Joanna Charton
email: joanna.charton@gmail.com

Vincent Jomelli

Irene Schimmelpfennig

Deborah Verfaillie

Vincent Favier

Fatima Mokadem

Adrien Gilbert

Fanny Brun

Georges Aumaitre

Didier L Bourlès

Georges Aumaître

Karim Keddadouche

A debris-covered glacier at Kerguelen (49°S, 69°E) over the past 15 000 years

Keywords: glacier fluctuations, in situ cosmogenic chlorine-36 dating, paleoclimate, Antarctic Cold Reversal, Holocene, sub-Antarctic

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Introduction

Debris-covered glaciers exhibit a debris-mantled glacial tongue, with rock debris ranging from a few centimetres to several metres in thickness [START_REF] Mayr | Debris-Covered Glaciers[END_REF]. This type of glaciers constitutes a substantial proportion of the glaciers worldwideboth in the Northern and Southern Hemispheres [START_REF] Scherler | Global assessment of supraglacial debris-cover extents[END_REF]. Therefore, several recent investigations have been conducted on debris-covered glaciers in order to document their surface changes [START_REF] Vincent | Reduced melt on debris-covered glaciers: Investigations from Changri Nup Glacier, Nepal[END_REF][START_REF] Brun | Ice cliff contribution to the tongue-wide ablation of Changri Nup Glacier, Nepal, central Himalaya[END_REF], and the morphological [START_REF] Salerno | Debris-covered glacier anomaly? Morphological factors controlling changes in the mass balance, surface area, terminus position, and snow line altitude of Himalayan glaciers[END_REF][START_REF] Brun | Heterogeneous influence of glacier morphology on the mass balance variability in High Mountain Asia[END_REF]) and climatic factors [START_REF] Ojha | Topographic controls on the debris-cover extent of glaciers in the Eastern Himalayas: Regional analysis using a novel high-resolution glacier inventory[END_REF] controlling their spatial distribution and response to climate fluctuations. It has been previously suggested that debriscovered glaciers are less sensitive to climatic variation than debris-free glaciers, owing to the thickness of the debris cover, which plays an isolating role and protects the ice from melting by absorbing incoming radiation [START_REF] Mayr | Debris-Covered Glaciers[END_REF]. However, recent studies on Himalayan glaciers have shown a similar thinning rate between debris-free and debriscovered glaciers facing regional pluriannual climatic variations [START_REF] Kääb | Contrasting patterns of early twenty-first-century glacier mass change in the Himalayas[END_REF][START_REF] Vincent | Reduced melt on debris-covered glaciers: Investigations from Changri Nup Glacier, Nepal[END_REF][START_REF] Brun | Ice cliff contribution to the tongue-wide ablation of Changri Nup Glacier, Nepal, central Himalaya[END_REF][START_REF] Brun | Heterogeneous influence of glacier morphology on the mass balance variability in High Mountain Asia[END_REF].

However, little is known about differences in the glacier responses to temperature and precipitation changes over longer time scales. This issue has rarely been investigated, and the sites of all known studies are located in the Northern Hemisphere and subject to alpine climate conditions. To our knowledge, so far only two studies using cosmic ray exposure (CRE) dating of supraglacial debris were specifically undertaken to constrain the Late Glacial and Holocene history of a fossil debris-covered glacier in Spain [START_REF] Fernández-Fernández | Chronological and geomorphological investigation of fossil debris-covered glaciers in relation to deglaciation processes: A case study in the Sierra de La Demanda, northern Spain[END_REF]) and of debris-covered glacier -rock glacier continua in Northern Iceland [START_REF] Fernández-Fernández | Constraints on the timing of debris-covered and rock glaciers: An exploratory case study in the Hólar area, northern Iceland[END_REF]. Debris-covered glaciers on the Kerguelen Archipelago in the sub-Antarctic region offer an excellent opportunity to document their evolution related to longterm regional and global climate changes. Moreover, recent chronologies of debris-free glaciers are available in different glacial valleys of the archipelago [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF]. This work presents 13 new 36 Cl cosmic-ray exposure ages from boulders collected on two sets of frontal morainic ridges (G1, G2) from the Gentil Glacier, a debriscovered glacier located in the south of the Kerguelen Archipelago. We then compare the evolution of Gentil Glacier to the evolution of the nearby debris-free glaciers investigated in previous studies [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF].

Study area

The French Kerguelen Archipelago is located in the southern Indian Ocean (49°S) and constitutes a 40-million-year-old part of an underwater basaltic plateau that has emerged. The archipelago is of major regional interest because it is the largest above-water glaciated area of the sub-Antarctic (552 km 2 ; Berthier et al. 2009) It is one of the few places in the Indian Ocean where glacial records are available, besides Heard Island (e.g. [START_REF] Thost | Glacier recession on Heard Island, Southern Indian Ocean[END_REF] and the recently investigated Marion Islands [START_REF] Rudolph | Early glacial maximum and deglaciation at sub-Antarctic Marion Island from cosmogenic 36 Cl exposure dating[END_REF]. The Cook Ice Cap (CIC, 410 km 2 in 2001) is the largest glaciated area on the Kerguelen Archipelago with marine and terrestrial termini. It deposited several glacial moraines on the eastern part of the main island, called Grande Terre, which were previously investigated [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF]. Smaller debris-free mountain glaciers are also present in different sectors of the archipelago: on the Rallier du Baty Peninsula, in the Presqu'île de la Société de Géographie and on the Gallieni Peninsula (Fig. 1).

Kerguelen has an oceanic subpolar climate, characterized by a low seasonality in temperatures and precipitations. The annual mean temperature is about 4.5°C and precipitation is ⁓ 800 mm per year on the eastern side of the archipelago, whereas the western side receives up to 4000 mm [START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF][START_REF] Verfaillie | Regional modeling of surface mass balance on the Cook Ice Cap[END_REF]. A permanent weather station has been continuously maintained since 1951 at the scientific station based at Portaux-Français on the Coubert Peninsula.

The debris-covered Gentil Glacier is an active glacier on the Gallieni Peninsula, which is located in the south of the main island and dominated by the Mount Ross (1850m a.s.l.), the highest peak of the archipelago (Fig. 1). The Mount Ross is a strato-volcano in which the caldera formed a glacial cirque from where the Buffon Glacier flows down on its western slope and reaches the sea (Fig. 1b). The Gentil Glacier constitutes a glacial lobe of the Buffon Glacier and is covered by debris over its entire ablation area. Glacier cross sections exposed at the numerous ice cliffs (Figs. 2a & 3c) indicate debris thicknesses ranging from about 20 cm up to 50-100 cm. Debris cover on the glacier originates from rockslides along the cirque headwalls, avalanches and subglacial bedrock erosion (Fig. 2a). In addition, supraglacial debris is laterally supplied from the catchment slopes and lateral moraines during the downstream flow of the glacier. As a result, the glacier transports subglacial and supraglacial debris, which are concentrated by the glacial flow into discrete subparallel lateral and frontal layered ridges atop the glacier (Figs. 2 & 3a). At the glacier's snout, the debris bulges isolate the subjacent ice and then form ice-cored moraines that are concentrically deposited following the delineation of the glacier (also called "controlled moraine", e.g. [START_REF] Evans | Controlled moraines: origins, characteristics and palaeoglaciological implications[END_REF]). Three geomorphological features can be distinguished (Fig. 2), (i) an active part, which is still fed by ice flow (where the investigated G2 moraine set is located); (ii) an inactive part, which is characterized by an ice-cored moraine layer that does not seem to be connected with the ice flow anymore (pink swath on Fig. 2); and (iii) the fossil part, where the ice has melted completely and only vegetated glacial moraine deposits remain (where the investigated G1 moraine set is located). In both active and inactive areas, the debris mantle surface is hilly and characterized by numerous ice cliffs and supraglacial lakes (Figs. 3a &c). The fossil part is characterized by a near continuous, hummocky rock pavement very similar to the current detrital cover of the glacier (Fig. 3b). Therefore, we assume that the glacier was already covered during one of its last advances and that this rocky pavement corresponds to the former debris-cover.

