Leandro Quadrana
email: leandro.quadrana@ens.psl.eu

Amanda Bortolini Silveira

Erwann Caillieux

Vincent Colot
email: vincent.colot@ens.psl.eu

Detection of transposable element insertions in Arabidopsis using sequence capture

Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape and consequences of transposition. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e. non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

INTRODUCTION

Transposable elements (TEs) are ubiquitous parasitic DNA sequences capable of self-propagating across genomes. Although designated by their discoverer Barbara McClintock in the 1940s as controlling elements because of their effect on genes, TEs have long been considered as mere selfish DNA. However, this situation has radically changed with the advent of genomics and TEs are now universally recognized as powerful engines of genome evolution. Nonetheless, despite the importance of TE mobilization in the creation of genetic novelty, the precise extent to which TE insertions can contribute to heritable phenotypic variation remains unknown. This lack of knowledge is largely due to the repetitive nature of TE sequences, which makes them less amenable than single copy DNA to genome-wide analyses, particularly using shortread sequencing technologies.. Moreover, TE mobilization is typically rare and therefore new TE insertions tend to be missed in small-scale population studies. To circumvent the difficulties to detect TE insertions de novo using short-read sequencing technologies, we have designed a TE-sequence capture method that concentrates the sequencing power on DNA sequences that immediately flank TE insertions [START_REF] Quadrana | The Arabidopsis thaliana mobilome and its impact at the species level[END_REF]. Here, we provide a detailed description of our TE-sequence capture design, which is aimed at enabling a comprehensive and cost-effective assessment of TE mobilization in A. thaliana (Figure 1).

Materials

Design of TE-sequence capture probes

1. Reference genome 2. High quality TE annotation 3. List of target TE families (i.e. potentially mobile TEs)

DNA Extraction and library preparation

1. High quality DNA extracted using CTAB method 2. 1.5 mL plastic tubes.

3. KAPA HTP/LTP library preparation kit (Roche, Ref. 07961871001) 4. Freshly prepared Ethanol 80% [START_REF]Place the tube in a magnet rack and once clear, remove the supernatant. Remove the tube from the magnet and resuspend the beads in 100 µl of 1X BWB[END_REF]. Oligos (see Table 1)

Oligo

Sequence (5'-3') searches among the set of Illumina short reads that cannot be completely mapped on the reference TAIR10 genome for so-called "split-reads" that contain TE extremities. Moreover, because most TE families generate short target site duplications (TSDs) of fixed size upon insertion, we looked for split-reads covering TE junctions that are absent from the reference genome and that produce, when mapped to the insertion site, a sequence overlap of the size of TSDs. An improved version of this bioinformatic approach can be found in [START_REF] Baduel | Efficient detection of transposable element insertion polymorphisms between genomes using short-read sequencing data. Methods in Molecular Biology[END_REF]. Overall, we identified non-reference TE insertions with TSDs for 131 TE families, of which 14 were not included in the TE-sequence capture design because of their very high copy numbers (>500 copies) in the reference A. thaliana genome, which would likely negatively impact the efficiency and evenness of the TE-sequence capture.

Based on these two complementary approaches, we considered a total of 318 reference TE copies belonging to 167 distinct families for TE-sequence capture. This list includes 75 families of LTR-retrotransposons, eight families of non-LTR retrotransposons, 65 families of DNA transposons and 19 families of helitrons (Supplementary Table 1). Probes covering the first and last 400bp of each of the 318 reference TE copies were chosen and produced by Roche using proprietary technologies. . Ultimately, these SeqCap Capture Oligos defined 634 capture targets, ranging from 53-322bp. Given the high sequence similarity between closely related TE families, TEs belonging to 181 TE families could in fact be captured with our design.

DNA Extraction and library preparation

This section describes the procedure to obtain high-quality genomic DNA and library preparation based on the CTAB method and KAPA HTP/LTP library preparation kit (Roche), respectively.

1. Extract DNA from plant material (1-2 g fresh weight, we use aerial parts of 3-week-old plants grown under long day conditions) with CTAB protocol [START_REF] Allen | A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide[END_REF] of Sample Library (from step 20) in a PCR tube. Amplify in thermocycler using the following program.

Step 1: 45 seconds at 98ºC

Step 2: 15 seconds at 98ºC

Step 3: 30 seconds at 60ºC

Step 4: 35 seconds at 72ºC

Step 5: Repeat steps 2-4 six times.

Step 6: 1 minute at 72ºC 17. Clean up amplified Library using a PCR purification kit, such as QIAquick PCR Purification kit (QIAGEN), and elute the sample in 50 µl of PCR grade water.

18. Quantify the concentration of the pre-captured amplified library using Nanodrop. If more than one sample will be pooled for multiplexing, quantify using Qubit and Qubit dsDNA HS Assay Kit according to the manufacturer indications.

TE-sequence capture, library amplification and sequencing

This section describes the procedure to capture TE sequences from a standard Illumina library prepared with the Kappa HTP/LTP kit (Figure 2). The TE sequence capture protocol presented here is based on a customized dual capture SeqCap EZ Library (Roche NimbleGen).

1. In case of multiplexing, mix together equal mass of each amplified DNA library to obtain a total combined mass of 1.25 µg. Up to 24 samples can be multiplexed. Step 1: 45 seconds at 98ºC

Step 2: 15 seconds at 98ºC

Step 3: 30 seconds at 60ºC

Step 4: 35 seconds at 72ºC

Step 5: Repeat steps 2-4 four times.

Step Step 1: 45 seconds at 98ºC

Step 2: 15 seconds at 98ºC

Step 3: 30 seconds at 60ºC

Step 4: 35 seconds at 72ºC

Step 5: Repeat steps 2-4 13 times.

Step may be calculated using the following formula:

𝐶𝐸 = 𝑒𝑓𝑓 &'()*& +,& -∏ / 0 𝑒𝑓𝑓 0102&'()*& 3 +,&, 0102&'()*& 3 6
where CE indicates the Capture efficiency, eff is the amplicon efficiency, ΔCt is the Ct difference between the post-capture and pre-capture samples, target and non-target indicate the capture and non-capture amplicons, respectively, and f represents the number of non-capture amplicons analyzed. A successful captured library has >500-fold enrichment.

Sequence 200pM of pooled library with 20% PhiX on Illumina sequencer, >76 cycles, paired-end reads. Note that 20 million paired-end reads per library is sufficient for most applications.

Bioinformatic analysis

The first step to identify non-reference TE insertions based on short-read sequencing data obtained by TEsequence capture is to detect informative reads partially mapping on the TE sequences covered by the probes. A multifasta file containing the complete set of captured TEs must be generated using the coordinates of full-length TE sequences annotated in the reference genome and the FastaFromBed command from Bedtools. This fasta file is used to builds a Bowtie2 index as detailed below:

FastaFromBed -i captured_TE_sequences.bed -g TAIR10.fa > captured_TE_sequences.fa Bowtie2-build captured_TE_sequences.fa captured_TE_sequences Detection of reads mapping partially over TE sequences.

After mapping short-reads on the collection of TE sequences, collect those reads mapping partially (with at least 20bp soft-clipped at 5' or 3' read's end).

###mapping pair-ends using bowtie2 in local mode (--local) and reporting as much as four equally possible mapping positions (-k 4).

Estimating sensitivity

This section describes how we calculated the sensitivity of our TE sequence capture approach using DNA extracted from plants with validated novel TE insertions.

In order to estimate empirically the sensitivity of our TE-sequence capture approach, we took advantage of a population of Arabidopsis TE accumulation (TEA) lines previously characterized using Illumina matepair libraries [START_REF] Quadrana | Transposition favors the generation of large effect mutations that may facilitate rapid adaption[END_REF]. The large physical distance (~5 kb) between mate-pair reads was used to determine the complete sequence of the insertions with high accuracy. We selected eight TEA lines carrying non-reference TE insertions for the LTR-retroelement ATCOPIA93 and we extracted DNA from four weeks-old plants as described in 3.2. Each DNA sample was serially diluted (from 1:50 to 1:6400) and then pooled together, leading to a differential representation of each DNA sample. The resulting pooled DNA was processed as indicated in 3.2 and 3.3. Data analysis was performed as in 3.4. The set of nonreference ATCOPIA93 insertions detected by TE-sequence capture was compared to the validated set of insertions previously identified using mate-pair WGS [START_REF] Quadrana | Transposition favors the generation of large effect mutations that may facilitate rapid adaption[END_REF]. TE-sequence capture identified 95% of ATCOPIA93 non-reference insertions for dilutions above 1:800, although the sensitivity of our TE-sequence capture drops <30% for dilutions below 1:800. Moreover, the number of reads supporting each ATCOPIA93 insertion strongly correlates with the dilution factor (Figure 2). Thus, he number of supporting reads is a reliable estimator of the TE insertion representation and can therefore be used to identify rare transposition events and to determine with reasonable accuracy the allele frequency of polymorphic TE insertions within populations of up to 1000 individuals.

Conclusions

We have described a comprehensive protocol for the detection of TE insertions in Arabidopsis using a custom-designed TE-sequence capture approach. This protocol uniquely combines all necessary steps from choosing target TE sequences, designing probes, performing sequence capture and analysing the short-read sequencing data. Thanks to the high sensitivity of TE-sequence capture, it offers a cost-effective option to investigate transposition as well as TE insertion polymorphisms (TIPs). Indeed, we have previously applied this protocol for the study of TIPs in a large panel of Arabidopsis natural accessions [START_REF] Quadrana | The Arabidopsis thaliana mobilome and its impact at the species level[END_REF] as well as of transposition in Arabidopsis TEA lines [START_REF] Quadrana | Transposition favors the generation of large effect mutations that may facilitate rapid adaption[END_REF]. As the sensitivity of TEsequence capture is determined by the number of target sequences and sequencing depth, this approach can readily be implemented for the study of transposition in species with large genomes, such as maize and wheat. Finally, because of the increasing interest in understanding the role of TEs in the generation of large effect mutations, we predict that TE-sequence capture will be a key component of genomic tool-kits in the future.

