Site-selective chemical cleavage of peptide bonds

Article abstract of DOI:10.1039/c6cc01509c

Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage, modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation) and mutations (d- and β-amino acids), which are a known cause of age related diseases.

Full text of DOI:10.1039/c6cc01509c

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DOI: 10.1039/C6CC01509C
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ARTICLE
Site-Selective Chemical Cleavage of Peptide Bonds
Hader E. Elashal and Monika Raj*
Received 00th January 20xx,
Accepted 00th January 20xx
Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides
invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for
therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively
recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building
on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue
in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage,
modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and
selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation)
and mutations (D- and β-amino acids), which are a known cause of age related diseases.
DOI: 10.1039/x0xx00000x
www.rsc.org
for N-terminal amino acid determination include the use of
Sanger’s method5 and dansyl chloride.6 However, due to the
non-selectivity of the abovementioned chemical methods, site-
selective methods are more desirable in peptide cleavage. Those
currently used for selective cleavage include cyanogen bromide
for Met,7 2-nitro-5-thio-cyanobenzoic acid,8 dehydroalanine9, 10
for Cys, and 2-iodosylbenzoic acid for Trp and Tyr.11 However,
these chemical reagents, sometimes, are inadequate in
providing site-selectivity, often require harsh reaction
conditions, and result in low yields.12 For example, cyanogen
bromide uses toxic conditions and has a limited scope due to
side reactions, which prevent cleavage in the presence of
Site-selective cleavage of a peptide backbone is an important
chemical modification which is used for protein sequencing and
has various applications in bioanalytical, biotherapeutic, and
bioengineering techniques.1 For the selective hydrolysis of
peptide bonds, enzymatic cleavage is commonly employed.
Proteases that are commonly used in protein hydrolysis include:
trypsin (cleaves at Arg and Lys), chymotrypsin (Phe, Trp, and
Tyr), and pepsin (Phe and Leu). While proteases cleave proteins
with high precision and specificity, they have several
disadvantages. They are sometimes limited because they
contaminate the protein digests and they require narrow ranges
of temperature and pH for optimal activity. Moreover, proteases
often have high site-selectivity that can limit their use for
proteins containing unnatural amino acid residues and post-
translational modifications. Therefore, chemical cleavage
methods have been developed as a complementary approach to
enzymes due to their applicability to a wide range of substrates.
Peptide bonds, nonetheless, are highly unreactive towards
hydrolysis; even nonselective cleavage is difficult to achieve. The
cleavage of peptides by water at neutral pH occurs with a half-
life of 250-600 years.2 This extreme stability of peptide bonds
limits the range of appropriate cleavage reagents.3 The most
common chemical cleavage method used in protein sequencing
is Edman’s degradation,4 which utilizes phenyl isothiocyanate for
non-selective N-terminal cleavage of peptides. Other methods
methionyl-serine
and
methionyl-threonine.13
Recently,
diacetoxyiodobenzene (DIB) has been reported for asparagine-
selective cleavage of peptide bonds but oxidative modifications
were observed at several reactive amino acid side chains.14
Metals have been intensively studied for hydrolysis of peptide
bonds, but their practical use in structure determination of
proteins is still in infancy.15-22
Here, we report a chemical methodology that selectively cleaves
the peptide bond at serine residues with high efficiency.
Selective cleavage was achieved by chemical modification of the
amide backbone chain to the cyclic urethane moiety (B, Scheme
1) that is susceptible to hydrolysis under neutral, aqueous
conditions. Most importantly, the reactive side chains of amino
acids remained unmodified under the reaction conditions,
preferred over existing cleavage methods which yield
irreversibly modified fragments.
