**1. Introduction**

## **1.1. 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

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receptors in tissues). Weak β-emitters such as tritium (<sup>3</sup> H, T) and carbon-14 (14C) 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 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 3 H 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 [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


**Table 1.** Nuclear characteristics of discussed radio-isotopes.

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].

#### **1.2. Brassinosteroids: a newest class of phytohormones**

receptors in tissues). Weak β-emitters such as tritium (<sup>3</sup>

12 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

advantages of 3

either 3

Specific activity–labeled drugs (1 atom per molecule)

Type of radiation (emission

Maximum penetration air/ water(tissue)/glass

Decay product <sup>3</sup>

Detection and measurement LSC

Energy Emax = 18.6 keV

probability, %)

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

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

H 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 [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

**Tritium Carbon-14**

1.066 TBq/mmol 2.309 GBq/mmol

29.1 Ci/mmol 0.0624 Ci/mmol

6 mm/6 µm/2 µm 24 cm/0.250 um/170 µm

(100%)

Emax = 156 keV Eavg = 49 keV

(stable)

Geiger-Mueller [10% efficiency]

None required—mCi quantities not an external radiation hazard

LSC

(100%) β−

(stable) <sup>14</sup>N+

(undetectable by portable survey

Radioactive half-life 12.33 years 5730 years

Specific activity—element 3.56 × 1014 Bq/g 1.66 × 1011 Bq/g 2.57 Ci/mL

Eavg = 5.7 keV

β−

He+

meters)

radiation hazard

Shielding None required—not an external

**Table 1.** Nuclear characteristics of discussed radio-isotopes.

H compared to 14C are much higher specific activity, significantly lower cost of

H, T) and carbon-14 (14C) are by far

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 22*R*, 23*R*-) turned out to be an essential factor for high biological activity of such BRs (**Figure 1**).

**Figure 1.** 24-epiBL (**1**) is the 24-(*R*)-epimer of the first isolated brassinosteroid, 24-epiCS (**2**), 28-homoCS (**3**).

This chapter is engaged in the recent developments in the synthesis of 3 H- and 14C-radiolabeled analogs of the brassinolide.
