L Ben Ali

N Bujan

S El Fekih

H Ben Ali

E Caire

N Douet-Guilbert

Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality

Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality

Introduction

There is a 2-20 times higher probability of having a structural chromosomal abnormality in the infertile male population than in the general population [START_REF] Hafez | Differential enrichment of sperm with no DNA strand breaks using magnetic activated cell sorting (MACS) in men with various categories of semen parameters[END_REF][START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF].

Robertsonian translocations are the most common structural chromosomal abnormalities observed with an incidence of 1/1085 newborns, and balanced reciprocal translocations are the second most common aberration with an incidence of 1/1175 births [START_REF] Braekeleer | Cytogenetic studies in male infertility: a review[END_REF]. Carriers of a structural chromosome abnormality are phenotypically normal but males can have spermatogenic disruption such as decreased sperm concentration [START_REF] Vincent | Cytogenetic investigations of infertile men with low sperm counts: A 25-year experience[END_REF][START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF]) which directs them towards assisted reproductive technologies (ART) through in vitro fertilization (IVF) with Intracytoplasmic Sperm Injection (ICSI).

However, the mobility and morphology parameters used to select spermatozoa for ICSI are not predictive of the quality of chromosomal equipment [START_REF] Nicopoullos | The role of sperm aneuploidy as a predictor of the success of intracytoplasmic sperm injection?[END_REF]. Men with chromosomal structural abnormality are at a higher risk of producing chromosomally unbalanced gametes. These gametes are the result of adjacent and 3:0 segregation modes in men with Robertsonian translocation and the result of adjacent I, adjacent II, 3:1 and 4:0 segregation in men with balanced reciprocal translocations. Therefore, these men have at a higher risk of repeated miscarriages and the birth of children with a chromosome abnormality [START_REF] Stern | Chromosome translocations in couples with in-vitro fertilization implantation failure[END_REF][START_REF] Munné | Outcome of preimplantation genetic diagnosis of translocations[END_REF]. It would thus be helpful to select spermatozoa with normal or balanced chromosomal content for ICSI in this group of men.

In a previously published study, we showed that the DNA fragmentation rate depended on the presence of a chromosomal abnormality, and that consequently a structural chromosomal abnormality predicted DNA fragmentation [START_REF] Perrin | DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality[END_REF]. Some studies have observed significantly higher rates of apoptotic markers in translocation carriers as compared to sperm donors [START_REF] Brugnon | Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients[END_REF]. Several authors have also reported higher aneuploidy levels in spermatozoa with fragmented DNA in infertile 46,XY men with disrupted semen parameters [START_REF] Muriel | Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis[END_REF]Perrin et al. 2011a;Perrin et al. 2011b;[START_REF] Brahem | Study of aneuploidy rate and sperm DNA fragmentation in large-headed, multiple-tailed spermatozoa[END_REF], suggesting that DNA fragmented spermatozoa were apoptotic cells [START_REF] Muriel | Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis[END_REF]. More recently, we showed that in men with a constitutional chromosomal abnormality, DNA fragmentation was significantly higher in chromosomally unbalanced spermatozoa than in spermatozoa with a normal or balanced chromosomal content (Perrin et al. 2011a;[START_REF] Perrin | DNA fragmentation is higher in spermatozoa with chromosomally unbalanced content in men with a structural chromosomal rearrangement[END_REF]. Different hypotheses are advanced to explain this DNA fragmentation: exposure to reactive oxygen species [START_REF] Agarwal | Clinical relevance of oxidative stress in male factor infertility: an update[END_REF][START_REF] Aitken | Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria[END_REF]), sub-protamination (McPHERSON & Longo 1992;McPherson & Longo 1993a;McPherson & Longo 1993b) or abortive apoptosis (Sakkas et al. 1999a;Sakkas et al. 1999b;[START_REF] Sakkas | Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis[END_REF][START_REF] Sakkas | Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis[END_REF]. Our results support this latter hypothesis. Indeed, since there are significantly more spermatozoa with fragmented DNA among chromosomally unbalanced gametes, we can speculate that these gametes, between the spermatocyte and spermatid stages, have been tagged for elimination by apoptosis because of their abnormal chromosomal equipment. These gametes would then have undergone an apoptotic process, but it would not have been completed for a number of them. Consequently, abortive apoptosis would explain why chromosomally unbalanced gametes are more fragmented than chromosomally normal or balanced gametes (Perrin et al. 2011a;[START_REF] Perrin | DNA fragmentation is higher in spermatozoa with chromosomally unbalanced content in men with a structural chromosomal rearrangement[END_REF]). In the apoptosis process, one of the earliest signs is the externalization of the phosphatidylserine (EPS) inner plasma membrane, a phospholipid with a high affinity for annexin V. This high affinity is guaranteed by the biological property of annexin V to bind to phospholipids in a Calcium (Ca2+) dependent manner [START_REF] Klee | Ca2+-dependent phospholipid-(and membrane-) binding proteins[END_REF][START_REF] Swairjo | Annexin structure and membrane interactions: a molecular perspective[END_REF][START_REF] Van Heerde | The complexity of the phospholipid binding protein Annexin V[END_REF]. Magnetic-activated cell separation (MACS) is a technique of spermatozoa selection based on super-paramagnetic microbeads conjugated with annexin V which labels the apoptotic and dead spermatozoa within an external magnetic field. MACS was used to separate sperm into 2 populations: an annexin V (-) fraction (spermatozoa without EPS) and an annexin V (+) fraction (spermatozoa with EPS) (de Vantéry [START_REF] De Vantéry Arrighi | Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential[END_REF][START_REF] Grunewald | Increased sperm chromatin decondensation in selected nonapoptotic spermatozoa of patients with male infertility[END_REF][START_REF] Huang | 25th Annual Meeting of the European Society on Human Reproduction and Embryology (ESHRE), Sperm preparation by magnetic-activated cell sorting improve the sperm-zona pellucida binding capacity and reduces apoptotic sperm[END_REF][START_REF] Buzzi | Magnetic activated cell sorting (MACS) improves oocytes donation results associated to severe male factor infertility[END_REF]. This is a very simple, specific and rapid technique which is known to reduce a large proportion of apoptotic spermatozoa in the processed samples [START_REF] Said | Evaluation of sperm recovery following annexin V magnetic-activated cell sorting separation[END_REF], and also to reduce the level of DNA fragmentation [START_REF] Rawe | ICSI outcome using Annexin V columns to select non-apoptotic spermatozoa[END_REF][START_REF] Lee | Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility[END_REF]Herrero et al. 2012;[START_REF] Delbes | The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting[END_REF][START_REF] Nadalini | Annexin V magnetic-activated cell sorting versus swim-up for the selection of human sperm in ART: is the new approach better then the traditional one?[END_REF][START_REF] Troya | Annexin V-MACS in infertile couples as method for separation of sperm without DNA fragmentation[END_REF][START_REF] Berteli | Magneticactivated cell sorting before density gradient centrifugation improves recovery of high-quality spermatozoa[END_REF]. As a consequence, spermatozoa that have undergone abortive apoptosis should be retained in the column when using annexin V coupled with MACS. Therefore, if the origin of the DNA fragmentation is due to abortive apoptosis, we expect the rate of chromosomally unbalanced spermatozoa to be decreased in the annexin V (-) population.

In this study, we separated spermatozoa of males with a chromosomal rearrangement into two populations using MACS, an annexin V (-) and annexin V (+) population, in order to study meiotic segregation and DNA fragmentation on the whole ejaculate and on selected spermatozoa (annexin V (-) versus annexin V (+)) in the same patient. The percentage of spermatozoa with EPS was also evaluated on the whole ejaculate and on selected spermatozoa to determine the purity rate of our samples after sorting.

The aim of the present study was to determine if magnetic-activated cell separation could select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men carrying a structural chromosomal abnormality.

Patients and methods

Patients and sperm parameters

Ten males with a structural chromosome abnormality were included in the study (Table 1).

Karyotyping was performed on peripheral blood cultures. For this study straws of cryopreserved semen samples were obtained with the collaboration of different national centers (3 samples from University Hospital of Clermont-Ferrand and 7 samples from the GERMETHEQUE national biobank, BB-0033-00081; coordination: Toulouse, France).

Sperm parameters were evaluated according to World Health Organization recommendations (WHO 2010).

Our study was approved by the regional Committee for the Protection of Persons (declaration number DC-2008-597;authorization AC-2009-886). All patients gave their informed consent for the study.

Study design and MACS separation

Spermatozoa from frozen semen samples were thawed for 30 min at room temperature, and were washed in 1 ml of BB 1X (Binding Buffer) (Miltenyi Bioec, Paris, France) at 400 x g for 10 minutes to eliminate the cryoprotectant. After centrifugation, the final pellets were resuspended in BB 1X. Sperm DNA slides were prepared for annexin V staining, TUNEL (TdT-mediated-dUTP nick-end labeling) and FISH (Fluorescence in situ Hybridation), and the remaining sample was processed by MACS separation.

For MACS separation, the washed spermatozoa were incubated with 100 µl of BB 1X and annexin V microbeads (Miltenyi Biotec) at room temperature for 15 minutes. Sperm DNA slides were prepared for TUNEL. The remaining spermatozoa-microbead suspension was placed on top of the separation column containing iron balls and the column placed in a magnetic field (MiniMACS, Miltenyi Biotec). The spermatozoa with intact membranes (without EPS) passed through the column and were labeled annexin V (-). The spermatozoa with EPS retained in the separation column were eluted using BB 1X after the column was removed from the magnetic field, and were labeled annexin V (+). Sperm DNA slides from annexin V (-) and annexin V (+) solutions were prepared for annexin V staining, TUNEL and FISH. The different steps of our experiment design are illustrated in Figure 1.

Evaluation of spermatozoa with EPS using annexin V-FITC For technical reason, to evaluate spermatozoa with EPS using annexin V-FITC, the separated spermatozoa populations were adjusted to a concentration of 200 000 spermatozoa. They were incubated in the dark with annexinV-FITC and 100µl of BB 1X for 15 minutes at room temperature, and centrifuged at 400 x g for 10 minutes. The final pellets were resuspended in 10 µl of BB 1X. Sperm slides were prepared and counterstained with DAPI. The fluorescence signals of labeled spermatozoa were analyzed using a Zeiss AxioPlan Microscope (Zeiss, Le Pecq, France). A total of 500 spermatozoa were analyzed for each sample. Spermatozoa were considered "annexin V (-)" (blue fluorescence) or "annexin V (+)" (green fluorescence).

Subsequent image acquisition was performed using a CCD camera with in situ imaging system (ISIS) (Metasystems, Altlussheim, Germany).

Analysis of DNA fragmentation using TUNEL assay

Sperm DNA slides were fixed with Carnoy solution (methanol + acetic acid). The slides were immersed in a jar containing a 2x SSC (Saline Sodium Citrate) /0.4% NP40 (Nonidet P40) solution and then immediately passed through an ethanol series of increasing concentration (70%/90%/100%). Sperm DNA fragmentation was detected by the ApopTag kit (Millipore, Saint Quentin en Yvelines, France) according to the manufacter's recommendations. Briefly, equilibration buffer was put on the slides and a mix of a reaction buffer and terminal deoxyribonucleotidyl transferase was added. The slides were incubated in a dark humidified chamber at 37°C. They were then incubated in a stop/wash buffer and washed with 1x phosphate-buffered saline (PBS). A mix of blocking solution and anti-digoxigenin-rhodamine was added to the slides before incubation at 37°C. The slides were washed in 1x PBS. They were air-dried and counterstained with DAPI (4', 6-diamidino-2-phenyl-indole). A total of 500 spermatozoa from each sample were analyzed using a Zeiss AxioPlan Microscope.

