Facile synthesis of borofragments and their evaluation in activity-based protein profiling

Article abstract of DOI:10.1039/c4cc09107h

The discovery of enzyme inhibitors relies on synthetic methods that enable rapid and modular construction of small molecules. Heterocyclic fragments designed to maximize enthalpic interactions with their protein targets represent a particularly desirable class of molecules. Here we describe a reagent that enables straightforward construction of borofragments , in which a heterocycle is separated from the boron center by two or three rotatable bonds. The stability of these molecules depends on the MIDA group which likely acts as a slow-release element under biological conditions. Borofragments can be used to discover inhibitors of enzymes that use catalytic oxygen nucleophiles. We have employed this method to identify inhibitors of ABHD10 and the predicted carboxypeptidase CPVL. This technique should be applicable to other classes of targets. This journal is

Full text of DOI:10.1039/c4cc09107h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Facile synthesis of borofragments and their
evaluation in activity-based protein profiling†
Shinya Adachi,a Armand B. Cognetta 3rd,b Micah J. Niphakis,b Zhi He,a
Adam Zajdlik,a Jeffrey D. St. Denis,a Conor C. G. Scully,a Benjamin F. Cravattb and
Andrei K. Yudin*a
Cite this: Chem. Commun., 2015,
51, 3608
Received 14th November 2014,
Accepted 12th January 2015
DOI: 10.1039/c4cc09107h
www.rsc.org/chemcomm
The discovery of enzyme inhibitors relies on synthetic methods that reactions with low functional group tolerance, we recently devel-
enable rapid and modular construction of small molecules. Hetero- oped a boron-containing isocyanide reagent that enabled us to
cyclic fragments designed to maximize enthalpic interactions with synthesize novel boromorpholinones and boropeptides under
their protein targets represent a particularly desirable class of mole- mild conditions.3 The N-methyliminodiacetic acid (MIDA) residue
cules. Here we describe a reagent that enables straightforward on boron4,5 was the main factor that contributed to the stability of
construction of ‘‘borofragments’’, in which a heterocycle is separated boropeptides and their successful one-step synthesis.
from the boron center by two or three rotatable bonds. The stability
Building on our approach to boropeptides, we became
of these molecules depends on the MIDA group which likely acts as a interested in advancing the concept of MIDA-containing boro-
slow-release element under biological conditions. Borofragments fragments, which are chimeric molecules where a boron-
can be used to discover inhibitors of enzymes that use catalytic containing group is connected to a recognition unit, most
oxygen nucleophiles. We have employed this method to identify commonly a heterocycle. As many biologically relevant hetero-
inhibitors of ABHD10 and the predicted carboxypeptidase CPVL. This cycles with high ligand efficiency have conjugate acids with
technique should be applicable to other classes of targets.
relatively low pKa’s,6 we were motivated to develop a mild
method for connecting a diverse set of heterocyclic nucleo-
Organoboron compounds have received considerable attention philes to the smallest possible C–B unit equipped with the
as valuable building blocks in chemical synthesis.1 Boron- MIDA substituent (furnishing the corresponding borofragments).
containing molecules have also been used as enzyme inhibitors.2 To facilitate navigation of the accessible chemistry space in
Both of these applications result from the accessibility of boron’s enzyme active sites, we were particularly intrigued by the boro-
empty p-orbital, which enables reversible interaction with fragments in which a heterocycle is separated from the boron
synthetic and biological nucleophiles. Our recent interest in center by two or three rotatable bonds. In this paper, we present
this area has led to an ongoing exploration of boron-based our efforts to synthesize electrophilic borofragments and their
electrophiles that engage nucleophilic residues in active sites evaluation as serine hydrolase inhibitors using competitive
of proteins. In contrast to aldehydes, acrylates and epoxides, activity-based protein profiling (ABPP). We show that the MIDA
which are widely used as ‘‘baits’’ for nucleophilic residues in group is compatible with the synthesis of a diverse collection of
active sites, small boron-containing electrophiles have received borofragments and that select members of this class inhibit
relatively little attention. This is mainly due to their lack of several poorly characterized serine hydrolases, including a/b
stability and relative difficulty of preparation, especially when it hydrolase domain-containing protein 10 (ABHD10) and predicted
comes to molecules with C(sp3)–B bonds.1 Because the corre- serine carboxypeptidase (CPVL). These findings should encourage
sponding synthetic protocols often depend on organometallic broader campaigns to append C(sp3)–B bonds in their MIDA
forms to various molecular recognition components.
