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Microwave spectroscopy of biomimetics molecules Isabelle KLEINER Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), Créteil, France Nice, 15-16 Sept 2009 What do we call « Biomimetic Molecules » ? Small molecules forming the elementary blocks of biomolecules: amino acids, small peptides, nucleic acids, sugars… Can serve as validation tools relatively small molecules are the favourite candidates for most oral drugs (so-called « Lipinsky rule »): -molecular weight of 500 or less, -not more than 5 hydrogen-bond donor sites, -not more than 10 hydrogen-bond acceptor sites Jorgensen, “Drug Discovery”, Science, 303, 1813 (2004) Today, what systems will we talk about? Proteins are formed by a reservoir of 20 amino acids. Amino acids are related by peptidic bondings to form polypeptides Backbone chain Side chain Residue 1 Residue 2 Residue3 Peptide link: rigid, planar Formation of peptide link by condensation and elimination of water Only certain values of the Ramachadran angles f and Y are possible Hydrogen Bond structure Primaire Secondaire Tertiaire Quaternaire g turns b feuillets a helice Primary structure Systems between 1 to 5 amino acids residues (up to a few hundred Daltons) Optical spectroscopic techniques (microwave, millimeter wave, terahertz, infrared, UV/visible). Determination of effective neutral molecular structures Comparison with quantum mechanical calculations at equilibrium. Advantages : functional-group and conformational specificity. Challenge : getting good signal-tonoise Secondary 5 to 30 amino-acid residues Tertiary Quaternary Above about 30 amino acids Experimental measurement of electric dipole moment or diffusion velocity in a gas (« ion mobility ») Mass spectrometry Such measurements can be coupled with “hybrid” calculation methods Advantages: Many proteins acquire their secondary or tertiary structures when they bond. Advantages: peptidic « maps » dipoles/mass to identify proteins Challenge: Mass spectrometry does not give structures directly. For macromolecular systems, modelling using a classical force field (AMBER and CHARMM softwares). Challenge: need a structure calculation Determination of complexation of the biomolecule by ligands. Coupling mass spectrometry with spectroscopy (Oomens, Meyer et al, 2005, Kapota , Maïtre, Ohanessian et al JACS, 2004).IR/UV or UV/UVhole-burning spectroscopy (Mons et al, Zwier et al, Gerhards et al, Simons et al…) Hydrogen Bond & Torsion Secondary and tertiary structure of proteins How is Microwave spectroscopy at high resolution going to contribute ??? : Internal rotation splittings can be used to obtain the structure/folding of molecules in gas phase WITHOUT doing isotopic substitution. Lavrich et al. JCP 2003 What is internal rotation? Microwave is a good spectral range to determine very accurately molecular structures but the size of the molecule is limited Erot JKaKc 110 111 101 Rigid rotor (zero order): Asymmetric top, rotation structure characterized by the quantum numbers J, Ka, Kc: 000 B+C A+C A+B Limit size of the molecule Detected by most of FourierTransform spectrometers (4-20 GHz) : 250-300 uma Peptidic bonding and torsion : a few examples of molecules studied in MW Formamide Astrophysical detection: Rubin et al, ApJ 1971, Brown et al JMS 1987 Acetamide Potential Barrier : V3 = 25 cm-1 ; Ilyushin et al, JMS 2003, 1 top low barrier, Cs frame Astrophysical detection : « The Largest Interstellar Molecule with a Peptide Bond », Hollis et al, ApJ 643 2006, L25 N-methylformamide Potential Barrier: V3 cis =60 cm-1, V3 