A Surface-Modified Dendrimer Set for Potential Application as Drug Delivery Vehicles: Synthesis, In Vitro Toxicity, and Intracellular Localization

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A Surface-Modified Dendrimer Set for Potential Application as Drug

Delivery Vehicles: Synthesis, In Vitro Toxicity, and Intracellular Localization

Sabine Fuchs,^[a]Timo Kapp,^[b]Henning Otto,^[c]Torsten Schˆneberg,^[d]Peter Franke,^[c]

Ronald Gust,*^[b]and A. Dieter Schl¸ter*^[a]

Abstract: The synthesis, cytotoxicity,

and behavior in cell culture of a new

set of first- (G1) and second-generation

(G2) dendrimers is reported. The sur-

face functionality of these dendrimers

has been varied to see whether struc-

ture/toxicity relations can be observed.

The outermost functional groups are

amines that are decorated either with

protons, tert-butoxycarbonyl (Boc) or

benzyloxycarbonyl (Cbz) protecting

groups, Boc-protected or unprotected

natural amino acid residues, ethylene-

diamine ligands, and/or dansyl fluores-

cence labels. The cytotoxicity was de-

termined in vitro in concentration-de-

pendent assays using the human MCF-

7

breast cancer cell line. Cellular

uptake and intracellular distribution

was monitored by confocal fluores-

cence microscopy after internalization

of the dansyl-labeled dendrimers by

HeLa cells.

Keywords: cellular

cytotoxicity dendrimers

delivery ¥ synthesis

uptake

¥

drug

Introduction

(PAMAM) family is now being used extensively as transfec-

23]

tion agents for DNA transfer into living cells,^[19

while

Dendrimers have attracted considerable attention in the last

two decades.^[1,2]They combine a monodisperse nanoscale

geometry with high endgroup density at their ™surface,∫

others are used as contrast agents for magnetic resonance

imaging (MRI),^[24,25]and in boron neutron capture therapy

29]

(BNCT) for cancer treatment.^[26Because of such applica-

5]

through which properties can be widely engineered.^[3

tions these and related behavior of dendrimers in living or-

ganisms, as well as in cell culture, is being researched but

little is known yet about their way into living cells, about

their intracellular distribution, and aspects such as immuno-

genic response and cell toxicity.

Larger dendrimers contain dynamic inner cavities in which

13]

guest molecules can be trapped and released.^[6Some rep-

resentatives possess charges in the periphery and, thus, are

soluble in polar and protic solvents, sometimes even in

16]

water.^[3,4,14In the last few years, water-soluble dendrimers

In 1996 Roberts et al. investigated the biological behavior

of PAMAM dendrimers of the third, fifth, and seventh gen-

eration in vitro and in vivo.^[30]The authors studied the be-

havior of dendrimers in V79 cells and in Swiss Webster

mice for a number of biological properties, including in vitro

and in vivo toxicity, immunogenicity, and biodistribution. No

evidence for immunogenicity was found, but like Duncan

et al.^[31]a few years later, they found a concentration- and

generation-dependent toxicity of the dendrimers. Especially

the seventh-generation dendrimers exhibited a considerably

larger toxicity than those of the fifth or third generation.

The differences between the lower generations were not

very pronounced, though an increase in toxicity with in-

creasing generation could be concluded. In mice, the G3

dendrimer showed the highest accumulation in kidney

tissue, while G5 and G7 preferentially localized in the pan-

creas. In 2000 Duncan and co-workers performed a broader,

more systematic investigation on five different dendrimer

types in vitro with three different cell lines (B16F19, CCRF,

and HepG2), in vivo with Wistar rats, and with freshly pre-

have gained more and more importance in biochemical and

biomedical applications.^[17,18]Especially the polyamidoamine

[a] Dipl. Biochem. S. Fuchs, Prof. Dr. A. D. Schl¸ter

Freie Universit‰t Berlin, Institut f¸r Chemie/Organische Chemie

Takustrasse 3, 14195 Berlin (Germany)

Fax : (+49)30-838-53357

E-mail: adschlue@chemie.fu-berlin.de

[b] Dipl. Lebensmittelchem. T. Kapp, Prof. Dr. R. Gust

Freie Universit‰t Berlin, Institut f¸r Pharmazie

Kˆnigin-Luise Strasse 2 4, 14195 Berlin (Germany)

Fax : (+49)30-838-56906

E-mail: rgust@zedat.fu-berlin.de

[c] Dr. H. Otto, Dr. P. Franke

Freie Universit‰t Berlin, Institut f¸r Chemie/Biochemie

Thielallee 63, 14195 Berlin (Germany)

[d] Prof. Dr. T. Schˆneberg

Universit‰t Leipzig, Institut f¸r Biochemie

Deutscher Platz 6, 04103Leipzig (Germany)

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pared rat erythrocytes for haemolysis assays.^[31]Dendrimers

tested were the polycationic PAMAMs and two kinds of

poly(propyleneimine) dendrimers with either a diaminobu-

tane (DAB) or a diaminoethane (DAE) core, respectively.

They also tested the polyanionic, carboxylate-terminated

™half-generation∫ PAMAMs and DABs, as well as un-

charged oligoethyleneoxide-terminated carbosilane (CSi-

PEO) dendrimers. All investigations were carried out for

various generations and concentrations. Furthermore, pre-

liminary biodistribution studies with ¹²⁵I-labeled PAMAM

dendrimers in vivo were performed. The authors found that

all cationic dendrimers were cytotoxic with the degree of

toxicity depending on dendrimer-type, cell-type, and genera-

tion. They also caused haemolysis and changes in erythro-

cyte morphology. In contrast, dendrimers with carboxylate

surfaces were neither haemolytic nor cytotoxic.^[31]These ex-

amples show that polycationic species appear to be more

problematic than polyanionic ones. Despite such advances,

one still has not reached a substantial understanding of the

inherent structure/toxicity relationships of dendrimers. It

may well be that each dendrimer type and each generation

within the types has its own behavior in biological systems

and mechanism of toxicity. It is, thus, obviously necessary to

more broadly explore the influence of different surface func-

tionalizations of otherwise identical dendrimers on their in

vitro and in vivo behavior. Such investigations are a prereq-

uisite for considering these branched macromolecules as

drug delivery systems, for example, as novel carriers for an-

ticancer drugs.

tems. More than ever it is now important to unravel the

import pathways of the dendrimer into living cells to get an

impression on how and where they act in living organisms.

Herein, we describe the synthesis of new sets of first- and

second-generation (G1 and G2) polyamidoamine (yet not

PAMAM) dendrimers, which differ only in surface motifs.

As such were used a) quaternized amines (set A dendrim-

ers), b) the natural, proteinogenic amino acids l-phenylala-

nine, l-methionine, and l-aspartic acid (set B dendrimers),

c) diaminopropionic acid, a bidentate ligand, for example,

for Ptⁱⁱ-binding (set C dendrimers), as well as d) the 5-dime-

thylaminonaphthalene-1-sulfonyl (dansyl) motif^[34]for partial

or complete fluorescence labeling of the dendrimers (set D

dendrimers). Some of the modified dendrimers carry chelat-

ing ligands for Ptⁱⁱ(l-methionine, diaminopropionic acid)

and could thus be potentially used for binding cisplatin-like

complexes.^[35,36]As a fluorescence tag the dansyl group was

chosen because of its high fluorescence intensity and polar

nature, which should not detrimentally interfere with the

dendrimers’ required water solubility. Dendrimer sets A C

were used to start a systematic exploration of the influence

of the surface functionalization on the dendrimer toxicity in

cell culture, and first results of this endeavor will be report-

ed here. Set D was employed in studies on cell uptake and

intracellular distribution using confocal fluorescence micro-

scopy. Initial conclusive findings will also be reported in this

study.

In this context it would be desirable to learn how den-

drimers distribute in cells. Such studies have already been

performed, mainly with PAMAM dendrimers aimed for use

as transfection or drug-delivery agents. Juliano and co-work-

ers visualized the subcellular distribution of fluorescence-la-

beled dendrimer oligonucleotide complexes by two-color

fluorescence microscopy.^[32]The authors found that the den-

drimer oligonucleotide complexes remained associated

during the process of uptake into vesicular compartments

and entry into the nucleus. A large majority of cells showed

strong fluorescence intensity in the nuclei after one hour of

incubation with dendrimers and oligonucleotides. Also cells

treated with Oregon green 488-conjugated dendrimers alone

showed nuclear fluorescence. It remained unclear in this

study whether nuclear localization of the dendrimer oligo-

nucleotide complexes was just an intermediate stage of in-

tracellular trafficking, or rather the final stage of intracellu-

lar distribution. The authors conclude from their nuclear

fractionations that a substantial amount of dendrimer mate-

rial was also located at other sites of the cell, presumably at

the plasma membrane and at different intracellular mem-

branes. Also, Baker et al. described the targeted uptake of

folic acid-conjugated dendrimers carrying a fluorescein iso-

thiocyanate (FITC) fluorescence tag and their intracellular

recognition using confocal fluorescence microscopy.^[33]After

24 h of incubation, a ’clumpy’ cytoplasmatic staining of the

cells could be observed, but the exact place of intracellular

localization remained unclear. These investigations clearly

demonstrate the necessity to get a better insight into the

structure/action relationship of dendrimers in biological sys-

Results and Discussion

Synthesis and purification of building blocks and dendrim-

ers: Schemes 1 7 contain all syntheses performed. Scheme 1

describes the steps leading to core 4, which was used as the

center piece for all dendrimers. Scheme 2 shows the steps

leading to tert-butyloxycarbonyl (Boc)-protected G1 (7) and

G2 dendrimers (9), which together with core 4, represent

the parent set of all dendrimers reported (set A). Scheme 3

and Scheme 4 depict the sequences to the protected [12a c

(G0) and 20a c (G1)] and deprotected amino acid terminat-

ed dendrimers [13a c (G0) and 21a c (G1)] (set B), and

Scheme 3and Scheme 5 the ones to the protected [ 15 (G0)

and 24 (G1)] and deprotected diaminopropionic acid termi-

nated dendrimers [16 (G0) and 25 (G1)] (set C) respectively.

Finally, in Scheme 6 and Scheme 7 one can see the syntheses

of the completely (26, 27, 28) and partially dansylated den-

drimers (33 36) (set D). The two main reactions used are

40]

Suzuki Miyaura cross-coupling^[37

to attach the branches

to the branching unit (phenyl) and standard peptide cou-

43]

pling reactions^[41

for dendron and dendrimer assembly.

Whenever possible, terminal amines were Boc protected

during the conversions, as this protecting group is stable

46]

under the applied reaction conditions.^[44

Purification of

the protected dendrimers in most cases could easily be per-

formed by standard column chromatography.

Although all synthetic steps are conventional, relatively

easy to perform, and gave products in good yield and high

purity, a few comments on synthetic issues seem neverthe-

less appropriate. For the triamine core molecule 4 1,3,5-tri-

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Surface-Modified Dendrimers

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bromobenzene (1) and tert-butyl allylcarbamate 2 were used

as starting components (Scheme 1). Their cross-coupling

gave the Boc-protected core molecule 3 in 94% yield, which

indicates a very high conversion per coupling step. Pure

product 4 was obtained on a 10 g scale and precipitated

from the reaction mixture as trishydrochloride. The known

dendrons 5 and 6^[47]were attached to 4 with the EDC/HOBt

strategy^[43]in good overall yields of 75 78% to the corre-

sponding set A G1 and G2 dendrimers 7 and 9, respectively

(Scheme 2).

The dendrimers of set B were obtained by starting from

the Boc- and, in case of l-aspartic acid, Boc- and tBu-pro-

Scheme 1. Synthesis of the triamine core molecule 4. Reagents and conditions: a) 1. 2, 9-BBN, toluene, room temperature, 12 h, 2. 1m KOH, 1,

[Pd(PPh₃)₄], 608C, 24 h (94%); b) 3, aqueous HCl, THF, room temperature, 18 h (96%).

Scheme 2. Synthesis of the basic dendrimer set A. Reagents and conditions: a) 1. CH₂Cl₂, EDC, HOBt, ꢀ208C/1 h, room temperature/2 h, 2) 4, DIPEA,

ꢀ308C/1 h, room temperature 16 h (78%); b) 1. CH₂Cl₂, EDC, HOBt, ꢀ208C/1 h, room temperature/2 h, 2. 4, DIPEA, ꢀ308C/1 h, room temperature

24 h (75%); c) CH₂Cl₂, CF₃CO₂H, room temperature, 1 h (98%); d) CH₂Cl₂, CF₃CO₂H, room temperature, 2 h (99%).

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tected hydroxysuccinimide esters of l-phenylalanine, l-me-

thionine, and l-aspartic acid. Isolated yields for the three

G0 dendrimers 12a c were 80 89% (Scheme 3). The G1

dendrons 18a,b (Scheme 4) were used to convergently

access the corresponding l-methionine and l-phenylalanine

G1 dendrimers 20a and 20b, respectively. In contrast to this

mode of synthesis, the l-aspartic acid G1 dendrimer 20c was

built divergently by utilizing the Boc- and tBu-protected l-

aspartic acid hydroxysuccinimide ester 11c and deprotected

parent G1 dendrimer 8. The reason for this change in strat-

egy was difficulties in saponifying the G1 dendron of struc-

ture 18 with R¹=CO₂tBu and R²=C₂H₅(not shown) at R²

without cleaving the R¹ester (Scheme 4). All reactions in-

volving the oxidatively sensitive l-methionine were per-

formed under nitrogen. The deprotections were carried out

in the presence of the ™cleavage cocktail∫ of ethane-

dithiol (EDT), thioanisole, and triisopropylsilane (TIPS)

as nucleophilic scavengers to avoid undesired alkylation

of the thioether. The MALDI-TOF mass spectra of

the G1 dendrimer 20a nevertheless showed signals of low

intensity due to the incorporation of up to three oxygen

atoms.

The non-proteinogenic d/l-diaminopropionic acid was

used for the synthesis of dendrimers 15 (Scheme 3) and 24

(Scheme 5) of set C. They ought to exhibit even better che-

lating properties than their methionine analogs of set B. The

synthetic route starts with the commercially available d/l-di-

aminopropionic acid, which was Boc-protected at both

amine functionalities to 14 in a slightly modified version of

the procedure described by Sergheraert et al.,^[36]and no at-

tempt was done to separate the two enantiomers. Com-

pound 14 was activated with O-(1H-benzotriazole-1-yl)-

N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU)^[48]

and reaction with core 4 gave the Boc-protected G0 (15)

and G1 dendrimers (24) in 80 and 57% isolated yield, re-

spectively (Scheme 3and Scheme 5). The Boc-protected in-

termediates possessed poor sol-

ubility in dichloromethane,

probably due to intramolecular

hydrogen bonding. In all cases

polar solvents such as DMF or

methanol had to be added to

render the diaminopropionic

acid modified compounds com-

pletely soluble.

The highly fluorescent dansyl

group was chosen for the fluo-

rescence-tagged dendrimers of

set D.^[34]To achieve the fully la-

beled series, the parent den-

drimers 4, 8, and 10 were treat-

ed with an 1.5 3-fold excess of

dansyl chloride per amine func-

tionality (Scheme 6). The re-

spective highly fluorescent den-

drimers 26, 27, and 28 were iso-

lated in yields of 65 92%. In

contrast to the procedures re-

cently described by Vˆgtle

et al.^[34g,h,i]for dansylation of

poly(propylene amine) and pol-

ylysine dendrimers, the reac-

tions proceeded fast and were

in most cases already finished

after one hour at room temper-

ature (TLC monitoring). This

finding is consistent with the re-

sults of Bartzatt, who described

dansylation of primary and sec-

ondary amines in aqueous

Na₂CO₃buffer to be complete

within one hour.^[34d]The degree

Scheme 3. Synthesis of the amino acid-terminated G0 dendrimers of set B and C. Reagents and conditions: a)

11a + 4, CH₂Cl₂, DIPEA, ꢀ108C/1 h, room temperature, 18 h (85%); 11b + 4, CH₂Cl₂/DMF, DIPEA, 08C/

1 h, room temperature, 18 h (80%); 11c + 4, CH₂Cl₂, DIPEA, ꢀ108C/1 h, room temperature, 18 h (89%); b)

CH₂Cl₂/CF₃CO₂H/EDT/thioanisole/methanol/TIPS, r.t., 1 h (99%); c) CH₂Cl₂/methanol, CF₃CO₂H, room tem-

perature, 1 h (99%); d) CH₂Cl₂/CF₃CO₂H, room temperature, 3h (98%); e) 1. CH ₂Cl₂/DMF, TBTU, DIPEA,

ꢀ208C/15 min, room temperature/30 min), 2. 4, DIPEA, ꢀ208C/30 min, room temperature/18 h (80%); f)

CH₂Cl₂, CF₃CO₂H, room temperature, 1 h (100%).

of dansylation was quantified

by NMR spectroscopy and

found to be beyond 95%. In ac-

cordance with this the MALDI-

TOF mass spectra showed clean

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Scheme 4. Synthesis of the amino acid terminated G1 dendrimers of set B. Reagents and conditions: a) 11a + 17, CH₂Cl₂, DIPEA, ꢀ108C/1 h, room

temperature/18 h (79%); 11b + 17, CH₂Cl₂/DMF, DIPEA, room temperature/18 h (82%); b) 1m KOH, methanol/water/THF, 408C, 12 h (80%); c) 1m

KOH, methanol/water/THF, 508C, 12 h (92%); d) 1. 19a, [19b], CH₂Cl₂/DMF, EDC, HOBt, ꢀ208C [58C]/1 h, room temperature 2 h , 2. 4, DIPEA,

ꢀ308C [08C]/1 h, room temperature 18 h (20a: 54%, 20b: 48%); e) CH₂Cl₂/DMF, DIPEA, ꢀ108C/1 h, room temperature 18 h (71%); f) CH₂Cl₂/

CF₃CO₂H/EDT/thioanisole/methanol/TIPS, room temperature, 1 h (97%); g) CH₂Cl₂, CF₃CO₂H, room temperature, 1 h (99%); h) CH₂Cl₂, CF₃CO₂H,

room temperature, 3h (100%).

molecular ion peaks. Dendrimers 26 28 were well soluble in

dichloromethane/methanol mixtures, but exhibited poor sol-

ubility in water. Compounds 27 and 28 tended to aggregate

in water and in cell culture media (with 28 forming the

larger aggregates under comparable conditions), whereas for

the small G0 dendrimer 26 no aggregation was observed.

Despite these properties dendrimers 26 and 27 showed cell

uptake (see below).

In addition, dendrimers 33 36 carrying a second function-

al group besides the fluorescence label were synthesized

(Scheme 7). The synthetic strategy applied differs from the

direct sulfonamide derivatization used by Vˆgtle et al. for

their labeling of light-harvesting dendrimers with different

chromophoric groups.^[34c]The orthogonally protected

branching unit 30 served as the key compound for the syn-

thetic route leading to dendrimers 33 36. The corresponding

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Scheme 5. Synthesis of the diaminopropionic acid terminated G1 dendrimer of set C. Reagents and conditions: a) 1. 14, CH₂Cl₂/DMF, TBTU, DIPEA,

ꢀ208C/30 min, room temperature/15 min, 2. 17, DIPEA, ꢀ208C/30 min, room temperature/12 h (90%); b) 1m KOH, methanol/THF, 508C, 12 h (87%);

c) 1. 23, CH₂Cl₂/DMF, TBTU, DIPEA, ꢀ108C/15 min, room temperature/30 min, 2. 4, DIPEA, 08C/15 min, room temperature/18 h (57%); d) CH₂Cl₂,

CF₃COOH, room temperature, 1 h (99%).

G1-dendrimer 31 was isolated in 63% yield. Its partial de-

protection to 32 and subsequent dansylation gave the highly

fluorescent dendrimer 33 in 99% yield.

be above 98%. Figure 1 (see p. 1175) shows the elution

curves of four typical cases (dendrimers 10, 20c, 24, and 35).

