Engineering Boron Hot Spots for the Site-Selective Installation of Iminoboronates on Peptide Chains

Article abstract of DOI:10.1002/chem.202002675

Boronic acids (BAs) are a promising bioconjugation function to design dynamic materials as they can establish reversible covalent bonds with oxygen/nitrogen nucleophiles that respond to different pH, ROS, carbohydrates and glutathione levels. However, the dynamic nature of these bonds also limits the control over the stability and site-selectivity of the bioconjugation, which ultimately leads to heterogeneous conjugates with poor stability under physiological conditions. Here we disclose a new strategy to install BAs on peptide chains. In this study, a “boron hot spot“ based on the 3-hydroxyquinolin-2(1H)-one scaffold was developed and upon installation on a peptide N-terminal cysteine, enables the site-selective formation of iminoboronates with 2-formyl-phenyl boronic acids (K_a of 58128±2 m^?1). The reaction is selective in the presence of competing lysine ?-amino groups, and the resulting iminoboronates, displayed improved stability in buffers solutions and a cleavable profile in the presence of glutathione. Once developed, the methodology was used to prepare cleavable fluorescent conjugates with a laminin fragment, which enabled the validation of the 67LR receptor as a target to deliver cargo to cancer HT29 cells.

Full text of DOI:10.1002/chem.202002675

Accepted Article
Accepted Article
Title: Engineering Boron Hot Spots for the Site-Selective Installation of
Iminoboronates on Peptide Chains
Authors: Roberto Russo, Rita Padanha, Fábio Fernandes, Luis Veiros,
Francisco Corzana, and Pedro Miguel pimenta Gois
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202002675
Link to VoR: https://doi.org/10.1002/chem.202002675
01/2020

10.1002/chem.202002675
Chemistry - A European Journal
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Engineering Boron Hot Spots for the Site-Selective Installation of
Iminoboronates on Peptide Chains
Roberto Russo,[a] Rita Padanha,[a] Fábio Fernandes,[b,c] Luis F. Veiros,[c] Francisco Corzana,[d] Pedro
M. P. Gois[a]*
[a]
R. Russo, R. Padanha, Prof. P. M. P. Gois
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa (Portugal)
E-mail: pedrogois@ff.ulisboa.pt
Homepage: https://sites.ff.ulisboa.pt/goislab/
[b]
[c]
[d]
[e]
Dr. F. Fernandes
Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology
IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa
Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
Prof. L. F. Veiros
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal.
Dr. Francisco Corzana
Departamento de Química, Universidad de La Rioja, Centro de Investigaciꢀn en Síntesis Química, 26006 Logroꢁo, Spain.
Supporting information for this article is given via a link at the end of the document
Abstract: Boronic acids (BAs) are a promising bioconjugation
function to design dynamic materials as they can establish reversible
covalent bonds with oxygen/nitrogen nucleophiles that respond to
different pH, ROS, carbohydrates and glutathione levels. However,
the dynamic nature of these bonds also limits the control over the
stability and site-selectivity of the bioconjugation, which ultimately
leads to heterogeneous conjugates with poor stability under
physiological conditions. Here we disclose a new strategy to install
BAs on peptide chains. In this study, a “Boron Hot Spot” based on the
3-hydroxyquinolin-2(1H)-one scaffold was developed and upon
installation on a peptide N-terminal cysteine, enables the site-
selective formation of iminoboronates with 2-formyl-phenyl boronic
acids (Ka of 58128 ± 2 M-1). The reaction is selective in the presence
of competing lysine ε-amino groups, and the resulting iminoboronates,
displayed improved stability in buffers solutions and a cleavable
profile in the presence of glutathione. Once developed, the
methodology was used to prepare cleavable fluorescent conjugates
with a laminin fragment, which enabled the validation of the 67LR
receptor as a target to deliver cargo to cancer HT29 cells.
