Ethoxyethylidene protecting group prevents N-overacylation in aminooxy peptide synthesis

Article abstract of DOI:10.1016/j.tet.2007.09.015

We report herein an improved synthetic route for the preparation of homogenous aminooxy peptides suitable for oxime ligation. Aminooxyacetic acid (Aoa) was protected with 1-ethoxyethylidene group (Eei) then incorporated either using PyBOP or as N-hydroxysuccinimidyl ester at N-terminal end or at a lysine side chain into model peptides in solution and on solid support. Due to the Eei protecting group, these new reagents prevent the N-overacylation side reaction in comparison with Boc-Aoa derivatives. Subsequent deprotection under mild acidic conditions gave the corresponding pure aminooxylated peptides.

Full text of DOI:10.1016/j.tet.2007.09.015

Tetrahedron 63 (2007) 11952–11958
Ethoxyethylidene protecting group prevents N-overacylation
in aminooxy peptide synthesis
*
Vincent Dulery, Olivier Renaudet and Pascal Dumy
*
ꢀ
´
Departement de Chimie Moleculaire, UMR-CNRS 5250 ICMG FR 2607, Universite Joseph Fourier, 38041 Grenoble Cedex 9, France
´
´
Received 24 July 2007; revised 29 August 2007; accepted 7 September 2007
Available online 12 September 2007
Abstract—We report herein an improved synthetic route for the preparation of homogenous aminooxy peptides suitable for oxime ligation.
Aminooxyacetic acid (Aoa) was protected with 1-ethoxyethylidene group (Eei) then incorporated either using PyBOP or as N-hydroxysuc-
cinimidyl ester at N-terminal end or at a lysine side chain into model peptides in solution and on solid support. Due to the Eei protecting group,
these new reagents prevent the N-overacylation side reaction in comparison with Boc–Aoa derivatives. Subsequent deprotection under mild
acidic conditions gave the corresponding pure aminooxylated peptides.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
to overcome this side reaction. For example, it was shown re-
cently that N-overacylation can be minimized either by con-
trolling the pH of the coupling reaction mixture containing
Aloc–Aoa–OH and uronium coupling reagents or by using
DCC, which does not require basic conditions for activa-
tion.25,26 However, incomplete reaction or traces of bis-acyl-
ated side product can still be observed even when a large
excess of reagents, repeated coupling reactions, or different
reagents are used. Since mono-protection of Aoa is not sat-
isfactory to prevent N-overacylation, an alternative strategy
would consist in using protecting groups, which mask com-
pletely the nucleophilicity of the nitrogen atom such as
phthaloyl protected Aoa.27 The bis-protected derivative
Boc2–Aoa–OH24 constitutes also an attractive building
block, but its instability under Fmoc deprotection conditions
precludes, however, the use of this reagent in SPPS, except at
the end of the peptide elongation.28
The synthesis of relevant functional macromolecules re-
mains a crucial challenge for the investigation of biological
processes. Among the large number of chemical ligation
methods developed for this purpose,1–5 chemoselective ox-
ime bond formation represents one of the most efficient syn-
thetic strategies. First developed by Rose for the preparation
of homogenous artificial proteins,6 the oxime-based strategy
has found increasing applications in the last few years, going
from the conjugation of peptides,7–9 carbohydrates,10–12 and
oligonucleotides,13,14 to the preparation of combinatorial li-
braries,15 the on-chips immobilization of biomolecules,16–19
or the chemical engineering.20,21
The oxime ligation requires the convergent preparation of
each counterpart bearing either an aldehyde or a keto group
and a super nucleophile aminooxy moiety, which react
mutually with high chemoselectivity under mild acidic con-
ditions to form a stable oxime linkage.22 Whereas the
methods to generate aldehydes in solid phase peptide synthe-
sis (SPPS) are well documented,23 and the incorporation of
aminooxy at N-terminal end or at a lysine side chain of pep-
tide remains tricky. Indeed, in most cases aminooxyacetic
acid (Aoa) building block bearing a single carbamate pro-
tecting group (e.g., Boc or Aloc) is introduced as N-hydroxy-
succinimidyl ester (OSu) or using in situ activation.
