Selective cleavage of an azaGly peptide bond by copper(II). Long-range effect of histidine residue

Article abstract of DOI:10.1002/psc.1211

Several reports have highlighted the interest of replacing Gly, a frequent amino acid within bioactive peptides, by azaGly (Agly) to improve their stability, activity or for the design of prodrugs. Because metal catalysis is increasingly used for tailoring peptide molecules, we have studied the stability of Agly peptides in the presence of metal ions. In this study, we show that Cu(II), unlike other metal ions such as Fe(II), Fe(III), Pd(II), or Pt(II), induces the cleavage of Agly peptides at room temperature and pH 7.3. The cleavage occurred in the absence of an anchoring His residue within the peptide but it was accelerated when this amino acid was present in the sequence. The influence of His residue on the cleavage rate was minimal when His and Agly were adjacent, whereas large effects were observed for distant His residues. The reaction between Cu(II) and Agly peptides induced the formation of Cu(I) species, which could be detected using bicinchoninic acid as a probe. The nature of products formed in this reaction allowed suggesting a mechanism for the Cu(II)-induced cleavage of Agly peptides. Copyright

Full text of DOI:10.1002/psc.1211

Research Article
Received: 11 September 2009
Revised: 30 November 2009
Accepted: 9 December 2009
Published online in Wiley Interscience: 3 February 2010
(www.interscience.com) DOI 10.1002/psc.1211
Selective cleavage of an azaGly peptide
bond by copper(II). Long-range effect
of histidine residue
Reda Mhidia and Oleg Melnyk∗
Several reports have highlighted the interest of replacing Gly, a frequent amino acid within bioactive peptides, by azaGly
(Agly) to improve their stability, activity or for the design of prodrugs. Because metal catalysis is increasingly used for tailoring
peptide molecules, we have studied the stability of Agly peptides in the presence of metal ions. In this study, we show that
Cu(II), unlike other metal ions such as Fe(II), Fe(III), Pd(II), or Pt(II), induces the cleavage of Agly peptides at room temperature
and pH 7.3. The cleavage occurred in the absence of an anchoring His residue within the peptide but it was accelerated when
this amino acid was present in the sequence. The influence of His residue on the cleavage rate was minimal when His and Agly
were adjacent, whereas large effects were observed for distant His residues. The reaction between Cu(II) and Agly peptides
induced the formation of Cu(I) species, which could be detected using bicinchoninic acid as a probe. The nature of products
c
formed in this reaction allowed suggesting a mechanism for the Cu(II)-induced cleavage of Agly peptides. Copyright ꢀ 2010
European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: azapeptides; azaglycine; Cu(II); cleavage; histidine; long-range effect
Introduction
reagents. The stability of Agly peptides in the presence of metal
ions, and particularly of Cu(II), has never been reported before and
is the subject of this communication.
Azapeptides are peptide analogs in which the α-CH moiety of
one or more amino acid residues in the peptide chain is replaced
by a nitrogen atom [1,2]. The β-turn geometry induced by the
aza residue [3–5] and the increased chemical stability of urea-type
bonds compared to amide bonds might explain the better stability
of azapeptides in biological medium relative to native peptides
[6]. In the same way, Aza-amino acids were used for the design of
Ser/Cys protease inhibitors [7]. Several reports have highlighted
theinterestofreplacingGly,afrequentaminoacidwithinbioactive
peptides, by azaGly (Agly) to improve their stability and/or activity
[6,8–11]. Another interesting application of Agly residue consists
in the design of prodrugs [12].
The synthesis of azapeptides is usually performed using
solid-phase methods [13–15]. In particular, Agly residue can easily
be introduced into peptides by reacting 1-Fmoc-2-oxoimidazole
hydrazine with a peptidyl resin assembled using Fmoc/tert-butyl
chemistry [8]. Another strategy relies on the site-specific ligation
of unprotected peptide fragments. We have recently reported
that Agly peptides can be assembled chemoselectively and
without racemization using unprotected peptide fragments
by silver catalyzed reaction of C-terminal peptide hydrazides
with N-terminal phenylthiocarbonyl peptides [16]. N-terminal
phenylthiocarbonyl peptides are readily synthesized using stan-
dard Fmoc/tert-butyl SPPS methods and commercially available
phenylthiochloroformate [17]. This ligation method, which opens
up the possibility to synthesize large Agly peptides, was also used
for the lipopeptides synthesis featuring an Agly residue between
the lipid and the peptide chain.
