Synthesis of DOTA-conjugated multivalent cyclic-RGD peptide dendrimers via 1,3-dipolar cycloaddition and their biological evaluation: Implications for tumor targeting and tumor imaging purposes

Article abstract of DOI:10.1039/b615940k

This report describes the design and synthesis of a series of α_Vβ₃ integrin-directed monomeric, dimeric and tetrameric cyclo[Arg-Gly-Asp-d-Phe-Lys] dendrimers using "click chemistry". It was found that the unprotected N-ε-azido derivative of cyclo[Arg-Gly-Asp-d-Phe-Lys] underwent a highly chemoselective conjugation to amino acid-based dendrimers bearing terminal alkynes using a microwave-assisted Cu(i)-catalyzed 1,3-dipolar cycloaddition. The α_Vβ ₃ binding characteristics of the dendrimers were determined in vitro and their in vivo α_Vβ₃ targeting properties were assessed in nude mice with subcutaneously growing human SK-RC-52 tumors. The multivalent RGD-dendrimers were found to have enhanced affinity toward the α_Vβ₃ integrin receptor as compared to the monomeric derivative as determined in an in vitro binding assay. In case of the DOTA-conjugated ¹¹¹In-labeled RGD-dendrimers, it was found that the radiolabeled multimeric dendrimers showed specifically enhanced uptake in α_Vβ₃ integrin expressing tumors in vivo. These studies showed that the tetrameric RGD-dendrimer had better tumor targeting properties than its dimeric and monomeric congeners. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b615940k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of DOTA-conjugated multivalent cyclic-RGD peptide dendrimers
via 1,3-dipolar cycloaddition and their biological evaluation: implications for
tumor targeting and tumor imaging purposes†‡
a,b
a
Ingrid Dijkgraaf,§ Anneloes Y. Rijnders,§ Annemieke Soede,b Annemarie C. Dechesne,a G. Wilma van Esse,a
Arwin J. Brouwer,a Frans H. M. Corstens,b Otto C. Boerman,b Dirk T. S. Rijkersa and Rob M. J. Liskamp*a
Received 2nd November 2006, Accepted 2nd January 2007
First published as an Advance Article on the web 29th January 2007
DOI: 10.1039/b615940k
This report describes the design and synthesis of a series of aVb3 integrin-directed monomeric, dimeric
and tetrameric cyclo[Arg-Gly-Asp-D-Phe-Lys] dendrimers using “click chemistry”. It was found that
the unprotected N-e-azido derivative of cyclo[Arg-Gly-Asp-D-Phe-Lys] underwent a highly
chemoselective conjugation to amino acid-based dendrimers bearing terminal alkynes using a
microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition. The aVb3 binding characteristics of the
dendrimers were determined in vitro and their in vivo aVb3 targeting properties were assessed in nude
mice with subcutaneously growing human SK-RC-52 tumors. The multivalent RGD-dendrimers were
found to have enhanced affinity toward the aVb3 integrin receptor as compared to the monomeric
derivative as determined in an in vitro binding assay. In case of the DOTA-conjugated 111In-labeled
RGD-dendrimers, it was found that the radiolabeled multimeric dendrimers showed specifically
enhanced uptake in aVb3 integrin expressing tumors in vivo. These studies showed that the tetrameric
RGD-dendrimer had better tumor targeting properties than its dimeric and monomeric congeners.
Structure–activity relationship studies on this cyclic pentapeptide
showed that the exchange of the valine by a lysine residue (Lys,
Introduction
Integrins are a class of heterodimeric transmembrane proteins1
which play an important role in cell-signaling, cell–cell adhesion,
apoptosis and cell-matrix interactions.2 Integrin aVb3, which binds
to the Arg-Gly-Asp (RGD) tripeptide motif containing ligands,3
plays a pivotal role in tumor angiogenesis2 and metastasis. aVb3
Integrin expressed on endothelial cells modulate cell migration
and survival during angiogenesis, while aVb3 integrin expressed
on carcinoma cells potentiate metastasis by facilitating invasion
and movement across blood vessels. The aVb3 integrin is expressed
on activated endothelial cells during tumor induced angiogenesis,
whereas it is absent on quiescent endothelial cells and normal
tissues. In addition, aVb3 is expressed on various tumor cell
types (e.g. breast, ovarian, and prostate cancers). Evidence exists
that inhibition of aVb3 integrin function prevents tumor growth
and induces tumor regression by antagonizing angiogenesis.4
Several peptidic5 and peptidomimetic6 aVb3 antagonists have
been synthesized. Among these, the cyclo[Arg-Gly-Asp-D-Phe-
Val] (c[RGDfV]), as developed by Kessler and coworkers, is one
of the most active and selective antagonists for the aVb3 integrin.7
K) did not significantly influence activity and selectivity.8 Because
the e-amino moiety of the lysine residue can be easily modified,
numerous applications of c[RGDfK] have been studied for tumor
targeting and imaging.9
Multivalency is a well accepted approach to increase the interac-
tion of weakly interacting individual ligands with their respective
receptors.10 Dendrimers are macromolecules consisting of multiple
perfectly branched monomers and this architecture makes them
versatile constructs for the simultaneous presentation of receptor
binding ligands and other biologically relevant molecules.11 Addi-
tionally, dendrimers might serve as promising molecular scaffolds
containing a number of ligands thereby inducing an apparent
increase of ligand concentration and increasing the probability
of statistical rebinding.10b–e,12 Alternatively, dendrimers may align
these ligands and induce multivalency when receptor clustering
occurs or is initiated after initial monovalent binding.10b–e To
improve tumor targeting efficacy and to obtain better in vivo
imaging properties, several studies explored the multivalency effect
by using dimeric and tetrameric RGD peptides with affinity
toward the aVb3 integrin.13 These studies clearly demonstrated the
multivalency effect, since the in vivo affinity significantly increased
going from monomer via dimer to tetramer. Moreover, also with
respect to tumor-uptake and tumor-to-organ ratios, a similar
increase was observed. These are promising results in view of the
development of integrin-targeted radionuclide therapy.12
aDepartment of Medicinal Chemistry and Chemical Biology, Utrecht In-
stitute for Pharmaceutical Sciences, Utrecht University, P. O. Box 80082,
3508 TB Utrecht, The Netherlands. E-mail: R.M.J.Liskamp@pharm.uu.nl;
Fax: +31 30 253 6655; Tel: +31 30 253 7396/7307
bDepartment of Nuclear Medicine, Radboud University Nijmegen Medical
Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
To decorate the dendrimer end-groups with biologically relevant
peptides as ligands, it is of crucial importance to have the disposal
of efficient and chemoselective conjugation chemistry to ensure
the complete attachment of the ligands to the dendrimer. In
cases of completely amino acid- or peptide-based dendrimers,14,15
† Parts of this research have been published earlier in ref. 22a.