trans = 279 cm-1, Kawashima, et al (Columbus 2002), Fantoni, Caminati, J. Mol. Struct., 2002 Examples : Acetamide derivatives N-Methylacetamide V3(1)=36 cm-1, V3(2)=42 cm-1 ; Ohashi, Hougen et al JMS 2004 2 tops problem, Cs frame N-Methylpropionamide V3(1)=796 cm-1, V3(2)=81 cm-1 ; Kawashima, Hirota et al JMS 2003 N-Ethylacetamide V3= 75 cm-1 ; Kawashima (Dijon 2003) This talk: dipeptidic derivatives Collaboration NIST (Gaithersburg, USA), PhLAM (Lille) N-acetyl-alanine N’- methylamide (AAMA) V3(1)=98 cm-1, V3(2)=81 cm-1 Observé Lavrich et al. JCP 2003 Ethyl AcetamidoAcetate (EAA) or N acetylglycine ethyl ester Alanine Dipeptide Methyl Ester (ADME) Methylcarbamate Collaboration with Institute for Radioastronomy of NASU (Ukraine), PhLAM (Lille), University of Eotvos (Budapest) METHYLCARBAMATE : isomer of glycine, Plausible candidate for an astrophysical detection because more stable than glycine Glycine Rotation-torsion MW spectrum: Ilyushin et al., J. Mol. Spectrosc., 240, 127 (2006). NH2COOCH3 Good candidate for validation of high level quantum chemical Calculations: Equilibrium vs. Ground-State Planarity of the CONH Linkage ? Demaison et al., J. Phys. Chem. A., 111, 2574-2586 (2007). HOW TO MODEL INTERNAL ROTATION? For one C3v top, and a frame with a plane of symetry Cs HRAM = Htor + Hrot + Hd.c + Hint 1) Diagonalization of the torsional part of the Hamiltonian in an axis system where torsion-rotation coupling is minimal (Rho Axis Method, RAM), Kirtman et al, Lees and Baker , Herbst et al: Htor= F (pa - r.Jz)2 + V(a) F: internal rotation constant r depends on Itop/Imolecule Eigenvalues = torsional energies 2) Eigenvectors are used to set up the matrix of the rest of the Hamiltonian: Hrot = ARAMJa2 + BRAMJb2 +CRAMJc2 + Dab(JaJb + JbJa) Hd.c usual centrifugal distorsion terms Hint higher order torsional-rotational interactions terms : cos3a et pa and global rotational operators like Ja, Jb , Jc Theoretical Model: the global approach RAM = Rho Axis Method (axis system) for a Cs (plane) frame HRAM = Hrot + Htor + Hint + Hd.c. Torsional operators and potential function V(a) Rotational Operators Constants 1 1-cos3a p2a Japa 1-cos6a p4a Jap3a V3/2 F r V6/2 k4 k3 J2 (B+C)/2* Fv Gv Lv Nv Mv k3J Ja2 A-(B+C)/2* k5 k2 k1 K2 K1 k3K Jb2 - Jc2 (B-C)/2* c2 c1 c4 c11 c3 c12 JaJb+JbJa Dab or Eab dab Dab dab dab6 DDab ddab 1 Kirtman et al 1962 Lees and Baker, 1968 Herbst et al 1986 a = angle of torsion, r = couples internal rotation and global rotation, ratio of the moment of inertia of the top and the moment of inertia of the whole molecule Hougen, Kleiner, Godefroid JMS 1994 Internal Rotation Programs http://info.ifpan.edu.pl/~kisiel/prospe.htm: programs for rotational spectroscopy (Z. Kiesel) Name authors what it does? Method _______________________________________________________________________ XIAM Hartwig up to 3 sym tops combined RAM-PAM Maeder up to one quad. (based on Woods method) nucleus Separate vt fit, sometimes separate A and E fits _______________________________________________________________________ ERHAM Groner one and two Effective, combined RAM-PAM internal rotors Separate vt states fit of sym.C3v or C2v J up to 120. acetone,diMEether 8191 lines max MeCarbamate intensities ________________________________________________________________________ BELGI Kleiner one C3v internal RAM method Godefroid, rotor. Frame can Global fit of vt states Hougen Cs or C1 A and E species fit together Xu, Ortigoso, J up to 70 Ilyushin, vt up to 11 acetaldehyde, acetic acid Carvajal intensities acetamide,MeFormate 1 or 2 different MeCarbamate, EAA