Efficient synthesis of an (aminooxy) acetylated-somatostatin derivative using (aminooxy)acetic acid as a 'carbonyl capture' reagent

Article abstract of DOI:10.1002/psc.1294

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones an

Full text of DOI:10.1002/psc.1294

Research Article
Received: 16 June 2010
Revised: 22 July 2010
Accepted: 5 August 2010
Published online in Wiley Online Library: 2 September 2010
(wileyonlinelibrary.com) DOI 10.1002/psc.1294
Efficient synthesis of an (aminooxy)
acetylated-somatostatin derivative using
(aminooxy)acetic acid as a ‘carbonyl capture’
reagent
a∗
a,b
c
a,b
´
¨
´
´
´
´
´
Gabor Mezo, Ildiko Szabo, Istvan Kertesz, Rozsa Hegedu¨s,
a,b
d
a
e
´
¨
´
Erika Orban, Ulrike Leurs, Szilvia Bosze, Gabor Halmos
and Marilena Manead,f∗
Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most
preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides
to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of
an (aminooxy)acetylated-somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a ‘carbonyl
capture’ reagent in the final cleavage step. This (aminooxy)acetylated compound was further used for the chemoselective
ligation (oxime bond formation) with daunorubicin and 4-fluorobenzaldehyde leading to the formation of conjugates with
c
potential applications in targeted cancer chemotherapy and positron emission tomography. Copyright ꢀ 2010 European
Peptide Society and John Wiley & Sons, Ltd.
Keywords: (aminooxy)aceticacid;somatostatin;chemicalligation;oximebondformation;peptideconjugates;carbonylcapturereagent;
drug targeting
Introduction
aminooxy group toward aldehydes and ketones leading to the
formation of unwanted side products [9]. (Aminooxy)acetylated
peptides can react with traces of acetone or formaldehyde (e.g.
from the softeners of the plastic tubes) not only during the
reaction steps (cleavage, ligation) but also under the HPLC
purification conditions and even during the storage of the
purified peptides. Bure´ et al. identified the main side products
that were formed during oxime ligation of peptides [10]. Using
Chemoselective ligation methods are commonly used for the
preparation of peptide conjugates, as well as for the condensa-
tion of unprotected peptide fragments. Oxime ligation is one of
the most preferred procedures, due to the high chemoselectiv-
ity between an aminooxy function and a carbonyl group [1,2].
Formation of chemoselective oxime bonds has been successfully
applied for the synthesis of various peptide conjugates [3,4], gly-
copeptides [5], conjugates of DNA with different biomolecules [6],
oligonucleotide–peptide conjugates [7,8], and proteins from pep-
tide fragments [9,10]. Moreover, oxime ligation has recently been
investigated for the preparation of 18F-labeled peptide conju-
gates [11,12] and drug-peptide conjugates [13–15]. Furthermore,
(aminooxy)acetic acid was incorporated into heterobifunctional
linkers for facile synthesis of bioconjugates [16,17].
∗
´
¨
Correspondence to: Gabor Mezo, Research Group of Peptide Chemistry,
¨
¨
´
´
HungarianAcademyofSciences, EotvosL. University, 1117Budapest, Pazmany
P. stny. 1/A, Hungary. E-mail: gmezo@elte.hu
Marilena Manea, University of Konstanz, Zukunftskolleg and Depart-
ment of Chemistry, Laboratory of Analytical Chemistry and Biopolymer
¨
Structure Analysis, Universitatsstrasse 10, 78457 Konstanz, Germany. E-
During the past decade, several side reactions have been
reported to occur during synthesis, purification, and work-up
procedures of (aminooxy)acetylated peptides [9,10,18]. One of
them is the N-overacylation of the NH–O function, either during
the incorporation of Boc-(aminooxy)acetic acid derivative (Boc-
Aoa) or through the peptide chain elongation. Carbodiimide-
mediated one-pot acylation without base [18] or the application
of Boc-Aoa-OSu active ester as an acylating agent [19], as well as
the use of a high excess (8 equiv.) of Boc-Aoa-OH and coupling
agents for a short acylation time (10 min) [20] might prevent the
overacylation.
