A solid-phase approach for the synthesis of α-Aminoboronic acid peptides

Article abstract of DOI:10.1039/c7ra13479g

A solid-phase synthesis of α-Aminoboronic acid peptides using a 1-glycerol polystyrene resin is described. Standard Fmoc solid-phase peptide chemistry is carried out to construct bortezomib and ixazomib. This approach eliminates the need for liquid-liquid extractions, silica gel column chromatography, and HPLC purifications, as products are isolated in high purity after direct cleavage from the resin.

Full text of DOI:10.1039/c7ra13479g

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A solid-phase approach for the synthesis of
a-aminoboronic acid peptides†
Cite this: RSC Adv., 2018, 8, 3343
*
Blake E. Daniels and Craig E. Stivala
Received 19th December 2017
Accepted 10th January 2018
A solid-phase synthesis of a-aminoboronic acid peptides using a 1-glycerol polystyrene resin is described.
Standard Fmoc solid-phase peptide chemistry is carried out to construct bortezomib and ixazomib. This
approach eliminates the need for liquid–liquid extractions, silica gel column chromatography, and HPLC
purifications, as products are isolated in high purity after direct cleavage from the resin.
DOI: 10.1039/c7ra13479g
rsc.li/rsc-advances
a-Aminoboronic acids are currently being investigated for their reported for the preparation of a-aminoboronic acid peptides.5
utility as reversible covalent inhibitors in a diverse range of This approach provides direct access to these compounds from
therapeutic applications (Fig. 1).1 These compounds' Lewis their parent peptide constructs but sacrices stereochemical
acidity enables the formation of stable tetrahedral adducts with integrity.
nucleophilic residues in biological targets (Fig. 2). In 2003, the
Regardless of the method, a-aminoboronic acid/ester
rst boronic acid drug, bortezomib, was approved for the peptides are difficult to prepare for a number of reasons.6
treatment of multiple myeloma.2 Ixazomib, a related a-amino- First, the C–B bond can be oxidatively labile.7 Second, a-ami-
boronic acid inhibitor, was later approved in 2015 for the same noboronic acids and esters containing an unsubstituted a-
indication.3
amino group can undergo a spontaneous 1,3-rearrangement
Peptidic a-aminoboronic acids, such as bortezomib and (Scheme 1, A); this process can be minimized or suppressed
ixazomib, have traditionally been assembled using standard entirely if the amino group is rapidly acylated or protonated.8
peptide coupling techniques,4 wherein an a-aminoboronic ester Third, boronic esters can be hydrolytically labile, especially at
is introduced onto a pre-constructed peptide and is subse- low pH (Scheme 1, B).6,9 Therefore, any multistep approach
quently deprotected to unmask the boronic acid. Metal- must entail careful extractive workups and purications to
catalyzed decarboxylative borylation strategies have also been ensure that the ester remains intact.
Fig.
1 a-Aminoboronic acids featured in various drug discovery
programs. Flaviviral protease inhibitor (1), HCV protease inhibitor (2),
LepB inhibitor (3).
Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080,
USA. E-mail: stivala.craig@gene.com
Fig. 2 Yeast 20S proteasome in complex with bortezomib (PDB ID
2F16). The boronic acid forms a stable tetrahedral adduct with the N-
terminal threonine (Thr1).
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra13479g
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Scheme 3 2-Step protocol for loading boroleucine onto a 1-glycerol
polystyrene resin.
the amine to remain protonated or acylated throughout the
loading process. These considerations lead to the design of
a two-step loading protocol (Scheme 3). Commercially available
boroleucine pinanediol ester 7 was hydrolysed with aqueous
HCl. The boroleucine salt (8) was isolated in quantitative yield,
free of pinanediol impurities, aer a simple liquid–liquid
extraction. The crude boroleucine salt was then shaken with the
1-glycerol polystyrene resin (loading capacity 0.6 mmol gꢀ1),13
Fmoc chloride, and N,N-diisopropylethylamine to provide resin-
bound Fmoc-protected boroleucine 9.
With the C-terminal a-aminoboronic acid resin in hand, we
used standard Fmoc SPPS coupling techniques16 to synthesize
bortezomib (Scheme 4). Fmoc deprotection (piperidine, DMF)
and amide coupling (Fmoc-Phe-OH, TBTU, N,N-diisopropyle-
thylamine, DMF) delivered intermediate 10. A subsequent Fmoc
deprotection/coupling sequence with pyrazinecarboxylic acid
produced resin-bound bortezomib 11. Hydrolysis of the resin
bound peptide was accomplished with gentle shaking in a THF/
water mixture.13 Filtration and concentration delivered borte-
zomib (7-steps from boroleucine pinanediol ester 7) in 54%
yield and in >95% purity.
