Ap4A can be created by a non-canonical activity of the Lysyl- tRNA synthetase  (LysRS). This function of LysRS is activated by the phosphorylation of LysRS on serine 207, its subsequent dissociation from the multi-synthetase complex (MSC). [ 2 ]  The molecule's role as a second messenger has recently been discovered in The  LysRS -Ap4A- MITF  signaling pathway. [ 3 ]  Ap4A binds to the MITF-HINT1 inhibitory complex, specifically to the molecule  histidine triad nucleotide–binding protein 1 (HINT1), releasing the Microphthalmia-associated transcription factor (MITF) and causing an increase in the transcription of its target genes. [ 4 ]   Ap4A also positively regulates the activity of the transcription factor  USF2  through a similar molecular mechanism to that of MITF. [ 5 ]


It has also been shown, that Ap4A plays a role in the functionality of  dendritic cells  (DCs). An increase in the intracellular amount Improves their motility and antigen presenting ability through alterations in small  GTPases  present in the cells. This was discovered by creating mice deficient in the enzyme  NUDT2,  which serves as an Ap4A  hydrolase  and thus controls the levels of Ap4A in the cell. [ 6 ]  Ap4A, however, has also been shown to cause  apoptosis  in several cell lines through an unknown mechanism, the degradation of Ap4A was necessary for the process as hydrolysis-resistant analogues of the molecule showed no apoptotic activity. [ 7 ]


In E. Coli, Ap4A has been shown to function as an alarmone, as the intracellular concentration of the molecule increases upon heat stress. [ 8 ]  Ap4A can also be incorporated into RNA as a  5' Cap  along with other dinucleoside polyphosphates. It serves as a substrate for the  RNA polymerase  and the intracellular levels of these capped RNAs increase upon stress, suggesting that the cap adds a level of stability to the  RNA . [ 9 ]


Myxococcus xanthus  is a type of  Gram-negative bacteria , and  M. xanthus  lysyl-tRNA synthetase (LysS) is an enzyme from the bacteria that synthesizes diadenosine tetraphosphates (Ap4A) when  adenosine triphosphate  (ATP) is present. Diadenosine pentaphosphate (Ap5A) is synthesized from Ap4A with ATP. [ 10 ]


Arabinofuranosylcytosine triphosphate  is a  nucleotide  that inhibits the  synthesis  of  DNA  by acting as an  antimetabolic agent  against  deoxycytidine  (a component of DNA).  It is the biologically active form of  cytarabine . [ 1 ]


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Bemnifosbuvir  ( AT-527 ,  RO7496998 ) is an  antiviral drug  invented by  Atea Pharmaceuticals  and licensed to  Roche  for clinical development, a novel  nucleotide analog   prodrug  originally developed for the treatment of  hepatitis C . [ 1 ] [ 2 ]  Bemnifosbuvir is the orally bioavailable hemisulfate salt of AT-511, which is metabolized in several steps to the active nucleotide triphosphate AT-9010, acting as an  RNA polymerase  inhibitor and thereby interfering with viral replication. Bemnifosbuvir has been researched for the treatment of  coronavirus  diseases such as that produced by  SARS-CoV-2 . [ 3 ]   It showed good results in early  clinical trials  but had inconsistent results at later stages. [ 4 ] [ 5 ]  Bemnifosbuvir's Phase III study ended early as it failed to meet its primary endpoint of symptom alleviation and did not decrease viral load. However, the drug was well-tolerated and reduced relative hospitalization risk by 71%. [ 6 ]


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A  bridged nucleic acid  ( BNA ) is a modified  RNA  nucleotide. They are sometimes also referred to as constrained or inaccessible RNA  molecules . BNA  monomers  can contain a five-membered, six-membered or even a seven-membered bridged structure with a "fixed" C 3 '-endo sugar puckering. [ 1 ]  The bridge is synthetically incorporated at the 2', 4'-position of the ribose to afford a 2', 4'-BNA monomer. The monomers can be incorporated into  oligonucleotide  polymeric structures using standard  phosphoramidite  chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability.


Chemical structures of BNA monomers containing a bridge at the 2', 4'-position of the ribose to afford a 2', 4'-BNA monomer as synthesized by Takeshi Imanishi's group. [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ]  The nature of the bridge can vary for different types of monomers. The 3D structures for A-RNA and B-DNA were used as a template for the design of the BNA monomers. The goal for the design was to find derivatives that possess high binding affinities with complementary RNA and/or DNA strands.


An increased conformational inflexibility of the sugar moiety in  nucleosides  (oligonucleotides) results in a gain of high binding affinity with complementary single-stranded RNA and/or double-stranded DNA. The first 2',4'-BNA (LNA) monomers were first synthesized by Takeshi Imanishi's group in 1997 [ 2 ]  followed independently by Jesper Wengel's group in 1998. [ 8 ]


BNA  nucleotides  can be incorporated into DNA or RNA  oligonucleotides  at any desired position. Such oligomers are synthesized chemically and are now commercially available. The bridged ribose conformation enhances base stacking and pre-organizes the backbone of the oligonucleotide significantly increasing their hybridization properties.


The incorporation of BNAs into oligonucleotides allows the production of modified synthetic oligonucleotides with
equal or higher binding affinity against a DNA or RNA complement with excellent single-mismatch discriminating power;
better RNA selective binding;
stronger and more sequence selective triplex-forming characters;
pronounced higher nuclease resistance, even higher than  Sp-phosphorothioate analogues; and
good aqueous  solubility  of the resulting oligonucleotides when compared to regular DNA or RNA oligonucleotides.


New BNA analogs introduced by Imanishi's group were designed by taking the length of the bridged  moiety  into account. A six-membered bridged structure with a unique structural feature (N-O bond) in the sugar moiety was designed to have a nitrogen atom. This atom improves the formation of duplexes and triplexes by lowering the repulsion between the negatively charged backbone phosphates. These modifications allow to control the affinity towards complementary strands, regulate resistance against nuclease degradation and the synthesis of functional molecules designed for specific applications in genomics. The properties of these analogs were investigated and compared to those of previous 2',4'-BNA (LNA) modified oligonucleotides by Imanishi's group. Imanishi's results show that "2',4'-BNA NC -modified oligonucleotides with these profiles show great promise for applications in antisense and antigene technologies."


Yamamoto et al. in 2012 [ 9 ]  demonstrated that BNA-based antisense therapeutics inhibited hepatic PCSK9 expression, resulting in a strong reduction of the serum LDL-C levels of mice. The findings supported the hypothesis that PCSK9 is a potential therapeutic target for hypercholesterolemia and the researchers were able to show that BNA-based antisense oligonucleotides (AONs) induced cholesterol-lowering action in hypercholesterolemic mice.  A moderate increase of aspartate aminotransferase, ALT, and blood urea nitrogen levels was observed whereas the histopathological analysis revealed no severe hepatic toxicities. The same group, also in 2012, reported that the 2',4'-BNA NC [NMe] analog when used in antisense oligonucleotides showed significantly stronger inhibitory activities which is more pronounced in shorter (13- to 16mer) oligonucleotides. Their data led the researchers to conclude that the 2',4'-BNA NC [NMe] analog may be a better alternative to conventional LNAs.


Some of the benefits of BNAs include ideal for the detection of short RNA and DNA targets; increase the thermal stability of duplexes; capable of single nucleotide discrimination; increases the thermal stability of triplexes; resistance to exo- and endonucleases resulting in a high stability for  in vivo  and  in vitro  applications; increased target specificity; facilitate Tm normalization; strand invasion enables detection of "hard to access" samples; compatible with standard enzymatic processes. [ citation needed ]


Application of BNAs include small RNA research; design and synthesis of RNA aptamers; siRNA; antisense probes; diagnostics; isolation; microarray analysis; Northern blotting; real-time PCR;  in situ  hybridization; functional analysis; SNP detection and use as antigens and many others  nucleotide base applications. [ 10 ] [ citation needed ]


8-Bromoadenosine 3',5'-cyclic adenosine monophosphate  (8-Br-cAMP) is a  brominated  derivative of  cyclic adenosine monophosphate  (cAMP). 8-Br-cAMP is an activator of  cyclic AMP-dependent protein kinase , and it is long-acting because it is resistant to degradation by  cyclic AMP phosphodiesterase . [ 2 ]


8-Bromoguanosine 3′,5′-cyclic monophosphate  is a  brominated  derivative of  cyclic guanosine monophosphate  (cGMP). [ 1 ]   It acts as an activator of cGMP-dependent  protein kinases . [ 2 ]


Bucladesine   is a cyclic nucleotide derivative which mimics the action of endogenous  cAMP  and is a  phosphodiesterase inhibitor .


Bucladesine is a cell permeable cAMP analog. The compound is used in a wide variety of research applications because it mimics cAMP and can induce normal physiological responses when added to cells in experimental conditions. cAMP is only able to elicit minimal responses in these situations.


The  neurite  outgrowth instigated by bucladesine in cell cultures has been shown to be enhanced by  nardosinone .


The effect of bucladesine as a cAMP analog has been studied on the pentylenetetrazol-induced seizure in the wild-type mice. The data showed that bucladesine (300nM/mouse) reduced the seizure latency and threshold. In addition they found that combination of bucladesine and pentoxyfillin has additive effect on seizure latency and threshold. [ 1 ]


Bucladesine (50-100nM/mouse) showed significant attenuation in the morphine withdrawal syndrome in the wild-type mice. In addition, its high dose (200nM/mouse) combination with  H-89 , as a protein kinase inhibitor, had additive attenuating effect on withdrawal syndromes. [ 2 ]


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Calcium guanylate  is a  compound  with formula Ca(C 10 H 12 O 4 N 5 PO 4 ). It is the  calcium   salt  of  guanylic acid . [ 1 ]  It is present in all living cells as part of RNA, and is commercially prepared from yeast extract or fish.


As a  food additive , it is used as a  flavor enhancer , particularly low-salt products [ 1 ]  and has the  E number  E629.


Calcium inosinate  is a calcium  salt  of the  nucleoside   inosine .  Under the  E number  E633, it is a  food additive  used as a  flavor enhancer .


Cangrelor , sold under the brand name  Kengreal  among others, is a  P2Y 12  inhibitor FDA approved as of June 2015 as an  antiplatelet drug [ 5 ]  for  intravenous  application. Some P2Y 12  inhibitors are used clinically as effective inhibitors of  adenosine diphosphate -mediated platelet activation and aggregation. [ 5 ]  Unlike  clopidogrel  (Plavix), which is a  prodrug , cangrelor is an active drug not requiring  metabolic  conversion.


