. 2019 Apr 12;84(10):6143–6157. doi:10.1021/acs.joc.9b00338

Synthesis of TerminalRibose Analogues of Adenosine5′-Diphosphate Ribose as Probes for the Transient ReceptorPotential Cation Channel TRPM2

Ondřej Baszczyňski^‡,Joanna M Watt^†,^‡,Monika D Rozewitz^§,Andreas H Guse^§,Ralf Fliegert^§,Barry V L Potter^†,^‡,^*

^†MedicinalChemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.

^‡WolfsonLaboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, U.K.

^§TheCalcium Signalling Group, Department of Biochemistry and MolecularCell Biology, University Medical CenterHamburg-Eppendorf, Martinistrasse52, 20246 Hamburg, Germany

E-mail:barry.potter@pharm.ox.ac.uk. Phone: ++44-1865-271945. Fax: ++44-1865-271853.

Received 2019 Feb 1; Issue date 2019 May 17.

This is an open access article published under a Creative Commons Attribution (CC-BY)License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

PMC Copyright notice

PMCID: PMC6528165 EMSID:EMS82738 PMID:30978018

Abstract

graphic file with name jo-2019-00338t_0008.jpg

TRPM2(transient receptor potential cation channel, subfamily M,member 2) is a nonselective cation channel involved in the responseto oxidative stress and in inflammation. Its role in autoimmune andneurodegenerative diseases makes it an attractive pharmacologicaltarget. Binding of the nucleotide adenosine 5′-diphosphateribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain activates the channel. A detailed understanding of how ADPRinteracts with the TRPM2 ligand binding domain is lacking, hamperingthe rational design of modulators, but the terminal ribose of ADPRis known to be essential for activation. To study its role in moredetail, we designed synthetic routes to novel analogues of ADPR and2′-deoxy-ADPR that were modified only by removal of a singlehydroxyl group from the terminal ribose. The ADPR analogues were obtainedby coupling nucleoside phosphorimidazolides to deoxysugar phosphates.The corresponding C2″-based analogues proved to be unstable.The C1″- and C3″-ADPR analogues were evaluated electrophysiologicallyby patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compoundwith all hydroxyl groups of the terminal ribose blocked as its 1″-β-O-methyl-2″,3″-O-isopropylidenederivative was evaluated. Removal of either C1″ or C3″hydroxyl groups from ADPR resulted in loss of agonist activity. Boththese modifications and blocking all three hydroxyl groups resultedin TRPM2 antagonists. Our results demonstrate the critical role ofthese hydroxyl groups in channel activation.

Introduction

The nonselective cationchannel TRPM2 (transient receptor potentialcation channel, subfamily M, member 2) is activated in a Ca²⁺-dependent manner after binding of the nucleotide ADP-ribose (ADPR)to its cytosolic C-terminal NUDT9H domain that shareshomology with a mitochondrial nucleotide pyrophosphatase NUDT9.¹ While earlier studies indicated that the NUDT9H domain also has a low pyrophosphatase activity, hydrolyzingADPR to AMP and ribose 5-phosphate (R5P),² a recent study showed that the Nudix box motif in TRPM2 does notsupport catalysis and the production of AMP might have been due tospontaneous hydrolysis of ADPR at alkaline pH.³

Reactive oxygen species (ROS) and genotoxic stress can resultinrelease of ADPR from the nucleus due to the activation of the poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase pathways.^4,5 The ADPR so generated can then activate TRPM2, resulting in prolongedCa²⁺-entry, mitochondrial Ca²⁺-overload, andapoptosis,⁶ thereby contributing to celldamage in post ischemic reperfusion injury during myocardial infarction⁷ and stroke.⁸

Besides this role in cell death, TRPM2 also participates in physiologicalprocesses like inflammation.⁹ In neutrophilgranulocytes and dendritic cells, TRPM2 contributes to chemotaxis.¹⁰⁻¹² The chemotaxis of murine neutrophils in response to fMLP is independentof PARP-1 but can be inhibited by 8-Br-ADPR, a compound that inhibitsactivation of TRPM2 by ADPR, and by knock-out of CD38, a glycohydrolasethat produces ADPR from NAD.¹⁰ In macrophagesand monocytes, TRPM2 is involved in secretion of chemokines and cytokinesin response to ROS and pro-inflammatory cytokines,^13,14 whereas in effector T cells, it plays a role in proliferation andsecretion of pro-inflammatory cytokines.¹⁵ Inhibition of TRPM2 has been shown to reduce tissue damage afterstroke by preventing invasion of neutrophils,¹⁶ and knock-out of TRPM2 ameliorates the symptoms of experimentallyinduced autoimmune encephalomyelitis, a model for multiple sclerosis.¹⁵ TRPM2 is thus an attractive pharmacologicaltarget for the treatment of neurodegenerative and autoimmune diseases.^17,18

Further investigations into the role of TRPM2 in physiologyandpathophysiology require specific modulators of channel function, andtheir rational design will benefit from a better understanding ofits structure–activity relationship (SAR). Recently, TRPM2structures from three different species were elucidated by cryo-EM.The structure of RPM2 from the sea anemoneNematostellavectensis nvTRPM2 lacks the NUDT9H domain, probably because of the flexibility of this part of themolecule.¹⁹ TRPM2 from zebrafish (Danio rerio) drTRPM2 has been solved in the apo stateand in an ADPR-bound state.²⁰ While ADPRcould not be localized in the NUDT9H domain, electrondensity corresponding to ADPR was found in theN-terminalMHR1/2 domain. Comparison of the conformations of the apo and theADPR-bound state and mutational analysis indicate that in drTRPM2,gating of the channel occurs via binding of ADPR to the MHR1/2 domaininstead of NUDT9H.²⁰ Structuresof human TRPM2 (hTRPM2) have been resolved for the apo state, an ADPRbound (primed) state and an open conformation bound to ADPR and Ca²⁺.²¹ Mutagenesis of the MHR1/2domain and removal of the NUDT9H domain show thatin the human TRPM2 channel, gating occurs after binding of ADPR tothe NUDT9H domain, but again poor resolution preventsthe placement of ADPR in the presumed binding pocket. Structure-baseddrug design therefore still awaits a high-resolution structure ofeither the full-length channel or the isolated NUDT9H domain of TRPM2.

Recently, we synthesized ADPR analogues toexplore the role ofthe adenosine, pyrophosphate, and terminal ribose motifs in activationof TRPM2.^22,23 The pyrophosphate-forming couplings usedemployed morpholidate- or CDI-mediated methodologies. Yields weregenerally low and reaction times were generally long and improvementsare warranted. To our surprise, the majority of structural modificationsof ADPR led to compounds that do not activate TRPM2, indicating thatall three moieties are required for channel opening.²³ These studies also revealed that hydroxyl group removalat C2′ resulted, in the case of ADPR, in a compound that isa TRPM2 agonist with significantly higher efficacy than ADPR itself²³ and also improved the antagonist 8-Ph-ADPR (IC₅₀ 11 μmol/L compared to 8-Ph-2′-deoxy-ADPR, IC₅₀ 3 μmol/L).²² We also highlightedthe importance of the terminal ribose for TRPM2 activation by evaluatinga series of modified ADPR analogues.²⁹ Whilesimple ADP neither activated nor antagonized the channel, introductionof substituents at the β-phosphate that increasingly resembledthe terminal ribose (Figure 1) returned antagonist properties, but none of the analoguesexhibited agonist activity. The fact that β-(tetrahydrofuran-2-yl)methyl-ADP,an analogue with the ribofuranose backbone, but lacking the hydroxylgroups, did not activate the channel but instead antagonized TRPM2indicated that one or more of the hydroxyl groups might be importantfor the gating of TRPM2. Previous studies have shown hydroxyl groupdeletion to be a valuable tool in SAR elucidation for complex bioactivemolecules.²⁴⁻²⁶ While such endeavors may be synthetically protractedin order to effect a desired precision edit to the parent molecule,they can divulge key mechanistic information.^27,28

Structureof ADPR and known terminal ribose analogues.²⁹

We report here the synthesis andelectrophysiological evaluationof novel terminal-ribose-modified analogues of the TRPM2 agonistsADPR and 2′-deoxy-ADPR to study the role of the individualhydroxyl groups of the terminal ribose in TRPM2 activation. The criticalstep of analogue formation was achieved by combining a sugar phosphatewith a P-activated nucleotide to form a single molecule linked bya pyrophosphate bond. A recently reported improved procedure for thepreparation of NDP sugars from nucleoside phosphorimidazolides³⁶ uses 2–4 equiv of magnesium chlorideto achieve high reaction yields and short reactions times for nucleoside5-phosphorimidazolide coupling to sugar phosphates.

Results and Discussion

The terminal ribose of ADPR is essential for the ligand-drivenactivation of the cation channel TRPM2.²⁹ To investigate the SAR of this part of ADPR in more detail, we focusedupon the synthesis of all three possible 1″-deoxy, 2″-deoxy,and 3″-deoxy terminal ribose ADPR derivatives2,3, and4 by selective deletion of theappropriate hydroxyl group (Figure 2). The similar reactivity of the ribose hydroxyl groupsand potential for ring opening of the cyclic hemi-acetal requireddevelopment of multistep synthetic routes with selective masking anddeprotection. The modified ribose building blocks were then coupledto either AMP or 2′-deoxy-AMP via pyrophosphate coupling reaction(Figure 2). We alsosynthesized a corresponding analogue1 with all hydroxylgroups present, but blocked with small alkyl groups to interrogatethe H-bond donating capability of the terminal ribose (Figure 2). The chemically stable analogueswere evaluated regarding their agonist and antagonist activity bypatch-clamp experiments in whole cell configuration using HEK cellswith stable expression of human TRPM2.

Pyrophosphate couplingreaction and ADPR analogues prepared inthis study.

At first, routes to therequired modified terminal ribose 5-phosphateswere designed. It was envisaged that such phosphates would be idealcoupling partners for activated nucleotide imidazolides. Protectedribose monophosphate9 was chosen to exploit the ligandspace around the terminal ribose, H-bond donating capability, andits good susceptibility for coupling with activated nucleosides. Theprecursor of9, compound8, was obtainedfrom protected ribose³⁰7 byphosphitylation with dibenzylN,N-diisopropylphosphoramidite and subsequent oxidation using hydrogenperoxide³¹ to afford8 (79%)(Scheme 1A). Compound8 was then hydrogenated using Pd/C to afford9 (82%). Ribose-5-monophosphate9 was converted to itstributylammonium salt and freeze-dried. Synthesis of 1-deoxyribose-5-phosphate10, to explore the role of the anomeric OH group of the terminalribose, started from 2,3-O-isopropylidene ribose13 that was protected at position 5 by a trityl group, toafford14 (65%) (Scheme 1B). Reduction of14 by sodium borohydrideled to compound15 (85%). Reaction of15 with tosyl chloride in pyridine gave no significant result at roomtemperature, but heating the reaction mixture up to 60 °C helpedto afford the protected 1-deoxyribose16 (80%). However,deprotection of16 with acetic acid failed after multipleattempts, as both the 5-O-trityl and 2,3-O-isopropylidene protecting groups were cleaved, leadingto the undesired fully deprotected 1-deoxyribose or to complex mixtures.Using the alternative deprotection of16 with formicacid in diethylether³² afforded compound17 (32%).³³ Phosphitylation andsubsequent oxidation of17 with di-tert-butylN,N-diisopropylphosphoramiditegave the phosphate derivative18 (41%). Careful deprotectionof18 with aqueous trifluoroacetic acid at low temperatureafforded the target 1-deoxyribose-5-phosphate10 (49%)that was subsequently converted to its tributylammonium salt.

Scheme 1 — Reagents and conditions: (a)5-Ph-1-H-tetrazole, dibenzylN,N-diisopropylphosphoramidite, DCM, 20 °C, 1 h; (b)triethylamine, H₂O₂, 0–20 °C, 1h; (c) hydrogen (balloon), Pd/C, 5 h, TEAB (1 M), 20 °C; (d)Dowex D50 (H⁺), tributylamine; (e) tritylchloride, pyridine,20 °C, 16 h; (f) sodium borohydride, ethanol, 0–20 °C,2 h; (g) tosyl chloride, pyridine, 60 °C, 16 h; (h) HCOOH/diethylether,20 °C, 16 h; (i) 5-Ph-1-H-tetrazole, di-*tert*-butylN,N-diisopropylphosphoramidite,DCM, 20 °C, 1 h; (j) triethylamine, H₂O₂, 0–20 °C, 1 h; (k) aqueous TFA, 0–20 °C,4 h; (l) Dowex D50 (H⁺), tributylamine.

