Combination of inverse electron-demand Diels-Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations

Article abstract of DOI:10.1039/c5cc03434e

A modular approach combining inverse electron-demand Diels-Alder coupling (DAR_inv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated

Full text of DOI:10.1039/c5cc03434e

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DOI: 10.1039/C5CC03434E
Journal Name
COMMUNICATION
Combination of inverse electron-demand Diels-Alder reaction
with highly efficient oxime ligation expands the toolbox of site-
selective peptide conjugations
Received 00th January 20xx,
Accepted 00th January 20xx
‡
a
‡a
a
b
c
a
DOI: 10.1039/x0xx00000x
S. Hörner, C. Uth, O. Avrutina, H. Frauendorf, M. Wiessler, and H. Kolmar*
www.rsc.org/
Abstract: A modular approach combining inverse electron-demand blocks10 either upon recombinant production11 or in the course of
Diels-Alder coupling (DARinv) and oxime ligation expands the chemical synthesis. Bioorthogonal reactions to target these non-
toolbox of bioorthogonal peptide chemistry. Applicability of natural functional groups often make use of rich ketone and
1
2
3, 13
versatile site-specific bifunctional building blocks is demonstrated aldehyde chemistry as well as numerous click-type reactions,
by generation of defined conjugates comprising linear, cystine- with the azide-alkyne cycloaddition being the most prominent
bridged and multi-disulfide functional peptides as well as their representative.14-16 During the last decade, a special class of
conjugation with hybrid silsesquioxane nanoparticles.
pericyclic reactions has got the highest priority as they utilize the
ring strain to promote increased reactivity upon cycloaddition.
1
7-19
Covalent linkage of certain peptidic units, being an alternative or a In particular, the Diels-Alder reaction with inverse electron-demand
2
0-22
23-25
21, 26,
complementary strategy to recombinant production, often (DAR )
between numerous dienophiles
and tetrazines
inv
2
7
becomes a method of choice for the synthesis of sophisticated
was found to be a valuable tool for effective bioorthogonal
macromolecular constructs with tailored properties.1 Indeed, to conjugations.28-30 Followed by a retro-Diels-Alder reaction to
date a vast number of bioconjugates and the respective techniques eliminate nitrogen gas, this so-called tetrazine ligation is
2
3
have been reported, ranging from relatively simple fluorescently
characterized by extremely fast kinetics with second-order rate
or small-molecule-labelled peptides4 to complex, multifunctional constants up to 2·10 M s and has been already used in a number
3
-1 -1
5
25, 31, 32
architectures like antibody-drug conjugates. Obviously, chemical of both in situ and in vivo studies.
transformations suitable for bioconjugations must satisfy at least In this study we present a modular approach to the conjugation of
6
two obligatory requirements, chemoselectivity and efficiency. In biomolecules based on the combination of two efficient chemical
view of the variety of inherent functional groups present in peptidic transformations, oxime ligation and DAR . For the proof-of-
inv
molecules, the development of a viable orthogonal chemistry for concept experiments we designed bifunctional building blocks to
their effective junction at a certain position still remains a incorporate the bioorthogonal DARinv coupling site in a peptide site-
challenge.
specifically via oximation of the respective aldehyde. Following this
7
Generally, the strategy towards site-specific bioconjugations relies step, attachment of a DARinv counterpart would accomplish a
on incorporation of a uniquely addressable group at the desired desired conjugate. The choice of the strategy was specified by two
position in the molecule of interest followed by its peculiar reaction arguments, feasible generation of required aldehydes in
with the respective counterpart. Such a uniquely addressable biomolecules8, 9, 33 and fast kinetics of an irreversible DAR . We
inv
moiety could be incorporated into peptidic molecules through a reasoned that our approach, initially investigated on peptides of
vast number of post-synthetic modifications, e.g. periodate different size and molecular complexity, could be further extended
8, 9
oxidation of β-aminoalcohols,
or via the non-natural building to orthogonal conjugations featuring
a broad spectrum of
biomacromolecules, i.e. proteins, sugars, or other biopolymers.
Here it is important to mention that, while having been used for
3
4, 35
numerous labeling approaches,
the DARinv reaction was
a.
Technische Universität Darmstadt, Clemens-Schöpf-Institut für Organische
Chemie und Biochemie, Alarich-Weiss Straße 4, 64287 Darmstadt, Germany.
Georg-August-Universität Göttingen, Institut für Organische und Biomolekulare
Chemie, Zentrale Analytik/Massenspektrometrie, Tammannstraße 2, 37077
Göttingen, Germany.
Deutsches Krebsforschungszentrum, Medizinische Physik in der Radiologie,
Projektgruppe Biologische Chemie E020, Im Neuenheimer Feld, 69120 Heidelberg,
Germany.
surprisingly rarely applied to connect functional peptides site-
This could be explained taking into account that
both the diene and the dienophile counterparts have been to date
2
8, 36
b.
c.
specifically.