All compounds were characterized by their fully assigned

¹H and ¹³C NMR spectra and molecular ion peaks in the

mass spectra. The composition of those compounds that did

not give correct data from elemental analysis was proven by

high-resolution EI, FAB, or isotopically resolved MALDI-

TOF mass spectrometry (see Experimental Section).

Figure 1 shows the enlargened molecular ion regions of mo-

lecular ion peaks of the MALDI-TOF MS spectra of den-

Deprotection of its remaining three benzyloxycarbonyl

(Cbz) protecting groups proved to be difficult, but could fi-

nally be realized by reaction with neat trifluoroacetic acid

over a period of seven days. Reaction of the dansylated and

deprotected dendrimer 34 with the Boc-protected diamino-

propionic acid 14 gave dendrimer 35 in 33% yield. Its quan-

titative deprotection finally resulted in dendrimer 36, which

carried both the diaminopropionic acid chelating ligands as

well as the dansyl imaging functions.

1

drimers 10, 20c, 24, and 35. The H NMR spectra of the de-

protected dendrimers 10 and 36 illustrate the achieved

In all cases, the purity of the dendrimers was checked by

analytical RP-HPLC in their protected form and found to

purity (Figure 2; see p. 1177). All deprotections were quanti-

fied by high-field H NMR integration and found to be vir-

1

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Scheme 6. Synthesis of completely dansylated set D dendrimers. Reagents and conditions: a) Dns-Cl, TEA, CH₂Cl₂, 1 h, room temperature (92%); b)

Dns-Cl, TEA, CH₂Cl₂, 6 h, room temperature (74%); c) Dns-Cl, TEA, CH₂Cl₂, 12 h, room temperature (65%).

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Scheme 7. Synthesis of set D dendrimers with two functional units. Reac-

tions and conditions: a) KOH, Boc₂O, Cbz-Cl THF/water, 08C, 1 h

(35%); b) 1m KOH, methanol/water, 508C, 12 h (98%); c) 1. CH₂Cl₂,

EDC, HOBt, ꢀ208C/1 h, room temperature 2 h , 2. 4, DIPEA, ꢀ208C8C/

1 h, room temperature 24 h (63%); d) CH₂Cl₂, CF₃COOH, room temper-

ature, 1 h (88%); e) Dns-Cl, TEA, CH₂Cl₂, 3h, room temperature

(99%); f) CF₃COOH, room temperature, 7 days (99%); g) 1. 14, TBTU,

DIPEA; CH₂Cl₂/DMF, ꢀ208C/30 min, room temperature/30 min, 2. 34,

DIPEA, ꢀ208C/1 h, room temperature/24 h (33%); h) CH₂Cl₂,

CF₃COOH_,room temperature, 1 h (97%).

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1167 1192

conditions are given in the Ex-

perimental Section. Despite

their charge differences all den-

drimers, by visual inspection,

gave molecularly dispersed sol-

utions in the cell experiments.

The only two exceptions were

compounds 28 and 36, whose

behavior in buffered cell media

was already described above.

The completely dansylated den-

drimers 26 and 27 of set D pos-

sessed only low solubility in

water, but caused no extensive

aggregate formation in fetal

calf serum (FCS) containing

cell culture media. Only the

dansylated G2 dendrimer 28 ex-

hibited a strong tendency to ag-

gregate even in cell culture

media and was thus not used in

the subsequent cell uptake

studies. Dendrimers 26, 27, and

36 were mixed with cell culture

media by low-energy ultrasoni-

fication prior to addition to the

cultured cells. This always re-

sulted in clear solutions for the

cytotoxicity and cell uptake ex-

periments as judged by visual

inspection. Only for dendrimers

27 and 36 fluorescence micros-

copy proved the formation of

Figure 1. A) Analytical HPLC traces of the Boc-protected dendrimers 9, 20c, 24, and 35. Purity of the com-

pounds was determined by UV detection (254 nm) and subsequent integration. Aa) dendrimer 20c; eluent:

methanol/H₂O (9:1/v:v), flow rate: 1 mLmin^ꢀ1; purity: 97.2% (aggregate: 2.6%). Substance eluting after

0.89 min (*) (2.6%) appeared within the void volume of the used column (for details see Experimental Sec-

tion); it is consequently likely to be an artefact or an aggregate of high molar mass. Ab) dendrimer 35; eluent:

methanol/H₂O (9:1/v:v), flow rate: 1 mLmin^ꢀ1; purity: 100%. (Ac) dendrimer 24; eluent: methanol/H₂O (9:1/

v:v), flow rate: 1 mLmin^ꢀ1; purity: 98.3%. (Ad) dendrimer 9; eluent: methanol/H₂O (9:1/v:v), flow rate:

1 mLmin^ꢀ1; purity: 100%. B) MALDI-TOF-MS spectra of dendrimers 9, 20c, 24, and 35; dithranol was used

as a matrix (for details see Experimental Section). From the whole spectra only the enlargend parts of the iso-

topically resolved [M+K]⁺and [M+Na]⁺signals are depicted. In no case signals of higher mass were ob-

served. Ba) dendrimer 20c; Bb) dendrimer 35; Bc) dendrimer 24; Bd) dendrimer 9.

tually quantitative with the conversion 33 to 34 being the

only exception. Traces of remaining Cbz (500 MHz NMR)

were still found even after repeated deprotection attempts.

The deprotected dendrimers of sets A D were fully solu-

ble in water and in buffered cell culture media as judged by

visual inspection, and could immediately been examined in

cell culture. The only exception was the mixed dansylated

dendrimer 36, which turned out to be highly water-soluble

but precipitated with phosphate in phosphate buffered

saline (PBS) buffer at a concentration of 1 mm.

Ammonium salts of most primary amines have pK_avalues

of 9 10. At pH 7 most of the amines are thus protonated. It

is reasonable to assume that most of the amines on the den-

drimer basically behave like independent primary amines

and, thus, are also protonated, though their degree of proto-

nation was not determined. Standard PAMAMs are only

partially protonated at physiological pH due to the surpris-

ingly low pK_avalues of their terminal (pK_a=6.9^[49]) and in-

ternal amines (pK_a=3.9^[49]).^[19]In the following the unpro-

tected dendrimers are therefore somewhat unspecifically re-

ferred to as being polycationic and no correlation to total

numbers of charges is attempted.

smaller aggregates. At 378C, however, at which all experi-

ments were performed, they never formed aggregates which

could have been precipitated by centrifugation. Mainly the

same procedure was performed for all Boc-protected den-

drimers of sets A C, except that only two concentrations

were tested (5 and 10 mm) because of the expected lower

water solubility. It has to be noted, however, that at none of

the tested concentrations precipitation of the dendrimers

was observed. Control experiments with similar concentra-

tions of the sodium salts of the used counterions (chloride

and trifluoroacetate) were performed and showed no effect

in MCF7 cell culture, which assures that all cytotoxic effects

were caused by the dendrimers.

The water-soluble dendrimers of set A exhibited a con-

centration- and generation-specific cytotoxicity (Figure 3b;

see p. 1178). While core 4 was inactive at every concentra-

tion tested, the G1 dendrimer 8 showed a concentration-de-

pendent reduction of cell proliferation at 10 mm. The maxi-

mum effect was reached for each concentration at the end

of the test (Figure 3b: after 220 h at 20 mm: T/C=15%). The

antiproliferative effect of its G2 analogue 10 was much

higher than for 8. At concentrations of 5 10 mm no surviving

cells were observed. Concentrations from 0.5 3 mm were

then chosen to examine the concentration-dependent cyto-

toxic effects of this dendrimer. Its absolute cytotoxicity was

the highest observed for all dendrimers examined; even a

In vitro cytotoxicity in MCF-7 cell culture: All dendrimers

were examined in MCF-7 cell culture at concentrations of

1 20 mm over a period of nine days (Figures 3 8). The exact

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Figure 2. ¹H NMR spectra (500 MHz) of the two deprotected dendrimers 10 (a) and 36 (b) in [D₄]methanol; protons 9 and 11 of dendrimer 36 show dia-

stereotopic coupling and are therefore split into two distinct signals. In both spectra [D₄]methanol is marked by (*), and remaining H₂O from lyophiliza-

tion is assigned by (+); the signal of proton 17 was reduced in size, as indicated by (ꢁ).

2 mm concentration lowered the viable cell mass under the

amount at the beginning of the test (Figure 3b).

proliferative at higher concentrations, and 21b exhibited the

highest level of cytotoxicity of this series without any ap-

pearance of resistance (Figure 4b).

The same experiments were also carried out with set B

dendrimers. Figure 4 (see p. 1179) depicts the results of the

deprotected G0 and G1 dendrimers 13a c and 21a c, and

Figure 5 those of their protected analogues 12a c and 20a

c, respectively. The cationic 13a and the zwitterionic 13c did

not show significant antiproliferative effects even at 20 mm

(Figure 4), while the cationic 13b exhibited a pronounced

cytotoxicity increase with increasing concentration (Fig-

ure 4a). It should be noted that in this case the highest activ-

ity was observed after an incubation time of 70 100 h, which

was lowered during the course of incubation. This can be in-

terpreted to mean that the cells become resistant to this

compound. The same trend was observed with the corre-

sponding G1 dendrimers. Whereas 21c did not show any an-

tiproliferative effects even at 20 mm, 21a was slightly anti-

The protected set

B dendrimers 12a c and 20a c

(Figure 5; see p. 1180) showed a somewhat unexpected be-

havior in that most, but not all, of them were not antiproli-

ferative. 12a and 12b turned out to be cytotoxic. While 12b

showed concentration dependency, no difference between

the two concentrations tested (5 and 10 mm) was observed

for 12a. This might indicate that the available amount of

12a in solution is not higher than 5 mm. The reason for this

unexpected behavior of the smaller protected dendrimers is

unclear. All other protected dendrimers showed no effect

on cell proliferation.

For the Boc-protected set C dendrimers 15 and 24 no an-

tiproliferative effect was observed. A rather unexpected and

potentially important result was found for the unprotected

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Surface-Modified Dendrimers

1167 1192

Figure 2. (Continued).

dendrimers 16 and 25. Despite their positive charges both

compounds had no effect on proliferation at all concentra-

tions tested (Figure 6; see p. 1180).

The set D dendrimers 26 28 showed no cytotoxicity at all

concentrations tested (Figure 7; see p. 1181). In contrast, the

partially dansylated dendrimers 34 and 36 were both cyto-

toxic at higher concentrations (10 and 20 mm) (Figure 8).

34 reached even cytocidal effects at 20 mm. Dendrimer

36, which carries both diaminopropionic acid and dansyl

substituents, can be considered

a

hybrid of the fully

dansylated G1 dendrimer 27 and dendrimer 25, which is

fully covered with diaminopropionic acid groups. In this

sense, the fact that only 36 shows toxicity is somewhat sur-

prising.

The findings regarding the deprotected representatives of

sets A and B correlate well with the observations by Rob-

erts et al.^[30]and Duncan et al.,^[31]who also found polycation-

ic dendrimers to be cytotoxic depending on their generation

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Figure 3. In vitro cytotoxicity of core molecule 4 and of the basic dendrimers 8 and 10. Comparison of the a) Boc-protected and the b) deprotected

forms. For detailed information on the investigation of the T/C_corr[%] and T[%] values see Experimental Section.

number and concentration in cell culture media. Since cyto-

toxic behavior of polycationic compounds was not only re-

ported for dendrimers, but especially also for positively

charged polyelectrolytes like poly(ethyleneimine) (PEI),

poly(l-lysine hydro bromide) (PLL), poly(diallyl-dimethyl-

ammonium chloride) (DAD-MAC), diethylaminoethyl

(DEAE)-dextran, and chitosans,^[50,51]it is conceivable that

toxicity correlates with the presence of positive charges,

though a detailed molecular picture of the toxic mechanism

is not yet available. The cytotoxic effect of polycations is

sometimes explained by means of charge density and flexi-

bility of the macromolecules.^[50,52]Ryser proposed that the

three-dimensional structure of compounds is important in

their biological response on cell membranes.^[53]Branched

molecules were found to be more efficient in neutralizing

the cell surface charge than polymers with linear or globular

structures, and rather rigid molecules like dendrimers were

less toxic than linear or branched polymers.^[50]Fischer et al.

concluded from their experiments that membrane leakage

occured first in cells exposed to cationic polymers and is fol-

lowed by a decrease in metabolic activity.^[50]In contrast to

these findings it is somewhat surprising that the completely

ethylenediamine-modified dendrimers of set C exhibited no

cytotoxic effects at all concentrations tested (Figure 6). It is

not yet clear whether the bidendate nature of the terminal

diaminopropionic amide substituents with its different pro-

tonation, metal ion complexation, and hydrogen bonding be-

havior is responsible for this finding. While the completely

dansylated dendrimers of set D did not exhibit antiprolifera-

tive effects, the observed cytotoxicity of the mixed dansylat-

ed (34 and 36) and phenylalanine dendrimers (13b and 21b)

suggests that ’tenside effects’ (disruption of cell membranes

by interaction with negatively charged groups and hydro-

phobic parts) are likely to be responsible for the observed

cell death. Also, induction of apoptosis by the added den-

drimers cannot be completely excluded, though indications

for such a mechanism have not yet been reported.^[50]It is

unlikely that details of the interior structure of the dendrim-

ers, in particular the phenyl branching groups, are responsi-

ble for the observed toxicity, because in the analogous stud-

ies under identical conditions the Boc-protected dendrimers

3, 7, and 9 (Figure 3) did not show cytotoxicity. It should be

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Surface-Modified Dendrimers

1167 1192

Figure 4. In vitro cytotoxicity of the a) amino acid-terminated G0 dendrimers 13a, 13b, 13c , and b) of the corresponding G1 dendrimers 21a, 21b, 21c.

noted, however, that these compounds have a lower solubili-

ty in cell culture media and, thus, their bioavailability may

be reduced.

is modulated by mitotic phosphorylation. The cells were

then analyzed by confocal fluorescence microscopy. Figure 9

and Figure 10 (see p. 1181 and 1182, respectively) present

typical confocal images of such experiments. Similar experi-

ments with human MCF-7 cells and mouse fibroblast cells

(NIH 3T3) performed under the same conditions resulted in

the same distribution pattern (not shown).

All experiments were reproducible, and the observed in-

tracellular patterns were representative for the cells of each

single experiment. At least 30 40% of all cells exhibited a

specific intracellular fluorescence, and 90% of the fluores-

cence-positive cases showed practically the same distibution

patterns as presented in Figure 9 and Figure 10.

Figure 9 shows the differential interference contrast

(DIC) and fluorescence images of the cells incubated with

dendrimer 26, obtained by confocal fluorescence microsco-

py. Already in the DIC image, large intracellular aggregates

with a granular structure were identified, (red arrows in Fig-

ure 9a). Upon excitation (364 nm), these aggregates exhibit-

ed a strong green-blue fluorescence and could, therefore,

easily be identified and located within cells (dendrimer fluo-

rescence encoded in blue in Figure 9c and Figure 10). Com-

pared with the fluorescence pattern of Cy2-immunolabeled

Nevertheless, the polycationic nature of compounds

cannot fully explain the observed cytotoxicity in every case.

The results of dendrimers 13b, 21b, 16, 25, 34, and 36 show

that other structural factors may also play a role. A correla-

tion of cytotoxicity data with structural motifs such as the

hydrophobic or ionic groups is tempting but does not seem

to be appropriate at the present state. An even more broad

study would be required.

Cellular uptake and localization: To study cellular uptake

and intracellular distribution of the dansylated dendrimers,

human HeLa cells were incubated for 20 h with dendrimers

26, 27, 34, and 36 at a final concentration of 5 mm. Then, the

cells were fixed and, for better orientation within the cell,

immunostained with an antibody raised against the mem-

brane forms of the lamina-associated polypeptide 2 (LAP-

2), which are enriched at the inner membrane of the nuclear

envelope and are partially also present in the endoplasmic

reticulum.^[54]Integral membrane proteins of the nuclear en-

velope interact with lamins and chromosomes, and binding

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Figure 5. In vitro cytotoxicity of a) the Boc-protected amino acid-terminated G0 dendrimers 12a, 12b, 12c, and b) of the corresponding G1 dendrimers

20a, 20b, 22c.

normally observed per cell, and

their diameters ranged from 2

to 4 mm. For dendrimer 27 the

size of the formed aggregates

was larger, and they appeared

more granular (Figure 10a).

The rather long incubation time

prior to observation in compari-

son with typical cellular traf-

ficking time-scales of dendrim-

57]

ers^[33,55

suggests that a stable

situation had been reached. The

water-soluble dendrimer 34, on

the other hand, showed a more

punctated distribution pattern,

with a diameter of the granules

of approximately 0.5 1 mm on

average. This pattern could be

Figure 6. In vitro cytotoxicity of the diaminopropionic acid G0 and G1 dendrimers 16 and 25.

LAP-2, the distribution of the aggregated dendrimers was

distinguishable from the nuclear envelope staining (Fig-

ure 9c). The granular structures were typically located near

the cell nucleus. Between one and three aggregates were

of endosomal or lysosomal origin, since distribution and size

agree well with that of compartments formed by fused

endo- or lysosomes (Figure 10b). The intracellular distribu-

tion pattern of dendrimer 36, finally, resembled that of 26

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Surface-Modified Dendrimers

1167 1192

Figure 7. In vitro cytotoxicity of the dansylated dendrimers 26, 27, and 28.

(NRK) cells after one hour of

incubation. In this case, endocy-

tosis was suggested to be the

mechanism

of

cellular

uptake.^[55]Juliano et al. also

found a slightly ™clumpy∫ cellu-

lar distribution of Oregon green

488-conjugated PAMAM den-

drimer/oligonucleotide

com-

plexes in HeLa cells. Dendrim-

ers were not only found inside

the nucleus but also at other

sites in the cell, presumably at

the plasma membrane and in

endomembrane

compart-

shown in

ments.^[32,58]

As

Figure 8. In vitro cytotoxicity of the mixed dendrimers 34 and 36 with two different surface moieties.

Figure 9 and Figure 10, none of

the dendrimers investigated in

this study displays a localization

inside of the nucleus. Instead,

they are rather always found di-

rectly next to the nucleus. This

could suggest

a

localization

within or close to the Golgi ap-

paratus. An example for such a

distribution was given by

Merlin et al. who found a se-

questration of daunorubicin in

the Golgi vesicles in drug-resis-

tent MCF-7 cells.^[59,60]Tanke,

Reedijk and co-workers report

a punctated staining of a cyto-

plasmatic region, when human

Figure 9. Confocal fluorescence microscopy images. HeLa cells were incubated with a final concentration of

5 mm of dendrimer 26 for 20 h at 378C, washed, fixed and immunostained for the nuclear envelope marker

LAP-2 (membrane isoforms) which was visualized with a Cy2-labeled secondary antibody. Dendrimer localiza-

tion is indicated by red arrows. a) DIC image; b) overlay of LAP-2 staining (green) and dendrimer fluores-

cence (blue); c) Dendrimer fluorescence (blue channel).

and 27. One to three large aggregates near the cell nucleus

were typically observed (Figure 10c).

A similar punctated intracellular distribution pattern has

been described previously for other dendrimers. Polypro-

line-based dendrimers of Giralt and co-workers exhibited a

more or less vesicular distribution in normal rat kidney

osteosarcoma (U2-OS) cells were incubated with fluoro-

phore-labeled platinum complexes.^[61]Co-localization experi-

ments with a Golgi apparatus-selective stain also indicated

the involvement of Golgi-vesicles in the intracellular proc-

essing. A related early endosomal or lysosomal localisation

also seems to be likely for dendrimer 34 because of its ob-

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drimer surface do not automati-

cally cause cell toxicity as one

may have been inclined to con-

clude both from literature re-

sults and those obtained in the

present work. It is not clear yet

whether the bidendate nature

of ethylenediamine plays a role

here, for example by chelating

metal ions.

