Hélène Falentin
email: helene.falentin@rennes.inra.fr

Stéphanie-Marie Deutsch

Valentin Loux

Amal Hammani

Julien Buratti

Sandrine Parayre

Victoria Chuat

Valérie Barbe

Jean-Marc Aury

Gwenaël Jan

Yves Le Loir

Permanent draft genome sequence of the probiotic strain Propionibacterium freudenreichii CIRM-BIA 129 (ITG P20)

Keywords: GRAS, QPS, probiotic, anti-inflammatory, immunomodulation, surface proteins

Propionibacterium freudenreichii belongs to the class Actinobacteria (Gram positive with a high GC content). This "Generally Recognized As Safe" (GRAS) species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production, (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties. Until now, only strain CIRM-BIA1T had been sequenced, annotated and become publicly available. Strain CIRM-BIA129 (commercially available as ITG P20) has considerable anti-inflammatory potential. Its gene content was compared to that of CIRM-BIA1 T. This strain contains 2384 genes including 1 ribosomal operon, 45 tRNA and 30 pseudogenes.

Introduction

Propionibacterium freudenreichii belongs to the class of Actinobacteria (Gram positive bacteria with a high GC content). This 'Generally Recognized As Safe' species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic properties (enhancing intestinal transit). A recent screening of 23 strains belonging to this species revealed the considerable antiinflammatory properties of strain CIRM-BIA129 (ITG-P20) [START_REF] Foligne | Tracking the microbiome functionality: focus on Propionibacterium species[END_REF]. Its anti-inflammatory potential is superior to that of the previously sequenced CIRM-BIA1 T strain. CIRM-BIA129 (ITG-P20) is currently used to make cheese at an industrial scale and indeed is consumed in large quantities because one gram of Swiss-type cheese contains at least 10 9 CFU. The genetic basis for the antiinflammatory properties of P. freudenreichii are still poorly understood. Sequencing CIRM-BIA129 (ITG-P20) would enable investigation of the genomic determinants responsible for the important anti-inflammatory properties of this strain. To our knowledge, CIRM-BIA129 is the second strain in this species to be sequenced, annotated and made publicly available.

Organism information

Classification and features P. freudenreichii CIRM-BIA129 (ITG P20) cells are Grampositive, microaerophilic, pleiomorphic (coccoid to rod shape forming 'Chinese characters') bacillae (1.0-1.5 μm × 0.5-0.8 μm wide) forming creamy-white colonies on YEL agar plates (Table 1). Cells grow at the bottom of liquid medium tubes (to escape oxygen) and tend to clot in liquid culture at the beginning of the stationary phase. Transmission electron microscopy pictures of liquidgrown cultures revealed a thick cell wall (Fig. 1) made of peptidoglycan. No exopolysaccharides were observed at the surface of the bacteria, unlike what is seen in numerous strains of P. freudenreichii, including the type strain CIRM-BIA1 T (Fig. 2).

A BLASTn of the 16S sequence of type strain CIRM-BIA1 T against the contigs of CIRM-BIA129 (ITG-P20) confirmed the affiliation of this strain to the species (100 % identity, 100 % coverage).

Representative genomic 16S rRNA sequences of the strains were compared with those of other type strains belonging to Actinobacteria present in the Ribosomal Database Project. (Figure 2) shows the phylogenetic tree.

CIRM-BIA129 (ITG P20) was found to be able to utilize glycerol, erythritol, L-arabinose, adonitol, galactose, D-glucose, D-fructose, D-mannose, inositol, arbutine, esculine, lactose, lactate and gluconate as carbon sources according to the gallery-API results.

CIRM-BIA129 (ITG P20) was shown to grow with up to 1 M NaCl in a chemically defined medium in presence of osmoprotectant (G. Jan, personal communication).

Genome sequencing information

Genome project history

Propionibacterium freudenreichii CIRM-BIA 129 (ITG P20) genome was sequenced to obtain information regarding mechanism(s) or molecule(s) responsible for anti-inflammatory properties of the strain. Project information and associated MIGS are shown in Table 2.