However, the mono-protection of Aoa does not ensure the
complete protection of the nitrogen, therefore providing N-
overacylated Aoa–Aoa-peptide as an undesirable side prod-
uct (Fig. 1, route A).24 Several methods have been reported
As part of our program related to the construction of syn-
thetic multiepitopic vaccine candidates, which requires
a highly clean chemistry to ensure the formation of
well-defined molecules,29 we focused recently our investi-
gations in using a new reagent providing aminooxy pep-
tides without trace of N-overacylated side products. We
presumed that an Aoa derivative protected with 1-ethoxy-
ethylidene group (Eei), which ensures the full protection
of aminooxy function might assume this purpose (Fig. 1,
route B). In this study, we thus investigated the incorpora-
tion of Aoa in different peptides by varying conditions
and reagents both in solution and on solid support
(Fig. 1). We prepared Aoa derivatives protected with
a Boc or an Eei then we compared the outcome of acyl-
ation of different model peptides. The Aoa building block
was either incorporated as active ester or by using in situ
* Corresponding authors. Tel.: +33 04 76 51 48 63; fax: +33 04 76 51 49 46;
e-mail: pascal.dumy@ujf-grenoble.fr
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.09.015

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V. Dulery et al. / Tetrahedron 63 (2007) 11952–11958
11953
Fmoc-AAn-....-AA2-AA1-X
route B
route A
5, PyBOP, DIPEA
or 6, DIPEA
1, PyBOP, DIPEA
or 2, DIPEA
H
N
AAn-....-AA2-AA1-X
AAn-....-AA2-AA1-X
O
N
O
O
O
O
O
+
O
H
O
N
Deprotection
O
O
O
O
AAn-....-AA2-AA1-Y
AAn-....-AA2-AA1-X
H2N
O
O
N
Pure aminooxy
peptide
O
O
O
Deprotection
with :
O
X = resin or CONH2
Y = CONH2 or CO2H
AAn-....-AA2-AA1-Y
H2N
O
H
O
Mono-acylated
peptide
N
R
O
1: R = -OH (Boc-Aoa-OH)
2: R = -OSu (Boc-Aoa-OSu)
O
O
O
H2N
+
Bis-acylated
by-product
AAn-....-AA2-AA1-Y
O
N
R
5: R = -OH (Eei-Aoa-OH)
6: R = -OSu (Eei-Aoa-OSu)
O
HN
O
O
O
O
Figure 1. General strategy for the functionalization of peptides with aminooxy moiety in solution and on solid support using Boc protected Aoa (route A) or
ethoxyethylidene (Eei) derivatives (route B).
activation to provide pendant and N-terminal aminooxy
moiety on linear, bridged, and branched peptides.
was performed using the procedure described previously for
2. The pure imidate Eei–Aoa–OSu 6 was obtained after flash
chromatographyandrecrystallizationassinglecrystals, which
were analyzed by X-ray diffraction (Fig. 2).32 Both reagents 5
and 6 were easily prepared in gram scale and were proved
completely stable at room temperature and under storage.
2. Results and discussion
2.1. Synthesis of Aoa derivatives
2.2. Stability of Eei protecting group
The commercially available aminooxyacetic acid 1 pro-
tected with a Boc was activated using N-hydroxysuccini-
mide and dicyclocarbodiimide (DCC) in a mixture of ethyl
acetate and dioxane. After removal of DCU by filtration
and precipitation, the active ester Boc–Aoa–OSu 230 was ob-
tained in 83% yield and was used without further purification
for the coupling reaction with peptides (Scheme 1).
We were next interested in studying the stability of the Eei
protecting group in various conditions used in the Fmoc/tBu
strategy (Table 1).
No deprotection was detected by RP-HPLC under the stan-
dard basic conditions used for coupling reaction and Fmoc
removal, therefore confirming the compatibility of Eei–
Aoa–OH or Eei–Aoa–OSu derivatives with peptide elonga-
tion. Interestingly, Eei was stable by treatment with mild
acidic solution such as 1% of TFA in CH2Cl2 or 50% aque-
ous solution of acetic acid, whereas the complete removal of
Eei was observed in 3 or 1 h with aqueous solution of 1% or
H
O
N
N
OH
OH
OH
+
O
i
I
O
3
O
O
O
O
4
1
ii
O
H
N
N
R
O
O
O
N
O
O
O
O
O
2
5: R =OH
6: R = OSu
i
Scheme 1. Reagents and conditions: (i) DCC, NHS, AcOEt/dioxane, 83%
for 2, 68% for 6; (ii) NaOH 40%, 80 ꢀC, 69%.
The synthesis of Aoa derivatives protected with 1-ethoxy-
ethylidene starts with the condensation between ethyl N-
hydroxyacetimidate 3 and iodoacetic acid 4 in sodium
hydroxide, which provides N-(1-ethoxyethylidene)2-amino-
oxyacetic acid 5 in good yield.31 The subsequent activation
Figure 2. X-ray structure of Eei–Aoa–OSu 6.

