Synthesis of peptide di-aldehyde precursor for stepwise chemoselective ligations via oxime bonds

Article abstract of DOI:10.1016/S0040-4039(00)01907-9

To synthezise a triple-function branched peptide in a modular way, we present a new strategy based on orthogonal generation of two aldehyde functions from an acetal and a 2-amino alcohol. Successive unmaskings of aldehyde functions of the stem peptide affords stepwise chemoselective ligations of two (aminooxy)acetyl peptides via oxime bonds.

Full text of DOI:10.1016/S0040-4039(00)01907-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 235–238
Synthesis of peptide di-aldehyde precursor for stepwise
chemoselective ligations via oxime bonds
Dominique Lelie`vre,* Corinne Bure´, Fabrice Laot and Agne`s Delmas*
Centre de Biophysique Mole´culaire, CNRS,† rue Charles Sadron, 45071 Orle´ans Cedex 2, France
Received 19 September 2000; accepted 25 October 2000
Abstract—To synthezise a triple-function branched peptide in a modular way, we present a new strategy based on orthogonal
generation of two aldehyde functions from an acetal and a 2-amino alcohol. Successive unmaskings of aldehyde functions of the
stem peptide affords stepwise chemoselective ligations of two (aminooxy)acetyl peptides via oxime bonds. © 2000 Elsevier Science
Ltd. All rights reserved.
Beside conventional protected segment condensation
which is limited by poor solubility of protected pep-
tides, chemoselective ligation has recently been deve-
loped for the synthesis of long, branched and cyclic
peptides.1 This methodology relies on the ligation of
unprotected peptides in aqueous buffer without any
activated step thanks to the chemoselective coupling
reaction of a C-electrophile on one peptide and a
Scheme 1. Modular synthesis of a tri-branched peptide through two successive chemoselective ligations.
Keywords: peptide aldehyde; chemoselective ligation; oxime; branched-peptide.
* Corresponding authors. Fax: 33 2 38 63 15 17; e-mail: lelievre@cnrs-orleans.fr; delmas@cnrs-orleans.fr
†
UPR 4301-CNRS affiliated to INSERM and the University of Orle´ans.
0040-4039/01/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01907-9

