Inhibition of mitosis by glycopeptide dendrimer conjugates of colchicine

Article abstract of DOI:10.1002/chem.200401294

Glycopeptide dendrimers have been prepared bearing four or eight identical glycoside moieties at their surface (β-glucose, α-galactose, α-N-acetyl-galactose, or lactose), natural amino acids within the branches (Ser, Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-diaminopropionic acid as the branching unit, and a cysteine residue at the core. These dendrimers have been used as drug-delivery devices for colchicine. Colchicine was attached to the dendrimers at the cysteine thiol group through a disulfide or thioether linkage. The biological activities of the glycopeptide dendrimer conjugates were evaluated in HeLa tumor cells and non-transformed mouse embryonic fibroblasts (MEFs). The concentrations of glycopeptide dendrimer drug conjugates required to achieve inhibition of cell proliferation by interference with the tubulin system were found to be higher (IC₅₀ > 1 μm) compared to the required colchicine concentration. On the other hand, the glycopeptide dendrimer conjugates inhibited the proliferation of HeLa cells 20-100 times more effectively than the proliferation of MEFs. In comparison, non-glycosylated dendrimers and colchicine itself showed a selectivity of 10-fold or less for HeLa cells.

Full text of DOI:10.1002/chem.200401294

FULL PAPER
Inhibition of Mitosis by Glycopeptide Dendrimer Conjugates of Colchicine
David Lagnoux, Tamis Darbre, M. Lienhard Schmitz,* and Jean-Louis Reymond*[a]
Abstract: Glycopeptide dendrimers
have been prepared bearing four or
eight identical glycoside moieties at
their surface (b-glucose, a-galactose, a-
N-acetyl-galactose, or lactose), natural
amino acids within the branches (Ser,
Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-
diaminopropionic acid as the branching
unit, and a cysteine residue at the core.
These dendrimers have been used as
drug-delivery devices for colchicine.
Colchicine was attached to the den-
drimers at the cysteine thiol group
through a disulfide or thioether link-
age. The biological activities of the gly-
copeptide dendrimer conjugates were
evaluated in HeLa tumor cells and
non-transformed mouse embryonic fi-
broblasts (MEFs). The concentrations
of glycopeptide dendrimer drug conju-
gates required to achieve inhibition of
cell proliferation by interference with
the tubulin system were found to be
higher (IC50 > 1 mm) compared to the
required colchicine concentration. On
the other hand, the glycopeptide den-
drimer conjugates inhibited the prolif-
eration of HeLa cells 20–100 times
more effectively than the proliferation
of MEFs. In comparison, non-glycosy-
lated dendrimers and colchicine itself
showed a selectivity of 10-fold or less
for HeLa cells.
Keywords: amino acids · antitumor
agents · carbohydrates · dendrimers ·
drug delivery · glycopeptides
Introduction
surfaces, where they mediate cell-cell recognition and the
uptake of macromolecules.[6] For example, galactose and N-
acetyl-galactosamine groups are known to bind to asialogly-
coprotein receptors and have been used to target anticancer
compounds to hepatic cancer cells.[7]
Dendrimers are tree-like macromolecules composed of a
core, a branched structure, and end groups that form the
dendrimer surface.[1] One particularly attractive potential
application of dendrimers is in drug delivery, whereby an in-
active dendrimer–drug conjugate is delivered to a diseased
cell and is degraded within the cell to release the active
principle, for example by a cascade release mechanism.[2]
Dendrimers are already in use as gene transfection agents.[3]
In principle, a dendritic drug-delivery system should include
a tissue- or cell-targeting molecule and an inactive prodrug
that can be released by a selective triggering mechanism.
Folic acid has been placed at the surface of dendrimers as a
targeting device for the release of methotrexate to cancer
cells.[4] Arrays of multiple carbohydrate moieties at a den-
drimer surface, which may be assembled by a variety of den-
drimer-assembly chemistries,[5] should provide optimal tar-
geting devices. Various glycoprotein receptors occur at cell
A key aspect of dendrimer-mediated drug delivery is that
the dendrimer should undergo biodegradation within the
target cell after delivery. Dendrimers consisting of peptide
building blocks would be optimally suited for this purpose
since they would be sensitive to proteolytic cleavage. We
have recently prepared peptide dendrimers consisting of al-
ternating sequences of proteinogenic a-amino acid and di-
amino acid building blocks.[8] These dendrimers can be pre-
pared by Fmoc-based solid-phase peptide synthesis and pro-
vide functional artificial protein models exhibiting enzyme-
like catalytic properties and binding affinities.[9] We have
also discovered that these dendrimers are readily degraded
by proteases, with cleavage occurring both at the surface
and within the branches of the dendrimers.[10] This suggests
that peptide dendrimers might be suitable for targeting
drugs to cancer cells. One interesting drug is the well-known
antimitotic agent colchicine, which is able to bind tubulin
and thus prevents its assembly during mitosis.[11] This process
precludes cell division and can induce apoptosis. The anti-
proliferative function of colchicine makes it an interesting
agent for tumor therapy, but its use is restricted as it lacks
specificity and inhibits proliferation of both tumor cells and
[a] D. Lagnoux, T. Darbre, M. L. Schmitz, Prof. J.-L. Reymond
Department of Chemistry and Biochemistry, University of Berne
Freiestrasse 3, 3012 Berne (Switzerland)
Fax : (+41)31-631-8057
E-mail: jean-louis.reymond@ioc.unibe.ch
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 3941 – 3950
DOI: 10.1002/chem.200401294
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healthy cells. Therefore, it is of potential interest to produce
colchicine derivatives that show a bias towards the inhibi-
tion of tumor cells.
gating colchicine to the core of a peptide dendrimer would
form a biologically inactive derivative, but that degradation
of the conjugate might release a smaller fragment carrying
colchicine, which should be biologically active. The peptide
dendrimer would be functionalized with glycosidic groups to
facilitate cellular uptake and provide optimal water solubili-
ty (Figure 1).
Synthesis of colchicine and glycoside components: Colchi-
cine 1 was first modified at C(7) to allow conjugation to
peptide dendrimers (Scheme 1). Deacetylation at C(7) was
performed by using a known procedure involving the inter-
Herein, we report the preparation of glycopeptide den-
drimer–drug conjugates of colchicine equipped with surface
glycosides as targeting devices. The dendrimers display mul-
tiple glycoside moieties at the surface and carry a covalently
bound colchicine unit at the core. The dendrimers are
shown to inhibit mitosis much more potently in cancer cells
as compared to non-transformed fibroblasts (as a model for
healthy cells), presumably by a mechanism involving cellular
uptake followed by intracellular degradation and release of
active colchicine, which can then bind to tubulin (Figure 1).
Figure 1. Hypothetical mechanism for mitosis inhibition by glycopeptide
conjugates of colchicine. The glycopeptide dendrimer drug conjugate is
taken into the cell (1), and is then degraded by intracellular proteases to
release colchicine (2). Colchicine binds to tubulin and inhibits its poly-
merization (3), thereby blocking mitosis.
Although less potent than colchicine itself, these glycopep-
tide dendrimer–drug conjugates show an enhanced selectivi-
ty towards cancer cells versus immortalized but non-trans-
formed cells.
Results and Discussion
Design: Colchicine (1) is a tricyclic plant alkaloid that has a
cytostatic effect triggered by a binding interaction to tubu-
lin, which prevents cell division.[12] Colchicine is abundant
and inexpensive, yet is not useful therapeutically due to its
toxicity, presumably as a result of a lack of selectivity. Its
usefulness as a drug might therefore be improved if it could
be deployed within a selective drug-delivery device. Colchi-
cine can be readily modified at various positions.[13] In par-
ticular, it is possible to append various groups at the C(7)
amide position and preserve bioactivity, as long as these
groups are relatively small in size. We envisaged that conju-
Scheme 1. Synthesis of colchicine and carbohydrate building blocks. Con-
ditions : a) 1) Boc2O, Et3N, DMAP, CH2Cl2, 3 h, 258C (30%), 2) NaOMe,
MeOH, 08C, 30 min (80%); b) CF3CO2H/CH2Cl2, 1:1, 258C, 3 h (100%);
c) ClCH2COCl, Na2CO3, acetone/H2O, 0 8C, 3 h (26%); d) PySSCH2CH2-
CONHS, DMAP, CH2Cl2, 2 h, 258C (54%); e) 1) HBr/AcOH, CH2Cl2,
À
258C, 3 h, 2) Bu4N+ HSO4
, N-hydroxyphthalimide, CH2Cl2/1m aq.
