Oxidative peptide bond formation of glycine-amino acid using 2-(aminomethyl)malononitrile as a glycine unit

Article abstract of DOI:10.1039/d1cc00130b

Amide linkage of glycine-amino acid was synthesized by coupling of substituted 2-(aminomethyl)malononitrile as a C-terminal glycine unit and N-terminal amine using CsOAc and O₂in an aqueous solution. This is a coupling reagent-free and catalyst-free peptide synthesisviaoxidative amide bond formation. Various tripeptides and tetrapeptides were synthesized efficiently and the sulfide moiety is inert even under an oxygen atmosphere.

Full text of DOI:10.1039/d1cc00130b

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Oxidative peptide bond formation of
glycine–amino acid using
2-(aminomethyl)malononitrile as a glycine unit†
Cite this: Chem. Commun., 2021,
57, 4283
Received 8th January 2021,
Accepted 22nd March 2021
Xiaoling Wang, Jing Li and Yujiro Hayashi
*
DOI: 10.1039/d1cc00130b
rsc.li/chemcomm
Amide linkage of glycine–amino acid was synthesized by coupling simple coupling methodology for peptide synthesis is still in great
of substituted 2-(aminomethyl)malononitrile as C-terminal demand.
glycine unit and N-terminal amine using CsOAc and O2 in an We have previously developed a protocol for the oxidative
a
aqueous solution. This is a coupling reagent-free and catalyst- synthesis of amide from a-substituted propanedinitrile and
free peptide synthesis via oxidative amide bond forma- amine in the presence of O2 and K2CO3 in anhydrous CH3CN
tion. Various tripeptides and tetrapeptides were synthesized (Scheme 1a).11c This method accommodates a broad range of
efficiently and the sulfide moiety is inert even under an oxygen both steric hindered propanedinitrile and amine in moderate
atmosphere.
to excellent yield. The only reagents needed are K2CO3 and
oxygen with the generation of KCN as a byproduct. An anhy-
Peptides and proteins are essential components in almost all drous condition was employed in order to suppress the unde-
living organisms and play various central functions.1 Their sired hydrolysis of acyl cyanide intermediate in the reaction of
abundant natural occurrence and unparalleled biological and sterically hindered substrates. If protected amino acids and
medicinal significance have made them a vital research topic in peptides can be employed as a nucleophile and a substituted
both biological and chemical research fields.2 The chemical 2-(aminomethyl)malononitrile can be employed as an electro-
synthesis represents the most reliable method to provide the phile, it would be a new method for the synthesis of an amide
desired peptides and proteins in both reasonable quantity and linkage between glycine and amino acid with the generation of
purity compared to the other two main pathways that are natural the elongated peptides, without coupling reagent and with
isolation and recombinant expressions in microorganisms.3 minimum waste generation (Scheme 1b). As amino acids and
There are many methods for amide bond synthesis.4 The direct peptides are soluble in an aqueous solvent, the challenge in the
amide formation between C-terminal carboxylic acid and present reaction is whether it can proceed efficiently in such an
N-terminal amine amino acids and peptides is well utilized by aqueous solvent. In this paper, we will describe the realization
employing coupling reagents,5 and recently excellent catalytic of this scenario.
methods have been reported.6 Other methods such as native
We began our initial investigation by optimizing the reac-
chemical ligation,7 Staudinger ligation,8 and ketoacid–hydroxyla- tion conditions using Boc-protected L-phenylalanine substi-
mine ligation9 have been developed. The amide bond formation tuted propanedinitrile 1a and L-phenylalanine methyl ester 2a
from aldehyde or alcohol with an amine in the presence of a
catalyst is also reported.10 Our group11 and Johnston’s group12
have reported oxidative amide bond formation recently. An ideal
synthetic method for peptides requires no coupling reagents,
catalysts, and toxic metals, often in aqueous solution and a simple
work-up, which has been a long-standing goal.13 Therefore, a new
Department of Chemistry, Graduate School of Science, Tohoku University,
6-3 Aramaki Aza-Aoba, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
E-mail: yujiro.hayashi.b7@tohoku.ac.jp
Scheme 1 Previous amide bond formation in our group and present
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
peptide synthesis.
