Proteo-dendrimers designed for complementary recognition of cytochrome c: Dendrimer architecture toward nanoscale protein complexation

Article abstract of DOI:10.1002/chem.200501131

"Proteo-dendrimers" in which polyanionic hepta(glutamic acids), fluorescent zinc porphyrinate cores, hydrophilic polyether surfaces, and nonpeptide hydrophobic dendrons are combined, were developed as a new series of synthetic receptors for protein recogn

Full text of DOI:10.1002/chem.200501131

DOI: 10.1002/chem.200501131
Proteo-Dendrimers Designed for Complementary Recognition of
Cytochrome c: Dendrimer Architecture toward Nanoscale Protein
Complexation
Dharam Paul, Hiroyuki Miyake, Satoshi Shinoda, and Hiroshi Tsukube*[a]
Dedicated to the late Emeritus Professor Kazuhiro Maruyama of Kyoto University
Abstract:
which
“Proteo-dendrimers”
polyanionic hepta(glutamic
in
observed in biological protein–protein
recognition systems. Stability constants
of the resulting supramolecular com-
plexes were determined in phosphate
buffer (pH 7) by monitoring the fluo-
rescence quenching of the zinc por-
phyrinates. These proteo-dendrimer re-
ceptors exhibited higher affinities with
cytochrome c proteins in aqueous solu-
tions than with biological cytochrome
b5. Furthermore, they effectively
blocked complexation of biological cy-
tochrome b5 with cytochrome c, indi-
cating that the proteo-dendrimers and
cytochrome b5 similarly occupy the
polycationic patch of cytochrome c.
acids), fluorescent zinc porphyrinate
cores, hydrophilic polyether surfaces,
and nonpeptide hydrophobic dendrons
are combined, were developed as a
new series of synthetic receptors for
protein recognition. They have polyan-
ionic “patch” structures on their sur-
faces and undergo complementary
electrostatic interactions with a posi-
tively charged cytochrome c patch, as
Keywords: cytochromes
· dendri-
mers · fluorescence · protein recog-
nition · supramolecular chemistry
Introduction
of cytochrome c.[2] Hirota et al. and Ogawa et al. reported
that polyanionic oligo(glutamic acids) and oligo(aspartic
acids) strongly bound cytochrome c.[3] Dendrimers have re-
ceived recent attention as more sophisticated receptors.[5]
Kluger and Zhang attached hemoglobin clusters onto the
dendrimer surface,[6] and Zimmerman et al. introduced the
molecularly imprinted cavity into the interior domains.[7]
Hirsch et al. first prepared polyanionic fullerene dendrimers
for cytochrome c complexation.[8] Although a few dendri-
mers have been constructed from combinations of different
dendritic blocks,[9] many biological examples suggest that
the dendrimers with asymmetrically distributed patch struc-
tures on their surface can offer nanoscale protein recogni-
tion.
We present a new series of “proteo-dendrimer”-type re-
ceptors for recognition of biological proteins (Scheme 1).
These dendrimers were designed to undergo complementary
electrostatic interactions with cytochrome c by including
several key features: 1) asymmetrically distributed polyan-
ionic hepta(glutamic acids) responsible for the interaction
with the polycationic patch on the cytochrome c surface,
2) a zinc porphyrinate core working as a fluorescence signal-
ing device, 3) a hydrophilic polyether surface for high water
solubility, and 4) nonpeptide dendritic components provid-
Biological electron-transfer reactions often involve highly
specific complexation between protein redox partners. Cyto-
chrome c and cytochrome b5 typically form a stable 1:1
supramolecular complex, in which two porphyrin centers are
arranged in a suitable geometry for efficient electron trans-
fer.[1] As cytochrome c has a positively charged “patch”
composed of four protonated lysines and cytochrome b5 has
several carboxylate anions on its surface, the two asymmetri-
cally distributed patches effectively dock to undergo com-
plementary electrostatic interactions for protein–protein
complexation. Several synthetic receptors have recently
been developed for cytochrome c complexation.[2–4] Hamil-
ton et al. demonstrated that tetraphenylporphyrins with four
ꢀ
-CO2 groups matched up well with the polycationic patch
[a]Dr. D. Paul, Dr. H. Miyake, Dr. S. Shinoda, Prof. H. Tsukube
Department of Chemistry, Graduate School of Science
Osaka City University, Sugimoto
Sumiyoshi-ku, Osaka 558-8585 (Japan)
Fax : (+81)6-6605-2560
E-mail: tsukube@sci.osaka-cu.ac.jp
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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Chem. Eur. J. 2006, 12, 1328 – 1338

FULL PAPER
type dendritic components of
different generations. Although
the same polyanionic peptide
was introduced, the generation
of the nonpeptide dendritic
component effectively regulat-
ed the supramolecular complex-
ation with cytochrome c. Due
to the size compatibility with
proteins, the present type of
proteo-dendrimers can work as
novel supramolecular receptors
of cytochrome c.
Scheme 1. Schematic illustration of proteo-dendrimers.
ing a peripheral, hydrophobic structure. As illustrated in
Figure 1, we linked an oligopeptide composed of seven glu-
tamic acid moieties to a zinc porphyrinate core. The result-
ing dendrimer has four pairs of -CO2 anions on its surface,
and provides a polyanionic patch to dock with the polycat-
Results and Discussion
Synthesis and characteristics of proteo-dendrimers: The
ester derivatives of proteo-dendrimers 1b–4b were obtained
by the coupling of peptide dendron 5 with zinc hepta-substi-
tuted tetraphenylporphyrinates 1c–4c (Scheme 2). Scheme 3
ꢀ
ionic patch of cytochrome c. We also attached benzyl ether
Figure 1. Proteo-dendrimers employed in this work.
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H. Tsukube et al.
Scheme 2. Synthesis of proteo-dendrimers 1a–4a. a) HOBt, HBTU, DIPEA, CH2Cl2; b) LiOH, H2O, THF-MeOH. Italic letters have been attached to
the structures for simplified descriptions of NMR assignments (see Experimental Section). * indicates the yields for (1st, 2nd, 3rd, and 4th generation).
Scheme 3. Synthesis of zinc porphyrinates 1c–4c. a) BF3·OEt2, CHCl3; then DDQ; b) BBr3, CH2Cl2, 08C to RT; c) Zn(OAc)2, CHCl3/MeOH, reflux;
d) K2CO3, [18]crown-6 ether, acetone, 508C; e) NaOH, H2O, THF/MeOH; f) Zn(OAc)2, CHCl3/MeOH, reflux; NaOH, H2O, THF/MeOH. Italic letters
have been attached to the structures for simplified descriptions of NMR assignments (see Experimental Section). * indicates the yields for (2nd, 3rd, and
4th generation).
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Proteo-Dendrimers
FULL PAPER
illustrates the synthesis of the zinc hepta-substituted tetra-
phenylporphyrinates. AB3-type porphyrin 6 was synthesized
by acid-catalyzed cross-condensation according to the Lind-
sey procedure.[10] After demethylation with BBr3,[11] zinc por-
phyrinate 8 was obtained by treatment with Zn(OAc)2 in
CHCl3/MeOH. Benzyl ether type dendritic branches with
three different generations were prepared as mesylates 9–11
starting from methyl 3,5-dihydroxybenzoate according to
the reported methods with some modifications.[11,12] Den-
drimers 2d–4d were obtained by reaction of zinc porphyrin
8 with 7–8 equivalents of the corresponding mesylate 9–11,
followed by hydrolysis to yield dendrimers 2c–4c, whereas
complex 1c was derived from 6. Peptide dendron 5 was
synthesized according to the literature[13] (see Scheme 4).
The ester dendrimer derivatives 1b–4b were synthesized
tochrome c (12173–12588 dalton and 4.0 nm, respectively):
for 2a, Mw =3680 dalton, d.=3.6 nm; for 3a, Mw =
6371 dalton, d.=5.0 nm; for 4a, Mw =11753 dalton, d.=
6.0 nm.[14] Table 1 summarizes absorption and fluorescence
Table 1. Spectroscopic profiles of proteo-dendrimers 2a–4a in aqueous
solution.[a] All values are lmax in nm.
UV
Fluorescence
Soret band
Q band
2a
3a
4a
429
436 (br)
434 (br)
559, 602
559, 599
559, 600
610, 658
610, 650
609, 648
[a]Conditions: dendrimer, 2.5”10 ꢀ7 moldmꢀ3
;
in 5”10ꢀ3 moldmꢀ3
sodium phosphate buffer, pH 7.0 (containing 10% DMF v/v); lex
558 nm.
=
spectral profiles of proteo-dendrimers 2a–4a. The com-
pounds exhibited almost the same spectra in aqueous DMF
(at pH 7.0), which indicated that the zinc porphyrinate core
of each dendrimer was located in a similar environment.
The conformation search of dendrimer 1a was carried out
by using CONFLEX 5 and subsequent Mulliken population
analysis was carried out by using Q-Chem (version 2.02,
RHF/STO-3G, Q-Chem. Inc., USA).[15] As illustrated in
Figure 2, the optimized structure reveals that three pairs of
Scheme 4. Synthesis of peptide dendron 5. a) CBZ-glutamic acid, HBTU,
DIPEA, CH2Cl2, 08C; b) ammonium formate, Pd/C, H2, MeOH.
by reactions of 1c–4c with an excess of peptide dendron 5
using 1-hydroxybenzotriazole (HOBt), O-(benzotriazol-
1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
(HBTU) and diisopropylethylamine (DIPEA). Esters 1b–
Figure 2. Optimized structure of zinc porphyrinate 1a.
1
4b were fully characterized by elemental analysis, H and
13C NMR spectroscopy, IR spectroscopy, and TOF-MS
methods (see Experimental Section). Hydrolysis of 1b–4b
with LiOH gave proteo-dendrimers 1a–4a that exhibited
ꢀ
-CO2 anions point toward the outside and the remaining
ꢀ
pair is located above the porphyrin plane. As one -CO2
1
broad H NMR signals, and their purities were confirmed by
anion of each pair can occupy the position matching the
protonated lysine residue of the cytochrome c, the intro-
duced oligopeptide is expected to provide a polyanionic
“patch” suitable for complementary electrostatic interac-
tions with cytochrome c.
capillary electrophoresis (see Figure S1 in the Supporting In-
formation). As the two -CO2H groups of parent glutamic
acid have pKa values of 2.19 and 4.25, all of the -CO2H
groups present in each proteo-dendrimer are expected to be
deprotonated at neutral pH.