We envisioned serine-selective cleavage of peptide bonds by the
reaction of their side chain hydroxymethyl group with N,N’-
disuccinimidyl carbonate (DSC) to generate an activated
intermediate (A) (Scheme 1). Intramolecular nucleophilic attack
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of the amide nitrogen of the peptide backbone on intermediate
(A) could produce a five-membered cyclic urethane ring (B)
through path a or a six-membered ring (B’) by following path b
(Scheme 1). Our studies suggest the high specificity for the
formation of the kinetically favorable five-membered cyclic
urethane ring (B), which is supported by Nuclear Magnetic
Resonance (NMR) analysis (Supporting Information). Earlier
attempts of utilizing DSC for the formation of the cyclic urethane
moiety on Fmoc/Boc peptides were unsuccessful and led to the
formation of dehydroalanine.23 Hydrolysis of the peptide bond
at the cyclic urethane moiety (B) produces an N-terminal
peptide fragment (C) and the cyclic urethane modified C-
terminal fragment (D).
modified C-terminal fragment can undergo ring opening under
DOI: 10.1039/C6CC01509C
basic conditions reverting back to unmodified serine at the
terminus (see Supporting Information). With the original
fragment in hand, it is possible to proceed with semi-synthesis
by further coupling with another peptide or protein.
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a)
100
OH
730.2
+
1a [M+H]
Fmoc-Gly-Ala
Phe-Ala-Gly
N
H
1aO
50
t = 0 h
0
0
b)
5
10
15
20
20
20
100
778.5
[M+Na]
+
Gly-Ala-Phe
O
O
2a
H
H
O
Fmoc-Gly-Ala
N
N
N
backbone
activation
N
N
H
2a
50
O
+
H
O
[M+H]
O
O
path a
OH
A
756.4
O
path b
t = 17 h
0
O
O
0
c)
5
10
15
406.2
[M+H]
369.2
N
Fmoc-Gly-Ala-OH
100
+
O
O
3a
O
+
3a
4
H
N
[M+H]
Phe-Ala-Gly
path b
O
4
+
path a
X
O
50
0
[M+Na]
428.2
O
H
N
H
N
t = 48 h
N
O
O
0
10
15
5
B’
O
O
retention time (min)
O
N
Fig. 1 (a) HPLC charts of the reaction mixture of 1a and DSC (10 equiv), DIEA (10
equiv), and a crystal of DMAP in DMF at t = 0 h (top), at t =17 h (middle), and after
addition of 0.1 M phosphate buffer (pH 7.5) at t = 48 h (bottom) at 37 oC. HPLC
conditions: 0.1 % FA (v/v) in water, 0.1 % FA in CH3CN, gradient: 0–80 % in 25 min,
B
O
O
1.0 mL min-1 flow rate, detected at 254 nm. (b) Insets show the LCMS spectrum
corresponding to tR at 16.9 min (1a, top), 17.9 min (2a, middle), 18.6 min (3a,
bottom) and 8.9 min (4, bottom) (Supporting Information).
hydrolysis
O
H
OH
N
+
N
H
O
To determine the effect of the side chain functionality of the
residue preceding serine on the cyclization and cleavage of the
peptides, various hexapeptides, Fmoc-Gly-Xaa-Ser-Phe-Ala-Gly
(1a-1j) with a different residue at the Xaa position were
screened (Table 1). Thus, after incubation in buffer (pH 7.5) for
48 h, C-terminal fragment, Oxd-Phe-Ala-Gly 4 was obtained in all
the cases with varying HPLC conversion (70 - >99%). The results
indicate that peptides with Xaa = Gly (1b), Met (1c), His (1d), Tyr
(1e), Trp (1f), and Asp (1i) (entries 2-6 and 9, Table 1) underwent
smooth conversion to cleavage products after 48 h similar to 1a
with Xaa = Ala (entry 1, Table 1). This occurs in contrast to
known chemical reagents, which result in over-oxidation in Tyr,
Trp, and Met containing peptides.14, 22 The peptide with a bulky
amino acid adjacent to serine, 1g (Xaa = Val), gave moderate
yield after four days in phosphate buffer (pH 7.8) (entry 7, Table
1). Peptide 1h (Xaa = Lys) with a free side chain amine, also
reacted with DSC and upon treatment with buffer underwent
desired cleavage at the Lys-Ser bond and generated the N-
terminal carboxylate lysine derivative and the C-terminal
O
C
D
O
Scheme 1. Rationale for the serine-selective modification and peptide bond
cleavage in neutral aqueous solution.