Metal-catalyzed reactions, in particular copper-catalyzed 1,3-
dipolar cycloadditions that deliver 1,2,3-triazoles from alkynes and
azides, are increasingly used for the assembly of peptide scaffolds,
the preparation of peptide-based conjugates or the linkage of
peptides to surfaces. These reactions are catalyzed by Cu(I), which
is typically generated in situ through reduction of Cu(II) ion by
ascorbate. Copper(II)/ascorbate-mediated oxidative damage to
peptides or proteins has been reported [18,19]. Cu(II)/ascorbate
generates oxygen-derived free radical species which mainly cause
a modification of histidine residues. Cu(II) as such is known to
promotethecleavageofsomepeptidebonds[20,21]. Forexample,
the peptide sequence DK226T227HT within a recombinant human
IgG1 is specifically cleaved by cupric ions between Lys226 and
Thr227, i.e. at the second amide bond upstream from His residue
(X–Y bond within X–Y-His sequence). Other metal ions such as
Pd(II) and Pt(II) also have the property to act as artificial peptidases
[22–27]. Overall, the cleavage of peptide bonds by metals such
as Cu(II), Pd(II), Pt(II) occurs one or two residues upstream or
downstream from the anchor residue (His or Met).
Agly peptides feature a 1,2-dicarbonyl hydrazine motif. The
interaction or reaction of Cu(II) salts with hydrazine derivatives
suchassemicarbazide[28],1,2-dialkylsemicarbazide[29],1-phenyl
∗
Correspondence to: Oleg Melnyk, Institut Pasteur de Lille, UMR 8161
´
CNRS/Universite de Lille Nord de France, 1 rue du Pr Calmette 59021 Lille
Cedex, France. E-mail: oleg.melnyk@ibl.fr
Agly peptides represent an important class of peptidomimetics.
Their use in chemical or biological research requires characteriza-
tion of their reaction or compatibility with well-known chemical
´
Institut Pasteur de Lille, UMR 8161 CNRS/Universite de Lille Nord de France, 1
rue du Pr Calmette 59021 Lille Cedex, France
c
J. Pept. Sci. 2010; 16: 141–147
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

MHIDIA AND MELNYK
water and lyophilized. The crude Agly peptide was purified by
RP-HPLC on a C18 Nucleosil column, 100 Å 5 µm, 10 × 300 mm,
using a linear water/acetonitrile gradient containing 0.05% TFA by
volume (6 ml/min, detection at 215 nm). Fractions containing the
Agly peptide were collected and lyophilized. The overall yield of
the synthesis varied from 33 to 42% depending on the sequence.
All peptides displayed the expected molecular ions by MALDI-TOF
MS (Table 1 for yields, and Supporting information for all peptide
analytical data).
Table 1. Agly peptides and control peptides used in this study. His
residue is highlighted in bold
Entry
Peptide
Number
Yield (%)a
1
2
H-YKGAAA-Agly-GILKEPVGA-NH2
H-YKGAAAGILKEPVGA-NH2
H-YKGAAH-Agly-GILKEPVGA-NH2
H-YKGAAHGILKEPVGA-NH2
H-YKGAHA-Agly-GILKEPVGA-NH2
H-YKGAHAGILKEPVGA-NH2
H-YKGHAA-Agly-GILKEPVGA-NH2
H-YKGHAAGILKEPVGA-NH2
H-YGA-Agly-GILKEPVHGA-NH2
H-YGAGILKEPVHGA-NH2
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
42
35
38
38
33
41
40
32
31
53
3
4
5
6
7
General Procedure for the Cleavage of Agly Peptides by Cu(II)
8
The Agly peptide (0.21 µmol, 10 mM) was dissolved in 20 µl of
a 0.2-M Tris, HCl buffer containing 47 µg or 470 µg (0.21 or
2.10 µmol) of CuBr2. The pH was adjusted with either Tris (for
pH >9 sodium carbonate was used instead) or HCl. The reaction
mixture was stirred at 22, 37, or 50 ◦C. Progression of the reaction
was monitored by RP-HPLC on a 100 Å 5 µm C18 Nucleosil column
using a linear water/acetonitrile gradient containing 0.05% TFA by
volume (1 ml/min, detection at 215 nm). Peaks were collected and
analyzed by MALDI-TOF MS (see Supporting information).