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V. Dulery et al. / Tetrahedron 63 (2007) 11952–11958
11954
Table 1. Stability of Eei protecting group under standard conditions of SPPS
AcOH/TFE/CH2Cl2, which preserved the Eei and other
acid labile protecting groups of the peptide.
Conditions
Deprotectionb
DIPEA/DMF (pH 8)a
No deprotection after 45 min
No deprotection after 30 min
No deprotection after 1 h
2.3. Functionalization using active ester derivatives
Piperidine/DMF (1:4)a
TFA/CH2Cl2 (1:99)a
AcOH/H2O (1:1)
TFA/CH3CN/H2O (1:49.5:49.5)
TFA/CH3CN/H2O (5:47.5:47.5)
TFA/TIS/H2O (95:2.5:2.5)
AcOH/TFE/CH2Cl2 (1:1:8)a
We first studied the acylation of different peptides with these
protected Aoa–OSu reagents. For this purpose, linear,
bridged, and branched peptides were chosen as model pep-
tides to incorporate aminooxy function either at the N-termi-
nal end of peptides A and B or at the side chain of Lys
residue of peptide C after removal of Aloc protecting group.
All the peptides were synthesized manually on solid phase
using the standard Fmoc/tBu strategy. The peptide B was ob-
tained after the formation of a disulfide bridge between two
Cys(S-Trt) by an iodine-mediated oxidation.33
No deprotection after 3 h
Complete deprotection after 3 h
Complete deprotection after 1 h
Complete deprotection after 1 h
No deprotection after 2 h
a
Dry solvents were used.
The stability of Eei was followed by analytical RP-HPLC of reaction
mixture.
b
5% of TFA in acetonitrile, respectively. This result denotes
a useful orthogonality between Eei and acid labile protecting
groups such as Boc or tBu. Moreover, the standard cleavage
cocktail containing TFA/TIS/H2O ensured the complete de-
protection of peptide, in contrast with cocktail containing
The coupling reactions of Aoa were performed at room tem-
perature both in solid phase and in solution using 1 or
2 equiv of Aoa derivative and DIPEA (pH adjusted at 8) in
Table 2. Acylation of peptides A, B, and C on solid support and of peptides A0, B0, and C0 in solution using protected Aoa–OSu 2 or 6
Linear peptides
Branched peptides
t
Boc-Ser( Bu)
t
t
A
H-Gly-Leu-Ser( Bu)-Asp( Bu)-Val-Gly-SASRIN
Lys
C
t
t
t
H-Gly-Leu-Ser( Bu)-Asp( Bu)-Val-Gly-OH
A'
Boc-Ser( Bu)
Lys-Ala-Lys-RINK AMIDE
t
Boc-Ser( Bu)
A"
H2NOCH2CO-Gly-Leu-Ser-Asp-Val-Gly-OH
Lys
t
Boc-Ser( Bu)
Bridged peptides
H-Ser
t
t
B
H-Gly-Gly-Cys-Asp( Bu)-Gly-Asp( Bu)-Gly-Cys-SIEBER AMIDE
C'
Lys
Lys
COCH2ONH2
Lys-Ala-Lys-NH2
H-Ser
H-Ser
t
t
B' H-Gly-Gly-Cys-Asp( Bu)-Gly-Asp( Bu)-Gly-Cys-NH2
B" H2NOCH2CO-Gly-Gly-Cys-Asp-Gly-Asp-Gly-Cys-NH2
H-Ser
Peptide
Conditionsa
H2N-peptide
MS foundc
Mono-acylated peptide
MS foundc
Bis-acylated peptide
MS foundc
b
%
b
%
b
%
A
A
A
1 equiv of 2
2 equiv of 2
2 equiv of 6
20
0
0
547.11 (547.27)
—
—
80
91
100
620.11 (620.29)
620.08
620.08
0
9
0
—
693.10 (693.31)
—
B
B
1 equiv of 2
2 equiv of 2
+2 equiv of 2
2 equiv of 6
+2 equiv of 6
50
41
0
40
0
792.28 (792.30)
792.28
—
792.23
—
50
55
88
60
965.09 (965.35)
965.13
965.03
935.08 (935.34)
934.91
0
4
12
0
—
1138.22 (1138.44)
1138.34
—
—
B
100
0
C
C
1 equiv of 2
2 equiv of 2
+2 equiv of 2
2 equiv of 6
+2 equiv of 6
d.d
d.