Na2CO3, 258C, 15 h (22%); f) MeNHNH2, 258C, 15 h (76%); g) N-hy-
droxyphthalimide, Et3N, Et2O·BF3, CH2Cl2, 258C, 3 h (64%); h) 1) H2,
Pd/C, MeOH/CH2Cl2, 258C, 2 h (52%); 2) MeNHNH2, 258C, 15 h
(80%); i) 4-pentenol, Et2O·BF3, 958C, 4 h, then Ac2O/pyridine, 258C,
24 h (60%); j) NaIO4, cat. RuCl3, CCl4/CH3CN/H2O, 25 8C, 3 h (73%);
k) ClCH2COCl, H2O/acetone, Na2CO3, 08C, 1 h (67%).
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Glycopeptide Dendrimer Conjugates
FULL PAPER
mediacy of the Boc derivative 2 to give deacetylcolchicine
3.[14] The amino group was then acylated to give either the
7-chloroacetamido derivative 4,[15] to be used for thioether
bond formation with the core cysteine of the dendrimers, or
the dithiopyridyl-propionyl derivative 5,[16] to be used for di-
sulfide coupling to cysteine. Penta-O-acetyl-a-d-glucopyran-
oside (6) was converted to the corresponding bromide by
reaction with hydrogen bromide,[17] and then glycosylated
with N-hydroxyphthalimide under phase-transfer conditions,
as described for the reaction with N-hydroxysuccinimide,[18]
to give intermediate 7. Deacetylation and removal of the
phthalimide protection was finally accomplished by treat-
ment with methylhydrazine to give the sensitive b-d-gluco-
pyranosyloxyamine (8). Oxyamine 8 was purified by precipi-
tation from ethanol/dichloromethane, and would be used in
oxime ligation for glycodendrimer synthesis. The related a-
d-galactopyranosyloxyamine (11) was prepared by using a
known procedure[19] involving a-selective glycosylation of
tetra-O-benzyl-a-d-galactose (9)[20] with N-hydroxyphthal-
imide to give intermediate 10, followed by a short hydroge-
nation for debenzylation, removal of the phthalimide pro-
tecting group by treatment with methylhydrazine, and purifi-
cation by precipitation as above. Glycosylation of N-acetyl-
galactosamine 12 with 4-penten-1-ol using Et2O·BF3 as cata-
lyst followed by acetylation gave the corresponding a-d-gal-
actopyranoside 13.[21] Oxidation of the vinyl group with
Scheme 2. Synthesis of oxime-linked glycopeptide colchicine conjugates
20 and 21. Conditions: a) 4, aq. phosphate buffer pH 8, 258C, 15 h
(58%); b) NaIO4, H2O, 25 8C, 2 h (62%); c) 8, H2O/AcOH, 258C, 15 h
(64%); d) 11, H2O/AcOH, 258C, 15 h (36%). Branching points in the
dendrimers consist of Dap ((S)-2,3-diaminopropionic acid).
(11) under catalysis by acetic acid to give the oxime-linked
glycopeptide dendrimer–colchicine conjugates 20 and 21, re-
spectively. In a similar sequence, the threonine-terminated
dendrimer 22 was prepared and coupled to dithiopyridylpro-
pionyl-colchicine 5 by disulfide exchange to yield the den-
drimer–colchicine conjugate 23 (Scheme 3). Subsequent oxi-
dation with sodium periodate produced the corresponding
glyoxamide-terminated dendrimer 24. As for dendrimer 18
above, MS analysis revealed a mass difference of +16,
[22]
NaIO4 and a catalytic amount of RuCl3 gave the corre-
sponding acid 14, ready for amide bond formation with N-
terminal amino acids on a solid support. Finally, chloroace-
tylation of amino-fluorescein 15 gave N-chloroacetamido-
fluorescein 16 for glycopeptide dendrimer labeling studies.
Synthesis of glycopeptide dendrimer–colchicine conju-
gates: The peptide dendrimers were prepared by solid-phase
peptide synthesis (SPPS). All of the dendrimers possessed a
cysteine residue at the core for the conjugation of colchi-
cine. Various combinations of hydrophilic (serine, threonine,
histidine, aspartate, glutamate) and hydrophobic (leucine,
valine, phenylalanine) amino acid residues were used within
the branches. Three different synthetic strategies were ex-
plored for the attachment of carbohydrates at the dendrimer
surface.
The first approach relied on chemoselective ligation[23] of
oxyamine glycosides to surface carbonyl groups generated
by periodate cleavage of N-terminal serine or threonine res-
idues (Scheme 2 and Scheme 3). This strategy has been used
previously for the glycosylation of peptides[24] and peptide
dendrimers.[25] The peptide dendrimer 17 was first prepared
by SPPS, then cleaved and purified by preparative HPLC.
Colchicine was then coupled to the cysteine thiol group as
the chloroacetamide derivative 4 to give conjugate 18
(Scheme 2). Oxidation of the N-terminal serine residues
gave the corresponding glyoxamides. MS analysis showed a
molecular mass with +16 relative to the value expected for
the peptide dendrimer. This was attributed to oxidation of
the thioether linkage to a sulfoxide.[26] The glyoxamide den-
drimer 19 thus obtained was combined with either b-d-glu-
copyranosyloxyamine (8) or a-d-galactopyranosyloxyamine
Scheme 3. Synthesis of glycopeptide dendrimer colchicine conjugate 25.
Conditions: a) 5, MeOH, CH3CN, 258C, 2 h (60%); b) NaIO4, H2O,
258C, 2 h (56%); c) d-GalNAc-a-ONH2, H2O, AcOH, 258C, 15 h (23%).
Branching points in the dendrimers consist of Dap ((S)-2,3-diaminopro-
pionic acid).
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M. L. Schmitz, J.-L. Reymond et al.
which was attributed to a single oxidation of the disulfide
bridge. Subsequent coupling with N-acetyl-a-d-galactopyran-
osyl-oxyamine[19] in the presence of acetic acid finally gave
peptide dendrimer conjugate 25. It should be noted that at-
tempts to functionalize the dendrimers on a solid support
using glycosyloxyamines and levulinate end groups were un-
successful.
In the second approach to glycopeptide dendrimer synthe-
sis, the carbohydrate was attached to the N-termini by re-
ductive amination (Scheme 4).[27] This approach is known to
Scheme 4. Synthesis of lactose-capped peptide dendrimer colchicine con-
jugate 27. Conditions: a) lactose, NaBH3CN, AcOH, MeOH, 708C, 15 h
(44%); b) 5, CH3CN, MeOH, 258C, 2 h (58%). Branching points in the
dendrimers consist of Dap ((S)-2,3-diaminopropionic acid).
modify whole proteins,[28] and has recently been applied by
Stoddart et al. for the preparation of glycodendrimers.[29]
Treatment of dendrimer 17 with lactose in the presence of
sodium cyanoborohydride in methanol resulted in clean con-
version to glycodendrimer 26 bearing eight galactose end
groups. The product was purified by semipreparative HPLC
and coupled with the thiopyridyl-activated colchicine 5 to
yield conjugate 27.
Scheme 5. Synthesis of (GalNAc)4 and (GalNAc)8 glycopeptide dendri-
mer colchicine conjugates 30 and 32. Conditions: a) 5, CH3CN/MeOH,
258C, 2 h (30%); b) NH4OH, MeOH/H2O, 25 8C, 12 h (79%); c) 5,
CH3CN/MeOH, 258C, 2 h (13%); d) 16, aq. phosphate buffer, pH 8,
MeOH, 258C, 15 h (20%). Branching points in the dendrimers consist of
Dap ((S)-2,3-diaminopropionic acid).