Notes

1-To prevent damage of oligos due to multiple freeze/thaw cycles, after resuspending the oligos in PCRgrade water they should be aliquoted into small volumes (20µl) and stored at -20°C.

  bowtie2 -x captured_TE_sequences -1 in_1.fastq -2 in_2.fastq -S in.sam --local --very-sensitive -k 4 samtools view -H in.sam > in-TE.sam ###collecting pair ends of reads with at least 20nt softclipped at 5' or 3' read's end.samtools view -F 4 -S $in.sam | awk '$6~/^[2-8][0-9]S/ || $6~/[2-8][0-9]S$/ || $6~/^1[0-9][0-9]S$/ || $6~/1[0-9][0-9]S$/ {print $0}' >> in-TE.sam ###collecting unmapped reads with the paired read mapped samtools view -f 4 -F 8 -S in.sam | awk '{print $0}' >> in-TE.sam samtools view -f 8 -F 4 -S in.sam | awk '{print $0}' >> in-TE.sam ### convert sam to bam samtools view -Sb in-TE.sam > in-TE.bam Identification of potential novel TE insertion sites ## extracting split-clipped mapping on specific TEs (i.e. ATCOPIA93) samtools view -F 4 in-TE.bam | grep ATCOPIA93 | awk '$6~/^[2-8][0-9]S/ || $6~/[2-8][0-9]S$/ || $6~/^1[0-9][0-9]S/ || $6~/1[0-9][0-9]S$/ {print $1"\t"$10"\t"$11}' | sort -k1,1 -k2,2 -u | awk '{print "@"$1"\n"$2"\n+\n"$3}' > in-ATCOPIA93-split.fastq ## extracting discordant-pair reads mapping on specific TEs (i.e. ATCOPIA93) samtools view -F 8 in-TE.bam | grep ATCOPIA93 | awk '{print $1}' | sort -u >> reads.name.disc samtools view -f 64 -u in-TE.bam > in-TE-disc-first.bam= samtools view -f 128 -u in-TE.bam > in-TE-disc-second.bam java -Xmx10g -XX:+UseSerialGC -Djava.io.tmpdir=./ -cp /usr/share/java/picard.jar net.sf.picard.sam.FilterSamReads INPUT=in-TE-disc-first.bam FILTER=includeReadList READ_LIST_FILE=reads.name.disc OUTPUT=in-ATCOPIA93-selected-disc-first.sam TMP_DIR=./ java -Xmx10g -XX:+UseSerialGC -Djava.io.tmpdir=./ -cp /usr/share/java/picard.jar net.sf.picard.sam.FilterSamReads INPUT=in-TE-disc-second.bam FILTER=includeReadListREAD_LIST_FILE=reads.name.disc OUTPUT=in-ATCOPIA93-selected-disc-second.sam TMP_DIR=./ cat in-ATCOPIA93-selected-disc-first.sam | awk '$1!~/^@/ {print $1"\t"$10"\t"$11}' | sort -u -k1,1 -k2,2 | awk '{print "@"$1"|1\n"$2"\n+\n"$3}' > in-ATCOPIA93-disc.fastq cat in-ATCOPIA93-selected-disc-second.sam | awk '$1!~/^@/ {print $1"\t"$10"\t"$11}' | sort -u -k1,1 -k2,2 | awk '{print "@"$1"|2\n"$2"\n+\n"$3}' >> in-ATCOPIA93-disc.fastq ##Mapping split-and discordant-reads on reference genome bowtie2 -x TAIR10_index -U in-ATCOPIA93-split.fastq, in-ATCOPIA93-disc.fastq -S in-ATCOPIA93-local.sam -local --very-sensitive samtools view in-ATCOPIA93-local.sam > in-ATCOPIA93-local.bam ##Identification of putative insertion sites BamToBed -i in-ATCOPIA93-local.bam -split > ATCOPIA93_bamtobed.bed mergeBed -i ATCOPIA93_bamtobed.bed -c 4 -o count_distinct -d 500 | awk '$4>1 && $3-$2<3000' > ATCOPIA93_putative_insertions.bed

Figure 1 .

 1 Figure 1. Description of the TE-capture workflow. Target TE sequences are indicated in red. After

Table 1 . List of oligos for TE-sequence capture

 1

	TS-PCR-1	AATGATACGGCGACCACCGAGA
	TS-PCR-2	CAAGCAGAAGACGGCATACGAG
	HE-Universal-oligo AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT
		TCCGATCT
	HE-Index-2-oligos	CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACG
		TGTGCTCTTCCGATCT

. Note that the sequence for only one HE-Index-2-oligos is provided. HE-Index-oligos for other indexes can be obtained by modifying the underlined sequence.

2.4. Bioinformatic analysis

1. Multifasta file with complete reference genome sequence 2. Desktop Linux workstation with at least 4Gb of memory and 4 CPUs. 3. Fasta file with a collection of TE sequences targeted by TE sequence capture 4. Bowtie2 v2.3.2 (available at https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/) 5. Samtools v1.2.1 (available at http://www.htslib.org/download/) 6. bedtools v2.29.3 (available at https://bedtools.readthedocs.io/en/latest/index.html) 7. Picardtools (available at https://broadinstitute.github.io/picard/)

. Design of TE-sequence capture probes

  This section describes the procedure used to select TE sequences to design capture probes. This protocol is based on the SeqCap EZ Prime Developer Probes application (Roche) and could be implemented for any species for which a reference genome sequence with well annotated TEs is available. The reference genome sequence of A.thaliana (TAIR10) contains ~32,000, mostly degenerate TE copies, which belong to 326 distinct families, split in equal parts into class I retrotransposons, which are mobilized using a "copy and paste" mechanism and class II DNA transposons, which transpose through a "cut and paste" mechanism

	(Ahmed et al., 2011; The Arabidopsis Genome Initiative, 2000). So far, transposition activity has been
	documented experimentally for eleven TE families, mainly based on studies carried out in the reference
	accession Col-0 (Ito & Kakutani, 2014; Tsay et al., 1993). To determine the set of TE sequences mobile or
	potentially mobile in A. thaliana we combined the results of two complementary approaches:
	-Bioinformatic analysis of consensus TE sequences
	A comprehensive repository of TE consensus sequences from diverse eukaryotic organisms is available
	through the Repbase Update database (Jurka, 2000; Jurka et al., 2005). These consensus sequences are
	routinely used in alignmentment-based algorithms (such as RepeatMasker) to detect and mask TEs from
	genome sequences. Out of 326 consensus sequences identified in A. thaliana, 83 contain intact open reading
	frames, suggesting that they are still capable of mobilization. Representatives of all 83 TE families, 31 of
	which were also detected as potentially mobile in the population genomics approach (see below), were
	included in the TE-sequence capture design.
	-Population genomics of TE insertion polymorphisms
	We used short-read resequencing data publically available for 211 natural accessions (Schmitz et al., 2013;
	Schneeberger et al., 2011) to detect TE insertions polymorphisms in nature, which may be indicative of
	recent transposition. To this end we deployed our SPLITREADER pipeline (Quadrana et al., 2016), which

  . 1.2 µg of DNA is needed for this protocol (includes sonication test). 2. Quantify DNA and place 1.1 µg in a final volume of 55 µL (complete with PCR-grade water if necessary). Sonicate to obtain fragment sizes around 300bp. We use a Covaris sonicator with Peak Incident Power 140, Duty Factor 10%, Cycles per burst 200 and time 100''. Remove the tubes from the magnet, resuspend the beads in 30µl of Elution Buffer (10mM Tris-Hcl pH 8.0) and incubate at room temperature for 2 minutes. Add 50µl PEG/NaCL SPRI solution and mix thoroughly by pipetting up and down until getting an homogenous solution. Incubate at room temperature for 10 minutes. 14. Repeat step 6. 15. Remove the tubes from the magnet, resuspend the beads in 25µl of Elution Buffer (10mM Tris-Hcl pH 8.0) and incubate at room temperature for 2 minutes. Place the tubes in the magnet and incubate until the solution is clear. Transfer the supernatant to a new tube and proceed with the pre-capture library amplification. 16. Mix 25µl of KAPA HiFi HotStart Ready Mix, 5µl of 100µM TS-PCR Oligo 1/2 (100 µM) and 20µl

	13.
	3. Run 100ng of sonicated and non-sonicated samples side by side in 1.5 % TAE gel. A smear should
	be visible between 200 and 600 bp.
	4. Prepare WGS Library for illumina sequencing without performing dual size selection. This protocol
	was optimized for KAPA HTP/LTP library preparation kit (Roche), however other library kits
	(such as Illumina TruSeq or NEB Nebnext) should also perform well. To prepare one library, mix
	8µl PCR-grade water, 7µl 10X KAPA End Repair Buffer and 5µl KAPA End Repair Enzyme. Add
	the 50µl of the fragmented DNA (from step 3.2.3). Mix and incubate at 20ºC for 30 minutes.
	9. Repeat step 6.
	10. Remove the tubes from the magnet and add 30µl of water, 10µl 5X KAPA Ligation Buffer, 5µl
	KAPA T4 DNA Ligase and 5µl of 15mM of TruSeq DNA Single Indexes adapter. Mix by pipetting
	up and down. Incubate at 20ºC for 15 minutes.
	11. Add 50µl PEG/NaCL SPRI solution and mix thoroughly by pipetting up and down until getting an
	homogenous solution. Incubate at room temperature for 10 minutes.
	12. Repeat step 6.

5. Add 120µl

Agencourt AMPure XP and mix by pipetting up and down several times. Incubate the tube at room temperature for 10 minutes. 6. Place the tube on a magnet until the liquid is clear and discard the supernatant. Wash the beads with 200µl ethanol 80% twice. Allow the beads to dry at room temperature and continue immediately to the next step. 7. Remove the tubes from the magnet and add 42µl of water, 5µl 10X KAPA A-Tailing Buffer and 3µl KAPA A-tailing Enzyme. Mix by pipetting up and down. Incubate at 30ºC for 30 minutes. 8. Add 90µl PEG/NaCL SPRI solution and mix thoroughly by pipetting up and down until getting an homogenous solution. Incubate at room temperature for 10 minutes.

  6: 1 minute at 72ºC 32. Combine the two PCR reactions and clean up using a PCR purification kit, such as QIAquick PCR Purification kit (QIAGEN), and elute the sample in 50 µl of PCR grade water.33. Quantify the library using NanoDrop. The library yield should be >500ng.

	34. Run 1µl post capture library on a Bioanalyzer DNA 1000 chip. A successfully constructed library
	should have an average fragment size between 150 and 500bp.
	35. Evaluate capture efficiency by qPCR pre-capture (step 3.2.23) and post-capture (3.3.37) libraries
	using primers spanning capture targets as well as non-captured sequences. Capture efficiency (CE)

Acknowledgements

We thank members of the Colot group for discussions, specially P. Baduel for critical reading of the manuscript. Support was from the Agence National de la Recherche (ANR-09-BLAN-0237, the Investissements d'Avenir ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL* Research University to V.C) and the Centre National de la Recherche Scientifique (MOMENTUM program, to L.Q.).

Figure 3. Sensitivity of TE sequence capture. a) Examples of TE insertions detected at different dilution factors. b) Number of supporting reads per insertion site as well as percentage of TE insertions detected.


Rama M Shakaroun

Hui Li

Philippe Jéhan

Marielle Blot

Ali Alaaeddine
email: alikassem.alaaeddine@ul.edu.lb

Jean-François Carpentier
email: francois.carpentier@univ-rennes1.fr

Sophie M Guillaume
email: sophie.guillaume@univ-rennes1.fr

Stereoselective Ring-Opening Polymerization of Functional β-Lactones: Influence of the Exocyclic Side-Group †

Keywords: cyclic ester, functional β-propiolactone, β-lactone, non-covalent interactions, polyester, poly(hydroxyalkanoate) (PHA), ring-opening polymerization (ROP), stereoselective catalysis, yttrium Graphical

FG s, and complementing NMR and MS spectra, TGA, DSC thermograms of the BPL FG s and PBPL FG s.