The proteo-dendrimers have comparable molecular
weights (Mw) and diameters (d.) to those of the targeted cy-
Complexation with cytochrome c: Supramolecular complex-
ation between cytochrome c and proteo-dendrimers 2a–4a
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H. Tsukube et al.
was spectroscopically monitored in 5.0”10ꢀ3 moldmꢀ3 phos-
phate buffer solution containing 10% DMF (pH 7.0). As de-
scribed above, their zinc porphyrin cores gave intense fluo-
rescence bands around 610 and 652 nm upon photoexcita-
tion at their Q bands (558 nm). Addition of horse heart cy-
tochrome c, which contains a nonfluorescent heme, progres-
sively decreased the fluorescence intensity of each zinc
porphyrinate dendrimer (Figure 3). The relative fluores-
Figure 3. Fluorescence quenching of zinc porphyrinates with horse heart
cytochrome c and its acetylated derivatives. Typical titration data for
*
each dendrimer are shown. : 2a + cytochrome c; &: 3a + cytochrome
~
^
c; : 4a + cytochrome c; : 3a + acetylated cytochrome c. Conditions:
cytochrome c, 0–1.75”10ꢀ6 moldmꢀ3; dendrimer, 2.5”10ꢀ7 moldmꢀ3; in
5.0”10ꢀ3 moldmꢀ3 sodium phosphate buffer, pH 7.0 (containing 10%
DMF v/v); lex =558 nm; lem =610 nm. Each solid line was drawn based
on nonlinear curve fitting.
Figure 4. Job plots of proteo-dendrimers 2a–4a versus cytochrome c in
5.0”10ꢀ3 moldmꢀ3 phosphate buffer, pH 7.0 (containing 10% DMF v/v);
x=[cytochrome c]/([cytochrome c] + [dendrimer]); total concentration
cence intensity of these dendrimers (Fx/Ft, for which Fx =
fluorescence at molar ratio x and Ft =fluorescence of den-
drimer alone) significantly decreased upon addition of
7 equivalents of cytochrome c, and their changes clearly de-
pended on the dendrimer structures: 0.33 for 2a, 0.53 for
3a, and 0.72 for 4a. As the generation of the nonpeptide
component increased (2a!4a), the zinc porphyrinate of the
dendrimer was confirmed to be positioned away from the
bound cytochrome c heme. Lysine-acetylated horse heart cy-
tochrome c and microperoxidase-8[16] have fewer protonated
lysines than wild cytochrome c. When seven equivalents of
one of these compounds were added to a solution of den-
drimer 3a, the fluorescence quenching efficiencies were
modestly suppressed: Fx/Ft =0.91 with the acetylated one
and 0.80 with microperoxidase-8 (the quenching curve was
not included in Figure 3) relative to 0.53 with horse heart cy-
tochrome c. Thus, the positively charged patch on cyto-
chrome c was essentially involved in the complexation with
the proteo-dendrimer.
of two species was held at a) 2a, 5.0”10ꢀ7 moldmꢀ3
, b) 3a, 5.0”
10ꢀ7 moldmꢀ3, and c) 4a, 2.5”10ꢀ6 moldmꢀ3. The fluorescence intensity
was monitored at 658 nm.
no clear indication of a single component mainly existing in
each system. The supramolecular complexation behaviors of
proteo-dendrimers with cytochrome c were analyzed by the
fluorescence titration experiments (see Figure 3). The con-
centration of cytochrome
c varied from 0 to 1.75”
10ꢀ6 moldmꢀ3, although that of the proteo-dendrimer was
kept at 2.5”10ꢀ7 moldmꢀ3. The titration curves with den-
drimers 2a and 3a gave better fits for mixtures of 1:1 and
1:2 complexes than for solely 1:1 complexes, while dendri-
mer 4a exhibited satisfactory results based on both assump-
tions. The stability constants were estimated by the nonlin-
ear-curve-fit method and are listed in Table 2. Although the
same hepta(glutamic acids) provided major binding sites for
cytochrome c, dendrimers 2a and 3a exhibited higher stabil-
ity (2a: logK1 =7.6, logK2 =7.2; 3a: logK1 =7.7, logK2 =
7.2) than dendrimer 4a (logK1 =6.5, logK2 =7.1). The bulky
nonpeptide dendritic component of dendrimer 4a seems to
sterically suppress the electrostatic interactions between the
polyanionic dendrimer and the polycationic cytochrome c
protein. Table 2 also lists fluorescence intensity ratios of cy-
Job plots for fluorescence quenching of dendrimers 2a–4a
with cytochrome c are illustrated in Figure 4.[17] Dendrimer
2a appeared to form both 1:1 and 1:2 complexes (cyto-
chrome c/dendrimer), whereas 3rd and 4th generation den-
drimers 3a and 4a, respectively, exhibited roundly bent
curves of around 1:1 stoichiometry. Thus, these plots gave
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Proteo-Dendrimers
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Table 2. Stability constants between proteo-dendrimers 2a–4a and cyto-
chrome c[a] and calculated fluorescence intensity ratios of supramolecular
complexes to free dendrimer.
and cytochrome b5 play important roles in protein recogni-
tion.[16] The proteo-dendrimers competed well with cyto-
chrome b5 and blocked biologically important complexation
with cytochrome c. Figure 5 illustrates the effects of cyto-
[b]
[c]
logK1
logK2
I1:1/Io
I1:2/Io
2a
3a
7.6Æ0.3
7.7Æ0.3
6.5Æ0.1
6.3Æ0.1
7.2Æ0.3
7.2Æ0.3
7.1Æ0.2
0.29Æ0.02
0.48Æ0.05
0.45Æ0.14
0.59Æ0.06
1.10Æ0.20
1.76Æ0.10
1.96Æ0.03
4a
4a[d]
[a]Conditions: horse heart cytochrome c, 0–1.75”10ꢀ6 moldmꢀ3; den-
drimer, 2.5”10ꢀ7 moldmꢀ3; in 5”10ꢀ3 moldmꢀ3 sodium phosphate buffer,
pH 7.0 (containing 10% DMF v/v); lex =558 nm; lem =610 nm. The aver-
aged values of four or five independent experiments are shown. [b]Cal-
culated fluorescence intensity ratio of a 1:1 complex to free dendrimer.
[c]Calculated fluorescence intensity ratio of a 1:2 complex to free den-
drimer. [d]The data was fitted considering only 1:1 complexation.
tochrome c bound dendrimer to free dendrimer. Each 1:2
complex contained two zinc porphyrinates, but its fluores-
cence was modestly quenched. The two zinc porphyrinates
of the bound dendrimers were located under different envi-
ronments. Because simple zinc porphyrinate 1a gave a parti-
ally insoluble complex with cytochrome c in the employed
neutral aqueous solution, nonpeptide dendritic substituents
must play a significant role in the solubilization and com-
plexation processes.
Figure 5. Competitive binding of cytochrome c between proteo-dendri-
mer 3a and cytochrome b5. a) b, 3a; b) c, 3a + cytochrome c;
c) g, 3a + cytochrome c + cytochrome b5). Conditions: dendrimer 3a,
2.5”10ꢀ7 moldmꢀ3; horse heart cytochrome c, 2.5”10ꢀ7 moldmꢀ3; cyto-
chrome b5, 3.5”10ꢀ6 moldmꢀ3; in 5.0”10ꢀ3 moldmꢀ3 phosphate buffer,
pH 7.0 (containing 10% DMF v/v); lex =558 nm.
Proteo-dendrimers contained chiral oligopeptide moieties,
but gave very small circular dichroism (CD) signals in the
Soret region. They exhibited CD signals of the same sign at
432 and 442 nm upon cytochrome c complexation, which
correspond to the zinc porphyrinate absorption bands. As
cytochrome c itself displayed CD signals around 406 nm, the
above-mentioned observed CD signals indicated asymmetric
orientation of the zinc porphyrinate upon complexation with
cytochrome c.[18] Proteo-dendrimer 2a gave four times larger
signals than dendrimer 3a; this suggests that the generation
of the nonpeptide component influenced the orientations
and distances between the two porphyrinates.[19] The dense
peripheral moieties of the higher generation dendrimer
were thought to cause steric hindrance around the polyan-
ionic patch and to prevent the docking with cytochrome c.
Pigeon breast and yeast cytochromes c also have polycation-
ic patches for complementary interactions with proteo-den-
drimers, though the latter has lower solubility in the aque-
ous medium. Dendrimer 3a typically formed a stable com-
plex with the former (logK1 =7.5, logK2 =6.5), but insoluble
material with the latter.
chrome c and cytochrome b5 on fluorescence of proteo-den-
drimer 3a. An equimolar addition of cytochrome c largely
decreased the fluorescence intensity of dendrimer 3a (see
Figure 5a!b). When 14 equivalents of cytochrome b5 were
added to this mixture, the fluorescence signals partially re-
covered (see Figure 5c). This indicates that approximately
20% of the dendrimer-bound cytochrome c was released
and then complexed with cytochrome b5. Such competitive
binding behavior supports the idea that proteo-dendrimer
3a formed a more stable complex with cytochrome c than
biological cytochrome b5, and also that both dendrimer and
cytochrome b5 occupied the polycationic patch of cyto-
chrome c in the same fashion. Supramolecular complexation
with crown ethers, calixarenes, and other synthetic receptors
have often been reported for modifying the structure and re-
activity of cytochrome c.[4] Although resonance Raman and
CD spectroscopic methods (in the a-helix region) were not
available for the present systems, the cytochrome c com-
plexes with proteo-dendrimers exhibited almost the same
UV signal intensity at 690 nm as that of free cytochrome c.
