Cindy Colson
email: colson-c@chu-caen.fr

Hervé Mittre

Adeline Busson

Antoine Leenhardt

Isabelle Denjoy

Véronique Fressard

Yann Troadec

Unusual clinical description of adult with Timothy syndrome, carrier of a new heterozygote mutation of CACNA1C

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

Introduction

Timothy syndrome 1 (TS1) is a rare genetic disorder characterized by associated features such as prolonged QT interval, cardiopathy, hand and foot syndactyly, variable facial dysmorphism, immune deficiency, intermittent hypoglycemia and neurocognitive trouble. This autosomal dominant syndrome is recurrently caused by a heterozygous p.Gly406Arg mutation, in the alternative spliced exon 8A of the CACNA1C gene, encoding for the main cardiac L-type calcium channel. Along with p.Gly402Ser, p.Gly406Arg is also responsible for Timothy Syndrome 2 (TS2) with usually absence of syndactyly [START_REF] Splawski | Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations[END_REF][START_REF] Fröhler | Exome sequencing helped the fine diagnosis of two siblings afflicted with atypical Timothy syndrome (TS2)[END_REF][START_REF] Diep | Long QT syndrome with craniofacial, digital, and neurologic features: Is it useful to distinguish between Timothy syndrome types 1 and 2?[END_REF][START_REF] Hiippala | Expanding the phenotype of Timothy syndrome type 2: an adolescent with ventricular fibrillation but normal development[END_REF][START_REF] Philipp | Cardiac arrest refractory to standard intervention in atypical Timothy syndrome (LQT8 type 2)[END_REF]. Other rare mutations have been reported: p.Ala1473Gly and p.Ile1166thr in TS1 [START_REF] Gillis | Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: expanding the spectrum of Timothy syndrome[END_REF][START_REF] Wemhöner | Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome[END_REF][START_REF] Boczek | Novel Timothy syndrome mutation leading to increase in CACNA1C window current[END_REF].

Usually, life expectancy is very short due to the severity of this syndrome for the patients who carry the homogeneous constitutional mutation [START_REF] Splawski | Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism[END_REF][START_REF] Gao | Inhibition of late sodium current by mexiletine: a novel pharmotherapeutical approach in timothy syndrome[END_REF]. One of the oldest patients alive published with TS is 24 years old and carries the canonic p.Gly406Arg mutation. She presented very mild TS1 with prolonged QTc and mild learning difficulties [START_REF] Walsh | A multicentre study of patients with Timothy syndrome[END_REF]. Attenuated phenotype has been exposed in mosaic form of TS [START_REF] Splawski | Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism[END_REF][START_REF] Etheridge | Somatic mosaicism contributes to phenotypic variation in Timothy syndrome[END_REF][START_REF] Dufendach | Maternal mosaicism confounds the neonatal diagnosis of type 1 Timothy syndrome[END_REF]14]. We describe a novel de novo heterozygous variant p.Glu407Ala in CACNA1C identified by the analysis of genes implicated in heart rhythm diseases, on a patient suffering of an first-degree atrioventricular block, ventricular tachycardia and important QT prolongation at 640ms. Phenotypical revision of the proband allowed to classify her in TS1. This case reports the natural history of an adult TS with novel de novo heterozygous CACNA1C variation and expands the clinical and molecular spectrum of TS. It also shows the significant contribution of targeted NGS in diagnosing rare or atypical diseases and the importance of clinicobiologic discussion leading to classify a variant, in order to conclude on its pathogenicity. Keyswords Timothy; CACNA1C; adult; c.1220A>C; PKP2

Clinical report

The proband is 22-year-old girl. She was born as a first of three children to a healthy young Caucasian couple. There was neither report of consanguinity nor premature sudden cardiac death nor arrhythmic disorders nor neurocognitive disorders in the medical family history. She was born eutrophic at term following good pregnancy. The neonatal follow-up was uncomplicated. At 6 months she presented sudden collapse several times a day, more or less associated with convulsive movements. At first, the diagnostic of epilepsy was retained and treatment with Depakine was instituted. However, the sudden collapses persisted and she was addressed to a cardiologist at three years old. Echocardiogram revealed normal cardiac structure and function. ECG identified: a corrected QT interval slightly prolonged to 440 ms and reaching 640 ms on Holter monitoring at night, an atrioventricular block and ventricular tachycardia episodes. Long QT syndrome (LQTS) was concluded. Beta-blockers were then prescribed.

The treatment was not completely effective with the persistence of syncopes. ECGs performed on both her brothers and parents were normal. Sanger sequencing of all introns close to the coding regions of KCNQ1, HERG (KCNH2) and SCN5A was negative. Because of the persistence of syncopes, an internal pacemaker was implanted at six and an automated internal cardioverter defibrillator (AICD) at seven years old.

Ethics statement

This study analysis was conducted according to the Declaration of Helsinki with written consent to use clinical data and photographs for research purposes obtained from the patient and her parents.

Genetic analysis

A rythmologic genes panel (CASQ2, CALM1, DSC2, DSG2, DSP, KCNE1, KCNE2, KCNQ1, KCNH2, KCNJ2, LMNA, NKX2-5, PKP2, RYR2, SCN5A, TRDN, CACNA1C genes) using next generation sequencing techniques on MiSeq (Illumina, San Diego CA), was performed on genomic DNA extracted from leucocyte of peripheral blood using standard techniques. It studied all exons and flanking intronic region of all genes and identified two heterozygous variations on CACNA1C (NM_199460.3) and PKP2 (NM_004572.3) genes. In the CACNA1C gene, a missense variant c.1220A>C in exon 9 was found, predicting an amino acid substitution of an alanine (hydrophilic amino acid) for a glutamine (hydrophobic amino acid with loss of a negative charge) at codon 407 (p.Glu407Ala). This mutation had never been reported before and was missing from the gnomAD exomes database. Assessments of pathogenicity obtained with PolyPhen indicated 100% probability of damaging (1 out of 1). It is the same with other sites (Varsome). Multiple sequence alignments of the mutated region of Cav1.2 protein (OMIM: Q13936-12) showed a high conservation in many available species. The mutation affects the first codon of the constitutive splicing exon 9 meaning the amino acid downstreams from the one involved in the recurrent mutation p.Gly406Arg. The topological position of the variation in channel architecture is very similar to the position of the original TS mutation. Indeed, the two variation p.Gly406arg and p.Glu407Ala are located in the cytoplasmic DI-DII loop of the channel. p.Glu407Ala leads to structural modifications in channel architecture with an additional alpha helix in the ion transport domain (Figure 1). The patient's mother and father did not carry the variant, indicating a de novo variant occurrence. In the PKP2 gene, c.2146-1G>C variation known as responsible for arrhythmogenic right ventricular dysplasia was found, inherited from her father (Figure 2). The two variations were validated by Sanger sequencing (Supplemental data, Figure 1). According to the American College of Medical Genetics 2015 guidelines, CACNA1C variation was classified as likely pathogenic (LP) and PKP2 variation as pathogenic.

A posteriori, clinical history was marked by mild learning difficulties requiring adapted school orientation with speech therapy and an history of seizures. Cerebral tomodensitometry was normal. To date, there are no signs of a psychiatric disorder. Her physical examination showed cutaneous syndactyly on both hands with hypermobility and partial syndactyly of the 2 nd -3 rd toes bilaterally. She does not suffer from other musculoskeletal abnormalities and does not have specific craniofacial dysmorphism. Furthermore, laboratory finding did not reveal hypoglycemia and neither hypocalcemia, hypothyroidism nor abnormal immunity. Fragile X testing and Array Comparative Genomic Hybridization were negative. The variant was interpreted as causing the disease and the patient was given the diagnosis of TS.

Discussion

We report an unusual case of homogeneous adult Timothy syndrome with de novo novel heterozygous CACNA1C variation. She presented severe cardiac phenotype with incomplete TS1 features and the known c.2146-1G>C heterozygous inherited variation of PKP2. The fact that the rythmologic genes panel was not targeted on the known deleterious regions of CACNA1C allowed us to diagnose this disease.

Next generation sequencing is useful for genetic etiology in patients with rare non-classic phenotypes but results could be difficult to analyze. The assessment of the pathogenicity of the variant on several sites (gnomAD exomes database, PolyPhen, Varsome), the neomutation and the topography of the variation in front of p.Gly406Arg are in favor of very high probability of a damaging effect of the mutation. However, functional expressional studies are required to definitively confirm the disease-causing effect of the p.Glu407Ala mutation. Moreover physical examination and the patient medical history helped to classify the disease as an atypical TS1 rather than a TS2.