Other procedures and analytical data are presented in Support-
ing information.
9
10
a After RP-HPLC purification.
semicarbazide[30],or1,2-diacylhydrazines[31]hasbeenreported.
In this context, the study of Agly peptides stability in the presence
of Cu(II) was worth studying. In this communication, we show that
Cu(II) induces cleavage of Agly peptides. The influence of a His
residue located upstream or downstream from the azaGly bond
was examined as this amino acid is known to be an anchor residue
for Cu(II). We describe a long-range effect of His residue on the
Agly bond rate of cleavage, an effect which has not been reported
before. The dependence of the rate and extent of cleavage on
the pH or temperature was studied. Evidence is provided for the
formation of Cu(I) during the cleavage reaction.
Results and Discussion
Agly peptides and control peptides used in this study are
presented in Table 1. A Tyr residue was inserted at the N-
terminus to facilitate UV detection of N-terminal fragments by
RP-HPLC. Agly peptides 1–5a were synthesized as described in
Scheme 1. Peptides were assembled using standard Fmoc/tert-
butyl SPPS protocols on a Rink-poly(ethylene glycol)-polystyrene
(PEG-PS) resin [33]. Incorporation of Agly residue was first
performed by reacting Fmoc-NHNH2 with CDI to produce 1-
Fmoc-2-oxoimidazole hydrazine derivative 6. Reaction of 6 with
protected peptidyl resin 7 gave resin 8, which was again subjected
to Fmoc/tert-butyl SPPS. Final deprotection and cleavage in
concentrated trifluoroacetic acid in the presence of appropriate
scavengers furnished Agly peptides 1–5a (Table 1 for the yields
after RP-HPLC purification).
Materials and Methods
Synthesis of Nꢀ-[(9H-fluoren-9-ylmethoxy) Carbonyl] 1H-
imidazole-1-carbohydrazide 6
(9H-fluoren-9-ylmethoxy)carbonylhydrazide (Fmoc-NHNH2) was
prepared as described elsewhere [32]. About 127.1 mg (0.5 mmol)
of Fmoc-NHNH2 and 81.0 mg (0.5 mmol) of CDI were dissolved in
5 ml of anhydrous DMF. The reaction mixture was stirred for 2 h at
rt under argon to give reagent 6. The resulting solution was used
directly for the next stage.
Agly peptides 1–5a and control peptides 1–5b were dissolved
in Tris, HCl buffer (pH 10.6) in the presence of 10 equiv of CuBr2
(Figure 1). Cu(II) induced the cleavage of Agly peptides 1–5a but
not of control peptides 1–5b lacking the Agly residue. Peptide
1a without His residue within its sequence was also cleaved in
the presence of Cu(II), showing that the presence of an anchoring
His residue is not required for the cleavage occurrence. However,
data obtained for peptides 2–5a (Figure 1) demonstrate that
the presence of a His residue within the peptide sequence had a
positiveeffectontherateofcleavage. Forpeptides2–4afeaturing
a His residue upstream from Agly residue, the rate of cleavage was
in order 2a > 3a > 4a, i.e. increased by the distance between
His and Agly residues. The rate of cleavage of Agly peptide 4a
was about twice higher than for Agly peptide 2a. This positive
effect on the rate of cleavage was also observed when His residue
was located downstream from Agly residue. Indeed, the rate of
cleavage of Agly peptide 5a (Figure 1) was about ten times the
rate of cleavage of peptide 1a. In this case, His residue is separated
from Agly residue by seven amino acids, showing a long-range
effect of His residue on the Cu(II)-induced degradation of Agly
peptide 5a.
