1. Microwave spectroscopy of biomimetics molecules Isabelle KLEINER Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), Créteil, France Nice, 15-16 Sept 2009

2. 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)

3. 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 Peptide link: rigid, planar Residue 1 Residue 2Residue3 Formation of peptide link by condensation and elimination of water Only certain values of the Ramachadran angles  and  are possible Backbone chain Side chain

4.  feuillets  helice structurePrimaire Secondaire Tertiaire Quaternaire Hydrogen Bond  turns

5. Primary structure Secondary Tertiary Quaternary residues 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-to- noise 5 to 30 amino-acid residues Experimental measurement of electric dipole moment or diffusion velocity in a gas (« ion mobility ») Such measurements can be coupled with “hybrid” calculation methods Advantages: peptidic « maps » dipoles/mass to identify proteins Challenge: need a structure calculation Above about 30 amino acids Mass spectrometry Determination of complexation of the biomolecule by ligands. Advantages: Many proteins acquire their secondary or tertiary structures when they bond. Challenge: Mass spectrometry does not give structures directly. For macromolecular systems, modelling using a classical force field (AMBER and CHARMM softwares). 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…)

6. 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

7. What is internal rotation?

8. Microwave is a good spectral range to determine very accurately molecular structures but the size of the molecule is limited Rigid rotor (zero order): Asymmetric top, rotation structure characterized by the quantum numbers J, Ka, Kc: Limit size of the molecule Detected by most of Fourier- Transform spectrometers (4-20 GHz) : 250-300 uma

9. 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 : V 3 = 25 cm -1 ; Ilyushin et al, JMS 2003, 1 top low barrier, C s frame Astrophysical detection : « The Largest Interstellar Molecule with a Peptide Bond », Hollis et al, ApJ 643 2006, L25 N-methylformamide Potential Barrier: V 3 cis =60 cm -1, V 3 trans = 279 cm -1, Kawashima, et al (Columbus 2002), Fantoni, Caminati, J. Mol. Struct., 2002

10. Examples : Acetamide derivatives N-Methylacetamide V 3 (1)=36 cm -1, V 3 (2)=42 cm -1 ; Ohashi, Hougen et al JMS 2004 2 tops problem, C s frame N-Methylpropionamide V 3 (1)=796 cm -1, V 3 (2)=81 cm -1 ; Kawashima, Hirota et al JMS 2003 N-Ethylacetamide V 3 = 75 cm -1 ; Kawashima (Dijon 2003)

11. This talk: dipeptidic derivatives N-acetyl-alanine N’- methylamide (AAMA) V 3 (1)=98 cm -1, V 3 (2)=81 cm -1 Lavrich et al. JCP 2003 Alanine Dipeptide Methyl Ester (ADME) Ethyl AcetamidoAcetate (EAA) or N acetylglycine ethyl ester Collaboration NIST (Gaithersburg, USA), PhLAM (Lille) Observé

12. Methylcarbamate METHYLCARBAMATE : isomer of glycine, Plausible candidate for an astrophysical detection because more stable than glycine 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). Glycine NH 2 COOCH 3 Rotation-torsion MW spectrum: Ilyushin et al., J. Mol. Spectrosc., 240, 127 (2006). Collaboration with Institute for Radioastronomy of NASU (Ukraine), PhLAM (Lille), University of Eotvos (Budapest)

13. HOW TO MODEL INTERNAL ROTATION? For one C 3v top, and a frame with a plane of symetry C s H RAM = H tor + H rot + H d.c + H int 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: H tor = F (p  - .J z ) 2 + V(  ) F: internal rotation constant  depends on I top /I molecule Eigenvalues = torsional energies 2) Eigenvectors are used to set up the matrix of the rest of the Hamiltonian: H rot = A RAM J a 2 + B RAM J b 2 +C RAM J c 2 + D ab (J a J b + J b J a ) H d.c usual centrifugal distorsion terms H int higher order torsional-rotational interactions terms : cos3  et p  and global rotational operators like J a, J b, J c

14. Theoretical Model: the global approach H RAM = H rot + H tor + H int + H d.c. RAM = Rho Axis Method (axis system) for a C s (plane) frame Constants1 1-cos3  p2p2 JapJap 1-cos6  p4p4 Jap3Jap3 1V 3 /2F  V 6 /2k4k4 k3k3 J2J2 (B+C)/2*FvFv GvGv LvLv NvNv MvMv k 3J Ja2Ja2 A-(B+C)/2*k5k5 k2k2 k1k1 K2K2 K1K1 k 3K J b 2 - J c 2 (B-C)/2*c2c2 c1c1 c4c4 c 11 c3c3 c 12 JaJb+JbJaJaJb+JbJa D ab or E ab d ab  ab  ab d ab6  ab  ab Torsional operators and potential function V(  ) Rotational Operators Hougen, Kleiner, Godefroid JMS 1994  = angle of torsion,  = couples internal rotation and global rotation, ratio of the moment of inertia of the top and the moment of inertia of the whole molecule Kirtman et al 1962 Lees and Baker, 1968 Herbst et al 1986

15. Internal Rotation Programs Nameauthors what it does? Method _______________________________________________________________________ XIAMHartwig up to 3 sym tops combined RAM-PAM Maeder up to one quad. (based on Woods method) nucleus Separate v t fit, sometimes separate A and E fits _______________________________________________________________________ ERHAMGronerone and two Effective, combined RAM-PAM internal rotorsSeparate v t states fit of sym.C 3v or C 2v J up to 120. acetone,diMEether 8191 lines max MeCarbamate intensities ________________________________________________________________________ BELGI Kleinerone C 3 v internal RAM method Godefroid,rotor. Frame can Global fit of vt states HougenC s or C 1 A and E species fit together Xu, Ortigoso,J up to 70 Ilyushin,vt up to 11 acetaldehyde, acetic acid Carvajalintensities acetamide,MeFormate 1 or 2 different MeCarbamate, EAA vibrational statesdipeptide alanine ester http://info.ifpan.edu.pl/~kisiel/prospe.htm: programs for rotational spectroscopy (Z. Kiesel)