To our knowledge, it is the first description of a variation in exon 9 of CACNA1C in TS. Another variation in exon 9 of CACNA1C (c.1368G>A, p.Met456Ile) had been detected in a patient with diagnosed long QT syndrome [START_REF] Fukuyama | Long QT syndrome type 8: novel CACNA1C mutations causing QT prolongation and variant phenotypes[END_REF]. Exon 9 is not alternatively spliced and is therefore C (likely constitutively expressed. It has multi-organic expression in multiple adult and fetal tissues including heart and brain. p.Ala1473Gly in exon 38 is also constitutively expressed, that may likely be in favor of the more severe phenotype seen in their patient who manifested prolonged QT interval, polymorphic ventricular tachycardia, dysmorphic facial features, syndactyly of the hands and feet, joint contractures, seizures and developmental delay [START_REF] Gillis | Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: expanding the spectrum of Timothy syndrome[END_REF]. p.Ile1166Thr in exon 27 was found by whole exome sequencing in case with severe atypical TS who exhibited QT prolongation, patent ductus arteriosis, seizures, facial dysmorphism, joint hypermobility, hypotonia, hand anomalies (clinodactyly and short thumbs), intellectual impairment and tooth decay [START_REF] Boczek | Novel Timothy syndrome mutation leading to increase in CACNA1C window current[END_REF]. Our case seems less severe, especially in musculoskeletal manifestations and development delay. Interestingly, functional analyses of Boczek et al. identified a novel mechanism of TS-mediated disease through increased window current [START_REF] Boczek | Novel Timothy syndrome mutation leading to increase in CACNA1C window current[END_REF]. They hypothesize it could be linked in signaling cascades in the cytoskeleton and dendrite behavior, which could be important for neuronal and skeletal phenotypes [START_REF] Boczek | Novel Timothy syndrome mutation leading to increase in CACNA1C window current[END_REF]. Despite the canonical mutation, TS1 cardiac and extracardiac features are not identical from one patient to another [START_REF] Sepp | Timothy syndrome 1 genotype without syndactyly and major extracardiac manifestations[END_REF]. Further analyses are necessary to explain the phenotype variability, which is not explained by alternatively splicing or mosaicism [START_REF] Splawski | Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations[END_REF][START_REF] Etheridge | Somatic mosaicism contributes to phenotypic variation in Timothy syndrome[END_REF][START_REF] Dufendach | Maternal mosaicism confounds the neonatal diagnosis of type 1 Timothy syndrome[END_REF]14]. This variability could be due to modifying genetic factors that remain to be identified. The c.2146-1G>C heterozygous variation of PKP2 is a known splice acceptor site mutation responsible for arrhythmogenic right ventricular cardiomyopathy [START_REF] Gerull | Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy[END_REF]. Usually, palpitations or ventricular tachyarrhythmias are the common clinical manifestations of the disease [START_REF] Syrris | Clinical expression of plakophilin-2 mutations in familial arrhythmogenic right ventricular cardiomyopathy[END_REF]. Low-penetrance in family members is also described [START_REF] Dalal | Penetrance of mutations in plakophilin-2 among families with arrhythmogenic right ventricular dysplasia/cardiomyopathy[END_REF]. Our patient had an echocardiogram, echocardiography and isotopic ventriculography. No data were disponible for her father. These examinations did not show anything in favor of this disease. However, we do not know the effect of this mutation associated with the one in CACNA1C. Cerrone et al. reported that PKP2 knock-out led to decrease the rate of L-type calcium channel inactivation and prolong L-type calcium current decay. This suggests that if the splicing variation PKP2 of the proband affects clinical expression, it may worsen the phenotype of Timothy syndrome. Unfortunately, further tests would be needed to show if the PKP2 variation could affect the phenotype or not [START_REF] Cerrone | Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm[END_REF].

Theses clinical and molecular findings expand the clinical and molecular spectrum of TS and show the interest of next generation sequencing or whole exome sequencing in rare disorders, atypical or novel phenotype. Further studies are needed to delineate the role of CACNA1C and to look for modifying genetic factors that could explain the phenotype variability of TS.


Juan José Rodríguez-Bencomo

Ahmad Mehdi

Removal of biogenic amines from hydroalcoholic solutions by functionalized silica

Keywords: Detoxification, Biogenic Amines, Hydroalcoholic Solutions, Synthetic Wine, Hybrid Silica

The structure and the cation-exchange functional groups of hybrid silica materials were evaluated for the effective detoxification of hydroalcoholic solutions containing eight toxic biogenic amines (BA) usually found in fermented beverages. Results show the effectiveness of the removal is related to the number of amino functions in the extracted molecule, retention by the solid being more effective in the case of multiple amino groups, since retention is stabilized through interaction with the material surface at several points. BA with one amino function (isoamylamine, tyramine, β-phenylethylamine), in general, showed a weak retention by the solids. For BA with more than two amine groups (spermine, spermidine), the removal rate was close to 100% for all studied materials. For histamine, cadaverine and putrescine, the removal percentages were higher with a lamellar structured sulfonic acid functionalized material and with bifunctional materials (SBA-15 type and macroporous) containing sulfonic/phosphonic acid groups obtained by co-condensation solgel route.

Introduction

The application of adsorption processes for the removal of toxic compounds is common in the industry. The effectiveness and selectivity requirements of the adsorption will depend on the type of application, so searching for suitable adsorbents can be complex. In the case of toxic compounds present in food products, the challenge is even more complex since the process should be compatible with maintaining the properties and the quality of the product [START_REF] Kammerer | Adsorption and ion exchange: basic principles and their application in food processing[END_REF][START_REF] Nageeb Rashed | Adsorption technique for the removal of organic pollutants from water and wastewater[END_REF].

In general, the origin of biogenic amines (BA) in food products is mainly due to the action of microorganisms (decarboxylase enzyme activity) that produce these compounds by the metabolism of the amino acids. Thus, amines such as histamine, putrescine, cadaverine, spermine, spermidine, tyramine, tryptamine and β-phenylethylamine are very common in cheese, fishery products and fermented beverages such as wine [START_REF] Smit | Biogenic amines in wine: understanding the headache[END_REF][START_REF] Naila | Control of biogenic amines in food--existing and emerging approaches[END_REF].

Regarding the toxicity of BA, the human body can tolerate low amounts, in fact, most of them are naturally present in the body due to the fact that they have numerous physiological functions; however, at high levels they can cause negative effects on health. An example is histamine, which is involved in the control of body temperature, learning and memory, immune response and allergic reactions; however, at toxic levels it can produce headaches, sweating, burning nasal secretion, facial flushing, bright red rashes, dizziness, urticaria, respiratory distress, tachycardia and blood pressure disorders [START_REF] Maynard | Monoamine oxidase inhibition by ethanol in vitro[END_REF][START_REF] Ladero | Toxicological effects of dietary biogenic amines[END_REF]. These negative effects can be increased by the presence of ethanol, so its control in alcoholic beverages is justified, though these are not the products with the highest contents in amines. [START_REF] Maynard | Monoamine oxidase inhibition by ethanol in vitro[END_REF]. For this reason, in recent years, some countries have implemented regulations based on maximum levels or maximum recommended levels in wine for some BA (for example, maximum recommended levels for histamine in wine: France, 8 mg/L; Germany, 2 mg/L) [START_REF] Smit | Nitrogen fertilisation increases biogenic amines and amino acid concentrations in Vitis vinifera var. Riesling musts and wines[END_REF][START_REF] Guo | Biogenic amines in wine: a review[END_REF].

aging. The final levels depend on viticulture and winemaking factors, such as soil fertilization, indigenous microorganisms in the vineyard (bacteria, yeast and fungi) or the winemaking technique [START_REF] Kiss | Study of amine composition of Botrytized grape berries[END_REF][START_REF] Moreno-Arribas | Amino acids and biogenic amines[END_REF][START_REF] Smit | Nitrogen fertilisation increases biogenic amines and amino acid concentrations in Vitis vinifera var. Riesling musts and wines[END_REF].

In particular, the main origin of these BA in wines is the malolactic fermentation (MLF) that is carried out by lactic acid bacteria (LAB) [START_REF] Moreno-Arribas | Amino acids and biogenic amines[END_REF]). Although inoculation with lactic acid bacteria starters is less common compared with the inoculation of yeasts in industrial winemaking, numerous studies have been carried out focusing on the selection of bacteria starters with low production of BA [START_REF] Berbegal | Lowering histamine formation in a red Ribera del Duero wine (Spain) by using an indigenous O. oeni strain as a malolactic starter[END_REF]. However, the effectiveness of this option is strongly dependent on the strain and the winemaking technique and conditions [START_REF] Beneduce | Biogenic amine in wines[END_REF][START_REF] Ancin-Azpilicueta | Comparative study of the amine concentration in wines obtained from the traditional fermentation and from a more anaerobic fermentation method[END_REF], since the production of BA is not controllable. On the other hand, another, less studied, approach is the degradation of BA by specific LAB (Garcia-Ruiz, Gonzalez-Rompinelli, [START_REF] Garcia-Ruiz | Potential of wine-associated lactic acid bacteria to degrade biogenic amines[END_REF][START_REF] Callejon | Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine[END_REF] or by other microorganisms such as fungi [START_REF] Cueva | Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking[END_REF]. However, the addition of other microorganisms could produce undesired effects due to the enzymatic activities and the microorganisms' metabolism.

There is a further option which has been explored much less than the aforementioned, an approach based on the removal of BA by selective adsorption by solids.

Vlasova and collaborators [START_REF] Vlasova | The adsorption of biogenic amines on the surface of highly dispersed silica from aqueous solutions[END_REF][START_REF] Vlasova | Adsorption of biogenic amines on albumin-modified silica surface[END_REF] studied the removal of histamine, tyramine and tryptamine from water solutions with non-functionalized silica and albumin-modified silica surface, evaluating the effect of the solution pH. However, these authors observed the best results at high pH, different from the typical pH of the wine (around 3.5). One of the most interesting studies, published by [START_REF] Amghouz | Biogenic amines in wine: Individual and competitive adsorption on a modified zirconium phosphate[END_REF], is based on the chemical state of amines in wines (as protonated substances), eliminating them through a cation exchange process with solids based on zirconium phosphate. In this case, the authors worked on synthetic wine media and obtained good extraction rates for putrescine, cadaverive and histamine. More recently, [START_REF] Bettini | A smart method for the fast and lowcost removal of biogenic amines from beverages by means of iron oxide nanoparticles[END_REF] proposed the use of iron oxide nanoparticles covered by a silica shell for the BA removal from alcoholic beverages. The results showed good extraction for putrescine and 2-phenylethylamine, but in the case of the last compound after application of several treatments. Moreover, they proposed the removal of the particles from the liquid by using a weak magnetic field due to the paramagnetic character of the particles.

