1. Elucidating the effects of amide side chain interactions in flexible biomolecules V. Alvin Shubert, Esteban E. Baquero, Jasper R. Clarkson, Timothy S. Zwier Department of Chemistry, Purdue University West Lafayette, IN 47907 M. J. Tubergen Department of Chemistry, Kent State University

2. Introduction Previous studies on the single conformation IR and UV spectroscopy and dynamics of conformational isomerization. Tryptamine Melatonin N-acetyl tryptophan methyl amide NATMA Above studies have led us to custom build molecules with different types of flexibility built in. p-methoxyphenethylacetamide 2-phenoxyethylacetamide O-(acetamidoethyl)-N-acetyltyramine MPEAmide POEA OANAT RI03 59 th MSS, WG02

3. Introduction Doubly substituted molecules Two branches to potential energy surface (PES) –Can branches be decoupled? –What types of chains allow for interchain interactions (e.g. H-bonding)? –What types of chains hinder interchain interactions? Dynamics studies –Breaking H-bonds –Put energy into one chain, observe effects on other chain

4. N-(2-phenylethyl)-acetamide N,N’-(1,4-phenylenedi-2,1- ethanediyl)bis acetamide 1,4-phenylenebis(oxy)bis- N-methylpropanamide- 2,2’-[1,4-phenylenebis(oxy)] bis-ethanamide 3,3’-(1,4-phenylenedi)bis-(N- methylpropanamide) O-(acetamidoethyl)-N- acetyltyramine 1,4 (N-methylpropanamide- N’-ethylacetamide)benzene N-methyl- benzenepropanamide 3-phenoxy-N- methylpropanamide 2-phenoxyethylacetamide Molecules under study

5. What types of low energy conformations will be formed for double chain molecules? How does the presence of the second chain affect the conformational preferences of the first chain and vice versa? Given n low energy structures for one chain and m for the other, might expect nXm conformations. However, this assumes two things: –Chains do not asymmetrize the benzene ring –Chains are non interacting Since chains do asymmetrize the benzene ring, # of conformations expected = nXmX # of spectroscopically distinct orientations If chains do interact, this may lead to structures containing individual chain conformations that were high energy in single chain molecules. The number of possible conformations gets very large very fast.

6. Disconnectivity Diagrams -44.0 -40.0 -36.0 -32.0 -28.0 -24.0 -20.0 -16.0 Energy (kcal/mol) POEA OANAT MPEAmide -415.0 -410.0 -405.0 -400.0 -395.0 -390.0 -385.0 -380.0 -375.0 -370.0 -365.0 -360.0 -355.0 -350.0 -89.0 -85.0 -81.0 -77.0 -73.0 -69.0 -65.0 -61.0 OPTIM.2.3 and Disconnect, David J. Wales, Cambridge University

7. Experimental methods Resonant 2 photon ionization (R2PI): Records spectra in mass selective fashion Biomolecule * (S 1 ) Biomolecule (S 0 ) Biomolecule + + e - Hole-burn Probe Conformer A Conformer B Hole-burn Probe UV Source fixed: Provides  selectivity IR Source tuned R2PI: Electronic Spectrum Resonant ion dip infrared spec- troscopy (RIDIRS): Conforma- tion specific IR spectrum UV-UV Hole-burning: Conformation specific electronic spectrum

8. Computational methods Search for lowest energy structures 1. Draw molecule in MacroModel, use conformation search. 2. Sift through structures found by MacroModel (Typically > 500). 3. Use unique structures as starting structures for optimizations using Gaussian03. Optimize at B3LYP/6-311+G* level of theory. 4. Using Gaussian03 and optimized structures, calculate infrared frequencies and intensities.

