Assembling ligands in situ using bioorthogonal boronate ester synthesis

Article abstract of DOI:10.1016/j.chembiol.2010.09.008

Many molecules that could manipulate cellular function are not practical due to their large size and concomitant undesirable pharmocokinetic properties. Here, we describe a bioorthogonal, highly stable boronate ester (HiSBE) synthesis and use this reactio

Full text of DOI:10.1016/j.chembiol.2010.09.008

Chemistry & Biology
Brief Communication
Assembling Ligands In Situ
Using Bioorthogonal Boronate Ester Synthesis
Sung Bin Y. Shin,1,3 Ramiro D. Almeida,1,3 Guillermo Gerona-Navarro,1,3 Clay Bracken,2 and Samie R. Jaffrey1,
*
1Department of Pharmacology
2Department of Biochemistry
Weill Medical College, Cornell University, New York, NY 10065, USA
3These authors contributed equally to this work
*Correspondence: srj2003@med.cornell.edu
DOI 10.1016/j.chembiol.2010.09.008
SUMMARY
wise required for this class of click reactions (Baskin et al.,
2007; Codelli et al., 2008). Bioorthogonal reactions have been
Many molecules that could manipulate cellular used to develop chemical reporters for labeling and visualizing
biomolecules in living cells (Prescher and Bertozzi, 2005). In
these reactions, bioorthogonal functional groups are metaboli-
cally incorporated into biomolecules, and then probes that
function are not practical due to their large size and
concomitant undesirable pharmocokinetic proper-
ties. Here, we describe a bioorthogonal, highly stable
boronate ester (HiSBE) synthesis and use this reac-
contain reactive moieties are covalently coupled by a sponta-
neous bioorthogonal reaction.
tion to synthesize a biologically active molecule
Current bioorthogonal reactions have limitations that affect
from smaller precursors in a physiological context.
The rapid rate of HiSBE synthesis suggests that it
may be useful for assembling a wide variety of bio-
their use. In the case of the Staudinger ligation, slow reaction
rates and oxidation of reactants affect its usefulness (Lin et al.,
2005). Although the copper-free click reaction exhibits signifi-
cantly higher reaction kinetics (9.0 3 10À2 M-1 s-1) (Baskin
et al., 2007; Codelli et al., 2008), the reaction rates are still slow
relative to the timescales used for biological experiments, and
the availability of additional bioorthogonal reactions would
provide additional versatility for chemical reactions in living cells.
Because of the limitations of existing bioorthogonal reactions,
logically active molecules in physiological solutions.
INTRODUCTION
Synthetic reactions are typically performed using catalysts,
temperatures, and organic solvents that are not compatible wesoughttoidentifywhetherotherpreviouslydescribedreactions
with living cells. However, certain chemical reactions can occur could be used for bioorthogonal chemistry. Phenylboronic acid
in biological solutions and physiological conditions, and are not (PBA) interacts with D-glucose to form boronate esters that are
affected by the complex mixture of small molecules and proteins readily hydrolyzed in water (Ferrier et al., 1965). However, Stolo-
that are characteristic of biological solutions. These reactions witz and colleagues recently showed that PBA interacts with sali-
are referred to as ‘‘bioorthogonal,’’ and provide the opportunity cylhydoxamic acid (SHA) to form considerably more stable boro-
to probe cellular function with organic synthesis (Prescher and nate esters (Stolowitz et al., 2001). The bioconjugate reactions of
Bertozzi, 2005). Bioorthogonal reaction strategies involve pre- PBA and SHA have been used to purify biomolecules such as
synthesized nonnative chemical moieties that typically perform proteins and nucleic acids modified with SHA residues (Stolowitz
the final synthetic step in the cellular environment.
et al., 2001). Unlike D-glucose, which interacts with boronic acid
Certain reactions have been shown to exhibit bioorthogonal- through vicinal diols (Ferrier et al., 1965), SHA interacts with
ity, such as copper-free ‘‘click’’ chemistry (Baskin et al., 2007), boronic acid through both an oxygen and a nitrogen, which
and the Staudinger ligation (Saxon and Bertozzi, 2000). These may account for the increased stability (Stolowitz et al., 2001).
