domain: info sdv

text:
Julien Leblanc

Olivier Boulle

Emeline Roux

Jacques Nicolas

Dominique Lavenier

Yann Audic

Lavenier Dominique

Nicolas Jacques

Leblanc Julien

Boulle Olivier

Roux Emeline

"In vitro construction and long read sequencing analysis of a 24 kb long artificial DNA sequence encoding the Universal Declaration of the Rights of Man and of the Citizen ."

Keywords: synthetic DNA, DNA assembly, long read sequencing, DNA storage

In absence of DNA template, the ab initio production of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. We propose in this report a fully in vitro protocol to generate very long double-stranded DNA molecule starting from commercially available short DNA blocks in less than 3 days. This innovative application of Golden Gate assembly allowed us to streamline the assembly process to produce a 24 kb long DNA molecule storing part of the Universal Declaration of Human rights and citizens. The DNA molecule produced can be readily cloned into suitable host/vector system for amplification and selection.

Introduction

For billions of years, double-stranded DNA molecules (dsDNA) have been the molecular support of choice for the storage of biological information and the support of life. In biological systems, DNA replication is a core biological process that occurs at high speed in prokaryotes (~ 700 nucleotide/sec, [START_REF] Mccarthy | DNA Elongation Rates and Growing Point Distributions of Wild-Type Phage T4 and a DNA-Delay Amber Mutant[END_REF] ). In eukaryotes, the process is slower (~ 15-30 nt/sec 2 ) but so highly parallelized that it allows to achieve replication of a 1,7 billion nt genome in less than 30 min in early Xenopus embryos (~ 9.4 x 10 5 nt/sec). It is also particularly efficient for the synthesis of collinear DNA molecules of up to hundreds millions of nucleotides such as lungfish chromosomes ( 3 ). This is made possible by the faithful copy of pre-existing nucleic acid polymers and template-specific DNA polymerases ( 4 ). On the other hand, the ab initio production of DNA molecules of only thousands of nucleotides of defined sequence remains a technological and scientific challenge. So far, commercial companies generally offer cost-effective chemical synthesis of oligonucleotides up to a few tens of nucleotides and advertise longer DNA molecules of desired sequences for up to 50,000 nt ( [START_REF] Hoose | DNA Synthesis Technologies to Close the Gene Writing Gap[END_REF][START_REF] Hughes | Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology[END_REF] ) albeit at a substantial cost (Table 1). Assemblies of impressively long DNA molecules such as the mouse mitochondrial genome (16.7 kb), T7 bacteriophage (39.9 kb) or even the Mycoplasma genitalium genome (583.0 kb) are documented in the literature but all rely on hierarchical assembly and molecular cloning steps which are time-consuming and laborious ( [START_REF] Gibson | Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma Genitalium Genome[END_REF][START_REF] Gibson | Chemical Synthesis of the Mouse Mitochondrial Genome[END_REF][START_REF] Pryor | Rapid 40 Kb Genome Construction from 52 Parts through Data-Optimized Assembly Design[END_REF] ). Furthermore, analysis and assessment of the accuracy of the assemblies relies on their cloning into an appropriate vector, which must be transferred into a host and biologically selected. A less laborious procedure has yet to be defined, aimed at producing in vitro and cost-effectively long dsDNA molecules of predefined sequences in the absence of a DNA template. This limitation is unfortunate considering the numerous applications of long DNA molecules in synthetic biology [START_REF] Zhang | Enabling Technology and Core Theory of Synthetic Biology[END_REF][START_REF] Kosuri | Large-Scale de Novo DNA Synthesis: Technologies and Applications[END_REF] ). Long synthetic DNA molecules are also a subject of interest in experimental biology, as they can provide insights into several areas of research such as the reconstruction of ancestral DNA genes, regulatory elements and proteins [START_REF] Chang | Ancestral Gene Reconstruction and Synthesis of Ancient Rhodopsins in the Laboratory[END_REF] or the investigation of alternative splicing regulation ( 13 ). Moreover, the production of large artificial or semi-artificial DNA molecules, including mutants or variants, is a widespread practice for the remodelling of genetic circuits, the evaluation of gene function, and the identification of functional domains within DNA or encoded proteins ( 6 ). Additionally, these DNA molecules are also useful as templates for RNA-vaccine production ( 14). Their main industrial applications concern in vitro screening of mutations for the development of therapeutics and chemical products, including drugs and biofuels [START_REF] Zhang | Enabling Technology and Core Theory of Synthetic Biology[END_REF][START_REF] Casini | Bricks and Blueprints: Methods and Standards for DNA Assembly[END_REF] ). Another important use of long DNA Molecules takes root in their dense and stable information content. Current data storage systems, whether magnetic or silicon-based, are indeed unable to respond to the rapid increase in archiving needs and have several drawbacks such as a limited lifespan, energy consumption, miniaturization restrictions and environmental impact. DNA molecules are nowadays envisaged as an alternative to store data because of their tremendous information density and high chemical stability as evidenced by the recovery and analysis of ancient DNA extracted from fossils ( [START_REF] Matange | DNA Stability: A Central Design Consideration for DNA Data Storage Systems[END_REF][START_REF] Orlando | Ancient DNA Analysis[END_REF] ). However, the main bottleneck for the storage of information on DNA is the DNA synthesis step itself, which is slow, expensive and can only generate short oligonucleotides [START_REF] Goldman | Towards Practical, High-Capacity, Low-Maintenance Information Storage in Synthesized DNA[END_REF][START_REF] Ezekannagha | Design Considerations for Advancing Data Storage with Synthetic DNA for Long-Term Archiving[END_REF] ). The reduced size of the oligonucleotides implies fragmenting the digital documents into a large number of small pieces which must necessarily include indexes allowing the reconstruction of the original document [START_REF] Bornholt | A DNA-Based Archival Storage System[END_REF]. The size of this index can be important compared to the size of a DNA oligonucleotide and thus significantly affect the effective amount of information stored and the synthesis costs. Incidentally, this fragmentation will also increase the difficulty of recovering the original documents. On the other hand, single molecules real-time sequencing (Pacific Bioscience) or Nanopore sequencing (Oxford Nanopore Technology) are now capable of reading individual long DNA molecules, which is compatible with storing of information on longer DNA fragments ( [START_REF] Song | Nanopore Detection Assisted DNA Information Processing[END_REF][START_REF] Rhoads | PacBio Sequencing and Its Applications[END_REF][START_REF] Weirather | Comprehensive Comparison of Pacific Biosciences and Oxford Nanopore Technologies and Their Applications to Transcriptome Analysis[END_REF] ).

Such long DNA molecules will only contain a small fraction of indexing information making them more archival-efficient.

Many methods have been developed to construct large DNA fragments from short chemically synthesized DNA oligonucleotides [START_REF] Hughes | Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology[END_REF][START_REF] Casini | Bricks and Blueprints: Methods and Standards for DNA Assembly[END_REF][START_REF] Shevelev | Modern Approaches to Artificial Gene Synthesis: Aspects of Oligonucleotide Synthesis, Enzymatic Assembly, Sequence Verification and Error Correction[END_REF]. The most common are Golden Gate assembly (GGA), Gibson cloning and polymerase cycling assembly. These methods are all based on userdefined overlapping ends, defined as overhangs, and generally allow the seamless joining of 5 to 10 fragments per reaction, the assembly is then cloned into a vector, transferred to an host, selected and amplified.

Herein we propose a fully in vitro iterative method based on GGA to faithfully construct a long synthetic DNA molecule starting from commercially available dsDNA. The synthetic DNA molecules could be used for any common biological purpose or to store information. The various assembly steps of our procedure were evaluated on an application for storing part of the Declaration of the Rights of Man and of the Citizen (26th August 1789) in a single long DNA fragment which was then sequenced using Oxford Nanopore Technology (ONT) to retrieve the original text. The different steps of the DNA construction were analysed using ONT long read sequencing technology to quantify the ordering of the building blocks, demonstrating that our protocol is highly effective in obtaining correctly assembled molecules.

Results and discussion

Articles 1 through 9 of the Declaration of Human and Citizen rights were encoded into a 23.4 kb long DNA sequence (Supplementary data 1). To fully build in vitro this dsDNA molecule we started from 50 commercially available 524 nt long dsDNA molecules (eBlocks, IDT™) that we assembled into 4,764 bp long dsDNA molecules (BigBlocks) that were then assembled into a 23,796 bp DNA molecule (MaxiBlock) (Figure 1). This fully in vitro strategy uses as building blocks commercial dsDNA molecules few hundreds of nucleotides long (eBlock, IDT™). The design of these building blocks relies on an architecture composed of, starting from the end, a 15 nt long buffer sequences that ensure the integrity of the two BsaI prefix and suffix sequences, the four bases cleavage sites that allow directional ligation and finally the cargo part of the DNA containing the encoded information (Figure 1A). Upon cleavage by BsaI, the DNA cargo part will remain solely framed by the predefined 4 nt overhangs to allow ordered assembly of 10 blocks in a single GGA reaction. Specific primer pairs targeting the extremities of the assembled BigBlocks (Figure 1B) are used to select and amplify the 4,764 bp long assembly products and flank them with sequence specific type IIS (BsaI) restriction sites to allow for the second in vitro assembly, to generate the MaxiBlocks (Figure 1C). As described in Supplementary data 2, the different assembly steps were controlled during the course of the experiment. The 23,796 bp MaxiBlocks are PCR amplified before utilization.

First assembly step: eBlocks to BigBlock

To design a 23,796 bp long DNA molecule, we split the sequence into fifty 524 bp parts, which were first assembled in groups of ten to generate five 4,764 bp BigBlocks. At the time of our study, purchasing the 524 bp eBlocks at Integrated DNA Technologies (IDT™) was the most cost-effective way to obtain these long dsDNA molecules (Table 1). The five GGA reactions aiming to obtain 4,764 bp long DNA molecules were directly controlled on agarose gels (Figure 2A).

Incomplete sequential ligation occurred and a scale of DNA molecules ranging from 480 bp, the initial size of the eBlocks after BsaI digestion, to 4,764 bp by incremental steps of about 500 bp could clearly be observed. We selected the desired 4,764 bp assembly using a PCR targeting the extremities of the BigBlocks. As presented in Figure 2B, size fractionation of the PCR product demonstrated that for each of the five BigBlocks we could specifically amplify the longer 4,764 bp DNA molecules. This demonstrated that ~ 5 kb long dsDNA molecules can be effectively assembled in vitro from commercial 524 bp dsDNA molecules. However so far this did not demonstrate that the fragments are correctly ordered in the assembly.