Synthesis of the 2-deoxy version of the terminal ribose (Scheme 2) started from commerciallyavailable 2-deoxyribose-5-phosphate sodium salt19 (Sigma-Aldrich)that was converted to its corresponding mono-tributylammonium salt11 by using Dowex resin (H⁺ form) followed by titrationwith tributylamine to pH ≈ 7 (Scheme 2A). Partial decomposition (20–30%)of19 was observed during the transformation to11 via³¹P NMR, suggesting that 2-deoxyribose-5-monophosphateis less stable toward changes in pH (seeSupporting Information, S14–16) compared to its 1-deoxy- and 3-deoxy-counterparts.Synthesis of 3-deoxyribose-5-monophosphate12 startedfrom 1,2-O-isopropylidene xylose20,³⁴ which was protected at the 5-hydroxyl groupas the TBDPS ether to give21 (83%) (Scheme 2B). Reaction of21 with 1,1′-thiocarbonyldiimidazole in DCM afforded compound22 (63%). Reductive deoxygenation of22 withtributyltin hydride led to the compound23 (76%) whichwas subsequently deprotected in buffered TBAF to give 1,2-O-isopropylidene-3-deoxyribose24 (82%). Phosphitylationand subsequent oxidation of24 gave the desired di-tert-butyl phosphate derivative25 (50%). However,several attempts to deprotect compound25 with aqueousTFA led to a complex mixture, suggesting that di-tert-butyl protected phosphate25 was not the optimal precursorfor12. Therefore,24 was phosphitylatedand subsequently oxidized to its dibenzylphosphate derivative26 (72%) that was successfully deprotected to give27 (68%). Hydrogenation of27 followed by neutralizationof the free phosphate using tributylamine afforded the target analogue3-deoxyribose-5-phosphate as its mono-tributylammonium salt12 (67%).

In previous work, we usedmorpholidate chemistry or 1,1′-carbonyldiimidazole-basedmethodology in coupling reactions, but these had been less than satisfactoryduring ADPR analogue preparation. To find the most suitable conditionshere for pyrophosphate bond formation between the corresponding ribose-5-monophosphateand either AMP or 2′-dAMP, two different methods were tried.Activation of AMP tributylammonium salt with 1,1′-carbonyldiimidazole³⁵ followed by addition of the triethylammoniumsalt of ribose9 did show formation of the desired pyrophosphateproduct1 by high-performance liquid chromatography (HPLC).However, several byproducts were also observed (seeSupporting Information Figure S1a, S2). In contrast, followingthe Dabrowski-Tumanski procedure³⁶ activationof the AMP tributylammonium salt using imidazole, 2,2′-dithiodipyridine(Aldrithiol) and triphenyl phosphine cleanly afforded the AMP-imidazolidethat was isolated by precipitation with a cold solution of NaI inacetone. When stirred with the triethylammonium salt of9 and magnesium chloride in dimethylformamide (DMF), the AMP-imidazolidegave almost exclusively the desired compound1 (seeSupporting Information Figure 1b, S2–S3).Finally, purified target ribose derivatives9,10,11, and12 were individuallycoupled to imidazolide-activated AMP or 2′-deoxy-AMP (Scheme 3). Adenosine-5′-monophosphate28 and 2′-deoxy-adenosine-5′-monophosphate29 were transformed to their imidazolides30 and31, by reaction of the corresponding mono-tributylammoniumsalt of28 and29 and imidazole using theAldrithiol and triphenylphosphine condensation protocol.³⁶ Imidazolides30 and31 were precipitated from the reaction mixture by addition of a 0.1M solution of sodium iodide in cold, anhydrous acetone and were directlyused for coupling. The target modified ADPR analogues1–6 were then prepared by coupling³⁶ the corresponding imidazolide30 or31 and mono-tributylammonium salt of the modifiedribose-5-phosphate9,10,11, or12 to give variable yields of the desired ADPRanalogues1 (24%),2 (66%),3 (8%),4 (17%),5 (42%), and6 (27%). Final compounds1–6 were purified bysemi-preparative HPLC using triethylammonium bicarbonate (TEAB) bufferand isolated as the corresponding triethylammonium salts.

Scheme 3 — Reagentsand conditions: (a)Aldrithiol, imidazole, triethylamine, triphenylphosphine, 20 °C,16 h; (b) NaI, acetone (precipitation), 0 °C; (c) MgCl₂, dimethylformamide, 20 °C, 3–16 h.

Stabilityof Terminal-Modified ADPR Analogues1–6

The stability of ADPR analogues1–6 was evaluated using analytical HPLC. Aqueous solutions of1–6 stored at 5 °C were comparedwith freshly prepared standard solutions of1–6 (from solid samples stored at −20 °C). All compoundsexcept3 were stable in aqueous solution for severaldays when stored in the fridge (5 °C). Compound3, 2″-deoxy-ADPR, was found to be unstable under these conditions.We also observed decomposition of3 during transportand sample preparation, so we could not do electrophysiological experimentswith3 (Supporting Information Figure 2, S4). The instability of3 may correspondwith the lower stability of 2-deoxyribose-5-monophosphate11 that was sensitive toward changes in pH during conversion of thecommercially available sodium salt to the tributylammonium salt (conversionof19 to11). This decomposition led tohydrolysis of the 5′-phosphate (approx. 20–30% de-phosphorylationof11 was observed via³¹P NMR). The correspondingdecomposition by de-phosphorylation in 2″-deoxy-ADPR3 might cleave the pyrophosphate bond and would be predictedto lead to inactive fragments.

Evaluation of Novel AnaloguesAgainst TRPM2

Recently,we investigated the importance of the ADPR terminal ribose for theactivation of TRPM2.²⁹ This can be investigatedby whole cell patch-clamp experiments. During these experiments, aglass pipette with a diameter much smaller than a cell and filledwith a solution mimicking the ionic composition of the cytosol isattached to the plasma membrane. By applying suction to the pipette,the membrane patch underneath the pipette is ruptured. In voltage-clampexperiments, a voltage is applied between an electrode in the pipettesolution and a bath electrode in the bath solution surrounding thecells. This voltage results in ions moving through channels in themembrane resulting in a current that can be recorded.^37,38 In HEK293 cells expressing TRPM2, addition of ADPR to the pipettesolution results in a current that is absent in wild type HEK293 cells.ADPR analogues can be tested for their ability to activate the channel,and they can also be tested for antagonist activity by adding themin addition to ADPR. We found that adenosine 5′-monophosphate(AMP) and adenosine 5′-diphosphate (ADP) neither activatedor inhibited the channel even when applied in large excess over ADPR.²⁹ Interestingly, replacement of the terminal riboseof ADPR with small substituents led to analogues that did not activatethe channel, but inhibited activation by ADPR, indicating that theycompete with ADPR for the ligand binding site of TRPM2. Thus, activationof TRPM2 may be attributed to specific interactions between the hydroxylgroups of the terminal ribose of ADPR and the ligand binding NUDT9H domain of TRPM2. We evaluated the deoxy analogues2 and4 and an ADPR analogue1 whereall hydroxyl groups of the terminal ribose are masked and could nolonger act as hydrogen bond donors (Figure 3). It was not possible to evaluate3 biologically because of instability issues (see above andSI). During the experiments, we included 8-Br-ADPRas a control. 8-Br-ADPR has previously been shown to inhibit activationof TRPM2 by ADPR most likely by competing with the agonist.¹⁰

Effect of ADPR analogues on whole cell currents in TRPM2expressingHEK293 cells. Conditions: (a) Effect of ADPR analogues on whole cellcurrents in TRPM2 expressing HEK293 cells. Experiments were done asoutlined in theExperimental Section. ADPRor the indicated ADPR analogue was added to the pipette solution ata concentration of 100 μmol/L. (b) Effect of ADPR analogueson TRPM2 whole cell currents elicited by ADPR. In this case, the pipettesolution contained either no nucleotide (buffer), 100 μM ADPRor a combination of 100 μM ADPR with 900 μM of the indicatedADPR analogue. 8-Br-ADPR was included as the inhibitor control. Pointsindicate the maximum current from individual cells. The number ofcells for each condition is indicated at the bottom. Bars representthe mean of the log-transformed currents (ns = non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Controlconditions on both panels include overlapping sets of data becauseon some days, both agonist and antagonist experiments were performed.

As expected, the masked analogue1″-β-O-Me-2″,3″-O-iPr-ADPR1 did not activateTRPM2 on its own (Figure 3a). Because it is more space-filling thanADPR, we were unsure whether it would compete with ADPR for bindingto the NUDT9H domain. When applied in excess overADPR, however, it indeed inhibited activation of TRPM2 (Figure 3b), showing that further stericconstraints than those simply at the 1″-position of the terminalribose are tolerated and may potentially be exploitable for TRPM2antagonist optimization.

Interestingly, an analogue32 (Figure 4) of thelow-affinity partial TRPM2 agonistα-β-methylene ADPR (AMPCPR)³⁹ masked in the same way as1 was recently describedto be a high affinity TRPM2 antagonist that inhibits ADPR-elicitedTRPM2 currents with an IC₅₀ of 5.7 μM.⁴⁰ Luo et al. here did not explicitly mention whetherthey tested this compound for TRPM2 activation but, because it fullyinhibits the channel at 100 μM, it is unlikely to exhibit agonistactivity. Methylene analogues may show increased stability towardcellular pyrophosphatases like NUDT9 and NUDT5 which may be retainedin the cell during whole cell patch-clamp experiments. Earlier reportsthat indicated an ADPR pyrophosphatase activity of the NUDT9H domain of TRPM2,¹ that mightcontribute to degradation of agonists with pyrophosphate bridge haverecently been disputed.³ The observed differencecould also result from different experimental conditions as we bufferedthe intracellular Ca²⁺ concentration to 200 nmol/L with10 mmol/L of EGTA, whereas Luo et al. left the Ca²⁺ concentrationlargely unbuffered (50 μmol/L EGTA) which would result in amuch steeper dependence of the current on the agonist concentrationand a more pronounced inhibition by the antagonist.

Structures of maskedADPR1, α-β-methyleneADPR (AMPCR)32, 2″-O-acetyl-ADPR33, and 3″-O-acetyl-ADPR34.

Contrary to our expectations,both ADPR analogues2 and4 lacking hydroxylgroups at the terminal ribosedid not induce significant whole cell currents in TRPM2-expressingHEK cells at 100 μmol/L (Figure 3a). This was unexpected asO-acetyl-ADPR,a product of NAD-dependent histone deacetylases of the Sir2 family,is an effective activator of TRPM2 that binds to the NUDTH domain with similar affinity as ADPR, as shown by UV cross-linkingexperiments and activates TRPM2 with a comparable concentration dependenceas ADPR.⁴¹ 2″-O-acetyl-ADPR33, the product of the deacetylases, undergoesin neutral solution rapid intramolecular transesterification resultingin roughly equal amounts of 2″-O-acetyl-ADPR33 and 3″-O-acetyl-ADPR34.⁴² Either the nucleotide binding siteof TRPM2 is indifferent to the position of the acetyl group, whichwould indicate that neither the 2″- nor the 3″-hydroxylgroup is essential as hydrogen bond donor, or there is a specificityfor one of the isomers which would mean that only half of theO-acetyl-ADPR molecules in solution are effective agonists.Because the latter seems unlikely, this may suggest that the 2″-OHand 3″-OH groups are hydrogen-bond acceptors, a feature thatcould be retained even when acetylated. We have previously shown thatα-1″-O-methyl-ADPR and β-1″-O-methyl-ADPR (Figure 1) do not activate TRPM2, but both inhibit channel activationby ADPR.²⁹ This indicates that the 1″-hydroxylgroup in ADPR could not be masked or that there is no space in thisregion to accommodate even a relatively small methyl group, withoutlosing agonist activity in contrast to the 2″- and 3″-hydroxylgroups. So why does 3″-deoxy-ADPR4 not activatethe channel? Possible explanations may be that the conformation ofthe furanose ring or the ratio between the two anomeric forms of theribose is affected by the absence of the 3″-hydroxyl groupin a way that prevents the proper hydrogen bonding necessary for channelactivation or that this hydroxyl is required as a hydrogen bond acceptor.

The five-membered ribose sugar ring is puckered because of nonbondedinteractions, and the energetically most stable conformation has allsubstituents as far apart as possible.⁴³ Thus, the different substitution patterns in the terminal riboseof ADPR, 1″-deoxy-ADPR2, 2″-deoxy-ADPR3, and 3″-deoxy-ADPR4, might be expectedto produce differing types of puckering. Extensive analysis of theribose and 2′-deoxy-ribose rings in RNA and DNA nucleotideshas shown that while ribose adopts a primarily C3′-endo configuration,2′-deoxy-ribose adopts a primarily C2′-endo form.⁴⁴ Such changes alter the orientation of the ribosering substituents, and this may affect the orientation of the substituentsand the way they are presented to the binding site, as well as thestability of a given ADPR analogue by bringing nucleophilic hydroxylgroups into proximity for intramolecular attack on the pyrophosphate.Unfortunately, the broad nature of the peaks in the¹HNMR spectra of the ADPR analogues means that it is not possible fullyto determine the coupling constants and hence to analyze fully eachterminal ribose ring conformation and suggests that rapid interconversionbetween configurations may be taking place in solution.

ADPRis a mixture of terminal ribose α- and β-anomersand it is not known whether one or both anomers activate TRPM2. Analysisof¹H NMR spectra demonstrates that both 2″-deoxy-ADPR3 and 3″-deoxy-ADPR4 are also mixturesof the α- and β-forms in the following ratios (α/β,2″-deoxy-ADPR3, 1:1.17; 3″-deoxy-ADPR4; 1:4). Both analogues still adopt cyclic (as opposed toopen chain form) structures for the terminal ribose. Given that bothanomers (α- and β-forms) are present in both cases, itwould seem unlikely that this could be the sole cause of their observedinactivity.

Surprisingly, di-deoxy ADPR analogues5 and6 did not have significant antagonist activity(data not shown).This was unexpected because the potency of our previous antagonist8-Ph-ADPR (IC₅₀ = 11 μM) was improved by the 2′-deoxymodification (8-Ph-2′-deoxy-ADPR IC₅₀ = 3 μM).

