The effect on cytokineand chemokine induction was also evaluated. The best activities wereobserved for CDNs bearing large aromatic substituents that point abovethe CDN molecule. We solved four X-ray structures of complexes ofnew CDNs with human STING. We observed π–π stackinginteractions between the aromatic substituents and Tyr240 that areinvolved in the stabilization of CDN-STING complexes.Cyclic dinucleotides (CDNs) are second messengers thatactivatestimulator of interferon genes (STING). The cGAS-STING pathway playsa promising role in cancer immunotherapy. Here, we describe the synthesisof CDNs containing 7-substituted 7-deazapurine moiety. We used mousecyclic GMP–AMP synthase and bacterial dinucleotide synthasesfor the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurineCDNs were prepared by Suzuki–Miyaura cross-couplings. New CDNswere tested in biochemical and cell-based assays for their affinityto human STING. Eight CDNs showed better activity than 2′3′- Due to the importance of the cGAS-STING pathway, there has beenan increased interest in identifying new STING agonists with improveddrug-like properties compared to the natural STING ligands. This attentionis highlighted by the fact that seven different STING agonists arepresently being investigated in different phases of clinical trials.22Human STING agonists20 7-deazapurine CDN  as previously shown orwith bacterial dinucleotide cyclase from Vibrio cholerae (DncV) and diadenylate cyclase from Bacillus thuringiensis (DisA).35 Otherwise, the compoundswere prepared by chemical synthesis, either by arylation of an enzymaticallyprepared precursor or by total synthesis. All compounds were testedby differential scanning fluorimetry (DSF) and in cell-based assays.For four of the compounds, we determined their structures in complexwith STING by X-ray crystallography to better understand their bindingmode.Herein, we report the design, synthesis,and biochemical and biologicalevaluation of 7-substituted 7-deazapurine CDNs that activate STINGsignaling. As we reported in our previous study,rine CDN 1 can be 34 This allows for the substitutionof a long and multi-step total synthesis of CDNs36 with a one-step enzymatic reaction. Only the enzymaticsynthesis of CDNs from nucleoside triphosphates (NTPs) bearing smallnucleobase modifications has been studied so far.33 Therefore, we decided to explore 7-substituted 7-deazaguanosinetriphosphates 2 (GRTPs) and 7-substituted7-deazaadenosine triphosphates 3 (ARTPs) assubstrates for these enzymes (4b (2′3′-cGRAMP), 5a-f,n,o (2′3′-cGARMPs), and 6f (2′3′-cGRARMP) indicates that mcGAS toleratessmall substituents in position 7 of 7-deazapurine NTPs including halogen,alkyl, cycloalkyl, alkynyl, small hetaryl, and phenyl groups (3g) and 2-benzothienyl(3q),37 caused a disruptionof the enzymatic reaction, leaving triphosphates unreacted. In somecases, such as CDN 5a, the conversions were quantitative;thus, we were able to enzymatically prepare more than 70 mg of thisCDN in one batch, which was further arylated as described below. Forthe synthesis of the 7-substituted 3′3′-CDNs 7a and 8a, we used the bacterial enzymes DncV and DisA and/or enzymatic synthesis . The chemical synthesis ofiodinated 7-deazaadenine CDN 5a started from 7-iodo-7-deazaadenosine(9). First, H-phosphonate 12 had to be synthesized(9 was N-benzoylated usingtransient silyl protection.38 The N-benzoylated intermediate was not isolated due to its poorsolubility that complicated its chromatographic purification. Thecrude product was directly used in a tritylation step to obtain 5′-O-DMTr-protected nucleoside 10 (68% over twosteps). Regioselective 2′-O-silylation usingsilver nitrate catalysis39 provided nucleoside 11. 3′-O-Silyl isomer was also formedduring the reaction as a minor product, but it could not be efficientlyseparated from the 2′-O-silyl isomer 11. However, the mixture of silylated products could be easilydeprotected using TBAF in THF to regenerate the starting material 10. 3′-H-Phosphonate moiety was installedby the reaction of 11 with diphenyl phosphite. The crudeproduct was directly used for the next step in order to avoid a lossof the material during column chromatography. After the removal ofthe DMTr-group by DCA, H-phosphonate 12 was obtained as a triethylammonium salt (77% over two steps).Chemical synthesis of 7-aryl-7-deazaadenineCDNs relied on a modular approach. The key intermediates, 7-iodo-7-deazaadenineCDNs thesized6. IodinaH-phosphonate building blockswith phosphoramidites is typically performed in acetonitrile in thepresence of a coupling activator,40 suchas py-TFA. However, due to the low solubility of 12 inacetonitrile, its reaction with guanosine phosphoramidite 13 proceeded poorly. Efforts to improve the reaction yield by increasingthe amount of py-TFA or by the use of other coupling activators, suchas ethylthio-1H-tetrazole (ETT), failed. However,when 12 was treated with DCA in order to convert thetriethylammonium salt to a standard H-phosphonate,the solubility in acetonitrile improved, and the reaction with guanosinephosphoramidite 13 proceeded even without any couplingactivator. After detritylation, linear dinucleotide 14 was partially purified using reverse phase flash chromatography.The crude linear product 14 was cyclized using 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane2-oxide (DMOCP).41 After oxidation withiodine and a partial purification on a reverse phase C18 column, theprotected CDN 15 was obtained as a crude material. Deprotectionof nucleobases and the phosphate using CH3NH2 and the removal of silyl groups by Et3N·3HF provided7-iodinated 7-deazaadenine CDN 5a in an overall yieldof 18%  was crucial in order to avoid excessivehydrolysis. The cross-coupling reactions were performed under Pd(OAc)2 catalysis in the presence of a water-soluble ligand, triphenylphosphan-3,3′,3″-trisulfonate(TPPTS), and Cs2CO3 in water–acetonitrilemixture (2:1). The reaction of iodinated CDN 5a with2 equiv of phenylboronic acid did not provide full conversion so thatphenyl derivative 5f was obtained in low yield  and 8f (78%),respectively . This methodprovides a useful insight into the STING binding properties of CDNsby determining their ability to improve the thermal stability of theprotein.48 Digitonin 293T cell-based assays were performedusing five major allelic variants 49 in the presence of digitonin A that facilitatesentry of CDNs into cells due to the permeabilization of cell membranes.20 The standard format of the 293T cell assay wascarried out only with cells expressing wild-type human STING and inthe absence of the permeabilizing agent.20In order to biochemicallyand biologically characterize prepared CDNs, all CDNs were testedusing DSF and 293T cell-based reporter assays. The selected compoundsthat showed activity were further evaluated for the induction of cytokinesusing human peripheral blood mononuclear cells (PBMCs). DSF was performedusing wild-type human STING protein (UniProt cGAMP showed that 7-methyl is much less tolerated on 7-deazaguanine(4b) than on 7-deazaadenine (5b) , cyclopropyl (5c), and ethynyl (5d),but also small aromatic substituents, that is, phenyl (5f), 2-furyl (5n), and 2-thienyl (5o) substituents,and even relatively large aromatic groups such as 2-naphthyl (5g), 4-biphenylyl (5k), 4-(2-naphthyl)phenyl(5l), 4-[(2-naphthyloxy)methyl]phenyl (5m), 2-benzofuryl (5p), and 2-benzothienyl (5q).Our initial experiments with 7-deaza substituted 2′3′-ine (5b) 1. 7-Deazer assay 1. This lTm, for 5c, it decreased by only 0.7 °C, whereas 5d showed a nearly 3 °C drop compared to 5b. Thisminor decrease suggests that in position 7 of 7-deazaadenine, thereis a space for the introduction of larger modifications than justa methyl group. When tested in a digitonin cell-based reporter assay,modifications on both 5c and 5d were tolerated.However, for 5d, the EC50 value obtained froman assay in cells expressing the REF allelic form increased by nearly6-fold, whereas at all other tested allelic forms, the increase wasnot higher than 3-fold. This suggests a disruption of interactionsbetween 5d and STING when R232 is replaced by H232. Ina standard cell-based reporter assay for compounds 5c and 5d, we observed a deterioration of cellular activity,which was not present when tested with a membrane-permeabilizing agent.When considering Δ5f), small heterocycles , and pent-1-ynyl (5e) into position 7 of 7-deazaadenine.Surprisingly, we found that ΔTm for 5f, 5n, and 5o decreased by lessthan 3 °C, and EC50 values in the digitonin assayfor these compounds did not increase by more than 2-fold comparedto 5c. Moreover, for compounds 5f, 5n, and 5o, we observed restored activity inthe standard assay, which was deteriorated for 5c and 5d , 3′3′-cGAPheMP (7f), and 3′3′-c-diAPheMP (8f). Unfortunately,substitutions with phenyl in these compounds did not show an improvementat STING in DSF or cell-based reporter assays compared to 5f. For 7f, we found a decrease in ΔTm by 3 °C, but in digitonin assay, EC50 values for allelic form HAQ and AQ remained nearly the same. Forother tested allelic forms, EC50 did not increase morethan 2-fold compared to 5f. However, we observed a deteriorationof activity in the standard cell-based reporter assay similar to compounds 5c and 5d. According to data from both DSF andcell-based reporter assays, substitutions in 6f causeda significant drop of STING activity and led to the inactivity of 8f. Substitutions in these compounds might potentially leadto the disruption of interactions with both R238 residues, which wehave already reported to significantly affect the binding of CDNs.47Considering the structure of 5n and 5o showed better activity thancompounds with phenylsubstituents 5–8f, we focused on larger heterocycleslike benzofuryl (5p), benzothienyl (5q),and dibenzofuryl (5r). For 5p and 5q, we observed an increase in ΔTm by more than 3 °C when compared to 5n and 5o, respectively. Higher ΔTm suggests increasing stabilization of complex STING–CDN.48 We