Glycopeptide dendrimer colchicine conjugates targeting cancer cells

Article abstract of DOI:10.1016/j.bmc.2010.04.026

Screening of a 65,536-member one-bead-one-compound (OBOC) combinatorial library of glycopeptide dendrimers of structure ((βGal)_{n +1}X⁸X⁷X⁶X⁵) ₂DapX⁴X³X²X¹(β-Gal) _m (βGal = β-galactosyl-thiopropionic acid, X^8-1= variable amino acids, Dap = l-2,3-diaminopropionic acid, n, m = 0, or 1 if X⁸= Lys resp. X¹= Lys) for binding of Jurkat cells to the library beads in cell culture, resynthesis and testing lead to the identification of dendrimer J1 (βGal-Gly-Arg-His-Ala)₂Dap-Thr- Arg-His-Asp-CysNH₂ and related analogues as delivery vehicles. Cell targeting is evidenced by FACS with fluorescein conjugates such as J1F. The colchicine conjugate J1C is cytotoxic with LD₅₀ = 1.5 μM. The β-galactoside groups are necessary for activity, as evidenced by the absence of cell-binding and cytotoxicity in the non-galactosylated, acetylated analogue AcJ1F and AcJ1C, respectively. The pentagalactosylated dendrimer J4 βGal₄(Lys-Arg-His-Leu)₂Dap-Thr-Tyr-His-Lys(βGal) -Cys) selectively labels Jurkat cell as the fluorescein derivative J4F, but its colchicine conjugate J4C lacks cytotoxicity. Tubulin binding assays show that the colchicine dendrimer conjugates do not bind to tubulin, implying intracellular degradation of the dendrimers releasing the active drug.

Full text of DOI:10.1016/j.bmc.2010.04.026

Bioorganic & Medicinal Chemistry 18 (2010) 6589–6597
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Glycopeptide dendrimer colchicine conjugates targeting cancer cells
Emma M. V. Johansson a, Joëlle Dubois b, Tamis Darbre a, Jean-Louis Reymond a,
*
a Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
b ICSN-CNRS, avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Screening of a 65,536-member one-bead-one-compound (OBOC) combinatorial library of glycopeptide
dendrimers of structure ((bGal)n + 1X8X7X6X5)2DapX4X3X2X1(b-Gal)m (bGal = b-galactosyl-thiopropionic
Received 3 November 2009
Revised 15 March 2010
Accepted 7 April 2010
Available online 11 April 2010
acid, X8–1 = variable amino acids, Dap = -2,3-diaminopropionic acid, n, m = 0, or 1 if X8 = Lys resp.
L
X
1 = Lys) for binding of Jurkat cells to the library beads in cell culture, resynthesis and testing lead to
the identification of dendrimer J1 (bGal-Gly-Arg-His-Ala)2Dap-Thr-Arg-His-Asp-CysNH2 and related ana-
logues as delivery vehicles. Cell targeting is evidenced by FACS with fluorescein conjugates such as J1F.
The colchicine conjugate J1C is cytotoxic with LD50 = 1.5 lM. The b-galactoside groups are necessary for
Keywords:
Dendrimers
activity, as evidenced by the absence of cell-binding and cytotoxicity in the non-galactosylated, acety-
lated analogue AcJ1F and AcJ1C, respectively. The pentagalactosylated dendrimer J4 bGal4(Lys-Arg-His-
Leu)2Dap-Thr-Tyr-His-Lys(bGal)-Cys) selectively labels Jurkat cell as the fluorescein derivative J4F, but
its colchicine conjugate J4C lacks cytotoxicity. Tubulin binding assays show that the colchicine dendrimer
conjugates do not bind to tubulin, implying intracellular degradation of the dendrimers releasing the
active drug.
Peptides
Drug delivery
Colchicine
Combinatorial chemistry
On-bead screening
Cytotoxicity
Targeting
N
HN
HN
O
O
NH
NH
O
O
O
HN
O
HN
HN
NH
NH
N
NH
O
HN
2N
OMe
HO
O
O
O
NH
NH
HN
HN
NH
H
HN
S
O
O
HN
NH2
HO
NH
NH
O
OH
HN
O
MeO
MeO
HO
NH2
NH
OH
HN
O
O
N
OH
O
O
O
NH
S
HN
MeO
S
O
OH
O
H2
N
HO
HO
OH
J1C (LD50 = 1.50 μM)
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
dendrimer or polymer carrier is typically loaded with multiple
copies of the drug,4 and may carry a targeting groups such as folic
acid,5 biotin,6 or integrin ligands.7 Ligand-based targeting is also
Dendrimers are tree-like synthetic macromolecules with various
applications in technology and medicine.1 One of the frequently
envisioned applications for dendrimers is targeted drug delivery of
cytotoxic drugs in cancer therapy, with the aim of increasing the
therapeutic ratio by avoiding unwanted toxicity in non-target
tissues.2 Targeting to cancer cells can be obtained by using macro-
molecular carriers with MW >40 kDa due to the enhanced perme-
ation and retention (EPR) effect.3 In such applications a PEGylated
used in non-dendritic delivery devices, for example based on anti-
9
bodies8 or vitamin B12
.
We have recently developed the chemistry of peptide dendri-
mers10 and glycopeptide dendrimers11 as artificial protein mod-
els.12 Peptide dendrimers resemble the molten globule state of
proteins,13 and can display various functions such as enzyme-like
catalysis,14 metal and cofactor binding,15 and inhibition of bacterial
biofilms mediated by multivalent lectin binding.16,17 We have also
started to probe the potential of glycopeptide dendrimers as drug
delivery agents by focusing on colchicine.18,19 Colchicine is a
relatively inexpensive natural product too toxic for use in cancer
* Corresponding author. Fax: +41 31 631 80 57.
E-mail address: jean-louis.reymond@ioc.unibe.ch (J.-L. Reymond).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.026

6590
E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
therapy and which might benefit from combination with a target-
ing device, or a prodrug strategy as shown for close analogues.20
Initial experiments with glycopeptide dendrimer conjugates of col-
chicine18 showed that they can be cytotoxic to cancer cells by dis-
rupting cell division by the tubulin binding mechanism of
2. Results and discussion
2.1. Combinatorial design and selection
A 65,536-membered combinatorial library was prepared fol-
lowing our previously reported combinatorial design allowing
decoding by amino acid analysis,26 featuring a first generation den-
drimer with three tetrapeptide arms separated by a 2,3-diamino-
propionic acid branching unit. We used four variable amino acids
at each of the eight variable positions X1–X8 and attached a galact-
ose residue by standard amide bond coupling of the known27 b-thi-
ogalactosyl propionic acid 1 at the N-terminus (Scheme 1). A side-
chain alloc protected lysine and a bis-Fmoc protected lysine were
placed as one of the four variable amino acids at position X1 and
X8, respectively, resulting in dendrimers displaying galactose resi-
dues in copy number two (no lysine at either X1 or X8, 56.25% of
the library), three (lysine at X1 but not at X8, 18.75% of the library),
four (lysine at X8 but not at X1, 18.75% of the library), or five (lysine
at both X1 and X8, 6.25% of the library). We have successfully used
this first generation dendrimer design with variable multivalency
previously to identify multivalent dendrimers binding to a fu-
cose-specific lectin.28 The relatively long tetrapeptide arms are
colchicine,21 however with only weak activities (LD50 >5
lM). In
our efforts to optimize the system, we now report a combinatorial
study of glycopeptide dendrimers carrying two to five copies of a
b-galactoside as targeting device and a colchicine molecule at their
core. On-bead screening for binding to Jurkat cells lead to identifi-
cation of dendrimer J1 as delivery device for colchicine as a cys-
teine-thioether conjugate J1C (Fig. 1). The corresponding
fluorescein conjugate J1F shows strong binding to these cells. The
cytotoxicity of the colchicine conjugate J1C is presumably medi-
ated by binding to galactose specific receptors such as galec-
tins,22,23 internalization, and degradation to release the active
drug, as for other glycosylated delivery devices based on polymeric
N-acetyl-galactosamine24 and galactosylated25 ligands.
