Synthesis of boronophenylalanine-like aza-amino acids for boron-containing azapeptide precursors

Article abstract of DOI:10.1016/j.tetlet.2020.152585

Aza-amino acids and an azapeptide with a boron-containing substituent were developed for the first time. We synthesized p-boronophenylalanine (BPA)-like aza-amino acid (aza-BPA) and its analogs in which the α-carbon of the peptide is replaced by nitrogen and the boronate ester is situated at the ortho, meta, or para position of the phenyl group. The N- and C-terminals of aza-BPA were linked to α-amino acids to afford an α/aza/α-tripeptide. These compounds are expected to be used in boron neutron capture therapy, chemotherapy, and synthesis of functional materials.

Full text of DOI:10.1016/j.tetlet.2020.152585

Journal Pre-proofs
Synthesis of boronophenylalanine-like aza-amino acids for boron-containing
azapeptide precursors
Kota Miyata, Airi Narita, Ryota Fujisawa, Makoto Roppongi, Satoshi Ito,
Tamesue Shingo, Toru Oba
PII:
S0040-4039(20)31084-4
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152585
TETL 152585
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
7 September 2020
16 October 2020
19 October 2020
Please cite this article as: Miyata, K., Narita, A., Fujisawa, R., Roppongi, M., Ito, S., Shingo, T., Oba, T.,
Synthesis of boronophenylalanine-like aza-amino acids for boron-containing azapeptide precursors, Tetrahedron
Letters (2020), doi: https://doi.org/10.1016/j.tetlet.2020.152585
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Graphical Abstract
Synthesis of boronophenylalanine-like aza-
amino acids for boron-containing azapeptide
precursors
Leave this area blank for abstract info.
Kota Miyata1, Airi Narita1, Ryota Fujisawa1, Makoto Roppongi2, Satoshi Ito1, Tamesue Shingo1, and Toru
Oba1,*
1: Graduate School of Engineering, Utsunomiya University, 2: Advanced Instrumental Analysis Department,
Center for Industry-University Innovation Support, Utsunomiya University

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Synthesis of boronophenylalanine-like aza-amino acids for boron-containing
azapeptide precursors
Kota Miyataa, Airi Naritaa, Ryota Fujisawaa, Makoto Roppongib, Satoshi Itoa, Tamesue Shingoa and Toru
Obaa, 
a
Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi, 321-8585,
Japan
b
Advanced Instrumental Analysis Department, Center for Industry-University Innovation Support, Utsunomiya University, 7-1-2 Yoto, Utsunomiya,Tochigi 321-
8585, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Aza-amino acids and an azapeptide with a boron-containing substituent were developed for the
first time. We synthesized p-boronophenylalanine (BPA)-like aza-amino acid (aza-BPA) and its
analogs in which the α-carbon of the peptide is replaced by nitrogen and the boronate ester is
situated at the ortho, meta, or para position of the phenyl group. The N- and C-terminals of aza-
BPA were linked to α-amino acids to afford an α/aza/α-tripeptide. These compounds are
expected to be used in boron neutron capture therapy, chemotherapy, and synthesis of functional
materials.
Available online
Keywords:
Aza-amino acid
Azapeptide
Boronic acid
BPA
2020 Elsevier Ltd. All rights reserved.
BNCT
1. Introduction
Azapeptides are peptidomimetics in which the α-carbon
–
atom(s) in the peptide is replaced by nitrogen (Figure 1).1 The
semicarbazide structure enhances the chemical stability and
protease resistance of the azapeptide more than amide bonds do.
The semicarbazide also prefers β-turn geometry due to the
planarity of the urea structure and electron repulsion between the
3
–
5
lone pairs of adjacent nitrogen atoms.1 These characteristics
have positioned azapeptides as attractive structures for the design
and synthesis of drugs. Atazanavir is a clinically approved
azapeptide containing a 4-(2-pyridyl)benzyl group on the
hydrazine moiety, and it has a high inhibitory activity against
HIV protease.6,7 Goserelin is used in the treatment of breast and
prostate cancers to suppress the production of testosterone and
estrogen, and it contains an aza-glycinamide at the C-terminal.8,9
An azapeptide analog of the growth hormone-releasing peptide 6
(GHRP-6) influenced angiogenesis through selective binding to
the cluster of differentiation-36 (CD36) receptor.10 The β-turn
conformation of the peptide may be important for receptor
binding.11 The “submonomer method” reported by Lubell et al. in
Figure 1. Structure of peptide and azapeptide.
