Recent Developments in a Radio-labeling of Brassinosteroids

The chapter provides a comprehensive overview on methodologies used for radio-label- ing of brassinosteroids as one of the newest class of phytohormones. Discussed labeling strategies are lined up in terms of reached specific activities (SA) of brassinosteroids (BRs) as a key parameter for further utilization of such labeled drugs. The chapter is focused on two key natural radio-isotopes (tritium and carbon-14) used for drug tracing in phar - maceutical research. The catalytic reductive dehalogenation of BR–chlorocarbonates was studied with deu terium in the system of 2 H 2 /Pd[0]/Et 3 N providing appropriate 3β-deutero-2,3-carbonates with 70–80% deuterium enrichment (based on 1 H NMR) at the C-3 position and with an isolated yield of up to 65% (initially on a cheap pregnane analog available in multi-game scale). The best results for deuterium dehalogenation were achieved with the molar ratio of PdO/CaCO 3 (5%)/Et 3 N/chlorocarbonate being 2:6:1 for every BR derivative at a short period of time (6 h). When the amount of the base was too high, it diminished the yield of [ 2 H]-labeled ethylene carbonate. Various catalysts such as Pd/C (either 5 or 30%), PdO/BaSO 4 (10%) used for reduction yielded lower yield of desired carbonate (15–19%). Authors disclosed very significant solvent effect with an impact on the isolated yield as well as the by-products formation. Briefly, the best results provided EtOAc (dry), giv ing up to a 65% yield of labeled carbonate with 80% 2 H-enrichment. Other solvents used for reduction provided both low conversion and isolated yield of carbonate (0–19%). Androstane chlorocarbonate employed for reductive dehalogenation under similar condi tion as for pregnane analog provided analogous results (58% yield, 75% 2 H-enrichment at C-3). Surprisingly, in addition to desired labeled carbonate two by-products were detected, isolated, and afterwards characterized in that experiment—the multi-labeled ketone and the multi-labeled alcohol, both in the yield of 15%. The reaction course toward formation of both by-products was further accelerated while protic solvent (MeOH) was used in the reaction [43% (ketone) and 40% (alcohol)]. The reaction conditions used for the labeling of 24-epiCS were the same as described above for the other two steroids. A full conversion of appropriate chlorocarbonate was obtained after a


Radio-labeling
Radioactive labels used for tracing of studied ligands have long been a part of the biological laboratory repertoire. Radioactivity gives a clear, unmistakable signal, and its use is fairly straightforward. Because of smooth traceability, visualization in organ tissues, quantification (liquid scintillation counting [LSC]), and unsurpassed sensitivity of radio-labeled molecules the radio-labeling is a powerful and practical tool to closely follow accurate mass balance and monitor the fate of a molecule on the molecular level and its biochemically transformed derivatives. Pharmacokinetic studies have traditionally used radio-labeled target compounds as a means for evaluating body absorption, distribution, metabolism, and excretion (ADME) [1,2]. The use of radioligands is essential tool in binding assays aimed at ligand-receptor structure-activity relationship studies, which however requires high-specific activity (SA-a qualitative parameter) of studied ligands (because of, in general, very low concentration of receptors in tissues). Weak β-emitters such as tritium ( 3 H, T) and carbon-14 ( 14 C) are by far the most versatile and convenient natural labels available [3]. These two isotopes preserve molecular structure (no added tags or pendant groups that alter or change the structure). The advantages of 3 H compared to 14 C are much higher specific activity, significantly lower cost of starting material, and environment friendly radioactive waste management (shorter half time, Table 1). Also, an introduction of radio-isotope in a later stage of synthetic sequence (often in the last step) is a critical benefit in terms of synthetic yield, safety handling, and waste disposal. In general, all above-mentioned issues come out in favor of tritium over carbon- 14. On the other hand, a relevant advantage of carbon-14 is lower potential of label loss. Label selection is usually at the discretion of the investigator and studies can be reported using either 3 H or 14 C label. For instance, tritium label could be applied to earlier stage development studies and then switched to a 14 C label for the later stage development studies, for example, an advanced human ADME [4]. Each compound radio-labeled at the non-exchangeable and metabolically stable position need to possess radiochemical purity (RCP) basically over 97%. Instability of all radioligands caused by self-radiolysis requires a need to check a radiochemical purity of studied radio-labeled material before a particular experiment is carried out. Such instability can be significantly suppressed by appropriate storage of labeled material. In general, samples stored at −196°C (Cryoflex-sealed vial immersed in liquid nitrogen) in alcoholreached medium last over 1-2 years in acceptable quality. Radiochemical purity (RCP) is then usually still over 95%. Samples in such conditions can be often used immediately for biological experiments and no further purification is needed. On the contrary, radio-labeled drugs stored in a refrigerator (+4°C) over 1 year rarely show better than 90% RCP. Foremost advantage of using radio-labeled drugs is that the radioactivity is easily detected and quantified using liquid scintillation techniques in a very low limit of detection (technically <1 Bq/L) [5,6].