Synthesis of Agly Peptides 1–5a
Peptide elongation
Peptide elongation was performed on Rink-PEG–PS resin
(NovaSyn TGR, 0.25 mmol/g, 0.38 g, 0.095 mmol) using stan-
dard Fmoc/tert-butyl chemistry on a Pioneer synthesizer (Applied
Biosystems, Courtaboeuf, France) using TBTU/HOBt/DIEA activa-
tion in DMF. A capping step was performed after each coupling
with Ac2O/DIEA. At the end of the synthesis, the Fmoc protecting
group of the last amino acid was removed using 20% piperidine
in DMF. The resin was washed with DMF (4 × 2 min).
The solution of reagent 6 was then added to the peptidyl
resin and the bead suspension was shaken for 20 h. The Fmoc
protecting group was removed using 20% piperidine and the
peptidyl resin was again subjected to SPPS as described above.
Final deprotection and cleavage from the solid support were
performed using 20 ml of TFA/anisole/TIS: 95/2.5/2.5 by volume
for1 h.ThecrudeAglypeptidewasprecipitatedin200 mlofdiethyl
ether/pentane: 1/1 by volume, solubilized in 10 ml of deionized
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CU(II) AGLY PEPTIDE CLEAVAGE
O
H
N
Fmoc
N
H
N
P
N
P
Fmoc/tBu SPPS
6
H2N
H-peptide
N
H
7
Rink PEG-PS resin
P
P
O
O
H
N
H
Fmoc/tBu SPPS
N
peptide
N
H
N
H-peptide
Fmoc
peptide
N
H
N
H
H
8
9
TFA/H2O/TIS
peptides 1-5a
P: side-chain protecting groups
Scheme 1. Solid phase synthesis of Agly peptides 1–5a.
90
80
70
60
50
40
30
20
10
0
O
O
H
N
H
N
H
N
LKEPVHGA-NH2
N
H
H-YG
N
H
O
O
Agly peptide 5a
H-YGA-OH
10
11
H-GILKEPVHGA-NH2
0
20
40
60
80
time (h)
O
LKEPVHGA-NH2
12
HN
Agly peptide 1a
Agly peptide 2a
Agly peptide 3a
N
O
Agly peptide 4a
Agly peptide 5a
O
Figure 1. CleavageofAglypeptides1–5a(10 mM)inthepresenceofCuBr2
(100 mM) in Tris, HCl buffer (0.2 M, pH 10.6, 22 ◦C). Native peptides 1–5b
were stable using the same experimental conditions (data not shown).
Percentage of cleavage was determined by RP-HPLC.
Scheme 2. Products formed in the Cu(II)-induced cleavage of Agly
peptides. Peptide 5a is used as an example.
significant effect on the rate of cleavage and led to a decrease of
half-life from 180 h to only 7 h.
Scheme 2 shows the products formed in the Cu(II)-induced
cleavage of peptides 1–5a, using Agly peptide 5a as an example.
Only three products were observed. The proposed structures are
in accordance with MALDI-TOF-post-source decay analyses (PSD).
In addition, peptide 12 formed in the Cu(II)-induced cleavage
of peptide 5a by RP-HPLC and MALDI-TOF-PSD was similar
to hydantoin peptide prepared by treating phenylthiocarbonyl
peptide 13 with base (Scheme 3).
Finally, we have examined the stability of Agly peptide 5a in
the presence of other metal ions (FeSO4, FeCl3, PdCl2, Pd(en)Cl2,
PtCl2, Pt(en)Cl2, CuSO4, CuBr2) in pH 7.3 Tris, HCl buffer at room
temperature for up to 48 h of incubation. The reaction mixtures
were monitored by RP-HPLC. Neither degradation nor complex
formation was observed in the presence of FeSO4 or FeCl3.
Complex formation occurred with all Pd(II) or Pt(II) complexes,
in particular PdCl2 or PtCl2, but no cleavage of Agly peptide 5a
was observed. Cleavage occurred only in the presence of CuSO4
or CuBr2, the latter being more efficient for inducing the cleavage
of Agly peptide 5a. PdCl2 and PtCl2 were also unable to induce the
cleavage of Agly peptide 5a at 50 ◦C for up to 24 h of incubation
(data not shown).
