De Novo Design of Boron-Based Peptidomimetics as Potent Inhibitors of Human ClpP in the Presence of Human ClpX

Article abstract of DOI:10.1021/acs.jmedchem.9b00878

Boronic acids have attracted the attention of synthetic and medicinal chemists due to boron's ability to modulate enzyme function. Recently, we demonstrated that boron-containing amphoteric building blocks facilitate the discovery of bioactive aminoboronic acids. Herein, we have augmented this capability with a de novo library design and a virtual screening platform modified for covalent ligands. This technique has allowed us to rapidly design and identify a series of α-aminoboronic acids as the first inhibitors of human ClpXP, which is responsible for the degradation of misfolded proteins.

Full text of DOI:10.1021/acs.jmedchem.9b00878

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Article
De novo design of boron-based peptidomimetics as potent
inhibitors of human ClpP in the presence of human ClpX
Joanne Tan, Julie G Grouleff, Yulia Jitkova, Diego B Diaz, Elizabeth Griffith, Wenjie Shao, Anastasia
F. Bogdanchikova, Gennady Poda, Aaron D Schimmer, Richard E Lee, and Andrei K Yudin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00878 • Publication Date (Web): 12 Jun 2019
Downloaded from http://pubs.acs.org on June 12, 2019
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De novo design of boron-based peptidomimetics as potent inhibitors of human ClpP
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in the presence of human ClpX
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Joanne Tan1, Julie J. Grouleff2, Yulia Jitkova3, Diego B. Diaz1, Elizabeth C. Griffith4, Wenjie
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Shao1, Anastasia F. Bogdanchikova1, Gennady Poda2,5, Aaron D. Schimmer3, Richard E. Lee4 and
Andrei K. Yudin1*
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1Davenport Research Laboratories, Department of Chemistry, University of Toronto, 80 St. George
Street, Toronto, ON, M5S 3H6, Canada, Email: ayudin@chem.utoronto.ca
2Drug Discovery Program, Ontario Institute for Cancer Research, MaRS Centre, 661 University Avenue,
Suite 510, Toronto, ON, M5G 0A3, Canada
3Princess Margaret Cancer Centre, University Health Network, 610 University Avenue, Toronto, ON,
M5G 2M9, Canada
4Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny
Thomas Place, Memphis, TN, 38105-3678, USA
5Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON, M5S 3M2,
Canada
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Abstract
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Boronic acids have attracted the attention of synthetic and medicinal chemists due to boron’s ability to
modulate enzyme function. Recently, we demonstrated that boron-containing amphoteric building blocks
facilitate the discovery of bioactive aminoboronic acids. Herein, we have augmented this capability with
a de novo library design and virtural screening platform modified for covalent ligands. This technique has
allowed us to rapidly design and identify a series of α-aminoboronic acids as the first inhibitors of human
ClpXP, which is responsible for the degradation of misfolded proteins.
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Introduction
Boron-containing molecules (BCMs) have garnered much attention over the years due to their recent
successes as chemical probes and therapeutic agents.1–7 The FDA approval of the multiple myeloma drug
bortezomib8 in 2008 led synthetic and medicinal chemists to reconsider the significance of boron in
therapeutics. The empty p orbital of boronic acids can interact with nucleophilic amino acid residues
forming a tetracoordinate “ate” complex. In addition, tricoordinate boron can adopt various coordination
modes upon biological target engagement.9 This stands in contrast to other electrophiles such as epoxides,
aziridines, and Michael acceptors, which display a singular type of interaction with active site
nucleophiles. Boron is also selective for oxygen nucleophiles, such as serine and threonine residues, which
prevents cross-reactivity with cysteine residues.10,11 Despite the versatility and recent successes of BCM-
driven medicinal chemistry, there are still few examples of boron-containing therapeutic agents. This can
be partially explained by the fact that synthetic technologies to site-selectively introduce boron into
heteroatom-rich environments remain underdeveloped. The thermodynamic preference of boron to migrate
from carbon to oxygen or nitrogen, further aggravated by the low kinetic barrier for these transformations,
also accounts for the dearth of available methods.12,13
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We recently developed a series of amphoteric molecules such as boryl aldehydes,14,15 acyl
boronates16 and boryl isocyanides17 and applied them for rapid construction of bioactive BCMs that
contain numerous heteroatoms in the vicinity of boron. For instance, we have reported the synthesis of
structurally novel boromorpholinone scaffolds from boryl isocyanides and demonstrated their role as
potent inhibitors of the 20S proteasome.17 We have also identified a novel β-aminoboronic acid chemotype
as a cell-active inhibitor of the serine hydrolase, (ox)lipid-metabolizing enzyme α/β-hydrolyase domain 3
(ABHD3), where the N-methyliminodiacetic acid (MIDA) ligand increased the cellular permeability of
the inhibitor.18 To fully leverage the synthetic utility of boron-based amphoteric reagents, we now report
on the integration of this chemistry with a de novo library design and virtual screening program and detail
the discovery of bioactive inhibitors of human caseinolytic protease P.
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Caseinolytic protease P (ClpP) is a highly conserved serine protease present in both bacteria and
eukaryotes.19,20 Its primary role is to maintain cellular homeostasis by working together with its AAA+
chaperones such as ClpA, ClpC and ClpX, to recognize, unfold, and translocate substrates into the
proteolytic chamber of ClpP for protein degradation. In the absence of its chaperones, ClpP can only
degrade small peptides. Thus far, research efforts in small molecule modulation of ClpP have been
primarily directed towards bacteria.19,21 This has led to the development of both activators and inhibitors
of bacterial ClpP which can cause bactericidal inhibition, and impact bacterial virulence. Human ClpP
(hClpP) is overexpressed in the mitochondria of a subset of acute myeloid leukemia (AML) cells and stem
cells.20 Therefore, inhibition or activation of hClpP has the potential to be an effective strategy for
therapeutic intervention of AML.21 In 2015, Sieber and coworkers identified phenyl ester AV167 as a low
micromolar inhibitor of hClpP (IC50 = 1.54 μM).22 More recently, Sieber has shown that TG42, an analogue
of AV167, is a more potent and selective inhibitor of hClpP (IC50 = 0.39 μM) compared to AV167.23
Additionally, TG42 was shown to inhibit the hClpP in the presence an acyldepsipeptide derived chaperone
mimic and Escherichia coli ClpX (EcClpX) complex in a fluorescein-isothiocyanate-labelled casein
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(FITC-casein) degradation assay and GFP degradation assay, respectively.23 Despite inhibiting ClpP in the
presence of its chaperone for the first time, the hClpP-EcClpX complex exhibits bacterial ClpXP substrate
specificity which is more relevant for the development of antibiotics than as a therapeutic strategy for
AML.24 Currently, there are no selective, small molecule inhibitors of hClpP-hClpX complex which may
be attributed to conformational changes in ClpP upon ClpX binding.25,26
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In order to develop boron-containing inhibitors of hClpP, we opted to utilize a de novo design
approach. Small molecule virtual screening has become a valuable tool in drug discovery. The benefit of
this approach is that it negates the need to manually synthesize libraries of structurally diverse molecules,
thus saving time and resources. For the docking of boronic acid-based ligands with hClpP, an important
part of the screening process is to select only compounds which bind in a manner that allows for formation
of a covalent bond between the boronic acid and the hydroxyl group of the catalytically active serine
residue. A number of different software solutions exist for performing covalent docking,27–30 however,
none of them are optimally suited for screening a large user-designed library of covalent ligands, such as
those synthesized using our amphoteric boron reagents. For many programs, only a small set of reactions
can be performed, which limits their applicability. In addition, the side chain conformation of the reactive
residue is often not optimized during the calculations, but instead treated as static, which means that the
covalent docking results are heavily dependent on the chosen conformation of the residue. Furthermore,
the majority of covalent docking methods are prohibitively slow when it comes to screening thousands of
compounds. In 2014, the Shoichet lab developed the webserver DOCKovalent as a covalent docking
program made for screening large libraries.28 However, while it is well-suited for screening predetermined
libraries of commercially available compounds, it cannot readily be used for screening user-designed
libraries, and is thus not suitable for our chemistry. In light of these limitations of covalent docking
programs, we developed a protocol for screening covalent inhibitors using a standard non-covalent
docking method.
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Herein, we show that chemistry derived from our boryl isocyanide building blocks combined with
a de novo library design and virtual screening leads to peptidomimetic boron-based inhibitors of hClpP
with low micromolar potency. The boron-based inhibitors are the first reported inhibitors of the more
therapeutically relevant hClpXP protease.
