Laser spectroscopic study of estrogen and its hydrated clusters in a supersonic jet ○ Fumiya Morishima, Yoshiya Inokuchi, Takayuki Ebata Graduate School of Science, Hiroshima Univ.

Introduction A D D Fukuzawa et al. Journal of Computational Chemistry In estrogen receptor Cegelski et al. Drug Development Research In membrane estriol(E 3 ) A D estrone(E 1 ) A D β-estradiol(E 2 ) A D estrogen B C B C B C

Introduction Investigate the structure of bare estrogen and its hydrated clusters to clarify their hydrogen bonding ability. estriol(E 3 ) A D estrone(E 1 ) A D β-estradiol(E 2 ) A D estrogen B C B C B C

Experimental and Computational vacuum chamber He gas IR laser UV laser ~4 atm Supersonic jet Vacuum chamber heater temperature estrone : ~150 ℃ estradiol : ~130 ℃ estriol : ~190 ℃ Electronic spectra LIF To differentiate bands UV-UV hole-burning(HB) Vibrational spectra IR-UV double resonance experimental Quantum chemical calculation M05-2X / G**

S 1 -S 0 Electronic Spectra of Bare Estrogen a bc d UV wavenumber / cm -1 LIF and UV-UV HB spectra of bare estrogen Number of conformer = 4 = 6 or more? = 2 Each conformer of estrogen originates from orientation of OH group(s) estrone estradiol estriol

IR Spectra of Bare Estrone UV wavenumber / cm IR wavenumber / cm -1 IR spectra in the region of OH stretching vibration IR spectra in the region of OH stretching vibration cis-estrone trans-estrone estrone(E 1 ) cis-estrone trans-estrone A D Number of conformer = 2 LIF

cis-estrone 1 2 UV wavenumber / cm -1 Assignment for cis- and trans- Conformation of Estrone trans-estrone Comparison of LIF spectra of estrone and results of TD-DFT calculation Comparison of LIF spectra of estrone and results of TD-DFT calculation cis-estrone trans-estrone

a bc d UV wavenumber / cm -1 estrone estradiol estriol Assignment for cis- and trans- Conformation of Estrogen cis-trans-

IR Spectra of Bare Estradiol UV wavenumber / cm -1 a b c d a b c d IR wavenumber / cm -1 β-estradiol(E 2 ) A-ring OH D-ring OH A-ring OH stretching vibrations… a, c show two bands b, d show only one band. A D estrone(E 1 ) A D IR spectra in the region of OH stretching vibration IR spectra in the region of OH stretching vibration Number of conformer = 4 cis- trans- LIF

16 17 Conformations of A-ring & D-ring anti: ~14cm -1 gauche(+): 0cm -1 gauche(-): ~73cm-1 cis trans anti gauche(+)

antigauche(+) Four conformers cis trans anti gauche(+) × Conformations of A-ring & D-ring 16 17

Assignment of D-ring OH Conformations a b c d IR wavenumber / cm -1 UV wavenumber / cm -1 a : anti b : gauche(+) c : anti d : gauche(+) cis -anti trans -gauche(+) cis -gauche(+) trans -anti

d: trans -gauche(+) b: cis -gauche(+) a: cis -anti c: trans -anti a b c d cis -anti trans -gauche(+) cis -gauche(+) trans -anti Assignment of bare estradiol

With water vapor Without water vapor Hydrated Cluster of Estradiol on A-ring UV wavenumber / cm -1 A B C D a b c d LIF and UV-UV HB spectra of monohydrated cluster of estradiol LIF and UV-UV HB spectra of monohydrated cluster of estradiol β-estradiol(E 2 ) Red shift = 350 cm -1 ~340 cm -1

IR Spectra of Hydrated Clusters of Estradiol on A-ring trans-anti-(H 2 O) 3641 band C trans-anti -(H 2 O) trans-anti ν 3 (H 2 O) ν 1 (H 2 O) trans-gauche(+) -(H 2 O) band D trans-gauche(+) ν 3 (H 2 O) ν 1 (H 2 O) trans-gauche(+)-(H 2 O) UV wavenumber / cm -1 C D LIF A B Each conformer forms hydrogen bond on A-ring OH as proton donor. ν(D-ring) ν(A-ring) ν(D-ring) ν(A-ring)

Another Isomer of Hydrated Cluster of Estradiol a b c d gauche(+) type proton donor anti type proton acceptor B C D Different patterns of hydrogen bond are conceivable for D-ring OH

IR Spectra of Bare Estriol UV wavenumber / cm -1 IR wavenumber / cm -1 IR spectra in the region of OH stretching vibration IR spectra in the region of OH stretching vibration estriol(E 3 ) A-ring OH cis region trans region IR wavenumber / cm -1 A-ring OH Band Bare estriol Band Hydrated cluster or its vibronic band A D cis- trans- LIF

Predicted Conformers of Bare cis-Estriol Conformational name Relative energy / cm c_16a_17a 810 3c_16g-_17a 208 3c_16g+_17a 740 3c_16a_17g c_16g-_17g c_16g+_17g c_16a_17g c_16g+_17g- 46.3

