Boronic Acid Pairs for Sequential Bioconjugation

Article abstract of DOI:10.1021/acs.orglett.1c01624

Boronic acids can play diverse roles when applied in biological environments, and employing boronic acid structures in tandem could provide new tools for multifunctional probes. This Letter describes a pair of boronic acid functional groups, 2-nitro-arylboronic acid (NAB) and (E)-alkenylboronic acid (EAB), that enable sequential cross-coupling through stepwise nickel- and copper-catalyzed processes. The selective coupling of NAB groups enables the preparation of stapled peptides, protein-protein conjugates, and other bioconjugates.

Full text of DOI:10.1021/acs.orglett.1c01624

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Letter
Boronic Acid Pairs for Sequential Bioconjugation
Mary K. Miller,† Michael J. Swierczynski,† Yuxuan Ding, and Zachary T. Ball*
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ABSTRACT: Boronic acids can play diverse roles when applied in
biological environments, and employing boronic acid structures in
tandem could provide new tools for multifunctional probes. This Letter
describes a pair of boronic acid functional groups, 2-nitro-arylboronic
acid (NAB) and (E)-alkenylboronic acid (EAB), that enable sequential
cross-coupling through stepwise nickel- and copper-catalyzed processes.
The selective coupling of NAB groups enables the preparation of stapled
peptides, protein−protein conjugates, and other bioconjugates.
hemical biology and biotechnology increasingly demand
profound effect on the reaction efficiency, kinetics, and
chemoselectivity. For instance, catalytic cysteine arylation
with arylboronic acids containing certain electron-withdrawing
ortho substituents occurs efficiently within minutes,33 whereas
simple arylboronic acids without ortho substitution afford no
product under identical conditions.33 In contrast, such simple
arylboronic acid reagents do readily participate in backbone
N−H arylation catalyzed by Cu2+,28,29 but the reactions are
sensitive to steric demand, and ortho substitution of any kind
is not tolerated. We therefore wondered if these methods with
significantly different structure−reactivity frameworks could
form the basis for sequential coupling partners for the
construction of complex bioconjugate architectures. Previous
efforts33 identified arylboronic acids containing certain
electron-withdrawing ortho substituents, such as 2-nitro-
arylboronic acids (NABs), as displaying especially fast
Chan−Lam product formation, indicating that they might
serve as effective first coupling partners in a sequential coupling
strategy (Figure 1).
C
complex, polyfunctional biopolymers for diverse “smart”
materials and molecules. Concepts such as theranostics1 rely
on multifunctional molecules with diverse, complementary,
and often orthogonal reactivity. As a result, diverse
biorthogonal chemistries facilitate the construction of complex
bioconjugates by sequential couplings.
Organoborane reagents are useful chemical tools with
remarkably diverse applications in chemical biology.2−6 Their
use as organometallic precursors in catalytic cross-coupling
reactions is widely appreciated.7 Organoboranes are also
utilized in remarkably diverse applications in biological
chemistry: recognition of poly hydroxy motifs,8−10 enzyme
inhibition,11,12 reactive oxygen species (ROS) sensing,13
bioconjugation4,14,15 (including facilitation of oxime forma-
tion16,17), and other concepts.18,19 These diverse possibilities
raise questions about how multiple boronic acid functional
groups might be used in tandem, playing complementary roles
in multifunctional reagents. This goal requires boronic acid
reagents that are mutually compatible so that the selective
activation of one organoboronate group is possible while a
second organoboronate remains inert. For small-molecule
synthesis in an organic solvent, the development of specific
boronic acid derivatives has enabled selective, sequential
coupling reactions by masking boronic acid reactivity.20−27
We envisioned an alternative strategy of boronic acids with
inherently differential reactivity in coupling reactions, over-
coming the limited hydrolytic stability of boronate esters.
These concepts were motivated by our own studies of
transition-metal-catalyzed bioconjugation with boronic acid
substrates, promoted by copper,28−31 rhodium,32 or nickel
salts.33,34
The second sequential coupling would require boronic acid
structures were are stable under the initial conditions. In
addition to cross-coupling, boronic acids are prone to a variety
of side reactions in water and under air, which can also be
catalyzed by transition metals, including protodeborylation,
oxidative hydroxylation, and C−C homocoupling.30,33 Appro-
priate “second” boronic acids would need to avoid these
Received: May 12, 2021
Published: July 2, 2021
In the course of these efforts, we found that the precise
substitution patterns of the boronic acid reagent had a
https://doi.org/10.1021/acs.orglett.1c01624
© 2021 American Chemical Society
Org. Lett. 2021, 23, 5334−5338
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analogous to 3. Conversely, there were profound differences
in the reactivity with nickel(II) catalysis (Figure 2, red).
