LETTER pubs.acs.org/JPCL
Unconventional Zwitterionic State of L-Cysteine E. Ataman,† C. Isvoranu,† J. N. Andersen,†,‡ J. Schnadt,*,† and K. Schulte*,‡ † ‡
Division of Synchrotron Radiation, Department of Physics, Lund University, Box 118, 22100 Lund, Sweden MAX-lab, Lund University, Box 118, 22100 Lund, Sweden
bS Supporting Information ABSTRACT: We report the signature of an unconventional zwitterion in thick (>40 Å) L-cysteine films grown at low temperature (100 K) by means of sublimation in ultrahigh vacuum. Using X-ray photoelectron spectroscopy (XPS) we find that protonated amino groups (57%) are less prevalent than in films grown at higher temperatures. A large majority of the protonated amino groups receive the hydrogen through deprotonation of the thiol group (45%) and, in contrast, only a small fraction of the carboxylic groups (13%) is deprotonated. This shows, for the first time, the unconventional zwitterion to be the dominant species in L-cysteine. We believe the unusual growth parameters are responsible for this novel observation. SECTION: Kinetics, Spectroscopy
A
mino acids form a fascinating class of small biological molecules with potent functional groups. In addition to the amino and carboxylic acid groups, other chemically relevant side groups (such as OH, SH, and aromatic rings) may influence the properties of the molecules. In structural biochemistry, the 20 universal amino acids form the building blocks of an endless variety of proteins, as their functional groups allow them to link to one another aided by the easy formation of hydrogen bonds. The different moieties can act either as acceptors or donors, which is also important in ligand coordination, as used in enzyme activity. It is therefore not surprising that these molecules have also stirred an interest in the fields of surface science and nanotechnology, where molecular self-organization and chemical surface modification play an important role. L-Cysteine (HS CH2 CH(NH2) COOH, Figure 1, top) offers a high degree of chemical complexity due to the presence of three different functional groups. Further complexity is added by the fact that it can assume both the neutral and zwitterionic forms. In the gas phase, the nonionic form is the most stable form of L-cysteine,1,2 while both in aqueous solution and in the solid state it is found in the usual zwitterionic state with the carboxylic group deprotonated in favor of a protonated amino group3 5 (see Figure 1, left). Recently, Tian and co-workers presented hydrogen deuterium exchange and gas phase acidity measurements alongside density functional theory (DFT) calculations, which indicate that, surprisingly, the standard enthalpy of acid dissociation involving the thiol group is 3.1 kcal/mol lower than that involving the carboxylic group, i.e., the thiol group is more acidic than the carboxylic group.6 Deprotonation of L-cysteine would therefore most likely lead to a thiolate ion in the gas phase. The further formation of a zwitterionic anion (i.e., with the carboxylic r 2011 American Chemical Society
and thiol groups deprotonated and the amino group protonated), would cost an extra 10.1 kcal/mol and is therefore deemed unfeasible in the gas phase, whereas Fernandez-Ramos et al. found a stable anionic form (without protonating the amino group). In aqueous solution, this anionic form as well as both zwitterionic forms gain stability.1 Similarly, Woo et al. combined DFT calculations and laser-induced ionization photoelectron spectroscopy of cysteine anions sprayed from solution and found a hydrogenbonded thiolate anion [ S CH2 CH(NH2) COOH] at 70 K to be the most prevalent form. The intramolecular O H 3 3 3 S hydrogen-bond strength in cysteine thiolate anion was estimated experimentally as 16.4 kcal/mol.7 In contrast to this, Oomens et al. obtained the carboxylate anion at room temperature, using gas-phase IR spectroscopy on electrosprayed cysteine molecules.8 In the solid state, two polymorphs have been found for crystals at adiabatic pressure,4,9 11 and phase transitions at 70 K (orthorhombic)12 and 150 K (monoclinic)13 have been observed. Both transitions are subtle and smeared out over a larger temperature range. The use of different experimental methods has shown that a series of small changes in dihedral angles involving, among others, the CH2SH side chain can affect the hydrogen bonding network and hence the crystal structure.13,14 The movement of different fragments of the molecule is activated at different temperatures, which makes an abrupt change unfeasible. Moreover, a dependence on the thermal history of the crystals and on the heating/cooling velocity during these experiments is also reported. In orthorhombic L-cysteine at room temperature, the thiol group is disordered, participating in both Received: May 23, 2011 Accepted: June 23, 2011 Published: June 23, 2011 1677
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Figure 1. Schematic drawings of the neutral and zwitterionic forms of L-cysteine, representing stable conformers in solution (see ref 1). The unconventional zwitterionic form has a deprotonated thiol, protonated amino, and an intact, protonated carboxylic group. Likely intramolecular hydrogen bonding is represented by thin dotted lines.
