PAMAM structure-based multifunctional fluorescent conjugates for improved fluorescent labelling of biomacromolecules

Article abstract of DOI:10.1002/chem.200800328

Fluorescent probes are of increasing interest in medicinal and biological applications for the elucidation of the structures and functions of healthy as well as tumour cells. The quality of these investigations is determined by the intensity of the fluorescence signal. High dye/carrier ratios give strong signals. However, these are achieved by the occupation of a high number of derivatisation sites and therefore are accompanied by strong structural alterations of the carrier. Hence, polyvalent substances containing a high number of fluorescent dyes would be favourable because they would allow the introduction of many dyes at one position of the compound to be labelled. A large number of different dyes have been investigated to determine the efficiency of coupling to a dendrimer scaffold and the fluorescence properties of the oligomeric dyes, but compounds that fulfil the requirements of both strong fluorescence signals and reactivities are rare. Herein we describe the synthesis and characterisation of dye oligomers containing dansyl-, 7-nitro-2,l,3-benzoxadiazol-4yl- (NBD), coumarin-343, 5(6)-carboxyfluorescein and sulforhodamine B2 moieties based on polyamidoamine (PAMAM) dendrimers. The PAMAM dendrimers were synthesised by an improved protocol that yielded highly homogeneous scaffolds with up to 128 conjugation sites. When comparing the fluorescent properties of the dye oligomers it was found that only the dansylated dendrimers met the requirements of enhanced fluorescence signals. The dendrimer containing 16 fluorescent dyes was conjugated to the anti-epidermal-growth-factor receptor (EGFR) antibody hMAb425 as a model compound to show the applicability of the dye multimer compounds. This conjugate revealed a preserved immunoreactivity of 54 %. We demonstrate the applicability of the dye oligomers to the efficient and applicable labelling of proteins and other large molecules that enables high dye concentrations and therefore high contrasts in fluorescence applications.

Full text of DOI:10.1002/chem.200800328

DOI: 10.1002/chem.200800328
PAMAM Structure-Based Multifunctional Fluorescent Conjugates
for Improved Fluorescent Labelling of Biomacromolecules
C. Wängler, G. Moldenhauer, R. Saffrich, E.-M. Knapp, B. Beijer, M. Schnçlzer,
B. Wängler, M. Eisenhut, U. Haberkorn, and W. Mier*[a]
Abstract: Fluorescent probes are of in-
creasing interest in medicinal and bio-
logical applications for the elucidation
of the structures and functions of
healthy as well as tumour cells. The
quality of these investigations is deter-
mined by the intensity of the fluores-
cence signal. High dye/carrier ratios
give strong signals. However, these are
achieved by the occupation of a high
number of derivatisation sites and
therefore are accompanied by strong
structural alterations of the carrier.
Hence, polyvalent substances contain-
ing a high number of fluorescent dyes
would be favourable because they
would allow the introduction of many
the efficiency of coupling to a dendri-
mer scaffold and the fluorescence
properties of the oligomeric dyes, but
compounds that fulfil the requirements
of both strong fluorescence signals and
reactivities are rare. Herein we de-
scribe the synthesis and characterisa-
tion of dye oligomers containing
dansyl-, 7-nitro-2,1,3-benzoxadiazol-4-
yl- (NBD), coumarin-343, 5(6)-carb-
conjugation sites. When comparing the
fluorescent properties of the dye oligo-
mers it was found that only the dansy-
lated dendrimers met the requirements
of enhanced fluorescence signals. The
dendrimer containing 16 fluorescent
dyes was conjugated to the anti-epider-
mal-growth-factor receptor (EGFR)
antibody hMAb425 as a model com-
pound to show the applicability of the
dye multimer compounds. This conju-
gate revealed a preserved immunoreac-
tivity of 54%.
We demonstrate the applicability of
the dye oligomers to the efficient and
applicable labelling of proteins and
other large molecules that enables high
dye concentrations and therefore high
contrasts in fluorescence applications.
ACHRTEoUNG xyCAHTREfUGN luorescein and sulforhodamine B2
moieties based on polyamidoamine
(PAMAM) dendrimers. The PAMAM
dendrimers were synthesised by an im-
proved protocol that yielded highly ho-
mogeneous scaffolds with up to 128
dyes at
ACHTREUNGone position of the compound
Keywords: biomacromolecules
dansyl chloride dendrimers
fluorescence · fluorescent probes
·
·
to be labelled.
ACHTREUNG
·
A large number of different dyes
have been investigated to determine
Introduction
ing, localisation, transport of arbitrary genes),[1] interactions
between proteins,[2] the influence, activity and function of
tumour-suppressor genes,[3,4] imaging of apoptosis by the de-
termination of caspase activity,[5] determination of chromatin
elimination,[6] dynamics of chromatin interactions[7] and the
activation of genes.[8] In oncology, in vitro fluorescence la-
belling techniques have become increasingly interesting be-
cause they allow the determination of tumour malignancy,[9]
chromosomal abnormalities,[10] metastatic disposition of tu-
mours,[11] tumour oxygenation and pH,[12] tumour-induced
angiogenesis,[13] gene deletions, duplications and amplifica-
tions[14] and also apoptosis versus necrosis imaging in cancer
cell lines.[15]
In the past decades, fluorescent dyes have increasingly
gained importance for various applications in biology and
medicine. They are widely used for non-invasive visualisa-
tion on the anatomic as well as on the cellular level.
Their numerous applications in vitro include the identifi-
cation of genotypes (mutations, expression, spatial process-
[a]Dr. C. Wängler, Dr. G. Moldenhauer, Dr. R. Saffrich, E.-M. Knapp,
Dr. B. Beijer, Dr. M. Schnçlzer, Dr. B. Wängler, Prof. M. Eisenhut,
Prof. Dr. U. Haberkorn, Dr. W. Mier
Universitätsklinikum Heidelberg
Im Neuenheimer Feld 400
69120 Heidelberg (Germany)
An emerging application of fluorescent compounds is di-
agnostic imaging in vivo, which allows an estimation of the
metastatic disposition of tumours,[16] the detection of
tumour-associated enzymes[17] and single cells in living or-
Fax : (+49)6221-565473
E-mail: walter.mier@med.uni-heidelberg.de
8116
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

FULL PAPER
ganisms,[18,19] the imaging of angiogenesis,[20] intra-operative
sentinel lymph node mapping,[21–23] the determination of pro-
tein kinase activity,[24] apoptosis imaging,[25] the functional
state of cells and the detection of tumour cells.[26]
which were analysed by HPLC and MALDI-TOF mass
spectrometry and showed high polydispersities of the com-
pounds.[46]
Therefore, these compounds were not chosen as building
blocks for the syntheses. Attempts to synthesise PAMAM
scaffolds according to literature methods[47–49] resulted in
products with higher quality but they also did not meet the
homogeneity requirements.
Therefore, improved synthesis protocols were developed
to achieve the low dispersity required. As reported
before,[50] a substantial modification of the reaction tempera-
tures had to be made to obtain highly homogeneous prod-
ucts: when synthesising the half and full generations by re-
action with ethylenediamine special care had to be taken
that the reaction temperature did not rise above room tem-
perature and 48C, respectively. Reactions that were not con-
trolled showed a high degree of fragmentation, elimination
and intramolecular ring formation and as a consequence a
high heterogeneity of the products. In contrast, the dendri-
A disadvantage of fluorescent labels over radioactive
labels is their relatively low sensitivity. Effective imaging
relies on fluorescent labels that produce strong signals and
thus high signal-to-noise ratios. Their applications are limit-
ed by the fluorescent properties of the particular dye. To
further improve the potential of fluorescent labels, it would
be advantageous to develop dyes that produce strong fluo-
rescence signals but do not lead to strongly altered proper-
ties of the compound to be labelled. For this purpose poly-
fluorescent dyes based on polymeric structures would be
suitable. These compounds should allow a facilitated intro-
duction into the compound to be labelled without severe
structural alteration of the carrier. The multimeric fluores-
cent substances synthesised so far are merely used as che-
mosensors for H+ or metal ions,[27–31] optical brighteners[32,33]
or potential compounds for organic light-emitting
diodes[34,35] because they are either insoluble in water-con-
taining systems or cannot be introduced into a biocompat-
ible carrier molecule.
Dendrimers, which represent a class of homogeneous mol-
ecules with a symmetrically branching structural motif, have
been shown to be suitable for the linkage of several effec-
tors. They have, for example, been shown to be effective
drug delivery vehicles for oligonucleotides,[36] methotrex-
ate,[37] folic acid,[38] taxol,[38] methylprednisolone,[39] doxoru-
bicin[40] and boron-rich compounds for boron neutron cap-
ture therapy (BNCT)[41–43] and are even the basis for ap-
proved pharmaceuticals such as the microbicide Vivagel[44]
and the diagnostic cardiac marker Stratus.[45]
AHCTREmUNG ers synthesised by the modified protocol were analysed by
HPLC and mass spectrometry, which showed they were ob-
tained in good yields and high purity (Figure 1).
A second crucial point that has to be considered to ensure
the quality of the products is the purification of the den-
Thus, well-characterised dendritic substances containing a
high number of fluorescent dyes and a functional group for
selective coupling to a carrier molecule should be advanta-
geous to the introduction of a high number of dyes concen-
trated at one position of the compound to be labelled.
Therefore, high dye concentrations that produce strong fluo-
rescence signals should become assessable for improved
fluorescence labelling.
Herein we describe the synthesis and characterisation of
polyfluorescent dendrimers containing a conjugation site for
the labelling of macromolecules. Their suitability as fluores-
cent-labelling probes is shown by the conjugation of an ex-
emplary dansylated dendrimer to the anti-epidermal-
growth-factor receptor (anti-EGFR) antibody hMAb425.
Results and Discussion
Improvement of polyamidoamine (PAMAM) dendrimer
synthesis: In general, a major advantage of dendrimers over
polymers is the low dispersity of the products. However, the
homogeneity of commercially available dendrimers was
found to be low. Similar results have recently been de-
scribed for commercially available PAMAM dendrimers,
Figure 1. HPLC chromatograms of the G3 and G4 dendrimers containing
8 (A) and 16 (B) amino functions synthesised on an S-trityl-mercaptoeth-
yl-pentaethylene-glycol-ethylamine core, as described in the Experimen-
tal Section. Chromatograms were obtained by using a Chromolith Perfor-
mance column and a gradient from 20–45 and 0–60% acetonitrile in
5 min, ACHTRErUGN espectively.
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8117