Figure 10. Confocal fluorescence microscopy images; overlay of DIC-image, and dendrimer fluorescence.

HeLa cells were incubated with dendrimers at a final concentration of 5 mm (see text for details). a) dendrimer

27; b) dendrimer 34; c) dendrimer 36.

Confocal fluorescence micros-

copy studies revealed that all

dansylated dendrimers are in-

ternalized by HeLa cells and

served distribution pattern (Figure 10), but this has not been

proven yet.

remain intracellularly present over a 20-hour incubation

period. Further kinetic studies will address the velocity of

the cellular uptake and the intracellular trafficking to their

final destination.

Hopefully, the results of such experiments, in combination

with studies on metabolic degradation and the biodistribu-

tion pattern in animals will finally enable the design of opti-

mized and nontoxic drug-carrier systems.

To finally answer the question of intracellular localization

of the dendrimers reported here after uptake by the cell,

further kinetic and co-localization studies have to be per-

formed. Initial kinetic studies indicate that cellular uptake

of the dendrimers is a rather fast process and probably fin-

ishes after 4 5 h (data not shown). If the dendrimers are

taken up by endocytosis, they should initially be localized in

clathrin-coated vesicles which subsequently fuse to larger

endosomes and then lysosomes. Some of the dendrimers

might also be directed towards the Golgi apparatus. There-

fore, the exact intracellular localization of the dendrimers

needs to be characterized in more detail, for example by co-

localization with known markers for the different intracellu-

lar compartments.^[62]

Experimental Section

Abbreviations: 9-BBN: 9-borabicyclononane; Boc: tert-butyloxycarbonyl;

Cbz: benzyloxycarbonyl; CCA: a-cyano-4-hydroxy-cinnamonic acid;

DCM: dichloromethane; DIPEA: N-ethyl-diisoropylamine; DMF: N,N-

dimethylformamide; DMSO: dimethyl sulfoxide; Dns: 5-dimethylamino-

naphthalene-1-sulfonyl; Dpa: diaminopropionic acid; EDC: N-(3-dime-

thylaminopropyl)-N’-ethyl-carbodiimide hydrochloride; EDTA: ethylene-

diamine tetraacetate; FCS: fetal calf serum; HOBt: hydroxy-benzotria-

zole hydrate; HRMS: high-resolution mass spectrometry; MNBA: m-ni-

trobenzyl alcohol; PBS: phosphate-buffered saline; RP-HPLC: reversed-

phase high-performance liquid chromatography; TBTU: O-(1H-benzo-

triazole-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate; THF: tet-

rahydrofuran.

Conclusion

With a series of simple and optimized steps a set of low gen-

eration polyamidoamine dendrimers was synthesized, many

of which are highly water-soluble. Convergent and divergent

strategies were applied in order to make accessible a bou-

quet of differently surface decorated representatives at the

least synthetic effort. The surfaces of the dendrimers were

decorated with natural amino acid and ethylenediamine

moieties, fluorescence labels, and simple amino-protecting

groups either as the only substituents or in combinations.

In vitro cytotoxicity experiments were performed with all

these dendrimers using the human breast cancer cell line

MCF-7. These experiments proved that surface-functionali-

zation grossly influences dendrimer toxicity. Based on the

data obtained a broader structure/toxicity correlation could

not be established yet. A few interesting conclusions can

nevertheless be drawn: The internal structure of the present-

ed dendrimers does not seem to play a profound role, de-

spite the common view that the interior of low-generation

dendrimers is accessible. The surface decoration, however, is

crucial for toxicity. Most of the examined noncharged den-

drimers (e.g., the protected and dansylated ones) are non-

toxic though they are clearly bioavailable as was proved by

cell uptake experiments, and all completely diamino pro-

pionic acid decorated dendrimers are also nontoxic. Espe-

cially this last case shows that positive charges on a den-

Synthesis and purification of the dendrimers: general remarks: All start-

ing materials were purchased from commercial sources and used without

further purification. Solvents were dried under standard conditions. Lyo-

philization from water was performed by using deionized, destilled,

’MilliQ’ (Millipore) (0.45 mm) filtered water. The following compounds

were prepared according to literature procedures: N-(tert-butyloxycarbo-

nyl)allylamine (2),^[63]5,^[47c]6,^[47b,c]14,^[36]and 17.^[47b]Compounds 29 and 30

are known,^[47b]but were prepared according to a different procedure.^[64]

Therefore their analytical and spectral data are not given. All other com-

pounds are new. Whenever possible, reactions were monitored by thin-

layer chromatography (TLC) using TLC silica gel coated aluminum

plates 60F₂₅₄(Merck). Compounds were detected by UV light (254 nm or

366 nm) and/or by treatment with a solution of ninhydrine in ethanol fol-

lowed by heating. ¹H and ¹³C NMR spectra were recorded using Bruker

AC 500 (500 MHz) and AB 250 (250 MHz) instruments; the solvent

signal was used for internal calibration. Mass spectra were recorded by

using a Varian MAT 711 and CH6 (EI) or Type CH5DF (FAB), and a

Bruker Reflex with delayed extraction source (MALDI-TOF). Elemental

analyses were performed by using a Perkin-Elmer EA 240. Because of

the polarity of the prepared compounds, it was generally difficult to

obtain correct data from elemental analysis. This was specifically so for

the Boc-protected and dansylated dendrimers, and for some of the free

carboxylic acids, for which the carbon values obtained differed from the

calculated ones by up to 1%. Analytical RP-HPLC was carried out using

an HPLC System consisting of a Gynkotek UVD 340 S Diode Array De-

tector, a Gynkotek Mod. 480 Pump, and a Knaur Eurosphere column

(C₁₈, 100 5 mm, 4î120 mm).

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Surface-Modified Dendrimers

1167 1192

For clarity reasons a simplified nomenclature has been added in front of

each compound name in the Experimental Section. For example, in

(Boc-N)₃3D0 the text in parentheses (here Boc-N) defines the functional

endgroup, the subscript number, here (₃), is number of those groups, the

regular sized number (3) gives the total number of modifiable endgroups,

and finally the letter D plus a number (here 0) stands for a dendrimer of

the corresponding specific generation.

(ArC), 156.09 (CON_Boc), 167.75 ppm (CON); MS (FAB+, MNBA/DCM/

DMSO): m/z (%): 1505 (17.8) [M+H]⁺, 1504 (17.8) [M]⁺, 1404 (57.1)

[MꢀC₅H₉O₂]⁺,

1304

1104

(22.8)

[Mꢀ2(C₅H₉O₂)]⁺,

1204

(23.3)

(21.5)

[Mꢀ3(C₅H₉O₂)]⁺,

[Mꢀ4(C₅H₉O₂)]⁺, 1004

[Mꢀ5(C₅H₉O₂)]⁺, 904 (100) [Mꢀ6(C₅H₉O₂)]⁺; MS (MALDI-TOF, di-

thranol): m/z: 1543[ M+K]⁺, 1527 [M+Na]⁺; monoisotopic mass calcd

for C₈₄H₁₂₉N₉NaO₁₅⁺: 1526.95, found: 1526.94.

(Boc-N)₃3D0:

1,3,5-tris-[(tert-butyloxycarbonylamino)propyl]benzene

(N)₆6D1: 1,3,5-tris[3,5-bis(3-aminopropyl)-N-propylbenzamide]benzene

hexatrifluoroacetate (8): The Boc-protected G1 dendrimer 7 (336 mg,

223 mmol) was dissolved in a small amount of dichloromethane (10 mL).

Trifluoroacetic acid (2 mL) was added, and the reaction mixture was stir-

red for 1 h at room temperature. After complete reaction (TLC) the sol-

vent was removed in vacuo to give the deprotected dendrimer (348 mg,

219 mmol; 98.2%) as a colorless oil which could be lyophilized from

water. M.p. 978C; ¹H NMR (250 MHz, [D₄]methanol): d=2.02 (m, 6H

+ 12H; CH₂), 2.69 (t, ³J(H,H)=7.4 Hz, 6H; CH₂Ar), 2.79 (t, ³J(H,H)=

7.8 Hz, 12H; CH₂Ar), 2.99 (t, ³J(H,H)=7.5 Hz, 12H; CH₂N), 3.44 (t,

³J(H,H)=7.1 Hz, 6H; CH₂N), 6.97 (s, 3H; ArH), 7.33 (s, 3H; ArH),

7.57 ppm (s, 6H; ArH); ¹³C NMR (63MHz, [D ₄] methanol): d=30.12

(CH₂), 32.21 (CH₂), 33.28 (CH₂), 34.33 (CH₂), 40.26 (CH₂), 40.87 (CH₂),

126.30 (ArC), 127.26 (ArC), 132.66 (ArC), 136.58 (ArC), 142.71 (ArC),

143.23 (ArC), 170.24 ppm (CON); MS (FAB+, DMSO/2-nitrophenol):

m/z (%): 927 (3.3) [M+Na]⁺, 905 (15.6) [M+H]⁺, 219 (100)

[C₁₃H₁₉N₂O]⁺; MS (MALDI-TOF, CCA): m/z: monoisotopic mass calcd

for C₅₄H₈₂N₉O₃⁺: 904.65, found: 904.85 [M+H]⁺.

(3): 9-BBN (15.4 g, 126 mmol) was added at 08C to a solution of tert-

butyl allylcarbamate 2 (16.5 g, 105 mmol) in dry toluene. The reaction

mixture was allowed to warm up to room temperature and stirred for an

additional 12 h. Then 1,3,5-tribromobenzene (1, 7.87 g, 25.0 mmol) and a

1m solution of KOH in water (225 mL) were added. After degassing the

reaction mixture, [Pd(PPh₃)₄] (1.16 g, 1.00 mmol) was added. The reac-

tion mixture was degassed repeatedly thereafter and stirred at 508C for

24 h. After complete reaction (TLC) the layers were separated, the or-

ganic phase was washed with brine once, and the aqueous phase was ex-

tracted with diethyl ether. The combined organic layers were dried over

magnesium sulfate and the solvent was removed in vacuo. Column chro-

matography (silica gel, hexane/ethyl acetate (3:1/v:v)) and subsequent re-

cristallization from hexane/ethyl acetate (4:1/v:v) yielded the Boc-pro-

tected core (12.9 g, 23.5 mmol; 94.0%) as a colorless solid. R_f=0.30

(hexane/ethyl acetate=2:1/v:v); m.p. 898C; ¹H NMR (500 MHz, CDCl₃):

d=1.41 (s, 27H; CH₃), 1.75 (q, ³J(H,H)=7.4 Hz, 6H; CH₂), 2.54 (t,

³J(H,H)=7.6 Hz, 6H; CH₂Ar), 3.08 (m, 6H; CH₂N), 4.65 (s, br, 3H;

NH), 6.78 ppm (s, 3H; ArH); ¹³C NMR (63MHz, CDCl ₃): d=28.38

(CCH₃), 31.58 (CH₂), 32.85 (CH₂Ar), 40.07 (CH₂N), 78.97 (CCH₃),

126.10 (ArC), 141.67 (ArC), 155.95 ppm (CO); MS (EI, 80 eV, 1808C);

m/z (%): 549 (3.2) [M]⁺, 449 (49.8) [MꢀC₅H₈O₂]⁺, 263(100.0); elemen-

tal analysis calcd (%) for C₃₀H₅₁N₃O₆(549.75): C 65.54, H 9.35, N 7.64;

found: C 65.47, H 9.06, N 7.51.

(Boc-N)₁₂12D2: 1,3,5-Tris(3,5-bis{3,5-bis[(tert-butyloxycarbonylamino)-

propyl]-N-propylbenzamide}-N-propylbenzamide)benzene (9): The G2

acid 4 (830 mg, 800 mmol) was dissolved in dry dichloromethane. HOBt

(130 mg, 851 mmol) was added, and the reaction mixture was stirred for

15 min at room temperature. The mixture was cooled down to ꢀ208C

and EDC (171 mg, 890 mmol) was added. The solution was stirred for an

additional 2 h and allowed to warm up to room temperature. After com-

plete reaction (TLC) the mixture was cooled down to ꢀ308C and

(381 mL, 290 mg, 2.24 mmol) DIPEA was added. The trisamine core 4

(65.0 mg, 180 mmol) was dissolved in a small amount of dry methanol

and slowly added to the reaction mixture under vigorous stirring. The sol-

ution was stirred for an additional 12 h and during that time allowed to

warm up to room temperature. Afterwards the mixture was washed twice

with sodium hydrogencarbonate solution and once with brine. The organ-

ic layer was dried over magnesium sulfate, and the solvent removed in

vacuo. The crude product was purified by column chromatography (silica

gel, dichloromethane containing 4% methanol as eluent) to afford

(526 mg, 154 mmol; 85.6%) of the G2 dendrimer 9 as a colorless foam.

R_f=0.19 (dichloromethane/methanol=19:1/v:v); m.p. 1068C; ¹H NMR

(500 MHz, CDCl₃): d=1.38 (s, 108H; CH₃), 1.69 (quin, ³J(H,H)=7.4 Hz,

24H; CH₂), 1.82 (m, 12H; CH₂), 1.86 (m, 6H; CH₂), 2.52 (m, 24H +

12H + 6H; CH₂Ar), 3.29 (m, 24H; CH₂N), 3.31 (m, 12H; CH₂N), 3.37

(m, 6H; CH₂N), 4.94 (s, br, 12H; NH), 6.83(s, 3H; Ar H), 7.00 (s, 3H;

ArH), 7.03(s, 6H; Ar H), 7.28 (s, br, 6H + 3H; NH), 7.31 (s, 6H; Ar H),

7.43ppm (s, 12H; Ar H); ¹³C NMR (126 MHz, CDCl₃): d=28.43(C CH₃),

29.68 (CH₂), 30.74 (CH₂), 31.33 (CH₂), 32.51 (CH₂), 32.85 (CH₂), 33.37

(CH₂), 39.32 (CH₂), 39.66 (CH₂), 53.40 (CH₂), 79.06 (CCH₃), 124.91

(ArC), 126.33 (ArC), 131.38 (ArC), 131.63 (ArC), 134.73 (ArC), 134.83

(ArC), 141.75 (ArC), 141.84 (ArC), 141.95 (ArC) 142.04 (ArC), 156.16

(CON_Boc), 167.86 (CON), 167.96 ppm (CON); MS (MALDI-TOF, IAA):

(N)₃3D0: 1,3,5-tris(propylamine)benzene trishydrochloride (4): The Boc-

protected core 3 (8.80 g, 16.0 mmol) was dissolved in THF and stirred

with a 25% HCl solution (28.0 mL, 192 mmol) for 12 h at room tempera-

ture. Part of the pure product already precipitated from the reaction mix-

ture, for the other part the solvent was removed in vacuo, the residue dis-

solved in ethanol and precipitated with diethyl ether. The procedure

yielded the trishydrochloride (5.49 g, 15.3mmol; 95.6%) as a colorless

1

solid. M.p. 2908C (decomp); H NMR (500 MHz, [D₄]methanol): d=2.04

(quin, ³J(H,H)=7.6 Hz, 6H; CH₂), 2.74 (t, ³J(H,H)=7.6 Hz, 6H;

CH₂Ar), 2.99 (t, ³J(H,H)=7.8 Hz, 6H; CH₂N), 7.05 ppm (s, 3H; ArH);

¹³C NMR (63MHz, [D ₄]methanol): d=30.16 (CH₂), 33.34 (CH₂Ar), 40.35

(CH₂N), 127.55 (ArC), 142.45 ppm (ArC); MS (EI, 80 eV, 2008C): m/z

(%): 249 (15.0) [M]⁺, 219 (100.0) [MꢀCH₂NH₂]⁺; HRMS: m/z: monoi-

sotopic mass calcd for C₁₅H₂₇N₃⁺: 249.22050, found: 249.22221.

(Boc-N)₆6D1: 1,3,5-tris-{3,5-bis[(tert-butyloxycarbonylamino)propyl]-N-

propylbenzamide}benzene (7): The free acid 5 (1.83g, 4.20 mmol) was

dissolved in dry dichloromethane, HOBt (674 mg, 4.40 mmol) was added,

and the solution was stirred for 15 min. Afterwards the reaction mixture

was cooled down to ꢀ208C, EDC (882 mg, 4.60 mmol) was added, and

the mixture was subsequently stirred for additional 2 h. During that time

the reaction mixture wass allowed to warm up to room temperature

slowly. After complete reaction (TLC), the mixture was cooled down to

ꢀ308C, and DIPEA (2.01 mL, 1.53g, 11.8 mmol) was added. The trisa-

mine core 4 (359 mg, 1.00 mmol) was dissolved in a small amount of dry

methanol and slowly added to the reaction mixture under vigorous stir-

ring. The solution was stirred for an additional 16 h, and during that time

allowed to warm up to room temperature slowly. After complete reaction

the solution was washed twice with sodium hydrogencarbonate solution

and once with brine. The organic layer was dried over magnesium sulfate,

the solvent removed in vacuo, and the crude product purified by column

chromatography (silica gel, dichloromethane containing 2% methanol as

eluent) to afford the G1 dendrimer (1.18 g, 784 mmol, 78.4%) as a color-

less foam. R_f=0.37 (dichloromethane/methanol=19:1/v:v); m.p. 898C;

¹H NMR (250 MHz, CDCl₃): d=1.40 (s, 54H; CH₃), 1.72 (m, 12H; CH₂),

1.94 (m, 6H; CH₂), 2.55 (m, 6H + 12H; CH₂Ar), 3.05 (m, 12H; CH₂N),

3.41 (m, 6H; CH₂N), 4.78 (m, br, 6H; NH), 7.85 (s, 3H; ArH), 7.02 (s,

br, 3H; NH), 7.03 (s, 3H; Ar H), 7.37 ppm (s, 6H; ArH); ¹³C NMR

(63MHz, CDCl ₃): d=28.41 (CCH₃), 30.85 (CH₂), 31.35 (CH₂), 32.48

(CH₂), 33.16 (CH₂), 39.58 (CH₂), 48.77 (CH₂), 79.10 (CCH₃), 124.79

(ArC), 126.28 (ArC), 131.45 (ArC), 134.91 (ArC), 141.71 (ArC), 141.78

m/z: 3452 [M+K]⁺,

3 ; monoisotopic mass calcd for

43 6M+[ Na]⁺

C₁₉₂H₂₈₅N₂₁NaO₃₃⁺: 2436.12, found: 2436.29.

(N)₁₂12D2:

1,3,5-Tris{3,5-bis[3,5-bis(3-aminopropyl)-N-propylbenza-

mide]-N-propylbenzamide}benzene dodecatrifluoroacetate (10): The

Boc-protected G2 dendrimer 9 (180 mg, 54.1 mmol) was dissolved in di-

chloromethane (8 mL). Trifluoroacetic acid (2 mL) was added, and the

reaction mixture was stirred for 2 h at room temperature. After complete

reaction (TLC) the solvent was removed in vacuo to give the deprotected

dendrimer (192 mg, 53.6 mmol, 99.1%) as a colorless oil which could be

1

lyophilized from water. M.p. 110 1128C; H NMR (500 MHz, [D₄]metha-

nol): d=1.97 (m, 18H; H-4

+ H-10), 2.02 (m, 24H; H-10’) 2.67 (t,

³J(H,H)=7.4 Hz, 6H; H-3), 2.73 (t, ³J(H,H)=7.6 Hz, 12H; H-9), 2.77 (t,

³J(H,H)=7.8 Hz, 24H; H-9’), 2.98 (t, J(H,H)=7.7 Hz, 24H; H-11’), 3.43

3

(m, 18H; H-11 + H-5), 6.96 (s, 3H; H-1), 7.30 (s, 3H; H-8), 7.32 (s, 6H;

H-8’), 7.50 (s, 6H; H-8), 7.55 ppm (s, 12H; H-7’); ¹³C NMR (126 MHz,

[D₄]methanol): d=30.11 (C-10’), 31.95 (C-10), 32.04 (C-4), 33.28 (C-9’),

1183

Chem. Eur. J. 2004, 10, 1167 1192

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

R. Gust, A. D. Schl¸ter et al.

FULL PAPER

(0.9) [M+H]⁺, 120 (100) [C₈H₁₀N]⁺; elemental analysis calcd (%) for

C₅₇H₇₈N₆O₉(991.3): C 69.06, H 7.93, N 8.48; found: C 69.10, H 7.83, N

8.29.

