Tubulin-guided dynamic combinatorial library of thiocolchicine- podophyllotoxin conjugates

Article abstract of DOI:10.1016/j.tet.2011.07.038

The use of tubulin as a target to influence the composition of the mixture from a dynamic combinatorial library, based on the disulfide bond exchange reaction, is described. ESI-FT-ICR-MS was used to determine the composition of the library. The heterodim

Full text of DOI:10.1016/j.tet.2011.07.038

Tetrahedron 67 (2011) 7354e7357
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Tubulin-guided dynamic combinatorial library of thiocolchicineepodophyllotoxin
conjugates
Graziella Cappelletti a, Daniele Cartelli a, Bruno Peretto b, Micol Ventura b, Marco Riccioli b,
,
Francesco Colombo b, John S. Snaith c, Stella Borrelli b, Daniele Passarella b
*
a
ꢀ
Dipartimento di Biologia, Universita degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
b
ꢀ
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
c School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2011
Received in revised form 21 June 2011
Accepted 12 July 2011
Available online 22 July 2011
The use of tubulin as a target to influence the composition of the mixture from a dynamic combi-
natorial library, based on the disulfide bond exchange reaction, is described. ESI-FT-ICR-MS was used
to determine the composition of the library. The heterodimeric compound amplified by this approach
was used to design the homologous derivative with a two-carbon spacer in place of the disulfide
function. The ability of the compounds to inhibit tubulin polymerization is reported and compared to
thiocolchicine.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Dynamic combinatorial chemistry
Target-guided synthesis
Disulfide-exchange reaction
1. Introduction
process that generates the tremendous molecular diversity of
natural products.7 In our earlier work, we have demonstrated the
Tubulin and microtubules remain one of the most interesting
targets in the design of new anticancer compounds.1 In addition,
increasing evidence supports a pathogenic role for tubulin in the
neurodegeneration underlying several diseases, including Alz-
heimer’s and Parkinson’s disease,2a,b and the therapeutic potential
of anti-tubulin drugs for treating neurodegeneration has been very
recently proposed.2c
potential of the disulfide-exchange reaction to generate a dynamic
library of thiocolchicineepodophyllotoxin adducts and the in-
fluence on the composition of this mixture induced by subtilisin or
albumin.8 Our next aim was to demonstrate the possibility of using
tubulin to guide the evolutionary formation of divalent binders by
favouring the structure that is thermodynamically most stabilized
by non-covalent interactions with the biological target (Fig. 1).
The discovery of new compounds that bind to tubulin is a chal-
lenging research aim with implications for the design and prepa-
ration of new high-quality collections of compounds useful for the
study of the regulation of different ailments of modern society. The
generation of divalent or multivalent compounds is a well-known
strategy inspired by nature, that has produced a number of in-
teresting results.3 In our lab we accomplished the preparation of
a number of multivalent conjugates, using as building blocks sev-
eral natural products with well-documented pharmacological
activity.4
Target-guided synthesis5 emerged in the last decade as an im-
portant strategy that increases the versatility of combinatorial
chemistry and in particular of Dynamic Combinatorial Libraries6
(DCL), and has provided a demonstration of the evolutionary
* Corresponding author. Tel./fax: þ39 02 50314081; e-mail address: daniele.
Fig. 1. General concept for the target-guided DCL based on the disulfide-exchange
reaction.
passarella@unimi.it (D. Passarella).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.07.038

G. Cappelletti et al. / Tetrahedron 67 (2011) 7354e7357
7355
In this paper we report the use of thiocolchicine and podo-
phyllotoxin (Fig. 2) for the generation of a dynamic combinatorial
mixture of homo- and heterodimeric compounds based on the
reversible formation of disulfide bonds.9 The building blocks for the
library were the homodimers obtained by condensation of thio-
colchicine and podophyllotoxin with dicarboxylic acids in-
corporating a disulfide bond.
solution at a concentration of 5ꢀ10ꢁ3 mM and maintained at room
temperature for 96 h. The composition of the mixture was then
evaluated by ESI-FT-ICR-MS (Supplementary data), which showed
all six possible compounds due to the formation of the hetero-
dimers 7e9 (Fig. 4). In parallel we prepared a second solution
containing a cytoskeleton fraction to give a 2.1ꢀ10ꢁ6 M solution of
tubulin. The analysis after 96 h revealed a marked alteration of the
mixture composition (Table 1) and, in particular, we observed the
amplification of the heterodimer 9.
