate vulnerability in amphibians[END_REF][START_REF] Lertzman-Lepofsky | Water loss and temperature interact to compound amphibian vulnerability to climate change[END_REF]. Some amphibians can reduce evaporative water loss by means of behavioural changes such as postural adjustments to reduce the cutaneous water-exchange surface, mucus production or the selection of moist microhabitats (e.g. [START_REF] Lillywhite | Integumentary structure and its relationship to wiping behaviour in the common Indian tree frog, Polypedates maculatus[END_REF][START_REF] Wolcott | Role of Behavior in Meeting Osmotic Challenges[END_REF]. There is good evidence that some amphibians perform active behavioural hydroregulation to maintain an optimal hydration state independently from thermoregulation [START_REF] Mitchell | Thermal and moisture habitat preferences do not maximize jumping performance in frogs[END_REF], but whether this behavioural flexibility is efficient during extreme weather events remains unknown. Behavioural data are even more elusive for dryskin ectotherms due to difficulties disentangling hydroregulation and thermoregulation mechanisms in these organisms [START_REF] Pintor | Hydroregulation in a tropical dry-skinned ectotherm[END_REF][START_REF] Rozen-Rechels | When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms[END_REF]. Dry-skin ectotherms often have a strong resistance to evaporative water loss, meaning that their hydration state changes more slowly than body temperature and that shifts caused by behavioural hydroregulation could take place over long time periods. Behavioural hydroregulation could be controlled by the internal hydration status, leading individuals to choose less desiccating microclimates as a water-saving strategy only when chronically stressed [START_REF] Ladyman | The influence of dehydration on the thermal preferences of the Western tiger snake, Notechis scutatus[END_REF][START_REF] Rozen-Rechels | Water restriction induces behavioral fight but impairs thermoregulation in a dry-skinned ectotherm[END_REF]. On the other hand, terrestrial organisms can perceive moisture gradients through hygrosensing [START_REF] Chown | Water loss in insects: An environmental change perspective[END_REF] and further rely on odours or other cues to locate free standing water [START_REF] Serrano-Rojas | Tadpole-transporting frogs use stagnant water odor to find pools in the rainforest[END_REF][START_REF] Wood | African elephants can detect water from natural and artificial sources via olfactory cues[END_REF], such that changes in air water vapour pressure conditions may also trigger rapid changes in their behaviours.

Herein, we experimentally studied hydroregulation behaviours independently from body temperature regulation in a temperate ectotherm, the asp viper (Vipera aspis). We used climatic chambers to simulate weather conditions akin to those of a summer dry spell. We removed access to water and manipulated air moisture (dry versus wet air conditions) for three weeks. Water-deprived vipers were either allowed to hydroregulate by providing them with a wet shelter (with a damp substrate) or not by providing them with a dry shelter. We examined the following hypotheses and predictions: first, water deprivation will most negatively impact hydration state in a dry air environment. We predict important mass loss and plasma osmolality increase in these conditions. Second, dehydration should induce compensatory physiological responses to mitigate water constraints. We predict, in particular, patterns of structural muscle catabolism to release bound water [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF] and a greater thirst reaction [START_REF] Dezetter | Water deprivation compromises maternal physiology and reproductive success in a cold and wet adapted snake Vipera berus[END_REF][START_REF] Dupoué | Effect of water deprivation on baseline and stress-induced corticosterone levels in the Children's python (Antaresia childreni)[END_REF]. Third, behavioural selection of wet microclimates should mitigate these effects by reducing evaporative water loss, especially when the simulated air is drier. We predict higher wet shelter use and lower physiological costs in this treatment group. The intensity of shelter use should also negatively correlate with physiological costs at the individual level.

Material and Methods

Study species

The asp viper is a typical sit-and-wait foraging and capital breeding snake distributed over southwestern Europe in sunny and warm microhabitats [START_REF] Lourdais | Capitalbreeding and reproductive effort in a variable environment: a longitudinal study of a viviparous snake[END_REF]. This diurnal species is an active thermoregulator that maintains a high body temperature (30 to 33°C) during daily activity [START_REF] Lorioux | Stage dependence of phenotypical and phenological maternal effects: insight into squamate reptile reproductive strategies[END_REF][START_REF] Lourdais | Hydric "Costs" of Reproduction: Pregnancy Increases Evaporative Water Loss in the Snake Vipera aspis[END_REF]. A functional trade-off exists between thermoregulatory needs and water loss in asp vipers: behavioural thermoregulation implies the use of warm microclimates, which increase evaporative water loss rates (Dupoué et al., 2015b;[START_REF] Guillon | Microclimate preferences correlate with contrasted evaporative water loss in parapatric vipers at their contact zone[END_REF][START_REF] Lourdais | Hydric "Costs" of Reproduction: Pregnancy Increases Evaporative Water Loss in the Snake Vipera aspis[END_REF]. Behavioural selection of warm and moist basking locations helps gravid females to mitigate the conflict between thermal needs and water loss [START_REF] Lourdais | Hydric "Costs" of Reproduction: Pregnancy Increases Evaporative Water Loss in the Snake Vipera aspis[END_REF]. Access to water can be limited in natural habitats, especially during summer dry spells, which are increasing in frequency and intensity over the range distribution of this species [START_REF] Spinoni | Will drought events become more frequent and severe in Europe?[END_REF]. This capital breeder and semi-arid adapted species can tolerate long periods of fast and water deprivation, but at the cost of lower body reserves, enhanced emaciation and physiological stress, and therefore potential reduction in future fitness (Dupoué et al., 2015a;Lorioux et al., 2016). Herein, we used 42 non-reproductive adults (27 females and 15 males) from a captive colony originating from western France and maintained at the Centre d'Etudes Biologiques de Chizé, France.

Manipulation of hydric conditions and air temperature

Our experimental design consisted of a strictly defined manipulation of hydric conditions independently from air temperature. We used 4 controlled environment chambers (Vötsch VP 600, Balingen, Germany) allowing realistic simulations of daily fluctuations in air temperature and humidity. Asp vipers were deprived of water for three weeks, which corresponds to a moderate duration for a dry spell in natural conditions [START_REF] Perkins | Increasing frequency, intensity and duration of observed global heatwaves and warm spells[END_REF].

The daily temperature cycle mimicked body temperature recorded during a typical heatwave with daytime temperature of 32°C for 14h and nighttime temperature of 26°C for 5h, with temperature either gradually increasing or decreasing for the remaining 5h [START_REF] Lourdais | Cold climate specialization: Adaptive covariation between metabolic rate and thermoregulation in pregnant vipers[END_REF]. We next manipulated air humidity (dry air vs. wet air) and microclimate (dry shelter vs. wet shelter) using a two-by-two factorial design. First, we compared two contrasted ambient water vapour pressure deficit (VPD) to expose individuals to either desiccant (VPD = -3.10 ± 0.08 kP a in dry air; Dupoué et al., 2015b) or humid conditions (VPD = -1.43 ± 0.08 kP a in wet air). Second, we manipulated microclimate conditions by providing either a dry or a wet shelter. Shelters consisted of small, custom made plastic containers (15 cm diameter, 11 cm height; one entrance of 3 cm diameter on the side) filled with an artificial sponge and microfiber cloths. Once accustomed, vipers will readily use shelters in the laboratory. The sponge and microfiber cloths were changed every 3 days and maintained dry in the dry shelters but damp in the wet shelters in order to enhance air humidity. In dry shelters, VPD were similar to the ambient conditions and therefore offered no humidity gradient. In turn, wet shelters provided high and constant humidity (VPD = -0.61 ± 0.09 kP a ), which simulates natural underground microhabitats that are typically moist with a stable and high humidity throughout the day. The differential between wet shelter and ambient air VPD was respectively of 2.49 kP a and 0.82 kP a for dry and wet air. Temperatures inside the shelters were similar to ambient temperatures. Note that we observed no water condensation inside the plastic shelters so drinking water was deemed extremely unlikely.