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Results and Discussion
Substrate-based hClpP inhibitor. The structures of the most celebrated boron-containing
chemotherapeutics currently on the market are based on the α-aminoboronic acid motif. In fact, α-
aminoboronic acids were shown to be selective inhibitors of Mycobacterium tuberculosis ClpP
(MtClpP).31,32 Therefore, we hypothesized that α-aminoboronic acids may also covalently inhibit the
activity of hClpP. We synthesized compound 1, which is an aminoboronic acid analogue of Phe-Ala-Pro-
His-Phe (Figure 1). The boronic acid can interact with the catalytic serine (Ser97) upon binding to the
active site. To synthesize boropeptide 1, we first prepared benzyl-α-amino(MIDA)boronate. Starting from
N-Cbz-benzyl-α-amino(MIDA)boronate, which was prepared according to literature procedures,33
a
hydrogenation with Pd/C was performed to obtain the benzyl-α-amino(MIDA)boronate. N-Ac-Phe-Ala-
Pro-His-B(OH)2 1 was then prepared by coupling benzyl-α-amino(MIDA)boronate and the tetrapeptide
N-Ac-Phe-Ala-Pro-His-OH (See Supporting Information, Scheme S1). Testing of 1 in a fluorescence-
based Ac-Trp-Leu-Ala-AMC (WLA-AMC) assay confirmed that the compound is capable of inhibiting
hClpP with an IC50 value of 27.27 ± 2.07 μM.
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L-Pro
HO
OH
B
O
H
N
L-Ala
O
N
N
H
N
O
O
HN
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N
H
L-His
NH
O
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L-Phe
1
Figure 1: Boronic acid analoge of Phe-Ala-Pro-His-Phe.
Library enumeration. With the knowledge that α-aminoboronic acids display weak inhibitory activity
towards hClpP, we realized that our amphoteric boron building blocks enabled us to synthesize
peptidomimetic α-aminoboronic acids. Using our boryl isocyanide reagents, we performed an Ugi 4-
component reaction (U4CR) with an aldehyde, amine and carboxylic acid.17 The resulting compounds
contain a similar molecular backbone to ClpP’s natural peptide substrates. Based on the U4CR reaction,
a four-component virtual library was enumerated using BIOVIA Pipeline Pilot34 and the available
chemical inventory in our laboratory: 89 aldehydes, 274 carboxylic acids and 8 boronic acid isocyanides
were used (See Supporting Information for structures). The fourth component was kept constant as NH3
in order to generate products that contain a secondary amide, which was shown to participate in
hydrogen bonding to the carbonyl of Ser125 in ClpP’s backbone. Among the aldehyde-containing
building blocks, we kept only the ones with a single aldehyde group and no carboxylic acid functional
groups. Likewise, among the carboxylic acid-containing building blocks, we kept only the ones with a
single carboxylic acid and no aldehyde functional groups. The library enumeration resulted in 127,032
unique chemical structures. To eliminate reactive, chemically unstable at acidic conditions compounds
and compounds with undesirable functional groups and unusual calculated physical properties, we
applied a set of in-house High-Throughput Screening (HTS) Filters, developed in the Drug Discovery
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group at Ontario Institute For Cancer Research as well as a set of lead-like filters based on calculated
physical properties. Application of the filters reduced the virtual library size to 85,063 compounds. In
order to determine which of these compounds would fit into the active site of hClpP, a virtual screen of
the compound library was performed. As previously mentioned, none of the available covalent docking
programs are well-suited for screening our custom library of α-aminoboronic acids. Therefore, we
developed a new protocol for screening covalent inhibitors using a standard non-covalent docking
method using Glide from Schrodinger, Inc.35 The protocol involves three changes as compared to a
regular virtual screening with Glide (Figure 2). For covalent ligands, it is essential that the docking
procedure produces complexes where the reactive group on the ligand is in close proximity of the
reactive residue in the target. However, complexes in which the reactive residue sidechain is as close to
the ligand as it would be in a covalent complex will invariably lead to steric clashes, and thus an
unfavorable docking score. To solve this, the reactive residue, Ser97, is mutated to a glycine prior to the
docking calculation. Second, a distance constraint between the Cα atom of the reactive residue and the
reactive group on the ligand is applied. This allows the reactive group in the ligand to be positioned in
close contact with the reactive residue without the introduction of steric clashes. Lastly, for addition
reactions, such as the reaction between a serine residue and a boronic acid, the geometry of the
attachment atom on the ligand changes. In the case of a boronic acid addition, the boron changes from a
trigonal to a tetrahedral geometry. To mimic this, an artificial conversion of each ligand is performed
prior to the docking, which consists of adding a hydrogen atom to the boron atom. This allows the
docking procedure to test whether the tetrahedral boronates that are formed upon interact with Ser97 will
able to fit into the binding pocket. We specifically chose a hydrogen atom over an oxygen atom because
an oxygen would be added in the form of a hydroxyl group (-OH) or hydroxide which is negatively
charged. In combination, the three described deviations from a standard docking protocol; mutation of
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reactive residue, distance constraint, and compound conversion, provide the basis for using a standard
non-covalent docking program to screen covalent ligands.
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Figure 2: Depiction of the three changes made to a standard covalent docking protocol that makes it
possible to use non-covalent docking to virtually screen large libraries of covalent ligands within a
reasonable time.
The main caveat of this approach is that although the resulting poses will have the reacting atoms
in the vicinity of each other, they will not necessarily be in an optimal position for covalent bond formation.
Therefore, additional visual inspection of the results is needed for determining the most promising hits.
Alternatively, geometric filters can be applied to the docking results, to only select poses that would allow
the reactive residue to adopt a favorable conformer and at the same time keep the distance between the
two reacting atoms close to the value of the equilibrium bond length for such an interaction. Ai et al. have
also published a method for using non-covalent docking for screening covalent ligands, known as Steric-
Clashes Alleviating Receptor (SCAR).36 In SCAR, the reactive residue is also mutated to glycine, however
there is no distance constraint between the reactive groups, and the geometry of the ligands is not changed
prior to the docking calculation. Instead, the docking poses are tested after the calculation and only ligands
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which have the two reactive groups in close vicinity are selected. This means that if the top-scoring poses
of a given ligand does not fulfill the distance criteria it is discarding even though it might also be able to
adopt a slightly less favorable mode in which the two groups do come close together. In our approach, we
avoid such issues, since the distance constraint is applied during the actual docking.
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Virtual screening and compound selection. The method described above was used to screen the U4CR
library against the active site of hClpP. The 1,000 top-ranked compounds based on Glide SP docking score
were subjected to further analysis in order to select compounds for synthesis. Due to the combinatorial
nature of the library, it is possible to separate the analysis into three parts, focusing at finding the most
favorable groups at the either the R1, R2 or R3 position (See Supporting Information, Figure S1). Thus, an
analysis was performed to determine the substituents that appeared most frequently at each of the three
positions. The results of this analysis, combined with a visual inspection of the top-scoring poses to ensure
that bond formation to Ser97 was possible for the chosen R1, R2 and R3 substituents, was used to select a
final set of substitutents predicted to be favorable for each position (See Supporting Information, Figure
S1). Based on the observed binding mode of the docked compounds, the R1 group was expected to impact
the hClpP potency the most, followed by the group at the R3 position. For the R1 position, aliphatic groups
such as isobutyl and n-butyl appeared to be well suited for filling out the S1 pocket. Phenyl and benzyl
groups were predicted to be less favorable at the R1 position compared to aliphatic groups. However, a
compound containing a benzylic group at R1 was still selected for synthesis in order to validate this
hypothesis. For the R2 position, aliphatic and phenyl groups were selected. For the R3 position, aromatic
groups dominated the top-scoring compounds, including phenylic compounds and bicyclic ring systems.
Overall, these results are consistent with Sieber’s findings on hClpP specificity derived from a fluorogenic
library screening.37 The molecules that were selected from this virtual library for synthesis were chosen
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based on this analysis as well as synthetic feasibility and structural diversity (See Supporting Information,
Figure S2).
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Synthesis of α-aminoboronic acids. Based on the in silico screening results and synthetic feasibility, 9
molecules were selected for synthesis (See Supporting Information, Figure S2). We began the synthesis
by subjecting isobutyl- or benzyl-substituted boryl isocyanide 2a and 2b, respectively, to an U4CR with
an aldehyde, carboxylic acid and ammonia in TFE (Scheme 1). From this reaction, we obtained a 1:1
diastereomeric mixture of the desired α-amino(MIDA)boronates 3 which could be separated by normal-
phase column chromatography. The compounds that were below 95% purity as determined by 1H NMR
were purified again by reverse-phase column chromatography. Out of the 9 molecules selected for
synthesis, we were only able to synthesize 6 of them using the U4CR. For more details on the substrate
tolerance of the reaction, please see the Supporting Information.
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MIDA-protected boronates are inactive as serine hydrolase inhibitors due to the tetracoordinate
environment around boron. Thus, MIDA is typically released in situ under the assays conditions or as a
separate synthetic step prior to assay submission. Although α-aminoboronic acids possess the ability to
inhibit serine hydrolases, they are susceptible to degradation through a 1,2-boryl migration.13 However,
acylation at this nitrogen atom engages the lone pair in an amide bond which eliminates the decomposition
pathway. Thus, α-amino(MIDA)boronates 3 should be stable upon MIDA hydrolysis. We began our
experiments by attempting to hydrolyze MIDA under basic conditions.18 We were pleased to see formation
of free boronic acids 4, however, we also saw evidence of boric acid. On the other hand, the hydrolysis
went smoothly with acid to provide the free boronic acid in quantitative yields. It is important to mention
that this deprotection only proceeded at reasonable rates in alcoholic solvents and when using other
solvents, degradation was observed over time. Unfortunately, the second diastereomer of compound 4d
did not readily undergo MIDA hydrolysis due to poor solubility.
