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Synthesis of triazole-linked analogues of c-diGMP and their interactions with diguanylate cyclase Silvia Fernicola, Ilaria Torquati, Alessandro Paiardini, Giorgio Giardina, Giordano Rampioni, Marco Messina, Livia Leoni, Fabio Del Bello, Riccardo Petrelli, serena rinaldo, Loredana Cappellacci, and Francesca Cutruzzolà J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01184 • Publication Date (Web): 01 Oct 2015 Downloaded from http://pubs.acs.org on October 8, 2015

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Journal of Medicinal Chemistry

Synthesis of triazole-linked analogues of c-di-GMP and their interactions with diguanylate cyclase

Silvia Fernicola1*, Ilaria Torquati2*, Alessandro Paiardini3, Giorgio Giardina1, Giordano Rampioni4, Marco Messina4, Livia Leoni4, Fabio Del Bello2, Riccardo Petrelli2, Serena Rinaldo1, Loredana Cappellacci2§, Francesca Cutruzzolà1§

1

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza

University of Rome, Italy. 2

School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino (MC), Italy

3

Department of Biology and Biotechnology “Charles Darwin” Sapienza University of Rome, Italy.

4

Department of Science, University Roma Tre, Rome, Italy.

Abstract Cyclic di-GMP (c-di-GMP) is a widespread second-messenger, that plays a key role in bacterial biofilm formation. The compound’s ability to assume multiple conformations allows it to interact with a diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs), and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric non-hydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for

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mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.

Keywords: c-di-GMP, biofilm, Pseudomonas aeruginosa, Caulobacter crescentus, WspR, PleD, RocR, inhibitors, copper(I)-catalyzed 1,3-dipolar cycloaddition, click chemistry, Huisgen cycloaddition, alkynes, azides, dinucleoside, heterodinucleoside, 1,2,3-triazole, purine, dimer.

Abbreviations: DGCs, diguanylate cyclases; PDEs, phosphodiesterase; I-site, inhibitory site, CuAAC, Copper(I)-catalyzed azide-alkyne cycloaddition.


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INTRODUCTION

Since its discovery in 1987 1, cyclic-di-GMP (c-di-GMP) has attracted growing attention and is now acknowledged as one of the most important second messengers controlling bacterial adaptation strategies, such as biofilm formation, persistence, cytotoxicity and cell-cycle progression 2. The intracellular levels of c-di-GMP are regulated by the opposing activities of diguanylate cyclases (DGCs), containing GGDEF domains, and phosphodiesterases (PDEs), containing either EAL or HD-GYP domains, which synthesizes and degrade c-di-GMP, respectively 2. In many cases, the activity of GGDEF and EAL or HD-GYP domains is modulated by environmental/metabolic signals or by neighbouring domains. The receptors sensing the cellular levels of this dinucleotide, which represent the end-nodes of cdi-GMP controlled networks, are quite heterogeneous, embracing riboswitches and transcription factors or enzymes containing PilZ domains, degenerate DGCs or PDEs, or other domains, and are yet to be fully structurally characterized 3. C-di-GMP shows a very rich conformational polymorphism 4-6 which accounts for its capability to interact with structurally unrelated binding sites 7-14, making the prediction of c-di-GMP-binding proteins very challenging 15. As an example, c-di-GMP forms an intercalated dimer to bind to GGDEF domains via the I-site, a widespread (but not ubiquitous) binding site for noncompetitive product inhibition, which switches the enzymes to an inactive conformation

16-18

. On the other hand, the binding mode of c-

di-GMP to the EAL active site involves a single molecule in the open conformation

19-22

, even

though structural polymorphism at the level of one of the two glycosidic bonds has also been reported 12. In recent years, c-di-GMP signalling systems have emerged as promising targets to hamper biofilm formation and virulence in many bacterial pathogens. However, since many bacterial genomes encode multiple proteins involved in c-di-GMP turnover and reception, we still need to 3 ACS Paragon Plus Environment


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deeply understand the mechanistic details governing its synthesis, degradation and signalling to efficiently develop strategies targeting c-di-GMP-dependent pathways. Up to now, a number of approaches have been described aimed at inhibiting biofilm formation by interfering with c-di-GMP pathways; these are mainly based on the screening of small-molecule libraries using whole-cell

23-25

, in vitro

26

, or in silico

27

methods, or on the rational design of

potential inhibitors (see 28 for an up-to-date review) including c-di-GMP analogues (see Tables SI-1 and SI-2) that are useful tools for investigating the molecular bases of c-di-GMP/protein interactions 28. Among the c-di-GMP analogues tested, the most promising compounds effective against DGCs display very conservative modifications of the starting chemical structure of c-di-GMP, involving the 2’-OH and the bridging oxygen in the phosphodiester linkage. The most potent so far is 2’deoxy-2’-F-c-diGMP (2’-F-c-di-GMP, IC50 = 11 µM, assayed on WspR from Pseudomonas aeruginosa, Table SI-1). However, this inhibitor lacks selectivity as it also strongly inhibits PDEs (IC50 = 0.7 µM, assayed on RocR from P. aeruginosa) 29. The relatively weak effect of these c-diGMP analogues on DGCs, as compared to PDEs, could be ascribed to the substantial entropy loss when such compounds bind as stacked dimers on the I-site. On the other hand, the three selective inhibitors for the PDE RocR, bearing 2’-OH substitutions (2’-H and 2’-OMe29) or phosphate linkage modifications (endo-S-c-di-GMP30), may still be hydrolysed to the linear product, since the phosphodiester moiety is conserved 30. The development of new compounds able to selectively inhibit DGCs without affecting the activity of PDEs is an ambitious goal with potential biomedical applications, and thus demands a systematic study focused on the role of the various moieties of the c-di-GMP scaffold in controlling its interaction with the target protein. Therefore, in the present work we have designed and synthetized an array of c-di-GMP-based molecules, encompassing linear derivatives with a completely novel scaffold, which have been tested for their ability to inhibit both DGCs and PDEs. This study allowed us to identify the 4 ACS Paragon Plus Environment

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minimal structural determinants required for specifically targeting the I-site of DGCs. In particular, identification of a linker able to hold the two guanine bases (and their derivatives) at the appropriate distance required for selective targeting of the DGCs’ I-site makes these novel molecules potent compounds, and possibly future therapeutic agents.

RESULTS AND DISCUSSION Chemical Synthesis Three series of compounds have been designed and synthetized as linear derivatives of c-diGMP. As was recently described

31

, the linear analogues may present features that are more

attractive from a drug development perspective, as compared to the cyclic counterpart: i) these compounds are in principle structurally simpler and therefore cost less to synthesize; ii) the different subset of conformations accessible to linear compounds, as compared to c-di-GMP, could favour the identification of molecules able to selectively target a subset of c-di-GMP-interacting proteins. We designed linear c-di-GMP-based compounds starting from 5,5’-triazolyl-dinucleosides (Chart 1, Scaffold A), which were further simplified to hybrid dimers (i.e. linking one nucleoside and one purine base by a triazole linker) (Chart 1, Scaffold B), and then to a third scaffold, where the sugar was completely removed (Chart 1, Scaffold C). The synthesis of all the compounds was carried out using a click chemistry approach 32, 33. This simple chemistry allowed us to replace the charged and hydrolizable phosphodiester linker of c-diGMP with the neutral 1,2,3-triazole bioisostere ring, thus preventing potential hydrolysis of the linkage portion in a cellular background; moreover, this moiety is able to form hydrogen bonds and π – π interactions with the amino acid side chains of the target proteins. As reported in Scheme 1, for scaffold A the click reaction of 2-N-isobutyryl-5’-azido-5’-deoxy-2’,3’-O-isopropylideneguanosine (4) with 2-N-isobutyryl-2’,3’-O-isopropylidene-5’-O-propargyl-guanosine (8) yielded the 5 ACS Paragon Plus Environment


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1,2,3-triazole intermediate 10. Similarly, the protected 5’-azido-5’-deoxy-guanosine 3 was reacted with N6-benzoyl-2’,3’-O-isopropylidene-5’-O-propargyl-adenosine (9) 34, to obtain the intermediate 11. The 5’-azido-5’-deoxy-2’,3’-O-isopropylidene-adenosine (5) 35, prepared starting from 2’,3’-Oisopropylidene-adenosine (2)

36-40

was clicked with 8 and 9 to furnish the 1,2,3-triazole

intermediates 12 and 13, respectively. Compound 10 was first deprotected in acidic conditions and then in saturated ammonium hydroxide solution at 50°C, to give, after purification, the triazolelinked dinucleoside DCI028. In contrast, the deprotection reactions of dinucleosides 11-13 were performed first by treatment with saturated methanolic ammonia solution at room temperature, followed by 70% formic acid aqueous solution at 40 °C, to furnish the triazole-linked dinucleosides DCI016, DCI021, DCI015 respctively. The 5’-azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (3) was synthesized starting from 2’,3’-O-isopropylidene-guanosine (1) 41. Compound 3 was then treated with isobutyryl-chloride in the presence of pyridine and trimethylsililchloride to obtain the 2-N-isobutyryl-5’-azido-5’-deoxy2’,3’-O-isopropylidene-guanosine (4) in 65% yield, according to the general procedure described by Kline et al. 42. 2-N-isobutyryl-2’,3’-O-isopropylidene-guanosine (6) 43 was converted into the 5’-Opropargyl derivative 8 by reaction with propargyl bromide and NaH in THF at room temperature (Scheme 1). The side product 8a (Scheme 2) was also obtained during the reaction, and characterized by 1H-NMR. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (Huisgen’s

cycloaddition)

between

2-N-isobutyryl-2’,3’-O-isopropylidene-5’-O-propargyl-

guanosine (8) and 2-amino-6-chloro-N9-(3-azidopropyl)-purine (16) yielded the triazole intermediate 20 (Scheme 3). Compound 20 was deprotected first by a mixture of TFA/H2O (3:1) followed by hydrolysis in basic conditions, to afford the hybride triazole-linked dimer DCI091 (Figure 1, Scaffold B). Compound DCI059 was prepared, as shown in Scheme 4 by Huisgen’s cycloaddition of 2-Nisobutyryl-5’-azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (4) with 2-amino-6-chloro-N9propargyl-purine (19). The resulting triazole intermediate 21 was deprotected as reported for 20 to 6 ACS Paragon Plus Environment

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obtain the hybride triazole-linked DCI059. The alkyl azides 23, 25, 26 were synthetized following the procedure reported by Wijtmans et al.