mail: marilena.manea@uni-konstanz.de
¨ ¨
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos
a
´
Lorand University, 1117 Budapest, Hungary
¨
¨
´
b
c
Institute of Chemistry, Eotvos Lorand University, 1117 Budapest, Hungary
DepartmentofNuclearMedicine,MedicalandHealthScienceCenter,University
of Debrecen, 4032 Debrecen, Hungary
d
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis,
Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany
e
f
Department of Biopharmacy, University of Debrecen, 4032 Debrecen, Hungary
Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany
Another side reaction which has been less investigated and
reported so far derives from the high reactivity of the free
c
J. Pept. Sci. 2011; 17: 39–46
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

¨
MEZO ET AL.
liquid chromatography-tandem mass spectrometry (LC-MS/MS),
ions corresponding to products having −15, +12, +26, +40, and
+96 Da difference than the calculated mass of the conjugate
were detected. These ions corresponded to the NH2 loss (H2N-
O-R to HO-R), to conjugates with formaldehyde, acetaldehyde,
acetone, and to trifluoroacetylated compounds, respectively. To
overcome this problem, the use of Boc-Aoa-peptide precursors
for chemical ligation in combination with in situ Boc cleavage
was suggested [12]. The efficiency of the in situ Boc cleavage
during the labeling reactions was demonstrated in the case
of the synthesis of [18F]FBOA-peptides [11] and of a template-
assembled cyclic RGD peptide conjugate [21]. However, it
was mentioned that a fivefold higher peptide concentration
was needed for the efficient labeling in comparison with the
unprotected (aminooxy)acylated peptides. Careful handling of
unprotected Aoa-containing peptides, such as working under
argon or nitrogen stream in an ‘acetone-free’ laboratory, using
freshly distilled diethylether for precipitation [22], shortening
the work-up procedure, using methanol or freshly prepared
acetonitrile solutions for HPLC purification, and storage of the
compounds at temperatures below −20 ◦C in glass tubes might
help avoid the unwanted side reactions. However, in some cases
all of these precautions are not enough to prevent the formation
of a large amount of side products.
(HOBt), 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), piperidine,
trifluoroacetic acid (TFA)], pentachlorophenol (PcpOH) as well
as daunorubicin (Dau), aminooxyacetic acid (Aoa), and Boc-
aminooxyacetic acid (Boc-Aoa-OH) were Fluka products (Buchs,
Switzerland). Solvents used both for synthesis and HPLC purifi-
cation [dichloromethane (DCM) N,N-dimethylformamide (DMF),
ethyl acetate, and methanol (MeOH)] were purchased from Molar
Chemicals (Budapest, Hungary). HPLC grade acetonitrile (MeCN),
2,5-dihydroxybenzoicacid(DHB), and4-fluorobenzaldehydewere
from Sigma-Aldrich Kft (Budapest, Hungary).
Synthesis of Boc-Aoa-OPcp (1)
Boc-Aoa-OH and PcpOH (10 mmol each) were dissolved in 50 ml
ethyl acetate and then an equivalent amount of DCC in 50 ml ethyl
acetate was added to the solution (under cooling). The reaction
mixture was stirred overnight at RT. The precipitated DCU was
filtered out prior to the evaporation of the solvent. The remaining
compound was recrystallized from methanol. The yield of the
pure product was 72%. Melting point: 165–166 ◦C. Elemental
analysis: C 35.28 (calc. 35.50); H 2.78 (2.72); N 3.03 (3.18); Cl 40.49
(40.39).
