# Fileset [STAM2026Murakami_SI_revision2_SK_KM_KM_nomark.pdf](https://mdr.nims.go.jp/filesets/a24bbbf2-f3f1-4d3c-812e-b398ccfa384b/download) ## Creator Koichi Murakami, Tomohiro Nobeyama, Yuta Koda, Takahiro Sasamori, Kentaro Shiraki, Yohei Yamamoto, Soh Kushida ## Rights [Creative Commons BY-NC Attribution-NonCommercial 4.0 International](https://creativecommons.org/licenses/by-nc/4.0/) ## Other metadata [Reorientational dynamics of amino acids in aqueous solution probed by ultrafast optical Kerr effect spectroscopy](https://mdr.nims.go.jp/datasets/f7eaf89c-9c54-4e53-9a2e-e1a1b65ce49b) ## Fulltext 1 Supporting Information Reorientational Dynamics of Amino Acids in Aqueous Solution Probed by Ultrafast Optical Kerr Effect Spectroscopy Koichi Murakamia, Tomohiro Nobeyamaa,b, Yuta Kodaa, Takahiro Sasamoria, Kentaro Shirakia, Yohei Yamamotoa,c*, and Soh Kushidaa* *corresponding author: Dr. Soh Kushida, Institute of Pure and Applied Sciences, University of Tsukuba, Tsukuba 305-8573, Japan Prof. Yohei Yamamoto, Institute of Pure and Applied Sciences, Tsukuba Research Center for Energy Materials Science (TREMS) and Hydrogen Boride Research Center, Tsukuba Institute for Advanced Research, University of Tsukuba, Tsukuba 305-8573, Japan Email: s-kushida@ims.tsukuba.ac.jp, yamamoto@ims.tsukuba.ac.jp Table of Contents 1. General Information ---------------------------------------------------------------------- S2 2. Experimental Section ---------------------------------------------------------------------- S2 3. Supporting Figures and Tables --------------------------------------------------------- S4 4. Single Crystal Analysis ----------------------------------------------------------------- S10 5. Calculations for collision frequency ------------------------------------------------- S13 6. DFT Calculation ------------------------------------------------------------------------- S14 7. Supporting Reference ------------------------------------------------------------------ S18 2 1. General Information Unless otherwise noted, all chemical compounds and solvents were used as received from either Merck Co., Ltd., WAKENYAKU CO.,LTD. or Tokyo Chemical Industry Co., Ltd. Optical components—including polarizers, wave plates, mirrors, beam splitters, filters, lenses, and others—were purchased from Thorlabs, Inc. Sigma Koki or Newport Corporation. UV–vis absorption spectra were recorded using a commercial spectrophotometer (JASCO V-570). OKE (Optical Kerr Effect) measurements were performed with a home-built apparatus. The delay stage and lock-in amplifier were controlled via Python scripts. Data processing and curve fitting were conducted using Python. 2. Experimental Section Sample Preparation Solutions: Each amino acid solution was prepared by dissolving the corresponding amino acid in distilled water. The initial amounts of amino acid and water were adjusted to yield a slightly higher concentration than the target value, as dissolution of the amino acid results in a change in the total solution volume and consequently affects the final concentration. The mixture was stirred until complete dissolution. The solution was then adjusted to the final volume using a graduated cylinder to obtain the desired concentration. Viscosity measurement The viscosities of the aqueous PVA solutions (0.2, 0.5, 