molecules

Review Synthetic Strategies for Dinucleotides Synthesis

Lucie Appy, Crystalle Chardet, Suzanne Peyrottes andand Béatrice Béatrice Roy Roy * *

Nucleosides & Phosphorylated EEffectors,ffectors, Institut desdes BiomolBiomoléculesécules MaxMax MousseronMousseron (IBMM),(IBMM), UMRUMR 52475247CNRS, CNRS,Universit Universitéé de Montpellier, de Montpellier, ENSCM, ENSCM, Campus Camp Triolet,us Triolet, cc 1705, cc Place 1705, Eug Placeène Eugène Bataillon, Bataillon, CEDEX 34095 5, Montpellier,34095 Montpellier, CEDEX France; 5, France; [email protected] [email protected] (L.A.);m (L.A.); [email protected] [email protected] (C.C.); (C.C.); [email protected]@umontpellier.fr (S.P.) * Correspondence: [email protected]; Tel.: ++33-4-6714387933-4-6714-3879 Academic Editor: György Keglevich  Academic Editor: György Keglevich  Received: 7 November 2019; Accepted: 25 November 2019; Published: 27 November 2019 Received: 7 November 2019; Accepted: 25 November 2019; Published: 27 November 2019

Abstract: DinucleosideDinucleoside 5′,5′ 5-polyphosphates0,50-polyphosphates (DNPs) (DNPs) are endogenous are endogenous substances substances that play important that play intra-important and extracellular intra- and extracellular roles in various roles biological in various processes, biological such processes, as cell proliferation, such as cell proliferation,regulation of enzymes,regulation neurotransmissio of enzymes, neurotransmission,n, platelet disaggregation platelet disaggregation and modulation and modulation of vascular of tone. vascular Various tone. methodologiesVarious methodologies have been have developed been developed over the past over fifty the years past fifty to access years these to access compounds, these compounds, involving enzymaticinvolving enzymaticprocesses or processes chemical orprocedures chemical based procedures either on based P(III) either or P(V) on chemistry. P(III) or P(V) Both chemistry. solution- phaseBoth solution-phaseand solid-support and strategies solid-support have strategiesbeen developed have beenand are developed reported and here. are Recently, reported green here. chemistryRecently, greenapproaches chemistry have approaches emerged, offering have emerged, attracting o ffalternatives.ering attracting This alternatives.review outlines This the review main syntheticoutlines thepathways main synthetic for the pathways preparation for theof preparationdinucleoside of 5 dinucleoside′,5′-polyphosphates, 50,50-polyphosphates, focusing on pharmacologicallyfocusing on pharmacologically relevant compounds, relevant compounds,and highlighting and highlighting recent advances. recent advances.

Keywords: phosphorylation;phosphorylation; dinucleoti dinucleotides;des; organophosphorus organophosphorus chemis chemistry;try; mechanochemistry; mechanochemistry; dry dry eye syndrome; ;

1. Introduction Introduction

Dinucleoside 5 ′0,5,5′0-polyphosphates-polyphosphates (DNPs), (DNPs), commonly commonly abbreviated abbreviated as as Np NpnnNs,Ns, are are essential to biological systems [[1,2].1,2]. They They contain contain two two ribonucleosi ,des, which are linked at the 5 ′0-position-position of their sugar moiety through n phosphate groups (Figure1 1).).

O O B' B O P O P O O O O n O HO OH HO OH B, B' = Ade, Gua, Ura n = 1-6

PURINES NH2 O NH2 O O 7 6 4 N 5 1 N 3 N NH 5 N NH NH 8 2 6 9N 4 N 2 N N NH O H H 2 N 1 N O N O 3 H H H (Ade) (Cyt) BH Nucleobases (Gua) (Ura) (Thy)

Figure 1. GeneralGeneral structure structure of natural dinucleoside 5 ′0,5-polyphosphates,5-polyphosphates and and canonical canonical nucleobases. nucleobases.

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Historically,Historically, dinucleoside 5 5′0,5,5′-polyphosphates0-polyphosphates (i.e., (i.e., Ap Ap2A2 Aand and Up Up2U)2 U)were were first first isolated isolated by bychemists chemists [3]. [In3]. 1976, In 1976,E. Rapaport E. Rapaport and P.C. and Zamecnik P.C. Zamecnik brought brought to light to the light presence the presence of P1,P of4- 1 4 Pdiadenosine-5,P -diadenosine-5′-tetraphosphate0-tetraphosphate () (Apin mammals4A) in mammals [4]. The authors [4]. The assumed authors that assumed this dinucleotide that this dinucleotidecould have acould role in have cellular a role communication in cellular communication as a “signal asnucleotide”. a “signal ”.Later, Up4A was Later, the Up first4A wasendogenous the first endogenous dinucleotide, dinucleotide, possessing possessingboth both and purine and pyrimidine moieties, moieties,to be identified to be identified in living inorganisms living organisms [5,6]. Currently, [5,6]. Currently, 17 symmetrical 17 symmetrical or mixed or Np mixednNs have NpnNs been have isolated been isolated from human from humantissues tissuesand characterized and characterized [2]. DNPs [2]. are DNPs released are released into the intoextracellular the extracellular space from space different from di typesfferent of typescells, ofsuch cells, as platelets such asplatelets and endothelial and endothelial cells, where cells, they where stimulate they stimulateseveral cell-surface several cell-surface purinergic purinergic receptors. receptors.They have Theya strong have physiological a strong physiological and pathophysiological and pathophysiological impact on the impact cardiovascular on the cardiovascular system [2,7], systemand may [2, 7also], and interact may also with interact enzymes, with acting enzymes, as inhi actingbitors as inhibitors (e.g., kinases) (e.g., kinases)or substrates or substrates [8–11]. [Three8–11]. Threeclasses classes of enzymes of enzymes are known are known to catalyze to catalyze the the de degradationgradation of of dinucleotides dinucleotides [10–12]: [10–12]: symmetrically cleavingcleaving dinucleosidedinucleoside polyphosphate polyphosphate hydrolases, hydrolas asymmetricallyes, asymmetrically cleaving dinucleoside cleaving polyphosphatedinucleoside hydrolases,polyphosphate and hydrolases, dinucleoside and polyphosphate dinucleoside polyphosphate phosphorylases. phosphor Over theylases. years, Over DNPs the haveyears, gained DNPs increasedhave gained attention increased and theirattention therapeutic and their potential ther hasapeutic been potential revealed. has Likewise, been structuralrevealed. analoguesLikewise, havestructural been analogues developed have and somebeen ofdeveloped them are and drug some candidates. of them are drug candidates. Herein,Herein, wewe firstfirst describedescribe purinergicpurinergic signalling,signalling, i.e.,i.e., thethe rolerole ofof nucleotidesnucleotides asas extracellularextracellular signalsignal molecules,molecules, withwith a a focus focus on on the the P2Y P2Y receptor subtypes subtypes that interactthat interact with dinucleotides. with dinucleotides. Then, we Then, present we thepresent synthetic the synthetic strategies strategies to obtain dinucleotides.to obtain dinucl Pyrophosphateeotides. Pyrophosphate bond formation bond has formation been addressed has been in someaddressed recent in reviews some andrecent book reviews chapters and [1, 13book–15 ].chap Theters current [1,13–15]. review The focuses current on methods review reportedfocuses foron themethods synthesis reported of dinucleoside for the 5synthesis0,50-polyphosphates, of dinucleoside especially 5′,5 those′-polyphosphates, of biological andespecially pharmacological those of interest.biological We and also pharmacological highlight recent interest. advances We in also the field,highlight such recent as green advances chemistry in the approaches. field, such It as should green bechemistry noted that approaches. methods It for should preparing be noted cyclic that dinucleotides methods for [16 preparing] are beyond cyclic the dinucleotides scope of this [16] review. are Furthermore,beyond the scope the synthesis of this review. of nicotinamide Furthermore, adenine the dinucleotidessynthesis of nicotinamide [17] and mRNA adenine cap analogues dinucleotides such 7 m[17]Gp andnN mRNA (see reviews cap analogues [18–20]) are such not m presented.7GpnN (see reviews [18–20]) are not presented.

2.2. Interaction of DNPs with Purine and Pyrimidine ReceptorsReceptors PurinergicPurinergic receptorsreceptors areare extracellularextracellular receptors, currently consisting of four subtypes of P1P1 (or(or )adenosine) receptors,receptors, seven seven subtypes subtypes of P2Xof P2X ion channelion channel receptors, receptors, and eight and subtypes eight subtypes of P2Y receptors of P2Y (Figurereceptors2). (Figure Specifically, 2). Specifically, P2Y receptors P2Y (P2YRs) receptors are G-protein-coupled(P2YRs) are G-protein-coupled receptors (GPCRs) receptors activated (GPCRs) by extracellularactivated by ,extracellular which nucleo aretides, divided which into are two divided groups into on two the basisgroups of on sequence the basis homology of sequence and thehomology type of and G protein the type they of areG protein primarily they coupled are primarily to [21– 23coupled]. to [21–23].

FigureFigure 2. The 2. classificationThe classification of purine of purine and pyrimidineand pyrimidi receptorsne receptors and theirand their main main physiological physiological agonists. agonists.

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Purinergic receptors are expressed in nearly all cell types, and have been found to play crucial roles in many many biological biological processes, processes, e.g., e.g., neurotransmission, neurotransmission, neuroprotection neuroprotection in in hypoxia hypoxia and and ischemia, ischemia, regulation of cardiovascular function, platelet aggregation, smooth muscle contraction, secretion of hormones, modulationmodulation of of immune immune response, response, control control of cell of proliferation, cell proliferation, differentiation, differentiation, and . and apoptosis.In this regard, In this diff erentregard, aspects different of purinergic aspects of signaling purinergic such signaling as the pathophysiology such as the pathophysiology and the therapeutic and thepotential therapeutic have beenpotential extensively have been reviewed extensively [21– reviewed28]. Several [21–28]. purinergic Several compounds purinergic compounds are already are on alreadythe market on the to treatmarket thrombosis to treat thrombosis and stroke, and and stroke, one dinucleotideand one dinucleotide is used to is treatused dryto treat eye dry disease eye disease(see Section (see 2.1Section). 2.1). DNPs are strong endogenous agonists of the purinergicpurinergic system [[2].2]. In particular, they interact with the P2YRs, aa familyfamily ofof highhigh therapeutictherapeutic relevance relevance [ 2[2,23,29,30].,23,29,30]. The The P2Y P2Y1,1, P2Y P2Y1111,, P2YP2Y1212,, andand P2YP2Y1313 receptors are activated by adenosine 50′-di--di- or or triphosphates triphosphates (ADP (ADP and and ATP), ATP), while uracil nucleotides (UDP and UTP)UTP) areare thethe endogenousendogenous agonistsagonists forfor thethe P2YP2Y44,, P2YP2Y66, andand P2YP2Y1414 receptors. On On the other hand, the P2Y 2 receptorreceptor responds responds to to both both ATP ATP and and UTP. UTP. Natural dinucleoside tetraphosphates Ap4A and Up 44UU have have agonist agonist potencies potencies comparable comparable to to those those of of ATP ATP and and UTP UTP at atP2Y P2Y2 receptors2 receptors [31–34]. [31–34 It]. shouldIt should be be notednoted that that dinucleotides dinucleotides are more are resistant more toresistant hydrolysis to thanhydrolysis their parent than mononucleotides, their parent mononucleotides,but are generally less but potent, are generally and lack selectivityless potent, in manyand lack cases selectivity [9,23]. Ligand in many development cases [9,23]. for theLigand class developmentof uracil nucleotide-activated for the class of uracil P2Y nucleotide-activ receptors has beenated extensively P2Y receptors reviewed has been by extensively Rahefi and Müllerreviewed in by2018 Rahefi [23]. and They Müller have compiled in 2018 [23]. existing They data have on, compiled inter alia, existing dinucleotides data on, such inter as alia, Ap ndinucleotidesA(n = 2–6), Up suchnU as(n Ap= 3–4)nA (n and = 2–6), structural UpnU ( analogues,n = 3–4) and as structural agonists analogues, for the uracil-activated as agonists for P2YRs. the uracil-activated Given the significant P2YRs. Givenclinical the potential significant of the clinical P2YRs, potential substantial of researchthe P2YRs, efforts substantial directed research towards developingefforts directed P2YR towards ligands developingfor use as pharmacological P2YR ligands for tools use and as pharmacological drugs have led to tools the discovery and drugs and have development led to the ofdiscovery a significant and developmentnumber of agonists of a significant but, so far, number only a of moderate agonists numberbut, so far, of antagonists.only a moderate Two number relevant of dinucleotides, antagonists. Twonamely relevant Up4U dinucleotides, and Up4dC, have namely undergone Up4U and clinical Up4 developmentdC, have undergone to cure severalclinical diseases development involving to cure the severalP2Y2 receptor diseases (Figure involving3). the P2Y2 receptor (Figure 3).

Figure 3. DinucleotidesDinucleotides with clinical developments.

2.1. Up U for the Treatment of Dry Eye Disease (DED) 2.1. Up44U for the Treatment of Dry Eye Disease (DED) P1 P4 Diquas® Diquafosol tetrasodium ((P1,, P4-diuridine 550′,5′0-tetraphosphate,-tetraphosphate, Up4U, NS365, Diquas®)) is is the the first first agonist of the P2Y2 receptorreceptor subtype subtype that that has has been been approved approved in in Japan as 3% ophthalmic solution (Santen Pharmaceutical Co, Ltd., Osaka, Japan) fo forr the management of dry eye disease (see reviews by Lau et al. [35], [35], and Keating [36]). [36]). This path pathologyology commonly commonly causes causes symp symptomstoms including including dryness, dryness, irritation, itching, itching, and and light light sensitivity. sensitivity. It It is is associ associatedated with with tear tear film film instability, increased tear film film osmolarity, andand ocular surface inflammation.inflammation. It impacts the daily life of patients and has a prevalence prevalence that varies from 5% to 35%.35%. DED results from eithereither decreased aqueous tear production (aqueous tear-deficienttear-deficient dry eye) or increased tear evaporationevaporation (evaporative dry eye), or both.both. Several studies demonstrated the presence of P2X and and P2Y P2Y receptors receptors in in ocular ocular tissues tissues (retina, (retina, ciliary ciliary body, body, and and lens), lens), and indicatedindicated thatthat P2Y P2Y2 2receptors receptors may may be be the the main main subtype subtype of purinergic of receptor located located at the ocularat the ocularsurface surface [33]. Clinical [33]. Clinical data show data that show diquafosol that diquafosol tetrasodium tetrasodium improves improves ocular surface ocular staining surface andstaining may andimprove may tearimprove film volumetear film and volume stability. and stability.

