{"paper_id":"31a0bea4-3f2f-4cb2-b6b2-d7567be733d4","body_text":"License and Terms: This document is copyright 2024 the Author(s); licensee Beilstein-Institut.\nThis is an open access work under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0). Please note that the reuse,\nredistribution and reproduction in particular requires that the author(s) and source are credited and that individual graphics may be subject to special legal provisions.\nThe license is subject to the Beilstein Archives terms and conditions: https://www.beilstein-archives.org/xiv/terms.\nThe definitive version of this work can be found at https://doi.org/10.3762/bxiv.2024.9.v1\nThis open access document is posted as a preprint in the Beilstein Archives at https://doi.org/10.3762/bxiv.2024.9.v1 and is\nconsidered to be an early communication for feedback before peer review. Before citing this document, please check if a final,\npeer-reviewed version has been published.\nThis document is not formatted, has not undergone copyediting or typesetting, and may contain errors, unsubstantiated scientific\nclaims or preliminary data.\nPreprint Title Synthesis of 1,4-azaphosphinine pyrimidine nucleosides and their\nevaluation as inhibitors of human cytidine deaminase and\nAPOBEC3A\nAuthors Maksim V. Kvach, Stefan Harjes, Harikrishnan M. Kurup, Geoffrey B.\nJameson, Elena Harjes and Vyacheslav V. Filichev\nPublication Date 08 Feb. 2024\nArticle Type Full Research Paper\nSupporting Information File 1 Experimental_part_submission.pdf;  1.2 MB\nORCID® iDs Stefan Harjes - https://orcid.org/0000-0001-6571-9057; Harikrishnan\nM. Kurup - https://orcid.org/0000-0001-5517-9907; Geoffrey B.\nJameson - https://orcid.org/0000-0003-4839-0784; Elena Harjes -\nhttps://orcid.org/0000-0002-3643-9432; Vyacheslav V. Filichev -\nhttps://orcid.org/0000-0002-7383-3025\n\n \n1 \nSynthesis of 1,4-azaphosphinine pyrimidine nucleosides and their evaluation as inhibitors of \nhuman cytidine deaminase and APOBEC3A  \nMaksim V. Kvach,[a] Stefan Harjes,[a] Harikrishnan M. Kurup,[a, b] Geoffrey B. Jameson,[a, b] Elena \nHarjes,[a, b]* Vyacheslav V. Filichev[a, b]* \nSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New \nZealand; \nE-mail: e.harjes@massey.ac.nz, v.filichev@massey.ac.nz \n[a] Dr. H. M. Kurup, Prof. Dr. G. B. Jameson, Dr. E. Harjes, Prof. Dr. V. V. Filichev \n[b] Maurice Wilkins Centre for Molecular Biodiscovery, Auckland 1142, New Zealand. \nTwitter/X accounts: \n@FilichevLab \nMassey University: @MasseyUni \nMaurice Wilkins Centre: @MWC_CoRE \nWorldwide Cancer Research: @WorldwideCancer \nKeywords: APOBEC3, cytidine deaminase, zebularine, enzyme activity, inhibitor, transition state \nanalogue, nucleoside, nucleotide \nAbstract: \nNucleoside and polynucleotide cytidine deaminases (CDA and APOBEC3) share similar mechanism of \ncytosine to uracil conversion. In 1984 phosphapyrimidine riboside was characterised as the most \npotent inhibitor of human CDA but its quick degradation in water limited its applicability as a \npotential therapeutic. To improve stability in water, we synthesised a derivative dPC of \nphosphapyrimidine nucleoside having CH2 group instead of the N3 atom in the nucleobase. A \ncharge-neutral phosphinamide dPC-NH2 and a negatively-charged phosphinic acid derivative dPC-OH \nhad excellent stability in water at pH 7.4 but only the charge-neutral dPC-NH2 inhibited human CDA \nsimilar to previously described 2’-deoxyzebularine (Ki = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). \nHowever, at basic conditions the charge-neutral phosphinamide dPC-NH2 was unstable which \nprevented its incorporation into DNA using conventional DNA chemistry. In contrast, the negatively \ncharged phosphinic acid derivative dPC-OH was incorporated into DNA instead of the target dC using \nan automated DNA synthesiser but no inhibition of APOBEC3A was observed for modified DNAs. \nAlthough this shows that negative charge is poorly accommodated in the active site of CDA and \n\n \n2 \nAPOBEC3, the synthetic route reported here provides opportunities for the synthesis of other \nderivatives of phosphapyrimidine riboside for potential development of more potent CDA and \nAPOBEC3 inhibitors.  \n  \n\n \n3 \n1. Introduction \nSpontaneous hydrolytic deamination of cytosine to uracil (Fig. 1A) is very slow under ambient \nconditions[1] but it is greatly accelerated by enzymes. These enzymes share a similar mechanism of \ncytosine deamination and a similar tertiary structure. Despite this similarity, individual enzymes are \nselective for their corresponding cytosine-containing substrates with little or no cross-reactivity. \nCytosine deaminase which is present in bacteria and fungi but not in mammalian cells acts only on \ncytosine. Cytidine deaminase (CDA) as a key enzyme in the pyrimidine salvage pathway in mammals \ndeaminates both cytidine and 2’-deoxycytidine. Members of the APOBEC family (apolipoprotein B \nmRNA editing enzyme, catalytic polypeptide-like), such as activation-induced deaminase (AID) and \nAPOBEC3, act preferentially on single-stranded DNA containing one or multiple cytosines. Although \nsome activity was detected on RNA, none was observed on cytidine or cytosine alone.  \nEach cytosine deaminase has an important biological function in an organism, but their activities can \nalso be detrimental. CDA is highly active in liver and spleen which results in deamination and \nconsequent deactivation of several chemotherapeutic agents, including the anti-cancer agents \ncytarabine, gemcitabine and decitabine.[2-5] Full inhibition of CDA leads to accumulation of toxic \npyrimidine catabolism intermediates.[6, 7] However, local and temporary inhibition of CDA in the \nliver provides a therapeutic benefit by allowing cytosine-like containing drugs to by-pass liver with \nan intact nucleobase. Recently, a combination of a CDA inhibitor cedazauridine, (4R)-2′-deoxy-2′,2′-\ndifluoro-3,4,5,6-tetrahydrouridine (Fig. 1B), with an anticancer drug decitabine was approved as an \noral pill (C-DEC or ASTX727) for the treatment of patients with intermediate/high-risk \nmyelodysplastic syndromes MDS and chronic myelomonocytic leukaemia (CMML).[8]  \nIn normal human cells the enzyme family  APOBEC3 (A3) [9-12] disables pathogens by scrambling \nsingle-stranded DNA (ssDNA) by cytidine to uridine mutation (Fig. 1A).[9, 13-15] However, several \nenzymes, particularly A3A, A3B, A3H and A3G, deaminate cytosine in human nuclear and \nmitochondrial genomes.[16] This A3-induced mutational activity is used by viruses and cancer cells to \nincrease the rates of mutagenesis, which allows them to escape adaptive immune responses, and \nbecome drug resistant[17-21] leading to poor clinical outcomes. A range of genetic, biochemical and \nstructural studies support a model in which this A3 -mediated mutagenesis promotes tumour \nevolution and strongly influences disease trajectories, including the development of drug resistance  \nand metastasis .[19-24] Of the seven A3 enzymes, three (A3A, A3B and A3H) are at least partially \nlocalised in the nucleus of cells and are genotoxic. [25] A3A and A3H are single -domain enzymes, \nwhereas A3B is a double -domain enzyme, in which only the C -terminal domain (CTD) has catalytic \nactivity and the N-terminal domain (NTD) is responsible for binding of DNA and for nuclear localisation.  \n\n \n4 \nInitially, A3B had been identified as the primary source of genetic mutations in breast[19-24, 26, 27] \nand other cancers.[28, 29] The breast cancers with high expression of A3B show a two-fold increase \nin overall mutational load. Elevated A3B expression correlates with reduced tamoxifen sensitivity of \ntumours in those patients [20] and poor survival rates for estrogen receptor-positive (ER+) breast \ncancer patients.[22, 30] In line with these observations, A3B overexpression accelerates the \ndevelopment of tamoxifen resistance in murine xenograft with ER+ breast cancer. In contrast, \nknockdown of A3B results in prolonged tamoxifen responses and leads to the survival of mice during \nthe experimental time (1 year).[20] More recent research points also to a prominent role of A3A in \nbreast[31] and other cancers.[31-34] Overexpression of A3A and A3B leads to tumourigenesis in \ntransgenic mouse models.[25, 29, 35, 36] High levels of A3A and A3B mRNA are linked also to the \nmore aggressive breast cancers including triple negative cancers.[37] Since A3B is not essential for \nhumans[38] and A3A does not take part in primary metabolism, inhibition of A3A and A3B offers a \npotent strategy to suppress cancer evolution and prolong efficacy of existing anticancer \ntherapies.[20, 39, 40] \nDespite of low sequence identity, cytidine deaminase (CDA) and A3 share a similar overall structural \ntopology and close structural homology for the Zn2+-containing active site. Since cytosine \ndeamination involves a nucleophilic attack at the C4 position by a Zn2+-activated water molecule,[41-\n43] it was proposed to employ transition state analogues and mimetics of the tetrahedral \nintermediate formed as inhibitors of these enzymes.[44-48] More than 30 compounds have been \nsynthesised in the past and evaluated as inhibitors targeting the active site of CDA. \nTetrahydrouridine (THU),[46, 49] diazepinone riboside,[43-45, 50] zebularine[48, 51, 52] and 5-\nfluorozebularine[48, 53] were among the most potent compounds (Fig. 1B). THU quickly converts \ninto inactive β-ribopyranosyl form in solution, but substituting hydrogen atoms with fluorine atoms \nin the 2’-position leads to cedazuridine, which is stable[54] and used now in clinics as a CDA inhibitor \nin the liver extending the life-time of co-administered decitabine (5-aza-2'-deoxycytidine).[8]  \n\n \n5 \n \nFigure 1. A) Cytosine deamination, R = H (cytosine) or 1-β-deoxyribofuranosyl (dC) and 1-β-\nribofuranosyl (C) as individual nucleosides or as a part of a polynucleotide chain; B) Previously \ndescribed CDA inhibitors and a structure of proposed inhibitors dPC. \nWe have recently developed the first rationally designed competitive inhibitors of A3 by \nincorporating 2-deoxy derivatives of zebularine (2'-deoxyzebularine, dZ and 5-fluoro-2'-\ndeoxyzebularine, FdZ, Fig. 1B)[55] and diazepinone riboside[56] into DNA fragments. We \ndemonstrated that dZ does not inhibit A3 enzymes as the free nucleoside but becomes a low µM \ninhibitor if it is used in single-stranded DNA (ssDNA) instead of the target dC in the recognition \nmotifs of A3A/A3B and A3G.[55] This observation supports a mechanism in which ssDNA delivers dZ \nto the active site for inhibition. By changing the nucleotides around dZ, we obtained the first A3B-\nselective inhibitor.[57] By inserting the fluoro-substituted FdZ into ssDNA we observed three times \nbetter inhibition of A3Bctd and wild-type A3A in comparison with the dZ-containing DNA,[58] which \n\n \n6 \ncorrelates with the trend reported for CDA inhibitors.[48, 53] We also demonstrated that dZ- and \nFdZ-containing DNAs also inhibit full-length wild type A3G with similar efficiency to that for the \nsingle catalytically active C-terminal domain.[58, 59] Recently, analysis of crystal structures revealed \nthat both dZ and FdZ form tetrahedral intermediates after hydrolysis of N3-C4 double bond in the \nactive sites of A3Gctd and A3A.[60, 61] The intermediates formed had the same R-stereochemistry \nat C4 atom of the nucleobase as previously observed for CDA thus confirming the general \nmechanism of cytosine deamination for A3 and CDA.[52, 60-65] \nThe fact that dZ, FdZ and diazepinone deoxynucleside used in the same DNA sequence had different \ninhibitory effect on individual A3 under identical conditions means that structure of the cytidine \nanalogue determines the inhibitory potential of the inhibitor-containing oligo.[56, 58] This also \nsupports our strategy of using more potent CDA inhibitors in DNA sequences for development of \nmore powerful A3 inhibitors. The most potent inhibitor of CDA reported so far is phosphapyrimidine \nriboside (P, Ki = 0.9 nM, Fig. 1B).[46] However, it is unstable in solution and thus cannot be used as \nCDA inhibitor and cannot be incorporated into ssDNA and evaluated as A3 inhibitor. Here, we report \nthe  synthesis of novel inhibitors of CDA and A3 based on the 1,4-azaphosphinine scaffold, \ncompounds dPC (Fig. 1B), in which N3 atom present in the nucleobase of P is replaced with CH2. We \nassumed that these changes should not significantly affect their inhibitory potential but should \nincrease stability of the target nucleosides in water and allow their chemical incorporation into \nssDNA.  \n \n2. Results \n Synthesis of nucleosides \nIt is more straightforward to start the synthesis of a modified nucleoside from assembly of a \nnucleobase that afterwards can be coupled to the sugar using the Hilbert-Johnson reaction or a silyl \nvariation of it as described in the literature.[66] Scheme 1 shows the synthesis of the target \nnucleobases. \nN-Boc-vinylamine 3 was synthesised from commercially available N-vinylformamide 1 as a stable \nsource of vinylamine by treatment of 1 with Boc2O in THF in the presence of a catalytic amount of \nDMAP followed by cleavage of the formyl moiety in basic conditions. Compound 3 was obtained in \nnearly 20 g scale in 89% yield after its purification by sublimation in vacuo. In the presence of \ncatalytic amount of AIBN compound 3 reacted with bis-(trimethylsilyloxy)phosphine (4) that was \nprepared in situ.[67] Treatment of the reaction mixture with MeOH/Et3N followed by silica gel \n\n \n7 \ncolumn chromatography led to triethylammonium salt of 2-N-boc-aminoethylphosphinic acid 5 in 50 \n% yield. Alkylation of acid 5 with methyl chloroacetate in the presence of TMSCl and Et3N took five \ndays at room temperature and compound 6 as triethylammonium salt was obtained in 84% yield \nafter silica gel purification. Removal of Boc-protection from 6 in the presence of trifluoroacetic acid \nin DCM at room temperature overnight followed by cyclisation in boiling pyridine/triethylamine led \nto 4-hydroxy-1,4-azaphosphinan-2,4-dione (7) in 84% yield. The free phosphinic acid 7 was further \nprotected with benzyl alcohol by a procedure adopted from reference[68] using TBTU (O-\n(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate) and Et3N in refluxing \ndichloroethane. Compound 8 was obtained in 65% yield after silica gel column chromatography.  \n \nScheme 1. i) Boc2O, DMAP, THF, r.t., overnight; ii) aq. 5M NaOH, r.t., 3h; iii) 3, azobisisobutyronitrile \n(AIBN), ACN, r.t. then 80-90 °C overnight under Ar followed by Et3N/MeOH work-up, r.t.; iv) methyl \nchloroacetate, Et3N, TMSCl, CH2Cl2, r.t., 5d; v) trifluoroacetic acid, CH2Cl2, r.t., overnight then 5h reflux in \npyridine/Et3N; vi) BnOH, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), \nEt3N, DCE, reflux, 3h; vii) BnOH, abs. Et2O, pyridine, -78 →0 °C; viii) chloroacetamide, \nhexamethyldisilazane (HMDS), ACN, 70 °C, 48h under argon; ix) trifluoroacetic acid, CH2Cl2, r.t., overnight. \nTo synthesise a nucleobase for nucleoside dPC we first obtained dichlorophosphane 9 from \ncommercially available PCl3 and ethylvinyl ether using previously published procedure.