To determine to which extent the assembly process is ordered, we took advantage of ONT sequencing technology to directly sequence ligation and PCR products for BigBlock 1 and 4. We reasoned that single molecule long read sequencing technology such as ONT or PacBio were the most effective in obtaining sequence information about the accuracy of long collinear assembly. For the assembly of BigBlock 1 (Figure 3A) or BigBlock 4 (Figure 3B) the graphics represent the percentage of transition from block (n) to another block, to the end of the read (end), or to an unrecognized sequence (Unknown). BigBlock 1 graphics were produced from 4,935 reads with median length of 1,435 nt and median quality of 10.9. For BigBlock 4, graphics were generated from 5,281 reads with median length of 1,898 nt and median quality of 11.1 (Supp data 3). These medians reads lengths are about one-third of the expected product size in agreement with size fractionation ( Figure 2), showing that the reaction is composed of partially assembled molecules. Directly analysing the assembly reaction products, we could note that the percentage of correct concatenations of two consecutive eBlocks ranged from 61 to 95% for the assembly of BigBlock 1 and from 74 to 94% for the assembly of BigBlock 4 (Left panel on Figure 3 A and B respectively). In both cases and as expected, eBlock 10 was most generally terminal, 99 and 96% respectively. In the case of BigBlock 1 assembly, the lower efficiency of assembly of eBlock 5 was associated with either incorrect assembly of eBlock 5 to eBlock 7 (~ 10%) or eBlock 5 being terminal (~ 20%). Similarly, in the assembly of BigBlock 4, the lower level of concatenation of eBlock 34 to eBlock 35 was due to eBlock 34 being terminal. This analysis indicates a faithful assembly process as most generally the assembly is correct from eBlock (n) to eBlock (n+1), or otherwise no assembly occurs.

We used PCR to select and amplify the full-length BigBlock assembly (see Figure 2B).

BigBlock 1 post-PCR graphics were produced from 3,000 reads with median reads length of 4,757 nt and median read quality of 10.9. For BigBlock 4 post-PCR, graphics were generated from 1,476 reads with median length of 4,752 nt and median quality of 10.9 (Supp data 3). PCR selection and amplification therefore led to a ~ 3-fold increase in median reads length compared to pre-PCR products, 4,757 nt compared to 1,435 nt for BigBlock 1 and 4,752 nt compared to 1,898 nt for BigBlock 4. This median size is close to the 4,796 nt size of the expected product. This illustrates how effective is the PCR amplification step in selecting full length assembly products.

After PCR, the BigBlock sequencing results show a frequency of correctly ordered eBlock pairs ranging from 87-99% for BigBlock 1 assembly and 92-100% for BigBlock 4 assembly, a neat increase in accuracy with respect to previous results (Right panel on Figure 3 A and B respectively).

The problematic assembly of eBlock 5 to eBlock 7 in BigBlock 1 before PCR remains at a similar level after, while the premature termination of the assembly at eBlock 5 becomes negligible as it cannot be amplified. Similarly, for BigBlock 4, only the premature termination of the assembly at eBlock 9 remains notable (7%), while being reduced by half compared to the assembly without PCR. This shows that when an eBlock is erroneously terminal this does not affect the final PCR-selected product, while if two eBlocks are erroneously assembled they will stay present in the PCR-selected assembly. It is therefore important to maximize individual ligation efficiency.

While the previous analysis focuses on the correct assembly of pairs of eBlocks, it is also important to estimate the overall accuracy of whole BigBlocks assembly. We used the above ONT sequencing data to estimate the distribution of the length of the DNA assembly and to determine the proportion of correct assembly for the different fragment sizes. This also enabled us to quantify the enrichment in correct assemblies following the PCR amplification and selection process (Figure 3C).

As observed by gel electrophoresis, the reaction generated all product sizes between 1 and 10 assembled eBlocks (0.5 to 5 kb). The target molecule composed of 10 eBlocks dominates the reaction products and represent 15 and 22% of the sequenced reads in BigBlock 1 and BigBlock 4 reactions respectively with less than 3% inaccurate assemblies. The proportion of molecules with more than 10 eBlocks is negligible (< 1%). Upon PCR amplification and selection, we have observed a drastic depletion of BigBlocks composed of less than 10 fragments. About 84% of the DNA is included in molecules composed of 10 fragments almost all in the right (~ 99% for both BigBlock 1 and BigBlock 4).

Taken together these analyses indicate that the combination of directed assembly without any intermediate purification step and with PCR selection allows to efficiently produce long DNA molecules of the expected structure.

Second assembly step: BigBlocks to MaxiBlock

Having established the reliable production of 4,796 nt dsDNA molecules (the BigBlocks), we set about assembling five of them into a 23,796 bp MaxiBlock. As shown above, the five BigBlocks were amplified using primers that introduced BsaI restriction sites together with tetramer sequences required to correctly order the fragments (see Figure 1B). The assembly reaction of the five BigBlocks shown in Figure 2 was performed using GGA. The reaction products were analyzed either directly after the reaction or after PCR amplification and selection using primers targeting the first (BigBlock 1) and the last (BigBlock 5) BigBlocks of the assembly. Because of the expected size of the DNA assembly, we analyzed the reaction products by capillary electrophoresis on a Tape Station (Agilent Technologies) (Figure 4). It is noteworthy that the resolution of the Tape Station does not allow for precise sizing of the DNA molecules in the higher size range. The products of the ligation reactions (Figure 4A To better evaluate the quality of the assembled fragments we again took advantage of ONT sequencing to compare the reaction products before and after amplification and selection of a 23,796 bp DNA assembly. ONT sequencing of MaxiBlock assembly produced 25,507 reads with median length of 3,091 nt and median quality of 11.7 before PCR and 3,215 reads with median length of 1,143 nt and median read quality of 11.9 after PCR (Supp data 3). The limited median size of the sequencing reads of the MaxiBlock assembly takes its root in the presence of a) numerous short sequencing reads composed of part of terminal eBlocks 01 or 50 and b) presence of sequences that were ill-attributed during demultiplexing (Supp data 4). As shown in Figure 5 A, left panel, the proportion of correctly ordered BigBlock pairs ranges from 56 to 85%. As designed, the BigBlock 5 is most often (97%) the terminal block. When BigBlock (n) is not ligated with BigBlock (n+1), then it is usually a terminal block of the assembly. Upon PCR selection the DNA molecules sequenced are mainly composed of an assembly of BigBlocks in the right order (Figure 5B, right panel). The proportion of correctly ordered BigBlock pairs ranges from 89 to 96% and BigBlock 5 is exclusively terminal. This indicates that upon PCR, full-length products are selected while a large proportion of partial assemblies are not amplified.

We analyzed the number of eBlocks and the correctness of their assembly for all sequenced reads. In the pre-PCR reaction products, a striking pattern of reads composed of 10, 20, 30, 40 and 50 eBlocks are present and represent the assembly of 1, 2, 3, 4 or 5 BigBlocks together. They respectively represent 12, 4, 11, 4 and 14% of the DNA content of the reaction products. Most notably the majority of these reads are composed of correctly ordered fragments (Figure 5B).

As done for the selection of the BigBlocks, PCR on the MaxiBlock aims to both amplify and select the full-length 23,796 bp assembly product. Comparing the PCR-amplified products and the pre-PCR assembly reaction there is a clear depletion of DNA molecules composed of 10, 20, 30 and 40 fragments while the full-length assembly product of 50 eBlock represents 12% of the total DNA content in the mix (Figure 5C). Taken together these analyses indicate that our fully in vitro iterative method is able to faithfully construct long synthetic DNA molecules by assembling commercially available short dsDNA.

The cost of construction of the 23,796 bp molecule including DNA and enzymatic reactions, is about $2,000 without negotiation on suppliers' list prices (see Table 1). One important aspect of our protocol is the relatively short hands-on time : once the materials have been received and the DNA sequences designed, the full-size construction process can be completed within 3-4 days. On day one, eBlocks are controlled by agarose gel electrophoresis and quantified. The assembly of the eBlocks into BigBlocks is carried out overnight. On day two, the assembled BigBlocks are controlled by agarose gel electrophoresis, followed by PCR selection. PCR products are then controlled by agarose gel electrophoresis and quantified by spectrometry. The MaxiBlock assembly is then realized overnight.

On day three, the MaxiBlock assembly is controlled on a TapeStation and PCR amplified. The final PCR products are ultimately controlled on TapeStation. Higher throughput is easily achievable by robotizing and parallelizing the assembly [START_REF] Storch | DNA-BOT: A Low-Cost, Automated DNA Assembly Platform for Synthetic Biology[END_REF] The length of the final DNA is limited by the capabilities of the DNA polymerase used for the final PCR selection. In fact, obtaining PCR products larger than 24 kb can be challenging with respect to efficiency and accuracy with standard DNA polymerase such as Taq polymerase. However, some DNA polymerases are known to have a higher processivity, extension rate, or error correction capabilities making them more suitable for amplifying longer DNA fragments. LongAmp ® Hot Start Taq DNA polymerase from NEB ® has the theoretical capacity to amplify DNA fragments up to 35 kb in length, while Platinum™ SuperFi II DNA Polymerase from Invitrogen™ can amplify up to 40 kb and TaKaRa LA Taq® DNA Polymerase from Takara Bio can amplify up to 48 kb. However, it is important to keep in mind that amplifying long DNA fragments can be a difficult task, and successfully obtaining a PCR product larger than 24 kb will depend on various factors such as DNA quality, reaction conditions, and the optimization of PCR conditions.

The final PCR product can be either directly used for molecular biology processes such as in vitro transcription with T7 RNA polymerase if a T7 promoter was included in the PCR primers. It must be highlighted that this product is not clonal and therefore contain a population of different molecules with potentially point substitutions or deletions. To obtain a clonal product, the DNA molecule produced may be purified by performing agarose gel electrophoresis and excising the appropriate size band, and cloned into a suitable host/vector system for amplification and selection.