Galactosyl-peptide drug
conjugate
A
b
b
cb
cb
X21X1
X22X1
X23X1
X24X1
X1
X1
X1
X1
1
2
3
4
a
H2N
a
cb
cb
b
Tentagel
(0.47 mmol/g)
1) uptake
b
O
S
AcO
AcO
CO2H
2) degradation
OAc
OH
OAc
colchicine
3) inhibition of tubulin
polymerization
1
HO
OH
OH
O
d-f
X8
X7
X6
X5
O
S
HO
HO
n
O
Dap
X4
HO
S
O
X5
X6
X3
OH
X7
O
X8
OH
HO
B
NH2
HN
X2
n
X1
S
OH
OH
HN
S
O
HO
O
HN
HN
O
O
OH
HO
N
NH
O
HO
OH
O
m
HN
NH2
NH2
S
HN
Library βGalL (n = 1 or 2, m = 0 or 1)
O
HN
O
NHO
HN
X8 X7 X6 X5 X4 X3 X2 X1
Lys* Asp Ser Thr Ser Arg Phe Lys**
Phe Asn His Ala Asn Gly Glu Asp
Gly Arg Ile Leu Thr Tyr His Val
Tyr Val Glu Pro Pro Ile Leu Ala
HN
NH
HN
O
N
O
O
O
NH
NH
HN
NH
OH
O
O
OMe
HN
O
O
HN
Scheme 1. Synthesis and structure of the library bGalL. The solid support is
tentagel and the branching unit is -2,3-diaminopropanoic acid (Dap). Lys* = alloc
protected -lysine, Lys** = bis-Fmoc protected -lysine. Reagents and conditions: (a)
NH
2 MeO
OMe
L
O
NH
MeO
L
L
HN
suspend the whole resin batch in NMP/DCM (2:1, v/v), mix via nitrogen bubbling for
15 min, and split the batch in four equal portions 1–4; (b) in each portion Xab (a = 1–
8; b = 1–4): 3.0 equiv Fmoc–Xab–OH, 3.0 equiv PyBOP, 6.0 equiv DIEA, DCM/
NMP(1:1, v/v), 1.5 h; (c) DMF/piperidine (4: 1, v/v), 2 ꢀ 10 min. Steps a–c are
repeated nine times, the fifth coupling being with only Dap; (d) Alloc/Fmoc
removal: 25 equiv PhSiH3, 0.2 equiv Pd(PPh3)4, DCM 2 ꢀ 20 min under nitrogen
followed by washing with 10 mL DCM, dioxane/H2O (9:1) DMF and DCM, then
DMF/piperidine (4:1, v/v), 2 ꢀ 10 min; (e) 5 equiv 1, 5 equiv DIC, 5 equiv HOBt,
DCM/NMP(1:1, v/v), 24 h, or for the control acetylated library: Ac2O/CH2Cl2 1:1,
30 min; (f) TFA/TIS/H2O (95:2.5:2.5), 4 h, then MeOH/NH4/H2O (v/v 8:1:1), 24 h.
NH
O
N
HO
O
O
OH
NH
H
N
HN
S
OH
OH
O
J1C (LD50 = 1.50 μM)
Figure 1. (A) Principle of degradable drug delivery device. (B) Structural formula of
glycopeptide dendrimer colchicine conjugate J1C.

E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
6591
A
B
+
Peptide library
Jurkat cells
C
Figure 2. On-bead assay for Jurkat cells binding to peptide dendrimers. (A) Schematic representation of the solid-phase test using Jurkat cells. (B) Images captured using a
microscope of library bGalL. Cells are seen as granulation on the surface of some of the beads (red arrow). (C) Picture of the acetylated library AcL. No cells are seen at the bead
surface.
thought to provide a conformationally flexible spacer displaying
various functional groups as the amino acid side-chains that
should be favorable for both carbohydrate multivalency and sec-
ondary binding interactions.17
acid analysis for sequence determination.30 Analysis of the
sequences showed a strong preference for Asp/Asn at position X7
(Table 1). On the other hand, the hits contained cationic, anionic,
and neutral sequences, as well as sequences displaying two, three,
four and five galactose residues.
The dendrimer library was assembled on a one gram batch of
non-cleavage tentagel (TG) resin (0.47 mmol/g, 90 lm U beads).
After coupling the last four amino acids at position X8, the library
was pooled and treated with Pd(PPh3)4/PhSiH3 to remove the alloc
protecting group of lysine residues at position X1 and with piperi-
dine to effect removal of the Fmoc protecting groups at the N-ter-
mini. The resin was then split into two portions and the free N-
termini were either coupled with the acetylated b-thiogalactosyl
propionic acid building block 1 or acetylated. Side-chain deprotec-
tion under acidic conditions and methanolysis of the galactosyl
acetate ester finally gave the libraries bGalL (Scheme 1) and the
control acetylated library AcL for testing.
The cell-binding potential of the dendrimers was assessed by an
on-bead assay consisting in simply adding the polymer beads to a
culture medium containing Jurkat cells. Jurkat cells were selected
as standard cancer cells because they grow as suspension and are
known to express galectins. Under optimized conditions, binding
of Jurkat cells to a few percent of the polymer beads of library
bGalL was visible under a light microscope after 24 h incubation
in the cell culture (Fig. 2). This procedure is similar to other re-
ported cell-binding assays with OBOC combinatorial libraries.29
Interestingly, the Jurkat cells did not bind at all to the beads of
the control library AcL, the empty beads, or a related focusylated
combinatorial dendrimer library16 under the same conditions,
showing that the presence of a galactose residue was essential
for cell-binding. The cell-decorated beads detected with the assay
of library bGalL were manually picked with a pipette, washed with
aqueous phosphate buffer and methanol to remove cell debris,
transferred to individual analysis tubes and subjected to amino
2.2. Synthesis of selected dendrimers and their colchicine and
fluorescein conjugates
Sequences J1–J10 displaying all valency possibilities and a
diversity of amino acid sequences were selected for resynthesis.