can only speculate that this stabilization could be related tothe introduction of a benzofused heterocycle, which might be in abetter position to form π–π stacking with guanine.Consistent with DSF, we observed that 5p showed lowerEC50 values than 5n, except for wild-typeSTING. Derivative 5q showed lower EC50 forREF and Q than 5o. Compound 5r did not showany improvement in DSF or in digitonin assay when compared to 5n, 5o, 5p, or 5q.Nevertheless, all 5p, 5q, and 5r compounds showed an improvement in a standard assay compared to 5n, 5o, and 2′3′-cGAMP. In the case of 5q, it was 3-fold better than 2′3′-cGAMP.When we considered the fact that the resultsfor cGARMP. When correlated with findings about improvedactivity in the standard assay for compounds with heterocyclic substituents,we designed compounds modified with large 2-naphthyl (5g), 1-naphthyl (5h), 5-acenaphthenyl (5i), and 9-phenanthrenyl (5j) substituents. Interestingly,derivatives 5h, 5i, and 5j showedlower ΔTm and considerably higherEC50s compared to 5n, 5o, 5p, 5q, and 5r. On the other hand, 5g showed a ΔTm of 14.4°C and a nearly 4-fold lowered EC50 in a standardcell-based reporter assay when compared to 2′3′-cGAMP. Unfortunately, 5g showed lower activationof HAQ, AQ, and REF allelic forms. These results show that interactionsin the STING binding pocket are more likely to tolerate substitutionspointing above the adenine moiety rather than substitutions that needadditional interactions and space around the cGAMPmolecule.We proved that there is a space for largermodifications withouta reduction in activity in position 7 of 7-deazaadenine in 2′3′-cGARMP (5k) by applying our findingsabout the preference of the STING protein in the site above AMP in2′3′-cGAMP and with the knowledge ofthe relative flexibility of the lid above the binding.51 The biphenylyl substituent brings additionallipophilicity to the compound, as in the case of 5g,but with the substitution pointing above the cGAMPmolecule, as in the case of 5f. This substitution causeda decrease of only 3 °C in DSF, and EC50s were comparableto those of 2′3′-cGAMP. Moreover, 5k showed nearly 10 times better activity than 2′3′-cGAMP in the standard assay.We designed a 4-biphenylyl-substituted 2′3′-5k, we designed a substitutionwith 4-(2-naphthyl)phenyl (5l). This modification resultedin nearly the same ΔTm and EC50s despite the change from phenyl to naphthyl, which mightcause more significant collisions with the lid of the STING bindingpocket.Considering the resultsfor 5m contained the largest modificationweintroduced, 4-[(2-naphthyloxy)methyl]phenyl. Despite the introductionof such a large modification that was expected to largely clash witha lid of the STING binding pocket, we observed similar complex stabilityfor 5m and 5l in DSF. Moreover, we observedcomparable activities in cell-based reporter assays. When comparingthe activities of derivatives 5k–m with thoseof the clinical candidate ADU-S100,50 similar orbetter activities were observed in digitonin assay. Moreover, in thestandard 293T cell-based assay, compounds 5k–m showed lower EC50 values than ADU-S100. To explain potentactivities of compounds 5k–m, we solved the X-raycrystallographic structure for the complex of STING and correspondingCDNs.Compound cGARMPs, we crystallized human wild-type STINGtruncated to residues 140–379 in complexes with 5f, 5k, 5l, and 5m using ourimproved STING crystallization protocols.52 Crystal structures were determined at resolution 2.69–1.89Å, and the asymmetric unit consisted of two STING heterodimers(chain A/B) and one molecule of the ligand (Table S1). Protein residues were modeled into a well-defined electrondensity map, except for several regions belonging to flexible surfaceexposed loops that could not be resolved owing to their dynamic disorder(Table S2). All ligands were modeled intothe binding site with full occupancy; however, some parts of the mapsfor substitutions of position 7 of 7-deazaadenine were not as welldefined, suggesting some flexibility of this part of the molecule. Ligandsare deeply buried in the binding site (cGAMP (PDB 4KSY).5 Most of the interactions observed in the binding site weresimilar to those described for a natural ligand : Tyr167 π–π stacking with nucleobases,Arg232 forming a salt bridge with phosphates, a side chain of Thr263forming a hydrogen bond with NH2 of guanine, Arg238 interactingthrough a hydrogen bond with phosphates, and Arg238 cation−πstacking with nucleobases on the opposite of the binding site. Ribosemoiety and the central part are linked to the protein residues througha network of water-mediated hydrogen bonds. In all our structures,we also observed Val239 carbonyl forming a hydrogen bond with NH2 at 7-deazaadenosine and Tyr240 π–π stackingwith introduced aromatic substituents (The binding site is formed at the interface oftwo STING monomersand is covered by two “lids” (residues 227–241).The electron density for the lid region was mostly well defined, withsome exceptions pointing to residues disordered by the binding ofa voluminous ligand phenyl substitution, wepreserved the interactions that were observed for 5k . Despite this clash, 5l exhibited a slightly better stabilization of complex inDSF when compared to 5k . Most of the tested compounds induced higher levelsof IFNγ, TNFα, and IFNα secretion when comparedto 2′3′-cGAMP. EC50 obtainedfrom testing at PBMCs correlated with data from a standard 293T cell-basedreporter assay for most of the tested compounds.Selected compounds were further tested forinduction of IFNγ, TNFα, and IFNα secretion usingPBMCs 2. Noneo53 In the optimizationprocess of biochemical binding potency of our compounds, SAR revealedan unexpected potential for modifications with large aromatic groups.This potential is applicable for 2′3′-cGAMP analogues with a preference for modifications that point abovethe CDN molecule. We observed π–π stacking interactionsbetween the aromatic substituents and Tyr240 that are involved instabilization of CDN–STING complexes. In the case of 3′3′-cGAMP and c-di-AMP, our modifications didnot lead to increased binding potency. Substitutions with large aromaticmoieties increased lipophilicity and thus potentially permeabilityinto cells. They might also be more efficiently transported by anSLC19A1 receptor or an LRRC8A transporter, which would result in improvedcellular activity.54Here, we report the design, synthesis,and biochemical and biologicalevaluation of a novel class of 7-substituted 7-deazaadenine containingCDNs capable of STING activation. During the preparation of this classof compounds, we demonstrated a mixed enzymatic–chemical syntheticapproach for the preparation of CDNs. This approach allowed us toefficiently overcome the total synthesis of the whole CDN, a multistepand time-consuming process.9 and 7-deazapurine NTPs 2b,f, 3a–d,f,n, and o were preparedaccording to the literature.55 The synthesis of 3e is described in the Supporting Information (Scheme S1). All chemical reactions were performed under anargon atmosphere. Reactions were monitored by thin layer chromatography(TLC) on TLC Silica gel 60 F254 (Merck) and detected byUV (254 nm). NMR spectra were measured on a Bruker AVANCE 500 MHzspectrometer  or on a Bruker 600 AVANCE IIIHD instrument equipped with 5 mm cryo-probe at 25 °C in D2O  = 3.75 ppm, δ(13C) = 67.19 ppm]) or in CD3OD  = 3.31 ppm, δ(13C) = 49.0 ppm]). 31P NMR spectra were referencedexternally to the signal of H3PO4. Chemicalshifts are given in ppm , and coupling constants (J) are given in Hz. The complete assignment of all NMR signalswas performed using a combination of H,H-COSY, H,H-ROESY, H,C-HSQC,and H,C-HMBC experiments. Low resolution mass spectra were measuredusing electrospray ionization (ESI). High resolution mass spectrawere measured on LTQ Orbitrap XL (Thermo Fisher Scientific) usingESI. High performance flash chromatography (HPFC) was performed withISCO Combiflash Rf system on RediSep Rf Gold Silica Gel columns orReverse Phase (C18) RediSep Rf Gold columns. Purification of CDNswas performed using HPLC (Waters modular HPLC system) on a columnpacked with 5 μm polar C18 reversed phase . The purity of all final compounds wasdetermined by clean NMR spectra and by UPLC. The identification ofCDNs was performed on ACQUITY UPLC HClass PLUS chromatographic systemwith MS SQ Detector 2  using Acquity UPLC BEHC18 column 50 mm × 2.1 mm, 1.7 μm , and 20 mM ammonium acetate buffer, pH 6.8, with a linear gradientof acetonitrile . Column temperature was kept at 40 °C.Negative ESI method was used for ionization. ULPC purity  is shown in Table S3.Unless otherwisenoted, all starting materials, solvents, and reagents were purchasedfrom commercial suppliers and used as received. 7-Iodo-7-adenosine 20 Briefly, the appropriate NTPs were mixed tothe final 2 mM concentration with 5 μM of mcGAS and 0.1 mg/mLherring testes DNA in 1 mL buffer containing 20 mM Tris–HCl[pH 8.0] and 20 mM MgCl2 and incubated for 16 h at 37 °Cin a heating shaker. The synthesis of 7a was performedby using 2 mM 3a, 2 mM GTP, and 2 μM DncV in 1mL of 50 mM HEPES buffer [pH 8.0] supplemented with 100 mM NaCl, 10mM MgCl2, and 1 mM DTT. The whole mixture was incubatedovernight at 37 °C in a heating shaker. The synthesis of 8a was done overnight at 50 °C in a 1 mL reaction mixturecontaining 4 mM 3a, 20 μM DisA, and 50 mM HEPESbuffer [pH 8.0] supplemented with 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT. The next morning, the reactions were spun at 25,000g for 15 min and supernatants were passed through Nanosep3K Omega . The purification of CDNs was continuedby adding 3 mL of ddH2O to the flow-through fractions,and CDNs were purified on a semipreparative C18 column (Luna 5 μmC18 250 mm × 10 mm) using a 60 min gradient at a flow rate of3 mL/min of 0–20% acetonitrile in 0.1 M TEAB buffer [pH 8.5].TEAB was removed from the collected fractions by 3 cycles of evaporation/dissolutionin 50% methanol.Enzymatically prepared 2′3′-CDNs3 and 4 w2b (2 μmol) and ATP (2 μmol) were enzymatically cyclizedusing mcGAS. The final product was purified by HPLC (5–30%MeCN in 0.1 M TEAB). The conversion to a sodium salt form on a Dowex50WX8 (in a Na+ cycle) provided CDN 4b . 