Introduction

Poly(β-hydroxyalkanoate)s (PHAs) are an important family of diversified natural and synthetic aliphatic polyesters renowned for their biodegradation in various environments. While natural PHAs are derived from biorenewable resources and produced by bacteria, synthetic PHAs are formed by ring-opening polymerization (ROP) of β-lactones. PHAs also differ one from another by the side-chain at the βposition of the repeating units along the backbone. Introduction of a pendant functional group (FG) along the polyester chains enables to access to chemically reactive PHAs and to polymers with different and tunable physico-chemical properties, thereby widening their range of applications. [START_REF]The Handbook of Polyhydroxyalkanoates[END_REF]2 Within a worldwide increasing market, PHA materials can partially substitute any of the traditional fossil-based polymer families, especially polypropylene (PP) and polyethylene (PE); thus, their uses range from injection molding, extrusion, thermoforming, 3D printing, to foams, non-wovens, fibers, coatings, glues, adhesives, and paints. Besides, owning to their biocompatibility and biodegradability, PHAs are also being used in pharmaceutical, biomedical, cosmetic and packaging applications. 3 PHAs are thus of topical interest especially as versatile green alternatives to single-use plastics with minimal environmental impact. 4 Natural PHAs are isotactic crystalline polymers, as exemplified with poly(3hydroxybutyrate) (PHB), the most ubiquitous PHA with a methyl group on the β-carbon, that displays a melting temperature (Tm) of 170-180 °C and a relatively low degradation temperature (ca. 256 °C), thus making difficult its processability and thereby somewhat limiting its commercial applications. 5 Several features have been reported to influence the thermal properties of PHAs. Besides the nature of the side-chain, tacticity is one of the most critical determinants. Isotactic or syndiotactic stereoregular polymers are typically crystalline materials that display improved thermal and mechanical properties relative to their atactic counterparts. Therefore, developing synthetic strategies toward stereoregular PHAs is of great interest.

In this regard, the stereoselective ROP of chiral cyclic esters is a highly topical research field. A challenging objective is the synthesis of functional PHAs with a microstructure different from the naturally occurring isotactic PHAs, namely the preparation of syndiotactic PHAs from readily available racemic β-lactone monomer(s).

Extensive research over the past decades has demonstrated that rare-earth complexes combined with tetradentate bis(phenolate) ligands are highly efficient catalysts/initiators for the controlled ROP of chiral cyclic esters/carbonates, thereby affording synthetic polyesters/polycarbonates with different tacticities. 6,7,8,9,10,11,12 In particular, achiral yttrium complexes with tripodal dianionic diamino-or aminoalkoxy-bis(phenolate) ligands {ONXO} 2-(X = NMe2, OMe) are, in the presence of an initiator (typically exogenous isopropanol), regarded as a "privileged" class of catalysts, due to their ability to promote the controlled ROP of heterocyclic monomers, their very high activity, and especially in light of their high stereoselectivity in the ROP of chiral cyclic esters such as of the ubiquitous racemic lactide, 12 or of racemic transcyclohexyl-ring-fused γ-butyrolactone (rac-GBL), 13 racemic trans-cyclohexene carbonate, 14 and of particular interest to our present research, the more reluctant-topolymerize four membered-ring racemic β-lactones (BPL FG ) 12,15,16 (Scheme 1). It was thus previously demonstrated that yttrium complexes incorporating bulky arylcontaining substituents at the ortho positions of the phenolate ligands enable to access highly syndio-enriched PBPL Me (i.e. PHB) from racemic β-butyrolactone (rac-BPL Me ) with a probability for syndiotactic enchainment (Pr = probability of racemic linkage; Pr = 1 for a perfectly syndiotactic polymer, Pr = 0 for an isotactic one, and Pr = 0.5 for an atactic one) up to 0.94. 12c These yttrium complexes also successfully promote the stereoselective ROP of higher functional cyclic esters such as the alkyl βmalolactonates (4-alkoxycarbonyl-β-lactones, BPL CO 2 R with R = allyl (All = CH2CH=CH2), benzyl (Bn = CH2Ph)). 15 Only the yttrium catalyst bearing small chloro, fluoro or methyl substituents installed at the ortho positions on the phenolate rings afford highly syndiotactic PBPL CO 2 R (Pr > 0.98), while the incorporation of sterically crowded alky/aryl substituents (typically CMe2t-Bu, CMe2Ph = cumyl, CPh3) on the aromatic rings provide significantly less syndio-enriched PBPL CO 2 R (Pr = 0.68-0.87).

Subsequently, the first highly stereoselective ROP of 4-alkoxymethylene-βpropiolactones (rac-BPL CH 2 OR s, R = Me, All, Bn) by such yttrium complexes giving PBPL CH 2 OR s was reported. 16 It was evidenced that a simple modification of the R ortho-substituents on the {ONXO R 2 } 2-platform of the yttrium catalyst from dicumyl to dichloro, induced a complete switch from a syndioselective (Pr up to 0.91) to an isoselective (Pm up to 0.93) polymerization of rac-BPL CH 2 OR s, respectively. DFT computations, focused on the first steps of these ROPs, attributed the origin of the isoselectivity to strong C-H … Cl "second-sphere" or "non-covalent" interactions (NCIs) between the methylene hydrogens in the ring-opened monomer of the growing chain (-CHCH2(OCH2CH=CH2)) and the chloro substituents of the ligand. Such NCIs arise from the attractive interactions induced by the halogen ortho-substituents on the phenolate ligand onto the methylene hydrogens of the last inserted monomer unit within the growing polymer chain. 8,12,15,16 In this contribution, we first report the synthesis of the unprecedented functional PBPL CH 2 ZPh s by the ROP of the corresponding newly synthesized racemic βpropiolactones BPL CH 2 ZPh s (except of BPL CH 2 OPh , vide infra) 17 , promoted by diamino-

O (R) O (S) O O n O (S) O (R) O O n R 2 R 1 O O R 1 R 2 Y X N N(Me2 HSi) 2 THF O (S) O (R) O O n O (S) O (S) O O O O O O CO 2 R O O OR CO 2 R CO 2 R RO RO RO RO 1 equiv iPrOH Toluene, 20°C rac-BPL Me rac-BPL CO2R rac-BPL CH2OR R = -CH 2 CH=CH 2 -CH 2 C 6 H 5 R = -CH 3 -CH 2 CH=CH 2 -CH 2 C 6 H 5 syndiotactic PBPL Me (aka PHB) syndiotactic PBPL CO2R syndiotactic PBPL CH2OR isotactic PBPL CH2OR or X = NMe 2 , OMe R 1 , R 2 = Cl, Br, Me, tBu, CMe 2 tBu, CMe 2 Ph, CPh 3 Y{ON(X)O R1,R2 } n (±) (±) (±)

Experimental section

Methods and materials

All manipulations involving organometallic catalysts were performed under inert atmosphere (argon, < 3 ppm O2) using standard Schlenk, vacuum line, and glovebox techniques. Solvents were freshly distillated from Na/benzophenone under argon and degassed thoroughly by freeze-thaw-vacuum cycles prior to use. Isopropyl alcohol (Acros) was distillated over Mg turnings under argon atmosphere and kept over activated 3-4 Å molecular sieves. Bisphenol {ONXO R 2 }H2 proligands and yttrium amide precursors of 1a-1g were synthesized according to the methods reported in the literature. 12b, 18 Racemic glycidyl phenyl ether (rac-G CH 2 OPh ) was dried onto and distilled from CaH2 and then stored over 3-4 Å activated molecular sieves (Sigma). MeOH solution of the cationizing agent (NaI, 10 mg mL -1 ) were prepared. The solutions were combined in a 1:1:1 v/v/v ratio of matrix-to-sample-to-cationizing agent.

The resulting solution (0.25-0.5 µL) was deposited onto the sample target (Prespotted AnchorChip PAC II 384 / 96 HCCA) and air or vacuum dried.

Chiral HPLC analysis of (S) or (R)-G FG s was performed on a Thermofisher Scientific chromatograph equipped with a Chiralcel-OD DAICEL column (250 mm × 4.6 mm, 5 µm) and a UV detector at 214 nm at 20 °C, using for (S)-G CH 2 OPh : hexane/isopropanol 90:10 v/v (1 mL.min -1 , 22 bar), (S)-G CH 2 SPh : hexane/isopropanol 99.5:0.5 v/v (0.9 mL.min -1 , 23 bar), and (R)-G CH 2 CH 2 OCH 2 Ph (0.9 mL.min -1 , 23 bar).

Thermal gravimetry analyses (TGA) were performed on a Metler Toledo TGA/DSC1 by heating the polymer samples at a rate of 10 °C min -1 from +25 to +600 °C in a dynamic nitrogen atmosphere (flow rate = 50 mL min -1 ).

Differential scanning calorimetry (DSC) analyses were performed with a Setaram DSC 131 apparatus calibrated with indium, at a rate of 10 °Cmin -1 , under a continuous flow of helium (25 mL.min -1 ), using aluminum capsules. The thermograms were recorded according to the following cycles: -80 to 200 °C at 10 °C min The carbonylation of (S)-phenyl glycidyl ether was performed similarly and gave (S)-BPL CH 2 OPh as an off-white solid (3.45 g, 74% yield) that displayed NMR spectra identical to those of rac-BPL CH 2 OPh . 17 Both rac-BPL CH 2 OPh and (S)-BPL CH 2 OPh were stored under argon in the fridge at -27 °C. Hz, 1He), 3.17 The carbonylation of (S)-G CH 2 SPh was performed similarly and gave (S)-BPL CH 2 SPh as a pale yellow viscous liquid (2.2 g, 47% yield) that displayed NMR spectra identical to those of rac-BPL CH 2 SPh . To prevent the degradation of rac-BPL CH 2 SPh and (S)-BPL CH 2 SPh (Figure S9), 17 they were both freshly prepared prior to use.

Carbonylation of racemic 2-((phenylthio)methyl)oxirane (rac-G CH2SPh ) to racemic 4-(phenylthio)methylene-β-propiolactone (rac-BPL CH2SPh

Carbonylation of racemic 2-(2-(benzyloxy)ethyl)oxirane rac-G CH2CH2OCH2Ph to racemic 4-(2-(benzyloxy)ethylene)-β-propiolactone (rac-BPL CH2CH2OCH2Ph ).

Following the aforementioned typical carbonylation procedure, starting from ESI-MS m/z observed=229.0836 vs m/z calculated = 229.0835 (Figure S11).

The carbonylation of (R)-G CH 2 CH 2 OCH 2 Ph was performed similarly and gave (R)-BPL CH 2 CH 2 OCH 2 Ph as a colorless liquid (1.5 g, 55% yield) that displayed NMR spectra identical to those of rac-BPL CH 2 CH 2 OCH 2 Ph . Both rac-BPL CH 2 H 2 OCH 2 Ph and (R)-BPL CH 2 CH 2 OCH 2 Ph were stored under argon in fridge at -27 °C.

Typical polymerization procedure. In a typical experiment (Table 1, entry 6), in the glovebox, a Schlenk flask was charged with [Y(N(SiHMe2)2)3](THF)2 (8.8 mg, 14 µmol)

and {ONNO tBu 2 }(1d, 7.4 mg, 14 µmol), and toluene (0.25 mL) was next added. To this solution, iPrOH (107 µL of a 1% (v/v) solution in toluene, 1 equiv vs. Y) was added under stirring at room temperature (ca. 20 °C). After 5 min of stirring, a solution of rac-BPL CH 2 OPh (150 mg, 0.84 mmol, 60 equiv) in toluene (0.5 mL) was added rapidly and the mixture was stirred at 20 °C for 1 h. The reaction was quenched by addition of acetic acid (ca. 0.5 mL of a 1.6 mol•L -1 solution in toluene). The resulting mixture was concentrated to dryness under vacuum and the conversion was determined by 1 H NMR analysis of the residue in CDCl3. The crude polymer was then dissolved in CH2Cl2 (ca.