44

and used to prepare the N-9 (azidoalkyl)-purine

derivatives 14-17 (Scheme SI-1, Supporting information). The commercial 6-chloropurine (22) was alkylated only with alkyl azide 23, while 2-amino-6-chloropurine (24) was alkylated with the alkyl azides 23, 25, and 26, following the procedure of Lindsell et al. 45. The reactions led to a mixture of N-9/N-7 isomers, with the N-9 isomers always as the major products. The N-9 isomer derivatives 14-17 were obtained with a 9.7:1 ratio, 5.8:1 ratio, 11.5:1 ratio and 6.5:1 ratio, respectively, along with their N-7 regioisomers 14a-17a. The chemical structures of the N-9 and N-7 isomers were assigned from spectroscopic data, on the basis of the chemical shift of the protons H-8 and -CH2-Nas reported by Lambertucci et al.

46

. The 1H-NMR spectrum of the N-7 isomers always showed a

downfield shift of the H-8 and -CH2-N- protons with respect to the same protons of the N-9 isomers. Alkynes 18 and 19 were prepared following the same procedure of azide 16 45 starting from commercially available 6-chloropurine (22) and 2-amino-6-chloropurine (24), respectively, and propargyl bromide (Scheme SI-1, Supporting information). The desired N-9 isomers 18 and 19 were obtained as major products (3.8:1 and 3.5:1 ratio) along with their N-7 regioisomers 18a and 19a, respectively. The chemical structures of N-7 or N-9 isomers were assigned as reported above. In Scheme 5 the synthesis of dinucleobases DCI058, DCI061, DCI070, DCI072, DCI095, DCI096, and DCI133-136 was reported (Figure 1, Scaffold C). The Huisgen’s cycloaddition between the 6chloro-N9-(3-azidopropyl)-purine (14) and the 6-chloro-N9-propargyl-purine (18) yielded the triazole intermediate DCI070. Then, the nucleophilic substitution of DCI070 with a saturated isopropanolic ammonia solution at 60 °C furnished the triazole-linked dimer DCI072. The click reactions of the azides 15-17 with the alkyne 19, resulted in the formation of triazole-linked dimers DCI133, DCI058 and DCI095, respectively. These intermediates were treated with a mixture of TFA/H2O to afford the homodimers DCI135, DCI061 and DCI096, respectively. Furthermore, 7 ACS Paragon Plus Environment


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DCI058 (linker n = 3) was treated with two different amines, methylamine and benzylamine, to obtain the N6-substituted derivatives DCI134 and DCI136, respectively.

Effect of linear c-di-GMP-based compounds on DGC and/or PDE activity All the compounds synthesized in the present study (Tables 1, 2, and SI-3) were tested at a final concentration of 100 µM as possible inhibitors of PleD from Caulobacter crescentus and of RocR from P. aeruginosa, as representatives of DGCs and PDEs, respectively 18, 47. Following a stepwise approach for the design of the compounds, we pursued the goal to obtain the simplest c-di-GMP linear analogue(s) effective against PleD and/or RocR. Scaffold A. First, the four compounds with scaffold A (Table 1, rows 1-4) were designed to assess the impact of nonconservative modification at the level of the linkage of the two sugar moieties; different combinations of purine bases have also been tested. Compound DCI028 significantly affects DGC activity (63% of residual activity of PleD, at 100 µM inhibitor), in contrast compounds DCI015 and DCI016, where one or both guanines were replaced by adenine(s), exhibited no inhibition. Compound DCI021 was not assayed due to the interference of its CD spectrum with that of c-di-GMP and co-migration with c-di-GMP on the C8-RP column. The guanine moiety of c-di-GMP is indeed crucial for promoting the formation of the intercalated dimer targeting the I-site of DGCs 48; more specifically, the carbonyl group, the –NH at position 1 of guanine and, to a lesser extent, the –NH2 moiety at position 2, which are absent in the adenine base, are crucial both for the formation of the intercalated dimer and for the interaction with the RxxD motif of the I-site. The carbonyl and the amino moieties establish H-bonding interactions with the two conserved arginines and aspartic residues of the I-site, respectively, while the -NH at position 1 stabilizes the two intercalated molecules by contributing an additional intramolecular Hbond. The driving force of c-di-GMP binding to the I-site of DGCs is the π-cation interaction of the stacked bases with a charged nitrogen atom on the arginine residue, while the cyclic phosphate lactone ring is not involved in polar interaction with protein residues15. 8 ACS Paragon Plus Environment

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Given that the two guanines are essential to form stacking interactions with the protein in the intercalated dimer

48

, it is likely that compound DCI028 targets the I-site of PleD. This model

would explain why compounds DCI015 and DCI016 failed to inhibit PleD, as their adenine moieties are not able to form dimers. In light of this result, we can speculate that replacement of the phosphodiester moiety with a triazole ring does not negatively affect the plasticity of this compound, in terms of its capability to mimic the intercalated dimer of c-di-GMP. On the other hand, lack of significant inhibition of RocR (Table 1) may indicate that the linker employed in scaffold A compounds disrupts targeting of the catalytic site of the EAL PDEs, where c-di-GMP binds as a linear monomer 22. It should be mentioned that, while the binding mode of cdi-GMP to the I-site of DGCs favours H-bonding between protein residues and the guanine moieties

48

, binding of c-di-GMP to the EAL active site mainly involves the phosphate moieties,

besides the stacking interactions of the bases (see also below) 22. Scaffold B. Based on these initial findings, a second series of compounds, lacking one of the two sugar moieties, was synthetized and tested. As reported in Table 1, row 5, removal of one of the two sugars in the sugar-triazol-sugar motif, as in compound DCI091, decreased the inhibitory effect on PleD observed with compound DCI028. Shortening of the linker length, as in compound DCI059 (Table 1, row 6), was not able to overcome this loss of activity. Interestingly, compound DCI091 appears to enhance the catalytic activity of RocR, though the mechanistic details of this effect have not been pursued. Scaffold C. A third series of compounds was then designed and synthetized in order to determine the minimal structural determinants controlling the DGC/c-di-GMP interaction (Table 1, rows 7-8). The main goal was to identify the simplest linker between the two purine bases able to target the Isite of DGCs and, possibly, to generate selectivity between this site and the catalytic site of PDEs. The sugar moiety was completely removed and the triazole-based linker was assayed. Compound DCI061, bearing two guanines, was found to significantly inhibit both PleD (12,5% of residual 9 ACS Paragon Plus Environment


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activity, at 100 µM inhibitor) and RocR (41% of residual activity, at 100 µM inhibitor). According to our structural hypothesis, compound DCI072, bearing two adenines, would not be a promising inhibitor of PleD, but could act on RocR, which demonstrates a less stringent requirement for guanine. In agreement with this prediction, DCI072 inhibits RocR, though to a lower extent than compound DCI061 (64% of residual activity, at 100 µM inhibitor). Binding of c-di-GMP to the PDE active site mainly involves interactions with the linker moiety of c-di-GMP (which is then hydrolysed, see below) rather than with the nitrogenous bases; accordingly, RocR is sensitive to both DCI061 and DCI072 but the overall inhibitory activity is lower than that exerted by DCI061 with PleD. The effect of compound DCI061 on both PleD and RocR was analysed in greater detail to determine the IC50, which was found to be 17.5±1.1 µM and 66.3±1.3 µM, respectively (Figure 1A). As mentioned above, we speculated that compound DCI061 specifically targets the I-site of PleD. To support this assumption, compound DCI061 was assayed on two other DGCs, from P. aeruginosa, namely WspR, containing a canonical I-site, and YfiNHAMP-GGDEF insensitive to c-diGMP feedback inhibition (due to the presence of a degenerate I-site,

49

). In agreement with our

hypothesis, WspR activity was strongly reduced in the presence of DCI061 (∼20% of residual activity, at 100 µM inhibitor), while the activity of YfiNHAMP-GGDEF was not affected by a large excess of DCI061 (Figure 1B), even up to 250 µM (data not shown). Literature data report the case of XCC4471GGDEF, the GGDEF domain of a DGC from Xanthomonas campestris, which, despite the absence of a canonical I-site, is competitively inhibited by c-di-GMP. Structural data showed a novel partially intercalated dimeric form of c-di-GMP in the active site, which accounts for the competitive inhibition observed

50

. It is likely that proteins able

to bind c-di-GMP in such a way would also be able to bind compound DCI061, as suggested by modelling studies (Figure SI-1).


