Synthesis and biological evaluation of a biotinylated paclitaxel with an extra-long chain spacer arm

Article abstract of DOI:10.1021/ml300149z

A biotinylated paclitaxel derivative with an extra-long chain (LC-LC-biotin) spacer arm was synthesized using an improved synthetic reaction sequence. The biotinylated paclitaxel analogue retained excellent microtubule stabilizing activity in vitro. Furth

Full text of DOI:10.1021/ml300149z

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Biological Evaluation of a Biotinylated Paclitaxel with
an Extra-Long Chain Spacer Arm
Lev. G. Lis,† Mary A. Smart,† Anna Luchniak,‡ Mohan L. Gupta, Jr.,‡ and Vadim J. Gurvich*,†
†Institute for Therapeutics Discovery and Development and Department of Medicinal Chemistry, University of Minnesota,
Minneapolis, Minnesota 55414, United States
‡Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, United States
S
* Supporting Information
ABSTRACT: A biotinylated paclitaxel derivative with an extra-long chain (LC-LC-biotin) spacer arm was synthesized using an
improved synthetic reaction sequence. The biotinylated paclitaxel analogue retained excellent microtubule stabilizing activity in
vitro. Furthermore, it was shown that this analogue can simultaneously engage streptavidin and the binding site on microtubules,
making it suitable for localization studies or for the attachment of paclitaxel to solid substrates via a streptavidin linkage.
KEYWORDS: paclitaxel, taxol, biotin, biotinylated
aclitaxel 1 (Taxol, Scheme 1)1,2 is a complex diterpene
natural product that was first isolated in 1971 from the
ends, is not known. One confounding factor has been that
although the paclitaxel binding site on tubulin is oriented
toward the interior lumen of the microtubule, paclitaxel can
diffuse through the 1−2 nm fenestrations present in the
microtubule lattice. Thus, paclitaxel can readily exchange into
preformed or existing polymer.24 Paclitaxel−biotin conjugates
that were previously reported by Rosen25 and Hwu26 contained
shorter linkers (16 and 4 atoms, respectively). To prevent
access through fenestrations or other similar access points that
may exist along the lattice, we sought to attach paclitaxel to a
relatively large molecule such as streptavidin. To facilitate the
ability of the paclitaxel−biotin conjugate to simultaneously
engage streptavidin and the binding site on tubulin within the
microtubule, we sought to maximize the intervening linker arm.
To achieve that goal, we took advantage of a commercially
available biotin with an extra-long chain spacer.
Our synthetic strategy was based on the approach earlier
reported by Nicolaou and co-workers27 for fluorescent taxoid
synthesis and later expanded by Rosen and co-workers25 for
biotin−taxol conjugation using N-hydroxysuccinimide (NHS)
esters of biotin as convenient biotinylation reagents. A similar
approach was reported by Lee and co-workers for paclitaxel−-
camptothecin conjugates.28 NHS is a good leaving group, and
NHS-activated biotins react easily with primary amino groups
forming stable amide bonds. The synthetic sequence is shown
P
stem bark of the western yew, Taxus brevifolia.3 It is widely used
as an anticancer chemotherapeutic drug to treat a variety of
solid tumors such as breast, ovarian, nonsmall cell lung, and
head and neck cancers.2,4−12 Biotin (vitamin B7, vitamin H) is
an essential cellular micronutrient responsible for various
normal cellular functions. Various cancer cell lines are known
to overexpress biotin receptors,13 and it has been suggested that
the sodium-dependent multivitamin transporter (SMVT) is the
primary transporter responsible for biotin uptake.14 SMVT
expression in several lung, renal, colon, and breast cancer cell
lines is higher than that of the folic acid receptor.15 Therefore, it
is possible that a biotin conjugate may be more efficient for
targeting tumors than using a folic acid conjugate.15 This
overexpression makes it possible to use biotinylation as a
strategy for selective delivery of biotinylated anticancer agents
to cancer cells.15−19
Paclitaxel 1 represents a class of clinically proven anticancer
agents that function by promoting microtubule assembly and
suppressing microtubule dynamics, leading to mitotic arrest and
apoptosis.20 Understanding how compounds like paclitaxel
exert influence over microtubule dynamics holds potential for
optimizing microtubule-based therapies. Under saturating
conditions, paclitaxel binds to microtubules at a 1:1 molar
ratio to tubulin.21 However, the dynamics of microtubules are
sharply suppressed when paclitaxel-like compounds are bound
at very low stoichiometry.22,23 Currently, the binding arrange-
ment of paclitaxel under these conditions, that is, spread
throughout the lattice or bound preferentially to microtubule
Received: June 13, 2012
Accepted: July 26, 2012
Published: July 30, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
degradation. At the previously reported substrate−catalyst ratio
(20:1 by weight),27 deprotection is likely to occur on the 2′-
position initially, producing monodeprotected compound 5
(Figure 1).33 Its structure was confirmed by nuclear magnetic
resonance (NMR) analysis (for NMR data for compounds 4
and 7 and main byproduct 5; see the Supporting Information).
