Coordination Chemistry of Nucleotides and Antivirally Active Acyclic Nucleoside Phosphonates, including Mechanistic Considerations

Considering that practically all reactions that involve nucleotides also involve metal ions, it is evident that the coordination chemistry of nucleotides and their derivatives is an essential corner stone of biological inorganic chemistry. Nucleotides are either directly or indirectly involved in all processes occurring in Nature. It is therefore no surprise that the constituents of nucleotides have been chemically altered—that is, at the nucleobase residue, the sugar moiety, and also at the phosphate group, often with the aim of discovering medically useful compounds. Among such derivatives are acyclic nucleoside phosphonates (ANPs), where the sugar moiety has been replaced by an aliphatic chain (often also containing an ether oxygen atom) and the phosphate group has been replaced by a phosphonate carrying a carbon–phosphorus bond to make the compounds less hydrolysis-sensitive. Several of these ANPs show antiviral activity, and some of them are nowadays used as drugs. The antiviral activity results from the incorporation of the ANPs into the growing nucleic acid chain—i.e., polymerases accept the ANPs as substrates, leading to chain termination because of the missing 3′-hydroxyl group. We have tried in this review to describe the coordination chemistry (mainly) of the adenine nucleotides AMP and ATP and whenever possible to compare it with that of the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA2− = adenine(N9)-CH2-CH2-O-CH2-PO32) [or its diphosphate (PMEApp4−)] as a representative of the ANPs. Why is PMEApp4− a better substrate for polymerases than ATP4−? There are three reasons: (i) PMEA2− with its anti-like conformation (like AMP2−) fits well into the active site of the enzyme. (ii) The phosphonate group has an enhanced metal ion affinity because of its increased basicity. (iii) The ether oxygen forms a 5-membered chelate with the neighboring phosphonate and favors thus coordination at the Pα group. Research on ANPs containing a purine residue revealed that the kind and position of the substituent at C2 or C6 has a significant influence on the biological activity. For example, the shift of the (C6)NH2 group in PMEA to the C2 position leads to 9-[2-(phosphonomethoxy)ethyl]-2-aminopurine (PME2AP), an isomer with only a moderate antiviral activity. Removal of (C6)NH2 favors N7 coordination, e.g., of Cu2+, whereas the ether O atom binding of Cu2+ in PMEA facilitates N3 coordination via adjacent 5- and 7-membered chelates, giving rise to a Cu(PMEA)cl/O/N3 isomer. If the metal ions (M2+) are M(α,β)-M(γ)-coordinated at a triphosphate chain, transphosphorylation occurs (kinases, etc.), whereas metal ion binding in a M(α)-M(β,γ)-type fashion is relevant for polymerases. It may be noted that with diphosphorylated PMEA, (PMEApp4−), the M(α)-M(β,γ) binding is favored because of the formation of the 5-membered chelate involving the ether O atom (see above). The self-association tendency of purines leads to the formation of dimeric [M2(ATP)]2(OH)− stacks, which occur in low concentration and where one half of the molecule undergoes the dephosphorylation reaction and the other half stabilizes the structure—i.e., acts as the “enzyme” by bridging the two ATPs. In accord herewith, one may enhance the reaction rate by adding AMP2− to the [Cu2(ATP)]2(OH)− solution, as this leads to the formation of mixed stacked Cu3(ATP)(AMP)(OH)− species, in which AMP2− takes over the structuring role, while the other “half” of the molecule undergoes dephosphorylation. It may be added that Cu3(ATP)(PMEA) or better Cu3(ATP)(PMEA)(OH)− is even a more reactive species than Cu3(ATP)(AMP)(OH)−. – The matrix-assisted self-association and its significance for cell organelles with high ATP concentrations is summarized and discussed, as is, e.g., the effect of tryptophanate (Trp−), which leads to the formation of intramolecular stacks in M(ATP)(Trp)3− complexes (formation degree about 75%). Furthermore, it is well-known that in the active-site cavities of enzymes the dielectric constant, compared with bulk water, is reduced; therefore, we have summarized and discussed the effect of a change in solvent polarity on the stability and structure of binary and ternary complexes: Opposite effects on charged O sites and neutral N sites are observed, and this leads to interesting insights

Comparison of the π-stacking properties of purine versus pyrimidine residues. Some generalizations regarding selectivity