In order to convert readily accessible nucleophilic small
molecules into borofragments, we pursued hydroxymethyl-
containing building block 5 with the goal of evaluating its
reactivity in redox-driven condensations. Despite the relative
bulk of the MIDA group, the mild and neutral conditions7 of
the Mitsunobu process were particularly attractive to us.
Compound 5 was synthesized according to Scheme 1. Starting
with dibromomethane (1), bromomethylpinacolborate (2) was
a Davenport Research Laboratories, Department of Chemistry, University of Toronto,
80 St. George St., Toronto, ON M5S3H6, Canada.
E-mail: ayudin@chem.utoronto.ca
b The Skaggs Institute for Chemical Biology and Department of Chemical
Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
† Electronic supplementary information (ESI) available. CCDC 1021097–1021099.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4cc09107h
3608 | Chem. Commun., 2015, 51, 3608--3611
This journal is ©The Royal Society of Chemistry 2015

View Article Online
Communication
ChemComm
Table 1 Synthesis of a-functionalized alkyl(MIDA)boronatesa
NuH
Product
Entry
1b
Yield (%)
99
6a
7a
Scheme 1 Preparation of hydroxymethyl(MIDA)boronate.
2b
3
6b
6c
7b
7c
94
97
prepared according to literature procedure.8 Treatment of 1
with n-butyllithium in the presence of triisopropoxyborate
followed by transesterification of the resulting bromomethyl-
diisopropoxyborane with pinacol furnished intermediate 2.
Subsequent treatment of crude product 2 with sodium benzyl-
oxide in DMSO afforded benzyloxymethylpinacolboronic ester
(3),9 which was converted to benzyloxymethyl(MIDA)boronate
(4) in situ by heating in the presence of MIDA. Most impor-
tantly, purification of compound 4 was achieved by trituration
with Et2O, which eliminated the need for chromatography.
Hydrogenolysis of 4 with Pd/C afforded hydroxymethyl(MIDA)-
boronate (5) as a bench-stable white solid in pure form (49%
isolated yield over 4 steps, 410 g).
We were pleased to observe that, despite the steric bulk
of the B-MIDA substituent, hydroxymethyl(MIDA)boronate (5)
reacted with various acidic pro-nucleophiles (NuH) in the
presence of diisopropyl azodicarboxylate (DIAD) and triphenyl-
phosphine to produce a variety of a-functionalized alkyl(MIDA)-
boronates in excellent yields (Table 1). Ester formation via
coupling of benzoic acid (6a) or cinnamic acid (6b) with 5
afforded the corresponding products 7a and 7b, respectively
(entries 1 and 2). N-Hydroxyphthalimide (6c) was easily converted
into the corresponding heterocyclic product 7c (entry 3). Etheri-
fication of 5 was also carried out. Thus, phenol 6d was used to
produce 7d, although 2.0 equivalents of 6d were required in this
case (entry 4). Nitrogen-containing acidic pro-nucleophiles were
also employed in this methodology (entries 5 and 6). The reaction
of phthalimide (6e) with 5 gave the corresponding product 7e
(entry 5). N,O-Bis(phenoxycarbonyl)hydroxylamine (6f)10 was
4c
5
6d
6e
7d
7e
91
99
6
6f
7f
78
7
8
6g
6h
7g
7h
98
96
9
6i
6j
7i
7j
85
92
10
a
Unless otherwise noted, reactions were conducted with alcohol
(1.0 equiv.), 1.0 equiv. of acidic pronucleophile, 1.2 equiv. of triphenylphos-
b
phine, and 1.2 equiv. of DIAD in THF at 23 1C for 30 min. 1.5 equiv. of
acidic pronucleophile, 1.5 equiv. of triphenylphosphine, and 1.5 equiv. of
DIAD were used. Reaction time: 30 min. Temp.: 23 1C. 2.0 equiv. of acidic
pronucleophile, 1.5 equiv. of triphenylphosphine, and 1.5 equiv. of DIAD
c
were used. Reaction time: 12 h. Temp.: 23 1C.
converted to 7f (entry 6). Purine derivatives 6g and 6h11 were neutral pH and room temperature, thus expanding the scope
found to react with 5 to afford N(9)-alkylated products 7g and of compatible functional groups, which includes biologically
7h, whose structures were confirmed by X-ray crystallography relevant heterocycles with low pKa’s.