In the third approach, the dendrimers were directly func-
tionalized with a carbohydrate during SPPS by amide bond
formation to the N-terminal residues (Scheme 5). O-Acetyl-
protected 4-(a-N-acetyl-galactosyloxy)butyric acid 14 was
used for amide bond formation to the N-terminal residues
at the dendrimer surface under standard peptide-coupling
conditions. Deprotection and cleavage from the resin gave
the O-acetyl-protected glycodendrimer 28, which was then
coupled with the thiopyridyl derivative of colchicine 5 to
give 29. The O-acetyl protecting groups were then removed
by treatment with methanolic ammonia to provide the gly-
copeptide dendrimer colchicine conjugate 30 bearing four
N-acetyl-galactosamine moieties at the surface. In a similar
approach, glycopeptide dendrimer 31 bearing eight N-
acetyl-galactosamine moieties was obtained after N-terminal
coupling with 14, followed by cleavage from the resin, de-
acetylation of the crude cleavage product with methanolic
ammonia, and purification. Dendrimer 31 was then coupled
with either thiopyridylcolchicine 5 or the chloroacetyl-fluo-
rescein derivative 16 to produce the glycopeptide dendrimer
conjugates 32 and 33, respectively.
Finally, chloroacetylcolchicine 4 was conjugated to pep-
tide dendrimers available from other projects,[9] bearing a
variety of non-carbohydrate surface groups (carboxylate,
ammonium, and acetyl groups), to provide dendrimer conju-
gates 34–38 (Scheme 6).
Biological evaluation: The biological activities of the vari-
ous colchicine derivatives were compared by using immor-
talized mouse embryonic fibroblasts (MEFs) and HeLa
cervix carcinoma cells, which are oncogenically transformed
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Glycopeptide Dendrimer Conjugates
FULL PAPER
hibition of cell division was observed at a concentration of
5 mm.[30] A survey of the cytostatic activities of the colchicine
glycopeptide dendrimer conjugates against MEFs and HeLa
tumor cells was therefore carried out at this concentration
(Table 1). While glycopeptide dendrimer 31 not bearing a
Table 1. Inhibition of mitosis by colchicine glycopeptide and peptide
dendrimer conjugates.
Compd Dend.
surf.[a]
Link[b] Colch.[c] HeLa
[%][d]
MEF
[%][e]
Selec.[f]
blank
1
4
100.0
0.2
0.2
100.0
2.4
0.6
1
12
3
20
21
25
27
30
32
31
34
35
36
37
38
8ꢁGlc
8ꢁGal
oxime S(O)
oxime S(O)
0.7
2.1
0.9
0.4
0.5
0.3
95.6
3
1
16
48
17
113.8
92.2
93.4
34.1
47.9
6.6
81.8
2
10
163
44
104
85
96
22
1
1
10
7
8ꢁGalNAc oxime SS(O)
8ꢁLac amine SS
4ꢁGalNAc amide SS
8ꢁGalNAc amide SS
8ꢁGalNAc amide
–
S
S
S
S
S
4ꢁglutarate
4ꢁglutarate
4ꢁlysine
4ꢁproline
4ꢁacetyl
–
–
–
–
–
106
86
84
2
5
Scheme 6. Peptide dendrimer conjugates of colchicine. Branching points
are formed by 1,3-diamino-2-propyloxy-acetic acid in dendrimers 34, 35,
37, 38 and by (S)-1,2-diaminopropionic acid in dendrimer 36.
[a] Glycoside type at dendrimer surface. [b] Type of glycopeptide link.
[c] Link between colchicine and cysteine thiol. [d] Percent HeLa tumor
cells after 3 days in culture with 5 mm of indicated compounds. [e] Percent
surviving MEF cells after three days in culture with 5 mm of indicated
compounds. [f] Ratio of percent surviving MEFs to HeLa cells.
by the papillomavirus proteins E6 and E7. As colchicine
binding to tubulin inhibits mitosis and thus cell division, the
determination of cell numbers allows a direct comparison of
the biological activities of colchicine and its derivatives.
Equal numbers of cells were plated and either left untreated
or incubated with various concentrations of some of the
compounds, including the lactose-terminated dendrimer 27
and the GalNAc-terminated dendrimers 30 and 32. After
three days, the cell number was determined. The dendrimers
were found to be generally much less active than colchicine
itself or its chloroacetyl analogue 4 (Figure 2). Optimal in-
colchicine substituent was not cytostatic, all glycopeptide
dendrimer conjugates of colchicine showed cytostatic activi-
ty. The glycopeptide conjugates generally showed a higher
selectivity against the HeLa tumor cells over the non-trans-
formed MEF cells when compared to colchicine itself. The
effect was most pronounced with dendrimer 20 bearing
eight oxime-linked b-glucose units at its surface. In future
studies, it will be interesting to determine whether dendri-
mer 20 also shows this bias for other tumor cells.
The non-glycosylated dendrimers 34–38 were generally
found to be less active than the glycopeptide dendrimers.
Only dendrimer conjugates 34 and 35 showed bioactivity
comparable to that of the glycopeptide dendrimer conju-
gates, albeit without noticeable selectivity towards HeLa
cells as observed for the glycopeptide dendrimers such as
20. The activity of 34 and 35 in comparison to the less active
dendrimers 36–38 might stem from the presence of the neg-
atively charged carboxylate groups at the surface, although
there are also other differences in amino acid composition.
The bioactivity of 34 and 35 further indicates that the
nature of the colchicine–dendrimer link (thioether, sulfox-
ide, disulfide, thiosulfoxide) is not the primary determinant
of bioactivity of the conjugates.
The effect of the tumor cell selective conjugate 20 was in-
vestigated in more detail. To test whether it affects the spin-
dle apparatus or acts by additional pathways, human lung
cancer H1299 cells were transiently transfected to express
green fluorescent protein (GFP)-tagged tubulin and re-
Figure 2. Concentration dependence of the cytostatic effect of colchicines
1 and 4 and of the glycopeptide dendrimer conjugates 27, 30, and 32. The
indicated concentrations of colchicine and its derivatives were added to a
constant number of HeLa cells. After three days, the cell number was de-
termined, which is given on the y axis.
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M. L. Schmitz, J.-L. Reymond et al.
mained either untreated or were incubated with colchicine
or conjugate 20. Immune fluorescence microscopy revealed
a normal cell morphology for control cells, while treated
cells often displayed double or inadequately separated
nuclei, which is indicative of a disturbed tubulin system
(Figure 3). These results suggest that the colchicine den-
colchicine conjugation step was necessary after the solid-
supported peptide dendrimer synthesis. Non-glycosylated
peptide dendrimer colchicine conjugates have also been pre-
pared.
Cytostatic properties have been investigated against HeLa
cells, as a model for cancer cells, and MEF cells, as a model
for immortalized but non-transformed cells. Although the
dendrimer conjugates were found to be less active than col-
chicine itself, the glycopeptide dendrimer colchicine conju-
gates showed enhanced selectivity towards HeLa cells over
MEF cells as compared to colchicine. Non-glycosylated pep-
tide dendrimer colchicine conjugates were generally found
to be less active and less selective than the glycosylated con-
jugates. Dendrimers without colchicine were inactive. The
bioactivity of glycopeptide dendrimer conjugate 20 has been
traced to the inhibition of mitosis and nuclear separation
during cell division, as observed for colchicine itself. There
was no degradation of the dendrimers in the cell culture
medium. The bioactivity of the glycopeptide dendrimer col-
chicine conjugates can best be explained in terms of inter-
nalization followed by degradation with the release of an
active colchicine derivative.
These experiments show that glycopeptide dendrimers
can be readily prepared and provide a suitable selective ve-
hicle for the delivery of cytotoxic compounds to cancer cells.
Dendrimer glycosylation induces a selective cytotoxicity to-
wards Hela cells over MEF cells. This effect might become
even more pronounced when applied to cell types express-
ing specific receptors for carbohydrates, such as hepatic
cancer cells. Optimization of the dendritic glycopeptide
array for optimal targeting to such cells and for the delivery
of drugs other than colchicine is currently under investiga-
tion.
Figure 3. H1299 cells expressing GFP-tubulin were treated for 30 h as in-
dicated and analyzed by immune fluorescence microscopy. The mal-
formed double nuclei are shown by arrows. Typical examples are shown.
drimer conjugate 20 acts in the same manner as colchicine
itself, that is, by disrupting tubulin function.
Glycopeptide dendrimer fluorescein conjugate 33 was in-
cubated with cultures of HeLa or MEF cells under the con-
ditions of the bioassay. HPLC analysis of the supernatant
did not reveal any noticeable degradation of the conjugate,
showing only unchanged dendrimer. When the cells were
washed and examined under a fluorescence microscope,
both the HeLa and MEF cells showed a homogeneous weak
fluorescein fluorescence, indicating that the dendrimers
were able to enter inside the cells during the course of the
experiment.
