Encoded combinatorial libraries for the construction of cyclic peptoid microarrays

Article abstract of DOI:10.1039/b812735b

A one bead two compound approach to the synthesis of encoded cyclic peptoid libraries is reported. The Royal Society of Chemistry.

Full text of DOI:10.1039/b812735b

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COMMUNICATION
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Encoded combinatorial libraries for the construction of cyclic peptoid
microarraysw
Yong-Uk Kwonab and Thomas Kodadek*a
Received (in College Park, MD, USA) 25th July 2008, Accepted 29th September 2008
First published as an Advance Article on the web 16th October 2008
DOI: 10.1039/b812735b
A ‘‘one bead two compound’’ approach to the synthesis of
encoded cyclic peptoid libraries is reported.
been developed.18–24 These studies have confirmed that pep-
toid libraries are indeed rich sources of protein ligands. We
have also shown that peptoids are, in general, far more cell
permeable than peptides due to their lack of polar main chain
N–H bonds.25 In this report, we describe a strategy that is
optimized for the creation of cyclic peptoid microarrays.
A key design issue has to do with the ability to determine the
sequence of hits after screening a one bead one compound
library. Since cyclic peptides or peptoids lack a free N-terminus,
Edman sequencing cannot be employed. Moreover, while
peptoids, like peptides, can be sequenced by tandem mass
spectrometry,26 cyclic molecules will fragment at multiple
positions, complicating interpretation of the MS/MS spectrum
severely. This issue has limited the development of synthetic
cyclic peptide libraries. Pei and co-workers addressed this
problem recently by developing a ‘‘two compound, one bead’’
approach in which each bead contains both a linear and cyclic
molecule containing the same peptide sequence.11 In other
words, the linear molecule encodes the cyclic molecule. This
was accomplished using the strategy of Lam27 in which
different solvents were employed to segregate beads into two
different domains (internal and surface-exposed) to which
were attached glutamic acid residues with differentially pro-
tected carboxylate side chains. The same peptide chain was
then extended from both the internal and external Glu resi-
dues. Finally, only the surface-exposed Glu side chains were
deprotected, allowing them to be cyclized with the terminal
amino group of the peptide. The peptides in the internal layer
remained linear and thus served as the encoding strand.
We have developed a different one bead two compound
strategy that is tailored to the creation of microarrays, a useful
platform for protein fingerprinting and library screening.28,29
The idea was to employ differential deprotection to create two
chains, both of which contain the peptoid of interest, but only
one of which contains both a glutamic acid residue to support
cyclization as well as a Cys residue to allow specific conjuga-
tion of only the cyclic peptoid molecule to a maleimide-
activated microscope slide30 (Fig. 1). The linear molecule
would not couple to the slide, but would be present to support
tandem MS-based sequencing.
Drug-like small molecules (o500 Daltons) generally do not
bind well to the relatively shallow surfaces of proteins involved
in protein-protein interactions. Thus, in order to develop
effective therapeutic agents against these increasingly impor-
tant targets, it is necessary to develop libraries of compounds
able to cover a greater surface area and engage in multiple
contacts with the target protein, as well as efficient methods to
screen these libraries. With regard to their protein-binding
properties, peptides are an attractive class of molecules, but
linear peptides have many undesirable features. They are
peptidase- and protease-sensitive, relatively cell impermeable
and generally form complexes with only modest dissociation
constants (KDs in the high nM to mid mM range). However,
cyclic peptides can exhibit enhanced cell permeability1,2 and
are much less sensitive to enzymatic degradation.3 Moreover,
it is presumed that the conformational restriction imposed by
cyclization may generally afford higher binding affinities,
though rigorous proof for this idea is lacking.4,5 Indeed, many
naturally occurring cyclic peptides and depsipeptides have
been found to display potent biological activities.6–10 This
interest has led to the development of facile methods for the
creation of either synthetic11 or genetically encoded12,13
libraries of cyclic peptides as potential sources of drug leads.
A limitation of peptide libraries, cyclic or linear, is that only
a relatively small number of building blocks are available.
Moreover, although cyclic peptides, can be more cell perme-
able than their linear counterparts, this appears to be depen-
dent on their ability to form intramolecular hydrogen bonds,2
a property that is likely to vary from compound to compound.
Therefore, we became interested in the development of
libraries of cyclic peptoids (N-substituted oligoglycines)14,15
as potential protein ligands. Large libraries of linear peptoids
with a wide variety of different side chains16–18 are readily
accessible using split and pool methods and efficient protocols
with which to screen these libraries for protein binding have
a Division of Translational Research, Departments of Internal
Medicine and Molecular Biology, University of Texas Southwestern
Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas
75390-9185, USA. E-mail: thomas.kodadek@utsouthwestern.edu;
Fax: 1-214-648-4156; Tel: 1-214-648-1239
To effect this strategy (see Fig. 2), a 7 : 1 ratio of
Fmoc-Cys(Trt)-OH and ivDde-b-Ala-OH was added to
b-Ala-primed Rink amide resin. This ratio was optimized
empirically to provide enough linear peptoid for tandem MS
sequencing from a single bead, but also produce as much cyclic
peptoid as possible. After selective deprotection of Fmoc,
Fmoc-b-Ala-OH was again attached to Cys followed, after
b Department of Chemistry and Nano Science, Ewha Womans
University, 120-750 Seoul, South Korea
w Electronic supplementary information (ESI) available: Experimental
details for synthesis and microarray construction, supplemental data.
See DOI: 10.1039/b812735b
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of the peptoid on a particular bead could be determined easily
by tandem MS analysis of the linear molecule. We therefore
turned our attention to the determination of the efficiency of
the cyclization reaction. For some of the molecules, mass
spectrometry and HPLC analysis showed that cyclization of
the Cys-Glu-containing molecule was clearly incomplete, as
linear starting material was clearly detectable. This was not
surprising, since a general problem in the creation of cyclic
libraries is that not all sequences cyclize with equivalent
efficiencies.31 One would presume that the nature of the
N-terminal residue would have the largest effect on cyclization.
Indeed, an analysis of more than 50 peptoids by MS/MS
Fig. 1 Schematic view of the general strategy employed to create a
library in which each bead carries a cyclic peptoid and an analogous
linear encoding strand. Only the cyclic molecule contains a thiol and
thus will couple to a maleimide-activated glass slide.
Fig. 2 Synthesis of the encoded cyclic peptoid library via the one
bead two compound strategy. The amines employed in the sub-
monomer peptoid synthesis are shown at the bottom of the figure
(one of the amines in 1,4-diaminobutane and a hydroxyl group in
ethanolamine were protected).
removal of this Fmoc, by addition of Fmoc-Glu(O-2-PhiPr)-OH.
At this point, both the Fmoc and ivDde protetecting groups
were removed and peptoid synthesis was carried out on both
strands. Seven peptoid residues were incorporated using
conventional sub-monomer chemistry and the nine amines
shown in Fig. 2. The 2-PhiPr protecting group on the Glu
side chain was then removed selectively with 1% TFA. Finally,
macrocyclization was carried out using the method of
Kirshenbaum and colleagues (PyBOP (3 eq), HOBt (3 eq)
and DIPEA (10 eq).15 Note that the linear molecule lacks two
residues present in the (presumed) cyclic molecule and thus the
mass peaks derived from each can be distinguished easily,
facilitating analysis and sequence determination.
Fig. 3 Attachment of Cys-containing cyclic peptoid to a maleimide-
activated glass slide. A. General structure of the cyclic and linear
molecules made on each bead before cleavage and deprotection of the
thiol side chain. Below: Sequences of the variable regions of five
peptoids picked for the spotting experiment. B. Fluorescent image of
microarrays in which each of the five peptoids have been spotted onto
the activated surface. A DMSO solution of each peptoid (E2 mM)
was spotted two times, the solution was diluted three-fold, spotted
again, etc. After washing and drying, the arrays were hybridized with
Cy3-conjugated streptavidin, washed and the slide was scanned with a
fluorescence scanner (see ref. 30 for details). The spots are false-
colored green. C. The Cys is essential for retention of the peptoid on
the microarray. Two peptoids were synthesized. Each had the se-
quence Fluorescein-Nlys-Nser-Nleu-Nser-Nall-Npip-Nlys-Nlys. One
peptoid also contained a C-terminal cysteine, while the other did
not. The two peptoids were spotted onto a maleimide-activated glass
slide. After washing, the slide was scanned using a fluorescence
scanner. The fluorescence intensity is false-colored blue.
To determine the efficacy of this procedure, individual beads
were separated and treated with acid to cleave the molecules
from the beads, followed by HPLC, MS and MS/MS analysis.
Gratifyingly, we found that in almost every case the sequence
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revealed that if the N-terminal residue was Nmea, the cycliza-
tion yield was almost quantitative. Therefore, this residue was
made invariant in all subsequent work.
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With this information in hand, we prepared ten peptoids of the
form: b-Ala-Cys-Glu(Biotin)-cyclo(Glu-X-X-X-X-X-X-Nmea)
(see Fig. 3), where biotin-Glu bears a side chain-conjugated biotin
and X was derived from one of the amines shown in Fig. 2. The
molecules were cleaved from the resin and analyzed by HPLC
and tandem MS. In each case, we were able to easily sequence the
linear species by tandem mass spectrometry (see Supplementary
Material). Moreover, all of the detectable Cys-containing mole-
cules were in the cyclic form (see Supplementary Material).
Serial dilutions of the five peptoids shown in Fig. 3 were
spotted robotically onto PEGylated, maleimide-activated glass
microscope slides30 and the slides were then washed rigorously.
In order to demonstrate the immobilization of the cyclic pep-
toids, the slides were incubated with Cy3-labeled Streptavidin
and scanned. As expected, the amount of protein captured
decreased as the amount of peptoid spotted decreased, confirm-
ing that the fluorescence is indeed due to specific capture of the
protein by the peptoid (Fig. 3B). To demonstrate that the Cys
residue is critical for retention of the cyclic peptoid to the
maleimide-derivatized slide, we synthesized two fluorescein-
conjugated linear peptoids that were identical except for the
presence and absence of Cys. These were spotted onto a slide,
which was then scanned after washing. As shown in Fig. 3C,
detectable fluorescence was seen only where the Cys-containing
peptoid was spotted. This experiment validates an important
feature of our strategy, which is that the linear encoding molecule
(see Fig. 1) will not be retained on the slide when the mixture of it
and the Cys-containing cyclic molecule are spotted onto the slide
and thus will not interfere with screening experiments.
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In summary, we have developed a new ‘one bead two
compound’ strategy for the creation of encoded cyclic peptoid
libraries. This particular scheme is useful for the creation of
cyclic peptoid microarrays since only the cyclic peptoid, not
the linear encoding molecule, contains a cysteine and thus
can be spotted onto a maleimide-activated microscope slide.
The use of these cyclic peptoid microarrays in screening
experiments will be reported in due course.
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This work was funded by a grant from the Welch Founda-
tion (I-1299) and a contract from the National Heart Lung
and Blood Institute for the UT Southwestern Center for
Proteomics Research (NO1-HV-28185). We thank Dr Reddy
Moola (University of Texas Southwestern Medical Center) for
assistance with printing the molecules on the microarray.
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Notes and references
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Kodadek, Chem. Commun., 2005, 581–583.
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5706 | Chem. Commun., 2008, 5704–5706