reactions are distinct from bioconjugation reactions, such as The boron-nitrogen bond has both ionic and covalent character
reactions of labeling agents with cysteine or tyrosine (Joshi (Plumley and Evanseck, 2007). These boronate esters exhibit
et al., 2004; Sletten and Bertozzi, 2009), or the formation of pH-dependent hydrolysis, with instability at low pHs (pH <5)
oximes by reaction of aminoxy moieties with aldehydes or (Stolowitz et al., 2001). Based on the increased resistance of these
ketones (Sletten and Bertozzi, 2009), since the nonnative boronate esters to hydrolysis at neutral pHs, we refer to this reac-
chemical moieties involved in bioorthogonal reactions are non- tion as highly stable boronate ester (HiSBE) synthesis. Due to its
perturbing to chemical functionalities found in living systems success inbioconjugationreactions, weconsideredthepossibility
and demonstrate high selectivity (Prescher and Bertozzi, 2005; that HiSBE chemistry would exhibit bioorthogonality.
Sletten and Bertozzi, 2009). For example, copper-free click
chemistry involves the reaction of an azide and an alkyne to RESULTS AND DISCUSSION
form a triazole in a [3 + 2] cycloaddition (Baskin et al., 2007).
The reactivity of the alkyne is promoted by ring strain and can HiSBE Synthesis between SHA and PBA
be enhanced by halogen substitution, resulting in rapid reaction We first asked if the HiSBE reaction was affected by biological
rates and an elimination of the need for copper, which is other- nucleophiles. To measure boronate ester formation between
Chemistry & Biology 17, 1171–1176, November 24, 2010 ª2010 Elsevier Ltd All rights reserved 1171

Chemistry & Biology
Bioorthogonal Boronate Ester Synthesis
Similarly, tissue culture media, which contains diverse small
molecule cofactors, amino acids, and ions found in biological
solutions, did not affect the interaction of PBA and SHA (Fig-
ure 1B). Together, these data indicate that the reaction of
PBA and SHA is not affected by molecules commonly found in
biological solutions.
We measured the association constants of PBA and SHA. The
coordination state of the boron nucleus changes from trigonal to
tertrahedral after complexation with SHA, which is detected by
the chemical shift of boron in 11B-NMR (Beachell and Beistel,
1964; Stolowitz et al., 2001). To monitor association, we
incubated 10 mM PBA with varying concentrations of SHA in
phosphate buffer. The observed association constant was
17,800 M-1 at pH 7.4 and 4 M-1 at pH 4.5, indicating that the com-
plexed form is highly favored at physiological pH. To measure
the association rate constant, we used UV spectrophotometry
to measure the reaction of SHA with PBA with respect to time.
Based on the linear relationship demonstrated in rate versus
[SHA]2[PBA] (see Figure S1 available online), the reaction of
SHA and PBA was modeled with a third order rate constant.
The plot of 1/([SHA][PBA]0) versus time was used to determine
the kinetic rate of (7.01 2.04) 3 106 M-2 s-1 (Figure 1C). These
data indicate that this reaction occurs considerably faster than
previously described bioorthogonal chemical reactions.
Assembly of Dimeric c-Mpl via HiSBE
We next wanted to determine if boronate ester synthesis can
occur in a physiologically relevant setting. To test the bioortho-
gonality of this reaction, we sought to generate biologically
active molecules in situ using HiSBE synthesis. Numerous
receptors are activated by ligands which bind and dimerize
their cognate receptors (Heldin, 1995). In the case of the throm-
bopoeitin (TPO) receptor c-Mpl, receptor dimerization and
activation is triggered by binding TPO (Cwirla et al., 1997). Pep-
tide ligands to c-Mpl, such as IEGPTLRQWLAARA, have been
identified, but due to their monomeric nature exhibit only weak
agonist activity (Cwirla et al., 1997). However, dimeric forms
of the peptide act as agonists of the receptor. In one case,
a c-Mpl-activating pseudosymmetric dimer was prepared on
a lysine scaffold: the COOH terminus of one IEGPTLRQWLAARA
was coupled to the 3-amine of the lysine, while the COOH
terminus of another IEGPTLRQWLAARA was coupled to the
a-amine of the lysine via a linker, resulting in a potent c-Mpl
agonist (Cwirla et al., 1997).
