Structure of the SEVOFLURANE-BENZENE complex as determined by CP-FTMW spectroscopy Nathan A. Seifert, Daniel P. Zaleski, Justin L. Neill, Brooks H. Pate University of Virginia Alberto Lesarri, Montserrat Vallejo Universidad de Valladolid Emilio J. Cocinero, Fernando Castano Universidad de País Vasco, Bilbao Isabelle Kleiner Laboratoire Interuniversitaire des Systèmes Atmosphériques

Sevoflurane Monomer Last year, Lesarri et al. 1 published a microwave and ab-initio study of the sevoflurane monomer. A DFT study of the torsional potential energy surface of the monomer reveals a single global minimum (CG), with two equivalent local minima (TR) corresponding to 180° degree rotations of the isopropyl group Global minimum structure fit experimentally, as well as 5 isotopologues corresponding to sevoflurane singly substituted with 13 C at all four positions, as well as the 18 O substitution. 1 A. Lesarri, et al. Phys. Chem. Chem. Phys. 2010, 12,

Previous Study Last year, Dom et al. published an IR/Raman study of the sevoflurane-benzene complex J. J. J. Dom et al. Phys. Chem. Chem. Phys., 2011, 13, Two clusters observed in Raman and IR slit jet and in liquid xenon cryosolution: Most abundant; formation enthalpies similar but isopropyl- bonded species is entropically favored

Why Benzene? Although the mechanism for anesthetic action is currently not well understood, studies suggest a path via direct binding to synaptic ligand/voltage-gate ion channel proteins 2. MD studies with the related ether anesthetic, halothane, suggest binding to hydrophobic residues, such as in nicotinic acetylcholine receptor (figure on left) 3. Additionally, a CD/NMR study from 2005 of sevoflurane with a four-α-helix bundle suggests binding interactions to phenylalanine residues 4 in the bundle. Conclusion Benzene provides a suitable choice to study non-covalent, potentially biochemically relevant, interactions with sevoflurane. 2 N.P. Franks, et al. Nature, 1998, 396, G. Brannigan, et al. PNAS. 2010, 107, R. Pidikiti, et al. Biochem. 2005, 44,

Experimental 2-8 GHz (top figure 5 ): Spectra taken with both perpendicular (as shown) and coaxial alignments. Coaxial alignment, although limited to one nozzle, narrows linewidths not only by minimizing Doppler broadening, but also by lengthening the T 2 dephasing time: FID collection time doubles from 40 to 80 μs Perpendicular limited to 2 nozzles. ~25 kHz FWHM in coaxial, ~120 kHz in perpendicular GHz (bottom figure 6 ): Only perpendicular arrangement used 3 nozzles 5 J.L. Neill, et al. J. Mol. Spec. 2011, 269, G.G. Brown, et al. Rev. Sci. Instru. 2008, 59, Gs/s AWG 50 Gs/s

Results M05-2X/6-31G+(d,p) calculations suggest a cluster structure containing the sevoflurane conformer predicted and fit by Lesarri, et al., with a C-H∙∙∙π interaction between the isopropyl hydrogen and the benzene ring, as shown below: ParameterM05-2XExperimental* A (MHz) B (MHz) C (MHz) μ a (D)2.34- μ b (D)0.33- μ c (D)1.68- * Experimental constants determined by averaging constants for A 1 and B 2 torsional symmetry states Problem: Internal rotation with six-fold symmetry

Internal Rotation C 6 symmetry, high barrier rotor Standard PAM fit with a minimum of parameters gets good results A, B, C fixed B, C fixed All float N138 (a) 86 (b) J max 11 (a) 9 (b) RMS (kHz)92 (a) 55 (b) 75 (a) 43 (b) 63 (a) 34 (b) I α fixed to benzene C constant (assume rotor axis normal to ring plane) A/B/C fixed to average of A and B state fits (RMS ~2 kHz for each) V 6 = 33(3) cm -1 MP2/6-311G++(d,p) predicts 48 cm -1 barrier

Internal Rotation Application of BELGI 7,8 improves fit dramatically 7 I. Kleiner and J. T. Hougen, J. Chem. Phys., 2003, 119, R. J. Lavrich, et al. J. Chem. Phys., 2003, 119, Fit by combining C 6 quartet into two pairs (B, E 2 ) and (A, E 1 ) in C 3 basis: (B, E 2 ): v t = 1←1 (A, E 1 ): v t = 0←0 In terms of input, functionally similar to a 3-fold rotor with two torsional states; These “virtual” torsional states are connected by fitting higher order torsion-rotation coupling terms 5.1 kHz fit for 135 lines up to J =11! ParameterOperatorValue A (MHz)Ja2Ja (5) B (MHz)Jb2Jb (7) C (MHz)Jc2Jc (1) D JK (kHz)-J 2 J a (2) D ab (MHz){J a, J b } 17.65(8) D ac (MHz)sin(3α){J a,J c } 24.1(1) N V (MHz)(1-cos(6α))J 2 5.5(1) V 6bc (MHz) (1-cos(6α))(J b 2 -J c 2 ) 14.1(4) K 2 (MHz)(1-cos(6α))J a 2 -76(5) ρ (unitless)PαJaPαJa (6) F (GHz)Pα2Pα2 [3.453] V 6 (cm -1 )(1-cos(6α))/ (3) # 135 RMS (kHz) 5.1

v t = 1←1 v t = 0←0 0.2% SF.2% Benzene Neon coax 258k avg Sevoflurane Monomer 0.2% SF 0.2% Benzene Neon coax 258k avg

Substitution Structure Experiment performed using.2% /.2% sevoflurane/benzene-d 1 in 100 psi Ne Using d 1 benzene breaks the symmetry of the rotor Rotor quartet per transition  6 singlets per transition, each representing a rigid rotor for a singly deuterated position on benzene 9 J. Kraitchman. Am. J. Phys., 21, 1953, * Average of A 1 and B 2 rotational constants NS*D(1)D(2)D(3)D(4)D(5)D(6) A (MHz) B (MHz) C (MHz) All but D(6) were assigned in 2-8 GHz spectrum. H(6) required sensitivity improvements in 6-18 GHz arrangement to detect.

Substitution Structure D(3) D(4) D(5) D(6) Sevoflurane coordinates taken from Lesarri et al. Benzene experimentally planar, with a small ~5° tilt relative to isopropyl C-H bond D(1) D(2) D(3) D(4) D(5) D(6) Sevoflurane positions adapted from Lesarri et al. 0.2% sevoflurane/0.2% benzene-d 1 Neon, coaxial, 316k avg D(6) transitions much weaker

92.35° 95.61° D(6) ° 2.34 Å

Conclusions & Future Work Substitution structure fit and calculated for the sevoflurane-benzene complex ~5 KHz fit for the high-barrier six-fold rotor, with a barrier of approximately 33 cm -1 Additional work needed on adapting RAM fit to the principal axis frame PAM fit still satisfactory on predicting barrier height, as compared to RAM fit Suggests that non-rigidity might not play a huge role in perturbing the torsion Expanding library to other volatile anesthetics No success with desflurane (Suprane)/benzene yet Isoflurane or halothane have yet to be studied in this context Conclusions: Future work: New binding candidates? Tyrosine has been suggested as a potential binding partner for halothane 10 – phenol? Other hydrophobic candidates might require laser ablation for gas-phase studies. 10 D.C. Chiara, et al. Biochem. 42, 2003,

Acknowledgements Thanks for listening! Research at UVa was supported by: National Science Foundation MRI-R2 project ( ).