‡ Electronic supplementary information (ESI) available: Experimental
details for the synthesis of 2–5, 7, 8, 10, 17, 18 and 20–22, as well as analysis
data for 23–25 (HPLC, MALDI-TOF). See DOI: 10.1039/b615940k
§ These authors contributed equally to this work.
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this is often achieved using peptide coupling reagents, however,
in most cases, the peptide ligands are attached to dendrimers
by chemoselective reaction of sulfhydryl groups of cysteine
residues with maleimide or iodoacetamide functionalities,16 by
thiol–disulfide exchange, by native chemical ligation17 or via
a chemoselective oxime13d–l,m respectively hydrazone18 ligation.
However, new bioconjugation reactions with mutually reactive
conjugation partners with increased efficiency and chemoselec-
tivity which are synthetically easily accessible would be very
welcome.
Recently, the well-known reaction between an alkyne and an
azide to yield 1,4-disubstituted 1,2,3-triazoles, was reinvestigated
independently by Meldal et al.19a and Sharpless et al.19b They found
that an alkyne and an azide in the presence of Cu(I) undergo
a 1,3-dipolar cycloaddition to the corresponding triazole under
very mild reaction conditions with very high chemoselectivity
and efficiency which make this reaction particularly suitable for
bioconjugations. So far, this 1,3-dipolar cycloaddition denoted
as a ‘click reaction’,20 has led to a plethora of applications in
the literature.21 Recently, we synthesized multivalent dendrimeric
peptides22a (up to octa- and hexadecavalent systems) respectively
triazole-linked glycodendrimers22b via a microwave-assisted 1,3-
dipolar cycloaddition between azido peptides respectively glycosyl
azides and dendrimeric alkynes as an alternative approach to
functionalize dendrimers.22c
Results and discussion
Synthesis
Schemes 1 and 2 illustrate our approach for the convergent
synthesis of amino acid based dendrimers23 and their corre-
sponding DOTA-conjugated derivatives. Monovalent compound
2 and divalent 3 respectively, were synthesized starting from 3-
hydroxy methyl benzoate or 3,5-dihydroxy methyl benzoate and
propargylbromide in the presence of K2CO3 as a base and were
obtained in 95 and 81% yield. Since these two compounds were
also used as synthons in further syntheses, the resulting methyl
esters 2 and 3 were treated with Tesser’s base24 to yield acids 4 and
5 in nearly quantitative yield. After treatment of the previously
described 623c with TFA to remove both Boc-functionalities, the
resulting bisamine TFA salt was coupled to acid 5 in the presence
of BOP–DIPEA to give the tetravalent dendrimer 7 with 75% yield.
To conjugate the tetravalent dendrimer with a DOTA-moiety at
a later stage of the synthesis, its methyl ester was saponified with
Tesser’s base and acid 8 was obtained quantitatively.
The DOTA-moiety was connected to the dendrimer core via a
short ethylene spacer. For this purpose, 1,2-diaminoethane was
converted into the mono-protected Boc derivative 10 which was
obtained in 56% yield. Unfortunately, although a large excess of
the amine was used, the bis-protected side product was obtained in
a considerable amount. Compound 10 was coupled in the presence
of BOP–DIPEA to either the monovalent, divalent or tetravalent
dendrimer acids 4, 5 or 8 to obtain the corresponding amides 11,
12 or 15, respectively, generally in yields higher than 90%. The
Boc-protected dendrimers were treated with TFA to obtain the
corresponding amines and they were treated with BOP–DIPEA in
the presence of 2-(4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-
tetraazacyclododecan-1-yl) acetic acid (DOTA(OtBu)3) to give the
DOTA-conjugated mono-, di- and tetravalent dendrimers 13, 14
Here we describe the synthesis of monomeric, dimeric and
tetrameric c[RGDfK] dendrimers via a microwave-assisted 1,3-
dipolar cycloaddition of dendrimeric alkynes with the N-e-azido
derivative of cyclo[Arg-Gly-Asp-D-Phe-Lys] and their subsequent
evaluation as aVb3 integrin antagonists. Additionally, the RGD
dendrimers were conjugated with a 1,4,7,10-tetraazadodecane-
N,Nꢀ,Nꢀꢀ,Nꢀꢀꢀ-tetraacetic acid (DOTA) moiety. These analogs were
radiolabeled with 111In to evaluate the in vitro receptor binding
characteristics and in vivo tumor targeting properties.
Scheme 1 Synthesis of the mono-, di- and tetravalent dendrimeric alkynes 2, 3 and 7.
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Scheme 2 Synthesis of the DOTA-conjugated dendrimeric alkynes 13, 14 and 16.
and 16 respectively. It is important to note that the solubility
of the DOTA-conjugated dendrimer is an important factor that
determines the yield of the coupling reaction. Compounds 13 and
14 were isolated in very high yields (>94%) but compound 16 was
isolated with a modest yield of 60% due to its low solubility in
solvents like EtOAc and CH2Cl2.
transfer as the final reaction step, did not substantially improve
the isolated yield.
At this stage of the synthesis, the challenge was the chemos-
elective coupling of the different dendrimeric alkynes (2, 3, 7,
13, 14, or 16) to the cyclic RGD azido peptide (19) to furnish
the DOTA-conjugated dendrimeric cyclo-RGD peptides as aVb3
integrin antagonists as shown in Scheme 4. Our first experiments
were based on the literature procedure19b in which acetylene 3
was coupled to azido glycine ethyl ester (ethyl 2-azidoacetate) in
the presence of CuSO4–Na-ascorbate–Cu-wire in tert-BuOH–H2O
for 16 h at room temperature. Monitoring the reaction by TLC
showed that formation of the monovalent cycloadduct proceeded
rapidly, but the conversion into the divalent product was sluggish.
However, a tremendous improvement was achieved by running this
reaction under microwave irradiation. After 10 min at 100 ◦C using
DMF–H2O as solvent in the presence of CuSO4–Na-ascorbate,
the divalent cycloaddition product was obtained in 96% yield.
This microwave-assisted cycloaddition of dendrimeric alkynes and
azido peptides was recently reported as a versatile approach to ob-
tain multivalent dendrimeric peptides.22a,c The optimized reaction
conditions were used to couple the cyclic RGD azido peptide (19)
to the different dendrimeric alkynes (2, 3, 7, 13, 14, or 16).
