P S Mello

S L F Trivelato

L Q Vieira

EXPLORING THE INQUIRY-BASED LEARNING STRUCTURE TO PROMOTE SCIENTIFIC CULTURE IN THE CLASSROOMS OF HIGHER EDUCATION SCIENCES

Keywords: Science higher education, Immunology, epistemic practices

This paper examines student engagement in an inquiry-based learning activity, planned to provide students with elements in the social, epistemic and conceptual dimensions related to the scientific practice in Immunology. The activity was applied to 39 groups of students enrolled in Immunology or Biochemistry courses in a public university in Brazil. Students performed data-collection through the execution of an in vitro assay. We analyzed how students represent data and use them to support their claims in their written constructs. To clarify which are the productive epistemologies in students' reports we developed a framework for epistemic practice analysis. Our findings point to a pattern of several epistemic practice categories in their written text, mostly related to the particular contingences of data analysis, rather than to theoretical concepts. In addition, we observed that students performed literary inscriptions to represent their data, however, they tended not to cite all data obtained in their written texts. These results suggest that Immunology education strategies should provide students with approaches that explore the role of data representation in the scientific text rhetoric.

INTRODUCTION

At the higher education level, conceptions of inquiry-based learning are appealing, since they provide opportunities for students engaged in academic and professional training to establish close contact with typical elements of the scientific communities [START_REF] Kelly | Toward a research agenda for epistemological studies in science education[END_REF].

The term "inquiry learning" has been defined in many forms and applied to many procedures throughout history [START_REF] Deboer | Historical Perspectives On Inquiry Teaching In Schools, in Scientific Inquiry and Nature of Science: Implications for Teaching, Learning, and Teacher Education[END_REF]. The definition most closely related to this work is that inquiry-based learning is an educational strategy that engages students in science matters [START_REF] Council | A Framework for K-12 Science Education: Practices, Crosscutting Concepts, and Core Ideas[END_REF] by having them perform tasks similar to those scientists perform routinely [START_REF] Keselman | Supporting inquiry learning by promoting normative understanding of multivariable causality[END_REF]. Several researchers consider the inquiry learning strategy more effective than traditional instruction [START_REF] Alfieri | Does discovery-based instruction enhance learning[END_REF][START_REF] Blanchard | Is Inquiry Possible in Light of Accountability?: A Quantitative Comparison of the Relative Effectiveness of Guided Inquiry and Verification Laboratory Instruction[END_REF][START_REF] Furtak | Experimental and Quasi-Experimental Studies of Inquiry-Based Science Teaching:A Meta-Analysis[END_REF], and other researchers associate inquiry-based learning with the promotion of skills related to processual, procedural, and conceptual scientific knowledge [START_REF] Lee | Do Different Levels of Inquiry Lead to Different Learning Outcomes? A comparison between guided and structured inquiry AU -Bunterm, Tassanee[END_REF].

Teaching science in higher education should contemplate specific features in order to reach the main educational goal: acquiring professional scientific skills. Thus, science education must train future professionals to apply their scientific knowledge to their everyday practices in order to benefit society. Therefore, research must be used as precondition for teaching future professionals from biomedical courses [START_REF] Lampert | O ensino com pesquisa: realidade, desafios e perspectivas na Universidade brasileira[END_REF]. After all, the professional activity that does not use scientific knowledge reverts to the simple reproduction of procedures for which academic formation is not necessary. Thus, using research as a methodological resource for teaching at universities is appealing, because it offers opportunities for students to use elements of the scientific practice to develop comprehension of science in the three dimensions of knowledge [START_REF] Kelly | Epistemic Practices and Science Education[END_REF].

It is crucial for students to learn how to observe what counts as valid knowledge for the specific community in which they are inserted [START_REF] Kelly | Toward a research agenda for epistemological studies in science education[END_REF]. Latour and Woolgar (1986, p. 60) name "literary inscription" all forms of data representation (points, codes, charts, maps, spectra, photos) that support claims when they are tied to the assertions in scientific texts. There is a different epistemic value for each literary inscription [START_REF] Roth | Pictures in Biology Education[END_REF], and then each knowledge community utilizes the literary inscriptions related to the specific knowledge under study, as a resource for agonistic debates. A professional in science-related fields should be proficient in representing and interpreting data, however, recognizing evidence in the results is not a trivial matter.

Epistemic practices are good resources for authors to analyze the productive epistemologies in student´s discourses. Epistemic practices appear whenever the social practices for knowledge establish a routine pattern [START_REF] Kelly | Epistemic Practices and Science Education[END_REF], allowing researchers to list and classify the sources used by the community upon which knowledge is built. Epistemic practices are social, cognitive and discursive activities given to a community that shares common values, meanings and tools [START_REF] Sandoval | Designing Knowledge Representations for Learning Epistemic Practices of Science[END_REF]. As a result, they are interactional, contextual, intertextual, consequential [START_REF] Kelly | Epistemic Practices and Science Education[END_REF], and contribute to shape the cognitive processes that lead to knowledge. The most recent framework for epistemic practices analysis sets them into four categories based on normative and educational goals: proposition, communication, evaluation and legitimation of knowledge. Because they vary according to the systematic ways that each community functions, the types of epistemic practices situated in each category will also vary for each area of knowledge [START_REF] Kelly | Epistemic Practices and Science Education[END_REF].

Immunology deals with themes of relevance for human health. However, there is a lack of studies on teaching and learning Immunology. Our group has been analyzing the written discourse produced by students while solving an Immunology problem [START_REF] Manzoni-De-Almeida | O desenvolvimento da escrita argumentativa nas aulas de imunologia do ensino superior por metodologias ativas[END_REF][START_REF] Manzoni-De-Almeida | Analysis of epistemic practices in reports of higher education students groups in carrying out the inquiry-based activity of immunology[END_REF]. After receiving active-based learning instruction in Toulmin's argument layout [START_REF] Toulmin | The Uses of Argument[END_REF], students deal with data previously obtained to make claims and justify them according to scientific conceptions. Our data suggest that students use categories of epistemic practices related to production of knowledge in their arguments [START_REF] Manzoni-De-Almeida | O desenvolvimento da escrita argumentativa nas aulas de imunologia do ensino superior por metodologias ativas[END_REF]. These epistemic practices are similar to those performed by scientists while analyzing data, suggesting that the activity favored use of skills related to science [START_REF] Manzoni-De-Almeida | Analysis of epistemic practices in reports of higher education students groups in carrying out the inquiry-based activity of immunology[END_REF]. Here, we seek to describe patterns related to data representation and use of evidence in the students' written text to clarify features that could be referenced in future projects aimed at teaching and learning Immunology.

We consider that exposing students to complex tasks is an important way to bring them closer to key elements of the scientific practice in Immunology. Our goal is to analyze student´s written constructs to answer three research questions: 1) What kind of literary inscriptions are performed by students to represent raw experimental data? 2) Do students use literary inscriptions in their written texts? 3) What are the categories of epistemic practices utilized by the students to explain their claims?

METHODOLOGY

Inquiry-based activity and its application to undergraduate classes

In this work, we describe and interpret interactional phenomena emerging in situ [START_REF] Kelly | Toward a research agenda for epistemological studies in science education[END_REF]. Thus, our methodological approach is drawn from qualitative research [START_REF] Denzin | Introduction: The Discipline and Practice of Qualitative Research[END_REF].

A detailed description of the inquiry-based activity is described in Mello et al. [START_REF] Mello | Elaboração de uma atividade investigativa experimental em imunologia sobre o sistema do complemento para ensino superior: Relato da construção da sequência didática[END_REF]. Due to the importance of the experimental activity in building knowledge in Immunology, we created an activity based on experimental procedures to collect data. Cells of the immune system express surface-receptors or secret immunomodulators, which bind to other molecules. Based on this principle and on the possibility of performing an assay that would be feasible within the available time and met ethical and financial limitations, we elected the "analysis of complement fixation" as our experimental approach.

We asked students to choose their own test substances or physical conditions that putatively would interfere with the complement cascade. By using this strategy, we expected to stimulate the curiosity of the students and to encourage them to apply the formal disciplinary theoretical knowledge or informal daily observations. Since they were free to choose any substance related to their personal and academic routines, we expected a link between everyday life and science.

According to the educational goals and because of time limitation, we adopted a model of "structured inquiry" [START_REF] Blanchard | Is Inquiry Possible in Light of Accountability?: A Quantitative Comparison of the Relative Effectiveness of Guided Inquiry and Verification Laboratory Instruction[END_REF]. An inquiry-learning approach is classified as such when the teacher proposes a general problem to the students. The problem posed was: "Are there substances (or physical conditions) that interfere with the complement cascade?". In our model, we moved beyond the basic layout of a structured inquiry by proposing that students create their own experimental question, which should necessarily associate the substance chosen by them with the problem posed by the teacher. Moreover, we also provided students with the method for data collection, but the data interpretation was a task they should perform by themselves.

