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Supporting Information for

Unconstrained peptoid tetramer exhibits a predominant conformation in aqueous solution

Leah T. Roe1, Jeffrey G. Pelton2, John R. Edison1, Glenn L. Butterfoss3, Blakely W. Tresca1,5, Bridgette A. LaFaye1, Stephen Whitelam1, David E. Wemmer4 and Ronald N. Zuckermann1,*

1The Molecular Foundry, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720.2QB3 Institute, University of California, Berkeley, CA 94720.3Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.4Department of Chemistry, University of California, Berkeley, CA 947205Department of Chemistry, Kalamazoo College, Kalamazoo, MI 49006

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Table of ContentsSupplemental NMR Figures.......................................................................................................... 3

Table S1. NMR assignments for all identified conformations.........................................................3

Figure S1. Peptoid 1 Full 1D 1H.....................................................................................................4

Figure S2. Peptoid 1 Full HSQC.....................................................................................................5

Figure S3. Peptoid 1 Full COSY.....................................................................................................6

Figure S4. Peptoid 1 Full HMBC....................................................................................................7

Figure S5. Peptoid 1 Full ROESY...................................................................................................8

Figure S6. Peptoid 2 Full 1D 1H Spectra.........................................................................................9

Figure S7. Peptoid 2 HSQC..........................................................................................................10

Figure S9. Peptoid 3 HSQC..........................................................................................................12

Figure S10. Peptoid 3 HMBC.......................................................................................................13

Figure S11. Peptoid 3 ROESY......................................................................................................14

Figure S12. Peptoid 4 Full 1D 1H Spectra.....................................................................................15

Figure S13. Peptoid 4 HSQC........................................................................................................16

Figure S14. Peptoid 5 HSQC........................................................................................................17

Figure S16. Peptoid 5 1H-13C HSQC-ROESY...............................................................................19

Figure S17. Peptoid 5 hCCH-TOCSY with 260 ms mixing time...................................................20

Figure S18. Peptoid 5 HcCH-TOCSY with 260 ms mixing time...................................................21

Figure S19. Peptoid 5 HcCH-TOCSY with 97.5 ms mixing time..................................................22

Figure S20. Peptoid 6 HSQC........................................................................................................23

Figure S21. Peptoid 6 HSQC-ROESY..........................................................................................24

Figure S22. Peptoid 7 HSQC........................................................................................................25

Figure S23. Peptoid 7 HSQC ROESY...........................................................................................26

Figure S24. Peptoid 8 HSQC........................................................................................................27

Figure S25. Peptoid 8 HSQC-ROESY..........................................................................................28

Figure S26. Peptoid 9 HSQC........................................................................................................29

Figure S27. Peptoid 9 HSQC-ROESY..........................................................................................30

Supplemental Quantum Mechanics Information..........................................................................31

Table S2........................................................................................................................................31

Figure S28.....................................................................................................................................31

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Supplemental NMR Figures

Table S1. NMR assignments for all identified conformations.

Conformer Abundance (%) R1 1H / 13C R2 1H/13C R3 1H/13C R4 1H/13C1 52.4 3.29 / 51.0 3.52 / 49.6 3.80/51.9 4.25/51.2

2 17.9 3.21/51.44 3.42/51.26 3.75/51.85 4.24/51.55**R4 overlap

3 10.5 3.18/50.95 3.97/ 49.6 4.29 / 52.48 4.16 / 51.74 8.8 3.07 / 50.79 4.31/ 50.0 3.85 / 50.84 4.05 / 51.955 3.8 3.18 / 51.35 3.79 / 51.45 4.19 / 52.25 Unassigned6 2.8 3.10 / 50.84 4.37 / 49.96 4.15 / 51.17 4.09 / 52.7

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Figure S1. Peptoid 1 Full 1D 1H

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Figure S2. Peptoid 1 Full HSQC

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Figure S3. Peptoid 1 Full COSY

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Figure S4. Peptoid 1 Full HMBC

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Figure S5. Peptoid 1 Full ROESY

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Figure S6. Peptoid 2 Full 1D 1H Spectra

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Figure S7. Peptoid 2 HSQC

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Figure S8. Peptoid 3 Full 1D 1H Spectra

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Figure S9. Peptoid 3 HSQC

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Figure S10. Peptoid 3 HMBC

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Figure S11. Peptoid 3 ROESY

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Figure S12. Peptoid 4 Full 1D 1H Spectra

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Figure S13. Peptoid 4 HSQC

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Figure S14. Peptoid 5 HSQC

Figure S14. 2D 1H-13C HSQC spectrum of peptoid 5 labeled at each backbone position with 13C. Peaks are labeled by form (F) and residue number (R).