Several mechanisms might be involved in the Cu(II)-induced
cleavageofAglypeptides.First,themetalmightcatalyzehydrolysis
of amide or urea-type bonds. Bivalent metal ions such as Cu(II),
Pd(II), or Pt(II) can indeed catalyze hydrolysis of peptide bonds,
whichareextremelynonreactivetowardhydrolysisunderstandard
conditions[21,23,24,26,27]. Usually,themetalioniscapturedbyan
anchoring residue such as His or Met, which promotes hydrolysis
The pH-dependence of the reaction at 22 ◦C was studied using
Aglypeptide5a(Figure 2,Table 2).Thecleavagereactionincreased
significantly with the reaction mixture pH. At pH 11.0, the cleavage
reaction was about seven times faster than at pH 7.3. Allen et al.
havestudiedthespecificcleavageofhistidine-containingpeptides
by Cu(II) [21]. pH-dependence of the Cu(II)-induced cleavage in
Tris, HCl buffer showed a bell-shape curve with a maximum at
pH 7. Thus, pH-dependence of the Cu(II)-induced cleavage of
Agly peptide 5a differs markedly from what was observed for
native peptides. The influence of the temperature on the rate of
cleavage of Agly peptide 5a was also examined (Figure 3, Table 2).
Increasing the temperature from 22 ◦C to 50 ◦C at pH 7.3 had a
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MHIDIA AND MELNYK
100
90
80
70
60
50
40
30
20
10
0
O
H
LKEPVHGA-NH2
13
N
PhS
N
H
O
O
pH 9.2
O
O
LKEPVHGA-NH2
12, quant.
HN
N
0
10
20
30
40
50
60
O
time (h)
37°C
22°C
50°C
Scheme 3. Synthesis of hydantoin peptide 12 by treating phenylthiocar-
bonylpeptide 13 with base.
Figure 3. Cleavage of Agly peptides 5a (10 mM) in the presence of CuBr2
(100 mM) in Tris, HCl buffer (0.2 M, pH 7.3) at 22, 37, or 50 ◦C. PdCl2 and
PtCl2 were unable to induce the cleavage of Agly peptide 5a at 50 ◦C for
up to 24 h of incubation (data not shown). Percentage of cleavage was
determined by RP-HPLC.
100
90
80
70
60
50
1.4
1.2
1
40
30
20
10
0
pH 7.3
pH 9.2
pH 11.0
0.8
0.6
0.4
0.2
0
0
100
200
300
400
time (h)
Figure 2. Influence of the Tris, HCl buffer pH on the cleavage rate of
peptide 5a. Control peptide 5b was stable using the same experimental
conditions (data not shown). Percentage of cleavage was determined by
RP-HPLC.
0
20
40
time (min)
Aglypeptide 1a
Peptide 5b
60
80
100
of adjacent peptide bonds. The anchored metal complex can bind
the oxygen atom of the scissile amide bond, thus activating the
carbonyl group toward the external attack by water molecule.
Another possible mechanism is the internal attack of the scissile
amide bond by an anchored aqua ligand. Both mechanisms are
known to promote the cleavage of amide bonds close to the
anchoring residue, typically the first or second peptide bond
upstream or downstream from the anchoring residue.
Aglypeptide 5a
Cu(II)-BCA
Peptidel 1b
Figure 4. Evidence for formation of Cu(I) species in the Cu(II)-induced
cleavage of Agly peptides. Peptides 1,5a and 1,5b (1 mM) were reacted
with CuBr2 in pH 9.0 Tris, HCl buffer containing BCA (2.2 mM). BCA forms a
stable complex with Cu(I) which absorbs at 562 nm.
