Ribosomal elongation of aminobenzoic acid derivatives

Article abstract of DOI:10.1021/jacs.0c05765

2-Aminobenzoic acid and heterocyclic amino acids are aromatic homologues of β-amino acids, but their chemical properties are quite distinct. Because of the poor nucleophilicity of the amino group conjugated with the aromatic ring, no literature reports of

Full text of DOI:10.1021/jacs.0c05765

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Ribosomal Elongation of Aminobenzoic Acid Derivatives
Takayuki Katoh* and Hiroaki Suga*
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ABSTRACT: 2-Aminobenzoic acid and heterocyclic amino acids are aromatic homologues of β-amino acids, but their chemical
properties are quite distinct. Because of the poor nucleophilicity of the amino group conjugated with the aromatic ring, no literature
reports of their successful ribosomal elongation in nascent peptide chains have appeared to date. Here we report for the first time
their incorporation in nascent peptide chains by means of a reconstituted translation system in which a designer tRNAPro1E2 capable
of recruiting the elongation factors EF-Tu and EF-P was charged with their derivatives and the corresponding peptides were
successfully expressed. We also demonstrate the expression of macrocyclic peptides containing exotic amino acids, including
aminobenzoic acid derivatives.
(Figure 1), for instance, with the amino group at the meta
position [e.g., 3-aminobenzoic acid (3NAbz)], with additional
2-Aminobenzoic acid (Abz) is an attractive building block of
peptidomimetics because of its constrained rigid conformation.
Since the amino nitrogen and carbonyl carbon of Abz are both
directly attached to the aromatic ring, all of them are aligned on a
flat surface with the fixed dihedral angle φ = 0°. The π-stacking
ability of the aromatic ring that induces intramolecular and
intermolecular interactions can also contribute to increasing the
structural rigidity of peptides containing Abz. It can also act as a
good turn/helix inducer, which property has applied to the
synthesis of poly-Abz derivatives, known as aromatic
foldamers.1−9 For instance, a crystal structure of D-Leu-D-Phe-
Abz-D-Ala peptide displayed a planar β-turn around Abz
stabilized by three hydrogen bonds.10 Alternating Pro-Abz
repeat peptides showed an unusual periodic pseudo-β-turn
comprising hydrogen-bonded nine-membered rings.11 Not only
synthetic foldamers but also naturally occurring peptides
containing Abz have been reported (e.g., psychrophilin,
cycloaspeptide, and asperterrestide).12−14
Figure 1. Structures of Abz derivatives tested for ribosomal elongation.
Although biosynthetic processes of the above examples of
Abz-containing natural peptides have not been well-understood,
their biosynthesis very likely relies on either nonribosomal
peptide synthetases15 or post-translational-modifying en-
zymes.16 However, if Abz can be directly incorporated into
nascent peptide chains by ribosomes, it will be a conventional
tool for the biosynthesis of foldamer-like peptides whose
sequences can be readily programmed by mRNA templates.
However, the amino group of Abz is supposedly a poorer
nucleophile than those of the 20 canonical L-α-amino acids (the
pKa values of their ammonium forms are 4.9 and 9.4,
respectively), likely resulting in its low reactivity in peptide
bond formation. Moreover, Abz is classified as a β-amino acid,
and the incorporation efficiency of β-amino acids is known to be
much lower than that of α-amino acids.17−20 To the best of our
knowledge, ribosomal incorporation of Abz derivatives has been
substituents on the aromatic ring [e.g., 2-amino-5-hydroxyben-
zoic acid (Abz5OH), 2-amino-5-methoxybenzoic acid (Abz5OMe),
and 2-amino-5-fluorobenzoic acid (Abz5F)], or with substitution
of the aromatic ring itself with heterocycles, such as pyridine,
thiophene, and thiazole [e.g., 3-aminopyridine-4-carboxylic acid
(Apy), 3-aminothiophene-2-carboxylic acid (Atp), and 5-
aminothiazole-4-carboxylic acid (Atz)]. These derivatives are
also attractive building blocks to construct foldamer peptides,
but it is yet unknown whether they can be incorporated into
peptide chains during elongation.
