A Modular Synthesis of Conformationally Preorganised Extended β-Strand Peptidomimetics

Article abstract of DOI:10.1002/chem.201501366

A promising strategy for mediating protein-protein interactions is the use of non-peptidic mimics of secondary structural protein elements, such as the α-helix. Recent work has expanded the scope of this approach by providing proof-of-principle scaffolds that are conformationally biased to mimic the projection of side-chains from one face of another common secondary structural element - the β-strand. Herein, we present a synthetic route that has key advantages over previous work: monomers bearing an amino acid side-chain were pre-formed before rapid assembly to peptidomimetics through a modular, iterative strategy. The resultant oligomers of alternating pyridyl and six-membered cyclic ureas accurately reproduce a recognition domain of several amino acid residues of a β-strand, demonstrated herein by mimicry of the i, i+2, i+4 and i+6 residues.

Full text of DOI:10.1002/chem.201501366

DOI: 10.1002/chem.201501366
Communication
&
Protein–Protein Interactions |Hot Paper|
A Modular Synthesis of Conformationally Preorganised Extended
b-Strand Peptidomimetics
Tohru Yamashita, Peter C. Knipe, Nathalie Busschaert, Sam Thompson,* and
[a]
Andrew D. Hamilton*
Abstract: A promising strategy for mediating protein–pro-
tein interactions is the use of non-peptidic mimics of sec-
ondary structural protein elements, such as the a-helix.
Recent work has expanded the scope of this approach by
providing proof-of-principle scaffolds that are conforma-
tionally biased to mimic the projection of side-chains from
one face of another common secondary structural ele-
ment—the b-strand. Herein, we present a synthetic route
that has key advantages over previous work: monomers
bearing an amino acid side-chain were pre-formed before
rapid assembly to peptidomimetics through a modular,
iterative strategy. The resultant oligomers of alternating
pyridyl and six-membered cyclic ureas accurately repro-
duce a recognition domain of several amino acid residues
of a b-strand, demonstrated herein by mimicry of the i, i+
2
, i+4 and i+6 residues.
The inhibition or stabilization of protein–protein interactions
PPIs) represents a potential goldmine for medicinal chemis-
(
[1]
try, yet the rational design of synthetic agents able to achieve
[
2]
this goal has been seen by many as intractable until recently.
There is growing interest in the synthesis of non-peptidic
[3]
[4]
mimics of secondary and supersecondary protein elements
and the side-chain residues they present for the recognition of
cognate partners. There has been considerable success in mim-
icking the a-helical structures that are ubiquitous at interfacial
peptide regions, prompting researchers to develop scaffolds
for the projection of groups that reproduce side-chain vectors
Figure 1. b-Strand peptidomimicry. A) Canonical b-strand; B) schematic of
side-chain projection; C) previous work: overlay of a three-residue mimic ri-
gidified through dipolar repulsion (green) with a strand (red); D) this work:
a modular synthesis from preformed monomers provides rapid access to fol-
damers of alternating six-membered rings. Increased linearity over extended
distances allows mimicry of longer peptide sequences. For example the six-
point (trimer) RMSD is reduced to 0.7 Š, and the eight-point value for the
tetramer is 1.1 Š. b-Strand from PDB: 3QXT, side-chains represented as
[
5]
[6]
from one or two faces of the helix.
PPIs mediated by extended regions of proteins, such as b-
[
7]
[8]
[12]
strands (Figure 1A), are also of great interest for our group
spheres for clarity, a- and b-carbons used for calculation.
[
9]
and others, inspiring strategies for the synthesis of minimalist
frameworks that replicate their key domains (Figure 1B).
Recently, we described a homochiral foldamer of alternating
bearing methyl groups, resulting in the mimicry of side-chains
[
10]
2
,6-disubstituted pyridyl groups linked by imidazolidin-2-ones
from the i, i+2, and i+4 positions of one face of a b-strand.
