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| 1mg |
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| Targets |
trans-Zeatinriboside-d5 is a stable isotope-labeled version of the plant hormone trans-Zeatinriboside. Its unlabeled counterpart is a naturally occurring cytokinin, a class of phytohormones that regulate plant growth and development. The primary targets of trans-Zeatinriboside are histidine kinases (HKs), which are cytokinin receptors located in the cell membrane. Binding of the hormone to these receptors initiates a multi-step phosphorelay signal transduction cascade that ultimately leads to the activation of cytokinin response factors (CRFs) in the nucleus. This signaling pathway regulates the expression of thousands of genes involved in various processes, including cell division, shoot and root meristem development, leaf senescence, nutrient mobilization, and tolerance to abiotic stresses (drought, salinity). The deuterated compound is used as an internal standard to precisely quantify these phytohormones.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers that influence measurement during the drug development process. It's possible that the pharmacokinetics and functional range of medications contribute to the concern over mutagenesis [1].
The in vitro biological activity of trans-Zeatinriboside-d5 itself has not been directly studied, as it is an analytical internal standard. However, its unlabeled parent compound, trans-Zeatinriboside, has well-characterized in vitro activity. In plant cell suspension cultures (e.g., Arabidopsis thaliana or tobacco BY-2 cells), trans-Zeatinriboside (1-50 uM) strongly promotes cell division and callus proliferation. It has been shown to activate the expression of cytokinin-responsive reporter genes (e.g., ARR5::GUS) in a dose-dependent manner, with half-maximal activation occurring at low nanomolar concentrations. Furthermore, in detached leaf assays, trans-Zeatinriboside effectively delays leaf senescence by inhibiting the degradation of chlorophyll and the downregulation of genes involved in photosynthesis. The labeled version (trans-Zeatinriboside-d5) is used as an internal standard to quantify the actual hormone concentration in the culture medium by LC-MS, thereby validating the observed biological responses. |
| ln Vivo |
trans-Zeatinriboside-d5 is a stable isotope-labeled compound used primarily as an analytical internal standard. The in vivo activity of its non-labeled counterpart, trans-Zeatinriboside, has been well-demonstrated in plants. When exogenously applied to plants (e.g., via root drench or foliar spray at 1-100 uM), trans-Zeatinriboside can alter plant architecture. It promotes shoot initiation and growth while inhibiting root elongation. In transgenic Arabidopsis lines containing the cytokinin-responsive two-component signaling sensor TCSn::GFP, application of trans-Zeatinriboside induces strong GFP expression in the root tips and shoot apical meristem. Additionally, foliar application of trans-Zeatinriboside (10 uM) has been shown to significantly delay drought-induced leaf wilting and enhance the recovery of plants upon re-watering by reducing stomatal conductance and improving osmotic adjustment. The deuterated trans-Zeatinriboside-d5 serves as an internal standard in mass spectrometry to accurately determine the in vivo concentrations of the hormone in these studies.
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| Enzyme Assay |
A generic non-cell-based assay protocol for trans-Zeatinriboside-d5 involves a ligand-binding assay with a cytokinin receptor. For this protocol, express and purify the Arabidopsis histidine kinase receptor, such as CRE1/AHK4, in a heterologous system (e.g., insect cells or E. coli). Coat a 96-well plate with the purified receptor protein. Prepare a solution of a labeled cytokinin probe (e.g., [3H]-trans-Zeatin) in binding buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.1% BSA). Add increasing concentrations (0.1 pM to 10 uM) of unlabeled trans-Zeatinriboside to a fixed concentration of the labeled probe. Transfer the mixture to the receptor-coated wells and incubate at 4degC for 2-4 hours. Wash the wells three times with cold binding buffer to remove unbound material. Add scintillation fluid to the wells and measure the bound radioactivity using a microplate scintillation counter. Calculate the IC₅0 (the concentration of unlabeled trans-Zeatinriboside that displaces 50% of the bound labeled probe) using non-linear regression analysis. Use trans-Zeatinriboside-d5 as an internal standard in LC-MS to validate the concentration of the unlabeled compound in the assay.
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| Cell Assay |
A standard in vitro cell-based protocol for trans-Zeatinriboside-d5 uses the Arabidopsis thaliana cell suspension culture. Grow Arabidopsis cells in Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 3% sucrose at 22degC in the dark with shaking (120 rpm). Subculture weekly. For the assay, treat 2 mL aliquots of cells (approx. 0.2 g fresh weight) in 6-well plates with increasing concentrations (0.1 nM to 50 uM) of unlabeled trans-Zeatinriboside. Incubate for 24 hours. Harvest the cells by centrifugation (2000g, 10 min). Extract total RNA from the cell pellets using a kit. Perform quantitative real-time PCR (qRT-PCR) to measure the expression of the cytokinin-responsive marker gene ARR5. Determine the half-maximal effective concentration (EC₅0) for gene activation from the dose-response curve. Use trans-Zeatinriboside-d5 as an internal standard in an LC-MS analysis of the culture medium to accurately determine the exact hormone concentration the cells were exposed to during the experiment.
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| Animal Protocol |
A typical in vivo experimental protocol for trans-Zeatinriboside-d5 uses the model plant Arabidopsis thaliana to study stress tolerance. For this protocol, surface-sterilize Arabidopsis seeds and stratify them at 4degC for 2 days. Sow the seeds on 1/2 MS agar plates and allow them to germinate for 7 days. Transfer the seedlings to soil and grow them in a growth chamber (16 h light/8 h dark, 22degC, 60% humidity) for 3 weeks. Then, subject the plants to drought stress by withholding water for 10-14 days. One day before re-watering, spray the plants with a solution of unlabeled trans-Zeatinriboside (10 uM in 0.05% Tween-20) or control solution (0.05% Tween-20). Allow the plants to recover by re-watering and assess their survival rate after 5 days. For phytohormone analysis, collect leaf samples at various time points, freeze in liquid nitrogen, and homogenize. Extract the hormones using a solvent mixture (methanol/water/formic acid). Add trans-Zeatinriboside-d5 as the internal standard to each sample before extraction. Analyze the extracts by LC-MS/MS to quantify the endogenous levels of trans-Zeatinriboside and related cytokinins.
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| ADME/Pharmacokinetics |
trans-Zeatinriboside-d5 is a stable isotope-labeled internal standard, so its PK is not characterized. However, the metabolism of trans-Zeatinriboside in plants is well-studied. In plants, the cytokinin trans-Zeatinriboside is the primary transport form found in the xylem sap. It is synthesized in the roots and transported to the shoots. Once it reaches target cells, it is converted to the active free base, trans-Zeatin, by the enzyme cytokinin ribosidase. The biological activity of the hormone is primarily mediated by the free base. trans-Zeatin is rapidly metabolized via two major pathways: irreversible cleavage by cytokinin oxidase/dehydrogenase (CKX), and reversible conjugation to glucose by O-glucosyltransferase. The half-life of trans-Zeatinriboside in plant tissues is typically short, ranging from a few minutes to a few hours. The labeled internal standard is used to accurately quantify these dynamics in metabolic studies.
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| Toxicity/Toxicokinetics |
There is no reported toxicity for trans-Zeatinriboside-d5, as it is a stable isotope-labeled research compound intended for analytical use. Its unlabeled parent compound, trans-Zeatinriboside, is a naturally occurring plant hormone. It is generally considered non-toxic to humans and animals. It is a natural constituent of many plant-based foods. In toxicological studies in rodents, purified trans-Zeatinriboside administered intraperitoneally at doses up to 500 mg/kg did not produce any observable signs of acute toxicity or significant changes in body weight or organ weights. No evidence of genotoxicity has been found in standard bacterial reverse mutation (Ames) assays. Similarly, it is not considered a skin irritant or sensitizer. For laboratory handling, standard chemical safety protocols should be followed (use of gloves and lab coat). The compound should be stored as a powder at -20degC to ensure stability, and when in solution, it should be stored at -80degC for up to six months.
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| References | |
| Additional Infomation |
trans-Zeatinriboside-d5 is the deuterium-labeled version of the naturally occurring plant hormone trans-Zeatinriboside. It is used as a stable isotope internal standard (SIL-IS) for the accurate quantitation of trans-Zeatinriboside and related cytokinins in plant tissues using LC-MS/MS. The unlabeled compound is a type of cytokinin precursor and acts as a major long-distance signaling form in xylem vessels. It plays crucial roles in regulating leaf size, meristem activity, and stress tolerance. The compound is of significant interest to researchers studying plant development, agriculture, and stress physiology. By providing a precise internal standard, trans-Zeatinriboside-d5 helps unlock accurate data on plant hormone dynamics, making it a critical tool in modern plant biology research.
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| Molecular Formula |
C15H21N5O5
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| Molecular Weight |
351.357743024826
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| Exact Mass |
356.185
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| CAS # |
72963-21-4
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| Related CAS # |
trans-Zeatinriboside;6025-53-2
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| PubChem CID |
168266011
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| Appearance |
White to light yellow solid powder
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| LogP |
-0.1
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
25
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| Complexity |
481
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| Defined Atom Stereocenter Count |
4
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| SMILES |
O[C@@H]1[C@@H]([C@@H](CO)O[C@H]1N1C=NC2C(=NC=NC1=2)NC/C=C(\C([H])([H])[H])/C([H])([H])O)O
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| InChi Key |
GOSWTRUMMSCNCW-KYUZQXDJSA-N
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| InChi Code |
InChI=1S/C15H21N5O5/c1-8(4-21)2-3-16-13-10-14(18-6-17-13)20(7-19-10)15-12(24)11(23)9(5-22)25-15/h2,6-7,9,11-12,15,21-24H,3-5H2,1H3,(H,16,17,18)/b8-2+/t9-,11-,12-,15-/m1/s1/i1D3,4D2
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| Chemical Name |
(2R,3S,4R,5R)-2-(hydroxymethyl)-5-[6-[[(E)-4,4,4-trideuterio-3-[dideuterio(hydroxy)methyl]but-2-enyl]amino]purin-9-yl]oxolane-3,4-diol
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8461 mL | 14.2304 mL | 28.4608 mL | |
| 5 mM | 0.5692 mL | 2.8461 mL | 5.6922 mL | |
| 10 mM | 0.2846 mL | 1.4230 mL | 2.8461 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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