1 mL) and precipitated in cold pentane (ca. 5 mL), filtered and dried. The PBPL CH2OPh , PBPL CH2SPh and PBPL CH2CH2OCH2Ph were recovered as white solid, yellow oil, and colorless oil, respectively. All recovered polymers were then analyzed by NMR spectroscopy, mass spectrometry, SEC, TGA and DSC analyses (Table 1, Figures 12345,S12-S25).

Results and discussion

The functional 4-substituted-β-propiolactones rac-BPL CH 2 ZPh s and the enantiopure (S)or (R)-BPL CH 2 ZPh s (Z = O, S, CH2OCH2) were first prepared in good yields (47-75%) on a multi-gram scale (1.5-5.3 g) from the carbonylation reaction of the corresponding epoxide rac-, (S)-or (R)-G CH 2 ZPh s, respectively, according to a typical procedure, 17 and next characterized by NMR spectroscopy and mass spectrometry (MS) analyses (Figures S1-S11). While the synthesis of BPL CH 2 OPh was already reported, 17 to our knowledge, its ring-opening homopolymerization has never been described. Only the random copolymerization of BPL CH 2 OPh with of BPL Me via anionic simultaneous ringopening copolymerization using the metal-free tetrabutylammonium acetate initiator was established to next study the resulting oligomers (Mn,SEC = 1200 g mol -1 ) by mass spectrometry. 23 In addition, for the first time to our knowledge, a functional β-lactone and PHA featuring a sulphur heteroatom within the exocyclic side-arm, namely BPL CH 2 SPh and PBPL CH 2 SPh , were prepared herein. Subsequently, the ROP of these original four-membered ring β-lactones was investigated, at room temperature in toluene, using the diamino-or amino-alkoxy-bis(phenolate) yttrium catalytic systems Y{ONXO R 2 }/(iPrOH) (1a-1g/(iPrOH)), the latter ones differing in terms of the phenolate ortho-substituents on the ancillary ligand (Scheme 2). Note that the influence of the para-substituents on the phenolate ligand of complexes 1 having been previously

shown not to significantly contribute to the performance (especially the activity and the stereoselectivity) of the yttrium catalysts/initiators, they were not considered further in the present studies. 12,15,16 These active catalysts were conveniently generated in situ upon addition of 1 equiv. of an exogeneous iPrOH, added as co-initiator to the mixture of the proligand and the yttrium amido precursor [Y(N(SiHMe2)2)3](THF)2, respectively (Scheme 2). 15,16 The kinetics of the NMR-scale ROP of rac-BPL CH 2 ZPh s was monitored by 1 H NMR spectroscopy. The most significant data for the thus synthesized PBPL CH 2 ZPh s are gathered in Tables 1 andS1. The macromolecular characteristics, and especially the microstructure of the recovered PBPL CH 2 ZPh s polyesters, were thoroughly assessed by 1 H, 13 polystyrene standards. g Pr is the probability of racemic linkages between BPL CH2ZPh units as determined by 13 C{ 1 H} NMR analysis of the isolated PBPL CH2ZPh s. h Glass transition temperature as determined by DSC. i Refer to the kinetic study (Table S1); j Not determined.

ROP of rac-BPL CH2ZPh s (Z = O, S, CH2OCH2) promoted by 1a-1g/(iPrOH) catalyst systems: insights into the catalyst activity and the kinetics

Yttrium catalyst systems based on ligands with small Me or Cl ortho-phenolate substituents (1a-1c), in the presence of iPrOH, proved poorly active regardless of the rac-BPL CH 2 ZPh , presumably due to the aggregated/dimeric nature of these metal complexes (Table 1, entries 1-4, 14-18,28-31). 8b The Me-substituted catalyst systems 1a,1b/(iPrOH) could achieve near complete conversion of ca. 30 monomer equiv. in a few hours (99-100% yield, i.e. 25-30 equiv. in 2-7.5 h), unlike the Cl-substituted one 1c which hardly proceeded to complete monomer consumption (57-95% yield, i.e. 17-28 equiv. in 14-60 h) (Table 1, entries 1,14,29;3,17,31). Correspondingly, the best activity achieved by the catalysts as expressed by turnover frequency (TOF) values remained sluggish: TOF1a-b = 4.8-15 h -1 vs TOF1c = 1.0-1.4 h -1 (Table 1 , entries 2,15,30;3,16,30). This poor activity of uncrowded alkyl or halogenated catalysts 1a-1c/(iPrOH) contrasted with the moderate-to-high activity recorded in the alike ROP of BPL CH 2 OAll/Bn (TOF = 20-30 h -1 ) or BPL CO 2 R (TOF = 1400 h -1 ) under the same operating conditions. 8b,15,16 On the other hand, yttrium catalyst systems with ancillary ligands bearing bulky substituents ( t Bu, cumyl, 1d-1g/(iPrOH)) revealed quite active for the ROP of rac-BPL CH 2 ZPh s, with the cumyl substituted derivatives being most efficient (Table 1, entries 5- 13,19-27,32-40). Nearly quantitative conversion of ca.

98-120 equivalents of rac-BPL CH 2 ZPh by 1d-1g was reached within 6-15 min (Table 1, entries 7,12,21,26,35,38). The overall slightly faster rate of polymerization observed for BPL CH 2 OPh as compared to BPL CH 2 SPh (Tables 1,S1; Figure 1), regardless of the catalytic system, most likely arose from the relative instability of BPL CH 2 SPh at 20 °C, the secondary products formed (products 2 and 3; Figure S9) presumably impeding the efficiency of the catalyst, hence altering the polymerization rate. Furthermore, the highest TOF values obtained for the ROP of BPL CH 2 OPh/CH 2 SPh/CH 2 CH 2 OCH 2 Ph promoted by the catalytic systems 1d-1g/(iPrOH) (450 < TOF (h -1 ) < 1840; Table 1, entries 7,9,12,21,26,34-39) were significantly higher than those reported for the closely related alkoxymethylene β-propiolactones BPL CH 2 OR s (R = Me, Bn, All) similarly polymerized by these same yttrium catalytic systems (TOF = 54-100 h -1 ), 8b,16 yet lower than those obtained from BPL CO 2 R monomers with pendant ester side-chains (R = Bn, All; TOF > 3000 h -1 ). 15b The overall trend for the monomers' ROP ability is thus BPL CH 2 OPh ≈ BPL CH 2 SPh > BPL CH 2 CH 2 OCH 2 Ph , while the catalysts' activity thus generally followed the order 1d-1g >> 1a ≈ 1b >> 1c, as previously reported for the previous ROP of various BPL FG s β-lactones (FG = Me, CH2OR, CO2R) promoted by these catalyst systems. 7,15,16 Monitoring of NMR-scale polymerizations of BPL 4.25 ± 0.43 × 10 -2 min -1 , BPL CH 2 CH 2 OCH 2 Ph /1c; 1.2 ± 0.1 × 10 -2 min -1 , BPL CH 2 SPh /1c

(Tables 1,S1, Figure 1). (Table 1, entries 2,15; Table S1, entry 1); 1c (Table 1, entries 4,18; Table S1, entry 2); plots for the ROPs mediated by 1d (Table 1, entries 6,20; Table S1, entry 3) and 1f

(Table 1, entries 11,25; Table S1, entry 4), all overlap due to similar high activity of these catalysts regardless of the monomer, and are represented as .

All the PBPL CH 2 ZPh s isolated showed, regardless of the catalytic system used, a quite good agreement between the theoretical molar mass values (Mn,theo) and the experimental values determined by NMR (Mn,NMR) and by SEC (Mn,SEC) analyses. The latter experimental molar mass values of the PBPL CH 2 ZPh s increased linearly with the BPL CH 2 ZPh monomer loading up to a degree of polymerization of ca. 450, as illustrated for the ROP of BPL CH 2 ZPh mediated by the 1d or 1g/(iPrOH) (1:1) catalytic system (Figure 2; Table S1). The dispersities of all PBPL CH 2 ZPh s remained generally narrow (Table 1, entries 5-9; 19-23;36-39). Motivated by the outstanding stereoselectivity of the achiral Y{ONXO R 2 }/(iPrOH) catalyst systems 1a-1g/(iPrOH) in the ROP of chiral β-lactones, 8a,8b,15,16 the stereoregularity of the herein synthesized PBPL CH 2 ZPh s was closely examined. Most commonly, the stereocontrol in the ROP of chiral cyclic esters mediated by achiral metal catalyst systems originates from a so-called chain-end stereocontrolled mechanism (CEM) in which the chirality of each next incoming monomer unit is tuned by the chirality of the last monomer unit inserted into the growing polymer chain. 8 Thus, syndiotactic polymers are most often formed from CEM, since the active species alternatively selects from a racemic monomer, the enantiomer with the opposite configuration in order to minimize the steric bulkiness in the transition state.

Macromolecular characterizations of PBPL

The tacticity of the recovered PBPL CH 2 ZPh s was thus assessed from 13 S2,S3).

First, no significant difference in terms of stereoselectivity of PBPL CH 2 ZPh s was observed to arise from the X donor group in the ''capping'' moiety of the {ONXO R 2 } 2- ligand, similarly to the previous alike ROP of BPL FG s with FG = Me, CH2OR, C(O)OR. 8 Regardless of the catalyst system implemented, exchanging oxygen with sulphur in FG = CH2ZPh (Z = O, S, i.e. BPL CH 2 OPh vs BPL CH 2 SPh ) did not affect the stereochemistry of the resulting syndiotactic PBPL CH 2 ZPh s. This was different with PBPL CH 2 CH 2 OCH 2 Ph which revealed either syndiotactic or atactic, depending on the yttrium complex used, namely 1d-1g or 1a-1c, respectively. Generally, 13 C NMR spectra of the PBPL CH 2 ZPh s indicated that when the yttrium complex incorporated bulky substituents at the ortho-positions of the phenolate rings (tBu or cumyl groups, 1d-1g), syndio-enriched PBPL CH 2 ZPh s (Pr up to 0.77-0.87) were produced from rac-BPL CH 2 ZPh s, (Table 1, entries 5-12, 19-26,32-39). In comparison, similarly highly syndiotactic PBPL CH 2 OCH 2 Ph s were reported from rac-BPL CH 2 OCH 2 Ph (Pr = 0.88-0.90). 16a The ROP of the β-lactones promoted by non-crowded non-halogenated yttrium catalysts, typically Me-substituted phenolate catalysts 1a-1b, provided atactic PBPL FG s, except with BPL CH 2 OPh/CH 2 SPh and the β-malolactonate monomers 15 which returned syndio-enriched-to-highly syndiotactic polymers, respectively. This latter S22). DSC thermograms only showed the presence of a glass transition temperature (Tg); no melting temperature was observed below 200 °C, suggesting amorphous polyesters (Tables 1,S2,S3; Figures S23-S25). This lack of crystallinity was similarly reported for the closely related syndiotactic or isotactic PBPL CH 2 OCH 2 Ph , while a melting temperature was only observed for the syndiotactic PBPL CH 2 OMe/All (Tm = 116, 85 °C) and PBPL CO 2 All/Bn (Tm = 51-117 °C). 15b,16a The Tg values were similarly observed to be closely related to the nature of the functional side-chain of the PHAs. The PBPL CH 2 OPh/CH 2 SPh displayed positive Tg values going from 8 to 14 °C for the phenoxymethylene substituent, up to 21 to 40 °C for the corresponding thiophenoxymethylene. On the other hand, the PBPL CH 2 CH 2 OCH 2 Ph polyester with the longer benzyloxyethylene pendant moieties, that most likely impart more mobility to the macromolecular chains, showed the lowest Tg (typically Tg = -12 °C)and Td as wellvalues.