Since we recently demonstrated significant enhancement of
the incorporation efficiency of β-amino acids using an
Received: June 6, 2020
accomplished only at the N-terminus of peptides using the
21−23
initiator tRNAfMet
.
Because of these issues, ribosomal
elongation of Abz via the amino group has never been reported
to date. There are more kinds of Abz derivatives available
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engineered tRNA named tRNAPro1E2 (Figure S1B),24,25 we here
explored the incorporation of Abz derivatives into nascent
peptide chains by the use of tRNAPro1E2. This tRNA has the T-
stem and D-arm motifs required for EF-Tu and EF-P binding,
respectively.26−29 The improved EF-Tu binding accelerates
accommodation of aminoacyl-tRNAPro1E2 onto the ribosomal A
site, whereas EF-P enhances the peptidyl transfer reaction.
To charge Abz derivatives onto tRNA, we decided to adopt
two methods. The first method is our standard flexizyme (eFx),
an aminoacylation ribozyme, that uses amino acid cyanomethyl
ester (CME).30,31 Prior to testing of various Abz derivatives on
tRNA, we attempted this method on a shortened tRNA
analogue called microhelix RNA (μhRNA). However, a long
incubation time (6−8 days) was required to complete the
flexizyme reaction (vide infra). This forced us to consider a
different method, chemical acylation using N-carboxyanhy-
drides (NCAs).32 Because of the high reactivity of the NCAs of
Abz derivatives and also commercial availability, we decided to
preferentially use NCAs for charging Abz, Abz5OH, Abz5OMe, and
Abz5F (Figure S2A). NCA aminoacylation was performed on
μhRNA at pH 9.0−10.5 (25 °C for 1−3 h) and confirmed by
acid-denaturing PAGE and MALDI-TOF mass spectrometry
(MS) (Figure S3 and S4). For Apy, Atp, and Atz, we applied the
eFx-catalyzing μhRNA acylation using their CME derivatives at
pH 9.0−10.5 (4 °C for 24−336 h; Figures S2B, S5, and S6).30,31
In all cases, we were able to observe satisfactory yields (25−
74%), which are sufficient to apply to the tRNA acylation.
The optimal conditions summarized in Table S1 were applied
for the preparation of aminoacyl-tRNAPro1E2CGG. The respective
Figure 2. Ribosomal incorporation of Abz derivatives into a model
peptide. (A) Sequences of an mRNA (mR1) and the corresponding
peptide (rP1). Abz derivatives were assigned at the CCG codon using
precharged aminoacyl-tRNAPro1E2CGG. The amino acid sequence of
“flag” is Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. (B) MALDI-TOF MS of
ribosomally synthesized rP1 peptides containing Abz derivatives. Red
and blue arrows indicate monovalent and divalent ions of the desired
peptides, respectively. “Obsd.” and “Calcd.” indicate observed and
calculated m/z values, respectively. Asterisks denote template-
independent impurities derived from the translation system. (C)
Expression levels of rP1 peptides estimated by autoradiography.
Numbers above the bars indicate the ratio of EF-P(+) to EF-P(−). See
Figure S7 for the raw data from tricine SDS-PAGE analyses of peptides.
aminoacyl-tRNAPro1E2
(Abz, Abz5OH, Abz5OMe, Abz5F, Apy,
CGG
Atp, and Atz) were introduced at a CCG codon of the mRNA
mR1 to synthesize the model peptide rP1 (Figure 2A).
Translation reactions were carried out using the Flexible In
vitro Translation (FIT) system,33 in which six amino acids (Met,
Tyr, Lys, Phe, Asp, and Gly) and the corresponding aminoacyl-
tRNA synthetases (ARSs) were included, whereas the other 14
proteinogenic amino acids and their cognate ARSs were
omitted. The identity of each peptide was confirmed by
MALDI-TOF MS, giving the desired m/z values of [M + H]+
and [M + 2H]2+ ions without side products (Figure 2B). The
same peptides were translated in the presence of [14C]-labeled
Asp and subjected to tricine SDS-PAGE, followed by
quantification by autoradiography (Figure S7). The presence
of EF-P enhanced the rP1 expression levels by modest (1.4-fold)
to high (6.8-fold) levels compared with those observed without
EF-P (Figure 2C). As a positive control, we also translated rP1-
Ala containing Ala in place of Abz derivatives, whose expression
level was not enhanced by EF-P.
that of rP1-Abz, suggesting that 3NAbz is a more challenging
substrate for the ribosome to elongate than Abz.