The recent cryo-EM structures of human TRPM2 show that in the absenceof ADPR interactions between the NUDT9H domain andMHR1/2 domains of the same and adjacent subunits lock the channelin a closed conformation.²¹ Binding ofADPR impacts the conformation of the NUDT9H domainof TRPM2 in a way that leads to disengagement of the inter-subunitinteractions, priming the channel for full activation by binding ofCa²⁺ ions to a cytosolic site near the pore region.²¹ The NUDT9H domain of TRPM2has a bi-lobed structure with the two lobes forming a cleft that issupposed to bind ADPR. While comparison of the apo and the ADPR boundstate shows a narrowing of this cleft upon binding of ADPR,²¹ the low local resolution of the structure inthe NUDT9H domain does not currently allow for localizationof ADPR in this binding pocket or for identification of the interactions,which would trigger the conformational changes leading to the primedstate. In the absence of a high-resolution structure, SAR data thereforeremain essential for ligand based drug design. Our current data furthersupport the role of interactions involving the terminal ribose ofADPR in the gating of TRPM2.

Recently, key differences betweenhTRPM2 and the invertebrateN. vectensis variant nvTRPM2 were identified. Thus,two reported synthetic ADPR analogue hTRPM2 antagonists were nvTRPM2agonists.⁴⁵ Moreover, a regulatory functionof NUDT9H in nvTRPM2 opposed to that in hTRPM2 wasuncovered through the action of another synthetic analogue, inosine5′-diphosphate ribose IDPR. Thus, ADPR analogues, such as thosereported here, are at the cutting edge of progress to unravel themechanism of TRPM2 function.

Conclusions

Buildingon our previous study that showed the essential role ofthe terminal ribose of ADPR in gating of the Ca²⁺-permeable,nonselective cation channel TRPM2, we synthesized ribose and 2′-deoxyriboseanalogues of ADPR1–6, each lackingone of the hydroxyl groups of the terminal ribose. The convergentsynthesis of the targets1–6 consistedof two separate steps: (a) synthesis of particular deoxyribose monophosphates9–12 and (b) coupling analogues9–12 to the AMP and 2′-deoxy-AMPimidazolides30–31, following theDabrowski-Tumanski procedure.³⁶ Such anapproach seems to be highly efficient in preparation of such complexmolecules as modified ADPR analogues, offering improved yields inour hands compared to pyrophosphate formation using AMP-morpholidatesor activation using CDI. While 2″-deoxy-ADPR3 proved to be unstable, analogues1,2,4,5, and6 showed acceptable stabilityand were tested electrophysiologically in patch-clamp cell experiments.Neither 1″-deoxy-ADPR2 nor 3″-deoxy-ADPR4 was able to activate TRPM2 significantly; instead, both2 and4 were weak antagonists of ADPR-mediatedTRPM2 activation in whole cell experiments, further highlighting thesensitivity of channel activation to structural changes of the terminalribose. Thus, 1″-β-O-Me-2″,3″-O-iPr-ADPR, 1″-deoxy-, and 3″-deoxy-ADPR(1,2 and4) were all antagonistsof ADPR-mediated Ca²⁺-release in whole cell patch-clampexperiments. Unlike in our previous observations, where includingan additional 2′-deoxy-modification generated a more potentTRPM2 antagonist, the corresponding 1″- and 3″, 2′-dideoxy-analogues5 and6 were less potent antagonists of TRPM2.Results further highlight the significance of the ADPR terminal ribosefor activation of TRPM2 and the use of synthetic ADPR analogues ingeneral. The synthesis of such ADPR analogues as chemical biologytools using the methods outlined here is proving invaluable in thewider TRPM2 field.⁴⁵ Further analoguesto interrogate individual hydroxyl group stereochemistry or individuallymask each of the three terminal hydroxyl groups to probe hydrogenbonding interactions at the TRPM2 binding site will undoubtedly shedmore light on the role of the terminal ribose.

ExperimentalSection

General

All reagents and solvents were of commercialquality and were used without further purification, unless describedotherwise. Triethylamine was dried over potassium hydroxide, distilled,and then stored over potassium hydroxide pellets. H₂O wasof MilliQ quality. Unless otherwise stated, all reactions were carriedout under an inert atmosphere of nitrogen. All¹H,¹³C, and³¹P NMR spectra of the final compoundswere collected, either on a Varian Mercury 400 MHz or Bruker AVANCEIII 500 MHz Spectrometer. Chemical shifts (δ) are reported inparts per million (ppm) and all¹H and¹³C NMRassignments are based on COSY, HSQC, HMBC, and DEPT experiments. Abbreviationsfor splitting patterns are as follows: br, broad; s, singlet; d, doublet;t, triplet; m, multiplet etc. High-resolution time-of-flight massspectra were obtained on a Bruker Daltonics micrOTOF mass spectrometerusing electrospray ionisation. Analytical HPLC analyses were carriedout on a Waters 2695 Alliance module equipped with a Waters 2996 PhotodiodeArray Detector (210–350 nm). The chromatographic system consistedof a Hichrom Guard Column for HPLC and a Phenomenex Synergi 4u MAX-RP80A column (150 × 4.60 mm), eluted at 1 mL/min with a gradientof MeCN in 0.05 M TEAB. Semi-preparative HPLC was carried out on aWaters 2525 pump with manual FlexInject. The chromatographic systemconsisted of a Phenomenex Gemini, 5u, C18, 110A column (250 ×10.00 mm), eluted at 5 mL/min.

1-O-Methyl-2,3-O-isopropylidene-β-d-ribofuranose (7)³⁰

Compound7 was preparedaccording to the literaturefromd-ribose. Yield (4.28 g, 63%). NMR and HRMS confirmed.¹H NMR (400 MHz, CDCl₃): δ 4.93 (s, 1H, H-1),4.78 (d,J = 6.0 Hz, 1H, H-2), 4.54 (d,J = 6.0 Hz, 1H, H-3), 4.38 (br s, 1H, H-4), 3.66–3.55 (m, 2H,H-5_a,b), 3.38 (s, 3H, OCH₃), 3.19 (br s, 1H,OH), 1.44 (s, 3H, CH₃), 1.27 (s, 3H, CH₃).

Dibenzyl 1-O-methyl-2,3-O-isopropylidene-β-d-ribofuranose-5-phosphate (8)

1-O-methyl-2,3-O-isopropylidene-β-d-ribofuranose7 (100 mg, 0.49 mmol) and 5-phenyl-1H-tetrazole (143 mg, 0.98 mmol) were co-evaporated withdry toluene (2×). Then, the solid mixture was suspended in dichloromethane(2 mL), and dibenzylN,N-diisopropylphosphoramidite(250 μL, 0.669 mmol) was added dropwise. The mixture was stirredat 20 °C for 2 h. TLC analysis (petroleum ether/EtOAc, 2:1 v/v)showed complete phosphitylation. The reaction was cooled to 0 °C,and triethylamine (407 μL, 2.92 mmol) was added followed byaqueous hydrogen peroxide (35%, 109 μL, 1.24 mmol). The resultingmixture was stirred at 20 °C for 1 h. The mixture was dilutedwith EtOAc (20 mL) and extracted with aqueous Na₂SO₃ (10%, w/v). The organic phase was dried (Na₂SO₄), solids were removed by filtration, and the solvent wasevaporated in vacuo. The crude product was purified by Isco-Flashchromatography using petroleum ether/EtOAc (1:0 → 0:1, v/v).This procedure afforded the title compound8 as a colorlessliquid (180 mg, 79%).¹H NMR (400 MHz, CDCl₃): δ 7.40–7.31 (m, 10H, Bn-2, 3, 4), 5.10–5.0(m, 4H, Bn-CH₂), 4.93 (s, 1H, H-1), 4.60 (dd, 1H,J = 6.0 Hz,J = 0.4 Hz, H-2), 4.50 (d,1H,J = 6.0 Hz, H-3), 4.28 (dt, 1H,J = 6.0 Hz,J = 0.4 Hz, H-4), 4.0–3.88 (m,2H, H-5), 3.26 (s, 3H, OMe), 1.46 and 1.28 (2 × s, 2 × 3H,CH₃iPr).³¹P NMR (160 MHz,CDCl₃): δ −1.23 (s, P).¹³C{¹H}NMR (100 MHz, CDCl₃): δ 135.7 and 135.6(2 × C-ipso-Bn), 128.5 and 127.9 (10 ×C-Bn-o,m,p),112.5 (C(CH₃)), 109.3 (C-1), 84.9 (C-3), 84.6(d,J = 9.0 Hz, C-4), 81.4 (C-2), 69.4 and 69.4 (CH₂-Bn), 67.1 (d,J = 6.0 Hz, C-5), 55.0 (OMe), 26.3 and 24.9 (CH₃iPr). HRMS (ES⁺) calcd for C₂₃H₂₉NaO₈P, 487.1492 [M + Na]⁺; found, 487.1501.

1-O-Methyl-2,3-O-isopropylidene-β-d-ribofuranose-5-phosphate (9)

Dibenzyl1-O-methyl-2,3-O-isopropylidene-β-d-ribofuranose-5-phosphate8 (180 mg, 0.39 mmol)was dissolved in MeOH–water (17:3 v/v, 8 mL). After vacuum/argondeoxygenation of the reaction mixture, a catalytic amount of palladiumon charcoal (10%, 20 mg) was added. The reaction was stirred underpositive pressure of hydrogen atmosphere (balloon) at 20 °C for5 h. Then, the reaction was flushed with argon and neutralized byaddition of TEAB buffer (1 mL, 1 M). The solvent was evaporated andthe crude product was co-evaporated (2×) with dry toluene anddried under high-vacuum to give a colourless solid film, compound9 (1.17 × Et₃N salt, 122 mg, 82%).¹H NMR (400 MHz, CD₃OD): δ 4.83 (s, 1H, H-1), 4.77(m, 1H, H-2), 4.53 (d, 1H,J = 8.0 Hz, H-3), 4.24–4.18(m, 1H, H-4), 3.83–3.72 (m, 2H, H-5), 3.24 (s, 3H, OMe), 3.08(q, 7H,J = 8.0 Hz, NCH₂CH₃), 1.37 (t, 3H, CH₃iPr), 1.27–1.21 (m, 13.5 H, NCH₂CH₃, CH₃iPr).³¹P NMR (160 MHz,d₄-MeOD): δ 0.87(s, P).¹³C{¹H}NMR (100 MHz, CD₃OD):δ 113.3 (C(CH₃)), 110.7 (C-1), 86.9(d,J = 9.0 Hz, C-4), 86.4 (C-3), 83.4 (C-2), 66.3(d,J = 5.0 Hz, C-5), 55.1 (OMe), 47.5 (NCH₂CH₃), 26.7 and 25.0 (CH₃iPr), 9.2 (NCH₂CH₃). HRMS (ES^–) calcd for C₉H₁₆O₈P 283.0588 [M – H]⁻; found 283.0575. (The product was transformed into its tributylammoniumsalt before the pyrophosphate coupling). Transformation of 9 to mono-tributylammoniumsalt: The triethylammonium salt of compound9 (89 mg,0.23 mmol) was dissolved in water (2 mL) and treated with Dowex D50(H⁺). Then, the resin was removed by filtration and thesolution of9 (free acid) was titrated with tributylamine(54 μL, 0.23 mmol, 1 equiv, to pH 7). The solution of9 was freeze-dried overnight to obtain a colorless film, compound9 (mono-tributylammonium salt, quant.).¹H NMR(400 MHz, H₂O, water suppression): δ 5.09 (s, 1H,H-1), 4.90 (d, 1H,J = 5.6 Hz, H-2), 4.65 (d, 1H,suppressed by H₂O, H-3), 4.43–4.35(m, 1H, H-4), 3.89–3.82 (m, 2H, H-5), 3.37 (s, 1H, OMe), 3.15–3.07(m, 6H, NCH₂), 1.69–1.59(m, 6H, NCH₂CH₂),1.49 (s, 3H, CH₃iPr), 1.40–1.30(m, 9H, N(CH₂)₂CH₃, CH₃iPr), 0.95–0.87 (m,9H, N(CH₂)₃CH₃).³¹P NMR (160 MHz, D₂O): δ 0.14(s, P).¹³C{¹H}NMR (100 MHz, D₂O):δ 113.2 (C(CH₃)), 108.5 (C-1), 86.4(C-3), 85.0 (d,J = 9.0 Hz, C-4, HSQC), 81.0 (C-2),65.2 (d,J = 7.0 Hz, C-5, HSQC), 54.8 (OMe), 52.8(NCH₂), 25.3 (CH₃iPr), 25.3 (NCH₂CH₂), 23.7 (CH₃iPr), 19.3 (N(CH₂)₂CH₂), 12.8(N(CH₂)₃CH₃).

2,3-O-Isopropylidene-5-O-trityl-d-ribofuranose (14)

2,3-O-Isopropylidene-D-ribofuranose13 (4 g, 21.1mmol) was dissolved in pyridine (10 mL). Trityl chloride (7.05 g,25.3 mmol) was added to the stirred solution, and the mixture wasstirred at 20 °C for 16 h. Water (30 mL) was added, and the mixturewas stirred additional 10 min. The aqueous phase was extracted withdichloromethane (3×). The combined organic phase was dried withMgSO₄, solids were removed by filtration, and the solventwas evaporated in vacuo. The crude product was purified by silicagel chromatography petroleum ether/EtOAc (1:0 → 0:1, v/v).This procedure afforded the title compound14 as a whiteamorphous solid (5.9 g, 65%).¹H NMR²⁵ (400 MHz, DMSO-d₆): δ7.42–7.32 and 7.30–7.24 (2 × m, 12 + 3H,o,m,p-Tr), 6.41 (d,1H,J = 4.0 Hz, OH), 5.15 (d, 1H,J = 4.0 Hz, H-1), 4.56 (dd, 1H,J = 6.0 Hz,J = 1.0 Hz, H-2), 4.36 (d, 1H,J = 6.0Hz, H-3), 4.11–4.05 (m, 1H, H-4), 3.16–3.05 (m, 2H,H-5), 1.37 and 1.23 (2 × s, 2 × 3H, CH₃iPr). HRMS (ES⁺) calcd for C₂₇H₂₈NaO₅, 455.1829 [M + Na]⁺; found, 455.1842.