(entries 7 and 8). Sulfur-containing pro-nucleophiles 6i, j
We were also able to incorporate the azide function via a
reacted with 5 to afford the corresponding products 7i,j (entries modified Mitsunobu reaction of hydroxymethyl(MIDA)boronate
9 and 10). For compounds 7a–e,g,j purification was achieved by (5) with diphenylphosphorylazide (DPPA) (8). This reaction pro-
trituration with Et2O due to the low solubility of the products in duced azidomethyl(MIDA)boronate (9) in 90% yield (Scheme 2).14
non-polar solvents. We note that Molander and co-workers have Compound 9 was also synthesized through an alternate route in
reported the nucleophilic substitution of a-halomethyltrifluoro- 75% yield from the reaction of sodium azide with iodomethyl-
borates with nucleophiles such as amines and organolithium/ (MIDA)boronate (10), which was in turn prepared by treatment
organosodium reagents.12 Their work also includes nucleo- of 5 with iodine, PPh3, and imidazole. The presence of an azide in
philic substitutions of a-halomethylboronic esters with amines compound 9, which can be elaborated via click chemistry,15 further
followed by addition of KHF2 to synthesize a number of expands the synthetic versatility of borofragment construction.
a-functionalized alkyl trifluoroborates.13 However, those sub-
While boronic acids and their derivatives have been explored
stitutions have been conducted under basic conditions and often as enzyme inhibitors for decades,2 their potency and selectivity
at high temperatures. In contrast, our reactions are carried out at across mechanistically conserved enzyme classes remains poorly
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 3608--3611 | 3609

View Article Online
ChemComm
Communication
Scheme 2 Synthesis of azidomethyl(MIDA)boronate.
understood. In large part this is due to the poor stability of free
boronic acids under biological conditions and the consequential
lack of compounds for analysis. We have found that the MIDA
ligand enables preparation of compounds unavailable in their
free boronic acid form, yet in a biological milieu would likely be
released to form active boronic acids. With this in mind, we
wondered if broadly profiling the activity of serine hydrolases in
the presence of our (MIDA)boronate fragments would elucidate
novel inhibitor–enzyme pairs that could aid functional annotation
of poorly characterized enzymes within this class. Using compe-
titive activity-based protein profiling (ABPP) with the serine
hydrolase-directed activity-based probe fluorophosphonate–
rhodamine (FP–Rh),16 we first assayed 10 library members
(Table 1) across the serine hydrolases natively expressed in a
human prostate cancer cell line (PC3). Briefly, a soluble PC3
cell proteomic lysate was treated with either DMSO or a
(MIDA)boronate (10 mM) for 30 min at 37 1C and then with
FP–Rh (1.0 mM) for 30 min. The enzymes labeled by FP–Rh
were then resolved by SDS-PAGE and their degree of labeling
was measured by fluorescence gel imaging. This initial screen
revealed that, while most of these compounds did not affect
FP-labeling for any detectable serine hydrolase, two thioether
boronates (7i and 7j) displayed moderate inhibition of a
Fig. 1 Competitive ABPP of (MIDA)boronates in PC3 cell lysates. (A) Gel-
based ABPP analysis of (MIDA)boronates in the soluble proteome of PC3
cells. The proteome was treated with either DMSO or compounds 7a–j (20
mM) for 2 h, then treated with FP–Rh (1.0 mM) for 30 min. Labeled serine
hydrolases were resolved by SDS-PAGE and analyzed by fluorescence gel
imaging. (B) Gel-based ABPP analysis of 7j (1.0 nM–100 mM) and DMSO in
the soluble fraction of PC3 cells showing dose-dependent inhibition of
ABHD10 and ACOT1/2. (C) ABPP-SILAC analysis of 7j at 25 mM (heavy)
versus DMSO (light) in whole PC3 cell proteomic lysates showing inhibition
of ABHD10, CPVL and, to a lesser extent, ACOT1/2. Data are presented as
the mean Æ standard deviations of heavy/light ratios for multiple unique
peptides from each serine hydrolase.