In case of alkynes 2, 3 and 7 the formation of the cycloadducts
20, 21 and 22 could be followed by TLC and LC-MS. It turned
The next step in the synthesis was the preparation of the N-
e-azido cyclo(Arg-Gly-Asp-D-Phe-Lys) peptide 19 (Scheme 3).
To obtain this compound, peptide resin 17 was synthesized
using Fmoc–tBu SPPS (solid phase peptide synthesis) based
on the protocol of Liu et al.25 It was decided to cleave the
26
protected peptide acid from the resin by HFIP–CH2Cl2 instead
of AcOH–TFE to avoid premature acetylation during the BOP–
DIPEA-mediated macrolactamization step. Cyclic peptide 18 was
obtained in 36% overall yield based on the initial resin loading
of 0.64 mmol g−1. Subsequently, the e-amine of the lysine residue
was selectively converted into the azide moiety by a diazotransfer.27
At pH 10, the e-amine can be deprotonated in the presence of a
guanidino functionality, since the latter is a much stronger base
and will not act as a nucleophile in the diazotransfer reaction.
Finally, the peptide N-e-azido cyclo(Arg-Gly-Asp-D-Phe-Lys) 19
was obtained in 21% yield after purification by HPLC and was
1
characterized by H-NMR (500 MHz) and mass spectrometry
(LC-MS). Incorporation of Fmoc-Lys(N3)-OH, to avoid the diazo
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Scheme 3 Synthesis of the N-e-azido cyclo(Arg-Gly-Asp-D-Phe-Lys) peptide 19.
out that the formation of 20 and 21 was complete after 10 to
20 min microwave irradiation at 100 ◦C, whereas the formation
of 22 was complete after 30 min. Although HPLC analysis of the
crude cycloaddition products evidenced a complete conversion as
judged by the absence of the alkyne starting material, the RGD-
dendrimers 20–22 were obtained in yields varying between 14
to 57%. Then, the DOTA-conjugated alkyne dendrimers 13, 14
and 16 were subjected to the cycloaddition reaction conditions in
the presence of azido peptide 19. It should be emphasized that
the carboxyl functionalities of the DOTA-moiety needed to be
protected by tert-butyl groups to avoid premature and irreversible
sequestering of the Cu2+ ions. Chelated copper(II) will result in
a lower efficiency of the Cu(II)–Cu(I) redox couple to generate
the active Cu(I)-catalyst. More importantly, it will hamper the
radiolabeling of the DOTA-moiety of compounds 23–25 with
trivalent radiometals such as 111In, 90Y or 177Lu. As a result, after
the click reaction an additional reaction step was needed in which
the partially protected cycloadducts were treated with TFA, in the
presence of suitable scavengers, to give the unprotected DOTA-
conjugated RGD-dendrimers 23–25.
followed by a TFA-treatment without isolation of the cycload-
dition intermediates. The isolated yield (13%) of monovalent 23
was rather disappointing. Recently, optimized conditions with
respect to the generation of the catalytic active Cu(I) species were
published28 and these conditions were applied in the cycloaddition
of 14 and 19. Unfortunately, an increase of the isolated yield
was not observed using these modified reaction conditions. As
was mentioned earlier, the cycloaddition reaction was complete
according to HPLC analysis, and the low isolated yield was mainly
due to the difficult purification. The DOTA-conjugated RGD-
dendrimers were obtained in yields varying between 11 and 36%.
Radiolabeling of the RGD dendrimers
Dendrimers 23, 24 and 25 were radiolabeled by dissolving these
compounds in an NH4OAc buffer of pH 6.0 and 22.2–37 MBq
111InCl3 was added to each of the reaction mixtures. The reaction
mixtures were degassed and subsequently heated at 100 ◦C for
15 min. Reversed phase-HPLC analysis showed a single peak for
each of the three 111In-labeled compounds with an elution time of
25.9 min, 29.5 min and 29.4 min for the 111In-labeled monovalent
23, divalent 24, and tetravalent 25, RGD peptide dendrimers
respectively.
The cycloaddition reaction of the DOTA-conjugated den-
drimeric alkynes 13, 14 and 16 was difficult to monitor by mass
spectrometry. As was described above, reaction times of 10 to
30 min were used and the cycloaddition reaction was directly
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Scheme 4 Synthesis of the mono-, di- and tetravalent cyclo[RGDfK] peptide dendrimers 20, 21 and 22 and their respective DOTA-conjugated counterparts
23, 24 and 25.
Solid phase aVb3 binding assay
tetravalent 25. The dendrimer containing four c[RGDfK] units
(25) showed an increased affinity for avb3 compared to the
dendrimers containing one (23) or two (24) c[RGDfK] units.
Multimerization of c[RGDfK] resulted in enhanced affinity for
avb3 as was evidenced by a decrease of the IC50 concentration.
The affinity of the DOTA-conjugated RGD dendrimers 23, 24,
and 25 for the avb3 integrin was determined in a competitive
binding assay. The results of these analyses are shown in Fig. 1.
Binding of the 111In-labeled dimeric peptide, 111In-DOTA-Glu-
(c[RGDfK])2,29 to avb3 was competed by unlabeled 23, 24, and
25 in a concentration dependent manner. The IC50 values were
212 nM for monovalent 23, 356 nM for divalent 24, and 50 nM for
Biodistribution studies
In athymic mice with subcutaneously (s.c.) growing SK-RC-
52 renal cell carcinoma, the tumor uptake of the 111In-labeled
tetrameric RGD dendrimer 25 at 2 h post-injection (p.i.; 7.27
2.06%ID/g) was significantly higher (P < 0.05) compared to
that of the 111In-labeled monomeric RGD dendrimer 23 (1.69
0.41%ID/g) as shown in Fig. 2A. At 2 h p.i., the tumor uptake
of tetrameric RGD dendrimer 25 was also significantly higher
(P < 0.05) than the dimeric analog 24 (3.15 0.51%ID/g). The
tumor-to-blood ratios of the tetramer 25 (5.66
1.74%ID/g,
34.73 5.95%ID/g) were significantly higher (P < 0.05)—both
at 2 h p.i. and at 24 h p.i.—than those of the monomer 23
(3.12 1.92%ID/g, 19.65 12.42%ID/g) and dimer 24 (1.70
0.50%ID/g, 14.66 0.25%ID/g). At 24 h post injection, the tumor
uptake of the tetrameric RGD dendrimer 25 (5.83 1.18%ID/g)
was significantly higher compared to the dimeric RGD dendrimer
24 (2.82
dendrimer 23 (1.19
in Fig. 2B. Co-injection of an excess of non-radiolabeled RGD
0.59%ID/g, P < 0.05) and the monomeric RGD
0.31%ID/g, P < 0.01) which is shown
Fig. 1 Competition of specific binding of 111In-DOTA-Glu-(c[RGDfK])2
with RGD dendrimers 23, 24, and 25.
