Article Cite This: J. Phys. Chem. A 2018, 122, 5978−5982
pubs.acs.org/JPCA
Terahertz Spectroscopy and Density Functional Theory Calculations of DL-Norleucine and DL-Methionine Jens Neu,† Heinrich Nikonow,†,§ and Charles A. Schmuttenmaer*,†,‡ †
Yale University, Department of Chemistry, New Haven, Connecticut 06520, United States Yale University, Energy Science Institute (ESI), New Haven, Connecticut 06520, United States
‡
Downloaded via UNIV OF WINNIPEG on January 28, 2019 at 03:47:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: We present terahertz (THz) measurements and density functional theory (DFT) calculations of two amino acid crystals: DL-norleucine and DL-methionine. Their molecular structures are very similar and therefore also their crystal structures. We report the absorption spectra for both amino acids, which have a strong resonance at 1.87 THz in DLnorleucine and 1.94 THz in DL-methionine. In addition, we find a higher frequency resonance at 2.49 THz in DLmethionine, which has no corresponding mode in DLnorleucine. The experimental data are supported by DFT calculations, which show that the origin of the two strongest vibrational modes in DL-norleucine and DL-methionine are based on the same underlying vibrational motions, whereas the 2.49 THz resonance in DL-methionine is due to the motion of the sulfur atom, which is not present in DL-norleucine.
■
replaced with norleucine,5 making this pair an ideal tool for bioengineering. It is also worth noting that methionine does not form sulfur−sulfur linkages as is the case with cystein, the other sulfur-containing proteinogenic amino acid. Therefore, the functionality changes are not due to changes in chemical bonding. The two pure crystals exhibit similar physical and chemical properties. Both amino acids in a racemic mixture form a crystalline structure consisting of eight molecules per unit cell. The two crystals have the same space group (C2/c), and the three lattice constants are within 2% of each other;8,9 see Table 1. As a result, crystallographic investigations demand highquality crystalline samples. This similarity also applies for IR spectroscopy, with both amino acids exhibiting similar absorption features between 1600 and 600 cm−1.10,11 Terahertz (THz) spectroscopy12−14 covers the frequency range between 10 and 100 cm−1 (0.3 to 3.0 THz)15 and has been demonstrated as a powerful technique to identify chemical compounds,16−18 different amino acids,19−22 and even different polymorphs of the same amino acids.23,24 Combined with density functional theory (DFT) calculations, THz spectroscopy can be used to understand the vibrational motions in ordered crystalline structures. We present THz spectroscopic measurements of two crystalline amino acids, DL-methionine and DL-norleucine. The low-frequency range between 0.3 and 3 THz is particularly sensitive to the geometry of the unit cell, and the vibrational
INTRODUCTION The human metabolic system requires nine essential amino acids. Additionally, 12 nonessential amino acids are found in naturally occurring organisms, and an even larger number of amino acids have been synthesized in the lab.1 These synthetic, non-natural amino acids are not found in any wild-type organism. However, in recent years, mutants have been grown in which synthetic amino acids are incorporated.2 It is interesting to consider two very similar amino acids: the essential methionine and the non-natural norleucine. The difference between these amino acids is found only in the second to last moeity in the R-group side chain, where it is a methylene group in norleucine and sulfur in methionine. (See Figure 1.) Replacing naturally occurring methionine with
Figure 1. Chemical structures of DL-norleucine and DL-methionine. In DL-methionine, the second to last CH2 group is replaced with sulfur.
norleucine has been demonstrated for proteins,3,4 enzymes,5 and bacteria.6 In some of these cases, the replacement can be achieved by simply growing the bacteria in a norleucine-rich and methionine-depleted environment.7 These impressive experiments demonstrate that the binding mechanism between the bacterial backbone and the two amino acids is independent of the sulfur atom. However, the resulting functionality of the bacteria or enzyme changes drastically when methionine is © 2018 American Chemical Society
Received: May 24, 2018 Revised: June 11, 2018 Published: June 12, 2018 5978
DOI: 10.1021/acs.jpca.8b04978 J. Phys. Chem. A 2018, 122, 5978−5982
Article
The Journal of Physical Chemistry A Table 1. Starting Geometry of the Optimization from the CCSD Database and DFT-Optimized Structure, Calculated Using SIESTA
a [Å] b [Å] c [Å] α [deg] β [deg] γ [deg] space group Z ref
DL-Nle
DL-Met
DL-Nle
DL-Met
DLNLUA02
DLMETA08
SIESTA
SIESTA
31.67 4.72 9.85 90.0 91.4 90.0 C2/c 8 8
9.88 4.69 32.60 90.0 106.3 90.0 C2/c 8 9
30.88 4.79 9.85 90.0 90.2 90.0 C2/c 8 this work
9.88 4.76 32.60 90.0 106.0 90.0 C2/c 8 this work
Figure 2. THz absorption spectra of DL-norleucine, measured at room temperature (red), 150 K (green), and 100 K (blue). The black line represents the calculated results convoluted with a 0.1 THz line width and frequency-scaled by 0.93. The main features of the measured spectra are reproduced, and the strength of the lower energy resonance was overestimated in the calculation. Inset: structure of DLnorleucine.
