Epimerization-Free C-Term Activation of Peptide Fragments by Ball Milling

Article abstract of DOI:10.1021/acs.orglett.0c03209

Peptides were produced in high yields and, if any, very low epimerization, by mechanochemical coupling of peptide fragments containing highly epimerization-prone and/or highly hindered amino acids at C-term. Ball milling was clearly identified as the key element enabling one to obtain such results.

Full text of DOI:10.1021/acs.orglett.0c03209

pubs.acs.org/OrgLett
Letter
Epimerization-Free C‑Term Activation of Peptide Fragments by Ball
Milling
́
́
́
Yves Yeboue, Marion Jean, Gilles Subra, Jean Martinez, Frederic Lamaty,* and Thomas-Xavier Metro*
Cite This: Org. Lett. 2021, 23, 631−635
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ABSTRACT: Peptides were produced in high yields and, if any,
very low epimerization, by mechanochemical coupling of peptide
fragments containing highly epimerization-prone and/or highly
hindered amino acids at C-term. Ball milling was clearly identified
as the key element enabling one to obtain such results.
hanks to their wide range of structural and physicochem-
(Phg) is known as being one of the most epimerization-prone
Tical properties, as well as their diverse biological activities, amino acid,9,12,27−29 while isoleucine (Ile) is a hindered amino
acid exhibiting low coupling speeds. Thus, Z-Ala-Phg-OH was
reacted with HCl·H-Ile-OMe (1.5 equiv) in the presence of
EDC·HCl (N-(3-(dimethylamino)propyl)-N′-ethylcarbodii-
mide hydrochloride, 1.2 equiv), Oxyma (ethyl cyano-
(hydroxyimino)acetate, 1.2 equiv) and a small amount of
peptides found numerous applications in material and life
sciences. In healthcare, they play a key role in the design of
active biological molecules, targeting agents, vectors and
diagnostic tools, such as that recently demonstrated in the
fight against COVID-19.1,2 Over the years, interest in this class
of compounds has increased,3−6 because of their good
biological activities at very low doses, high selectivity, and
high probability of success at clinical trial stages.3 Yet, the
chemical synthesis of long peptides and proteins is a highly
challenging task. Indeed, the efficiency of coupling and
deprotection reactions decreases with the length of the peptide
chain, leading to amino acid deletions and uncompleted
sequences. Significant efforts have been deployed by peptide
scientists to tackle this challenge, mainly by using convergent
fragment coupling approaches, such as native chemical ligation
(NCL).7,8 These approaches suffer from a limited scope:
peptide bond disconnections cannot be performed at any point
of the peptide chain. Limitations are even more constraining
when coupling a C-term activated peptide fragment with a free
N-term fragment; the risk of epimerization can be extremely
high, leading to the production of highly undesirable
diastereomers.9 Some examples are reporting peptide fragment
couplings with low to null epimerization levels, but they
remain scarce.10−15 In this context, and based on our
knowledge and on previous work in the literature on peptide
synthesis under solvent-less/solvent-free conditions by mecha-
nochemical means,16−25 we considered evaluating the capacity
of ball mills to reduce or eliminate epimerization during C-
term activation of peptide fragments, and to compare these
results with the classical synthesis in solution.26
b
DMF (η = 0.45 μL/mg), 30 in a 5 mL polytetrafluoroethylene
(PTFE) vibrating ball mill (vbm) reactor containing three
stainless steel balls (5 mm diameter), for 10 min at 25 Hz.31
Here, the amount of dimethyl formamide (DMF) was not
sufficient to solubilize the reaction mixture but improved its
,
c
homogeneity. Although being a highly problematic solvent,
DMF is the most widely used solvent for peptide synthesis. It
appeared as the liquid additive of choice to ensure a rigorous
comparison between the ball-milling and the classical solution-
phase approach. Following the same idea, the η parameter was
calculated to facilitate the comparison of the quantity of
b
solvent that were used. After workup, the corresponding
tripeptide Z-Ala-Phg-Ile-OMe was isolated in 93% yield, with
excellent purity and no detectable traces of the Z-Ala-D-Phg-
Ile-OMe epimer as indicated by high-performance liquid
chromatography (HPLC) (Table 1, entry 1; tR(LLL) = 5.08
min and tR(LDL) = 5.20 min; see the Supporting Information
for details).