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radiolabeled multimeric dendrimers showed specifically enhanced
uptake in aVb3 integrin expressing tumors in vivo. These studies
showed that the tetrameric RGD-dendrimer had better tumor
targeting properties than its dimeric and monomeric congeners.
Experimental
Instruments and methods
Peptides were synthesized on an ABI 433A automatic Peptide Syn-
thesizer using the FastMoc solid phase peptide synthesis protocols.
Microwave-assisted reactions were carried out in a Biotage mi-
crowave reactor. Analytical HPLC runs were carried out on a Shi-
madzu HPLC system and preparative HPLC runs were performed
on a Gilson HPLC workstation. Analytical HPLC runs were
performed on Alltech Prosphere C4 or C8 and Adsorbosphere
˚
XL C18 columns (250 × 4.6 mm, pore size 300 A, particle size:
5 lm) or on a Merck LiChroCART CN column (250 × 4.6 mm,
−1
˚
pore size 100 A, particle size: 5 lm) at a flow rate of 1.0 mL min
using a linear gradient of buffer B (0–100% in 25 min) in buffer A
(buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA in CH3CN–H2O
95 : 5 v/v). Preparative HPLC runs were performed on an Alltech
˚
Prosphere C4 or C8 column (250 × 22 mm, pore size 300 A,
particle size: 10 lm), and semi-prep HPLC runs were performed
on an Alltech Adsorbosphere XL C18 column (250 × 10 mm,
Fig. 2 A Biodistribution of 111In-labeled monomer 23, dimer 24, and
tetramer 25 at 2 h p.i. in athymic mice with s.c. SK-RC-52 tumors. B
Biodistribution of 111In-labeled monomer 23, dimer 24, and tetramer 25 at
24 h p.i. in athymic mice with s.c. SK-RC-52 tumors.
˚
pore size 300 A, particle size: 10 lm) or on a Merck LiChroCART
˚
CN column (250 × 10 mm, pore size 100 A, particle size: 10 lm)
at a flow rate of 10.0 mL min−1 (semi-prep HPLC: 4.0 mL min−1)
using a linear gradient of buffer B (0–100% in 50 min) in buffer
A (buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA in CH3CN–
H2O 95 : 5 v/v). Liquid chromatography electrospray ionization
mass spectrometry was measured on a Shimadzu LCMS-QP8000
single quadrupole bench-top mass spectrometer operating in a
positive ionization mode. LC/MS(MS) runs were performed on a
Finnigan LCQ Deca XP MAX LC/MS equipped with a Shimadzu
10A VP analytical HPLC system. The samples were dissolved in
10% formic acid in CH3CN–H2O 1 : 1 v/v and analyzed using a
peptide (DOTA-Glu-(c[RGDfK])2) to saturate all avb3 receptors
in vivo, resulted in a significantly reduced tumor uptake of each of
the three compounds: 23: 0.46
0.31%ID/g (24 h p.i.), 24: 0.76
0.04%ID/g (2 h p.i.), 0.36
0.09%ID/g (2 h p.i.), not
determined (24 h p.i.) and 24: 1.56
0.02%ID/g (2 h p.i.),
1.19 0.03%ID/g (24 h p.i.), indicating that each of the three
RGD dendrimers of this study showed receptor mediated uptake
in the tumor. These in vivo results were in line with the in vitro
binding assay. The tetrameric RGD dendrimer showed enhanced
affinity for avb3, as compared to the monomeric and dimeric RGD
dendrimer, respectively. The results of this study correlated nicely
with the results observed in a previous study in which we evaluated
multimeric RGD peptides in the same animal model.13o
The affinity of the dendrimers as determined in an in vitro
binding assay are in agreement with the results obtained from the
in vivo experiment: the IC50 concentration of the tetrameric RGD
dendrimer 25 was lower compared to those of the monomeric 23
and dimeric 24 analogs, resulting in a significantly higher uptake
of the former in avb3-expressing tumors and better tumor-to-blood
ratios compared to the monomeric and dimeric RGD dendrimers.
In conclusion, a series of aVb3 integrin-directed monomeric,
dimeric and tetrameric cyclo[Arg-Gly-Asp-D-Phe-Lys] dendrimers
using “click chemistry” was successfully synthesized, since the un-
protected N-e-azido derivative of cyclo[Arg-Gly-Asp-D-Phe-Lys]
underwent a highly chemoselective conjugation to amino acid-
based dendrimers bearing terminal alkynes using a microwave-
assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition. The aVb3 bind-
ing characteristics and aVb3 targeting properties of the dendrimers
were determined both in vitro and in vivo. In the case of the DOTA-
conjugated 111In-labeled RGD-dendrimers, it was found that the
Phenomenex Gemini C18 column (150 × 4.6 mm, particle size:
−1
˚
3 lm, pore size: 110 A) at a flow rate of 1.0 mL min using a
linear gradient of 100% buffer A (0.1% TFA in H2O–CH3CN 95 :
5 v/v) to 100% buffer B (0.1% TFA in CH3CN–H2O 95 : 5 v/v)
in 50 min. MALDI-TOF analysis was performed on a Kratos
Axima CFR apparatus with bradykinin(1–7) (monoisotopic [M +
H]+ 757.399), human ACTH(18–39) (monoisotopic [M + H]+
2465.198) and bovine insulin oxidized B chain (monoisotopic
[M + H]+ 3494.651) as external references and a-cyano-4-
hydroxycinnamic acid or sinapinic acid as matrices. 1H NMR
spectra were recorded on a Varian G-300 (300 MHz) spectrometer
and chemical shifts are given in ppm (d) relative to TMS. 13C NMR
spectra were recorded on a Varian G-300 (75.5 MHz) spectrometer
and chemical shifts are given in ppm relative to CDCl3 (77.0 ppm).