Figure 1. HiSBE Synthesis Is Facile and Orthogonal to Common
Biological Nucleophiles
(A) Time-dependent boronate ester formation between SHA and PBA immobi-
lized on agarose in 100 mM phosphate buffer at pH 7.4. Reaction solutions
containing 6 mM SHA (0.0036 mmol, 0.6 ml) were incubated with PBA agarose
(0.1 ml, 0.002 mmol of immobilized PBA). The SHA concentration in the
supernatant was quantified by absorption spectrophotometry at 214 nm.
(B) The efficiency of HiSBE synthesis in the presence of common biological
nucleophiles. Reaction solutions containing
6 mM SHA (0.0036 mmol,
0.6 ml) in 6% DMEM/100 mM phosphate buffer at the indicated pH and with
the indicated nucleophiles were incubated with PBA agarose (0.1 ml,
0.002 mmol of immobilized PBA) for 5 min. After incubation, the resin was
washed to remove uncomplexed SHA, and the complexed SHA was eluted
with 100 mM phosphate buffer at pH 4.5. Eluted SHA was quantified by
absorption spectrophotometry at 214 nm.
(C) Kinetic measurement of 0.1 mM SHA with 0.1, 0.2, and 0.5 mM PBA in
100 mM phosphate buffer at pH 7.4. The reaction rates were modeled
after third-order reaction kinetics and were determined by plotting 1/(([SHA]-
[SHA]f) 3 [PBA]0)) versus time. The slopes were used to approximate the
rate constants. k = (7.01 2.04) 3 106 M-2s-1
.
To assess the bioorthogonality of HiSBE synthesis, we
decided to determine if a dimeric c-Mpl agonist could be synthe-
sized in situ, and if the synthesized compound could lead to the
See also Figure S1.
SHA with PBA, we incubated SHA with PBA immobilized on activation of c-Mpl signaling in cells. Rather than using a lysine
agarose at pH 7.4. After incubation, the complexed SHA was linker, we decided to use a ‘‘dimerizer’’ comprising two SHA
eluted with low pH buffer (pH 4.5), which hydrolyzes SHA-PBA moieties connected by a linker (Figure 2A). This dimerizer is
complexes (Stolowitz et al., 2001). Eluted SHA was quantified expected to generate a dimeric c-Mpl ligand by interacting
by absorption spectrophotometry. A linear increase in boronate with two IEGPTLRQWLAARA, each synthesized with a PBA
ester formation was observed during the first 1 min of incubation, appended to the COOH terminus. We reasoned that the
with saturation observed after 5 min (Figure 1A).
c-Mpl-binding peptide would exhibit increased agonist activity
To determine the effect of common biological nucleophiles if it became dimerized upon application to cells which contained
such as serine, cysteine or lysine, as well as D-glucose, on the dimerizer.
HiSBE synthesis, we measured the effect of these compounds
We first confirmed that this dimerizer could simultaneously
on boronate ester synthesis measured at 5 min. None of these bind two PBA molecules. The dimerizer was titrated into a solu-
treatments significantly reduced the ester formation (Figure 1B). tion of 10 mM PBA in 100 mM phosphate buffer at pH 7.4, and
1172 Chemistry & Biology 17, 1171–1176, November 24, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Bioorthogonal Boronate Ester Synthesis
We next addressed whether the dimerizer could recruit two
peptides to form a dimeric peptide ligand. When the PBA moiety
on the peptide forms a complex with the SHA moiety on the
dimerizer, the hydroxamate NH proton is removed and a N-B
bond is formed. The formation of dimeric c-Mpl ligand was
demonstrated by the complete disappearance of the hydroxa-
mate NH proton NMR signal in a 2:1 mixture of PBA-modified
IEGPTLRQWLAARA and dimerizer (Figure 2C). We next sought
to determine the mass of the complex. Standard MS approaches
were not successful due to hydrolysis of the boronate ester by
the acidic buffers used in ESI and the acidic matrices commonly
used in MALDI. We therefore measured the size using 2D
DOSY-NMR. The molecular size of the 2:1 ratio of PBA-modified
IEGPTLRQWLAARA and the dimerizer measured by 2D DOSY
NMR was consistent with the expected hydrodynamic size of a
complex composed of two peptide molecules and a single dimer-
izer (Figure S3A). Together, these data indicate that the dimerizer
recruits two peptides to form a dimeric peptide complex.
Formation of a c-Mpl-Activating Ligand In Situ
We next addressed whether HiSBE chemistry could be used to
synthesize c-Mpl agonists in tissue culture. Consistent with
previous studies (Kaushansky et al., 1994; Yamada et al.,
1995), BaF3-cMpl cells, which overexpress c-Mpl, exhibit in-
creased p42/44 MAPK phosphorylation in response to TPO
(5 ng/ml), as measured by anti-p42/44 MAPK western blotting
(Figures 3A and 3B). Application of the PBA-modified
IEGPTLRQWLAARA also increased p42/44 MAPK activity,
consistent with earlier data showing that this monomeric peptide
exhibits weak agonist activity (Cwirla et al., 1997). However,
inclusion of the dimerizer along with the peptide resulted in a
doubling of p42/44 MAPK levels (Figures 3A and 3B). This effect
was mediated by c-Mpl, as BaF3 cells that did not express c-Mpl
did not exhibit an increase in p42/44 MAPK upon TPO or peptide
treatments, but remained responsive to IL-3, a cytokine that
activates p42/44 MAPK through an independent pathway (Pala-
cios and Steinmetz, 1985; Figure 3C). Furthermore, this effect
was dependent on the dimerizer, as SHA, which functions as a
monomeric control, did not elicit a statistically significant
increase in p42/44 MAPK relative to peptide alone (Figures 3D
and 3E). Last, we examined the effect of a control peptide that,
when dimerized, activates an unrelated receptor, TrkB (O’Leary
and Hughes, 2003). Application of either the control peptide
alone or the peptide in the presence of the dimerizer resulted
in no detectable activation of p42/44 MAPK (Figure 3F).
Together, these data indicate that the dimerizer and the peptide
react to form a c-Mpl-activating ligand in tissue culture media in
the presence of cells.
Figure 2. Assembly of PBA on a Dimerizing Scaffold
(A) Synthetic scheme of ‘‘dimerizer’’ synthesis.
(B) Titration of a dimerizer reveals stoichiometric complexation with PBA.
11B-NMR spectra of 10 mM PBA and 0, 2.5, 5, 10, and 20 mM of the dimerizer
in 6% DMSO-d6/ 100 mM of deuterated phosphate buffer in D2O (pD = 7.4).
PBA is largely complexed when present at a 2:1 ratio with dimerizer, as
evidenced by the appearance of the tetrahedral coordination state of boron
(6.7 ppm), and loss of the free PBA, which exhibits tetrahedral boron coordina-
tion geometry (30.8 ppm).
(C) 1H NMR spectra of dimerizer and PBA-modified c-Mpl-activating peptide
at various relative concentrations in DMF-d7. The inset shows the structure
of one of the two SHA present on the dimerizer. The hydroxamate O-H proton
is labeled (A) and this proton does not participate in the complex formation.
The hydroxamate N-H proton is labeled (B) and this proton is removed
when the nitrogen atom forms a dative coordinate bond with boron in the
peptide. The numbers below peaks (A) and (B) represent the relative area
under each peak. With increasing concentrations of the peptide, the area
under peak (B) is diminished, indicating the complex formation.
The peak assignments for protons (A) and (B) were made based on the
previous reports (Garcia et al., 2007).
