Serine-selective aerobic cleavage of peptides and a protein using a water-soluble copper-organoradical conjugate

Article abstract of DOI:10.1002/anie.201402618

The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copper-organoradical conjugate. The method is applicable to the site-selective cleavage of polypeptides that possess various functional groups. Peptides comprising D-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

Full text of DOI:10.1002/anie.201402618

Angewandte
Chemie
DOI: 10.1002/anie.201402618
Peptide Bond Cleavage
Serine-Selective Aerobic Cleavage of Peptides and a Protein Using
a Water-Soluble Copper–Organoradical Conjugate**
Yohei Seki, Kana Tanabe, Daisuke Sasaki, Youhei Sohma, Kounosuke Oisaki,* and
Motomu Kanai*
Abstract: The site-specific cleavage of peptide bonds is an
important chemical modification of biologically relevant
macromolecules. The reaction is not only used for routine
structural determination of peptides, but is also a potential
artificial modulator of protein function. Realizing the substrate
scope beyond the conventional chemical or enzymatic cleavage
of peptide bonds is, however, a formidable challenge. Here we
report a serine-selective peptide-cleavage protocol that pro-
ceeds at room temperature and near neutral pH value, through
mild aerobic oxidation promoted by a water-soluble copper–
organoradical conjugate. The method is applicable to the site-
selective cleavage of polypeptides that possess various func-
tional groups. Peptides comprising d-amino acids or sensitive
disulfide pairs are competent substrates. The system is extend-
able to the site-selective cleavage of a native protein, ubiquitin,
which comprises more than 70 amino acid residues.
tides[12] has been intensively studied as a potential alternative
to the enzymatic method. The establishment of practical
protocols and generalization to protein cleavage, however,
remain challenging because of the chemical robustness of
amides.[13]
Herein we report a serine(Ser)-selective cleavage of
peptides (including a native protein) through mild aerobic
oxidation of the hydroxymethyl group of Ser, promoted by
a water-soluble Cu/N-oxyl radical system.
We envisioned a Ser-selective cleavage of the peptide
bond initiated by aerobic chemoselective oxidation of its
hydroxymethyl moiety (see Scheme 1).[8c,d] Oxidation of the
hydroxymethyl moiety of a Ser residue (A) to a formyl group
would produce an a-formylglycineamide intermediate B.
Further oxidation of B would produce oxalimide C through
oxidative deformylation. Hydrolysis of the latent precursor C
under mild conditions should then be possible, because the
carbonyl groups of the oxalimide are more electrophilic than
those of simple amides, giving peptide bond cleaved frag-
ments D and D’. Using molecular oxygen as a terminal
oxidant, water and a C1 molecule (possibly HCO2H) are
stoichiometric side products. This strategy is distinct from
Lewis acid promoted Ser-selective peptide hydrolysis through
N-to-O rearrangement.[14]
To realize the designed reaction, we focused on an
oxidation of a Ser hydroxymethyl moiety with molecular
oxygen catalyzed by a Cu/N-oxyl radical.[15–17] This catalysis is
tolerant to water and Lewis basic functionalities.[16b] The
initial optimization was conducted using N-Cbz-serine tert-
butylamide as a model substrate (0.1–0.2m, see Tables S1 and
S2 in the Supporting Information for details). Optimized
catalytic conditions for the hydroxymethyl oxidation of Ser
include the use of CuI, bathophenanthrolinedisulfonic acid
disodium salt (bathophen salt), keto-ABNO[16b] (10 mol%
each), and NaNO2 (two additions: 15 mol% at the start of the
reaction and 15 mol% after five hours) in CH3CN/H2O/
AcOH (9/9/2) at room temperature under O2 atmosphere
(1 atm).