Thus, the methionine-coordinated heme center was accom-
modated in the protein matrix as observed in biological sys-
tems.[16]
Competitive binding with cytochrome b5: Proteo-dendrimers
2a–4a formed stable complexes with horse heart cyto-
chrome c at neutral pH. Their stability constants are larger
than those reported with cytochrome b5 (logK=4.8)[20] and
fullerene dendrimer (logK=5.2),[8] though direct compari-
sons are difficult under different ionic strength and buffer
conditions. The competitive binding of cytochrome c be-
tween proteo-dendrimer and cytochrome b5 was carried out
by monitoring the fluorescence of zinc porphyrinate den-
drimer at 610 nm. It has already been established that com-
plementary electrostatic interactions between cytochrome c
Conclusion
A new series of proteo-dendrimers were successfully devel-
oped, in which polyanionic peptide binding sites, neutral
dendritic structures, and zinc porphyrinate cores were com-
bined in a nanosize three-dimensional skeleton. These struc-
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H. Tsukube et al.
reported in ref. [10]. Yield: 90%. 1H NMR (400 MHz, CD3OD): d=4.08
(s, 3H; COOMe), 6.70 (m, 3H; Hp), 7.16 (m, 6H; Hq), 8.32 (d, J=8.0 Hz,
2H; Hy), 8.42 (d, J=8.0 Hz, 2H; Hx), 8.76 (d, J=4.4 Hz, 2H; pyrrole-b),
8.98 (s, 4H; pyrrole-b) 8.99 ppm (d, J=4.4 Hz, 2H; pyrrole-b); 13C NMR
(75 MHz, CD3OD): d=52.85, 102.62, 115.87, 119.79, 121.99, 122.13,
128.55, 130.17, 131.72, 132.52, 132.57, 132.83, 135.77, 146.66, 150.27,
150.62, 151.09, 151.30, 151.37, 157.43, 168.92 ppm; MS (FAB): m/z: 830.2
[M]+.
tures undergo effective complementary electrostatic interac-
tions with the polycationic surface of cytochrome c. The re-
sulting supramolecular complexes were confirmed to be
more stable than a biological protein pair of cytochrome c
and cytochrome b5 in aqueous solution. The asymmetrically
distributed patch structures on the dendrimer surfaces effi-
ciently offered sites for nanoscale protein recognition.
Compound 1c: Porphyrin 6 was metallated with Zn(OAc)2 by using a
procedure similar to that reported[10] in the literature, and the isolated
product was dissolved in THF (5 mL) and MeOH (5 mL), and NaOH
(50 mg in 0.5 mL water) was added to the reaction mixture. The progress
of hydrolysis was monitored by TLC (silica plate, 2% EtOAc in dichloro-
methane). After completion of the reaction (5 h), the solvents were re-
moved under vacuum and the residue was dissolved in dichloromethane
and washed with water, 2% citric acid, water, and brine (with gentle
shaking). The organic layer was separated, dried over Na2SO4, and
evaporated. The residue was purified by silica gel chromatography using
10% EtOAc in dichloromethane (to remove unhydrolyzed ester deriva-
tive) and then 10% MeOH in acetone, giving 1c (92%). 1H NMR
(300 MHz, CD3OD): d=3.94 (s, 12H; OMe), 3.95 (s, 6H; OMe), 6.90–
6.92 (m, 3H; Hp), 7.36–7.37 (m, 6H; Hq), 8.26 (d, J=8.0 Hz, 2H; Hy),
8.40 (d, J=8.2 Hz, 2H; Hx), 8.80 (d, J=4.7 Hz, 2H; pyrrole-b), 8.91 ppm
(m, 6H; pyrrole-b); MS (FAB): m/z: 900.3 [M]+.
Experimental Section
Materials: Cytochromes c from horse heart (Wako, Mw =12384), pigeon
breast muscle (Sigma-Aldrich, Mw =12173), and Saccaromyces cerevisiae
yeast (Sigma-Aldrich, Mw =12588) are commercially available. As their
absorbance at the Soret band region was only slightly enhanced by oxida-
tion with K3[Fe(CN)6], we used them as received. Acetylated horse heart
cytochrome c was obtained from Sigma-Aldrich, in which 60% of the
lysine residues were derivatized. Microperoxidase-8 was also obtained
from Sigma-Aldrich. This is partially hydrolyzed material of the horse
heart cytochrome c and has the heme portion with amino acids 14–21 at-
tached.[16] Cytochrome b5 was purchased from Wako Pure Chemical In-
dustries and used after dialysis.
Synthesis of dendritic methanesulfonates 9–11[11,12]
1
All the reaction mixtures containing porphyrin derivatives and dendri-
mers were protected from light by covering reaction flasks with alumi-
num foil. K2CO3 was dried in an oven at 1508C for 5 h and cooled under
a dry N2 atmosphere. Dry acetonitrile and THF were freshly distilled
over P2O5 and sodium benzophenone, respectively. Stabilizer-free THF
was used in the hydrolysis of ester dendrimers. Chloroform was used as
an eluting solvent for gel permeation chromatography (GPC) purification
of dendrimers using JAIGEL 1H-2H or 2H-3H column combinations
(Japan Analytical Industry Co., Ltd.). Phosphate buffer was prepared to
adjust the pH to 7.0 by mixing 5.0”10ꢀ3 moldmꢀ3 solutions of mono- and
dibasic sodium phosphate in deionized water containing 10% DMF.
1H and 13C NMR spectra were recorded on JEOL LA-300 and LA-400
spectrometers. The details of their assignments refer to the structures de-
scribed in Schemes 2–4. IR and CD spectra were obtained on Perkin–
Elmer SpectrumOne FTIR and Jasco J-720 spectrometers, respectively.
FAB mass spectra were measured on a JEOL JMS-700T spectrometer
using 3-nitrobenzyl alcohol as a matrix. MALDI-TOF mass experiments
were carried out by the Shimadzu Corporation, Tsukuba, Japan, using
Compound 9: H NMR (400 MHz, CDCl3): d=2.91 (s, 3H; SO2Me), 3.40
(s, 6H; OMe), 3.58 (AA’BB’ multiplet, 4H; CH2), 3.72 (AA’BB’ multip-
let, 4H; CH2), 3.85 (brt, J=4.8 Hz, 4H; CH2), 4.12 (brt, J=4.8 Hz, 4H;
CH2), 5.15 (s, 2H; benzylic-CH2), 6.51 (t, J=2.4 Hz, 1H; ArH), 6.56 ppm
(d, J=2.4 Hz, 2H; ArH); 13C NMR (100 MHz, CDCl3): d=38.23, 58.90,
67.42, 69.44, 70.57, 71.30, 71.74, 102.13, 107.19, 135.22, 160.05 ppm; MS
(FAB): m/z: 422.2 [M+H]+.
Compound 10: 1H NMR (400 MHz, CDCl3): d=2.86 (s, 3H; SO2Me),
3.39 (s, 12H; OMe), 3.58 (AA’BB’ multiplet, 8H; CH2), 3.72 (AA’BB’
multiplet, 8H; CH2), 3.85 (brt, J=4.8 Hz, 8H; CH2), 4.12 (brt, J=
4.8 Hz, 8H; CH2), 4.96 (s, 4H; benzylic-CH2), 5.15 (s, 2H; benzylic-CH2),
6.45 (t, J=2.4 Hz, 2H; ArH), 6.57–6.60 ppm (m, 7H; ArH); 13C NMR
(100 MHz, CDCl3): d=38.19, 58.88, 67.29, 69.48, 69.79, 70.53, 71.24,
71.74, 100.92, 102.80, 105.82, 107.40, 135.36, 138.62, 159.89, 159.93 ppm;
MS (FAB): m/z: 871.4 [M+H]+.
Compound 11: 1H NMR (400 MHz, CDCl3): d=2.86 (s, 3H; OSO2Me),
3.38 (s, 24H; OMe), 3.57 (AA’BB’ multiplet, 16H; CH2), 3.71 (AA’BB’
multiplet, 16H; CH2), 3.84 (brt, J=4.8 Hz, 16H; CH2), 4.12 (brt, J=
4.8 Hz, 16H; CH2), 4.95 (brs, 8H; benzylic-CH2), 4.98 (brs, 4H; benzyl-
ic-CH2), 5.16 (s, 2H; benzylic-CH2), 6.45 (brt, J=1.6 Hz, 4H; ArH), 6.54
(brt, J=1.6 Hz, 2H; ArH), 6.59 (s, 10H; ArH), 6.63–6.65 ppm (m, 5H;
ArH); 13C NMR (100 MHz, CDCl3): d=37.75, 58.56, 67.03, 69.20, 69.46,
69.51, 70.22, 71.00, 71.46, 100.64, 101.15, 102.39, 105.60, 105.91, 107.13,
135.24, 138.56, 138.65, 159.62, 159.64 ppm; MS (FAB): m/z: 1766.8 [M]+.
Shimadzu AXIMA CFRplus apparatus:
a trans-3-indoleacrylic acid
matrix in THF for 2b–3b; a sinapic acid matrix in acetonitrile/water for
1a–4a. The calculated molecular masses are the average molecular
masses. Capillary electrophoresis was carried out with Otsuka CAPI-3200
apparatus using a bare fused-silica capillary (75 mm (i.d.)”45 cm).
Synthesis of zinc porphyrinate 1c
Compound 6: This AB3-type porphyrin was synthesized by acid-catalyzed
cross-condensation according to the Lindsey procedure[10] in a 12% yield.
1H NMR (300 MHz, CDCl3): d=ꢀ2.83 (s, 2H; NH), 3.96 (s, 18H; OMe),
4.11 (s, 3H; COOMe), 6.90 (brt, 3H; Hp), 7.39 (brd, 6H; Hq), 8.30 (d,
J=8.2 Hz, 2H; Hy), 8.44 (d, J=8.2 Hz, 2H; Hx), 8.77 (d, J=4.7 Hz, 2H;
pyrrole-b), 8.95 (s, 4H; pyrrole-b) 8.96 ppm (d, J=4.7 Hz, 2H; pyrrole-
b); 13C NMR (75 MHz, CDCl3): d=52.42, 55.62, 100.15, 113.85, 118.61,
120.03, 120.09, 127.91, 129.56, 131.8, 134.52, 143.89, 147.00, 158.85,
167.33 ppm; MS (FAB): m/z: 853.4 [M+H]+.