The 13C NMR spectra were recorded using the attached proton
test (APT) sequence. 1H NMR spectra in H2O–D2O 9 : 1 v/v were
recorded on a Varian Inova-500 (500 MHz) spectrometer and
chemical shifts are given in ppm (d) relative to 3-(trimethylsilyl)-
1-propanesulfonic acid sodium salt (0.00 ppm). Peak assignments
are based on DQF-COSY, TOCSY (mixing times: 20 or 60 ms) and
ROESY (mixing times: 150 or 250 ms) spectra. HSQC and HMBC
spectra were measured on a Varian Inova-500 spectrometer and
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chemical shifts are given in ppm (d) relative to 3-(trimethylsilyl)-1-
propanesulfonic acid sodium salt (0.00 ppm). Fourier transform
infrared spectra (FTIR) were measured on a Bio-Rad FTS-25
spectrophotometer. Melting points were measured on a Bu¨chi
Schmelzpunktbestimmungsapparat and are uncorrected. Ele-
mental analyses were done by Kolbe Mikroanalytisches La-
bor (Mu¨lheim/Ruhr, Germany). Rf values were determined by
thin layer chromatography (TLC) on Merck precoated silica
gel 60F254 plates. Spots were visualized by UV-quenching,
ninhydrin or Cl2–TDM.30 The 2-chlorotrityl chloride resin
(Hecheng Science & Technology Company) was used in all solid
phase syntheses. The coupling reagents 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and
benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) were obtained from Biosolve. N-Hydroxy-
benzotriazole (HOBt) was from Advanced ChemTech and Na-9-
fluorenylmethyloxycarbonyl (Fmoc) amino acids were obtained
from MultiSynTech. The side-chain protecting groups were
chosen as tert-butyl for aspartic acid, tert-butyloxycarbonyl
(Boc) for lysine and 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-
sulfonyl (Pbf) for arginine. Peptide-grade tert-butanol (tBuOH),
dichloromethane, N,N-dimethylformamide (DMF), 1,1,1,3,3,3-
hexafluoroisopropanol (HFIP), tert-butyl methylether (MTBE),
N-methylpyrrolidone (NMP), and trifluoroacetic acid (TFA) and
HPLC-grade acetonitrile were purchased from Biosolve. 2-(4,7,10-
Tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-
yl) acetic acid (DOTA(OtBu)3) was purchased from Macrocyclics.
Piperidine, N,N-diisopropylethylamine (DIPEA), CuSO4 and
sodium ascorbate were obtained from Acros Organics. Triiso-
propylsilane (TIS) and HPLC-grade TFA were obtained from
Merck. Triflic anhydride and propargylbromide were purchased
from Aldrich.
111In-labeled DOTA-Glu-(c[RGDfK])2 (3 MBq lg−1) was prepared
as described above and was used as the tracer in the assay.
Microtiter 96-well vinyl assay plates (Corning B.V., Schiphol-Rijk,
The Netherlands) were coated with 100 lL/well of a solution
of purified human integrin avb3 (150 ng mL−1) in Triton X-100
Formulation (Chemicon International, Temecula, CA, USA) in
coating buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
◦
CaCl2, 0.5 mM MgCl2 and 1 mM MnCl2) for 17 h at 4 C. The
plates were washed twice with binding buffer (0.1% bovine serum
albumin (BSA) in coating buffer). The wells were blocked for
2 h with 200 lL blocking buffer (1% BSA in coating buffer). The
plates were washed twice with binding buffer. Then 100 lL binding
buffer containing 11.1 kBq of 111In-DOTA-Glu-(c[RGDfK])2 and
appropriate dilutions of non-labeled monovalent 23, divalent
24 and tetravalent 25 RGD dendrimers in binding buffer were
incubated in the wells at 37 ◦C for 1 h. After incubation,
the plates were washed three times with binding buffer. The
retained radioactivity in each well was determined in a c-counter
(1480 Wizard, Wallac, Turku, Finland). The IC50 values of the
RGD dendrimers were calculated by nonlinear regression using
GraphPad Prism (GraphPad Prism 4.0 Software, San Diego, CA,
USA). Each data point represents the average of three individual
determinations.
Biodistribution studies
In the right flank of 6–8 weeks old female nude BALB/c mice,
0.2 mL of a cell suspension of 8.5 × 106 cells/mL SK-RC-
52 cells was injected subcutaneously (s.c.). Two weeks after
inoculation of the tumor cells, mice were randomly divided into
three groups. The mice were injected with 0.25–0.29 MBq of the
111In-labeled dendrimers 23, 24, or 25 via a tail vein. The mice
were euthanized by CO2 asphyxiation, 2 and 24 h postinjection
(p.i.) (2–5 mice/group). Blood, tumor, and the major organs and
tissues were collected, weighed, and counted in a c-counter. The
percentage injected dose per gram (%ID/g) was determined for
each sample. To investigate whether the uptake of each of the
three RGD dendrimers is avb3-mediated, a separate group of mice
was co-injected with an excess (50 lg) of non-radiolabeled DOTA-
Glu-(c[RGDfK])2 to saturate all the avb3 integrin receptors.
Radiolabeling of the RGD dendrimers
Dendrimers 23 (25 lg, 20 nmol), 24 (25 lg, 13 nmol), and
25 (120 lg, 33 nmol) were radiolabeled by dissolving these
compounds in 500 lL 0.5 M NH4OAc buffer, pH 6.0, containing
0.6 mg mL−1 gentisic acid. Then 22.2–37 MBq 111InCl3 was added
to each of the reaction mixtures. The reaction mixtures were
degassed and subsequently heated at 100 ◦C for 15 min. The
111In-labeled dendrimers were further purified on a Waters C-18
SepPak cartridge (Milford, MA). After applying the sample on
the methanol-activated cartridge, the cartridge was washed with
5 mL 25 mM NH4OAc and eluted with 25% CH3CN in 25 mM
NH4OAc. The radiochemical purity was determined by reversed-
phase HPLC (HP 1100 series, Hewlett Packard, Palo Alto, CA,
USA) using a Zorbax RX-C18 column (250 × 4.6 mm) eluted
with a linear gradient of buffer B (8–20% in 25 min or 8–100% in
30 min in buffer A (buffer A: 25 mM NH4OAc, buffer B: CH3CN)
at a flow rate of 1 mL min−1. The radioactivity of the eluate was
monitored using an in-line radiodetector (Flo-One Beta series,
Radiomatic, Meriden, CT, USA).
Statistical analysis
All mean values are given standard deviation (S.D.). Statistical
analysis was performed using the One-way Analysis of Variance.
Tukey corrections for multiple comparisons were applied. The
level of significance was set at P < 0.05.
