A multifaceted secondary structure mimic based on piperidine-piperidinones

Article abstract of DOI:10.1002/anie.201400927

Minimalist secondary structure mimics are typically made to resemble one interface in a protein-protein interaction (PPI), and thus perturb it. We recently proposed suitable chemotypes can be matched with interface regions directly, without regard for secondary structures. Here we describe a modular synthesis of a new chemotype 1, simulation of its solution-state conformational ensemble, and correlation of that with ideal secondary structures and real interface regions in PPIs. Scaffold 1 presents amino acid side-chains that are quite separated from each other, in orientations that closely resemble ideal sheet or helical structures, similar non-ideal structures at PPI interfaces, and regions of other PPI interfaces where the mimic conformation does not resemble any secondary structure. 68 different PPIs where conformations of 1 matched well were identified. A new method is also presented to determine the relevance of a minimalist mimic crystal structure to its solution conformations. Thus dld-1-faf crystallized in a conformation that is estimated to be 0.91 kcal-mol^-1 above the minimum energy solution state. Do we know, when designing a new peptidomimetic scaffold like the one shown, how it can resemble secondary structures? Design and modular synthesis of this elongated mimic is reported, and the structure is related to ideal and real structures at PPI interfaces.

Full text of DOI:10.1002/anie.201400927

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DOI: 10.1002/anie.201400927
Protein–Protein Interactions
A Multifaceted Secondary Structure Mimic Based On Piperidine-
piperidinones**
Dongyue Xin, Lisa M. Perez, Thomas R. Ioerger, and Kevin Burgess*
Abstract: Minimalist secondary structure mimics are typically
made to resemble one interface in a protein–protein interaction
(PPI), and thus perturb it. We recently proposed suitable
chemotypes can be matched with interface regions directly,
without regard for secondary structures. Here we describe
a modular synthesis of a new chemotype 1, simulation of its
solution-state conformational ensemble, and correlation of that
with ideal secondary structures and real interface regions in
PPIs. Scaffold 1 presents amino acid side-chains that are quite
separated from each other, in orientations that closely resemble
ideal sheet or helical structures, similar non-ideal structures at
PPI interfaces, and regions of other PPI interfaces where the
mimic conformation does not resemble any secondary struc-
ture. 68 different PPIs where conformations of 1 matched well
were identified. A new method is also presented to determine
the relevance of a minimalist mimic crystal structure to its
solution conformations. Thus dld-1 faf crystallized in a con-
formation that is estimated to be 0.91 kcalmolÀ1 above the
minimum energy solution state.
indicate how a minimalist mimic can optimally fit on one
secondary structure relative to another, and which side-chain
spacings are best represented. This is valuable information
because otherwise, for example, it is unclear which of the so-
called ideal a-helical mimics actually fit better on other
secondary structures, and which ones best present a given
side-chain combination (eg i, i + 3, i + 4 or i, i + 4, i + 5).[7]
M
inimalist mimics of secondary structures display amino
acid side-chains on backbones more rigid than, and structur-
ally different to, polypeptide chains.[1–3] Mimics that can be
made conveniently with a variety of amino acid side-chains
tend to be the most valuable, hence modular syntheses
involving amino acid-derived starting materials are ideal.[4–6]
Besides these synthetic considerations, it is also important to
gain an understanding of how well ideal secondary structures
can be represented by the best-fitting accessible conformer
from the mimicꢀs conformational ensemble. Such analyses
Figure 1. Pyrrolidine–pyrrolidinone mimic A can snake between various
S- and crescent-shaped conformers, whereas all the inter-ring s-
rotations in 1 together represent only one significant degree of
freedom making the scaffold overall more linear.
