Chemoselective fragment condensation between peptide and peptidomimetic oligomers

Article abstract of DOI:10.1039/c3ob40606g

We report the first example of chemoselective fragment condensation, through native amide bond formation, between peptoid and peptide oligomers. Peptoid oligomers bearing C-terminal salicylaldehyde esters were synthesized and ligated to peptides containin

Full text of DOI:10.1039/c3ob40606g

Organic &
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Chemoselective fragment condensation between
peptide and peptidomimetic oligomers†
Paul M. Levine,a Timothy W. Craven,b Richard Bonneaub,c and Kent Kirshenbaum*a
Cite this: Org. Biomol. Chem., 2013, 11,
4142
Received 26th March 2013,
Accepted 7th May 2013
DOI: 10.1039/c3ob40606g
www.rsc.org/obc
We report the first example of chemoselective fragment conden- routes for ligating abiotic and peptide oligomers through
sation, through native amide bond formation, between peptoid native amide linkages.2 Discovering new methods for conduct-
and peptide oligomers. Peptoid oligomers bearing C-terminal ing chemoselective fragment condensation to incorporate
salicylaldehyde esters were synthesized and ligated to peptides abiotic constituents into protein constructs will facilitate the
containing N-terminal serine or threonine residues. We investigate development of hybrid biomacromolecules that can establish
the ligation efficiency of peptoid oligomers varying in length, unprecedented structural motifs, enhanced stability, and novel
sequence, and C-terminal steric bulk. These protocols enhance functions.
accessibility of structurally complex peptoid–peptide hybrids and
N-Substituted glycine oligomers, or ‘peptoids,’ are an
will facilitate the design new semi-synthetic proteins with unique important class of foldamer compounds composed of tertiary
attributes.
amide linkages (Fig. 1).3 The ability to incorporate extensive
chemical diversity into the peptoid side chains facilitates
design strategies that allow for a wide range of applications,
such as enantioselective catalysis, molecular recognition, and
intracellular delivery.4–6 Importantly, numerous studies have
identified peptoid side chains that can control backbone con-
formational ordering, establishing the potential to fold pep-
toids into distinct secondary structures that are not populated
by polypeptides.7 More recently, two well-ordered peptoid sec-
ondary structure modules were assembled via triazole linkages
to generate rudimentary peptoid tertiary structures.8 Although
this strategy permits the design of peptoids that begin to
resemble small folded proteins, we seek the capability to craft
functional peptoid-peptide hybrids joined through native
amide bonds.
Native chemical ligation (NCL) has proven to be a valuable
technique for the construction of synthetic proteins of extra-
ordinary complexity.9 NCL typically involves the reaction
between a C-terminal peptide thioester and a peptide bearing
an N-terminal cysteine residue. Following a selective trans-
thioesterification reaction, a spontaneous S→N acyl transfer
affords the native amide bond at the ligation site. Extensive
developments in cysteine-based NCL have allowed a multitude
A general route for the synthesis of structurally complex
sequence-specific heteropolymers capable of performing
sophisticated functions, such as enzyme-like catalysis, remains
a fundamental challenge. While significant attention has been
devoted to total chemical synthesis of native proteins, recent
advances have also established synthetic routes to generate
hybrid biomacromolecules, such as proteins bearing abiotic
oligomer constituents.1 These semi-synthetic biomolecules
have the potential to exhibit new modes of molecular recog-
nition and enhanced thermodynamic stability. Previously,
single-atom or small abiotic substitutions have been incorpor-
ated into native protein frameworks.2 We now seek to establish
new efficient routes to hybrids between diverse abiotic oligo-
mers and polypeptide sequences.
Conventional linear solid-phase peptide synthesis (SPPS)
protocols can routinely generate large polypeptides of sizable
chain lengths (20–50 monomers). Limitations in the size and
structural diversity of the synthetic polypeptide products can
restrict their ability to exhibit particular functions, such as
enzymatic catalysis. There are limited chemoselective synthetic
aDepartment of Chemistry, New York University, New York, New York 10003, USA.
