NMR Structure Note: Solution Structure of a Novel C2-Symmetrical Bifunctional Bicyclic

03a9507f6074d606c306bac4579dbbba.doc

Supplementary material

NMR structure note: Solution structure of a novel C2-

symmetrical bifunctional bicyclic inhibitor based

on SFTI-1

Agnès M. Jaulenta, Arnd B.E. Brauera,b, Stephen J. Matthewsc & Robin J.

Leatherbarrow1*

aDepartment of Chemistry and cDepartment of Biological Sciences, Imperial College

London, South Kensington Campus, London SW7 2AZ, U.K.

b Freie Universität Berlin, Institut für Chemie – Biochemie, Otto-Hahn-Bau,

Thielallee 63, D-14195 Berlin (Dahlem), Germany

*Email: [email protected]

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Table 1. Assignment table for the chemical shifts in ppm of the CTKSIPP’I’ repeat unit of BiKK, including coupling constants and derived dihedral restraints.

3 3 HN     other H JHNH JHH  1 in Hz in Hz in o in o Cys 8.92 5.89 2.94 S 10.6 2.8 -120±20 -60±30 3.04 R 12.4 Thr 8.71 4.43 4.61 1.43 7.0 2.9 -120±60 +60±30

Lys 8.51 4.50 2.02 1.51 1.70  3.00 8.3 7 -120±45 2.06 1.43  HN7.49 Ser 7.29 4.39 3.79 7.8 3.7 -120±45 +60±30 3.90 3.3 Ile 8.21 4.31 1.80 γ1 1.45 0.84 10.0 11 -120±20 -60±30 1.07 γ2 0.84 Pro - 5.10 2.46 S 1.85 S 3.63 S - 8.9 -70±20 +26±15 2.04 R 1.96 R 3.54 R 3.0 Pro′ - 4.22 2.45 S 2.04 S 3.83 S - 9.6 -70±20 1.87 R 2.11 R 3.72 R 4.5 Ile′ 7.87 4.34 1.93 γ1 1.46 0.81 10.0 11 -120±20 -60±30 1.16 γ2 0.87

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Table 2. Statistics of the family of 20 lowest-energy structures calculated for the bifunctional trypsin inhibitor

Distance restraints for each C-T-K-S-I-P-P'-I'- segment Intraresidue 34 i + 1 19 i + 2 3 i + 3 1 i + 4 1 i + 5 1 i + 6 5 i + 7 1

Dihedral restraints for each C-T-K-S-I-P-P'-I'- segment  8  8 1 6

Hydrogen bond restraints 0

Ramachandran analysis Residues in favoured regions 83.2 % Residues in additional allowed regions 16.8 % Residues in generously allowed regions 0 % Residues in disallowed regions 0 %

Pairwise root mean square deviations (Å) All residues over backbone atoms 0.24  0.08 (0.00…0.47) All residues over heavy atoms 1.05  0.25 (0.00…1.63) Backbone atoms of the two C-T-K-S-I-P-P'-I'- segments 0.18  0.07 (0.00…0.33) Heavy atoms of the two C-T-K-S-I-P-P'-I'- segments 0.98  0.30 (0.00…1.71)

Pairwise root mean square deviations from SFTI-1 over C- T-K-S-I-P-P'-I'-C loop (Å) Crystal structure in complex with trypsin (1SFI) Backbone atoms 0.45  0.03 (0.42…0.51) Heavy atoms 1.16  0.25 (0.77…1.54) NMR solution structure (1JBL) Backbone atoms 0.31  0.15 (0.06…0.61) Heavy atoms 0.98  0.25 (0.37…1.60) 11-mer BBI mimic (1GM2) Backbone atoms 0.4  0.13 (0.08…0.70)