Growth conditions and genomic DNA preparation

The P. freudenreichii strain CIRM-BIA129 (ITG-P20), isolated from Emmental cheese by Actalia Dairy Products (Institut Technique du Gruyère, Actalia, Rennes, France), was provided by the CIRM-BIA Biological Resource Centre (Centre International de Ressources Phylum Actinobacteria TAS [START_REF] Stackebrandt | Proposal for a New Hierarchic Classification System, Actinobacteria classis nov[END_REF][START_REF] Whitman | s Manual of Systematic Bacteriology: Volume 5a: The Actinobacteria[END_REF] Class Actinobacteria TAS [START_REF] Stackebrandt | Proposal for a New Hierarchic Classification System, Actinobacteria classis nov[END_REF][START_REF] Whitman | s Manual of Systematic Bacteriology: Volume 5a: The Actinobacteria[END_REF] Order Actinomycetales TAS [START_REF] Stackebrandt | Proposal for a New Hierarchic Classification System, Actinobacteria classis nov[END_REF] Family Propionibacteriaceae TAS [START_REF] Zhi | An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa[END_REF] Genus Propionibacterium TAS [START_REF] Charfreitag | Inter-and Intrageneric Relationships of the Genus Propionibacterium as Determined by 16S rRNA Sequences[END_REF] Species Propionibacterium freundenreichii TAS [START_REF]Necessary corrections to the Approved Lists of Bacterial Names according to Rule 40d (formerly Rule 46)[END_REF] Strain Microbiennes-Bactéries d'Intérêt Alimentaire, INRA, Rennes, France). It was cultivated at 30 °C without shaking in YEL [START_REF] Malik | Evaluation of the taxonomy of the Propionibacterium[END_REF]. Growth was monitored spectrophotometrically at 650 nm, as well as by counting colony-forming units in YEL medium containing 1.5 % agar.

A cell pellet (equivalent to 2 × 10 10 CFU) was obtained by centrifugation for 10 min at 5000 × g from a one-day exponentional phase culture of CIRM-BIA129 (ITG P20). DNA was extracted using the Blood & Cell Culture DNA Midi Kit (Qiagen) according to the manufacturer's recommendations with the following modifications. Briefly, complete bacterial lysis was obtained by adding 220 mg of lysozyme powder (Qbiogene) to 3.5 ml of B1 buffer (Qiagen) followed by 2.5 h of incubation at 37 °C. High molecular weight genomic DNA was purified by gravity flow and anion exchange chromatography, eluted in 5 ml QF buffer (Qiagen) and precipitated with 3.5 ml of isopropanol. DNA was collected by centrifugation for 10 min at 4 °C and 15000 g and then air dried. DNA was resuspended in 100 μL TE 1X buffer at pH8 (Sigma) for Fig. 2 The evolutionary history of the strain was inferred using the Neighbor-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The optimal tree with a sum of branch lengths = 0.80 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [START_REF] Tamura | Prospects for inferring very large phylogenies by using the neighbor-joining method[END_REF] and are in the units of the number of base substitutions per site. The analysis involved 18 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1376 positions in the final dataset. Evolutionary analyses were performed under MEGA5 [START_REF] Tamura | MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods[END_REF] two hours at 55 °C. DNA integrity was checked on an 0.8 % agarose gel. The OD 260/280nm was 1.9.

Genome sequencing and assembly

The genome of the CIRM-BIA129 (ITG-P20) strain was obtained using a whole-genome strategy based on Illumina 36 nucleotide paired-end sequencing (Illumina genome analyser II) (Table 2). After filtration and trimming, the number of reads was downsized to 50 million of 34.8 bases mean read length, representing a 681-fold genome coverage. The reads were assembled using Velvet version 1.1.03 [START_REF] Zerbino | Velvet: Algorithms for de novo short read assembly using de Bruijn graphs[END_REF] with a k-mer size of 31. The assembly resulted in 59 scaffolds of a length greater than 1 kb, with an N50 of 123 kb.