Syntheses
Details of the synthetic procedures for compounds 2–5, 7, 8, 10,
17, 18 and 20–22 are given in the ESI‡.
tert-Butyl-2-(3-(prop-2-ynyloxy)benzamido)ethylcarbamate (11).
Acid 4 (774 mg, 4.40 mmol) and amine 10 (704 mg, 4.40 mmol)
were dissolved in CH2Cl2 (25 mL) and BOP (1.95 g, 4.41 mmol)
followed by DIPEA (1.77 mL, 10 mmol, 2.27 equiv) were added
and the obtained reaction mixture was stirred for 16 h. Then,
the solvent was removed by evaporation and the residue was
redissolved in EtOAc (50 mL) and subsequently washed with H2O
Solid phase aVb3 binding assay
The affinity of the DOTA-conjugated monovalent 23, divalent
24 and tetravalent 25 RGD dendrimers for the avb3 integrin
was determined using a solid-phase competitive binding assay.
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(3 × 20 mL), 1 N KHSO4 (3 × 20 mL), H2O (3 × 20 mL), 5%
NaHCO3 (3 × 20 mL) and brine (3 × 20 mL), dried (Na2SO4)
and evaporated to dryness. The residue was purified by column
chromatography (eluents: EtOAc–hexane 1 : 1 v/v) and was
obtained as a white solid with 98% yield (1.38 g). Mp: 118–121 ◦C;
Rf (EtOAc–hexane 1 : 1 v/v): 0.20; 1H NMR (CDCl3) d: 1.42 (s, 9H,
(CH3)3 Boc), 2.54 (s, 1H, CH), 3.38 (m, 2H, ∼NH–CH2–CH2∼),
3.54 (m, 2H, ∼CH2–CH2–NH∼), 4.70 (s, 2H, ∼O–CH2), 5.35 (m,
1H, NH urethane), 7.10–7.47 (broad m, 5H, arom H/NH amide);
13C NMR (CDCl3) d: 28.3, 39.9, 41.7, 55.8, 75.7, 78.1, 79.7, 113.4,
118.3, 119.8, 129.4, 135.6, 157.3, 157.6, 167.5; MS analysis: calcd
for C17H22N2O4 318.16, found ES-MS 319.27 [M + H]+, 341.33
[M + Na]+; Elemental analysis: calcd for C17H22N2O4 C 64.13, H
6.97, N 8.80 found C 63.81, H 6.81, N 8.63%.
tert-Butyl-2,2ꢀ,2ꢀꢀ-(10-(2-(2-(3,5-bis(prop-2-ynyloxy)benzamido)-
ethylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
triyl)triacetate (14). This compound was synthesized as
described for 13 starting from 12 (107 mg, 0.30 mmol).
Compound 14 was obtained as a yellowish solid with nearly
◦
quantitative yield (250 mg). Mp: 74–84 C; Rf (CHCl3–MeOH–
1
AcOH 95 : 20 : 3 v/v/v): 0.45; Rt: 18.70 min (C8); H NMR
(CDCl3) d: 1.43 (s, 27H, (CH3)3 tBu), 2.04–3.70 (broad s, 28H,
CH2 DOTA (24H)/∼NH–CH2–CH2–NH∼(4H)), 2.52 (s, 2H,
CH), 4.73 (s, 4H, ∼O–CH2), 6.73 (m, 2H, arom H4/NH), 7.10
(s, 2H, arom H2/H6), 7.11 (m, 1H, NH); 13C NMR (CDCl3) d:
27.9, 39.6, 50.0*, 55.6, 55.8, 56.2, 75.6, 78.4, 81.9, 106.4, 106.5,
136.4, 158.7, 167.2, 172.0, 172.4 (*broad signal: CH2 DOTA);
MS analysis: calcd for C43H66N6O10, 826.48, found ES-MS 827.65
[M + H]+; Elemental analysis: calcd for C43H66N6O10·K2SO4 C
51.58, H 6.64, N 8.39 found C 52.05, H 6.54, N 8.04%.
tert-Butyl-2-(3,5-bis(prop-2-ynyloxy)benzamido)ethylcarbamate
(12). This compound was synthesized using acid 5 (506 mg,
2.20 mmol) and amine 10 (352 mg, 2.20 mmol) as described
for 11. Compound 12 was obtained in 96% yield (760 mg) after
column chrom◦atography with EtOAc–hexane 8 : 2 v/v as eluents.
tert-Butyl-2-(3,5-bis(2-(3,5-bis(prop-2-ynyloxy)benzamido)-
ethoxy)benzamido)ethylcarbamate (15). This compound was
synthesized as described for 11 using amine 10 (292 mg, 2.0 mmol)
and acid 8 (460 mg, 2.0 mmol). Compound 15 was obtained as a
pale yellow solid with 91% yield (1.46 g). Mp: 110 ◦C; Rf (EtOAc–
hexane 4 : 1 v/v): 0.53; Rt: 18.38 min (C8); 1H NMR (DMSO-d6)
d: 1.37 (s, 9H, (CH3)3 Boc), 3.10 (m, 2H, ∼NH–CH2–CH2–NH∼),
3.28 (m, 2H, ∼NH–CH2–CH2–NH∼), 3.58 (s, 4H, CH), 3.65 (m,
4H, ∼O–CH2–CH2–NH∼), 4.17 (m, 4H, ∼O–CH2–CH2–NH∼),
4.85 (s, 8H, ∼O–CH2), 6.72 (m, 1H, arom H4), 6.80 (m, 2H, arom
H2/H6), 6.90 (m, 1H, NH urethane), 7.05 (m, 2H, arom H4ꢀ), 7.15
(m, 4H, arom H2ꢀ/H6ꢀ), 8.44 (m, 1H, NH amide), 8.68 (m, 2H, NH
amide); 13C NMR (DMSO-d6) d: 28.0, 39.2, 55.9, 75.8, 75.9, 77.8,
79.6, 104.7, 105.4, 105.9, 106.7, 135.9, 136.1, 157.2, 158.5, 159.5,
167.7, 167.8, 168.0, 168.1; MS analysis: calcd for C44H46N4O11,
806.32, found ES-MS 807.65 [M + H]+, 707.55 [(M–C5H8O2) +
H]+; Elemental analysis: calcd for C44H46N4O11 C 65.50, H 5.75, N
6.94 found C 65.28, H 5.71, N 6.80%.