The size of the final product ( ~ 24 kb) remains compatible with cloning into plasmid or cosmid vectors. The in vitro process described here allows for the time-efficient construction of long dsDNA molecules. The absence of any subcloning, transformation and selection step allows to streamline the process of long dsDNA molecules construction. Yet, they can be readily subcloned and selected on the basis of a double PCR screen using 2 pairs of primers located in the vector backbone and the building blocks at the extremity. Therefore, we feel this protocol may be an approach of choice to obtain artificial building blocks of thousands of nucleotides long that can be used in downstream applications.

Methods

Encoding of a binary file to DNA alphabet

We encoded the first articles of the Declaration of Human and Citizen Rights (DDHC) as a binary string of 4.2 Ko (Supp data 1). The binaries were first randomized by performing bitwise XOR operation between the input binary and the output of the hash function (SHA-256) over a constant in a process similar to what is described in ( 26 ). The randomized binary is converted to DNA alphabet using an algorithm that respects synthesis rules of the DNA blocks, briefly, 60 > %GC > 40, no homopolymer > 3 nt (see Supp data 5). This encoding allows to alter the DNA sequence at the βencoded bits to ensure the absence of BsaI restriction sites and of inverse repeat regions longer than 10 nt. The 23,400 nt long DNA sequence (Figure 1C and Supp data 1) is partitioned into forty blocks of 472 nt (eight internals eBlocks for each of the five BigBlocks) and ten blocks of 452 nt (one eBlock for each BigBlock extremity). Each internal block is then framed by a DNA sequence composed of a 15 nt buffer sequence and a specific BsaI restriction site. Similarly, each external block is framed by a DNA sequence that includes a 20 nt region following the BsaI restriction site, which serves as a target for PCR selection of the BigBlock. Digestion with BsaI generates a 5' overhang of 4 nt which allows ordered assembly. Overhangs are selected with the NEBridge GetSet ™ Tool. Overhangs used are CGCT, ACGA, GCAA, CACC, CCTA, CGGA, TGAA, ACTC, ACAT. Primers used for amplification of assembly products were defined with ITHOS, a submodule of Genofrag( 27 , with parameters defined in Supp data 6). A 20 nt flanking sequence composed of a BsaI site and a 9 nt external buffer sequence was added to each primer to allow for the assembly of BigBlocks to MaxiBlocks ( primer sequences are available in Supp data 7). The sequence was controlled to comply to IDT™ eBlock synthesis rules.

DNA fragments

Fifty DNA blocks were ordered as 524 bp long eBlocks™ from IDT™ (Coralville, IA). The eBlocks™ were delivered in a 96 well plate at 10 nmol/µL in TE buffer pH 8.0 (10 mM Tris-HCl/0.1 mM EDTA). Each eBlock™ was quantified with AccuGreen™ High Sensitivity dsDNA Quantitation Kit on a Qubit® Fluorometer (Invitrogen™ Q32851), aliquoted and stored at -20°C. The eBlock™ integrities were individually verified by electrophoretic analysis on 3% agarose-TBE gels stained with GelRed ® (Biotium).

BigBlock assemblies

Each of the 5 BigBlocks is composed of 10 oriented eBlocks™ described above. The assembly reaction is conducted in a 25.0 µL assembly reaction mix composed of 1.0 µL NEBridge ® Golden Gate Assembly Kit BsaI-HF ® v2 (NEB ® E1601), 2.5 µL T4 DNA ligase buffer 10X (NEB ® B0202), 0.04 pmol of each eBlock™. Reactions were conducted for 65 cycles (37°C, 5' ; 16°C, 5') and stopped by incubation at 60°C for 5'. The expected assembly size is 4,764 bp.

BigBlocks were PCR-amplified with dedicated primer pairs in a 25.0 µL PCR reaction mix composed of 0.25 µL Q5 ® Hot Start High-Fidelity DNA Polymerase 2 U/µL (NEB ® M0493), 5.0 µL Q5 ® 5X buffer (NEB ® B9027), 0.75 µL dNTP 10 mM Mix (NEB ® N0447), 1.25 µL of each primer (10 µM) and 1.5 ng of BigBlock as template. Amplification was conducted at 98°, 30'' ; 5x (98°C, 10" ; Thyb, 20" ; 72°C, 2'30) followed by 10x (98°C, 10" ; 72°C, 2'30) and a terminal extension at 72°C, 2'00. The annealing temperature (Thyb) was 62°C except for BigBlock 1, 66°C and BigBlock 3, 66°C.

The expected size of each amplified BigBlock is 4,796 bp. BigBlock assemblies and PCR products were analyzed by electrophoresis on 1-2% agarose-TBE gels stained with GelRed ® (Biotium).

MaxiBlock assembly

The MaxiBlock is assembled from the five BigBlocks (BigBlock 1 to 5) in a 20.0 µL reaction mix composed of 2.0 µL NEBridge ® Golden Gate Assembly Kit BsaI-HF ® v2 (NEB ® E1601), Amplification was conducted at 94°, 30'' ; 15x (48°C, 20" ; 55°C, 20" ; 65°C, 22') and a terminal extension at 65°C, 15'. The expected size of each amplified MaxiBlock is 23,796 bp.

MaxiBlock assembly and PCR products were analyzed on TapeStation 4200 (Agilent-Technologies) following the supplier protocol using Genomic DNA reagents (Agilent-Technologies 5067-5366) loaded onto a Genomic DNA ScreenTape (Agilent-Technologies 5067-5365).

DNA sequencing

BigBlock and MaxiBlock assemblies and PCR products were sequenced using the Oxford Nanopore Technologies (ONT) MinION Mk1C and GridION Mk1 sequencer hardware respectively. The sequencing libraries were prepared according to the manufacturer protocol (Version: NBE_9065_v109_revAD_14Aug2019) with Ligation Sequencing Kit (ONT SQK-LSK109) and PCRfree Multiplexing Native Barcoding Kit (ONT EXP-NBD104). Sequencing libraries were quantified with AccuGreen™ High Sensitivity dsDNA Quantitation Kit on a Qubit ® Fluorometer (Invitrogen™ Q32851) and loading on Flowcell Flongle (ONT FLO-FLG001) for a run time of 24 hours.

Data processing

Raw sequencing data were preprocessed with ONT Guppy software (V6.0.1 and V6.3.9, [START_REF] Wick | Performance of Neural Network Basecalling Tools for Oxford Nanopore Sequencing[END_REF] for super accuracy base calling (see config file Supp data 8) and demultiplexing (front and rear score set > 60). Sequencing quality of each demultiplexed samples were controlled with Nanoplot (V1.38.0, [START_REF] De Coster | NanoPack: Visualizing and Processing Long-Read Sequencing Data[END_REF] ). Scripts are available as Supp data 9. Sequencing data are made available on the European Nucleotide Archive under accession number PRJEB62556.

Bioinformatics results analysis

Each quality passed long read is analysed to identify the order and the identity of the blocks that compose it. We used the Smith-Waterman algorithm ( 30 ) to test the local alignment of a read to all the 50 original block sequences. The highest scoring block is identified and removed from the read.

The alignment procedure is then recursively applied to the other parts of the read before and after the aligned sub-sequence of the read, until the entire read is identified in blocks or not. To quantify the association between each block, we determined whether each individual block is followed by an identified block, an unrecognized block or the end of the read. To analyse the global assembly, we classified reads as either correct if all the blocks composing it are correctly ordered, or as incorrect if at least one block is misplaced.

  , MB1) ranged in size from 4 kb to more than 15 kb in a manner compatible with partial and complete assembled molecules in the reaction mix. After PCR amplification ( Figure 4B, MB2) using primers targeting the extremity of the Maxiblock and LongAmp ® Hot Start Taq (NEB ® ) DNA polymerase, a single PCR products of size between 15 and 48 kb is detected and in agreement with the expected 24 kb DNA product.

2. 0

 0 µL T4 DNA ligase buffer 10X (NEB ® B0202) and 200 ng of each BigBlock. Reaction was conducted for 65x (37°C, 5' ; 16°C, 5') and stopped by incubation at 60°C for 5'. The MaxiBlock size is 23,796bp. The MaxiBlock was PCR-amplified with primers MaxiBlock_PCR_Fw and MaxiBlock_PCR_Rv in a 25.0 µl reaction composed of 1.0 µL LongAmp® Hot Start Taq DNA polymerase, 2.5 U/µL (NEB ® M0534), 5.0 µL LongAmp ® Taq 5X buffer (NEB ® B0323), 0.75 µL dNTP 10mM Mix (NEB ® N0447), 0.25 µL primers pair 5,0 µM and 1.0 ng MaxiBlock assembly.

Julien Leblanc is supported by the Labex CominLabs (dnarXiv project), Olivier Boulle is supported by the PEPR MolecularXiv (ANR-22-PEXM-003). We gratefully acknowledge Edouard Cadieu and Thomas Derrien (IGDR) for their help with ONT sequencing and data processing.

Supporting Information:

Supplementary data 1 -Text and DNA encoding of the Universal Declaration of Human rights and citizens, sequences of the ordered geneblocks. (TXT) Supplementary data 2 -supplementary figure presenting the time table of the assembly of a 24 Kb artificial dsDNA fragment. (PDF) Supplementary data 3 -ONT sequencing quality metrics. (TXT) Supplementary data 4 -Supplementary figure presenting the analysis of unidentified reads. (PDF) Supplementary data 5 -Supplementary figure presenting the DNA encoding scheme of a text file.

(PDF) Supplementary data 6 -primer selection parameters. (TXT) Supplementary data 7 -oligonucleotides sequences. (TXT) Supplementary data 8 -configuration file for ONT Guppy basecaller software (TXT) Supplementary data 9 -Script file for basecalling, demultiplexing and quality control


Leonardo De

Silva Munoz

Régine Basséguy

Alain Bergel

Study of the influence of phosphoric acid in steel anaerobic corrosion via

Study of the influence of phosphoric acid in steel anaerobic corrosion via

the hydrogen evolution reaction.

