E-olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: Probing the requirements for Alzheimer's amyloidogenesis

Article abstract of DOI:10.1021/ja0551382

Herein, we report a stereospecific E-olefin dipeptide isostere synthesis that can be used to make gram quantities of the Phe-Phe isostere desired for eliminating a specific backbone H-bond donor and acceptor in the Alzheimer's disease related Aβ peptide.

Full text of DOI:10.1021/ja0551382

Published on Web 10/14/2005
E-Olefin Dipeptide Isostere Incorporation into a Polypeptide Backbone
Enables Hydrogen Bond Perturbation: Probing the Requirements for
Alzheimer’s Amyloidogenesis
Yanwen Fu, Jan Bieschke, and Jeffery W. Kelly*
Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, BCC 506, La Jolla, California 92037
Received July 28, 2005; E-mail: jkelly@scripps.edu
Intermolecular backbone-backbone hydrogen bonding is ob-
served in quaternary structures and is believed to be an important
stabilizing force in protein-protein interactions. It is possible to
eliminate both a backbone H-bond acceptor (CdO) and a donor
(N-H) by replacing the amide functionality in a single dipeptide
substructure within a protein with an E-alkene moiety (Figure 1).
The idea of incorporating E-olefin dipeptide isosteres into polypep-
tides to perturb backbone H-bonding is not new. However, this
strategy has been rarely realized in large part because of the
Figure 1. The backbone amide-to-E-olefin mutation eliminates both a
difficulties involved in the chemical synthesis of E-alkene dipeptide
isosteres and the challenge of incorporating these dipeptide isosteres
into proteins.1 Protein backbone H-bond perturbation has been
realized using amide-to-ester backbone mutations.2 Like ester
perturbations, amide-to-E-alkene substitutions are ideal in that the
conformational and geometrical preferences of the backbone are
maintained (Figure 1). Moreover, unlike the situation in esters,
where the non-carbonyl oxygen experiences an unfavorable elec-
trostatic interaction with the amide carbonyl the NH once H-bonded
to, E-olefin perturbations do not introduce unfavorable electrostatic
interactions, making it easier to extract H-bond energies from ∆∆G
measurements.
H-bond acceptor (CdO) and a donor (N-H) (left). The X-ray structure of
the Phe-Phe E-olefin dipeptide isostere synthesized herein (right).
Scheme 1. Synthesis of E-Olefin Isosterea
Herein, we describe a stereoselective E-olefin dipeptide isostere
synthesis (Scheme 1) that can be used to make gram quantities of
the amino acid required for eliminating a specific H-bond donor
and acceptor in a protein while maintaining side-chain structures.
The Phe-Phe E-olefin dipeptide isostere (5) was incorporated
into the 40 residue Alzheimer’s amyloid peptide, referred to as
Aâ(1-40), in place of phenylalanines 19 and 20 utilizing a Boc/
benzyl solid-phase synthesis strategy. The aim is to examine the
role of intermolecular H-bonding in the process of amyloidogenesis
thought to cause Alzheimer’s disease. This amide-to-E-alkene
backbone mutation precludes amyloid formation, but not spherical
aggregate formation, providing insight into the structural require-
ments of amyloidogenesis.
Previously reported synthetic approaches to E-olefin isosteres
often relied on SN2′ substitutions or rearrangements of allylic
substrates3 or generation of the double bond via 1,2 elimination.4
In the E-olefin dipeptide isostere synthesis described herein, the
amino terminal CR stereocenter comes from an amino aldehyde
(derived from an amino acid), whereas the C-terminal stereocenter
is produced via the diastereoselective (11:1) Evans alkylation of
an oxazolidinone enolate (Scheme 1). The synthesis commenced
with a Wittig reaction, followed by a hydroboration-oxidation,
affording the N-Boc-Phe-Gly dipeptide E-olefin isostere using
established procedures.5 The acid within 2 was then condensed with
Evans’ chiral oxazolidinone6 utilizing a mixed anhydride approach.
Diastereoselective alkylation of the C-terminal CR carbon in 3 is
the key step in this approach. To avoid formation of Ν-alkylated
or Cγ-alkylated side products and decomposition of the enolate,
a Conditions: (a) Ph3PCH2CtCSiMe3Br, n-BuLi, E/Z 7:1, 99% ee, 70%.
(b) (i) cyclohexene, BH3; (ii) H2O2, 82% for two steps. (c) (i) t-BuCOCl,
Et3N; (ii) oxazolidinone, n-BuLi, 85% for two steps. (d) LDA, PhCH2Br,
70%, dr 11:1. (e) LiOH, H2O2, 88%. (f) TMS-CHN2, 92%.
the reaction was conducted under kinetically controlled conditions,
where 3 was added to 2 equiv of freshly prepared LDA at -78 °C.
Addition of excess benzyl bromide afforded the desired product
with a high diastereoselectivity ratio (11:1) and in good chemical
yield (70%). Precursor 4 was treated with basic hydrogen peroxide
to remove the oxazolidinone chiral auxiliary affording 5, ready for