To achieve cyclization at serine, reaction conditions were
optimized
using
varying
amounts
of
DSC,
4-
dimethylaminopyridine (DMAP), and N,N-diisopropylethylamine
(DIEA) on a model hexapeptide, Fmoc-Gly-Ala-Ser-Phe-Ala-Gly
1a (retention time (tR) = 16.9 min, Figure 1, Table S1, Figure S1
Supporting Information). After 17 h, one sharp peak 2a, with
retention time tR = 17.9 min was observed, indicating serine
cyclization as analyzed by a mass spectrometer (MS) (b, Figure
1). After incubating the resulting sample in phosphate buffer (pH
7.5) for 31 h, the sharp peak 2a at 17.9 min disappeared and two
new peaks appeared with retention times tR = 8.9 min and tR
=
18.6 min (c, Figure 1). MS analysis of these peaks corresponded
to the cleavage products at the N-terminal side of the serine
residue, with N-terminal fragment 3a at 18.6 min, and the
modified C-terminal fragment, 4 at 8.9 min (c, Figure 1). The
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Table 2. Substrate scope of site-selective cleavage of pepVtiiedwe AbrotincldesO.anline
fragment, Oxd-Phe-Ala-Gly-NH2 4. N-terminal carboxylated
lysine derivative 3h eventually underwent decarboxylation to
generate N-terminal fragment, Fmoc-Gly-Lys-OH 3h’ (entry 8,
Table 1, Figure S2 see Supporting Information). In the case of
peptide (1j) with a Pro residue next to serine, easy cyclization
and cleavage at the Pro-Ser bond was observed, which is in
contrast to enzymatic degradation, where Pro at a subsequent
position nearly blocks the cleavage completely independent of
the amino acid residue.21
DOI: 10.1039/C6CC01509C
Entry
Substrate
Convb (%)
1
2
Fmoc-Gly-Ala-Thr-Phe-Arg-Phe-Gly-NH2 (5)
78
75
Fmoc-Ala-Ser-Phe-Val-Gly-Ala-Thr-Phe-Arg-
Phe-Gly-NH2 (6)c
3
4
5
6
7
8
Fmoc-Ala-Val-Arg-Ser-Phe-Ser-Ala-Arg-Gly-
Phe-Gly-NH2 (7)c
75
79
80
82
72
70
Fmoc-Arg-Ala-Gly-Ala-Ser-Val-Arg-Phe-Ala-
Ser-Phe-Gly-NH2 (8)c
Fmoc-D-Val-D-Arg-D-Lys-D-Ala-D-Ser-D-Arg-
D-Ala-D-Ala-NH2 (9)
Table 1 Serine-selective amide bond cleavage of Fmoc-Gly-Xaa-Ser-Phe-
Ala-Gly (1a-j).a
Fmoc-D-Val-D-Arg-D-Lys-D-Ala-L-Ser-D-Arg-
D-Ala-D-Ala-NH2 (10)
Entry
Substrate
Xaa
Ala
Gly
Met
His
Convb (%)
>99
>99
98
1
2
1a
1b
1c
1d
1e
1f
1gc
1hc
1i
Fmoc-Cys-Gly-Arg-Arg-Ala-Cys-Gly-Ser-Phe-
Ala-Gly-NH2, disulfide bond (11)
3
Fmoc-Arg-Ala-Glu-Ala-Gly-Ser-Gly-Phe-NH2
(12)d
4
98
aReaction conditions: aPeptide (1 equiv) was reacted with DSC (20 equiv), DIEA
(20 equiv), and a crystal of DMAP in DMF followed by cleavage with 0.1 M
phosphate buffer (pH 7.5) at 37 oC for 48 h unless otherwise noted. bN-terminal
fragment. cThree fragments were detected in the HPLC trace for cleavage at Ser
and Thr at both the Ser residues. dThree fragments were detected in the HPLC
trace for cleavage at both Ser and Glu.