[*] D. Xin, Prof. K. Burgess
Reported here are modular syntheses of novel minimalist
mimics 1 from amino acid-derived starting materials. We
hypothesized scaffold 1 could present three amino acid side-
chains in orientations resembling extended secondary struc-
tures with wide side-chain separations. To test our hypothesis,
a large number of accessible (< 3 kcalmolÀ1) conformations
for each stereomer of 1 were simulated, then systematically
matched with the common secondary structures by using the
data mining technique we call Exploring Key Orientations on
Secondary structures (EKOS).[7] A solid-state structure of
a compound 1 was also obtained, and EKOS was used to
relate that conformation to ideal secondary structures, and to
the predicted conformational ensemble for scaffold 1 in
solution. Finally, some preferred conformations of 1 were
statistically related to more than 120000 structurally charac-
terized PPIs, the 68 that best matched side-chain orientations
of any accessible conformer of 1 were selected, and the
secondary structures at these interfaces were analyzed[8] to
Department of Chemistry, Texas A & M University
Box 30012, College Station, TX 77841-3012 (USA)
E-mail: Burgess@tamu.edu
Dr. L. M. Perez
Laboratory for Molecular Simulation, Texas A & M University
Box 30012, College Station, TX 77842 (USA)
Prof. T. R. Ioerger
Department of Computer Science, Texas A & M University
College Station, TX 77843-3112 (USA)
[**] Financial support for this work was provided by the National
Institutes of Health (GM087981) and the Robert A. Welch Foun-
dation (A-1121). We thank Dr. Joe H. Reibenspies for help with the
X-ray crystallography.
Supporting information for this article (experimental procedures
and spectroscopic data for compounds 1–11, procedures and
results of the overlays on secondary structures, data of protein
database mining and X-ray crystallographic data for compound
1 faf, 9gg and 11af) is available on the WWW under http://dx.doi.
org/10.1002/anie.201400927.
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explore how this structure is predicted to best match side-
chain orientations at PPI interfaces. Throughout, these data
are compared with the previously reported[8–10] mimics A
(Figure 1).
A vulnerability in syntheses of substrates A is the
enolizable, and therefore stereochemically delicate, CH.[9]
Conversely, an attribute of scaffolds 1 is that they are based
on b-substituted b-amino acids where a methylene group
insulates the chiral center from stereomutation. Another
problem in syntheses of scaffolds A is that fragments bearing
bulkier amino acid side-chains tend to retard coupling
reactions. We postulated this steric effect would be less
pronounced in construction of 1 where the side-chains are one
methylene removed from the electrophilic coupling site.
Syntheses of molecules 1 began with preparation of b-
substituted b-amino acid derivatives 2 using a novel combi-
nation of literature procedures[11,12] that allowed us to obtain
multi-gram amounts, without chromatography in most cases
(see Supporting Information). These amino acids 2 were
reacted with Meldrumꢀs acid, a known reaction for a-amino
acids,[13] to form six-membered homologs of tetramic acids
(Scheme 1). Removal of the Boc-protecting group gave the
Scheme 1. Syntheses of the electrophilic N-caps 4. Boc=tert-butoxy-
carbonyl; EDCl=N’-(3-dimethylaminopropyl)-N-ethylcarbodiimide;
DMAP=4-dimethylaminopyridine; TFA=trifluoroacetic acid; Bn=ben-
zyl; Cbz=carbobenzyloxy.
piperidinediones 3 that were converted to the vinylic chlor-
ides 4. Those analogs with threonine side-chains have an
inherent marker for loss of stereochemical fidelity; as
anticipated, no epimerization was observed, at least in the
syntheses of the compounds containing Thr.
Scheme 2. Syntheses of the target materials 1.
Nitriles 5, intermediates in the syntheses of the b-amino
acids 2, were simultaneously N-deprotected and hydrolyzed,
then reductively coupled with the known, and commercially
available, synthons B[14] or C[15] to give the amines 6
(Scheme 2). Reaction of these b-amino esters with Best-
mannꢀs ylide[16] gave the protected intermediates 7. Com-
pounds 5 to 7 were isolated, without chromatography, on
multigram scales. N-Deprotection of the intermediates 7 gave
the nucleophiles 8. Amines 8 were then condensed with the
electrophiles 4 to give the scaffolds 9 bearing two side-chains.
As anticipated, this process seems largely unaffected by steric
demands of the side-chains since the coupling yields were
uniformly high.
In a divergent step, intermediates 7 were C-deprotected,
then converted to the vinylogous chlorides 10. Convergence
was then possible by coupling of the nucleophiles 8 with the
electrophiles 10 to give the extended systems 11 that were
then N-deprotected and capped with the electrophiles 4.