Brassinosteroids: a newest class of phytohormones
The entire evolutionary process in plants is regulated by the changes of hormonal concentration, tissue sensitivity, and their interaction during the entire life cycle of plants. One of the most recent groups of phytohormones represents brassinosteroids. They occur at low levels distributed throughout the plant kingdom [7]. The ability of plants to biosynthesize a large variety of such steroids were discovered in 1970 by Mitchell et al. who first from 40 kg of beecollected rape pollen of Brassica napus L. isolated and characterized a steroidal lactone named brassinolide [8]. Few years later, castasterone was found in insect galls of Castanea crenata spp. Up to date, more than 70 structurally and functionally related polyhydroxylated sterol derivatives are known and this group of compounds has been identified as the natural brassinosteroids (BRs) [9]. All BRs have a 5α-cholestane skeleton, with functional variations due to differences in orientations of oxygenated functions on the skeleton [10,11].
The highest concentration of BRs in plants is detected in the reproductive organs (pollen and seeds; 1-100 ng/g). They were also detected in other plant organs from roots to leaves. They are involved in various kinds of regulatory actions on growth and development, that is, stimulation of cell expansion, cell division, stress tolerance, accretion of biomass, yield and quality of seeds, and plant adaptability [9]. The metabolism of nucleic acids and proteins and the gene expression is changed by BRs at the molecular level.
The extremely high activity of brassinosteroids has attracted the attention of many specialists in the field of analytical and synthetic chemistry, biochemistry, plant physiology, and agriculture. Recently, it has been reported that some natural BRs have (besides early reported, e.g., antibacterial, antiviral, antifungal, and neuroprotective) potent cell growth inhibitory activities in animal and human cancer cell lines without affecting the normal cell growth (BJ fibroblasts) [12,13]. The presence of a lactone or ketone moiety in ring B and diol functions (2α, 3α-and 22R, 23R-) turned out to be an essential factor for high biological activity of such BRs (Figure 1). This chapter is engaged in the recent developments in the synthesis of 3 H-and 14 C-radiolabeled analogs of the brassinolide.  (Figure 2). The labeling strategy is designed to employ a radio-labeling step at the later stage of the synthetic sequence.

Synthesis of tritium-labeled
In 1998, Seto et al. described a fairly elegant strategy for the deuterium-multi-labeling of brassinolide in its side-chain [15]. The five-step reaction sequence was started by full protection of hydroxyl groups on the BR. The C-25 carbon was oxidized by freshly generated trifluoromethyldioxirane (TFD) that yielded appropriate hydroxy derivative. Its consecutive dehydration led to a mixture of Δ 25(26) and Δ 24(25) (2) was synthesized [14].

Stability of BRs possessing very high SA
The free BRs with a high SA are extremely sensitive to radiolysis if stored improperly. Authors reported one representative example-when a sample was evaporated to dryness and used for the NMR analysis (DMSO-d 6 ), the signals in 3 H NMR spectrum were difficult to assign [14]. Hence, the particular NMR sample was re-checked for purity by radio-HPLC, and indeed, only 12% of activity of desired 24-[24, 25, 26, 27-3 H]epiCS ([ 3 H]-2) was left whereas 88% of radioactivity was found in a broaden peak with higher retention on the column (Figures 3  and 4). Nevertheless, authors reported secure procedure to remove chromatographic solvents and formulate the high-SA BRs for application in biochemical experiments. Combined HPLC fractions (each about 2 mL) were first enriched with glycerol (300 mL) (acts as an antioxidant and also prevents risk of getting dryness of labeled samples) and 10 mg of (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (acts as additional antioxidant with no harm in eventual biological experiments) [14]. When methanol and water evaporated on

Reductive tritium-dehalogenation of generated chlorocarbonates (6 Ci/mmol)
To get polyhydroxylated steroid regio-and enantio-specifically labeled on the un-exchangeable position of C-3, a general procedure can be effectively used ( Figure 5) [17]. A suitable precursor for the introduction of tritium, 3β-chloro-2,3-carbonate derivative, is synthetically affordable by a short-reaction sequence from a 2α,3α-dihydroxy steroid to be labeled (Figure 6) [18].  (Figure 5) [19]. A crucial aspect in the reductive dehalogenation of the chloro derivative is the choice of a solvent that would provide a reasonable yield [17]. The optimized reaction conditions turned out to be PdO/CaCO 3 (5%)/Et 3 N/chlorocarbonate (2:6:1) dissolved in dry EtOAc for all investigated steroids. The successful 3 H-labeling experiments have proven the stereo selectivity of the reductive dehalogenation and afforded a product with high specific activity (5.8 Ci/mmol).
The synthetic procedure starts with transformation of the vicinal 2α,3α-diols of appropriate BR to α-hydroxy ketone by oxidation with a freshly generated dimethyldioxirane (DMD). Such α-hydroxy ketone moiety proved to be an excellent substrate for high-yield enantiospecific formation of 3β-chloro-2,3-carbonate by a reaction with easy-to-handle triphosgene.
The key substrate for reductive dechlorination-3β-chloro-2,3-carbonate-was synthesized by a three-step reaction sequence in an overall yield of 46-55% (Figure 6; representative synthesis of 24-[ 3 H]epiCS). To improve the solubility of the starting steroid (to be labeled) in a  [20]. The reaction carried out in CH 2 Cl 2 overnight at 4°C in dark afforded the desired α-hydroxy ketone 13 accompanied by its regioisomer 3-hydroxy ketone, in a ratio of 10:1 in favor of isomer 13. In general, α-hydroxy ketone 13 is supposed to be isolated in a higher yield when the oxidation of 2,3-unprotected diols takes place; a selective deprotection of acetonide by Ce(NH 4 ) 2 (NO 3 ) 6 in borate buffer followed by the DMD oxidation of the vicinal 2,3-diol group used to provide a higher overall yield of about 5% [21]. However, in the case of the 28-HCS derivative, a partial deprotectionoxidation reaction sequence afforded a drop of an isolated yield of about 15% compared to direct oxidation of the protected derivative. The 3β-chloro-2,3-carbonate 14 was synthesized by a stereospecific reaction of 13 with triphosgene in dry benzene providing a quantitative yield in 3 h (Figure 7).  