2,3-O-Isopropylidene-5-O-trityl-d-ribitol (15)

2,3-O-Isopropylidene-5-O-trityl-d-ribofuranose14 (5.2 g,11.98 mmol) was dissolved in EtOH and cooled to 0 °C. Sodiumborohydride (460 mg, 12.1 mmol) was added to the solution in threeportions over the period of 30 min. The mixture was stirred at 20°C for 2 h. Water (80 mL) was added, and the solution was carefullyacidified with acetic acid to pH 6. Then, the aqueous phase was extractedwith dichloromethane (3×). The combined organic phase was dried(Na₂SO₄), solids were removed by filtration,and the solvent was evaporated in vacuo. The crude product was purifiedby Isco-Flash chromatography using CH₂Cl₂/acetone(1:0 → 1:1, v/v). This procedure afforded the title compound15 as an amorphous white solid (4.35 g, 83%).¹H NMR (400 MHz, DMSO-d₆): δ 7.48–7.41(m, 6H,o-Tr), 7.35–7.28 (m, 6H,m-Tr), 7.26–7.20 (m, 3H,p-Tr), 5.15 (d,J = 5.6 Hz, OH-4), 4.80 (t,J = 5.6 Hz,OH-1), 4.17–4.10 (m, 1H, H-2), 4.08–4.01 (m, 1H, H-3),3.78–3.66 (m, 2H, H-4 and H-1_a), 3.51–3.43(m, 1H, H-1_b), 3.15–3.11 (m, 1H, H-5_a), 3.06–3.01 (m, 1H, H-5_b), 1.20 (s, 6H, CH₃iPr).¹³C{¹H}NMR (100MHz, DMSO-d₆): δ 144.0 (3 ×C-ipso-Tr), 128.4 (6 × C-o-Tr),127.7 (6 × C-m-Tr), 126.8 (3 × C-p-Tr), 107.4 (C(CH₃)), 85.7 (C(Tr)), 78.2 (C-2), 76.4 (C-3), 68.2 (C-4), 66.1 (C-1), 60.0(C-5), 27.8 and 25.4 (CH₃iPr). HRMS (ES⁺) calcd for C₂₇H₃₀NaO₅, 457.1985[M + Na]⁺; found, 457.2004.

1-Deoxy-2,3-O-isopropylidene-5-O-trityl-d-ribofuranose(16)

2,3-O-Isopropylidene-5-O-trityl-d-ribitol15 (1.89 g, 4.35mmol) was dissolved in pyridine (15 mL) andtosyl chloride (2.49 g, 13.06 mmol) was added. The mixture was stirredat 60 °C for 16 h. TLC analysis showed that the reaction wascomplete. The tosyl chloride surplus was decomposed by addition ofwater (50 mL) and the aqueous phase was extracted with dichloromethane(3×). The combined organic phase was dried (Na₂SO₄), solids were removed by filtration, and the solvent wasevaporated in vacuo. The crude product was purified by Isco-Flashchromatography using petroleum ether/EtOAc (1:0 → 1:1, v/v),affording the title compound16 as an amorphous whitesolid (1.44 g, 80%).¹H NMR (400 MHz, CDCl₃):δ 7.44–7.39 (m, 6H,o-Tr), 7.33–7.27(m, 6H,m-Tr), 7.26–7.21 (m, 3H,p-Tr), 4.91–4.86 (m, 1H, H-2), 4.65 (dd, 1H,J = 6.4 Hz,J = 1.2 Hz, H-3), 4.24–4.19 (m,1H, H-4), 4.15 (dd, 1H,J = 10.4 Hz,J = 4.4 Hz, H-1_a), 4.05 (dd, 1H,J = 10.0Hz,J = 1.0 Hz, H-1_b), 3.27 (dd, 1H,J = 10.0 Hz, J = 4.0 Hz, H-5_a), 3.11 (dd, 1H,J = 10.0 Hz,J = 4.4 Hz, H-5_b), 1.51 and 1.34 (2 × s, 6H, CH₃iPr).¹³C{¹H}NMR (100 MHz, CDCl₃):δ 143.6 (3 × C-ipso-Tr), 128.6 (6 ×C-o-Tr), 127.8 (6 × C-m-Tr),127.2 (3 × C-p-Tr), 112.3 (C(CH₃)), 87.1 (C(Tr)), 84.0 (C-4), 83.0 (C-3), 81.6(C-2), 74.3 (C-1), 64.5 (C-5), 26.6 and 25.1 (CH₃iPr). HRMS (ES⁺) calcd for C₂₇H₂₈NaO₄, 439.1880 [M + Na]⁺; found, 439.1868.

1-Deoxy-2,3-O-isopropylidene-d-ribofuranose(17)

1-Deoxy-2,3-O-isopropylidene-5-O-trityl-d-ribofuranose16 (100 mg,0.24 mmol) was dissolved in diethylether–HCOOH (2 mL, 1:1,v/v) and stirred at 20 °C for 16 h. TLC analysis (petroleum ether/EtOAc,1:1, v/v) indicated the complete conversion of reaction and the mixturewas evaporated to dryness. The dry residue was suspended in waterand the suspension was filtered through a cotton pad. Evaporationof water afforded the title compound17 as a colorlessviscous liquid (14.5 mg, 32%).¹H NMR (400 MHz, CDCl₃): δ 4.83–4.78 (m, 1H, H-2), 4.59 (dd, 1H,J = 6.4 Hz,J = 2.0 Hz, H-3), 4.13–4.09(m, 1H, H-4), 3.98–3.95 (m, 2H, H-1_a,b), 3.65 (dd,1H,J = 11.6 Hz,J = 4.0 Hz, H-5_a), 3.58 (dd, 1H,J = 11.6 Hz,J = 6.4 Hz, H-5_b), 1.51 and 1.33 (2 × s, 6H, CH₃iPr).¹³C{¹H}NMR (100MHz, CDCl₃): δ 113.0 (C(CH₃)), 84.8 (C-4), 81.8 (C-3), 81.0 (C-2), 72.8 (C-1), 61.8 (C-5), 26.7and 25.0 (CH₃iPr). HRMS (ES⁺) calcd for C₈H₁₄NaO₄ 197.0784 [M+ Na]⁺; found 197.0791.

1-Deoxy-2,3-O-isopropylidene-d-ribofuranose-5-O-di-tert-butylphosphate (18)

1-Deoxy-2,3-O-isopropylidene-d-ribofuranose17 (94 mg, 0.49 mmol) and 5-phenyl-1H-tetrazole (145mg, 0.99 mmol) were co-evaporated withdry toluene (2×). Then, the dry mixture was dissolved in dichloromethane(2 mL), and di-tert-butylN,N-diisopropylphosphoramidite (234 μL, 0.74 mmol) wasadded dropwise. The mixture was stirred at 20 °C for 2 h afterwhich TLC analysis (petroleum ether/EtOAc, 2:1, v/v) showed the reactionwas complete. It was cooled to 0 °C and triethylamine (411 μL,2.97 mmol) was added, followed by hydrogen peroxide (35% aq, 109 μL,1.24 mmol). The resulting mixture was stirred at 20 °C for 1h. The mixture was diluted with EtOAc (20 mL) and extracted with aqueousNa₂SO₃ (10%, w/v). The organic phase was dried(Na₂SO₄), solids were filtered off, and thesolvent was evaporated in vacuo. The crude product was purified byIsco-Flash chromatography using petroleum ether/EtOAc (1:0 →0:1, v/v), to afford the title compound18 as a colorlessliquid (74 mg, 41%).¹H NMR (400 MHz, CDCl₃):δ 4.85–4.81 (m, 1H, H-2), 4.76 (dd, 1H,J = 6.4 Hz,J = 1.2 Hz, H-3), 4.23–4.18 (m,1H, H-4), 4.2–3.94 (m, 4H, H-1, H-5), 1.51 (s, 3H, CH₃iPr), 1.48 (s, 18H, CH₃tBu), 1.33 (s, 3H, CH₃iPr).³¹P NMR (160 MHz, CDCl₃): δ −10.1 (s, P).¹³C{¹H}NMR (100 MHz, CDCl₃): δ112.6 (C(CH₃)), 83.1 (d,J = 8.0 Hz,C(tBu)), 82.6 (d,J = 7.0 Hz, C-4), 82.3 (C-3), 81.2 (C-2), 73.9 (C-1), 66.6(d,J = 7.0 Hz, C-5), 29.8–29.7 (m, CH₃tBu), 26.5 and 24.9 (CH₃iPr)). HRMS (ES⁺) calcd for C₁₆H₃₁NaO₇P, 389.1700 [M + Na]⁺; found, 389.1699.

Tributylammonium 1-deoxy-2,3-O-isopropylidene-d-ribofuranose-5-phosphate (10)

1-Deoxy-2,3-O-isopropylidene-D-ribosfuranose-5-O-di-tert-butyl phosphate18 (36 mg,0.098 mmol) was dissolved in aqueous methanol (1:1, v/v, 2 mL), andthe solution was cooled to 0 °C. Trifluoroacetic acid (2 mL)was added dropwise and the solution was allowed to warm up to 20 °Cand stirred for an additional 4 h. The solution was evaporated todryness and the residue co-evaporated with water (3×) and withmethanol (3×). The procedure afforded the pure phosphate derivativeas a colorless gum (21 mg, quantitative) which was directly dissolvedin water (2 mL), neutralized with tributylamine (33 μL, 0.14mmol), the solvent evaporated, and the residue co-evaporated withEtOH (2×). The title compound10 was obtained asthe mono-tributylammonium salt (18.7 mg, 49%).¹H NMR (400MHz, MeOH): δ 4.21–4.16 (m, 1H, H-2), 4.12–4.07(m, 1H, H-3), 4.03–3.87 (m, 4H, H-1_a, 4, 5_a,b), 3.69 (dd, 1H,J = 9.6 Hz,J =4.0 Hz, H-1_b), 3.10–3.03 (m, 6H, NCH₂), 1.73–1.64 (m, 6H, NCH₂CH₂), 1.41 (m, 6H, N(CH₂)₂CH₂), 1.00 (t, 9H,J = 7.6 Hz, CH₃Bu).³¹P NMR (160 MHz, MeOH):δ 1.09 (s, P).¹³C{¹H}NMR (100 MHz, MeOH):δ 83.3 (d,J = 8.0 Hz, C-4), 73.7 (C-1), 73.6(C-3), 72.5(C-2), 66.3 (d,J = 5.0 Hz, C-5), 54.0(NCH₂), 27.0 (NCH₂CH₂), 21.0 (N(CH₂)₂CH₂), 14.0 (CH₃Bu). HRMS (ES^–) calcd for C₅H₁₁O₇P, 213.0170 [M– H]⁻; found, 213.0162.

2-Deoxy-d-ribofuranose-5-phosphate sodium Salt (19)

Compound19 (sodium salt) was purchasedfrom Sigma-Aldrich (UK) and found to be a mixture of α and βanomers in the ratio 1/1.17 α/β).¹H NMR (500MHz, D₂O): δ 5.52–5.47 (m, 1.6H, H-1α,β, deuterium exchange), 4.51–4.46 (m, 1H, H-3β),4.38–4.34 (m, 1H, H-3α), 4.25–4.20 (m, 1H, 4α),4.04–3.99 (m, 1H, H-4β), 3.85–3.81 (m, 2H, H-5β),3.81–3.75 (m, 2H, H-5α), 2.51–2.42 (m, 1H, H-2α),2.22–2.14 (m, 2H, H-2β), 1.91–1.83 (m, 1H, H-2α).³¹P NMR (202 MHz, D₂O): δ 3.65 (P-β)and 3.68 (P-α).¹³C{¹H}NMR (126 MHz, D₂O): δ 98.4 (C-1β), 98.0 (C-1α), 84.9 (d,J = 10.08 Hz, C-4β), 84.6 (d,J =8.82 Hz, C-4α), 71.6 (C-3β), 71.1 (C-3α), 64.6 (d,J = 5.0 Hz, C-5β), 63.7 (d,J = 3.8Hz, C-5α), 40.8 (C-2β), 40.6 (C-2α). Compound19 was transformed to its tributylammonium salt.

