Bioactive unnatural somatostatin analogues through bioorthogonal iodo-and ethynyl-disulfide intercalators

Article abstract of DOI:10.1002/chem.201100287

Iodo-and ethynyl-containing bisalkylating bioconjugation agents 5 and 8 were achieved and allow the introduction of reactive unnatural substituents into proteins and peptides whilst the bioactive 3D structure is retained. Derivatives of the peptide hormon

Full text of DOI:10.1002/chem.201100287

FULL PAPER
DOI: 10.1002/chem.201100287
Bioactive Unnatural Somatostatin Analogues through Bioorthogonal Iodo-
and Ethynyl-Disulfide Intercalators
Anne Pfisterer,[b] Klaus Eisele,[a, b] Xi Chen,[a] Manfred Wagner,[b] Klaus Mꢀllen,[b] and
Tanja Weil*[a]
Abstract: Iodo- and ethynyl-containing
bisalkylating bioconjugation agents 5
and 8 were achieved and allow the in-
troduction of reactive unnatural sub-
stituents into proteins and peptides
whilst the bioactive 3D structure is re-
tained. Derivatives of the peptide hor-
mone somatostatin bearing a single
iodo or ethynyl group were prepared
through intercalation into the disulfide
bridge. For the first time, the exact re-
action mechanism of the intercalation
was elucidated by applying 2D NMR
experiments and it was shown that,
during the reaction, somatostatin dia-
stereomers were formed. Site-directed
modification of the ethynyl-modified
peptide with a coumarin chromophore
was achieved through a [1,3] dipolar
Huisgen cycloaddition reaction; this
suggests that such a derivative could
serve as an attractive platform to pre-
pare artificial somatostatin compound
libraries. The biological activity and
specificity of a representative modified
somatostatin derivative was demon-
strated and efficient receptor-mediated
cell uptake occurred in a dose-depen-
dent manner into receptor positive
cells only. The iodo and ethynyl bio-
conjugation reagents presented herein
could be applied for introducing such
substituents into alternative peptides
and proteins and, in principle, could fa-
cilitate the efficient design of a broad
variety of artificial protein and peptide
analogues with previously unknown
bioactivities.
Keywords: antitumor agents · bio-
organic chemistry · intercalations ·
Michael addition · peptides
Introduction
amino acids bearing bioorthogonal groups such as ethynyl
groups[10,11] have successfully been introduced. However, the
main challenges of these approaches include low product
yields or the loss of bioactivity after modification or muta-
genesis.
In this context, it has been shown that the intercalation of
tailored alkylation agents into native disulfide bonds pro-
vides an efficient way to selectively modify biomolecules.[12]
Up to now, mainly poly(ethylene oxide) chains have been
attached to antibodies or Fab fragments[13] in a site-directed
fashion. From the inspiration of these published bisalkylat-
ing reagents, two bisthiol-reactive labels bearing a single bi-
oorthogonal ethynyl or iodo group have been designed. Bio-
orthogonality is essential to target only one functionality, re-
gardless of the presence of other functional groups in the
amino acid side chains.
We have selected the cyclic tetradecapeptide somatosta-
tin-14, due to its important biological role, its relatively
small size, and the presence of only a single disulfide
bond.[14] Somatostatin is a hormonal neuropeptide that was
found to inhibit growth-hormone secretion from the pituita-
ry gland[15] and that also plays a decisive role in other phys-
iological functions, including the inhibition of gastric release,
insulin, glucagon, and various intestinal hormones.[16] In ad-
dition, several cancer cells, such as endocrine tumors like
pancreatic tumors, express a specific class of somatostatin
receptors in their cell membranes, which thus facilitates so-
matostatin uptake through receptor-mediated endocyto-
sis.[15,17] Therefore, radiolabeled somatostatin derivatives
Protein- or peptide-based drugs are emerging therapeutics
that combine high biological activities, biocompatibilities,
and specificities. However, their pharmacokinetic profile
often represents a major limitation that makes their preclini-
cal and clinical development challenging.[1] Therefore, a
wide range of posttranslational modifications have been ex-
plored in order to improve their proteolytic stability, mem-
brane and tissue penetration, and oral and bioavailability.[2,3]
Up to now, only a few chemistry approaches have been re-
ported that allow the introduction of functional groups at a
specific location on the protein surface or into the peptide
backbone in a reproducible fashion. Tailored reagents, as
well as enzymes, have been applied to specifically modify
the N terminus of proteins,[4–6] and cysteine point mutations
allowing site-directed modifications[7–9] or noncanonical
[a] Dr. K. Eisele, X. Chen, Prof. Dr. T. Weil
Institute of Organic Chemistry III
University of Ulm
Albert-Einstein-Allee 11, 89069 Ulm (Germany)
Fax : (+49)731-5022883
E-mail: Tanja.Weil@uni-ulm.de
[b] A. Pfisterer, Dr. K. Eisele, Dr. M. Wagner, Prof. Dr. K. Mꢀllen
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100287.