9. R2PI and UV-UV hole-burning spectra of OANAT Six conformations resolved to date. Four conformations (C,D,E,F) have origins within 85 cm -1 of 35,630 cm -1. Two conformations (A,B) are red shifted by more than 1000 cm -1 from the other four. A B C D E F

10. RIDIR spectra in amide NH stretch region RIDIR spectra indicate three classes of conformations for OANAT. Two stretches seen, one from each chain MPEAmide POEA OANAT

11. Summary of Experimental Results 2 major conformations for alkyl chain 1 major conformation for alkoxy chain Expected 8 conformations in OANAT, only found 6 Two classes of conformations: –H-bonding between chains –Independent chains Only 3 independent chain conformations found, not 8 as expected Interacting chains Independent chains

12. Computational results Single chain molecules NPEA, NMBPA, PNMP, and POEA Generally, find few low energy structures (< 0.2 kcal/mol, relative) (3, 2, 1, and 1, respectively) Most significant differences seen in vibrational frequencies are those associated with the ether linkages of PNMP and POEA MPEAmide, NPEBA, NMPNEA, PBNMP, PBOBEA, and OANAT Generally, find many low energy structures (6-10) Many have non-interacting chains and can be additively constructed from conformations found for single chain molecules Little shift seen in vibrational frequencies compared to single chain molecules when chains are non-interacting If chains interact (i.e. form an interchain H-bond), shifts are seen in frequencies, especially for NH and CO stretches Double chain molecules OCH 3

13. Combining single chains onto one molecule: Structures NPEA 0.000 kcal/mol 0.172 kcal/mol NMBPA 0.000 kcal/mol + NMPNEA 0.283 kcal/mol 0.329 kcal/mol 0.240 kcal/mol 0.414 kcal/mol

14. Computational results Combining single chains onto one molecule: Vibrational frequencies NPEA (0.000 kcal/mol) + NMBPA (0.000 kcal/mol) NPEA (0.172 kcal/mol) + NMBPA (0.000 kcal/mol) NMPNEA (0.283kcal/mol) NMPNEA (0.240 kcal/mol) NMPNEA (0.329 kcal/mol) NMPNEA (0.414 kcal/mol) NPEA NMBPA NMPNEA

15. What if chains do interact? Example, OANAT Interacting chains Independent chains A B C D E F B3LYP/6-31+G*, 6-311+G* Gaussian98 and Gaussian03 Alkyl NH · · · Alkoxy COAlkyl CO · · · Alkoxy NH Alkyl NH · · · Alkoxy CO Alkyl CO · · · Alkoxy NH

16. NPEBA: H-bond conformation at 0.006 kcal/mol, 3 more with H-bonds at < 0.75 kcal/mol PBNMP: Weak H-bonding in 2 conformations > 1.4 kcal/mol PBOBEA: No conformations with H-bonds found NMPNEA: Lowest energy conformation contains H- bond between NH of CONH and CO of NHCO Discussion What types of chains lead to interactions? –NHCO ordering leads to more interaction than CONH –Alkyl chains allow interaction between chains –O connecting chain to ring hinder interaction between chains –Different orderings of amide groups on opposite chains allows for more interaction How does interaction effect number of conformations? –Interchain interaction tends to increase number of low energy conformations Best design for a molecule with two decoupled branches to PES is to use CONH ordering for amide group and use O to connect chain to ring

17. Summary Six conformers of OANAT have been resolved to date Doubly substituted molecules are more than sum of their parts – simply building structures based on low energy conformations of single arm molecules is not adequate. Interactions between chains act to lower the energy of some chain configurations. Calculations are only a rough guide at our level of theory. Future Work Spectra in the 1000-2000 cm -1 region SEP and SEP hole-filling IR hole-filling: Selective excitation of amide in each chain Complete search for low energy conformations, disconnectivity diagrams Build different types of chains (i.e. vary number of carbons between amide group and chromophore, use different chromophores, and/or substitute into different positions, ortho or meta)

18. Acknowledgements People Prof. Timothy S. Zwier The Zwier Group Jasper R. Clarkson Esteban Baquero Tracy LeGreve Bill James Jaime Stearns Talitha Selby Josh Newby Prof. Michael J. Tubergen Prof. David Wales Dr. Dave Evans Prof. Mark Lipton Kevin Worrel Funding National Science Foundation