Scheme 1. Synthesis of 7-(β-Alanyl)paclitaxel 4
Figure 1. Partially deprotected compound 5.
Compound 5 is far less soluble than substrate 3 and the
product 4 and precipitated during the reaction. As a result, the
process significantly slowed down, increasing the level of
degradation of the substrate or product. Therefore, accelerating
the hydrogenation rate is critical for this reaction. To achieve
that goal, we increased the amount of the Pd−C catalyst 5-fold
and used shaking instead of stirring to increase the efficiency of
mixing. A slow stream of hydrogen gas in the reaction vessel
allowed rapid removal of the carbon dioxide formed from the
reaction. This protocol achieved a 90% yield of compound 4
within a 2 h period.
In the next step, compound 4 was reacted with the
hydroxysuccinimide ester of LC-LC-biotin 6 (NHS-LC-LC-
biotin) (Scheme 2). The reaction was carried out in in
Scheme 2. Biotinylation of 7-(β-Alanyl)paclitaxel 4
in Scheme 1. In the first step, the 2′-hydroxy group of paclitaxel
1 was protected by carboxybenzylation. The resultant 2′-
carboxybenzyl derivative 2 was subjected to esterification at the
7-hydroxy group. The 2′-hydroxy group is protected, and the 1-
hydroxy group is unreactive; therefore, the subsequent
acylation takes place selectively at the 7-hydroxy group. It
was previously reported that α-amino esters at the 7-hydroxy
group of paclitaxel are very unstable.29 As an alternative, β-, γ-,
or ω-amino acid esters, which are far more stable, can be
used.27,30,31 We employed N-carboxybenzoyl-β-alanine32 for
esterification of the 7-hydroxy group, resulting in compound 3.
The next step, deprotection of both carboxybenzyl groups by
hydrogenation, led to the formation of key intermediate 4.
Our study revealed that deprotection of compound 3 by
hydrogenation is very sensitive to the reaction time, amount of
solvent, and amount of catalyst. A significant formation of
degradation products was observed when the reaction time
exceeded 2.5 h. Using a more diluted solution (4.2−6.7 mM) of
substrate 3 (as compared to the previously reported 21 mM)27
increased the reaction time from 2 to 18 h and the level of
anhydrous mixture of dichloromethane (DCM) and dimethyl-
formamide (DMF) in the presence of N-ethylmorpholine at 4
°C for 24 h to produce paclitaxel-LC-LC-biotin 7. Because the
thin-layer chromatography (TLC) mobility of the product 7 is
very similar to that of NHS-LC-LC-Biotin 6, preparative HPLC
was employed for the final purification of the product.
The 1H NMR spectrum shows broad multiplets at 3.08−3.32
and 3.6 ppm (total seven protons), which are characteristic for
the three CH2NC(O) groups and the Hβ of the CH2S group
of biotin. The signals at 4.25 and 4.45 ppm represent the two
biotin CHN protons. The group of signals at 1.14−1.69 ppm is
associated with the aliphatic protons of the spacer arm.
