Labeled Taxols

The synthesis of labeled analogs of taxol has been an important aspect of research on the drug. Radioactively labeled analogs are needed for metabolism and related studies, while photoaffinity-labeled and fluorescently labeled analogues have been used to study the interaction of taxol with tubulin. This section will focus on the synthetic aspects of this work the biochemical studies of the taxol-tubulin interaction will be discussed in a later section. 

Several syntheses of carbon-14 labeled taxols have been reported. N-3 -[Carbonyl- C]-taxol 11.1.1 was prepared by coupling a [carbo-nyl- C]-A -benzoyl- 5-lactam with 7-(triethylsilyl)baccatin III (378), while the same group prepared [3 - C]-taxol 11.1.2 by using a / -lactam prepared from carbonyl- C-benzaldehyde (379). [3 - C]-Docetaxel 11.1.3 was prepared by attachment of a labeled cinnamoyl side chain to 7,10-ditroc-10-deacetylbaccatin III, followed by hydroxyamination of the side chain (380). A slightly different approach was used to prepare [2, 3 - C2]taxol, in that the required doubly labeled /3-lactam synthon was prepared using a doubly labeled Oppolzer s bromoacylbomanesul-tam intermediate (381). 7-([carbonyl- C]-acetyl)taxol was prepared by simple acylation of 2 -(triethylsilyl)taxol with [carbonyl--acetic anhydride and deprotection (382). The taxol analog PNU-105298 (4.1.3.12) was also prepared in both deuterium and carbon-14 labeled forms in both cases the label was placed on the side chain (383). 

The [ I]-labeled taxol derivative 11.1.5 was prepared by reacting the stannane 11.1.4 with sodium [ I]-iodide (384). 

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The location of the binding site of taxol on tubulin has been determined by photoaffinity labeling using several differently labeled taxol analogs. An early study showed that taxol labeled -tubulin as opposed to a-tubulin 466), and this result was confirmed with the azidophenyl taxol analog 15.1.1 467). 

Walker DG, Swigor JE, Kant J, Schroeder DR (1994) Synthesis of Carbon-14 Labeled Taxol (Paclitaxel). J Labelled Cpd Radiopharm 34 973... 

Nicholov R, Kingston DGI, Chordia MC, DiCosmo F (1997) Molecular Mobility of Nitroxyl-labelled Taxol During Tubulin Assembly. FEBS Lett 405 73... 

By using an elegant 13C-labeling study that involved incubation of Phoma sp. with 1-13C and 1,2-13C acetate, Oikawa et al. [9], were able to isolate the proposed biosynthetic intermediate phomactatriene (or Sch 49026), with 13C incorporation from singly labeled acetate units as indicated by in Fig. 8.3. Phomactatriene is strikingly reminiscent of taxadiene, a biosynthetic intermediate for Taxol . The net biosynthesis for both involves geranylgeranyl diphosphate (GGDP) cyclization [9]. It is noteworthy that prior to isolation of phomactins, the only known related structure is cleomeolide, a diterpene from the herb Cleome viscosa [10] that remarkably resembles phomactin H. 

Fig. 5. 3D EM shows how kinesin and tau bind to microtubules. (A) Reconstruction of a microtubule decorated with kinesin heads (ochre). One kinesin head binds per afi-tubulin heterodimer (grey) and, due to its asymmetric form, can be used to distinguish between the subunits. (B) Inside view of a microtubule that was coassembled with gold-labeled tau and decorated with kinesin heads. The kinesin heads can be seen on the outside through the holes between the protofilaments. The labeled repeat motif of tau binds to the inside face of microtubule. The averaged nanogold density (yellow), which is attached to a repeat motif of tau through a linker, can only be seen near the Taxol binding site of -tubulin, but not on the a subunit (Kar et al, 2003a). The ribbon diagram of the refined zinc-sheet structure is also shown for reference (see Figure 3).