vibrational states dipeptide alanine ester Internal Rotation Programs (suite) Name authors what it does? Method ______________________________________________________________________ JB95 Plusquellic one internal rotor PAM Separate vt states, separate A and E fits graphical interface alanine dipeptide and many other molecules http://physics.nist.gov/Divisions/Div844/facilities/uvs/jb95userguide.htm ______________________________________________________________________ SPFIT/ Pickett one or two internal Combined RAM-PAM SPCAT rotors, sym or asym. Separate vt states, separate A and E fits propane, pyruvic acid acetaldehyde (more recent) ______________________________________________________________________ Results : Ethyl AcetamidoAcetate 1. R. J. Lavrich, A. R. Hight Walker, D. F. Plusquellic, I. Kleiner, R. D. Suenram, J. T. Hougen, and G. T. Fraser, JCP 119 (2003) 5497 Experimental problems : Biomolecules Properties Spectrometer MWFT NIST (9-18 GHz) Liquid or solid Injection with reservoir nozzle Low vapor pressure Heated reservoir nozzle (135-155°C) Thermal instability Injection with inert material Multi-conformations Jet at 1K to simplify the spectra Internal rotation splittings Large spectral range investigated Nitrogen quadrupole Synthesis of 15N isotopomers Microwave spectra of EAA T = 150°C Two conformers identificated : CI and CII CII: « non planar ? Structures MP2/6-311G(d,p) CI : « planar » EAA (15N) : a good case for comparing the JB95 and BELGI codes J up to 20, K up to 6 JB95 «High barrier, perturbative approach» BELGI « Global approach » CI 160 A lines, rms = 1.7 kHz 197 E lines, rms = 1.8 kHz 160A+197E lines, rms = 1.8 kHz CII 165 A lines, rms = 1.4 kHz 203 E lines, rms = 1.3 kHz 165A+203E lines, rms = 1.7 kHz For the CII conformer (non-planar), a C1 global code was written (JCP 119, 5505 (2003) EAA: CH3 group orientations in PAS V3(1) determined ; V3(2) too high, not determined BELGI A,B,C (EAA) JB95 BELGI JB95 Comparisons with ab initio calculations do not predict the correct experimentally observed energy ordering for the two conformers ! problem of data basis/method ? : MP2/6-311G(d,p) Ab initio qcalc qcalc-qobs planar non planar Alanine Dipeptide Methyl Ester I. Kleiner, J. Demaison, D. F. Plusquellic, R. D. Suenram, R. J. Lavrich, F. J. Lovas, G. T. Fraser, V. V. Ilyushin, JCP (2006) Theoretical problems: Develop new models for molecules which has no plane of symmetry for the frame(1) AND have more than one methyl internal rotation groups Deal with the hyperfine structure Deduce structural informations and compare them with the ab initio calculations results (1) I. Kleiner and J.T. Hougen, J. Chem. Phys. 119 (2003) 5505, voir EAA. ADME: 2methyl tops Fits: for each internal rotor about 120 lines RMS: 2 kHz N-methylacetamide: N. Ohashi, J. T. Hougen, R. D. Suenram, F. J. Lovas, Y. V3(3) high Kawashima, M. Fujitake, and J. Pyka, JMS JKaKc 3 sets of torsional splittings: V3 = 68 cm-1 D1 = 2 cm-1 (AA,AE). V3 = 400 cm-1 (AA,EA). D2 = 0.01 cm-1 Interaction between the 2 tops: very small splittings. NOT TREATED (AA,EE). ADME MW spectrum Experimentally deduced molecular parameters for ADME Good agreement between the global and perturbation approaches Torsional parameters better determined when V3 is smaller AR / MHz BR / MHz CR / MHz Rot. Tors Eab / cm-1 Ebc / cm-1 Eac / cm-1 Global Fit :BELGI (AA,EA) States (AA,AE) States LOW Barrier HIGH Barrier 2998.7(2) 669.23(6) 596.97(2) -0.0163809(2) 0.000528077(6) 0.fixed -0.00714(9) 0.00031(6) 0.fixed ρ V3 / cm-1 F / cm-1 0.013375(1) 64.96(4) 5.341(2) 0.01768(2) 396.45(7) 5.30fixed θa / ° θb / ° θc / ° 44.86(1) 46.75(1) 80.36(1) 22.5(1.5) 67.7(1.0) 87.0(3.5) PERTUR. JB95 (AA,EA) States (AA,AE) States LOW Barrier HIGH Barrier 2998.1(7) 670.05(8) 596.29(2) -0.0147019(1) 0.0019754(1) 0.00717fixed 0.01336(5) 66.35(5) 5.30fixed 44.94(5) 46.59(5) 80.58(5) 0.fixed 0.fixed 0.fixed 0.01719(1) 402(4) 5.30fixed 25.0(7.7) 65.2(8.5) 86.4(1.2) Conformational searches, Structure and hydrogen bond 13 stable conformers of ADME located, full geometry optimisations with B3LYP/6-31G(d) et G3MP2B3 Comparison of ab initio structure for AAMA (alanine dipeptide) et ADME (N-acetyl alanine methyl ester) AAMA ADME φ ψ C5 C7 Ramachandran angles Ψ 75° φ -82° Similar to a g-turn structure Ramachandran angles Ψ 171° φ -159° Similar to a b-sheet structure Ab initio calculations : structural comparisons of ADME φ ψ MP2 et B3LYP: base cc-p-VTZ, Gaussian03 ; PW91 et HCTH: double numerical basis, DMol Expt. MP2 B3LYP PW91 HCTH A / MHz 2998.4(3) -16.1 +54.6 +114.0 +138.6 B / MHz 669.6(4) -5.6 +2.4 +3.1 +5.2 C / MHz 596.6(3) -2.9 +1.6 +1.3 -1.8 θa / ° d 44.9(1) +1.9 +2.1 +1.9 +3.1 θb / ° d 46.7(1) -1.4 -1.8 -1.4 -2.9 θc / ° d 80.5(1) -1.4 -1.0 -1.5 -1 V3 / cm θa / ° θb / ° d θc / ° d d -1 V3 / cm φ/° ψ/° rN-H---O=C θN-H---O=C V3 / cm-1 65.6(7) -2.7 +20.1 23.7(1.2) 66.5(1.2) -2.8 +3.0 -2.0 +2.2 -2.9 +3.1 86.7(3) -1.6 -3.1 -3.2 399.2(3.0) +0.3 -159.4 171.1 2.218 105.4 +145.6 -155.5 169.8 2.239 105.2 -22.2 +166.3 -0.4 e -52.6 e -1.2 +1.4 -2.2 e -153.3 169.2 2.234 105.7 +99.9 e -150.8 166.9 2.291 103.9 1229.7 DFT (B3LYP) gives rotational constants too small and MP2 too big. DFT overestimates the structure, MP2 underestimate it ! Methylcarbamate Equilibrium structure of Methyl carbamate is not planar! Method B3LYP B3LYP MP2_FC CCSD(T)_AE Basis VTZ AVTZ 6-311 VTZ AVTZ VQZ V(D,T)Z --------------------------------------------------------------------------------------------------- H9N1C2O3 Method Basis 13.12 10.18 12.59 17.59 16.02 15.88 16.52 a b c tot 0.163(2) 2.294(9) 0a 2.300(9) VTZ 0.222 2.412 0.757 2.538 AVTZ 0.204 2.462 0.671 2.560 VQZ 0.238 2.459 0.673 2.560 MP2 6-31G* 0.115 2.089 0.862c 2.263 CCSD(T) B3LYP V(T,D)Z VTZ 0.234 0.347 2.215 2.353 0.710 0.512 2.338 2.433 AVTZ 0.200 2.410 0.374 2.447 Exp. MP2 Ground state is planar: no out-of-plane terms needed to fit the spectrum, no c type transitions, c = 0 Ilyushin, Alekseev, Demaison, Kleiner JMS 2006 J up to 20, Ka up to 10 Methyl Carbamate Syn configuration Equilibrium vs. Ground-State Planarity of the CONH Linkage ? Jean Demaison, Attila G. Császár, Isabelle Kleiner, and Harald Møllendald Formamide (X = Y = H), carbamic acid (X = OH, Y = H), urea (X = NH2, Y = H), acetamide (X = CH3, Y = H), and methyl carbamate (MC, X = OCH3, Y = H): all except formamide have a pyramidalized N at equilibrium with a very small inversion barrier ! The effective structure (ground state) (determined by experimental microwave work) is however planar ALL ab initio optimizations indicate that the amide group is non planar (difference between planar and non planar is 53 cm-1 CCSD(T)/V(T,D)Z in apparent contradiction with experimental results (c is zero) WHAT’s GOING ON? MC behaves like other molecules containing the amino group: small barrier between planar and non planar and the ground torsional state is above this barrier. Kydd and Rauk, J. Mol. Struct. 