Maher Abla
email: maher.abla@ul.edu

Catherine Ladavière
email: catherine.ladaviere@univ-lyon1.fr

Stéphane Trombotto
email: stephane.trombotto@univ-lyon1.fr

Impact of HILIC amino-based column equilibration conditions on the analysis of chitooligosaccharides

Keywords: chitosan, oligomer, HILIC, chromatography, anomer, counter-ion

Obtaining well-defined Chitooligosaccharides (COS) structures is very important as their physicochemical and biological properties depends strongly on their degree of polymerization (DP) and degree of N-acetylation (DA).

Hydrophilic Interactions Liquid Chromatography (HILIC), that is commonly used for COS analyses, suffers from several drawbacks, related to their complex structure, leading to broadening/splitting of the chromatogram signals.

We investigated, herein, on the role of the equilibration step of an amino-based HILIC column on the separation of COS DP≤ 5. We demonstrated that COS could be separated according to their DP after equilibrating the column by a NaHCO3 buffer solution (100 mM, pH=10) and mild elution chromatographic parameters (neutral mobile phase, r.t.); or according to their DA after equilibrating the column by an NH4Ac buffer solution (50 mM, pH=4.5).

Also, the nature of the counterion of the column stationary phase was found to affect both retention times and signal profiles of analyzed COS.

remains challenging, several studies have shown that physicochemical and biological properties of COS are strongly dependent on their DP and DA. [START_REF] Xia | Biological activities of chitosan and chitooligosaccharides[END_REF][START_REF] Dos Santos | Partially Acetylated Chitosan Oligo-and Polymers Induce an Oxidative Burst in Suspension Cultured Cells of the Gymnosperm Araucaria angustifolia[END_REF] Therefore, it seems to be crucial to find out novel methods of preparation controlling both DA and DP. To this purpose, two main pathways were investigated: (i) the chemical synthesis of COS starting from GlcN as building block, although this pathway still requires complicated and multisteps synthetic processes which limit the preparation of higher COS or their preparation at gram scale; [START_REF] Yang | Recent advances in the synthesis of chitooligosaccharides and congeners[END_REF] (ii) the development of robust analytical technical methodologies that could improve the separation or identification of these COS entities according to their DA and/or DP. Several chromatography techniques have been employed for the separation of COS. For instance, size exclusion chromatography, [START_REF] Chambon | Efficient chemoenzymatic synthesis of lipo-chitin oligosaccharides as plant growth promoters[END_REF] adsorption chromatography, [START_REF] Semeňuk | Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1[END_REF] metal affinity chromatography, [START_REF] Dévédec | Separation of chitosan oligomers by immobilized metal affinity chromatography[END_REF] or ion exchange chromatography. [START_REF] Xiong | Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro[END_REF] More particularly, hydrophilic interaction liquid chromatography (HILIC) has been widely used for the analysis of highly polar compounds, especially simple and complex carbohydrates. [START_REF] Fu | Carbohydrate separation by hydrophilic interaction liquid chromatography on a 'click' maltose column[END_REF][START_REF] Lowenthal | Separation of monosaccharides hydrolyzed from glycoproteins without the need for derivatization[END_REF][START_REF] Nagy | Recent Liquid Chromatographic Approaches and Developments for the Separation and Purification of Carbohydrates[END_REF]. In comparison to other HPLC techniques, HILIC offers several advantages such as: (i) to separate highly polar compounds that cannot be well-retained on reverse phase liquid chromatography (RPLC); (ii) to overcome poor solubility drawbacks of some polar analytes, encountered with normal phase liquid chromatography (NPLC), and finally, (iii) to use no expensive ion pairs reagents as required for ion-exchange chromatography (IC). [START_REF] Buszewski | Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique[END_REF] A large variety of HILIC columns exists and each one has its own mechanism of retention. One of these columns is the amino-based one which results from grafting amino groups onto a silica-based, [START_REF] Fu | Separation of carbohydrates using hydrophilic interaction liquid chromatography[END_REF] or a polymeric based stationary phase. [START_REF] Honda | Novel glucoamylase-resistant glucooligosaccharides with adjacent α-1, 6 branches at the non-reducing end discovered in Japanese rice wine, sake[END_REF] The latter one presents many advantages in terms of reproducibility, durability, and high resistance to both alkaline and acidic eluents [START_REF] Hirata | New, stable polyaminebonded polymer gel column[END_REF] and has been commonly adopted for COS analysis. [START_REF] Yoon | Enzymatic synthesis of chitooligosaccharides in organic cosolvents[END_REF][START_REF] Dong | Key Technologies of Enzymatic Preparation for DP 6-8 Chitooligosaccharides[END_REF] Despite all the progress accomplished in the analysis of carbohydrates; these biomolecules remain challenging to analyze due to their complex spatial structure and their multiple isomers. The subtle modifications between isomers, mainly in reducing carbohydrates, complicate the analysis and lead sometimes to broadening and splitting of the chromatogram signals. Overcoming such a complication requires using high temperature or high pH mobile phase. These conditions could reduce the column life. We report herein, the influence of the equilibration conditions of an amino-based HILIC column on the analysis of acetylated and deacetylated COS (DP ≤ 5) under room temperature and neutral mobile phase acetonitrile/H2O (7:3 v/v).

Materials and Methods

Chemicals.

All deacetylated COS used were commercially available under their chloride salt form. (GlcN).HCl, (GlcN)2.2HCl, (GlcNAc)1, and (GlcNAc)2 were purchased from sigma Aldrich Corporation (purity >99%). (GlcN)3.3HCl and (GlcN)5.5HCl (purity >98%) were purchased from Seigakaku Business Corporation ® and characterized by 1 The synthesis of the (GlcNAc)5 and tetra-N-acetylchitopentaose (D1A4) was carried out following Abla et al. [START_REF] Abla | Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer[END_REF] and fully characterized by 1 H NMR and MALDI-TOF MS (Supplementary Information Fig S3 -S5). Water was obtained from a Milli-Q Water Purification. All commercially chemicals and solvents were used directly without any further purification.

Chromatography analyses and sample preparation

The chromatographic analysis was performed on a Shimadzu HPLC Model CBM-20A equipped with a shodex Asahipak NH2P-50 4E column (5 μm granulometry, 250 mm × 4.6 mm), a shodex Asahipak NH2P-50G 4A guard column and Shimadzu RID-10A detector. The mobile phase was composed of a mixture of acetonitrile/H2O (7:3 v/v). The stationary phase was equilibrated by eluting the adequate pH solution under a constant flow rate of 0.2 mL/min for a period of 72 hours. Then the mobile phase (ACN/H2O 7:3 v/v) was eluted at a flow rate of 0.8 mL/min for two hours before injections. All COS samples were prepared in a similar way, by dissolving the accurate amount of the solid in a mixture of acetonitrile/H2O (1:1 v/v) at 25 mg/mL and injected (10 µL) at room temperature, at a constant flow rate of 0.8 mL/min.

To assess the repeatability of the chromatography analysis, relative standard deviation (RSD) values were calculated for all the samples and under different chromatography conditions. It is worthy to note that the measurements were performed on 5 different measurements for each oligomer samples and these measurements were performed on several days. The RSD obtained are presented in Table 1 and were satisfactory (<5%).

(Supplementary Information Table S1-S3)

Results and Discussion.

Despite a very complex retention mechanism of HILIC, it is mainly considered as a hydrophilic partitioning between a highly organic rich mobile phase and a semi-immobilized water enriched layer neighboring to the stationary phase. [START_REF] Buszewski | Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique[END_REF][START_REF] Greco | Main Interactions and Influences of the Chromatographic Parameters in HILIC Separations[END_REF][START_REF] Jandera | Stationary and mobile phases in hydrophilic interaction chromatography: a review[END_REF] HILIC systems possess a diversity of stationary phases (neutral or ionic) onto which a variety of ligands could be attached, and several types of mobile phases could also be used. Therefore, in order to better understand the retention mechanism of analytes, other types of interactions such as hydrogen bonds, dipoledipole, or ionic attractive or repulsive interactions should also be taken into consideration. [START_REF] Buszewski | Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique[END_REF][START_REF] Greco | Main Interactions and Influences of the Chromatographic Parameters in HILIC Separations[END_REF] The shodex Asahipak NH2P column used in this study is an amino-based stationary phase composed of polyvinyl alcohol hydrophilic polymer onto which secondary ethyl-amino groups are grafted. [START_REF] Hirata | New, stable polyaminebonded polymer gel column[END_REF] Thus the ratio of protonated/unprotonated amino groups of the stationary phase highly depends on the pH of the solution used to equilibrate the column, as well as the pH of the eluent, if this latter is different. This ratio could have a great influence on the interactions between the stationary phase and the analytes, therefore, the separation of COS should take into consideration four main experimental parameters: (i) the nature and the pH of the solution used to equilibrate the stationary phase; (ii) the amphoteric character of the secondary amino groups (pKa = 10, data given by the supplier) of the stationary phase; (iii) the cationic or neutral form of the COS (intrinsic dissociation constant for fully deacetylated COS pK0 ~ 6.5-7.5; [START_REF] Delas | Effects of Chain Length of Chitosan Oligosaccharides on Solution Properties and Complexation with siRNA[END_REF] and finally, (iv) the pH of the eluent. In order to ensure that analytes will remain in their initial form as they were injected, a neutral mobile phase composed of a mixture of acetonitrile and water (7:3; v/v) was used in all the experiments. For the sake of clarity in this work, COS were abbreviated as DxAy Fig 1 as it was previously published [START_REF] Haebel | Quantitative Sequencing of Complex Mixtures of Heterochitooligosaccharides by vMALDI-Linear Ion Trap Mass Spectrometry[END_REF] where "x" and "y" are the number of deacetylated (D=GlcN) and acetylated (A=GlcNAc) units, respectively.

