Chemoselective Nitrile Oxide-Alkyne 1,3-Dipolar Cycloaddition Reactions from Nitroalkane-Tethered Peptides

Article abstract of DOI:10.1021/acs.orglett.7b01498

Synthesis and incorporation of a new amino acid with a nitroalkane side chain into peptides, in situ transformation of a nitroalkane side chain into nitrile oxide, and chemoselective 1,3-dipolar cycloaddition reactions between in situ generated nitrile oxide and different alkynes are reported. The nitroalkane-mediated nitrile oxide-alkyne cycloaddition was found to be orthogonal to the copper(I)-catalyzed azide-alkyne cycloaddition reaction. The combination of orthogonal nitrile oxide-alkyne and azide-alkyne cycloaddition reactions can be explored to tailor different 1,2,3-triazole and 3,5-isoxazoles, respectively, on the peptide backbone.

Full text of DOI:10.1021/acs.orglett.7b01498

Letter
pubs.acs.org/OrgLett
Chemoselective Nitrile Oxide−Alkyne 1,3-Dipolar Cycloaddition
Reactions from Nitroalkane-Tethered Peptides
Rahi M. Reja, Sereena Sunny, and Hosahudya N. Gopi*
Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pune 411 008, India
S
* Supporting Information
ABSTRACT: Synthesis and incorporation of a new amino acid
with a nitroalkane side chain into peptides, in situ transformation of
a nitroalkane side chain into nitrile oxide, and chemoselective 1,3-
dipolar cycloaddition reactions between in situ generated nitrile
oxide and different alkynes are reported. The nitroalkane-mediated
nitrile oxide−alkyne cycloaddition was found to be orthogonal to
the copper(I)-catalyzed azide−alkyne cycloaddition reaction. The
combination of orthogonal nitrile oxide−alkyne and azide−alkyne
cycloaddition reactions can be explored to tailor different 1,2,3-
triazole and 3,5-isoxazoles, respectively, on the peptide backbone.
n his pioneering work, Huisgen has established a wide range
compounds into nitrile oxides mediated by phenyl isocyanate.13
̈
Iof three-atom, four-electron cycloaddition reactions with However, 1,3-dipolar cycloaddition reactions between nitrile
dipolarophiles.1 The recent introduction of copper(I) as a
oxide and dipolarophiles on the peptide backbone have scarcely
been studied. In addition, orthogonality of 1,3-dipolar cyclo-
addition reaction between in situ generated nitrile oxide and
azide has not been systematically investigated. Nitroalkanes are
neutral and versatile functional groups.14 Besides their
transformation into nitrile oxides, the nitroalkanes can be
transformed into various other functional groups such as
amines, carboxylic acids, aldehydes, and ketones. We
hypothesized that the introduction of a neutral nitroalkane
functionality on peptide backbone could be immediately
utilized for 1,3-dipolar cycloaddition reaction with alkynes
through in situ generated reactive nitrile oxide. In addition, as
the reaction conditions for azide−alkyne and nitrile oxide−
alkyne cycloaddition are quite different, they may be orthogonal
to one another. Nevertheless, no amino acids with nitroalkane
side chains are commercially available to test our hypothesis. In
this context, we sought to investigate the introduction of amino
acids with nitroalkane side chains into peptides, generation of
nitrile oxide on the peptide backbone, and the possibility of 1,3-
dipolar cycloaddition reactions with alkynes. Herein, we report
the synthesis and incorporation of nitroalkane-functionalized
new amino acid into peptides and its utility in efficient and
chemoselective 1,3-dipolar nitrile oxide−alkyne cycloaddition
reactions in the presence of azide functionality on peptides. The
nitroalkane mediated nitrile oxide−alkyne cycloaddition
reactions were found to be compatible in the solution phase
as well as on resin. Importantly, the nitroalkane functionality
was found to unaffected while performing CuAAC reactions.