Experimental design

Prior to the experiment, for the purpose of acclimation to general maintenance conditions, we housed all individuals for one week in climatic chambers using individual plastic boxes (35 x 20 x 17 cm) with one dry shelter, a sheet of newspaper as a substrate and a natural day light cycle (circa 12:12). During this period, vipers had ad-libitum access to drinking water and were exposed to a standard daily temperature cycle (6h daytime at 30°C, 13h nighttime at 22°C, and mean VPD of -1.20 ± 0.10 kP a ). Then, we randomly allocated individuals to each treatment group while ensuring a balanced sex ratio. Each individual was placed in a climatic chamber with the corresponding air humidity treatment (dry air or wet air) and we applied the following protocol over 24 days: from Day 0 to Day 2 (3 days), we provided all individuals with a dry shelter in order to quantify baseline shelter use; from Day 3 to Day 20 (18 days), we exposed each individual to its microclimate treatment (dry shelter or wet shelter) in order to quantify the combined effects of air humidity and microclimate; from Day 21 to Day 23 (3 days), we provided all individuals with a wet shelter to test for change in shelter use in individuals previously exposed to dry shelters; and finally, on Day 24, we provided all individuals with water ad-libitum to assess post treatment water intake from drinking. We then transferred them to standard housing conditions. We did not feed the snakes in order to avoid confounding effects of digestion and energy intake on body mass [START_REF] Stahlschmidt | Consequences of complex environments: Temperature and energy intake interact to influence growth and metabolic rate[END_REF].

Variables collected and parameters measured

Shelter use over time

Changes in body mass and muscle condition

In the absence of food intake, body mass (BM) change provides a non-invasive proxy of the whole body hydration state in snakes [START_REF] Dupoué | Effect of water deprivation on baseline and stress-induced corticosterone levels in the Children's python (Antaresia childreni)[END_REF]. We therefore weighed (± 0.1 g) all individuals every 3 days to assess BM changes during the treatment period, and then weighed them again at the end of Day 24 to assess mass uptake and rehydration after the manipulation. From this, we calculated total ΔBM as the difference between BM at Day 20 and at Day 0. We also analysed mass changes over each 3 days' period during the combined manipulation of air humidity and microclimate (Day 3 to Day 20, 6 repetitions per individual) to investigate the relation between individual shelter use and body mass changes during that period. Finally, post-treatment water intake was inferred from the mass difference between Day 24 and Day 23.

Bound water is a significant component of all body tissues that can be mobilised to support water demands during periods of water deprivation, especially from wet protein of muscles in reptiles [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. In snakes, skeletal muscles can be mobilised to support water requirements [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. To quantify skeletal muscles loss during the treatment period, we measured tail width (TW ± 0.01 mm, average from triplicates) on Day 0 and on Day 20 using an electronic pressure-sensitive specimeter (Absolute Digimatic, Mitutoyo, Japan) at the position of the sixth subcaudal scale (Lorioux et al., 2016). We calculated ΔTW as the difference between mean TW at Day 20 and Day 0.

Changes in plasma osmolality

We collected blood samples on Day 0 and Day 20 to assay plasma osmolality, which measures the whole-body electrolyte and water balance [START_REF] Peterson | Temporal, population, and sexual variation in hematocrit of free-living desert tortoises: Correlational tests of causal hypotheses[END_REF]. We sampled individuals in a random order by sex and treatments and collected 150 µL of blood through cardiocentesis, using a 1mL syringe and a heparinized 29-gauge needle. We immediately centrifuged the blood at 2000g, separated plasma from the red blood cells and stored the samples at -28°C. Plasma osmolality (± 1 mOsm kg -1 ) was measured from 10 µL duplicates (intra-individual CV: 0.36%) using a vapour pressure osmometer (model 5500, Wescor, Logan, UT, USA). We calculated ΔOsmolality as the difference between mean plasma osmolality before and after manipulation.

Statistical analyses

We conducted all analyses using the R software version 3.6.3 (R Core Team, 2020). To investigate the effects of treatments on behavioural hydroregulation, we first built a generalized linear model with a binomial error distribution and a logit link function to examine cumulative shelter use (success = total number of observation when shelter was used; failure = total number of observation when shelter was not used) between Day 3 to Day 20., We used sex, air humidity, microclimate treatment and the interaction between both treatments as explanatory factors in the full model. This full model was built with glm function of the stats package (R Core Team, 2020) and fitted well with the data (overdispersion test: X² = 43.9, dispersion coefficient = 1.1, df = 39, P = 0.27). We used two similar models to analyse the baseline shelter use before the start of the microclimate manipulation (between Day 0 and Day 2) and the wet shelter use at the end of the treatment (between Day 21 and Day 23). Because of significant overdispersion of the binomial distribution in these two data sets, we compared treatment group using a betabinomial approach (Harrison, 2015). The betabinomial model for proportions was fitted with betabin function of the aod package (Lesnoff & Lancelot, 2012). We further investigated repeated measures through time of daily shelter use according to treatments between Day 0 and Day 20 using a generalized linear mixed effects model with a binomial error distribution (success = number of observations per day when shelter was used; failure = number of observation per day when shelter was not used) and a logit link function. We included sex as a fixed factor, and air humidity, microclimate treatment, time (number of days) and their three-way interaction as explanatory variables. Individual identity was included as a random term to control for repeated measures on the same individuals. Because data were skewed toward zero at Day 0 and shelter use was identical between treatment groups at the beginning of the manipulation, we forced a similar intercept for the four treatments in our full model. This model was built with glmer function of the lme4 package (Bates et al., 2015) and fitted well with the data (overdispersion test: X² = 816.4, dispersion coefficient = 0.9, df = 896, P = 0.92). In all cases, we performed backward elimination model selection starting from the full models to remove non-significant effects using likelihood ratio tests. We report here P values from Wald tests based on z-score of the parameter estimates of the models.

To analyse physiological and morphological responses, wee used linear models fitted with the lm function (stats package) and univariate linear mixed effects models fitted with the lme function of the nlme package [START_REF] Pinheiro | An introduct nlme: Linear and Nonlinear Mixed Effects Models[END_REF]. We performed backward elimination model selection starting from the full models after checking that the residuals of our models fulfilled a Gaussian distribution using Shapiro-Wilk tests and had homogeneous variances across treatment groups. We further explored statistical differences between treatment groups using pairwise post-hoc Tukey tests (emmeans, package emmeans ; Lenth et al., 2019).

Regarding morphological and osmolality data, we first built independent linear models to confirm that there were no treatment differences in BM, TW or plasma osmolality at the beginning of the experiment. Models included the initial value as the dependent variable and air humidity, microclimate treatment, and their interaction as fixed effects. There was no difference in BM, TW, plasma osmolality among treatments before the manipulation (all P ˃ 0.05, Table S1 & S2). To investigate the effects of the treatments on body mass loss over time, we used a mixed effects model for repeated measures with BM as the dependent variable, sex as a fixed factor, air humidity treatment, microclimate treatment, time (i.e. days on which individuals were weighed) and their three-way interaction as explanatory variables.

Individual identity was included as a random, intercept term.

We next built three independent linear models to investigate the effects of the treatments on total ΔBM, ΔTW and ΔOsmolality and post-treatment ΔBM. We included air humidity, microclimate treatment and their interaction as explanatory variables, sex as a categorical factor and the initial value as a linear covariate. We further examined whether ΔBM, ΔTW and post-treatment ΔBM were correlated with ΔOsmolality or final osmolality using linear regressions. To determine whether ΔOsmolality or final osmolality better explained physiological changes, we used the Akaike Information Criterion to compare models (AIC, package stats). We also investigated whether post-treatment ΔBM was correlated with total ΔBM using a similar model construction and comparison.

We also wanted to investigate the relationship between wet shelter use and mass change of snakes. To achieve this, we considered mass changes across individuals in the dry air treatment with access to wet shelter and applied the statistical methods from van de Pol and Wright, (2009).We calculated an individual-centred shelter use score at each 3-day period for each individual. We built a linear mixed effects model with the change in body mass per 3-day period as the dependent variable and shelter use score together with individual-centred shelter use score as explanatory variables. Individual identity was set as a random factor.

Results

Quantification of hydroregulation behaviour

At the onset of the study (from Day 0 to Day 2), there was no difference in shelter use among treatment groups (Air humidity: z = -0.55, 𝑃 = 0.58; Microclimate treatment: z = 0.55, 𝑃 = 0.58). During the cumulated manipulation period (from Day 3 to Day 20), the total proportion of shelter use was influenced by the interaction between air humidity and microclimate treatment(𝑧 = -2.1, 𝑃 = 0.03). Supporting our prediction, shelter use was mainly observed in vipers maintained in dry air with a wet shelter, while other individuals almost never selected the shelter (Table 1). The daily shelter use gradually increased over time in the dry air and wet microclimate treatment group, while it remained stable and low in other groups (Air humidity × Microclimate treatment × Time: z = -2.1, 𝑃 = 0.03; Fig. 1a). When all individuals were provided with a wet shelter at the end of the experiment (from Day 21 to Day 23), the proportion of shelter use was then additively influenced by air humidity (𝑧 = -3.5, 𝑃 < 0.001) and former microclimate treatments (𝑧 = -2.4, 𝑃 = 0.02). This was due to a behavioural shift where vipers maintained in the dry microclimate treatment rapidly increased their use of the shelter when it became wet (Fig. 1a).