Former studies on Kerguelen glaciers

Recent investigations conducted on Kerguelen glaciers have shown a significant retreat of debris-free glaciers on the archipelago in recent decades, in particular for the Cook Ice Cap (CIC), which lost 20% of its surface from 1963 to 2001 [START_REF] Berthier | Ice wastage on the Kerguelen Islands (49°S, 69°E) between 1963 and 2006[END_REF]. Direct surface mass balance and energy balance measurements combined with satellite image analyses revealed an even more drastic shrinkage over the last decade [START_REF] Verfaillie | Recent glacier decline in the Kerguelen Islands (49°S, 69°E) derived from modeling, field observations, and satellite data[END_REF][START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]. The current CIC wastage is mainly attributed to an atmospheric drying since 1960 caused by a shift of the storm track (positive phase of the Southern Annular Mode (SAM); [START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF] due to the greenhouse gases concentration increase and the Antarctic ozone hole [START_REF] Thompson | Signatures of the Antarctic ozone hole in Southern Hemisphere surface climate change[END_REF].

In addition, long-term paleo glacier changes have been investigated through 36 Cl dating. At ~40 ka ago, i.e. during Marine Isotopic Stage 3 (MIS 3), glaciers covered a greater part of the archipelago and 36 Cl chronologies obtained from moraines and erratic boulders revealed that CIC outlet glaciers were generally retreating from ~ 40 ka to ~ 15 ka ago [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF]. This indirectly implies that Kerguelen glaciers might have experienced an early local Last Glacial Maximum during MIS 3 in agreement with recent findings on Marion Island [START_REF] Rudolph | Early glacial maximum and deglaciation at sub-Antarctic Marion Island from cosmogenic 36 Cl exposure dating[END_REF]. The deglaciation trend was interrupted ~ 15-12 ka ago in at least two locations on the archipelago, probably during the Antarctic Cold Reversal (ACR; [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF]. One of the locations revealing a glacial advance or stillstand at this time is the Bontemps moraine dated at 13.6 ± 1.5 ka. It is located 26 km from the current ice margin of the Explorateur Glacier on the eastern slope of CIC (Fig. 1). The other location is the Belvedere moraine situated at 2.7 km from the watershed crest and dated at 15.5 ± 1.8 ka (Fig. 1).

There is little evidence of glacial fluctuations during the Holocene period apart from radiocarbon ages [START_REF] Frenot | Datation de quelques sédiments tourbeux holocènes et oscillations glaciaires aux îles Kerguelen[END_REF]) and 36 Cl ages from the Ampere Glacier proglacial margin [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF], located on the southern slope of CIC (Fig. 1). The current glacial retreat of the Ampere Glacier uncovered peat bogs upstream of the Ampere Lake, from which samples were radiocarbon dated to the early, mid and late Holocene, likely attesting to the presence of smaller glaciers during most of the Holocene period than during the last millennium [START_REF] Frenot | Datation de quelques sédiments tourbeux holocènes et oscillations glaciaires aux îles Kerguelen[END_REF]. During this time, the Ampere Glacier experienced a last advance, which overlaid the peatland and formed a series of moraines, including one dated to 1310 ± 320 CE [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF].

Methods

Sampling

Fieldwork was conducted in January 2012 in the Radioleine glacial valley, on the eastern slope of Mount Ross (Fig. 1b). Thirteen samples were collected on moraine boulders from two groups of moraines (G1 and G2; Fig. 2 & 3) that were identified as Gentil Glacier's morainic deposits. Since the Gentil Glacier is a debris-covered glacier, morainic deposits originate from supraglacial and subglacial rock materials, which are transported atop the ice by the ice flow [START_REF] Evans | Controlled moraines: origins, characteristics and palaeoglaciological implications[END_REF]. Thus, boulders that were sampled for 36 Cl dating might already have been exposed at the surface for an unknown duration of time before the moraine formation. Moreover, chaotic topography at the glacier surface and heterogeneous rock debris thickness may cause a variation in the exposure to the cosmic ray flux due to the possible rotation of the boulders or burying, as observed on the active part of the glacier. In addition, moraines that were deposited at the glacier terminus -both at the active or inactive areas -are still ice-cored. Therefore, further ice movements and melting processes have certainly occurred locally, even after moraine formation. Thus, each boulder has its own exposure and stability history [START_REF] Heyman | Too young or too old: Evaluating cosmogenic exposure dating based on an analysis of compiled boulder exposure ages[END_REF]. The specific character of these glacial moraines has several implications that differ from the investigation of debris-free glacier moraines: i). Moraine ridges on the still active part of the glacier indicate that the glacier advanced at some time in the past, but these ridges are not yet stabilized, because after the glacier advance the insulating debris cover prevents the ice from melting for several millennia; ii). Some variability in the 36 Cl ages may be observed from a single landform due to boulder pre-exposure during supraglacial transport and post-depositional remobilisation of earlier exposed boulders (nuclide inheritance), leading to overestimated 36 Cl ages. Moreover, melting of the ice-cored moraine may cause post-depositional rotation or gradual exhumation of boulders that can stretch over several centuries or millennia causing underestimated 36 Cl ages;

iii). Each ridge does not necessarily correspond to the front of the glacier at a specific time, i.e. several parallel ridges might have formed during the same glacial advance.

Samples were, therefore, taken from both the fossil and the active parts of the glacial debris. Each of the two moraine groups sampled in this study is composed of several nearby moraine ridges, which we assume to be formed during the same glacier advance period. We targeted the moraine boulders located on the top of the moraine ridges thus minimizing the risk of post-depositional rotation and exhumation. The samples were collected with a hammer and a chisel from the uppermost ~ 3-4 cm of flat basaltic boulders, avoiding, as much as possible, edges or eroding surfaces (Fig. 4). A clinometer was used to determine topographic shielding and a handheld GPS device to record geographic coordinates and elevations, which are shown in Table I.

Due to the difficulty of collecting samples and transporting them to the scientific base on Kerguelen without helicopter, a limited number of samples were collected. Ten samples were taken from the G1 moraine group located on the fossil moraine deposit at a distance of around 0.7 and 1.5 km downstream from the present-day frontal area. G1 is formed by ~ 8 moraine ridges, 4 of which were sampled for dating. Ker-56 and -57 were sampled on the most distal moraine ridge (G1a) at ~ 402 m a.s.l. Ker-47, -49, -50, -58, -65, and -66 were extracted from boulders on 2 moraine ridges (G1b and G1c) bulged close to the G1a moraine ridge, at the average altitude of 341 m a.s.l. and 207 m a.s.l., respectively. Ker-67, -68 were collected on the G1 innermost moraine ridge (G1d) at 187 m a.s.l.

Three samples, which are Ker-42, -44, -45, were taken on the two ridges (G2a and G2b) that form the G2 moraine group located on the active moraine deposit (current ice front), at an average altitude of 268 m a.s.l.

Field observations were also performed upstream of G1 and G2 in order to find evidence of pumice that would have been deposited at the beginning of the last millennium due to the Allouarn volcanic eruption [START_REF] Arnaud | Extensive lake sediment coring survey on Sub-Antarctic Indian Ocean Kerguelen Archipelago (French Austral and Antarctic Lands)[END_REF].

In situ-produced 36 Cl cosmic ray exposure dating Basaltic whole rock samples were processed and measured at CEREGE, Aix-en-Provence, France, for in situ-produced 36 Cl cosmic ray exposure (CRE) dating, following the same methods described in [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF]. Age calculation methods and parameters are also the same as in these previous studies at Kerguelen. For completeness, details and the according references are presented in the online Supplemental Material 1 together with the compositional data used for the age calculations (Tables S1 I & S1 II). The resulting individual ages are presented in Table II with their 1 σ uncertainties, which take into account the full propagation of all uncertainties (analytical and production rate uncertainties). Analytical uncertainties only are also reported in Table II. In order to facilitate internal comparison between the ages, the individual ages are indicated with their analytical uncertainties in the text and on the figures. Arithmetic means are mentioned with their full uncertainties to allow for comparison with other paleorecords. This full error results for each age population from the calculation of the standard deviation added by quadrature propagation to the average analytical and production rate uncertainties. Each statistical population was tested using a Chi 2 test, thus enabling the identification of potential outliers.

During sampling, surfaces without direct evidence of frost, salt or wind impacts were preferred, since no quantitative erosion data are available for Kerguelen. A snow cover correction did not seem necessary, due to glaciological model showing a short modern annual duration of snow cover (∼1.5 months at 90 m altitude and ∼3 weeks at 35 m altitude [START_REF] Verfaillie | Recent glacier decline in the Kerguelen Islands (49°S, 69°E) derived from modeling, field observations, and satellite data[END_REF][START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]).

