Ronan Larget

Brian Lockhart

Pierre Renard

Martine Largeron

A CONVENIENT EXTENSION OF THE WESSELY-MOSER REARRANGEMENT FOR THE SYNTHESIS OF SUBSTITUTED ALKYLAMINOFLAVONES AS NEUROPROTECTIVE AGENTS IN VITRO

Keywords: Antioxidants, Neuroprotective agents, Radical scavengers, Substituent effects

A series of 8-alkylamino-5,7-dihydroxyflavones was prepared from chrysine via a seven step sequence. The synthesis of their 6-alkylamino isomers could be subsequently accomplished through a convenient extension of the Wessely-Moser rearrangement. These compounds were found to be efficient neuroprotective agents in vitro.

disease and Alzheimer's disease. [START_REF] Halliwell | Free Radicals in Biology and Medicine, Third Edition[END_REF] Consequently, supplementation with exogenous antioxidants can represent an important therapeutic potential to minimize central nervous system damage. So, there is considerable interest in the discovery and development of efficient antioxidants.

In the course of our search for new neuroprotective agents, [START_REF] Fleury | [END_REF]3 we recently reported the synthesis of novel 8-alkylamino-3-substituted-1,4-benzoxazine derivatives I as well as 3-alkylamino-2,4-dihydroxybenzophenones II (Figure). [4][5][6]

N O O N H R 3 H R 2 R 1 R HO N O H O H II I Figure
From their capacity to inhibit oxidative stress-mediated neuronal degeneration in vitro, these compounds were found to be potent neuroprotective agents with activity close to that of standard tocopherol. 7,8 From these studies, substituted-1,4-benzoxazines I were identified as the most promising compounds for therapeutic potential owing to their very low intrinsic cytotoxicity. 8,9 Although 3alkylaminophenols II were found to be at least ten fold more toxic than the series of 1,4-benzoxazines, they *Fax : 01 43 29 05 92, E-mail : largeron@pharmacie.univ-paris5.fr remained potent neuroprotective agents. 7 So, we envisioned potentially useful structural modifications in order to enhance antioxidant activity. We thought that it would be then possible to determine a concentration range for which antioxidant effects could be achieved without the manifestation of intrinsic toxicity. Toward this goal, we planned to synthesize 6-alkylamino-5,7-dihydroxyflavones 10a-e (Scheme 1). Flavonoids, in particular polyhydroxyflavones such as quercetin, morin, myricetin and kempferol, are indeed well known to exhibit antioxidant activity. [10][11][12] This raised the question of whether or not 6-alkylaminoflavones 10a-e in general would be more promising derivatives than their benzophenone analogues II.

In this paper, we wish to report the synthesis of 6-alkylamino-5,7-dihydroxyflavones 10a-e that has been successfully accomplished from their 8-alkylamino isomers through a convenient extension of the Wessely-Moser rearrangement. We also disclose the neuroprotective properties of these compounds as part of our continuing research efforts to find safe and effective neuroprotective agents.

Chemistry

At the ouset, our synthetic strategy for the preparation of compounds 10a-e involved the intermediary synthesis of 5,7-dihydroxy-6-nitroflavone, which appeared deceptively simple. This should be further reduced to 6-amino-5,7-dihydroxyflavone, and then alkylated by the appropriate methods leading to the expected compounds 10a-e (Scheme 1). Currently available methods to prepare 5,7-dihydroxy-6-nitroflavone from 2,4,6-trihydroxy-3nitroacetophenone including Baker-Venkataraman rearrangement, 13 synthesis from chalcones 14 and synthesis via an intramolecular Wittig strategy 15 met with total failure. Furthermore, newer methods for directly converting 2,4,6-trihydroxy-3-nitroacetophenone into the required 1,3-diketone intermediate, such as organolithium reagents 16 , or phase-transfer catalysis 17 , proved to be ineffective. At this juncture, an alternative route which involved the functionalization of chrysine 1 was followed for the preparation of 5,7dihydroxy-6-nitroflavone (scheme 2). Unfortunately, the nitration of chrysine 1 with fuming HNO 3 in acetic acid gave the desired 5,7-dihydroxy-6-nitroflavone as a minor product (2% yield), while the unwanted 8nitro isomer 2 was isolated as the main product (70% yield). 18 Nevertheless, in view of the relative ease of preparation of 8-alkylamino-5,7-dihydroxyflavones from 8-nitrochrysine 2, the synthesis of isomers 8a-e was pursued for two reasons. First, encouraged by the results of Wessely and Moser which previously observed the conversion of 5,7,8-trimethoxyflavone into the 5,6,7-trihydroxy compound on demethylation, 19 we thought that 8-alkylaminoflavones 8a-e possibly could serve as starting materials for the preparation of their 6-isomers 10a-e. Second, compounds 8a-e could be interesting to explore the relationship between the position of the alkylamino chain and the neuroprotective activity. Accordingly, 8alkylaminoflavones 8a-e were synthesized from 8-nitrochrysine 2 via a six step sequence, which involved protection and deprotection steps as reported in scheme 2. Alkylation of 8-nitrochrysine 2 with Me 2 SO 4 and K 2 CO 3 , followed by reduction of the nitro group with stannous choride in ethanol, 20 afforded 4 in roughly 60% overall yield. The latter was alkylated, after protection with the 2,4-dinitrobenzenesulfonyl group that could be further cleaved under mild conditions. 21 N-monosubstituted 2,4-dinitrobenzenesulfonamide 5, readily prepared in 88% yield from 8-amino-5,7-dimethoxyflavone 4 and 2,4-dinitrobenzenesulfonyl chloride, could be efficiently N-alkylated with appropriate primary alkyl halides by the conventional methods, in roughly 85% yield. For the introduction of the isopropyl chain, compound 5 was alkylated under the Mitsunobu conditions, 22 leading to compound 6a in 77% yield. Facile deprotection of N,N-disubstituted-2,4-dinitrobenzenesulfonamides 6a-e was achieved by treatment with mercaptoacetic acid and triethylamine in dichloromethane, giving the required compounds 7a-e (71-94% yields). 21 Finally, complete demethylation of compounds 7a-d was successfully performed using 5 equiv. of AlCl 3 , in toluene heated at reflux, to afford compounds 8a-d, in yields ranging from 68 to 92%. In the specific case of compound 7e, bearing an allyl chain, demethylation with 5 equiv. of BBr 3 in dichloromethane at room temperature led to compound 8e in 70% yield. Having established the seven step sequence for the synthesis of 8-alkylamino-5,7-dihydroxyflavones 8a-e, we next turned our attention to the hypothetical conversion of these compounds to their 6-alkylamino isomers 10a-e. It has been found for a long time that flavonoids having the 5,7,8-arrangement of methoxy groups suffered isomeric change, on demethylation, yielding a product with the 5,6,7-arrangement. This conversion, known as the Wessely-Moser rearrangement, 19 was essentially used for the elucidation of structures of flavonoids and was further extended to chromones, xanthones and derivatives. [23][24][25][26] Surprisingly, little attention has been devoted to this rearrangement, as a surrogate of direct synthesis, when the latter was cumbersome and inefficient. 26,27 Furthermore, use of the Wessely-Moser rearrangement has been restricted in general to compounds bearing hydroxy or methoxy groups. Initial attempts to perform the Wessely-Moser rearrangement on 8-nitroflavone 3 were to no avail: in no cases, were we able to detect a trace of the expected 6-nitro isomer. So, we decided to investigate the conversion of 8-alkylamino derivatives 7a-e, by the action of boiling hydrochloric acid. In all cases, the isomeric change took place leading to 6-alkylaminoflavones 9a-e, in good yields ranging from 60 to 74%.

Finally, synthesis of 6-alkylamino isomers 10a-e could be achieved through demethylation at the 7-position, under the conditions reported above 28 (scheme 3).

In vitro biological results

The intrinsic neurotoxicity as well as the neuroprotective activity of 8-alkylaminoflavones 8a-e and 6-alkylaminoflavones 10a-e were assessed in vitro on murine HT-22 hippocampal cell cultures 29 and compared with those of benzophenone analogues II. The results are presented in Table .   In order to determine potential pro-oxidant effects and to select a concentration range lacking intrinsic toxicity suitable for studying the neuroprotective activity of the compounds, the intrinsic neurotoxic effects of each compound was evaluated following two different methods. Neurotoxicity was monitored either by quantification of cellular lysis (death), after measurement of the lactate dehydrogenase (LDH) activity released from damaged cells into the culture supernatant, or by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reduction assay), that allows an evaluation of the "redox state" of the cells and emphazises oxidative stress. The maximum tolerated concentration (MTC), and the concentration producing 50% toxicity (TC 50 ), were estimated for each tested compounds using both MTT and LDH determination.

Neuroprotective properties of alkylaminoflavone derivatives were estimated through their protective effects against L-homocysteic acid (L-HCA) cytotoxicity. The latter has been reported to provoke, on immature primary neurons or on certain neuronal cell lines, oxidative-stress-mediated neuronal degeneration that could be attenuated with antioxidants. The concentration leading to 50% protection (PC 50 ) was estimated for both MTT and LDH determinations.

Taking into account that optimum activity was displayed by alkyl substituents in the benzophenone series II, 8 only were evaluated substituted alkylaminoflavones bearing isopropyl, n-propyl, isobutyl, isopentyl and allyl chains. As reported in Table, all tested flavonoids showed significant in vitro neuroprotective activity, with PC 50 (MTT) values between 4.6 and 7.0 M and PC 50 (LDH) values between 4.2 and 10.0 M. It could be noted that 6-alkylaminoflavones in general seemed to be slightly more active than their 8-isomers, (for example, compare 10b and 8b). Nevertheless, in view of the PC 50 values, it appeared that 6-alkylaminoflavones 10a-d, as well as their benzophenone analogues IIa-d, gave similar results, (for instance, compare 10a and IIa). Furthermore, as they did not exhibit a lower toxicity than that showed by their benzophenone analogues IIa-d, it could be concluded that alkylaminoflavone derivatives could not be considered as more promising compounds for therapeutic potential. The synthesis of the title compounds, as neuroprotective agents, has been successfully accomplished via the Wessely-Moser rearrangement of their 8-isomers.

