The Adsorption of Neutral Glycine Molecules on Ice Nanolayers∗

We report first results about the ability of low density amorphous ice nanolayers to behave as a “matrix” and trap monomeric neutral glycine molecules at 125 K, a temperature much greater than the commonly used liquid He temperature region. FTIR-RAS spectra of those monomeric neutral glycine molecules adsorbed on low dense amorphous ice were obtained for the first time and indicate that glycine adsorbs molecularly with the carboxylic group and the nitrogen atom hydrogen-bonded to the ice surface. [DOI: 10.1380/ejssnt.2009.693]


I. INTRODUCTION
An atomic scale understanding of the way how proteins interact with real surfaces is far from being totally explained.A reason for that status is the complexity of both systems: proteins and of real surfaces.For instance it is well known that the conformational characteristics of proteins strongly depend on one hand on the type of interactions they have with their surrounding environment and on the other hand on the complex interactions among side chains of amino acids, which dictates their chemical properties.That is why, investigating smaller biomolecules such as amino acids (protein's building blocks) and how they adsorb onto surfaces opens up a way to understanding the behaviour of larger biomolecules such as the protein molecules [1,2].Moreover, it has also been discovered that amino acids may have played a role in the early evolution of life on our planet [3,4].
Netzer and co-workers [17,18] studied the interaction of glycine with ice nanolayers, by means of temperatureprogrammed desorption (TPD), x-ray photoelectron spectroscopy, and work function measurements.It was found that low coverages of adsorbed glycine molecules on amorphous ice surfaces suppress the amorphous-to-crystalline phase transition in the temperature range 140-160 K and in near-surface regions the desorption of water is signif-icantly influenced by the presence of glycine molecules, whereas the TPD of glycine is unaffected.
So far only a few techniques have been providing access to the neutral form of amino acid molecules like glycine.Among them, matrix isolation technique was the only successful method for accurately trapping monomeric neutral amino acids [21,[31][32][33][34].
Owing to the usually strong correlation between infrared spectra and structure, and to the potentiality of the ice surface, which enables to bind molecules, we have undertaken in this work the investigation of individual neutral glycine (NH 2 CH 2 COOH) molecules adsorption onto low-density amorphous LDA ice nanolayers.
This is an ideal model system because, it constitutes a first step in understanding the adsorption of proteins, the constituent of the so-called primary film, which enables the further growth of biofilms [35].We present the first study aiming at the IRAS spectral features of the neutral form of glycine deposited onto LDA ice nanolayers.

II. EXPERIMENTAL
Details of the experimental setup and procedure have been discussed elsewhere [36].Briefly, the FTIR-RAS experiments were performed in a UHV chamber with a base pressure better than 5.0×10 −10 Torr to which vapors of water (Millipore) can be introduced through a home made spray-on nozzle dosing system [37] after further purification via several freeze-pump-thaw cycles.
The polycrystalline OFHC copper disk serving as substrate was cleaned before water vapor deposition by sputtering with argon ions.Glycine deposition was carried out by exposing the LDA ice surface to the vapor of glycine powder (¿99.5% purity, from Aldrich Co.).The glycine powder (with a melting point of 182 • C) was held in a newly developed cryogenic dosing system for the UHV deposition of organic materials like amino acids and DNA base pairs [38].The vacuum chamber contained a quadrupole mass spectrometer (Stanford Research Systems SRS RGA 100) which enabled the mass fragments to be monitored during deposition.
Prior to deposition, the glycine was carefully outgassed for several hours at a temperature of ∼400 K, with the shutter of the molecular beam doser closed.
The minimal temperature at which the flow of glycine was found sufficient for the sample preparation was ∼446 K (173 • C); the corresponding average partial pressure of the 30 amu mass fragment of glycine (NH 2 CH + 2 ) was in the range of 2.4-5×10 −9 Torr during the experiments.A Fourier transform infrared spectrometer (Bruker Equinox 55) was used for IRAS in the reflection mode (the IR beam specularly reflected off the sample at 83 • from the surface normal) with a mercury cadmium telluride detector (MCT).FTIR-RA spectra of the deposited sample were obtained over the spectral range of 620-4000 cm −1 by coadding 250 scans at 40 kHz scanner frequency at a preset spectral resolution of 4 cm −1 .The reference was the reflected intensity of the copper sample directly before exposure obtained by coadding 500 scans at the same resolution setting.The exposure is given in langmuirs (1 L = 1×10 −6 Torr.s).