During the coupling under ball-milling conditions, a slight
temperature increase was observed: right after milling was
Received: September 24, 2020
Published: January 4, 2021
The coupling between Z-Ala-Phg-OH and HCl·H-Ile-OMe
a
was considered as a probe of choice. Indeed, phenylglycine
https://dx.doi.org/10.1021/acs.orglett.0c03209
© 2021 American Chemical Society
Org. Lett. 2021, 23, 631−635
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Letter
a
Table 1. Comparative Study between Vibrating Ball Milling and Solution Synthesis of Z-Ala-Phg-Ile-OMe
b
b
entry
reagents
temperature (°C)
time, t (min)
yield (%)
purity (%)
LDL (%)
1
2
3
4
5
6
7
8
EDC·HCl/Oxyma
EDC·HCl/HOBt·H2O
EDC·HCl/HOAt
DIC/HOAt
DIC/Oxyma
HATU/Et3N
33 (33)
34 (34)
34 (34)
30 (31)
31 (31)
34 (34)
33 (33)
10 (30)
10 (30)
10 (30)
10 (40)
10 (40)
10 (60)
10 (20)
30
93 (88)
90 (90)
88 (90)
n.d. (n.d.)
n.d. (n.d.)
85 (88)
86 (82)
96
>99 (32)
70 (48)
95 (59)
67 (39)
46 (<10)
88 (58)
71 (55)
>99
<1 (9)
25 (35)
<1 (26)
17 (33)
c
c
c
<1 (n.d.)
1 (<1)
2 (9)
<1
HBTU/Et3N
EDC·HCl/Oxyma
d
c
n.d.
a
Milling reactions were performed in a 5 mL PTFE jar with three stainless steel balls (5 mm diameter) at 25 Hz. The total mass of reactants was 50
b
c
d
mg. The values in parentheses correspond to solution reactions. Determined by HPLC. n.d. = not determined. Reaction performed on a total
mass of 250 mg of reactants in a 15 mL PTFE jar with one stainless steel ball (10 mm diameter) at 25 Hz for 30 min using 1.7 equiv of EDC·HCl.
EtOAc was used as a liquid additive instead of DMF.
stopped, the temperature of the milled material reached 33
°C.32 For the sake of comparison, the same temperature was
applied to the synthesis of Z-Ala-Phg-Ile-OMe under classical
magnetic stirring conditions, while using the minimal amount
of DMF enabling proper mixing (η = 20 μL/mg). Under these
experimental conditions, Z-Ala-Phg-Ile-OMe was produced in
88% yield, 32% purity, and 9% of the LDL epimer (Table 1,
entry 1; see values given in parentheses). These results clearly
showed that liquid-assisted ball milling could minimize
epimerization of a highly epimerization-prone C-term activated
peptide fragment. In addition, the coupling was complete after
10 min of ball milling, while 30 min were required under
classical solution conditions. This latter observation could be
explained by the higher concentration of the reaction mixture
in the ball mill, compared to that in solution. Other coupling
reagents and additives were screened to establish the broadness
of our observations. When couplings were performed in
solution, the quantity of DMF was kept as low as possible
while enabling proper mixing under classical magnetic stirring
(11 μL/mg < η < 26 μL/mg). When Oxyma was replaced with
HOBt·H2O, ball milling conditions led to 25% of the LDL
epimer and 70% purity, while 35% of the same epimer and 48%
purity were obtained for the synthesis in solution (Table 1,
entry 2). The use of HOAt gave better results than HOBt·
H2O: only 1% of the LDL epimer was produced in the ball-
milling conditions while 26% LDL epimer were obtained after
synthesis in solution (Table 1, entry 3). Using DIC with HOAt
under ball milling conditions furnished the tripeptide with 67%
purity along with 17% of the LDL epimer, while synthesis in
solution led to lower purity (39%) and higher epimerization
(33%) (Table 1, entry 4). These results obtained with DIC
were not improved when replacing HOAt with Oxyma (Table
1, entry 5). Concerning the stand-alone coupling reagents
HBTU and HATU, their use induced low epimerization levels
both by ball milling and in solution (Table 1, entries 6 and 7).