W. Mier et al.
drimers. Attempts to purify the reaction mixtures of the
higher generation dendrimers by column chromatography,
ultrafiltration or dialysis resulted in low yields or impure
products. In contrast to this, HPLC purification provided
the best results, yielding products of high homogeneity.
To be suitable for selective introduction into proteins and
other macromolecules that are to be conjugated with high
efficiency, the polyfluorescent dendrimers have to contain a
reactive site for selective coupling reactions. Therefore the
PAMAM dendrimers were synthesised on a pentaethylene
glycol linker containing a trityl-protected thiol. The linker
was introduced to increase the accessibility of the free thiol
during the coupling reaction. The PAMAM dendrimers
were grown on the terminal amino function of the linker by
alternating the reaction with methyl acrylate and ethylenedi-
amine to give half and full generations of the dendrimer
(Scheme 1).
Derivatisation of the PAMAM dendrimers with fluorescent
dyes: The synthesis of the dendrimer was followed by the
derivatisation of the amino functions with fluorescent dyes.
A large number of different fluorescent dyes were tested
with respect to their reactivity with the terminal amino func-
tions of the PAMAM dendrimer. When introducing two or
more dyes, a high variation of the coupling efficiencies
could be observed. This low reactivity can be attributed to
steric hindrance because it has been observed that smaller
fluorescent dyes can be coupled with higher efficiencies and
give more homogeneous products than bulky ones. Poor
coupling yields were observed for sulforhodamine Q4 fluo-
ride, sulforhodamine Q5 fluoride, sulforhodamine B chlo-
ride, rhodamine B isothiocyanate, rhodamine 101, rhoda-
mine 101 aminohexanoic acid, fluorescein isothiocyanate, 5-
carboxyfluorescein, naphthofluorescein, 6-[fluorescein-5(6)-
carbamido]hexanoic acid, NIR 820 chloride,[53] NIR 820 iso-
thiocyanate, NIR 820 thiol, NIR 797 chloride,[54] NIR 797
aminohexanoic acid, Py-5[55] and Py-5 aminohexanoic acid.
The reactions with these dyes led to dendritic structures that
were not fully derivatised or heterogeneous mixtures that
could not be purified (Table 1).
Table 1. Yields from the coupling of the fluorescent dyes to dendrimers
containing one (G0), two (G1) and four (G2) amino groups.
Fluorescent dye
Yield[a] [%]
G1
G0
G2
sulforhodamine Q4 fluoride
sulforhodamine Q5 fluoride
sulforhodamine B chloride
rhodamine B isothiocyanate
rhodamine 101
rhodamine 101 aminohexanoic acid
fluorescein isothiocyanate
5-carboxyfluorescein
naphthofluorescein
6-[fluorescein-5(6)-carbamido]hexanoic acid
NIR 820 chloride
NIR 820 isothiocyanate
NIR 820 thiol
NIR 797 chloride
NIR 797 aminohexanoic acid
Py-5
Py-5 aminohexanoic acid
dansyl chloride
NBD chloride
63 (45)[b]
2
2
2
3
2
4
56
3
6
0
0
0
0
0
0
0
0
0
27
0
0
0
0
0
0
40
15
45 (55)[b]
32 (39)[b]
70
68 (49)[b]
45 (60)[b]
13
5
73 (79)[b] 52
42 (20)[b]
3
2
8
5
7
74
12
12
52
Scheme 1. Synthesis of the pentaethylene glycol linker and the PAMAM
dendrimers.
39 (44)[b] 56
67 (26)[b] 49
63[c]
43[c]
48[c]
58[c] 40[c]
63[c] 46[c]
54[c] 56[c]
89[c] 81[c]
76[c] 46[c]
To synthesise the linker, tetraethylene glycol ditosylate[51]
was first treated with S-tritylmercaptoethanol[52] and subse-
quently with 2-(2-aminoethoxy)ethanol. The PAMAM den-
drimers were subsequently built by alternating the reaction
with methyl acrylate and ethylenediamine.
coumarin-343
5(6)-carboxyfluorescein pentafluorophenyl ester 89[b]
sulforhodamine B2 acid fluoride
71[c]
[a]Yields [%]for the coupling reaction with dendrimers G 0, G1 and G2.
[b]Monosubstituted. [c]Yields of isolated products.
By these methods, we were able to synthesise PAMAM
dendrimers of up to 128 amino functions on a pentaethylene
glycol linker that contains a thiol for selective coupling to
antibodies or other macromolecules. The dendrimers ob-
tained were found to be of high homogeneity and are there-
fore suitable as building blocks for the synthesis of the fluo-
rescent dye oligomers.
Five fluorescent dyes, dansyl chloride, 7-nitro-2,1,3-ben-
zoxadiazol-4-yl (NBD) chloride, coumarin-343, 5(6)-carb-
ACHRTEoGNU xyACHTREfUGN luorescein pentafluorophenyl ester and sulforhodamine
B2 acid fluoride, could be introduced into G0, G1 and G2
dendrimers containing one, two and four amino functions,
respectively. Comparison of the fluorescence properties of
the dendrimers derivatised with these dyes should reveal the
8118
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

Fluorescent Labelling of Biomacromolecules
FULL PAPER
By using dansyl chloride as
the fluorescent dye, the amino
functions of the dendritic sys-
tems could be derivatised quan-
titatively to yield highly homo-
geneous products. The com-
pounds obtained produce fluo-
rescence signals that vary
roughly as a function of the
number of dyes per dendrimer
with only a minor self-quench-
ing of the fluorescence.
Conjugation of the dansyl16-
PAMAM
dendrimer
to
hMAb425: To show the applica-
bility of the synthesised mole-
cules for fluorescence labelling
of antibodies and other macro-
molecules, the dansyl16-SH den-
drimer was conjugated to the
anti-EGFR antibody hMAb425
Figure 2. Fluorescence spectra of the compounds containing one, two and four amino functions derivatised
with NBD chloride, coumarin-343, 5(6)-carboxyfluorescein pentafluorophenyl ester and sulforhodamine B2
acid fluoride.
(Matuzumab) as
AHCTREcGNU ompound.
a
model
most suitable compounds for fluorescence imaging
ACHTREUNG
with NBD chloride, coumarin-343, 5(6)-carboxyfluorescein
pentafluorophenyl ester and sulforhodamine B2 acid fluo-
ride unambiguously revealed that the fluorescence intensi-
ties decreased with increasing number of coupled dyes
(Figure 2). This has been attributed to the self-quenching of
the fluorescence, which is due to the relatively small Stokes
shift of the compounds and their small distance from each
other within the molecules.[56]
In contrast, the compounds derivatised with dansyl chlo-
ride showed only relatively low self-quenching. This is due
to the large Stokes shift of the dansyl dye of approximately
195 nm. As dansyl chloride is the only fluorescent dye pres-
ently available that shows a sufficiently large Stokes shift
and because the dansylated dendrimers show an increase in
fluorescence with higher numbers of dyes, larger generations
of dendrimers were synthesised with an increasing number
of dyes. The fluorescence intensities of the dye monomer
and the multivalent dyes containing 1–128 dansyl moieties
are shown in Figure 3. In these dendrimers the fluorescence
intensity varies roughly as a function of the number of dyes
per dendrimer.
To enable a dansyl quantisation that cannot be influenced
by self-quenching effects, the number of dyes per dendrimer
was determined by MALDI-TOF MS and NMR spectrosco-
py. The results of these measurements are listed in Table 2.
The accuracy of the MS and NMR data for the largest
multimeric system containing 128 dansyl moieties was rela-
tively low. Thus, the results of these analyses are not given.
Figure 3. Fluorescence spectra of the dye monomer and the multivalent
dyes containing 1, 2, 4, 8, 16, 32, 64 and 128 dansyl moieties.
Therefore, the antibody was derivatised with sulfo-SMCC
(sulfo-SMCC=sulfosuccinimidyl-4-(N-maleimidomethyl)cy-
clohexane-1-carboxylate) to introduce maleimide functions.
The conjugation reaction was performed in phosphate
buffer with a seven-fold excess of the maleimide-N-hydroxy-
succinimide ester following the protocol of Samoszuk
et al.[57] with minor modifications.
The number of maleimides per antibody molecule was de-
termined by treating the maleimides with an excess of
MESNA (MESNA=sodium 2-mercaptoethanesulfonate).
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8119

W. Mier et al.
Table 2. Characterisation of the dansylated dendrimers by MS and NMR
spectroscopy.
rescence signals when used for optical imaging by exhibiting
only a minor structural alteration compared with a polysub-
stituted antibody molecule.
To prove the exact ratio of one fluorescent dendritic
structure per protein, the conjugate was characterised by
mass spectrometry (Figure 5).
Generation
(number of
amine func-
tions)
Theoretical mass Number of
of the dansylated dansyl moieties
Number of dansyl
moieties deter-
mined by NMR
compound
determined by
MS
G0 (1)
G1 (2)
G2 (4)
G3 (8)
G4 (16)
G5 (32)
G6 (64)
816
1278
1958
4048
7736
1.0
2.0
4.0
8.0
14.9
28.4
–
1.0
2.0
3.9
7.8
15.9
31.2
59.7
15111
28967
The amount of reacted thiol corresponds to the amount of
maleimide and was found to be between 1.4 and 1.7 per an-
tibody. This low number of maleimides represents a minor
derivatisation of the antibody molecule, and therefore, it
should not influence the integrity of the protein. Despite
this low number of reactive sites, an efficient coupling reac-
tion with the thiol-containing dendrimer was possible when
the maleimide was allowed to react with a 25-fold excess of
the dendritic thiol. The size-exclusion chromatogram of the
reaction mixture of the maleimide-derivatised antibody with
dansyl16-SH is given in Figure 4. The derivatised antibody
Figure 5. Mass spectra of underivatised hMAb425 and hMAb425 cova-
lently linked to dansyl16-SH. The shift of 7661.6 Da of the derivatised an-
tibody molecule with one and two charges corresponds to the introduc-
tion of exactly one fluorescent dendrimer into each protein.
To prove that the functionality of the antibody is pre-
served, its immunoreactivity was determined. HT29 cells
were incubated for 1 h with the untreated antibody or the
dansyl16-antibody conjugate at concentrations of 0.001,
0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 mgmLÀ1. The amount of an-
tibody or antibody conjugate bound to the cells was quanti-
fied by incubating the cells with a secondary fluorescein iso-
thiocycanate (FITC)-labelled F(ab)’2 goat-anti-human anti-
body for 1 h. The fluorescence of the secondary antibody
was quantified by fluorescence-activated cell sorting (FACS)
analysis.
By plotting the mean fluorescence intensity (MFI) versus
the antibody concentration, binding curves could be ob-
tained for the untreated antibody as well as for the dansyl16-
antibody conjugate. From these curves, the immunoreactivi-
ty of the dansylated antibody conjugate and the untreated
antibody could be determined.
It was found that the immunoreactivity of the dansyl16-an-
tibody conjugate was 54% of that of the non-derivatised
protein, which shows that the dendritic system exerts a
minor modification. The dansyl1-antibody conjugate synthes-
ised for comparison showed a preserved immunoreactivity
of 61%. An antibody containing 16 isolated dansyl moieties
could not be obtained because the protein lost its integrity
upon derivatisation.
The relatively highly preserved immunoreactivities of the
dansyl1- and dansyl16-antibody conjugates show that the
dansyl multimers exert only a minor effect on the biological
properties of the antibody molecule. In contrast, the intro-
duction of 16 isolated dansyl moieties leads to a loss of in-
tegrity of the antibody, which shows the superiority of the
approach of introducing multimers into an antibody mole-
Figure 4. Size-exclusion chromatogram of the reaction mixture of sulfo-
SMCC-derivatised hMAB425 with an excess of dansyl16-SH obtained by
using a Superdex 75 10/300 GL column with 0.1m phosphate buffer
(pH 7.0) as eluent at a flow rate of 1 mLminÀ1. The FPLC analysis was
performed after a reaction time of 5 min.
was isolated by size-exclusion chromatography and desalted
by using a NAP-5 column.
The molar extinction coefficient of the antibody contain-
ing 16 dansyl moieties was determined and compared with a
conjugate containing only one dansyl dye. A molar extinc-
tion coefficient (e) of (31874.94Æ644.16) LmolÀ1 cmÀ1 was
found. In the case of the conjugate containing one dansyl
dye, e was determined to be (3092.95Æ92.82) LmolÀ1 cmÀ1,
which is comparable to other dansyl-labelled proteins.[58]
Consequently, the antibody labelled with the dendrimer con-
taining 16 dansyl moieties should give much stronger fluo-
8120
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