During the experimental class, students found on their workbench all the labelled reagents, necessary equipment and a standardized experimental protocol according to Morgan (2000, p.63). Briefly, the protocol instructed them to first pipette different concentrations of the substance with a fixed volume of mouse serum aiming to build a curve, and incubate the tubes in physiological conditions of pH, temperature and osmolarity. Then, they should transfer an amount of that solution to a microplate containing rabbit erythrocytes in a concentration previously determined. They also had extra samples as "control groups": mouse serum with erythrocytes; substance with erythrocytes; water with erythrocytes ("100% lysis") and saline solution with erythrocytes (spontaneous background lysis). After incubation, students should centrifuge the microplate to concentrate whole erythrocytes in the the bottom of the plates. They also took a picture of the plate to have a qualitative interpretation of the results. After that, they gently transferred a volume of the supernatant to another plate, to perform absorbance measurements. Using the numeric values obtained, they must calculate the percentage lysis in each condition. We planned the template for a written report, called "Lab notebook" [START_REF] Manzoni-De-Almeida | Analysis of epistemic practices in reports of higher education students groups in carrying out the inquiry-based activity of immunology[END_REF] to favor the assembly of assertions leading to scientific arguments. Thus, we prepared a set of commands that students should fill with the experimental question, hypothesis, expected results, motivation for choosing the test conditions, and the concentration of the substance solution or specifics of the test conditions. In addition, after performing the lab tasks, they should get back to the "Lab notebooks" and fill other commands such as the description of methodology, their data representation, description of results, discussion and conclusion.

We devised two protocols with different levels of complexity. In both protocols, teacher and students interacted in a first moment to establish conceptual knowledge (conceptual goal) of the complement system, but only one protocol included an extra step (Table SI, activity 3) planned to improve the achievement on the goals: we provided students with seminal scientific papers on the complement system, which they should read and present the strategies used by the authors to convince the reader of the interpretation of the results.

To guide students through the reading task, we provided them with a set of contents that we expected to see in the presentation: 1) "After reading the paper, select a figure that you think it is very import to the work"; 2) "Present the information that can be extracted from the figure"; 3) "Clarify the previous knowledge the authors already had, as well as the purpose of the experiment performed by them"; 4) "Discuss the manners by which the authors tied their own evidences with the knowledge generated by others.

Both protocols of the inquiry-based sequence are presented in Table SI.

Groups of students engaged in Immunology and Biochemistry classes in a public university in Brazil performed the two models of inquiry. Of the 39 groups, 15 did not perform activity 3 described in Table SI, and 24 performed all the activities in Table SI. To favor our descriptions, we named each of the protocols as A (the one in which activity 3 is absent) and B (the one in which activity 3 was performed). To avoid the identification of the participants we codded all the 39 "Lab notebooks" with the letter G followed by a number, from G1 to G39. We previously submitted this study to the local ethics committee, which reviewed and approved our protocol (CAAE 58960416.6.0000.5149 / protocol number: 1.717.179). Participants signed an informed consent allowing the analysis and publication of the information collected in classes. For all the activities in both protocols, students received some degree of instruction by the teacher, who avoided providing them with ready answers, but asked questions to direct them to reasoning, as showed in Table SII.

Data analysis

Our framework for epistemic practices analysis was built according to Manzoni-de-Almeida et al. (2016), and [START_REF] Kelly | Epistemic Practices and Science Education[END_REF], to contain epistemic practices categories related to the ones found in scientific texts describing experimental results.

We went through students' written constructs looking for the epistemic practices. We divided each text into sentences, which were considered our unit of analysis. The end of the sentence is marked by a period (.) or a colon (:). The sentences were analyzed according to our framework shown in Table SIII.

RESULTS

Engagement of students while performing the protocol A of the inquiry-based activity

During the planning phase, students engaged in protocol A chose test conditions, e.g.. acetic acid, albumin, vodka, dipyrone, chalk dust, glucose, saliva, fermented milk, dry baking yeast. Only one group chose to test variations of a physical condition: temperature.

We considered as valid the experimental questions in which we found some speculation about the effects caused by the test substance on hemolysis mediated by complement. As showed in figure 1A, eight out of 15 groups formulated valid experimental questions. For example, group 11 asked "Can dipyrone bind to the complement system?".

As to the analysis of hypothesis and expected results, we found that some groups registered the hypothesis under the command originally planned for expected results registration. This fact suggests that the distinction between these elements was not consolidated in the pre-experimental phase. The framework of epistemic practice categories allowed us to localize the sentence corresponding to hypothesis even when students registered them in a command other than the one intended by us. The analysis of the students' writings showed that 12 out of 15 groups formulated valid hypothesis (Figure 1B), and six groups formulated valid expected results (Figure 1C).

From the inquiry phase III (Table SI), students should deal with raw data obtained to produce their assertions. According to Latour (1987), the production of non-verbal inscriptions and the use of the evidences extracted by them in the written text is a strategy to transmit reliability to claims.

We then decided to check out how students represented their data in the "Lab notebooks". Eleven out of 15 groups represented their data using non-verbal inscriptions (Figure 2A), such as images, tables and calculations (Figure 2B). The prevalence of calculations in detriment of other forms of representation is not surprising, since the protocol contained guidance on how to calculate the percent lysis using the absorbance values. No groups used charts to represent data (Figure 2B). [START_REF] Kelly | Toward a research agenda for epistemological studies in science education[END_REF] point out that the social practices of science provide information on how communities shape knowledge in their specific contexts. Thus, categorization of epistemic practices in students' written material reflects resources used by them to formulate and support their assertions. Thus, we performed the analysis of epistemic practices in each sentence of the five last commands in the "Lab notebooks". Figure 3 shows the occurrence of different categories of epistemic practices in the students' constructs. The analysis suggests that the activity favored the appearance of all the categories from our framework, distributed in texts averaging 10 sentences, with the predominance of the categories developing hypothesis, making literary inscriptions, explaining the scheme of investigation. We relate the high appearance of this last category to one of the commands of "Lab notebooks", which asked students to describe the methods used in the experiment.

We observed a low number of groups performing the categories related to theoretical knowledge (conceptualize and relating data and theory), compared to the number of groups performing the categories related to the specific data analysis (describing data, comparing data, interpreting data, explaining data). We had expected this occurrence, since the inquirybased activity demanded the analysis of experimental data. However, we expected to see a higher number of groups performing categories related to the data analysis (describing data, comparing data, interpreting data, explaining data). It is possible that the low number of each of the data analysis categories is simply due to the choice of strategy to analyze data: describing data in detriment of comparing data, for example.

From Figures 2A and3, it is possible to note that 11 groups made literary inscriptions to represent raw data, but only half of the groups mentioned the inscriptions explicitly in their texts. To investigate how students use evidences obtained from their analysis, we looked up the sentences in which their claims were clearly supported by data, and then we graded the written texts as "always", "partially" and "never". Groups classified as "always" built all of their constructs upon data information, and groups classified as "partially" made at least one assertion based on personal beliefs or on observations from data not attached to the "Lab notebook". Groups classified as "never" did not support any of their claims with evidences.

Because we noticed that some groups performed more than one type of literary inscription, we checked how many of the inscriptions were used to formulate their assertions. Then, we graded the texts as "all", "partial use", or "none", depending on how many inscriptions made by them were used to making the verbal constructs. Some groups produced claims not supported by data, as seen in Figure 4A. These groups are the ones which did not perform literary inscriptions, in addition to the ones that described information not attached to the reports. For example: many groups performed calculations, but described features related to the macroscopic observations (e.g. pellet presence/absence, or solution color and turbity). However, they did not attach the image to the "Lab notebooks". Interestingly, none of the groups used all the literary inscriptions in the text (Figure 4B). These data suggest that the groups failed to develop the comprehension of data representation as a resource of rhetoric, but used literary inscriptions as an instructional and illustrative component.

To illustrate our analysis, we present in Table SIV an excerpt extracted from G14's "Lab notebook". They tested the possibility that chalk dust interfered in hemolysis mediated by the complement system, to associate a putative interaction with the complement system to allergic reaction to chalk dust. The analysis of epistemic practices shows that they appealed to several descriptions and comparisons to relate the evidences obtained from the "experimental groups" and "control groups" in both literary inscriptions: calculation and plate design. They then used all those evidences to claim that chalk dust does not bind to the complement components but promotes hemolysis by itself (sentence 19).

The engagement of students while subjected to Protocol B of the inquiry-based activity

Students who performed this form of inquiry engaged in scientific paper presentations primarily (inquiry-phase I: activity 3 -table SI). We planned this extra step to provide them with the comprehension of how immunologists build models based on their own data and communicate knowledge. We also wanted to provide them with concept and data source to favor construction of a structured scientific text.

These students chose aspirin, glucose, Ibuprofen, sheep blood (courtesy of the Veterinary School, UFMG), Peumus boldus' extract, Biomphalaria hemolymph (courtesy of the Parasitology Department, UFMG, and obtained from healthy snails), saliva, skim milk and honey.

All 24 groups that formulated valid experimental questions (Figure 5A) and hypothesis (Figure 5B). Only four groups did not formulate expected results in the preexperimental phase (Figure 5C). These results suggest that the guided presentation of scientific papers contributed to their comprehension of these three scientific practices.

Analysis of data representation shows that 22 out of 24 groups performed literary inscriptions (Figure 6A), and most of the non-verbal inscriptions were also identified by legends. In addition, we observed that students incremented their data representations by performing more than one literary inscription in "Lab notebooks". There was a prevalence of images, calculation and tables, as well as charts (Figure 6B).

Figure 7 shows the profile of epistemic practice prevalence in the students' written constructs. The analysis suggests the activity favored the appearance of all the categories of epistemic practices, with the predominance of making literary inscriptions, asking questions, making hypothesis, and explaining the scheme of investigation. This last category is related to the command that asked for a methodological description, and in this case, we also observed that students usually described elements of the experimental procedure to find explanations for data that did not fit an expected pattern (monitoring anomalous results).

A great number of groups performed comparisons (comparing data) and interpretations (interpreting data). On the other hand, a low number of groups performed the categories related to theoretical knowledge (conceptualize and relating data and theory).