To test whether this approach also allows us to incorporate
Abz in consecutive or multiple fashion, we prepared the mRNAs
mR2−4 to express the peptides rP2−4 (Figure 3A) in the
Some cyclic γ-amino acids (cγAAs), such as 3-amino-
cyclobutanecarboxylic acid, could be elongated in the
combination of cγAA-tRNAPro1E2 and EF-P in a similar manner
to β-amino acids.34 This fact motivated us to test whether 3NAbz,
a γ-amino acid version of Abz, could be elongated into a nascent
peptide chain (Figure 1). Despite the report by Ad et al. where
the yield of 3NAbz acylation using eFx was 0%,21 we were able to
aminoacylate 3NAbz at the level of 45% under our optimized
conditions (pH 10.0, 144 h, and 20 mM 3NAbz-CME; Figures
5D and 6D). By the use of 3NAbz-tRNAPro1E2CGG, we attempted
the incorporation of 3NAbz into rP1, and the product was
analyzed by MALDI-TOF MS (Figure 2B). To our surprise, the
correct peak corresponding to rP1-3NAbz was indeed observed,
but its peak intensity over the background signals was lower than
Figure 3. Incorporation of multiple Abz into model peptides. (A)
Sequences of mRNAs (mR2−mR4) and the corresponding peptides
(rP2−rP4). (B) Expression levels of rP1−rP4 peptides estimated by
autoradiography. See Figure S8 for the raw data from tricine SDS-
PAGE. (C) MALDI-TOF MS of ribosomally synthesized rP4. The red
arrow indicates the monovalent ion of the desired peptide. “Obsd.” and
“Calcd.” indicate observed and calculated m/z values, respectively.
B
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Figure 4. Incorporation of Abz derivatives into model macrocyclic peptides. (A) Sequences of mRNAs (mR5−mR10) and the corresponding peptides
(rP5−rP10). The thiol group of D-Cys attacks the N-terminal chloroacetyl group to form a thioether bond. (B) MALDI-TOF MS of rP5−rP10. Red
and blue arrows indicate monovalent and divalent ions of the desired peptides, respectively. “Obsd.” and “Calcd.” indicate observed and calculated m/z
values, respectively. Asterisks denote template-independent impurities derived from the translation system. (C) Structure of rP8 containing Abz or
Abz5OMe
.
presence of Abz-tRNAPro1E2
and EF-P. rP2 has two
(CUU in mR6), or Apy (AUU in mR7), each of which was
charged onto tRNAPro1E2NNN (where NNN is the corresponding
anticodon), was introduced into a sequence of peptide rP5, rP6,
or rP7, respectively (Figure 4A). The respective rP5−rP7 were
analyzed by MALDI-TOF MS (Figure 4B), which showed that
the correct macrocyclic peptides were expressed. More
aggressive reprogramming was conducted to express rP8 (Figure
4A−C), where N-methyl-L-α-Ser (MeSer), (1S,2S)-2-ACPC,25
and Abz or Abz5OMe were embedded between the cyclizing ClAcD-
Phe/D-Cys. Those amino acids were introduced using MeSer-
tRNAPro1E2GAU, (1S,2S)-2-ACPC-tRNAGluE2CGG, and Abz- or
CGG
consecutive Abz residues, while rP3 and rP4 have one and two
Phe insertions between two Abz residues, respectively. The
respective expression of rP2−4 labeled with [14C]-Asp was
analyzed by tricine SDS-PAGE. We observed a discrete band in
the rP4 expression (Figures 3B and S8) with a level comparable
to that of rP1 (0.065 vs 0.072 pmol/μL), and the identity of rP4
with the expected molecular mass was confirmed by MALDI-
TOF MS (Figure 3C). On the other hand, no obvious bands
corresponding to rP2 and rP3 were detected in the PAGE
analysis. These results indicate that the consecutive incorpo-
ration of Abz is far more difficult than for saturated β-amino
acids, but when two Abz residues are separated by two or more L-
α-amino acid residues, their multi-incorporation is allowed.