C
hemical modifications of proteins[1,2] have a great impact
on a broad range of research fields, such as chemical biology,[3]
chemical genetics,[4] protein engineering,[5] proteomics,[6] and
drug discovery.[7] Site-specific cleaving reactions of peptide
bond are among such important chemical modifications. The
method is routinely used when structural determination or
fragmentation of peptides plays a pivotal role,[8] and it is also
regarded as a new strategy for modulating the physiological
functions of proteins for medical applications.[9,10] Although
peptidases hydrolyze peptide bonds at specific sites under
mild conditions with high fidelity,[11] the scope of the scissile
substrates is restricted in principle to genetically encoded
amino acid sequences. Unnatural or structurally modified
peptides and proteins are out of the scope of peptidase
digestion. Metal-catalyzed site-selective hydrolysis of pep-
[*] Y. Seki, Dr. K. Tanabe, Dr. D. Sasaki, Dr. Y. Sohma, Dr. K. Oisaki,
Prof. Dr. M. Kanai
Graduate School of Pharmaceutical Sciences
The University of Tokyo
Bunkyo-ku, Tokyo 113-0033 (Japan)
E-mail: oisaki@mol.f.u-tokyo.ac.jp
kanai@mol.f.u-tokyo.ac.jp
Next we applied the thus-optimized catalytic conditions to
the Ser-selective cleavage of pentapeptide 1a (Fmoc-Gly-Ser-
Asn-Lys-Gly-OH, Fmoc was used for ease of analysis) in
a diluted concentration (10 mol% of the catalyst and 5 mm of
1a). Only small amounts of N-terminal fragments were
produced as a mixture of Fmoc-Gly-OH and Fmoc-Gly-
NH2 (ca. 5% combined yield), but most of 1a remained
unchanged. Probably, the generation of the catalytically
active species was inefficient because of the small concen-
trations of each catalytic component and the substrate. To
overcome the low reactivity, we used stoichiometric condi-
Dr. K. Tanabe, Dr. D. Sasaki, Dr. Y. Sohma, Prof. Dr. M. Kanai
Japan Science and Technology Agency (JST), ERATO
Kanai Life Science Catalysis Project
Bunkyo-ku, Tokyo 113-0033 (Japan)
[**] This work was supported by a Grant in-Aid for Young Scientist B and
Scientific Research C from JSPS (for K.O.), and ERATO from JST (for
M.K.). We thank T. Sonobe, Dr. Y. Aoi, and Dr. S. Kawashima for
fruitful discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201402618.
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Table 1: Cu/keto-ABNO/NOx/O2-promoted Ser-selective chain
observed by high-performance liquid chromatography
(HPLC) analysis (entries 6 and 7). In the case of Met-
containing peptides (entry 5), oxidized C-terminal fragments
(+ 16 Da) were detected as major products by liquid chro-
matography/mass spectroscopy (LC/MS). Tyr- and Trp-con-
taining peptides (entries 6 and 7) produced complex mixtures
of C-terminal-fragment derivatives as a result of overoxida-
tion.[17e] We speculate that side reactions consumed active
components and decreased the efficiency of the cleavage. The
reaction was also applicable to substrates that contain Thr
(secondary alcohol; entries 9 and 10). The oxidation of a Thr
side chain was much slower (5% yield) than the oxidative
cleavage at Ser (52% yield, entry 10). Other pentapeptides
with greater steric hindrance around the Ser residue also
provided good results (entries 11 and 12). Longer hexapep-
tides 1m and 1n, which contain a Ser residue at an internal
position, gave comparable results (entries 13 and 14). Deca-
peptide 1o, a fragment of the Alzheimer disease associated
amyloid-b peptide, produced the cleaved chain in 93% yield
(entry 15). A biorelevant oligopeptide, bradykinin, was
cleaved in 96% yield (entry 16). Substrate 1q, which contains
d-amino acids and is out of the scope for enzymatic digestion,
was successfully cleaved (entry 17). This method is compat-
ible with disulfide pairings.[18] Peptide 1r, which contains an
intramolecular disulfide bond, afforded (X = NH2 and OH)
cleavage.[a]
Entry Substrate
Yield [%][b]
1
2
3
4
5
6
7
8
9
Fmoc-Gly-Ser-Asn-Lys-Gly-OH
1a 94
Fmoc-Gly-Ser-Asn-Arg-Gly-OH
Fmoc-Gly-Ser-Asn-Asp-Gly-OH
Fmoc-Gly-Ser-Asn-Cys-Gly-OH
Fmoc-Gly-Ser-Asn-Met-Gly-OH
Fmoc-Gly-Ser-Asn-Tyr-Gly-OH
Fmoc-Gly-Ser-Asn-Trp-Gly-OH
Fmoc-Gly-Ser-Asn-His-Gly-OH
Fmoc-Gly-Ser-Asn-Thr-Gly-OH
1b quant.