Fluorescent Labelling of Biomacromolecules
FULL PAPER
cule, which leads to only a minor structural modification
and therefore to a minor decrease in immunoreactivity.
To show the applicability of the antibody-multimer conju-
gates and the multimerisation concept, fluorescence micro-
scopy studies of the dansyl1- and dansyl16-antibody conju-
gates were carried out by using A431 cells. The results of
these studies are shown in Figure 6. The images show a sig-
The majority of the dyes studied were found to be unsuit-
able for this purpose. However, we have demonstrated the
conjugation of dansyl chloride, NBD chloride, coumarin-
343, 5(6)-carboxyfluorescein pentafluorophenyl ester and
sulforhodamine B2 acid fluoride to dendritic structures con-
taining up to 128 conjugation sites. Analysis of the fluores-
cence properties of these substances showed that the synthe-
sis of fluorescent oligomers producing favourably strong
fluorescence signals is possible. As shown here, dansyl-oligo-
mers based on dendritic structures are highly effective inten-
sive fluorescent probes and can be coupled efficiently to
biomacromolecules, for example, antibodies, with a highly
preserved functionality of the carrier that enables high dye
concentrations and the production of strong fluorescence
signals for optical imaging applications.
Because of the favourable signal amplification that can be
achieved, oligomeric dyes are a valuable tool for macromo-
lecule labelling. However, as the dansyl moiety shows sub-
optimal fluorescence properties, this approach could be fur-
ther improved by the development of a monomeric near-in-
frared-emitting fluorescent dye that has a higher molar ex-
tinction coefficient and a larger quantum yield than dansyl
but a similarly high reactivity.
Experimental Section
General: All commercially available chemicals were of analytical grade
and used without further purification.
The analytical HPLC system used was an Agilent 1100 system and a
Chromolith Performance (RP-18e, 100”4.6 mm, Merck, Germany)
column. Semipreparative HPLC purifications were performed with a
Gyncotech P-580 system (Germering, Germany) equipped with a varia-
ble SPD 6A UV detector and a C-R5A integrator (both Shimadzu, Duis-
burg, Germany). The column used was a Chromolith (RP-18e, 100”
10 mm, Merck, Germany).
Figure 6. Fluorescence microscopy images of A431 cells treated with the
dansyl1-antibody conjugate (top) and the dansyl16-antibody conjugate
(bottom).
Fluorescence spectra were obtained by using an AMINCO-Bowman
Series 2 Luminescence Spectrometer (Thermo Electron Corporation).
MALDI-TOF-TOF mass spectrometry was performed by using a Kratos
Analytical Compact Maldi III system or a Bruker-Daltonik Reflex II
time-of-flight instrument (Bremen, Germany). ESI mass spectra were ob-
nificant increase in the fluorescence signal produced by the
dansyl16-antibody conjugate (bottom) relative to the dansyl1-
antibody conjugate (top). This demonstrates that even
though dansyl has no optimal fluorescence properties, the
concept of dye multimerisation can be exploited to obtain
strongly enhanced fluorescence signals of labelled biomacro-
molecules.
tained by using
a Triple-Quadrupole TSQ 7000 mass spectrometer
(Thermo Fisher Scientific, Bremen). NMR spectra were recorded by
using Varian Mercury Plus 300 MHz and Varian NMR System 500 MHz
spectrometers.
Thin-layer chromatography was performed by using Polygram SIL G/
UV254 TLC plates (Macherey-Nagel, Düren, Germany).
Size-exclusion gel chromatography was carried out by using NAP-5 col-
umns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and size-ex-
clusion fast protein liquid chromatography (FPLC) was performed on a
Superdex 200 10/30 GL column (Amersham Biosciences AB, Uppsala,
Sweden).
Conclusion
Fluorescence microscopy images were acquired by using an Olympus
IX70 inverted fluorescence microscope equipped with a digital image ac-
quisition and processing system (analySIS 3.2, Soft Imaging System,
Münster, Germany). The appropriate fluorescence filter set for DAPI
was used for the detection of dansyl-labelled antibodies.
Fluorescent dyes are highly important for various applica-
tions in biology and medicine. Due to the fact that imaging
is only possible with dyes that produce strong fluorescence
signals, these applications are limited by the fluorescent
properties of the particular dye. Therefore, the development
of new oligomeric fluorescent labelling compounds that pro-
duce strong fluorescence signals is desirable.
S-Trityl-mercaptoethyl-tetraethylene glycol tosylate: Powdered KOH
A
45 mmol), S-tritylmercaptoethanol (10 g, 31 mmol) and tetrabutylammo-
nium bromide (TBAB; 1.5 g, 4.5 mmol) in toluene (200 mL) After 2 h
the mixture was filtered and the resulting solution evaporated. The re-
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8121

W. Mier et al.
maining oil was purified by chromatography on silica gel with n-hexane/
acetone 2:1 as eluent (Rf =0.4). The product was obtained as a light-
yellow oil (11 g, 17 mmol, 55%).
ature. Subsequently the ethylenediamine was removed by evaporation.
The product was clean enough for further use and was obtained as a
yellow oil (13.9 g, 17.2 mmol, 100%).
1H NMR ([D6]DMSO): d=7.31 (m, 12H; 2-H+3-H), 7.24 (m, 3H; 1-H),
3.47–3.33 (m, 24H; 7-H to 18-H), 3.21 (t, 3J=6.7 Hz, 4H; 20-H), 3.03 (q,
3J=6.1 Hz, 4H; 19-H), 2.64 (t, 3J=7.2 Hz, 4H; 23-H), 2.54 (m, 10H; 21-
H, 23-H, 26-H), 2.27 (t, 3J=6.7 Hz, 2H; 6-H), 2.17 ppm (t, 3J=7.2 Hz,
4H; 25-H); 13C NMR ([D6]DMSO): d=172.30 (C-22), 144.34 (C-4),
129.06 (C-2), 127.89 (C-3), 126.59 (C-1), 69.69, 69.66, 69.64, 69.53, 69.50,
69.37, 68.60, 68.44, 65.91 (C-7 to C-18), 52.20 (C-20), 48.80 (C-24), 48.56
(C-25), 36.51 (C-19), 33.13 (C-21), 31.25 ppm (C-6); MS (MALDI-TOF):
m/z calcd for [M+H]+: 812.5; found: 810.8.
1H NMR ([D6]DMSO): d=7.80–7.75 (m, 2H; 4-H), 7.49–7.44 (m, 2H; 3-
H), 7.33–7.20 (m, 15H; 18-H to 20-H), 4.12–4.08 (m, 2H; 6-H), 3.57–3.53
(m, 2H; 7-H), 3.45 (brs, 4H; 10-H+11-H), 3.42 (brs, 4H; 8-H+9-H),
3.35–3.26 (m, 4H; 12-H+13-H), 3.23 (t, 3J=6.6 Hz, 2H; 14-H), 2.40 (s,
3H; 1-H), 2.28 ppm (t, 3J=6.6 Hz, 2H; 15-H); 13C NMR ([D6]DMSO):
d=150.37 (C-2), 138.09 (C-5), 135.64 (C-3), 134.67 (C-19), 133.53 (C-4),
133.12 (C-18), 132.25 (C-17), 75.94, 75.48, 75.31, 75.28, 75.20, 75.12, 75.06,
74.13, 73.44, 71.63 (C-6 to C-14), 36.97 (C-15), 26.62 ppm (C-1); MS
(MALDI-TOF): m/z calcd for [M+H]+: 651.2; found: 650.2.
G1.5 dendrimer: A solution of the G1 dendrimer (12.5 g, 15.4 mmol) in
methanol (30 mL) was added to methyl acrylate (280 mL, 3.1 mol) and
stirred for 3 d at room temperature. The volatile components were
evaporated and the crude product was purified by chromatography on
silica gel with ethanol/acetone (5:6) as eluent (Rf =0.4). The product was
obtained as a light-yellow oil (6.5 g, 5.6 mmol, 37%).
S-Trityl-mercaptoethyl-pentaethylene glycol-ethylamine (G0 dendrimer):
Powdered KOH (ꢀ5 g) was added to a solution of S-trityl-mercaptoeth-
yl-tetraethylene glycol-tosylate (28.5 g, 59 mmol) and 2-(2-aminoeth-
ACHTREUNGoxy)ethanol (31 g, 296 mmol) in toluene (100 mL) and DMF (10 mL).
After 5 h the mixture was filtered and the resulting solution evaporated.
The remaining oil was purified by chromatography on silica gel with
CHCl3/ethanol (5:2 as eluent (Rf =0.37)). The product was obtained as a
yellow oil (15.5 g, 27 mmol, 45%).
1H NMR ([D6]DMSO): d=7.32 (d, 3J=4.4 Hz, 12H; 2,3-H), 7.24 (m,
3H; 1-H), 3.49–3.32 (m, 24H; 8-H to 18-H+20-H); 3.22 (t, 3J=6.6 Hz,
2H; 7-H), 2.69 (t, J=5.7 Hz, 2H; 19-H), 2.27 ppm (t, J=6.5 Hz, 2H; 6-
H); 13C NMR ([D6]DMSO): d=145.13 (C-4), 129.76 (C-2), 128.66 (C-3),
127.35 (C-1), 70.47, 70.44, 70.31, 70.29, 70.19, 69.38, 69.24, 66.73 (C-7 to
C-18), 41.49 (C-19), 32.08 ppm (C-6); MS (MALDI-TOF): m/z calcd for
[M+H]+: 584.3; found: 582.8, 605.3, 621.2.
1H NMR ([D6]DMSO): d=7.33–7.31 (m, 12H; 2-H+3-H), 7.26–7.21 (m,
3H; 1-H), 3.56 (s, 12H; 29-H), 3.48–3.39 (m, 22H; 8-H to 18-H), 3.21 (t,
3J=6.6 Hz, 2H; 7-H), 3.07–3.01 (q, 3J=6.3 Hz, 4H; 24-H), 2.69–2.62 (m,
16H; 20-H+25-H+26-H), 2.54 (t, 3J=6.1 Hz, 2H; 19-H), 2.43–2.36 (m,
12H; 21-H+27-H), 2.27 ppm (t, 3J=6.6 Hz, 2H; 6-H); 13C NMR
([D6]DMSO): d=172.35 (C-22), 171.02 (C-28), 144.37 (C-4), 129.08 (C-
2), 127.91 (C-3), 126.61 (C-1), 69.70, 69.68, 69.65, 69.59, 69.52, 69.40,
68.61, 68.45, 65.95 (C-7 to C-18), 55.93 (C-25), 52.21 (C-20), 51.09 (C-29),
49.85 (C-26), 48.80 (C-24), 36.53 (C-19), 33.16 (C-21), 31.93 (C-27),
31.28 ppm (C-6); MS (ESI): m/z calcd for [M+H]+: 1156.6; found:
1156.8.
3
3
G0.5 dendrimer: A solution of the G0 Dendrimer (14.5 g, 25 mmol) in
methanol (20 mL) was added to methyl acrylate (450 mL, 5.0 mol) and
stirred for 3 d at room temperature. The volatile components were
evaporated and the crude product was purified by chromatography on
silica gel with n-hexane/acetone (4:5) as eluent (Rf =0.55). The product
was obtained as a light-yellow oil (12 g, 16 mmol, 64%).
G2 dendrimer: A solution of the G1.5 dendrimer (6.5 g, 5.6 mmol) in
methanol (20 mL) was added dropwise to a cooled solution of ethylene-
diamine (600 mL, 9 mol) over 0.5 h and stirred for 5 d at 48C. Subse-
quently the ethylenediamine was evaporated. The product was clean
enough for further use and was obtained as a yellow oil (6.7 g, 5.3 mmol,
95%).
1H NMR ([D6]DMSO): d=7.33–7.30 (m, 12H; 2-H+3-H), 7.27–7.21 (m,
3H; 1-H), 3.55 (s, 6H; 23-H), 3.48–3.31 (m, 22H; 8-H to 18-H), 3.22 (t,
3J=6.6 Hz, 2H; 7-H), 2.69 (t, 3J=6.9 Hz, 4H; 20-H), 2.54 (t, 2H; 3J=
6.1 Hz, 2H; 19-H), 2.38 (t, 3J=6.9 Hz, 4H; 21-H), 2.27 ppm (t, 2H; 3J=
6.6 Hz, 6-H); 13C NMR ([D6]DMSO): d=172.20 (C-22), 145.10 (C-4),
129.75 (C-2), 128.68 (C-3), 127.34 (C-1), 70.48, 70.45, 70.31, 70.16, 69.38,
69.17 (C-7 to C-18), 52.17 (C-20), 50.97 (C-23), 36.50 (C-19), 33.14 (C-
21), 32.12 ppm (C-6); MS (ESI): m/z calcd for [M+H]+ 756.4; found:
756.4.
1H NMR ([D6]DMSO): d=7.33–7.31 (m, 12H; 2-H+3-H), 7.26–7.21 (m,
3H; 1-H), 3.47–3.39 (m, 22H; 8-H to 18-H), 3.34–3.31 (m, 2H; 7-H), 3.21
(t, 3J=6.7 Hz, 2H; 23-H), 3.10–3.00 (m, 12H; 21-H+27-H), 2.70–2.50
G1 dendrimer: A solution of the G0.5 dendrimer (13 g, 17.2 mmol) in
methanol (20 mL) was added dropwise to a cooled solution of ethylene-
diamine (918 mL, 13.8 mol) over 0.5 h and stirred for 5 d at room temper-
8122
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