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R1
O
R1
O
4
10 eq. HCl
MeOH/MeCN
R1
H
N
H
N
O
O
NH3 in dioxane
TFE, rt, 24 h
R3
HO
R3
[B]
N
H
B
N
H
H
R2
HO
R3
rt, O/N
quant.
[B]
NC
R2
O
OH
R2
O
2a = iBu
2b = Bn
3
7
8
9
OMe
F
O
O
O
H
N
H
N
H
N
HO
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HO
HO
B
N
H
B
N
H
B
N
H
NO2
OH
O
OH
O
Cl
OH
O
4a, 45%, dr 1:1a
4b, 92%, dr 1:1a
4c, 61%, dr 1:1a
OMe
OH
OMe
O
H
O
H
O
H
HO
N
HO
N
HO
N
B
N
H
B
N
H
B
N
H
OH
O
OH
O
OH
O
Cl
4e, 63%, dr 1:1a
4f, 45%, dr 1:1b
4d, 80%, dr 1:1a,c
Scheme 1: Synthesis of α-aminoboronic acids from an U4CR reaction with our boryl isocyanide building
blocks. aSynthesized from compound 2a. bSynthesized from compound 2b. cUnable to access the boronic
acid through MIDA hydrolysis due to poor solubility of the starting material.
Initial testing for the inhibition of human ClpP. The racemic α-aminoboronic acids 4 were screened for
hClpP inhibition in a fluorescence-based WLA-AMC assay in independent triplicates (Table 1). Ac-WLA-
AMC has been recently reported as a substrate for human ClpP that is used for measuring its peptidase
activity by monitoring release of fluorescent amino-methylcoumarin.38 The syn and anti configurations of
compounds 4 were assigned based on the relative positioning of the R1 and R2 side chains. The relative
stereochemistry was assigned after a crystal structure of compound anti-4a bound to Staphylococcus
aureus ClpP (SaClpP) was obtained as described in the next paragraph (Figure 3). Interestingly, only the
second diastereomer anti-4 of this series of compounds exhibited activity towards hClpP whereas the syn-4
diastereomer was inactive. This suggests that stereochemistry plays an important role in binding.
Compounds anti-4a, anti-4b, anti-4c and anti-4e displayed IC50 values of 41.9 ± 2.8, 18.4 ± 5.5, 28.9 ±
5.0 and 34.27 ± 7.84 μM, respectively. Compound anti-4f, which was synthesized to probe the P1 pocket
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of hClpP, was significantly less potent than anti-4a (IC50 = 103.0 ± 31.1 μM). This result confirms that
aliphatic groups are more favorable than benzylic groups at the R1 position as predicted from the virtual
screening results.
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Table 1: IC50 values for the inhibition of hClpP by compounds 4 in WLA-AMC assay. The assay was
performed in independent triplicates.
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hClpP IC50 (μM)
Compound Name
syn-4 (Diastereomer 1)
No inhibition
anti-4 (Diastereomer 2)
41.9 ± 2.8
4a
4b
No inhibition
18.4 ± 5.5
4c
No inhibition
28.9 ± 5.0
4d
No inhibition
N/Aa
4e
No inhibition
34.27 ± 7.84
117.6 ± 1.01
4f
No inhibition
AV167b
4.44 ± 0.18
aUnable to synthesize boronic acid. bAV167 was tested as a positive control.
Crystal structure of ligand-bound bacterial ClpP. Crystallization trials to produce structural data
depicting compound anti-4a bound to hClpP were undertaken but ultimately failed to produce data.
SaClpP and hClpP display high protein sequence homology (See Supporting Information, Figure S3). As
an alternative we utilized previously established SaClpP conditions to co-crystalize anti-4a with SaClpP
to a resolution of 2.0 Å (PBD 6N80; See Supporting Information, Table S1). The structure displayed clear
electron density for anti-4a confirming its binding mode (See Supporting Information, Figure S4). A
comparison of the X-ray structure of anti-4a bound to SaClpP with the docking pose of anti-4a in hClpP
from the non-covalent virtual screening reveals striking similarities (Figure 3). The predicted hydrogen
bonds between the amide group in the compound and the backbone atoms in the protein are also observed
in the X-ray structure. There is also excellent agreement between the predicted and observed interactions
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of the boronic acid group with the protein. The main difference is observed at the R3 position where the
rotation angle of the phenyl ring differs by ~90°. This change is likely due to residue differences between
the SaClpP and hClpP in this part of the binding site, including Thr146/Leu145 and His142/Glu141 (See
Supporting Information, Figure S3, black). Overall, the similarity in binding mode for the docking pose
and the X-ray structure show that our non-covalent protocol for docking covalent compounds is a valuable
tool for predicting protein-ligand interactions and design of covalent inhibitors.
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Figure 3: Binding modes of anti-4a. Left: X-ray structure of anti-4a (purple) bound to the active site of
SaClpP (yellow, PDB:3STA). Right: Predicted binding mode of anti-4a (purple) in the active site of hClpP
(blue, PDB: 1TG6). The compound was docked using the non-covalent docking protocol described in this
paper. The dashed lines repesent hydrogen bonds between the ligand and the protein. The RMSD of
SaClpP and hClpP protein backbone is 1.01 Å. The RMSD of heavy atoms between co-crystallized anti-
4a with SaClpP and docked anti-4a with hClpP is 1.45 Å.
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Synthesis of enantiomerically pure α-aminoboronates. Based on the results of the initial hClpP
screening, compounds anti-4a, anti-4b and anti-4c were chosen for follow up studies. We decided to
pursue the synthesis of the enantiopure variants given that is it likely we will see improvements in the
potency. We began the synthesis with the protection of isobutyl boronic acid with (+)-pinanediol and (-)-
pinanediol to afford (S)-pinanediol-2-methylpropane-1-boronate and (R)-pinanediol-2-methylpropane-1-
boronate, respectively. Next, the Matteson homologation was performed using dichloromethane and n-
butyllithium followed by zinc chloride addition to obtain the (S)-chloroboronate ester and (R)-
chloroboronate ester in high diastereoselectivity (>95:5 based on HPLC of compounds 7) from (S)-
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pinanediol-2-methylpropane-1-boronate
and
(R)-pinanediol-2-methylpropane-1-boronate,
respectively.39,40 The (S)-chloroboronate ester was then converted to the (R)-(N-TMS)2-aminoboronate
ester through an SN2 reaction with LHMDS. Likewise, the (R)-chloroboronate ester was converted to the
(S)-(N-TMS)2-aminoboronate ester. The trimethylsilyl groups were removed using excess trifluoroacetic
acid to provide (R)-aminoboronate ester 5a and (S)-aminoboronate 5b as the TFA salts (See Supporting
Information, Scheme S2). At this point, we presumed there was no erosion of the enantiopurity and carried
on to the next steps. The corresponding carboxylic acids 6 were prepared using standard Fmoc solid-phase
synthesis with 2-chlorotrityl resin, HATU and DIPEA for the coupling, 30% piperidine in DMF for the
Fmoc removal, and 25% HFIP in DCM for the resin cleavage (See Supporting Information for a list of
carboxylic acids). Carboxylic acids 6a-c were coupled to (R)-aminoboronate ester 5a using TBTU and
DIPEA in DCM to yield α-aminoboronates 7a-c (Scheme 2). Carboxylic acid 6d was coupled to (S)-
aminoboronate ester 5b using the same protocol to afford α-aminoboronate 7d as the enantiomer of 7a.
The diastereoselectivity of α-aminoboronates 7 was analyzed by HPLC to be >95:5 which indicates there
was no epimerization during the coupling step. Lastly, the α-aminoboronates 7a-d were deprotected with
10 equivalents of aqueous HCl and phenylboronic acid for effective pinanediol transfer in hexanes:MeOH
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(1:1) at room temperature to provide the α-aminoboronic acids 8a-d in excellent yields.
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8
9
NH2·TFA
O
O
10 eq. HCl
PhB(OH)2
H
R2
H
TBTU, DIPEA
DCM, 0 °C
O
8
O
N
O
N
R2
B
H
B
N
R2
HO
HO
N
H
Hexanes:MeOH (1:1)
rt, O/N
O
R1
O
B
N
O
R1
6
O
H
OH
R1
O
5
7
a: (+)-pinanediol
b: (-)-pinanediol
a: (+)-pinanediol
b: (-)-pinanediol
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OMe
F
OMe
O
H
O
H
N
O
O
H
H
HO
N
HO
HO
N
HO
N
B
N
B
N
B
N
NO2
B
N
H
H
H
H
OH
O
OH
O
OH
O
Cl
Ph
OH
O
Cl
8ac, 78%a, >95:5 erb
8bc, 62%a, >95:5 erb
8cc, 34%a, >95:5 erb
8dd,e, 78%a, >95:5 erb
Scheme 2: Synthesis of enantiopure α-aminoboronates and boronic acids. aYield is reported over two steps
b
c
d
from 5. er is based on the dr of 7 from the HPLC trace. Synthesized from 5a. Synthesized from 5b.
eSynthesized from 6d containing D-leu.