Genome annotation

Automatic and manual annotations were carried out with the AGMIAL platform [START_REF] Bryson | AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system[END_REF], using P. freudenreichii CIRM-BIA 1 T as the reference [START_REF] Falentin | The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications[END_REF]. Sub-cellular localization was predicted using SurfG+ [START_REF] Barinov | Prediction of surface exposed proteins in Streptococcus pyogenes, with a potential application to other Gram-positive bacteria[END_REF], a software specific to sub-cellular prediction in Gram-positive bacteria. A comparison of the predicted proteome of CIRM-BIA129 with that of the sequenced type strain CIRM-BIA1 was performed with Koriblast software (Korilog, France) with its default parameters. Genes present in CIRM-BIA129 and not in the type strain CIRM-BIA1 were searched by tblastn of the CIRM-BIA129 predicted proteome against the complete genome of CIRM-BIA1. No hit proteins were specific to CIRM-BIA129. In order to search for genomic islands specific to CIRM-BIA129, a blastp of the CIRM-BIA129 proteome against the CIRM-BIA1 proteome was performed. Proteins filtered for a 'hit coverage' of less than 75 % and those corresponding to locus-tags colocalized along the chromosome were attributed to genomic islands. To search for probable pseudogenes in CIRM-BIA1, a blastp analysis of CIRM-BIA1 protein against CIRM-BIA129 proteins filtered on a 'hit coverage' of less than 75 % was performed. Among them, the corresponding genes were declared as pseudogenes in the event of two adjacent genes having the same 'best hit'. S-layer homology domains inside the proteins were sought from the SLH.hmm pattern using hmmer with default parameters.

Genome properties

The genome was found to be composed of 111 contigs for a total size of 2,588,969 bp (67.3 % GC content). The contig length was encompassed between 158 bp and 179,656 bp with an average at 23,324 bp. The N50 is 46,655 bp. One ribosomal operon (containing one 16S rRNA gene and one 23S rRNA gene) was present, on contig 24, A total of 2354 complete genes were predicted, 2,307 of them protein-coding genes and 45 tRNA encoding genes. Thirty genes were pseudogenes. According to the COG results, 1,236 protein coding genes were assigned to a putative function, the remainder being annotated as hypothetical proteins. The properties and statistics of the genome are summarized in (Tables 3 and4).

Insights into the genome sequence

Surface proteins encoding genes

In a previous research study [START_REF] Foligne | Promising immunomodulatory effects of selected strains of dairy propionibacteria evidenced in vitro and in vivo[END_REF], the authors observed that the removal of surface-associated proteins led to a marked decrease in anti-inflammatory properties. Numerous studies dealing with the anti-inflammatory properties of food or probiotic bacteria have also identified the S-layer proteins or other surface compounds responsible for this trait [START_REF] Konstantinov | S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions[END_REF][START_REF] Taverniti | S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity[END_REF]. The S-layer proteins were found to be paracrystalline mono-layered assemblies of proteins which coat the surface of bacteria. S-layer proteins were associated with the cell wall via an SLH domain, with a cell wall polymer serving as the anchoring structure. This SLH domain comprised about 40-50 amino-acids, and could be found as one or more copies in the protein. Proteins other than S-layer proteins are known to be anchored by SLH domains to the cell wall of bacteria and are called S-layer associated proteins [START_REF] Johnson | Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM[END_REF]. As P. freudenreichii CIRM-BIA129 is known for its anti-inflammatory properties [START_REF] Foligne | Promising immunomodulatory effects of selected strains of dairy propionibacteria evidenced in vitro and in vivo[END_REF][START_REF] Marechal | Surface proteins of Propionibacterium freudenreichii are involved in its antiinflammatory properties[END_REF], it was necessary to determine the presence of protein sequences containing SLH domains. This search led to the identification of eight genes: slpB (PFCIRM129_00700) that contained five SLH domains; slpE, (PFCIRM129_05460) containing four SLH domains; slpF (PFCIRM129_01545), slpG (PFCIRM129_09890), slpA (PFCIRM129_09350) and inlA (PFCIRM129_12235) that contained three SLH domains at the C-terminal part, and slh2 (PFCIRM129_03800) and slpD (PFCIRM129_11775), containing two SLH domains. Interestingly, CIRM-BIA129 (ITG-P20) did not present a typical, thick S-layer at its surface, so these genes most probably code for SLAPs.