To introduce nitroalkane functionality on the peptide
backbone, we have synthesized a new nitroalkane-function-
catalyst in the azide−alkyne cycloaddition (CuAAC or click
chemistry) reaction has exponentially increased its applications
in chemistry, biology, and materials sciences.2,3 The mild and
broad substrate specificity of CuAAC has been extensively
utilized in the conjugation of proteins, nucleic acids, and
carbohydrates in vitro and in vivo.4 In continuation, Bertozzi et
al. developed a copper-free and strain-promoted azide−alkyne
cycloaddition reaction (SPAAC) to avoid the toxicity of
copper(I) in biological systems.5 Besides the biomolecules,
click chemistry has been widely utilized to functionalize small
molecules and synthetic peptides. The click-conjugated
peptides have found applications in therapeutics, diagnostics,
tissue engineering, and drug delivery.6 In spite of its widespread
applications, click chemistry is also associated with inherent
limitations. The selective azide−alkyne cycloaddition in a
molecule containing two or more azides has been found to be a
difficult task. Nevertheless, Wolfbeis and colleagues have
demonstrated the dual labeling of biomolecules with and
without copper using alkynes and strained alkynes, respectively,
on a peptide.7 In addition, Carell and colleagues demonstrated
a triple modification of DNA by incorporating protected
alkynes on the DNA backbone.8 In addition to the double- and
triple-click conjugations on peptides and nucleic acids, the click
reaction was also found to be orthogonal to many other organic
reactions. Recently, Boturyn and colleagues demonstrated the
orthogonality of click reaction with oxime formation9 as well as
amide bond synthesis10 involving thioacids11 on a peptide
template.
In addition to azides, nitrile oxides have also been extensively
explored as precursors in the 1,3-dipolar cycloaddition reactions
to derive medicinally important isoxazoles.12 Mukaiyama and
Hoshino demonstrated the transformation of alkyl nitro
Received: May 23, 2017
© XXXX American Chemical Society
A
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Letter
alized amino acid 4 starting from the commercially available α-
tert-butyl ester of Boc-Glu(COOH)-OH (1). The schematic
representation of the reaction is shown in Scheme 1. The
azide functionality on peptides. In this context, we designed
peptides P1−P4 by incorporating either nitro amino acid alone
or along with azide as well as alkyne. The sequences of the
peptides are shown in Scheme 3.
Scheme 1. Synthesis of Amino Acid with a Nitroalkane Side
Chain from Protected Glutamic Acid
Scheme 3. Sequences of Peptides
unprotected γ-carboxylic acid (1) was reduced to alcohol (2)
through a mixed anhydride method using NaBH4. The alcohol
was transformed to iodide (3) through a modified Apple
reaction.15 The alkyl iodide was finally subjected to a
substitution reaction with NaNO2 in DMF16 to obtain amino
acid 4, which was further transformed to TFA salt 5 (δ-nitro
ornithine) through the deprotection of Boc and tert-butyl
esters.17 The free amine of amino acid 5 was again protected
with Fmoc and Boc groups to obtain amino acids 6 and 7,
respectively. Synthetic details are given in the Supporting
Information. The X-ray structure of protected amino acid 4 is
shown in Figure S1. We adopted a tert-butyl ester strategy
instead of ethyl/methyl esters to avoid the complications of
nitro functionality during the saponification of esters.18
First to understand whether the nitro amino acids can be
incorporated into peptides through solid-phase peptide syn-
thesis, we synthesized peptide P1 on Rink amide resin using
nitro amino acid 6. All coupling reactions were performed using
HBTU/HOBt conditions. The Fmoc group was deprotected
using 20% piperidine in DMF. All coupling reactions were
monitored using the Kaiser test. The mass spectral analysis of
the final peptide, cleaved from the resin using TFA, suggests
the incorporation of nitro amino acid into the peptide. The
resin-bound peptide P1 was treated with phenylacetylene in the
presence of phenyl isocyanate and triethylamine to understand
the feasibility of 1,3-dipolar cycloaddition reaction between the
in situ generated nitrile oxide and phenylacetylene. The
progress of the reaction was monitored using MALDI-TOF.
Complete transformation of the primary nitroalkane group into
the isoxazole derivative (P5) was achieved in 12 h with slow
stirring of the resin beads at room temperature. The excess
phenylacetylene and urea byproducts20 were removed by
washing the resin with DMF. The schematic representation
of the reaction is shown in Scheme 4. Peptide P5 was cleaved
from the resin and purified by RP-HPLC. The HPLC analysis
suggested the neat and quantitative transformation of P1 to P5.
Motivated by the mild 1,3-dipolar cycloaddition on solid
support, we synthesized peptide P2 by incorporating both
nitroamino acid 5 and azido lysine to understand the
orthogonality of 1,3-dipolar cycloadditions with alkynes. The
resin-bound peptide P2 was subjected to nitroalkane-mediated
nitrile oxide−alkyne cycloaddition reaction. In the process of in
situ generation of nitrile oxide through the dehydration using
phenyl isocyanate and subsequent 1,3-dipolar cycloaddition
with N-Cbz-propargylamine, we observed no cycloaddition
product from azide functionality on peptide. The formation of
isoxazole and unreacted azide on peptide P6 was confirmed by
the mass spectral analysis. Concurrently, we subjected P2 to
CuAAC with phenylacetylene in the presence of nitroalkane
functionality. We observed no cycloaddition from the nitro
functionality while performing the click reaction on azide (P7).