B30 kDa band which we have previously identified17 as compound across a broad concentration range using gel-based
ABHD10 (Fig. S1, ESI†). Considering that enzyme inhibition ABPP (Fig. 1B). From this analysis, we observed near complete
might necessitate decomposition of the (MIDA)boronate frag- inhibition of ABHD10 at 10 mM and acyl-coenzyme A thioesterase
ments to an active boronic acid (Scheme 3), we retested each 1/2 (ACOT1/2)20 (acyl-CoA thioesterases which are involved in
compound at 20 mM with an extended 2 h preincubation time fatty acid metabolism21) at 100 mM. Although 7j is less potent for
prior to adding FP–Rh, allowing more time for the formation ABHD10 relative to previously described inhibitors,17,19a
of the putative boronic acid (Fig. 1A). Notably, this preincubation it should be emphasized that the ABPP platform used herein,
time was sufficient to produce 495% conversion of 7j to its which relies on an irreversible FP probe to report target occu-
corresponding boronic acid in DPBS (see ESI†). Using this new pancy, may underestimate the degree of enzyme inhibition for
assay protocol, 7i and 7j displayed enhanced activity, completely reversible inhibitors, such as boronates/boronic acids, unless the
inhibiting ABHD10 with few observable off-targets. We and kinetics of FP probe reactivity is carefully monitored using
others have recently shown that ABHD10, a poorly characterized competitive ABPP experiments. This question can be addressed
serine hydrolase, is also potently inhibited by b-lactones17,18 and in future studies using established protocols.19b
aza-b-lactams,19 but to our knowledge, targeting this enzyme
To definitively identify the targets of 7j, we used a quanti-
with boron-based inhibitors has not been reported. To further tative mass spectrometry (MS)-based platform termed ABPP-
assess the potency and selectivity of 7j, we next tested this SILAC.22 Following previously described protocols, cell lysates
were prepared from PC3 cells cultured in the presence of
isotopically light and heavy lysine and arginine. These lysates
were then treated with either DMSO (light) or 25 mM 7j (heavy)
for 2 h and subsequently treated with a biotinylated FP probe
(FP-biotin)23 for 30 min. After mixing the heavy and light
proteomes in equal proportions, the biotinylated proteins were
enriched by streptavidin chromatography, trypsinized on bead
and analyzed by LC-MS/MS to quantify the extent of inhibition
Scheme 3 In situ hydrolysis of 7j to the boronic acid 11 and its covalent
reaction with the active site nucleophile of ABHD 10.
3610 | Chem. Commun., 2015, 51, 3608--3611
This journal is ©The Royal Society of Chemistry 2015

View Article Online
Communication
ChemComm
S. Y. Chang and M. D. Burke, Angew. Chem., Int. Ed., 2011, 50,
7862–7864; (m) J. Li and M. D. Burke, J. Am. Chem. Soc., 2011, 133,
13774–13777; (n) E. M. Woerly, J. R. Struble, N. Palyam, S. P. O’Hara
and M. D. Burke, Tetrahedron, 2011, 67, 4333–4343; (o) G. R. Dick,
E. M. Woerly and M. D. Burke, Angew. Chem., Int. Ed., 2012, 51,
2667–2672; (p) E. M. Woerly, J. E. Miller and M. D. Burke, Tetra-
hedron, 2013, 69, 7732–7740; (q) E. M. Woerly, J. Roy and M. D.
Burke, Nat. Chem., 2014, 6, 484–491.
5 (a) Z. He and A. K. Yudin, J. Am. Chem. Soc., 2011, 133, 13770–13773;
(b) Z. He, A. Zajdlik, J. D. St. Denis, N. Assem and A. K. Yudin, J. Am.
