Céline Plainvert

Magalie Longo

Elise Seringe

Benjamin Saintpierre

Elisabeth Sauvage

Laurence Ma

Johann Beghain

Nicolas Dmytruk

Gislène Collobert

Eric Hernandez

Pascal Astagneau

Philippe Glaser

Frédéric Ariey

Claire Poyart
email: claire.poyart@aphp.fr

Agnès Fouet

A clone of the emergent Streptococcus pyogenes emm89 clade

Keywords: Group A Streptococcus, emm89, emerging clade, phylogeny, biofilm, bacterium-cell interaction, outbreak

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

INTRODUCTION

Group A Streptococcus (GAS, Streptococcus pyogenes) is a human pathogen causing a broad range of diseases, non-invasive and invasive diseases as well as autoimmune sequelae, with an estimated 517,000 deaths per year [START_REF] Carapetis | The global burden of group A streptococcal diseases[END_REF]. These can be community acquired or be the consequence of outbreaks in human settings with high proximity such as schools, military barracks or hospital wards. Residents of the latter are at increased risk of acquiring GAS infections because of underlying medical conditions favorable to streptococcal infections [START_REF] Deutscher | Investigation of a group A streptococcal outbreak among residents of a long-term acute care hospital[END_REF].

GAS strains are extremely diverse and currently the most widely used typing-method for GAS strains relies on the 5'end of the emm gene sequence that encodes the hypervariable amino-terminus region of the M protein, a surface exposed virulence factor [START_REF] Beall | Sequencing emm-specific pcr products for routine and accurate typing of group A streptococci[END_REF]. Among more than 200 emm-types defined, the most frequently associated with invasive infections in Europe are emm1, emm28 and emm89, with variable distribution worldwide [START_REF] Plainvert | Invasive group a streptococcal infections in adults, france (2006-2010)[END_REF][START_REF] Steer | Global emm type distribution of group A streptococci: Systematic review and implications for vaccine development[END_REF].

Epidemiological studies have evidenced the emergence of given genotypes and more precisely of clones as leading cause of disease in Europe or the United States. Such was the case with the emm1 type and the M1T1 clone in the late 1980s ( [START_REF] Nasser | Evolutionary pathway to increased virulence and epidemic group a Streptococcus disease derived from 3,615 genome sequences[END_REF] and references herein) and more recently of the emm89 type exemplified by the emergent clade or clade 3 variant [START_REF] Turner | Emergence of a new highly successful acapsular group a Streptococcus clade of genotype emm89 in the united kingdom[END_REF][START_REF] Zhu | Trading capsule for increased cytotoxin production: Contribution to virulence of a newly emerged clade of emm89 Streptococcus pyogenes[END_REF].

The emergence of emm89 strains, most probably of the same clone, has been described in multiple geographic locations [START_REF] Friaes | Emergence of the same successful clade among distinct populations of emm89 Streptococcus pyogenes in multiple geographic regions[END_REF][START_REF] Musser | Reply to "emergence of the same successful clade among distinct populations of emm89 streptococcus pyogenes in multiple geographic regions[END_REF][START_REF] Turner | Reply to "emergence of the same successful clade among distinct populations of emm89 Streptococcus pyogenes in multiple geographic regions[END_REF][START_REF] Latronico | Genomic characteristics behind the spread of bacteremic group a Streptococcus type emm89 in finland, 2004-2014[END_REF][START_REF] Beres | Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes[END_REF][14][START_REF] Chochua | Population and whole genome sequence based characterization of invasive group a streptococci recovered in the united states during 2015[END_REF][START_REF] Teatero | Rapid emergence of a new clone impacts the population at risk and increases the incidence of type emm89 group a Streptococcus invasive disease[END_REF]. Although emm89 strain recovery from outbreaks has been described previously [START_REF] Falkenhorst | Outbreak of group a streptococcal throat infection: Don't forget to ask about food[END_REF][START_REF] Thigpen | Nursing home outbreak of invasive group a streptococcal infections caused by 2 distinct strains[END_REF][START_REF] Yang | A probable food-borne outbreak of pharyngitis after a massive rainstorm in beijing, caused by emm89 group a streptococcus rarely found in china[END_REF], the newly emerging emm89 clone has not been decisively implicated in major outbreaks.

In 2012 in a follow-up care and rehabilitation post-surgery oncology unit in France, GAS infections were recorded at an unusual level and, although state-of-the-art prophylaxis measures were taken, this outbreak lasted twenty months. We investigated at the molecular level the cause of this outbreak by analyzing all isolates, be they from invasive or noninvasive diseases or colonization. The strains were genetically characterized and, using biological and date criteria, fifty-six strains were chosen and sequenced. To shed light on possible reasons for this long-lasting outbreak we investigated whether the GAS strains involved had specific properties in terms of biofilm formation capacity, interactions with human lung epithelial cells or human macrophages.

MATERIALS AND METHODS

Case definition

Invasive disease is defined through the isolation of GAS from normally sterile sites or the isolation of GAS from non-sterile sites (e.g., sputum, wounds) in the presence of either streptococcal toxic shock syndrome or necrotizing fasciitis and non-invasive disease through the isolation of GAS from a non-sterile site and which developed a clinical syndrome consistent with GAS infection. We defined a carrier as a patient or health care worker with GAS cultured from a non-sterile site and no clinical evidence of infection at the time of culture.

Bacterial strains

All GAS isolates collected during the outbreak were characterized by the French national reference center for streptococci (CNR-Strep) (Table S1). emm sequence type was determined by sequencing the variable 5'end of the emm gene and comparing sequences with database of the Center for Disease Control and Prevention (http://www.cdc.gov/ncdidod/biotech/strep/doc.htm) [START_REF] Beall | Sequencing emm-specific pcr products for routine and accurate typing of group A streptococci[END_REF]. All strains were tested by a multiplex PCR method for the presence of genes encoding the toxins or superantigens SpeA, SpeB, SpeC, Ssa and SmeZ [START_REF] Plainvert | Invasive group a streptococcal infections in adults, france (2006-2010)[END_REF]. A prior cluster analysis was carried out by pulsed-field gel electrophoresis (PFGE) with SmaI restriction enzyme and interpreted according to Tenover criteria [START_REF] Tenover | Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing[END_REF]. Other GAS strains collected by the CNR and similarly characterized were used as controls (Table S2). Antibiotic resistance profiles were determined according to the European Committee on Antimicrobial Susceptibility Testing guidelines (http://www.eucast.org).

Bacterial growth conditions

GAS isolates were grown at 37ºC without agitation in Todd-Hewitt broth supplemented with 0.5 % Yeast extract (THY) or in Brain Heart Infusion (BHI) (Difco). Bacteria were collected in mid-log phase, washed once with sterile phosphate-buffered saline (PBS), and diluted to the required inoculum; the number of viable bacteria was determined by counting the colony forming units (CFUs) after plating dilutions on THY agar.

GAS strain sequencing

Chromosomal DNA was extracted using the MasterPure TM Gram-positive DNA purification kit (Tebu-Bio) and sequenced using the Illumina technology, with read length of 100 nt and a more than 100 fold-coverage. Libraries were constructed by using the Illumina TrueSeq kit following the manufacturer's instructions. Illumina short reads were assembled by using the Velvet software [START_REF] Zerbino | Velvet: Algorithms for de novo short read assembly using de bruijn graphs[END_REF]. Strain 20120499 considered as the outbreak index case was used as a reference for SNPs calling. Contigs of strains 20120499 were ordered by aligning them to the complete genome sequence of strain MGAS315 (emm1, accession number NC_004070.1, no emm89 complete sequence being available at the time of this analysis) using the Mauve software [START_REF] Darling | Mauve: Multiple alignment of conserved genomic sequence with rearrangements[END_REF]. Each strain was analyzed for SNPs, small indels and deletions compared to strain 20120499 by using the BRESEQ software (pmid 24838886), which allowed the identification of errors in the Velvet assembly. Sequence specific to each strain was analyzed by assembling unmapped reads with Velvet. Accession numbers pending.

Reconstruction of large insertion from fastq files.

A Perl software named iSeGWalker was newly developed to accomplish a de novo genome reconstruction from the reads file in fastq format [START_REF] Karadjian | Highly rearranged mitochondrial genome in nycteria parasites (haemosporidia) from bats[END_REF]. The search step is an exact-matching reads selection using a regular expression (seed) and a simple Perl script, reading each sequence one by one. Once all matching reads of the index case genome (20120456) had been selected, a consensus sequence was determined; then, a new seed, composed by the 30 last consecutive nucleotides, was obtained and a new search was performed.

Phylogeny analysis

Phylogenetic analyses were performed after alignment of all strains against the new reconstructed sequence as reference. The evolutionary history was inferred using the Neighbor-Joining method [24]. 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 rate variation among sites was modeled with a gamma distribution (shape parameter = 1). The differences in the composition bias among sequences were considered in evolutionary comparisons [START_REF] Tamura | Evolutionary distance estimation under heterogeneous substitution pattern among lineages[END_REF]. All positions containing gaps and missing data were removed for each sequence pair. Evolutionary analyses were conducted in MEGA7 [START_REF] Kumar | Mega7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF].

PCR amplification

The DNA sequence surrounding the has operon was amplified using the oligonucleotides Has-M89_F CTGTGCCACTAAGACTCTTT and Has-M89_R CGGACAGGTGCAGTTGG-TTT using the GoTaq® Green Master Mix with an elongation time of 4 min.

Biofilm formation assay

Overnight cultures grown in BHI, for optimal abiotic biofilm formation as described in [START_REF] Koller | Typing of the pilus-protein-encoding fct region and biofilm formation as novel parameters in epidemiological investigations of Streptococcus pyogenes isolates from various infection sites[END_REF],

were diluted to an optical density at 600 nm (OD600) of 0.06 and 180 µL were distributed in quadruplet in 96-well plates. After 24 h static growth at 37°C, one plate was used to record the OD595 after resuspension while the other one was treated with crystal violet as described by Köller et al. [START_REF] Koller | Typing of the pilus-protein-encoding fct region and biofilm formation as novel parameters in epidemiological investigations of Streptococcus pyogenes isolates from various infection sites[END_REF] except that the aqueous 1% SDS solution was replaced by a 80/20 ethanol/acetone solution. Biofilm formation capacity was defined as the ratio of the OD595 elicited by crystal violet and the OD595 due to growth on the control plate. The experiment was carried out at least in triplicate.

