Towards Constrained Alpha-Helical Peptides

Beyer, Renee (2008). Towards Constrained Alpha-Helical Peptides PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Beyer, Renee
Thesis Title Towards Constrained Alpha-Helical Peptides
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2008-07-10
Thesis type PhD Thesis
Supervisor Professor David Fairlie
Professor Trevor Appleton
Dr Yogendra Singh
Total pages 338
Total colour pages 55
Total black and white pages 283
Subjects 270000 Biological Sciences
Formatted abstract The peptide α-helix is a common secondary structural motif within proteins and plays
both a structural and functional role in biology. The study of isolated helices in aqueous
solution however is difficult due to their thermodynamic instability. Chapter 1 introduces the
features of the α-helix and methods for constraining short peptides into α-helical
conformations including side chain constraints, hydrogen bond replacement techniques and
helix nucleating templates. Non-peptide mimetics are introduced along with templates for
helix bundles.
Helix induction in short peptides by coordination of histidine residues, that potentially
reside on the same face of an α-helix, to a metal ion has been previously reported, particularly
when the metal was at the N-terminus of a peptide. In Chapter 2, metal ions have been
coordinated to two histidines either at the C-terminus of, or within, short peptide sequences
which have been investigated for α-helical structure by circular dichroism and NMR
spectroscopy. Reaction of [Pd(en)(ONO2)2] in the aprotic solvent DMF with a peptide
corresponding to the DNA binding region of transcription factor Zif268
(AcRSDDELTRH*IRIH*T-NH2) produces a metallopeptide with two histidines both
coordinated via N1 nitrogens to the Pd metal. The metallopeptide showed evidence from
NMR spectroscopy of α-helicity within the pentapeptide segment containing both histidines,
and this extended one helix turn towards the N-terminus in DMF.
To test whether additional α-helicity could be induced in a short peptide in the protic
solvent water, which competes more effectively for the intramolecular hydrogen bonding
amide CO and NH groups that define peptide helicity, we decided to focus on the effect of
metal binding on a peptide with both N- and C- terminal coordinating atoms. We chose the
unstructured peptide AcH*AAH*H*ELH*-NH2 with multiple histidines, positioned at i, i+3,
i+4, i+7 thereby providing us with an opportunity to compare favoured binding sites for bothchelation to metals and helix induction. The peptide reacted with two equivalents of
[Pd(en)(ONO2)2] in water to produce initially the metallopeptide with (1,4)(5,8) coordination
(i, i+3) featuring two non-helical turn motifs in the peptide backbone. This transformed to a
metallopeptide with (1,5)(4,8) coordination (i, i+4) with an α-helical peptide backbone
structure. Thus while (i, i+3) chelation was kinetically preferred, (i, i+4) chelation was clearly
more thermodynamically favoured, a factor that is attributed to stabilisation of an α-helical
peptide backbone featuring multiple adjacent intramolecular hydrogen bonds.
A handful of organic compounds have been reported to increase or stabilize α-helicity
in short peptides. These observations have usually been made on the basis of CD spectral
measurements that have often been recorded under varying conditions (solvent, temp, ionic
strength, buffer, etc) making comparisons of the effectiveness of different helix nucleators
quite difficult. Chapter 3 compares a range of N-terminal helix nucleators for their capacity
to induce α-helicity in a 17-residue RNA-binding peptide fragment
(TRQARRNRRRRWRERQR) of the viral protein HIV-1 REV. This protein is essential for
replication of HIV by acting as a transporter that exports mRNA from the nucleus of a host-
infected cell to the cytoplasm. The RNA-binding fragment REV34-50 has very little α-helical
structure in water, but is α-helical when bound to a piece of RNA known as the Rev Response
Element (RRE). Methods for inducing α-helicity may lead to higher affinity binding to RNA
and could potentially guide design of REV-RNA antagonists as antiviral drugs. At present
there are no drugs that act by this mechanism. We have found that helix nucleating templates
can induce very high proportions of α-helicity in REV34-50, although the extent of helix
induction is quite variable between the nucleators.
Chapter 4 investigates the synthesis and structure of cyclic tetrapeptides with main
chain to main chain or side chain to main chain cyclisation constraints, using circular
dichroism and NMR spectroscopic characterisation techniques, with a view to producing the
smallest peptide α-helical turn. Since 1-turn of an alpha helix is defined by just 3.6 residues,we wondered whether an alpha helix could be stabilised in 4 residues. The first series of
cyclic tetrapeptides cyclo[ARAX] varied the length of the linker (X = β-Ala, 4-Abu and 5-
Ava), joining the N- and C-termini to produce 13- to 15- membered rings. The effect of an N-
methylated amino acid (N-methyl-β-alanine) was also investigated.
The next series of cyclic tetrapeptides cyclo[ARAX]-NH2 contained side chain
carboxylic acid (X = Asp, Glu, Homoglu, Homohomoglu) to main chain N-terminal amine
constraints resulting in 13- to 16- membered rings. Cyclic tetrapeptides cycloAc[XARA] (X =
Dap, Dab, Orn, Lys) with side chain amine to main chain C-terminal carboxylic acid linkages
produced 13- to 16- membered rings. Appendage of three extra residues beyond the cycle
allowing the potential for three or more hydrogen bonds, required for an α-helix to form, was
examined for α-helix induction. None of the cycles presented in this chapter formed a
‘classical’ helical α-turn, however the 14-membered ring cycles, formed with a side chain
carboxylic acid to N-terminal amine constraint, cyclo[ARAE]-NH2 and cyclo[ARAE]LAH-
NH2 produced a non classical type II-αLU turn first reported by Pavone et al.
Chapter 5 explores the cyclooligomerisation reaction as a vehicle to create
discontinuous surfaces of protein secondary structure. In this study a constrained α-helix was
appended to a peptide which carried this motif unchanged via cyclooligomerisation to create a
series of macrocycles, each projecting multiple helices into different regions and fixed
positions in space. Tetrapeptide H-[Ile-Ser-Lys(Ox)]-OH, containing an oxazole turn-
inducing constraint with an appended constrained helical peptide (cyclo-
4,8)AcLRL[KARAD](Aib), connected via the C-terminus to the side chain lysine of the
template via β-alanine, underwent a cyclooligomerisation reaction using BOP and DIPEA in
DMF, with orthogonal protection of arginine and serine, to produce after deprotection a series
of macrocycles (cyclic dimer to cyclic hexamer) in the crude mixture and cyclic dimer and
cyclic trimer were isolated pure.
Additional Notes 1, 19-23, 31, 32, 40, 41, 48, 97, 103, 104, 112, 152, 164, 171, 173, 192, 193, 196-198, 204, 205, 209-211, 213-219, 222, 223, 228-230, 232, 268-270, 280, 287, 290, 291, 294, 316-318, 333, 336.

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