Column equilibration under acidic conditions

First, the stationary phase was equilibrated, with a continuous flow of an H2SO4 solution (50mM; pH = 1). Under these conditions, the secondary amino groups of the stationary phase were converted into their ammonium sulfate form. Then, different COS with well-defined DP, ranging from 1 to 5, fully acetylated (D0Ay), fully deacetylated (DxA0) or partially acetylated (D1A4) were analyzed and the results are summarized in Table 1. In this case, the retention of these neutral COS to the cationic stationary phase was likely controlled through hydrophilic interactions. [START_REF] Greco | Main Interactions and Influences of the Chromatographic Parameters in HILIC Separations[END_REF] Following this assumption, it was obvious to note that the retention time increased with the number of repeating units as the hydrophilic interactions are stronger with higher DP COS. Besides, the signal of each fully acetylated COS (D0Ay) was split into two peaks is sufficiently low to permit the resolution of the two anomers. [START_REF] Bennett | Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography[END_REF] However, in order to confirm experimentally this assumption, a sample of D0A1 was injected under the same chromatographic conditions and a portion of the ascending slope of its first peak was separated. This latter portion was then injected in a different run and showed again two separated peaks having the same integration ratio and same retention times as for the two peaks of the former sample injection (Supplementary Information Fig S7 and Table S4). It is worthy to note, that for all COS analyzed in this study, α anomers showed shorter retention times than β isomers indicating a weaker interaction of α anomers with the stationary phase. For each COS, the anomer/peak assignment was based on the peak area integration ratio of 2:1 assigned for α: β anomers, respectively, as found in the literature for D-glucosamine, [START_REF] Bertuzzi | General Protocol to Obtain D-Glucosamine from Biomass Residues: Shrimp Shells, Cicada Sloughs and Cockroaches[END_REF][START_REF] Pedrali | Determination of N-acetylglucosamine in cosmetic formulations and skin test samples by hydrophilic interaction liquid chromatography and UV detection[END_REF] which is also in agreement with the α: β peak integration ratio calculated from 1 H NMR spectra of fully acetylated and deacetylated COS (Supplementary Information Fig S1 -S3).

The mutarotation phenomenon limits the use of HILIC systems regarding the quantitative and qualitative analysis of reducing carbohydrates. In order to overcome such a problem, chemical modifications of the carbohydrate analytes are proposed by some authors. [START_REF] Harvey | Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry[END_REF] However, these methods are not suitable for preparative chromatography separation. In order to avoid the separation of anomer peaks, it is usually recommended to work at high temperature (e.g., above 45°C) where the mutarotation rate becomes much faster than the timescale for chromatography separation. [START_REF] Fu | Separation of carbohydrates using hydrophilic interaction liquid chromatography[END_REF][START_REF] Ortner | Interconversion and chromatographic separation of carbohydrate stereoisomers on polystyrene-divinylbenzene resins[END_REF] Another way to make the two anomer peaks collapse is by adding some additives to the mobile phase. For instance, the addition of tertiary amines (e.g. TEA) was found to suppress the split of the two anomer peaks of reducing monosaccharides and small oligosaccharides. [START_REF] Bennett | Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography[END_REF] The rate constant of the mutarotation could also be improved by raising the pH of the mobile phase. [START_REF] Bawazeer | A method for the analysis of sugars in biological systems using reductive amination in combination with hydrophilic interaction chromatography and high resolution mass spectrometry[END_REF][START_REF] Yan | Neutral monosaccharide composition analysis of plant-derived oligo-and polysaccharides by high performance liquid chromatography[END_REF] Kahle et al. [START_REF] Kahle | Separation of saccharides and their anomers by high-performance liquid chromatography[END_REF] have shown that converting an amino-based silica gel column into sulfate or phosphate forms (strong kosmotropic ions) led to anomers separation whereas after converting it to perchlorate form (strong chaotropic ion) the two anomer peaks collapsed. Kosmotropic ions are known for their strong hydrogen bonds with water molecules which leads to an increase in water viscosity, while chaotropic ions act oppositely. [START_REF] Winkworth-Smith | Polysaccharide structures and interactions in a lithium chloride/urea/water solvent[END_REF] Accordingly, we intended to replace the sulfate ion by a less kosmotropic ion, for instance an acetate ion, by equilibrating the stationary phase with an ammonium acetate buffer solution (50mM, pH = 4.5). Due to its relatively high solubility in acetonitrile, its volatility and compatibility with other techniques such as mass spectrometry, the ammonium acetate salt is typically used in HILIC studies. [START_REF] Greco | Main Interactions and Influences of the Chromatographic Parameters in HILIC Separations[END_REF][START_REF] Guo | Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography[END_REF] As a result, fully deacetylated COS (DxA0) were not well retained while fully acetylated COS (D0Ay) were successfully well separated according to their DP with a Rs ≥1.2 Table 2, and were eluted in one single peak Fig 3 . In addition, we noted that, for a given single COS, the retention time in presence of acetate ions was shorter than in presence of sulfate ions. For instance, the tR of D0A3 is 10.5 min in presence of acetate ions vs 12.0/14.5 min, (for α/β anomers, respectively) in presence of sulfate ions Table 1. Table 2 Selectivity (α) and peak resolutions (Rs) between different COS pairs; retention times of D0A4, D2A0 and D4A0 were theoretically calculated from the standard curves of their corresponding DxAy series (r 2 > 0.9980) in order to calculate their selectivity and peak resolution (see Supplementary Information).

The difference between the two counter ions, regarding both the signal profile and the retention time, could be related to their degree of hydration as it is already described in the literature. Indeed, several studies [START_REF] Guo | Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography[END_REF][START_REF] Alpert | Effect of salts on retention in hydrophilic interaction chromatography[END_REF][START_REF] Bicker | Retention and selectivity effects caused by bonding of a polar urea-type ligand to silica: A study on mixed-mode retention mechanisms and the pivotal role of solute-silanol interactions in the hydrophilic interaction chromatography elution mode[END_REF] have demonstrated that the presence of salts promotes the retention of ionic and nonionic analytes. The common explanation suggests that, the presence of solvated salt ions in the water-enriched layer, increases its thickness. Thus, the partitioning of polar analytes into this layer is reinforced and the analyte-ligand interactions, for instance hydrogen bonds, are strengthened. This would increase the retention time. [START_REF] Bicker | Retention and selectivity effects caused by bonding of a polar urea-type ligand to silica: A study on mixed-mode retention mechanisms and the pivotal role of solute-silanol interactions in the hydrophilic interaction chromatography elution mode[END_REF] The thickness of the water-enriched layer adjacent to the stationary phase plays, therefore, an important role in the modulation of the retention characteristics. [START_REF] Greco | Study of the retention behavior in zwitterionic hydrophilic interaction chromatography of isomeric hydroxy-and aminobenzoic acids[END_REF] Alpert et al., [START_REF] Alpert | Effect of salts on retention in hydrophilic interaction chromatography[END_REF] showed that the nature of the counter ion has an influence on the thickness of the water-enriched layer. They have demonstrated that a highly hydrated kosmotropic ions such as sulfates have markedly promoted the retention time, and a weakly hydrated chaotropic ions, for instance perchlorate ions, had opposite effects. A similar trend was also observed by Bicker et al. [START_REF] Bicker | Retention and selectivity effects caused by bonding of a polar urea-type ligand to silica: A study on mixed-mode retention mechanisms and the pivotal role of solute-silanol interactions in the hydrophilic interaction chromatography elution mode[END_REF] by comparing the influence of a more hydrated ions (e.g. acetate ions) to less hydrated ions (e.g. trifluoroacetate ions) on the retention times under HILIC separations. These results are in good agreement with our observations and would suggest that as the degree of hydration of sulfates ions (strong kosmotropic ions) is higher than the acetates ions (weak kosmotropic ions), the water-enriched layer containing the former ions should be thicker than the one with the latter ions. This would be a suitable hypothesis to explain the higher retention times of COS obtained in presence of sulfate ions as compared to acetate ions Table 1. Concerning the signal profile and the anomeric separation, one could hypothesize that in the more viscous water-enriched layer containing sulfate ions, the mutarotation rate would be slower than in presence of acetate ions, which results in the separation of the two anomers. This reminds the results obtained by Kahle et al. [START_REF] Kahle | Separation of saccharides and their anomers by high-performance liquid chromatography[END_REF] up mentioned and comparing the effect of counter ions of the stationary phase on the anomeric separation. Our results showed that the anomers resolution is affected by the nature of the counter ion of the stationary phase even when using a neutral mobile phase.