The catalytic reductive dehalogenation of BR-chlorocarbonates was studied with deuterium in the system of 2 H 2 /Pd[0]/Et 3 N providing appropriate 3β-deutero-2,3-carbonates with 70-80% deuterium enrichment (based on 1 H NMR) at the C-3 position and with an isolated yield of up to 65% (initially on a cheap pregnane analog available in multigame scale). The best results for deuterium dehalogenation were achieved with the molar ratio of PdO/CaCO 3 (5%)/Et 3 N/chlorocarbonate being 2:6:1 for every BR derivative at a short period of time (6 h). When the amount of the base was too high, it diminished the yield of [ 2 H]-labeled ethylene carbonate. Various catalysts such as Pd/C (either 5 or 30%), PdO/BaSO 4 (10%) used for reduction yielded lower yield of desired carbonate (15-19%). Authors disclosed very significant solvent effect with an impact on the isolated yield as well as the by-products formation. Briefly, the best results provided EtOAc (dry), giving up to a 65% yield of labeled carbonate with 80% 2 H-enrichment. Other solvents used for reduction provided both low conversion and isolated yield of carbonate (0-19%). Androstane chlorocarbonate employed for reductive dehalogenation under similar condition as for pregnane analog provided analogous results (58% yield, 75% 2 H-enrichment at C-3). Surprisingly, in addition to desired labeled carbonate two by-products were detected, isolated, and afterwards characterized in that experiment-the multi-labeled ketone and the multi-labeled alcohol, both in the yield of 15%. The reaction course toward formation of both by-products was further accelerated while protic solvent (MeOH) was used in the reaction [43% (ketone) and 40% (alcohol)]. The reaction conditions used for the labeling of 24-epiCS were the same as described above for the other two steroids. A full conversion of appropriate chlorocarbonate was obtained after a 6-h reaction [PdO/CaCO 3 (5%)/Et 3 N/substrate 2:6:1] in dry EtOAc. The isolated yield of labeled carbonate was determined to be 31% [D/H at C-3 = 70:30]. Both the by-products, ketone (20%) and alcohol (13%), were isolated too. The use of DMF as solvent reduced the conversion of chlorocarbonate to 45%, the yield of labeled carbonate down to 19% and the formation of by-products to 11 and 10%, respectively.
As was already mentioned, traces of water (partly synthesized by the reduction of PdO with D 2 gas) play a crucial role in the suggested mechanism of by-product formation (Figure 8) [17]. The initial oxidative addition of in situ generated Pd  elimination of H 2 O leading to 19. Authors declare a statement that theoretical pathway B was not supported by any analytical findings. First, the α-hydroxyketone was not detected in the reaction mixture. Moreover, the single experiment using synthesized α-hydroxyketone and reaction conditions simulating conditions used for reductive deuterium dehalogenation, yielded ultimately 2α,3β-dihydroxy-3α[ 2 H] derivative in an isolated 85% yield and no other product was identified in the reaction mixture. In advance, the absolute configuration at C-2 and C-3 of 2α,3β-dihydroxy derivative was confirmed by was confirmed by X-ray analysis affording ORTEP diagram. A regular ESI-MS analysis of 20 and 21 measured in CH 3 OH has confirmed the structure of multi-labeled ketones with the distribution of deuterium 1D/2D being 70:30. The second by-product, multi-labeled alcohols 22 and 23, are most likely formed by a Pd-catalyzed reduction of the ketones 20 and 21 by D 2 . The structure of 22 and 23 was confirmed by 1 H NMR as well as ESI-MS with a deuterium distribution 1D/2D/3D being 10:50:40.