1H NMR : 1.81 ; 4.14 ; 4.23 ; 4.23 ; 4.37 ; 4.47 ; 4.49 ; 4.62 ; 4.75 ;4.99 ; 5.33 ; 6.01 ; 6.19 ; 6.83 ; 8.23 ; 8.27 . 13C NMR : 12.90 (5-CH3); 65.61 (C5′-A); 68.75 (C5′-G); 73.55 (C3′-A);74.20 (C3′-G); 76.54 (C2′-A); 79.46 (C2′-G);83.02 (C4′-A); 85.95 (C4′-G); 87.70 (C1′-G);92.47 (C1′-A); 103.55 (C5-G); 118.24 (C4a-G); 119.52 (C6-G);121.67 (C5-A); 141.42 (C8-A); 150.64 (C4-A); 154.81 (C7a-G); 155.07(C2-G); 155.40 (C2-A); 158.26 (C6-A); 31P NMR : −0.20 and −1.02.ESI MS m/z (rel. %): 342 (100) [M–2H]2–, 686 (37) [M–H]−, 708 (8)[M–2H + Na]−. HR MS (ESI): for C22H25N9O13P2 [M–H]−, calcd 686.11308; found, 686.11212.NTP 3a  and GTP (107 μmol) were enzymatically cyclizedusing mcGAS. The final product was purified by HPLC (5–30%MeCN in 0.1 M TEAB). The conversion to a sodium salt form on a Dowex50WX8 (in a Na+ cycle) provided CDN 5a  as a white lyophilizate (water).Enzymatic synthesis: NTP 12  was dissolved in DCM(2.5 mL). Water  and dichloroacetic acid inDCM  were added, and the solution was stirredfor 10 min. Then, pyridine  was added, andthe mixture was evaporated under reduced pressure and co-evaporatedwith anhydrous acetonitrile (3×). The residue was dissolved inanhydrous acetonitrile (480 μL), and a solution of phosphoramidite 13  in anhydrous acetonitrile (1.45 mL)was added. The mixture was stirred at rt for 10 min, then t-butylhydroperoxide  was added, and the stirring continued for another 30 min.Then, the solution was cooled to 0 °C, and a solution of NaHSO3  was added. The mixturewas stirred for 10 min at 0 °C and then for 5 min at rt. Then,solvent was removed in vacuo, and the residue was dissolved in DCM(3.22 mL). Water  and dichloroacetic acidin DCM  were added. The mixture was stirredfor 10 min, then pyridine  was added, andthe mixture was evaporated under reduced pressure and co-evaporatedwith water. Product 14 was partially purified by flashchromatography .Chemical synthesis:Phosphonate 14 was co-evaporated with anhydrous pyridine(3 × 2 mL) and dissolved in anhydrous pyridine (2.6 mL). DMOCPwas added , and the mixture was stirred for 110min at rt. Then, water  and iodine  were added, and the stirring continued for another 10 minat rt. The mixture was cooled to 0 °C, and an aqueous solutionof NaHSO3  was added. After stirringfor 5 min at 0 °C, another portion of NaHSO3 solution was added. The clear solution was evaporatedunder reduced pressure and co-evaporated with water. Crude 15 was partially purified by flash chromatography .Crude 15 wasdissolved in ethanolic solution of CH3NH2 , and the solutionwas stirred for 3 h at rt. Then, the mixture was evaporated underreduced pressure, and the residue was co-evaporated with anhydrouspyridine (3 × 2 mL). A mixture of anhydrous pyridine, Et3N , and Et3N·3HF  was added, and the mixture was stirred at 50 °C for3.5 h. Then, aqueous ammonium acetate  was added, and thesolvents were removed in vacuo. The residue was co-evaporated withwater. After HPLC purification (0–15% MeCN in 0.1 M TEAB) andconversion to a sodium salt form on a Dowex 50WX8 (in a Na+ cycle), CDN 5a  was obtainedas a white lyophilizate (water). 1H NMR : 4.14 ; 4.22 ; 4.23 ; 4.41 ; 4.44 ;4.52 ; 4.61 ; 4.67 ; 4.92 ; 5.62 ; 5.94 ; 6.10; 7.71 ; 7.88 ; 8.09. 13C NMR : 51.15(C5-A); 65.23 ;68.48 ; 72.88; 73.75 (C3′-G);76.68 (C2′-A); 77.43 ; 82.55 ; 85.14 ; 89.10 ; 92.50 (C1′-A);106.80 (C4a-A); 120.60 (C5-G); 129.42 (C6-A); 143.38 (C8-G); 150.09(C7a-A); 153.95 (C2-A); 154.71 (C4-G); 155.84 (C2-G); 159.59 (C4-A);161.74 (C6-G). 31P NMR : −0.32 and −1.41. ESI MS m/z (rel. %): 398 (100) [M–2H]2–, 798 (97) [M–H]−, 820 (30) [M–2H+ Na]−. HR MS (ESI): for C21H23O13N9IP2 [M–H]−, calcd 797.99407; found, 797.99335.Crude 3b (2 μmol) and GTP (2μmol) were enzymatically cyclized using mcGAS. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5b . 1H NMR : 1.98 ; 4.17 ; 4.18 ; 4.26 ; 4.43 ;4.44 ; 4.63 ; 4.71 ; 5.06 ; 5.62 ; 5.96 ; 6.23; 7.36 ; 7.92 ; 8.19 . 13C NMR  and shifts of C4-A, C2-G,and C6-G were not determined): 13.29 (CH3); 64.98 (C5′-A);68.33 (C5′-G); 73.32 (C3′-A); 73.85 (C3′-G);77.08 (C2′-A); 77.49 (C2′-G); 82.64 (C4′-A);86.11 (C4′-G); 88.85 (C1′-G); 92.20 (C1′-A);105.17 (C4a-A); 114.97 (C5-A); 119.83 (C5-G); 123.61 (C6-A); 143.19(C8-G); 147.84 (C2-A); 149.48 (C7a-A); 154.89 (C4-G). 31P NMR : −0.27and −1.18. ESI MS m/z (rel.%): 342 (100) [M–2H]2–, 353 (3) [M–3H+ Na]2–, 686 (36) [M–H]−, 708 (8) [M–2H + Na]−. HR MS (ESI): forC22H27N9O13P2, calcd 686.11308; found, 686.11212.NTP 3c (2 μmol) and GTP (2μmol) were enzymatically cyclized using mcGAS. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5c . 1H NMR : 0.16 m and 0.74 m ; 0.31m and 0.47 m ; 1.52 ; 4.18; 4.24 ; 4.26 ;4.41 ; 4.44 ; 4.45 ; 4.66 ; 4.70;5.05 ; 5.65 ; 5.98 ; 6.22; 7.10 ; 7.96 ; 8.18. 13C NMR : 6.49 (C2-cyclopropyl); 8.97 (C1-cyclopropyl); 10.80(C3-cyclopropyl); 65.31 ; 68.48 ;73.09 ; 73.66(C3′-G); 76.99 (C2′-A); 77.63 ; 82.42 ; 86.08 ; 89.02 ; 91.94 (C1′-A); 105.76 (C5-A); 121.01 (C6-A);121.77 (C4a-A); 143.31 (C8-G); 149.79 (C7a-A); 161.15 (C4-A). 31P NMR : −0.26and −1.09. ESI MS m/z (rel.%): 356 (100) [M–2H]2–, 367 (6) [M–3H+ Na]2–, 712 (7) [M–H]−, 734 (8) [M–2H + Na]−. HR MS (ESI): forC24H28O13N9P2, calcd 712.12873; found, 712.12814.NTP 3d (2 μmol) and GTP (2μmol) were enzymatically cyclized using mcGAS. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5d . 1H NMR : 3.41 ; 4.17 ; 4.22 ; 4.24 ; 4.42 ; 4.46 ; 4.50; 4.63 ; 4.66 ; 4.99 ; 5.69 ; 5.96 ; 6.26; 7.86 ; 7.91 ; 8.23. 13C NMR  and shifts of C2-G and C6-G not determined): 61.36 (-C≡CH); 65.02 (C5′-A); 68.40 (C5′-G); 72.82 (C3′-A);73.82 (C3′-G); 76.91 (C2′-A); 77.38 (C2′-G);82.47 (C4′-A); 84.18 (-C≡CH); 85.94 (C4′-G);88.89 (C1′-G); 92.31 (C1′-A); 97.30 (C5-A); 105.67 (C4a-A);120.22 (C5-G); 130.16 (C6-A); 143.10 (C8-G); 149.06 (C7a-A); 154.62(C4-G); 151.83 (C2-A); 157.90 (C4-A). 31P NMR : −0.18 and −1.14.ESI MS m/z (rel. %): 348 (100) [M–2H]2–, 696 (21) [M–H]−, 718 (16)[M–2H + Na]−. HR MS (ESI): for C23H23O13N9P2, calcd 696.09743;found, 696.09637.NTP 3e (2 μmol) and GTP (2μmol) were enzymatically cyclized using mcGAS. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5e . 1H NMR : 0.91 ; 1.47 ;2.29 ; 4.15 ; 4.23 ; 4.25 ;4.41 ; 4.45 ;4.51 ; 4.62 ; 4.63 ; 4.98 ; 5.75 ; 5.95 ; 6.22; 7.67 ; 7.88 ; 8.18. 13C NMR  and shifts of C2-G, C4-G, C5-G, and C6-G not determined): 15.55(C5-pentynyl); 23.44 (C3-pentynyl); 24.04 (C4-pentynyl); 65.09 (C5′-A);68.48 (C5′-G); 72.73 (C3′-A); 73.80 (C3′-G);74.50 (C1-pentynyl); 76.83 (C2′-A); 77.11 (C2′-G); 82.28(C4′-A); 85.73 (C4′-G); 89.00 (C1′-G); 92.02(C1′-A); 96.60 (C2-pentynyl); 98.09 (C5-A); 105.93 (C4a-A);127.64 (C6-A); 143.12 (C8-G); 149.52 (C7a-A); 154.41 (C2-A); 159.83(C4-A); 31P NMR : −0.15 and −0.83. ESI MS m/z (rel. %): 368 (100) [M–2H]2–,738 (15) [M–H]−, 760 (9) [M–2H + Na]−. HR MS (ESI): for C26H30O13N9P2, calcd 738.14438; found, 738.14313.NTP 3f (2 μmol)and GTP (2 μmol) were enzymatically cyclized using mcGAS. Thefinal product was purified by HPLC (5–30% MeCN in 0.1 M TEAB).The conversion to a sodium salt form on a Dowex 50WX8 (in a Na+ cycle) provided CDN 5f .Enzymatic synthesis: NTP 5a ,phenylboronic acid , and cesium carbonate were mixed with MeCN–H2O  in an argon purged vial. In a separatevial, Pd(OAc)2  and TPPTS  were dissolved in MeCN–H2O, and the solution was sonicated under an argon atmospherefor 30 s. Then, 200 μmol  of this solution was transferredinto the mixture containing the CDN 5a, and the reactionwas stirred at 100 °C for 30 min. Then, the reaction mixturewas cooled to rt, diluted with water to approx. 3 mL, and filteredthrough a 5 μm nylon syringe filter. The filtrate was directlyapplied on HPLC for purification. After two HPLC separations (0–15%MeCN in 0.1 M TEAB) and conversion to a sodium salt form on a Dowex50WX8 (in a Na+ cycle), CDN 5f was obtained as a white lyophilizate (water). 1H NMR : 4.15 ; 4.25 ; 4.29 ;4.43 ; 4.49 ;4.51 ; 4.66 ; 4.79 ; 5.02 ; 5.73 ; 6.01 ; 6.29; 7.09 ; 7.24; 7.25 ;7.63 ; 7.89 ; 8.22 . 13C NMR : 65.53 ; 68.66 ; 73.07 ; 73.93 (C3′-G); 76.96 (C2′-A);77.87 ; 82.61; 86.04; 88.70 ; 92.27 (C1′-A);103.79 (C4a-A); 119.40 (C5-A); 119.88 (C5-G); 122.74 (C6-A); 129.47(p-Ph-A); 130.27 (o-Ph-A); 131.61 (m-Ph-A); 135.65 (i-Ph-A); 142.42 (C8-G); 151.35 (C7a-A);153.88 (C2-A); 154.58 (C4-G); 155.99 (C2-G); 160.08 (C4-A); 161.17(C6-G). 31P NMR : −0.19 and −0.90. ESI MS m/z (rel. %): 373 (100) [M–2H]2–,385 (5) [M–3H + Na]2–, 748 (2) [M–H]−, 770 (6) [M–2H + Na]−. HRMS (ESI): for C27H28O13N9P2 [M–H]−, calcd 748.12873; found,748.12862.Chemical synthesis: CDN 5a ,naphthalene-2-boronic acid , and cesiumcarbonate  were mixed with MeCN–H2O  in an argon purged vial. In a separatevial, Pd(OAc)2  and TPPTS  were dissolved in MeCN–H2O, and the solution was sonicated under argon atmospherefor 30 s. Then, 173 μmol of this solution was transferred intothe mixture containing the CDN 5a, and the reaction mixturewas stirred at 100 °C for 30 min. Then, the reaction mixturewas cooled to rt, diluted with water to approx. 3 mL, and filteredthrough a 5 μm nylon syringe filter. The filtrate was directlyapplied on HPLC for purification (5–25% MeCN in 0.1 M TEAB).After HPLC repurification (9–24% MeCN in 0.1 M TEAB) and conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle),CDN 5g  was obtained as a white lyophilizate(water). 1H NMR : 4.13 ; 4.26 ; 4.36 ; 4.45 ; 4.53 ; 4.57 ; 4.73 ;4.81 ;5.03 ; 5.67 ; 6.07 ; 6.34 ; 7.32 ; 7.53 ; 7.545 ;7.64 ;7.74 ; 7.75 ; 7.77 ; 7.82 ; 7.90 ; 8.25 . 13C NMR : 65.66 ; 68.76 ;73.16 ; 73.91(C3′-G); 76.98 (C2′-A); 79.00 (C2′-G); 82.58; 86.13; 87.80 (C1′-G);92.32 (C1′-A); 103.93 (C5-A); 119.40 (C4a-A); 119.61 (C5-G);123.09 (C6-A); 128.19 (C1-naphth); 128.51 (C3-naphth); 128.70 (C7-naphth);129.24 (C6-naphth); 130.26 (C8-naphth); 130.89 (C5-naphth); 131.18(C4-naphth); 133.19 (C2-naphth); 134.43 (C4a-naphth); 135.96 (C8a-naphth);141.25 (C8-G); 151.60 (C7a-A); 153.89 (C2-A); 154.34 (C4-G); 155.90(C2-G); 160.32 (C4-A); 160.72 (C6-G). 