In the present study, we provide evidence that free
(aminooxy)acetic acid could serve as an efficient ‘carbonyl cap-
ture’ reagent if it is added to the cleavage mixtures used for
the cleavage of (aminooxy)acetylated peptides from the resin.
We employed this ‘carbonyl capture’ reagent for the synthesis of
an (aminooxy)acetylated-somatostatin derivative which was fur-
ther used for the preparation of daunorubicin–somatostatin and
FBOA–somatostatin bioconjugates.
Synthesis of linear somatostatin derivative (H-D-Phe-Cys-Tyr-
D-Trp-Lys(ivDde)-Val-Cys-Thr-NH2, 2)
The linear somatostatin analog was synthesized manually on
a Rink-Amide MBHA resin (0.65 mmol/g coupling capacity)
by Fmoc/tBu strategy. The following Fmoc-protected amino
acid derivatives were used: Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-
OH, Fmoc-Val-OH, Fmoc-Lys(ivDde)-OH, Fmoc-D-Trp-OH, Fmoc-
Tyr(tBu)-OH, and Fmoc-D-Phe-OH. The synthetic protocol was as
follows:(i) DMFwashing(4×1 min);(ii) Fmocdeprotectionwith2%
DBU, 2% piperidine in DMF (4 times: 2 + 2 + 5 + 10 min); (iii) DMF
washing (8 × 1 min); (iv) coupling of 3 equiv. of Fmoc-amino
acid/DIC/HOBt in DMF (60 min); (v) DMF washing (4 × 1 min);
and (vi) ninhydrine assay. The peptide was cleaved from the
resin using a mixture of TFA-phenol-EDT-thioanisole-water (10 ml,
0.75 g, 0.25 ml, 0.5 ml, 0.25 ml) at RT for 1.5 h, then precipitated
with cold diethyl ether, washed three times with diethyl ether,
and solubilized in 100% acetic acid prior to freeze drying. By
this cleavage procedure, all side-chain protecting groups were
removed except for the ivDde from the side chain of Lys. The crude
peptide was characterized by analytical RP-HPLC (Rt: 36.3 min) and
ESI-MS (MWcalc/ exp: 1253.60/1254.05). The compound was used in
the next synthetic step without purification.
Recently, we have demonstrated the in vitro and in vivo an-
titumor activity of a daunorubicin – GnRH-III derivative biocon-
jugate (GnRH: gonadotropin-releasing hormone) containing an
oxime linkage between the components [13]. The formation
of a side product (M + 40 Da) was observed after the cleav-
age of the GnRH-III derivative (Glp-His-Trp-Ser-His-Asp-Trp-Lys(H-
Aoa-Gly-Phe-Leu-Gly)-Pro-Gly-NH2) from the resin. However, the
(aminooxy)acetylated peptide could be purified and further used
for the attachment of daunorubicin via oxime ligation. In contrast,
the synthesis of H-Aoa-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-
NH2, an N-terminal Aoa-elongated version of RC-121 somatostatin
analog developed in Schally’s laboratory [23] was unsuccessful.
Several synthetic procedures were employed; however, none of
them led to the formation of the desired cyclic peptide. In spite
of the careful work, oximes of the somatostatin derivative with
acetone, acetaldehyde, and formaldehyde were almost quantita-
tively formed. Finally, this highly sensitive (aminooxy)acetylated
peptide could efficiently be prepared in the presence of free Aoa
as a ‘carbonyl capture’ reagent which was used during the removal
of the N-terminal Boc protecting group.