Results

36

Cl CRE ages span from 1760 ± 540 to 17500 ± 2200 years. These ages are distributed among two statistically different populations corresponding to the two sets of moraines G1 and G2 on which the samples were collected (Fig. 2 &7).

Cl CRE ages from G1 moraine boulders (n = 10)

The first outer and older group G1 is composed of several moraine ridges within up to 300 m of distance from each other. Ker-56, -57 were sampled on the external moraine ridge G1a located around 1.5 km from the current ice front and are respectively dated to 11.8 ± 1.5 and 30.1 ± 2.9 ka. The 36 Cl CRE ages of Ker-47, -49, -50, -58, -65, -66, collected on 2 moraines ridges G1b and G1c close to the external moraine, are 15.4 ± 1.7, 12.9 ± 1.3, 12.8 ± 1.5, 12.7 ± 3.7, 12.5 ± 1.7 and 16.2 ± 1.9 ka. Ker-67, -68 were extracted from boulders on the inner ridge G1d and are respectively dated to 17.5 ± 2.2 and 16.9 ± 1.8 ka. The nominal ages are not consistent with the stratigraphic order, but considering their internal (analytical) uncertainties, all ages except Ker-57, seem to belong to the sample population based on the Chi 2 test (Fig. 5). Ker-57 has been rejected as an outlier and might have been pre-exposed to cosmic-rays in the past. G1 samples yield a mean age and full uncertainty of 14.3 ± 2.3 ka (n = 9) for the entire fossil moraine deposit. However, the 36 Cl CRE age probability density plot (Fig. 5) shows two statistical subpopulations within the G1 group of samples. The older group is centred around 16.5 ± 2.2 ka (n = 4), whereas the younger group is clustered around 12.5 ± 2.1 ka (n = 5).

36

Cl CRE ages from G2 moraine boulders (n = 3) Ker-42, -44, -45 were sampled on two moraine ridges (G2a and G2b) close to the front position named group G2 (Fig. 2 & 3). The 36 Cl CRE ages are 2.34 ± 0.48 ka, 3.77 ± 0.70 ka and 1.76 ± 0.54 ka, respectively (Fig. 6). According to the Chi 2 test, these 3 samples from the G2 moraines are indistinguishable and yield a mean age of 2.62 ± 0.97 ka.

Discussion

36

Cl CRE dating of the moraine groups G1 and G2 of the Gentil Glacier provides new insights into understanding the responses of debris-covered glaciers to climate variations in the Southern Hemisphere. It allows us, for the first time, to document the evolution of a debris-covered glacier on Kerguelen during the Late Glacial period and the Holocene and to compare it to the previously investigated 36 Cl chronologies of debris-free glaciers on the archipelago [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF].

Glacial fluctuation history of the Gentil Glacier

Evidence of the Late Glacial glacier advance comes from 36 Cl ages of boulders from several ridges of the fossil G1 moraine deposit. The individual ages are dispersed over a time span of several thousand years between ~18 ka and ~12 ka, and are not in chronological order with regard to the position of the dated ridges. Considering the specific depositional processes leading to debris-covered glacier landforms and the related implications for their dating (see Methods section), it is inferred that moraines G1a to G1d were formed during the same glacier advance period. Taken together, the age distribution of the G1 group exhibits two distinct subgroups, whose arithmetic means and full errors yield exposure ages of 16.5 ± 2.2 ka and 12.5 ± 2.1 ka, respectively (Fig. 5). This large age spread can be explained by the high probability of boulder pre-exposure and post-depositional processes in this specific setting leading to variable exposure histories of the boulders within the same debris deposit (see Methods section). Similar to the interpretation of 36 Cl boulders ages from a fossil rock glacier deposit in the Austrian Alps, which are subject to comparable dating challenges [START_REF] Moran | Rock glacier development in the Northern Calcareous Alps at the Pleistocene-Holocene boundary[END_REF], it is thus proposed that (i) the oldest 36 Cl ages of the G1 moraine deposit are affected by some nuclide inheritance during boulder transportation before the glacier advance, (ii) the youngest 36 Cl ages represent most likely a late stabilization of the moraine deposit that postdates the glacial advance, notably accompanied by rotation and exhumation of debris during ice ablation, and (iii) the glacier advance occurred sometime during this time span. We further assume that the best estimate for the timing of this glacial advance is represented by the arithmetic mean and full error determined from the set of ages from the G1 moraine group, 14.3 ± 2.3 ka, which largely overlaps with the ACR (14.5 -12.9 ka) period, which suggests that glacier advance occurred during this period. The large G1 advance is probably not the oldest one in this area. Aerial photographs and field investigations revealed some moraine remains located at a distance of ~ 2.5 km from the investigated moraines and suggest older and larger ice extents. Dating of the three boulders from the G2 moraine deposit, yielded ages spread between ~3.8 ka and ~1.7 ka, thus suggesting another glacial advance occurred during the Late Holocene. Following the same rationale as above, we estimate the timing of this advance at 2.62 ± 0.97 ka (arithmetic mean and full error).

Between the Late Glacial and the Late Holocene, the glacier evolution remains unknown. At least 3 moraine ridges can be identified in between the 2 sets of dated ridges (Fig. 2, 3 &7). Samples from boulders of these moraines were used for an unsuccessful trial of in situ cosmogenic 3 He dating and no rock material remains from the field expedition for further 36 Cl dating. However, we assume that two of these moraines, which are the closest to the G1 moraine group, belong to the Late Glacial glacier advance, because of the similar eroded and revegetated topography. The other moraine ridge is located close to G2 in the area we described as inactive, which seems to still be ice-cored and is not yet revegetated. This moraine could belong to the Late Holocene advance dated on the G2 moraines or to an anterior one.

Pumice stones deposited at several locations on the western part of the Archipelago during a volcanic eruption dated to the beginning of the last millennium [START_REF] Arnaud | Extensive lake sediment coring survey on Sub-Antarctic Indian Ocean Kerguelen Archipelago (French Austral and Antarctic Lands)[END_REF] were observed on the G1 moraines, G2 moraines and in between, but not on morainic bulges upstream of G2 ridges. It is thus suggested that the glacier advanced again bulging new ridges after this volcanic eruption, i.e. during the last millennium.

Glacier evolution on Kerguelen Archipelago: the debris-covered Gentil Glacier in comparison with debris-free glaciers

Kerguelen Archipelago offers an opportunity to compare the possible synchrony or asynchrony between the two different glaciers types (debris-covered versus debris free).

Despite the limited number of dated moraines and high degree of uncertainties in the ages related to the method and the specific challenges in dating boulders of debris-covered glaciers, the distribution of the 36 Cl ages covering the Late Glacial period from the Gentil Glacier overlaps with other debris-free glacier chronologies such as Bontemps moraine formed by Explorateur Glacier and a smaller local glacier located on Presqu'île Jeanne D'Arc (Belvedere moraine) (Jomelli et al. 2017) (Fig. 8 & 9). Comparing the timing of moraine formation at these different locationsi.e. 13.6 ± 1.5 ka at Bontemps moraine, 15.5 ± 1.8 ka at Belvedere moraine and 14.3 ± 2.3 ka at Gentil G1 morainesuggest a broadly synchronous advance (Fig. 9), potentially due to the same climate forcing.

Moreover, none of the existing glacier chronologies provides evidence of large advances between the Late Glacial and the Late Holocene. The advance of the Gentil Glacier at ~ 2.6 ka has so far not been directly documented for debris-free glaciers of the archipelago.

In addition, the age and standard deviation of G2 moraine (2.62 ± 0.97 ka) is distinguishable from the age of the last-millennium moraine of Ampere Glacier (0.70 ± 0.32 ka) [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF], suggesting an advance of Gentil glacier that occurred ~2 ka earlier than that of Ampere glacier. However, this does not exclude that these glaciers experienced a comparable trend, as moraines of Ampere Glacier may have been overrun by more the recent advances.

Besides, the Gentil Glacier might also have experienced a glacial advance during the last millennium as suggested by the observed lack of pumice stones on moraine ridges upstream of the G2 moraine set.

Climate sensitivity

The CIC has experienced a glacial shrinkage over the last few decades [START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF] owing to a dramatic drought occurring since the mid 1960's in the archipelago. To put this observation in a long-term context, we aim to understand whether the unusual current Kerguelen glacier sensitivity to precipitation changes differs from the past response of the different types of glaciers to climate fluctuations at a regional scale.