The influence of the stereoenrichment of PBPL CH 2 ZPh s on Tg showed, as anticipated, higher Tg values for the most syndiotactic polymers as compared to the less syndioenriched ones (Tables 1,S1; Figures S23-S25). Typically, for PBPL CH 2 OPh s with Pr = 0.75-0.79, the Tg values varied from 21 to 30 °C, while a higher syndio-enrichment with Pr = 0.83-0.87 or an almost purely isotactic sample (Pr < 0.05) resulted in higher Tg values ranging from 36 to 40 °C. The same trend was observed with PBPL CH 2 SPh s that displayed Tg values varying from 8-9 °C for samples with Pr = 0.73-0.76, while more syndio-enriched polymer samples with Pr = 0.83-0.87 or an almost purely isotactic sample (Pr < 0.05) gave higher Tg values ranging from 12 to 14 °C. The trend is, as expected, similar to the previously reported thermal behavior of the related PBPL CH 2 OCH 2 Ph that showed a Tg value going from -6 °C to -2 and to 0 °C upon improving the stereoregularity of the polyesters from atactic (Pr ca. 0.50) to syndiotactic (Pr ca. 0.85 -0.90) and isotactic (Pr ca. 0.10), respectively. 16 No distinct tendency in the Tg values of PBPL CH 2 CH 2 OCH 2 Ph samples was however observed.

Conclusion

The unprecedented functional PBPL CH 2 ZPh s with Z = O, S, CH2OCH2 have been easily

and successfully prepared from the controlled ROP of the corresponding racemic 4substituted-β-propiolactones rac-BPL CH 2 ZPh s, mediated by diamino-or amino-alkoxybis(phenolate) yttrium amido complexes 1a-1g in the presence of isopropanol as coinitiator. All yttrium complexes revealed active, affording well-defined high molar mass PHAs with a good control of the polymerization in terms of molar mass values and limited undesirable side reactions (Mn,NMR up to 86,400 g mol -1 , ÐM < 1.23).

The best activities reached by the yttrium complexes 1d-1g with the most crowded substituents installed on the tetradentate bisphenolate ligand (e.g. tBu, CMe2Ph), in the ROP of these β-lactones (typically 225 < TOF (h -1 ) < 1840), revealed significantly higher than those achieved with the related rac-4-alkoxymethylene-β-propiolactones BPL CH 2 OMe/All/Bn (1 < TOF (h -1 ) < ca. 100), 16 yet lower than the record value established with the ROP of rac-β-malolactonates BPL CO 2 R (TOF > 3000 h -1 ). 15 On the other hand, catalysts with less-crowded phenolate substituents (e.g. Me, Cl) revealed significantly less active in the ROP of rac-BPL CH 2 ZPh s, with the chloro derivative 1c being sluggish, as previously observed in the ROP of BPL CH 2 OAll/Bn . 8b

Highly syndiotactic PBPL CH 2 OPh and PBPL CH 2 SPh s were always obtained (Pr up to 0.87), unrelatedly to the O or S heteroatom and regardless of the yttrium catalyst, and similarly to syndiotactic PBPL Me , PBPL CH 2 OMe/All/Bn , and PBPL CO 2 All/Bn . 8,12,15,16 On the other hand, the simple modification of ortho-R substituents on the {ONOO R 2 } 2-ligand platform from uncrowded to crowded, allowed a complete reversal from a nonselective (Pr ca.0.50 with R = Cl, Me) to highly syndioselective (Pr up to 0.86 with R = cumyl, ter-butyl) ROP of BPL CH 2 CH 2 OCH 2 Ph , respectively. The intimate relationship between the chemical nature of the exocyclic functional side-group on the β-lactone and the stereoelectronic tuning arising from the phenolate ligand ortho-substituents on the yttrium coordination sphere, is evidenced in the control of the stereoselectivity of the ROP through the implementation -or not -of NCIs. These achiral rare earth catalyst systems thus afforded a remarkable stereocontrol in the ROP of original functional racemic β-lactones, among which the first sulphur-substituted PHAs, ultimately providing PHAs with tunable thermal properties, and further highlighting their valuable potential.

Conflicts of interest

There are no conflicts to declare.

  Scheme 2. Stereoselective ROP of chiral 4-substituted-β-propiolactones rac-BPL CH 2 ZPh s (Z = O, S, CH2OCH2) catalyzed by diamino-or amino-alkoxybis(phenolate) yttrium complexes Y{ONXO R 2 } 1a-1g in the presence of iPrOH, investigated in the present work.

[

  Int.PBPL(CH2SPh) + Int.(2) + Int.(3)] / [Int.PBPL(CH2SPh) + Int.BPL(CH2SPh) + Int.(2) + Int.(3)] (compounds 2 and 3 correspond to the decomposed and rearrangement products of rac-BPL CH 2 SPh , respectively; vide infra) of the methine hydrogens of BPL CH 2 ZPh and PBPL CH 2 ZPh (corresponding methine hydrogen signal of the polymers (see above), and of the monomers: δ (ppm) 4.85 BPL CH 2 OPh , 4.62 BPL CH 2 SPh , and 4.71 BPL CH 2 CH 2 OCH 2 Ph ). Mass spectra were recorded at CRMPO-ScanMAT (Rennes, France). ESI mass spectra were recorded on an orbitrap type Thermofisher Scientific Q-Exactive instrument with an ESI source in positive mode by direct introduction with a flow rate of 5-10 µL min -1 . Samples were prepared in CH2Cl2 at 10 µg mL -1 . High resolution Matrix Assisted Laser Desorption Ionization -Time of Flight, MALDI-ToF, mass spectra were recorded using an ULTRAFLEX III TOF/TOF spectrometer (Bruker Daltonik Gmbh, Bremen, Germany) in positive ionization mode. Spectra were recorded using reflectron mode and an accelerating voltage of 25 kV. A mixture of a freshly prepared solution of the polymer in THF or CH2Cl2 (HPLC grade, 10 mg mL -1 ) and DCTB (trans-2-(3-(4-tert-butylphenyl)-2methyl-2-propenylidene) malononitrile, and a

- 1 ; 1 .

 11 200 to -80 °C at 10 °C min -1 ; -80 °C for 5 min; -80 to 200 °C at 10 °C min -1 ; 200 to -80 °C at 10 °C min -Procedure for the carbonylation of the epoxides into the corresponding βlactones 17Carbonylation of phenyl glycidyl ether (rac-G CH2OPh ) into racemic 4phenyloxymethylene-β-propiolactone (rac-BPL CH2OPh). In a typical experiment, in the glovebox, a Schlenk flask was charged with [Salph(Cr(THF)2)][Co(CO)4] (235 mg, 0.26 mmol). On a vacuum line, dry DME (15 mL) was syringed in and the resulting solution was cannulated into a degassed high-pressure reactor, which was pressurized with carbon monoxide to 20 bars, and stirred for 15 min before depressurization. A solution of racemic-phenyl glycidyl ether (rac-2-(phenyloxymethyl)oxirane) (rac-G CH 2 OPh ) (3.88 g, 25.9 mmol, 100 equiv.) in dry DME (15 mL) was transferred into the reactor, which was then pressurized with CO to 40 bars. The reaction mixture was stirred for 2 days at 20 °C. The reactor was then vented to atmospheric pressure, volatiles were removed under vacuum and the crude product was purified through a silica column (CH2Cl2, 2 × 300 mL). Evaporation of volatiles afforded rac-BPL CH 2 OPh as a white solid that was crystallized from diethyl ether to give pure 4phenoxymethylene-β-propiolactone (rac-BPL CH 2 OPh ) (3.5 g; 75% yield).[START_REF]The Handbook of Polyhydroxyalkanoates[END_REF] H NMR (500 MHz, CDCl3, 25 °C) δ (ppm): δ 7.34-7.28 (m, 2Hg), 7.03-6.98 (m, 1Hh), 6.95-6.91 (m, 2Hf), 4.88-4.81 (m, 1Hc), 4.33 (dd, J = 11, 3 Hz, 1He), 4.23 (dd, J = 11, 4 Hz, 1Hd), 3.62-3.53 (m, 2Hab) (Figure S6-top). 13 C NMR (125 MHz, CDCl3, 25 °C) δ (ppm): 167.3 (C1=O), 158.1 (ipso-C5), 129.8 (m-C7H), 121.9 (p-C8H), 114.8 (o-C6H), 68.4 (C3HOC(O)), 67.4 (C4H2OPh), 40.2 (C2H2C(O)O) (Figure S6-bottom).

  -3.11 (m, 2Hab) (Figure S7-top). 13 C NMR (101 MHz, CDCl3, 25 °C) δ (ppm): 167.3 (C1=O), 133.9 (ipso-C5), 131.2 (o-C6H), 129.4 (m-C7H), 127.7 (p-C8H), 68.9 (C3HOC(O)), 43.0 (C4H2SPh), 38.0 (C2H2C(O)O) (Figure S7-bottom). ESI-MS m/z observed = 217.0293 vs m/z calculated = 217.0299 (Figure S8).

Figure 1 .

 1 Figure 1. Semi-logarithmic first-order plots for the ROP of rac-BPL FG s mediated by 1a,1c,1d,1f/(iPrOH) (20 °C, toluene; [BPL CH 2 ZPh ]0/{[1]0/[iPrOH]0} = 60:1:1): 1a

  1.05-1.23), supporting a relatively fast initiation (compared to propagation) along with some limited undesirable side reactions (classically inter-and intramolecular transesterification reactions (i.e. reshuffling and backbiting, respectively), or other transfer or termination reactions). Such dispersity values fell within the range of those typically obtained for the related racemic 4-substituted-β-propiolactones BPL FG s.8,15,16 All these characteristics highlighted the controlled feature and to some extent the livingness of the ROP of the BPL CH 2 ZPh s mediated by 1a-1g/(iPrOH) catalytic systems.

Figure 2 .