Physiological and morphological changes across treatments

Vipers significantly lost body mass over time during the experiment (𝐹 1,290 = 1674.8, 𝑃 < 0.001, Fig. 1b), and air humidity and microclimate treatments interactively influenced BM loss (Air humidity × Microclimate treatment × Time: 𝐹 1,290 = 26.2, 𝑃 < 0.001). Vipers from the dry air and dry shelter lost BM at a much higher rate than those maintained in other conditions (Fig. 1b). In the dry air treatments, vipers with access to a wet shelter lost BM at a slower rate than those with a dry shelter. The total ΔBM over the experimental period was also influenced by the interaction between both treatment types (𝐹 1,37 = 4.5, 𝑃 = 0.04; Table 1, mean ΔBM = -13.2 ± 5.5 % of initial BM).

Plasma osmolality increased in all groups indicating physiological dehydration, and the increase in osmolality was influenced by sex (𝐹 1,37 = 23.7, 𝑃 < 0.001), air humidity treatment (𝐹 1,37 = 15.5, 𝑃 < 0.001) but only marginally by microclimate treatment (𝐹 1,37 = 2.9, 𝑃 = 0.09). Individuals had a higher increase in osmolality in dry conditions, and tended to have a lower increase in osmolality when a wet shelter was available (Table 1). However, one male from the dry air and wet microclimate treatment almost never used the wet shelter (shelter use score of 10%). Excluding this individual, we found that ΔOsmolality was significantly influenced by the interaction between air humidity and microclimate treatments (𝐹 1,35 = 6.6, 𝑃 = 0.01) and by sex (𝐹 1,35 = 23.5, 𝑃 < 0.001). Increase in plasma osmolality was highest in vipers maintained in dry air with a dry shelter and lowest and similar in the other three groups (Table 1). Overall, males had a higher increase in plasma osmolality than females. Tail width change ΔTW was additively influenced by air humidity (𝐹 1,38 = 5.7, 𝑃 = 0.02, Fig. 2a) and microclimate treatments (𝐹 1,38 = 5.6, 𝑃 = 0.02, Fig. 2b). Vipers had increased TW loss in dry air conditions and when they only had access to a dry shelter (Table 1; Fig. 2a).

Benefits of hydroregulation and post-treatment water intake

Focusing on the dry air and wet shelter treatment, we found that body mass loss was negatively correlated with the individual-centred shelter use score (𝐹 1,65 = 26.1, 𝑃 < 0.001).

Higher behavioural selection of the wet shelter led to reduced body mass loss (Fig. 3). Posttreatment water intake was additively influenced by air humidity (𝐹 1,37 = 32.5, 𝑃 < 0.001, Fig. 2c) and microclimate treatments (𝐹 1,37 = 8.7, 𝑃 = 0.005, Fig. 2D). Vipers exposed to dry air conditions and vipers with access to a dry shelter drank more on Day 24 (Table 1; Fig. 2c).

Correlations between physiological responses

Overall, BM loss was positively correlated with both ΔOsmolality (𝐹 1,39 = 55.9, 𝑃 < 0.001, Fig. S1a) and final osmolality (𝐹 1,39 = 4.99, 𝑃 < 0.001), but most influenced by ΔOsmolality (Table S8). TW loss was also positively correlated with both ΔOsmolality (𝐹 1,39 = 17.9, 𝑃 < 0.001, Fig. S1b) and final osmolality (𝐹 1,39 = 19.2, 𝑃 < 0.001; Fig. S6) with similar strength of correlation (Table S8). Post-treatment water intake was positively correlated with ΔOsmolality (𝐹 1,39 = 66.0, 𝑃 < 0.001, Fig. S1c), final osmolality (𝐹 1,39 = 80.0, 𝑃 < 0.001) and ΔBM (𝐹 1,39 = 34.3, 𝑃 < 0.001), but most influenced by final osmolality (Table S8).

Discussion

We provide the first experimental evidence that behavioural hydroregulation can mitigate physiological consequences of a simulated dry spell in a terrestrial ectotherm. Wet shelters were used more frequently when individuals were exposed to dry air conditions. This pattern was gradual, reaching 75% of the time by the end of the manipulation, compared to less than 10% in other treatment groups. This demonstrates microclimate selection based on water vapour gradient independently from temperature, thus providing unambiguous evidence of behavioural hygroregulation sensu stricto [START_REF] Mitchell | Thermal and moisture habitat preferences do not maximize jumping performance in frogs[END_REF][START_REF] Wolcott | Role of Behavior in Meeting Osmotic Challenges[END_REF]. This behaviour allowed individuals to attenuate mass loss and dehydration risks in more desiccating conditions. Concurrent changes in morphology, plasma osmolality and post-treatment drinking behaviour all indicated that more desiccating air led to more severe physiological dehydration. Body mass loss throughout the treatment period was higher in individuals exposed to dry air and these individuals also had the greatest increase in plasma osmolality. Muscle loss and posttreatment water intake were greater in individuals exposed to dry air and both traits were correlated with physiological dehydration, here assessed by final osmolality values. Therefore, dry air conditions increased muscle catabolism, probably in order to release the bound water that makes up a significant portion of skeletal muscles [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF] and resulted in increased thirst levels [START_REF] Edwards | Thirst and drinking in North American watersnakes (Nerodia spp.)[END_REF][START_REF] Lillywhite | Drinking by sea snakes from oceanic freshwater lenses at first rainfall ending seasonal drought[END_REF]. Dehydrationinduced muscle catabolism can be beneficial to support water needs and reduce the immediate survival risks associated with hyperosmolality in vertebrates [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF][START_REF] Gerson | House sparrows (Passer domesticus) increase protein catabolism in response to water restriction[END_REF][START_REF] Mccue | Dehydration Causes Increased Reliance on Protein Oxidation in Mice: A Test of the Protein-for-Water Hypothesis in a Mammal[END_REF]. However, such compensatory mechanisms directly alter structural muscles and associated locomotor capacities (Lorioux et al., 2016;[START_REF] Lourdais | Protein catabolism in pregnant snakes (Epicrates cenchria maurus Boidae) compromises musculature and performance after reproduction[END_REF]. It is well-known that dehydration can impair locomotor performance as demonstrated in amphibians and some lizards [START_REF] Greenberg | Hydrothermal physiology and climate vulnerability in amphibians[END_REF][START_REF] Anderson | Trading heat and hops for water: Dehydration effects on locomotor performance, thermal limits, and thermoregulatory behavior of a terrestrial toad[END_REF][START_REF] Wilson | Dehydration Reduces the Endurance Running Capacity of the Lizard Uta stansburiana[END_REF]. Although we did not measure behavioural capacities or functional properties such as locomotion or foraging, the high levels of dehydration and muscle mobilization observed in some of our snakes are most likely associated with impaired locomotor abilities. Altogether, these results indicate that a more desiccating air in the absence of free standing water caused greater dehydration, likely through increased evaporative water loss (Dupoué et al., 2015b). Such hygrometric constraints are likely even more prominent at high ambient temperatures because desiccation risks rise exponentially with temperature (increased magnitude of VPD, see Riddell et al., 2019) and may be even stronger during key life history events when thermal requirements are elevated such as gestation in female vipers (see Dupoué et al., 2015b;[START_REF] Stahlschmidt | The role of temperature and humidity in python nest site selection[END_REF].

Supporting our initial predictions, preferential wet shelter use buffered the effects of dry air conditions on physiological dehydration. When a wet shelter was available, individuals had on average a lower body mass loss especially in dry air, tended to have a lower increase in plasma osmolality and had a lower post-treatment water intake. Interestingly, when we excluded one viper whose behaviour was not responsive to wet shelter availability, the influence of a wet microclimate on osmolality and body mass changes was only significant under the dry air condition. Individuals with access to a wet shelter also had lower muscle loss rates compared to those with a dry shelter, which suggests that they had less of a need to catabolize muscle protein. In addition, within the dry air and wet shelter treatment, the more a viper used the shelter, the less body mass it lost. Behavioural selection of a moist microclimate thus allowed vipers to attenuate dehydration risks and specific associated morphological responses. Altogether, snakes that had access to a wet shelter could thus mitigate the direct performance costs possibly induced by hyperosmolality [START_REF] Greenberg | Hydrothermal physiology and climate vulnerability in amphibians[END_REF][START_REF] Anderson | Trading heat and hops for water: Dehydration effects on locomotor performance, thermal limits, and thermoregulatory behavior of a terrestrial toad[END_REF], and also minimised the indirect performance costs of muscle catabolism [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]Lorioux et al., 2016).