Aromatic-ring stacking is pronounced among the noncovalent interactions occurring in biosystems and therefore some pertinent features regarding nucleobase residues are summarized. Self-stacking decreases in the series adenine>guanine>hypoxanthine>cytosine~uracil. This contrasts with the stability of binary (phen)(N) adducts formed by 1,10-phenanthroline (phen) and a nucleobase residue (N), which is largely independent of the type of purine residue involved, including (N1)H-deprotonated guanine. Furthermore, the association constant for (phen)(A)0/4− is rather independent of the type and charge of the adenine derivative (A) considered, be it adenosine or one of its nucleotides, including adenosine 5′-triphosphate (ATP4−). The same holds for the corresponding adducts of 2,2′-bipyridine (bpy), although owing to the smaller size of the aromatic-ring system of bpy, the (bpy)(A)0/4− adducts are less stable; the same applies correspondingly to the adducts formed with pyrimidines. In accord herewith, [M(bpy)](adenosine)2+ adducts (M2+is Co2+, Ni2+, or Cu2+) show the same stability as the (bpy)(A)0/4− ones. The formation of an ionic bridge between -NH3 + and -PO3 2−, as provided by tryptophan [H(Trp)±] and adenosine 5′-monophosphate (AMP2−), facilitates recognition and stabilizes the indole-purine stack in [H(Trp)](AMP)2−. Such indole-purine stacks also occur in nature. Similarly, the formation of a metal ion bridge as occurs, e.g., between Cu2+ coordinated to phen and the phosphonate group of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA2−) dramatically favors the intramolecular stack in Cu(phen)(PMEA). The consequences of such interactions for biosystems are discussed, especially emphasizing that the energies involved in such isomeric equilibria are small, allowing Nature to shift such equilibria easily

Metal Ion-Coordinating Properties in Aqueous Solution of the Antivirally Active Nucleotide Analogue (S)-9-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA). Quantification of Complex Isomeric Equilibria

Acyclic nucleoside phosphonates are of medical relevance and deserve detailed chemical characterization. We focus here on ( S )‐9‐[3‐hydroxy‐2‐(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA), as well as the nucleobase‐free (phosphonomethoxy)ethane (PME) and ( R )‐hydroxy‐2‐(phosphonomethoxy)propane (HPMP). The acidity constants of H 3 (HPMPA) + were determined and compared with those of the related phosph(on)ate derivatives; they are also needed to understand the properties of the metal ion complexes. Given that in vivo nucleotides and their analogues participate in reactions typically as divalent metal ion (M 2+ ) complexes, the stability constants of the M(H;HPMPA) + and M(HPMPA) species with M 2+ = Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , and Cd 2+ were measured. Comparisons between the results for HPMPA 2- and the previous data for PMEA 2- , HPMP 2- and PME 2- revealed that for most M(HPMPA) complexes the enhanced stability (the enhancement relative to the stability of a simple phosphonate‐M 2+ coordination), can solely be explained by the formation of 5‐membered chelates involving the ether oxygen. These chelates occur in equilibrium with simple ′open′ phosphonate‐M 2+ species, the phosphonate group being the primary binding site. The only exceptions are the M(HPMPA) complexes of Ni 2+ , Cu 2+ , and Zn 2+ , which show an additional stability enhancement; in these instances not only the indicated 5‐membered chelates are formed, but M 2+ coordinates in addition to N3 of the adenine residue forming a 7‐membered chelate ring. This observation regarding N3 is important because it emphasizes the metal ion affinity of this site (which is often ignored). Note that in the DNA double helix N3 is exposed to the solvent in the minor groove. The stability data for the monoprotonated M(H;HPMPA) + complexes suggest that these carry H + at the phosphonate group whereas M 2+ is partly at the nucleobase and partly also at the phosphonate group. The ratios of these isomers depend on the metal ion involved, e.g., for Cu(H;HPMPA) the ratio of the isomers is about 1:1

Metal Ion Complexes of Nuceloside Phosphorothioates Reflecting the Ambivalent Properties of Lead (II)