Yet, the HPLC purity profiles of the products obtained by ball
milling were much better than when synthesized in solution.
The comparison between the ball milling and solution-phase
processes clearly showed the superiority of the mechanochem-
ical approach over the classical synthesis in solution, since
lower epimerization rates and better purity profiles were
obtained with almost all tested coupling agents and additives,
while yields remained comparable. In addition, performing the
coupling with the best coupling agent and additive (EDC·
HCl/Oxyma) on a larger scale (250 mg total mass instead of
50 mg) with larger reactor and ball (15 mL reactor with one
10-mm-diameter ball) did not affect the yield (96%) or
epimerization rate (<1% LDL; Table 1, entry 8) (see the
Supporting Information for performances of other combina-
tions of jar and balls).
We next applied the best ball-milling experimental
conditions (EDC·HCl/Oxyma) to the synthesis of other
challenging peptides. Here, EtOAc was used instead of DMF
to improve the overall environmental impact of our ball-milling
conditions. Thus, ball milling Z-Ala-Phg-OH with HCl·H-Phe-
OMe produced the tripeptide Z-Ala-Phg-Phe-OMe in 89%
yield with full conservation of the stereogenic centers (Table 2,
entry 1). When Z-Ala-D-Phg-OH was used, the tripeptide Z-
Ala-D-Phg-Phe-OMe was isolated with a similar yield of 92%
and excellent purity (Table 2, entry 2). Cysteine, which is
another epimerization-prone amino acid,9,33 was not racemized
during the synthesis of Z-Ala-Cys(Bn)-Ala-OMe and Z-Ala-
Cys(Bn)-Phe-OMe under our conditions. In both cases,
tripeptides were obtained in excellent yields (>94%) and
diastereomeric excesses (>99%) (Table 2, entries 3 and 4).
Like isoleucine, valine is a β-branched amino acid known to be
more difficult for coupling than most other amino acids.34 Yet,
the reaction between Z-Phe-Val-OH and HCl·H-Cys(Bn)-
OMe led to the desired tripeptide, Z-Phe-Val-Cys(Bn)-OMe,
in almost quantitative yield after 30 min of ball milling (Table
2, entry 5). Importantly, no trace of the LDL epimer could be
detected by HPLC analysis.
Similar results were obtained starting with the Z-Phe-D-Val-
OH (Table 2, entry 6). When HCl·H-Cys(Bn)-OMe was
replaced with HCl·H-Ser(tBu)-OtBu, excellent yields were
obtained, again with no epimerization (Table 2, entries 7 and
632
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Letter
a
results suggest that the absence of epimerization could be
explained by the highly concentrated reaction conditions
enabled by ball milling, which would prevent the transient
formation of an oxazolone ring (see the Supporting
Information for a scheme of the proposed mechanism).
Table 2. Scope of the Peptide Couplings by Ball Milling
To conclude, using the combination of EDC·HCl, Oxyma,
and small amounts of a liquid additive (DMF or EtOAc) under
ball-milling conditions permitted us to perform peptide
fragment couplings with high yields and, if any, very low
epimerization. Noteworthy, very good results were obtained
with peptide fragments containing highly epimerization-prone
and/or highly hindered amino acids at C-term, such as
phenylglycine, cysteine, and valine. Ball milling was clearly
identified as the key element to obtain both high yield and
purity, along with low epimerization. Indeed, the ball milling
conditions proved to be superior to the classical solution
synthesis approach on a various array of widely used coupling
agents. These results open avenues for the development of
highly efficient, convergent and flexible peptide synthesis
strategies based on peptide fragment couplings. Further
investigations aiming at (i) deciphering the mechanism
enabling epimerization suppression during ball milling, when
compared to synthesis in solution and (ii) applying this
strategy to longer peptide fragments are currently being
explored in our laboratory and will be reported in due course.
