Mechanoenzymatic peptide and amide bond formation

Article abstract of DOI:10.1039/c7gc00615b

Mechanochemical chemoenzymatic peptide and amide bond formation catalysed by papain was studied by ball milling. Despite the high-energy mixing experienced inside the ball mill, the biocatalyst proved stable and highly efficient to catalyse the formation of α,α- and α,β-dipeptides. This strategy was further extended to the enzymatic acylation of amines by milling, and to the mechanosynthesis of a derivative of the valuable dipeptide L-alanyl-l-glutamine.

Full text of DOI:10.1039/c7gc00615b

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Mechanoenzymatic peptide and amide bond formation
José G. Hernández,*a Karen J. Ardila-Fierro,a Deborah Crawford,b Stuart L. James,b,c and Carsten
Bolm.a
Received 00th January 20xx,
Accepted 00th January 20xx
Mechanochemical chemoenzymatic peptide and amide bond formation catalysed by papain was studied by ball milling.
Despite the high-energy mixing experienced inside the ball mill, the biocatalyst proved stable and highly efficient to
catalyse the formation of α,α- and α,β-dipeptides. This strategy was further extended to the enzymatic acylation of
DOI: 10.1039/x0xx00000x
www.rsc.org/
amines by milling, and to the mechanosynthesis of a derivative of the valuable dipeptide L-alanyl-L-glutamine.
pharmaceutical chemistry.11 Hence, it is not surprising that
syntheses of amides12 and peptides13 have also been the focus
of systematic mechanochemical studies (Scheme 1a).
Introduction
The use of mechanochemical activation by milling, grinding or
shearing have experienced a steady growth in recent years.1
The increasing popularity and success of mechanosynthesis
within the scientific community - particularly from the green
For instance, coupling of several amino acids has been
successfully achieved by ball milling activated urethane-
protected α- or β-amino acid N-carboxyanhydrides (UNCAs)
(a) Previous works; mechanochemical coupling
chemistry perspective
- has to do with the fact that
mechanochemical reactions are often conducted under
solventless conditions or only in the presence of catalytic
volumes of organic solvents [liquid-assisted grinding (LAG)],2
thereby drastically minimizing waste production by having a
direct impact in the E factor of chemical processes.3 Moreover,
a variety of other advantages of mechanochemistry have also
been experienced by scientists in fields covering organic,4
inorganic,5 organometallic,6 medicinal7 and supramolecular
chemistry8 among others, where the possibility of bypassing
solubility concerns in chemical reaction design, together with
the precise stoichiometry control, shorter reaction times and
higher yielding procedures have encouraged the utilization of
ball-milling techniques to develop more beneficial protocols
compare to the solution-based counterparts. Besides offering
an alternative for conducting more sustainable chemical
syntheses, mechanochemical activation has also led to the
discovery of new chemical reactivity, affording materials and
products difficult or impossible to access in solution.9
ꢀ UNCA approach (ref.13a)
ꢀ UNCA approach (ref. 13b)
O
PG
N
AHA—H2N
or
OR4
R1
R1
O
O
R2
O
+
AHA—H N
+
R3
PG N
2
O
OR4
O
O
R2
OR4
O
AHA—H2N
O
NaHCO3
NaHCO3
Ball milling
R1
Ball milling
O
H
PG
N
OR4
N
H
R2
O
ꢀ TCT approach (ref. 13f)
ꢀ Suꢀester approach (ref. 13c)
α,αꢀdipeptides
O
H
O
H
O
H
H
N
N
N
N
PG
OH
PG
OSu
OR4
TCT, K2CO3
PPh3
Grinding
OR4
PG
R1
+
R1
+
NaHCO3
R1
R2
O
O
Ball milling
β,αꢀdipeptides
O
AHA—H2N
AHA—H2N
OR4
R2
H
H
N
OR4
R2
N
PG
Oxyma
R1
β,βꢀdipeptides
R3
O
O
NaHPO4
DMAP
.
.