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have been used as attractive diagnostic tools for identifying
and imaging certain cancer cells.[18]
The biological activity of somatostatin originates from the
loop sequence Phe7-Trp8-Lys9-Thr10, which is crucial for
high-affinity
G-protein-coupled
membrane
receptor
uptake.[19] This loop is maintained by Cys3 and Cys14 forming
a disulfide bridge, which is essential to maintain biological
integrity.[20] Unfortunately, somatostatin has a very low
plasma stability of only a few minutes[21] due to disulfide
cleavage, which greatly limits its medicinal applications.
Therefore, a few synthetic somatostatin analogues with
longer plasma stability, such as octreotide, have been pre-
pared.[15,22–24] The preparation of bioactive somatostatin de-
rivatives bearing unnatural functionalities might reveal un-
usual in vivo bioactivities, as well as an altered pharmacoki-
netic profile, which could be attractive for the design of im-
proved somatostatin drug conjugates.
Scheme 1. Synthesis of iodo-functionalized monosulfone reagent 5. a) Pi-
peridine HCl, HCl, paraformaldehyde, EtOH, 1058C; b) piperidine, for-
malin, 4-methylbenzenethiol, EtOH, 1058C; c) oxone, EtOAc/CH3CN/
H2O; d) KOtBu, tetrahydrofuran (THF).
Herein, the design and synthesis of iodo- and ethynyl-con-
taining intercalation agents, the intercalation mechanism
into the disulfide bridge of somatostatin, and the bioactivity
of the synthesized derivatives will be discussed. Such novel
bioorthogonal conjugation reagents offer an attractive plat-
form for the introduction of desirable artificial substituents
into peptides and proteins and thus offer an efficient alter-
native for reproducible and site-directed protein modifica-
tions.
In addition, the triisopropylsilyl (TIPS) protected ethynyl-
bis-sulfide 6 was obtained in excellent yields from iodo-bis-
sulfide 3 by treatment with TIPS acetylene in the presence
of PdCl2ACHTUNTRGNEUNG(PPh3) and Cu2I2 (Scheme 2) As the deprotected
ethynyl-bis-sulfide 7 was highly sensitive to the presence of
oxygen, it was advisable to perform the oxidation of the sul-
fide groups prior to removal of the TIPS protecting group.
The highly basic system generated by the addition of TBAF
to the oxidized TIPS-protected ethynyl-bis-sulfide 6 dis-
solved in THF facilitated the deprotection of the ethynyl
group and, at the same time, the elimination of p-toluene
sulfinic acid to afford the ethynylmonosulfone 8 in one reac-
tion step only (Scheme 2).
The intercalating reagents iodomonosulfone 5 and ethy-
nylmonosulfone 8 both allow covalent modification of
native disulfide bonds through reductive liberation of the
cysteine thiol groups or the introduction of bioorthogonal
reactive groups suitable for bioconjugation reactions, even
in the presence of unprotected amino acid residues. These
tailored organic molecules provide an attractive platform
with novel bioorthogonal tags that could be further treated
with a broad range of reagents in a highly selective fashion.
Results and Discussion
Synthesis of bioorthogonal iodo- and ethynyl-functionalized
intercalation reagents: The introduction of iodo substituents
is attractive because high-yield palladium(0)-catalyzed cross-
coupling reactions, such as the Heck, Suzuki, or Sonogashira
reactions,[25] allow a broad range of further chemical modifi-
cations. Due to recent success in synthesizing ligands that
allow palladium-catalyzed reactions in water, we have de-
signed a synthetic pathway towards the iodo-containing
monosulfone protein or peptide intercalator 5, as outlined in
Scheme 1. Compound 5 was prepared from p-iodoacetophe-
none (1) in a four-step synthesis. First, preparation of the
Mannich salt 2 proceeded in the
presence of piperidine hydro-
chloride and paraformaldehyde
with acceptable yields. There-
after, 2 was treated with 4-meth-
ylbenzenethiol, piperidine, and
formalin to yield the iodo-bis-sul-
fide 3. Compound 3 was further
oxidized to the iodo-bis-sulfone 4
and, after elimination of p-tolu-
ene sulfinic acid with potassium
tert-butoxide, the iodo-function-
alized monosulfone 5 was isolat-
ed
in
acceptable
yields
Scheme 2. Synthesis of ethynyl-functionalized monosulfone reagent 8. a) Triisopropylsilylacetylene, copper(I)
iodide, PdCl2ACTHNUTRGNEUNG(PPh3)2, THF/Et3N; b) 1. oxone, dichloromethane, 2. tetrabutylammonium fluoride (TBAF), THF.
(Scheme 1).
