5-Propargylamino-ddUTP
[CAS# 114748-59-3]

Identification

• Classification: Biochemical >> Nucleoside drugs >> Deoxynucleotides and their analogues
• Name: 5-Propargylamino-ddUTP
• Synonyms: [[(2S,5R)-5-[5-(3-aminoprop-1-ynyl)-2,4-dioxopyrimidin-1-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate
• Molecular Formula: C₁₂H₁₈N₃O₁₃P₃
• Molecular Weight: 505.20
• CAS Registry Number: 114748-59-3
• SMILES: C1C[C@@H](O[C@@H]1COP(=O)(O)OP(=O)(O)OP(=O)(O)O)N2C=C(C(=O)NC2=O)C#CCN

Properties

• Density: 1.9±0.1 g/cm³ Calc.^*
• Index of refraction: 1.63 (Calc.)^*

^* Calculated using Advanced Chemistry Development (ACD/Labs) Software.

Discovory and Applicatios

5-Propargylamino-2',3'-dideoxyuridine 5'-triphosphate (5-Propargylamino-ddUTP) is a chemically modified nucleotide analog derived from 2',3'-dideoxyuridine triphosphate (ddUTP), in which a propargylamino group is introduced at the 5-position of the uracil base. This structural modification enables the molecule to function as a reactive and versatile tool in nucleic acid chemistry, especially in labeling and detection technologies. The key functional feature of this analog is the terminal alkyne group, which permits selective conjugation through click chemistry reactions.

The parent compound, ddUTP, is a well-established chain terminator in DNA synthesis due to the absence of a 3'-hydroxyl group, which precludes the formation of phosphodiester bonds. Like other dideoxynucleotides, ddUTP plays a foundational role in chain-termination DNA sequencing techniques, most notably in the Sanger method. Modifying ddUTP to carry a propargylamino moiety expands its utility beyond sequencing by introducing a chemically addressable site for post-synthetic modifications.

The synthesis of 5-Propargylamino-ddUTP typically involves the selective substitution of the hydrogen atom at the 5-position of the uracil ring with a propargylamino group. This step is followed by a multi-step phosphorylation process to introduce the triphosphate moiety. The resulting molecule retains sufficient affinity for DNA polymerases, allowing it to be incorporated into DNA strands in place of thymidine or uridine analogs during polymerase-mediated DNA synthesis. Once incorporated, the alkyne handle on the base is exposed and available for covalent attachment to azide-bearing probes via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).

The incorporation of 5-Propargylamino-ddUTP into oligonucleotides has been widely utilized in applications requiring site-specific labeling of DNA. One of the major uses is in fluorescent labeling, where azide-functionalized fluorophores such as Cy3, Cy5, Alexa Fluor, or fluorescein can be conjugated to the DNA. This technique is especially valuable in fluorescence in situ hybridization (FISH), real-time PCR, and DNA microarray technologies, where sensitivity and specificity of detection are critical.

Besides fluorescent labeling, 5-Propargylamino-ddUTP also supports conjugation with biotin, digoxigenin, and other haptens, enabling downstream detection with streptavidin or antibody-based systems. This versatility makes the molecule particularly useful in non-radioactive labeling protocols. The modification at the 5-position does not interfere with Watson-Crick base pairing, and incorporation fidelity is generally preserved, ensuring compatibility with standard DNA polymerases such as Taq, Pfu, and Klenow fragments.

Commercially available versions of 5-Propargylamino-ddUTP, either unlabeled or pre-conjugated with a range of reporter molecules, have facilitated its broad use in molecular biology and biotechnology research. It is employed in single-nucleotide labeling techniques, primer extension assays, and DNA mapping strategies, where the terminal alkyne serves as a modular site for probe addition after DNA synthesis. The compound is particularly suitable for multiplex assays where distinct fluorescent signals are required for different DNA targets.

In the context of synthetic biology and nucleic acid therapeutics, 5-Propargylamino-ddUTP provides a platform for introducing functional groups into DNA strands without disrupting base recognition or helix stability. It supports the development of novel biomolecular constructs such as DNA-based sensors, functional nanostructures, and chemically addressable gene probes. Moreover, its alkyne functionality is orthogonal to native biological functionalities, making it ideal for selective modification in complex biological environments.

5-Propargylamino-ddUTP exemplifies the advancement of nucleotide chemistry aimed at combining biological activity with chemical reactivity. Its ability to act as a chain terminator while offering a modular point for conjugation makes it a valuable reagent in modern molecular biology, diagnostics, and imaging.