Protonation–deprotonation of the glycine backbone as followed by Raman scattering and multiconformational analysis Belén Hernández a , Fernando Pflüger a , Sergei G. Kruglik b , Mahmoud Ghomi a,⇑ a Groupe de Biophysique Moléculaire, UFR Santé-Médecine-Biologie Humaine, Université Paris 13, Sorbonne Paris Cité, 74 rue Marcel Cachin, 93017 Bobigny cedex, France b Laboratoire Jean Perrin, FRE 3231, Université Pierre et Marie Curie (Paris 6), Case courrier 138, 75252 Paris Cedex 05, France article info Article history: Received 13 December 2012 In final form 14 August 2013 Available online 29 August 2013 Keywords: Glycine Protonation–deprotonation Raman spectra Multiconformational approach abstract Because of the absence of the side chain in its chemical structure and its well defined Raman spectra, gly- cine was selected here to follow its backbone protonation–deprotonation. The scan of the recorded spec- tra in the 1800–300 cm 1 region led us to assign those obtained at pH 1, 6 and 12 to the cationic, zwitterionic and anionic species, respectively. These data complete well those previously published by Bykov et al. (2008) [16] devoted to the high wavenumber Raman spectra (>2500 cm 1 ). To reinforce our discussion, DFT calculations were carried out on the clusters of glycine + 5H 2 O, mimicking reasonably the first hydration shell of the amino acid. Geometry optimization of 141 initial clusters, reflecting plau- sible combinations of the backbone torsion angles, allowed exploration of the conformational features, as well as construction of the theoretical Raman spectra by considering the most stable clusters containing each glycine species. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Glycine (Gly) is the only achiral a-amino acids (a-AAs), because of the absence of a side chain in its chemical composition (Scheme 1). Thanks to its structural simplicity, Gly has been often used as a simple model compound of a-AAs in numerous structural and vibrational studies in solid, liquid and gas phases [1–24]. Par- ticularly, the aqueous solution analysis of this AA has been pro- moted by its remarkable water solubility (251 g/L at 25 °C) [25]. Although the Gly backbone adopts a zwitterionic form in the conditions close to the physiological ones, it can however fol- low a protonation–deprotonation process upon increasing/ decreasing pH [25]. More precisely, taking into account the two known pK a values of the Gly backbone [25], namely pK 1 (2.34) and pK 2 (9.58), three different forms corresponding to the cationic (N t H þ 3 =C t OOH), zwitterionic (N t H þ 3 =C t OO  ) and anionic (N t H 2 =C t OO  ) species, as shown in Scheme 1, are expected in aque- ous solutions. H-bond networks of the Gly backbone with surrounding water molecules are stabilized by the rotation of the backbone terminal groups with respect to the central N t –C a –C t plane. As a consequence, the variation of the two torsion angles, re- ferred to as u and w (Scheme 1), permits the analysis of water rear- rangement around the AA backbone. Raman scattering is considered as an efficient tool for probing the characteristic vibrational modes which are sensitive to proton transfer, hydrogen bonding, as well as to conformational transi- tions. Furthermore, the joint use of this spectroscopic technique with quantum mechanical (QM) approaches can bring interesting information on the interaction of water molecules with the polar groups of an organic molecule such as an AA. As far as the applica- tion of this combined protocol to Gly is concerned, in a previous study, Bykov et al. [16] have reported the high wavenumber region Raman spectra (above 2500 cm 1 ) by focusing particularly on the C a H 2 bond stretching modes and their coupling with the backbone vibrational motions. To achieve this goal, deuterated Gly samples were studied as a function of pD, and the observed Raman shifts were analyzed on the basis of the DFT calculations with an implicit hydration model, i.e. a polarizable continuum. The effect of the backbone conformation (especially w angle) on the C a H 2 bond stretching modes could thus be fully discussed. More recently, Vyas et al. [22] have presented the simulation of the Raman spec- trum of the zwitterionic form in the 1800–400 cm 1 spectral re- gion, by means of a mono-conformation approach based on the clusters containing 1–5 water molecules. Finally, in a preliminary report from our group, aqueous solution (H 2 O and D 2 O) Raman and FT-IR spectra of the zwitterionic form were reported in the middle wavenumber spectral region along with the theoretical assignments derived from a mono-cluster/mono-conformer ap- proach (Gly + 12H 2 O) [15]. It should be emphasized that the experimental and theoretical analyses of Gly in hydrated media constitutes an important topic in the framework of the physicochemical properties of amino acids. For instance, the number of water molecules stabilizing a 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.08.008 ⇑ Corresponding author. Tel.: +33 1 48388927; fax: +33 1 48388928. E-mail address: mahmoud.ghomi@univ-paris13.fr (M. Ghomi). Chemical Physics 425 (2013) 104–113 Contents lists available at ScienceDirect Chemical Physics journal homepage: www.elsevier.com/locate/chemphys