One-pot, two-step synthesis of unnatural α-amino acids involving the exhaustive aerobic oxidation of 1,2-diols

Article abstract of DOI:10.1039/c9cc07889d

Herein, we report the nor-AZADO-catalyzed exhaustive aerobic oxidations of 1,2-diols to α-keto acids. Combining oxidation with transamination using dl-2-phenylglycine led to the synthesis of free α-amino acids (AAs) in one pot. This method enables the rapid and flexible preparation of a variety of valuable unnatural AAs, such as fluorescent AAs, photoactivatable AAs, and other functional AAs for bioorthogonal reactions.

Full text of DOI:10.1039/c9cc07889d

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One-pot, two-step synthesis of unnatural a-amino
acids involving the exhaustive aerobic oxidation
of 1,2-diols†
Cite this: Chem. Commun., 2019,
55, 15105
Received 9th October 2019,
Haruki Inada, Keisuke Furukawa, Masatoshi Shibuya * and
Yoshihiko Yamamoto
Accepted 20th November 2019
DOI: 10.1039/c9cc07889d
rsc.li/chemcomm
Herein, we report the nor-AZADO-catalyzed exhaustive aerobic
oxidations of 1,2-diols to a-keto acids. Combining oxidation with
transamination using DL-2-phenylglycine led to the synthesis of free
a-amino acids (AAs) in one pot. This method enables the rapid and
flexible preparation of a variety of valuable unnatural AAs, such as
fluorescent AAs, photoactivatable AAs, and other functional AAs for
bioorthogonal reactions.
The incorporation of functional a-amino acids (AAs) into peptides
is a powerful strategy for probing or modulating their biological
properties.1–4 More specifically, fluorescent AAs can be used to
visualize intracellular processes involving peptides,2,3 and photo-
activatable AAs enable the crosslinking of ligands and interacting
proteins.4 AAs bearing azides and alkynes (for click chemistry),
alkenes (for olefin cross metathesis), and aryl halides (for
Pd-catalyzed cross coupling) enable the introduction of various
functional groups into peptides in flasks as well as in living
cells by bioorthogonal chemistry.5 Such functional AAs are usually
synthesized by one of the following two synthetic strategies: (1) the
modification of a natural AA or commercially available AA3,4
(Fig. 1a); and (2) the alkylation of a glycine equivalent6 (Fig. 1b).
However, the structures of the AAs accessible by the former
strategy are limited by the availability of AAs as starting materials.
In terms of the latter strategy, an alkyl halide with a carbon–
halogen bond adjacent to an unsaturated bond is generally used as
an alkylating reagent owing to the efficiency of an SN2 reaction.
These limitations are obstacles to obtaining more-suitable
functional AAs according to the intended use. Hence, a de novo
synthetic method that enables the preparation of a wide range
of unnatural AAs will strengthen research using functional AAs.
We previously developed a three-step method to directly
synthesize free AAs,7 which involves the chemoselective oxidation
Fig. 1 The conventional synthetic methods of AAs and this study. NBD =
7-nitrobenz-2-oxa-1,3-diazol-4-yl, Dap = 2,3-diaminopropionic acid.
of 1,2-diols to a-hydroxy acids,8 the chemoselective oxidation of
a-hydroxy acids to a-keto acids,9 and transamination of a-keto
acids with DL-2-phenylglycine to AAs. Unfortunately, we were
unable to apply this method to the synthesis of a fluorescent
coumarinyl AA in our preliminary study. Owing to the poor
solubility of the corresponding 1,2-diol in toluene, the first-step
Department of Basic Medicinal Sciences Graduate School of Pharmaceutical
Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
E-mail: m-shibu@ps.nagoya-u.ac.jp
† Electronic supplementary information (ESI) available: Experimental details,
NMR spectra, and other characterisation data. See DOI: 10.1039/c9cc07889d
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Table 1 Optimization of the exhaustive aerobic oxidations of 1,2-diols to
a-keto acids
reaction does not efficiently proceed. The undesired oxidative C–C
bond cleavage easily occurred during the oxidation of a 1,2-diol to
the corresponding a-keto acid.10 To suppress the side reaction, the
first step uses two-phase conditions involving hydrophobic
toluene and phosphate buffer. Therefore, the physical properties
of 1,2-diols and a-hydroxy acids are critical for reaction efficiency.
However, the preliminary result suggests that control through
phase separation is not applicable to the synthesis of fluorescent
and other functional AAs, because their relatively large side
chains strongly influence the physical properties of the corres-
ponding 1,2-diols and a-hydroxy acids. Hence, a substantially
improved oxidation method is required in order to prepare such
functional AAs.