An important conformational feature of this motif is preorgani-
zation of the scaffold by dipolar repulsion between the pyridyl
nitrogen lone pair and the carbonyl groups of adjacent imida-
zolidinones. The resultant projection of three groups for side-
chain mimicry is in good agreement with those of one face of
a canonical strand (Figure 1C).
[
a] Dr. T. Yamashita, Dr. P. C. Knipe, Dr. N. Busschaert, Dr. S. Thompson,
Prof. Dr. A. D. Hamilton
Chemistry Research Laboratory, University of Oxford
1
2 Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: sam.thompson@chem.ox.ac.uk
andrew.hamilton@chem.ox.ac.uk
Extending the scope of this design for the mimicry of amino
acid sequences over longer stretches of peptide requires two
challenges to be overcome: 1) the alternation of five- and six-
Homepage: http://hamilton.chem.ox.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501366.
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membered rings introduces a backbone curvature that is in-
creasingly divergent from that of a canonical strand; and 2) a
linear synthesis, in which each side-chain mimic is formed on
the growing oligomeric strand, that is inherently inefficient. A
potential solution is to pre-form pyridyl–urea monomers that
mimic a given amino acid side-chain prior to oligomerization.
As one-carbon homologues of the imidazolidin-2-one, the re-
sultant foldamer is much more linear—thus, more closely re-
Monomers 4 and 8 were coupled under Buchwald–Hartwig
conditions to give two-residue mimic 9 in an excellent yield of
96%. The oligomer can be rapidly extended to a mimic of
three- 11 and four-residues 13 by an iterative two-step se-
quence of N-tosyl deprotection with magnesium in methanol
followed by Buchwald–Hartwig cross-coupling with bromide 8
(Scheme 2).
[11]
producing the side-chain vectors of a b-strand. Calculation
suggests a reduction in six-point root-mean-square deviation
(
RMSD) value from 1.1 to 0.7 Š for the five- and six-membered
[12a,b]
urea mimics, respectively.
Pre-forming each monomer has
the potential to drastically reduce the longest linear synthetic
sequence and more readily allows a peptidomimic of several
residues to be varied at a particular position and generated in
an iterative fashion (Figure 1D). There are numerous commer-
cially available derivatives of enantiomerically pure a-amino al-
cohols bearing a wide variety of side-chains, making them an
ideal starting material for monomer construction.
As a representative amino acid found commonly in b-strands
we chose to explore a proof-of-principle strategy based on
phenylalanine. One-carbon homologation of N-Boc-l-phenyl-
alaninol (1; Boc= tert-butyloxycarbonyl) through O-mesylation
and displacement with sodium cyanide gave nitrile 2, which
was transformed to the aniline derivative 3 by diisobutylalumi-
nium hydride (DIBAL-H) mediated reduction followed by re-
ductive amination with sodium cyanoborohydride and aniline.
Trifluoroacetic acid mediated removal of the Boc group and
cyclization with triphosgene gave terminally capped monomer
4
(Scheme 1A).
The iterative monomer 8 was synthesized from commercially
Scheme 2. Oligomer homologation to form conformationally rigidified pepti-
domimetics of two (9, 10), three (11, 12) and four (13) side-chain residues
from one face of a natural b-strand. Reagents and conditions: a) condi-
tions X, 96%; b) conditions Y, 56%; c) conditions X, 72%; d) conditions Y,
available aziridine 5. Opening of the three-membered ring
with sodium cyanide gave 6, followed by nickel boride mediat-
ed reduction and cyclization with triphosgene to produce 7 in
1
0%; e) conditions X, 52%. Conditions X: XantPhos (30 mol%), [Pd
10 mol%), CsCO (2 equiv), dioxane, 908C, 16 h; conditions Y: Mg (25 equiv),
MeOH, sonication, RT, 10 min.
2 3
dba ]
4
2
0% overall yield. Subsequent Buchwald–Hartwig amination of
(
3
,6-dibromopyridine with 7 gave tosyl-protected monomer 8
in 54% yield (Scheme 1B).