Each dendrimer was prepared by solid-phase peptide synthesis
on rink-amide resin incorporating a cysteine residue as an addi-
tional residue at the first position. The dendrimers were then con-
verted to the colchicine and fluorescein conjugates by coupling the
cysteine side-chain with chloroacetyl-colchicine (4)31 and iodoace-
tyl-fluorescein (5), respectively, as described earlier.18 An acety-
lated version of J1 was also prepared as a control. All products
were obtained in sufficient yields and purities for biological studies
after purification by RP-HPLC (Table 2). The chloroacetyl-colchicine
(4) was synthesised by a modification of a reported procedure as
shown in Scheme 2.31 Direct deacetylation of colchicine results in
isomerisation of the carbonyl and methoxy group in the adjacent
unsaturated cycloheptane. Therefore, the acetyl group is activated
by addition of a Boc-protecting group followed by stepwise re-
moval of the acetyl group (to 2) and Boc-protecting group to give
the free amine derivative (3). In the final step, the free amine deriv-
ative is reacted with chloroacetic anhydride in dichloromethane to
give chloroacetyl-colchicine 4 in an overall yield of 34% from col-
chicine. In the published Boc protection step31 the yield was 65%
and around 25% of the starting material was recovered. We found
that changing the solvent from dichloromethane to THF and having
a constant temperature of 45 °C improved the yield to 90%.

6592
E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
Table 1
icantly higher with galactosylated ligands compared to non-
galactosylated ligands, with cells incubated with the pentavalent
dendrimer J4F showing the strongest fluorescence intensity. Selec-
tive staining by the galactosylated dendrimers over fluorescein
was also evident under the fluorescent microscope (Fig. 4).
Amino acid sequences of peptide dendrimers binding to Jurkat cells selected for
resynthesis
No.a
nGalb
X8
X7
X6
X5
X4
X3
X2
X1
J1
J2
J3
J4
J5
J6
J7
J8
J9
J10
2
2
2
5
4
4
3
3
4
4
Gly
Phe
Phe
Lys
Lys
Lys
Gly
Gly
Lys
Lys
Arg
Asp
Asn
Arg
Asp
Asn
Asp
Asn
Asp
Asn
His
Ser
Ser
His
Glu
Glu
Glu
Glu
Glu
Glu
Ala
Ala
Ala
Leu
Pro
Pro
Thr
Thr
Pro
Pro
Thr
Thr
Thr
Thr
Thr
Thr
Asn
Asn
Pro
Pro
Arg
Tyr
Tyr
Tyr
Gly
Gly
Arg
Arg
Ile
His
Leu
Leu
His
Phe
Phe
Phe
Phe
Glu
Glu
Asp
Asp
Asp
Lys
Asp
Asp
Lys
Lys
Val
Val
2.4. Cytotoxicity studies
Having ascertained that the glycopeptide dendrimers selected
in the on-bead cell-binding assay bound to Jurkat cells as the fluo-
rescein conjugates, we next investigated the cytotoxicity of the
corresponding colchicine–dendrimer conjugates. The colchicine
Ile
dendrimer conjugates were added at 5 lM final concentration to
a
Dendrimers J2/J3, J5/J6, J7/J8 and J9/J10 represent the Asp and Asn version at
an overnight culture of cells that had a starting concentration of
50,000 cells per mL. After incubation for 48 h, the cells were
stained with propidium iodide (PI) and healthy and dead cells were
counted by FACS. LD50 values were subsequently determined for all
dendrimers showing significant cytotoxicity (Fig. 5 and Table 3).
Strikingly, only the colchicine dendrimer conjugates displaying
two galactose residues, namely J1C, J2C, and J3C, showed measur-
able cytotoxicity. The acetylated control dendrimer AcJ1C was not
cytotoxic, in agreement with the weak cell-binding of the fluores-
cein conjugate AcJ1F (Fig. 3) and suggesting that cell internaliza-
tion of the galactosylated dendrimers was dependent on
galactose recognition and not caused by passive diffusion as is
the case for colchicine itself. More surprisingly, the dendrimers dis-
playing three, four or five galactose residues did not show measur-
able cytotoxicity. This is surprising considered that the
corresponding fluorescein-labeled dendrimers showed strong
binding to Jurkat cells (Fig. 3). It should be noted that J2C bearing
an aspartate residue at X7 was threefold more active than its aspar-
agine analogue J3C, in line with the stronger labeling of Jurkat cells
position X7, which were both possible sequences from the amino acid analysis
results.
b
nGal = number of b-thiogalactosyl-propionyl groups on the dendrimers.
2.3. Binding of peptide dendrimers to Jurkat cells
The binding to Jurkat cells was investigated by fluorescence
activated cell sorting (FACS) using fluorescein and its dendrimer
conjugates J1F, AcJ1F, J2F, J3F, and J4F, displaying between zero
and four b-thiogalactoside residues. The cells were plated at a con-
centration of 50,000 cells per mL and grown overnight in order to
produce a healthy growing population, treated with the various
ligands to a final concentration of 5 lM, and incubated for further
two days before analysis. The results from these experiments are
shown in Figure 3 as single parameter histograms. Co-staining
with propidium iodide (PI) showed no increase in dead cells upon
addition of ligands, implying that the fluorescein–dendrimer con-
jugates were non-cytotoxic. The fluorescence intensity was signif-
Table 2
Synthesis of peptide dendrimers and conjugates
No.
Sequencea
Yieldb mg, %
MS calcd
MSc obsd
J1
(bGalGRHA)2DapTRHDCNH2
15 mg, 12%
1.8 mg, 46%
0.9 mg, 38%
35 mg, 36%
2.3 mg, 53%
2.2 mg, 42%
5 mg, 4%
0.5 mg, 42%
1.1 mg, 46%
6 mg, 5%
0.2 mg, 17%
0.8 mg, 34%
11 mg, 6%
1.6 mg, 31%
0.7 mg, 13%
13.1 mg, 9%
2.0 mg, 56%
25 mg, 16%
2.8 mg, 84%
11 mg, 8%
2058.86
2457.01
2446.93
1642.78
2040.93
2030.85
2040.7
2962.2
2426.8
2038.77
2964.2
2424.5
3056.3
3453.43
3443.36
2565.7
2963.2
2587.7
2961.2
2306.4
2703.9
2305.5
2701.9
2583.8
2981.3
2582.9
2978.3
2059.0
2457.0
2446.0
1642.9
2040.0
2030.0
2039.2
n.d.
J1C
J1F
AcJ1
AcJ1C
AcJ1F
J2
J2C
J2F
J3
J3C
J3F
J4
J4C
J4F
J5
J5C
J6
J6C
J7
J7C
J8
J8C
J9
(bGalGRHA)2DapTRHDC(Col)NH2
(bGalGRHA)2DapTRHDC(Fluo)NH2
(AcGRHA)2DapTRHDCNH2
(AcGRHA)2DapTRHDC(Col)NH2
(AcGRHA)2DapTRHDC(Fluo)NH2
(bGalFDSA)2DapTYLDCNH2
(bGalFDSA)2DapTYLDC(Col)NH2
(bGalFDSA)2DapTYLDC(Fluo)NH2
(bGalFNSA)2DapTYLDCNH2
(bGalFNSA)2DapTYLDC(Col)NH2
(bGalFNSA)2DapTYLDC(Fluo)NH2
bGal4(KRHL)2DapTYHK(bGal)CNH2
bGal4(KRHL)2DapTYHK(bGal)C(Colch)NH2
bGal4(KRHL)2DapTYHK(bGal)C(Fluo)NH2
bGal4(KDEP)2DapTGFDCNH2
2427.0
2037.5
n.d.
