Gram-Scale Synthesis of a Hexapeptide by Fragment Coupling in a Ball Mill

Article abstract of DOI:10.1002/ejoc.202100839

Synthesis of long peptides is generally considered as a challenge to peptide chemists, in addition to producing significant amounts of toxic waste, such as DMF. Here we show that using solvent-less methods, such as ball milling, enabled the production of

Full text of DOI:10.1002/ejoc.202100839

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Gram-Scale Synthesis of a Hexapeptide by Fragment Coupling in
a Ball Mill
Authors: Yves Yeboue, Nadia Rguioueg, Gilles Subra, Jean Martinez,
Frédéric Lamaty, and Thomas-Xavier Métro
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100839
Link to VoR: https://doi.org/10.1002/ejoc.202100839
01/2020

10.1002/ejoc.202100839
European Journal of Organic Chemistry
COMMUNICATION
Gram-Scale Synthesis of a Hexapeptide by Fragment Coupling in
a Ball Mill
Yves Yeboue,[a] Nadia Rguioueg,[a] Gilles Subra,[a] Jean Martinez,[a] Frédéric Lamaty[a]* and Thomas-
Xavier Métro[a]*
[a]
IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France
E-mail: frederic.lamaty@umontpellier.fr, thomas-xavier.metro@umontpellier.fr
Homepage URL: https://greenchem.cnrs.fr/
Supporting information for this article is given via a link at the end of the document.
Abstract: Synthesis of long peptides is generally considered as being
a challenge to peptide chemists, in addition to producing significant
amounts of toxic waste, such as DMF. Here we show that using
solvent-less methods, such as ball milling, enabled the production of
the hexapeptide Boc-(Ala-Phe-Gly)2-OBn at the gram scale with high
overall yield (77%, 5 linear steps). This is the longest peptide chain
synthesized in a ball mill to date, in which the amino acid sequence is
precisely controlled. This study complements the current fundamental
knowledge required to synthesize longer and more difficult peptide
chains (or small proteins) by using peptide fragment couplings in a
ball mill.
process, by the dependence of expression systems on the type of
peptide and by the production of peptides containing mainly
natural amino acids. As a consequence, there is still a large space
and need for alternative peptide coupling strategies. Based on our
knowledge and on previous studies reported in the literature on
peptide synthesis under solvent-less/solvent-free conditions by
mechanochemical means,[8] we have considered evaluating the
possibility to synthesize long peptides by using a ball mill and a
fragment coupling approach. In order to avoid epimerization,
tripeptide fragments presenting a C-terminal glycine (Gly) were
preferentially selected. In addition, coupling such C-terminal
glycine fragments could be a model for the synthesis of collagen
oligomer mimics. Indeed, the collagen structures are mainly
composed of a tripeptide (X-Y-Gly)n repeat in which X or Y
represent in most cases proline or hydroxyproline, some
structures having alanine or phenylalanine.[9] Although the
presence of amino acids bearing aromatic side chains such as
phenylalanine is known to destabilize the collagen triple helix,[10]
it allows an easy monitoring and optimization of the reactions by
HPLC. This is critical for the main objective of this study that is to
rapidly access the longest possible peptide chain. For this reason,
we aimed at synthesizing oligomers of (Ala-Phe-Gly)n, first
targeting the monomer Boc-Ala-Phe-Gly-OMe 6 (Scheme 1).
Thus, Boc-Phe-OH was reacted with HCl∙H-Gly-OMe (1.2 eq) in
a vibratory ball mill (vbm), in the presence of Oxyma (1.2 equiv),
Peptides can be found in many and diverse applications as
nutriments, cosmetics and pharmaceuticals. In the field of
medicinal chemistry, they can act as therapeutics and diagnostic
agents, including to fight the Covid-19 disease.[1] Among the
peptide-based drugs, we can highlight Macrilen, which was
discovered in our institute (IBMM) in 1999. Macrilen was recently
approved by both FDA and EMA as diagnostic test of patients with
adult growth hormone deficiency (AGHD),[2] and is actually in
phase 2 clinical trial for its use in pediatrics. While the stepwise
synthesis of low molecular weight peptides does not generally
present any difficulty, the chemical synthesis of longer peptides
and proteins is a more challenging task. One of the strategies to
their access is the coupling of partially protected peptide
fragments. However, when protected peptide fragments are
synthesized by SPPS,[3] their hydrophobicity generally increases
with the number of side-chain protecting groups and with the size