Coupling of Boc-Aoa-OPcp to the semiprotected linear
somatostatin derivative (Boc-Aoa-D-Phe-Cys-Tyr-D-Trp-
Lys(ivDde)-Val-Cys-Thr-NH2, 3)
Boc-Aoa-OPcp (1.5 equiv.) and H-D-Phe-Cys-Tyr-D-Trp-Lys(ivDde)-
Val-Cys-Thr-NH2 (1 equiv.) were dissolved in DMF and then 1.5
equiv. DIEA was added to the reaction mixture. The coupling
reactionwascarriedoutatRTfor1 day. Afterremoving thesolvent,
the peptide was dissolved in a mixture of eluent A (0.1% TFA in
water) and B (0.1% TFA in acetonitrile–water (80 : 20, v/v) and
purified by RP-HPLC. The application of Boc-Aoa-OPcp prevented
the overacylation of the compound completely and the yield
was 75%. The purified compound was characterized by analytical
RP-HPLC (Rt: 44.5 min) and ESI-MS (MWcalc/ exp: 1427.7/1427.4).
Materials and Methods
Chemicals
All amino acid derivatives and Rink-Amide MBHA resin were pur-
chasedfromIRISBiotechGmbH(Marktredwitz,Germany)orReanal
(Budapest, Hungary). Scavengers, coupling agents, and cleavage
reagents [N,Nꢁ-diisopropylcarbodiimide (DIC), 1,2-ethanedithiol
(EDT), N-diisopropyl-ethylamine (DIEA), 1-hydroxybenzotriazole
c
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J. Pept. Sci. 2011; 17: 39–46

EFFICIENT SYNTHESIS OF AN AOA-SOMATOSTATIN DERIVATIVE
Figure 1. Structures of daunorubicin–somatostatin (A) and FBOA–somatostatin conjugates (B).
Figure 2. HPLC chromatogram (A) and ESI-ion trap mass spectrum of crude H-D-Phe-Cys-Tyr-D-Trp-Lys(ivDde)-Val-Cys-Thr-NH2 (B).
Simultaneous cleavage of ivDde protecting group
and disulfide bond formation (Boc-Aoa-D-Phe-
c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, 4)
Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 with 95% TFA – 5% H2O in the
presence of 10 equiv. of free (aminooxy)acetic acid at RT for
30 min. The mixture of cyclic peptide and Aoa was precipitated
with diethylether, then the precipitate was redissolved in 100%
acetic acid and lyophilized. The Boc-cleaved compound was
characterized by analytical RP-HPLC (Rt: 26.4 min) and ESI-MS
(MWcalc/ exp: 1118.6/1118.8). The Aoa-derivatized somatostatin
analogcouldbestoredforseveralmonthsat4 ◦Cinthepresenceof
the‘carbonylcapture’reagentwithoutdetectinganymodification.
Priortotheoximeligation,theexcessofAoawasseparatedbyHPLC
or by using a Sep-Pak Plus C18 cartridge (Waters, Milford, MA).
From the Boc-protected linear peptide, the ivDde group was
cleaved with 2% hydrazine in DMF. Under these cleavage
conditions (peptide concentration of 1 mg/ml, RT, 60 min), not
only was the protecting group removed but the disulfide bridge
betweenthethiolgroupsofcysteineswasalsoformed. Thesolvent
was evaporated in vacuo and the crude peptide was purified by
semipreparative RP-HPLC (47% yield). The purified compound
was characterized by analytical RP-HPLC (Rt: 33.1 min) and ESI-MS
(MWcalc/ exp: 1218.6/1218.5). The cyclic peptide containing ivDde
protecting group(Rt:43.7 min)andtheunprotectedlinearpeptide
(Rt: 30.9 min) were identified as main side products.
Oxime ligation of daunorubicin (Dau) to Aoa-somatostatin
derivative (Dau=Aoa-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-
Thr-NH2, 6)
Boc removal in the presence of (aminooxy)acetic acid
as a ‘carbonyl capture’ reagent (H-Aoa-D-Phe-c[Cys-
Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, 5)
The mixture of H-Aoa-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-
NH2 and (aminooxy)acetic acid was dissolved in eluent A
and separated by RP-HPLC. The solvent of the collected
(aminooxy)acetylated peptide fraction was evaporated in a
weighed flask. The product was dissolved in 0.2 M NaOAc
In order to prevent the side reactions by oxime formation, the
Boc protecting group was removed from Boc-Aoa-D-Phe-c[Cys-
c
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¨
MEZO ET AL.