incorporation into a polypeptide chain by solid-phase peptide
synthesis. The methyl ester 6, afforded by TMSCHN2 treatment,
was employed to confirm the structure of the Phe-Phe dipeptide
E-olefin isostere by X-ray crystallography (Figure 1).
The misassembly of Aâ, ultimately affording insoluble amyloid
fibrils, is genetically linked to the pathogenesis of Alzheimer’s
disease.7 Aâ(1-40) contains two hydrophobic subsequences which
are underlined in one letter code below: DAEFRHDSGYEVH-
HQKLVFFAEDVGSNKGAIIGLMVGGVV. The central hydro-
phobic core LVFFA (17-21) is essential for amyloidogenesis.8
Since it has been argued that it is primarily the backbone that
enables so many peptides to form cross-â-sheet-based amyloid
fibrils,9
it follows that intermolecular H-bonding should be essential.
An amide-to-E-olefin substitution was therefore introduced into the
key Phe19-Phe20 subsequence, removing both a hydrogen-bond
donor and acceptor thought to make H-bonds with different
neighboring Aâ molecules.10
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10.1021/ja0551382 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
EM measurements reveal a ∼10 nm diameter, similar to the
dimensions seen in diffusible oligomer aggregation intermediates
(“ADDLs”)12 and in cholesterol metabolite induced spherical
aggregates.11 There is substantial evidence that Aâ aggregation
proceeds first by forming spherical aggregates which transform into
protofilaments and then into fibrils having a cross-â-sheet structure.7b
It is striking that removal of 1 of the 39 amide bonds in Aâ, and
thus one H-bond donor and one acceptor, could prevent the typical
progression of spherical aggregates into protofilaments and fibrils.
The results herein indicate that the 19-20 amide bond is critical
for protofilament and fibril formation, but not for spherical
aggregate formation. Ongoing studies to discern whether replace-
ment of other amides will influence amyloidogenesis will be
reported in due course. The discovery of an Aâ analogue that can
form spherical aggregate morphologies but cannot progress to
protofilaments or fibrils is very valuable for delineating the structure,
toxicity, and antigenicity of spherical Aâ aggregates.
In summary, we report a synthesis of E-olefin isosteres that can
be prepared on a scale that enables their incorporation into
polypeptide sequences to examine the role of individual hydrogen
bonds in protein structure acquisition.
Figure 2. (A) TfT fluorescence of wt Aâ and the EOAâ analogue after
agitated incubation at 37 °C for 25 h. (B) Mean residue ellipticity of
Aâ(1-40) (solid lines) and EOAâ (dotted lines) before aggregation (black),
after 25 h (red), and 6 weeks (green). AFM images (scale bars: 200 nm)
of aggregated Aâ (C) and the EOAâ after 25 h (D) and 6 weeks (E).
Acknowledgment. We gratefully acknowledge financial support
from the NIH (GM 51105), the Lita Annenberg Hazen Foundation,
and the Bundy Foundation, and thank I. Yonemoto for EM images.
The Phe19-Phe20 E-olefin dipeptide isostere was incorporated
into Aâ by manual solid-phase peptide synthesis using HATU
activation. The reverse phase HPLC purified Phe19-Phe20 E-olefin
analogue of Aâ(1-40), hereafter referred to as EOAâ, was
subjected to high pH treatment and membrane filtration to mono-
merize the sample.11 TfT is an environmentally sensitive fluor that
binds and fluoresces when bound to spherical and cross-â-sheet
aggregates of Aâ.11 Incubation of EOAâ (50 µM) with agitation
on a rocker (30/min) in the buffer (150 mM NaCl, 50 mM
phosphate, pH 7.4, 37 °C) that typically leads to the aggregation
of Aâ(1-40) resulted in a strong thioflavin T signal after 25 h,
comparable to that exhibited by wt Aâ (Figure 2A), indicating that
EOAâ was able to self-assemble. TfT fluorescence remained
essentially constant for both peptides over the course of 6 weeks.
To examine whether aggregated EOAâ adopts a â-sheet rich
structure analogous to that exhibited by wt Aâ, far-UV circular
dichroism (CD) was employed. The CD spectra of unaggregated
Aâ peptides display strong minima around 200 nm, whereas
aggregated wt Aâ exhibits a strong minimum at 217 nm, charac-
teristic of cross-â-sheet structure. The amplitude at 217 nm was
very weak if present at all in EOAâ (25 h), but increased very
slowly over 6 weeks, long after the TfT signal had plateaued (Figure
2B). Absorbance at 280 nm revealed that the EOAâ concentrations
remained very similar to those of wt Aâ.
Supporting Information Available: Experimental details and
characterization data for the synthesis of 6; crystal structure of 6;
procedures for peptide assembly. This material is available free of
charge via the Internet at http://pubs.acs.org.
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