2-Deoxy-D-ribofuranose-5-phosphate Mono-tributylammoniumSalt (11)

Commercially available 2-deoxy-D-ribofuranose-5-phosphate sodium salt19 (Sigma-Aldrich)(8 mg, 0.029 mmol) was dissolved in water (2 mL) and acidified topH 1–2 by using the ion-exchange resin Dowex (D50 H⁺). The resin was filtered off and the free phosphate was neutralizedby addition of one equivalent of tributylamine (6.9 μL, 0.029mmol). The aqueous solution was freeze-dried (quant.). (NMR analysisshowed partial decomposition (approx. 20–30%), of 2′-deoxyribose.The compound itself is sensitive to basic conditions (pH 9)).¹H NMR (500 MHz, D₂O): δ 5.50–5.47(m, 0.54H, H-1β), 5.43–5.41 (dd, 0.46H,J = 5.5 Hz,J = 2.5 Hz, H-1α), 4.43–4.39(m, 0.53H, H-3β), 4.29–4.25 (m, 0.47H, H-3α), 4.15–4.1(m, 0.46H, H-4α), 4.02–3.97 (m, 0.7H, H-5β), 3.94–3.85(m, 1.32H, H-4β, 5α, 5β), 3.84–3.77 (m, 0.52H,H-5α), 3.09–3.01 (m, 10H, NCH₂(CH₂)₂CH₃), 2.38–2.30(m, 0.5H, H-2α), 2.12–1.99 (m, 1H, H-2β), 1.86–1.79(m, 0.5H, H-2α), 1.72–1.60 (m, 10H, NCH₂CH₂CH₂CH₃),1.47–1.35 (m, 10H, NCH₂CH₂CH₂CH₃), 1.04–0.94 (m, 15H,NCH₂(CH₂)₂CH₃).³¹P NMR (202 MHz, D₂O): δ1.10 (P-β) and 1.03 (P-α).¹³C{¹H}NMR (126 MHz, D₂O): δ 100.0 (C-1β), 99.5(C-1α), 86.3 (d,J = 8.4 Hz, C-4β), 85.6(d,J = 8.7 Hz, C-4α), 73.5 (C-3β), 73.0(C-3α), 67.3 (d,J = 5.4 Hz, C-5β), 66.1(d,J = 5.3 Hz, C-5α), 53.8 (NCH₂(CH₂)₂CH₃), 43.0 (C-2β), 42.6 (C-2α), 26.9 (NCH₂CH₂CH₂CH₃),21.1 (NCH₂CH₂CH₂CH₃), 14.0 (NCH₂(CH₂)₂CH₃).

1,2-O-Isopropylidene-d-xylofuranose(20)³⁴

Finely powderedd-xylose (10 g, 67 mmol) was suspended in acetone (260 mL) containingsulphuric acid (10 mL) and stirred for 30 min until it was dissolved.The solution was cooled to 0 °C, and a solution of Na₂CO₃ (13 g in 112 mL H₂O) was added. The mixturewas stirred at 20 °C for 1 h, and then solid Na₂CO₃ (7 g) was added. The slurry was stirred at 20 °C foran further 30 min. Solids were removed by filtration and washed withacetone. Acetone was evaporated, and the water phase was extractedwith EtOAc (3×) and dried with Na₂SO₄.Isco-Flash chromatography using petroleum ether/EtOAc (1:0 →1:1, v/v) afforded the title compound20 as a colorlessviscous oil (4.35 g, 34%) and doubly protected 1, 2-O-isopropylidene-3,5-O-isopropylidene-d-xylofuranose(3.29 g, 21%).³⁴¹H NMR (400MHz, CDCl₃): δ 5.98 (d, 1H,J =3.6 Hz, H-1), 4.52 (d, 1H,J = 3.6 Hz, H-2), 4.32(d, 1H,J = 2.4 Hz, H-4), 4.19–4.11 (m, 2H,H-3, 5_a), 4.05 (dd, 1H,J = 12.4 Hz,J = 2.4 Hz, H-5_b), 1.48 and 1.32 (CH₃iPr).¹³C{¹H}NMR (100 MHz,CDCl₃): δ 112.0 (C(CH₃)₂), 105.1 (C-1), 85.9 (C-2), 78.7 (C-3), 77.3 (C-4), 61.5 (C-5),26.9 and 26.3 (CH₃iPr). HRMS (ES⁺) calcd for C₈H₁₄NaO₅, 213.0733[M + Na]⁺; found, 213.0787.

5-O-tert-Butyl(diphenyl)silyl-1,2-O-isopropylidene-d-xylofuranose (21)

1,2-O-Isopropylidene-d-xylofuranose20 (3.63 g,19.08 mmol) and DMAP (5 mg) were dissolved inpyridine (15 mL).tert-Butyl(diphenyl)silyl chloride(4.89 mL, 19.08 mmol) was added dropwise, and the solution was stirredat 20 °C for 16 h. Solvent was evaporated and the crude productwas purified by Isco-Flash chromatography using petroleum ether/EtOAc(1:0 → 1:1, v/v). This procedure afforded the title compound21 as a white solid (6.58 g, 83%).¹H NMR (400MHz, CDCl₃): δ 7.74–7.65 (m, 4H,o-Ph), 7.48–7.38 (m, 6H,m,p-Ph), 6.00 (d, 1H,J = 3.6 Hz, H-1), 4.54 (d, 1H,J = 3.6 Hz, H-2), 4.39–4.35 (m, 1H, H-4), 4.16–4.12(m, 1H, H-3), 4.12–4.09 (m, 2H, H-5), 3.99 (d, 1H,J = 2.4 Hz, 3-OH), 1.47 and 1.33 (CH₃iPr), 1.06 (CH₃tBu).¹³C{¹H}NMR (100 MHz, CDCl₃): δ 135.8 and135.7 (o-Ph), 132.6 and 132.1 (C-ipso-Ph), 130.2 and 128.1 (2 × d,J = 2.2 Hz,J = 1.7 Hz,m,p-Ph),111.8 (C(CH₃)₂), 105.2 (C-1), 85.6(C-2), 78.6 (C-3), 77.0 (C-4, obstructed by CDCl₃ signal),62.9 (C-5), 27.0 and 26.3 (CH₃iPr), 26.9(CH₃tBu), 19.2 (C(CH₃)₃). HRMS (ES⁺) calcd for C₂₄H₃₂NaO₅Si, 451.1911 [M + Na]⁺; found,451.1929.

5-O-tert-Butyl(diphenyl)silyl-1,2-O-isopropylidene-3-thiocarbonylimidazolo-d-xylofuranose(22)

A mixture of 5-O-tert-butyl(diphenyl)silyl-1,2-O-isopropylidene-d-xylofuranose21 (3.0 g, 7.01 mmol) and 1,1′-thiocarbonyldiimidazole(2.37 g, 13.32 mmol) was gently refluxed in dichloroethane at 85 °Cfor 2 h. The solvent was evaporated, and the crude product was purifiedby Isco-Flash chromatography using petroleum ether/EtOAc (1:0 →0:1, v/v) to afford the title compound22 as a whitesolid (2.4 g, 63%).¹H NMR (400 MHz, CDCl₃):δ 8.24–8.21 (m, 1H, imidazole), 7.64–7.60 (m,2H, Ph), 7.55–7.51 (m, 2H, Ph), 7.47 (t, 1H,J = 1.6 Hz, imidazole), 7.46–7.33 (m, 4H, Ph), 7.29–7.23(m, 2H, Ph), 7.04 (q, 1H,J = 0.8 Hz, imidazole),5.98 (d, 1H,J = 2.8 Hz, H-3), 5.94 (d, 1H,J = 4.0 Hz, H-1), 4.72 (d, 1H,J = 4.0Hz, H-2), 4.61 (dq, 1H,J = 8.8 Hz,J = 2.8 Hz, H-4), 4.02 (dd, 1H,J = 10.0 Hz,J = 4.4 Hz, H-5_a), 3.82 (dd, 1H,J = 10.0 Hz,J = 8.4 Hz, H-5_b), 1.60 and1.36 (2 × s, 2 × 3H, CH₃iPr),1.02 (s, 9H, CH₃tBu).¹³C{¹H}NMR (100 MHz, CDCl₃): δ 182.6 (C=S),136.9 (imidazole), 135.6 and 135.4 (Ph), 132.8 and 132.7 (C-ipso-Ph), 131.2 (imidazole), 130.1 (Ph), 127.9 and 127.8(Ph), 117.8 (Imz), 112.8 (C(CH₃)₂), 105.0 (C-1), 84.2 (C-3), 82.7 (C-2), 78.9 (C-4), 60.3 (C-5), 26.9(CH₃tBu), 26.7 and 26.4 (CH₃iPr), 19.2 (C(CH₃)₃). HRMS (ES⁺) calcd for C₂₈H₃₄N₂NaO₅SSi, 561.1850 [M + Na]⁺; found,561.1837.

5-O-tert-Butyl(diphenyl)silyl-1,2-O-isopropylidene-3-deoxy-d-ribofuranose (23)

Tributyltin hydride(1.07 mL, 3.99 mmol) wasadded into boiling toluene (90 mL) under argon followed by AIBN (13mg, 0.08 mmol). Then, 5-O-tert-butyl(diphenyl)silyl-1,2-O-isopropylidene-3-thiocarbonylimidazolo-d-xylofuranose22 (1.26 g, 2.35 mmol) was added dropwise in toluene (20 mL).The mixture was refluxed for 3 h. Solvent was evaporated and the crudeproduct was purified by Isco-Flash chromatography using petroleumether/EtOAc (1:0 → 0:1, v/v) to afford the title compound23 as a white solid (731 mg, 76%).¹H NMR (400MHz, CDCl₃): δ 7.71 (m, 4H,o-Ph),7.45–7.35 (m, 6H,m,p-Ph),5.83 (d, 1H,J = 3.6 Hz, H-1), 4.75 (t, 1H,J = 4.0 Hz, H-2), 4.33 (dq, 1H,J = 10.4Hz,J = 4.0 Hz, H-4), 3.82 (dd, 1H,J = 11.2 Hz,J = 4.4 Hz, H-5_a), 3.76 (dd,1H,J = 11.2 Hz,J = 4.0 Hz, H-5_b), 3.08 (dd, 1H,J = 13.6 Hz,J = 4.8 Hz, H-3_a), 1.88 (ddd, 1H,J =13.6 Hz,J = 10.4 Hz,J = 4.8 Hz,H-3_b), 1.52 and 1.34 (2 × s, 2 × 3H, CH₃iPr), 1.06 (s, 9H, CH₃tBu).¹³C{¹H}NMR (100 MHz, CDCl₃):δ 135.8 and 135.7 (o-Ph), 133.7 and 133.5 (C-ipso-Ph), 129.8 and 129.8 (p-Ph), 127.8and 127.8 (m-Ph), 111.3 (C(CH₃)₂), 105.9 (C-1), 80.6 (C-2), 78.7 (C-4), 64.9 (C-5),35.1 (C-3), 27.1 (CH₃iPr), 27.0 (CH₃tBu), 26.5 (CH₃iPr), 19.4 (C(CH₃)₃). HRMS (ES⁺) calcd for C₂₄H₃₂NaO₄Si,435.1962 [M + Na]⁺; found, 435.1995.

1,2-O-Isopropylidene-3-deoxy-d-ribofuranose(24)

A mixture of TBAF·3H₂O(1.55 g, 5.0 mmol) and acetic acid (300 μL, 5.25 mmol) in DMF(10 mL) was stirred at room temperature for 30 min. The mixture wascooled to 0 °C and 5-O-tert-butyl(diphenyl)silyl-1,2-O-isopropylidene-3-deoxy-d-ribofuranose23 (687 mg, 1.67 mmol) was addeddropwise in DMF (5 mL). The resulting mixture was stirred at 20 °Cfor 3 h. Solvent was evaporated and the crude product was purifiedby Isco-Flash chromatography using petroleum ether/EtOAc (1:0 →0:1, v/v). This procedure afforded the title compound24 as white solid (260 mg, 82%).¹H NMR (400 MHz, CDCl₃): δ 5.79 (d, 1H,J = 3.6 Hz, H-1α),4.73 (t, 1H,J = 4.4 Hz, H-2), 4.31 (dtd, 1H,J = 10.8 Hz,J = 4.4 Hz,J = 3.2 Hz, H-4), 3.84 (dd, 1H,J = 12.0 Hz,J = 2.8 Hz, H-5_a), 3.54 (dd, 1H,J = 12.0 Hz,J = 4.4 Hz, H-5_b), 1.97 (dd,1H,J = 13.6 Hz,J = 4.8 Hz, H-3_a), 1.85–1.76 (m, 1H, H-3_b), 1.48 and 1.29(2 × s, 2 × 3H, CH₃iPr).¹³C{¹H}NMR (100 MHz, CDCl₃): δ111.4 (C(CH₃)₂), 105.7 (C-1α),80.9 (C-2), 78.7 (C-4), 63.1 (C-5), 34.0 (C-3), 26.9 and 26.3 (CH₃iPr). HRMS (ES⁺) calcd for C₈H₁₄NaO₄, 197.0784 [M + Na]⁺; found, 197.0793.