Fig. 2. Structure of deacetylthiocolchicine 1, podophyllotoxin
deacetylthiocolchicine 3.
2 and glycinoyl-
Although thiocolchicine10 and podophyllotoxin11 recognize the
same binding site, previous work in other areas has shown that
homo- and heterodimers can have biological characteristics that are
not merely the sum of the properties of the single entities.3a For this
Fig. 4. Structures of compounds 7e9.
Table 1
reason, we cannot predict if the interaction will be with single
a-
Comparison of the percentages of dimeric structures based on the relative intensity
of the mass peaks in the presence or absence of tubulin
or -tubulin, with the heterodimer or with the microtubule
b
a-b
assembly and this makes it of interest to consider conjugate
compounds.
Compound
Mass peak
No biol. target
With biol. target
4
5
6
7
8
9
951.28422
1033.27563
1065.32572
992.27994
1049.29456
1008.30411
58.3
3.8
27.6
1.3
2.5
6.4
37.5
d
44.6
d
2. Results and discussion
d
We needed to generate a water soluble library, allowing us to
work in aqueous conditions that would maintain the coherent
structure of the biological target. Thus, our first task was to prepare
a range of water soluble homodimeric starting compounds. We
designed three different homodimers (Fig. 3) in which the spacer
that connects the two entities presents primary amino and amide
groups. They were obtained by condensation between podo-
17.8
We then undertook the synthesis of compound 12 that re-
sembles the structure of the selected compound 9 but with an
ethylene group in the place of the liable disulfide bond to guar-
antee the binary structure in the cell medium. The condensation
of the dicarboxylic acid 104a with deacetylthiocolchicine 1 gave
adduct 11 that was subsequently reacted with glycinoylth-
iocolchicine to obtain, after Boc removal, the desired compound
12 (Scheme 1).
phyllotoxin 2, deacetylthiocolchicine
1 and deacetylglycinyl-
thiocolchicine 3 with N-Boc-cystine. The removal of the Boc
group generated, respectively, compounds 4,4a 54a and 6, all of
which had a satisfactory water solubility (1.2e1.5 mg/mL, as
ditartrate salts). The three homodisulfides were mixed in a buffered
Scheme 1. Synthesis of compound 12.
Fig. 3. Structures of compounds 4e6.

7356
G. Cappelletti et al. / Tetrahedron 67 (2011) 7354e7357
To get an insight into the potential biological activity of 12 we
4. Experimental section
4.1. General
investigated its ability to affect tubulin polymerization in vitro in
comparison with the homodimers 4e6 and with the symmetric
compound 13 that is missing the glycine framework. Bovine tu-
bulin (purified from brain) was mixed with a standard solution of
each sample in the absence of GTP. The solutions were incubated
at 37 ꢂC to allow slow-binding compounds to bind to the tubulin,
and after 15 min GTP was added to initiate microtubule assembly.
After 30 min the polymerized and the unpolymerized fractions
were collected by centrifugation, separated by SDS-PAGE and
quantified by densitometry as previously described.4a Compound
12 proved more active than two of the starting disulfide homo-
dimers (4 and 5, Table 2), although the homodimer 6 had the
highest activity. Comparison with analogue 13 underlines the
importance of the glycine spacer. Furthermore, we demonstrated
that inhibition of tubulin polymerization by compound 12 is dose-
dependent (Fig. 5).