(Boc/tBu-Asp)₃3D0: 1,3,5-tris-(l-3-tert-butoxycarbonylamino-N-propyl-

succinamic acid tert-butyl ester)benzene (12c): The hydroxysuccinimide

ester 11c (2.67 g, 6.90 mmol) was dissolved in dry dichloromethane and

cooled down to ꢀ108C. DIPEA (1.02 mL, 775 mg, 6.00 mmol) and a solu-

tion of core 4 (718 mg, 2.00 mmol) in dry methanol were added dropwise.

The reaction mixture was stirred for additional 12 h, and during that time

allowed to warm up to room temperature. After complete reaction the

mixture was washed twice with sodium hydrogencarbonate solution and

once with brine, dried over magnesium sulfate, and the solvent was

evaporated in vacuo. Column chromatography (silica gel, dichlorome-

thane containing 3% methanol as eluent) afforded pure 12c (1.88 g,

1.77 mmol; 88.5%) as a colorless foam. R_f=0.39 (dichloromethane/meth-

anol=19:1/v:v); m.p. 808C; ¹H NMR (500 MHz, CDCl₃): d=1.42 (s,

27H; CH₃), 1.43(s, 27H; CH ₃), 1.79 (quin, ³J(H,H)=7.2 Hz, 6H; CH₂),

2.55 (t, ³J(H,H)=7.4 Hz, 6H; CH₂Ar), 2.62 (dd, ²J(H,H)=16.7 Hz,

³J(H,H)=6.7 Hz, 3H; CHCH₂), 2.80 (dd, ²J(H,H)=16.7 Hz, ³J(H,H)=

5.0 Hz, 3H; CHCH₂), 3.19 (q, ³J(H,H)=6.6 Hz, 6H; CH₂NH), 4.42 (m,

br, 3H; CH), 5.79 (s, br, 3H; CHN H), 6.71 (s, br, 3H; CONH), 6.78 ppm

(s, 3H; ArH); ¹³C NMR (126 MHz, CDCl₃): d=28.02 (CCH₃), 28.32

(CCH₃), 30.81 (CH₂), 32.56 (CH₂Ar), 37.49 (CHCH₂), 38.75 (CH₂N),

50.85 (CH), 80.26 (CCH₃), 81.45 (CCH₃), 126.31 (ArC), 141.42 (ArC),

155.57 (CON_Boc), 170.93(CO t_Bu), 171.09 ppm (CON); MS (FAB+,

MNBA/DCM): m/z (%): 1064 (0.4) [M+H]⁺, 964 (2.3) [MꢀC₅H₈O₂]⁺, 57

(100) [C₄H₉]⁺; elemental analysis calcd (%) for C₅₄H₉₀N₆O₁₅(1063.3): C

61.00, H 8.53, N 7.90; found: C 60.84, H 8.22, N 7.73.

34.19 (C-9), 34.35 (C-3), 40.27 (C-11’), 40.72 (C-11), 40.86 (C-5), 126.02

(C-7), 126.30 (C-7’), 127.33 (C-1), 132.69 (C-8’), 132.91 (C-8), 136.01 (C-

2 2’’’’), 136.50 (C-2 2’’’’), 142.72 (C-2 2’’’’), 143.25 (C-2 2’’’’), 143.70 (C-2

2’’’’), 170.22 (C-6,6’), 170.48 ppm (C-6’,6); MS (MALDI-TOF; CCA):

m/z: 2252 [M+K]⁺, 2236 [M+Na]⁺, 2214 [M+H]⁺; monoisotopic mass

calcd for C₁₃₂H₁₉₀N₂₁O₉⁺: 2213.51, found: 2213.55.

(Boc-Met)₃3D0:

1,3,5-tris-[l-(3-methylsulfanyl-1-propylcarbamoylpro-

pyl)carbamic acid tert-butyl ester]benzene (12a): The hydroxysuccini-

mide ester 11a (3.74 g, 10.8 mmol) was dissolved in dry dichloromethane

under a nitrogen atmosphere, and the solution was cooled down to

ꢀ108C. Afterwards DIPEA (1.57 mL, 1.16 g, 9.00 mmol) and a solution

of core 4 (1.08 g, 3.00 mmol) in dry methanol was added dropwise. The

reaction mixture was stirred for an additional 12 h, and during that time

allowed to warm up to room temperature. After complete reaction the

mixture was washed twice with sodium hydrogencarbonate solution and

once with brine, and dried over magnesium sulfate. The solvent was re-

moved under reduced pressure, and the crude product was subsequently

purified by column chromatography (silica gel, dichloromethane contain-

ing 2% methanol as eluent) under a nitrogen atmosphere. The procedure

yielded 12a (2.40 g, 2.54 mmol, 84.7%) as a colorless foam. R_f=0.32 (di-

chloromethane/methanol=19:1/v:v); m.p. 1628C; ¹H NMR (500 MHz,

CDCl₃): d=1.41 (s, 27H; CCH₃), 1.80 (m, 6H; CH₂), 1.98 (m, 3H;

CHCH₂), 2.03(m, 3H; CHC H₂), 2.08 (s, 9H; SCH₃), 2.55 (m, 6H + 6H;

CH₂Ar + CH₂S), 3.17 (m, 6H; CH₂N), 4.34 (m, br, 3H; CH), 5.80 (s, br,

3H; NH), 6.77 (s, 3H; Ar H), 7.17 ppm (s, br, 3H; NH); ¹³C NMR

(126 MHz, CDCl₃): d=15.31 (SCH₃), 28.36 (CCH₃), 30.26 (CH₂S), 30.51

(CH₂), 31.98 (CHCH₂), 32.28 (CH₂Ar), 38.34 (CH₂N), 53.61 (CH), 79.91

(CCH₃), 126.45 (ArC), 141.24 (ArC), 155.90 (CON_Boc), 171.98 ppm

(CON); MS (FAB+, MNBA/ethanol): m/z (%): 965 (1.5) [M+Na]⁺, 943

(Met)₃3D0: 1,3,5-tris(l-2-amino-4-methylsulfanyl-N-propylbutyramide)-

benzene tristrifluoroacetate (13a): Under a nitrogen atmosphere den-

drimer 12a (755 mg, 800 mmol) was dissolved in a ’cleavage cocktail’ mix-

ture of dichloromethane/trifluoroacetic acid/ethanedithiol/thioanisole/

methanol/triisopropylsilane (50:37.5:5:5:2:0.5/v:v), and stirred for 1 h at

room temperature. After complete reaction (TLC) the ’cleavage cocktail’

mixture was removed in vacuo to give the deprotected methionine G0

dendrimer 13a (778 mg, 790 mmol; 98.8%) as a colorless oil, which could

1

be lyophilized from water. M.p. 84 868C; H NMR (500 MHz, [D₄]meth-

anol): d=1.87 (quin, ³J(H,H)=7.4 Hz, 6H; CH₂), 2.16 (s, 9H; SCH₃),

2.17 (m, 6H; CHCH₂), 2.62 (m, 6H; SCH₂), 2.65 (m, 6H; CH₂Ar), 3.25

(m, 3H; CH₂N), 3.34 (m, 3H; CH₂N), 3.99 (t, ³J(H,H)=6.6 Hz, 3H;

CH), 6.92 ppm (s, 3H; ArH); ¹³C NMR (127 MHz, [D₄]methanol): d=

15.16 (SCH₃), 29.93(SCH ₂), 32.10 (CH₂), 32.19 (CHCH₂), 34.10

(CH₂Ar), 40.37 (CH₂NH), 53.87 (CH), 127.28 (ArC), 143.06 (ArC),

169.68 ppm (CON); MS (EI, 80 eV, 2908C): m/z (%): 642 (6.5) [M]⁺, 53 9

(88.2) [MꢀC₄H₉NS]⁺, 350 (100) [MꢀC₁₂H₂₆N₃OS₂]⁺; HRMS: m/z: mon-

oisotopic mass calcd for C₂₇H₄₈N₆O₃S₂⁺: 568.32366 [MꢀC₃H₆S]⁺, found:

568.32294.

(2.4) [M+H]⁺, 57 (100) [C₄H₉]⁺

; elemental analysis calcd (%) for

C₄₅H₇₈N₆O₉S₃(943.3): C 57.29, H 8.33, N 8.91, S 10.20; found: C 57.24, H

8.29, N 8.81, S 10.26.

(Phe)₃3D0:

1,3,5-Tris(l-2-amino-3-phenyl-N-propylpropionamide)ben-

zene tristrifluoroacetate (13b): The l-phenylalanine G0 dendrimer 12b

(991 mg, 1.00 mmol) was dissolved in dichloromethane/methanol=19:1/

v:v (40 mL). Trifluoroacetic acid (5 mL) was added, and the reaction mix-

ture was stirred at room temperature for 1 h. After complete reaction

(TLC) the solvent was removed in vacuo to afford the deprotected l-

phenylalanine dendrimer 13b (1.02 g, 987 mmol; 98.7%) as a colorless

solid. M.p. 115 1178C; ¹H NMR (500 MHz, [D₄]methanol): d=1.72

(quin, ³J(H,H)=7.5 Hz, 6H; CH₂), 2.49 (t, ³J(H,H)=7.6 Hz, 6H;

CH₂Ar), 3.15 (m, 3H; CH₂N), 3.16 (m, 3H; CH₂N), 3.26 (m, 6H;

(Boc-Phe)₃3D0: 1,3,5-tris-[l-(2-phenyl-1-propylcarbamoylethyl)-carbam-

ic acid tert-butyl ester]benzene (12b): The hydroxysuccinimide ester 11b

(3.91 g, 10.8 mmol) was dissolved in dry dichloromethane under a nitro-

gen atmosphere and cooled down to 08C. Subsequently DIPEA

(1.57 mL, 1.16 g, 9.00 mmol) and a solution of 4 (1.08 g, 3.00 mmol) in

dry methanol were added dropwise. The reaction mixture was stirred for

additional 12 h, and during that time allowed to warm up to room tem-

perature. After complete reaction the mixture was washed twice with

sodium hydrogencarbonate solution and once with brine, dried over mag-

nesium sulfate, and the solvent was evaporated in vacuo. Column chro-

matography (silica gel; dichloromethane containing 3% methanol as

eluent) afforded 12b (2.37 g, 2.39 mmol, 79.7%) as a colorless solid. R_f=

0.46 (dichloromethane/methanol=19:1/v:v); m.p. 1908C; ¹H NMR

(500 MHz, CDCl₃): d=1.34 (s, 27H; CH₃), 1.65 (m, 6H; CH₂), 2.39 (t,

³J(H,H)=6.4 Hz, 6H; CH₂Ar), 3.02 (m, 6H; CH₂N), 3.02 (m, 6 H +

3H; CH₂N + CHCH₂), 3.10 (m, 3H; CHCH₂), 4.44 (m, br, 3H; CH),

5.64 (s, br, 3H; NH), 6.67 (s, 3H; Ar H), 6.81 (s, br, 3H; NH), 7.19 ppm

(m, 15H; ArH_phe); ¹³C NMR (127 MHz, CDCl₃): d=28.31 (CCH₃), 30.45

(CH₂), 32.25 (CH₂Ar), 38.29 (CHCH₂), 38.95 (CH₂N), 55.42 (CH), 79.84

(CCH₃), 126.31 (ArC), 126.65 (ArC_phe), 128.40 (ArC_phe), 129.35 (ArC_phe),

137.05 (ArC_phe), 141.05 (ArC), 155.52 (CON_Boc), 171.69 ppm (CON); MS

(FAB+, MNBA/methanol/DMSO): m/z (%): 1013(0.3) [ M+Na]⁺, 991

3

CHCH₂), 4.09 (t, J(H,H)=7.5 Hz, 3H; CH), 6.81 (s, 3H; Ar H), 7.32 (m,

9H; ArH_phe), 7.35 ppm (m, 6H; ArH_phe); ¹³C NMR (127 MHz, [D₄]meth-

anol): d=32.16 (CH₂), 34.22 (CH₂Ar), 39.02 (CH₂N), 40.54 (CHCH₂),

56.15 (CH), 127.48 (ArC), 129.08 (ArC_phe), 130.33 (ArC_phe), 130.80

(ArC_phe), 136.02 (ArC_phe), 143.21 (ArC), 169.73ppm (CON); MS (EI,

80 eV, 150 1808C): m/z (%): 690 (2.8) [M]⁺, 513(59.3) [ MꢀC₁₀H₁₃N₂O]⁺

,

44 (100) [C₃H₈]⁺

; HRMS: m/z: monoisotopic mass calcd for

C₄₂H₅₄N₆O₃: 690.42622, found: 690.42572.

(Asp)₃3D0: 1,3,5-Tris(l-3-amino-N-propylsuccinamic acid)benzene tris-

trifluoroacetate (13c): Trifluoroacetic acid (4 mL) was added to a solu-

tion of 12c (106 mg, 100 mmol) in dichloromethane (10 mL), and the re-

action mixture was stirred for 2 h at room temperature. After complete

reaction (TLC) the solvent was removed in vacuo to yield the deprotect-

ed aspartic acid G0 dendrimer (92 mg, 98 mmol; 98%) as a colorless oil,

1184

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 1167 1192

Surface-Modified Dendrimers

1167 1192

which could be lyophilized from water. M.p. 106 1098C; ¹H NMR

(500 MHz, [D₄]methanol): d=1.86 (quin, ³J(H,H)=7.3Hz, 6H; CH ₂),

2.64 (t, ³J(H,H)=7.6 Hz, 6H; CH₂Ar), 2.87 (dd, ²J(H,H)=17.5 Hz,

³J(H,H)=7.8 Hz, 3H; CHCH₂), 2.94 (dd, ²J(H,H)=17.6 Hz, ³J(H,H)=

5.2 Hz, 3H; CHCH₂), 3.23 (m, 3H; CH₂N), 3.31 (m, 3H; CH₂N), 4.19

(m, 3H; CH), 6.92 ppm (s, 3H; Ar H); ¹³C NMR (63MHz, [D ₄]metha-

nol): d=31.92 (CH₂), 33.85 (CH₂), 36.41 (CH₂), 40.27 (CH₂), 51.22 (CH),

127.39 (ArC), 143.00 (ArC), 169.19 (CON), 173.12 ppm (COO); MS

(FAB-, MNBA/methanol): m/z (%): 593(2.)3 [ MꢀH]^ꢀ, 113(89.9)

[C₄H₅N₂O₂]^ꢀ; monoisotopic mass calcd for C₂₇H₄₁N₆O₉^ꢀ: 593.3, found:

593.3 [MꢀH]^ꢀ.

(Boc-Dpa)₃3D0: 1,3,5-tris[(2-tert-butoxycarbonylamino-1-propylcarba-

moylethyl)carbamic acid tert-butyl ester]benzene (15): A solution of 14

(4.11 g, 13.5 mmol) in a mixture of dry dichloromethane/DMF (14:1/v:v)

was cooled down to - 208C. DIPEA (2.30 mL, 1.74 g, 13.5 mmol) and a

solution of TBTU (4.43g, 14.1 mmol) in DMF were added slowly and al-

lowed to warm to room temperature. The reaction mixture was stirred at

room temperature for additional 30 min, cooled down to ꢀ208C again,

followed by the dropwise addition of DIPEA (3.80 mL, 2.91 g;

thane containing 2 3% methanol as eluent) to afford the desired product

(1.15 g, 1.58 mmol, 79.0%) as a colorless foam. R_f=0.09 (dichlorome-

thane/methanol=49:1/v:v); m.p. 758C; ¹H NMR (500 MHz, CDCl₃): d=

1.34 (t, ³J(H,H)=7.1 Hz, 3H; CH₂CH₃), 1.37 (s, 18 H CCH₃), 1.81 (quin,

³J(H,H)=7.0 Hz, 4H; CH₂), 1.92 (m, 2H; CHCH₂), 2.02 (m, 2H;

3

CHCH₂), 2.05 (s, 6H; SCH₃), 2.51 (t, J(H,H)=7.3Hz, 4H; CH ₂Ar), 2.61

(m, 4H; SCH₂), 3.15 (m, 4H; CH₂N), 4.29 (m, br, 2H; CH), 4.31 (q,

³J(H,H)=7.1 Hz, 2H; CH₂CH₃), 5.69 (d, br, ³J(H,H)=6.1 Hz, 2H;

CHNH), 7.05 (s, br, 2H; CONH), 7.14 (s, 1H; ArH), 7.62 ppm (s, 1H;

ArH); ¹³C NMR (63MHz, CDCl ₃): d=14.26 (CH₂CH₃), 15.24 (SCH₃),

28.28 (CCH₃), 30.23 (CH₂), 30.44 (CH₂), 32.00 (CH₂), 32.11 (CH₂), 38.16

(CH₂), 53.62 (CH), 60.83 (CH₂CH₃), 79.83( CCH₃), 127.16 (ArC), 130.78

(ArC), 133.60 (ArC), 141.42 (ArC), 155.89 (CON_Boc), 166.63(CON),

172.09 ppm (COO); MS (FAB+, MNBA/DCM): m/z (%): 727 (10.3)

[M+H]⁺, 627 (22.2) [MꢀC₅H₉O₂+H]⁺, 527 (62.0) [Mꢀ2(C₅H₉O₂)+H]⁺,

57 (100) [C₄H₉]⁺; elemental analysis calcd (%) for C₃₅H₅₈N₄O₈S₂(727.0):

C 57.82, H 8.04, N 7.71; found: C 57.54, H 7.94, N 7.60.

3,5-Bis[l-(2-phenyl-1-propylcarbamoylethyl)carbamic acid tert-butyl es-

ter]benzoic acid ethyl ester (18b): A solution of the Boc-l-phenylalanine

hydroxysuccinimide ester 11b (5.22 g, 14.4 mmol) in dry dichloromethane

was cooled down to 58C. DIPEA (2.09 mL, 1.55 g, 12.0 mmol) and the

bishydrochloride dendron 17 (2.02 g, 6.00 mmol), dissolved in dry metha-

nol, were added slowly. The reaction mixture was allowed to warm up to

room temperature slowly and was stirred for an additional 12 h. After

complete reaction (TLC) the organic layer was washed twice with

sodium hydrogencarbonate solution and once with brine. The organic

phase was subsequently dried over magnesium sulfate. The solvent was

evaporated under reduced pressure, and column chromatography (silica

gel, dichloromethane containing 2% methanol as eluent) afforded the

Boc-l-phenylalanine dendron (3.72 g, 4.90 mmol; 81.7%) as a colorless

solid. R_f=0.59 (dichloromethane/methanol=19:1/v:v); m.p. 1218C;

¹H NMR (250 MHz, CDCl₃): d=1.35 (s, 18H; CCH₃), 1.36 (t, ³J(H,H)=

7.3Hz, 3H; CH ₂CH₃), 1.69 (m, 4H; CH₂), 2.47 (t, ³J(H,H)=7.0 Hz, 4H;

CH₂Ar), 3.03 (m, 4H; CH₂N), 3.05 (m, 4H; CHCH₂), 4.40 (m, br, 2H;

CH), 4.33 (q, ³J(H,H)=7.1 Hz, 2H; CH₂CH₃), 5.61 (d, br, ³J(H,H)=

5.9 Hz, 2H; CHNH), 6.68 (s, br, 2H; CONH), 7.05 (s, 1H; ArH), 7.19

(m, 10H; ArH_Phe), 7.60 ppm (s, 2H; ArH); ¹³C NMR (63MHz, CDCl ₃):

d=14.33 (CH₂CH₃), 28.31 (CCH₃), 30.42 (CH₂), 32.11 (CH₂), 38.25

(CH₂), 38.82 (CH₂), 52.00 (CH), 60.87 (CH₂CH₃), 79.98 (CCH₃), 126.78

(ArC), 127.16 (ArC), 128.51 (ArC), 129.34 (ArC), 130.84 (ArC), 133.57

(ArC), 136.98 (ArC), 141.47 (ArC), 155.61 (CON_Boc), 166.69 (CON),

171.64 ppm (COO); MS (FAB+, MNBA/DCM): m/z (%): 781 (0.2)

[M+Na]⁺, 759 (5.0) [M+H]⁺, 659 (11.2) [MꢀC₅H₉O₂+H]⁺, 559 (35.8)

[Mꢀ2(C₅H₉O₂)+H]⁺, 120 (100) [C₈H₁₀N]⁺, 57 (70.1) [C₄H₉]⁺; elemental

analysis calcd (%) for C₄₃H₅₈N₄O₈(758.9): C 68.05, H 7.70, N 7.38;

found: C 67.79, H 7.66, N 7.31.

