Boronic Acid Mediated Coupling of Catechols and N-Hydroxylamines: A Bioorthogonal Reaction to Label Peptides

Article abstract of DOI:10.1021/acs.orglett.7b01198

An irreversible, three-component assembly with 2-formylphenylboronic acid, catechol, and N-hydroxylamines was achieved in aqueous media. The boronate ester product was formed with substituted catechols including l-DOPA. Assembly was found to be orthogonal to common biological functional groups and both copper(I)-catalyzed alkyne-azide cycloaddition and aminoether/carbonyl condensations. Boronate ester formation and aminoether condensation were achieved in one pot with a hexameric peptide.

Full text of DOI:10.1021/acs.orglett.7b01198

Letter
pubs.acs.org/OrgLett
Boronic Acid Mediated Coupling of Catechols and
N‑Hydroxylamines: A Bioorthogonal Reaction to Label Peptides
Margaret K. Meadows,†,§ Emily K. Roesner,† Vincent M. Lynch,† Tony D. James,*,‡ and Eric V. Anslyn*,†
†Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
‡Department of Chemistry, University of Bath, Bath BA2 7AY, U.K.
S
* Supporting Information
ABSTRACT: An irreversible, three-component assembly with
2-formylphenylboronic acid, catechol, and N-hydroxylamines
was achieved in aqueous media. The boronate ester product
was formed with substituted catechols including L-DOPA.
Assembly was found to be orthogonal to common biological
functional groups and both copper(I)-catalyzed alkyne−azide
cycloaddition and aminoether/carbonyl condensations. Boronate ester formation and aminoether condensation were achieved in
one pot with a hexameric peptide.
Scheme 1. Boronate Ester Assembly As Published by James
et al.21
here are currently a wide variety of coupling reactions
1−5
Tbeing explored for applications in materials chemistry,
organometallic catalysis,6−10 and chemical biology.11,12 When
these methods proceed under mild conditions, are high
yielding, and do not generate noxious byproducts, they have
been termed “click” reactions.13 Those that do not involve or
react with common biological functional groups act in a
bioorthogonal fashion.14,15 Developing bioorthogonal reactions
that label peptides necessarily entails the incorporation of an
unnatural amino acid or chemical modification of one of the
common 20 amino acids.16−18 Further, the popular coupling
methods, except for those that are metal-catalyzed, such as the
Cu(I)-catalyzed Sharpless−Huisgen cycloaddition,19 involve
reactions that commence immediately upon mixing of the
components. Thus, overall common bioorthogonal reactions
cannot be easily controlled in a spatial or temporal manner, and
chemical synthesis is required prior to their utilization in vitro
or in vivo.
together catechol-functionalized biological compounds, such as
the amino acid L-DOPA, with virtually any moiety that carried a
hydroxylamine. Although L-DOPA is not one of the common
20 amino acids, it has been incorporated into both peptides and
proteins24 and therefore provides a readily available coupling
partner in a semibioorthogonal fashion.
Another avenue we were interested in exploring was the
difference in reactivity between N-functionalized hydroxyl-
amines (RNHOH) and O-functionalized hydroxylamines,
hereafter referred to as aminoethers (RONH2). Aminoethers
have been previously reported in bioorthogonal couplings25 but
have different reactivity than hydroxylamines; we hypothesized
that these two isomeric functional groups could potentially be
paired together while retaining their orthogonal reactivity. We
demonstrate herein that the boronic acid-triggered coupling of
catechols and hydroxylamines indeed works well in water, has
broad catechol substrate scope, can be used to fluorescently
label L-DOPA-containing peptides in vitro, and is compatible
with couplings involving aminoethers.
While not bioorthogonal, we have recently described a
method for a chemically triggered reversible click reaction that
yields the original coupling partners without modification.20 In
our continued pursuit of methods to trigger chemical coupling
and decoupling upon demand, we explored the use of 2-
formylphenylboronic acid (1) as a reagent to combine two
otherwise unreactive components. This work was inspired by
the recent Bull and James report that 1 can be used to form an
assembly that acts as a 1H NMR chiral shift reagent to measure
the enantiomeric excess values of chiral hydroxylamines when
in the presence of BINOL in aprotic, organic media (Scheme
1).21
Phenylboronic acids have been briefly studied in bioorthog-
onal labeling via boronate ester formation with dopamine22 and
Pd-catalyzed Suzuki couplings.15,23 Because boronic acids
generally have even greater affinities to catechols than alkyl
diols, and because they bind catechols relatively well in water,
we realized that the method had the potential to “click”
Previous assemblies with 1 and standard amines have been
shown to form reversibly and are capable of exchanging both
diol and amine under equilibrium conditions.26,27 These
assemblies were also shown to be stabilized by solvent insertion
in protic media (2).28 We hypothesized that the hydroxyl group
Received: May 2, 2017
Published: June 5, 2017
© 2017 American Chemical Society
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Letter
of the hydroxylamine would occupy the same geometric
position as the oxygen of an inserted solvent and increase the
stability of these complexes in water (Scheme 2). Since we
Scheme 3. Substrate Scope of the Three-Component
Assembly with N-Hydroxylamine in Water
Scheme 2. (a) Formation of a Three-Component Assembly
with Amine and Solvent insertion (2); (b) N-Hydroxylamine
(3) in Water with the Stabilizing Oxygen Atoms Highlighted
in Red
began work on this system, other groups have similarly found
that these complexes are water stable.29,30 We found that the
assembly with catechol (3) formed in water under a variety of
conditions: neutral water, pH 7 phosphate buffer, pH 5.5
acetate buffer, and pH 9 bicarbonate buffer. Although the
addition of Na2CO3 or a similar base is not necessary, it does
speed assembly formation considerably. A crystal of 3 grown
from slow evaporation of CH2Cl2 shows the coordination of
the oxygen from the hydroxylamine to the boron, as predicted
(Figure 1).
were unable to out compete assembly formation with water,
yielding 4.
Once compound 4 forms, it does not equilibrate with any
added catechols to give a different assembly. This gives insight
into the sequence of steps. To achieve the desired product, the
catechol must condense with the boronic acid prior to the
addition of the hydroxylamine into the aldehyde. To support
this hypothesis, we examined the order of addition of the three
components to this assembly. When 1 and catechol or t-
BuNHOH and catechol are combined prior to the addition of
the third component, the assembly with catechol is formed
(Schemes 4a and 4b). When 1 and t-BuNHOH are combined,
the condensation occurs prior to catechol addition and is
unreactive to the catechol (Scheme 4c).
Boronic acid complexes are well-known for their ability to
reversibly bind sugars;31 however, the hydroxylamine assembly
using 1 did not show the same affinity. If fructose or glucose
was combined with 1 and t-BuNHOH, the only product was 4.
We hypothesize that this is due to the low binding constants of
boronic acids with sugars without the assistance of an o-
aminomethyl group to increase binding,32 and once 4 is
formed, it will not then exchange with an added diol.
With our desire to transition this assembly to a
biocompatible platform, we tested the robustness of our
complex to the presence of common biological functional
groups with no interferences found (Scheme 5). Additionally,
the assembly can be run in parallel with other common
bioorthogonal coupling reactions (Scheme 6); CuAAC
chemistry can be used to label azidolysine residues, and
aldehyde and ketone residues can be labeled using aminoethers.
The latter is of particular interest due to the similarity between
Figure 1. Crystal structure of 3, grown from CH2Cl2.
Additionally, we found that assembly 3 is not nearly as
reversible as the previously reported amine assembly (2). Once
formed and isolated, 3 is stable to neutral methanol/water
1
mixtures for more than 72 h with no change by H NMR
spectroscopy. Although the catechol is slowly replaced with
hydroxyl groups under strongly basic conditions (pH 13), the
hydroxylamine addition is irreversible and stable to acid (pH
1), base (pH 13), and heat (50 °C) over 24 h.
Varying the substitution on the catechol provided a range of
assemblies; we were also able to complex α-hydroxy acids and
cis-1,2-cyclopentanediol (Scheme 3). The determining factors
in assembly appear to be solubility of the diol and strength of
its binding to 1. Those that were less soluble or bound poorly
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Scheme 4. Order of Addition Studies Showing (a)
Combination of t-BuNHOH and Catechol Prior to Addition
of 1, (b) Combination of 1 and Catechol Prior to Addition
of t-BuNHOH, and (c) Combination of 1 and t-BuNHOH
Prior to Addition of Catechol
Scheme 7. Dual, One-Pot Labeling of Peptide 14
In summary, we extended the utility of a three-component
assembly to the purpose of labeling peptides containing L-
DOPA. We further expanded the substrate scope as well as
uncovered the sequence of formation in aqueous conditions
and irreversibility of the coupling. The three-component
assembly discussed herein is thus a semi-bioorthogonal
coupling reaction, showing no cross-reactivity with other
common “click” reactions when simultaneously performed in
the same pot.
Scheme 5. Assembly Formation in the Presence of Various
Functional Groups
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b01198.
Synthesis and characterization of compounds 3−15 and
reversibility, order of addition, and bioorthogonal studies
(PDF)
Scheme 6. Assembly Formation Alongside (a) CuAAC and
(b) Aminoether Condensation
Single-crystal X-ray data for 3 (CIF)
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: t.d.james@bath.ac.uk.
*E-mail: anslyn@austin.utexas.edu.
Present Address
§(M.K.M.) Department of Chemistry, Northwestern Univer-
sity, Evanston, IL, 60208.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Steve Sorey (UT Austin) for his help with the 11B
NMR studies. We thank Dr. Brette Chapin (University of
Durham) for her help with manuscript revisions and Prof.
hydroxylamine and aminoether structures, as discussed above.
In order to further explore this opportunity for orthogonal
reactivity, we moved to a peptide scaffold.
Jeffrey Bode and Yi-Chung Dzeng (ETH Zurich) for providing
̈
Peptide 14 was synthesized using an automated solid-phase
synthesis. The benzaldehyde moiety was incorporated to allow
for conjugation of a commercially available aminoether
fluorescent dye, CF488A. PEG ethers have been used in
pharmaceuticals to increase hydrophilicity and label proteins to
increase water solubility and stability.33−36 The dual-labeling of
14 was accomplished in a one-pot fashion (Scheme 7); 14,
CF488A, catalytic aniline, and the PEGylated hydroxylamine
were dissolved in water at room temperature. After 1 h, 1 was
added. The dual-labeled peptide 15 was isolated after 4 h and
characterized by fluorescence and 11B NMR spectroscopies and
MS.
the PEG hydroxyl amine. Financial support from the NSF
(CHE-1212971) and the Welch Foundation (F-0046) is
gratefully acknowledged.
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