Promoting peptide α-helix formation with dynamic covalent oxime side-chain cross-links

Article abstract of DOI:10.1039/c1cc12010g

Covalent side-chain cross-linking has been shown to be a viable strategy to control peptide folding. We report here that an oxime side-chain linkage can elicit α-helical folds from peptides in aqueous solution. The bio-orthogonal bridge is formed rapidly

Full text of DOI:10.1039/c1cc12010g

Chemical Communications
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Volume 47 | Number 39 | 21 October 2011 | Pages 10841–11156
ISSN 1359-7345
COMMUNICATION
W. Seth Horne et al.
Promoting peptide ꢀ-helix formation
with dynamic covalent oxime
side-chain cross-links
1359-7345(2011)47:39;1-6

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Promoting peptide a-helix formation with dynamic covalent oxime
side-chain cross-linksw
Conor M. Haney, Matthew T. Loch and W. Seth Horne*
Received 8th April 2011, Accepted 2nd June 2011
DOI: 10.1039/c1cc12010g
Covalent side-chain cross-linking has been shown to be a viable
strategy to control peptide folding. We report here that an oxime
side-chain linkage can elicit a-helical folds from peptides in
aqueous solution. The bio-orthogonal bridge is formed rapidly
under neutral buffered conditions, and the resulting cyclic
oximes are capable of dynamic covalent exchange.
cyclic peptides have been reported;10 however, neither the
dynamics of the linkage in that context nor its ability to
influence folding has been studied.
We selected peptide 1 (Fig. 1) as a model system to explore
the ability of oxime side-chain cross-links to promote an
a-helical conformation in aqueous solution. This 17-residue
sequence, partially helical at pH 7, has previously been used to
examine the energetic contributions of non-covalent interactions
(e.g., salt bridges) to a-helix formation.11 Peptides 2–5 are
derivatives of 1 designed to probe the relationship between the
structure of an oxime bridge (residue spacing, side-chain
length) and its ability to stabilize the a-helix fold. Residues
X and Z (isosteres of Orn and Lys, respectively) bear aminooxy
groups attached to the peptide backbone by either 2 or 3
carbons. Residue U has a Ser-acylated amine side chain that
can be efficiently converted to a glyoxyl aldehyde in neutral
aqueous buffer by treatment with a mild oxidant.10a,b,12
Peptides 1–5 were prepared by standard microwave-assisted
Fmoc solid-phase peptide synthesis (SPPS). Monomers for
incorporation of X and Z were synthesized by modification
a-Helical secondary structures are commonly found at protein-
protein binding interfaces.1 The need for chemical agents that
selectively disrupt protein-protein interactions has motivated
extensive efforts to mimic a-helical segments of proteins with
peptides, peptide mimetics and small molecules.2 The use of
short peptide sequences derived from natural proteins for this
purpose is limited by their tendency to be minimally structured
when removed from the native protein context. One of the first
chemical methods examined for promoting a-helix formation
in short peptides was the introduction of a covalent lactam
bridge.3 Recent years have seen significant developments in
covalently-constrained helical peptides, including new side-chain
cross-linking chemistries4 and complementary strategies based
on replacement of backbone hydrogen bonds with covalent
isosteres.5 These technologies have been applied to produce
potent, bioactive helix mimics.6
of known routes to related derivatives (Scheme S1w).13
A
protected form of residue U suitable for use in SPPS is
known.10b Two synthetic observations about the aminooxy-
functionalized peptides are noteworthy. We found that
TFA-mediated cleavage of peptides containing X or Z from
a Rink-linker-based resin without addition of ethanedithiol as
a nucleophilic scavenger led to no isolatable product. Addition
of the nucleophilic scavenger provided yields and crude
purities similar to those obtained for the natural sequence 1
A subset of linkages examined to date as side-chain ‘‘staples’’
can be formed chemoselectively in physiological conditions.4a,d–f
We are interested in exploring the ability of reactions that are
(1) bio-orthogonal and (2) dynamically reversible in water to
control peptide folding. Such technologies have the potential
to enable the discovery of bioactive cross-link topologies in
polycyclic peptides by combining the benefits of peptide
stapling4–6 with template-assisted amplification as demon-
strated in the field of dynamic combinatorial chemistry.7 As
a first step toward this goal, we report here the ability of
dynamic covalent oxime side-chain cross-links to promote
a-helix formation in aqueous solution. Oximes are widely used
in chemoselective ligations involving proteins8 and have been
used in dynamic combinatorial libraries.7,9 A few oxime-based
Department of Chemistry, University of Pittsburgh, Pittsburgh,
PA 15260, USA. E-mail: horne@pitt.edu; Fax: +1 412 624 8611;
Tel: +1 412 624 8700
w Electronic supplementary information (ESI) available: Fig. S1–S6,
Table S1–S2, Scheme S1 and experimental methods. See DOI:
10.1039/c1cc12010g
Fig. 1 Structures of peptides 1–5 and residues U, X and Z.
Chem. Commun., 2011, 47, 10915–10917 10915
c
This journal is The Royal Society of Chemistry 2011