All reagents and solvents were reagent grade or were purified by
standard methods before use. Column chromatography was carried
out on flash silica gel (Merck 230e400 mesh). NMR spectra were
recorded at 300/400 MHz (1H) and at 75/100 MHz (13C). FABþ mass
spectra were recorded at an ionizing voltage of 6 Kev. ESI mass
spectra were recorded on FT-ICR instrument.
4.1.1. Thiocolchicine derivative (3). To a solution of 1 (100 mg,
0.27 mmol) in CH2Cl2 (15 mL), N-Boc-glycine (95 mg, 0.59 mmol),
DCC (334 mg, 1.62 mmol) and DMAP (66 mg, 0.54 mmol) were
added. The reaction mixture was stirred at room temperature for
16 h, then filtered through Celite. The solvent was concentrated in
vacuo and the residue was purified by column chromatography on
silica gel (AcOEt/hexane 20:1) to afford the desired intermediate as
a yellow amorphous solid (64 mg, 47%). 1H NMR (300 MHz, CDCl3):
Table 2
d
7.49 (1H, s), 7.35e7.38 (1H, m), 7.07e7.11 (1H, m), 6.54 (1H, s),
Ratios of polymerized/unpolymerized tubulin obtained in the presence of com-
pounds 4, 5, 6, 12 and 13
4.61e4.89 (3H, m), 3.41e3.93 (11H, m), 2.32e2.57 (5H, m), 1.94 (2H,
m), 1.45 (9H, s). 13C NMR (100 MHz, CDCl3, detected signals):
Compound (10
m
M)
P/U tubulin (meanꢄSEM)
d
182.3, 169.6, 158.2, 156.3, 154.5, 151.0, 141.8, 139.1, 135.5, 131.4,
Control
10.2ꢄ0.36
5.9ꢄ0.14b
7.2ꢄ0.18a
3.9ꢄ0.12c
5.4ꢄ0.43c
9.2ꢄ0.21
128.1, 107.2, 80.6 (3C), 61.5, 61.3, 56.1, 52.1, 45.0, 36.4, 29.9, 28.3,
15.3. Anal. Calcd for C27H34N2O7S: C, 61.11; H, 6.46; N, 5.28. Found:
C, 61.14; H, 6.41; N, 5.30. To a freshly prepared saturated solution of
HCl in dioxane (21 mL) the previously obtained intermediate
(130 mg, 0.26 mmol) was added and the reaction mixture was
stirred at 0 ꢂC for 1 h. The solvent was removed in vacuo and the
residue was dissolved in CH2Cl2 and washed with saturated
NaHCO3. The combined organic layers were dried over sodium
sulfate and concentrated in vacuo to afford 3 as a yellow amorphous
4
5
6
12
13
a
p<0.05.
p<0.002.
b
c
p<0.001 versus control, according to ANOVA, Fisher LSD post-hoc.
solid (107 mg, 99%). 1H NMR (300 MHz, CDCl3):
d 8.51 (1H, s),
7.91e7.92 (2H, m), 7.35e7.52 (2H, m), 3.93 (9H, s), 3.73 (2H, m), 3.47
(4H, m), 2.79e2.91 (4H, m), 1.94 (1H, m), 1.74 (1H, m). 13C NMR
(100 MHz, CDCl3, detected signals):