 2 Figure 2. Variation of Mn,NMR , Mn,SEC , and Mn,theo (solid line) values of PBPL CH 2 ZPh s synthesized from the ROP of rac-BPL CH 2 ZPh s mediated by 1d or 1g/(iPrOH) (1:1) catalytic system as a function of the BPL CH 2 ZPh monomer loading

  CH2ZPh s synthesized by ROP of rac-BPL CH2ZPh s (Z = O, S, CH2OCH2) promoted by 1a-1g/(iPrOH) catalyst systems The PBPL CH 2 ZPh s were unambiguously characterized by 1D and 2D NMR spectroscopy and mass spectrometry (Tables 1,S1; Figures 3-5,S12-S18). All 1 H and 13 C{ 1 H}and J-MOD NMR spectra clearly displayed the characteristic signals corresponding to both the BPL CH 2 ZPh repeating units, especially the backbone methine and methylene signals, and the typical pendant CH2ZPh moieties, respectively (refer to the Experimental Section). The distinctive isopropoxycarbonyl chain-end group resonances were also clearly observed (δ (ppm): ca. 4.95 (CH3)2CHO-, ca. 1.19 (CH3)2CHO-), supporting the propagation from the Y{ONXO R 2 }/(iPrOH) active species (Figures 3-5,S12-S18).

Figure 3 . 1 H

 31 Figure 3. 1 H NMR (500 MHz, CDCl3, 25 °C) (top) and J-MOD (125 MHz, CDCl3, 25 °C) (bottom) spectra of syndiotactic PBPL CH 2 OPh prepared from the ROP of rac-BPL CH 2 OPh mediated by complex 1d in the presence of iPrOH and precipitated twice in cold pentane (Table1, entry 6) (*: residual grease).

Figure 4 . 1 H

 41 Figure 4. 1 H NMR (500 MHz, CDCl3, 25 °C) (top) and J-MOD (125 MHz, CDCl3, 25 °C) (bottom) spectra of syndiotactic PBPL CH 2 SPh prepared from the ROP of rac-BPL CH 2 SPh mediated by complex 1f in the presence of iPrOH and precipitated twice in cold pentane (Table 1, entry 26) (*: minor peaks correspond to the degradation product 3 of BPL CH 2 SPh ).

Figure 5 . 1 H

 51 Figure 5. 1 H NMR (500 MHz, CDCl3, 25 °C) (top) and J-MOD (100 MHz, CDCl3, 25 °C) (bottom) spectra of syndiotactic PBPL CH 2 CH 2 OCH 2 Ph prepared from the ROP of rac-BPL CH 2 CH 2 OCH 2 Ph mediated by complex 1b in the presence of iPrOH and precipitated twice in cold pentane (Table 1, entry 29).

  stereocontrol might then originate from the coordination to the yttrium center of the heteroatom (O, S) atom of the phenoxide (CH2OPh), thiophenoxide (CH2SPh) or alkoxycarbonyl (C(O)OR) of the side-group of the last inserted monomer unit of the growing chain, thereby forming a highly coordinated -and thus bulkier and more prone to stereoselectivity -rare earth active species. Finally, when implementing crowded substituted phenolate ligands, i.e CMe2Ph and tBu, onto the yttrium active center 1d-1g/(iPrOH), syndiotactic polymers were commonly produced regardless of the BPL FG exocyclic substituent (FG = Me, CH2OPh, CH2SPh or CH2CH2OCH2Ph, CO2All/Bn, CH2OMe/All/Bn), in line with a CEM mechanism being at play. The results we report herein thus further highlight the strong dependence of the resulting PHA's stereocontrolled microstructure on the couple formed by the functional β-lactone and the yttrium catalyst, and more specifically on the chemical nature of the β-lactone side-arm and of the substituents on the metal surrounding ancillary.

Figure 6 .

 6 Figure 6. Regions of the 13 C{ 1 H} NMR spectra (125 MHz, CDCl3, 23 ˚C) of PBPL CH 2 ZPh s prepared by ROP of rac-BPL CH 2 ZPh s (Z = O, S, CH2OCH2), except for the

Table 1 .

 1 C{[START_REF]The Handbook of Polyhydroxyalkanoates[END_REF] H} and 2D (COSY, HMBC) NMR spectroscopy (Tables1,S1; FiguresS12-S18), MALDI-ToF MS (FiguresS19-S21), SEC (Tables1,S1), TGA and DSC (Table1; FiguresS22-S25) analyses. Characteristics of the PBPL CH 2 ZPh s synthesized by ROP of rac-BPL CH 2 ZPh s with

		Z = O, S, CH2OCH2 mediated by 1a-1g/(iPrOH) catalytic systems in toluene at room
		temperature.
	Entry	BPL CH2ZPh	Cat.	[BPL CH2ZPh ]0/ [1]0/[iPrOH]0 a	Time b (min)	Conv. c (%)	Mn,theo d (g.mol -1 )	Mn,NMR e (g.mol -1 )	Mn,SEC f (g.mol -1 )	ÐM f	Pr g	Tg h (°C)
	1	CH2OPh	1a	30:1:1	120	100	5400	5200	6200	1.18 0.76	nd j
	2 i	CH2OPh	1a	60:1:1	240	95	10 200	10 000	11 100	1.13 0.79	30
	3	CH2OPh	1c	30:1:1	30 h	95	5100	5000	5200	1.14 0.77	22
	4 i	CH2OPh	1c	60:1:1	50 h	54	5800	5400	6200	1.15 0.75	21
	5	CH2OPh	1d	20:1:1	120	100	3600	3200	4100	1.21 0.84	nd j
	6 i	CH2OPh	1d	60:1:1	8	100	10 700	10 600	12 200	1.14 0.86	37
	7	CH2OPh	1d	120:1:1	8	98	21 000	23 000	25 600	1.06 0.86	37
	8	CH2OPh	1d	250:1:1	15	100	44 600	44 100	51 200	1.16 0.87	38
	9	CH2OPh	1d	500:1:1	15	92	81 900	80 500	95 000	1.20 0.86	40
	10	CH2OPh	1f	28:1:1	120	100	5000	4700	5400	1.20 0.81	nd j
	11 i	CH2OPh	1f	60:1:1	30	100	10 700	10 000	12 200	1.15 0.84	38
	12	CH2OPh	1f	100:1:1	8	100	17 900	17 800	19 000	1.11 0.83	40
	13	(S)-CH2OPh	1d	60:1:1	60	100	10 700	10 500	11 700	1.10 <0.05 36
	14	CH2SPh	1a	30:1:1	240	100	5900	5800	6600	1.14	0.74	9
	15 i	CH2SPh	1a	60:1:1	480	99	11 600	11 300	13 900	1.21	0.76	8
	16	CH2SPh	1c	20:1:1	240	38	1500	1 400	1700	1.08	nd j	nd j
	17	CH2SPh	1c	30:1:1	60 h	57	3400	3400	3600	1.14 0.73	9
	18 i	CH2SPh	1c	60:1:1	96 h	48	5600	5100	6400	1.17 0.74	9
	19	CH2SPh	1d	40:1:1	240	100	7800	7400	8900	1.19 0.83	nd j
	20 i	CH2SPh	1d	80:1:1	60	100	15 600	15 200	17 900	1.15 0.87	13
	21	CH2SPh	1d	120:1:1	15	100	23 200	23 300	25 700	1.07 0.86	14
	22	CH2SPh	1d	250:1:1	60	95	46 100	48 300	51 400	1.15 0.87	14
	23	CH2SPh	1d	500:1:1	120	90	87 400	86 400	96 700	1.17 0.86	12
	24	CH2SPh	1f	20:1:1	60	100	3900	3400	4400	1.19 0.85	nd j
	25 i	CH2SPh	1f	60:1:1	60	100	11 700	11 300	13 300	1.12 0.85	13
	26	CH2SPh	1f	120:1:1	15	95	22 200	23 200	24 600	1.14 0.84

a Reactions performed with [BPL CH2ZPh ]0 = 1.0 M. b Reaction time was not necessarily optimized. c Conversion of BPL CH2ZPh as determined by 1 H NMR analysis of the crude reaction mixture. d Molar mass calculated according to Mn,theo = ([BPL CH2ZPh ]0/[1]0 × conv.BPL(CH2ZPh) × MBPL(CH2ZPh)) + MiPrOH, with MBPL(CH2OPh) = 178 g.mol -1 , MBPL(CH2SPh) = 194 g.mol -1 , MBPL(CH2CH2OCH2Ph) = 206 g.mol -1 , MiPrOH = 60 g.mol -1 . e Molar mass determined by 1 H NMR analysis of the isolated polymer, from the resonances of the terminal OiPr group (refer to Experimental section). f Number-average molar mass and dispersity (Mw/Mn) determined by SEC analysis in THF at 30 °C vs.

  CH 2 OPh/CH 2 SPh/CH 2 CH 2 OCH 2 Ph

	performed with 1a-1g/(iPrOH) confirmed the kinetics trend derived from batch
	experiments, and linear semi-logarithmic plots established that all reactions were first
	order in monomer (apparent rate constant kapp = 67.3 ± 3.1 min -1 ,
	BPL CH 2 CH 2 OCH

2 Ph /1d; 32.93 ± 0.01 min -1 , BPL CH 2 OPh/CH 2 SPh /1d,1f; 15.05 ± 0.38 min -1 , BPL CH 2 CH 2 OCH 2 Ph /1f; 0.76 ± 0.07 min -1 , BPL CH 2 OPh /1a; 0.58 ± 0.11 min -1 , BPL CH 2 SPh /1a; 9.05 ± 0.06 × 10 -2 min -1 , BPL CH 2 OPh /1c; 6.69 ± 0.62 × 10 -2 min -1 , BPL CH 2 CH 2 OCH 2 Ph /1a;

  (Figure6, Table1). It is worth pointing out that the rac-BPL CH 2 ZPh s (Z = O, S, CH2OCH2) herein investigated ''simply'' differ from the previously studied rac-BPL CH 2 OCH 2 Ph by one less methylene group in the phenoxide/phenylthiol moiety in the case of BPL CH 2 OPh and BPL CH 2 SPh , or by the replacement in the exocyclic pendant group of a methylene by an ethylene moiety in the case of BPL CH 2 CH 2 OCH 2 Ph . The stereoselectivity of the yttrium catalysts incorporating variously ortho-substituted phenolate ligands revealed different from one class of BPL FG monomer to another; the stereoselectivity was strongly affected by the nature of the R ortho-substituents installed on the phenolate ligands and by the nature of the exocyclic functional group FG of the β-lactone monomer (Tables

	C NMR
	spectra analyses (upon comparison with the corresponding spectra of the isotactic
	PBPL CH 2 ZPh s synthesized from the enantiopure monomers, respectively (Figures
	S12-14,S18)) in close relationship with the nature of the exocyclic side-chain and the
	nature of the substituted phenolate ligand framework of the yttrium complexes
	1a-1g/(iPrOH)

Table 1 ,

 1 entries 13,27,40), mediated by 1a, 1c, 1d, or 1f /(iPrOH): (left) PBPL CH 2 OPh (Table1, entries 2,4,6,9), (middle) PBPL CH 2 SPh (Table1, entries 15,18,20,25), (right) PBPL CH 2 CH 2 OCH 2 Ph (Table1, entries 28,31,32,34).