  Scheme 2

  Scheme 3

  i , nPr, Bu i , Pe i , CH 2 =CHCH 2

Table .

 . In vitro neuroprotective activity of compounds 8a-e and 10a-e.

	N-CH 2 , nPr, J = 6 Hz), 6.33 (s, 1H, 6-H), 6.96 (s, 1H, 3-H), 7.60 (m, 3H, H meta and H para, Ph),
	8.10 (m, 2H, H ortho, Ph), 12.50 (s , 1H, 5-OH); MS (DCI) : m/z = 312 (MH + ).
	5,7
		O	H	O		H	O	H	O		H	O	H	O
					R	N				R	N
	HO			O	HO			O	HO
	R	N	H
				8a-e					10a-e			II
							Toxicity		Protection vs
											2mM L-HCA
	Compd		R	MTC (M)		TC 50 (M)	PC 50 (M)
					MTT		LDH	MTT	LDH	MTT	LDH
	IIa		Pr i	25		100		118	>250	5.5	5.9
	IIb		nPr	25		100		78	>250	5.1	5.1
	IId		Pe i	10		50		20	82	4.0	5.5
	8b		nPr	5		25		14	28	7.0	8.0
	8c			Bu i	10		10		12	12	4.6	4.2
	8d		Pe i	10		10		12	16	5.3	5.5
	8e			CH 2 =CHCH 2	5		10		18	11	>5	>10
	10a		Pr i	10		100		18	152	5.1	6.6
	10b		nPr	5		100		10	176	5.2	5.5
	10d		Pe i	5		100		16	116	5.2	5.2

-dihydroxy-2-phenyl-6-propylamino-4H-1-benzopyran-4-one 10b: 1

  H NMR (300 MHz, DMSO D 6 ): 0.87 (t, 3H, Me, J = 7 Hz), 1.43 (sextet, 2H, CH 2 , J = 7 Hz), 3.20 (t, 2H, N-CH 2 , J = 7 Hz), 6.62 (s, 1H, 8-H), 6.97 (s, 1H, 3-H), 7.56 (m, 3H, H meta and H para, Ph), 8.03 (m, 2H, H ortho, Ph), 12.50 (s , 1H, 5-OH); MS (DCI) : m/z = 312 (MH + ). 29. In vitro biological assays are extensively detailed in references 7 and 8.

	A

Convenient Extension of the Wessely-Moser Rearrangement for the Synthesis of Substituted Alkylaminoflavones as Neuropro- tective Agents in Vitro

  Ronan Larget a , Brian Lockhart b , Pierre Renard C and Martine Largeron a* a Laboratoire de Chimie Analytique et Electrochimie, UMR 8638 CNRS-Université René Descartes, 4, avenue de l'Observatoire, 75270 Paris Cedex 06, b Institut de Recherche Servier, 125 chemin de Ronde, 78290 Croissy-sur-Seine and c ADIR, 1 rue Carle Hébert, 92415 Courbevoie Cedex, France

Acknowledgment : We would like to thank Professor M.-B. Fleury, in whose laboratory this work was carried out, for his help and encouragement.

Abbreviations : L-HCA, L-homocysteic acid; MTC, maximum tolerated concentration; TC 50 , concentration producing 50% toxicity; PC 50 , concentration producing 50% protection; Bu i , isobutyl; Pe i , isopentyl; nPr, npropyl; Pr i , isopropyl.

In summary, we have synthesized a series of novel 8-alkylaminoflavone derivatives from chrysine via a seven step sequence. The synthesis of their 6-alkylamino isomers could be then successfully accomplished through a convenient extension of the Wessely-Moser rearrangement. To the best of our knowledge, such a rearrangement had not been as yet reported on alkylaminoflavones. Finally, the results of our biological evaluation revealed that replacement of the benzophenone skeleton by the flavone ring did not markedly affect the neuroprotective activity in vitro.


Rabiâa Merrouche

Amine Yekkour
email: amineyek@gmail.com

Yannick Coppel

Noureddine Bouras

Abdelghani Zitouni

Florence Mathieu

Nasserdine Sabaou

Saccharothrix algeriensis NRRL B-24137, the first non-Streptomyces actinobacterium, produces holomycin after cystine feeding

Keywords: Dithiolopyrrolone antibiotics, Holomycin, Saccharothrix algeriensis NRRL B-24137, L-cystine, Antimicrobial activity

Saccharothrix algeriensis NRRL B-24137 is an actinobacterium isolated from Algerian Saharan soil. This strain has the ability to produce several dithiolopyrrolone antibiotic derivatives depending on the precursors added to the culture medium. This group of antibiotics is known for their potent antimicrobial and anticancer activities. Holomycin is a member of the dithiolopyrrolone group of antibiotics, and has already been isolated from several species of actinobacteria belonging to the genus Streptomyces and also from some Gram-negative bacteria. In this study, holomycin was produced for the first time in the culture broth of a non-Streptomyces actinobacteria. This antibiotic was induced by adding 5 mM of L-cystine as precursor to the semi-synthetic fermentation broth of Sa. algeriensis NRRL B-24137 and then fully identified after HPLC purification. The minimum inhibitory concentrations (MIC) of holomycin were determined against several pathogenic microorganisms, including Escherichia coli ATCC 10536 Klebsiella pneumoniae CIP 82.91, Listeria monocytogenes CIP 82110, Staphylococcus aureus CIP 7625, Aspergillus carbonarius M333, Fusarium culmorum FC1, Candida albicans IPA 200. This antibiotic showed a broad-spectrum antimicrobial activity, inhibiting a variety of Gram-positive and Gram-negative bacteria, and micro-fungi.

Introduction

Dithiolopyrrolones, which possess a unique pyrrolinonodithiole skeleton linked to two variable acyl groups, are a group of natural antibiotics that have showed strong broad-spectrum activities towards various Gram-positive and Gram-negative bacteria, protozoa, yeast, pathogenic micro-fungi, and also against different cell lines involved in human cancers [START_REF] Jimenez | Mode of Action of Thiolutin, an inhibitor of macromolecular synthesis in Saccharomyces cerevisiae[END_REF][START_REF] Oliva | Antimicrobial properties and mode of action of the pyrrothine holomycin[END_REF][START_REF] Li | Dithiolopyrrones: biosynthesis, synthesis and activity of unique class of disulfide-containing antibiotics[END_REF][START_REF] Merrouche | Efficiency of Saccharothrix algeriensis NRRL B-24137 and its produced antifungal dithiolopyrrolones compounds to suppress Fusarium oxysporum-induced wilt disease occurring in some cultivated crops[END_REF]. Thiolutin was shown to potently inhibit developmental of angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures by inhibition of endothelial cell adhesion [START_REF] Jia | Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton[END_REF]). In the context of emerging antibiotic-resistant microorganisms, holomycin, one of the three subgroups of dithiolopyrrolones, received great interest regarding its activity against rifamycin-resistant bacteria as well as against methicillin-resistant Staphylococcus aureus N315 [START_REF] Qin | Dithiolopyrrolone natural products: isolation, synthesis and biosynthesis[END_REF]. This activity was determined to be involved in the inhibition of bacterial RNA polymerase [START_REF] Oliva | Antimicrobial properties and mode of action of the pyrrothine holomycin[END_REF][START_REF] Qin | Dithiolopyrrolone natural products: isolation, synthesis and biosynthesis[END_REF]. Holomycin was first discovered in Streptomyces griseus [START_REF] Ettlinger | Stoffwechselprodukter von Actinomyceten, holomycin[END_REF] and later was reported to be isolated from a number of Streptomyces species [START_REF] Okamura | Holomycin and N-propionylholothin, antibiotics produced by a cephamycin C producer[END_REF][START_REF] Kenig | Holomycin and an antibiotic (MM 19290) related to tunicamycin, metabolites of Streptomyces clavuligerus[END_REF][START_REF] Ding | Derivatives of holomycin and cyclopropaneacetic acid from Streptomyces sp[END_REF][START_REF] Wei | Identification and characterization of Streptomyces flavogriseus NJ-4 as a novel producer of actinomycin D and holomycin[END_REF][START_REF] Zhang | Antibacterial activity and composition of the fermentation broth of Streptomyces parvus 33[END_REF]. The Gram-negative bacteria Photobacterium halotolerans and Yersinia ruckeri were also found to produce holomycin [START_REF] Wietz | Antibacterial compounds from marine Vibrionaceae isolated on a global expedition[END_REF][START_REF] Qin | Dithiolopyrrolone natural products: isolation, synthesis and biosynthesis[END_REF].

Saccharothrix algeriensis NRRL B-24137 is a rare actinobacterium first isolated from the soil of a palm grove in Southern Algerian Sahara [START_REF] Zitouni | Saccharothrix algeriensis sp. nov., isolated from a Saharan soil[END_REF]. This strain was shown to produce several dithiolopyrrolones depending on the incorporated precursors available in the culture medium. Five dithiolopyrrolone derivatives were produced in complex ISP2 medium (thiolutin, senecioylpyrrothine, tigloyl-pyrrothine, iso-butyryl-pyrrothine and butanoyl-pyrrothine) (Lamari et al. 2002a, b). Interestingly, the selection of appropriate sources of carbon and nitrogen was shown to have a significant effect on the production levels of these five known dithiolopyrrolones, and also on the induction of biosynthesis of new dithiolopyrrolone derivatives in fermentations that had been supplemented with valeric, benzoic or cinnamic acids (Bouras et al. 2006a(Bouras et al. , b, 2007[START_REF] Bouras | Precursor-directed biosynthesis of new dithiolopyrrolone analogs by Saccharothrix algeriensis NRRL B-24137[END_REF]. Thus, eight novel dithiolopyrrolones have been produced and characterized after addition of different carbon sources in culture broth of Sa. algeriensis (valeryl-pyrrothine, isovaleryl-pyrrothine, formyl-pyrrothine, crotonyl-pyrrothine, sorbyl-pyrrothine, 2-hexonyl-pyrrothine, 2-methyl-3-pentenyl-pyrrothine and benzoyl-pyrrothine) [START_REF] Merrouche | Dithiolopyrrolone antibiotic formation induced by adding valeric acid to the culture broth of Saccharothrix algeriensis[END_REF][START_REF] Merrouche | New dithiolopyrrolone antibiotics induced by adding sorbic acid to the culture medium of Saccharothrix algeriensis NRRL B-24137[END_REF][START_REF] Merrouche | Effective biosynthesis of benzoyl-pyrrothine dithiolopyrrolone antibiotic by cinnamic acid-precursor addition in culture of Saccharothrix algeriensis NRRL B-24137[END_REF].