We next expressed macrocyclic peptides containing multiple
exotic amino acids, including Abz derivatives. We designed
mRNA templates mR5−mR10 to express peptides rP5−rP10
under the reprogrammed genetic codes (Figure 4A−C). All of
the genetic codes were reprogrammed to assign N-chloroacetyl-
Abz5OMe-tRNAPro1E2
(Figure S1B−D). MALDI-TOF MS
GGU
analysis of rP8-Abz and rP8-Abz5OMe also showed the desired
peak (Figure 4B), indicating successful expression.
We finally conducted incorporation of two Abz derivatives
into macrocyclic scaffolds of rP9 and rP10. In this experiment,
Abz-tRNAPro1E2
was assigned to the CCG codon in both
CGG
mR9 and mR10, and Apy-tRNAPro1E2GGU and (1S,2S)-2-ACPC-
tRNAGluE2GAU were assigned to ACU and AUU, respectively, in
mR10. We were able to observe clean expression of rP9 as
confirmed by MALDI-TOF MS analysis (Figure 4B). On the
other hand, the correct peak of rP10 was also observed, but its
peak intensity over the background peaks was much lower than
for rP9, indicating that the expression would be less efficient
(Figure 4B). Nevertheless, these results clearly indicate that the
incorporation of multiple Abz derivatives into macrocyclic
peptide scaffolds is also possible.
D-tyrosine (ClAcD-Tyr) or N-chloroacetyl-D-phenylalanine
ClAc
(
D-Phe) as an initiator (charged onto tRNAfMet
by
CAU
eFx/ClAcD-Tyr-CME or eFx/ClAcD-Phe-CME), and D-Cys was
introduced at a downstream position (charged onto
tRNAPro1E2
by dFx/D-Cys-DBE, where DBE is 3,5-
GUG
dinitrobenzyl ester), where the ClAc and sulfhydryl groups on
these D-amino acids autocyclized via a thioether bond when the
entire peptide was synthesized (Figure 4A).35 Between these
cyclizing residues, a single residue of Abz (UUU in mR5), Atp
C
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In our recent studies, we reported ribosomal incorporation of
aromatic oligoamide foldamers containing 8-amino-2-quinoli-
necarboxylic acid and its derivatives as building blocks at the N-
Japan; orcid.org/0000-0002-5298-9186; Email: hsuga@
chem.s.u-tokyo.ac.jp
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c05765
termini of peptides by means of the initiator tRNAfMet 36,37 We
.
also demonstrated elongation of amino acids bearing such
aromatic oligoamides at their side chains.38 Ad et al. also
reported ribosomal incorporation of Abz derivatives at the N-
terminus. However, those molecules were not introduced in the
main chain in the middle of peptide sequences, i.e., not by the
elongation process. The present study has demonstrated for the
first time that the ribosome is able to catalyze peptide bond
formation on the amino group of Abz derivatives despite its poor
nucleophilicity. This is likely the case because even if the
nucleophilic attack is slow, the following deprotonation of the
Abz’s amino group of the tetrahedral intermediate is fast enough
to afford the peptide bond.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a Japan Society for the Promotion
of Science (JSPS) Grant-in-Aid for Scientific Research (B)
(18H02080) to T.K. and a JST CREST for Molecular
Technologies grant (JPMJCR12L2) to H.S.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c05765.
■
sı
Figures S1−S8 and Materials and Methods (PDF)
Table S1 (PDF)
Table S2 (XLS)
AUTHOR INFORMATION
Corresponding Authors
Takayuki Katoh − Department of Chemistry, Graduate School of
Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033,
Japan; Email: katoh@chem.s.u-tokyo.ac.jp
■
Hiroaki Suga − Department of Chemistry, Graduate School of
Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033,
D
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