Spermatozoa were considered to contain either normal (blue fluorescence) or fragmented (red fluorescence) DNA [START_REF] Perrin | DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality[END_REF].

Analysis of meiotic segregation by FISH

Meiotic segregation was analyzed by FISH with a mix of appropriate probes according to the chromosomal breakpoints involved in the rearrangement (Table 2). The hybridization techniques have been previously published [START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF][START_REF] Perrin | DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality[END_REF]. Sperm DNA preparations were fixed on a slide with Carnoy solution. Before hybridization sperm nuclei were decondensed in 1M NaOH solution and then washed in SSC 2X for 5-10 min at room temperature. They were immersed in a jar of SSC 2X/0.4%NP40 solution for 30 min at 37°C and then passed through an ethanol series of increasing concentration (70%/90%/100%) and allowed to air dry. The denaturation was performed simultaneously on spermatozoa and probes for 1 min at 70°C using a ThermoBrite System (Abbott, Rungis France). The slides were incubated overnight in a dark humidified chamber at 37°C. They were washed for 30 s in SSC 0.4X /0.3% NP40 at 72°C and 15 s in SSC 2X /0.1% NP40 at room temperature.

Finally, they were counterstained with DAPI. The chromosomal content in spermatozoa was determined according to the combination of fluorescent spots observed. A minimum of 2000 spermatozoa were analyzed for each sample using a Zeiss AxioPlan Microscope.

Statistical analyses

An independent chi-square test was used to compare the percentages of spermatozoa stained with annexin V-FITC before and after MACS separation. The same test was used to compare:

-the percentage of spermatozoa with DNA fragmentation -the number of spermatozoa with normal/balanced chromosomal content and chromosomally unbalanced content before and after MACS separation.

To compare the number of chromosomally unbalanced spermatozoa produced by different segregation modes we used an independent chi-square test or the Fisher's exact test if the expected numbers were below 5. The data was analyzed online using the BiostaTGV software (http://marne.u707.jussieu.fr/biostatgv/). A value of p < 0.05 was considered to be significant.

Results

Patients

Ten male carriers of a structural chromosomal abnormality were included in the study. The karyotypes of the patients are shown in Table 1. There were 3 patients with a Robertsonian translocation (P1 to P3), and 7 patients with a simple balanced reciprocal translocation (P4 to P10).

Semen analysis

The summary of the basic semen parameters on raw semen samples before freezing are presented in Table 3. Sperm concentration ranges were between 14.4 millions/mL (P9) and 151 millions/mL (P2), the percentage of motility (a+b+c) ranged from 35% (P3, P7, P9 and P10) to 60% (P8), the percentage of normal forms according to the classification of David ranged from 10% (P3 and P4) to 70% (P8).

Evaluation of spermatozoa with EPS using annexin V-FITC Data concerning the percentage of spermatozoa with EPS before MACS separation and in the annexin V (-) and V (+) fractions after MACS separation are summarized in Table 4. For all patients, the percentage of spermatozoa with EPS is lower in the annexin V (-) fraction (average: 29.72 ± 7.45%) and higher in the annexin V (+) fraction (average: 80.58 ± 7.44) as compared to the frozen-thawed semen sample (average: 59.32 ± 9.34).

Analysis of DNA fragmentation using TUNEL assay

Data concerning the percentage of DNA fragmentation for each patient at each step of the study are summarized in Table 5. For the 10 male carriers of a structural chromosomal abnormality, the percentage of DNA fragmentation after freeze-thaw varied between 7.2% (P2) and 20.6% (P6). For 6 patients, the rates of DNA fragmentation were statistically significantly higher after incubation with BB 1X + annexin V microbeads than for the frozenthawed semen sample. The rates of DNA fragmentation were statistically significantly lower in the annexin V (-) fraction than after incubation with BB1X + annexin V microbeads except for P6, and conversely, we observed a statistically significantly higher rate in the annexin V (+) fraction than after incubation with BB 1X + annexin V microbeads for 6 patients (P2, P3, P4, P7, P9 and P10) and a significant decrease for one patient (P5). There was a statistically significant difference for DNA fragmentation rates in the annexin V (-) fraction as compared to the annexin V (+) fraction except for P6.

Analysis of meiotic segregation by FISH

Regarding the normal/balanced spermatozoa products of alternate segregation, the frequencies after freeze-thaw varied from 78.06% (P3) to 83.37% (P2) for patients with a Roberstonian translocation and from 36.39% (P10) to 48.36% (P5) for patients with a balanced reciprocal translocation (Table 6). When compared to raw semen, there was a statistically significant increase of normal/balanced spermatozoa in the annexin V (-) fraction for all patients (p < 0.05) after MACS. On the other hand, we observed a statistically significant decrease in the annexin V (+) fraction for P1, P4, P6, P7, P8, P9 and P10. For all patients, we observed a statistically significant difference (p < 0.05) between the annexin V (-) and annexin V (+) fractions with a majority of normal or balanced spermatozoa in the first group, and a majority of unbalanced spermatozoa in the second group.

Concerning the population of unbalanced spermatozoa, the results from FISH studies are summarized in Table 6. After MACS, the proportion of unbalanced spermatozoa from each of the segregation modes decreased significantly in the annexin V (-) fraction and increased significantly in the annexin V (+) fraction.

Discussion

In this study, we analyzed spermatozoa with an externalization of the phosphatidylserine using annexin V-FITC, DNA fragmentation and the meiotic segregation on total ejaculate and on selected spermatozoa after MACS, in 10 men with a structural chromosomal abnormality.

Concerning the analysis using annexin V-FITC, we found an average of 29.72% spermatozoa with EPS in the annexin V (-) fraction versus 80.58% in the annexin V (+) fraction with a significant difference when compared to the frozen-thawed semen samples. Even if for certain patients the annexin V-FITC spermatozoa percentage was still high in the annexin V (-) fraction, on average MACS reduced this percentage by 49.90%. These data obtained considering them as a simple good functioning check tool of MACS. Moreover, our results are similar to those described in the literature [START_REF] De Vantéry Arrighi | Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential[END_REF][START_REF] Lee | Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility[END_REF][START_REF] Vendrell | Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients[END_REF]. Paasch et al. analyzed the separation effect of MACS in donor sperm with flow cytometry using FITC-conjugated anti-annexin V antibodies and found an average of 5.2% ± 1.0% labeled spermatozoa in the annexin V (-) fraction versus 72.2% ± 2.7% labeled spermatozoa in the annexin V (+) fraction [START_REF] Paasch | Deterioration of plasma membrane is associated with activated caspases in human spermatozoa[END_REF]. The presence of annexin V-FITC+ spermatozoa in the annexin V (-) fraction could be due to unspecific binding or an insufficient number of beads to retain them in the column. Glander and Schaller showed that the primary structural membrane of spermatozoa influences the ability of annexin V to detect externalization of phosphatidylserine [START_REF] Glander | Hidden effects of cryopreservation on quality of human spermatozoa[END_REF].

Concerning the analysis of DNA fragmentation, some authors have reported that cryopreservation might induce DNA damage [START_REF] Santo | Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART[END_REF]. In particular, many authors support the hypothesis of less susceptibility to freezing DNA damage in the sperm cells of fertile men, than those of infertile men [START_REF] Donnelly | The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences[END_REF]. In order to be able to compare the data of the different patients in this study, we used cryopreserved samples for all patients.

Data showed that for 6 patients the rates of DNA fragmentation were statistically significantly higher after incubation with BB 1X (+ annexin V microbeads) than for the frozen-thawed semen sample. These results confirm those previously obtained [START_REF] Fekih | Impact des facteurs maternels et paternels sur les résultats de FIV / ICSI et investigations génétiques des spermatozoïdes d'hommes infertiles[END_REF]. In a previous study, we evaluated the rate of spermatozoa with EPS and the DNA fragmentation at each stage of magnetic sorting (after frozen-thawed semen sample, after incubation with BB 1X (+ annexin V microbeads) and after sorting in the annexin V (-) fraction) [START_REF] Fekih | Impact des facteurs maternels et paternels sur les résultats de FIV / ICSI et investigations génétiques des spermatozoïdes d'hommes infertiles[END_REF].

Concerning the rate of spermatozoa with EPS, we have shown that it remains unchanged between frozen-thawed semen sample and after incubation with BB 1X (+ annexin V microbeads). After sorting, the rate significantly decreases in the annexin V (-) fraction.

Concerning the rate of DNA fragmentation, we have identified a significant increase of this rate between frozen-thawed semen sample and after incubation with BB 1X (+ annexin V microbeads). Moreover, we have identified a significant decrease after sorting in the annexin V (-) fraction compared to after incubation with BB 1X (+ annexin V microbeads). Thus, these results show that the BB1X has no influence on apoptosis -it does not induce DNA fragmentation. BB1X allows better accessibility and better integration of TdT and labeled dUTP during TUNEL (El Fekih 2018).

Although for certain patients the percentage of DNA fragmentation was still not low in the annexin V (-) fraction after column selection, on average MACS reduced the percentage by 46.15%. Our results are comparable with those described in the literature, with the rate of DNA fragmentation statistically significantly lower after MACS separation in the annexin V (-) fraction than before MACS separation and conversely, statistically significantly higher in the annexin V (+) fraction than before MACS separation [START_REF] Rawe | ICSI outcome using Annexin V columns to select non-apoptotic spermatozoa[END_REF][START_REF] Lee | Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility[END_REF]Herrero et al. 2012).

The presence of fragmented DNA spermatozoa in the annexin V (-) fraction may be due to an apoptotic process sequence different from that known in somatic cells. In fact, in previous studies, we showed that chromosomally unbalanced spermatozoa are more fragmented than chromosomally normal or balanced sperm in men with chromosomal abnormalities and we hypothesized that abortive apoptosis might explain this association (Perrin et al. 2011a;[START_REF] Perrin | DNA fragmentation is higher in spermatozoa with chromosomally unbalanced content in men with a structural chromosomal rearrangement[END_REF]. Nevertheless, the sequence of the apoptotic process in spermatozoa is still unclear. A hypothetical sequence was recently developed by Vendrell et al. [START_REF] Vendrell | Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients[END_REF]. Indeed, these authors hypothesized that during spermatogenesis an alteration of meiotic segregation would trigger an apoptotic process leading to the fragmentation of DNA.

Nevertheless, the early stages of apoptosis do not involve externalization of phosphatidylserine there are therefore fragmented spermatozoa without externalization of phosphatidylserine. These spermatozoa would thus not be retained in the column and be found in annexin V (-) [START_REF] Vendrell | Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients[END_REF].