We also found significant differences among individuals in their wet shelter use irrespective of treatment conditions and sex suggesting wide variation in hydroregulation behaviours likely exist. Within natural populations, such variation might translate in individual differences in physiological sensitivity to extreme climate events. Since flexible hydroregulation behaviours buffer physiological consequences of desiccating air, they should be beneficial for populations facing more frequent and intense dry spells, such as those located at the warm edge of the distribution. At the end of the experiment, most vipers that had been housed with a dry shelter immediately selected the wet shelter at a frequency similar to that of vipers that were maintained with a wet shelter and dry air for several days. This sudden shift suggests that these snakes had reached a significant dehydration threshold, which induced an immediate behavioural response once a wet microclimate was available.

Hygrosensing and behavioural hydroregulation have been previously observed in dryskinned ectotherms both in the field [START_REF] Guillon | Microclimate preferences correlate with contrasted evaporative water loss in parapatric vipers at their contact zone[END_REF][START_REF] Lourdais | Hydric "Costs" of Reproduction: Pregnancy Increases Evaporative Water Loss in the Snake Vipera aspis[END_REF] and in laboratory conditions (Dupoué et al., 2015b;[START_REF] Stahlschmidt | Parental behavior in Pythons is responsive to both the hydric and thermal dynamics of the nest[END_REF][START_REF] Stahlschmidt | The role of temperature and humidity in python nest site selection[END_REF]. Our findings extend these earlier observations and highlight that microclimate selection within a "hygrometric landscape" caused by spatial heterogeneity in humidity promotes water balance regulation and minimises the costs of dehydration. We posit that such behavioural adjustments are frequent in terrestrial ectotherms and call for a better characterisation of microclimate moisture gradients in the context of climate change [START_REF] Pincebourde | Fine-Scale Microclimatic Variation Can Shape the Responses of Organisms to Global Change in Both Natural and Urban Environments[END_REF][START_REF] Woods | Extended phenotypes: buffers or amplifiers of climate change?[END_REF]. Our results indicate that the use of moist microhabitats may allow ectotherms to avoid desiccation during extreme climate events, similar to the key role of behavioural thermoregulation in limiting exposure to acute thermal extremes and overheating [START_REF] Huey | Can behavior douse the fire of climate warming?[END_REF][START_REF] Kearney | The potential for behavioral thermoregulation to buffer "cold-blooded" animals against climate warming[END_REF]. For example, following the classic terminology for thermoregulation, some species may actively "hygroregulate" while others may behave as "hygroconformer". The former may efficiently seek microclimates minimising the deviance to optimal moisture [START_REF] Moore | Under the weather?-The direct effects of climate warming on a threatened desert lizard are mediated by their activity phase and burrow system[END_REF][START_REF] Székely | Dryness affects burrowing depth in a semi-fossorial amphibian[END_REF][START_REF] Lourdais | Hydric "Costs" of Reproduction: Pregnancy Increases Evaporative Water Loss in the Snake Vipera aspis[END_REF], which would allow individuals to maintain their hydration state, performance and activity [START_REF] Greenberg | Hydrothermal physiology and climate vulnerability in amphibians[END_REF][START_REF] Anderson | Trading heat and hops for water: Dehydration effects on locomotor performance, thermal limits, and thermoregulatory behavior of a terrestrial toad[END_REF].

However, their behavioural capacity to do so ultimately depends on habitat "hygrometric quality". This emphasizes the critical importance of the availability of moist microhabitats for dry-skinned ectotherms, which can decrease with anthropogenic disturbances [START_REF] Gorissen | A skink out of water: Impacts of anthropogenic disturbance on an Endangered reptile in Australian highland swamps[END_REF] or climate change [START_REF] Dundas | Some like it hot: Droughtinduced forest die-off influences reptile assemblages[END_REF]. In addition, we posit that a behavioural capacity to maintain optimal hygrometric conditions may dampen long-term exposure to dehydration risks along gradients of climate or habitat aridity. This may reduce selection on physiological tolerance to dehydration, similar to the concept of the "Bogert effect" for thermoregulatory behaviour [START_REF] Muñoz | The Bogert effect, a factor in evolution[END_REF]. More generally, the occurrence of behavioural hygroregulation may have implications for our understanding of the evolution of ecological niches of organisms [START_REF] Encarnación-Luévano | Burrowing habit in Smilisca frogs as an adaptive response to ecological niche constraints in seasonally dry environments[END_REF][START_REF] Farallo | Scaling between macro-to microscale climatic data reveals strong phylogenetic inertia in niche evolution in plethodontid salamanders[END_REF][START_REF] Woods | Extended phenotypes: buffers or amplifiers of climate change?[END_REF].

Understanding and predicting organismal responses to climate change is currently a major challenge in ecology and conservation (Kearney et al., 2018;Riddell et al., 2019). Our experimental study demonstrates that hygrometric constraints and behavioural hydroregulation capacity must be considered in this context. Recent individual-based models have used energy and heat budgets to predict fine-scale movements within microhabitats in response to thermal microclimate conditions [START_REF] Malishev | An individual-based model of ectotherm movement integrating metabolic and microclimatic constraints[END_REF][START_REF] Sears | Configuration of the thermal landscape determines thermoregulatory performance of ectotherms[END_REF], but those approaches often overlooked water constraints. Future mechanistic models should include the effects of air moisture and behavioural hydroregulation on the water budget of organisms (see [START_REF] Huang | Evaporative water loss simulation improves models' prediction of habitat suitability for a high-elevation forest skink[END_REF][START_REF] Peterman | Spatial variation in water loss predicts terrestrial salamander distribution and population dynamics[END_REF]. One challenge to this approach is that, in contrast to behavioural thermoregulation where individuals generally immediately adjust their behavioural activity in response to well-known temperature thresholds or thermal performance curves [START_REF] Angilletta | Thermal Adaptation[END_REF], behavioural hygroregulation likely changes more gradually in response to a shift in individual hydration state. The physiological costs of desiccant conditions should also vary among species depending on their resistance to water loss, thus influencing the timing of behavioural changes. For example, some dry-skinned ectotherms may adjust their hydroregulation behaviour over days or weeks, as shown here, while most wet-skinned ectotherms may adjust over minutes or hours [START_REF] Peterman | Spatial variation in water loss predicts terrestrial salamander distribution and population dynamics[END_REF][START_REF] Székely | Dryness affects burrowing depth in a semi-fossorial amphibian[END_REF]. In addition, empirical data suggest that the performance costs of dehydration increase abruptly only after a significant dehydration threshold is reached in amphibians [START_REF] Cheuvront | Dehydration: Physiology, Assessment, and Performance Effects[END_REF][START_REF] Greenberg | Hydrothermal physiology and climate vulnerability in amphibians[END_REF].

Such nonlinear, threshold effects should trigger more or less rapid behavioural adjustment depending on the shape of hydric performance response curve [START_REF] Edwards | Thirst and drinking in North American watersnakes (Nerodia spp.)[END_REF][START_REF] Lillywhite | Drinking by sea snakes from oceanic freshwater lenses at first rainfall ending seasonal drought[END_REF]. Future studies should therefore examine the strength and shape of the relationships between dehydration and performance capacities for which we currently lack data [START_REF] Rozen-Rechels | When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms[END_REF].

In conclusion, our study emphasizes the importance of air humidity, as well as behavioural hydroregulation, when investigating the effects of climate change on ectotherms.

Microhabitat quality and heterogeneity appear to be essential to support behavioural responses and mitigate deleterious effects of extreme weather events. However, current habitat simplifications associated with human activities are likely to compromise these important components of microhabitat [START_REF] Guiller | Response of farmland reptiles to agricultural intensification: Collapse of the common adder Vipera berus and the western green lizard Lacerta bilineata in a hedgerow landscape[END_REF].

Acknowledgement

Table 1. Summary statistics of the traits measured on asp vipers from each treatment group. Values written in italic are from the Dry air & Wet shelter group when excluding the individual that almost never selected the shelter (see details in main text). Different superscript letters are used to show significant differences between treatment groups for each variable.