The different strategies for minimizing the contents of BA in wines present drawbacks and advantages. The degradation of the BA by using microorganism or the inoculation of selected starters of LAB presents the advantage that they are easy to integrate on the winemaking process without complex operations. However, the main drawbacks are related to the variable effectiveness and the possible generation of undesired compounds and/or organoleptic deviations in the final products. On the other hand, the approaches based on the removal of BA by the adsorption on solid presents the advantage that there is no risk of generating undesired degradation products, however, the collateral removal of other components could affect the characteristics of the final products. Moreover, depending on the way to use the adsorbent (in batch or in packed columns), the process could be carried out and integrate into common winery operations. However, due to the scarce bibliography related the BA removal from finished wines, until now, the organization international of vine and wine (OIV) has not approved any method or technique to this purpose.

Focusing on the removal of BA from finished wines, although the studies carried out until now show the great potential of the use of solids (or functionalized solids), however, in general, these works are focused on a single type of adsorbent and/or on minimal modifications of these, so, really no systematic studies have carried out considering structural and chemical aspects of the adsorbents. The main novelty of this study is the approach for a systematic searching the adequate adsorbent for the BA removal considering of two important characteristics of the adsorbents, its structure and the functional groups on the surface. In addition, two methods for the introduction of the functional groups have been considered in order to establish the most adequate synthesis method of the adsorbents.

Therefore, the aim this work was to evaluate different hybrid silica structures and cation exchange functional groups for the removal of eight usual BA found in wines.

Experimental Section

Reagents

The eight BA studied in this work were (the size of the molecule is indicated in brackets and expressed as the longer length (nm) (obtained from http://molcalc.org/)) (purity>97%): histamine (0.789), putrescine (0.783), cadaverine (0.777), spermidine (0.937), spermine (1.062), 2-phenylethylamine (0.812), tyramine (0.875) and isoamylamine (0.673).

BA were obtained from TCI chemicals (Zwijndrecht, Belgium) and Across chemicals (Geel, Belgium). Tetraethyl orthosilicate (TEOS) (98%) from Alfa-Aesar (Kandel, Germany), 3mercaptopropyltrimethoxysilane (MPTS) (95%), (Diethylphosphatoethyl)triethoxysilane (DPETS) (95%) and 3-cyanopropyltriethoxysilane (CPTS) (95%) from Abcr.de (Karlsruhe, Germany), were used as precursors for the synthesis of functionalized silica. Bis [3-(silsequioxy)propyl] tetrasulfide was kindly donated by Sikemia (Montpellier, France).

Triblock copolymer Pluronic P123 (Poly(ethylene glycol)-block-poly(propylene glycol)-blockpoly(ethylene glycol)) and cetyltrimethyl ammonium bromide (CTAB) (99%) from Aldrich (St.

Louis, USA), and polyethylenglycol (PEG) 20.000 from Alfa-Aesar, were used as surfactants for the synthesis of silica by sol-gel route. Finally, hydrochloric acid (37%), ethanol (>99.8%), and nitric acid (65%) and dichloromethane (>99.8%) from VWR (Fontenay sous Bois, France), toluene (>99.7%) and sulfuric acid (98%), Triphenylphosphine (99%), dioxane (99.8%) and meta-chloroperoxybenzoic acid (99%) from Sigma-Aldrich (Steinheim, Germany), and ammonia (30%) from Carlo-Erba (Val de Reuil, France) were obtained.

Synthesis of materials

A scheme of the synthesis of the cation exchange materials used in this study is presented in Figure S1 in supplementary information section.

SBA-15 and mesoporous xerogel materials (direct synthesis sol-gel route)

For SBA-15 synthesis, TEOS or the adequate mixture of TEOS and the functionalized precursors (DPETS and/or MPTS or CPTS) were added to a mixture of H2O, HCl and P123 in the following molar ratio 1:140:13.3:0.015. The mixture was heated and stirred at 40 °C for 20 h. The aging process was carried out at 60 °C and 110 °C (depending on the type of silica desired) for 24 h. After washing with water, surfactant was removed by soxhlet extraction with ethanol [START_REF] Margolese | Direct syntheses of ordered SBA-15 mesoporous silica containing sulfonic acid groups[END_REF]Mouawia, Mehdi, Reye & Corriu, 2008;[START_REF] Pan | Probing the nature and local structure of phosphonic acid groups functionalized in mesoporous silica SBA-15[END_REF].

For the synthesis of mesoporous xerogel materials, the ratio of H2O and HCl was the same than in the case SBA-15 at 60 °C but without surfactant. In addition, 1% of NaF (in weight respect to TEOS) was added in order to accelerate the aging process [START_REF] Tilgner | Effect of acidic, basic and fluoride-catalyzed sol-gel transitions on the preparation of sub-nanostructured silica[END_REF][START_REF] Carre | Use of the 2,6bis[(dimethylamino)methyl]phenyl ligand for the study of nucleophilic substitution at hexacoordinate silicon centers. Evidence suggestive of a heptacoordinate silicon transition state[END_REF].

MCM-41 materials (direct synthesis sol-gel route)

TEOS or the adequate mixture of TEOS and the functionalized precursors (DPETS) were added to a mixture of H2O, NaOH and CTAB in the molar ratio 1:100:0.3:0.12. The mixture was stirred for 24 h at 30 °C and 20 h at 60 °C. After washing with water, ethanol and acetone, the surfactant was removed by soxhlet extraction with acidified ethanol (HCl) [START_REF] Qian | Exploring meso-/microporous composite molecular sieves with core-shell structures[END_REF].

Macroporous xerogel materials (direct synthesis sol-gel route)

TEOS or the adequate mixture of TEOS and the functionalized precursors (DPETS and MPTS) were added to a mixture (previously stirred 30 min in an ice bath) of H2O, HNO3 and PEG in the molar ratio 1:13.4:0.26:0.0013. The mixture was cooled in an ice bath and stirred for 30 min and then was aged for 72 h. at 40 °C. The solid was washed with water and then was treated with ammonium solution 0.1 M for 24 h at 40 °C. Surfactant was removed by soxhlet extraction with ethanol [START_REF] Galarneau | Hierarchical porous silica monoliths, a novel class of microreactors for process intensification in catalysis and adsorption[END_REF]).

Post-grafting route

For the addition of the second functional groups by post-grafting route, the dry materials (functionalized solids obtained by sol-gel route) were treated with the adequate precursor (MPTS or CPTS) in toluene for 24 h at 120 °C (under reflux). The precursor percentage was fixed at 15% (in molar ratio). After reflux, solids were filtered, washed with toluene and dried under vacuum at 80 °C [START_REF] Chaudhuri | SBA-15 functionalised with high loading of amino or carboxylate groups as selective adsorbent for enhanced removal of toxic dyes from aqueous solution[END_REF].

Lamellar sulfonic acid-functionalized material

Bis [3-(silsequioxy))propyl]-tetrasulfide was treated with triphenylphosphine and water (molar ratio 1:6.5:8.1) in dioxane acidized with 10 drops of HCl (37%). The mixture was stirred for 3 h at 40 °C under argon. The product was washed with dioxane and acetone and then was treated with 2 equivalents of meta-Chloroperoxybenzoic acid in dichloromethane for 12 h at room temperature. The lamellar sulfonic acid-functionalized material obtained was washed with dichloromethane and was dried under vacuum [START_REF] Alauzun | Synthesis of new lamellar materials by self-assembly and coordination chemistry in the solids[END_REF].

Chemical transformations of functionalized materials

For the transformation of the thiol group of MPTS into sulfonic acid, 4.0 g of synthesized solid were treated with 50 mL of H2O2 (33%) for 24 h at room temperature. After that, the solid was treated with H2SO4 (2M) for 20 min at room temperature and then was washed with water. For the transformation of the diethylphosphonate groups into phosphonic acid, 4.0 g of the synthesized solid were treated with 80 ml of HCl (37%) at 110 °C for 24 h (under reflux). After that, the solid was washed with water. For the transformation of nitrile group of the CPTS into carboxylic acid, 4.0 g of synthesized solid were treated with 50 mL of H2SO2 (9M) for 12 h at 130ºC (under reflux) and then was washed with water. All products were dried under vacuum at 60 °C.

Characterization of materials

Surface area and pore size of the materials were measured using the nitrogen adsorption/desorption method on a Micromeritics TriStar instrument (Micromeritics Instrument Co., Norcross, GA) at -196 °C (77 K). Before analysis, all samples were outgassed under vacuum at 100ºC for 4 h. 2.4. Removal of BA from hydroalcoholic solutions by adsorption on solids Concentrated individual solutions of each amine (between 1 and 5 g/L) were prepared in HCl (0.1 M). For the experiments evaluating the effectiveness of the removal of BA by the synthesized solids, a hydroalcoholic solution (12% ethanol v/v; 3.5 g/L of tartaric acid; pH=3.5) were spiked with BA at a level of 8 mg/L for each one. Experiments evaluating the BA removal capacity of each synthesized material (at a solid dose of 5 g/L) were carried out in 5 mL vials by adding 4 mL of hydroalcoholic solution and 20 mg of the functionalized solid.

Extraction time was initially fixed at 24 h (with continuous stirring) in order to ensure reaching the extraction equilibrium. After extraction time, samples were filtered (0.2 µm). 1 mL of sample and 50 µL of internal standard solution of 160 mg/L (L-2-Aminoadipic acid (TCI chemicals)) were added to a HPLC vial which was stored at 4 °C until analysis. All experiments were carried out in duplicate.