Testing of enantiomerically pure α-aminoboronic acids for inhibition of human ClpP. With the
enantiomerically pure α-aminoboronic acids 8 in hand, we began to screen them along with the
corresponding racemate in the WLA-AMC assay. The IC50 values of compounds 8a, 8b and 8c were 10.2
± 3.7, 8.4 ± 2.6 and 12.4 ± 0.1 μM, respectively (Figure 4a). Therefore, the enantiomerically pure α-
aminoboronic acids are up to four times more potent than the racemate. Compound 8d, which is the
enantiomer of 8a, was unable to inhibit hClpP peptidase activity. This suggests that stereochemistry is
important for binding to the hClpP active site.
Testing for inhibition of hClpP in the presence of hClpX. ClpXP performs protein degradation in cells20
whereas ClpP can only degrade small peptides.19 In 2015, Sieber and coworkers showed that AV167 was
a potent inhibitor of hClpP; however, it was unable to inhibit hClpXP proteolysis.22 More recently, TG42
was shown to inhibit the hClpP-EcClpX complex;23 however, the hClpP-EcClpP complex displays
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bacterial ClpXP substrate specificity and therefore, cannot degrade casein.24,25 Thus, the development of
selective, small molecule inihbitors of hClpP in the presence of hClpX is important in order to study the
therapeutic relevance of hClpXP. We began by subjecting the enantiopure compounds 8a, 8b and 8c and
racemates anti-4a, anti-4b and anti-4c to a hClpXP FITC-casein assay in independent triplicates. ClpXP-
induced proteolysis occurs through binding of a chaperone such as ClpX to the proteolytic component
ClpP whereupon a functional ClpXP protease complex is formed. This assay is aimed at detecting the
presence of protease activity of ClpXP by using casein labeled with fluorescein isothiocyanate as a
substrate. The fluorescein label on the FITC-casein is highly quenched until it is digested into smaller
peptides by ClpXP.41 The IC50 values for inhibition of hClpXP protease activity of the enantiopure α-
aminoboronic acids 8a, 8b and 8c are 0.8 ± 0.3 μM , 0.9 ± 0.4 μM and 1.0 ± 0.3 μM, respectively (Figure
4b). These results indicate that α-aminoboronic acids are potent inhibitors of hClpXP and are unique in
their ability to inhibit the holoenzyme.26
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Inhibition of peptidase activity of hClpP
a)
Inhibition of protease activity of hClpP
b)
in the presence of hClpX
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0
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Figure 4: a) Inhibition of hClpP by racemic and enantiopure α-aminoboronic acids anti-4 and 8,
respectively, in a WLA-AMC assay. The assay was performed in independent triplicates. b) Inhibition of
hClpXP by racemic and enantiopure α-aminoboronic acids anti-4 and 8, respectively, in a FITC-casein
assay. The assay was performed in independent triplicates.
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Conclusions
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For the first time, we have exemplified the power of combining our boron-based amphoteric reagents with
a de novo library design and virtual screening in order to rapidly identify and generate bioactive boron-
containing molecules for human ClpP and hClpP-hClpX complex. More specifically, we utilized our boryl
isocyanides in a multicomponent Ugi reaction to synthesize peptidomimetic α-aminoboronic acids while
the de novo virtual screening effectively analyzed and filtered all possible α-aminoboronic acid candidates.
By integrating these two capabilities, we have successfully identified three α-aminoboronic acids as potent
inhibitors of hClpP and hClpXP with IC50 values in the low micromolar range. These IC50 values were
comparable to AV167 in our testing against hClpP but as opposed to AV167 and TG42, the α-
aminoboronic acids also inhibited hClpXP. To the best of our knowledge, these α-aminoboronic acids are
the first reported inhibitors of hClpXP. This work serves as a starting point for the development of more
synthetically complex α-aminoboronic acid inhibitors of hClpXP.
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Experimental Section
Chemistry. Synthesis. Methylene chloride (DCM), methanol (MeOH) and triethylamine were distilled
from CaH2 under nitrogen. Acetonitrile (MeCN) was distilled from activated 4Å MS under nitrogen.
Tetrahydrofuran (THF) was freshly distilled from sodium benzophenone ketyl. Amino acid reagents were
sourced from Chem-Impex International Inc. Peptide grade DIPEA was sourced from Sigma Aldrich.
Peptide grade DMF were sourced from Caledon Laboratories Ltd. All other solvents were of reagent grade
quality and dried over 4Å MS prior to use. All reagents were purchased from commercial sources and used
as received. Flash column chromatography was carried out using Silicycle 230-400 mesh silica gel, or
ISCO Teledyne Combiflash Rf 200 Flash system. Thin-layer chromatography (TLC) was performed on
Macherey Nagel pre-coated glass backed TLC plates (SIL G/UV254, 0.25 mm) and visualized using a UV
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lamp (254 nm), KMnO4 or curcumin stain (preparation: 200 mg curcumin in 100 mL ethanol). Reverse-
phase chromatography was carried out using RediSep Rf Gold C18 Columns. 1H NMR, 13C, and 2D NMR
spectra were recorded on Varian Mercury 300 MHz, 400 MHz, 500 MHz, 600 MHz or 700 MHz
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spectrometers. B NMR were recorded using Bruker 400/500 MHz spectrometer at 128/160 MHz and
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referenced to an external standard of BF3·Et2O (δ = 0 ppm). F NMR were recorded using Bruker 400
1
MHz spectrometer at 376 MHz and referenced to an external standard of CFCl3 (δ = 0 ppm). H NMR
spectra chemical shifts (δ) are reported in parts per million (ppm) referenced to residual protonated solvent
peak (CD3CN δ = 1.94, DMSO-d6, δ = 2.49, CD3OD δ = 3.31, CDCl3 δ = 7.26; center line). Spectral data
is reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd =
doublet of doublets, dt = doublet of triplets, ddt = doublet of doublet of triplets, dtd = doublet of triplet of
doublets, m = multiplet, br = broad), coupling constant (J) in Hertz (Hz), and integration. 13C NMR spectra
chemical shifts (δ) are reported in parts per million (ppm) were referenced to carbon resonances in the
NMR solvent (CD3CN δ = 118.3, DMSO-d6, δ = 39.5, CD3OD δ = 49.0, CDCl3 δ = 77.2; center line).
Carbons exhibiting significant line broadening brought about by boron substituents were not reported
(quadrupolar relaxation). High resolution mass spectra were obtained on a VG 70- 250S (double focusing)
mass spectrometer at 70 eV or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and
accurate mass capabilities. Low resolution mass spectra were obtained on an Agilent Technologies 1200
series HPLC paired to a 6130 Mass Spectrometer. Low-resolution mass spectra (ESI) were collected on
an Agilent Technologies 1200 series HPLC paired to a 6130 Mass Spectrometer. Compounds were
resolved on Phenomenex’s Kinetex 2.6u C18 50x4.6mm column at room temperature with a flow of 1
mL/min. The gradient consisted of eluents A (0.1% formic acid in double distilled water) and B (0.1%
formic acid in HPLC-grade acetonitrile). Method A: The flow rate was held at 1.0 mL/min. From 0 min to
4 min, was used a linear gradient starting from 5% of B to 95% of B. From 4 min to 5 min, the solvent
was held at 95% of B. From 5 min to 5.5 min, was used a linear gradient from 95% of B to 5% of B. From
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5.5 min to 6.5 min, the solvent was held at 5% of B. Method B: The flow rate was held at 1.5 ml/min. From
0 min to 15 min, was used a linear gradient starting from 5% of B to 95% of B. From 15 min to 19.5 min,
the solvent was held at 95% of B. From 19.5 min to 20 min, the solvent was held at 5% of B. Method C:
A linear gradient starting from 5% of B to 95% over 15 min at a flow rate of 1.0 mL/min. All compounds
synthesized in this manuscript are >95% in purity unless otherwise stated. The purity was assessed using
1H NMR and/or HPLC as listed above.
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Synthesis of N-Ac-Phe-Ala-Pro-His(N-Trt)-Phe-B(OH)2 (1). To a solution of N-Cbz-benzyl-α-
amino(MIDA)boronate33 (649 mg, 1.58 mmol, 1.0 equiv) in acetic acid (15 mL) was added palladium
hydroxide 20% on carbon (111 mg, 0.158 mmol, 0.1 equiv). The reaction mixture was stirred at room
temperature under an atmosphere of H2(g) for 3 h until the reaction was complete as indicated by LC-MS.
The reaction mixture was filtered through a pad of Celite, rinsing with a total of 100 mL of acetonitrile.