Fluorescent Labelling of Biomacromolecules
FULL PAPER
3
(m, 36H; 20-H+26-H+29-H+30-H+32-H), 2.41 (t, J=6.7 Hz, 4H; 24-
H), 2.27 (t, 3J=6.6 Hz, 2H; 6-H), 2.18 ppm (t, 3J=6.9 Hz, 12H; 25-H+
31-H); 13C NMR ([D6]DMSO): d=171.33 (C-27), 171.08 (C-22), 144.36
(C-4), 128.98 (C-2), 127.89 (C-3), 126.60 (C-1), 69.67, 69.58, 69.52, 69.39,
68.65, 68.45 (C-7 to C-18), 52.17 (C-5), 49.89 (C-30), 49.60 (C-31), 44.35
(C-26), 42.05 (C-19), 41.18 (C-25), 36.79 (C-21+C-27), 33.23 ppm (C-6);
MS (MALDI-TOF): m/z calcd for [M+H]+: 1268.8; found: 1267.6,
1290.1, 1306.2.
water containing 0.1% TFA and 50% MeCN in water also containing
0.1% TFA as eluent (Rf =0.1). The product was obtained as a dark-
yellow oil (3.8 g, 568 mmol, 52%) after lyophilisation.
G5 dendrimer: A solution of the G4.5 dendrimer (3.8 g, 568 mmol) in
methanol (15 mL) was added dropwise to a cooled solution of ethylene-
diamine (485 mL, 7.3 mol) over 0.5 h and stirred for 16 d at 48C. Subse-
quently the ethylenediamine was evaporated. The product was clean
enough for further use and was obtained as an orange oil (4 g, 522 mmol,
92%).
G2.5 dendrimer: A solution of the G2 dendrimer (6.7 g, 5.3 mmol) in
methanol (30 mL) was added to methyl acrylate (95 mL, 1.1 mol) and
stirred for 5 d at room temperature. The volatile components were
evaporated and the crude product was purified by chromatography on
silica gel with ethanol/acetone (5:2) followed by 20% MeOH in CH2Cl2
as eluent (Rf =0.2). The product was obtained as an orange oil (3.1 g,
1.6 mmol, 30%).
G5.5 dendrimer: A solution of the G5 dendrimer (4 g, 522 mmol) in metha-
nol (15 mL) was added to methyl acrylate (602 mL, 6.7 mol) and stirred
for 24 d at room temperature. The volatile components were evaporated
and the crude product was purified by semipreparative HPLC with
AHCTREUNG
AHCTREUNG
AHCTREUNG
G6 dendrimer: A solution of the G5.5 dendrimer (2.6 g, 214 mmol) in
methanol (15 mL) was added dropwise to a cooled solution of ethylene-
diamine (365 mL, 5.5 mol) over 0.5 h and stirred for 34 d at 48C. Subse-
quently the ethylenediamine was evaporated. The crude product was pu-
rified by semipreparative HPLC with 0–100% MeCN+0.1% TFA in
7 min as gradient (Rt =2.1 min). The product was obtained as an orange
oil (2.1 g, 150 mmol, 70%) after lyophilisation.
5(6)-Carboxyfluorescein pentafluorophenyl ester: A solution of N,N-dicy-
clohexylcarbodiimide (DCC) (603 mg, 2.93 mmol) in pyridine (5 mL) was
added to a solution of 5(6)-carboxyfluorescein (1 g, 2.66 mmol) and pen-
tafluorophenol (538 mg, 2.93 mmol) in DMF (10 mL) over 10 min and
stirred for 1 h. After removal of urea and the solvent, the crude product
was purified by chromatography on
silica gel with water/MeCN (1:1) and
0.1% TFA as eluent (Rf =0.45 and
0.55; isomers). The product was ob-
tained as an orange powder after lyo-
philisation (977 mg, 1.8 mmol, 68%).
1H NMR ([D6]DMSO): d=10.05 (brs,
1H NMR ([D6]DMSO): d=7.33–7.30 (m, 12H; 2-H+3-H), 7.27–7.19 (m,
3H; 1-H), 3.65 (s, 24H; 33-H), 3.60–3.24 (m, 30H; 7-H to 18-H+24-H+
29-H), 2.80 (brt, 3J=6.7 Hz, 8H; 26-H), 2.73 (t, 3J=6.7 Hz, 16H; 31-H),
2.61–2.51 (m, 16H; 20-H+25-H+30-H), 2.43–2.33 (m, 28H; 21-H+27-
H+32-H), 2.26 ppm (t, 3J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO):
d=173.01 (C-22+C-28), 172.26 (C-33), 144.78 (C-4), 129.54 (C-2), 127.80
(C-3), 126.54 (C-1), 70.50, 70.46, 70.37, 70.26, 70.08, 69.54 (C-7 to C-18),
53.79 (C-25), 52.90 (C-30), 52.53 (C-20), 51.60 (C-34), 50.16 (C-31), 49.86
(C-26), 49.21 (C-24+C-29), 37.41 (C-32), 37.15 (C-19), 33.74 (C-21),
32.64 (C-27), 31.28 ppm (C-6); MS (MALDI-TOF): m/z calcd for
[M+H]+: 1957.07; found: 1957.08.
0.7H; 15-H), 8.64–8.63 (m, 0.5H; 10-
H), 8.49 (dd, 3J=8.1, 4J=1.6 Hz,
0.5H; 12-H), 8.44 (dd, 3J=8.0, 4J=
1.4 Hz, 0.3H; 11-H), 8.23 (d, 3J=8.1,
0.5H; 12-H), 7.98 (m, 0.3H; 11-H), 7.54 (d, 0.5H; 3J=8.1 Hz, 13-H),
6.70–6.65 (m, 4H; 4-H+5-H), 6.58–6.50 ppm (m, 2H; 2-H); 13C NMR
([D6]DMSO): d=168.53 (C-3), 168.50 (C-16), 167.91 (C-14), 166.69 (C-
1), 161.79 (C-3), 160.50 (C-18), 160.36 (C-18), 158.70 (C-1), 156.73 (C-
20), 153.35 (C-8), 152.57 (C-19), 152.52 (C-19), 152.49 (C-7), 137.96 (C-
12), 137.58 (C-5), 133.53 (C-11), 131.54 (C-4), 130.09 (C-10), 129.88 (C-
10), 129.84 (C-12), 128.61 (C-9), 128.17 (C-6), 127.74 (C-11), 127.48 (C-
6), 126.20 (C-5), 125.26 (C-13), 125.09 (C-13), 113.37 (C-4), 109.66 (C-
17), 102.96 ppm (C-2); MS (MALDI-TOF): m/z calcd for [M+H]+:
543.1; found: 542.5; MS (ESI): m/z calcd for [M+H]+: 543.1; found:
543.1.
G3 dendrimer: A solution of the G2.5 dendrimer (3.1 g, 1.6 mmol) in
methanol (15 mL) was added dropwise to a cooled solution of ethylene-
diamine (340 mL, 5.1 mol) over 0.5 h and stirred for 9 d at 48C. Subse-
quently the ethylenediamine was evaporated. The product was clean
enough for further use and was obtained as a yellow oil (3.5 g, 1.6 mmol,
99%). MS (MALDI-TOF): m/z calcd for [M+H]+: 2181.4; found:
2183.2.
G3.5 dendrimer: A solution of the G3 dendrimer (3.5 g, 1.6 mmol) in
methanol (15 mL) was added to methyl acrylate (462 mL, 5.1 mol) and
stirred for 7 d at room temperature. The volatile components were
evaporated and the crude product was purified by chromatography on
silica gel with 55% MeCN in water containing 0.1% trifluoroacetic acid
(TFA) as eluent (Rf =0.25). The product was obtained as an orange oil
(4.8 g, 1.3 mmol, 83%) after lyophilisation. MS (MALDI-TOF): m/z
calcd for [M+H]+: 3558.0; found: 3559.7.
G0 5(6)-carboxyfluorescein-1: A solution of the G0 dendrimer (20 mg,
34 mmol) in DMF (200 mL) was added to a solution of 5(6)-carboxyfluor-
escein pentafluorophenyl ester (37 mg, 69 mmol) in DMF (200 mL) and al-
lowed to react overnight. MeCN (1 mL) and 1m NaOH (300 mL) were
added to this mixture and the solvents were evaporated. After acidifica-
tion with 1m HCl (500 mL) the product was purified by semipreparative
HPLC with 40–100% MeCN+0.1% TFA in 10 min as gradient (Rt =
3.3 min). The product was isolated as a yellow powder (28 mg, 30 mmol,
89%) after lyophilisation.
G4 dendrimer: A solution of the G3.5 dendrimer (4.8 g, 1.3 mmol) in
methanol (15 mL) was added dropwise to a cooled solution of ethylene-
diamine (570 mL, 8.5 mol) over 0.5 h and stirred for 13 d at 48C. Subse-
quently the ethylenediamine was evaporated. The product was clean
enough for further use and was obtained as a yellow oil (5.2 g, 1.3 mmol,
97%). MS (ESI): m/z calcd for [M+H]+: 4006.7; found: 4007.4.
1H NMR ([D6]DMSO): d=8.88 (t, 3J=5.3 Hz, 0.5H; 26-H), 8.73 (t, 3J=
5.4 Hz, 0.5H; 26-H), 8.46 (brs, 0.5H; 23-H), 8.23 (dd, J=8.1, J=1.5 Hz,
3
4
G4.5 dendrimer: A solution of the G4 dendrimer (5.5 g, 1.1 mmol) in
methanol (15 mL) was added to methyl acrylate (1.3 L, 14.4 mol) and
stirred for 17 d at room temperature. The volatile components were
evaporated and the crude product was purified by chromatography on
silica gel with MeCN containing 0.1% TFA followed by 75% MeCN in
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8123