Through early feeding experiments, L-cystine appeared to be the precursor of dithiolopyrrolone biosynthesis [START_REF] Fukuda | Function of plasmid in the production of aureothricin 1. Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis[END_REF], but the biosynthesis pathway remained poorly understood until the last decade. Works on Sa. algeriensis NRRL B-24137 have determined that the transfer of the acyl group from acyl-CoA to a pyrrothine core, during dithiolopyrrolone biosynthesis, was linked to enzymatic activity of pyrrothine N-acyltransferase [START_REF] Chorin | Expression of pyrrothine N-acyltransferase activities in Saccharothrix algeriensis NRRL B-24137: new insights into dithiolopyrrolone antibiotic biosynthetic pathway[END_REF][START_REF] Saker | Enzymatic synthesis of dithiolopyrrolone antibiotics using cell-free extract of Saccharothrix algeriensis NRRL B-24137 and biochemical characterization of two pyrrothine N-acyltransferases in this extract[END_REF]. [START_REF] De La Fuente | Mutants of Streptomyces clavuligerus with disruptions in different genes for clavulanic acid biosynthesis produce large amounts of holomycin: possible cross-regulation of two unrelated secondary metabolic pathway[END_REF] has also shown that an N-acetyltransferase could be involved in the holomycin biosynthesis in cell-free extracts of the holomycin producer, Streptomyces clavuligerus. More recently, the gene cluster for holomycin biosynthesis was first characterized in S. clavuligerus ATCC 27064 [START_REF] Li | Identification of the gene cluster for the dithiolopyrrolone antibiotic holomycin in Streptomyces clavuligerus[END_REF] and later in Pseudoalteromonas sp. [START_REF] Fukuda | Function of plasmid in the production of aureothricin 1. Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis[END_REF] and Yersinia ruckeri [START_REF] Qin | Dithiolopyrrolone natural products: isolation, synthesis and biosynthesis[END_REF].

The present study showed that the addition of 5 mM of L-cystine in a semi-synthetic medium as a nitrogen source has allowed the induction of holomycin biosynthesis by Sa. algeriensis NRRL B-24137. This antibiotic has been isolated for the first time from a non-Streptomyces genus member of actinobacteria.

Materials and methods

Producing strain

Saccharothrix algeriensis NRRL B-24137 was grown and maintained at 4 °C on slants of ISP2 (International Streptomyces Project 2) solid medium [START_REF] Shirling | Methods for characterization of Streptomyces species[END_REF]. The pH of the medium was adjusted to 7.0 with 2-M NaOH before autoclaving at 120 °C for 20 min.

Fermentation conditions

The basal semi-synthetic medium (SSM) developed by Bouras et al. (2006a) was used for the antibiotic production. This medium consisted of 10-g d-glucose (Fisher Scientific, Illkirch, France), 2-g (NH 4 ) 2 SO 4 (Prolabo, Paris, France), 2-g NaCl (Fisher Scientific), 0.5-g KH 2 PO 4 (Acros, Geel, Belgium), 1-g K 2 HPO 4 (Acros), 0.2-g MgSO 4 7H 2 O (Acros), 5-g CaCO 3 (Prolabo) and 2-g yeast extract (Difco, Detroit, MI, USA) in 1-L distilled water. The pH of the medium was adjusted to 7 using 2-M NaOH before autoclaving. L-cystine (Fluka, Buchs, Switzerland) was autoclaved separately and added to the basal SSM at a concentration of 5 mM which is the optimal concentration for antibiotic production as reported by [START_REF] Bouras | Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137[END_REF]. A mature slant culture of Sa. algeriensis was used to inoculate autoclaved 500-mL Erlenmeyer flasks, containing 100 mL of basal SSM. Concurrently, a control (SSM without L-cystine) was also prepared. The cultures were incubated on a rotary shaker (240 rpm) at 30 °C for 10 days.

Kinetics of growth, pH, antimicrobial products and antibiotics production

The changes in dry cell weights (DCWs), antimicrobial activity, pH, and antibiotic production were examined daily during 10 days of fermentation. DCWs were determined as described by Bouras et al. (2006a), briefly, the DCWs were determined by centrifuging (Microlitre Centrifuges, Heraeus Instruments, Biofuge, Hanau, Germany) 4 mL of homogenized culture broth in pre-weighed Eppendorf tubes for 10 min at 16,000×g. The pellet was washed three times with HCl (0.35 N, Sigma-Aldrich, Saint-Quentin-Fallavier, France) to eliminate CaCO 3 , followed by distilled water. The Eppendorf tubes containing pellet were dried at 105 °C for 24 h, cooled in a desiccator, and weighed. The results were expressed as gram per liter. The pH value was measured with a pH meter (Consort C 832, Consort, NY, USA). The activity against Bacillus subtilis ATCC 6633 and Umbelopsis ramanniana NRRL 1829, used as test microorganisms, was regularly recorded each day by the agar diffusion method. Briefly, the microbial suspensions were prepared in sterile distilled water and adjusted at a concentration of 1.2 × 10 8 CFU/mL and 0.4 × 10 6 CFU/mL for bacteria and micro-fungi, respectively. A volume of 20-mL nutrient agar (Difco) was inoculated with 20 μL of the considered microbial suspension and then poured into a Petri dish. After solidification, wells of 10 mm diameter were aseptically bored into the culture medium, and 200 μL of the culture supernatant was loaded in each well. The antimicrobial activities were evaluated by measuring the diameter of inhibition corresponding to the clear zones around the wells after 24 h and 48 h of incubation for bacteria and microfungi, respectively.

Concomitantly, the quantification of the induced dithiolopyrrolones was performed daily using a thiolutin standard calibration curve, since the molar extinction coefficient (ɛ) of thiolutin is nearly the same for all others dithiolopyrrolones (ɛ390 = 8317-9333 L/mol/cm) as described by Lamari et al. (2002b). All tests were repeated two times from two separate cultures.

HPLC analysis and purification of the induced compound

The analysis of antibiotics induced by addition of L-cystine (at 5 mM) in the SSM was carried out according to the procedure described earlier by [START_REF] Merrouche | New dithiolopyrrolone antibiotics induced by adding sorbic acid to the culture medium of Saccharothrix algeriensis NRRL B-24137[END_REF]. After mycelium was separated, the culture filtrate broth was extracted with an equal volume of dichloromethane (Fisher Scientific) and the organic layer was dried with anhydrous sodium sulfate (Acros). The extract was concentrated to dryness under vacuum on a Rotavapor (Laborata 4000, Heidolph, Illkirch, France), and then re-dissolved in 1 mL of methanol (Fisher Scientific) as crude extract. The analysis of antibiotics induced by addition of L-cystine in the SSM was carried out by a HPLC system equipped with a C18 reverse phase column (Zorbax SB, 150 × 4.6 mm; BioTek Instruments, Milan, Italy). The crude extracts were analyzed by linear gradient elution using methanol-water as mobile phase. The methanol separation gradient was 0-30% for 5 min, then 30-100% for 25 min with a flow rate of 0.8 mL/ min. The formation of the new dithiolopyrrolone analog was monitored by comparison of the peak retention times and UV spectra with those of already known dithiolopyrrolone standards (produced by Sa. algeriensis) for distinction, since appearing dithiolopyrrolone products could be easily detected by HPLC analysis due to their intense absorption at 390 nm (Lamari et al. 2002b;[START_REF] Bouras | Precursor-directed biosynthesis of new dithiolopyrrolone analogs by Saccharothrix algeriensis NRRL B-24137[END_REF].

To obtain a sufficient pure quantity of the induced new compound, for subsequent characterization, cultures were combined to obtain 15 l. The culture broth was centrifuged to remove the biomass and extracted with an equal volume of dichloromethane on the seventh day of fermentation.

The organic extract was concentrated to dryness. The crude extract was dissolved in methanol and subjected to semipreparative HPLC purification on a Waters system using a C18 column (UP5ODB, 250 × 7.8 mm, 227 Waters, Milford, MA). The analysis was done using a linear gradient of methanol-water (20-100% for 30 min), as the mobile phase, with a flow rate of 1.5 mL/min. The detection of compounds was carried out at 390 nm.

Chemical characterization of induced compound

The UV spectrum was determined with a Shimadzu UV1605 spectrophotometer (Shimadzu, Kyoto, Japan). The molecular weight of holomycin was obtained by electron impact MS (EI-MS) recorded at 70 eV with a Nermag R-10-10C spectrometer. The nuclear magnetic resonance (NMR) sample was prepared by dissolving the pure molecule cyst1 in 600 mL of CD 2 Cl 2 . 1D 1 H experiment was recorded on a Bruker Avance 500 spectrometer equipped with a 5-mM triple resonance inverse Z-gradient probe (TBI 1 H, 31 P, BB). All chemical shifts for 1 H are in comparison with tetramethylsilane (TMS) using 1 H (residual) of the solvent as a secondary standard. The temperature was set at 298 K.

Antimicrobial MIC of induced compound

The MIC of the induced antibiotic was determined by a conventional agar dilution method [START_REF] Oki | Pradimicins A. B and C: new antifungal antibiotics II. In vitro and in vivo biological activities[END_REF]). Twenty target micro-organisms (Table 1) were used and inoculated onto nutrient agar medium containing different concentrations of the purified induced antibiotic compound (1,2,3,5,8,10,15,20,25,30,40,50,60,80 and 100 µg/mL). The antimicrobial activity was observed after a 24-48-h incubation at 37 °C for bacteria and a 48-72-h incubation at 28 °C for micro-fungi and yeasts. Medium without the antibiotic compound and inoculated with target microorganisms was used as a control.