The absence of Agly peptide 5a cleavage in the presence of
Pd(II) or Pt(II) complexes at room temperature can be explained by
the mild conditions used. Indeed, Pd(II) or Pt(II) complexes were
proved to act as synthetic peptidases at acidic pH under thermal or
microwave heating [22,26]. The stability of control peptides 1–5b
in the presence of Cu(II) at room temperature might be due to
both the mild experimental conditions used in this study and the
peptide sequence chosen. The rate of cleavage of peptide bonds
by Cu(II) is indeed known to be highly dependent on peptide
sequence, in other words, the presence of a His residue in the
sequence is not a sufficient element to induce significant cleavage
rates [21]. Moreover, as for Pd(II) or Pt(II) complexes, significant
rates of cleavage for Cu(II)-sensitive His-X peptide bonds were
observed under thermal heating, typically at 50 ◦C [21].
In this work, the presence of a histidine residue favored the
cleavage reaction of Agly peptides (Agly peptides 2–5a, Figure 1).
However, unlike what has been observed previously for metal-
based synthetic peptidases, the largest effect was observed for
distant His residues separated from Agly residue by more than
Table 2. Half-life of Agly peptide 5a (10 mM) as a function of pH or
temperature in the presence of CuBr2 (100 mM) in Tris, HCl 0.2 M buffer
Temperature (◦C)
pH
t1/2 (h)
22
22
22
37
50
7.3
9.2
180
48
25
32
7
11.0
7.3
7.3
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CU(II) AGLY PEPTIDE CLEAVAGE
O
H
N
O
N
H
O
NH
HN
LKEPVHGA-NH2
H-YG
N
H
O
Agly peptide 5a
base, Cu(II)
H
N
H
N
H-YG
H-YG
NH
N
O
N
N
O
Cu2+
Cu2+
Cu2+
O
O
O
O
HN
LKEPVHGA-NH2
HN
LKEPVHGA-NH2
N
H
N
H
O
O
14
15
H
N
GA-NH2
H
N
HN
H-YG
V
NH
P
NH
N
Cu2+
O
O
E
16
O
K
O
L
N
H
HN
O
NH
LKEPVHGA-NH2
H-YGA
+
N
N
H
N
O
Cu(I)
O
17
18
O
H2O
10
11 or 12
Scheme 4. Hypothetical mechanism for the Cu(II)-induced cleavage of Agly peptides. Peptide 5a is used as an example.
two amino acids (Agly peptides 4a and 5a). These data, combined
with the fact that cleavage of Agly peptides occurred as well in the
absence of an anchoring His residue (Agly peptide 1a, Figure 1),
suggest that the catalysis of amide bond hydrolysis by Cu(II) is
not the main pathway involved in Cu(II)-induced cleavage of Agly
peptides.
Analternativemechanismmightinvolvethepotentialoxidation
of Agly moiety by Cu(II). Indeed, interaction and/or reaction
of hydrazino compounds with Cu(II) have been thoroughly
studied [34]. For example, oxidation of 1-aryl-2-acylhydrazines
by Cu(II) into corresponding aryl diazene is on the basis of
the safety-catch linker developed by Millington et al. [35]. In
this reaction, Cu(II) is reduced to Cu(I) which is then oxidized
to Cu(II) by molecular oxygen, making the reaction catalytic in
Cu(II). N,Nꢁ-diacylhydrazines are less susceptible to oxidation into
corresponding diazenes and require stronger oxidants such as
fuming nitric acid, mercuric chloride, or silver nitrate combined
with iodine or bromine or N-bromosuccinimide [34]. Reaction of
semicarbazide (SC) with Cu(II) affords stable complexes of types
Cu(SC)2X2 andCu(SC)2X2,(X=Cl,Br,NO3,ClO4,or1/2SO4),whereas
reaction of 1-phenylsemicarbazide with copper leads to oxidation
of the hydrazino group and formation of azo derivatives [30]. Little
attention has been devoted to reaction of 1-oxosemicarbazide
derivatives with Cu(II) or other metal ions.
Oxidation of Agly residue by Cu(II) might result in the formation
of Cu(I). To test the potential formation of Cu(I) species during the
Cu(II)-induced cleavage of Agly peptides, Agly peptides 1a and 5a
and the corresponding control peptides 1b and 5b were reacted
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MHIDIA AND MELNYK
with Cu(II) as usual in the presence of bicinchoninic acid (BCA).
BCA is a sensitive, stable, and highly specific reagent for Cu(I) [36].
It forms a BCA/Cu(I): 2/1 complex that absorbs strongly at 562 nm.