2.2. Up4dC for the Treatment of

Denufosol tetrasodium (P1--P4-2′- 5′,5′-tetraphosphate, Up4dC, dCp4U, INS37217) is a mixed dinucleoside tetraphosphate. Along with Ap4A and Up4U, dCp4U acts as a P2Y2 agonist [29,37]. It has been developed by Inspire Pharmaceuticals (now part of Merck) as an inhaled

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2.2. Up4dC for the Treatment of Cystic Fibrosis 1 4 Denufosol tetrasodium (P -uridine-P -20-deoxycytidine 50,50-tetraphosphate, Up4dC, dCp4U, INS37217)Molecules 2019 is, 24 a, x mixed dinucleoside tetraphosphate. Along with Ap4A and Up4U, dCp4U acts4 of as 27 a P2Y2 agonist [29,37]. It has been developed by Inspire Pharmaceuticals (now part of Merck) as an inhaleddrug for drug the fortreatment the treatment of cystic of cysticfibrosis fibrosis [38]. [Th38is]. Thisrecessive recessive genetic genetic disease disease is characterized is characterized by bypulmonary pulmonary and and reproductive reproductive tract tract dysfunctions, dysfunctions, involving involving abnormal abnormal ion ion transport transport and defective mucociliary clearance. Denufosol acts on P2Y recept receptorsors expressed on the surface of airway epithelia to stimulatestimulate chloridechloride secretion independentindependent of thethe chloridechloride channel,channel, whichwhich isis dysfunctionaldysfunctional inin cysticcystic fibrosis.fibrosis. It was shown to significantlysignificantly enhance trac trachealheal transport in an animal model [39,40]. [39,40]. The firstfirst clinical trials established a good safety profile.profile. It was evaluated in two phase 3 clinical trials, TIGER-1 and TIGER-2, as a therapytherapy forfor cysticcystic fibrosisfibrosis patientspatients [[41,42].41,42]. In the firstfirst trialtrial (TIGER-1),(TIGER-1), it waswas foundfound toto significantlysignificantly improve lung function in cystic fibrosisfibrosis patients with normal to mildly impairedimpaired lunglung function. function. Unfortunately, Unfortunately, less less than than 3 weeks 3 weeks after after publication publication of the of TIGER-1 the TIGER-1 data, Inspire data, PharmaceuticalsInspire Pharmaceuticals announced announced that the 466-patients, that the 466- 48-weekpatients, placebo-controlled 48-week placeb phaseo-controlled 3 TIGER-2 phase clinical 3 trial,TIGER-2 had clinical failed to trial, demonstrate had failed any to demonstrate benefit [41,42 any]. benefit [41,42].

3. Synthesis Based on P(V) Chemistry To date,date, thethe mostmost widely used methods to access dinucleotides involve P(V) chemistry. They are based on thethe activationactivation ofof aa 550′-nucleotide-nucleotide (i.e.,(i.e., nucleosidenucleoside 550′-mono,-mono, 550′-di and 50′-triphosphate) and the reaction ofof thethe correspondingcorresponding intermediate intermediate with with a a second second 50 5-nucleotide′-nucleotide or or inorganic inorganic pyrophosphate pyrophosphate to formto form dinucleotides dinucleotides containing containing up to up six bridgingto six br phosphateidging phosphate groups. Thesegroups. steps These are usuallysteps are performed usually inperformed dry, aprotic in dry, solvents aprotic (mostly solventsN,N (mostly-dimethylformamide, N,N-dimethylformamide, DMF), and, DMF), therefore, and, requiretherefore, the require use of nucleotides,the use of nucleotides, as well as inorganic as well phosphateas inorganic or pyrophosphate,phosphate or pyrophos in their tri-phate, or tetra- in theirn-butylammonium tri- or tetra-n- formsbutylammonium due to solubility forms issues. due to Divalent solubility cations, issues. especially Divalent Mg cations,2+ and especially Zn2+, are sometimesMg2+ and addedZn2+, are as catalystssometimes for added anhydride as catalysts bond formation. for anhydride It is assumed bond formation. that the metal It is assumed ion could that serve the two metal roles, ion namely, could theserve activation two roles, of thenamely, electrophilic the activation P(V) center of the and electrophilic templating P(V) the incomingcenter and phosphate templating nucleophile the incoming and thephosphate P(V) electrophilic nucleophile center and the [13 ].P(V) electrophilic center [13].

3.1. Synthesis via a Phosphoromorpholidate Intermediate The firstfirst descriptiondescription of a chemicalchemical synthesis of dinucleotides was reported in the mid-60s by MoMoffattffatt andand KhoranaKhorana [[43,44].43,44]. This strategy relies on the conversion of 50′-monophosphates (NMPs)(NMPs) intointo theirtheir phosphoromorpholidatephosphoromorpholidate derivatives, followed by reaction with a phosphate salt, i.e.,i.e., orthophosphateorthophosphate or pyrophosphate (Scheme 11).). Briefly,Briefly, thethe acidicacidic formform ofof thethe nucleotidesnucleotides werewere activated with N,N’-dicyclohexylcarbodiimide (DCC) in the presence of morpholine, leading to the nucleoside 550′-phosphoromorpholidates as 4-morpholine N,N’-dicyclohexylcarboxamideN,N’-dicyclohexylcarboxamide salts 1–3 (Scheme(Scheme1 1))[ [45].45]. In the the second second step, step, addition addition of of pyrophosphate pyrophosphate afforded afforded the symmetrical dinucleoside tetraphosphates. Similarly, Similarly, dinucleoside dinucleoside tripho triphosphatessphates were were obtained when pyrophosphate was replaced byby orthophosphate.orthophosphate.

Scheme 1. Synthesis via phosphoromorpholidate intermediates [[44].44].

Since then, this approach has been adapted by several research groups to access Np2N, Np3N, as well as some analogues. For example, Ap2A was obtained in 90% yield, through the condensation of the bis-(tri-n-butylammonium) salt of AMP and the adenosine-5′-phosphoromorpholidate-4- morpholine-N,N’-dicyclohexylcarboxamide salt in anhydrous pyridine [46]. Mixed dinucleoside triphosphates were obtained in 10–30% yields, by reacting nucleoside 5′-phosphoromorpholidates with nucleoside 5′-diphosphates (NDPs), possibly in the presence of tetrazole [31,47,48]. However, due to long reaction times and generally low yields, this method has lost its relevance.

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Since then, this approach has been adapted by several research groups to access Np2N, Np3N, as well as some analogues. For example, Ap2A was obtained in 90% yield, through the condensation of the bis-(tri-n-butylammonium) salt of AMP and the adenosine-50- phosphoromorpholidate-4-morpholine-N,N’-dicyclohexylcarboxamide salt in anhydrous pyridine [46]. Mixed dinucleoside triphosphates were obtained in 10–30% yields, by reacting nucleoside 50-phosphoromorpholidates with nucleoside 50-diphosphates (NDPs), possibly in the presence of tetrazole [31,47,48]. However, due to long reaction times and generally low yields, this method has lostMolecules its relevance. 2019, 24, x 5 of 27

3.2. Synthesis via a Phospho Phosphorimidazoliderimidazolide Intermediate Phosphorimidazolide derivativesderivatives exhibit exhibit high high reactivity reactivity toward toward various nucleophiles.various nucleophiles. Therefore, theyTherefore, have beenthey have extensively been extensively used as intermediates used as intermediates for pyrophosphate for pyrophos bondphate formation. bond formation. Typically, activationTypically, activation of a 50-nucleotide of a 5′-nucleotide (NMP, NDP (NMP, or NDP NTP) or with NTP)N ,withN’-carbonyldiimidazole N,N’-carbonyldiimidazole (CDI) in(CDI) DMF, in followedDMF, followed by the by in the situ in situ condensation condensation with with a seconda second 50 -nucleotide5′-nucleotide or or pyrophosphate,pyrophosphate, affords affords dinucleotides containingcontaining two two to fourto bridgingfour bridging phosphate phosphate groups ingroups 10–60% in yields 10–60% [47– 50yields]. Accordingly, [47–50]. theAccordingly, tri-n-butylammonium the tri-n-butylammonium salt of UMP was salt activated of UMP with was CDI activated to form awith phosphorimidazolide CDI to form a intermediatephosphorimidazolide4, which intermediate reacted with 4, thewhich remaining reacted nucleotidewith the remaining to form Upnucleotide2U (Scheme to form2)[ 47Up,492U]. Slight(Scheme modifications 2) [47,49]. Slight of this modifications protocol allowed of this access protocol to mixed allowed dinucleotides access to mixed [50]. dinucleotides First, activation [50]. of the bis(tri-n-butylammonium) salt of CMP was performed using 2 equiv of CDI in DMF for 2 h First, activation of the bis(tri-n-butylammonium) salt of CMP was performed≈ using ≈ 2 equiv of CDI atin rt,DMF followed for 2 h by at the rt,addition followed of by dry the methanol addition to of quench dry methanol the remaining to quench CDI. the Treatment remaining with CDI. the bis(tri-Treatmentn-butylammonium) with the bis(tri-n salt-butylammonium) of GMP for 2 h salt at rt of a ffGMPorded for Gp 2 2hC at in rt 54% afforded yield. Gp Addition2C in 54% of yield. GDP insteadAddition of of GMP GDP in instead the second of GMP step in aff theorded second Gp3C, step albeit afforded in only Gp 3%3C, yield. albeit Similarly, in only 3% activation yield. Similarly, of NDPs withactivation CDI followed of NDPs bywith reaction CDI followed with a NMP by reaction allowed with the isolationa NMP allowed of dinucleoside the isolation triphosphates of dinucleoside in low yieldstriphosphates [48]. in low yields [48].

Scheme 2.2. Activation of UMP by CDI to access Up22UU[ [47,49].47,49].

Alternatively, symmetricalsymmetrical dinucleosidedinucleoside tetraphosphatetetraphosphate UpUp44U was obtained, either by activation of UMP with CDI followed by coupling with bis(tri-n-butylammmonium) pyrophosphate [47,49] [47,49] or dimerization of UDP in the presencepresence ofof CDICDI [[51]51] (Scheme(Scheme3 3).).

Scheme 3. Dimerization of nucleotides to access symmetrical Up44UU[ [49,51].49,51].

Activation of in the presence of an excess of CDI usually results in the carbonation of the moiety (Scheme 4). In 1H-decoupled 31P NMR spectroscopy, adenosine 5′- phosphorimidazolide is characterized by a singlet at –7.56 ppm, whereas phosphorimidazolide 5 exhibits a singlet at –7.76 ppm [52]. However, in some of the above-mentioned publications [47,49], carbonation was not observed, and may be due to the experimental conditions (number of equivalents of CDI, reaction time, temperature, adventitious presence of water). Nonetheless, the removal of the 2′,3′-carbonate protecting group can be easily performed under basic conditions [52,53].

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Activation of ribonucleotides in the presence of an excess of CDI usually results in the carbonation of the ribose moiety (Scheme4). In 1H-decoupled 31P NMR spectroscopy, adenosine 5 -phosphorimidazolide is characterized by a singlet at 7.56 ppm, whereas phosphorimidazolide 5 0 − exhibits a singlet at 7.76 ppm [52]. However, in some of the above-mentioned publications [47,49], − carbonation was not observed, and may be due to the experimental conditions (number of equivalents of CDI, reaction time, temperature, adventitious presence of water). Nonetheless, the removal of the Molecules 2019, 24, x 6 of 27 Molecules20,30-carbonate 2019, 24, x protecting group can be easily performed under basic conditions [52,53]. 6 of 27

Scheme 4. Reaction of AMP31 with 4 equiv CDI [52]. Scheme 4. Reaction of AMPScheme with 4. 4 equivReaction CDI of [ 52AMP]. withP chemical 4 equiv shifts CDI are[52]. indicated in ppm together 31P chemical shifts are indicated in ppm together with their multiplicity. with their multiplicity.31P chemical shifts are indicated in ppm together with their multiplicity. In 2011, a variant of the CDI method was developed by Yanachkov and co-workers [54], where In 2011, a variant of the CDI method was developed by by Yanachkov Yanachkov and co-workers [54], [54], where the activation of pyrophosphate by CDI gave rise to1 P12,P2-di(1-imidazolyl)pyrophosphate (Scheme the activation ofof pyrophosphatepyrophosphate by by CDI CDI gave gave rise rise to toP P,P1,P-di(1-imidazolyl)pyrophosphate2-di(1-imidazolyl)pyrophosphate (Scheme (Scheme5). 5). Using13 13C-labeled CDI and monitoring the reaction by31 31P NMR13 (13C-3131P and1 1H-3131P couplings), the 5).Using UsingC-labeled 13C-labeled CDI CDI and and monitoring monitoring the the reaction reaction by by P31P NMR NMR ( (13C-C-31PP andand 1H-H-31P couplings), the authors proposed a reaction mechanism involving the fast formation of mixed anhydrides, which authors proposed a reaction mechanism involving the fast formation of mixed anhydrides, which react slowly with imidazole to give diimidazolyl pyrophosphate. This activated pyrophosphate was react slowly with imidazole to give diimidazolyl pyrophosphate. This activated pyrophosphate was isolated as its disodium salt 6 by precipitation with perchlorate in acetone, and then reacted isolated as its disodium salt salt 6 by precipitation with sodium perchlorate in acetone, and then reacted with AMP oror UMPUMP toto aaffordfford thethe symmetricalsymmetrical 55,5′,5′-dinucleoside-dinucleoside tetraphosphatestetraphosphates ApAp4A and Up4U in with AMP or UMP to afford the symmetrical 50′,50′-dinucleoside tetraphosphates Ap44A and Up4U in good yields (Scheme 5). Noteworthy, the use of 1H-tetrazole or chloride during the second step good yields (Scheme 55).). Noteworthy,Noteworthy, thethe useuse ofof 11H-tetrazole-tetrazole or or zinc zinc chloride chloride during during the the second step allowed the reaction time to be shortened from 24–48 h to 3–16 h. allowed the reaction time to be shortened from 24–48 h to 3–16 h.