[69] \nCompound 9 reacted with 1 equivalent of benzyl alcohol in absolute Et2O and pyridine at -78 °C \n\n \n8 \nfollowed by quenching of the reaction mixture with H2O. This procedure provided phosphinate 10 in \nquantitative yield with more than 90% purity according to 31P NMR; compound 10 was used in the \nnext step without further purification. A linear amide 11 was obtained in 47% yield by reacting \nphosphinate 10 with chloroacetamide in the presence of large excess of HMDS in acetonitrile at 70 \n°C for two days. A cyclisation of the linear amide 11 was performed in DCM using 10-fold excess of \ntrifluoroacetic acid at room temperature providing 1,4-azaphosphinine 12 in 68% yield. \nVarious conditions used for coupling of nucleobase 8, such as sylilated (HMDS or BSA) derivative or \nas a salt obtained after treatment with the base (NaH, t-BuOK), with Hoffer’s chlorosugar 13 in the \npresence or absence of Lewis acids (TMSOTf, SnCl4) did not result in formation of a reasonable \namount of nucleoside 14. Nucleobase 12 could not be converted to the corresponding sylilated \nderivative by using HMDS, TMSCl or combination of both. Difficulties in the Hilbert-Johnson reaction \nand the low yield observed for nucleoside 14 prompted us to use an alternative option for the \nsynthesis of the target nucleosides based on the assembly of a nucleobase on the 2-\ndeoxyribofuranos-1-yl scaffold.  \nHydrogenation of azide 15[70] followed by treatment of 2-deoxyribofuranosyl amine formed in s itu \nwith chloroacetyl chloride and Et3N led to 2-deoxyribofuranosyl 2-chloroacetamide 16 in 38% with β/α \nratio about 1:1 (Scheme 2). Phosphinate 10 was then alkylated with compound 16 in the presence of \nHMDS at elevated temperatures providing a linear nucleoside 17 as a mixture of two anomers which \nwere successfully separated on a silica gel column. Finally, cyclisation of a linear nucleoside was \naccomplished in the presence of a catalytic amount of a Lewis acid (TMSOTf)  in 64% yield . \nUnfortunately, cyclisation precedes by racemisation and nucleoside 14 with the same α/β ratio of 3:2 \nforms from either anomerically pure 17 or mixture of its anomers. \n \n\n \n9 \nScheme 2. i) NaN3, n-Bu4NHSO4, NaHCO3/CHCl3 (1:1), r.t., 20 min; ii) a) H2, Pd/C, CH2Cl2, r.t.; 3h; b) \nchloroacetyl chloride, Et3N, 0 °C, overnight; iii) 10, HMDS, DCE, 90 °C, 24h; iv) TMSOTf, ACN, 40 °C, 2.5h. \nCatalytic hydrogenation is usually used for removal of benzyl protecting group. However, standard \nhydrogenation conditions using 10% Pd/C led to reduction of the C-C double bond in the nucleobase \nproviding nucleoside 26 (Scheme 3). To circumvent this problem, we used poisoned Pd-catalyst \n(Lindlar’s catalyst, 5%Pd/CaCO3/3%Pb) and obtained the desired nucleoside 18. Individual anomers \nof nucleosides 18 and 26 were separated on a C18 column in a gradient of CH3CN in H2O. Removal of \ntoluoyl groups was accomplished in aq. NH3 providing pure α- and β-nucleosides 20 and 27 carrying \na negative charge on the phosphinic group. These compounds were found to be stable in sodium \nphosphate buffer at pH 7.0 as no decomposition was observed in NMR samples for several days.  \nTo synthesise a charge-neutral nucleoside 22 (compound dPC-NH2 as shown in Figure 1), a \nphosphinic acid 18 was converted to the phosphinic chloride followed by ammonolysis in CHCl3 \n(Scheme 3). The resulting toluoyl-protected compound 19 was obtained in 46% yield but was found \nto be unstable in basic media required to remove toluoyl groups in the next step. This unfortunate \ninstability of nucleoside 19 in basic media repelled us from the idea to introduce the charge-neutral \ncompound 22 into DNA because basic conditions are used for DNA cleavage and deprotection. To \nobtain 22 for experiments with human CDA (hCDA), we used deprotected nucleoside 20 as a mixture \nof anomers and converted it to 22 through a four-step (silylation, treatment with oxalyl chloride, \nammonolysis, removal of silyl groups) one-pot synthesis. Purified phosphinamide 22 was obtained as \na mixture of anomers with α/β ratio of 2:1 as estimated by 1H and 13C NMR spectra.  \nDeprotected nucleosides 20, 22 but not 27 exhibited absorbance in UV-region with ε258 = 4230 L·mol-\n1·cm-1 and ε262 4730 L· mol-1·cm-1, respectively. This most likely a result of presence of a double bond \nnext to the P=O in nucleosides 20 and 22, whereas there is no double bond in the nucleobase of \ncompound 27.  \n\n \n10 \n \nScheme 3. i) H2, 5%Pd/CaCO3/3%Pb, Et3N, CH2Cl2, r.t.; 1.5 h; ii) H2, 10%Pd/C, Et3N, CH2Cl2, r.t.; overnight; \niii) oxalyl chloride, CHCl3, r.t., 15 min then satd. NH3 in CHCl3, r.t., 10 min; iv) 28% aq. NH4OH, r.t., \novernight; v) tert-butyldiphenylsilyl chloride (TBDPSCl), Et3N, CHCl3, r.t., 2h then oxalyl chloride, CHCl3, \nr.t., 15 min followed by satd. NH3 in CHCl3, r.t., 10 min; vi) n-Bu4NF·3H2O, THF, r.t.; 1h; vii) 4,4'-\ndimethoxytrityl chloride (DMTCl), dry pyridine, 0 °C to rt, overnight; viii) 3-hydroxypropionitrile, TBTU, \nEt3N, CH2Cl2, reflux, 1h; ix) N,N-diisopropylamino-2-cyanoethoxychlorophosphine (CEP-Cl), Et3N, dry \nCH2Cl2, 0 °C to rt, 1h. \nFor incorporation of nucleoside 20 into DNA, we need to equip it with standard 5'-O-DMT and 3'-O-\nN,N-diisopropylamino-2-cyanoethoxyphosphanyl groups and also eliminate a negative charge on the \nnucleobase as it might interfere with automated DNA synthesis. Starting from compound 14 as a \nmixture of anomers, compound 20 was obtained using above-described steps and after installation \nof 5'-O-DMT group, individual anomers of 23 were isolated on reverse-phase column (C18 media). \nThen, α- or β-anomer of salt 23 was converted to 2-cyanoethoxy-derivative 24 using 3-\nhydroxypropionitrile and TBTU which was further transformed into the required phosphoramidite 25 \nas individual α- and β-anomers which were used in preparation of modified DNA sequences on DNA \nsynthesiser. \n Evaluation of 1,4-azaphosphinine derivatives as inhibitors of human CDA, \nengineered APOBEC3B and wild-type APOBEC3A \n2.2.1. Evaluation of hCDA inhibition  \nWe monitored human CDA (hCDA)-catalysed deamination of dC at 286 nm[71] and analysed kinetic \nprofiles at various inhibitor concentrations using a global regression analysis of the kinetic data using \nLambert’s W function.[72] This method provides better estimates for Km and Vmax than non-linear \nregression analysis of initial rate or any of the known linearised transformations of the Michaelis-\n\n \n11 \nMenten equation, such as Lineweaver-Burk, Hanes-Woolf and the Eadie-Hofstee \ntransformations.[72] Then Michael-Menten constant (KM) for the substate and the inhibition \nconstant (Ki) for each inhibitor were calculated (Table 1) assuming competitive nature of inhibitors. \n \n  \n\n \n12 \nTable 1. Km of the substrate dC and Ki of dZ and 1,4-azaphosphinine nucleosides against hCDA. \nInhibitor pH Km of dC \n(μM)a) \nKi (μM) Km/Ki \ndZ 7.4 260 ± 40 10.7 ± 0.5 24 \nβ-anomer of \n22b) \n7.4 240 ± 150 8.0 ± 1.9 30 \nβ-anomer of \n20 \n7.4  No inhibition  \ndZ 6.0 270 ± 60 49 ± 13 5.5 \nβ-anomer of \n20 \n6.0  No inhibition  \nβ-anomer of \n20 \n4.7 90 ± 20 560 ± 100  \na) KM was fitted in each experiment independently (see Supplementary Information). b) Concentration of \nβ-anomers in solutions was determined by NMR (see Supplementary Information) and was used as \ninhibitor concentration assuming that α-anomers were not inhibiting hCDA. \nInitially, we performed this assay in 50 mM sodium phosphate buffer at pH 7.4 (25 °C) and observed \nthat β-anomer of charge-neutral nucleoside 22 exhibited similar inhibition of hCDA as control dZ. \nPresence of a negative charge in nucleoside 20 led to lack of inhibition at pH 7.4. We assumed that \nprotonation of 20 might result in some inhibition of hCDA. However, pKa of 20 was estimated to be ≤ \n1.5 (see Supplementary Information). This means that pH of the assay should be close to pH = 1.5 to \nsee any meaningful effect of partially protonated compound 20 but hCDA will be denatured at this \npH. By lowering pH to 6.0, dZ started to lose potency against hCDA (Table 1) which might be a result \nof protonation of the pyrimidine ring in dZ. Some inhibition of hCDA by β-anomer of 20 was \nobserved at pH 4.7 with Ki value of 560 μM. At this pH less than 1 in 1000 molecules of 20 might be \nprotonated which could mean that protonated phosphininic acid 20 is a potent hCDA inhibitor.  \n2.2.2. Evaluation of inhibitors against engineered A3A-mimic and wild-type A3A by 1H-\nNMR assay \nWe used the 1H NMR assay to test our short oligodeoxynucleotides (ODNs), linear and hairpins, \ncontaining individual α- and β-anomers of nucleoside 20 as inhibitors of A3. This real-time NMR \nassay is a direct assay; it uses only A3 enzymes and ODNs in a buffer, unlike many fluorescence-\nbased assays where a secondary enzyme and a fluorescently modified oligo are used.[73] The NMR-\n\n \n13 \nbased assay provides the initial velocity of deamination of ssDNA substrates, including the modified \nones,[57] in the presence of A3 enzymes. Consequently, the Michaelis–Menten kinetic model can be \nused to characterise substrates and inhibitors of A3. Both anomers of nucleoside 20 were \nindividually incorporated instead of the target dC in the preferred DNA motif (TCA) of A3A and A3B \non linear DNA. A previously described A3 inhibitor 5'-T4FdZAT, was used as a control.[55, 57, 58] The \nengineered A3A-mimic was used in our initial experiments. This is a well-characterised and active \nderivative of C-terminal domain of A3B (A3BCTD), originally called A3BCTD-QM-∆L3-AL1swap[55], in \nwhich loop 3 is deleted and loop 1 is replaced with the corresponding loop 1 from A3A. The residual \nactivity of A3A-mimic on the unmodified oligo (5'-T4CAT) as a substrate in the presence of a known \nconcentration of inhibitors was measured using the NMR assay (Fig. 2). \n \n \nFigure 2. Initial rate of A3A-mimic catalysed deamination of 5'-T4CAT in the absence (no inhibitor) and \npresence of inhibitors at concentrations indicated.  \nConditions: 400 µM of 5'-T4CAT, 36 µM of seven-membered ring containing oligos and 8 µM of FdZ-\ncontaining ODN, 300 nM of A3A-mimic in a 50 mM sodium phosphate buffer (pH 6.0) containing 100 mM \nNaCl, 2.5 mM β-mercaptoethanol, 50 µM 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid (TSP) and \n20% D2O at 25 °C. Error bars are estimated standard deviations from triplicate measurements. \n \n\n\n \n14 \nThe results revealed that both anomers of 20 do not inhibit engineered A3A-mimic even at elevated \nconcentrations in comparison with a control ODN containing FdZ at pH 6.0. It is very likely that a \nnegative charge on nucleobase 20 prevents binding to the enzyme.  \nRecently, it was reported that A3A prefers deaminating cytosines present in the short loops of DNA \nhairpins rather than linear DNA at pH 7.[74-76] We assumed that placing nucleoside β-20 in much \nmore preferred substrate would allow us to detect inhibitory potential of the resulting DNA hairpin. \nNucleoside β-20 was introduced instead of the target dC in the DNA hairpin with TTC loop and tested \nin the 1H NMR assay monitoring A3A-catalysed deamination of dC-hairpin (T(GC)2TTC(GC)2T, bold C is \ndeaminated) at 150 mM salt concentration, pH 7.4. Recently, FdZ, 5-methyl-2'-deoxyzebularine and \ndiazepinone deoxyriboside inserted in loops of DNA hairpins have shown selective inhibition of A3A \nwith IC50 and Ki in low nM range.[56, 61, 77, 78] Unfortunately, no inhibition of A3A by the DNA \nhairpin carrying β-20 was detected at concentrations used (20 and 100 µM of inhibitor DNA, 1 mM \ndC-hairpin as a substrate, 600 nM wtA3A-His6 in 50 mM Na+/K+ phosphate buffer, supplemented \nwith 100 mM NaCl, 1 mM TCEP, 100 µM sodium trimethylsilylpropanesulfonate (DSS) and 10% D2O; \nat pH 4.7). \n3. Discussion and Conclusion \nNucleoside and polynucleotide (A3) cytidine deaminases share a universal mechanism of target \nnucleobase engagement, deamination, and inhibition.