Reversible lysine modification on proteins by using functionalized boronic acids

Article abstract of DOI:10.1002/chem.201500127

Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC₅₀ value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells. Easy installation: The use of iminoboronates is a successful strategy to install diverse functions on proteins (see picture; GSH=glutathione, PEG=polyethylene glycol) and to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ.

Full text of DOI:10.1002/chem.201500127

DOI: 10.1002/chem.201500127
Full Paper
&
Protein Modifications
Reversible Lysine Modification on Proteins by using
Functionalized Boronic Acids
Pedro M. S. D. Cal,[a] Raquel F. M. Frade,[a] Carlos Cordeiro,[b] and Pedro M. P. Gois*[a]
Abstract: Iminoboronates have been utilized to successfully
install azide and alkyne bioorthogonal functions on proteins,
which may then be further reacted with their bioorthogonal
counterparts. These constructs were also used to add poly-
ethylene glycol (PEG) to insulin, a modification which has
been shown to be reversible in the presence of fructose. Fi-
nally, iminoboronates were used to assemble a folic acid/pa-
clitaxel small-molecule/drug conjugate in situ with an
IC50 value of 20.7 nm against NCI-H460 cancer cells and neg-
ligible cytotoxicity against the CRL-1502 noncancer cells.
Introduction
areas such as the selective delivery of drugs to cancer cells, in
which fine control over the deconjugation mechanism is essen-
tial to carry out the function of these drugs.[7] In this context,
we recently developed the reversible functionalization of lysine
residues and the N-termini by using 2-carbonyl-benzenoboron-
ic acids (Scheme 1).[8a,b] This simple reaction yields stable alkylic
iminoboronates in aqueous media, which undergo hydrolysis
in the presence of fructose, dopamine, and glutathione (GSH),
Posttranslational modifications, such as acylation, methylation,
phosphorylation, ubiquitination, or glycosylation, are part of
Nature’s “toolbox”, which is devised to extend the range of
protein functions and to control fundamental cellular process-
es, such as differentiation, trafficking, or signaling.[1] Apprecia-
tion for this strategy has led chemical biologists to develop
many different genetic and chemical methods to mimic the
proficiency of Nature to attach chemical handles onto proteins,
although the complex structure and marginal stability of pro-
teins in vitro still constitute a formidable challenge for biocon-
jugation.[2] Nevertheless, a plethora of innovative strategies
have been developed to modify specific residues selectively
without disturbing the protein architecture or function.[3] This
synthetic “toolbox” has been greatly expanded in recent years
by benefiting from the nucleophilicity of the thiol and e-amino
groups of cysteine and lysine residues, respectively, although
tyrosine, tryptophan, and disulfide bridges have also become
important bioconjugation hotspots.[3,4] Interestingly, most of
the scaffolds for protein modification were engineered to yield
stable constructs that can endure complex conditions such as
the biological milieu. However, protein functionalization based
on reversible covalent bonds[5] has been rather overlooked,
notwithstanding the potential of this strategy to generate con-
structs that respond to a predetermined stimulus. These
“smart bioconjugates”[6] may play a very important role in
Scheme 1. Proposed R-chain functionalization with azide and alkyne bioor-
thogonal functions, PEG chains, and a cytotoxic drug for protein modifica-
tion through the formation of iminoboronates.
presumably due to the disruption of the BÀN bond.[8c–g] Based
on these findings, we present herein the use of iminoboro-
nates to install reversible handles onto biomolecules that fea-
ture azide and alkyne bioorthogonal functions and polyethy-
lene glycol (PEG) chains and to assemble cancer-cell-targeting
drug conjugates.
[a] Dr. P. M. S. D. Cal, Dr. R. F. M. Frade, Dr. P. M. P. Gois
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL)
Faculty of Pharmacy, University of Lisbon
Av. Prof. Gama Pinto, 1649-003 Lisbon (Portugal)
Fax: (+351)217946470
Results and Discussion
Among the arsenal of chemical reactions available for the
modification of biomolecules,[3,4] selective ligation between
functional groups that are not naturally present in biological
systems (bioorthogonal ligation) offers tantalizing opportuni-
ties for ligation under complex conditions.[9] This strategy gen-
erally involves the installation of a bioorthogonal handle onto
the target biomolecule, which then selectively reacts with its
bioorthogonal counterpart. Many of these reactions have been
E-mail: pedrogois@ff.ulisboa.pt
[b] Prof. C. Cordeiro
Chemistry and Biochemistry Centre
Faculty of Science, University of Lisbon
1749-016 Lisbon (Portugal)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201500127.
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reported over the last 20 years, among which azide-related li-
gation strategies, such as the strain-promoted azide–alkyne cy-
cloaddition (SPAAC) and the copper-catalyzed azide–alkyne cy-
cloaddition (CuAAC), have been extensively used in many dif-
ferent applications.[9] Based on this background, we set out to
investigate whether iminoboronates could be used to install
bioorthogonal azide and alkyne functions onto biomolecules.
To test this hypothesis, 2-acetylbenzeneboronic ester 2 was
readily prepared from 2’,4’-dihydroxyacetophenone[8b] and fur-
ther treated with 2-azidoethanol and propargyl alcohol to gen-
erate 3 and 4 in yields of 74 and 75% yields, respectively. The
pinacol group of compounds 3 and 4 was subsequently re-
moved by using a boronic acid resin under acidic conditions,
thus affording boronic acids 5 and 6 in 68 and 61% yield of
the isolated products, respectively (Scheme 2).
mixture in which the presence of cycloaddition product 9 was
detected (see the Supporting Information), which was con-
firmed by means of high-resolution mass-spectrometric analy-
sis (Scheme 3).
Encouraged by these results, we attempted the installation
of an alkyne function onto the protein. Similar to the previous
case, upon the addition of 6 (1 mm) to the model-protein lyso-
zyme (10 mm), the iminoboronate construct 10 was readily
formed in ammonium acetate buffer (50 mm, pH 7.0;
Scheme 3). Subsequently, construct 10 was treated with the
protected azido sugar 11 under standard CuAAC reaction con-
ditions.[9] This protocol afforded a complex mixture in which
the presence of the expected cycloaddition product 12 was
detected (see the Supporting Information; characterization
was carried out in situ by using HRMS; Scheme 4).
The covalent modification of peptides and proteins with
PEG chains is a widely used technology to prevent degradation
by proteolytic enzymes, to reduce immunogenicity and recep-
tor-mediated uptake by the reticuloendothelial system, and to
increase solubility, stability, and biodistribution.[10] In addition,
PEGylation of biomolecules has also been explored to generate
therapeutic bioconjugates suitable for oral administration. This
strategy has been actively pursued to generate orally bioavail-
able insulin.[11] Based on this strategy, we conceived that imino-
boronates could be used to generate PEGylate/insulin con-
structs that regenerate free insulin in the presence of fructose.
To test this hypothesis, 2-acetylbenzeneboronic ester 2 was
successfully converted into the PEGylated boronic acid 13 in
60% overall yield. Once prepared, an aqueous solution of 13
(1 mm) was added to a solution of insulin in ammonium ace-
tate buffer (10 mm, 50 mm, pH 9.0) at room temperature and
the resulting mixture was analyzed by means of ESI-MS analy-
sis. Compound 13 completely modified the insulin to yield PE-
Gylated iminoboronate constructs of the peptide, one of m/
z 6200 and two of m/z 6592 (Figure 1A and Scheme 5).
Finally, fructose was added to the reaction mixture to see
whether construct 14 could be reverted in the presence of car-
bohydrates. Gratifyingly, although the reversibility was not
complete, the concentration of the PEGylated constructs in so-
lution clearly diminished under these conditions, thus regener-
ating the free insulin (m/z 5808.0; Figure 1B).
Scheme 2. Synthetic routes to install two bioorthogonal handles on a 2-ace-
tylbenzeneboronic acid core structure. DCM=dichloromethane, DMAP=4-
dimethylaminopyridine, EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide.
Recently, we have shown that alkylic iminoboronates are re-
versible in the presence of GSH.[8a] This constitutes a very ap-
pealing property to develop a delivery system for cancer cells
that typically exhibit an increased concentration of GSH (milli-
molar range) in the cytoplasm.[12] Consequently, GSH may be
targeted to promote the conjugate dissociation upon internali-
zation. Therefore, we next addressed the possibility of assem-
bling an iminoboronate small-molecule/drug conjugate
(SMDC) that may selectively target and deliver a cytotoxic drug
to a tumor-cell line of non-small-cell lung cancer (which is re-
ported to have increased levels of GSH).[13]
Folic acid (FA) is an essential vitamin for cell functioning,[14]
and many cancer cell lines overexpress receptors for this small
vitamin due to their fast cell division and growth; hence, FA
has been extensively used as a recognition moiety in many
SMDCs.[7,15] In this context, we have reported that FA can be
Once prepared, compound 5, which features an azide func-
tion, was immediately tested for its ability to conjugate with ly-
sozyme. Gratifyingly, 5 (1 mm) effectively functionalized the
model-protein lysozyme (10 mm) and yielded the expected imi-
noboronate construct 7 in ammonium acetate buffer (50 mm,
pH 7.0) at room temperature (confirmed by means of electro-
spray ionization Fourier transform ion cyclotron resonance
mass spectrometry (ESI FTICR-MS); Scheme 3). Upon the instal-
lation of the azide function onto the protein, we addressed the
possibility of performing a SPAAC reaction with these con-
structs. Therefore, a fluorescein-PEG-cyclooctyne model 8 was
used to treat the reaction mixture containing construct 7 in
ammonium acetate buffer. This reaction afforded a complex
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Scheme 3. Reaction of 5 in DMSO (1 mm) with lysozyme in ammonium acetate buffer (10 mm, 50 mm, pH 7) at room temperature. An enlargement of the
(8+) charge state of the ESI FTICR mass spectrum that resulted from this reaction. A SPAAC reaction between cyclooctyne/fluorescein derivative 8 in DMSO
(5 mm) and the azide function on the protein surface.
used to promote the selective delivery of fluorescent 2-acetyl-
benzeneboronic acids to human non-small-cell lung-cancer
cells (NCI-H460).[8b] Based on these results, we selected FA and
the mitotic inhibitor paclitaxel to construct the SMDC to target
NCI-H460 cancer cells. With this objective in mind, 2-acetylben-
zeneboronic ester 2 was successfully functionalized with pacli-
taxel, thus affording 15 in 74% overall yield (Scheme 6). The
SMDC was assembled in situ by adding 15 (100 mm) in DMSO
to N-(2-aminoethyl)folic acid (EDA-FA) 16 (100 mm) in DMSO in
ammonium acetate buffer (50 mm, pH 7.0) and the formation
of the expected SMDC 17 was confirmed by HR mass-spectro-
ties on proteins and peptides by the generation of reversible
iminoboronates with lysine residues or at the N-termini. 2-Ace-
tylbenzeneboronic acids functionalized with azide and alkyne
bioorthogonal handles were used to modify lysozyme-yielding
protein constructs with up to six modifications. Once installed
on the protein, these functionalities were successfully subject-
ed to SPAAC and CuAAC reactions, thus yielding the expected
cycloaddition products, which were characterized in situ by
high-resolution mass-spectrometric analysis. 2-Acetylbenzene-
boronic acid functionalized with PEG chains was used to effi-
ciently modify insulin. The insulin–PEG iminoboronates were
shown to be reversible and sensitive to the addition of fruc-
tose. The reversible nature of these constructs offers tantalizing
opportunities to develop a monosaccharide-responsive insulin
delivery system to tackle diabetes. Finally, these reversible imi-
noboronates were used to design a folic acid/paclitaxel SMDC
with an IC50 value of 20.7 nm against NCI-H460 cancer cell line
and a negligible cytotoxicity against the noncancer human-
skin normal-fibroblast cell line.
1
metric and H NMR spectroscopic analysis (see the Supporting
Information). A solution of SMDC 17 was tested against NCI-
H460 cancer cells and the noncancer human-skin normal-fibro-
blast cell line CRL-1502. Very gratifyingly, the solution of imino-
boronate SMDC 17 was shown to be quite cytotoxic toward
the NCI-H460 cancer cell line with an IC50 value of 20.7 nm (Fig-
ure 2A), whereas the boronic acid precursor 15 was less
potent (IC50 =33.0 nm) under the same conditions. Conjugate
17 (concentration=30 nm) did not elicit any relevant cytotox-
icity against the noncancer human-skin normal-fibroblast cell
line (Figure 2B).
Experimental Section
General procedure for the esterification of 2:[16] In a round-
bottom flask, 2 (1 equiv),[8b] EDC (3 equiv), and DMAP (0.1 equiv)
were mixed at 08C in a previously distilled solvent (0.11m) and an
inert atmosphere, stirred for 30 min. An alcohol derivative (2 equiv)
in a previously distilled solvent (0.22m) was added through a sy-
Conclusion
In this study, we have shown that 2-acetylbenzeneboronic
acids can be simply modified to install important functionali-
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Scheme 4. Reaction of 6 in DMSO (1 mm) with lysozyme in ammonium acetate buffer (10 mm, 50 mm, pH 7) at room temperature. An enlargement of the
(8+) charge state of the ESI FTICR mass spectrum that resulted from this reaction. CuAAC between azide sugar 11 in DMSO (5 mm) and the terminal alkyne
unit on the protein surface under standard CuAAC conditions.
Figure 1. A) Deconvoluted low-resolution mass spectrum of the reaction of
13 in DMSO (1 mm) with insulin (10 mm) in ammonium acetate buffer
(50 mm, pH 9) at room temperature (obtained constructs with one and two
modifications and the respective dehydration products). B) Deconvoluted
low-resolution mass spectrum of the same sample in the presence of 20 mm
of d-fructose (observed regeneration of free insulin (5808 Da)).
Scheme 5. Synthetic route to install a PEG chain on a 2-acetylbenzeneboron-
ic acid core and reaction of 13 in DMSO (1 mm) with insulin in ammonium
acetate buffer (10 mm, 50 mm, pH 9) at room temperature.
ringe, and the solution was stirred for a determined time at room
temperature. The solvent was concentrated in vacuo and the resi-
temperature gave different yields of boronic acids after evapora-
dues were purified by means of flash column chromatography to
tion of the solvents and isolation by flash column chromatography.
afford the different esters.
These compounds were immediately used for conjugation after de-
protection.
General procedure for the deprotection of pinacolyl boronate
esters:[17] Treatment of the ester compounds with polystyrene/bor-
onic acid (5 equiv) in acetonitrile/1m HCl (9:1) for 20 h at room
General procedure for the lysozyme conjugation: A 4-substituted
2-carbonylbenzene boronic acid in DMSO (1 mm) was treated with
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with 10% fetal bovine serum (FBS), antibiotic solution, and antimy-
cotic solution and kept in a humidified atmosphere with 5% CO2
and at 378C.
Anti-proliferative assay: NCI-H460 cells were plated in 96-well
plates at a density of 5ꢁ104 cellsmLÀ1. The plates were incubated
overnight and treated the next day with the samples to be tested.
The conjugate of 15 and 16 was obtained by mixing equal micro-
molar amounts of 15 with 16 and leaving the mixture at room
temperature for approximately 1 h. This solution and an equimolar
solution of 15 were diluted with the cell-culture media with only
0.5% FBS (the percentage of the organic solvent is !1% (v/v)
after dilution). The cells were incubated for 48 h with several con-
centrations of the samples to be tested. After incubation, the
media was removed and the cells were washed with the Hank bal-
anced salt solution (HBSS). A fresh 0.5% FBS cell-culture media
with 50 mgmLÀ1 neutral red was added to the plate and left for
3 h. The cells were washed with HBSS and the amount of neutral
red retained by the cells was extracted and dissolved with an or-
ganic solution (distilled water (19.96 mL), ethanol (20 mL), and gla-
cial acetic acid (400 mL)). The plate was gently shaken and read at
l=540 nm in a plate reader. Viability was determined by the ratio
of the absorbance of the treated/nontreated cells (control). Three
duplicates were done for each experimental condition. The
IC50 values were determined by using the GraphPad Prism soft-
ware.
Toxicity assays: CRL-1502 cells were plated in 96-well plates and
grown until a monolayer was formed. The samples were prepared
as described previously. Incubation continued for 24 h and the via-
bility was determined as explained before. The results are shown
in Figure 2 and Figure S30 in the Supporting Information.
Scheme 6. Synthetic route to install a cytotoxic drug (paclitaxel) on a 2-ace-
tylbenzeneboronic acid core and reaction of 15 in DMSO (100 mm) with
EDA-FA in ammonium acetate buffer (100 mm, 50 mm, pH 7) at room tem-
perature.
Acknowledgements
Fundażo para a CiÞncia e Tecnologia (SFRH/BD/72376/2010
PTDC/QUI-QUI/118315/2010; Pest-OE/SAU/UI4013/2011; P.M.P.
Gois, a FCT Investigator, is thanked for financial support.
Keywords: boronic acids · conjugation · cycloaddition · drug
design · protein modifications
Figure 2. A) Toxicity curve obtained for the NCI-H460 cell model (incuba-
tion=48 h). B) Viability assay for the CRL-1502 cell model (incuba-
tion=24 h); black squares=compound 15; black dots=conjugate 17.
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Received: January 12, 2015
Published online on && &&, 0000
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FULL PAPER
&
Protein Modifications
P. M. S. D. Cal, R. F. M. Frade, C. Cordeiro,
P. M. P. Gois*
&& – &&
Reversible Lysine Modification on
Proteins by using Functionalized
Boronic Acids
Easy installation: The use of iminoboro-
nates is a successful strategy to install
diverse functions on proteins (see pic-
ture; GSH=glutathione, PEG=polyethy-
lene glycol) and to assemble a folic
acid/paclitaxel small-molecule/drug con-
jugate in situ.
Chem. Eur. J. 2015, 21, 1 – 7
www.chemeurj.org
7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