HCl EDC
HCl EDC
Ball milling
Ball milling
H
R3 R4
AHA—H2N
N
CO2H
+
PG
CO2R4
R1 R2
Peptide and amide bond formation is a fundamental synthetic
tool in chemical and biosynthesis,10 which has been widely
utilised by organic and medicinal chemists in fields such as
ꢀ EDC approach (ref. 13d)
ꢀ EDC approach (ref. 13e)
(b) This work; mechanoenzymatic strategy
R1
R2
O
O
H
Papain
OR2
HCl—H2N
PG
+
PG
N
NH2
N
N
NH2
Ball milling
H
H
R3
R3
O
O
Scheme 1 Mechanochemical peptide bond formation: (a) selected
chemical approaches; (b) chemoenzymatic strategy.
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Table
mechanoenzymatic synthesis of PG-
1
Screening of the reaction conditions for the
(a)
with hydrochloride salts derived from
α- and β-amino esters
(UNCA approach; Scheme 1a).13a-b Up to the present time, the
UNCA strategy has enabled the solvent-free mechanochemical
preparation of diverse short peptides, including illustrative
applications such as the mechanosyntheses of the sweetener
aspartame (Asp-Phe-OMe),13a and the protected derivative of
L
-Ala-L-Phe-NH2 (4a-5a)
the natural dipeptide
L-carnosine (Boc-β
-Ala-His-OMe).13b
Furthermore, mechanical milling of N-Boc-protected α-amino
acid N-hydroxysuccinimide esters and amino esters led to the
high-yielding formation of peptides (Su-ester approach;
Scheme 1a), including the relatively complex natural peptide
Leu-enkephalin.13c Similarly, screening by milling of commonly
used peptide coupling agents such as carbodiimides13d-e and
benzotriazoles13f revealed that those popular coupling
reagents also tolerated mechanochemical conditions, and
could be implemented into solvent-free or LAG peptide
synthesis protocols (EDC approach; Scheme 1a). Analogously,
in the presence of triphenylphosphine, 2,4,6-trichloro-1,3,5-
Entry
PG-
L
-Ala-OR
Enzyme/additive/base
–/–/Na2CO3 or NaHCO3
Papain/–/Na2CO3
PG-
L
-Ala-
L
-Phe-NH2 (%)
4a) 0
4a) 57
1
2
3
Z-
Z-
Z-
L-Ala-OEt (1b
L-Ala-OEt (1b
L-Ala-OEt (1b
)
)
)
(
(
(
Papain/
Papain/
Na2CO3·H2O
Papain/ -Cys/
Na2CO3·10H2O
Papain/ -Cys/
Na2CO3·10H2O
Papain/ -Cys/
Na2CO3·10H2O
Papain/ -Cys/
Na2CO3·10H2O
Papain/ -Cys/
Na2CO3·10H2O
Papain/ -Cys/
Na2CO3·10H2O
Papain(c)
-Cys/
Na2CO3·10H2O
Papain latex/ -Cys/
Na2CO3·10H2O
Papain(c)
-Cys/
Na2CO3·10H2O
L
-Cys(b)/Na2CO3
4a) 63
4a) 82
L
-Cys/
4
Z-
Z-
L
-Ala-OEt (1b
)
)
(
L
5
6
L-Ala-OEt (1b
(4a) 90
(4a) 89
(4a) 93
(5a) 59
(5a) 86
(5a) 67
(4a) 99
(4a) 89
L
Z-
L-Ala-OMe (1a
)
L
7
Z-
L-Ala-OBn (1c)
triazine (TCT) was also found to activate N-protected α-amino
acids by manual grinding to generate activated TCT-esters,
L
8
Boc-L-Ala-OMe (2a)
which upon further grinding with amino esters afforded a
library of α,α
-dipeptides (TCT approach; Scheme 1a).13g
L
9
Boc-L-Ala-OEt (2b)
Despite all the advantages of the aforementioned
mechanochemical protocols, including solvent waste
reduction, rapid coupling reactions and broad reaction scope,
most of the approaches shown in Scheme 1a still suffer from
drawbacks such as the use of harmful reagents and additives
(DMAP, DCM, MeNO2, EDC, HOBt, cyanuric chloride, PPh3
etc.)14 or cumbersome and time-consuming synthetic
L
10
11
12
13
Boc-L-Ala-OBn (2c)
/
L
Z-
Z-
Z-
L-Ala-OEt (1b
L-Ala-OEt (1b
L-Ala-OEt (1b
)
)
)
L
/
L
(
4a) 93d (23)e
procedures to access the activated starting materials (e.g. β-
(a) Reaction conditions: a mixture of 1b (40 mg; 0.16 mmol), 3a (48 mg; 0.24 mmol),
enzyme (10 mg), additive (3.9 mg; 0.032 mmol) and the base (0.24 mmol) was milled
in a mixer mill at 25 Hz, using a 10 mL ZrO2 milling jar with one ZrO2 ball of 10 mm in
diameter for 2h; (b) the use of HCl·L-Cys·H2O gave 4a in 58%; (c) 20 mg of papain and
(7.7 mg; 0.064 mmol) of L-Cys were used; (d) after 30 min of milling; (e) the mixture
UNCAs).13b As an alternative, chemoenzymatic catalysis has
been widely utilised in solution and solid-phase peptide
chemistry due to the mild reaction conditions required, lower
use of toxic chemicals, higher yields, minimal side-chain was ground in a mortar with a pestle for 30 min.