22.5 mmol) and

a solution of the trishydrochloride core 4 (1.08 g,

3.00 mmol) in dry methanol. The mixture was stirred for additional 12 h

and during that time allowed to warm to room temperature. After com-

plete reaction (TLC), the mixture was washed twice with sodium hydro-

gencarbonate solution, once with brine, and dried over magnesium sul-

fate. The solvent was removed under reduced pressure and column chro-

matography (silica gel, dichloromethane containing 4% methanol as

eluent) afforded the desired product (2.66 g, 2.40 mmol, 80.0%) as a col-

orless foam. R_f=0.21 (dichloromethane/methanol=19:1/v:v); m.p.

1

1138C; H NMR (500 MHz, CDCl₃): d=1.38 (s, 27H; CH₃), 1.40 (s, 27H;

CH₃), 1.79 (m, 6H; CH₂), 2.54 (t, ³J(H,H)=7.0 Hz, 6H; CH₂Ar), 3.14

(m, 6H; CH₂N), 3.44 (m, 6H; CHCH₂), 4.26 (m, br, 3H; CH), 5.56 (s, br,

3H; NH_Boc), 6.05 (s, br, 3H; NH_Boc), 6.79 (s, 3H; ArH), 7.08 ppm (s, br,

3H; CONH); ¹³C NMR (127 MHz, CDCl₃): d=28.30 (CCH₃), 30.39

(CH₂), 32.08 (CH₂Ar), 38.14 (CH₂N), 42.56 (CHCH₂), 55.60 (CH), 77.25

(CCH₃), 79.66 (CCH₃), 126.52 (ArC), 141.14 (ArC), 156.10 (CON_Boc),

157.05 (CON_Boc), 170.75 ppm (CON); MS (FAB+, MNBA/DCM/metha-

nol): m/z (%): 1131 (0.9) [M+Na]⁺, 1009 (4.8) [MꢀC₅H₈O₂+H]⁺, 609

(2.2) [Mꢀ5(C₅H₈O₂)+H]⁺, 508 (21.2) [Mꢀ6( C₅H₈O₂)]⁺, 57 (100)

[C₄H₉]⁺; elemental analysis calcd (%) for C₅₄H₉₃N₉O₁₅(1108.4): C 58.52,

H 8.46, N 11.37; found: C 58.31, H 8.23, N 11.33.

(Dpa)₃3D0: 1,3,5-tris(2,3-diamino-N-propylpropionamide)benzene hexa-

trifluoroacetate (16): Trifluoroacetic acid (5 mL) was added to a solution

of the Boc-protected diaminopropionic acid G0 dendrimer 15 (554 mg,

500 mmol) in dichloromethane (10 mL), and the mixture was stirred for

1 h at room temperature. After complete reaction (TLC) the solvent and

the excess of trifluoroacetic acid were removed in vacuo. Lyophilization

from water yielded deprotected 16 (595 mg, 499 mmol; 99.8%) as a color-

less solid. M.p. 101 1038C; ¹H NMR (500 MHz, [D₄]methanol): d=1.89

(quin, ³J(H,H)=7.4 Hz, 6H; CH₂), 2.66 (t, ³J(H,H)=7.7 Hz, 6H;

CH₂Ar), 3.25 (m, 3H; CH₂N), 3.38 (m, 3H; CHCH₂), 3.40 (m, 3H;

CH₂N), 3.47 (dd, ²J(H,H)=13.7 Hz, ³J(H,H)=5.5 Hz, 3H; CHCH₂), 4.19

(t, ³J(H,H)=6.0 Hz, 3H; CH), 6.94 ppm (s, 3H; Ar H); ¹³C NMR

(63MHz, [D ₄]methanol): d=31.77 (s, CH₂), 33.97 (s, CH₂Ar), 40.65 (s,

CH₂N), 41.13(s, CHCH ₂), 52.23(s, CH), 118.03(q, ¹J(C,F)=294.4 Hz,

3,5-Bis[l-(3-methylsulfanyl-1-propylcarbamoylpropyl)carbamic acid tert-

butyl ester]benzoic acid (19a): The l-methionine G1 dendron 18a

(2.18 g, 3.00 mmol) was dissolved in methanol/water/THF (3:1:1/v:v)

under a nitrogen atmosphere, a 1m KOH solution (24.0 mL, 24.0 mmol)

was added, and the reaction mixture was subsequently heated to 408C

for 12 h. After complete reaction (TLC), acetic acid was added to give

pH 5, and the product subsequently extracted with dichloromethane. The

united organic layers were dried over magnesium sulfate and the solvent

was removed in vacuo. Chromatographic workup (silica gel, dichlorome-

thane containing 4% methanol as eluent) gave the desired product

(1.68 g, 2.40 mmol; 80.0%) as a colorless foam. R_f=0.11 (dichlorome-

thane/methanol=19:1/v:v); m.p. 898C; ¹H NMR (250 MHz, [D₄]metha-

nol): d=1.48 (s, 18H; CCH₃), 1.88 (m, 4H; CH₂), 1.91 (m, 2H; CHCH₂),

2.03(m, 2H; CHC H₂), 2.08 (s, 6H; SCH₃), 2.53(m, 4H; SCH ₂), 2.67 (t,

³J(H,H)=7.8 Hz, 4H; CH₂Ar), 3.22 (m, 4H; CH₂N), 4.14 (t, ³J(H,H)=

6.7 Hz, 2H; CH), 7.30 (s, 1H; ArH), 7.69 (s, 2H; ArH), 8.05 ppm (m, br,

2H; CONH); ¹³C NMR (63MHz, [D ₄]methanol): d=15.39 (SCH₃), 28.71

(CCH₃), 31.15 (CH₂), 31.15 (CH₂), 31.85 (CH₂), 32.95 (CH₂), 33.59

(CH₂), 55.12 (CH), 80.68 (CCH₃), 128.31 (ArC), 131.98 (ArC), 134.37

(ArC), 143.22 (ArC), 157.51 (CON_Boc), 169.98 (CON), 174.43ppm

(COO); MS (FAB+, MNBA/DCM): m/z (%): 699 (4.8) [M+H]⁺, 599

(7.9) [MꢀC₅H₉O₂+H]⁺, 499 (23.5) [Mꢀ2(C₅H₉O₂)+H]⁺, 57 (100)

[C₄H₉]⁺; monoisotopic mass calcd for C₃₃H₅₅N₄O₈S₂⁺: 699.4, found: 699.5

[M+H]⁺.

2

CF₃CO₂H), 127.34 (s, ArC), 142.97 (s, ArC), 163.35 (q, J(C,F)=34.2 Hz,

CF₃CO₂H), 166.99 ppm (s, CON); MS (FAB+, MNBA/DMSO): m/z

(%): 640 (1.1) [M+Cs]⁺, 508 (3.2) [M+H]⁺; elemental analysis calcd

(%) for C₃₆F₁₈H₅₁N₉O₁₅(1191.8): C 36.28, H 4.31, N 10.58; found: C

35.89, H 4.38, N 10.70.

3,5-Bis[l-(3-methylsulfanyl-1-propylcarbamoylpropyl)carbamic acid tert-

butyl ester]benzoic acid ethyl ester (18a): The Boc-l-methionine hydrox-

ysuccinimide ester 11a (1.59 g, 4.60 mmol) was dissolved in dry dichloro-

methane under a nitrogen atmosphere, and cooled down to ꢀ108C.

DIPEA (680 mL, 517 mg; 4.00 mmol) and the G1 hydrochloride 17

(675 mg, 2.00 mmol) dissolved in dry methanol were added dropwise.

The reaction mixture was allowed to warm up to room temperature

while being stirred for additional 12 h. After complete reaction (TLC)

the organic layer was washed twice with sodium hydrogencarbonate solu-

tion and once with brine. The organic phase was subsequently dried over

magnesium sulfate, and the solvent was removed in vacuo. The crude

product was purified by column chromatography (silica gel, dichlorome-

1185

Chem. Eur. J. 2004, 10, 1167 1192

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

R. Gust, A. D. Schl¸ter et al.

FULL PAPER

3,5-Bis[l-(2-phenyl-1-propylcarbamoylethyl)carbamic acid tert-butyl es-

ter]benzoic acid (19b): A 1m KOH solution (13.6 mL, 13.6 mmol) was

added to a solution of the phenylalanine G1 dendron 18b (1.29 g,

1.70 mmol) in methanol/water/THF (3:1:1), and the reaction mixture was

stirred at 508C for 12 h. After complete saponification (TLC), acetic acid

was added to give pH 5. The product was subsequently extracted with di-

chloromethane, and the organic phases were dried with magnesium sul-

fate. Evaporation of the solvent and subsequent chromatographic purifi-

cation (silica gel, dichloromethane containing 4% methanol) afforded

the G1 acid 19b (1.15 g, 1.57 mmol, 92.4%) as a colorless foam. R_f=0.25

(dichloromethane/methanol=19:1/v:v); m.p. 1528C; ¹H NMR (250 MHz,

[D₄]methanol): d=1.41 (s, 18H; CCH₃), 1.75 (quin, ³J(H,H)=7.3Hz,

magnesium sulfate. Evaporation of the solvent under reduced pressure

and subsequent purification by column chromatography (silica gel, di-

chloromethane containing 3% methanol as eluent) gave the Boc-protect-

ed phenylalanine-G1-dendrimer 20b (575 mg, 241 mmol, 48.2%) as a col-

orless foam. R_f=0.32 (dichloromethane/methanol=19:1/v:v); m.p.

1738C; ¹H NMR (500 MHz, CDCl₃/[D₄]methanol): d=1.37 (s, 54H;

CCH₃), 1.72 (quin, br, 12H; CH₂), 1.96 (quin, ³J(H,H)=7.3Hz, 6H;

CH₂), 2.51 (t, br, 12H; CH₂Ar), 2.66 (t, ³J(H,H)=7.5 Hz, 6H; CH₂Ar),

2.96 (m, 6H; CHCH₂), 3.03 (m, 6H; CHCH₂), 3.11 (m, 12H; CH₂N),

3.42 (m, 6H; CH₂N), 4.31 (m, br, 6H; CH), 6.92 (s, 3H; ArH), 7.06 (s,

3H; ArH), 7.19 (m, 18H; ArH_phe), 7.25 (m, 12H; ArH_phe), 7.38 ppm (s,

6H; ArH); ¹³C NMR (127 MHz, [D₄]methanol): d=28.50 (CCH₃), 30.78

(CH₂), 31.30 (CH₂), 32.86 (CH₂), 33.61 (CH₂), 38.94 (CH₂), 39.21 (CH₂),

40.15 (CH₂), 56.47 (CH), 80.44 (CCH₃), 125.39 (ArC), 126.68(ArC),

127.20 (ArC), 128.86 (ArC), 129.43(Ar C), 129.70 (ArC), 132.18 (ArC),

135.16 (ArC), 137.32 (ArC), 142.27 (ArC), 156.41 (CON_Boc), 169.40

(CON), 172.90 ppm (CON); MS (MALDI-TOF, dithranol): m/z: 2425

3

4H; CH₂), 2.59 (t, J(H,H)=7.7 Hz, 4H; CH₂Ar), 2.90 (m, 2H; CHCH₂),

3.06 (m, 2H; CHCH₂), 3.19 (m, 4H; CH₂N), 4.32 (t, ³J(H,H)=7.3Hz,

2H; CH), 7.25 (s, 1H; ArH), 7.28 (m, 10H; ArH_phe), 7.70 (s, 2H; ArH),

8.00 ppm (m, br, CONH); ¹³C NMR (63MHz, [D ₄]methanol): d=28.67

(CCH₃), 31.88 (CH₂), 33.65 (CH₂), 39.52 (CH₂), 39.84 (CH₂), 57.62 (CH),

80.66 (CCH₃), 127.75 (ArC), 128.37 (ArC), 129.44 (ArC), 130.39 (ArC),

132.11 (ArC), 134.46 (ArC), 138.55 (ArC), 143.46 (ArC), 157.46

(CON_Boc), 170.09 (CON), 174.17 ppm (COO); MS (FAB+, MNBA/

DCM): m/z (%): 753(6.9) [ M+Na]⁺, 731 (14.2) [M+H]⁺, 631 (19.6)

[MꢀC₅H₉O₂+H]⁺, 531 (67.8) [Mꢀ2(C₅H₉O₂)+H]⁺, 120 (100) [C₈H₁₀N]⁺,

57 (38.2) [C₄H₉]⁺; monoisotopic mass calcd for C₄₁H₅₄N₄NaO₈⁺: 753.4,

found: 753.5 [M+Na]⁺.

[M+K]⁺,

2409

[M+Na]⁺

;

monoisotopic

mass

calcd

for

C

₁₁₄H₁₈₃N₁₅NaO₂₁S₆⁺: 2409.36, found: 2409.36.

(Boc/tBu-Asp)₆6D1: 1,3,5-tris-[3,5-bis(l-3-tert-butoxycarbonylamino-N-

propylsuccinamic acid tert-butyl ester)-N-propylbenzamide]benzene

(20c): A solution of the l-aspartic acid hydroxysuccinimide ester 11c

(522 mg, 1.35 mmol) in dry dichloromethane was cooled down to ꢀ108C.

The G1 dendrimer 8 (238 mg, 150 mmol), dissolved in dry methanol

(3mL), and DIPEA (153 mL, 116 mg, 900 mmol) were added dropwise.

The solution was stirred at ꢀ108C for 1 h, and was afterwards allowed to

warm up to room temperature slowly while being stirred for an addition-

al 18 h. After complete reaction (TLC) the mixture was washed twice

with sodium hydrogencarbonate solution and once with brine. The organ-

ic phase was dried over magnesium sulfate, the solvent subsequently

evaporated in vacuo, and the crude product was purified by column chro-

matography (silica gel, dichloromethane containing 3% methanol as

eluent) to afford pure 20c (269 mg, 106 mmol; 70.7%) as a colorless

foam. R_f=0.15 (dichloromethane/methanol=19:1/v:v); m.p. 1078C;

¹H NMR (500 MHz, CDCl₃): d=1.39 (s, 54H; CCH₃), 1.41 (s, 54H;

CCH₃), 1.75 (quin, ³J(H,H)=7.0 Hz, 12H; CH₂), 1.95 (quin, ³J(H,H)=

7.2 Hz, 6H; CH₂), 2.55 (t, ³J(H,H)=7.2 Hz, 12H; CH₂Ar), 2.64 (t,

³J(H,H)=7.7 Hz, 6H; CH₂Ar), 2.44 (dd, ²J(H,H)=16.8 Hz, ³J(H,H)=

6.3Hz, 6H; CHC H₂), 2.79 (dd, ²J(H,H)=16.6 Hz, ³J(H,H)=5.2 Hz, 6H;

CHCH₂), 3.13 (m, 6H; CH₂N), 3.19 (m, 6H; CH₂N), 3.42 (m, 6H;

(Boc-Met)₆6D1:

1,3,5-tris-{3,5-bis[l-(3-methylsulfanyl-1-propylcarba-

moylpropyl)carbamic acid tert-butyl ester]-N-propylbenzamide}benzene

(20a): The G1 acid 19b (1.10 g, 1.58 mmol) was dissolved in dry dichloro-

methane under a nitrogen atmosphere. HOBt (246 mg, 1.61 mmol) was

added, and the mixture was stirred at room temperature for 15 min. Af-

terwards the reaction mixture was cooled down to ꢀ208C, EDC (323 mg,

1.68 mmol) was added, and the flask was allowed to warm up to room

temperature while being stirred for an additional 2 h. After the active

ester had formed (TLC), the reaction was cooled down to ꢀ308C,

DIPEA (726 mL, 552 mg, 4.30 mmol), and the trishydrochloride core 4

(123mg, 340 mmol), dissolved in dry methanol (5 mL), were added drop-

wise. The reaction mixture was stirred for additional 18 h, and during

that time was allowed to warm up to room temperature slowly. After

complete reaction (TLC), the mixture was washed twice with sodium hy-

drogencarbonate solution and once with brine, and the organic layer was

dried over magnesium sulfate. The solvent was evaporated under reduced

pressure and the crude product purified by column chromatography

(silica gel, dichloromethane containing 2% methanol as eluent) to afford

the desired product (421 mg, 184 mmol, 54.1%) as a colorless foam. R_f=

0.21 (dichloromethane/methanol=19:1/v:v); m.p. 1718C; ¹H NMR

(500 MHz, [D₄]methanol): d=1.39 (s, 54H; CCH₃), 1.80 (m, 12H; CH₂),

1.83(m, 6H; CHC H₂), 1.92 (m, 6H; CH₂), 1.97 (m, 6H; CHCH₂), 2.05

(s, 18H; SCH₃), 2.49 (m, 12H; SCH₂), 2.63(m, 6 H + 12H; CH₂Ar +

CH₂S), 3.17 (m, 12H; CH₂N), 3.38 (m, 6H; CH₂N), 4.18 (m, br, 6H;

CH), 6.95 (s, 3H; ArH), 7.21 (s, 3H; ArH), 7.46 ppm (s, 6H; ArH);

¹³C NMR (63MHz, [D ₄]methanol): d=15.51 (SCH₃), 28.71 (CCH₃), 30.94

(CH₂), 31.34 (CH₂), 31.65 (CH₂), 32.84 (CH₂), 33.32 (CH₂), 33.95 (CH₂),

39.37 (CH₂), 40.48 (CH₂), 54.79 (CH), 80.66 (CCH₃), 125.75 (ArC),

126.96 (ArC), 132.53 (ArC), 135.56 (ArC), 142.67 (ArC), 142.74 (ArC),

157.09 (CON_Boc), 169.86 (CON), 173.90 ppm (CON); MS (MALDI-TOF,

dithranol): m/z: 23 3 0M[ +K]⁺, 2313 [M+Na]⁺; monoisotopic mass calcd

for C₁₁₄H₁₈₃N₁₅NaO₂₁S₆⁺: 2313.19, found: 2313.55.

3

CH₂N), 4.42 (m, br, 6H; CH), 5.80 (d, br, J(H,H)=7.7 Hz, 6H; CHNH),

6.75 (t, br, ³J(H,H)=5.6 Hz, 6H; CONH), 6.90 (s, 3H; ArH), 7.05 (s,

3H; ArH), 7.17 (t, br, 3H; CONH), 7.37 ppm (s, 6H; Ar H); ¹³C NMR

(127 MHz, CDCl₃): d=28.02 (CCH₃), 28.33 (CCH₃), 30.64 (CH₂), 30.78

(CH₂), 32.28 (CH₂), 33.07 (CH₂), 37.54 (CH₂), 38.43 (CH₂), 39.61 (CH₂),

51.01 (CH), 80.23( CCH₃), 81.55 (CCH₃), 125.07 (ArC), 126.34 (ArC),

131.56 (ArC), 134.86 (ArC), 141.51 (ArC), 141.69 (ArC), 155.59

(CON_Boc), 167.95 (CON), 170.97 (CO_tBu), 171.06 ppm (CON); MS

(MALDI-TOF, dithranol): m/z: 2570 [M+K]⁺, 2554 [M+Na]⁺; monoiso-

topic mass calcd for C₁₃₂H₂₀₇N₁₅NaO₃₃⁺: 2553.49, found: 2553.59 .