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Based on these results, the chemical scaffold of compound DCI061 was used as a starting point to design and synthetize optimized inhibitors.

Derivatives of compound DCI061 and selective inhibition of DGCs As reported in Table 2, eight compounds were synthesized to improve the selectivity and, possibly, the activity of compound DCI061. First, a series of four compounds bearing the same linker of compound DCI061 but harbouring guanine modifications were assayed (Table 2, rows 14). Among the substitutions of the carbonyl moiety in the guanine ring assayed (Table 2, rows 1-3), the chloride derivative (compound DCI058) is the most promising, given that it retains the inhibitory property on PleD (IC50 = 25.5±1.2 µM, Figure 2) and, more importantly, does not significantly inhibit RocR (96% of residual activity, at 100 µM inhibitor). The chlorine atom retains the electrophilic character at the C-6 position, a feature which is crucial for targeting the I-site of DGCs; on the other hand, the replacement of the carbonyls with chlorine most likely abolishes the H-bonding of the nitrogenous base with the EAL active site (see below). Since position 6 is crucial for the interaction of guanine with both DGCs and PDEs, it is not surprising that compounds DCI134 and DCI136, where the carbonyl moiety has been replaced by the methylamino- or the bulky phenylmethylamino- substituents, respectively, are ineffective against PleD and exhibit only slight activity against RocR. It has been previously shown by crystal structure analysis that the 2-amino group of the guanine moiety is involved in the H-bonding of c-di-GMP with highly conserved residues in I-sites of DGCs

29, 49

, while it is not crucial for the stabilization of substrate binding to the EAL site (see

below). Despite this, in vitro data on the Slr1143 DGC

51

indicated that cyclic di-inosinylic acid,

corresponding to the c-di-GMP analogue lacking the 2-amino group, inhibits DGC activity (IC50 = 68.9 µM), being more effective than c-di-GMP itself on the same enzyme (IC50 >> 100 µM). In light of these opposite observations, the 2-amino group of compound DCI058 was removed, leading to compound DCI070 (Table 2, row 4), which is unable to inhibit either PleD or RocR. 11 ACS Paragon Plus Environment


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These data indicate that, in agreement with structural evidence, the conservation of position 2 of guanine is crucial for the class of analogues presented in this work. The mild inhibitory effect of cyclic di-inosinylic acid reported in

51

could be related to the peculiarity of the DGC analysed, in

which the feedback inhibition of c-di-GMP presents unconventional features (IC50 >> 100 µM vs PleD IC50 = 5 µM 18 or WspR IC50 = 49 µM,

29

).

Finally, as compounds DCI061 and DCI058 proved to be the most interesting molecules, they were further modified to contain longer or shorter linker regions (Table 2, rows 5-8). However, no inhibition of either PleD or RocR was observed, thus suggesting that the optimal linker length is that contained in compounds DCI061 and DCI058. It is worth noting that this linker length most closely mimics both that of the intercalated dimer of c-di-GMP and its monomeric and linear counterpart (as in the case of the EAL active site). In Vivo Studies Preliminary in vivo analyses revealed that, despite the strong repressive effect that compounds DCI061 and DCI058 exert on DGCs in vitro, they are not effective in decreasing biofilm formation in P. aeruginosa and E. coli (Supporting information, Figure SI-2). Also the evidence that neither DCI058 nor DCI061 alter the expression of c-di-GMP regulated genes in P. aeruginosa cells argues against their ability to decrease intracellular levels of c-di-GMP in vivo (Supporting information, Figure SI-3). This lack of efficacy on bacterial cells is not surprising when considering that among the c-di-GMP analogues with DGC inhibitory activity published so far (see supporting informations), none has been reported to decrease biofilm formation, which is very likely due to a lack of permeability with these compounds. It is not ruled out that once we will find optimized compounds able to exert their effect not only in vitro but also in vivo, they may also target other kinds of c-di-GMP receptors, given that c-diGMP is flexible enough to bind its partners with different conformations via only minor adjustments15. Molecular Modeling 12 ACS Paragon Plus Environment

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Modeling of DCI061 into the I-site of PleD gave additional clues regarding the possible binding mode of triazole derivatives. DCI061 is predicted to bind in the same way as the intercalated dimer of c-di-GMP, with two molecules bound at the interface between the DGC and D2 domains of PleD by a multitude of specific interactions (Figure 3). The latter mimic the common base-arginine pairing motif involving O6 and N7 of the guanyl groups of c-di-GMP and arginine residues (Arg390, Arg359, and Arg178). The length of the linker connecting the two purine moieties appears to be optimal to place the aromatic rings at stacking distance and provides a structural rationale to the finding that longer/shorter linkers generally result in poor inhibition (Table 2, rows 5-8). The obtained model is also able to explain why small electrophilic groups are the most favoured at position 6 of the purine base. Indeed, the partial negative charges of O- or Cl- atoms are placed at a suitable distance to interact with Arg178 and Arg359, and possibly with the triazole ring in an anion/π-like interaction. Amines or bulkier groups, as observed in some of the triazole derivatives tested in this study, would disrupt such favorable interactions. Finally, the importance of an amine group at position 2 of the ring for PleD inhibition, as assessed in this study, is justified by the interactions of this moiety with residues Asp362, Gly174 and His177 of PleD. c-di-GMP is expected to bind to RocR in a manner similar to BlrP1 and TBD1265, since the key residues that bind c-di-GMP are conserved among the three homologous proteins

47

. When

modelled in the EAL active site of RocR using the crystal structure of BlrP1 as a template 21, c-diGMP appears in the correct conformation to bind to the same residues that are conserved in RocR and BlrP1. When docked into the active site of RocR, DCI061 adopts an extended conformation, which again resembles the binding mode of c-di-GMP (Figure 4A and 4B, respectively). Base-arginine and base-tyrosine pairing motifs involving Arg319 and Tyr374 stabilize one of the guanyl groups of DCI061. The latter group is further stabilized by the electrostatic interaction with Glu355, and by a hydrogen-bond between the main-chain of Tyr374 and the carbonyl moiety of the guanine ring. The 2,3 N- atoms of the triazole ring of DCI061 appear at suitable distance to coordinate at least one of 13 ACS Paragon Plus Environment


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the two metallic centers of RocR (2.9Å and 3.5Å, respectively), while they seem too distant to interact with the other centre (3.9Å and 4.8Å, respectively); this may account for the poorer inhibitory activity of DCI061 against PDEs than against DGCs. The second guanyl moiety of DCI061 is deeply buried into a cleft of the EAL domain of RocR, as observed in the crystal structure of the c-di-GMP/BlrP1 complex 21. Three polar interactions take place in this case: Glu116 and His235 with the O6 of the guanyl group, and a hydrogen-bond between a water molecule (403) and the amino group of the guanyl ring. CONCLUSIONS In the present paper a new class of c-di-GMP-based compounds able to selectively target DGCs is reported. The idea was to simplify the complex molecule of c-di-GMP and to substitute the charged and hydrolyzable phosphate linker of c-di-GMP with a neutral and stable triazole-based linker, in order to obtain a small molecule able to discriminate between the I-site of DGCs and the active site of PDEs. Two compounds are reported to inhibit DGCs targeting the I-site, namely compound DCI061 and DCI058, the latter being selective for this class of enzyme (See Figure 5 for a scheme summarizing the proposed mechanism of action). Among c-di-GMP analogues characterized so far, their effect on DGCs is comparable to those of 2’-F-c-di-GMP 29, but, contrary to the latter, they do not significantly inhibit PDEs. The array of compounds designed, synthesized and tested in this study revealed new information as to the specific role of each of the guanine substituents in the two different conformations required for binding to the I-site or to the PDE active site. These compounds also represent precious tools for future mechanistic characterization of DGCs and PDEs. In particular, compound DCI058 will be useful for studying hybrid proteins containing both GGDEF and EAL motifs, which, in spite of their involvement in biofilm formation and bacterial pathogenesis, are still poorly characterized biochemically. Indeed, many of the biophysics and biochemical analyses required to study kinetic properties, binding affinity and site-directed mutants of such proteins could take advantage of the 14 ACS Paragon Plus Environment

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capability of DCI058 to selectively target GGDEF domains without altering the catalytic activity of EAL domains. We demonstrated that the linker region of c-di-GMP (and the analogues characterized herein) does not interact directly with the I-site, but rather serves as a flexible spacer to allow the correct stacking of the four guanines (or their chloride derivatives). In conclusion, these results lay the foundation for the future development of cell permeable analogues of these compounds, based on modification of the linker region, which could be used to hamper c-di-GMP signalling in bacteria cells, and hence biofilm formation.

EXPERIMENTAL SECTION Chemical Synthesis. Materials and Instrumentation All reagents and solvents were purchased from Sigma-Aldrich Chemical Co and were analytical grade. Thin layer chromatography (TLC) was run on silica gel 60 F254 plates; silica gel 60 (70–230 mesh, Merck and 200-400 mesh, Merck) for column chromatography was used. Preparative thin layer chromatography was run on silica gel GF (20 x 20 cm, 1000 µ, Analtech). The final compounds were characterized by 1H-NMR,

13

C-NMR, MS and elemental analyses.