To further examine the bioorthogonality of HiSBE synthesis, we
assessed the effects of the reactants on cell proliferation and
viability. Neither the dimerizer or the PBA-modified peptide
reduced cell proliferation as measured by the MTT viability assay
(Figure 3B), consistent with previous data suggesting the compat-
See also Figure S2.
the formation of PBA-SHA complexes was measured by the ibility of the boronic acid and salicylhydroxamic acid pharmaco-
appearance of the tetrahedral boron hybridization state as phoresinlivingsystems(Barnicoat etal., 1981;Dennisetal., 2003).
measured by 11B-NMR. At a 2:1 PBA:dimerizer ratio, the boron
Our data suggest that HiSBE synthesis can be used to
was essentially completely observed to be in the tetragedral assemble bioactive compounds in a physiologic setting. Multiva-
configuration (Figure 2B), indicating that each dimerizer was lent presentation of receptor ligands is a well-established
simultaneously complexed to two PBA molecules.
approach to switch inactive or relatively inactive ligands to
Chemistry & Biology 17, 1171–1176, November 24, 2010 ª2010 Elsevier Ltd All rights reserved 1173

Chemistry & Biology
Bioorthogonal Boronate Ester Synthesis
Figure 3. Synthesis of an Active c-Mpl
Ligand In Situ
(A–F) BaF3-cMpl and BaF3-WT (parental) cells
were transferred to media lacking IL-3, and
cultured for 5 hr. The cells were then stimulated
with the indicated compounds for 10 min at 37ꢀC
and analyzed by immunoblot against phospho-
p44/42 MAPK (upper panel) and total p44/42
MAPK (lower panel). In each experimental
condition, the immunoreactivity obtained was
calculated as a percentage of the total signal
obtained. Data are presented as mean
SEM
of four independent experiments and each immu-
noblot is representative of four independent
experiments.
(A) The agonist activity of the c-Mpl peptide is
enhanced by the dimerizer. BaF3-cMpl cells
were stimulated with TPO and IL-3, resulting in
enhanced phospho-p44/42 MAPK levels as
measured by western blotting. The PBA-modified
c-Mpl peptide (P) is a weak agonist of c-Mpl
signaling, which is enhanced by treatment with
the dimerizer (D). The dimerizer does not exert
any agonist activity on its own.
(B) Quantification of results in (A). Groups were
compared using a one-way ANOVA and New-
man-Keuls post-test: **p < 0.001 (n = 4) of P alone
relative to P + D. Data are presented as mean
SEM of four independent experiments.
(C) The effects of peptide-dimer complexes are
mediated through c-Mpl. BaF3-WT cells, which
are the parental line for the BaF3-cMpl cells, do
not express c-Mpl. Under the same conditions
as (A), the c-Mpl peptide and the dimer fail to
activate c-Mpl signaling as assayed by phospho-
p44/42 MAPK western blotting.
(D) The c-Mpl-binding peptide is not significantly
activated by a monomerizer. SHA, a monomeric
control (M) to the dimerizer used in (A), was used
to form complexes with the PBA-modified c-Mpl
binding peptide. Treatment of cells with SHA along
with the c-Mpl peptide failed to significantly
increase phospho-p44/42 MAPK levels. These
data support the idea that the dimerizer induces
the formation of a dimeric complex which is the
most efficacious ligand to activate c-Mpl
signaling.
(E) Quantification of the results in (D). Groups were compared using a one-way ANOVA and Newman-Keuls post-test: n.s. = non significant (n = 4) of P alone
relative to P + M. Data are presented as mean SEM of four independent experiments.
(F) Activation of c-Mpl signaling requires a c-Mpl-binding peptide. When a PBA-modified peptide that targets TrkB was used, no phospho-44/42 MAPK activation
was detected with either the peptide alone or in conjunction with the dimerizer. These results suggest that activation of c-Mpl signaling is mediated by
a sequence-specific interaction with c-Mpl by a dimerized peptide.
