Microwave spectroscopy of biomimetics molecules

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Microwave spectroscopy of biomimetics molecules

• Isabelle KLEINER
• Laboratoire Interuniversitaire des Systèmes
  Atmosphériques (LISA), Créteil, France

Nice, 15-16 Sept 2009
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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)

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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
4
Hydrogen Bond
structure Primaire Secondaire
Tertiaire Quaternaire
g turns
b feuillets
a helice
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Primary structure
Secondary Tertiary
Quaternary
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
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
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)
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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
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What is internal rotation?
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Microwave is a good spectral range to determine
very accurately molecular structures but the
size of the molecule is limited
Limit size of the molecule Detected by most of
Fourier- Transform spectrometers (4-20 GHz)
250-300 uma
Rigid rotor (zero order) Asymmetric top,
rotation structure characterized by the quantum
numbers J, Ka, Kc
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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

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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)

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This talk dipeptidic derivatives
Collaboration NIST (Gaithersburg, USA), PhLAM
(Lille)

• Ethyl AcetamidoAcetate (EAA) or N acetylglycine
  ethyl ester

• N-acetyl-alanine N-methylamide (AAMA)
• V3(1)98 cm-1, V3(2)81 cm-1
• Lavrich et al. JCP 2003
• Alanine Dipeptide
• Methyl Ester (ADME)

Observé
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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).
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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
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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)
Constants 1 1-cos3a p2a Japa 1-cos6a p4a Jap3a
1 V3/2 F r V6/2 k4 k3
J2 (BC)/2 Fv Gv Lv Nv Mv k3J
Ja2 A-(BC)/2 k5 k2 k1 K2 K1 k3K
Jb2 - Jc2 (B-C)/2 c2 c1 c4 c11 c3 c12
JaJbJbJa Dab or Eab dab Dab dab dab6 DDab ddab
Rotational Operators

• 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

Kirtman et al 1962 Lees and Baker, 1968 Herbst
et al 1986
Hougen, Kleiner, Godefroid JMS 1994
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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
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Internal Rotation Programs (suite)
Name authors what it does?
Method _________________________________
_____________________________________ JB95 Plusque
llic one internal rotor PAM Separate
vt states, separate A and E fits
alanine dipeptide graphical
interface and many other molecules http//physics
.nist.gov/Divisions/Div844/facilities/uvs/jb95user
guide.htm _______________________________________
_______________________________ SPFIT/ Pickett on
e 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) ____________
__________________________________________________
________
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Results Ethyl AcetamidoAcetate

• 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
(135-155C) Injection with inert material Jet at
1K to simplify the spectra Large spectral range
investigated Synthesis of 15N isotopomers
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Microwave spectra of EAA

Two conformers identificated CI and CII
T 150C
CII  non planar 
?
Structures MP2/6-311G(d,p)
CI  planar 
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EAA (15N) a good case for comparing the JB95
and BELGI codes

• J up to 20, K up to 6
• JB95 BELGI
• High barrier, perturbative approach
   Global approach 
• CI 160 A lines, rms 1.7 kHz 160A197E lines,
  rms 1.8 kHz
• 197 E lines, rms 1.8 kHz
• CII 165 A lines, rms 1.4 kHz 165A203E lines,
  rms 1.7 kHz
• 203 E lines, rms 1.3 kHz

For the CII conformer (non-planar), a C1 global
code was written (JCP 119, 5505 (2003)
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EAA CH3 group orientations in PAS
V3(1) determined V3(2) too high, not determined
BELGI JB95 BELGI
JB95
A,B,C (EAA)
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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
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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.

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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. Kawashima, M. Fujitake,
  and J. Pyka, JMS

• 3 sets of torsional splittings
• (AA,EA). V3 68 cm-1
• D1 2 cm-1
• (AA,AE). V3 400 cm-1
• D2 0.01 cm-1
• (AA,EE). Interaction between the 2 tops very
  small splittings. NOT TREATED

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ADME MW spectrum
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Experimentally deduced molecular parameters for
ADME

• Good agreement between the global and
  perturbation approaches
• Torsional parameters better determined when V3 is
  smaller

Rot.
Tors
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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
f
?
C5
C7
Ramachandran angles ? ?171 f
?-159 Similar to a b-sheet structure
Ramachandran angles ? ?75 f
?-82 Similar to a g-turn structure
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Ab initio calculations structural comparisons
of ADME
f
?

• MP2 et B3LYP base cc-p-VTZ, Gaussian03 PW91 et
  HCTH double numerical basis, DMol

DFT (B3LYP) gives rotational constants too small
and MP2 too big. DFT overestimates the
structure, MP2 underestimate it !
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Methylcarbamate
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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 13.12 10.18 12.59 17.59 16.02 15.88 16.52
  

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Ground state is planar no out-of-plane terms
needed to fit the spectrum, no c type
transitions, mc 0
Ilyushin, Alekseev, Demaison, Kleiner JMS 2006
J up to 20, Ka up to 10
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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
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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 (mc is zero) WHATs 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.
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Kydd and Rauk, J. Mol. Struct. 1981
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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.

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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!

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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

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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
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Under course Microwave Study of Phenyl Alanine
Methyl Ester Reducing the Complexity of
Confomational SearchesDouglass, Roe,
Plusquellic, Pratt and Pate
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)
Lowest energy conformers MP2/6-311G
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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!

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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 Eugene Alekseev
Physical Chemistry, RWTH Aachen (Germany) W.
Stahl L. Nguyen D. Jelisavac L. Sutikdja D.
Cortés Gómez , H. Mouhib