Poor interim results led to the abandonment of the two CHAMPION clinical trials in mid-2009. [ 6 ]   The BRIDGE study, for short term use prior to surgery, continues. [ 7 ]  The CHAMPION PHOENIX trial was a randomized study of over 11,000 patients published in 2013.  It found usefulness of cangrelor in patients getting cardiac stents.  Compared with clopidogrel given around the time of stenting, intravenous ADP-receptor blockade with cangrelor significantly reduced the rate of stent thrombosis and myocardial infarction. [ 8 ]  Reviewers have questioned the methodology of the trial. [ 9 ]


According to phase III randomized trials, a cangrelor– clopidogrel  combination is safe and has been found to be more effective than standard clopidogrel treatment at reducing ischemic events in the heart, without increasing major bleeding in the treatment of stenotic coronary arteries. [ 10 ]  The advantages of this drug combination are most prominent in patients with myocardial infarction. [ 10 ]


Available antiplatelet drugs have delayed onset and offset of action. [ 10 ]  Since cangrelor's effects are immediate and quickly reversed, it is a more desirable drug for elective treatment of stenotic coronary arteries, high risk acute coronary syndromes treated with immediate coronary stenting, and for bridging those surgery patients who require P2Y 12  inhibition. [ 10 ]


Evidence regarding cangrelor therapy is limited by the lack studies assessing cangrelor administration in conjunction with either prasugrel or ticagrelor. [ 10 ]


Cangrelor been approved for adults undergoing  percutaneous coronary intervention  (PCI). [ 11 ]


Cangrelor is a high-affinity, reversible inhibitor of P2Y 12  receptors that causes almost complete inhibition of ADP-induced platelet aggregate. [ 12 ]  It is a modified ATP derivative stable to enzymatic degradation. [ 12 ]  It does not require metabolic conversion to an active metabolite. This allows cangrelor's immediate effect after infusion, [ 12 ]  and the therapeutic effects can be maintained with continuous infusion. [ 13 ]  The pharmacokinetics of cangrelor has allowed it to rapidly achieve steady-state concentrations with a clearance of 50 L/h and a half-life of 2.6 to 3.3 minutes. Cessation of its administration is associated with rapid removal, and normal platelet function is restored within 1 hour. [ 12 ] [ 13 ]


Despite fewer bleeding events during cardiac surgery, cangrelor carries the risk of potential autoimmune reactions manifesting as breathlessness. [ 14 ]  Potential mechanisms for dyspnea following cangrelor treatment include: repeated binding and unbinding cycles, impaired platelet turnover, and lung sequestration or apoptosis of overloaded destructive platelets. [ 14 ]  The dyspnea risks following cangrelor treatment, suggest a common mechanism linking transfusion-related acute lung injury, dyspnea, and reversible platelet inhibition. [ 14 ]


The risk of  breathlessness  after intravenous cangrelor is smaller when compared with other reversible platelet  P2Y 12  receptor inhibitors , however, it is still significantly higher when compared to irreversible oral antiplatelet drugs or intravenous glycoprotein IIb/IIIa inhibitors; which do not increase the incidence of breathlessness at all. [ 14 ]


Cangrelor is synthesized starting from 2-thiobarbituric acid and peracetyl-D- ribofuranose . [ 15 ] [ 16 ]


The synthesis starts with the selective S-alkylation of 2-thiobarbituric acid, followed by  nitration  with  nitric acid , leading to the nitrated dihydroxypyrimidine. Treatment with  phosphorus oxychloride  affords the corresponding dichloropyrimidine. Subsequently, the  nitro group  is reduced using  iron  as the reductant, yielding the aniline derivative. This is cyclized to the  purine  using  triethyl orthoformate  and  hydrochloric acid .


N , O -bis-(trimethylsilyl)acetamide  is used to protect the anilinic nitrogen, allowing for the selective N9-alkylation of the compound with peracetyl-D-ribofuranose using  trimethylsilyl triflate . [ 17 ]


The 5'-OH is converted to a phosphodichloridate using phosphorus oxychloride in triethyl phosphate as the solvent. [ 18 ]  This is converted to Cangrelor without isolation by reaction with dichloromethylenebis(phosphonic acid) and tributylamine as the base.


Citicoline  ( INN ), also known as  cytidine diphosphate-choline  ( CDP-choline ) or  cytidine 5'-diphosphocholine  is an intermediate in the generation of  phosphatidylcholine  from  choline , a common biochemical process in  cell membranes . Citicoline is naturally occurring in the  cells  of human and animal tissue, in particular the organs.


Citicoline is available as a supplement in over 70 countries under a variety of brand names: CereBleu, Cebroton, Ceraxon, Cidilin, Citifar, Cognizin, Difosfocin, Hipercol, NeurAxon, Nicholin, Sinkron, Somazina, Synapsine, Startonyl, Trausan, Xerenoos, etc. [ 1 ]  When taken as a supplement, citicoline is hydrolyzed into  choline  and  cytidine  in the  intestine . [ 2 ]  Once these cross the  blood–brain barrier  it is reformed into citicoline by the rate-limiting enzyme in  phosphatidylcholine  synthesis,  CTP-phosphocholine cytidylyltransferase . [ 3 ] [ 4 ]


Studies suggest, but have not confirmed, potential benefits of citicoline for cognitive impairments. [ 5 ]


Some preliminary research suggested that citicoline may reduce the rates of death and disability following an  ischemic stroke . [ 6 ] [ 7 ]
However, the largest citicoline clinical trial to date (a randomised, placebo-controlled, sequential trial of 2,298 patients with moderate-to-severe acute ischaemic stroke in Europe), found no benefit of administering citicoline on survival or recovery from stroke. [ 8 ]  A meta-analysis of seven trials reported no statistically significant benefit for long-term survival or recovery. [ 9 ]


The effect of citicoline on visual function has been studied in patients with  glaucoma , with possible positive effect for protecting vision. [ 10 ]


Citicoline may have neuroprotective effects due to its preservation of  cardiolipin  and  sphingomyelin , preservation of  arachidonic acid  content of  phosphatidylcholine  and  phosphatidylethanolamine , partial restoration of phosphatidylcholine levels, and stimulation of  glutathione  synthesis and  glutathione reductase  activity. Citicoline's effects may also be explained by the reduction of  phospholipase A2  activity. [ 11 ]
Citicoline increases phosphatidylcholine synthesis. [ 12 ] [ 13 ] [ 14 ]  The mechanism for this may be:


The brain preferentially uses   choline  to synthesize  acetylcholine . This limits the amount of choline available to synthesize phosphatidylcholine. When the availability of choline is low or the need for acetylcholine increases, phospholipids containing choline can be catabolized from neuronal membranes. These phospholipids include sphingomyelin and  phosphatidylcholine . [ 11 ]  Supplementation with citicoline can increase the amount of choline available for acetylcholine synthesis and aid in rebuilding membrane  phospholipid  stores after depletion. [ 16 ]
Citicoline decreases  phospholipase  stimulation. This can lower levels of  hydroxyl radicals  produced after an  ischemia  and prevent  cardiolipin  from being catabolized by  phospholipase A2 . [ 17 ] [ 18 ]  It can also work to restore cardiolipin levels in the  inner mitochondrial membrane . [ 17 ]


Citicoline may enhance cellular communication by increasing levels of neurotransmitters. [ 19 ]  The choline component of citicoline is used to create acetylcholine, which is a neurotransmitter in the human brain. Clinical trials have found that citicoline supplementation might improve focus and attention. [ 20 ]


Citicoline lowers increased  glutamate  concentrations and raises decreased  ATP  concentrations induced by  ischemia . Citicoline also increases  glutamate  uptake by increasing expression of  EAAT2 , a  glutamate transporter , in vitro in rat astrocytes. It is suggested that the neuroprotective effects of citicoline after a  stroke  are due in part to citicoline's ability to decrease levels of  glutamate  in the brain. [ 21 ]


Citicoline is water-soluble, with more than 90% oral  bioavailability . [ 16 ]  Plasma levels of citicholine peak one hour after oral ingestion, and a majority of the citicoline is excreted as  CO 2  in respiration with the remaining citicoline being excreted through  urine . [ 22 ]  The pharmacokinetic profile of citicholine cannot be described by a single smooth exponential decrease over time. [ 22 ]  However, the elimination half-life for citicholine has been reported as approximately 50 hours for citicholine removed via respiration and approximately 70 hours for citicholine removed via urine. [ 22 ]  Plasma levels of choline peak about four hours after ingestion. [ 23 ]


Citicoline has a very low toxicity profile in animals and humans. Clinically, doses of 2000 mg per day have been observed and approved. Minor transient adverse effects are rare and most commonly include stomach pain and diarrhea. [ 13 ] [ 24 ]  A 2020 study reported that concerns had emerged that chronic citicoline use may have adverse psychiatric effects, however, the study's meta-analysis of the relevant literature did not support this hypothesis. [ 25 ] [ 24 ]  Citicoline may exacerbate psychotic episodes or interact with antipsychotic medication.


Phosphatidylcholine is a major phospholipid in eukaryotic cell membranes. Close regulation of its biosynthesis, degradation, and distribution is essential to proper cell function. Phosphatidylcholine is synthesized  in vivo  by two pathways


Cyclic adenosine monophosphate  ( cAMP ,  cyclic AMP , or  3',5'-cyclic  adenosine monophosphate ) is a  second messenger , or cellular signal occurring within cells, that is important in many biological processes. cAMP is a derivative of   adenosine triphosphate  (ATP) and used for intracellular  signal transduction  in many different organisms, conveying the  cAMP-dependent pathway .


Earl Sutherland  of  Vanderbilt University  won a  Nobel Prize in Physiology or Medicine  in 1971 "for his discoveries concerning the mechanisms of the action of hormones", especially epinephrine, via  second messengers  (such as cyclic adenosine monophosphate, cyclic AMP).


The synthesis of cAMP is stimulated by trophic hormones that bind to receptors on the cell surface. cAMP levels reach maximal levels within minutes and decrease gradually over an hour in cultured cells. [ 1 ]
Cyclic  AMP  is synthesized from  ATP  by  adenylate cyclase  located on the inner side of the plasma membrane and anchored at various locations in the interior of the cell. [ 2 ]  Adenylate cyclase is  activated  by a range of signaling molecules through the activation of adenylate cyclase stimulatory G ( G s )-protein-coupled receptors. Adenylate cyclase is  inhibited  by agonists of adenylate cyclase inhibitory G ( G i )-protein-coupled receptors. Liver adenylate cyclase responds more strongly to glucagon, and muscle adenylate cyclase responds more strongly to adrenaline.


cAMP decomposition into  AMP  is catalyzed by the enzyme  phosphodiesterase .


cAMP is a  second messenger , used for intracellular signal transduction, such as  transferring  into cells the effects of  hormones  like  glucagon  and  adrenaline , which cannot pass through the plasma membrane. It is also involved in the activation of  protein kinases .  In addition, cAMP  binds to  and regulates the function of  ion channels  such as the  HCN channels  and a few other  cyclic nucleotide-binding proteins  such as  Epac1  and  RAPGEF2 .


cAMP is associated with kinases function in several biochemical processes, including the regulation of  glycogen ,  sugar , and  lipid   metabolism . [ 3 ]


In eukaryotes, cyclic AMP works by activating protein kinase A (PKA, or  cAMP-dependent protein kinase ). PKA is normally inactive as a tetrameric  holoenzyme , consisting of two  catalytic  and two regulatory units (C 2 R 2 ), with the regulatory units blocking the catalytic centers of the catalytic units.


Cyclic AMP binds to specific locations on the regulatory units of the protein kinase, and causes dissociation between the regulatory and catalytic subunits, thus enabling those catalytic units to  phosphorylate  substrate proteins.


The active subunits catalyze the transfer of phosphate from ATP to specific  serine  or  threonine  residues of protein substrates. The phosphorylated proteins may act directly on the cell's ion channels, or may become activated or inhibited enzymes. Protein kinase A can also phosphorylate specific proteins that bind to promoter regions of DNA, causing increases in transcription. Not all protein kinases respond to cAMP. Several classes of  protein kinases , including protein kinase C, are not cAMP-dependent.


Further effects mainly depend on  cAMP-dependent protein kinase , which vary based on the type of cell.


Still, there are some minor PKA-independent functions of cAMP, e.g., activation of  calcium channels , providing a minor pathway by which  growth hormone-releasing hormone  causes a release of  growth hormone .


However, the view that the majority of the effects of cAMP are controlled by PKA is an outdated one. In 1998 a family of cAMP-sensitive proteins with  guanine nucleotide exchange factor  (GEF) activity was discovered. These are termed Exchange proteins activated by cAMP (Epac) and the family comprises  Epac1  and  Epac2 . [ 4 ]  The mechanism of activation is similar to that of PKA: the GEF domain is usually masked by the N-terminal region containing the cAMP binding domain. When cAMP binds, the domain dissociates and exposes the now-active GEF domain, allowing Epac to activate small Ras-like GTPase proteins, such as  Rap1 .