Carriers of a constitutional structural chromosomal abnormality are a particular group of infertile men who produce a high rate of chromosomally unbalanced spermatozoa in their ejaculate [START_REF] Vegetti | Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men[END_REF]. For Robertsonian translocations the alternate mode, which is the only mode which leads to the production of chromosomally normal or balanced spermatozoa, prevailed. Our results are comparable to the more than 75% normal or balanced spermatozoa produced reported in the literature [START_REF] Scriven | Robertsonian translocations-reproductive risks and indications for preimplantation genetic diagnosis[END_REF][START_REF] Simpson | Genetics in Obstetrics and Gynecology[END_REF][START_REF] Blanco | Sperm FISH studies in seven male carriers of Robertsonian translocation t(13;14)(q10;q10)[END_REF][START_REF] Roux | Segregation of chromosomes in sperm of Robertsonian translocation carriers[END_REF]. Reciprocal translocation carriers produced between 18.6% and 80.7% normal or balanced spermatozoa [START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF][START_REF] Vidal | Role of sperm FISH studies in the genetic reproductive advice of structural reorganization carriers[END_REF][START_REF] Wiland | Successful pregnancy after preimplantation genetic diagnosis for carrier of t(2;7)(p11.2;q22) with high rates of unbalanced sperm and embryos: a case report[END_REF][START_REF] Perrin | DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality[END_REF]) which corroborates our result for our seven other patients. FISH technique is frequently used for the analysis of meiotic segregation in spermatozoa of translocation carriers [START_REF] Estop | The meiotic segregation pattern of a reciprocal translocation t(10;12)(q26.1;p13.3) by fluorescence in situ hybridization sperm analysis[END_REF][START_REF] Van Assche | Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22)[END_REF]Escudero et al. 2000;[START_REF] Frydman | Assisting reproduction of infertile men carrying a Robertsonian translocation[END_REF] to estimate the number of unbalanced spermatozoa in each patient, thus providing information about the reproductive risks. Indeed translocation carriers are at a high risk of repeated miscarriages and the birth of children with chromosomal abnormalities [START_REF] Stern | Chromosome translocations in couples with in-vitro fertilization implantation failure[END_REF][START_REF] Munné | Outcome of preimplantation genetic diagnosis of translocations[END_REF]. It thus seems important to eliminate unbalanced spermatozoa in order to reduce the reproductive risk associated with repeated miscarriages and the birth of children with chromosomal abnormalities in this population of infertile men.

To the best of our knowledge, only five publications have reported techniques for the selection of normal or balanced spermatozoa in translocation carriers. Two studies evaluated the potential of motile sperm organelle morphology examination (MSOME) for selecting normal or balanced spermatozoa [START_REF] Cassuto | Decreasing birth defect in children by using high magnification selected spermatozoon injection[END_REF][START_REF] Chelli | High-magnification sperm selection does not decrease the aneuploidy rate in patients who are heterozygous for reciprocal translocations[END_REF]. Both studies concluded that MSOME could not be used to select normal or balanced spermatozoa in men with a constitutional chromosomal abnormality. In 2012, Vozdova et al. did not find a difference in mean frequencies of unbalanced segregation products in the ejaculated and swim-up processed samples in men with a constitutional chromosomal abnormality but they found a significantly lower frequency of unbalanced spermatozoa after selection by the hyaluronan-binding technique [START_REF] Vozdova | The effect of the swim-up and hyaluronan-binding methods on the frequency of abnormal spermatozoa detected by FISH and SCSA in carriers of balanced chromosomal translocations[END_REF]. More recently, Rouen's teams obtained a decrease in the proportion of unbalanced spermatozoa in translocation carriers after selection using discontinuous gradient centrifugation [START_REF] Rouen | First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier[END_REF] or the hypo-osmotic swelling test [START_REF] Rouen | Potential selection of genetically balanced spermatozoa based on the hypo-osmotic swelling test in chromosomal rearrangement carriers[END_REF].

To the best of our knowledge, only two studies have worked on the effects of MACS on sperm aneuploidy rate in patients with a normal karyotype [START_REF] Vendrell | Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients[END_REF][START_REF] Esbert | Spermatozoa with numerical chromosomal abnormalities are more prone to be retained by Annexin V-MACS columns[END_REF]. These studies reported that processing samples through MACS decreased sperm aneuploidy rates in the annexin V (-) fraction. Moreover they compared the aneuploidy rate in the annexin V (-) and V (+) fractions and concluded that aneuploid sperm cells are preferentially retained in the MACS column [START_REF] Esbert | Spermatozoa with numerical chromosomal abnormalities are more prone to be retained by Annexin V-MACS columns[END_REF]. Our study is the first of its kind to evaluate the rate of normal or balanced spermatozoa and unbalanced spermatozoa after MACS in the two fractions of annexin V (-) and annexin V (+) for patients carrying a constitutional structural chromosomal abnormality. We observed a significant increase of normal or balanced spermatozoa in the annexin V (-) fraction and a significant decrease in the annexin V (+) fraction and there is always a significant difference when comparing the two fractions. Even if unbalanced spermatozoa are found in the annexin V (-) fraction, on average MACS reduced this percentage by 32.14%. The presence of unbalanced spermatozoa in the annexin V (-) fraction could be explained by the hypothetical sequence recently developed by [START_REF] Vendrell | Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients[END_REF] We also analyzed the impact of MACS on the different segregation modes in unbalanced spermatozoa. In the literature, for Robertsonian translocation carriers the segregation mode producing unbalanced spermatozoa with disomies and nullisomies for chromosomes involved in the translocation are adjacent and 3:0 [START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF]. In reciprocal translocation carriers, the unbalanced spermatozoa result mainly from the adjacent I, adjacent II, 3:1 and 4:0 [START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF]. Our results are concordant with these data, except for P5 and P8, where the predominant segregation mode for unbalanced spermatozoa was 3:1. This phenomenon has already been reported in previous studies [START_REF] Morel | Meiotic segregation of translocations during male gametogenesis[END_REF][START_REF] Brugnon | Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients[END_REF]. Regarding the different meiotic segregation modes producing unbalanced spermatozoa, we always found a significant difference between the annexin V (-) and annexin V (+) fractions, always in favor of annexin V (-) with a significant decrease of unbalanced spermatozoa. We concluded that there is no preferential selection depending on the type of chromosomal imbalance.

MACS is a simple and inexpensive technique, without deleterious or magnetic effects detectable on the spermatozoa due to their passage in the separation columns [START_REF] Grunewald | Increased sperm chromatin decondensation in selected nonapoptotic spermatozoa of patients with male infertility[END_REF]. Microbeads do not affect cell viability and are biodegradable [START_REF] Miltenyi | High gradient magnetic cell separation with MACS[END_REF]).

Since 2010, many healthy offspring have been reported with the use of MACS as a sperm selection technique with ICSI (Polak de Fried & Denaday 2010; [START_REF] Rawe | Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI[END_REF]Herrero et al. 2013;[START_REF] Ugozzoli | Neonatal outcome after ICSI with MACS for selection of non apoptotic spermatozoa: first report[END_REF]. Moreover in 2017 Romany et al. did a randomized control trial on 237 couples with liveborn children conceived through the association of MACS and ICSI. They concluded that there were no noticeable differences in obstetric or perinatal events when MACS is used [START_REF] Romany | Obstetric and perinatal outcome of babies born from sperm selected by MACS from a randomized controlled trial[END_REF]. However, further research is needed to verify the presence of freely floating microbeads in the annexin V (-) fraction on the plasma membrane of the spermatozoa, and long term effects on the health and development of children.

Despite the small number of cases in our study we conclude that after MACS separation we observed a significant decrease of spermatozoa with an externalization of phosphatidylserine, DNA fragmentation and chromosomally unbalanced spermatozoa in the annexin V (-) fraction and inversely a significant increase in the annexin V (+) fraction. In order to improve this selection, in a future study it would be interesting to combine the MACS technique with another selection method such as density gradient centrifugation. The results obtained in this study consolidate the hypothesis that MACS tends to retain unbalanced spermatozoa and improve the selection of normal or balanced spermatozoa for ART. It strengthens our hypothesis of an abortive apoptosis initiated in the testis.

MACS is thus a promising tool for use in selecting healthy spermatozoa with a normal or balanced chromosomal content which can be used in assisted reproduction technique (ART) such as ICSI in males with a structural chromosomal abnormality and also for use in selecting spermatozoa with a normal or balanced chromosomal content before preimplantation genetic diagnosis in these patients.

The risk of picking up an unbalanced spermatozoon for ICSI after MACS separation remains and selection by MACS cannot replace preimplantation diagnosis which is the best method for embryo selection after ICSI. However, MACS carries the benefit of selecting spermatozoa of better quality with respect to apoptosis, DNA fragmentation and chromosomal content and thus increasing the chance of achieving a successful pregnancy.

A study on the data of offspring obtained through this technology would certainly be interesting but remains subject to prevailing legislation and at the present time French legislation does not authorize this kind of study.

FIGURES

Table 1: Karyotype performed on peripheral blood cultures of ten male carriers of Robertsonian or reciprocal translocations Table 2: Mix, spectrums and localization of probes used to perform FISH on spermatozoa of ten male carriers of Robertsonian or reciprocal translocations Table 3: Data on sperm parameters for raw semen samples from each individual (volume, concentration, mobility, normal forms) Table 4: Percentage of spermatozoa with externalization of phosphatidylserine in frozenthawed semen sample, in the annexin V (-) and V (+) fractions for each patient Table 5: Percentage of DNA fragmentation in frozen-thawed semen sample, after incubation, in the annexin V (-) and V (+) fractions for each patient Table 6: Different modes of meiotic segregation in semen samples, in the annexin V (-) and V (+) fractions for each patient

P2

BACs pool on 14q32.33 (Green), BACs pool on 15q26.3 (Orange), CEP8 (Aqua)

P3

BACs pool on 13q33.3 (Green), BACs pool on 14q32.33 (Orange), CEP8 (Aqua)

P4

BACs pool on 4q22.1 (Aqua), BACs pool on 4q34.1 (Green), CEP12 (Orange)

P5

BACs pool on 9q34 (Green), BACs pool on 12p13.33 (Aqua), CEP12 (Orange)

P6

BACs pool on 1p36.33 (Orange), BACs pool on 1q24.1-24.2 (Aqua), BACs pool on 15q26.3 (Green)

P7

BACs pool on 11q25 (Orange), BACs pool on 13q14.3 (Green), BACs pool on 13q33.3 (Aqua)

P8

BACs pool on 13q33.3 (Green), BACs pool on 19p12-p13.11 (Orange), BACs pool on 19q13.41 (Aqua)

P9

BACs pool on 1p36.33 (Orange), BACs pool on 1q24.1-24.2 (Aqua), BACs pool on 22q11.23 (Green)

P10

BACs pool on 2p25.3 (Green), BACs pool on 2q37.3 (Orange), BACs pool on 9q34.12-q34.13 (Aqua)

Figure 1 :

 1 Figure 1: Flow diagram of the overall experiment design

Table 1 :

 1 Karyotype performed on peripheral blood cultures of ten male carriers of Robertsonian or reciprocal translocations

	Patient	Karyotype
	P1	45,XY,rob(13;14)(q10;q10)
	P2	45,XY,rob(14;15)(q10;q10)
	P3	45,XY,rob(13;14)(q10;q10)
	P4	46,XY,t(4;12)(q31;q23)
	P5	46,XY,t(9;12)(q31;p12)
	P6	46,XY,t(1;15)(p22.2;q21.1)
	P7	46,XY,t(11;13)(q22.1;q21.2)
	P8	46,XY,t(13;19)(q12;q13.3)
	P9	46,XY,t(1;22)(p11;p11.2)
	P10	46,XY,t(2;9)(p24;p21)

