Design and synthesis of a novel photoaffinity taxoid as a potential probe for the study of paclitaxel-microtubules interactions

Article abstract of DOI:10.1016/S0040-4039(00)00627-4

A novel and non-radioactive bifunctional photoaffinity probe (BPP) for the study of paclitaxel-microtubules interactions is designed and synthesized. This new BPP-taxoid bears a 3-nitro-5-trifluoromethyl- diazirinyl-phenoxyacetyl group at C-3'N for photoaffinity/photocleavage and a biotin subunit at C-7 for affinity chromatography. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00627-4

Tetrahedron Letters 41 (2000) 4287±4290
Design and synthesis of a novel photoanity taxoid as a
potential probe for the study of paclitaxel±microtubules
interactions
Songnian Lin,a Kan Fang,b Masaru Hashimoto,b Koji Nakanishib and Iwao Ojimaa,*
aDepartment of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA
bDepartment of Chemistry, Columbia University, New York, NY 10027, USA
Received 17 February 2000; revised 4 April 2000; accepted 5 April 2000
Abstract
A novel and non-radioactive bifunctional photoanity probe (BPP) for the study of paclitaxel±micro-
tubules interactions is designed and synthesized. This new BPP-taxoid bears a 3-nitro-5-tri¯uoromethyl-
diazirinyl-phenoxyacetyl group at C-30N for photoanity/photocleavage and a biotin subunit at C-7 for
anity chromatography. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: taxoid; bifunctional probe; photoanity label; biotin; diazirine.
Taxol1 (paclitaxel) and taxotere1 (docetaxel), are extremely important new chemotherapeutic
agents for the treatment of various cancers.1 4 Since the ®rst characterization of paclitaxel in
1971,5 its complex structure, unique mechanism of action,6 and potent antineoplastic activity7,8
have served as the impetus for intensive studies not only in clinical oncology, but also in the wide
range of biomedical research arena. Searching for the three-dimensional biologically active
structures (binding structures) of paclitaxel has been one of the active directions along this line.9
Clarifying the binding site of paclitaxel at microtubules will provide a full understanding of
paclitaxel±microtubule interactions when paclitaxel binds to microtubules, and develop a rational
basis for improving drug design. Although X-ray analysis or electron crystallography may eventually
provide this information,10 photoanity labeling and protein sequencing can provide direct
information about the structure of the drug binding site, and considerable progress has been
made in this area.11,12 Recently, a new and ecient photoanity labeling protocol for ligand/
receptor interaction studies has been developed in these laboratories.13 We describe here the
design and synthesis of a novel photoreactive taxoid as the probe for paclitaxel±microtubule
interactions applying the new protocol.
* Corresponding author. Tel: 1-631-632-7890; fax: 1-631-632-7942; e-mail: iojima@notes.cc.sunysb.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00627-4

4288
As Fig. 1 shows, a bifuntional photoanity probe (BPP) with a photoanity label (site A) and
a photocleavable moiety (site B) can be used to streamline the frequently tedious photoanity
labeling process. The new process will include: (i) the BPP-taxoid is bound to the receptor
(microtubules) and photolyzed; (ii) the receptor is cleaved enzymatically or chemically; (iii) the
crosslinked peptide fragments (with biotin tag) are separated from non-crosslinked ones; (iv)
taxoid is detached from the crosslinked peptides by site B photocleavage; (v) the mixture of peptides
with nitrophenolic marker is sequenced by tandem MS. Tandem MS should be able to directly
sequence the respective peptide fragment at femtomole level without separation of mixture.
Moreover, no radioisotope is required.
Figure 1. Proposed BPP protocol
In order to assess the ecacy of the proposed BPP protocol, a novel, bifunctional photoanity
probe of paclitaxel with a biotin tag (1, Fig. 2) was designed. The BPP moiety for photoanity/
photocleavage, 3-nitro-5-tri¯uoromethyldiazirinyl-phenoxyacetyl group, is connected to the C-30
position, and the biotin tag for anity chromatography, is attached to the C-7 position. BPP-taxoid
1 was synthesized through the following three steps: (a) LiHMDS-mediated coupling of modi®ed
baccatin III 4 with b-lactam 3; (b) attachment of biotin tether (9) to the C-7 position of taxoid 6
by a succinyl linkage, and (c) connecting the BPP moiety 14 to the C-30N position of taxoid 12.
Figure 2. Taxoid photoanity labeling probe 1
(3R,4S)-3-TIPSO-4-phenyl-b-lactam 5 with 96% ee was prepared in excellent yield through a
highly ecient chiral ester enolate±imine cyclocondensation reaction.14 16 Protection of the N-H
group with Cbz-Cl gave N-Cbz-b-lactam 3. Taxoid 5 was then synthesized using the ring-opening
coupling protocol developed in these laboratories.1,11,14 18 Thus, the couplings of 7-TES-baccatin
(4)19 with b-lactam 3 was carried out to a€ord taxoid 5. 7-TES group was selectively removed by
0.1N HCl/EtOH to give 7-OH-taxoid 6. Treatment of 6 with succinic anhydride in the presence of
Et3N and DMAP a€orded taxoid-acid 7 bearing a monosuccinic acid moiety at the C-7 position
(Scheme 1).