2425.4
3056.0
3453.3
3443.4
2566.0
2965.0
2586.5
2961.0
2308.0
2708.0
2305.0
2703.0
2584.0
2982.0
2582.8
2980.0
bGal4(KDEP)2DapTGFDC(Col)NH2
bGal4(KNEP)2DapTGFDCNH2
bGal4(KNEP)2DapTGFDC(Col)NH2
(bGalGDET)2DapNRFK(bGal)CNH2
(bGalGDET)2DapNRFK(bGal)C(Col)NH2
(bGalGNET)2DapNRFK(bGal)CNH2
(bGalGNET)2DapNRFK(bGal)C(Col)NH2
bGal4(KDGP)2DapPIEVCNH2
bGal4(KDGP)2DapPIEVC(Col)NH2
bGal4(KNGP)2DapPIEVCNH2
bGal4(KNGP)2DapPIEVC(Col)NH2
1.1 mg, 34%
20 mg, 14%
1.4 mg, 40%
18.6 mg, 13%
1.9 mg, 55%
9.0 mg, 6%
0.52 mg, 23%
J9C
J10
J10C
a
Peptide sequences with one-letter code amino acids, see Scheme 1 for detailed structural formulae. bGal = b-thiogalactosyl-propionyl. K indicates
L-lysine acylated at both
amino groups at the N-terminus, Dap is the L-2,3-diaminopropanoic acid branching unit. K(bGal) indicates lysine with a b-thiogalactosyl-propionyl group acylated on the
e-amino group. C(Col) indicates cysteine alkylated at the thiol group with chloroacetyl-colchicine. C(Fluo) indicates cysteine alkylated at the thiol group with iodoacetyl-
fluorescein.
b
Isolated yields from calculated resin loading after cleavage and purification by preparative RP-HPLC.
ESI MS, see Section 4 for details.
c

E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
6593
O
Cl
dation or the formation of inactive fragments due to the particular
core residues present in these dendrimers. On the other hand, mul-
tivalent tight binding to galactose receptors inhibiting internaliza-
tion and degradation might also play a role.
MeO
MeO
MeO
MeO
NHR
O
NH
MeO
MeO
O
OMe
3. Conclusion
OMe
4
colchicine (R = Ac)
2 (R = Boc)
Although the peptide dendrimers may be considered as rela-
tively complex structures compared to other dendrimer types, it
should be stressed that they are relatively easy to prepare, not only
as combinatorial library, which is not possible with other dendri-
mer types, but also as single purified products, as illustrated by
the synthesis of 27 different dendrimers for the present study (Ta-
ble 1). Thus, glycopeptide dendrimers selected from the combina-
torial library by an on-bead cell-binding assay showed good
affinity to Jurkat cells mediated by b-thiogalactoside residues at
the branches ends, as evidenced by FACS using fluorescein-labeled
dendrimers. The presence of the b-galactoside groups was neces-
sary for efficient cell-binding. When considering the cytotoxicity
of the corresponding colchicine conjugates however, only the three
peptide dendrimers J1C, J2C, and J3C, each carrying two b-galacto-
side residues in their branches, showed activity. Dendrimers with
different sequences including higher multivalency of galactose or
non-galactosylated dendrimers were not cytotoxic. Tubulin poly-
merization inhibition assays showed no tubulin binding activity
for the complete dendrimers but weak binding for fragments.
The inactive glycopeptide dendrimers are thus probably degraded
intracellularly to release small fragments whose affinity might be
significantly weaker than colchicine itself, explaining the higher
LD50 values of the glycopeptide dendrimers conjugates compared
to free colchicine. Although the galactose-dependent cell-binding
and cytotoxicity of the dendrimer–cochicine conjugates excludes
significant proteolytic degradation prior to cell uptake, future
improvements of the system will need to address the question of
degradation by serum proteases for an in vivo situation. Tubulin
binding itself could be optimized by searching for cell-internalizing
glycopeptide dendrimer colchicine conjugates retaining strong
tubulin binding without the need for degradation, or by designing
conjugates that can release colchicine itself.
a
b
c
H
N
3 (R = H)
HO
I
O
O
CO2H
O
5
Scheme 2. Synthesis and structure of ligands for conjugation. Reagents and
conditions: (a) (i) Boc2O, Et3N, DMAP, THF, 45 °C, 24 h (95%); (ii) NaOMe, MeOH,
0 °C, 1.5 h (quant); (b) TFA: CH2Cl2 (1:10), 4 h (80%); (c) 3 equiv (ClCH2CO)2O, Et3,
DMAP, CH2Cl2, 45 °C, 2 h (45%).
with J2F than with J3F. The higher activity of J2C suggests that the
bead selected in the cell-binding assay carried the sequence of J2
with an aspartate at X7, and highlights the critical role of the amino
acid sequence in activity.
Additional cytotoxicity studies were performed with mouse
embryonic fibroblasts (MEF) cells as a model of healthy cells. FACS
analysis was not reliable with these cells due to aggregation, and
cytotoxicity was estimated instead by visual cell counting under
the microscope. The data indicated that both free colchicine and
the dendrimer conjugates exhibited a similarly slightly higher
cytotoxicity against Jurkat cells compared to MEF (Table 4), show-
ing that the galactose-dependent toxicity of the dendrimer conju-
gates did not result in a more selective toxicity.
2.5. Tubulin binding assay and mechanistic hypothesis
Considering that only the bis-galactosylated, colchicine conju-
gated dendrimers show measurable cytotoxicity, one must assume
that this activity involves a selective uptake and intracellular re-
lease of the dendrimers followed by a colchicine-type inhibition
of mitosis as previously observed with related glycopeptide den-
drimer colchicine conjugates.18 A tubulin polymerization inhibi-
tion assay was performed to test the ability of the glycopeptide
dendrimer conjugates to directly interact with tubulin. While col-
chicine itself exhibits micromolar activity in this assay
4. Experimental section
4.1. Synthesis of the split-and-mix combinatorial library bGalL
The peptide dendrimer library bGalL was prepared from a 1 g
resin batch of TentagGel HL NH2 (loading: 0.47 mmol/g) divided
equally in four reactors. In four reactors the resin was acylated
with one of the four amino acids (3 equiv) in the presence of PyBOP
(3 equiv) and N,N-diisopropylethylamine (DIEA) (5 equiv). Amino
acids were acylated for 1.5 h, and Dap was coupled in all reactors
for 2 h. After each coupling, the four resin batches were mixed to-
gether and again split into four parts, equally introduced in the
four reactors. These split-and-mix steps were repeated after each
amino acid coupling. After each coupling the resin was succes-
sively washed with N-methylpyrrolidone (NMP), MeOH, CH2Cl2
(3 times with each solvent) and subsequently checked for free ami-
no groups with the trinitrobenzene sulfonate (TNBS test). Proline
coupling was checked with the chloranile test. If the test indicated
the presence of free amino groups, the coupling was repeated. The
Fmoc protecting groups were removed with a solution of 20%
piperidine in DMF (two times 10 min) and the solvent was re-
moved by filtration. At the end of the synthesis the Fmoc protected
resin was dried and stored at -20 °C. A random selection of 25 of
beads were picked, analysed and decoded to determine the dendri-
mer sequence to confirm the distribution of amino acids in each
(IC50 = 1.6 lM), there was no detectable inhibition with dendrimer
conjugates such as J1C or J4C. However partial polymerization
inhibition was observed with the tripeptide colchicine conjugate
His-Asp-Cys(S-Colch)NH2 corresponding to the core sequence of
J1C (33% at 1 mg/ml), as well as with the chloroacetyl-colchicine
4 used to prepare the conjugates (IC50 = 17 lM). These data suggest
that the glycopeptide dendrimers are degraded after internaliza-
tion to release short peptide conjugates of colchicine capable of
tubulin binding and cytotoxicity, albeit more weakly than colchi-
cine itself. The degradation process should not be problematic if
proteases are present, as we have recently shown in a systematic
reactivity study.32 Weaker tubulin binding by dendrimer frag-
ments compared to colchicine might explain the difference in cyto-
toxicity between colchicine (LD50 = 0.02
lM) and the dendrimers
(LD50 = 1.5–5.3 M) (Table 3). A degradation mechanism for activa-
l
tion suggests that the lack of activity observed in tri-, tetra- and
pentavalent dendrimers might reflect either the absence of degra-

6594
E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
Figure 3. FACS of Jurkat cells binding to fluorescein and the fluorescein–dendrimer conjugates. One parameter distribution plots of fluorescence intensity versus cell count.