In the species  Dictyostelium discoideum , cAMP acts outside the cell as a secreted signal.  The  chemotactic  aggregation of cells is organized by periodic waves of cAMP that propagate between cells over distances as large as several centimetres.  The waves are the result of a regulated production and secretion of extracellular cAMP and a spontaneous biological oscillator that initiates the waves at centers of territories. [ 5 ]


In  bacteria , the level of cAMP varies depending on the medium used for growth.  In particular, cAMP is low when glucose is the carbon source.  This occurs through inhibition of the cAMP-producing enzyme,  adenylate cyclase , as a side-effect of glucose transport into the cell.  The transcription factor  cAMP receptor protein  (CRP) also called  CAP (catabolite gene activator protein)  forms a complex with cAMP and thereby is activated to bind to DNA.  CRP-cAMP increases expression of a large number of genes, including some encoding  enzymes  that can supply energy independent of glucose.


cAMP, for example, is involved in the positive regulation of the  lac operon . In an environment with a low glucose concentration, cAMP accumulates and binds to the allosteric site on CRP ( cAMP receptor protein ), a transcription activator protein. The protein assumes its active shape and binds to a specific site upstream of the lac promoter, making it easier for RNA polymerase to bind to the adjacent promoter to start transcription of the lac operon, increasing the rate of lac operon transcription. With a high glucose concentration, the cAMP concentration decreases, and the CRP disengages from the lac operon.


Since cyclic AMP is a second messenger and plays vital role in cell signalling, it has been implicated in various disorders but not restricted to the roles given below:


Some research has suggested that a deregulation of cAMP pathways and an aberrant activation of cAMP-controlled genes is linked to the growth of some cancers. [ 6 ] [ 7 ] [ 8 ]


Recent research suggests that cAMP affects the function of higher-order thinking in the  prefrontal cortex  through its regulation of ion channels called  hyperpolarization-activated cyclic nucleotide-gated channels  (HCN). When cAMP stimulates the HCN, the channels open, This research, especially the cognitive deficits in age-related illnesses and ADHD, is of interest to researchers studying the brain. [ 9 ]


cAMP is involved in activation of trigeminocervical system leading to neurogenic inflammation and causing migraine.  [ 10 ]


Disrupted functioning of cAMP has been noted as one of the mechanisms of several bacterial exotoxins.


They can be subgrouped into two distinct categories: [ 11 ]


Forskolin  is commonly used as a tool in biochemistry to raise levels of cAMP in the study and research of  cell  physiology. [ 15 ]


Cyclic ADP-ribose , frequently abbreviated as  cADPR , is a cyclic adenine nucleotide (like  cAMP ) with two phosphate groups present on 5' OH of the  adenosine  (like  ADP ), further connected to another  ribose  at the 5' position, which, in turn, closes the cycle by  glycosidic bonding  to the nitrogen 1 (N 1 ) of the same  adenine  base (whose position N 9  has the glycosidic bond to the other  ribose ). [ 1 ] [ 2 ]  The N 1 -glycosidic bond to adenine is what distinguishes cADPR from  ADP-ribose  (ADPR), the non-cyclic analog. cADPR is produced from  nicotinamide adenine dinucleotide  (NAD + ) by ADP-ribosyl cyclases ( EC 3.2.2.5 ) as part of a  second messenger system .


cADPR is a cellular messenger for  calcium signaling . [ 3 ]  It stimulates calcium-induced calcium release at lower cytosolic concentrations of Ca 2+ . The primary target of cADPR is the  endoplasmic reticulum  Ca 2+  uptake mechanism. cADPR mobilizes Ca 2+  from the endoplasmic reticulum by activation of  ryanodine receptors , [ 4 ]  a critical step in muscle contraction. [ 5 ]


cADPR also acts as an  agonist  for the  TRPM2  channel, but less potently than  ADPR . [ 6 ]  cADPR and ADPR act  synergistically , with both molecules enhancing the action of the other molecule in activating the TRPM2 channel. [ 7 ]


Potentiation of Ca 2+  release by cADPR is mediated by increased accumulation of Ca 2+  in the  sarcoplasmic reticulum . [ 8 ]


cADPR and ADPR are synthesized from NAD +  by the bifunctional ectoenzymes of the  CD38  family (also includes the  GPI -anchored  CD157  and the specific, monofunctional ADP ribosyl cyclase of the mollusc  Aplysia ). [ 9 ] [ 10 ] [ 11 ]  The same enzymes are also capable of hydrolyzing cADPR to  ADPR . Catalysis proceeds via a covalently bound intermediate. The hydrolysis reaction is inhibited by  ATP , and cADPR may accumulate. Synthesis and degradation of cADPR by enzymes of the CD38 family involve, respectively, the formation and the hydrolysis of the N 1 -glycosidic bond. In 2009, the first enzyme able to hydrolyze the phosphoanhydride linkage of cADPR, i.e. the one between the two phosphate groups, was reported. [ 12 ]


SARM1  and other  TIR domain -containing proteins also catalyze the formation of cADPR from NAD + . [ 13 ] [ 14 ]


Variants of cADPR that differ in their  HPLC  retention times compared to canonical cADPR have been identified as products of bacterial and plant  TIR domain -containing enzymes. [ 14 ] [ 15 ]  v-cADPR (also referred to as 2'cADPR or 1''-2' glycocyclic ADPR (gcADPR)) and v2-cADPR (also referred to as 3'cADPR or 1''-3' gcADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. [ 16 ] [ 17 ]  3'cADPR produced by bacterial  TIR domain -containing proteins can act as an activator of bacterial antiphage defense systems and as a suppressor of plant immunity. [ 16 ]


Cyclic di-AMP  (also called  c-di-AMP  and  c-di-adenosine monophosphate ) is a  second messenger  used in signal transduction in  bacteria  and  archaea . [ 1 ] [ 2 ] [ 3 ]  It is present in many  Gram-positive  bacteria, some  Gram-negative  species, and archaea of the  phylum   Euryarchaeota . [ 2 ] [ 3 ]


It is one of many ubiquitous nucleotide second messengers including  cyclic adenosine monophosphate  (cAMP),  cyclic guanosine monophosphate  (cGMP),  guanosine pentaphosphate  ((p)ppGpp), and  cyclic di-GMP  (c-di-GMP). c-di-AMP is a signaling nucleotide used in signaling pathways that trigger outputs by using receptor or target proteins to sense c-di-AMP concentrations in the cell.


In bacteria, cyclic di-AMP has been implicated in the control of growth, cell wall homeostasis, bacterial biofilm formation and virulence gene expression, heat and osmotic stress regulation and responses, sporulation, potassium transport, lysis, and antibiotic resistance. [ 2 ] [ 4 ]


In humans, cyclic di-AMP has been implicated in the control of innate immune response and antiviral response against pathogens. The dinucleotide is also produced by numerous human pathogens, prompting the exploration of numerous c-di-AMP-regulating pathways both in humans and in bacteria.


Cyclic di-AMP is synthesized by a membrane-bound  diadenylate cyclase  (also called diadenylyl cyclase, CdA, and DAC) enzyme called CdaA (DacA). DacA condenses two ATP molecules to make c-di-AMP, releasing 2  pyrophosphates  in the process. [ 5 ] [ 6 ]  DacA requires a manganese or cobalt metal ion cofactor. [ 7 ]  Most bacteria possess only one DAC enzyme, but some bacteria like  B. subtilis  possess two additional DAC enzymes (DisA and CdaS). [ 2 ]


Cyclic di-AMP synthesis is inhibited by the GImM I154F mutation in the  Lactococcus lactis  bacterium. GImM is the phosphoglucosamine mutase enzyme that interconverts glucosamine-6-phosphate to glucosamine-1-phosphate to later form cell wall  peptidoglycan  and other polymers. [ 4 ]  The I154F mutation inhibits CdA activity by binding to it more strongly than wild-type GImM binds. [ 4 ]  Thus, GImM modulates c-di-AMP levels.


Synthesis is regulated a number of ways, including negative  feedback  inhibition and  upregulation  through a decrease in phosphodiesterase. [ 2 ]


Phosphodiesterase  (PDE) enzymes degrade cyclic di-AMP to the linear molecule 5’-pApA (phosphadenylyl adenosine). 5'-pApA is also involved in a feedback inhibition loop that limits GdpP gene-dependent c-di-AMP hydrolysis, leading to elevated c-di-AMP levels. [ 8 ]


Since cyclic di-AMP is a signaling nucleotide, it is presumed to adhere to the same regulation pathways, where environmental changes are sensed by synthesis or degradation enzymes, which modulate enzyme concentration. Regulation of c-di-AMP is critical because high c-di-AMP levels lead to abnormal physiology, growth defects, and reduced virulence in infection. [ 9 ]  In some bacteria, loss of the phosphodiesterases that degrade c-di-AMP leads to cell death. [ 9 ] [ 10 ] [ 11 ]


It is possible that in addition to enzymatic regulation, intracellular c-di-AMP levels can be regulated by active transport via multidrug resistance transporters that secrete c-di-AMP from the cytoplasm.  Listeria monocytogenes  has demonstrated such an effect. [ 9 ]


At high concentrations, cyclic di-AMP binds to receptor and target proteins to control specific pathways. Elevated c-di-AMP levels have also been linked to increased resistance toward cell wall-damaging antibiotics (e.g.  β-lactams ) and reduced cellular  turgor . [ 12 ] [ 13 ]


Cyclic di-AMP has been linked to fatty acid synthesis regulation in  Mycobacterium smegmatis , the growth of  S. aureus  in conditions of low potassium, the sensing of DNA integrity in  B. subtilis , and cell wall homeostasis in multiple species. [ 14 ] [ 15 ] [ 16 ] [ 17 ]


Cell wall precursor, and thus peptidoglycan precursor, biosynthesis activity can also affect c-di-AMP levels in the cell. [ 4 ]  Similarly, c-di-AMP levels affect peptidoglycan precursor synthesis, suggesting a strong link between the c-di-AMP and peptidoglycan synthetic pathways. [ 17 ]


It is suggested that cyclic di-AMP is involved in the regulation of cell lysis. Studies have shown that bacterial mutant strains with low c-di-AMP levels lysed significantly faster than their parent strains. [ 4 ] [ 18 ]


Cyclic di-AMP has also been linked to bacterial RNA synthesis inhibition. c-di-AMP stimulates the production of (p)ppGpp, an  alarmone  involved in bacterial  stringent response . [ 19 ]


In eukaryotic cells, c-di-AMP is sensed and subsequently elicits a  type I interferon  (IFN) response, leading to the activation of defense mechanisms against viral infection. This detection and activation pathway involves STING, TBK1, and IRF3. [ 20 ] [ 21 ]  c-di-AMP may also stimulate dendritic cells, leading to T cell activation. [ 22 ]


c-di-AMP activates the innate immune pathway STING ( stimulator of interferon genes ) to detect damaged DNA. The nucleotide either binds to the helicase DDX41, which in turn activates the STING pathway, or directly binds to the STING protein. [ 23 ]  Cyclic di-AMP has been identified (along with 2’3’-cGAMP) as a ligand that induces closing of the STING dimer, leading to STING polymerization and pathway activation. [ 24 ]  When a type I IFN response is not induced in response to c-di-AMP, STING is unable to relocate from the endoplasmic reticulum to the cytoplasm for pathway activation, suggesting that c-di-AMP is a predominant ligand in STING polymerization, and thus activation, via intracellular translocation. [ 24 ] [ 25 ]


Cyclic di-GMP  (also called  cyclic diguanylate  and  c-di- GMP ) is a  second messenger  used in signal transduction in a wide variety of  bacteria . [ 1 ]  Cyclic di-GMP is not known to be used by  archaea , and has only been observed in  eukaryotes  in  Dictyostelium . [ 2 ]  The biological role of cyclic di-GMP was first uncovered when it was identified as an allosteric activator of a cellulose synthase found in  Gluconacetobacter xylinus  in order to produce  microbial cellulose . [ 3 ]


In structure, it is a cycle containing only two  guanine  bases linked by  ribose  and  phosphate .