A possible mechanism of action of our dendrimer–colchi-
cine conjugates involves either passive or active uptake of
the dendrimer-bound colchicine into the cells, followed by
intracellular proteolytic degradation of the peptide structure
to release an active colchicine compound, which then inter-
feres with tubulin and blocks mitosis. The proposed pro-
drug-type mechanism of action involving dendrimer degra-
dation would also explain the fact that structurally diverse
glycopeptide dendrimer conjugates show similar activities.
Experimental Section
General: Preparative HPLC purifications were performed on a Waters
Prepak cartridge packed with 500 g of RP-C18, particle size 20 mm, pore
size 300 ꢂ, flow rate 100 mLminÀ1, eluent A: 0.1% TFA in water;
eluent B: acetonitrile/water (3:2) + 0.1% TFA; gradient +1% B minÀ1
detection by UV at l = 214 nm.
,
Semipreparative RP-HPLC purifications were performed on a Vydac
218TP510, C18, 1.0 cmꢁ25 cm, flow rate 4 mLminÀ1, eluent A: 0.1%
TFA in water; eluent B: acetonitrile/water (3:2) + 0.1% TFA, gradient
+1% B minÀ1, detection by UV at l = 220 nm.
Conclusion
N-[(tert-Butoxy)carbonyl]colchicine:
Di(tert-butyl)
pyrocarbonate
A series of glycopeptide dendrimer colchicine conjugates
has been prepared using different strategies for glycoside at-
tachment. The glycoside attachment methods used have in-
cluded oxime bond formation to N-terminal glyoxyamide
generated by periodate cleavage of N-terminal serine or
threonine residues, reductive alkylation of peptide amino
termini with lactose/NaBH3CN, and amide bond formation
with an acetylated carboxypropyl glycoside directly on a
solid support as the last step of the peptide dendrimer syn-
thesis followed by cleavage and deacetylation. The latter
strategy proved to be the most practical because only the
(1.55 g, 7.10 mmol, 6.0 equiv) was added portionwise to a mixture of col-
chicine 1 (0.40 mg, 1.25 mmol), Et3N (0.2 mL, 1.43 mmol, 1.1 equiv), and
4-(dimethylamino)pyridine (0.18 mg, 1.46 mmol, 1.2 equiv) in CH2Cl2
(3 mL), and the mixture was stirred for 3 h at 258C. The solvent was then
removed in vacuo and the residue was purified by flash chromatography
on silica gel (AcOEt/acetone, 4:1, Rf = 0.28) to give N-Boc-colchicine
(0.187 mg, 0.38 mmol, 30%) as a yellow solid. 1H NMR (400 MHz,
CDCl3): d = 7.55 (s, 1H), 7.18 (d, J = 10.88 Hz, 1H), 6.74 (d, J =
10.88 Hz, 1H), 6.51 (s, 1H), 5.13 (q, J = 6.11 Hz, 1H), 3.95 (s, 3H), 3.92
(s, 3H), 3.88 (s, 3H), 3.64 (s, 3H), 2.70–2.43 (m, 3H), 2.26 (s, 3H), 1.95
(m, 1H), 1.54 ppm (s, 9H); 13C NMR (75 MHz, CDCl3): d
164.7, 154.2, 154.1, 151.8, 151.2, 142.3, 137.5, 136.3, 134.7, 133.3, 126.7,
113.7, 107.8, 85.5, 62.2, 62.1, 58.5, 57.0, 56.7, 33.3, 30.8, 30.3, 28.5,
= 179.9,
3946
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 3941 – 3950

Glycopeptide Dendrimer Conjugates
FULL PAPER
26.2 ppm; EI-MS: m/z: 500 [M+]; HRMS: calcd for C27H33NO8 500.2284;
found 500.2295.
lution of NaHCO3. The organic phase was then dried with Na2SO4 and
concentrated in vacuo to give tetra-O-acetyl-a-d-glucopyranosyl bromide.
This product was dissolved in CH2Cl2 (100 mL) and treated with tetrabu-
tylammonium hydrogen sulfate (4.41 g, 0.013 mol, 1.0 equiv) and N-hy-
droxyphthalimide (10.6 g, 0.065 mol, 5 equiv) dissolved in 1m aqueous
Na2CO3 solution (100 mL) and the resulting mixture was vigorously stir-
red overnight at 258C. Thereafter, CH2Cl2 was added and the mixture
was washed with water, aqueous NaHCO3 solution, and saturated aque-
ous NaCl solution. The organic phase was dried with Na2SO4 and concen-
trated. The residue was purified by flash chromatography on silica gel
(CH2Cl2/AcOEt, 9:1, Rf = 0.40) to furnish 7 (1.44 g, 2.9 mmol, 22%) as a
white solid. 1H NMR (300 MHz, CDCl3): d = 7.88–7.77 (m, 4H), 5.32–
5.24 (m, 3H), 5.11 (dd, J = 6.30 and 1.59 Hz, 1H), 4.34 (dd, J = 12.42
and 4.89 Hz, 1H), 4.14 (dd, J = 12.42 and 2.64 Hz, 1H), 3.80–3.74 (m,
1H), 2.20 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.03 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d = 171.3, 170.8, 170.2, 170.0, 163.3, 135.5, 129.5,
124.6, 105.8, 73.1, 73.1, 70.3, 68.8, 62.5, 21.4, 21.3, 21.3 ppm.
N-[(tert-Butoxy)carbonyl]deacetylcolchicine (2): A mixture of N-Boc-
colchicine (89.3 mg, 0.18 mmol) and NaOMe (36.2 mg, 0.67 mmol,
3.7 equiv) in MeOH (2 mL) was stirred for 30 min at 08C. It was then
neutralized by the addition of NH4Cl and the solvent was evaporated.
The residue was purified by flash chromatography on silica gel (AcOEt/
acetone, 4:1, Rf = 0.39) to give 2 (65.8 mg, 0.144 mmol, 80%) as a pale-
1
yellow solid. H NMR (300 MHz, CDCl3): d = 7.48 (s, 1H), 7.26 (d, J =
10.74 Hz, 1H), 6.79 (d, J = 10.74 Hz, 1H), 6.51 (s, 1H), 4.98 (d, J =
7.72 Hz, 1H), 4.38 (m, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.88 (s, 3H), 3.63
(s, 3H), 2.52–2.21 (m, 3H), 1.66 (m, 1H), 1.35 ppm (s, 9H); 13C NMR
(75 MHz, CDCl3): d
= 180.2, 164.6, 154.9, 154.1, 151.8, 142.2, 136.6,
135.6, 134.9, 131.8, 126.3, 112.7, 107.8, 80.5, 62.1, 61.2, 61.0, 56.9, 56.7,
53.7, 38.3, 30.6, 28.9 ppm; MS (ES+): m/z: 458.14 [M+]; HRMS: calcd
for C25H31NO7 458.2178; found 458.2175.
Deacetylcolchicine (3):
A
solution of compound
2
(158.1 mg,
O-b-d-Glucopyranosyl-oxyamine (8): Compound 7 (170 mg, 0.34 mmol)
was dissolved in methylhydrazine (2.0 mL, 37.59 mmol) and the solution
was stirred overnight at 258C. The solvent was then evaporated in vacuo
and the residue was purified by precipitation from the minimum volume
of MeOH and CH2Cl2. After filtration, compound 8 (51 mg, 0.26 mmol,
76%) was obtained as a white solid. TLC: CH2Cl2/EtOH, 1:1, Rf = 0.38;
1H NMR (300 MHz, D2O): d = 4.49 (d, J = 8.46 Hz, 1H), 3.86 (dd, J =
12.51 and 2.19 Hz, 1H), 3.65 (dd, J = 12.51 and 5.90 Hz, 1H), 3.46–
3.19 ppm (m, 4H); 13C NMR (75 MHz, D2O): d = 107.5, 78.3, 78.3, 74.1,
0.346 mmol) in CH2Cl2 (4 mL) containing TFA (0.4 mL) was stirred for
3 h. Toluene was then added, the mixture was concentrated in vacuo, and
the residue was purified by flash chromatography on silica gel (CH2Cl2/
MeOH, 9:1, Rf = 0.34) to give compound 4 (123.1 mg, 0.345 mmol, quan-
1
titative) as a yellow solid. H NMR (300 MHz, CDCl3): d = 7.66 (s, 1H),
7.33 (d, J = 11.11 Hz, 1H), 6.88 (d, J = 11.11 Hz, 1H), 6.57 (s, 1H), 4.12
(m, 1H), 3.92 (s, 3H), 3.91 (s, 6H), 3.57 (s, 3H), 2.71–2.59 (m, 2H), 2.48–
2.28 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d = 179.6, 164.7, 154.7,
151.6, 147.4, 142.2, 137.3, 137.2, 134.4, 132.0, 125.1, 114.4, 108.3, 61.9,
61.9, 57.2, 56.7, 54.5, 36.4, 30.2 ppm; EI-MS: m/z: 357 [M+]; HRMS:
calcd for C20H23NO5 357.157623; found 357.157530.
+
72.0, 63.2 ppm; HRMS: calcd for C6H14NO6 196.0821; found 196.0824.
O-(2,3,4,6-Tetra-O-benzyl-a-d-galactopyranosyl)-N-oxyphthalimide (10):
Et2O·BF3 (9.0 mL, 71.66 mmol, 4 equiv) was added to a solution of tetra-
N-[2-(Chloromethyl)carbonyl]deacetylcolchicine (4): A solution of chlo-
roacetyl chloride (30 mL, 0.376 mmol, 1.1 equiv) in acetone (0.1 mL) was
added dropwise to a solution of 3 (135.0 mg, 0.378 mmol) and Na2CO3
(20 mg) in water/acetone (1 mL, 1:1) at 08C. The resulting mixture was
stirred for 1 h and then the solvent was removed in vacuo. The residue
was extracted with AcOEt, the solvent was removed from the extract,
and the crude product was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 19:1, Rf = 0.37) to give 4 (42.8 mg, 0.097 mmol, 26%)
O-benzyl-d-galactose
9 (9.74 g, 18.00 mmol), N-hydroxyphthalimide
(2.92 g, 18.00 mmol, 1.0 equiv), and Et3N (2.5 mL, 17.93 mmol, 1 equiv)
in CH2Cl2 (70 mL) at 08C. The mixture was stirred for 3 h at 258C and
then washed with water and a saturated solution of NaHCO3. The organ-
ic phase was concentrated and the residue was purified by flash chroma-
tography on silica gel (hexane/AcOEt, 7:3, Rf = 0.46) to yield 10 (7.89 g,
11.50 mmol, 64%) as a white solid. 1H NMR (300 MHz, CDCl3): d =
7.86–7.73 (m, 4H), 7.61–7.29 (m, 20H), 5.71 (d, J = 3.78 Hz, 1H), 5.13
(d, J = 11.49 Hz, 1H), 5.04–4.79 (m, 5H), 4.66–4.51 (m, 3H), 4.33 (dd, J
= 10.56 and 3.87 Hz, 1H), 4.22–4.17 (m, 2H), 3.70–3.57 ppm (m, 2H);
13C NMR (75 MHz, CDCl3): d = 164.0, 139.4, 139.2, 138.9, 138.6, 135.1,
129.6, 129.0, 128.9, 128.9, 128.9, 128.8, 128.4, 128.3, 128.2, 128.2, 128.1,
124.2, 103.3, 78.8, 76.0, 75.6, 75.6, 74.1, 73.8, 73.5, 71.6, 68.8 ppm.
1
as a yellow solid. H NMR (400 MHz, CDCl3): d = 7.40 (s, 1H), 7.32 (d,
J = 10.84 Hz, 1H), 7.12 (d, J = 7.16 Hz, NH), 6.83 (d, J = 10.84 Hz,
1H), 6.54 (s, 1H), 4.63 (m, 1H), 4.03 (m, 2H), 4.00 (s, 3H), 3.94 (s, 3H),
3.91 (s, 3H), 3.64 (s, 3H), 2.56 (m, 1H), 2.44 (m, 1H), 2.85 (m, 1H),
1.91 ppm (m, 1H); 13C NMR (100 MHz, CDCl3): d = 180.1, 166.4, 164.8,
154.3, 151.9, 150.8, 142.5, 136.8, 136.1, 134.6, 131.3, 126.2, 113.2, 108.1,
62.1, 62.1, 57.1, 56.8, 53.3, 43.1, 37.5, 30.4 ppm; EI-MS: m/z: 434 [M+];
HRMS: calcd for C22H24ClNO6 434.1370; found 434.1382.
O-a-d-Galactopyranosyl-oxyamine (11):
A mixture of compound 10
(1.70 g, 2.48 mmol) and 10% Pd/C (214 mg, 1.98 mmol, 0.8 equiv) in
CH2Cl2/MeOH (1:1, 5 mL) was stirred under a hydrogen atmosphere for
2 h at 258C. The catalyst was then removed by filtration through Celite,
and the filtrate was concentrated. The solid residue was purified by flash
chromatography on silica gel (CH2Cl2/MeOH, 8.5:1.5; TLC: CH2Cl2/
N-[3-(2-Pyridyldithio)propionyl]deacetylcolchicine (5): A solution of 3
(46.7 mg, 0.131 mmol) in CH2Cl2 (0.5 mL) was treated with N-succinimid-
yl 3-(2-pyridyldithio)propionate (80%, 51.1 mg, 0.131 mmol, 1.0 equiv)
and DMAP (16.6 mg, 0.131 mmol, 1.0 equiv). The resulting mixture was
stirred for 2 h at 258C. Thereafter, the solvent was removed in vacuo and
the residue was purified by flash chromatography on silica gel (CHCl3/
MeOH, 94:6, Rf = 0.54) and subsequently precipitated from CH2Cl2/pe-
MeOH, 9:1, Rf
= 0.23) to yield O-a-d-galactopyranosyl-N-oxyphthal-
imide (0.42 g, 1.28 mmol, 52%) as a white solid. This product (0.809 g,
2.49 mmol) was dissolved in methylhydrazine (22.2 mL, 0.42 mol) and the
solution obtained was stirred overnight at 258C. It was then concentrated
in vacuo and the residue was purified by precipitation from the minimum
volume of MeOH and CH2Cl2. After filtration, compound 11 (0.387 mg,
1.98 mmol, 80%) was obtained as a white solid; TLC: CH2Cl2/EtOH, 1:1,
Rf = 0.38; 1H NMR (300 MHz, D2O): d = 4.96 (d, J = 3.93 Hz, 1H),
3.92–3.88 (m, 2H), 3.82–3.68 ppm (m, 4H); 13C NMR (75 MHz, D2O): d
= 104.3, 73.4, 71.8, 71.7, 70.3, 63.6 ppm; LSI-MS (DTT/DTE): m/z: 196
troleum ether to give the thiopyridyl-activated colchicine
5 (39.5 g,
0.071 mmol, 54%) as an orange solid. 1H NMR (300 MHz, CDCl3): d =
8.48 (m, J = 4.89 Hz, 1H), 7.65–7.56 (m, 3H), 7.47 (s, 1H), 7.28 (d, J =
10.82 Hz, 1H), 7.14–7.10 (m, NH), 6.81 (d, J = 10.82 Hz, 1H), 6.53 (s,
1H), 4.67 (m, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.90 (s, 3H), 3.65 (s, 3H),
3.01 (m, 2H), 2.69 (m, 2H), 2.68–2.25 (m, 3H), 1.93–1.83 ppm (m, 1H);
13C NMR (75 MHz, CDCl3): d = 180.1, 171.1, 164.7, 160.1, 154.1, 151.9,
151.6, 150.2, 142.4, 137.8, 136.8, 135.8, 134.8, 131.6, 126.4, 121.9, 121.1,
112.9, 108.0, 62.2, 62.1, 57.0, 56.8, 53.0, 37.6, 36.4, 35.8, 30.6 ppm; LSI-
MS: m/z: 555 [M+]; HRMS: calcd for C28H30N2O6S2 555.1623; found
555.1632.
+
[M]; HRMS: calcd for C6H13NO6 196.0821; found 196.0815.