All the chemicals used were supplied at a purity level ≥ 99%.

Results and discussion

Effect of L-cystine addition on growth, pH, antibiotics production and antimicrobial activity in Sa. algeriensis

To evaluate the effect of 5-mM L-cystine supplementation to SSM, the kinetic evolution of fermentation parameters (antibiotics production, DCW and pH) was monitored in comparison to non-supplemented control medium (Fig. 1) along with the analysis of the produced antibiotics by HPLC (Fig. 2). With regard to retention times and UV characteristics of HPLC-obtained fractions profi le from the 7-day-old crude fermentation extract, Sa. algeriensis NRRL B-24137 was shown to produce five known dithiolopyrrolones (thiolu tin, iso-butyryl-pyrrothine, butanoyl-pyrrothine, tigloyl pyrrothine and senecioyl-pyrrothine) in the basal control SSM (without addition of precursors) (Fig. 2a) as reported by Lamari et al. (2002b). Remarkably, addition of 5 mM of L-cystine in SSM induced the appearance of a new peak named Cystl (Fig. 2b), which bas a different retention time of 11.10 min in comparison to those of known dithiolopyr rolone produced in the control.

The production of the induced compound Cystl started at the 2nd day and reached a maximum after 4 days of fermentation, with a production of 0.21 mg/L (Fig. l b). The L-cystine supplementation triggered also modifica tion in growth kinetic and pH in comparison with the con trol (Fig. le,d). In fact, without supplementation (control While, addition of L-cystine permitted to obtain a greater maximal biomass (0.75 ± 0.08 g/L) after a fermenta tion time of 3 days. Moreover, the final pH at the end of fermentation seemed slightly less alkaline for L-cystine supplemented conditions in comparison to control. These results were in agreement with the previous findings that demonstrated for all the described Sa. algeriensisderived dithiolopyrrolones a production in the decline phase of growth with alkaline pH condition (Lamari et al. 2002a;[START_REF] Merrouche | New dithiolopyrrolone antibiotics induced by adding sorbic acid to the culture medium of Saccharothrix algeriensis NRRL B-24137[END_REF][START_REF] Merrouche | Effective biosynthesis of benzoyl-pyrrothine dithiolopyrrolone antibiotic by cinnamic acid-precursor addition in culture of Saccharothrix algeriensis NRRL B-24137[END_REF].

Purification and characterization of induced compound

After 7 days of fermentation in L-cystine-supplemented SSM, 15 L of the culture broth was extracted by dichloromethane and the yellow organic phase was concentrated to dryness. Through semi-preparative HPLC, the new Cyst1 compound was repetitively recovered and then finally purified after two successive re-injections in the HPLC system (Fig. 2c). This induced compound was subject to spectroscopic and spectrometric analyses to elucidate its structure.

The UV-visible spectrum of Cyst1 showed three absorption maxima at 245, 300 and 384 nm (Supplementary Fig. 1).

Through EI-MS analysis, compound Cyst1 exhibited a mass unit of m/z 214 (by EI-MS) (Fig. 3), that is similar to the molecular weight of formyl-pyrrothine, a known dithiolopyrrolone produced by Sa. algeriensis [START_REF] Merrouche | Dithiolopyrrolone antibiotic formation induced by adding valeric acid to the culture broth of Saccharothrix algeriensis[END_REF]. However, in contrast to formyl-pyrrothine, which exhibits a prominent fragment ion at m/z 186, Cyst1 showed a prominent fragment ion at m/z 172, indicating an extra hydrogen in the heterocyclic ring as reported for other dithiolopyrrolones [START_REF] Okamura | Holomycin and N-propionylholothin, antibiotics produced by a cephamycin C producer[END_REF][START_REF] De La Fuente | Mutants of Streptomyces clavuligerus with disruptions in different genes for clavulanic acid biosynthesis produce large amounts of holomycin: possible cross-regulation of two unrelated secondary metabolic pathway[END_REF]. While, a prominent fragment ion at m/z 186 indicated an extra methyl group in the heterocyclic ring instead of a hydrogen [START_REF] Merrouche | Dithiolopyrrolone antibiotic formation induced by adding valeric acid to the culture broth of Saccharothrix algeriensis[END_REF].

By the analysis of the 1 H NMR spectrum (Supplementary Fig. 2), it is possible to discern one olefinic group (δ H 6.83) and NH groups (δ H 8.03/7.93) typical of dithiolopyrrolone derivatives. Furthermore, compound Cyst1 shows one additional 1 H methyl signal (δ H 2.14). However, a signal at 3.4 ppm corresponding to a methyl linked to Nitrogen of the cycle (N-CH 3 ) was not observed. The methyl was replaced by a hydrogen (N-H). The 1 H chemical shifts of the compound were as follows: 1 H NMR (CD 2 Cl 2 , 500 MHz) δ Fig. 2 Effect of L-cystine addition to the SSM on dithiolopyrrolones production displayed by HPLC analysis. a On SSM without L-cystine (control). b After addition of 5-mM L-cystine to SSM. c Purification of the compound Cyst1 after 2 successive re-injections. HPLC analysis at 390 nm was done on dichloromethane extract obtained from an 8-day-old Sa. algeriensis culture. Formation of dithiolopyrrolone analogs was monitored by comparison of the peak retention times and UV (390 nm) spectra with those of known dithiolopyrrolone standards. The retention times were as follows: Cyst1,11.10 min;thiolutin,12.50 min;16.80 min;17.60 min;18.50 min;18.80 min dithiolopyrrolone derivative identifi ed for the fi rst time in Sa. algeriensis and more generally in a non-Streptomyces strain.

Accordingly, this study showed that the addition of 5 mM of L-cystine in SSM as precursor induced biosynthesis of holomycin by Sa. algeriensis. Bouras et al. (2006a, b) have already suggested that sulfur-containing amino acids, such L-cystine, can serve as a tool for studying the mechanism of dithiolopyrrolones biosynthesis in Sa. algeriensis.

As known, Sa. algeriensis bas a remarkable ability to pro duce a wide range of dithiolopyrrolone derivatives de pe nd ing on the composition of the culture medium, and both the nature and concentration of precursors added (Lamari et al. 2002b;Bouras et al. 2006aBouras et al. , b, 2007)). This extraordinary flexibility of the dithiolopyrrolone biosynthetic pathway in Sa. algeriensis may be rendered by the enzymatic sys tem, which was reported to be responsible for this diversity [START_REF] Saker | Enzymatic synthesis of dithiolopyrrolone antibiotics using cell-free extract of Saccharothrix algeriensis NRRL B-24137 and biochemical characterization of two pyrrothine N-acyltransferases in this extract[END_REF]. This pro pe rty was explored by intro duction of various organic acids into fermentation media and led to generate 10 new dithiolopyrrolone analogs by precursor-directed biosynthesis (PDB) process in Sa. algeriensis [START_REF] Bouras | Precursor-directed biosynthesis of new dithiolopyrrolone analogs by Saccharothrix algeriensis NRRL B-24137[END_REF][START_REF] Merrouche | Dithiolopyrrolone antibiotic formation induced by adding valeric acid to the culture broth of Saccharothrix algeriensis[END_REF][START_REF] Merrouche | New dithiolopyrrolone antibiotics induced by adding sorbic acid to the culture medium of Saccharothrix algeriensis NRRL B-24137[END_REF][START_REF] Merrouche | Effective biosynthesis of benzoyl-pyrrothine dithiolopyrrolone antibiotic by cinnamic acid-precursor addition in culture of Saccharothrix algeriensis NRRL B-24137[END_REF].

The PDB is the simplest derivatization of a secondary metabolite by feeding biosynthetic precursor analogs into the fermentation broth of the producing microorganisms [START_REF] Thiericke | Biological variation of microbial metabolites by precursor-directed biosynthesis[END_REF]. Moreover, PDB provides a potent method to introduce non-native starting materials into biosynthetic pathways. It should be possible to reprogram the biosynthesis of natural products to produce new molecules with altered and possibly improved biological activities. PDB has proven to be a powerful tool for the rational synthesis of structural analogs of erythromycin and sansanmycin [START_REF] Cane | Precursor-directed biosynthesis: biochemical basis of the remarkable selectivity of the erythromycin polyketide synthase towards unsaturated triketides[END_REF][START_REF] Zhang | Precursor-directed biosynthesis of new sansanmycin analogs bearing para-substituted-phenylalanines with high yields[END_REF]) and for the production of borrelidin analog in Streptomyces rochei MB037, which presented low cytotoxicity and a potent inhibition of angiogenesis [START_REF] Li | Comprehensive optimization of precursor-directed production of BC194 by Streptomyces rochei MB037 derived from the marine sponge Dysidea arenaria[END_REF].

MIC of induced compound

The antimicrobial activity of the pure antibiotic compound Cyst1 is shown in Table 1. This antibiotic showed a broad antibacterial spectrum, inhibiting a variety of Gram-positive and Gram-negative bacteria tested, except Agrobacterium tumefaciens 2410 LB, Salmonella enterica CIP 81.3 and Pseudomonas aeruginosa CIPA22 which were resistant. The latter Gram-negative bacterium has been previously noted to be resistant to holomycin [START_REF] Oliva | Antimicrobial properties and mode of action of the pyrrothine holomycin[END_REF][START_REF] Qin | Dithiolopyrrolone natural products: isolation, synthesis and biosynthesis[END_REF]. Cyst1 antibiotic showed a strong to moderate activity against all filamentous fungi tested, the most sensitive micro-fungi were Aspergillus carbonarius M333 and Fusarium moniliforme FM1 (CMI = 8 µg/mL). This antibiotic lacked activity against the yeasts Saccharomyces cerevisiae ATCC 4226 and Candida albicans IPA 200, in accordance with [START_REF] Oliva | Antimicrobial properties and mode of action of the pyrrothine holomycin[END_REF] results, that already reported an absence of holomycin activity against S. cerevisiae and Candida kefyr.

In conclusion, this work demonstrates that Saccharothrix algeriensis NRRL B-24137 produced holomycin, as the first known non-Streptomyces actinobacteria exhibiting this ability, in a cystine-amended semi-synthetic culture medium.