Comparison with the reference strain

A tblastn analysis revealed 77 new genes (no hit) that were not present in the type strain CIRM-BIA1. Most of them were of unknown function except for four, which encoded two transporters PFCIRM129_07475 (pseudogene) and PFCIRM129_08355, an amidohydrolase PFCIRM129_03910 and a transcription factor PFCIRM129_03595. The blastp results revealed three genomic islands that were present in CIRM-BIA129 but absent from CIRM-BIA1: (i) from PFCIRM129_09365 to 09660 encompassed by conjugal transfer protein gene PFCIRM_09215 and conjugative transfer gene complex protein PFCIRM129_09665, probably corresponding to an integrative conjugal element, (ii) from _10870 to _10910 comprising several relaxase genes and an exopolyphosphatase gene PFCIRM129_10900, and (iii) from PFCIRM129_10830 to 10905 containing three alpha, beta hydrolase genes (PFCIRM129_10860, 10890, 10895).

A blastp analysis revealed a gene encoding a ribulokinase PFCIRM129_07785 which was pseudogenized in CIRM-BIA129 but appeared to be functional in CIRM-BIA1.

By contrast, the glucokinase gene PFREUD_00950 involved in gluconate degradation was pseudogenized in CIRM-BIA1 but complete in CIRM-BIA129. This difference may explain the ability of CIRM-BIA129 to degrade gluconate. In the same way, inlA (containing three SLH domains, see above) was pseudogenized in CIRM-BIA1 but complete in CIRM-BIA129. This suggests a better ability to interact with eukaryotic cells, as internalin A was described as a bacterial adhesin.

Conclusion

The genome of CIRM-BIA129 revealed new genes that had never previously been described in the species. Some of them, which probably encode surface exposed proteins, may be of considerable importance to the adaptation of the bacterium to the intestinal tract. Its ability to degrade gluconate may enable it to survive in the intestine where this sugar is abundant. Genes including the SLH domain may be candidates for the immune properties of the strain because SLH domains enable the protein to be anchored in the cell wall. The total is based on the total number of protein coding genes in the genome

Fig. 1 S000264635

 1 Fig. 1 Transmission electron microscopy pictures of liquid-grown cultures of CIRM-BIA129 (ITG P20)

Table 1

 1 Classification and general features of P. freudenreichii CIRM-BIA129 (ITG-P20) according to MIGS guidelines and the Catalogue of Life database

	MIGS ID	Property	Term

Table 3

 3 Nucleotide content and gene count levels of the genome

	Attribute	Value	% of total (a)
	Genome size (bp)	2,588969	100
	DNA coding (bp)	2,209128	85.3
	DNA G + C (bp)	1,741869	67.3
	DNA scaffolds	59	100
	Total genes	2,388	100
	Protein-coding genes	2,307	96.6
	RNA genes	3	0.2
	Pseudo genes	30	1.2
	Genes in internal clusters	Not reported	Not reported
	Genes with function prediction	1190	49.8
	Genes assigned to COGs	1,325	55.5
	Genes with Pfam domains	1148	48.1
	Genes with signal peptides	247	10.3
	Genes with transmembrane helices	521	21.8
	CRISPR repeats	Not reported	Not reported

