Nuclear characteristics of discussed radio-isotopes.
The chapter provides a comprehensive overview on methodologies used for radio-labeling 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 pharmaceutical research.
- radio-isotope 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 (3H, T) and carbon-14 (14C) are by far the most versatile and convenient natural labels available . These two isotopes preserve molecular structure (no added tags or pendant groups that alter or change the structure). The advantages of 3H compared to 14C 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 3H or 14C label. For instance, tritium label could be applied to earlier stage development studies and then switched to a 14C label for the later stage development studies, for example, an advanced human ADME . 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 alcohol-reached 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].
|Radioactive half-life||12.33 years||5730 years|
|Specific activity–labeled drugs (1 atom per molecule)||1.066 TBq/mmol||2.309 GBq/mmol|
|29.1 Ci/mmol||0.0624 Ci/mmol|
|Specific activity—element||3.56 × 1014 Bq/g||1.66 × 1011 Bq/g|
|Type of radiation (emission probability, %)||β− (100%)||β− (100%)|
|Energy||Emax = 18.6 keV
Eavg = 5.7 keV
|Emax = 156 keV
Eavg = 49 keV
|Maximum penetration air/water(tissue)/glass||6 mm/6 µm/2 µm||24 cm/0.250 um/170 µm|
|Decay product||3He+ (stable)||14N+ (stable)|
|Detection and measurement||LSC (undetectable by portable survey meters)||LSC Geiger-Mueller [10% efficiency]|
|Shielding||None required—not an external radiation hazard||None required—mCi quantities not an external radiation hazard|
1.2. 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 . 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 bee-collected rape pollen of
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 . 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 22
This chapter is engaged in the recent developments in the synthesis of 3H- and 14C-radio-labeled analogs of the brassinolide.
2. Synthesis of tritium-labeled BRs
2.1. BRs with very high SA of tritium (~99 Ci/mmol)
For binding assays, study aimed at ligand-receptor-activity relationship is a high specific activity (SA), a bottom line requirement. The SA of such radio-labeled drugs need to be in scale of tenths of Ci/mmol. This critical precondition used to be an obstacle in the way of BR’s studies for decades. The state-of-art strategy for such a labeling was reported by Marek et al. . The methodology yields tritium-labeled BRs bearing a very high SA of 99.4 and 98 Ci/mmol (approx. 3.4 tritium enrichment per molecule), respectively. Convenient, a six-step synthetic sequence starting with the brassinosteroid
In 1998, Seto et al. described a fairly elegant strategy for the deuterium-multi-labeling of brassinolide in its side-chain . 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 Δ25(26) and Δ24(25) regiomers in the 65:35 ratio that was possible to separate after deprotection. The deuteration of Δ25(26) regioisomer by deuterium gas catalyzed by Pd/C (1 atm, 25°C, 1 h) yielded [24, 25, 26, 27-2H]brassinolide with 60% deuterium enrichment calculated from MS data. The ratio of the individual multi-deuterated species in the cluster was 2H2:2H3:2H4:2H6:2H7 = 3:8:14:15:60. The basic idea of this methodology for usage at labeling with radioactive isotope tritium was waiting almost for two decades—then 24-[3H]epiBL (
The protocol of Seto et al. paved the way for the synthesis of an unsaturated precursor for the intended synthesis of 3H-labeled 24-epiCS [15, 16]. First, the 2,3-22,23-bisisopropylidene derivative
To get [24, 25, 26, 27-3H]epibrassinolide ([3H]-
2.1.1. 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-
2.2. BRs with reasonable high SA of tritium
2.2.1. 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 (
) . 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
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% (
; representative synthesis of 24-[3H]epiCS). To improve the solubility of the starting steroid
The catalytic reductive dehalogenation of BR–chlorocarbonates was studied with deuterium in the system of 2H2/Pd/Et3N providing appropriate 3β-deutero-2,3-carbonates with 70–80% deuterium enrichment (based on 1H 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/CaCO3(5%)/Et3N/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 [2H]-labeled ethylene carbonate. Various catalysts such as Pd/C (either 5 or 30%), PdO/BaSO4 (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% 2H-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% 2H-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/CaCO3 (5%)/Et3N/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 D2 gas) play a crucial role in the suggested mechanism of by-product formation (
) . The initial oxidative addition of
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/CaCO3 (5%)/Et3N/substrate 2:6:1, dry EtOAc]. 3H-labeled 24-epiCS and 24-epiBL, was synthesized when the appropriate 3β-chloro-2,3-carbonate
The SA of isolated [3H]-
2.2.2. 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. . [3H]-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 (
). Reduction of dolichosterone afforded two epimers 24-[24, 28-3H]castasterone (8.1 mCi, SA = 2.2 Ci/mmol) and 24-[24, 28-3H]epicastasterone [3H]-(
2.3. BRs with very low SA of tritium (10−3 Ci/mmol)
Tritium-labeled protected 24-epiCS and 24-epiBL [3H]-
A base-catalyzed exchange was used for labeling of biogenetic brassinosteroids precursors . 24-[5, 7, 7-3H]epiteasterone (SA = 1.5 10−3 Ci/mmol), 6-oxo-24β-methyl-22-dehydro[5, 7, 7-3H]cholestenol (SA = not indicated), and 6-oxo-24-[5, 7, 7-3H]epicampestanol (SA = 3.5 10−3 Ci/mmol ), respectively, were partly labeled on positions of C-5 and C-7 by reaction in sealed 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 Et3N ( 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.
2.4. BRs labeled by carbon-14 (SA = 56.8 × 10−3 Ci/mmol)
Till now, only one report on 14C-labeled brassinosteroids is available in the literature. In 1989, Seo et al. described the synthesis of [14C]-labeled (22
3. 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, 3H, 14C, 32P, 33P, 35S, 51Cr, 54Mn, 55Fe, 99Tc and 125I. 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, 3He-free tritium for tritiation by simply heating the UT3-bed, also allowing the recovery of surplus gas after completion of a reaction ( Figure 16 ). During the operation of the manifold, the internal atmosphere of the glove box is continually decontaminated by a scrubber equipped with catalytic oxidation of gaseous tritium to tritiated water, which is trapped on a molecular sieve. 3H 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.
The authors thank the Czech Academy of Sciences for the financial support within the program RVO: 61388963.
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