linkers with mechanisms to respond to disease associated
chemical environments.[10,11] In this context, boronic acids (BAs)
emerged as a particularly promising function to design responsive
materials, because they can establish reversible covalent bonds
with oxygen/nitrogen nucleophiles, that are sensitive to pH, ROS
carbohydrates and glutathione (GSH) levels.[12] Due to this stimuli
responsiveness, BAs have been used in the construction of
prodrugs, sensors, nanoparticles, polymers, among other
functional materials. However, their use as a bioconjugation
handle for the design of dynamic bioconjugates has been
considerably more troublesome, mainly due to the poor stability
and unfavorable formation of boronates esters in dilute aqueous
conditions.[13-15] Some of these limitations were circumvented with
the discovery that 2-formyl (or acetyl)-phenyl boronic acids
(2FPBA or 2APBA) could rapidly promote the dynamic
functionalization of peptides and proteins, by forming B-N
stabilized imines (iminoboronates) with exposed lysine and N-
terminal residues (Scheme 1A).[12,16,17]
Introduction
Bioconjugation emerged in recent years as an invaluable tool to
generate bioconjugates in which biomolecules like peptides and
proteins are endowed with the properties of the attached
entities.[1–3] The assembly of bioconjugates by direct chemical
installation of the molecular payload onto the peptide chain is
often impossible due to the lack of suitable chemical functions that
may be targeted in the bioconjugation process, therefore the use
of chemical linkers featuring bioconjugation warheads are an
invaluable tool to prepare these type of hybrid materials.[4–6]
Extensive research in the engineering of chemical linkers
revealed that these structures are not just molecular spacers
required to connect both functions,[7] but perhaps as importantly,
are instrumental structures to control the conjugate’s
homogeneity and stimuli-responsiveness.[8,9] Therefore, many
efforts have been made to devise synthetic methods to equip
Scheme 1. Proposed strategy to site-selectively install an iminoboronate in a
peptide chain using a Boron-Hot-Spots (BHS) strategy. 2-formyl-phenyl boronic
acids (2FPBA).
1
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This reactivity triggered much interest on the use of this reaction
in the functionalization of biomolecules, bacterial and eukaryotic
cell walls, and in the assembly of functional materials.[18-23]
However, the impossibility to control the site of the iminoboronate
formation and the poor stability of these constructs, limited the use
mode. Hence, heterocycles 2 and 3 were prepared to probe the
important functional groups for the coordination. As shown in
Scheme 2C, while the N-methylation had no impact over the BA
coordination, the O-methylation clearly impaired the reaction.
These results suggest that the hydroxyl group is a requisite to the
of this technology in the assembly of homogeneous bioconjugates. formation of the complex. Therefore, we anticipated that the
Therefore, we conceived that a possible strategy to overcome the
lack of stability and site selectivity in the iminoboronate formation,
would be to design a boron ligand (boron hot spot - BHS) that,
upon installation at a precise site of the peptide chain, would direct
the 2FPBA carbonyl function towards a specific amino group of
the peptide chain (Scheme 1B). Considering this design, we
initiated a study to identify a structure that could yield a fast and
reversible BA ligation to integrate in the structure of the BHS.
insertion of an amide function at position 4 of the heterocycle
could also improve the reactivity, by increasing the OH acidity,
while offering a site to add the chemical handle needed to install
the BHS onto the peptide chain.
Based on this rationale, the model 4-benzylamide-3HQ 4 was
prepared and tested in the reaction with PBA. Unexpectedly, this
reaction generated a complex reaction mixture, indicating that the
insertion of the 4-carboxamido substituent sterically perturbed the
BA coordination. However, by increasing the electron withdrawing
nature of the boron center and the acidity of the 3-hydroxyl group
in 5, the reaction efficiency was restored (Scheme 3).
Results and Discussion
The 3-hydroxyquinolin-2(1H)-one (3HQ) is an important
heterocycle that reversibly binds enzyme metal co-factors under
physiological conditions (Scheme 1A).[24] Inspired by this
coordination mode, we reasoned that 3HQ could also be a
valuable boron ligand to incorporate in the BHS because this core
exhibits an OH group that can establish a B-O bond and stabilize
the charged boronate species by coordinating its carbonyl group
to the boron atom.