The filtrate was concentrated to dryness to afford the crude product as a yellowish solid in 87% yield
(462 mg, 1.37 mmol). Benzyl-α-amino(MIDA)boronate was used in the next step without further
purification. The N-acylated tetrapeptide [N-Ac-Phe-Ala-Pro-His(N-Trt)-OH] was assembled on 2-
chlorotrityl chloride resin with manual loadings of each amino acid. The linear peptide was synthesized
by conventional Fmoc solid phase-based peptide synthesis using single coupling steps with HATU in
DIPEA. The peptide was cleaved from the resin by three successive washes, 0.5 h apart, with 1:4
HFIP/DCM. The cleavage layers were then evaporated and the tetrapeptide precipitated from diethyl
ether. The tetrapeptide (102 mg, 0.135 mmol, 1.0 equiv) was added to a 2 Dram vial equipped with a
magnetic stir and was dissolved in 2 mL of anhydrous DMF. In a separate vial, benzyl-α-
amino(MIDA)boronate (56 mg, 0.167 mmol, 1.23 equiv) was stirred with Amberlite IRA743 free base in
1 mL of DMF for 20 minutes. The benzyl-α-amino(MIDA)boronate solution was then transferred via
syringe to the vial containing the tetrapeptide. HATU (83.9 mg, 0.203 mmol, 1.5 equiv) and 2,6-lutidine
(47 L, 0.406 mmol, 3.0 equiv) were then added, and the reaction mixture was left to stir at room
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temperature for 14 h. The reaction mixture was concentrated to about 1 mL of DMF and subsequently
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purified by C18 reverse-phase chromatography using a 0%  75% acetonitrile gradient in water over 45
minutes. The fractions containing desired product were then lyophilized to afford the MIDA-protected
boropeptide [N-Ac-Phe-Ala-Pro-His(N-Trt)-Phe-B(MIDA)] (51 mg, 50.3 µmol) in 37.2% yield as a
white powder. NMR and LC-MS analysis confirmed that the MIDA-protected boropeptide was produced
in a 1:1 mixture of diastereomers. The purity of N-Ac-Phe-Ala-Pro-His(N-Trt)-Phe-B(MIDA) was
assessed by HPLC using Method A. Quantitative deprotection of the N-Trt-protected His side chain and
the MIDA-group of N-Ac-Phe-Ala-Pro-His(N-Trt)-Phe-B(MIDA) was achieved using a solution of 3 M
HCl (105 L, 10 equiv) in 5:1 MeOH:H2O (1 mL) solvent mixture with stirring for 12 h. The reaction
mixture was then purified by C18 reverse-phase chromatography using a 0%  50% acetonitrile
gradient in water over 45 minutes. The fractions containing desired product were then lyophilized to
afford N-Ac-Phe-Ala-Pro-His(N-Trt)-Phe-B(OH)2 1 (20.8 mg, 31.6 mol) in quantitative yield as a white
powder. The purity of 1 was assessed by HPLC using Method C.
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Synthesis of boryl isocyanides (2). Boryl isocyanides were prepared using the following modified
procedure.17 To a flame-dried, two-neck round-bottom flask equipped with a magnetic stir bar under with
nitrogen atmosphere, was added freshly distilled DCM (8 mL) which was cooled to 0 °C. Freshly
distilled trichlorosilane (3.31 mmol, 0.33 mL, 2.0 eq.) was added followed by dropwise addition of
triethylamine (4.13 mmol, 0.58 mL, 2.5 eq.). In a separate flame-dried flask flushed with nitrogen was
added α-boryl isocyanate (1.65 mmol, 0.50 g, 1.0 eq.) and triethylamine (4.13 mmol, 0.58 mL, 2.5 eq.) in
DCM (8 mL) and the flask was cooled to 0 °C. The α-boryl isocyanate solution was slowly transferred to
the trichlorosilane flask via cannula while maintaining the temperature at 0 °C. The resulting suspension
was stirred vigorously at 0°C for 30 min. The resulting solution was allowed to warm to rt. Stirring was
continued until starting material was completely consumed as indicated by TLC or crude 1H NMR. The
reaction solution was cooled to 0 °C and then 1 mL of triethylamine was added. Next, saturated aq.
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NaHCO3 was added slowly. The resulting suspension was stirred at 0 °C until bubbling ceased at which
point it was allowed to warm to rt. The DCM layer was removed in vacuo. The aqueous layer was
extracted with EtOAc (x3). The combined organic layers were washed with saturated aq. NaHCO3/H2O
(50/50) and saturated aq. NaHCO3/brine (50/50), dried over Na2SO4, filtered and concentrated. The
crude residue was purified by flash column chromatography on neutralized silica using hexanes:ethyl
acetate with 1% triethylamine to yield the desired product 2. Characterization of 2a matched previous
literature reports.17
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MIDA (1-isocyano-2-phenylethyl)boronate (2b). White solid; yield = 296 mg, 63%; Rf = 0.22 (4:1
DCM:Ace). 1H NMR (500 MHz, Acetonitrile-d3) δ 7.38 – 7.32 (m, 4H), 7.29 – 7.26 (m, 1H), 4.13 (dd, J
= 17.2, 11.9 Hz, 2H), 3.98 (dd, J = 17.2, 9.9 Hz, 2H), 3.51 (brd, J = 11.4 Hz, 1H), 3.09 (s, 3H), 3.04 (dt,
J = 14.2, 3.9 Hz, 1H), 2.80 (dd, J = 14.2, 11.9 Hz, 1H). 13C NMR (126 MHz, Acetonitrile-d3) δ 168.4,
168.4, 158.3 (t, J = 4.8 Hz), 139.9, 130.0, 129.3, 127.6, 63.9, 63.7, 47.2, 37.2. 11B NMR (160 MHz,
Acetonitrile-d3) δ 10.1. HRMS (DART-TOF+): m/z for C14H16BN2O4: calcd 287.1203, observed
287.1198 [M+H].
General procedure for the synthesis of racemic B(MIDA) compounds from boryl isocyanides (3).
In a 20 mL vial equipped with a magnetic stir bar under nitrogen atmosphere, was added boryl
isocyanide 2a or 2b (1.0 eq.), a carboxylic acid (2.0 eq.), an aldehyde (2.0 eq.) and ammonia (0.5 M in
1,4-dioxane, 2.0 eq.) in TFE (4 mL, 0.1 M). The reaction was allowed to stir at room temperature for up
to 3 days or until the reaction went to completion. The reaction was monitored by TLC and LC-MS. The
solvent was removed in vacuo and then loaded onto Celite. The crude mixture was purified by flash
column chromatography or Combiflash using hexanes:ethyl acetate to afford syn-3 and anti-3 as pure
diastereomers.
MIDA (1-(2-(2-chloro-4-methoxybenzamido)-4-methylpentanamido)-3-methylbutyl)boronate (3a).
White solid; yield = 47 mg, 45% (1:1 dr); syn-3a: Rf = 0.18 (1:9 hex:EtOAc). 1H NMR (500 MHz,
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Acetonitrile-d3) δ 7.41 (d, J = 8.6 Hz, 1H), 7.04 (brd, J = 7.0 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H), 6.90 (dd,
J = 8.6, 2.5 Hz, 1H), 6.39 (brd, J = 10.2 Hz, 1H), 4.25 (ddd, J = 10.5, 7.1, 4.6 Hz, 1H), 4.19 (d, J = 16.6
Hz, 1H), 3.94 (d, J = 17.3 Hz, 1H), 3.90 (d, J = 16.6 Hz, 1H), 3.82 (d, J = 17.2 Hz, 1H), 3.82 (s, 3H),
3.67 (ddd, J = 12.0, 10.2, 3.1 Hz, 1H), 2.90 (s, 3H), 1.79 – 1.71 (m, 1H), 1.65 – 1.53 (m, 2H), 1.50 –
1.44 (m, 2H), 1.28 (ddd, J = 13.9, 10.5, 3.1 Hz, 1H), 0.94 (d, J = 6.5 Hz, 3H), 0.93 (d, J = 6.4 Hz, 3H),
0.90 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.5 Hz, 3H). 13C NMR (126 MHz, Acetonitrile-d3) δ 173.1, 169.2,
169.0, 167.9, 162.2, 132.7, 131.6, 128.7, 116.2, 113.7, 63.7, 63.0, 56.5, 54.3, 46.6, 41.4, 41.0, 25.6, 25.2,
24.1, 23.4, 21.7, 21.4. 11B NMR (160 MHz, Acetonitrile-d3) δ 11.7. anti-3a: Rf = 0.14 (EtOAc). 1H
NMR (500 MHz, Acetonitrile-d3) δ 7.50 (d, J = 8.6 Hz, 1H), 7.01 (m, 2H), 6.92 (dd, J = 8.6, 2.5 Hz,
1H), 6.25 (brd, J = 10.1 Hz, 1H), 3.94 (d, J = 17.2 Hz, 1H), 3.92 (d, J = 16.8 Hz, 1H), 3.86 (d, J = 17.2
Hz, 1H), 3.83 (s, 3H), 3.77 (d, J = 16.8 Hz, 1H), 3.73 – 3.67 (m, 1H), 2.96 (s, 3H), 1.76 (ddq, J = 13.0,
7.5, 6.6 Hz, 1H), 1.62 – 1.59 (m, 2H), 1.56 (dp, J = 10.2, 3.5 Hz, 1H), 1.45 (ddd, J = 14.1, 12.1, 3.3 Hz,
1H), 1.28 (ddd, J = 13.9, 10.5, 3.1 Hz, 2H), 0.94 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.5 Hz, 4H), 0.88 (d, J
= 6.7 Hz, 4H), 0.87 (d, J = 6.5 Hz, 4H). 13C NMR (126 MHz, Acetonitrile-d3) δ 172.5, 168.9, 168.9,
167.9, 162.4, 132.6, 132.0, 128.4, 116.1, 113.8, 63.5, 63.1, 56.5, 54.5, 46.7, 41.5, 41.3, 25.6, 25.0, 24.2,
23.3, 21.6, 21.5. 11B NMR (160 MHz, Acetonitrile-d3) δ 11.6. HRMS (ESI, positive): m/z for
C24H36BClN3O7: calcd 523.2366, observed 523.2365 [M+H].