S H 3 3 3 O and S H 3 3 3 S hydrogen bonds, and it only freezes out at low temperatures where the sulfur hydrogen bonds remain.4 The opposite appears to be the case for the monoclinic form, where one of the two molecules of the unit cell prefers S H 3 3 3 O bonding, whereas the other is involved in S H 3 3 3 S bonding and disorder is not reported.11,13 Most information on the electronic configuration of the L-cysteine molecules in the solid state have been obtained through diffraction studies, aided by modeling calculations. Locating the atoms and their relative distances to each other has afforded the determination of hydrogen bonds and changes in conformational angles of the molecular backbone. Similarly, recording IR or Raman spectra and comparing them to a database of characteristic vibrational frequencies allows for an analysis of the motion of the individual side groups as a function of temperature, signaling the onset of phase transitions and their driving forces. Alternatively, in X-ray photoelectron spectroscopy (XPS), the characteristic elemental core levels are influenced by the chemical surrounding of the atom and can therefore also indicate the presence of hydrogen bonds and zwitterions. For cysteamine (HS CH2 CH2 NH2), a sister molecule to L-cysteine, but with the carboxylic group removed, XPS spectra on gas phase, monolayers on Au, and thick layers have revealed the presence of a zwitterionic form S CH2 CH2 NH+3 in the thick film, whereas the gas phase shows the neutral form and the monolayer shows the sulfur bonding to the gold substrate.15 Similarly, we have performed an extensive study of adsorption of L-cysteine on the TiO2(110) surface, where the thicker layers, grown at room temperature or around 200 K, have similarly revealed the presence of the (normal) zwitterion only.16 Furthermore, we observed a ratio of zwitterion to neutral molecule that increases with thickness, being close to 9:1 for the thickest multilayers at 200 K. In this article, we mainly focus on XPS results obtained on films grown with the substrate held at 100 K, and here a different scenario is observed. Figure 2 displays the O 1s, C 1s, N 1s, and S 2p XPS spectra for multilayers of L-cysteine deposited onto the TiO2(110) surface at 100 and 200 K. At first glance, the appearance of a second sulfur doublet, a second nitrogen peak, and a redistribution of weight from the oxygen peak at 531.9 eV to a higher binding energy peak centered at 533.4 eV clearly distinguishes the 100 K from the
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200 K data. Furthermore, a noticeable change in the binding energy of the composite carbon 1s peak can be seen. In order to further elucidate these changes, Voigt curve fitting was employed. The parameters obtained from the curve fits are given in Table 1. The best fit for the C 1s spectrum in both cases is obtained by using four components, where the carboxylic carbon contribution is denoted by the label c and that of the amino carbon by R. The two remaining peaks imply the presence of carbon atoms related to both hydrogen dissociated thiol groups (Cd) as well as those bound to intact thiol groups (Cβ). At 100 K, the CR peak is broader, which is consistent with the presence of amino and protonated amino groups in nearly equal amounts. The weight of the overarching peak has furthermore shifted to lower binding energies due to