949.11 (949.58)
949.02
—
949.07
—
d.
d.
d.
d.
d.
1022.08 (1022.59)
1022.07
1022.17
1022.09
1022.14
n.d.
d.
d.
n.d.
n.d.
1095.2 (1095.61)
1095.21
1095.31
—
n.d.d
d.
C
n.d.
—
A0
A0
A0
B0
B0
B0
1 equiv of 2
2 equiv of 2
2 equiv of 6
1 equiv of 2
2 equiv of 2
2 equiv of 6
30
0
0
7
0
547.06
—
—
792.34
792.28
—
70
91
100
86
80
100
620.12
620.08
620.12
965.36
965.89
934.75 (935.34)
0
9
0
7
20
0
—
693.12
—
1138.78 (1138.44)
1138.89
—
0
a
Reactions were performed in DMF in the presence of DIPEA (2 equiv, pH 8) for 45 min at room temperature.
Percentage of each compound is determined by analytical RP-HPLC by the integration of each peak after cleavage (peptides A, B, and C) or by analyzing the
reaction mixture (peptides A0 and B0).
Mass spectrometry analysis was performed by electrospray ionization method in positive mode (ESI-MS). The mass found is given for either protected (B) or
fully deprotected peptides (A and C). Calculated mass is given in brackets.
b
c
d
The high polarity of both mono- and bis-acylated peptide C prevents the identification of the corresponding peaks in our HPLC analysis conditions. Thus, no
percentage can be calculated from HPLC profile and only a qualitative result obtained by MS analysis is given as detection (d.) or no detection (n.d.).

´
V. Dulery et al. / Tetrahedron 63 (2007) 11952–11958
11955
A
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Mono-acylated
peptide (peptide A’’)
Bis-acylated
peptide
Starting
material
(peptide A)
A + 2 eq. of 6
A + 2 eq. of 2
A + 1 eq. of 2
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes
B
[M+H]+
1+
1.25
1.00
0.75
0.50
0.25
0.00
620.1
[M+2H]2+
400
[M+Na]+
[M+K]+
2+
329.4
200
300
500
600
700
800
900
m/z
Figure 3. (A) Analytical RP-HPLC chromatogram (C18 column, linear gradient 5–60% B in 15 min, detection: l¼214 nm) of crude peptide A00 obtained after
solid phase functionalization of peptide Awith Boc–Aoa–OSu 2 or Eei–Aoa–OSu 6 followed by deprotection. (B) Mass spectrum (electrospray analysis, posi-
tive mode) of crude peptide A00 obtained after solid phase functionalization of peptide A with Eei–Aoa–OSu 6 followed by deprotection.
DMF. The outcome of functionalization given in Table 2 was
determined by analytical RP-HPLC by integrating the peaks
corresponding to the mono- or bis-acylated peptide as well
as by electrospray ionization mass spectrometry (ESI-MS)
as illustrated in Figure 3.
conversion of peptides A, B, and C into the corresponding
mono-acylated peptide. As expected, this new reagent al-
lows the efficient preparation of pure aminooxy derivatives
A0, B0, and C0 after deprotection since no trace of starting
material and overacylated side product was detected by
RP-HPLC and mass analysis (Fig. 3). The results obtained
in solution with peptides A0 and B0 were very similar. As
2 equiv of Boc–Aoa–OSu 2 was necessary for the total con-
version of starting material, 9% and 20% of the correspond-
ing bis-acylated side products were obtained. On the other
hand, the coupling reaction with Eei–Aoa–OSu 6 provided
the desired aminooxy peptides A0 and B0 quantitatively.
On solid phase, we first observed that 1 equiv of Boc–Aoa–
OSu 2 did not ensure a complete conversion since starting
peptide A, B, or C was still observed after 45 min, therefore
suggesting that an excess of reagent might be required. RP-
HPLC chromatogram analysis of crude reaction mixtures
(Fig. 3A) shows that 2 equiv of 2 was necessary to complete
the reaction with peptide A, however, a significant quantity
of overacylated compound (9%) was detected as previously
reported.25,26 Presumably due to the low accessibility of the
amino anchoring site, one additional cycle was even neces-
sary to get full reaction with peptides B and C, affording
also the desired mono-acylated peptides B0 and C0 contami-
nated with an increased quantity of undesired bis-acylated
side products. In contrast, the use of excess of imidate
Eei–Aoa–OSu 6 (2 or 4 equiv) ensured the complete
2.4. Functionalization using PyBOP coupling reagent
We further investigated the convenience of Eei protection for
the functionalization of peptide with aminooxy moiety on
solid phase using PyBOP coupling reagent. Eei–Aoa–OH
5 was introduced at the N-terminal end of peptide A and
the outcome of N-overacylation was compared with the
mono-protected Boc–Aoa–OH 1 (Table 3).