Growth kinetics.

Overnight cultures grown in THY were diluted 1:100 in fresh THY and 150 µL were distributed in quadruplet in 96-well plates. The plates were incubated at 37°C in a Thermo Scientific Multiskan™ GO microplate spectrophotometer. OD600 was measured every 10 min after agitation.

Human lung epithelial cell culture and adhesion and invasion assays

A549 cells were grown in DMEM-Glutamax 10% Fetal Calf Serum (FCS) in 24-well plates during 2 days, washed with PBS and infected with mid-logarithmic GAS resuspended in RPMI cell-culture medium at a multiplicity of infection (MOI) of 1 bacterium / cell. For adhesion assays, the plates were centrifuged 5 min at 1000 rpm and incubated 1 h at 4°C; the non-adherent extracellular bacteria were then eliminated removing the culture medium and washing three times with sterile PBS. For internalization assays, the plates were further incubated 45 min at 37°C, 5% CO2, and the adherent extracellular bacteria were subsequently killed by incubation with fresh medium containing 50 U/mL penicillin/ and 50 g/mL streptomycin for 30 min. After washing with PBS A549 cells were lysed with 1 mL cold sterile distilled water and serial dilutions were plated on THY plates and the number of CFUs was determined after 24-48 hours growth at 37ºC. For all experiments, 4 or 5 independent assays in duplicate were carried out for each bacterial isolate.

Human macrophage culture and infection assays

To differentiate the THP-1 into macrophages, 2.10 5 to 4.10 5 cells were inoculated in RPMI GluMax supplemented with 10% FCS and 200 nM PMA (Phorbol 12-myristate 13-acetate) (Sigma, P1585). After 3 days at 37°C, 5% CO2, the medium was replaced with RPMI GluMax supplemented with 10% FCS and the incubation extended for 5 days. Cells were then infected with mid-logarithmic GAS resuspended in RPMI medium at a MOI of 1. The phagocytosis and survival assays were carried out as previously described [START_REF] Dinis | The innate immune response elicited by group a Streptococcus is highly variable among clinical isolates and correlates with the emm type[END_REF].

Statistical analyses.

Significance of differences in biofilm formation capacities were assessed by Student's t-test.

That of cellular adhesion, invasion, phagocytosis and survival were assessed by Repeated measures Anova and Turkey's multiple comparison test, GraphPad software.

RESULTS

Outbreak description

From 16 th April through 7 th May 2012, 4 cases of invasive GAS infections were recorded in a 100-bed post-surgery oncology unit (Fig. 1). To limit secondary cases, epidemiological investigations were started among the patients and healthcare workers to determine their status against GAS. Although appropriate hygiene measures were taken this outbreak lasted until 23 rd December 2013 concerned 66 cases and ended after mass chemoprophylaxis by azithromycin 500 mg per os once daily for 3 days and enhanced infection control such as mask wear, hand hygiene and the implementation of appropriate disinfection measures for tracheostomy cannula.

Almost all the patients had surgery for an oral cancer and were in second line of treatment with radiotherapy. Men accounted for 83% (55/66) of the patients and the mean age were 61 year-old (range 24-87) and 67.5 year-old (52-83) for men and women, respectively (Table S1 and data not shown).

During the outbreak, 26 cases of GAS invasive infections were identified, 13 bacteremia, 11 pneumonias and 2 necrotizing fasciitis, among which 5 (19.2%) died. During the epidemiological surveillance, 57 non-invasive GAS strains were isolated; 44 from patients and 13 from healthcare workers with direct patient-care responsibilities (Table S1).

GAS patients' strains, were isolated from blood cultures (n=13), oropharynx (n=9), wounds (n=13), gastrostomy sites (n=8), tracheostomy cannula (n=8), respiratory specimen (n=18), and lymphadenectomy (n=1). All thirteen GAS-positive cultures from healthcare workers were from the oropharynx. A total of 83 GAS strains were thus isolated between April 2012 and December 2013 including 26 invasive, 38 non-invasive, and 19 from colonization. An invasive respiratory device was present in 13/26 (50%) patients with an invasive infection and in only 12/44 (27%) patients colonized or without an invasive infection, p=0.049.

Genetic characterization of the GAS strains

All strains were genotyped (Table S1). All invasive but one (25/26) and 51 out of 57 noninvasive or colonization strains shared the same emm89 genotype. Sixty-nine/76 (91%)

emm89 GAS strains, be they invasive, non-invasive or colonization strains, were indistinguishable by PFGE displaying an 89-A4 pattern according to our nomenclature (Table S1 and data not shown). Patterns of the seven emm89 remaining strains showed a maximum of four differences. All 76 emm89 strains displayed a low-level resistance to aminoglycosides (kanamycin and gentamicin) and were susceptible to β-lactams, vancomycin, macrolides and tetracycline except two non-89-A4 strains that were resistant to tetracycline due to the presence of tet(M) determinant located on Tn916 related conjugative transposon.

Thus, whereas non-89-A4 PFGE and non-emm89 strains were occasionally isolated, 69/83 GAS strains isolated throughout the considered period shared the same emm89 genotype 89-A4 PFGE pattern. This argued for an outbreak origin of these strains; the other strains were considered outside the outbreak. To further analyze these emm89 89-A4 strains, a total of 56 were randomly chosen for sequencing among a sub-collection selected according to date and clinical criteria as follows: a balanced distribution, 22/26 and 34/50 isolated in 2012 and 2013, respectively, including the first, index case in 2012, and last of each year, and 9 and 11 invasive infections from 2012 and 2013, respectively. For sake of comparison, we added an unrelated emm89 invasive strain isolated in 2012 (20120456) as well as three clinical isolates isolated at the same post-surgery oncology unit but in 2014 (20141462, 20141463 and 20140469; Table S2).

Sequence analysis and phylogeny of the GAS outbreak isolates

The alignment of three fastq files, including that of the index case strain (20120499), to H293 emm89 genomic sequence (NC_002737.2), and the use of the Tablet software [START_REF] Milne | Using tablet for visual exploration of secondgeneration sequencing data[END_REF], led to the identification of 5 areas of possible chromosomal rearrangement sites based on a relative increase in the diversity of these loci. We then reconstructed the new reference genome (see For phylogenetic analyses we used the whole genome alignment of 61 strains: we combined genomic data obtained from 56 isolates of the 2012-2013 outbreak, one emm89 control strain independent of this outbreak (20120256), three strains isolated in 2014 at a time-related distance from the outbreak, and the reference MGAS27061 an emm89 clade 3 strain [START_REF] Turner | Emergence of a new highly successful acapsular group a Streptococcus clade of genotype emm89 in the united kingdom[END_REF][START_REF] Zhu | Trading capsule for increased cytotoxin production: Contribution to virulence of a newly emerged clade of emm89 Streptococcus pyogenes[END_REF] (Fig. 2; Table S2). This tree (Fig. 2) clearly shows the monophyletic character of the 2012/2013 outbreak, well separated from the isolates collected at the same site but in time away from the outbreak (2014 strains). The control strain 20120456 is phylogenetically more distant to the index case strain (42 SNPs compared to the 20120499 index strain) than all other strains. Furthermore, the fact that the reference strain MGAS26761 is positioned between the branches 2012/2013 and 2014, reinforces the independence of the two events and strongly suggests the extinction of the outbreak episode of 2012/2013 (Fig. 2).

The tree covering the 55 outbreak strains (Fig. 3) presents three separate subclones linked to their date of isolation. Of the three main subclones, two are composed solely of 2012 strains (subclones 1 and 2 2012). Among strains belonging to subclone 3 2013, two, 20121368 and 20121198, were the last isolated in 2012, in fact during the last trimester of 2012.

Also they have lost the phage harboring speC, however loss of a phage being compatible with a convergence event. The strain 20130948 appears to have a quite peculiar evolutionary history and lies apart from all subclones. Despite these exceptions this topography is highly compatible with an epidemic signature: most strains deriving from a unique colonization event occurring in 2012. The emm89 outbreak subclone was termed emm89-F.

Forty-three genetic profiles were defined (Table S3). At the gene level, four mutations stabilized over the outbreak period; two occurred in intergenic regions and the two others in genes involved in bacterial metabolism, the oxaloacetate decarboxylase beta chain (ODC), found in 36 strains from 8 th September 2012 onward, and the shikimate 5-dehydrogenase I alpha (S5D), found in 33 strains from 25 th January 2013 onward. Ten non-stabilized mutations were also found in the gene encoding the serum opacity factor (SOF) among 29 strains. Six non-fixed mutations were also found among 8 strains in the gene encoding the CovS regulatory protein. No mutation or group of mutations could be related to the strain invasive status.

Phenotypic characterization of the emm89-F strains

Isolates of the emm89-F clone were characterized for phenotypes relevant to the clinical settings of this outbreak, i. e. growth capacities, abiotic biofilm formation, interactions with lung epithelial cells and macrophages. Strains were chosen to cover all the branches of the genetic tree and to, altogether, harbor all mutation profiles (Fig. 3, Table S1).

Biofilm formation

As mentioned a significant correlation between the occurrence of invasive GAS infections and the presence of a medical respiratory device was observed among infected patients.

(Table S1). The recognition that biofilm formation by GAS is involved in persistence (for review [START_REF] Young | Streptococcus pyogenes biofilm[END_REF]) and that biofilm formation varies among strains within emm genotypes [START_REF] Lembke | Characterization of biofilm formation by clinically relevant serotypes of group a streptococci[END_REF] prompted us to assay the capacity of these clinical isolates to form biofilm on abiotic surface (Fig. 4). All strains were able to form biofilms and most of them in similar amounts; no difference was observed between strains sampled from tracheostomy cannula and from other sites. The stronger biofilm producers are mutated in either the global positive regulator gene mga, yielding values slightly above those of the other strains, or, and more impressively, in covS, the sensor of the CovRS two component system known to negatively regulate the expression of multiple virulence genes [START_REF] Churchward | The two faces of janus: Virulence gene regulation by covr/s in group a streptococci[END_REF].