Scheme 1. Outline of the synthesis of daunorubicin–and FBOA–somatostatin conjugates.
(pH 5.0) and 50% excess of Dau was added to the solution
(the oxime ligation was carried out for 3 h). The oxime bond
containing Dau–peptide conjugate was formed almost quantita-
tively. The excess of Dau was separated from the conjugate by
RP-HPLC. The oxime bond-linked Dau–somatostatin conjugate
was characterized by analytical HPLC (Rt: 31.5 min) and ESI-MS
(MWcalc/xp: 1628.0/1628.1).
andeluentB[0.1%TFAinacetonitrile–water(80 : 20,v/v)]wasused
at a flow rate of 1 ml/min. Peaks were detected at λ = 220 nm. The
crude products were purified on a semipreparative Phenomenex
Jupiter C18 column (250 mm × 10 mm) with 10 µm silica (300 Å
pore size). For purification, methanol–water (90 : 10, v/v) solvent
mixture was used at a flow rate of 4 ml/min. However, in the case of
Boc-Aoa-somatostatin derivatives, 0.1% TFA in acetonitrile–water
(80 : 20, v/v)) was used as eluent B.
For the purification of FBOA–somatostatin derivative, Waters
XterraMSC18(50 mm×10 mm), 2.5 µmcolumn(Milford, MA)was
used. Linear gradient elution (0 min 15% B; 2 min 15% B; 15 min
60% B) with eluent A [0.1% TFA in acetonitrile-water (5 : 95, v/v)]
and eluent B [0.1% TFA in acetonitrile–water (95 : 5, v/v)] was used
at a flow rate of 2 ml/min. The purity of the FBOA–somatostatin
conjugate was determined by analytical HPLC using Supelco
Discovery Bio Wide Pore C18 (150 × 2.1 mm), 5 µm (Supelco,
Sigma-Aldrich, Bellefonte, PA). The same eluents were used with
the following gradient elution: 0 min 15% B, 2 min 15% B, 15 min
60% B. The flow rate was 0.35 ml/min.
Oxime ligation of 4-fluorobenzaldehyde to Aoa-somatostatin
derivative (FBOA-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-
NH2, 7)
A mixture of 10 mg somatostatin analog and Aoa was dissolved
in 0.8 ml of 0.1% TFA (aqueous solution). The peptide was
immobilized on a Sep-Pak Plus C18 cartridge and the cartridge
was flushed with 8 ml of 0.1% TFA in water. The peptide was eluted
with 75% MeOH/0.1% TFA. The first fraction (0.6 ml) was discarded
and the fractions containing the peptide analog were combined
(1 ml). A solution of 0.62 mg 4-fluorobenzaldehyde in 25 µl MeOH
was added (5 µmol) to this mixture and stirred for 60 min at 60 ◦C.
The solution was cooled down to room temperature and then
diluted with water to a final volume of 5 ml. The labeled peptide
analog was purified by semipreparative RP-HPLC. The purity was
higherthan97%andtheamountofpurifiedpeptideconjugatethat
resulted was 3.2 mg. The yield could not be calculated due to the
unknown composition of the starting mixture of the somatostatin
analog and Aoa. The conjugate was characterized by analytical
HPLC (Rt: 12.9 min) and MALDI-MS (MWcalc/ exp: 1224.5/1224.6).
Mass spectrometry (MS)
MassspectrometricanalyseswerecarriedoutonaBrukerDaltonics
Esquire 3000+ ion trap mass spectrometer (Bremen, Germany).
Spectra were acquired in the 50–2000 m/z range. Samples
were dissolved in acetonitrile–water = 1 : 1 (v/v) solvent mixture
containing 0.1% acetic acid.