Table 3. Acylation of peptide A (H-Gly-Leu-Ser(tBu)-Asp(tBu)-Val-Gly-SASRIN) with protected-Aoa–OH and PyBOP
Conditionsa
H2N-peptide
MS foundc
Mono-acylated peptide
MS foundc
Bis-acylated peptide
MS foundc
b
%
b
%
b
%
1 equiv of 1
2 equiv of 1
2 equiv of 5
7
0
0
547.10 (547.27)
—
—
60
59
100
620.08 (620.29)
620.11
620.08
33
41
0
693.07 (693.31)
693.08
—
a
Reactions were performed using 1 or 2 equiv of PyBOP in DMF in the presence of DIPEA (2 equiv, pH 8) for 45 min at room temperature.
See Table 2.
Mass spectrometry analysis was performed by ESI in positive mode. The mass found is given for fully deprotected peptide. Calculated mass is given in
brackets.
b
c

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V. Dulery et al. / Tetrahedron 63 (2007) 11952–11958
11956
We observed that 1 equiv of 2 led to a mixture of compounds
corresponding to the starting material, the mono-, and bis-
acylated peptides with 7%, 60%, and 33% ratio, whereas
the use of 2 equiv of 2 dramatically increased the quantity
of bis-acylated side product (41%). As expected, the effi-
ciency of the Eei protection group was confirmed by the re-
action with excess of Eei–Aoa–OH 5 and PyBOP since the
aminooxy functionalized peptide A0 was obtained cleanly.
This interesting result suggests that the imidate 5 could be
incorporated manually or automatically during the solid
phase peptide elongation.
The analysis was performed in the positive mode for peptide
derivatives using 50% aqueous acetonitrile as an eluent.
3.2. Synthesis of Aoa derivatives
3.2.1. N0-Boc-aminooxyacetyl N-hydroxysuccinimide es-
ter (2). To a stirred solution of N-Boc 2-aminooxyacetic
acid 1 (0.500 g, 2.6 mmol) in EtOAc/dioxane (1:1, 10 mL)
at 0 ꢀC were added N-hydroxysuccinimide (0.310 g,
2.7 mmol) and DCC (0.563 g, 2.7 mmol). The mixture was
stirred at room temperature for 5 h and then filtered through
a pad of Celite. After evaporation of the solvent, the residue
was dissolved in EtOAc and washed with aqueous NaHCO3,
water, and brine. The organic phase was dried over Na2SO4
and evaporated to dryness. The crude solid was recrystal-
lized from CH2Cl2/ether/pentane thereby providing pure
and white solid (0.618 g, 2.14 mmol, 83%). 1H NMR
(300 MHz, CDCl3): d 7.66 (s, 1H), 4.78 (s, 2H), 2.87 (s,
4H), 1.49 (s, 9H); 13C NMR (75 MHz, CDCl3): d 171.8,
171.1, 168.7, 165.1, 70.9, 28.1, 25.6, 25.4; DCI-MS calcd
for C11H20N3O7: 306.1; found: m/z 305.8 [M+NH4]+.