  Every day from Day 0 to Day 23, we recorded the position of each individual 3 times per day during daytime (9:00, 13:00 and 17:00). Individual position in the box was categorised as either using the shelter or being outside. First, we considered the cumulative shelter use from Day 0 to Day 2 (proportion of shelter use out of 9 observations) to record the baseline shelter use. To investigate responses to combined air humidity and microclimate treatments (Day 3 to Day 20), we first examined the cumulative shelter use during that period (proportion of shelter use out of 54 observations). Second, we considered more specifically changes in daily shelter use (proportion of shelter use out of the 3 daily observations) over time (Day 0 to Day 20). We also calculated an individual shelter use score over a 3-day period between Day 3 and Day 20 (total number of times inside the shelter per 9 observations, 6 repetitions) to investigate the relation between shelter use behaviour and body mass changes (recorded every 3 days, see below). Finally, we also calculated cumulative shelter use from Day 21 to Day 23 (proportion of shelter use out of 9 observations) to investigate wet shelter use at the end of the treatment.

Figure 1 .

 1 Figure 1. Effects of experimental treatments on behavioural hydroregulation and body mass

Figure 2 .

 2 Figure 2. Additive effects of air humidity and microclimate treatment on changes in tail width

Figure 3 .

 3 Figure 3. Correlation between body mass change (g) and individual-centred shelter use score

Figure 1 .

 1 Figure 1.

Figure 3 .

 3 Figure 3.

The authors thank Rémi Ksas (Venom World) for providing the snakes for this experiment, and for his contribution through his ideas on wet microclimates, as well as Bruno Michaud for logistic support. We thank Andréaz Dupoué for his advices and help with the laboratory analysis. We also thank Sydney Hope for helpful comments on the English of the manuscript.

Declarations

Funding

Funding support was provided by the Agence Nationale de la Recherche under the 'Aquatherm' project (ANR-17-CE02-0013 to J.-F.L.G.), by the Conseil Régional de Nouvelle-Aquitaine under the 'Aquastress' project (2018-1R20214 to O.L.) and by the Climate Sentinels Program 2016 -2021 coordinated by the "Cistude Nature" association (Phd Fanny Mallard, funded by the European Regional Development Fund, the region Nouvelle Aquitaine, the department of Gironde and Pyrénées-Atlantiques). M. D. is currently supported by a doctoral grant from ED 227 Sorbonne University.

Availability of data and material

Data and associated material are available in a public repository at doi:10.5281/zenodo.7118362.

Code availability

Not applicable

Conflict of interest

The authors declare no competing or financial interests.

Ethics approval

All experimentation procedures with animals listed in this study were approved by an independent ethical committee (Apafis#23238-2019121010574211_V3).

Consent to participate

Not applicable

Consent for publication

Not applicable


Dr Michel Nguyen

Dr Youzhi Li

Anne Robert
email: anne.robert@lcc-toulouse.fr

Dr Yan Liu
email: yanliu@gdut.edu.cn

Dr Bernard Meunier
email: bmeunier@lcc-toulouse.fr

Dr A Robert

Oxidation of TDMQ20, a Specific Copper Chelator as Potential Drug Against Alzheimer's Disease

Keywords: Alzheimer's disease, catalytic oxidation, copper chelator, metabolism

Developed as a regulator of copper homeostasis in the brain of patients affected by Alzheimer's disease (AD), TDMQ20 is a specific copper(II) chelator able to restore short term memory in AD mice. To anticipate on metabolic studies of this drug-candidate, here we report the catalytic oxidation of TDMQ20 by a biomimetic system reminiscent of P450 oxidation, using a metalloporphyrin and hydrogen persulfate as oxidant. Oxidation products were mainly characterized by HPLC with UV-visible spectrometry or high-resolution mass spectrometry detection. In these conditions, TDMQ20 was readily oxidized by aromatic hydroxylation, oxidation of 1,4-diphenol or 1,4aminophenol to 1,4-quinone or quinone-imine, respectively, or oxidative chlorination. These putative metabolites were water-soluble, suggesting that the drug-candidate might be easily metabolized and excreted in vivo.

Introduction

With the increase in life expectancy, Alzheimer's disease (AD), age-related disease, is one of the major public health issues in the world. Unfortunately, very few treatments have been approved until recently, [START_REF] Cummings | [END_REF]2] and these drugs are not curative, offering only short-term symptomatic relief, a high level of side effects and questionable efficiency/cost ratios. [3] Then, there is an urgent need to enlarge the drug spectrum with molecules acting by different mechanisms, in order to stop the neurodegenerative process and the cognitive decline at the early stages of the disease. The destruction of neurons in brains affected by AD has been attributed to an oxidative stress mainly caused by rupture of the homeostasis of redox-active metal ions, such as copper and iron, trapped by β-amyloid peptide (Aβ). [4][5][6] Then, we designed specific copper chelators named TDMQ (Scheme 1), able to inhibit the catalytic reduction of dioxygen produced by copperloaded amyloids (Cu-Aβ). [4,[7][8][9] Based on an 8-aminoquinoline skeleton with a chelating side chain at C2, these ligands have the required structure to generate Cu(II)-TDMQ complexes with a tetradentate N4-square planar scaffold. [10] Due to this coordination pattern, TDMQ20 (Scheme 2), exhibits a high chelation specificity for Cu(II) with respect to Cu(I) or to other divalent biologically relevant metal ions such as Fe(II) or Zn(II). [11,12] Due to its high affinity for Cu(II), TDMQ20 is able to transfer copper from Cu-amyloid complexes to the Cu-glutathione complex which is a natural provider of copper to copper proteins, without disturbing the activities of copper proteins such as Cu,Zn-SOD or tyrosinase. [13] Oral administration of TDMQ20 to three different murine models of AD indicated that this specific regulator of copper homeostasis reduced the oxidative stress in mouse cortex, and was able to rescue the declarative memory and to inhibit cognitive impairment of the early stages of AD, without any detectable toxic effect. [14,15] TDMQ20 can therefore be considered as a promising drug-candidate. In this context, to document the possible oxidative metabolism of this drug, we investigated its oxidation by biomimetic systems. a Mn(Por)Cl stands for a metalloporphyrin complex as catalyst, for example Mn(TMPyP)Cl 5 . b The single oxygen atom donor is hydrogen persulfate (HSO 5 -) or meta-chloroperbenzoic acid (mCPBA).

Mn III (Por )Cl = Mn III (TMPyP)Cl 5

Cl

N-dealkylation…

N Cl Cl NH 2 • 3 HCl N H N (a) (b)
The biomimetic oxidation of drugs by redox-active metalloporphyrin catalysts associated with a single oxygen atom donor has been reported to efficiently mimic the biotransformation performed in vivo by cytochromes P450 or peroxidases, two families of major enzymes involved in xenobiotic metabolism. [16- 18] In fact, these catalytic systems can efficiently oxidize a variety of substrates and perform, among other reactions, biologically relevant hydroxylation of saturated structures, oxidation of aromatics, or N-demethylation of substituted amines. We report here the catalytic oxidation of TDMQ20 in such oxidative conditions.

Results and Discussion

The selection of the different metalloporphyrin catalysts and associated oxidants was based on their known abilities to mimic the reactions mediated by cytochrome P450 enzymes. The activation of metal complexes of porphyrins by single oxygen atom donors (hypochlorite, hydrogen persulfate, metachloroperbenzoic acid, magnesium monoperoxyphthalate...), involves a highly electrophilic metal-oxo active species which is reminiscent of cytochrome P450 active species generated in the presence of dioxygen associated with NADPH/P450 reductase. [16] The general equation of these catalytic biomimetic reactions is depicted in Scheme 2. First, the non-catalytic oxidation of base form of TDMQ20 by m-chloroperbenzoic acid (mCPBA) or tetrabutylammonium hydrogen monopersulfate (NBu4HSO5) was carried out, in order to characterize expected N-oxide derivatives of the drug. Then, oxidation of TMDQ20 was carried out using manganese(III) tetramethylpyridiniumylporphyrin, pentachloride [Mn(TMPyP)Cl5, MnTMPyP for short] as catalyst and potassium hydrogen persulfate (KHSO5) as oxidant in a acetonitrile/water mixture. [16] To get a fairly broad overview of the possible metabolites of TDMQ20, various oxidative conditions were explored: different oxidants, aqueous or organic media, variable concentrations of TDMQ20 (in the range 50 M-1 mM) and reaction times, etc. All reactions were monitored by HPLC with UV-visible detection at 260 and 340 nm, and by LC-MS [TDMQ20: tR = 16.8 min, m/z = 327.1 amu (MH + )].