DE SILVA MUÑOZ Leonardo, BASSEGUY Régine, BERGEL Alain Laboratoire de Génie Chimique, UMR5503, 5 rue Paulin Talabot, 31106 Toulouse France Leonardo.DeSilvaMunoz@ensiacet.fr, Regine.Basseguy@ensiacet.fr, Alain.Bergel@ensiacet.fr

Although it is well known that the phosphate species are quiet electrochemically inert when considering their calculated redox potential, few studies since the end of 80's reported electrochemical phenomenon observed in the reduction domain which were linked to the presence of phosphate species. Some authors proposed that phosphate ions and weak acids in general catalysed the electrochemical reduction of water [1-3] following the kind of reactions written above:

Electrochemical reactions H 2 PO 4 -+ e - H ad + HPO 4 2- (1) H ad + H 2 PO 4 -+ e - H 2 + HPO 4 2- (2) Acido-basic equilibrium H 2 PO 4 -+ OH -- H 2 O + HPO 4 2- (3)
Combining the reactions, the reduction of water was obtained 2

H 2 O + 2e - H 2 + 2OH - (4)
Marinovic and Despic [1] assumed that the hydrogen evolution was more likely to occur from species other than water, since the bond between the hydrogen atom and the rest of the molecule is weaker in weak acid compared with the hydrogen-oxygen bond in a water molecule. This was shown on silver electrodes [2] and also on platinum ones [1]. In a previous work [3], we showed that phosphate ions exhibited the same electrochemical behaviour on stainless steel. A theoretical model was proposed according to the experimental data the model showed that a significant quantity of molecular hydrogen was produced by the mechanism presented above.

In the present paper, the influence of phosphoric acid on hydrogen evolution reaction has been studied by voltammetry on platinum and stainless steel rotating disc electrodes. A linear correlation between acid concentration and the absolute value of the current at certain reduction potential has been verified. The effect of the pH value of the solution in the obtained current has demonstrated to be strongly related to the dissociation state of the acid.

In a second part, as the hydrogen evolution reaction is the principal cathodic reaction of metal corrosion in anaerobic aqueous environments, the role that weak acids can play in this process was studied. Corrosion experiments on carbon steel were carried out in phosphoric acid solution under anaerobic conditions. The influence of hydrogen in the solution was also studied. In the absence of oxygen, a substantial increase of corrosion rate was found when carbon steel samples were submerged in low phosphoric acid concentration solution compared to that obtained in distilled water.


Ngoc Anh

MPH Thu Nguyen

Any Pascal Auquier

Beltran Anzola

Annie Harroche

Sabine Castet

Yoann Huguenin

Sandrine Meunier

A multicentric French national observational cross-sectional study based on the FranceCoag registry

Keywords: Adherence, Haemophilia, Transition, Quality of life, Adolescents, Young adults

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

9 NGUYEN Ngoc Anh Thu 1,2 ; AUQUIER Pascal 1,2,3 ; BELTRAN ANZOLA Any 1,2 ; HARROCHE Annie 4 ; 10 CASTET Sabine 5 ; HUGUENIN Yoann 5 ; MEUNIER Sandrine 6 ; REPESSE Yohann [START_REF] Ringenbach Stéphanie | RISSO Marini[END_REF] ; D'OIRON Roseline [START_REF] Roche Martine | ROJAS[END_REF] ; 11 RAUCH Antoine 9 ; DESPREZ Dominique 10 ; SPIEGEL Alexandra 11 ; CHAMOUNI Pierre [START_REF] Vanessa | Haemophilia Treatment Centre[END_REF] ; SCHNEIDER 12 Pascale [START_REF] Vanessa | Haemophilia Treatment Centre[END_REF] ; BAUMSTARCK Karine 1,2,3 ; BOUCEKINE Mohamed 1,2,3 ; TABELE Clemence 1,2,3 ; VIPREY 13 Marie 3 ; LEROY Tanguy 13 ; ROQUES Marie-Anaïs 14,15 ; SANNIE Thomas 16 ; GIRAUD Nicolas 16 ; 14 CHAMBOST Hervé [START_REF] Palamaringue Pascale | PRADINES Bénédicte[END_REF]15 ; RESSEGUIER Noémie [START_REF] Palamaringue Pascale | PRADINES Bénédicte[END_REF] FranceCoag Network, Marseille, France 22 4 AP-HP, Haemophilia Treatment Centre, Hospital Necker, Paris, France 23 5 University Hospital of Bordeaux, Haemophilia Treatment Centre, Bordeaux, France 24 6 Hospices Civils de Lyon -Haemophilia Treatment Centre, Groupement Hospitalier Est, Bron, France 25 [START_REF] Ringenbach Stéphanie | RISSO Marini[END_REF] University Hospital of Caen, Haemophilia Treatment Centre, Caen, France 26 [START_REF] Roche Martine | ROJAS[END_REF] AP-HP, Haemophilia Treatment Centre, Hospital Bicêtre, Paris, France 27 9 University Regional Hospital of Lille, Haemophilia Treatment Centre, Lille, France 28 [START_REF] Santos | SAVARY Isabelle[END_REF] University Regional Hospital of Strasbourg, Haemophilia Treatment Centre, Strasbourg, France 29 11 University Regional Hospital of Strasbourg, Pediatric Onco-Haematology Service, Strasbourg, France 30 [START_REF] Vanessa | Haemophilia Treatment Centre[END_REF] University Hospital of Rouen, Haemophilia Treatment Centre, Rouen, France 31 13 3 (PwSH), especially during the transition from pediatric to adult care, which is an important phase in lives 4 of young people with childhood chronic disease. This adherence can be considered as a marker of successful 5 transition.

6 Objectives: The main objective of the quantitative phase of the TRANSHEMO project was to compare the 7 adherence to healthcare between adolescents and young adults (YAs) with severe haemophilia. The 8 secondary objective was to identify the determinants (facilitators and barriers) of this adherence and 9 associations between these determinants. 7 Non-adherence to healthcare might have serious consequences, such as hemarthrosis, which may lead to 8 impairment in physical functioning and quality of life (QoL). [START_REF]The authors would like to thank all collaborators who participate in the study: 3 AIT CHEKDHIDH Tafat (Hospital of Simone Veil, Haemophilia Treatment Center, Eaubonne-4 Montmorency, France); AMICHI Kahéna (AP-HM, France)[END_REF] A study on adherence to prophylaxis of people 9 with haemophilia (PwH) aged 12-25 years found a higher number of hemarthroses in people who were less 10 adherent to prophylaxis. [START_REF] Palamaringue Pascale | PRADINES Bénédicte[END_REF] It is necessary to gain insights into adherence to healthcare during the transition 11 from pediatric to adult care, since this is an important phase of young PwH. [4][5][6] Previous studies assessed 12 adherence to the prescribed therapeutic regimen and found a decrease in the level of adherence when PwH 13 reached adulthood. In a study on practice patterns for the management of haemophilia A, the rates of high 14 adherence in people aged 13-18 years and those aged 19-28 years were 54% and 36%, respectively. [START_REF] Ringenbach Stéphanie | RISSO Marini[END_REF] In 15 another study, 83% of adolescents aged 13-17 years adhered to prophylaxis, while this rate was 53% in 16 young adults (YAs) aged 18-25 years. [START_REF] Roche Martine | ROJAS[END_REF] Similarly, a lower level of adherence in adults compared to children, 17 as well as in adolescents compared to children with haemophilia was found. 9,10 As the decrease in adherence 18 to treatment regimens from adolescence to adulthood exists, maintaining the adherence to healthcare 19 remains one of the issues in the transition process. 20 In the context of transition to adult care in adolescents and YAs with serious pediatric illness conditions, 21 the SMART (Social-ecological Model of adolescents and young adults Readiness for Transition) 11 22 incorporates pre-existing objective factors (sociodemographics, medical status) and modifiable subjective 23 factors (knowledge, skills, beliefs, goals, relationships, psychosocial functioning). 24 In the general context of haemophilia outside the transition process, determinants of adherence to healthcare 25 were identified as barriers (absence of or infrequent symptoms, older age) and motivators (belief in necessity 26 of treatment, good relationship with healthcare provider, experience of symptoms). [START_REF] Vanessa | Haemophilia Treatment Centre[END_REF][13][14] Regarding the 27 transition process in young PwH, younger age, positive emotions, high self-esteem, sufficient school 28 attendance, adequate disease management, family and social support were found to be positively associated 29 with adherence to treatment in quantitative studies. [START_REF] Roche Martine | ROJAS[END_REF]15,16 30 However, the number of quantitative studies [START_REF] Roche Martine | ROJAS[END_REF]15,16 3 The main objective of the quantitative phase of the TRANSHEMO project was to compare the adherence 4 to healthcare, which was considered as a marker of the successful transition, between adolescents and YAs 5 with severe haemophilia. The secondary objective was to identify the determinants (facilitators and barriers)

6 of this adherence and associations between these determinants by structural equation modeling (SEM).

7 12 Comparison of the adherence to healthcare between adolescents and YAs. The evaluation criteria were 13 compared between adolescents and YAs using the chi-squared test.

F
14 18 analyze the association of each characteristic with the adherence to healthcare. Odds ratios (ORs) and 95%

19 confidence intervals (CIs) were calculated for this analysis.

20

Structural equation modeling (Figure 1). Based on the results from previous studies presented in 21 Table 2 and results of the univariate analysis, a theoretical model was built consistently with SMART. The 22 model specification is shown in Figure 1.

23 Confirmatory factor analysis (CFA) and mediation analysis were carried out to estimate the effects of the 24 determinants on adherence to healthcare. As the main evaluation criterion was a binary variable, logistic 25 regression in SEM was performed. There were two mediators (mental health concerns and haemophilia-26 specific QoL) for the effects on the outcome. "Mental health concerns" was a latent variable manifested by 27 past-negative perspective, 19,20 avoidance coping strategy [START_REF] Srivastava | Guidelines for the management of 3 hemophilia[END_REF][START_REF] Dupre Charlène | Hospices Civils de Lyon, 23 Groupement Hospitalier Est, France); DURIEUX Fréderique (AP-HM, France)[END_REF][23] and mental health-related quality of life 28 (HRQoL). [START_REF] Rivaud | [END_REF] "Haemophilia-specific QoL" was a latent variable manifested by six items in the Haemo-QoL 29 Index 25,26 (Item 2-feeling, Item 3-view, Item 4-family, Item 5-friends, Item 6-others, Item 7-sports).

30 "Autonomy" was a latent variable manifested by functional, emotional, and attitudinal autonomy. 27 All 31 latent variables were verified by CFA.