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MIDA (1-(2-([1,1'-biphenyl]-2-carboxamido)-4-methylpentanamido)-3-methylbutyl)boronate (3b).
White solid; yield = 90 mg, 92% (1:1 dr); syn-3b: Rf = 0.71 (EtOAc). 1H NMR (500 MHz, Acetonitrile-
d3) δ 7.51 (td, J = 7.4, 1.7 Hz, 1H), 7.46 – 7.43 (m, 2H), 7.42 – 7.38 (m, 5H), 7.37 – 7.33 (m, 1H), 6.67
(brd, J = 6.7 Hz, 1H), 6.23 (brd, J = 10.2 Hz, 1H), 4.13 (d, J = 16.6 Hz, 1H), 3.99 (ddd, J = 11.0, 6.8, 4.1
Hz, 1H), 3.90 (d, J = 17.2 Hz, 1H), 3.82 (d, J = 16.5 Hz, 1H), 3.78 (d, J = 17.2 Hz, 1H), 3.63 (ddd, J =
12.0, 10.2, 3.1 Hz, 1H), 2.78 (s, 3H), 1.52 (dtt, J = 13.3, 6.6, 3.1 Hz, 1H), 1.48 – 1.33 (m, 2H), 1.28 –
1.23 (m, 2H), 1.20 – 1.10 (m, 1H), 0.87 (dd, J = 16.2, 6.6 Hz, 6H), 0.77 (dd, J = 15.6, 6.5 Hz, 6H). 13C
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NMR (126 MHz, Acetonitrile-d3) δ 173.2, 171.1, 169.3, 168.9, 141.4, 140.7, 137.0, 131.1, 130.8, 129.5
(2C), 129.4 (2C), 129.1, 128.4, 128.2, 63.7, 62.9, 54.2, 46.45, 41.4, 40.8, 25.2, 25.0, 24.1, 23.5, 21.6,
21.4. 11B NMR (128 MHz, Acetonitrile-d3) δ 11.5. anti-3b: Rf = 0.53 (EtOAc). 1H NMR (400 MHz,
Acetonitrile-d3) δ 7.56 (td, J = 7.4, 1.4 Hz, 1H), 7.52 (dd, J = 7.5, 1.5 Hz, 1H), 7.46 – 7.38 (m, 7H), 6.43
(brd, J = 6.3 Hz, 1H), 6.07 (brd, J = 10.2 Hz, 1H), 4.00 – 3.95 (m, 3H), 3.83 (dd, J = 17.0, 10.8 Hz, 2H),
3.67 (ddd, J = 12.9, 10.4, 3.1 Hz, 1H), 2.94 (s, 3H), 1.51 – 1.41 (m, 2H), 1.28 (dtd, J = 14.3, 11.1, 10.3,
6.3 Hz, 2H), 1.16 (ddd, J = 13.7, 10.2, 4.7 Hz, 1H), 1.03 – 0.91 (m, 1H), 0.85 (dd, J = 12.7, 6.5 Hz, 6H),
0.72 (dd, J = 16.9, 6.5 Hz, 6H). 13C NMR (126 MHz, Acetonitrile-d3) δ 172.6, 171.3, 169.1, 168.9,
141.5, 140.6, 136.6, 131.0, 131.0, 129.6 (2C), 129.4 (2C), 129.4, 128.5, 128.3, 63.5, 63.1, 54.6, 46.7,
41.4, 41.3, 25.0, 24.8, 24.1, 23.3, 21.9, 21.4. 11B NMR (128 MHz, Acetonitrile-d3) δ 11.5. HRMS (ESI,
positive): m/z for C29H39BN3O6: calcd 535.2963, observed 535.2962 [M+H].
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MIDA (1-(2-(4-fluoro-3-nitrobenzamido)-4-methylpentanamido)-3-methylbutyl)boronate (3c).
Yellow solid; yield = 62 mg, 61% (1:1 dr); syn-3c: Rf = 0.19 (1:9 hex:EtOAc). 1H NMR (500 MHz,
Acetonitrile-d3) δ 8.49 (ddd, J = 7.2, 2.3, 1.2 Hz, 1H), 8.15 (ddd, J = 8.7, 4.2, 2.3 Hz, 1H), 7.52 – 7.45
(m, 2H), 6.43 (brd, J = 9.7 Hz, 1H), 4.31 – 4.27 (m, 1H), 4.15 (d, J = 16.6 Hz, 1H), 3.95 (dd, J = 10.1,
1.2 Hz, 1H), 3.92 (dd, J = 9.5, 1.2 Hz, 1H), 3.83 (dd, J = 17.2, 1.1 Hz, 1H), 3.66 (ddd, J = 12.0, 10.1, 3.1
Hz, 1H), 2.87 (s, 3H), 1.81 – 1.70 (m, 2H), 1.60 – 1.44 (m, 3H), 1.28 (ddd, J = 13.9, 10.4, 3.1 Hz, 1H),
0.97 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.5 Hz, 4H), 0.90 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.5 Hz, 3H). 13C
NMR (126 MHz, Acetonitrile-d3) δ 173.1, 169.2, 169.0, 165.6, 157.9 (d, 1JC-F = 266.7 Hz), 138.0 (d, 4JC-
F = 8.0 Hz), 135.9 (d, 3JC-F = 10.0 Hz), 131.9 (d, 2JC-F = 3.9 Hz), 126.5 (d, 3JC-F = 2.0 Hz), 119.6 (d, 2JC-F
= 21.6 Hz), 63.7, 63.0, 54.7, 46.4, 41.4, 40.8, 25.6, 25.3, 24.1, 23.4, 21.7, 21.5. 11B NMR (128 MHz,
Acetonitrile-d3) δ 11.8. 19F NMR (470 MHz, Acetonitrile-d3) δ -115.7 (ddd, J = 11.2, 7.3, 4.0 Hz). anti-
3c: Rf = 0.19 (EtOAc). 1H NMR (400 MHz, Acetonitrile-d3) δ 8.56 (dd, J = 7.2, 2.3 Hz, 1H), 8.19 (ddd,
J = 8.7, 4.3, 2.3 Hz, 1H), 7.49 (dd, J = 10.9, 8.8 Hz, 1H), 7.47 (brd, J = 6.5 Hz, 1H), 6.23 (brd, J = 10.1
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Hz, 1H), 4.31 (ddd, J = 9.6, 6.6, 5.3 Hz, 1H), 3.95 – 3.73 (m, 4H), 3.68 (ddd, J = 12.1, 10.2, 3.3 Hz, 1H),
2.96 (s, 3H), 1.80 – 1.73 (m, 1H), 1.71 – 1.63 (m, 2H), 1.53 (ddt, J = 13.4, 6.5, 3.6 Hz, 1H), 1.46 (ddd, J
= 14.0, 12.1, 3.2 Hz, 1H), 1.28 – 1.23 (m, 1H), 0.96 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H), 0.86
(dd, J = 6.6, 5.6 Hz, 6H). 13C NMR (126 MHz, Acetonitrile-d3) δ 172.6, 169.1, 168.7, 165.8, 158.0 (d, J
= 266.8 Hz), 136.2 (d, J = 10.0 Hz), 131.6 (d, J = 3.8 Hz), 126.8 (d, J = 2.1 Hz), 119.5 (d, J = 21.6 Hz),
63.6, 63.1, 55.3, 46.78, 41.1, 41.1, 25.6, 25.0, 24.1, 23.1, 21.7, 21.4. 11B NMR (128 MHz, Acetonitrile-
d3) δ 11.6. 19F NMR (377 MHz, Acetonitrile-d3) δ -115.5 (q, J = 5.6 Hz). HRMS (ESI, positive): m/z for
C23H33BFN4O8: calcd 522.2406, observed 522.2397 [M+H].
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MIDA (3-methyl-1-(4-methyl-2-(1-phenylcyclopropane-1-
carboxamido)pentanamido)butyl)boronate (3d). White solid; yield = 48 mg, 80% (1:1 dr); syn-3d: Rf
= 0.33 (EtOAc). 1H NMR (500 MHz, Acetonitrile-d3) δ 7.43 – 7.36 (m, 4H), 7.36 – 7.30 (m, 1H), 6.26
(brd, J = 10.1 Hz, 1H), 5.76 (brd, J = 7.3 Hz, 1H), 4.08 (q, J = 7.4 Hz, 1H), 4.05 (d, J = 16.6 Hz, 1H),
3.96 (d, J = 17.2 Hz, 1H), 3.90 (d, J = 16.6 Hz, 1H), 3.83 (d, J = 17.2 Hz, 1H), 3.60 (ddd, J = 11.9, 10.0,
3.1 Hz, 1H), 2.85 (s, 3H), 1.50 (ddq, J = 13.3, 6.6, 3.2 Hz, 1H), 1.45 (ddd, J = 9.8, 6.4, 3.2 Hz, 1H), 1.41
(dd, J = 5.3, 1.8 Hz, 1H), 1.40 – 1.38 (m, 1H), 1.36 (dt, J = 6.6, 3.3 Hz, 1H), 1.34 – 1.30 (m, 2H), 1.25
(ddd, J = 13.9, 10.5, 3.1 Hz, 1H), 1.06 (ddd, J = 9.7, 6.4, 3.2 Hz, 1H), 1.00 (ddd, J = 9.4, 6.4, 3.2 Hz,
1H), 0.88 (d, J = 6.7 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H), 0.81 (d, J = 1.3 Hz, 3H), 0.80 (d, J = 1.3 Hz, 3H).