Scheme 1 General considerations for preparing a-aminoboronic acid
peptides.
While solid-phase peptide synthesis (SPPS) has become
a standard method for the construction of peptides,10 this
technology has remained underexplored for the preparation of
a-aminoboronic acid peptides.11 An approach of this type could
eliminate liquid–liquid extractions and HPLC purications and
could enable high-throughput access to this class of
compounds. To the best of our knowledge, there has only been
one report of C-terminal SPPS to generate a-aminoboronic acid
peptides (Scheme 2).12 Although this study provides a critical
conceptual foundation, the approach it describes lacks the
simplicity of a traditional solid-phase approach, requiring
a complex 8-step synthesis to prepare resin-bound a-amino-
boronic ester 6 for SPPS. This limitation may preclude its use as
a general strategy for the preparation of a-aminoboronic acid
peptides.
We sought to identify an approach that could enable access
to resin-bound a-aminoboronic acids for SPPS in a limited
number of steps using the emerging supply of commercially
available a-aminoboronic acid building blocks. The Klein group
recently described the use of a 1-glycerol polystyrene resin that
could be used for Fmoc SPPS to construct boronic acid-con-
taining peptides.13–15 These results prompted us to explore the
use of this resin for preparation of a-aminoboronic acid
peptides, specically bortezomib and ixazomib.
Considering the unique reactivity of a-aminoboronic acids,
we needed to devise a concise loading strategy that would
suppress the potential for C to N boron migration. This required
Scheme 2 8-Step preparation of a resin-bound a-aminoboronic acid
for C-terminal SPPS.
Scheme 4 Solid-phase synthesis of bortezomib.
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singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling
constant (Hz), and integration; 13C-NMR – chemical shi (d,
ppm). High-resolution mass spectra (HRMS) were recorded on
a Thermo Scientic Orbitrap Q Exact mass spectrometer.
Reactions were monitored by a Shimadzu LCMS/UV system with
LC-30AD solvent pump, 2020 MS, Sil-30AC auto sampler, SPD-
M30A UV detector, CTO-20A column oven, using a 2 – 98%
acetonitrile/0.1% formic acid (or 0.001% ammonia) gradient
over 2.5 minutes. Purity was determined by LCMS analysis with
an Agilent 1290 UHPLC coupled with Agilent MSD (6140) mass
spectrometer using ESI as ionization source. The LC separation
used a Phenomenex XB-C18, 1.7 mm, 50 ꢁ 2.1 mm column with
a 0.4 mL minꢀ1 ow rate. Solvent A was water with 0.1% FA and
solvent B was acetonitrile with 0.1% FA. The gradient consisted
of 2 – 98% solvent B over 7 min and hold 98% B for 1.5 min
following equilibration for 1.5 min. The LC column tempera-
ture was 40 ꢂC. UV absorbance was collected at 220 nm and
254 nm.
Scheme 5 Solid-phase synthesis of ixazomib.
Synthesis of boronic acid 8. (R)-Boroleucine-(1S,2S,3R,5S)-
(+)-2,3-pinanediol ester triuoroacetate (1.00 g, 2.64 mmol, 1.00
equiv.) and isobutylboronic acid (297 mg, 2.77 mmol, 1.05
equiv.) were suspended in heptane (5.86 mL, 0.45 M). Hydro-
chloric acid (5.86 mL, 0.45 M) was added, and the reaction
mixture was stirred at room temperature for two hours. Upon
consumption of the starting material, the reaction mixture was
diluted with water and heptane (both 10 mL). The aqueous layer
was separated and concentrated under reduced pressure to
afford (R)-boroleucine hydrochloride 8 (442 mg, quantitative
The synthesis of ixazomib (Scheme 5) was accomplished in
an analogous manner. Fmoc deprotection (piperidine, DMF)
and amide coupling (Fmoc-Gly-OH, TBTU, N,N-diisopropyle-
thylamine, DMF) delivered intermediate 12. The deprotection/
coupling sequence was repeated with 2,5-dichlorobenzoic acid
to generate resin-bound ixazomib 13. Finally, boronic ester
hydrolysis (THF/water) provided ixazomib in 49% yield and in
>95% purity.
1
yield) as a white solid. H NMR (400 MHz, methanol-d4) d 2.84
(br s, 1H), 1.78–1.64 (m, 1H), 1.53 (m, 2H), 0.97 (d, J ¼ 6.6 Hz,
3H), 0.96 (d, J ¼ 6.4 Hz, 3H). HRMS (ESI) calcd for C5H15O2NB
[M + H]+: 132.1190, found 132.1188.