See also Figure S3.
receptor agonists (Fournel et al., 2005), and combinatorial cules within cells. In this approach, at least two cell-permeable
libraries of symmetric multivalent ligands as potential receptor molecules, one containing a PBA moiety and the other contain-
agonists have attracted considerable attention (Boger et al., ing a SHA moiety would be applied to cells, allowing bioactive
1997). However, these molecules and molecules with similarly complexes to form in the cytoplasm. This approach may enable
high molecular weights are typically accompanied by poor the formation of large and otherwise impermeable molecules to
solubility or undesirable pharmacokinetics (Lipinski, 2000). accumulate and exert effects in cells.
Synthesis of these compounds in situ provides a previously unex-
Assembly of molecules in situ requires rapid reaction kinetics
plored avenue to alleviate some of these problems, providing in order to provide temporal control over ligand synthesis. One
a strategy to facilitate the therapeutic use of multivalent ligands. prominent feature of HiSBE synthesis is its rapid reaction rate.
Although our focus is the design of dimeric and multimeric The copper-free click reaction utilizing difluorinated cyclooctyne
receptor agonists that act on the cell surface, in situ ligand (DIFO) exhibits rate constants (Baskin et al., 2007; Codelli et al.,
assembly could also conceivably be used to synthesize mole- 2008; Lin et al., 2005) that are several orders of magnitude slower
1174 Chemistry & Biology 17, 1171–1176, November 24, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Bioorthogonal Boronate Ester Synthesis
SUPPLEMENTAL INFORMATION
than the rate of HiSBE synthesis. The rapid rate of the HiSBE
synthesis suggests that bioorthogonal boronate ester formation
will be useful for temporally controlled, rapid assembly of
molecules in vivo. Another feature of HiSBE synthesis is that
the reaction is reversible by ester hydrolysis. This reversibility
allows HiSBE-linked molecules to be hydrolyzed to lower
molecular weight components, providing a route to temporally
control the duration of action of HiSBE-linked molecules.
Although the copper-free click reactions using DIFO exhibits
slower reaction kinetics compared with the HiSBE reaction, it
may also be suitable for in situ ligand synthesis, especially in
applications where a nonhydrolyzable product is desired.
The major application of bioorthogonal chemistry thus far is for
labeling biomolecules in vivo with reporter groups (Prescher and
Bertozzi, 2005). In these experiments, the cellular metabolic
machinery is exploited to incorporate bioorthogonal functional
groups within biological molecules. Reporter probes are then
covalently coupled to these functional groups by spontaneous
bioorthogonal reactions. Copper-free click chemistry has been
particularly useful for these reactions since alkynes and azides,
the bioorthogonal functional groups in this reaction, are relatively
small allowing modified glycans, lipids and amino acid precur-
sors to be readily incorporated into biomolecules (Prescher
and Bertozzi, 2005). HiSBE synthesis may also be suitable for
bioorthogonal chemical reporters. PBA moieties have been
swapped for aromatic rings in both nucleotides and amino acids,
resulting in only mild perturbations of structure (Lin et al., 2007;
Snyder et al., 1958). Additionally, HiSBE formation can easily
be conducted with linkers that contain multiple SHA moieties,
potentially leading to multivalent bioactive ligands. Combina-
tions of PBA-modified ligands and SHA linkers raise the exciting
possibility of rapidly generative diverse combinatorial libraries.
Also, HiSBE formation leads to products containing tetrahedral
boron coordination states, which can be imaged in living animals
by 11B-magnetic resonance imaging (Kabalka et al., 1988),
raising the possibility of monitoring bioorthogonal reactions
in vivo.
Supplemental Information includes Supplemental Experimental Procedures
and three figures and can be found with this article online at doi:10.1016/j.
chembiol.2010.09.008.
ACKNOWLEDGMENTS
The BaF3-cMPL expressing cell line was a kind gift of Dr. Kenneth Kaushansky
(University of California, San Diego). We thank D. Gin (Sloan Kettering Institute)
for helpful comments. This project was supported by the Starr Cancer
Consortium Award I1-A42 and NIH R21AI068512 (S.R.J.), Training Grant
T32CA062948 from the National Cancer Institute (S.Y.S.), and a postodoctoral
ˆ
fellowship from Fundac¸ a˜ o para a Ciencia e Tecnologia, Portugal (R.D.A.). The
authors declare no competing financial interests.