Synthesis of ester dendrimers 2d–4d
Compound 2d: Zinc 5-(4-methoxycarbonylphenyl)-10,15,20-tris(3,5-dihy-
droxyphenyl)porphinate
8 (100 mg, 0.12 mmol), mesylate 9 (355 mg,
0.84 mmol), anhydrous K2CO3 (200 mg, 1.24 mmol) and [18]crown-6
(20 mg, 0.08 mmol) were added to dry acetone (20 mL). The reaction
mixture was stirred at 508C under an N2 atmosphere. The progress of the
reaction was monitored by TLC by using acetone/dichloromethane (3:7).
After completion of the reaction (20 h), the solvents were removed
under vacuum and the residue was dissolved in dichloromethane (30 mL)
and filtered. The separated solid was washed with dichloromethane. The
filtrate and washings were collected and evaporated. The residue was dis-
solved in a minimum amount of dichloromethane (ca. 5 mL), and MeOH
(ca. 30 mL) was added with stirring. The product precipitated as a thick
gum and the solvents were separated by decantation. The residue was pu-
rified by silica gel column chromatography using EtOAc (to remove me-
sylate), dichloromethane, and then 30% acetone in dichloromethane as
eluent, giving 2d (76%). 1H NMR (CDCl3, 400 MHz): d=3.12 (s, 12H;
OMe’), 3.13 (s, 24H; OMe), 3.28–3.31 (m, 24H; Ha and Ha’), 3.47–3.50
(m, 24H; Hb and Hb’), 3.73 (brt, J=4.4 Hz, 24H; Hc and Hc’), 4.06 (brt,
Compound 7: Demethylation of 6 was carried out by using BBr3 by fol-
lowing the procedure similar to that reported in ref. [11]to give 7 (35%).
1H NMR (400 MHz, CD3OD): d=4.07 (s, 3H; COOMe), 6.72–6.73 (m,
3H; Hp and Hp’), 7.15 (d, J=2.2 Hz, 4H; Hq), 7.16 (d, J=2.2 Hz, 2H;
Hq’), 8.29 (d, J=8.3 Hz, 2H; Hy), 8.41 (d, J=8.3 Hz, 2H; Hx), 9.00 ppm
(brm, 8H; pyrrole-b); 13C NMR (75 MHz, CD3OD): d=52.90, 103.23,
115.77, 119.51, 121.74, 121.88, 128.92, 130.82, 135.67, 144.96, 148.32,
157.93, 168.62 ppm; MS (FAB): m/z: 769.2 [M+H]+.
Compound 8: Porphyrin metalation was carried out with Zn(OAc)2 in a
mixture of chloroform and MeOH by using a procedure similar to that
1334
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 1328 – 1338

Proteo-Dendrimers
FULL PAPER
J=4.8 Hz, 24H; Hd and Hd’), 4.11 (s, 3H; COOMe), 5.14 (s, 12H; Hg and
Hg’), 6.42–6.44 (m, 6H; He and He’), 6.64–6.66 (m, 12H; Hf and Hf’), 7.02–
7.04 (m, 3H; Hp and Hp’), 7.46 (d, J=2.4 Hz, 4H; Hq), 7.48 (d, J=2.4 Hz,
2H; Hq’), 8.30 (d, J=8.4 Hz, 2H; Hy), 8.43 (d, J=8.4 Hz, 2H; Hx), 8.81
(d, J=4.4 Hz, 2H; pyrrole-b), 8.94–8.97 ppm (m, 6H; pyrrole-b);
13C NMR (CDCl3, 100 MHz): d=52.17, 58.65, 67.24, 69.36, 70.14, 70.30,
70.33, 71.47, 71.49, 101.10, 101.64, 106.08, 114.99, 119.17, 120.50, 120.54,
127.52, 128.96, 131.20, 131.89, 132.02, 134.38, 139.00, 144.65, 144.70,
147.93, 149.26, 149.63, 149.78, 149.81, 157.59, 157.61, 159.89, 167.19 ppm;
IR (film between KBr discs): n˜ =1718 cmꢀ1 (ester C=O); UV/Vis
10 mL). The organic layer was separated, dried over MgSO4, filtered, and
evaporated. The residue was purified by silica gel chromatography using
acetone and then 10% MeOH in acetone to give 2c (92%). 1H NMR
(CDCl3, 400 MHz): d=3.12 (s, 24H; OMe), 3.14 (s, 12H; OMe’), 3.27–
3.33 (m, 24H; Ha and Ha’), 3.45–3.51 (m, 24H; Hb and Hb’), 3.69–3.74 (m,
24H; Hc and Hc’), 4.03–4.08 (m, 24H; Hd and Hd’), 5.14 (brs, 8H; Hg),
5.16 (brs, 4H; Hg’), 6.42 (brt, J=2.0 Hz, 4H; He), 6.44 (brt, J=2.0 Hz,
2H; He’), 6.64 (brd, J=2.0 Hz, 8H; Hf), 6.66 (brd, J=2.0 Hz, 4H; Hf’),
7.03–7.05 (brm, 3H; Hp and Hp’), 7.48 (brd, J=1.6 Hz, 4H; Hq), 7.49
(brd, J=1.6 Hz, 2H; Hq’), 8.34 (d, J=8.2 Hz, 2H; Hy), 8.49 (d, J=
8.2 Hz, 2H; Hx), 8.84 (d, J=4.8 Hz, 2H; pyrrole-b), 8.95 ppm (m, 6H;
pyrrole-b); 13C NMR (CDCl3, 100 MHz): d=58.70, 58.72, 67.37, 67.39,
69.46, 69.47, 70.27, 70.39, 70.42, 71.57, 71.60, 101.26, 101.83, 106.22,
115.14, 119.19, 120.60, 120.65, 128.16, 128.61, 131.28, 131.92, 132.12,
134.57, 139.12, 144.73, 144.78, 148.60, 149.34, 149.74, 149.89, 149.91,
157.70, 157.72, 160.00, 160.01, 170.32 ppm; IR (film between KBr discs):
n˜ =1718 cmꢀ1 (acid C=O); UV/Vis (CHCl3): lmax (loge)=427 (5.76), 555
(4.38), 595 nm (3.78); elemental analysis calcd (%) for C147H184N4O44Zn:
C 63.59, H 6.68, N 2.02; found: C 63.76, H 6.82, N 2.00.
(CHCl3)
: lmax (loge)=427 (5.78), 555 (4.39), 595 nm (3.79); MS
(MALDI-TOF): m/z: 2786.3 [M]+; elemental analysis calcd (%) for
C148H186N4O44Zn: C 63.70, H 6.72, N 2.01; found: C 63.53, H 6.72, N 1.94.
Compound 3d: Dendrimer 3d was obtained by coupling of zinc por-
phyrinate 8 with mesylate 10, by using the same procedure as that used
1
for 2d (66%). H NMR (CDCl3, 400 MHz): d=3.18 (s, 24H; OMe’), 3.21
(s, 48H; OMe), 3.32–3.35 (m, 16H; Ha’), 3.36–3.39 (m, 32H; Ha), 3.46–
3.49 (m, 16H; Hb’), 3.51–3.53 (m, 32H; Hb), 3.64 (brt, J=4.8 Hz, 16H;
Hc’), 3.68 (brt, J=4.8 Hz, 32H; Hc), 3.96 (brt, J=4.8 Hz, 16H; Hd’), 4.00
(brt, J=4.8 Hz, 32H; Hd), 4.11 (s, 3H; COOMe), 4.92 (brs, 8H; Hg’),
4.93 (brs, 16H; Hg), 5.14 (s, 12H; Hj and Hj’), 6.34 (brt, J=2.4 Hz, 4H;
He’), 6.36 (brt, J=2.4 Hz, 8H; He), 6.49 (d, J=2.0 Hz, 8H; Hf’), 6.51 (d,
J=2.0 Hz, 16H; Hf), 6.53 (brs, 6H; Hh and Hh’), 6.73 (brd, J=2.0 Hz,
12H; Hi and Hi’), 7.06 (brt, J=2.0 Hz, 3H; Hp and Hp’), 7.49 (brd, J=
2.0 Hz, 6H; Hq and Hq’), 8.24 (d, J=8.2 Hz, 2H; Hy), 8.39 (d, J=8.2 Hz,
2H; Hx), 8.80 (d, J=4.6 Hz, 2H; pyrrole-b), 8.98–9.03 ppm (m, 6H; pyr-
role-b); 13C NMR (CDCl3, 100 MHz): d=52.18, 58.69, 58.71, 67.14, 67.18,
69.32, 69.35, 69.79, 70.04, 70.29, 70.33, 71.53, 71.56, 100.88, 101.16, 101.62,
105.81, 106.35, 106.41, 115.13, 119.12, 120.41, 120.52, 127.50, 128.93,
131.18, 131.95, 134.38, 138.90, 139.02, 139.06, 144.78, 147.94, 149.23,
149.60, 149.74, 149.81, 157.59, 159.80, 159.81, 159.89, 167.22 ppm; IR
(film between KBr discs): n˜ =1719 cmꢀ1 (ester C=O). UV/Vis (CHCl3):
lmax (loge)=427 (5.78), 555 (4.38), 595 nm (3.77); MS (MALDI-TOF):
m/z: 5476.5 [M]+; elemental analysis calcd (%) for C292H378N4O92Zn: C
63.98, H 6.95, N 1.02; found: C 63.92, H 7.06, N 1.03.