4-Pentenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-d-galactopyranoside
(13): Dry 4-pentenol (8 mL, 78 mmol, 34 equiv) and freshly distilled
Et2O·BF3 (0.05 mL, 0.42 mmol, 0.19 equiv) were added to N-acetyl-galac-
tosamine 12 (501 mg, 2.26 mmol) and the mixture was stirred for 4 h at
958C. The excess 4-pentenol was then removed in vacuo and the residue
was dissolved in Ac2O (5 mL) and pyridine (10 mL). After allowing the
reaction to proceed for 24 h, toluene was added and the solution was
concentrated. The residue was purified by flash chromatography on silica
O-(2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl)-N-oxyphthalimide (7):
A
30% solution of HBr in AcOH (8.2 mL, 0.142 mol, 11 equiv) was added
dropwise to a solution of a-d-glucose pentaacetate 6 (5.06 g, 0.013 mol)
in CH2Cl2 (30 mL) at 08C. The resulting mixture was stirred at 08C for
30 min and then at room temperature for 3 h. After the addition of fur-
ther CH2Cl2, the solution was washed with cold water and with a cold so-
gel (AcOEt/hexane, 4:1, Rf
=
0.33) to yield 13 (552 mg, 1.33 mmol,
Chem. Eur. J. 2005, 11, 3941 – 3950
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3947

M. L. Schmitz, J.-L. Reymond et al.
60%) as an orange oil. 1H NMR (400 MHz, CDCl3): d = 5.85–5.75 (m,
1H), 5.58 (d, J = 9.68 Hz, 1H), 5.36 (d, J = 2.44 Hz, 1H), 5.15 (dd, J =
11.4 and 3.30 Hz, 1H), 5.05–4.98 (m, 2H), 4.85 (d, J = 3.68 Hz, 1H),
4.59–4.30 (m, 1H), 4.16–4.03 (m, 3H), 3.72–3.66 (m, 1H), 3.47–3.41 (m,
1H), 2.15 (s, 3H), 2.14–2.04 (m, 2H), 2.03 (s, 3H), 1.99 (s, 3H), 1.95 (s,
3H), 1.74–1.67 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d = 171.6,
171.0, 171.0, 170.5, 138.3, 115.9, 98.3, 69.1, 68.4, 68.0, 67.3, 62.6, 48.4, 30.9,
29.1, 23.9, 21.4, 21.4, 21.3 ppm; EI-MS: m/z: 416 [M+]; HRMS: calcd for
C6H13NO6 416.1920; found 416.1906.
rification by semipreparative RP-HPLC gave 18 (5.1 mg, 1.93 mmol,
58%). MS (ES+): calcd for C108H174N34O41S: 2635.23, found: 2636.39.
[{(CHO-CO)2DapLeu}2DapAsp]2DapCys(S(O)-Colch)HisNH2 (19): Den-
drimer 18 (1.7 mg, 0.45 mmol) was dissolved in H2O (100 mL) and NaIO4
(4.9 mg, 22.9 mmol, 51 equiv) was added. The solution was stirred for 2 h
under nitrogen and then worked-up by semipreparative RP-HPLC. The
fraction corresponding to the main peak (tR = 14.4 min; A/B = 70:30 to
40:60 in 30 min) was collected and lyophilized to give 19 (0.7 mg,
0.28 mmol, 62%). MS (ES+): calcd for C100H133N25O43S: 2403.87, found:
2403.36.
3-Carboxypropyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-d-galactopy-
ranoside (14): NaIO4 (1.66 g, 7.74 mmol, 4.1 equiv) and RuCl3 (20 mg,
[{(d-Glc-b-ONCH-CO)2DapLeu}2DapAsp]2DapCys(S(O)-Colch)HisNH2
(20): Dendrimer 19 (1.0 mg, 0.40 mmol) was dissolved in H2O (0.1 mL)
0.076 mmol, 0.04 equiv) were added to
a solution of 13 (783 mg,
1.89 mmol) in CCl4 (3.5 mL)/CH3CN (3.5 mL)/H2O (5 mL) and the mix-
ture was stirred for 3 h at room temperature. It was then extracted with
CH2Cl2 and the extracts were washed with H2O. The combined organic
layers were concentrated and the residue was purified by flash chroma-
tography on silica gel (Et2O/CH2Cl2/MeOH, 7:2.5:0.5 + 1% AcOH, Rf
containing 2 drops of AcOH, and oxyamine
8 (7.7 mg, 39.5 mmol,
12 equiv/aldehyde function) was added. The solution was kept overnight
under nitrogen at 258C and then worked-up by semipreparative HPLC.
Collection of the fraction corresponding to the main peak (tR = 9.6 min;
A/B = 70:30 to 40:60 in 30 min) and lyophilization gave 20 (1.0 mg,
0.25 mmol, 64%). MS (ES+): calcd for C148H222N34O82S: 3819.4, found:
3820.25.
1
= 0.48) to yield 14 (599 mg, 1.38 mmol, 73%) as a colorless oil. H NMR
(400 MHz, CDCl3): d = 6.14 (d, J = 9.68 Hz, 1H), 5.33 (d, J = 2.48 Hz,
1H), 5.10 (dd, J = 11.32 and 3.24 Hz, 1H), 4.83 (d, J = 3.52 Hz, 1H),
4.57–4.50 (m, 1H), 4.14–4.05 (m, 3H), 3.80–3.75 (m, 1H), 3.47–3.41 (m,
1H), 2.50–2.32 (m, 2H), 2.13 (s, 3H), 2.11–2.05 (m, 2H), 2.02 (s, 3H),
1.96 (s, 3H), 1.96 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d = 177.7,
171.6, 171.6, 171.1, 170.9, 98.4, 69.0, 68.1, 67.9, 67.3, 62.5, 48.3, 31.9, 25.4,
23.4, 21.3, 21.2, 21.2 ppm; EI-MS: m/z: 434 [M+]; HRMS: calcd for
C6H13NO6Na 456.1481; found 456.1495.
[{(d-Gal-a-ONCH-CO)2DapLeu}2DapAsp]2DapCys(S(O)-Colch)HisNH2
(21): Treating dendrimer 19 (0.7 mg, 0.28 mmol) as above, but using oxy-
amine 11 (3.9 mg, 20.00 mmol, 9 equiv/aldehyde function), followed by
purification by semipreparative HPLC and isolation of the fraction corre-
sponding to the main peak (tR = 18.4 min; A/B = 80:20 to 50:50 in
30 min) gave dendrimer 21 (0.4 mg, 0.10 mmol, 36%). MS (ES+): calcd
for C148H222N34O82S: 3819.4, found: 3820.88.
Chloroacetamido-fluorescein (16): A solution of chloroacetyl chloride
(23 mL, 0.29 mmol, 1.1 equiv) in acetone (0.1 mL) was added dropwise to
a solution of fluoresceinamine 15 (90.1 mg, 0.26 mmol) and Na2CO3
(14.4 mg) in water/acetone (1 mL, 1:1) at 08C. The resulting mixture was
stirred for 1 h and then the solvent was removed in vacuo. After extrac-
tion with AcOEt and removal of the solvent, the crude product was puri-
fied by flash chromatography on silica gel (acetone/AcOEt/hexane,
3:2:5) to give 16 (74 mg, 0.17 mmol, 67%) as a yellow solid. 1H NMR
(400 MHz, [D6]acetone): d = 8.42 (dd, J = 2.02 and 0.48 Hz, 1H), 7.95
(dd, J = 8.31 and 2.02 Hz, 1H), 7.24 (dd, J = 8.31 and 0.48 Hz, 1H),
[{(NH2-Thr)2DapLeu}2DapAsp]2DapCysHisNH2 (22): Starting with
50 mg of NovaSyn TGR resin (0.25 mmolgÀ1), the dendrimer was pre-
pared by standard Fmoc-SPPS (see the preparation of 17). Cleavage, pre-
cipitation, and purification by preparative HPLC gave, after lyophiliza-
tion of the fraction corresponding to the main peak (tR = 20.0 min; A/B
= 90:10 to 60:40 in 30 min), dendrimer 22 (20.3 mg, 16%) as a colorless
foamy solid. MS (ES+): calcd for C94H167N33O35S: 2350.20, found:
2351.25.