	Thermal characteristics of PBPL CH2ZPh s synthesized by ROP of rac-BPL CH2ZPh s (Z
	= O, S, CH2OCH2) promoted by 1a-g catalyst systems
	The thermal behavior of the synthesized PBPL CH 2 ZPh s was analyzed by thermal
	gravimetry analysis (TGA) and differential scanning calorimetry (DSC) (Figures
	S22-S25). The PBPL CH 2 OPh and PBPL CH 2 SPh samples were found to thermally degrade
	at the same temperature (Td onset	PBPLCH2OPh = 272 °C, Td onset	PBPLCH2SPh = 271 °C) that is
	significantly higher than the one measured for PBPL Me (Td onset = 256 °C) and
	PBPL CH 2 CH 2 OCH 2 Ph (Td onset PBPLCH2CH2OCH2Ph = 226 °C) (Figure

(a) W. F. Edgell and J. Lyford IV, Inorg.Chem. 1970Chem. , 9, 1932Chem. -1933;; (b) J.

Acknowledgements

This research was financially supported by the Lebanese University (Ph.D. grant to R.S.), the China Scholarship Council (CSC Ph.D. grant No. 201804910783 to H.L.), and Université de Rennes 1 (Ph.D. grant to R.S.). This manuscript is in honor of the 50 year anniversary of the French Polymer Group (Groupe Francais des Polymeres -GFP).

Further support of the macromolecular structure of the PBPL CH 2 ZPh s was gained from ESI or MALDI-ToF mass spectrometry analyses (Figures S19-S21). The spectra recorded for low molar mass samples of PBPL CH 2 ZPh s prepared from 1d,1f/iPrOH catalytic systems, showed a single population of macromolecules having a repeating unit of m/z 178, 194, or 206 (MBPL(CH2OPh), MBPL(CH2SPh), MBPL(CH2CH2OCH2Ph)), respectively, corresponding to α-isopropoxy,ω-hydroxyl telechelic PBPL S2,S3. 15,16 Considering the catalyst system based on 1c flanked with an halogenated non-crowded ancillary ligand, the presence of two "CH2" moieties apart from the central oxygen of the pendant FG = CH2OR (R = Me (CH3), All (CH2CH=CH2), Bn (CH2Ph)), i.e. "CH2OCH2" in 4-alkoxymethylene-β-propiolactones BPL CH 2 OR s, was evidenced to be essential to afford highly isotactic PBPL CH 2 OMe/All/Bn s that arose from the presence of strong Cl…C-H non-covalent interactions (NCIs) between the chloro substituents and the two methylene groups. 16 The stereocontrol was then switched to syndiotacticity, either upon replacement of one of these methylenes into FG = C(O)OR of the β-malolactonates, or upon its removal into FG = CH2OPh or CH2SPh of the BPL CH 2 ZPh s (Z = O, S). Moreover, increasing the length of the pendent chain from alkoxymethylene "CH2OCH2" in BPL CH 2 OR s to alkoxyethylene "CH2CH2OCH2" in BPL CH 2 CH 2 OCH 2 Ph seem to affect the Cl…CH2s interactions. We assume this is possibly due to a different conformation in the monomer/active species interactions, and/or due to the decrease in acidity of the methine-adjacent methylenes of the side-chain in -CH2CH2OCH2Ph vs -CH2OCH2Ph, ultimately weakening the NCIs and leading to the loss of sterecontrol to afford atactic polymers, similarly to those obtained with BPL Me . 12b,c


Peter M Chapple

Ghanem Hamdoun

Thierry Roisnel

Jean-François Carpentier

Hassan Oulyadi

Yann Sarazin
email: yann.sarazin@univ-rennes1.fr

Bis(imino)carbazolate lead(II

Bis(imino)carbazolate lead(II) fluoride and related halides †

Peter M. Chapple, a Ghanem Hamdoun, b Thierry Roisnel, a Jean-François Carpentier, a Hassan Oulyadi c and Yann Sarazin* a

The versatility of a bulky bis(imino)carbazolate ligand in lead(II) chemistry is illustrated by the syntheses of a soluble, heteroleptic lead(II) fluoride and of several halide (Cl, Br I), amide and hydrocarbyl congeners. All complexes have been structurally authenticated, and a full set of 207 Pb NMR data is discussed.

Since the seminal report of a stable σ-bonded organolead(II) complex, [Pb{CH(SiMe3)2}2], 1 low-valent lead(II) compounds have been attracting some attention, although on the whole they remain less developed that their lighter Ge(II) and Sn(II) analogues. The introduction of bulky ligands to the field has enabled the syntheses of hitherto elusive species. The molecular Pb(II)-fluoride [{BDI DiPP }PbF], where {BDI DiPP } -is the versatile β-diketiminate CH[C(Me)N-2,6i Pr2-C6H3]2 -, was reported in 2011. 2 It is only in 2017 that the first Pb(II)-hydride, the dimeric [{2,6-Trip2-C3H3}Pb(μ-H)]2 (Trip = 2,4,6i Pr3-C6H2), was described. 3 [{Carb tbp }Pb] + .[Al(OC4F9)4] -, featuring a onecoordinate Pb(II) cation encapsulated in the 1,8-bis(3,5-di-tertbutylphenyl)-3,6-di-tert-butyl-carbazolate ligand (aka {Carb tbp } - ) having bulky flanking aromatic groups, was obtained in 2019. 4 Although Pb(II) complexes supported with amidinate and guanidinate 5 ligands exist, the β-diketiminato ligand {BDI Dipp } -is arguably the most commonly used chelating amide ligand in this area due to its simple synthesis, useful NMR resonances and relative kinetic and thermodynamic stability arising from its delocalised charge and from the chelate effect. It has been used to support a range of heteroleptic Pb(II) complexes, e.g. amides, 6 alkyls, 7 triflates, 7a halides, 2,6a phosphides, 6b,8 chalcogenides 9 and alkoxides. 10 Developing new stable soluble sources of Pb(II) is potentially of interest to assist in not only expanding the range of uses for Pb(II) materials, but also to gain further understanding about the fundamental organometallic chemistry and bonding with lead. In this regard, 207 Pb NMR spectroscopy is a useful tool in order to gain information about the chemical environment of the Pb(II) atom. 207 Pb NMR has many desirable characteristics; 207 Pb is a reasonably abundant isotope (22.6%), and has a ½ spin and good NMR receptivity (11.9 vs 13 C). Despite this, 207 Pb NMR data are often not recorded for organometallic complexes, usually due to problems with chemical shift anisotropy. For instance, the set of halide complexes [{BDI DiPP }PbX] is available for X = F, Cl, Br and I, 2,6a but the 207 Pb NMR chemical shift has only been reported for the fluoride 2 and the chloride 11 (δ207Pb 787 and 1413 ppm, respectively).

In this context, we are reporting here on a complete series of easily prepared and soluble lead(II) halides supported by a bulky bis(imino)carbazolate, and on their full set of high resolution 207 Pb NMR and crystallographic data. It is also shown how they can be used as convenient precursors to access organolead(II) derivatives.

We have shown that the bis(imino)carbazolate {Carb DiPP } - ligand (DiPP = 2,6i Pr2-C6H3) effectively stabilises heteroleptic complexes of the large alkaline-earth metals. 12 The equimolar reaction in thf of [{Carb DiPP }K•(thf)] with PbI2 and PbBr2 yielded the corresponding heteroleptic, non-solvated [{Carb DiPP }PbX] in good yields (X= I, 1, 75%; Br, 2, 79%; Scheme 1). The chloride [{Carb DiPP }PbCl] (3) was obtained in 82% yield upon stoichiometric reaction in thf of the lithium precursor [{Carb DiPP }Li.(thf)2] and PbCl2. The complexes were isolated as bright yellow powders that are very soluble in aromatic solvents and ethers. However, salt metathesis reactions with PbF2 failed to deliver the sought Pb(II)-fluoride derivative, likely due to the Please do not adjust margins Please do not adjust margins relative insolubility of PbF2 in thf or due to the relatively high crystal lattice energy of PbF2 (estimated by the Kapustinskii 13 and Born-Mayer 14 equations to be -2495 kJ mol -1 and -2392 kJ mol -1 , respectively). [START_REF]The lattice energy in PbX2 decreases with halide size. Other Kapustinskii and Born-Mayer energies[END_REF] The identity and purity of 1-3 was established on the basis of X-ray diffraction crystallography and NMR spectroscopic data recorded in benzene-d6. Their 1 H NMR spectra all display the same multiplicity patterns. The CH imine resonance (δH: 1, 8.37 ppm; 2, 8.44 ppm; 3, 8.45 ppm) is increasingly shielded with halogen size. The isopropyl groups in the DiPP substituents are non-equivalent at room temperature, resulting in two CH heptets (1, δH 3.88 and 3.07 ppm; 2, 3.84 and 3.03 ppm; 3, 3.79 and 3.01 ppm). The 207 Pb NMR spectra each display a welldefined singlet [δPb (ppm) and Δν1/2 (Hz): 1, 1542/239; 2, 1054/186; 3, 791/190], with increased shielding correlating with decreasing halogen size. 207 Pb NMR has a range of ca. 17000 ppm, from + 11000 to -6000 ppm, with the most deshielded signals generally being from alkyl plumbylenes, while the most shielded are attributed to plumbocenes. [START_REF] Wrackmeyer | [END_REF] There is a typically strong relation between coordination number and δPb, with higher coordination numbers resulting in lower chemical shifts. The 207 Pb NMR chemical shift for 3 is noticeably upfield shifted from other known heteroleptic Pb(II) chlorides supported by monoanionic N-type ligands, e.g. the threecoordinate [{BDI DiPP }PbCl] (1413 ppm) 11 and [{N^N DiPP }PbCl (1317 ppm, where {N^N DiPP } -is a iminoanilide bearing a DiPP group), 17 or also the two-coordinate [{Carb tbp }PbCl] (2398 ppm). 4 Moreover, the 207 Pb resonance for the Pb(II) bromide 2 is also shifted highfield compared to the three-coordinate [Pb{μ-SAr Me6 }Br]2 (δPb 4283 ppm; SAr Me6 = C6H3-2,6-(C6H2-2,4,6-Me3)2). These observations suggested that 1-3 form fourcoordinate complexes. We did not find in the literature any comparative 207 Pb NMR data for Pb(II) iodides relevant to 1. For instance, [{BDI DiPP }PbI] 3a and [{CH[C(Me)N-4i Pr-C6H4]2}PbI] 18 have both been structurally authenticated, but their 207 Pb NMR data are not available.