III. RESULTS AND DISCUSSIONS
A. Preparation of LDA ice at 125 K Ice nanolayers (17 nm) have been grown on a polycrystalline copper substrate at 125 K under controlled ultrahigh vacuum conditions (UHV) to generate clean LDA ice surfaces.
The IRAS spectrum (Fig. 1) exhibits a strong broad band at around 3394 cm −1 , a strong peak at 898 cm −1 (libration mode), a feature at ∼1656 cm −1 , a weaker band at ∼2240 cm −1 and a small sharp peak at 3696 cm −1 .The condensed water film grown at 125 K can be characterized as amorphous dense ice [39,40].
The OH stretching mode of water molecules that are fully (four-coordinated) involved in hydrogen bonding gives rise to the strong broad peak, while the small sharp peak is due to OH stretching modes in water molecules that have an OH group not involved in hydrogen bonding.H 2 O molecules giving rise to this 'free-OH' vibration at 3696 cm −1 have been characterized by Buch and Devlin using spectroscopic and computational methods.They concluded that the 'free-OH' band at 3696 cm −1 comes from three-coordinated water molecules in amorphous ice (with two hydrogen bonds via O, and one via H) [40].
The features near 1656 and 2240 cm −1 @have been attributed to the OH bending band possibly coupled with the first overtone of the hindered rotational lattice mode (898 cm −1 ) and to the combination band of the OH bending and hindered rotational mode [41,42].The estimation of the ice thickness has been obtained by spectral simulation of the experimental reflection absorption spectra using the "three layer model" [43].The assignments are based on a comparison with infrared data available in the literature from matrixisolation technique experiments of neutral monomeric glycine [31] and are summarized in Table I and Table II.Furthermore, in view of differences which exist in the literature concerning some of the assignments in the fingerprint region, we arbitrarily choose the most accurate one from Fausto [31], as already done in Ref. [8].
The broad peak observed in the interval 3015-3530 cm −1 comes mainly from both readsorbed water-ice from the gas phase and from the asymmetric (3324 cm −1 ) and symmetric streching modes (3242 cm −1 ) of NH 2 [21].These NH 2 vibration modes are convoluted in the broad peak.
The NH 2 functional group exhibits also an extremely weak scissoring NH 2 mode at 1636 cm −1 and a strong wagging vibration at 882 cm −1 .
We observe both the asymmetric (2980 cm −1 ) and symmetric (2896 cm −1 ) streching modes of the CH 2 side group of glycine.The peaks observed at 1385 cm −1 and 1442 cm −1 represent the contributions of the CH 2 wagging mode ω(CH 2 ) and of the CH 2 scissoring mode δCH 2 ), respectively.
Finally, the peak at 1720-1740 cm −1 can be attributed to C=O stretching motion of neutral glycine hydrogenbonded to the ice surface [6,44,45] TABLE I: Spectra assignment for the neutral form of glycine.ν: stretching, δ: bending, ω: wagging, ρ: rocking, tw: twisting, t: torsion, s: symmetric, and as: asymmetric, respectively.Band Assignement Fausto [31] Kikuchi [30]  The distinct feature observed at 3696 cm −1 and ascribed to the dangling-H of three coordinated surface water molecules in LDA ice [40] is observed with a negative value.This indicates that an interaction of the glycine molecules with the d-H sites of LDA ice has occured.
The observed values confirm that the deposited glycine molecules are in their neutral form.Moreover the band positions and shapes are different from those obtained for multilayers of glycine deposited on Cu at 131 K (Fig. 3(A)) and from those obtained previously by Barlow et al. [6], Gomez-Zavaglia et al. [21] and Swiderek et al. [8].For those multilayers of glycine prepared at low temperatures, it is well known that their infrared spectra show the presence of neutral carboxylic acid-like glycine dimers with contribution of zwitterionic glycine.On the contrary, our data perfectly agree with the infrared data of neutral monomeric glycine molecules obtained by matrix-isolation technique [31][32][33][34].The conclusion which can be drawn from these data is that the deposited glycine molecules do not have internal hydrogen bond [31,32], and are not involved in hydrogen bonded dimers [21].