In Figure 8A, we show that 15 out of 24 "Lab notebooks" contained claims based on the obtained data. Groups classified as "never" are the ones who did not attach any kind of data in their "Lab notebooks", and the five groups classified as "partially" sometimes mentioned data descriptions that were not attached to "Lab notebooks". When an author does not use data to support the information under debate, it is impossible to make inferences about the reliability of the information. Thus, texts with this characteristic drift from the concept of a scientific text. Figure 8B shows that eight groups used all the literary inscriptions. Most groups, however, registered a great number of literary inscriptions (Figure 6B) but did not use all of them in their written constructs (Figure 8B). Taken together, these data suggest that active learning-based activities that focuses on data usage in scientific texts is needed to improve the achievement of this skill by students.

We found evidences that students developed a line of scientific reasoning while analyzing experimental data. To illustrate this observation, we present an excerpt from G38 in Table SV. This group chose to test sodium hydroxide on the complement system, based on the information that a chemical reaction occurs between sodium hydroxide and the magnesium ion. Their hypothesis was that the product of the reaction would remove magnesium from the medium. Since magnesium is an important co-factor in the complement cascade, its removal would interrupt the chain of reactions.

We categorized about 40 sentences in their "Lab notebook". The categories were: discussion supported by several data sources (sentences 18 and 28), by theoretical knowledge (sentences [START_REF] Kelly | Epistemic levels in argument: An analysis of university oceanography students' use of evidence in writing[END_REF][START_REF] Goodwin | Professional Vision[END_REF][START_REF] Jiménez-Aleixandre | Determinism and Underdetermination in Genetics: Implications for Students' Engagement in Argumentation and Epistemic Practices[END_REF][22] and by selection of evidences (sentences 28 and 29). The epistemic practices categories for this group are presented in Table SV.

DISCUSSION

This work presents the analysis of a level I [START_REF] Blanchard | Is Inquiry Possible in Light of Accountability?: A Quantitative Comparison of the Relative Effectiveness of Guided Inquiry and Verification Laboratory Instruction[END_REF] inquiry-based learning activity, planned to offer opportunities in the three different dimensions of knowledge [START_REF] Kelly | Epistemic Practices and Science Education[END_REF] related to Immunology. Experimental activity comprises the resource used by immunologists (social dimension) to test the influence of a substance on the complement system cascade (conceptual knowledge) and, by performing this task, understand the basis of the conceptual knowledge (epistemic knowledge).

Our analysis showed that both the inquiry-learning protocols (A and B) promoted engagement in scientific practices, which can be confirmed by the appearance of different categories of epistemic practices related to experimental data analysis in the written constructs (Figures 4 and8). This work raises important issues related to the role of data representation and use of evidences in the students written constructs. Some groups did not used in their discussion all the literary inscriptions that they performed, even the ones who executed the protocol B, with guided reading and presentation of scientific papers (Figure 5 and9). We think it is possible that students used tables, pictures and charts without realizing the importance of the systematization and representation of data. It seemed that students were using data representation as illustrations rather than realizing that data representation is actually the core of a scientist paper. These observations suggest a need for more activelearning strategies focused on debating the elements of rhetoric and persuasiveness of the scientific text. These results corroborate other observations that claim for more training on these abilities [18].

In addition, it is thought that scientific training must consider core elements used for inquiry in their disciplines of interest [START_REF] Kelly | Epistemic levels in argument: An analysis of university oceanography students' use of evidence in writing[END_REF][START_REF] Goodwin | Professional Vision[END_REF][START_REF] Jiménez-Aleixandre | Determinism and Underdetermination in Genetics: Implications for Students' Engagement in Argumentation and Epistemic Practices[END_REF]. The lower number of sentences categorized in the epistemic practices conceptualize and relating data and theory corroborate this conclusion. According to [START_REF] Kelly | Epistemic levels in argument: An analysis of university oceanography students' use of evidence in writing[END_REF], one criterion to estimate the strength of students' arguments based on scientific rhetoric is the ratio of data statements to theory statements. In our analysis, we found more sentences classified in epistemic practices related to data analysis (comparing, interpreting, describing and explaining data), than in epistemic practices related to theory statements, suggesting that students need to be trained in the genre of scientific texts.

FINAL CONSIDERATIONS

Our strategy to teach immunology by doing science was a complex activity that included: 1) dealing with unknown conditions that entailed technical difficulties, such as pipetting viscous substance, solid particles in suspension, turbidity which affected reading, etc; Students had to, in a short time, solve these difficulties. 2) Results were obtained indirectly, that is, lysis or no lysis had to be interpreted as complement integrity or consumption. These are typical Immunology practices, and the performance of such practices should be encouraged in classrooms so that students are more aware of how textbook knowledge is obtained. We also determined the several categories of epistemic practices that emerged during problem solving. Our findings contribute to the improvement of the knowledge in science education. We estimated the number of (A) constructs supported in data for each "Lab notebook"; and (B) the number of literary inscriptions utilized that were related to the number of literary inscriptions performed in each "Lab notebook"

List of captions

Figure 1 :

 1 Figure1: Analysis of experimental questions, hypothesis and expected results produced in students' written material while submitted to protocol A.

Figure 2 :

 2 Figure 2: Data representation by groups of students in protocol A. (A) Number of groups that performed non-verbal inscriptions. (B) Qualitative profile comparing the frequency of types of literary inscriptions in students' writings.

Figure 3 :

 3 Figure 3: Appearances of each epistemic practice in the 15 written constructs from students submitted to protocol A.

Figure 4 :

 4 Figure4: Articulation of literary inscriptions. Estimated number of (A) constructs supported in data for each "Lab notebook"; and (B) literary inscriptions utilized, related to number of literary inscriptions performed in each "Lab notebook".

Figure 5 :

 5 Figure 5: Analysis of experimental question, hypothesis and expected results produced in protocol B students' written material.

Figure 6 :

 6 Figure 6: Data representation by groups of students subjected to protocol B. (A) Number of groups that performed non-verbal inscriptions. (B) Qualitative profile comparing the frequency of types of literary inscriptions in students' writings.

Figure 7 :Figure 8 :

 78 Figure 7: Appearances of each epistemic practices in 24 written constructs from students submitted to protocol B


Nicolas Folliet

Christel Gervais

Dominique Costa

Guillaume Laurent

Florence Babonneau

Lorenzo Stievano

Jean-François Lambert

Frederik Tielens

Jean-Francois Lambert

A Molecular Picture of the Adsorption of Glycine in Mesoporous Silica through NMR Experiments Combined with DFT-D Calculations

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

INTRODUCTION

An increased interest has been shown in the past for the synthesis of templated inorganic materials due to their potential applications in catalysis and controlled release. [START_REF] Cauvel | Hydrophobic and Hydrophilic Behavior of Micelle-Templated Mesoporous Silica[END_REF]2 These applications are closely related to the binding of biomolecules on inorganic surfaces, but questions related to the nature of interfacial interactions still need to be addressed. Studies on simple model systems are thus needed for a thorough understanding of more complex systems. This is the reason why building block approaches are developed by some groups in order to understand the adsorption of biomolecules (from amino acids to proteins) on solid surfaces. [START_REF] Vallee | Peptide Interactions with Metal and Oxide Surfaces[END_REF] To this respect, glycine, the smallest and simplest amino acid, simultaneously exhibits both carboxylate/carboxylic and amine/ammonium moieties and, thus, represents a good reference object for other more complex biological molecules. Indeed, a number of papers appeared in recent years concerning the adsorption of glycine or other amino acids on oxide surfaces, [START_REF] Lambert | Adsorption and Polymerization of Amino Acids on Mineral Surfaces: A Review[END_REF] and a review specially devoted to amino acids on silica has recently been published. [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] Recent developments in solid-state NMR open promising perspectives for the detailed characterization of such interactions between small molecules and inorganic surfaces. [START_REF] Bonhomme | Advanced Solid State NMR Techniques for the Characterization of Sol-Gel-Derived Materials[END_REF] This local spectroscopic technique can establish "dialogues" between nuclear spins. In that sense, solid-state NMR is a perfect tool of investigation for the hybrid organic-inorganic interfaces in nanomaterials. NMR spectroscopy and more particularly dipolar recoupling experiments were used recently to identify interacting moieties in alanine adsorbed on a mesoporous silica SBA-15 surface, [START_REF] Ben Shir | Molecular Level Characterization of the Inorganic-Bioorganic Interface by Solid State NMR: Alanine on a Silica Surface, a Case Study[END_REF] and later in glycine adsorbed on the same support. [START_REF] Ben Shir | Binding Specificity of Amino Acids to Amorphous Silica Surfaces: Solid-State NMR of Glycine on SBA-15[END_REF] In both cases, the authors show that the ammonium group interacts directly with silanols (predominantly terminal ones) on the surface while the carboxylates seem to freely reorient.