The study of the effect of the dose of solid was carried in vials of 5 mL with 4 mL of synthetic solution (spiked with BA at a level of 8 mg/L) and increasing amounts of solid (from 5 to 12.5 g/L). After the extraction (24 h), the samples were prepared for HPLC analysis.

The evaluation of extraction kinetics was carried out with 80 mL of hydroalcoholic solution spiked with 8 mg/L of each amine and a dose of 5g/L of solid (Macroporous xerogel 10% phosphonic acid). Samples of 1.5 mL were taken at different times (0, 0.5, 1, 3, 5, 8, 12, 24, 48 hours) and were prepared for HPLC analysis as previously described.

Biogenic amine Analysis.

Samples were analyzed by Liquid chromatography (LC) coupled to a mass spectrometer detector (MS). LC-MS analysis were performed on an Ultimate 3000 UPLC system (Thermo), equipped with a Biphenyl 50x2.1mm, 2.6µm column (Phenomenex)

Column temperature was 30 ºC and 10 µL of sample were injected. The elution gradient was maintaining 100% of A (Formic acid 0.01%) for 3 minutes, then from 0 to 50% of B (Acetonitrile + formic acid 0.01%) in 2 minutes, then from 50% to 100% of B in 1 minute, 100% of B is maintained for 2 minutes before returning to initial conditions. Flow rate was set at 0.5 ml/min. Mass spectrometer coupled with the UPLC system was an AmaZon Speed Ion

Trap system (Bruker) with electrospray ionization source operating in SIM or MRM mode.

The m/z ions for SIM quantification and the MRM transitions were: histamine (112); putrescine (89); isoamylamine (88); cadaverine (103→86); spermidine (146→129); spermine (203→129); tyramine (138→121); 2-phenylethylamine (122→105); L-2-aminoadipc acid (162→145). In these analytical conditions, acceptable and adequate repeatability and linearity were observed in the concentration range 0 -10 mg/L. BA contents were expressed as relative peak area and the extracted percentage was calculated with respect to a reference sample for each experiment.

Statistics

One way-ANOVA and least significant difference (LSD) were used to evaluate the differences in amine removal results. STATISTICA program for Windows version 7.1 was used for data processing (StatSoft, Inc., 2005, www.statsoft.com).

Results and Discussion

3.1. Structure and functionalization of the silica.

The selection of silica as base material was based on the possibility of easily synthesizing different structures (with different functional groups) and on its chemical stability at acid pH. In addition, thanks to the high stability of Si-C bond, the functionalization of the silica was obtained by using organotrialkoxisilane precursors that allow generating very stable immobilization of functional groups on the silica structure [START_REF] Mehdi | From molecular chemistry to hybrid nanomaterials. Design and functionalization[END_REF][START_REF] Borges | Silica, hybrid silica, hydride silica and non-silica stationary phases for liquid chromatography. part II: chemical and thermal stability[END_REF]. Considering that BA at the pH of wine (around 3.5) are protonated, the more logical interaction mechanism for its removal from wines would be the cationic exchange. Preliminary experiments with non-functionalized silica or functionalized with groups without cation exchange capacity (diethylphosphatoethyl group) showed a practically null BA removal capacity from hydroalcoholic solutions (data not shown). These results are in accordance with those obtained by [START_REF] Vlasova | The adsorption of biogenic amines on the surface of highly dispersed silica from aqueous solutions[END_REF] that did not observe amine removal capacity from water solutions at acid pH by non-functionalized silica.

Therefore, several functional groups (phosphonic, sulfonic and carboxylic acids) with cation exchange capacity were considered in order to functionalize the silica. On the other hand, taking into account the selectivity and effectiveness requirements of the removal process, and to avoid the possible effects on the wine quality (color, aroma, etc.), several types of structures of silica were considered (ordered hexagonal mesoporous silica materials (SBA-15 and MCM-41), mesoporous and macroporous xerogels and lamellar structures).

A first set of materials functionalized with a 10% of phosphonic acid was obtained by sol-gel co-condensation route (direct synthesis) and its characteristics are presented in Table 1. As can be seen, the porosity ranged from less than 10 nanometers (SBA-15, MCM-41 and mesoporous xerogel structures) to more than 500 nanometers (macroporous xerogel structure). With the phosphonic acid set fixed as a primary functional group, the introduction of other functional groups, such as carboxylic or sulfonic acids, was carried by cocondensation and post-grafting. Taking into account the distortion of the porous structure that the sulfonic acid precursor (hydrophilic character of thiol group that could distort the micelles) can produce during sol-gel synthesis [START_REF] Margolese | Direct syntheses of ordered SBA-15 mesoporous silica containing sulfonic acid groups[END_REF], the post-grafting route of this functional group was also considered using 5% phosphonic acid functionalized silica. The reduction of the percentage of co-condensate phosphonic acid to 5% was based on avoiding steric hindrance effects on the introduction of the second functional group by post-grafting.

Finally, a lamellar material functionalized with sulfonic acid was also considered. The characteristics of the bi-functional materials synthesized are also presented in Table 1. As can be seen, the introduction of a second functional group by co-condensation sol-gel route and the variation of the percentages of the organic functions (for SBA-15 structures) produced variations of the pore size, being more intense in the case of sulfonic acid functionalization.

3.2. Removal of Biogenic Amines from hydroalcoholic solutions 3.2.1. Silica structure effect For the first evaluation of the silica structure effect on BA removal, the 5 types of silica (SBA-15 60°C , SBA-15 110°C , MCM-41, and macroporous and mesoporous xerogel structures) functionalized with 10% of phosphonic acid were compared for the removal of the 8 BA from a hydroalcoholic solution (with a dose of solid of 5 g/L). Table 2 shows the removal percentage for each amine. As can be seen, the extraction of spermine and spermidine was close to 100 % for all solids assayed. However, histamine, putrescine and cadaverine presented a clear effect of the silica structure. Thus, the solid with the narrow pores, MCM-41, showed the lowest extractions for these three compounds. The maximum removal percentage for putrescine, cadaverive and histamine ranged from of 85.6 to 96.3%. In the case of monoamines, tyramine, isoamylamine and 2-phenylethyamine, removal was lower than 35% and not showing statistical differences among solids.

These results show an effect of the type of amine; in particular, of the number of amino functions in the molecules, polyamines being better retained by the solids than monoamines. These differences between monoamines and polyamines have previously observed by [START_REF] Amghouz | Biogenic amines in wine: Individual and competitive adsorption on a modified zirconium phosphate[END_REF] that suggested, in addition to the cationic exchange, the interaction of N with the OH (of the functional group of the solid (zirconium phosphate sodium salt)) that could stabilize the amine retention. Moreover, these authors also suggest the interaction of tyramine is mainly due to hydrogen bonds. Considering our type of solid (silica) and the type of functionalization (phosphonic acid), probably the interaction mechanisms of BA would be similar to described by [START_REF] Amghouz | Biogenic amines in wine: Individual and competitive adsorption on a modified zirconium phosphate[END_REF]. At the normal pH of wine, between 3 and 4, silanol groups of silica are not dissociated, so an interaction by cation exchange cannot be produced between them and the cationic amines [START_REF] Vlasova | The adsorption of biogenic amines on the surface of highly dispersed silica from aqueous solutions[END_REF]).

However, hydrogen bond type interactions would be possible and could contribute to the stabilization of the amine retention. Regarding the differences observed due to the solid structure, taking into account the size of BA molecules (< 1.1 nm of length) (section 2.1) and the pore size of the solids (> 2 nm), a steric hindrance effect due to pore size is perhaps not logical, considering the longest molecules (spermine and spermidine) were the best retained by all solids. However, the results suggest that the distance between functional groups and its distribution on the solids and separation of the functional groups in the amine molecules could play an important role in the stabilization of retained compounds [START_REF] Mouawia | Bifunctional ordered mesoporous materials: direct synthesis and study of the distribution of two distinct functional groups in the pore channels[END_REF][START_REF] Amghouz | Biogenic amines in wine: Individual and competitive adsorption on a modified zirconium phosphate[END_REF]).

Effect of the dose of the solid and extraction kinetics

According to the results of Table 2, no statistical differences were observed for BA removal with SBA-15 and mesoporous and macroporous xerogels materials. In order to evaluate the solid dose effect and the extraction kinetic, only one of them was chosen, the macroporous xerogel material (10% phosphonic acid).

The study of the dose effect on the BA extraction for the range 5.0 to 12.5 g/L of solid (Figure 1) showed almost total extraction in the case of spermine and spermidine for the entire dose range evaluated. For the rest of amines, as expected, the increase of the dose of solid produced an increase in the amount retained, but not in a totally proportional relation.

Monoamines were not removed from the solutions at percentages higher than 50% with the highest dose (12.5 g/L), corroborating the slight interaction between these amines and the functionalized solids. In the case of putrescine, cadaverine and histamine, the percentage extracted showed a slight increase in relation to the dose added, achieving an increase of the retention lower than 10% when the dose is doubled. Although the doses assayed in this study are quite high, is important take into account that the application to BA removal in real alcoholic beverages, for example wine, must consider the maximum legal limits (or suggested limits) of each country for the amine content of wines. Therefore, depending on the initial value of the beverage, the dose would be adapted and, in most cases, doses lower than 5 g/L would probably be necessary. Considering that the treatment of real alcoholic beverages must avoid affecting its organoleptic characteristics, the minimum dose (5 g/L) was fixed for the following studies.

With the same macroporous xerogel silica (10% phosphonic acid) (at a dose of 5 g/L), a study of the adsorption kinetics was carried out in synthetic media from 30 min to 48 hours of extraction time. The behaviors for all studied compounds are presented in Figure 2. As expected, spermine and spermidine showed extractions higher than 98% from the first 30 min of treatment. For other amines, the extraction equilibrium was reached, in general, after 1 or 2 hours of contact with the solid. At a practical level, for the use of the solid in batch applications, the characteristics of the solid should allow a suspension of the solid for sufficient time to permit the equilibrium to be reached.