We also assessed whether GAS biofilm formation capacities increased during the outbreak.

To that aim we calculated a trend line, after removing the strains mutated in regulatory genes (Fig. S1). It slightly raises (y = 0.0362 x + 8.8) but displays no great change in biofilm formation throughout the outbreak.

To assess whether the outbreak isolates were weak or robust biofilm formers, their capacity to form biofilms was compared to that of other unrelated clinical isolates (Fig. 4, Table S2).

Biofilm formation being dependent on the CovRS regulatory system, we included only covR covS wild-type isolates from other studies and calculated the mean value for the outbreak isolates excluding all regulatory mutants [START_REF] Cho | Patterns of virulence gene expression differ between biofilm and tissue communities of streptococcus pyogenes[END_REF]. The clinical isolates collected during the outbreak formed substantial more biofilms compared to isolates from other emm types, (p = 0.001). In contrast, no difference was observed when comparison was established with other emm89 isolates, also belonging to the emergent clade 3 [START_REF] Turner | Emergence of a new highly successful acapsular group a Streptococcus clade of genotype emm89 in the united kingdom[END_REF][START_REF] Zhu | Trading capsule for increased cytotoxin production: Contribution to virulence of a newly emerged clade of emm89 Streptococcus pyogenes[END_REF], (data not shown) indicating that this may be a specific trait to the emerging emm89 clones.

In conclusion these results show that emm89 outbreak isolates are more robust biofilm formers than non-emm89 isolates; this property could account and contribute for the current emergence and spread of this clone.

Adhesion and invasion into lung epithelial cells

The pathologies observed during the outbreak such as respiratory infections prompted us to analyze the adhesion and invasion of selected clinical isolates into human A549 lung epithelial cells. Four strains were selected, in addition to the index case (IC; 120499), using different criteria to ensure a representative sampling: dispersion throughout the genetic tree and the presence of different mutations, SOF (120811), ODC (120747), S5D (130922) and the regulator CovS (120619) (Fig. 3 and Fig. S1 and Table S1). We first assessed that these strains displayed no growth difference in laboratory conditions (data not shown).

Adhesion capacities of these strains were determined and compared to that of an unrelated emm89 strain previously studied [START_REF] Dinis | The innate immune response elicited by group a Streptococcus is highly variable among clinical isolates and correlates with the emm type[END_REF]. No significant difference in the adhesion capacity of these strains was observed (Table 1). In contrast, a difference was noted when invasion was compared: the invasion of the covS mutant isolate was lower than that of all the other strains and was significantly lower than that of the emm89 control strain (Table 1). This suggests that these clinical isolates possessed no specific properties in their interaction with pulmonary cells compared to other unrelated GAS emm89 clinical isolates and did not acquire any, through their mutations detected.

Phagocytosis and survival within macrophages

Macrophages are critical host defense cells. We have previously shown that emm89 isolates were more phagocytozed by murine macrophages than non-emm89 strains, that they were differentially phagocytozed depending on their invasive status and that there was a variability within the invasive and non-invasive strains [START_REF] Dinis | The innate immune response elicited by group a Streptococcus is highly variable among clinical isolates and correlates with the emm type[END_REF]. Consequently, we analyzed the behavior of the outbreak clinical isolates towards uptake and survival in the physiologically relevant THP-1 human macrophages and compared it to that of the emm89 control strain (Fig. 5).

Except for the covS mutant strain, which seemed less phagocytozed than all other strains and was significantly less phagocytozed than the SOF and S5D strains, all outbreak isolates and the control strain were similarly phagocytozed by THP1 macrophages (Fig. 5A).

We then tested the survival of these isolates in THP-1 (Fig. 5B). Only the covS mutant strain survived, at T2, less than other strains, and significantly less than the index isolate.

This indicates that the outbreak strains and the emm89 control isolate behaved similarly towards the host defense elicited by the human macrophages.

DISCUSSION

This is, to our knowledge, one of the largest outbreaks of GAS infection in a single health care facility among highly vulnerable patients with oral cancer. Here we describe a remarkably long outbreak linked to the persistence and spread of a clone in both patients and healthcare workers. Almost all isolates from patients with GAS infection and those from healthcare personnel were subtype emm89, suggesting that one GAS strain was circulating throughout the healthcare facility. However, we could not determine whether a patient or an infected colonized healthcare worker initiated the outbreak since investigations began three weeks after notification to the authorities of the four cases of invasive infections. In France, emm89 is now among the top three leading emm types, accounting for 19 % of all invasive infections in 2017 and the first one (23 %) for GAS clustered cases since 2011 (https://cnrstrep.fr/index.php/bilans-dactivite/rapports-dactivite).

Using whole-genome sequencing we demonstrated that this clone belongs to the emerging emm89 clade 3 [START_REF] Turner | Emergence of a new highly successful acapsular group a Streptococcus clade of genotype emm89 in the united kingdom[END_REF][START_REF] Zhu | Trading capsule for increased cytotoxin production: Contribution to virulence of a newly emerged clade of emm89 Streptococcus pyogenes[END_REF]14]. Interestingly, most but not all isolates could be phylogenetically grouped on the basis of their sampling year (Fig. 2). The exceptions suggest that some clones reappeared after a few months during which they were asymptomatically carried. Several data report that this emm89 emerged in many countries in a closely similar time frame during the first decade of the 2000s, accompanied by a swift from clade 1 to clade 2 and subsequent emergence of clade 3 [START_REF] Latronico | Genomic characteristics behind the spread of bacteremic group a Streptococcus type emm89 in finland, 2004-2014[END_REF].

The extent of this outbreak and the case-fatality rate of 19 % among patients with invasive disease prompted us to shed light on the advantages brought by this specific clone; we studied phenotypic traits such as biofilm formation and interaction with human pulmonary cells or macrophages. The similar high level of biofilm formation displayed by the outbreak and other emm89 clones compared to other emm-type strains could be a consequence of all strains belonging to the emerging emm89 clade. Indeed, emm89 strains were not described as high biofilm producer by Koller et al. [START_REF] Koller | Typing of the pilus-protein-encoding fct region and biofilm formation as novel parameters in epidemiological investigations of Streptococcus pyogenes isolates from various infection sites[END_REF], and this could be linked to their strain panel, sampled between 2001 and 2006, that is before the full emergence of this clone [START_REF] Turner | Emergence of a new highly successful acapsular group a Streptococcus clade of genotype emm89 in the united kingdom[END_REF][START_REF] Koller | Typing of the pilus-protein-encoding fct region and biofilm formation as novel parameters in epidemiological investigations of Streptococcus pyogenes isolates from various infection sites[END_REF]. Here we show that the emm89-F clone, which is uncapsulated, is a robust biofilm former confirming that capsule is dispensable for abiotic biofilm formation [START_REF] Cho | Patterns of virulence gene expression differ between biofilm and tissue communities of streptococcus pyogenes[END_REF][START_REF] Fiedler | Streptococcus pyogenes biofilms-formation, biology, and clinical relevance[END_REF]. The emm89-F mga mutants, Mga being a global transcriptional activator including of the M and M-like encoding genes, yielded slightly higher biofilm production than the wild-type emm89-F isolates. M and M-like proteins most often contribute to biofilm formation, which is nevertheless emm-type dependent [START_REF] Cho | Patterns of virulence gene expression differ between biofilm and tissue communities of streptococcus pyogenes[END_REF][START_REF] Fiedler | Streptococcus pyogenes biofilms-formation, biology, and clinical relevance[END_REF][37]; a weaker M and M-like protein production in an emm89 background may have, as in the emm2 background, little influence on biofilm formation [37]. Furthermore, a variation in the abundance of Mga-controlled products, other than the M and M-like proteins, could account for the biofilm increase. CovRS is a two-component system that controls, mainly repressing, the expression of 15 % of the GAS genome. Several covS-mutant strains produce less, and others more biofilm than their wild-type counterparts, suggesting that the consequences of mutations in covS on biofilm formation are strain-dependent [START_REF] Cho | Patterns of virulence gene expression differ between biofilm and tissue communities of streptococcus pyogenes[END_REF]38]. The emm89-F covS mutant isolates were particularly robust biofilm formers supporting the straindependent contribution of CovS to biofilm formation and demonstrating that CovRScontrolled products other than the capsule are involved in biofilm formation.

Several reports suggested that acapsular emm89 clade-associated GAS strains may have acquired potential for long-term colonization [START_REF] Turner | Emergence of a new highly successful acapsular group a Streptococcus clade of genotype emm89 in the united kingdom[END_REF][START_REF] Beres | Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes[END_REF]. Moreover, the increased expression of SLO and NADase by this clade could enhance internalization and intracellular bacterial survival in epithelial cells, theoretically providing protection from natural antimicrobial peptides and antibiotic treatment. Adhesion and invasion experiments on human epithelial lung cells performed on a representative sampling did not reveal major difference among strains except for the covS strain for which, if adhesion was similar to other strains, its capacity to invade cells was affected. Furthermore, there was a tendency for covS strain to be less phagocytozed and to survive less than the others in the macrophage. Altogether this meant a disadvantage for this strain for which epithelial cells and macrophages would not act as a reservoir and promote a carrier state. This result is consistent with the report that covS strains are badly transmitted [39], thus accounting for sporadic apparition of covS mutations during this outbreak.

In this outbreak the majority of the patients had undergone surgery associated with radiotherapy for oral cancer and belongs to a disadvantaged and highly vulnerable population.

These features, could explain the very high rate of transmission in fragile patients where promiscuity was highly problematic.