1

H NMR

spectra were determined at 400 MHz with a Varian Mercury AS400 instrument. The chemical shift values are expressed in δ values (ppm), and coupling constants (J) are in Hertz; TMS was used as an internal standard. The presence of all exchangeable protons was confirmed by addition of D2O. The purity of final compounds was checked using an Agilent 1100 Series instrument equipped with Synergi Polar-RP 80A 4 µm analytical column (150 mm x 4.6 mm, Phenomenex, Torrance, CA). Mobile phase consisted of a mixture of water/methanol (50:50) at a flow rate of 0.6 mL/min. Peaks were detected by UV adsorption with a diode array detector (DAD) at 230, 254, and 280 nm. All derivatives tested for biological activity showed ≥ 96% purity by HPLC analysis (detection at 254 nm). Mass spectra were recorded on an HP 1100 series instrument.



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All measurements were performed in the positive ion mode using atmospheric pressure electrospray ionization (API-ESI). Elemental analyses (C, H, and N) were determined on ThermoFisher Scientific FLASH 2000 CHNS analyzer and are within 0.4% of theoretical values. 5’-Azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (3). 2’,3’-O-isopropylidene-guanosine (1) 41 (1 mmol) was suspended in dry 1,4-dioxane (4 mL) under nitrogen atmosphere. DPPA (2 mmol) and DBU (3 mmol) were added dropwise and the mixture was stirred at room temperature for 4 h. Sodium azide (5 mmol), tetrabutylammonium iodide (0.1 mmol) and 15-crown-5 (0.1 mmol) were added and the reaction mixture was refluxed for 5 h. The reaction was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel (0–2% MeOH in CHCl3) to give compound 3 (65% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.49 (dd, J = 4.8, 13.0 Hz, 1H, H-5′a), 3.58 (dd, J = 7.6 Hz, 13.0 Hz, 1H, H-5′b), 4.24-4.17 (m, 1H, H-4’), 5.03 (dd, J = 3.3, 6.2 Hz, 1H, H-3′), 5.29 (dd, J = 2.2, 6.3 Hz, 1H, H-2′), 5.99 (d, J = 2.2 Hz, 1H, H-1′), 6.54 (brs, 2H, NH2), 7.88 (s, 1H, H-8), 10.70 (s, 1H, NH). MS (API-ESI): m/z calcd for C13H16N8O4 [M+ H]+ 349.13; found 349.12. 2-N-Isobutyryl-5’-azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine

(4).

5’-Azido-5’-deoxy-

2’,3’-O-isopropylidene-guanosine 3 (1 mmol) was suspended in pyridine (1 mL) and the suspension was evaporated. Pyridine (3 mL) was added and the mixture was cooled at 0 °C. Trimethylsilyl chloride (4.9 mmol) and isobutyryl chloride (5 mmol) were added dropwise and the mixture was stirred at room temperature for 3 h. The mixture was cooled to 0 °C, water (1 mL) and ammonium hydroxide solution (1.5 mL) were added and the solution was allowed to warm to room temperature for 1 h. The pyridine was evaporated, water was added (3 mL) to residue, then the water phase was extracted 3 times with chloroform. The combined organic phases were dried over Na2SO4 and concentrated. The crude was purified by column chromatography on silica gel (0–2% MeOH in CHCl3) to give compound 4 (65% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.11 (s, 3H, CH3-isobutyryl), 1.13 (s, 3H, CH3-isobutyryl), 1.25 (s, 3H, CH3), 1.50 (s, 3H, CH3), 2.702.80 (m, 1H, CH-isobutyryl), 3.56-3.52 (m, 2H, H-5’), 4.25-4.20 (m, 1H, H-4’), 5.15 (dd, J = 3.4, 16 ACS Paragon Plus Environment

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6.4 Hz, 1H, H-3′), 5.35 (dd, J = 2.5, 6.2 Hz, 1H, H-2′), 6.02 (d, J = 2.2 Hz, 1H, H-1′), 8.20 (s, 1H, H-8), 11.50 (s, 1H, NH-isobutyryl), 12.10 (s, 1H, NH). MS (API-ESI): m/z calcd for C17H22N8O5 [M+ H]+ 419.17; found 419.20. 2-N-isobutyryl-2’,3’-O-isopropylidene-5’-O-propargyl-guanosine (8). 2-N-isobutyryl-2’,3’-Oisopropylidene-guanosine (6) 43 (1 mmol) was dissolved in dry THF (10 mL), cooled to 0 °C before the addition of NaH (1.79 mmol, 60% dispersion in mineral oil). The mixture was stirred for 30 minutes and propargyl bromide (1.79 mmol) was added dropwise. After 26 h of stirring at room temperature, the reaction was evaporated to dryness and the crude was purified by column chromatography on silica gel (0–2% MeOH in CHCl3) to give 8 (31% yield) as a white solid and the side product 8a (17% yield). 1H NMR (400 MHz, DMSO-d6): δ 1.11 (s, 3H, CH3-isobutyryl), 1.13 (s, 3H, CH3-isobutyryl), 1.31 (s, 3H, CH3), 1.51 (s, 3H, CH3), 2.73-2.82 (m, 1H, CHisobutyryl), 3.41 (t, J = 2.4 Hz, 1H, CH-propargyl), 2.50-2.65 (m, 2H, H-5’), 4.13 (d, J = 2.4 Hz, 2H, OCH2-propargyl), 4.27 (dd, J = 5.0, 8.3 Hz, 1H, H-4’), 5.07 (dd, J = 3.2, 6.2 Hz, 1H, H-3′), 5.27 (dd, J = 2.4, 6.2 Hz, 1H, H-2′), 6.05 (d, J = 2.3 Hz, 1H, H-1′), 8.15 (s, 1H, H-8), 11.53 (brs, 1H, NH-isobutyryl), 12.10 (brs, 1H, NH). MS (API-ESI): m/z calcd for C20H25N5O6 [M+ H]+ 432.18; found 432.21. 2-N-isobutyryl-N1-propargyl-2’,3’-O-isopropylidene-5’-O-propargyl-guanosine (8a). 1H NMR (400 MHz, DMSO-d6): δ 1.03 (s, 3H, CH3-isobutyryl), 1.04 (s, 3H, CH3-isobutyryl), 1.29 (s, 3H, CH3), 1.51 (s, 3H, CH3), 2.48 (brs, 1H, CH-isobutyryl), 3.24 (brs, 1H, CH-N1-propargyl), 3.38-3.42 (m, 1H, CH-propargyl), 3.50-3.64 (m, 2H, H-5’), 4.10 (d, J = 2.3 Hz, 2H, OCH2- propargyl), 3.264.32 (m, 1H, H-4’), 4.54-4.58 (m, 2H, NCH2- propargyl), 4.94 (dd, J = 2.9, 6.4 Hz, 1H, H-3′), 5.305.35 (m, 1H, H-2′), 6.11 (d, J = 2.2 Hz, 1H, H-1′), 8.25 (s, 1H, H-8), 12.84 (brs, 1H, NHisobutyryl). MS (API-ESI): m/z calcd for C23H27N5O6 [M+ H]+ 470.19; found 470.18. N6-Benzoyl-2’,3’-O-isopropylidene-5’-O-propargyl-adenosine isopropylidene-adenosine (7)

34

(9).

N6-Benzoyl-2’,3’-O-

(1 mmol) was dissolved in dry DMF (5 mL), cooled to 0 °C before

the addition of NaH (5 mmol, 60% dispersion in mineral oil). The mixture was stirred for 30 17 ACS Paragon Plus Environment


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minutes and propargyl bromide (1.2 mmol) was added dropwise. After 1.5 h of stirring at 0 °C, the reaction was quenched with ethyl acetate (1 mL) and concentrated. The residue was treated with a mixture of CH2Cl2/H2O; the organic phase was separated and the aqueous phase was extracted with CH2Cl2 (3 times). The combined organic phases were dried over Na2SO4 and concentrated. The crude was purified by column chromatography on silica gel (0–2% MeOH in CH2Cl2) to give compound 9 (85% yield) as a pale solid. 1H NMR (400 MHz, DMSO-d6): δ 1.34 (s, 3H, CH3), 1.55 (s, 3H, CH3), 3.42 (s, 1H, CH-propargyl), 3.50-3.70 (m, 2H, H-5’), 4.03-4.18 (m, 2H, OCH2propargyl), 4.28-4.44 (m, 1H, H-4’), 5.01 (dd, J = 2.7, 6.0 Hz, 1H, H-3′), 5.47 (dd, J = 2.4, 6.0 Hz, 1H, H-2’), 6.29 (d, J = 2.3 Hz, 1H, H-1’), 7.54 (t, J = 7.5 Hz, 2H, benzoyl), 7.64 (t, J = 7.4 Hz, 1H, benzoyl), 8.03 (d, J = 2.3 Hz, 2H, benzoyl), 8.60 (s, 1H, H-2), 8.77 (s, 1H, H-8), 11.22 (s, 1H, NHbenzoyl). MS (API-ESI): m/z calcd for C23H23N5O5 [M+ H]+ 450.17; found 450.19. General procedure for the synthesis of 1,2,3-triazole derivatives via CuACC. To a mixture of alkyne (1 mmol) in 10 mL of H2O/tBuOH (3:2) was added CuSO4·5H2O (0.2 mmol), sodium ascorbate (0.04 mmol), and then azide (1.3 mmol) 52. The heterogeneous reaction mixture was stirred at room temperature for 2-3 days and monitored by TLC and MS. After the completion, the mixture was evaporated to dryness and the intermediates were used directly for following reactions or purified by column chromatography on silica gel. 5’-Deoxy-(4-(2-N-isobutyryl-2’,3’-O-isopropylidene-guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1yl)-2-N-isobutyryl-2’,3’-O-isopropylidene guanosine (10). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-N-isobutyryl-2’,3’O-isopropylidene-5’-O-propargyl-guanosine (8) and