The x-axis represents emission intensity (excited at 488 nm, emission observed at 510–560 nm) and the y-axis is the number of cells. Md = median fluorescence intensity.
Fluorescence levels were negligible without added ligand (Md = 2.8) or with the non-labeled dendrimer J1 (Md = 3.0).
position as expected. Immediately before screening, the Fmoc pro-
4 h to give a library with acetylated N-termini and amine side-
tecting groups were removed and the library was capped with a b-
chain groups.
D-thiogalactoside residue 1 (prepared following the procedure of
Magnusson et al.27 5 equiv) of DIC (5 equiv) and HOBt (5 equiv)
in CH2Cl2/NMP (2:1, v/v) overnight. The side-chain protecting
groups were removed with TFA/TIS/H2O (95/2.5/2.5) for 4 h. Then
the carbohydrate was deacetylated with a solution of MeOH/
NH4/H2O (v/v 8:1:1) for 24 h, resulting in the glycopeptide dendri-
mer library on beads bGalL.
4.3. Cell culture
Jurkat cells were grown in RPMI medium with 10% (v/v) FCS, 1%
(v/v) penicillin/streptomycin, and 1% (v/v) L-glutamine. Mouse
embryonic fibroblasts (MEFs) were grown in DMEM (Dulbeccos
modified Eagle medium) with 10% (v/v) FCS, 1% (v/v) penicillin/
streptomycin, 1% (v/v) HEPES buffer, and 1% (v/v) L-glutamine. All
cells were grown in a humidified incubator at 37 °C and 5% CO2.
Passage of the cells were performed every 2–3 days to preserve
the exponential growth of the cells. The MEFs cells were split by
removing the media and then wash with sterile PBS buffer. Antiad-
herent Trypsin/EDTA solution was added and the cells were incu-
bated at 37 °C for 20 min.
4.2. Synthesis of N-acetylated control library AcL
Fmoc and Alloc protecting groups were removed and the library
was capped with Ac2O/CH2Cl2 1:1 for 30 min. The side-chain pro-
tecting groups were removed with TFA/TIS/H2O (95:2.5:2.5) for

E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
6595
Table 3
LD50 values for cytotoxic colchicine dendrimer conjugates against Jurkat cells
No.
Sequence
nGala
LD50 (lM)
—
Colchicine
0.020 0.004
1.50 0.04
—
1.53 0.06
5.52 0.15
J1C
AcJ1C
J2C
J3C
J4C
J5C
J6C
J7C
J8C
J9C
J10C
(bGalGRHA)2DapTRHDC(Col)NH2
(AcGRHA)2DapTRHDC(Col)NH2
(bGalFDSA)2DapTYLDC(Col)NH2
(bGalFNSA)2DapTYLDC(Col)NH2
bGal4(KRHL)2DapTYHK(bGal)C(Colch)NH2
bGal4(KDEP)2DapTGFDC(Col)NH2
bGal4(KNEP)2DapTGFDC(Col)NH2
(bGalGDET)2DapNRFK(bGal)C(Col)NH2
(bGalGNET)2DapNRFK(bGal)C(Col)NH2
bGal4(KDGP)2DapPIEVC(Col)NH2
bGal4(KNGP)2DapPIEVC(Col)NH2
2
0
2
2
5
5
4
3
3
4
4
b
b
—
—
—
—
—
—
—
b
b
b
b
b
b
a
Number of b-thiogalactosyl-propionyl groups in the dendrimer.
These dendrimer conjugates showed negligible cytotoxicity (2–14% dead cells)
b
at 5 lM.
4.4. Solid phase screening with Jurkat cells
The glycodendrimer library bGalL (50 mg), was washed three
times with 3 mL of PBS buffer. The cells were pelleted and resus-
pended in new RPMI medium with 10% (v/v) FCS, 1% (v/v) penicil-
lin/streptomycin and 1% (v/v) L-glutamine. The cells were added to
Figure 4. Fluorescence image of Jurkat cells stained with dendrimer J2F at 2.5 lM
the library in a sterile Petri dish and incubated at 37 °C, 5% CO2 for
24 hours, first gently shaken (60 rpm) for 4 h then static incubation
for the remaining 24 h. Resin beads with cells attached were man-
and fluorescein at 10
lM for 4 h indicating the increased affinity of the dendrimers
to the cell.
800
600
400
200
0
AcJ1C
J1C
AcJ1C
J1C
200
150
100
50
0
7.24
84.9
92.2
14.4
0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
FL2-H
10
10
FL2-H
100
J1C, LD50
1.50 0.04
=
80
60
40
20
0
1
Concentration Dendrimer (μm)
Figure 5. Cytotoxicity assays. FACS analysis of Jurkat cells treated with colchicine dendrimer conjugates at 5
l
M for 48 h followed by staining with propidium iodide. The x-
axis is the intensity of emitted light by the propidium iodide–DNA complex, measured at 580–650 nm, from excitation at 488 nm. Propidium iodide penetrates only into dead
cells and complexes the DNA, leading to a strong fluorescence only in dead cells. An LD50 curve obtained by this assay for J1C is shown as a representative example.

6596
E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
Table 4
0.189 mmol) was added drop wise and the resulting mixture was
stirred for 2 h. The reaction was quenched with ice water and or-
ganic phase was washed with brine. The organic phase was dried
over MgSO4 and then removed to give 4 as yellow oil. Preparative
RP-HPLC was used to obtain 4 as a pale yellow powder 74 mg,
47%). Analytical-HPLC: tr = 8.4 (k = 350 nm, A/B = 70:30 to 0:100
in 15 min); 1H NMR (300 MHz, CD3OD): d = 8.98 (d, J = 6.0 Hz, 1H,
NH), 7.43 (d, J = 12.0 Hz, 1H), 7.38 (s, 1H), 7.22 (d, J = 12.0 Hz,
1H), 6.75 (s, 3H), 4.51 (m, 1H), 4.09 (s, 2H), 4.01 (s, 3H), 3.90 (s,
3H), 3.88 (s, 3H), 3.60 (s, 3H), 2.70–2.60 (m, 1H), 2.50–2.25 (m,
2H), 2.08–1.95 (m, 1H); MS (ES+): calcd for C22H5ClNO6 [M+H]+:
434.1, found: 434.2.