W. Mier et al.
0.5H; 27-H), 8.16 (dd, 3J=8.0, 4J=1.4 Hz, 0.5H; 27-H), 7.68 (brs, 0.5H;
23-H), 7.36–7.28 (m, 12H; 2-H+3-H), 7.25–7.18 (m, 3H; 1-H), 6.69 (t,
4J=1.8 Hz, 2H; 34-H), 6.59–6.51 (m, 4H; 31-H+32-H), 3.58–3.29 (m,
24H; 8-H to 19-H), 3.19 (t, 3J=6.6 Hz, 2H; 7-H), 2.25 ppm (t, 3J=
6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=168.84 (C-28), 168.72 (C-
21), 165.38 (C-33), 165.26 (C-33), 160.35 (C-35), 155.25 (C-35), 153.28 (C-
30), 152.54 (C-30), 145.12 (C-4), 141.24 (C-25), 136.85 (C-25), 135.30 (C-
31), 130.09 (C-27), 129.90 (C-32), 129.78 (C-23), 128.66 (C-23), 127.37 (C-
31), 125.53 (C-22), 124.89 (C-26), 124.02 (C-26), 122.99 (C-32), 113.39 (C-
34), 109.86 (C-29), 109.80 (C-24), 102.92 (C-34), 70.42, 70.28, 70.21, 70.16,
69.43, 69.30, 69.22, 66.72 (C-7 to C-18), 32.05 ppm (C-6); MS (ESI): m/z
calcd for [M+H]+: 942.3; found: 942.2.
G1 5(6)-carboxyfluorescein-2: A solution of the G1 dendrimer (20 mg,
25 mmol) in DMF (200 mL) was added to a solution of 5(6)-carboxyfluor-
escein pentafluorophenyl ester (67 mg, 124 mmol) in DMF (200 mL) and
allowed to react overnight. MeCN (1 mL) and 1m NaOH (300 mL) were
added to this mixture and the solvents were evaporated. After acidifica-
tion with 1m HCl (500 mL) the product was purified by semipreparative
HPLC with 40–100% MeCN+0.1% TFA in 10 min as gradient (Rt =
2.3 min). The product was isolated as a yellow powder after lyophilisation
(34 mg, 22 mmol, 89%).
70.24, 70.13, 69.21, 66.72, 64.89 (C-7 to C-18), 49.97 (C-20), 32.05 ppm
(C-6); MS (MALDI-TOF): m/z calcd for [M+H]+: 2701.0; found:
2704.2; MS (ESI): m/z calcd for [M+H]+: 2701.0; found: 2701.0.
G0 sulforhodamine B2 acid fluoride-1: A solution of the G0 dendrimer
(20 mg, 34 mmol) in DMF (200 mL) was added to a solution of sulforhoda-
mine B2 acid fluoride (58 mg, 103 mmol) and N,N-diisopropylethylamine
(DIPEA) (10 mL) in DMF (200 mL) and allowed to react overnight. After
acidification with 1m HCl (100 mL) the product was purified by semipre-
parative HPLC with 0–100% MeCN+0.1% TFA in 10 min as gradient
(Rt =7.1 min). The product was isolated as a violet oil after lyophilisation
(27 mg, 24 mmol, 71%).
1H NMR ([D6]DMSO): d=8.86 (t, 3J=5.1 Hz, 1H; 32-H), 8.72 (t, 3J=
5.0 Hz, 1H; 32-H), 8.44 (brs, 1H; 29-H), 8.24–8.20 (m, 1H; 33-H), 8.16–
8.13 (m, 1H; 33-H), 7.64 (brs, 1H; 29-H), 7.37–7.28 (m, 12H; 2-H+3-
H), 7.25–7.18 (m, 3H; 1-H), 6.69–6.68 (m, 4H; 40-H), 6.59–6.51 (m, 8H;
37-H+38-H), 3.73–3.17 (m, 42H; 7-H to 21-H+24-H+25-H), 2.25 ppm
(t, 3J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=170.25 (C-22),
168.87 (C-34), 168.72 (C-27), 165.61 (C-39), 165.50 (C-39), 160.35 (C-41),
155.37 (C-41), 153.40 (C-36), 152.50 (C-36), 145.12 (C-4), 141.33 (C-31),
136.94 (C-31), 135.36 (C-38), 130.05 (C-33), 129.90 (C-38), 129.77 (C-29),
128.67 (C-29), 127.37 (C-37), 125.54 (C-28), 124.89 (C-32), 124.01 (C-32),
122.93 (C-38), 113.42 (C-40), 109.77 (C-35), 109.71 (C-30), 102.93 (C-40),
70.42, 70.36, 70.26, 70.13, 69.21, 66.72, 64.91 (C-7 to C-18), 52.67 (C-5),
50.16 (C-20), 32.05 ppm (C-6); MS (ESI): m/z calcd for [M+H]+: 1528.6;
found: 1528.6.
1H NMR ([D6]DMSO): d=8.41 (d, 4J=1.5 Hz, 1H; 22-H), 8.00 (dd, 3J=
7.9, 4J=1.6 Hz, 1H; 24-H), 7.42 (d, 3J=7.9 Hz, 1H; 25-H), 7.31–7.28 (m,
12H; 2-H+3-H), 7.25–7.18 (m, 3H; 1-H), 7.07–6.99 (m, 4H; 29-H+30-
H), 6.93 (d, 4J=2.0 Hz, 2H; 32-H), 3.62 (q, 3J=7.0 Hz, 8H; 34-H), 3.43–
3.25 (m, 22H; 8-H to 18-H), 3.19 (t, 3J=6.6 Hz, 2H; 7-H), 2.78 (m, 2H;
19-H), 2.25 (t, 3J=6.6 Hz, 2H; 6-H), 1.18 ppm (t, 3J=7.0 Hz, 12H; 35-
H); 13C NMR ([D6]DMSO): d=157.85 (C-26), 155.96 (C-31), 155.82 (C-
33), 151.08 (C-23), 145.11 (C-4), 140.81 (C-21), 132.41 (C-29), 131.57 (C-
25), 131.00 (C-27), 129.75 (C-2), 128.68 (C-3), 127.38 (C-1), 126.42 (C-
24), 114.87 (C-30), 114.22 (C-28), 96.35 (C-32), 70.40, 70.27, 70.20, 70.16,
69.75, 69.20, 66.72 (C-7 to (C-18), 46.01 (C-34), 42.84 (C-19), 32.06 (C-6),
13.12 ppm (C-35); MS (ESI): m/z calcd for [M+H]+: 1124.4; found:
1124.4.
G2 5(6)-carboxyfluorescein-4: A solution of the G2 dendrimer (20 mg,
16 mmol) in DMF (200 mL) was added to a solution of 5(6)-carboxyfluor-
escein pentafluorophenyl ester (85 mg, 158 mmol) in DMF (200 mL) and
allowed to react overnight. MeCN (1 mL) and 1m NaOH (300 mL) were
added to this mixture and the solvents were evaporated. After acidifica-
tion with 1m HCl (500 mL) the product was purified by semipreparative
HPLC with 40–100% MeCN+0.1% TFA in 10 min as gradient (Rt =
2.0 min). The product was isolated as a yellow powder after lyophilisation
(35 mg, 13 mmol, 81%).
G1 sulforhodamine B2 acid fluoride-2: A solution of the G1 dendrimer
(20 mg, 25 mmol) in DMF (200 mL) was added to a solution of sulforhoda-
mine B2 acid fluoride (83 mg, 148 mmol) and DIPEA (20 mL) in DMF
(200 mL) and allowed to react overnight. After acidification with 1m HCl
(100 mL) the product was purified by semipreparative HPLC with
100% MeCN+0.1% TFA in 10 min as gradient (Rt =4.0 min). The
product was isolated as a violet powder (36 mg, 19 mmol, 76%) after
lyophilisation.
ACHTREUNG40–
AHCTREUNG
1H NMR ([D6]DMSO): d=8.89 (t, 3J=4.8 Hz, 2H; 38-H), 8.74 (t, 3J=
4.8 Hz, 2H; 38-H), 8.44 (brs, 2H; 35-H), 8.24–7.96 (m, 4H; 39-H), 7.65
(brs, 2H; 39-H), 7.38–7.29 (m, 12H; 2-H+3-H), 7.25–7.18 (m, 3H; 1-H),
6.70 (brs, 8H; 46-H), 6.60–6.51 (m, 16H; 43-H+44-H), 3.73 (brs, 2H;
23-H), 3.52–2.52 (m, 74H; H-7 to 21-H+24-H to 27-H+30-H+31-H),
AHCTREUNG
3
2.25 ppm (t, J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=170.75 (C-
22), 170.21 (C-28), 168.87 (C-40), 168.73 (C-33), 165.66 (C-45), 165.53 (C-
45), 160.37 (C-47), 155.39 (C-47), 152.51 (C-42), 145.11 (C-4), 141.30 (C-
37), 136.92 (C-37), 135.33 (C-44), 130.03 (C-39), 129.88 (C-44), 129.78 (C-
35), 128.66 (C-35), 127.36 (C-43), 125.51 (C-34), 124.89 (C-38), 124.05 (C-
38), 113.38 (C-46), 109.75 (C-41), 109.62 (C-36), 102.97 (C-46), 70.38,
8124
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

Fluorescent Labelling of Biomacromolecules
FULL PAPER
1H NMR ([D6]DMSO): d=8.39 (d, 4J=1.4 Hz, 2H; 28-H), 8.01 (dd, 3J=
7.9, 4J=1.4 Hz, 2H; 30-H), 7.45 (d, 3J=7.9 Hz, 2H; 31-H), 7.31–7.28 (m,
12H; 2-H+3-H), 7.26–7.18 (m, 3H; 1-H), 7.08–6.94 (m, 12H; 35-H, 36-
1H NMR ([D6]DMSO): d=8.48 (d, 3J=8.9 Hz, 1H; 23-H), 7.34–7.19 (m,
15H; 1-H to 3-H), 6.46 (d, 3J=9.0 Hz, 1H; 22-H), 3.56–3.30 (m, 24H; 8-
H to 18-H), 3.20 (t, J=6.6 Hz, 2H; 7-H), 2.25 ppm (t, J=6.6 Hz, 2H; 6-
H); 13C NMR ([D6]DMSO): d=145.11 (C-4), 129.77 (C-2), 128.68 (C-3),
127.38 (C-1), 70.53, 70.42, 70.28, 70.16, 69.21, 68.56, 66.72 (C-7 to C-18),
44.10 (C-19), 32.05 ppm (C-6); MS (ESI): m/z calcd for [M+H]+: 747.3;
found: 747.3.
3
3
3
3
H, 38-H), 3.72 (t, J=4.7 Hz, 2H; 26-H), 3.62 (q, J=7.0 Hz, 16H; 40-H),
3.54–3.29 (m, 30H; 8-H to 18-H+20-H+21-H), 3.19 (t, 3J=6.6 Hz, 2H;
3
3
7-H), 3.01 (q, J=5.7 Hz, 4H; 25-H), 2.76 (q, J=5.8 Hz, 4H; 24-H), 2.57
3
3
3
(t, J=6.3 Hz, 2H; 19-H), 2.25 (t, J=6.6 Hz, 2H; 6-H), 1.19 ppm (t, J=
7.0 Hz, 24H; 41-H); 13C NMR ([D6]DMSO): d=170.12 (C-22), 157.82
(C-32), 155.84 (C-37), 155.67 (C-39), 150.97 (C-29), 145.11 (C-4), 140.51
(C-27), 132.31 (C-35), 131.89 (C-31), 131.24 (C-33), 130.03 (C-28), 129.76
(C-2), 128.69 (C-3), 127.40 (C-1), 126.10 (C-30), 114.98 (C-36), 114.17 (C-
34), 96.41 (C-38), 70.42, 70.27, 70.16, 69.21, 66.72, 64.92 (C-7 to C-18),
52.73 (C-5), 50.03 (C-20), 46.04 (C-40), 42.44 (C-19), 32.06 (C-6),
13.12 ppm (C-41); MS (ESI): m/z calcd for [M+H]+: 1892.7; found:
1893.8.
G1 NBD-2: A solution of the G1 dendrimer (20 mg, 25 mmol) in DMF
(250 mL) was added to a solution of NBD chloride (20 mg, 99 mmol) and
DIPEA (100 mL) in DMF (250 mL) and allowed to react overnight. After
acidification with 1m HCl (250 mL) the product was purified by semipre-
parative HPLC with 40–100% MeCN+0.1% TFA in 10 min as gradient
(Rt =3.7 min). The product was isolated as a brown oil after lyophilisa-
tion (18 mg, 16 mmol, 63%).
G2 sulforhodamine B2 acid fluoride-4: A solution of the G2 dendrimer
(20 mg, 16 mmol) in DMF (200 mL) was added to a solution of sulforhoda-
mine B2 acid fluoride (88 mg, 158 mmol) and DIPEA (20 mL) in DMF
(200 mL) and allowed to react overnight. After acidification with 1m HCl
(100 mL) the product was purified by semipreparative HPLC with 40–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =3.8 min). The prod-
uct was isolated as a violet powder after lyophilisation (25 mg, 7.3 mmol,
46%).
1H NMR ([D6]DMSO): d=8.48 (d, 3J=8.9 Hz, 2H; 28-H), 7.32–7.29 (m,
3
12H; 2-H+3-H), 7.26–7.21 (m, 3H; 1-H), 6.40 (d, J=8.9 Hz, 2H; 27-H),
3.57–3.13 (m, 38H; 8-H to 18-H+20-H+21-H+24-H+25-H), 3.21 (t,
3J=6.6 Hz, 2H; 7-H), 2.59 (t, 3J=6.9 Hz, 2H; 19-H), 2.26 ppm (t, 3J=
6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=168.91 (C-22), 149.77 (C-
30), 144.36 (C-4), 129.01 (C-2), 127.92 (C-3), 126.62 (C-1), 69.65, 69.51,
69.40, 68.46, 67.86, 64.13 (C-7 to C-18), 31.29 ppm (C-6); MS (ESI): m/z
calcd for [M+H]+: 1138.5; found: 1138.7.
G2 NBD-4: A solution of the G2 dendrimer (20 mg, 16 mmol) in DMF
(250 mL) was added to a solution of NBD chloride (38 mg, 190 mmol) and
DIPEA (100 mL) in DMF (250 mL) and allowed to react overnight. After
acidification with 1m HCl (250 mL) the product was purified by semipre-
parative HPLC with 40–100% MeCN+0.1% TFA in 10 min as gradient
(Rt =3.4 min). The product was isolated as a brown oil after lyophilisa-
tion (14 mg, 7.3 mmol, 46%).
1H NMR ([D6]DMSO): d=8.38 (d, 4J=1.2 Hz, 4H; 34-H), 8.02 (dd, 3J=
7.9, 4J=1.3 Hz, 4H; 36-H), 7.46 (d, 3J=7.9 Hz, 4H; 37-H), 7.31–7.27 (m,
12H; 2-H+3-H), 7.24–7.19 (m, 3H; 1-H), 7.07–6.94 (m, 24H; 41-H, 42-
H, 44-H), 3.75 (brs, 2H; 23-H), 3.62 (q, 3J=6.5 Hz, 32H; 46-H), 3.56–
2.60 (m, 72H; 8-H to 21-H+24-H to 27-H+30-H+31-H), 2.24 (t, 3J=
6.6 Hz, 2H; 6-H), 1.18 ppm (t, 3J=6.7 Hz, 48H; 47-H); 13C NMR
([D6]DMSO): d=170.16 (C-22+C-28), 157.82 (C-38), 155.84 (C-43),
155.54 (C-45), 150.63 (C-35), 145.10 (C-4), 140.55 (C-33), 132.30 (C-41),
131.99 (C-37), 131.39 (C-39), 129.76 (C-2), 128.69 (C-3), 127.39 (C-1),
126.09 (C-36), 114.96 (C-42), 114.15 (C-40), 96.43 (C-44), 70.41, 70.26,
70.15, 69.21 (C-7 to C-18), 46.04 (C-47), 13.12 ppm (C-48); MS (ESI): m/
z calcd for [M+H]+: 3429.3; found: 3430.3.
G0 NBD-1: A solution of the G0 dendrimer (20 mg, 34 mmol) in DMF
(250 mL) was added to a solution of NBD chloride (17 mg, 86 mmol) and
DIPEA (100 mL) in DMF (250 mL) and allowed to react overnight. After
acidification with 1m HCl (250 mL) the product was purified by semipre-
parative HPLC with 40–100% MeCN+0.1% TFA in 10 min as gradient
(Rt =3.9 min). The product was isolated as a brown oil after lyophilisa-
tion (11 mg, 15 mmol, 43%).
1H NMR ([D6]DMSO): d=8.49–8.44 (m, 4H; 35-H), 7.32–7.28 (m, 12H;
2-H+3-H), 7.25–7.20 (m, 3H; 1-H), H6.38 (d, 3J=8.9 Hz, 4H; 34-H),
3.74 (brt, 3J=4.7 Hz, 2H; 20-H), 3.55–3.30 (m, 62H; 8-H to 18-H+20-
H+21-H+24-H to 27-H+30-H), 3.20 (t, 3J=6.6 Hz, 2H; 7-H), 2.64 (t,
3J=6.8 Hz, 2H; 19-H), 2.59 (t, 3J=6.6 Hz, 8H; 31-H), 2.25 ppm (t, 3J=
6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=169.73 (C-22+C-28),
144.34 (C-4), 129.00 (C-2), 127.91 (C-3), 126.62 (C-1), 69.63, 69.48, 69.37,
68.44, 65.95, 64.17 (C-7 to C-18), 51.09 (C-31), 49.20 (C-20+C-26), 48.80
(C-5), 33.52 (C-27), 31.27 ppm (C-6); MS (ESI): m/z calcd for [M+H]+:
1920.8; found: 1921.8.
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8125