time
isolated yield
(%)
de
entry
peptides
(min)
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Z-Ala-Phg-Phe-OMe
20
20
30
20
30
30
30
20
15
10
10
25
25
30
30
89
92
98
94
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
98
Z-Ala-D-Phg-Phe-OMe
Z-Ala-Cys(Bn)-Ala-OMe
Z-Ala-Cys(Bn)-Phe-OMe
Z-Phe-Val-Cys(Bn)-OMe
Z-Phe-D-Val-Cys(Bn)-OMe
Z-Phe-Val-Ser(tBu)-OtBu
Z-Phe-D-Val-Ser(tBu)-OtBu
Boc-Trp-Phe-Glu(Bn)-OBn
Boc-Trp-Phe-Gly-OBn
Boc-Trp-D-Phe-Gly-OBn
Z-Phe-Val-Leu2-OBn
b
98
97
97
b
97
92
84
95
95
91
c
c
Z-Phe-D-Val-Leu2-OBn
Z-Phe-Val-Leu3-OBn
Z-Phe-D-Val-Leu3-OBn
cd
,
93
91
>99
99
cd
,
a
Milling reactions were performed in a 15-mL PTFE jar with one
stainless steel ball (10 mm in diameter). The total mass of reactants
was 250 mg. 1.2 equiv of EDC·HCl were used. 1.0 equiv of HCl·H-
AAn-OR were used. 2.2 equiv of EDC·HCl, η (EtOAc) = 0.9 μL/mg
b
c
ASSOCIATED CONTENT
* Supporting Information
■
d
sı
and total mass = 267 mg.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c03209.
8). Of note, the t-butyl ester was stable in these experimental
conditions. In classical peptide synthesis in solution, Z and Boc
N-protecting groups are commonly used. Thus, Boc-Trp-Phe-
Glu(Bn)-OBn, Boc-Trp-Phe-Gly-OBn, and Boc-Trp-D-Phe-
Gly-OBn were synthesized and successfully isolated in good
to excellent yields (84%−95%), without any detectable
epimerization (Table 2, entries 9−11). These results supported
that the presence of Boc and benzyl groups was not an obstacle
to the success of these syntheses. We next aimed at
demonstrating that peptide fragment couplings mediated by
ball milling could be applied to longer peptide fragments. For
this, activation of dipeptides, bearing the hindered valine at C-
term, and couplings with dipeptides and tripeptides were
performed. When the optimal experimental reaction conditions
were applied to the coupling of Z-Phe-Val-OH with HCl·H-
Leu2-OBn, the expected tetrapeptide was obtained in 95%
yield without epimerization (Table 2, entry 12). The same
conditions applied to the synthesis of the diastereomer Z-Phe-
D-Val-Leu2-OBn yielded the desired tetrapeptide in 91%, yet
containing 2% of the epimer (Table 2, entry 13). To our
delight, when the tripeptide HCl·H-Leu3-OBn was reacted
with Z-Phe-Val-OH and Z-Phe-D-Val-OH, the corresponding
pentapeptides Z-Phe-Val-Leu3-OBn and Z-Phe-D-Val-Leu3-
OBn were successfully isolated with high yields (>91%) and
had negligible level of epimerization (Table 2, entries 14 and
15). So far, the longest peptide synthesized by ball milling was
the pentapeptide Leu-enkephalin.17 The successful synthesis of
these two pentapeptides by a fragment coupling approach
consolidates the potential of mechanochemistry to produce
oligopeptides with high efficiency. In classical solvent-based
conditions, epimerization is often attributed to the intra-
molecular formation of an oxazolone ring during activation,9
which is a process that is favored under diluted conditions. Our
Operating procedures and characterization of all
compounds, including NMR spectra and HPLC
chromatograms (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
́
́
Frederic Lamaty − IBMM, Univ Montpellier, CNRS,
ENSCM, Montpellier, France; Email: frederic.lamaty@
umontpellier.fr
́
Thomas-Xavier Metro − IBMM, Univ Montpellier, CNRS,
ENSCM, Montpellier, France; orcid.org/0000-0003-
2280-3595; Email: thomas-xavier.metro@umontpellier.fr
Authors
Yves Yeboue − IBMM, Univ Montpellier, CNRS, ENSCM,
Montpellier, France
Marion Jean − Aix-Marseille Université, CNRS, Centrale
Marseille, iSm2, Marseille, France; orcid.org/0000-0003-
0524-8825
Gilles Subra − IBMM, Univ Montpellier, CNRS, ENSCM,
Montpellier, France; orcid.org/0000-0003-4857-4049
Jean Martinez − IBMM, Univ Montpellier, CNRS, ENSCM,
Montpellier, France; orcid.org/0000-0002-4551-4254
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c03209
Author Contributions
Y.Y. performed the syntheses and the chemical analyses. Y.Y.
