Doctor of Philosophy (PhD)



Document Type



In Skp1 of Dictyostelium discoideum (Dd), Pro143 is located at the N-terminus of an α-helix with four consecutive Glu residues immediately following Pro. Preceeding Pro143 is a segment of random coil. The proline residue undergoes post-translational modifications: hydroxylation and glycosylation. A cytoplasmic prolyl hydroxylase (P4H1) delivers a hydroxyl group to Pro143 and N-acetylglucosamine transferase 1 (Gnt1) transfers GlcNAc from UDP to Hyp143 of Skp1. The installation of the first GlcNAc residue in Skp1 in Dictyostelium is important for the organism to differentiate into a fruiting body to disperse spores. We describe herein some structural and synthesis studies of the Pro143 region of the Skp1 protein. We report the synthesis of the bisubstrate analog Ac-Thr-[(α-1,4-GlcNAc)-2S,4R-4-thioproline]-OH (17). Enzyme assays showed that 17 was not an inhibitor for Gnt1 under assay conditions optimized for the full length Skp1 substrate. Cis → trans isomerization about the prolyl amide bond was studied by NMR for a series of peptides. Equilibrium constants (Kt/c, 25 °C) increased in the order: Ac-Thr-Pro-NHMe (61) (2.3) < Ac-[(α-1,4)GlcNAc]-NHMe (89) (3.25) < Ac-Thr-Hyp-NHMe (62) (8.7) < Ac-[(α-1,4)GlcNAc]-NHMe (89) (13.2). Hydroxylation, glycosylation and extension of the peptide in the N-terminal direction all favor the trans conformation. Magnetization transfer experiments were possible for Ac-Thr-Hyp-NHMe (62) (kc/t = 0.94 s-1, 60 °C) and Ac-[(α-1,4)GlcNAc]-NHMe (89) (kc/t =0.45 s-1, 60 °C) at 60-75 °C. Insufficient resolution of signals for the other two compounds precluded kinetic analysis. Synthesis of dipeptides Ac-Thr-Hyp-NHMe (62) and Ac-Thr-hyp-NHMe (106) enabled us to determine the Cγ stereochemistry of the Skp1 Hyp143 by comparing the 1H NMR spectra of both dipeptides with that of a 13-mer obtained by enzymatic degradation of the native protein. The lack of inhibition of Gnt1 by bisubstrate analog 17 may be attributable to an adjacent glutamic acid rich α-helix recognition element in the full length protein. The synthesis of a 15-mer substrate incorporating an α-helical mimetic is advanced to test this hypothesis. Three fragments are presented: Fmoc-IKNDFT-OBn (171), Cbz-[HypE]*EEE-OAll (253), and Fmoc-QIRK-NH2 (132) where [HypE]* represents turn inducing mimetic for the HypE dipeptide.



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Committee Chair

Taylor, Carol M.

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Chemistry Commons