1.0, and 2.0 wt%) and of the 1 M amino-acid solutions (L-alanine, L-proline, and L-lysine monohydrochloride) were measured with a rotational rheometer (Anton Paar, MCR 301) at 25 °C. Flow curves were recorded as a function of shear rate, and the viscosity of each solution was taken from the Newtonian region. Single Crystal Analysis: Single crystals of racemi_a, D, and L were obtained after recrystallization from water solution at room temperature. Intensity data of racemi_a, D, and L were collected on a Bruker APEX-II system using Mo-Ka radiation (l = 0.71073 Å). The structures were solved using SHELXT-2018S1 and refined by a full-matrix least-squares method on F2 for all reflections using SHELXL-2018.[1] All non-hydrogen atoms were refined anisotropically, and the all hydrogen atoms were refined isotropically. Crystallographic data for racemi_a: C6H15ClN2O2, FW 182.65, crystal size 0.25×0.15×0.10 mm3, temperature –170 ˚C, monoclinic, space group P21/a (#14), a = 8.5308(1) Å, b = 3 11.1431(1) Å, c = 9.1365(1) Å, b = 105.8894(14)˚, V = 835.327(16) Å3, Z = 4, Dcalcd = 1.452 g cm–3, µ = 0.412 mm–1, qmax = 33.629˚, 33741 reflections measured, 3206 independent reflections (Rint = 0.0148), 160 parameters refined, GOF = 1.115, completeness = 99.3%, R1 [I > 2s(I)] = 0.0201, wR2 (all data) = 0.0560, largest diff. peak and hole 0.510 and –0.227 e Å–3. D: C6H19ClN2O4, FW 218.68, crystal size 0.20×0.15×0.12 mm3, temperature –170 ˚C, monoclinic, space group P21 (#4), a = 5.8646(1) Å, b = 13.2180(2) Å, c = 7.4120(1) Å, b = 97.9459(12)˚, V = 569.049(15) Å3, Z = 2, Dcalcd = 1.276 g cm–3, µ = 0.326 mm–1, qmax = 33.609˚, 15909 reflections measured, 4084 independent reflections (Rint = 0.0106), 194 parameters refined, GOF = 1.089, completeness = 99.5%, R1 [I > 2s(I)] = 0.0166, wR2 (all data) = 0.0448, largest diff. peak and hole 0.275 and –0.190 e Å–3. L: C6H19ClN2O4, FW 218.68, crystal size 0.20×0.18×0.05 mm3, temperature –170 ˚C, monoclinic, space group P21 (#4), a = 5.8691(1) Å, b = 13.2195(3) Å, c = 7.4146(2) Å, b = 98.010(2)˚, V = 569.66(2) Å3, Z = 2, Dcalcd = 1.275 g cm–3, µ = 0.326 mm–1, qmax = 33.540˚, 19051 reflections measured, 4034 independent reflections (Rint = 0.0122), 194 parameters refined, GOF = 1.133, completeness = 100.0%, R1 [I > 2s(I)] = 0.0176, wR2 (all data) = 0.0475, largest diff. peak and hole 0.233 and –0.267 e Å–3. 4 3. Supporting Figures and Tables Figure S1. Calibration curves of (a) alanine (207 nm), (b) valine (207 nm), (c) arginine (221 nm), (d) phenylalanine (257 nm), (e) proline (213 nm), and (f) lysine (217 nm) obtained by UV–Vis spectroscopy. The data were fitted using least-squares linear regression. 5 Figure S2. Solubilities of enantiopure (L and D) and racemic amino acids in aqueous solution. The amino acids are (a) alanine, (b) valine, (c) arginine, (d) phenylalanine, and (e) proline, (f) lysine monohydrochloride. The molecular structures of each amino acid are shown above the corresponding plots. Error bars represent standard deviations obtained from repeated measurements. Figure S3. Photographs of aqueous lysine monohydrochloride solutions prepared at an initial concentration of 2.8 M: (a) enantiopure (L-) and (b) racemic. 