Compound 3c: Dendrimer 3d was hydrolyzed to dendrimer 3c by using
the same procedure as that used for 2c. Yield: 85%; 1H NMR (CDCl3,
400 MHz): d=3.19 (s, 24H; OMe’), 3.20 (s, 48H; OMe), 3.34–3.38 (m,
48H; Ha and Ha’), 3.48–3.52 (m, 48H; Hb and Hb’), 3.64–3.69 (m, 48H; Hc
and Hc’), 3.96–4.01 (m, 48H; Hd and Hd’), 4.93 (s, 24H; Hg and Hg’), 5.13–
5.21 (m, 12H; Hj and Hj’), 6.35 (brt, J=2.4 Hz, 4H; He’), 6.37 (brt, J=
2.4 Hz, 8H; He), 6.49 (brd, J=2.0 Hz, 8H; Hf’), 6.50 (brd, J=2.0 Hz,
16H; Hf), 6.54 (brt, J=2.0 Hz, 6H; Hh and Hh’), 6.74 (brd, J=1.6 Hz,
12H; Hi and Hi’), 7.04–7.06 (m, 3H; Hp and Hp’), 7.46 (brd, J=2.4 Hz,
4H; Hq), 7.48 (brd, J=2.0 Hz, 2H; Hq’), 8.14 (d, J=7.8 Hz, 2H; Hy), 8.29
(d, J=7.8 Hz, 2H; Hx), 8.70 (d, J=4.8 Hz, 2H; pyrrole-b), 8.80 (d, J=
4.4 Hz, 2H; pyrrole-b), 9.00 (d, J=4.8 Hz, 2H; pyrrole-b), 9.05 ppm (d,
J=4.4 Hz, 2H; pyrrole-b); 13C NMR (CDCl3, 100 MHz): d=58.74, 67.21,
69.37, 69.40, 69.83, 70.06, 70.33, 70.50, 71.60, 100.92, 101.57, 101.66,
105.78, 105.85, 106.43, 115.20, 115.41, 119.24, 120.45, 120.51, 127.85,
128.86, 131.27, 131.97, 134.44, 138.97, 139.12, 139.19, 144.73, 144.86,
147.98, 149.24, 149.65, 149.73, 149.81, 157.56, 157.63, 159.85, 159.86,
159.94, 159.98, 168.62 ppm; IR (film between KBr discs): n˜ =1716 cmꢀ1
(acid C=O); UV/Vis (CHCl3): lmax (loge)=427 (5.75), 555 (4.35), 595 nm
(3.77); elemental analysis calcd (%) for C291H376N4O92Zn: C 63.93, H
6.93, N 1.02; found: C 63.75, H 7.08, N 0.99.
Compound 4d: Dendrimer 4d was obtained by coupling of zinc por-
phyrinate 8 with mesylate 11 (8.0 molar equiv) by using the same proce-
dure as that for 2d and was finally purified with 2H-3H JAIGEL GPC.
Yield: 64%; 1H NMR (CDCl3, 400 MHz): d=3.22 (s, 48H; OMe’), 3.26
(s, 96H; OMe), 3.36–3.39 (m, 32H; Ha’), 3.41–3.44 (m, 64H; Ha), 3.50–
3.53 (m, 32H; Hb’), 3.56–3.58 (m, 64H; Hb), 3.67 (brt, J=4.8 Hz, 32H;
Hc’), 3.72 (brt, J=4.8 Hz, 64H; Hc), 3.96 (brt, J=4.4 Hz, 32H; Hd’), 4.01
(brt, J=4.8 Hz, 64H; Hd), 4.06 (s, 3H; COOMe), 4.82 (s, 16H; Hg’), 4.87
(s, 32H; Hg), 4.91 (brs, 8H; Hj’), 4.92 (brs, 16H; Hj), 5.13 (brs, 12H; Hm
and Hm’), 6.33 (brt, J=2.4 Hz, 8H; He’), 6.37 (brt, J=2.4 Hz, 16H; He),
6.44 (brt, J=2.4 Hz, 4H; Hh’), 6.46 (brd, J=2.4 Hz, 24H; Hf’ and Hh),
6.50 (d, J=2.4 Hz, 32H; Hf), 6.56 (brt, J=2.4 Hz, 6H; Hk and Hk’), 6.59
(brd, J=2.4 Hz, 8H; Hi’), 6.62 (brd, J=2.4 Hz, 16H; Hi), 6.77 (brm,
12H; Hl and Hl’), 7.08 (brs, 3H; Hp and Hp’), 7.54 (brs, 6H; Hq and Hq’),
8.23 (d, J=8.2 Hz, 2H; Hy), 8.36 (d, J=8.2 Hz, 2H; Hx), 8.81 (d, J=
4.8 Hz, 2H; pyrrole-b), 9.02 (d, J=4.8 Hz, 2H; pyrrole-b), 9.07–9.10 ppm
(m, 4H; pyrrole-b); 13C NMR (CDCl3, 100 MHz): d=52.05, 58.63, 58.67,
67.07, 67.12, 69.26, 69.31, 69.54, 69.60, 69.71, 70.25, 70.30, 71.50, 71.54,
100.78, 101.25, 105.70, 105.75, 106.11, 106.25, 106.46, 114.92, 119.07,
120.37, 120.48, 127.42, 128.81, 131.12, 131.86, 134.30, 138.79, 138.91,
139.03, 144.75, 147.80, 149.14, 149.54, 149.67, 149.74, 157.59, 159.68,
159.72, 159.85, 167.05 ppm; IR (film between KBr discs): n˜ =1718 cmꢀ1
(ester C=O); UV/Vis (CHCl3): lmax (loge)=427 (5.78), 555 (4.38),
595 nm (3.78); MS (MALDI-TOF): m/z: 10863.8 [M]+; elemental analy-
sis calcd (%) for C580H762N4O188Zn: C 64.12, H 7.07, N 0.52; found: C
63.81, H 7.12, N 0.62.
Compound 4c: Ester dendrimer 4d was hydrolyzed to dendrimer 4c
(83%) by using the same procedure as that used for dendrimer 2c.
1H NMR (CDCl3, 300 MHz): d=3.23 (s, 48H; OMe’), 3.27 (s, 96H;
OMe), 3.38–3.40 (m, 32H; Ha’), 3.43–3.45 (m, 64H; Ha), 3.51–3.54 (m,
32H; Hb’), 3.57–3.59 (m, 64H; Hb), 3.68 (brt, J=4.2 Hz, 32H; Hc’), 3.73
(brt, J=5.1 Hz, 64H; Hc), 3.97 (brt, J=5.1 Hz, 32H; Hd’), 4.02 (brt, J=
5.1 Hz, 64H; Hd), 4.83 (brs, 16H; Hg’), 4.87 (s, 32H; Hg), 4.91 (brs, 8H;
Hj’), 4.93 (brs, 16H; Hj), 5.13 (brs, 12H; Hm and Hm’), 6.34 (brt, J=
2.1 Hz, 8H; He’), 6.38 (brt, J=2.1 Hz, 16H; He), 6.45–6.48 (brm, 28H;
Hh’, Hf’ and Hh), 6.52 (brd, J=2.1 Hz, 32H; Hf), 6.57 (brs, 6H; Hk and
Hk’), 6.60 (brd, J=1.8 Hz, 8H; Hi’), 6.62 (brd, J=1.8 Hz, 16H; Hi), 6.77
(brd, J=1.8 Hz, 12H; Hl and Hl’), 7.06–7.08 (brm, 3H; Hp and Hp’), 7.54
(brs, 6H; Hq and Hq’), 8.20 (d, J=8.0 Hz, 2H; Hy), 8.35 (d, J=8.0 Hz,
2H; Hx), 8.81 (brd, J=4.8, 2H; pyrrole-b), 8.99 (d, J=4.8 Hz, 2H; pyr-
role-b), 9.06 (brd, J=4.8 Hz, 2H; pyrrole-b), 9.10 ppm (brd, J=4.8 Hz,
2H; pyrrole-b); 13C NMR (CDCl3, 100 MHz): d=58.76, 58.79, 67.24,
67.27, 69.40, 69.45, 69.71, 69.76, 69.87, 70.39, 70.43, 71.63, 71.68, 100.91,
100.96, 101.40, 105.87, 105.93, 106.27, 106.50, 106.61, 115.15, 119.42,
120.48, 120.54, 127.83, 129.18, 131.39, 132.01, 134.42, 138.94, 139.03,
139.10, 139.14, 144.89, 147.73, 149.32, 149.73, 149.86, 157.74, 159.83,
159.86, 159.88, 160.00, 167.72 ppm; IR (film between KBr discs): n˜ =
1718 cmꢀ1 (ester C=O); UV/Vis (CHCl3): lmax (loge)=427 (5.78), 555
(4.38), 595 nm (3.76); MS (MALDI-TOF): m/z: 10849.9 [M]+; elemental
analysis calcd (%) for C579H760N4O188Zn: C 64.10, H 7.06, N 0.52; found:
C 63.68, H 7.04, N 0.56.
Synthesis of proteo-dendrimers 2c–4c
Compound 2c: Dendrimer 2d (200 mg, 0.072 mmol) was dissolved in sta-
bilizer-free THF (5 mL) and MeOH (2 mL). Aqueous NaOH solution
(40 mg, 1.0 mmol, in 1.0 mL water) was added, and the reaction mixture
was stirred at room temperature. After 7 h, the solvents were removed
under vacuum and the residue dissolved in dichloromethane was washed
with water (2”10 mL), 2% citric acid (10 mL), and then with water (2”
Synthesis of oligopeptide ester 5:[13] Oligopeptide ester 5 was synthesized
by repeated condensation of N-CBZ-glutamic acid (CBZ=carbobenzyl-
oxy) and glutamic acid diethyl ester followed by deprotection as shown
Chem. Eur. J. 2006, 12, 1328 – 1338
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1335

H. Tsukube et al.
in Scheme 4. [a]2D5 =ꢀ1.2 (c=2.5 in CHCl3), ꢀ19.9 (c=0.26 in MeOH);
1H NMR (CDCl3, 400 MHz): d=1.21–1.32 (m, 24H; ester CH3), 1.77–
2.68 (m, 28H; CH2), 2.96 (dd, 1H; CH), 4.05–4.28 (m, 18H; ester CH2
and CH), 4.63–4.71 (m, 2H; CH), 4.81–4.88 (m, 2H; CH), 7.78 (d, J=
8.8 Hz, 1H; amide NH), 7.81 (d, J=8.8 Hz, 1H; amide NH), 8.21 (d, J=
6.4 Hz, 1H; amide NH), 8.24 (d, J=9.2 Hz, 1H; amide NH), 8.29 (d, J=
9.2 Hz, 1H; amide NH), 8.41 ppm (d, J=6.4 Hz, 1H; amide NH);
13C NMR (CDCl3, 100 MHz): d=14.01, 14.13, 14.20, 26.45, 26.51, 27.00,
27.21, 27.23, 27.34, 30.03, 30.18, 30.70, 31.89, 31.97, 32.02, 33.15, 51.41,
51.46, 51.63, 51.67, 53.01, 53.30, 53.40, 60.46, 60.52, 60.60, 62.13, 62.17,
62.32, 172.37, 172.45, 172.52, 172.59, 172.67, 173.03, 173.92, 173.99, 174.11,
174.20, 175.59 ppm; MS (FAB): m/z: 1146.5 [M+H]+; elemental analysis
calcd (%) for C51H83N7O22: C 53.44, H 7.30, N 8.55; found: C 53.33, H
7.29, N 8.44.