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D. Lelie`6re et al. / Tetrahedron Letters 42 (2001) 235–238
N-nucleophile on another peptide. Depending on the
organic reaction used, peptide-to-peptide ligations are
achieved through formation of amide, oxime, hydra-
zone, thioester, thioether or thiazolidine bonds.1c For
most of the surrogate bonds, the C-electrophile partner
is an aldehyde. It is classically obtained by periodate
oxidation of a 2-amino alcohol (Thr or Ser) which
could be incorporated at any position in a peptidic
sequence through acylation of a selectively deprotected
No-lysine by a serine.2 Alternatively, many studies have
been devoted to the solid phase synthesis of peptide
C-terminal aldehyde which could be obtained either
free3–6 or masked.7,8 Among the latter, we have recently
elaborated a strategy to synthesize a peptide with the
aldehyde protected as an acetal.8 The key step was the
cleavage of the ester bond between the peptide and the
commercially available PAM9 resine by aminolysis with
aminoacetaldehyde-dimethylacetal. In order to develop
modular methods to synthesize triple-function branched
peptides, we decided to explore the compatibility of our
strategy8 with the generation of a second aldehyde by
periodate oxidation.
tected peptides which were precipitated with diethyl
ether. Diethyl ether has to be devoid of any trace of
acetaldehyde as this quantitatively reacts with the
aminooxy group to form the N-ethyloxime peptide. The
(aminooxy)acetyl peptides B and C were purified to
homogeneity by HPLC with a 62 and 67% overall yield,
respectively, based on the resin substitution. They were
characterized by ESI-MS (calculated mass: 504.5 and
462.5, experimental mass: 504.4 and 462.4 for
(aminooxy)acetyl peptide B and (aminooxy)acetyl pep-
tide C, respectively).
First ligation: The aldehyde of the peptide A(Ser)-
CH(OCH3)2 (11.5 mmol, 1 equiv.) masked as an acetal
was readily unprotected to afford the peptide A(Ser)-
CHO with 95% TFA containing 5% H2O at 20°C for 6
min.12 After evaporation of TFA in vacuo and stabili-
sation of the pH at 4.6 with 0.1 M acetate buffer, the
(aminooxy)acetyl peptide B (17.25 mmol, 1.5 equiv.)
was added. The peptide aldehyde A(Ser)-CHO was
totally depleted after 60 min and there was no signifi-
cant formation of by-products. The conjugate A(Ser)-B
was HPLC-purified with a 82% yield (experimental
mass: 1251.9, calculated mass: 1252.3).
We present here the completion of two stepwise ortho-
gonal chemoselective ligations to an unprotected stem
peptide. The modular strategy depicted in Scheme 1 is
based on the successive generation of two aldehydes
from an acetal and a 2-amino alcohol (Ser) borne on
the stem peptide A. Stepwise chemoselective ligation of
the (aminooxy)acetyl peptide B and the (aminooxy)-
acetyl peptide C affords the tri-branched chimeric pep-
tide A(C)-B with two oxime bonds. To assess the
potential of this approach, we synthesized a conjugate
composed of the following model sequences:
Second ligation: NaIO4 oxidation of the No-terminal
serine of the conjugate A(Ser)-B provided
a
ketoaldehyde2 without affecting the first oxime bond.
The conjugate A(CHO)-B was isolated with a 52% yield
after HPLC purification without special caution. To
afford the second ligation, the conjugate A(CHO)-B
(3.57 mmol,
1
equiv.) was reacted with the
(aminooxy)acetyl peptide C (5.35 mmol, 1.5 equiv.) in
0.1 M sodium acetate, pH 4.6. The coupling reaction
was followed by analytical HPLC. The desired conju-
gate A(C)-B (calculated mass: 1664.7, experimental
mass: 1665.3) appeared rapidly as a double peak after 2
min. It took 24 h for the aldehyde partner (by default)
to be depleted. During the time course of the reaction,
a second double peak emerged which corresponds to
the by-product A(C)-C (calculated mass: 1622.7, experi-
mental mass: 1623.1). It increased up to 43%13 con-
comitantly with the decrease of the target conjugate
A(C)-B. This is consistent with a transoximation reac-
tion that likely occurs between the desired conjugate
A(C)-B and the (aminooxy)acetyl peptide C, which was
1.5-fold in excess, leading to the conjugate A(C)-C.
According to the kinetics, the target conjugate A(C)-B
was maximum after 6 h (84%13), the conjugate A(C)-C
being kept at 14%.13 The corresponding UV chro-
matogram is depicted in Fig. 1. Under these conditions,
the conjugate A(C)-B was purified with a 52% yield.
Starting from the peptide di-aldehyde precursor A(Ser)-
CH(OCH3)2, the target conjugate was obtained with a
17% overall yield.
-peptide A: H-Ala-Tyr-Asp-Ala-Lys(Ser)-Ala-NH-CH2-
CH(OCH3)2, so-called A(Ser)-CH(OCH3)2,
-peptide B: H2NO-CH2-CO-Asp-Ala-Arg-Ala-OH,
-peptide C: H2NO-CH2-CO-Trp-Lys-Gly-OH.
Peptide synthesis: The peptide A(Ser)-CH(OCH3)2 was
synthesized using Boc-Ala-PAM-resin and Fmoc elon-
gation. The lysine was introduced as Fmoc-Lys(Dde)-
OH and the N-terminal alanine as Boc-Ala-OH. After
complete elongation of the peptide, hydrazinolysis of
the Dde group was followed by introduction of Boc-
Ser(tBu)-OH (Bachem, Switzerland) on the No-lysine.
TFA treatment10a gave the unprotected peptidyl resin.
Aminolysis of the ester bond between the peptide and
the resin with aminoacetaldehyde-dimethylacetal8
(Aldrich) afforded the crude peptide A(Ser)-
CH(OCH3)2 in 92% yield according to UV spec-
troscopy. The peptide A(Ser)-CH(OCH3)2 was then
isolated in a pure form with a 78% yield following
HPLC purification11 and characterized by ESI-MS (cal-
culated mass: 811.8, experimental mass: 811.8).
A third conjugate with a molecular mass of 1706.9 was
attributed to the conjugate A(B)-B (calculated mass:
1706.7). It was stable during the time course of the
reaction suggesting that it was formed before being
mixed with the (aminooxy)acetyl peptide C. To verify
this point, the peptide A(CHO)-B alone was left stirring
in 0.1 M acetate buffer, pH 4.6. The presence of the
conjugate A(B)-B was characterized in the reaction
The (aminooxy)acetyl peptides B and C were assembled
using conventional Fmoc/tBu chemistry on a Wang
resin. The side chain protecting groups were Arg, Pbf;
Asp, OtBu; Lys, Boc; Trp, Boc. Aminooxyacetic acid
(Aoa) was introduced as Boc-Aoa-OH after being dis-
solved in DMSO, and the coupling was controlled by
the Kaiser’s test. TFA treatment10b afforded the unpro-