[{(NH2-Thr)2DapLeu}2DapAsp]2DapCys(SS(CH2)2Colch)HisNH2
(23):
6.74 (d, J
= 2.32 Hz, 2H), 6.72 (s, 1H), 6.70 (s, 1H), 6.64 (d, J =
Dendrimer 22 (4.7 mg, 1.39 mmol) was dissolved in degassed MeOH/
CH3CN (1:1, 0.2 mL) and dithiopyridylpropionylcolchicine 5 (8.0 mg,
14.4 mmol, 10 equiv) was added. The solution was kept at 258C for 2 h
under nitrogen. Purification by semipreparative RP-HPLC, isolation of
2.44 Hz, 1H), 6.62 (d, J = 2.44 Hz, 1H), 4.32 ppm (s, 2H); 13C NMR
(75 MHz, [D6]acetone): d = 206.9, 206.9, 206.9, 166.7, 160.9, 154.0, 153.9,
146.3, 141.7, 130.9, 130.8, 128.1, 127.0, 126.0, 122.4, 115.9, 115.0, 114.0,
113.9, 112.3, 104.0, 44.7 ppm; EI-MS: m/z: 423 [M+]; HRMS: calcd for
C22H14ClNO6 424.0587; found 424.0572.
the fraction corresponding to the main peak (tR
= 10.8 min; A/B =
80:20 to 50:50 in 30 min), and lyophilization gave dendrimer 23 (3.2 mg,
0.84 mmol, 60%). MS (ES+): calcd for C117H192N34O41S2: 2793.34, found:
2793.75.
[{(NH2-Ser)2DapLeu}2DapAsp]2DapCysHisNH2 (17): The dendrimer syn-
thesis was carried out by means of standard Fmoc SPPS. In each step, the
resin was acylated with 3 equivalents of the suitably protected Fmoc-
amino acid or Fmoc-protected (S)-2,3-diaminopropanoic acid in the pres-
ence of 3 equiv of BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phos-
phonium hexafluorophosphate) and 5 equiv of DIEA (N,N-diisopropyl-
ethylamine). After reaction for 30 min, the resin was successively washed
with DMF, MeOH, and CH2Cl2 (2ꢁ with each solvent) and then checked
for free amino groups by carrying out a TNBS (2,4,6-trinitrobenzenesul-
fonic acid) test. The Fmoc protecting group was removed by treating the
resin with a 20% solution of piperidine in DMF for two periods of
10 min, and then the resin was separated from the solution by filtration.
The resin was again washed with DMF, MeOH, and CH2Cl2 (2ꢁ with
each). At the end of the synthesis, the resin was dried and the product
was cleaved using trifluoroacetic acid/1,2-ethanedithiol/water/triisopro-
pylsilane (94:2.5:2.5:1) for 4 h. The peptide was precipitated with methyl
tert-butyl ether and then dissolved in a water/acetonitrile mixture. Start-
ing with 50 mg of NovaSyn TGR resin (0.25 mmolgÀ1), dendrimer 17 was
obtained as a colorless foamy solid after purification by preparative
HPLC (16.2 mg, 13%). MS (ES+): calcd for C86H151N33O35S: 2238.08,
found: 2238.50.
[{(CHO-CO)2DapLeu}2DapAsp]2DapCys(S(O)S(CH2)2Colch)HisNH2
(24): Dendrimer 23 (3.2 mg, 0.84 mmol) was dissolved in H2O (0.2 mL)
and NaIO4 (9.2 mg, 43 mmol, 51 equiv) was added. The solution was kept
at 258C for 2 h under nitrogen. Purification by semipreparative RP-
HPLC and isolation of the fraction corresponding to the main peak (tR
=
23.6 min; A/B = 80:20 to 50:50 in 30 min) gave dendrimer 24 (1.2 mg,
0.47 mmol, 56%), which was used directly for the next step.
[{(d-GalNAc-a-ONCH-CO)2DapLeu}2DapAsp]2DapCys(S(O)S-
(CH2)2Colch)HisNH2 (25): Dendrimer 24 (1.2 mg, 0.40 mmol) was dis-
solved in H2O (0.15 mL) containing 2 drops of AcOH, and a-d-N-acetyl-
galactosyloxyamine (3.5 mg, 14.8 mmol, 4.6 equiv/aldehyde function) was
added. The solution was kept at 258C overnight under nitrogen. Purifica-
tion by semipreparative RP-HPLC, isolation of the fraction correspond-
ing to the main peak (tR = 20.8 min; gradient: A/B = 80:20 to 50:50 in
30 min), and lyophilization gave dendrimer 25 (0.4 mg, 0.09 mmol, 23%).
MS (ES+): calcd for C165H248N42O82S2: 4193.60, found: 4196.00.
[{(Lactyl-NH-Ser)2DapLeu}2DapAsp]2DapCysHisNH2 (26): Dendrimer
17 (4.5 mg, 1.38 mmol) was dissolved in MeOH (1 mL) containing 3 drops
of AcOH. Lactose (12.2 mg, 33.9 mmol, 25 equiv) and NaBH3CN (1.9 mg,
30.2 mmol, 22 equiv) were added and the mixture was stirred overnight at
708C. The methanol was then evaporated and the residue was purified by
semipreparative RP-HPLC. Collection of the fraction corresponding to
the main peak (tR = 26.0 min; gradient: A/B = 100:0 to 70:30 in 30 min)
[{(NH2-Ser)2DapLeu}2DapAsp]2DapCys(S-Colch)HisNH2 (18): Dendri-
mer 17 (7.8 mg, 3.32 mmol) was dissolved in an oxygen-free phosphate
buffer solution (0.3 mL, pH 8, 0.1 mm) and a solution of chloroacetylcol-
chicine 4 (1.4 mg, 3.23 mmol, 1 equiv) in degassed MeOH (50 mL) was
added. The mixture was stirred overnight under nitrogen. Subsequent pu-
3948
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 3941 – 3950

Glycopeptide Dendrimer Conjugates
FULL PAPER
and lyophilization gave 26 (3.0 mg, 0.60 mmol, 44%) as a white solid. MS
(ES+): calcd for C182H327N33O115S: 4847.05, found: 4848.13.
gradient: A/B = 80:20 to 60:40 in 20 min) gave 33 (0.7 mg, 0.11 mmol,
20%). MS (ES+): calcd for C254H392N58O125S: 6286.58, found: 6290.75.
[{(Lactyl-NH-Ser)2DapLeu}2DapAsp]2DapCys(SS(CH2)2Colch)HisNH2
(27): Dendrimer 26 (2.4 mg, 0.48 mmol) was dissolved in degassed
MeOH/CH3CN (1:1, 0.2 mL) and dithiopyridylpropionylcolchicine
(2.6 mg, 4.69 mmol, 10 equiv) was added. The solution was kept at 258C
for 2 h under nitrogen. Purification by semipreparative RP-HPLC, collec-
tion of the fraction corresponding to the main peak (tR = 16.0 min; gra-
dient: A/B = 80:20 to 50:50 in 30 min), and lyophilization gave dendri-
mer 27 (1.5 mg, 0.28 mmol, 58%). MS (ES+): calcd for C205H352N34O121S2:
5290.19, found: 5291.63.
[(HO2C(CH2)3CO-His)2DipPhe]2DipCys(S-Colch)IleNH2 (34): A sample
of the purified peptide dendrimer (3.6 mg, 1.54 mmol) was dissolved in an
oxygen-free phosphate buffer solution (0.3 mL, pH 8, 0.1 mm) and a solu-
tion of chloroacetylcolchicine 4 (1.0 mg, 2.3 mmol, 1.5 equiv) in degassed
MeOH (50 mL) was added. The mixture was stirred overnight under ni-
5
trogen. Subsequent purification by semipreparative RP-HPLC (tR
=
18.0 min; A/B = 70:30 to 50:50 in 20 min) gave 34 (2.5 mg, 0.90 mmol,
58%). MS (ES+): calcd for C108H142N24O32S: 2318.99, found: 2319.50.
[(HO2C(CH2)3CO-His)2DipPhe]2DipCys(S-Colch)PheNH2
(35):
A
[(d-Ac3GalNAc-a-O(CH2)3CO-Leu)2DapHis]2DapCysAspNH2
(28):
sample of the purified peptide dendrimer (6.1 mg, 2.53 mmol) was dis-
solved in an oxygen-free phosphate buffer solution (0.6 mL, pH 8,
0.1 mm) and a solution of chloroacetylcolchicine 4 (1.0 mg, 2.3 mmol,
0.9 equiv) in degassed MeOH (50 mL) was added. The mixture was stir-
red overnight under nitrogen. Subsequent purification by semipreparative
RP-HPLC (tR = 37.6 min; A/B = 90:10 to 40:60 in 50 min) gave 35
(2.1 mg, 0.75 mmol, 30%). MS (ES+): calcd for C111H140N24O32S: 2352.98,
found: 2354.00.
Starting with 100 mg of NovaSyn TGR resin (0.25 mmolgÀ1), the den-
drimer was prepared by standard Fmoc-SPPS (see the preparation of 17).
