Hubert Casajus

Eric Dubreucq

Sylvain Tranchimand

Véronique Perrier

Caroline Nugier- Chauvin

Sandrine Cammas-Marion

Lipase catalyzed ring-opening polymerization of benzyl malolactonate: an unusual mechanism?

ABSTRACT: The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of β-substituted β-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzymesubstrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such ε-caprolactone, propiolactone and lactide. Enzymatic-catalyzed ROPs of MLABe in presence of the lipase/acyltransferase CpLip2 and its serine knock-out (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight average molar mass of about 3,600 g/mol (Ð = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in presence of inactivated CpLip2 (heated at 100°C). Moreover, the ROP of MLABe in presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in presence of CpLip2 and CpLip2_180A nor its chemical ROP in presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of (R) and (S)-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.

INTRODUCTION. Aliphatic polyesters belong to a polymer family with outstanding biocompatibility and biodegradability properties particularly appreciated when such polymeric materials are designed for applications in the biomedical field. Such polymers, being in contact with human bodies, must follow very strict specifications: they must be non-toxic, nonimmunogenic, non-carcinogenic and non-thrombogenic [START_REF] Vert | Aliphatic Polyesters: Great degradable polymers that cannot do everything[END_REF] . Moreover, their biodegradation must lead to low molar mass molecules, which are respectful of the previously cited specifications and/or eliminated/excreted from the body. These polyesters are usually synthesized by polymerization reactions using chemical catalysts through either the direct polycondensation of a diacid with a diol, for example, or ring opening polymerization (ROP) of lactones or lactide [START_REF] Allcock | Polymerization of Cyclic Organic Compounds[END_REF] . The ROP technique allows the obtention of polymers having high and controlled molar masses as well as defined structures [START_REF] Jérôme | Recent Advances in the Synthesis of Aliphatic Polyesters by Ring-Opening Polymerization[END_REF] . On the other hand, the ROP reactions in presence of cationic or anionic initiators often requires the addition of organometallic catalysts whose presence even as traces might lead to unwanted toxicity [START_REF] Tanzi | Cytotoxicity of Some Catalysts Commonly Used in the Synthesis of Copolymers for Biomedical Use[END_REF] . Consequently, alternative synthetic methods are developed to overcome this major drawback, more particularly when such (co)polymers are synthesized for human health applications. Therefore, the use of biological catalysts such as lipases is of growing interest. Indeed, enzymes allow polymerization reactions under mild conditions (temperature, pH and pressures), avoid the use of organic solvents and most importantly are generally not toxic [START_REF] Shoda | Enzymes as Green Catalysts for Precision Macromolecular Synthesis[END_REF] . The synthesis of polyesters through ROP of their corresponding cyclic monomers in the presence of lipases was described for the first time in 1993 by Kobayashi et al. [START_REF] Uyama | Synthesis of Polyesters by Enzymatic Ring-Opening Copolymerization Using Lipase Catalyst[END_REF] and Kohn et al. [START_REF] Knani | Enzymatic Polyesterification in Organic Media. Enzyme-Catalyzed Synthesis of Linear Polyesters. I. Condensation Polymerization of Linear Hydroxyesters. II. Ring-Opening Polymerization of ϵ-Caprolactone[END_REF] , who synthesized a poly(ε-caprolactone) thanks to the ROP of ε-caprolactone in the presence of Pseudomonas fluorescens lipase and porcine pancreatic lipase (PPL), respectively, as catalysts. These preliminary results have opened the way towards enzymatic-catalyzed polymerization reactions leading to "greener" and more biocompatible polyesters such as poly(ε-caprolactone) [START_REF] Kobayashi | Enzymatic Ring-Opening Polymerization and Polycondensation for the Green Synthesis of Polyesters[END_REF][START_REF] Champagne | Recent Developments and Optimization of Lipase-Catalyzed Lactone Formation and Ring-Opening Polymerization[END_REF] . In this context, we have evaluated the possibilities of using lipase-based ROP of a special family of lactones, i.e. the β-substituted β-lactones also called alkyl malolactonates, to synthesize poly(malic acid) and its derivatives [START_REF] Casajus | Optimization of Lipase-Catalyzed Polymerization of Benzyl Malolactonate through a Design of Experiment Approach[END_REF][START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF] . In a first step, we have established, through a Design of Experiment (DoE) procedure, the optimal experimental conditions to reproducibly synthesize poly(benzyl malate) with weight average molar masses varying from 5,000 to 13,000 g/mol, dispersities between 1.2 and 1.6 (depending on the conditions used for the polymerization) and hydrolysis rates lower than 5%, using PPL as catalyst [START_REF] Casajus | Optimization of Lipase-Catalyzed Polymerization of Benzyl Malolactonate through a Design of Experiment Approach[END_REF] . PMLABe obtained through lipasecatalyzed ROP of MLABe were then formulated and the resulting nanoparticles were involved in several in vitro assays whose results have highlighted the influence of the polymer synthesis method on cell viability in particular [START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF] . Moreover, physico-chemical characteristics of PMLABe obtained through lipase-catalyzed ROP of racemic and optically active MLABe led us to question the mechanism of this enzymatic polymerization [START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF] . Indeed, we have previously shown that the