Whereas 2-acetyl- (2b) and 2-nitro-phenylboronic (NAB, 2a)
are consumed quickly under these conditions, simple aryl-
(2h−2j) and (E)-alkenylboronic acids (EABs) (2k) were
substantially stable under these conditions, showing <1%
conversion after 30 min. These results implied that boronic
acid reagents reactive with copper could be utilized
sequentially after a nickel-catalyzed process.
In a more stringent and relevant test, we next examined
mixtures of putative “reactive” and “unreactive” boronic acids
in the nickel(II)-mediated arylation of N-acetylcysteine. The
stability of the “unreactive” boronic acids (2k−m) was
monitored by 1H NMR analysis, with dimethylformamide
(DMF) as an internal standard (Figure 3a). Again, we found
that an arylboronic acid (Figure 3b) and alkenylboronic acid
Figure 1. Schematic depiction of the sequential cross-coupling of
NAB and EAB boronic acid pairs.
decomposition pathways. We compared the consumption of a
variety of boronic acids under catalytic conditions. Boronic
acids 2a−k were subjected to a reaction with either copper(II)
or nickel(II) and a coupling partner, N-acetylcysteine (Figure
2). No suitably stable boronic acids were discovered under
copper(II) catalysis conditions; all boronic acid reagents were
consumed to a significant extent (Figure 2, blue), even for
compounds (e.g., 2f, 2g, 2i, 2j) for which previous studies33
demonstrated no productive coupling to give products
Figure 2. Conversion of boronic acid reagents 2a−k in the presence
of Ni2+ and Cu2+ salts. The total conversion to products 3 and 4 is
shown for Ni2+ (red) and Cu2+ (blue). Conditions: boronic acid (2
mM), N-acetylcysteine (0.2 mM), and M(OAc)2 (1 mM) in aqueous
N-methylmorpholine (NMM) buffer (10 mM).
Figure 3. (a) Stability of aryl- and alkenyl-boronic acid reagents in the
nickel(II)-mediated reaction. (b,c) NMR monitoring of the stability
of “bystander” boronic acids 2l and 2m in an NAB-type Ni2+ coupling
with boronic acid 2a.
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(Figure 3c) showed minimal conversion after 30 min. An
alkenylboronic acid reagent that could be utilized to design a
diboronic acid scaffold was also tested (Figure 3d) and was
likewise found to survive the reaction conditions.
We next applied this sequential coupling concept to the
preparation of a stapled peptide with a nonsymmetrical linkage
of defined orientation (Figure 4). Stapled peptides can confer
Cyclization of the linear intermediate 6 was then accomplished
by copper-catalyzed N−H alkenylation, providing the cyclic 7
(crude reaction analysis: Figure 4c). This result was
particularly enlightening, as it demonstrated the success of
aqueous copper-mediated N−H alkenylation where the
intramolecular nature of the cyclization necessitates 1:1
stoichiometry.
Sequential reactivity of diboronic acid reagents with protein
substrates could also be realized. We have previously shown
that the cysteine arylation chemistry could be used to obtain
protein−polymer conjugates,35 and we therefore wondered if
sequential conjugations could be used to create large
biopolymer structures. Peptide and protein heterodimers find
use in vaccine development and targeted drug delivery.36,37
Both cysteine and pyroglutamate-histidine tags38 are readily
incorporated into the protein substrate from E. coli expression
systems. To explore these possibilities, a trifunctional reagent
(2o) containing a desthiobiotin affinity purification handle was
synthesized (Scheme 1). A model protein, T4 lysozyme
V131C (T4L), bearing a single cysteine residue, reacted with
2o under nickel conditions to afford an alkenylboronic-acid-
labeled protein (Figure 5).
Scheme 1. Synthesis of Di-boronic Acid Reagent 2o
Figure 4. Peptide stapling with sequential boronic acid coupling. (a)
Synthetic scheme for the preparation of the peptide staple 7.
Conditions: cysteine modification: 5 (0.2 mM), 2n (2 mM), and
Ni(OAc)2 (1 mM) in NMM buffer (10 mM, pH 7.5), TCEP (1 mM),
37 °C, 5 h; pEH alkenylation: 6 (0.2 mM) and Cu(OAc)2 (1 mM) in
NMM buffer pH 7.5 for 2 h. (b) LCMS chromatogram of 6. Insert:
mass chromatogram of 6. (c) LCMS of the crude reaction mixture for
the conversion of 6 to 7. Insert: mass chromatogram of the new peak
7.
This boronic-acid-labeled T4L protein could then be directly
coupled to afford protein−peptide conjugates. Using an excess
of pEH-containing peptides leuprolide (LE, 13, pEHWSY-
LLRP) or LHRH (LH, 14, pEHWSYGLRPG) in the presence
of copper(II), efficient conjugation was achieved (Figure 5b).
