Protease-mediated ligation of abiotic oligomers

Article abstract of DOI:10.1021/ja055105m

We demonstrate that proteases can catalyze the ligation of peptidomimetic oligomers. The enzyme clostripain was used to facilitate the native ligation of N-substituted glycine oligomers, or peptoids. In addition to mediating the efficient condensation of

Full text of DOI:10.1021/ja055105m

Published on Web 11/10/2005
Protease-Mediated Ligation of Abiotic Oligomers
Barney Yoo and Kent Kirshenbaum*
Department of Chemistry, New York UniVersity, 100 Washington Square East, New York, New York 10003-6688
Received July 27, 2005; E-mail: kent@nyu.edu
Scheme 1
N-substituted glycine oligomers, or peptoids, are an important
class of peptidomimetics that demonstrate a propensity to form
stable secondary structures.1 These “foldamers”2 can display
rudimentary biological activities3 along with resistance to proteoly-
sis.4 Ongoing studies seek to discover peptoids that are capable of
autonomous folding into compact tertiary structures.1c,d Such efforts
have been hampered by the difficulty in synthesizing sequence-
specific heteropolymers of the requisite chain lengths. Segment
condensation or ligation is an approach that can address this
challenge.5 A number of studies have shown that proteases can be
Scheme 2 a
coerced to run in reverse, catalyzing the formation of peptide
bonds.6-8 One major impediment to the use of proteases for peptide
synthesis is the sequence limitation imposed by the proteasesthe
product itself can be a target for proteolysis. In comparison,
protease-catalyzed ligation of peptoid segments is an attractive
strategy due to their inherent resistance to proteolytic degradation.
Here, we report enzyme-mediated ligation for the generation of
biomimetic macromolecules through the condensation of peptoid
fragments.
Our protocols utilize a protease in concert with its corresponding
substrate mimetic,9 a chemical moiety resembling the protease’s
natural substrate. Clostripain, from Clostridium histolyticum, is a
cysteine protease that catalyzes the hydrolysis of the amide bond
following an arginine residue.10 This enzyme was selected for its
broad amino acid sequence tolerance, particularly in the positions
adjacent to the scissile bond.11 p-Guanidinophenyl esters have been
reported as substrate mimetics for trypsin and, more recently, for
clostripain.7 In this ligation approach, the p-guanidinophenyl ester
serves as a recognition element for the protease and also acts as an
efficient leaving group. Catalyzed formation of the amide bond is
conducted in competition with the hydrolysis of the p-guanidi-
nophenyl ester.
To determine the compatibility of the substrate mimetic approach
with peptoid fragments, two model oligomers were evaluated for
ligation (Scheme 1). A peptoid tetramer was synthesized and
N-acetylated on chlorotrityl resin. The oligomer was then cleaved
from the solid support and modified with p-guanidinophenol to form
the acyl donor 1. The acyl acceptor 2 was prepared using a
combination of standard peptoid “submonomer” chemistry12 and
Fmoc chemistry on Rink amide resin. An unsubstituted glycine
residue was positioned at the N-terminus of 2, followed by an
N-methylglycine (sarcosine) at the subsequent residue. The ligation
of 1 and 2 progressed efficiently in the presence of clostripain, as
monitored by analytical HPLC (Figure 1). Complete conversion
of the acyl donor was observed within 4 h, yielding primarily
decamer 3 along with some hydrolysis product. Identity of 3 was
confirmed by electrospray mass spectrometry ([M + H+], calcd
m/z 1236.63; obs. m/z 1237.0). No ligation was observed in the
absence of enzyme.
a Reaction conditions: 0.2 M HEPES, pH 8.0, 100 mM NaCl, 1 mM
CaCl2, 12% DMF, 5 mM 1, 10 mM 2 or 4, 1.6 µM clostripain, rt, 4 h
(Scheme 1) or 8 h (Scheme 2).
side chains were used to generate peptoid products of 10, 16, and
32 residues (see Supporting Information). HPLC profiles and mass
spectrometry of the reaction mixtures revealed the anticipated
fragment condensations with minimal side product formation.
Product hydrolysis or degradation was not observed, even following
overnight incubation in the presence of clostripain. These results
demonstrate that protecting groups may not be required for
potentially reactive side chains and further highlight the resistance
of peptoids to protease-catalyzed degradation.
The ligation of oligomers possessing N-substituent side chains
at all residues was investigated (Scheme 2). Acyl donor 1 and acyl
acceptor 4 were used for this reaction. Peptoid 4 incorporates two
sarcosine monomer units at the N-terminal positions. The ligation
reaction successfully generated oligomer 5 ([M + H+], calcd m/z
1250.65; obs. m/z 1250.9), but with a marked decrease in yield.
Steric constraints at or near the active site of clostripain may alter
the efficiency of amide bond formation between the acyl donor
and acceptor.11 Overall, these reactions underscore the compatibility
Experiments were undertaken to probe sequence tolerance and
fragment length requirements for ligation. Oligopeptoid acyl
acceptors bearing unprotected amino- or guanidino-functionalized
Figure 1. Peptoid ligation reaction monitored by reversed-phase HPLC.
1, 2, and 3 correspond to Scheme 1 entries.
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10.1021/ja055105m CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
natural oligomers as substrates. Protease-catalyzed peptide synthesis
has been well documented for both L- and D-amino acids as well
as for small peptide isosteres.7,9,13 Additionally, ribosome-based
translation systems have been utilized for the incorporation of
abiotic residues into short peptide sequences.14 The study presented
here further broadens the possibilities for appropriating biological
catalysts in order to generate nonnatural products. The synthesis
of peptoid concatenation products 7 suggests that the ligation
approach will indeed prove to be generally suitable for assembling
peptoid macromolecules. We believe the products reported here
are among the largest sequence-specific peptidomimetics generated
to date.
Figure 2. Peptoid concatenation reaction. (a) HPLC of peptoid concatena-
tion products 7. (b) MALDI mass spectrometry of concatemer products 7.
Average ∆ m/z ) 581, corresponding to mass of pentamer repeat units.
Scheme 3 a
Acknowledgment. This work was supported by the NYSTAR’s
James D. Watson Investigator Program. We acknowledge the
NCRR/NIH for the Research Facilities Improvement Grant (C06
RR-16572) at NYU.
Supporting Information Available: Experimental procedures;
HPLC data; mass spectrometry data. This material is available free of
charge via the Internet at http://pubs.acs.org.
a Reaction conditions: 0.2 M HEPES, pH 8.0, 100 mM NaCl, 1 mM
CaCl2, 12% DMF, 5 mM 6, 1.6 µM clostripain, rt, 12 h; n ) 1,2,3,...>30.
References
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