13C NMR (126 MHz, Acetonitrile-d3) δ 174.7, 172.9, 169.2, 169.0, 140.6, 131.6 (2C), 129.9 (2C), 128.8,
63.7, 63.0, 54.1, 46.4, 41.4, 41.0, 31.0, 25.6, 25.2, 24.1, 23.3, 22.0, 21.4, 15.8, 15.7. 11B NMR (128
MHz, Acetonitrile-d3) δ 11.5. anti-3d: Rf = 0.14 (EtOAc). 1H NMR (500 MHz, Acetonitrile-d3) δ 7.46 –
7.44 (m, 2H), 7.42 – 7.38 (m, 2H), 7.36 – 7.33 (m, 1H), 5.93 (brd, J = 10.3 Hz, 1H), 5.79 (brd, J = 6.7
Hz, 1H), 4.07 – 4.03 (m, 1H), 3.95 (d, J = 17.2 Hz, 1H), 3.94 (d, J = 16.7 Hz, 1H), 3.84 (d, J = 17.2 Hz,
1H), 3.72 (d, J = 16.8 Hz, 1H), 3.61 (ddd, J = 12.1, 10.2, 3.1 Hz, 1H), 2.88 (s, 3H), 1.48 – 1.20 (m, 8H),
1.10 (ddd, J = 7.6, 5.8, 2.8 Hz, 1H), 0.99 (ddd, J = 9.4, 5.9, 2.5 Hz, 1H), 0.87 (d, J = 6.7 Hz, 3H), 0.83
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(d, J = 6.5 Hz, 3H), 0.81 (d, J = 4.4 Hz, 3H), 0.80 (d, J = 4.3 Hz, 3H). 13C NMR (126 MHz, Acetonitrile-
d3) δ 175.3, 172.8, 169.0, 168.8, 140.4, 132.0 (2C), 129.9 (2C), 128.8, 63.6, 63.2, 54.9, 46.67, 41.5, 40.9,
31.0, 25.6, 24.9, 24.1, 23.2, 21.7, 21.4, 16.5, 16.0. 11B NMR (128 MHz, Acetonitrile-d3) δ 11.6. HRMS
(DART, TOF+): m/z for C26H39BN3O6: calcd 500.2931, observed 500.2928 [M+H].
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MIDA (1-(2-(2-hydroxy-5-methoxybenzamido)-4-methylpentanamido)-3-methylbutyl)boronate
(3e). White solid; yield = 74 mg, 63% (1:1 dr); syn-3e: Rf = 0.53 (EtOAc). 1H NMR (400 MHz,
Acetonitrile-d3) δ 11.55 (brs, 1H), 7.69 (brd, J = 6.9 Hz, 1H), 7.19 (d, J = 3.0 Hz, 1H), 6.99 (dd, J = 9.0,
3.0 Hz, 1H), 6.83 (d, J = 9.0 Hz, 1H), 6.52 (brd, J = 10.1 Hz, 1H), 4.37 (ddd, J = 10.2, 6.9, 4.7 Hz, 1H),
4.03 (d, J = 16.3 Hz, 1H), 3.98 (d, J = 3.6 Hz, 1H), 3.93 (d, J = 2.9 Hz, 1H), 3.83 (d, J = 17.2 Hz, 1H),
3.76 (s, 3H), 3.69 (ddd, J = 11.9, 10.1, 3.0 Hz, 1H), 2.89 (s, 3H), 1.81 – 1.72 (m, 2H), 1.63 – 1.53 (m,
1H), 1.53 – 1.43 (m, 1H), 1.29 (ddd, J = 13.8, 10.4, 3.0 Hz, 1H), 0.96 (d, J = 6.3 Hz, 3H), 0.91 (d, J =
6.4 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H). 13C NMR (126 MHz, Acetonitrile-d3) δ
172.9, 171.1, 169.1, 168.8, 156.1, 152.9, 122.7, 119.6, 115.0, 111.3, 63.5, 63.0, 56.6, 54.2, 46.4, 41.3,
40.8, 25.6, 25.3, 24.1, 23.4, 21.6, 21.4. 11B NMR (128 MHz, Acetonitrile-d3) δ 11.8. anti-3e: Rf = 0.10
(EtOAc). 1H NMR (400 MHz, Acetonitrile-d3) δ 11.37 (brs, 1H), 7.55 (brd, J = 6.9 Hz, 1H), 7.20 (d, J =
3.0 Hz, 1H), 7.04 (dd, J = 9.0, 3.0 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 6.26 (brd, J = 10.1 Hz, 1H), 4.40
(ddd, J = 9.8, 6.9, 5.0 Hz, 1H), 3.94 (d, J = 17.4 Hz, 1H), 3.88 – 3.81 (m, 1H), 3.78 (s, 3H), 3.72 – 3.66
(m, 1H), 3.67 (d, J = 16.8 Hz, 1H), 2.95 (s, 3H), 1.79 – 1.68 (m, 2H), 1.66 – 1.60 (m, 1H), 1.59 – 1.51
(m, 1H), 1.46 (ddd, J = 14.1, 12.0, 3.3 Hz, 1H), 1.26 (ddd, J = 13.9, 10.5, 3.1 Hz, 1H), 0.95 (d, J = 6.2
Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 4.2 Hz, 3H), 0.85 (d, J = 4.0 Hz, 3H). 13C NMR (126
MHz, Acetonitrile-d3) δ 172.7, 170.9, 169.1, 168.5, 155.8, 152.8, 122.5, 119.4, 115.0, 111.8, 63.5, 63.0,
56.6, 54.4, 46.7, 41.3, 41.0, 25.6, 25.0, 24.1, 23.1, 21.7, 21.4. 11B NMR (128 MHz, Acetonitrile-d3) δ
11.7. HRMS (ESI, positive): m/z for C24H37BN3O8: calcd 505.2705, observed 505.2701 [M+H].
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MIDA (1-(2-(2-chloro-4-methoxybenzamido)-4-methylpentanamido)-2-phenylethyl)boronate (3f).
White solid; yield = 78 mg, 40% (1:1 dr); syn-3f: Rf = 0.11 (EtOac). 1H NMR (500 MHz, Acetonitrile-
d3) δ 7.37 (d, J = 8.6 Hz, 1H), 7.25 – 7.21 (m, 4H), 7.17 – 7.14 (m, 1H), 6.98 (d, J = 2.5 Hz, 1H), 6.88
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(d, J = 16.6 Hz, 1H), 4.03 (ddd, J = 10.9, 7.0, 4.3 Hz, 1H), 3.98 (d, J = 17.2 Hz, 1H), 3.95 – 3.90 (m,
1H), 3.92 (d, J = 16.6 Hz, 1H), 3.86 (d, J = 17.2 Hz, 1H), 3.80 (s, 3H), 3.02 (dd, J = 14.1, 3.5 Hz, 1H),
2.92 (s, 3H), 2.61 (dd, J = 14.1, 12.3 Hz, 1H), 1.41 (dddd, J = 13.2, 11.5, 9.4, 6.6 Hz, 1H), 1.13 (ddd, J =
14.0, 10.7, 5.0 Hz, 1H), 0.85 (td, J = 9.5, 4.7 Hz, 1H), 0.81 (d, J = 4.9 Hz, 3H), 0.80 (d, J = 4.9 Hz, 3H).
13C NMR (126 MHz, Acetonitrile-d3) δ 172.9, 169.3, 168.9, 167.9, 162.2, 141.2, 132.7, 131.6, 130.4
(2C), 128.9 (2C), 126.7, 116.2, 113.6, 63.8, 63.0, 56.5, 54.2, 46.6, 40.5, 38.0, 25.3, 23.4, 21.6. 11B NMR
(128 MHz, Acetonitrile-d3) δ 11.6. anti-3f: Rf = 0.08 (EtOAc). 1H NMR (500 MHz, Acetonitrile-d3) δ
7.51 (d, J = 8.7 Hz, 1H), 7.25 – 7.20 (m, 4H), 7.17 – 7.12 (m, 1H), 7.02 (d, J = 2.5 Hz, 1H), 6.94 (dd, J =
8.6, 2.5 Hz, 1H), 6.77 (brd, J = 6.6 Hz, 1H), 6.23 (brd, J = 9.8 Hz, 1H), 4.15 (ddd, J = 9.8, 6.5, 5.1 Hz,
1H), 3.98-3.96 (m, 1H), 3.92 (d, J = 16.3 Hz, 2H), 3.88 (d, J = 5.6 Hz, 2H), 3.85 (s, 3H), 3.81 (d, J =
16.7 Hz, 2H), 2.99 (s, 3H), 1.59 (dddd, J = 13.2, 12.0, 9.0, 6.6 Hz, 1H), 1.25 – 1.22 (m, 2H), 0.87 (d, J =
6.6 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H). 13C NMR (126 MHz, Acetonitrile-d3) δ 172.6, 168.9, 168.9, 168.2,
163.0, 141.4, 132.6, 130.7, 130.7, 129.2, 129.1, 127.1, 116.6, 114.3, 64.0, 63.6, 56.9, 55.2, 47.2, 41.8,
38.6, 25.9, 23.5, 21.9. 11B NMR (128 MHz, Acetonitrile-d3) δ 11.4. HRMS (ESI, positive): m/z for
C27H34BClN3O7: calcd 557.2209, observed 557.2204 [M+H].