1 2 SchemeScheme 5.5. Np44Ns synthesis using P1,,PP2-di(1-imidazolyl)pyrophosphate-di(1-imidazolyl)pyrophosphate 66 [54]. [54]. Scheme 5. Np4Ns synthesis using P1,P2-di(1-imidazolyl)pyrophosphate 6 [54]. Interestingly, this approach allows the preparationpreparation ofof dinucleotidedinucleotide analogues,analogues, starting from Interestingly, this approach allows the preparation of dinucleotide analogues, starting from modifiedmodified pyrophosphate (the central oxygen beingbeing replaced with monofluoro,monofluoro, monochloro or modified pyrophosphate (the central oxygen being replaced with monofluoro, monochloro or dichloromethylene). These DNPs analogue analoguess were obtained in 70–80% yields. dichloromethylene). These DNPs analogues were obtained in 70–80% yields. While these methods involving phosphoromidazolid phosphoromidazolideses are straightforward and rapid, they all While these methods involving phosphoromidazolides are straightforward and rapid, they all require drydry conditionsconditions to to prevent prevent side-product side-product formation formation and and most most often, often, use trialkylammoniumuse trialkylammonium salts. require dry conditions to prevent side-product formation and most often, use trialkylammonium Tosalts. avoid To thisavoid drawback, this drawback, our research our groupresearch has grou recentlyp has developed recently adeveloped one-pot synthesis a one-pot of dinucleotides, synthesis of salts. To avoid this drawback, our research group has recently developed a one-pot synthesis of dinucleotides,either in aqueous either solution in aqueous or mechanochemically, solution or mechanochemically, through the use of through phosphorimidazolides the use of dinucleotides, either in aqueous solution or mechanochemically, through the use of phosphorimidazolidesintermediates [55,56]. intermediates These strategies [55,56]. will These be detailed strategies in will Section be 5detailed and are in relatedSection to5 and green are phosphorimidazolides intermediates [55,56]. These strategies will be detailed in Section 5 and are relatedchemistry to green approaches. chemistry approaches. related to green chemistry approaches. 3.3. Synthesis via a Cyclic Trimetaphosphate Intermediate 3.3. Synthesis via a Cyclic Trimetaphosphate Intermediate 3.3. Synthesis via a Cyclic Trimetaphosphate Intermediate The initial synthesis of trimetaphosphate esters from ATP was described by Khorana [57]. The initial synthesis of trimetaphosphate esters from ATP was described by Khorana [57]. Later, Later,The Ng initial and synthesis Orgel reported of trimetap thathosphate adenosine esters 5 -polyphosphates from ATP was described can react by with Khorana 1-ethyl-3-(3-dim [57]. Later, Ng and Orgel reported that adenosine 50′-polyphosphates can react with 1-ethyl-3-(3- Ng and Orgel reported that adenosine 5′-polyphosphates can react with 1-ethyl-3-(3- dimethylaminopropyl)carbodidimide (EDC) in water to form diadenosine 5′,5′-polyphosphates as dimethylaminopropyl)carbodidimide (EDC) in water to form diadenosine 5′,5′-polyphosphates as the main products [58]. Yields were improved by performing the reaction in organic solvents such as the main products [58]. Yields were improved by performing the reaction in organic solvents such as DMF or DMSO, in the presence of DCC and using the tris or tetrakis(tri-n-butylammonium) salts of DMF or DMSO, in the presence of DCC and using the tris or tetrakis(tri-n-butylammonium) salts of nucleotides [31,47]. The activation of nucleoside 5′-triphosphates (NTPs) with DCC proceeds via a nucleotides [31,47]. The activation of nucleoside 5′-triphosphates (NTPs) with DCC proceeds via a cyclic nucleoside 5′-trimetaphosphate intermediates (7–8), which can further react with another NMP cyclic nucleoside 5′-trimetaphosphate intermediates (7–8), which can further react with another NMP to produce the corresponding dinucleoside tetraphosphates (Scheme 6). Accordingly, Up4U and to produce the corresponding dinucleoside tetraphosphates (Scheme 6). Accordingly, Up4U and Up4A were obtained in low yields starting from UTP and ATP, respectively. One should note that Up4A were obtained in low yields starting from UTP and ATP, respectively. One should note that N,N’-dicyclohexylurea (DCU) may be removed as a precipitate either after the first or the second step N,N’-dicyclohexylurea (DCU) may be removed as a precipitate either after the first or the second step of the reaction. of the reaction.

Molecules 2019, 24, 4334 7 of 27

ethylaminopropyl)carbodidimide (EDC) in water to form diadenosine 50,50-polyphosphates as the main products [58]. Yields were improved by performing the reaction in organic solvents such as DMF or DMSO, in the presence of DCC and using the tris or tetrakis(tri-n-butylammonium) salts of nucleotides [31,47]. The activation of nucleoside 50-triphosphates (NTPs) with DCC proceeds via a cyclic nucleoside 50-trimetaphosphate intermediates (7–8), which can further react with another NMP to produce the corresponding dinucleoside tetraphosphates (Scheme6). Accordingly, Up 4U and Up4A were obtained in low yields starting from UTP and ATP, respectively. One should note that N,N’-dicyclohexylurea (DCU) may be removed as a precipitate either after the first or the second step ofMoleculesMolecules the reaction. 20192019,, 2424,, xx 77 ofof 2727

SchemeScheme 6.6.6. SynthesisSynthesis ofof NpNp44NsNs starting starting from from nucleoside nucleoside 5 5′0′-triphosphates-triphosphates (NTPs) (NTPs) [47]. [[47].47].

Similarly,Similarly, thethe activationactivation ofof UTPUTP oror ATPATP withwith DCCDCC inin anhydrousanhydrous DMF,DMF, followedfollowed byby thethe additionaddition ofof thethe tris(tri-tris(tri-nn-butylammonium)-butylammonium) salt salt of of UDP UDP or or ADP ADP in in th thethee second second step step of of thethe reaction,reaction, afforded aaffordedfforded UpUp55UU and and Ap Ap55A,A, inin 10–60% 10–60% yields yields [8,49,50]. [[8,49,50].8,49,50]. Reactions Reactions mu mustmustst be performed performed under under strictly strictly anhydrousanhydrous conditionsconditions andand longlong reactionreaction timestimes areare required.required.required. InIn 2013,2013, the the Taylor Taylor researchresearch groupgroupgroup reportedreportedreported aaa high-yieldinghigh-yieldinghigh-yielding synthesissynthesis ofof dinucleosidedinucleosidedinucleoside pentaphosphatespentaphosphates (Np(Np 555N)N)N) usingusing using thethe the tri(tetra-tri(tetra- tri(tetra-nn-butylammonium)-butylammonium)n-butylammonium) saltsalt salt ofof cycliccyclic of cyclic trimetaphosphatetrimetaphosphate trimetaphosphate 99 asas 9aa asphosphorylatingphosphorylating a phosphorylating agentagent agent [59].[59]. [ 59TheThe]. Thelatterlatter latter waswas waspreparedprepared prepared inin almostalmost in almost quantitativequantitative quantitative yieldyield yield byby conversionconversion by conversion ofof thethe of thetrisodiumtrisodium trisodium saltsalt salt ofof trimetaphosphatetrimetaphosphate of trimetaphosphate 1010 10 intointointo itsits its pyridiniumpyridinium pyridinium form,form, form, andand and thenthen then byby by titrationtitration titration ofof of thethe solution solution toto pHpH 7.07.0 withwith aa dilutedilute solutionsolution ofofof tetrabutylammoniumtetrabutyltetrabutylammoniumammonium hydroxidehydroxidehydroxide andandand freeze-dryingfreeze-dryingfreeze-drying (Scheme(Scheme(Scheme7 7).).7).

SchemeScheme 7.7. PreparationPreparation of of the the cyclic cyclic trimetaphosphate trimetaphosphate 99 [[59].[59].59].

Then,Then, thethe the electrophilicelectrophilic electrophilic reagentreagent reagent 99 waswas9 was functionalizedfunctionalized functionalized byby aa sulfonylsulfonyl by a sulfonyl leavingleaving leavinggroupgroup (LG)(LG) group usingusing (LG) NN-- usingmethylimidazolemethylimidazoleN-methylimidazole (NMI)(NMI) inin (NMI) thethe inpresencepresence the presence ofof eithereither of either 2-mesitylenesulfonyl2-mesitylenesulfonyl 2-mesitylenesulfonyl chloridechloride chloride 1111 or11or or1-1- 1-benzenesulfonyl-3-methyl-imidazoliumbenzenesulfonyl-3-methyl-imidazoliumbenzenesulfonyl-3-methyl-imidazolium triflatetriflate triflate 121212 ininin DMFDMF DMF (Scheme (Scheme 8 ).8).8). CompoundCompoundCompound 1111 waswas commerciallycommercially available,available,available, whereas whereaswhereas12 12was12 waswas prepared preparedprepared in two inin steps twotwo startingstepssteps startingstarting from benzenesulfonyl fromfrom benzenesulfonylbenzenesulfonyl chloride 31 andchloridechloride imidazole andand imidazoleimidazole [60]. Based [60].[60]. on BasedBasedP on NMRon 3131PP NMR monitoringNMR monitoringmonitoring of the ofof reaction, thethe reaction,reaction, the authors thethe authorsauthors proposed proposedproposed that thatthat the activationthethe activationactivation proceeds proceedsproceeds via aviavia mixed aa mixedmixed anhydride anhydrideanhydride13, which1313,, whichwhich reacts reactsreacts with withwith NMI NMINMI to give toto givegive rise riserise to intermediate toto intermediateintermediate14. 1414.. Addition of sub-stœchiometric amounts of NMPs to the activated cyclic trimetaphosphate 9 allowed their conversion to intermediates 15–18, as suggested by 31P NMR (Scheme9). Addition of slight excess of the second NMP and anhydrous MgCl2 gave rise to the corresponding dinucleoside pentaphosphates. In the absence of Mg2+ ions, the reaction was extremely slow.

SchemeScheme 8.8. ActivationActivation ofof thethe cyccycliclic trimetaphosphatetrimetaphosphate 99 [59].[59].

AdditionAddition ofof sub-stœchiometricsub-stœchiometric amountsamounts ofof NMPsNMPs toto thethe activatedactivated cycliccyclic trimetaphosphatetrimetaphosphate 99 allowedallowed theirtheir conversionconversion toto intermediatesintermediates 15–1815–18,, asas suggestedsuggested byby 3131PP NMRNMR (Scheme(Scheme 9).9). AdditionAddition ofof slightslight excessexcess ofof thethe secondsecond NMPNMP andand anhydrousanhydrous MgClMgCl22 gavegave riserise toto thethe correspondingcorresponding dinucleosidedinucleoside pentaphosphates.pentaphosphates. InIn thethe absenceabsence ofof MgMg2+2+ ions,ions, thethe reactionreaction waswas extremelyextremely slow.slow.

Molecules 2019, 24, x 7 of 27

Scheme 6. Synthesis of Np4Ns starting from nucleoside 5′-triphosphates (NTPs) [47].

Similarly, the activation of UTP or ATP with DCC in anhydrous DMF, followed by the addition of the tris(tri-n-butylammonium) salt of UDP or ADP in the second step of the reaction, afforded Up5U and Ap5A, in 10–60% yields [8,49,50]. Reactions must be performed under strictly anhydrous conditions and long reaction times are required. In 2013, the Taylor research group reported a high-yielding synthesis of dinucleoside pentaphosphates (Np5N) using the tri(tetra-n-butylammonium) salt of cyclic trimetaphosphate 9 as a phosphorylating agent [59]. The latter was prepared in almost quantitative yield by conversion of the trisodium salt of trimetaphosphate 10 into its pyridinium form, and then by titration of the solution to pH 7.0 with a dilute solution of tetrabutylammonium hydroxide and freeze-drying (Scheme 7).

Scheme 7. Preparation of the cyclic trimetaphosphate 9 [59].

Then, the electrophilic reagent 9 was functionalized by a sulfonyl leaving group (LG) using N- methylimidazole (NMI) in the presence of either 2-mesitylenesulfonyl chloride 11 or 1- benzenesulfonyl-3-methyl-imidazolium triflate 12 in DMF (Scheme 8). Compound 11 was commercially available, whereas 12 was prepared in two steps starting from benzenesulfonyl chloride and imidazole [60]. Based on 31P NMR monitoring of the reaction, the authors proposed that theMolecules activation2019, 24 proceeds, 4334 via a mixed anhydride 13, which reacts with NMI to give rise to intermediate8 of 27 14.

Molecules 2019, 24, x Scheme 8. Activation of the cyc cycliclic trimetaphosphate 9 [59].[59]. 8 of 27

Addition of sub-stœchiometric amounts of NMPs to the activated cyclic trimetaphosphate 9 allowed their conversion to intermediates 15–18, as suggested by 31P NMR (Scheme 9). Addition of slight excess of the second NMP and anhydrous MgCl2 gave rise to the corresponding dinucleoside pentaphosphates. In the absence of Mg2+ ions, the reaction was extremely slow.

SchemeScheme 9.9. Synthesis of dinucleoside pentaphosphates via via an an activated activated cyclic cyclic trimetaphosphate trimetaphosphate [59]. [59]. 31P chemical shifts31P chemical are indicated shifts inare ppm, indicated together in ppm with, theirtogether multiplicity. with their multiplicity.

ThisThis method constitutes an an interesting interesting approach approach as as it itallows allows formation formation of ofsymmetric symmetric as aswell well as asmixed mixed Np Np5Ns5Ns in inhigh high yields, yields, and and uses uses inexpensive inexpensive reagents reagents (sodium (sodium cyclic trimetaphosphate, mesitylenemesitylene chloride, NMI). It has recently beenbeen appliedapplied forfor thethe synthesissynthesis ofof dicaptides,dicaptides, usedused asas substratessubstrates forfor polymerasespolymerases [[61].61]. However, it requires thethe preparationpreparation ofof thethe tetra-tetra-nn-butylammonium-butylammonium saltssalts ofof thethe cycliccyclic trimetaphosphatetrimetaphosphate and thosethose ofof thethe nucleotides,nucleotides, reactionreaction timestimes areare longlong ((>> 3 days), andand mustmust bebe performedperformed underunder strictlystrictly anhydrousanhydrous conditions.conditions.