C. Discussion
In the IRAS difference spectrum we note the absence of the strong and broad absorption band between 3200 and 2300 cm −1 , which is found in the spectrum of crystalline zwitterionic glycine (Fig. 3(B)) [20,31].This very broad absorption appears for most amino acids and is characteristic of the intermolecular hydrogen bonding NH + 3 . . .− OOC in the crystalline (zwitterionic) state.Another convincing evidence for a non-zwitterionic structure of glycine is the absence of the band at 1525 cm −1 , which Thiam and Ebrahimi TABLE II: Spectra assignment for the zwitterionic form of glycine.ν: stretching, δ: bending, ω: wagging, ρ: rocking, tw: twisting, t: torsion, s: symmetric, and as: asymmetric, respectively.Band Assignement Fausto [31] Machida [29]   corresponds to the symmetric NH + 3 bending mode [20,21].Given the strong intensity of ν as (CH 2 ) compared to the weak ν s (CH 2 ), we can deduce that the direction of the dipole moment for the asymmetric stretch is almost perpendicular to the ice surface; see Fig. 4.
The observation of a weak but discernable wagging mode (ω(CH 2 ) = 1385 cm −1 ) and of a scissoring mode (δCH 2 ) =1442 cm −1 ) with a very small amplitude supports the idea that the molecular plane of the CH 2 group lays tilted with respect to the ice surface.
The NH 2 functional group exhibits also an extremely weak scissoring NH 2 mode at 1636 cm −1 and a strong wagging vibration at 882 cm −1 , superposed to the libration mode of LDA ice at ∼898 cm −1 .This indicates that, the NH 2 group has such an orientation that the plane con-taining the two hydrogen and the nitrogen atoms as well as the axis passing through the two hydrogen atoms lay tilted with respect to the ice surface.
It implies that, the NH 2 group is bound to the ice surface with the nitrogen atom bound to a dangling-H; see Fig.  tively).This finding can be considered as a convincing indication that hydrogen bonding has occurred between C-O-H and a dangling-O on one hand and between C-N and a dangling-H on the other hand, as can be seen in Fig. 4.
Finally, the peak at 1720 and 1740 cm −1 can be attributed to the C=O stretching motion of glycine interacting with some dangling-H at the ice surface, it is shifted to lower wavenumbers compared to the gas-phase value of 1774 cm −1 and to Ar matrix isolation value of 1781 cm −1 [21,[31][32][33][34]46].The observation of this absorption band is a clear evidence that glycine adsorbs molecularly on LDA ice.

IV. CONCLUSIONS
We have presented the first study aiming at the IRAS spectral features of the neutral form of individual glycine molecules deposited onto LDA ice nanolayers.Our results demonstrate the viability of using ultrathin films of ice to mimick water in biological environment.Of primary concern in these studies was the nature (neutral form, zwitterionic, cationic or anionic) of glycine adsorption on LDA ice as well as its orientation and structure.We found that the most favorable bonding of glycine with the LDA ice surface occurs through the COOH and NH 2 moieties, forming a cycle in which the CO group is an Hbond acceptor whereas the acidic OH group is an H-bond donor and the N atom an H-bond acceptor.

FIG. 1 :
FIG.1: Infrared spectrum of a 17 nm amorphous LDA ice film prepared at 125 K and water pressure of 1.4×10 −8 Torr.The Spectrum was obtained with FTIRAS.