The XRD structures of 1-3 all reveal a single molecule in the asymmetric unit cell (plus a molecule of toluene for 2 and 3), with very similar four-coordinate distorted see-saw geometries around the central Pb atom. The structure of the iodide 1 is displayed in Fig. 1 with its relevant metric parameters (see SI for Complex 52 dissolves well in aromatic hydrocarbons and ethers; as such, it is a rare example of soluble Pb(II) fluoride. 2 Diffusion coefficient-molecular weight analysis by DOSY NMR spectroscopy in benzene-d6 (see SI) indicated with a great level of confidence that the dimer splits in solution to generate the monomeric [{Carb DiPP }PbF] (5). All other complexes 1-4 remain monomeric in solution. The 19 F NMR spectrum of 5 exhibits a resonance at δF -110 ppm, with clearly detectable satellites due to coupling with the Pb atom ( 1 JF-Pb ~ 3350 Hz). Accordingly, its 207 Pb NMR spectrum reveals a doublet centred on δPb 332 ppm with a 1 JPb-F scalar coupling constant of 3350 Hz. The 207 Pb data is consistent with a monomeric complex and Pb(II) in a fourcoordinate environment. The resonance is deshielded compared to [{BDI DiPP }PbF] (δPb 784 ppm, 1 JPb-F = 2792 Hz). 2 The chemical shift of δPb 332 ppm for 5 (halide = F) fits well with the trend for the other Pb-halides, that is, δPb 1542 (1 -I), 1054 (2 -Br) and 791 (3 -Cl) ppm. This trend, which strongly suggests that there is a clear upfield chemical shift with increasing electronegativity of the halide, is the opposite of expectations based on σ-inductive effects. Yet, it is known that the electron-donating or -withdrawing ability of the 'X' reactive ligand does not dictate chemical shift in 207 Pb NMR. 7b Instead, 207 Pb NMR chemical shifts δ are heavily influenced by the different relative contributions to the isotropic shielding (σ) of the 207 Pb nucleus from diamagnetic (σ D ), paramagnetic (σ P ) and spin-orbit (σ SO ) shieldings: 7b,20 σ= σ D + σ P + σ SO (1)

δ = σreference -σ (2)
The σ P and σ SO terms are those with the largest effect on δ for 207 Pb NMR. The diamagnetic shielding σ D results from core terms depending on the density of electrons at the nucleus (associated to the circulation of electrons in orbitals determined by the ground state wave function) that seldom vary across different lead(II) complexes, and therefore are cancelled out in the final expression of δ. The spin-orbit contribution σ SO is significantly affected by changes in the atomic number of the elements directly bonded to the Pb atom, and is particularly important for elements with large spin-orbit constant (Br, I). Its value increases with atomic number, and it is likely to play a significant role in the chemical shift for 1-3 and 5. The paramagnetic term σ P is related to the movement of electrons between ground and excited states, with the main contribution coming from excitation from S0 to S1. Increasingly electronegative elements lower the level of the ground state S0, resulting in a drop of σ P . The outcome is a decrease of the overall isotropic shielding σ and, as a consequence, shielding of the Pb nucleus and a more highfield chemical shift. 7b,20 Overall, the trend of increased shielding seen upon descending group 14 in the series of complexes 1-3 and 5 is therefore likely to be set by both the changing contributions of σ P and σ SO . Further comments on this trend will require a detailed DFT analysis. The dimeric geometry of 5 was a surprise, as we expected the compound would crystallise with a terminal fluoride ligand, as for the heavier analogues 1-3 and for [{BDI DiPP }PbF]. 2 It was also our general assumption that the {Carb DiPP } -ligand was more Please do not adjust margins Please do not adjust margins sterically cumbersome than {BDI DiPP } -, on account of its wider bite angle, as well as from the empirical evidence gathered from related barium chemistry. 12 To better quantify the difference in steric profiles of the {Carb DiPP } -and {BDI DiPP } -ligands, buried volume calculations were carried out on a range of comparable Pb(II) halide complexes (Fig. 4), using the freeware Sambvca 2.1. 21 The different percentages of buried volume (%Vbur) were calculated from the crystal structure for each of the halides [{BDI Dipp }PbX] 6a and 1-3 for X = I, Br, Cl; the fluorides were left out on account of the dimeric structure of 52. %Vbur is a single value parameter used to access steric bulk as a percentage of the first coordination sphere (of radius r) around the central metal atom occupied by a ligand. 21 The qualitative results give an indication about the relative steric profiles of the ligands and show that %Vbur for {BDI DiPP } -is substantially smaller than for the {Carb DiPP } -ligand, compare the values for 1 (%Vbur = 54.5%), 2 (55.0%), 3 (54.9%) and for the corresponding [{BDI DiPP }PbX] complexes 6 where X = I (47.4%), Br (48.5%) and Cl (49.0%). The {Carb DiPP } -ligand hence lends itself better to a spatially tight control of the coordination sphere around a metallic centre.

The Pb(II)-halides 1-3 and 5 constitute valuable synthetic precursors. For instance, the reaction of 3 with [LiCH(SiMe3)2] afforded the lead(II)-alkyl [{Carb DiPP }PbCH(SiMe3)2] (6) in a nonoptimised 44% yield. Complex 6 is an orange solid which crystallised from a petroleum ether solution spiked with toluene. Its identity was established by XRD analysis (Fig. S32), and purity was confirmed by NMR. Its four-coordinate molecular structure resembles closely that of 4. The 207 Pb NMR spectrum of 6 exhibits a resonance at δPb 2950 ppm (Δv1/2 = 651 Hz), i.e. towards the highfield limit of chemical shifts for heteroleptic lead(II)-alkyl complexes. 7 The diagnostic resonance for CH(SiMe3)2 in the 1 H NMR spectrum appears at -0.97 ppm.

In summary, the bis(imino)carbazolate {Carb DiPP } -is a new addition to the set of tools available to organometallic and coordination synthetic chemists for the preparation of molecular lead(II) compounds. It is a versatile ligand that allows for the preparation of stable heteroleptic lead(II) halides, alkyls and amides, and as such it is a useful complement to the ever popular {BDI DiPP } -β-diketiminate. In line with our initial insight in the chemistry of s-block metals, 12 the results disclosed herein suggest that {Carb DiPP } -affords improved steric control over the coordination sphere around large metal ions. The synthesis and characterisation, including by 207 Pb NMR in solution, of a stable Pb(II) fluoride, has been achieved, and hence a full comparative set of 207 Pb NMR data for Pb(II) halides is available for the first time. We will next endeavour to capitalise on these spectroscopic data with a DFT analysis of the observed chemical shifts and deconvolution of the constitutive shielding constants.

The authors thank the Agence Nationale de la Recherche (ANR-17-CE07-0017-01 and ANR-11-LABX-0029) for support.

Notes and references

2 and 3 ,Fig. 1

 31 Fig. 1 ORTEP view of the molecular solid-state structure of [{Carb DiPP }PbI] (1). Ellipsoids at the 30% probability level. All H atoms omitted for clarity. Selected bond lengths (Å): Pb1-I1 = 2.9441(5), Pb1-N1 = 2.294(2), Pb1-N11 = 2.532(3), Pb1-N41 = 2.547(3). Selected angles (°): I1-Pb1-N1 = 88.24(7), I1-Pb1-N11 = 95.05(6), I1-Pb1-N41 = 98.35(6), N1-Pb1-N11 = 79.23(9), N1-Pb1-N41 = 80.72(9), N11-Pb1-N41 = 155.45(9), N11-N1-N41 = 105.76(10). Distance from Pb1 to N1N11N41plane: 0.379(2) Ǻ.

Fig. 2

 2 Fig. 2 ORTEP view of the molecular solid-state structure of [{Carb DiPP }PbN(SiMe3)2] (4). Ellipsoids at the 30% probability level. All H atoms omitted for clarity. Selected bond lengths (Å): Pb1-N1 = 2.2413(19), Pb1-N8 = 2.3427(17), Pb1-N11 = 2.6203(18), Pb1-N41 = 2.6494(18). Selected angles (°): N1-Pb1-N8 = 102.79(7), N1-Pb1-N11 = 94.23(6), N1-Pb1-N41 = 95.51(6), N8-Pb1-N11 = 78.56(6), N8-Pb1-N41 = 95.51(6), N11-Pb1-N41 = 158.10(6), N11-N8-N41 = 108.55(7). Distance from Pb1 to N8N11N41-plane: 0.150(1) Ǻ.

A c c e p t e d M

 d a n u s c r i p t Please do not adjust margins Please do not adjust margins hexamethyldisilazide ligand. The Pb-Nsilazide bond length in 4 (2.2413(19) Å) is half-way between those in the two-coordinate [{Carb tbp }PbN(SiMe3)2] (2.2008(27) Ǻ 19 ) and the threecoordinate [{BDI DiPP }PbN(SiMe3)2] (2.281(3) Ǻ 6a ). The 207 Pb NMR spectrum of 4 features a broad singlet at δPb 1347 ppm (Δν1/2 = 517 Hz), i.e. much more highfield than these two complexes [δPb 3345 (Δν1/2 = 450 Hz) and 1824 ppm, respectively 6b,19 ]. The lead(II) fluoride analogue of the halide complexes 1-3 was eventually synthesised by reacting the fluorinating reagent Me3SnF with compound 4. The reaction proceeds smoothly (though slowly, requiring 7 days to reach completion) at 85 °C to give [{Carb DiPP }PbF], which crystallised as the unusual dimer [{Carb DiPP }Pb(µ-F)]2 (52) (Scheme 2). The C2-symetric molecular solid-state structure displayed in Fig. 3 reveals a near-perfect square pyramidal arrangement (τ5 = 0.02), with the Ncarb atom in apical position, about each of the five-coordinate Pb atoms. At a distance of 1.369(2) Å, the Pb atom sits very significantly out of the ligand plane defined by N2N11N41, with relatively long Pb-Nimine distances (2.641(2) and 2.700(2) Å). This geometry is starkly different from that in the monomeric 1-3, where the Pb atom sits only slightly out of the NNN ligand plane (see above); this distance is also much larger than in the amide parent 4 (0.2269(18) Å). As expected for a bridging ligand, the Pb-F interatomic distances in 52 (2.3042(17) and 2.3265(17) Å) are much longer than in the monomeric [{BDI DiPP }PbF] (2.088(17) Å), 2 the only other know molecular lead(II)-fluoride.

Scheme 2

 2 Scheme 2 Syntheses of [{Carb DiPP }Pb(µ-F)]2 (52). DiPP = 2,6i Pr2-C6H3.

Fig. 3

 3 Fig. 3 ORTEP view of the molecular solid-state structure of [{Carb DiPP }Pb(μ-F)]2 (52). Ellipsoids at the 30% probability level. All H atoms and i Pr groups omitted for clarity. Selected bond lengths (Å): Pb1-F1 = 2.3042(17), Pb1-F1 i = 2.3265(17), Pb1-N2 = 2.321(2), Pb1-N11 = 2.641(2), Pb1-N41 = 2.700(2). Selected angles (°): F1-Pb1-F1 i 70.67(7), F1-Pb1-N2 = 91.79(7), F1-Pb1-N11 = 78.54(7), F1-Pb1-N41 = 147.75(7), F1 i -Pb1-N2= 91.50(7), F1 i -Pb1-N11 = 146.40(7), F1 i -Pb1-N41 = 81.66(7), N2-Pb1-N11 = 76.06(8), N2-Pb1-N41 = 72.25(7), N11-Pb1-N41 = 122.05(7), Pb1-F1-Pb1 i = 109.33(7). Distance from Pb1 to N2N11N41-plane: 1.369(2) Ǻ.

Fig. 4

 4 Fig. 4 Topographic steric map of the %Vbur bulk distribution, defined as the percent of the total volume of a sphere (r = 3.5 Å) occupied by the ligand. Left: {Carb DiPP } - ligand in 2. Right: {BDI DiPP } -ligand in [{BDI DiPP }PbBr]. 6a Isocontour scheme, in Ǻ, displayed on the right. The maps are a 2D isocontour representation of the interaction surface S(x,y) = ʐB, as defined in reference 21d. See SI for details.

A c c e p t e d M

 d a n u s c r i p t

J. Name., 2013, 00, 1-3 | 3