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Iodo and Ethynyl Somatostatin Analogues
FULL PAPER
Bioorthogonal functionalized somatostatin derivatives: So-
matostatin represents an ideal model peptide for assessing
the potential of both bioorthogonal intercalation agents 5
and 8 because 1) only a single disulfide group is present for
chemical modification, 2) reaction control and product and
side-product characterization could be realized through
mass spectrometry and NMR spectroscopy due to the small
size of the peptide, and 3) preservation of the bioactive 3D
conformation could be accessed through suitable cell
models.
and 8 with retention of the 3D geometry represents a key
concern. The disulfide bridge of somatostatin acetate (9)
was first reduced by adding tris(2-carboxyethyl)phosphine
hydrochloride (TCEP HCl) to form reduced somatostatin
10, which was then conjugated to the iodomonosulfone re-
agent 5 to yield pure iodosomatostatin 11 after purification
by
filtration
and
reversed-phase
chromatography
(Scheme 3). The corresponding ethynyl derivative 12 was
obtained and purified by the same approach (see the Exper-
imental Section).
As described above, somatostatin bears a single disulfide
bridge that is essential for biological activity. Therefore,
modification of this disulfide bridge by intercalation of 5
Figure 1 reveals the analytical HPLC trace of native so-
matostatin 9, reduced somatostatin 10, and iodo-modified
somatostatin 11 with traces of 10. Interestingly, the retention
Scheme 3. Synthesis of bioorthogonally functionalized somatostatins 11 and 12.
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T. Weil et al.
identified according to mass spectrometry studies with dif-
ferent quantities of the alkylating reagents. The use of
1.5 equivalents of ethynylmonosulfone reagent (Figure 3)
yielded monosubstituted somatostatin derivative 12, whereas
traces of unmodified somatostatin 9 were also detected by
MALDI-TOF mass spectrometry with the application of
only 1.2 equivalents of the reagent. Higher quantities such
as 2 or 3 equivalents yielded the monosulfone along with
traces of the doubly substituted, linear peptide. Obviously, a
second intermolecular addition reaction occurs if the bio-
conjugation reagent is used in excess.
The mechanism of this reaction has been suggested by
Lawton and co-workers[26] as an in situ addition–elimination
procedure (Scheme 4). However, this reaction mechanism
has never been supported by experimental data. The first
addition of the nucleophilic thiol group to the Michael ac-
ceptor system of the reagent is followed by elimination of p-
toluene sulfinic acid to generate the second addition system
(Scheme 4, top). Covalent rebridging proceeds by attack of
the second thiol group. This mechanistic pathway for the
cross-linking of sulfhydryl groups was proposed by Mitra
and Lawton,[12] but the structural identity of the three-
carbon bridge formed due to intercalation has never been
proven.
Therefore, NMR studies have been undertaken in order
to elucidate this reaction mechanism. After purification of
iodosomatostatin 11 by reversed-phase HPLC, the conjugate
was freeze-dried and different deuterated solvents were in-
vestigated for the NMR experiments. The best resolution of
the signals was achieved in D2O with suppression of the
HDO signal. Additionally, proton exchange of the amide
and amino protons facilitated the analysis of the spectra and
Figure 1. Illustration of the intercalation reaction by reversed-phase
HPLC: Chromatograms for native somatostatin (9; solid line a), disul-
fide-reduced somatostatin 10 (dashed line b), and iodo-modified somatos-
tatin 11 with traces of 10 (dotted line c). The two cyclic peptides (lines a
and c) have similar retention times,
whereas the reduced linear peptide
(line b) elutes faster
times of 9 and 10 differ, where-
as 9 and 11 display nearly equal
retention times. This observa-
tion suggests that the cyclic
structure of somatostatin was
retained after modification,
which is crucial for keeping the
biological activity after biocon-
jugation.
The identities of the modified
peptides 11 and 12 were also
confirmed by MALDI-TOF and
ESI-TOF mass spectrometry.
As representative examples, the
spectra of native somatostatin
9, iodo-modified somatostatin
11, and ethynyl-modified soma-
tostatin 12 are shown in
Figure 2.
Figure 2. MALDI-TOF mass spectra: a) Native somatostatin (9) with its salt adducts, b) somatostatin deriva-
tive 11 after modification with the iodomonosulfone, and c) somatostatin derivative 12 after modification with
The optimal conditions for
somatostatin modification were the ethynylmonosulfone.
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Iodo and Ethynyl Somatostatin Analogues
FULL PAPER
Figure 4. The formation of diastereomers of iodo-modified somatostatin
1
11 was confirmed by H NMR spectrometry. a) 1H NMR spectrum of the
aromatic region of native somatostatin (9). b) 1H NMR spectrum of the
aromatic region of iodo-modified somatostatin 11. The relative intensities
of the signals of the aromatic reagent protons of the two diastereomers
of iodo-modified somatostatin 11 (1,1* and 2,2* in the structures, respec-
tively) were compared to the relative intensity of all of the aromatic sig-
nals.
the signals of the aromatic protons of native somatostatin to
be assigned, the elucidation of the spectrum of iodosomatos-
tatin 11 is much more complex due to the existence of two
somatostatin diastereomers with different magnetic behav-
iors. Obviously, during the second addition step, a new
chiral center is created due to keto–enol tautomerism of the
newly generated ketone (Scheme 4). This racemization leads
to the formation of diastereomers and no enantiomerically
Figure 3. MALDI-TOF mass spectra of reaction mixtures of somatostatin
with different amounts of ethynylmonosulfone 8. With 1.2 equivalents,
unmodified somatostatin (labeled a) is
still present, whereas traces of double-
labeled somatostatin (labeled c)
appear with
2 or 3 equivalents of
linker. With 1.5 equivalents of mono-
sulfone linker, neither native nor
double-modified somatostatin, but
only the cyclic modified peptide (la-
beled b), were detected.
the assignment of the signals.