2-N-isobutyryl-5’-azido-5’-deoxy-2’,3’-O-

isopropylidene-guanosine (4) as starting materials. The reaction mixture was stirred at room temperature for 2 days and evaporated to dryness. The crude was purified by column chromatography on silica gel (CHCl3/MeOH 98:2) to give compound 10 (85% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.09-1.14 (m, 12H, CH3-isobutyryl), 1.29 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.49 (s, 3H, CH3), 1.50 (s, 3H, CH3), 2.71-2.80 (m, 2H, CH-isobutyryl), 3.4718 ACS Paragon Plus Environment

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3.58 (m, 2H, H-5’), 4.18-4.26 (m, 1H, H-4’), 4.42-4.50 (m, 3H, OCH2-triazolyl, H-4’), 4.55-4.70 (m, 2H, H-5’), 5.01 (dd, J = 3.2, 6.2 Hz, 1H, H-3′), 5.24 (dd, J = 2.3, 6.2 Hz, 1H, H-3′), 5.27-5.34 (m, 2H, H-2′), 6.02 (d, J = 2.3 Hz, 1H, H-1’), 6.13 (d, J = 1.4 Hz, 1H, H-1’), 7.90 (s, 1H, =CH-N), 8.08 (s, 1H, H-8), 8.09 (s, 1H, H-8), 11.43 (brs, 1H, NH-isobutyryl), 11.51 (brs, 1H, NHisobutyryl), 12.10 (brs, 2H, NH). MS (API-ESI): m/z calcd for C37H47N13O11 [M+ H]+ 850.35; found 850.33. 5’-Deoxy-(4-(N6-benzoyl-2’,3’-O-isopropylidene-adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)2’,3’-O-isopropylidene-guanosine (11). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using N6-benzoyl-2’,3’-O-isopropylidene5’-O-propargyl adenosine (9) and 5’-azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (3) as starting materials. The reaction mixture was stirred at room temperature for 3 days and the suspension was filtered. The solid was washed with ethanol, dried and used directly for following reaction. 5’-Deoxy-(4-(2-N-isobutyryl-2’,3’-O-isopropylidene-guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1yl)-2’,3’-O-isopropylidene-adenosine (12). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-N-isobutyryl-2’,3’-Oisopropylidene-5’-O-propargyl-guanosine

(8)

and

5’-azido-5’-deoxy-2’,3’-O-isopropylidene-

adenosine (5) as starting materials. The reaction mixture was stirred at room temperature for 3 days and evaporated to dryness. The crude was purified by column chromatography on silica gel (CHCl3/MeOH 95:5) to give compound 12 (56% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.10 (d, J = 1.5 Hz, 3H, CH3-isobutyryl), 1.11 (d, J = 1.5 Hz, 3H, CH3-isobutyryl), 1.29 (s, 6H, CH3), 1.49 (s, 3H, CH3), 1.50 (s, 3H, CH3), 2.72-2.81 (m, 1H, CH-isobutyryl), 3.473.58 (m, 2H, H-5’), 4.34-4.39 (m, 1H, H-4’), 4.44 (s, 2H, OCH2-triazolyl), 4.47-4.52 (m, 1H, H-4’), 4.59-4.76 (m, 2H, H-5’), 4.96 (dd, J = 3.1, 6.1 Hz, 1H, H-3′), 5.11 (dd, J = 3.2, 6.3 Hz, 1H, H-3′), 5.23 (dd, J = 2.6, 6.2 Hz, 1H, H-2′), 5.43 (dd, J = 2.3, 6.2 Hz, 1H, H-2’), 6.02 (d, J = 2.4 Hz, 1H, H-1’), 6.20 (d, J = 2.3 Hz, 1H, H-1’), 7.35 (brs, 2H, NH2), 7.92 (s, 1H, =CH-N), 8.11 (s, 1H, H-2), 19 ACS Paragon Plus Environment


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8.17 (s, 1H, H-8), 8.26 (s, 1H, H-8), 11.52 (brs, 1H, NH-isobutyryl), 12.10 (brs, 1H, NH). MS (API-ESI): m/z calcd for C33H41N13O9 [M+ H]+ 764.31; found 764.35. 5’-Deoxy-(4-(N6-benzoyl-2’,3’-O-isopropylidene-adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)2’,3’-O-isopropylidene-adenosine (13). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using N6-benzoyl-2’,3’-O-isopropylidene5’-O-propargyl-adenosine (9) and 5’-azido-5’-deoxy-2’,3’-O-isopropylidene-adenosine (5) as starting materials. The reaction mixture was stirred at room temperature for 3 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (CHCl3/MeOH 95:5) to give compound 13 (40% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.27 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.53 (s, 3H, CH3), 3.50-3.64 (m, 2H, H-5’), 4.34-4.39 (m, 1H, H-4’), 4.44 (s, 2H, OCH2-triazolyl), 4.47-4.52 (m, 1H, H-4’), 4.59-4.76 (m, 2H, H-5’), 4.94 (dd, J = 2.6, 6.1 Hz, 1H, H-3′), 5.09 (dd, J = 3.4, 6.2 Hz, 1H, H-3′), 5.38-5.43 (m, 2H, H-2’), 6.19 (d, J = 2.2 Hz, 1H, H-1’), 6.24 (d, J = 2.4 Hz, 1H, H-1’), 7.35 (brs, 2H, NH2), 7.53 (t, J = 7.6 Hz, 2H, benzoyl), 7.63 (t, J = 7.4 Hz, 1H, benzoyl), 7.93 (s, 1H, =CH-N), 8.03 (d, J = 7.4 Hz, 2H, benzoyl), 8.16 (s, 1H, H-2), 8.24 (s, 1H, H-2), 8.55 (s, 1H, H-8), 8.72 (s, 1H, H-8), 11.21 (brs, 1H, NH). MS (API-ESI): m/z calcd for C36H39N13O8 [M+ H]+ 782.30; found 782.31. 5’-Deoxy-(4-(guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-guanosine (DCI028). Compound 10 (1 mmol) was treated with 10 mL of formic acid aqueous solution (70%) and the reaction was stirred at 40 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The residue was purified by column chromatography on silica gel (CHCl3/MeOH/NH3 8.2:1.6:0.2) to give the intermediate N-iBu-protected as a white solid (46% yield). The intermediate N-iBuprotected (1 mmol) was then treated with an ammonium hydroxide solution 33% (20 mL) and the reaction was stirred at 50 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated with ethanol and diethyl ether (2 times), filtered and purified by preparative thin-layer chromatography (iPrOH/NH3/H2O 8:1.5:0.5) to give compound DCI028 (20% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 3.50-3.70 (m, 2H, H-5’), 3.86-3.98 20 ACS Paragon Plus Environment

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(m, 1H, H-4’), 3.99-4.06 (m, 1H, H-4’), 4.09-4.26 (m, 2H, H-5’), 4.37-4.42 (m, 1H, H-3’), 4.434.49 (m, 1H, H-3’), 4.52 (s, 2H, OCH2-triazolyl), 4.65-4.67 (m, 1H, H-2′), 4.69-4.72 (m, 1H, H-2′), 5.19 (brs, 1H, OH), 5.42 (brs, 2H, OH), 5.55 (d, J = 5.8 Hz, 1H, OH), 5.66 (d, J = 5.9 Hz, 1H, H1’), 5.68 (d, J = 6.1 Hz, 1H, H-1’), 6.47 (brs, 2H, NH2-guanosine), 6.48 (brs, 2H, NH2-guanosine), 7.78 (s, 1H, H-8), 7.83 (s, 1H, H-8), 7.97 (s, 1H, =CH-N), 10.44 (brs, 2H, NH).