The syntheses of mimics 1 may seem complicated, but
they are driven by the simple modularity concept expressed in
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Figure 2. N-Capping, N-protected, and C-protected synthons like 4, 10,
and 8, are pivotal in divergent–convergent routes to scaffolds 1.
Figure 2. Overall, the divergent–convergent syntheses of
compounds 1 pivot around synthons 7; these were converted
to nucleophiles and electrophiles that were joined to elongate
the scaffold. Fragments 8 and 10 in Scheme 2 are similar to C-
and N-protected amino acids in peptide syntheses.
No spectroscopic technique can characterize conforma-
tional ensembles of small molecules like 1 or A because they
equilibrate between hundreds solution states that have
significantly different side-chain orientations. Techniques
like circular dichroism and NOESY/ROESY NMR spectros-
copy give a conformational average, that does not correspond
to any real state.[6] An X-ray structure shows whatever mimic
conformation happened to crystallize, determined, at least in
part, by crystal packing forces. Those packing forces are
significant enough that some solid-state conformations may
not be preferred in solution. Indeed, a solid-state mimic
conformation that does not resemble the target secondary
structure has been reported by Hamilton et al.,[17] and this
type of occurrence should not be surprising because crystal
packing forces are relatively large.
A problem that has not been addressed in studies of
minimalist mimics is how to evaluate the relevance of a solid-
state structure to a conformational ensemble in solution. Here
we suggest a strategy similar to EKOS can be used to
determine if a solid-state conformation from an X-ray
structure is present in the simulated conformational ensemble
and, if it is, provide an estimate of the energy difference
between the simulated solution-state conformer closest to the
solid-state structure and the lowest energy one overall. Here
we call this strategy EKOX. EKOX makes it possible to
overlay the single conformer represented in a solid-state
structure on all ideal secondary structures to find the one it
best matches and the preferred side-chain combination. This
information is generated free from the bias of the research-
er(s) who may have designed the scaffold to mimic a particular
secondary structure and side-chain conformation.
An example of the use of EKOX as described above is as
follows. Figure 3a shows data for a solid-state structure of
dld-1faf. That structure overlaid on an ideal a-helix with an
RMSD (root-mean-square deviation) of 1.5 ꢁ (based on the
six Ca + Cb coordinates of the side-chains; Figure 3b). To
estimate the energy of the conformer represented by the X-
ray structure, but in solution, EKOX was used to systemati-
cally overlay the simulated conformational ensemble on the
X-ray structure to find the one with the best fit. In fact, that
conformer (matched to 0.46 ꢁ RMSD; Figure 3c) had
a simulated energy of 0.91 kcalmolÀ1 relative to the lowest
one generated, i.e., it is predicted to be significantly
Figure 3. a) X-Ray crystal structure of dld-1 faf. That structure is
shown overlaid upon: b) an ideal a-helix and c) the simulated confor-
mer best matching the X-ray structure. d) The simulated conformer
best matching an ideal a-helix is overlaid on an ideal a-helix. e) Two
copies of the X-ray structure overlapping on the C- and N-termini
illustrates the helical structure is anticipated to repeat every 3 nm and
5 residues.
populated in solution. The simulated conformer that overlaid
most closely on the ideal a-helix did so with a similar RMSD
and energy (0.50 ꢁ, 0.84 kcalmolÀ1, Figure 3d). Overall,
these data indicate there is a conformer in the simulated
ensemble that represents an ideal a-helix better than the one
that crystallized. Moreover, the simulated solution-state
conformers closest to the solid state structure and to an
ideal a-helix seem to have about the same relative energies.
The conformer from the X-ray and the most a-helical one
from the simulation both had the same side-chain corre-
spondence: i, i + 5, i + 10.
Figure 3e shows copies of the X-ray structure arranged
such that the C-terminal piperidinone of one is superimposed
on the N-terminal piperidinone of the other. This illustrates
how the solid-state conformer is anticipated to coil in a helical
way, repeating every 5 residues and 3 nm. Parenthetically,
Whitesides and co-workers communicated repeated reductive
couplings of unfunctionalized 4-piperidinones to give “linker
rods”.[18] A “rod” with four piperidine repeats was 1.6 nm long
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in the solid state (X-ray), comparable to 1.7 nm for 1 faf which
has five rings (Figure 3a). Whitesideꢀs structure is like
a straight rod whereas 1 twists to accommodate the chiral
piperidinones making it shorter between the N- and C-
termini.