There has been a continuous increase in the number of ab initio theoretical studies on the adsorption of amino acids or organic molecules on several oxide surfaces such as amorphous or crystalline silica, [START_REF] Costa | Microsolvation of Glycine by Silanol Ligands: A DFT Study[END_REF][START_REF] Costa | DFT Study of the Adsorption of Microsolvated Glycine on a Hydrophilic Amorphous Silica Surface[END_REF][START_REF] Lomenech | Theoretical and Experimental Study of the Adsorption of Neutral Glycine on Silica from the Gas Phase[END_REF][START_REF] Rimola | Interaction of Glycine with Isolated Hydroxyl Groups at the Silica Surface: First Principles B3LYP Periodic Simulation[END_REF][START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] alumina, [START_REF] Arrouvel | DFT Periodic Study of the Adsorption of Glycine on the Anhydrous and Hydroxylated (0001) Surfaces of Alpha-Alumina[END_REF] ZnO, [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF][START_REF] Irrera | Molecular Modelling by DFT of 1,2-Diaminoethane Adsorbed on the Zn-Terminated and O-Terminated, Anhydrous and Hydroxylated Zno[END_REF] Cr 2 O 3 , [START_REF] Costa | Biomolecule-Biomaterial Interaction: A DFT-D Study of Glycine Adsorption and Self-Assembly on Hydroxylated Cr2o3 Surfaces[END_REF][START_REF] Garrain | Biomaterial-Biomolecule Interaction: DFT-D Study of Glycine Adsorption on Cr2o3[END_REF] TiO 2 , [START_REF] Ojamae | Ir and Quantum-Chemical Studies of Carboxylic Acid and Glycine Adsorption on Rutile TIO2 Nanoparticles[END_REF][START_REF] Tonner | Adsorption of Proline and Glycine on the TIO2(110) Surface: A Density Functional Theory Study[END_REF][START_REF] Guo | DFT Study of the Adsorption of Aspartic Acid on Pure, N-Doped, and Ca-Doped Rutile (110) Surfaces[END_REF][START_REF] Koppen | Simulation of Adhesion Forces and Energies of Peptides on Titanium Dioxide Surfaces[END_REF][START_REF] Langel | Simulation of the Interface between Titanium Oxide and Amino Acids in Solution by First Principles Md[END_REF][START_REF] Fleming | Probing the Reaction Pathways of Dl-Proline on TIO2(001) Single Crystal Surfaces[END_REF][START_REF] Szieberth | Adsorption of Glycine on the Anatase (101) Surface: An Ab Initio Study[END_REF] and hydroxyapatite. [START_REF] Rimola | Ab Initio Modelling of Protein-Biomaterial Interactions: Influence of Amino Acid Polar Side Chains on Adsorption at Hydroxyapatite Surfaces[END_REF][START_REF] Almora-Barrios | Density Functional Theory Study of the Binding of Glycine, Proline, and Hydroxyproline to the Hydroxyapatite (0001) and (01(1)over-Bar0) Surfaces[END_REF][START_REF] Almora-Barrios | A Density Functional Theory Study of the Interaction of Collagen Peptides with Hydroxyapatite Surfaces[END_REF][START_REF] Rimola | Toward a Surface Science Model for Biology: Glycine Adsorption on Nanohydroxyapatite with Well-Defined Surfaces[END_REF][START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Even glycine, the simplest amino acid, may present different adsorption mechanisms on an oxide surface: first, it may be adsorbed in neutral, cationic, zwitterionic, or anionic forms according to the protonation state of the amine and carboxylate groups (NH 2 -CH 2 -COOH, NH 3 + -CH 2 -COOH, NH 3 + -CH 2 -COO -, NH 2 -CH 2 -COO -, respectively). Second, adsorption may be driven by covalent bonding, H-bonding with the surface, ionic interactions, self-assembly between the biomolecules, and any combination of those possibilities. [START_REF] Lambert | Adsorption and Polymerization of Amino Acids on Mineral Surfaces: A Review[END_REF] Most of the ab initio studies of glycine adsorption were performed in the absence of solvent; that is, they are relevant to adsorption from the gas phase. However, the presence of solvent molecules (e.g., water) may significantly influence glycine adsorption equilibria and kinetics. On the one hand, the solvation of the biomolecule may hinder its interaction with the surface. On the other hand, the coadsorption of water and biomolecule may actually contribute to stabilizing the adsorbed form. Water may also lower the kinetic barriers for conformational changes or proton transfers. Only few ab initio studies of the adsorption of amino acids on surfaces in the presence of water as a solvent have been undertaken, due to the extensive cost of ab initio molecular dynamics (MD) calculations and of the very short time periods simulated during a calculation (a few picoseconds). Marx and co-workers studied the adsorption of glycine on pyrite at the solid-liquid interface. [START_REF] Boehme | Glycine on a Wet Pyrite Surface at Extreme Conditions[END_REF][START_REF] Nair | Glycine at the Pyrite-Water Interface: The Role of Surface Defects[END_REF][START_REF] Schreiner | Peptide Synthesis in Aqueous Environments: The Role of Extreme Conditions and Pyrite Mineral Surfaces on Formation and Hydrolysis of Peptides[END_REF][START_REF] Pollet | Ab Initio Simulations of Desorption and Reactivity of Glycine at a Water-Pyrite Interface at ″Iron-Sulfur World″ Prebiotic Conditions[END_REF] In this series of studies, glycine was found to be preferentially solvated in the liquid phase and not adsorbed on the ideal surface of pyrite, while the presence of sulfur vacancies caused a specific adsorption involving coordination of the carboxylate to iron ions, with or without coadsorption of water. On rutile TiO 2 , Langel and Menken 23 investigated the adsorption of glycine, methionine, cysteine and serine. MD simulation revealed that the most stable adsorbed form of the amino acids involved coordination of the carboxylate moiety to two Ti 4+ centers, in a bridging position (in addition, cysteine could also coordinate through its deprotonated thiol group and thus behave as a bidentate ligand). An adsorption mechanism involving carboxylate coordination to Ti 4+ was also found to be operating for glycine (Gly) on rutile (110). [START_REF] Li | Journey toward the Surface: How Glycine Adsorbs on Titania in Water Solution[END_REF] We have recently explored the adsorption mechanism of glycine on Boehmite (AlOOH). [START_REF] Motta | Aimd Evidence of Inner Sphere Adsorption of Glycine on a Stepped (101) Boehmite AlOOH Surface[END_REF] Inner-sphere adsorption (by unidentate carboxylate coordination to Al 3+ ) was favored over outer-sphere, with adsorption energies of -160 and -20 kJ•mol -1 , respectively.

On silica, in contrast, the favored amino acid adsorption mechanisms do not involve coordination, or covalent bond formation. Recently, Nonella and Seeger 36 investigated the alanine-silica interaction in the presence of bulk water by means of DFT-MD. They found that the strongest interaction occurred for a positively charged alanine ion and a negatively charged silica surface. The formation of H-bonds with isolated silanols on Gly/SiO 2 systems was found to be weakly exothermic, [START_REF] Costa | Microsolvation of Glycine by Silanol Ligands: A DFT Study[END_REF][START_REF] Lomenech | Theoretical and Experimental Study of the Adsorption of Neutral Glycine on Silica from the Gas Phase[END_REF] whereas the formation of a H-bond network with a specific silanol nest could induce strong bonds of glycine with the surface. [START_REF] Costa | DFT Study of the Adsorption of Microsolvated Glycine on a Hydrophilic Amorphous Silica Surface[END_REF] "Microsolvating" water molecules were added to the system to understand the effect of water coadsorption. Several first principles molecular dynamics runs indicated that in the dominant conformation the COO -moiety is directly H-bonded to one geminal SiOH group on the surface, whereas NH 3 + interacts with water molecules far from the surface. [START_REF] Costa | DFT Study of the Adsorption of Microsolvated Glycine on a Hydrophilic Amorphous Silica Surface[END_REF] Zhao et al. [START_REF] Zhao | An SCC-DFTB/MD Study of the Adsorption of Zwitterionic Glycine on a Geminal Hydroxylated Silica Surface in an Explicit Water Environment[END_REF] and Walsh et al. 38 obtained very similar results: the dominant configuration exhibits the COO - group pointing toward the surface in direct contact with geminal SiOH groups, while the NH 3 + prefers to interact with bulk water, away from the surface. Interesting data concerning the effect of increasing numbers of microsolvating water molecules on the structure of glycine interacting with an edingtonite SiO 2 surface can also be found in the work of Rimola et al. [START_REF] Rimola | Neutral Vs Zwitterionic Glycine Forms at the Water/Silica Interface: Structure, Energies, and Vibrational Features from B3LYP Periodic Simulations[END_REF] The ideal way to thoroughly understand adsorption at the solid liquid interface, however, is to couple the experimental and the theoretical approaches by performing an ab initio assisted interpretation of spectroscopic data. For instance, the spectroscopic data obtained by NMR and the theoretical structural models of glycine adsorption can be associated by performing first principles calculations of the NMR parameters, starting from structural models based on DFT. For that purpose, the GIPAW (Gauge Included Projected Augmented Wave) method, first proposed by Pickard and Mauri, [START_REF] Pickard | All-Electron Magnetic Response with Pseudopotentials: NMR Chemical Shifts[END_REF] is now considered as a tool of choice for solid state NMR calculations (in the frame of DFT and under periodic boundary conditions). [START_REF] Charpentier | The PAW/GIPAW Approach for Computing NMR Parameters: A New Dimension Added to NMR Study of Solids[END_REF] The combined theoretical/experimental approach [START_REF] Tielens | The Synergistic Power of Theory and Experiment in the Field of Catalysis Preface[END_REF][START_REF] Bonhomme | First-Principles Calculation of NMR Parameters Using the Gauge Including Projector Augmented Wave Method: A Chemist's Point of View[END_REF] and in particular the DFT structural modeling/ solid state NMR/GIPAW approach represents a very useful tool for the detailed characterization of organic-inorganic interfaces [START_REF] Folliet | Investigation of the Interface in Silica-Encapsulated Liposomes by Combining Solid State NMR and First Principles Calculations[END_REF] and seems therefore adequate for a detailed study of the glycine-silica interface at an atomic level.