The diastereomers are not mag-
netically equivalent, so the
cyclic identity of the modified
peptide was proven for the first
time by 1H NMR techniques.
In Figure 4, the 1H NMR
spectra of native somatostatin
(a) and iodo-modified somatos-
tatin (b) are compared. Where-
Scheme 4. Proposed addition–elimination mechanism for the intercalation of iodomonosulfone 5 into the re-
duced form of somatostatin to yield iodo-functionalized somatostatin 11. Racemization in the second addition
as 2D-NMR techniques allowed step leads to formation of diastereomers.
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T. Weil et al.
pure product is obtained, which might be of great relevance
because chiral centers are important in biological systems.
The signals of the iodophenyl protons (H1, H1*, H2, and
H2*; Figure 4) were assigned and their relative intensity was
1:1:1:1, whereas the ratio of the relative intensity of these
protons versus the relative intensity of all aromatic signals
was 1:6. This factor is consistent with the fact that conjuga-
tion provided a total of twenty-four aromatic protons and
with the assumption that two diastereomers are formed in a
1:1 ratio.
In addition, the signal for the aromatic methyl group and
the two signals corresponding to the nonequivalent hydro-
gen atoms of the double bond were no longer present in the
spectrum of 11. Moreover, the COSY cross-peaks at d=3.78
and 2.73 ppm and the NOESY cross-peaks at d=3.78 and
2.73 ppm confirm the connectivity between the CH signal
(H3,3*; new chiral center) and the CH2 signals (H4,4*;
Figure 5).
the presence of unprotected amino acids in the somatostatin
side chains. The [2+3] cycloaddition “click” reaction of an
ethynyl bond with an azide functionality has been widely in-
vestigated for biological applications because this reaction
can be carried out in aqueous solution at room temperature
if a CuI catalyst is added. 3-Azido-7-hydroxycoumarine was
applied, which represents a profluorophore that reveals high
fluorescence only after a successful “click” reaction and the
formation of the triazole moiety. Therefore, this chromo-
phore is ideally suited to verify the accessibility of bioor-
thogonal triple bonds of engineered biomacromolecules. By
the reaction of 3-azido-7-hydroxycoumarine with ethynylso-
matostatin 12, the “click” product was obtained and the
characteristic increase in fluorescence emission was ob-
served; this reaction demonstrates selective modification of
the ethynyl group (Figure 6). The successful modification
was furthermore visualized and verified by gel electrophore-
sis and MALDI-TOF mass spectrometry, as illustrated in
Figure 7. This reaction points out the value of intercalator 8
as a reagent for introducing unnatural residues that allow
further chemical modifications.
Modification of bioorthogonally functionalized somatostatin
through a click reaction: Subsequently, we investigated the
opportunity to selectively address the ethynyl group of 12 in
Figure 5. The cyclic geometry of 11 was proven by 1H NMR spectrometry. a) 1H-COSY spectrum of native somatostatin (9). b) 1H-COSY and c) 1H-
NOESY spectra of iodo-modified somatostatin 11 showing cross-peaks of the signals of the newly generated methylene protons (4, 4*) to the racemic
proton (3, 3*).
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Iodo and Ethynyl Somatostatin Analogues
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Figure 6. Absorbance (X360, c) and emission (a) spectra of coumar-
in-attached somatostatin 13.
Somatostatin–dye conjugates are particularly suitable to
prove whether the biologically active 3D conformation of
somatostatin was retained after modification. Even though
coumarin-modified somatostatin 13 is very attractive to
prove the successful cycloaddition reaction, it is also sensi-
tive to photobleaching and does not reveal sufficient photo-
stability for cell experiments. Therefore, the rhodamine dye
intercalator 15 (Scheme 5) with increased photostability was
prepared; this would allow investigation of the cell-uptake
characteristics of modified somatostatin. First, commercially
available lissamine rhodamine B was coupled to the active
ester 14, which was obtained by a procedure described pre-
viously.[27] Afterwards, native somatostatin was treated with
the LRB intercalator 15 to yield the highly fluorescent so-
matostatin conjugate 16.