Simultaneously with the refinement of the EC structure, additional data on the conformation of taxol bound to MT came from ss-NMR REDOR measurements [84]. As introduced above, the REDOR experiment exploits the dipolar interaction between nuclear spins to determine distances between atoms closer than 12 A. A PTX analogue with suitable spin labeling for REDOR was prepared and complexed to MT. Labeling of the amide carbonyl and C3 methine with 13C, and position p- of the 2-O-benzoyl with 19F (Fig. 9, compound 3), allowed the measurement of the two 19F-13C distances I and II (Fig. 9), that could serve, in principle, to discriminate among candidates for PTX conformation. The respective values of 9.8 A 0.5 A and 10.3 A 0.5 A were interpreted by the authors as supportive of the hydrophobi-cally collapsed polar conformation for PTX [84]. 

More recently, additional 2H "I J REDOR measurements on new PTX derivatives bound to MT provided three new interatomic distances that helped to define more precisely the bound conformation of PTX [87], The two PTX analogues labeled with fluorine and deuterium were designed to determine the three key distances based on the measurement of 2II— "I distances, as indicated in Fig. 9 (compounds 4 and 5). These interatomic distances rule out the polar and nonpolar conformers and further support the T-taxol conformation (Tablel). The polar conformation is rejected on the basis of distance IV, as the 4.5 A is much shorter than the > 8 A determined by REDOR. The nonpolar conformation can be ruled out on the basis of distances I and II, that are too short in the nonpolar conformer compared to the experimental ones (1.8 and 3.6 A short). Besides, bridged taxanes resembling the nonpolar conformation displayed no activity in the tubulin assembly assay [88], PTX-NY closely matches the REDOR distances. Nevertheless, it is discarded as the bioactive form of PTX due to its poor fit to the EC density [87],... 

Discodermolide (DDM, Fig. 16) is a marine natural product that promotes MT assembly more potently than PTX and is active against some PTX-resistant cell lines [118-120], The photoaffmity analogue C19-BPC-DDM labels (3-tubulin in close proximity to the taxol binding site [121] and DDM itself is a competitive inhibitor of PTX binding to MT [120], suggesting that DDM also binds to the PTX binding site on P-tubulin. 

Fig. 30 Taxoid site on (3-tubulin and predicted peloruside site on a-tubulin. a Surface representation (view from the inner side of the microtubule) of a tubulin dimer with FIX (red) bound to P-tubulin (green) and peloruside A (orange) bound to the predicted site in a-tubulin (blue), b View of the peloruside binding site. Hydrogen bonds are represented as yellow dashed lines, and the residues involved in these bonds are labeled. Some secondary structure elements are also labeled, c View of the taxol binding site. Some secondary structure elements are labeled. In panels b and c, H7 is colored in orange, and the N-terminal and intermediate domains are colored in green and blue, respectively. (Reprinted with permission from [17]. Copyright 2006 American Chemical Society)...

Georg, G. I. Harriman, G. C. B. Himes, R. H. MejiUano, M. R. Taxol photoaflinity label 7-(p-azidobenoyl) taxol synthesis and biological evaluation. Bioorg. Med. Chem. Lett., 1993, 2 735-738. 

Recently, we proposed a new bioactive conformation of paclitaxel, RKDOR-Taxol [50], based on (i) the 19F-13C distances obtained by the REDOR experiment [49], (ii) the photoaffinity labeling of microtubules [51], (iii) the crystal structure (PDB code 1TUB) of the Zn2+-stabilized aP-tubulin dimer model determined by cryo-electron microscopy (cryo-EM) [52], and (iv) molecular modeling (Monte Carlo Macromodel) [50], In this computational biology analysis, we first docked a paclitaxel-photoaffinity label molecule to the position identified by our photoaffinity labeling study and then optimized the... 

Klecker, R.W. Jamis-Dow, C.A. Egorin, M.J. Erkmen, K Parker, R.J. Stevens, R. Collins, J.M. Effect of cimetidine, probenecid, and ketoconazole on the distribution, biliary secretion, and metabolism of [3H]taxol in the Sprague-Dawley rat. Drug Metab.Dispos., 1994, 22, 254-258 [rat plasma hile urine lung spleen liver kidney heart muscle brain testes fat UV detection radioactivity detection tritium labeled]... 

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