13

C NMR (101

MHz, DMSO-d6): δ 51.83, 64.15, 70.51, 70.98, 71.42, 72.86, 73.67, 82.75, 83.41, 86.78, 87.36, 117.02, 117.33, 125.03, 135.71, 136.30, 144.03, 151.57 (x2), 154.06 (x2), 157.12 (x2). MS (APIESI): m/z calcd for C23H27N13O9 [M+ H]+ 630.20; found 630.20. Elem. Anal: C, 43.88; H, 4.32; N, 28.92; Found: C, 43.90; H, 4.33; N, 28.90. 5’-Deoxy-(4-(adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-guanosine (DCI016). Compound 11 (1 mmol) was treated with a saturated methanolic ammonia solution (50 mL) and the reaction was stirred at room temperature overnight. After full conversion the solvent was removed under reduced pressure and the residue was purified by preparative thin-layer chromatography (CHCl3/MeOH 8:2) to give intermediate 2’,3’-O-isopropylidene-protected

as a white solid (48% yield). The

intermediate 2’,3’-O-isopropylidene-protected (1 mmol) was then treated with 10 mL of formic acid aqueous solution (70%) and the reaction was stirred at 40 °C for 2 days. After full conversion the solvent was removed under reduced pressure followed by coevaporation with MeOH (3 times). The residue was purified by preparative thin-layer chromatography (iPrOH/NH3/H2O 8:1:1) to give compound DCI016 (25% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 3.58-3.71 (m, 2H, H-5’), 3.99-4.03 (m, 1H, H-4’), 4.10-4.21 (m, 3H, H-4’, H-5’), 4.44-4.56 (m, 4H, H-3′, H-3′, OCH2-triazolyl), 4.69-4.72 (m, 2H, H-2’, H-2’), 5.38 (s, 1H, OH), 4.45-4.49 (m, 2H, OH), 5.59 (s, 1H, OH), 5.69 (d, J = 5.6 Hz, 1H, H-1’), 5.87 (d, J = 5.6 Hz, 1H, H-1’), 6.53 (brs, 2H, NH2guanosine), 7.26 (brs, 2H, NH2-adenosine), 7.79 (s, 1H, =CH-N), 7.97 (s, 1H, H-2), 8.12 (s, 1H, H8), 8.29 (s, 1H, H-8), 10.47 (brs, 1H, NH). 13C NMR (101 MHz, DMSO-d6): δ 51.96, 66.25, 69.30, 70.67, 72.89, 72.95, 74.50, 84.23, 87.30, 86.78, 88.65, 117.10, 117.34, 125.10, 136.20, 136.45, 144.07, 151.40, 152.25, 154.20, 155.63, 157.12, 157.30. MS (API-ESI): m/z calcd for C23H27N13O8 21 ACS Paragon Plus Environment


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[M+ H]+ 614.20; found 614.40. Elem. Anal: C, 45.03; H, 4.44; N, 28.68; found: C, 45.02; H, 4.42; N, 28.65. 5’-Deoxy-(4-(guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-adenosine (DCI021). Compound 12 (1 mmol) was treated with a saturated methanolic ammonia solution (50 mL) and the reaction was stirred at room temperature overnight. After full conversion the solvent was removed under reduced pressure and the crude was treated with 10 mL of formic acid aqueous solution (70%). The reaction was stirred at 40 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The residue was purified by preparative thin-layer chromatography (iPrOH/NH3/H2O 8:1:1) to give the compound DCI021 (25% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 3.53-3.66 (m, 2H, H-5’), 3.90-3.97 (m, 1H, H-4’), 3.97-4.04 (m, 1H, H-4’), 4.19-4.31 (m, 2H, H-5’), 4.364.53 (m, 4H, H-3′, H-3′, OCH2-triazolyl), 4.62-4.69 (m, 1H, H-2’), 4.70-4.78 (m, 1H, H-2’), 5.175.59 (m, 4H, OH), 5.66 (d, J = 5.8 Hz, 1H, H-1’), 5.89 (d, J = 5.2 Hz, 1H, H-1’), 6.57 (brs, 2H, NH2-guanosine), 7.29 (brs, 2H, NH2-adenosine), 7.86 (s, 1H, =CH-N), 7.96 (s, 1H, H-2), 8.13 (s, 1H, H-8), 8.25 (s, 1H, H-8), 10.50 (brs, 1H, NH). 13C NMR (101 MHz, DMSO-d6): δ 51.83, 64.48, 70.36, 71.03, 72.56, 72.83, 74.52, 83.66, 85.28, 86.66, 87.43, 117.15, 117.33, 125.10, 135.88, 136.58, 144.07, 151.48, 153.73, 154.29, 155.63, 157.43, 157.55. MS (API-ESI): m/z calcd for C23H27N13O8 [M+ H]+ 614.21; found 614.30. Elem. Anal: C, 45.03; H, 4.44; N, 29.68; found: C, 45.04; H, 4.45; N, 29.65. 5’-Deoxy-(4-(adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-adenosine (DCI015). Compound 13 (1 mmol) was treated with a saturated methanolic ammonia solution (50 mL) and the reaction was stirred at room temperature overnight. After full conversion the solvent was removed under reduced pressure and the crude was treated with 10 mL of formic acid aqueous solution (70%). The reaction was stirred at 40 °C for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The residue was purified by preparative thin-layer chromatography (EtOAc/AcOH/EtOH/H2O 6:2:2:1) to give the compound DCI015 (24% yield) as a white solid. 1H NMR (400 MHz, DMSOd6): δ 3.57-3.70 (m, 2H, H-5’), 3.97-4.02 (m, 1H, H-4’), 4.08-4.15 (m, 1H, H-4’), 4.20-4.29 (m, 22 ACS Paragon Plus Environment

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2H, H-5’), 4.47-4.54 (m, 4H, H-3′, H-3′, OCH2-triazolyl), 4.61-4.67 (m, 1H, H-2’), 4.70-4.77 (m, 1H, H-2’), 5.23-5.30 (m, 1H, OH), 5.49-4.54 (m, 1H, OH), 5.54-4.58 (m, 1H, OH), 5.62-4.68 (m, 1H, OH), 5.86-5.91 (m, 2H, H-1’, H-1’), 7.26 (brs, 2H, NH2-adenosine), 7.30 (brs, 2H, NH2adenosine), 7.96 (s, 1H, =CH-N), 8.12 (s, 1H, H-2), 8.13 (s, 1H, H-2), 8.24 (s, 1H, H-8), 8.28 (s, 1H, H-8).

13

C NMR (101 MHz, DMSO-d6): δ 52.05, 64.58, 70.58, 71.05, 71.39, 72.82, 73.50,

82.37, 83.10, 86.72, 87.26, 117.04, 117.39, 125.08, 135.88, 136.23, 144.01, 151.49 (x2), 153.84 (x2), 157.56 (x2). MS (API-ESI): m/z calcd for C23H27N13O7 [M+ H]+ 598.21; found 598.22. Elem. Anal: C, 46.23; H, 4.55; N, 30.47; found: C, 46.22; H, 4.57; N, 30.45. 2-Amino-6-chloro-N9-(3-(4-(2-N-isobutyryl-2’,3’-O-isopropylidene-guanosin-5’-yl)-methyl1H-1,2,3-triazol-1-yl)propyl)-purine (20). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-amino-6-chloro-N9-(3azidopropyl)-purine (16) and 2-N-isobutyryl-5’-O-propargyl-5’-deoxy-2’,3’-O-isopropylideneguanosine (8) as starting materials. The reaction mixture was stirred at room temperature for 3 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (CHCl3/MeOH 93:7) to give 20 (53% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.10 (s, 3H, CH3-isobutyryl), 1.11 (s, 3H, CH3-isobutyryl), 1.30 (s, 3H, CH3), 1.49 (s, 3H, CH3), 2.30-2.39 (m, 2H, CH2-CH2-CH2), 2.72-2.80 (m, 1H, CH-isobutyryl), 3.51-3.62 (m, 2H, H-5’), 4.05 (t, J = 6.8 Hz, 2H, -CH2-N-purine), 4.23-4.29 (m, 1H, H-4’), 4.36 (t, J = 7.0 Hz, 2H, CH2-Ntriazolyl), 4.50 (s, 2H, OCH2-CH2), 5.01-5.04 (m, 1H, H-3′), 5.23-5.26 (m, 1H, H-2′), 6.02 (d, J = 2.2 Hz, 1H, H-1′), 6.89 (s, 2H, NH2), 8.05 (s, 1H, =CH-N), 8.10 (s, 2H, H-8, H-8), 11.53 (brs, 1H, NH-isobutyryl), 12.10 (brs, 1H, NH). MS (API-ESI): m/z calcd for C28H34ClN13O6 [M+ H]+ 684.22; found 684.20. N9-(3-(4-(guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-guanine (DCI091). Compound 20 (1 mmol) was treated with a mixture of TFA/H2O (3:1, 10 mL) and the reaction was stirred at room temperature for 24 hours, followed by evaporation and coevaporation with MeOH (3 times). The residue was treated with an ammonium hydroxide solution 33% (20 mL) and the reaction was 23 ACS Paragon Plus Environment


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stirred at 60 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated with methanol and diethyl ether (2 times), filtered and purified by preparative thin-layer chromatography (iPrOH/NH3/H2O 8:1.5:0.5) to give compound DCI091 (30% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.24-2.34 (m, 2H, CH2-CH2-CH2), 3.58 (dd, J = 4.8, 10.6 Hz, 1H, H-5’a), 3.66 (dd, J = 3.8, 10.7 Hz, 1H, H-5’b), 3.94 (t, J = 6.6 Hz, 2H, -CH2-Npurine), 4.00-4.06 (m, 1H, H-4’), 4.29-4.42 (m, 4H, CH2-N-triazolyl, H-2′, H-3′), 4.54 (s, 2H, OCH2-CH2), 5.19 (d, J = 4.9 Hz, 1H, OH), 5.42 (d, J = 6.0 Hz, 1H, OH), 5.66 (d, J = 5.8 Hz, 1H, H-1’), 6.42 (brs, 2H, NH2-guanosine), 6.46 (brs, 2H, NH2-guanine), 7.66 (s, 1H, =CH-N), 7.81 (s, 1H, H-8), 8.11 (s, 1H, H-8), 10.56 (brs, 2H, NH). 13C NMR (101 MHz, DMSO-d6): δ 30.60, 47.51, 64.50, 70.74, 71.33, 74.05, 83.70, 87.00, 117.26, 117.32, 124.77, 135.03, 136.04, 138.02, 144.45, 151.85, 152.10, 154.22, 154.37, 157.42, 157.50. MS (API-ESI): m/z calcd for C21H25N13O6 [M+ H]+ 556.20; found 556.22. Elem. Anal: C, 45.40; H, 4.54; N, 32.78; found: C, 45.42; H, 4.55; N, 32.79. 5’-Deoxy-(4-(2-amino-6-chloro-9H-purin)-methyl-1H-1,2,3-triazol-1-yl)-2-N-isobutyryl-2’,3’O-isopropylidene-guanosine (21). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-amino-6-chloro-N9-propargyl-purine (19)