D. Lelie`6re et al. / Tetrahedron Letters 42 (2001) 235–238
237
Figure 1. Analytical RP-HPLC of the second ligation mixture after 6 h at rt, C18 Lichrospher column (1 ml/min, linear gradient
3–30% acetonitrile in water, 0.1% TFA over 43 min).
mixture by HPLC and ESI-MS. This by-product would
be the result of an intermolecular transoximation, with
the aldehyde of the conjugate A(CHO)-B reacting on
the oxime bond of another conjugate A(CHO)-B.
mental conditions, the methyloxime-forming ligation
proceeded at a faster rate than the ketooxime. This is
likely due to the a-substituent of aldehyde function,
methyl and carbonyl, respectively. It could modulate
the electrophilicity of the carbonyl. However, we can-
not rule out that steric hindrance engendered by the
longer peptide aldehyde A(CHO)-B as compared to the
shorter one A(Ser)-CHO diminished the rate of oxime
formation. Among all the ligation reactions realized in
our laboratory, peptides containing a methyloxime ex-
hibited a double peak on the HPLC traces, whereas
peptides containing a ketooxime produced a single
peak. This is consistent with the literature where syn
and anti isomers of the oxime bond have been reported
The formation of the by-products A(B)-B and A(C)-C
underlines that transoximation reactions could occur
when the methyloxime-containing peptide was in solu-
tion in the presence of either the aldehyde or the
aminooxy partner. These side reactions were main-
tained at a minimum level. Moreover, it is worth stress-
ing that oxime-containing conjugates studied in this
work are quite stable upon storage at −20°C in the
lyophilized state, even the conjugate A(CHO)-B.
for conjugates composed of
a
a
sugar and an
steroid and an
As expected, the (aminooxy)acetyl peptide C is less
stable.14 LC-MS/MS analysis of the purified
(aminooxy)acetyl peptide C revealed that peaks 1, 2, 3
and 4 correspond to alterations of the peptide C at the
level of the aminooxy group. Peak 1 (Fig. 1) corre-
sponds to the hydroxyacetyl peptide C, probably
formed by acidic hydrolysis of the NꢀO bond of the
(aminooxy)acetyl peptide due to the presence of TFA in
HPLC-collected fractions. Peaks 2, 3 and 4 correspond
to compounds resulting from the addition of formalde-
hyde, acetaldehyde and acetone, respectively, to the
aminooxy function of the peptide. These side products
likely appeared during the last steps of purification or
during storage. We precise here consequences of the
reactivity of aminooxy compounds, briefly mentioned
by others.14
(aminooxy)acetyl peptide,15 or
(aminooxy)alkylamine.16 To the best of our knowledge,
such isomers have never been mentioned about conju-
gate containing a ketooxime.
In summary, we have developed a new strategy to
efficiently achieve two stepwise chemoselective ligations
for modular synthesis of tri-branched chimeric peptide.
It proceeds under mild conditions in aqueous buffer
and provides ligated compounds in good and repro-
ducible yields. The successive generation of two alde-
hyde functions on a stem peptide offers a further level
of orthogonality to chemoselective formation of oxime
bonds beside those based on the NH2-protecting group
chemistry for selective deprotection of aminooxy
group.15 As an oxime bond is compatible with the
immune response,17 we are currently applying this
methodology for the preparation of a subunit vaccine
composed of a universal T-helper, a B-cell and a T-cy-
totoxic epitopes needed to induce a complete immune
response.
Differences have been noted in the behaviour of the
two oxime bonds handled in this study, i.e. methyl-
oxime as the result of the first ligation and ketooxime
as the result of the second ligation. Under our experi-

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D. Lelie`6re et al. / Tetrahedron Letters 42 (2001) 235–238
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