protection, and the possibility to strictly control the
dipeptides mediated by cysteine proteases initiates with the
stereoselectivity of the peptide formed. In that sense,
esterases and proteases have displayed outstanding peptide
and amide bond formation activity,15 despite in nature these
biocatalysts carry out completely different tasks. For instance,
under standard conditions the main role of proteases consists
in hydrolysing peptide bonds (proteolysis), nevertheless by
tuning of the reaction conditions, proteases can exhibit
peptide bond activity under thermodynamic or kinetic
conditions.15a,16
formation of an acyl-enzyme intermediate with an amino acid
(aa1-E), which is later transfer to a nucleophile (aa2) to form
the dipeptide (aa1-aa2). In peptide synthesis, papain is known
for having flexibility with respect to the nature of aa1, but it
exhibits a preference for bulky hydrophobic amino acid
residues in the aa2 position (e.g. aa2 = Leu, Ile, Phe).
Results and discussion
Recently, we have reported the compatibility between lipases
and mechanochemical activation.17 The high catalytic activity
and exceptional resilience by enzymes under high mechanical
stress, made us wonder about the potential to utilise
proteases in the ball mill to mediate the formation of peptide
bonds (Scheme 1b). Therefore, motivated by our initials
findings and the previous reports on biocatalysis under
solvent-free conditions18 we focused our attention on studying
in depth the use of papain, a cysteine protease found in
papaya latex (Carica papaya), to catalyse peptide and amide
formation under kinetically controlled conditions. Synthesis of
To test the idea of a mechanoenzymatic formation of the
peptide bond, we selected
NH2 (3a) as benchmark system to be studied in the ball mill. To
begin, we milled a mixture of Z- -AlaOEt (1b), 3a and solid
L-Ala esters (1-2) and the HCl·L-Phe-
L
bases commonly used under milling conditions (Na2CO3 or
NaHCO3) (1.0:1.5:1.5 equiv).19 After 2 h of mixing at 25 Hz, the
analysis of the reaction mixture by 1H NMR spectroscopy
showed just the presence of the reactants and none of the
product 4a (entry 1 in Table 1). In contrast, repeating the
reaction in the presence of papain from Carica papaya powder
and Na2CO3 as the base, pleasingly afforded the dipeptide Z-L-
Ala-L-Phe-NH2 (4a) in 57% yield after filtration (entry 2 in Table
2 | J. Name., 2012, 00, 1-3
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1). In peptide synthesis, low-molecular-mass thiol compounds,
such as
-Cysteine are known to activate cysteine proteases,20
improving its activity towards the peptide bond formation.
Indeed, adding -Cys to the reaction was observed to have a
L
L
positive effect on the activity of papain raising the yield of 4a
from 57% to 63% (entries 2 and 3 in Table 1). Similarly, the
presence of water is recognised to be vital in enzymatic
synthesis to guarantee the correct enzyme structural activity.
However, in a system containing a protease, an excess of
water could compete with the nucleophile 3a for the acyl-
enzyme intermediate (aa1-E), and lead to the formation of the
hydrolysis product 1b-OH. Experimentally, in comparison to
the use of anhydrous Na2CO3 (entry 3 in Table 1), milling of 1b
2a, papain, -Cys and Na2CO3 monohydrate led to a substantial
rise in the yield of 4a (82%; entry in Table 1).