100
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40
20
0
150
200
250
300
m/z
Scheme 3. Evolution of the 3HQ scaffold and model reactions with BAs with
relative ESI-MS spectra. See SI (S10, S11) for full ESI-MS spectra.
Scheme 2. Known reactivity of 3HQs with divalent metals and expected BA
coordination mode (A). ESI-MS spectrum of the model reaction of 3HQ 1 and
phenyl BA (PBA) (B). Reaction of methylated 3HQ derivatives 2 and 3 with PBA
(C). See also SI (S1-9) for full ESI-MS spectra and additional experiments with
BAs.
Considering the structure of the 4-carboxamido 3HQs 4 and 5, we
realized that the introduction of the amide function generated an
additional site for potential BA coordination. Therefore, 3HQ 6
featuring a secondary amide was prepared to study if the
boronate species was preferentially formed through an O-B-O or
a N-B-O coordination. As shown in Scheme 3, the 3HQ 6
performed as well as 5 with a primary amide, which suggests that
in these 4-carboxamido 3HQs, the O,O-bidentate ligation
continues to be the preferential coordination site. Next, a glycine
residue was added to the 3HQ to act as a chemical spacer to
facilitate the BA coordination. As shown in Scheme 4, this
Based on these promising features, 3HQ 1 was prepared[25] and
reacted with 10 equiv. of phenyl BA (PBA) in ammonium acetate
solution at pH 7. Under these conditions, the 3HQ effectively
yielded the boronate species (248.1 m/z) in a process that was
completed within 5 min. at 25 ºC (Scheme 2B). After validating the
3HQ 1 scaffold as a BA ligand, we addressed the modification of
the heterocycle, envisioning the installation on a peptide chain.
The first step in this direction was to confirm the 3HQ-BA binding
2
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modification was quite successful and the 4-Gly-3HQ 7 not only
reacted with 4-CF3-PBA, but also, with other BAs exhibiting
distinct electronic profiles (Scheme 4). Based on these results,
the core 7 was submitted to the alizarin competitive binding
fluorescence assay,[26] which confirmed its affinity for PBA (Ka =
699 ± 2 M-1; see SI, S17-22).[27]
the assembly generates a tetrahedral sp3 boronate species (11B-
NMR 1 ppm) with a OH group (1H-NMR 10 ppm), and a strongly
deshielded nitrogen atom (15N-NMR 529 ppm), when compared
with the imine obtained in the reaction with benzaldehyde (15N-
NMR 364 ppm). These results were further corroborated by DFT
calculations performed on this system. The reaction between the
BHS-Cys 9 and 2FPBA generates the imine in a process that
corresponds to an energy gain (G) of -8 Kcal/mol (SI). The
formation of the B-N bond further stabilized the assembly by -4.2
Kcal/mol and supports the formation of the iminoboronate as the
likely driving force in the reaction with the 2FPBA (see SI for DFT
details).
100
80
60
40
20
0
300
400
m/z
500
Scheme 4. Model reaction of 3HQ Glycine derivative 7 with different boronic
acids. See SI (S12-16) for full ESI-MS spectra.
Considering the results obtained with the 4-Gly-3HQ core, we
next addressed the insertion of a chemical handle to install the
BHS on the peptide chain. With this objective in mind, the N-
terminal cysteine (Cys) residue was selected as the
functionalization site, as it presents a sulfhydryl side chain that
can be site-selectively modified, and an amino group that can be
targeted to generate the iminoboronate. Hence, the 4-Gly-3HQ
core was equipped with a maleimide handle (compound 8,
Scheme 5) and used in the conjugation with a Cys methyl ester.
The reaction afforded the thiosuccinimide adduct 9 quantitatively,
which was then used to assess the ability of the BHS to promote
the iminoboronate formation on the adjacent free amine. The
incubation of the BHS-Cys 9 with 2FPBA in ammonium acetate
solution (20 mM, pH 7.0) afforded the desired imine within 2h at
37 °C with a Ka of 58128 ±2 M-1 (See SI S24-26). This result was
further supported by the reaction of BHS-N-acetyl Cys with
2FPBA, in which the presence of N-acetyl group completely
inhibited the assembly (see SI, S37). Next, we questioned the
importance of the B-N bond formation in the assembly, by testing
the 3- and 4-FPBA isomers that cannot generate iminoboronates.