Contact with surfaces increases c-di-GMP which increases  transcription ,  translation , and  post translation  of  exopolysaccharides  (EPSs) and other  extracellular polymeric substance  matrix components (see the review by Jenal et al 2017). [ 4 ]  In bacteria, certain signals are communicated by synthesizing or degrading cyclic di-GMP. Cyclic di-GMP is synthesized by  proteins  with  diguanylate cyclase  activity. These proteins typically have a characteristic  GGDEF  motif, which refers to a conserved sequence of five  amino acids . Degradation of cyclic di-GMP is affected by proteins with  phosphodiesterase  activity. These proteins have either an EAL or an HD-GYP amino acid motif. Processes that are known to be regulated by cyclic di-GMP, at least in some organisms, include  biofilm  formation (such as EPS matrices found by Steiner et al 2013), [ 4 ]   motility  (especially the motile-to-sessile transition, see the review by Jenal et al 2017) [ 4 ]  and  virulence  factor production.


Cyclic di-GMP levels are regulated using a variety of mechanisms. Many proteins with GGDEF,  EAL  or HD-GYP domains are found with other domains that can receive signals, such as  PAS domains . Enzymes that degrade or synthesize cyclic di-GMP are believed to be localized to specific regions of the cell, where they influence receivers in a restricted space. [ 1 ]  In  Gluconacetobacter xylinus,  c-di-GMP stimulates the polymerization of glucose into cellulose as a high affinity allosteric activator of the enzyme cellulose synthase. [ 3 ]  Some diguanylate cyclase enzymes are allosterically inhibited by cyclic di-GMP.


Cyclic di-GMP levels regulate other processes via a number of mechanisms. The  Gluconacetobacter xylinus  cellulose synthase is allosterically stimulated by cyclic di-GMP, presenting a mechanism by which cyclic di-GMP can regulate cellulose synthase activity. The PilZ domain has been shown to bind cyclic di-GMP and is believed to be involved in cyclic di-GMP-dependent regulation, but the mechanism by which it does this is unknown. Recent structural studies of PilZ domains from two bacterial species have demonstrated that PilZ domains change conformation drastically upon binding to cyclic di-GMP. [ 5 ] [ 6 ]  This leads to the strong inference that conformational changes in PilZ domains allow the activity of targeted effector proteins (such as cellulose synthase) to be regulated by cyclic di-GMP.  Riboswitches  called the  cyclic di-GMP-I riboswitch  and  cyclic di-GMP-II riboswitch  regulate gene expression in response to cyclic di-GMP concentrations in a variety of bacteria, but not all bacteria that are known to use cyclic di-GMP.


Cyclic di-GMP has been linked to host-association in multiple  Pseudomonas  species. In an experiment where  Pseudomonas lurida  was grown in association with the nematode host  Caenorhabditis elegans  genetic mutations were observed in certain genes that upregulated c-di-GMP, causing a host specialist morphotype to emerge. The mutations affecting c-di-GMP regulation were uncovered with whole genome sequencing. Genes  wspE  and  wspF  from the wsp operon exhibited mutations that upregulated c-di-GMP. Additionally, mutations in the gene  rph  were determined to also affect c-di-GMP expression, which is a novel discovery because  rph  has not previously been linked to c-di-GMP regulation. [ 7 ]


For a review of c-di-GMP roles in  Caulobacter crescentus ,  Pseudomonas aeruginosa ,  Komagataeibacter xylinus / Gluconacetobacter xylinus ,  Myxococcus xanthus ,  Bdellovibrio bacteriovorus  and  Pseudomonas fluorescens  see Jenal et al 2017. [ 4 ]


Cyclic guanosine monophosphate–adenosine monophosphate  (cyclic GMP-AMP, cGAMP) is the first  cyclic di-nucleotide  found in  metazoa . [ 1 ]
In mammalian cells, cGAMP is synthesized by  cyclic GMP-AMP synthase  ( cGAS ) from  ATP  and  GTP  upon cytosolic DNA stimulation. [ 2 ]  cGAMP produced by cGAS contains mixed phosphodiester linkages, with one between 2'-OH of GMP and 5'-phosphate of AMP and the other between 3'-OH of AMP and 5'-phosphate of GMP. [ 3 ] [ 4 ] [ 5 ] [ 6 ]


This molecule, referred to as 2′3′-cGAMP (cyclic [G(2’,5’)pA(3’,5’)p]), functions as an endogenous  second messenger  inducing  STING -dependent  type I interferon  response. [ 1 ] [ 3 ]  cGAMP has also been shown to be an effective adjuvant that boosts the production of antigen-specific antibodies and T cell responses in mice. [ 7 ]   cGAMP exercises antiviral functions in the cell where it is produced, but can also cross  cell membranes  by  passive diffusion  through gap junctions to exert effects on neighboring cells. [ 8 ] [ 9 ]   It may even be packaged into  lentivirus  (such as  HIV-1 ),  poxvirus  and  herpes virus , and under  cell culture  conditions has been found to transmit an antiviral signal to the cells infected with these viruses; however, there is reason to think that at least HIV is capable of evading this mechanism by some means. [ 8 ]
 [ 9 ]


In recent years, cGAMP signalling has been identified in  prokaryotes  including  Vibrio cholerae  which express a bacterial analogue of cGAS. In these organisms, cGAS is encoded as part of an operon alongside cGAMP-activated phospholipases (e.g. CapV in  V. cholerae ). During infection by  bacteriophage , cGAS is activated and cGAMP is synthesised, activating CapV which degrades the cell's membrane and triggers cell death before the phage can complete its replicatory cycle. [ 10 ]


Cyclic guanosine monophosphate  ( cGMP ) is a  cyclic nucleotide  derived from  guanosine triphosphate  (GTP). cGMP acts as a  second messenger  much like  cyclic AMP .  Its most likely mechanism of action is activation of intracellular  protein kinases  in response to the binding of  membrane -impermeable  peptide hormones  to the external cell surface. [ 1 ]  Through protein kinases activation, cGMP can relax smooth muscle. [ 2 ]  cGMP concentration in urine can be measured for kidney function and diabetes detection. [ 3 ]


Cyclic guanosine monophosphate (cGMP) research began after cGMP and  cyclic adenosine monophosphate (cAMP)  were identified as cellular components and potentially involved with cellular regulation. [ 4 ]  Upon the synthesis of cGMP in 1960, [ 4 ]  progress rapidly spread in the understanding of regulation and effects of cGMP.  Earl W. Sutherland  received the 1971  Nobel Prize in Medicine  for his work with  cAMP  and  secondary messengers . This award sparked extensive research into  cAMP , while cGMP received less attention, with its biological functions largely unknown until the 1980s. [ 5 ]  During this period, two pivotal discoveries highlighted cGMP’s role in cellular signaling:  atrial natriuretic peptide (ANP)  was found to stimulate cGMP synthesis through the particulate  guanylyl cyclase  (pGC) receptor, and  nitric oxide  (NO), identified as the endothelium-derived relaxing factor, was shown to activate  soluble guanylyl cyclase  (sGC), producing cGMP to mediate  vasodilation  in smooth muscle cells. [ 5 ]  Further components involved with the cGMP were also identified such as cGMP-hydrolyzing  phosphodiesterases  (PDEs) and cGMP-binding proteins. [ 5 ]  The awarding of the 1998  Nobel Prize  to  Robert F. Furchgott ,  Louis J. Ignarro , and  Ferid Murad  for their discoveries in the NO-cGMP pathway renewed interest in cGMP research with the 1st International Conference on cGMP being held in 2003. [ 5 ]


Guanylate cyclase  (GC)  catalyzes  cGMP synthesis.  This enzyme converts  GTP  to cGMP.  Peptide hormones such as the  atrial natriuretic factor  activate membrane-bound GC, while soluble GC (sGC) is typically activated by  nitric oxide  to stimulate cGMP synthesis. sGC can be inhibited by ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). [ 6 ]


cGMP acts as a regulator of ion channel conductance, glycogenolysis, cellular apoptosis, and platelet inhibition. cGMP relaxes smooth muscle tissue leading to vasodilation which increases blood flow. Additionally, cGMP is involved with neurogenesis and  neuroplasticity . At presynaptic terminals in the  striatum , cGMP controls the efficacy of  neurotransmitter  release. [ 7 ]


cGMP is a secondary messenger in  phototransduction  in the eye. In the photoreceptors of the mammalian eye, the presence of light activates  phosphodiesterase , which degrades cGMP. The  sodium ion channels  in photoreceptors are cGMP-gated, so degradation of cGMP causes sodium channels to close, which leads to the hyperpolarization of the photoreceptor's plasma membrane and ultimately to visual information being sent to the brain. [ 8 ]


cGMP is also seen to mediate the switching on of the attraction of  apical dendrites  of  pyramidal cells  in  cortical layer V  towards  semaphorin-3A  (Sema3a). [ 9 ]  Whereas the  axons  of pyramidal cells are repelled by Sema3a, the apical dendrites are attracted to it. The attraction is mediated by the increased levels of  soluble guanylate cyclase  (sGC) that are present in the apical dendrites. sGC generates cGMP, leading to a sequence of chemical activations that result in the attraction towards Sema3a. The absence of sGC in the axon causes the repulsion from Sema3a. This strategy ensures the structural polarization of pyramidal neurons and takes place in embryonic development.


cGMP, like cAMP, gets synthesized when olfactory receptors receive odorous input.  cGMP is produced slowly and has a more sustained life than cAMP, which has implicated it in long-term cellular responses to odor stimulation, such as  long-term potentiation .  cGMP in the olfactory is synthesized by both membrane guanylyl cyclase (mGC) as well as soluble guanylyl cyclase (sGC).  Studies have found that cGMP synthesis in the olfactory is due to sGC activation by nitric oxide, a neurotransmitter. cGMP also requires increased intracellular levels of cAMP and the link between the two  second messengers  appears to be due to rising intracellular calcium levels. [ 10 ]


The  nitric oxide  (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) pathway has become a target in developing treatments for heart failure. A deficit in cGMP levels has been associated with adverse  cardiovascular  outcomes, promoting factors like  myocardial fibrosis ,  vasoconstriction , and  inflammation , all of which accelerate  heart failure  progression. [ 11 ]  Some soluble  guanylate cyclase  (sGC) stimulators, have yielded promising outcomes in reducing cardiovascular events. [ 11 ]  Their effectiveness is thought to result from increased sensitivity of sGC to endogenous NO.


Elevated plasma cGMP levels, regulated predominantly by  natriuretic peptides  (NP) rather than  nitric oxide  (NO), were found to correlate with a higher risk of heart failure, atherosclerotic cardiovascular disease, and  coronary heart disease . [ 12 ]


The cGMP signaling pathway plays a role in the regulation of  neuroplasticity , an area of interest in understanding the pathophysiology of  major depressive disorder (MDD) . [ 13 ]  The cGMP signaling pathway in the brain operates as a  second messenger system , amplifying  neurotransmitter  signals, influencing  gene expression  and neuronal function. Within neurons, cGMP levels are modulated by guanylate cyclase  enzymes , which synthesize cGMP, and by PDEs, which degrade cGMP. [ 13 ]


Enhancing cGMP levels, either by stimulating guanylate cyclase or inhibiting PDEs, promotes  neurogenesis  and synaptic  plasticity , particularly in brain regions implicated in  MDD , such as the  hippocampus  and  prefrontal cortex . [ 13 ]  Animal studies also demonstrate that chronic  antidepressant  treatment can elevate cGMP levels in these areas. [ 13 ]  Genetic research has further highlighted specific polymorphisms in PDE genes associated with MDD susceptibility and treatment response. [ 13 ]


Certain pathogens, such as  Enterotoxigenic Escherichia coli  (ETEC) , elevate cGMP to evade host immune defenses and establish infection. ETEC’s heat-stable toxin induces significant cGMP production within intestinal epithelial cells, and this cGMP is often secreted into the extracellular space, where it serves as a signaling molecule. [ 14 ]  Extracellular cGMP, in turn, triggers the release of IL-33 release which modulate inflammation and impact the immune system’s ability to mount effective responses, dampening both innate and adaptive immunity. [ 14 ] [ 15 ]


Numerous cyclic nucleotide  phosphodiesterases  (PDE) can degrade cGMP by  hydrolyzing  cGMP into 5'-GMP. PDE 5, -6 and -9 are cGMP-specific while PDE1, -2, -3, -10 and -11 can hydrolyse both cAMP and cGMP.


