ACS Combinatorial Science
Research Article
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Scheme 1. Developed General Amide Coupling Conditions for Single Nα-Fmoc-Amino Acid Building Blocks
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¥: Certain amino acids were not soluble in MeCN and had to be prepared in DMSO. *: Reactions were typically complete after 2 h but can be
boosted for higher yields with the addition of 150 equiv of DMTMM at 2 h for reaction until 3 h; the chemical structure of the DNA tag (in
Biological display methodsphage, ribosome, and mRNA
displayare the protagonists of de novo peptide ligand
discovery, but their main lacuna has been the inability to fully
implement the lessons learned from pharmacokinetic opti-
mization studies due to limited synthetic access to such
analogs.1 It is true, however, that the expansion of the genetic
code to encode for noncanonical amino acids20 and the
engineering of flexible ribosomes21 have significantly enhanced
such access. An illustration is the 1014-member mRNA-display
library produced by Szostak and co-workers22 who were able to
incorporate 12 unnatural amino acids into the design.
Additionally, post translational modifications, such as cycliza-
tion and stapling, have been made possible in mRNA-display.23
The chemical space accessible to biological display methods
therefore promises to broaden as synthetic and recombinant
methods converge.1 However, such convergence is incomplete,
which limits the chances of discovery and is incompatible with
the pressing need for new peptide therapeutics.
DNA-encoded chemical libraries (DECLs) provide a
potential solution. The value of DECLs in this context lies in
the synergy of large numbers (106−109-member libraries)24,25
and in their broader, ribosome-independent synthetic access.
Theoretically, they could include any unnatural amino acid
in addition to all natural onesas well as allow for a larger set
of postsynthetic modifications thanks to a larger and expanding
chemical toolkit.26−35 An illustration of the advantage of
DECLsone of particular interest to uslies in all-hydro-
carbon stapled peptide drug discovery, whereby peptides are
stapled into proteolytically stable, cell-permeable forms using
the ring-closing metathesis reaction.15 This area has borne the
clinical candidate ALRN-6954, an all-hydrocarbon stapled α-
helical peptide capable of inhibiting the p53-MDM2/MDMX
PPIs in p53-dependent cancer therapy.4 Such peptides require
the incorporation of α,α-disubstituted unnatural amino acids,
which are not currently accessible to biological display
methods. Similarly, the ring-closing metathesis reaction
remains unreported on the latter platform. In contrast, the
latter reaction has been developed for DECLs,35 while the
incorporation of α,α-disubstituted amino acids is investigated
and demonstrated in this work.
strategy and reaction conditions for the coupling of α,α-
disubstituted alkenyl amino acids as a tool for the generation of
all-hydrocarbon stapled peptide DECLs, and (5) the synthesis
of a fully deprotected DNA-decamer conjugate from amino
acid monomers to illustrate the overall efficacy of the
developed conditions.
RESULTS AND DISCUSSION
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Solid-phase peptide synthesis (SPPS) is one of the most
established chemical processes owing to decades of develop-
ment,36−38 and the pleiotropy of protecting group strategies39
and coupling reagents40 available today enable the most
synthetically challenging endeavors. Neri and co-workers30
reported the relative efficiency of various amide coupling
reagents for the conjugation of carboxylic acids to DNA.
However, few of the substrates tested were amino acids, and
the recommended amide coupling conditionsEDC/HOAt/
DIPEAproved inadequate, as explained below (see section
on glutamine). Similarly, while amide coupling conditions have
been reported for the generation of macrocyclic peptide and
polyketide DECLs,41,42 the amino acids/amino acid oligomers
used were mostly hydrophobic, offering no significant
protecting group strategies. Of note, DNA is sensitive to
harsh conditions43,44 (acidic and oxidative, for example),
making Boc chemistry problematic and protecting group
strategies limited.
Developed Amide Coupling Reaction Conditions.
DECL builds are based on combinatorial chemistry, and
high-yielding, substrate tolerant reaction conditions are
desirable. Such conditions were developed, as summarized in
Scheme 1.
The main challenge we faced was the limited solubility of
amino acid/amino acid oligomers in aqueous media. Indeed,
peptide insolubility is recognized as one of the most significant
problems of peptide chemistry.38 Moreover, since a protic
medium is best for the suppression of nucleophilicity45 and
hence for the protection of the DNA tag from reagents,44
solution-phase DECL reactions are ideally performed in
cosolvent mixtures of high water proportion. A balance
between amino acid/amino acid oligomer solubility and
protection of DNA integrity was therefore empirically struck.
Interestingly, most amino acids remained soluble in a buffered
(pH ∼9.5) 40% acetonitrile (MeCN) aqueous solution. We
hypothesized the ionization of the α-carboxyl group of the
amino acid residues (pKa ∼ 2−3) enabled such solubility,
which is not achievable if preactivation is used as a coupling
strategy. Indeed, preactivated amino acids generally precipi-
tated from the same medium and exhibited poor coupling
Hence, as a bridge to building peptide DECLs, we, herein,
report (1) solution-phase, aqueous reaction conditions for the
coupling of amino acid monomers to DNA and DNA-amino
acid conjugates, (2) a screen of coupling efficiency between
amino acid residues and DNA-amino acid conjugates to
demonstrate the substrate tolerance of the developed
conditions, (3) DNA-compatible protecting group strategies
and the limitations thereof for relevant amino acids, (4) a
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ACS Comb. Sci. XXXX, XXX, XXX−XXX