Cytotoxicity of colchicine-linked dendrimers against Jurkat cells and MEF cells
Ma
MEF cells (%)
% Dead cells at 5
Jurkat cells (%)
l
Colchicine
J1C
J2C
85
91
91
19
57
51
68
6
J3C
a
The cells were incubated for 2 days at 37 °C in the presence of 5
or dendrimer. Live and dead cells were quantified by counting.
lM colchicine
ually picked and washed with 70% ethanol (10 1 mL), water
(10 mL), MeOH (two times), CH2Cl2 (five times), MeOH (two times),
and water (five times) in order to remove bound cells. Picked beads
were analysed by amino acid analysis to determine their sequence.
4.9. Dendrimer synthesis
Peptide syntheses were performed manually in a glass reactor
or plastic syringes (5 or 10 ml). The resin NovaSyn TGR (loading:
0.18–0.29 mmol/g) was acylated with each amino acid or diamino
acid (3 equiv) in the presence of BOP or PyBOP (3 equiv) and DIEA
(5 equiv) for 1.5 h (3 h after the first generation). After each cou-
pling, the resin was successively washed with NMP, MeOH, and
CH2Cl2 (three times with each solvent), then checked for free ami-
no groups with the TNBS test. If the TNBS test indicated the pres-
ence of free amino groups, the coupling was repeated. After each
coupling, the potential remaining free amino groups were capped
with acetic anhydride/CH2Cl2 for 10 min. The Fmoc protecting
groups were removed with a solution of 20% piperidine in DMF
(2310 min) and the solvent was removed by filtration. In the end
of the sequence the resin was capped with b-thiogalactosyl propi-
onic acid (1) (5 equiv) in the presence of DIC (5 equiv) and HOBt
(5 equiv) or DIEA (5 equiv) and HCTU (3 equiv) in NMP overnight.
The carbohydrate was deprotected with a solution of MeOH/NH3/
H2O (v/v 8:1:1) for 24 h. The resin was dried and the cleavage
was carried out with TFA/TIS/H2O (95:2.5:2.5) for 4 h. The peptide
was precipitated with methyl tert-butyl ether then dissolved in a
water/acetonitrile mixture. All dendrimers were purified by pre-
parative HPLC with detection at k = 214 nm. Eluent A contained
water and TFA (0.1%); eluent B contained acetonitrile, water, and
TFA (3/2/0.1%). The yields (mg, %) and MS data are given in Table 2.
4.5. N-((tert-Butoxy)carbonyl)-colchicine
Colchicine (0.60 g, 1.5 mmol) was dissolved in anhydrous THF.
Di(tert-butyl)pyrocarbonate
(1.5 g,
7 mmol),
triethylamine
(0.2 mL, 1.43 mmol) and DMAP (0.15 g, 1.2 mmol) was added.
The mixture was stirred at 45 °C for 24 h. The solvent was removed
under reduced pressure to give a yellow oil. Purification using col-
umn chromatography (silica gel, ethyl acetate) and concentration
of the appropriate fractions gave N-((tert-butoxy)carbonyl)-colchi-
cine (0.56 g, 95%); 1H NMR (300 MHz, CDCl3): d = 5.52 (s, 1H,), 7.26
(d, J = 10.6 Hz, 1H), 6.72 (d, J = 10.7 Hz, 1H), 6.49 (s, 1H) 5.10 (q,
J = 6.0 Hz, 1H), 3.91 (s, 1H), 3.88 (s, 1H), 3.84 (s, 1H), 3.61 (s, 1H),
2.70–2.45 (m, 3H), 2.22 (s, 3H), 1.98–1.80 (m, 1H), 1.51 (s, 9H);
MS (ES+): calcd for C27H34NO8 [M+H]+: 499.6, found: 500.2.
4.6. N-((tert-Butoxy)carbonyl)-deacetylcolchicine (2)
An excess of sodium (500 mg, 22 mmol) carefully dissolved in
methanol (5 mL) was added to compound N-((tert-butoxy)car-
bonyl)-colchicine (0.042 g, 0.084 mmol) and the solution was stir-
red for 2 h at 0 °C. The solvent was removed under reduced
pressure and the residue was re-dissolved in ethyl acetate (10 mL).
The organic phase was washed with H2O (10 mL) and brine
(10 mL), dried over MgSO4 and the solvent was removed under re-
duced pressure to give-((tert-butoxy)carbonyl)-deacetylcolchicine
(2) as a yellow oil, which was used without further purification.
4.10. Synthesis of dendrimer conjugates
Conjugation of chloroacetyl-colchicine 4 and iodoacetyl-fluo-
rescein 5 derivative to the dendrimers was performed in PB-buffer
(50 mM, pH 8.5) acetonitrile (70:30), stirring at 40 °C for 24–48 h
under nitrogen. The reaction took place in the minimum amount
of solvent required to dissolve starting materials and always with
an excess of either 4 (2–4 mg) or 5 (4 mg) compared to the dendri-
mer (1–5 mg). The final dendrimer conjugates were obtained after
semi-preparative HPLC. The yields (mg, %) and MS data are given in
Table 2.
4.7. Deacetylcolchicine (3)
The crude N-((tert-butoxy)carbonyl)-deacetylcolchicine (0.10 g,
0.22 mmol) was dissolved in anhydrous dichloromethane (10 mL)
and concentrated HCl (1 mL) was added. The reaction mixture
was stirred at 45 °C for 3 h. Toluene was added as a co-solvent
and removed under reduced pressure to give crude deacetylcolch-
icine as a dark orange oil. Preparative RP-HPLC was used to obtain
deacetylcolchicine (3) as a yellow powder (0.063 g, 80%). Analyti-
cal-HPLC: tr = 7.4 (k = 214 nm, A/B = 70:30 to 0:100 in 15 min);
1H NMR (300 MHz, CD3OD): d = 7.44 (d, J = 10.7 Hz, 1H,), 7.25 (d,
J = 10.7 Hz, 1H), 7.20 (s, 1H), 6.78 (s, 1H), 4.04 (s, 3H), 3.91 (s,
3H), 3.87 (s, 3H), 3.69 (s, 3H), 2.80–2.60 (m, 1H), 2.50–2.35 (m,
2H), 2.08–1.95 (m, 1H); MS (ES+): calcd for C20H23NO5 [M+H]+:
358.16, found: 358.4.
5. Cytotoxicity and fluorescein staining FACS experiments
One day prior to the addition of test compounds (colchicine or
fluorescein derivatives), cells (5 ꢀ 104 cells mLꢁ1) were seeded on
to six-well plates (2 mL/well). A volume of a stock solution in ster-
ile MilliQ water of the test compound (1–50 lL) was added to the
wells with a micropipette. After another two days of incubation the
medium with cells were transferred to a FACS tube. In the case of
4.8. Chloroacetyl-colchicine (4)
the cytotoxicity experiment, 50
l
L
of propidium iodide
(0.4 mg.mLꢁ1) was added 2 min before the sample was analysed.