modes are delocalized among many atoms in the unit cell. As a result, replacing a CH2 group in the unit cell with a sulfur affects the vibrational modes in this low-energy range. We present experimental spectra that show a frequency shift between DL-methionine and DL-norleucine. We also present and discuss a THz mode that is present only in DL-methionine, allowing for its easy identification. The DFT calculations are used to explain the origin of the different modes and to demonstrate the influence of the sulfur atom compared with CH2.
■
EXPERIMENTAL AND COMPUTATIONAL RESULTS Temperature-dependent THz absorption spectra for DLnorleucine are plotted in Figure 2. At room temperature (red line) the spectrum shows a broad absorption around 1.87 THz and multiple unresolved absorptions around 1.4 and 1.6 THz. These absorption features are broadened due to the high temperature of the crystal (290 K, or 25 meV) compared with the THz photon energy (2−8 meV). Hence, a clear identification of characteristic absorptions at room temperature is only feasible if the absorption is particularly strong. A second measurement was performed with the sample cooled to 150 K (green line). The measured spectrum displays sharper features compared with the room-temperature spectrum. The strong resonance at 1.87 THz is narrower with a full width at half-maximum (fwhm) of 0.34 THz. Furthermore, a lower frequency resonance can now be clearly identified at 1.4 THz. This change in the spectrum is due to the narrowing of absorption lines with decreasing temperature.28 The third measurement at 100 K (blue line) clearly shows the same features as that at 150 K. However, the characteristic absorptions are more pronounced and allow a clear identification of the resonance frequencies at 1.87 and 1.44 THz. DFT calculations were performed to help interpret the experimental results. The atomic coordinates for these calculations were obtained from single-crystal XRD data measured at 120 K,8 as archived in the Cambridge Crystal Structure Database (CCSD, Database Identifier DLNLUA02). The unit cell was then optimized using SIESTA-DFT calculations (at 0 K), and the vibrational modes were then calculated. The calculated spectra were normalized to the strongest peak and are plotted as a black line in Figure 2 using a Lorentzian line shape with a width of 0.1 THz (full width at half-maximum). There is excellent agreement between the primary experimental peak and that of the calculation. The calculation overestimates the intensity of the smaller feature near 1.4 THz. This is because the calculations correspond to 0 K, whereas the experimental spectra were obtained at 100 K (≅ 8.6 meV ≅ 2 THz) or higher. Therefore, a lower energy resonance will have a contribution from hot
■
EXPERIMENTAL SETUP AND SAMPLE PREPARATION The apparatus used in this experiment is a standard THz timedomain spectrometer (THz-TDS). The pulse was generated with an interdigitaded photoconductive antenna25 that was biased with a 5 kHz, ±10 square-wave function and photoexcited by a 50 fs pulse with 100 mW optical power. The THz beam was routed via four off-axis paraboloidal mirrors through the sample onto the detector. The detector was a photoconductive dipole antenna with a 5 μm gap. The usable bandwidth spanned 0.5 THz to ∼3 THz. The sample was mounted in a temperature-controlled sample-in-vapor nitrogen cryostat, as described in more detail in previous publications.23,26 The structure and vibrational modes were calculated using the SIESTA v3.2r462 package, assuming periodic boundary conditions. We employed the vdW-DF-cx pseudopotentials27 and triple-ζ, doubly polarized (TZDP) basis set, as described in previous publications.23,28 DL-Methionine was purchased from Fluka (99% pure) and DL-norleucine was purchased from Alfa Aesar (98%). The amino acids were ball-milled to ensure grain sizes of