In summary, we have investigated the use of 1-ethoxyethyl-
idene protected aminooxy acetic acid for the synthesis of
aminooxy peptides suitable for oxime ligation. Two stable
reagents prepared in one or two steps were incorporated
into peptide sequence either using PyBOP coupling reagent
or as N-hydroxysuccinimidyl ester (Eei–Aoa–OH 5 and Eei–
Aoa–OSu 6) and were easily deprotected under mild aque-
ous acidic conditions. In contrast with the corresponding
Boc protected Aoa, the use of Eei derivatives prevents the
formation of N-overacylated side product. Indeed, we have
demonstrated that these new reagents ensure the efficient
preparation of homogenous linear, bridged, or branched pep-
tides bearing N-terminal or pendent oxyamine both in solu-
tion and on solid support. These results might find broad
interests for bio-organic chemists who are interested in the
synthesis of functional macromolecules by chemoselective
methods. Particularly, this strategy is currently used in our
laboratory for the preparation of multiepitopic anticancer
vaccines29 and molecular conjugate vectors.34
3.2.2. N-(1-Ethoxyethylidene)2-aminooxyacetic acid (5).
To a stirred solution of iodoacetic acid 4 (5.00 g,
26.9 mmol) in water (10 mL) was added an aqueous solution
of NaOH (1.6 mL, 40%) at 0 ꢀC. After reaching room
temperature, ethyl N-hydroxyacetimidate
3
(4.16 g,
40.3 mmol) was added followed by an aqueous solution of
NaOH (2.5 mL, 40%) and water (10 mL) to get a basic pH
(>12). The mixture was stirred for 5 h at 80 ꢀC and cooled
at room temperature. Water was then added (50 mL) and
the aqueous solution was extracted successively with Et2O
and CH2Cl2. The aqueous phase was brought to pH 2–3
with concentrated hydrochloric acid. The acidified aqueous
phase was then extracted with AcOEt and these combined
organic layers were washed with brine (50 mL), dried over
Na2SO4, and concentrated in vacuum. The compound 5
was obtained as a colorless oil (2.99 g, 18.6 mmol, 69%).
1H NMR (300 MHz, CDCl3) d 4.48 (s, 2H), 4.00 (q, 2H,
J¼7.2 Hz), 2.01 (s, 3H), 1.27 (t, 3H, J¼7.2 Hz); 13C NMR
(75 MHz, CDCl3): d 174.5, 70.2, 62.8, 14.2, 14.0; ESI-MS
calcd for C6H12NO4: 162.2; found: m/z 162.0 [M+H]+.
3. Experimental
3.1. General
Protected amino acids, Sasrin, Sieber Amide, and Rink
Amide MBHA resin were obtained from Advanced Chem-
Tech Europe (Brussels, Belgium), Bachem Biochimie
SARL (Voisins-Les-Bretonneux, France), and France Bio-
chem S.A. (Meudon, France). PyBOP was purchased from
France Biochem and other reagents were obtained from
either Aldrich (Saint Quentin Fallavier, France) or Acros
(Noisy-Le-Grand, France). RP-HPLC was performed on
Waters equipment equipped with a 600 controller and a
Waters 2487 Dual Absorbance Detector. The purity of pep-
tide derivatives were analyzed on an analytical column
3.2.3. N0-(1-Ethoxyethylidene)2-aminooxyacetyl N-hy-
droxysuccinimide ester (6). The activated ester 6 was
obtained following the procedure described for 2. After fil-
tration of DCU and evaporation, the residue was purified
by a flash chromatography on silica gel with AcOEt/hexane
(1:1) and recrystallized in CH2Cl2/pentane to get 6 as single
crystals (3.3 g, 12.7 mmol, 68%). 1H NMR (300 MHz,
CDCl3) d 4.78 (s, 2H), 4.01 (q, 2H, J¼7.2 Hz), 2.84 (s,
4H), 1.98 (s, 3H), 1.28 (t, 3H, J¼7.2 Hz); 13C NMR
(75 MHz, CDCl3) d 168.8, 165.7, 164.8, 68.5, 62.8, 25.6,
14.2, 13.9; DCI-MS (positive mode) calcd for
C10H14N2O6: 259.1; found: m/z 259.2 [M+H]+.
˚
(Macherey–Nagel Nucleosil 120 A 3 mm C18 particles,
30ꢁ4.6 mm, flow rate of 1.3 mL minꢂ1 for 15 min gradient,
˚
Nucleosil 100 A 5 mm C18 particles, 250ꢁ4.6 mm, flow rate
of 1 mL minꢂ1 for 30 min gradient) using linear gradient and
the following solvent system: solvent A, water containing
0.09% TFA; solvent B, acetonitrile containing 0.09% TFA
and 9.91% H2O. UV absorbance was monitored at 214 and
250 nm simultaneously. Semi-preparative column (Delta-
˚
PakÔ 100 A 15 mm C18 particles, 200ꢁ2.5 mm) was used
3.3. Peptides synthesis
to purify crude peptides (when necessary) by using an iden-
tical solvent system at a flow rate of 22 mL minꢂ1. The direct
chemical ionization (DCI) mass spectra were obtained on
a Thermo Filligan PolarisQ with NH3/isobutane as the re-
agent gas. The electrospray ionization mass spectrometry
(ESI-MS) was recorded on a VG Platform II (Micromass).