37, 38
installed into partner biomolecules using either amide
or
3
9
maleimide chemistry
which cannot provide the required
orthogonality, especially with regard to cysteine/cystine-bearing
molecules. Though the installation of DARinv blocks into peptide-like
molecules on solid support has been reported, this approach did
not find broad application. Moreover, the on-resin assembly is very
rarely used for the production of full-size proteins.
† ‡
These authors contributed equally to this work.
*
Corresponding author
This work was partially funded by the DFG priority program SPP 1623. The authors
thank Peter Lorenz (DKFZ, Heidelberg) for his advice regarding tetrazine synthesis.
Electronic Supplementary Information (ESI) available: Combination of inverse
electron-demand Diels-Alder reaction with highly efficient oxime ligation expands
the toolbox of site-selective peptide conjugations. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
Since oxime ligation has been applied to couple exo-norbornene as Thus, mono-Boc-protected miniPEG 1 was reactedVieww iAtrhticleROepnlpinee
a dienophile handle onto aldehydes of the reducing ends of anhydride 2 or tetrazine derivative 6, respecDtOivIe: l1y0,.1a0n3d9/tCh5eCrCe0su34lt3in4Eg
4
0
oligosaccharides and taking into consideration that this moiety is constructs 3 and 7 were transformed into N-ethoxyethylidene(Eei)-
easily generated in both recombinant and synthetic peptides41 protected aminooxy building blocks 5 and 8 upon acidolytic
without interference with functional side chains, we decided to use cleavage of N-Boc protection followed by the N-acylation with N-
this highly efficient reaction to decorate the peptides of interest hydroxysuccinimide-activated (NHS) Eei-protected aminooxy acetic
4
8
with the respective diene and dienophile partners.
acid 4 (Fig. 1, ESI 1.2.1-1.2.8). The oxime ligation was performed
in 50 % (v:v) aqueous TFA overnight leading to peptide-tetrazines
1
5-17 and peptide-dienophiles 18-20 (Fig. 2, ESI 1.2.12).
After the conjugation partners have been decorated with the
respective DARinv building blocks, the resulted diene and dienophile
counterparts were reacted with each other in 10 % aq. acetonitrile
containing 0.1 % TFA overnight at ambient temperature giving
conjugates 21-29 (Fig. 3, 4, ESI 1.2.15). The exemplified monitoring
of DARinv reaction progress presented in Fig. 4 and in ESI 1.2.15
clearly indicates that conversion into the desired products has been
achieved for all constructs. Interestingly, upon evaluation of the
mass spectrometric data, we observed for some DARinv conjugates
the recovery of the aromatic system in the tetrazine, as previously
4
9, 50
reported.
The mass spectra can be seen in Fig. 5 as well as in
ESI 1.2.21-1.2.29.
Fig. 1: Synthesis of DARinv building blocks.
Our modular approach relies on the tailor-made bifunctional
building blocks equipped with an aminooxy moiety for the primary
incorporation into peptidic counterparts along with the DARinv site
for the successive ligation (Fig. 1). In our proof-of-concept study we
installed the DARinv building blocks 5 and 8 into the next functional
4
2, 43
peptides: a Sortase A recognition heptapeptide 12,
an integrin-
1
2
binding RGD decapeptide 9, an antimicrobial decapeptide Jelleine
derivative 13, a disulfide-bridged pentadecapeptide matriptase
inhibitor 10, and two cystine knots comprising three disulfides and
a backbone of more than thirty amino acids 11, 14 (Scheme ESI-
). An additional N-terminal serine was introduced into each
4
4
4
5
4
6
2
peptide to provide the orthogonally addressable site upon post-
synthetic modification. The resulted glyoxylyl moieties generated
Fig. 3: Schematic representation of peptide conjugates synthesized
by DARinv between the counterparts depicted on the left and the
right panels.
9
by periodate oxidation (ESI Scheme-1) were oximated by the
aminooxy-bearing derivatives of Reppe anhydride 5 and tetrazine 8,
respectively (Fig. 1, 2). The dienes 15-17 were synthesized from
peptide-glyoxals 9-11, and the dienophiles 18-20 - from peptide-
glyoxals 12-14 (Fig. 2, ESI 1.2.12). Peptides were assembled by
As the proof-of-concept study showed the viability of our approach,
it was further expanded towards a more sophisticated (in view of
architecture and functional properties) molecular construct. Thus,
we attached a peptidic cargo to the smallest nanoparticle known, a
cell-penetrating organic-inorganic hybrid molecule comprising
4
7
microwave-assisted Fmoc-SPPS. Following cleavage from the
support with successive oxidative folding if required, the N-terminal
serines were oxidized by NaIO
4
to generate the aminooxy-reactive
44
cube-octameric silsesquioxane (COSS). This highly symmetric
glyoxals 9-14 for further conjugations (ESI 1.2.9-1.2.11).