The recent evolution of debris-covered glaciers at Kerguelen is difficult to assess because remote sensing techniques, which are often used to determine surface mass balance through elevation changes of the glacial tongue [START_REF] Vincent | Reduced melt on debris-covered glaciers: Investigations from Changri Nup Glacier, Nepal[END_REF][START_REF] Brun | Ice cliff contribution to the tongue-wide ablation of Changri Nup Glacier, Nepal, central Himalaya[END_REF], are difficult to apply here due to a consistent high degree of cloud cover at Kerguelen. Without mass and energy balance measurements in the field, a comparison between debris-free and debris-covered ice wastage on the archipelago is difficult to assess.

Paleoglacial studies based on approaches such as 36 Cl CRE dating represent a useful tool to compare the response of both glacier types to multi-millennial climate variation. Based on the results presented here it is postulated that the Gentil Glacier has been sensitive to multi-millennial climate fluctuations, with a behaviour that is broadly synchronous to debrisfree glaciers on the Kerguelen archipelago, at least since the Late Glacial, most likely during the abrupt cold ACR event and probably also during the Holocene.

The ACR event was recorded in the WDC (WAIS Divide ice core) Antarctica ice core (Fig. 9b), where more depleted δ 18 O values and a decrease in snow accumulation rate (WAIS Divide Project [START_REF] Members | Onset of deglacial warming in West Antarctica driven by local orbital forcing[END_REF][START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF] are observed. This event influenced most of the Southern Hemisphere glaciers and even those in the tropical Andes [START_REF] Jomelli | A major advance of tropical Andean glaciers during the Antarctic cold reversal[END_REF]. Some of the still glaciated sub-Antarctic areas seem to have experienced more or less synchronous glacial advances in association with the ACR period (see for instance [START_REF] Putnam | Glacier advance in southern middle-latitudes during the Antarctic Cold Reversal[END_REF][START_REF] Pedro | The spatial extent and dynamics of the Antarctic Cold Reversal[END_REF][START_REF] Darvill | The timing and cause of glacial advances in the southern mid-latitudes during the last glacial cycle based on a synthesis of exposure ages from Patagonia and New Zealand[END_REF][START_REF] Graham | Major advance of South Georgia glaciers during the Antarctic Cold Reversal following extensive sub-Antarctic glaciation[END_REF][START_REF] Sagredo | Trans-pacific glacial response to the Antarctic Cold Reversal in the southern mid-latitudes[END_REF]). In addition, multi-proxy analysis of peat sequences on the eastern part of the archipelago indicate increased moisture and wind during the ACR (van der Putten et al. 2015), which supports the hypothesis of cold and wet conditions over Kerguelen, owing to a potential shift of the westerlies belt [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF]. This would have led to glacial advances of debrisfree as well as debris-covered glaciers, meaning that both types of Kerguelen glaciers were sensitive to the wide-ranging temperature fluctuations of the ACR [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF].

The role of climate changes on Holocene glacier behaviour is more difficult to assess as Late Holocene moraine deposits could reflect a climate-driven glacier advance and a nonclimatic ice dynamics signature. The amplitude of temperature change documented from WDC ice core records (WAIS Divide Project Members 2013) (Fig. 9b) remains rather limited compared to the ACR period. A general SAM-negative-like state (associated with higher precipitation) occurred during the Late Holocene (⁓2.7 -0.1 ka) (Fig. 9c), as inferred from the Lago Cipreses non-arboreal pollen (NAP) record in Patagonia [START_REF] Moreno | Onset and evolution of Southern Annular Mode-Like changes at centennial timescale[END_REF]) (Fig. 9d). This SAM-negative-like state supports more moisture during the Late Holocene that may have partly compensated for possible warmer temperatures until 2.5 ka thus suggesting the importance of precipitation in the formation of the G2 moraine set. This behaviour would be supported by current observations that have revealed the major role of atmospheric drying related to high-index SAM conditions on current glacier retreat at Kerguelen [START_REF] Verfaillie | Recent glacier decline in the Kerguelen Islands (49°S, 69°E) derived from modeling, field observations, and satellite data[END_REF][START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]. The prominent role of precipitation in debris-covered glacier behaviour seems plausible, because supraglacial debris insulate the underlying glacier ice and possibly lower the glacier's sensitivity to low-ranging temperature changes. Increased precipitation and temperature decrease around 2.5 ka ago may also have favoured glacier expansion of Ampere Glacier. In that case, it is possible that Ampere Glacier deposited moraines, which however would have been obliterated by more extensive advances during the last millennium. On the contrary, the Gentil Glacier has probably also expanded several times during the Late Holocene. However, the low terminus velocity of debris-covered glaciers and their external moraine being usually much bigger and broader than that of debris-free glaciers, owing to the high debris load, prevent the obliteration of earlier moraine ridges. In addition, given that Gentil Glacier is a marginal glacier lobe of Buffon Glacier, its ice dynamics might have been partly controlled by the behaviour of the ocean-terminating main glacier tongue, leading to fluctuations that are less directly related to climate changes than that of Ampere Glacier.

Conclusion

This study provides new insights into the multi-millennial evolution of debris-covered glaciers, which are very scarce in the Southern Hemisphere. The here established chronology of the Gentil Glacier on the Kerguelen Archipelago, constrained by in situ-produced 36 Cl CRE dating, allows for documenting periods of large advances of this debris-covered glacier and to compare them to existing chronologies of debris-free Kerguelen glaciers [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF]. Evidence of two glacial advances of the Gentil Glacier is provided: (i) the first during the Late Glacial (~ 14.3 ka) which could coincide with the ACR event; (ii) the second during the Late Holocene (~ 2.6 ka). As debris-free glaciers experienced advances that can probably also be assigned to the ACR, this broadly synchronous behaviour suggests that both debris-free and debris-covered glaciers were sensitive to the multi-millennial temperature fluctuations recorded in West Antarctica ice core (WDC, WAIS divide Project [START_REF] Members | Onset of deglacial warming in West Antarctica driven by local orbital forcing[END_REF]. The late Holocene advance experienced by the Gentil Glacier has not yet been observed for the debris-free glaciers. This advance could be a specific response of debris-covered glaciers to a significant precipitation increase resulting from infra-millennial SAM-like changes. Thus, the debris-covered Gentil Glacier was probably sensitive to large amplitude temperature changes, but also to precipitation changes. On the basis of current qualitative observations and paleoglacial investigations, it appears that all types of Kerguelen glaciers have been sensitive to the same climate forcings, but the amplitudes of their glacial responses might be distinct.

Authors contributions

V.J., D.V., V.F. and A.G. conducted the field work on the Islands; I.S. and F.M. participated in producing the cosmogenic data; G.A., D.L.B. and K.K. (ASTER Team) performed AMS measurements; J.C., I.S. and V.J. interpreted the cosmogenic ages; J.C., V.J. and I.S. prepared figures and J.C., V.J., I.S., D.V., V.F., F.B., A.G. and D.L.B. contributed to writing the paper. [START_REF] Moreno | Onset and evolution of Southern Annular Mode-Like changes at centennial timescale[END_REF]). e. and f. 36 Cl age probability density distributions with their analytical uncertainties (coloured curves) and summed probabilities (shaded areas) of debris-free glaciers (blue) [START_REF] Jomelli | Sub-Antarctic glacier extensions in the Kerguelen region (49°S, Indian Ocean) over the past 24,000 years constrained by 36 Cl moraine dating[END_REF][START_REF] Jomelli | Glacier extent in sub-Antarctic Kerguelen archipelago from MIS 3 period: Evidence from 36 Cl dating[END_REF] and debris-covered Gentil glacier (red and purple).

Figures captions

Fig. 1

 1 Fig. 1. a. Map of the Kerguelen Archipelago and its location. b. The study area, Gentil

Fig. 2 .

 2 Fig. 2. a. Glacial geomorphological map of Gentil Glacier, Kerguelen Archipelago. Rectangle

Fig. 3

 3 Fig. 3. a. Picture of active debris-covered Gentil glacier, with the Latest Holocene moraine

Fig. 4 .

 4 Fig. 4. Pictures of boulders on the fossil G1 moraine sampled during the field campaigns.

Fig. 5 .