3.3.1. Standard procedures for solid phase synthesis of
peptides. The synthesis of all protected peptides was carried
out manually using Fmoc/tBu strategy in a glass reaction
vessel fitted with a sintered glass frit. The coupling reac-
tions were performed using, relative to the resin loading,

´
V. Dulery et al. / Tetrahedron 63 (2007) 11952–11958
11957
2 equiv of N-a-Fmoc protecting amino acids activated in
situ with 2 equiv of PyBOP and 4 equiv of DIPEA in
DMF (10 mL/g of resin) for 30 min. After washing with
DMF (4ꢁ1 min) and CH2Cl2 (2ꢁ1 min), the completeness
of the coupling reactions was controlled by TNBS test.
N-a-Fmoc protection was removed by treatment with a
piperidine/DMF solution (1:4, 10 mL/g of resin) for 10 min.
The process was repeated two times. After washing the
resin with DMF (6ꢁ1 min), the loading of the resin was
quantified by measuring the absorption of washing solutions
at 299 nm.
mode) of fully deprotected peptide calcd for
C39H77N14O13: 949.57; found: m/z 949.23 [M+H]+.
3.4. Standard procedure for functionalization of pep-
tides with Aoa derivatives
3.4.1. Solid phase functionalization with Boc–Aoa–OH 1
and Eei–Aoa–OSu 5 with PyBOP. A solution of 1 equiv of
Boc–Aoa–OH 1 or 2 equiv of Eei–Aoa–OH 5 (relative to the
resin loading of peptide A), 1 equiv of PyBOP (or 2 equiv for
5) and 2 equiv of DIPEA (or 2 equiv for 5, pH 8) in dry DMF
was added to the resin. After shaking for 45 min at room tem-
perature, the solution was filtered and the resin was washed
with DMF (5ꢁ1 min) and CH2Cl2 (2ꢁ1 min). The complete-
ness of the coupling reaction was controlled by TNBS test.
Each peptide was finally obtained upon acidic cleavage and
deprotection by treatment of the resin with a mixture of
TFA/TIS/H2O (95:2.5:2.5) for 2 h. After evaporation of the
cleavage solution, the peptides were precipitated with diethyl
ether and analyzed by RP-HPLC and ES-MS. Deprotected
aminooxy peptide A0: analytical RP-HPLC: tR¼5.9 min
(5–60% B in 15 min); ESI-MS (positive mode) calcd for
C24H42N7O12: 620.29; found: m/z 620.11 [M+H]+.
3.3.2. H-Gly-Leu-Ser(tBu)-Asp(tBu)-Val-Gly-OH (A0).
The linear protected peptide A was synthesized using the
Sasrin resin (450 mg, loading: 0.68 mmol/g) following the
standard procedure. One part of the resin was used for
the solid phase functionalization with Aoa derivatives (pep-
tide A). With the second part of the resin, the peptide was
cleaved by successive treatments with a solution of 1%
TFA in CH2Cl2 until the resin became dark purple. The com-
bined washings were concentrated under reduced pressure
and white solid peptides were obtained by precipitation
from ether (135 mg, 0.2 mmol, 68%). The protected linear
peptide A0 was used for functionalization with Aoa deriva-
tives without further purification. Analytical RP-HPLC:
tR¼6.1 min (5–60% B in 15 min); ESI-MS (positive mode)
calcd for C30H55N6O10 659.39; found: m/z 659.11 [M+H]+.
3.4.2. Solid phase functionalization with Boc–Aoa–OSu 2
and Eei–Aoa–OSu 6. A solution of 1 equiv of Boc–Aoa–
OSu 2 or 2 equiv of Eei–Aoa–OSu 6 (relative to the resin
loading of peptide A, B, or C) and 1 equiv of DIPEA (pH
8) in dry DMF was added to the resin. After shaking for
45 min at room temperature, the solution was filtered and
the resin was washed with DMF (5ꢁ1 min) and CH2Cl2
(2ꢁ1 min). The completeness of the coupling reaction was
controlled by TNBS test. If necessary, this process is repeated
once. Each peptide was finally obtained upon acidic cleavage
and deprotection by treatment of the resin with a mixture of
TFA/TIS/H2O (95:2.5:2.5) for 2 h. After evaporation of
the cleavage solution, the peptides were precipitated with
diethyl ether and analyzed by ES-MS. Deprotected amino-
oxy peptide A0: see Section 3.4.1; deprotected aminooxy
peptide B0: analytical RP-HPLC: tR¼12.6 min (0–30%
3.3.3. H-Gly-Gly-c[Cys-Asp(tBu)-Gly-Asp(tBu)-Cys]-
NH2 (B0). The protected cyclic peptide B was synthesized
on Sieber Amide resin (1 g, loading: 0.72 mmol/g) follow-
ing the standard procedure. A solution of iodine (1.97 mg,
7.7 mmol) in DMF (10 mL) was added to the resin and
stirred for 4 h. The solution was filtered and the resin was
washed with DMF (10ꢁ1 min), CH2Cl2 (10ꢁ1 min), and
DMF until washing solution became colorless. One part of
the resin was used for the solid phase functionalization
with Aoa derivatives (peptide B). With the second part of
the resin, the peptide was cleaved by successive treatments
with a solution of 1% TFA in CH2Cl2 until the resin became
dark purple. The combined washings were concentrated un-
der reduced pressure and white solid peptides were obtained
by precipitation from ether (402 mg, 0.52 mmol, 71%). This
protected cyclic peptide B0 was used for functionalization
with Aoa derivatives after semi-preparative RP-HPLC puri-
fication (5–100% B in 30 min). Analytical RP-HPLC:
tR¼6.3 min (5–100% B in 15 min); ESI-MS (positive
mode) calcd for C30H50N9O12S2: 792.28; found: m/z
792.30 [M+H]+.