69.49, 70.29, 70.44, 70.45, 71.60, 71.61, 101.27, 101.84, 101.92, 106.23,
114.99, 115.07, 119.45, 120.55, 125.30, 131.36, 131.91, 132.02, 133.05,
134.40, 139.12, 139.14, 144.73, 146.49, 149.53, 149.72, 149.86, 149.88,
157.71, 160.01, 166.57, 171.75, 172.38, 172.47, 172.53, 172.62, 172.67,
173.27, 173.36, 173.66, 173.81, 173.97, 174.06, 174.15 ppm; IR (film be-
tween KBr discs): n˜ =1651 (amide C=O), 1733 cmꢀ1 (ester C=O); UV/
Vis (CHCl3): lmax (loge)=427 (5.79), 555 (4.38), 595 nm (3.76); MS
(MALDI-TOF): m/z: 3903.2 [M]+; elemental analysis calcd (%) for
C198H265N11O65Zn + 4H2O: C 59.80, H 6.92, N 3.87; found: C 59.81, H
6.94, N 3.85.
Compound 3b: Dendrimer 3b was prepared by coupling acid dendrimer
3c with peptide dendron 5 using the same procedure as that for 1b (reac-
tion time=60 h). Yield: 80%; 1H NMR (CDCl3, 400 MHz): d=1.19–1.34
(m, 24H; Glu-ester CH3), 1.90–2.79 (m, 28H; Glu-CH2), 3.17 (s, 24H;
OMe’), 3.20 (s, 48H; OMe), 3.32–3.34 (m, 16H; Ha’), 3.36–3.38 (m, 32H;
Ha), 3.46–3.48 (m, 16H; Hb’), 3.50–3.53 (m, 32H; Hb), 3.63 (brt, J=
4.8 Hz, 16H; Hc’), 3.68 (brt, J=4.8 Hz, 32H; Hc), 3.96 (brt, J=5.2 Hz,
16H; Hd’), 4.00 (brt, J=5.2 Hz, 32H; Hd), 4.07–4.48 (m, 19H; Glu-ester
OCH2 and Glu-CH), 4.66–4.73 (m, 2H; Glu-CH), 4.79–4.85 (m, 1H; Glu-
CH), 4.92 (s, 8H; Hg’), 4.94 (s, 16H; Hg), 5.00–5.05 (m, 1H; Glu-CH),
5.14 (brs, 12H; Hj and Hj’), 6.34 (brt, J=2.4 Hz, 4H; He’), 6.36 (brt, J=
2.4 Hz, 8H; He), 6.49 (brd, J=2.4 Hz, 8H; Hf’), 6.52 (brd, J=2.4 Hz,
16H; Hf), 6.53 (brt, J=2.0 Hz, 6H; Hh and Hh’), 6.73–6.75 (m, 12H; Hi
and Hi’), 6.94 (d, J=7.2 Hz, 1H; CONH), 7.05–7.07 (m, 3H; Hp and Hp’),
7.50 (brm, 6H; Hq and Hq’), 7.75 (d, J=9.6 Hz, 1H; CONH), 7.88 (d, J=
9.6 Hz, 1H; CONH), 8.15 (d, J=8.0 Hz, 2H; Hy), 8.21–8.24 (m, 3H; Hx
and CONH), 8.31 (d, J=10.0 Hz, 1H; CONH), 8.42 (d, J=7.2 Hz, 1H;
CONH), 8.81 (d, J=4.4 Hz, 2H; pyrrole-b), 8.86 (d, J=6.6 Hz, 1H;
CONH), 9.01 (s, 4H; pyrrole-b), 9.02 ppm (d, J=4.4 Hz, 2H; pyrrole-b);
13C NMR (CDCl3, 100 MHz): d=14.02, 14.12, 14.16, 14.19, 26.48, 26.63,
26.93, 27.23, 27.61, 29.59, 30.15, 30.42, 30.60, 30.69, 31.39, 32.00, 32.14,
51.57, 51.68, 51.72, 51.76, 52.00, 52.83, 53.41, 58.77, 58.79, 60.29, 60.53,
60.55, 60.59, 61.98, 62.07, 62.29, 67.26, 67.30, 69.41, 69.45, 69.91, 70.14,
70.38, 70.42, 71.63, 71.66, 101.02, 101.73, 105.92, 105.95, 106.51, 115.04,
115.20, 119.38, 120.44, 120.55, 125.28, 131.32, 131.99, 132.83, 134.43,
139.02, 139.07, 139.15, 144.93, 144.98, 146.61, 149.50, 149.70, 149.85,
149.90, 157.69, 157.73, 159.90, 159.91, 159.98, 166.66, 172.72, 172.36,
172.47, 172.50, 172.55, 172.62, 172.66, 173.17, 173.60, 173.64, 173.95,
170.01, 174.06 ppm; IR (film between KBr discs): n˜ =1651 (amide C=O),
1732 cmꢀ1 (ester C=O) ; UV/Vis (CHCl3): lmax (loge)=427 (5.79), 555
(4.39), 595 nm (3.79); MS (MALDI-TOF): m/z: 6594.8 [M]+; elemental
analysis calcd (%) for C342H457N11O113Zn + 6H2O: C 61.27, H 7.05, N
2.30; found: C 61.27, H 7.14, N 2.20.
Synthesis of ester dendrimers 1b–4b
Compound 1b: Acid dendrimer 1c (25.0 mg, 0.028 mmol), HOBt
(6.0 mg, 0.039 mmol), HBTU (15 mg, 0.039 mmol), and DIPEA (10 mg,
0.078 mmol) were added to dry dichloromethane (10 mL) and stirred at
08C for 20 min. Peptide dendron 5 (45 mg, 0.039 mmol) was added and
the reaction mixture was stirred at 08C for 45 min and the temperature
was allowed to rise to room temperature slowly. The progress was moni-
tored by TLC (20% EtOAc in dichloromethane). The reaction was
found to be complete after stirring for 36 h. The solvent was removed
under vacuum and the residue was dissolved in dichloromethane and
washed with 2% citric acid (10 mL), water (2”10 mL), aqueous
NaHCO3 (10 mL), water (10 mL), and brine (10 mL). The organic layer
was separated, dried over MgSO4, filtered, and evaporated. The residue
was purified by alumina column chromatography, followed by 1H-2H
JAIGEL GPC, giving 1b (70%). M.p. 117–1188C; 1H NMR (CDCl3,
400 MHz): d=1.20–1.34 (m, 24H; Glu-ester CH3), 1.85–2.85 (m, 28H;
Glu-CH2), 3.94 (s, 18H; OMe), 4.10–4.30 (m, 17H; Glu-ester OCH2 and
Glu-CH), 4.32–4.38 (m, 1H; Glu-CH), 4.44–4.50 (m, 1H; Glu-CH), 4.65–
4.73 (m, 2H; Glu-CH), 4.83–4.89 (m, 1H; Glu-CH), 5.01–5.07 (m, 1H;
Glu-CH), 6.82 (d, J=8.4 Hz, 1H; CONH), 6.88 (brt, 3H; Hp), 7.39 (brd,
6H; Hq), 7.77 (d, J=9.0 Hz, 1H; CONH), 7.90 (d, J=8.8 Hz, 1H;
CONH), 8.15 (d, J=8.4 Hz, 2H; Hx), 8.21–8.34 (m, 5H; 3”CONH and
Hy), 8.85 (d, J=4.6 Hz, 2H; pyrrole-b), 8.87 (brd, 1H; CONH), 9.04 ppm
(brs, 6H; pyrrole-b); 13C NMR (CDCl3, 75 MHz): d=14.02, 14.06, 14.13,
14.14, 14.20, 14.21, 26.44, 26.61, 27.00, 27.20, 27.44, 29.62, 30.15, 30.39,
30.59, 30.65, 31.39, 31.91, 32.01, 51.51, 51.62, 51.71, 51.79, 52.95, 53.29,
55.50, 60.27, 60.55, 60.57, 62.07, 62.26, 99.90, 113.76, 119.65, 120.80,
125.35, 131.45, 131.95, 132.10, 133.10, 134.34, 144.58, 146.31, 149.66,
149.83, 149.97, 158.50, 158.55, 166.51, 171.71, 172.34, 172.42, 172.51,
172.53, 172.61, 173.26, 173.36, 173.49, 173.74, 173.78, 173.92, 174.05 ppm;
IR (film between KBr discs): n˜ =1657 (amide C=O), 1736 cmꢀ1 (ester C=
O); UV/Vis (CHCl3): lmax(loge)=425 (5.77), 553 (4.37), 593 nm (3.68);
HRMS (FAB): m/z calcd for C102H121N11O29Zn: 2027.7623 [M]+; found:
2027.7596; elemental analysis calcd (%) for C102H121N11O29Zn + H2O: C
59.80, H 6.05, N 7.52; found: C 59.80, H 6.05, N 7.44.