the growth of the thiolate peak. A more detailed discussion of the differences between the two carbon spectra can be found in the Supporting Information. In the S 2p region of the 100 K spectrum, two doublets are observed. The high and low binding energy components are attributed to thiol and deprotonated thiol groups, respectively. The observed intensities in both the carbon and sulfur spectra indicate that 45% of the thiol groups are deprotonated. We have observed that the difference in binding energy between the two sulfur doublets is essentially the same for all preparations, at all different temperatures. We therefore find it unlikely that the thiol group is broken off from the molecule at the C S bond, and that atomic sulfur would be formed in this low temperature preparation. The O 1s spectrum at 200 K consists of a small TiO2 substrate oxygen contribution at 530.3 eV and a single peak representing deprotonated carboxylic (carboxylate) oxygen atoms at 531.9 eV. The spectrum at 100 K was curve fitted with three components, which, in order from lower to higher binding energy, are attributed to deprotonated carboxylic (COO ), carbonyl (CdO), and hydroxyl (OH) oxygen atoms, respectively. We find a binding energy difference of 0.6 eV between the carbonyl and hydroxyl oxygen peaks. In the literature, somewhat larger values are reported, ranging from 1.8 eV (amino acid molecules, gas phase17) and 1.0 1.2 eV for thick films of isonicotinic, nicotinic, picolinic, and bi-isonicotinic acid adsorbed on the TiO2(110) surface18,19 to 0.8 eV for glycine on Si(111).20 Hydrogen bonding, where the hydroxyl and carbonyl groups act as donors and acceptors, respectively, could contribute to a significant lowering of the binding energy difference. We also considered the inclusion of a significant amount of water molecules in the grown layer as an explanation,21,22 but the intensity ratio of the combined oxygen peaks to that of nitrogen, measured at the same photon energy and corrected for photon flux and cross sections, is entirely conformed to stoichiometry. It is also important to mention at this stage that, according to calculations, even with the inclusion of up to five water molecules per L-cysteine, the neutral form is still the most favorable, followed by the normal zwitterionic state.23 The unconventional zwitterion is stabilized by the presence of water molecules, but remains energetically unfavorable. Similarly, studies of different sequential and codeposited mixtures of glycine (NH2 CH2 COOH) and water, grown at low temperatures (110 and 150 K) have shown a remarkably small influence of water on the formation of the NH+3 CH2 COO zwitterion, as the ratio of NH2 to NH+3 seems quite unaffected.22 We may therefore safely assume that the signal originates from the molecule only, and hence, from the intensity ratio of the deprotonated carboxylic peak to the combined carbonyl and hydroxyl peaks, we can deduce that only 13% of all carboxylic groups are deprotonated. 1678
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Figure 2. (a) Oxygen, (b) carbon, (c) nitrogen, and (d) sulfur XPS spectra with component fits as indicated. For carbon labels, see Figure 1. The 200 K data were taken from ref 16.
Table 1. Summary of Binding Energies for the Components in the XPS Spectra and Their Relative Intensities spectrum C 1s
component
BE200 K
rel int.
BE100 K
rel int.