In this contribution, a mesoporous MCM-41 silica [START_REF] Kresge | Ordered Mesoporous Molecular-Sieves Synthesized by a Liquid-Crystal Template Mechanism[END_REF][START_REF] Beck | A New Family of Mesoporous Molecular-Sieves Prepared with Liquid-Crystal Templates[END_REF] was prepared and loaded with glycine. Solid-state NMR was then used for the investigation of the adsorbed amino acid and in particular for investigating the proximities between silanols, water molecules and glycine molecules using two-dimensional 1 H-X HETCOR CP MAS experiments (X = 13 C, [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N, and [START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si). The amorphous silica slab model we previously developed [START_REF] Tielens | Ab Initio Study of the Hydroxylated Surface of Amorphous Silica: A Representative Model[END_REF] was used to study various configurations of glycine adsorption, taking explicitly into account possible interfacial water molecules. Finally, and in order to determine the relevance of the models, first principles calculations of NMR parameters were performed by using the GIPAW method for all involved nuclei and compared with experimental data. The mixture was heated at 90 °C for 24 h. A solid white powder formed in the reaction mixture; it was washed with distilled water, dried at 60 °C overnight, and then heated at 540 °C for 8 h under air flux to remove the surfactant.

EXPERIMENTAL DETAILS

2.1.2. Gly Deposition on MCM-41. The calcined MCM-41 sample was loaded with glycine according to the "incipient wetness" procedure: 0.300 g of MCM-41 was impregnated with a small amount (2 mL) of 0.1 M aqueous glycine solution, allowing the complete filling of the pore volume of MCM-41 (without formation of a bulk liquid phase). This resulted in the deposition of 0.67 mmol Gly per gram of MCM-41, or a loading of 5.0 wt %. The sample was then dried at 60 °C for 3.5 days. In order to increase the signal-to-noise ratio of the NMR spectra, glycine isotopically enriched in [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N and [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C (the latter on the carboxyl function) was employed. This sample will be called Gly/MCM.

Nitrogen adsorption/desorption isotherms were recorded at 77 K with a Micromeritics ASAP2000 apparatus, after degassing under vacuum (1 × 10 -3 Torr) at 250 °C for pure calcined MCM-41 or 40 °C for the glycine-loaded sample.

2.2. Solid-State NMR Experiments. The 13 C, [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N CP (Cross-Polarization) MAS experiments were recorded on a Bruker Avance 400 spectrometer (9.4 T, ν 0 ( 1 H) = 400.78 MHz, ν 0 ( 13 C) = 100.61 MHz, ν 0 ( 15 N) = 40.56 MHz), and 2D HETCOR (HETeronuclear CORrelation) experiments were recorded on a Bruker Avance III 300 spectrometer (7.0 T, ν 0 ( 1 H) = 300.29 MHz, ν 0 ( 13 C) = 75.51 MHz, ν 0 ( 15 N) = 30.44 MHz, ν 0 ( 29 Si) = 59.66 MHz). At both fields, Bruker 4 mm double resonance MAS probes were used, and the samples were spun at the magic angle at 14 kHz.

2D 1 H-X HETCOR CP MAS experiments (X = 13 C, [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N, and [START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si) were recorded with ramped-amplitude cross-polarization (RAMP-CP) [START_REF] Metz | Ramped-Amplitude Cross-Polarization in Magic-Angle-Spinning NMR[END_REF] in the 1 H channel to transfer magnetization from 1 H to X (typically, 64 increments of 63 μs were used with 600 scans per acquisition). The 1 H field was ramped from 50 to 100% while the X RF field was set around 65 kHz. RD = 3 s, t 90°( 1 H) = 3 μs, CP contact time = 1 ms, optimized 1 H spinal-64 decoupling. A single pulse [START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si NMR MAS spectrum was also recorded at 7.0 T with a Bruker 7 mm double resonance MAS probe, spinning at 5 kHz, 5.0 μs π/2 pulses, and a recycle delay of 60 s. Spectra deconvolutions were performed using the Dmfit software. [START_REF] Massiot | Modelling One-and Two-Dimensional Solid-State NMR Spectra[END_REF] 1 H, [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C, and [START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si chemical shifts were referenced to external TMS, while [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N was referenced to external CH 3 NO 2 .

2.3. Computational Details. 2.3.1. Ab Initio Geometrical Configurations and Molecular Dynamics Calculations. All calculations were performed using the ab initio plane-wave pseudopotential approach as implemented in the VASP code. [START_REF] Kresse | Ab-Initio Molecular-Dynamics for Liquid-Metals[END_REF][START_REF] Kresse | Ab-Initio Molecular-Dynamics Simulation of the Liquid-Metal Amorphous-Semiconductor Transition in Germanium[END_REF] The Perdew-Burke-Ernzerhof (PBE) functional [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF][START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF] was chosen to perform the periodic DFT calculations with an accuracy on the overall convergence tested elsewhere. [START_REF] Tielens | Ab Initio Study of the Hydroxylated Surface of Amorphous Silica: A Representative Model[END_REF][START_REF] Calatayud | Reactivity of Gas-Phase, Crystal and Supported V2O5 Systems Studied Using Density Functional Theory Based Reactivity Indices[END_REF][START_REF] De Bocarme | Oxygen Adsorption on Gold Nanofacets and Model Clusters[END_REF][START_REF] Tielens | Molecular Oxygen Adsorption on Electropositive Nano Gold Tips[END_REF][START_REF] Tielens | What Do Vanadium Framework Sites Look Like in Redox Model Silicate Zeolites?[END_REF][START_REF] Tielens | Nature of Vanadium Species in V Substituted Zeolites: A Combined Experimental and Theoretical Study[END_REF] The valence electrons were treated explicitly and their interactions with the ionic cores are described by the Projector Augmented-Wave (PAW) method, [START_REF] Blochl | Improved Tetrahedron Method for Brillouin-Zone Integrations[END_REF][START_REF] Kresse | From Ultrasoft Pseudopotentials to the Projector Augmented-Wave Method[END_REF] which allows one to use a low energy cutoff equal to 400 eV for the plane-wave basis. The integration over the Brillouin zone was performed on the Γ-point, in all calculations.

The potential energy surfaces (PESs) were systematically explored by ab initio molecular dynamics at 300 K, starting from several configurations. The time step was set at 1.5 fs; several starting conformations were considered (but no statistic was performed on the starting conformations), and the run was stopped when an average stable conformation was reached during 500 fs or more; we used a microcanonical ensemble in the NVE (constant number of molecules, constant volume and constant energy) approach. To avoid fluctuations due to the large time step chosen, the mass of the hydrogen atom was set to 3. The local minima found in the PES were then optimized at 0 K. It is noticeable that this MD approach allowed us to find the minima of the PES showed in the present paper, which were not intuited beforehand. Glycine was adsorbed in its zwitterionic form (ZG).

In the geometry optimizations at 0 K, the positions of all atoms in the supercell are relaxed in the potential energy determined by the full quantum mechanical electronic structure until the total energy differences between the loops is less than 10 -4 eV. In a second step, the DFT-D approach [START_REF] Wu | Towards Extending the Applicability of Density Functional Theory to Weakly Bound Systems[END_REF] was applied as implemented in VASP, which consists in adding a semiempirical dispersion potential to the conventional Kohn-Sham DFT energy. In particular we used the DFT-D method of Grimme [START_REF] Grimme | Semiempirical GGA-Type Density Functional Constructed with a Long-Range Dispersion Correction[END_REF] up to D2 correction.

The energies of interaction/adsorption of glycine on the surface were calculated following different reactions: First, neutral glycine (NG) was considered as the adsorbant. The adsorption energy ΔE ads was calculated as follows:

Δ = - - - E E E E nE (G, Sil) (NG) (Sil) (H O) ads 2 (1)
where E(NG), E(Sil), and E(H 2 O) are the total electronic energies of NG, silica surface, and one water molecule obtained after separate geometry optimization. E(G, Sil) is the energy of the optimized (glycine + silica + n water) system with glycine adsorbed on the silica surface. The number of water molecules in the models runs up to 3.

The adsorption energy can be decomposed into interaction and deformation energies, as follows:

Δ = Δ -Δ -Δ - - E E E E ads binding deform mol deform surface (2)
where ΔE deform-mol of the molecule and ΔE deform-surface of the surface indicates the energy required for the molecule (respectively, the surface) to adapt to the constraints induced by the surface (respectively, the molecule), and ΔE binding is the energy of the bond between the surface and the molecule, in the geometries of the adsorption configuration. ΔE ads is negative, indicating an exothermic process, when the binding energy between the molecule and the surface is more important than the induced respective deformations. 2.3.2. Ab initio Calculations of NMR Parameters. The first principles NMR calculations were performed within Kohn-Sham DFT using the QUANTUM-ESPRESSO software (available online). [START_REF] Giannozzi | Quantum Espresso: A Modular and Open-Source Software Project for Quantum Simulations of Materials[END_REF] The PBE generalized gradient approximation [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF][START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF] was used. and the valence electrons were described by norm conserving pseudopotentials [START_REF] Troullier | Efficient Pseudopotentials for Plane-Wave Calculations[END_REF] in the Kleinman-Bylander [START_REF] Kleinman | Efficacious Form for Model Pseudopotentials[END_REF] form. The core definition for O, C, and N is 1s 2 and 1s 2 2s 2 2p 6 for Si. The core radii are 1.2 au for H, 1.5 au for O, 1.6 au for C, 1.45 au for N, and 2.0 au for Si. The wave functions were expanded on a plane wave basis set with a kinetic energy cutoff of 1088 eV. The integral over the first Brillouin zone was performed using a Monkhorst-Pack 1 × 1 × 1 k-point grid for the charge density and chemical shift tensor calculation. The shielding tensor is computed using the GIPAW [START_REF] Pickard | All-Electron Magnetic Response with Pseudopotentials: NMR Chemical Shifts[END_REF] approach which permits the reproduction of the results of a fully converged all-electron calculation. The isotropic chemical shift δ iso is defined as