31P NMR : −0.12 and −0.96.ESI MS m/z (rel. %): 398 (100) [M–2H]2–, 798 (47) [M–H]−, 820 (35)[M–2H + Na]− HR MS (ESI): for C31H29O13N9P2 [M–2H]2–, calcd 398.56855; found, 398.56823.CDN 5h was prepared as describedfor 5g from iodinated CDN 5a  and naphthalene-1-boronic acid .The final product was purified by HPLC (5–30% MeCN in 0.1 MTEAB). After HPLC repurification (5–50% MeOH in 0.1 M TEAB)and conversion to a sodium salt form on a Dowex 50WX8 (in a Na+ cycle), CDN 5h  was obtained asa white lyophilizate (water). The final product was a mixture of twodiastereomers (63:37) due to hindered rotation (at 298 K). Chemicalshifts of the resolved signals of minor isomer are given in italics. 1H NMR : 4.125 ; 4.13 ; 4.23 ; 4.24 ; 4.25 ; 4.28; 4.385 ; 4.39 ; 4.44 ; 4.47 ; 4.50 ; 4.615 ; 4.64 ; 5.025 ; 5.04 ;5.09 ; 5.34 ; 5.79 ; 5.87 ;5.97 ; 5.98 ; 6.38 ;6.55 ; 6.895 ; 7.18 ; 7.21 ;7.36 ; 7.38 ; 7.38 ; 7.41 ; 7.485 ;7.52 ; 7.59 ; 7.64 ; 7.70 ; 7.86 ; 7.90 ; 7.905 ; 7.91 ; 7.995 ; 8.20 ; 8.235 . 13CNMR : 65.48 ; 68.92 ; 65.98 ; 68.79; 73.59 ; 73.71 ; 74.19(C3′-G); 74.32 (C3′-G); 76.70 (C2′-A); 76.84 (C2′-A); 77.34 ; 77.50 ; 82.55 ; 82.83 ; 85.72 ; 85.75; 88.63 ;89.02 ; 90.32 (C1′-A); 91.67 (C1′-A); 105.81 (C4a-A); 106.86 (C4a-A); 117.04 (C5-A); 118.96 (C5-A);119.75 (C5-G); 119.79 (C5-G); 123.68 (C6-A); 123.73 (C6-A); 127.65 (C5-naphth); 127.82(C5-naphth); 128.11 (C3-naphth); 128.24 (C3-naphth);128.92 (C7-naphth); 129.03 (C7-naphth); 129.64 (C6-naphth); 129.69 (C6-naphth); 130.82 (C2-naphth); 130.97 (C4-naphth);131.06 (C8-naphth); 131.26 (C4-naphth); 131.45 (C2-naphth); 131.48 (C8-naphth); 132.48 (C4a-naphth); 132.73 (C4a-naphth); 134.11 (C8a-naphth); 134.28 (C8a-naphth); 136.12 (C1-naphth); 136.20 (C1-naphth); 142.91 (C8-G); 142.91 (C8-G); 151.38 (C7a-A); 152.67 (C7a-A); 154.21 (C2-A); 154.40 (C4-G); 154.40 (C4-G); 154.59 (C2-A); 156.04 (C2-G); 156.89 (C2-G);159.90 (C4-A); 160.06 (C4-A); 160.28 (C6-G); 160.94 (C6-G). 31P NMR : −0.08; −0.25;−0.32; −0.61. ESI MS m/z (rel. %): 398 (100) [M–2H]2–, 798 (25) [M–H]−. HR MS (ESI): for C31H30O13N9P2 [M–H]−, calcd 798.114438; found, 798.14472.CDN 5i was prepared as described for 5g from iodinatedCDN 5a  and acenaphthene-5-boronicacid . The final product was purified byHPLC (5–30% MeCN in 0.1 M TEAB). The conversion to a sodiumsalt form on a Dowex 50WX8 (in a Na+ cycle) provided CDN 5i  as a white lyophilizate (water). The finalproduct was a mixture of two diastereomers (78:22) due to hinderedrotation (at 298 K). The hindered rotation resulted in the line broadeningof most NMR signals. Only a few resolved proton signals of minor isomercould be detected, and their chemical shifts are given in italics. 1H NMR : 3.38 ;3.46 ; 4.11 ; 4.23 ; 4.30 ;4.38 ; 4.43 ; 4.48 ; 4.62 ; 4.99 ;5.13, 5.30 ; 5.85 ; 5.96, 5.90 ; 6.44, 6.52 ; 7.03, 6.96 ; 7.15;7.24 ;7.26 ;7.29 ; 7.44 ; 7.85; 8.21 . 13C NMR : 32.48 (C1-acenaphtylene); 32.83 (C2-acenaphtylene);65.48 (C5′-A); 68.89 ; 73.69 (C3′-A); 74.29 (C3′-G); 76.93(C2′-A); 77.37 ; 82.27 (C4′-A); 85.62 ; 88.92 ; 91.06 (C1′-A); 105.17 (C4a-A);117.20 (C5-A); 119.55 (C5-G); 121.73 (C4-acenaphtylene); 122.41 (C6-acenaphtylene);122.58 (C8-acenaphtylene); 123.48 (C6-A); 127.73 (C5a-acenaphtylene);131.66 (C7-acenaphtylene); 132.01 (C3-acenaphtylene); 132.25 (C5-acenaphtylene);141.70 (C8b-acenaphtylene); 142.56 (C8-G); 148.94 (C8a-acenaphtylene);149.81 (C2a-acenaphtylene); 151.60 (C7a-A); 154.28 (C2-A); 154.24(C4-G); 155.89 (C2-G); 160.11 (C4-A); 160.68 (C6-G); 31P NMR : −0.22and −0.45. ESI MS m/z (rel.%): 411 (100) [M–2H]2–, 824 (22) [M–H]−, 846 (26) [M–2H + Na]−. HRMS (ESI): for C33H32O13N9P2 [M–H]−, calcd 824.16003; found,824.15900.CDN 5j was prepared as describedfor 5g from iodinated CDN 5a  and phenanthrene-9-boronic acid .The final product was purified by HPLC (5–30% MeCN in 0.1 MTEAB). The conversion to a sodium salt form on a Dowex 50WX8 (in aNa+ cycle) provided CDN 5j  asa white lyophilizate (water). The final product was a mixture of twodiastereomers (63:37) due to hindered rotation (at 298 K). Chemicalshifts of the resolved signals of minor isomer are given in italics. 1H NMR : 4.07 ; 4.125 ; 4.22 ; 4.25 ; 4.30 ; 4.33 ; 4.35 ; 4.415 ; 4.47 ; 4.49 ; 4.53 ; 4.66 ; 4.67 ; 5.09 ;5.10 ; 5.12 ; 5.47 ; 5.80 ;5.86 ; 5.92 ; 6.06 ; 6.35 ; 6.62 ; 7.305 ; 7.32 ; 7.34 ; 7.515 ; 7.61 ; 7.63 ; 7.635 ; 7.65 ; 7.725 ; 7.75 ; 7.765 ; 7.77 ;7.785 ; 7.79 ; 7.805 ; 7.90 ; 8.21 ; 8.25 ; 8.76 ; 8.79 ; 8.795 ; 8.84. 13C NMR : 65.52 (C5′-A);66.17 (C5′-A); 68.79 (C5′-G); 69.00(C5′-G); 73.73 (C3′-A); 74.17 (C3′-G); 74.29 (C3′-G); 74.66 (C3′-A); 76.43 (C2′-A); 76.89 (C2′-A); 77.91 (C2′-G); 77.91 (C2′-G); 82.66 (C4′-A);82.73 (C4′-A); 85.34 (C4′-G); 85.88 (C4′-G); 88.17 (C1′-G); 88.67 (C1′-G);90.19 (C1′-A); 91.83 (C1′-A); 105.86(C4a-A); 106.05 (C4a-A); 116.93 (C5-A);118.78 (C5-G); 119.24 (C5-A); 119.48 (C5-G); 123.85 (C6-A); 124.16 (C6-A); 125.23 (C4-phen);125.34 (C4-phen); 125.60 (C5-phen); 126.24 (C5-phen);128.53 (C8-phen); 128.79 (C8-phen); 129.70 (C7-phen); 129.76 (C7-phen); 129.84 (C3-phen); 129.84 (C6-phen); 130.00 (C2-phen); 130.03 (C2-phen); 130.88 (C8a-phen); 131.18 (C10-phen); 131.48 (C10-phen); 131.49 (C8a-phen); 131.74 (C1-phen); 132.33 (C4a-phen); 132.35 (C1-phen); 132.45 (C4a-phen); 132.84 (C4b-phen); 133.02 (C9-phen); 133.09 (C4b-phen);133.18 (C9-phen); 141.58 (C8-G); 142.58 (C8-G); 151.23 (C7a-A); 152.86 (C7a-A); 153.68 (C4-G); 154.05 (C2-A); 154.37 (C4-G); 154.57 (C2-A); 155.96 (C2-G); 155.98 (C2-G); 159.90 (C4-A); 159.68 (C6-G); 159.74 (C6-G); 160.72 (C4-A). 31P NMR : −0.07; –0.23;−0.44; –0.68. ESI MS m/z (rel. %): 423 (100) [M–2H]2–, 856 (9) [M–2H + Na]−. HR MS (ESI): forC35H32O13N9P2 [M–H]−, calcd 848.16003; found, 848.15924.CDN 5k was prepared as describedfor 5g from iodinated CDN 5a  and 4-biphenylboronic acid .The final product was purified by HPLC (5–25% MeCN in 0.1 MTEAB). The conversion to a sodium salt form on a Dowex 50WX8 (in aNa+ cycle) provided CDN 5k  asa white lyophilizate (water). 1H NMR : 4.13 ; 4.24 ; 4.32 ; 4.42 ; 4.50 ;4.56 ; 4.68 ;4.74 ; 4.97 ; 5.64 ; 6.02 ; 6.25 ; 7.11 ; 7.41 ; 7.48 ; 7.51 ; 7.66; 7.71 ; 7.82 ; 8.18 . 13C NMR : 65.54 ;68.72 ; 73.09; 73.80 (C3′-G);76.97 (C2′-A); 78.50 (C2′-G); 82.55 ;86.10 ; 88.07(C1′-G); 92.24 (C1′-A); 103.65 (C4a-A); 119.08 (C5-A);119.67 (C5-G); 122.79 (C6-A); 129.37 (C2′-Ph + C6′-Ph);129.67 (C2-phenylene + C6-phenylene); 130.31 (C4′-Ph); 130.49(C3-phenylene + C5-phenylene); 131.82 (C3′-Ph + C5′-Ph);134.81 (C4-phenylene); 140.79 (C1-phenylene); 141.33 (C8-G); 142.49(C1′-Ph); 151.25 (C7a-A); 153.39 (C2-A); 154.49 (C4-G); 155.98(C2-G); 159.95 (C4-A); 160.98 (C6-G). 31P NMR : −0.14 and −0.93.ESI MS m/z (rel. %): 411 (100) [M–2H]2–, 824 (43) [M–H]−, 846 (17)[M–2H + Na]−. HR MS (ESI): for C33H32O13N9P2 [M–H]−, calcd 824.16003; found, 824.16012.CDN 5l was prepared as described for 5g from iodinatedCDN 5a  and 4-phenylboronicacid pinacol ester . The final product waspurified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5l  as a white lyophilizate(water). 1H NMR : 4.14 ; 4.22 ; 4.35 ; 4.44 ; 4.52 ; 4.58 ; 4.70 ;4.76 ; 4.98 ;5.61 ; 6.03 ; 6.30 ; 7.16 ; 7.53 ; 7.54 ;7.57 ; 7.65 ; 7.81 ; 7.84 ; 7.90 ;7.91 ; 7.94 ; 8.12 ; 8.12 . 13C NMR : 65.71 ; 68.78 ; 73.19 ;73.96 (C3′-G); 76.98 (C2′-A); 78.87 (C2′-G);82.58 ; 86.12 ; 87.90 ; 92.18 (C1′-A); 103.64 (C4a-A);119.16 (C5-A); 119.61 (C5-G); 122.71 (C6-A); 127.76 (C1-naphth); 127.81(C3-naphth); 128.93 (C7-naphth); 129.24 (C6-naphth); 129.89 (C3 andC5-phenylene); 130.24 (C8-naphth); 130.57 (C2 and C6-phenylene); 130.82(C5-naphth); 131.15 (C4-naphth); 134.88 (C1-phenylene); 134.99 (C4a-naphth);136.05 (C8a-naphth); 139.85 (C2-naphth); 140.55 (C4-phenylene); 141.08(C8-G); 151.23 (C7a-A); 153.51 (C2-A); 154.49 (C4-G); 156.00 (C2-G);159.89 (C4-A); 160.98 (C6-G). 31P NMR : −0.14 and −0.93. ESI MS m/z (rel. %): 436 (100) [M–2H]2–, 874 (17) [M–H]−, 896 (14)[M–2H + Na]−. HR MS (ESI): for C37H34O13N9P2 [M–H]−, calcd 874.17568; found, 874.17462.CDN 5m was prepared as described for 5g from iodinatedCDN 5a  and [(1-naphthyloxy)methyl]phenylboronicacid . The final product was purified byHPLC (5–32.5% MeCN in 0.1 M TEAB). The conversion to a sodiumsalt form on a Dowex 50WX8 (in a Na+ cycle) provided CDN 5m  as a white lyophilizate (water). 1H NMR : 4.13 ;4.24 ; 4.30 ;4.42 ; 4.49 ; 4.53 ; 4.66 ; 4.72 ; 4.97 ; 5.26 ; 5.30 ; 5.68 ;5.97 ; 6.11 ; 7.09 ; 7.12 ;7.34 ; 7.47 ;7.54 ; 7.54 ; 7.76 ; 7.87 ;8.03 ; 8.17 . 13C NMR : 65.51 ;68.67 ; 73.02(CH2O); 73.15 ; 73.91 (C3′-G); 76.88 (C2′-A); 78.06 ; 82.56 ; 85.95 ;88.51 ;92.09 (C1′-A); 103.57 (C4a-A); 109.29 (C2-naphth); 118.94 (C5-A);119.81 (C5-G); 122.80 (C6-A); 123.49 (C4-naphth); 124.15 (C8-naphth);127.72 (C4a-naphth); 128.35 (C7-naphth); 129.04 (C3-naphth); 129.41(C6-naphth); 130.23 (C5-naphth); 130.46 (C2 and C6-phenylene); 130.98(C3 and C5-phenylene); 136.61 (C1-phenylene); 136.86 (C8a-naphth-A);137.84 (C4-phenylene); 142.06 (C8-G); 150.92 (C7a-A); 153.37 (C2-A);154.50 (C4-G); 155.92 (C2-G); 156.43 (C1-naphth-A); 159.61 (C4-A);161.05 (C6-G). 31P NMR : −0.21 and −0.96. ESI MS m/z (rel. %): 451 (100) [M–2H]2–, 462 (5) [M–3H + Na]2–, 904 (6) [M–H]−. HR MS (ESI): for C38H36O14N9P2 [M–H]−, calcd 904.18624; found, 904.18469.CDN 3n (2 μmol) and GTP (2μmol) were enzymatically cyclized using mcGAS. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5n . 1H NMR : 4.13 ; 4.23 ; 4.34 ; 4.42 ; 4.50 ;4.61 ; 4.66;4.66 ; 5.01 ; 5.74 ; 5.93 ; 6.27; 6.26 ; 6.28 ; 7.42 ;7.75 ; 8.00 ; 8.19 . 13C NMR : 65.48 ; 68.56 ; 72.66 ; 73.70 (C3′-G); 76.92 (C2′-A);77.59 ; 82.34 ; 85.82; 88.84; 92.50(C1′-A); 102.40 (C5-A); 106.58 (C3-furyl); 109.14 (C4a-A);114.75 (C4-furyl); 119.79 (C5-G); 120.95 (C6-A); 142.52 (C8-G); 143.61(C5-furyl); 150.48 (C2-furyl); 150.75 (C7a-A); 154.03 (C2-A); 154.47(C4-G); 155.82 (C2-G); 159.59 (C4-A); 161.11 (C6-G). 31P NMR : −0.16and −1.18. ESI MS m/z (rel.%): 368 (100) [M–2H]2–, 379 (6) [M–3H–Na]2–, 738 (14) [M–H]−, 760 (14)[M–2H + Na]−. HR MS (ESI): for C25H26O14N9P2 [M–H]−, calcd 738.10799; found, 738.10760.NTP 3o (2 μmol) and GTP (2μmol) were enzymatically cyclized using mcGAS. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 5o . 1H NMR : 4.15 ; 4.29 ; 4.31 ; 4.44 ; 4.49 ;4.52 ; 4.66 ; 4.79 ; 5.00 ; 5.70 ; 5.99 ; 6.28; 6.82 ; 6.98; 7.24 ;7.65 ; 7.87 ; 8.24 . 13C NMR : 65.53 ; 68.66 ; 73.03 ; 73.86 (C3′-G); 76.84 (C2′-A);77.74 (C2′-G); 82.62 ; 85.96 ; 88.78 ; 92.20 (C1′-A); 103.70 (C5-A); 112.08 (C4a-A);120.00 (C5-G); 123.16 (C6-A); 128.07 (C3-thienyl); 128.26 (C5-thienyl);130.70 (C4-thienyl); 137.46 (C2-thienyl); 142.56 (C8-G); 151.16 (C7a-A);154.12 (C2-A); 154.69 (C4-G); 155.97 (C2-G); 160.00 (C4-A); 161.29(C6-G). 31P NMR : −0.24 and −0.93. ESI MS m/z (rel. %): 376 (100) [M–2H]2–,387 (4) [M–H + Na]2–, 754 (4) [M–H]−, 776 (10) [M–2H + Na]−. HRMS (ESI): for C25H26O13N9P2S [M–H]−, calcd 754.08515;found, 754.08502.NTP 5p was prepared as describedfor 5g from iodinated CDN 5a  and benzofuran-2-ylboronic acid .After two HPLC purifications (5–25% MeCN in 0.1 M TEAB and5–50% MeOH in 0.1 M TEAB) and conversion to a sodium salt formon a Dowex 50WX8 (in a Na+ cycle), CDN 5p  was obtained as a white lyophilizate (water). 1H NMR : 4.07 ;4.20 ; 4.35 ; 4.38 ; 4.51 ; 4.59 ; 4.65 ; 4.67 ;4.93 ; 5.62 ; 5.90 ; 6.18 ; 6.65 ; 7.22 ; 7.24;7.42 ; 7.47 ; 8.09; 8.10 . 13C NMR : 65.53 (C5′-A); 68.59 ; 72.68 ; 73.74 (C3′-G); 78.30; 78.86 (C2′-A);82.33 ; 85.83 ; 88.02 ; 92.25 (C1′-A); 102.44 (C4a-A);102.51 (C3-benzofuryl); 108.58 (C5-A); 113.48 (C7-benzofuryl); 119.31(C5-G); 122.77 (C6-A); 123.46 (C4-benzofuryl); 125.99 (C5-benzofuryl);126.32 (C6-benzofuryl); 131.49 (C3a-benzofuryl); 141.30 (C8-G); 150.77(C7a-A); 152.57 (C2-benzofuryl); 153.71 (C2-A); 154.26 (C4-G); 155.77(C2-G); 156.02 (C7a-benzofuryl); 159.60 (C4-A); 160.54 (C6-G). 31P NMR : −0.10and −1.10. ESI MS m/z (rel.%): 393 (100) [M–2H]2–, 788 (44) [M–H]−, 810 (21) [M–2H + Na]−. HRMS (ESI): for C29H28O14N9P2 [M–H]−, calcd 788.12364; found,788.12413.CDN 5q was prepared as describedfor 5g from iodinated CDN 5a  and benzothiophen-2-ylboronic acid .After two HPLC purifications (5–25% MeCN in 0.1 M TEAB and9–24% MeCN in 0.1 M TEAB) and conversion to a sodium salt formon a Dowex 50WX8 (in a Na+ cycle), CDN 5q  was obtained as a white lyophilizate (water). 1H NMR : 4.12 ;4.25 ; 4.35 ; 4.44 ; 4.52 ; 4.57 ; 4.71 ; 4.77 ; 4.95 ; 5.60 ; 6.03 ; 6.30; 7.10 ; 7.36 ; 7.41;7.72 ; 7.75 ; 7.78; 7.82 ; 8.23 . 13C NMR : 65.61 ;68.75 ; 72.96; 73.90 (C3′-G);76.89 (C2′-A); 78.91 (C2′-G); 82.52 ; 86.08 ; 87.84 (C1′-G); 92.23(C1′-A); 103.70 (C5-A); 112.55 (C4a-A); 119.66 (C5-G); 123.70(C6-A); 124.04 (C3-benzothienyl); 124.89 (C7-benzothienyl); 126.22(C4-benzothienyl); 126.99 (C6-benzothienyl); 127.37 (C6-benzothienyl);141.14 (C8-G); 138.36 (C2-benzothienyl); 141.78 (C7a-benzothienyl);142.94 (C3a-benzothienyl); 151.46 (C7a-A); 154.26 (C2-A); 154.59 (C4-G);156.00 (C2-G); 160.18 (C4-A); 160.87 (C6-G). 31P NMR : −0.16 and −0.88.ESI MS m/z (rel. %): 436 (100) [M–2H]2–, 874 (16) [M–H]−, 896 (14)[M–2H + Na]−. HR MS (ESI): for C29H28O14N9P2 [M–H]−, calcd 874.17568; found, 874.17462.CDN 5r was prepared as described for 5g from iodinated CDN 5a  and dibenzofuran-4-ylboronic acid. After two purifications (5–25% MeCNin 0.1 M TEAB) and conversion to a sodium salt form on a Dowex 50WX8(in a Na+ cycle), CDN 5r  wasobtained as a white lyophilizate (water). As a byproduct, compound 16 was isolated and converted to a sodium salt form on a Dowex50WX8 (in a Na+ cycle). Open isomer 16  was obtained as a white lyophilizate (water). CDN 5r: 1H NMR : 4.10 ; 4.22 ; 4.33 ; 4.38 ; 4.48 ; 4.49 ; 4.64 ; 4.85 ; 5.10 ; 5.84 ; 5.92 ; 6.40; 7.04 ;7.14 ; 7.40 ; 7.44 ; 7.49; 7.59 ; 7.68 ; 7.76 ; 8.00 ; 8.19 . 13C NMR : 65.59 ; 68.80 ; 73.79 ; 74.22 (C3′-G); 77.14(C2′-A); 77.50 ; 82.84 ; 85.76 ; 88.79 ; 91.31 (C1′-A);104.21 (C4a-A); 113.84 (C5-A); 114.61 (C6-dibenzofuryl); 119.57 (C4-dibenzofuryl);119.60 (C5-G); 122.52 (C1-dibenzofuryl); 123.72 (C9-dibenzofuryl);124.05 (C6-A); 125.81 (C8-dibenzofuryl); 126.08 (C2-dibenzofuryl);126.44 (C9a-dibenzofuryl); 127.01 (C9b-dibenzofuryl); 130.15 (C7-dibenzofuryl);130.40 (C3-dibenzofuryl); 142.45 (C8-G); 151.79 (C7a-A); 154.22 (C4-G);154.27 (C2-A); 155.08 (C4a-dibenzofuryl); 155.88 (C2-G); 158.29 (C5a-dibenzofuryl);160.13 (C4-A); 160.59 (C6-G). 31P NMR : −0.19 and −0.44. ESI MS m/z (rel. %): 418 (100) [M–2H]2–, 838 (28) [M–H]−, 860 (13)[M–2H + Na]−. HR MS (ESI): for C33H30O14N9P2 [M–H]−, calcd 838.13929; found, 838.13778. Open isomer 16: 1H NMR : 3.34 ;3.46 ;3.50 ; 3.76; 4.12 ; 4.19 ; 4.42 ; 4.58 ;5.19 ; 5.42 ; 5.60 ; 6.34 ; 7.00 ;7.25 ; 7.39 ; 7.47 ;7.49 ; 7.50 ; 7.56 ; 7.74 ; 7.92 ; 8.01 . 13C NMR : 62.76 (C5′-G);67.72 ; 71.28(C3′-G); 79.98 ; 80.78 ;84.00 ; 85.52(C4′-G); 86.02 ; 89.95 ;90.99 ; 103.36(C4a-A); 114.12 (C5-A); 114.35 (C6-dibenzofuryl); 118.64 (C5-G); 119.57(C4-dibenzofuryl); 122.29 (C1-dibenzofuryl); 123.51 (C9-dibenzofuryl);124.43 (C6-A); 125.85 (C8-dibenzofuryl); 126.16 (C2-dibenzofuryl);126.20 (C9a-dibenzofuryl); 127.03 (C9b-dibenzofuryl); 129.87 (C3-dibenzofuryl);130.34 (C7-dibenzofuryl); 139.26 (C8-G); 152.46 (C7a-A); 152.86 (C4-G);154.06 (C2-A); 154.96 (C4a-dibenzofuryl); 155.58 (C2-G); 158.12 (C5a-dibenzofuryl);159.44 (C4-A); 160.77 (C6-G). 31P NMR : 0.66 and 20.92. ESI MS m/z (rel. %): 418 (100) [M–2H]2–, 838 (12) [M–H]−, 860 (17) [M–2H+ Na]−, 882 (4) [M–3H+2Na]− HR MS (ESI): for C33H30O14N9P2 [M–H]−, calcd 838.13929;found, 838.13853.CDN 2f (2 μmol) and 3f (2 μmol) were enzymatically cyclized using mcGAS. The finalproduct was purified by HPLC (5–30% MeCN in 0.1 M TEAB). Theconversion to a sodium salt form on a Dowex 50WX8 (in a Na+ cycle) provided CDN 6f . 1H NMR : 4.14 ;4.26 ; 4.36 ; 4.46 ;4.475 ; 4.52 ; 4.53 ; 4.76 ; 4.99; 5.28 ;6.22 ; 6.34 ; 6.98 ; 7.11 ; 7.26 ; 7.31 ; 7.37 ; 7.54 ; 7.98 . 13C NMR : 66.04(C5′-A); 69.04 (C5′-G); 73.11 (C3′-A); 74.29(C3′-G); 77.29 (C2′-A); 81.50 (C2′-G); 82.55(C4′-A); 85.90 (C1′-G); 86.40 (C4′-G); 92.42(C1′-A); 104.12 (C5-A); 117.34 (C6-G); 119.53 (C4a-A); 122.67(C6-A); 123.20 (p-Ph-G); 124.93 (C4a-G); 129.29 (o-Ph-G); 129.85 (o-Ph-A); 130.10 (m-Ph-G); 131.27 (m-Ph and p-Ph-A); 151.02 (C7a-A); 153.39 (C2-A); 155.51 (C7a-G); 160.00 (C4-A);163.10 (C4-G). 31P NMR : 0.28 and −1.33. ESI MS m/z (rel. %): 411 (100) [M–2H]2–,422 (13) [M–3H + Na]2–, 823 (11) [M–H]−, 845 (19) [M–2H + Na]−. HRMS (ESI): for C34H33N8O13P2 [M–H]−, calcd 823.16478; found,823.16454.NTPs 3a (13 μmol) and GTP (13μmol) were enzymatically cyclized using DncV. The final productwas purified by HPLC (5–30% MeCN in 0.1 M TEAB). The conversionto a sodium salt form on a Dowex 50WX8 (in a Na+ cycle)provided CDN 7a  as a white lyophilizate(water). 1H NMR : 4.11 ;4.12 ; 4.36 ; 4.39 ; 4.42 ; 4.55; 4.73; 4.79 ;4.85 ; 6.01 ; 6.24 ; 7.75 ; 8.05 ; 8.10. 13C NMR : 53.44(C5-A); 64.77 ;64.92 ; 73.32; 73.34 ; 76.57 (C2′-A);76.74 (C2′-G); 81.85 ; 82.43 ; 91.90 (C1′-A); 92.86 ; 106.75 (C4a-A); 119.66 (C5-G); 129.22(C6-A); 139.65 (C8-G); 151.10 (C7a-A); 153.43 (C4-G); 154.33 (C2-A);158.62 (C2-G); 159.91 (C4-A); 161.62 (C6-G). 31P NMR : −0.56 and −0.89.ESI MS m/z (rel. %): 398 (100) [M–2H]2–, 798 (45) [M–H]−, 820 (27)[M–2H + Na]−. HR MS (ESI): for C21H22O13N9IP2 [M–2H]2–, calcd 398.49340; found, 398.49324.NTP 7f was prepared as describedfor 5g from iodinated CDN 7a  and phenylboronic acid . Thefinal product was purified using HPLC (0–15% MeCN in 0.1 MTEAB). The conversion to a sodium salt form on a Dowex 50WX8 (in aNa+ cycle) provided CDN 7f  asa white lyophilizate (water). 1H NMR : 4.10 ; 4.13 ; 4.37 ; 4.41 ;4.43 ; 4.44 ;4.52 ; 4.61 ; 4.78 ; 4.80 ; 5.98 ; 6.42 ;7.18 ;7.28 ; 7.47 ; 8.03; 8.18 . 13C NMR : 64.73 ; 64.77 ;72.94 ; 73.55; 76.74 (C2′-A);76.86 (C2′-G); 81.62 ; 82.11 ; 91.51 (C1′-A);92.46 (C1′-G); 103.77 (C4a-A); 119.48 (C5-G); 120.80 (C5-A);122.27 (C6-A); 129.37 (p-Ph); 130.68 (2× o-Ph); 131.16 (2× m-Ph); 135.81 (i-Ph); 138.74 (C8-G); 152.17 (C7a-A); 152.78 (C4-G); 154.17(C2-A); 156.67 (C2-G); 160.35 (C4-A); 161.40 (C6-G). 31P NMR : −0.36and −0.75. ESI MS m/z (rel.%): 373 (100) [M–2H]2–, 748 (6) [M–H]−, 770 (5) [M–2H + Na]−. HRMS (ESI): for C27H28O13N9P2 [M–H]−, calcd 748.12873; found,748.12726.CDN 3a (26 μmol) was enzymaticallycyclized using DisA. The final product was purified by HPLC (5–30%MeCN in 0.1 M TEAB). The conversion to a sodium salt form on a Dowex50WX8 (in a Na+ cycle) provided CDN 8a  as a white lyophilizate (water). 1H NMR : 4.15 ;4.42 ; 4.47 ; 4.60 ; 4.69 ; 6.28; 7.91 ; 8.12 . 13C NMR : 52.38 (C5-A); 64.69 ; 73.45 ; 77.15 (C2′-A);81.80 ; 92.67 (C1′-A); 107.78 (C4a-A);128.45 (C6-A); 150.57 (C7a-A); 154.11 (C2-A); 160.03 (C4-A). 31P NMR : −2.00.ESI MS m/z (rel. %): 452 (100) [M–2H]2–, 906 (28) [M–H]−, 928 (35)[M–2H + Na]−. HR MS (ESI): for C22H23O12N8I2P2 [M–H]−, calcd 906.90055; found, 906.90021.NTP 8a ,phenylboronic acid , and cesium carbonate were mixed with MeCN–H2O  in an argon-purged vial. In a separate vial,Pd(OAc)2  and TPPTS  were dissolved in MeCN–H2O , and the solution was sonicated under argon atmospherefor 30 s. Then, 353 μmol of this solution was transferred intothe mixture containing CDN 8a, and the reaction was stirredat 100 °C for 30 min. Then, the reaction mixture was cooled tort, diluted with water to approx. 3 mL, and filtered through a 5 μmnylon syringe filter. The filtrate was directly applied on HPLC forpurification (0–17.5% MeCN in 0.1 M TEAB). The conversion toa sodium salt form on a Dowex 50WX8 (in a Na+ cycle) providedCDN 8f  as a white lyophilizate (water). 1H NMR : 4.18 ;4.28 ; 4.45 ; 4.79 ; 6.37 ; 6.90 ; 7.04 ;7.61 ; 8.16 . 13C NMR : 64.78 ; 73.41 ; 77.32 (C2′-A); 81.62 ; 91.84 (C1′-A); 103.96 (C4a-A);120.61 (C5-A); 121.74 (C6-A); 129.62 (p-Ph); 130.32(o-Ph); 130.85 (m-Ph); 135.28 (i-Ph); 151.20 (C7a-A); 153.87 (C2-A); 159.85 (C4-A). 31P NMR : −0.84.ESI MS m/z (rel. %): 403 (100) [M–2H]2–, 807 (3) [M–H]−, 829 (4)[M–2H + Na]−. HR MS (ESI): for C34H33O12N8P2 [M–H]−, calcd 807.16986; found, 807.16888.CDN 9, ref  provided nucleoside 10  as a white amorphous solid. 1H NMR : 3.39 ; 3.42 ; 3.757,3.760 ; 4.19 ; 4.46 ; 4.72 ; 6.37 ; 6.81–6.88; 7.21; 7.25–7.32; 7.30–7.37; 7.42–7.48; 7.54–7.59; 7.64 ; 7.86 ; 8.08–8.12 ; 8.65 . 