Single crystals suitable for X-ray diffraction were obtained
[
13]
for monomer 8. The solid-state conformation is consistent
with dipolar repulsion between the urea carbonyl group and
the pyridine nitrogen atom favouring an anti-relationship (Fig-
ure 2A). The largely planar cyclic urea adopts a puckered con-
formation placing the benzyl group in a pseudo-axial position
[
14]
(
Figure 2B and the Supporting Information). To probe the
preferences of the higher order homologues 9–13, we con-
ducted a computational search of their lowest-energy confor-
Scheme 1. Synthesis of monomeric cyclic ureas bearing an amino acid side-
chain mimic for: A) uni-directional functionalization; and B) bi-directional
[
12b,c]
mations.
The validity of this method was confirmed by the
functionalization. Reagents and conditions: (a, i) MsCl (1.2 equiv), Et
1.5 equiv), CH Cl
, 08C!rt, 10 min; (ii) NaCN (2.5 equiv), DMF, 608C, 3 h,
6% over two steps, (b, i) DIBAL-H (2.5 equiv), CH Cl
, À788C!08C, 1 h;
ii) aniline (2 equiv), NaBH CN (3 equiv), AcOH, MeOH, rt, 16 h, 19% over two
steps, (c, i) trifluoroacetic acid, CH Cl , rt, 0.5 h; (ii) triphosgene (0.5 equiv),
iPr NEt (4 equiv), rt, 1 h, 47% over two steps, (d) NaCN (1.35 equiv), MeCN,
O, reflux, 1.5 h, 84%, (e, i) NiCl (2 equiv), NaBH (10 equiv, portion-wise),
MeOH, 08C!rt, 10 min; (ii) triphosgene (0.5 equiv), iPr NEt (2 equiv), rt, 2 h,
7% over two steps, (f) 2,6-dibromopyridine (2 equiv), 4,5-bis(diphenylphos-
phino)-9,9-dimethylxanthene (XantPhos; 30 mol%), [Pd dba ] (10 mol%;
3
N
close agreement between the solid-state structure of mono-
(
6
(
2
2
mer 8 and the lowest-energy conformer by calculation (RMSD
2
2
0
.5 Š; see the Supporting Information). Calculations for mimics
–13 gave lowest-energy structures adopting fully extended
3
8
2
2
2
conformations with urea carbonyl groups anti to pyridine ni-
trogens, and with R groups in pseudo-axial positions (see the
Supporting Information). Consequently, the phenylalanine side-
chain mimics are projected from a common face, and are thus
in good agreement with those of a canonical b-strand. Trimer
H
2
2
4
2
4
2
3
dba=dibenzylideneacetone), CsCO
3
(2 equiv), dioxane, 808C, 1.5 h, 54%.
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Figure 2. Conformational behaviour of b-strand mimics 8–13. A) X-ray crystal
structure of monomer 8 showing the benzyl substituent in a pseudo-axial
position; B) equilibrium between ring conformers placing the Bn side-chain
mimic in pseudo-axial and pseudo-equatorial positions. Selected inter-hydro-
[12b,c,14,15]
gen distances given for each conformer based on MM calculations.
The difference in inter-hydrogen distance allows the conformers to be distin-
guished by NOE observations.
11 mimics the i, i+2, i+4 residues with a six-point RMSD
value of 0.7 Š, whereas tetramer 13 adds the i+6 residue and
has an eight-point RMSD value of 1.1 Š (Scheme 2 and the
[12a]
Supporting Information).
Solution-phase conformational behaviour was probed with
NOESY and ROESY NMR experiments in CDCl (Figure 3). NOE
3
signals between the pyridine and cyclic urea hydrogens, for ex-
ample, H8$H15 and H20$H17 for dimer 9, were not ob-
served for compounds 8–13 (Figure 3B, dashed red arrows).