(a)The total is based on the size of the genome in base pairs

Table 2

 2 Project information

	MIGS ID Property	Term
	MIGS-31 Finishing quality	Improved high quality draft
	MIGS-28 Libraries used	300 bp-insert Illumina library
	MIGS-29 Sequencing	36 nucleotide paired-end sequencing
	platforms	(Illumina genome analyser II).
	MIGS-31.2 Fold coverage	681x
	MIGS-30 Assemblers	Velvet version 1.1.03 k-mer
	MIGS-32 Gene calling method Show program
	Locus Tag	PFCIRM129
	EMBL ID	CCBE010000001-CCBE010000111
	EMBL Date of Release 20-Jun-2014
	BIOPROJECT	PRJEB4826
	MIGS-13 Source Material	CIRM-BIA 129
	Identifier
	Project relevance	Probiotic, anti-inflammatory

Table 4

 4 Number of genes associated with 25 general COG functional categories (2337 proteins)

	Code Value %	Description
			age
	J	116 4.96 Translation, ribosomal structure and biogenesis
	A	1	0.04 RNA processing and modification
	K	83	3.55 Transcription
	L	76	3.25 Replication and repair
	B	0	0	Chromatin structure and dynamics
	D	14	0.60 Cell cycle control and mitosis
	V	30	1.28 Defence mechanisms
	T	32	1.37 Signal transduction mechanisms
	M	58	2.48 Cell wall/membrane biogenesis
	N	0	0	Cell motility
	U	16	0.68 Intracellular trafficking and secretion
	O	51	2.18 Post-translational modification, protein turnover,
				chaperone functions
	C	88	3.77 Energy production and conversion
	G	119 5.09 Carbohydrate metabolism and transport
	E	121 5.18 Amino acid metabolism and transport
	F	47	2.01 Nucleotide metabolism and transport
	H	88	3.77 Coenzyme metabolism
	I	38	1.63 Lipid metabolism
	P	88	3.77 Inorganic ion transport and metabolism
	Q	21	0.90 Secondary structure
	R	149 6.38 General function prediction only
	S	89	3.81 Function unknown
	-	1012 43.3 Not in COG

Acknowledgements

We would like to thank Actalia for supplying the CIRM-BIA129 (ITG P20) strain. Work on sequencing was funded by INRA through the "AIP-Bioressource" call for projects.

Abbreviations YEL: yeast extract lactate.

Competing interests

Authors do not disclose any competing interests.

Authors' contributions HF extracted the DNA, annotated the genome, supervised the project and wrote the paper. SMD annotated surface proteins and provided pictures. VL cured the database and deposited the genome in the EMBL database. AH, JB and JMA assembled the genome. SP phenotyped the strain. VC stored the strain and checked its identity. VB sequenced the strain. GJ and YLL participated to the writing and provided funding for the research. All authors read and approved the final manuscript.


Dr Apurba Ranjan Sahoo

Dr Gummidi Lalitha

Dr V Murugesh

Dr Christian Bruneau

V M Gangavaram

Sharma

Surisetti Suresh

Dr Mathieu Achard
email: mathieu.achard@univ-rennes1.fr

Professor Jonathan M J Williams

Dr G V M Sharma

Prof. P. C J J Cossy

Kamer

Direct Access to (±)-10-Desbromoarborescidine A from Tryptamine and Pentane-1,5-diol

Keywords: indoloquinolizine, green chemistry, natural products, homogeneous catalysis, hydrogen transfer

A single step synthetic strategy for (±)10-Desbromoarborescidine A is described. Starting from tryptamine and pentane-1,5-diol, this acceptorless dehydrogenative condensation process is efficiently catalyzed by a ruthenium complex featuring proton-responsive phosphine pyridone ligand.

Scheme 1. Dehydrogenative couplings in the presence of Ru-1 precatalyst.