W. Mier et al.
G0 coumarin-343-1:
A
solution of benzotriazol-1-yloxy)tripyrrolidino-
(200 mL) the product was purified by semipreparative HPLC with 0–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =6.3 min). The prod-
uct was isolated as a brown solid after lyophilisation (21 mg, 9 mmol,
56%).
phosphonium hexafluorophosphate (PyBOP; 51 mg, 98 mmol) in DMSO
(200 mL) and DIPEA (50 mL) were added to a solution of coumarin-343
(30 mg, 103 mmol) in DMSO (200 mL). After 2 min a solution of the G0
dendrimer (30 mg, 51 mmol) in DMSO (250 mL) was added and the mix-
ture was allowed to react overnight. After acidification with 1m HCl
(200 mL) the product was purified by semipreparative HPLC with 0–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =8.1 min). The prod-
uct was isolated as a brown oil after lyophilisation (21 mg, 25 mmol,
48%).
1H NMR ([D6]DMSO): d=8.78 (t, 4J=5.4 Hz, 1H; 25-H), 8.48 (s, 1H;
23-H), 7.31–7.28 (m, 12H; 2-H, 3-H), 7.24–7.20 (m, 3H; 1-H), 3.51–3.27
3
(m, 32H; 8-H to 19-H+29-H+30-H), 3.19 (t, J=6.6 Hz, 2H; 7-H), 2.68
3
3
(q, J=6.6 Hz, 4H; 27-H+32-H), 2.25 (t, J=6.6 Hz, 2H; 6-H), 1.84 ppm
(dt, J=5.9 Hz, 4H; 28-H+31-H); 13C NMR ([D6]DMSO): d=162.39 (C-
3
21), 161.77 (C-35), 151.99 (C-34), 147.89 (C-36), 147.39 (C-23), 144.37 (C-
4), 129.01 (C-2), 127.91 (C-3), 127.04 (C-1), 126.62 (C-25), 119.33 (C-22),
107.68 (C-26), 107.27 (C-24), 105.94 (C-33), 69.74, 69.68, 69.61, 69.53,
69.40, 68.99, 68.46, 65.96 (C-7 to C-18), 49.44 (C-19), 48.91 (C-5), 31.29
(C-6), 26.70 (C-29+C-30), 20.43 (C-28+C-31), 19.48 ppm (C-27+C-32);
MS (MALDI-TOF): m/z calcd for [M+H]+: 851.4; found: 849.4; MS
(ESI): m/z calcd for [M+H]+: 851.4; found: 851.4.
1H NMR ([D6]DMSO): d=8.73 (t, 4J=5.6 Hz, 4H; 37-H), 8.41 (s, 4H;
35-H), 7.32–7.30 (m, 12H; 2-H+3-H), 7.26–7.21 (m, 3H; 1-H), 3.54–3.18
(m, 88H; 7-H to 18-H+20-H+21-H+24-H to 27-H+30-H+31-H+41-
H+42-H), 2.68–2.60 (m, 28H; 19-H+39-H+44-H), 2.25 (t, 3J=6.6 Hz,
2H; 6-H), 1.89–1.79 ppm (m, 16H; 40-H+43-H); 13C NMR
([D6]DMSO): d=169.57 (C-33), 162.77 (C-22+C-28), 161.67 (C-47),
151.91 (C-46), 147.84 (C-48) 147.25 (C-35), 144.34 (C-4), 129.00 (C-2),
127.91 (C-3), 126.98 (C-1), 126.63 (C-37), 119.26 (C-34), 107.63 (C-38),
107.24 (C-36), 104.44 (C-45), 69.63, 69.49, 69.39, 68.46 (C-7 to C-18),
49.45 (C-19), 48.89 (C-5), 26.70 (C-41+C-42), 20.43 (C-40+C-43),
19.48 ppm (C-39+C-44); MS (MALDI-TOF): m/z calcd for [M+H]+:
2337.1; found: 2339.9.
G1 coumarin-343-2: A solution of PyBOP (92 mg, 178 mmol) in DMSO
(200 mL) and DIPEA (50 mL) were added to a solution of coumarin-343
(56 mg, 197 mmol) in DMSO (200 mL). After 2 min a solution of the G1
dendrimer (40 mg, 50 mmol) in DMSO (250 mL) was added and the mix-
ture was allowed to react overnight. After acidification with 1m HCl
(200 mL) the product was purified by semipreparative HPLC with 0–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =7.6 min). The prod-
uct was isolated as a brown oil after lyophilisation (37 mg, 27 mmol,
54%).
G0 dansyl-1: A solution of the G0 dendrimer (30 mg, 51 mmol) and
DIPEA (50 mL) in MeCN (200 mL) was added to a solution of dansyl
chloride (56 mg, 206 mmol) in MeCN (300 mL). After 0.5 h and acidifica-
tion of the mixture with 1m HCl (100 mL) the product was purified by
semipreparative HPLC with 0–100% MeCN+0.1% TFA in 10 min as
gradient (Rt =6.8 min). The product was isolated as a yellow oil after lyo-
philisation (26 mg, 32 mmol, 63%).
1H NMR ([D6]DMSO): d=8.73 (t, 4J=5.8 Hz, 2H; 31-H), 8.44 (s, 2H;
29-H), 7.32–7.29 (m, 12H; 2-H+3-H), 7.25–7.21 (m, 3H; 1-H), 3.75 (t,
3J=4.7 Hz, 2H; 23-H), 3.56–3.18 (m, 48H; 7-H to 18-H+21-H+22-H+
24-H+25-H+35-H+36-H), 2.69–2.60 (m, 10H; 19-H+33-H+38-H),
2.26 (t, 3J=6.6 Hz, 2H; 6-H), 1.89–1.82 ppm (m, 8H; 34-H+37-H);
13C NMR ([D6]DMSO): d=169.54 (C-27), 162.80 (C-22), 161.72 (C-41),
151.97 (C-40), 147.90 (C-42), 147.31 (C-29), 144.39 (C-4), 129.02 (C-2),
127.91 (C-3), 127.02 (C-1), 126.65 (C-31), 119.33 (C-28), 107.72 (C-32),
107.27 (C-30), 104.50 (C-39), 69.68, 69.53, 69.45, 69.42, 68.48, 65.99, 64.19
(C-7 to C-18), 49.49 (C-19), 48.93 (C-5), 31.31 (C-6), 26.71 (C-35+C-36),
20.44 (C-34+C-37), 19.49 ppm (C-33+C-38); MS (MALDI-TOF): m/z
calcd for [M+H]+: 1346.6; found: 1347.6; MS (ESI): m/z calcd for
[M+H]+: 1346.6; found: 1346.9.
1H NMR ([D6]DMSO): d=8.45 (d, 3J=8.5 Hz, 1H; 24-H), 8.32 (d, 3J=
8.7 Hz, 1H; 29-H), 8.11 (m, 1H; 22-H), 7.59 (m, 2H; 23-H+28-H), 7.30
3
(d, J=4.2 Hz; 12H; 2-H+3-H), 7.22 (m, 4H; 1-H+27-H), 3.44–3.17 (m,
25H; 7-H to 18-H+20-H), 2.93 (q, 3J=5.9 Hz, 2H; 19-H), 2.84 (s, 6H;
31-H), 2.25 ppm (t, 3J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=
151.00 (C-26), 145.12 (C-4), 137.11 (C-21), 129.76 (C-2), 129.45 (C-25+
C-30), 128.77 (C-24+C-28), 128.68 (C-3), 128.40 (C-22), 127.39 (C-1),
124.45 (C-23), 120.55 (C-29), 116.13 (C-27), 70.43, 70.40, 70.28, 70.20,
70.16, 69.64, 69.21, 66.72 (C-7 to C-18), 45.86 (C-31), 42.92 (C-19),
32.06 ppm (C-6); MS (ESI): m/z calcd for [M+H]+ 817.4; found: 817.4.
G1 dansyl-2: A solution of the G1 dendrimer (25 mg, 31 mmol) and
DIPEA (50 mL) in MeCN (200 mL) was added to a solution of dansyl
chloride (67 mg, 247 mmol) in MeCN (300 mL). After 0.5 h and acidifica-
tion of the mixture with 1m HCl (100 mL) the product was purified by
semipreparative HPLC with 0–100% MeCN+0.1% TFA in 10 min as
gradient (Rt =5.8 min). The product was isolated as a yellow solid after
lyophilisation (23 mg, 18 mmol, 58%).
G2 coumarin-343-4: A solution of PyBOP (62 mg, 120 mmol) in DMSO
(200 mL) and DIPEA (50 mL) were added to a solution of coumarin-343
(36 mg, 126 mmol) in DMSO (200 mL). After 2 min a solution of the G2
dendrimer (20 mg, 16 mmol) in DMSO (250 mL) was added and the mix-
ture was allowed to react overnight. After acidification with 1m HCl
8126
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