E-mail: kent@nyu.edu; Tel: +1 212-998-8486
bCenter for Genomics and Systems Biology, New York University, New York, New York
10003, USA
cCourant Institute of Mathematical Sciences, Computer Science Department,
New York University, New York, New York 10012, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob40606g
Fig. 1 Comparison of peptide and peptoid structures.
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Fig. 2 Synthesis of C-terminal peptoid salicylaldehyde esters and the library of fragments utilized in ligation studies. Iterated steps include bromoacetylation using
DIC as a coupling agent, followed by displacement with a primary amine. (A) Peptoids bearing C-terminal salicylaldehyde esters are synthesized from 2-chlorotrityl
resin. (B) Library of peptoid and peptide fragments utilized in chemoselective fragment condensation reactions.
of variations in this methodology, including the use of C-term- Reversible imine formation with an N-terminal serine or threo-
inal phenyl-esters and hydrazide thioester surrogates.10,11 nine residue produces a stable N,O-benzylidene acetal inter-
However, the rare presence of cysteine (1.4% content in pro- mediate followed by irreversible amide bond formation
teins) and challenges associated with synthesizing thioesters through a 1,5 O→N acyl shift (ESI Fig. 1†). Upon deprotection,
have limited the utility of cysteine-based NCL.12 For this the intermediate is readily converted into a native serine/threo-
reason, new protocols have been introduced that include post- nine linkage (12.7% content in proteins) at the ligation site.16
ligation desulfurization to establish native amide linkages at Importantly, β-branched amino acids (e.g., Val, Pro, and Ile) at
additional amino acid residues (i.e., Arg, Gln, Val, Leu, Lys, the C-terminus do not hinder rapid coupling kinetics, a
and Pro).13
common drawback observed with cysteine-based NCL (which
In order to circumvent difficulties associated with cysteine- can require coupling times exceeding 48 h). Importantly, the
based NCL, alternative strategies to achieve chemoselective chemoselective serine/threonine-based ligation reaction is
fragment condensation, through native amide bond formation, orthogonal to cysteine-based NCL and has been utilized for
have been introduced. These include α-ketoacid-hydroxylamine total protein chemical synthesis.16
(KAHA) ligation and traceless Staudinger ligation.14,15 Of par-
Although initial synthetic strategies for peptoid ligation
ticular interest is a chemoselective serine/threonine-based liga- have been reported, such as disulfide bond formation and Cu-
tion that utilizes a C-terminal peptide salicylaldehyde ester. catalyzed azide–alkyne [3 + 2] cycloaddition (CuAAC) ‘click’
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Fig. 3 Fragment condensation between C-terminal peptoid salicylaldehyde ester 1 and peptide 4. Upper traces of peptoid 1 (purple), intermediate (blue), and
product containing native serine (red) are offset in y-direction for clarity. Reaction monitored by HPLC (214 nm) and LCMS. AU indicates absorbance units.
reactions, none have provided native amide bond for- based NCL with a small set of peptide fragments (Fig. 2B).
mation.17,18 Protease-mediated ligation of peptoid oligomers Beginning with a simple trimer model system, we coupled
has yielded sequence-defined macromolecular polymers, but peptoid trimer 1 to tripeptide 4, forming the corresponding
of heterogeneous chain lengths.19 Recently, cysteine-based N,O-benzylidene acetal intermediate. This reaction was moni-
NCL was attempted to develop peptoid–peptide hybrids of tored by HPLC and LCMS, which established that coupling
bovine pancreatic ribonuclease A.20 This study included was complete after 1 hour (Fig. 3 and ESI Table 2†). Upon
attempts to synthesize peptoid C-terminal thioesters, which removal of the acetal group under acidic conditions, the inter-
were unsuccessful using either MBHA or hydrazinobenzoyl mediate was rapidly and quantitatively converted to the native
resin due to incompatibilities with standard peptoid synthesis amide product.
protocols (e.g., prolonged exposure to primary amines or
Because C-terminal β-branched amino acids commonly
bromoacetic acid). Instead, the authors successfully utilized hinder peptide coupling in cysteine-based NCL reactions,
the thiazolidine ligation strategy (which does not form we evaluated ligation efficiency of peptoid salicylaldehyde
native amide bonds) to generate semi-synthetic mimics of esters that include steric bulk at the C-terminus (Table 1,
bovine pancreatic ribonuclease A and assess their catalytic entries 1–4). Ligation reactions were conducted between
activity.