13C NMR : 52.84 (C-5); 55.75, 55.76 (CH3O-DMTr); 64.74 (CH2-5′); 72.42 (CH-3′); 76.35 (CH-2′); 85.43(CH-4′); 88.01 (C-DMTr); 89.41 (CH-1′); 114.23 (CH-m-C6H4-DMTr); 114.68 (C-4a); 127.99(CH-p-C6H5-DMTr); 128.96 (CH-m-C6H5-DMTr); 129.23 (CH-o-Bz); 129.41 (CH-o-C6H5-DMTr);129.90 (CH-m-Bz); 131.29, 131.40 (CH-o-C6H4-DMTr); 133.30 (CH-6); 133.78 (CH-p-Bz); 134.94 (C-i-Bz); 136.87, 137.15(C-i-C6H4-DMTr); 146.04 (C-i-C6H5-DMTr); 152.24 (CH-2); 152.41(C-4); 153.87 (C-7a); 160.17, 160.19 (C-p-C6H4-DMTr); 169.21 (C-i-Bz). ESI MS m/z (rel. %): 799 (23) [M + H]+, 821 (100) [M+ Na]+, 837 (9) [M + K]+. HR MS (ESI): for C39H36O7N4I [M + H]+, calcd 799.16232; found, 799.16154.7-Iodo-7-deazaadenosine  was co-evaporated with anhydrous pyridine (2× 5 mL) and dissolved in anhydrous THF (10 mL). Anhydrous pyridine(485 μL) and silver nitrate  were added.The reaction flask was covered with aluminum foil, and the mixturewas stirred at rt for 15 min. Then, TBDMSCl  wasadded, and the mixture was stirred overnight in the dark. The reactionmixture was then filtered through a pad of Celite. The filtrationpad was washed with DCM, and the filtrate was evaporated. Flash chromatographyon silica (5–50% EtOAc + 1% Et3N in cyclohexane)provided nucleoside 11  as a white foam. 1H NMR : −0.13, 0.01 ; 0.81 3C); 3.43 ;3.46 ; 3.779, 3.782; 4.29 ; 4.38 ;4.80 ; 6.39;6.85–6.90 ; 7.24 ; 7.28–7.33 ; 7.34–7.39 ; 7.45–7.49 ; 7.55–7.60 ; 7.66 ;7.91 ; 8.09–8.13 ;8.65 . 13C NMR :−4.94, −4.68 (CH3Si); 18.99 ((CH3)3C); 26.15 ((CH3)3C); 53.05 (C-5); 55.80 (CH3O-DMTr); 64.80(CH2-5′); 72.98 (CH-3′); 78.48 (CH-2′);86.02 (CH-4′); 88.27 (C-DMTr); 89.24 (CH-1′); 114.32(CH-m-C6H4-DMTr); 114.57 (C-4a);128.11 (CH-p-C6H5-DMTr); 129.05(CH-m-C6H5-DMTr); 129.23 (CH-o-Bz); 129.35 (CH-o-C6H5-DMTr); 129.93 (CH-m-Bz); 131.33, 131.43(CH-o-C6H4-DMTr); 133.16 (CH-6);133.82 (CH-p-Bz); 134.94 (C-i-Bz);136.74, 136.99 (C-i-C6H4-DMTr);145.99 (C-i-C6H5-DMTr); 152.29(CH-2); 152.55 (C-4); 153.92 (C-7a); 160.25, 160.26 (C-p-C6H4-DMTr); 169.22 (C-i-Bz).ESI MS m/z (rel. %): 913 (100) [M + H]+. HR MS (ESI): for C45H50O7N4ISi [M + H]+, calcd 913.24880; found, 913.24853.Nucleoside 11  was co-evaporated with anhydrous pyridine(2 × 5 mL) and dissolved in anhydrous pyridine (5 mL), and diphenylphosphite  was added. The reaction mixturewas stirred for 1 h, and then, water (8 mL) was added. The reactionmixture was stirred for 5 min, and it was then diluted with EtOAcand washed with brine. Aqueous phase was extracted twice with EtOAc.Combined organic phases were dried over MgSO4 and evaporated.The residue was dissolved in DCM (7 mL), and then, water (104 μL)and a solution of dichloroacetic acid in DCM were added. The solution was stirred at rt for 15 min, and then, triethylsilane was added. The reaction mixture was stirredfor another 30 min. The reaction was quenched with pyridine (823 μL),and solvents were removed in vacuo. Flash chromatography on C18 column(gradient 5–100% MeCN in 0.1 M TEAB) provided phosphonate 12  as a white foam. 1H NMR : −0.24, 0.01 ; 0.77 3C); 1.31; 3.20 ; 3.87 ; 4.33 ;4.73–4.79 ; 6.33 ; 6.96; 7.54–7.59; 7.65 ; 8.07 ; 8.10–8.15 ; 8.67 . 13C NMR : −5.22, −4.60 (CH3Si); 9.22 (CH3CH2N); 18.84 ((CH3)3C); 26.13 ((CH3)3C); 47.86 (CH3CH2N); 52.61 (C-5);62.66 (CH2-5′); 75.43 ; 77.09 ; 87.07 ; 89.77 (CH-1′); 114.84 (C-4a);129.24 (CH-o-Bz); 129.89 (CH-m-Bz);133.74 (CH-p-Bz); 133.93 (CH-6); 135.03 (C-i-Bz); 152.05 (CH-2); 152.68 (C-4); 153.65 (C-7a); 169.10(C-i-Bz). 31P{1H} NMR : 5.24. ESI MS m/z (rel. %):673 (100) [M–H]−. HR MS (ESI): for C24H31O7N4IPSi [M–H]−, calcd 673.07498; found, 673.07515.Nucleoside 47 Briefly, thermal denaturation was performed in a 20 μLmixture consisting of 0.1 mg/mL wt hSTING, 150 μM CDN, 100 mMTris–HCl [pH 7.4], 150 mM NaCl, and 1:500 (v/v) SYPRO Orange. Measurements were taken in a 96-wellformat on a LightCycler480 Instrument II . Thermalshift (ΔTm) for each CDN was determinedby subtracting the melting temperature (Tm) of the reference sample (ligand-free STING) from the melting temperatureof the STING–CDN complex.Measurements wereperformed with wt hSTING as described earlier.20 Briefly, the day before testing, the cells wereseeded into 96-well transparent plates in a DMEM High glucose mediumcontaining 10% (v/v) heat inactivated fetal bovine albumin (FBS).The medium was removed the next day, and the cells were incubatedfor 7 h  with serially diluted compounds.After incubation, the collected cell culture medium was mixed withBright-Glo Luciferase Assay System reagent  in white96-well plates . Luminescence readout was performedon a SPARK instrument , and the values were used tocalculate EC50 values using nonlinear regression and softwarePrism .The generationof 293T reporter cells expressing different full-length STING proteinallelic variants and the assay procedure itself was described previously.2 in buffer containing 50 mM HEPES [pH 7.0], 100 mM KCl, 3mM MgCl2, 0.1 mM DTT, 85 mM sucrose, 0.2% (w/w) BSA, 1mM ATP, 0.1 mM GTP, and 10 μg/mL digitonin A. Next, the bufferwas removed, and the cells were washed with a cell culture mediumtwice and then incubated for 5 h  withfresh medium. Finally, the levels of Lucia Luciferase in medium weredetermined using Bright Glo , and EC50s werecalculated as described above.Cells wereplated as described above for a standard 293T cell-based reporterassay. The following day, the medium was removed, and serially dilutedcompounds were incubated with cells for 30 min at 37 °C, 5% CO54 Briefly, theisolation of PBMCs was performed using Ficoll density gradient centrifugation in SepMate tubes . Next, isolated PBMCs wereseeded into a 96-well U-shape plate in RPMI 1640 media containing10% (v/v) heat inactivated fetal bovine serum . Serially diluted CDNs were added, and the plates were incubatedfor a predetermined 16 h . Finally,secreted IFNα, IFNγ, and TNFα were determined inthe collected medium using ProcartaPlex Assays using a MAGPIX System according to manufacturer’s instruction. Thecytotoxic effect of CDNs on PBMCs was determined using CellTiter-GloLuminescent Cell Viability Assay  accordingto manufacturer’s instruction.Buffy coats from healthy individuals wereobtained from the Institute of Hematology and Blood Transfusion . An informed written consent was obtainedfrom each individual enrolled. The study was approved by the InstitutionalReview Board of IHBT (ev. nb. 13/06/2012). The assay was performedas previously described.20 with minor changes. The recombinant plasmidwas transformed in Escherichia coli BL21 (DE3) cells . The bacteria were grown at37 °C until OD600nm of 0.6–0.8 units and cooleddown to 20 °C, and then, cGAS expression was induced with 0.4mM IPTG for 16 h in an orbital shaker. After cell collection, thepellet was resuspended in ice-cold lysis/wash buffer  glycerol, 3 mM β-mercaptoethanol).The cells were lysed by sonication, and cell debris was removed bycentrifugation. The supernatant was incubated with equilibrated Ni-NTAbeads . The beads were loaded into a gravityflow column and washed with lysis/wash buffer. Next, the resin waswashed with lysis/wash buffer supplemented with NaCl to 1 M concentration,then washed with lysis/wash buffer. The mcGAS was eluted with lysis/washbuffer supplemented with imidazole to a 300 mM concentration. Thecollected protein was further purified on a size-exclusion chromatographycolumn HiLoad 26/60 Superdex 200 pg in buffer containing 50 mM Tris–HCl[pH 7.4] and 150 mM NaCl. The eluate was concentrated to about 5 mg/mLand stored in 20% glycerol at −80 °C.The expressionand purification of mouse cGAS were performed as previously described33 Briefly, proteins were overexpressedin E. coli BL21 (DE3) cells . Purification steps consisted of cell disintegration, Ni-NTAchromatography, and SEC chromatography using HiLoad 26/60 Superdex200 pg. The DncV and DisA proteins were concentrated using an AmiconUltra-15 10K device (Merck Millipore Ltd), and enzymes were flash-frozenin liquid nitrogen.Expressionand purification of DncV and DisA proteins were performed as describedpreviously.47 Briefly, the expression,lysis, and purification using Ni-NTA resin were done following mcGASprotocol. Eluate from the Ni-NTA resin was supplied with Ulp1 proteaseand incubated for 2 h at 4 °C. Digested wt hSTING protein wasfurther purified by size exclusion with HiLoad 26/60 Superdex 200pg  equilibrated in buffer containing 50 mM Tris–HCl[pH 7.4] and 150 mM NaCl. Finally, the purified protein was concentratedto about 5 mg/mL and stored in 20% glycerol at −80 °C,for usage in DSF. For the crystallography experiments, wt hSTING wasfurther purified on HiTrap Capto S , equilibratedin 50 mM Tris–HCl [pH 8] buffer, and eluted with a gradientof 50–1000 mM NaCl in the same buffer. Fractions correspondingto wt hSTING were concentrated to about 20 mg/mL and stored at −80°C without glycerol supplement.The expression and purification of wt hSTINGwere performed asdescribed previously.52 wt hSTING-5f was co-crystalized at 18 °Cusing sitting drop vapor diffusion in a condition composed of 200mM ammonium nitrate, 20% (w/v) PEG 3350, with additive 10 mM EDTA.Crystals were cryoprotected in mother liquor supplemented with 20%(v/v) glycerol. A complete diffraction data set was collected fora single crystal to 2.69 Å. wt hSTING-5k was co-crystalizedat 18 °C using sitting drop vapor diffusion in a condition composedof 100 mM tri-sodium citrate [pH 5.0] and 20% (w/v) PEG 6000, withadditives of 10 mM EDTA and 100 mM tri-sodium citrate. Crystals werecryoprotected in mother liquor supplemented with 20% (v/v) ethyleneglycol. A complete diffraction data set was collected for a singlecrystal to 2.32 Å. wt hSTING-5l was co-crystalizedat 18 °C using sitting drop vapor diffusion in a condition composedof 100 mM citric acid, 20% (w/v) PEG 6000, with additive 10 mM EDTA.Crystals were cryoprotected in mother liquor supplemented with 20%(v/v) ethylene glycol. A complete diffraction data set was collectedfor a single crystal to 2.16 Å. wt hSTING-5m wasco-crystalized at 18 °C using sitting drop vapor diffusion ina condition composed of 100 mM PIPES [pH 7.0], 100 mM magnesium formatedihydrate, 100 mM rubidium chloride, 25% (w/v) PEG smear high, withadditives 10 mM EDTA and 100 mM tri-sodium citrate. Crystals werecryoprotected in mother liquor supplemented with 20% (v/v) ethyleneglycol. A complete diffraction data set was collected for a singlecrystal to 1.89 Å. Measurements for hSTING-5f andhSTING-5k were carried out at an in-house diffractometer at 100 K. Measurements for hSTING-5l andhSTING-5m were carried out at MX14.1 operated by theHelmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring at 100 K.56 Data were reduced and processed by XDS.57 The structures were solved by the molecularreplacement method with MOLREP(REFMAC from CCP4 suite,59PHENIX,60 and Coot.61 Parameters for structuresand their geometry are summarized in Table S1.Crystallizationswere done using our previously optimized protocol.th MOLREP using th