Fluorescent Labelling of Biomacromolecules
FULL PAPER
1H NMR ([D6]DMSO): d=8.44 (d, 3J=8.5 Hz, 2H; 30-H), 8.27 (d, 3J=
8.7 Hz, 2H; 35-H), 8.16–8.01 (m, 2H; 28-H), 7.59 (m, 4H; 29-H+34-H),
7.30 (d, 3J=4,3 Hz, 12H; 2-H+3-H), 7.23 (m, 5H; 1-H+33-H), 3.68 (t,
3J=4.5 Hz, 2H; 26-H), 3.50–3.22 (m, 24H; 7-H to 18-H), 3.19 (t, 3J=
6.6 Hz, 4H; 20-H), 3.06 (q, 3J=6.4 Hz, 4H; 19-H), 2.81 (s, 12H; 37-H),
3
2.25 ppm (t, J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=170.10 (C-
22), 151.57 (C-32), 145.11 (C-4), 136.47 (C-27), 130.03 (C-30), 129.77 (C-
2), 129.60 (C-31+C-36), 128.92 (C-34), 128.68 (C-3), 128.57 (C-28),
127.37 (C-1), 124.35 (C-29), 119.91 (C-35), 116.01 (C-33), 70.38, 70.25,
70.14, 69.20, 66.72, 64.83 (C-7 to C-18), 52.72 (C-5), 45.71 (C-37), 42.27
(C-19), 32.05 ppm (C-6); MS (ESI): m/z calcd for [M+H]+: 1278.6;
found: 1278.7.
7.26 (m, 12H; 2-H+3-H), 7.24–7.19 (m, 8H; 1-H+45-H), 3.52–3.04 (m,
64H; 7-H to 18-H+20-H+21-H+24-H to 27-H+30-H to 33-H+36-H+
37-H), 2.80 (s, 47H; 49-H), 2.67–2.51 (m, 2H; 19-H), 2.24 ppm (t, 3J=
6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=170.38 (C-22), 169.92 (C-
28), 151.74 (C-44), 145.10 (C-4), 136.46 (C-39), 130.08 (C-42), 129.77 (C-
2), 129.65 (C-43+C-48), 128.87 (C-46), 128.67 (C-3), 128.56 (C-40),
127.37 (C-1), 124.29 (C-41), 119.84 (C-47), 115.94 (C-45), 70.36, 70.23,
70.12, 69.20 (C-7 to C-18), 51.80 (C-5), 49.60, 49.56, 49.46 (C-20+C-24 to
C-26+C-30 to C-32+C-36+C-37), 45.72 (C-49), 42.28 (C-19), 32.04 ppm
(C-6); MS (ESI): m/z calcd for [M+H]+: 4045.8; found: 4048.0.
G2 dansyl-4: A solution of the G2 dendrimer (30 mg, 24 mmol) and
DIPEA (50 mL) in MeCN (200 mL) was added to a solution of dansyl
chloride (102 mg, 379 mmol) in MeCN (300 mL). After 0.5 h and acidifica-
tion of the mixture with 1m HCl (100 mL) the product was purified by
semipreparative HPLC with 30–100% MeCN+0.1% TFA in 10 min as
gradient (Rt =3.4 min). The product was isolated as a yellow solid after
lyophilisation (21 mg, 9.5 mmol, 40%).
G4 dansyl-16: A solution of the G4 dendrimer (15 mg, 3.7 mmol) and
DIPEA (50 mL) in DMF (200 mL) was added to a solution of dansyl chlo-
ride (49 mg, 180 mmol) in DMF (300 mL). After 30 min 1m NaOH
(200 mL) was added and after acidification of the mixture with 1m HCl
(150 mL) the product was purified by semipreparative HPLC with 0–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =5.7 min). The prod-
uct was isolated as a brown solid after lyophilisation (14 mg, 1.9 mmol,
51%).
1H NMR ([D6]DMSO): d=8.43 (d, 3J=8.5 Hz, 4H; 36-H), 8.26 (d, 3J=
8.7 Hz, 4H; 41-H), 8.19–8.01 (m, 4H; 34-H), 7.58 (m, 8H; 35-H+40-H),
7.30 (d, 3J=4.1 Hz, 12H; 2-H+3-H), 7.24 (m, 6H; 1-H+39-H), 3.70
(brs, 4H; 32-H), 3.51–2.91 (m, 52H; 7-H to 18-H+20-H+21-H,+23-H
to 27-H+30-H+31-H), 2.80 (s, 23H; 42-H), 2.61 (t, 3J=7.1 Hz, 2H; 19-
3
H), 2.25 ppm (t, J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=170.67
(C-22), 169.98 (C-28), 151.81 (C-38), 145.11 (C-4), 136.47 (C-33), 130.11
(C-36), 129.74 (C-2), 129.67 (C-37+C-42), 128.84 (C-40), 128.68 (C-3),
128.58 (C-34), 127.30 (C-1), 124.28 (C-35), 119.79 (C-41), 115.91 (C-39),
70.38, 70.24, 70.13, 69.20, 66.72, 64.88 (C-7 to C-18), 52.67 (C-5), 51.82,
49.95, 49.67 (C-20+C-24 to C-26+C-30+C-31), 45.72 (C-43), 42.29 (C-
19), 32.05 ppm (C-6); MS (ESI): m/z calcd for [M+H]+: 2201.0; found:
2202.5.
G3 dansyl-8: A solution of the G3 dendrimer (20 mg, 9.2 mmol) and
DIPEA (50 mL) in DMF (200 mL) was added to a solution of dansyl chlo-
ride (50 mg, 183 mmol) in DMF (300 mL). After 30 min 1m NaOH
(200 mL) was added and after acidification of the mixture with 1m HCl
(150 mL) the product was purified by semipreparative HPLC with 0–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =6.0 min). The prod-
uct was isolated as a yellow solid after lyophilisation (22 mg, 5.4 mmol,
59%).
1H NMR ([D6]DMSO): d=8.55–8.41 (m, 17H; 48-H), 8.28–8.19 (m, 16H;
53-H), 8.08–8.01 (m, 18H; 46-H), 7.61–7.54 (m, 32H; 47-H+52-H), 7.30–
7.27 (m, 12H; 2-H+3-H), 7.23–7.18 (m, 19H; 1-H+51-H), 3.50–3.03 (m,
133H; 7-H to 18-H+20-H+21-H+24-H to 27-H+30-H to 33-H+36-H
to 39-H+42-H+43-H), 2.79 (s, 95H; 55-H), 2.69–2.51 (m, 2H; 19-H),
1H NMR ([D6]DMSO): d=8.47–8.42 (m, 8H; 42-H), 8.28–8.18 (m, 9H;
2.24 ppm (t, J=6.6 Hz, 2H; 6-H); 13C NMR ([D6]DMSO): d=170.34 (C-
3
47-H), 8.08–8.00 (m, 9H; 40-H), 7.61–7.54 (m, 15H; 41-H+46-H), 7.30–
22), 169.91 (C-28), 151.68 (C-50), 145.09 (C-4), 136.46 (C-45), 130.06 (C-
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8127

W. Mier et al.
48), 129.76 (C-2), 129.65 (C-49+C-54), 128.89 (C-52), 128.64 (C-3),
128.56 (C-46), 127.36 (C-1), 124.28 (C-47), 119.87 (C-53), 115.95 (C-51),
70.35, 70.21, 70.10 (C-7 to C-18), 51.79 (C-5), 49.59, 49.54, 49.42 (C-20+
C-24 to C-26+C-30 to C-32+C-36 to C-38+C-42+C-43), 45.70 (C-55),
42.28 (C-19), 32.04 ppm (C-6); MS (ESI): m/z calcd for [M+H]+: 7741.8;
found: 7741.0.
G5 dansyl-32: A solution of the G5 dendrimer (30 mg, 3.9 mmol) and
DIPEA (50 mL) in DMF (200 mL) was added to a solution of dansyl chlo-
ride (85 mg, 314 mmol) in DMF (300 mL). After 30 min 1m NaOH
(200 mL) was added and after acidification of the mixture with 1m HCl
(150 mL) the product was purified by semipreparative HPLC with 20–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =5.0 min). The prod-
uct was isolated as a brown solid after lyophilisation (17 mg, 1.1 mmol,
29%).
5.2 mmol, 98%). MS (MALDI-TOF): m/z calcd for [M+H]+: 7495.2;
found: 7490.4.
Derivatisation of hMAb425 (Matuzumab) with sulfo-SMCC: A freshly
prepared solution of sulfo-SMCC (42 mg, 9.7”10À8 mol) in DMF/water
(1:1, 10 mL) was added to a fresh solution of hMAb425 (2 mg, 1.39”
10À8 mol) in phosphate buffer (PB; 0.05m, pH 7.2, 500 mL). After 1 h of
incubation, the derivatised antibody was isolated and desalted by size-ex-
clusion chromatography using NAP-5 columns (GE Healthcare Bio-Sci-
ences AB, Uppsala). The derivatised antibody was obtained in high
yields (95–98%). The number of maleimide functions per antibody mole-
cule was determined by reaction of the maleimides with a known excess
of MESNA (sodium 2-mercaptoethanesulfonate) and subsequent quan-
tification of the remaining thiol by Ellmanꢁs assay. The number of malei-
mides per antibody molecule was found to be between 1.4 and 1.7.
1H NMR ([D6]DMSO): d=8.55–8.40 (m, 25H; 10-H), 8.26–8.18 (m, 29H;
15-H), 8.06–8.00 (m, 33H; 8-H), 7.59–7.52 (m, 62H; 9-H+14-H), 7.31–
7.20 (m, 47H; 1-H to 3-H+13-H), 3.52–2.99 (m, 139H), 2.79 (s, 187H;
17-H), 2.68–2.51 (m, 2H), 2.22 ppm (t, 3J=6.6 Hz, 2H; 6-H); 13C NMR
([D6]DMSO): d=169.88; 151.92 (C-12), 136.43 (C-7), 130.05 (C-10),
129.76 (C-2), 129.53 (C-11+C-16), 128.85 (C-14), 128.63 (C-3), 128.56
(C-8), 124.22 (C-9), 119.70 (C-15), 115.83 (C-13), 70.33, 51.74 (C-5),
50.21, 49.55, 49.52, 49.31, 45.67 (C-17), 42.28 ppm; MS (MALDI-TOF):
m/z calcd for [M+H]+: 15112.4; found: ꢀ13950.
G6 dansyl-64: A solution of the G6 dendrimer (48 mg, 3.4 mmol) and
DIPEA (50 mL) in DMF (200 mL) was added to a solution of dansyl chlo-
ride (119 mg, 439 mmol) in DMF (300 mL). After 30 min 1m NaOH
(200 mL) was added and after acidification of the mixture with 1m HCl
(150 mL) the product was purified by semipreparative HPLC with 25–
100% MeCN+0.1% TFA in 10 min as gradient (Rt =4.1 min). The prod-
uct was isolated as a brown solid after lyophilisation (30 mg, 1.0 mmol,
30%).
1H NMR ([D6]DMSO): d=8.55–8.39 (m, 60H; 10-H), 8.27–8.18 (m, 61H;
15-H), 8.06–8.00 (m, 63H; 8-H), 7.58–7.52 (m, 125H; 9-H+14-H), 7.26–
7.16 (m, 79H; 1-H to 3-H+13-H), 3.48–2.93 (m, 207H), 2.79 (s, 359H;
17-H), 2.61 ppm (brs, 12H); 13C NMR ([D6]DMSO): d=170.31, 169.88,
151.78 (C-12), 136.44 (C-7), 130.20 (C-10), 129.66 (C-2), 128.87 (C-14),
128.54 (C-3), 124.22 (C-9), 119.79 (C-15), 115.87 (C-13), 51.79 (C-5),
49.53, 45.65 (C-17), 42.28 ppm (C-19).
hMAb425-dansyl16: A freshly prepared solution of dansyl16-SH (4.0 mg,
2.78”10À7 mol) in DMSO (100 mL) was added to a solution of hMAb425
derivatised with sulfo-SMCC (1.6 mg, 1.11”10À8 mol) in PB (0.05m,
pH 7.2, 1 mL). The reaction was complete after 2 min and the product
was purified by size-exclusion FPLC with phosphate buffer saline (PBS;
0.05m, 0.15m NaCl, pH 7.0) as eluent (Rt =7.6 min) and desalted on a
NAP-5 sephadex column. The product was obtained in moderate yield
(23–31%) and characterised by MALDI-TOF MS and gel electrophore-
sis. MS (MALDI-TOF): m/z calcd for [M+H]+: 157449.6; found:
157396.0.
Immunoreactivities of the antibody–dendrimer conjugates
Cells: The human colon adenocarcinoma cell line HT29 expressing con-
siderable amounts of epidermal growth factor receptor was maintained in
RPMI 1640 medium supplemented with 2 mm l-glutamine, 1 mm pyru-
vate and 10% heat-inactivated foetal calf serum. Prior to use, cells were
detached from the surface of the tissue culture flask by treatment with
0.25% trypsin plus 0.25% ethylenediaminetetraacetic acid (EDTA) in
PBS and counted.
Deprotection of G4 dansyl-16 todansyl 16-SH: G4 dansyl-16 (41 mg,
5.3 mmol) was dissolved in triisopropylsilane (TIS; 200 mL) and TFA
(2 mL). After 2 min the volatile components of the mixture were evapo-
rated and the crude product was purified by semipreparative HPLC with
20–100% MeCN+0.1% TFA in 7 min as gradient (Rt =2.7 min). The
product was isolated as a brownish solid after lyophilisation (39 mg,
Flow cytometry: Serial dilutions of dendrimer-conjugated Matuzumab
were compared with unmodified Matuzumab for binding to HT29 cells.
For this, antibody dilutions ranging from 5 mgmLÀ1 to 1 ngmLÀ1 were
prepared in FACS buffer (Dulbeccoꢁs PBS with 2% foetal calf serum
and 0.1% sodium azide). Staining was performed in a 96-well microtitre
plate (U bottom). Antibody (100 mL per well) was mixed with a 50 mL
8128
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130