32 The total indirect effect via two mediators was obtained by adding two indirect effects. To estimate the total 33 effect, the direct effect was added to the total indirect effect. The model was built using diagonally weighted 34 least squares estimation with robust standard errors. ORs and 95% CIs were calculated for the direct, indirect 4 There were 266 non-participants who did not send back the questionnaires and 277 participants (107 5 adolescents and 170 YAs).

6 Regarding sociodemographic and clinical characteristics, the non-participants were more frequently YAs 11 not significantly differ between the participants and non-participants (Table 3).

12 13 Adherence to healthcare 14 The results concerning the main objective are presented in Table 4.

15 The adherence to healthcare in adolescents was 21% higher than in YAs (82.2% vs. 61.2%, p<0.001).

16 The adolescents also had better results than the YAs in all subcriteria. Wider differences between 17 adolescents and YAs were found in the physician-reported adherence to clinical follow-up (+22.9%, 18 p<0.001) and in the adherence to prophylaxis if under this treatment (+26.6%, p<0.001).

19 In the univariate analysis ( 25 The results of SEM are presented in Figure 2, Table 6 3 In this study, the adherence to healthcare in adolescents was 21% higher than in YAs. The adolescents also 4 had better results than the YAs in all subcriteria. This result is consistent with the findings of previous 5 studies on adherence to prophylaxis of young PwH. Geraghty et al. showed that 54% of their participants 6 aged 13 to 18 years had high adherence, while this rate was 36% in the 19-28 age group. [START_REF] Ringenbach Stéphanie | RISSO Marini[END_REF] Witktop et al. also 7 reported a higher percentage of adherence to prophylaxis in adolescents (13-17 years) compared to YAs 8 (18-25 years) (83% vs. 53%, p=0.01). [START_REF] Roche Martine | ROJAS[END_REF] Van Os et al. used VERITAS-Pro (Validated Hemophilia Regimen 9 Treatment Adherence Scale-Prophylaxis) and found better adherence in adolescents (12-17 years) than in 10 YAs (18-25 years). 15 These studies focused on adherence to prophylaxis, without including adherence to 11 on-demand treatment and to clinical follow-up.

12 13 Determinants of the adherence to healthcare 14 Being YA and having repeated at least one school grade had negative direct effects on adherence. Presenting 15 mental health concerns was considered simultaneously as a determinant and a mediator in this model. Its 16 direct effect on the outcome was found, whereas the indirect effects through this mediator were not 17 significant.

18 Being YA had a negative direct effect on the adherence to healthcare. During the transition from adolescence 19 to adulthood, like other people, young PwSH may have pressures and changes in their life when they move 20 out of their parents' house, adapt to lifestyle changes, have new relationships and make a career. 4-6,29, [START_REF] Djamel | [END_REF] 21 However, they have an additional difficulty, which is disease management. It seems that the shift in 22 responsibilities for disease management from parents is the most common reason for the non-adherence of 23 YAs. [START_REF] Roche Martine | ROJAS[END_REF] They need to take responsibility for building a relationship with a new medical team when transferring 24 from pediatric to adult services, communicating with caregivers, managing to attend clinic appointments, 25 adapting to a complex treatment plan, performing self-injection and reordering medicine. 5,6,31 These 26 healthcare-related challenges can make them feel uncomfortable following treatment plans and avoid 27 regular medical visits.

28 The negative effect of having repeated at least one school grade on adherence to healthcare has not been 29 analyzed previously, and school-related problems have been rarely studied. A positive association between 30 sufficient school attendance and adherence to prophylaxis was found in a study on QoL in children and 31 adolescents with haemophilia A. 16 Concerning childhood chronic disease like haemophilia, academic 32 difficulty may be a potential marker for the difficulties in seeking and applying health information.

33 Knowledge about the disease, sufficient abilities and skills in treatment management have been considered

34 to be important for adequate adherence to healthcare and successful transition to adult care. [START_REF] Vanessa | Haemophilia Treatment Centre[END_REF]15,31,32 This 3 Mental health concerns (avoidance coping strategies, past negative perceptions, mental HRQoL) directly 4 impaired the adherence to healthcare. Avoidance coping strategies can be considered as a lack of disease 5 acceptance. Previous studies 6,15,31,32 found that struggling with haemophilia acceptance was associated with 6 a lower level of adherence to healthcare, since this adherence was perceived as a burden. PwSH may avoid 7 coping with difficulties in disease management since they think that prophylaxis and clinical follow-up 8 would interfere in daily activities, preventing them from integrating into life with other people. [4][5][6]31,33 9 Avoidance, which is less adaptive for long-term psychological well-being, 34 may not be a suitable way to 10 eliminate psychological distress in a complex phase like the transition process from adolescence and 11 adulthood. Certain concerns, such as stress, worries about understanding health information, fear of lifestyle 12 changes and fear of the unknown, are very common in young PwSH during the transition process and may 13 have negative impacts on the adherence to healthcare. 5,15,16,[START_REF] Djamel | [END_REF]31,35 These concerns stem from their negative 14 experiences with the disease, which may constrain social-emotional functioning, making it difficult for them 15 to prepare for taking responsibility, to maintain a healthy mental state and good QoL. Acquiring autonomy, 16 for example by gradually assuming responsibility, and appropriate social support, such as therapeutic 17 education programs, accompaniment of medical team, support from family, friends and peers, can be 18 effective to enhance mental health and QoL. 6 19 Autonomy was not significantly associated with adherence to healthcare. However, it was associated with 20 improvement in mental health and QoL, which may help young people adapt better to the condition.

21 Autonomy during the transition from adolescence to adulthood can be developed by adjusting life (i.e.,

22 taking new opportunities and responsibilities) for self-regulation and independent decision-making. 36,37 23 Helping young PwSH to acquire autonomy may be a good way to calm their worries about treatment 24 management and gain confidence in making decisions and assuming responsibility.

25 Use of social support coping strategies was positively associated with the presence of mental health concerns.

26 This result showed that people with mental health concerns needed more social support from others (e.g., 27 help, comfort, understanding, advice), probably because they do not have enough personal resources to 28 lighten the burden. Although they are frequently supported by others, this support may not be sufficient for 29 them. These findings may suggest the essential role of family and social support in enhancing mental health 30 for young PwSH during their transition process.

31 Although we assumed that comorbidities and complications of severe haemophilia might have impacts on 32 the adherence to healthcare, significant associations between these clinical factors and the adherence were 2 Certain limitations need to be acknowledged. This study was designed as a cross-sectional study to provide 3 a general understanding of the adherence to healthcare in young PwSH. Future longitudinal study allowing 4 to follow PwSH from adolescence to adulthood could be proposed to evaluate changes during the transition 5 process and shed light on the findings of our study.

6

The validity of the results might be affected by the non-inclusion of some eligible people, especially those 7 who did not return the questionnaire. The non-participants were more often followed by the HTCs in the 8 same hospital and same medical service from adolescence to adulthood. This might suggest that they were 9 less interested in the transition issue.

10 Although the main outcome of this study is still debatable as a measure of the successful transition, it allows 11 to assess the adherence to healthcare more completely than previous studies, as it combines physician-and 12 patient-reported adherence to treatment regimens and to clinical follow-up, as well as the number of 13 prophylactic injections, number of follow-up visits and number of hemorrhagic events collected in the 14 FranceCoag registry database. Moreover, the level of adherence to healthcare is in the top five of healthcare 15 transition outcomes identified by the Delphi process with an interdisciplinary group of medical and 16 psychosocial professionals. 38 It can apply for adolescents and YAs, allowing for a more specific assessment 17 of the potential impact on the transition from adolescence to adulthood. Since the distance between Likert-18 type response options might not be equal, , 39 and since some of the subcriteria assessing the adherence in 19 our study were binary criteria and some others were based on 5-point Likert-scales, the Likert-type 20 responses were dichotomized to make all the subcriteria similar in order to create a composite endpoint .

21 To the best of our knowledge, the sample size of this study is larger than that of previous studies on 22 adherence to healthcare in young PwSH. The participants were included from the exhaustive national cohort 23 of PwSH in France (FranceCoag registry).

24 Although not all the associations between the potential determinants and the adherence to healthcare 25 hypothesized in the theoretical model were significant, this model suited to explore the determinants of the 26 adherence to healthcare, as recommended indicators for goodness of fit were acquired. Moreover, the SEM 27 had higher statistical power than that of traditional regressions and allowed to analyze associations between 28 the determinants. 40 Additionally, the determinants were chosen based on data from the literature and 29 consistent with SMART, which can be considered as an ideal framework to identify the determinants of

If under prophylaxis:

-Having no difficulties in adherence to prophylaxis or having difficulties but missing treatment less than once a week / Having difficulties and missing treatment once a week or more

If under on-demand treatment:

-Having no difficulties in recognizing early signs of a: associations of the determinants with two mediators are presented in Table 6.

b: factor loadings of the latent variable "Autonomy" manifested by attitudinal, functional and emotional autonomy of the Noom questionnaire are 0.80; 0.74; 0.47, respectively.

2 3 8

 28 Title of paper: 4 Determinants of adherence and consequences of the transition from adolescence to adulthood among young 5 people with severe haemophilia (TRANSHEMO): A multicentric French national observational cross-6 sectional study based on the FranceCoag registry 7 Authors:

7

  and had a history of major orthopedic interventions more often than the participants. Regarding 8 organizational factors, the non-participants more often stayed in the same hospital and service from 9 adolescence to adulthood. The non-participants came from the HTCs without education activities for 10 transition more often than the participants. The remaining sociodemographic and clinical characteristics did

  academic achievement emphasizes the essential role of adapted and targeted education programs, 2 during transition process.5,6

12 1

 12 Strengths and limitations

  Very high / High to very low Patient-reported adherence to clinical follow-up Visiting Haemophilia Treatment Centre (HTC) at least once a year / Not visiting HTC every year If under prophylaxis, number of prophylactic injections over the last three months (FranceCoag registry) Quantitative variable In agreement / Not in agreement with the recommended number If under prophylaxis, physicianreported adherence to prophylaxis

Figure 1 .Figure 2 .

 12 Figure 1. Model specification for the determinants of the adherence to healthcare. Rectangles represent observed variables, ovals represent latent variables, single-headed arrows represent direct effects, and double-headed row represents correlation.