These compounds were even more selective than genistein alone, the reference drug (5-FU) and the equimolar mixture of genistein plus 5-FU. In addition, hybrids 4a and 4g induced time- and concentration-dependent antiproliferative activity and cell cycle arrest at the S-phase and G2/M. It was also observed that hybrid 4a induced apoptosis in SW620 cells probably triggered by the extrinsic pathway in response to the activation of p53, as evidenced by the increase in the levels of caspases 3/8 and the tumor suppressor protein (Tp53). Molecular docking studies suggest that the most active compound 4a would bind efficiently to proapoptotic human caspases 3/8 and human Tp53, which in turn could provide valuable information on the biochemical mechanism for the in vitro cytotoxic response of this compound in SW620 colon carcinoma cell lines. On the other hand, molecular dynamics (MD) studies provided strong evidence of the conformational stability of the complex between caspase-3 and hybrid 4a obtained throughout 100 ns all-atom MD simulation. Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) analyses of the complex with caspase-3 showed that the interaction between the ligand and the target protein is stable. Altogether, the results suggest that the active hybrids, mainly compound 4a, might act by modulating caspase-3 activity in a colorectal cancer model, making it a privileged scaffold that could be used in future investigations.A series of 5-FU-Genistein hybrids were synthesized and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated in human colon adenocarcinoma cells (SW480 and SW620) and non-malignant cell lines (HaCaT and CHO-K1). Hybrid  Colorectal cancer (CRC) is the second deadliest and widely diagnosed cancer in the world, accounting for 10% of all cancers . For theConsidering the potential of the individual molecules, molecular hybridization has emerged as a promising strategy in medicinal chemistry to combine more than two pharmacophores, aiming the search for new therapeutic alternatives to treat colorectal cancer. The pharmacophores of the hybrid molecules exhibit different biological functions, and it is suggested they may display multiple activities, although each unit does not necessarily act on the same biological target ,12.A1–A8, named Fuplatin, was evaluated against human cancer cells HeLa, MCF-7, CaCo-2, LoVo, HCT-116, A549 and over normal cells MRC-5. In general, these hybrids, were more active than free drugs  or their physical mixtures. The cytotoxicity of the Fuplatin hybrids was selective against the cancer cells tested, as evidenced by lower IC50 values on these cells in comparison to normal human lung cells. Hybrid A5 exhibited the highest activity over human colon carcinoma HCT-116 cells with an IC50 value of 0.13 µM, which was 64-fold lower than oxaliplatin and 41-fold lower than the physical mixture + peaks corresponding to their molecular weights. The assignments of all the signals to individual H or C-atoms was performed based on typical δ-values and J-constants. The 1H-NMR spectra of hybrids dissolved in DMSO showed around 8.12 ppm, a signal corresponding to 5-FU. The chromone (-O-CH=C-) signal was observed around 8.39 ppm. 13C-NMR spectra of the hybrids showed around 180.84 ppm a signal corresponding to the carbonyl groups (C=O) of the chromone. The triazolyl ring exhibited signals around 142 and 124 ppm. Finally, the signal corresponding to F-C-C=O of 5-FU was observed around 140.8 and 139.4 ppm.The structures of all compounds were established by a combined study of HRMS-ESI (50). The results are summarized in 4a displayed cytotoxicity against both malignant cells evaluated with IC50 values of 62.73 ± 7.26 µM and 50.58 ± 1.33 µM for SW480 and SW620 cells, respectively. These values were slightly lower than in non-malignant cells which favored the selectivity indices. In a similar way, compound 4g induced cytotoxicity in SW620 cells with IC50 value of 36.84 ± 0.71 µM. This compound even displayed selective activity as evidenced by higher IC50 values in the non-malignant HaCaT and CHO-K1 cells, with greater selectivity indices (>2.71). This selectivity was even better compared to the parental compounds alone and the equimolar mixture, which could be explained by those physicochemical properties of the hybrid that are defined according to its polarity, while in the mixture, the physicochemical properties of the entities must be considered separately. On the other hand, the pharmacokinetic profile of the hybrid is more predictable and the absorption capacity of one part of the structure can be used to facilitate cell permeation of the whole entity ..4a was tSimilar to the docking procedure for caspase-3, our docking protocol was validated through the self-docking simulation of the co-crystallized inhibitor MMX-9 inside the catalytic pocket of caspase-8. As shown in 4a , and both the 5-OH and 7-OR substituents on the chromone moiety formed with Asp455 and Tyr365 two hydrogen bonds at a distance of 4.63 and 5.89 Å, respectively. In addition, one π−cation contact was observed between the positively charged side chain of Arg413 and the 5-FU ring, and two intramolecular π-π T-shaped contacts were observed between the fused aromatic ring in chromone and the important residue Tyr412. It was also noticed that the fluorine atom attached to the 5-FU ring interacted with the Ser316 and Ala359 residues located in the caspase-8 active site. Finally, multiple hydrophobic interactions were observed in the 4a-caspase-8 complex, which would play important roles in stabilizing the binding complex. The observed interactions between promising 4a and those key amino acid residues in caspase-8 could provide a reasonable explanation for the experimental observations.The validated docking procedure was then applied to the compound le sites C. First,4a was fitted inside the human p53 core DNA-binding domain. To accomplish this goal, protein–ligand docking simulations of this compound were conducted in the crystal structure of the human p53 in complex with DNA (PDB ID: 1tsr). Likewise, two potent quinazoline-based p53-DNA-binding inhibitors, SCH529074 and NSC194598 NSC194598 were also evaluated. Although the p53/DNA-binding interface is usually composed of several amino acid residues, only ten “hot spot” sites located in the DNA-binding domain of p53  play a critical structural and functional role for the biological function of human p53 + calcd. for C24H19FN5O7 [M + H]+ 508.1263.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4b): Yield 82%; White solid, m.p. 175–178; 1H NMR  δ 8.40 , 8.20 , 8.14 , 7.39 , 6.83 , 6.65 , 6.39 , 4.91 , 4.79 , 4.54 . 13C NMR  δ 180.87 (C=O chromone), 164.65 (Ar-O), 162.22 (Ar-O), 157.99 and 157.94 (F-C-C=O), 157.90 (Ar-O), 157.79 (Ar-O), 154.85 , 149.84 (N-C=O), 142.87 (triazolyl), 140.89 and 139.37 (F-C), 130.62 (3C-Ar), 130.43 and 130.21 (CH-C-F), 124.30 (triazolyl), 122.97, 121.51, 115.55 (2C-Ar), 105.96, 98.80, 93.31, 66.06, 47.05, 43.21, 29.54. HRMS-ESI (m/z): 522.1447 [M+H]+ calcd. for C25H21FN5O7 [M + H]+ 522.1419.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4c): Yield 84%; White solid, m.p. 200–203; 1H NMR  δ 8.40 , 8.18 , 8.14 , 7.40 , 6.83 , 6.64 , 6.40 , 4.91 , 4.43 , 4.11 , 2.01–1.94 , 1.75–1.68 . 13C NMR  δ 180.85 (C=O chromone), 164.91 (Ar-O), 162.22 (Ar-O), 157.99 (F-C-C=O), 157.94 (Ar-O), 157.82 (Ar-O), 154.82 , 149.84 (N-C=O), 142.60 (triazolyl), 140.89 and 139.37 (F-C), 130.61 (2C-Ar), 130.50 and 130.28 (CH-C-F), 124.02 (triazolyl), 122.96, 121.53, 115.55 (2C-Ar), 105.86, 98.80, 93.30, 68.26, 49.52, 43.24, 26.83, 25.88. HRMS-ESI (m/z): 536.1601 [M+H]+ calcd. for C26H23FN5O7 [M + H]+ 536.1576.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4d): Yield 70%; White solid, m.p. 211–214; 1H NMR  δ 9.60 , 8.39 , 8.18 , 8.12 , 7.40 , 6.83 , 6.63 , 6.38 , 4.90 , 4.37 , 4.08 , 1.92–1.85 , 1.79–1.73 , 1.43–1.36 . 13C NMR  δ 180.84 (C=O chromone), 165.02 (Ar-O), 162.21 (Ar-O), 157.99 (F-C-C=O), 157.94 (Ar-O), 157.82 (Ar-O), 154.81 , 149.84 (N-C=O), 142.58 (triazolyl), 140.88 and 139.36 (F-C), 130.62 (3C-Ar), 130.49 and 130.27 (CH-C-F), 123.94 (triazolyl), 122.95, 121.53, 115.54 (2C-Ar), 105.82, 98.79, 93.28, 68.70, 49.77, 43.23, 29.77, 28.18, 22.88. HRMS-ESI (m/z): 550.1756 [M+H]+ calcd. for C27H25FN5O7 [M+H]+ 550.1733.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)pentyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4e): Yield 82%; White solid, m.p. 220–223; 1H NMR  δ 8.39 , 8.17 , 8.12 , 7.40 , 6.83 , 6.63 , 6.39 , 4.90 , 4.35 , 4.07 , 1.88–1.80 , 1.76–1.67 , 1.48–1.39 , 1.34–1.27 . 13C NMR  δ 180.84 (C=O chromone), 165.07 (Ar-O), 162.21 (Ar-O), 157.99 (F-C-C=O), 157.95 (Ar-O), 157.82 (Ar-O), 154.80 , 149.83 (N-C=O), 142.58 (triazolyl), 140.88 and 139.36 (F-C), 130.61 (3C-Ar), 130.49 and 130.27 (CH-C-F), 123.90 (triazolyl), 122.95, 121.53, 115.54 (2C-Ar), 105.80, 98.79, 93.25, 68.83, 49.81, 43.24, 30.00, 28.63, 26.01, 25.26. HRMS-ESI (m/z): 564.1907 [M+H]+ calcd. for C28H27FN5O7 [M+H]+ 564.1889.