Table 2 :

 2 Mix, spectrum and localization of probes used to perform FISH on spermatozoa of ten male carriers of Robertsonian or reciprocal translocations

	Patient	Mix of probes used for FISH analyses
	P1	BACs pool on 13q33.3 (Green), BACs pool on 14q32.33 (Orange), CEP18 (Aqua)

Table 3 :

 3 Data on sperm parameters for raw semen samples from each individual(volume, concentration, motility, normal forms)

	Patient	Volume (mL) Concentration (M/mL) Motility (a+b+c)	Normal forms (%)
	P1	3.2	50	55	4*
	P2	2.1	151	45	13**
	P3	3.2	19.6	35	10**
	P4	3.2	117	41	10**
	P5	5.3	50	55	26**
	P6	2.8	108	40	31**
	P7	4.2	42	35	14**
	P8	2.5	49	60	70**
	P9	8.1	14.4	35	NR
	P10	3.5	15	35	15**

*according to the classification of Kruger, ** according to the classification of David, NR: unrealized

Table 4 :

 4 Percentage of spermatozoa with externalization of phosphatidylserine in frozenthawed semen sample, in the annexin V (-) and V (+) fractions for each patient

	Patient	Frozen-thawed	Fraction of	Fraction of	Between fraction of
		semen sample (%)	annexin V (-) (%)	annexin V (+) (%)	annexin V (-) and V (+)
	P1	57.4	19.6*	84.2*	**
	P2	56.0	32.6*	83.6*	**
	P3	54.6	33.2*	71.4*	**
	P4	38.0	14.2*	85.6*	**
	P5	72.8	36*	88*	**
	P6	67.2	35.4*	83.4*	**
	P7	58.0	30.6*	69.8*	**
	P8	63.2	28.4*	84.8*	**
	P9	64.8	37.6*	86.2*	**
	P10	61.2	29.2*	68.8*	**
	Average ± SD	59.32 ± 9.34	29.72 ± 7.45*	80.58 ± 7.44*	**

Table 5 :

 5 Percentage of DNA fragmentation in frozen-thawed semen sample, after incubation, in the annexin V (-) and V (+) fractions for each patient

	Patient	Frozen-thawed	After	Fraction of	Fraction of	Between fraction of
		semen sample	Incubation	annexin V (-)	annexin V (+)	annexin V (-) and V (+)
		(%)	(%)	(%)	(%)
	P1	15	23*	9.2**	28	***
	P2	7.2	21.8*	7.4**	31.2**	***
	P3	18.8	20.6	14**	34.8**	***
	P4	16.2	23.4*	16.2**	36.2**	***
	P5	18.4	37*	18.6**	25.4**	***
	P6	20.6	23.8	20	23.4	NS
	P7	18.2	22.8	9.4**	34**	***
	P8	15	21.2*	10**	24	***
	P9	14.8	25.4*	13**	37.6**	***
	P10	14	17.2	9.4**	32.8**	***
	Average ± SD	15.82 ± 3.93	23.62 ± 5.19	12.72 ± 4.36**	30.74 ± 5.20**	***

Table 6 :

 6 Different modes of meiotic segregation in semen samples, in the annexin V (-) and V (+) fractions for each patient

Modes of meiotic segregation Semen sample Fraction of annexin V (-) Fraction of annexin V (+) Between fraction of annexin V (-) and V (+) P1

		Alternate (%)	37.75	54.76*	27.63*	**
		Adjacent I (%)	32.49	30.13	36.21	**
	P6	Adjacent II (%)	16.12	10.06*	22.15*	**
		Alternate (%) 3 : 1 (%)	79.99 12.75	88.03* 4.76*	75* 12.64*	** **
		Adjacent (%) Diploidy or 4 : 0 (%)	18.82 0.79	11.14* 0.19 *	21.77* 1.08	** **
		3 : 0 (%) Others (%)	0.47 0.1	0.43 0.1	1.87* 0.29	** NS
	Diploid (%) No of spermatozoa analysed	0.47 2016	0.2* 2078	1.1* 2041	**
		Others (%) Alternate (%)	0.24 43.13	0.2 60.54*	0.26 33.07*	NS **
	No of spermatozoa analysed Adjacent I (%)	2534 35.63	2531 31.84*	2728 43.6*	**
	P2 P7 No of spermatozoa analysed Alternate (%) Adjacent II (%) Adjacent (%) 3 : 1 (%) 3 : 0 (%) Diploidy or 4 : 0 (%) Diploid (%) Others (%) Others (%) No of spermatozoa analysed Alternate (%) P3 Alternate (%) Adjacent I (%) Adjacent (%) P8 Adjacent II (%) 3 : 0 (%) 3 : 1 (%) Diploid (%) Diploidy or 4 : 0 (%) Others (%) Others (%) No of spermatozoa analysed No of spermatozoa analysed P4 Alternate (%) Alternate (%) Adjacent I (%) Adjacent I (%) Adjacent II (%) P9 Adjacent II (%) 3 : 1 (%) 3 : 1 (%) Diploidy or 4 : 0 (%) Diploidy or 4 : 0 (%) Others (%)	83.37 7.35 14.4 12.04 0.2 1.45 1.29 0.4 0.74 2001 2021 37.24 78.06 1.12 19.55 5.2 0.4 54.01 1.74 2.14 0.25 0.29 2010 2057 44.27 44.07 36.43 20.1 8.64 19.66 7.5 14.9 0.91 0.49 0.78	90.2* 3.47* 8.5* 3.47* 0.1 0.53* 1.05 0.14 0.15* 2073 2002 59.84* 88.64* 0.1* 10.72* 2.42* 0.4 35.77* 0.1* 1.58* 0.15 0.3 2024 2027 62.77* 58.02* 27.58* 18.42 4.77* 16.93* 3.66* 6.19* 0.59 0.35 0.1*	82.43 6.95 14.23* 14.44* 0.15 1.52 2.1* 0.41 1.1 2431 30.17* 2003 80.93* 0.35* 16.72* 9.76* 0.83 54.61 1.03 3.37* 0.49 1.73* 2254 2045 31.63* 39.21* 51.99 17.31* 7.19 24.67* 6.81* 17.18* 2.16* 1.19* 0.44	** ** ** NS ** NS NS ** ** ** NS ** ** ** ** NS ** ** ** NS ** ** NS ** ** ** ** ** ** ** **
	Others (%) No of spermatozoa analysed	2.25 2040	0.63* 2020	0.21* 2270	**
	No of spermatozoa analysed Alternate (%)	2627 36.39	2705 53.78*	2864 31.65*	**
		Alternate (%) Adjacent I (%)	48.36 31.51	66.16* 26.41*	50.77 32.28	** **
	P5 P10	Adjacent I (%) Adjacent II (%)	0.15 21.01	0.04 15.44*	0.16 24.33*	NS **
		Adjacent II (%) 3 : 1 (%)	0.18 9.86	0.19 4.04*	0.2 10.86	NS **
		3 : 1 (%) Diploidy or 4 : 0 (%)	50.83 0.5	33.38* 0.05*	48.64* 0.39	** **
		Diploidy or 4 : 0 (%) Others (%)	0.07 0.75	0.19 0.28*	0.24 0.48	NS NS
	Others (%) No of spermatozoa analysed	0.41 2009	0.04* 2105	0* 2063	NS
	No of spermatozoa analysed	2707	2651	2531	NS

* Significant difference when compared to the semen sample (p < 0.05) ** significant difference when comparing the annexin V (-) and V (+) fractions (p < 0.05) and NS: no significant.

Acknowledgements:

Some spermatozoa samples were provided by "GERMETHEQUE national biobank (BB-0033-00081; coordination, Toulouse, France".

The authors also thank Dr G LEFORT for her help with the translation of the manuscript.

Funding information:

This research was supported by the "Agence de la BioMédecine, France".

Disclosure:

The authors have no conflict of interest to declare.

Author's contribution:

The authors have all contributed to the study (Sahar El Fekih and Elodie Caire-Tetauru: technical assistance, result analysis and article writing; Corinne Tous and Nadia Gueganic: technical assistance; Florence Brugnon, Habib Ben Ali, Louis Bujan, Nathalie Moinard, Mounir Ajina, Nathalie Douet-Guilbert, Frédéric Morel and Aurore Perrin: expertise and article review).


Maria I Lapuh

Sara Mazeh

Tatiana Besset
email: tatiana.besset@insa-rouen.fr

Chiral Transient Directing Groups in Transition-Metal-Catalyzed Enantioselective C-H Bond Functionalization

Keywords: Transition Metal, Chiral Transient Directing Group, Asymmetric Transformations, Dual Catalysis, Synthetic Methodology, C-H Bond Functionalization

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INTRODUCTION

In the last decades, the transition metal-catalyzed C-H bond functionalization has emerged as a powerful strategy for achieving unprecedented transformations in a step-and atom-economical way. 1 This was illustrated, for instance, by the huge number of applications of this synthetic tool for the preparation of highly complex molecules. 2 Despite the unarguably advances, some limitations still remain: the selectivity is one of the most relevant challenges to be addressed since C-H bonds are ubiquitous on a molecule. This issue has been elegantly circumvented with the aid of a directing group (DG), which is able to coordinate to the metal center and to place it close to the C-H bond to be functionalized. 3 Hence, various monodentate and bidentate directing groups have been efficiently used for the selective functionalization of different positions over aromatic [START_REF] Yu | Direct Functionalization with Complete and Switchable Positional Control: Free Phenol as a Role Model[END_REF] and aliphatic derivatives. [START_REF] Schranck | Functionalization of Remote C─H Bonds: Expanding the Frontier[END_REF] Nevertheless, solutions to avoid the extra synthetic steps required to install and cleave the directing group on the molecules were needed. Therefore, several strategies have arisen, such as the use of traceless directing groups [START_REF] Bhadra | Synthesis of Aryl Ethers from Benzoates through Carboxylate-Directed C─H-Activating Alkoxylation with Concomitant Protodecarboxylation[END_REF] and transient mediators. [START_REF] Fontana | Pd/Norbornene: A Winning Combination for Selective Aromatic Functionalization via C-H Bond Activation[END_REF][START_REF] Wang | Catalytic Intermolecular C-Alkylation of 1,2-Diketones with Simple Olefins: A Recyclable Directing Group Strategy[END_REF] More recently, other promising alternatives emerged, based on the use of non-covalent interactions between the substrates and designed catalysts 5e,9 and transient directing groups (TDGs). [START_REF] Sun | Advances in the development of catalytic tethering directing groups for C-H functionalization reactions[END_REF][START_REF] Wu | Pd-Catalyzed γ-C(sp 3 )-H Arylation of Free Amines Using a Transient Directing Group[END_REF] Since the pioneering example described by Jun and co-workers in 1997, [START_REF] Jun | Chelation-Assisted Intermolecular Hydroacylation: Direct Synthesis of Ketone from Aldehyde and 1-Alkene[END_REF] the latter strategy was successfully applied in several transition metal-catalyzed regioselective C-H bond functionalization, appearing as a modern and sustainable solution. Encouraged by this new synthetic tool, the scientific community showed a real endeavor towards the design and the use of chiral transient directing groups to achieve the highly challenging enantioselective C-H bond functionalization. 1a,13 Indeed, the chiral transient directing group (CTDG) strategy, which relied on the reversible and temporary formation of a chiral directing group from a functional group, was particularly appealing. Taking benefit from the fact that the transient directing group also played the role of a chiral ligand, the number of species in the reaction media remained limited, reducing the possibilities for side reactions. Conceptually, a suitable functional group (FG A ) on the substrate would first react with a chiral organic catalyst to afford the aforementioned CTDG (I). After its coordination with the transition metal center in a monodentate or bidentate fashion, the metallacycle II would be formed. This latter would then react with the coupling partner leading to the species III and allowing the regeneration of the transition metal catalyst. A final hydrolysis would release the product along with the organo-catalyst (Scheme 1). Scheme 1. The chiral transient directing group strategy in transition metal-catalyzed C-H bond activation reactions.