  Inserm, U1296 Unit, Radiation: Defense, Health and Environment, Lumière Lyon 2 University, Lyon, Centre d'Études et de Recherche sur les Services de Santé et la Qualité de Vie It is necessary to gain insights into adherence to healthcare in people with severe haemophilia

	1 Correspondence 1 ABSTRACT
	2 RESSEGUIER Noémie 2 Introduction:
	3 27 Boulevard Jean Moulin
	4 UR 3279 -5 Faculté de Médecine, Campus Timone, 13385 Marseille Cedex 05
	6 Phone Number: 0033 4 91 32 42 72
	7 E-mail: noemie.resseguier@univ-amu.fr
	8
	9 Key words: Adherence, haemophilia, transition, quality of life, adolescents, young adults
	10
	11 Word count: 3188 12	F o r F o r
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	32 France
	33 14 Laboratory of Clinical Psychology, Psychopathology and Psychoanalysis, UR 3278, Aix-Marseille
	34 University, Aix-en-Provence, France
	35 15 AP-HM, Haemophilia Treatment Centre, Children Hospital La Timone, Marseille, France
	36 16 French Patients' Association for People with Haemophilia (AFH), Paris, France
	37
	38
		2 3

* Coauthors from the TRANSHEMO Study Group are listed in Appendix

10

  Methods: A multicenter, observational, cross-sectional study was conducted in 2017-2019 on PwSH aged 11 between 14 and 17 years (adolescents) or between 20 and 29 years (YAs), included in the FranceCoag

12 registry and having completed the questionnaires. The adherence to healthcare (treatment regimens and 13 clinical follow-up) was compared between adolescents and YAs using the chi-squared test. The 14 determinants of this adherence were analyzed by structural equation modeling.

15 Results: There were 277 participants, 107 adolescents and 170 YAs. The rate of adolescents adhering to 16 healthcare was 82.2%, while the rate of YAs was 61.2% (p<0.001). The barriers to the adherence to 17 healthcare were being YA, having repeated at least one school grade and presenting mental health concerns.

  Haemophilia is a rare and inherited bleeding disorder affecting mainly men due to deficiency in clotting 3 factors.1 People with severe haemophilia (PwSH) have less than 1% blood clotting factors, which can cause 4 serious hemorrhagic events (e.g., intracranial hemorrhage, hemarthrosis) if insufficiently treated. It is 5 possible to avoid these events by haemophilia care, such as substitutive therapies and lifelong regular 6 clinical follow-up, whose major issue is adherence.

	1 INTRODUCTION
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18 Conclusion: Adolescents had better adherence to healthcare than YAs. According to the determinants 19 enlightened in this project, targeted supportive strategies and adapted therapeutic education programs can 20 be developed for young PwSH to facilitate their adherence to healthcare.

MATERIALS AND METHODS 2 Study design and study population

  The measurements are presented in Table1. International validated scales and ad hoc items were used to 3 assess sociodemographic, clinical, psycho-behavioral and organizational (HTC-related) aspects. They were 4 completed by the referring physicians of the participants and/or by the participants themselves. Descriptive analysis. Categorical variables were described by counts and percentages. Quantitative 8 variables were described by mean values and standard deviations (SD). Sociodemographic and clinical data were 10 compared using the chi-squared test or Fisher's test for categorical variables, and Student's t test or 11 Mann-Whitney U test for quantitative variables.

	1 Other measurements 1 3 The study protocol of the TRANSHEMO project was previously published. 17 4 It was designed as a multicenter, observational, cross-sectional study involving 29 Haemophilia Treatment 5 Centers (HTCs) in France. The inclusion criteria were PwSH A or B, aged between 14 and 17 years 2 5
	6 (adolescent group) or between 20 and 29 years (YA group), followed in one of the participating HTCs, 6 Statistical analyses
	7 included in the national registry FranceCoag, 18 affiliated with the French social security system and having 7
	8 no reading or writing difficulties. Written consent was obtained from the YAs and the parents (or legal
	9 representatives) of adolescents. Participants were those who met all the inclusion criteria, consented to
	10 participate and sent back the questionnaires. The inclusion period started in February 2017 and ended in
	o r 14 The study was approved by the French Ethical Research Committees, by the French National Agency for 11 February 2019. 12 13 Ethical approval F o r
	P e 15 the Safety of Medicines and Health Products (reference number ID RCB: 2016-A01034-47), and by French 16 Data Protection Authority, whose principles are in line with those of the General Data Protection Regulation. P e
	e r 17 The protocol was registered in ClinicalTrials.gov (NCT02866526). 18 e r
	R e v i e w 20 Sociodemographic and psycho-behavioral data were collected by a self-administered questionnaire sent by 19 Data collection R e 21 post. There were two versions of the questionnaire: one for adolescents and one for YAs. The questionnaires v 22 for adolescents were completed by the adolescents themselves. People who agreed to participate completed i e 23 it at home and sent it back in a prepaid envelope. Clinical data were extracted from FranceCoag's database 24 and filled in a short questionnaire by the referring physician of each participant. Organizational data were w
	25 collected by an ad hoc questionnaire completed by the physician in charge of each HTC.
	26
	27 Measurements
	28 Main evaluation criterion
	29 The adherence to healthcare was assessed using the seven dichotomized criteria described in Table 1.
	30 A composite quantitative endpoint was created by adding these criteria (7 for people under prophylaxis, 5
	31 for people under on-demand treatment). Adherent people were defined as those having 4 to 7 points if under
	32 prophylaxis, and as those having 3 to 5 points if under on-demand treatment.
	33
	6 7

9 Comparison between participants and non-participants.

Analyses of determinants of adherence to healthcare 15

Univariate analysis. The characteristics of participants were compared between adherent and non-16 adherent people by using the chi-squared test or Fisher's test for categorical variables, and Student's t test 17 or Mann-Whitney U test for quantitative variables. The univariate logistic regression was performed to

  Model fit. Indicators to assess the model fit were Comparative Fit Index (CFI), Tucker Lewis Index 4 (TLI), Root Mean Square Error of Approximation (RMSEA), Standardized Root Mean Square Residual 5 (SRMR). Recommended cutoffs for good fit are CFI ≥ 0.90; TLI ≥ 0.95; RMSEA and SRMR < 0.08.[START_REF] Hacker | IDDIR Hayet[END_REF] 6 All statistical tests were two-sided with p-value < 0.05 as statistically significant and performed with 7 RStudio version 3.4.1 (RStudio, Boston, MA). The Rstudio "lavaan" package was used to conduct the SEM. According to FranceCoag's database and considering the inclusion criteria, 543 questionnaires were sent.

	1 RESULTS
	2 Participants
	3
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	9

8 1 and total effects. Standardized coefficients (𝝱) were calculated for the association between the two mediators 2 and the other determinants.

3

Table 5

 5

), people having repeated at least one school grade (OR 0.31; 95% CI 20 0.18-0.53) and people having history of major orthopedic interventions (OR 0.40; 95% CI 0.18-0.92) were 21 less likely to adhere to healthcare, while members of the French association for people with haemophilia 22 (OR 1.74; 95% CI 1.04-2.97) and people under prophylaxis (OR 2.51; 95% CI 1.27-4.94) were more likely 23 to adhere to healthcare. No significant differences were found in comorbidities, psycho-behavioral and 24 organizational factors.

Comparison of the adherence to healthcare between adolescents and YAs

	1 DISCUSSION
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	and Table 7. Being YA (OR 0.64; 95% CI 0.41-0.99),
	26 having repeated at least one school grade (OR 0.47; 95% CI 0.31-0.71) and presenting mental health
	27 concerns (OR 0.37; 95% CI 0.15-0.94) had negative effects on adherence to healthcare. No significant direct
	28 effects of the remaining factors on the adherence, neither indirect effects were found.
	29 Regarding the association between the determinants, higher level of autonomy was associated with fewer
	30 mental health concerns (𝝱= -0.10, p<0.001) and better haemophilia-specific QoL (𝝱= -0.09, p<0.001).
	31 Higher frequency of using social support coping strategies was associated with more mental health concerns
	32 (𝝱= 0.09, p=0.022).
	33 Indicators verifying good fit of the model were CFI=0.919, TLI=0.964, RMSEA=0.031, SRMR=0.054.
	34
	10

Table 1 . Measurements Dichotomized criteria assessing the adherence to healthcare Original Dichotomized

 1 Based on these barriers, healthcare professionals can develop targeted supportive strategies and adapted 2 therapeutic education programs, which will be proposed to young PwSH according to their profiles. The 3 TRANSHEMO project is based on a mixed methods design with quantitative and qualitative phases. The 4 qualitative phase will shed light on the results from the quantitative phase and will be published later. Nicolas GIRAUD, Thomas SANNIE) contributed with the communication actions for 2 participants. The members of the FranceCoag registry (Karine BAUMSTARCK, Mohamed BOUCEKINE, 3 Vanessa MILIEN, Clemence TABELE, Marie VIPREY, Romain VOLTZENLOGEL) contributed by 4 supplying datasets of the registry. Ngoc Anh Thu NGUYEN and Noémie RESSEGUIER performed the 5 statistical analyses. Ngoc Anh Thu NGUYEN, Noémie RESSEGUIER, Pascal AUQUIER, Hervé 6 CHAMBOST and Any BELTRAN ANZOLA wrote the draft of the manuscript. All authors critically 7 reviewed the manuscript, provided input on data interpretation, and approved the final version of manuscript. The project was funded by a grant from the French Ministry of Social Affairs and Health (Ministère des 10 Affaires sociales et de la Santé, grant number: PREPS-15-0597) and was supported by the 'Filière MHEMO'

	30 transition for people with serious pediatric illness conditions. 11
	31
	32 CONCLUSION

33 Adolescents with severe haemophilia adhere better to healthcare than YAs. The barriers to the adherence to 34 healthcare were being YA, having repeated at least one school grade and presenting mental health concerns.

14 Data sharing statement

15

The data that support the findings of this study are available on request from the corresponding author.