Received: January 20, 2010
Revised: August 19, 2010
Accepted: September 9, 2010
Published: November 23, 2010
REFERENCES
Barnicoat, A.J., van’t Hoff, W.G., Morrison, P.J., and Rogers, H.J. (1981).
Observations on salicyl hydroxamic acid, an experimental trypanocide.
Experientia 37, 1290–1291.
Baskin, J.M., Prescher, J.A., Laughlin, S.T., Agard, N.J., Chang, P.V., Miller,
I.A., Lo, A., Codelli, J.A., and Bertozzi, C.R. (2007). Copper-free click chemistry
for dynamic in vivo imaging. Proc. Natl. Acad. Sci. USA 104, 16793–16797.
Beachell, H.C., and Beistel, D.W. (1964). Nuclear magnetic resonance spectra
of phenylboronic acids. Inorg. Chem. 3, 1028–1032.
Boger, D.L., Ozer, R.S., and Andersson, C.-M. (1997). Generation of targeted
C2-symmetrical compound libraries by solution-phase combinatorial chem-
istry. Bioorg. Med. Chem. Lett. 7, 1903–1908.
Codelli, J.A., Baskin, J.M., Agard, N.J., and Bertozzi, C.R. (2008). Second-
generation difluorinated cyclooctynes for copper-free click chemistry. J. Am.
Chem. Soc. 130, 11486–11493.
Cwirla, S.E., Balasubramanian, P., Duffin, D.J., Wagstrom, C.R., Gates, C.M.,
Singer, S.C., Davis, A.M., Tansik, R.L., Mattheakis, L.C., Boytos, C.M., et al.
(1997). Peptide agonist of the thrombopoietin receptor as potent as the natural
cytokine. Science 276, 1696–1699.
Dennis, G., Jr., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C.,
and Lempicki, R.A. (2003). DAVID: database for annotation, visualization,
and integrated discovery. Genome Biol. 4, 3.
SIGNIFICANCE
We demonstrate that the reaction of phenyl boronic acid and
salicylhydroxamic acid, HiSBE synthesis, exhibits bioortho-
gonality, and unusually rapid reaction kinetics. HiSBE
synthesis leads to boronate esters that are acid labile, and
which therefore can produce products that are expected
to be biodegradable under cellular conditions. Because of
the rapid reaction kinetics of HiSBE synthesis, we show
that this reaction can be used to generate bioactive
compounds from relatively inert precursors. Assembling
ligands in situ using bioorthogonal chemistry provides a
strategy to bypass problems of membrane permeability
and poor pharmacokinetics of many high molecular weight
compounds by allowing these compounds to be formed in
a cellular context.
Ferrier, R.J., Hannaford, A.J., Overend, W.G., and Smith, B.C. (1965).
Boric acid derivatives as reagents in carbohydrate chemistry: part IV. The
interaction of phenylboronic acid with hexopyranoid compounds.
Carbohydr. Res. 1, 38–43.
Fournel, S., Wieckowski, S., Sun, W., Trouche, N., Dumortier, H., Bianco, A.,
Chaloin, O., Habib, M., Peter, J.C., Schneider, P., et al. (2005). C3-symmetric
peptide scaffolds are functional mimetics of trimeric CD40L. Nat. Chem. Biol.
1, 377–382.
Garcia, B., Secco, F., Ibeas, S., Mun˜ oz, A., Hoyuelos, F.J., Leal, J.M., Senent,
M.L., and Venturini, M. (2007). Structural NMR and ab initio study of salicylhy-
droxamic and p-hydroxybenzohydroxamic acids: evidence for an extended
aggregation. J. Org. Chem. 72, 7832–7840.
Heldin, C.H. (1995). Dimerization of cell surface receptors in signal transduc-
tion. Cell 80, 213–223.
