Identifier

etd-11132013-132906

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

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.

Date

2013

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Taylor, Carol M.

Included in

Chemistry Commons

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