Correspondingly, the use of Na2CO3·10H2O had more
,
L
4
a
pronounced impact on the yield giving the dipeptide 4a in
90%, showing than under the applied mechanochemical
conditions, aminolysis had outdone the unwanted hydrolysis
background reaction (entry 5 in Table 1). Besides Z-
L-AlaOEt
(
1b), the methyl and benzyl ester derivatives 1a and 1c where
also found suitable as acyl donors, exhibiting similar reactivity
under mechanochemical conditions (entries 6 and 7 in Table
1). Next, Boc-L-Ala-OR acyl donors were tested in the ball mill.
After 2 h of milling, the reaction between Boc-amino esters,
papain, base and the activator did generate the dipeptide Boc-
L-Ala-L-Phe-NH2
(5a), although to a lesser extend compare to
Scheme
between Z-
mill.
2
Mechanochemical enzymatic peptide bond formation
-Ala-OEt (1b) and various amino amides (3a-h) in the ball
the Z-L-Ala-derivatives, revealing a higher preference of the
L
biocatalyst for the benzyloxycarbonyl (Z) moiety (entries 8-10
in Table 1). Then, in order to improve the yield of 4a, the
protease loading was doubled in an experiment using 1b. After
this adjustment in the reaction conditions, milling and
posterior filtration of the reaction mixture led to the recovery
of the Z-L-Ala-L-Phe-NH2 (4a) quantitatively (entry 11 in Table
1). Subsequently, papain from papaya latex (crude powder)
was also tested as mediator for the peptide bond formation.
After 2 h of milling, this lower-cost papain source did favour
the formation of 4a, although less successfully compare to
papain from Carica papaya powder (entries 11 and 12 in Table
1). Finally, manual grinding was explored as an alternative to
carry out the enzymatic reaction. Pleasingly, this simpler
approach also generated product 4a. As expected, however,
the automated ball milling was more efficient, outperforming
the grinding by hand (entry 13 in Table 1).
the corresponding dipeptides 4d
afforded in lower yields compare to the more hydrophobic or
bulkier counterparts (Scheme 2). Next, HCl· -glycine amide (3g
-f where obtained, these were
L
)
was also reacted with 1b in the ball mill. After the standard
milling time just trace quantities of the dipeptide Z-L-Ala-L-Gly-
1
NH2 (4g) were detected by H NMR spectroscopy, along with
small quantities of the hydrolysis product Z-L-Ala-OH. On the
other hand, the HCl·β-alanine amide (3h) showed to be a
better nucleophile giving the
α,β-dipeptide 4h in 48% yield
(Scheme 2).
Next, we focused on studying the effect of the acyl donor aa1
on the mechanoenzymatic peptide formation. For this, amino
acid methyl esters derived from
1a) and -Gly (1f), were subjected to mechanical milling in the
presence of HCl· -Phe-NH2 (3a), papain and the corresponding
additives (Scheme 3). After 2 h of milling at 25 Hz, all the
above amino esters bearing side-chains of different
L-Phe (1d), L-Leu (1e), L-Ala
(
L
With the optimized reaction condition, the influence of the
nucleophile in the acyl acceptor residue (aa2) was studied
L
experimentally. For this, Z-
acyl donor (aa1) and it was reacted with various
amides (3a ) bearing diverse side chains. After 2 h of milling
HCl· -tryptophanamide 3b and HCl· -leucine amide (3c)
L
-AlaOEt (1b) was chosen again as
3,
α
-amino
bulkiness and hydrophobicity were positively recognized by
the biocatalyst and transferred to the acyl acceptor affording
-g
L
(
)
L
the α,α-dipeptides (4a, 4i-k) in yields up to 98 % (Scheme 3).
proved to be excellent nucleophiles and afforded the
dipeptides 4b and 4c in high yields, 97% and 95% respectively
(Scheme 2).