6 Figure S4. Viscosity of 1 M aqueous L-alanine (green), L-proline (blue), and L-lysine monohydrochloride (red) at 25 °C, measured by rheometer as a function of shear rate. The three solutions have nearly identical viscosities. Figure S5. Arrhenius plots of the reorientation time for L-lysine monohydrochloride (red), L-proline (blue), and L-alanine (green); the solid lines are linear fits, yielding apparent activation energies of 14.0, 12.4, and 9.5 kJ mol-1 for alanine, proline, and lysine monohydrochloride, respectively. 7 Figure S6. OKE signals at different concentrations for (a) D-, (b) L-, and (c) Rac-lysine monohydrochloride solutions. The concentrations are 0.25, 0.50, 1.0, and 2.8 M, indicated from dark to light colors. (d) τ as a function of concentration. Table S1. Solubilities of enantiopure (L and D) and racemic amino acids in aqueous solution. Amino acids SL (M) SD (M) SRac (M) Alanine 1.691 ± 0.009 1.640 ± 0.009 1.680 ± 0.009 Valine 0.560 ± 0.009 0.553 ± 0.009 0.567 ± 0.009 Arginine 0.907 ± 0.007 1.062 ± 0.016 1.085 ± 0.020 Phenylalanine 0.163 ± 0.0005 0.164 ± 0.0005 0.0772 ± 0.0004 Proline 6.046 ± 0.010 6.278 ± 0.001 6.089 ± 0.010 Lysine・HCl 2.756 ± 0.005 2.759 ± 0.005 1.198 ± 0.001 8 Table S2. Reorientational relaxation time of pure water and aqueous PVA solutions. Sample τ (ps) Water 0.7 ± 0.01 PVA 2wt% 0.8 ± 0.01 PVA 1wt% 0.7 ± 0.01 PVA 0.5wt% 0.7 ± 0.01 PVA 0.2wt% 0.7 ± 0.01 Table S3. DFT-derived polarizability parameters at 700 nm. The polarizability anisotropy γ was obtained from the frequency-dependent polarizability tensor α(−ω;ω) at 700 nm. Sample γ / au γ² / au² L-lysine 23.63 558.52 L-alanine 18.8 353.45 L-proline 20.35 414 water 0.335 0.112 Table S4. Reorientational relaxation time of 1 M aqueous L-alanine, L-proline, and L-lysine monohydrochloride at 25, 45, 65, and 85 °C. Sample 25 ℃ (ps) 45 ℃ (ps) 65 ℃ (ps) 85 ℃ (ps) L-lysine 25.8 ± 0.8 21.6 ± 0.6 17.5 ± 0.4 13.5 ± 0.4 L-alanine 11.3 ± 0.5 8.2 ± 0.3 5.4 ± 0.2 4.6 ± 0.3 L-proline 11.8 ± 0.4 8.4 ± 0.4 6.2 ± 0.2 5.2 ± 0.2 Table S5. Reorientational relaxation time of lysine monohydrochloride aqueous solutions with different enantiomeric ratios and concentrations. Sample 0.25 M (ps) 0.5 M (ps) 1 M (ps) 2.8 M (ps) L-lysine 33.6 ± 2.0 37.7 ± 1.8 46.3 ± 0.6 76.4 ± 1.6 D-lysine 37.2 ± 3.9 37.7 ± 1.5 46.9 ± 0.8 77.1 ± 1.5 Rac-Lysine 39.8 ± 3.1 37.1 ± 1.1 45.2 ± 1.2 79.6 ± 2.65 9 Table S6. Slow-component amplitudes A obtained from single-exponential fits to the unnormalized OKE traces of D-, L-, and racemic lysine monohydrochloride solutions at different concentrations. Sample 0.25 M (a.u.) 0.5M (a.u.) 1M (a.u.) 2.8M (a.u.) L-lysine 0.150 ± 0.008 0.198 ± 0.006 0.591 ± 0.007 0.730 ± 0.011 D-lysine 0.066 ± 0.004 0.259 ± 0.008 0.402 ± 0.006 0.727 ± 0.011 Rac-Lysine 0.108 ± 0.005 0.271 ± 0.005 0.645 ± 0.012 0.714 ± 0.016 Table S7. Effective hydrodynamic volumes derived from the SED analysis and DFT-estimated van der Waals volumes of alanine, proline, and lysine monohydrochloride. Sample Slope S (ps·K·mPa-1·s-1) Veff (Å3)a Vvdw (Å3)b L-alanine 3384.7 ± 281 46.7 ± 3.9 83.4 L-proline 3277.9 ± 106 45.3 ± 1.5 107 L-lysine monohydrochloride 5711.3 ± 1010 78.9 ± 13.9 145.5 a: Estimated from OKE experiments. b: Estimated from DFT calculations. 