16 The data are not publicly available due to privacy or ethical restrictions.

Table 2 . Previous findings on adherence to treatment regimens of PwH Findings References Comparison between adolescents and young adults

 2

	hemorrhage / Having difficulties in recognizing early signs of
	hemorrhage

Table 3 . Characteristics of the participants and non-participants

 3

	Participants (n=277)	Non-(n=266) participants	p-value

Table 4 . Comparison of the adherence to healthcare between adolescents and young adults

 4

	All participants (n=277)	Adolescents (n=107)	Young (n=170) adults	p-value

Table 5 . Characteristics of the adherent and non-adherent people

 5

		Non-adherent	Adherent people
		people (n=85)	(n=192)	p-value	OR (95% CI)
		n/mean (%/SD)	n/mean (%/SD)
	Sociodemographics
	Group			<0.001
	Adolescents	19 (17.8)	88 (82.2)
	Young adults	66 (38.8)	104 (61.2)		0.34 (0.19-0.60)
	Age, years	22.9 (4.4)	20.6 (5.2)	<0.001	0.91 (0.86-0.96)
	Age group (if adolescent)			0.073
	14-15	5 (10.4)	43 (89.6)
	16-17	14 (23.7)	45 (76.3)		0.37 (0.11-1.07)
	Age group (if young adult)			0.888
	20-24	31 (38.3)	50 (61.7)
	25-29	35 (39.3)	54 (60.7)		0.96 (0.51-1.77)
	Sex			>0.999
	Male	84 (30.8)	189 (69.2)
	Female	1 (25.0)	3 (75.0)		1.33 (0.17-27.17)
	Living near the HTC			0.440
	No	44 (28.8)	109 (71.2)
	Yes	41 (33.1)	83 (66.9)		0.82 (0.49-1.37)
	Socioeconomic status of the family			0.059
	Low or medium	48 (35.8)	86 (64.2)
	High	36 (25.4)	106 (74.6)		1.64 (0.98-2.77)
	Academic difficulty (at least one
	grade repetition at school)			<0.001
	No	29 (19.3)	121 (80.7)
	Yes	55 (43.7)	71 (56.3)		0.31 (0.18-0.53)
	Member of AFH			0.037
	No	54 (36.0)	96 (64.0)
	Yes	31 (24.4)	96 (75.6)		1.74 (1.04-2.97)
	Clinical characteristics
	Type of haemophilia			0.712
	Haemophilia A	73 (30.3)	168 (69.7)
	Haemophilia B	12 (33.3)	24 (66.7)		0.87 (0.42-1.89)
	At least one comorbidity			0.546
	No	76 (30.2)	176 (69.8)
	Yes	9 (36.0)	16 (64.0)		0.77 (0.33-1.89)
	HIV infection			-
	No	85 (30.7)	192 (69.3)
	Yes	0	0		-
	HBV infection			0.442
	No	82 (30.4)	188 (69.6)
	Yes	3 (42.9)	4 (57.1)		0.58 (0.13-3.01)
	HCV infection			0.555
	No	85 (30.7)	189 (69.3)
	Yes	0	3 (100.0)		3.16 (0.30-426.43)

Number of injections per week (if under prophylaxis)

  OR, odds ratio; CI, confidence interval; HTC, haemophilia treatment center; HIV, human immunodeficiency virus; HBV, hepatitis B virus; HCV, hepatitis C virus; FAD, Family Assessment Device; AFH, French association for people with haemophilia; HRQoL, health-related quality of life; PCS, physical component summary; MCS, mental component summary; Brief COPE, Brief Coping Orientation to Problems Experienced; ZTPI, Zimbardo Time Perspective Inventory.

	Social support	3.8 (1.1)	3.7 (0.9)	0.251	0.86 (0.66-1.11)
	Problem solving	5.1 (1.3)	5.0 (1.4)	0.803	0.98 (0.81-1.18)
	Avoidance	3.6 (0.8)	3.4 (0.7)	0.147	0.77 (0.53-1.10)
	Positive thinking	5.2 (1.1)	5.4 (1.2)	0.190	1.16 (0.93-1.44)
	Autonomy
	(scores of Noom questionnaire)
	Attitudinal autonomy	17.3 (4.0)	17.4 (4.4)	0.883	1.00 (0.94-1.07)
	Functional autonomy	18.5 (3.8)	18.2 (4.1)	0.550	0.98 (0.92-1.05)
	Emotional autonomy	18.1 (2.9)	18.0 (3.9)	0.910	1.00 (0.93-1.07)
	Time perspective (scores of ZTPI)
	Negative past	2.75 (0.7)	2.68 (0.7)	0.426	0.86 (0.59-1.24)
	Future	3.29 (0.5)	3.29 (0.6)	0.973	0.99 (0.62-1.58)
	Satisfaction with the clinical follow-
	up of the HTC			0.228
	No	6 (46.2)	7 (53.8)
	F o r P e e r R e v i e w 79 (29.9) 185 (70.1) Organizational (HTC-related) factors Yes Type of healthcare transition in the HTC Same hospital, two different services (pediatric/adult) 27 (27.0) 73 (73.0) Same hospital and service for adolescent and adults 58 (32.8) 119 (67.2) Pediatrician in the medical team of the HTC No 24 (34.8) 45 (65.2) Yes 61 (29.3) 147 (70.7) Setup of therapeutic education activities for transition by the HTC No 67 (30.9) 150 (69.1) Yes 18 (30.0) 42 (70.0) o r P e e r R e v Abbreviations: F i e w	0.438 0.317 0.395 0.896	2.01 (0.63-6.23) 0.76 (0.44-1.03) 1.29 (0.71-2.28) 1.04 (0.57-1.98)
	1 injection	5 (20.8)	19 (79.2)
	2 or more than 2 injections	60 (28.3)	152 (71.7)		0.67 (0.21-1.74)
	Psycho-behavioral factors
	Family functioning (score of FAD)	1.87 (0.6)	1.78 (0.6)	0.248	0.78 (0.51-1.19)
	Participation in therapeutic
	education activities			0.949
	No	28 (30.4)	64 (69.6)
	Yes	57 (30.8)	128 (69.2)		0.98 (0.57-1.68)
	Quality of life
	Physical health (PCS)	46.3 (10.7)	48.2 (10.2)	0.154	1.02 (0.99-1.04)
	Mental health (MCS)	47.8 (10.7)	49.0 (9.2)	0.344	1.01 (0.99-1.04)
	Haemophilia-specific quality of life (Haemo-QoL Index)	33.4 (24.9)	32.9 (21.9)	0.889	1.00 (0.99-1.01)
	Coping strategies
	(scores of Brief COPE)

Table 6 . Association of the determinants with two mediators of the structural equation model

 6 French association for people with haemophilia. † Latent variable manifested by Past-negative perspective of ZTPI, Avoidance of Brief COPE, Mental component summary (MCS) of SF-12. ‡ Latent variable manifested by attitudinal, functional and emotional autonomy of the Noom questionnaire. § Latent variable manifested by item 2-feeling, item 3-view, item 4-family, item 5-friends, item 6others, item 7-sports of the Haemo-QoL Index.

	Standardized coefficient (𝝱)	p-value

Table 7 . Determinants of the adherence to healthcare

 7

	Direct Effect	Total Indirect Effect †	Total Effect
	OR (95% CI)	OR (95% CI)	OR (95% CI)

Clinical characteristics


Alexandre Bergounioux

Romane Lhotellier

Thierry Roisnel

Nicolas Gouault

Gilles Argouarch
email: gilles.argouarch@univ-rennes.frclaudia.lalli@univ-rennes.fr

Claudia Lalli

Aza-Prins Cyclization on Ketones to Access Piperidines with Tetrasubstituted Carbon Stereocenters

Keywords: Aza-Prins, Ketones, 2-Spiropiperidines, tetrasubstituted stereocenter, Titanium

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

The modern Prins cyclization involves simple starting materials such as a homoallylic alcohol or amine and an aldehyde, to provide tetrahydropyrans or piperidines in only one step with the concomitant carbon-carbon and carbon-heteroatom bond formation. While the Prins cyclization is very well documented, [1] its nitrogen-based version is significantly less developed, [2] although the piperidine ring is more widespread than tetrahydropyrans in natural products and drugs. [3] Despite the great potential of aza-Prins cyclization for the synthesis of natural and/or bioactive compounds, particular attention has been paid solely to the diversification of the homoallylic amines. In particular, aza-silyl Prins reactions [4] have been developed to obtain 3,4dehydropiperidines with loss of the silicon group, alkynyl aza-Prins annulations [START_REF] Abdul-Rashed | [END_REF] have provided similar compounds with a nucleophile in position C-4, and finally cascade aza-Prins reactions [START_REF] Cuprova | Advances in heterocyclic chemistry[END_REF] have been reported for the formation of fused bi-and tricyclic systems. Concerning the carbonyl partner, apart from aldehydes, only two examples have been published to date: one with a very narrow range of ketones [START_REF] Liu | [END_REF] and the other limited to 1,2-dicarbonyl substrates, [8] both leading to the synthesis of piperidines containing a tetrasubstituted carbon stereocenter in position 2.

To the best of our knowledge, an effective aza-Prins cyclization with non-activated ketones has not been reported so far. In continuation with our interest in Prins processes, [9] we disclose here the first aza-Prins cyclization with aliphatic, aromatic, and heterocyclic ketones. Noteworthy, the present method competes well with other accesses to 2-spiranic piperidines. [10] We have already reported the effectiveness of a synergistic combination of Lewis and Brønsted acids in the aza-Prins cyclization between secondary alkyl homoallylic amines and aldehydes. [11] Therefore, we first investigated the reactivity between N-benzyl homoallylamine 1 and cyclohexanone (2a) in the presence of 1 equiv. of TiCl 4 and 0.1 equiv. of p-TsOH•H 2 O in DCM at 60 °C in a sealed Schlenk tube for 24 h (Table 1, entry 1). Unfortunately, a disappointing lack of reactivity has been observed. We next investigated the effect of the Brønsted and Lewis acids alone (Table 1, entries 2 and 3 respectively), and only 9% conversion of the desired 2-spiropiperidine 3a could be observed in the presence of 1 equiv. of TiCl 4 by 1 H-NMR analysis of the crude reaction mixture. Encouraged by this result, we decided to explore the effect of different additives such as molecular sieves, water and also PPh 3 (Table 1, entries 4-6) without further improvements. Gratifyingly, the use of 1 and then of 2 equiv. of triethylamine (TEA) (Table 1, entries 7 and8) led to respectively 25% and 52% conversion into the desired product. This last result prompted us to examine diamines, in particular N,N,N',N'tetramethylethylenediamine (TMEDA), N,N,N',N'tetraethylethylenediamine (TEEDA), 1,4diazabicyclo [2,2,2]octane (DABCO), and 1,2dipiperidinylethane. As depicted in entries 9-12 in Table 1, TEEDA was found to be superior, leading to 2-spiropiperidine 3a in 61% conversion, and was retained as the additive of choice for the rest of this study. In order to further improve the reactivity, the reaction was next performed in 1,2-DCE at 80 °C and indeed the analysis of the crude mixture showed 75% conversion (Table 1, entry 13). Increasing again the temperature to 100 °C proved detrimental to the reaction as by-products arising from the selfcondensation/enolization of ketone 2a became too abundant. Finally, the stepwise addition of an excess of ketone during the reaction, i.e. 1.5 equiv. at the start followed by 0.5 equiv. after 8 hours, allowed us to obtain 3a in 90% conversion (Table 1, entry 14). d) Reaction was performed with 1.5 + 0.5 equiv. of 2a.

