Cyclic dinucleotides (CDNs) are pivotal small-molecule second messengers with profound roles in bacterial signaling and host innate immunity. In immunotherapy applications, along with vaccine development and targeted drug delivery, these molecules have become a significant focus of study in both academic research and industrial settings. Alfa Chemistry provides researchers with a wide selection of high-purity cyclic dinucleotides to advance pharmaceutical and immunological research projects.
CDNs are intracellular signaling molecules consisting of two nucleotides linked by a unique 3′-5′ and/or 2′-5′ phosphodiester bond to form a ring structure. The discovery of CDNs began with studies of the mechanism of cellulose synthesis in Acetobacter xylinum (now known as Gluconacetobacter xylinus), when the discovery of bis(3′→5′)-cyclic diguanylate (c-di-GMP) was found to be a variable activator of cellulose synthase. ′→5′)-cyclic diguanosine monophosphate (c-di-GMP) was found to be a variable activator of cellulose synthase. This molecule is synthesized from guanosine triphosphate (GTP) by di-guanosine glycosyl synthase (DGC), and its cyclic dinucleotide configuration was confirmed by X-ray crystallographic structure analysis.
Figure 1. Chemical structures of naturally occurring cyclic dinucleotides known to date. The structure of 2′3′-cGAMP was originally misassigned as 2′2′-cGAMP shown in brackets[1].
The currently characterized cyclic dinucleotides include:
| CDN Name | Structure | Biological Source | Key Functions |
| c-di-GMP | cyclic di-guanosine monophosphate | Bacteria | Regulates biofilm formation, motility |
| c-di-AMP | cyclic di-adenosine monophosphate | Bacteria | Controls potassium homeostasis, DNA repair |
| cGAMP (2′3′) | cyclic GMP–AMP (mixed linkage) | Eukaryotic cells (cGAS enzyme) | Activates STING in innate immunity |
These molecules function as signaling entities in prokaryotic stress responses and are key activators of the stimulator of interferon genes (STING) pathway in eukaryotic immune cells.
In bacteria, CDNs act as universal second messengers that mediate responses to external environmental stimuli. Their intracellular levels are precisely regulated by synthetic enzymes (e.g., DGCs containing the GGDEF structural domain) versus degradative enzymes (e.g., PDEs with the EAL or HY-GYP structural domains). Despite the large number of homologs of related enzymes, only a fraction of them possess catalytic activity.
CDNs regulate a wide range of phenotypes by interacting with effector proteins and RNA components such as riboswitches. The following are some typical CDN effector proteins:
| Effector Protein | Source Strain | Identifying CDNs | Biological Functions |
| Cellulose Synthase | G. xylinus | c-di-GMP | Cellulose biosynthesis |
| PilZ Domain Proteins | Multi-bacteria | c-di-GMP | Motility, toxicity |
| PelD | P. aeruginosa | c-di-GMP | Polysaccharide synthesis |
| VpsT | V. cholerae | c-di-GMP | Biofilm formation |
| FleQ | P. aeruginosa | c-di-GMP | Flagellar regulation |
In addition, CDN-mediated riboswitches can feedback regulate DGC and PDE gene expression.
Figure 2. Processes regulated by the c-di-GMP signaling network[1].
In mammals, CDNs are important activators of the innate immune system, mainly through the activation of the STING pathway. Binding of CDNs to STING triggers a conformational change that initiates the TBK1/IRF3 phosphorylation cascade, inducing the production of type I interferons and pro-inflammatory cytokines.
In addition to the STING pathway, CDNs can be sensed by the cellular oxidoreductase RECON, which regulates NF-κB signaling and controls inflammatory responses. In addition, CDNs can activate Th1, Th2, and Th17 responses through TNF-α-dependent pathways independently of IFN-I.
Figure 3. Bacterial cyclic dinucleotides and microbial DNA are sensed by innate immunity through the cGAS-STING pathway. ER: endoplasmic reticulum; TBK-1: TANK-binding kinase 1; IRF3: interferon regulatory factor 3; NF-κB: nuclear factor κB; IFN-β: interferon-β[2].
CDNs are currently considered highly promising immunotherapeutic agents due to their ability to effectively activate innate immune responses. Their main applications include:
Figure 4. Activation of STING positively regulates each step of the cancer immune cycle[3].
CDNs, as vaccine adjuvants, have shown excellent immune activation in both systemic and mucosal immunity. Animal experiments demonstrated that CDNs such as c-di-GMP, c-di-AMP, and cGAMP significantly enhanced specific antibody (e.g., IgG) levels and increased CTL cell activity.
| CDNs | Immune response characteristics | Characteristic description |
| c-di-GMP | Th1-biased | High IFN-γ, TNF-α expression |
| c-di-AMP | Balanced | Activates Th1/Th2/Th17 |
| 3′3′-cGAMP | High IFN stimulation | Strong STING affinity |
| 2′3′-cGAMP | Endogenous messenger | Activates human and mouse STING |
In mucosal immunization, CDNs induced efficient secretory IgA responses and provided mucosal protection. CDNs induce stronger germinal center formation and durable immune memory compared to traditional adjuvants (e.g., CpG, LPS, and aluminum hydroxide).
Figure 5. Cellular mechanisms of lung dendritic cell (DC) action on CDN adjuvants[4].
Q1: What makes c-di-GMP a universal bacterial second messenger?
A: It regulates motility, biofilm, and virulence by binding to a variety of conserved structural domains (e.g., PilZ) for a wide range of bacteria.
Q2: How do CDNs activate the STING signaling pathway?
A: CDNs bind directly to STING, triggering a conformational change that initiates the TBK1/IRF3 cascade and the production of type I interferons.
Q3: How are cyclic dinucleotides different from other immune adjuvants?
Cyclic dinucleotides directly activate the STING pathway to induce a potent and specific type I interferon response, which is critical for antiviral and antitumor immunity, whereas conventional adjuvants typically rely on systemic immune stimulation.
Q4: Do all CDNs behave the same in immune adjuvants?
Different CDNs induce different immune responses. For example, c-di-GMP favors Th1, while cGAMP induces a more balanced Th1/Th2 response.
Q5: Are cyclic dinucleotides safe for humans?
Natural cyclic dinucleotides degrade rapidly, while synthetic analogs focus on stability and tolerability. Clinical trials are evaluating their safety in cancer and infectious disease applications.
Q6: Can CDNs be used in human therapy?
Yes. Several CDN analogs are in the preclinical or clinical stages of cancer immunotherapy and vaccine adjuvants.
Q7: How do CDNs enter host cells?
Some CDNs enter cells through transporter proteins, endocytosis, or mechanisms such as A2B adenosine receptor-mediated entry.
Q8: Why are cyclic dinucleotide analogs modified with phosphorothioate?
Phosphorothioate substitution enhances resistance to nuclease and phosphodiesterase, extends the half-life of the molecule, and improves efficacy.
Q9: Can cyclic dinucleotides be used orally or topically?
Due to their charged and hydrophilic nature, cyclic dinucleotides are usually administered by injection or encapsulated in a carrier. Novel formulations for oral and mucosal delivery are under development.
Q10: What CDN-related services does Alfa Chemistry offer?
A: Including custom synthesis of CDNs and their analogs, analytical characterization, and consulting for immunological study packages.
Alfa Chemistry is a trusted research partner for high-purity CDNs for immunology research and drug discovery. Please contact us for more technical information.
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