Effect of silica functionalization on BA removal

Table 3 shows BA removal percentages for the different bifunctional materials synthesized, based on the structure of SBA-15 110°C (phosphonic acid + sulfonic acid or carboxylic acid), the initial SBA-15 110°C (10% phosphonic acid) and the lamellar sulfonic material. As can be seen, the effect of the introduction of a second functional group depended on the type of amine and the type of functional group. In general, the introduction of carboxylic acid group showed less extraction of BA than in the case of sulfonic acid group.

In the particular case of the lamellar sulfonic material, for 2-phenylethylamine, isoamylamine and tyramine, the removal percentages were much higher than those obtained with the other materials. This fact is probably related to the different structure of this lamellar material in comparison with the others.

With regard to the polyamines spermine and spermidine, both of them presented removal percentages close to 100% for all solids, showing no statistical differences between them. Histamine only showed extraction percentages lower than 99% for the initial 10% phosphonic, and putrescine presented the best extractions with all solids that contain sulfonic acid functions. A similar trend to putrescine was observed for cadaverine, although there were no statistical differences. In general, there were no important differences between removal percentages of mixtures of phosphonic/sulfonic acids obtained by co-condensation and post-grafting.

Although in some cases, the lamellar sulfonic material showed the best removal capacities, it is important to take into account that this type of material could extract other compounds in the wine and therefore could strongly affect quality. In fact, the use of bentonite (a lamellar clay material) is very common in white winemaking for the removal of proteins in order to avoid turbidity after bottling. In this sense, the negative effect that high bentonite doses produce on wine aroma due to the removal of volatile compounds is wellknown [START_REF] Waters | Preventing protein haze in bottled white wine[END_REF]. For this reason, to achieve higher selectivity of BA removal based on the controlled pore structure and pore size, the two structures sulfonic and phosphonic acid mixtures have also been chosen as the most adequate solids.

Thus, in addition to these bifunctionalized (phosphonic + sulfonic acids) mesoporous SBA-15 110°C materials, the two macroporous xerogel equivalent structures have been synthesized in order to evaluate if the structural differences (for example the distances between active functional groups) affect the effectiveness of BA removal. Figure 3 shows the BA removal results for the mesoporous (SBA-15 110°C ) and macroporous xerogel bifunctionalized materials. As can be seen, all monoamines (isoamylamine, 2phenylethyamine and tyramine) showed no statistical differences between materials, although it is possible to observe a trend indicating better extractions with SBA-15 110°C structures than with macroporous xerogels ones. As commented in the previous section, the interaction of these monoamines with the functional groups is not strong enough and this fact could lead to a greater influence of environmental conditions during extraction. By contrast, spermine and spermidine showed extractions higher than 99% for all materials. For histamine, cadaverive and putrescine, on the other hand, statistical differences were observed for BA removal with the different solids. Thus, macroporous xerogel materials synthesized by post-grafting of sulfonic acid showed a lower capacity of extraction than the other three solids. These results suggest, once again, that the distribution of the functional groups on the solids and the distance between them could play an important role in the stabilization of the retained molecules [START_REF] Mouawia | Bifunctional ordered mesoporous materials: direct synthesis and study of the distribution of two distinct functional groups in the pore channels[END_REF][START_REF] Amghouz | Biogenic amines in wine: Individual and competitive adsorption on a modified zirconium phosphate[END_REF].

Moreover, in the post-grafting treatments of the materials, in addition to the possible steric hindrance due to phosphonic groups, the distribution obtained are probably less homogeneous than in the case of the co-condensation sol-gel process [START_REF] Lim | Comparative studies of grafting and direct syntheses of inorganic-organic hybrid mesoporous materials[END_REF], and this also could explain the lower BA removal with the post-grafted materials.

Conclusions

The results obtained establish the hybrid silica functionalized with phosphonic and/or sulfonic acids as an interesting option to remove the biogenic amines from hydroalcoholic solutions.

In addition, the more open structures such as meosoporous SBA-15, macroporous and mesoporous xerogels, and lamellar materials, showed a better amine removal capacity. On the other hand, the effectiveness of the removal strongly depended on the number of amino functions in the molecule, retention by the solid being more effective in the case of multiple amino functional groups. In the same way, extraction kinetics also depended on the number of amine functions in the biogenic amine molecule, achieving faster equilibrium for the molecules with more than 2 amino functions. With regard to the way to synthesize the bifunctionalized materials (phosphonic and sulfonic acid functions), ordered hexagonal mesoporous (SBA-15) and macroporous xerogel structures obtained by co-condensation achieved greater effectiveness in amine removal than post-grafted structures. Taking into account the selectivity needs to avoid undesired effects in the application in real wines, this study supply valuable information about the characteristics of the hybrid silica structures for its evaluation in real wine samples. In addition, the synthesis of this type of materials is not expensive and, depending on the way to its build, also would allow different approaches for its application, from discontinuous batch application to continuous processes by using packed columns. Histamine 95.0 ± 0.4 a 99.9 ± 0.1 c 99 ± 0.1 b 99.2 ± 0.0 bc 99.6 ± 0.5 bc Putrescine 82.0 ± 3.9 a 97.6 ± 1.6 b 94.9 ± 0.1 b 86.6 ± 3.0 a 98.0 ± 0.3 b Cadaverine 88.7 ± 3.9 97.7 ± 2.0 91.0 ± 0.9 81.4 ± 8.0 96.5 ± 5.0 Spermidine 100 ± 0 100 ± 0 99.7 ± 0.3 99.7 ± 0.2 99.9 ± 0.2 Spermine 100 ± 0 100 ± 0 100 ± 0 100 ± 0 99.9 ± 0.1 2-Phenylethylamine 22.1 ± 8.1 a 46.5 ± 3.0 a 37.9 ± 0.2 a 33 ± 2.2 a 78.9 ± 1.

Figure captions Figure 1 .

 captions1 Figure captionsFigure1. Effect of the dose of the solid (functionalized silica) on the biogenic amine removal from hydro-alcoholic solutions. Solid type: Macroporous xerogel (10% phosphonic acid). Different letters indicate statistics differences in ANOVA (p< 0.05) and the LSD test. Removal values of 100 ± 0 indicate that the amine was not detected in the treated sample.

Figure 2 .

 2 Figure 2. Adsorption kinetic of each biogenic amine on the functionalized silica material from hydro-alcoholic solutions. Solid type: Macroporous xerogel (10% phosphonic acid). Solid dose: 5 g/L.

Figure 3 .

 3 Figure 3. Biogenic amine removal (%) from hydro-alcoholic solutions with SBA 15 110°C and Macroporous xerogel silica materials functionalized with sulfonic (sulf) and phosphonic (pho) acids. (1) Co-condensation sol-gel route. (2) Post-grafting route. Different letters indicate statistics differences in ANOVA (p< 0.05) and the LSD test. Removal values of 100 ± 0 indicate that the amine was not detected in the treated sample.

a

  Removal values of 100 ± 0 indicate that the amine was not detected in the treated sample. Different letters indicate statistics differences in ANOVA (p< 0.05) and the LSD test. b Pho: Phosphonic acid. Sulf: Sulfonic acid. Carbox: Carboxylic acid. (1) Direct synthesis (co-condensation) sol-gel route. (2) Postgrafting route (15% molar ratio for the post-grating process).

Table 1 .

 1 Characteristics of the synthesized functionalized silica materials.

	Materials a			Synthesis method and precursors b	Surface Area (m 2 /g)	Pore diameter (nm)	Pore Volume (cm 3 /g)
	10% phosphonic acid functionalized materials
	15 % °	( )		DS: TEOS (90%), DPETS (10%)	560.3	≈ 6.2	0.662
	15 % °	( )		DS: TEOS (90%), DPETS (10%)	557.5	≈ 7.5	0.922
	41 %	( )		DS: TEOS (90%), DPETS (10%)	840.4	≈ 2.5	0.877
		. %	( )	DS: TEOS (90%), DPETS (10%)	681.7	>500	1.179
	. %	( )	DS: TEOS (90%), DPETS (10%)	603.6	≈ 7	0.942
	Bi-functionalized materials
	15 % ° % !"# ( ) ( )		DS: TEOS (80%), DPETS (10%) MPTS (10%)	587.4	≈ 4.0	0.549
	15 $% !"# (%) ° ( )		DS: TEOS (95%), DPETS (5%). P: MPTS.	526.7	≈ 8.5	0.867
	15 % ° % &'( ) ( ) ( )		DS: TEOS (80%), DPETS (10%), CPTS (10%)	569.3	≈ 5.0	0.512
		. % !"# ( ) ( )	DS: TEOS (80%), DPETS (10%) MPTS (10%)	601.2	> 500	0.788
		. $% !"# (%) ( )	DS: TEOS (95%), DPETS (5%). P: MPTS.	506.1	> 500	1.299

a Pho: Phosphonic acid. Sulf: Sulfonic acid. Carbox: Carboxylic acid. X: xerogel. (1) indicate functional group introduced by direct synthesis (2) indicate functional group introduced by post grafting. b Precursors: Tetraethyl orthosilicate (TEOS), 3-mercaptopropyltrimethoxysilane (MPTS), (Diethylphosphatoethyl)triethoxysilane (DPETS) and 3cyanopropyltriethoxysilane (CPTS). DS: indicate direct synthesis sol-gel route. Percentage in brackets indicate he molar ratio in direct synthesis. P: indicate post-grafting route (15% molar ratio for the MPTS precursor).

