ARTICLE pubs.acs.org/JPCA
Rotational Spectra and Computational Analysis of Two Conformers of Leucinamide Andrew R. Conrad,† Heather L. Seedhouse,† Richard J. Lavrich,‡ and Michael J. Tubergen*,† † ‡
Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44242, United States Department of Chemistry and Biochemistry, College of Charleston, 66 George Street, Charleston, South Carolina 29424, United States
bS Supporting Information ABSTRACT: Rotational spectra were recorded for two isotopic species of two conformers of the amide derivative of leucine in the range of 10.521 GHz and fit to a rigid rotor Hamiltonian. Ab initio calculations at the MP2/6-311þþG(d,p) level identified the low energy conformations with different side chain configurations; the rotational spectra were assigned to the two lowest energy ab initio structures. We recorded 16 a- and b-type rotational transitions for conformer 1; the rotational constants of the normal species are A = 2275.6(2), B = 1033.37(2) and C = 911.71(5) MHz. We recorded 23 a- and b-type rotational transitions for conformer 2; the rotational constants of the normal species are A = 2752.775(8), B = 843.502(1) and C = 796.721(1) MHz. The rotational spectra of the 15Namide isotopomer of each conformer were recorded and the atomic coordinates of the amide nitrogen were determined by Kraitchman’s method of isotopic substitution. The experimentally observed structures are significantly different from the crystal structures of leucinamide and the gas-phase structures of leucine, and a natural bond orbital analysis revealed the donoracceptor interactions governing side chain configuration.
’ INTRODUCTION The gas-phase structures of small biomolecules such as amino acids reveal their intrinsic structural preferences, which are often dominated by intramolecular hydrogen bonding interactions, without the interfering effects of condensed-phase interactions. Gas-phase structures also yield important data for refinement of computational chemical methods, and a detailed understanding of the intramolecular interactions in these systems may provide insight into the interactions of large biochemical systems. Amino acids are some of the simplest biomolecules that exhibit intramolecular hydrogen bonding. They also have a great degree of conformational flexibility resulting in many low energy structures. Recent advances in laser-vaporization microwave spectroscopy made possible the spectroscopic study of a number of amino acids in the gas phase including glycine,16 alanine,7 proline,8 valine,9 and leucine.10 The conformational preferences of these molecules are governed by intramolecular hydrogen bonding. The aliphatic amino acids glycine, alanine, valine, and leucine all exhibit the same backbone structure, and the most stable structures have a bifurcated hydrogen bond between the amine and carboxylic oxygen and a cis-COOH interaction.17,9,10 The second most stable energy backbone configuration of the aliphatic amino acids is characterized by a hydrogen bond between the carboxyl hydrogen and the amine nitrogen and a trans-COOH configuration.17,9,10 To investigate how these backbone preferences may change in peptides, we consider the amide derivatives of amino acids. r 2011 American Chemical Society
Peptides are joined together by amide linkages, and the amide derivatives of amino acids serve as simple models of peptides. The conformational structures of prolinamide,11 alaninamide,12 and valinamide13 have been investigated by microwave spectroscopy. In contrast with the corresponding amino acids, only one conformer was observed for each amino amide species. Additionally, the conformational preferences of amino amides are dominated by hydrogen bonding between an amide proton and the amine nitrogen, similar to the second lowest energy conformers of the amino acids. The isopropyl side chain has the same configuration in both valine and valinamide.9,13 Leucinamide, shown in Figure 1, has an isobutyl side chain and three torsional degrees of freedom: the backbone dihedral angle Ψ (NamideC0 CRNamine) and the side chain dihedral angles χ1 (C0 CRCβCγ) and χ2 (CRCβCγHγ). Two conformers of leucinamide were identified by X-ray crystallography and crystal-phase NMR and FTIR spectroscopies.14,15 The two solid-phase structures have different backbone configurations resulting from different intermolecular hydrogen bonding interactions; the side chain configurations in the crystal structures are similar.14,15 Because intermolecular hydrogen bonding is crucial Special Issue: David W. Pratt Festschrift Received: January 28, 2011 Revised: April 18, 2011 Published: May 10, 2011 9676
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energy than one of the observed conformers—no spectra were found for any of these additional conformers.10 The motivation for this work is to extend our previous studies of the amide derivatives of amino acids and to explore the backbone and side chain configurations of leucinamide for comparison to the crystal phase and to the parent amino acid.