In recent years, organoboron compounds have attracted much
attention not only as synthetic intermediates but also as
biologically
active
substances.24,25
Boron-containing
peptidomimetics are particularly important. Bortezomib, an
anticancer drug against multiple myeloma approved by the FDA
in 2003, is a boronic acid that substitutes the C-terminal
carboxylic acid moiety (Figure 2).26,27 This boronic acid interacts
with threonine residues located in the chymotrypsin-like active
site in the 20S proteasome and inhibits its protease activity.28
Some boronic acid-containing peptidomimetics have also been
–
31
reported as protease inhibitors.29
Another example is L-p-
–
18
2009 facilitated the successful synthesis of an azapeptide,12
boronophenylalanine (BPA), which has been clinically studied as
–
23
and it has since been improved by several groups. 19
———
 Corresponding author. Tel.: +81-28-689-6147; fax: +81-28-689-6147; e-mail: tob_p206@cc.utsunomiya-u.ac.jp (T. Oba)

2
Tetrahedron
a drug for boron neutron capturer therapy (BNCT).32 BPA can
be taken up into cancer cells by L-type amino acid transporter 1
(LAT1) overexpressed in cancer cells.33,34 Additionally,
According to the above method, we synthesized the isomers of
5a with one and two boronate groups at the meta positions of the
benzene ring (Scheme 2). Compound 2 reacted with 3-
bromobenzyl bromide and 3,5-dibromobenzyl bromide to afford
the corresponding aza-amino acids 3b and 3c in 98% yield.
Miyaura-Ishiyama borylations of 3b and 3c afforded 4b and 4c in
73% and 91% yields, respectively. For its efficient borylation,
3,5-dibromo derivative 4c required twice the amount of
Pd(OAc)2, DPPF, AcOK, and B2pin2 than 4b. The hydrazone
moieties of 4b and 4c were deprotected as in 4a to yield 5b
(80%) and 5c (82%), respectively. Overall, compounds 5b and 5c
were obtained in 43% and 56% yields from 1, respectively.
a
dipeptide containing BPA (Tyr-BPA) was taken up by cancer
cells via peptide transporter 1 (PEPT1).35
Boron-containing aza-amino acids and azapeptides are
expected to function as BNCT drugs, chemotherapeutic agents,
or antiviral agents with protease resistance, specific molecular
recognition, and boron-delivery ability. Boron-containing
azapeptides may also be employed as synthetic precursors of
bioactive compounds. However, there has most likely been no
report on introducing boron-containing functional groups (such
as boronic acid) to azapeptides. Herein, we report the synthesis of
aza-amino acids and azapeptides that possess boron-containing
functional groups for the first time.
Figure 2. Structure of boron-containing peptidomimetics.
Scheme 2. Preparation of aza-BPA analogs with boronate
esters at the meta positions.a
2. Results and Discussion
Aza-BPA 5a was synthesized in four steps from commercially
available Boc-hydrazine (1). Hydrazone 2 was obtained in 76%
yield by reaction of 1 with benzophenone in methanol in the
presence of a catalytic amount of acetic acid. When 2 was mixed
with potassium tertiary butoxide (t-BuOK) in dry tetrahydrofuran
(THF), the solution turned yellowish due to the deprotonation of
hydrazone. 4-Bromobenzyl bromide was added to this yellow
solution, and the mixture was stirred for 5 h at 60°C. Compound
3a was obtained in 98% yield. The bromine atom of 3a was
Conversely, ortho-boronate derivative 4d could not be
prepared using the above method. The Miyaura-Ishiyama
borylation of 3d was unsuccessful; instead, it afforded
dehalogenated products. Modification of the catalysts, bases, and
boron sources failed to induce the desired conversion (Scheme
3a). Additionally, ortho-analog 4d could not be obtained via an
alternative synthetic pathway using borylated benzyl bromide
(Scheme 3a). We hypothesized that the reaction was influenced
by steric hindrance between the diphenylhydrazone moiety of 2
and the pinacol ester group of the reactant. Then, we changed the
amine-protecting reagent of Boc-hydrazine (1) from
benzophenone to benzaldehyde (Scheme 3b). The resulting 2′
substituted by
borylation. Compound 3a was mixed with 5 mol% palladium(II)
acetate (Pd(OAc)2), 1,1′-bis(diphenylphosphino)ferrocene
a boronate ester via a Miyaura-Ishiyama
(DPPF), potassium acetate (AcOK), and bis(pinacolato)diborane
(B2pin2), and the mixture was stirred overnight at 80°C to obtain
boronate ester 4a in 97% yield. The hydrazone deprotection of 4a
was achieved via transimination using hydroxylamine
hydrochloride in pyridine to afford compound 5a in 96% yield.