10 4. Single Crystal Analysis Table S8. Crystal structure, crystal data and structure refinement for L-lysine. Identification code L Empirical formula C6 H19 Cl N2 O4 Formula weight 218.68 Temperature 103(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P21 (#4) Unit cell dimensions a = 5.86910(10) Å a= 90°. b = 13.2195(3) Å b= 98.010(2)°. c = 7.4146(2) Å g = 90°. Volume 569.66(2) Å3 Z 2 Density (calculated) 1.275 Mg/m3 Absorption coefficient 0.326 mm-1 F(000) 236 Crystal size 0.200 x 0.180 x 0.050 mm3 Theta range for data collection 2.774 to 33.540°. Index ranges -9<=h<=9, -20<=k<=20, -11<=l<=11 Reflections collected 19051 Independent reflections 4034 [R(int) = 0.0122] Completeness to theta = 25.242° 100.0 % Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4034 / 1 / 194 Goodness-of-fit on F2 1.133 Final R indices [I>2sigma(I)] R1 = 0.0176, wR2 = 0.0473 R indices (all data) R1 = 0.0180, wR2 = 0.0475 Absolute structure parameter 0.004(8) Extinction coefficient n/a Largest diff. peak and hole 0.233 and -0.267 e.Å-3 11 Table S9. Crystal structure, crystal data and structure refinement for D-lysine. Identification code D Empirical formula C6 H19 Cl N2 O4 Formula weight 218.68 Temperature 103(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P21 (#4) Unit cell dimensions a = 5.86460(10) Å a= 90°. b = 13.2180(2) Å b= 97.9459(12)°. c = 7.41200(10) Å g = 90°. Volume 569.049(15) Å3 Z 2 Density (calculated) 1.276 Mg/m3 Absorption coefficient 0.326 mm-1 F(000) 236 Crystal size 0.200 x 0.150 x 0.120 mm3 Theta range for data collection 2.775 to 33.609°. Index ranges -9<=h<=9, -20<=k<=19, -11<=l<=10 Reflections collected 15909 Independent reflections 4084 [R(int) = 0.0106] Completeness to theta = 25.242° 99.5 % Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4084 / 1 / 194 Goodness-of-fit on F2 1.089 Final R indices [I>2sigma(I)] R1 = 0.0166, wR2 = 0.0448 R indices (all data) R1 = 0.0167, wR2 = 0.0448 Absolute structure parameter 0.008(8) Extinction coefficient n/a Largest diff. peak and hole 0.275 and -0.190 e.Å-3 12 Table S10. Crystal structure, crystal data and structure refinement for Rac-lysine Identification code racemi_a Empirical formula C6 H15 Cl N2 O2 Formula weight 182.65 Temperature 103(2) K Wavelength 0.71073 Å Crystal system monoclinic Space group P21/a (#14) Unit cell dimensions a = 8.53080(10) Å a= 90°. b = 11.14310(10) Å b= 105.8894(14)°. c = 9.13650(10) Å g = 90°. Volume 835.327(16) Å3 Z 4 Density (calculated) 1.452 Mg/m3 Absorption coefficient 0.412 mm-1 F(000) 392 Crystal size 0.250 x 0.150 x 0.100 mm3 Theta range for data collection 2.952 to 33.629°. Index ranges -13<=h<=13, -17<=k<=17, -14<=l<=14 Reflections collected 33741 Independent reflections 3206 [R(int) = 0.0148] Completeness to theta = 25.242° 99.3 % Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 3206 / 0 / 160 Goodness-of-fit on F2 1.115 Final R indices [I>2sigma(I)] R1 = 0.0201, wR2 = 0.0557 R indices (all data) R1 = 0.0205, wR2 = 0.0560 Extinction coefficient n/a Largest diff. peak and hole 0.510 and -0.227 e.Å-3 13 5. Calculations for collision frequency To estimate the frequency of intermolecular encounters in concentrated lysine solutions, we employed a diffusion-controlled collision model based on the Stokes–Einstein relation. The translational diffusion coefficient 𝐷 was approximated using 𝐷 =𝑘!