Figure 7. a) Gel electrophoresis picture taken under UV irradiation, with
different concentrations of coumarin-attached somatostatin 13 (lanes 2–
5) as well as native and ethynyl-modified somatostatins (lanes 6 and 7).
b) MALDI-TOF mass spectrum for coumarin-attached somatostatin 13
with its salt adducts.
medium. After incubation for 24 h, the cells were washed
and the cell uptake was investigated with photometric and
confocal microscopy methods. In the case of A-549 cells,
fluorescence staining was mainly localized at the cell mem-
brane and no vesicles were present. By contrast, significantly
increased cell uptake was clearly observed in the CAPAN-2
cells. In addition, several small vesicles were detected inside
the cells, which indicates receptor-mediated cell uptake
(Figure 8). These findings indicate that the biological activi-
ty of somatostatin was retained regardless of the intercala-
tion and attachment of the large rhodamine chromophore.
Photometric investigation of cell uptake of the rhoda-
mine-modified somatostatin in CAPAN-2 cells revealed that
a dose-dependent increase in cell uptake could be detected
in receptor-positive CAPAN-2 cells with increasing amounts
of 16. However, only very low emission intensities were re-
corded after incubation of 16 with A-549 cells, which sug-
gests a low tendency for nonspecific cell uptake.
Cell uptake of modified somatostatin 16: It has been de-
scribed previously that somatostatin efficiently interacts
with somatostatin G-protein-coupled receptors only in its
cyclic 3D architecture. Therefore, receptor-mediated endo-
cytosis of the representative fluorescent somatostatin deriva-
tive 16 was assessed to prove that biologically active soma-
tostatin conjugates were achieved. CAPAN-2 cells, a human
pancreas carcinoma cell line, were used as receptor-positive
cells because it is known that they express somatostatin re-
ceptors that recognize somatostatin and induce its uptake.
As a negative control, the A-549 cell line, which does not
express somatostatin receptors, was applied. First, 16
(1 mgmLÀ1) was dissolved in water in the presence of 0.1%
trifluoroacetic acid (TFA) was added to the cells in culture
In order to visualize and explain these experimental re-
sults, 3D visualization of native somatostatin was achieved
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T. Weil et al.
has been obtained so far, so a
3D visualization of somatosta-
tin and iodosomatostatin 11 was
achieved by connecting the re-
spective amino acids according
to the amino acid sequence, as
described in the Supporting In-
formation. The respective 3D
structures are depicted in
Figure 9. After intercalation of
5 into 9, the distance between
the sulphur atoms increases
from about 2.1 ꢂ to 3.9 ꢂ,
which leads to widening and,
therefore, changes in the geom-
etry near to the intercalator.
However, the amino acid se-
quence Phe7-Trp8-Lys9-Thr10 in
the
b loop of somatostatin,
which is responsible for phar-
macological activity, is located
on the opposite side to the di-
sulfide bridge. In comparison
with the situation in native so-
matostatin, the distance and
Scheme 5. Synthesis of lissamine rhodamine B (LRB) bis-sulfone reagent 15. a) Lissamine rhodamine B, dime-
thylformamide/dichloromethane.
orientation of the four amino acid motif remained un-
changed, which is in accordance with the bioactivity data.
However, according to the 3D visualization of 11, changes
Figure 8. Confocal and fluorescence microscopy images of CAPAN-2 and
A-549 cells incubated with rhodamine-modified somatostatin 16 for 24 h.
a) The CAPAN-2 cell line that expresses somatostatin receptors showed
an increased uptake relative to that of b) the A-549 human lung carcino-
ma cell line that did not express somatostatin receptors. Cells showed
very weak background fluorescence. c) Clear round vesicle-like structures
could be detected at a higher magnification. d) Nuclear staining with the
commercially available dye DAPI showed a clear correlation between 16
and CAPAN-2 cells. White bar: 10 mm according to magnification.
Images (a–c) were recorded after laser excitation (confocal), whereas
image (d) was obtained after fluorescence excitation by a mercury lamp
(nonconfocal).
Figure 9. a) Photometric investigation of cell uptake of rhodamine-modi-
fied somatostatin 16 in CAPAN-2 cells. With increasing concentrations of
16, increased uptake was detected in receptor-positive CAPAN-2 cells.
As a control, the emission of 16 (10 mg) in A-549 cells was tested; very
low emission was detected. 3D visualizations of b) somatostatin (9) and
c) iodo-modified somatostatin 11.