45

and 2-N-isobutyryl-5’-azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (4) as starting materials. The reaction mixture was stirred at room temperature for 2 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (CHCl3/MeOH 9:1) to give compound 21 (71% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.12 (dd, J = 3.6, 6.8 Hz, 6H, CH3-isobutyryl), 1.31 (s, 3H, CH3), 1.50 (s, 3H, CH3), 2.69-2.78 (m, 1H, CH-isobutyryl), 4.44-4.60 (m, 2H, H-5’), 4.67 (dd, J = 4.6, 14.3 Hz, 1H, H-4’), 5.26-5.33 (m, 4H, NCH2-triazolyl, H-3′, H-2′), 6.12 (s, 1H, H-1’), 6.93 (s, 2H, NH2), 7.95 (s, 1H, =CH-N), 8.11 (s, 1H, H-8), 8.13 (s, 1H, H-8), 11.44 (brs, 1H, NH-isobutyryl), 12.10 (brs, 1H, NH). MS (API-ESI): m/z calcd for C25H28ClN13O5 [M+ H]+ 626.20; found 626.22. 5’-Deoxy-(4-(9H-guanin)-methyl-1H-1,2,3-triazol-1-yl)-guanosine (DCI059). Compound 21 (1 mmol) was treated with a mixture of TFA/H2O (3:1, 10 mL) and the reaction was stirred at room 24 ACS Paragon Plus Environment

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temperature for 20 hours, followed by evaporation and coevaporation with MeOH (3 times). The residue was treated with an ammonium hydroxide solution 33% (20 mL) and the reaction was stirred at 60 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated with methanol and diethyl ether (2 times), filtered and purified by preparative thin-layer chromatography (iPrOH/NH3/H2O 8:1.5:0.5) to give the compound 5 (30% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 4.03-4.22 (m, 2H, H-5’), 4.42-4.49 (m, 1H, H-4’), 4.61-4.71 (m, 2H, H-2′, H-3′), 5.18 (s, 2H, NCH2-triazolyl), 5.40 (brs, 1H, OH), 5.56 (brs, 1H, OH), 5.68 (d, J = 6.0 Hz, 1H, H-1’), 6.47 (brs, 2H, NH2-guanosine), 6.51 (brs, 2H, NH2-guanine), 7.66 (s, 1H, =CH-N), 7.81 (s, 1H, H-8), 7.91 (s, 1H, H-8), 10.58 (brs, 2H, NH).

13

C NMR (101 MHz,

DMSO-d6): δ 44.50, 56.38, 72.30, 75.26, 80.66, 89.54, 117.18, 117.68, 123.36, 136.58, 137.89, 145.02, 151.68, 152.17, 154.32, 154.44, 157.48, 157.63. MS (API-ESI): m/z calcd for C18H19N13O5 [M+ H]+ 498.16; found 498.18. Elem. Anal: C, 43.46; H, 3.85; N, 36.61; found: C, 43.48; H, 3.87; N, 36.63. 6-Chloro-N9-(4-(6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-purine (DCI070). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 6-chloro-N9-propargyl-purine (18) and 6-chloro-N9-(3-azidopropyl)-purine (14) as starting materials. The reaction mixture was stirred at room temperature for 2 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (CHCl3/MeOH 95:5) to give the compound DCI070 (40% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.38-2.46 (m, 2H, CH2-CH2-CH2), 4.30 (t, J = 6.9 Hz, 2H, -CH2-N-purine), 4.38 (t, J = 7.0 Hz, 2H, CH2-N-triazolyl), 5.59 (s, 2H, NCH2-C), 8.14 (s, 1H, =CH-N), 8.65 (s, 1H, H-2), 8.74 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.79 (s, 1H, H-8). 13C NMR (101 MHz, DMSO-d6): δ 30.23, 43.90, 45.67, 49.73, 122.67, 123.56, 123.87, 144.67, 145.70, 145.93, 150.09, 150.56, 151.67, 151.78, 156.32, 156.87. MS (API-ESI): m/z calcd for C16H13Cl2N11 [M+ H]+ 430.07; found 430.20. Elem. Anal: C, 44.67; H, 3.05; N, 35.81; found: C, 44.68; H, 3.07; N, 35.83.



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N9-(4-(adenine)-methyl-1H-1,2,3-triazol-1-yl)propyl)-adenine (DCI072). DCI070 (1 mmol) was treated with a saturated isopropanol ammonia solution (25 mL) and the reaction was stirred at 60 °C overnight. After this time the suspension was filtered and the solid was treated with methanol and diethyl ether (2 times), dried to give the compound DCI072 (55% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.28-2.38 (m, 2H, CH2-CH2-CH2), 4.12 (t, J = 6.9 Hz, 2H, -CH2-Npurine), 4.33 (t, J = 7.0 Hz, 2H, CH2-N-triazolyl), 5.41 (s, 2H, NCH2-C), 7.19 (s, 2H, NH2), 7.21 (s, 2H, NH2), 8.09 (s, 2H, =CH-N, H-2), 8.12 (s, 2H, H-8, H-2), 8.17 (s, 1H, H-8).

13

C NMR (101

MHz, DMSO-d6): δ 30.15, 45.67, 46.78, 49.63, 121.75, 122.46, 122.81, 139.37, 143.46, 143.58, 149.97, 149.99, 152.45, 152.68, 156.56, 156.79. MS (API-ESI): m/z calcd for C16H17N13 [M+ H]+ 392.18; found 392.22. Elem. Anal: C, 49.10; H, 4.38; N, 46.52; found: C, 49.11; H, 4.36; N, 46.53. 2-Amino-6-chloro-N9-(4-(2-amino-6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-ethyl)-purine (DCI133). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-amino-6-chloro-N9-propargyl-purine (19) and 2-amino-6-chloroN9-(2-azidoethyl)-purine (15) as starting materials. The reaction mixture was stirred at room temperature for 5 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (CHCl3/MeOH 95:5) to give the compound DCI133 (22% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 4.49 (t, J = 5.7 Hz, 2H, -CH2-N-purine), 4.77 (t, J = 5.7 Hz, 2H, -CH2-N-triazolyl), 5.29 (s, 2H, NCH2-C), 6.93 (s, 2H, NH2), 6.94 (s, 2H, NH2), 7.71 (d, J = 1.5 Hz, 1H, =CH-N), 7.99 (s, 1H, H-8), 8.10 (d, J = 1.5 Hz, 1H, H-8). 13C NMR (101 MHz, DMSO-d6): δ 38.53, 42.76, 46.53, 123.3, 128.24, 130.73, 142.96, 143.67, 144.54, 149.53, 150.03, 154.33, 154.98, 160.41, 161.04. MS (API-ESI): m/z calcd for C15H13Cl2N13 [M+ H]+ 446.08; found 446.10. Elem. Anal: C, 40.37; H, 2.94; N, 40.80; Found: C, 40.38; H, 2.91; N, 40.83. 2-Amino-6-chloro-N9-(4-(2-amino-6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-propyl)purine (DCI058). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-amino-6-chloro-N9-propargyl-purine (19) and 2-amino-6chloro-N9-(4-azidopropyl)-purine (16) as starting materials. The reaction mixture was stirred at 26 ACS Paragon Plus Environment