𝑇6π𝜂𝑟" where 𝑘! is the Boltzmann constant, 𝑇 is the absolute temperature (298 K), 𝜂 is the solution viscosity, and 𝑟" is the hydrodynamic radius of lysine. For the 2.8 M lysine solution, we assumed 𝜂 = 10 mPa·s and 𝑟" = 0.5nm,[2] yielding 𝐷 ≈ 4.4 × 10#$$m² s⁻¹. The diffusion-controlled collision rate constant for identical molecules was estimated using the Smoluchowski expression: 𝑘diff = 8π𝐷𝑅 where R is the encounter distance, approximated as R = 2r%. Under these assumptions, 𝑘diff ≈ 1.1 × 10#$&m³ s⁻¹. The collision frequency per molecule at concentration (𝐶) was then estimated as 𝑓 ≈ 𝑘diff 𝑛 where 𝑛 = 1000 𝑁'𝐶 is the number density with 𝑁' denoting Avogadro’s constant (6.022 × 10²³ mol⁻¹). For 𝐶 = 2.8 mol L⁻¹, this yields 𝑓 ∼ 2 × 10( s⁻¹, corresponding to an average intermolecular encounter every ~ 0.5 ns. This estimated collision timescale is many orders of magnitude shorter than the characteristic timescale of crystallization (typically ~hours), indicating that nucleation cannot be limited by simple diffusive encounters but instead requires rare, orientation-specific configurations. 14 6. DFT calculation All ab initio and density functional theory (DFT) calculations were performed by using Gaussian 16 Rev.A.03 or Rev.B.01 and results were visualized with Gaussview63, 4. All geometries were optimized using density functional theory (DFT) at the B3LYP/aug-cc-pvtz level of theory without any symmetry assumptions. Harmonic vibration frequency analysis was performed at the same level of theory to verify all stationary points as local minima (with no imaginary frequency). The size of molecule was calculated from the optimized coordination by getting diameter value as the longest distance between the center of gravity and atoms for each molecule. As for the polarity, we conducted a single point calculation with the following keywords: Polar, pop=full, CPHF=Rdfreq, int=grid=ultrafine. L-alanine Optimized Cartesian coordinates (Å) N 1.23818300 -1.15346700 0.12910900 C 0.58092100 0.17916900 0.40804000 C 1.25996300 1.30480000 -0.35384100 C -0.92409800 -0.01041100 0.03744900 O -1.22438300 -1.15702700 -0.39783500 O -1.66377700 0.97019700 0.21551900 H 0.42124700 -1.72200000 -0.20543900 H 1.94823900 -1.10479900 -0.59791500 H 1.65555300 -1.57775600 0.95292000 H 0.64763200 0.35078700 1.47948300 H 0.74057000 2.23627400 -0.14418500 H 2.30056900 1.41523900 -0.04986900 H 1.22347600 1.12980800 -1.43011500 Frequency-dependent polarizability tensor at 700 nm Alpha(-ω;ω), ω = 700 nm Dipole polarizability tensor in the Gaussian molecular coordinate system. (au) (10**-24 esu) (10**-40 SI) iso 0.636504D+02 0.943202D+01 0.104945D+02 aniso 0.188002D+02 0.278591D+01 0.309974D+01 xx 0.514999D+02 0.763149D+01 0.849118D+01 yx 0.134635D+01 0.199509D+00 0.221984D+00 yy 0.688172D+02 0.101977D+02 0.113464D+02 15 zx 0.147947D+01 0.219234D+00 0.243931D+00 zy -0.150418D+01 -0.222896D+00 -0.248006D+00 zz 0.706342D+02 0.104669D+02 0.116460D+02 L-proline Optimized Cartesian coordinates (Å) N 0.70763700 -1.02510600 0.57897900 C 1.98636600 -0.60838300 -0.10497600 C 1.61771400 0.70293800 -0.78753900 C 0.65546200 1.36087400 0.20940100 C -0.18091400 0.19405500 0.75172300 C -1.48557400 -0.09143900 -0.05161400 O -2.35288600 0.79737100 -0.01719800 O -1.51760300 -1.20237800 -0.64833000 H 0.88318500 -1.51508600 1.45118700 H 0.10628300 -1.62382100 -0.02917000 H 2.73738700 -0.46091000 0.66643800 H 2.30674100 -1.40165300 -0.77305700 H 1.11908900 0.50969900 -1.73774300 H 2.49938900 1.30744900 -0.98609900 H 0.01801400 2.11519400 -0.24327400 H 1.21415900 1.83021400 1.01916100 H -0.43211800 0.30643900 1.80195900 Frequency-dependent polarizability tensor at 700 nm Alpha(-ω;ω), ω = 700 nm Dipole polarizability tensor in the Gaussian molecular coordinate system. (au) (10**-24 esu) (10**-40 SI) iso 0.843808D+02 0.125039D+02 0.139125D+02 aniso 0.203471D+02 0.301513D+01 0.335478D+01 xx 0.739220D+02 0.109541D+02 0.121881D+02 yx -0.258368D+01 -0.382862D+00 -0.425991D+00 yy 0.857067D+02 0.127004D+02 0.141311D+02 zx 0.542775D+01 0.804310D+00 0.894916D+00 zy -0.214191D+01 -0.317398D+00 -0.353153D+00 zz 0.935138D+02 0.138573D+02 0.154183D+02 16 L-lysine Optimized Cartesian coordinates (Å) N 1.72186400 1.78677300 -0.03799100 C 1.50327100 0.49090900 0.71126900 C 0.02819800 0.10090000 0.78179100 C -0.62934600 -0.20728700 -0.56804400 C -2.08322100 -0.68140700 -0.45408000 C -3.07246600 0.39868300 -0.02529000 N -4.44337600 -0.13493300 0.00161300 C 2.41846200 -0.56075400 0.00675100 O 3.08215900 -0.11931600 -0.97214500 O 2.41015100 -1.70586300 0.48552300 H 2.08829200 2.53149100 0.54841700 H 2.43180300 1.49818600 -0.75444800 H 0.88459100 2.13089800 -0.50168500 H 1.88550900 0.64236400 1.71776000 H -0.02256700 -0.78520700 1.41480900 H -0.51360500 0.88914100 1.30770400 H -0.04972500 -0.98353700 -1.07142600 H -0.60130100 0.66822200 -1.22542100 H -2.40482000 -1.07244300 -1.42180000 H -2.13453200 -1.51785000 0.25077000 H -3.04818400 1.21626400 -0.74966800 H -2.77252900 0.82154800 0.94044200 H -5.09669300 0.59711100 0.25509000 H -4.52352100 -0.84390700 0.72270100 Frequency-dependent polarizability tensor at 700 nm Alpha(-ω;ω), ω = 700 nm Dipole polarizability tensor in the Gaussian molecular coordinate system. (au) (10**-24 esu) (10**-40 SI) iso 0.116349D+03 0.172411D+02 0.191833D+02 aniso 0.236330D+02 0.350205D+01 0.389656D+01 xx 0.120859D+03 0.179095D+02 0.199270D+02 yx 0.104114D+02 0.154281D+01 0.171660D+01 yy 0.111433D+03 0.165127D+02 0.183729D+02 zx -0.744537D+01 -0.110329D+01 -0.122758D+01 17 zy 0.836782D-01 0.123998D-01 0.137967D-01 zz 0.116753D+03 0.173011D+02 0.192500D+02 Water Optimized Cartesian coordinates (Å) H 0.00000000 -0.77781100 1.52378900 O 0.00000000 -0.02950700 0.91724400 H 0.00000000 0.74613100 1.48881800 Frequency-dependent polarizability tensor at 700 nm Alpha(-ω;ω), ω = 700 nm Dipole polarizability tensor in the Gaussian molecular coordinate system. (au) (10**-24 esu) (10**-40 SI) iso 0.105481D+02 0.156306D+01 0.173914D+01 aniso 0.335288D+00 0.496845D-01 0.552815D-01 xx 0.104277D+02 0.154523D+01 0.171930D+01 yx 0.000000D+00 0.000000D+00 0.000000D+00 yy 0.107714D+02 0.159615D+01 0.177596D+01 zx 0.000000D+00 0.000000D+00 0.000000D+00 zy 0.129327D-02 0.191642D-03 0.213231D-03 zz 0.104451D+02 0.154781D+01 0.172217D+01 The polarizability anisotropy was calculated from the frequency-dependent polarizability tensor at 700 nm as γ) =12 [>α** − α++A𝟐 + (>α++ − α--A𝟐 + (α-- − α**)𝟐] + 3>α*+) + α*-) + α+-)A The orientational OKE contribution was then estimated using the approximate scaling A./0 ∝ cγ), where c is the molar concentration. 18 7. References [1] Usón I, Sheldrick GM. An introduction to experimental phasing of macromolecules illustrated by SHELX ; new autotracing features. Acta Crystallogr D Struct Biol. 2018;74(2):106–116. [2] Patil, KC, Gowardipe A. 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