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A c c e p t e d M a n u s c r i p t lipase-catalyzed ROP of R-MLABe and S-MLABe occurred through an O-alkyl bound cleavage with an inversion of configuration of the asymmetric carbon of the lactone's ring, as in the case of anionic ROP of MLABe for which the initiator attacks on the asymmetric carbon of the lactone's ring leading to a ring opening through an O-alkyl bound cleavage and an inversion of configuration of the asymmetric carbon [START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF] . On the contrary, the mechanism described, and well-accepted, for the lipase-catalyzed ROP of unsubstituted and α,β-alkyl substituted lactones (ε-caprolactone, butyrolactone, lactide, etc.) involved an O-acyl bound cleavage as the result of the attack of the activated catalytic serine onto the carbonyl of the lactone and the formation of an enzymemonomer intermediate (canonical mechanism) [START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF][START_REF] Matsumura | In Enzyme-Catalyzed Synthesis of Polymers[END_REF][START_REF] Uyama | Enzymatic Ring-Opening Polymerization of Lactones Catalyzed by Lipase[END_REF][START_REF] Henderson | Enzyme-Catalyzed Polymerizations of ε-Caprolactone: Effects of Initiator on Product Structure, Propagation Kinetics, and Mechanism[END_REF][START_REF] Macdonald | Enzyme-Catalyzed Epsilon-Caprolactone Ring-Opening Polymerization[END_REF][START_REF] Svirkin | Enzyme-Catalyzed Stereoelective Ring-Opening Polymerization of α-Methyl-β-Propiolactone[END_REF][START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF] . Therefore, it seems that the lipase-catalyzed ROP of MLABe does not follow the canonical mechanism described for other lactones and lactides, but rather the one described for its anionic ROP [START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF] . Consequently, we have realized several experiments involving the use of the lipase/acyltransferase CpLip2 enzyme (a lipase with high affinity for nucleophiles other than water [START_REF] Subileau | Lipases/acyltransferases for lipid modification in aqueous media[END_REF] ), a modified CpLip2 which catalytic serine was mutated into an alanine residue (serine-KO), an inactivated CpLip2 obtained by heating at 100°C, a MLABe derivative on which the two "acidic" protons of the lactone's ring have been changed by two methyl groups, synthetic mimics of the three amino acids of the lipase's catalytic triad and several molecular modeling experiments as well as DFT quantum-chemical calculations. The obtained results described in the present paper allowed us to propose a mechanism for the lipase-catalyzed ROP of malolactonates. MATERIALS AND METHODS. All chemicals were used as received. Porcine pancreatic lipase (PPL) was purchased from Sigma-Aldrich Co. Dimethyl benzyl malolactonate (diMeMLABe) was a gift from Kymia Nova (Saclay, France). Benzyl malolactonate (MLABe) was synthesized according to literature [START_REF] Cammas | Poly(β-malic acid: obtaining high molecular weights by improvement of the synthesis route[END_REF] .

Nuclear Magnetic Resonance spectroscopy:

The standard temperature was adjusted to 298K. NMR spectra were recorded on a Bruker Avance III 400 spectrometer operating at 400.13 MHz for 1 H, equipped with a BBFO probe with a Z-gradient coil and a GREAT 1/10 gradient unit. The zg30 Bruker pulse program was used for 1D 1 H NMR, with a TD of 64 k, a relaxation delay d1 = 2 s and 8 scans. The spectrum width was set to 18 ppm. Fourier transform of the acquired FID was performed without any apodization in most cases.

Size Exclusion Chromatography (SEC):

Weight average molar mass (Mw) and dispersity (Đ = Mw/Mn) values were measured by SEC in THF at 40 °C (flow rate = 1.0 mL/min) on a GPC2502 Viscotek apparatus equipped with a refractive index detector Viscotek VE 3580 RI, a guard column Viscotek TGuard, Org 10x4.6mm, a LT5000L gel column (for samples soluble in organic medium) 300 x 7.8 mm and a GPC/SEC OmniSEC Software. The polymer samples were dissolved in THF (2 mg/mL). All elution curves were calibrated with polystyrene standards (weight average molar masses: 1,240; 2,630; 6,220; 13,700; 30,100 and 78,200 g/mol). Preparation of the CpLip2 and CpLip2_S180A (serine KO) lipases: Recombinant lipase/acyltransferase CpLip2 from Candida parapsilosis CBS 604 was produced by heterologous expression in Komagataella phaffii (formerly named Pichia pastoris) as described previously [START_REF] Brunel | Highlevel expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris[END_REF] . The serine KO mutant CpLip2_S180A was obtained by site-directed mutagenesis of serine S180 into an alanine residue. The mutation was performed by Life Technologies (Regensburg, Germany) on the gene of CpLip2 in fusion with the signal peptide of the alpha mating factor of Saccharomyces cerevisiae within the pPICZαB plasmid, that was used to transform K. phaffii according to the work already described elswhere [START_REF] Neang | Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties[END_REF] . The protein was produced in a 1L bioreactor and purified as described earlier [START_REF] Neang | Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties[END_REF] .

Enzymatic polymerization of MLABe:

The different PMLABe were synthesized as described previously [10] by ring-opening polymerization of the MLABe in presence of the enzyme (PPL, CpLip2 or CpLip2_S180A) in Tris-HCl buffer pH7. Experiments were realized in a 24-multi reactor Büchi Syncore Line. In a 30 mL-tube, enzyme (8.4 mg) was mixed with pure MLABe (336 mg) and Tris-HCl buffer (125 µL, pH7, 105 mM). The mixture was stirred at 390 rpm and 60 °C. After 72 h, reaction was stopped by the addition of 3 mL of THF and 3 mL of chloroform. The mixture was then transferred into a separating funnel. Separations were realized by adding 15 mL of distilled water and 15 mL of chloroform. Organic phases were dried over MgSO4 and filtered. Finally, solvents were evaporated under reduced pressure. The crude polymers were analyzed by 1 H NMR to determine monomer's conversion (from 70 to 90%) and hydrolysis rate (below 3%). Then, they were dissolved in chloroform and precipitated in a large excess of cold diethyl ether to eliminate unreacted monomer and impurities with a recovering yield of 70 to 90%. After elimination of the supernatant, polymers were dried under vacuum and analyzed by 1 H NMR and SEC.

Polymerization of MLABe in presence of the synthetic catalytic triad:

-Synthesis of imidazolium trifluoroacetate salt [START_REF] Coulembier | An Imidazole-Based Organocatalyst Designed for Bulk Polymerization of Lactide Isomers: Inspiration from Nature[END_REF] : In a round-bottomed flask, 505 mg (7.42 mmol, 1 eq.) of imidazole were dissolved in 50 mL of chloroform. This solution was cooled to 0°C, then, 1.25 mL (16.32 mmol, 2.2 eq.) of trifluoracetic acid (TFA) was added dropwise. After 24 h stirring, the precipitated salt was filtered, washed with chloroform and dried overnight at 80°C. 578 mg of imidazolium salt were obtained (yield = 43 %). Melting point: Pm, ref = 127.2 °C [20], Pm, measured = 128 °C.

-Polymerization reactions: Experiments were realized in a 24-multi reactor Büchi Syncore Line. In a 30 mL-tube, imidazolium salt and/or imidazole, and/or TFA, and/or benzyl alcohol (composition depending of the entry as shown by data gathered in Table 1) were mixed with MLABe (200 mg, 0.97 mmol, 1eq.).