(eV)
(200 K)
(eV)
(100 K)
Cc, carboxyl
289.02
0.21
288.97
0.26
CR, amino
287.07
0.40
286.52
0.37
Cβ, thiol
286.49
0.37
286.05
0.21
N 1s
Cd thiolate NH+3
285.55 401.37
0.02 0.86
285.42 401.54
0.16 0.57
399.39
0.14
399.88
0.43
O 1s
COOH
533.57
0.435
COOH
532.98
0.435 0.13
NH2
S 2p3/2
COO
531.90
1.00
531.93
SH
164.46
0.96
164.25
0.55
S
162.12
0.04
162.22
0.45
The low- and high-binding energy peaks observed in the N 1s spectrum can be attributed to photoemission from the amino and protonated amino groups, respectively. The intensity ratio at 100 K shows that the amino groups of 57% of the molecules were protonated, against 86% at 200 K. The carboxylic groups show a complete deprotonation at 200 K. When we now look at all the relevant percentages deduced from the 100 K spectra, we find that 45% of the thiol groups are deprotonated, compared to only 13% of the carboxylic groups. The combined hydrogen donation
corresponds nearly exactly to the 57% of protonated amino groups observed. Hence we must conclude that the thiol groups act as the primary donors of protons to the amino groups. This in turn implies that, surprisingly, the unconventional zwitterion seems to be the favored state in this case, which stands in sharp contrast to films grown at room temperature and around 200 K, as well as any observations on crystals grown at room temperature, and simply cooled down to temperatures as low as 2 K (monoclinic)13 and 6 K (orthorhombic).12 To our knowledge, this is also the first experimental observation of the unconventional zwitterion in L-cysteine, or indeed in any molecule possessing both a carboxylic and thiol group available for hydrogen donation. A possible explanation might lie in the unusual growth conditions of this sample. It has been shown for isonicotinic acid (4-pyridine carboxylic acid) that rapid growth of a multilayer at low substrate temperatures suppresses the formation of a hydrogen bonding network in the film.18 This effectively means that the molecules experience a kinetic barrier, which hampers the formation of a long-range hydrogen bonded network. In our case, this would be consistent with the observation that only 57% of the L-cysteine molecules appear to exist in a zwitterionic form, compared to 86% for the thickest 200 K film, and that one-fourth of these are still accounted for by the formation of a conventional zwitterion, seeing as the acidity values for the carboxylic and thiol groups are similar. Alternatively, a certain lability of the side groups, especially of the SH group, has been observed throughout wide temperature and pressure ranges.24 This is indicative of a frustrated system with more than one suitable candidate for the 1679
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The Journal of Physical Chemistry Letters groundstate conformer, depending on the precise parameters of the location on the thermodynamic landscape. Tzvetkov et al. report a similar morphology change above 170 K in glycine layers grown at lower temperatures.22 In summary, we have presented the first observation of the unconventional zwitterionic form of L-cysteine with a deprotonated thiol, protonated amino, and intact, protonated carboxylic group, through the analysis of XPS measurements of thick films. We believe that the explanation for this unusual state can be found in the growth conditions of the film, favoring more disorder, aided by the intrinsic nature of the molecule with its labile side groups and the nearly equal acidity levels of the thiol and carboxylic moieties.
’ EXPERIMENTAL SECTION In all our experiments, L-cysteine (powder, 99.5% pure, Sigma-Aldrich) was sublimated at ∼120 °C for 2 h, after degassing up to 120 °C for 12 h. During deposition, the pressure in the preparation chamber increased to around 7 10 9 mbar (from low tens). Deposition onto a cleaned TiO2(110) substrate held at 100 K was employed to ensure a thick layer growth. Afterward, no signal from the substrate could be observed, and the layer was determined to be at least 40 Å thick. All spectra were calibrated with respect to the position of the molecular O 1s peak at 531.9 eV obtained for thicker films deposited at 200 K.16 During measurement, the sample was kept moving at a speed such that no difference due to X-ray irradiation damage between successive scans (of around 30 s each) was observed.15 XPS measurements were performed at beamline I311 of the MAX-lab synchrotron using a hemispherical Scienta SES200 electron energy analyzer. The S 2p, C 1s, N 1s, and O 1s lines were measured with photon energies of 305, 400, 520, and 675 eV, respectively, at total energy resolutions of approximately 0.2, 0.15, 0.22, and 0.25 eV, respectively. A Shirley-type background was removed from all spectra, and for peak fitting Voigt profiles were used. For an in-depth explanation of the assignment of the different spectral components, we kindly refer the reader to the discussion in our previously published article.16 ’ ASSOCIATED CONTENT
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Supporting Information. Detailed discussion of the differences between the two carbon spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected];
[email protected].
’ ACKNOWLEDGMENT The authors would like to thank the staff of MAX-lab for technical support. We also would like to acknowledge fund (VR, Grant Nos. 2004-4404 and ing from Vetenskapsradet 2010-5080), and the European Commission through the Early Stage Researcher Training Network (Grant MEST-CT-2005020908).
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