3.4. Synthesis via a Phosphoromethylimidazolium Intermediate 3.4. Synthesis via a Phosphoromethylimidazolium Intermediate Reacting the free acids of canonical nucleoside 5 -monophosphates with a large excess of NMI Reacting the free acids of canonical nucleoside 50′-monophosphates with a large excess of NMI in the presence of triphenylphosphine/2,2 -dipyridyl sulfide as coupling agents and triethylamine in in the presence of triphenylphosphine/2,20′-dipyridyl sulfide as coupling agents and triethylamine in DMF or DMSO allowed dimerization to Np2N in about 85% yields [62]. Unsymmetrical dinucleotides DMF or DMSO allowed dimerization to Np2N in about 85% yields [62]. Unsymmetrical dinucleotides could also be obtained, albeit in much lower yields, by using a equimolar amount of the two NMPs to could also be obtained, albeit in much lower yields, by using a equimolar amount of the two NMPs be coupled. to be coupled. Another methodology to provide symmetrical and mixed dinucleoside polyphosphates Another methodology to provide symmetrical and mixed dinucleoside polyphosphates NpnN Np N(n = 2–4) was inspired by the Bogachev procedure for the synthesis of (n n= 2–4) was inspired by the Bogachev procedure for the synthesis of deoxynucleoside 5′- deoxynucleoside 5 -triphosphates (dNTPs) [63]. It involves the reaction of nucleoside triphosphates (dNTPs)0 [63]. It involves the reaction of nucleoside 5′-monophosphates-N- 5 -monophosphates-N-methylimidazolium as donors with an NMP, NDP or NTP to obtain methylimidazolium0 as donors with an NMP, NDP or NTP to obtain dinucleoside di, tri and dinucleoside di, tri and tetraphosphates (Scheme 10)[64]. These donors were prepared by treatment tetraphosphates (Scheme 10) [64]. These donors were prepared by treatment of the NMPs (disodium of the NMPs (disodium salts dihydrate or free acid hydrate) with a 16-fold excess of trifluoroacetic salts dihydrate or free acid hydrate) with a 16-fold excess of trifluoroacetic anhydride (TFAA) in anhydride (TFAA) in acetonitrile (ACN) in the presence of excess triethylamine. This step resulted acetonitrile (ACN) in the presence of excess triethylamine. This step resulted in the temporary protection of the hydroxyl and the amino (if present) groups and formation of a mixed anhydride, as shown by 31P NMR (δ = 2 ppm for the mixed anhydride formed within a few minutes). It offered the additional benefit of drying the starting materials without deleterious effects on the reaction outcome. However, in the case of GMP, these conditions resulted in the complete decomposition of the starting material. This limitation could be circumvented by using GMP in its tri-n-butylammonium form, together with reducing the number of equivalent of TFAA.

Molecules 2019, 24, 4334 9 of 27 in the temporary protection of the hydroxyl and the amino (if present) groups and formation of a mixed anhydride, as shown by 31P NMR (δ = 2 ppm for the mixed anhydride formed within a few minutes). It offered the additional benefit of drying the starting materials without deleterious effects on the reaction outcome. However, in the case of GMP, these conditions resulted in the complete decomposition of the starting material. This limitation could be circumvented by using GMP in its tri-Moleculesn-butylammonium 2019, 24, x form, together with reducing the number of equivalent of TFAA. 9 of 27

SchemeScheme 10.10. SynthesisSynthesis ofof dinucleotidesdinucleotides viavia phosphoromethylimidazoliumphosphoromethylimidazolium intermediatesintermediates [[64].64].

AfterAfter removalremoval ofof unreactedunreacted TFAA,TFAA, thethe mixedmixed anhydrideanhydride waswas reactedreacted withwith excessexcess NMINMI inin thethe presencepresence ofof triethylamine triethylamine to quantativelyto quantatively afford afford the highlythe highly reactive reactive N-methylimidazolium N-methylimidazolium salt donors salt 19–22donorswithin 19–22 10 within min. Formation10 min. Formation of the N-methylimidazolium of the N-methylimidazolium salts could salts also could be easily also followedbe easily 31 by P NMR31 spectroscopy as these species exhibit chemical shifts of approximately 9 to 10 ppm. followed by P NMR spectroscopy as these species exhibit chemical shifts of approximately− − −9 to −10 Theppm. crude The crude acetonitrile acetonitrile solutions solutions were were used used directly direct inly the in the next next step. step. Thus, Thus, addition addition of of the thedonors donors solutionssolutions toto thethe bis(tri-bis(tri-nn-butylammonium)-butylammonium) saltssalts ofof thethe nucleotidenucleotide acceptorsacceptors inin DMFDMF (in(in acetonitrileacetonitrile precipitationprecipitation of of the the acceptors acceptors was wa observed)s observed) led toled partially to partially protected prot products.ected products. The latter The were latter treated were withtreated aqueous with aqueous ammonium ammonium acetate to acetate remove to the remove protecting the groups,protecting providing groups, the providing desired compounds.the desired Forcompounds. dinucleotides For dinucleotides involving guanine involving nucleotides, guanine nucleotides, N,N-dimethylaniline N,N-dimethylaniline was found towas be found beneficial. to be Usingbeneficial. this procedure,Using this procedure, up to nine dinucleotidesup to nine dinucleotides were obtained were in obtained yields of in 51–68%. yields of It 51–68%. should beIt should noted thatbe noted this method that this cannot method be cannot used to be activate used to nucleoside activate nucleoside 50-di- or triphosphates. 5′-di- or triphosphates. While the While reactions the werereactions performed were performed under argon, under the argon, authors the pointed authors out pointed that vigorous out that drying vigorous of the drying donor of precursors the donor wasprecursors not always was necessary not always when necessary preparing when the dinucleoside preparing the di- anddinucleoside triphosphates. di- and Advantages triphosphates. of this one-potAdvantages method of this are one-pot short reaction method time are andshort good reaction yields. time and good yields. TheThe samesame groupgroup alsoalso reportedreported thethe synthesissynthesis ofof symmetricalsymmetrical di-di- andand tetraphosphatestetraphosphates byby dimerizationdimerization of nucleotides, nucleotides, using using sulfonyl sulfonyl imidazolium imidazolium salt salt 12 12as asa reagent a reagent (Scheme (Scheme 11) [60,65].11)[60 ,The65]. Thetetra- tetra-n-butylammoniumn-butylammonium salts salts of nucleoside of nucleoside 5′-mono 50-mono or 5′ or-diphosphates 50-diphosphates were were activated activated in DMF in DMF with with0.6–0.75 0.6–0.75 equiv equiv of 12 of in12 presencein presence of of1–3 1–3 equiv equiv of of NMI NMI and and MgCl MgCl22 (Scheme(Scheme 11).11). Dimerization Dimerization ofof thethe nucleotidesnucleotides ledled toto NpNp22NN andand NpNp44N in high yields. 31P NMR analyses showed that the reaction proceeds via the formation of the N-methylimidazolium salt intermediates 23–27 (Figure4, 31P δ = 9.2 ppm), which are able to react with the remaining − nucleotide to form the corresponding dimer. Mixed dinucleotides could also be obtained in high yields, by introducing minor modifications into the protocol. Accordingly, activation was performed in the presence of a small excess of sulfonyl imidazolium salt 12, and NMI was replaced by diisopropylethylamine (DIPEA) to prevent dimerization (Scheme 12). Moreover, the time of reaction of the first step was reduced to 1 min. Cp2A and Ap3U were obtained in similar yields starting from AMP and UMP, respectively.

Scheme 11. Synthesis of symmetrical Np2N and Np4N [60].

31P NMR analyses showed that the reaction proceeds via the formation of the N- methylimidazolium salt intermediates 23–27 (Figure 4, 31P δ = –9.2 ppm), which are able to react with the remaining nucleotide to form the corresponding dimer.

Molecules 2019, 24, x 9 of 27

Scheme 10. Synthesis of dinucleotides via phosphoromethylimidazolium intermediates [64].

After removal of unreacted TFAA, the mixed anhydride was reacted with excess NMI in the presence of triethylamine to quantatively afford the highly reactive N-methylimidazolium salt donors 19–22 within 10 min. Formation of the N-methylimidazolium salts could also be easily followed by 31P NMR spectroscopy as these species exhibit chemical shifts of approximately −9 to −10 ppm. The crude acetonitrile solutions were used directly in the next step. Thus, addition of the donors solutions to the bis(tri-n-butylammonium) salts of the nucleotide acceptors in DMF (in acetonitrile precipitation of the acceptors was observed) led to partially protected products. The latter were treated with aqueous ammonium acetate to remove the protecting groups, providing the desired compounds. For dinucleotides involving guanine nucleotides, N,N-dimethylaniline was found to be beneficial. Using this procedure, up to nine dinucleotides were obtained in yields of 51–68%. It should be noted that this method cannot be used to activate nucleoside 5′-di- or triphosphates. While the reactions were performed under argon, the authors pointed out that vigorous drying of the donor precursors was not always necessary when preparing the dinucleoside di- and triphosphates. Advantages of this one-pot method are short reaction time and good yields. The same group also reported the synthesis of symmetrical di- and tetraphosphates by dimerization of nucleotides, using sulfonyl imidazolium salt 12 as a reagent (Scheme 11) [60,65]. The tetra-n-butylammonium salts of nucleoside 5′-mono or 5′-diphosphates were activated in DMF with Molecules0.6–0.752019 equiv, 24, 4334of 12 in presence of 1–3 equiv of NMI and MgCl2 (Scheme 11). Dimerization 10of ofthe 27 nucleotides led to Np2N and Np4N in high yields.

Molecules 2019, 24, x 10 of 27

Molecules 2019, 24, x SchemeScheme 11.11. SynthesisSynthesis ofof symmetricalsymmetrical NpNp22N and Np4NN[ [60].60]. 10 of 27 Figure 4. Phosphoromethylimidazolium intermediates [60]. 31P NMR analyses showed that the reaction proceeds via the formation of the N- Mixed dinucleotides could also be obtained in high yields, by introducing minor modifications methylimidazolium salt intermediates 23–27 (Figure 4, 31P δ = –9.2 ppm), which are able to react with into the protocol. Accordingly, activation was performed in the presence of a small excess of sulfonyl the remaining nucleotide to form the corresponding dimer. imidazolium salt 12, and NMI was replaced by diisopropylethylamine (DIPEA) to prevent dimerization (Scheme 12). Moreover, the time of reaction of the first step was reduced to 1 min. Cp2A and Ap3U were obtainedFigure in similar 4. Phosphoromethylimidazolium yields starting from AMP intermediates and UMP, respectively. [[60].60].

Mixed dinucleotides could also be obtained in high yields, by introducing minor modifications into the protocol. Accordingly, activation was performed in the presence of a small excess of sulfonyl imidazolium salt 12, and NMI was replaced by diisopropylethylamine (DIPEA) to prevent dimerization (Scheme 12). Moreover, the time of reaction of the first step was reduced to 1 min. Cp2A and Ap3U were obtained in similar yields starting from AMP and UMP, respectively.

Scheme 12. Synthesis of mixed dinucleotides [[60].60].

Finally, mixed mixed adenine adenine dinucleotides dinucleotides were were obtained obtained in high in highyields yieldsby activating by activating the tetra- then- tetra-butylammoniumn-butylammonium salt of ATP salt ofwith ATP sulfonyl with sulfonyl imidazolium imidazolium salt 28, saltfollowed28, followed by reaction by reaction with a second with a 31 secondnucleotide nucleotide (Scheme (Scheme 13). Investigation 13). Investigation of the first of step the first by 31 stepP NMR by revealedP NMR that revealed the reaction that the proceeds reaction proceedsvia a cyclic via adenosine a cyclic adenosine trimetaphosphate trimetaphosphate intermediate intermediate 8. 8. Scheme 12. Synthesis of mixed dinucleotides [60].

Finally, mixed adenine dinucleotides were obtained in high yields by activating the tetra-n- butylammonium salt of ATP with sulfonyl imidazolium salt 28, followed by reaction with a second nucleotide (Scheme 13). Investigation of the first step by 31P NMR revealed that the reaction proceeds via a cyclic adenosine trimetaphosphate intermediate 8.

Scheme 13. Synthesis of mixed dinucleotides starting from ATP [[60].60].

The advantages of this methodmethod usingusing sulfonylimidazoliumsulfonylimidazolium salts as key reagents, includeinclude short reaction times,times, accessaccess toto aa widewide rangerange ofof NNppNs ((nn == 2–6) and high yields. However, careful attention must be paid to maintain anhydrousanhydrous conditions.conditions.

3.5. Synthesis via a PhosphoropiperidateScheme 13. Synthesis Intermediate of mixed dinucleotides starting from ATP [60]. In 2014, the Sun research group developed an approach based on the activation of nucleoside 5′- The advantages of this method using sulfonylimidazolium salts as key reagents, include short phosphoropiperidates with 4,5-dicyanoimidazole (DCI) as activator. The phosphoropiperidates 29– reaction times, access to a wide range of NpNs (n = 2–6) and high yields. However, careful attention 32 were obtained beforehand from unprotected NMPs via a redox condensation involving must be paid to maintain anhydrous conditions. triphenylphosphine and 2,2′-dithiodianiline (Scheme 14) [66].

3.5. Synthesis via a Phosphoropiperidate Intermediate In 2014, the Sun research group developed an approach based on the activation of nucleoside 5′- phosphoropiperidates with 4,5-dicyanoimidazole (DCI) as activator. The phosphoropiperidates 29– 32 were obtained beforehand from unprotected NMPs via a redox condensation involving triphenylphosphine and 2,2′-dithiodianiline (Scheme 14) [66].

Molecules 2019, 24, 4334 11 of 27

3.5. Synthesis via a Phosphoropiperidate Intermediate In 2014, the Sun research group developed an approach based on the activation of nucleoside 50-phosphoropiperidates with 4,5-dicyanoimidazole (DCI) as activator. The phosphoropiperidates 29–32 were obtained beforehand from unprotected NMPs via a redox condensation involving triphenylphosphineMolecules 2019, 24, x and 2,20-dithiodianiline (Scheme 14)[66]. 11 of 27

SchemeScheme 14.14. Synthesis of nucleoside 50′-phosphoropiperidates-phosphoropiperidates [66]. [66].

As shown shown in in Scheme Scheme 15, 15 the, the reaction reaction of nucleoside of nucleoside 5′-phosphoropiperidates 50-phosphoropiperidates with withthe tri- then- tri-butylammoniumn-butylammonium salts salts of nucleotides of nucleotides (NMPs, (NMPs, NDPs, NDPs, NT NTPs)Ps) in inthe the presence presence of of 2.5–3 2.5–3 equiv equiv of of DCI, DCI, led to thethe formation formation of of more more than than 20 symmetrical20 symmetrical or mixed or mixed dinucleoside dinucleoside di-, tri- di-, and tri- tetraphosphates and tetraphosphates [67,68]. Yields of NpnN’s dropped significantly along with the increase of the polyphosphate chain. [67,68]. Yields of NpnN’s dropped significantly along with the increase of the polyphosphate chain.

Scheme 15.15. Synthesis of dinucleotides from nucleoside 550′-phosphoropiperidates [67,68]. [67,68].