Under the above optimized conditions, compound 3a was isolated in 63% yield and the scope of the reaction was first evaluated on various other cyclic ketones (Scheme 1). Cyclopentanone (2b) and cycloheptanone (2c) gave satisfactory results albeit both reaction needed to be carried out at 100 °C, and products 3b and 3c were isolated in 30% and 49% yields, respectively. Next, a series of cyclohexanone derivatives substituted at the C-4 position 2d-f were screened, and regardless the presence of an alkyl or aryl group in this position, these substrates readily participated in the reaction leading to 3d, 3e, and 3f in 87%, 57%, and 58% yields, respectively. It has to be stressed that cyclanic isomerism has been observed for this series of 2spiropiperidines and that the formation of the most stable trans isomer is favoured, if not exclusive, as in the case of 3e possessing the bulky t-Bu group. When present, the two isomers are separable by column chromatography on silica gel and the relative stereochemistry of 3f-trans has been unambiguously confirmed by single crystal X-ray analysis, as shown in Figure 1. [12] Scheme 1. Scope of the aza-Prins cyclization. Reaction conditions: 1 (1 mmol), 2 (1.5 + 0.5 equiv.), TiCl 4 (1 equiv.), TEEDA (1 equiv.) in 1,2-DCE at 80 °C in a sealed Schlenk tube for 24 h. Yields were determined after purification by column chromatography on silica gel (General procedure A). a) Reactions performed with 2 (1.5 + 1.5 equiv.) at 100 °C (General procedure B). b) Reactions performed without TEEDA (General procedure C). The aza-Prins cyclization was then applied to more challenging acyclic substrates. In the presence of 3pentanone (2g), 4-heptanone (2h), and benzophenone (2i), we were pleased to observe the formation of compounds 3g-i. Given the difficulty of constructing tetrasubstituted carbon stereocenters from acyclic systems due to their inherent conformational mobility, [13] the yields obtained here, ranging from 30% to 34%, are still acceptable.

Encouraged by those results, we next examined the reaction between N-benzyl homoallylamine 1 and various activated (di)ketones. We were satisfied to conclude that this panel efficiently underwent aza-Prins cyclization with a very marked diastereoselectivity in favour of the trans isomer. As depicted in Scheme 1, benzil (2j) and its analog 4,4'dimethylbenzil (2k) gave separable mixtures of diastereomers 3j and 3k with overall yields of 64% and 50%, respectively. The use of keto-ester 2l allowed to recover 3l-trans and 3l-cis in 70% yield, whereas trifluoromethylated ketone 2m gave piperidines 3m-trans and 3m-cis in 60% overall yield. Moreover, single crystal X-ray analysis of 3l-trans allowed to establish the relative stereochemistry of the two stereogenic centers that were formed during the reaction (Figure 1). [12] Finally, we looked at the reactivity of Nheterocyclic ketones. Piperidones in particular were considered because their reaction could provide an access to spiro-fused dipiperidines, a class of compounds withacc strong biological activities. [14] Therefore, the aza-Prins cyclization between 1 and 1methyl-4-piperidone (2n) was attempted under the optimized conditions but led to a loss of the reactivity. It was assumed that, if TEEDA is required to carry out the reaction, the combination of this additive and a nitrogen-containing substrate could deactivate the titanium active species. Indeed, without adding TEEDA to the reaction medium, the reactivity was recovered and product 3n was isolated in 60% yield (Scheme 1). This trend could be extended to 1benzyl-4-piperidone (2o), leading to the corresponding dipiperidine 3o in 49% yield.

In summary, we have developed a titanium-TEEDA promoted aza-Prins cyclization of aliphatic, aromatic, and heterocyclic ketones. Our methodology provides an access to piperidines bearing tetrasubstituted or even spiranic carbon centers at the C-2 position, which are common naturally occuring scaffolds. The reaction proceeds diastereoselectively in favour of the trans isomer, both when nonsymmetric substrates are used and when cyclanic isomerism is present. Further applications and substrate variations are in progress in our laboratory.

Experimental Section

General procedure A To a solution of N-benzyl homoallylamine 1 (163 mL, 1 mmol) and the ketone (1.5 mmol) in dry 1,2-DCE (10 mL) was added TEEDA (213 mL, 1 mmol). The mixture was stirred for 10 min and a solution of TiCl 4 (1 mL, 1.0 M in DCM, 1 mmol) was added dropwise. The solution was stirred at 80 °C for 8 hours. After cooling to room temperature, the ketone (0.5 mmol) was again added to the mixture and the solution was further stirred at 80 °C overnight. After cooling to room temperature, the reaction was quenched with sat. NaHCO 3 (15 mL) and filtered on a Buchner funnel (1 layer of filter paper). The product was extracted with DCM (2 x 20 mL), the combined organic phases were washed with water (1 x 20 mL), dried over anhydrous MgSO 4 , and evaporated to dryness. The residue was purified by column chromatography on silica gel.

General procedure B

To a solution of N-benzyl homoallylamine 1 (163 mL, 1 mmol) and the ketone (1.5 mmol) in dry 1,2-DCE (10 mL) was added TEEDA (213 mL, 1 mmol). The mixture was stirred for 10 min and a solution of TiCl 4 (1 mL, 1.0 M in DCM, 1 mmol) was added dropwise. The solution was stirred at 100 °C for 8 hours. After cooling to room temperature, the ketone (1.5 mmol) was again added to the mixture and the solution was further stirred at 100 °C overnight. After cooling to room temperature, the reaction was quenched with sat. NaHCO 3 (15 mL) and filtered on a Buchner funnel (1 layer of filter paper). The product was extracted with DCM (2 x 20 mL), the combined organic phases were washed with water (1 x 20 mL), dried over anhydrous MgSO 4 , and evaporated to dryness. The residue was purified by column chromatography on silica gel.

General procedure C

A solution of N-benzyl homoallylamine 1 (163 mL, 1 mmol) and the ketone (1.5 mmol) in dry 1,2-DCE (10 mL) was stirred for 10 min before adding a solution of TiCl 4 (1 mL, 1.0 M in DCM, 1 mmol) dropwise. The solution was stirred at 80 °C for 8 hours. After cooling to room temperature, the ketone (0.5 mmol) was again added to the mixture and the solution was further stirred at 80 °C overnight. After cooling to room temperature, the reaction was quenched with sat. NaHCO 3 (15 mL) and filtered on a Buchner funnel (1 layer of filter paper). The product was extracted with DCM (2 x 20 mL), the combined organic phases were washed with water (1 x 20 mL), dried over anhydrous MgSO 4 , and evaporated to dryness. The residue was purified by column chromatography on silica gel.

COMMUNICATION

Aza-Prins Cyclization on Ketones to Access Piperidines with Tetrasubstituted Carbon Stereocenters

Figure 1 .

 1 Figure 1. ORTEP representations of 3f-trans (top) and 3ltrans (bottom) at 50% thermal ellipsoids. Hydrogen atoms are omitted for clarity.



  one-step  3 bonds formation  quaternary/spiranic stereocenter  diastereoselective Adv. Synth. Catal. Year, Volume, Page -Page Alexandre Bergounioux, a Romane Lhotellier, a Thierry Roisnel, a Nicolas Gouault, a Gilles a, * and Claudia Lalli a, *

Table 1 .

 1 Optimization of the reaction conditions between N-benzyl homoallylamine 1 and cyclohexanone (2a).

	Entry a) Additive	Solvent	T	Conv.
		(equiv.)		(°C)	(%) b)
	1	p-TsOH•H 2 O	DCM	60	0
		(0.1)
	2 c)	p-TsOH•H 2 O	DCM	60	0
		(1)
	3	none	DCM	60	9
	4	MS (3Å)	DCM	60	0
	5	H 2 O (1)	DCM	60	0
	6	PPh 3 (1)	DCM	60	0
	7	TEA (1)	DCM	60	25
	8	TEA (2)	DCM	60	52
	9	TMEDA (1)	DCM	60	58
	10	TEEDA (1)	DCM	60	61
	11	DABCO (1)	DCM	60	43
	12	1,2-	DCM	60	55
		dipiperidinyl
		ethane (1)
	13	TEEDA (1)	1,2-DCE 80	75
	14 d)	TEEDA (1)	1,2-DCE 80	90

a) Unless otherwise noted, reactions were performed with homoallylamine 1 (1 mmol, 1 equiv.), 2a (1 mmol, 1 equiv.), and TiCl 4 (1 mmol, 1 equiv.) in a sealed Schlenk tube for 24 h. b) Conversions were calculated by 1 H-NMR analysis of the crude reaction mixtures. c) Reaction was performed without TiCl 4 .

Acknowledgements

The authors thank Université de Rennes and CNRS for financial support. The CRMPO of the Institut des Sciences Chimiques de Rennes for mass measurement, the Centre de Diffractométrie X for X-ray analyses and FEDER funds for acquisition of singlecrystal equipment are gratefully acknowledged. Part of this work has been performed using the PRISM core facility (Biogenouest©, UMS Biosit, Université de Rennes 35000 RENNES, FRANCE). Finally, Solenn Ferron is acknowledged for running 19 F NMR experiments.