Full Paper
doi.org/10.1002/chem.202102042
Chemistry—A European Journal
An Optimized Protocol for the Synthesis of Peptides
Containing trans-Cyclooctene and Bicyclononyne
Dienophiles as Useful Multifunctional Bioorthogonal
Probes
Agustina La–Venia,[a, b] Rastislav Dzijak,[a] Robert Rampmaier,[a] and Milan Vrabel*[a]
Abstract: Despite the great advances in solid-phase peptide
synthesis (SPPS), the incorporation of certain functional
groups into peptide sequences is restricted by the compati-
bility of the building blocks with conditions used during
SPPS. In particular, the introduction of highly reactive groups
used in modern bioorthogonal reactions into peptides
remains elusive. Here, we present an optimized synthetic
protocol enabling installation of two strained dienophiles,
trans-cyclooctene (TCO) and bicyclononyne (BCN), into differ-
ent peptide sequences. The two groups enable fast and
modular post-synthetic functionalization of peptides, as we
demonstrate in preparation of peptide-peptide and peptide-
drug conjugates. Due to the excellent biocompatibility, the
click-functionalization of the peptides can be performed
directly in live cells. We further show that the introduction of
both clickable groups into peptides enables construction of
smart, multifunctional probes that can streamline complex
chemical biology experiments such as visualization and pull-
down of metabolically labeled glycoconjugates. The pre-
sented strategy will find utility in construction of peptides for
diverse applications, where high reactivity, efficiency and
biocompatibility of the modification step is critical.
Introduction
approach is the compatibility of the modified building blocks
with SPPS. In this regard, the most challenging part of the
synthesis involves the deprotection/cleavage steps, which
require the use of cocktails containing as much as 95%
trifluoroacetic acid (TFA). The synthetic or commercial avail-
ability of modified building blocks, which are usually used in an
excess, can be also a limiting factor. Another approach for
synthesis of functional peptides is based on the attachment of
the functional moiety after the synthesis of the peptide
backbone. This post-synthetic modification approach takes
advantage of the unique reactivity of functional groups present
on natural amino acids[6] or, alternatively, involves a two-step
process where an artificial reactive group, compatible with
SPPS, is introduced into the sequence first, and is then used for
the functionalization by selective chemical click reaction in the
second step.[7]
Peptides represent a unique class of macromolecules with wide
range of properties, functions and applications.[1] It is the great
structural diversity on one hand, and synthetic accessibility on
the other, which make peptides an attractive functional modal-
ity that in many aspects combines the advantages of small
molecules and larger biomolecules. Indeed, the last decades
have witnessed a huge increase in the popularity of peptides as
therapeutics,[2] diagnostics,[3] delivery agents[4] and chemical
biology probes.[5] Many of these conjugates require the attach-
ment of additional functional moieties to the peptide backbone
to fulfill their function. Owing to the chemical complexity of
peptides, the development of methods enabling their selective
functionalization is not trivial. The most straightforward method
for construction of functionalized peptides is based on the use
of modified building blocks, unnatural amino acids, during
standard SPPS. The obvious requirement for the success of this
Among the bioorthogonal click reactions available,[8] the
strain-promoted alkyne-azide cycloaddition (SPAAC)[9] and the
inverse electron-demand Diels-Alder reaction (iEDDA)[10] of
1,2,4,5-tetrazines with strained dienophiles are especially valued
due to high reaction rates, good biocompatibility and no need
for additional catalysis. For application of SPAAC in peptide
chemistry, the main strategy is based on the introduction of the
azide moiety during SPPS.[11] Examples of peptides functional-
ized with different cyclooctyne moieties also exist.[12] The
strained dienophile/dipolarophile was in most of the cases
installed via active ester chemistry on the N-terminus or single
lysine residue within the peptide sequence. A general method
for the de novo synthesis of a bicyclononyne amino acid (BCN-
AA) has been published, however, the compound was not used
in SPPS.[13] Recently, a copper-protection strategy was devised
[a] Dr. A. La–Venia, Dr. R. Dzijak, R. Rampmaier, Dr. M. Vrabel
Institute of Organic Chemistry and Biochemistry
Czech Academy of Sciences
Flemingovo nám. 2, 16000, Prague (Czech Republic)
E-mail: vrabel@uochb.cas.cz
[b] Dr. A. La–Venia
Current address:
Instituto de Química Rosario
Facultad de Ciencias Bioquímicas y Farmacéuticas
Universidad Nacional de Rosario-CONICET
Suipacha 531, S2002LRK, Rosario (Argentina)
Supporting information for this article is available on the WWW under
Chem. Eur. J. 2021, 27, 1–11
1
© 2021 Wiley-VCH GmbH
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