Among the the polar polar aprotic aprotic solvents, solvents, NN-methylpyrrolidone-methylpyrrolidone exhibited exhibited the the best best solvency solvency for forthe thetri- tri-n-butylammoniumn-butylammonium salts salts of ofboth both NDPs NDPs and and NTPs, NTPs, while while DMF DMF was was preferred preferred for for NMPs. NMPs. Considering Considering the costcost ofof nucleosidenucleoside 550′-polyphosphates and their similarsimilar chromatographychromatography properties with thethe products, onlyonly 0.50.5 equivequiv ofof NDPsNDPs oror NTPsNTPs werewere used.used. This P(V) method is an eefficientfficient one, allowing access to a large rangerange ofof NpNpnnNs ( n = 2–4)2–4) in in short short reaction reaction times times and and good good to to high high isolated isolated yields. Symmetrical NpNpnnN(N n(n= 3–5)= 3–5) could could also bealso obtained be obtained by reacting by nucleosidereacting nucleoside 50-phosphorop 5′- iperidatesphosphoropiperidates with sub-stœchiometric with sub-stœchiometri amounts (0.35–0.4c amounts equiv) of inorganic(0.35–0.4 phosphorylating equiv) of inorganic agents in thephosphorylating presence of excess agents DCI in [the69]. presence In this alternative of excess DCI protocol, [69]. bis(tetra-In this alternativen-butylammonium) protocol, bis(tetra- hydrogenn- phosphate,butylammonium) tris(tetra- hydrogenn-butylammonium) phosphate, tris(tetra- hydrogenn pyrophosphate-butylammonium) or tris(tetra-hydrogenn-butyl-ammonium) pyrophosphate or 31 dihydrogentris(tetra-n-butyl-ammonium) triphosphate were useddihydrogen instead oftriphosphate a nucleotide we (Schemere used 16 instead). P NMR of atracking nucleotide experiments (Scheme showed16). 31P thatNMR pyrophosphate tracking experiments reacts first showed with the that starting pyrophosphate phosphoropiperidate reacts first to formwith anthe NTP, starting and then,phosphoropiperidate with the remaining to form phosphoropiperidate an NTP, and then, with to give the remaining rise to Np 4phosphoropiperidateN. This methodology towas givealso rise adaptedto Np4N. to This the methodology synthesis of nucleotides was also adapted with a higherto the synthesis number ofof phosphatenucleotides groups with a (such higher as number Np5N), usingof phosphate inorganic groups triphosphate (such as insteadNp5N), ofusing pyrophosphate. inorganic triphosphate instead of pyrophosphate.

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Scheme 16. Synthesis of symmetrical NpN by dimerization [67,68]. SchemeScheme 16. SynthesisSynthesis of of symmetrical symmetrical NpN NpN by by dimerization dimerization [67,68]. [67,68]. Interestingly, this strategy can be adapted to the synthesis of nucleotide analogues such as 2 3 dinucleosideInterestingly,Interestingly, P ,Pthis this-(dihalo)methylene strategy strategy can can be be adapted adaptedtetraphosphates, to to the the synthesis synthesis by ofreplacing ofnucleotide nucleotide pyrophosphate analogues analogues such suchwith as 2 3 dinucleosideasbisphosphonate dinucleoside P2 ,reagents.PP3-(dihalo)methylene,P -(dihalo)methylene tetr tetraphosphates,aphosphates, by byreplacing replacing pyrophosphate pyrophosphate with bisphosphonatebisphosphonate reagents. 4. Synthesis Based on P(III) Chemistry 4. Synthesis Based on P(III) Chemistry 4. SynthesisSeveral Based methods on involveP(III) Chemistry P(III) reag ents to provide phosphite or phosphoramidite intermediates, Several methods involve P(III) reagents to provide phosphite or phosphoramidite intermediates, whichSeveral are further methods oxidized involve to P(III) P(V). reag Initiallyents to developped provide phosphite for the orsynthesis phosphoramidite of , intermediates, they which are further oxidized to P(V). Initially developped for the synthesis of oligonucleotides, they whichhave beenare further adapted oxidized to dinucleotides to P(V). Initially by introducing developped minor for the modifications synthesis of inoligonucleotides, the last steps ofthey the have been adapted to dinucleotides by introducing minor modifications in the last steps of the reaction havereaction been sequences. adapted toBecaus dinucleotidese of the high by reactivityintroducing of P(III)minor spec modificationsies, most of thesein the approaches last steps requireof the sequences. Because of the high reactivity of P(III) species, most of these approaches require the use of reactionthe use sequences. of N-, and Becaus O-protectinge of the high groups reactivity on the of P(III)starting spec nucleos(t)idesies, most of these and approaches strictly anhydrous require N-, and O-protecting groups on the starting nucleos(t)ides and strictly anhydrous conditions. theconditions. use of N-, and O-protecting groups on the starting nucleos(t)ides and strictly anhydrous conditions. 4.1.4.1. SynthesisSynthesis viavia aa SalicylphosphiteSalicylphosphite IntermediateIntermediate 4.1. SynthesisThisThis strategystrategy via a Salic isis basedbasedylphosphite onon thethe Intermediate well-knownwell-known Ludwig–EcksteinLudwig–Eckstein procedureprocedure forfor NTPNTP synthesissynthesis [[70],70], and allows the one-pot preparation of symmetric dinucleoside tetra- and pentaphosphates andThis allows strategy the one-pot is based preparation on the well-known of symmetric Ludwig–Eckstein dinucleoside tetra-procedure and pentaphosphates for NTP synthesis starting [70], starting from protected [71]. Phosphitylation of triacetyl adenosine 33 with andfrom allows protected the one-pot nucleosides preparation [71]. Phosphitylation of symmetric dinucleoside of triacetyl adenosinetetra- and pentaphosphates33 with 2-chloro-4 startingH-l,3,2- 2-chloro-4H-l,3,2-benzo-dioxaphosphorin-4-one (salicylchlorophosphite) led to intermediate 34, then frombenzo-dioxaphosphorin-4-one protected nucleosides [71]. (salicylchlorophosphite) Phosphitylation of triacetyl led toadenosine intermediate 33 with 34 , 2-chloro-4then additionH-l,3,2- of addition of bis(tri-n-butylammonium) pyrophosphate gave rise to the cyclic intermediate P(III)–P(V) benzo-dioxaphosphorin-4-onebis(tri-n-butylammonium) pyrophosphate (salicylchlorophosphite) gave rise to the led cyclic to intermediate intermediate 34P(III)–P(V), then addition 35 (Scheme of 35 (Scheme 17). Oxidation to the cyclic trimetaphosphate, intermediate 36, followed by reaction with bis(tri-17). Oxidationn-butylammonium) to the cyclic pyrophosphate trimetaphosphate, gave intermediaterise to the cyclic 36, intermediatefollowed by reaction P(III)–P(V) with 35 adenosine (Scheme adenosine 50-monophosphate (AMP) or adenosine 50-diphosphate (ADP) in dry DMF in the presence of 17).5′-monophosphate Oxidation to the (AMP) cyclic trimetaphosphate,or adenosine 5′-diphosphate intermediate (ADP) 36, followed in dry DMF by reaction in the presence with adenosine of ZnCl 2 ZnClas catalyst,2 as catalyst, afforded afforded 37 and37 38and, respectively.38, respectively. Removal Removal of the of acetyl the acetyl groups groups under under basic basic conditions, conditions, ion 5′-monophosphate (AMP) or adenosine2+ 5′-diphosphate (ADP) in dry DMF in the presence of ZnCl2 ion exchange to remove the Zn2+ cations and purification led to Ap4A and Ap5A as ammonium salts. asexchange catalyst, toafforded remove 37 the and Zn 38, cationsrespectively. and purification Removal of ledthe toacetyl Ap4 Agroups and Ap under5A as basic ammonium conditions, salts. ion It It should be noted that symmetrical Ap A was obtained in much better yield than Ap A. should be noted that symmetrical Ap4A4 was obtained in much better yield than Ap5A.5 exchange to remove the Zn2+ cations and purification led to Ap4A and Ap5A as ammonium salts. It should be noted that symmetrical Ap4A was obtained in much better yield than Ap5A.

SchemeScheme 17. 17.Synthesis Synthesis of of the the symmetrical symmetrical dinucleotides dinucleotides Ap Ap4A4A andand ApAp55AA byby phosphitylationphosphitylation [[71].71].

Scheme 17. Synthesis of the symmetrical dinucleotides Ap4A and Ap5A by phosphitylation [71].

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The derivatives Gp4G and Ap4G were prepared in a similar manner, starting from The guanosine derivatives Gp4GG and Ap 4GG were were prepared prepared in in a similar manner, starting from 20′,3,3′0--OO-2--2-NN-triacetylguanosine-triacetylguanosine 3939 (Scheme(Scheme 18).18).

Scheme 18. Synthesis of dinucleotides Gp4G and Ap4G by phosphitylation [71]. SchemeScheme 18.18. Synthesis ofof dinucleotidesdinucleotides GpGp44G and Ap4G by phosphitylation phosphitylation [71]. [71].

4.2. Synthesis via a Cyclosaligenyl Phosphite Intermediate This strategy initially developed by Meier and co-workers for the synthesis of prodrugs of nucleotides, calledcalledcyclo cycloSalSal (for (forcyclo cyclosaligenyl)saligenyl) pronucleotides pronucleotides [72,73 [72,73],], has been has applied been applied to the synthesis to the ofsynthesis nucleoside of 5nucleoside0-polyphosphates, 5′-polyphosphates, sugar nucleotides sugar and nucleotide mixed dinucleotidess and mixed [74 ,75dinucleotides]. Phosphochloridite [74,75]. 40Phosphochloriditewas prepared from 40 thewascorresponding prepared from salicyl the alcoholcorrespond anding phosphorus salicyl trichloride and (Schemephosphorus 19). Reactiontrichloride with (Scheme 30-O-acetylthymidine 19). Reaction with followed 3′-O-acetylthymidine by oxidation withfollowed oxone by (2 oxidation KHSO5.KHSO with oxone4.K2SO (24) aKHSOfforded5.KHSO 5-nitro-4.Kcyclo2SO4Sal-3) afforded-O-acetylthymidine 5-nitro-cycloSal-3 phosphotriester′-O-acetylthymidine41. phosphotriester 41. KHSO5.KHSO4.K2SO4) afforded0 5-nitro-cycloSal-3′-O-acetylthymidine phosphotriester 41.

Scheme 19. Synthesis of a cycloSal phosphotriester deriva derivativetive of [[74].74].

This phosphotriester was too sensitive to moisture to be purified by chromatography. Therefore, This phosphotriester was too sensitive to moisture to be purified by chromatography. Therefore, the crude material (obtained after liquid-liquid extraction) was directly engaged in the next step, i.e., the crude material (obtained after liquid-liquid extraction) was directly engaged in the next step, i.e., reaction with rigorously dried tetra-n-butylammonium UMP and tris(tetra-n-butylammonium) ATP in reaction with rigorously dried tetra-n-butylammonium UMP and tris(tetra-n-butylammonium) ATP + + + + DMFin DMF (Scheme (Scheme 20). 20). After After removal removal of the of acetylthe acetyl group group and ion-exchangeand ion-exchange (n-NBu (n4-NButo NH4+ to4 NH), Up4+),2 TUp and2T in DMF (Scheme 20). After removal of the acetyl group and ion-exchange (n-NBu4+ to NH4+), Up2T Apand4 TAp were4T were obtained obtained in their in their ammonium ammonium form. form. and Ap4T were obtained in their ammonium form.

SchemeScheme 20.20. SynthesisSynthesis ofof thethe dinucleotidesdinucleotides UpUp22T and Ap4TT[ [74].74]. Scheme 20. Synthesis of the dinucleotides Up2T and Ap4T [74]. This approach has been adapted to solid phase [76]. The key intermediates of the synthesis are This approach has been adapted to solid phase [76]. The key intermediates of the synthesis are the 50-substituted cycloSal nucleotides linked to a polystyrene support through the 30-OH group and a the 5′-substituted cycloSal nucleotides linked to a polystyrene support through the 3′-OH group and succinyl linker (42–44, Figure5). a succinyl linker (42–44, Figure 5).

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Figure 5. Structure of the supported nucleoside phosphotriesters [76]. Figure 5. Structure of the supported nucleoside phosphotriesters [[76].76]. Figure 5. Structure of the supported nucleoside phosphotriesters [76]. Preparation of of these these intermediates intermediates started started with the with anchoring the anchoring of the succinyl of the linker succinyl to the linker 5′′-O- (4,4′-dimethoxytrityl)-protected nucleosides, and removal of the protecting group with trifluoroacetic to(4,4 the′-dimethoxytrityl)-protectedPreparation 50-O-(4,40 -dimethoxytrityl)-protectedof these intermediates nucleosides, started and nucleosides, with removal the anchoring of andthe protecting removal of the succinyl group of the with protectinglinker trifluoroacetic to the group 5′-O- acid (TFA). The 3′-O-succinylnucleosides were then treated with the appropriate chlorophosphites, withacid(4,4′-dimethoxytrityl)-protected (TFA). trifluoroacetic The 3′-O-succinylnucleosides acid (TFA). nucleosides, The 3were0-O and-succinylnucleosides then removal treated of thewith protecting the were appropriate group then with treatedchlorophosphites, trifluoroacetic with the appropriatefollowedacid (TFA). by Theoxidation chlorophosphites, 3′-O-succinylnucleosides with oxone, followed to give wererise by oxidationto then compounds treated with with 45 oxone,– 47the (Scheme(Scheme appropriate to give 21).21). rise Coupling Couplingchlorophosphites, to compounds withwith thethe 45aminomethylfollowed–47 (Scheme by oxidation polystyrene 21). Coupling with support oxone, with towas give the achieved aminomethylrise to compoundsusing 1-hydroxybenzotriazole polystyrene 45–47 (Scheme support 21). was (HOBt)Coupling achieved and with usingN,N’ the- 1-hydroxybenzotriazolediisopropylcarbodiimideaminomethyl polystyrene (HOBt)(DIC) support as andco wasuplingN,N’ achieved -diisopropylcarbodiimideagents. using Alternatively, 1-hydroxybenzotriazole 2-(1H (DIC)-benzotriazole-1-yl)-1,1,3,3- as (HOBt) coupling and agents. N,N’- Alternatively,tetramethyluroniumdiisopropylcarbodiimide 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (DIC) as coupling (TBTU) agents. and 4-ethylmorpholine Alternatively, 2-(1 tetrafluoroborate Hmay-benzotriazole-1-yl)-1,1,3,3- be used. Conversion (TBTU) and to Up2T and Up2dA was carried out by adding a large excess of tetra-n-butylammonium UMP, and then 4-ethylmorpholineUptetramethyluronium2T and Up2dA was may tetrafluoroborate carried be used. out by Conversion adding (TBTU) a tolarge and Up excess2 4-ethylmorpholineT and of Up tetra-2dAn was-butylammonium carriedmay be out used. by UMP, addingConversion and alarge then to cleavage under basic conditions (70–78% purity, no overall yield was given). Compared to the excesscleavageUp2T and of tetra- underUp2dAn -butylammoniumbasic was carriedconditions out by UMP,(70–78% adding and purity,a then large cleavage excessno overall of under tetra- yield basicn-butylammonium was conditions given). (70–78%Compared UMP, purity, and to then the no overallsolutioncleavage yield phase under was synthesis, basic given). conditions this Compared supported (70–78% tothe approach solutionpurity, is no phaselessless overall straightforwardstraightforward synthesis, yield thiswas andand supported given). involvesinvolves Compared approach multiplemultiple toissteps.steps. lessthe straightforwardsolution phase synthesis, and involves this supported multiple steps. approach is less straightforward and involves multiple steps.

