Angewandte Chemie International Edition
10.1002/anie.201908593
RESEARCH ARTICLE
[
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measure of its activity.
The formation of
a
2,3-
for this methodology. Very high conversions to protein
bioconjugates were observed under the reaction conditions
as shown in Figure 6 and 7. This approach leads to the
nitroalkylation of proteins in a selective manner thus mimic a
key PTM of proteins. Such nitroalkylated proteins can be
used to investigate the role of these PTMs in various cellular
processes and enzyme functions. The nitroalkylated proteins
enable rapid detection of labeled peptides using MS/MS
fragmentation even if they are present in low abundance due
to the unique fingerprint pattern for the parent peptide due to
loss of the nitro species. This is of high significance because
nitroalkane peptides can be identified in a complex mixture
without the need for enrichment of the nitroalkane-bound
peptides. Importantly, di-nitroalkanes react exclusively with
dialdehyde peptides to form peptide staples of different sizes.
Considering the easy synthesis of a variety of nitroalkane
derivatives (fluorescent tag, NMR tag, and an affinity tag) and
their ability to carry out rapid and highly selective
bioconjugation of proteins and peptide stapling with very high
conversions (99%), we anticipate that this method will
become a highly useful tool in many fields, including chemical
biology and medicinal chemistry.
diaminophenazine was monitored at 426 nm by UV
spectroscopy. The freshly prepared myoglobin, nitromethane-
labeled myoglobin, o-phenylenediamine, and hydrogen
peroxide (30 %) solutions were used for the assay. The
results confirmed that installation of a nitromethane does not
alter the activity of myoglobin (Figure S14, Supporting
Information). Together, these results reaffirm that the
nitroreagents provide access to the single-site installation of a
probe to
a protein while conserving its structure and
bioactivity.
Nitroreagents for late stage diversification of
peptides and stapling of peptides
Late stage diversification of peptides (e.g., labeling,
glycosylation, lipidation, etc.) and peptide stapling has a great
potential as biological tools, and thus the development of
[
28]
such reagents is of high significance.
The capability of
nitroalkanes for selective modification of aldehydes prompted
us to explore their application in late-stage diversification of
peptides and stapling of peptides. First, we introduced an
aldehyde group on the side chain of
modification of a side-chain of a glutamic acid
treated completely unprotected peptide 7 with nitromethane
a under physiological conditions (phosphate buffer 50 mM,
a
peptide
7
by
[
29]
and then
Experimental Section
1
See the supporting information for materials, instruments, synthetic
procedures, and supporting figures
pH 7.5). Higher conversion to the modified peptide 8 was
observed as analyzed by HPLC and MS (Figure 8a, Figure
S15, Supporting Information). This study indicates that our
methodology is independent of the nature and position of
aldehydes (N-terminus and side-chain position).
Acknowledgements
For peptide stapling, we synthesized dinitrohexane 9 (see
synthesis; Figure S16, Supporting Information) and carried
out reactions with completely unprotected peptides 10
containing two aldehyde groups at varying positions (i,i + 2 to
i,i + 7) under neutral aqueous conditions. High conversions to
peptide staples 11 were observed for all dialdehyde peptides
This research was supported by NSF (Grant No. CHE-
1
752654) granted to M.R.
Keywords: Nitroalkylation, Protein Bioconjugation,
Chemoselective, Peptide Stapling, Selective Fluorination
1
0, as determined by HPLC and MS analysis (Figure 8b,
[
1] a) S. I. van Kasteren, H. B. Kramer, H. H. Jensen, S. J. Campbell, J.
Figure S17, Supporting Information). Our stapling method
introduces two nitro groups in a peptide chain, which are
considered to be a versatile and unique functional group in
Kirkpatrick, N. J. Oldham, D. C. Anthony, B. G. Davis, Nature 2007, 446,
1
105-1109; b) M. Zheng, L. Zheng, P. Zhang, J. Li, Y. Zhang, Molecules
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[
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medicinal chemistry.
These nitrofunctionalized peptide
2
staples can be used to investigate the target selectivity of
3] a) J. I. MacDonald, H. K. Munch, T. Moore, M. B. Francis, Nat. Chem.
Biol. 2015, 11, 326-331; b) L. Ducry, B. Stump, Bioconjugate Chem.
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stapled peptides for the development of new therapeutics.
2
010, 21, 5-13.
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4] a) P. V. Chang, J. A. Prescher, E. M. Sletten, J. M. Baskin, I. A. Miller,
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Conclusion
1
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075-1101.
In summary, we have demonstrated that bioinspired
nitroalkanes are highly efficient for bioconjugation reaction
and exhibit excellent selectivity for a single functional group
that can be easily installed by both chemical and biochemical
approaches, in different proteins. Peptide and protein studies
have shown that a diverse array of stable bioconjugates can
be obtained rapidly, under mild reaction conditions. Although
some aldehyde bioconjugations often suffer from low
conversion and slow reaction rate, no such effect was seen
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