Joshi, N.S., Whitaker, L.R., and Francis, M.B. (2004). A three-component
Mannich-type reaction for selective tyrosine bioconjugation. J. Am. Chem.
Soc. 126, 15942–15943.
EXPERIMENTAL PROCEDURES
Kabalka, G.W., Davis, M., and Bendel, P. (1988). Boron-11 MRI and MRS of
intact animals infused with a boron neutron capture agent. Magn. Reson.
Med. 8, 231–237.
Details of experimental procedures can be found in the Supplemental
Experimental Procedures.
Chemistry & Biology 17, 1171–1176, November 24, 2010 ª2010 Elsevier Ltd All rights reserved 1175

Chemistry & Biology
Bioorthogonal Boronate Ester Synthesis
Kaushansky, K., Lok, S., Holly, R.D., Broudy, V.C., Lin, N., Bailey, M.C.,
Forstrom, J.W., Buddle, M.M., Oort, P.J., Hagen, F.S., et al. (1994).
Promotion of megakaryocyte progenitor expansion and differentiation by the
c-Mpl ligand thrombopoietin. Nature 369, 568–571.
Plumley, J.A., and Evanseck, J.D. (2007). Covalent and ionic nature of the
dative bond and account of accurate ammonia borane binding enthalpies.
J. Phys. Chem. A 111, 13472–13483.
Prescher, J.A., and Bertozzi, C.R. (2005). Chemistry in living systems. Nat.
Chem. Biol. 1, 13–21.
Lin, F.L., Hoyt, H.M., van Halbeek, H., Bergman, R.G., and Bertozzi, C.R.
(2005). Mechanistic investigation of the Staudinger ligation. J. Am. Chem.
Soc. 127, 2686–2695.
Saxon, E., and Bertozzi, C.R. (2000). Cell surface engineering by a modified
Staudinger reaction. Science 287, 2007–2010.
Sletten, E.M., and Bertozzi, C.R. (2009). Bioorthogonal chemistry: fishing for
Lin, N., Yan, J., Huang, Z., Altier, C., Li, M., Carrasco, N., Suyemoto, M.,
Johnston, L., Wang, S., Wang, Q., et al. (2007). Design and synthesis of
boronic-acid-labeled thymidine triphosphate for incorporation into DNA.
Nucleic Acids Res. 35, 1222–1229.
selectivity in a sea of functionality. Angew. Chem. Int. Ed. Engl. 48, 6974–6998.
Snyder, H.R., Reedy, A.J., and Lennarz, W.J. (1958). Synthesis of aromatic
boronic acids. aldehydo boronic acids and a boronic acid analog of tyrosine.
J. Am. Chem. Soc. 80, 835–838.
Lipinski, C.A. (2000). Drug-like properties and the causes of poor solubility and
Stolowitz, M.L., Ahlem, C., Hughes, K.A., Kaiser, R.J., Kesicki, E.A., Li, G.,
Lund, K.P., Torkelson, S.M., and Wiley, J.P. (2001). Phenylboronic acid-
salicylhydroxamic acid bioconjugates. 1. A novel boronic acid complex for
protein immobilization. Bioconjug. Chem. 12, 229–239.
poor permeability. J. Pharmacol. Toxicol. Methods 44, 235–249.
O’Leary, P.D., and Hughes, R.A. (2003). Design of potent peptide mimetics of
brain-derived neurotrophic factor. J. Biol. Chem. 278, 25738–25744.
Yamada, M., Komatsu, N., Okada, K., Kato, T., Miyazaki, H., and Miura, Y.
(1995). Thrombopoietin induces tyrosine phosphorylation and activation of
mitogen-activated protein kinases in a human thrombopoietin-dependent
cell line. Biochem. Biophys. Res. Commun. 217, 230–237.
Palacios, R., and Steinmetz, M. (1985). Il-3-dependent mouse clones that
express B-220 surface antigen, contain Ig genes in germ-line configuration,
and generate B lymphocytes in vivo. Cell 41, 727–734.
1176 Chemistry & Biology 17, 1171–1176, November 24, 2010 ª2010 Elsevier Ltd All rights reserved