Oxidation of TDMQ20 by mCPBA

In the absence of catalyst, TDMQ20 or its base form were readily oxidized in aqueous acetonitrile or in dichloromethane, respectively. For example, oxidation of TDMQ20 (base form,10 mM) by mCPBA (1.2 molar equiv) in CH2Cl2 resulted in a 99 % conversion after 15 min, and products 1 (tR = 18.6-18.7 min) and 2 (tR = 21.7 min), were characterized by mass increments of +16 amu and +32 amu, respectively (Figure S1). The structure of these products resulting from N-oxidation of the distal tertiary amine or N-oxidation of the two amines of the side chain, respectively, were fully characterized by NMR after chromatographic purification (Schemes 3 and 4). The The UV-visible characteristics of isolated compound 1 were very similar to that ones of TDMQ20, with max = 261 and 345 nm, and 260 = 30,000 L x mol -1 x cm -1 . Consequently, the yields of products 1 and 2 were calculated directly using the UV-vis trace of HPLC chromatograms. In these conditions, after 15 min, the yields of 1 and 2 were 70% and 7%, respectively, with respect to the starting amount of TDMQ20. When TDMQ20 (base form,10 mM) was oxidized by hydrogen persulfate (1 mol equiv, NBu4HSO5) instead of mCPBA in CH2Cl2, the conversion of TDMQ20 was 57% after 10 min. The N-oxide derivative 1 was detected as main product (yield = 52% with respect to starting amount of TDMQ20). The di-N-oxide 2 was also detected in very low yield (≤ 2%). In the presence of an excess of oxidant, (2 mol equiv), compound 2 was no more detected. In these conditions, a minor product 3 was detected but not isolated (tR = 20.3-20.4 min, 2% yield). Its chromatographic peak exhibited maximum wavelengths at 260-261 nm and 345 nm, indicating that the 8-aminoquinoline ring of TDMQ20 was unchanged, and a m/z value = 256.0 amu, that should correspond to oxidative dealkylation of the proximal secondary amine (Scheme 4). UV-vis monitoring of the oxidation of TDMQ20 (50 M) by KHSO5 (4 mol equiv) in aqueous acetonitrile (CH3CN/H2O = 1/1, v/v) confirms that the spectrum of the reaction mixture did not significantly change upon oxidation of TDMQ20 after 1 h (Figure 1). Then, the substrate conversion and yield of products 1-3 reported in Scheme 4 were roughly calculated from the areas of the HPLC peaks upon monitoring of the reaction at 260 nm. The isotope distribution of the three oxidation products 1-3 detected by LC-MS, is consistent with the presence of two chlorine atoms in these products, like in TDMQ20 [intensity ratio of (m/z + 2)/(m/z) = 66/100, see Figure S1]. Oxidation of TDMQ20 (hydrochloride form, 1 mM) by KHSO5 (4 mol equiv) in aqueous acetonitrile (1/1, v/v) in the absence of catalyst, also produced the expected terminal N-oxide 1 as major product (conversion of TDMQ20 = 77%, and yield of 1 = 46% after 1 h). No significant amount of other products was detected.

Oxidation of TDMQ20 by hydrogen persulfate (NBu4HSO5 or KHSO5)

Catalytic oxidation of TDMQ20 by KHSO5 in the presence of MnTMPyP

In a preliminary experiment, the oxidation of TDMQ20•3HCl (50 M) in the presence of a catalytic amount of MnTMPyP (1 mol% with respect to TDMQ20) associated to KHSO5 (2 mol equiv added at 5 min) was monitored by kinetic UV-visible analysis. In these conditions, TDMQ20 (max = 261 nm) was converted in one or several compounds with absorbance at 251 nm (Figure S2a). This significant hypsochromic shift indicated that oxidation was not restricted to the side chain but also occurred on the quinoline moiety. This reaction has reached a plateau after roughly 15 min in these conditions (20 min after recording start Figure S2b), but started again after addition of 2 additional equiv of KHSO5 (Figure 2a). The maximum wavelength was then shifted at 251 nm with a shoulder at 240-242 nm, indicating that the amount of KHSO5 was the limiting factor of the reaction (Figure 2b and2c). The oxidation of TDMQ20 was complete after addition of 2 x 2 mol equiv of KHSO5, and no further change was observed after the addition of a third aliquot of oxidant (Figure 2a). In attempts to isolate the oxidation products of TDMQ20•3HCl by the biomimetic system MnTMPyP/KHSO5, the reaction was carried out at higher concentration (1 mM), and monitored by HPLC with detection by UV-visible (Figure S4) or HRMS (Figure S3). Measured on UV-vis. chromatogram at 260 nm (Figure S4), the conversion of the substrate was very fast, with a substrate conversion being 52% or 97% after only 1 min in the presence of 1 or 4 mol equiv of oxidant, respectively. After 10 min in presence of 2 mol equiv of KHSO5, the conversion of TDMQ20 was nearly complete (Table 1). In these conditions, N-oxidation of the substrate (compounds 1 and 2) was not detected upon monitoring of the reaction by LC-MS. In fact, six new peaks of this biomimetic oxidation (4-9) were detected, with retention times ranging from 7.0 to 21.0 min (Figure S4). Consistently with the detection of welldefined isosbestic points at 251, 274 and 330 nm in Figure 2b, UV-visible spectra of all these products were similar (Figure S4), suggesting the formation of several products having similar aromatic moieties. This allowed to calculate their respective proportions according to the reaction time (1 min, 10 min or 1 h) and the amount of oxidant (1 or 4 mol equiv with respect to TDMQ20). Results are reported in Table 1 (and Figure S5) and suggest that both competitive and consecutive reactions occurred in these biomimetic conditions. The identification of compounds 4-9 responsible for the most part of these HPLC peaks was carried out using ESI + -HRMS (Figure S3). In fact, consistency of HRMS, associated to the specific isotopic patterns of compounds containing one to 5 chlorine atoms, allowed unambiguous determination of the molecular formulas of compounds 4-9. Then, owing to the already reported oxidative reactivity of metalloporphyrins towards aniline and phenol derivatives, reasonable structures could be assigned to these products. The first series of oxidation products corresponds to monoquinoline derivatives 4a-4d, 5 and 6 (Scheme 5). These compounds result from one-electron oxidation of the 8aminoquinoline ring by the high-valent metalloporphyrin catalyst, leading to a radical cation species. Then, nucleophilic substitution by HO -leads to hydroquinone B, Scheme 6 (compounds 4b, 4c, Scheme 5), through 1,4-chlorophenol derivative A, Scheme 6, and followed by oxidation of hydroquinone B to 1,4-benzoquinone C (4a, 4d and 6, Scheme 5). Oxidative chlorination (structure D, Scheme 6; 4d, 5 and 6, Scheme 5) or aromatic hydroxylation (structure E, Scheme 6; 4b, Scheme 5) also occurred, probably at C4 which is expected to be the more electrophilic position of the quinoline ring (however, the formation of several position isomers cannot be unambiguously ruled out). The metal catalyzed oxidation of aniline was previously reported to generate 1,4aminophenol, hydroquinone and p-benzoquinone. [19] The oxidative dechlorination at C5, the para position of NH2, is reminiscent from the first step of oxidative degradation of 2,4,6trichlorophenol by similar catalytic systems, [20] while oxidative halogenation by biomimetic catalysts has been already reported. [21] In addition, the 2-vinyl derivative 4d (Scheme 5) was detected. This compound may result from catalytic hydroxylation of the highly oxidizable benzylic position (Quin-C2-CH2; F, Scheme 6), followed by dehydration either in the reaction mixture or under mass spectrometry conditions. x Table 1. Oxidation of TDMQ20 in aqueous acetonitrile, in presence of MnTMPyP associated to KHSO5 according to the reaction time and the number of molar equiv of KHSO5.

Number of mol equiv. of KHSO5

Conversion of TDMQ20 [a] Proportions [b] of chromatographic peaks at 100% nd [c] nd [c] 37% nd [c] nd [c] 50%

Rt

[a] Calculated with respect to starting amount of TDMQ20, on the basis of HPLC peaks areas at 260 nm; [b] Calculated as (amount of peak n)/ amounts of product peaks x 100, based on areas of HPLC peaks at 260 nm (Figure S4). [c] No significant amount (< 1%). A second series of oxidation products 7-9 results from the nucleophilic attack of the aniline moiety of TDMQ20 on one of the carbonyl functions of the 5,8-benzoquinone ring resulting from the two-electron oxidation of quinoline ring (C, Scheme 6). This reaction yields a series of TDMQ20 dimers linked by a 5,8'-or 8,8'-imine function, and bearing three chlorine substituents (H and H', respectively, Scheme 6, compound 7, Scheme 5).