These isomers yielded only traces of the expected products upon
reaction with BHS-Cys, suggesting that the imine formation is not
sufficient to secure the assembly (see SI, S31-34).
Scheme 5. Model reaction of the BHS-CysOMe adduct and 2FPBA along with
DFT-derived structures for the generated complex. For NMR details see SI
(S27-29)
After confirming the BHS-promoted iminoboronate formation on
the N-terminal Cys, we studied its compatibility with neighboring
amino acids that could potentially disrupt the reaction. Importantly,
as shown in Scheme 6, the BHS succeeded in promoting the
assembly with all dipeptides tested. When using the N-Cys-Lys
dipeptide, MS/MS experiments performed on the product suggest
that only the N-terminal Cys iminoboronate was formed (13) (see
SI, S48 and S49).
Encouraged by these results, the next step was to study more
complex peptides, such as c-Ovalbumin. This peptide, which
features both a N-terminal Cys and a Lys residue, was
successfully functionalized with the BHS (compound 15, Scheme
7). The subsequent reaction of c-Ovalbumin with 10 equiv. of
2FPBA gave exclusively the iminoboronate at the N-terminal
residue (compound 16). Interestingly, when a large excess of
2FPBA was used, both N-terminal and Lys’ ε-amino groups were
modified (derivative 17), though upon dialysis, only the tighter-
binding BSH-iminoboronate remained installed on the peptide
chain (Scheme 7).
In order to probe the structure of the construct resulting from this
assemblage, we studied by NMR the reaction of 2FPBA and
benzaldehyde with 9, prepared with a 15N-enriched cysteine
(Scheme 5). The 11B and 1H-NMR of the reaction of 2FPBA with
9 strongly suggests the formation of an iminoboronate, because
3
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The Cys-RGD peptide, exhibiting an N-terminal and an in-chain
Cys residues, was used to install 2 BHS units, but only the N-
terminal modification promoted the assembly with 2FPBA. Finally,
the anti-nucleolin F3 peptide, was used to establish if a more
complex structure could promote the reaction between the BHS
and an in-chain amino group. However, this 32-amino-acid
peptide modified with the BHS at the C-terminal residue, was
unable to form a BHS-stabilized iminoboronate with 2FPBA.
Altogether, these results clearly demonstrate that the installation
of the BHS at the N-terminal Cys is instrumental to promote the
site-selective iminoboronate formation.
hydrolysis of 14 after 8h. These results confirmed that, although
BHS-iminoboronate display an improved stability when compared
with iminoboronates formed between 2FPBA and primary amino
groups, it still offers a controlled mechanism of reversibility.
Once the site-selective iminoboronate formation was established
and the cleavable nature of this linkage was confirmed, the BHS-
technology was used to validate the 67 KDa laminine receptor
(67LR) as a target to selectively deliver cargo to cancer cells.
100
80
60
40
20
0
700
750
800
850
900
m/z
100
80
60
40
20
0
700
750
800
850
900
m/z
100
80
60
40
20
0
Scheme 7. Reaction of BHS-modified c-Ovalbumin fragment 15 with different
amounts of 2FPBA and relative ESI-MS spectra of the resulting reaction
mixtures. See SI for full spectra (S55-60)
The 67LR is a well characterized cell-surface receptor that is over
expressed in cancer cells.[29,30] This receptor displays high affinity
for laminin and is involved in the cell adhesion to the basement
membrane and in the metastasis of cancer cells. Therefore, to
study the ability of the 67LR protein to promote the delivery of
cargo to cancer cells, we prepared fluorescent conjugates with
the laminin fragment CDPGYIGSR containing the YIGSR motif,
which is the minimal sequence that is recognized by the
receptor.[29,30] As shown in Scheme 8, the peptide was selectively
600
700
800
m/z
Scheme 6. Reaction of BHS-modified N-terminal Cys dipeptides with 2FPBA
and ESI-MS spectrum of product 14. See SI for full spectra (S38-47).