 5 Fig. 5. Probability plots of 36 Cl boulder CRE ages from the G1 moraines set. Individual 36 Cl

Fig. 6 .Fig. 7 .

 67 Fig. 6. Probability plots of 36 Cl boulder CRE ages from G2 moraine set. Individual 36 Cl CRE

Fig. 8 .

 8 Fig. 8. Summary of 36 Cl CRE ages covering the investigated period obtained at different sample sites (this study and Jomelli et al. (2017, 2018)). Reported mean 36 Cl CRE boulder

Fig. 9 .

 9 Fig. 9. Kerguelen paleoglacier records compared with paleoclimatic proxies. a. Glacier

  Figure 1

Figure 9

 9 Figure 9

Acknowledgements

This work has received financial support from the LabEx DynamiTe (ANR-11-LABX-0046) Les Envahisseurs, as part of the "Investissements d'Avenir" program. This paper was also supported by the French INSU LEFE Glacepreker project and by the IPEV Kesaaco 1048 project. The 36 Cl measurements were performed at the ASTER AMS national facility (CEREGE. Aix-en-Provence) that is supported by the INSU/CNRS, the ANR through the "Projets thématiques d'excellence" program for the "Equipements d'excellence" ASTER-CEREGE action and IRD. We are thankful to the two anonymous reviewers for their positive and constructive comments.

Details of data deposit

This 36 Cl data will be found on open access repository on the informal cosmogenic-nuclide exposure-age database (ICE-D: Alpine; http://alpine.ice-d.org/)


Gregorio Cullia

Roberto Fanelli

Romé Voulhoux

Pascal Arnoux

Florine Cavelier

Synthesis of Two Epimers of Pseudopaline

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

INTRODUCTION

Opines constitute a large family of highly polar molecules with different structures. A substantial part of these molecules belongs to the octopine (1) and nopaline (2) classes (fig. 1), characterized by the presence of a N-1-carboxylalkyl amino acid group. [START_REF] Petit | Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF] Figure 1. Structures of octopine (1) and nopaline (2). The characteristic moiety of opines is highlighted. Such molecules were firstly identified in plant crown gall tumors, caused by Agrobacterium spp, [START_REF] Petit | Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF] and in lower marine invertebrate phyla and middle phyla of Protostomia. [START_REF] Kan-No | Tauropine dehydrogenase from the starfish Asterina pectinifera (Echinodermata: Asteroidea): presence of opine production pathway in a deuterostome invertebrate[END_REF] Opines are synthesized by opine dehydrogenases (ODHs), which catalyse the reductive amination between different amino acids and ketoacids (often pyruvate, the dead-end product of glycolysis) while consuming nucleotinamide adenine dinucleotide (phosphate) (NAD(P)H). [START_REF] Thompson | Biosynthesis and distribution of N-carboxyalkyl amino acids (opines) in bacteria[END_REF] Different species of the genus Agrobacterium use opines as a selective advantage in their invasion of a variety of plants. These bacteria transfer their tumor-inducing (Ti) plasmid, which contains the genes encoding for opine synthesis, to plants cells. The latter transform in crown gall tumors and start producing opines which represent specific growth substrates for the bacteria; this is known as t e " pine c ncept". [START_REF] Petit | Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF][START_REF] Thompson | Biosynthesis and distribution of N-carboxyalkyl amino acids (opines) in bacteria[END_REF][START_REF] Vladimirov | Opine biosynthesis and catabolism genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes[END_REF] On the other hand, marine invertebrates (e.g. Antarctica islandica and Asterina pectinifera) synthesize opines in order to balance the cellular redox equilibrium. Similarly to the lactic acid fermentation, the synthesis of opines allows to re-oxidize NADH in hypoxic conditions, granting a continuous flux of glycolysis. [START_REF] Kan-No | Tauropine dehydrogenase from the starfish Asterina pectinifera (Echinodermata: Asteroidea): presence of opine production pathway in a deuterostome invertebrate[END_REF][START_REF] Harcet | Opine de ydr genases in arine invertebrates[END_REF] Recently, two new opines, staphylopine (3) [START_REF] Ghssein | Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus[END_REF] and pseudopaline (4), [START_REF] Mcfarlane | Biosynthesis of an Opine Metallophore by Pseudomonas aeruginosa[END_REF] have been identified in Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Compounds 3 and 4 have similar structures as depicted in figure 2: both molecules present a histidine (His) residue (with R or S configuration, respectively), a 2-aminobutyrate residue, and an end portion deriving from pyruvate (compound 3) or from -ketoglutarate (compound 4). These two molecules are related to nicotianamine (compound 5, fig 2), an important plant siderophore. [START_REF] Curie | Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters[END_REF] Compounds 3 and 4 do not participate in metabolism as the other opines, but are , like 5, soluble metallophores involved in a system which also comprehends the biosynthetic machinery and membrane reporters, whose function is to grant the supply of divalent metal cations to the bacteria. 6, 7b Figure 2. Structures of the three related metallophores staphylopine (3), pseudopaline (4) and nicotianamine (5).

In P. aeruginosa, pseudopaline is synthetized by two cytoplasmic enzymes encoded by the cntL and cntM elements of a four gene operon. The cnt operon also encodes the CntI and CntO proteins respectively involved in pseudopaline export and recovery by the bacteria. 7b, 9 In vivo, the pseudopaline operon is induced under zinc limitation in line with the in-volvement of pseudopaline in zinc uptake in metal scarce conditions (it is also involved in manganese uptake). 7b The role of pseudopaline is crucial for P. aeruginosa growth in such metal scarce conditions recovered during infections where the nutritional immunity framework (basically the host-guest competition for micronutrients) renders particularly difficult the supplying of metals. 7b, [START_REF] Mastropasqua | Growth of Pseudomonas aeruginosa in zinc poor environments is promoted by a nicotianaminerelated metallophore[END_REF][START_REF] Gi | A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus[END_REF] The interest around this molecule is particularly relevant in the case of cystic fibrosis (CF): P. aeruginosa causes lung infections in the 60-70% of adult patients, leading to a progressive pulmonary insufficiency, the principal cause of death. [START_REF] Folkesson | Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective[END_REF] Given the impact of the Cnt systems in P. aeruginosa and S. aureus infections, knowledge on metallophores production and control of metal level are highly relevant. After our recent characterization of SaCntM (the ODH of S. aureus), and in particular its peculiar activity modification in presence of different divalent metals, [START_REF] Hajjar | Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis[END_REF] we focused on the P. aeruginosa case. The full understanding of this metal acquisition system might disclose the route to novel antimicrobial therapies that could improve the life expectation of CF or other immunocompromise patients.

Recently, Zhang et al. proposed a synthesis for 3 and 4. [START_REF] Zhang | Total Syntheses of Natural Metallophores Staphylopine and Aspergillomarasmine A[END_REF] Their strategy, similarly to the synthesis that we previously published for compound 5, [START_REF] Bouazaoui | Efficient Synthesis of Nicotianamine and Non-Natural Analogues[END_REF] relies on the conversion of amines in sulfonamides, followed by N-alkylations for the construction of the main chain of the molecule. This approach implies drastic conditions for the final deprotection reaction (i.e. the use of triflic acid). To approach the synthesis of pseudopaline 4 we designed a synthesis that could afford both epimers of 4 (namely compounds 4a and 4b, fig. 3) as single enantiomers at one time, in a convenient way, both in terms of number of steps and overall yield.

RESULTS AND DISCUSSION

From a retrosynthetic point of view (scheme 1), 4 can be obtained from its protected version, ideally with all protecting groups with the same lability in order to facilitate the final deprotection. This intermediate can be disconnected in an αhaloglutarate ester and a primary amine. Its protection PG 3 at the previous stage must have an orthogonal stability compared to the other protecting groups. This fully protected intermediate can derive from its corresponding unprotected secondary amine, which could be finally disconnected in a protected histidine and an aspartic semi-aldehyde analogue.

Scheme 1. Proposed retrosynthesis of 4.

We planned to protect all carboxylic acids as tert-butyl ester for two practical reasons: i) they are easily cleaved under strong acidic condition resulting in a neat reaction with only volatile by-products and ii) the steric hindrance prevent that the secondary amine in position γ attacks the carbonyl to generate an undesired γ-lactam by intramolecular reaction (scheme 2).

Scheme 2. Intramolecular side reaction.