However, strong correlations were observed between urea hy-
drogens and the terminal phenyl/tosyl hydrogens by way of
comparison, for example, H6 $H3 and H22 $H29 (Figure 3B
eq
eq
[
15c,16]
and D, green arrows and the Supporting Information).
These data are consistent with the previously reported five-
[10]
membered imidazolidin-2-one analogues, indicating that the
predominant species in solution adopts the desired extended
conformation, in which the urea carbonyl groups are anti- to
the pyridine nitrogen atoms as depicted in Figures 3B and D.
This result highlights the utility of dipolar repulsion as a confor-
mational determinant for pre-organisation of peptidomimics.
Further inspection of the NOESY NMR spectra of 8–13 re-
vealed interesting features regarding the position of the phe-
nylalanine mimic. For dimer 9, the absence of trans-annular
NOE correlations between H6 and H8, and between H20 and
H22, was consistent with their occupying pseudo-equatorial
Figure 3. Solution-phase conformational analysis of dimer 9. A) Selected
region of the NOESY NMR spectrum focusing on cross-peaks with the pyri-
dine signals (py); B) conformation based on NOE data; C) selected region of
the NOESY NMR spectrum focusing on cross-peaks between the cyclic urea
hydrogens; and D) perspective view of conformation based on NOE data.
Key observed (green) and absent (red, dashed) NOE correlations are indicat-
[
15a]
positions,
Figure 3C and D). Interestingly, the only NOE signal in this
region, corresponds to two hydrogens (H8 and H20) located
with the side-chain mimic in a pseudo-axial site
(
[16]
[15b]
3
ed (CDCl , 400 MHz, 298 K).
on adjacent cyclic ureas.
Additional evidence for this ring
conformation was given by cross-peaks between the cyclic
urea and benzylic hydrogens. For example, the NOE signal be-
tween benzylic H9 and one of the diastereotopic H6 hydro-
gens (Figure 3D, green arrows) is much stronger than it is with
the H7 hydrogen (49:1 by direct integration of cross-peaks).
Moreover, one of the diastereotopic H7 hydrogens has no
cross-peak with the aromatic region, indicative of occupation
[
16]
of a pseudo-axial position.
In conclusion, we have designed a synthetic route to an
oligomer consisting of alternating pyridyl and six-membered
cyclic urea groups. The resultant foldamer is conformationally
Chem. Eur. J. 2015, 21, 14699 – 14702
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Communication
pre-organized by dipolar repulsion and ring puckering to proj-
ect groups along the same vectors as the side-chains of amino
acid residues from one face of a b-strand, as was shown by X-
ray crystallography, NMR and MM calculations. As proof-of-
principle, we demonstrated this for mimics of two, three and
four phenylalanine side-chains. A particularly attractive aspect
of the approach is that an iterative two-step process allows
the foldamer to be extended by incorporation of pre-formed
monomers. Work is underway to evaluate the conformational
behaviour of mimics of specific peptide sequences containing
a range of proteinogenic amino acids, and to assess their effi-
cacy in mediating protein–protein interactions in aqueous
media.
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[
We thank The University of Oxford for funding and Dr.
Amber L. Thompson for assistance with X-ray crystallography.
T.Y. thanks Takeda Pharmaceutical Company Limited for their
generosity in providing financial and logistical support during
a sabbatical position as Visiting Scientist in The University of
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Keywords: dipolar repulsion
foldamers · inhibitors · protein–protein interactions
·
extended conformations
·
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[12]
15] Calculations give inter-nuclear hydrogen distances of: a) ꢀ4.18 Š be-
tween 1,3-di-pseudo-equatorial positions; b) ꢀ2.8 Š between pseudo-
equatorial positions on neighbouring ureas; c)>4 Š between the pyri-
dine and urea hydrogens (see the Supporting Information).
1
16] H NMR and NOESY spectra for dimer 9 were chosen for Figure 3 due
[
[
to superior resonance dispersion. Observations for trimer 11 and tetra-
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Published online on August 17, 2015
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