Prior to the installation into synthetic peptides, the bifunctional
DARinv building blocks were synthesized following a two-step
procedure as illustrated in Fig. 1.
octavalent compound has recently attracted keen attention, being
used as a scaffold in a number of biomedical applications, including
delivery of bioactive payloads into cancer cells and development of
44
novel tailor-made conjugates of pharmacological interest.
Fig. 2: Synthesized peptidic ligands for successive DARinv conjugations.
2
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A)
macromolecular constructs of undefined stoichiometry.34, 36 Our
approach offers the advantage of being suDiOtaI:b1l0e.1f0o3r9/aCll5CsCyn0t3h4e3t4iEc
View Article Online
peptides, particularly the recombinant ones.
B)
15
16
17
18
19
20
21 (73.3%)
22 (85%)
23 (92.5%)
24 (87.7%)
25 (100%)
26 (87%)
27 (n.d.)
28 (93.8%)
29 (n.d.)
Fig. 4: A) HPLC monitoring of an exemplified DARinv conjugation
between Jelleine-Reppe construct 19 and SDMI-3 tetrazine partner
1
6. B) Conversion rate for DARinv conjugations calculated from the
HPLC traces (n.d.: not determined).
Compared to the modification of peptides with a dienophile, a
different procedure was applied. Thus, Reppe anhydride 2 was
equipped with
a
linker comprising the NHS-activated
γ-aminobutyric acid (ESI 1.2.13). Building block 30 was attached to a
single corner of octaamino COSS through an amide bond (Fig. 5, ESI
Fig. 5: Top: Formation of conjugate 32 by DARinv between cell-
penetrating COSS 31 and peptide 16. Bottom: Exemplary ESI-
HR-MS analysis of conjugate 32 [M+4H] .
1
.2.14) resulting in COSS-dienophile 31 which was reacted with
peptide-diene 16 giving conjugate 32. The reaction was carried out
in dry DMSO to assure stability of the siloxane core in the presence
of the pendant propylamine groups (Fig. 5).
4+
In our hands, being incorporated into peptides and hybrid
silsesquioxanes, the strained four-membered ring system of the
Reppe anhydride reacted with the respective tetrazine, smoothly
converting it into the desired product. This reactivity can be easily
explained considering the non-reversible character of DARinv
coupling and the fact that cyclobutene is amongst the most reactive
Conclusion
The combination of oxime ligation and DARinv is a convenient
method for site-specific conjugation of complex peptidic molecules.
What is the rationale behind using the set of two transformations
rather than the individual reactions, each of which is bioorthogonal,
selective, and fast per se? First, at physiological pH oxime ligation is
rather slow and requires catalysis. This could pose a problem if two
functional biological macromolecules are ligated directly, whereas
the introduction of small bifunctional linker proceeds unhampered,
and the next step is irreversible and uncatalyzed. Second, although
the direct introduction of DARinv moieties on solid support has been
5
3
species classified via their activation free energies.
Interestingly, for some peptide conjugates we observed the
regeneration of the aromatic system within the tetrazine moiety
after DARinv reaction. This observation has previously been
4
9, 50
reported, but detailed study of the mechanism is still required.
To demonstrate modularity and versatility of our approach, we
4
5
applied it to covalently graft a bioactive peptide onto a cell-
penetrating COSS nanoparticle. The success of this reaction opens
new avenues for the facile generation of peptide-COSS conjugates
for intracellular delivery, thus expanding the toolbox for the
chemical modifications of these promising compounds. In a
perspective, our system could be applied to the generation of
2
1, 30, 36, 51
reported for short peptides,
their introduction in
recombinant proteins requires extensive modification of the cellular
1
0, 32, 52
translation machinery.
And, third, our approach provides
certain modularity, giving an option to choose between the reactive
sites according to reaction environment and nature of the
conjugation partners.
In general, our bifunctional building blocks are readily synthetically
accessible and can be introduced into peptides site-selectively,
without affecting their architecture. The generation of the required
5
4
peptide-protein conjugates under physiological conditions. Taking
into consideration that stability of oxime linkage is context-
dependent and at physiological pH a catalyst is required to assure
efficient transformation, an adequately high-performance
chemistry should be considered to replace this ligation step. To this
carbonyl moieties in peptidic macromolecules is
a routine
5
5
end, recently reported trapped Knoevenagel condensation could
procedure as it proceeds smoothly both upon post-synthetic
8
33
be applied as an alternative strategy.
modification and in the course of recombinant production.
Successive oxime ligation was highly efficient and orthogonal to the
side-chain functionalities. Subsequently, the generated DARinv
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361.
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