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General procedure for the synthesis of racemic boronic acids (4). In a small vial equipped with a
magnetic stir bar under nitrogen atmosphere, was added MIDA boronate syn-3 or anti-3 (1.0 eq.) and
HCl (1.25 M in MeOH, 10 eq.), in MeOH (0.5 M). The reaction was left to stir overnight at room
temperature. The reaction was monitored by TLC and LC-MS. Upon completion, the solvent was
removed under a gentle stream of nitrogen. The crude mixture was loaded onto Celite and purified by
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flash column chromatography* [Acetone  MeCN  DCM:MeOH (10:1)] to afford pure product. The
pure compound was dissolved in MeCN:H2O (1:1) and lyophilized to provide the boronic acid syn-4 or
anti-4 as a fluffy white solid. The racemic boronic acids were checked for purity by LC-MS prior to
assay submission. Full characterization were provided for the enantiopure boronic acids instead.
Note: The boronic acids are streak on silica gel and reverse-phase C18. In order to recover appreciable
amounts of boronic acid using normal-phase column chromatography, it is necessary to flush with ~6-8
column volumes of 15% MeOH in DCM. It is also important to use a smaller diameter column (~1 cm
for 100 mg of starting material) to prevent the product from eluting too dilute.
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Synthesis of (1R)-(S)-pinanediol 1-ammonium-3-methylbutane-1-boronate trifluoroacetate salt
(5a). (1R)-(S)-pinanediol 1-ammonium-3-methylbutane-1-boronate trifluoroacetate salt was prepared
using a modified procedure.4 In a 100 mL round-bottom flask equipped with a magnetic stir bar under
nitrogen atmosphere, was added (2-methylpropyl)boronic acid (2.50 g, 24.5 mmol, 1.0 eq.),
(1S,2S,3R,2S)-(+)-pinanediol (4.58 g, 26.9 mmol, 1.1 eq.) and MgSO4 (ca. 5 g) in Et2O (24.5 mL, 1.0
M). The reaction was left to stir at room temperature overnight. The reaction was monitored by TLC.
Upon completion, the mixture was filtered and the solvent was removed in vacuo. The crude oil was
purified by flash column chromatography using hexanes:Et2O to afford pure (S)-pinanediol-2-
methylpropane-1-boronate in 87% yield. Characterization matched previous literature reports.3 In a
flame-dried, 250 mL two-neck round-bottom flask equipped with a magnetic stir bar under nitrogen
atmosphere, was added (S)-pinanediol-2-methylpropane-1-boronate (5.00 g, 21.2 mmol, 1.0 eq.) and
DCM (9.38 g, 110.4 mmol, 5.2 eq.) in THF (30 mL). The flask was cooled to -60 ºC. In a second flame-
dried, round-bottom flask under nitrogen atmosphere, was added freshly distilled diisopropylamine (3.9
mL, 27.6 mmol, 1.3 eq.) in THF (15 mL). The flask was cooled to -10 ºC. nBuLi (10.5 mL, 26.3 mmol,
2.5 M in hexanes, 1.24 eq.) was added to diisopropylamine over 15 min while maintaining the
temperature at -10 ºC. The LDA-mixture was stirred for 15 min -10 ºC before it was used. In a third
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flame-dried, round-bottom flask under nitrogen atmosphere, was added ZnCl2 (5.0 g, 37.1 mmol, 1.75
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eq.) in THF (34 mL). The reaction flask was heated to 40 ºC and stirred for 1 h and 15 min before it was
used. The LDA-mixture was added over 15 min to flask containing (S)-pinanediol-2-methylpropane-1-
boronate, while maintaining the temperature at -60 ºC. A THF rinse (6 mL) was used to complete the
addition. The reaction mixture was stirred for another 10 min at -60 ºC. The reaction mixture was
warmed to -50 ºC over 5 min. The ZnCl2-mixture was added over 15 min to the flask containing (S)-
pinanediol-2-methylpropane-1-boronate and LDA-mixture, while maintaining the temperature at -50 ºC.
A THF rinse (6 mL) was used to complete the addition. The reaction mixture was stirred for 15 min at -
45 ºC and then warmed to 10 ºC over 30 min. 10% (v/v) H2SO4 solution (30 mL) was added over 10
min, while maintaining the temperature at 10 ºC to 20 ºC. The reaction was stirred for 5 minutes at room
temperature before the aqueous phase was separated. The organic phase was washed with deionized
water (15 mL) and then with 10% (w/v) NaCl solution (15 mL). The organic phase was collected and
dried over Na2SO4. The organic layer was filtered and then the solvent was removed in vacuo to afford
crude (1S)-(S)-pinanediol 1-chloro-3-methylbutane-1-boronate which was used in the next step without
further purification. If the reaction did not go to completion, the crude mixture can still be carried
through to the next step. In a flame-dried, round-bottom flask equipped with a magnetic stir bar under
nitrogen atmosphere, was added crude (1S)-(S)-pinanediol 1-chloro-3-methylbutane-1-boronate (3.53 g,
12.4 mmol, 1.0 eq.) in THF (19 mL). It is possible to extrapolate the amount of (1S)-(S)-pinanediol 1-
chloro-3-methylbutane-1-boronate in the crude mixture using 1H NMR. In a second flame-dried, 150 mL
two-neck round-bottom flask under nitrogen atmosphere, was added LiHMDS (11.9 mL, 1.0 M in THF,
0.96 eq.). The flask was cooled to -20 ºC. The crude (1S)-(S)-pinanediol 1-chloro-3-methylbutane-1-
boronate in THF was added over 30 min to the flask containing the LiHMDS, while maintaining the
temperature at -18 ºC. A THF rinse (1.7 mL) was used to complete the addition. The reaction was stirred
for an additional 30 min at -15 ºC and then warmed to room temperature over 15 min. The solvent was
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removed in vacuo. The crude oil was re-dissolved in Et2O (40 mL) and the solids were filtered over a
pad of Celite. The filtrate was collected and filtered through a plug of silica. The silica plug was washed
with Et2O (500 mL). The solvent was removed in vacuo to afford (1R)-(S)-pinanediol 1-
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bis(trimethylsilyl)amino-3-methylbutane-1-boronate as a crude oil. In a 250 mL round-bottom flask
equipped with a magnetic stir bar under a nitrogen atmosphere, was added trifluoroacetic acid (1.9 mL,
25 mmol, 2.0 eq.) in Et2O (25 mL). The flask was cooled to -10 ºC. In a separate round-bottom flask
under nitrogen atmosphere, was added (1R)-(S)-pinanediol 1-bis(trimethylsilyl)amino-3-methylbutane-1-
boronate (5.07 g, 12.4 mmol, 1.0 eq.) in Et2O (25 mL). The (1R)-(S)-pinanediol 1-
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bis(trimethylsilyl)amino-3-methylbutane-1-boronate mixture was added over 15 min to the flask
containing trifluoroacetic acid causing product precipitation, while maintaining the temperature at -10
ºC. An Et2O rinse was used to complete the addition. The reaction mixture was stirred overnight at 4 ºC.
The solid was collected by vacuum filtration and washed with Et2O to afford pure (1R)-(S)-pinanediol 1-
ammonium-3-methylbutane-1-boronate trifluoroacetate salt 5a.
(1R)-(S)-pinanediol 1-ammonium-3-methylbutane-1-boronate trifluoroacetate salt (5a). White
solid; yield = 1.309 g, 18%; LC-MS retention time = 3.23 min (Method A) 1H NMR (600 MHz,
Chloroform-d) δ 7.77 (brs, 3H), 4.33 (dd, J = 8.8, 1.8 Hz, 1H), 2.91 (brd, J = 6.4 Hz, 1H), 2.31 – 2.26
(m, 1H), 2.24 – 2.19 (m, 1H), 2.03 (dd, J = 6.0, 4.9 Hz, 1H), 1.91 – 1.86 (m, 2H), 1.79 (dt, J = 13.5, 6.8
Hz, 1H), 1.65 – 1.56 (m, 2H), 1.38 (s, 3H), 1.27 (s, 3H), 1.07 (d, J = 11.2 Hz, 1H), 0.92 (d, J = 5.7 Hz,
3H), 0.91 (d, J = 5.7 Hz, 3H), 0.81 (s, 3H). 13C NMR (151 MHz, Chloroform-d) δ 162.3 (q, J = 35.7 Hz),
116.5 (q, J = 291.8 Hz), 87.8, 78.9, 51.2, 39.5, 38.6, 3836, 34.9, 28.2, 27.1, 26.4, 24.8, 24.0, 22.4, 22.2.
11B NMR (192 MHz, Chloroform-d) δ 31.2. 19F NMR (564 MHz, Chloroform-d) δ -75.6. HRMS
(DART-TOF+): m/z for C15H29BNO2: calcd 266.2291, observed 266.2285 [M+H].
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