Finally, another feature of the presence of the acetate counter ions in proximity of the stationary phase exist in the behavior of the fully deacetylated COS D5A0 in its chloride form. Indeed, under these conditions, the COS D5A0 was weakly retained (tR = 7.3 min) as if the electrostatic repulsions are weaker than in the case of sulfate ions. This result is very interesting as it allows the separation of COS according their DA. Indeed, Fig 4-a show a large difference in retention times between COS pentamers with different DA, i.e. D5A0, D1A4 and D0A5 (7.3 min, 10.6 min and 16.8 min, respectively) even when the three COS were injected together (Fig. 4-b). The calculation of RS for these three COS pentamers (ranging between 2.5 and 5.2, table 2) confirms their perfect separation under these conditions. In order to confirm the efficiency of the separation according to the DA, we carried out the selective tetra N-acetylation of the commercially available D5A0 according to the method previously described by Abla et al. [START_REF] Abla | Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer[END_REF] Following this method, two acetylated derivatives of the pentamer starting material were obtained and perfectly separated on a Shodex Asahipak NH2P-50 4E column after its equilibration by an NH4Ac buffer solution

Column equilibration under alkaline conditions

In order to decrease the repulsive electrostatic interactions, and to improve the retention of fully deacetylated COS (DxA0), the shodex Asahipak NH2P column was equilibrated with a highly alkaline buffer solution of sodium In both series, a very satisfactory peak resolution (Rs) was observed (Rs ≥ 1.8, Table 2) and the retention time highly depended on the DP. The Rs values are higher than in the case of ammonium acetate equilibration (50 mM, pH=4.5). As it could be expected, no splitting of the peaks was observed. 2. These results show the efficiency of an alkaline equilibration on the separation of COS according to their DP but not their DA. In this study, the effect of both the pH and the nature of the salt of the solution used for equilibrating the aminobased stationary phase of HILIC column was investigated. The results showed that a high pH equilibrating solution, for instance a sodium bicarbonate buffer solution (100 mM, pH=10), is the most appropriate for both completely acetylated and deacetylated COS analysis (Rs values ≥ 1.8) using ACN/H2O (7:3 v/v) as mobile phase.

Indeed, the retention times as well as the separation of these oligomers were DP dependent. Although the resolution of anomers is considered as a major limitation concerning the analysis of carbohydrates (mainly when it comes to preparative HPLC analysis of reducing carbohydrates), we showed that the choice of the counter ion (e.g., acetate

or sulfate) of ammonium groups of the column stationary phase is crucial. For instance, sulfate ions favor the separation of anomers, while the presence of acetate ones favors the merging of the two anomer peaks and leads to lower retention times. Whereas other published methods preconize the use of high pH mobile phases to merge the anomer peaks, which could affect the state of the analytes and therefore complicate the analysis, our method should be considered to be interesting in terms of simplifying the chromatographic conditions of COS analysis. It permits to overcome the anomerization drawbacks while working under room temperature and neutral mobile phase. We hypothesized that the degree of hydration of the counter ion of ammonium groups of the column stationary phase would affect the thickness of the immobilized water-enriched layer which in turn would influence the retention time as well as the interaction between the analytes and the column stationary phase ligands. In addition, the results obtained in this study showed that equilibrating the stationary phase by an ammonium acetate buffer solution (50 mM, pH = 4.5) could be used for the separation of COS according to their DA. This latter result was demonstrated in this work with COS pentamers (Rs ≥ 2.5) and likely requires further investigations for COS with other DP.

  H NMR (Supplementary Information Fig S1 and Fig S2). (GlcNAc)3 (purity >90%) was purchased from Elicityl.

Fig 1

 1 Fig 1 Chemical structure of COS (DxAy with DP = x +y ≤ 5) studied in this work.

Fig 2

 2 Fig 2 Chromatograms of fully acetylated COS (D0Ay with y=1, 2, 3 or 5) after equilibrating the column stationary phase by a sulfuric acid solution (50 mM, pH=1).

Fig 2 .

 2 This observation, in agreement with the literature,[START_REF] Xu | Large scale preparation of single chitin oligomers by the combination of homogeneous acid hydrolysis and reversed phase preparative chromatography[END_REF][START_REF] Lopes | Simultaneous chromatographic separation of enantiomers, anomers and structural isomers of some biologically relevant monosaccharides[END_REF][START_REF] Fu | Separation of carbohydrate isomers and anomers on poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase by hydrophilic interaction chromatography as well as determination of anomer interconversion energy barriers[END_REF] is a consequence of the mutarotation phenomenon. The interconversion between α and β anomers is commonly observed when analyzing reducing sugars on HILIC and appears when the anomerism rate

Fig 3

 3 Fig 3 Chromatograms of fully acetylated COS (D0Ay with y=1, 2, 3 or 5) after equilibrating the column stationary phase by an ammonium acetate buffer solution (50 mM, pH=4.5).

(Fig 4

 4 Fig 4 Analyses of three COS pentamers (D5A0, D0A5 and D1A4) after equilibrating the column stationary phase by an ammonium acetate buffer solution (50 mM, pH=4.5): a) chromatogram of the three pure COS pentamers; b) chromatogram of a mixture composed of the three COS pentamers; c) chromatogram of a mixture composed of D1A4 and D0A5 obtained after the N-acetylation of D5A0 described by Abla et al.[START_REF] Abla | Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer[END_REF]

  mM, pH=10). After that, two series of fully deacetylated COS (DxA0 in their hydrochloride form) and fully acetylated COS (D0Ay) were injected. The results, shown in Fig 5 and Fig 6 for D0Ay and DxA0, respectively and summarized in Table1, demonstrated a good affinity of both types of COS to the stationary phase.

Fig 5

 5 Fig 5 Chromatograms of fully acetylated COS (D0Ay with y=1, 2, 3 or 5) after equilibrating the column stationary phase by a sodium bicarbonate buffer solution (100 mM, pH=10).

Fig 6

 6 Fig 6 Chromatograms of fully deacetylated COS (DxA0 with x=1, 3 or 5) after equilibrating the column stationary phase by a sodium bicarbonate buffer solution (100 mM, pH=10).

Fig 7

 7 Fig 7 Chromatogram of a mixture of COS monomers (D1A0 and D0A1; mass ratio D1A0:D0A1 2:3) after equilibrating the column stationary phase by a sodium bicarbonate buffer solution (100 mM, pH=10).

Fig 8

 8 Fig 8 Chromatograms of three COS pentamers (D5A0, D0A5 and D1A4) after equilibrating the column stationary phase by a sodium bicarbonate buffer solution (100 mM, pH=10).

Table 1

 1

	pH (a)		D1A0 D3A0 D5A0	D0A1		D0 A2	D0A3	D0A5	D1A4
						α	β	α	β	α	β	α	β
	1	tR (b) SD (c)	--	--	--	7.3 8.7 0.1 0.3	8.8 0.3	10.5 0.4	12.0 0.3	14.5 0.4	21.8 0.1	26.6 0.3	--
	4.5	tR (b) SD (c)	--	--	7.3 0.2	7.5 0.3			8.7 0.3	10.5 0.6	16.8 0.1	10.6 0.1
	10	tR (b) SD (c)	7.6 0.2	9.6 0.2	13.3 0.4	7.1 0.3			8.1 0.3	9.9 0.5		13.6 0.5	14.0 0.6
	These experiments showed that fully deacetylated COS (DxA0), injected in their chloride salt form, were not
	retained by the column (Supplementary Information Fig S6), likely as a consequence of electrostatic repulsions
	with the cationic stationary phase. On the other hand, a better retention was observed when fully acetylated COS
	(D0Ay) were analyzed Fig 2.

Summary of the HILIC analyses of different fully acetylated (D0Ax), fully deacetylated (DxA0) and partially acetylated (D1A4) COS: (a) pH of the solution used for the equilibration of the column; (b) retention time (in min); (c) standard deviation (in min); Relative standard deviation (in %) were calculated and found <5%.

Present address: Department of Chemistry and Biochemistry, Laboratoire d'Innovation Thérapeutique (LIT), Lebanese University, Faculty of Sciences II, Fanar, Lebanon.

Acknowledgments

Financial support from Bayer CropScience (La Dargoire, France), the University of Lyon, and the Agence Nationale de la Recherche ("BIOCOS" 10-CD2I-008 research project) are greatly acknowledged. We also thank Mrs. Agnès Crépet for her technical help in HILIC analyses.

Methodology/Investigation/ Formal analysis/ Writing-Original Draft.  Stéphane Trombotto: Conceptualization/ Methodology/ Validation/ Writing-Review and Editing/ Funding acquisition/ Project administration/ Supervision.  Catherine Ladavière: Conceptualization/ Methodology/ Validation/ Writing-Review and Editing/ Funding acquisition/ Project administration/ Supervision. Ethics approval: not applicable Consent to participate: not applicable

Funding: Bayer CropScience (La Dargoire, France), the University of Lyon, and the Agence Nationale de la Recherche ("BIOCOS" 10-CD2I-008 research project)

Availability of data and material: yes (see supporting data file)

Code availability: not applicable

Authors contribution:

Conflict of interest/competing interest:

The authors declare no conflict of interest nor competing interest.