Inspired by the previous finding that the undesired oxidative
cleavage of a labile a-keto acid does not proceed under nitroxyl
radical-catalyzed aerobic oxidation conditions,9 we envisaged
that the development of a nitroxyl radical-catalyzed aerobic
oxidation protocol that converts 1,2-diols to the corresponding
a-keto acids would enable the synthesis of fluorescent and
other functional AAs. Such an aerobic oxidation protocol would
also enable the step-economical synthesis of AAs. On the other
hand, a highly efficient aerobic oxidation method capable of
triply oxidizing a single molecule is required for the desired
transformation. To develop the desired reaction, the use of a
highly efficient nitroxyl radical catalyst bearing an a-hydrogen,
such as 2-azaadamantane N-oxyl (AZADO) and 9-azabicyclo[3.3.1]-
nonane N-oxyl (ABNO) is needed.11,12 On the other hand, because
these catalysts have low chemoselectivities for primary alcohols
over secondary alcohols,13 the non-chemoselective oxidation
of a 1,2-diol will generate several intermediates, such as the
corresponding a-hydroxyaldehyde, a-hydroxyketone, a-hydroxy
acid, a-ketoaldehyde, and the hydrates of these aldehydes.
Therefore, a method that exhaustively oxidizes 1,2-diols to the
corresponding a-keto acids irrespective of the intermediate
species formed is required. Herein, we established an exhaustive
aerobic oxidation method. Moreover, by combining oxidation with
transamination, we developed a one-pot, two-step method for the
synthesis of a-amino acids that provides facile access to a variety of
functional AAs (Fig. 1c). This method is also distinguishable from
conventional synthetic methods in that functional AAs can
be synthesized from the corresponding 1,2-diols without any
protecting groups, and the AA moiety is constructed onto the
carbon chain of the starting material without increasing or
decreasing the length of the carbon chain.
Yieldb,c [%]
Entry Cat., additive, and solventsa
1b 1c 1d
1a
1
2
AZADOL MeCN/pH 3.9 acetate buffer
AZADO MeCN/pH 3.9 acetate buffer
37
68
6
3
3
1
14
3
3
4
AZADO MeCN/pH 6.8 phosphate buffer n.d. n.d. 1
AZADO MeCN/pH 2.1 phosphate buffer 35
TEMPO MeCN/pH 3.9 acetate buffer n.d. n.d. 6
96
13
70
3
2
5d
6d
7
DMN-AZADO MeCN/pH 3.9 acetate buffer 48 12 Trace Trace
nor-AZADO MeCN/pH 3.9 acetate buffer 77
3
1
3
8d,e nor-AZADO MeCN/pH 3.9 acetate buffer 82 n.d. n.d. n.d.
9d,e nor-AZADO, AcOH (2 equiv.) MeCN/H2O 87 n.d. n.d. n.d.
a
b
c
d
v/v = 1/1. NMR yields. n.d. = not detected. Cat. (10 mol%) and
e
NaNO2 (40 mol%) were used. Reaction time is 12 h.
reaction resulted in the recovery of a large amount of 1a, whereas
the DMN-AZADO-catalyzed reaction afforded the desired product
1b in moderate yield (entries 5 and 6).13 We eventually found that
nor-AZADO efficiently catalyzed the desired exhaustive oxidation
(entries 7 and 8).14 Although small amounts of 1c, 1d, and 1a
remained in the presence of 5 mol% nor-AZADO, the reaction was
complete within 12 h in the presence of 10 mol% nor-AZADO. The
desired oxidation proceeded efficiently in MeCN and H2O in the
presence of acetic acid (2 equiv.) as well as in MeCN and pH 3.9
acetate buffer (entry 9). The reaction conditions of entry 9 were
optimal for the synthesis of AAs, because the use of the acetate
buffer sometimes results in the AA precipitate being contaminated
with AcONa (vide infra).
With the exhaustive oxidation of 1,2-diols to the corresponding
a-keto acids established, the one-pot conversion to AAs employing
transamination with DL-2-phenylglycine was examined in order to
maximize operational simplicity (Scheme 1, see the ESI† for
details).7 After 1,2-diol 1a had been exhaustively oxidized to a-keto
acid 1b, DL-2-phenylglycine (0.9 equiv.) and additional MeCN were
added to the reaction mixture prior to any workup operation. After
refluxing the reaction mixture for 24 h and the following addi-
tion of diethyl ether, the precipitate was simply collected by
filtration. The desired AA 1e was obtained in 62% isolated yield
At the outset, we examined the aerobic oxidation of 1,2-diol
1a in MeCN and pH 3.9 acetate buffer using commercially
available AZADOL as the catalyst (entry 1, Table 1) (see the ESI†
for details). The desired a-keto acid 1b was produced in 37%
yield together with small amounts of a-hydroxy acid 1c, a-hydroxy-
aldehyde 1d, and several unidentified byproducts after 24 h. The
unidentified byproducts are presumably partially oxidized inter-
mediates. The use of AZADO instead of AZADOL improved the
yield of 1b to 68%, although the reason for this improvement is
unclear (entry 2). The reaction in pH 6.8 or 2.1 phosphate buffer
and MeCN did not proceed efficiently (entries 3 and 4), suggesting
that weakly acidic conditions are optimal. The TEMPO-catalyzed
Scheme 1 One-pot synthesis of AA 1e.