Phosphodiesterase inhibitors  prevent the degradation of cGMP, thereby enhancing and/or prolonging its effects. For example,  Sildenafil  (Viagra) and similar drugs enhance the vasodilatory effects of cGMP within the  corpus cavernosum  by inhibiting PDE 5 (or PDE V). This is used as a treatment for  erectile dysfunction . However, the drug can inhibit PDE6 in retina (albeit with less affinity than PDE5). This has been shown to result in loss of visual sensitivity but is unlikely to impair common visual tasks, except under conditions of reduced visibility when objects are already near visual threshold. [ 16 ]  This effect is largely avoided by other PDE5 inhibitors, such as  tadalafil . [ 17 ]


The cGMP-dependent protein kinase (PKG) activation pathway begins with the production of cGMP by  guanylyl cyclase  enzymes, which can be activated by signaling molecules such as  nitric oxide  (NO) or  natriuretic peptides . Elevated cGMP levels then lead to the activation of some protein-dependent kinases like PKG. [ 5 ]  For example, PKG ( protein kinase G ) is a  dimer  consisting of one  catalytic  and one regulatory unit, with the regulatory units blocking the  active sites  of the catalytic units.


cGMP binds to sites on the regulatory units of PKG and activates the catalytic units, enabling them to phosphorylate their substrates. Unlike with the activation of some other protein kinases, notably PKA, the PKG is activated but the catalytic and regulatory units do not disassociate.


Once activated, PKG  phosphorylates  various target proteins, altering their function and contributing to cellular processes such as smooth muscle relaxation, ion channel regulation, and inhibition of platelet aggregation. This pathway is also significant in cardiovascular physiology, where it helps maintain vascular tone and  blood pressure . [ 11 ]


A  cyclic nucleotide  (cNMP) is a single- phosphate   nucleotide  with a cyclic bond arrangement between the  sugar  and phosphate groups.  Like other nucleotides, cyclic nucleotides are composed of three functional groups: a sugar, a  nitrogenous base , and a single phosphate group.  As can be seen in the  cyclic adenosine monophosphate  (cAMP) and  cyclic guanosine monophosphate  (cGMP) images, the 'cyclic' portion consists of two bonds between the phosphate group and the 3' and 5'  hydroxyl  groups of the sugar, very often a  ribose .


Their biological significance includes a broad range of  protein - ligand  interactions.  They have been identified as  secondary messengers  in both  hormone  and  ion-channel  signalling in  eukaryotic  cells, as well as  allosteric effector  compounds of  DNA  binding proteins in  prokaryotic  cells.  cAMP and cGMP are currently the most well documented cyclic nucleotides, however there is evidence that  cCMP  (with  cytosine ) is also involved in eukaryotic cellular messaging.  The role of cyclic uridine monophosphate (cUMP) is even less well known.


Discovery of cyclic nucleotides has contributed greatly to the understanding of  kinase  and  phosphatase  mechanisms, as well as protein regulation in general.  Although more than 50 years have passed since their initial discovery, interest in cyclic nucleotides and their biochemical and physiological significance continues.


The understanding of the concept of second messengers, and in particular the role of cyclic nucleotides and their ability to relay physiological signals to a  cell , has its origins in the research of  glycogen  metabolism by  Carl  and  Gerty Cori , for which they were awarded a  Nobel Prize in Physiology or Medicine  in 1947. [ 1 ]   A number of incremental but important discoveries through the 1950s added to their research, primarily focusing on the activity of  glycogen phosphorylase  in dog  liver .  Glycogen phosphorylase catalyzes the first step in  glycogenolysis , the process of breaking  glycogen  into its substituent  glucose  parts. [ 2 ]   Earl Sutherland  investigated the effect of the hormones  adrenaline  and  glucagon  on glycogen phosphorylase, earning him the Nobel Prize in Physiology or Medicine in 1971. [ 1 ]


In 1956  Edwin Krebs  and  Edmond Fischer  discovered that  adenosine triphosphate  (ATP) is required for the conversion of  glycogen phosphorylase  b to glycogen phosphorylase a. While investigating the action of adrenaline on  glycogenolysis  the next year, Sutherland and Walter Wosilait reported that inorganic phosphate is released when the  enzyme  liver phosphorylase is inactivated; but when it is activated, it incorporates a phosphate. [ 1 ]  The “active factor” that the hormones produced [ 2 ]   was finally purified in 1958, and then identified as containing a  ribose , a phosphate, and an  adenine  in equal ratios. Further, it was proved that this factor reverted to 5’-AMP when it was inactivated. [ 1 ]


Evgeny Fesenko, Stanislav Kolesnikov, and Arkady Lyubarsky discovered in 1985 that  cyclic guanosine monophosphate  (cGMP) can initiate the photoresponse in  rods . Soon after, the role of cNMP in gated ion channels of chemosensitive  cilia  of  olfactory sensory neurons  was reported by Tadashi Nakamura and Geoffrey Gold. In 1992 Lawrence Haynes and King-Wai Yau uncovered cNMP’s role in the light-dependent cyclic-nucleotide-gated channel of  cone photoreceptors . [ 3 ]  By the end of the decade, the presence of two types of intramembrane receptors was understood: Rs (which stimulates  cyclase ) and Ri (which inhibits cyclase).  Wei-Jen Tang and James Hurley reported in 1998 that adenylyl cyclase, which synthesizes cAMP, is regulated not only by  hormones  and  neurotransmitters , but also by  phosphorylation ,  calcium ,  forskolin , and guanine nucleotide-binding proteins ( G proteins ). [ 2 ]


The two most well-studied cyclic nucleotides are cyclic AMP (cAMP) and cyclic GMP (cGMP), while cyclic CMP (cCMP) and cyclic UMP (cUMP) are less understood.  cAMP is 3’5’-cyclic adenosine monophosphate, cGMP is 3’5’-cyclic guanosine monophosphate, cCMP is cytidine 3',5'-monophosphate, and cUMP is uridine 3',5'-cyclic phosphate. [ 4 ] [ 5 ]


Each cyclic nucleotide has three components. It contains a nitrogenous base (meaning it contains nitrogen): for example,  adenine  in cAMP and  guanine  in cGMP. It also contains a sugar, specifically the five- carbon  ribose. And finally, a cyclic nucleotide contains a phosphate.  A double-ring  purine  is the nitrogenous base for cAMP and cGMP, while cytosine,  thymine , and  uracil  each have a single-ring nitrogenous base ( pyrimidine ).


These three components are connected so that the nitrogenous base is attached to the first carbon of ribose (1’ carbon), and the phosphate group is attached to the 5’ carbon of ribose. While all nucleotides have this structure, the phosphate group makes a second connection to the ribose ring at the 3’ carbon in cyclic nucleotides.  Because the phosphate group has two separate bonds to the ribose sugar, it forms a cyclic ring. [ 6 ]


The  atom  numbering convention is used to identify the carbons and nitrogens within a cyclic nucleotide.  In the pentose, the carbon closest to the  carbonyl  group is labeled C-1.  When a pentose is connected to a nitrogenous base, carbon atom numbering is distinguished with a prime (') notation, which differentiates these carbons from the atom numbering of the nitrogenous base. [ 7 ]


Therefore, for cAMP, 3’5’-cyclic adenosine monophosphate indicates that a single phosphate group forms a cyclic structure with the ribose group at its 3’ and 5’ carbons, while the ribose group is also attached to adenosine (this bond is understood to be at the 1’ position of the ribose).


Cyclic nucleotides are found in both prokaryotic and eukaryotic cells.  Control of intracellular concentrations is maintained through a series of enzymatic reactions involving several families of proteins.  In higher order mammals, cNMPs are present in many types of tissue.


Cyclic nucleotides are produced from the generic reaction NTP → cNMP + PP i , [ 8 ]  where N represents a nitrogenous base.  The reaction is catalyzed by specific nucleotidyl cyclases, such that production of cAMP is catalyzed by  adenylyl cyclase  and production of cGMP is catalyzed by  guanylyl cyclase . [ 2 ]   Adenylyl cyclase  has been found in both a transmembrane and  cytosolic  form, representing distinct protein classes and different sources of cAMP. [ 9 ]


Both cAMP and cGMP are degraded by  hydrolysis  of the 3'  phosphodiester bond , resulting in a 5'NMP.  Degradation is carried out primarily by a class of enzymes known as  phosphodiesterases  (PDEs).  In mammalian cells, there are 11 known PDE families with varying  isoforms  of each protein expressed based on the cell's regulatory needs.  Some phosphodiesterases are cNMP-specific, while others can hydrolyze non-specifically. [ 10 ]  However, the cAMP and cGMP degradation pathways are much more understood than those for either cCMP or cUMP.  The identification of specific PDEs for cCMP and cUMP has not been as thoroughly established. [ 11 ]


Cyclic nucleotides can be found in many different types of eukaryotic cells, including photo-receptor rods and cones,  smooth muscle cells  and  liver cells .  Cellular concentrations of cyclic nucleotides can be very low, in the 10 −7 M  range, because  metabolism  and function are often localized in particular parts of the cell. [ 1 ]   A highly conserved  cyclic nucleotide-binding domain  (CNB) is present in all proteins that bind cNMPs, regardless of their biological function.  The domain consists of a beta sandwich architecture, with the cyclic nucleotide binding pocket between the  beta sheets .  The binding of cNMP causes a conformational change that affects the protein's activity. [ 12 ]   There is also data to support a synergistic binding effect amongst multiple cyclic nucleotides, with cCMP lowering the effective concentration (EC 50 ) of cAMP for activation of  protein kinase A  (PKA). [ 13 ]


Cyclic nucleotides are integral to a communication system that acts within cells. [ 1 ]   They act as "second messengers" by relaying the signals of many first messengers, such as hormones and neurotransmitters, to their physiological destinations. Cyclic nucleotides participate in many physiological responses, [ 14 ]  including receptor-effector coupling, down-regulation of drug responsiveness, protein-kinase cascades, and transmembrane signal transduction. [ 1 ]


Cyclic nucleotides act as second messengers when first messengers, which cannot enter the cell, instead bind to receptors in the cellular membrane.  The receptor changes conformation and transmits a signal that activates an enzyme in the cell membrane interior called adenylyl cyclase.  This releases cAMP into the cell interior, where it stimulates a protein kinase called cyclic AMP-dependent protein kinase.  By phosphorylating proteins, cyclic AMP-dependent protein kinase alters protein activity.  cAMP's role in this process terminates upon hydrolysis to AMP by phosphodiesterase. [ 2 ]


Cyclic nucleotides are well-suited to act as second messengers for several reasons. Their synthesis is energetically favorable, and they are derived from common metabolic components (ATP and GTP). When they break down into AMP/GMP and inorganic phosphate, these components are non-toxic. [ 14 ]  Finally, cyclic nucleotides can be distinguished from non-cyclic nucleotides because they are smaller and less  polar . [ 2 ]


The involvement of cyclic nucleotides on biological functions is varied, while an understanding of their role continues to grow. There are several examples of their biological influence. They are associated with long-term and short-term memory. [ 20 ]   They also work in the liver to coordinate various enzymes that control  blood glucose  and other  nutrients . [ 21 ]  In  bacteria , cyclic nucleotides bind to catabolite gene activator protein (CAP), which acts to increase metabolic enzymatic activity by increasing the rate of  DNA   transcription . [ 5 ]  They also facilitate relaxation of smooth muscle cells in  vascular  tissue, [ 22 ]  and activate cyclic CNG channels in  retinal  photoreceptors and  olfactory sensory neurons . In addition, they potentially activate cyclic CNG channels in:  pineal gland  light sensitivity, sensory neurons of the  vomeronasal organ  (which is involved in the detection of  pheromones ),  taste receptor  cells,  cellular signaling  in  sperm , airway  epithelial  cells,  gonadotropin-releasing hormone  (GnRH)-secreting  neuronal  cell line, and  renal   inner medullary collecting duct . [ 3 ]


Examples of disruptions of cNMP pathways include: mutations in CNG channel  genes  are associated with degeneration of the retina and with  color blindness ; [ 3 ]  and  overexpression  of cytosolic or  soluble adenylyl cyclase  (sAC) has been linked to human  prostate carcinoma .  Inhibition of sAC, or knockdown by  RNA interference  (RNAi)  transfection  has been shown to prevent the  proliferation  of the prostate carcinoma cells.  The regulatory pathway appears to be part of the EPAC pathway and not the PKA pathway. [ 9 ]


Phosphodiesterases, principle regulators of cNMP degradation, are often targets for therapeutics.  Caffeine is a known PDE inhibitor, while drugs used for the treatment of erectile dysfunction like  sildenafil  and  tadalafil  also act through inhibiting the activity of phosphodiesterases. [ 10 ]


Cytidine diphosphate , abbreviated  CDP , is a  nucleoside diphosphate . It is an  ester  of  pyrophosphoric acid  with the  nucleoside   cytidine . CDP consists of the  pyrophosphate   group , the  pentose   sugar   ribose , and the  nucleobase   cytosine .