by applying the MOE software package of the Chemical
Computing Group.[28] No crystal structure of somatostatin
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Iodo and Ethynyl Somatostatin Analogues
FULL PAPER
nyl)methyl]acetyl}benzoic acid N-hydroxysuccinimide (NHS) ester (14)[30]
were synthesized according to published procedures. The structures of
the molecules were investigated on a Bruker Avance III 500 spectrome-
ter or a Bruker Avance III 700 NMR spectrometer, both with a 5 mm z-
gradient BBI 1H/X probe. The proton NMR spectra were measured in
D2O at 298.3 K. The temperature was kept at 298.3 K and regulated by a
standard 1H-methanol NMR sample. The spectra were referenced with
the residual HDO signal at d(1H)=4.8 ppm. The 1H,1H 2D NOESY ex-
periment was recorded on the 500 MHz system, with a mixing time of
0.45 s and with presaturation of the water signal during the relaxation
delay and the mixing time. The 1H,1H 2D COSY spectra were acquired
on the 500 and 700 MHz spectrometers with presaturation on the residual
water signal during relaxation delay and gradient pulses for selection. p/
2 pulse widths for 1H protons of 10 and 9 ms were chosen on the 500 and
700 MHz systems, respectively. Mass spectra were recorded in order to
determine molecular weights with matrix-assisted laser desorption/ioniza-
tion time-of-flight (MALDI-TOF) or field desorption (FD) mass spec-
trometry on Bruker Reflex and VG ZAB 2-SE-FPD Spectrofield instru-
ments. Electrospray ionization (ESI) mass spectrometry was measured
on a Navigator 1 instrument from ThermoQuest or a Finnigan MAT 95
spectrometer. Reversed-phase chromatography purification was per-
formed on a Resource RPC 3 mL column from GE Healthcare at a flow
rate of 2 mLminÀ1 with a linear gradient of buffer B (0–60% over 8 min
and 60–100% over 6.5 min) in buffer A (buffer A: 0.1% TFA in H2O;
buffer B: 0.1% TFA in CH3CN). HPLC purification was performed on a
Jasco HPLC 2000 series system at a flow rate of 10 mLminÀ1 with a
linear gradient of buffer B (10% for 3 min, 10–70% over 21 min, 70–
100% over 0.5 min, and 100% for 9.5 min) in buffer A (buffer A: 0.1%
TFA in H2O; buffer B: 0.1% TFA in 95% CH3CN).
in the biological activity are expected if the disulfide bridge
is in close vicinity to a binding motif, which should be con-
sidered if disulfide bridges are targeted by this approach.
Conclusion
We have presented the successful synthesis of bioorthogonal
conjugation reagents that facilitate the introduction of iodo
and ethynyl groups into peptides by intercalation into disul-
fide bridges. The successful intercalation of these novel re-
agents was demonstrated by application to the peptide hor-
mone somatostatin, which bears a single disulfide bridge, as
a model peptide. The reaction mechanism was elucidated by
2D NMR experiments and it was shown that somatostatin
diastereomers were formed due to keto–enol tautomerism
of the intermediate. Both the iodo and the ethynyl group
are bioorthogonal. Site-directed modification of the ethynyl-
modified somatostatin and the introduction of a coumarin
chromophore bearing an azido group were achieved through
a [1,3] dipolar Huisgen cycloaddition reaction. The biologi-
cal activity of a fluorescent somatostatin derivative bearing
a single bulky rhodamine substituent was investigated in cell
experiments. CAPAN-2 cells expressing somatostatin recep-
tors revealed a high uptake of rhodamine-labeled somatos-
tatin in a dose-dependant manner and vesicle formation in-
dicated receptor-mediated uptake as the major pathway. By
contrast, receptor-negative A-549 cells showed only very
low nonspecific uptake of somatostatin conjugate 16. The
high bioactivity of 16 was explained by the retention of the
bioactive conformation around the tetrapeptide receptor-
binding motif and might also be due to a somewhat higher
stability of the modified somatostatin in contrast to native
somatostatin.
In summary, the novel bisalkylating bioconjugation agents
5 and 8 represent attractive labels for the introduction of
iodo and ethynyl groups into proteins and peptides while
keeping the bioactive 3D structure. Somatostatin derivatives
bearing a single iodo or ethynyl group represent a valuable
platform for designing an artificial somatostatin library with
so-far unknown bioactivities. In addition, the attachment of
further bioactive small molecules entities, such as anticancer
drugs, might allow efficient and selective cell-specific deliv-
ery into cancer cells expressing somatostatin receptors.
Future work will focus on increasing the solubility of 5 and
8 in aqueous media and thus facilitating their use in the ab-
sence of organic solvents; this would open up the opportuni-
ty to decorate a broad range of proteins with iodo and eth-
ynyl groups.
Detailed descriptions of the syntheses of 1-[3’-(4’’-iodophenyl)-3’-oxo-
propyl]piperidinium hydrochloride (2), 2,2-bis[(4’’-tolylthio)methyl]-4’-
iodo-acetophenone (3), and 2,2-bis[(4’’-tolylthio)methyl]-4’-triisopropylsi-
lylethynyl-acetophenone (6) can be found in the Supporting Information.
