CONFORMATIONAL ANALYSIS

1. (b) (a) (c) Figure 1. (a) The structure of g-turn; (b) The crystal structure of HIF (light pink) in the active site of FIH (sphere) together with Fe(II) (orange) and 2-oxoglutarate (cyan); and (c) Model of cyclic peptide design. Compound 1 Compound 3 Compound 2 Conformer A Conformer A Figure 4. Tr-ROESY of 2 in [2H]5-pyridine at 238K – showing relevant NOEs and an expansion of the weak NOE of NH4B to H2’B, whereas NH1B has NOEs to H2’B (strong) and H2 (medium). Figure 8. NOESY of 3 in CDCl3 at 298K – expanding the region of strong NOE between H10’  H7’. Figure 2. Tr-ROESY of 1 in [2H]5-pyridine at 238K – showing NOEs which define confermers A and B. Figure 7. Temperature dependence of 3 in CDCl3. Figure 8. [2H]6-DMSO titration of 3 at 298K. Figure 3. Temperature dependence of conformers A and B of 1 in CDCl3. Conformer A NH chemical shift/ppm NH chemical shift/ppm Figure 5. Temperature dependence of conformers A and B of 2 in CDCl3. Temperature/K [2H]6-DMSO-CDCl3 ratio NH chemical shift/ppm Conformer B Conformer A Temperature/K NH chemical shift/ppm Temperature/K NH chemical shift/ppm Conformer B Temperature/K NH chemical shift/ppm Temperature/K Design, Synthesis, and Conformational Analysis of Inverse g-Turn Cyclic Peptidomimetics of HIF Morakot Kaewpet, Biswadip Banerji, Barbara Odell, Timothy D. Claridge, and Christopher J. Schofield Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK INTRODUCTION Conformationally constrained analogues of biologically important peptides can be useful mechanistic probes. Although much attention has been focused on peptidomimetics based on b-turns,1 our search for peptidomimetic inhibitors of hydroxylase enzymes has led us to consider peptides based on g-turns2 (Fig 1a). A study of the three-dimensional requirements involved in the binding of hypoxia inducible factor (HIF) to FIH (Factor Inhibiting HIF) based on crystal structures3 (Fig 1b) revealed an inverse g-turn in the HIF residues Val802-Asn803-Ala804 (Fig 1c), the design target for our peptidomimetics. SYNTHESIS The synthesis of glycine- and proline-containing cyclic peptides 1-3 was achieved by standard amide coupling procedure and ring closure metathesis (RCM) reaction as shown in Scheme 1. Scheme 1.Synthesis of cyclic peptides CONFORMATIONAL ANALYSIS Results for 3 • Only one conformer in CDCl3 at rt. • NH2 involved in intramolecular H-bonding. • NH2 & H13a are on the same face, due to strong NOE. • H8 has strong NOEs between both d-proline (H10 slightly stronger), thus a trans-Pro. • Strong NOE between H10’  H7’, together with weak NOEs between H10’  H5/H6, suggest the aliphatic chain is folded towards the d-proline moiety. Conformer B Conformer B Results for 1 • A:B (2:1) in [2H]5-pyridine at 238K. • Similar temp-dependence behaviour. -0.009 ppm/K for NH1 -0.005 ppm/K for NH4 • NH4 has more of a propensity for H-bonding than NH1. • Torsional rotations involving in the conversion of A to B. Results for 2 • A:B (3:1) in [2H]5-pyridine at 238K. • In A, NH4 has the lowest temp coeff, suggesting intramolecular H-bonding. • In B, no intramolecular H-bonding. • A has both trans-amide bonds, pointing to the opposite faces due to strong NOEs between NH1  H2’ and NH4  H2. • B has one cis-amide bond due to strong NOE between NH1  H2’, and weak NOEs between NH4  H2 and NH4  H2’. CONCLUSIONS • REFERENCES • Fink, B. E.; Kym, P. R.; Katzenellenbogen, J. A. Journal of the American Chemical Society 1998, 120(18), 4334-4344. • Bystrov, V. F.; Portnova, S. L.; Tsetlin, V. I.; Ivanov, V. T.; Ovchinnikov, Y. A. Tetrahedron 1969, 25(3), 493-515. • Elkins, J. M.; Hewitson, K. S.; McNeill, L. A.; Seibel, J. F.; Schlemminger, I.; Pugh, C. W.; Ratcliffe, P. J.; Schofield, C. J. J Biol Chem 2003, 278(3), 1802-6. • This work has focused on peptidomimetics of HIF designed to form inverse g-turns, initiated from the X-ray structure of HIF bound to FIH. Ring-closing olefin metathesis was shown to be an effective method for closing medium-ring amides with good yields, representing the first example of cyclisation to form eleven-membered peptides using RCM reaction The following conclusions were observed for this family of cyclic tripeptides; • ·  • There is a structural propensity of compounds 1-3 to form inverse g-turns.   • Restraints can either be introduced at the (i+1)th residue using proline as in 3 or on the alkyl chain side of the molecule employing unsaturation as in 1. • In 2, there is increased flexibility in the saturated system as noted by two conformers in which the predominant one is still an inverse g-turn, but the minor conformer has a cis-amide bond and is therefore no longer an inverse g-turn • 3 is an interesting example where there is only one observable conformation containing a stable inverse g-turn with the alkyl chain folded towards the proline side chain. • An inverse g-turn might still be expected to play an important role for inhibition of FIH, but the core motif is not a sufficient requirement for activity. We conclude that appropriate functionality of the side chains must be involved. The next stage of this work will focus on designing proline analogues that contain extended functional groups which will be capable of binding to other strategic parts of the active site of FIH. ACKNOWLEDGEMENT : The Royal Thai Government