Thus, absorbance of the reaction mixture at 562 nm reveals the
in situformationofCu(I)species.Figure 4showsthatabsorbanceof
the reaction mixture at 562 nm increased significantly for peptides
1a and 5a but not for control peptides 1b and 5b or CuBr2/BCA
mixture without peptide. Interestingly, absorbance at 562 nm
was significantly higher for peptide 5a than for peptide 1a, in
accordance with the higher rate of cleavage observed for the
former due to the long-range effect of His residue. This experiment
showed the formation of Cu(I) species during the Cu(II)-induced
cleavageofAglypeptides(Scheme 4). Thus, thereactioncouldfirst
involve formation of type 14 or 15 complexes. A type 14 complex
was proposed as an intermediate in the reaction between 1-
phenylsemicarbazide and Cu(II) [30]. Formation of the type 15
complex can be proposed as well because similar complexes are
formed when 1,2-diacylhydrazines are reacted with Cu(II) in the
presence of a base [31]. Formation of these complexes is probably
favored by the ease of 1,2-diacylhydrazines deprotonation (pKa
10.9 for 1,2-diacetylhydrazine) [37]. By comparison, the pKa for
deprotonation of the amide group in N-methylacetamide, a model
for the peptide bond, has been estimated to 18 [38]. This pKa
explains why the biuret reaction between polypeptides and Cu(II)
is performed at high pH, typically in 0.1 M aqueous NaOH [39].
Moreover, deprotonation of the peptide bond is facilitated by
coordination of Cu(II) to the amide oxygen [40]. Precoordination
of Cu(II) to the Agly moiety might lower the pKa of hydrazine
nitrogens similarly allowing the reaction to proceed at pH 7.3
(Figure 2).
species, which could be detected using BCA as a probe. A cleavage
mechanism is proposed based on previous reports and on data
presented. Besides the usefulness of this report for those involved
in azapeptide synthesis, Agly peptides might be used as cleavage
linkers whose cleavage is triggered by the presence of Cu(II).
Acknowledgements
We are grateful to the CNRS, Universite´ de Lille Nord de France,
Institut Pasteur de Lille, IFR 142, Re´gion Nord Pas de Calais, the
European Community (FEDER), and from Cance´ropoˆle Nord-Ouest
for their financial support. We warmly thank Herve´ Drobecq for
MALDI-TOF MS and Ge´rard Montagne for NMR experiments. This
research was performed using the Chemistry Systems Biology
platform (http://csb.ibl.fr).
Supporting information
Supporting information may be found in the online version of this
article.
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The biuret complex between polypeptides and Cu(II) involves
deprotonation of four peptide bonds, resulting in the binding
of four imidic nitrogens to Cu(II) [41]. The mild experimental
conditions used in this work do not favor such complex formation
because peptides 1–5b were unable to react with Cu(II) (Figures 2
and 3). Assuming that Cu(II) binds to a deprotonated form of the
1,2-dioxohydrazine moiety of Agly peptides 1–5a only and that
no deprotonated peptide bonds participate in the ligand shell of
Cu(II), there is place for the imidazole ring of His residue to bind
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16, Scheme 4). The conformational constrains imposed at the
Agly moiety in complex 14 or 15 are expected to disfavor the
participation of neighboring His residues as observed in this work
and might explain the long-range effect of His residue on the rate
of cleavage. The next step is the oxidation of the hydrazo moiety
by Cu(II), which has been demonstrated to generate Cu(I) species
(Figure 4). Formation of isocyanate 18 is proposed based on the
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semicarbazides with CuCl2 was shown to produce isocyanates by
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In conclusion, we show that Cu(II) but not other metal ions
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peptides at room temperature and pH 7.3. The cleavage occurs
in the absence of an anchoring His residue within the peptide
but it is accelerated when this amino acid is present in the
sequence. The distance between the His and Agly residues has an
important impact on the rate of cleavage. The highest rates were
observed for His residues distant from Agly moiety. The reaction
between Cu(II) and Agly peptides leads to the formation of Cu(I)
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 141–147

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c
J. Pept. Sci. 2010; 16: 141–147 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc