Solid-phase synthesis of peptoid-like oligomers containing diverse diketopiperazine units

Article abstract of DOI:10.1039/c4ob00829d

Diketopiperazine (DKP) units are found in many bioactive small molecules. Here we report facile chemistry for incorporating diverse DKP units within peptoid and peptoid-like libraries made by solid-phase split and pool synthesis. This journal is the Partn

Full text of DOI:10.1039/c4ob00829d

Organic &
Biomolecular Chemistry
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Solid-phase synthesis of peptoid-like oligomers
containing diverse diketopiperazine units†
Cite this: Org. Biomol. Chem., 2014,
12, 5831
Sujit Suwal and Thomas Kodadek*
Received 22nd April 2014,
Accepted 23rd June 2014
DOI: 10.1039/c4ob00829d
www.rsc.org/obc
Diketopiperazine (DKP) units are found in many bioactive small
molecules. Here we report facile chemistry for incorporating
diverse DKP units within peptoid and peptoid-like libraries made
by solid-phase split and pool synthesis.
Large combinatorial libraries of peptides, peptoids and other
synthetic oligomers are a valuable source of protein ligands.
There is continued interest in increasing the chemical diversity
of the building blocks that can be included in such oligomer
libraries by solid-phase synthesis. We have extended the utility
of the “sub-monomer” synthesis of peptoids1 by developing a
variety of different units that can be used in this general
process, which involves addition of the activated ester of
bromoacetate to an amine followed by displacement of the
bromide by a primary amine. In particular, we have been
focused on building blocks that incorporate significant confor-
mational constraints into the molecule with the idea that
“stiffer” molecules will bind to proteins with higher affinity.
For example, substitution of 2-bromoacetate with more
complex γ-halo acids has allowed the incorporation of hetero-
cycles,2 chiral centers and unsaturation3,4 into the oligomer
main chain. We also demonstrated that 2-oxopiperazines can
be assembled on-resin through a multi-step processes,5 all of
which proceed efficiently enough to be compatible with the
creation of high-quality combinatorial libraries. Here we report
another example of this type of multi-step on-resin construc-
Scheme 1
linear intermediate 3, which we postulated should cyclize in
high yield under basic conditions. A variety of substituents
could be readily attached to the DKP through the use of
diverse amino acid and amine building blocks.
To test this scheme, compounds 4a–f were synthesized
using the conditions shown in Scheme 1 on Rink amide
MBHA resin. The compounds were released from the resin and
analyzed by NMR. DKPs 4a and 4b were formed cleanly with
no significant side products or starting material apparent (ESI
Fig. S1–S10†). To address possible racemization of the chiral
center in the DKP, compounds 4c and 4d were synthesized,
which differ in the absolute stereochemistry at this position
and also have a chiral center external to the ring that renders
the two compounds diastereomeric. Analysis of 4c and 4d
showed that both were stereochemically pure (ESI Fig. S11–
S15†). To further test the scope and limitation of our strategy,
DKPs 4e and 4f were prepared, which have a chiral, branched
center adjacent to the nitrogen undergoing cyclization. This
might be expected to slow ring closure, possibly leading to
some racemization in the ring. While the proton NMR of crude
4e showed small amounts of other products (∼10%) these were
not the diastereomer 4f, as evidenced by comparing their
spectra (ESI Fig. S20†). Thus, even in this case of a difficult
cyclization, the DKP-forming reaction proceeds with excellent
stereochemical purity.
tion of a cyclic unit, specifically a diketopiperazine (DKP),6–8
structural feature found in many bioactive molecules.9
a
The synthetic scheme that we imagined could be employed
for the solid-phase synthesis of diverse DKPs is shown in
Scheme 1. Addition of the activated ester of bromoacetic acid
to a resin-bound amine would be followed by displacement of
the bromide with an α-amino acid ester. Another round of
peptoid synthesis on the N-terminal nitrogen would provide
Department of Chemistry and Cancer Biology, The Scripps Research Institute,
130 Scripps Way, Jupiter, FL 33458, USA. E-mail: Kodadek@scripps.edu
†Electronic supplementary information (ESI) available: Full experimental proto-
cols and compound characterization. See DOI: 10.1039/c4ob00829d
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Scheme 2
To further test the scope of this process, DKPs 7a–i, derived
from nine different amino acid esters, were synthesized on-
resin (Scheme 2). In each case, the DKP ring was assembled
onto compound 5. To establish rigorously the efficiency of the
DKP-forming steps, further chain elongation was attempted,
which should not proceed if the ring-forming reaction incor-
porates the nucleophilic nitrogen into an amide linkage. In
the event, after cleavage of the compounds from the resin,
HPLC analysis indicated that the expected DKP products were
formed with purities ranging from 80–98%. No significant
material from unwanted chain extension was observed by
mass spectrometry in any of the nine cases (ESI Fig.
S26–S34†).
Fig. 1 Synthesis of a library containing a DKP unit inserted into the
main chain. (a) General synthetic scheme for the construction of a com-
binatorial library containing internal DKP residues. The library was con-
structed on 150 µm TentaGel MB-NH2 resin. (b) Top: MALDI mass
spectrum of a crude library member (from a single bead) after release
form the bead with CNBr ([MH+]
= 1206.5) obtained after CNBr
mediated single bead cleavage. Top-right: Structure of the peptoid
identified after MALDI-TOF based sequencing. Bottom: MALDI-TOF MS/
MS of the 1206.5 peak.
The chemistry discussed above results in a DKP unit that
cannot be extended further and thus represents a terminating
modification for the synthesis of oligomers. We also examined
the use of mono-protected diamines in this scheme since,
after deprotection, this would afford the opportunity to further
extend the molecule and allow the DKP unit to be flanked by
diverse elements. As is shown the ESI,† this worked well. We
synthesized two different compounds on Rink amide resin
where the protected diamine was either incorporated as a sub-
monomer prior to DKP formation or used as a part of the DKP
ring. In each case, the alloc protecting group was removed
after DKP formation and the chain was extended from this
nitrogen, providing oligomers in which the DKP was an
internal element in excellent yield and purity (see ESI
S35–S38†) (Fig. 1).
Having validated the efficiency of the DKP-forming chem-
istry in the context of single compounds, we proceeded to con-
struct a combinatorial library with the general structure shown
in Fig. 2 using the split and pool strategy.10,11 Following an
invariant linker and one diversified peptoid unit, DKPs derived
from nine different amino acid esters were incorporated into
the second position of the main chain. A N-tBoc mono-
protected ethylene diamine was employed as the primary
amine in the formation of the DKP ring, allowing the addition
of two additional residues following the DKPs. Both were con-
structed using the peptoid sub-monomer route. In the first of
these positions, both (S)-2-bromopropionic acid and bromo-
acetic acid were employed as sub-monomers. In the last posi-
tion, only bromoacetic acid was employed. 12 amines were
Fig. 2 Synthesis of a library of peptoids containing DKP side chains. (a)
General synthetic scheme of DKP-SC peptoid library. (b) Top-left: MS of
a peptoid ([MH+] = 1240.7) obtained after CNBr mediated single bead
cleavage. Top-right: Structure of the peptoid identified after MALDI-TOF
based sequencing. Bottom: MS/MS of a peptoid obtained from MALDI-
TOF with the precursor mass 1240.5.
used to construct the peptoid units, resulting in a library con-
taining approximately 27 000 compounds. To test the quality
of the library, 20 beads were chosen randomly from the popu-
lation and cleaved from the resin at the invariant methionine
using CNBr. In each case, a single, strong molecular ion was
observed in the MALDI mass spectrum and the molecules could
be sequenced unambiguously by tandem MALDI MS/MS, a
result comparable to that obtained for most standard peptoid
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Fig. 3 Synthesis of a complex DKP-terminated library. (a) Left: General structure of the library. Middle: Structures of the five different main chain
diversity elements used at second position of the library. Right: DKP units employed as a capping group in the library. (b) Top-left: MS of 11 ([MNa+]
= 1241.6) obtained after CNBr-mediated cleavage from a single bead picked from the library. Top-right: Structure of the peptoid identified after
MALDI-TOF based sequencing. Bottom: MS/MS of 11 (from fragmentation of 1241.6 ion).