2,2-Bis[(4’’-tolylsulfonyl)methyl]-4’-iodo-acetophenone (4): Iodo-bis-sul-
fide
3 (0.2 g, 0.4 mmol) and potassium peroxomonosulfate (0.95 g,
1.54 mmol) were suspended in EtOAc/CH3CN/H2O (6:6:1, 8 mL). The
reaction mixture was stirred at ambient temperature overnight. The solu-
tion was filtered and diluted with ethanol/water (1:1, 100 mL) and then
the aqueous phase was washed twice with ethyl acetate (50 mL). The or-
ganic solution was dried with magnesium sulphate. Gravity filtration fol-
lowed by removal of volatiles gave 4 as a colorless solid product: yield:
1
0.21 g (90%); H NMR (300 MHz, CDCl3, tetramethylsilane (TMS)): d=
7.76 (d, J=8.6 Hz, 2H), 7.72 (d, J=8.3 Hz, 4H), 7.40–7.25 (m, 6H), 4.33
(quintet, J=6.3 Hz, 1H), 3.55 (qd, J=14.3, 6.3 Hz, 4H), 2.50 ppm (s,
6H); 13C NMR (75 MHz, CDCl3): d=200.09, 145.49, 138.24, 137.63,
135.39, 130.16, 129.81, 128.33, 112.02, 55.68, 21.72 ppm; FT-IR: n˜ =740,
802, 868, 928, 1000, 1020, 1090, 1140, 1170, 1200, 1240, 1300, 1390, 1420,
1490, 1580, 1680, 2910, 2980, 3020, 3090 cmÀ1; UV/Vis (acetonitrile): lmax
(e)=225 (14210), 274 nm (3877 molÀ1 dm3 cmÀ1); FD MS: m/z: 581.8
[M]+; HRMS (ESI): calcd: 604.9929; found: 604.9958.
2-[(4’’-Tolylsulfonyl)methyl]-3-(4’-iodophenyl)-3-oxo-prop-1-ene (5): Po-
tassium tert-butoxide (0.076 g, 0.68 mmol) was dissolved in tetrahydrofur-
an (34 mL). Iodo-bis-sulfone 4 (0.1 g, 0.17 mmol) was dissolved in tetra-
hydrofuran (10 mL) and slowly added to the first solution. The resulting
mixture was stirred at ambient temperature for 1 hour. The solution was
diluted with dichloromethane/water (1:1, 100 mL), extracted with di-
chloromethane, and dried with magnesium sulfate. After filtration, drying
in vacuo gave 5 as colorless solid and the crude product was purified by
chromatography (silica gel, hexane/acetone 3:1): yield: 0.052 g (72%);
1H NMR (250 MHz, CD2Cl2, TMS): d=7.83 (d, J=8.4 Hz, 2H), 7.75 (d,
J=8.3 Hz, 2H), 7.40 (d, J=8.4 Hz, 2H), 7.34 (d, J=8.2 Hz, 2H), 6.14 (s,
1H), 5.94 (s, 1H), 4.31 (s, 2H), 2.41 ppm (s, 3H); 13C NMR (75 MHz,
TMS): d=194.30, 145.64, 138.03, 136.28, 136.13, 136.07, 133.94, 131.36,
130.25, 128.64, 100.51, 58.23, 21.75 ppm; FT-IR: n˜ =665, 710, 789, 839,
Experimental Section
899, 987, 1090, 1150, 1250, 1290, 1400, 1480, 1580, 1650, 2380, 2980 cmÀ1
;
Unless otherwise stated, reagents and chemicals were obtained from
commercial suppliers and used without further purification. Somatostatin
acetate was ordered from Chengdu CP Biochem Co., Ltd. Tris(3-hydro-
xypropyltriazolylmethyl)amine (THPTA)[29] and 4-{2’,2’-bis[(4’’-tolylsulfo-
UV/Vis
(acetonitrile):
lmax
(e)=271
(24400),
225 nm
(6000 molÀ1 dm3 cmÀ1); FD MS: m/z: 425.4 [M]+; HRMS (ESI): calcd:
448.9684; found: 448.9691.
Chem. Eur. J. 2011, 17, 9697 – 9707
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9705

T. Weil et al.
2-[(4’’-Tolylsulfonyl)methyl]-3-(4’-ethynylphenyl)-3-oxo-prop-1-ene (8):
TIPS-protected ethynyl-bis-sulfone (6; 0.105 g, 0.18 mmol) and oxone
(0.443 g, 0.72 mmol) were suspended in dichloromethane (10 mL). The
suspension was allowed to stir at ambient temperature for 24 h and after-
wards filtered to take out the salts. The volatiles were removed in vacuo
and the residue was dissolved in dry THF (10 mL). TBAF (0.094 g,
0.36 mmol) was added to the solution and, after 10 min of stirring, the re-
action was quenched by adding water (10 mL). The aqueous phase was
extracted twice with dichloromethane (20 mL) and the combined organic
solutions were dried with magnesium sulphate. After filtration and re-
moval of the volatiles in vacuo, the crude product was purified by chro-
matography (silica gel, hexane/acetone 3:1): yield: 0.041 g (70%);
1H NMR (250 MHz, CD2Cl2, TMS): d=7.75 (d, J=8.3 Hz, 2H), 7.64 (d,
J=8.6 Hz, 2H), 7.54 (d, J=8.6 Hz, 2H), 7.35 (d, J=8.6 Hz, 2H), 6.15 (s,
1H), 5.95 (s, 1H), 4.31 (s, 2H), 3.32 (s, 7H), 2.41 ppm (s, 3H); 13C NMR
(75 MHz, TMS): d=193.97, 136.06, 135.96, 135.75, 133.80, 131.97, 129.85,
129.55, 128.34, 126.55, 107.54, 82.64, 80.29, 57.84, 21.62 ppm; FT-IR: n˜ =
665, 710, 789, 839, 899, 987, 1090, 1150, 1250, 1290, 1400, 1480, 1580,
and filtered through a 0.22 mm membrane filter. The modified peptide
was obtained after purification by reversed-phase chromatography and
freeze-drying of the pooled fractions: MALDI-TOF MS: m/z: 2412.11
[M]+.