The aim of this review is to showcase and discuss the recent advances made in the cutting-edge C-H activation field based on the use of chiral transient directing groups to address transition metal-catalyzed enantioselective C-H bond functionalization. First, major advances regarding the atroposelective C(sp 2 )-H functionalization for the synthesis of enantioenriched chiral biaryls will be described. Then, the application of the transient directing group strategy to the preparation of enantioenriched heterocycles and carbocyles along with miscellaneous reactions will be highlighted. Finally, a last part of the review will be dedicated to the recent developments on the transition metal-catalyzed enantioselective C(sp 3 )-H bond functionalization.

2. Asymmetric C(sp 2 )-H bond activation for atropisomer synthesis 2.1. from biaryl derivatives Given the significance and the abundance of the axially chiral biaryls in bioactive natural products, [START_REF]For selected examples[END_REF] advanced material sciences, [START_REF] Wu | 1,2-Di(phenylethynyl)ethenes with Axially Chiral, 2,2'-bridged 1,1'-dinaphthyl Substituents: Potent Cholesteric Liquid-Crystal Inducers[END_REF] chiral ligands [START_REF] Noyori | BINAP: an efficient chiral element for asymmetric catalysis[END_REF] and catalysts, [START_REF] Akiyama | Recent Progress in Chiral Brønsted Acid Catalysis[END_REF] groundbreaking advances were made towards a straightforward access to these scaffolds. 14c,18 Existing methods to synthesize such derivatives generally relied on stereoselective cross-coupling reactions, [START_REF] Luo | Novel Achiral Biphenol-Derived Diastereomeric Oxovanadium(IV) Complexes for Highly Enantioselective Oxidative Coupling of 2-Naphthols[END_REF] asymmetric cycloadditions, 20 transition metal-catalyzed atroposelective C-H bond functionalization, [START_REF] Hayashi | Catalytic asymmetric synthesis of axially chiral biaryls by palladium-catalyzed enantioposition-selective cross-coupling[END_REF] among others. [START_REF] Guo | Enantioselective Synthesis of Biphenols from 1,4-Diketones by Traceless Central-to-Axial Chirality Exchange[END_REF] Recently, the chiral transient directing group strategy appeared as a potent tool for the atroposelective synthesis of chiral biaryls. Therefore, the major advances made to access these high-value added axially chiral biaryl backbones thanks to this approach will be highlighted and discussed in this section.

a) Olefination and allylation reactions

In 2017, Shi and co-workers studied the chiral transient directing group-assisted Pd-catalyzed atroposelective olefination of biaryl aldehydes based on the following working hypothesis [START_REF] Yao | Atroposelective Synthesis of Axially Chiral Biaryls by Palladium-Catalyzed Asymmetric C-H Olefination Enabled by a Transient Chiral Auxiliary[END_REF] First, reaction of the racemic biaryl aldehyde (rac-1) with the chiral amino acid A would reversibly afford the two imines I and II. Then, formation of the axially enantioenriched biaryl palladacycle III derived from the diastereoisomer imine I would take place, presumably driven by steric interactions. The latter intermediate III would then undergo a Heck-type reaction with the alkene. After a reductive elimination and an in situ hydrolysis, the enantioenriched product 2 would be released along with the organocatalyst A. Finally, the palladium (II) catalyst would be regenerated after oxidation. Using the readily available chiral amino acid, L-tert-leucine (A), as a pivotal transient directing group precursor, a panel of aromatic aldehdyes (rac-1) were olefinated through a dynamic kinetic resolution pathway (Scheme 2). When using substrates bearing electron-donating groups, the expected alkenylated products (2a-d) were obtained in yields up to 94% and high enantioselectivities (up to >99%). The transformation was tolerant to functional groups such as a halogen (1e) and an ester (1f). Pleasingly, a styrene derivative was a suitable coupling partner as showed by the synthesis of the compound 2g. In the case of biaryl derivatives bearing bulky substituents at both 6-and 2'-positions, the reaction proceeded via a kinetic resolution pathway, furnishing the enantiopure products (2h-k) in 30-45% yields along with the enantioenriched starting biaryl aldehydes (1h-k). It is worth to mention that in this case the reaction also went smoothly with other acrylate and electron-poor styrene derivatives as demonstrated with the synthesis of compounds 2l-n. Scheme 2. Pd(II)-catalyzed atroposelective C-H olefination reaction by using a chiral transient directing group.

A year later, a Pd-catalyzed asymmetric allylation of biaryl aldehydes via a (dynamic) kinetic resolution reaction was developed by the same group (Scheme 3). [START_REF] Liao | Pd-Catalyzed Atroposelective C-H Allylation through ß-O Elimination: Diverse Synthesis of Axially Chiral Biaryls[END_REF] Using the L-tert-leucine (A) as a chiral organo-catalyst, an array of biaryl aldehydes rac-3 was functionalized with the allylic acetate reagent in the presence of benzoquinone in a HFIP/AcOH mixture. It turned out that the presence of an oxidant under acidic reaction conditions was crucial for the efficiency of the reaction. With substrates bearing a substituent at either the phenyl (3a-e) or the naphthyl part (3f-g), the corresponding allylated products were obtained efficiently through a dynamic kinetic resolution pathway in yields ranging between 33% and 71% and in high enantioselectivities (96% to >99% ee). Allylated biaryls bearing either halogens or electronwithdrawing groups (4a-c) as well as electron-donating groups (4d-f) were efficiently synthesized. In addition, the substitution pattern did not have a significant impact on the outcome of the reaction (4a, 4b and 4g). Worth mentioning that other coupling partners were also successfully used (4h-i) and replacing the leaving group of the allylation reagent with OBz or OBoc led to the corresponding product (4j) in 65% and 23% yields, respectively. With 2'and 6-disubstituted racemic biaryls, the reaction proceeded in the presence of the allylic acetate reagent via a kinetic resolution process, affording the enantioenriched allylated products (4k-n) along with the enantioenriched starting materials (3k-n). Then, a Pd-catalyzed olefination of non-C2-symmetric biaryl aldehydes was achieved using the vinyl ethylene carbonate followed by a reduction step, offering an efficient access to the corresponding diols 6a-g in moderate to good yields and high enantioselectivities (>99%) (Scheme 3). Scheme 3. Synthesis of axially chiral biaryls via a Pd(II)-catalyzed asymmetric allylation reaction.

a A (30 mol%). b The leaving group of the coupling partner (OAc) was replaced by a OBz group. c The leaving group of the coupling partner (OAc) was replaced by a OBoc group.

Applying the enantioselective C-H functionalization strategy to natural products synthesis, 2a,c,d,k,25 the group of Shi reported a concise and highly enantioselective total synthesis of TAN-1085, [START_REF] Fan | Asymmetric Total Synthesis of TAN-1085 Facilitated by Pd-Catalyzed Atroposelective C-H Olefination[END_REF] an angucycline antibiotic isolated from Streptomyces species (Scheme 4). 27 Taking benefit from their pioneering work, [START_REF] Yao | Atroposelective Synthesis of Axially Chiral Biaryls by Palladium-Catalyzed Asymmetric C-H Olefination Enabled by a Transient Chiral Auxiliary[END_REF] a scalable and efficient construction of the axially chiral biaryl scaffold 8 was achieved in 75% yield with a high level of enantioselectivity (99% ee). In 2019, Zhang and Xie extended this appealing strategy to the synthesis of unprecedented N-C axially chiral N-arylindoles by means of a transition metal-catalyzed atroposelective C-H alkenylation reaction (Scheme 5). [START_REF] Zhang | Construction of N-C Axial Chirality through Atroposelective C-H Olefination of N-Arylindoles by Palladium/Amino Acid Cooperative Catalysis[END_REF] Using a catalytic amount of Pd(OAc)2 and L-Valine (B) in the presence of benzoquinone, the olefination of a panel of aryl-1H-indole-2-carbaldehyde derivatives rac-9 with the n-butyl acrylate was achieved in a high regio-and stereocontrol leading to the corresponding alkenylated products 10a-g. Various N-arylindoles bearing electron-donating and electron-withdrawing groups at either the indole (9a-c) or the phenyl part (9d-g) were functionalized in good yields via a desymmetrization process. The transformation also went smoothly with a phenyl vinyl sulfone, a styrene derivative and with the 3-methylenedihydrofuran-2(3H)-one affording the compounds 10h-j. Moreover, a dynamic kinetic resolution pathway was reported allowing the functionalization of N-arylindoles bearing a halogen or an alkyl chain in good yields with excellent enantioselectivities (10kl). The transformation was not restricted to the use of the n-butyl acrylate as several olefins were suitable (10m-n). The olefination of the bulkier substrates rac-11a-i with various activated olefins as coupling partners was achieved in the presence of L-tert-leucine and silver triflate via a kinetic resolution process furnishing a range of enantioenriched heterobiaryl derivatives in moderate to good yields and excellent enantioselectivities (12a-i). Scheme 5. Atroposelective Pd(II)-catalyzed olefination of N-arylindoles.

a Pd(OAc)2 (10 mol%), A (20 mol%), AgTFA (2 equiv), NaHCO3 (2 equiv), TFE/AcOH (1:1), 60 °C, N2.