Table 2 .

 2 Removal of BA by the 10% phosphonic acid functionalized silica materials a (expressed as removed %) b . Obtained by direct synthesis (co-condensation) sol-gel route. Pho: phosphonic acid. Macro. X: Macroporous Xerogel. Meso: X.: Mesoporous Xerogel. Removal values of 100 ± 0 indicate that the amine was not detected in the treated sample. b Different letters indicate statistics differences in ANOVA (p< 0.05) and the LSD test.

	Biogenic Amine	15 % . °	15 % . °	41 % .	. . % .	. . % .
	Histamine	96.3 ± 0.8 c	95.0 ± 0.4 bc	70.6 ± 2.8 a	92.4 ± 0.5 b	95.5 ± 0.2 bc
	Putrescine	77.3 ± 0.6 b	82.0 ± 3.9 b	64.2 ± 2.3 a	77.0 ± 2.4 b	85.6 ± 9.0 b
	Cadaverine	88.8 ± 3.1 b	88.7 ± 3.9 b	68.8 ± 9.6 a	88.0 ± 5.3 b	90.5 ± 2.2 b
	Spermidine	100 ± 0	100 ± 0	98.2 ± 1.0	99.3 ± 0.5	99.7 ± 0.3
	Spermine	100 ± 0	100 ± 0	100 ± 0	100 ± 0	99.9 ± 0.1
	2-Phenylethylamine	32.3 ± 3.1	22.1 ± 8.1	25.7 ± 2.5	27.1 ± 1.9	33.1 ± 0.6
	Tyramine	20.0 ± 1.3	13.3 ± 8.8	13.2 ± 3.9	10.9 ± 8.5	21.0 ± 2.0
	Isoamylamine	33.5 ± 4.7	23.8 ± 9.5	21.9 ± 3.0	24.4 ± 3.0	31.5 ± 0.5

a

Table 3 .

 3 Removal of BA from hydro-alcoholic solutions by silica material functionalized with different types of functional groups (expressed as removed %) a

	Solid Type		Mesoporous materials based on SBA15 110ºC	Lamellar
	Functionalization and route of synthesis b	*+, -. -/% 012 (-) --/°3	*+, -. -/% 012 (-) -/% *456 (-) --/°3	*+, -. .% 012 (-) *456 (7) --/°3	*+, -. -/% 012 (-) -/% 389:2; (-) --/°3	<8=>5589 *456

Acknowledgements.

This study has been financed by the European Union (Horizon 2020) framed in the Marie S.

Curie Individual Fellowships program (Reference: 701912). http://cordis.europa.eu/project/rcn/200992_en.html Authors declare no competing interests.

Supplementary Information description

Figure S1 shows a scheme of the synthesis of the hybrid silica functionalized with cation exchange groups used in this study.


Claude Pouchan
email: claude.pouchan@univ-pau.fr

Quantum molecular dynamic approach to modeling the vibrational spectra of propynenitrile and its 1:1 complexes with water

Keywords: Density functional theory, DFT Molecular Dynamic Simulation, IR spectroscopy, propynenitrile or cyanoacetylene molecules, complexes 1:1 with water

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Introduction

Vibrational spectroscopies (Infrared and Raman) and IR spectroscopy particularly are commonly used in laboratories to identify and know the quality of a product. To do this, it is sufficient to choose the most intense characteristic bands without worrying about the globality and the complexity of all the signals appearing in the spectrum. Indeed, all the IR bands of a molecule are not easy to assign because we can find in addition to fundamental bands, overtones and combinations whose position and intensity varies according to the physical state of the product which can be gaseous, liquid, in solution, solid or trapped in a matrix [START_REF] Wilson | Molecular Vibrations Dover[END_REF]. To help in the full identification of an IR spectrum, and to take into account the effects of the environment, modelling through theoretical chemistry methods must be used very often [START_REF] Pulay | Modern Electronic Structure Theory[END_REF]. When we are only interested in the fundamental bands, quantum mechanics methods easily provide harmonic frequencies that are corrected by scale factors to take into account the anharmonicity and thus get closer the calculated spectrum to the observed data [START_REF] Scott | Harmonic Vibrational Frequencies: An evaluation of Hartree-Fock, Moller-Plesset, Quadratic Configuration Interaction, Density Functional Theory and Semi-Empirical Scale Factors[END_REF]. When we want to know in order to explain or predict the whole spectrum (fundamentals, overtones, combinations) and to account for the effects due to the environment (liquid, solution, solid, adsorption…) we must use much more sophisticated quantum methods. Two main types of methods make it possible to obtain a theoretical IR spectrum that can be classified into 'static' and 'dynamic' methods and whose main characteristics are briefly recalled in the paragraph 'Methods and Computational Details' Several 'static' models have been developed and implemented to explicitly take into account anharmonicity. In particular, for small molecules converged rovibrational levels can be obtained by fully variational treatments, as Vibrational Configuration Interaction (VCI) [START_REF] Dunn | Vibrational energy levels of hydrogen cyanide[END_REF][START_REF] Carter | Rovibrational Bound States in Polyatomic Molecules[END_REF][START_REF] Cassam-Chenaï | The VMFCI method: A flexible tool for solving the molecular vibration problem[END_REF][START_REF] Ribeiro | A Jacobi-Wilson description coupled to a block-Davidson algorithm: An efficient scheme to calculate highly excited vibrational levels[END_REF][START_REF] Begue | A comparison of two methods for selecting vibrational interaction spaces on a heptatomic system: Ethylene oxyde[END_REF][START_REF] Mielke | Vibrational Configuration Interaction Using a Tiried Multimode Scheme and Tests of Approximate Treatments of Vibrational Angular Momentum Coupling : A case Study for Methane[END_REF] or (VCI-P) [START_REF] Pouchan | Ab Initio Configuration Interaction determination of the overtone vibrations of methyleneimine in the region 2800-3200 cm -1[END_REF][START_REF] Carbonniere | The VCI-P code: an iterative variation scheme for efficient computations of anharmonic vibrational levels and IR intensities of polyatomic molecules[END_REF][START_REF] Rauhut | Configuration selection as aroute towards efficient vibrational configuration interaction calculations[END_REF] methods when the configuration interaction is guessed by a perturbative treatment and more recently Vibrational Coupled Cluster method (VCC) [START_REF] Godtliebsen | Tensor Decomposition and Vibrational Coupled Cluster Theory[END_REF][START_REF] Madsen | Efficient algorithms for solving the non-linear vibrational coupled-cluster equations using full and decomposed tensors[END_REF]. However, to conveniently handle large isolated systems it becomes unavoidable to introduce some approximations concerning both the form of the potential and the rovibrational treatment .Today to modelling vibrational spectra in the mid infrared region the most successful approaches are based on truncated three-mode potentials by using the effective second order perturbative (VPT2) [START_REF] Bloino | General Perturbative Approach for Spectroscopy, Thermodynamics, and Kinetics: Methodological Background and Benchmarks Studies[END_REF] treatment, fast and easy, which tends to correct the numerical instabilities of a traditional perturbative approach due to the presence of random degeneracy.

Beside these static approach and starting from the pioneer work of Wilson et al. [START_REF] Berens | Molecular dynamics and spectra. I. Diatomic rotation and vibration[END_REF], vibrational spectra can be calculated in an alternative way using a 'molecular dynamic' approach and statistical mechanic models based on the Fourier transform analysis of the time correlation function of atomic dipole moments. These molecular dynamic (MD) approaches in principle yield to a full description of the modes activated at a given energy or temperature and describe their couplings and finally allow the complete description of the IR properties as position and intensity for all bands. Although several quantum corrections to the classical time-correlation functions have been proposed, they don't affect the calculated wavenumbers but only the shape and the width of the bands [START_REF] Ramirez | Quantum corrections to classical time-correlation functions: Hydrogen bonding and anharmonic floppy modes[END_REF].

This work aims to compare the spectra obtained by the two approaches for an isolated molecule and a molecule interacting with water in order to describe, as a first approximation, the effects of the environment related to micro-hydration. For MD simulations, DFT has been applied for the description of the electronic structure and gradients 's computations using the very popular Becke three parameter hybrid functional (B3LYP) [START_REF] Becke | Density-functional thermochemistry. III. The role of exact exchange[END_REF] and a localized Gaussian type basis set [6-31+G(p,d)] known to give excellent IR spectra for organic molecules [START_REF] Carbonniere | Vibrational computations beyond the harmonic approximation: Performances of the B3LYP density functional for semi rigid molecules[END_REF].

The DFT/MD results based on the propagation of the density matrix will be compared, for the valence modes, to the computed data obtained at CCSD(T)/aug-cc-pVTZ anharmonic wavenumbers issued from a VPT2 treatment also known to provide excellent results in the static method when the couplings between the normal modes are not very important.

The molecule chosen for this study is a linear system the propynenitrile HC 3 N since it is the simplest cyanopolyyne discovered in interstellar environments. Detected in the atmosphere of Titan it was identified by means of the knowledge of the fundamental bands and has been extensively studied by means of careful laboratory studies of high resolution infra-red spectra since 1990 [START_REF] Uyemura | Infrared intensities of Bending Fundamentals in Gaseous HCCCN and DCCCN[END_REF][START_REF] Khlifi | Absolute Intensity of the IR Bands of Propynenitrile[END_REF][START_REF] Khlifi | Integrated band intensity versus temperature for v 1 ,v 2 , v 5 , and v 6 bands of cyanoacetylene[END_REF][START_REF] Jolly | New infrared integrated bands intensities for HC 3 N and extensive line list for the v 5 aand v 6 bending modes[END_REF][START_REF] Bizzochi | Rotational and High-resolution Infrared Spectrum of HC 3 N: Global Ro-vibrational Analysis and Improved Line Catalog for Astrophysical Observations[END_REF]. Recently, Bizzochi et al. [START_REF] Borget | Cyanoacetylene Adsorption on Amorphous and Crystalline Water Ice Films: Investigation through Matrix Isolation and Quantum Study[END_REF] reinvestigate the IR spectrum up to 1100 cm -1 and improve all the data by an extensive list of highly accurate rest frequencies. Taking into account these experimental data, our objective will be to know what is the most appropriate approach to modelling the experimental spectrum.

After a brief description of the computational methods applied (section 2), the DFT/MD results obtained for the propynenitrile will be discussed and compared both with the experimental data and the results issued from CCSD(T)/VPT2 computations. A first approach of the IR spectra for the complexes HC 3 N:H 2 O will complete this theoretical study. (section 3).

Methods and Computational details

Structural optimization, harmonic frequencies, IR intensities, and anharmonic frequencies issuing from second order perturbative approach (VPT2) and ab-initio molecular dynamic simulations were performed with Gaussian 09 program [START_REF] Frisch | Gaussian 09[END_REF]. Spectral analysis from the MD simulations was obtained using the VAMD (Vibrational Asignments in Molecular Dynamic) program developed in our group and described in reference [START_REF] Carbonniere | Vibrational analysis of glycine radical: a comparative ab initio static and dynamic study[END_REF]. Electronic structure calculations were performed at the DFT level using the most common three parameter hybrid functional of Becke (B3LYP) [START_REF] Becke | Density-functional thermochemistry. III. The role of exact exchange[END_REF] and a valence double zeta Pople basis set including diffuse and polarization functions, 6-31+G(d,p). This model chemistry was chosen since it has been previously shown that, for the prediction of harmonic and anharmonic force constants and in the case of small organic [START_REF] Becke | Density-functional thermochemistry. III. The role of exact exchange[END_REF][START_REF] Carbonniere | Performances of different density functionals in the computation of vibrational spectra beyond the harmonic approximation[END_REF][START_REF] Carbonniere | Vibrational spectra for P(4)O(6) and P(4)O(10) systems: Theoretical study from DFT quartic potential and mixed perturbation-variation method[END_REF] and radical [START_REF] Barone | Accurate vibrational spectra and magnetic properties of organic free radicals: The case of H2CN[END_REF], systems it is able to recover the results obtained using the more expensive CCSD(T) [START_REF] Ragavarachri | A fifth-order perturbation comparison of electron correlation theories[END_REF]/Aug-ccp-VTZ [START_REF] Kendall | Electron affinities of the first-row atoms revisited. Systematic basis sets and wave functions[END_REF] approach.

Static treatment of anharmonicity

The static approach used to compute anharmonic effects using the perturbative (VPT2) approach is briefly recalled. From a minimum energy structure of a given system, a quartic force field is built in which the third and fourth derivatives are computed by 6N-11 (N=number of atoms) numerical differentiations of analytical or numerical (in case of CCSD (T) calculations) second derivatives from a least square fitting procedure [START_REF] Carbonniere | Construction of an accurate quartic force field by using generalized least-square fitting and experimental design[END_REF]. In the quartic approximation, the potential is given by:

l k j i l k j i ijkl k j i k j i ijk i i i q q q q k q q q k q V       , , , , , 2 ! 2 1



where q i the i-th dimensionless normal coordinate, and  i , k ijk , k ijkl the harmonic, cubic and quartic force constant respectively expressed in cm -1 . Note that only the anharmonic force constants involving the tri-mode interactions (k=l) are determined by numerical differentiation.

Next, the vibrational frequencies can be evaluated in the VPT2 approach by using the well-know formula of Willets and coworkers [START_REF] Miller | Ab initio calculation of anharmonic constants for a transition state, with application to semi classical transition state tunneling probabilities[END_REF] derived from second order perturbation theory:

From a technical point of view, it can be shown [START_REF] Barone | Anharmonic vibrational properties by a fully automated second-order perturbative approach[END_REF] that the best setup is obtained using a step size of 0.01Å for the numerical differentiation of harmonic frequencies. For DFT computations very tight convergence criteria for structural optimizations and fine grids for integral evaluation (that is at least 99 radial and 590 angular points) are necessary.

Vibrational analysis from ab-initio Molecular dynamic simulations.

Ab-initio molecular dynamic simulations were performed using the Atom-centered Density Matrix (ADMP) formalism [START_REF] Schlegel | Ab initio molecular dynamics: Propagating the density matrix with Gaussian orbitals[END_REF][START_REF] Iyengar | Ab initio molecular dynamics: Propagating the density matrix with Gaussian orbitals. II. Generalizations based on mass-weighting, idempotency, energy conservation and choice of initial conditions[END_REF][START_REF] Schlegel | Ab initio molecular dynamics: Propagating the density matrix with Gaussian orbitals. III. Comparison with Born-Oppenheimer dynamics[END_REF] which uses localized Gaussian basis sets, thus allowing for sampling on the same potential used for the static methods. This is of particular importance when both the potential energy surface and the observables are sensitive to the choice of the DFT computational level and, in particular, it allows for the use of hybrid functionals [START_REF] Becke | Density-functional thermochemistry. III. The role of exact exchange[END_REF][START_REF] Pavone | First-principle molecular dynamics of the Berry pseudorotation: Insights on 19F NMR in SF4[END_REF][START_REF] Joubert | Static and dynamic descriptions of bond breaking/formation: A complementary view?[END_REF][START_REF] Ciofini | Static and Dynamic approaches for the calculation of NMR parameters: Permanganate ion as a case study[END_REF]. Furthermore, in our case, this choice let to a direct comparison of the results of anharmonic calculations obtained from static and dynamic approaches. Molecular dynamic simulations were performed in the microcanonical subspace [START_REF] Martinez | Extracting effective normal modes from equilibrium dynamics at finite temperature[END_REF] with a time-step of 0.2 fs over an overall period of 10 ps, the initial nuclear velocities being set to perform simulations averaging the temperatures of 150K, 300K, 450K and 600K. A suite of programs developed in our lab is used to perform the spectral analysis from ADMP DFT trajectories [START_REF] Carbonniere | Vibrational analysis of glycine radical: a comparative ab initio static and dynamic study[END_REF]. Several papers report in detail a simple procedure [START_REF] Schmitz | Vibrational spectra from atomic fluctuations in dynamics simulations: I. Theory, limitations, and a sample application[END_REF][START_REF] Schmitz | Vibrational spectra from atomic fluctuations in dynamics simulations. II. Solventinduced frequency fluctuations at femtosecond time resolution[END_REF] to get the IR spectrum as Fourier transform of the dipole autocorrelation function [START_REF] Boatz | Decomposition of normal-coordinate vibrational frequencies[END_REF] that is: Table 1 reports the harmonic vibrational wavenumbers obtained at different levels of theory. The harmonic values obtained at the CCSD and DFT levels for the valence vibrations v 1 , v 2 , v 3 , v 4 and the CCH bending mode v 5 are in fairly good agreement. However, it should be noted that for v 7 and v 6 modes, the B3LYP values are overestimated by about 12 and 16 % respectively compared to CCSD frequencies. Except for the modes v 5 and v 1 (this latter associated with the CH valence mode), the introduction of triple excitations reduces harmonic frequencies by 3 to 5%, bringing them closer to the experimental data.

  t i t e M M dt I       0 M
Table 2 reports the anharmonic vibrational values obtained for the five modes v 1 , v 2 , v 3 , v 4 and v 5 from VPT2

and DFT/MD approaches. At VPT2 level, modes v 6 and v 7 that have large differences according to their CCSD or B3LYP harmonic determination are decoupled from the other five modes to compute anharmonic wavenumbers. In addition, it should be noted that the quartic force constants are not often adapted for the description of the soft modes that are here v 6 and v 7 leading to inappropriate VPT2 results to describe these modes. As reported by Handy and coworkers [START_REF] Burel | On the representation of potential energy surfaces of polyatomic molecules in normal coordinates. II Parametrisation of the force field[END_REF], the simplest VPT2 treatment generally provides, in absence of strong anharmonic couplings, reasonable vibrational wavenumbers of stretching motions: this can be the case for HC 3 N. Also the comparison between VPT2 and DFT/MD values appears quite reasonable to describe the five highest modes (valence modes v 1 to v 4 and the bending mode v 5 ) reported in Table 2, against the experimental data [START_REF] Jolly | New infrared integrated bands intensities for HC 3 N and extensive line list for the v 5 aand v 6 bending modes[END_REF][START_REF] Bizzochi | Rotational and High-resolution Infrared Spectrum of HC 3 N: Global Ro-vibrational Analysis and Improved Line Catalog for Astrophysical Observations[END_REF][START_REF] Borget | Cyanoacetylene Adsorption on Amorphous and Crystalline Water Ice Films: Investigation through Matrix Isolation and Quantum Study[END_REF]. At the VPT2 level, the CCSD(T) wavenumbers are much closer to the experimental values than the DFT results . The mean average deviation varies from 11 to 48 cm -1 . Molecular dynamic simulations show a relative weak influence of temperature with an average deviation ranging from 26 cm -1 (300K) to 19 cm -1 (600K). As a general rule, the CCSD(T)/VPT2 and B3LYP/MD frequencies lead to convergent results in fair agreement with the experimental data with a more favorable mean average deviation for the CCSD(T)/VPT2 static determination (11cm -1 ) than for the B3LYP/MD dynamic one (19 cm -1 ).

3-1 Propynenitrile in interaction with water.