The dimerization/polymerization of p-aminophenol derivatives in the presence of manganese porphyrins associated to KHSO5, was previously reported. [22] The concomitant oxidative chlorination of 7 yielded 8 and 9. Note that the position of the chlorine atom at C4 and/or C4' is a proposal consistent with the expected reactivity of this position trans of the pyridine nitrogen. However, other possible regio-isomers cannot be excluded. The loss of HCl from chlorinated aromatics 8 and 9 in electrospray mass spectrometry generated compounds 10 and 11, respectively. [23] Noteworthy, all these dimeric quinone-imine derivatives exhibit similar UV-vis spectra with absorbances at 247-250 nm, 280 nm, 316-317 nm, and a broad band at 497-500 nm (Figure S4). This latter one is in the range reported for a quinone-imine structure. [24] The various involved reactions (aromatic hydroxylation, oxidation of 1,4-diphenol or 1,4-aminophenol to 1,4-quinone or 1,4-quinone-imine, respectively, oxidative chlorination) are usually performed by P450 enzymes and biomimetic catalytic systems.

Faced to complex reaction mixtures, we tried to isolate the two more abundant products obtained in the presence of 4 mol equiv of KHSO5, in order to characterize them by NMR. After 1 h of reaction time and elimination of the excess of KHSO5 by addition of sodium dithionite (Na2S2O4, 4 mol equiv with respect to TDMQ20), the reaction mixture was extracted with dichloromethane, and precipitated by addition of hexane. Alternatively, the reaction mixture was submitted to preparative HPLC. However, in both conditions, products were labile upon work-up conditions. One can notice that the aniline function of TDMQ20 disappeared in all the oxidation products that have been characterized. Since this amine function was essential in the specific chelation of Cu(II) by TDMQ20, [7,10] the affinity of oxidized derivatives 4-9 for Cu might be significantly lower than that of TDMQ20 itself. However, pharmacokinetic studies indicated that, after oral administration of TDMQ20 to rat, bioavailability and brain penetration brain were very high. [25] Conclusion TDMQ20 hydrochloride is easily oxidized by a catalytic system mimicking cytochrome P450 and the resulting oxidation products 4-9 are fully soluble in aqueous experimental conditions. These data suggest that the drug-candidate TDMQ20 will be metabolized in vivo avoiding a possible accumulation in tissues. The isolated distal N-oxide derivative 1 was also highly soluble in aqueous medium at physiological pH. All these data collected on the oxidation products of TDMQ20 might be useful for the identification of the drugcandidate metabolites in future in vivo studies.

Experimental Section

Materials and methods.

All solvents and commercially available reagents were purchased from Sigma-Aldrich, Fluka, Acros or Carlo Erba, and used without further purification. Reactions were monitored by thin-layer-chromatography (TLC) by using neutral aluminum oxide plates (Merck). Aqueous solutions were obtained using ultrapure milliQ water. Depending on solvent conditions, two sources of oxone (hydrogen persulfate, named also monopersulfate, HSO5 -) salt were used for oxidation reactions.

Oxone tetrabutylammonium salt [triple salt of 2NBu4HSO5•NBu4HSO4•(NBu4)2SO4] for reactions in dichloromethane and oxone potassium salt [triple salt 2KHSO5•KHSO4•K2SO4] for reactions in water/acetonitrile. Mn(TMPyP)Cl5 (MnTMPyP for short) was obtained from Enzo. 1 H and 13 C NMR spectra were recorded on Bruker Avance 600 and Avance 400 spectrometers using tetramethylsilane (TMS) as the external standard. UV-visible conditions. UV/Visible spectra were recorded from 200 to 800 nm on an Agilent Cary 3500 spectrophotometer equipped with magnetic stirrer. For kinetic experiments, spectra were acquired per cycle time of 15 sec or 1 min, under reaction time collection of 1 h. HPLC conditions. HPLC analyses were carried out using an Agilent 1200 system equipped with a diode array detector and a Waters XBridge C18 column (4.6 x 150 mm, 3.5 μm), eluted with a linear gradient methanol/water/formic acid from 5/95/0.1, v/v/v, to 95/5/0.1, v/v/v, in 24 min; flow rate: 0.5 mL/min, UV detection: 260 and 340 nm. LC-MS conditions. LC-MS analyses were performed on a Thermo Scientific LCQ Fleet or Waters Xevo-G2QTOF mass spectrometer in positive ionization mode (ESI + ) with the same column under the same gradient conditions as applied for HPLC analysis (see above). The spectra were acquired in m/z range 200-1200 or 200-2000 amu; flow rate was 0.5 mL/min. Preparative HPLC conditions. Purifications were carried out using an autopurification Water 2767 semi-preparative HPLC system equipped with a Waters 2998 photodiode array detector and a Waters XBridge C18 column (19 x 150 mm, 5 μm), eluted with a linear gradient methanol/water/formic acid from 5/95/0.1, v/v/v to 100/0/0.1, v/v/v, in 36 min; flow rate: 20 mL/min, UV detection: 260 nm. Neutralization of TDMQ20 TDMQ20 free base was obtained by dissolving the TDMQ20•3HCl salt (202 mg, 0.45 mmol) in 30 mL of 5% NaHCO3 aqueous solution, followed by extraction with dichloromethane (3 x 30 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to afford the free base as yellow oil (146 mg, 99 % yield). [START_REF] Cummings | [END_REF] H NMR (600 MHz, CDCl3): δ (ppm) 8.36 (d, 3 J = 8.6 Hz, 1H), 7.46 (s, 1H), 7.39 (d, 3 J = 8.6 Hz, 1H), 3.24 (t, 3 J = 6 Hz, 2H), 3.19 (t, 3 J = 6 Hz, 2H), 2.82 (t, 3 J = 6 Hz, 2H), 2.50 (t, 3 J = 6 Hz, 2H), 2.25 (s, 6H). 13 Oxidation of TDMQ20 by mCPBA or NBu4HSO5 in CH2Cl2 in the absence of catalyst Oxidation of TDMQ20 was carried out by reacting TDMQ20 base (2.78 mg, 8.5 µmol) with 1.2 mole equiv of mCPBA (10.2 µmol) in dichloromethane (850 µL, initial [TDMQ20] = 10 mM), at room temperature. Reaction progress was monitored by TLC on aluminum oxide plates eluted with CH2Cl2/4M NH3 in MeOH (9/1). Alternatively, the reaction was performed with oxone tetrabutylammonium in dichloromethane. Two molar equiv of NBu4HSO5 (16.2 µmol) were reacted with TDMQ20 base (2.65 mg, 8.1 µmol) in dichloromethane (810 µL) at room temperature. After 45 min of stirring, full conversion of the substrate was checked by TLC, and the mixture was analyzed by HPLC and LC-MS. Product yields were determined by comparison with TDMQ20 relative absorbance obtained by HPLC with UV detection at 260 nm.

Preparation of TDMQ20 N-oxide derivatives 1 and 2

To a solution of TDMQ20 free base (119 mg, 0.36 mmol) in dichloromethane (7 mL) at 0°C was added 1.3 equiv of mCPBA (0.47 mmol). The mixture was stirred at room temperature for 1 h and, then, evaporated under vacuum. The crude yellow residue obtained was purified by chromatography over a neutral alumina column (dichloromethane/4 M NH3 in MeOH, from 100/0 to 90/10, v/v) to afford the N-oxide derivative 1 as a yellow oil (81 mg, 65% yield). [START_REF] Cummings | [END_REF] To a cuvette containing TDMQ20•3HCl (15 µL of a 5 mM stock solution, 50 µM in the cuvette, 1 equiv) in presence of MnTMPyP (0.8 µL of a 1 mM stock solution, 0.5 µM in the cuvette, 1 mol% with respect to TDMQ20) in water/acetonitrile, 1/1, v/v, under stirring at room temperature, was added 2 mol equiv or 4 mole equiv of KHSO5 (10 µL of 0.015 M or 0.03 M stock solutions, 100 µM or 200 µM in the cuvette, corresponding to 2 mol equiv or 4 mol equiv, respectively), 5 min after the start of the run. Final volume in the cuvette was 1.5 mL. All stock solutions were prepared in water. UV-visible spectra were recorded as a function of time (cycle time 15 sec, time collection 1 h) from 200 to 800 nm. Similar experiments were also performed with all components except MnTMPyP.