N-terminal serine/threonine peptide fragments and peptoid
Due to the challenges associated with NCL between peptoid oligomers incorporating N-alkyl or N-aryl side chains at their
and peptide fragments (vide supra), we decided to investigate C-termini (Table 1). The presence of the bulky C-terminal
the applicability of serine/threonine-based NCL to achieve peptoid side-chains does not abrogate rapid coupling kinetics,
chemoselective condensation using small peptoid and peptide as these reactions were conducted with ∼99% conversion
fragments. Employing modified solid-phase peptoid synthesis after
1 h. These N-alkyl or N-aryl peptoid side-chains
protocols, we synthesized linear peptoid oligomers containing can promote polyproline I- or polyproline II-type helix
C-terminal free acids from 2-chlorotrityl resin (Fig. 2A).19 Fol- formation, respectively, establishing the ability to generate
lowing C-terminal phenolysis conversion in solution and puri- conformationally ordered architectures, including secondary
fication, a small library of peptoid salicylaldehyde esters were structure motifs that may be of marginal stability for
evaluated for ligation efficiency utilizing serine/threonine- polypeptides.21,22
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Table 1 Ligation efficiency of peptoid salicylaldehyde esters displaying steric bulk at the C-terminus with peptides containing N-terminal serine or threonine
residues
Entry
Peptoid
Peptide
Aa (%)
Bb (%)
B calc. m/z
B obs. m/z
1
2
3
4
2
2
3
3
4
5
4
5
99
99
99
99
99
99
99
99
787.4
801.4
759.4
773.4
787.9
801.7
759.9
773.9
a Reaction conversion after 1 h. The conversion was monitored by HPLC and was calculated based on the consumption of the salicylaldehyde.
b Deprotection conversion after 10 min. The conversion was monitored by HPLC and was calculated based on the consumption of the N,O-
benzylidene acetal intermediate. X denotes an (S)-N-1-phenylethyl glycine (peptoid 2) or an N-aryl moiety (peptoid 3).
Fig. 4 Chemical structures of peptoid–peptide hybrids 9 (ligation product between peptoid 6 and peptide 7) and 10 (ligation product between peptoid 2 and
peptide 8). Red circle indicates native amide linkage formed by the ligation reaction.
In order to demonstrate that peptoid oligomers, including sequence-specific heteropolymers capable of performing soph-
diverse side chain functional groups, can be ligated using isticated protein-like functions.
unprotected peptide fragments, coupling was conducted
with sequence-diverse peptoid 6 and peptide 7. Electrospray
ionization mass spectrometry confirmed the initial formation
of the N,O-benzylidene acetal ligation intermediate (calc. m/z:
Conclusions
1842.0; obs. m/z: 1842.0) and the peptoid–peptide hybrid
product 9 (Fig. 4, calc. m/z: 1738.9; obs. m/z: 1738.8, see ESI
Fig. 2†). In addition, peptide 8, which incorporates numerous
reactive side-chains (i.e., Lys, Glu, Tyr, and Ser) was success-
fully coupled to peptoid 2, affording the desired ligated
product 10 (Fig. 4, calc. m/z: 2145.0; obs. m/z: 2144.9). Based
on previous studies, the expectation is that cysteine is not com-
patible with this ligation method.16 The ability to conduct frag-
ment condensation with peptoids bearing diverse side chains
will further assist in the development of structurally complex
In this report, we describe a facile chemoselective synthetic
route to establish native amide bond formation between
peptoid and peptide oligomers. Fragment condensation was
rapid, including peptoids containing β-branched side-chains
at their C-termini, and the intermediates were quantitatively
converted to native amide products. In addition, unprotected
oligomer fragments varying in length and sequence were
efficiently coupled, establishing the ability to generate highly
complex peptoid–peptide hybrids. We anticipate this method
will be used to synthesize macromolecules that integrate
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structural and functional attributes from both polypeptides
and peptoids.
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Acknowledgements
This work was supported by the NSF (Award CHE-1152317 to
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