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We determined the relative activity of compound 7 in a
tubulin assembly assay (Figure 2). The EC50 for stimulation of
Visualization of Cy3-labeled streptavidin confirmed that
biotinylated paclitaxel 7 bound to streptavidin could also bind
to tubulin during microtubule polymerization and be retained
during sedimentation through the glycerol cushion and dilution
prior to visualization. Microtubules assembled with paclitaxel in
the presence of identical concentrations of fluorescent
streptavidin did not contain any Cy3 signal associated with
the polymer, demonstrating that streptavidin was not simply
constrained within the microtubules during polymerization but
was bound to biotinylated paclitaxel 7 during polymerization
and isolation of the microtubules.
In summary, we developed an improved process for the
synthesis of the key intermediate amine 4 that is commonly
used25,26,27,28,30,31,34,35 in biotinylation of taxol derivatives.
Using this process, we synthesized a biotinylated paclitaxel
derivative with the longest spacer arm reported as of yet. This
long spacer arm did not significantly disrupt the microtubule
stabilizing activity of paclitaxel, yet allowed microtubule-bound
paclitaxel to engage streptavidin through the biotin moiety.
This compound, bound to streptavidin, can be utilized in
biochemical studies to both stabilize microtubules and restrict
paclitaxel access through microtubule fenestrations. Biotiny-
lated paclitaxel 7 could also potentially be used to attach
paclitaxel to solid supports or for selective delivery into cells.
Figure 2. Tubulin assembly assay for paclitaxel (circles) and
biotinylated paclitaxel 7 (squares). The EC50 was 0.52 0.1 μM for
paclitaxel 1 and 0.68 0.1 μM for biotinylated paclitaxel 7. The data
points are from two independent experiments.
microtubule polymerization in vitro was 0.52 0.1 and 0.68
0.1 μM for paclitaxel 1 and the biotinylated paclitaxel 7,
respectively. Thus, the presence of the long linker biotin moiety
at position 7 did not significantly impact the microtubule
polymerizing and stabilizing activity of paclitaxel.
To determine whether biotinylated paclitaxel 7 could
simultaneously bind to streptavidin and microtubules, we
used biotinylated paclitaxel 7 prebound to fluorescent
streptavidin to drive microtubule assembly. The microtubules
were sedimented through a glycerol cushion to remove
unbound streptavidin and paclitaxel before being visualized by
fluorescent microscopy (Figure 3). Oregon green-labeled
tubulin demonstrated that both paclitaxel and biotinylated
paclitaxel 7 stabilized microtubules during this procedure.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental priocedures, copies of 1H NMR and 13
C
NMR, MS, and UPLC chromatogram for the final product 7,
and analytical data for key intermediate 4 and main byproduct
5. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: vadimg@umn.edu.
■
Funding
We gratefully acknowledge a National Institutes of Health
Grant (GM094313), Cancer Research Foundation Young
Investigator Grant, and American Cancer Society Institutional
Research Grant (#IRG-58-004-48) to M.L.G. for providing
financial support for this study.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are thankful to Professor Gunda I. Georg, University of
Minnesota, for helpful discussions in the course of this study.
■
ABBREVIATIONS
■
Cy3, 3H-indolium, 2-[3-[1-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-
6-oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-yli-
dene]-1-propen-1-yl]-1-ethyl-3,3-dimethyl-5-sulfo-, inner salt;
DCM, dichloromethane; DMF, dimethylformamide; NMR,
nuclear magnetic resonance; TLC, thin-layer chromatography
Figure 3. Biotinylated paclitaxel 7 can simultaneously engage
microtubules and streptavidin. Microtubules were polymerized from
Oregon green-labeled tubulin in the presence of Cy3-labeled
streptavidin and either paclitaxel 1 (bottom row) or biotinylated
paclitaxel 7 (top row). Microtubules were sedimented through a 40%
glycerol cushion before being visualized by fluorescence microscopy
for microtubules (left) and streptavidin (center).
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