The mass spectrometric analysis of FBOA-somatostatin con-
jugate was performed on a Bruker Biflex-III MALDI-TOF mass
spectrometer (Bremen, Germany) using 2,5-dihydroxy benzoic
acid (DHB) as a matrix.
High performance liquid chromatography (HPLC)
Analytical RP-HPLC was performed on a Knauer (H. Knauer, Bad
Homburg, Germany) system using a Phenomenex Jupiter C18
column (250 mm × 4.6 mm) with 5 µm silica (300 Å pore size)
(Torrance, CA) as a stationary phase. Linear gradient elution (0 min
0% B; 5 min 0% B; 50 min 90% B) with eluent A (0.1% TFA in water)
Cells
MCF-7 human breast adenocarcinoma cell line was maintained in
DMEM (Sigma Ltd. St. Louis, MO) containing 10% FCS (fetal calf
c
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EFFICIENT SYNTHESIS OF AN AOA-SOMATOSTATIN DERIVATIVE
Figure 3. HPLC chromatogram of crude Boc-Aoa-D-Phe-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 (A); HPLC chromatogram of the compound after Boc
cleavage in the absence of free Aoa (B); HPLC chromatogram of the compound after Boc cleavage in the presence of 10 equiv. free Aoa (C); ESI-ion trap
mass spectrum of H-Aoa-D-Phe-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 (D).
serum, Sigma Ltd.), L-glutamine (2 mM), gentamicin (160 µg/ml)
at 37 ◦C in a humidified 5% CO2 atmosphere. HT-29 human
colon adenocarcinoma cell line was cultured in RPMI-1640 (Sigma
Ltd.) containing 10% FCS, L-glutamine (2 mM), and gentamicin
(160 µg/ml) at 37 ◦C in a humidified 5% CO2 atmosphere. NCI-
H358 human non-small cell lung cancer cell line was maintained
in RPMI-1640 containing 10% FCS and L-glutamine (2 mM) at 37 ◦C
in a humidified 5% CO2 atmosphere.
37 ◦C, cells were washed twice with serum-free medium and
cultured in serum-containing medium for 72 h. The amount of the
living cells was determined by MTT assay using 0.367 mg/ml final
concentration of MTT in each well. After 3.5 h incubation with MTT,
cells were centrifugated at 2000 rpm for 5 min, supernatant was
removedandcrystalsweredissolvedinDMSO.Theabsorbancewas
measured using an ELISA-reader (Labsystems MS Reader, Finland)
at λ = 540 nm and at λ = 620 nm as reference wavelength.
Statistical data analysis was performed by Student’s t-test of
Origin7.5 at the 95% confidence level.
In vitro cytostatic effect of compound 6 determined by MTT
assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay)
Results and Discussion
Tostudythein vitrocytostaticeffectofcompound6, MCF-7human
breast, HT-29 human colon and NCI-H358 human non-small cell
lung cancer cells were plated into 96-well tissue-culture plate in
100 µl culture medium with the initial cell number of 5 × 103
cells/well. The compounds used in the 5 × 10−4 − 2 × 102 µM
concentration range were dissolved in fresh culture medium and
added to the cells. After treatment and incubation (6 h in case
of NCI-H358 cells or 16 h in case of MCF-7 and HT-29 cells) at
Hormonal peptides such as somatostatin, GnRH, and their deriva-
tives are applied for targeting drugs and radionuclides to cancer
cells [24,25]. One approach to attach drugs or isotope-containing
organic compounds to peptides is oxime ligation between an
(aminooxy)acetylated peptide and an oxo group. However, the
sensitivity of the aminooxy moiety to other oxo group contain-
ing compounds like acetone, formaldehyde and so on makes
c
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¨
MEZO ET AL.
