1. The Rotational Spectrum of N-Acetyl Phenylalanine Methyl Ester Measured with a Medium Bandwidth (100 MHz) Chirped-Pulse Fourier Transform Microwave Spectrometer Kevin O. Douglass, Francis Lovas, Kevin Davis, Karen Siegrist, David F. Plusquellic National Institute of Standards and Technology Biophysics Group Brooks H. Pate Department of Chemistry, University of Virginia David W. Pratt Department of Chemistry, University of Pittsburg

2. N-Acetyl-Phenylalanine Methyl Ester Single Conformer Observed 1,2 1. Gerhards, M. and Unterberg, C.; Phys.Chem.Chem.Phys. 2002, 4, 1760-1765. 2. Gerhards, M.; Unterberg, C.; Gerlach, A.; Jansen, A., Phys.Chem.Chem.Phys. 2004, 6, 2682-2690. 3. Lavrich, R. J.; Plusquellic, D. F.; Suenram, R. D.; Fraser, G. T.; Walker, A. R. H.; Tubergen, M. J., J.Chem.Phys.2004, 118, 1253-1265. Methyl groups T 1 and T 2 have low barriers to internal rotation. -Fit AA, AE and EA states  methyl top angles provide added structural information -Ramachandran angles 3 φ and ψ -Side Chain dihedral angles χ 1 and χ 2 φ ψ χ1χ1 χ2χ2 T1T1 T2T2

3. Challenges for studying peptide systems Low signal intensity –Population spread over many levels –Population potentially further spread by multiple conformers –Low vapor pressure –Sample decomposition Many variable search –Temperature, MW power, pulse timing, line position

4. Microwave Spectrometer Designs Bandwidth 0.5 MHz 1 Extremely high sensitivity on a single shot (typ. 10 avgs.) Requires low MW input power (~0.1 mW) Very slow scan speed (14 MHz/min) –12,000 steps (5 GHz) Bandwidth 12000 MHz 2 Equivalent Sensitivity reached at 10,000 avgs Requires high input power (~1 kW) Fast data acquisition time –1 Step (12 GHz) –17 min. Bandwidth ~100 MHz Equivalent Sensitivity reached at ~100 avgs. Requires moderate MW input power (~1 W) 170 MHz/min ~80 Steps (5 GHz 30 min) Q=10,000 Q=~100 Q=1 Broad search window with the cavity enhancement For large systems there is high probability of observing a line in a single step 1.Balle, T. J.; Flygare, W. H., Rev.Sci.Instrum. 1981, 52, 33-44. 2 G. G. Brown, B. C. Dian, K. O. Douglass, S. M. Geyer, B. H. Pate, Rev. Sci. Inst, 79 053103 (2008).

5. Semi-Confocal CP-FTMW 1,2,3 1. G. G. Brown, K. O. Douglass, B. C. Dian, S. M. Geyer and B. H. Pate, “SEMI-CONFOCAL CAVITY AND OTHER EXPERIMENTS IN BROADBAND FOURIER TRANSFORM MICROWAVE (FTMW) SPECTROSCOPY”, 60 th International Symposium on Molecular Spectroscopy, Columbus OH 2005 2. Brown, G. G.; Dian, B. C.; Douglass, K. O.; Geyer, S. M.; Pate, B. H., J.Mol.Spectrosc., 238, 200-212. (2006) 3. G. G. Brown, B. C. Dian, K. O. Douglass, S. M. Geyer, S.T. Shipman, B. H. Pate, Rev. Sci. Inst, 79 053103 (2008). 4. Hansen N, Andresen U, Dreizler H, Grabow JU, Mader H, Temps F, Chem. Phys. Lett. 289, 311-318 (1998) Directly Digitize IF signal (Time domain 100 – 200  s) Fourier Transform to Frequency Domain DC – 200 MHz - 100 MHz Synthesizer 1 L.O. 10-18 GHz 1-100 MHz Chirp (1 μs) 1 GS/s 2 Channel 500 MHz Digital Oscilloscope Synthesizer 2 Down Convert Coaxial nozzle alignment Arbitrary Function Generator 2GS/s DC-240 MHz ± 1-100 MHz Semi-confocal cavity 4 10 MHz Ru clock Up convert chirped pulse (upper and lower sidebands) LNA IF FFT 1 W Amp

6. Chirped Pulse: Generation and Diagnostics Obs. Signal 100 - 300 MHz - 200 MHz Synthesizer 1 L.O. 1-100 MHz Chirp (1 μs) Synthesizer 2 Down Convert Arbitrary Function Generator 2GS/s 240 MHz ± 1-100 MHz 10 MHz Ru clock 2 GS/s 4 Channel 500 MHz Digital Oscilloscope Relative Power (dB)

7. Mode Analysis MW foam

8. Cavity Design 9.50” 20” Rad. of CurvatureMirror separation ~12.5” Heated reservoir nozzle

9. Microwave Spectrum of Ac-Phe-OMe 1000 avgs/step 60 MHz step size ~1 GHz/hr scan speed Digitized for 20 μs SC-CP-FTMW Simulation 3K Nozzle Temp. 190 C

10. Microwave Spectrum of Ac-Phe-OMe 50 avgs/step 400 μs 2.5 kHz Semi-Confocal Cavity vs. High Q mini-FTMW Spectrometer 1000 avgs/step 100 μs 10 kHz

11. Cavity Design 9.50” 20” Rad. of CurvatureMirror separation ~12.5” Heated reservoir nozzle

12. Large vs. Small Aperture Setup 200 μs record length Q ~ 200 Q ~ 9000 250 avgs 2500 avgs EA AE AA 200 μs record length