1,2-O-Isopropylidene-3-deoxy-d-ribofuranose-5-O-di-tert-butylphosphate (25)

1,2-O-Isopropylidene-3-deoxy-d-ribofuranose24 (100mg, 0.52 mmol) and 5-phenyl-1H-tetrazole (154 mg,1.05 mmol) were co-evaporated withdry toluene (2×). The solid mixture was then dissolved in dichloromethane(2 mL) and di-tert-butylN,N-diisopropylphosphoramidite (249 μL, 0.79 mmol) wasadded dropwise. The mixture was stirred at 20 °C for 2 h. TLCanalysis (petroleum ether/EtOAc, 2:1, v/v) showed complete phosphitylation.The reaction was cooled to 0 °C and triethylamine (437 μL,3.15 mmol) added followed by hydrogen peroxide (35% aq, 116 μL,1.32 mmol). The resulting mixture was stirred at 20 °C for 1h. The mixture was diluted with EtOAc (20 mL) and extracted with aqueousNa₂SO₃ (10%, w/v). The organic phase was dried(Na₂SO₄), solids were filtered off, and thesolvent was evaporated in vacuo. The crude product was purified byIsco-Flash chromatography using petroleum ether/EtOAc (1:0 →0:1, v/v), followed by a second chromatography in dichloromethane/acetone(1:0 → 1:1, v/v). This procedure afforded the title compound25 as a colorless viscous liquid (97 mg, 50%).¹H NMR (400 MHz, CDCl₃): δ 5.81 (d, 1H,J = 4.0 Hz, H-1), 4.73 (t, 1H,J = 4.4 Hz, H-2),4.04 (ddd, 1H,J = 8.8 Hz,J = 4.4Hz,J = 1.2 Hz, H-4), 4.12–4.06 (m, 1H, H-5a),4.02–3.96 (m, 1H, H-5_b), 2.1 (dd, 1H,J = 13.6 Hz,J = 4.8 Hz, H-3_a), 1.84–1.76(m, 1H, H-3_b), 1.50 (s, 3H, CH₃iPr), 1.48 (s, 9H, CH₃tBu) 1.31 (s, 3H,CH₃iPr).³¹P NMR (160 MHz,CDCl₃): δ −9.91 (s, phosphate).¹³C{¹H}NMR (100 MHz, CDCl₃): δ 111.4 (C(CH₃)₂), 105.8 (C-1), 82.7 and 82.6(2 × d,J = 2.1 Hz,C(CH₃)₃), 80.7 (C-2), 76.6 (d,J = 8.7 Hz,C-4), 67.0 (d,J = 6.0 Hz, C-5), 35.2 (C-3), 30.01and 29.97 (2 × CH₃tBu), 27.0 and26.4 (2 × CH₃iPr). HRMS (ES⁺) calcd for C₁₆H₃₂O₇P, 367.1880[M + H]⁺; found, 367.1877; calcd for C₁₆H₃₁NaO₇P, 389.1700 [M + Na]⁺; found, 389.1737.

1,2-O-Isopropylidene-3-deoxy-d-ribofuranose-5-O-dibenzylphosphate (26)

1,2-O-Isopropylidene-3-deoxy-d-ribofuranose24 (120 mg, 0.63 mmol) and 5-phenyl-1H-tetrazole (185mg, 1.26 mmol) were co-evaporated with dry toluene (2×). Then,the solid mixture was dissolved in dichloromethane (3 mL) and dibenzylN,N-diisopropylphosphoramidite (353 μL,0.95 mmol) was added dropwise. The mixture was stirred at 20 °Cfor 2 h. TLC analysis (petroleum ether/EtOAc, 2/1, v/v) showed completephosphitylation. The reaction was cooled to 0 °C and triethylamine(524 μL, 3.79 mmol) added followed by hydrogen peroxide (35%aq, 139 μL, 1.58 mmol). The resulting mixture was stirred at20 °C for 1 h. The mixture was diluted with EtOAc (20 mL) andextracted with Na₂SO₃ (10% aq, w/v). The organicphase was dried (Na₂SO₄), solids were removedby filtration, and the solvent was evaporated in vacuo. The crudeproduct was purified by Isco-Flash chromatography using dichloromethane/acetone(1:0 → 1:1, v/v) followed by a second chromatography in petroleumether/EtOAc (1:0 → 0:1, v/v) to afford the title compound26 as a colorless viscous liquid (218 mg, 73%).¹H NMR (400 MHz, CDCl₃): δ 7.37–7.32 (m, 10H,Bn), 5.76 (d,J = 3.6 Hz, H-1), 5.10–5.00(m, 4H,CH₂Bn), 4.68 (t, 1H,J = 4.4 Hz, H-2), 4.35 (ddd, 1H,J = 8.8Hz,J = 4.4 Hz,J = 0.8 Hz, H-4),4.16 (dq, 1H,J = 3.6 Hz,J = 2.8Hz, H-5_a), 4.00 (dq, 1H,J = 4.4 Hz,J = 2.8 Hz, H-5_b), 2.00 (dd, 1H,J = 13.2 Hz,J = 4.4 Hz, H-3_a), 1.72–1.64(m, 1H, H-3_b), 1.48 and 1.31 (2 × s, 2 × 3H,CH₃iPr).³¹P NMR (160 MHz,CDCl₃): δ −1.00 (s, phosphate).¹³C{¹H}NMR (100 MHz, CDCl₃): δ 136.0 (d,J = 2.0 Hz, C-ipso-Bn), 135.9 (d,J = 3.0 Hz, C-ipso-Bn), 128.8–128.7and 128.2–128.1 (2 × m, C-Bn-o,m,p), 111.5 (C(CH₃)₂), 105.8 (C-1), 80.5 (C-2), 76.4 (d,J = 8.0 Hz, C-4), 69.6 and 69.5 (2 × d,J =1.3 Hz andJ = 1.4 Hz,CH₂Bn), 68.8 (d,J = 5.6 Hz, C-5), 34.7(C-3), 27.0 and 26.3 (CH₃iPr). HRMS (ES⁺) calcd for C₂₂H₂₈O₇P, 435.1567 [M + H]⁺; found, 435.1571;calcd for C₂₂H₂₇NaO₇P, 457.1387 [M+ Na]⁺; found, 457.1392.

3-Deoxy-d-ribofuranose-5-O-dibenzylphosphate(27)

An aqueous solution (4 mL) of 1, 2-O-isopropylidene-3-deoxy-d-ribofuranose-5-O-dibenzylphosphate26 (67 mg, 0.154 mmol)was cooled to 0 °C and trifluoroacetic acid (4 mL) was added.The solution was stirred at 0 °C for 1.5 h and then allowed towarm to 20 °C. The solvent was evaporated in vacuo at 30 °Cand co-evaporated with water. Crude material was purified by silicagel chromatography using petroleum ether/EtOAc (1:0 → 0:1,v/v) to give a colorless solid (mixture of α/β anomers1:4, 41 mg, 68%).¹H NMR (400 MHz, CD₃OD): δ7.39–7.35 (m, 10H, Bn), 5.18 (s, 0.2H, C-1α), 5.17 (s,0.8H, C-1β), 5.09–5.04 (m, 4H,CH₂Bn), 4.45–4.39 (m, 0.8H, H-4β), 4.39–4.35(m, 0.2H, H-4α), 4.13–4.07 (m, 1H, H-2α, β),4.06–3.99 (m, 1.8H, H-5α_a, β_a,b), 3.90 (ddd, 0.2H,J = 12.8 Hz,J = 5.6 Hz,J = 4.0 Hz, H-5α_b),1.96–1.85 (m, 2H, H-3α, β).³¹P NMR(160 MHz, CD₃OD): δ −1.30 (s, α-phosphate),−1.54 (s, β-phosphate).¹³C{¹H}NMR(100 MHz, CD₃OD): δ 137.2 and 137.2 (2 × C-ipso-Bn), 129.8–129.7 and 129.3–129.2 (2 ×m, 10 × C-Bn-o,m,p), 104.3 (C-1β), 98.4 (C-1α), 78.6 (d,J = 6.2 Hz, C-4β), 77.1 (C-2β), 75.8 (d,J = 6.2 Hz, C-4α), 72.4 (d,J = 4.8Hz, C-5β), 72.2 (C-2α), 71.0 (d,J =4.8 Hz, C-5α), 70.9–70.8 (m,CH₂Bn), 34.7 (C-3β), 33.9 (C-3α). HRMS (ES⁺) calcd for C₁₉H₂₄O₇P, 395.1254[M + H]⁺; found, 395.1256; calcd for C₁₉H₂₃NaO₇P, 417.1074 [M + Na]⁺; found, 417.1079.

3-Deoxy-d-ribofuranose-5-phosphate TributylammoniumSalt (12)

3-Deoxy-d-ribofuranose-5-O-dibenzylphosphate27 (39 mg, 0.098 mmol)was dissolved in MeOH–water (8 mL, 17:3 v/v). After vacuum/argondeoxygenation of the reaction mixture, palladium on charcoal (Pd/C10%, 10 mg) was added. The reaction was stirred under positive pressureof hydrogen atmosphere (balloon) at 20 °C for 4 h. Then, thereaction was flushed with argon and solids were filtered off and washedwith water. The aqueous solution (pH 1–2) was carefully titratedby portionwise addition of tributylamine (5 × 4 μL, 0.084mmol, pH 6–7). The solvent was evaporated, and the crude productwas co-evaporated with dry toluene (2×) and dried under highvacuum for 16 h to give a colorless solid film, compound12 (mixture of α/β anomers 1:4, 1 × Bu₃N salt, 29 mg, 67%).¹H NMR (500 MHz, CD₃OD):δ 5.20 (d, 0.2H,J = 4.5 Hz, H-1α), 5.12(s, 0.8H, H-1β), 4.48–4.41 (m, 0.8H, H-4β), 4.41–4.36(m, 0.2H, H-4α), 4.25–4.20 (m, 0.2H, H-2α), 4.08(d, 0.8H,J = 4.5 Hz, H-2β), 3.96–3.84(m, 1.6H, H-5β), 3.83–3.79 (m, 0.4H, H-5α), 3.14–3.08(m, 6H, NCH₂), 2.12 (ddd, 0.2H,J = 12.5Hz,J = 5.5 Hz,J = 2.5 Hz, H-3α_a), 2.04 (ddd, 0.8H,J = 13.5 Hz,J = 8.5 Hz,J = 5.0 Hz, H-3β_a), 2.01–1.97 (m, 0.2H, H-3α_b), 0.92(dd, 0.8H,J = 14.0 Hz,J = 7.0Hz, H-3β_a), 1.73–1.64 (m, 6H, NCH₂CH₂), 1.47–1.38 (m, 6H,N(CH₂)₂CH₂), 10.14 (t, 9H,J = 7.5 Hz, N(CH₂)₃CH₃).³¹PNMR (202 MHz, CD₃OD): δ 1.04 (s, α-phosphate),0.97 (s, β-phosphate).¹³C{¹H}NMR (126MHz, CD₃OD): δ 104.3 (C-1β), 98.2 (C-1α),79.7 (d,J = 8.8 Hz, C-4β), 77.4 (C-2β),76.8 (d,J = 8.8 Hz, C-4α), 72.3 (C-2α),69.7 (d,J = 5.03 Hz, C-5β), 68.4 (d,J = 5.0 Hz, C-5α), 53.8 (NCH₂), 35.3 (C-3β), 34.3 (C-3α), 26.8 (NCH₂CH₂), 21.0 (N(CH₂)₂CH₂), 14.0(N(CH₂)₃CH₃). HRMS (ES^–) calcd for C₅H₁₀O₇P, 213.0170 [M – H]⁻; found,213.0159.

AMP Imidazolide: Adenosine-5′-phosphorylImidazolide(30)

AMP sodium salt (50 mg, 0.128 mmol, Sigma-Aldrich)was dissolved in water (2 mL) and treated with Dowex D50 (H⁺) until all the phosphate was transformed to free acid (pH 1–2).Then, the resin was filtered off and washed with water, and the solutionof AMP (free acid) was titrated with tributylamine (30 μL, 0.13mmol, 1 equiv, pH 7). The solution of adenosine-5′-phosphatemono-tributylammonium salt (28) was freeze-dried to obtaina light white powder (quantitative).

AMP tributylammonium salt(15 mg, 0.028 mmol) and imidazole (19 mg, 0.28 mmol) were co-evaporatedwith EtOH (2×) and with toluene (2×). Aldrithiol (18.6 mg,0.085 mmol) was added followed by DMF (220 μL), and the suspensionstirred until all solid matter was dissolved. Triethylamine (16 μL,0.113 mmol, distilled-dry-stored over KOH in the dark) and triphenylphosphine(22.2 mg, 0.085 mmol) were added, and the solution was stirred at20 °C for 16 h. Product precipitation: The reaction mixture wascooled to 5 °C and a cold solution of NaI (33.8 mg) in dry acetone(2.2 mL) was added at 5 °C. The white precipitate was filteredoff and washed with cold acetone. The product was dried under highvacuum for 16 h and stored under argon (crude compound, hygroscopic,yield was not quantified).¹H NMR (400 MHz, CD₃OD): δ 8.38 (s, H-8), 8.19 (s, H-2), 7.88–7.87 (m, 1H,imidazole), 7.28–7.26 (m, 1H, imidazole), 6.99–6.97(m, 1H, imidiazole), 6.04 (d, 1H,J = 6.0 Hz, H-1′),4.65 (t, 1H,J = 5.6 Hz, H-2′), 4.25 (dd,1H,J = 5.2 Hz,J = 3.6 Hz, H-3′),4.18–4.13 (m, 1H, H-4′), 4.06–3.96 (m, 2H, H-5_a,b^′).³¹P NMR (160 MHz, CD₃OD): δ −9.03 (s,P).¹³C{¹H}NMR (100 MHz, CD₃OD):δ 157.4 (C-6), 153.9 (C-2), 150.9 (C-4), 140.9 (C-8), 140.8(d,J = 5.5 Hz, imidazole), 129.7 (d,J = 10.4 Hz, imidazole), 121.3 (d,J = 5.7 Hz, imidazole),120.3 (C-5), 89.2 (C-1′), 85.3 (d,J = 8.8Hz, C-4′), 75.8 (C-2′), 72.19 (C-3′), 66.8 (d,J = 5.9 Hz, C-5′). HRMS (ES^–) calcdfor C₁₃H₁₅N₇O₆P, 396.0827[M – H]⁻; found, 396.0853.