B.Figure 2
Figure2is an IRAS difference spectra of ice films covered with 0.4 L of glycine, where the initial clean ice film served as a background.Initially, at an exposure of 0.4 L, adsorbate-induced features come to full evidence by displaying five intense vibrational modes at 882, 1052, 1096, 2980 and 3696 cm −1 , and six weak bands at 1281, 1342, 1385, 1442, 1463 and 2896 cm −1 .These vibrational modes are accompanied by three broad peaks (725-1025 cm −1 ), (1652-1780 cm −1 ) and (3015-3530 cm −1 ).The assignments are based on a comparison with infrared data available in the literature from matrixisolation technique experiments of neutral monomeric glycine[31] and are summarized in TableIand TableII.Furthermore, in view of differences which exist in the literature concerning some of the assignments in the fingerprint region, we arbitrarily choose the most accurate one from Fausto[31], as already done in Ref.[8].The broad peak observed in the interval 3015-3530 cm −1 comes mainly from both readsorbed water-ice from the gas phase and from the asymmetric (3324 cm −1 ) and symmetric streching modes (3242 cm −1 ) of NH 2[21].These NH 2 vibration modes are convoluted in the broad peak.The NH 2 functional group exhibits also an extremely weak scissoring NH 2 mode at 1636 cm −1 and a strong wagging vibration at 882 cm −1 .We observe both the asymmetric (2980 cm −1 ) and symmetric (2896 cm −1 ) streching modes of the CH 2 side group of glycine.The peaks observed at 1385 cm −1 and 1442 cm −1 represent the contributions of the CH 2 wagging mode ω(CH 2 ) and of the CH 2 scissoring mode δCH 2 ), respectively.Two other peaks, observed at 1052 cm −1 and at 1096 cm −1 , are assigned to ν(C-O) and ν(C-N), respectively.Finally, the peak at 1720-1740 cm −1 can be attributed to C=O stretching motion of neutral glycine hydrogenbonded to the ice surface[6,44,45].
Figure2is an IRAS difference spectra of ice films covered with 0.4 L of glycine, where the initial clean ice film served as a background.Initially, at an exposure of 0.4 L, adsorbate-induced features come to full evidence by displaying five intense vibrational modes at 882, 1052, 1096, 2980 and 3696 cm −1 , and six weak bands at 1281, 1342, 1385, 1442, 1463 and 2896 cm −1 .These vibrational modes are accompanied by three broad peaks (725-1025 cm −1 ), (1652-1780 cm −1 ) and (3015-3530 cm −1 ).The assignments are based on a comparison with infrared data available in the literature from matrixisolation technique experiments of neutral monomeric glycine[31] and are summarized in TableIand TableII.Furthermore, in view of differences which exist in the literature concerning some of the assignments in the fingerprint region, we arbitrarily choose the most accurate one from Fausto[31], as already done in Ref.[8].The broad peak observed in the interval 3015-3530 cm −1 comes mainly from both readsorbed water-ice from the gas phase and from the asymmetric (3324 cm −1 ) and symmetric streching modes (3242 cm −1 ) of NH 2[21].These NH 2 vibration modes are convoluted in the broad peak.The NH 2 functional group exhibits also an extremely weak scissoring NH 2 mode at 1636 cm −1 and a strong wagging vibration at 882 cm −1 .We observe both the asymmetric (2980 cm −1 ) and symmetric (2896 cm −1 ) streching modes of the CH 2 side group of glycine.The peaks observed at 1385 cm −1 and 1442 cm −1 represent the contributions of the CH 2 wagging mode ω(CH 2 ) and of the CH 2 scissoring mode δCH 2 ), respectively.Two other peaks, observed at 1052 cm −1 and at 1096 cm −1 , are assigned to ν(C-O) and ν(C-N), respectively.Finally, the peak at 1720-1740 cm −1 can be attributed to C=O stretching motion of neutral glycine hydrogenbonded to the ice surface[6,44,45].

4
FIG. 4: Orientation of glycine molecule adsorbed on low dense amorphous ice nanolayers at 125 K.