1c quant.
1d 77
1e 93
1 f 24
1g 47
1h 81
1i quant.
1j 52[d]
1k quant.
1l 92
10[c] Fmoc-Gly-Thr-Asn-Ser-Gly-OH
11
12
13
14
15
Fmoc-Gly-Ser-Gln-Phe-Gly-OH
Fmoc-Ile-Ser-Asn-Lys-Gly-OH
Fmoc-Ile-Gly-Ser-Asn-Lys-Gly-OH
Fmoc-Gly-Ile-Ser-Asn-Lys-Gly-OH
Fmoc-Ab (21-30)-OH: Fmoc-Ala-Glu-Asp-Val-
Gly-Ser-Asn-Lys-Gly-Ala-OH
1m 98
1n 97
1o 93
16
Fmoc-Bradykinin-OH: Fmoc-Arg-Pro-Pro-Gly-
Phe-Ser-Pro-Phe-Arg-OH
1p 96
17
Fmoc-Gly-d-Ser-d-Asp-d-Phe-Gly-OH
1q quant.
1r 68
18[e]
19[f] Fmoc-Gly-Ser-Gly-Asn-Gly-Lys(w-FmocNH)-
1s 83 (2s)[b]
82 (3s)[g]
Gly-OH
[a] Standard conditions: CuI, bathophen salt, keto-ABNO (100 mol%
each), and NaNO2 (150 mol% at the start of the reaction and 150 mol%
after 5 h) in CH3CN/H2O/AcOH (9/9/2, 5 mm) at room temperature for
20 hours under O2 atmosphere (1 atm). [b] Combined yield of N-terminal
fragments (Fmoc-peptide-NH2 and Fmoc-peptide-OH) calculated from
the absorbance at 301 nm (maximum absorbance of the Fmoc group)
using reverse-phase HPLC analysis. [c] Reaction time was 3 h. [d] N-
terminal fragments possessing oxidized Thr were observed in 5% yield.
[e] Reaction was conducted in the presence of Me2S (100 mol%). [f] CuI,
bathophen salt, keto-ABNO (300 mol% each), and NaNO2 (450 mol%
at the start of the reaction, 450 mol% after 5 h) in CH3CN/H2O/AcOH
(9/9/2, 5 mm) at 378C for 51 h under O2 atmosphere (1 atm).
[g] Combined yield of C-terminal fragments (3s: X-COCO-Gly-Asn-Gly-
Lys(w-FmocNH)-Gly-OH; X=NH2 and OH) calculated from the
absorbance at 301 nm in reverse-phase HPLC analysis.
with the disulfide bond remaining intact (entry 18). To
achieve a higher and cleaner conversion, the addition of
Me2S as a peroxide scavenger was essential. Time-course
HPLC analysis of the oxidative cleavage reaction of hepta-
peptide 1s, which contains two Fmoc groups at both the N-
and C-terminal sides of the Ser residue, demonstrated that the
C-terminal fragments were recovered in equal combined
amounts to the sum of N-terminal fragments (entry 19 and
Figure S20 in the Supporting Information). This result
supports our reaction design (Scheme 1), in which the N-
terminal fragments 2s and C-terminal fragments 3s were
simultaneously produced in a 1:1 ratio from the same
precursor (oxalimide C).
keto-ABNO=9-azabicyclo[3.3.1]nonan-3-one N-oxyl.
tions (CuI, bathophen salt, keto-ABNO (100 mol% each),
and NaNO2 (150 mol% at the start of the reaction and
150 mol% after five hours) in CH3CN/H2O/AcOH (9/9/2,
5 mm of 1a) at room temperature under O2 atmosphere (1
atm)), and the expected cleaved products were predomi-
nantly obtained in 94% combined yield (Table 1, entry 1).