Scheme 1 Conversion of peptides 2–5 to cyclic oximes 2b–5b, and
exchange of 2b to linear oxime 2c.
Fig. 2 HPLC analysis of the oxidation and cyclization of peptide 2.
The reaction, 100 mM peptide in 100 mM phosphate pH 7, was
monitored (a) before and (b) 10 min after addition of 2 equiv. NaIO4.
prepared by the same method. We attribute loss of material in
the absence of ethanedithiol to the irreversible reattachment of
peptide to resin during cleavage by linker alkylation of the
aminooxy side chain.14 We also found that some C18 HPLC
stationary phases irreversibly bind aminooxy-functionalized
peptides (i.e., injected material does not elute). We were able to
find C18 analytical and preparative columns free of this
problem (see supporting information).
(Fig. S3w). The above data suggest that the system is in
dynamic exchange in pH 7 buffer but is kinetically trapped
at a non-equilibrium state.
In order to further characterize the dynamic covalent nature
of the cyclic peptide oximes, we prepared an equilibrating
mixture of 2a/2b at pH 7 as described above and added
1000 equiv. of buffered O-methylhydroxylamine to compete
with the peptide X residue aminooxy group for reaction with
the glyoxyl aldehyde; aniline was added to a final concentration
of 10 mM to facilitate exchange.16 The above conditions set up
a dynamic equilibrium between linear peptide 2a, cyclic oxime
2b (E and Z isomers) and linear oxime 2c (E and Z isomers).
HPLC/MS analysis after 24 h showed formation of a small
amount of product with mass corresponding to the linear
oxime 2c (Fig. S4w); the amount of 2c increased slightly at
48 h to a final conversion of B3%, suggesting the system is
slow to reach equilibrium under these conditions. At pH 4,
exchange was much faster, and B10% of the material converted
to linear oxime 2c after 24 h.
With peptides 1–5 in hand, we next sought to test the
efficiency of the oxidation of the U residue in 2–5 to an
aldehyde and subsequent oxime formation (Scheme 1). We
carried out cross-linking reactions in conditions that are
typical for biophysical characterization of peptide folding or
receptor binding. We prepared a 100 mM solution of each
peptide in 100 mM phosphate buffer at pH 7 and added
sodium periodate to cleave the 1,2-aminoalcohol in each U
residue to the corresponding glyoxyl aldehyde.10a,b,12 HPLC/MS
analyses of the reaction mixtures minutes after addition of
periodate showed complete consumption of starting material
in all cases (Fig. 2, S1).
The oxidized peptides are capable of establishing an equilibrium
between the linear aminooxy-aldehyde (2a–5a) and cyclic
oxime (2b–5b) forms. While the oxime is thermodynamically
favored,8,9,15 the kinetics of intermolecular oxime ligation
reactions are typically extremely slow at neutral pH; acidic
conditions and/or nucleophilic catalysts are used to achieve
reasonable rates.16 No evidence was observed for the linear
peptides 2a–5a at the first HPLC time point taken to monitor
the reaction (B10 min). Instead, the majority (85–90%) of the
starting material was converted to two new species with mass
corresponding to oximes 2b–5b. We assign the two major
products observed by HPLC in each case as resulting from
the E- and Z-oxime isomers; the E/Z ratio varied among
2b–5b. For peptide 2b, we assigned the absolute configuration
of the two oxime isomers by 1HNMR spectroscopy (Fig. 2b, S2w).
The distribution of products observed at 10 min was
unchanged after 24 h. For peptide 2b, we isolated the E- and
Z-oxime isomers by HPLC and examined the isomerization of
each. The isomers are able to interconvert in pH 7 phosphate,
albeit slowly; isomerization is faster under acidic conditions
We employed circular dichroism (CD) spectroscopy to
compare the a-helicity of control peptide 1 to that of linear
peptides 2–5 and cyclic analogues 2b–5b (Fig. 3). All samples
were measured at 100 mM peptide concentration in pH 7
phosphate buffer. Control peptide 1 showed modest helicity
under the conditions of the experiment, indicated by the
negative peaks at 208 and 222 nm. CD spectra of peptides
2–5 prior to oxidation showed almost no a-helical secondary
structure (Fig. 3a). The loss of helicity in 2–5 relative to 1 is
likely due to the introduction of the bulky, cationic U residue
side chain in place of an Ala in parent sequence 1.
Addition of 2 equiv. of periodate to peptides 2–5, conditions
where the cyclic oximes 2b–5b are the predominant species by
HPLC/MS (vide supra), led to significant changes in the CD
spectra (Fig. 3b). The a-helical population among 2b–5b
varied as a function of the structure of the oxime bridge
(Table S1w): an i- i+4 spacing of aldehyde and aminooxy
residues in the sequence (2b, 3b) leads to more helical
population than the corresponding i-i+3 spacing (4b, 5b).
c
10916 Chem. Commun., 2011, 47, 10915–10917
This journal is The Royal Society of Chemistry 2011