’ EXPERIMENT
Figure 1. Structures of leucinamide conformers 1, 2, and 3 from the MP2/6-311þþG(d,p) calculations.
to the conformational preferences in the crystal phase, we expect differences in the monomer gas-phase structures where these forces are absent. Two conformers of the amino acid leucine were identified in the gas phase recently using Fourier transform microwave (FTMW) spectroscopy.10 The principal difference between the two observed structures is the backbone configuration: the lower energy conformer is stabilized by a bifurcated hydrogen bond between the amine and the carboxyl oxygen,and the higher energy conformer (ΔE = 4.20 kJ mol1) is stabilized by a hydrogen bond between the carboxyl proton and the amine.10 Both observed conformers adopt the same side chain configuration, labeled “b1” in ref 10 (τ(HβCβCRHR) ≈ 60°), with a stretched configuration that separates the terminal side chain atoms from the backbone.10 Three additional conformational minima with different side chain configurations were identified by MP2/6-311þþG(d,p) calculations, but—despite having lower (2.681.79 kJ mol1)
Microwave Spectroscopy. Microwave spectra of leucinamide were recorded using a mini-cavity Fourier-transform microwave spectrometer.16,17 The instrument consists of a FabryPerot resonant cavity residing in a vacuum chamber formed by a 6-way cross pumped by a Varian VHS-6 diffusion pump (2400 L s1) and backed by a two-stage Edwards E2M30 rotary pump. The resonant cavity is formed by two 7.5 in. diameter diamond-tip finished aluminum mirrors, each with a 30.5 cm spherical radius of curvature. One mirror is fashioned on the vacuum flange mounted on the 6-way cross; the second mirror is mounted on steel rails that pass through ball bearing brackets and is positioned to tune the resonant frequency by a motorized micrometer with a 5 cm range of travel. Microwave radiation is generated by an Agilent Technologies E8247C PSG CW synthesizer and coupled into the cavity using an L-shaped antenna. Molecular emission is detected through the same antenna, is frequency reduced by a heterodyne circuit, and is digitized by a National Instruments NI 5112 digitizing board. Details of the irradiation and heterodyne circuitry can be found in ref 17. Custom Labview software controls the frequency and timing, performs signal averaging, displays and saves spectral data, and scans the spectrometer by stepping the frequency source and cavity. The digital frequency resolution, limited by sampling rate and length of the free induction decay record, is 2.5 kHz. Rotational transitions are split into Doppler doublets centered at the transition frequency due to the parallel arrangement of gas expansion with respect to the cavity axis. The fwhm of each Doppler component is typically 13 kHz. Samples of leucinamide were prepared by liberation of the HCl salt (Aldrich 99%) with an equimolar amount of NaOH. Leucinamide was extracted from the aqueous mixture with organic solvent and recrystallized. The resulting sample was entrained in a 70/30% Ne/He carrier gas at 1 atm and expanded into the cavity using a reservoir nozzle18 made from a modified Series-9 General Valve. The expansion passes through a 0.182 in diameter orifice into the resonant cavity via the reservoir nozzle which is externally mounted in a recessed region of the mirror flange. The vapor pressure of leucinamide was increased by heating the reservoir nozzle to 145 °C using a Watlow STB1A1A3-A12 band heater and an Omega CN8201 temperature regulator. Rotational transitions were measured between 10.5 and 21 GHz. 15 N-labeled samples were prepared using a four-step synthesis also used for preparation of prolinamide, alaninamide, and valinamide.1113,19 These reactions protect the amine, activate the carbonyl, form the amide, and deprotect the amine. The intermediate products were characterized by melting points and NMR spectroscopy. The most abundant isotopomer of leucinamide was prepared from leucine using this synthetic scheme, and the product was identified as leucinamide using microwave spectroscopy. DL-Leucine (Sigma) and 15NH4Cl (Cambridge Isotope Laboratories 98%) were used to prepare the 15Namide isotopomer. Computational Methods. Theoretical model structures of leucinamide were calculated to provide approximate rotational 9677
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Figure 2. Leucinamide conformer models from the RHF/6-311þþG(d,p) calculations.
constants and relative energies to aid in the assignment of the microwave spectra and characterization of the structures. To determine the lowest energy conformers, 18 model structures were optimized at the RHF/6-311þþG(d,p) level using Gaussian9820 on an R10000 workstation. Structural optimizations of the resulting lowest energy conformers were preformed using second-order MøllerPlesset perturbation theory and the same basis set using the GAUSSIAN0321 suite of programs on the Glenn cluster at the Ohio Supercomputer Center. Zero-point energy corrections were made to improve the reliability of the relative energy data, and the hydrogen bonding interactions were investigated via natural bond orbital (NBO) calculations at the MP2/6-311þþG(d,p) level using the NBO 3.122 implementation in GAUSSIAN03.21
’ RESULTS The structural optimization calculations at the RHF/6311þþG(d,p) level converged to 16 unique molecular conformations; the 6 lowest energy structures are shown in Figure 2.
The relative energies, rotational constants, and dipole moments for the six lowest energy structures are summarized in Table 1. The root-mean-square averages of the percent differences between observed and calculated moments of inertia23 (% ΔIrms where % ΔI = (Ix(obs) Ix(calc))/Ix(obs) and x = a, b, c) are also listed in Table 1. The three lowest energy structures, which also had the smallest % ΔIrms values, were then optimized at the MP2/6-311þþG(d,p) level and are shown in Figure 1. The resulting rotational constants, dipole moments, relative energies, structural parameters, and % ΔIrms comparisons to spectroscopic constants are summarized in Table 2. The microwave spectrum of leucinamide was recorded and two rotational spectra (A and B), corresponding to two unique structures of leucinamide, were assigned. We observed 16 a- and b-type transitions for spectrum A and 23 a- and b-type transitions for spectrum B. The corresponding spectra for the 15Namide isotopomers were also recorded; we observed 16 a- and b- type transitions for 15Namide spectrum A and 25 a- and b- type transitions for 9678
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Table 1. Relative Energies, Rotational Constants, Dipole Moments, and % ΔIrms Values of the Six Lowest Energy Leucinamide Theoretical Structures at the RHF/6-311þþG(d,p) Level conformer
a
ΔE/kJ mol1 a
A/MHz
B/MHz
C/MHz
μa/D
μb/D
% ΔIrmsA
% ΔIrmsB
1
0.66
2278.8
1014.6
907.8
0.7
3.4
0.4
1.1
17.0
2 3
0.00 2.53
2773.8 2711.8
837.0 835.2
796.9 800.7
1.4 0.9
3.6 2.9
0.8 1.6
19.0 18.4
0.6 1.1
4
6.56
2774.2
878.3
753.7
1.4
3.3
1.3
18.9
4.0
5
6.59
2179.5
1010.9
923.1
0.7
2.2
2.7
2.9
19.6
6
9.03
2293.6
1083.3
870.2
0.6
3.1
1.5
3.9
17.9
Relative to conformer 2, includes zero-point energy correction.
Table 2. Rotational Constants, Dipole Moments, Relative Energies, Structural Parameters, and % ΔIrms Values of the Leucinamide Theoretical Structures from the MP2/6311þþG(d,p) Calculations parameter
a
μc/D
Table 3. Spectroscopic Parameters of Leucinamide Parent and 15Namide Isotopomers spectrum A parameter
parent
15
spectrum B
Namide
parent
15
Namide
conformer 1
conformer 2
conformer 3
A/MHz
2229.2
2774.6
2717.2
A/MHz
2275.6(2)
2263.0(3)
2752.775(8)
2743.64(5)
B/MHz
1058.2
851.4
837.8
B/MHz
1033.37(2)
1020.71(3)
843.502(1)
832.750(8)
C/MHz
934.0
797.7
812.2
C/MHz
911.71(5)
903.352(8)
796.721(1)
787.610(6)
μa/D
0.9
1.68
1.0
N Δνrms/kHz
16 147
16 233
23 41
25 238
μb/D
3.6
3.5
2.5
μc/D
0.1
1.2
2.1
ΔE/kJ mol1 a
0.00
2.14
7.76
Ψ/deg χ1/deg
36.5 53.7
14.5 175.7
146.4 179.7
χ2/deg
59.8
65.4
61.4
%ΔIrmsA
2.3
18.1
17.9
%ΔIrmsB
19.8
0.7
1.4
Relative to conformer 1, includes zero-point energy correction.
15
Namide spectrum B. The observed transitions for the parent and Namide isotopomers of spectra A and B are listed in the Supporting Information. The congested hyperfine patterns arising from the two 14 N nuclei in the parent species and single 14N nucleus in the 15 Namide isotopomer were not resolved. Approximate line centers were estimated near the center of each hyperfine cluster, and the estimated uncertainty of the rotational transition centers is 200 kHz. The observed rotational transitions for the parent and 15Namide isotopomers were fit to a rigid rotor Hamiltonian24 using Pickett’s SPFIT program,25 and the resulting rotational constants are listed in Table 3. 15
’ DISCUSSION The structure of leucinamide is characterized by the shape of its backbone and the orientation of the side chain. The lowest energy (at the MP2 level) theoretical structures, conformers 1 and 2, adopt a backbone structure similar to that seen in the other aliphatic amino amides where there is a hydrogen bonding interaction between the amide proton and the amine nitrogen and a cis arrangement of the amide. The backbone of the third lowest energy conformer, conformer 3, has a bifurcated hydrogen bond between the amine protons and the carbonyl and a cis arrangement of the amide characteristic of the backbone configuration in the lowest energy conformers of the aliphatic amino acids.7,9,10 Because the two lowest energy conformers of leucinamide have similar backbone configurations, the energy ordering is largely dependent on the side chain orientation. Conformer 1 has
a side chain with χ1 = 53.7° and χ2 = 59.8° and is labeled the “c2” conformation in ref 10. Conformers 2 and 3 have similar side chain orientations with χ1 = 175.7° and 179.7° and χ2 = 65.4° and 61.4°, respectively, and are labeled the “b1” conformation in ref 10. This stretched side chain configuration keeps the terminal atoms of the side chain far removed from the backbone. The energy difference of 2.14 kJ mol1 between conformers 1 and 2 must largely depend on the side chain configuration; to explore this effect, we performed a natural bond orbital analysis of the three lowest energy conformers. While the c1 and b2 side chain configurations have many similar donoracceptor interactions between bonding orbitals, lone pairs, and antibonding orbitals, the important interaction differences are the losses of occupancy from CβH bonding orbitals to C0 CR antibonding orbitals and donoracceptor interactions between CβH bonding orbitals and CRNamine antibonding orbitals. The analysis revealed that the c2 configuration of conformer 1 is stabilized by an interaction between a CβH bonding orbital and a C0 CR antibonding orbital. The estimates of hyperconjugative energy E(2) of the donoracceptor interaction is 14.5 kJ mol1. The b2 configuration of conformers 2 and 3 is stabilized by a donor acceptor interaction between a CβH bonding orbital and CRNamine antibonding orbital where E(2) = 21.2 and 18.7 kJ mol1, respectively. This interaction is also present in the c2 configuration, but the interaction is much less important with E(2) = 2.8 kJ mol1. While conformers 2 and 3 have similar side chain configurations, they have different backbone structures resulting in the 5.62 kJ mol1 energy difference between them. The NBO analysis revealed the donoracceptor interactions of the lone pair of the amine nitrogen atom to be significant for both conformers. Conformers 1 and 2 have a donoracceptor interaction between the lone pair on Namine and the NamideH antibonding orbital with E(2) = 8.0 and 7.8 kJ mol1 for conformers 1 and 2, respectively; this interaction is not present in conformer 3. Also, conformers 1 and 2 have a significant interaction between the 9679
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The Journal of Physical Chemistry A Namine lone pair and the C0 H antibonding orbital (E(2) = 36.1 and 39.4 kJ mol1, respectively) that is much reduced in conformer 3 (E(2) = 5.5 kJ mol1). Conformer 3 has a significant interaction between the Namine lone pair and the C0 CR antibonding orbital (E(2) = 42.0 kJ mol1), which is significantly reduced in conformers 1 and 2 (E(2) = 15.1 and 15.8 kJ mol1, respectively). Conformer 3 also has an interaction between the Namine lone pair and the C0 O antibonding orbital (E(2) = 2.9 kJ mol1) not present in conformers 1 and 2. Conformer 3 has a significant interaction between a NamineH bonding orbital and a C0 H antibonding orbital (E(2) = 16.0 kJ mol1) that is smaller in conformers 1 and 2 (E(2) = 2.6 and 2.4 kJ mol1, respectively). Finally, conformers 1 and 2 have an interaction between the NamineH bonding orbital and the C0 CR antibonding orbital (E(2) = 11.8 and 12.5 kJ mol1, respectively). Two rotational spectra were recorded for leucinamide, spectra A and B, corresponding to two conformers. Because comparison of absolute differences in model and experimental moments of inertia (ΔIrms) is biased toward larger values of these parameters,23 the root-mean-square average of the percent relative differences of moments of inertia (% ΔIrms) was used to compare the model structures to the experimental spectra. The % ΔIrms values for spectra A and B with the RHF/6-311þþG(d,p) and MP2/6311þþG(d,p) structures are listed in Tables 1 and 2, respectively. The lowest energy MP2 structure, conformer 1, has the smallest % ΔIrms value for spectrum A, and therefore spectrum A is assigned to conformer 1. The second and third lowest energy structures both have small % ΔIrms values for spectrum B (0.7 and 1.4, respectively). Conformer 3 is 5.62 and 2.53 kJ mol1 higher in energy at the MP2 and RHF levels, respectively. With the smaller % ΔIrms value and lower relative energy, spectrum B is assigned to conformer 2. Analysis of the coordinates of the amide nitrogen provides additional evidence supporting the assignment of the spectra to the theoretical structures. Using the rotational constants of the 15 Namide spectra in Kraitchman’s equations for single isotopic substitution,24,26 the absolute values of the coordinates of the amide nitrogen atoms were calculated in the principal axis system of the parent isotopic species. The Kraitchman coordinates with Costain estimates of uncertainty are given in Table 4 with the atomic coordinates from the MP2/6-311þþG(d,p) theoretical structures. The signs of the Kraitchman coordinates were taken to be the same as the theoretical structures. The theoretical (MP2) and experimental values of the amide nitrogen atomic coordinates for conformer 1 have large discrepancies, but the atomic coordinates of the amide nitrogen at the RHF/6311þþG(d,p) level, a = 2.209, b = 0.364, and c = 1.094, reproduce the experimental coordinates significantly better. The Kraitchman coordinates of conformer 2 agree well with the atomic coordinates of the amide nitrogen of the MP2 structure, as shown in Table 4. The Kraitchman coordinates differ significantly from the nitrogen atomic coordinates calculated for conformer 3, confirming the assignment of spectrum B to conformer 2. The small differences between the experimental and RHF structures (conformer 1) and between the experimental and MP2 structures (conformer 2) are attributed to zero-point vibrational effects. Surprisingly, the % ΔIrms value for conformer 1 is smaller at the RHF level than at the MP2 level; the reverse is true for conformer 2. Also, the energy ordering of conformers 1 and 2 is reversed from the RHF level, where conformer 2 is lowest in energy, to the MP2 level, where conformer 1 is the lowest energy
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Table 4. Inertial Principal-Axis Atomic Coordinates (Å) of Namide from Kraitchman Analysis and MP2/6-311þþG(d,p) Calculations conformer 1
conformer 2
coordinate Kraitchman ab initio Kraitchman ab initio
conformer 3 ab initio
a
2.2396(7) 2.067
2.7015(6)
2.709
2.401
b
0.389(4)
0.329(5)
0.295
1.315
c
1.055(1)
1.183 0.721(2)
0.714
0.620
0.352
Figure 3. Relative energy (MP2/6-311þþG(d,p)) as a function of the change in χ1 from the MP2 optimized structure of leucinamide conformer 1.
conformer. The principal differences between the two levels of theory for conformer 1 are the dihedral angles Ψ, which are 33.8° at the RHF level and 36.5° at the MP2 level, and χ1, which are 58.2° and 53.7° at the RHF and MP2 levels, respectively (see Figure 1). Single-point energy calculations were performed at the MP2/6-311þþG(d,p) level (without zero point correction) on a series of intermediate structures with incremental values of χ1; the calculations revealed that the potential energy surface is relatively flat along the χ1 coordinate (the relative energy rises