Figure 4. ESI-ion trap mass spectra of the HPLC fractions obtained after the separation of the reaction mixture derived from Boc deprotection in the
absence of Aoa.
the synthesis and storage of Aoa-peptides difficult. The major
goal of our work was to prepare oxime bond-linked daunoru-
bicin–somatostatin and FBOA–somatostatin conjugates for tar-
geted cancer chemotherapy and positron emission tomography
(PET) (Figure 1) using daunorubicin or 4-fluorobenzaldehyde and
H-Aoa-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2,anN-terminal
Aoa-elongatedversionofRC-121somatostatinanalog.Forthispur-
pose, the peptide was initially purchased from a supplier; however,
the received compound had a molecular mass with 40 Da higher
than the calculated one, modification that might derive from
the oxime bond formation between acetone and Aoa-peptide
(according to the literature data [10]). Therefore, we decided to
synthesize the peptide by ourselves.
The somatostatin analog was built up by SPPS on a Rink-Amide
MBHA resin followed by the cleavage with TFA in the presence
of appropriate scavengers and reducing agents (water, phenol,
thioanisole, EDT). Surprisingly, the crude product obtained after
lyophilization (liquid nitrogen was used for freezing the sample)
was characterized by one main HPLC peak corresponding to the
acetone-modified peptide (M + 40 Da mass). In the next trial, the
somatostatin derivative without Aoa was prepared using ivDde
protecting group on the side chain of Lys (Figure 2). The peptide
was cyclized by air oxidation, followed by the coupling of Boc-Aoa-
OH to the cyclic peptide using BOP reagent. The reaction resulted
in the Aoa-containing cyclic somatostatin derivative with a very
low yield. Finally, Boc-Aoa-OPcp prepared in our laboratory was
connected to the linear semiprotected peptide chain (Boc-Aoa-
D-Phe-Cys-Tyr-D-Trp-Lys(ivDde)-Val-Cys-Thr-NH2)(Scheme1). The
cleavage of ivDde protecting group with 2% hydrazine in DMF
resulted not only in the removal of the protecting group but
also in the cyclization of the somatostatin derivative (Figure 3(A);
compound with a retention time of 33.1 min). The Boc group from
the cyclic compound was cleaved with TFA in the presence of 5%
water using argon flushed glass wears followed by precipitation
with diethylether. The RP-HPLC profile of the crude product
after lyophilization showed four peaks that corresponded to the
expected cyclic peptide (Rt: 26.4 min) and oxime derivatives with
+12 Da, +26 Da, and +40 Da molecular masses (Figures 3(B) and
4). The crude peptide was purified by RP-HPLC using methanol
as a major component of eluent B. However, the separated
fraction corresponding to the expected product (fraction 1, Rt:
26.4 min) turned to the acetone-modified one very fast and no
other compound could be detected by MS after lyophilization
(Figure 4). The chemical modification of the expected product
was also confirmed by the change of the retention time (Rt:
29.8 min), which was identical with that of the acetone-modified
compound (fraction 4, Figure 4). To overcome the unwanted
oxime bond formation under the cleavage conditions and during
the work-up procedure, 10 equiv. of free (aminooxy)acetic acid
were added to the cleavage mixture used to remove the Boc
group of the cyclic somatostatin derivative (Scheme 1). The HPLC
profile of the resulted crude product showed only one main
peak (Figure 3(C)) which corresponded to the expected product
as determined by mass spectrometry (Figure 3(D)). The work-up
procedure (precipitation with dry diethylether, solubilization in
acetic acid, and lyophilization) did not result in the removal of
c
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EFFICIENT SYNTHESIS OF AN AOA-SOMATOSTATIN DERIVATIVE
Figure 5. HPLC chromatogram of the reaction mixture of oxime ligation after 3 h and ESI-ion trap mass spectra of the corresponding HPLC fractions:
daunorubicin ([M+H]+ = 528.2 Da) and Dau–somatostatin derivative conjugate ([M+H]+ = 1628.7 Da).