[52, 60-65] We have recently demonstrated \nfirst inhibition of A3A-induced mutagenesis in cells using a DNA hairpin carrying FdZ instead of the \ntarget C in the TTC loop.[61] To further improve potency of DNA-based inhibitors of A3 one can use \nmore potent inhibitors of cytosine deamination than previously characterised FdZ, dZ and \ndeazepinone. There are two obvious choices based on the literature on CDA inhibitors, THU and \nphosphapyrimidine nucleoside P (Fig. 1). However, hemiaminal functionality in the nucleobase and \nfast transformation into pyranose in THU along with instability of P in water prevent their \nincorporation into DNA fragments using conventional DNA synthesis chemistry. Here, we \nhypothesised that aqueous stability of P can be significantly improved by changing of the N3 atom in \nthe nucleobase to the methylene group providing nucleosides dPC with and without a double bond \nbetween C5-C6 atoms, respectively (Fig 1). Towards this end we developed a synthetic strategy for \nthese nucleosides and identified that assembly of the nucleobase on the sugar was more viable that \ncoupling of the final nucleobase to the Hoffer’s sugar. It is interesting that only the charge-neutral \nphosphinamide 22 inhibited hCDA similarly to dZ at pH 7.4 whereas negatively charged phosphinic \nacid 20 showed some inhibition of hCDA only at pH 4.7. Unfortunately, due to low stability of \ncharge-neutral phosphinamide 22 towards nucleophiles we could not incorporate it into DNA. \n\n \n15 \nSynthesis of DMT-protected phosphoramidite of nucleoside 20 and its incorporation into DNA was \nmore straightforward but no inhibition of A3A was observed for these oligodeoxynucleotides. These \nresults suggest that negatively charged nucleobases cannot be accommodated in the active site of \nhCDA and A3A and other options need to be considered for the development of new nucleobases \nmimicking transitions states and an intermediate of cytosine deamination to improve potency of \nDNA-based A3 inhibitors.  \n \nAuthor Contributions: M.V.K., G.B.J., E.H., and V.V.F. designed the research, M.V.K. performed the \nsynthesis of nucleosides and linear DNA, H.M.K. performed the synthesis of hairpin DNA, S.H. and \nH.M.K. performed enzymatic assays. All authors analysed the data, wrote the article, and have read \nand agreed to the published version of the manuscript. \n \nAcknowledgements \nNMR and mass spectrometry facilities at Massey University and the assistance of Dr Patrick J. B. \nEdwards and Mr David Lun are gratefully acknowledged. We thank Prof. Reuben S. Harris (HHMI and \nUniversity of Texas Health, San Antonio, TX, USA) and members of his cancer research program for \nmany helpful discussions. We are grateful for the financial support provided by the Worldwide \nCancer Research (grant 16–1197), breast cancer partnership of Health Research Council of New \nZealand with Breast Cancer Cure, and Breast Cancer Foundation NZ (grant 20/1355), Palmerston \nNorth Medical Research Foundation, Maurice Wilkins Centre for Molecular Biodiscovery, Massey \nUniversity Research Fund (MURF 2015, 7003 and RM20734), Kiwi Innovation Network (KiwiNet) and \nMassey Ventures Ltd (MU002391) and the School of Natural Sciences, Massey University. \n \nSupplementary information:  \nSupplementary experimental details about the synthesis of nucleosides and modified ODNs and \nenzymatic assays; 1H, 13C, 31P NMR, IR and HRMS (ESI) spectra of new compounds synthesised, RP-\nHPLC profiles and HRMS (ESI) spectra of ODNs.  \n \nData Availability Statement: \nOriginal NMR spectra, HPLC profiles and details of enzymatic assays are available from \ncorresponding authors upon request. \n \n\n \n16 \nReferences: \n1. 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