Dr Baptiste Rugeri

Dr Hassib Audi

Dr Pawel Jewula

Dr Radouane Koudih

Raluca Malacea-Kabbara

Dr Delphine Vimont

Pr Jürgen P Schulz

Philippe Fernandez

Sylvain Jugé
email: sylvain.juge@u-bourgogne.fr

Pr S Jugé

Fernandez

Designing Silylated L-Amino Acids using a Wittig Strategy: Synthesis of Peptide Derivatives and 18 F-Labeling

Keywords: Wittig reagent, phase transfer, amino acid derivatives, hydrosilanes, fluorosilanes, 18 F-labeling

An efficient hemisynthesis of silylated L-amino acids is described by reaction of silylated benzaldehydes with a phosphonium L-amino acid used as Wittig reagent. The efficiency of the silylated L-amino acids in peptide synthesis was investigated by coupling both carboxylic acid or amino moiety with L-alanine and phenylalanine derivatives, respectively. The silylated derivatives were reacted with KF or TBAF to afford the corresponding fluorosilyl derivatives without racemization. The resistance to hydrolysis of the fluorosilylated derivatives was checked in phosphate buffer at pH 7.2. Finally, the 18 F-labeling of the di-t-butylsilylated saturated and unsaturated dipeptides was achieved in hot DMSO with a mixture of K[ 18 F]/cryptand (K2.2.2) and acetic acid. The radiolabeled 18 F-labeled dipeptides were obtained with radiochemical yields and molar activities up to 27 % (decay uncorrected) and 410 GBq/µmol, respectively. Consequently, the hemisynthesis via the Wittig reaction offers a new and efficient route for the design and the 18 F-labeling of Si-based amino acids and peptide derivatives, applicable in PET imaging .

Introduction

The use of radioisotopic, optical, fluorescent, electrochemical or nanomaterials tags to label bioactive molecules is now used extensively to explore metabolic pathways, to establish diagnosis and follow diseases progression, to treat pathologies or to guide surgery. [1,2] The development of new methods to track biomolecules like peptides, proteins or antibodies is a constant challenge, because these molecules occupy a prominent position in all living processes. Thus, visualizing and characterizing them in their native environment is essential to understand the biological processes and to focus new diagnostic method. [3] Usually the labelling of peptides and proteins is achieved using either fluorescent probes or metal radioisotopes such as 64 Cu, 68 Ga, 177 Lu, 99m Tc or 186 Re complexed to polydentate chelators. [START_REF] Lee | [END_REF] Due to the increasing use of 2-[ 18 F]fluoro-2-deoxy-D-glucose (FDG) for cancer diagnosis via positron emission tomography (PET) during the last decades, the [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-radionuclide has become more accessible for the labeling of amino acids and peptides. [2,[START_REF] Lee | [END_REF]5] Figure 1. Representative Si-based prosthetic group for [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-labeling of peptides

The presence of such prosthetic groups usually increase the peptide lipophilicity giving a negative influence on the pharmacokinetics of the labeled peptide and thus a nonspecific binding. Further designing is therefore needed in order to reduce the lipophilicity, for example by introduction of polyethylene glycol (PEG) or charged auxiliaries.

These strategies require multi-step sequences which are often time consuming. This is the reason why it is highly interesting to perform fast direct [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-labeling of amino acids which could be used in peptide synthesis or as substrates for multi amino acid transport systems in order to improve the specificity with regard to their targets. [6] Such labeling methods offer the possibility to change the position of the modified amino acid inside the sequence in order to reduce the difference with the native peptide. [START_REF] Lee | [END_REF]6] The synthesis of silicon-containing amino acids was intensively developed for maintaining or reinforcing the biological activity of peptides and to enhance their resistance toward enzyme degradation, [12] but very few examples concerning their use in [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] Flabeling have been reported. This is explained by in vivo hydrolysis of [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-fluorosilane moiety unless bulky groups such as i-propyl or t-butyl substitute the silicon center (Figure 1). [9a,10c] In this context, Wängler/Jurkschat et al have described the synthesis of (S)-4-[(di-tbutyl)fluorosilyl]phenylalanine derivative 7 by asymmetric Rh-catalyzed hydrogenation of dehydroamino acid substrate 6. The Siamino acid was then incorporated in Tyr 3 -octreotate derivative for radiolabeling study (Scheme 1a). [13] In addition, the (±)-(di-tbutyl)hydrosilylated amino acid 10, obtained by Huisgen cycloaddition of azidoethylsilane 8 with racemic propargyl glycine 9, has been reported by Ametamey et al for the labeling of peptides (Scheme 1b). [14] The 18 F-labeling of the silicon-based amino acids 7 and 10 was then achieved either by isotopic exchange according to the SiFA procedure [13] or by direct fluorination of the hydrosilyl moiety, [14,[START_REF] Balentova | ethoxysilylamino acid derivative prepared according to Huisgen cyclisation[END_REF] respectively (Scheme 1).

Scheme 1. Synthesis of silylated amino acid applied to [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-labeling of peptides

In connection with our ongoing investigations in phosphorus-and boron-derivatives useful in [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-labeling, [16,17] we investigated a new stereoselective synthesis of silylated (Si-)amino acids and studied their efficiency as potential imaging agents. For this, we developed an hemisynthetic approach of Si-amino acids 15 bearing a free carboxylic acid function, by C=C bond forming on the side chain using a key Wittig reaction. This synthesis allows an easy designing of the silyl moiety by changing the substituents R or the living group Y (Scheme 2). The efficiency of the Si-amino acids in peptide synthesis was demonstrated by coupling both acid and amino group with alanine or phenylalanine derivatives (Scheme 3). The fluorination, the kinetics and the resistance to hydrolysis of the Si-amino acids 15 and dipeptide derivatives 18 was then investigated in phosphate buffer (Table 1; Figures 3 and4). Finally, the [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-labeling of the Si-peptides 18a and 20a was studied. (Table 2).

Results and Discussion

Stereoselective synthesis of silylated amino acid derivatives using a Wittig reaction

The silylated amino acids 15a-c were synthesized by Wittig reaction of the corresponding aldehydes 13a-c with the phosphonium salt 14, in hot chlorobenzene (or dioxane) with K3PO4 as base according to the described procedure (Scheme 2). [17] The silylated aldehydes 13a-c were prepared in yields up to 91% by halogen metal exchange on commercially available 4bromobenzaldehyde diethyl acetal 12, and trapping with the appropriate chloro or dichlorosilane, followed by hydrolysis (Scheme 2). The Wittig reagent 14 was prepared in 30% overall yield starting from L-aspartic acid according to a previous established procedure. [17] Scheme 2. Synthesis of silylated amino acid derivatives 15a-c by Wittig reaction

The silylated amino acids 15a-c were initially obtained with a free carboxylic acid group and as a Z/E mixture in 3:7 ratio, as shown by the NMR signals of the silane moiety of compound 15a (Figure 2a). Further heating in THF with diphenyldisulfide trigger isomerization to afford pure (E)-15a in 88% yield (Figure 2b). While in the same conditions the silylated amino acid 15b led to the pure (E)-isomer, in the case of the silanol derivative (Z/E)-15c no isomerization was observed under these conditions (Scheme 2).

Application of silylated amino acids 15a-c in peptide synthesis

The efficiency of silylated amino acids 15a-c in peptide synthesis via both carboxylic acid or amino function was investigated by coupling with L-alanine 16 and L-phenylalanine derivative 17, respectively (Scheme 3). Silylated compound (E)-15a (or (E)-15b) bearing a free carboxylic function was reacted with methyl alaninate 16 at room temperature using HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxid hexafluorophosphate) as coupling agent,as coupling agent, to afford the corresponding dipeptide 18a (or 18b) in yield up to 93%. Under similar conditions the silanol amino acid (Z/E)-15c led to the peptide 18c as a (Z/E) mixture in 38% yield (Scheme 3). The coupling reaction proceeded without racemisation as shown the NMR spectra of the dipeptide 18a with an epimeric sample previously prepared starting from racemic Wittig reagent (±)-14 (see Supporting Information). After deprotection of the Boc group by reaction with HCl 1M in AcOEt, the silyl dipeptide 18a was coupled with the phenylalanine derivative 17 using the previous HATU procedure, to afford the corresponding tripeptide 19 in 72% yield (Scheme 3). Finally, dipeptide 18a was quantitatively hydrogenated in MeOH in the presence of Pd/C as catalyst, to give the corresponding saturated methoxysilane 20a, [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] whereas under the same conditions compound 18c led to a mixture of silanol 20b and methoxysilane 20c in 2:3 ratio (Scheme 3). It is interesting to note that silanol amino acids and peptides are scarcely described in the literature to date. [12,[START_REF] Smith | For example of silanol amino acids[END_REF] Considering the half-life of 18 F (110 min), [10d] both fluorination kinetics and stability are crucial for the development of radiolabeled agents in PET medical imaging. [START_REF] Schirrmacher | Fluorine-18 Radiochemistry: Theory and Practice in Pharmaceutical Radiochemistry[END_REF] In first attempt, the fluorination of the silylated amino acid derivative 15a was investigated according to literature conditions, [10c] by reaction with KF in refluxing THF and the presence of cryptand 1,10-diaza- 4,7,13,16,21,24-hexa-oxabicyclo[8.8.8] hexacosane (K2.2.2) and a small amount of acetic acid. The Kryptofix ® K2.2.2 (2.2.2 cryptand [4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-(8.8.8)hexacosane]) is a sequestering agent of the potassium counter ion, used in order to enhance the nucleophily of F -. Under these conditions the fluorosilylated product 21a was obtained in 97% yield as a stable compound after chromatography on silica gel, exhibiting 29 Si and [START_REF] Smith | For example of silanol amino acids[END_REF] F NMR signals at +13.9 ppm (d, 1 JSi-F = 298 Hz) and -188 ppm, respectively (Table 1, entry 1). On the other hand, the fluorination of the di-i-propylsilyl amino acid analogue (15b) was achieved by reaction with tetrabutylammonium fluoride (TBAF) in THF at room temperature for 20 min, to afford the fluorosilylated derivative 21b in 79% isolated yield (entry 2). Finally, under similar conditions as for 15a, the fluorination of dipeptides 18a and 18b led to the corresponding fluorosilylated dipeptides 21c and 21d in 96% and 92% yields, respectively (entries 3, 4). These results demonstrate that hydrosilylated amino acids and peptides could be quickly fluorinated under mild conditions which offers an efficient alternative method for the direct 18 F-labeling.

SiH (Z) SiH (E) [a] (Z,E)-15a 1 H NMR [b] (E)-15a After isomerisation 29 Si NMR SiH (Z) SiH (E)

Resistance to hydrolysis in phosphate buffer of fluorosilylated dipeptides 21c-d

The aqueous behavior the fluorosilylated dipeptides 21c and 21d (chosen as examples) was investigated in an aqueous phosphate buffer (pH 7.2) /acetonitrile-D3 (8:2) mixture at room temperature. The monitoring was performed using [START_REF] Smith | For example of silanol amino acids[END_REF] F-NMR analysis of the Si-F signal (Figures 3 and4). Under these conditions, the hydrolysis of 21c exhibited only traces of the free fluoride F -ion after 10 days (Figure 3). [START_REF] Smith | For example of silanol amino acids[END_REF] F NMR By contrast, the di-i-propylfluorosilane dipeptide analogue 21d released 40% of F -within 5 min, simultanously with the rise of a slightly high field shifted signal at -191.8 ppm (vs -191.4 ppm), the intensity of 21d decreased then steadily with time (Figure 4). This transient signal can be explained by the formation of an solvated hypervalent intermediate such as 22, in equilibrium with the fluorosilylated precursor 21d, which leads to the silanol product 18c and the free fluoride F -(Figure 4). [9b] It should also be noted that the formation of solvated hypervalent intermediate analogue to 22 has been previously reported by DFT calculations as intermediate in hydrolysis of fluorosilane. The 18 F-labeling of organosilylated compounds is currently achieved using K[ 18 F] in the presence of cryptand K2.2.2. [START_REF] Al-Huniti | For recent progresses in radiofluorination using crown ether with both chelating and leaving group properties[END_REF] The fluorination of the hydrosilylated "prosthetic compounds" such as 3 (X= H) or 4, has been carried out by heating between 30 and 100°C in the presence of a small quantity of acid such as AcOH. [10,11] Under these conditions 18 F-incorporation (73%) was achieved and specific activities up to 62 GBq/μmol were observed. [10d,22] In this study, the direct radiofluorination was investigated with the di-tbutylsilylated dipeptides 18a and 20a bearing hydrosilane and methoxysilane moieties, respectively (Table 2). The labeling was performed by heating the dipeptide for 15 min with a mixture of K[ 18 F], cryptand K2.2.2 and AcOH in DMSO. The initial activity of the [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F solution at cyclotron output was in the range 103-127 Gbq and the radiolabeling conditions and results are reported in Table 2. [18 F]-21c was obtained in 18% radiochemical yield (Decay uncorrected RCY/ radiochemical purity > 97%) and with a molar activity (Am) of 410 GBq/µmol when the radiolabeling of the unsaturated dipeptide 18a was performed by heating at 100°C with 120 GBq as starting 18 F (Table 2, entry 1). Different RCY and molar activities of [ 18 F]-21c were obtained by changing the quantities of acetic acid and the heating conditions (entries 2-4). (the measurement time for the molar activity determination must be stated)

Results showed that performing the radiolabeling at 80°C afforded a lower SA but a slightly higher RCY (entry 4 vs entry 1). It was also shown that the radiolabeling was not facilitated by higher acidic conditions (entry 3), possibly because the dipeptide was partially degraded either by reaction of the unsaturation or by deprotection of the Boc group.

However contrary to 21c, [ 18 F]-21c evolved toward a new radiolabeled species with similar HPLC retention time. This unexpected reaction was slowed down by diluting [ 18 F]-21c in saline solution (NaCl, 0.9%) and nearly stopped using aqueous ascorbic acid (0.1%). It was identified as resulting from an addition reaction on the C=C double bond with radical intermediates due to ionizing conditions (see Supporting Information). [23] Since stability over time is mandatory for potential PET imaging, the radiolabeling was considered in a second instance with the saturated methoxysilylated dipeptide 20a, obtained by hydrogenation of 18a (Scheme 3). When the radiolabeling of the saturated dipeptide 20a was performed by heating at 100°C for 15 min with 111 GBq as starting K[ 18 F], the labeled product [ 18 F]-21e was obtained in 10% RCY and with a SA of 82 GBq/µmol (entry 5). By heating at 80°C, the radiolabelled dipeptide [ 18 F]-21e was obtained in only 9% RCY and with a SA of 104 GBq/µmol (entry 6). HPLC analyses of dipeptide [ 18 F]-21c and [ 18 F]-21e were compared to those of corresponding non-radioactive references 21c and 21e, the latter prepared by hydrogenation of 21c. This proved that no degradation was observed in the case of the saturated dipeptide [ 18 F]-21e for at least 5h40 (see Supporting Information). While RCY are moderate, it should be pointed out that high specific activities are requested for receptor binding studies in order to avoid receptor saturation. [START_REF] Lien | [END_REF]

Conclusions

In this manuscript we have demonstrated that unsaturated silylated L-amino acids can be synthesized based on the Wittig reaction between silylated benzaldehydes with a phosphonium L-amino acid reagent. The silane fragment tolerates the conditions of peptide coupling allowing synthesis of several dipeptides. Although tBu2SiH compounds are more difficult to fluorinate than the corresponding iPr derivatives, the resulting fluorinated tBu2SiF dipeptides are more resistant in aqueous conditions. Dipeptides carrying silane and methoxysilane moieties were easily labeled with [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F, however better radiochemical yields and specific activities were obtained for the SiH fluorination. Whereas the radiolabeled unsaturated dipeptide turned out to be radiosensitive, this was not the case of the corresponding saturated analogue.

Experimental Section General

All reactions were carried out using standard Schlenk techniques under inert atmosphere. Solvents were dried and purified by conventional methods prior to use. The chlorobenzene, toluene, diethyl ether, n-hexane, ethyl acetate, petroleum ether (EP), methanol, dimethylsulfoxyde (DMSO) and acetic acid were purchased in anhydrous form. The reagents t-butyllithium,

4-bromobenzaldehyde diethylacetal, di-t-butylchlorosilane, chloro-di-i- propylsilane, dichloro-di-t-butylsilane, cryptand K2.2.2 (Kryptofix ® ), diphenyldisufide, ethyl-di-i-propylamine (DIPEA), O-(7-Aza-1H- benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexa- fluorophosphate (
HBTU), L-Boc-Phe-OH, L-Alanine methyl ester hydrochloride, hydrogen chloride 1M in AcOEt, tetrabutylammonium fluoride 1M in THF, were purchased from commercial sources. The iodide (S)-2-(t-butyloxycarbonyl)amino]-4-triphenylphosphonium-butanoic acid 14 was prepared according to the published procedure. [17] The flash chromatography was performed with the indicated solvents using silica gel 60 (60AAC, 35-70 µm; SDS). The 1 H, 13 C, [START_REF] Smith | For example of silanol amino acids[END_REF] F and 29 Si NMR spectra were recorded on Bruker Avance 600, 500 or 300 MHz spectrometers at ambient temperature using TMS as internal reference for 1 H, 13 C and 29 Si NMR, and CCl3F for [START_REF] Smith | For example of silanol amino acids[END_REF] F NMR. Data are reported in ppm as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad signal, coupling constants in Hertz, integration. The mass spectra and accurate mass measurements (HRMS) were performed under (ESI) conditions with a Bruker micro Q-TOF detector or Thermo Orbitrap detector. HPLC analyses were performed either on a Shimadzu chromatograph equipped with a UV detector at λ = 210 nm and λ = 254 nm, or a JASCO system equipped a MD-2010 Plus photodiode array detector, or a Dionex P680 system equipped with UV multiwavelength and Raytest Gabi Star detectors. High-performance liquid chromatography (HPLC) analyses were performed using a Phenomenex Luna C18 column (250 × 4.6 mm, 5 µm, flow 1 mL/ min) and the indicated conditions. The infra-red spectra were recorded on a FT-IR Bruker Vector 22 or Vertex 70v instruments and the data are given in cm -1 . The buffer solution pH = 7.2 (0.2M) was prepared by mixing in 100 mL H2O, Na2HPO4•7H2O (5.3 g) and NaH2PO4•H2O (40 mg). Melting points were measured on a Kofler melting point apparatus and are uncorrected. Optical rotation values were measured on Perkin Elmer 241 polarimeter at 589 nm (sodium lamp). All radiofluorination experiments were run on a commercial TRACERLab FxF-N ® (Ge Healthcare).

4-[Hydro(di-t-butyl)silyl]benzaldehyde 13a.

tBuLi in hexane (1.84M, 11 mL, 20.2 mmol) was added dropwise at -78°C under argon to a solution of 4-bromobenzaldehyde diethylacetal 12 (2 mL, 10 mmol) in 20 mL of dry THF in a Schlenk tube. After 30 min, di-tbutylchlorosilane (1.8 mL, 11.4 mmol) was added. The mixture was stirred 2h at -78°C and then let warm up to room temperature overnight. After

4-[Hydro(di-i-propyl)silyl]benzaldehyde 13b.

This compound was prepared according to a new procedure with regard to the literature. [25] tBuLi in hexane (2.23M, 1.8 mL, 4 mmol) was added dropwise at -78°C under argon to a solution of 4-bromobenzaldehyde diethyl acetal 12 (0.5 mL, 2 mmol) in 5 mL of dry THF in a Schlenk tube.

After 30 min, chloro(di-i-propyl)silane (2 mL, 2.2 mmol) was added. The mixture was stirred for 2h at -78°C and then let warm up to room temperature overnight. The mixture was acidified to pH = 3 with HCl 1M and stirred at room temperature for 1h. The reaction mixture was extracted with 3 x 30 mL of CH2Cl2, the organic phases were gathered, dried over MgSO4, filtrated and concentrated under reduced pressure. The residue was purified by chromatography on silica gel using a mixture petroleum ether/AcOEt (9:1) as eluent. The 4-[hydro(di-i-propyl)

4-[Hydroxy(di-i-propyl

)silyl]benzaldehyde 13c.

tBuLi in hexane (1.7M, 5.9 mL, 10 mmol) was added dropwise at -78°C under argon to a solution of 4-bromobenzaldehyde diethyl acetal 12 (1.0 mL, 5 mmol) in 5 mL of dry THF in a Schlenk tube. After 30 min, the mixture was cannulated over a solution of dichloro(di-i-propyl)silane (1.2 mL, 6.6 mmol) in 5 mL of dry THF. The mixture was stirred 2h at -78°C then let warm up to room temperature overnight. The reaction mixture was quenched with 10 mL of water and extracted with 3 x 30 mL of CH2Cl2, the organic phases were then gathered, dried over MgSO4, filtrated and concentrated under reduced pressure. The residue was purified by chromatography on column using petroleum ether/AcOEt (6:4) as eluent, to afford the Mixture (Z,E): Aldehyde 13a (500 mg, 2.0 mmol) and K3PO4 (1,08 g, 5.1 mmol) previously dried by heating under vacuum, were added to a solution of phosphonium salt 14 (500 mg, 0.85 mmol) in chlorobenzene (6.5 mL).

The reaction mixture was stirred for 16h at 90°C, then hydrolyzed with distilled water (5 mL) and extracted with diethyl ether (3 x 5 mL). The resulting aqueous phase was acidified with KHSO4 (1M) to pH = 3 and extracted with ethyl acetate (3 x 5 mL). The organic phase was then dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using petroleum ether/AcOEt (8:2 + 1% AcOH) as eluent, to afford the silylated amino acid 15a (330 mg, 0.76 mmol, 90% yield) as a (Z/E) mixture in the ratio 3: (2S,4E)-Isomer 15a. The isomerization of the Z/E mixture 15a (275 mg, 0.63 mmol) in THF (10 mL) was performed by heating under argon for 15h with diphenyldisulfide (27.7 mg, 0.127 mmol) according to a described procedure. [17] After cooling and removing the solvent, the crude product was purified by chromatography on silica gel using a mixture petroleum ether/AcOEt (7:3) + 1% AcOH as eluent to afford the trans-15a (±)-(4E)-Isomer 15a. The racemic silylated amino acid trans-15a was synthesized from (±)-amino acid Wittig reagent 14, previously prepared from (±)-aspartic acid, according to the procedure described above.

(

(2S)-2-(t-Butoxycarbonyl)amino-5-[4-hydro(di-i-propyl)silylphenyl] pent-4-enoique acid 15b.

Mixture (Z,E). Aldehyde 13b (240 mg, 1.09 mmol) and K3PO4 (600 mg, 2.83 mmol) previously dried by heating under vacuum, were added to a solution of phosphonium salt 14 (260 mg, 0.44 mmol) in dry dioxane (5 mL). The reaction mixture was heated at 80°C under stirring for 12h. After cooling and filtration on a celite pad, the residue was washed with CH2Cl2 (10 mL). The filtrate was concentrated under reduced pressure. The crude product was then purified by chromatography on silica gel using a mixture petroleum ether/AcOEt (9:1) + 1% AcOH as eluent, to afford the amino acid 15b (158 mg, 0.39 mmol, 89% yield) as a Z/E mixture in 3:7 ratio. (R,S+S,S)-(di-t-Butyl)hydrosilylated dipeptide 18a. The epimeric mixture of the silylated dipeptide 18a was prepared from (±)-trans-15a and methyl Lalaninate 16 using the same procedure as described above. The epimeric mixure 18a was obtained in 81% yield as colorless oil; Rf: 0. (S,S)-Hydro(di-i-propyl)silylated dipeptide 18b.

This dipeptide was prepared using the same procedure described above for 18a, starting from the L-amino acid 15b (40 mg, 0.100 mmol). DIPEA (90 µL, 0.55 mmol), HATU (46 mg, 0.12 mmol) and methyl L-alaninate.HCl 16

(

(E,Z)-(S,S)-Hydroxy(di-i-propyl)silylated dipeptide 18c.

This dipeptide was prepared using the same procedure as described above for 18a, from the (E,Z)-L-amino acid 15c (270 mg, 0.64 mmol). The reaction of 15c with DIPEA (0.56 mL, 3.2 mmol), HBTU (291 mg, 0.77 mmol) and methyl L-alaninate.HCl 16 (135 mg, 0.96 mmol) in DMF (10 mL) led to the dipeptide 18c as an isomeric mixture (55:45) (122 mg, 0.24 mmol, 38% yield) and as a colorless solid. Rf: 0.31 (petroleum ether/ AcOEt 1:1); [α]D 20 = +4.8 (c = 0.8 in CHCl3); 1 H NMR (300 MHz, CDCl3): δ = 0.80 (d, 3

(S,S,S)-(di-t-Butyl)hydrosilylated tripeptide 19.

A solution of (S,S)-di-t-butylhydrosilylated dipeptide 18a (70 mg, 0.135 mmol) in HCl (1M in AcOEt, 3 mL) was stirred at room temperature under argon for 12h. After removal of solvent under vacuum, the residue was dissolved in dry DMF (1mL) under argon and DIPEA (60 µL, 0.340 mmol) was added. The mixture was stirred for 1h. Meanwhile, a solution of (S)-Boc-Phe-OH 17 (36.1 mg, 0.136 mmol) in dry DMF (2 mL) was reacted with HATU (62.1 mg, 0.163 mmol) and DIPEA (60 µL, 0.340 mmol). The mixture was stirred at 0°C for 10 min and was then added to the activated dipeptide solution. The reaction mixture was stirred for 2h and subsequently treated with KHSO4 (1M) and extracted with CH2Cl2. The organic phases were gathered, dried over MgSO4 and then filtrated and the solvent removed under vacuum. The residue was purified by chromatography on silica gel using a mixture AcOEt/petroleum ether (1:1) as eluent to afford the silylated tripeptide 19 (65 mg, 97.6 µmol, 72% yield) as an uncrystallized compound. Rf: 0.30 (AcOEt/petroleum ether 1:1); [α]D 20 = -3.4 (c = 1 in CHCl3); 1 H NMR (300 MHz, CDCl3): δ = 1.02 (s, 18 H; C(CH3)3), 1.30 (s, 9 H; OC(CH3)3), 1.38 (d, 3 (S,S)-Hydroxy-and methoxy(di-i-propyl)silylated saturated dipeptide 20b and 20c.

These dipeptides were obtained using the same procedure described above for 20a. Pd/C 10% (15 mg, 13 µmol) was added to the enantiopure (E/Z)isomeric mixture peptide 18c (92 mg, 0.18 mmol) in dry MeOH (1.8 mL) and the mixture was stirred under H2 (10 bar) overnight. After filtration over celite and removing the solvent under vacuum, a mixture of saturated dipeptide 20b and 20c was obtained in 2:3 ratio (79 mg, 0.15 mmol, 85%) as a colorless foam. Rf: 0.50 (petroleum ether/AcOEt 1:1); [START_REF] Smith | For example of silanol amino acids[END_REF] F NMR (470 MHz, CDCl3): δ = -188.9 (s, 0.95F, 28/30 Si-F), -188.9 ppm (d, 0.05F, 1 J(Si,F) = 298 Hz, 29 Si/F); 29 Si NMR (99 MHz, CDCl3): δ = +13.9 ppm ( (d, 3 J(H,H) = 7.5 Hz, 1 H; NHCHCH3), 7.17 (d, 3 J(H,H) = 7.8 Hz, 2 H; ArH), 7.50 ppm (d, 3 J(H,H) = 8.1 Hz, 2 H; ArH). 13 C NMR (75 MHz,

19

F NMR spectroscopic kinetic analyses: A small quantity of the fluorosilylated compound (5 mg) was introduced into the NMR tube and dissolved in 0.1 mL acetonitrile D3. At the beginning of the solvolysis (t = 0 min), 0.4 mL of phosphate buffer solution pH 7.2 (0.2M) was added and the hydrolysis was monitored by [START_REF] Smith | For example of silanol amino acids[END_REF] F NMR spectroscopy. [START_REF] Smith | For example of silanol amino acids[END_REF] F NMR spectra were acquired at different times and the integration was determined from spectra. The ratio of [START_REF] Smith | For example of silanol amino acids[END_REF] F-signal existing as the fluorosilylated compound to the total [START_REF] Smith | For example of silanol amino acids[END_REF] F signals was plotted against time to determine the kinetic of the hydrolysis.

Radiofluorination of compounds 18a and 20a into [ 18 F]-21c and [ 18 F]-21e:

No-carrier-added [ 18 F]-fluoride was produced via the 18 O(p,n) [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F nuclear reaction by irradiation of enriched [ 18 O]H2O. [ 18 F]-Fluoride production: Bombarded 30-60 min; 60 µA; target volume 1.8-2.5 mL, 100-130 GBq are obtained. [ 18 F]-Fluoride was trapped on an anion-exchange resin cartridge (Sep-Pak QMA light, Waters). The cartridge was eluted with a solution of Kryptofix (K2.2.2, 22 mg) and potassium carbonate (7 mg) in H2O (0.3 mL) and CH3CN (0.3 mL). Solvents were removed by heating at 100°C for 8 min applying a gentle stream of nitrogen. During this time, dry K[ 18 F]F/ K2.2.2 complex was prepared by azeotropic drying using CH3CN (4 x 0.25 mL). Silylated compound 18a (or 20a) (4.8-5.4 mg) diluted in a mixture of DMSO (500 µL) and acetic acid (3 µL) was added to the dry K[ 18 F]F/K2.2.2 complex and then allowed to react for 15 min at 60-100°C under constant stirring before to be diluted with CH3CN (3 mL) and H2O (2 mL). This solution was injected into a semi preparative HPLC 85/15 (CH3CN/ (H2O/TFA, 0.1%, v/v)), 2.5 mL/min. Then [ 18 F]-21c (or [ 18 F]-21e) was formulated in an aqueous physiological solution (0.9% NaCl solution) and purity was checked by analytical HPLC 85/15 (CH3CN/(H2O/TFA, 0.1%, v/v)) with 1 mL/min flow, using non-radioactive molecule 21c (or 21e) as reference. Results showed that [ 18 F]-21c and [ 18 F]-21e were obtained with radiochemical purity > 97%, with RCY (Decay uncorrected) in the range 9-27% and with SA from 82 to 410 GBq.μmol -1 (Table 2).

Figure 2 .

 2 Figure 2. 1 H-and 29 Si-NMR of compound 15a: (a) (Z/E)-mixture; (b) (Z)-mixture after isomerization

  Scheme 3. Synthesis of silylated peptides 18-20 Fluorination of the hydrosilylated amino acids 15a-b and dipeptides 18a-b

Figure 3 .

 3 Figure 3. Hydrolysis of fluorosilylated dipeptide 21c at pH 7 monitored by 19 F NMR

  [10c]

Figure 4 .

 4 Figure 4. Hydrolysis of fluorosilylated dipeptide 21d at pH 7.2 monitored by 19 F NMR 18 F-Labeling of dipeptides derivatives 18a and 20a

  );29 Si NMR (99 MHz, CDCl3): δ = +12.7 ppm (s); HRMS (ESI-Q-TOF) calcd. for C28H46N2O5Si [M+Na] + : 541.30682; found: 541.30724.

CDCl3

  171.5 (CON), 173.2 ppm (CO2CH3); 19 F NMR (CDCl3, 282 MHz): δ = -189.0 (s, 0.95F, 28/30 Si-F), -189.0 ppm (d, 1 J(Si,F) = 296 Hz, 0.05F, 29 Si-F); 29 Si NMR (CDCl3, 59 MHz); δ = +14.0 ppm (d, 1 J(Si,F) = 296 Hz); HRMS (ESI-Q-TOF) calcd. for C28H48FN2O5Si [M+H] + : calcd 539.33110; found: 539.33081.

Table 1 .

 1 Preparation of fluorosilylated amino acid and dipeptide derivatives 21

	Entry	R2Si-substrate R	R 1	Conditions	R2SiF-Product Yield [%] [a}
						KF/ K2.2.2
	1	15a	tBu		H	AcOH/ THF	21a	97 %
						reflux 48h
	2	15b	iPr		H	TBAF/ THF rt/ 20 min	21b	79 %
	3	18a	tBu	(L)-Ala-OMe	KF/ K2.2.2 reflux 24h AcOH/ THF	21c	96 %
	4	18b	i-Pr	(L)-Ala-	"	21d	92 %
				OMe
	[a] Isolated yields.

Table 2 .

 2 [START_REF] Hirakue | For the preparation of methoxysilane from hydrosilane[END_REF] F-labeling of dipeptide derivatives 18a and 20a HCl 1M until pH = 3 and stirring at room temperature for 1 h, the reaction mixture was extracted with 3 x 50 mL of CH2Cl2. The organic phases were gathered, dried over MgSO4, filtrated and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using a mixture petroleum ether/ AcOEt (19:1) as eluent. After removing the solvent under vacuum the 4-[hydro(di-t-butyl)silyl] benzaldehyde 13a was obtained as a colorless oil with an intense almond-

	Entry	Dipeptide Y	Initial activity [GBq]	Conditions T[°C]
	1	18a	H	120	100	[ 18 F]-21c	18	410
	2	18a	H	103	80	[ 18 F]-21c	27	260
	3	18a	H	122	80 [d]	[ 18 F]-21c	13	117
	4	18a	H	117	60	[ 18 F]-21c	19	158
	5	20a MeO	111	100	[ 18 F]-21e	10	82
	6	20a MeO	127	80	[ 18 F]-21e	9	104

[a] Decay uncorrected radiochemical yield.

[b]

Radiochemical purity > 97%.

[c]

Molar activity.

[d]

50 µL AcOH instead of 3 µL addition of

  = 8.0 Hz, 2 H; ArH), 7.41 ppm(d, 3 J(H,H) = 8.0 Hz, 2 H; ArH);13 C NMR (CDCl3, 125 MHz): δ = 11.2 (d, 2 J(C,F) = 13.7 Hz; CH(CH3)2),15.6 (d, In an autoclave, Pd/C 10% (15 mg, 13 µmol) was added to a solution of fluorosilylated dipeptide 21c (82 mg, 0.15 mmol) in dry MeOH (1.5 mL). The mixture was stirred under H2 (10 bar) overnight. After filtration over celite and removing the solvent under vacuum, the fluorosilyl-saturated dipeptide 21e was quantitatively obtained (82 mg, 0.15 mmol) as a colourless oily compound. Rf: 0.30 (petroleum ether/acetone 8:2); [α]D 20 = -5.8 (c = 1 in CH2Cl2); 1 H NMR (300 MHz, CDCl3): δ = 1.05 (s, 18 H; C(CH3)3), 1.40(d, 3 J(H,H) = 7.5 Hz, 3 H; CHCH3), 1.44 (s, 9 H; OC(CH3)3), 1.69-1.71 (m, 4 H, (CH2)2 overlapping signals), 2.63-2.65 (m, 2 H; ArCH2), 3.73 (s, 3 H; OCH3), 4.10 (brs, 1 H; CHN), 4.57 (q, 3 J(H,H) = 7.5 Hz, 1H; NCHCH3), 4.95 (brs, 1 H; NH), 6.48