δ iso = -[σ -σ ref ],
where σ is the isotropic shielding and σ ref is the isotropic shielding of the same nucleus in a reference system as previously described. [START_REF] Gervais | Combined First-Principles Computational and Experimental Multinuclear Solid-State NMR Investigation of Amino Acids[END_REF][START_REF] Gervais | Combined Ab Initio Computational and Experimental Multinuclear Solid-State Magnetic Resonance Study of Phenylphosphonic Acid[END_REF] 2.3.3. Description of the Model. The adsorption of glycine was studied using a silica model structure described and characterized in a previous work. [START_REF] Tielens | Ab Initio Study of the Hydroxylated Surface of Amorphous Silica: A Representative Model[END_REF] In summary, the model consists of a Si 27 O 54 slab hydrated with 13 H 2 O molecules. The silanol density at the surface is 5.8 nm -2 , and unit cell dimensions are 12.77 × 17.64 × 25.17 Å [START_REF] Vallee | Peptide Interactions with Metal and Oxide Surfaces[END_REF] . The slab has a density of approximately 1.7 g/cm [START_REF] Vallee | Peptide Interactions with Metal and Oxide Surfaces[END_REF] . The number of Si atoms belonging to tetrahedra exposed on the surface is approximately 8 nm -2 . The same slab has been used in studies in which the model was used to describe a catalyst support. [START_REF] Wojtaszek | Gold Grafted to Mesoporous Silica Surfaces, a Molecular Picture[END_REF][START_REF] Wojtaszek | The Formation of Gold Clusters Supported on Mesoporous Silica Material Surfaces: A Molecular Picture[END_REF][START_REF] Islam | Characterization of Supported Vanadium Oxide Species on Silica: A Periodic DFT Investigation[END_REF][START_REF] Guesmi | Chromium Oxide Species Supported on Silica: A Representative Periodic DFT Model[END_REF] Despite the fact that the global silanol density is higher than the one usually reported for MCM-41, [START_REF] Shenderovich | Pyridine-N-15 -a Mobile NMR Sensor for Surface Acidity and Surface Defects of Mesoporous Silica[END_REF] locally, the different expected types (terminal and geminal) are present on the model surface, enabling, thus, a correct, local, description of the interaction sites.

RESULTS AND DISCUSSION

3.1. Surface Area and Porosity. Nitrogen adsorption/ desorption isotherms of the calcined MCM-41 and the glycineloaded sample (see the Supporting Information for details, Figure S1 and Table S1) provided specific surface areas, evaluated by BET calculations, [START_REF] Brunauer | Adsorption of Gases in Multimolecular Layers[END_REF] of 1060 and 830 m 2 •g -1 , respectively. Both the mean pore diameter estimated by the BJH method [START_REF] Barrett | The Determination of Pore Volume and Area Distributions in Porous Substances. 1. Computations from Nitrogen Isotherms[END_REF] and the mesoporous volume decrease after loading, in line with the successful incorporation of glycine in the mesopores of MCM-41.

3.2. Solid-State NMR Investigation. In the carboxylate region, the [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C CP MAS spectrum of glycine encapsulated in mesoporous silica (Figure 1) exhibits a sharp signal at 176.4 ppm and a distribution of broader ones ranging from 168 to 175 ppm, with one maximum at 173.6 ppm. Similar results have already been observed for glycine adsorbed on nonporous silica nanoparticules (Aerosil 380): [START_REF] Ben Shir | Binding Specificity of Amino Acids to Amorphous Silica Surfaces: Solid-State NMR of Glycine on SBA-15[END_REF][START_REF] Lopes | Adsorption of Amino Acids on Oxide Supports: A Solid-State NMR Study of Glycine Adsorption on Silica and Alumina[END_REF] the sharp line was assigned to bulk α-glycine 76 ( 13 C CP MAS spectra of the α, β and γ polymorphs of glycine are presented in Figure 1), while the broader line could correspond to a population of glycine molecules interacting with silica surface groups (most likely silanols) and/or adsorbed water molecules. A [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C single-pulse (SP) MAS NMR spectrum of the sample was also recorded (Supporting Information Figure S2): this experiment corresponds to direct [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C detection, without CP from 1 H to [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C. The signal assigned to crystalline glycine is very similar in the two spectra (SP and CP), but important differences are visible in the 168-175 ppm range. Instead of a distribution of broad signals in the CP spectrum, only one sharper signal at 173.5 ppm is present in the SP spectrum, with no clear sign of any of the broader components present in the CP spectrum. This suggests the existence of two populations of glycine molecules: one mobile enough not to be detected by CP due to an efficient averaging of the 1 H- [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C dipolar interactions, and one more rigid detected by CP, a priori corresponding to the molecules strongly interacting with the surface (apart from crystalline forms of glycine). Similar observations were already reported for encapsulated molecules in MCM-41. [START_REF] Bonhomme | Advanced Solid State NMR Techniques for the Characterization of Sol-Gel-Derived Materials[END_REF][START_REF] Azais | Solid-State NMR Characterization of Drug-Model Molecules Encapsulated in MCM-41 Silica[END_REF] The population detected by CP is thus the one of interest for the present study.

A two-dimensional 1 H- [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C HETCOR CP MAS NMR experiment was recorded in order to discriminate between [START_REF] Cauvel | Hydrophobic and Hydrophilic Behavior of Micelle-Templated Mesoporous Silica[END_REF] H sites according to their proximities to the different carbon atoms (Figure 2a). The 13 C signal at 176.4 ppm assigned to bulk α-glycine clearly shows two cross peaks with proton signals at 3.2 ppm and 8.4 ppm corresponding to the methylene group bonded to the carboxylate and to the ammonium group in interaction with the carboxylate of a neighboring molecule through NH-OOC hydrogen bonds in the bulk state. Two cross peaks centered at 4.3 and 7.2 ppm are observed for the [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C signal at 173.6 ppm. The first 1 H chemical shift is possibly due to the superposition of methylene and water signals while the second is consistent with an ammonium group or with silanols/water in interaction with the carboxylate. Finally, the larger [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C signal centered at 171.5 ppm only correlates with a proton signal at 4.7 ppm. The latter is usually assigned to the protons of silanols in fast exchange with adsorbed water on imperfectly dehydrated silica, be it mesoporous or nonporous. [START_REF] Trebosc | Solid-State NMR Study of MCM-41-Type Mesoporous Silica Nanoparticles[END_REF] The conventional 1 H MAS spectrum (Supporting Information Figure S3) shows indeed a predominant large signal centered at 5 ppm characteristic of the presence of water.

The [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N CP MAS spectrum of Gly/MCM (Figure 3) shows a narrow signal at -346.6 ppm corresponding to the chemical shift of ammonium groups in α-glycine and a second component centered at -348.5 ppm most probably corresponding to the glycine in interaction with the surface. A twodimensional 1 H- [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N HETCOR CP MAS NMR experiment was then recorded to identify 1 H chemical shifts of the ammonium groups of these two types of glycine (Figure 2b). Consistently with the [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C results, the [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N signal at -346.6 ppm shows a proton cross peak at 8.6 ppm, assigned to the ammonium protons of bulk glycine [START_REF] Stievano | Density Functional Theory Modeling and Calculation of NMR Parameters: An Ab Initio Study of the Polymorphs of Bulk Glycine[END_REF] while the [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N peak at -346.6 ppm is correlated to a 1 H signal at 6.4 ppm possibly corresponding to -NH protons involved in a H-bond network with the silica surface. The weak cross peak at 3.7 ppm can be assigned to the protons of the CH 2 linked to the ammonium.

Finally, the 2D 1 H- [START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si HETCOR CP MAS NMR spectrum of the sample (Figure 2c) confirms that the protons with chemical shift values centered at about 5 ppm are strongly correlated with surface silicon atoms (they were assigned above to silanols in fast exchange with water). Overall, the HETCOR results are very similar to those published by Ben Shir et al. for Gly/SBA-15 in the "intermediate" hydration state. [START_REF] Ben Shir | Binding Specificity of Amino Acids to Amorphous Silica Surfaces: Solid-State NMR of Glycine on SBA-15[END_REF] 3.3. Theoretical Investigations of Adsorption Geometries. Several combinations of H-bonds between glycine and the surface were investigated. We refer to our previous paper for the description of the model silica surface, [START_REF] Tielens | Ab Initio Study of the Hydroxylated Surface of Amorphous Silica: A Representative Model[END_REF] which exhibits vicinal and geminal silanols and their combinations into silanol nests. Using this model, we explore the possibility of cooperative H-bond formation between glycine, silanols, and water.

First, glycine interaction with two types of surface sites (vicinal, and silanol nest containing geminal silanol groups) was investigated. Then water was coadsorbed with glycine. The adsorption energies were calculated using eq 1.

Zwitterionic glycine (ZG) adsorption through one or two Hbonds with the silanols results in spontaneous neutralization to neutral glycine (NG), yielding adsorption energies of -55 and -62 kJ/mol (Figure 4a andb). It must be underlined that, in this configuration, the surface does not "solvate" the glycine molecule, in the sense that the interaction is not sufficient to stabilize the zwitterionic form with respect to the neutral form as happens, for example, in water, in conformity with previously calculated results. [START_REF] Costa | Microsolvation of Glycine by Silanol Ligands: A DFT Study[END_REF][START_REF] Lomenech | Theoretical and Experimental Study of the Adsorption of Neutral Glycine on Silica from the Gas Phase[END_REF] When glycine is allowed to make three Hbonds to the surface (Figure 4c,d), the ZG form is stabilized, although the energy of adsorption (-64 and -69 kJ/mol) is not significantly more negative than in the previous case. The rather weak interaction of glycine with vicinal silanols was confirmed with MD runs at 300 K. For all the previous structures, the glycine molecule spontaneously desorbs from the surface in its neutral form.