of the fragment, which creates solubility issues and hence
synthesis difficulties. These solubility issues are one of the most
common challenges encountered by peptide chemists,[4] along
with the risk of epimerization of the peptide C-terminal activated
amino acid. Indeed, it is common to see solubility issues forcing
chemists to modify the synthetic routes to access targeted
peptides.[5] The removal of protective groups from amino acid side
chains and the use of water as a solvent has allowed native
chemical ligation techniques (NCL)[6] to push the limits of these
solubility problems. Yet, NCL is still limited by the lack of freedom
in the choice of the peptide bond disconnections, and solubility
issues are still present for hydrophobic peptide fragments. All
these difficulties may also explain why recombinant protein
strategy is generally preferred when sequences of more than 60
amino acid residues are targeted.[7] Unfortunately, recombinant
protein strategy is also limited by the time required to develop the
NaH2PO4
(4.0
equiv),
N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide (EDCI, 1.4 equiv), and EtOAc (0.11 µL/mg) as
a liquid additive, following experimental conditions previously
described.[8e] In order to reach the longest (Ala-Phe-Gly)n peptide
chain possible, we aimed at synthesizing this first fragment at the
gram scale. The coupling was first performed on a total mass of
reagents of 9 g, leading to the isolation of 2.2 g (91% yield) of the
expected dipeptide, Boc-Phe-Gly-OMe 1 (Scheme 1). The
reaction was performed in a 25 mL PTFE reactor with one PTFE
ball (20 mm diameter). Although we’ve never observed an
incident before, the use of PTFE milling material was preferred
over stainless steel for safety reasons. Indeed, the potential
formation of sparks arising from violent shock between the balls
and the surface of the reactor both made of stainless steel is not
desirable when working with liquid additives having a low flash
point such as EtOAc (flash point -4°C). The pure Boc-Phe-Gly-
OMe 1 was then submitted to gaseous hydrochloric acid for
removal of the Boc group.[11] This solvent-free solid-gas reaction
quantitatively yielded the Boc-deprotected peptide (HCl∙H-Phe-
Gly-OMe 2) as a white powder.
1
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10.1002/ejoc.202100839
European Journal of Organic Chemistry
COMMUNICATION
Scheme 1. First strategy to synthesize Boc-(Ala-Phe-Gly)2-OR (6; R = Me)
This peptide was then reacted with Boc-Ala-OH under similar
experimental conditions than for the first coupling of Boc-Phe-OH
with HCl∙H-Gly-OMe, yielding 1.6 g of the desired tripeptide Boc-
group would be more resistant in our reaction conditions.[14] The
synthetic pathway for Boc-(Ala-Phe-Gly)2-OBn 12 was similar to
that of Boc-(Ala-Phe-Gly)2-OMe 6. Thus, HCl∙H-Ala-Phe-Gly-OBn
11 was obtained in 4 steps in 92% overall yield from pTsOH∙H-
Gly-OBn (Scheme 2). When Boc-Ala-Phe-Gly-OH 4 (1.0 equiv)
was reacted with HCl∙H-Ala-Phe-Gly-OBn 11 (1.1 equiv) in the
presence of Oxyma (1.2 equiv), NaH2PO4 (4.0 equiv), EtOAc
(0.45 µL/mg) and EDCI (1.2 equiv) in a ball mill for 30 min, 1.7 g
of the expected hexapeptide Boc-(Ala-Phe-Gly)2-OBn 12 (84%
yield) was isolated, without visible traces of the corresponding
nonapeptide impurity. Remarkably, despite the violent impacts of
the ball movement in the ball mill reactor, the hexapeptide
structure was not degraded. Additionally, the environmental
impact of the coupling steps was evaluated by calculating their
simple E factor (noted sEF).[15] For each coupling step, the sEF
Ala-Phe-Gly-OMe
3 (87% yield). This tripeptide was then
submitted to saponification, also performed in a ball mill, using
lithium hydroxide monohydrate (2.0 equiv) in the presence of
H2O/MeOH (50/50 v/v; 0.4 µL/mg) as a liquid additive. This
procedure furnished 1.5 g of Boc-Ala-Phe-Gly-OH 4 after work-up
(94% yield).a,[8l, 12] Boc-Ala-Phe-Gly-OMe 3 was also submitted to
the Boc-deprotection by using gaseous hydrochloric acid
producing 1.0 g of HCl∙H-Ala-Phe-Gly-OMe 5 in quantitative yield.
Next, Boc-Ala-Phe-Gly-OH 4 (1.0 equiv) and HCl∙H-Ala-Phe-Gly-
OMe 5 (1.0 equiv) were reacted together in a ball mill in the
presence of Oxyma (1.2 equiv), NaH2PO4 (4.0 equiv), EtOAc
(0.45 µL/mg) and EDCI (1.0 equiv) for 30 min. Although an LC/MS
analysis of the crude revealed the expected hexapeptide Boc-
(Ala-Phe-Gly)2-OMe 6 was the major compound that formed, this
analysis also showed the presence of ~ 5% of an impurity with a