No side reactions were observed during the deprotection. The
total yield of the four steps from Boc hydrazine 1 to 5a was 69%.
reacted
with
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl bromide to afford compound 4d′ in 43% yield.
Deprotection of the phenyl hydrazone-protected amino group of
4d′ by hydroxylamine afforded the desired compound 5d. The
total yield of these processes (1→5d) was 28%.
Scheme 1. Preparation of aza-BPA 5a.
Scheme 3. Preparation of aza-BPA analog with a boronic
ester at the ortho position.

3
We further investigated the synthesis of boron-containing
azapeptides using aza-BPA 5a (Scheme 4). N-Acetyl proline
(Ac-Pro-OH) reacted with isobutyl chloroformate (IBCF) in
dichloromethane to activate the carboxy group. Then, 5a was
added to the system to afford α/aza-dipeptide 6. The Boc group
of compound 6 was easily deprotected by trifluoroacetic acid
(TFA)/dichloromethane for 1 h at room temperature. Moreover,
α/aza/α-tripeptide 7 was obtained by condensation between 6 and
N-activated leucine (SI-Leu-OMe), prepared by disuccinimidyl
carbonate and leucine methyl ester. The boronic ester was not
hydrolyzed through these processes. While racemization was not
fully controlled in this trial (data not shown), these
transformations indicated that aza-BPA can be used as building
blocks for boron-containing azapeptides.
to the solution. The mixture was refluxed until the reaction was
completed (4–7 h) and monitored with thin-layer
chromatography. After cooling to room temperature, the reaction
mixture was chilled in an ice bath. The resulting white precipitate
was collected via filtration, washed with hexane, and dried under
vacuum. Compounds 2 and 2′ were used without further
purification.
4.2.2. tert-Butyl 2-
(diphenylmethylene)hydrazinecarboxylate (2)3 6
1
Yield of 76%, white powder. H-NMR (500 MHz, CDCl3) δ:
7.68 (brs, 1H, NH), 7.62–7.25 (m, 10H, Ph), 1.50 (s, 9H, Boc).
4.2.3. tert-Butyl 2-benzylidenehydrazinecarboxylate
(2′)3 7
1
Yield of 87%, white powder. H-NMR (500 MHz, CDCl3) δ:
7.90 (brs, 1H, NH), 7.83 (brs, 1H, CH), 7.69–7.67 (m, 2H, Ph),
7.38–7.35 (m, 3H, Ph), 1.54 (s, 9H, Boc).
4.3. Synthesis of compounds 3a–3d and 4d′
4.3.1. General procedure
Compound 2 (1.0 equiv.) was dissolved in dry THF and
cooled in an ice bath. Then, we added t-BuOK (1.5 equiv.) and
various benzylbromides (1.1 equiv.) to this solution. The reaction
mixture was stirred at 60°C under argon (4–7 h). The solvent was
evaporated to dryness and the residue was extracted with ethyl
acetate (AcOEt). The organic phase was washed with saturated
NH4Cl(aq), water, and brine. The solution was dried over Na2SO4
and evaporated to dryness. The residue was chromatographed to
obtain the desired compounds.
Scheme 4. Synthesis of boron-containing azapeptide 7 using
an aza-BPA 5a.