A detailed description of the visualization of somatostatin derivative 11
in comparison to somatostatin and the experimental details of the cell-
culture experiments can be found in the Supporting Information.
Acknowledgements
Financial support from the DFG (SFB 625) is gratefully acknowledged.
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1650, 2380, 2980 cmÀ1
; UV/Vis (acetonitrile): lmax (e)=271 (24400),
225 nm (6000 molÀ1 dm3 cmÀ1); FD MS: m/z: 323.4 [M]+; HRMS (ESI):
calcd: 325.0898; found: 325.0908.
LRB intercalating agent (15): 4-{2’,2’-Bis[(4’’-tolylsulfonyl)methyl]acetyl}-
benzoic acid NHS ester (14; 17 mg, 0.034 mmol) and LRB (10 mg,
0.017 mmol) were dissolved in dichloromethane (3 mL) and dimethylfor-
mamide (1 mL). The solution was allowed to stir at ambient temperature
for 72 h and afterwards was directly loaded on an aluminium oxide
column and eluted with ethyl acetate/ethanol (1:1). After removal of the
volatiles in vacuo, the residue was redissolved in a small quantity of ace-
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MALDI-TOF MS: m/z: 1083.42 [M]+.
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was incubated at room temperature for 30 min. Iodomonosulfone
5
(3.5 mg, 6 mmol) was dissolved in acetonitrile (8 mL) and slowly added to
the first solution. The resulting mixture was incubated at ambient tem-
perature for 24 h, concentrated in vacuo, and filtered through a mem-
brane filter (0.22 mm). The modified peptide was obtained after purifica-
tion by reversed-phase chromatography and freeze-drying of the pooled
fractions: yield: 43% (gravimetric determination after HPLC purifica-
tion); MALDI-TOF MS: m/z: 1909.5 [M]+; HRMS (ESI-TOF): calcd:
1909.6987; found: 1909.6993.
Ethynyl-modified somatostatin (12): The preparation and purification
were carried out in the same way as for the synthesis of 11; for the modi-
fication, ethynylmonosulfone 8 (2 mg, 6 mmol) was added in acetonitrile
(8 mL): yield: 56% (gravimetric determination after HPLC purification);
MALDI-TOF MS: m/z: 1809.24 [M+H]+.
Coumarin-modified somatostatin (13): Ethynyl-modified somatostatin 8
(0.05 mg, 0.03 mmol) was dissolved in H2O (90 mL) and 0.05m sodium
phosphate (pH 7.8; 298 mL) was added. 3-Azido-7-hydroxycoumarin
(0.01 mg, 0.05 mmol) in dimethylsulfoxide (50 mL) was added to this mix-
ture. Copper sulphate (0.008 mg, 0.05 mmol) in H2O (2.3 mL) was pre-
mixed
with
tris(3-hydroxypropyltriazolylmethyl)amine
(0.11 mg,
0.25 mmol) in deionized water (34.3 mL) and then added to the reaction
solution. After addition of aminoguanidine (0.19 mg, 2.5 mmol) in water
(4.8 mL) and sodium ascorbate (0.5 mg, 2.5 mmol) in water (34.3 mL), the
mixture was mixed and incubated at ambient temperature for 24 h, con-
centrated in vacuo, and filtered through a membrane filter with 0.22 mm
pore size. Modified peptide 13 was obtained after purification by re-
versed-phase chromatography and freeze-drying of the pooled fractions:
MALDI-TOF MS: m/z: 2012.09 [M+H]+.
LRB-modified somatostatin (16): Somatostatin acetate (9, 1 mg,
0.6 mmol) was dissolved in 0.05m sodium phosphate at pH 7.8 (2.4 mL).
Tris(2-carboxyethyl)phosphine hydrochloride (0.34 mg, 6 mmol) was
added and the solution was incubated at room temperature for 30 min.
LRB-bis-sulfone (15; 3.25 mg, 3 mmol) was dissolved in acetonitrile
(1.6 mL) and slowly added to the first solution. The resulting mixture
was incubated at ambient temperature for 24 h, concentrated in vacuo,
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9706
www.chemeurj.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 9697 – 9707

Iodo and Ethynyl Somatostatin Analogues
FULL PAPER
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Received: January 26, 2011
Revised: April 26, 2011
Published online: July 11, 2011
Chem. Eur. J. 2011, 17, 9697 – 9707
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9707