The same year, the Shi group depicted the atroposelective Pd-catalyzed allylation and olefination of biaryl aldehydes, offering a straightforward access to the challenging five-membered axially chiral biaryls (Scheme 6). [START_REF] Chen | Pd-Catalyzed Atroposelective C-H Allylation and Alkenylation: Access to Enantioenriched Atropisomers Featuring Pentatomic Heteroaromatics[END_REF] For this purpose, a catalytic amount of L-tert-leucine and Pd(OAc)2 along with chloranil as the oxidant were used for the allylation of a broad range of heterocycles rac-13 with the allylic acetate reagent leading to enantioenriched atropoisomers featuring a heteroaryl part. Importantly, it is worth to mention that the position of the heteroarenes on the biaryl scaffold did have an influence on the mechanism pathway involved in the transformation. When the benzothiophene was at the upper part of the biaryls (13a-b), the transformation proceeded via a kinetic resolution affording the expected products (14a-b) in good yields and high selectivities. While for substrates bearing heteroarenes in the lower part (13c-d), the enantiopure allylated biaryls (14c-d) were obtained through a dynamic kinetic resolution pathway in moderate to good yields. The synthesis of the allylated 3,3'-bisbenzothiophene (14e) was straightforward (77% yield, 92% ee), while no enantioselectivity was observed with a biaryl bearing a benzothiophene and a benzofuran residue (14f). This approach was then applied to the alkenylation of heteroarenes with various acrylate derivatives and styrene under similar reaction conditions. While a set of biaryls bearing a benzothiophene part (15a-e) were functionalized via a kinetic resolution process, the olefination of the benzofuran-based biaryl 15f and the 3,3'-bisbenzothiophene 15g proceeded according to a dynamic kinetic resolution pathway. Scheme 6. Synthesis of enantioenriched atropoisomers featuring a heteroaryl part by Pd(II)-catalysis. This year, the Ackermann group described the first atroposelective palladoelectro-catalyzed olefination of biaryl aldehydes by C-H bond activation under mild reaction conditions (Scheme 7). [START_REF] Dhawa | Enantioselective Palladaelectro-Catalyzed C-H Activations by Transient Directing Groups: Expedient Access to Helicenes[END_REF] A panel of biaryl derivatives rac-17 was olefinated using the L-tert-Leucine as chiral organo-catalyst and LiOAc as the electrolyte, furnishing the corresponding products in moderate to good yields (up to 71%) and high enantioselectivities (up to 98% ee). Both electron-rich (17a) and electronpoor (17b) substrates reacted well under these conditions showcasing the robustness of this approach. Besides, disubstituted biphenyls proved to be suitable substrates for this functionalization (17c-d). Notably, a series of electrondeficient olefins was successfully used as coupling partners offering an atroposelective access to the corresponding biaryls in up to 68% yield (18e-l). More interestingly, this transformation turned out to be efficient also with N-arylpyrrole derivatives. Hence, an access to N-C axially chiral heteroaryls 20a-e was achieved in a complete regio-and stereoselective manner. Finally, the strategy was further applied to access key compounds such as chiral BINOLs, dicarboxylic acids and helicenes. Scheme 7. Electrochemical enantioselective olefination of biaryl aldehydes by Pd(II)-catalyzed C-H bond activation using the chiral transient directing group strategy.

b) Naphthylation reaction

In 2019, the Shi group reported the atroposelective Pd-catalyzed ortho C-H naphthylation of racemic biaryl aldehydes rac-21 with 7-oxabenzonorboranadienes (Scheme 8) [START_REF] Liao | Synthesis of Chiral Aldehyde Catalysts by Pd-Catalyzed Atroposelective C-H Naphthylation[END_REF] leading to key scaffolds, known to be efficient chiral catalysts. 17d,32 For this transformation, a four-step process was designed: insertion of a 7-oxabenzonorboranadiene derivative within the palladacycle intermediate (I) followed by a -oxygen elimination would lead to the intermediate II. The latter would then undergo a protonolysis to afford the dihydronaphthol III, which after a dehydration step would furnish the expected product 22. By employing L-tert-leucine, 1-adamantaneacetic acid and sodium butyrate, an array of the desired naphthylated products (22a-h) was obtained efficiently in good yields and enantioselectivities. Indeed, this reaction was compatible with substrates bearing at the aryl ring either an electron-donating functional group (21a) or an electron-withdrawing one (21b) and with difunctionalized substrates (21c-d). Also, the methodology was successfully applied to substrates with electronrich naphthyl parts (21e-f). Nonetheless, a lower reactivity was observed once the naphthalene ring is functionalized with an electron-withdrawing group (22g). Besides, an access to the chiral poly-substituted biaryl compound 22h was possible by this methodology. Notably, the desymmetrization of prochiral substrates was achieved in good to moderate yields and high enantioselectivities (22i-j). When several analogues of the 7-oxabenzonorboranadiene were used, the desired products (22k-l) were successfully synthesized with electron-rich partners in contrast to electron-poor ones, which only afforded the dihydronaphthol products (22m-n). Scheme 8. Atroposelective Pd(II)-catalyzed ortho C-H naphthylation of biaryl aldehydes.

c) Alkynylation reaction

Aiming at having an efficient access towards advanced intermediates for the synthesis of the bioactive dibenzocyclooctadiene lignans, [START_REF] Chang | Progress on the Chemistry of Dibenzocyclooctadiene Lignans[END_REF] Shi et al. developed a novel Pd-catalyzed C-H alkynylation reaction in the presence of a TIPS-protected alkynyl bromide (Scheme 9). [START_REF] Liao | Stereocontrolled Formal Syntheses of (+)-Isoschizandrin and (+)-Steganone: Development and Applications of Palladium(II)-Catalyzed Atroposelective C-H Alkynylation[END_REF] The approach displayed a high functional group tolerance since products bearing electron-withdrawing groups and halogens (24a-c) as well as electron-donating groups (24d-e) were obtained through a dynamic kinetic resolution process. Poly-substituted biaryl aldehydes 23f-h also underwent this reaction successfully. Moreover, different silylated-protected alkynyl bromides turned out to be suitable for this transformation leading to the corresponding compounds 24i-j in up to 74% yield and high selectivities (up to 99% ee). Furthermore, prochiral substrates were desymmetrized in good yields and high enantioselectivities (24k-m) and both halogenated 23l and alkylated biaryl aldehydes 23m were suitable substrates. In case of biaryl aldehydes, substituted at both 6-and 2'positions (25a-d), the Pd-catalyzed alkynylation reaction proceeded via a kinetic resolution pathway under slightly modified reaction conditions (Scheme 9). Scheme 9. Atroposelective synthesis of alkynylated biaryl scaffolds.

Then, the same group extended their methodology to the challenging atroposelective alkynylation of biaryls featuring at least one five-membered heteroarene (Scheme 10). [START_REF] Zhang | Enantioselective Synthesis of Atropisomers Featuring Pentatomic Heteroaromatics by Pd-Catalyzed C-H Alkynylation[END_REF] Under Palladium catalysis, the enantioselective alkynylation of various biaryl aldehydes bearing a C-N and a C-C chiral axis with a TIPS-protected alkyne bromide was realized in an efficient manner. Different N-arylpyrroles were functionalized (28a-d) and different alkynes were efficient partners in the reaction (28e-g), albeit N-arylindoles were unreactive. The alkynylation of the thiophene derivatives (27h,i) was achieved in moderate to good yields and with enantiomeric excesses ranging from 21% to 76%. Moreover, replacing the benzothiophene in the lower part by a benzofuran had a deleterious effect on the enantioselectivity (28j vs 28k and 28l vs 28m), whereas the nature of the heteroaryl in the upper part of the biaryls had no significant impact on the outcome of the reaction (28n-q). Interestingly, with this approach, the alkynylation of substrates having two five-membered heteroarenes was possible and it turned out that the enantioselectivity of the reaction was dependent on the nature of the heteroarene in the lower part (28r vs 28s). Scheme 10. Pd(II)-catalyzed alkynylation of five-membered containing biaryls by C-H bond activation.

from styrene derivatives

Recently in 2020, Shi and co-workers investigated the challenging synthesis of axially chiral styrenes. To that purpose, the Pd-catalyzed atroposelective C-H olefination of racemic styrenes (rac-29) using the bulky amino acid C was developed in the presence of benzoquinone and cobalt acetate in a mixture of AcOH and DMSO, this latter presumably acting as a ligand (Scheme 11). [START_REF] Song | Synthesis of Axially Chiral Styrenes through Pd-Catalyzed Asymmetric C-H Olefination Enabled by an Amino Amide Transient Directing Group[END_REF] With substrates bearing a substituent at the ortho position, the expected products were obtained with a good enantiocontrol (30a-c) except in the case of 30d. By replacing the aryl part by a naphthyl one, the olefination of several derivatives (30e-i) went smoothly. Moreover, several activated olefins and styrenes were suitable coupling partners leading to the synthesis of axially chiral compounds (30j-o) in high enantioselectivities. Interestingly, using acrylates derived from natural products, an access to added-value styrenes (30p-q) was possible with high diastereomeric excesses. Notably, axially chiral styrenes 30 were oxidized to the corresponding carboxylic acids, which showed higher efficiency as chiral ligands in the Co(III)-catalyzed enantioselective (sp 3 )-H amidation of a thioamide compared to their biaryl counterparts. Scheme 11. Synthesis of axially chiral styrenes via the transient directing group strategy.

Asymmetric synthesis of chiral carbocycles and heterocycles by C-H bond activation

The preparation of chiral carbocycles and heterocycles through transition metal-catalyzed direct C-H bond activation has been considerably studied in the last decades. Indeed, the use of Rhodium complexes, especially those containing cyclopentadienyl (Cp)-derived ligands, allowed many successful enantioselective transformations employing different strategies to induce asymmetry. 13g,37 In contrast, the utilization of chiral transient directing groups to this end is still in its infancy despite the indisputable advances made in the recent years. In 2012, in the course of their study regarding the Rh(I)-catalyzed intramolecular hydroacylation of disubstituted alkenes, Douglas and co-workers provided the first example applied to the enantioselective cyclization of the aldehyde 31. [START_REF] Beletskiy | Cooperative Catalysis Approach to Intramolecular Hydroacylation[END_REF] To this purpose, a chiral derivative of 2-amino-3-picoline (D) was used to achieve the in situ formation of the aldimine species from 31 (Scheme 12). Although a unique example was depicted with a modest enantioselectivity, this seminal work paved the way for further developments in catalytic asymmetric transformations using the chiral transient directing group strategy. Note that a theoretical mechanistic study was then published by Zhang, Lei and co-workers in 2014. [START_REF] Wang | A theoretical study on the mechanisms of intermolecular hydroacylation of aldehyde catalyzed by neutral and cationic rhodium complexes[END_REF] Scheme 12. Rh(I)-catalyzed enantioselective hydroacylation using a chiral TDG.

In 2019, Wang and co-workers took benefit from the use of cyclopentadienyl-containing Rh(III) catalysts for the asymmetric synthesis of phthalides. [START_REF] Li | Introducing the Chiral Transient Directing Group Strategy to Rhodium(III)-Catalyzed Asymmetric C-H Activation[END_REF] By employing a combination of the [Cp* ipr RhCl2]2 catalyst and the chiral amine E, a methodology was developed for the enantioselective homo-and hetero-coupling of benzaldehyde derivatives, offering an access to a large panel of chiral phthalides (yields up to 73% and enantiomeric excesses ranging from 61% to 99.9 %, Scheme 13). [START_REF] Tan | Rhodium(III)-Amine Dual Catalysis for the Oxidative Coupling of Aldehydes by Directed C-H Activation: Synthesis of Phthalides[END_REF] For compounds 35a-f, resulting from a homocoupling reaction, the best yields were obtained with phenyl-and alkylsubstituted aromatic aldehydes (33a and 33c) as well as benzaldehyde (33e). On the contrary, the presence of a CF3 group (33b) or halogens (33d and 33f) had a deleterious effect on the outcome of the reaction. Notably, when metasubstituted aldehydes reacted, the functionalization at the less hindered position occurred (35c-d). Regarding the heterocoupling reactions (36a-i), the observed selectivity stemmed from the fact that the transition metal-catalyzed C-H bond activation event was favored with substrates substituted with electron-neutral or electron-donating groups (alkyl, phenyl, OBn), which then reacted with aromatic aldehydes bearing electron-withdrawing substituents (halogens, CF3, SO2Me, CO2Et, NO2). Hence, a selective access to only one of the four potential products was possible in most cases. Moreover, heterocoupling of 2,4-dimethoxybenzaldehyde with aliphatic aldehydes was also achieved, leading to the corresponding products 36j-l with good to high enantioselectivities. Regarding the mechanism of the transformation, a plausible pathway was suggested to explain the observed enantioselectivities for both homo-and hetero-couplings. First, the precatalyst [Cp* ipr RhCl2]2 would react with the silver tetrafluoroboate to afford the active species [Cp* ipr Rh III ]. Then, the rhodacycle II formation would take place from the aldimine I via a reversible C-H activation event, followed by the coordination of the second coupling partner (intermediate III). Subsequently, the intermediate IV would be obtained after a stereoselective addition of the arene to the second aldehyde, followed by an intramolecular attack of the resulting alkoxide at the imine carbon. After a β-hydride elimination, Rh(I) species would be released, which after oxidation with the silver salt would regenerate the active Rh(III) catalyst along with the imine V. This latter would be easily hydrolyzed to provide the desired chiral phthalides 35 or 36 and the organo-catalyst. Scheme 13. Rh(III)-catalyzed preparation of chiral phthalides from aldehydes using a chiral TDG.

a Absolute configuration determined by X-ray diffraction.