1″-β-O-Methyl-2″,3″-O-isopropylideneadenosine diphosphoriboside (1)

Compound9 (4.3 mg, 0.009 mmol) and MgCl₂ (1.8 mg, 0.018mmol) were dissolved in DMF (1 mL), co-evaporatedwith dry toluene (2×) and evaporated to dryness. Dry DMF (500μL) was added to the solid residue and the mixture was stirredat 20 °C until all solids were dissolved (5–10 min). Then,imidazolide30 (3.5 mg, 0.008 mmol) was added dropwiseto the mixture in DMF (400 μL). The mixture was stirred at 20°C for 3 h, after which conversion to product was 80–90%by HPLC. Solvent was evaporated in vacuo and crude product was dissolvedin TEAB buffer (3 mL, 0.1 M) and purified by semi-preparative, reverse-phaseHPLC using a gradient of TEAB (0.1 M)–acetonitrile (95:5 →35:65, v/v). The title compound1 was obtained as a colorlessglass, 1.7 × Et₃N salt, (2 μmol, 1.57 mg, 24%).¹H NMR (500 MHz, D₂O): δ 8.48 (s, 1H, H-8),8.20 (s, 1H, H-2), 6.06 (d, 1H,J = 5.0 Hz, H-1′),4.89 (s, 1H, H-1″), 4.70–4.63 (m, 2H, H-2′, 2″),4.50 (d, 1H,J = 5.0 Hz, H-3″), 4.45 (t, 1H,J = 5.0 Hz, H-3′), 4.33–4.28 (m, 1H, H-4′),4.21–4.11 (m, 3H, H-4″, 5_a,b^′), 3.82–3.75 (m, 2H, H-5_a,b), 3.21(s, 3H, OMe), 3.12 (m, 9H, NCH₂CH₃), 1.31 (s, 3H, CH₃-iPr),1.19 (m, 16.5H, NCH₂CH₃, CH₃-iPr).³¹P NMR (202 MHz,D₂O): δ −11.47 (m, pyrophosphate).¹³C{¹H}NMR (126 MHz, D₂O): δ 155.1 (C-6),152.0 (C-2), 149.1 (C-4), 140.0 (C-8), 118.3 (C-5), 112.9 (C(CH₃)₂), 108.4 (C-1″), 87.0 (C-1′),84.7 (d,J = 10.1 Hz, C-4″), 84.1 (C-3″),83.9 (d,J = 6.3 Hz, C-4′), 80.9 (C-2″),74.3 (C-2′), 70.3 (C-3′), 65.8 (d,J = 3.8 Hz, C-5″), 65.1 (d,J = 6.3 Hz, C-5′),54.7 (OMe), 46.6 (NCH₂CH₃), 25.1 and 23.5 (CH₃-iPr), 8.2(NCH₂CH₃). HRMS (ES^–) calcd for C₁₉H₂₈N₅O₁₄P₂, 612.1114 [M – H]⁻; found, 612.1143. UV (H₂O, pH 7.4) λ_max 259 nm (ε 16 900 L/mol·cm). HPLCt_R = 5.52 min.

1″-Deoxyadenosine diphosphoriboside(1″-deoxy-ADPR)(2)

1-Deoxy-d-ribofuranose-5-phosphatetributylammonium salt10 (5.8 mg, 0.0145 mmol) and MgCl₂ (2.8 mg, 0.029 mmol) were dissolved in DMF (1 mL), co-evaporatedwith dry toluene (2×) and evaporated to dryness. Dry DMF (500μL) was added to the solid residue and the mixture was stirredat 20 °C until all solids were dissolved (5–10 min). Then,AMP-imidazolide30 (5.5 mg, 0.013 mmol) was added dropwiseto the mixture in DMF (400 μL). The mixture was stirred at 20°C for 3 h. Solvent was evaporated in vacuo and the crude productwas dissolved in TEAB buffer (3 mL, 0.1 M) and purified by semi-preparative,reverse-phase HPLC using a gradient of TEAB (0.1 M)–acetonitrile(95:5 → 35:65, v/v). The title compound2 wasobtained as a colorless glass, 1.7 × Et₃N salt, (9.6μmol, 6.86 mg, 66%).¹H NMR (500 MHz, D₂O): δ 8.54 (s, 1H, H-8), 8.26 (s, 1H, H-2), 6.14 (d, 1H,J = 5.0 Hz, H-1′), 4.79–4.76 (m, 1H, H-2′,obstructed by H₂O peak), 4.56–4.52 (m, 1H, H-3′),4.42–4.38 (m, 1H, H-4′), 4.26–4.21 (m, 3H, H-5_a,b, H-2″), 4.17–4.14 (m, 1H, H-3″), 4.13–4.07(m, 1H, H-5″_a), 4.03–3.92 (m, 3H, H-1″_a, 4″, 5″_b), 3.74 (dd, 1H,J = 8.5 Hz,J = 3.5 Hz, H-1_b^″), 3.10(m, 10H, NCH₂CH₃),1.18 (m, 15H, NCH₂CH₃).³¹P NMR (202 MHz, D₂O): δ −11.35(m, pyrophosphate).¹³C{¹H}NMR (126 MHz, D₂O): δ 155.2 (C-6), 152.3 (C-2), 149.1 (C-4) HMBC, 140.0(C-8), (C-5) not observed (HMBC), 86.8 (C-1′), 83.9 (d,J = 7.6 Hz, C-4′), 80.3 (d,J =7.6 Hz, C-4″), 74.2 (C-2′), 72.2 (C-5_a,b), 71.6 (C-3″),70.9 (C-2″), 70.4 (C-3′), 65.5 (d,J = 3.8 Hz, C-5_a,b^″), 65.2 (d,J = 3.8 Hz, C-5′),46.6 (NCH₂CH₃), 8.2(NCH₂CH₃). HRMS (ES^–) calcd for C₁₅H₂₂N₅O₁₃P₂, 542.0695 [M – H]⁻; found, 542.0716. UV (H₂O, pH 7.4) λ_max 259 nm (ε 15 958 L/mol·cm). HPLCt_R = 2.93 min.

1″-α/β-2″-Deoxyadenosinediphosphoriboside(2″-deoxy-ADPR) (3)

2-Deoxyribose-5-phosphatetributylammonium salt11 (8 mg, 0.029 mmol) and MgCl₂ (5.6 mg, 0.058 mmol) were dissolved in DMF (1.5 mL), co-evaporatedwith dry toluene (2×), and evaporated to dryness. Dry DMF (500μL) was added to the solid residue and the mixture was stirredat 20 °C until all solids dissolved (5–10 min). Then,AMP-imidazolide30 (9 mg, 0.0215 mmol) was added dropwiseto the mixture in DMF (700 μL). The mixture was stirred at 20°C for 16 h (HPLC showed a complex reaction mixture comprising3–4 products, including AMP and AMP dinucleotide). The solventwas evaporated in vacuo and the crude product was dissolved in TEABbuffer (3 mL, 0.1 M) and purified by semi-preparative, reverse-phaseHPLC using a gradient of TEAB (0.1 M)–acetonitrile (95:5 →35:65, v/v). The title compound was obtained as a colorless glassas a mixture of anomers β/α 0.45:0.55, 1.7 × Et₃N salt, (1.72 μmol, 1.23 mg, 8%).¹H NMR(500 MHz, D₂O): δ 8.43 (s, 1H, H-8), 8.19 (s, 1H,H-2), 6.06 (d, 1H,J = 10.0 Hz, H-1′), 5.49–5.46(m, 0.45H, H-1″β), 5.42–5.39 (m, 0.55H, H-1″α),4.70–4.68 (m, 1H, H-2′, obstructed by H₂Opeak), 4.47–4.43 (m, 1H, H-3′), 4.37–4.33 (m,0.45H, H-3″β), 4.33–4.29 (m, 1H, H-4′),4.23–4.19 (m, 0.55H, H-3″α), 4.16–4.10(m, 2.45H, H-4″α and H-5′), 3.93–3.89 (m,1.45H, H-4″β and H-5″β), 3.87–3.83(m, 1H, H-5″α), 3.12 (q, 10H,J = 7.5Hz, NCH₂CH₃), 2.30–2.23(m, 0.55H, H-2_a^″-α), 2.06–2.01 (m, 0.9H, H-2_a,b-β), 1.77–1.70 (m,0.55H, H-2_b^″-α), 1.19 (t, 15H,J = 7.5 Hz, NCH₂CH₃).³¹P NMR (202MHz, D₂O): δ −11.34 (m, pyrophosphate).¹³C{¹H}NMR (126 MHz, D₂O): δ 155.7(C-6), 152.9 (C-2), 149.1 (C-4), 139.8 (C-8), 118.6 (C-5), 98.4 (C-1″β),98.1 (C-1″α), 86.7 (C-1′, 84.0 and 83.9 (m, C-4′,4″α, 4″β), 74.2 (C-2′), 71.4 (C-3″β),71.0 (C-3″α), 70.4 (C-3′), 66.4 (d,J = 3.8 Hz, C-5″β), 65.6 (d,J = 2.5Hz, C-5″α), 65.2 (d,J = 3.8 Hz, C-5′),46.6 (NCH₂CH₃), 40.6 (C-2″α,β), 8.3 (NCH₂CH₃). HRMS (ES^–) calcd for C₁₅H₂₂N₅O₁₃P₂, 542.0695 [M – H]⁻; found 542.0690; (ES⁺) calcd for C₁₅H₂₂N₅NaO₁₃P₂,565.0587 [M + Na]⁺; found, 565.0579. HPLCt_R = 3.10 min.

1″-α/β-3″-Deoxyadenosinediphosphoriboside(3″-deoxy-ADPR) (4)

3-Deoxy-d-ribofuranose-5-phosphate tributylammonium salt12 (10mg, 0.025 mmol) and MgCl₂ (4.9 mg, 0.05 mmol) were dissolvedin DMF (1 mL), co-evaporated with dry toluene (2×), and evaporatedto dryness. Dry DMF (600 μL) was added to the solid residue,and the mixture was stirred at 20 °C until all solids were dissolved(5–10 min). Then, AMP-imidazolide30 (9.4 mg,0.023 mmol) was added dropwise to the mixture in DMF (400 μL).The mixture was stirred at 20 °C for 16 h. Solvent was evaporatedin vacuo, and the crude product was dissolved in TEAB buffer (3 mL,0.1 M) and purified by semi-preparative, reverse-phase HPLC usinga gradient of TEAB (0.1 M)–acetonitrile (95:5 → 35:65,v/v). The title compound4 was obtained as a colorlessglass, 1.7 × Et₃N salt, (3.88 μmol, 2.8 mg,17%).¹H NMR (500 MHz, D₂O, water suppressionexp.): δ 8.37 (s, 1H, H-8), 8.11 (s, 1H, H-2), 5.98 (d, 1H,J = 6.0 Hz, H-1′), 5.10 (d, 0.3H,J = 4.0 Hz, H-1″α), 5.06 (s, 0.7H, H-1″β),4.63 (t, 1H,J = 5.5 Hz, H-2′), 4.40–4.37(m, 1H, H-3′), 4.34–4.27 (m, 1H, H-4″α,β), 4.26–4.22 (m, 1H, H-4′), 4.12–4.04(m, 2.3H, H-2″α, 5_a,b^′), 4.04–4.01 (m, 0.7H, H-2″β),3.91–3.85 (m, 0.7H, H-5″β_a), 3.81–3.71(m, 1H, H-5″α_a, β_b), 3.69–3.63(m, 0.3H, H-5″α_b), 3.04 (q, 10H,J = 7.5 Hz, NCH₂), 1.90 (ddd,0.3H,J = 13.0 Hz,J = 7.5 Hz,J = 2.0 Hz, H-3″α_a), 1.84–1.78(m, 1.7H, H-3″α_b, β_a,b),1.12 (t, 15H,J = 7.5 Hz, NCH₂CH₃).³¹P NMR (202 MHz, D₂O): δ −11.17 (m, α-pyrophosphate), −11.46(m, β-pyrophosphate).¹³C{¹H}NMR (126MHz, D₂O): δ 155.1 (C-6), 152.1 (C-2), 149.0 (C-4),139.9 (C-8), 118.5 (C-5), 102.0 (C-1″β), 96.5 (C-1″α),86.7 (C-1′), 83.9 (d,J = 8.7 Hz, C-4′),78.2 (d,J = 8.4 Hz, C-4″β), 75.5 (d,J = 8.4 Hz, C-4″α), 75.3 (C-2″β),74.2 (C-2′), 70.3 (C-3′), 70.3 (C-2″α),68.8 (d,J = 5.2 Hz, C-5″β), 67.8 (d,J = 4.8 Hz, C-5″α), 65.1 (d,J = 5.0 Hz, C-5′), 46.5 (NCH₂), 32.3 (C-3″β), 31.5 (C-3″α), 8.1(NCH₂CH₃). HRMS (ES^–) calcd for C₁₅H₂₂N₅O₁₃P₂, 542.0695 [M – H]⁻; 542.0716. HPLCt_R = 2.87 min.

2′-DeoxyAMP Imidazolide: 2′-Deoxy-adenosine-5′-phosphorylImidazolide (31)

2′-Deoxy-AMP was treatedin the same way as AMP to generate the mono Bu₃N salt (29).