Compound 4b: Dendrimer 4b was prepared by coupling acid dendrimer
4c with peptide dendron 5 using the same procedure as that for 1b (reac-
tion time=85 h; purified by 2H-3H JAIGEL GPC followed by silica gel
flash chromatography). Yield: 52%; 1H NMR (CDCl3, 400 MHz): d=
1.17–1.33 (m, 24H; Glu-ester CH3), 1.80–2.80 (m, 28H; Glu-CH2), 3.22
(s, 48H; OMe’), 3.25 (s, 96H; OMe), 3.36–3.39 (m, 32H; Ha’), 3.41–3.43
(m, 64H; Ha), 3.50–3.53 (m, 32H; Hb’), 3.55–3.58 (m, 64H; Hb), 3.66 (brt,
J=4.8 Hz, 32H; Hc’), 3.71 (brt, J=4.8 Hz, 64H; Hc), 3.96 (brt, J=
4.8 Hz, 32H; Hd’), 4.01 (brt, J=4.8 Hz, 64H; Hd), 4.05–4.27 (m, 17H;
Glu-ester OCH2 and Glu-CH), 4.38–4.45 (m, 2H; Glu-CH), 4.62–4.71 (m,
2H; Glu-CH), 4.75 (m, 1H; Glu-CH), 4.82 (s, 16H; Hg’), 4.87 (s, 32H;
Hg), 4.90 (brs, 8H; Hj’), 4.93 (brs, 16H; Hj), 5.00 (m, 1H; Glu-CH), 5.13
(brs, 12H; Hm and Hm’), 6.33 (brt, J=2.0 Hz, 8H; He’), 6.37 (brt, J=
2.0 Hz, 16H; He), 6.44 (brt, J=1.6 Hz, 4H; Hh’), 6.46 (brd, J=2.0 Hz,
24H; Hf’ and Hh), 6.50 (d, J=2.0 Hz, 32H; Hf), 6.55 (brm, 6H; Hk and
Hk’), 6.59 (brd, J=2.0 Hz, 8H; Hi’), 6.61 (brd, J=2.0 Hz, 16H; Hi), 6.76
(brd, J=2.0 Hz, 4H; Hl’), 6.78 (brd, J=2.0 Hz, 8H; Hl), 7.03 (brd, J=
7.2 Hz, 1H; CONH), 7.07 (brs, 1H; Hp’), 7.10 (brs, 2H; Hp), 7.55 (brs,
6H; Hq and Hq’), 7.72 (d, J=6.8 Hz, 1H; CONH), 7.84 (d, J=6.8 Hz,
1H; CONH), 8.15–8.28 (m, 6H; Hy, Hx, and CONH), 8.46 (brd, J=
6.8 Hz, 1H; CONH), 8.82 (brm, 3H; CONH and pyrrole-b), 9.05–
9.08 ppm (m, 6H; pyrrole-b); IR (film between KBr discs): n˜ =1652
(amide C=O), 1734 cmꢀ1 (ester C=O); UV/Vis (CHCl3): lmax (loge)=427
(5.78), 555 (4.39), 595 nm (3.75); MS (MALDI-TOF): m/z: 10967.86
[M]+; elemental analysis calcd (%) for C630H841N11O209Zn + 8H2O: C
62.42, H 7.13, N 1.27; found: C 62.38, H 7.19, N 1.22.
Compound 2b: Dendrimer 2b was prepared by coupling 2c with peptide
dendron 5 using the same procedure as that for 1b (reaction time=24 h).
Yield: 77%; 1H NMR (CDCl3, 400 MHz): d=1.21–1.35 (m, 24H; Glu-
ester CH3), 1.80–2.85 (m, 28H; Glu-CH2), 3.13 (s, 12H; OMe’), 3.15 (s,
24H; OMe), 3.29–3.33 (m, 24H; Ha and Ha’), 3.48–3.52 (m, 24H; Hb and
Hb’), 3.72–3.76 (m, 24H; Hc and Hc’), 4.06–4.08 (m, 24H; Hd and Hd’),
4.09–4.49 (m, 19H; Glu-ester OCH2 and Glu-CH), 4.67–4.74 (m, 2H;
Glu-CH), 4.80–4.86 (m, 1H; Glu-CH), 5.00–5.06 (m, 1H; Glu-CH), 5.15
(brs, 12H; Hg and Hg’), 6.44 (brt, J=2.4 Hz, 6H; He and He’), 6.65 (brd,
J=2.4 Hz, 12H; Hf and Hf’), 6.94 (d, J=7.6 Hz, 1H; CONH), 7.04 (brs,
3H; Hp and Hp’), 7.46–7.48 (m, 6H; Hq and Hq’), 7.78 (d, J=10.4 Hz, 1H;
CONH), 7.89 (d, J=8.4 Hz, 1H; CONH), 8.17 (d, J=8.4 Hz, 2H; Hy),
8.24–8.27 (m, 3H; CONH and Hx), 8.34 (d, J=9.6 Hz, 1H; CONH), 8.42
(d, J=6.4 Hz, 1H; CONH), 8.80 (d, J=4.6 Hz, 2H; pyrrole-b), 8.89 (d,
J=6.8 Hz, 1H; CONH), 8.94 (brs, 4H; pyrrole-b), 8.97 ppm (d, J=
4.6 Hz, 2H; pyrrole-b); 13C NMR (CDCl3, 100 MHz): d=14.02, 14.05,
14.15, 14.19, 14.24, 26.50, 26.66, 27.00, 27.24, 27.30, 27.56, 29.77, 30.17,
30.47. 30.67, 30.72, 31.45, 32.06, 32.16, 51.60, 51.64, 51.74, 51.79, 51.85,
52.97, 53.44, 58.73, 58.75, 60.30, 60.55, 60.60, 62.06, 62.09, 62.32, 67.39,
1336
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 1328 – 1338

Proteo-Dendrimers
FULL PAPER
Synthesis of proteo-dendrimers 1a–4a
LiOH (3.2 molar equiv) was added and stirred, and the reaction was
found to be completed after 24 h. The excess of LiOH was removed by
ultrafiltration using a 3000 molecular weight cut-off membrane using dis-
tilled water three times. Finally, the residue was dried under vacuum,
giving 4a as a hygroscopic semisolid. 1H NMR (CD3OD, 400 MHz): d=
1.90–2.55 (brm, 28H; Glu CH2), 3.16 (brs, 144H; OMe’ and OMe), 3.29–
3.31 (brs, 96H; CH2O), 3.41 (brs, 96H; CH2O), 3.51 (brm, 96H; CH2O),
3.78 (brs, 96H; CH2O), 4.28–4.70 (brm + 2brs, 189H; benzylic CH2 and
Glu-CH), 6.13–6.60 (brm, 126H; ArH), 6.91 (brs, 3H; ArH), 7.43 (brs,
6H; ArH), 8.26 (brs, 4H; ArH), 8.77, 8.99 ppm (2brs, 8H; pyrrole-b);
IR (film between KBr discs): n˜ =1595 cmꢀ1 (broad with shoulder at
1656 cmꢀ1); UV/Vis (5.0”10ꢀ3 moldmꢀ3 sodium phosphate buffer con-
taining 10% DMF, pH 7.0): lmax (loge)=434 (5.53), 558 (4.34), 599 nm
(3.81); MS (MALDI-TOF, linear): m/z calcd for C614H809N11O209Zn:
11753.6 [M]+; found: broad peak centered at 11774.7.
Compound 2a: Dendrimer 2b (25 mg, 0.0064 mmol) was dissolved in
THF (1 mL) and MeOH (1 mL). LiOH (8.8 molar equiv, 0.056 mmol)
dissolved in water (0.5 mL) was added to reaction mixture and stirred at
room temperature. The progress of hydrolysis was followed by capillary
electrophoresis by withdrawing small aliquots (see Figure S1 in the Sup-
porting Information). After 36 h the solvents were removed under
vacuum and the residue was dissolved in water (1 mL) and LiOH
(1.6 molar equiv) was added. The reaction mixture was stirred at room
temperature for
a further 24 h. The solvents were removed under
vacuum and the residue was dried under high vacuum, offering 2a as a
1
hygroscopic semisolid. H NMR (CD3OD, 400 MHz): d=1.90–2.55 (brm,
28H; Glu CH2), 3.04 (s, 12H; OMe’), 3.12 (s, 24H; OMe), 3.13–3.16 (m,
8H; CH2O), 3.21–3.27 (brm, 24H; CH2O), 3.30–3.40 (brm, 24H; CH2O),
3.51 (brt, 16H; CH2O), 3.62 (brs, 8H; CH2O), 3.84 (brs, 16H; CH2O),
4.25–4.74 (4brm + 1brs, total 11H; benzylic CH2 and Glu-CH), 5.05
(brs, 8H; benzylic CH2), 6.20 (s, 2H; ArH), 6.29 (s, 4H; ArH), 6.35 (s,
4H; ArH), 6.53 (brs, 8H; ArH), 6.68 (brs, 1H; ArH), 6.96 (brs, 2H;
ArH), 7.37 (s, 2H; ArH), 7.42 (s, 4H; ArH), 8.31 (brs, 4H; ArH), 8.75
(brs, 6H; pyrrole-b), 8.86 ppm (brd, 2H; pyrrole-b); IR (between KBr
discs): n˜ =1596 cmꢀ1 (broad with shoulder at 1656 cmꢀ1); UV/Vis (5.0”
10ꢀ3 moldmꢀ3 sodium phosphate buffer containing 10% DMF, pH 7.0):
lmax (loge)=429 (5.52), 558 (4.34), 599 nm (3.81); MS (MALDI-TOF,
linear): m/z calcd for C182H233N11O65Zn: 3680.3 [M]+; found: broad peak
centered at 3682.4.