(Met)₆6D1: 1,3,5-tris-[3,5-bis(l-2-amino-4-methylsulfanyl-N-propylbutyr-

amide)-N-propylbenzamide]benzene hexatrifluoroacetate (21a): The

Boc-protected G1 dendrimer 20a (150 mg, 65.4 mmol) was dissolved in a

’cleavage cocktail’ mixture (10 mL) containing dichloromethane/trifluoro-

acetic

acid/ethanedithiol/thioanisole/methanol/triisopropylsilane

(50:37.5:5:5:2:0.5/v:v) under a nitrogen atmosphere, and stirred for 1 h at

room temperature. After complete deprotection (TLC), the ’cleavage

cocktail’ mixture was removed by repeated evaporation under reduced

pressure by using methanol as solvent. The residue was dried under high

vacuum, and lyophilization from water afforded the desired product

(150 mg, 63.1 mmol, 96.5%) as a colorless solid. M.p. 91 928C; ¹H NMR

(500 MHz, [D₄]methanol): d=1.91 (quin, ³J(H,H)=7.4 Hz, 12H; CH₂),

1.97 (quin, ³J(H,H)=7.7 Hz, 6H; CH₂), 2.14 (s, 18H; SCH₃), 2.13(m,

6H; CHCH₂), 2.17 (m, 6H; CHCH₂), 2.61 (m, 12H; CH₂S), 2.69 (m, 6H;

CH₂Ar), 2.72 (m, 12H; CH₂Ar), 3.28 (m, 6H; CH₂N), 3.33 (m, 6H;

CH₂N), 3.43 (t, ³J(H,H)=7.1 Hz, 6H; CH₂N), 4.00 (t, ³J(H,H)=6.6 Hz,

6H; CH), 6.97 (s, 3H; ArH), 7.28 (s, 3H; ArH), 7.51 ppm (s, 6H; ArH);

¹³C NMR (127 MHz, [D₄]methanol): d=15.47 (SCH₃), 30.25 (SCH₂),

32.16 (CH₂), 32.41 (CH₂), 32.46 (CHCH₂), 34.22 (CH₂Ar), 34.61

(CH₂Ar), 40.55 (CH₂N), 41.12 (CH₂N), 54.08 (CH), 126.37 (ArC), 127.61

(ArC), 133.13 (ArC), 136.49 (ArC), 143.51 (ArC), 143.75 (ArC), 170.04

(Boc-Phe)₆6D1: 1,3,5-tris-{3,5-bis[l-(2-phenyl-1-propylcarbamoylethyl)-

carbamic acid tert-butyl ester]-N-propylbenzamide}benzene (20b): HOBt

(297 mg, 2.20 mmol) was added to

a solution of the G1 acid 19b

(1.53mg, 2.10 mmol) in a mixture of dry dichloromethane/DMF (19:1/

v:v), and the reaction mixture was stirred for 15 min at room tempera-

ture. The flask was cooled down to 08C, EDC (441 mg, 2.30 mmol) was

added, and the solution was stirred for additional 2 h while warming up

to room temperature. When the formation of the active ester was com-

plete (TLC), the reaction mixture was cooled down to 58C again, and

DIPEA (1.03mL, 763mg, 5.90 mmol) and the trishydrochloride core

4

(179 mg, 500 mmol), dissolved in dry methanol (5 mL), were added

slowly. The reaction mixture was allowed to warm up to room tempera-

ture slowly while being stirred for additional 18 h. After complete reac-

tion (TLC) the solution was washed twice with sodium hydrogencarbon-

ate solution and once with brine, and the organic layer was dried over

1186

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 1167 1192

Surface-Modified Dendrimers

1167 1192

(CON), 170.77 ppm (CON); MS (MALDI-TOF, CCA): m/z: monoisotop-

ic mass calcd for C₈₄H₁₃₆N₁₅O₉S₆⁺: 1690.90, found: 1690.91 [M+H]⁺.

(CH₂CH₃), 28.44 (CCH₃), 31.88 (CH₂), 33.16 (CH₂), 39.15 (CH₂), 42.98

(CH₂), 56.37 (CH), 61.30 (CH₂CH₃), 78.74 (CCH₃), 78.99 (CCH₃), 127.39

(ArC), 131.19 (ArC), 134.25 (ArC), 143.47 (ArC), 156.28 (CON_Boc),

157.08 (CON_Boc), 166.89 (COO), 171.04 ppm (CON); MS (FAB+,

MNBA/DCM): m/z (%): 859 (1.8) [M+Na]⁺, 837 (1.1) [M+H]⁺, 73 7

(6.1) [MꢀC₅H₉O₂+H]⁺, 637 (2.0) [Mꢀ2^.(C₅H₉O₂)+H]⁺, 537 (3.9)

[Mꢀ3(C₅H₉O₂)+H]⁺, 438 (15.2) [Mꢀ4(C₅H₉O₂)]⁺, 57 (100) [C₄H₉]⁺; el-

emental analysis calcd (%) for C₄₁H₆₈N₆O₁₂(837.0): C 58.83, H 8.19, N

10.04; found: C 58.62, H 7.96, N 10.05.

(Phe)₆6D1:

1,3,5-tris-[3,5-bis(l-2-amino-3-phenyl-N-propylpropiona-

mide)-N-propylbenzamide]benzene hexatrifluoroacetate (21b): Trifluoro-

acetic acid (1.5 mL) was added to a solution of the Boc-protected G1

dendrimer 20b (24 mg, 10 mmol) in dichloromethane (4 mL), and the re-

action mixture was stirred for 1 h at room temperature. When cleavage

of the Boc protecting group was complete (TLC), the solvent was re-

moved in vacuo to yield the deprotected G1l-phenylalanine dendrimer

(25 mg, 9.9 mmol, 99%) after lyophilization from water. M.p. 126 1278C;

¹H NMR (500 MHz, [D₄]methanol): d=1.74 (quin, ³J(H,H)=7.4 Hz,

12H; CH₂), 1.96 (quin, ³J(H,H)=7.3Hz, 6H; CH ₂), 2.56 (t, ³J(H,H)=

7.6 Hz, 12H; CH₂Ar), 2.69 (t, ³J(H,H)=7.5 Hz, 6H; CH₂Ar), 3.13 (m,

12H; CHCH₂), 3.16 (m, 6H; CH₂N), 3.27 (m, 6H; CH₂N), 3.43 (t,

3,5-Bis-[(2-tert-butoxycarbonylamino-2-propylcarbamoylethyl)carbamic

acid tert-butyl ester]benzoic acid (23): KOH (2.08 g, 37.0 mmol) was

added to a solution of the G1 ester 22 (3.87 g, 4.60 mmol) in methanol/

THF/water (3:1:1), and the reaction mixture was stirred for 12 h at 50 8C.

After complete reaction (TLC) acetic acid was added to give pH 5, and

the desired product was subsequently extracted with dichloromethane.

The combined organic layers were dried over magnesium sulfate, and the

solvent was evaporated in vacuo. Chromatographic separation (silica gel,

dichloromethane containing 5% methanol as eluent) gave the G1 acid

(3.22 g, 3.98 mmol, 86.5%) as a colorless solid. R_f=0.38 (dichlorome-

thane/methanol=9:1/v:v); m.p. 1188C; ¹H NMR (500 MHz, CDCl₃): d=

1.37 (s, 18H; CCH₃), 1.40 (s, 18H; CCH₃), 1.80 (quar, br, 4H; CH₂), 2.60

(t, br, 4H; CH₂Ar), 3.16 (m, 4H; CH₂N), 3.42 (m, 2H; CHCH₂), 3.47 (m,

2H; CHCH₂), 4.32 (m, 2H; CH), 5.61 (s, br, 2H; NH), 6.13 (d, br, 2H;

NH), 7.19 (s, 1H; ArH), 7.30 (s, br, 2H; NH), 7.65 ppm (s, 2H; ArH);

¹³C NMR (127 MHz, CDCl₃): d=28.30 (CCH₃), 30.33 (CH₂), 32.09

(CH₂), 38.14 (CH₂), 42.48 (CH₂), 55.41 (CH), 79.75 (CCH₃), 80.14

(CCH₃), 127.66 (ArC), 130.14 (ArC), 134.25 (ArC), 141.51 (ArC), 156.14

(CON_Boc), 157.07 (CON_Boc), 169.53(COO), 170.96 ppm (COO); MS

(FAB+, MNBA/DCM/methanol): m/z (%): 831 (1.3) [M+Na]⁺, 809

3

³J(H,H)=7.1 Hz, 6H; CH₂N), 4.08 (t, J(H,H)=7.5 Hz, 6H; CH), 6.97 (s,

3H; ArH), 7.16 (s, 3H; ArH), 7.29 (m, 18H; ArH_phe), 7.35 (m, 12H;

ArH_phe), 7.45 ppm (s, 6H; ArH); ¹³C NMR (127 MHz, [D₄]methanol):

d=31.95 (CH₂), 32.49 (CH₂), 34.08 (CH₂Ar), 34.63 (CH₂Ar), 39.05

(CHCH₂), 40.44 (CH₂N), 41.13(CH ₂N), 56.18 (CH), 126.32 (ArC),

127.61 (ArC), 129.14 (ArC_phe), 130.37 (ArC_phe), 130.80 (ArC_phe), 133.08

(ArC), 136.01 (ArC_phe), 136.42 (ArC), 143.54 (ArC), 143.68 (ArC), 169.75

(CON), 170.80 ppm (CON); MS (MALDI-TOF, CCA): m/z: 1825

[M+K]⁺, 1809 [M+Na]⁺, 1787 [M+H]⁺; monoisotopic mass calcd for

C

₁₀₈H₁₃₆N₁₅O₉⁺: 1787.06, found: 1787.21.

(Asp)₆6D1: 1,3,5-tris[3,5-bis(l-3-amino-N-propylsuccinamic acid)-N-pro-

pylbenzamide]benzene hexatrifluoroacetate (21c): Trifluoroacetic acid

(2 mL) was added to a solution of dendrimer 20c (101 mg, 40.0 mmol) in

dichloromethane (2 mL), and the solution was stirred at room tempera-

ture for 1 h. When the deprotection was complete (TLC), the solvent was

repeatedly removed in vacuo. Lyophilization from water afforded the de-

protected l-aspartic acid dendrimer (91 mg, 40 mmol, 100%) as a color-

less solid. M.p. 155 1568C; ¹H NMR (500 MHz, [D₄]methanol): d=1.89

(quin, ³J(H,H)=7.2 Hz, 12H; CH₂), 1.97 (quin, ³J(H,H)=7.3Hz, 6H;

CH₂), 2.71 (m, 18H; CH₂Ar), 2.93(dd, ²J(H,H)=17.8 Hz, ³J(H,H)=

7.9 Hz, 6H; CHCH₂), 3.00 (dd, ²J(H,H)=17.7 Hz, ³J(H,H)=5.0 Hz, 6H;

CHCH₂), 3.26 (m, 6H; CH₂N), 3.32 (m, 6H; CH₂N), 3.38 (t, ³J(H,H)=

7.1 Hz, 6H; CH₂N), 4.17 (m, 6H; CH), 6.92 (s, 3H; ArH), 7.23(s, 3H;

ArH), 7.45 ppm (s, 6H; ArH); ¹³C NMR (127 MHz, [D₄]methanol): d=

32.08 (CH₂), 32.38 (CH₂), 34.07 (CH₂Ar), 34.65 (CH₂Ar), 36.52

(CHCH₂), 40.54 (CH₂N), 41.22 (CH₂N), 51.51 (CH), 126.37 (ArC),

127.62 (ArC), 133.26 (ArC), 136.41 (ArC), 143.52 (ArC), 143.77 (ArC),

169.41 (CON), 170.83(CON), 17.302 ppm (COO); MS (MALDI-TOF,

CCA): m/z: 1594.9; monoisotopic mass calcd for C₇₈H₁₁₂N₁₅O₂₁⁺: 1594.82,

found: 1594.85 [M+H]⁺.

(0.1)

[M+H]⁺,

709

(1.5)

[MꢀC₅H₉O₂+H]⁺,

609

(0.8)

[Mꢀ2(C₅H₉O₂)+H]⁺, 509 (1.7) [Mꢀ3(C₅H₉O₂)+H]⁺, 409 (20.8)

[Mꢀ4(C₅H₉O₂)+H]⁺, 57 (100) [C₄H₉]⁺; elemental analysis calcd (%) for

C₃₉H₆₄N₆O₁₂(837.0): C 57.90, H 7.97, N 10.39; found: C 57.64, H 7.96, N

10.28.

(Boc-Dpa)₆6D1: 1,3,5-tris{3,5-bis-[(2-tert-butoxycarbonylamino-2-propyl-

carbamoylethyl)carbamic acid tert-butyl ester]-N-propylbenzamide}ben-

zene (24): The G1 acid 23 (1.46 g, 1.80 mmol) were dissolved in a solvent

mixture of dry dichloromethane/DMF (14:1/v:v), and cooled down to

ꢀ108C. TBTU (607 mg, 1.90 mmol), dissolved in a small amount of

DMF, and DIPEA (306 mL, 233 mg, 1.80 mmol) were added slowly under

vigorous stirring. The reaction mixture was allowed to warm up to room

temperature, and stirred at room temperature for additional 30 min.

After complete formation of the active ester (TLC), the reaction mixture

was cooled down to 08C, and the trishydrochloride core 4 (144 mg,

400 mmol), dissolved in a small amount of dry methanol, and DIPEA

(510 mL, 388 mg, 3.00 mmol) were added dropwise. The mixture was al-

lowed to warm up to room temperature slowly, and was then stirred for

additional 18 h. After the reaction was finished (TLC), the solution was

washed once with sodium hydrogencarbonate solution, and once with

brine. The organic phase was dried with magnesium sulfate. After evapo-

ration of the solvent under reduced pressure, the crude product was puri-

fied by column chromatography (silica gel, dichloromethane containing

5% methanol as eluent) to yield the G1 dendrimer (601 mg, 229 mmol;

57.3%) as a colorless solid. R_f=0.23(dichloromethane/methanol =9:1/

v:v); m.p. 1418C; ¹H NMR (500 MHz, [D₄]methanol): d=1.44 (s, 54H;

CCH₃), 1.47 (s, 54H; CCH₃), 1.86 (quin, ³J(H,H)=7.1 Hz, 12H; CH₂),

1.97 (quin, ³J(H,H)=7.2 Hz, 6H; CH₂), 2.68 (m, 18H; CH₂Ar), 3.22 (m,

12H; CH₂N), 3.32 (m, 6H; CHCH₂), 3.41 (m, 6H; CHCH₂), 3.43 (m,

6H; CH₂N), 4.20 (m, br, 6H; CH), 6.96 (s, 3H; ArH), 7.26 (s, 3H; ArH),

7.48 ppm (s, 3H; ArH); ¹³C NMR (127 MHz, [D₄]methanol): d=29.08

(CCH₃), 32.23 (CH₂), 32.43 (CH₂), 33.98 (CH₂Ar), 34.61 (CH₂Ar), 40.04

(CH₂N), 41.07 (CH₂N), 43.52 (CHCH₂), 57.21 (CH), 80.77 (CCH₃), 81.10

(CCH₃), 126.44 (ArC), 127.64 (ArC), 133.36 (ArC), 136.31 (ArC), 143.49

(ArC), 143.73 (ArC), 157.98 (CON_Boc), 158.96 (CON_Boc), 170.71 (CON),

173.25 ppm (CON); MS (MALDI-TOF, dithranol): m/z: 2660 [M+K]⁺,

2644 [M+Na]⁺; monoisotopic mass calcd for C₁₃₂H₂₁₃N₂₁NaO₃₃⁺: 2643.55,

found: 2643.77 [M+Na]⁺.

Ethyl-3,5-bis-[(2-tert-butoxycarbonylamino-2-propylcarbamoylethyl)car-

bamic acid tert-butyl ester]benzoate (22): A solution of the Boc-protect-

ed diaminopropionic acid 14 (3.96 g, 13.0 mmol) in a solvent mixture of

dry dichloromethane/DMF (29:1/v:v) was cooled down to ꢀ208C. Under

vigorous stirring TBTU (4.34 g, 13.5 mmol), dissolved in DMF, and

DIPEA (2.21 mL, 1.68 g, 13.0 mmol) were added slowly. The reaction

mixture was stirred at ꢀ208C for additional 30 min, and was then allowed

to warm up to room temperature. After complete formation of the active

ester (TLC), the reaction mixture was cooled down to ꢀ208C again. The

G1 hydrochloride 17 (1.69 g, 5.00 mmol) was dissolved in a small amount

of dry methanol, and, together with DIPEA (3.91 mL, 2.97 g, 23.0 mmol),

was added to the reaction mixture slowly. The solution was stirred for an

additional 30 min at ꢀ208C, and was then allowed to warm up to room

temperature slowly. After being stirred for additional 12 h the solution

was washed twice with sodium hydrogencarbonate solution, and once

with brine. The organic phase was dried over magnesium sulfate, the sol-

vent removed in vacuo, and the crude product was purified by column

chromatography (silica gel, dichloromethane containing 3% methanol as

eluent) to give the desired product (3.79 g, 4.52 mmol, 90.4%) as a color-

less solid. R_f=0.31 (dichloromethane/methanol=19:1/v:v); m.p. 1478C;

¹H NMR (250 MHz, [D₇]DMF): d=1.40 (t, ³J(H,H)=7.2 Hz, 3H;

CH₂CH₃), 1.43(s, 18H; CCH₃), 1.45 (s, 18H; CCH₃), 1.86 (quin,

³J(H,H)=7.3Hz, 4H; CH ₂), 2.75 (t, ³J(H,H)=8.2 Hz, 4H; CH₂Ar), 3.27

(m, 4H; CH₂N), 3.46 (m, 4H; CHCH₂), 4.25 (m, 2H; CH), 4.39 (q,

³J(H,H)=7.3Hz, 2H; C H₂CH₃), 6.73(d, br, ³J(H,H)=7.3Hz, 2H;

CHNH), 6.86 (t, br, 2H; NH_Boc), 7.45 (s, 1H; ArH), 7.74 (s, 2H; ArH),

8.14 ppm (t, br, 2H; CONH); ¹³C NMR (127 MHz, [D₇]DMF): d=14.52

(Dpa)₆6D1:

1,3,5-tris[3,5-bis-(2,3-diamino-N-propyl-propionamide)-N-

propylbenzamide]benzene dodecatrifluoroacetate (25): Trifluoroacetic

acid (5 mL) was added to a vigorously stirred solution of dendrimer 24

(105 mg, 40.0 mmol) in dichloromethane (5 mL). The reaction mixture

1187

Chem. Eur. J. 2004, 10, 1167 1192

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

R. Gust, A. D. Schl¸ter et al.

FULL PAPER

was stirred for 1 h at room temperature. When deprotection was com-

plete (TLC), the solvent was removed in vacuo and the desired product

could be lyophilized from water. The procedure yielded the deprotected

G1 dendrimer (110 mg, 39.4 mmol, 98.5%) as a colorless solid. M.p. 119

1218C; ¹H NMR (500 MHz, [D₄]methanol): d=1.92 (m, 18H; CH₂), 2.68

125.00 (ArC), 126.15 (ArC), 128.26 (ArC_dns), 129.33 (ArC_dns), 129.38

(ArC_dns), 129.57 (ArC_dns), 129.99 (ArC_dns), 131.42 (ArC), 134.64 (ArC),

135.07 (ArC_dns), 141.45 (ArC), 141.93(Ar C), 151.51 (ArC_dns), 167.81 ppm

(CON); MS (MALDI-TOF, dithranol): m/z: 2341 [M+K]⁺, 2325

+

[M+Na]⁺, 2303 [M+H]⁺; monoisotopic mass calcd for C₁₂₆H₁₄₈N₁₅O₁₅S₆

(t, ³J(H,H)=7.4 Hz, 6H; CH₂Ar), 2.73(t,

³J(H,H)=7.6 Hz, 12H;

: 2302.96, found: 2303.08.