Fluorescent Labelling of Biomacromolecules
FULL PAPER
cell suspension containing 5”105 HT-29 cells and incubated for 1 h on
ice. Cells were washed twice with FACS buffer by centrifuging the plate
and siphoning off the supernatant. Subsequently, F(ab)’2 goat-anti-human
IgG (H+L)-FITC (100 mL per well; Jackson ImmunoResearch, West
Grove, PA) diluted by 1.100 in FACS buffer was added as the second
step reagent. The plate was incubated for 1 h on ice in the dark and cells
were washed again twice as above. Dead cells were discriminated by
staining with propidium iodide. Analysis was carried out on a FACScan
cytometer (Becton Dickinson, Heidelberg, Germany) using the CELL-
QUEST software.
[18]E. I. Galanzha, V. V. Tuchin, V. P. Zharov, World J. Gastroenterol.
2007, 13, 192–218.
[19]N. A. Christian, M. C. Milone, S. S. Ranka, G. Li, P. R. Frail, K. P.
Davis, F. S. Bates, M. J. Therien, P. P. Ghoroghchian, C. H. June,
D. A. Hammer, Bioconjugate Chem. 2007, 18, 31–40.
[20]N. D. Lawson, B. M. Weinstein, Dev. Biol. 2002, 248, 307–318.
[21]S. Kim, Y. T. Lim, E. G. Soltesz, A. M. De Grand, J. Lee, A. Na-
kayama, J. A. Parker, T. Mihaljevic, R. G. Laurence, D. M. Dor,
L. H. Cohn, M. G. Bawendi, J. V. Frangioni, Nat. Biotechnol. 2004,
22, 93–97.
[22]E. G. Soltesz, S. Kim, R. G. Laurence, A. M. DeGrand, C. P. Parun-
go, D. M. Dor, L. H. Cohn, M. G. Bawendi, J. V. Frangioni, T. Mihal-
jevic, Ann. Thorac. Surg. 2005, 79, 269–277.
For each dendrimer conjugate as well as for unconjugated Matuzumab a
binding curve was obtained by plotting the mean fluorescence intensity
(MFI) versus the antibody concentration.
[23]S. Ohnishi, S. J. Lomnes, R. G. Laurence, A. Gogbashian, G. Maria-
ni, J. V. Frangioni, Mol. Imaging 2005, 4, 172–181.
[24]M. D. Shults, B. Imperiali, J. Am. Chem. Soc. 2003, 125, 14248–
14249.
[25]M. Ranji, S. Kanemoto, M. Matsubara, M. A. Grosso, J. H. Gorman,
R. C. Gorman, D. L. Jaggard, B. Chance, J. Biomed. Opt. 2006, 11,
064036.
Fluorescence microscopy studies of dansyl-labelled antibodies: A431
cells expressing considerable amounts of epidermal growth factor recep-
tor were used for the experiments. 5”104 cells were applied on cover
slips and incubated for 24 h. After washing, the cells were incubated with
solutions of the labelled antibodies (0.3 nmolmLÀ1) for 30 min at room
temperature. The cells were washed and subsequently fixed with parafor-
maldehyde (4%) for 15 min. Fluorescence microscopy was carried out
after 12 h of desiccation.
[26]A. Zaheer, T. E. Wheat, J. V. Frangioni, Mol. Imaging 2002, 1, 354–
364.
[27]J. Kang, M. Choi, J. Y. Kwon, E. Y. Lee, J. Yoon, J. Org. Chem.
2002, 67, 4384–4386.
[28]C.-T. Chen, W.-P. Huang, J. Am. Chem. Soc. 2002, 124, 6246–6247.
[29]A. Bencini, A. Bianchi, C. Giorgi, B. Valtancoli, J. Inclusion
Phenom. Macrocyclic Chem. 2001, 41, 87–93.
[30]M. Berton, F. Mancin, G. Stoccereo, P. Tecilla, U. Tonellato, Lang-
muir 2001, 17, 7521–7528.
[31]A. Almutairi, S. J. Guillaudeu, M. Y. Berezin, S. Achilefu, J. M. Frꢂ-
chet, J. Am. Chem. Soc. 2008, 130, 444–445.
[32]I. Grabchev, V. Bojinov, J. Photochem. Photobiol. A 2001, 139, 157–
160.
[33]M. K. Sharma, R. H.-S. Wang, J. J. Krutak (Eastman Chemical Co.,
Kingsport TN), US6162869, 2000; [Chem. Abstr. 2000, 134, 57466].
[34]J. G. Dominguez-Chꢃvez, I. Lijanova, I. Moggio, E. Arias, R. A.
Vazquez-Garcia, I. Reyes-Valderrama Ma, T. Klimova, M. Gutier-
rez-Nava, M. Martꢄnez-Garcꢄa, J. Nanosci. Nanotechnol. 2007, 7,
2758–2766.
Acknowledgements
The authors would like to thank T. Timmermann, Faculty of Pharmacy at
the University of Heidelberg, and G. Erben, German Cancer Research
Center, for recording NMR spectra and ESIMS, respectively. We would
also like to thank F. Kiessling, German Cancer Research Center, for pro-
viding access to the fluorescence spectrometer and H. Faulstich, Max-
Planck-Institut für Medizinische Forschung, Heidelberg, for his support
with Ellmanꢁs assay.
[1]A. Tsourkas, G. Bao, Briefings Funct. Genomics Proteomics 2003, 1,
372–384.
[35]I. V. Lijanova, I. Moggio, E. Arias, R. Vazquez-Garcia, M. Martꢄnez-
Garcꢄa, J. Nanosci. Nanotechnol. 2007, 7, 3607–3614.
[36]H. Kang, R. DeLong, M. H. Fisher, R. L. Juliano, Pharm. Res. 2005,
22, 2099–2106.
[37]G. Wu, R. F. Barth, W. Yang, S. Kawabata, L. Zhang, K. Green-
Church, Mol. Cancer Ther. 2006, 5, 52–59.
[2]A. Gertler, E. Biener, K. V. Ramanujan, J. Djiane, B. Herman, J.
Dairy Res. 2005, 72, 14–19.
[3]Y. Yin, M. A. Tainsky, F. Z. Bischoff, L. C. Strong, G. M. Wahl, Cell
1992, 70, 937–948.
[4]W. W. Zhang, S. Labrecque, E. Azoulay, R. Dudley, G. Matlashew-
ski, J. Biotechnol. 2001, 84, 79–86.
[5]H. Hug, M. Los, W. Hirt, K. M. Debatin, Biochemistry 1999, 38,
13906–13911.
[38]I. J. Majoros, A. Myc, T. Thomas, C. B. Mehta, J. R. Baker, Jr., Bio-
macromolecules 2006, 7, 572–579.
[39]J. Khandare, P. Kolhe, O. Pillai, S. Kannan, M. Lieh-Lai, R. M.
Kannan, Bioconjugate Chem. 2005, 16, 330–337.
[6]S. Kubota, M. Kuro-o, S. Mizuno, S. Kohno, Chromosoma 1993, 102,
163–173.
[40]O. L. Padilla De Jesus, H. R. Ihre, L. Gagne, J. M. Frechet, F. C. Szo-
ka, Jr., Bioconjugate Chem. 2002, 13, 453–461.
[41]W. Yang, R. F. Barth, G. Wu, A. K. Bandyopadhyaya, B. T. Thiruma-
magal, W. Tjarks, P. J. Binns, K. Riley, H. Patel, J. A. Coderre, M. J.
Ciesielski, R. A. Fenstermaker, Appl. Radiat. Isot. 2004, 61, 981–
985.
[7]M. Wijgerde, F. Grosveld, P. Fraser, Nature 1995, 377, 209–213.
[8]S. Kawamoto, H. Niwa, F. Tashiro, S. Sano, G. Kondoh, J. Takeda,
K. Tabayashi, J. Miyazaki, FEBS Lett. 2000, 470, 263–268.
[9]M . Fukuda, A. Sun, Eur. J. Histochem. 2005, 49, 11–26.
[10]J. M. Hoovers, C. H. Mellink, N. J. Leschot, Ned. Tijdschr. Geneeskd.
1999, 143, 2265–2268.
[42]R. F. Barth, G. Wu, W. Yang, P. J. Binns, K. J. Riley, H. Patel, J. A.
Coderre, W. Tjarks, A. K. Bandyopadhyaya, B. T. Thirumamagal,
M. J. Ciesielski, R. A. Fenstermaker, Appl. Radiat. Isot. 2004, 61,
899–903.
[43]M. V. Backer, T. I. Gaynutdinov, V. Patel, A. K. Bandyopadhyaya,
B. T. Thirumamagal, W. Tjarks, R. F. Barth, K. Claffey, J. M. Backer,
Mol. Cancer Ther. 2005, 4, 1423–1429.
[44]T. D. McCarthy, P. Karellas, S. A. Henderson, M. Giannis, D. F.
OꢁKeefe, G. Heery, J. R. A. Paull, B. R. Matthews, G. Holan, Mol.
Pharmaceutics 2005, 2, 312–318.
[45]P. Couck, R. Claeys, E. Vanderstraeten, F. K. Gorus, Acta Clin. Belg.
2005, 60, 75–78.
[11]H. C. Lichtenbeld, F. Yuan, C. C. Michel, R. K. Jain, Microcircula-
tion 1996, 3, 349–357.
[12]G. R. Martin, R. K. Jain, Cancer Res. 1994, 54, 5670–5674.
[13]C. Y. Li, S. Shan, Q. Huang, R. D. Braun, J. Lanzen, K. Hu, P. Lin,
M. W. Dewhirst, J. Natl. Cancer Inst. 2000, 92, 143–147.
[14]A. Kallioniemi, O. P. Kallioniemi, D. Sudar, D. Rutovitz, J. W. Gray,
F. Waldman, D. Pinkel, Science 1992, 258, 818–821.
[15]G. Imreh, M. Beckman, K. Iverfeldt, E. Hallberg, Exp. Cell Res.
1998, 238, 371–376.
[16]G. N. Naumov, S. M. Wilson, I. C. MacDonald, E. E. Schmidt, V. L.
Morris, A. C. Groom, R. M. Hoffman, A. F. Chambers, J. Cell Sci.
1999, 112, 1835–1842.
[17]J. O. McIntyre, B. Fingleton, K. S. Wells, D. W. Piston, C. C. Lynch,
S. Gautam, L. M. Matrisian, Biochem. J. 2004, 377, 617–628.
[46]M. T. Islam, X. Shi, L. Balogh, J. R. Baker, Jr., Anal. Chem. 2005,
77, 2063–2070.
Chem. Eur. J. 2008, 14, 8116 – 8130
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8129

W. Mier et al.
[47]J. M. J. Frꢂchet, D. A. Tomalia, Dendrimers and other dendritic poly-
mers, Wiley, Chichester, 2002, pp. 590–604.
[54]M. Lipowska, G. Patonay, L. Strekowski, Synth. Comm. 1993, 23,
3087–3094.
[48]D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J.
Roeck, J. Ryder, P. Smith, Macromolecules 1986, 19, 2466–2468.
[49]J. Peterson, A. Ebber, V. Allikmaa, M. Lopp, Proc. Est. Acad. Sci.
Chem. 2001, 50, 156–166.
[50]A. D. Meltzer, D. A. Tirrell, A. A. Jones, P. T. Inglefield, D. H. Hed-
strand, D. A. Tomalia, Macromolecules 1992, 25, 4541–4548.
[51]M. Maltese, J. Org. Chem. 2001, 66, 7615–7625.
[52]Y. Chen, L. Baker, J. Org. Chem. 1999, 64, 6870–6873.
[53]W. Pham, W.-F. Lai, R. Weissleder, C.-H. Tung, Bioconjugate Chem.
2003, 14, 1048–1051.
[55]B. K. Hçfelschweiger, Ph. D. Thesis, Universität Regensburg (Ger-
many), 2005.
[56]U. Mahmood, R. Weissleder, Mol. Cancer Ther. 2003, 2, 489–496.
[57]M. K. Samoszuk, A. Petersen, M. Lo-Hsueh, C. Rietveld, Antibody,
Immunoconjugates Radiopharm. 1989, 2, 37–45.
[58]R. F. Chen, Anal. Biochem. 1968, 25, 412–416.
Received: February 21, 2008
Revised: June 24, 2008
Published online: August 27, 2008
8130
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8116 – 8130