In conclusion, this report adds France to the growing list of European countries and North America where, by whole genome sequencing, emm89 clade 3 strains have been demonstrated

to an emergent clone eliciting GAS infections. It strengthens the fact that the spreading of this clone is worldwide and emphasizes the importance of epidemiological monitoring by reference centers and health authorities. It confirms the relevance, power and robustness of the whole genome sequencing approach for epidemiological analysis of major outbreaks and its routine use for surveillance. S2). For each strain, the mean value of its S2, [START_REF] Dinis | The innate immune response elicited by group a Streptococcus is highly variable among clinical isolates and correlates with the emm type[END_REF]) as described in Material and Methods and results are expressed as the percentages of (A) bacterial CFUs recovered after 30 min antibiotic treatment relative to the initial inoculum and (B) the percentage of phagocytozed bacteria that survived. The results represent the mean ± SD of 5 independent experiments carried out in triplicate for each isolate, with significance levels indicated between a given strain and the covS mutant strain types (*p ≤ 0.05; **p ≤ 0.01).

Figure S1.

The outbreak emm89-F isolates form substantial biofilms compared to other non-emm89 GAS strains. A tendency curve was calculated using the means shown in Fig. 4, y = 0.0362 x + 8.8 excluding the mutants in regulatory genes. The sampling year is indicated below. Symbols are the same as in Fig. 1. are expressed as the percentages of, for the adhesion, bacterial CFUs recovered after three PBS washing relative to the initial inoculum and, for the invasion, bacterial CFUs recovered after 30 min antibiotic treatment relative to the adherent CFUs. The results represent the mean ± SD of 3 and 5 independent experiments, respectively, carried out in triplicate for each isolate, with significance levels indicated between a given strain and the covS mutant strain types (**p ≤ 0.01) The patient's initials, in bold, was added when more than one strain was collected from the same individual b S, susceptible to all antibiotic tested; TcR, tetracycline resistant c + = biofilm formation capacity studied; initials refer to the strains whose interactions with A549 and THP1 have been further studied

Table 1. Adhesion and invasion of human epithelial lung cells (A549) by emm89-F isolates

  Materials and Methods) (1 747 907 base pairs) and realigned all the fastq files on it. The genomes are 99 % identical on a 99% coverage to that of MGAS27061 (accession number NZ_CP013840.1), indicating that these emm89 clinical isolates belong to the new emm89 clade 3, lacking the has operon and harboring the same mutation in the slo-nga promoter region[START_REF] Beres | Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes[END_REF][START_REF] Watanabe | Complete genome sequence of Streptococcus pyogenes strain jmub1235 isolated from an acute phlegmonous gastritis patient[END_REF]. To get insight into the emergence of this new clade in France we sought, in a collection of emm89 French strains, when the has operon loss could have occurred. The DNA fragment encompassing the has operon was amplified from 15 unrelated strains received by the CNR-Strep during the last 15 years (data not shown). In three of these strains, including one from 2003 the has operon was absent, suggesting the presence of the new clade in France already at that time.

37

  Courtney HS, Ofek I, Penfound T, et al. Relationship between expression of the family of m proteins and lipoteichoic acid to hydrophobicity and biofilm formation in Streptococcus pyogenes. PLoS One. 2009; 4: e4166. 38 Sugareva V, Arlt R, Fiedler T, Riani C, Podbielski A, Kreikemeyer B. Serotype-and strain-dependent contribution of the sensor kinase covs of the covrs two-component system to Streptococcus pyogenes pathogenesis. BMC Microbiol. 2010; 10. 39 Alam FM, Turner CE, Smith K, Wiles S, Sriskandan S. Inactivation of the covr/s virulence regulator impairs infection in an improved murine model of Streptococcus pyogenes naso-pharyngeal infection. PLoS One. 2013; 8: e61655.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Evolutionary relationships of taxa of the sequenced isolates.

Figure 3 .

 3 Figure 3. Evolutionary relationships of emm89 outbreak isolates.

Figure 4 .

 4 Figure 4. The outbreak emm89-F isolates form substantial biofilms compared to other non-

  figure. Grey closed circles 2012 strains; black closed circles, 2013 strains; symbols for the

Figure 5 .

 5 Figure 5. Phagocytosis (A) and survival (B) of GAS emm89-F isolates in human

Table S1 .

 S1 Clinical and microbiological characteristics of Streptococcus pyogenes strains isolated during the outbreak

	table S1
		20131102	17/09/13	M	brush Wound	Non-invasive				speB, speC, smeZ	emm89	89-A4	S
		20131103	13/02/13 65 17/09/13	F M	DHN/CE Gastrostomy site	Invasive Non-invasive				speB, smeZ speB, speC, smeZ	emm104 104-A emm89 89-A4	TcR S	+	+
	a	Strain 20131134 20131144 20120499 20131179 20120583 20120584 20120585 20120619 20120665 20120686 20120691 20120713 20120727 20120747 20131188 20131200 20131201 20131202 20131268 20131331 20131380 20131421	Isolation Date 15/02/13 83 Age Sexe Anatomical origin / Patient initials a F DHN 23/09/13 F Pharynx/GV 15/02/13 58 M Tracheostomy cannula Colonization Infection status Invasive Colonization 27/09/13 F Pharynx Non-invasive 16/04/12 M Blood culture Invasive 15/02/13 52 F Pharynx Non-invasive 06/10/13 M Blood culture/BP Invasive 04/05/12 M Blood culture Invasive 03/05/12 F Blood culture Invasive 07/05/12 M Blood culture Invasive 14/05/12 F Pharynx Colonization 16/05/12 M Gastrostomy site Non-invasive 22/05/12 M Pharynx Non-invasive 18/05/12 F Pharynx Non-invasive 26/05/12 M Pharynx Colonization 29/05/12 M Sputum Colonization 02/06/12 M Pharynx Colonization 22/02/13 59 M Tracheostomy cannula Non-invasive 15/02/13 68 M Protected specimen Invasive 12/10/13 M Sputum Colonization brush 20/02/13 66 F Tracheostomy cannula Non-invasive 15/10/13 M Pharynx Colonization 19/02/13 84 M Wound Non-invasive 16/10/13 F Pharynx Colonization 19/02/13 49 M Wound Non-invasive 17/10/13 F Blood culture Invasive 19/02/13 54 M Wound Non-invasive 08/11/13 M Gastrostomy site Colonization 19/02/13 65 M Pharynx Non-invasive 25/11/13 M Blood culture Invasive 20/02/13 60 M 09/12/13 F Sputum Colonization Tracheostomy Non-invasive 23/12/13 M Tracheostomy cannula Colonization cannula/BP 25/02/13 72 M brush Invasive Protected specimen	Death +	Tracheos tomy cannula + + + + + + + +	HCW Molecular markers speB, speC, smeZ + speB, speC, smeZ speB, speC, smeZ + speB, speC, smeZ speB, speC, smeZ speB, smeZ speB, speC, smeZ speB, spec, smeZ speB, speC, smeZ speB, speC, smeZ + speB, speC, smeZ speB, speC, smeZ speB, smeZ + speA, speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ + speB, speC, ssa, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ	emm genotype emm89 emm89 emm89 emm89 emm89 emm104 104-A PFGE pattern 89-A4 89-A4 89-A4 89-A4 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A emm6 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm87 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4 emm89 89-A4	S S ATB b S S S TcR S S S S S S S S S S S S S S S S S S S S S S S S S S S S	+ + WGS + + + + + + + + + + + + + + + + + + + +	biofilm / + + cellular + analysis c + IC + + + CovS + + + ODC + + + + + +
		20120748 20120749	05/06/12 02/06/12 24/02/13 69 25/02/13 23	M M M F	Pharynx Protected specimen brush Protected specimen brush Pharynx	Colonization Invasive Invasive Non-invasive	+	+ +	+	speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ	emm89 emm89 emm89 emm89	89-A14 89-A4 89-A4 89-A4	TcR S S S	+ +	+
		20120772	09/06/12 04/03/13 73	F F	Pharynx Wound	Non-invasive Non-invasive			+	speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+ +	+
		20120773	07/06/12 06/03/13 74	M f	Pharynx Wound	Colonization Colonization				speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A21 89-A4	TcR S	+
		20120774	06/06/12 12/03/13 24	F m	Gastrostomy site Pharynx	Non-invasive Non-invasive		+		speB, speC, smeZ speB, speC, smeZ	emm89 emm75	89-A4	S S	+
		20120775 20120776	05/06/12 11/06/12 11/03/13 60	F F m	Pharynx Pharynx Tracheostomy cannula/BP	Non-invasive Non-invasive Non-invasive		+	+ +	speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ	emm89 emm89 emm89	89-A4 89-A4 89-A4	S S S	+ + +	+ +
		20120811	14/06/12 18/03/13 66	M F	Blood culture Wound/CE	Invasive Colonization	+			speB, speC, smeZ speB, smeZ	emm89 emm104	89-A4	S TcR	+	FBP
		20120824	21/06/12 20/03/13 22	F M	Pharynx Pharynx	Colonization Non-invasive			+ +	speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+	+
		20120825	24/06/12 19/03/13 21	M F	Sputum Pharynx	Colonization Non-invasive		+	+	speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+ +	+ +
		20120849	29/06/12 17/03/13 87	M M	Protected specimen brush Protected specimen brush	Invasive Invasive		+ +		speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+ +	+
		20120852 20121026 20121198 20121368	28/06/12 02/08/12 03/04/13 61 08/09/12 08/04/13 78 20/10/12 15/04/13 53	M M M M M M M	Tracheostomy cannula Non-invasive Wound Non-invasive Tracheostomy Non-invasive cannula/BP Blood culture Invasive Blood culture Invasive Sputum Non-invasive Protected specimen brush Invasive	+	+ + + + + +		speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ speB, smeZ speB, speC, smeZ	emm89 emm89 emm89 emm89 emm89 emm89 emm89	89-A4 89-A4 89-A4 89-A 89-A4 89-A 89-A4	S S S S S S S	+ + + + + +	+ + + + +
		20130016 20130148	14/12/12 30/04/13 61 25/01/13 06/05/13 69 01/07/13 65	M M M M M	Protected specimen Wound brush Wound Protected specimen brush Sputum	Invasive Non-invasive Non-invasive Invasive Non-invasive		+		speB, speC, smeZ speB, speC, smeZ speB, speC, ssa, smeZ speB, speC, smeZ speB, speC, smeZ	emm89 emm89 emm4 emm89 emm89	89-A4 89-A4 89-A4 89-A4	S S S S S	+ + + +	+ + + +
		20130149	25/01/13 12/07/13 28	M F	Adenopathy discharge Non-invasive Pharynx/GV Non-invasive		+	+	speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+	+
		20130150	25/01/13 20/07/13 80	M M	Blood culture Gastrostomy site	Invasive Non-invasive		+ +		speB, speC, smeZ speB, smeZ	emm89 emm89	89-A4 89-A	S S	+	+
		20130243	12/02/13 22/07/13 60	M M	Blood culture Sputum	Invasive Colonization	+	+		speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+ +	+ +
		20130244	11/02/13 30/07/13 74 10/08/13 NA	M M M	Protected specimen brush Blood culture Gastrostomy site	Invasive Invasive Non-invasive		+		speB, speC, smeZ speB, speC, smeZ speB, speC, smeZ	emm89 emm89 emm89	89-A4 89-A4 89-A4	S S S	+ +	S5D +
		20130245	11/02/13 28/08/13 56	M F	Gastrostomy site Gastrostomy site	Non-invasive Non-invasive				speB, speC, smeZ speB, speC, smeZ	emm89 emm89	89-A4 89-A4	S S	+ +	+
		20130246	11/02/13 29/08/13 75	M M	Protected specimen Wound	Invasive Non-invasive		+		speB, speC, smeZ speB, smeZ	emm89 emm89	89-A4 89-A	S S

Table S2 .

 S2 Control strains used

	CNR Strain number	Anatomical origin	emm genotype	Molecular markers a	Antibiotic resistance b
		Blood culture	emm89	speB, speC	S
		Bronchial aspiration	emm89	speB, speC	S
		Wound	emm89	speB, speC	S
	201410469 Wound	emm89	speB, speC	S
		Blood culture	emm1	speA, speB, speJ S
		Blood culture	emm1	speA, speB, speC S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm1	speA, speB	S
		Blood culture	emm101.2 speB	Tc R
		Blood culture	emm2	speB, speC	S
		Blood culture	emm2	speB, speC	S
		Knee hygroma emm28	speA, speB	S
		Blood culture	emm28	speA, speB	Kan R , Str R , Ery R , Cli R , Tc R
		Blood culture	emm28	speB, speC	S
		Blood culture	emm28	speB, speC	S
		Vagina	emm28	speB, speC	Kan R , Str R , Ery R , Cli R
		Vagina	emm28	speB, speC	S
		Blood culture	emm3	speA, speB	S
		Blood culture	emm90,2	speB, speC	Tc R

a The presence of smeZ was not assayed. b S, susceptible to all antibiotics tested. R , resistant; Cli, clindamycin; Ery, erythromycin; Kan, kanamycin; Str, streptomycin; Tc, tetracycline

table S2 Table S3 .

 S2S3 Relevant mutations occuring in the sequenced isolates

	table S3
				D	Yes
			AQ	Yes
	position 13953 21763 22538	AH	Yes 14733 21964 37183	4334	21044 37290	7957 22055	21074 50763	9031 22168
	type of mutation mutation transcription orientation of the gene AB Yes X ← ← → → → → S5D Δ1 bp C → T coding (189/1284 nt) A142T (GCC → ACC) duplication G → A C → A G16D (GGC → GAC) D525Y (GAT → TAT) → ← → → → ← 11 bp x 2 Δ2 bp C → T A → G coding (802-803/1503 nt) Q335* (CAA → TAA) D365G (GAT → GGT) C → T C → T G → A C → A → C → A A38D (GCC → GAC) 22 bp x 2 duplication → → Δ5 bp coding (1207-1211/1503 nt) T147I (ACT → ATT) S79F (TCC → TTC) A115T (GCT → ACT) D107Y (GAT → TAT) X
	Strain name Dihydroorotate mutation profile a F AH -C FIG009439: Cytosolic protein biofilm / cellular analysis b Yes Yes Yes L-xylulose 5-phosphate 3-hypothetical protein X Transmembrane histidine kinase Dipeptide-binding ABC transporter, periplasmic substrate-binding component DppA (TC 3.A.1.5.2) Transmembrane histidine kinase Fibronectin-binding protein Transmembrane histidine kinase Transmembrane histidine kinase Transmembrane histidine kinase Transmembrane histidine kinase dehydrogenase, catalytic Topoisomerase IV subunit Topoisomerase IV subunit Response regulator of the containing multiple CBS (EC 1.3.3.1) domains epimerase (EC 5.1.3.-) CovS CovS CovS CovS CovS CovS subunit A (EC 5.99.1.-) A (EC 5.99.1.-) competence regulon ComE
			AP A AL B AM AO E G AK AA AK A A A	Yes IC Yes Yes Yes Yes Yes CovS Yes Yes Yes			X	X
	a	AG AK The profile is not indicated for the strains outside the epidemic clone ODC X
	b	U yes, biofilm formation assayed; strains further analyzed are indicated by their acronymes Yes	X
			AP
			AK
			AP
			AK
			AP	Yes
			AN	FBP
				T	Yes				X
			AP
			AJ	Yes
				K	Yes			X
			X	V	Yes
			AF	Yes
			AE	Yes
			X	J	Yes
			AO	Yes
				R	Yes
			AO
				Q	Yes				X
				S	Yes
			AD	Yes
				I	Yes				X
				P	Yes
				L	Yes
				H	Yes				X
				Z	Yes
				Y	Yes
				Z	Yes
				I					X
			AH	Yes
				W	Yes	X

Acknowledgements

This work was supported by Santé Publique France, INSERM, CNRS, Université Paris Descartes and by the High Council for Scientific and Technological Cooperation between France-Israel "Complexity in Biology" program. Authorship/Contributor Conceived and designed the study: Céline Plainvert, Agnès Fouet and Claire Poyart. Provided clinical and epidemiological data Elise Seringe, Eric Hernandez and Pascal Astagneau. Performed the bacteriological analyses Magalie Longo, Nicolas Dmytruk and Gislaine Collobert. Performed biofilms and phagocytosis analyses Magalie Longo. Performed Whole genome sequencing of bacterial strains Benjamin Saintpierre, Elisabeth Sauvage, Laurence Ma and Johann Beghain. Analysed genomes data Philippe Glaser, Frédéric Ariey and Agnès Fouet. Wrote the manuscript Claire Poyart, Céline Plainvert and Agnès Fouet.

Conflict of interest statement

The other authors declare no competing financial interests.

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Fibronectin-binding protein

Thymidine kinase (EC 2.7.1.21)

Click here to download Electronic Supplementary Material Fig. S1.tiff


Nicolas De

Marion Jean

Nicolas Vanthuyne

Thierry Buffeteau

Thierry Brotin
email: thierry.brotin@ens-lyon.fr

Enantioselective Complexation of Chiral Oxirane Derivatives by an Enantiopure Cryptophane in Water

In this article we show that optically active cryptophane 1 bearing phenol functions can efficiently bind the two enantiomers of epichlorohydrin, 1,2-butyloxirane, and bioxirane derivatives in aqueous solution. The binding process is characterized by the presence of high-field shifted 1 H NMR signals, which are specific of the encapsulated species.

In all cases, an enantioselective effect has been measured and a relationship between the molecular volume and the affinity constants can be clearly established, even though the flexibility of the guest molecule seems also to play a role. In this series, the bioxirane guest derivative is a particularly interesting example since this compound possesses two stereogenic centers. Thus, in solution, three different couple of diastereomers can be obtained in presence of the optically active host 1. Electronic Circular Dichroism spectroscopy (ECD) has also been used to characterize these complexes. As previously observed with the methyloxirane derivative, the ECD spectra recorded for the 1 L b region show specific cotton bands, which are characteristic of each diastereomers formed in solution.

Introduction: cryptophane derivatives are a well-known family of organic covalently bound capsules whose binding properties have been studied for nearly 40 years. 1 In the past, these molecules have received a lot of attention because these organic systems can accommodate different atoms or molecules within their cavity. For instance, it has been shown that neutral species (methane, xenon, halogenomethanes) or charged species (alkali cations, ammonium, anions) could easily enter the cavity of the cryptophane derivatives depending on the cavity size of the host and the nature of the substituents grafted on the benzene rings. 2 , 3 Interestingly, the encapsulation process strongly modifies the physical properties of both guest and host molecules thus leading to important spectroscopic changes.

For instance, the encapsulated guest usually possesses new original physical properties that can be used for practical applications. As an example, the Xe@cryptophane complexes have received a lot of attention over the last few years since these systems show remarkable physical properties that can be exploited for MRI biosensing applications. 4 Beside their interesting binding properties, it can also be noticed that the overwhelming majority of the cryptophane derivatives show an inherently chiral structure. This second characteristic has not been studied as much as the binding properties of cryptophanes probably because these molecules are difficult to obtain in their enantiopure form. Collet and co-workers pioneered this work in 1987 but this is only in 2003 that new approaches have been proposed to obtain new enantiopure cryptophane derivatives. 5,6 Combined together, these two characteristics lead to original organic capsules that can be used for the complexation of optically active guest molecules. 7 However, despite the high interest for these chiral capsules the examples reported in the literature are rare. The difficulties to find small chiral guest molecules that show a good size matching with the cavity of these hosts are the main reasons that explain the scarcity of the examples reported in the literature. Collet and co-workers were the first to take advantage of the chiral structure of a cryptophane derivative to bind the enantio-enriched CHFClBr derivative. Later in 2012, we have shown that optically active methyloxirane 2 (also called propylene-oxide), could easily enter the cavity of the enantiopure cryptophane 1 in aqueous solution. 8 The association between (R)-(+) and (S)-(-)-methyloxirane and 1 gave rise to two diastereomers that could be easily distinguished by 1 H NMR spectroscopy, thanks to the strong shielding effect induced by the chiral cavity. [START_REF] Herein | the sign of the polarimetric measurement R-(+) and R-(-) of methyloxirane refers to the specific optical rotation (SOR) of the neat compound. For this compound, it has been shown that the sign of the SOR values is solvent dependent[END_REF] A moderate enantioselective effect was also observed between the two enantiomers of methyloxirane and specific ECD signals have been obtained for the two pair of diastereomers.

These interesting results prompted us to investigate other oxirane derivatives with host 1. For instance small oxirane derivatives such as Epichlorohydrin 3 (V vdw = 72.1 Å 3 ), 1,2-Butyloxitrane 4 (V vdw = 74 Å 3 ) and Bioxirane 5 (V vdw = 70.6 Å 3 ) have retained our attention because they possess small Vn der Waals volume ad they are therefore able to enter the cavity of 1 (Scheme 1). However, these molecules possess a somewhat larger molecular volume than methyloxirane (V vdw = 56.9 Å 3 ) and their ability to enter the cavity of host 1 is questionable.

In this series, compound 5 is particularly interesting since this compound possesses two stereogenic centers. Thus, three different types of diastereomers can be formed in solution in presence of the chiral host 1. In this article, we examine the ability of these enantiopure derivatives to enter the cavity of host 1 under different experimental conditions. For this study, 1 H NMR spectroscopy seems to be the method of choice to study these complexes since a slow exchange regime is usually observed between the guest molecules and the cryptophane derivatives. The ECD spectra of the different diastereomers are also reported at 278 K.

Scheme 1: structure of cryptophane 1 and chiral oxiranes (methyloxirane-2, Epichlorohydrin-3; epoxybutane-4; Bioxirane-5) used in this study. Only a single stereomer is shown for each compound.

Results and Discussion:

Optical resolution of compound 1: the preparation of compound 1 in its racemic form has been previously reported in the literature. 10 The separation of the two enantiomers of 1 has also been reported from a multi-steps synthesis using cryptophanol-A as a starting material. 11,12 This approach allowed us to obtain the two enantiomers of 1 in fair quantities and with very high enantiomeric excess (ee ~ 100%). However, this method is time consuming and in this study we have preferred to use another approach based on the separation of the two enantiomers of compound 6 by HPLC chromatography on a semipreparative column. Compound 6 is the protected version of 1. The separation of this compound was preferred because it shows higher solubility in organic solvents. The two enantiomers of 1 can then be easily recovered by hydrolysis under basic solution of the two enantiomers separation of 6 (scheme 2).

Scheme 2: synthetic procedure use for the separation of the two enantiomers of 1. The synthesis of (rac)-6 is

reported ref X.
The two enantiomers of compound 6 have been successfully separated on a semi-preparative Welk-O1 column chromatography using a chloroform/ethanol mixture (see experimental section for more details). This allowed us to obtain these two enantiomers in fair quantities and very high enantiomeric excess (ee > 99.5 % for both enantiomers; see Supp. Info S1-S2).

Finally, hydrolysis of the two enantiomers of 6 under basic conditions, gives rise to the two enantiomers MM-1 and PP-1.

H NMR Spectroscopy of the oxirane complexes:

The previous study performed in aqueous solution with the methyloxirane derivative has shown that 1 H NMR spectroscopy is a valuable tool to extract information about these complexes. Indeed, in aqueous solution the host-guest association occurs under a slow exchange regime, even at room temperature. This means that the encapsulated species can be clearly identified on the 1 H NMR spectrum. In addition, thanks to the strong shielding effect induced by the six aromatic rings, the 1 H NMR signals of the encapsulated molecule appears at negative chemical shift (reference TMS). For instance, At 275 K, the efficient binding of the (rac)-Epichlorohydrin by host PP-1 occurs in LiOH (0.1 M). The association between guest 3 and host 1 gives rise to additional 1 H NMR signals. As expected, these signals appear at lower frequencies and at negative chemical shifts with respect to TMS taken as a reference. Thus, several 1 H NMR signals located between +0.4 ppm and -1.0 ppm can be clearly identified on the spectrum of the (rac)-3@PP-1 complexes.

The presence of numerous 1 H NMR signals and the large difference in intensity observed between some of these signals suggest that the two diastereomers can be distinguished and that some enantioselectivity takes place. To confirm these results, the two enantiomers (R)-(-)3 and (S)-(+)3 have been introduced separately using a single enantiomer of 1. For instance, under the same conditions the (R)-(-)-3@PP-1 diastereomer shows a simplified 1 H NMR spectrum compared to that of (rac)-3@PP-1. Thus, three sets of protons are observed for this complex at 0.15 ppm, -0.3 and -0.8 ppm, respectively. In contrast, the (S)-(+)-3@PP-1 diastereomer shows five well-resolved signals, located at 0.2, -0.35, -0.55, -0.65 and -0.95 ppm, respectively. Assuming a 1:2.5 ratio in favour of the (S)-(+)-3@PP-1 diastereomer, the summation of these two spectra reproduce very well the 1 H NMR spectrum observed with the (rac)-3@PP-1. It is noteworthy that the 1 H NMR spectrum of the (S)-(+)3@PP-1 diastereomer is identical to that observed for the (R)-(-)-3@MM-1 diastereomer. Similarly, the 1 H NMR spectrum of the (R)-(-)-3@PP-1 is identical to that of the (S)-(+)-3@PP-1 diastereomer. A change of the experimental conditions does not change significantly the shape of the 1 H NMR spectra. For instance, in NaOD/D 2 O (0.1 M) with the two enantiomers PP-1 or MM-1 we observe the same high-field shifted 1 H NMR signals (see Supp. Info. S3). However, in KOD/D 2 O (0.1 M) we can notice a significant decrease in intensity of these signals but these spectra remain identical to those previously recorded in NaOD/D 2 O (see Supp. Info. S4). In CsOD/D 2 O no signal is detected in that region suggesting that complexation of guest 3 does not take place under these conditions. It is noteworthy that a similar solvent effect was previously observed with methyloxirane under the same experimental conditions. Indeed, It has been shown that host 1 shows a good affinity for the K + cation and a binding constant K = 1000 M -1 has been measured in LiOH/H 2 O (0.1 M) by titration experiments. Other alkali cations such as Rb + and Cs + cations are also well recognized in LiOH/H 2 O or NaOH/H 2 O solutions and exhibit higher affinity for this host. (REF 10) Thus, herein the K + cation acts as a competitor and a significant decrease in affinity of guest 3 for the host 1 is observed in KOH/H 2 O solution. In CsOH/H 2 O, it has been shown that the affinity of 1 for the Cs + cation is about 10 6 larger than the association constant observed with the K + cation. Thus, in these conditions, no complexation takes place even in presence of a large excess of guest 3.

The association constants between optically active guest 3 and enantiopure host 1 are reported as a function of the nature of the solvents (table 1 in Supp. Info. S5). Since a slow exchange regime is observed in all cases, the determination of the binding constants is facilitated and the different concentration needed to calculate the binding constant K can be directly extracted from the 1 H NMR spectrum. A repetition of these experiments led to an uncertainty value of 20 % on the measure of the binding constant K. The calculation of the binding constants reveals that MM-1 shows some enantioselectivity in presence of 3. For instance, the binding constants for the two diastereomers (R)-(-)-3@MM-1 and (S)-(+)-3@MM-1 have been measured to be K = 117 M -1 and 45 M -1 , respectively at 278 K. This leads to an enantioelective ratio (R)-(-)-3@MM-1/(S)-(+)-3@MM-1 = 2.6, which is similar to that measured previously from the 1 H NMR spectrum of the (rac)-3; (R)-(-)-3@MM-1/(S)-(+)-3@MM-1 ~ 3.0. This ratio is similar to that calculated with methyloxirane. However, in the case of compound 3, the binding constants are significantly smaller than those calculated with the two enantiomers of the methyloxirane derivative. This difference of affinity between these two guests can be explained by the large difference in their molecular volume. Indeed, methyloxirane (V vdw = 57.0 Å 3 ) has a smaller volume than the eipchlorohydrin derivative (V vdw = 72.1 Å 3 ) and it shows a better size matching with the cavity of host 1 (V vdw ~ 95 Å 3 ).

The replacement of the LiOD/D 2 O solution by NaOD/D 2 O (0.1 M) gives similar results. However, in this solvent slightly higher binding constants K = 170 M -1 and K = 70 M -1 have been calculated for the two (R)-(-)-3@MM-1 and (S)-(+)-3@MM-1 complexes, respectively.

A similar result was also observed in the case of methyloxirane. In KOD/D 2 O (0.1 M), the association constants for these two complexes decrease significantly but some enantioselectivity is still observed in these conditions.

In NaOD/D 2 O (0.1 M), the 1,2-butyloxirane-4 derivative is also well recognized by host 1 (see figure 2). For instance, in presence of host MM-1, (rac)-4 reveals several high-field shifted 1 H NMR signals between 0.0 and -2.5 ppm. The use of a single enantiomer of 4 leads to a simplification of the 1 H NMR spectrum. For instance, the 1 H NMR spectrum of the (R)-(+)4@MM-1 complex is reduced to five well-resolved signals. In contrast, the other diastereomer complex (S)-(-)4@MM-1 exhibits in the same region six 1 H NMR signals. The sum of these two spectra allows us to reproduce the 1 H NMR spectrum obtained for (rac)-4@MM-1. Interestingly, we have noticed that host 1 does not accommodate 2,3-butyloxirane, an isomer of compound 4, whereas these two molecules have the similar molecular volume.

The higher rigidity of 2,3-butyloxirane can explain the difference of behaviour between the two isomers and the difficulties for this compound to enter the cavity of host 1. A measure of the binding constants shows a weak enantioselectivity in favour of the (S)-(-)4@MM-1 diastereomer (see Table 2 in Supp. Info. S4). Indeed, a weak binding constant K = 16 M -1 has been measured for this complex whereas the other diastereomer (R)-(+)4@MM-1 shows a lower association constant K = 9 M -1 . The difference of affinity between guests 3 and 4 is large is probably a consequence of the larger molecular volume of guest 4 (V vdw = 74 Å 3 ) that does fit well with the cavity of the host.

Compound 5 represents the most interesting compound for these complexation studies. Indeed, 2,2'-bioxirane (5) (CAUTION; toxic) possesses two stereogenic centers. Thus, three different stereomers can be obtained: the (2R,2'R)-(-)-2,2'-bisoxirane, the (2S,2'S)-(+)-2,2'-bisoxirane and the meso-derivative, which is achiral. In presence of the chiral host PP-1, three different diastereomers can thus be formed in solution. To our knowledge, this is the first time that a chiral guest with two stereogenic centers is used for complexation studies with an enantiopure cryptophane. This compound has been chosen because it possesses a van der Waals volume (V vdw =70.6 Å 3 ), which is very close to that of guest 3. 13 Thus, this compound is expected to enter the cavity of 1 under the same conditions. It is noteworthy that, even though the two enantiomers of 5 are commercially available, we have encountered a lot of difficulties to purchase these compounds. Thus, we decided to prepare the two enantiomers (2R, 2'R)-(-)-2,2'-bisoxirane and (2S, 2'S)-(+)-2,2'-bisoxirane from a known procedure. 14 The two enantiomers (ee ~ 100%) have been purified by distillation and obtained as crystals with low melting points (mp = 23°C). Buffeteau and co-workers have recently reported the chiroptical properties of the two enantiomers of compound 5. 15 On the other hand, the meso stereoisomer has been purchased from a chemical company.

The 1 H NMR spectra (high-field region) of the three diastereomers recorded in NaOD/D 2 O at 275 K are reported in figure 3. These spectra show large difference between them that depends on the stereochemistry of guest 5. For instance, in this solvent the meso-stereomer shows six well-resolved 1 H NMR signals with the same intensity and the encapsulation of (meso)-5 by host PP-1 represents an interesting textbook case. Indeed, the (meso)-5 derivative possesses a plane of symmetry and it is therefore achiral. Consequently, each pair of protons (H a , H a' ; H b , H b' and H c , H c' ) of this stereoisomer is enantiotopic and the 1 H NMR spectrum of the free guest is characterized by three sets of protons signals (spectrum not shown). However, a different situation occurs when this molecule is introduced into a chiral environment such as the cavity of PP-1. Thus, under these conditions, a splitting of each pair of proton takes place thus leading to six well-resolved 1 H NMR signals for the encapsulated (meso)-stereomer, as observed in figure 3. A change of the nature of the solvent gives rise to similar results and in LiOD/D 2 O similar spectra are obtained. A different situation occurs with the two optically active (2R,2'R)-(-)-5 and (2S,2'S)-(+)-5 enantiomers. Indeed, each pair of protons (H a , H a' ; H b , H b' and H c , H c' ) present on these two derivatives is homotopic. Thus, the two (2R,2'R)-(-)-5@PP-1 and (2S,2'S)-(+)-5@PP-1 diastereomers show a simplified 1 H NMR spectra with respect to the spectrum observed with the (meso)-5@PP-1. Indeed, when placed in a chiral environment, each pair of protons is still characterized by a single 1 H NMR signal. However, it can be easily seen that the two diastereomers (2R,2'R)-(-)-5@PP-1 and (2S,2'S)-(+)-5@PP-1 can be easily distinguished since they present large chemical shift differences. In LiOD/D 2 O (0.1 M) at 275 K, the calculated binding constants shows a higher affinity (K = 70 M -1 ) of the PP-1 enantiomer for the (2R,2'R)-(-)-5. A similar value K = 80 M - 1 has been calculated with the (2S,2'S)-(+)-5@MM-1 complex (see Supp. Info. S6). In contrast, the the diastereomers (2S,2'S)-(+)-5@PP-1 and (2R,2'R)-(-)-5@MM-1 show lower association constants that have been estimated to be K = 20 M -1 and K = 30 M -1 , respectively.

On another hand, the determination of the binding constants for (meso)-5@PP-1 and (meso)-5@MM-1, gave similar K = 70 M -1 values. As previously observed with methyloxirane and guest 3, slightly higher binding constants have been calculated in NaOD/D 2 O (see Supp. Info.

X).

All combined together, the 1 H NMR spectroscopy shows that guests 3-5 are well recognized by the enaniopure hosts PP-1 and MM-1 in LiOD/D 2 O, NaOD/D 2 O. In KOD/D 2 O, the K + cation is a competitor and a decrease of the association constants is observed with guest 3 and 5. For guests 3-5, an enantioselectivity effect is measured in these solvents. The binding constants measured in all cases are moderate but the differences observed between the different guest molecules show that a clear correlation could be established between the association constants K and their molecular volume. For instance, for a given solvent, epichlorhydrin (3, V vdw = 72.1 Å 3 ) shows higher binding constants than 1,2-butyloxirane (4, V vdw = 74 Å 3 ). It is noteworthy that these values are significantly smaller than those previously calculated for the two enantiomers of methyloxirane (V vdw = 57 Å 3 ). (ref 9) The two enantiomers of 5 (V vdw = 70.6 Å 3 ) have a slightly smaller volume than guest 3 and show also lower affinity for host 1. However, it can be noticed that guest 5 is made of two oxirane cycles and is therefore more rigid than guest 3 that possesses a single oxirane cycle.

Consequently, guest 5 cannot change its conformation as easily as guest 3 to match with the cavity of the host 1. A similar effect has also been observed with the two isomers of butyloxirane. Indeed, the 2,3-butyloxirane appears to be more rigid than 1,2-butyloxirane (4) and it cannot enter the cavity of the host, even though the molecular volumes of these two molecules are expected to be identical.

ECD Spectroscopy of the oxirane complexes 3, 4 and 5: as previously demonstrated with the complexation of the two enantiomers of methyloxirane with host 1, Electronic Circular Dichroism (ECD) spectroscopy is a valuable tool to characterize these supramolecular complexes in solution. Indeed, with this technique it is possible to visualize the spectral modifications of the host upon encapsulation. In the case of host 1, the ECD signals originating from the solvent or the oxirane guest is not a problem since both species only absorb light below 220 nm. Thus, there is no overlap between these signals and the ECD signals of the host, at least for the two 1 L a and 1 L b transitions located between 240 and 300 nm. Previously, we have reported that at 293 K, the complexation of the two enantiomers of methyl-oxirane (2) with the chiral host 1 gives rise to specific ECD spectra in the 1 L b region of the UV-visible spectrum. The differences observed between these ECD signals are small but they appear to be characteristic of each diastereomers and are not the consequence of artefacts. Interestingly, a comparison of these spectra with the ECD spectrum of the racemic mixture allowed us to confirm the higher affinity of the (R)-(+)-2 enantiomer for PP-1. At 278 K the difference between the two spectra is even more pronounced (see Supp. Info. S7).

Based on these interesting results, we decided to record at 278 K the ECD spectra of optically active guests 3, 4 and 5 molecules with the two enantiomers of 1.

The ECD spectra of PP-1 and MM-1 enantiomers recorded in NaOD (0.1 M) at 278 K in presence of (rac)-3, (R)-(-)-3, (S)-(+)-3 guests are reported in figure 4. Another series of ECD spectra recorded at 278 K in LiOD (0.1 M) are also shown in Supp. Info. S8. As observed for the methyloxirane guest, the ECD spectra of guest 3 recorded in NaOH (0.1 M) show clear difference between the different diastereomers, at least for the 1 L b region of the UV-vis spectrum. For instance, between 270 and 340 nm, we observe that the (S)-3@PP-1 diastereomer exhibits two ECD bands in this region: a weak ECD signal could be clearly detected with a maximum at 320 nm and a bigger ECD band is also present at 295 nm. As expected for enantiopure materials, the same ECD signals with the same intensities are obtained for the (R)-3@MM-1 diastereomer. Interestingly, it can be notice that different spectra could be obtained with the two other (R)-(-)-3@PP-1 and (S)-(+)-3@MM-1 diastereomers. With these two complexes, the ECD spectra show a single broad ECD band with a shoulder at 310 ppm. A superposition of these spectra shows clear differences and these differences cannot be attributed to artefacts and are characteristic of these complexes.

Interestingly, we noticed that the ECD of the rac-3@PP-1 and rac-3@MM-1 diastereomers, showed a lot of similarities with the ECD spectra recorded for the (S)-(+)-3@PP-1 and (R)-(-)-3@MM-1 diastereomers. Thus, it can be deduced that host PP-1 shows a clear preference for the (S)-(+)-3 enantiomer. These experiments demonstrate that enantioselective complexation of guest 3 can also be detected by ECD spectroscopy. These experiments nicely confirm our results previously obtained by 1 H NMR spectroscopy (see table 1). In LiOH/H 2 O (0.1 M), similar ECD spectra have been obtained (see Supporting Information S8).

Figure 4: ECD spectra of (R)-(-)-3@PP-1 (red curve); (S)-(+)-3@PP-1 (blue curve); (rac)-3@PP-1 (green curve) recorded in NaOH/H 2 O (0.08 M) at 278 K. The ECD spectra of (S)-(+)-3@MM-1 (red dashed curve); (R)-(-)-3@MM-1 (blue dashed curve); (rac)-3@PP-1 (green dashed curve) are also shown.

Interestingly, the ECD spectra recorded at T = 293 K also show clear difference between the different diastareomers (spectra not shown). At this temperature 1 H NMR spectroscopy is not useful to extract useful information since these spectra only reveals broads signals with small intensities due to a faster in-out exchange dynamics (spectra not shown).

Interestingly, the ECD spectra recorded in KOH/H 2 O (0.1 M) for the same complexes show complete different spectra at 293 K and 278 K (see supporting information S9). For instance, at 278K an ECD band is first observed at 310 nm ( ~ 28 L mol -1 cm -1 ) and a second ECD band with a lower intensity is also visible at 290 nm ( ~ 10 L mol -1 cm -1 ). Compared to the ECD spectra recorded in LiOH/H 2 O and NaOH/H 2 O, The difference observed in this solvent can be explained by the ability of host 1 to accommodate a K + cation within its cavity. The affinity of host 1 for this small cation forces the cage to modify the conformation of the three linkers in order to maximize interactions with the cation. Consequently, the ECD spectrum of 1 is strongly affected by this conformational change. It is noteworthy that, in contrast to what is observed at 293 K (see supporting information X), the two spectra recorded for the two pair of diastereomers are not exactly identical at 278 K and very small differences can be observed at this temperature. This result supports the idea that even in presence of this competitor the two enantiomers of 3 do not behave similarly with host 1. The measures of small but different K values support this assumption.

The ECD spectra of (R)-(+) and (S)-(-)-1,2-butyloxirane (4) also show some differences in presence of host 1. For instance, in NaOH (0.1 M), these ECD spectra are very similar to those previously observed for guest 3. However, for guest 4 a comparison of these spectra with those obtained for the (rac)-4@PP-1 and the (rac)-4@MM-1 did not allow us to detect with confidence an enantioselective effect (see Supp. Info. S10).

As previously mentioned compound 5 that contains two stereogenic centers is the most interesting guest molecule to be studied in this series. Indeed, in presence of host 1, three different pairs of diastereomers can be obtained in solution. It is important to note that, in contrast to guest 2, 3 and 4, different experimental conditions have been used to record the ECD spectra with guest 5. Indeed, in the case of 5 we are limited by the amount of enantiopure material and we do not have the possibility to use this material in large excess as this was achieved with guest 2, 3 and 4. The experimental procedure used for recording ECD spectra with guest 5 is described in detail in the experimental section. The ECD spectra recorded in NaOH/H 2 O (0.08 M) for the different diastereomers are shown in figure 5. The ECD spectra recorded in LiOH/H 2 O (0.08 M) are also reported in supporting information (Supp. Info S11). Thanks to the high enantiomeric purity of host PP-1 and guests 5, the ECD spectra of the different diastereomers reveal clear differences between them even though these differences are small. These spectra also differ from that of the ECD spectra obtained with the meso-5 derivative that show an intermediate behaviour. The ECD spectra recorded with the other enantiomer MM-1 also reveal perfect mirror images thus excluding the possibility that these spectral differences could be due to artefacts. It can be noticed that these three pair of diastereomers differ greatly from the guest free host PP-1 and MM-1 recorded in the same condition. Thus, this suggests that the encapsulation of the different stereomers of guest 5 have a strong impact on the conformation of the linkers of the host. In LiOH/H 2 O (0.08 M) identical spectra have been obtained even though in this solvent it is more difficult to distinguish the meso-5@PP-1 (meso-5@MM-1) and the (2S,2'S)-(+)-5@PP-1 ((2R,2'R)-(-)-5@MM-1) diastereomers.

Figure 5: ECD spectra of (2R,2'R)-(-)-5@PP-1 (red curve); (2S,2'S)-(+)-5@PP-1 (blue curve); (meso)-5@PP-1 (green curve) and guest free PP-1 (black curves). ECD spectra of (2S,2'S)-(+)-5@MM-1 (red dashed curve); (2R,2'R)-(-)-5@MM-1 (blue dashed curve);

(meso)-5@MM-1 (green dashed curve) and guest free MMP-1 (black curves). Spectra recorded in NaOH/H 2 O (0.08 M) at 278 K.

All combined together, our results reveal that ECD spectroscopy is a valuable and extremely sensitive chiroptical tool to distinguish the different diastereomers present in solution.

Interestingly, even when these experiments are performed at room temperature, ECD spectroscopy still reveals large differences between the different species. In contrast, at the same temperature, 1 H NMR spectroscopy does provide much informations due to a fast in-out exchange kinetics. Thus in this case only very broad signals are observed at this temperature.

In the case of the chiral oxirane 3, 4 and 5, we observe that ECD spectroscopy gives rise to specific ECD signals in the 1 L b region of the UV-vis spectrum (270 -300 nm). In aqueous solution, this spectral region is known to be very sensitive to the encapsulation process since it has a strong impact on the conformation of the three linkers. Herein, even tereomers that show identical molecular volume but different spatial arrangements show different ECD spectra.

Conclusion:

In this article we report a complexation study of small chiral oxirane derivatives (1,2-epichlorhydrin -3, 1,2-epxybutane -4, andbis A slow in-out exchange dynamics is observed by 1 H NMR spectroscopy in all cases. In addition, a small enantioselective effect is observed for guests 3, 4 and 5. A correlation between the affinity of the host for these oxirane molecules and their molecular volume can be established. The flexibility of the guest seems also to play a role since 2,3-epoxybutane is not recognized whereas 1,2-epoxybutane (4) shows some affinity for the cavity of 1. For this study, ECD spectroscopy appears to be a valuable tool for characterizing the different species formed in solution since specific ECD signals are obtained in the 1 L b region of the spectrum.

These results nicely confirm the previous data obtained with the chiral methyloxirane (2) in solution.

Experimental section:

Synthesis of optically active cryptophan-ol 1: the two enantiomers MM-1 and PP-1 have been prepared by hydrolysis under basic conditions of the two enantiopure PP-2 and MM-2 derivatives, respectively. Compounds MM-2 and PP-2 have been separated by HPLC on a semi-preparative chiral chromatography and they have been obtained with an enantiomeric purity (ee  99.5 %). Details on the separation of compounds MM-2 and PP-2 by HPLC are reported in the Supporting Information section. The synthesis of optically active cryptophanol-1 has been previously reported from a multi-steps synthesis. The 1 H NMR spectra of MM-1 and PP-1 were found identical for these two approaches.

1 H NMR spectra of guest 3 and 4 have been recorded on a Varian 500 MHz at 278 K. 1 H NMR spectra of guest 5 have been recorded on a Bruker 400 MHz at 278 K. The measurement of the binding constants was achieved as follow: compound 1 was weighed in an NMR tube and a known amount of solvent (400 L) was introduced in the tube. Then a very small amount of oxirane derivative was introduced in the NMR tube in order to have guest signals of the same intensity than the molecular host. By integration, the knowledge of the host concentration allows us to determine the concentration of both the free guest and the complex. A repetition of these experiments gives an uncertainty of 20% on the calculated binding constants.

For the two enantiomers of compounds 3 and 4 in presence of hosts PP-1 and MM-1, the ECD spectra were recorded in aqueous solution (LiOH/H 2 O, NaOH/H 2 O and KOH/H 2 O) at a concentration of 0.1 M. All the spectra were recorded at 278 K. A path length quartz cell of 1 cm (concentrations were in the range 5 × 10 -5 -1 × 10 -4 M) has been used for these experiments. Spectra were recorded in the wavelength ranges of 270 -400 nm with a 0.5 nm increment and a 1s integration time. Spectra were processed with standard spectrometer software, baseline corrected and sometimes slightly smoothed by using a third order least square polynomial fit.

For the two enantiomers of compound 5 the ECD spectra were recorded in LiOHH 2 O and NaOH/H 2 O at a concentration of 0.08 M according to the following procedure: to be described. A path length quartz cell of 1 cm (concentrations were in the range 5 × 10 -5 -1 × 10 -4 M) has been used for these experiments. Spectra were recorded in the wavelength ranges of 270 -400 nm with a 0.5 nm increment and a 1s integration time.

  in host 1 give rise to several sets of 1 H NMR signals between 0.0 and -2.0 ppm. Compound 1 is only soluble in H 2 O (D 2 O) under basic conditions. Thus the 1 H NMR spectra of 1 with the different chiral guests have been recorded in LiOD/D 2 O, NaOH/D 2 O, KOH/D 2 O and CsOH/D 2 O at a concentration of 0.1 M. At this concentration the oxirane molecules are stable and no decomposition was observed during the time of our experiments. The spectra have been recorded at 278 K in order to decrease the exchange dynamics and to detect more easily the 1 H NMR signals corresponding to the encapsulated species.

Figure 1 :

 1 Figure 1: 1 H NMR spectrum of PP-1 in presence of (S)-(+)-3, (R)-(-)-3 and (rac)-Epichlorhydrin-3 recorded in LiOD/D 2 O (0.1 M) at 275 K. Only the spectral region corresponding to the encapsulated guest is shown.

Figure 2 :

 2 Figure 2: 1 H (500 MHz) NMR spectra of the (S)-4@MM-1, (R)-4@MM-1, (rac)-4@MM-1 complexes recorded in NaOD/D 2 O (0.1 M) at 275 K. Only the high-field region (0.0 to -2.5 ppm) is shown.

Figure 3 :

 3 Figure 3: 1 H (400 MHz) NMR spectra of the (2R,2'R)-5@PP-1, (2S,2'S)-5@PP-1 and (meso)-5@PP-1 complexes recorded in NaOD/D 2 O (0.1 M) at 275 K. Only the high-field region (1.0 to -2.0 ppm) is shown. A signal (methyl group) corresponding to diethyl ether has been removed for clarity.

  -oxirane -5) with the watersoluble cryptophane 1. The two enantiomers of compound 1 have been isolated by HPLC on chiral stationary phase and have been obtained with very high enantiomeric excess (ee ~ 100 %). 1 H NMR spectroscopy reveals that efficient complexation of guests 3, 4 and 5 takes place in aqueous solution in LiOD/D 2 O, NaOD/D 2 O and KOD/D 2 O. Compound 5 is a particularly interesting guest for this study since this molecule contains two stereogenic centers. Thus, three different pair of diastereomer can be obtained in solution in presence of the chiral host 1. For this study, the two enantiomers (2R,2'R)-5 and (2S,2'S)-5 have been prepared according to a known procedure.

Acknowledgments:

The authors are indebted to the CNRS (Chemistry Department) and to Région Aquitaine for financial support in VCD equipments. The GDR 3712 Chirafun is acknowledged for allowing a collaborative network between some partners of this project.