In  Bacillus subtilis  and  Staphylococcus aureus , CDP-activated  glycerol  and  ribitol  are necessary to build wall  teichoic acid . [ 1 ]


In  Rhodothermus marinus , CDP-activated  inositol  is necessary to form the  phospholipid  dialkylether glycerophosphoinositide, which contains inositol  phosphate  and  ether -linked  alkyl  chains. [ 2 ]


CDP is commonly formed in the reaction  dolichol  +  cytidine triphosphate  (CTP) ⟶  dolichol-phosphate  + CDP, which is prevalent in many  biochemical pathways . [ 3 ]


Cytidine diphosphate glucose , often abbreviated  CDP-glucose , is a  nucleotide-linked sugar  consisting of  cytidine diphosphate  and  glucose . [ 1 ] [ 2 ]  This nucleotide saccharide participates in the synthesis of  deoxy sugars  such as  paratose  and  tyvelose . [ 3 ]


CDP-glucose is produced from  CTP  and  glucose-1-phosphate  by the enzyme  glucose-1-phosphate cytidylyltransferase . [ 3 ]


CDP-glucose is an important metabolite in certain bacteria, which synthesize  O antigens  from it. [ 3 ]  CDP-glucose can also be used as a substrate for  glycogenin , along its native substrate,  UDP-glucose . The same is true for  TDP-glucose . [ 4 ]


Cytidine monophosphate , also known as  5'-cytidylic acid  or simply  cytidylate , and abbreviated  CMP , is a  nucleotide  that is used as a  monomer  in  RNA . [ 1 ]  It is an  ester  of  phosphoric acid  with the  nucleoside   cytidine . CMP consists of the  phosphate   group , the  pentose   sugar   ribose , and the  nucleobase   cytosine ; hence, a  ribonucleoside monophosphate .
As a  substituent  it takes the form of the prefix  cytidylyl- .


CMP can be phosphorylated to  cytidine diphosphate  by the enzyme  CMP kinase , with  adenosine triphosphate  or  guanosine triphosphate  donating the phosphate group. Since  cytidine triphosphate  is generated by amination of  uridine triphosphate , the main source of CMP is from RNA being decomposed by  RNAse .


Cytidine triphosphate  ( CTP ) is a  pyrimidine   nucleoside triphosphate . CTP, much like  ATP , consists of a ribose sugar, and three phosphate groups. The major difference between the two molecules is the base used, which in CTP is  cytosine .


CTP is a  substrate  in the synthesis of  RNA .


CTP is a  high-energy molecule  similar to ATP, but its role as an energy coupler is limited to a much smaller subset of metabolic reactions.
CTP is a  coenzyme  in metabolic reactions like the synthesis of  glycerophospholipids , where it is used for activation and transfer of diacylglycerol and lipid head groups, [ 1 ]  and  glycosylation  of proteins.


CTP acts as an inhibitor of the enzyme  aspartate carbamoyltransferase , which is used in  pyrimidine biosynthesis . [ 2 ]


Denufosol  ( INN ) is an inhaled  drug  for the treatment of  cystic fibrosis , being developed by  Inspire Pharmaceuticals  and sponsored by the  Cystic Fibrosis Foundation . It was tested in two Phase III  clinical trials , TIGER-1 and TIGER-2.  Initially, in the first Phase III trial, TIGER-1, the compound showed significant results as compared with  placebo . [ 1 ]   In the second Phase III trial, TIGER-2, the compound did not meet the primary endpoint, a significant change in baseline  FEV1  (forced expiratory volume in one second) at the week 48 endpoint as compared to placebo. [ 2 ]  As of 2011, no additional clinical studies are being conducted with the compound.


The drug was also investigated for the treatment of  retinal detachment  and other  retinal  diseases, but trials were terminated in 2006. [ 3 ]


In Phase III studies, denufosol was orally inhaled by patients with cystic fibrosis three times a day using a jet  nebulizer .  To be effective, it had to reach the deeper parts of the lung ( bronchioles ), making it unsuitable for children under five years of age. [ 4 ]


Cystic fibrosis is characterised by a defect of the  chloride channel   CFTR  (cystic fibrosis transmembrane conductance regulator) on  epithelial cells  in the lungs. The CFTR regulates the components of sweat, digestive juices, and  mucus . Defects lead to a  viscous ,  dehydrated  mucus, hindering  mucociliary clearance . Denufosol is an  agonist  at the  P2Y 2  subtype of  purinergic receptors  which via its associated G protein leads to activation of alternative chloride channel.  Activating this alternate chloride channel theoretically increases ion transport in cystic fibrosis patients which compensates the effects caused by the non-functioning CFTR. [ 5 ]


Denufosol consists of two  nucleosides  (composed of a  nucleobase  and a sugar each),  deoxycytidine  and  uridine , linked on their sugars by four units of   phosphoric acid . It is used in form of its tetra sodium  salt. [ citation needed ]


Deoxyadenosine diphosphate  is a  nucleoside diphosphate .  It is related to the common  nucleic acid  ATP, or  adenosine triphosphate , with the -OH ( hydroxyl ) group on the 2' carbon on the nucleotide's  pentose  removed (hence the deoxy- part of the name), and with one fewer  phosphoryl  group than ATP. This makes it also similar to  adenosine diphosphate  except with a hydroxyl group removed.


Deoxyadenosine diphosphate is abbreviated dADP.


Deoxyadenosine monophosphate  ( dAMP ), also known as  deoxyadenylic acid  or  deoxyadenylate  in its  conjugate acid  and conjugate base forms, respectively, is a derivative of the common  nucleotide  AMP, or  adenosine monophosphate , in which the -OH ( hydroxyl ) group on the 2' carbon on the nucleotide's  pentose  has been reduced to just a hydrogen atom (hence the "deoxy-" part of the name).  Deoxyadenosine monophosphate is abbreviated dAMP.  It is a  monomer  used in  DNA .


Deoxyadenosine triphosphate  ( dATP ) is a  nucleotide  used in cells for  DNA synthesis  (or replication), as a substrate of  DNA polymerase . [ 1 ]


Deoxyadenosine triphosphate is produced from  DNA  by the action of  nuclease  P1,  adenylate kinase , and  pyruvate kinase . [ 2 ]


High levels of dATP can be  toxic  and result in impaired  immune function , since dATP acts as a  noncompetitive inhibitor  for the DNA synthesis  enzyme   ribonucleotide reductase . Patients with  adenosine deaminase deficiency  (ADA) tend to have elevated  intracellular  dATP concentrations because  adenosine deaminase  normally curbs  adenosine  levels by converting it into  inosine . [ 3 ] [ 4 ]  Deficiency of this deaminase also causes immunodeficiency. [ 5 ]


In  cardiac   myosin , dATP is an alternative to ATP as an energy substrate for facilitating  cross-bridge formation . [ 6 ] [ 7 ]


Deoxycytidine diphosphate  is a  nucleoside diphosphate .  It is related to the common  nucleic acid  CTP, or  cytidine triphosphate , with the -OH ( hydroxyl ) group on the 2' carbon on the nucleotide's  pentose  removed (hence the deoxy- part of the name), and with one fewer  phosphoryl  group than CTP .


2'-Deoxycytidine diphosphate is abbreviated as  dCDP . [ 1 ]


Deoxycytidine diphosphate is synthesized through the oxidation-reduction reaction of  cytidine 5'-diphosphocholine  which is catalyzed by the presence of  ribonucleoside-diphosphate reductase . [ 2 ]  Additionally, ribonucleoside-diphosphate reductase is capable of binding and catalyzing both the formation of deoxyribonucleotides from ribonucleotide. [ 3 ]


Deoxycytidine monophosphate  ( dCMP ), also known as  deoxycytidylic acid  or  deoxycytidylate  in its  conjugate acid  and conjugate base forms, respectively, is a  deoxynucleotide  and one of the four  monomers  that make up  DNA . In a DNA double helix, it will base pair with  deoxyguanosine monophosphate . [ 1 ]


Deoxycytidine monophosphate is a molecule of interest in treating genetic disorders such as thymidine kinase 2 ( TK2 ) deficiency. In the case of  TK2  deficiency, dCMP in conjunction with  deoxythymidine monophosphate  (dTMP) to bypass the deficiency in the  TK2  enzyme. [ 2 ]  Additionally dCMP is an indicator of several metabolite deficiency pathways such as UMP synthase deficiency or  orotic aciduria . [ 3 ]


Deoxycytidine triphosphate  ( dCTP ) is a  nucleoside triphosphate  that contains the  pyrimidine  base  cytosine .  The triphosphate group contains high-energy phosphoanhydride bonds, which liberate energy when  hydrolized .


DNA polymerase  enzymes use this energy to incorporate  deoxycytidine  into a newly synthesized strand of  DNA .  A  chemical equation  can be written that represents the process:


That is, dCTP has the PP i  ( pyrophosphate ) cleaved off and the  dCMP  is incorporated into the DNA strand at the 3' end.
Subsequent hydrolysis of the PP i  drives the equilibrium of the reaction toward the right side, i.e. incorporation of the nucleotide in the growing DNA chain.


Like other nucleoside triphosphates, manufacturers recommend that dCTP be stored in aqueous solution at −20 °C. [ 1 ]


Deoxyguanosine diphosphate  ( dGDP ) is a  nucleoside diphosphate .  It is related to the common  nucleic acid   guanosine triphosphate  (GTP), with the  -OH group  on the 2' carbon on the nucleotide's  pentose  removed (hence the deoxy- part of the name), and with one fewer  phosphoryl  group than GTP. [ 1 ]


Deoxyguanosine monophosphate  ( dGMP ), also known as  deoxyguanylic acid  or  deoxyguanylate  in its  conjugate acid  and conjugate base forms, respectively, is a derivative of the common  nucleotide   guanosine triphosphate  (GTP), in which the –OH ( hydroxyl ) group on the 2' carbon on the nucleotide's  pentose  has been reduced to just a hydrogen atom (hence the "deoxy-" part of the name).  It is used as a  monomer  in  DNA . [ 1 ]


Deoxyguanosine triphosphate [ 1 ]  ( dGTP ) is a  nucleoside triphosphate , and a  nucleotide  precursor used in cells for  DNA synthesis . The substance is used in the  polymerase chain reaction  technique, in  sequencing , and in  cloning . It is also the competitor of inhibition onset by  acyclovir  in the treatment of  HSV   virus . [ 2 ]


Deoxyinosine monophosphate  ( dIMP ) is a  nucleoside monophosphate  and a derivative of  inosinic acid . It can be formed by the  deamination  of the  purine  base in  deoxyadenosine monophosphate  (dAMP). The enzyme deoxyribonucleoside triphosphate pyrophosphohydrolase, encoded by YJR069C in  S. cerevisiae  and containing (d)ITPase and (d)XTPase activities,  hydrolyses   dITP , resulting in the release of  pyrophosphate  and dIMP. [ 1 ]


A  deoxyribonucleotide  is a  nucleotide  that contains  deoxyribose . They are the  monomeric  units of the informational  biopolymer , deoxyribonucleic acid ( DNA ). Each deoxyribonucleotide comprises three parts: a deoxyribose sugar ( monosaccharide ), a  nitrogenous base , and one  phosphoryl group . [ 1 ]  The nitrogenous bases are either  purines  or  pyrimidines ,  heterocycles  whose structures support the specific  base-pairing  interactions that allow nucleic acids to carry information. The base is always bonded to the 1'-carbon of the deoxyribose, an analog of  ribose  in which the  hydroxyl group  of the 2'-carbon is replaced with a hydrogen atom. The third component, the phosphoryl group, attaches to the deoxyribose monomer via the  hydroxyl group  on the 5'-carbon of the sugar.