The study of 1:1 complexes of propynenitrile with water is undertaken to simulate as a first approximation the study of hydration and explain the expected vibration frequency shifts on the IR spectra. These calculated displacements will be compared to the experimental values from FT-IR matrix isolation spectroscopy reported by Borget et al [START_REF] Borget | Cyanoacetylene Adsorption on Amorphous and Crystalline Water Ice Films: Investigation through Matrix Isolation and Quantum Study[END_REF]. For these 1:1 complexes, two structures are possible (fig 2). The most stable is the formation of a hydrogen bond between the oxygen in the water and the hydrogen of HC 3 N (OH structure), the other about 2 kcal mol -1 less stable combines the hydrogen in the water with the nitrogen in HC 3 N (NH structure). From a Boltzmann population point of view at 300K the former represents 96%. To best modelize the hydrogen bonds, calculations are also performed using the B3LYP-D/6-31G+(d,p) method. From a static point of view, the quartic force field is calculated on all complexes modes (molecule + water) but not by coupling for HC 3 N the two lowest deformation modes (v 6 and v 7 ) as for the study of the isolated molecule. From a dynamic point of view, DFT or DFT-D/MD calculations are performed with energies corresponding to temperatures around 300K and 450 K for the OH structure where the largest band shifts relative to the isolated molecule are expected and around 300K for the NH structure. All theoretical results are reported in Tables 3 and4 for each of the complexes and compared to the experimental data reported by Borget et al [START_REF] Borget | Cyanoacetylene Adsorption on Amorphous and Crystalline Water Ice Films: Investigation through Matrix Isolation and Quantum Study[END_REF].

When examining Tables 3 and4, we can note a slight improvement brought by the consideration of dispersion effects (DFT-D better than DFT) as well as the relatively small influence of temperature in dynamic simulations with the exception of the v 1 mode for the OH form largely affected by the hydrogen bond. The results provided by DFT-D/VPT2 and DFT-D/MD approaches are both qualitatively reliable, but globally, the mean average deviation (MAD) between theory and experiment is better for molecular dynamics (respectively 19 and 13 cm -1

for OH and NH forms) than for the static DFT/VPT2 approach (42 and 52 cm -1 respectively). However, we can emphasize that this difference is mainly due to the incorrect description of the v 2 mode in the DFT or DFT-D/VPT2 computations. When we look at the displacements of the IR bands of the OH and NH forms relatively to the isolated molecule, we can see that the largest shifts concern the OH form. Indeed for this form the wavenumber of the CH valence mode (v 1 ) is downshifted by about hundred cm -1 (-98 to -115 cm -1 according to our calculations) while the frequency of the CCH bending mode v 5 is upshifted from 95 to 100 cm -1 . These results are in excellent agreement with the experimental values reported by Borget et al (-96 and +93 cm -1 respectively). The large decrease observed for the CH stretching mode is likely due to the presence of hydrogen bond which increase the CH bond with respect to the isolated molecule, whereas the upshift of the bending mode is essentially due to the coupling between the bending mode v 5 and the CC valence mode v 4 . The frequencies of the other modes are little modified by the presence of the hydrogen bond except for v 3 upshifted of about ten cm - 1 (13 cm -1 in our calculations versus 10 cm -1 experimentally). For the NH structure, the shifts brought by solvation are lower and never exceed about ten cm -1 as confirmed by our calculations.

Conclusion

We have investigated the vibrational infra-red spectra of propynenitrile and its 1:1 complexes with water in the framework of quantum mechanics. Building on a B3LYP/6-31G+(d,p) level of theory, two different approaches have been tested to account for vibrational anharmonicity: a static calculation with second order vibrational perturbation theory (DFT/VPT2) and a dynamic through quantum molecular dynamics (DFT/MD). Comparing the results of these anharmonic quantum-mechanical approaches with experimental data reveals that if the

3 . Results and discussions 3 - 1 2 ( 1 .

 33121 being the total dipole moment, M  the derivative dt dM and    I the intensity at the  wavenumber.To assign the vibrational bands we used the Fourier Transform of Time Correlation Function (FTTCF) of the velocity (V(t)) obtained together with the dipole moment at each time step of the simulation, this function projected along the normal modes or better the internal or local modes giving as described in reference[START_REF] Carbonniere | Vibrational analysis of glycine radical: a comparative ab initio static and dynamic study[END_REF] a good representation of the band assignment.This procedure has been used in our computational strategy to modelling successfully DFT/MD infra-red spectra for gaseous and micro-hydrated forms of nucleobases[START_REF] Carbonniere | Vibrational analysis beyond the harmonicity from Ab initio molecular dynamics: Case of cytosine in its anhydrous and aqueous forms[END_REF][START_REF] Thicoipe | Structural investigation of microhydrated thymine clusters and vibrational study of isolated and aqueous forms of thymine using DFT level of theory[END_REF][START_REF] Thicoipe | Comparison of static and dynamic methods of treatment of anharmonicity for the vibrational study of isolated and aqueous forms of guanine[END_REF][START_REF] Thicoipe | DFT modelling of the infrared spectra for isolated and aqueous forms of adenine[END_REF][START_REF] Pouchan | DFT modelling of the infrared spectra for the isolated and the micro-hydrated forms of uracil[END_REF] Propynenitrile as exemple of linear molecule.At B3LYP/6-31 G+(d,p) and CCSD(T)/Aug-cc-pVTZ levels the calculated geometries are very similar for C 1 -C 211 Å vs 1.204 Å) and C 2 -H 3 (1.067 Å vs 1.063 Å). A small difference of about 0.01 angstroms can be noted for the other two lengths C 1 -C 4 (1.373 Å vs 1.386 Å) and C 4 -N 5 (1.167 Å vs 1.157) Å. It should be emphasized the weak influence of the triple excitation at CCSD level for the computed bond lengths.

TABLE 3 :

 3 Wavenumbers (in cm-1) calculated following VPT2 and DFT/MD approaches for HC3N:H20 (OH form)

TABLE 4 :Fig 1 :Fig 2 :

 412 Fig 1 : Propynenitrile atomic numbering

Fig 1 :Fig 2 :

 12 Fig 1 : Propynenitrile atomic numbering

TABLE 1 : Computed harmonic wavenumbers (in cm-1) for propynenitrile in vacuo at different levels of theory. Comparison with experimental values

 1

	Mode	Main	CCSD(T)/	CCSD /	B3LYP /6-
		Assignment	aug-cc-	Aug-cc-	31+G(d,p) Experimental
			pVTZ	pVTZ
						
	 	(C-CC-N) a	218	226	244	222 c
	 	(CCN) a	487	509	563	504 d ; 499 c ; 500 b
	 	(CCH) a	664	702	706	667 d ; 663 c ; 664 b
	   	(C-C) (C≡C)	875 2100	883 2164	907 2174	862 c ; 861 b 2077 d ; 2079 c
	 	(CN)	2306	2393	2374	2269 d ; 2274 c
	(CH) a Double degenerated modes  	3445	3472	3474	3316 d ; 3327 c

TABLE 2 : Wavenumbers (in cm-1) calculated for prpynenitrile in vacuo following VPT2 and DFT/MD approaches. Comparison with experimental values

 2

	Mode	VPT2 a :CCSD(T)/	VPT2 :		DFT/MD		Experiment
		aug-cc-pVTZ	B3LYP /6-
			31+G(d,p)
				T=300K T=450K T=600K	
	 	218 a	244 a	252	251	248	222 c
	 	487 a	563 a	567	559	563	504 d ; 499 c ; 500 b
	 	657	698	652	652	657	667 d ; 663 c ; 664 b
	 	867	897	903	900	903	862 c ; 861 b
	   	2078 2279	2145 2344	2093 2252	2088 2247	2083 2242	2077 d ; 2079 c 2269 d ; 2274 c
	 	3337	3365	3363	3354	3346	3316 d ; 3327 c

a Order 2 : CCSD(T)/aug-cc-pVTZ -Orders 3 and 4 : B3LYP/6-31+G(d,p) Modes   and    are totally decoupled from others: their harmonic values are reported in the

table 2 b

 2 Ref[START_REF] Bizzochi | Rotational and High-resolution Infrared Spectrum of HC 3 N: Global Ro-vibrational Analysis and Improved Line Catalog for Astrophysical Observations[END_REF]; c Ref[START_REF] Jolly | New infrared integrated bands intensities for HC 3 N and extensive line list for the v 5 aand v 6 bending modes[END_REF]; d Ref[START_REF] Borget | Cyanoacetylene Adsorption on Amorphous and Crystalline Water Ice Films: Investigation through Matrix Isolation and Quantum Study[END_REF]

TABLE 1 : Computed harmonic wavenumbers (in cm-1) for propynenitrile in vacuo at different levels of theory. Comparison with experimental valuesTABLE 2 : Wavenumbers (in cm-1) calculated for propynenitrile in vacuo following VPT2 and DFT/MD approaches. Comparison with experimental valuesTABLE 3 : Wavenumbers (in cm-1) calculated following VPT2 and DFT/MD approaches for

 123

	HC	N:H	O (OH form). Comparison with experimental values
		3	2

TABLE 4 : Wavenumbers (in cm-1) calculated following VPT2 and DFT-D/MD approaches for HC 3 N:H 2 O (NH form). Comparison with experimental values

 4

Caption for Figures:

perturbative treatment at the CCSD(T) /VPT2 level of theory offers a more reliable description of both stretching modes for the isolated molecule, the DFT/MD approach (extremely time consuming however) is better than the DFT/VPT2 (fastest when the quartic force constants are obtained) one, by using the same basis set, to describe the IR spectra of propynenitrile and its two 1:1 complexes with water.