5
Tyr
Trp
Val
Lys
Asp
Pro
97
6
97
7
70
8
80
9
95
Table 3. Site-selective cleavage of mutated and post-translationally
modified peptides.
10
1j
98
aReaction conditions: a Peptide (1 equiv) was reacted with DSC (5-20 equiv), DIEA
(5-20 equiv) and a crystal of DMAP in DMF followed by cleavage with 0.1 M
phosphate buffer (pH 7.5) at 37 oC for 48 h. bConversion to N-terminal fragment,
Fmoc-Gly-Xaa-OH (3a-j), was calculated from the absorbance at 254 nm using
HPLC. cCleavage with 0.1 M phosphate buffer (pH 7.8) at 37 oC for 4 days.
dAcylation of the side chain of the lysine with DSC generated acylated lysine
fragment 2h and after buffer treatment, generated fragments 3h’ and 4 (Figure S2,
see Supporting Information).
Entry
1
Substrate
Conva (%)
Fmoc-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-NH2
(13)
80
77
73
75
79
70
2
3
4
5
6
Fmoc-Leu-Asn-Asp-Arg-Leu-Ala-Ser-Tyr-Leu-NH2
(14)
OAc-Ala-Val-Ala-Pro-Ala-Ala-Ser-Ile-Val-Ala-NH2
(15)
OAc-Ala-Val-Ala-Pro-βAla-Ala-Ser-Ile-Val-AlaNH2
(16)
The substrate scope of this reaction was further evaluated (Table
2). The reaction was also applicable to substrates containing
threonine 5 and 6 (entries 1 and 2, Table 2). After treatment with
buffer, cleavage was observed at the N-terminal side of both
serine and threonine, which is expected due to the similar side
group functionality exhibited by both residues. Threonine side
chain cyclization required a higher amount of DSC than the
cyclization at serine residue (entry 2, Table 2). Longer 11-mer and
12-mer peptides, 7 and 8 with multiple serine residues gave
comparable results (75-79%) (entries 3 and 4, Table 2, see
Supporting Information). Peptides 9 and 10, which contain D-
amino acid residues and are unsuitable for enzymatic
degradation, were successfully cleaved under the reaction
conditions (entries 5 and 6, Table 2). Peptide 11, comprising an
intramolecular disulfide bridge, afforded the cleavage product at
the Ser with an intact disulfide bond (entry 7, Table 2). Thus, this
methodology allows for determination of disulfide pairing
Fmoc-Leu-Arg-Arg-Ala-Ser-(N-methyl)Leu-Gly-
NH2 (17)
Fmoc-Ile-βAla-Gly-Lys-Asn-Ser-Gly-Val-NH2 (18)
aN-terminal fragment.
Surprisingly, peptide 12 containing Glu along with serine
underwent cleavage at the N-terminal of both Glu and Ser to
generate three fragments under the reaction conditions (entry 8,
Table 2, Figure S4 see Supporting Information). The activation of
Glu by DSC for scission takes place by the formation of a five
membered pyroglutamyl imide moiety (pGlu) (Figure S4).