2

CH₂Ar), 3.25 (m, 6H; CH₂N), 3.43 (m, 12H; CH₂N), 3.45 (dd, J(H,H)=

13.9 Hz, ³J(H,H)=6.3Hz, 6H; CHCH ₂), 3.52 (dd, ²J(H,H)=13.9 Hz,

³J(H,H)=5.4 Hz, 6H; CHCH₂), 4.29 (t, ³J(H,H)=5.8 Hz, 6H; CH), 6.96

(s, 3H; ArH), 7.29 (s, 3H; ArH), 7.51 ppm (s, 3H; ArH); ¹³C NMR

(127 MHz, [D₄]methanol): d=31.86 (s, CH₂), 32.37 (s, CH₂), 34.15 (s,

CH₂Ar), 34.61 (s, CH₂Ar), 40.85 (s, CH₂N), 41.13(s, CH ₂N), 41.47 (s,

CH₂CH), 52.55 (s, CH), 118.36 (q, ¹J(C,F)=292.5 Hz, CF₃CO₂H), 126.36

(s, ArC), 127.61 (s, ArC), 133.21 (s, ArC), 136.43 (s, ArC), 143.51 (s,

ArC), 143.73 (s, ArC), 163.49 (q, ²J(C,F)=35.4 Hz, CF₃CO₂H), 167.48 (s,

CON), 170.84 ppm (s, CON); MS (MALDI-TOF, CCA): m/z: 1459

[M+K]⁺, 1443[ M+Na]⁺, 1421 [M+H]⁺; monoisotopic mass calcd for

C₇₂H₁₁₈N₂₁O₉⁺: 1420.94, found: 1420.93.

(Dns)₁₂12D2: 1,3,5-tris-(3,5-bis{3,5-bis[3-(5-dimethylaminonaphthalene-

1-sulfonylamino)propyl]-N-propylbenzamide}-N-propylbenzamide)ben-

zene (28): A solution of dry triethylamine (166 mL, 121 mg, 1.20 mmol)

and the deprotected G2 dendrimer 10 (72 mg, 20 mmol) in dry methanol

were added dropwise to a vigorously stirred solution of dansyl chloride

(194 mg, 720 mmol) in dry dichloromethane at room temperature. While

the solution was stirred in the dark for an additional 12 h the reaction

was continuously monitored by TLC. After complete reaction the solu-

tion was washed once with brine, once with a saturated sodium carbonate

solution, and once again with brine. The organic phase was dried over

magnesium sulfate, the solvent removed in vacuo, and the crude product

purified by column chromatography (silica gel, dichloromethane contain-

ing 2 4% methanol as eluent). The procedure afforded the desired G2

dendrimer (65 mg, 13 mmol; 65%) as a bright yellow-greenish oil which

could be lyophilized from dioxane. R_f=0.44 (dichloromethane/metha-

nol=19:1/v:v); m.p. 132 1348C; ¹H NMR (500 MHz, [D₄]methanol,

308C): d=1.63(quin, ³J(H,H)=7.0 Hz, 24H; CH₂), 1.80 (m, 6H; CH₂),

1.86 (m, 12H; CH₂), 2.45 (m, 24 H + 6H; CH₂Ar), 2.46 (t, ³J(H,H)=

6.9 Hz, 12H; CH₂Ar), 2.59 (m, 24H; CH₂N), 2.92 (s, 72H; NCH₃), 3.31

(m, 6H; + 12H; CH₂N), 6.75 (s, 3H; ArH), 6.78 (s, 6H; ArH), 7.12 (s,

3H; ArH), 7.26 (s, 6H; ArH), 7.29 (s, 12H; ArH), 7.38 (d, ³J(H,H)=

5.5 Hz, 12H; ArH_dns), 7.49 (m, 24H; ArH_dns), 8.13(d, ³J(H,H)=7.3Hz,

12H; ArH_dns), 8.41 (d, ³J(H,H)=8.0 Hz, 12H; ArH_dns), 8.55 ppm (d,

³J(H,H)=8.7 Hz, 12H; ArH_dns); ¹³C NMR (127 MHz, [D₄]methanol,

308C): d=30.04 (CH₂), 30.61 (CH₂), 31.18 (CH₂), 32.51 (CH₂Ar), 33.34

(CH₂Ar), 33.62 (CH₂Ar), 39.87 (CH₂N), 40.15 (CH₂N), 42.50 (CH₂N),

46.02 (NCH₃), 116.46 (ArC_dns), 123.50 (ArC_dns), 124.31 (ArC_dns), 125.27

(ArC), 125.38 (ArC), 128.49 (ArC_dns), 129.55 (ArC_dns), 129.70 (ArC_dns),

129.98 (ArC_dns), 130.03 (ArC_dns), 132.08 (ArC), 132.19 (ArC), 134.85

(ArC), 134.91 (ArC), 135.88 (ArC_dns), 140.23(Ar C), 142.06 (ArC), 142.26

(ArC), 142.56 (ArC), 152.53(Ar C_dns), 169.06 (CON), 169.09 ppm (CON);

(Dns)₃3D0: 1,3,5-tris-(5-dimethylaminonaphthalene-1-sulfonic acid pro-

pylamide)benzene (26): Dry triethylamine (2.00 mL , 1.46 g, 14.4 mmol)

and the trishydrochloride core 4 (431 mg, 1.20 mmol), dissolved in a

small amount of dry methanol, were added dropwise at room tempera-

ture to a vigorously stirred solution of 5-dimethylamino-naphthalene-1-

sulfonyl chloride (dansyl chloride) (1.46 g, 5.40 mmol) in dry dichlorome-

thane. The reaction was stirred for 1 h in the dark and was continuously

checked by TLC. After complete reaction the organic layer was washed

once with brine, once with a saturated sodium carbonate solution, and

once again with brine. Column chromatography (silica gel, dichlorome-

thane containing 1% methanol as eluent) gave the dansylated G0 den-

drimer (1.04 g, 1.10 mmol, 91.7%) as a bright yellow-greenish solid. R_f=

0.46 (dichloromethane/methanol=49:1/v:v); m.p. 968C; ¹H NMR

(500 MHz, CDCl₃, 3 08C): d=1.57 (quin, ³J(H,H)=7.1 Hz, 6H; CH₂),

2.31 (t, ³J(H,H)=7.5 Hz, 6H; CH₂Ar), 2.84 (q, ³J(H,H)=6.6 Hz, 6H;

CH₂N), 2.85 (s, 18H; NCH₃), 4.97 (t, br, ³J(H,NH)=5.4 Hz, 3H; NH),

6.40 (s, 3H; ArH), 7.14 (d, ³J(H,H)=7.6 Hz, 3H; ArH_dns), 7.45 (t,

³J(H,H)=7.9 Hz, 3H; ArH_dns), 7.50 (t, ³J(H,H)=8.1 Hz, 3H; ArH_dns),

8.19 (d, ³J(H,H)=7.6 Hz, 3H; ArH_dns), 8.30 (d, ³J(H,H)=8.7 Hz, 3H;

ArH_dns), 8.51 ppm (d, ³J(H,H)=8.4 Hz, 3H; ArH_dns); ¹³C NMR

(127 MHz, CDCl3): d=31.03 (CH₂), 32.34 (CH₂Ar), 42.64 (CH₂N), 45.44

(NCH₃), 115.28 (ArC_dns), 119.01 (ArC_dns), 123.30 (ArC_dns), 126.11 (ArC),

128.35 (ArC_dns), 129.57 (ArC_dns), 129.64 (ArC_dns), 129.77 (ArC_dns), 130.28

(ArC_dns), 134.93 (ArC_dns), 141.14 (ArC), 151.70 ppm (ArC_dns); MS (EI,

80 eV, 3208C); m/z (%): 948 (2.0) [M]⁺, 714 (1.4) [MꢀC₁₂H₁₂NO₂S]⁺,

171 (100) [C₁₂H₁₂N]⁺, 64 (38.3) [SO₂]⁺; HRMS: m/z: monoisotopic mass

calcd for C₅₁H₆₀N₆O₆S₃⁺: 948.37365, found: 948.37652.

MS (MALDI-TOF, dithranol): m/z: 5048 [M+K]⁺, 5032 [M+Na]⁺, 5010

+

[M+H]⁺

;

monoisotopic mass calcd for C₂₇₆H₃₂₂N₃₃O₃₃S₁₂

: 5010.12,

found: 5010.21 [M+H]⁺.

Ethyl-3-[3-(3-Benzyloxycarbonylaminopropyl)-5-(3-tert-butoxycarbonyla-

mino)propyl]benzoate (29): Dendron 17 (12.0 g, 35.6 mmol) was suspend-

ed in 1000 mL THF. An aqueous 1m KOH solution (300 mL, 300 mmol)

was added, and the mixture was cooled down to 08C. A solution of di-

tert-butyl dicarbonate (31.0 g, 142 mmol) and benzyl chloroformate

(6.07 g, 35.6 mmol) in THF was added slowly, and the reaction mixture

was stirred for 1 h. After complete reaction (TLC) the layers were sepa-

rated, the organic phase was washed with brine once, and the aqueous

layer was extracted with diethyl ether. The combined organic phases

were dried over magnesium sulfate, and the solvent was removed in

vacuo. Chromatographic separation (silica gel, dichloromethane contain-

ing 1% methanol as eluent) yielded the mixed-protected dendron 29

(6.13g, 12.3mmol, 34.6%) as a colorless oil. R_f=0.71 (dichloromethane/

methanol=19:1/v:v).

(Dns)₆6D1: 1,3,5-Tris{3,5-bis-[3-(5-dimethylaminonaphthalene-1-sulfony-

lamino)propyl]-N-propylbenzamide}benzene (27): A solution of dry trie-

thylamine (1.26 mL, 923mg, 9.12 mmol) and the G1 dendrimer

8

(477 mg, 300 mmol) in dry methanol were added slowly to a vigorously

stirred solution of dansyl chloride (1.17 g, 4.32 mmol) in dry dichlorome-

thane at room temperature. The reaction mixture was continuously moni-

tored by TLC and stirred in the dark for 6 h. After complete reaction the

solution was washed once with brine, once with a saturated sodium car-

bonate solution, and once again with brine. The organic phase was dried

over magnesium sulfate, the solvent removed in vacuo, and the crude

product purified by column chromatography (silica gel, dichloromethane

containing 2 3% methanol as eluent). The procedure yielded the dansy-

lated G1 dendrimer as a bright yellow-greenish oil (512 mg, 222 mmol;

74.0%) that could be lyophilized from dioxane. R_f=0.50 (dichlorome-

3-(3-Benzyloxycarbonylaminopropyl)-5-[3-(tert-butoxycarbonylamino)-

propyl]benzoic acid (30): A solution of the mixed-protected dendron 29

(6.00 g, 12.0 mmol) and KOH (3.08 g, 77.0 mmol) in methanol/water (3:1/

v:v) was stirred at 508C for 14 h. When the reaction was finished (TLC),

acetic acid was added to give pH 5. The product was extracted with di-

chloromethane, and the combined organic layers dried over magnesium

sulfate. After evaporation of the solvent under reduced pressure the

mixed-protected acid (5.50 g, 11.7 mmol, 97.5%) was received as a color-

less solid. R_f=0.35 (dichloromethane/methanol=19:1/v:v); m.p. 1288C.

1

thane/methanol=19:1/v:v); m.p. 114 1178C; H NMR (500 MHz, CDCl₃,

508C): d=1.61 (quin, ³J(H,H)=6.8 Hz, 12H; CH₂), 1.83(quin,

3

³J(H,H)=6.8 Hz, 6H; CH₂), 2.42 (t, J(H,H)=7.2 Hz, 12H; CH₂Ar), 2.53

(t, ³J(H,H)=6.9 Hz, 6H; CH₂Ar), 2.80 (q, ³J(H,H)=6.3Hz, 12H;

CH₂N), 2.84 (s, 36H; NCH₃), 3.32 (q, ³J(H,H)=5.9 Hz, 12H; CH₂N),

5.52 (s, br, 6H; SO₂NH), 6.75 (s, 3H; ArH), 6.77 (t, br, ³J(H,NH)=

5.4 Hz, 3H; CONH), 6.82 (s, 3H; Ar H), 7.12 (d, ³J(H,H)=7.3Hz, 6H;

ArH_dns), 7.26 (s, 6H; ArH), 7.42 (m, 12H; ArH_dns), 8.14 (d, ³J(H,H)=

(Cbz-N)₃(Boc-N)₃6D1: 1,3,5-tris-{3-[3-(benzyloxycarbonylamino)prop-

yl]-5-[3-(tert-butyloxycarbonylamino)propyl]-N-propylbenzamide}ben-

zene (31): HOBt (1.63g, 12.1 mmol) was added to a solution of the

mixed-protected G1 acid 30 (5.41 g, 11.5 mmol) in dry dichloromethane

under a nitrogen atmosphere, and the suspension was stirred at room

temperature for 20 min. Thereafter, the mixture was cooled down to

ꢀ208C, EDC (2.43g, 12.7 mmol) was added, and the reaction was al-

lowed to warm up to room temperature slowly while being stirred for ad-

3

7.2 Hz, 6H; ArH_dns), 8.33 (d, J(H,H)=8.7 Hz, 6H; ArH_dns), 8.50 ppm (d,

³J(H,H)=8.5 Hz, 6H; ArH_dns); ¹³C NMR (127 MHz, CDCl₃): d=30.56

(CH₂), 30.77 (CH₂), 32.09 (CH₂Ar), 33.26 (CH₂Ar), 39.78 (CH₂N), 42.34

(CH₂N), 45.51 (NCH₃), 115.50 (ArC_dns), 119.62 (ArC_dns), 123.47 (ArC_dns),

1188

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 1167 1192

Surface-Modified Dendrimers

1167 1192

ditional 2 h. After complete formation of the active ester (TLC), the re-

action mixture was cooled down to ꢀ208C again. A solution of the trish-

ydrochloride core 4 (918 mg, 2.56 mmol) and DIPEA (7.6 mL, 5.6 g,

43mmol) in dry methanol was added dropwise while the solution was

stirred at ꢀ208C for 1 h. The reaction was allowed to warm up to room

temperature slowly and was stirred for additional 24 h. After complete

reaction (TLC) the solution was washed twice with sodium hydrogencar-

bonate solution, and once with brine. The organic phase was dried over

magnesium sulfate, and the solvent removed in vacuo. Column chroma-

tography (silica gel, dichloromethane containing 3% methanol as eluent)

afforded the mixed-protected G1 dendrimer (2,61 g, 1.62 mmol, 63.3%)

as a colorless solid. R_f=0.43(dichloromethane/methanol =19:1/v:v); m.p.

668C; ¹H NMR (500 MHz, CDCl₃): d=1.40 (s, 27H; CCH₃), 1.69 (m,

6H; CH₂), 1.74 (m, 6H; CH₂), 1.89 (quin, ³J(H,H)=6.8 Hz, 6H; CH₂),

2.54 (m, 12H; CH₂Ar), 2.58 (t, ³J(H,H)=7.2 Hz, 6H; CH₂Ar), 3.02 (m,

6H; CH₂N), 3.10 (m, 6H; CH₂N), 3.37 (m, 6H; CH₂N), 4.72 (s, br, 3H;

NH), 5.03(s, 6H; CH ₂Ar_Cbz), 5.17 (s, br, 3H; NH), 6.83 (s, 3H; Ar H),

7.02 (s, 3H; ArH), 7.11 (s, br, 3H; NH), 7.29 (m, 15H; ArH_Cbz), 7.36 (s,

3H; ArH), 7.38 ppm (s, 3H; ArH); ¹³C NMR (127 MHz, CDCl₃): d=

28.41 (CCH₃), 30.70 (CH₂), 31.12 (CH₂), 31.31 (CH₂), 32.44 (CH₂Ar),

33.13 (CH₂Ar), 39.63 (CH₂N + CH₂N), 40.15 (CH₂N), 66.55 (CH₂Ar_Cbz),

79.17 (CCH₃), 124.82 (ArC), 124.90 (ArC), 126.27 (ArC), 127.97

(ArC_Cbz), 128.02 (ArC_Cbz), 128.45 (ArC_Cbz), 131.56 (ArC), 134.74 (ArC),

136.59 (ArC_Cbz), 141.68 (ArC), 141.75 (ArC), 141.82 (ArC), 156.10

(CON_Boc), 156.55 (CON_Cbz), 167.86 ppm (CON); MS (MALDI-TOF, di-

thranol): m/z: 1645 [M+K]⁺, 1629 [M+Na]⁺; monoisotopic mass calcd

for C₉₃H₁₂₃N₉NaO₁₅⁺: 1628.90, found: 1628.99.

8.13(d, ³J(H,H)=7.3Hz, 3H; Ar H_dns), 8.36 (d, ³J(H,H)=8.7 Hz, 3H;

ArH_dns), 8.52 ppm (d, ³J(H,H)=8.5 Hz, 3H; ArH_dns); ¹³C NMR

(127 MHz, CDCl₃): d=30.72 (CH₂), 30.83 (CH₂), 31.19 (CH₂), 32.12

(CH₂Ar), 32.55 (CH₂Ar), 33.31 (CH₂Ar), 39.80 (CH₂N), 40.36 (CH₂N),

42.28 (CH₂N), 45.60 (NCH₃), 66.65 (CH₂Ar_Cbz), 115.59 (ArC_dns), 119.40

(ArC_dns), 123.50 (ArC_dns), 123.58 (ArC_dns), 124.93(Ar C), 125.21 (ArC),

126.31 (ArC), 128.05 (ArC_Cbz), 128.11 (ArC_Cbz), 128.30 (ArC_dns), 128.56

(ArC_Cbz), 129.46 (ArC_dns), 130.16 (ArC_dns), 131.53 (ArC), 134.94 (ArC),

135.23 (ArC_dns), 136.70 (ArC_Cbz), 141.42 (ArC), 141.90 (ArC), 142.00

(ArC), 151.65 (ArC_dns), 156.69 (CON_Cbz), 167.90 ppm (CON); MS

(MALDI-TOF, dithranol): m/z: 2028 [M+Na]⁺, 2006 [M+H]⁺; monoiso-

topic mass calcd for C₁₁₄H₁₃₃N₁₂O₁₅S₃⁺: 2005.92, found: 2005.94.

(N)₃(Dns)₃6D1: 1,3,5-tris{3-(aminopropyl)-5-(5-dimethylaminonaphtha-

lene-1-sulfonic acid propylamide)-N-propylbenzamide}benzene (34): A

solution of the mixed dansylated dendrimer 33 (401 mg, 200 mmol) in tri-

fluoroacetic acid (8 mL) was stirred for seven days at room temperature.