EKOS was used to systematically overlay simulated
conformers in the ensemble of 1aaa (all stereomers) on the
most common ideal secondary structure elements, as pre-
viously described.[7] Figure 4 features data for two stereomers
of 1; it shows the RMSDs for the best fitting conformers
corresponding to each element of secondary structure (com-
plete data is given in Figure S1 in the Supporting Informa-
tion). These data are calibrated relative to the average
RMSDs for all the best fitting conformers for a given
stereomer. Thus for lll-1aaa, 0.65 ꢁ was the average for
the seven best fitting conformers on the seven secondary
structures indicated. This graphic indicates the lll-isomer is
predicted to mimic a sheet–turn–sheet closely, ideal helical
structures less well, and extended parallel b-sheet- and b-
strand structures very poorly (Figure 4a). Comparing this
with the plot for the dld-isomer (selected because the X-ray
data in Figure 3 was accumulated for that isomer) shows
a similar tendency towards helical conformations. Overlays
on sheet–turn–sheet and antiparallel b-sheet structures have
identical RMSD values because the antiparallel b-sheet
region is part of the sheet–turn–sheet motif, and this is
where the mimic overlaid.
X-ray structures of two other analogs in this series were
also obtained and showed variance at the piperidine–piper-
idone dihedral angles (see Figures S3 and S4). This is
consistent with the mimics 1 populating a diversity of
conformational states.
Figure 4c shows the corresponding data for lll-Aaaa.
Scaffold A has a strong tendency to mimic b-sheet structures,
but not helical ones, and that trend is in fact consistent for all
the stereomers of A.
Minimalist mimics are usually designed to perturb real
PPIs by displacing a secondary structure at the interface.
However, interfaces rarely have ideal secondary structures,
and many do not feature secondary structures at all. Explor-
ing Key Orientations (EKO) is data mining to compare
simulated conformational ensembles of minimalist mimics
with crystallographically characterized PPI regions, irrespec-
tive of the secondary structures involved.[8] Here EKO
analyses for lll-1aaa and lll-Aaaa were performed to
determine if the biases for ideal secondary structures
predicted above (using EKOS) are consistent with the
statistical match of accessible conformations on real interface
secondary structures. To do this we developed a script to
assess the secondary structure content of the interface regions
that best match simulated conformers. After analyses of tens
of thousands of PPI structures, EKO found 68 interface
regions that matched a simulated conformer of 1 with RMSDs
of < 0.3 ꢁ, and 32 that similarly matched conformers of A.
Figure 5 shows only lll-1aaa matched on helical regions at
PPI interfaces though it found relatively more sheet interface
regions. lll-1aaa and lll-Aaaa had a similar proclivity to
match with contiguous or non-contiguous motifs of no
particular secondary structure (called “single segment” and
“multiple segments” in Figure 5). Overall, these observations
are consistent with those from comparisons of the simulated
conformational ensembles with ideal secondary structures in
Figure 4. Four representative overlays for lll-1aaa at
interfaces are illustrated in Figure S5.
In summary, a route to mimics 1 that is not vulnerable to
epimerization, and involves facile couplings, was devised.
Only six methylene groups differentiate the structures of 1aaa
and Aaaa but that drastically changes side-chain orientations
in their preferred solution conformers. Overall, we assert that
even though none of the conclusions about the secondary
Figure 4. Predicted mimicry of secondary structures for: a,b) two
selected stereomers of 1aaa (full data is given in supporting) and
c) lll-Aaaa.
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systematically overlaid on a huge database of PPI structures
using EKO; in other words, these two scaffold designs
complement each other. Applications of mimics 1 in pertur-
bation of a particular PPI based on EKO analyses will be
reported in due course.
Received: January 27, 2014
Published online: March 3, 2014
Keywords: amino acids · computational chemistry ·
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helical structures · peptidomimetics · protein–
protein interactions · secondary structures
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