In contrast to Rhodium based-catalysts, Ruthenium ones have been less employed in asymmetric C-H activation reactions. 21b,42 In 2019, Cui and co-workers reported the first Ruthenium-catalyzed synthesis of chiral indoline derivatives by means of the chiral transient directing group strategy. Using the [Ru(p-cymene)Cl2]2 catalyst combined with the chiral amine F and an additional chiral acid G, the enantioselective intramolecular hydroarylation of aromatic aldehydes 37 was achieved leading to N-Tosyl protected indoline derivatives with yields up to 92% and enantiomeric excesses between 69% and 96% (Scheme 14). [START_REF] Li | Ruthenium-Catalyzed Enantioselective C-H Functionalization: A Practical Access to Optically Active Indoline Derivatives[END_REF] Several benzaldehyde derivatives 37a-i bearing either an electron-donating (OMe, 37a, 37c, 37g) or an electron-withdrawing (CF3, 37e and 37h) group were functionalized with moderate to high yields. The substitution pattern on the aromatic aldehydes did not have a strong impact on the outcome of the reaction, except in the presence of a methoxy group at the ortho position, as the corresponding product 38g was isolated in only 34% yield with a high enantiomeric excess. The substitution over the alkene counterpart was also investigated (37j-r) and it turned out that various (E)-styrenyl groups containing both electron-donating and electron-withdrawing groups (OMe and F, among others) were suitable (37j-o). Moreover, alkenes substituted with different substituents (Me, CH2OTBDMS, CO2Et, 37p-r) were well tolerated. Note that the nitrogen-protecting group was not only restricted to tosyl, since the N-nosyl indoline 38s was synthesized. Notably, when both E and Z diastereoisomers of the same alkene were employed as substrates (37t-u), the same product 38t was obtained with high enantiomeric excesses (91% and 92%, respectively), showing that the alkene geometry did not have any influence on the stereocontrol of the transformation. The following plausible mechanism was suggested for the enantioselective synthesis of indoline scaffolds (Scheme 14). First, the active catalyst species (Ru(pcymene)(O2CR*)2) would be generated from (Ru(p-cymene)(Cl2)2) followed by the formation of the ruthenacycle II from the transient aldimine I through a reversible C-H activation step. Coordination of the alkene to the Ruthenium metal center would lead to the cationic intermediate III as an ion pair with chiral carboxylate G, crucial for the efficiency and the enantioselectivity of the reaction. Then, the insertion of the alkene would occur selectively through the less hindered face of the chiral imine (intermediate IV) presumably due to its conformationally rigid structure. Finally, the product 38f would be obtained after the hydrolysis of the aldimine V and F would be regenerated along with the Ruthenium-based catalyst. Scheme 14. Preparation of chiral indolines through an enantioselective Ru(II)-catalyzed intramolecular hydroarylation.

a Absolute configuration determined by X-ray diffraction.

In 2020, Wang and co-workers reported another application of the chiral transient directing group strategy in a Rucatalyzed transformation for the synthesis of 2,3-dihydrobenzofurans through an asymmetric intramolecular hydroarylation reaction. [START_REF] Li | Ruthenium(II)-Catalyzed Asymmetric Inert C-H Bond Activation Assisted by a Chiral Transient Directing Group[END_REF] To this end, the combination of [Ru(p-cymene)Cl2]2 catalyst with the chiral amine F and trifluoroacetic acid were found to be the best for this transformation. Several olefin-tethered aromatic aldehydes were smoothly converted into the corresponding 2,3-dihydrobenzofurans 40 with yields up to 98% and enantiomeric excesses up to >99% (Scheme 15). The reaction proceeded successfully with an unsubstituted aldehyde 39a as well as with aromatic aldehydes bearing halogens, electron-donating (eg. OMe, Me) and electron-withdrawing groups (NO2), either at the para-, meta-or orthoposition (39b-m). Nevertheless, the presence of a methoxy group at the ortho position led to a lower yield (40j). Substrates 39n-s, bearing various substituents at the allylic position were also tested, leading to the expected products 40n-s with very good yields and enantioselectivities, except for the chlorine-containing product 40q. Extension of their methodology to the synthesis of the indoline derivative 40u turned out to be feasible but less efficient (18% yield, 70% ee). A similar mechanism to the one depicted in the Scheme 14 was suggested for this transformation, although the use of a chiral acid was not required to improve yields and enantioselectivities. Scheme 15. Synthesis of chiral 2,3-dibenzofuranes through a Ru(II)-catalyzed enantioselective intramolecular hydroarylation.

a F (40 mol%), TFA (40 mol%).

Application of the chiral transient directing group strategy for miscellaneous reactions

In 2018, Xu, Jin and co-workers applied the chiral transient directing group strategy in the enantioselective C(sp 2 )-H arylation of ferrocenyl ketones by Palladium catalysis in order to obtain enantioenriched planar-chiral ferrocenes with yields up to 75%, enantiomeric excesses ranging from 92% to 98% and a ratio of mono-vs di-functionalized products between 88:12 and 99:1 (Scheme 16). [START_REF] Xu | Palladium-catalyzed enantioselective C(sp 2 )-H arylation of ferrocenyl ketones enabled by a chiral transient directing group[END_REF] It was found that the reaction of the acetylferrocene with the 1-iodo-4-nitrobenzene was successfully achieved using the L-tert-leucine as the transient directing group precursor leading to the corresponding product 42a. Both the pivalic acid and the sodium bicarbonate were essential for the efficiency and the enantioselectivity of the transformation. In particular, the pivalic acid had a dual function as it promoted the transient aldimine formation and played a key role in the rate determining C-H cleavage event. The arylation of 41 was also realized with an array of paraand meta-substituted aryl iodides (42b-i); electron-deficient ones being more efficient coupling partners (42b-e and 42h-i). Aryl iodides bearing electron-donating substituents (OMe, tBu) led to the expected products 42f-g in lower yields. Note that the reaction went smoothly with disubstituted aryl iodides containing one electron-withdrawing group as demonstrated with the compound 42j. Additionally, when heteroaryl iodides were used, the expected products 42k-l were obtained in moderate to good yields and high enantioselectivities (up to 97% ee). Notably, in all the cases, an excellent selectivity towards the monoarylation was observed. Substituents over the ferrocene ring were also tolerated, affording the corresponding arylated products 42m-n with moderate to good yields. Finally, the n-propyl ketone 41o led to the arylated product 42o in 40% yield and 92% enantiomeric excess. It is worth mentioning that a dimeric palladacycle intermediate was presumably formed in the course of the transformation. Scheme 16. Enantioselective Pd(II)-catalyzed C(sp 2 )-H arylation of ferrocenyl ketones.

Ratio of mono:di arylated products are indicated in parenthesis. a The absolute configuration was assigned to Rp on the basis of X-ray diffraction.

In 2020, Liu, Engle and co-workers reported another application of the chiral transient directing group strategy for the enantioselective Pd-catalyzed β-hydroarylation of alkenyl benzaldehydes. [START_REF] Oxtoby | Transient Directing Group Strategy Enables Enantioselective Reductive Heck Hydroarylation of Alkenes[END_REF] It is worth to mention that the enantioselective β-hydroarylation of substituted alkenyl arenes [START_REF] Saxena | Enantioselective rhodium-catalyzed arylation of electron-deficient alkenylarenes[END_REF] is still underdeveloped compared to the asymmetric α-hydroarylation reaction. [START_REF] Podhajsky | Asymmetric palladium-catalyzed hydroarylation of styrenes and dienes[END_REF] Using L-tert-leucine and in the presence of a hydride donor (TMA.HCO2), the Pd(0)-catalyzed reductive hydroarylation of alkenyl benzaldehydes with a panel of (hetero)aromatic iodides was achieved with yields up to 94% and enantiomeric excesses between 92% and >99% (Scheme 17). The reaction of the (E)-2-(prop-1-en-1-yl)benzaldehyde with several aryl iodides bearing electron-donating groups, electron-withdrawing ones and halogens led to the corresponding products 44a-i in moderate to excellent yields. Interestingly, various functional groups were tolerated such as a hydroxyl group (44b), a N-Boc-protected amine (44c) and a free carboxylic acid (44f). Heteroaryl iodides were suitable as well (44j-l), although the pyridine derivative 44l was synthesized with a very low yield. Regarding the alkene part, both E and Z isomers were hydroarylated and it turned out that E alkenes gave the best enantiomeric excesses but with lower yields (44m-n). Moreover, when the size of the substituent on the alkene part was increased, lower yields were generally obtained (44g vs. 44m). Finally, benzaldehydes with different substitution patterns were tested leading to the corresponding products 44o-u in good yields. Note that ortho-substituted benzaldehyde derivatives 43t-u were also functionalized. The authors suggested the following pathway for the enantioselective hydroarylation. First, aldimine I formation followed by the coordination to the Pd(0) would lead to the species II, which would undergo an oxidative addition with the aryl iodide to afford the intermediate III. Then, the enantioselective carbopalladation would take place (intermediate IV), which would be the key step for enantioinduction. Subsequently, the formate salt would coordinate to the metal center (intermediate V) and after a decarboxylation reaction, a Pd-H species VI would be obtained. A final reductive elimination would regenerate the Pd(0)-catalyst and would furnish VII, which after hydrolysis would release the product 44a along with L-tert-leucine (A). Scheme 17. Pd(0)-catalyzed enantioselective reductive Heck arylation of alkenyl benzaldehydes.

a From Z-alkene. b The absolute configuration was assigned through X-ray diffraction.

Enantioselective functionalization of C(sp 3 )─H bonds

Despite their lower reactivity in comparison with C(sp 2 )-H bonds, groundbreaking advances were made in the enantioselective functionalization of C(sp 3 )-H bonds by transition metal catalysis in the last decades. 1a,13g,h,k . Among the existing approaches employed, the use of chiral ligands in Pd-catalyzed directed C-H bond functionalization reactions was broadly exploited. [START_REF] Shi | Pd II -Catalyzed Enantioselective Activation of C(sp 2 )─H and C(sp 3 )─H Bonds Using Monoprotected Amino Acids as Chiral Ligands[END_REF] Therefore, the quest for new approaches to reach highly challenging asymmetric transformations was particularly attractive. In this context, although restricted so far to handful of examples, the use of the chiral transient directing group strategy turned out to be an efficient tool as highlighted in this section.