A c c e p t e d M a n u s c r i p t

In parallel, ROP of MLABe (m = 331 mg, 1.6*10 -3 mol) in presence of imidazole (n = 1.73*10 -7 mol, 113 µL of a stock solution at a concentration of 0.1g/L) in a catalytic amount, i.e. the same imidazole mole number than the one corresponding to 8.3 mg of PPL (Molar mass = 48,000 g/mol, n = 1.73*10 -7 mol), was conducted in 172 µL of Tris-HCl buffer at pH7. After 72 h at 60 °C and 390 rpm, reactions were stopped by the adding 5 mL of chloroform. The mixture was then filtered and transferred in a separating funnel. Separation was realized by adding 15 mL of distilled water and 15 mL of chloroform. The aqueous phase was extracted twice by 7 mL of chloroform. Organic phases were dried over MgSO4 and filtered. Finally, chloroform was evaporated under vacuum. The crude polymers were analyzed by [START_REF] Vert | Aliphatic Polyesters: Great degradable polymers that cannot do everything[END_REF] H NMR to determine monomer's conversion and hydrolysis rate. Then, they were dissolved in chloroform and precipitated in a large excess of cold diethyl ether. After elimination of the supernatant, polymers were dried under vacuum at room temperature, and characterized by SEC and 1 H NMR.