d1cc00130b
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Table 1 The effect of base and solvent in the reaction of 1a and 2aa
was synthesized by applying various N-terminal free amines of
amino acid esters with acceptable yields (Table 2a). Not only
Boc-protected L-phenylalanine substituted propanedinitrile 1a
but also Cbz-protected leucine substituted and Boc-protected
serine benzyl ether substituted propanedinitriles are suitable C-
terminal glycine units. Suitable nucleophilic amino acids are
Phe-OMe, Phe-Ot-Bu (Bu = butyl), Tyr-OMe, Leu-OMe, Ile-OMe,
Met-OMe, Ser(Ot-Bu)-Ot-Bu, Thr(Ot-Bu)-Ot-Bu, Glu(OMe)-OMe.
Entry
Base
Solvent
Time [h]
Yieldb [%]
1
2
3
4
5
6
7c
8
9
K2CO3
Cs2CO3
KOAc
CsOAc
CsOAc
CsOAc
CsOAc
CsOAc
CsOAc
DMF/H2O = 9/1
DMF/H2O = 9/1
DMF/H2O = 9/1
DMF/H2O = 9/1
DMF/H2O = 6/1
DMF/H2O = 3/1
DMF/H2O = 6/1
CH3CN/H2O = 6/1
DMSO/H2O = 6/1
17
18
15
12
12
24
4
o5
o5
52
62
56
42
47
Trp-OMe, Cys(STr)-OMe (Tr
=
trityl), Lys(NHBoc)-OMe,
His(NTr)-OMe and Arg(Pbf)-OMe.15 Thus, the tripeptides such
as Boc-Phe-Gly-Phe-OMe (3a), Boc-Phe-Gly-Phe-Ot-Bu (3b), Boc-
Phe-Gly-Tyr-OMe (3c), Boc-Phe-Gly-Leu-OMe (3d), Boc-Phe-Gly-
Ile-OMe (3e), Boc-Phe-Gly-Met-OMe (3f), Boc-Phe-Gly-Ser
(Ot-Bu)-Ot-Bu (3g), Boc-Phe-Gly-Thr(Ot-Bu)-Ot-Bu (3h), Boc-
Phe-Gly-Glu(OMe)-OMe (3i), Boc-Phe-Gly-Trp-OMe (3j), Boc-
Phe-Gly-Cys(STr)-OMe (3k), Boc-Phe-Gly-Lys(NHBoc)-OMe (3l),
Boc-Phe-Gly-His(NTr)-OMe (3m), Boc-Phe-Gly-Arg(Pbf)-OMe (3n),
Cbz-Leu-Gly-Phe-OMe (3o), Cbz-Leu-Gly-Met-OMe (3p) and Cbz-
Leu-Gly-Trp-OMe (3q) and Boc-Ser(OBn)-Gly-Lys(NHBoc)-OMe (3r)
were synthesized in good yield, in which Gly–amino acid bonds
were constructed oxidatively. Moreover, tetrapeptides such as Boc-
Phe-Gly-Leu-Phe-Ot-Bu (3s), Cbz-Leu-Gly-Leu-Phe-Ot-Bu (3t), Boc-
Phe-Leu-Gly-Phe-OMe (3u), Boc-Phe-Leu-Gly-Ile-OMe (3v), and
Boc-Phe-Leu-Gly-Tyr-OMe (3w) were also synthesized in a moder-
ate yield, in which Gly–amino acid bonds were prepared
(Table 2b).