The reaction was continuously monitored by TLC. After complete reac-

tion the solvent was repeatedly removed in vacuo to afford the partially

deprotected dendrimer 34 as a yellow-greenish solid (379 mg, 198 mmol,

99.0%) after lyophilization from water. M.p. 119 1208C; ¹H NMR

(500 MHz, [D₄]methanol): d=1.68 (quin, ³J(H,H)=7.1 Hz, 3H; CH₂),

1.96 (m, 12H; CH₂), 2.56 (t, ³J(H,H)=7.4 Hz, 6H; CH₂Ar), 2.66 (t,

³J(H,H)=7.6 Hz, 6H; CH₂Ar), 2.71 (t, ³J(H,H)=7.7 Hz, 6H; CH₂Ar),

2.84 (t, ³J(H,H)=6.7 Hz, 6H; CH₂N), 2.87 (s, 18H; NCH₃), 2.95 (t,

3

³J(H,H)=7.7 Hz, 6H; CH₂N), 3.40 (t, J(H,H)=5.6 Hz, 6H; CH₂N), 6.96

3

(s, 3H; ArH), 7.09 (s, 3H; ArH), 7.27 (d, J(H,H)=6.9 Hz, 3H; ArH_dns),

7.34 (s, 3H; ArH), 7.49 (s, 3H; ArH), 5.53(t, ³J(H,H)=7.9 Hz, 3H;

(Cbz-N)₃(N)₃6D1:

1,3,5-tris{3-[3-(benzyloxycarbonylamino)propyl]-5-

3

ArH_dns), 7.58 (t, J(H,H)=8.1 Hz, 3H; ArH_dns), 8.14 (d, J(H,H)=7.3Hz,

3H; ArH_dns), 8.39 (d, ³J(H,H)=8.8 Hz, 3H; ArH_dns), 8.53ppm (d,

³J(H,H)=8.5 Hz, 3H; ArH_dns); ¹³C NMR (127 MHz, [D₄]methanol): d=

30.39 (CH₂), 32.39 (CH₂), 32.42 (CH₂), 33.54 (CH₂Ar), 33.55 (CH₂Ar),

34.63 (CH₂Ar), 40.53(CH ₂N), 41.12 (CH₂N), 43.37 (CH₂N), 46.13

(NCH₃), 116.81 (ArC_dns), 120.90 (ArC_dns), 124.66 (ArC_dns), 126.37 (ArC),

126.54 (ArC), 127.63(Ar C), 128.28 (ArC_dns), 129.46 (ArC_dns), 130.42

(ArC_dns), 131.37 (ArC_dns), 133.07 (ArC), 136.50 (ArC_dns), 137.32 (ArC_dns),

142.61 (ArC), 142.99 (ArC), 143.55 (ArC), 143.81 (ArC), 153.25 (ArC_dns),

170.57 ppm (CON); MS (FAB+, MNBA/methanol): m/z (%):1604 (1.2)

[M+H]⁺, 170 (42.5) [C₁₂H₁₂N]⁺; MS (MALDI-TOF, dithranol): m/z:

monoisotopic mass calcd for C₉₀H₁₁₅N₁₂O₉S₃⁺: 1603.81, found: 1603.81.

(aminopropyl)-N-propylbenzamide}benzene tristrifluoroacetate (32): Tri-

fluoroacetic acid (25 mL) was added to a solution of the mixed-protected

G1 dendrimer 31 (2.41 g, 1.50 mmol) in dichloromethane (100 mL), and

the reaction mixture was stirred for 1 h at room temperature. After com-

plete reaction (TLC) the solvent was repeatedly removed in vacuo to

afford the partially deprotected dendrimer 32 (2.09 g, 1.35 mmol, 90.0%)

as a colorless oil. ¹H NMR (500 MHz, [D₄]methanol): d=1.81 (quin,

3

³J(H,H)=7.6 Hz, 6H; CH₂), 1.91 (quin, J(H,H)=7.1 Hz, 6H; CH₂), 1.96

(quin, ³J(H,H)=7.4 Hz, 6H; CH₂), 2.64 (m, 12H; CH₂Ar), 2.71 (t,

³J(H,H)=7.6 Hz, 6H; CH₂Ar), 2.91 (t, ³J(H,H)=7.7 Hz, 6H; CH₂N),

3.10 (t, ³J(H,H)=6.7 Hz, 6H; CH₂N), 3.37 (t, ³J(H,H)=7.1 Hz, 6H;

CH₂N), 5.05 (s, 6H; CH₂Ar_Cbz), 6.91 (s, 3H; ArH), 7.22 (s, 3H; ArH),

7.29 (m, 15H; ArH_Cbz), 7.44 (s, 3H; ArH), 7.48 ppm (s, 3H; ArH);

¹³C NMR (127 MHz, [D₄]methanol): d=30.38 (CH₂), 32.38 (CH₂), 32.69

(CH₂), 33.55 (CH₂Ar), 33.92 (CH₂Ar), 34.61 (CH₂Ar), 40.51 (CH₂N),

41.11 (CH₂N), 41.37 (CH₂N), 67.66 (CH₂Ar_Cbz), 126.38 (ArC), 126.56

(ArC), 127.61 (ArC), 128.96 (ArC), 129.26 (ArC), 129.77 (ArC), 133.11

(ArC), 136.67 (ArC), 138.74 (ArC), 142.64 (ArC), 143.52 (ArC), 144.13

(ArC), 159.22 (CON_Cbz), 170.60 ppm (CON); MS (MALDI-TOF, dithra-

nol): m/z: monoisotopic mass calcd for C₇₈H₁₀₀N₉O₉⁺: 1306.76, found:

1306.76 [M+H]⁺.

(Cbz-N)₃(Dns)₃6D1: 1,3,5-tris-{3-[3-(benzyloxycarbonylamino)propyl]-5-

(5-dimethylaminonaphthalene-1-sulfonic acid propylamide)}-N-propyl-

benzamide}benzene (33): A solution of the G1 trifluoroacetate 32 (2.09 g,

1.35 mmol) and dry triethylamine (2.82 mL, 2.05 g, 20.3 mmol) in dry

methanol (20 mL) was added dropwise to a vigorously stirred solution of

dansyl chloride (2.18 g, 8.10 mmol) in dry dichloromethane (250 mL).

The reaction was monitored by TLC. After complete reaction, the mix-

ture was washed once with brine, once with a saturated sodium carbonate

solution, and once again with brine. The organic layer was dried over

magnesium sulfate, and the solvent was removed in vacuo. Chromato-

graphic purification (silica gel, dichloromethane containing 4% methanol

as eluent) afforded the partially dansylated G1 dendrimer (2.68 g,

1.34 mmol; 99.3%) as a bright yellow solid. R_f=0.38 (dichloromethane/

methanol=19:1/v:v); m.p. 98 1018C; ¹H NMR (500 MHz, CDCl₃): d=

(Boc-Dpa)₃(Dns)₃6D1: 1,3,5-tris{3-[(2-tert-butoxycarbonylamino-2-pro-

pylcarbamoylethyl)carbamic acid tert-butyl ester]-5-(5-dimethylamino-

naphthalene-1-sulfonic acid propylamide)-N-propylbenzamide}benzene

(35): A solution of the Boc-protected diaminopropionic acid 14 (274 mg,

900 mmol) in dry dichloromethane/DMF (18:2/v:v) was cooled down to

ꢀ208C. DIPEA (153 mL, 116 mg, 900 mmol), and TBTU (303 mg, 945

mmol), dissolved in dry DMF (8 mL), were added slowly under vigorous

stirring. The reaction mixture was allowed to warm up to room tempera-

ture and stirred for an additional 30 min. After complete formation of

the active ester (TLC), the solution was cooled down to ꢀ208C again.

DIPEA (204 mL, 155 mg, 1.20 mmol) and the deprotected dansylated

3

1.61 (quin, J(H,H)=6.9 Hz, 6H; CH₂), 1.67 (quin, J(H,H)=7.3Hz, 6H;

CH₂), 1.82 (quin, ³J(H,H)=6.8 Hz, 6H; CH₂), 2.47 (m, 12H; CH₂Ar),

2.52 (t, ³J(H,H)=7.1 Hz, 6H; CH₂Ar), 2.77 (q, ³J(H,H)=6.2 Hz, 6H;

CH₂N), 2.85 (s, 18H; NCH₃), 3.06 (q, ³J(H,H)=6.7 Hz, 6H; CH₂N), 3.31

3

(q, J(H,H)=6.0 Hz, 6H; CH₂N), 5.02 (s, 6H; CH₂Ar_Cbz), 5.15 (t, br, 3H;

NH), 5.82 (s, br, 3H; NH), 6.80 (s, 3H; Ar H), 6.86 (s, 3H; ArH), 7.00 (s,

br, 3H; NH), 7.14 (d, ³J(H,H)=7.3Hz, 3H; Ar H_dns), 7.26 (m, 15H;

ArH_Cbz), 7.28 (s, 3H; ArH), 7.36 (s, 3H; ArH), 7.42 (m, 6H; ArH_dns),

1189

Chem. Eur. J. 2004, 10, 1167 1192

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

R. Gust, A. D. Schl¸ter et al.

FULL PAPER

dendrimer 34 (195 mg, 100 mmol), dissolved in a small amount of metha-

nol (2 mL), were added dropwise to the vigorously stirred solution. The

reaction mixture was stirred at ꢀ208C for one hour, and was then al-

lowed to warm up to room temperature slowly. Afterwards the solution

was stirred at room temperature for an additional 24 h. When the reac-

tion was complete (TLC), the solution was washed twice with a saturated

sodium carbonate solution, and once with brine. The organic phase was

dried over magnesium sulfate, the solvent removed in vacuo, and the

crude product purified by column chromatography (silica gel, dichloro-

methane containing 4% methanol as eluent). Lyophilization from diox-

ane gave the mixed dansylated and diaminopropionic acid functionalized

dendrimer 35 (81 mg, 33 mmol; 33%) as a brilliant yellow-greenish solid.

R_f=0.16 (dichloromethane/methanol=19:1/v:v); m.p. 122 1238C;

¹H NMR (500 MHz, [D₄]methanol): d=1.42 (s, 27H; H-18,18’), 1.44 (s,

27H; H-18’,18), 1.61 (quin, ³J(H,H)=7.0 Hz, 6H; H-12’), 1.78 (quin, br,

6H; H-12), 1.92 (quin, ³J(H,H)=6.8 Hz, 6H; H-4), 2.46 (t, br, 6H; H-

11’), 2.58 (t, br, 6H; H-11), 2.64 (t, br, 6H; H-3), 2.84 (t, ³J(H,H)=

6.5 Hz, 6H; H-13’), 2.96 (s, 18H; H-26), 3.19 (m, 6H; H-13), 3.30 (m,

H-12,12’), 8.48 (d, ³J(H,H)=8.7 Hz, 3H; H-16), 8.53ppm (d, ³J(H,H)=

8.5 Hz, 3H; H-12’,12); ¹³C NMR (127 MHz, [D₄]methanol): d=31.86

(CH₂), 32.31 (CH₂), 32.45 (CH₂), 33.59 (CH₂Ar), 34.11 (CH₂Ar), 34.61

(CH₂Ar), 40.87 (CH₂N), 41.10 (CH₂N), 41.28 (CH₂N), 43.42 (CH₂CH),

46.35 (NCH₃), 52.42 (CH), 117.29 (ArC_dns), 121.80 (ArC_dns), 125.09

(ArC_dns), 126.24 (ArC), 126.41 (ArC), 127.65 (ArC), 129.40 (ArC_dns),

130.59 (ArC), 130.86 (ArC_dns), 131.22 (ArC_dns), 133.14 (ArC), 136.28

(ArC_dns), 137.03 (ArC_dns), 137.53 (ArC_dns), 143.54 (ArC), 143.57 (ArC),

143.58 (ArC), 151.91 (ArC_dns), 166.95 (CON), 170.73ppm (CON); MS

(MALDI-TOF, CCA): m/z: 1900 [M+K]⁺, 1884 [M+Na]⁺, 1862

[M+H]⁺; monoisotopic mass calcd for C₉₉H₁₃₃N₁₈O₁₂S₃⁺: 1861.95, found:

1862.02.

Cell culture: The human MCF-7 breast cancer cell line was obtained

from the American Culture Collection (ATCC, Rockville, Md.). The

¬

MCF-7 cells were maintained in MEM Eagles medium containing l-glu-

tamine, supplemented with NaHCO₃(2.2 gL^ꢀ1), sodium pyrovate

(110 mgL^ꢀ1), gentamycin (50 mgL^ꢀ1), and 10% fetal calf serum (FCS)

using 75 cm²culture flasks in a water-saturated atmosphere (95% air/5%

CO₂) at 3 78C. The cells were serially passaged weekly following trypsini-

zation using 0,05% trypsin/0,02% ethylenediaminetetraacetic acid

(EDTA). Mycoplasma contamination was routinely monitored, and only

mycoplasma-free cultures were used.

3

3H; H-15a), 3.38 (m, 3H; H-15b), 3.40 (m, 6H; H-13), 4.17 (t, J(H,H)=

5.9 Hz, 3H; H-14), 6.93(s, 3H; H-1), 6.97 (s, 3H; H-10), 7.26 (s, 3H; H-

8,8’), 7.40 (m, 6H; H-8’,8 + H-23), 7.60 (m, 6H; H-21 + H-24), 8.18 (d,

³J(H,H)=7.3Hz, 3H; H-20,20’), 8.51 ppm (m, 6H; H-25 + H-20’,20);

¹³C NMR (127 MHz, [D₄]methanol): d=29.04 (C-18,18’), 29.06 (C-18’,18),

32.24 (C-12), 32.35 (C-4), 32.45 (C-12’), 33.65 (C-11’), 33.97 (C-11), 34.62

(C-3), 40.11 (C-13’), 41.08 (C-5), 43.53 (C-13 + C-15), 46.50 (C-26), 57.26

(C-14), 80.81 (C-17,17’), 81.14 (C-17’,17), 117.42 (C-23), 120.29 (C-25),

125.32 (C-21), 126.35 (C-8’), 126.36 (C-8), 127.66 (C-1), 129.37 (C-24),

130.65 (C-20,20’), 131.23 (C-20’,20), 133.14 (C-10), 136.23 (C-19,19’,19’’),

137.78 (C-19,19’,19’’), 137.89 (C-19,19’,19’’), 143.46 (C-9,9’), 143.52 (C-

9’,9), 143.70 (C-7), 151.80 (C-22), 158.02 (C-16,16’), 158.98 (C-16’,16),

170.64 (C-6), 173.25 ppm (C-6’); MS (MALDI-TOF, dithranol): m/z:

Human HeLa-cells were cultured at 378C in a humidified atmosphere

with 5% CO₂in RPMI 1640 containing 10% fetal bovine serum,

100 mgmL^ꢀ1penicillin, 100 mgmL^ꢀ1streptomycin, and additional gluta-

mine and nonessential amino acids with the following final concentra-

tions: 4 mm glutamine, 1 mm alanine, 1.5 mm aspartic acid, 1.2 mm aspara-

gine, 1.2 mm glutamic acid, 1.2 mm glycine, 1.2 mm proline, and 1.3m m

serine.

In vitro chemosensitivity assay: The in vitro testing of the dendrimers on

the cytotoxic activity was carried out on exponentially dividing MCF-7

2500 [M+K]⁺, 2484 [M+Na]⁺

;

monoisotopic mass calcd for

67]

cells according to a previously published microtiter assay.^[65

Briefly, in

C₁₂₉H₁₈₀N₁₈NaO₂₄S₃⁺: 2484.25, found: 2484.21.

96-well microtiter assay plates (Nunc), 100 mL of a cell suspension at

7000 cellsmL^ꢀ1culture medium were plated into each well and incubated

at 378C for 2 3days in a humidified atmosphere (5% CO ₂). By addition

of an adequate volume of a stock solution of the respective compound

(solvent: DMF) to the medium the desired test concentration was ob-

tained. For each test concentration and for the control, which contained

the corresponding amount of DMF, 16 wells were used. After the proper

incubation time the medium was removed, the cells were fixed with a

glutaraldehyde solution and stored at 48C. Cell biomass was determined

by a crystal violet staining technique. The influence of the dendrimers on

cell growth, was obtained by corrected T/C values according to Equa-

tions (1) and (2).

Cytostatic effect : T=C_corr½% ¼ ½ðTꢀC₀Þ=ðCꢀC₀Þ ꢂ 100

Cytocidal effect : T ½% ¼ ½ðTꢀC₀Þ=C₀ ꢂ 100

ð1Þ

ð2Þ

In Equations (1) and (2), T (test) and C (control) are the optical densities

at 578 nm of the crystal violet extract of the cell lawn in the wells (i.e.

the chromatin-bound crystal violet extracted with ethanol 70%), and C₀

is the density of the cell extract immediately before treatment.Equa-

tion (2) allows the automatic estimation of the optical density of the crys-

tal violet extract in the wells of a Flashscan S19 microplatereader (Analy-

tikjena, Jena, Germany).

(Dpa)₃(Dns)₃6D1: 1,3,5-tris-[3-(2,3-diamino-N-propylpropionamide)-5-

(5-dimethylaminonaphthalene-1-sulfonic acid propylamide)-N-propylben-

zamide]benzene (36): Trifluoroacetic acid (2 mL) was added to a solution

of dendrimer 35 (37.0 mg, 15.0 mmol) in dichloromethane (3mL), and the

solution was stirred for 1 h at room temperature. After complete reaction

(TLC) the solvent was repeatedly removed in vacuo. Lyophilization from

water yielded the deprotected mixed dansylated and diaminopropionic

acid modified dendrimer 36 as a brilliant yellow-greenish solid (37.0 mg,

14.5 mmol; 96.7%). M.p. 110 1128C; ¹H NMR (500 MHz, [D₄]methanol):

d=1.66 (quin, ³J(H,H)=7.1 Hz, 6H; H-8’), 1.88 (m, 6H; H-8), 1.94 (m,

6H; H-3), 2.53 (t, ³J(H,H)=7.4 Hz, 6H; H-7’), 2.66 (m, 12H; H-7 + H-

Fluorescence microscopy: The day before exposition to the dendrimers,

HeLa cells were seeded in 12-well plates containing a 12 mm glass cover

slip in such a density that the next day about 60% confluency was ob-

tained. Dendrimers were added to the cells in a concentration of 5 mm.

20 h after adding the dendrimer, cells were washed in PBS, fixed with

4% paraformaldehyde and probed with the rabbit polyclonal antibody

R1^[68]raised against the membrane isoforms of LAP-2 and a Cy2-conju-

gated goat anti-rabbit IgG antibody as secondary antibody following

standard procedures.^[69]The fluorescence images of the cells were ob-

tained with the Zeiss laser-scanning microscope LSM 510 and a plan-neo-

fluar 63î1.25 oil-immersion objective. The dyes were excited at 364 nm

(dansyl-labelled dentrimers) or 488 nm (Cy2) and fluorescent light was

collected by a photomultiplier after passage through a 505 530 nm BP

filter. Differential interference contrast (DIC) images were taken in parallel.

3

2), 2.84 (t, J(H,H)=6.7 Hz, 6H; H-9’), 2.96 (s, 18H; H-17), 3.21 (m, 3H;

H-9a), 3.39 (m, 6H; H-4), 3.41 (m, 3H; H-9b), 3.46 (dd, ²J(H,H)=

14.0 Hz, ³J(H,H)=6.2 Hz, 3H; H-11a), 3.53 (dd, ²J(H,H)=13.9 Hz,

³J(H,H)=5.4 Hz, 3H; H-11b), 4.30 (t, ³J(H,H)=5.6 Hz, 3H; H-10), 6.96

(s, 3H; H-1), 7.07 (s, 3H; H-6), 7.29 (s, 3H; H-5’), 7.40 (d, ³J(H,H)=

7.6 Hz, 3H; H-14), 7.45 (s, 3H; H-5), 7.58 (t, ³J(H,H)=7.9 Hz, 3H; H-

13), 7.63 (t, ³J(H,H)=8.2 Hz, 3H; H-15), 8.18 (d, ³J(H,H)=7.3Hz, 3H;

1190

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 1167 1192

Surface-Modified Dendrimers

1167 1192

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This work was supported by the Deutsche Forschungsgemeinschaft

(DFG, SFB 448), which is gratefully acknowledged. We especially thank

Dr. Sabine Koch and Stefan Jehle for their significant contribution in the

synthesis of some intermediates, and Gabriela Hertel for technical sup-

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Revised: September 10, 2003[F5836]

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Article Doi

A Surface-Modified Dendrimer Set for Potential Application as Drug Delivery Vehicles: Synthesis, In Vitro Toxicity, and Intracellular Localization

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