Given the great Sa. algeriensis ability to produce a wide range of dithiolopyrrolone derivatives depending on the composition of the culture medium, our study used the "one strain many compounds (OSMAC)" approach as a simple and effective method to generate a maximum number of bioactive secondary metabolites from a single well-characterized microbial genome. In accordance, we have observed that Sa. algeriensis produces other unknown dithiolopyrrolones when supplied with other different amino acids such as arginine (data not shown). Further studies on the characterization of these new dithiolopyrrolones are currently underway.

  microorganisms without an accession number were from our laboratory collection Compared to the control, the antimicrobial activity started earlier, at the 4th day of fermentation, in the presence of L-cystine against both Bacillus subtilis ATCC 6633 and Umbelopsis ramanniana NRRL 1829. Moreover, the Sa. algeriensis antimicrobial activity seemed better after the addition of L-cystine, and reached its maximum between the 7th and 8th days of fermentation (Fig. l a).

Fig. 1

 1 Fig. 1 Effect of L-cystine addition to tbe SSM on evolution of: a antimicrobial activity; b production of a new induoed dithiolopyr rolone (Cyst l); c biomass; and d pH. Umbelopsis ramanniana NRRL 1829 (line), Bacillus subtilis ATCC 6633 (dotted line); (open trian gle) SSM without adding L-cystine (control), (open circle) SSM with L-cystine at 5 mM

FigFig. 4

 4 Fig. 3 Electron impact mass spectrum of the purified Cystl antibiotic compound

Table 1

 1 Minimum inhibitory concentrations (µg/mL) of holomy cin antibiotic produced by Saccharothrix algeriensis against several microorganisms Target organism

		MIC (µg/mL)
	Agrobacterium tumefaciens 2410 LB*	80
	Escherichia coli ATCC 10536	75
	Klebsiella pneumoniae CIP 82.91	8
	Salmonella enterica CIP 81.3	80
	Pseudomonas aeruginosa CIPA22	60
	Bacillus subtilis ATCC 6633	8
	Bacillus coagulans CIP 6625	8
	listeria monocytogenes CIP 82110
	Micrococc11s l11teus ATCC 9314
	Staphylococc11s a11reus CIP7625
	Aspergillus carbonarius M333*
	Fusarium oxyspor11m f. sp. lini Foin 3*
	Fusarium moniliforme FMI*
	Fusarium equiseti FEI*
	Fusarium culmor11m FC I *
	Fusarium graminearum FG I *
	Umbelopsis ramanniana NRRL 1829
	Penicillium expansum PEI*
	Candida albicans IPA 200
	Saccharomyces cerevisiae ATCC 4226

Institut National de Recherche Agronomique d'Algérie, Station Mehdi Boualem-Baraki, Alger, Algeria

Laboratoire de Chimie de Coordination, CNRS, Université de Toulouse, UPS, INPT, Toulouse, France

Département de Biologie, Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre, Université de Ghardaia, Ghardaïa, Algeria

Laboratoire de Génie Chimique, Université de Toulouse, CNRS, Toulouse, France

Supplemental Fig. 1 UV spectrum of compound Cyst1. Supplemental Fig. 2 1 H NMR spectrum of compound Cyst1. Supplemental Fig. 1 Supplemental Fig. 2


K A K Gadallah
email: k.gadallah@azhar.edu.eg

A Sow

E Congiu

S Baouche

F Dulieu

Variation of the sticking of methanol on low temperature surfaces as a possible obstacle to freeze out in dark clouds

Keywords: ISM, molecules -methods, laboratory, solid state -methods, laboratory, molecular

Sticking of gas phase methanol on different cold surfaces -gold, 13 CO, and Amorphous Solid water ice (ASW) -was studied as a function of surface temperature (7 -40 K). In an ultrahigh-vacuum system, RAIRS and TPD methods were simultaneously used to measure methanol sticking efficiency. Methanol band strengths obtained by RAIRS vary greatly depending on the type of the surface. Nevertheless, both methods indicate that the sticking of methanol on different surfaces varies with surface temperature. The sticking efficiency decreases by 30% as the surface temperature goes from 7 to 16 K, then gradually increases until the temperature is 40 K, reaching approximately the initial value found at 7 K. The sticking of methanol differs slightly from one surface to another. At low temperature it has the lowest values on gold, intermediate values on water ice and the highest values are found on CO ice, although these differences are smaller than those observed with temperature variation. There exists probably a turning point during the structural organisation of methanol ice at 16 K, which makes the capture of methanol from the gas phase less efficient. We wonder if this observation could explain the surprising high abundance of gaseous methanol observed in dense interstellar cores, where it should accrete on grains. In this regard, a 30% reduction of the sticking is not sufficient in itself but transposed to astrophysical conditions dominated by cold gas (∼ 15 K), it could reduce the sticking efficiency by two orders of magnitude.

INTRODUCTION

Methanol is the prototypical interstellar complex organic molecule (Herbst & van Dishoeck 2009;Caselli & Ceccarelli 2012), and is considered as a proxy for many others molecules (Ceccarelli et al. 2017). It is often used as an ideal tool for determining the physical conditions (Kristensen et al. 2010). Moreover, It has been observed in many different environments such as in dark clouds (Friberg et al. 1988), stellar cores in molecular clouds (Bisschop et al. 2007;Punanova et al. 2018), proto-stellar envelopes, (Taquet et al. 2015), circumstellar regions (Charnley et al. 1995), protoplanetary disks (Walsh et al. 2016[START_REF] Walsh | IAU Symposium[END_REF], young stellar objects (Bottinelli et al. 2010;Penteado et al. 2015), comets (Bockelee-Morvan et al. 1991), and extragalactic environments (Galametz et al. 2016). Often, in warm environments, ice mantles can release methanol to the gas-phase by means of thermal desorption. But in the coldest and darkest places such as pre-stellar cores, gas phase COMs are not expected to be as abundant as observations suggest (Bacmann et al. 2012;Jiménez-Serra et al. 2016, 2018;Punanova et al. 2018). The enigma of the presence of COMs in the gas phase at low temperature in the dense phases of the interstellar medium stems from the fact that that COMs and any other molecule (except H 2 ) should condense onto dust grains within a short timescale (Bergin & Tafalla 2007).

There are different possibilities to explain the unexpected high abundances of COMs. One of them is that there are different non-thermal mechanism at work desorbing the adsorbed species. One possible mechanism is the chemical desorption (Dulieu et al. 2013). Indeed, after a reaction on a cold surface, the excess of energy can induce the return in the gas phase of the products. Therefore, methanol can be released in the gas phase during its formation on dust grains, and this process can be specifically efficient in the case of CO hydrogenation (Minissale et al. 2016a). Vasyunin & Herbst (2013) have demonstrated that methanol gas-phase abun-dance in different astrophysical environments can be explained by the desorption of a few per cent of methanol formed on the grain surfaces. Using revised experimental values of Minissale et al. (2016b), Vasyunin et al. (2017) successfully reproduced the abundance and the position of COMs of the L1544 prestesellar core.

Another possibility of non-thermal desorption is the sputtering by cosmic rays of solid methanol, as measured by Dartois et al. (2019) which releases intact molecules into the gas phase, contrarily to UV photons which easily photolyses large molecules (e.g., Cruz-Diaz et al. 2016;Bertin et al. 2016), and therefore mostly returns fragments in the gas phase. Similarly, electron irradiation of water ice (Gadallah et al. 2017;Marchione & McCoustra 2017) releases simple molecules such as H 2 , HD and D 2 more efficiently than larger ones. Latter authors have shown that H 2 formation is more significant for organic-based ices, such as methanol ice, than in water ice. This might indicate that processes driven via electronic excited states produced by dipole scattering of the CR in solid methanol do not produce significant molecular methanol.

In addition to the formation of small molecules (e. g. formaldehyde, methane, carbon monoxide, carbon dioxide, and water), the VUV photo-processing of methanol ice (Paardekooper et al. 2016) has also produced COMs such as methyl formate, ethylene glycol, dimethyl ether. This process reveals a reverse relationship between the rest of methanol and other outcomes with increasing the VUV fluence. Similar VUV photolysis with much longer irradiation of methanol ice (Abou Mrad et al. 2016) has produced small molecules and COMs with a variety of carbon chain, where abundances of these COMs decrease with increasing their carbon chain.

The other possibility to solve the enigma of the presence of certain molecules in the gas phase in dark and cold media is that there exist selective mechanisms to inhibit the accretion of some molecules. Nguyen et al. (2018) have studied the case of surface CO molecules that change the adsorption properties of N 2 , but it cannot apply for larger molecules. A difference of sticking probability for some specific molecules or under specific conditions would also greatly affect the gas phase composition and would explain how some molecules can be maintained in the gas phase. This is the starting point of the present study. However, the sticking of molecules like H 2 O, CH 4 , N 2 and CO has already been studied (Kimmel et al. 2001;Acharyya et al. 2007;Bisschop et al. 2006), and it has always been claimed to be "close to unity", and as such rounded to one in models. There is an exception in the case of H 2 and D 2 (Matar et al. 2010;Chaabouni et al. 2012) since it has been measured to be as low as 25%. This is due to the fact that a molecule has to lose some initial kinetic energy when impinging the surface, and if the energy loss is not efficient enough, the impactor just bounces back. Therefore, the sticking probability is dominated by the ability of the surface to evacuate the excess energy of the impactor. In the case of H 2 the mass match is very poor and this is why the sticking coefficient is rapidly decreasing with the temperature of the gas (the impactor). But the sticking coefficient, extrapolated at 0 K is not 100%. This is not obvious because one could consider that the probability of capture of the impactor should be unity since there is no energy to dissipate and/or since the interaction time is infinite, and that the energetic balance is favourable to adsorption. But this is only true if there is no entrance barrier, in this case some kinetic energy of the impactor is required to pass the saddle point. In other terms, sometimes the substrate has to be slightly geometrically reorganised and this requires some energy, which may be provided by the kinetic energy of the impactor.

Sometimes, the molecules can react with the surface as it is the case of methanol on a platinum surface (Diekhöner et al. 1998). On platinum, also the probability of its decomposition decreases as a function of both the surface temperature (400-1000 K) and the incidence energy (<0.5 eV) of methanol.However, metallic surfaces are of low interest for astrophysics. To date, there is no complete study of the sticking of methanol at cold temperatures. On the contrary there are many studies about its desorption (Sandford & Allamandola 1993;Collings et al. 2004;Wolff et al. 2007;Bahr et al. 2008;Green et al. 2009;Smith et al. 2014;Doronin et al. 2015;Luna et al. 2018). In these experiments we can learn that the methanol ice structuring can be complex.

In this paper, we study the sticking of methanol on cold surfaces as a function of surface temperature (T s ) in the range 7 -40 K. The surfaces are made of gold, 13 CO, and pours amorphous solid water (ASW). We measure the total molecules adsorbed by using both temperature-programmed desorption (TPD) and, in situ, using the Reflection Absorption Infrared Spectroscopy (RAIRS). The experimental setup will be described in Sec. 2, while the results will be shown and discussed in detail in Sec. 3. The astrophysical implications and conclusions will be given in Sec. 4 and 5, respectively.

EXPERIMENTAL SET-UP

Apparatus

To conduct our experiments, we used the new apparatus called VENUS (VErs de NoUvelles Synthèses), an Ultra-High Vacuum (UHV) system based in the LERMA laboratory at the University of Cergy-Pontoise (Congiu et al. 2020). VENUS consists of three stainless steel chambers. The first two (chambers 1 and 2) have a pressure of 10 -8 and 10 -9 mbar, respectively, and are used as differential pumping stages for the beamlines. The third one is the main chamber in which the pressure never exceed 2×10 -10 mbar. Inside the main chamber, a sample holder is mounted onto the cold head of a closed-cycle He cryostat. The sample holder is made of a circular copper mirror coated with gold. Its temperature can be varied in the range 7 -350 K and it is computer-controlled by a variable resistive heater. The main chamber is also equipped with a quadrupole mass spectrometer (QMS) that can translate vertically and be placed at 5 mm in front of the sample holder. Simultaneously, the beam of a Fourier Transform InfraRed Spectrometer (FTIRS) falls on the sample surface with a grazing angle of 85 • . In addition to the background line used for direct water vapour dosing inside the main chamber, the apparatus also includes several independent beam lines (up to 5 beam lines) passing through the expansion chambers 1 and 2, and finally to the main chamber via five 2-mm diaphragms aligned with the surface sample. One of beam lines was used for injecting an effusive molecular beam of CH 3 OH.

Experiments

Under the UHV conditions of VENUS, many experiments were performed to form very thin layers of methanol on three different cold surfaces (gold, 13 CO, and porous ASW) at very low surface temperatures (T s ). The gold surface consists simply of the polycrystalline gold layer coating the sample holder. 13 CO and ASW ice substrates are obtained by depositions onto the gold surface previously to methanol dosing. As for deposition techniques, both 13 CO and methanol were injected using two different beam lines, while water was introduced in the main chamber using the background line. Prior to each experimental run, both methanol and water were purified by doing multiple freeze-pump-thaw cycles.

For all gas injections, whether via the beam lines or the background line, the thickness of the deposited species was calibrated relative to a given pressure and injection duration. For this calibration, TPD techniques were used to determine one monolayer (1 ML) using the method of multilayer peak appearance (Noble et al. 2012;Nguyen et al. 2018). The 13 CO layer was deposited using a gas flow of 0.3 sccm (standard cubic centimeter per minute) giving a pressure of 2 × 10 -4 mbar within the beam line before entering into chamber 1. Under these physical conditions, a 1-ML layer of 13 CO is formed every 6.5 minutes. To deposit an ASW ice layer via the background line, a leak valve is used to inject water vapour into the main chamber. 1 ML of ASW ice is formed by injecting 10 -8 mbar of water vapour per 2 minutes. The methanol is dosed via one of the beam lines and 1 ML is formed every 8 minutes by using a pressure of ∼ 2 × 10 -4 mbar of methanol gas in the injection chamber. We first have grown films of methanol on the golden mirror at 14 different values of T s ranging from 7 to 40 K. During each experiment, FTIRS measurements were carried out every two minutes while the TPD measurement was done at the end of the deposition phase. These experiments were repeated two more times to form a film of methanol on predeposited samples of 13 CO and ASW ices. The T s values were chosen taking into account a small variation (1 K) at lower temperatures, that is T s =7, 8, 9 K and then with a larger variation (5 K) at higher values. When the 13 CO layer is deposited as an underlayer for methanol films, both species are deposited at the same T s . On the other hand, the ASW ice substrate was deposited at 42 K, so as to ensure that the morphology of the ice remained unchanged during all the experiments carried out at all T s values. Of course, the ASW ice structure evolves during the TPD above 42 K, but not during the deposition phase of methanol, which is key for studying sticking properties.

The TPD mass spectra are primarily used to measure the desorption of methanol molecules from the cold surfaces. The peak integrated area (A TPD ) as a function of the surface temperature is used to measure the methanol sticking. All TPD experiments were performed with a heating ramp of β = 0.2 K s -1 , and FTIRS spectra were baseline corrected.

RESULTS AND DISCUSSION

TPD experiments provided many mass spectra of methanol (m/z=32) as QMS signals (cps) or desorption rate (molecules cm -2 s -1 ). Of relevance to the sticking of methanol, the results of TPD and RAIRS spectra were quantitatively analysed. In the following subsections, we show in detail the sticking profiles derived from CH 3 OH desorption under different temperature initial conditions and from different surfaces.

Methanol beam flux

For all experiments, methanol gas was injected at the same pressure (∼ 2 × 10 -4 mbar) that was used for calibrating the methanol layer thickness. Keeping this pressure in the injection chamber, the beam flux (Φ) was measured with the QMS facing the beam and placed in front of the surface. QMS signals in counts per seconds (cps) were measured before and after each experiment and vary slowly from one experiment to the next. We attribute the methanol flux instability to fluctuations of ambient temperature of the room. For all T s , the values of Φ are shown in Fig. 1 as red open circles on the gold surface (left panel), on the 13 CO substrate (central panel) and on the ASW substrate (right panel). Also the integrated areas of CH 3 OH TPD peaks (A TPD ) are presented in this figure (black open squares). TPD profiles will be described in the following subsection according to thickness and T s . The horizontal dashed-line on Fig. 1 represents a reference of 700 cps corresponding to the normalisation of Φ.

First, we note that the relative variations of Φ are much smaller than the relative variations of A TPD . The measurements are not directly comparable, since the TPDs measure a signal area over the entire desorption time, while the flux is a simple average per unit time. That being said, we notice that the A TPD follow approximately the fluctuations of the Φ: low fluxes corresponding to low desorption areas, and vice versa. There are, however, some data against this general trend, for example on the gold surface (left panel) the highest A TPD is at 40 K (around 4600 cps K), whereas for a similar flux the point at 14 K is around 3500 cps K. Moreover, if we repeat the experiment at the same temperature, but using a different flux, the ratio A TPD /Φ is remarkably constant. Thus, if we consider that the ratio of the incoming molecules (measured by the flux) and of the desorbing molecules (measured by the TPD area) represents the sticking coefficient S, we can calculate S(T s ) after normalising the incoming flux Φ to the average value of 700 cps, and A TPD to its highest value at T s = 40 K. By doing so, we assume a sticking coefficient of unity at 40 K. The variation of S(T s ) will be analysed later.

Fig. 2 shows the ratio of the non-normalized A TPD over S(T s ) as a function of Φ. As expected, it shows a linear trend, and we would like to point out that the experimental deviation from this straight line (determined by repeated experiments) is less than 2%. Moreover, this ratio runs linearly to zero indicating that our measurements of both Φ and A TPD are exempt from noise, and that the normalisation of the flux allows us to extract the sticking coefficients as a function of temperature, in a very larger extent than our experimental uncertainty.

Methanol TPD profiles

QMS measurements have two essential properties for this study: they are stable and have a very good detection sen- sitivity. The drawback of this method is that it cannot give direct information on the solid phase, but only measures what is being deposited (Φ) and what is desorbing (TPD). Fig. 3 shows some TPD profiles of methanol at coverages of 0.5, 0.75, 1, 2, and 3 ML on the gold substrate held at T s =10 K. They present a maximum of desorption around 135-138 K in agreement with published results, given our temperature ramp, coverages and substrate. The curves for 1, 2 and 3 ML (magenta, red and green curves, respectively) present a common leading edge characteristic of the multilayer regime. The inset of the figure shows A TPD (normalised by the flux) as a function of coverage. It shows a proportional increase of the desorption rate with the dose. The deviation of the experimental points from the linear fit is very small and indicates a constant sticking, independent from the coverage.

At a specific thickness of 1 ML of methanol, TPD profiles were carried out at different deposition temperatures (T s between 7 and 40 K) on the three cold substrates. In Fig. 4, we show some examples of these profiles (taken at T s = 7, 16, 40 K) from gold (top panel), 13 CO (middle panel) and ASW (bottom panel). The overview on the top and mid-dle panels corresponding to gold and 13 CO surfaces, respectively, shows that methanol has a similar desorption peak at (T d ) max ∼ 137 K from both substrates. This peak position is marked by the orange dotted line in Fig. 4. In addition to this feature, another additional peak of methanol desorption appears having (T d ) max ∼ 152 K on ASW surface as marked by a dashed violet line. This second peak is the signature of co-desorption of methanol and water. This was expected since the morphology of water ice grown at 42 K is porous, and some trapping and mixing between CH 3 OH and H 2 O is very likely to occur during the linear heating of the ices. Moreover, we can see that the balance between the peak at (T d ) max =137 K and the peak at 152 K changes with deposition temperature. At the lowest deposition temperatures there is less co-desorption of water and methanol, that is less mixing, while at T s =40 K we observe the largest co-desorption effect. This indicates that the spreading of methanol on water ice is different depending on the surface temperature at the time of deposition. At the lowest T s , methanol molecules find adsorption sites on the outer surface of the water film, while they can rearrange more at higher temperatures and are in stronger interaction with the porous icy substrate. This is a known phenomenon that has been described during the study of the variation in water morphology with deposition temperature by B. Kay's group Kimmel et al. (2001). From all three different surfaces, however, it is clear that the desorption of 1 ML of methanol deposited at T s =16 K shows the lowest peak area, whilst in the experiments where methanol is deposited at 7 and 40 K the desorption peaks have the largest and similar areas. This leads us to conclude that at 16 K it is very likely that fewer molecules have stuck, since fewer molecules have eventually desorbed. Therefore, the sticking of molecules would depend on the surface temperature at the time of deposition, even if such temperature is much lower than the sublimation temperature.