d 182.3, 158.2, 156.3, 154.5,
151.0, 141.8, 139.1, 135.5, 131.4, 128.1, 107.2, 61.5, 61.3, 56.1, 52.1, 45.0,
36.4, 29.9, 15.3. Anal. Calcd for C22H26N2O5S: C, 61.38; H, 6.09; N,
6.51. Found: C, 61.42; H, 6.08; N, 6.48.
4.1.2. Thiocolchicine derivative (6). To a solution of (Boc-cys-OH)2
(114 mg, 0.26 mmol) in dry THF/CH3CN 8:1 (9 mL), compound 3
(236 mg, 0.055 mmol), DCC (163 mg, 0.79 mmol) and DMAP
(32 mg, 0.26 mmol) were added. The reaction mixture was stirred
at 50 ꢂC for 5 h. The solvent was removed in vacuo and the residue
was purified by column chromatography on silica gel (CH2Cl2/
MeOH 14:1) to afford the desired intermediate as a yellow amor-
Fig. 5. Ratios of polymerized/unpolymerized tubulin obtained in the presence of dif-
phous solid (307 mg, 92%). ½a D20
ꢃ
ꢁ5.4 (c 0.84, CHCl3); 1H NMR
ferent concentrations of thiocolchicine 1a (1, 5 and 10
and 50
experiments. *p<0.05, **p<0.002, ***p<0.001 versus control, according to ANOVA,
mM) and compound 12 (1, 5, 10
M). Data are expressed as meansꢄSEM of values of at least three independent
(400 MHz, CDCl3): d 8.55 (2H, d, J 6.85 Hz), 7.88 (2H, s), 7.66 (2H, s),
m
7.38 (2H, d, J 10.5 Hz), 7.16 (2H, d, J 10.5 Hz), 6.53 (2H, s), 5.89 (2H, d,
J 4.90 Hz), 4.72e4.77 (4H, m), 4.09e4.16 (2H, m), 3.95 (6H, s), 3.91
(6H, s), 3.82e3.85 (2H, m), 3.65 (6H, s), 3.23e3.26 (2H, m),
3.01e3.02 (2H, m), 2.46 (6H, s), 2.45e2.47 (2H, m), 2.24e2.27 (4H,
m), 1.84e1.88 (2H, m), 1.35 (18H, s); 13C NMR (100 MHz, CDCl3):
Fisher LSD post-hoc.
3. Conclusion
d
182.7, 171.6, 169.3, 159.4, 156.2, 154.6, 152.9, 151.9, 142.6, 139.8,
Tubulin has been used as a biological target to guide the as-
sembly of a binary compound in a DCL approach based on the
disulfide-exchange reaction. The structure of the selected hetero-
dimeric entity, derived from the precursor mixture of homodimers,
inspired the design of the corresponding compound lacking the
disulfide bond. Replacing the disulfide bond with an ethylene
spacer resulted in a compound that inhibits the polymerization of
tubulin in a dose-dependent manner. This result further demon-
strates the power of target-guided synthesis used in conjunction
with DCLs and underlines the pivotal role of weak interactions with
biomacromolecules.
135.8, 135.0, 129.6, 127.8, 126.3, 108.4, 80.8, 55.1, 52.8, 44.5, 43.8,
37.1, 30.7, 29.0, 15.9. Anal. Calcd for C60H76N6O16S4: C, 56.94; H,
6.05; N, 6.64. Found: C, 56.98; H, 6.01; N, 6.62. To a freshly prepared
saturated solution of HCl in dioxane the previously obtained in-
termediate (43 mg, 0.03 mmol) was added and the reaction mix-
ture was stirred for 2 h at 0 ꢂC. The solvent was removed in vacuo
and the residue was dissolved in CH2Cl2 and washed with saturated
NaHCO3. The combined organic layers were dried over sodium
sulfate and concentrated in vacuo to afford 6 as a yellow amorphous
solid (33.7 mg, 94%). ½a D20
ꢃ
þ0.2 (c 0.33, CHCl3); 1H NMR (400 MHz,
CDCl3): d 9.07 (2H, d, J 0.32 Hz), 8.58 (2H, br s), 7.68 (2H, s), 7.32 (2H,

G. Cappelletti et al. / Tetrahedron 67 (2011) 7354e7357
7357
d, J 10.3 Hz), 7.10 (2H, d, J 10.3 Hz), 6.52 (2H, s), 4.60e4.66 (6H, m),
4.39e4.43 (2H, m), 3.94 (6H, s), 3.90 (6H, s), 3.77e3.81 (2H, m), 3.68
(6H, s), 3.63e3.65 (2H, m), 3.16e3.19 (2H, m), 2.72e2.76 (2H, m),
2.45e2.53 (2H, m), 2.44 (6H, s), 2.22e2.29 (4H, m), 1.87e1.89 (2H,
4$tartrate, 5$tartrate and 6$tartrate were dissolved in water (10 mL)
and DMSO (1 mL) and pH was adjusted to pH 8 by addition of
Na2CO3 (satd sol) to obtain Solution C. A tubulin preparation that
contained the microtubule-enriched fraction was prepared from
PC12 cells following Triton-X 100 extraction as previously de-
m); 13C NMR (100 MHz, CDCl3, detected signals):
d 182.3, 175.6,
169.6, 158.2, 153.5, 152.8, 151.0, 141.8, 139.1, 135.5, 134.5, 129.1, 127.1,
125.9, 107.2, 61.5, 54.9, 52.7, 45.0, 43.3, 35.5, 30.1, 15.3. Anal. Calcd
for C50H60N6O12S4: C, 56.37; H, 5.68; N, 7.89. Found: C, 56.40; H,
5.63; N, 7.86.