Acquisition and analysis was performed using a FAC-Scan Flow
cytometer and the Cell Quest software. List mode data are acquired
using the Flow Cytometry Standard 2.0 (FCS). The excitation wave-
Deacetylcolchicine (3) (129 mg, 0.36 mmol) was dissolved in
anhydrous dichloromethane (5 mL) together with triethylamine
(65 lL, 4.6 mmol) at 0 °C. Chloroacetic anhydride (500 mg,

E. M. V. Johansson et al. / Bioorg. Med. Chem. 18 (2010) 6589–6597
6597
9. Waibel, R.; Treichler, H.; Schaefer, N. G.; Van Staveren, D. R.; Mundwiler, S.;
Kunze, S.; Küenzi, M.; Alberto, R.; Nüesch, J.; Knuth, A.; Moch, H.; Schibli, R.;
Schubiger, P. A. Cancer Res. 2008, 68, 2904.
length was 488 nm and the emission was collected at 550 nm. The
raw data was processed further with WinMDI 2.9 software (Joseph
Trotter, The Scripps Research Institute, USA). The MEF cells med-
ium was removed to the FACS tube. The cells were washed with
sterile PBS and antiadherent Trypsin/EDTA solution (0.5 mL) was
added and incubated at 37 °C for 20 min after which the solution
was added to the FACS tube.
10. (a) Crespo, L.; Sanclimens, G.; Pons, M.; Giralt, E.; Royo, M.; Albericio, F. Chem.
Rev. 2005, 105, 1663; (b) Sadler, K.; Tam, J. P. Rev. Mol. Biotechnol. 2002, 90, 195.
11. Niederhafner, P.; Sebestik, J.; Jezek, J. J. Pept. Sci. 2008, 14, 44.
12. (a) Esposito, A.; Delort, E.; Lagnoux, D.; Djojo, F.; Reymond, J.-L. Angew. Chem.,
Int. Ed. 2003, 42, 1381; (b) Darbre, T.; Reymond, J.-L. Acc. Chem. Res. 2006, 39,
925; (c) Darbre, T.; Reymond, J.-L. Curr. Top. Med. Chem 2008, 8, 1286–1293.
13. (a) Javor, S.; Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2007, 129,
13238; (b) Javor, S.; Natalello, A.; Doglia, S. M.; Reymond, J.-L. J. Am. Chem. Soc.
2008, 130, 17248; (c) Javor, S.; Reymond, J.-L. J. Org. Chem. 2009, 74, 3665.
14. (a) Lagnoux, D.; Delort, E.; Douat-Casassus, C.; Esposito, A.; Reymond, J.-L.
Chem. Eur. J. 2004, 10, 1215; (b) Douat-Casassus, C.; Darbre, T.; Reymond, J.-L. J.
Am. Chem. Soc. 2004, 126, 7817; (c) Clouet, A.; Darbre, T.; Reymond, J.-L. Adv.
Synth. Catal. 2004, 346, 1195; (d) Kofoed, J.; Darbre, T.; Reymond, J.-L. Org.
Biomol. Chem. 2006, 3268; (e) Javor, S.; Reymond, J.-L. Isr. J. Chem. 2009, 49, 129.
15. (a) Sommer, P.; Uhlich, N. A.; Reymond, J.-L.; Darbre, T. ChemBioChem 2008, 9,
689; (b) Duléry, V.; Uhlich, N. A.; Maillard, N.; Fluxá, V. S.; Garcia, J.; Dumy, P.;
Renaudet, O.; Reymond, J.-L.; Darbre, T. Org. Biomol. Chem. 2008, 6, 4134; (c)
Uhlich, N. A.; Sommer, P.; Bühr, C.; Schürch, S.; Reymond, J.-L.; Darbre, T. Chem.
Commun. 2009, 6237; (d) Uhlich, N.; Natalello, A.; Kadam, R. U.; Doglia, S. M.;
Reymond, J.-L.; Darbre, T. ChemBioChem 2010, 11, 358.
16. (a) Kolomiets, E.; Johansson, E. M. V.; Renaudet, O.; Darbre, T.; Reymond, J.-L.
Org. Lett. 2007, 9, 1465; (b) Johansson, E. M. V.; Crusz, S. A.; Kolomiets, E.; Buts,
L.; Kadam, R. U.; Cacciarini, M.; Bartels, K.-M.; Diggle, S. P.; Camara, M.;
Williams, P.; Loris, R.; Nativi, C.; Rosenau, F.; Jaeger, K.-E.; Darbre, T.; Reymond,
J.-L. Chem. Biol. 2008, 15, 1249; (c) Kolomiets, E.; Swiderska, M. A.; Kadam, R.
U.; Johansson, E. M. V.; Jaeger, K.-E.; Darbre, T.; Reymond, J.-L. ChemMedChem
2009, 4, 562.
17. (a) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321; (b) Mammen, M.; Choi, S.-
K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754; (c) Lundquist, J. J.;
Toone, E. J. Chem. Rev. 2002, 102, 555; Pieters, R. J. Org. Biomol. Chem. 2009, 7,
2013.
5.1. Tubulin Polymerization Assay
Sheep brains were obtained from the INRA (Institut National de
Recherche Agronomique) from freshly slaughtered animals and
tubulin isolation was started in the next hour. Thus, sheep brain
microtubule proteins were purified by two cycles of assembly/dis-
assembly at 37 °C/0 °C in MES buffer: 100 mM MES (2-[N-morpho-
lino]-ethanesulfonic acid, pH 6.6), 1 mM EGTA (ethyleneglycol-
bis[b-aminoethyl ether]-N,N,N0,N0-tetraacetic acid), 0.5 mM Mg
Cl2. All samples were dissolved in DMSO. The evaluated compound
(1 lL) was added to microtubular solution (150 lL) that was incu-
bated at 37 °C for 10 min and at 0 °C for 5 min before evaluation of
the tubulin assembly rate. The tubulin assembly assay was realized
according to a slightly modified Guénard’s protocol33 using colchi-
cine as reference compound.
Acknowledgments
18. Lagnoux, D.; Darbre, T.; Schmitz, M. L.; Reymond, J.-L. Chem. Eur. J. 2005, 11,
3941.
19. (a) Zhang, S. X.; Feng, J.; Kuo, S. C.; Brossi, A.; Hamel, E.; Tropsha, A.; Lee, K. H. J.
Med. Chem. 2000, 43, 167; (b) Bensel, N.; Lagnoux, D.; Niggli, V.; Wartmann, M.;
Reymond, J.-L. Helv. Chim. Acta 2004, 87, 2266.
This work was supported financially by the Swiss National Sci-
ence Foundation and the University of Berne.
Supplementary data
20. Goto, H.; Yano, S.; Zhang, H.; Matsumori, Y.; Ogawa, H.; Blakey, D. C.; Sone, S.
Cancer Res. 2002, 62, 3711.
21. (a) Hastie, S. B. Pharmacol. Ther. 1991, 512, 377; (b) Skoufias, D.; Wilson, L.
Biochemistry 1992, 31, 738; (c) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain,
I.; Lachkar, S.; Sobel, A.; Knossow, M. Nature 2004, 428, 198.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.026.
22. (a) Pieters, R. J. ChemBioChem 2006, 7, 721; (b) Lau, K. S.; Partridge, E. A.;
Grigorian, A.; Silvescu, C. I.; Reinhold, V. N.; Demetriou, M.; Dennis, J. W. Cell
2007, 129, 123.