When deoxyribonucleotides polymerize to form DNA, the phosphate group from one nucleotide will bond to the 3' carbon on another nucleotide, forming a  phosphodiester bond  via  dehydration synthesis . New nucleotides are always added to the 3' carbon of the last nucleotide, so synthesis always proceeds from 5' to 3'.


Just as a  nucleoside  can be considered as a  nucleotide  without a phosphate group, so too a  deoxyribonucleoside  is a deoxyribonucleotide without a phosphate. [ 2 ]  An example is  deoxycytidine .


Deoxyuridine monophosphate  ( dUMP ), also known as  deoxyuridylic acid  or  deoxyuridylate  in its  conjugate acid  and conjugate base forms, respectively, is a  deoxynucleotide .


It is an intermediate in the metabolism of  deoxyribonucleotides .


Deoxyuridine monophosphate (dUMP) is the deoxygenated form of  uridine monophosphate  (UMP), and is the precursor to deoxythymidine monophosphate (dTMP), a component of DNA  nucleotide  biosynthesis. [ 1 ]  By replacing the  hydroxyl group  at the 2' carbon of ribose with a hydrogen, UMP becomes deoxygenated to dUMP.


The synthesis of deoxyuridine monophosphate (dUMP) is a multi-step process that begins with  uridine monophosphate  (UMP), the product of  pyrimidine  biosynthesis. [ 2 ]  The enzyme  nucleoside monophosphate kinase  converts UMP and  ATP  to  uridine diphosphate  (UDP) and  ADP .


In the presence of excess ATP, the enzyme  ribonucleotide reductase  initiates a chain reaction with UDP, which catalyzes the formation of deoxyuridine diphosphate (dUDP), which is then converted to deoxyuridine triphosphate (dUTP), then deoxyuridine monophosphate (dUMP) via the addition or removal of phosphate groups. [ 3 ]


Click on genes, proteins and metabolites below to link to respective articles. [ § 1 ]


Dideoxynucleotides  are chain-elongating inhibitors of  DNA polymerase , used in the  Sanger method for DNA sequencing . [ 1 ]  They are also known as 2',3' because  both the 2' and 3' positions on the  ribose  lack  hydroxyl groups , and are abbreviated as  ddNTPs  (ddGTP, ddATP, ddTTP and ddCTP). [ 2 ]


The Sanger method is used to amplify a target segment of DNA, so that the DNA sequence can be determined precisely. The incorporation of ddNTPs in the reaction valves are simply used to terminate the synthesis of a growing DNA strand, resulting in partially replicated DNA fragments. This is because  DNA polymerase  requires the 3' OH group of the growing chain and the 5' phosphate group of the incoming dNTP to create a  phosphodiester bond . [ 2 ]  Sometimes the DNA polymerase will incorporate a ddNTP and the absence of the 3' OH group will interrupt the  condensation reaction  between the 5' phosphate (following the cleavage of  pyrophospate ) of the incoming  nucleotide with the 3' hydroxyl group of the previous nucleotide on the growing strand. This condensation reaction would normally occur with the incorporation of a non-modified dNTP by DNA polymerase. In the simplest of terms, the nucleophilic attack of the 3' OH group leads to the addition of a nucleotide onto a growing chain. The absence of the 3' hydroxyl group inhibits this nucleophilic attack from happening, disabling the DNA polymerase's ability to continue with its function. [ 2 ]


This discovery led to its appropriate name "Chain-terminating nucleotides". [ 2 ]  The dideoxyribonucleotides do not have a 3' hydroxyl group, hence no further chain elongation can occur once this dideoxynucleotide is on the chain. This can lead to the termination of the DNA sequence. Thus, these molecules form the basis of the  dideoxy chain-termination method  of  DNA sequencing , which was reported by  Frederick Sanger  and his team in 1977 [ 3 ]  as an extension of earlier work. [ 4 ]   Sanger's approach was described in 2001 as one of the two fundamental methods for sequencing DNA fragments [ 1 ]  (the other being the  Maxam–Gilbert method [ 5 ] ) but the Sanger method is both the "most widely used and the method used by most automated DNA sequencers." [ 1 ]   Sanger won his second  Nobel Prize in Chemistry  in 1980, sharing it with  Walter Gilbert  ("for their contributions concerning the determination of base sequences in nucleic acids") and with  Paul Berg  ("for his fundamental studies of the biochemistry of nucleic acids, with particular regard to  recombinant DNA "), [ 6 ]  and discussed the use of dideoxynucleotides in his Nobel lecture. [ 7 ]


Dideoxynucleotides are useful in the sequencing of DNA in combination with  electrophoresis .  A DNA sample that undergoes PCR ( polymerase chain reaction ) in a mixture containing all four  deoxynucleotides  and one dideoxynucleotide will produce strands of length equal to the position of each base of the type that complements the type having a dideoxynucleotide present. That the  taq polymerase  used in PCR favors the ddGNTP, is a pattern observed in various research. [ 8 ]  That is, each nucleotide base of that particular type has a probability of being bonded to not a deoxynucleotide but rather a dideoxynucleotide, which ends chain elongation.  Therefore, if the sample then undergoes electrophoresis, there will be a band present for each length at which the  complement  of the dideoxynucleotide is present. It is now common to use fluorescent dideoxynucleotides such that each one of the four has a different fluorescence that can be detected by a sequencer; thus only one reaction is needed.


In a patented method,  Tetrahydrofuran  was used in a solvent containing 50 g (0.205 mols) of  uridine  (or, presumably, any unprotected  nucleoside ), 112 ml (1.03 mols) of  methyl orthoformate  and 10 g (52.6 mmols) of  paratoluenesulfonic acid  at room temperature with stirring. After stirring at room temperature for 24 hours, the reaction mixture was poured into an aqueous sodium bicarbonate solution followed by extraction with chloroform 5 times. The extract was dried over sodium sulfate and concentrated to give 49.5 g (0.173 mols) of 2',3'-o-methoxymethylideneuridine (yield, 84.5%). After this reaction, the uridine example product  2',3'-o-methoxymethylideneuridine is then dissolved in 50 ml of  acetic anhydride  at room temperature with stirring. The solution was heated to 140 °C. and kept at this temperature for 5 hours under reflux of the solvent. After cooling to room temperature, the solvent was removed by distillation under reduced pressure, 50 ml of water were added, and the mixture was extracted 3 times with 100 ml of chloroform. The extract was concentrated under reduced pressure, 30% ammonia water to obtain an 82.3% yield of 2',3'-dideoxy-2',3'-didehydrouridine. This product example is then hydrated to remove the double bond through dissolving it in methanol (10 ml) containing a catalyst (wet 5% palladium on carbon) (400 mg) in an atmosphere of hydrogen for 1 h to obtain the resultant dideoxynucleoside ( 2',3'-dideoxyuridine in this case). [ 6 ]  The dye nucleo tide  to be used will likely occur by treatment with a phosphorylation enzyme and  biotinylation  and reaction of the biotinylated substance with the dye. It is possible that immediate reaction with the dye may also occur, but extending the arm is claimed to increase efficiency in the case of using a mutant form of DNA polymerase. [ 5 ]


Dipotassium guanylate  is a  compound  with formula K 2 (C 10 H 12 O 4 N 5 PO 4 ). It is a  potassium   salt  of  guanylic acid . [ 1 ]


As a food additive, it is used as a  flavor enhancer  and has the  E number  E628. [ 1 ]


Diquafosol  (tradename  Diquas ) is a pharmaceutical drug for the treatment of  dry eye disease . It was approved for use in Japan in 2010. [ 1 ]  It is formulated as a 3% ophthalmic solution of the  tetrasodium   salt .


Its  mechanism of action  involves  agonism  of the  P2Y2 purinogenic receptor . [ 2 ]


Disodium guanylate , also known as  sodium 5'-guanylate  and  disodium 5'-guanylate , is a natural  sodium   salt  of the  flavor enhancing  nucleotide  guanosine monophosphate  (GMP). Disodium guanylate is a  food additive  with the  E number  E627. [ 2 ]  It is commonly used in conjunction with  glutamic acid .


As it is a fairly expensive additive, it is usually not used independently of  glutamic acid ; if disodium guanylate is present in a list of ingredients but MSG does not appear to be, it is likely that glutamic acid is provided as part of another ingredient such as a processed  soy protein   complex . It is often added to foods in conjunction with  disodium inosinate ; the combination is known as  disodium 5'-ribonucleotides .


Disodium guanylate is produced by fermentation. [ 3 ] [ 4 ]  It is often added to  instant noodles ,  potato chips  and other  snacks , savory  rice , tinned  vegetables , cured  meats , and packaged  soup .


Disodium inosinate  ( E631 [ 2 ] ) is the  disodium   salt  of  inosinic acid  with the chemical formula C 10 H 11 N 4 Na 2 O 8 P.  It is used as a  food additive  and often found in  instant noodles ,  potato chips , and a variety of other snacks.


Commercial disodium inosinate may either be obtained from bacterial fermentation of sugars or prepared from animal products. The  Vegetarian Society  reports that production from meat or fish is more widespread, [ 3 ]  but the Vegetarian Resource Group reports that all three "leading manufacturers" claim to use fermentation. [ 4 ]


Disodium inosinate is used as a  flavor enhancer , in synergy with  monosodium glutamate  (MSG) to provide the  umami  taste. It is often added to foods in conjunction with  disodium guanylate ; the combination is known as  disodium 5′-ribonucleotides .


As a relatively expensive product, disodium inosinate is usually not used independently of  glutamic acid ; if disodium inosinate is present in a list of ingredients, but MSG does not appear to be, it is possible that glutamic acid is provided as part of another ingredient or is naturally occurring in another ingredient like tomatoes, Parmesan cheese, or  yeast extract .


Inosinate is naturally found in meat and fish at levels of 80–800 mg/100 g. [ 5 ]  It can also be made by fermentation of sugars such as  tapioca starch . [ 6 ]


Some sources claim that industrial levels of production are achieved by extraction from animal products, making E631 non-vegetarian. [ 3 ]  However, an interview by the Vegetarian Resource Group reports that all three "leading manufacturers" (one being  Ajinomoto ) claims to use an all-vegetarian fermentation process. [ 4 ]  Producers are generally open to providing information on the origin. E631 is in some cases labeled as "vegetarian" in ingredients lists when produced from plant sources. [ 7 ]


In the United States, consumption of added 5′-ribonucleotides averages 4 mg per day, compared to 2 g per day of naturally occurring  purines . A review of literature by an FDA committee found no evidence of  carcinogenicity ,  teratogenicity , or adverse effects on reproduction. [ 8 ]


In 2004, disodium inosinate was proposed to be removed from the food additive list by  Codex Alimentarius Commission . [ 9 ]  This change did not go through: it is still present in the 2009 Codex Alimentarius list. [ 10 ]


In  biochemistry ,  flavin adenine dinucleotide  ( FAD ) is a  redox -active  coenzyme  associated with various  proteins , which is involved with several enzymatic reactions in  metabolism . A  flavoprotein  is a protein that contains a  flavin group , which may be in the form of FAD or  flavin mononucleotide  (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex,  α-ketoglutarate dehydrogenase , and a component of the  pyruvate dehydrogenase complex .