In view of the favorable results of deuterium experiments, this protocol was followed using tritium gas. Tritium dehalogenation experiment was designed following the optimized reaction conditions [PdO/CaCO 3 (5%)/Et 3 N/substrate 2:6:1, dry EtOAc]. 3 H-labeled 24-epiCS and 24-epiBL, was synthesized when the appropriate 3β-chloro-2,3-carbonate 14 was used for a reaction with carrier-free tritium gas (600 mbar), PdO/CaCO 3 (5%), and in presence of base Et 3 N. The reaction proceeded at 25°C for 17 h (Figure 6). The reduction yielded 5.9 mCi of the desired 3 H-labeled carbonate 15, and two further unidentified by-products (21.3 and 12.4 mCi) were detected. By 1 H NMR was determined a specific activity of 15 at 5.8 Ci/mmol (which accounts for 0.2 tritium atom per molecule). Compared to previous deuterium comprehensive study, the obtained tritium enrichment was lower than expected. Authors speculate that the significant drop in SA was caused by the reduced pressure of tritium gas (600 mbar 3 H 2 ) compared to deuterium gas (950 mbar of 2 H 2 ) used for the reduction. The only signal in the 3 H NMR spectrum (the singlet at δ 4.8 ppm) explicitly determined the regio-and stereo-specificity of the reduction. The deprotection of the isopropylidene group in the side chain was  is accompanied by the formation of tritiated water. The suggested mechanism of the by-products formation explained how the traces of water significantly reduce the yield of the labeled product desired (Figure 8). Hence, to suppress synthesis of multi-labeled by-products Pd on charcoal was employed at synthesis of 28-[3β-3 H]homocastasterone [19]. The catalyst/base/substrate ratio for the tritium experiment was kept identical to the previous experiment of the tritium labeling of 24-epiCS-2:6:1.
Carrier-free tritium released over the reaction mixture (738 mbar, 11.5 Ci, 180 µmol) was left to react for 24 h at room temperature. Then, the analytical radio-HPLC proved the formation of the desired 3 H-labeled carbonate (13.3 mCi) and two other by-products (42.8 and 24.7 mCi) were detected. The SA of 3 H-carbonate was determined by 1 H NMR as 5.8 Ci/mmol (based on the decrease of the corresponding 1 H signal intensity in the labeled position). The deprotection of the 22,23-isopropylidene group by treatment with wet FeCl 3 in CH 2 Cl 2 was completed within 10 min. The crude 3β-tritio-2,3-carbonate was directly hydrolyzed by 0.5 M aqueous NaOH in 1,4-dioxane. The crude product was purified on the semi-prep radio-HPLC, affording 5.3 mCi of [ 3 H]-(3) of SA 5.8 Ci/mmol with radiochemical purity (RCP) >97%. Both by-products were isolated, and 3 H and 1 H NMR measured. The structure of multi-labeled by-products is believed to be similar to those that were recently described (Figure 9) for androstane and 24-epiCS (i.e., labeled 24-epityphasterol and 3-dehydro-24-epiteasteron, respectively) derivatives. In this case, these by-products are supposed to be 22,23-isopropylidene-protected multi-labeled [1, 2, 3-3 H]-28-homotyphasterol and [1, 2-3 H]-3-dehydro-28-homoteasterone derivatives (Figure 9).

Catalytic reduction of 24-methylene BRs (SA = 2 Ci/mmol)
An elegant and fast strategy to get BRs labeled by tritium was briefly communicated by Yokota et al. [22]. [ 3 H]-BRs synthesized on demand at Amersham International (Amersham, UK) were used for comparative analysis in stems and seeds by radioimmunoassay. The labeling strategy was based on platinum-catalyzed reduction of 24-methylene position of available BR (dolichosterone and dolicholide) in carrier-free tritium atmosphere (Figure 10)

BRs with very low SA of tritium (10 −3 Ci/mmol)
Tritium-labeled protected 24-epiCS and 24-epiBL [ 3 H]-1 were for the first time prepared by Kolbe et al. [23]. By very simple procedure of base-catalyzed exchange reaction with tritiated water (SA = 20.10 −3 Ci/mmol-accounts for 0.7 × 10 −3 tritium atom per molecule HTO) to the enolizable alpha positions of C-6 ketone of tetracetate 24-epiCS 27 (Figure 11). By this method, it is theoretically possible to exchange hydrogen on three distinctive positions (C-5α, C-7α, and C-7β). However, the obtained SA of BRs was very low (6.10 −3 Ci/mmol), about 3-4 orders lower than that provided by methods described above. Because of low SA of such prepared material cannot be used in receptor studies at all. The other drawback of this procedure is chemical exchangeability of labels, which inevitably leads to loss of label in protic solvents (water) during biological experiments and disqualifies this approach from use in ADME studies. Three-step reaction sequence provided 24-[5, 7, 7-3 H]epiBL with SA 6.10 −3 Ci/mmol; labeling step leading to protected 24-[ 3 H]epiCS 27, followed by a Baeyer-Villiger oxidation with CF 3 COOOH and sequence was accomplished by hydroxy group deprotection [20] . The same sequence was also performed with 2,3,22,23-bis-isopropylidenedioxy-24-epiCS 29 as starting compound. The advantage of the use of isopropylidene protecting groups over acetate group is the deprotection step. The lactone ring is stable under acidic hydrolysis conditions used for isopropylidene deprotection, which occurs simultaneously with Baeyer-Villiger oxidation.