For the final coupling step, the resin was treated with 3 equiv of 14 for
5 h. Cleavage from the resin, precipitation, and purification by prepara-
tive HPLC gave, after lyophilization of the fraction corresponding to the
main peak (tR = 6.8 min; gradient: A/B = 40:60 to 10:90 in 30 min), gly-
codendrimer 28 (13.4 mg, 19%) as a colorless foamy solid. MS (ES+):
calcd for C124H189N23O53S: 2880.25, found: 2881.25.
[(Lys)2DapLeu]2DapCys(S-Colch)LeuNH2 (36): A sample of the purified
peptide dendrimer (4.3 mg, 2.53 mmol) was dissolved in an oxygen-free
phosphate buffer solution (0.3 mL, pH 8, 0.1 mm) and a solution of chlo-
roacetylcolchicine 4 (1.0 mg, 2.3 mmol, 0.9 equiv) in degassed MeOH
(50 mL) was added. The mixture was stirred overnight under nitrogen.
Subsequent purification by semipreparative RP-HPLC (tR = 30.0 min;
A/B = 100:0 to 50:50 in 50 min) gave 36 (2.9 mg, 1.39 mmol, 55%). MS
(ES+): calcd for C76H130N20O17S: 1626.96, found: 1628.00.
[(d-Ac3GalNAc-a-O(CH2)3CO-Leu)2-DapHis]2DapCys(SS(CH2)2Colch)-
AspNH2 (29): Dendrimer 28 (5.0 mg, 1.67 mmol) was dissolved in de-
gassed MeOH/CH3CN (1:1; 0.2 mL) and dithiopyridylpropionylcolchicine
5 (9.2 mg, 16.6 mmol, 10 equiv) was added. The solution was kept at 258C
for 2 h under nitrogen and purified by semipreparative RP-HPLC. Col-
lection of the fraction corresponding to the main peak (tR = 22.8 min;
gradient: A/B = 50:50 to 20:80 in 30 min) and lyophilization gave den-
drimer 29 (1.7 mg, 0.49 mmol, 30%). MS (ES+): calcd for
C147H214N24O59S2: 3323.39, found: 3324.25.
[(Pro)2DipAsp]2DipCys(S-Colch)AspNH2 (37): A sample of the purified
peptide dendrimer (4.3 mg, 2.53 mmol) was dissolved in an oxygen-free
phosphate buffer solution (0.3 mL, pH 8, 0.1 mm) and a solution of chlo-
roacetylcolchicine 4 (1.0 mg, 2.3 mmol, 0.9 equiv) in degassed MeOH
(50 mL) was added. The mixture was stirred overnight under nitrogen.
Subsequent purification by semipreparative RP-HPLC (tR = 15.4 min;
A/B = 80:20 to 40:60 in 40 min) gave 37 (2.5 mg, 1.19 mmol, 47%). MS
(ES+): calcd for C72H104N16O26S: 1640.70, found: 1641.50.
[(d-GalNAc-a-O(CH2)3CO-Leu)2-DapHis]2DapCys(SS(CH2)2Colch)AspNH2
(30): A mixture of MeOH/H2O/NH4OH (100 mL, 8:1:1) was added to
dendrimer 29 (0.9 mg, 0.25 mmol). The solution was stirred overnight at
258C. Subsequent evaporation of the solvent and semipreparative HPLC
purification gave, after lyophilization of the fraction corresponding to the
main peak (tR = 8.2 min; gradient: A/B = 60:40 to 30:70 in 30 min),
dendrimer 30 (0.6 mg, 0.20 mmol, 79%) as a yellow solid. MS (ES+):
calcd for C123H190N24O47S2: 2819.27, found: 2819.63.
[(AcSer)2DipPhe][(AcHis)(AcAsp)DipLeu]DipCys(S-Colch)HisNH2
(38): A sample of the purified peptide dendrimer[9c] (4.1 mg, 2.53 mmol)
was dissolved in an oxygen-free phosphate buffer solution (0.3 mL, pH 8,
0.1 mm) and a solution of chloroacetylcolchicine 4 (1.0 mg, 2.3 mmol,
0.9 equiv) in degassed MeOH (50 mL) was added. The mixture was stir-
red overnight under nitrogen. Subsequent purification by semipreparative
RP-HPLC (tR = 32.6 min; A/B = 90:10 to 40:60 in 50 min) gave 38
(1.8 mg, 0.73 mmol, 30%). MS (ES+): calcd for C85H118N20O28S: 1898.81,
found: 1899.25.
[{(d-GalNAc-a-O(CH2)3COSerThr)2DapGlyGlu}2DapLeuHis]2Dap-
CysAspNH2 (31): Starting with 150 mg of NovaSyn TGR resin
(0.25 mmolgÀ1), the dendrimer was prepared by standard Fmoc-SPPS
(see the preparation of 17). For the final coupling step, the resin was
treated with 3 equiv of 14 for 5 h. The resin was dried and the cleavage
was carried out using CF3CO2H/1,2-ethanedithiol/H2O/triisopropylsilane
(94:2.5:2.5:1) for 4 h. The peptide was precipitated with methyl tert-butyl
ether and then dissolved in a water/acetonitrile mixture. The dendrimer
was directly deacetylated with MeOH/H2O/NH4OH (2 mL, 8:1:1). The
solution was stirred overnight at 258C and then the methanol was evapo-
rated in vacuo. Purification by preparative HPLC gave, after lyophiliza-
tion of the fraction corresponding to the main peak (tR = 23.4 min; gra-
dient: A/B = 100:0 to 70:30 in 30 min), glycodendrimer 31 (14.6 mg,
7%) as a colorless foamy solid. MS (ES+): calcd for C232H379N57O119S:
5899.51, found: 5901.63.
Cell culture experiments: HeLa cervix carcinoma cells, mouse embryonic
fibroblasts (MEFs), and human lung cancer H1299 cells were grown at
378C in DMEM (Dulbeccoꢃs modified Eagle medium), 10% heat-inacti-
vated foetal calf serum, and 1% (v/v) penicillin/streptomycin (all from
Life Technologies, Grand Island, NY). All cells were grown in a humidi-
fied incubator in an atmosphere containing 5% CO2. One day prior to
the addition of colchicine or glycopeptide dendrimer conjugates, equal
numbers of cells were seeded on 6-well plates. After three days of incu-
bation, cells were trypsinized and the cell number was determined in du-
plicate by counting in a Neubauer chamber. H1299 cells were transiently
transfected with 50 ng of GFP-tubulin[31] using Rotifect (Roth) according
to the instructions of the manufacturer, and were analyzed by fluores-
cence microscopy.
[{(d-GalNAc-a-O(CH2)3COSerThr)2DapGlyGlu}2DapLeuHis]2DapCys-
(SS(CH2)2Colch)-AspNH2 (32): Dendrimer 31 (4.2 mg, 0.69 mmol) was
dissolved in degassed MeOH/CH3CN (1:1, 0.2 mL) and dithiopyridylpro-
pionylcolchicine 5 (3.6 mg, 6.5 mmol, 10 equiv) was added. The solution
was stirred at 258C for 2 h under nitrogen and purified by semiprepara-
tive RP-HPLC. Collection of the fraction corresponding to the main
peak (tR = 24.0 min; gradient: A/B = 100:0 to 30:70 in 30 min) and lyo-
philization gave dendrimer 32 (0.6 mg, 0.09 mmol, 13%). MS (ES+):
calcd for C255H404N58O125S2: 6342.65, found: 6345.00.
Acknowledgements
[{(d-GalNAc-a-O(CH2)3COSerThr)2DapGlyGlu}2DapLeuHis]2Dap-Cy-
s(SCH2CONH-Fluorescein)AspNH2 (33): Dendrimer 31 (3.4 mg,
0.57 mmol) was dissolved in an oxygen-free phosphate buffer solution
(0.1 mL, pH 8, 0.1 mm) and a solution of 16 (0.7 mg, 1.65 mmol, 2.9 equiv)
in degassed MeOH (40 mL) was added. The mixture was stirred overnight
under nitrogen. Subsequent purification by semipreparative RP-HPLC,
collection of the fraction corresponding to the main peak (tR = 12.0 min;
This work was supported by the University of Berne and the Swiss Na-
tional Science Foundation.
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Received: December 16, 2004
Published online: April 29, 2005
3950
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 3941 – 3950