4289
Scheme 1. Synthesis of taxoid-acid 7. Reaction conditions: (i) Cbz-Cl (4 equiv.), Et3N/DMAP, CH2Cl2, re¯ux, 20 h; (ii)
LiHMDS (1.5 equiv.), THF, ^40 to ^30ꢀC, 30 min; (iii) HCl (0.1N), EtOH, rt, 24 h; (iv) succinic anhydride (10 equiv.),
Et3N (excess), DMAP (6 equiv.), CH2Cl2, re¯ux 5 h
Alcohol 9 was prepared in two steps from 2-[(2-azidoethoxy)ethoxy]ethanol and biotin p-nitro-
phenol ester,20 which was smoothly coupled with taxoid acid 7 using DCC/DMAP to a€ord
taxoid 10 with a biotin tag. It was found that the Cbz group at the C-30N position cannot be
removed by hydrogenolysis in the presence of the TIPS group at the C-20 position. Thus, TIPS
group was removed ®rst. Hydrogenolysis of the resulting C-20-OH taxoid 11 on Pd±C a€orded
taxoid-amine 13, which is ready for coupling with the BPP moiety (Scheme 2).
Scheme 2. Synthesis of taxoid-amine 12
BPP-aldehyde 14 was synthesized from 3-allyloxy-5-methyloxycarbonylbenzaldehyde (13)
using the method reported previously.13 Oxidation of aldehyde 14 with Ag2O provided BPP-acid
15. The coupling of BPP-acid 15 with taxoid-amine 12 using EDC/HOBt a€orded desired BPP-
taxoid 121 bearing a biotin tag at C-7 (Scheme 3).
Scheme 3. Final synthesis of taxoid probe 1
In conclusion, we have designed and synthesized a new bifunctional photoanity probe
of paclitaxel for the study of paclitaxel±microtubules interactions based on a non-radioactive

4290
photoanity labeling protocol. The results of photoanity experiments with BPP-taxoid probe 1
will be reported in due course.
Acknowledgements
This research was supported by grants from the NIH (NIGMS, to I.O. and AI10187, to K.N.).
Generous support from Indena, SpA, Italy is also gratefully acknowledged.
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20
D
21. Mp 112±124ꢀC; ‰ꢀŠ ^13.5 (c 0.52, CHCl3); 1H NMR (500 MHz, CDCl3) ꢁ 1.15 (s, 3H), 1.17 (s, 3H), 1.45 (m, 2H),
1.67 (m, 4H), 1.80 (s, 3H), 1.85 (s, 3H), 1.86 (m, 1H), 2.16 (s, 3H), 2.20 (m, 2H), 2.25 (m, 2H), 2.31 (s, 3H), 2.50
(m, 1H), 2.60 (m, 4H), 2.72 (m, 1H), 2.90 (dd, J=12.5, 5.0 Hz, 1H), 3.15 (m, 1H), 3.46 (m, 2H), 3.57±3.75 (m, 8H),
3.85 (d, J=6.5 Hz, 1H), 4.16 (d, J=8.5 Hz, 1H), 4.29 (d, J=8.5 Hz, 1H), 4.36 (dd, J=8.0, 5.0 Hz, 1H), 4.41 (m, 1H),
4.57 (dd, J=7.5, 5.0 Hz, 1H), 4.63 (d, J=14.5 Hz, 1H), 4.69 (d, J=14.5 Hz, 1H), 4.74 (d, J=3.5 Hz, 1H), 4.91 (d,
J=10.0 Hz, 1H), 5.56 (dd, J=10.0, 7.0 Hz, 1H), 5.61 (dd, J=8.5, 3.0 Hz, 1H), 5.65 (d, J=6.5 Hz, 1H), 6.06 (t, J=9.0
Hz, 1H), 6.16 (s, 1H), 6.81 (bs, 1H), 7.08 (s, 1H), 7.29 (t, J=7.0 Hz, 1H), 7.37 (t, J=7.5 Hz, 2H), 7.43 (t, J=7.5 Hz,
2H), 7.49 (t, J=7.0 Hz, 2H), 7.63 (t, J=7.5 Hz, 1H), 7.68 (s, 1H), 7.86 (t, J=2.0 Hz, 1H), 8.06 (d, J=7.5 Hz, 2H),
8.30 (d, J=9.0 Hz, 1H); 19F NMR (CDCl3) ꢁ ^65.51.; HRMS (FAB) m/z calcd for C70H82N7O24F3S.H+:
1494.5162. Found: 1494.5177 (Á=^1.0 ppm).