The same trend of neutralization was observed on the geminal silanol pair with glycine forming two H-bonds, with an energy of adsorption of -64 kJ/mol (Figure 5a). The geminal pair behaves similarly to the vicinal silanols, in agreement with previous results regarding water adsorption on silanols. [START_REF] Tielens | Ab Initio Study of the Hydroxylated Surface of Amorphous Silica: A Representative Model[END_REF] These results suggest that geminal silanols do not exhibit an increased acidity as compared to vicinal ones. Then, a minimum in the potential energy surface (glycine/silanol nest) was searched by means of molecular dynamics followed by geometry optimization. Figure 5b shows the most stable configuration obtained, with three H-bonds formed. The COO moiety is two times H-bond receiver from the geminal pair; in addition, the NH 3 moiety is one time H-bond donor to a vicinal silanol. Subsequently, the ZG form was stabilized and an adsorption energy of -111 kJ/mol was calculated, exhibiting a more exothermic adsorption than on vicinal silanols.

The most stable configuration in dehydrated conditions, (Figure 5b), shows the glycine molecule parallel to the surface with three H-bonds, an additional bond being formed between the NH 3 + moiety and a vicinal silanol. Moreover, a Si-O-H O-C-OH-O-Si-O cycle is formed. The energy of glycine adsorption is now -111 kJ/mol (148 kJ/mol vdW corrected). This energy of adsorption is much more exothermic than that calculated in other configurations. To better understand this result, the adsorption energy was decomposed into interaction and deformation energies as in eq 2.

Table 1 reports those energies for the most stable configurations obtained for glycine adsorbed on vicinal silanols and on a silanol nest. The H-bond network of silica has slightly changed in the presence of adsorbed glycine, with reorientations of H-bonds. The average deformation energy of the silica model is rather weak (38.5 ± 1 kJ/mol), which is not surprising since this solid is known to be very flexible. The average deformation energy of the glycine molecule is 68 ± 5 kJ/mol. Thus, deformation energies do not vary significantly between both configurations, and they cannot explain the differences in adsorption energies. It should be noted that the energy trends in Table 1 are not altered after inclusion of dispersion forces in a same set of model systems (i.e., the same number of atoms), as all energies are uniformly increased by about 30-50 kJ/mol depending on the degree of hydration. This correction is similar for both types of silanols, i.e. geminal and terminal. The most favorable adsorption configuration is found on geminal silanols, confirming our former result. [START_REF] Costa | DFT Study of the Adsorption of Microsolvated Glycine on a Hydrophilic Amorphous Silica Surface[END_REF] We performed the following additional calculations: for the most stable configurations obtained in each case (vicinal silanols/silanol nest), the atoms of glycine and the oxygens surrounding it were kept frozen. The other atoms of the structure were eliminated. Oxygen atoms were saturated with H atoms to form water molecules; those H atoms were optimized, converging into the structures shown in Figure 6. It was noticed that the interaction of glycine with the water clusters increases with the size and H-bond connectivity in the water cluster, from -53 to -114 kJ/mol. Indeed, water molecules help to screen the glycine dipole, and thus to stabilize the negative and positive charges of zwitterionic glycine. In other words, the high local silanol density, rather than the nature of the silanols, explains the increased (i.e., more negative) adsorption energy of glycine on silica. Such an increased reactivity of silanol nests was observed on other models (molecular and peri-odic). [START_REF] Tielens | What Do Vanadium Framework Sites Look Like in Redox Model Silicate Zeolites?[END_REF][START_REF] Wojtaszek | Gold Grafted to Mesoporous Silica Surfaces, a Molecular Picture[END_REF][START_REF] Wojtaszek | The Formation of Gold Clusters Supported on Mesoporous Silica Material Surfaces: A Molecular Picture[END_REF][START_REF] Islam | Characterization of Supported Vanadium Oxide Species on Silica: A Periodic DFT Investigation[END_REF][START_REF] Guesmi | Chromium Oxide Species Supported on Silica: A Representative Periodic DFT Model[END_REF][START_REF] Duchateau | Silsesquioxane Models for Silica Surface Silanol Sites with Adjacent Siloxide Functionalites and Olefin Polymerization Catalysts Thereof[END_REF][START_REF] Dijkstra | Silsesquioxane Models for Geminal Silica Surface Silanol Sites. A Spectroscopic Investigation of Different Types of Silanols[END_REF] In order to obtain a benchmark for the experimental NMR analysis showing the presence of a non-negligible amount of water, water molecules bridging glycine and the surface were considered, to calculate the corresponding NMR shifts (vide infra). This was achieved by adding three water molecules to the model system. Figure 7 shows the two most stable configurations obtained after MD followed by geometry optimization of the local minima. In both the silanol nest and vicinal silanol cases, the water molecules bridging the COO - moiety to the silanols are H-bond donors to COO -and Hbond acceptors from silanols. In contrast, water H-bond acceptors from NH 3 + are H-bond donor to silanols/siloxane oxygens. The energies of glycine+water coadsorption are -253 (-305) and -193 (-233) kJ/mol for silanol nest and vicinal silanols respectively, at the DFT (DFT-D) level. Again, we notice that the inclusion of van der Waals forces enhances the adsorption energy, by about 45 kJ/mol.

When only one water molecule is added to the model system as is the case in the last configuration, the glycine is found to interact directly with silanol groups through the carboxylate group whereas the ammonium group interacts via the water molecule. The adsorption energy obtained is -168 (-219) kJ/ mol (See Figure 8). We notice that the addition of one water molecule to the NH 3 + end is exothermic by -72 kJ/mol. Adding water molecules at the COO -end is exothermic by -43 kJ/mol per water molecule. Those values fall to -60 (one water molecule) and -31 (two more water molecules) kJ/mol, respectively, considering the ZPE differences between the gas phase water and water adsorbed on the surface. [START_REF] Hemeryck | Submitted to PhysChemChemPhys[END_REF] It seems thus that the addition of one water molecule at the ammonium end is more exothermic than at the carboxylate end. This may be due to the better screening of the charge of the proton in NH 3 + by an additional water molecule. Such a trend was already observed in a previous work. [START_REF] Costa | DFT Study of the Adsorption of Microsolvated Glycine on a Hydrophilic Amorphous Silica Surface[END_REF] Moreover, it was recently shown by molecular dynamics studies that the NH 3 + moiety does not interact directly with the surface silanols, whereas the carboxylate end does, at the silica-water interface. [START_REF] Zhao | An SCC-DFTB/MD Study of the Adsorption of Zwitterionic Glycine on a Geminal Hydroxylated Silica Surface in an Explicit Water Environment[END_REF][START_REF] Wright | Facet Selectivity of Binding on Quartz Surfaces: Free Energy Calculations of Amino-Acid Analogue Adsorption[END_REF] The results present a first order approximation for systems considering a real silica-water interface on which a molecular thermodynamical approach can be conducted in order to estimate the number of water molecules coadsorbed with glycine. [START_REF] Bouzoubaa | Insight of DFT and Atomistic Thermodynamics on the Adsorption and Insertion of Halides onto the Hydroxylated NiO(111) Surface[END_REF] This can be achieved by calculating free energies of adsorption from the liquid phase with an a posteriori treatment based on atomistic thermodynamics. In this approach chemical potentials of the reactants and products in the liquid phase are estimated. [START_REF] Hemeryck | Submitted to PhysChemChemPhys[END_REF] The free energies of glycine and water coadsorption are estimated to -16 and +12 kJ/mol in the case of addition of one water molecule at the NH 3 + end, and addition of two water molecules at the carboxylate end, respectively. To summarize, the interaction of glycine with amorphous silica is predicted to occur via its ammonium group through one water molecule, whereas a direct interaction via the carboxylate group with the surface is favored.

It is interesting to notice that Ben Shir et al. [START_REF] Ben Shir | Binding Specificity of Amino Acids to Amorphous Silica Surfaces: Solid-State NMR of Glycine on SBA-15[END_REF] concluded from NMR experiments that the carboxylate group is mobile and does not interact with the silica surface, while the ammonium group is tightly bond via three hydrogen bonds to the silanols of the silica surface. They described their rate of hydration to be intermediate, whereas in relatively dry conditions the carboxylate group is energetically predicted to be adsorbed on the surface. The theoretical model presented in this work, aimed to describe low hydration rates (microsolvation), might not be hydrated enough to represent the experimental adsorption conditions. The presence of water around the glycine molecule and on the silica surface might generate entropically favored configurations, ruling over the enthalpically favored ones, a situation already proposed in the interaction of a phospholipid on a silica surface. [START_REF] Folliet | Investigation of the Interface in Silica-Encapsulated Liposomes by Combining Solid State NMR and First Principles Calculations[END_REF] The present results point out that (i) geminal silanols "per se" do not have an increased reactivity in comparison to vicinal silanols; (ii) instead they participate in creating a "silanol nest", a local region of high silanol density, which exhibits an increased reactivity toward glycine; (iii) glycine adsorption on a "silanol nest" is significantly more exothermic, than on vicinal silanols; (iv) this result is valid also when water is coadsorbed with glycine, and (v) water coadsorbs preferentially at the NH 3 + moiety of glycine, in agreement with previous studies. [START_REF] Costa | DFT Study of the Adsorption of Microsolvated Glycine on a Hydrophilic Amorphous Silica Surface[END_REF][START_REF] Zhao | An SCC-DFTB/MD Study of the Adsorption of Zwitterionic Glycine on a Geminal Hydroxylated Silica Surface in an Explicit Water Environment[END_REF] 3.4. NMR Calculations. Calculations of NMR parameters were performed on four configurations of glycine adsorption on the surface (Figure 9): (i) Glycine carboxylate forming two Hbonds with two geminal silanols (config I, Figure 5b); (ii) glycine carboxylate forming two H-bonds with two vicinal silanols (config II, Figure 4c); (iii) glycine carboxylate forming two H-bonds with two geminal silanols via two interfacial water molecules (config III, Figure 7a); (iv) glycine carboxylate forming two H-bonds with two vicinal silanols via two interfacial water molecules (config IV, Figure 7b).