Kinetic oxidation of TDMQ20 by MnTMPyP/KHSO5 with sequential addition of KHSO5

To a cuvette containing TDMQ20•3HCl (15 µL of a 5 mM stock solution, 50 µM in the cuvette, 1 equiv) and MnTMPyP (0.8 µL of a 1 mM stock solution, 0.5 µM in the cuvette, 1 mol% with respect to TDMQ20) in H2O/CH3CN, 1/1, v/v, under stirring at room temperature, was added KHSO 5 in three portions (3 x 5 µL of a 0.03 M stock solution, 3 x 100 µM in the cuvette, 3 x 2 equiv), at 5 min, 20 min and 35 min after the start of the run (15 min between each addition). Final volume in the cuvette was 1.5 mL. All stock solutions were prepared in water. UVvisible spectra were recorded as a function of time (cycle time 1 min, time collection 1 h) from 200 to 800 nm. Oxidation of TDMQ20 by MnTMPyP/KHSO5 or by KHSO5 alone in H2O/CH3CN, 1/1, v/v To a solution TDMQ20•3HCl (0.25 µmol, 1 mM, 1 equiv) in presence of MnTMPyP (2.5 nmol, 10 µM, 1 mol % with respect to TDMQ20) in H2O/CH3CN, 1/1, v/v, was added 1 equiv, 2 equiv or 4 equiv of KHSO5 (0.25 µmol = 1 mM, 0.50 µmol = 2 mM or 1 µmol = 4 mM). Final volume was 250 µL. Reaction mixture was stirred at room temperature. Stock solutions were prepared in water. After 1 min, 10 min or 1 hour, the mixture was analyzed by HPLC and LC-MS. Experiments without catalyst were also performed in the same conditions. Table 2. Matching of retention times of the oxidation products of TDMQ20 upon detection in an Agilent 1200 system (detection UV-vis.), and in a Waters Xevo-G2QTOF (detection HRMS). Aromatic oxidation, followed by dimerization

Matching of Rt

Scheme 1 .Scheme 2 .

 12 Scheme 1. General structure of the Cu(II) specific TDMQ chelators.

Scheme 4 .

 4 Scheme 4. Oxidation of TDMQ20 by tetrabutylammonium hydrogenpersulfate or by m-chloroperbenzoic acid in CH2Cl2, in the absence of catalyst, after 15 min.

Figure 1 .

 1 Figure 1. (a) UV-visible kinetic spectra of oxidation of TDMQ20 (50 M) by KHSO5 4 mol equiv in H2O/CH3CN, 1/1, v/v; (b) Absorbance at 261 nm according to the reaction time after addition of KHSO5.

Figure 2 .

 2 Figure 2. (a)Absorbance at 261 nm and 345 nm according to the reaction time upon three successive additions of KHSO5 (2 mol equiv each) at 5 min, 20 min and 35 min; Spectra at t = 0, 18 min, 32 min and 50 min (red crosses) are depicted in Figure2c; (b) UV-visible kinetic spectra of the catalytic oxidation of TDMQ20 (50 M) by three successive additions of KHSO5 2 mol equiv in the presence of MnTMPyP 1 mol% in H2O/CH3CN, 1/1, v/v; time interval between two successive spectra was 1 min; (c) Spectra at t = 0 (blue), 18 min (orange), 32 min (grey) and 50 min (yellow). Absorbance at 462 nm is due to MnTMPyP catalyst.

Scheme 5 . 9 Scheme 6 .

 596 Scheme 5. Proposed structures of the products resulting from oxidation of TDMQ20 in the presence of 1 mol% of MnTMPyP and 1 mol equiv of KHSO5 in H2O/CH3CN, 1/1, v/v, 1 h, RT. All compounds were identified by LC-ESI + -HRMS, except 8a and 8b that were identified by ESI + -low resolution MS. Calculated exact mass values and experimental detected m/z vaIues are provided.

  C NMR (151 MHz, CDCl3): δ (ppm) 159.44 (Quin-C2), 139.65 (Quin-C8), 137.88 (Quin-C9), 133.45 (Quin-C4), 126.73 (Quin-C6), 123.74 (Quin-C10), 122.69 (Quin-C3), 117.64 (Quin-C5), 113.64 (Quin-C7), 58.68 [CH2-N(CH3)2], 48.56 (Quin-CH2-CH2-N), 47.05 (NH-CH2-CH2-N), 45.63 [N(CH3)2], 37.84 (Quin-CH2). HRMS (ESI+) calcd for C15H21N4Cl2 (M+H + ) 327.1143, found: 327.1145.

  H NMR (600 MHz, CDCl3): δ (ppm) 8.34 (d, 3 J = 8.6 Hz, 1H, H4), 7.44 (s, 1H, H6), 7.37 (d, 3 J = 8.6 Hz, 1H, H3), 3.45 [t, 3 J = 5.4 Hz, 2H, (CH2-N(O)(CH3)2)], 3.27-3.22 [m, 8H, N(O)-(CH3)2) + NH-CH2], 3.22-3.17

  [m, 4H, (Quin-CH2-CH2-N)].

.8 55.57 Dd = +8.5 69.00 Dd = +10.3 58.78

		Cl		8.36
	7.46 126.73 117.64 123.74	133.45	7.39 122.69
	Cl 113.64	NH 2 139.65	N	3.24 159.44 37.84	3.19 48.56	N H	2.82 47.05 58.68 2.50	N	2.25 45.63
		Cl
	7.44 126.72 117.59 123.71 NH 2 Cl 139.85 113.71 138.01 8.34 133.37 N 159.14 38.11 7.37 3.21 122.63 48.64 3.19 Dd = +0.1 N H	3.24 Dd = +0.42 Dd = +0.95 3.45 44.80 Dd = +10.9 69.60 Dd = -2.3	O N	3.24 Dd = +0.99 Dd = +14.1 59.77
		Cl
				8.32
	7.43 126.54 117.83 113.50	133.16 137.86 123.63	7.47 123.16	3.25 59.39		3.24 Dd = +0.42	N	3.27 Dd = +1.02
	Cl	NH 2 139.48	N 159.79	3.33 36.42		N H	3.63 Dd = +1.13	O	Dd = +13.2
								O

Dd = +10

for the Table of Contents TDMQ20

  , a drug-candidate against Alzheimer's disease, is easily oxidized in biomimetic conditions, suggesting that it should be readily metabolized in vivo, and that potentially deleterious accumulation in tissues should be avoided.

	Entry
				[min]
	Upon
	detection					Depicted in
	by
	LC-UV	7.0-7.3	12.5	14.0 15.4 17.5-17.7	21.0	Table 1 Figure S4
	LC-HRMS	5.4-5.6	10.7-11.0	12.1 13.5 15.6-15.8	19.1	Scheme S5 Figure S3

Acknowledgements and funding sources

This work has been supported by the CNRS, France, and by the University of Technology of Guangdong, China. Nathalie Martins (Mass spectrometry facilities of the Institute of chemistry of Toulouse, ICT, UAR 2599), and Isabelle Fabing (UMR 5068 CNRS-University of Toulouse) are acknowledged for help with LC-MS analyses and preparative HPLC, respectively.

Supporting Information Summary

Supporting Information contains Figure S1: Oxidation of TDMQ20 by mCPBA 1.2 mol equiv, 15 min. LC-MS monitoring with UV-vis detection at 260 nm. Figure S2: UV-visible kinetic spectra of the catalytic oxidation of TDMQ20 (50 μM) by KHSO5 2 mol equiv in the presence of MnTMPyP 1 mol% in H2O/CH3CN, 1/1, v/v. Figure S3: Catalytic Oxidation of TDMQ20 by Mn(TMPyP)Cl5 (1 mol%) associated to KHSO5 (2 mol equiv). LC-ESI + -HRMS for compounds 4-9 after 1 h of reaction. Figure S4: Catalytic Oxidation of TDMQ20 by Mn(TMPyP)Cl5 (1 mol%) associated to KHSO5 (2 mol equiv). LC-UV-vis. for compounds 4-9 after 1 h of reaction. Figure S5: Catalytic Oxidation of TDMQ20 by Mn(TMPyP)Cl5 (1 mol%) associated to KHSO5. Quantification of reaction products on the basis of HPLC chromatograms with detection at 260 nm.