FAD can exist in four redox states, which are the  flavin-N(5)-oxide ,  quinone ,  semiquinone , and  hydroquinone . [ 1 ]  FAD is converted between these states by accepting or donating electrons. FAD, in its fully oxidized form, or  quinone  form, accepts two electrons and two protons to become FADH 2  (hydroquinone form). The semiquinone (FADH · ) can be formed by either reduction of FAD or oxidation of FADH 2  by accepting or donating one electron and one proton, respectively. Some proteins, however, generate and maintain a superoxidized form of the flavin cofactor, the flavin-N(5)-oxide. [ 2 ] [ 3 ]


Flavoproteins  were first discovered in 1879 by separating components of cow's milk. They were initially called lactochrome due to their milky origin and yellow  pigment . [ 4 ]  It took 50 years for the scientific community to make any substantial progress in identifying the molecules responsible for the yellow pigment. The 1930s launched the field of  coenzyme  research with the publication of many  flavin  and  nicotinamide  derivative structures and their obligate roles in redox catalysis. German scientists  Otto Warburg  and Walter Christian discovered a yeast derived yellow  protein  required for  cellular respiration  in 1932. Their colleague  Hugo Theorell  separated this yellow enzyme into  apoenzyme  and yellow pigment, and showed that neither the enzyme nor the pigment was capable of  oxidizing   NADH  on their own, but mixing them together would restore activity. Theorell confirmed the pigment to be  riboflavin 's phosphate ester,  flavin mononucleotide  (FMN) in 1937, which was the first direct evidence for  enzyme   cofactors . [ 5 ]  Warburg and Christian then found FAD to be a cofactor of  D-amino acid oxidase  through similar experiments in 1938. [ 6 ]  Warburg's work with linking nicotinamide to hydride transfers and the discovery of flavins paved the way for many scientists in the 40s and 50s to discover copious amounts of redox biochemistry and link them together in pathways such as the  citric acid cycle  and  ATP  synthesis.


Flavin adenine dinucleotide consists of two portions: the  adenine   nucleotide  ( adenosine monophosphate ) and the  flavin mononucleotide  (FMN) bridged together through their  phosphate  groups. Adenine is bound to a cyclic  ribose  at the  1'  carbon, while phosphate is bound to the ribose at the  5'  carbon to form the adenine nucledotide.  Riboflavin  is formed by a carbon-nitrogen (C-N) bond between the  isoalloxazine  and the  ribitol . The phosphate group is then bound to the terminal ribose carbon, forming a FMN. Because the bond between the isoalloxazine and the ribitol is not considered to be a  glycosidic bond , the flavin mononucleotide is not truly a nucleotide. [ 7 ]  This makes the dinucleotide name misleading; however, the flavin mononucleotide group is still very close to a nucleotide in its structure and chemical properties.


FAD can be  reduced  to FADH 2  through the addition of 2 H +  and 2 e − . FADH 2  can also be  oxidized  by the loss of 1 H +  and 1 e −  to form FADH. The FAD form can be recreated through the further loss of 1 H +  and 1 e − . FAD formation can also occur through the reduction and dehydration of flavin-N(5)-oxide. [ 8 ]  Based on the oxidation state, flavins take specific colors when in  aqueous solution .   flavin-N(5)-oxide  (superoxidized) is yellow-orange, FAD (fully oxidized) is yellow, FADH (half reduced) is either blue or red based on the  pH , and the fully reduced form is colorless. [ 9 ] [ 10 ]  Changing the form can have a large impact on other chemical properties. For example, FAD, the fully oxidized form is subject to  nucleophilic attack , the fully reduced form,  FADH 2  has high  polarizability , while the half reduced form is unstable in aqueous solution. [ 11 ]  FAD is an  aromatic  ring system, whereas FADH 2  is not. [ 12 ]  This means that FADH 2  is significantly higher in energy, without the stabilization through  resonance  that the aromatic structure provides.  FADH 2  is an energy-carrying molecule, because, once oxidized it regains aromaticity and releases the energy represented by this stabilization.


The  spectroscopic  properties of FAD and its variants allows for reaction monitoring by use of  UV-VIS absorption  and  fluorescence  spectroscopies. Each form of FAD has distinct absorbance spectra, making for easy observation of changes in oxidation state. [ 11 ]  A major local absorbance maximum for FAD is observed at 450 nm, with an extinction coefficient of 11,300 M −1  cm −1 . [ 13 ]  Flavins in general have fluorescent activity when unbound (proteins bound to flavin nucleic acid derivatives are called  flavoproteins ). This property can be utilized when examining protein binding, observing loss of fluorescent activity when put into the bound state. [ 11 ]  Oxidized flavins have high absorbances of about 450 nm, and fluoresce at about 515-520 nm. [ 9 ]


In biological systems, FAD acts as an acceptor of H +  and e −  in its fully oxidized form, an acceptor or donor in the FADH form, and a donor in the reduced FADH 2  form. The diagram below summarizes the potential changes that it can undergo.


Along with what is seen above, other reactive forms of FAD can be formed and consumed. These reactions involve the transfer of electrons and the making/breaking of  chemical bonds . Through  reaction mechanisms , FAD is able to contribute to chemical activities within biological systems. The following pictures depict general forms of some of the actions that FAD can be involved in.


Mechanisms 1 and 2 represent  hydride  gain, in which the molecule gains what amounts to be one hydride ion. Mechanisms 3 and 4  radical formation  and hydride loss. Radical species contain unpaired electron atoms and are very chemically active. Hydride loss is the inverse process of the hydride gain seen before. The final two mechanisms show  nucleophilic addition  and a reaction using a carbon radical.


FAD plays a major role as an enzyme  cofactor  along with  flavin mononucleotide , another molecule originating from riboflavin. [ 8 ]  Bacteria, fungi and plants can produce  riboflavin , but other  eukaryotes , such as humans, have lost the ability to make it. [ 9 ]  Therefore, humans must obtain riboflavin, also known as vitamin B2, from dietary sources. [ 14 ]  Riboflavin is generally ingested in the small intestine and then transported to cells via carrier proteins. [ 9 ]   Riboflavin kinase  (EC 2.7.1.26) adds a phosphate group to riboflavin to produce flavin mononucleotide, and then  FAD synthetase  attaches an adenine  nucleotide ; both steps require  ATP . [ 9 ]  Bacteria generally have one bi-functional enzyme, but  archaea  and eukaryotes usually employ two distinct enzymes. [ 9 ]  Current research indicates that distinct  isoforms  exist in the  cytosol  and  mitochondria . [ 9 ]  It seems that FAD is synthesized in both locations and potentially transported where needed. [ 11 ]


Flavoproteins utilize the unique and versatile structure of flavin moieties to catalyze difficult redox reactions.  Since flavins have multiple redox states they can participate in processes that involve the transfer of either one or two electrons, hydrogen atoms, or  hydronium  ions.  The N5 and C4a of the fully oxidized flavin ring are also susceptible to  nucleophilic attack . [ 15 ]   This wide variety of ionization and modification of the flavin moiety can be attributed to the isoalloxazine ring system and the ability of flavoproteins to drastically perturb the kinetic parameters of flavins upon binding, including flavin adenine dinucleotide (FAD).


The number of flavin-dependent protein encoded genes in the genome (the flavoproteome) is species dependent and can range from 0.1% - 3.5%, with humans having 90 flavoprotein encoded genes. [ 16 ]   FAD is the more complex and abundant form of flavin and is reported to bind to 75% of the total flavoproteome [ 16 ]  and 84% of human encoded flavoproteins. [ 17 ]   Cellular concentrations of free or non-covalently bound flavins in a variety of cultured mammalian cell lines were reported for  FAD (2.2-17.0 amol/cell) and FMN (0.46-3.4 amol/cell). [ 18 ]


FAD has a more positive  reduction potential  than  NAD+  and is a very strong oxidizing agent. The cell utilizes this in many energetically difficult oxidation reactions such as dehydrogenation of a C-C bond to an  alkene . FAD-dependent proteins function in a large variety of metabolic pathways including electron transport, DNA repair, nucleotide biosynthesis,  beta-oxidation  of fatty acids, amino acid catabolism, as well as synthesis of other cofactors such as  CoA ,  CoQ  and  heme  groups.  One well-known reaction is part of the  citric acid cycle  (also known as the TCA or Krebs cycle);  succinate dehydrogenase  (complex II in the  electron transport chain ) requires covalently bound FAD to catalyze the oxidation of  succinate  to  fumarate  by coupling it with the reduction of  ubiquinone  to  ubiquinol . [ 11 ]  The high-energy electrons from this oxidation are stored momentarily by reducing FAD to FADH 2 . FADH 2  then reverts to FAD, sending its two high-energy electrons through the electron transport chain; the energy in FADH 2  is enough to produce 1.5 equivalents of  ATP [ 19 ]  by  oxidative phosphorylation . Some redox flavoproteins non-covalently bind to FAD like  Acetyl-CoA-dehydrogenases  which are involved in  beta-oxidation  of fatty acids and catabolism of amino acids like  leucine  ( isovaleryl-CoA dehydrogenase ),  isoleucine , (short/branched-chain acyl-CoA dehydrogenase),  valine  (isobutyryl-CoA dehydrogenase), and  lysine  ( glutaryl-CoA dehydrogenase ). [ 20 ]   Additional examples of FAD-dependent enzymes that regulate metabolism are  glycerol-3-phosphate dehydrogenase  (triglyceride synthesis) and  xanthine oxidase  involved in  purine  nucleotide catabolism. [ 21 ]   Noncatalytic functions that FAD can play in flavoproteins include as structural roles, or involved in blue-sensitive light  photoreceptors  that regulate  biological clocks  and development, generation of light in  bioluminescent  bacteria. [ 20 ]


Flavoproteins  have either an  FMN  or FAD molecule as a prosthetic group, this prosthetic group can be tightly bound or covalently linked. Only about 5-10% of flavoproteins have a covalently linked FAD, but these enzymes have  stronger redox power. [ 11 ]  In some instances, FAD can provide structural support for active sites or provide stabilization of intermediates during catalysis. [ 20 ]  Based on the available structural data, the known FAD-binding sites can be divided into more than 200 types. [ 22 ]


90 flavoproteins are encoded in the human genome; about 84% require FAD, and around 16% require FMN, whereas 5 proteins require both to be present. [ 17 ]  Flavoproteins are mainly located in the  mitochondria  because of their redox power. [ 17 ]  Of all flavoproteins, 90% perform redox reactions and the other 10% are  transferases ,  lyases ,  isomerases ,  ligases . [ 16 ]


Monoamine oxidase  (MAO) is an extensively studied flavoenzyme due to its biological importance with the  catabolism  of  norepinephrine ,  serotonin  and  dopamine . MAO oxidizes primary, secondary and tertiary amines, which nonenzymatically hydrolyze from the  imine  to  aldehyde  or  ketone . Even though this class of enzyme has been extensively studied, its mechanism of action is still being debated. Two mechanisms have been proposed: a radical mechanism and a nucleophilic mechanism. The radical mechanism is less generally accepted because no spectral or  electron paramagnetic resonance  evidence exists for the presence of a radical intermediate. The nucleophilic mechanism is more favored because it is supported by  site-directed mutagenesis  studies which mutated two tyrosine residues that were expected to increase the nucleophilicity of the substrates. [ 23 ]


Glucose oxidase  (GOX) catalyzes the oxidation of β-D-glucose to D-glucono-δ-lactone with the simultaneous reduction of enzyme-bound flavin. GOX exists as a homodimer, with each subunit binding one FAD molecule. Crystal structures show that FAD binds in a deep pocket of the enzyme near the dimer interface. Studies showed that upon replacement of FAD with 8-hydroxy-5-carba-5-deaza FAD, the stereochemistry of the reaction was determined by reacting with the  re  face  of the flavin. During turnover, the neutral and anionic semiquinones are observed which indicates a radical mechanism. [ 23 ]