We then investigated the substrate scope of the cleavage
reaction (Table 1). Pentapeptides 1a–k, which contain various
amino acids, were investigated to assess the functional-group
tolerance of this reaction (entries 1–11). Substrates that
contain Lys, Arg (amine), Asp (carboxylic acid), Cys (thiol),
Met (sulfide), or His (Lewis basic heterocycle) produced the
N-terminal fragments (Fmoc-Gly-X: X = NH2 and OH) with
yields ranging from 77% to quantitative (entries 1–5, 8, and
9). In contrast, substrates that contain Tyr or Trp residues (i.e.,
electron-rich aromatic rings) gave the cleaved products in
moderate yields, and some unidentified by-products were
Finally, to demonstrate the substrate scope of the present
method, we extended the current conditions to the scission of
a
native protein, ubiquitin (UQ(1-76), exact mass =
8559.6 Da; Figure 1), which comprises 76 amino acid residues,
including three serines (Ser20, Ser57, Ser65).[19] The conversion
of UQ was monitored by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE; Figure 1, inset). UQ
was consumed completely after five hours under conditions A
(CuI, bathophen salt, keto-ABNO (500 mol% each) and
NaNO2 (750 mol% at the start of the reaction and 750 mol%
after five hours) in CH3CN/H2O/AcOH (9/9/2, 1 mm) at room
temperature under O2 atmosphere (1 atm)). Coomassie
Brilliant Blue staining of the gel showed three distinct
bands that were different from that of UQ. On the other
hand, UQ remained unchanged under conditions B in the
absence of keto-ABNO and conditions C in the absence of
CuI, bathophen salt, and keto-ABNO. The reaction was
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slower with the decreased loading of the Cu/keto-ABNO
conjugate (Figure S1 in the Supporting Information).
Matrix-assisted laser desorption–ionization time-of-flight
mass spectrometry (MALDI-TOF-MS) analysis of the crude
reaction mixture obtained under conditions A showed the
presence of a UQ(58-64) fragment in a monooxidized form
([M+O+H]+; observed m/z = 996.8). However, no other
fragments were detected, probably because of their poor
ionizability. To gain more insight into the product compo-
nents, we extracted the products obtained under conditions A
from the SDS-PAGE bands and conducted MALDI-TOF-MS
analysis (Figure 1). The extract of band 1 (> 7 kDa by SDS-
PAGE) contained the UQ(1-64) fragments ([M+2O+H]+; m/
z = 7289.8 by MS). The extract of band 2 (< 7 kDa by SDS-
PAGE) contained the UQ(1-56) ([M+O+H]+; m/z = 6295.4
by MS) and UQ(21-56) ([M+H]+; m/z = 4132.4 by MS)
fragments as an inseparable mixture because of the resolution
limit of the employed gel. The extract of band 3 (< 4 kDa by
SDS-PAGE)
contained
the
UQ(1-19)
fragments
([M+O+H]+; m/z = 2164.1 by MS). Mass peaks that corre-
Scheme 1. A plausible mechanism of Ser-selective chain cleavage.
spond to mono- and dioxidized fragments were observed,
Figure 1. a) Ser-selective peptide chain cleavage of ubiquitin (UQ). Inset shows time-course of SDS-PAGE analysis of the reaction with the
Coomassie Brilliant Blue stain. SeeBlue pre-stained standard was used as the molecular weight marker in the left lane. Conditions A=CuI,
bathophen salt, keto-ABNO (500 mol% each), and NaNO2 (750 mol% at 0 h and 750 mol% at 5 h) in CH3CN/H2O/AcOH (9/9/2, 1 mm) at room
temperature under O2 (1 atm); conditions B=conditions A without keto-ABNO; conditions C=conditions A without catalyst components (CuI,
bathophen salt, and keto-ABNO). Primary sequence and 3D crystallographic structure of UQ are available from the Protein Data Bank (ID:
1UBQ). b) MALDI-TOF-MS spectra corresponding to each fragment in the gel extract.
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I
À
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ambient temperature and near neutral pH value. This is the
first example of the site-selective cleavage of peptides and
proteins using molecular oxygen as a stoichiometric oxidant.
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Received: February 20, 2014
Published online: May 14, 2014
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Keywords: aerobic oxidation · copper · peptide bonds ·
proteins · serine
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