libraries (ESI S42–S57†). This demonstrates that high quality first peptoid residue under basic conditions. After coupling of
combinatorial libraries can be created with DKPs inserted into the benzoyl and sulfonyl groups the beads were split and the
the main chain.
halides were displaced with 12 different amines.
Another library was created in which diverse DKPs were
In total, 27 different structures were included in the library
included as a side chain in an otherwise standard peptoid at this position (A in Fig. 3). Following a diversified unit A,
library. To do this, a mono-tBoc-protected ethylene diamine conventional peptoid units were introduced at the third posi-
was employed as the invariant sub-monomer at the second tion of the library. Finally, 56 different 2,5-DKPs were incorpor-
position in the library. After construction of the diverse DKP ated into the terminal position by using seven different amino
side chains using five amino acid esters and 12 amines, the acid esters and eight primary amines. In the design of this
other nitrogen of the ethylene diamine-derived unit was depro- library, peptoid units were placed between the A units and the
tected and an additional two peptoid units were added. This terminating DKP in order to simplify interpretation of the
resulted in a library of approximately 100 000 compounds MS/MS spectrum since peptoids fragment quite efficiently. The
(Fig. 3). Again, the high quality of the library was established quality of the tetrameric complex peptoid library was con-
by MALDI MS and MS/MS analysis of the beads chosen ran- firmed by CNBr-mediated cleavage at methionine in the invar-
domly (ESI S58–S68†). This demonstrates that this chemistry is iant linker region of 20 beads randomly selected from the pool
compatible with generating a high quality of library with DKP of the library. More than 80% of the compounds were identi-
unit at a variety positions within the compounds.
fied by MALDI-MS/MS (ESI S69–S84†). Further, HPLC analysis
With these encouraging results from the syntheses of rela- of several molecules synthesized on Rink amide resin with a
tively simple libraries, we constructed the more complex variety of monomers at the second position indicated purities
library shown in Fig. 3. The goal here was to begin to move of 80–99%. (ESI S39–S41†). We conclude that this chemistry
towards more diverse main chain chemistry, then cap this is efficient enough to create highly complex libraries of high
library was diverse DKPs. A tetrameric hybrid library of quality.
approximately 135 000 compounds was created on Tentagel-
MB-NH2 resin. A detailed protocol for the incorporation of the
various units at second position of the chain (A in Fig. 3) is
provided in the ESI.† Briefly, for the piperazine units, after
Conclusions
coupling 2-bromopropionic acid to the first peptoid residue,
In summary, we have demonstrated a facile approach for the
the bromide was either displaced with mono-N-tBoc-piperazine on-resin synthesis of creating a highly substituted, enantio-
or mono-N-t-Boc-(S)-2-methyl piperazine, separately (see ESI†). merically pure 2,5-DKP units. High quality combinatorial
The t-Boc group was deprotected before further chain exten-
libraries containing this unit can be created using the split
sion with bromoacetic acid/DIC. (S)-3-Benzyl-2-oxopiperazine and pool strategy. Depending on the precise nature of the
was introduced through tBoc-chemistry as reported pre- reagents employed, the DKP can be incorporated as a terminat-
viously.5 4-Bromomethyl benzenesulfonyl chloride or 3-chloro-
ing group,
a side chain element, or inserted into the
methyl benzoyl chloride were coupled with the nitrogen of the main chain of the oligomer and flanked by other units. We
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anticipate that these libraries will serve as a useful source of
bioactive compounds.
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Acknowledgements
This work was supported by a grant from the NIH (NIDDK)
DP3 DK094309.
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