the Aoa excess from the peptide; therefore, the compounds could
Table 1. Cytostatic effect (IC50 values) of the oxime bond-linked
daunorubicin–somatostatin conjugate 6 on various cancer cell lines
be kept for months at +4 ◦C without detecting any significant
modification. However, the excess of Aoa should be removed
prior to the chemical ligation in order to avoid the reaction of
Dau-somatostatin
Cell line
conjugate (6)
Daunorubicin
carbonyl-containing compounds with free Aoa. For this purpose,
the mixture containing Aoa-peptide and free Aoa was purified by
RP-HPLC using methanol as eluent B, followed by an immediate
evaporation of the solvent from the collected peptide fraction. The
resulting material was dissolved in 0.2 M NaOAc, pH = 5 and added
to daunorubicin. The ligation was completed in 3 h (Figure 5; it is
also important to note the fragmentation of the glycosidic bond
during the mass spectrometric analysis resulting in the loss of
daunosamine).
In another application, the peptide was dissolved in 0.1%
TFA (aqueous solution) and immobilized on a Sep-Pak Plus
C18 cartridge. The somatostatin derivative was eluted with 75%
MeOH solution containing 0.1% TFA and the peptide containing
fraction was mixed directly with the MeOH solution of 4-
fluorobenzaldehyde. The ligation continued at 60 ◦C for 60 min
resulting in the expected FBOA–somatostatin derivative.
In vitro cytostatic activity of oxime bond-linked daunoru-
bicin–somatostatin conjugate was determined on MCF-7 human
breast, HT-29 human colon, and NCI-H358 human lung cancer
cell lines by MTT assay. The cytostatic effect of the daunoru-
bicin–somatostatinconjugate(presentedasIC50 valuesinTable 1)
was one order of magnitude lower on all investigated cell types
MCF-7
4.3 µM
20.6 µM
4.5 µM
0.2 µM
2.0 µM
0.3 µM
HT-29
NCI-H358
(1–20 µM) than that of free daunorubicin (0.1–2 µM). The data are
similar to those obtained in the case of oxime bond containing
Dau-GnRH-III conjugates [13,26] that have been already shown to
have in vivo antitumor effect without significant toxic side effects.
The results reported here are promising and provide a basis for the
further investigation of the in vivo antitumor activity of this novel
Dau–somatostatin conjugate.
Conclusions
Oxime ligation is a chemoselective procedure most commonly
used for the preparation of peptide conjugates. However, the
sensitivity of (aminooxy)acetylated peptides to ketones and
aldehydes makes their synthesis and storage difficult. Therefore,
in order to prevent the unwanted modifications of highly sensitive
c
J. Pept. Sci. 2011; 17: 39–46
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/psc

¨
MEZO ET AL.
Aoa-peptide derivatives, we investigated the application of free
(aminooxy)acetic acid as a ‘carbonyl capture’ reagent to be used
in the final cleavage step and during the work-up procedure
and storage. In the presence of free (aminooxy)acetic acid, an
Aoa-somatostatin derivative could be efficiently prepared and
used for further chemoselective ligation with daunorubicin and
4-fluorobenzaldehyde leading to the formation of conjugates with
potential applications in targeted cancer chemotherapy and PET.
The oxime bond-linked daunorubicin–somatostatin conjugate
has in vitro cytostatic activity on various cancer cell lines, such as
MCF-7 human breast, HT-29 human colon, and NCI-H358 human
lung cancer cell lines.
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methodology for high-yield routine radiohalogenation of peptides:
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12 Schottelius M, Berger S, Poethko T, Schwaiger M, Westar H-J.
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Acknowledgements
This study was supported by grants from the Hungarian National
Science Fund (OTKA NK 77485, F 67884, K 81596), the University of
Konstanz (Zukunftskolleg, Project 879/08), and GVOP-3.2.1.-2004-
04-0005/3.
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A new method for conjugation
of carbohydrates to proteins using an aminooxy-thiol
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