Computational methods: The 3D structure of CpLip2 lipase/acyltransferase [START_REF] Subileau | The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipases superfamily[END_REF] was refined by molecular dynamics and energy minimization in an explicit TIP3P water box as described earlier 25- 27 . The initial geometry of R-and S-MLABe was determined by energy minimization using the density functional tight binding (DFTB) quantum chemical method with the GFN1-xTB model [START_REF] Grimme | A robust and accurate tight-binding quantum chemical method for structures, vibrational frequencies, and noncovalent interactions of large molecular systems parametrized for all spd-block elements (Z = 1-86)[END_REF] and the COSMO [START_REF] Klamt | A new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient[END_REF] water solvation model implemented in the DFTB module of AMS 2019 (SCM, Amsterdam, NL). Molecular docking was performed with Autodock Vina v1.1.2 [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[END_REF] using the UCSF Chimera v1.14.1 [START_REF] Pettersen | UCSF Chimera -A visualization system for exploratory research and analysis[END_REF] interface. The energy of the structures of the two best poses was minimized within Chimera using the AMBER ff14SB and the AM1-SCC force fields for atomic partial charge calculations in the protein and the ligand, respectively, with the protein backbone constrained to fixed positions, allowing the selection of the conformation with the lowest estimated energy. For each ligand, sub-models restricted to amino acid residues with at least one atom closer than 5 Å from the ligand (475-480 atoms) were constructed. The broken amide bonds of the protein backbone were restored by capping with -CO-CH3 and -NH-CH3 groups on the amino and carboxyl sides, respectively, in line with the backbone of the former neighboring residues. The geometry of the sub-models was then refined using DFTB (GFN1-xTB and COSMO water implicit solvation models) for energy minimization, with the position of C and N atoms of the protein backbone constrained to fixed positions. A final optimization used DFT for energy minimization, with MLABe and all the atoms (and bound hydrogen atoms) closer than 3Ǻ from the ligand being free to move and the other atoms being fixed as a bloc. DFT calculations were performed using the BP86 GGA functional with D4(EEQ) dispersion correction [START_REF] Caldeweyher | A generally applicable atomic-charge dependent London dispersion correction[END_REF] , the DZ double ζ basis set with a large frozen core and the COSMO water solvation model within the ADF module of AMS. The interactions between MLABe and the protein sub-model, both defined as closed-shell molecular fragments, were then analyzed at the same level of DFT by the extended transition state-natural orbitals for chemical valence (ETS-NOCV) energy decomposition method [START_REF] Mitoraj | Natural orbitals for chemical valence as descriptors of chemical bonding in transition metal complexes[END_REF] using ADF. Graphical representations were obtained with UCSF Chimera v1.14 [START_REF] Pettersen | UCSF Chimera -A visualization system for exploratory research and analysis[END_REF] . α/β hydrolases which have the particularity of being active in presence of an interface between an aqueous phase (water or buffer) and an organic phase (hydrophobic substrate and/or organic solvent). Present in plants, insects, animals and microorganisms, lipases play a key role in the degradation and synthesis of lipids, and catalyze, under physiological conditions, the hydrolysis of mono-, di-and triglycerides [START_REF] Casas-Godoy | Lipases: An Overview[END_REF] . The Protein Data Bank (PDB) records more than 100 threedimensional structures of lipases highlighting the following highly conserved structural elements specific to these enzymes [START_REF] Rauwerdink | How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: The Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes[END_REF] : i). The α/β folding composed by eight β sheets and six α helices; ii). The catalytic triad composed by a nucleophilic serine, an aspartate/glutamate and a histidine; iii). The oxyanion hole formed by two active amino acids (or more depending on the lipase) of the active site, stabilizing the enzyme/substrate reaction intermediate through hydrogen bonds between the substrate and the nitrogen of the amide function; iv). The lid formed by one or more α helices which is opened in presence of the activation interface thus allowing the positioning of the substrate into the active site; v). The active site, mostly composed of hydrophobic amino acids, and varying from enzyme to another thus explaining the difference in specificity observed in lipases [START_REF] Rauwerdink | How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: The Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes[END_REF] . The mechanism of ester functions hydrolysis, called canonical mechanism, involves the three amino acids (aspartate, histidine and serine) of the catalytic triad: briefly, the serine, activated by the aspartate and histidine, forms a covalent acyl-enzyme intermediate (acylation step) which is then hydrolyzed thanks to the attack of water molecules activated by the histidine (deacylation step) [START_REF] Rauwerdink | How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: The Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes[END_REF] . The structure of the acyl-enzyme intermediate has been observed for a deacylation step slower than the acylation one and, together with detailed kinetic studies, validated and confirmed the canonical mechanism [START_REF] Rauwerdink | How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: The Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes[END_REF] . Lipase-catalyzed ROP mechanism of unsubstituted lactones (such as ε-caprolactone, lactide) and α/β-alkyl substituted β-lactones (such as butyrolactone, α-methyl propiolactone) has been already described in the literature [START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF][START_REF] Matsumura | In Enzyme-Catalyzed Synthesis of Polymers[END_REF][START_REF] Uyama | Enzymatic Ring-Opening Polymerization of Lactones Catalyzed by Lipase[END_REF][START_REF] Henderson | Enzyme-Catalyzed Polymerizations of ε-Caprolactone: Effects of Initiator on Product Structure, Propagation Kinetics, and Mechanism[END_REF][START_REF] Macdonald | Enzyme-Catalyzed Epsilon-Caprolactone Ring-Opening Polymerization[END_REF][START_REF] Svirkin | Enzyme-Catalyzed Stereoelective Ring-Opening Polymerization of α-Methyl-β-Propiolactone[END_REF][START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF] . It uses the canonical mechanism as a basis, and is widely accepted by the entire scientific community (Scheme 1). In a first step, the lactone ring is opened by the lipase (Scheme 1.1): the activated serine attacks the carbonyl of the lactone, thus forming a tetrahedral intermediate, and the reformation of the carbon-oxygen double bond leads to the opening of the lactone and to the formation of an acyl-enzyme intermediate. Then, three possibilities exist for the de-acylation of the enzyme (Scheme 1.2): i). Initiation: the initiator (here water) attacks the enzyme-substrate complex leading to the formation of an activated open monomer. Conversely, the formation of an enzyme-substrate complex can be observed when the terminal acid function of the growing monomer or polymer reacts with catalytic serine. ii). Propagation: a growing monomer or polymer attacks the enzyme-substrate complex leading to the growth of the macromolecular chain. Conversely, lipase can randomly hydrolyze an ester function of the polymer, thereby forming two polymers with smaller degrees of polymerization. iii). Cyclization: the terminal alcohol function of the polymer reacts with the ester function between the lipase to the polymer. Conversely, lipase can hydrolyze an ester function of a cyclic polymer and thus leading to the corresponding linear polymer. On the other hand, and contrary to the other lactones mentioned above, the mechanism of enzymatic polymerization of alkyl malolactonates is still not elucidated. The question "Does the lipase-catalyzed ROP of alkyl malolactonate follow the same mechanism as the one described for chemical ROP of alkyl malolactonate (Scheme 2) or the one described by several authors for unsubstituted lactones (Scheme 1)?" is still unanswered right now. opening might occur through an O-alkyl bound cleavage, as for the anionic ROP of MLABe for which the initiator (tetraethylammonium benzoate or other carboxylate salts) attacks on the asymmetric carbon of the alkyl malolactonate thus leading to an O-alkyl ring opening [START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF][START_REF] Cammas | Poly(β-malic acid: obtaining high molecular weights by improvement of the synthesis route[END_REF] . Indeed, the polymers synthesized from R-MLABe using either PPL as catalyst or tetraethylammonium benzoate as initiator have identical rotational powers with the same sign whatever the route of synthesis [START_REF] Casajus | Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages[END_REF] . If this result seems logical in view of the alkyl malolactonate reactivity, it is much less if the reaction mechanism is considered as a whole. Indeed, according to literature and canonical mechanism, the catalytic serine should react with the carbonyl group of the monomer ring to form an acyl-enzyme intermediate through a hydrolyzable ester bond [START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF][START_REF] Matsumura | In Enzyme-Catalyzed Synthesis of Polymers[END_REF][START_REF] Uyama | Enzymatic Ring-Opening Polymerization of Lactones Catalyzed by Lipase[END_REF][START_REF] Henderson | Enzyme-Catalyzed Polymerizations of ε-Caprolactone: Effects of Initiator on Product Structure, Propagation Kinetics, and Mechanism[END_REF][START_REF] Macdonald | Enzyme-Catalyzed Epsilon-Caprolactone Ring-Opening Polymerization[END_REF][START_REF] Svirkin | Enzyme-Catalyzed Stereoelective Ring-Opening Polymerization of α-Methyl-β-Propiolactone[END_REF][START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF] . In the case of lipase catalyzed ROP of alkyl malolactonate, if the attack of the catalytic serine on the asymmetric carbon of the lactone is considered, an alkyl-enzyme intermediate through hardly hydrolyzable ether bound should be obtained. However, we never observed such intermediate in [START_REF] Vert | Aliphatic Polyesters: Great degradable polymers that cannot do everything[END_REF] H NMR spectra and SEC chromatograms of both the crude and purified products. Therefore, it would seem that the canonical mechanism cannot be applied in the case of lipase-catalyzed ROP of alkyl malolactonate. In an attempt to understand the mechanism involved in the lipase-catalyzed ROP of this unusual lactones' family, several experiments were conducted. Coulembier et al. [START_REF] Coulembier | An Imidazole-Based Organocatalyst Designed for Bulk Polymerization of Lactide Isomers: Inspiration from Nature[END_REF] designed and used a synthetic catalytic triad to study lipase-catalyzed polymerization of D-and L-lactide. As shown by Figure 1, this synthetic catalytic triad is composed by trifluoroacetate mimicking the aspartate with however a lower nucleophilic character, imidazole for the histidine and benzyl alcohol mimicking the catalytic serine. Therefore, such synthetic catalytic triad was synthesized and tested for the polymerization of MLABe. The reaction between the MLABe and the synthetic catalytic triad, complete or partial, was carried out at 60 °C and 390 rpm (conditions similar to those of the enzymatic polymerization). Six polymerization conditions were tested as summarized in Table 1 ("Materials and Methods"). In this experiment, imidazole can be considered as the "synthetic" pendant of histidine of the lipase catalytic triad. From the results obtained, it seems logical to ask whether histidine alone is able to polymerize MLABe, without the intervention of serine and, therefore, of the canonical mechanism. An acid-base mechanism excluding the serine would therefore be possible. Indeed, this kind of mechanism has already been described for PPL-catalyzed aldolization reaction between an aldehyde and a ketone in the presence of water [START_REF] Li | Biocatalytic Promiscuity: The First Lipase-Catalysed Asymmetric Aldol Reaction[END_REF] . Moreover, O-alkyl ring-opening has been described for unsubstituted lactones when they are polymerized in the presence of a weak base as initiator: the carboxylate ion thus formed would then be the species responsible for the propagation [START_REF] Albertsson | Recent Developments in Ring Opening Polymerization of Lactones for Biomedical Applications[END_REF][START_REF] Kricheldorf | The Role of Deprotonation in the Anionic Polymerization of β-Propiolactone[END_REF][START_REF] Hofman | Structure of Active Centers and Mechanism of the Anionic Polymerization of Lactones[END_REF] . However, the formation of a carboxylate in this case would come from side reactions, including deprotonations of the monomer or the growing polymer chains [START_REF] Kricheldorf | The Role of Deprotonation in the Anionic Polymerization of β-Propiolactone[END_REF] . Indeed, Kricheldorf et al. have shown that anionic initiators, such as alcoholate, or basic initiators, such as tertiary amine, could abstract one of the two "acidic" protons of the lactone's ring in α position of the carbonyl to form the corresponding acrylate (Scheme 3).

RESULTS AND

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A c c e p t e d M a n u s c r i p t Scheme 3. Mechanism of the propiolactone ROP in presence of a tertiary amine as initiator [START_REF] Kricheldorf | The Role of Deprotonation in the Anionic Polymerization of β-Propiolactone[END_REF] .

This acrylate would continue the polymerization of the remaining monomers thus leading to a polymer terminated at one end by a double bond. Furthermore, Guérin et al. have shown that these deprotonation reactions also take place during the anionic ROP of alkyl malolactonates [START_REF] Mabille | Obvious complexity of the anionic polymerization of malolactonic acid esters[END_REF] .

If this deprotonation reaction is considered for the lipase-catalyzed polymerization of MLABe, the first step of the mechanism would consist in the abstraction of an "acidic" proton of the lactone's ring in α position of the carbonyl by the basic nitrogen group of imidazole (Figure 2A). The carboxylate thus formed would then continue the polymerization of the remaining MLABe. Therefore, if the two "acidic" protons of MLABe's ring are replaced by methyl groups, which is the case for dimethyl-MLABe, then this first step could no longer take place and the polymerization would not start (Figure 2B). The enzymatic polymerization of benzyl dimethyl malolactonate (diMeMLABe) was therefore carried out using the optimized protocol established for the polymerization of MLABe. PPL was mixed with diMeMLABe (1/40 PPL/diMeMLABe ratio) in Tris-HCl buffer (105 mM, pH 7), and the mixture was stirred at 390 rpm during 72h at 60°C. The obtained crude product was analyzed by proton NMR and the spectrum showed that no polymerization reaction took place. Indeed, no characteristic peak of a polymer was observed while all the peaks of diMeMLABe ( The impossibility to polymerize the diMeMLABe using PPL as catalyst might be in favor of the proposed mechanism (abstraction of one of the two "acidic" protons from the lactone's ring by the catalytic histidine), even if we cannot ignore that this impossibility might be also due to steric hindrance brought by the presence of the methyl groups on the lactone ring. Therefore, we carefully analyzed the proton NMR spectra of PMLABe obtained by PPL-catalyzed ROP of MLABe in Tris-HCl buffer pH7 (Figure 3). The expansion of the proton NMR spectrum between 6.8 and 7.0 ppm has highlighted the presence of peaks corresponding to ethylenic protons, thus seeming to confirm the hypothetic mechanism described in Figure 2A. Moreover, molar masses of PMLABe calculated using the relative integrations of these ethylenic protons and of the macromolecular chain protons (Figure 3) agree with those measured by SEC in THF (MNMR = 6,800 g/mol; Mw = 7,570 g/mol; Ð = 1.12). Therefore, these results tend to demonstrate the validity of the proposed lipase-catalyzed ROP mechanism of MLABe involving the abstraction of one of the two "acidic" proton of the lactone's ring probably by the catalytic histidine without the intervention of the catalytic serine.

In order to demonstrate that the lipase-catalyzed ROP of malolactonates does not involve the catalytic serine, we realized the enzymatic ROP of MLABe in presence of a serine knock-out (serine-KO) enzyme. However, PPL mutants being very difficult to obtain, we have selected an easy modifiable recombinant model, the CpLip2 enzyme. Using site-directed mutagenesis of

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Biomacromolecules A c c e p t e d M a n u s c r i p t serine S180 into an alanine residue, we obtained the serine KO mutant CpLip2_S180A. This mutant was confirmed to show no detectable lipase activity. If our hypothesis is valid then the CpLip2-Serine KO should lead to the formation of a polymer. Therefore, the polymerizations of MLABe in the presence of CpLip2 and CpLip2_S180A in Tris-HCl buffer pH7 (optimized conditions for PPL) have been conducted. As shown by results gathered in Table 3 and Figure 4, PMLABe were obtained in both cases, with, in addition, the presence of the peaks corresponding to ethylenic protons formed by the abstraction of one of the two "acidic" protons of the lactone's ring as shown in Figure 2A. Finally, the CpLip2 has been inactivated by heating at 100°C and enzymatic polymerization of MLABe has been conducted with this inactivated enzyme: the 1 H NMR spectrum of the crude product has highlighted a monomer conversion of less than 7% and the absence of polymer. Such results showed that the catalytic histidine certainly plays an important role in the polymerization of malolactonates.

To support further these experimental results, in silico calculations were performed: Figure 5 shows the optimized geometry of S-MLABe and R-MLABe and of the active site of CpLip2 obtained after molecular docking, molecular dynamics equilibration, energy minimization by DFTB and DFT quantum chemical calculations as described in the Computational methods section. ETS-NOCV analysis (extended transition state energy decomposition analysis combined with the theory of natural orbitals for chemical valence) [START_REF] Mitoraj | Natural orbitals for chemical valence as descriptors of chemical bonding in transition metal complexes[END_REF] by DFT calculations (BP86-D4(EEQ)/DZ level of theory) indicated that the main orbital interaction energy contribution between MLABe and the active site residues was associated to a strong hydrogen bond (-29 kJ/mol and -23 kJ/mol with S-and R-MLABe, respectively) between the ε N atom of histidine 365 and one of the "acidic" protons of MLABe (Figure 5). This result corroborates the hypothesis of a preferred interaction between MLABe and the catalytic histidine instead of the catalytic serine 180, and supports the proposed mechanism of polymerization of MLABe involving histidine, thanks to the presence of "acidic" protons (Figure 2A).

Finally, we tried to polymerize the diMeMLABe in the presence of CpLip2, CpLip2_S180A and imidazole in a catalytic amount in Tris HCl buffer pH7. In all these cases, we did not observe the formation of the corresponding PdiMeMLABe, and unpolymerized and undegraded diMeMLABe was recovered (Figures SI.2C and SI.2D). It is to be noted that molecular docking simulations with (R) and (S)-diMeMLABe and the active site of CpLip2 showed that the steric hindrance brought by the two methyl groups of the lactone ring should prevent the substrate from getting close enough to the catalytic residues (Figure 6). Nevertheless, the impossibility of polymerizing diMeMLABe in the presence of imidazole as initiator, i.e. without the steric hinderance problem encountered with lipases, demonstrates that the absence of "acidic" protons prevents the enzymatic polymerization of these lactones.

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  DISCUSSION. Lipases are enzymes belonging to the family of hydrolases, class 3 in the EC nomenclature (Enzyme Commission numbers). More specifically, they are serine ACS Paragon Plus Environment Biomacromolecules A c c e p t e d M a n u s c r i p t

Biomacromolecules

2 .Scheme 1 .

 21 Scheme 1. Canonical mechanism of lipase-catalyzed ROP of unsubstituted and α/β-alkyl substituted β-lactones[START_REF] Shoda | Enzymes as Green Catalysts for Precision Macromolecular Synthesis[END_REF][START_REF] Johnson | Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization[END_REF] .

Scheme 2 .

 2 Scheme 2. Mechanism of the chemical ROP of alkyl malolactonate[START_REF] Cammas | Poly(β-malic acid: obtaining high molecular weights by improvement of the synthesis route[END_REF] .

Figure 1 .

 1 Figure 1. The lipase (A) and synthetic (B) catalytic triad.

  The crude PMLABe were analyzed by proton NMR (conversion rate and hydrolysis rate) and purified PMLABe were analyzed by SEC (dispersity and weight average molar mass, Figure SI.1). As shown by the results gathered in

Figure 2 .

 2 Figure 2. Proposed first step for the lipase-catalyzed ROP of: A. MLABe, and B. diMeMLABe.

  Figure SI.2A) were present thus highlighting both the non-degradation and non-polymerization of this monomer by PPL (Figure SI.2B).

Figure 3 .

 3 Figure 3. Proton NMR spectrum (CD3COCD3, 400MHz) of the purified PMLABe obtained by PPL-catalyzed ROP of MLABe in Tris-HCl buffer pH7 at 60°C under 390 rpm stirring speed during 72h.

Figure 4 .

 4 Figure 4. Proton NMR spectrum (CD3COCD3, 400 MHz) of purified PMLABe obtained: A. by CpLip2-catalyzed ROP of MLABe in Tris-HCl buffer pH7; and B. CpLip2_S180A-catalyzed ROP of MLABe in Tris-HCl buffer pH7.

Figure 5 .

 5 Figure 5. Optimized geometry of the active site of CpLip2 in complex with S-MLABe (left) and R-MLABe (right). The two amino acid residues evidenced are the catalytic serine (S180) and histidine (H365). Interaction energies between the ligand and the active site residues were calculated by DFT (BP86-D4(EEQ)/DZ with water as implicit solvent) according to the ETS-NOCV theory. The colored meshes show the deformation of the electron density (depletion in red and accumulation in blue; cut-off 0.007 a.u.) associated to the main NOCV, corresponding to a hydrogen bond between H365 and a proton of MLABe (-29 kJ/mol with S-MLABe and -23 kJ/mol with R-MLABe).

Biomacromolecules

A c c e p t e d M a n u s c r i p tFigure 6 .For

 6 Figure 6. Geometry of the catalytic pocket of CpLip2 in complex with S-diMeMLABe (left) and R-diMeMLABe (right) obtained by molecular docking and energy minimization by molecular dynamics of the whole CpLip2 structure in explicit water. The amino acid residues displayed correspond to the catalytic triad (S180, H365 and D332) and to the D90 residue interacting with the catalytic serine in CpLip2.

  1. Univ Rennes, ENSCR, CNRS, ISCR (Institut des Sciences Chimiques de Rennes) -UMR 6226, F-35000 Rennes, France. 2. L'Institut Agro -Montpellier SupAgro, Univ Montpellier, INRA, CIRAD -UMR IATE, F-34060 Montpellier, France 3. Univ Rennes, INSERM, INRA, Institut NUMECAN (Nutrition Metabolisms and Cancer) UMR_A 1341, UMR_S 1241, F-35000 Rennes, France.

	KEYWORDS: Lipase-Catalyzed Polymerization, Ring-Opening Polymerization, Mechanism,
	Benzyl Malolactonate, CpLip2.

Table 1 .

 1 Composition of the reactive mixture.

	Entry	Imidazolium salt	Benzyl alcohol	TFA	Imidazole
	1	35.3mg/0.194mmol/0.2eq. 4µL/0.039mmol/0.04eq.	0	0
	2	35.3mg/0.194mmol/0.2eq	0	0	0
	3	0	4µL/0.039mmol/0.04eq.	0	0
	4	0	0	14.8µL/0.194mmol/0.2eq.	0
	5	0	0	0	13.2mg/0.194mmol/0.2eq.
	6	0	0	0	113µL/0.

173µmol/1.07*10 -4 eq.

Table 2

 2 , the complete synthetic catalytic triad (Table2, Entry i) and the mixture trifluoroacetic acid/imidazole (Table2, Entry ii) are able to polymerize MLABe, but the weight average molar masses of the resulting polymers are very low (600 g/mol and 500 g/mol). Moreover, no polymerization reaction was observed when MLABe was brought together with benzyl alcohol (Table2, Entry iii) or trifluoroacetic acid (Table2, Entry iv) alone. On the other hand, 0.2 equivalent of imidazole (Table2, Entry v) and catalytic amount of imidazole equivalent to the one of histidine into the catalytic triad (Table2, Entry vi) are able to effectively polymerize MLABe with 100% monomer conversion leading to PMLABe with quite high weight average molar masses and low dispersities (3,350 g/mol, dispersity of 1.50 for Entry v, and 7,370 g/mol, dispersity of 1.15 for Entry vi). It should be noted that no monomer's hydrolysis has been observed whatever the experimental conditions used.

Table 2 .

 2 Characteristics of the PMLABe obtained by ROP of MLABe with the synthetic catalytic triad, complete or partial.

	Entry	Conditions	Conversion rate a (%)	M w b (g/mol)	Ð b
	(i)	Imidazolium salt (0.2 eq.) + benzyl alcohol (0.04 eq.)	100	600	1.40
	(ii)	Imidazolium salt (0.2 eq.)	100	500	1.40
	(iii)	Benzyl alcohol (0.04 eq.)	0	---	---
	(iv)	Trifluoroacetate (0.2 eq.)	0	---	---
	(v)	Imidazole (0.2 eq.)	100	3,350	1.50
	(vi)	Imidazole (1.07*10 -4 eq.)	100	7,370	1.15
	a. Determined from 1 H NMR spectra of crude products; b. Measured by SEC (THF, Polystyrene standards, 1
		mL/min, 40°C; Figure SI-1)

Table 3 .

 3 Characteristics of the PMLABe obtained by ROP of MLABe in presence of CpLip2 and CpLip2_S180A enzyme in Tris-HCl buffer pH7 at 60°C under 390 rpm stirring during 72 h.

	M RMN (g/mol)	M w (g/mol) a	Đ a

a. Measured by SEC in THF at 40°C, Standards Polystyrenes, Flow Rate = 1 mL/min.

All these observations are thus in favor of the proposed mechanism, namely the abstraction of one of the two "acidic" protons of the lactone's ring by the catalytic histidine leading to the formation of a monobenzyl fumarate which then initiates the polymerization of the remaining monomers.

CONCLUSIONS. The obtained results lead us to conclude that the lipase-catalyzed polymerization of MLABe does not follow the canonical mechanism, but results from the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the formation of an activated monomer, i.e. monobenzyl fumarate (Figure 2B), responsible for the polymerization of MLABe. In other words, the lipase acts only as a catalyst to form an activated monomer. However, it would also be responsible for particular conformation adopted by PMLABe chains, thus inducing the presence of crystalline zones even when racemic MLABe are polymerized, conclusion confirmed by the observed melting temperatures measured for PMLABe obtained by enzymatic polymerization of racemic MLABe [unpublished results]. However, additional studies (X-rays, for example) still need to be carried out to elucidate the organization of macromolecular chains induced by the presence of the lipase.

ACKNOWLEDGMENT

The authors gratefully thank the "Ministère de l'Enseignement Supérieur et de la Recherche" (MESR) for financial support of this research (Ph.D grant to H.C.) ABBREVIATIONS diMeMLABe: benzyl dimethylmalolactonate MLABe: benzyl malolactonate PdiMeMLABe: poly(benzyl dimethylmalate) PMLABe: poly(benzyl malate) ROP: Ring Opening Polymerization SUPPORTING INFORMATION SEC chromatograms of PMLABe obtained under different experimental conditions (Table 2 and Table 3), 1 H NMR spectra of pure diMeMLABe and after reaction under various conditions.

Funding Sources

This work was funded by the « Ecole Nationale Supérieure de Chimie de Rennes » (ENSCR, France), the Centre National de la Recherche Scientifique (CNRS).

AUTHOR INFORMATION

Corresponding Author * sandrine.marion.1@ensc-rennes.fr Present Addresses † Université Clermont Auvergne, CNRS, SIGMA Clermont, ICCF (Institut de Chimie de Clermont-Ferrand) -UMR 6296, 63000 Clermont-Ferrand, France Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Newton Silva Lima
email: newtonulbra@gmail.com

Aldemir Malveira De Oliveira

Atmospheric conditions in the Amazon River region the dry season (July 2016)

Keywords: Amazon River, atmosphere, Amazonia

This paper is a brief report of the period in the dry season of the Amazon River from Iquitos (Peru) and Manaus (Brazil), from the 13 th to the 30 th of July, 2016, focusing on the Amazonian biosphere and atmosphere. The expedition collected georeferenced data for water samples, measurements of temperature, humidity, atmospheric pressure, surface temperature of the river, and wind direction. Preliminary results show that the temperature at the surface of the river and the environment in general points to possible climate normality.

INTRODUCTION

The Amazon Forest has rich biodiversity resources, such as fresh water and oxygen,and plays an important role in the global carbon cycle by the fact that along with the oceans it has large carbon (C) stocks captured from the atmosphere. The absorption of C by the trees is equal to approximately 17% greenhouse gas emissions [1]. However, the role of the forests in maintaining the climate of South America cannot be forgotten due to the hydrological cycle of interaction and humidity regulation within the tropical forest basin. After the rains the intense evaporation production and humidity recycling returns to the forest as rain again, and between 30% and 50% of the rainfall in Amazon is due to the evaporation recycling, [START_REF] Molion | Amazon rainfall and its variability[END_REF].

To understand the interactions between the forest and the atmosphere, it is necessary to understand the turbulent changes in the Atmospheric Boundary Layer evolves continuously in response to the surface heating or to the cooling, assuming different states that can be described accurately by phase transitions (from day to night / or vice versa) [START_REF] Kaimal | Atmospheric Boundary Layer Flows (Their structure and measurement)[END_REF]. According to [START_REF] Sellers | Modeling Effects of Vegetation on Climate[END_REF], there are three main ways of exchanges between the surface and the atmosphere: radiation, momentum transference and latent heat transference and sensitive heat.

In recent research on the turbulent flows in Central Amazonia, [START_REF] Molion | Dynamic Climatology of the Amazon region: precipitation mechanisms[END_REF] points out that the quantification of turbulent changes and their analysis should be well detailed in tropical forests, so it is necessary to perform micrometeorological measures with great representativeness of the regional climate. This allows obtaining good information about these turbulent flows. In [1] and [START_REF] Molion | Amazon rainfall and its variability[END_REF], studies were done on the macro circulations and mesoscale operating in the Amazon and the dynamic processes that organize and promote the rainfall in the area. According to [1] and [START_REF] Molion | Amazon rainfall and its variability[END_REF], the mechanisms that trigger rain in the Amazon can be grouped into three types:

• Resulting daytime convection from surface heating and favorable large-scale conditions;

• Instability lines originating on the north-northeast of the Atlantic coast;

• Convective clusters of medium and large scales, associated with the penetration of frontal systems in the S / SE region of Brazil and interacting with the Amazon region.

II. MATERIAL AND METHODS

This work was conducted during the first part of the Peru-Brazil River Expedition (Figure 1). Micrometeorology measurements were done using an order of magnitude flow analysis (Method of Covariances) as done according to [START_REF] Foken | Micrometeorology[END_REF], pages 105 and 106, as well as the graphics of rhythmic analysis of the types of the climate and systems, responsible for atmospheric dynamics of the period according to satellite images with information from [START_REF]Weather of Forecast Center and Climate Studies[END_REF]. Analysis of the Wavelet Transform from Morlet, following the methodology of [START_REF] Torrence | A Practical Guide to Wavelet Analysis[END_REF], as the spectrum energy associated with the flow measures.

Wavelet Transform

The term "wavelet" refers to a set of functions in the shape of small waves generated by

    L 2 
, that is, it should have finite energy. The fact that its average energy is zero constitutes the admissibility condition of the function.

Mathematically, the wavelet function in scale a and position b is expressed by:

           a b t a b a 2 / 1 , ) ( (1)
in which a and b are real and a>0. It should be noted that the equations ( 1) and ( 2), include the normalization term a -1/2 . Wavelet Transform (WT) is defined by:

            dt a b t t f a b a f W   ) ( 1 , 2 / 1 , (2)
in which the temporal function f(t) is the series of data to be analysed. The equations ( 1) and ( 2), are similar and the only difference is the nucleus (kernel) of the equations, i.e., the nucleus in the FT is given by an exponential function and in the WT by a wavelet function.

There is the possibility of using different types of wavelet functions for WT depending only on the need. This way, these different types of wavelet functions can be classified into two large groups, namely: continuous wavelets and discrete wavelets. Among the best-known discrete wavelets are [START_REF] Gao | Wavelet analysis of coherent structures at the atmosphere-forest interface[END_REF], [START_REF] Meyer | Ondelettes -Ondelettes et opérateurs[END_REF] and [START_REF] Mak | Orthogonal wavelet analysis: Interannual variability in the sea surface temperature[END_REF]. The best-known continuous wavelet is Morlet, which also allows the analysis of the phase and the signal module when it is complex [START_REF] Farge | The Wavelet Transform and its applications to turbulence[END_REF]. The Mexican hat wavelet [START_REF] Davis | Multifractal characterizations of non-stationary and intermittency in geophysical fields: Observed, retrieved, or simulated[END_REF], [START_REF] Farge | Wavelets and turbulence[END_REF] and [START_REF] Chen | Coherent eddies and temperature structure functions for three contrasting surfaces. Part I: Ramp model with finite microfront time[END_REF] is also mentioned in the literature; however, it is generally operated without a complex part. In the present study, we will use Morlet wavelet function and, therefore, it deserves greater attention.

The Morlet function is a complex wavelet that provides a wealth of information about the signal, such as the module and the phase [START_REF] Farge | The Wavelet Transform and its applications to turbulence[END_REF] and [START_REF] Weng | Wavelets, period doubling, and time-frequency localization with application to organization of convection over the tropical western pacific[END_REF]. This function has the following equation:

) 2 / ( 2 ) ( t t iK e e t     (3)
With the choice of Morlet WT and the measurements of 120-hours temporal series for obtaining the power wavelet spectrum, temperature, and global wavelet spectrum, we obtained results within 95% confidence. The support used was [START_REF] Meyer | Ondelettes -Ondelettes et opérateurs[END_REF], WT modified routines.

III. RESULTS AND DISCUSSION

In Amazon Climate [1] it is said that the availability of the solar energy through the energy balance is 30.7 MJ.m -2 .per day -1 in June / July. These values reduced by the atmospheric transmission being on average of the order of 15 MJ.m -2 .per day -1 .

Figure 4: Satellite images between days 26-30 July 2016, identifying the atmospheric dynamics in the north of South America at 05 h 30 min (LT), time series below the average temperature of the Amazon River to the observed period and Morlet wavelet transform for characterizing the length (h) of occurrence of the most significant event, its duration (h) and their potential intensity warmer colors (red) at a reliability of 95% [START_REF] Gao | Wavelet analysis of coherent structures at the atmosphere-forest interface[END_REF] Figure 4 shows the satellite images panel [23], and the atmospheric dynamics between July, 26 th to 30 th , 2016 between Iquitos (Peru) and Manaus (Brazil) (yellow trace / Amazon river), at 05 h 30 min (LT -Local Time, UTC -5 and -4 UTC). There is a strong influence of the Intertropical Convergence Zone (ITCZ) over northern South America, with rainfall during the period, down from the satellite images it is possible to see the time series of the temperature from the period of 120 uninterrupted hours between Iquitos (Peru) and Manaus (Brazil). The average temperature was 27.45 ° C on the surface of the river, as data from obtained flows through a weather station (Davis -Vantage Vue K6250) coupled on the top deck of the boat M / Monteiro II (Figure 2). The total average temperature of the surface of the river, in Peru, is higher by approximately 2 0 C, which is a normal condition since this part of the river is the beginning and the distance to Manaus is the triple of this extension, where the average is finished. The wind direction seen in the graphic is a feature of many changes of direction caused by the river curves.

IV. CONCLUSION

This time on the Amazon River in the period of July 26-30, 2016 was within the climate normality in the Western Amazon. The observed period was of river drought from Iquitos (Peru) and Manaus (Brazil) already in the Central Amazon.

We cannot yet define the actual present climate because of the first phase of the Expedition, although preliminary results already indicate the real Amazon climate tendency in the drought period. The authors hope to portray the profile from this period in details in December, when the estuary of the river reaches the mouth in the Atlantic Ocean.

Figure 1 .

 1 Figure 1. Conceptual Map of the Expedition -Amazon River (Peru-Brazil), 1 st. Part 26 to 30 July 2016.

Figure 2 :

 2 Figure 2: Lagragian Footprint Model [18], Weather Station -DAVIS -Vantage -Vue, the boat M / Monteiro II, 300 m  R 500 m, between unstable and stable conditions in July 2016.

Figure 3 :

 3 Figure 3: Weather Station -DAVIS -Vantage -Vue, on the Amazon River in July 2016.

Figure 5 :

 5 Figure 5: Data and hours with GPS coordinates of the locations in Peru, with graphics of average temperatures the river's surface and environment and later wind direction between the Brazilian towns of Tabatinga to São Paulo de Olivença on 2016, 27 to 28 July. The speed and wind direction can be observed (Figure 5) (Left) with geo-referencing of the locations where water samples were taken in Peru. (Center) Average Temperature Charts (environment and the river's surface, Peru -July 2016) (Right) Wind direction between the