1
Mp: 128–134 C; Rf(EtOAc–hexane 7 : 3 v/v): 0.31; H NMR
(CDCl3) d: 1.42 (s, 9H, (CH3)3 Boc), 2.55 (s, 2H, CH), 3.37 (m,
2H, ∼NH–CH2–CH2∼), 3.52 (m, 2H, ∼CH2–CH2–NH∼), 4.68
(s, 4H, ∼O–CH2), 5.30 (m, 1H, NH urethane), 6.72 (s, 1H, arom
H4), 7.06 (s, 2H, arom H2/H6), 7.44 (m, 1H, NH amide); 13C
NMR (125 MHz, CDCl3) d: 28.3, 40.0, 41.7, 56.0, 75.9, 78.0, 79.8,
105.5, 106.6, 136.4, 157.3, 158.6, 167.2; MS analysis: calcd for
C20H24N2O5 372.17, found ES-MS 373.24 [M + H]+, 395.27 [M +
Na]+; Elemental analysis: calcd for C20H24N2O5 C 64.50, H 6.50,
N 7.52 found C 63.61, H 6.21, N 7.05%.
tert-Butyl-2,2ꢀ,2ꢀꢀ-(10-(2-oxo-2-(2-(3-(prop-2-ynyloxy)benzamido)-
ethylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-
acetate (13). To a solution of compound 11 (100 mg, 0.31 mmol)
in CH2Cl2 (5 mL), TFA (5 mL) was added to remove the Boc
protecting group. After 1 h of stirring at room temperature,
the volatiles were removed by evaporation and the residue was
coevaporated with CH2Cl2 to remove any residual TFA. The
obtained solid was used without further purification. Then, the
TFA-salt was dissolved in CH2Cl2 (10 mL) and 2-(4,7,10-tris(2-
tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)ace-
tic acid (DOTA(OtBu)3; 177 mg, 0.31 mmol), BOP (137 mg,
0.31 mmol) followed by DIPEA (220 lL, 1.24 mmol, 4 equiv)
were added and the obtained reaction mixture was stirred for 16 h
at room temperature. Subsequently, the solvent was removed by
evaporation and the residue was redissolved in EtOAc (50 mL)
and this solution was washed with H2O (3 × 20 mL), 1 N KHSO4
(3 × 20 mL), H2O (3 × 20 mL), 5% NaHCO3 (3 × 20 mL),
brine (3 × 20 mL) and dried (Na2SO4). Finally, the solvent was
evaporated in vacuo after which 13 was obtained as a pale yellow
oil with 94% yield (227 mg). Rf (CH2Cl2–MeOH 9 : 1 v/v): 0.49;
tert-Butyl-2,2ꢀ,2ꢀꢀ-(10-(2-(2-(3,5-bis(2-(3,5-bis(prop-2-ynyloxy)-
benzamido)ethoxy)benzamido)ethylamino)-2-oxyethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetate (16). Compound 16
was synthesized as described for 13 starting from 15 (242 mg,
0.30 mmol). After workup, the crude product was purified by
column chromatography (eluents DCM–MeOH 98 : 2 v/v →
DCM–MeOH 9 : 1 v/v) to yield a white solid (227 mg, 60%).
Mp: 108–114 ◦C; Rf (CH2Cl2–MeOH 9 : 1 v/v): 0.30; Rt:
19.70 min (C8); 1H NMR (CDCl3) d: 1.45 (s, 27H, (CH3)3
tBu), 2.04–4.50 (broad s, 36H, CH2 DOTA (24H)/∼NH–CH2–
CH2–NH∼(4H)/∼O–CH2–CH2–NH∼(8H)), 2.55 (s, 4H, CH),
4.72 (m, 8H, ∼O–CH2), 6.60 (m, 1H, arom H4), 6.70 (m, 2H,
arom H2/H6), 7.18–7.33 (m, 6H, arom H2ꢀ/H4ꢀ/H6ꢀ), 7.80 (m,
2H, NH), 8.75 (m, 2H, NH); 13C NMR (CDCl3) d: 27.9, 28.0,
38.8, 39.3, 39.7, 55.7, 55.9, 56.2, 66.5, 75.8, 76.0, 78.3, 82.0,
106.0, 106.3, 106.5, 106.7, 136.3, 136.8, 158.6, 159.4, 166.7,
166.9, 171.5, 172.3; MS analysis: calcd for C67H88N8O16, 1260.63,
found ES-MS 1261.75 [M + H]+; Elemental analysis: calcd for
C67H88N8O16·H2SO4 C 59.19, H 6.67, N 8.24 found C 59.60, H
6.82, N 7.71%.
1
Rt: 18.10 min (C8); H NMR (CDCl3) d: 1.42 (s, 27H, (CH3)3
tBu), 2.20–3.70 (broad s, 28H, CH2 DOTA (24H)/∼NH–CH2–
CH2–NH∼(4H)), 2.52 (s, 1H, CH), 4.75 (s, 2H, ∼O–CH2), 6.85
(m, 1H, NH), 7.08 (m, 1H, arom H), 7.25–7.32 (m, 2H, arom H),
7.51 (m, 2H, arom H/NH); 13C NMR (CDCl3) d: 27.9, 39.6, 39.7,
55.6, 55.8, 56.0, 75.5, 78.4, 81.8, 112.9, 118.9, 120.3, 129.6, 135.5,
157.6, 167.4, 172.0, 172.4; MS analysis: calcd for C40H64N6O9,
772.47, found ES-MS 773.90 [M + H]+.
N-e-Azido cyclo(Arg-Gly-Asp-D-Phe-Lys) (19). Cyclic peptide
18 (200 mg, 0.33 mmol) was dissolved in tert-BuOH–H2O (5 mL;
1 : 1 v/v) and the pH was adjusted to 10 by the addition of
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1 N NaOH. To this solution were added: CuSO4·5H2O (8 mg,
0.03 mmol, 0.1 equiv) and a solution of triflic azide (587 mg,
3.3 mmol, 10 equiv) in CH2Cl2 (freshly prepared from triflic
anhydride (555 lL, 3.3 mmol, 10 equiv) and NaN3 (975 mg,
15 mmol, 4.5 equiv) in CH2Cl2–H2O (13 mL; 10 : 3 v/v).27
The obtained two-phase reaction mixture was firmly stirred for
16 h at room temperature. Then, the solvents were removed by
evaporation and the residue was mixed with tert-BuOH–H2O and
subsequently lyophilized to yield 202 mg (97%) crude reaction
product. Pure azido peptide 19 was obtained in 21% yield (44 mg)
after purification by HPLC (C8). Rf (CHCl3–MeOH–AcOH 90 :
20 : 3 v/v/v): 0.25; Rt: 16.81 min (C4); Rt: 17.33 min (CN); FTIR
DOTA-conjugated divalent cyclo[RGDfK] peptide dendrimer
28
(24).