scribed.12 The tubulin preparation (194
mL of Triton-X 100 insoluble
fraction containing 2.25 mg of tubulin) was added to solution A
(final concentration of tubulin was 2.1ꢀ10ꢁ6 M). Solutions B and C
were used as references. The solutions were maintained at room
temperature for 96 h then submitted to ESI-FT-ICR-MS.
4.1.3. Thiocolchicine derivative (12). To a solution of 1 (18 mg,
0.051 mmol) in dry CH2Cl2 (4 mL) compound 10 (20 mg,
0.051 mmol), DCC (15 mg, 0.072 mmol) and DMAP (6 mg,
0.048 mmol) were added. The reaction mixture was stirred at room
temperature for 20 h, then filtered through Celite and washed with
saturated K2CO3. The combined aqueous layers were acidified and
extracted with CH2Cl2. The combined organic layers were dried
over sodium sulfate and concentrated in vacuo to afford compound
11 as a yellow amorphous solid (11 mg, 56%). 1H NMR (300 MHz,
Acknowledgements
This research has been developed under the umbrella of
CM0602 COST Action ‘Inhibitors of Angiogenesis: design, synthesis
and biological exploitation’.
Supplementary data
CDCl3):
d 7.74e7.76 (1H, m), 7.53e7.57 (1H, m), 7.00e7.10 (1H, m)
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2011.07.038.
6.52 (1H, s), 4.18e4.22 (2H, m), 3.93 (1H, m), 3.89 (3H, s), 3.65 (6H,
s), 3.42 (1H, m), 2.41 (3H, s), 2.26e2.30 (2H, m), 1.71e1.93 (2H, m),
1.43e1.68 (4H, m), 1.24 (18H, s), 0.81e0.93 (4H, m). Anal. Calcd for
C38H53N3O11S: C, 60.06; H, 7.03; N, 5.53. Found: C, 60.09; H, 7.00; N,
5.51. To a solution of 11 (30 mg, 0.039 mmol) in dry CH2Cl2 (6 mL),
compound 3 (18 mg, 0.042 mmol), DCC (13 mg, 0.06 mmol) and
DMAP (5 mg, 0.042 mmol) were added. The reaction mixture was
stirred at room temperature for 20 h then filtered through Celite.
The solvent was removed in vacuo and the residue was purified by
column chromatography on silica gel (AcOEt/hexane 8:2) to afford
the desired intermediate as a yellow amorphous solid (28 mg, 59%).
References and notes
1. (a) Kanthou, G.; Tozer, G. M. Int. J. Exp. Pathol. 2009, 90, 284; (b) Hait, W. N.;
Hambley, T. W. Cancer Res. 2009, 69, 1263; (c) De Rycker, M.; Rigoreau, L.;
Dowding, S.; Parker, P. J. Chem. Biol. Drug Des. 2009, 73, 599; (d) Jordan, M. A.;
Wilson, L. Nat. Rev. Cancer 2004, 4, 253; (e) Carlson, R. O. Expert Opin. Invest.