Scheme 21. Solid-phase synthesis of dinucleoside 5′,5′-diphosphates [76]. Scheme 21. Solid-phase synthesis of dinucleoside 5′,5′-diphosphates [76]. SchemeScheme 21.21. Solid-phaseSolid-phase synthesissynthesis ofof dinucleosidedinucleoside 550′,5,50′-diphosphates [76]. [76]. 4.3. Synthesis via a Supported Phosphite Intermediate 4.3. Synthesis via a Supported Phosphite Intermediate 4.3. Synthesis via a Supported Phosphite Intermediate Another P(III) strategy combined with a solid-phase approach was reported by Ahmadibeni and Another P(III) strategy combined with a solid-phase approach was reported by Ahmadibeni and ParangAnother [77]. Using P(III) strategythese polymer- combinedbound with phosphitylating a solid-phase approach reagents, was symmetrical reported by 5‘,5‘-dinucleoside Ahmadibeni and Parang [77]. Using these polymer-bound phosphitylating reagents, symmetrical 5‘,5‘-dinucleoside mono,Parang di, [77]. tri, Usingand tetraphospha these polymer-tes werebound prepared. phosphitylating Compounds reagents, 48–51 symmetrical werewere obtainedobtained 5‘,5‘-dinucleoside inin 22 toto 55 stepssteps mono, di, tri, and tetraphosphates were prepared. Compounds 48–51 were obtained in 2 to 5 steps startingmono, di, from tri, andphosphorus tetraphospha trichloridetes were (Figure prepared. 6). CouplingCompounds with 48 –aminomethyl51 were obtained polystyrene in 2 to 5 resin- steps starting from phosphorus trichloride (Figure6). Coupling with aminomethyl polystyrene resin-bound boundstarting p -acetoxybenzylfrom phosphorus alcohol trichloride yielded (Figure four classe 6). Couplings of supported-phosphi with aminomethyltylating polystyrene reagents 52resin-–55 p-acetoxybenzyl alcohol yielded four classes of supported-phosphitylating reagents 52–55 (Figure6). (Figurebound p6).-acetoxybenzyl alcohol yielded four classes of supported-phosphitylating reagents 52–55 (Figure 6).

Figure 6. Synthesis of the immobilized phosphitylating reagents [[77].77]. Figure 6. Synthesis of the immobilized phosphitylating reagents [77]. Figure 6. Synthesis of the immobilized phosphitylating reagents [77].

Molecules 2019, 24, x 15 of 27 Molecules 2019, 24, 4334 15 of 27 Molecules 2019, 24, x 15 of 27 These supported reagents were reacted with unprotected nucleosides (e.g., thymidine, adenosine,TheseThese , supportedsupported inos reagentsreagentsine or the werewere analogue reactedreacted 3 ′ with-azido-3with unprotectedunprotected′-deoxythymidine) nucleosidesnucleosides in the (e.g.,(e.g., presence thymidine,thymidine, of 5- (ethylthio)-1adenosine,adenosine, cytidine, H-tetrazole inos toine afford or or the thecompounds analogue analogue 356 3′-azido-30-azido-3–59 (Scheme′-deoxythymidine)0-deoxythymidine) 22). Polymer-bound in inthe the presence presencenucleotides of of5- underwent5-(ethylthio)-1(ethylthio)-1 oxidationHH-tetrazole-tetrazole with to to tertafford aff-butylord compounds compounds hydroperoxide 56–59 to (Scheme (Scheme60–63, then 2222).). removal Polymer-boundPolymer-bound of the cyanoethoxy nucleotidesnucleotides groupsunderwentunderwent with oxidation oxidation1,8-diazabicyclo[5.4.0]undec-7-en with withtert tert-butyl-butyl hydroperoxide hydroperoxide (DBU) to was60 to–63 performed.60, then–63, removalthen Acidic removal of thetreatment cyanoethoxyof the ofcyanoethoxy 60 and groups 64– with66groups gave 1,8-diazabicyclo[5.4.0]undec-7-en withrise 1,8-diazabicyclo[5.4.0]undec-7-ento the corresponding symmetrical (DBU) was (DBU) dinu performed. wascleotides performed. Acidic in 59–78% treatment Acidic yields treatment of 60startingand of64 60 from– 66andgave the64– supportedrise66 gave to the rise correspondingreagents to the 52 corresponding–55. symmetrical symmetrical dinucleotides dinu incleotides 59–78% in yields 59–78% starting yields from starting the supported from the reagentssupported52 –reagents55. 52–55.

Scheme 22. Synthesis of symmetrical dinucleotides using polymer-bound phosphitylating reagents. Yields are given for the adenine derivatives [77]. SchemeScheme 22. 22.Synthesis Synthesis ofof symmetricalsymmetrical dinucleotidesdinucleotides using polymer-bound phosphitylating reagents. reagents. Yields are given for the adenineYields are derivatives given for [the77]. adenine derivatives [77]. This strategy allowed the synthesis of symmetrical dinucleotides starting from unprotected nucleosidesThisThis strategystrategy without allowedallowed the need thethe to synthesissynthesis purify the ofof symmetricalsymmetrintermediates.ical dinucleotidesdinucleotides It also offers startingstarting the advantage fromfrom unprotectedunprotected of facile recoverynucleosidesnucleosides of withoutfinalwithout products the the need need by to fi purifytoltration purify the (trapped intermediates.the intermediates. linkers It alsoon Itthe o ffalso ersresins). theoffers advantage However, the advantage of it facile requires recoveryof facile the multistepofrecovery final products ofsynthesis final byproducts of filtration the supported by (trappedfiltration polyphosphite linkers(trapped on linkers the reagents. resins). on theItHowever, should resins). be However,it highlighted requires it the requiresthat multistep part theof thissynthesismultistep work ofcouldsynthesis the not supported beof reproducedthe supported polyphosphite by otherspolyphosphite reagents. [78]. Itreagents. should beIt should highlighted be highlighted that part of that this part work of couldthis work not becould reproduced not be reproduced by others [ 78by]. others [78]. 4.4. Synthesis via a 5′-H-Phosphonate Intermediate 4.4. Synthesis via a 50-H-Phosphonate Intermediate 4.4. SynthesisThis one-pot via a strategy5′-H-Phosphonate developed Intermediate by Sun et al. [79], and based on their previous work on the This one-pot strategy developed by Sun et al. [79], and based on their previous work on the synthesis synthesisThis ofone-pot nucleoside strategy triphosphates developed [80], by Suninvolv et esal. the [79], formation and based of a on pyridinium their previous phosphoramidate work on the of nucleoside triphosphates [80], involves the formation of a pyridinium phosphoramidate intermediate intermediatesynthesis of nucleosidestarting from triphosphates a nucleoside [80], 5′-H involv-phosphonatees the formation monoester. of a Nucleosidepyridinium 5 phosphoramidate′-H-phosphonate starting from a nucleoside 5 -H-phosphonate monoester. Nucleoside 5 -H-phosphonate monoesters monoestersintermediate 67starting–70 were from aobtained0 nucleoside from 5′-H -phosphonate2′,3′-O-isopropylidene monoester. ribonucleosides Nucleoside0 5′-H -phosphonate71–74 using 67–70 were obtained from 2 ,3 -O-isopropylidene ribonucleosides 71–74 using diphenylphosphite diphenylphosphitemonoesters 67–70 aswere a reagent obtained0 0 (Scheme from 23).2′,3 Th′-Oen,-isopropylidene removal of the ribonucleosides isopropylidene 71group–74 usingwith as a reagent (Scheme 23). Then, removal of the isopropylidene group with aqueous TFA afforded aqueousdiphenylphosphite TFA afforded as 5a′- Hreagent-phosphonate (Scheme monoesters 23). Then, 75 removal–78 in yields of the ranging isopropylidene from 68% togroup 82% overwith 5 -H-phosphonate monoesters 75–78 in yields ranging from 68% to 82% over two steps. twoaqueous0 steps. TFA afforded 5′-H-phosphonate monoesters 75–78 in yields ranging from 68% to 82% over two steps.

SchemeScheme 23.23. Synthesis of nucleoside 50′-H-phosphonates from the 20′,3,3′0-protected-protected nucleosides nucleosides [79]. [79]. Scheme 23. Synthesis of nucleoside 5′-H-phosphonates from the 2′,3′-protected nucleosides [79].

Molecules 2019, 24, x 16 of 27

MoleculesMolecules 20192019,, 2424,, 4334x 1616 of 2727 H-phosphonates were silylated with trimethylsilyl chloride (TMSCl) in pyridine, and in situ oxidizedH-phosphonates with iodine wereto generate silylated the with highly trimethylsilyl reactive zwitterionicchloride (TMSCl) pyridinium in pyridine, phosphoramidate and in situ oxidizedintermediatesH-phosphonates with 79 iodine–82 (Scheme wereto generate silylated 24). Indeed, the with highly even trimethylsilyl tracereactive amounts zwitterionic chloride of water (TMSCl) pyridinium may result in pyridine, inphosphoramidate the formation and in situ of oxidizedintermediatespolyphosphate with 79 iodine–by-products.82 (Scheme to generate 24).The Indeed, theauthors highly even hypoth reactivetrace amountsesized zwitterionic that of water if 50% pyridinium may of result the in phosphoramidatephosphoramidate the formation of intermediatespolyphosphateintermediate could79 –by-products.82 be(Scheme hydrolyzed 24 The). Indeed, withauthors precise even hypoth trace control, amountsesized NMP that ofgenerated water if 50% may in of resultsitu the would inphosphoramidate the react formation with the of polyphosphateintermediateremaining phosphoramidate could by-products. be hydrolyzed The to authors afford with hypothesizedpreciseNp2N control,as a thatmajor NMP if 50%product. generated of the phosphoramidateAccordingly, in situ would hydrolysis react intermediate with and the could be hydrolyzed with precise control,31 NMP generated in situ would react with the remaining remainingcoupling steps phosphoramidate were optimized to usingafford NpP NMR2N as trackinga major experimentproduct. Accordingly,s and accomplished hydrolysis by andtwo phosphoramidatecouplingsequential steps additions were to aofoptimizedff ordH2O Npin 2DMF.N using as aAfter major31P purificationNMR product. tracking Accordingly,and experimention exchange, hydrolysiss and symmetrical accomplished and coupling dinucleoside by steps two were optimized using 31P NMR tracking experiments and accomplished by two sequential additions sequentialdiphosphates additions sodium of saltsH2O inwere DMF. obtained After purification in 68–91% and yields ion exchange,starting from symmetrical the nucleoside dinucleoside 5′-H- ofdiphosphatesphosphonates. H2O in DMF. sodium Advantages After purification salts of were this and methodobtained ion exchange, are in the 68–91% sh symmetricalort reactionyields startingtime, dinucleoside simple from purification diphosphatesthe nucleoside procedure sodium 5′-H- saltsphosphonates.and werehigh obtainedyields. Advantages However, in 68–91% of it yieldsthis requires method starting the are frompreliminary the theshort nucleoside reaction synthesis time, 50- Hof-phosphonates. simple the H purification-phosphonate Advantages procedure starting of thisandmaterials. methodhigh yields. are the However, short reaction it requires time, simplethe preliminary purification synthesis procedure of andthe highH-phosphonate yields. However, starting it requiresmaterials. the preliminary synthesis of the H-phosphonate starting materials.

Scheme 24. Synthesis of symmetrical dinucleotides from nucleoside 5′-H-phosphonates [79]. SchemeScheme 24.24. SynthesisSynthesis ofof symmetricalsymmetrical dinucleotidesdinucleotides fromfrom nucleosidenucleoside 550′--H-phosphonates-phosphonates [[79].79]. 4.5. Synthesis via a Phosphoramidite Intermediate 4.5. Synthesis via a Phosphoramidite Intermediate 4.5. SynthesisIn 2015, viathe a JessenPhosphoramidite research Intermediategroup developed a very efficient strategy based on an iterative In 2015, the Jessen research group developed a very efficient strategy based on an iterative phosphoramiditeIn 2015, the approachJessen research to access group nucleoside developed 5′-polyphosphates a very efficient [81,82] strategy and basedsome derivativeson an iterative [83]. phosphoramidite approach to access nucleoside 5 -polyphosphates [81,82] and some derivatives [83]. phosphoramiditeThe phosphoanhydride approach bond to accessis formed nucleoside by coupling 50′-polyphosphates a phosphoramidite [81,82] (the and donor) some derivatives to a phosphate [83]. The phosphoanhydride bond is formed by coupling a phosphoramidite (the donor) to a phosphate (the The(the phosphoanhydrideacceptor). In contrast bond to isthe formed other byP(III) coupling methods, a phosphoramidite all steps were (theperformed donor) underto a phosphate ambient acceptor). In contrast to the other P(III) methods, all steps were performed under ambient conditions, (theconditions, acceptor). i.e., In in contrast open flasksto the andother using P(III) non- methods,dried allsolvents. steps wereRegarding performed of the under synthesis ambient of i.e., in open flasks and using non-dried solvents. Regarding of the synthesis of dinucleotides, conditions,dinucleotides, i.e., 2 ′,3in′- Oopen-diacetyl flasks uridine and 83using was treatednon-dried with solvents. phosphorodiamidite Regarding of84 inthe the synthesis presence of 2 ,3 -O-diacetyl uridine 83 was treated with phosphorodiamidite 84 in the presence of 1H-tetrazole to dinucleotides,10H-tetrazole0 to 2 afford′,3′-O-diacetyl phosphoramidite uridine 83 85 was (Scheme treated 25). with Addition phosphorodiamidite of the tetra-n-butylammonium 84 in the presence salt of afford phosphoramidite 85 (Scheme 25). Addition of the tetra-n-butylammonium salt of UMP and 1ofH UMP-tetrazole and DCI,to afford followed phosphoramidite by oxidation 85with (Scheme mCPBA, 25). treatment Addition with of the piperidine, tetra-n-butylammonium and cleavage of saltthe DCI, followed by oxidation with mCPBA, treatment with piperidine, and cleavage of the acetyl groups ofacetyl UMP groups and DCI, under followed basic by conditions,oxidation with gave mCPBA, rise totreatment Up2U withas a piperidine,mixed piperidinium/tetra- and cleavage of then- under basic conditions, gave rise to Up2U as a mixed piperidinium4 /tetra-n-butylammonium salt. The acetylbutylammonium groups under salt. basicThe mixedconditions, dinucleotide gave rise Ap toU wasUp2 Ualso as obtaineda mixed bypiperidinium/tetra- using the tetra-n- mixed dinucleotide Ap4U was also obtained by using the tetra-n-butylammonium salt of ATP and butylammonium saltsalt. of The ATP mixed and 5-ethylthio-1 dinucleotideH-tetrazole Ap4U was (ETT) also as anobtained activator by in using the second the tetra- step nof- 5-ethylthio-1H-tetrazole (ETT) as an activator in the second step of the reaction (Scheme 25). It should butylammoniumthe reaction (Scheme salt 25).of ATP It should and 5-ethylthio-1 be noted thatH-tetrazole these experiments (ETT) as anwere activator performed in the on second a small step scale of be noted that these experiments were performed on a small scale (10 mg). the(10 mg).reaction (Scheme 25). It should be noted that these experiments were performed on a small scale (10 mg).