Fig. 3 Circular dichroism scans at 20 1C (a,b) and thermal melts (c) for peptides 1–5 and oxime-bridged cyclic peptides 2b–5b. Measurements
were carried out on 100 mM concentration peptide solutions in 100 mM phosphate pH 7 in absence (a) or presence (b,c) of 200 mM sodium
periodate. Measurements of 2b–5b were carried out on unseparated E/Z oxime mixtures.
Molecular modelling suggests that the oxime macrocycles in
4b and 5b cannot be accommodated into an idealized a-helix
without significant side-chain distortion (Fig. S5w). The best
folded oxime-constrained peptides 2b and 3b are more helical
than the control sequence 1 under identical conditions.
Replacement of the Z residue in 3 with Lys (i.e., O - CH2
substitution on the side chain) abolished the enhanced helicity
observed for 3 after periodate addition (Fig. S6w), supporting
the essential role of the oxime bridge in the observed CD data.
In thermal melts monitored at 222 nm, 2b and 3b show a
B15 1C increase in Tm relative to 1 (Fig. 3c, Table S1w).
Interestingly, the oxime side-chain linkages impacted the
reversibility of the folding process; neither 2b nor 3b recovered
their initial helicity after cooling a thermally unfolded sample.
The unfolding of control peptide 1 was fully reversible under
these conditions.
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In summary, we have demonstrated that a bio-orthogonal
oxime side-chain linkage can promote a-helical folding in a
medium-length peptide in aqueous solution. The covalent
bridge is formed rapidly and in high yield under neutral
buffered conditions. Spacing and side-chain length of the
residues bearing the aminooxy and aldehyde precursor
functional groups are important determinants of a-helicity in
the cyclic oxime. We see evidence that the oxime peptides are
capable of isomerization and dynamic exchange with an
aminooxy-functionalized small molecule; however, the rate
of exchange is slow at neutral pH.16 It will be interesting to
see how the rate of oxime exchange compares when the
competing aminooxy groups reside on the same molecule
(i.e., a longer peptide with n U residues and n+1 Z residues).
We see such species as being useful for the template-assisted
discovery of new bioactive cross-link topologies and folding
patterns in polycyclic peptides and proteins. Efforts to analyse
the kinetics and thermodynamics of folding and oxime
exchange in these and more complex systems are ongoing.
We thank the University of Pittsburgh for financial support.
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c
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Chem. Commun., 2011, 47, 10915–10917 10917