This Perspective outlines the coordinating properties of lead( II ), to some extent in comparison with related metal ions like Ca 2+ , Zn 2+ or Cd 2+ . It is worth noting that the affinity of Pb 2+ towards phosphate residues corresponds to that of Cu 2+ . Furthermore, the binding tendency of Pb 2+ towards thiophosphate groups as present in methyl thiophosphate (MeOPS 2− ) or uridine 5′- O -thiomonophosphate (UMPS 2− ) is compared with that of the parent ligands, that is, methyl phosphate (CH 3 OPO 3 2− ) and uridine 5′-monophosphate (UMP 2− ). The replacement of an O by a S atom makes the monoprotonated thiophosphate group considerably more acidic [compared to ROP(O) 2 − (OH)], but at the same time its affinity for Pb 2+ increases tremendously: more than 99% of Pb 2+ is S-bound. This is very different if the coordinating properties of uridylyl-(5′→3′)-[5′]-uridylate (pUpU 3− ) and P -thiouridylyl-(5′→3′)-[5′]-uridylate (pUp (S) U 3− ) are compared. The phosphate-coordinated Pb 2+ forms a 10-membered chelate with one of the two terminal O atoms of the phosphodiester linkage, which reaches a formation degree of about 90% in Pb(pUpU) − . However, in Pb(pUp (S) U) − the formation degree of the chelate is reduced to about half in accordance with the fact that now only one terminal O atom is available in the thiophosphate diester bridge, that is, Pb 2+ coordinates to this O showing no affinity for S in ROP(O)(S) − OR′. These observations are ascribed to the properties of the Pb 2+ lone pair, which shapes the Pb 2+ coordination sphere; its role is discussed further in this Perspective and a caveat is made regarding Pb 2+ binding to a thiophosphate diester linkage

Extent of intramolecular π stacks in aqueous solution in mixed-ligand copper(II) complexes formed by heteroaromatic amines and the anticancer and antivirally active 9-[2-phosphonomethoxy)ethyl]guanine (PMEG).✩ a comparison with related acyclic nucleotide analogues

The acyclic nucleoside phosphonate (ANP2– ) 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) is anticancer and antivirally active. The acidity constants of the threefold protonated H3(PMEG)+ were determined by potentiometric pH titrations (aq. sol.; 25°C; I = 0.1 M, NaNO3). Under the same conditions and by the same method, the stability constants of the binary Cu(H;PMEG)+ and Cu(PMEG) complexes as well as those of the ternary ones containing a heteroaromatic N ligand (Arm), that is, of Cu(Arm)(H;PMEG)+ and Cu(Arm)(PMEG), where Arm = 2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), were measured. The corresponding equilibrium constants, taken from our earlier work for the systems with 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-[2-(phosphonomethoxy)ethyl]-2,6-diamino-purine (PMEDAP) as well as those for Cu(PME) and Cu(Arm)(PME), where PME2– = (phosphonomethoxy)ethane = (ethoxymethyl)phosphonate, were used for comparisons. These reveal that in the monoprotonated ternary Cu(Arm)(H;PE)+ complexes, the proton and Cu(Arm)2+ are at the phosphonate group; the ether oxygen of the -CH2-O-CH2-P(O) 2 ! (OH) residue also participates to some extent in Cu(Arm)2+ coordination. Furthermore, the coordinated Cu(Arm)2+ forms a bridge with the purine moiety undergoing π-π stacking which is more pronounced with H·PMEDAP– than with H·PMEA– . Most intense is π stack formation (st) with the guanine residue of H·PMEG– ; here the bridged form Cu(Arm)(H·PMEG) st + occurs next to an open (op), unbridged (binary) stack, formulated as Cu(Arm)2+/(H·PMEG) op ! . – The unprotonated and neutral ternary Cu(Arm)(PE) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO3 2! represents a phosph(on)ate ligand with a group R that is unable to participate in any intramolecular interaction. The observed stability enhancements are mainly due to intramolecular stack formation (st) between the aromatic rings of Arm and the purine residue in the Cu(Arm)(PE) complexes and also, to a smaller extent, to the formation of fivemembered chelates involving the ether oxygen of the -CH2-O-CH2-PO 3 2! residue (cl/O) of the PE2– species. The quantitative analysis of the intramolecular equilibria reveals three structurally different Cu(Arm)(PE) isomers; e.g., of Cu(Phen)(PMEG) ca. 1.1% exist as Cu(Phen)(PMEG)op, 3.5% as Cu(Phen)(PMEG)cl/O, and 95% as Cu(Phen)(PMEG)st. Comparison of the various 3 formation degrees reveals that within a given Cu(Arm)(PE) series the stacking tendency decreases in the order PMEG2– ≥ PMEDAP2– > PMEA2– . Furthermore, stacking is more pronounced in the acyclic Cu(Arm)(PE) complexes compared with that in the Cu(Arm)(NMP) species, where NMP2– = corresponding parent (2'-deoxy)nucleoside 5'-monophosphate. Here is possibly one of the reasons for the biological activity of the ANPs. One is tempted to speculate that the pronounced stacking tendency of PMEG2– , together with a different H-bonding pattern, leads to enhanced binding in the active site of nucleic acid polymerases, thus being responsible for the pronounced anticancer and antiviral activity of PMEG

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Compete with the Intestinal Microbiota

Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10−/−, VILLIN-HACL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen