7-Deaza-7-propargylamino-ddATP
[CAS# 114748-69-5]

Identification

• Classification: Biochemical >> Nucleoside drugs >> Deoxynucleotides and their analogues
• Name: 7-Deaza-7-propargylamino-ddATP
• Synonyms: [[(2S,5R)-5-[4-amino-5-(3-aminoprop-1-ynyl)pyrrolo[2,3-d]pyrimidin-7-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate
• Molecular Formula: C₁₄H₂₀N₅O₁₁P₃
• Molecular Weight: 527.26
• CAS Registry Number: 114748-69-5
• SMILES: C1C[C@@H](O[C@@H]1COP(=O)(O)OP(=O)(O)OP(=O)(O)O)N2C=C(C3=C(N=CN=C32)N)C#CCN

Properties

• Density: 2.1±0.1 g/cm³ Calc.^*
• Boiling point: 879.8±75.0 ºC 760 mmHg (Calc.)^*
• Flash point: 485.9±37.1 ºC (Calc.)^*
• Index of refraction: 1.768 (Calc.)^*

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

Discovory and Applicatios

7-Deaza-7-propargylamino-2',3'-dideoxyadenosine 5'-triphosphate (7-deaza-7-propargylamino-ddATP) is a modified nucleotide analog that combines three distinct structural features: a 7-deazapurine base replacing the natural adenine, a propargylamino group attached at position 7 of the purine ring, and a sugar moiety lacking the 2' and 3' hydroxyl groups. These alterations collectively confer unique chemical and functional properties that make the compound highly useful in the fields of nucleic acid labeling, sequencing, and chemical biology.

The 7-deaza modification replaces the nitrogen atom at position 7 of adenine with a carbon atom. This structural change is known to improve the stability of the purine ring under acidic or depurinating conditions and can also affect base-pairing and hydrogen-bonding interactions in nucleic acid duplexes. The addition of a propargylamino group at this position introduces a terminal alkyne functionality, making the nucleotide amenable to copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a form of bioorthogonal click chemistry. This feature enables the covalent attachment of various reporter molecules after the nucleotide has been incorporated into a DNA strand.

The dideoxyribose sugar in 7-deaza-7-propargylamino-ddATP lacks both the 2' and 3' hydroxyl groups, a hallmark of dideoxynucleotides. The absence of the 3' hydroxyl prevents further nucleotide extension once it is incorporated by DNA polymerases, thereby causing chain termination. This makes it suitable for Sanger-type DNA sequencing and other applications that depend on selective termination of DNA synthesis.

The synthesis of 7-deaza-7-propargylamino-ddATP typically begins with a 7-deazaadenosine core, which is selectively functionalized at the 7-position via nucleophilic substitution or palladium-catalyzed coupling with a propargylamine derivative. The triphosphorylation step, often conducted using phosphorylating reagents such as POCl₃ or salicyl phosphorochloridite followed by oxidation, yields the final triphosphate form of the molecule. The product is purified using ion-exchange chromatography and characterized with techniques such as NMR spectroscopy and high-resolution mass spectrometry.

Functionally, 7-deaza-7-propargylamino-ddATP is a substrate for many DNA polymerases, including both thermostable and mesophilic enzymes. When incorporated into a DNA strand during primer extension, the nucleotide causes termination due to its 3'-deoxy structure. The terminal alkyne introduced by the propargylamino group remains available for subsequent chemical modification. This allows for precise labeling of DNA at defined positions using azide-functionalized dyes, biotin, or other affinity tags.

One of the major applications of 7-deaza-7-propargylamino-ddATP is in the development of fluorescently labeled DNA for capillary electrophoresis sequencing, real-time PCR, and hybridization-based assays. The incorporation of this nucleotide provides a method for introducing functional groups at defined sites in the DNA sequence, facilitating post-reaction labeling without the need for modified primers or pre-synthesized labeled oligonucleotides. The alkyne group ensures high specificity and efficiency of labeling through click chemistry, which is widely used due to its robustness and compatibility with aqueous environments.

In addition to its labeling functions, the nucleotide analog serves as a tool for studying polymerase fidelity and substrate specificity. The effects of the 7-deaza and propargyl modifications on polymerase recognition and incorporation efficiency can yield insights into enzyme mechanisms and aid in the engineering of polymerases for specialized applications.

7-Deaza-7-propargylamino-ddATP has also found utility in the preparation of nucleic acid-based probes and biosensors. The clickable handle facilitates the attachment of immobilization groups or signaling moieties, making it suitable for incorporation into DNA nanostructures or surface-bound DNA arrays. Because the 7-deaza modification maintains base-pairing compatibility with thymidine, the presence of this analog in DNA does not significantly impair hybridization, ensuring its effectiveness in downstream molecular recognition processes.

As with other modified dideoxynucleotides, the careful design of 7-deaza-7-propargylamino-ddATP has expanded the chemical toolkit available for DNA manipulation. It enables the site-specific introduction of chemical functionalities without disrupting the biological function of DNA, thereby supporting a range of advanced techniques in molecular diagnostics, sequencing, and synthetic biology.