Recently, we have reported the site-specific activation of
glutamic acid for hydrolysis of a peptide bond by using PyBrop.25
The cyclization of Glu under reaction conditions occurs in contrast
to the peptide containing Asp 1i (entry 9, Table 1), which also has
a free carboxylic acid side chain but cleavage was observed only
at serine residue (Figure S5, Supporting Information). Next, to
demonstrate the compatibility of this methodology with a free
main chain carboxylic group, a peptide FmocGly-Ser-Gly-Phe-OH
was synthesized on Wang resin.26 Under the reaction conditions,
cleavage was observed only at the N-terminus of serine residue
positions in a peptide chain, which is in contrast to other
24
chemical reagents.4,
As expected, peptide Fmoc-Gly-Ala-Cys-
Phe-Arg-Phe-Gly-NH2 with a free cysteine residue underwent
cleavage at the N-terminus of cysteine and generated
a
thiazolidinone modified C-terminal fragment Thz-Phe-Arg-Phe-
Gly-NH2 and the N-terminal fragment Fmoc-Gly-Ala-OH (Figure
S3, Supporting Information).
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and generated an N-terminal fragment, Fmoc-Gly and a C-
terminal fragment, Oxd-Gly-Phe-OH (Figure S6, Supporting
Information). This experiment in addition to Table 1 confirms that
most of the reactive side chains remained unmodified under the
reaction conditions, contrary to other cleavage methods which
lead to the oxidation of reactive amino acid side chains.14, 22
Subsequently, bioactive peptides, Bradykinin 13,27 and type I hair
keratin fragment 14, were cleaved successfully with (77-80 %)
yield (entries 1-2, Table 3, see Supporting Information).
Furthermore, we extended the current conditions to the scission
of bioactive peptides with various posttranslational modifications
such as N-acetylated antimicrobial peptide AP00011 15, N-
acetylated-β amino acid containing antimicrobial peptide 16, and
N-methylated bioactive peptide 17, which acts as a substrate for
cAMP-dependent protein kinase28 (entries 3-5, Table 3, see
Supporting Information). All these modified peptide 15, 16, and
17 were cleaved selectively at the Ser with ease and high yields.
This is in contradiction to proteases, where enzymes are unable
to recognize and cleave these modified peptides selectively.
Notes and references
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peptide,
Fmoc-Ile-βAla-Gly-Lys-Asn-Ser-Gly-Val-NH2
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containing a mutated alanine β-amino acid residue, a well-known
mutation responsible for various age related disorders such as
cataracts and Alzheimer’s (entry 6, Table 3, see Supporting
Information Figure S7).29 Finally, we carried out the reaction with
fully unprotected peptide SGISGPLS, a fragment of antimicrobial
Bovine β-defensin 13. As expected, treatment with DSC followed
by hydrolysis generated the N-terminal fragment OxdGI, C-
terminal fragments OxdGPL and Oxd by cleavage at the N-
terminus of serine residues (Figure S8, Supporting Information).
17. L. Zhu, L. Qin, T. N. Parac and N. M. Kostic, J. Am. Chem. Soc.,
A site-selective approach for cleavage of peptides at the N-
terminal of serine residues by modification of serine using metal-
free organic reagents, under mild conditions, has been
developed. The reactive side chains of amino acids remained
unmodified under the reaction conditions, which is in contrast to
known chemical cleavage methods that generate modified
fragments due to over-oxidation. Disulfide bonds are stable to
the reaction conditions, enabling the application of this method
for determining the position of disulfide pairings in a peptide.
This method also demonstrated broad substrate scope including
cleavage of a Pro-Ser bond, which is resistant to enzymatic
digestion. In addition, the method was applicable to threonine
cleavage due to the presence of similar side group functionality
as serine, therefore mimicking protease selectivity for similar
substrates. Since cleavage proceeded smoothly on mutated and
post-translationally modified peptides (D-amino-acid residues and
β-amino acids) which are unsuitable substrates for proteases, this
method is potentially applicable for determining the mutations
responsible for various age related disorders. Furthermore, the
modified C-fragment obtained after cleavage can be converted
into the original fragment with serine residue at the terminus.
These results provide a firm basis for subsequent studies aimed
at developing artificial chemical proteases for a target protein.
Work in this direction is underway in our laboratory.
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Acknowledgements
We thank Ryan Cohen at Merck for structural analysis by NMR.
M.R. thanks Paramjit S. Arora at NYU for useful discussions.
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