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4f): Yield 60%; White solid, m.p. 212–215; 1H NMR  δ 8.39 , 8.17 , 8.11 , 7.39 , 6.83 , 6.63 , 6.39 , 4.90 , 4.33 , 4.07 , 1.84–1.77 , 1.75–1.67 , 1.42–1.35 , 1.34–1.27 , 1.27–1.20 . 13C NMR  δ 180.84 (C=O chromone), 165.11 (Ar-O), 162.20 (Ar-O), 157.97 (Ar-O), 157.93 (F-C-C=O), 157.82 (Ar-O), 154.80 , 149.82 (N-C=O), 142.75 (triazolyl), 140.88 and 139.35 (F-C), 130.61 (3C-Ar), 130.49, 130.27, 123.99 (triazolyl), 122.94, 121.53, 115.54 (2C-Ar), 105.79, 98.79, 93.25, 68.93, 49.87, 43.24, 30.06, 28.97, 28.79, 28.74, 26.24, 25.74. HRMS-ESI (m/z): 592.2216 [M+H]+ calcd. for C30H31FN5O7 [M+H]+ 592.2202.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)octyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4g): Yield 84%; White solid, m.p. 191–194; 1H NMR  δ 9.60 , 8.39 , 8.17 , 8.11 , 7.39 , 6.83 , 6.63 , 6.39 , 4.89 , 4.32 , 4.07 , 1.85–1.76 , 1.75–1.68 , 1.45–1.18 . 13C NMR  δ 180.83 (C=O chromone), 165.11(Ar-O), 162.20 (Ar-O), 157.95 (F-C-C=O and Ar-O), 157.81 (Ar-O), 154.77 , 149.82 (N-C=O), 143.25 (triazolyl), 140.88 and 139.35 (F-C), 130.60 (3C-Ar), 130.47, 130.25, 124.18 (triazolyl), 122.94, 121.53, 115.54 (2C-Ar), 105.78, 98.78, 93.23, 68.93, 49.88, 43.27, 30.05, 29.22, 29.05, 28.82, 28.73, 26.26, 25.79. HRMS-ESI (m/z): 606.2370 [M+H]+ calcd. for C31H33FN5O7 [M+H]+ 606.2358.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)nonyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione (4h): Yield 80%; White solid, m.p. 190–193; 1H NMR  δ 8.39 , 8.16 , 8.10 , 7.39 , 6.83 , 6.63 , 6.38 , 4.89 , 4.31 , 4.08 , 1.81–1.75 , 1.75–1.69 , 1.43–1.36 , 1.34–1.18 . 13C NMR  δ 180.83 (C=O chromone), 165.11 (Ar-O), 162.21 (Ar-O), 157.95 (F-C-C=O and Ar-O), 157.80 (Ar-O), 154.78 , 149.82 (N-C=O), 142.59 (triazolyl), 140.88 and 139.35 (F-C), 130.59 (3C-Ar), 130.46, 130.24, 123.92 (triazolyl), 122.94, 121.51, 115.54 (2C-Ar), 105.78, 98.78, 93.23, 68.94, 49.86, 43.20, 34.71, 30.08, 29.41, 29.37, 29.30, 29.15, 28.84, 28.81, 26.28, 25.83. HRMS-ESI (m/z): 648.2833 [M+H]+ calcd. for C34H39FN5O7 [M+H]+ 648.2828.5-fluoro-1-((1-(3-((5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4-dione  and non-malignant cell lines  were obtained from The European Collection of Authenticated Cell Cultures . For biological evaluations, cells were cultured in Dulbecco’s Modified Eagle Medium, supplemented with 10% heat-inactivated (56 °C) horse serum, 1% penicillin/streptomycin and 1% non-essential amino acids . During the treatments, the serum was reduced to 3%, and the medium was supplemented with insulin (10 mg/mL) transferrin (5 mg/mL) and selenium (5 ng/mL)  ,52. To a2. After the adaptation time (24 h), cells were treated with increasing concentrations of the hybrid molecules or DMSO (1%). At the end of each treatment, cells were fixed with trichloroacetic acid  for one hour at 4 °C. Subsequently, cell proteins were stained with SRB  and washed with acetic acid (1%). After drying at room temperature, Tris-base (10 mM) was added to solubilize protein bound stain. Absorbance was read in a microplate reader (Mindray MR-96A) at 492 nm [The hybrid compounds evaluated together with the parental compounds, the reference drug (5-FU) and the equimolar mixture (this was obtained by mixing 6.03 mg of genistein and 2.90 mg of 5-FU in 1 mL of solvent) were evaluated through the Sulforhodamine B (SRB) assay to demonstrate the probable cytotoxic and antiproliferative potential. Briefly, cells were seeded at 20,000 cells/well (for cytotoxicity assay) and 2500 cells/well (for the antiproliferative test) in 96-well tissue culture plates, incubating at 37 °C in a humidified atmosphere with 5% COt 492 nm . All exp50 value of the hybrids 4a, 4g and DMSO (1%) as a vehicle control, cells were collected by scraping and centrifugation, resuspending the cell pellet in a versene buffer. Thereafter, cells were fixed with 1.8 mL cold ethanol (70%) at 4 °C overnight. Alcohol was removed by subsequent washing twice with versene buffer. The final pellet was resuspended in 300 µL of PBS with 0.25 mg/mL RNAse  and 0.1 mg/mL PI and incubated for 30 min in the dark at room temperature. FACS Canto II flow cytometer  was used to read the PI fluorescence of 10,000 events. The analysis of data was performed with the software FlowJo 7.6.2  [Cell cycle analysis was evaluated by flow cytometry using propidium iodide (PI). This experiment was carried out as previously described by Nicoletti et al. (1991) . Then, 4OR, USA) .5 cells/well were seeded in 6-well tissue culture plates and allowed to grow for 24 h. Then, these were treated with hybrids 4a and 4g . Cells were harvested by scrapping 48 h after treatment, stained with DiOC6 and propidium iodide (PI) at room temperature for 30 min in darkness. Flow cytometric analysis of 10,000 events was carried out to quantify cells with mitochondrial depolarization, excitation at 488 nm and detection of the emission with the green (530/15 nm) and the red (610/20 nm) filters [The fluorescent dyes DiOC6  and propidium iodide (PI) were used to evaluate possible changes in mitochondrial membrane potential induced by the hybrid molecules. Then, 2.5 × 10 filters .4a and 4g to induce cell death. SW480 and SW620 cells were treated for 48 h and further collected by scraping. After centrifugation, the cell pellet was resuspended in Annexin V/FITC–PI and incubated in darkness for 20 min. Data were obtained through flow cytometry and analyzed with FlowJo 7.6.2 . Cells in early apoptosis showed single staining for Annexin V/FITC. Cells with positive staining for PI were considered dead cells, late apoptotic cells, necroptotic cells or secondary necrotic cells. All assays were repeated twice [Using double staining with Annexin V/FITC and PI (Roche Diagnostics), we evaluated the ability of the hybrids ed twice .4a  and 4g  for 48 h. Afterwards, cells were scraped and lysed with Cell Lysis Buffer . The supernatant was used for the determination of apoptotic biomarkers after the treatment with the hybrid molecules. Enzyme-linked immunosorbent assay (ELISA) kits were provided by Elabscience Biotechnology Co.,  (caspase-8) and Cell-Signaling Technology  (caspase-3 and p53), following the manufacturer’s instructions [Tumor cell lines (SW480 and SW620) were cultured as previously described and then treated with hybrids ructions .p values lower than 0.05 were considered significant. Data were analyzed with GraphPad Prism version 8.0.1 for .The experimental design included at least two repetitions for each experiment. Data were reported as mean ± SE (standard error). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test. 4a was drawn in the ChemDraw 17.0 software  and then saved as MDL MoL files. Chem3D 17.0 software  was used to generate the 3D structure of the ligand and optimization was performed using the MM2 Force-Field in Chem3D Ultra 8.0 Software CS, ChemOffice Chem3D Ultra 8.0, CambridgeSoft, AutodockTools (ADT) was used to parameterize the ligand: non-polar hydrogens were merged, rotatable bonds assigned, full hydrogens added, and Kollman united partial atom charges were added to the individual protein atoms. The 3D protein structure of the Caspase-3 (PDB ID: 5I9B), Caspase-8 (PDB code: 3kjn) and p53 (PDB ID: 1tsr) was downloaded from the Protein Data Bank website (accessed on 1 June 2022). Co-crystallized ligand, ions and water molecules were removed from the protein structure by using the DS Visualizer 2.5 program. For the docking analysis, grid map dimensions (32 × 32 × 32 Å) were set surrounding the active site at x, y, and z coordinates of x = 1.5, y = −8.1, z = −13.4 (for caspase-3), x = −10.032, y = 35.318, z = 41.367 (for caspase-8) and x = 56.907, y = 28.404, z = 85.947 (for p53), at exhaustiveness 20 for each protein-compound pair and 1Å for grid spacing. AutoDock Vina software package [The 2D chemical structure of  package  was usedThe protocol employed and procedural details for MD simulations (100 ns) have been described in a recent publication .The protocols employed for the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations have been described in a recent publication .4a and 4g displayed cytotoxicity against SW480 and SW620 cells with IC50 values between 36.84 ± 0.71 and 62.73 ± 7.26 μM. These compounds were even more selective than genistein alone, the reference drug (5-FU) and the equimolar mixture. Furthermore, we also demonstrated that hybrids 4a and 4g produced time- and concentration-dependent antiproliferative activity and cell cycle arrest at the S-phase and G2/M, inducing morphological changes in the cell. Our results allowed us to conclude that the probable mechanism of action of hybrid 4a in SW620 cells could probably be triggered by extrinsic apoptosis in response to the activation of Tp53. Finally, the molecular docking analysis showed that promising 4a bound effectively to caspases-3/7 and Tp53 protein obtaining binding affinities ranging from −7.9 to −9.7 kcal/mol compared to those current inhibitors. MD simulations not only validated the stability and rationality of docking solutions, but also showed that the complex between the hybrid 4a and caspase-3 achieved great stability in 100 ns. In addition, the MM-PBSA-based analysis showed that compound 4a binds to the caspase-3 enzyme with high affinity . Therefore, combined experimental and computational findings indicated that the modulation of these proteins, particularly caspase-3, may be a possible molecular mechanism to understand the cytotoxic response of 4a in SW620 colon carcinoma cell lines. However, further experimental and computational studies are needed to clearly delineate the cytotoxic mechanism associated with the most promising hybrid (4a).We showed for the first time the synthesis and biological evaluation of 5-FU/Genistein hybrids. We demonstrated that the synthesized hybrids exhibited selective chemopreventive potential towards the human colon adenocarcinoma cells SW480 and SW620. The most active hybrids