Chem. Soc., 2012, 134, 9926–9929; (c) Z. He, P. Trinchera, S. Adachi,
J. D. St. Denis and A. K. Yudin, Angew. Chem., Int. Ed., 2012, 51,
11092–11096; (d) J. D. St. Denis, A. Zajdlik, J. Tan, P. Trinchera,
C. F. Lee, Z. He, S. Adachi and A. K. Yudin, J. Am. Chem. Soc., 2014,
136, 17669–17673.
6 (a) D. A. Erlanson, R. S. McDowell and T. O’Brien, J. Med. Chem., 2004,
47, 3463–3482; (b) P. Bamborough, M. J. Brown, J. A. Christopher,
C.-W. Chung and G. W. Mellor, J. Med. Chem., 2011, 54, 5131–5143;
(c) R. M. Miller, V. O. Paavilainen, S. Krishnan, I. M. Serafimova and
J. Taunton, J. Am. Chem. Soc., 2013, 135, 5298–5301.
7 (a) O. Mitsunobu, Synthesis, 1981, 1–28; (b) K. C. K. Swamy,
N. N. B. Kumar, E. Balaraman and K. V. P. P. Kumar, Chem. Rev.,
2009, 109, 2551–2651.
8 N. Murai, M. Yonaga and K. Tanaka, Org. Lett., 2012, 14, 1278–1281.
9 R. P. Singh and D. S. Matteson, J. Org. Chem., 2000, 65, 6650–6653.
10 A. O. Stewart and D. W. Brooks, J. Org. Chem., 1992, 57, 5020–5023.
11 S. Dey and P. Garner, J. Org. Chem., 2000, 65, 7697–7699.
12 G. A. Molander and J. Ham, Org. Lett., 2006, 8, 2031–2034.
13 (a) G. A. Molander and M.-A. Hiebel, Org. Lett., 2010, 12, 4876–4879;
(b) G. A. Molander and I. Shin, Org. Lett., 2012, 14, 4458–4461.
14 B. Lal, B. N. Pramanik, M. S. Manhas and A. K. Bose, Tetrahedron
Lett., 1977, 18, 1977–1980.
15 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
16 M. P. Patricelli, D. K. Giang, L. M. Stamp and J. J. Burbaum, Proteomics,
2001, 1, 1067–1071.
for the identified serine hydrolases. Among B30 serine hydro-
lases only two targets were maximally inhibited (495%): ABHD10
and the predicted carboxypeptidase CPVL. While the former target
was inferred from the gel profiles, we had not detected CPVL by
gel-based profiling. Given that CPVL remains poorly annotated
with regard to its endogenous substrates and physiological
function, there is a clear need for the development of selective
inhibitors of this enzyme, which to our knowledge are still
lacking. Our data suggest that the (MIDA)boronates described
herein are promising leads for CPVL inhibitor development and
may facilitate studying the role of this enzyme in human biology.
In summary, the hydroxymethyl(MIDA)boronate reagent
disclosed in this paper allows for straightforward conjugation
of boron with heterocycles of biological significance. Signifi-
cantly, the MIDA group stabilizes borofragments against pre-
mature decomposition and is likely released under biological
conditions to unmask an active boronic acid. This chemistry
allowed us to generate new boron-containing inhibitors of
ABHD10 and the predicted carboxypeptidase CPVL. Given the
diversity of heterocycles that are either commercially available or
exist in the historical collections of the pharmaceutical industry,
our straightforward method of linking molecular recognition
units with stabilized boron electrophiles should enable facile
exploration of previously uncharted covalent inhibitor space.
We thank NSERC and NIH (DA033760) for financial support
and Dr A. Lough for the X-ray structural analysis of 7f, 7g, and 7h.
Notes and references
17 N. J. Lajkiewicz, A. B. Cognetta, III, M. J. Niphakis, B. F. Cravatt and
J. A. Porco, J. Am. Chem. Soc., 2014, 136, 2659–2664.
18 A. List, E. Zeiler, N. Gallastegui, M. Rusch, C. Hedberg, S. A. Sieber
and M. Groll, Angew. Chem., Int. Ed., 2014, 53, 571–574.