To understand whether the nitroalkane amino acid can
undergo a 1,3-dipolar cycloaddition reaction with alkyne
through in situ generated nitrile oxide, the amino acid 4 was
treated with protected mannose alkyne in the presence of
phenyl isocyanate and triethylamine (Scheme 2). The reaction
Scheme 2. 1,3-Dipolar Cycloaddition Reaction on Amino
Acid 4
was monitored using TLC, and complete transformation of
nitro amino acid 4 into its isoxazole derivative 8 was achieved in
12 h at room temperature. The 1,3-dipolar cycloaddition
product was isolated in excellent yield. The reaction was found
to be regioselective, and we isolated only 3,5-disubstituted
isoxazole derivative.19
Inspired by the mild cycloaddition reaction between alkyne
and amino acid 4, we sought to investigate the compatibility of
nitroalkane-mediated nitrile oxide−alkyne cycloaddition reac-
tion on peptide backbone as well as its chemoselectivity with
B
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selective 1,3-dipolar cycloaddition with N-Cbz-propargylamine
with a nitro group in the presence of phenyl isocyanate and
Et3N. Separately, P3 was also subjected to copper-catalyzed
click reaction with phenylacetylene to obtain monoconjugated
peptide P10. Finally, both P9 and P10 were separately
subjected to cycloaddition reactions with phenylacetylene and
N-Cbz-propargylamine, respectively, to obtain double-conju-
gated peptide P11. The selective cycloaddition reactions are
shown in the Scheme 6. All three peptides were purified by RP-
HPLC and characterized by 1H NMR and mass spectral
analysis.
Scheme 4. 1,3-Dipolar Cycloaddition Reaction of the
Peptide on Solid-Phase Support
Scheme 6. Orthogonal Cycloaddition Reaction with Azide in
Solution Phase
The cycloaddition reactions on P2 are shown in Scheme 5.
Both peptides P6 and P7 containing nitro and azide functional
Scheme 5. Orthogonal Cycloaddition Reaction with Azide
on Solid-Phase Support
Finally, to understand whether the nitro group is compatible
with alkyne functionality as well as alkyne−azide cycloaddition,
we have synthesized peptide P4 by incorporating both nitro
and alkyne functionalities. Pure P4 gave X-ray quality single
crystals after slow evaporation in aqueous MeOH solution, and
its X-ray structure is shown in Figure S2.
The peptide P4 was subjected to copper(I)-catalyzed 1,3-
dipolar cycloaddition reaction with 2-deoxyazido-1,3,4,6-
tetraacetyl glucopyranoside in the presence of the nitro
functionality on peptide. Selective azide−alkyne cycloaddition
product P12 was achieved without affecting the nitro
functionality (Scheme 7). The peptide was purified by RP-
HPLC and characterized by MALDI-TOF mass spectral
analysis. These results suggest that alkynes can also be used
along with the nitro alkane functional groups for orthogonal
groups, respectively, were further subjected to cycloaddition
reactions separately with alkynes to obtain doubly conjugated
product P8 on resin. The RP-HPLC and mass spectral analysis
suggested the quantitative conversion of both nitro and azide
into isoxazole and 1,2,3-triazole products, respectively. The
peptide P8 was cleaved from the resin, purified by RP-HPLC,
and characterized.
Scheme 7. Orthogonal Cycloaddition Reaction with Alkyne
in Solution Phase
Inspired by the orthogonal cycloaddition of nitro and azide
on a solid support, we sought to investigate whether the same
reaction can be performed in solution. In this context, we
synthesized peptide P3 by incorporating both azido lysine and
nitro amino acid 5 at the N- and C-terminus, respectively. The
nitro amino acid 7 was used in the solution-phase synthesis.
Peptide P3 was subjected to chemoselective azide−alkyne and
nitrile oxide−alkyne 1,3-dipolar cycloaddition reactions sepa-
rately. Monoconjugated product P9 was isolated through
C
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In conclusion, we have demonstrated the synthesis and
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b01498.
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1H and 13C NMR spectra, mass spectra, synthetic details
of amino acids and peptides, and crystallographic data of
amino acid 4 and P4 (PDF)
X-ray data for compound 4 (CIF)
X-ray data for compound P4 (CIF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: hn.gopi@iiserpune.ac.in.
ORCID
Hosahudya N. Gopi: 0000-0001-8156-9271
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
R.M.R. is thankful to IISER Pune for a research fellowship. We
thank DST, Government of India, and IISER Pune for financial
support.
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