In 2016, Yu and co-workers pioneered the use of a chiral transient directing group for achieving the Pd-catalyzed enantioselective arylation of benzylic C(sp 3 )-H bonds. [START_REF] Zhang | Functionalization of C(sp 3 )-H bonds using a transient directing group[END_REF] In the course of their study related to the arylation of C(sp 3 ) centers of ketones and aldehydes by Pd-catalysis, the authors developed a methodology for the preparation of enantioenriched products by using L-tert-leucine (A) with yields up to 88% and enantiomeric ratios between 95:5 and 98:2 (Scheme 18). Taking benefit from the steric interactions between the bulky tert-butyl group of the L-tert-leucine and the benzylic R 2 group, good enantiomeric ratios were reached for this transformation. With this approach, an array of meta-and parasubstituted aryl iodides was successfully employed for the functionalization of the 2-ethyl-5-(trifluoromethyl)benzaldehyde. Halogens as well as different electron-withdrawing (CO2Me, NO2, CF3) and electron-donating groups (OMe) were well tolerated, as demonstrated with the synthesis of compounds 46a-i. Several ortho-and meta-substituted benzaldehydes were suitable substrates in the reaction with 4-fluoroiodobenzene and the corresponding products 46j-n were obtained in 60-71% yields and enantiomeric ratios up to 97:3. Note that the transformation turned out to tolerant to functional groups such as an ester (46j) and halogens (46l-n). Moreover, the substituent over the benzylic position (R 2 ) was not restricted to a methyl group since substrates bearing other alkyl chains were also smoothly functionalized (46o-p). Note that later in 2018, Dang and co-workers reported a theoretical study to get more insight about the regio-and stereoselectivity observed for the Pd-catalyzed arylation of aldehydes and ketones by C(sp 3 )-H activation using the chiral transient directing group strategy. [START_REF] Liu | How Does Palladium-Amino Acid Cooperative Catalysis Enable Regioand Stereoselective C(sp 3 )-H Functionalization in Aldehydes and Ketones? A DFT Mechanistic Study[END_REF] Scheme 18. Pd(II)-catalyzed enantioselective benzylic C(sp 3 )-H arylation of benzaldehyde derivatives using the L-tertleucine as the chiral transient directing group precursor.

a Reaction performed at 110 °C. b 5 mol% Pd(OAc)2, 10 mol% A.

In 2019, during their investigations towards the arylation of phenylacetaldehyde derivatives with aryl iodides under Palladium catalysis, the group of Chen and Zhou applied a similar methodology than the one depicted by the Yu group. [START_REF] Gou | Palladium-Catalyzed Site-Selective C(sp 3 )-H Arylation of Phenylacetaldehydes[END_REF] A single example of Pd(II)-catalyzed enantioselective arylation of the 2-bromo-α,α-dimethylbenzaldehyde 47 using the imine derived from L-tert-leucine as the transient directing group was achieved leading to the corresponding product 48 in 43% yield and an enantiomeric ratio of 70:30 (Scheme 19). Scheme 19. Pd(II)-catalyzed C(sp 3 )-H asymmetric arylation of the phenylacetaldehyde derivative 47.

In 2020, Yu and co-workers provided a key contribution to the field by developing the Pd-catalyzed enantioselective arylation of nonactivated secondary C(sp 3 ) centers. By employing a combination of Pd(OAc)2, D-valine (H), pyridone and Ag2PO3, the authors were able to perform the arylation of cyclobutyl ketones 49, providing the corresponding products 50a-s with yields up to 82% and enantiomeric ratios up to 99:1 (Scheme 20). [START_REF] Xiao | Pd II -Catalyzed Enantioselective C(sp 3 )-H Arylation of Cyclobutyl Ketones Using a Chiral Transient Directing Group[END_REF] Remarkably, by replacing Ag2PO3 by AgTFA, the products were obtained with a reversed enantioselectivity. Moreover, in this transformation, pyridone ligand, usually known for fastening the cleavage of C-H bonds, 4k,7f,11k,54 was crucial to achieve the reaction with high enantioselectivities. The arylation proved to be efficient with several para-and meta-substituted aryl iodides bearing electron-withdrawing groups such as CO2Me, NO2, Ac, CN, CF3 and COC6H5 (50a-g). Disubstituted electron-poor aryl iodides were also suitable coupling partners (50h-i). Although the functionalization was not efficient with electron-neutral or electron-rich iodoarenes, it went smoothly with heteroaromatic iodides (50k-l). Moreover, different cyclobutyl alkyl ketones were arylated at the α-position (50m-r) and only in the case of the ketone 49m, a competitive arylation of the methyl group of the alkyl chain was also observed. Note that even the spiro-derivative 50s was successfully functionalized. After mechanistic studies, a plausible pathway was suggested. First, coordination of the imine I derived from 49a to the palladium catalyst in the presence of the pyridone would afford the intermediate II, followed by the formation of the palladacycle III. This latter would undergo an oxidative addition with the aryl iodide (intermediate IV) and subsequently the iodine abstraction by the silver salt would take place (intermediate V). After reductive elimination, the catalyst would be regenerated and the ketimine VI would be obtained. A final hydrolysis of V would furnish the product 50a (or ent-50a) and H. Worth to mention that depending on the nature of the silver salt (AgTFA or Ag2PO3), either the C-H activation event or the iodine abstraction was suggested to be the rate determining step. Scheme 20. Pd(II)-catalyzed enantioselective C(sp 3 )-H arylation of cyclobutyl ketones.

a Using AgTFA instead of Ag3PO4, ent-50a was obtained in 34% yield (14:86 er) along with the corresponding diarylated product (50% yield). b D-tert-leucine was used as TDG precursor. c 1 equivalent of H was used. d Ratio of the products resulting from the arylation of the cyclobutane ring and the terminal methyl group.

The asymmetric formation of C(sp 3 )-F bonds has been achieved in the last two decades through different strategies. [START_REF] Yang | Advances in catalytic enantioselective fluorination, mono-, di-, and trifluoromethylation, and trifluoromethylthiolation reactions[END_REF] In sharp contrast, the transition metal-catalyzed enantioselective direct fluorination of a C(sp 3 )-H bond is a highly difficult and appealing task and needs to be further investigated. To tackle this synthetic challenge, Yu and co-workers developed a new methodology based on the Pd(II)-catalyzed enantioselective fluorination of C(sp 3 ) centers using a chiral transient directing group. [START_REF] Park | Controlling Pd(IV) reductive elimination pathways enables Pd(II)-catalysed enantioselective C(sp 3 )─H fluorination[END_REF] They anticipated that the design and use of a suitable ligand in this Pd(II)/Pd(IV) process would be crucial to successfully promote the C(sp 3 )-F bond formation in an enantioselective fashion over the side-competitive reductive eliminations (C-C or C-X bond formation) from Pd(IV) species, 57 and the undesirable SN2-type reactions over C(sp 3 )-Pd(IV)-F intermediates. 58 Instead of using a traditional amino acid, it was discovered that the combination of the amide I (derived from L-tert-leucine) along with 2,3,4,5,6-pentafluorobenzoic allowed the fluorination of the trifluoromethylated benzaldehyde derivative 51a on a secondary C(sp 3 ) center in the presence of N-fluoro-2,4,6trimethylpyridinium tetrafluoroborate as the fluorinating reagent (Scheme 21). A panel of aromatic aldehydes was functionalized leading to the expected products 52b-j in yields up to 61% and enantiomeric excesses ranging between 86-99%. Even though the competitive pathway that led to the formation of the undesired C(sp 3 )-O bond formation by an SN2type process could not be completely circumvented, moderate to excellent ratios in favor to the fluorinated products were obtained (5:1 to >20:1). Benzaldehydes bearing longer alkyl chains than an ethyl group at the ortho position were also suitable and furnished the best enantiomeric excesses and the best C(sp 3 )-F/C(sp 3 )-O ratio (>20:1), albeit with lower yields (52b-c). Moreover, benzaldehydes substituted with electron-withdrawing groups (CO2Me, COiPr, NO2, 51d-f) at the meta position were successfully functionalized. Although electron-rich substrates turned out to be inefficient in this transformation, the fluorination was achieved when the disubstituted benzaldehyde 51g was used. Note that the tetraline derivative 51h, the ortho-fluorinated aldehyde 51i and the pyridine 51j were also suitable substrates in this transformation. On the basis of mechanistic and literature data, 59 the authors suggested that a cationic Pd(IV) intermediate would be the key intermediate in this transformation and would favor the fluorination reaction over the C(sp 3 )-O bond formation. Scheme 21. Pd(II)-catalyzed enantioselective fluorination secondary C(sp 3 )-H bonds. Isolated yields were reported as a mixture of the product 52 with the starting material 51. The numbers in parenthesis indicated the ratio of products resulting from either a C(sp 3 )-F or C(sp 3 )-O bond formation and were determined by 1 H NMR analysis. a 1 H NMR yields were given due to the volatility of the products. b 1-Fluoro-2,4,6-trimethylpyridinium hexafluorophosphate (1.5 equiv) was used as the fluorine source. c CH2Cl2 was used instead of benzene. d 25 mol% of I were used.

SUMMARY

Over the past few years, transition metal-catalyzed enantioselective transformations by C-H bond activation have been developed as a powerful tool for the construction of interesting chiral molecules. In particular, the use of the chiral transient directing group strategy was appealing, offering potent synthetic solutions to the challenging asymmetric transformations. In this review, a general overview of the recent groundbreaking advances made in this field was given. Original methodologies based on the astute use of chiral transient directing groups for the enantioselective synthesis of biaryls, as well as chiral heterocycles and carbocycles were developed. In addition, this strategy was successfully applied to the challenging enantioselective C(sp 3 )-H bond functionalization. Even though major breakthroughs were achieved, this modern and sustainable strategy is still in its infancy. Indeed, so far only aldehyde and ketone derivatives were used as substrates to provide the chiral transient directing group by the reversible formation of an imine and in most cases, acidic reaction conditions were necessary; which hampered the general use of this strategy. To widen the scope of these transformations, the use of amine derivatives as substrates and more generally, the reversible formation of other functional groups playing the role of chiral transient directing groups are expected to be explored. Moreover, most of the transformations were limited to the formation of C-C or C-F bonds, although in the last case, only a single example was reported. Therefore, there is an urgent need to further explore other transformations. Finally, the enantioselective functionalization of unactivated aliphatic derivatives and the use of earth-abundant 3d transition metals were not achieved yet, and any advances using the chiral transient directing group strategy will bring significant breakthroughs. We do believe that this novel tool will be inspiring for the scientific community and milestones will be reached in the forthcoming years, expanding the chemical space for asymmetric transformations.

Scheme 4 .

 4 Scheme 4. Application of the transient directing group strategy to access a key intermediate 8 for the total synthesis of TAN-1085.

Acknowledgements

This work was partially supported by Normandie Université (NU), the Région Normandie, the Centre National de la Recherche Scientifique (CNRS), Université de Rouen Normandie (URN), INSA Rouen Normandie, Labex SynOrg (ANR-11-LABX-0029) and Innovation Chimie Carnot (I2C). M.I.L., S.M. and T.B. thank the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 758710).