Alkyne 14 (4.9 mg, 5.9 lmol) and azido peptide 19
(11 mg, 14.8 lmol, 1.3 equiv) were dissolved in DMF–2,6-lutidine
(1 mL, 7 : 3 v/v) and to this solution the following reagents were
subsequently added: CuOAc (1.8 mg, 14.7 lmol, 2.5 equiv), Na-
ascorbate (5.9 mg, 29.8 lmol, 5.1 equiv) and DIPEA (9.8 lL,
7.1 lmol, 1.2 equiv). The obtained reaction mixture was heated
by microwave irradiation to 100 ◦C for 3 × 5 min. Then, the
solvents were removed under reduced pressure and the residue
was dissolved in tert-BuOH–H2O 1 : 1 v/v and lyophilized. The
obtained fluffy solid was dissolved in TFA–H2O (1 mL; 95 : 5 v/v)
and stirred for 4 h at room temperature. Subsequently, the reaction
mixture was concentrated in vacuo and the residue was redissolved
in tert-BuOH–H2O 1 : 1, lyophilized and purified by semi-prep
HPLC (C18) to obtain compound 24 in 11% yield (1.3 mg). Rt:
1
(KBr) t: 2100 cm−1; H NMR (500 MHz, H2O–D2O 9 : 1 v/v,
293 K, 6.4 mM, pH 4): Arg, d: 1.43 (m, 2H, cCH2), 1.65/1.86
(double m, 2H, bCH2), 3.18 (m, 2H, dCH2), 4.36 (m, 1H, aCH),
7.20 (t (J 5.8 Hz), 1H, dNH), 8.04 (d (J 8.7 Hz), 1H, aNH); Gly,
d: 3.49 (dd (J 4.5 Hz, J 14.8 Hz), 1H, aCH2), 4.21 (dd (J 7.7 Hz,
J 14.8 Hz), 1H, aCH2), 8.33/8.36 (dd, (J 4.7 Hz, J 7.4 Hz), 1H,
aNH); Asp, d: 2.63/2.66 (dd (J 6.7 Hz, J 16.4 Hz), 1H, bCH2),
2.79/2.83 (dd (J 7.7 Hz, J 16.4 Hz), 1H, bCH2), 4.73 (m, 1H, aCH),
8.12 (d, (J 8.8 Hz), 1H, aNH); D-Phe, d: 2.93/2.98 (dd (J 10.3 Hz,
J 13.2 Hz), 1H, bCH2), 3.07/3.10 (dd (J 5.9 Hz, J 13.2 Hz), 1H,
bCH2), 4.45 (m, 1H, aCH), 7.25 (d (J 7.3 Hz), 2H, arom H), 7.33–
7.38 (m, 3H, arom H), 8.42 (d (J 5.9 Hz), 1H, aNH); azido Lys,
d: 0.95 (m, 2H, cCH2), 1.46/1.65 (double m, 2H, bCH2), 1.49 (m,
2H, dCH2), 3.24 (t (J 7.1 Hz), 2H, eCH2), 3.85 (m, 1H, aCH), 8.44
(d (J 5.6 Hz), 1H, aNH); 13C NMR (H2O–D2O 9 : 1 v/v, 293 K,
6.4 mM, pH 4): Arg, d: 29.8 cC, 30.0 bC, 43.3 dC, 55.2 aC, 176.0
aCO, 176.3 guanidino C; Gly, d: 46.3 aC, 172.9 aCO; Asp, d: 38.5
bC, 52.9 aC, 175.1 aCO, 178.6 bCO; D-Phe, d: 39.6 bC, 58.1 aC,
130.0 arom CH, 131.5 arom CH, 131.9 arom CH, 138.8 arom qC,
176.5 aCO; azido Lys, d: 25.1 cC, 27.2 dC, 32.6 bC, 53.3 eC, 58.2
aC, 177.9 aCO; MS analysis: calcd for C27H39N11O7, 629.30, found
ES-MS 630.55 [M + H]+, 652.70 [M + Na]+, 668.25 [M + K]+.
12.1 min (C18); MS analysis: calcd for C85H120N28O24, 1918.067
+
(Mave ), found MALDI-TOF 1918.431 [M + H]ave
.
DOTA-conjugated tetravalent cyclo[RGDfK] peptide dendrimer
(25). Alkyne 16 (3.8 mg, 3.0 lmol) and azido peptide 19
(11 mg, 14.8 lmol, 1.2 equiv) were dissolved in DMF (500 lL)
and to this solution, 0.05 M Na-ascorbate (30 lL, 1.5 lmol,
0.50 equiv) followed by 6 mM CuSO4·5H2O (25 lL, 0.15 lmol,
0.05 equiv) were added. The obtained reaction mixture was heated
by microwave irradiation to 100 ◦C for 2 × 5 min. Then, the
solvents were removed under reduced pressure and the residue
was dissolved in tert-BuOH–H2O 1 : 1 v/v and lyophilized. The
obtained fluffy solid was dissolved in TFA–H2O (1 mL; 95 : 5 v/v)
and stirred for 4 h at room temperature. Subsequently, the reaction
mixture was concentrated in vacuo and the residue was redissolved
in tert-BuOH–H2O 1 : 1 v/v, lyophilized and purified by semi-
prep HPLC (C18) to give compound 25 in 36% yield (3.9 mg). Rt:
11.6 min (C18); MS analysis: calcd for C163H220N52O44, 3611.873
+
(Mave ), found MALDI-TOF 3612.646 [M + H]ave
.
General procedure for the microwave-assisted click reaction.
The alkyne (1 equiv) and the azide (1.3 equiv per arm)
Acknowledgements
22
1
We thank Dr Hans Ippel for measuring the 500 MHz H NMR
spectra of compound 19.
were dissolved in DMF–H2O. To this solution, CuSO4·5H2O
(0.05 equiv) and Na-ascorbate (0.50 equiv) were added. The
reaction mixture was placed in a microwave reactor and irradiated
during 10–30 min at 100 ◦C. The cycloaddition was monitored on
TLC and LC-MS for completion of the reaction.
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13, 3763; (e) I. Dijkgraaf, J. A. W. Kruijtzer, C. Frielink, A. C. Soede,
(C18); MS analysis: calcd for C55H79N17O16, 1234.340 (Mave ), found
+
MALDI-TOF 1234.807 [M + H]ave
.
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Org. Biomol. Chem., 2007, 5, 935–944 | 943
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