Conclusions
This proof-of-concept study details the use of the 1-glycerol
polystyrene resin for C-terminal SPPS of a-aminoboronic acid
peptides. The a-aminoboronic acid used in this study was
loaded onto the resin in 2 steps from a commercially available
building block. This loading protocol represents a dramatic
improvement to what has been reported previously. While the
efficiency of the solid-phase synthesis of bortezomib (7 steps
from 7, 54% yield) and ixazomib (7 steps from 7, 49% yield) is
comparable to other standard synthetic approaches,4,5 the use
of a solid support enables a-aminoboronic acid peptides to be
constructed rapidly in high purity and eliminates the need for
workup and purication.
General procedure for solid-phase synthesis of a-
aminoboronic acids
Step 1. Resin loading – 1-glycerol polystyrene resin (loading
capacity 0.6 mmol gꢀ1, 1.50 g, 20 mmol) was swollen in CH2Cl2
for 30 minutes and then dried via vacuum ltration. Fmoc
chloride (556 mg, 2.15 mmol, 3.6 equiv.), (R)-boroleucine
hydrochloride (100 mg, 0.597 mmol, 1.0 equiv.), THF (10 mL,
0.06 M), and then N,N-diisopropylethylamine (0.38 mL,
2.15 mmol, 3.6 equiv.) were added to the resin and the reaction
mixture was shaken overnight. The resin was dried by vacuum
ltration and washed sequentially with THF, CH2Cl2, and DMF
(3 ꢁ 10 mL each). Note – Quantitative loading of the a-amino-
boronic acid is assumed.
Experimental procedures
General
All commercial reagents and anhydrous solvents were used
Step 2. Fmoc deprotection – piperidine (10% in DMF, 20 mL,
without additional purication. Boroleucine pinanediol ester 7 21.8 mmol) was added to the resin and the suspension was
was purchased from Ark Pharm (Cat. # AK-44948, CAS # 179324- shaken for 20 minutes. The resin was dried by vacuum ltration
87-9). 1-Glycerol polystyrene resin was purchased from Iris and washed with DMF (2 ꢁ 10 mL). This procedure was
Biotech (Cat. # BR-5206.0025). Nuclear magnetic resonance repeated to ensure complete deprotection.
(NMR) spectra were acquired on a Bruker Avance DPX400
Step 3. Peptide coupling – N,N-diisopropylethylamine (0.42
operating at 400 and 100 MHz for 1H and 13C, respectively, and mL, 2.39 mmol, 4.0 equiv.), an Fmoc-amino acid (1.79 mmol,
are referenced internally according to residual solvent signals. 3.0 equiv.), and TBTU (587 mg, 1.79 mmol, 3.0 equiv.) were
NMR data were processed using MNova soware and recorded added to the resin in CH2Cl2 (10 mL) and DMF (10 mL). The
1
as follows: H-NMR – chemical shi (d, ppm), multiplicity (s, reaction mixture was shaken for two hours. The resin was dried
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Step 4. Cleavage from resin – the resin was dried by vacuum
ltration, washed with DMF, CH2Cl2, and Et2O (3 ꢁ 10 mL for
each), and dried under vacuum. The dried resin was treated
with 9 : 1 THF/water (40 mL) and stirred overnight at room
temperature. The resin was ltered and the ltrate concentrated
under vacuum to afford the pure a-aminoboronic acid.
Bortezomib. Bortezomib – recovered as a white solid in 54%
yield (124 mg). 1H NMR (400 MHz, methanol-d4) d 9.13–9.06 (m,
1H), 8.77–8.68 (m, 1H), 8.65–8.58 (m, 1H), 7.36–7.14 (m, 5H),
4.84–4.67 (m, 1H), 3.23–3.14 (m, 1H), 3.12–3.01 (m, 1H), 2.97–
2.87 (m, 1H), 1.47–1.28 (m, 3H), 1.28–1.17 (m, 1H), 0.82–0.73
(m, 6H). HRMS (ESI) calcd for C19H24O3N4B [M ꢀ OH]+:
367.1936, found 367.1925.
Ixazomib. Ixazomib – recovered as a white solid in 49% yield
(106 mg). 1H NMR (400 MHz, methanol-d4) d 7.60 (dd, J1 ¼ J2 ¼
1.5 Hz, 1H), 7.49–7.46 (m, 2H), 4.23 (s, 2H), 2.83–2.75 (m, 1H),
1.74–1.61 (m, 1H), 1.42–1.32 (m, 2H), 0.97–0.90 (m, 6H). HRMS
(ESI) calcd for C14H18O3N2BCl2 [M ꢀ OH]+: 343.0782, found
343.0770.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors would like to thank Baiwei Lin and Kewei Xu for
obtaining analytical data.
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