20
12
o5
54
a
Unless otherwise shown, the reaction was performed by employing 1a
(0.2 mmol), 2a (0.4 mmol), base (0.4 mmol), and solvent (total
volume = 2 mL) under O2 atmosphere at room temperature at the
b
c
indicated time. Isolated yield. The reaction temperature is 40 1C.
en-route to a Boc-Phe-Gly-Phe-OMe tripeptide 3a (Table 1). The
starting substituted propanedinitrile 1a was easily prepared
from Boc-protected L-phenylalanine and aminomethylene
propanedinitrile14 by the coupling reaction, followed by the
reduction with BH3ÁNH3 (eqn (1)).
(1)
It should be noted that there is no necessity to protect the
phenol moiety of tyrosine (3c, 3w), and the reaction is
compatible with methyl sulfide of methionine (3f, 3p) even
though the reaction was conducted under an oxygen atmo-
Previous oxidative amide bond formation was conducted sphere. Indole of tryptophan (3j, 3q) and ester moiety of
under anhydrous conditions, and in some cases in the presence glutamic methyl ester (3i) did not disturb the reaction.
of MS4Å to remove water completely.11c However, as the solu- Protected functionalized amino acids such as cysteine (3k),
bility of protected amino acids and peptides toward an organic lysine (3l, 3r), histidine (3m), and arginine (3n) can be
solvent is generally poor, the aqueous solvent has to be successfully employed. As all the substrates are soluble in
employed. We chose a solvent system of DMF/H2O = 9/1 in an aqueous solvent and aqueous condition did not affect the
the initial investigation. Although K2CO3 and Cs2CO3 are reaction, the reaction proceeded efficiently to afford elon-
suitable bases under previous anhydrous conditions, a complex gated peptides in a good yield. More important, unprotected
mixture was obtained (entries 1 and 2). We were happy to find leucine (3y), phenylalanine (3x, 3aa) and proline (3z) were
that the reaction proceeded even under aqueous conditions to successfully coupled demonstrating the orthogonality of the
afford product 3a in 52% yield when KOAc was employed as a current method with condensative peptide couplings
base (entry 3). The yield was further improved to 62% in the (Table 2c).
presence of CsOAc (entry 4). Next, we screened the amount of
Although the amide bond formation using 2-(aminomethyl)-
water using CsOAc as a base. A mixture of DMF and H2O (6/1) malononitrile as a glycine unit was successful, we do not have a
also gave an acceptable yield, but the yield decreased when the good result in the reaction using 2-substituted 2-(amino-
amount of water increased such as DMF/H2O (3/1) (entry 5 and methyl)malononitrile as the other amino acid surrogate
6). DMSO gave a similar result with DMF (entry 9), but CH3CN because of the racemization.
was found to be a poor solvent although it was the best
In conclusion, an oxidative synthetic method of the pep-
solvent under anhydrous conditions (cf. entry 8 and ref. 11c). tide bond of glycine–amino acid has been developed under
Therefore, the optimal reaction conditions were found an O2 atmosphere in the presence of CsOAc in an aqueous
to use CsOAc as a base in a solvent of a mixture of DMF and solution without coupling reagents and catalyst. Substituted
H2O at room temperature. We selected the ratio (6/1) of DMF 2-(aminomethyl)malononitrile acts as a glycine unit to react
and H2O considering the better solubility of other amino with a wide variety of amino acids to afford tripeptides and
acids.
tetrapeptides under mild reaction conditions. Although the
With the optimal conditions in hand, the generality of the present method is limited to the generation of an amide
reaction was investigated (Table 2). A wide range of tripeptides linkage between glycine and other amino acids, it offers an
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Table 2 Oxidative peptide synthesisa
a
Unless otherwise shown, the reaction was performed by employing 1 (0.2 mmol), 2 (0.4 mmol), CsOAc (0.4 mmol), DMF/H2O = 6/1 (total volume: 2 mL)
b
under an O2 atmosphere at room temperature at the indicated time. The yield is an isolated yield. The reaction was conducted with 0.5 mmol of 1.
c
d
DMF/H2O = 9/1 was used. The reaction was performed by using amino acid lithium salt in DMF/H2O = 7/1. See the ESI for more detail.
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Conflicts of interest
There are no conflicts to declare.
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