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Fig. 2 Substrate scope of the one-pot AA synthesis protocol. aAn inseparable mixture with 8% of N-iminyl amino acid. bPurified by ion-exchange chromatography.
cPurified by filtration and silica gel column chromatography. dDL-2-phenylglycine (0.8 equiv.) was used. eThe reaction was carried out in 0.50 mmol scale.
with high purity. In this manner, we established a one-pot, two- light-shielded conditions in 58% and 70% yields, respectively.
step method for the synthesis of AAs from 1,2-diols.
Notably, the relatively hydrophilic side chain of 10 or the hydro-
To investigate the scope of the one-pot, two-step AA-synthesis phobic side chains of 11–13 did not detrimentally affect the reaction
method, we examined the synthesis of several AAs (Fig. 2). efficiency. An alkyl azide and alkyne required for the Huisgen
Simple AA 2e bearing an alkyl chain was prepared in 69% yield, reaction, an alkene required for cross-olefin metathesis, and an aryl
and the transamination of a-keto acid 3b was selectively halide required for Pd-catalyzed cross coupling were also compa-
promoted with the phenylketone moiety intact to afford 3e in tible, affording AAs 14e–17e in high yields (55–73%), and high-
68% yield. The exhaustive oxidations of 1,2-diols 4a–6a to the lighting the mildness of the reaction conditions. These functional
corresponding a-keto acids 4b–6b also proceeded effectively, and AAs are used to modify peptides by bioorthogonal chemistry. During
AAs 4e–6e were obtained in high yields following transamination. the synthesis of 16e, aerobic oxidation in MeCN and pH 3.9 acetate
We note that AAs 4e–6e cannot be synthesized by the previously buffer instead of MeCN/H2O/AcOH (2 equiv.) led to contamination
reported three-step method. The initial chemoselective oxidations of of the AA precipitate with AcONa, affording a 5 : 1 mixture of the
diols 4a–6a to the corresponding a-hydroxy acids were unsuccessful desired AA and AcONa.
because of their low solubilities in toluene or highly hydrophilic
properties.8 These results suggest that the efficiency of the aerobic
oxidation is less influenced by the physical properties of 1,2-diols
and a-hydroxy acids than that of the previous stepwise oxidations.
Fluorophilic AA 7e bearing the perfluoroalkyl side chain was also
synthesized, albeit in moderate yield (17%). Exhaustive oxidations of
triols 8a and 9a proceeded with the oxidation of the side-chain
alcohols to afford AAs 8e (bearing a carboxyl group) and 9e (bearing
a ketone moiety). These results suggest that this novel method,
based on exhaustive aerobic oxidation, is robust.
We next examined the synthesis of functional AAs. The
coumarin and pyrene moieties were found to be compatible
with the reaction conditions, affording the fluorescent AAs 10e
and 11e15 in 51% and 49% yields, respectively. Photoactivatable
AAs 12e and 13e, bearing a benzophenone moiety and 4-azido-
Scheme 2 Large-scale synthesis of (Æ)-11e and its subsequent chemo-
enzymatic resolution.
2,3,5,6-tetrafluorophenyl group were effectively synthesized under
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Finally, to demonstrate the further utility of this synthetic Notes and references
method, we prepared fluorescent AA (Æ)-11e on the gram scale and
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acetylation, kinetic resolution using L-aminoacylase (EC 3.5.1.14,
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isolated yield and 499% ee. The recovered acetate D-19 was
isolated as benzyl ester D-20 in 35% yield and 99% ee.
In conclusion, we developed a practical one-pot, two-step
method for the synthesis of AAs that involves exhaustive aero-
bic oxidation and transamination. Precipitation in the final
step is the only purification operation required to obtain AAs
with high purities in most cases. Since aerobic oxidation of
1,2-diols to the corresponding a-keto acids proceeds under
mono-phasic conditions using MeCN and H2O as the solvents,
the hydrophilic/hydrophobic nature of the side chain does not
influence the efficiency of the reaction. Owing to its robustness,
this method facilitates the preparation of functional AAs such
as fluorescent AAs, photoactivatable AAs, and a variety of other
AAs. These results suggest that this synthetic method is useful
for the preparation of valuable unnatural AAs in chemical
biology research.
This work was partially supported by JSPS KAKENHI (No.
JP19K06973), the Platform Project for Supporting Drug Discovery
and Life Science Research (BINDS, No. JP19am0101099) from
AMED, the Research Foundation for Pharmaceutical Sciences,
and Grant for Basic Science Research Projects from the Sumitomo
Foundation. We are grateful to Amano Enzyme Inc. for the gift of
acylase H ‘‘Amano’’.
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the solvents.
Conflicts of interest
There are no conflicts to declare.
15108 | Chem. Commun., 2019, 55, 15105--15108
This journal is ©The Royal Society of Chemistry 2019