The corresponding calculated 1 H, [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C, and [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N NMR chemical shift values are summarized in Table 2. [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N chemical shift values calculated for the different adsorption configurations (shown in Figure 9) were positioned on a theoretical two-dimensional map, in regard with the experimental [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N spectra (Figure 10). It must be underlined that this figure is not an experimental 2D NMR map. The calculated chemical shifts of the three polymorphs of glycine are also shown as references. It appears that all adsorption configurations yield NMR parameters consistent with the experimental signals previously assigned to glycine in interaction with the silica surface, and separate from the parameters of bulk glycine (at least along the [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C chemical shift dimension), giving confidence in the used methodology. Additional data are necessary to determine whether one of the various models is more relevant than the others from the NMR point of view. Therefore, attention was then focused on the calculated 1 H chemical shifts of ammonium protons of glycine and on silanol/water protons interacting with glycine (Figure 11). Apart from configuration III, the -NH 3 + protons show chemical shifts smaller than in glycine polymorphs [START_REF] Dijkstra | Silsesquioxane Models for Geminal Silica Surface Silanol Sites. A Spectroscopic Investigation of Different Types of Silanols[END_REF] (8.0 ppm for αand γ-glycine, and 8.9 ppm for β-glycine), in good agreement with the experimental value of 6.4 ppm extracted from the 1 H- [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N HETCOR CP MAS NMR experiment. For configurations I and III, the δ iso ( 1 H) of silanols H-bonded to the carboxyl oxygens of glycine are very high (between 9.4 and 9.7 ppm) which is most probably related to very short OH-O distances (between 1.56 and 1.66 Å). Indeed, a correlation between proton chemical shift of silanols and the H-bond network has been reported previously. [START_REF] Tielens | Ab Initio Study of the Hydroxylated Surface of Amorphous Silica: A Representative Model[END_REF] For the vicinal configurations, δ iso ( 1 H) of silanols or water molecules Hbonded to the COO-group are smaller (between 5.1 and 6.5 ppm) and the corresponding OH-O distances are longer (between 1.70 and 1.74 Å). The latter values are in better agreement with the experimental values extracted from the 1 H- [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and 1 H- [START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si HETCOR CP MAS NMR experiment.

All in all, the best agreement between experimental and calculated NMR data is obtained for configurations II and IV, S4). Nonetheless, considering the accuracy of experimental results, all calculated NMR shifts fall within the experimental range suggesting that a distribution of adsorption configurations cannot be excluded.

CONCLUSIONS

DFT-D calculations have been performed on a model amorphous silica surface, in order to help interpreting experimental NMR data. Several typical configurations were considered, where glycine was adsorbed on vicinal or geminal silanols, without and with water coadsorption. [START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and [START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N NMR signal corresponding to the relatively rigid glycine molecules interacting with the silica surface could be clearly identified by combining CP experiments and NMR calculations on the models. Moreover, it appears that the best site for adsorption configuration was the silanol nest, and that water coadsorption was favored, especially concerning the ammonium entity. Calculated NMR shifts fall into the experimental range with a slightly better agreement for adsorption configurations on vicinal silanols, which are present in majority on this silica. However, contributions from zones of high local density of silanols cannot be excluded. Finally, we notice that our representative silica model exposes sites of very different reactivity, and is thus useful to explore different possibilities of adsorption, at low computational cost. Comparison with experimental data may be done by extracting data on particular sites that are known to be present on real surfaces. Obviously, our results are more indicative of tendencies than precise assignments. In particular, the electronic energy of the system is not the only criteria to discriminate between different adsorption scenarios. One might describe a very stable site and adsorption configuration on the basis of its energy but, due to its small probability of presence on the surface (as for example geminal silanols in MCM-41 sample), it may not be the most representative adsorption situation. The presence of the surface sites is dependent on the synthesis protocol of the silica material. Another unknown condition of adsorption is the accessibility of the surface site for the glycine molecule. The adsorbent may be blocked by steric hindrance. The role of the solvent is also a parameter to be considered, and will be the subject of future works.

■ ASSOCIATED CONTENT

* S Supporting Information

■ AUTHOR INFORMATION

Corresponding Author *E-mail: christel.gervais_stary@upmc.fr (C.G.); frederik. tielens@upmc.fr (F.T.).

Notes

The authors declare no competing financial interest.

■

  ACKNOWLEDGMENTSThis work was performed using HPC resources from GENCI-[CCRT/CINES/IDRIS] (Grant 2012-[x2012082022]) and the CCRE of UniversitéPierre et Marie Curie. N.F. benefitted from a Ph.D. grant from the CNano program of the Ile-de-France region. The "Bonus QualitéRecherche" (BQR) 2008 program of UniversitéPierre et Marie Curie is gratefully acknowledged for financing the purchasing of the isotopically enriched molecules.

Figure 1 .

 1 Figure 1. 13 C CP MAS NMR spectra of the glycine polymorphs (αglycine, blue; β-glycine, green; γ-glycine, red) and Gly/MCM (black line).

Figure 2 .

 2 Figure2. HETCOR CP MAS NMR spectra: (a)[START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C-{ 1 H}, (b)[START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N-{ 1 H}, and (c)[START_REF] Rimola | Does Adsorption at Hydroxyapatite Surfaces Induce Peptide Folding? Insights from Large-Scale B3LYP Calculations[END_REF] Si-{ 1 H} of Gly/MCM.[START_REF] Cauvel | Hydrophobic and Hydrophilic Behavior of Micelle-Templated Mesoporous Silica[END_REF] H projections for specific[START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C,[START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N, or 29 Si chemical shifts are presented on the right side of the figure.

Figure 3 .

 3 Figure 3. 15 N CP MAS NMR spectra of the glycine polymorphs: αglycine (blue line), β-glycine (green line), γ-glycine (red line), and glycine encapsulated in mesoporous silica (black line).

Figure 4 .

 4 Figure 4. Configurations obtained for glycine in interaction with a pair of vicinal silanols, forming (a) one H-bond, (b) two H-bonds, and (c,d) three H-bonds with the surface.

Figure 5 .

 5 Figure 5. Configurations obtained for glycine in interaction with geminal silanols, forming two H-bonds from the COO -end with the geminal pair (a), and an additional bond from the NH 3 + end with a more distant silanol (b).

Figure 6 .

 6 Figure 6. Configurations corresponding to zwitterionic glycine on silica (Figures 4e and 5b), but where the SiOH have been replaced by water molecules, the O atoms being fixed and the "healing" H atoms relaxed. The stabilization energy of glycine in those water clusters is given in kJ/mol.

Figure 7 .

 7 Figure 7. Optimized geometries of glycine coadsorbed with three water molecules on silica (a) on geminal silanols and (b) on a pair of vicinal silanols. (Dispersion corrected energies in parentheses.)

Figure 8 .Figure 9 .

 89 Figure 8. Optimized geometries of glycine coadsorbed with 1 water molecule on silica. (Dispersion corrected energies in parentheses.)

Figure 10 .

 10 Figure10. Theoretical two-dimensional map correlating[START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and[START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N spectra of the encapsulated glycine and positioning calculated[START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and[START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N chemical shift values for the different adsorption configurations and for glycine polymorphs.

Figure 11 .

 11 Figure 11. Calculated 1 H NMR parameters of silanols/water molecules in interaction with the carboxylate for various configurations of glycine adsorption on the silica surface: (a) glycine carboxylate in interaction with geminal silanols, (b) glycine carboxylate in interaction with vicinal silanols, (c) glycine carboxylate in interaction with geminal silanols in the presence of interfacial water, and (d) glycine carboxylate in interaction with vicinal silanols in the presence of interfacial water.

Table 1 .

 1 Adsorption Energies of Glycine (G) on Different Types of Silanols and Interaction Energy of Glycine with Water Clusters of Identical Geometry, Calculated at the DFT (DFT-D) Levels Following eqs 1 and 2 a

				vicinal			geminal
			NG	NG	ZG	ZG	NG	ZG
	dry	Figure	4a	4b	4c	4d	5a	5b
		# H-Bonds	1	2	3	3	2	3
		ΔE ads	-55	-62	-64	-69	-64	-111
					(-96)			(-148)
								-142 10
	1 water molecule	Figure						8
		# H-Bonds						3 + 2 water
		ΔE ads						-168
								(-219)
	3 water molecules	Figure					7a	7b
		# H-Bonds					8	8
		ΔE ads					-193	-253
							(-233)	(-304)

a Values in kJ/mol.

Table 2 .

 2 Comparison between Experimental and Calculated NMR 1 H,[START_REF] Rimola | Affinity Scale for the Interaction of Amino Acids with Silica Surfaces[END_REF] C and[START_REF] Irrera | DFT Periodic Study of Adsorption of Glycine on the (0001) Surface of Zinc Terminated Zno[END_REF] N Chemical Shift Values for Various Adsorption Configurations of Glycine on the Silica Surface

	δ iso (ppm)

  Table containing porosity characteristics for loaded and unloaded MCM-41; additonal figures showing nitrogen adsorption-desorption isotherms and NMR spectra of MCM-41 samples. This material is available free of charge via the Internet at http://pubs.acs.org.