2′-Deoxy-AMP mono-tributylammonium salt29 (145 mg, 0.28 mmol) and imidazole (191 mg, 2.8 mmol) wereput into the flask and co-evaporated with EtOH (2×) and withtoluene (2×). Aldrithiol (186 mg, 0.84 mmol) was added to theflask followed by dry DMF (1.5 mL) and stirred until all solid matterwas dissolved. Triethylamine (156 μL, 1.13 mmol, distilled-dry-storedover KOH in the dark) and triphenylphosphine (221 mg, 0.85 mmol) wereadded to the mixture, and the mixture was stirred at 20 °C for16 h. The reaction mixture was cooled to 5 °C, and a cold solutionof NaI (337 mg, 2.25 mmol) in dry acetone (15 mL) was added at 5 °C.The white precipitate was filtered off and washed with cold acetone.The product was dried under high vacuum and stored under argon (crudecompound, hygroscopic, yield was not quantified).¹H NMR(400 MHz, CD₃OD): δ 8.36 (s, H-8), 8.18 (s, H-2),7.87–7.86 (m, 1H, imidazole), 7.26–7.24 (m, 1H, imidazole),6.98–6.96 (m, 1H, imidazole), 6.44 (dd, 1H,J = 7.6 Hz,J = 1.2 Hz, H-1′), 4.49 (q, 1H,J = 2.8 Hz, H-3′), 4.08–4.04 (m, 1H, H-4′),3.99–3.95 (m, 2H, H-5′), 2.75 (ddd, 1H,J = 13.6 Hz,J = 7.6 Hz,J = 1.6Hz, H-2_a^′), 2.42 (dq, 1H,J = 13.6 Hz,J = 2.8 Hz, H-2_b).³¹P NMR (160 MHz, CD₃OD): δ −9.00(s, P).¹³C{¹H}NMR (100 MHz, CD₃OD):δ 157.3 (C-6), 153.8 (C-2), 150.5 (C-4), 140.9 (C-8), 140.7(d,J = 5.5 Hz, imidazole), 129.6 (d,J = 10.5 Hz, imidazole), 121.3 (d,J = 5.7 Hz, imidazole),120.3 (C-5), 87.5 (d,J = 8.7 Hz, C-4′), 85.5(C-1′), 72.9 (C-3′), 67.0 (d,J = 5.9Hz, C-5′), 41.2 (C-2′). HRMS (ES^–)calcd for C₁₃H₁₅N₇O₅P,380.0878 [M – H]⁻; found, 380.0883.

1″,2′-Dideoxyadenosinediphosphoriboside (1″,2′-deoxyADPR) (5)

1-Deoxy-d-ribofuranose-5-phosphatetributylammonium salt10 (12 mg, 0.03 mmol) and MgCl₂ (5.9 mg, 0.06 mmol) were dissolved in DMF (1 mL), co-evaporatedwith dry toluene (2×), and evaporated to dryness. Dry DMF (600μL) was added to the solid residue and the suspension was stirredat 20 °C until all solids were dissolved (5–10 min). Then,2′-dAMP-imidazolide31 (10.8 mg, 0.027 mmol) wasadded dropwise in DMF (400 μL). The mixture was stirred at 20°C for 3 h. HPLC showed consumption of all starting material.The solvent was evaporated in vacuo, and the crude product was dissolvedin TEAB buffer (10 mL, 0.1 M) and purified by semi-preparative, reverse-phaseHPLC using a gradient of TEAB (0.1 M)–acetonitrile (95/5 →35/65, v/v). The title compound5 was obtained as a colorlessamorphous solid (11.3 μmol, 8.02 mg, 42%, 1.8 × Et₃N salt).¹H NMR (500 MHz, D₂O): δ8.47 (s, 1H, H-8), 8.23 (s, 1H, H-2), 6.50 (t, 1H,J = 5.0 Hz, H-1′), 4.79–4.74 (m, 1H, H-3′, obstructedby water peak), 4.31–4.28 (m, 1H, H-4′), 4.25–4.21(m, 1H, H-2″), 4.19–4.11 (m, 3H, H-3″, 5_a,b^′), 4.10–4.05(m, 1H, H-5_a), 4.02–3.92 (m, 3H, H-1_a^″, 4″, 5_b), 3.74 (dd, 1H,J = 10.0 Hz,J = 2.5 Hz, H-1_b^″), 3.19 (q, 10H,J = 7.5 Hz, NCH₂CH₃), 2.83 (quint, 1H,J = 7.0 Hz, H-2_a), 2.59(dq, 1H,J = 14.0 Hz,J = 3.0 Hz,H-2_b^′), 1.27 (t, 15H,J = 7.0 Hz, NCH₂CH₃).³¹P NMR (202 MHz,D₂O): δ −11.33 (m, pyrophosphate).¹³C{¹H}NMR (126 MHz, D₂O): δ 155.5 (C-6),152.7 (C-2), 148.7 (C-4), 139.9 (C-8), 118.6 (C-5), 85.7 (d,J = 8.6 Hz, C-4′), 83.6 (C-1′), 80.3 (d,J = 8.4 Hz, C-4″), 72.2 (C-1″), 71.5 (C-3″),71.2 (C-3′), 70.9 (C-2″), 65.5 and 65.4 (2 × d,J = 5.3 Hz,J = 4.7 Hz, C-5′, 5″),64.6 (NCH₂CH₃), 39.0(C-2′), 8.2 (NCH₂CH₃). HRMS (ES^–) calcd for C₁₅H₂₂N₅O₁₂P₂, 526.0746 [M –H]⁻; found, 526.0768. HPLCt_R = 3.7 min.

1″-α/β-3″,2′-Dideoxyadenosinediphosphoriboside (3″,2′-dideoxy-ADPR) (6)

3-Deoxy-d-ribofuranose-5-phosphate tributylammoniumsalt12 (12 mg, 0.03 mmol) and MgCl₂ (5.9mg, 0.06 mmol) were dissolved in DMF (1 mL), co-evaporated with drytoluene (2×), and evaporated to dryness. Dry DMF (600 μL)was added to the solid residue, and the mixture was stirred at 20°C until all solids were dissolved (5–10 min). Then, 2′-deoxy-AMP-imidazolide31 (10.8 mg, 0.027 mmol) was added dropwise in DMF (400 μL).The mixture was stirred at 20 °C for 16 h. HPLC showed consumptionof all starting material. The solvent was evaporated in vacuo andthe crude product was dissolved in TEAB buffer (10 mL, 0.1 M) andpurified by semi-preparative, reverse-phase HPLC using a gradientof TEAB (0.1 M)–acetonitrile (95/5 → 35/65, v/v). Thetitle compound6 was obtained as a colorless amorphoussolid (7.5 μmol, 5.32 mg, 27%, 1.8 × Et₃N salt).¹H NMR (500 MHz, D₂O): δ 8.48 (s, 1H, H-8),8.24 (s, 1H, H-2), 6.51 (t, 1H,J = 7.0 Hz, H-1′),5.26 (d, 0.3H,J = 4.0 Hz, H-1″α), 5.21(s, 0.7H, H-1″β), 4.78–4.75 (m, 1H, H-3′,obstructed by water signal), 4.49–4.39 (m, 1H, H-4″α,β), 4.31–4.23 (m, 1.3H, H-2″α, 4′),4.20–4.11 (m, 2.7H, H-2″β, 5_a,b^′), 4.05–3.98 (m,0.7H, H-5″β_a), 3.94–3.84 (m, 1H, H-5″α_a, β_b), 3.82–3.76 (m, 0.3H, H-5″α_b), 3.19 (q, 11H,J = 7.5 Hz, NCH₂), 2.84 (ddd, 1H,J = 7.5Hz,J = 6.5 Hz,J = 1.0 Hz, H-2_a), 2.63–2.57(m, 1H, H-2_b^′), 2.10–2.01 (m, 0.3H, H-3″α_a), 2.00–1.92(m, 1.7H, H-3″α_b, β_a,b),1.27 (t, 16.5H,J = 7.5 Hz, NCH₂CH₃).³¹P NMR (202 MHz,D₂O): δ −11.12 (m, α-pyrophosphate),−11.38 (m, β-pyrophosphate).¹³C{¹H}NMR (126 MHz, D₂O): δ 155.6 (C-6), 152.7 (C-2),148.7 (C-4), 139.9 (C-8), 118.6 (C-5), 102.1 (C-1″β),97.6 (C-1″α), 85.8 (d,J = 8.8 Hz, C-4′),83.6 (C-1′), 78.3 (d,J = 8.2 Hz, C-4″β),75.5 (d,J = 8.4, C-4″α), 75.4 (C-2″β),71.3 (C-3′), 70.4 (C-2″α), 68.8 (d,J = 5.4 Hz, C-5″β), 67.9 (d,J = 5.5Hz, C-5″α), 65.4 (d,J = 4.9 Hz, C-5′),46.6 (NCH₂), 39.0 (C-2′),32.4 (C-3′β), 31.7 (C-3′α), 8.2 (NCH₂CH₃). HRMS (ES^–) calcd for C₁₅H₂₂N₅O₁₂P₂, 526.0746 [M – H]⁻; 526.0761.HPLCt_R = 3.68 min.

Pharmacology

Materials

ADPR was obtained from Sigma-Aldrich.

Cell Culture

HEK293cells were kept at 37 °C and5% CO₂ in complete medium (DMEM with 4.5 g/L glucose, Glutamax-I,10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin)at 37 °C. For the maintenance of HEK293 clones with stable expressionof TRPM2 400 μg/mL G418 sulfate was added to the medium.

Transfectionand Generation of Cell Lines

Generationof the clonal HEK293 cell line with stable expression of human TRPM2has been described previously.¹⁶ In brief,HEK293 were transfected with an expression vector encoding the full-lengthof human TRPM2 and EGFP (pIRES2-EGFP-TRPM2). Cells that successfullyintegrated the expression vector were then enriched by selection with400 μg/mL G418 sulfate (Biochrom). Clonal cell lines were establishedfrom these cells by limiting dilution. Expression of TRPM2 was confirmedby Ca²⁺ measurement and whole cell patch clamp.

Patch-ClampMeasurements

The day before the experiments,cells from a clonal HEK293 cell line with stable expression of humanTRPM2 were seeded to 35 mm tissue culture dishes at a low density.For the whole cell patch-clamp experiments, the culture medium wasreplaced by a bath solution containing 1 mM CaCl₂, 140mM NMDG, 5 mM KCl, 3.3 mM MgCl₂, 1 mM CaCl₂,5 mMd-glucose, and 10 mM HEPES, pH 7.4. Patch pipettes werepulled from borosilicate glass capillaries with an outer diameterof 1.5 mm and an inner diameter of 1.05 mm using a Sutter P-97 andfilled with a pipette solution containing 120 mM KCl, 8 mM NaCl, 1mM MgCl₂, 10 mM HEPES, 10 mM EGTA, and 5.6 mM CaCl₂ (resulting in 200 nM free [Ca²⁺] as calculatedby CaBuf software (G. Droogmans, formerly available fromftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip). These pipettes had a resistance between 1.5 and 3.5 MΩ.Data were recorded using an EPC-10 amplifier and PatchMaster software(HEKA Elektronik, Germany). Fast and slow capacity transients werecompensated, series resistance was compensated by 70%. After establishingwhole cell configuration, cells were held at −50 mV and channelactivation was followed by applying voltage ramps from −85to +20 mV over 140 ms every 5 s for a total of 450 s. For furtheranalysis, the maximum outward current at +15 mV during the courseof the recording was taken as a measure of channel activity. To testfor agonist activity, ADPR analogues were included in the pipettesolution at 100 μM. To test for antagonist activity, the pipettesolution contained 100 μM ADPR and 900 μM of the ADPRanalogue under test. All experiments were performed at room temperature.

Statistical analysis

The data from patch-clamp experimentswere analyzed using GraphPad Prism (version 7.04, GraphPad SoftwareInc.) Because the distribution of currents is skewed toward highervalues, data were log-transformed. Log-transformed data were testedfor significant differences using one-way-Anova followed by post hoctesting against the respective control (buffer for agonist experiments,ADPR for antagonist experiments) using Bonferroni correction for multipletesting (α = 0.05). In the charts, the horizontal bar indicatesthe mean of the log-transformed data.

Acknowledgments

B.V.L.P. is a Wellcome Trust Senior Investigator(grant 101010). This study was supported by the Deutsche Forschungsgemeinschaft(GU 360/16-1 and Projektnummer 335447717-SFB1328 project A01 to A.H.G.,Projektnummer 335447717-SFB1328 project A05 to R.F.) and LandesforschungsförderungHamburg (Research Group ReAd Me, project 01, to A.H.G.). The authorsthank Andreas Bauche for technical support.

Supporting Information Available

The Supporting Information isavailable free of charge on theACS Publications website at DOI:10.1021/acs.joc.9b00338.

¹HNMR,¹³C NMR, and³¹P NMR spectra for compounds(PDF)

Author Present Address

^∥ Department of Organic Chemistry, Faculty of Science, Charles University,Hlavova 2030/8, 128 43 Prague 2, Czech Republic.

Author Contributions

O.B. and J.M.W.equally contributed. R.F. and B.V.L.P. equally contributed. B.V.L.P.and A.H.G. devised the overall work area. B.V.L.P., J.M.W. and R.F.devised the focused strategy. O.B. synthesized the ADPR analoguessupervised by J.M.W. M.D.R. carried out patch-clamp experiments withR.F., O.B., J.M.W., R.F. and B.V.L.P. wrote the manuscript with inputfrom all authors.

The authors declare nocompeting financial interest.

Supplementary Material

jo9b00338_si_001.pdf^{(6.4MB, pdf)}

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