In accordance to the tert-butyl ester protection strategy, trityl (Trt) has been chosen as protecting group for the N of His. Trt is more robust than other acid labile groups: for example, N Boc protection was not stable even in presence of weak bases (i.e. K 2 CO 3 , NaHCO 3 ) at room temperature (r.t.). [START_REF] Abdo | Brucella suis histidinol dehydrogenase: Synthesis and inhibition studies of a series of substituted benzylic ketones derived from histidine[END_REF] H-His(Trt)-OtBu (7), despite commercially available, was prepared in a three step procedure (overall yields 40-50%) starting from Cbz-His-OH (6, scheme S1). Following a similar reduction/selective oxidation strategy to the one that we recently reported, [START_REF] Hajjar | Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis[END_REF] we synthesized protected aspartic acid semi aldehyde (Asa), Cbz-Asa-OtBu (9) starting from Cbz-Asp-OtBu•dicyclohexylamine (DCHA) (8, scheme S2). Reductive amination between synthons 7 and 9 proceeded in good yields (80%, scheme 3) without the formation of any potential byproduct, such as tertiary amine deriving from a second reductive amination, or γ-lactam deriving from the intramolecular attack of the secondary amine to the ester group of 10. Compound 10 was submitted to hydrogenolysis affording 11 in good yields, which was engaged in alkylation reaction (scheme 3) with di-tert-butyl 2-iodoglutarate 12 (prepared starting from L-glutamic acid, scheme S3). The reactivity of 12 is somehow limited (probably for steric reasons), as alkylation proceeded in a regioselective fashion at the more accessible primary amine of 11, as was verified by bidimensional nuclear magnetic resonance (NMR) analysis (see S.I.). After high performance liquid chromatography (HPLC) purification, compound 13 was obtained as an epimeric mixture in 47% yield. We also performed the same transformations starting from Boc protected 10 (scheme S5), invariantly obtaining the same epimeric mixture, but in lower yields.

Scheme 3. Synthesis of 4a and 4b.

Reagents and conditions: a) i. anh. MgSO 4 , dry MeOH, r.t., 3h; ii sodium triacetoxyborohydride (STAB), r.t., 16h; b) H 2 (atm. press.), 10% Pd/C, iPrOH, r.t., 4 h; c) K 2 CO 3 , dry N,N-dimethylformamide (DMF), 40°C; d) 20% HCO 2 H/MeOH, cat. TIS, 40 °C, overnight (o.n.); e) 37% aq. HCl, r.t., 3h.

With the aim of isolating each epimers, compound 13 was treated with diluted formic acid in presence of triisopropylsilane (TIS) as scavenger to selectively remove the Trt group, leading to epimers 14a and 14b which were separated by silica gel column chromatography. However, 14a required additional HPLC purification to increase its chemical purity (low preparative HPLC recovery is ascribed to the reduced yield for 14a). Finally, treatment of both 14a and 14b with concentrated HCl afforded the two epimers of pseudopaline, 4a (6 mg, [α] 20 D -9.1 (c 0.55 g/dL, H 2 O)) and 4b (22 mg,

[α] 20 D +18.9 (c 0.55 g/dL, H 2 O)) in 24% overall yields (starting from the three building blocks, scheme 3). The two molecules however revealed instability upon standing at 0-4 °C forming lactams by condensations, as suggested both by MS analysis (the molecular ion peak has a loss of 18 in m/z compared from 4, compatible with the loss of a water molecule)

and NMR spectra (the signals of the α amino acidic protons moves at lower fields, compatible with the presence of a lactam, see S.I.). This undesired conversion is probably prompted by acidic conditions, so in future work, different deprotection condition should be considered. A possible alternative would be represented by the hydrolysis of the esters under basic conditions, which would afford 4 in its deprotonated form, unreactive to lactamisation.

Remarkably, the two epimers present 1 H NMR spectra (fig. 4) with peculiar signals, especially around 4.1 ppm. For the attribution of the absolute configuration of 4a and 4b, we needed enantiopure 4 with a known configuration. Starting from commercial lacton (S)-15, we synthesized enantiopure alkyl iodide (R)-16 (scheme 4). The carboxylic acids were protected as methyl esters since attempts of obtaining the corresponding di-tert-butyl ester failed. (R)-16 was reacted with 11 affording 17 (scheme 4), a fully protected equivalent of (S,S,S)-4.

The crude product presented an impurity with the same molecular mass of 17 (with a ratio 3:1 17/impurity, see S.I.). As a S N 1 mechanism is unlikely, we speculated that the presence of the less hindered methyl esters in (R)-16, compared to the tert-butyl esters in compound 12, might allow alkylation of the secondary amine of 11, resulting in the regioisomeric byproduct. As confirmation, deprotection of the byproduct never afforded 4 (in any form). Compound 17 was deprotected by hydrolysis followed by acidic treatment, affording (S,S,S)-4 (scheme 4). Comparison of the 1 H NMR spectra unambiguously showed the correspondence between (S,S,S)-4 and 4b (fig. 4); by consequence, we attributed the (S,S,R) configuration to 4a.

Scheme 4. Synthesis of (S,S,S)-4

Reagents and conditions: a) By the time this article was prepared, Zhang et al. [START_REF] Zhang | De novo synthesis, structural assignment and biological evaluation of pseudopaline, a metallophore produced by Pseudomonas aeruginosa[END_REF] published the asymmetric synthesis of 4a and 4b, along with the attribution of the (S,S,S) configuration (the one of 4b) to the natural product. This synthesis relies, as the one the same authors proposed for compound 3, [START_REF] Zhang | Total Syntheses of Natural Metallophores Staphylopine and Aspergillomarasmine A[END_REF] on the conversion of the amino groups in sulfonamides which are engaged, in turn, in an asymmetric Tsuji-Trost and a Fukuyama-Mitsunobu reactions. This elegant strategy presents anyhow some drawbacks, as it requires harsh deprotection conditions and a series of oxidations involving highly toxic reagents (i.e. OsO 4 , Jones Reagent).

1 H NMR (D 2 O) comparison. The spectra of (S,S,S)-4 (registered at 600 MHz, blue) does not correspond to the one of 4a (400 MHz, green), but it does to the one of 4b (400 MHz, red). The little difference in the δ of H 2 O signa s (≤ 0.2 pp ) is within acceptable limits, considering the different condition of the acquisitions.

CONCLUSIONS

In this paper, we present a new synthesis of the two epimers of pseudopaline (4). This approach represents an alternative strategy for the synthesis of pseudopaline (and, in general, of opine metallophores) to the one exploited by Zhang et al., [START_REF] Zhang | De novo synthesis, structural assignment and biological evaluation of pseudopaline, a metallophore produced by Pseudomonas aeruginosa[END_REF] thanks to a reduced number of steps (5, starting from commercially available 7, vs 8), comparable overall yields (24% vs 22%), and more green conditions avoiding the use of transition metals, toxic oxidating reagents, and of non-atom efficient reactions (i.e. Fukuyama-Mitsunobu). Epimers 4a and 4b, differing at the absolute configuration of the glutarate portion, were obtained as single enantiomers after resolution by column chromatography at the n-1 step of the synthesis.

Starting from chiral building block (S)-15 we synthesized (S,S,S)-4, which allowed us to assign the absolute configuration of 4a and 4b. This second approach is less efficient than the first synthetic strategy (8% vs 24%), due to a side reaction in the alkylation step. A different protection for the diester (R)-16 (with larger groups), or the protection of the secondary amine of 10 before the hydrogenolysis of the Cbz (benzyloxycarbonyl) group, might avoid this problem. Our approach has different advantages, as a reduced number of steps, scale-up easiness (the main problem might be the purification of intermediates 14a and 14b) and flexibility (using different building blocks it is possible to obtain different analogues of 4).

This strategy appears thus to us suitable for an eventual drug discovery campaign and for chelation studies of pseudopaline with different metals.

EXPERIMENTAL SECTION

General procedures

Synthesis and characterization of compounds 7, 9, 12, and (R)-16 are reported in the supporting informations. All reagents and solvents were purchased from commercial suppliers (Merck, Fluorochem, TCI Europe, Carlo Erba, IRIS biothech, Fluka and AlfaAesar). All reaction involving air-sensitive reagents were performed under argon atmosphere.