16. Internal Rotation Programs (suite) Nameauthors what it does? Method ______________________________________________________________________ JB95Plusquellic one internal rotorPAM Separate v t states, separate A and E fits alanine dipeptide graphical interfaceand many other molecules http://physics.nist.gov/Divisions/Div844/facilities/uvs/jb95userguide.htm ______________________________________________________________________ SPFIT/Pickettone or two internalCombined RAM-PAM SPCATrotors, sym or asym. Separate vt states, separate A and E fits propane, pyruvic acid acetaldehyde (more recent) ______________________________________________________________________

17. 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 Liquid or solid Low vapor pressure Thermal instability Multi-conformations Internal rotation splittings Nitrogen quadrupole Spectrometer MWFT NIST (9-18 GHz) Injection with reservoir nozzle Heated reservoir nozzle (135-155°C) Injection with inert material Jet at 1K to simplify the spectra Large spectral range investigated Synthesis of 15 N isotopomers

18. Microwave spectra of EAA Two conformers identificated : CI and CII CI : « planar » CII: « non planar » T = 150°C Structures MP2/6-311G(d,p) ?

19. EAA ( 15 N) : a good case for comparing the JB95 and BELGI codes J up to 20, K up to 6 JB95BELGI «High barrier, perturbative approach» « Global approach » CI160 A lines, rms = 1.7 kHz160A+197E lines, rms = 1.8 kHz 197 E lines, rms = 1.8 kHz CII165 A lines, rms = 1.4 kHz165A+203E lines, rms = 1.7 kHz 203 E lines, rms = 1.3 kHz For the CII conformer (non-planar), a C 1 global code was written (JCP 119, 5505 (2003)

20. EAA: CH 3 group orientations in PAS V 3 (1) determined ; V 3 (2) too high, not determined A,B,C (EAA) BELGI JB95

21. 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  calc  calc -  obs planar non planar

22. 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.

23. ADME: 2methyl tops N-methylacetamide: N. Ohashi, J. T. Hougen, R. D. Suenram, F. J. Lovas, Y. Kawashima, M. Fujitake, and J. Pyka, JMS JK a K c 3 sets of torsional splittings: (AA,EA). V 3 = 68 cm -1  1 = 2 cm -1 (AA,AE). V 3 = 400 cm -1  2 = 0.01 cm -1 (AA,EE). Interaction between the 2 tops: very small splittings. NOT TREATED Fits: for each internal rotor about 120 lines RMS: 2 kHz V 3 (3) high

24. ADME MW spectrum

25. Experimentally deduced molecular parameters for ADME Good agreement between the global and perturbation approaches Torsional parameters better determined when V 3 is smaller Rot. Tors

26. 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 C5C5 C7C7 ψ Ramachandran angles Ψ  171° φ  -159° Similar to a  -sheet structure φ Ramachandran angles Ψ  75° φ  -82° Similar to a  -turn structure

27. Ab initio calculations : structural comparisons of ADME MP2 et B3LYP: base cc-p-VTZ, Gaussian03 ; PW91 et HCTH: double numerical basis, DMol ψ φ DFT (B3LYP) gives rotational constants too small and MP2 too big. DFT overestimates the structure, MP2 underestimate it !

28. Methylcarbamate

29. Equilibrium structure of Methyl carbamate is not planar! MethodB3LYPB3LYP MP2_FC CCSD(T)_AE BasisVTZAVTZ 6-311 VTZ AVTZ VQZV(D,T)Z --------------------------------------------------------------------------------------------------- H9N1C2O313.1210.1812.5917.5916.0215.8816.52

30. 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 2006J up to 20, K a up to 10

31. 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

32. 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.

33. Kydd and Rauk, J. Mol. Struct. 1981

34. Conclusions : EAA and ADME The internal rotation splittings in v t = 0 from different peptide mimetics containing one or more CH 3 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 CH 3 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.

35. 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!

36. 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 ground- state structure Acetamide or methyl carbamate : good model for this

37. UNDER COURSE : Trans and gauche conformer of ethyl acetate. trans conformer gauche conformer Jelisavac et al. JMS 2009 Collaboration with Institute of Physical Chemistry, RWTH Aachen (Germany) W. Stahl, L. Nguyen, D. Jelisavac, L. Sutikdja, D. Cortés Gómez, H. Mouhib Very few esters (even simple) have been studied so far by MW spectroscopy: - many atoms for isotopic substitution - Large internal rotation splittings - Different conformers

38. Under course :Microwave Study of Phenyl Alanine Methyl Ester: Reducing the Complexity of Confomational Searches Douglass, Roe, Plusquellic, Pratt and Pate Lowest energy conformers MP2/6-311++G** Previous works: IR-R2PI spectroscopy and DFT ab initio (Gerharts et al) 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)

39. 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 CH 3 ). Methyl acetate CH 3 COOCH 3 : 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!

40. National Institute For Standards And Technology (NIST, USA) Jon Hougen David Plusquellic Richard Lavrich Richard Suenram Frank Lovas Gerald Fraser Angela Hight Walker Institute of Radio Astronomy of NASU (Kharkov, Ukraine) Vadim Ilyushin Eugene Alekseev 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 Physical Chemistry, RWTH Aachen (Germany) W. Stahl L. Nguyen D. Jelisavac L. Sutikdja D. Cortés Gómez, H. Mouhib