Indoloquinolizidines and harmicines featuring a tetracyclic core structure constitute an important part of the attractive indole alkaloids family. [1][2][3][4][5] Owing to their structural diversity, these compounds display broad biological activities including opioid agonistic, blood pressure lowering and antitumoral activities. [6][7][8][9] Isolated in 1966, (±)-10-desbromoarborescidine A represents the simplest indoloquinolizidine alkaloid. [10] Reported in the early 50's, the pioneering syntheses of (±)-10-desbromoarborescidine A involve, amongst others, Fisher indole type synthesis of 1ketoquinolizidine with phenylhydrazine. [11][12][13] In 1972, Gribble reported the synthesis of (±)-10-desbromoarborescidine A from tryptamine hydrochloride with glutaraldehyde by using sodium borohydride as a reducing agent with ten days of reaction time to prevent dimerization of the starting dialdehyde. [14][15] More recently, the same author reported an interesting condensation starting from tryptophan and 3,4-dihydro-2H-pyran. [16] These approaches lie on the transient formation of an iminium intermediate which undergoes condensation via the famous Pictet-Spengler condensation. [START_REF] Gholamzadeh | Advances in Heterocyclic Chemistry[END_REF] This latter transformation, became the method of choice for the synthesis of tetrahydro-βcarboline derivatives.

Other syntheses (±)-10desbromoarborescidine A involving Pictet-Spengler condensation have been reported. [START_REF] Yamanaka | [END_REF][19][20][21][22][23][24] Alternative approaches involve amongst others imino-Stetter and Polonovski type reactions. [25][26][27][28] Several enantioselective syntheses have also been reported. [29][30][31][32][33][34][35][36][37][38] However, these approaches suffer from one or more drawbacks like the requirement of specially designed starting materials, multi-step synthetic protocols, and generation of noxious wastes. Therefore, from a medicinal chemistry perspective, due to the promising biological activities of indoloquinolizidines, a sustainable approach towards their synthesis involving minimum steps and wastes is highly desirable.

Hydrogen transfer processes ranging from borrowing hydrogen to acceptorless dehydrogenation represent sustainable strategies for the formation of carbon-carbon and carbon-heteroatom bonds. [39][40][41][42] These tandem transformations involve the activation of saturated substrates such as alcohols and amines through a formal dehydrogenation generating unsaturated reactive carbonyl or imine/ium intermediates acting as alkylating reagents. Depending on the catalytic species/reaction conditions, further reduction or liberation of molecular hydrogen can occur. These methodologies have been efficiently used for the construction of N-heterocycles from primary amines and diols. [41][42][43][44][45][46] In 2019, Yang, Ma, Tang, and coworkers reported the synthesis of tetrahydro-β-carboline from tryptamine and alcohol in the presence of methyl cinnamate acting as sacrificial hydrogen acceptor to ensure good conversion. [47] In 2005, Williams and coworkers, reported the Nalkylation of tryptamine with pentane-1,5-diols leading to the selective formation of N-heterocycles without formation of the indoloquinolizidine Pictet-Spengler adduct. [46] Based on our recent contributions on tandem hydrogen transfer processes for the synthesis and functionalization of aza-heterocycles [48][49][50][51] , we postulated that our recent Ru-1 precatalyst (Scheme 1) would enable N-alkylation and be acidic enough to play the role of Pictet-Spengler surrogate to efficiently afford indoloquinolizidine in one step generating hydrogen and water as the only side products of the transformation (Scheme 2). [52][53]

Scheme

2.

Proposed metallo-catalyzed to (±)-10desbromoarborescidine A from diol and tryptamine.

Herein, we report the one step tunable formation of Nalkylated tryptamine derivatives and (±)-10desbromoarborescidine A from tryptamine and pentane-1,5-diol through acceptorless dehydrogenative condensation.