SchemeScheme 25.25. SynthesisSynthesis ofof dinucleotidesdinucleotides viavia aa phosphoramiditephosphoramidite intermediateintermediate [[83].83]. Scheme 25. Synthesis of dinucleotides via a phosphoramidite intermediate [83].

Molecules 2019, 24, 4334 17 of 27 MoleculesMolecules 20192019,, 2424,, xx 1717 ofof 2727

SymmetricalSymmetrical Np NpnnNsNs ( (nn == 3,3, 5, 5, 7) 7) could could also also bebe accessedaccesseaccessedd viavia chemoselectivechemoselectivechemoselective homologativehomologative dimerizationdimerization ofof twotwo phosphatephosphate monoestersmomonoestersnoesters withwith phosphorodiamiditesphosphorodiamiditephosphorodiamiditess [[84].[84].84]. The The gene generalgeneralral strategy strategy for for thethe synthesissynthesissynthesis of ofof dinucleoside dinucleosidedinucleoside triphosphates triphosphatestriphosphates is isis shown shownshown in inin Figure FigureFigure7. First,7.7. FirsFirs phosphordiamiditest,t, phosphordiamiditesphosphordiamidites86 86and86 andand87 (donors)8787 (donors) (donors) were were were coupled coupled coupled with with with a NMPa a NMP NMP (the (the (the acceptor), acceptor acceptor),), a affording ffaffordingording terminal terminal terminalmixed mixed mixed phosphoricphosphoric phosphoric anhydrideanhydride phosphoramiditesphosphoramidites 8888––8989 asas unstableunstableunstable intermediates.intermediates.intermediates. Reaction Reaction with with another another phosphatephosphate monoestermonoester V III V resultedresulted inin thethe formationformation ofof 90 9090 containing containingcontaining aaa P PPVV–P–PIIIIII–P–PVV bridge,bridge,bridge, whichwhich which waswas was thenthen oxidizedoxidized toto yieldyield 91.9191.. TheThe correspondingcorresponding NpNp333NN was was obtainedobtained afterafter aa finalfinalfinal deprotectiondeprotection step.step.

FigureFigure 7.7. GeneralGeneral approachapproach forfor thethe preparationpreparation ofof symmetricalsymmetrical NpNp33NsNs basedbased onon dimerizationdimerization [84].[[84].84].

TheThe tetra-tetra-nn-butylammonium-butylammonium salts salts ofof theththee canonicalcanonical nucleosidenucleoside 550′′-monophosphates-monophosphates were were treated treated inin DMFDMF withwith 9-fluorenylmethyl-9-fluorenylmethyl-9-fluorenylmethyl-NN,,NN,,NN’,’,NN’-tetraalkyl’-tetraalkyl’-tetraalkyl phos phosphorodiamiditesphosphorodiamiditesphorodiamidites 8686––8787 ininin the the presence presence of of 31 ETT,ETT, followedfollowed byby oxidationoxidation withwithwith m mmCPBACPBACPBA (Scheme(Scheme(Scheme 26 26).26).). ProgressProgress ofofof thethethe reactionreactionreaction waswaswas monitored monitoredmonitored by byby 3131PP NMR.NMR. TheThe Fm-protectedFm-protected intermediatesintermediates werewewerere isolatedisolated byby precipitationprecipitation withwith EtEt22O/hexane,OO/hexane,/hexane, treatedtreated withwith piperidine,piperidine, andand purifiedpurifiedpurified byby anionanion exchangeexchange chromatographychromatography toto isolateisolate thethe symmetricalsymmetrical dinucleosidedinucleoside triphosphatestriphosphates asas ammoniumammonium saltssalts inin goodgood yields.yields.

SchemeScheme 26.26. SynthesisSynthesisSynthesis of of dinucleotides dinucleotides viavia didimerizationdimerizationmerization ofof phosphatesphosphates [84].[[84].84].

ThisThis methodologymethodology was was applied applied to to thethe synthesissynthesis ofof symmetricalsymmetrical dinucleosidedinucleoside penta-penta- andand heptaphosphatesheptaphosphates by byby using usingusing as asas acceptor acceptoracceptor a NDP aa NDPNDP or a or NTP,respectively.or aa NTP,NTP, respectively.respectively. However, However,However, due to theduedue water-sensivity toto thethe water-water- sensivityofsensivity the intermediates, ofof thethe intermediates,intermediates, reactions had reactionsreactions to be performed hadhad toto bebe under performedperformed anhydrous underunder conditions, anhydrousanhydrous with conditions,conditions, a shorter reaction withwith aa shortertimeshorter and reactionreaction low temperature timetime andand lowlow (coupling temperaturetemperature with (coupling ETT(coupling 30 s at withwith 0 ◦ C,ETTETT oxidation 3030 ss atat 00 5°C,°C, min oxidationoxidation at 0 ◦C). 55 Accordingly,minmin atat 00 °C).°C). ApAccordingly,Accordingly,5A and Ap Ap7ApA5 were5AA andand obtained ApAp77AA werewere in 50% obtainedobtained and 18% inin 50%50% yields, andand respectively. 18%18% yields,yields, respectively.Therespectively. chemoselective TheThe chemoselectivechemoselective aspect of this aspectmethodaspect ofof is thisthis a great methodmethod advantage isis aa greatgreat as it allowsadvantageadvantage to prevent asas itit allowsallows the use toto of preventprevent nucleoside thethe protectinguseuse ofof nucleosidenucleoside groups. Comparedprotectingprotecting groups.groups. ComparedCompared toto otherother proceduresprocedures usingusing P(III)P(III) chchemistry,emistry, itit offersoffers attrattractiveactive features,features, notablynotably thesethese rapidrapid reactionsreactions cancan bebe runrun underunder ambientambient conditions.conditions.

Molecules 2019, 24, 4334 18 of 27

Moleculesto other 2019 procedures, 24, x using P(III) chemistry, it offers attractive features, notably these rapid reactions18 of can 27 beMolecules run under 2019, 24 ambient, x conditions. 18 of 27 5. Green Chemistry Approaches 5. Green Chemistry Approaches Most of the reported methods present drawbacks, such as the use of non-volatile and toxic solventsMost (DMF, ofof the the reported pyridine,reported methods methodsN-methylpyrrolidone), present present drawbacks, drawbacks, high such such assensitivity the as use the of to non-volatileuse moisture, of non-volatile and preparation toxic and solvents toxic of (DMF,substratessolvents pyridine, (DMF, or phosphorus Npyridine,-methylpyrrolidone), reagents N-methylpyrrolidone), with highlipophilic sensitivity counterionshigh tosensitivity moisture, due toto preparation solubility moisture, issues ofpreparation substrates in organic orof phosphorusmedia,substrates anhydrous or reagents phosphorus conditions with lipophilicreagents (i.e., dry counterionswith reagents lipophilic dueand counterions tosolvents), solubility fastidious due issues to insolubility organicpurification media,issues and anhydrousin possibly organic priorconditionsmedia, synthesis anhydrous (i.e., of dry the conditions reagents activated and(i.e., nucleotide solvents), dry reagents substrates. fastidious and solvents),An purification ideal procedure fastidious and possibly wouldpurification meet prior theand synthesis following possibly of requirements:theprior activated synthesis nucleotideApplicable of the activated substrates.to a wide nucleotide range An ideal ofsubstrates. substr procedureates An where ideal would protectingprocedure meet the wouldgroups following meetare not requirements:the necessary, following Applicableandrequirements: giving torise a Applicable wideto the range products to of a substrates widein short range time where of and substr protecting highates yields. where groups protecting are not necessary, groups are and not giving necessary, rise to theand products givingIn 2017, rise inour shortto researchthe timeproducts andgroup highin reposhort yields.rted time a andone-pot high synthesis, yields. in water medium, of nucleoside 5′- polyphosphatesInIn 2017, our our and research research dinucleotides group group repo reportedstartingrted froma aone-pot one-pot the corresponding synthesis, synthesis, in in waterNMPs water medium, [55,56]. medium, This of ofnucleoside method nucleoside uses 5′- 2-chloro-1,3-dimethylimidazolinium5polyphosphates0-polyphosphates and and dinucleotides dinucleotides starting startinghexafluoro from from thphosphate thee corresponding corresponding (DMP) NMPs NMPsand [55,56]. [imidazole55,56]. This This asmethod coupling uses reagents2-chloro-1,3-dimethylimidazolinium to activate the NMP into hexafluorophosphatetheir hexafluoro phosphorimidazolides.phosphate (DMP) (DMP) and Dinucleoside imidazoleand imidazole asdiphosphates coupling as coupling reagents were obtainedtoreagents activate byto the self-dimerizationactivate NMP intothe theirNMP of phosphorimidazolides. into the NMPstheir phosphorimidazolides. (Scheme 27). Dinucleoside Dinucleoside diphosphates diphosphates were obtained were by self-dimerizationobtained by self-dimerization of the NMPs of (Scheme the NMPs 27). (Scheme 27).

Scheme 27. Preparation of symmetrical dinucleoside diphosphates in aqueous media [56]. Scheme 27. Preparation of symmetrical dinucleoside diphosphates in aqueous media [[56].56]. Our work originated from the small-scale synthesis of NDP-sugars described by Tanaka et al. [85]. OurThe authorswork originated originated performed from from the thecoupling the small-scale small-scale of a sugar-1-phosphate synthe synthesissis of of NDP-sugars NDP-sugars with a nucleotide described described inby D byTanaka2O, Tanaka using et al.2- et chloro-1,3-dimethylimidazoliniumal.[85]. [85 The]. The authors authors performed performed the the coupling chloride coupling of (DMC) ofa sugar-1-phosphate a sugar-1-phosphate and imidazole with (Scheme with a nucleotide a nucleotide28). Based in inonD2 D O,1H2 O,using and using 31 2-P 1 31 NMR2-chloro-1,3-dimethylimidazoliniumchloro-1,3-dimethylimidazolinium spectroscopies, the reaction chloride chloride was (DMC) (DMC) shown and and imidazole to proceed (Scheme via 2828). ).a BasedBased 2-imidazolyl-1,3- onon 1HH andand 31P dimethylimidazoliniumNMR spectroscopies,spectroscopies, the chloride reactionthe reaction wasintermediate shown was to proceed92,shown formed via in ato 2-imidazolyl-1,3-dimethylimidazolinium situproceed by reaction via of aDMC 2-imidazolyl-1,3- and imidazole (Schemechloridedimethylimidazolinium intermediate28). This key92 chloride,intermediate formed intermediate in situ was by able reaction92, formedto ofactivate DMCin situ an andby reactionNMP imidazole into of DMC (Schemeits correspondingand 28imidazole). This phosphorimidazolidekey(Scheme intermediate 28). This was key 93 able (viaintermediate to intermediate activate an was NMP 94 ),able which into to its then correspondingactivate reacted an with NMP phosphorimidazolide a sugar-1-phosphate into its corresponding 93salt(via to formintermediatephosphorimidazolide a sugar nucleotide.94), which 93 then (via reactedintermediate with a 94 sugar-1-phosphate), which then reacted salt towith form a sugar-1-phosphate a sugar nucleotide. salt to form a sugar nucleotide.

Scheme 28. ProposedProposed activation mechan mechanismism of UMP to UMP-Im by 90 [[85].85]. Scheme 28. Proposed activation mechanism of UMP to UMP-Im by 90 [85]. The main advantage of these reactions, which occur in D2O [85] or in H2O/CH3CN [55,56] instead of theThe usual main organic advantage solvents, of these is that reactions, the NMPs which are occur used in in D their2O [85] commercially or in H2O/CH available3CN [55,56] and insteadwater- solubleof the usual forms, organic i.e., sodium solvents, and is potassium that the NMPs salts. areThis used greatly in their simplifies commercially the handling available of the and reaction. water- soluble forms, i.e., sodium and potassium salts. This greatly simplifies the handling of the reaction.

Molecules 2019, 24, 4334 19 of 27

The main advantage of these reactions, which occur in D O[85] or in H O/CH CN [55,56] Molecules 2019, 24, x 2 2 3 19 of 27 instead of the usual organic solvents, is that the NMPs are used in their commercially available and water-solubleMoreover, protecting forms, i.e., groups sodium on and the potassium nucleotide salts. are This not greatly required simplifies and no the handlingcarbonation of theof reaction.ribonucleotides Moreover, are observed, protecting in contrast groups onto the the use nucleotide of CDI. are not required and no carbonation of ribonucleotidesAlternatively, are mechanochemistry observed, in contrast has to emerged the use of in CDI. the field of nucleosides and, to a lesser extent, nucleotidesAlternatively, [86]. This mechanochemistry technique enables has solid/so emergedlid in reactions the field through of nucleosides mechanical and, to grinding a lesser extent,under nucleotidessolvent-free [86conditions]. This technique [87–91]. enablesIn 2011, solid Ravalic/solido reactions et al. reported through mechanicalthe mechanosynthesis grinding under of solvent-freedinucleotides, conditions namely [Ap87–n91A ].(with In 2011, n = 2–4), Ravalico Ap2 etdT al. and reported nicotinamide the mechanosynthesis adenine dinucleotide of dinucleotides, (NAD+), + namelystarting ApfromnA adenosine (with n = 2–4),5′-phosphoromorpholidate Ap2dT and nicotinamide 2, in adenine the presence dinucleotide of (NAD ), startingchloride, from 1H- adenosinetetrazole as 50 -phosphoromorpholidatethe acidic promoter and2, inwater the presence (Scheme of 29) magnesium [92]. Although chloride, advantageous, 1H-tetrazole as this the acidicmethodology promoter requires and water the use (Scheme of costly 29)[ reagents92]. Although, namely advantageous, tetrazole and this the methodology phosphoromorpholidate requires the usesubstrate, of costly the reagents, latter may namely be prepared tetrazole beforehand and the phosphoromorpholidate in solution using conventional substrate, solution the latter synthesis may be prepared[45]. beforehand in solution using conventional solution synthesis [45].