These results agree with the observation made in solution,
that papain exhibits higher flexibility with regard to the nature
of the acyl donor aa1.21
The abovementioned preference of papain for bulky lipophilic
Subsequently, the scope of the mechanochemical peptide
bond formation reaction was expanded. The enzymatic
or aromatic aa2 residues was clearly observed when HCl·
L-Ile-
NH2 (3d), HCl·L-Val-NH2 (3e) and HCl·L-Ala-NH2 (3f) were used
coupling between Z-L-Phe-OMe (1d) and Z-L-Leu-OMe (1e) with
in the mechanochemical coupling. Although, in all the cases
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Scheme 4 Amide bond formation catalysed by papain in the ball mill.
This result clearly indicates the preference of papain for
natural L-amino acids, but at the same time it evidences a
degree of flexibility exhibited by the enzyme with regard to the
stereochemistry of the aa1 residue. In contrast, experiments
utilizing the HCl·
D-Phe-NH2 (3d) as nucleophile, either with L-
or D-alanine methyl ester failed to afford the corresponding
dipeptides 4q or 4r, highlighting the higher specificity of
papain for natural acyl acceptors (Scheme 3). With the aim to
test a potential mechanochemical acylation of amines with N-
protected amino acid ester as acyl donors, we studied the
coupling between Z-
L
-AlaOMe
(
1a)
and benzylamine
hydrochloride (3i) in the ball mill. Delightfully, after 2 h of
milling the reaction afforded the product 4s in 90% yield,
proving the benzylamine as an appropriate nucleophile that
also fulfilled the steric demands by the papain (Scheme 4).
Then, chiral amine hydrochlorides such as (S)- and (R)-3j were
subjected to the mechanochemical enzymatic coupling with
Scheme 3 Scope of the mechanoenzymatic peptide bond approach.
1a. As expected from the previous results using the L- or D-
phenylalanine amides 3a and 3d (Scheme 3), both enantiomers
of 3j exhibited different chemical reactivity in the presence of
amino amides HCl·
generated three
59% to 98% (Scheme 3). Once again, the use of the HCl·
L
-Leu-NH2
(
3b
)
and HCl·
L-Ile-NH2 (3c)
α
,
α
-dipeptides (4l
-n) in yields ranging from
the protease. For example, the HCl·(R)-1-phenylethylamine (3j
)
L
-
reacted faster with the protected L-alanine methyl ester 1a to
isoleucine amide (3d) as nucleophile proved challenging,
generating the product 4m in lower yield compare to the
reaction with the isomeric leucine derivative (3b) (Scheme 3).
On the other hand, the amino amide derived from the
give the product (S,R)-4t in 73 % yield, whereas the (S)-3j
enantiomer was recognized to a lesser extent by papain,
yielding the diastereoisomer (S,S)-4t in 49% (Scheme 4). This
difference in reactivity among the enantiomers of 3j made us
wonder about the potential of applying the mechanochemical
unnatural β-alanine (3h) proved also suitable as acyl acceptor
in the coupling with 1d. After the mechanical milling in the
protocol to resolve
phenylethylamine 3j in the ball mill. Indeed, under the
standard reaction conditions, milling a mixture of rac-3j, Z-
AlaOMe (1a), papain, base, and -Cys afforded the product 4t
a
racemic mixture of HCl·1-
presence of papain, the
43% yield (Scheme 3).
α,β-dipeptide 4o was separated in
L-
Finally, in order to study the preference of the enzyme for
natural -amino acids, a set of experiments using -amino acids
and -amino amides was conducted. Reacting Z-
with HCl· -Phe-NH2 (3a) in the ball mill afforded the Z-
L
L
D
as a diastereomeric mixture where the (S,R)-stereoisomer was
favoured [(S,R):(S,S)-4t, 80:20; Scheme 4]. Further modification
of the reaction conditions e.g. stoichiometry, enzyme loading
D
D
-AlaOMe (1g
)
-
L
D
- Ala-
L
Phe-NH2 (4p) in lower yield compare to the reaction with the
natural methyl ester derivative 1a (33% vs. 98%, Scheme 3).