Drugs 2008, 17, 707; (f) Li, W. T.; Hwang, D. R.; Song, J. J.; Chen, C. P.; Chuu, J. J.;
Hu, C. B.; Lin, H. L.; Huang, C. L.; Huang, C. Y.; Tseng, H. Y.; Lin, C. C.; Chen, T. W.;
Lin, C. H.; Wang, H. S.; Shen, C. C.; Chang, C. M.; Chao, Y. S.; Chen, C. T. J. Med.
Chem. 2010, 53, 2409.
2. (a) Feng, J. Neuroscientist 2006, 12, 469; (b) Cartelli, D.; Ronchi, C.; Maggioni, M.
G.; Rodighiero, S.; Giavini, E.; Cappelletti, G. J. Neurochem. 2010, 115, 247; (c)
Bruden, K. R.; Zhang, B.; Carroll, J.; Yao, Y.; Potuzak, J. S.; Hogan, A.-M. L.; Iba,
M.; James, M. J.; Xie, S. X.; Ballatore, C.; Smith, A. B., III; Lee, V. M.-Y.; Troja-
nowski, J. G. J. Neurosci. 2010, 30, 13861.
1H NMR (300 MHz, CDCl3):
d 8.16e8.21 (2H, m), 7.65e7.78 (2H, m),
7.38e7.46 (2H, m), 6.98e7.12 (2H, m), 6.57 (2H, s), 4.68e4.75 (4H,
m), 4.19e4.31 (4H, m), 3.85e4.04 (6H, m), 3.58e3.78 (12H, m), 2.88
(2H, s), 2.53 (6H, s), 1.9e2.35 (4H, m), 1.5e1.72 (4H, m), 1.37e1.49
(6H, m), 1.24 (18H, s). Anal. Calcd for C60H77N5O15S2: C, 61.47; H,
6.62; N, 5.97. Found: C, 61.51; H, 6.59; N, 5.98. To a freshly prepared
saturated solution of HCl in dioxane the previously obtained in-
termediate (7.5 mg, 0.0062 mmol) was added and the reaction
mixture was stirred for 1 h at 0 ꢂC. The solvent was removed in
vacuo and the residue was dissolved in CH2Cl2 and washed with
saturated NaHCO3. The combined organic layers were dried over
sodium sulfate and concentrated in vacuo to afford 12 as a yellow
3. (a) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37,
2754; (b) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L.
J. Am. Chem. Soc. 2002, 124, 14922; (c) Zhang, Y.; Li, Q.; Rodriguez, L. G.; Gil-
dersleeve, J. C. J. Am. Chem. Soc. 2010, 132, 9653.
4. (a) Passarella, D.; Peretto, B.; Blasco, R.; Cappelletti, G.; Cartelli, D.; Ronchi, C.;
Snaith, J. S.; Fontana, G.; Danieli, B.; Borlak, J. Eur. J. Med. Chem. 2010, 45, 219; (b)
Passarella, D.; Comi, D.; Cappelletti, G.; Cartelli, D.; Gertsch, J.; Quesada, A. R.;
Borlak, J.; Altmann, K.-H. Bioorg. Med. Chem. 2009, 17, 7435; (c) Passarella, D.;
Comi, D.; Vanossi, A.; Paganini, G.; Colombo, F.; Ferrante, L.; Zuco, V.; Danieli, B.;
Zunino, F. Bioorg. Med. Chem. Lett. 2009, 19, 6358; (d) Passarella, D.; Giardini, A.;
Peretto, B.; Fontana, G.; Sacchetti, A.; Silvani, A.; Ronchi, C.; Cappelletti, G.;
Cartelli, D.; Borlak, J.; Danieli, B. Bioorg. Med. Chem. 2008, 16, 6269; (e) Danieli,
B.; Giardini, A.; Lesma, G.; Passarella, D.; Silvani, A.; Appendino, G.; Noncovich,
A.; Fontana, G.; Bombardelli, E.; Sterner, O. Chem. Biodiversity 2004, 1, 327; (f)
Riva, E.; Comi, D.; Borrelli, S.; Colombo, F.; Danieli, B.; Borlak, J.; Evensen, L.;
Lorens, J. B.; Fontana, G.; Gia, O. M.; Dalla Via, L.; Passarella, D. Bioorg. Med.