and M.J. performed the HPLC analyses aiming at measuring
diastereomeric excesses. T.X.M. devised the presented idea,
obtained the funding, supervised the project and wrote the
633
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Letter
Patel, R.; Lantos, I.; Stevenson, D.; Tubman, K. D.; Undheim, K.
Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647
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commented on the manuscript.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
̈
(12) Popovic, S.; Bieraugel, H.; Detz, R. J.; Kluwer, A. M.; Koole, J.
́
Universite de Montpellier, Centre National de la Recherche
A. A.; Streefkerk, D. E.; Hiemstra, H.; van Maarseveen, J. H.
Epimerization-Free C-Terminal Peptide Activation. Chem. - Eur. J.
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Scientifique (CNRS), and LabEx CheMISyst (through ANR
program ANR-10-LABX-05-01) are acknowledged for financial
̀
support. Monique Calmes (CNRS, Univ Montpellier) is
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Liu, Z.; Zhao, J.; Liu, J. Kilogram-Scale Synthesis of Osteogenic
Growth Peptide (10−14) Using a Fragment Coupling Approach. Org.
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acknowledged for assistance in chiral HPLC analysis.
ADDITIONAL NOTES
■
(14) Zhang, T.; Song, W.; Zhao, J.; Liu, J. Full Solution-Phase
Synthesis of Acetyl Hexapeptide-3 by Fragments Coupling Strategy.
Ind. Eng. Chem. Res. 2017, 56, 11697−11704.
(15) Gurjar, M. K.; Tripathy, N. K.; Pramanik, C. M.; Deshmukh, S.
S.; Mahajan, U. S.; Deshpande, A. P. Process for preparation of
octreotide acetate via an improved 4 + 4 soln. phase synthesis of octreotide.
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a
For sake of clarity, absolute configuration indications of L
amino acids have been omitted throughout the manuscript.
Only D configurations have been indicated. Thus, Z-Ala-Phg-
OH corresponds to Z-L-Ala-L-Phg-OH.
b
Adding a liquid additive to a mechanochemical reaction,
known as liquid-assisted grinding (LAG), is known to have
beneficial effects during peptide couplings under mechano-
chemical conditions. (See refs 17 and 18.) The η parameter is
defined by the ratio of the volume of liquid (in μL) to the total
mass of solids (in mg). (See ref 30.)
(16) Declerck, V.; Nun, P.; Martinez, J.; Lamaty, F. Solvent-Free
Synthesis of Peptides. Angew. Chem., Int. Ed. 2009, 48, 9318−9321.
́
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Environmentally benign peptide synthesis using liquid-assisted ball-
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cN,N-dimethylformamide (DMF) is classified as presenting
reproductive toxicity, according to Regulation (EC) No. 1272/
2008.
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Lamaty, F. Peptide Mechanosynthesis by Direct Coupling of N-
Protected α-Amino Acids with Amino Esters. Eur. J. Org. Chem. 2016,
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NOTE ADDED AFTER ASAP PUBLICATION
References 22−25 were added January 25, 2021.
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