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Figure S15. Peptoid 5 Full 1,n Adequate

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Figure S16. Peptoid 5 1H-13C HSQC-ROESY

Figure S16. 2D 1H-13C HSQC-ROESY spectrum of peptoid 5 with each backbone position labeled with 13C. Single-bond 1H-13C correlations are labeled by form (F) and residue number R. ROESY peaks (green) between backbone protons of F1 R2 and F1 R3, and between F3 R3 and F3 R4 indicate the conformation about the amide bond is cis at these positions.

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Figure S17. Peptoid 5 hCCH-TOCSY with 260 ms mixing time

Figure S17. 3D figure. hCCH-TOCSY 2D 1H-13C plane of a 3D hCCH-TOCSY spectrum of peptoid 5 labeled with 13C at each backbone position. The single bond 1H-13C correlations for R1 and R3 appear in this plane (13C chemical shift 49.8 ppm), and are denoted by asterisks. Residue numbers denote correlations to the other 13C resonances of each conformer. Note that the carbon chemical shifts of R1 and R4 were similar, and not resolved in this spectrum. The full three-dimensional spectrum was used to identify the backbone 13C resonances for each conformer.

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Figure S18. Peptoid 5 HcCH-TOCSY with 260 ms mixing time

Figure S18. 3D HcCH-TOCSY spectrum – long mixing time2D 1H-13C plane of a 3D HcCH-TOCSY spectrum of peptoid 5 labeled with 13C at each backbone position. The spectrum was acquired with a mixing time of 260 ms. Single-bond 1H-13C correlations for R2 are denoted with asterisks. Reside numbers denote correlations to the other 1H resonances of each conformer. The full three-dimensional spectrum was used to identify the backbone 1H resonances of each conformer.

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Figure S19. Peptoid 5 HcCH-TOCSY with 97.5 ms mixing time

Figure S19. HcCH-TOCSY spectrum – shorter mixing time 2D 1H-13C plane of a 3D HcCH-TOCSY spectrum of peptoid 5 labeled with 13C at each backbone position. The spectrum was acquired with a mixing time of 97.5 ms. Single-bond 1H-13C correlations for R2 are denoted with asterisks. Reside numbers denote correlations to the 1H resonances of adjacent residues within each conformer. The full three-dimensional spectrum along with a 1H-13C HSQC spectrum of RN3 labeled only at position R1, were used together to obtain backbone sequential assignments for each of the conformers.

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Figure S20. Peptoid 6 HSQC

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Figure S21. Peptoid 6 HSQC-ROESY

Figures S21. 2D 1H-13C HSQC-ROESY spectrum of peptoid 6 (13C at position C1 only), One-bond 1H-13C1 correlations are in green, and ROE cross peaks are in blue.

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Figure S22. Peptoid 7 HSQC

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Figure S23. Peptoid 7 HSQC ROESY

Figure S23. 2D 1H-13C HSQC-ROESY spectrum of peptoid 7 (13C at position C2 only), One-bond 1H-13C2 correlations are in green, and ROE cross peaks are in blue.

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Figure S24. Peptoid 8 HSQC

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Figure S25. Peptoid 8 HSQC-ROESY

Figures S25. 2D 1H-13C HSQC-ROESY spectrum of peptoid 8 (13C at position C3 only), One-bond 1H-13C3 correlations are in green, and ROE cross peaks are in blue.

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Figure S26. Peptoid 9 HSQC

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Figure S27. Peptoid 9 HSQC-ROESY

Figure S27. 2D 1H-13C HSQC-ROESY spectrum of peptoid 9 (13C at position C4 only), One-bond 1H-13C4 correlations are in green, and ROE cross peaks are in blue.

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Supplemental Quantum Mechanics Information

We analyzed the Nae side chain conformational preferences with DFT in a simplified model of the local peptoid backbone: protonated N-(2-aminoethyl)-N-methylacetamide. We genertated starting structures in both cis and trans amide geometries and at 3 torsional states of χ2 (the amide nitrogen to side chain nitrogen dihedral) and then optimized at the B3LYP/6-311++G(2d,2p) level of theory with solvent water represented with the Polarizable Continuum Model (PCM). Energies are listed in Table S2. The cis omega conformations are generally more favorable, and a cis rotamer featuring an interaction between the side chain and the backbone carbonyl oxygen (Figure 10) has a predicted energy ~5 kcal/mol lower than the next most favorable structure. Although solvent is represented implicitly in this system, it is worth noting that the high preference for this particular geometry might be altered were the side chain ammonium proximal to alternative hydrogen bonding partners.

Table S2. Relative energies of the Nae side chain in a model compound (N-(2-Aminoethyl)-N-methylacetamide)

Omega Chi2 (degrees) Relative energy (kcal/mol)cis -83.1 0.00cis 58.3 4.94cis 179.2 5.08

trans -63.8 5.97trans 178.2 6.29trans 66.0 7.51

Figure S28. Lowest energy conformer of protonated N-(2-Aminoethyl)-N-methylacetamide shows a favorable polar interaction between the side chain ammonium group and the backbone carbonyl oxygen (B3LYP/6-311++G(2d,2p) with PCM solvent water).