Thus, we first evaluated the reaction of tryptamine 1a and pentane-1,5-diol 2a in toluene as solvent with various precatalysts (Table 1). Common ruthenium precursors such as [Ru(p-cymene)Cl2]2, [Ru3(CO)12] were found to be inactive with a 1a/2a ratio of 1:1.3 (entries 1 and 3). Notably, [RuCl2(PPh3)3] bearing phosphine ligand was also inefficient (entry 2). We then focused our attention on iridium precursors. [Ir(COD)Cl]2 precursor failed to afford any conversion (entry 6), confirming the previous findings of Williams who demonstrated the requirement of additional diphosphine ligand such as dppf to ensure N-alkylation of tryptamine with diols. [46] In contrast, the use of piano stool iridium precatalysts [Cp*IrCl2]2 and [Cp*Ir(H2O)3(SO4)] afforded up to 70% conversion (entries 4 and 5). [45] Analyses of these reactions revealed the formation of the N-alkylated adduct 3 as the major compound along with two side products. GC/MS analyses indicated the side formation of the (±)-10-desbromoarborescidine A (4) and the N-monoalkylated tryptamine, thus confirming our working hypothesis. [48] Astonishingly, our ruthenium precatalyst Ru-1 containing phosphine-pyridone ligand under similar reaction conditions led to complete conversion of the tryptamine 1a again with the major formation of the tricyclic product 3 along with 4 as the only side product in an interesting 7:3 ratio (entry 7). [52] It should be mentioned that under refluxing conditions with a Schlenk tube equipped with a condenser and a bubbler, in the presence of precatalyst Ru-1, product 3 can be more selectively obtained with complete conversion and a 9:1 ratio (entry 8). Isolation of

Entry

Cat. Ratio 3/4 [b] conv. (%) [c] 1 [a] [Ru(p-cymene)Cl2]2 -<1

2 [a] [RuCl2(PPh3)3] 99:1 2

3 [a] [Ru3(CO)12] - 0 4 [a,d] [Cp*IrCl2]2 9:1 70
5 [a,d] [Cp*Ir(H2O)3(SO4)] 9:1 68

6 [a] [Ir(COD)Cl]2 - 0 7 [a]
Ru-1 7:3 100

8 [e] Ru-1 9:1 100(87;80) [c] [a] Experimental conditions: all reactions were performed in closed Schenk tube under an inert atmosphere of argon and carried out with tryptamine 1 (1 mmol), pentane-1,5-diol 2 (1.3 mmol), catalyst (1 mol% with respect to metal atom) in toluene (1 mL) for 16 h at 140°C.

[b] Ratio was determined by GC analysis on the crude reaction mixture.

[c] Conversions were determined by GC. First number in parentheses corresponds to the GC yield of the product 3 using tetradecane as internal standard and second to the isolated yield after column chromatography over alumina gel.

[d] 5-((2-(1H-indol-3-yl)ethyl)amino)pentan-1-ol side product was also detected during GC analysis.

[e] Reaction was performed in a Schlenk tube equipped with a condenser and a bubbler and heated under toluene reflux.

Accepted Manuscript

the product 3 was achieved by chromatography on alumina gel affording 80% isolated yield. Successful crystallization confirmed the tricyclic structure (Figure 1). Under refluxing conditions, which are known to favor the formation of tricyclic adducts, we briefly examined the effect of the substituents toward product distribution (Scheme 3). Replacement of pentane-1,5-diol 2a by 3-methylpentane-1,5-diol 2b led to similar ratio and conversion, and 5 was isolated in 80% isolated yield. As expected, the presence of the electron-donating methoxy group in 1b enhancing the nucleophilicity at position 2 of the indole nucleus resulted in the decrease of the selectivity and N-alkylated products 7 and 9 were obtained in 68% and 65% isolated yields, respectively. Noteworthy that for 3-methylpentane-1,5-diol 2b, indoloquinolizidines 6 and 10 were observed as 6.5:3.5 diastereoisomeric mixtures.

Scheme 3. Effect of the substituents toward product distribution under refluxing conditions.

Having established the superior activity of Ru-1, we next turned our attention on the challenging selective formation of (±)-10-desbromoarborescidine A by modification of the reaction conditions (Table 2). The ratio of products 3 and 4, and the conversion were greatly affected by the ratio of the substrates.