4 | J. Name., 2012, 00, 1-3
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molten state,25 which enabled the papain to perform in short
milling times to generate a library of and -dipeptides in
α
,
α
α,β
good to high yields, and without the need for using organic
solvents. The value of the mechanochemical approach was
also demonstrated in the acylation of amines by milling and
during the mechanosynthesis of the valuable
L-alanyl-L-
glutamine derivative 4v. In order to improve our current
mechanochemical approach, studies with immobilised
enzymes to facilitate the easy recovery of the biocatalyst is on
going in our laboratories. Finally, from a more general
perspective, we believe the compatibility between
mechanochemistry and enzymes (e.g. lipases,17 proteases and
potentially many others), will have a significant impact in
increasing the complexity in chemical reaction design by
complementing, and connecting biocatalysts with the well-
established metal-4d and organocatalysed26 mechanochemical
approaches.
Scheme 5 (a) Papain catalysed mechanosynthesis of Z-L-Ala-L-Gln-NH2
(4v); (b) mechanochemical enzymatic synthesis of 4a catalysed by the
protease alcalase.
or milling time had
a
negligible impact on the
diastereoselectivity of the process. On the other hand, the
bulkier rac-1-(1-naphthyl)ethylamine salt (3k) was also found
suitable for the mechanoenzymatic acylation yielding the
product 4u in 85%, although with lower diastereoselectivity
(Scheme 4).
Acknowledgements
Next, in order to demonstrate the applicability of the
mechanoenzymatic peptide synthesis we attempted the
We thank RWTH Aachen University for support from the
Distinguished Professorship Program funded by the Excellence
Initiative of the German federal and state governments. We kindly
acknowledge Hannah Blasius and Chenlu Li for their experimental
work in this project, and Marcus Frings for the HPLC analysis of the
sample 4t (RWTH Aachen University). D.C. and S.L.J. thank the
agency EPSRC, grant no. EP/L019655/1.
preparation of the Z-L-Ala-L-Gln-NH2
dipeptide -alanyl- -glutamine ( ), a product widely used as an
important nutrient supplement and in cell culture (Scheme
5a).22 To test this idea, five solids (1a
3l, papain, -Cys,
(4v), a derivative of the
L
L
6
,
L
Na2CO3·10H2O) were ground in the ball mill for 2 h. After this
time, the analysis of the reaction mixture by 1H NMR
spectroscopy using internal standard revealed the formation of
the product 4v in 45% (Scheme 5a). The moderate yield in the
formation of 4v could be associated with a lower degree of
recognition of the glutamine amide (3l) by the enzyme.22b
Besides, trace quantities of unidentified by-products in the
reaction mixture could have been the result of background
reactions caused by the presence of the unprotected
glutamine side-chain.23
Notes and references
1
S. L. James, C. J. Adams, C. Bolm, D. Braga, P. Collier, T.
Friščić, F. Grepioni, K. D. M. Harris, G. Hyett, W. Jones, A.
Krebs, J. Mack, L. Maini, A. G. Orpen, I. P. Parkin, W. Ch.
Shearouse, J. W. Steed and D. C. Waddell, Chem. Soc. Rev.,
2012, 41, 413–447.
2
(a) T. Friščić, S. L. Childs, S. A. A. Rizvi and W. Jones,
CrystEngComm, 2009, 11, 418–426; (b) G. A. Bowmaker,
Chem. Commun., 2013, 49, 334–448.
Finally, with the aim of evaluating the generality of the
mechanoenzymatic approach, the reaction between 1a and 3a
was again conducted. This time, however, the reactants were
milled in the presence of the protease alcalase, a serine
protease which has been used in solution for chemoenzymatic
peptide and ester formation.24 Pleasingly, under the standard
mechanochemical conditions, this new enzyme did also favour
the formation of the dipeptide Z-L-Ala-L-Phe-NH2 (4a), proving
the compatibility of biocatalysts and ball milling (Scheme 5b).
3
4
R. A. Sheldon, Green Chem., 2017,19, 18-43.
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Conclusions
In summary, we have studied the mechanoenzymatic peptide
bond formation catalysed by the cysteine protease papain.
Under mechanochemical conditions, the biocatalyst proved to
be not only resilient to the mechanical stress experienced
inside the ball mill, but also very effective to mediate the
peptide and amide bond formation. The excellent mixing of
the solid reactants in the milling vessels, in combination with
the presence of the crystalline water contained in the
Na2CO3·10H2O led to the formation of a more homogenous
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DOI: 10.1039/C7GC00615B
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O
HCl—H2N
!High&yields&
!Broad&scope&
!Applica3ons&
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Graphical abstract
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