Intramolecular hydrogen bond Intramolecular hydrogen bond 3c_16a_17a 810 3c_16g-_17a 208 3c_16g+_17a 740 3c_16a_17g c_16g-_17g c_16g+_17g c_16a_17g c_16g+_17g Conformational name Relative energy / cm -1 Predicted Conformers of Bare cis-Estriol

IR Spectra of Bare cis-Estriol IR wavenumber / cm -1 UV wavenumber / cm estriol(E 3 ) 3c_16g-_17g+ 3c_16g-_17a 3c_16g+_17g Comparison of IR spectra of cis-estriol and results of frequency calculation Comparison of IR spectra of cis-estriol and results of frequency calculation 3c_16g-_17g c_16g+_17g c_16g-_17a 208 Bare estriol Form the intramolecular hydrogen bond between16OH and 17OH LIF cis-

Summary estrone(E 1 )β-estradiol(E 2 )estriol(E 3 )  monomer Assign the conformation of bare estrogen. Estriol forms intramolecular hydrogen bond  1:1 hydrated cluster of estradiol On A-ring Each conformer forms hydrogen bond on A-ring OH. On D-ring Different pattern of hydrogen bond are conceivable.  Future work Hydrated clusters of estrone / estriol Methoxy estrogen

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Relative energy of bare estrone / cm -1

anti gauche(-) gauche(+) Relative energy of bare estradiol / cm -1

≈ estradiol(A)-H 2 O estradiol(monomer) estradiol(D)-H 2 O Relative energy of clusters / cm -1

3c_16g-_17g c_16g+_17g c_16g-_17a208 3c_16a_17g-243 3c_16g+_17g+466 3c_16g+_17a740 3c_16a_17g+796 3c_16a_17a810 3t_16g-_17g t_16g+_17g t_16g-_17a207 3t_16a_17g-249 3t_16g+_17g+464 3t_16g+_17a730 3t_16a_17a796 3t_16a_17g+805 Relative energy of bare estriol Intramolecular hydrogen bond 16g-_17g+16g+_17g- 16g-_17a 16a_17g- 16g+_17g+16g+_17a 16a_17a 16a_17g+ / cm -1

IR Spectra of Bare Estrogen estradiol estrone estriol a b c d IR wavenumber / cm -1 IR spectra in the region of OH stretching vibration

cis-anti trans-anti trans-gauche(+) cis-gauche(+) Predicted Conformers M05-2X / G**

~410 cm -1 ~260 cm -1 Barrier between each D-ring conformation of estradiol ~820 cm -1 Without ZPE correction

810 cm -1 cis trans Rotation of A-ring OH Without ZPE correction

π (HOMO) π* (LUMO) cis-anti trans-anti HOMO and LUMO of Estradiol

cis -anti trans -gauche(+) cis -gauche(+) trans -anti a b c d cis -anti trans -gauche(+) cis -gauche(+) trans -anti Assignment of A-ring OH conformations

estradiol(D)-H 2 O Estradiol(D)-H 2 O cis-anti trans-gauche(+) cis-gauche(+) trans-anti cis-anti trans-gauche(+) cis-gauche(+) trans-anti gauche(+) type proton donor anti type proton acceptor red shift blue shift

1-cis-naphthol 1-trans-naphthol 2-trans-naphthol 2-cis-naphthol M05-2X / G** TD-DFT calculation

TD-DFT Calculation for conformers of bare estriol UV wavenumber / cm -1 trans- 16g+_17g-16g+_17a16g+_17g+16a_17g-16a_17a16g-_17a16a_17g+16g_17g+ Position / cm Relative energy / cm cis- 16g+_17g-16g+_17a16g+_17g+16a_17g-16a_17a16g-_17a16a_17g+16g_17g+ Position / cm Relative energy / cm cis- trans-

Lifetime Time / ns Monomer Cluster τ = 5 ns τ = 15 ns

Predicted Conformers of Bare trans-Estriol Conformational name Relative energy / cm t_16a_17a 796 3t_16g-_17a 207 3t_16g+_17a 730 3t_16a_17g t_16g-_17g t_16g+_17g t_16a_17g t_16g+_17g- 46.3

Predicted Conformers of Bare trans-Estriol 3t_16a_17a 796 3t_16g-_17a 207 3t_16g+_17a 730 3t_16a_17g t_16g-_17g t_16g+_17g t_16a_17g t_16g+_17g Conformational name Relative energy / cm -1

IR wavenumber / cm IR spectra of Bare Estriol 16g-_17g+ 16g+_17g- 16g-_17a 16a_17g- 16g+_17g+ 16g+_17a 16a_17a 16a_17g+ 16g-_17g+ 16g+_17g- 16g-_17a 16a_17g- 16g+_17g+ 16g+_17a 16a_17a 16a_17g+ cis region trans region Intramolecularhydrogen bond

Relative Energy of Monohydrated Estriol Isomers 3t_16g+_17g-_(A)-H 2 O3t_16g+_17g-_(D)-H 2 O 0.00 cm -1 ~1,000 cm -1 More stable

Relative Energy of Monohydrated Estradiol Isomers cis-gauche(+)-(A)-H 2 O cis-gauche(+)-(D)-H 2 O cis-anti-(D)-H 2 O 0.00 cm -1 ~330 cm -1 ~460 cm -1 More stable cis-anti-(A)-H 2 O 62.9cm -1