13. Parameter AA State AE State EA State A / MHz 571.86288(4)571.86127(7)471.181(4) B / MHz 480.58355(5)480.58164(8) 529.058 (2) C / MHz 309.16440(3)309.16420(6) 360.315 (2) D a / MHz ----0.5093(2) 355.094 (9) D b / MHz ----0.50(4)---- D c / MHz ----0.1849(2)---- E ab / MHz -------- -54.201 (2) E ac / MHz -------- -86.540 fixed E bc / MHz ---------3.818(3) Δ JK / kHz -0.9673(4) -0.9667(6)0.79(4) Δ J / kHz 0.2148(1)0.2147(1)0.25(1) Δ K / kHz 1.3162(3) 1.3153(5) 1.3153(5)-1.79(1) δ J / kHz 0.1001(1)0.10011(5)0.134(2) δ K / kHz -0.0974(1)-0.0967(2)-0.367(4) G b / kHz --------4.96(9) G c / kHz ---------5.14(6) G aab / kHz ---------1.14(6) G aac / kHz ----3.46(5) G cca / kHz ----0.67(1) eQq aa /MHz -0.098(9)cc eQq bb /MHz -1.498(9)cc eQq cc /MHz 1.596(9)cc σ RMS / kHz 1.552.82.2 96 lines 8 par. 126 lines, 11 par. 137 line, 17 par. Fit Results 1 Conformer assigned φ χ1χ1 χ2χ2 T1T1 ψ T2T2 β L (g+) Lavrich, R. J.; Plusquellic, D. F.; Suenram, R. D.; Fraser, G. T.; Walker, A. R. H.; Tubergen, M. J., J.Chem.Phys.2004, 118, 1253-1265. Jb95 spectral fitting program

14. γ L g+ β L (g-) δd(g+) β L (a) β L (g+) 112 cm -1 (724 cm -1 ) 370 cm -1 (826 cm -1 ) 0 cm -1 475 cm -1 532 cm -1 B3LYP/6-31G(d,p) ZPE corr.* MP2 ZPE corr. cc-vdz *Gerhards, M. and Unterberg, C.; Phys.Chem.Chem.Phys. 2002, 4, 1760-1765. Lowest energy Conformers

15. Experiment vs. Theory Exp-Calc. B3LYPMP2MP2 Exp. Exp.6-311++G**6-311++G**cc-vtz A / MHz 571.8629-0.86 -38.7 -38.7-6.4 B / MHz 480.583536 14 14-17 C / MHz 309.164415.4 -7 -7-10 T1T1T1T1 V 3 /cm -1 433 (5) 130 -36 (15) -7 θaθaθaθa53(3)-12810 θbθbθbθb46(5)10-11-13 θcθcθcθc66(2)234 T2T2T2T2 V 3 /cm -1 46.39(5)-69 -8 (12) -16 θaθaθaθa62.59(8)0.52.83.5 θbθbθbθb56.7(1)1.91.7-0.3 θcθcθcθc45.7(1)-2.1-3.8-1.3 φ χ1χ1 χ2χ2 T1T1 ψ T2T2 β L (g+)

16. Experiment vs. Theory Exp-Calc. MP2 MP2 optimized Exp. Exp.cc-vtzGeometry A / MHz 571.8629-6.4-0.09 B / MHz 480.5835-17-0.075 C / MHz 309.1644-101.04 T1T1T1T1 θaθaθaθa53(3)102 θbθbθbθb46(5)-133 θcθcθcθc66(2)43 T2T2T2T2 θaθaθaθa62.59(8)3.50 θbθbθbθb56.7(1)-0.30 θcθcθcθc45.7(1)-1.30 Chi squared analysis: 9 observables 5 variables 4 dihedrals: ψ, φ, χ 1, χ 2 Ring to backbone angle: A φ χ1χ1 χ2χ2 T1T1 ψ A T2T2

17. Chi Squared Error Surface φ χ1χ1 χ2χ2 T1T1 ψ T2T2 MP2/cc-vtz MP2-OptDFT-Opt Chi 2 27 1043 DFT 60 64(8) χ 1 175173(10) ψ -168-171(4) φ - -87-98(4) 2 χ -119(9) 57(6) 183(1) -155(10) 111117(4) A 108(3) -85 63 173 -153 115

18. Conclusions Demonstration of a low and high Q Semi-Confocal Chirped-Pulse FTMW spectrometer 1.Low Q mode  Rapid scan to locate and optimize signal 2.High Q mode  increased sensitivity Demonstrated sensitivity for measuring the dipeptide analogue N-Ac-Phe-OMe N-Ac-Phe-OMe The microwave spectra of Ac-Phe-OMe has been fit. –The AA, AE, and EA states of a single conformer of have been assigned Barriers to internal rotation are –T 1 V 3 = 432(5) cm -1 –T 2 V 3 = 46.42(4) cm -1 –From analysis of the methyl top angles the peptide backbone (Ramachandran angles) and side chain have been determined

19. Acknowledgements The Pate Lab –Justin L. Neil –Dr. Steven T. Shipman Dr. Richard Suenram Dr. Gordon G. Brown National Research Council

20. Full calibration range

22. Large vs. Small Aperture Setup 200 μs record length Q ~ 200 Q ~ 9000 250 avgs 2500 avgs EA AA AE 20 μs record length AE

23. 30 MHz step size (could be more like 60 or 90)  30 or 20 min / 5GHz 20 microsecond data

26. 30 MHz step size Section of Suprane Scan Each color is a single step

27. Section of Suprane Scan 30 MHz 60 MHz 90 MHz 120 MHz Scan Step Size