3. Conclusion
We synthesized BPA-like aza-amino acid (aza-BPA) 5a and
the analogs (5b-5d) as potential BNCT drugs. Azapeptide 7
containing 5a was also successfully obtained, indicating that aza-
BPA can be used as a building block for azapeptides. We are
currently studying the applications of aza-BPA and aza-BPA-
containing peptidomimetics in BNCT, molecular targeting, and
preparation of bioactive molecules for anticancer and antiviral
therapies.
4.3.2. tert-Butyl 1-(4-bromobenzyl)-2-
(diphenylmethylene)hydrazinecarboxyla te (3a)
Purification via flash column chromatography (AcOEt/hexane
= 1:5). Light-yellow oil, 95% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.57–7.00 (m, 14H, Ph), 4.63 (s, 2H, CH2), 1.27 (s, 9H, Boc);
13C-NMR (125 MHz, CDCl3) δ: 172.6, 152.3, 137.8, 136.6,
135.7, 131.4, 130.7, 130.5, 129.2, 128.9, 128.4, 128.1, 127.8,
121.2, 80.9, 54.0, 28.1; HRMS (ESI) found: m/z 465.1161, calcd.
for C25H25BrN2O2: MH+, 465.1178
4. Experimental
4.1. General information
4.3.3. tert-Butyl 1-(4-bromobenzyl)-2-
(diphenylmethylene)hydrazinecarboxylate (3b)
Reagents were purchased from Kanto Chemical Co., Inc.,
Tokyo Chemical Industry Co., Ltd., and Wako Pure Chemical
Industries Ltd. and were used as provided. H-NMR (500 MHz),
Purification via flash column chromatography (AcOEt/hexane
= 1:5). Light-yellow oil, 98% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.57–7.02 (m, 14H, Ph), 4.65 (s, 2H, CH2), 1.29 (s, 9H, Boc);
13C-NMR (125 MHz, CDCl3) δ: 172.7, 152.4, 139.9, 137.7,
135.7, 131.8, 130.7, 130.3, 129.8, 129.1, 128.9, 128.3, 128.1,
127.7, 127.3, 122.3, 81.0, 54.2, 28.1; HRMS (ESI) found: m/z
465.1163, calcd. for C25H25BrN2O2: MH+, 465.1178
1
13C-NMR (125 MHz), and 11B-NMR (160 MHz) spectra were
measured with a Varian VNMR-500 spectrometer. Chemical
shifts are reported in parts per million (ppm) with reference to
tetramethylsilane. High-resolution mass spectrometry (HRMS)
[electrospray ionization (ESI)] was performed using a SCIEX
Triple TOF 5600+ System. Melting points were measured using a
Yanaco MP-S3 melting point analyzer.
4.3.4. tert-Butyl 1-(3,5-dibromobenzyl)-2-
(diphenylmethylene)hydrazinecarboxylate (3c)
Purification via flash column chromatography (AcOEt/hexane
= 1:5). Light-yellow oil, 98% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.57–7.06 (m, 13H, Ph), 4.60 (s, 2H, CH2), 1.30 (s, 9H, Boc);
13C-NMR (125 MHz, CDCl3) δ: 173.2, 152.4, 141.7, 137.6,
135.7, 133.0, 131.0, 130.6, 130.2, 129.3, 129.2, 128.4, 128.3,
4.2. Synthesis of compounds 2 and 2′
4.2.1. General procedure
Boc-hydrazine 1 (1.0 equiv.) was dissolved in MeOH/AcOH
(5:1), and benzophenone or benzaldehyde (1.0 equiv.) was added

4
Tetrahedron
128.0, 122.8, 81.5, 54.0, 28.2; HRMS (ESI) found: m/z
Purification via flash column chromatography (AcOEt/hexane