References and notes
23. (a) Gabius, H.-J.; Siebert, H.-C.; Andre, S.; Jimenez-Barbero, J.; Ruediger, H.
ChemBioChem 2004, 5, 740; (b) Roseman, D. S.; Baenziger, J. U. J. Biol. Chem.
2001, 276, 17052; (c) Higashi, N.; Fujioka, K.; Denda-Nagai, K.; Hashimoto, S.;
Nagai, S.; Sato, T.; Fujita, Y.; Morikawa, A.; Tsuiji, M.; Miyata-Takeuchi, M.;
Sano, Y.; Suzuki, N.; Yamamoto, K.; Matsushima, K.; Irimura, T. J. Biol. Chem.
2002, 277, 20686.
24. (a) Veprek, P.; Jesek, J. J. Pept. Sci. 1990, 203; (b) Molema, G.; Jansen, R. W.;
Visser, J.; Herdewijin, P.; Moolenaar, F.; Meijer, D. K. F. J. Med. Chem. 1991, 34,
1137.
25. David, A.; Kopecková, P.; Minko, T.; Rubinstein, A.; Kopecek, J. Eur. J. Cancer
2004, 40, 148.
26. (a) Clouet, A.; Darbre, T.; Reymond, J.-L. Angew. Chem., Int. Ed. 2004, 43, 4612;
(b) Clouet, A.; Darbre, T.; Reymond, J.-L. Biopolymers 2006, 84, 114; (c) Maillard,
N.; Darbre, T.; Reymond, J.-L. J. Comb. Chem. 2009, 11, 667.
27. Magnusson, G. N. G.; Noori, G.; Dahmn, J.; Frejd, T.; Lave, T. Acta. Chim. Scand. B
1981, 35, 213.
28. Johansson, E. M. V.; Kolomiets, E.; Rosenau, F.; Jaeger, K.-E.; Darbre, T.;
Reymond, J.-L. New J. Chem. 2007, 31, 1291.
29. (a) Pennington, M. E.; Lam, K. S.; Cress, A. E. Mol. Divers. 1996, 2, 19; (b) Peng, L.;
Liu, R.; Marik, J.; Wang, X.; Takada, Y.; Lam, K. S. Nat. Chem. Biol. 2006, 2, 381;
(c) Gomika Udugamasooriya, D.; Dineen, S. P.; Brekken, R. A.; Kodadek, T. J. Am.
Chem. Soc. 2008, 130, 5744; (d) Aina, O. H.; Liu, R.; Sutcliffe, J. L.; Marik, J.; Pan,
C.-X.; Lam, K. S. Mol. Pharmacol. 2007, 4, 631.
30. (a) Kofoed, J.; Reymond, J.-L. J. Comb. Chem. 2007, 9, 1046; (b) Maillard, N.;
Clouet, A.; Darbre, T.; Reymond, J.-L. Nat. Prot. 2009, 4, 132; (c) Fluxa, V. S.;
Reymond, J.-L. Bioorg. Med. Chem. 2009, 17, 1018.
1. (a) Newkome, G. R.; Moorefield, C. N.; Vögtle, F. Dendritic Molecules: Concepts,
Synthesis, Applications; VCH: Weinheim, 2001; (b) Tomalia, D. A.; Dvornic, P. R.
Nature 1994, 372, 617; (c) Smith, D. K.; Diederich, F. Top. Curr. Chem. 2000, 210,
183; (d) Helms, B.; Fréchet, J. M. J. Adv. Synth. Catal. 2006, 348, 1125; (e) Smith,
D. K.; Diederich, F. Chem. Eur. J. 1998, 4, 1353; (f) Kofoed, J.; Reymond, J.-L. Curr.
Opin. Chem. Biol. 2005, 9, 656; (g) Bosman, A. W.; Jansen, H. M.; Meijer, E. W.
Chem. Rev. 1999, 99, 1665; (h) Lee, C. C.; MacKay, J. A.; Frechet, J. M. J.; Szoka, F.
C. Nat. Biotechnol. 2005, 23, 1517.
2. Patri, A. K.; Kukowska-Latallo, J. F.; Baker, J. R., Jr. Adv. Drug Delivery Rev. 2005,
57, 2203.
3. (a) Duncan, R. Nat. Rev. Cancer 2006, 6, 688; (b) Carlson, C. B.; Mowery, P.;
Owen, R. M.; Dykhuizen, E. C.; Kiessling, L. L. ACS Chem. Biol. 2007, 2, 119; (c)
Dijkgraaf, I.; Rijnders, A. Y.; Soede, A.; Dechesne, A. C.; van Esse, G. W.; Brouwer,
A. J.; Corstens, F. H. M.; Boerman, O. C.; Rijkers, D. T. S.; Liskamp, R. M. J. Org.
Biomol. Chem. 2007, 5, 935; (d) Vicent, M. J.; Duncan, R. Trends Biotechnol. 2006,
24, 39; (e) Greco, F.; Vincent, M. J. Adv. Drug Delivery Rev. 2009, 61, 1131.
4. Lee, C. C.; Gillies, E.; Fox, M. E.; Dy, E.; Frechet, J. M. J.; Szoka, F. C. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 16649; (b) Fox, M. E.; Guillaudeu, S.; Frechet, J. M. J.;
Jerger, K.; Macaraeg, N.; Szoka, F. C. Mol. Pharmacol. 2009, 6, 1562; (c) Fox, M.
E.; Szoka, F. C.; Frechet, J. M. J. Acc. Chem. Res. 2009, 42, 1141.
5. (a) Ren, Y.; Wong, S. M.; Lim, L.-Y. Bioconjugate Chem. 2007, 18, 836; (b) Singh,
P.; Gupta, U.; Asthana, A.; Jain, N. K. Bioconjugate Chem. 2008, 19, 2239.
6. Yang, W.; Cheng, Y.; Xu, T.; Wang, X.; Wen, L. P. Eur. J. Med. Chem. 2009, 44, 862.
7. (a) Hill, E.; Shukla, R.; Park, S. S.; Baker, J. R., Jr. Bioconjugate Chem. 2007, 18,
1756; (b) Foillard, S.; Sancey, L.; Coll, J.-L.; Boturyn, D.; Dumy, P. Org. Biomol.
Chem. 2009, 7, 221.
31. Ducray, P.; Lebeau, L.; Mioskowski, C. Helv. Chim. Acta 1996, 79, 2346.
32. Sommer, P.; Fluxa, V. S.; Darbre, T.; Reymond, J.-L. ChemBioChem 2009, 10,
1527.
8. (a) Sievers, E. L.; Appelbaum, F. R.; Spielberger, R. T.; Forman, S. J.; Flowers, D.;
Smith, F. O.; Shannon-Dorcy, K. Blood 1999, 93, 3678; Lo-Coco, F.; Cimino, G.;
Breccia, M.; Noguera, N. I.; Diverio, D.; Finolezzi, E.; Pogliani, E. M.; Di Bona, E.;
Micalizzi, C.; Kropp, M.; Venditti, A.; Tafuri, A.; Mandelli, F. Blood 2004, 104,
1995.
33. Zavala, F.; Guénard, D.; Robin, J.-P.; Brown, E. J. Med. Chem. 1980, 23, 546.