Scheme 29.29. Ball-millingBall-milling route route using using commercially commercially available available nucleotide nucleotide salts and salts morpholidate and morpholidate reaction partnersreaction partners [92]. [92].

In the course of our investigations on new stra strategiestegies for phosphoanhydride bond formation, we also investigated the ball-milling technique [[93].93]. To To begin with, activation of UMP was performedperformed according to the conditions previously reported in water medium (DMP(DMP/imidazole)/imidazole) [[55,56],55,56], without adding any solvent in a vibratory ball-mill (vbm) at 30 Hz. A complete conversion to Up U was adding any solvent in a vibratory ball-mill (vbm) at 30 Hz. A complete conversion to Up22U was obtained within 30 min, suggesting that UMP was activated to its phosphorimidazolide and reacted promptly with the remaining UMP to form the dimer. Alternatively, we tested the use of CDI, which has recently gained interest in the field field of mechanosynthesis due to the safety of its by-products (i.e., imidazole and carbon dioxide), its its efficacy efficacy in N-acylation-acylation reactions reactions and and relatively relatively low low cost cost [94–97]. [94–97]. Activation of UMP (acidic form)form) toto thethe correspondingcorresponding 550′-phosphorimidazolide 220′,3-carbonate 95 was 1 complete byby grinding,grinding, forfor 11 hh atat 3030 Hz, Hz, in in the the presence presence of of 4 4 equiv. equiv. of of CDI CDI and and acetonitrile acetonitrile (0.3 (0.3µL.mg µL.mg− )- as1) as a liquida liquid assistant assistant (Scheme (Scheme 30). 30). The The absence absence of reactivity of reactivity observed observed using using the disodium the disodium salt of salt UMP of suggestsUMP suggests that the that acid the form acid is requiredform is required to protonate to protonate the imidazole the imidazole of CDI and of to CDI facilitate and itsto substitutionfacilitate its bysubstitution the phosphate by the monoester. phosphate This monoester. activation This step activation was applied step successfully was applied to severalsuccessfully ribonucleotides to several andribonucleotides a 20- and a 2′-deoxyribonucleotide (dTMP). (dTMP). In the second step, a nucleoside mono, di or triphosphate was added onto the activated NMP 95–96 in order to form the desired DNPs (Scheme 30). Thus, the mixed dinucleoside 50,50-diphosphates were successfully obtained by adding a slight excess of a NMP and a small amount of acetonitrile (0.6–0.95µL/mg) in the jar, and then ball-milling for 2 h at 30 Hz. Remarkably, the second grinding step also removed the carbonate protection. The reaction was less efficient with GMP, UDP and ATP, and, thus, required a larger excess of reagent to form the corresponding dinucleotides Gp2U, Up3U and Ap4U, respectively. This user-friendly mechanochemical approach only requires a slight excess of reagents and limits the formation of side products. While isolated yields of symmetrical and mixed dinucleoside polyphosphates are similar to other approaches, the set-up of the experiments and the

Scheme 30. Mechanochemical approach to obtain DNPs starting from NMPs [93].

Molecules 2019, 24, x 19 of 27

Moreover, protecting groups on the nucleotide are not required and no carbonation of ribonucleotides are observed, in contrast to the use of CDI. Alternatively, mechanochemistry has emerged in the field of nucleosides and, to a lesser extent, nucleotides [86]. This technique enables solid/solid reactions through mechanical grinding under solvent-free conditions [87–91]. In 2011, Ravalico et al. reported the mechanosynthesis of dinucleotides, namely ApnA (with n = 2–4), Ap2dT and nicotinamide adenine dinucleotide (NAD+), starting from adenosine 5′-phosphoromorpholidate 2, in the presence of magnesium chloride, 1H- tetrazole as the acidic promoter and water (Scheme 29) [92]. Although advantageous, this methodology requires the use of costly reagents, namely tetrazole and the phosphoromorpholidate substrate, the latter may be prepared beforehand in solution using conventional solution synthesis [45].

Scheme 29. Ball-milling route using commercially available nucleotide salts and morpholidate reaction partners [92].

In the course of our investigations on new strategies for phosphoanhydride bond formation, we also investigated the ball-milling technique [93]. To begin with, activation of UMP was performed according to the conditions previously reported in water medium (DMP/imidazole) [55,56], without adding any solvent in a vibratory ball-mill (vbm) at 30 Hz. A complete conversion to Up2U was obtained within 30 min, suggesting that UMP was activated to its phosphorimidazolide and reacted promptly with the remaining UMP to form the dimer. Alternatively, we tested the use of CDI, which has recently gained interest in the field of mechanosynthesis due to the safety of its by-products (i.e., imidazole and carbon dioxide), its efficacy in N-acylation reactions and relatively low cost [94–97]. MoleculesActivation2019 ,of24 UMP, 4334 (acidic form) to the corresponding 5′-phosphorimidazolide 2′,3-carbonate 9520 ofwas 27 complete by grinding, for 1 h at 30 Hz, in the presence of 4 equiv. of CDI and acetonitrile (0.3 µL.mg- 1) as a liquid assistant (Scheme 30). The absence of reactivity observed using the disodium salt of workupUMP suggests are greatly that simplified,the acid form saving is required a considerable to protonate amount the ofimidazole time. In addition,of CDI and compared to facilitate to the its previouslysubstitution reported by the mechanochemicalphosphate monoester. synthesis This [ 92activation], this one-pot step twowas stepsapplied reaction successfully starts from to several NMPs, whichribonucleotides are readily and available. a 2′-deoxyribonucleotide (dTMP).

SchemeScheme 30.30. Mechanochemical approach toto obtainobtain DNPsDNPs startingstarting fromfrom NMPsNMPs [[93].93].

In addition to their simplicity, the mechanochemical approaches alleviate issues associated with the low solubility of reagents in solution phase synthesis. Therefore, nucleotides and inorganic salts can be used in their commercially available sodium or potassium forms.

6. Purification and Characterization

6.1. Purification In this section, we provide a brief overview of the purification procedures for dinucleotides. Due to their high polarity, they cannot be separated by normal-phase chromatography like most organic compounds. In addition, the byproducts or side products formed during synthesis share similar characteristics, resulting in a challenging separation from the desired compounds. The two main purification methods, reported in Table1, use ion-exchange or reverse-phase (RP) chromatography.

Table 1. Main methods to isolate dinucleotides.

Types of Chromatography Eluants References Anion-exchange DEAE-Sephadex® 0.02 M TEAB, pH 8; 10% ACN [54] TOYOPEARL® DEAE-650M 0.2 M TEAB, pH 8; 10% ACN [54] ® DEAE-Sephadex Gradient of aq. NH4HCO3 [67,69] ® Q Sepharose Fast Flow Gradient of aq. NH4HCO3 [83,84] ® DEAE Sepharose Fast Flow Gradient of aq. NH4HCO3 [83,84] C-18 reverse-phase HPLC 0.01 M NH4HCO2, pH 4, 50% MeOH [46] Semi-preparative HPLC TEAAc pH 7 or 9; 6–12% ACN [59,60,64] Preparative HPLC 0.1 M NH4HCO3; ACN [71] Preparative HPLC TEAB pH 7.8; 65% ACN [92] RP-18 silica gel column Water [74] RP-18 silica gel column 0.1 M TEAB, pH 7.5; 20% ACN [55,56] RP-18 silica gel column 0.1 M TEAAc, pH 7; 5–10% ACN [93] Gel filtration Sephadex® LH-20 Deionized water [79]

The first type of ion-exchange support is positively charged, such as diethylaminoethyl (DEAE) Sephadex, and allows the chemical species to elute according to their number of charges. Elution is often performed using a gradient of triethylammonium hydrogen carbonate (TEAB) or ammonium hydrogen Molecules 2019, 24, 4334 21 of 27

carbonate (NH4HCO3) buffers. Regarding C18 reverse-phase chromatography, either preparative or semi-preparative high pressure liquid chromatography (HPLC) or chromatography on RP-18 silica gel may be used. Compounds are often eluted using a gradient from an aqueous buffered solution, such as triethylammonium acetate (TEAAc) or TEAB, to acetonitrile or methanol. Alternatively, gel filtration was described for the purification of symmetrical Np2N. Then, the samples are usually freeze-dried and converted to their sodium form by ion exchange using a Dowex-50W-Na+ resin. The fact that purification is a time-consuming step is one of the reasons that pushed some research groups to develop solid phase strategies [76,77]. Indeed, those methods were designed in order to simplify purification of the final products, which can be collected by simple filtration. However, the recovered materials showed moderate purity and an additional purification step was required [76]. Consequently, solution-phase approaches are still predominant.

6.2. Physico-Chemical Properties NMR, UV spectroscopy, and mass spectrometry (MS) are the main tools for the analysis of dinucleotides. Since the sugar and the phosphate moieties have no significant absorption above 230 nm, dinucleotides exhibit UV absorption profiles similar to those of their parent nucleosides with λ max values close to 260 nm [1]. MS analysis of nucleotides and derivatives such as dinucleotides has been extensively reviewed by Banoub et al. [98]. Alternatively, 31P NMR is a very convenient tool to monitor reaction courses, characterize reaction intermediates, and study the conformation of dinucleotides and final compounds [1,50]. The chemical shift (δ) for most endogenous dinucleotides covers 8 to − 24 ppm, and exhibits pH and counter ion dependence [1]. Table2 summarizes the literature data − 1 31 for representative NpN(n =1–7). In H-decoupled P NMR, the phosphorus atoms closest to the nucleosides are characterized by a singlet at approximately 10 ppm, while the phosphorus atoms − inside the polyphosphate chain exhibit resonance signals around 22 ppm. − 31 Table 2. P Chemical shift ranges of dinucleoside polyphosphates in D2O.

NpnN δ Ranges References Np N’ 1 singlet (P1 + P2) from 9.6 to 11.5 ppm [47,49,50,53,56,60,64,67,74,76,77,79,83,93] 2 − − 1 doublet (P1 + P3) from 9.2 to 11.7 ppm Np N − − [47,49,50,54,60,64,67,69,84,93] 3 1 triplet (P2) from 21.0 to 23.4 ppm − − 1 multiplet or 1 broad singlet or 1 doublet Np N [47,49,50,54,60,64,67,69,71,74,93] 4 (P1 + P4) from 8.8 to 11.5 ppm − − 1 multiplet or 1 br singlet or 1 doublet (P2 + P3) from 20.6 to 23.1 ppm − − 1 multiplet or 1 br singlet or 1 doublet Np N [49,50,59,69,71,84] 5 (P1 + P5) from 8.7 to 11.4 ppm − − 1 multiplet or 1 br singlet or 1 doublet (P2 + P3 + P4) from 20.2 to 22.5 ppm − − 1 doublet at 10.1 ppm (P1 + P6) Np N − [49] 6 1 multiplet at 21.7 ppm (P 2+ P3 + P4 + P5) − 1 broad singlet or 1 doublet from 10.1 to Np N − [49,84] 7 11.6 ppm (P1 + P7) − 1 multiplet from 21.7 to 23.5 ppm − − (P2 + P3 + P4 + P5 + P6)

Interestingly, the study of dinucleotides using NMR techniques (1H, 13C and 31P) can determine the conformation (syn/anti, sugar puckering), properties (tautomerism, H-bonds and π-stacking interactions), as well as the anomeric α/β configuration. Such data are crucial for understanding the structure–activity relationship of either natural or synthetic dinucleotides as enzyme inhibitors and receptor ligands, and the design of potent therapeutic agents based on a dinucleotide scaffold. In this regard, Stern et al. have performed conformational studies on physiologically active NpnN0 sodium salts (N = A, G, U, C; N0 = A, G, U, C; n = 2–5) in neutral aqueous solution, using NMR and computational Molecules 2019, 24, 4334 22 of 27 techniques [50,98]. While no predominant conformation was observed for the ribose moiety in any of the studied dinucleotides, natural dinucleoside polyphosphates were more frequently found in a folded (stacked) rather than an extended conformation. Purine dinucleotides showed greater stacking interactions than pyrimidine dinucleotides. In addition, dinucleotides with longer phosphate chains were found to have weaker stacking interactions. Crystal structures of Ap4A sodium salt [99], and some dinucleotides bound to kinases or hydrolases are available [100,101]. In protein-bound dinucleotides, the polyphosphate chain adopts various conformations: extended [100–103], S-shaped [101] or folded [100]. In some cases, the protein-bound dinucleotides coordinate with a Mg2+ ion. Finally, Chen and Kohler investigated the dynamics of excited electronic states formed by UV excitation of the several diadenosine polyphosphates by femtosecond transient absorption (TA) spectroscopy [104]. They found that the excimer states seen in TA experiments on nucleobase dimers are only observed in π-stacked conformations, but the lifetimes of these states are insensitive to how the stacked bases are oriented.

7. Conclusions

Naturally occurring dinucleoside 50,50-polyphosphates are involved in a variety of cellular processes. Among them, symmetrical purine-containing dinucleotides are the most known representatives, whereas pyrimidine-containing and mixed derivatives are still intensively studied. Therefore, the development of synthetic methods required to obtain these compounds in few steps, good yields, high purity and comfortable amounts generated interest from the chemists and remains a field of intensive research. Herein, we have focused our attention on the synthesis of dinucleoside 50,50-polyphosphates, either symmetrical or mixed, and including non-modified pyrophosphate bonds. These methods may involve P(III) and/or P(V) intermediates and solution-phase or supported chemistry. P(V) synthetic approaches are usually based on the activation of a 50-nucleotide using inexpensive reagents but require the conversion of the substrates, as well as the phosphate reactants, into the corresponding tri- or tetra-alkyl salts due to solubility issues. P(III) synthetic approaches are often effective processes. However, the presence of protecting groups on the nucleoside is often mandatory, thus increasing the overall number of steps. In most cases, strictly anhydrous conditions are essential and the length of the poly-phosphate chain affects the yields of the synthesis. Recently, environmental friendly alternatives were investigated and may constitute an area of substantial and growing interest for the preparation of these derivatives in the coming years.

Author Contributions: Conceptualization: S.P. and B.R.; Writing—original draft: B.R.; Writing—review & editing: L.A., C.C., S.P. and B.R. Funding: This research received no external funding Conflicts of Interest: The authors declare no conflict of interest.

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