	dipeptide 21d (67 mg, 0.132 mmol, 92% yield) as a colorless foam. Rf: 0.25
	(petroleum ether/Et2O 1:1); 1 H NMR (500 MHz, CDCl3): δ = 0.93 (d, 3 J(H,H)
	= 7.5 Hz, 6 H; CH(CH3)2), 1.00 (d, 3 J(H,H) = 7.5 Hz, 6 H; CH(CH3)2), 1.18
	(m, 2 H; CH(CH3)2), 1.33 (d, 3 J(H,H) = 7.25 Hz, 3 H, CH(CH3)), 1.34 (s, 9 H;
	OC(CH3)3), 2.60 (m, 2 H; CH2), 3.63 (s, 3 H, OCH3), 4.20 (brs, 1 H; CHN),
	4.51 (m, 1 H; NCH(CH3), 5.08 (brs, 1 H; NH), 6.17 (m, 1 H; ArCH=CH),
	6.42 (d, 3 J(H,H) = 16.0 Hz, 1 H; ArCH=CH), 6.70 (brs, 1 H; NH), 7.29 (d,
	3 J(H,H) 3 J(C,F) = 19 Hz; CH(CH3)2), 17.3 (CHCH3), 27.3 (OC(CH3)3), 35.2 (CH2),
	47.1 (CHN), 51.4 (OCH3), 53.1 (CHN), 79.3 (OC(CH3)3), 124.4 (ArCH=CH),
	124.7 (Carom), 130.9 (Carom), 132.7 (ArCH=CH), 133.1 (Carom), 137.4
	(Carom), 155.6 (NCO2), 170.0 (CON), 172.0 ppm (CO2CH3); 19 F NMR (470
	MHz, CDCl3): δ = -187.2 (s, 0.95F, 28/30 Si-F), -187.2 ppm (d, 0.05F, 1 J(Si,F)
	= 294 Hz, 29 Si-F); HRMS (ESI-Q-TOF) calcd. for C26H41FN2O5SiNa	d, 1 J(Si,F) = 298 Hz); HRMS (ESI-Q-TOF) calcd. for
	[M+Na] + : 531.26610; found: 531.26647.	C24H38FNO4SiNa [M+Na] + : 474.24463; found: 474.24364.
	Hydrogenation of the di-t-butylfluorosilyl dipeptide 21c into saturated	(2S,4E)-2-(t-Butoxycarbonylamino)-5-[4-fluoro-(di-i-propyl)fluorosilyl)
	derivative 21e (see Supporting Information).	phenyl]-4-pentenoic acid 21b.
		TBAF 1M in THF (0.2 mL, 200 µmol) was added under argon to a solution
		of 15b (35 mg, 86 µmol) in dry THF (2 mL). After 20 min under stirring the
		solvent was removed under vacuum and the residue was purified by
		chromatography on silica gel using petroleum ether/AcOEt 9:1 + 1% AcOH
		as eluent to afford the compound 21b (28.7 mg, 68 µmol, 79% yield) as a
		colorless solid. Rf: 0.20 (petroleum ether/AcOEt 9:1 + 1% AcOH); [α]D 20 =
		+11.4 (c = 0.5 in CH2Cl2); 1 H NMR (300 MHz, CDCl3): δ = 0.93 (dd, 3 J(H,H)
		= 7.4 Hz, 3 J(H,H) = 1.5 Hz, 6 H; CH(CH3)2), 1.00 (dd, 3 J(H,H) = 7.4 Hz,
		3 J(H,H) = 1.5 Hz, 6 H; CH(CH3)2), 1.15-1.21 (m, 2 H; CH(CH3)2), 1.42 (s, 9
		H; OC(CH3)3), 2.63-2.68 (m, 2 H; CH2), 4.41 (brs, 1 H; CHN), 5.20 (brs, 1
		H; NH), 6.09-6.14 (m, 1 H; ArCH=CH), 6.43 (d, 0 Hz; CH(CH3)2), 28.4
		(C(CH3)3), 36.0 (CH2), 80.6 (OC(CH3)3), 124.7 (ArCH=CH), 125.9 (Carom),
		132.3 (Carom), 134.2 (ArCH=CH), 134.3 (Carom), 138.4 (Carom), 155.7
		(NCO2), 176.3 ppm (CO2H), 1C not observed; 19 F NMR (282 MHz, CDCl3):
		δ = -187.2 ppm; HRMS (ESI-Q-TOF) calcd. for C22H34FNO4SiNa [M+Na] + :
		446.21333; found: 446.214333.
		di-t-Butylfluorosilylated dipeptide 21c.
		KF (36.8 mg, 0.63 mmol), K2.2.2 (94 mg, 0.251 mmol) and acetic acid (80
		µL, 1.4 mmol) were added successively under argon to a solution of (S,S)-
		di-t-butylhydrosilylated dipeptide 18a (130 mg, 0.251 mmol) in dry THF (6.5
		mL). The reaction mixture was heated to reflux of THF for 24h. After cooling,
		the solvent was evaporated under vacuum and the residue was purified by
		chromatography on silica gel using a mixture petroleum ether/Et2O (1:1) as
		eluent, to afford the fluorinated dipeptide 21c (130 mg, 0.242 mmol, 96%
		yield) as a colorless foam. Rf: 0.25 (petroleum ether/Et2O 1:1); [α]D 20 = +8.46
		(c = 1.9 in CHCl3); 1 H NMR (500 MHz, CDCl3): δ = 1.04 (s, 18 H; SiC(CH3)3),
		1.39 (d, 3 J(H,H) = 7.0 Hz, 3 H; CHCH3), 1.41 (s, 9 H; OC(CH3)3), 2.62-2.71
		(m, 2 H; CH2), 3.70 (s, 3 H; OCH3), 4.26 (m, 1 H; CHN), 4.55-4.61 (m, 1 H;
		NCHCH3), 5.11 (d, 3 J(H,H) = 7.0 Hz, 1 H; NH), 6.18-6.24 (m, 1 H; ArCH=CH),
		6.48 (d, 3 J(H,H) = 15.5 Hz, 1 H; ArCH=CH), 6.71 (br d, 3 J(H,H) = 7.0 Hz, 1
		H; NH), 7.34 (d, 3 J(H,H) = 8.0 Hz, 2 H; ArH), 7.53 ppm (d, 3 J(H,H) = 8.0 Hz,
		2 H; ArH); 13 C NMR (125 MHz, CDCl3): δ = 18.3 (CHCH3), 20.2 (d, 2 J(C,F)
		= 12.5 Hz; C(CH3)3), 27.3 (C(CH3)3), 28.2 (OC(CH3)3), 36.2 (CH2), 48.1
		(CHN), 52.4 (OCH3), 53.7 (CHN), 80.3 (OC(CH3)3), 125.2 (ArCH=CH),
		125.5 (Carom), 132.9 (Carom), 133.8 (ArCH=CH), 134.2 (Carom), 138.0
		(Carom), 155.6 (NCO2), 170.9 (CON), 173.0 (CO2CH3); 19 F NMR (470 MHz,
		CDCl3): δ = -188.9 (s, 0.95F, 28/30 Si-F), -188.9 ppm (d, 0.05F, 1 J(Si,F) = 298 Hz, 29 Si-F); 29 Si NMR (99 MHz, CDCl3): δ = +13.9 (d, 1 J(Si,F) = 297 Hz);
		HRMS (ESI-Q-TOF) calcd. for C28H45FN2O5SiNa [M+Na] + : 559.29740;
		found: 559.29540.
		Fluoro-di-i-propylsilylated dipeptide 21d.

3 J(H,H) = 15.9 Hz, 1 H; ArCH=CH), 7.29

(d, 3

J(H,H) = 7.8 Hz, 2 H; ArH), 7.41 ppm

(d, 3

J(H,H) = 7.8 Hz, 2 H; ArH), H acid not observed;

13

C NMR (CDCl3, 75 MHz): δ = 12.4 (d, 2 J(C,F) = 12.8 Hz; CH(CH3)2), 16.8

(d, 3

J(C,F) = 10.KF (16.6 mg, 0.285 mmol), K2.2.2 (53.7 mg, 0.143 mmol) and acetic acid (70 µL) were successively added to a solution of (S,S)-18b (70 mg, 0.143 mmol) in dry THF (2.4 mL) under argon. The reaction mixture was heated at reflux of THF for 24h. After cooling the solvent was removed under vacuum and the residue was purified by chromatography on silica gel using a mixture petroleum ether/Et2O (1:1) as eluent, to afford the fluorosilylated

F-Labeled dipeptide RCY[a,b] Am[c] [%][GBq/µmol]

Acknowledgements

The authors are grateful for H.A, B.R. and R.K. fellowship and postdoctoral grants provided by the "Conseil Régional de Bourgogne" (research programs 3MIM and PARI-santé), the financial support from the "Ministère de l'éducation nationale de l'enseignement supérieur et de la recherche" and the CNRS (Centre National de la Recherche Scientifique). The authors would like also to thank M. J.-B. Deloye and the Sté Cyclopharma for their support to B.R. fellowship. Ms. M. J. Penouilh and F. Chaux at the Welience/Pôle Chimie Moléculaire, Ms. C. Moulatsa, M.-J. Eymin and M. J.-C. Chambron at the ICMUB-StéréochIM, and Ms L. Jugé at the Neurosciences Research and School of Medical Sciences, University of New South Wales, Australia, are warmly thanked for their technical assistance or their help to the preparation of this manuscript.