Chem. 2010, 18, 8660.
amorphous solid (4 mg, 57%). ½a D24
ꢃ
ꢁ3.1 (c 0.17, CHCl3); 1H NMR
(300 MHz, CDCl3):
d 8.16e8.21 (2H, m), 7.65e7.78 (2H, m),
7.38e7.46 (2H, m), 6.98e7.12 (2H, m), 6.57 (2H, s), 4.68e4.75 (4H,
m), 4.19e4.31 (4H, m), 3.85e4.04 (6H, m), 3.58e3.78 (12H, m), 2.88
(2H, s), 2.53 (6H, s), 1.9e2.35 (4H, m), 1.5e1.72 (4H, m), 1.37e1.49
(6H, m). 13C NMR (100 MHz, CDCl3) detected signals:
d 181.1, 171.1,
5. Hu, X.; Manetsch, R. Chem. Soc. Rev. 2010, 39, 1316.
6. For recent reviews see: (a) Ladame, S. Org. Biomol. Chem. 2008, 6, 219; (b) Lehn,
J. M. Chem. Soc. Rev. 2007, 36, 151; Corbett, P. T.; Leclaire, J.; Vial, L.; West, K. R.;
Wietor, J.-L.; Sanders, J. K. M.; Otto, S. Chem. Rev. 2006, 106, 3652.
7. Del Amo, V.; Philp, D. Chem.dEur. J. 2010, 16, 13304 and references cited
therein.
157.3, 155.3, 153.6, 52.7, 151.1, 141.6, 138.7, 133.6, 129.7, 125.9, 125.6,
108.3, 61.7, 60.8, 58.7, 56.1, 54.3, 44.0, 36.1, 34.5, 29.1, 22.8 15.5;
HRMS (ESI): m/z: calcd for C50H61N5O11S2Naþ: 1023.2192. Found:
1022,6579.
8. Danieli, B.; Giardini, A.; Lesma, G.; Passarella, D.; Peretto, B.; Sacchetti, A.; Sil-
vani, A.; Pratesi, G. J. Org. Chem. 2006, 71, 2848.
9. For other recent use of disulfide exchange reaction in DCL see: (a) Orillo, A. G.;
Escalante, A. M.; Furlan, R. L. E. Chem. Commun. 2008, 5298; (b) von Delius, M.;
Geertsema, E. M.; Leigh, D. A.; Slawin, A. M. Z. Org. Biomol. Chem. 2010, 8, 4617.
10. Sharma, S.; Poliks, B.; Chiauzzi, C.; Ravindra, R.; Blanden, A. R.; Bane, S. Bio-
chemistry 2010, 49, 2932 and reference cited therein.
4.1.4. Disulfide-exchange reaction in the presence of tubulin.
5ꢀ10ꢁ3 mmol of 4$tartrate, 5$tartrate and 6$tartrate were dis-
solved in 20 mL of a 1:1 mixture of PEM buffer [10% glycerol,
2
mM EGTA, 1
mM MgCl2, 10 mM piperazine-N,N0-bis(2-
ꢀ
11. Fortin, S.; Lacroix, J.; Cote, M.-F.; Moreau, E.; Petitclerc, E.; Gaudreault, R. C. Biol.
ethanesulfonicacid)] and water (HPLC purity grade). DMSO (1 mL)
was added to the solution. The solution was divided in two equal
amounts to give Solution A and Solution B. 5ꢀ10ꢁ3 mmol of
Proced. Online 2010, 12, 113 and reference cited therein.
12. Cappelletti, G.; Maggioni, M. G.; Ronchi, C.; Maci, R.; Tedeschi, G. Neurosci. Lett.
2006, 401, 159.