543.0273, calcd. for C25H24Br2N2O2: MH+, 543.0283
= 1:7). Light-yellow oil, 73% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.77–7.00 (m, 14H, Ph), 4.71 (s, 2H, CH2), 1.34 (s, 12H,
Bpin), 1.31 (s, 9H, Boc); 13C-NMR (125 MHz, CDCl3) δ: 172.3,
152.5, 138.1, 136.8, 136.0, 135.6, 133.7, 131.9, 130.6, 129.3,
128.8, 128.5, 128.1, 127.8, 127.7, 83.8, 80.8, 54.8, 28.2, 25.0
(The signal of the ipso-carbon bonded to the boron atom was not
observed); 11B-NMR (160 MHz, CDCl3) δ: 29.6 (br) ; HRMS
(ESI) found: m/z 513.2907, calcd. for C31H37BN2O4: MH+,
513.2925
4.3.5. tert-Butyl 1-(2-bromobenzyl)-2-
(diphenylmethylene)hydrazinecarboxylate (3d)
Purification via flash column chromatography (AcOEt/hexane
= 1:10). Light-yellow oil, 76% yield. 1H-NMR (500 MHz,
CDCl3) δ: 7.60–7.07 (m, 14H, Ph), 4.78 (s, 2H, CH2), 1.30 (s,
9H, Boc); 13C-NMR (125 MHz, CDCl3) δ: 172.9, 152.7, 137.7,
136.7, 135.8, 132.6, 130.7, 130.1, 129.2, 128.9 128.6, 128.4,
128.1, 127.8, 127.4, 123.7, 80.9, 54.2, 28.2; HRMS (ESI) found:
m/z 465.1169, calcd. for C25H25BrN2O2: MH+, 465.1178
4.4.4. tert-Butyl 1-(3,5-bis(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzyl)-2-
(diphenylmethylene)hydrazinecarboxylate (4c)
Purification via flash column chromatography (AcOEt/hexane
= 1:7→1,2-dichoroethane). Light-yellow oil, 91% yield. 1H-
NMR (500 MHz, CDCl3) δ: 8.17–6.99 (m, 13H, Ph), 4.69 (s, 2H,
CH2), 1.32 (s, 24H, Bpin), 1.31 (s, 9H, Boc); 13C-NMR (125
MHz, CDCl3) δ: 172.1, 152.4, 140.1, 138.6, 138.0, 135.9, 135.8,
130.4, 129.2, 128.5, 128.4, 127.9, 127.6, 83.6, 80.7, 54.7, 28.1,
24.8 (The signal of the ipso-carbon bonded to the boron atom
was not observed); 11B-NMR (160 MHz, CDCl3) δ: 30.3 (br);
HRMS (ESI) found: m/z 639.3794, calcd. for C37H48B2N2O6:
MH+, 639.3736
4.3.6. tert-Butyl 2-benzylidene-1-(2-
bromobenzyl)hydrazinecarboxylate (4d′)
Purification via flash column chromatography (AcOEt/hexane
= 1:5). Light-yellow oil, 43% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.87 (d, 1H, J = 7.5 Hz, Ph), 7.60 (brs, 1H, CH), 7.59 (d, 2H, J
= 7.4 Hz, Ph), 7.35 (t, 1H, J = 7.5 Hz, Ph), 7.32–7.27 (m, 3H,
Ph), 7.23 (t, 1H, J = 7.5 Hz, Ph), 7.07 (d, 1H, J = 7.4 Hz, Ph),
5.46 (s, 2H, CH2), 1.59 (s, 9H, Boc), 1.39 (s, 12H, Bpin); 13C-
NMR (500 MHz, CDCl3) δ: 154.0, 142.4, 140.3, 136.6, 135.3,
131.7, 129.1, 128.4, 127.1, 126.3, 125.1, 84.0, 81.7, 47.6, 28.3,
25.0 (The signal of the ipso-carbon bonded to the boron atom
was not observed); 11B-NMR (160 MHz, CDCl3) δ: 31.1 (br);
HRMS (ESI) found: m/z 437.2598, calcd. for C25H33BN2O4:
MH+, 437.2612
4.5. Synthesis of compounds 5a–5d
4.5.1. General procedure
Compound 4 (1.0 equiv.) was dissolved in pyridine and
treated with NH2OH·HCl (2.0 equiv.). The mixture was stirred
overnight at 80°C under argon. The solvent was azeotropically
evaporated with CH2Cl2.