peak at 958 m/z, corresponding to the nonapeptide Boc-(Ala-Phe-
Gly)3-OMe 7. Unfortunately, our efforts to separate the
was lower than
5 indicating a low environmental impact,
especially when compared to peptide synthesis in solution and on
solid support (see SI for details of the sEF values and
calculations).[8f] In order to access Boc-(Ala-Phe-Gly)4-OBn by
coupling of Boc-(Ala-Phe-Gly)2-OH with HCl∙H-(Ala-Phe-Gly)2-
OBn, Boc-(Ala-Phe-Gly)2-OBn 12 was submitted to gaseous
hydrochloric acid for removal of the Boc group. Yet, a partial
hydrolysis of the benzyl group was observed. Unfortunately, we
did not succeed to separate the corresponding impurity HCl∙H-
(Ala-Phe-Gly)2-OH from the desired HCl∙H-(Ala-Phe-Gly)2-OBn.
We then decided to give up with the collagen oligomer mimic as
a target to reach the longest peptide possible by using ball milling.
hexapeptide Boc-(Ala-Phe-Gly)2-OMe
nonapeptide Boc-(Ala-Phe-Gly)3-OMe
6
from the undesired
by column
7
chromatography were unsuccessful. Although the mechanism
behind the formation of this compound could not be clearly
identified, it seemed likely that the fragility of the methyl ester
function could be responsible for the formation of this nonapeptide
impurity.[13]
To address this question, a second strategy was tested: using a
benzyl ester instead of the methyl ester, expecting that the benzyl
2
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10.1002/ejoc.202100839
European Journal of Organic Chemistry
COMMUNICATION
Scheme 2. Second strategy to synthesize Boc-(Ala-Phe-Gly)2-OR (12; R = Bn).
Thus, in hopes of avoiding the hydrolysis of the C-terminal ester,
the C-terminal glycine was replaced with amino acids containing
a side chain to increase steric hindrance. The corresponding
study was published recently and has shown that although this
type of amino acid is strongly prone to epimerization during
fragment coupling, no epimerization has been observed in these
experimental conditions.[8l]
To conclude, our quest to synthesize the longest peptide chain by
using a ball mill led us to test two strategies: Boc/Me and Boc/Bn.
The first strategy led to the formation of the hexapeptide Boc-(Ala-
Phe-Gly)2-OMe 6 together with a nonapeptide impurity, likely
related to the fragility of the methyl ester function in our reaction
conditions.[13] To overcome this problem, the benzyl ester group
was installed as a replacement. Thus, 1.7 g of the hexapeptide
Boc-(Ala-Phe-Gly)2-OBn 12 was isolated in pure form in five linear
steps (77% overall yield). To the best of our knowledge, this is the
longest peptide chain synthesized in a ball mill, in which the amino
acid sequence was precisely controlled.b, [8k, 16] Together with our
recent study on epimerization-free C-term activation of peptide
fragments by ball milling,[8l] this study lays the fundamental
knowledge that will be needed for the synthesis of longer and
more difficult peptide chains (or small proteins) by using peptide
fragment couplings in a ball mill.
Acknowledgements
Université de Montpellier, Centre National de la Recherche
Scientifique (CNRS), and LabEx CheMISyst (through ANR
program ANR-10-LABX-05-01) are acknowledged for financial
support.
Keywords: Amino Acids
•
Ball Mill
•
Collagen
•
Mechanochemistry • Peptides
a
For previous examples of ester hydrolysis in basic conditions in a
ball mill, please see references [8l, 12]
.
b
For references describing the synthesis by ball milling of longer
peptide chains where the amino acid sequence is not precisely
controlled, see references [8k] and [16]
.
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European Journal of Organic Chemistry
COMMUNICATION
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10.1002/ejoc.202100839
European Journal of Organic Chemistry
COMMUNICATION
Entry for the Table of Contents
Ph
O
O
O
O
no DMF, no toxicsolvents
gram scale
BocHN
NH
BocHN
NH
OBn
OH
NH
NH
NH
O
O
O
Ph
Ph
high overall yield
Boc-(Ala-Phe-Gly)2-OBn
Solvent-lesstechniques
1.7 g, 77% overall yield
By using solvent-less techniques such as ball milling, a hexapeptide was synthesized at the gram scale without using any toxic
solvents. To date, this hexapeptide is the largest precisely controlled amino acid sequence ever synthesized in a ball mill. This study
paves the way to future developments for the synthesis of longer peptides (and proteins) by using ball milling.
Institute and researcher Twitter usernames: @YeboueYves @GillesSubra @FredLamaty @txmetro34 @umontpellier
@enscmchimiemtp @INC_CNRS @chimiebalard @iCarnot_CBC
5
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