B
in 30 min); ES-MS (positive mode) calcd for
C24H37N10O14S2: 753.19; found: m/z 753.01 [M+H]+. De-
protected aminooxy peptide C0: analytical RP-HPLC: tR¼
7.5 min (5–40% B in 30 min); ESI-MS (positive mode) calcd
for C41H80N15O15: 1022.59; found: m/z 1022.07 [M+H]+.
3.4.3. Functionalization with Boc–Aoa–OSu 2 and Eei–
Aoa–OSu 6 in solution. The functionalization reaction
was realized using 1 equiv of Boc–Aoa–OSu 2 (relative to
quantity of peptide A0 or B0), 1 equiv of DIPEA (pH 8) in
dry DMF. The reaction was monitored by RP-HPLC. After
45 min, the solution was evaporated and the peptide precip-
itated in diethyl ether. The peptides were finally deprotected
by treatment with a solution of TFA/TIS/H2O (95:2.5:2.5)
for 2 h. After evaporation of the cleavage solution, the pep-
tides were precipitated with diethyl ether and analyzed by
RP-HPLC and ESI-MS (see Section 3.4.2).
3.3.4. BocSer(tBu)-Lys[BocSer(tBu)]-Lys{BocSer(tBu)-
Lys[BocSer(tBu)]}-Ala-Lys-Rink amide (C). Peptide C
protected with an Alloc on the C-terminal lysine side chain
was synthesized on Rink Amide MBHA resin (450 mg,
loading: 0.69 mmol/g). The Alloc protecting group was re-
moved by adding a solution of phenylsilane (3.9 mL,
31.5 mmol) and tetrakis trisphenyl palladium (91 mg,
0.08 mmol) in dry CH2Cl2 (10 mL) to the resin, and the so-
lution was mixed under argon for 20 min. The resin was
washed with CH2Cl2 (2ꢁ1 min), dioxane/water (9:1,
2ꢁ1 min), DMF (2ꢁ1 min) and CH2Cl2 (2ꢁ1 min). For
analysis an aliquot of the resin was cleaved with a solution
of TFA/TIS/H2O (95:2.5:2.5) for 2 h. ESI-MS (positive
Acknowledgements
This work was supported by the Centre National pour la Re-
ꢀ
cherche Scientifique (CNRS), the Universite Joseph Fourier

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V. Dulery et al. / Tetrahedron 63 (2007) 11952–11958
11958
19. Defrancq, E.; Hoang, A.; Vinet, F.; Dumy, P. Bioorg. Med.
Chem. Lett. 2003, 13, 2683.
ꢁ
(UJF), and COST D-34. We also acknowledge the Ministere
ꢀ
ꢀ
de la Recherche for ACI-2003 ‘Molecules et Cibles Thera-
20. Chen, X.; Lee, G. S.; Zettl, A.; Bertozzi, C. R. Angew. Chem.,
Int. Ed. 2004, 43, 6112.
peutiques’ and for Grant No. 15432-2004 to V.D.
21. Prescher, J. A.; Bertozzi, C. R. Cell 2006, 126, 851.
22. Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475.
23. Melnyk, O.; Fehrentz, J. A.; Martinez, J.; Gras-Masse, H.
Biopolymers 2000, 55, 165.
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number CCDC-644968. Copies of the data can be obtained
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