4.4. Synthesis of compounds 4a–4c
4.4.1. General procedure
Compound 3 (1.0 equiv.) was dissolved in 1,4-dioxane and
degassed under 4 cycles of the freeze-pump-thaw process. AcOK
4.5.2. tert-Butyl 1-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)hydrazinecarboxylate (5a)
(3.0
equiv.),
Pd(OAc)2
(5.0
(5.0
mol%),
mol%),
1,1′-
bis(diphenylphosphino)ferrocene
and
Purification via flash column chromatography (1,2-
dichloroethane→AcOEt). Light-yellow oil, 96% yield. H-NMR
bis(pinacolato)diborane (1.0 equiv.) were added to the solution,
followed by another 2 freeze-pump-thaw cycles. The reaction
mixture was stirred overnight at 80°C under argon. The mixture
was diluted with CH2Cl2 and filtered through Celite to remove
the Pd catalyst and AcOK. The filtrate was concentrated using an
evaporator and purified via flash column chromatography to
afford the desired compounds.
1
(500 MHz, CDCl3) δ: 7.78 (d, 2H, J = 8.0 Hz, Ph), 7.27 (d, 2H, J
= 8.0 Hz, Ph), 4.57 (s, 2H, CH2), 4.00 (brs, 2H, NH2), 1.47 (s,
9H, Boc), 1.34 (s, 12H, Bpin); 13C-NMR (125 MHz, CDCl3) δ:
157.0, 141.2, 135.1, 127.2, 83.9, 80.9, 54.6, 28.5, 25.0 (The
signal of the ipso-carbon bonded to the boron atom was not
observed); 11B-NMR (160 MHz, CDCl3) δ: 31.0 (br); HRMS
(ESI) m/z 349.2307, calcd. for C18H29BN2O4: MH+, 349.2299
4.4.2. tert-Butyl 2-(diphenylmethylene)-1-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl)hydrazine -carboxylate (4a)
4.5.3. tert-Butyl 1-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)hydrazinecarboxylate (5b)
Purification via flash column chromatography (AcOEt/hexane
= 1:2). Light-yellow oil, 97% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.75–7.01 (m, 14H, Ph), 4.70 (s, 2H, CH2), 1.27 (s, 12H,
Bpin), 1.26 (s, 9H, Boc); 13C-NMR (125 MHz, CDCl3) δ: 172.0,
152.5, 140.8, 138.0, 135.8, 134.9, 130.7, 129.3, 129.0, 128.6,
128.2, 128.1, 127.8, 83.9, 80.8, 54.9, 28.2, 25.0 (The signal of the
ipso-carbon bonded to the boron atom was not observed); 11B-
NMR (160 MHz, CDCl3) δ: 29.9 (br); HRMS (ESI) found: m/z
513.2911, calcd. for C31H37BN2O4: MH+, 513.2925
Purification via flash column chromatography (1,2-
dichloroethane). Light-yellow oil, 80% yield. 1H-NMR (500
MHz, CDCl3) δ: 7.75 (s, 1H, Ph), 7.71 (d, 1H, J = 7.5 Hz, Ph),
7.37–7.32 (m, 2H, Ph), 4.56 (s, 2H, CH2), 4.00 (brs, 2H, NH2),
1.49 (s, 9H, Boc), 1.33 (s, 12H, Bpin); 13C-NMR (125 MHz,
CDCl3) δ: 157.1, 137.3, 134.6, 133.9, 130.8, 128.0, 83.9, 81.0,
54.6, 28.5, 25.0 (The signal of the ipso-carbon bonded to the
boron atom was not observed); 11B-NMR (160 MHz, CDCl3) δ:
30.0 (br); HRMS (ESI) m/z m/z 349.2292, calcd. for
C18H29BN2O4: MH+, 349.2299
4.4.3. tert-Butyl 2-(diphenylmethylene)-1-(3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl)hydrazinecarboxylate (4b)
4.5.4. tert-Butyl 1-(3,5-bis(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-
yl)benzyl)hydrazinecarboxylate (5c)

5
Purification via flash column chromatography (1,2-
dichloroethane). Light-yellow oil, 82% yield. 1H-NMR (500
MHz, CDCl3) δ: 8.18 (s, 1H, Ph), 7.83 (s, 2H, Ph), 4.56 (s, 2H,
CH2), 4.00 (brs, 2H, NH2), 1.50 (s, 9H, Boc), 1.33 (s, 24H, Bpin);
13C-NMR (125 MHz, CDCl3) δ: 157.2, 140.4, 137.4, 136.5, 83.9,
81.0, 54.7, 28.5, 25.0 (The signal of the ipso-carbon bonded to
the boron atom was not observed); 11B-NMR (500 MHz, CDCl3)
δ: 30.0 (br); HRMS (ESI) m/z m/z 475.3140, calcd. for
C24H40B2N2O6: MH+, 475.3151
1H-NMR (500 MHz, CDCl3) (as a mixture of cis- and trans-
Pro isomers38) δ: 8.86 (brs, 1H, NH), 7.76 (d, 2H, J = 8.0 Hz, Ph),
7.25 (d, 2H, J = 8.0 Hz, Ph), 4.81–4.39 (m, 3H), 3.50–3.30 (m,
2H), 2.51–2.42 (m, 1H), 2.12–1.92 (m, 2H), 1.95 (s, 3H), 1.80–
1.69 (m, 1H), 1.45 (s, 9H, Boc), 1.26 (s, 12H, Bpin); 13C-NMR
(125 MHz, CDCl3) δ: 170.9, 169.3, 154.9, 139.9, 134.9, 127.9,
83.8, 61.1, 57.6, 53.9, 52.3, 48.1, 46.6, 28.2, 28.1, 26.6, 25.0,
24.9, 22.3 (The signal of the ipso-carbon atom bonded to the
boron atom was not observed); 11B-NMR (160 MHz, CDCl3) δ:
31.3 (br); HRMS (ESI) m/z 488.2918, calcd. for C25H38BN3O6:
MH+, 488.2932; Mp = 108 °C.
4.5.5. tert-Butyl 1-(2-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)hydrazinecarboxylate (5d)
4.7.2. Methyl 2-(2-(1-acetylpyrrolidine-2-
carbonyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl) -benzyl)hydrazinecarboxamido)-
4-methylpentanoate (7)
Purification via flash column chromatography (AcOEt/hexane
= 1:7). Light-yellow oil, 75% yield. 1H-NMR (500 MHz, CDCl3)
δ: 7.80 (d, 1H, J = 7.5 Hz, Ph), 7.41 (t, 1H, J = 7.5 Hz, Ph), 7.25
(t, 1H, J = 7.5 Hz, Ph), 7.18 (d, 1H, J = 7.5 Hz, Ph), 4.91 (s, 2H,
CH2), 4.02 (brs, 2H, NH2), 1.47 (s, 9H, Boc), 1.34 (s, 12H, Bpin);
13C-NMR (125 MHz, CDCl3) δ: 157.0, 144.1, 136.0, 131.1,
126.2, 126.1, 83.7, 80.5, 53.3, 28.4, 24.9 (The signal of the ipso-
carbon bonded to the boron atom was not observed); 11B-NMR
(160 MHz, CDCl3) δ: 29.8 (br); HRMS (ESI) m/z m/z 349.2294,
calcd. for C18H29BN2O4: MH+, 349.2299
Compound 7 (0.10 mmol) was dissolved in CH2Cl2 (2.0 mL)
and cooled in an ice bath. N,N-diisopropylethylamine (3.0 equiv.,
0.30 mmol) and SI–Leu–OMe (1.0 equiv., 0.10 mmol) were
added to the solution, and the mixture was stirred for 24 h at
room temperature. The solvent was removed to dryness, and the
residue was dissolved in AcOEt. The organic phase was washed
with saturated NH4Cl(aq), saturated NaHCO3(aq), and brine. The
solution was dried over Na2SO4, and the solvent was removed to
dryness. The residue was purified via recrystallization
(AcOEt/hexane) to afford compound 7 as a white solid in 61%
yield.
4.6. Methyl 2-((2,5-dioxopyrrolidin-1-yloxy)carbonylamino)-4-
methylpentanoate (SI–Leu–OMe)
Leucine methyl ester hydrochloride (0.50 mmol) was
dissolved in CH3CN (2.0 mL), followed by the addition of N,N-
diisopropylethylamine (1.0 equiv., 0.50 mmol). The solution was
cooled in an ice bath. Disuccinimidyl carbonate (1.0 equiv., 0.50
mmol) was added to this solution dissolved in 2.0 mL of CH3CN.
The mixture was stirred overnight at room temperature. After
removal of the solvent, the residue was extracted with AcOEt.
The organic phase was washed with 10 wt% aqueous critic acid
and brine. The solution was dried over Na2SO4 and evaporated to
dryness. The obtained SI–Leu–OMe (white solid, 83% yield) was
used in the next step without further purification.
1H-NMR (500 MHz, CDCl3) δ: 8.10 (1H, s, NH), 7.76 (2H, d,
J = 8.0 Hz, Ph), 7.26 (2H, d, J = 8.0 Hz, Ph), 6.37 (1H, s, NH),
5.17–4.97 (1H, br), 4.58–4.32 (3H, m), 4.16–4.04 (1H, m), 3.73
(s, 3H), 3.56–3.37 (2H, m), 2.20–2.07 (2H, m), 2.03–1.86 (2H,
m), 1.96 (3H, s), 1.70–1.57 (3H, m), 1.34 (12H, s, Bpin), 0.93
(3H, d, J = 6.2 Hz), 0.91 (3H, d, J = 6.2 Hz); 13C-NMR (125
MHz, CDCl3) δ: 174.5, 170.9, 170.6, 157.3, 140.0, 135.1, 128.2,
83.9, 58.4, 52.3, 52.2, 51.1, 48.3, 41.2, 28.2, 25.3, 25.0, 24.6,
23.1, 22.4, 21.7 (The signal of the ipso-carbon atom bonded to
the boron atom was not observed); 11B-NMR (500 MHz, CDCl3)
δ: 31.1 (br); HRMS (ESI) m/z 559.3321, calcd. for C28H43BN4O7:
MH+, 559.3303; Mp = 227°C.
1H-NMR (500 MHz, CDCl3) δ: 5.72 (d, 1H, J = 8.0 Hz, NH),
4.43–4.34 (m, 1H), 3.78 (s, 3H), 2.82 (s, 4H), 1.95 (s, 3H), 1.78–
1.58 (m, 3H,), 0.96 (d, 3H, J = 6.2 Hz), 0.95 (d, 3H, J = 6.2 Hz) ;
13C-NMR (125 MHz, CDCl3) δ: 172.2, 169.8, 151.1, 53.4, 52.6,
41.8, 25.5, 24.6, 22.7, 21.8; HRMS (ESI) m/z 287.1231, calcd.
for C12H18N2O6: MH+, 287.1243; Mp = 94°C.
Funding
This work was supported by Grants-in-Aid for Scientific
Research (C) (Nos. 25410082, 16k05740, and 19k05729) from
the Japan Society for the Promotion of Science and by
collaboration research programs 1-b and 3-c from Utsunomiya
University.
4.7. Synthesis of boron-containing azapeptide using aza-BPA
analog 5a
Acknowledgments
4.7.1. tert-Butyl 2-(1-acetylpyrrolidine-2-
carbonyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)hydrazinecarboxylate (6)
We would like to acknowledge the Center for Industry-
University Innovation Support at Utsunomiya University for the
NMR and MS measurements.
N-Acetyl proline (1.2 equiv., 0.51 mmol) was dissolved in 4.0
mL of dry THF and cooled to −10°C. N-Methylmorpholine (1.5
equiv., 0.64 mmol) and isobutyl chloroformate (1.2 equiv., 0.51
mmol) were added to this solution. The mixture was stirred for 5
min in a cooling bath (ice/salt = 1:3) under argon, and compound
5a (1.0 equiv., 0.43 mmol) was added to the system dissolved in
4.0 mL of dry THF. After stirring the mixture for another 1 h, the
solvent was removed in vacuo. The residue was dissolved in
AcOEt, and the organic layer was washed with saturated
NH4Cl(aq), saturated NaHCO3(aq), and brine. The solution was
dried over Na2SO4 and evaporated to dryness. The residue was
purified via recrystallization (CH3Cl/hexane) to afford compound
6 as a white solid in 69% yield.
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7
Highlights
 Boron-containing aza-amino acids were
developed for the first time.
 These p-boronophenylalanine-analogs can be
building blocks for azapeptides.
 These are potential BNCT drugs, as well as
anticancer and antiviral agents.