1981 Conclusions : EAA and ADME The internal rotation splittings in vt = 0 from different peptide mimetics containing one or more CH3 groups have been analyzed with two different theoretical methods : “perturbative” and “global ” . Spectroscopic results were compared to quantum chemical calculations. Very good agreement for the internal rotor with a low potential barrier (larger splittings) Care for conclusions concerning the CH3 with a high barrier as no excited torsional states measured (small spittings, thus spectroscopic parameters less well determined). Higher order terms not taken into account Ab initio calculations relatively more precise for higher barriers; the choice of methods/bases must be pertinent. Conclusions: validation of ab initio calculations Torsional barriers at the MP2/cc-pVTZ level are in good agreement with experimental values. DFT barriers are 8 to 80% off! DFT overestimates the structure, MP2 underestimates (same discrepancy found with crystalline peptides : trialanine, THz absorption spectrum agrees with X-ray but not with DFT calculations, Siegrist et al, JACS, 128, 5764, 2006) Ab initio calculations at high level are very useful for Spectroscopists, since they can calculate precisely internal rotation parameters High resolution spectroscopy can be used to guide the choice/optimization of ab initio calculations! Conclusions : methyl carbamate formamide should not be considered as a general model of the amide linkage ! several molecules containing the CONH linkage seem to have a pyramidalized nitrogen at equilibrium and a double-minimum inversion potential with a very small inversion barrier allowing for an effectively planar groundstate structure Acetamide or methyl carbamate : good model for this UNDER COURSE : Trans and gauche conformer of ethyl acetate. Collaboration with Institute of Physical Chemistry, RWTH Aachen (Germany) W. Stahl, L. Nguyen, D. Jelisavac, L. Sutikdja, D. Cortés Gómez , H. Mouhib gauche conformer trans conformer Very few esters (even simple) have been studied so far by MW spectroscopy: - many atoms for isotopic substitution - Large internal rotation splittings - Different conformers Jelisavac et al. JMS 2009 Under course :Microwave Study of Phenyl Alanine Methyl Ester: Reducing the Complexity of Confomational Searches Douglass, Roe, Plusquellic, Pratt and Pate Previous works: IR-R2PI spectroscopy and DFT ab initio (Gerharts et al) γLg+ E βL(g-) βL(a) δd(g+) βL(g+) Lowest energy conformers MP2/6-311++G** Now: - mini-FTMW (NIST) :12-18 GHz - Semi-Confocal Chirped-Pulse FTMW : 12.6-18 GHz, makes possible the recording of the complete microwave spectrum of a gas phase sample using a single 1 μs pulse. -assignment of overlapping sub-bands : genetic algorithms (L. Meerts) Perspectives: towards larger biomimetic molecules? Experimental challenge: -nondestructively vaporizing fragile biomimetics: laser ablation Theoretical challenge : -extend present modeling using effective Hamiltonians and codes to describe more complicated system (containing two or more internal rotors CH3). Methyl acetate CH3COOCH3 : collaboration with Jon Hougen Sonia Melandri (Bologna), Lilian Sutikdja …. -transfer the information obtained by gas phase MW high resolution spectroscopy to biomolecules in a cell environnement! National Institute For Standards And Technology (NIST, USA) Jon Hougen David Plusquellic Richard Lavrich Richard Suenram Frank Lovas Gerald Fraser Angela Hight Walker Laboratory of Molecular Spectroscopy (Budapest, Hungary) Attila G. Császár Laboratoire de Physique des Lasers, Atomes, et Molécules (Lille, France) Jean Demaison, L. Margulès, Th. Huet, R. Motyenko, M. Tudorie Institute of Radio Astronomy of NASU (Kharkov, Ukraine) Vadim Ilyushin Physical Chemistry, RWTH Aachen (Germany) Eugene Alekseev W. Stahl L. Nguyen D. Jelisavac L. Sutikdja D. Cortés Gómez , H. Mouhib