This observation demonstrates the robust nature of individual
boronic acid labels to survive nickel-mediated conjugation,
purification, and routine protein manipulations and handling.
It also highlights the remarkable efficiency of this process, in
which the complex protein−boronic acid acts as the limiting
reagent in an intermolecular coupling process.
Protein−protein conjugates were also constructed using this
approach. When the T4L−boronic acid was treated with excess
green fluorescent protein (GFP, 5 equiv) bearing a
pyroglutamate-histidine tag, substantial conversion (46% by
gel densitometry) was achieved in 30 min under copper-
functional and stability advantages in vivo, and we have
previously demonstrated the stability of these arylation
linkages in serum and proteolytic stability assays.33 A peptide
with a cysteine site for S−H coupling with an NAB and a
“second” pyroglutamate−histidine (pEH) dipeptide sequence
for N−H coupling with EAB was prepared and tested (5). The
diboronic acid reagent 2n was prepared with both NAB and
EAB moieties. Nickel-catalyzed coupling of the peptide 5 with
the diboronic acid reagent 2n afforded the S-arylation product
6. Matrix-assisted laser desorption/ionization tandem mass
spectrometry (MALDI-MS/MS) confirmed cysteine as the site
of reactivity, and the material could be purified by reverse-
phase high-performance liquid chromatography (RP-HPLC).
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bearing green fluorescent protein (sfGFP) with a pyrogluta-
mate-histidine GFP (68% conversion, Figure 5e).
In conclusion, many simple boronic acids (including EAB)
are stable and inert to aqueous nickel-catalyzed S−H arylation
with NAB moieties. This observation allows sequential
aqueous coupling reactions with peptides and proteins. The
approach allows the simple two-step construction of diverse
bioconjugates and indicates that NAB coupling reactions may
be possible in the presence of a wide range of functional
boronic acid groups, permitting the creative implementation of
boronic-acid-based applications in protein chemistry.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c01624.
General experimental details, methods for peptide and
protein modification and product analysis, and charac-
terization of new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Zachary T. Ball − Department of Chemistry, Rice University,
Houston, Texas 77005, United States; orcid.org/0000-
0002-8681-0789; Email: zb1@rice.edu
Authors
Mary K. Miller − Department of Chemistry, Rice University,
Houston, Texas 77005, United States
Michael J. Swierczynski − Department of Chemistry, Rice
University, Houston, Texas 77005, United States
Yuxuan Ding − Department of Chemistry, Rice University,
Houston, Texas 77005, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c01624
Figure 5. (a) Overview of the conjugation of T4L with
pyroglutamate−histidine-tagged structures using diboronic acid 2o.
Conditions: (1) T4L (20 μM), 2o (200 μM), tris(2-carboxyethyl)-
phosphine (TCEP) (0.2 mM), Ni(OAc)2 (0.4 mM), and 6,6′-
dimethyl-2,2′-bipyridine (L1) (0.4 mM) in NMM buffer (50 mM, pH
7.5) at 37 °C for 30 min. (2) Boronic-acid-modified T4L (10 μM),
pGlu-His-peptide/protein (50 μM), and Cu(OAc)2 (0.25 mM) in
NMM buffer (50 mM, 150 mM NaCl, pH 7.5) at rt for 18 h. (b−e)
Gel images for the conjugation of T4L-boronic acid with (b) pGlu-
His peptides and (c) pGlu-His GFP. (d) Conjugation of T4L-boronic
acid to pGlu-His-GFP with excess T4L. (e) Conjugation of sfGFP-
boronic acid with pGlu-His-GFP. Conversion was measured by gel
densitometry (ImageJ) relative to the starting material and was
uncalibrated.
Author Contributions
†M.K.M. and M.J.S. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge support from the Robert A. Welch
Foundation Research Grant C-1680 (Z.T.B.) and the National
Science Foundation under grant number CHE-1904865. We
thank Raphael Hofmann and Jeffrey Bode (ETH Zurich) for
the T4L(V131C) and sfGFP(S14C).
catalyzed conditions (Figure 5c). The anticipated conjugate
was further confirmed by MALDI-MS (Figure S17). After 18
h, 93% conversion was observed without significant side
products. Conjugation was also performed with tagged GFP as
the limiting reagent. In this case, 81% conversion was observed
within 2 h (Figure 5d). A minor product was also observed,
with a molecular weight consistent with homocoupled T4L. It
is interesting to note that this minor homocoupled protein
product does not appear when tagged GFP is used in excess
(Figure 5c). Nonetheless, the reaction is compatible with
either coupling partner in excess. This protein−protein
conjugation protocol was further utilized to link a cysteine-
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