Catalytic hydrogenations were performed at atmospheric pressure. Solvent ratios are intended as a v/v ratios. Reaction were checked by TLC on commercial silica gel 60 F254 aluminum sheets (spots were further evidenced by spraying with a dilute alkaline solution of KMnO 4 ) or by RP-Analytic HPLC performed on an Agilent 1220 using a 50 x 4.6 mm Chromolith® High Resolution column. Compounds were separated using solvent A (H 2 O + 0.1 % TFA) and solvent B (MeCN + 0.1 % TFA) with a linear gradient of solvent B from 0 to 100% in 3 min using a constant flow rate of 3 mL min -1 .

Purifications were performed with column chromatography using silica gel (Merck 60, 230-400 mesh) or by preparative HPLC performed on a Gilson PLC2020 apparatus equipped with a Phenomenex Luna 5 μm C18(2) 100 Å, 75 x 21.2 cm column coupled to an UV detector (detection fixed at 215 and 245 nm). Solvent A (H 2 O + 0.1 % HCO 2 H) and solvent B (MeCN + 0.1 % HCO 2 H) were used with a constant gradient of solvent B from 0% to 100% in 12.8 min and a constant flow rate of 25.5 mL min -1 . Fractions of interest were collected, neutralized with solid NaHCO 3 and volume was reduced under reduced pressure. Product was extracted with DCM, pooled organic phase was dried over anhydrous Na 2 SO 4 , filtered and solvent removed under reduced pressure obtaining the desired prouct. Nuclear magnetic resonance spectra were recorded on a Bruker spectrometer avance 300 (300 MHz), on a Bruker avance III spectrometer 400 (400 MHz), or on a Bruker

Avance III spectrometer 600 (600 MHz). Chemicals shifts (δ) are reported from tetramethylsilane with the solvent resonance as the internal standard (according to literature data [START_REF] Gottlieb | NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities[END_REF] ). Data are reported as follows: chemical shift (δin ppm), multiplicity (s= singlet, d= doublet, t= triplet, b= broad, m= multiplet), coupling constants (J in Hz), integration, attribution. Low resolution electrospray ionization (ESI) mass spectra were recorded on a micromass platform electrospray mass spectrometer. Spectra were recorded in the positive mode (ESI+) or negative mode (ESI-, when specified). LC-MS system consisted of a Waters Alliance 2690 HPLC, coupled to a ZQ spectrometer (Manchester, UK), fitted with an electrospray source operated in the positive (ESI+) or negative (ESI-, when specified) ionization mode. All the analyses were carried out using a C18 Chromolith Flash 25 mm × 4.6 mm column operated at a flow rate of 3 mL/min. A gradient of 0% or 0.1% aqueous TFA (solvent A) to 100% of acetonitrile containing 0.1% TFA (solvent B) was developed over 3 min. Positive-ion electrospray mass spectra were acquired at a solvent flow rate of 100-200 μL/min. Nitrogen was used for both the nebulizing and drying gas. Rotary power determinations were carried out using a PerkinElmer Polarimeter 341; reported concentrations have the dimensions of g/dL. High-res ti n ass spectra (H MS) were perf r ed by t e "Lab rat ire de Mes res P ysiq es" f M ntpe ier University n a Micr ass Q-Tof spectrometer equipped with electrospray source ionization (ESI), using phosphoric acid as internal standard.

(S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-4-(((S)-1-(tert-butoxy)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-

Di-tert-butyl 2-(((S)-1-(tert-butoxy)-4-(((S)-1-(tert-butoxy)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2yl)amino

)-1-oxobutan-2-yl)amino)pentanedioate (13) Amine 11 (342 mg, 0.560 mmol) was dissolved in dry DMF (1 mL). K 2 CO 3 (101 mg, 0.728 mmol) and a solution of iodide 12 (269 mg, 0.728 mmol) in dry DMF (1.8 mL) were added. The reaction was stirred at 45°C until complete conversion. Water was added and extracted twice with EtOAc. The pooled organic phase was dried over anhydrous Na 2 SO 4 , filtered and the solvent was removed under reduced pressure. The crude was firstly purified by column chromatography on SiO 2 gel with 96:4 DCM/MeOH + 2% of 28% aq. NH 3 and the obtained compound (344 mg) was further purified by prep. HPLC (see general procedures). The product was obtained as a 1:1 mixture of epimers as a pale yellow oil (245 mg, 47% yield). R f = 0.39 and 0.29 (95:5 DCM/MeOH + 2 drops of 28% aq. NH 3 ).

Synthesis of epimers 14a and 14b

The mixture of 13 (110 mg, 0.129 mmol) was dissolved in DCM (0.8 mL). TIS (39.4 μL, 0.195 mmol) and formic acid (0.2 mL) were added and the mixture was stirred at 40 °C o.n. Reaction was diluted with DCM, sat. aq. NaHCO 3 was added, phases were separated and the aqueous one was extracted with DCM. The pooled organic phase was dried over anhydrous Na 2 SO 4 , filtered and the solvent was removed under reduced pressure. The crude was purified by column chromatography on SiO 2 gel with 95:5 DCM/MeOH + 2% of 28% aq. NH 3 affording a fraction containing 14a and pure 14b (49 mg, 62% yield). 14a was further purified by prep HPLC (see general procedures) affording pure 14a (13 mg, 16% yield). The products were obtained as colourless oils.

General procedure for the synthesis of 4a and 4b

Compound 14a or 14b was dissolved in 37% aq. HCl (1.5 mL) and the reaction was stirred at r.t. for 4 h. The solution was freeze-dried affording the title compound as a white powder in quantitative yields (4a: 6 mg; 4b: 22 mg).

(R)-2-(((S)- The reaction was stirred at 45 °C until complete conversion. Water was added and extracted twice with EtOAc. The pooled organic phase was dried over anhydrous Na 2 SO 4 , filtered and the solvent was removed under reduced pressure.

Figure 3 .

 3 Figure 3. Structure of the two epimers of pseudopaline 4a and 4b.

  yl)amino)pentanedioate (14a) R f = 0.58 (92:8 DCM/MeOH + 2 drops of 28% aq. NH 3

17 )

 17 Amine 11 (255 mg, 0.417 mmol) was dissolved in dry DMF (1 mL). NaHCO 3 (42 mg, 0.500 mmol) and a solution of iodure (R)-16 (143 mg, 0.500 mmol) in dry DMF (1 mL) were added.

  Amine 7 (300 mg, 0.661 mmol) and aldehyde 9 (300 mg, 0.661 mmol) were dissolved in dry MeOH. Anhydrous MgSO 4 (120 mg) was added and the mixture was stirred at r.t. for 1 h. STAB (420 mg, 1.98 mmol) was added in small portions over 1 h. The reaction mixture was stirred at r.t. o.n., then it was filtered over a celite pad and the solvent was removed under reduced pressure. The residue was dissolved in DCM and washed with sat. aq. NaHCO 3 , dried over anhydrous Na 2 SO 4 , filtered and the solvent was removed under reduced pressure. The crude was purified by column 9H).[START_REF] Zhang | Total Syntheses of Natural Metallophores Staphylopine and Aspergillomarasmine A[END_REF] C NMR (75 MHz, CDCl 3 ): δ 173.5, 171.6, 156.2, 142.6, 138.6, 137.3, 136.7, 129.9, 128.5, 128.1, 119.2, 81.7, 80.9, 75.2, 66.7, 61.9, 53.6, 44.1, 32.1, 31.9, 28.2, 28.1. MS(ESI): m/z calcd for C 45 H 52 N 4 O 6 : 744.4; found: 745.2(38) [M+H] + , 503.2(85) [M-Trt+H] + , 447.1(5) [M-Trt-tBu+H] + , 243.1(100) [Trt] + . [α] 20 D -0.95 (c 1.16, CHCl 3 ). Compound 10 (154 mg, 0.207 mmol) was dissolved in iPrOH (2.5 mL) in a sealed round bottom flask under argon. 10% Pd/C (36 mg) was added and the reaction mixture was stirred at r.t. under hydrogen for 4 h. The mixture was filtered over a celite pad then the solvent was removed under reduced pressure. The crude was purified by column chromatography on SiO 2 gel with affording the title compound as a pale yellow oil (103 mg, 82% yield). R f = 0.27 (95:5 DCM/MeOH + 2 drops 9H). 13 C NMR (101 MHz, CDCl 3 ): δ 175.3, 173.8, 142.6, 138.5, 137.5, 129.8, 128.0, 119.3, 80.8, 80.8, 75.1, 61.9, 53.7, 44.9, 34.9, 32.3, 28.1, 28.2, 28.1. MS(ESI): m/z calcd for C 37 H 46 N 4 O

	(m, 14H), 7.17-7.04 (m, 6H), 6.59 (s, 1H), 6.25 (d, J = 7.7 Hz, 1H), 5.05 (s, 2H), 4.26 (ddd, J = 5.5, 7.3, 7.7 Hz, 1H), 3.42 (dd, J =
	6.2, 6.4 Hz, 1H), 2.89 (dd, J = 6.2, 14.5 Hz, 1H), 2.83-2.67 (m, 2H), 2.60 (m, 1H), 2.07 (bs, 1H), 1.98-1.73 (m, 2H), 1.43 (s, 9H),
	1.35 (s, (S)-tert-Butyl 2-amino-4-(((S)-1-(tert-butoxy)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2-yl)amino)butanoate
	(11) of 28% aq NH 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7.31 (s, 1H), 7.31-7.26 (m, 9H), 7.15-7.05 (m, 6H), 6.60 (s, 1H), 3.43 (dd, J =
	5.8, 7.0 Hz, 1H), 3.34 (dd, J = 4.5, 8.3 Hz, 1H), 2.90 (dd, J = 5.8, 14.4 Hz, 1H), 2.82-2.69 (m, 2H), 2.59 (m, 1H), 1.84 (m, 1H),
	1.71 (bs, 3H), 1.57 (m, 1H), 1.42 (s, 9H), 1.36 (s,
	33-7.27

2-yl)amino)butanoate (10)

chromatography on SiO 2 gel with 3:7 cHx/EtOAc + 2% of 28% aq. NH 3 affording the title compound as a white foam (393 mg, 80% yield). R f = 0.38 (3:7 cHx/EtOAc + 2 drops of 28% aq. NH 3 ).

[START_REF] Petit | Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF]

H NMR (300 MHz, CDCl 3 ): δ 7.34 (s, 1H), 7.4 : 610.3; found: 611.3(38) [M+H] + , 369.2(60) [M-Trt+H] + , 243.1(100) [Trt] + . [α] 20 D +2.7 (c 1.04, CHCl 3 ).

1-carboxy-3-(((S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl)amino)propyl)amino) pentanedioic acid tri- hydrochloride (4a*3HCl

  )[START_REF] Petit | Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF] H NMR (400 MHz, D 2 O): δ 8.69 (d, J = 1.2 Hz, 1H), 7.45 (s, 1H, H4), 4.28 (dd, J = 5.2, 7.3 Hz, 1H, H2), 4.20-4.13 (m, 2H, H15+H12), 3.54 (dd, J = 5.2, 15.9 Hz, 1H, H1), 3.49-3.39 (m, 3H, H'1+H10+H'10), 2.68 (t, J = 7.0 Hz, 2H, H18+H'18), 2.40 (dt, J = 6.8, 7.4 Hz, 2H, H11+H'11), 2.27 (m, 2H, H17+H'17). 13 C NMR (101 MHz, D 2 O): δ 179.7 (C19), 176.4 J = 1.4 Hz, 1H, H6), 7.43 (s, 1H, H4), 4.30 (dd, J = 5.2, 7.5 Hz, 1H, H2), 4.16 (t, J = 6.5 Hz, 1H, H15), 4.11 (t, J = 6.5 Hz, 1H, H12), 3.52 (dd, J = 5.2, 15.9 Hz, 1H, H1), 3.48-3.39 (m, 3H, H'1+H10+H'10), 2.69 (dd, J = 7.1, 13.2 Hz, 2H, H18+H'18), 2.43 (m, 2H, H11+H'11), 2.29 (dd, J = 3.7, 7.0 Hz, 1H, H17), 2.26 (dd, J = 4.1, 7.0 Hz, 1H, H'17). 13 C NMR (101 MHz, D 2 O): δ 176.4 (C19), 170.9 (C16), 170.3 (C14), 169.8 (C8), 134.2 (C6), 126.1 (C3), 118.2 (C4), 59.9 (C15), 59.4 (C2), 58.2 (C12), 43.6 (C10), 29.5 (C18), 26.3 (C11), 24.7 (C17), 24.4 (C1). MS(ESI): m/z calcd for C 15 H 22 N 4 O 8 : 386.1; found: 387.1(67) [M+H] + , 369.1(83) [M-18+H] + , 351.0(100) [M-36+H] + . [α] 20 D +18.9 (c 0.55, H 2 O). HRMS(ES+): m/z calcd for C 15 H 23 N 4 O 8 : 387.1516; found: 387.1517 [M+H] + (Δ = 0.3 ppm).

	(C16), 172.4 (C14), 169.9 (C8), 134.2 (C6), 126.2 (C3), 118.2 (C4), 60.5 (C2), 59.2 (C15), 53.9 (C12), 44.0 (C10), 29.0 (C18), 25.5
	(C11), 24.4 (C1), 23.7 (C17). MS(ESI): m/z calcd for C 15 H 22 N 4 O 8 : 386.1; found: 387.1(30) [M+H] + , 369.1(95) [M-18+H] + ,
	351.0(98) [M-36+H] + , 153.2(100). [α] 20	D -9.1 (c 0.55, H 2 O). HRMS(ES+): m/z calcd for C 15 H 23 N 4 O 8 : 387.1516; found: 387.1517
	[M+H] + (Δ = 0.3 ppm).
	(S)-2-(((S)-1-carboxy-3-(((S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl)amino)propyl)amino) pentanedioic acid trihy-
	drochloride (4b*3HCl) 1 H NMR (400 MHz, D 2 O): δ 8.67 (d,

  Purification column chromatography on SiO 2 gel with 98:2 to 95:5 DCM/MeOH + 2 drops of 28% aq. NH 3 afforded pure title compound as a yellow oil (76 mg, 24% yield). R f = 0.32 (96:4 DCM/MeOH + 2 drops of 28% aq. NH 3 ).[START_REF] Petit | Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF] H NMR (400 MHz, CDCl 3 ): δ 7.35-7.28 (m, 10H), 7.14-7.08 (m, 6H), 6.60 (d, J = 1.1 Hz, 1H), 3.67 (s, 3H), 3.62(s, 3H), 3.44 (m, 1H), 3.22 (dd, J = 5.5, 7.1 Hz, 1H), 3.13 (dd, J = 5.0, 7.7 Hz, 1H), 2.90 (dd, J = 6.4, 14.5 Hz, 1H), 2.80 (dd, J = 6.4, 14.5 Hz, 1H), 2.76-2.66 (m, 1H), 2.65-2.54 (m, 1H), 2.48-2.32 (m, 2H) 2.10 (bs, 2H), 2.01-1.81 (m, 2H), 1.80-1.68 (m, 1H), 1.68-1.57 (m, 1H), 1.42 (s, 9H), 1.36 (s, 9H).[START_REF] Zhang | Total Syntheses of Natural Metallophores Staphylopine and Aspergillomarasmine A[END_REF] C NMR (101 MHz, CDCl 3 ): δ 174.5, 173.8, 173.7, 142.5, 138.4, 137.2, 129.8, 128.0, 119.3, 81.3, 81.0, 75.2, 61.8, 59.2, 59.1, 51.9, 51.6, 44.6, 33.8, 32.0, 30.1, 28.1, 28.1. MS(ESI): m/z calcd for C 44 H 56 N 4 O

8 : 768.4; found: 769.5(30) [M+H] + , 527.4(100) [M-Trt+H] + .

 (S)-2-(((S)-1-Carboxy-3-(((S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl)amino)propyl)amino)pentanedioic acid trihydrochloride [(S,S,S)-4*3HCl] Compound 17 (49 mg, 63.7 µmol) was dissolved in THF (0.14 mL) and 1 M aq. LiOH (0.14 mL) was added. After stirring at room temperature for 2 h, the crude was purified by HPLC. The obtained product was dissolved in 37% aq HCl 0.5 mL). TIS (2 drops) was added and the reaction was stirred at r.t. for 4 h. The solution was freeze-dried affording the title compound as a white powder (15 mg, 48% yield).

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Synthetic procedures and characterization of compounds (PDF)

AUTHOR INFORMATION

Corresponding Author * F.C., e-mail: florine.cavelier@umontpellier.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

We thank the association Vaincre la Mucoviscidose (VLM, grant RFI20160501495) for financial support.