19 (a) A. M. Zuhl, J. T. Mohr, D. A. Bachovchin, S. Niessen, K.-L. Hsu,
1 (a) Science of Synthesis: Boron Compounds, ed. D. E. Kaufmann and
D. S. Matteson, Georg Thieme Verlag, Stuttgart-New York, 2004,
vol. 6; (b) Boronic Acids: Preparation and Applications in Organic
Synthesis, Medicine and Materials, ed. D. G. Hall, Wiley-VCH,
Weinheim, 2nd edn, 2011.
2 (a) S. J. Baker, J. W. Tomsho and S. J. Benkovic, Chem. Soc. Rev.,
2011, 40, 4279–4285; (b) R. Smoum, A. Rubinstein, V. M. Dembitsky
and M. Srebnik, Chem. Rev., 2012, 112, 4156–4220.
´
J. M. Berlin, M. Dochnahl, M. P. Lopez-Alberca, G. C. Fu and
B. F. Cravatt, J. Am. Chem. Soc., 2012, 134, 5068–5071; (b) D. Leung,
C. Hardouin, D. L. Boger and B. F. Cravatt, Nat. Biotechnol., 2003, 21,
687–691.
20 We have previously suggested that this band corresponds to either
ACOT1, ACOT2 or both (see ref. 17), by correlating gel-based to
MS-based ABPP profiling along with the predicted molecular weight
and gel migration profile of these enzymes. However, the high
sequence identity between these two distinct enzymes has ham-
pered a definitive assignment.
3 A. Zajdlik, Z. Wang, J. L. Hickey, A. Aman, A. D. Schimmer and
A. K. Yudin, Angew. Chem., Int. Ed., 2013, 52, 8411–8415.
4 (a) E. P. Gillis and M. D. Burke, J. Am. Chem. Soc., 2007, 129,
6716–6717; (b) S. J. Lee, K. C. Gray, J. S. Paek and M. D. Burke,
J. Am. Chem. Soc., 2008, 130, 466–468; (c) E. P. Gillis and M. D. Burke,
J. Am. Chem. Soc., 2008, 130, 14084–14085; (d) D. M. Knapp, E. P.
Gillis and M. D. Burke, J. Am. Chem. Soc., 2009, 131, 6961–6963;
(e) S. G. Ballmer, E. P. Gillis and M. D. Burke, Org. Synth., 2009, 86,
344–359; ( f ) E. P. Gillis and M. D. Burke, Aldrichimica Acta, 2009, 42,
17–27; (g) B. E. Uno, E. P. Gillis and M. D. Burke, Tetrahedron, 2009,
65, 3130–3138; (h) J. R. Struble, S. J. Lee and M. D. Burke, Tetra-
hedron, 2010, 66, 4710–4718; (i) G. R. Dick, D. M. Knapp, E. P. Gillis
and M. D. Burke, Org. Lett., 2010, 12, 2314–2317; ( j) S. J. Lee,
T. M. Anderson and M. D. Burke, Angew. Chem., Int. Ed., 2010, 49,
8860–8863; (k) E. M. Woerly, A. H. Cherney, E. K. Davis and
M. D. Burke, J. Am. Chem. Soc., 2010, 132, 6941–6943; (l) S. Fujii,
21 B. Kirkby, N. Roman, B. Kobe, S. Kellie and J. K. Forwood, Prog. Lipid
Res., 2010, 49, 366–377.
22 (a) A. Adibekian, B. R. Martin, C. Wang, K.-L. Hsu, D. A. Bachovchin,
S. Niessen, H. Hoover and B. F. Cravatt, Nat. Chem. Biol., 2011, 7,
469–478; (b) D. A. Bachovchin, J. T. Mohr, A. E. Speers, C. Wang,
J. M. Berlin, T. P. Spicer, V. Fernandez-Vega, P. Chase, P. S. Hodder,
S. C. Schu¨rer, D. K. Nomura, H. Rosen, G. C. Fu and B. F. Cravatt,
Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 6811–6816.
23 Y. Liu, M. P. Patricelli and B. F. Cravatt, Proc. Natl. Acad. Sci. U. S. A.,
1999, 96, 14694–14699.
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 3608--3611 | 3611