LogK determination: The complexation experiments were carried out at
pH 7.0 (5.0”10ꢀ3 moldmꢀ3 sodium phosphate buffer containing 10%
DMF v/v). A dendrimer solution (2.5”10ꢀ7 moldmꢀ3, 2.5 mL) was added
to a 1 cm cuvette and its fluorescence spectra was recorded by excitation
at 558 nm. Cytochrome
c
solution (1.25”10ꢀ5 moldmꢀ3 and 6.25”
10ꢀ5 moldmꢀ3) prepared in the same buffer was added in small aliquots
of 5–30 ml. After each addition, the sample was stirred for 5 min before
the fluorescence spectrum was measured. The emission intensity at 610
or 650 nm was plotted against the molar ratio x [cytochrome c]/[den-
drimer]. The stability constants were calculated by using nonlinear curve
fitting as described below. We use the following abbreviations: R=
proteo-dendrimer; S=cytochrome c; [R]t and [S]t =total concentrations
of the proteo-dendrimer and cytochrome c, respectively. Total concentra-
Compound 1a: Dendrimer 1b was hydrolyzed to proteo-dendrimer 1a
by using a procedure similar to that used for 2a using 9.6 molar equiva-
lent of LiOH. M.p.: >3008C; 1H NMR (CD3OD, 400 MHz): d=1.9–2.5
(brm, 28H; Glu CH2), 3.94 (s, 18H; OMe), 4.24–4.63 (4brm, 7H; Glu-
CH), 6.89 (brt, 3H; ArH), 7.36 (brt, 6H; ArH), 8.26 (brq, 4H; ArH),
8.75 (d, J=5.4 Hz, 2H; pyrrole-b), 8.91 ppm (m, 6H; pyrrole-b); IR
(KBr): n˜ =1590 cmꢀ1 (broad with shoulder at 1654 cmꢀ1); UV/Vis (5.0”
10ꢀ3 moldmꢀ3 sodium phosphate buffer containing 10% DMF, pH 7.0):
lmax (loge)=425 (5.78), 557 (4.33), 597 nm (3.90); MS (MALDI-TOF, re-
flectron): m/z calcd for C86H89N11O29Zn: 1806.1 [M]+; found: 1807.7.
tion of proteo-dendrimer [R]t was kept constant (2.5”10ꢀ7 moldmꢀ3
)
during titration and the concentration of cytochrome was varied from 0
to 17.5”10ꢀ7 moldmꢀ3. The molar ratio x of two species is defined as
x ¼ ½SŠt=½RŠ
ð1Þ
t
Stepwise formation constants for 1:1 and 1:2 (cytochrome c/proteo-den-
drimer) complexes are defined in Equations (2) and (3):
Compound 3a: Dendrimer 3b (25 mg) was dissolved in THF (1 mL) and
MeOH (1 mL). LiOH (9.6 molar equiv, dissolved in 0.5 mL of water) was
added to the reaction mixture and stirred at room temperature. The
progress of the hydrolysis was followed by capillary electrophoresis by
withdrawing small aliquots (see Figure S1 in the Supporting Information).
After 48 h the solvents were removed under vacuum and the residue was
dissolved in water (1 mL) and LiOH (3.2 molar equiv) was added. The
reaction mixture was stirred at room temperature for another 48 h and
the reaction was completed. The excess of LiOH was removed by ultrafil-
tration using a 3000 molecular weight cut-off membrane using distilled
water three times. Finally, the residue was dried under high vacuum, of-
fering 3a as a hygroscopic semisolid. 1H NMR (CD3OD, 400 MHz): d=
1.90–2.55 (brm, 28H; Glu CH2), 3.09 (s, 24H; OMe’), 3.13 (s, 48H;
OMe), 3.22 (brm, 16H; CH2O), 3.25–3.31 (brm, 48H; CH2O), 3.36
(brm, 48H; CH2O), 3.47 (brm, 32H; CH2O), 3.60 (brm, 16H; CH2O),
3.75 (brm, 32H; CH2O), 4.27–4.70 (3brm + brs, 35H; benzylic CH2 and
Glu-CH), 5.01 (brs, 8H; benzylic CH2), 6.13–6.60 (6brs, 54H; ArH), 6.80
(brs, 1H; ArH), 6.96 (brs, 2H; ArH), 7.37 (brs, 2H; ArH), 7.42 (brs,
4H; ArH), 8.26 (brs, 4H; ArH), 8.76–8.80 (2 brs, 6H; pyrrole-b),
8.93 ppm (brd, 2H; pyrrole-b); IR (film between KBr discs): n˜ =
1598 cmꢀ1 (broad with shoulder at 1655 cmꢀ1); UV/Vis (5.0”
10ꢀ3 moldmꢀ3 sodium phosphate buffer containing 10% DMF pH 7.0):
lmax (loge)=436 (5.53), 558 (4.34), 599 nm (3.82); MS (MALDI-TOF,
linear): m/z calcd for C326H425N11O113Zn: 6371.4 [M]+; found: broad peak
centered at 6388.7.
½RSŠ
ð2Þ
ð3Þ
R þ S ¼ RS
K1
¼
½RŠ½SŠ
½R2SŠ
RS þ R ¼ R2S
K2
¼
½RSŠ½RŠ
Total concentrations of proteo-dendrimer and cytochrome c are ex-
pressed as Equations (4) and (5):
½RŠ ¼ ½RŠ þ ½RSŠ þ 2 ½R2SŠ
ð4Þ
ð5Þ
t
½SŠ ¼ ½SŠ þ ½RSŠ þ ½R2SŠ
t
From Equations (1)–(5), the concentration of free dendrimer [R]can be
obtained by solving the following equation [Eq. (6)]:
3
2
K1K2½RŠ þ ðK1ꢀK1K2½RŠt þ 2K1K2½RŠtxÞ½RŠ
ð6Þ
þð1ꢀK1½RŠt þ K1RtxÞ½RŠꢀ½RŠ ¼ 0
t
The concentration of other species can then be expressed as shown in
Equations (7)–(9):
½RŠtx
ð7Þ
½SŠ ¼
2
1 þ K1½RŠ þ K1K2½RŠ
Compound 4a: Dendrimer 4b (25 mg) was dissolved in THF (1 mL) and
MeOH (1 mL). LiOH (14.4 molar equiv, dissolved in 0.5 mL of water)
was added to the reaction mixture and stirred at room temperature. The
progress of the hydrolysis was followed by capillary electrophoresis by
withdrawing small aliquots (see Figure S1 in the Supporting Information).
After 48 h the solvents were removed under vacuum and the residue was
dissolved in water (1 mL) and LiOH (3.2 molar equiv) was added. The
reaction mixture was stirred at room temperature for another 48 h. More
½RSŠ ¼ K1½RŠ½SŠ
ð8Þ
ð9Þ
2
½R2SŠ ¼ K1K2½RŠ ½SŠ
The observed fluorescence can be expressed as shown in Equation (10):
Fx ¼ I0½RŠ þ I1:1½RSŠ þ I1:2½R2SŠ
ð10Þ
Chem. Eur. J. 2006, 12, 1328 – 1338
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1337

H. Tsukube et al.
In the absence of cytochrome c, the fluorescence can be written as shown
in Equation (11):
[5]a) F. Diederich, B. Felber, Proc. Natl. Acad. Sci. USA 2002, 99,
4778–4781; b) M. T. Morgan, M. A. Carnahan, C. E. Immoos, A. A.
Ribeiro, S. Finkelstein, S. J. Lee, M. W. Grinstaff, J. Am. Chem. Soc.
2003, 125, 15485–15489.
[6]R. Kluger, J. Zhang, J. Am. Chem. Soc. 2003, 125, 6070–6071.
[7]S. C. Zimmerman, M. S. Wendland, N. A. Rakons, I. Zharov, K. S.
Suslick, Nature 2002, 418, 399–403.
Ft ¼ I0½RŠ
ð11Þ
t
From Equations (10) and (11), Equation (12) can be formed:
[8]M. Braun, S. Atalik, D. M. Guldi, H. Lanig, M. Bettreich, S. Bur-
ghardt, M. Hatzimarinaki, E. Ravanelli, M. Prato, R. van Eldik, A.
Hirsch, Chem. Eur. J. 2003, 9, 3867–3875.
Fx
Ft
½RŠ I1:1 ½RSŠ I1:2 ½R2SŠ
ð12Þ
¼
þ
þ
I0
t
I0
½RŠ
t
½RŠ
½RŠ
t
[9]a) P. Furuta, J. M. J. Frꢁchet, J. Am. Chem. Soc. 2003, 125, 13173–
13181; b) V. Percec, C. M. Mitchell, W.-D. Cho, S. Uchida, M.
Glodde, G. Ungar, X. Zeng, Y. Liu, V. S. K. Balagurusamy, P. A.
Heiney, J. Am. Chem. Soc. 2004, 126, 6078–6094.
[10]J. S. Lindsey, S. Prathapan, T. E. Johnson, R. W. Wagner, Tetrahe-
dron 1994, 50, 8941–8968.
[11]K. W. Pollak, J. W. Leon, J. M. J. Frꢁchet, M. Maskus, H. D. Abruna,
Chem. Mater. 1998, 10, 30–38.
[12]Z. Bo, C. Zhang, N. Severin, J. P. Rabe, A. D. Schluter, Macromole-
cules 2000, 33, 2688–2694.
in which I1:1/I0 and I1:2/I0 are the fluorescence intensity ratios of com-
plexes RS and R2S to free dendrimer, respectively.
For fitting the titration curve, Fx/Ft was plotted against molar ratio x, and
K1, K2, I1:1/I0, and I1:2/I0 were treated as variables to be determined. These
parameters were determined by applying the least-square method using
the curve-fit function of IGOR Pro software (version 4.08). The equa-
tions used for 1:1 complexation between 4a and cytochrome c were ob-
tained similarly.
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Acknowledgements
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Fujitsu, Ltd., Japan), which were comparable to the reported values
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The authors are grateful to Dr. J. C. Mitchell of the University of Green-
wich for his valuable comments on peptide synthesis, and to Professors J.
Teraoka of Osaka City University and J. L. Beauchamp of the California
Institute of Technology for their kind support regarding cytochrome c
complex characterizations.
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[19]The hepta(glutamic acids) of the dendrimers 1a–4a themselves ex-
1
hibited the same H and 13C NMR and IR spectra.
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Received: September 13, 2005
Published online: November 25, 2005
1338
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