RAIRS measurements of methanol layers

Even if RAIRS has lower sensitivity than that of the QMS, infrared spectra have provide the great additional advantage of measuring the quantity of molecules adsorbed on the surface. RAIRS spectra were acquired under UHV conditions as mentioned in Sec. 2. Fig. 5 shows the RAIRS spectra obtained during the growth of the methanol film on the gold surface at T s =10 K. They are characterized by the growing methanol featrures; the O-H stretching vibrational mode around 3235 cm -1 , the C-H asymmetric stretching plateau around 2940 cm -1 , the C-H symmetric stretching mode at 2830 cm -1 , the broad C-H bending modes around 1460 cm -1 and the C-O stretching band at 1045±5 cm -1 . The plateau from 1080 to 1200 cm -1 is rather noisy, as can already be seen in the reference spectra. The C-O stretching feature was the band used to evaluate the thickness of the methanol layer. We used the Lorentzian fitting method to calculate its integrated area, A IR (1045±5 cm -1 ). A RAIR spectrum was acquired every two minutes during the deposition. We remind that 1 ML corresponds to 8 minutes of deposition time under our experimental conditions. The top panel of Fig. 6 shows the fitted A IR (1045±5 cm -1 , of a continuous deposition of methanol on gold surface held at 10 K. We have calculated the slope (α) as a function of the layer thickness as shown in Fig. 6 (bottom panel). The error bars are calculated as standard deviations of the linear fit. The first point has no uncertainty since a unique line connect two points, and so give a unique slope. We see that α decreases with the methanol thickness until nearly 1 ML, then it becomes stable with increasing thickness. This reduction is probably due only to the decrease in the error bar as α converges towards its average value. Therefore, we conclude that with a thickness of 1 ML we can obtain a good estimate of α, which remains constant beyond this value of the coverage. As we want to study the effect of the substrates, this is also the appropriate thickness that should enhance the differences between each of them.

We systematically measured the RAIRS spectra during the deposition of ∼ 1 ML (8 minutes) of methanol and analyzed the growth of the peak area at 1045 cm -1 , for the three types of surfaces and at various deposition temperatures from T s =7 to 40 K. Some of these spectra (at T s =7, 10, 16, 20, 30 and 40 K) are shown in Fig. 7 on the substrates of gold (top panel), 13 CO (middle panel), and ASW ice (bottom panel). The 13 CO underlayer can be seen by its absorption peak at 2094 cm -1 , whereas the water substrate spectral signature is not visible because we have reset the reference spectrum prior to injecting the methanol, due to the necessary delay between water ice deposition and return to the best UHV conditions. The calculation of the coefficients α versus T s for the different substrates makes it possible to compare the different growth behaviour of methanol. The trend profiles of α as a function of T s are displayed in the top panel of Fig. 8. We can see that their behaviours are similar and vary with increasing T s for the three surfaces. These profiles decrease with increasing T s until 16 K on all substrates. Above this temperature, α gradually increases again with T s . Obviously, the α profiles show a parallel trend for all surfaces, namely they decrease at lower temperatures between 7 and 16 K then they invert the trend and increase from 16 K to 40 K. An exception appears marginally on the profile α on 13 CO to T s > 25 K (empty triangles, Fig. 8) where a slight negative deviation is observed. This is probably due to the fact that above 25 K the 13 CO sublayer begins to desorb. Therefore, for temperatures between 25 and 40 K, there is only a fraction of a monolayer of CO on the surface, which is thus made up of a growing part of gold. In fact, we can see that α values on 13 CO tend towards those measured on bare gold.

The three trends are parallel, and there is a maximum absolute difference of a factor of two. We can then ask ourselves if it is because there are twice as many molecules or if it is because of the band strengths -which we know are very sensitive to the molecular environment (Palumbo 2006;Öberg et al. 2007;Fulvio et al. 2009;Palumbo et al. 2010) -are twice as strong, especially on a water substrate. The QMS measurements that will be discussed below suggest that the number of molecules is identical, so we conclude here that methanol band strengths differ by a factor of two between a gold surface and an ASW ice. We may also ask if our method of analysis (fit of the 1045 cm -1 band) is to be questioned. Indeed it can be sensitive to the deformations of the band, and not only to its change in intensity. We point out here that a direct integration, given the weakness of our signals, gave noisier results. But in any case, we used the same method, and the only difference lies in the type of substrate. This reminds us of how much the infrared band strengths of the same molecule can vary from one system/ environment to another. Finally, we note here that unlike many other infrared measurements done in similar studies, we did not use thick ices, whose bulk averages environmental effects.

In order to compare in detail the effect of the substrate, we have normalized α profiles relative to the value at 40 K (α max ). The bottom panel of Fig. 8 shows a remarkable difference as T s decreases. At the lowest T s , the 13 CO layer shows the highest sticking probability while the gold surface shows the lowest one. However, the variation with the temperature is more important than the effect of the substrate. As a result of the trend profiles of α, the sticking of methanol shows approximately a 30% reduction around 16 K compared to the values obtained at 7 and 40 K.

Sticking versus surface temperature

The RAIRS data clearly show that the α coefficients vary with T s . On the other hand, because the band strength may vary according to the environment, and since the morphology/topology of the methanol film is different according to the deposition temperature, as we have seen previously by examining the fraction of the co-desorption with water, we do not know if it is a variation of the sticking or only a variation in the band strength. Unlike RAIRS spectra, QMS data are not affected by variations of detection efficiency.

The profiles of A TPD are shown in the the top panel of Fig. 9 as a function of T s (7 -40 K). They have been corrected to take into account the variations of the methanol flux. The areas of the TPD peaks of show a variation with T s . The behaviour of this variation is in agreement with what is observed in the trend of α profiles derived from RAIRS spectra. Moreover, the spread of the values for the different substrate is low compared to the variation with T s .

The bottom panel of Fig. 9 shows quantitatively the sticking of methanol molecules as a function of T s , assuming that the sticking is one at 40 K on all substrates. Like with RAIRS data, we also observe a reduction of methanol molecules of around 30% at 16 K. Among all surfaces, methanol sticking profiles have slight differences in the sticking at lower surface temperatures: the lowest sticking is found on gold and the highest one is found on the 13 CO surface. We now compare the two bottom panels of Fig. 8 and Fig. 9, and find that they are very similar. The trend presented in Fig. 8 is consistent with that in Fig. 9, which comes from the TPD. This perhaps can be seen as further evidence that the trend is real. There are slight differences in the temperature range 12 to 14 K. The initial decrease with T s appears to be slightly steeper with RAIRS data, maybe indicating that the bands strengths may have slightly varied there, but this is too close to our experimental uncertainties to be conclusive.

The trend in methanol sticking profiles raises an open question about the origin of the reduction in methanol sticking around T s =16 K. To explain this point simply, it seems that there are two antagonistic mechanisms concerning the methanol sticking. One mechanism increases the sticking efficiency with surface temperature and the other decreases it, and their effect is reversed around 16 K.

The origin of these two mechanisms might lie in the bonding of methanol on cold surfaces. Methanol is composed of a methyl -CH 3 group and an hydroxyl group -OH. The binding properties of the two groups are different, and when approaching the surface the accommodating properties of methanol may be different according to which of these different groups faces the substrate. However, this does not explain why there is a dependence with the surface temperature. The surface energy transfer (phonons) is supposed to vary with the temperature, but again phonons are very dependent on the type of the surface, and therefore the difference between the surfaces should be more pronounced if it were mostly induced by energy transfer. Nonetheless, on that point we can see that the CO film has the highest sticking coefficient, and the gold has the lowest one. This confirms that the rigidity of the surface, or its mass match, is an important point, and it probably explains why 13 CO (m/z=29), having the best mass match with CH 3 OH (m/z=32), turns out to have the highest sticking coefficient.

The common point for the three experiments is the building of the methanol film itself. From the TPD profiles of Fig. 3, we can see that there is only one desorption peak, which means that there is no specific binding site for methanol in a sub-monlayer regime on the surface, as can be seen for example for lighter molecules on crystalline surfaces (Ulbricht et al. 2002). This means that the major contribution of binding energy is the molecular film itself, in other words, methanol prefers to cluster or make islands rather than making a structure depending on the interaction with the substrate. Considering the asymmetry of methanol, it is clear that the positioning of one molecule with respect to another (or a set of other molecules that constitute the growing film) significantly changes its binding energy. In addition, to minimize its energy, a cluster of a few molecules must rearrange itself. This rearrangement cannot be done without a small excess of energy, that is an activation bar-rier. It is therefore possible that around 16 K, the methanol film no longer has enough degrees of freedom to reorganize itself in the short time that corresponds to the approach of a methanol molecule from the gas phase, and therefore cannot propose to the new molecule a favourable adsorption site. For this reason, a molecule arriving in a configuration with only a low binding energy, would have a better chance of bouncing back, at equal dissipation of its incident kinetic energy. Another way to say is that at 16 K, the reconstruction of the methanol film is hindered and only low coordinated adsorption sites are available. When the temperature decreases below this critical point, these low coordinated adsorption sites are anyway sufficient to be maintained on the surface. So the possible mechanism that increases the sticking efficiency with T s is the restructuring of the methanol film, and it counterbalances the usual mechanism making lower temperatures more favorable to trapping due to the longer interaction time and faster energy dissipation.

Nevertheless, whatever our explanations may be, this variation in sticking with surface temperature remains a surprise, and deserves to be studied in more detail. Even more so, the relatively low surface effect adds to the mystery, but it also may indicate that its origin is in the structuring of the methanol film itself. The role of methanol asymmetry could also be investigated in other complex molecules, such as CH 3 C 3 or CH 3 CN. Molecular dynamics calculations, and perhaps even quantum calculations given the temperature ranges, could also provide valuable insight into our experimental measurements.