In the presence of one equivalent or more of tryptamine 1a, the reaction was inhibited, presumably due to the coordination of the nitrogen atoms to the catalyst (entries 1 and 2). However, up to 1:9 ratio in favor of the (±)-10-desbromoarborescidine A (4) was observed. Increasing the amount of diol 2a resulted in the increase of the reaction efficiency (entries 3 and 4). It should be noted that an excess of diols resulted in the selective formation of the compound 3 demonstrating that pentan-1,5-diol plays the role of both reagent and hydrogen donor generating lactone/polyesters along with hydrogen. [52,[54][55][56][57][58][59] Interestingly, decrease of catalyst loading resulted in the selective formation of the (±)-10-desbromoarborescidine A (4) (entries 3 and 5). Similarly, increasing dilution favored the formation of the latter (entries 3,6,7 and 9). These results tend to demonstrate that the lifetime of the iminium intermediates is inversely proportional to the loading of precatalyst. Thus, low catalyst loading is required to suppress reduction of the iminium in order to favor the attack by the indole nucleus. Lowering the amount of diol 2a to 1.1 equivalent led to higher selectivity but lower conversion (entry 11). Finally, at higher reaction temperature with a 1:1.2 substrate ratio, a ratio of 2:8 was obtained in favor of 4 produced in 65% GC yield (entry 10). The tedious separation of 3 and 4 over alumina gel resulted in a promising 45% isolated yield. Successful crystallization confirmed the tetracyclic structure (Figure 1).

One may argue that the formation of (±)-10desbromoarborescidine A (4) can also occur from the dehydrogenation/oxidation of uncyclized compound 3 as a formal monoamine oxidase/polonovski type reaction. [26] As demonstrated in Table 2, the ratio of 3:4 was not affected by reaction time (entries 12 and 13). Moreover, it should be pointed out that during the treatment of the isolated 3 in the presence of various hydrogen acceptor or under air (±)-10desbromoarborescidine A (4) was not detected even as traces (Scheme 4). Taken together, these results demonstrate that 4 arose from a Pictet-Spengler condensation on the last iminium intermediate (scheme 2). Table 2. Toward the selective formation of (±)-10-desbromoarborescidine A Entry [a] (mol %) Ratio 1a:2a Conc. T °C Conv. [b] Ratio 3/4 [a] Experimental conditions: all reactions were performed under an inert atmosphere of argon in a closed Schlenk tube in degassed dried toluene.

[b] Conversions were determined by GC. First number in parentheses corresponds to the GC yield of the product 4 using tetradecane as internal standard and second to the isolated yield after column chromatography over alumina gel.

[c] Ratio was determined by GC analysis on the crude reaction mixture.

[d] Reaction time 24 h.

Accepted Manuscript

[e] Reaction time 36 h.

In conclusion, it is now possible to access to (±)-10desbromoarborescidine A (4) via a formal dehydrogenative dehydrative coupling of tryptamine and pentane-1,5-diol. To the best of our knowledge, this is the first one-step synthesis of (±)-10-desbromoarborescidine A. The lack of reactivity of 3 demonstrated the trapping of the iminium intermediate resulting from the second condensation and not the subsequent oxidation/dehydrogenation of 3. Results also demonstrated that minor modification of the reaction conditions (ratio of reagents, concentration, catalyst loading, etc.) greatly affects the outcome of the reaction. Taken together, this strategy offers a powerful and promising way to repurpose the conventional syntheses of various indoloquinolizines and harmicine alkaloids. Current efforts are focused on the extension of this transformation with a second generation of precatalysts.

Figure 1 .

 1 Figure 1. X-ray structures of isolated N-alkylated compound 3 and 10desbromoarborescidine A 4. CCDC 1965973, 1965972 numbers contain the supplementary crystallographic data for compounds 3 and 4, respectively.

Scheme 4 .

 4 Scheme 4. Attempts to convert tricyclic heterocycle 3 to indoloquinolizidine 4.

Table 1 .

 1 Evaluation of precatalysts towards product distribution.

Acknowledgements ((optional))

gratefully acknowledged for the fellowships to A.R.S., G.L and the funding of this project. Prof.

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Institute and/or researcher Twitter usernames: @apurbarsahoo, @achardmathieu, @chimie_ISCR, @csiriict,