The basic hydrolysis needed for the acetate group cleavage leads to lactone hydrolysis and acid catalyzed re-lactonization is needed (Figure 11).
A base-catalyzed exchange was used for labeling of biogenetic brassinosteroids precursors [24]. 24-[5, 7, 7- ampoule with tritiated water (SA = 14.10 −3 Ci/mmol-accounts for 0.5 × 10 −3 tritium atom per molecule HTO) in presence of base Et 3 N (Figure 12). As mentioned above this simple methodology affords poor SA (in order of 10 −3 Ci/mmol) of BRs labeled on an exchangeable positions thus prone to loss of label later on. On the other hand, this approach provides high yield (>45%) of desired material.

Equipment of the radio-isotope laboratory (IOCB)
The author of this chapter is a member of the Radio-isotope laboratory, a service group of the IOCB CAS, working as a synthetic radiochemist in the production of radioactive molecular tracers. The laboratory is classified for handling of the open sources of ionizing radiation in quantities authorized for laboratories of II category according to Czech bylaw 307/2002 Sb for research and development and educational purposes. It is currently authorized to work with the main radioactive isotopes used in research, for example, 3 H, 14 C, 32 P, 33 P, 35 S, 51 Cr, 54 Mn, 55 Fe, 99 Tc and 125 I. The main purpose of the laboratory is to provide a series of highly specific facilities, equipment and services fully adapted to researchers' needs and maintained in optimum conditions, always in line with applicable legislation to ensure that all personnel is fully protected and ensuring the physical safety of the materials used and the environment. The laboratory has an equipment, stuff, and knowledge to cooperate with other chemists and biologists to provide them with custom synthesis of radio-labeled compounds, especially commercially unavailable compounds. Following time-depending stability of synthesized radio-labeled compounds is one of our basic services provided to biologists. The key instrument of the laboratory is a glove box with tritiation manifold from RC-TRITEC AG (Teufen, Switzerland) suitable for handling 100-1000 Ci of carrier-free tritium gas (Figure 15). Tritiation manifold is based on U-Bed Technology to provide fresh, Figure 15. The equipment for safe handling of tritium gas; the tritium manifold (RC-TRITEC AG) placed in the glove box; a scrubber for decontamination placed behind the glove box.
on a molecular sieve. 3 H NMR measurements were performed on a Bruker Avance II 300 MHz in the laboratory. Liquid scintillation analyser Tri-Carb 2900TR (Perkin Elmer) was used for detecting small amounts of α, β, and γ radioactivity. Mobile contamination monitor CoMo 170 (GRAETZ Strahlungsmeßtechnik GmbH) was used for the high-sensitive and nuclide referred measurement of surfaces with regard to α-, β-, and γ-contaminations when handling ionizing radiation. Analytical -preparative radio-HPLC (pump Waters 600, UV detector Waters 2487, radio chromatogram detector Ramona with analytical cell (LSC) and solid scintillator preparative cell (Raytest, Germany), data management software Empower 2 from Waters). Basic radiation protection equipment and waste disposal management.