ASTROPHYSICAL IMPLICATIONS

In our experiments, we find that at T s ∼ 16 K there is a decrease of the sticking coefficient by ∼ 30% and an effective value of S(T s )=0.7 . Let's assume that this is due to an activation barrier. Under our experimental condition the gas is at room temperature (∼ 293 K), and if the probability to overcome the barrier is p = exp(-E a /k B T g ) = 0.7, we find that the equivalent activation barrier is E a 124 K/k B . If we consider the physical conditions of a dark cloud having a gas and dust temperature of T=15 K, the sticking probability is exp(-E a /k B T) 10 -3 . We can apply this to all our measurements, using the CO-substrate values as a reference. The choice of CO instead of H 2 O is motivated by the astrophysical context of accretion at later times. Anyhow, since the sticking coefficients of the different substrates are close, it would not change the main conclusion. These results are presented in Fig 10 . The sticking is close to unity at very low temperatures and at the highest temperature (T s >30 K). We can see that the sticking efficiency drops by two orders of magnitude under conditions between 10 K and 20 K, which are typical temperatures of molecular clouds. Moreover, the minimum in the sticking efficiency is shifted to lower temperatures, especially when the calculations are done using RAIRS data. This is due to the exponential re-duction of the probability to stick with temperature, since the kinetic energy may be an efficient way to overcome the activation barrier.

Reasoning in terms of activation barrier is probably an oversimplification. Indeed, this activation barrier varies with surface temperature. Nevertheless, this approach shows the potential impact of this variation in sticking, if directly transposed to the conditions of the interstellar medium. A reduction of 2-3 orders of magnitude in the sticking of methanol would extend accretion times by as much, and thus make it possible to understand why this molecule can be observed in dark and cold environments, even if non-thermal desorption mechanisms were not efficient. Simply, methanol freeze-out may possibly be hindered.

Experimentally, it is very difficult to produce a cold beam of methanol, as it would condense on the cold parts of the apparatus well before entering the main chamber. Alternatively, it may be possible to play with the angle of deposition to reduce the perpendicular component of the kinetic energy, but unfortunately our set-up does not allow us to change the angle of deposition and adopt this technique. Therefore, the solution of the problem may come from the theory and/or the simulations. Molecular dynamic is possibly a good tool to explore this matter.

Molecular dynamics calculations (Bahr et al. 2008;Kong et al. 2019) show that the adsorption of a methanol on a layer of water occurs preferentially via the OH bond, leaving the methyl group facing upwards. But it appears that although the methanol-methanol interaction is weaker than with a water film, it remains important, of the order of 25 kJ/mol per H bond. In a methanol film, molecules tend to make several H bonds, and on graphite, methanol molecules are able to make clusters even at a very low coverage rate (10 -6 ML) at temperatures of about 180 K. These informations may then allow us to understand why the different surfaces do not seem to play a significantly different role. It is likely that the formation of a methanol cluster, or at least the interaction of one methanol with another methanol, and the formation of H bounds, plays a very important role in the formation of the film or surface clusters. It is questionable whether the presence of upwardly facing methyl groups does not prevent other molecules from sticking, or whether it is the difficulty of orienting and making the right H bonds that limits the capture rate around 15 K. As quoted by our anonymous referee, "assuming that the strongest potential interaction with a methanol molecule and the methanol ice surface would a single hydrogen bond interaction, the absence of any dangling OH on the methanol surface would mean that scattering from the surface might be more likely than adsorption unless partial accommodation of the incoming molecule on the surface provides sufficient energy to reorientate surface methanol species to expose OH groups". This point has to be compared with accurate calculations to see if it corresponds to the order of magnitude of the barrier we have derived here.

Until now, we do not yet understand the underlying reasons of the sticking variation as a function of surface temperature, and those of a minor impact of the substrate type. That is why it would not be wise to draw definitive conclusions on its astrophysical implications. With this paper, we simply wish to demonstrate that a more careful look, using both an experimental and a theoretical approach, has to be taken at the problem of sticking of COMs at low temperatures.

CONCLUSIONS

Our experimental study of methanol sticking as a function of surface temperature (7 -40 K) shows its variation with surface temperature. On three different cold surfaces (gold, 13 CO, and ASW ice), a similar variation in methanol sticking can be observed. This variation has two antagonistic tendencies: the sticking of methanol 1) decreases rapidly with increasing the surface temperature from 7 to about 16 K, 2) increases slowly from 16 K to 40 K where it returns to a value similar to that found at 7 K. Depending on the type of the surface, a slight difference in the degree of sticking is observed at low temperatures. The gold surface has the lowest sticking efficiency while the 13 CO surface has the largest one, in agreement with mass matching arguments. The results obtained from TPD and RAIRS are consistent similar in their trend profiles as a function of surface temperature on different cold surfaces. In fact, the reduction in methanol sticking over a surface temperature of 16 K has been proven but raises questions about its origin. This may be due to the structuring properties of the methanol film, because of the asymmetry of this molecule. If our results obtained for a impinging gas at room temperature are abruptly extrapolated under dark cloud conditions, we find that the sticking would be reduced by two orders of magnitude. However, before ap- plying this conversion law in space, it would be wiser to understand the physical origin of the variation in methanol sticking at low temperatures.

Figure 1 .Figure 2 .

 12 Figure 1. The beam flux (red circles) and integrated TPD area (A TPD ) of methanol (black squares) on the right and left vertical axes, respectively, versus the surface temperature T s , on gold (left panel), 13 CO (middle panel) and ASW ice (right panel).

Figure 3 .

 3 Figure 3. The TPD mass spectra of various doses of methanol (at the normalised flux Φ=700 cps) from gold hold at T s =10 K. Inset: methanol A TPD normalised by the flux, as a function of deposition thickness.

Figure 4 .

 4 Figure 4. Some of methanol TPD mass spectra normalized to depositions at Φ=700 cps, deposited at different values of T s on surfaces of gold (top panel), 13 CO (middle panel) and ASW ice (bottom panel). The orange dotted line refers to methanol peak desorption at approximately (T d ) max =137 K and the violet dashed line refers to desorption at approximately (T d ) max =152 K appearing only on ASW surface.

Figure 5 .

 5 Figure5. RAIRS spectra that show the growth of methanol IR bands with thickness up to 3 ML at a normalized flux Φ=700 cps and T s =10 K. All spectra were baseline corrected.

Figure 6 .

 6 Figure6. Top panel: integrated area of the 1045±5 cm -1 band of methanol after deposition at the normalised flux Φ=700 cps at T s =10 K versus film thickness. Bottom panel: α-thickness dependence derived from IR spectra taken every two minutes on gold.

Figure 7 .

 7 Figure7. RAIRS spectra of 1 ML of methanol deposited at the normalized flux Φ=700 cps at various surface temperatures (T s ) on the substrate of gold (top panel), of 13 CO ice (middle panel), and of ASW ice (bottom panel).

Figure 8

 8 Figure8. α-profiles of methanol versus T s on surfaces (gold, 13 CO, and ASW) at the normalized beam flux at Φ=700 cps (top panel). α-profiles normalized with respect to the value of α max at T s = 40 K (bottom panel).

Figure 9 .

 9 Figure9. Integrated TPD peak area of methanol corresponding to depositions at the normalized beam flux (Φ=700 cps) versus T s on surfaces of gold, 13 CO, and ASW ice(top panel). Calculated sticking coefficients S(T s ) versus the surface temperature (bottom panel).

Figure 10 .

 10 Figure10. Calculated sticking coefficient assuming an activation barrier measured at T gas = 293 K, and in astrophysical conditions where T gas =T dust = 15 K. In blue are represented the values extracted from QMS data, S(T s ), and in orange the values derived from RAIRS spectra, α(T s ).

MNRAS 000, 1-12 (2020)

ACKNOWLEDGEMENTS

This work was financially supported by the Egyptian government through the co-financed fellowship granted by the Science & Technology Development Fund (STDF; Project ID: 30565) and the Institut Français d' Égypte au Caire (IFE). So, authors acknowledge both STDF and IFE for supporting this research. KG thanks the Institute of Advance Studies of the university of Cergy Pontoise for its support. This work was supported by the Programme National "Physique et Chimie du Milieu Interstellaire (PCMI)" of CNRS/INSU with INC/INP co-funded by CEA and CNES and the DIM ACAV a funding programme of the Region Ile de France. CH 3 OH is a target molecule of SOLIS, a NOEMA key program led by P. Caselli & C. Ceccarelli, and this work is related to its scientific purposes.


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Abstract. The fatigue microstructure of an Al-2.5 wt %Li alloy with small precipitates has been investigated both by TEM and STEM (nanodiffraction and electron energy loss spectroscopy). Specific features of strain localization have been evidenced which are made of lithiumdepleted precipitate-free bands. A model is proposed to explain the redissolution of δ precipitates inside the bands, based on an increase of their free energy due to shear-induced antiphase boundaries.

  Experimental (TEM and STEM) investigation and theoretical approach to the fatigue-induced dissolution of delta'-precipitates in a 2.5 wt % Al-Li alloy Yves Brechet, François Louchet, C. Marchionni, J.-L. Verger-Gaugry To cite this version: Yves Brechet, François Louchet, C. Marchionni, J.-L. Verger-Gaugry. Experimental (TEM and STEM) investigation and theoretical approach to the fatigue-induced dissolution of delta'-precipitates in a 2.5 wt % Al-Li alloy. Philosophical Magazine a, 1987, 56 (3), pp.353-366. 10.1080/01418618708214391. hal-03134559Experimental (TEM and STEM) investigation and theoretical approach to the fatigue-induced dissolution of δ precipitates in a 2.5 wt % Al-Li alloy Y. Bréchet, F. Louchet, C. Marchionni and J.-L. Verger-Gaugry LTPCM/ENSEEG/INPG (CNRS UA 29), BP 75, Domaine Universitaire, 38402 Saint Martin d'Hères, France