A key feature of iminoboronates is their inherent dynamic nature.
Therefore, the stability and reversibility profiles of the
iminoboronates generated with the BHS technology was studied.
Using 14 as a model BHS-iminoboronate, the construct stability
was monitored in ammonium acetate solution (pH 7.0). Under
these conditions, the BHS-iminoboronate 14 maintained the
structural integrity over 4 weeks. This stability profile was further
confirmed by incubating 14 with 10% BSA and by lowering the
buffer pH to 4.5. In this case, the BHS-iminoboronate was stable
over a 24h period. Differently, the BHS-iminoboronate 14 was
sensitive to glutathione (GSH), which promoted a significant
functionalized with an NBD probe using
a
cleavable
iminoboronate linker (18, 19) and a more stable thiosuccinimide
linkage (20). Once prepared, both conjugates were incubated with
67LR overexpressing HT29 cancer cells[31] for 1h and then, after
washing with fresh medium, the cells were controlled by confocal
microscopy. As shown in Figure 1, the conjugate 20 was unable
to promote the labeling of the cells, while the iminoboronate
derived conjugate, effectively promoted the probe internalization.
The non-targeted BA-NBD probe (18) that may undergo a BA
assisted permeation,[32] was also able to label the cells, though
4
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with considerable less efficacy than when conjugated with to the
laminin fragment (Scheme 8D). Although is not yet clear the
mechanism by which the probe is internalized, the cleavable
conjugate 20 promotes a more efficient labeling and supports the
targeting of this receptor to deliver cargo to cancer cells.
Ovalbumin. NMR and DFT calculations performed on this system
reveled the importance of the B-N bond formation in the
stabilization of the construct which was shown stable in buffers at
pH 7 and 4.5 or in the presence of BSA, though reversible in the
presence of glutathione. The selective formation of
iminoboronates and their controlled reversibility were applied in
the construction of fluorescent cleavable homogeneous
conjugates that highlighted the 67LR as a promising target to
deliver cargo to cancer cells.
Acknowledgements
The authors acknowledge Fundação para a Ciência e Tecnologia
(PTDC/QEQ-QOR/3644/2014;
PTDC/QUI-QOR/29967/2017;
100
80
60
40
20
UID/DTP/04138/2013, SAICTPAC/0019/2015 (iMed.ULisboa),
UID/QUI/00100/2013 (L.F.V.), PPBI-POCI-01-0145-FEDER-
022122). FF thanks FCT (IF/00386/2015) and the European
Structural
&
Investment Funds through the COMPETE
Programme and FCT (SAICTPAC/0019/2015). F.C. thanks the
Ministerio de Ciencia, Innovación y Universidades and the
Agencia Estatal de Investigación (project RTI2018-099592-B-
C21).
0
Irreversible NBD-FBBA Reversible
conjugate
probe
conjugate
Scheme 8. Fluorescent NBD-FBBA probe 18. Reversible Laminin-NBD
conjugate based on the BHS-iminoboronate platform 19. Covalent Laminin-
NBD maleimide conjugate 20. Normalized fluorescence observed in HT29 cells
after incubation with the reported probes (C). 19 shows a 6.8 ± 0.93 fold
increase in internalization relative to 20, and 3.42 ± 0.93 fold increase relative
to the NBD-FBBA probe 18. See SI for full spectra and data treatment.
Keywords: Iminoboronate • Bioconjugation • Boronic Acids •
Laminin • 67LR
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Entry for the Table of Contents
Boron Hot Spot: Site-selective installation of iminoboronates on N-terminal cysteines using a 3-hydroxyquinolin-2(1H)-one scaffold
as a directing unit and the use of this Boron Hot Spot in the synthesis of a cleavable fluorescent laminin conjugate targeting the 67LR
receptor in HT29 cancer cells.
Institute and/or researcher Twitter usernames: @GoisLab
7
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