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room temperature for 2 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (CHCl3/MeOH 95:5) to give the compound DCI058 (66% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.26-2.38 (m, 2H, C-CH2-C), 4.02 (t, J = 6.9 Hz, 2H, -CH2-N-purine), 4.38 (t, J = 7.0 Hz, 2H, CH2-CH2-N-triazolyl), 5.54 (s, 2H, NCH2-C), 6.97 (s, 2H, NH2), 6.98 (s, 2H, NH2), 8.05 (s, 1H, =CH-N), 8.09 (s, 1H, H-8), 8.15 (s, 1H, H-8). 13C NMR (101 MHz, DMSO-d6): δ 30.05, 38.93, 41.15, 47.60, 123.84, 124.05, 124.33, 142.96, 143.68, 143.83, 150.02, 150.08, 154.57, 154.78, 160.41, 160.56. MS (API-ESI): m/z calcd for C16H15Cl2N13 [M+ H]+ 460.09; found 460.20. Elem. Anal: C, 41.75; H, 3.28; N, 39.56; found: C, 41.77; H, 3.31; N, 39.54. 2-Amino-6-chloro-N9-(4-(2-amino-6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-butyl)-purine (DCI095). The reaction was carried out according to the general procedure for the synthesis of 1,2,3-triazole derivatives, using 2-amino-6-chloro-N9-propargyl-purine (19) and 2-amino-6-chloroN9-(3-azidobutyl)-purine (17) as starting materials. The reaction mixture was stirred at room temperature for 3 days and evaporated to dryness. The crude was purified by column chromatography on a silica gel (0–5% MeOH in CH2Cl2) to give the compound DCI095 (40% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.68-1.74 (m, 4H, C-CH2-CH2-C), 4.014.06 (m, 2H, -CH2-N-purine), 4.30-4.35 (m,, 2H, -CH2-N-triazolyl), 5.31 (s, 2H, NCH2-C), 6.89 (s, 2H, NH2), 6.93 (s, 2H, NH2), 8.02 (s, 1H, =CH-N), 8.10 (s, 1H, H-8), 8.14 (s, 1H, H-8). 13C NMR (101 MHz, DMSO-d6): δ 26.38, 28.77, 38.93, 43.86, 49.73, 124.70, 125.03 (x2), 143.04, 144.45, 144.48, 150.46, 150.83, 154.23, 154.61, 159.89, 159.93. MS (API-ESI): m/z calcd for C17H17Cl2N13 [M+ H]+ 474.11; found 474.13. Elem. Anal: C, 43.05; H, 3.61; N, 38.39; found: C, 43.02; H, 3.63; N, 38.38. N9-(4-(guanine)-methyl-1H-1,2,3-triazol-1-yl)-ethyl)-guanine (DCI135). DCI133 (1 mmol) was treated with a mixture of TFA/H2O (3:1, 10 mL) and the reaction was stirred at room temperature for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated with methanol and diethyl ether (2 times), filtered and dried to give compound DCI135 (58% yield) 27 ACS Paragon Plus Environment


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as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 4.37 (t, J = 5.7 Hz, 2H, -CH2-N-purine), 4.73 (t, J = 5.7 Hz, 2H, -CH2-N-triazolyl), 5.19 (s, 2H, NCH2-C), 6.52 (brs, 4H, NH2), 7.41 (d, J = 7.9 Hz, 1H, =CH-N), 7.80 (d, J = 11.0 Hz, 1H, H-8), 7.91 (s, 1H, H-8), 10.65 (brs, 2H, NH).

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C NMR

(101 MHz, DMSO-d6): δ 43.15, 44.70, 54.53, 115.25, 116.04, 123.89, 137.23, 139.03, 145.83, 151.23, 151.55, 153.89, 154.20, 157.37, 157.68. MS (API-ESI): m/z calcd for C15H15N13O2 [M+ H]+ 410.15; found 410.17. Elem. Anal: C, 44.01; H, 3.69; N, 44.48; found: C, 44.02; H, 3.66; N, 44.49. N9-(4-(guanine)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-guanine (DCI061).

DCI058 (1 mmol)

was treated with a mixture of TFA/H2O (3:1, 10 mL) and the reaction was stirred at room temperature for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated with methanol and diethyl ether (2 times), filtered and dried to give the compound DCI061 (76% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.24-2.36 (m, 2H, CH2CH2-CH2), 3.99 (t, J = 6.9 Hz, 2H, -CH2-N-purine), 4.35 (t, J = 7.0 Hz, 2H, -CH2-N-triazolyl), 5.24 (s, 2H, N-CH2-CH2), 6.58 (s, 2H, NH2), 6.67 (s, 2H, NH2), 7.96 (s, 1H, H-8), 8.05 (s, 1H, =CH-N), 8.14 (s, 1H, H-8), 10.77 (brs, 1H, NH), 10.89 (brs, 1H, NH).

13

C NMR (101 MHz, DMSO-d6): δ

30.12, 38.97, 41.53, 47.52, 114.05, 115.24, 124.46, 137.86, 137.96, 143.03, 151.33, 151.43, 154.76, 154.91, 156.29, 156.80. MS (API-ESI): m/z calcd for C16H17N13O2 [M+ H]+ 424.16; found 424.30. Elem. Anal: C, 45.39; H, 4.05; N, 43.01; found: C, 45.41; H, 4.08; N, 42.98. N9-(4-(guanine)-methyl-1H-1,2,3-triazol-1-yl)-butyl)-guanine (DCI096). DCI095 (1 mmol) was treated with a mixture of TFA/H2O (3:1, 10 mL) and the reaction was stirred at room temperature for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated with methanol and diethyl ether (2 times), filtered and dried to give the compound DCI096 (93% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 1.64-1.77 (m, 4H, CH2-CH2-CH2), 3.934.00 (m, 2H, -CH2-N-purine), 4.29-4.36 (m, 2H, -CH2-N-triazolyl), 5.22 (s, 2H, N-CH2-CH2), 6.54 (s, 2H, NH2), 6.60 (s, 2H, NH2), 7.88 (s, 1H, H-8), 8.00 (s, 1H, =CH-N), 8.05 (s, 1H, H-8), 10.70 (brs, 1H, NH), 10.80 (brs, 1H, NH). 13C NMR (101 MHz, DMSO-d6): δ 27.43, 29.06, 40.47, 44.53, 49.79, 115.25, 116.63, 123.83, 135.64, 136.15, 142.03, 150.65, 151.68, 154.34, 154.76, 157.29, 28 ACS Paragon Plus Environment

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157.85. MS (API-ESI): m/z calcd for C17H19N13O2 [M+ H]+ 438.18; found 438.30. Elem. Anal: C, 46.68; H, 4.38; N, 41.63; found: C, 46.67; H, 4.40; N, 41.62. 2-Amino-(N6-methyl)-N9-(4-(2-amino-(N6-methyl)-purin)-methyl-1H-1,2,3-triazol-1-yl)propyl)-purine (DCI134). To a solution of DCI058 (1 mmol) in ethanol (20 mL) was added methylamine (33% in absolute ethanol, 10 mmol) and the reaction was stirred at 80 °C for 4 h. After this time the suspension was filtered and the solid was treated with ethanol and diethyl ether (2 times), dried to give the compound DCI134 (68% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.22-2.31 (m, 2H, CH2-CH2-CH2), 2.85 (brs, 6H, NH-CH3), 3.93 (t, J = 6.9 Hz, 2H, CH2-N-purine), 4.31 (t, J = 7.0 Hz, 2H, CH2-N-triazolyl), 5.22 (s, 2H, NCH2-C), 5.86 (brs, 4H, NH2), 7.17 (brs, 2H, NH-CH3), 7.65 (s, 1H, H-8), 7.70 (s, 1H, H-8), 8.07 (s, 1H, =CH-N). 13C NMR (101 MHz, DMSO-d6): δ 27.24, 27.33, 30.06, 40.63, 41.42, 49.53, 118.46, 118.78, 123.75, 136.23, 136.55, 142.79, 149.55, 150.27, 156.73, 156.91, 158.43, 158.90. MS (API-ESI): m/z calcd for C18H23N15 [M+ H]+ 450.23; found 450.23. Elem. Anal: C, 48.10; H, 5.16; N, 46.74; found: C, 48.08; H, 5.17; N, 46.76. 2-Amino-(N6-benzyl)-N9-(4-(2-amino-(N6-benzyl)-purin)-methyl-1H-1,2,3-triazol-1-yl)propyl)purine (DCI136). To a solution of DCI058 (1 mmol) in ethanol (20 mL) was added benzylamine (10 mmol) and the reaction was stirred at 80 °C for 4 h. After this time the suspension was filtered and the solid was treated with ethanol and diethyl ether (2 times), dried to give the compound DCI136 (68% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 2.22-2.33 (m, 2H, C-CH2C), 3.93 (t, J = 6.4 Hz, 2H, -CH2-N-purine), 4.32 (t, J = 6.8 Hz, 2H, CH2-N-triazolyl), 4.61 (brs, 4H, -CH2-benzyl), 7.17 (brs, 2H, NH), 5.23 (s, 2H, N-CH2-CH2), 5.86 (brs, 4H, NH2), 7.14-7.20 (m, 2H, arom), 7.23-7.29 (m, 4H, arom), 7.29-7.33 (m, 4H, arom), 7.68 (d, J = 1.5 Hz, 1H, H-8), 7.73 (d, J = 1.5 Hz, 1H, H-8), 8.07 (s, 1H, =CH-N).

13

C NMR (101 MHz, DMSO-d6): δ 30.54, 41.67

(x2), 41.60 (x2), 49.63, 119.67, 119.34, 122.89, 126.89 (x6), 127.69 (x4), 130.67, 140.63 (x2), 143.59, 145.84, 150.46, 150.81, 157.96, 157.98, 159.43, 159.67. MS (API-ESI): m/z calcd for



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C30H31N15 [M+ H]+ 602.29; found 602.40. Elem. Anal: C, 59.89; H, 5.19; N, 34.92; found: C, 59.87; H, 5.21; N, 43.93. DGC expression and purification: Expression and purification of PleD from Caulobacter crescentus, WspR, YfiNHAMP-GGDEF and RocR from Pseudomonas aeruginosa were performed as previously published 16, 48, 49, 53. Kinetic studies The diguanylate cyclase activity was assayed by circular dichroism (CD) as previously published48. Compound DCI061 was also tested on WspR and YfiNHAMP-GGDEF from Pseudomonas aeruginosa. PDE activity of RocR was assayed as reported in

48

both in the presence or in the absence of

inhibitors. Inhibition assay was performed under the same conditions tested on PleD (See SI for details). For those compounds whose CD spectra interfere with CD spectrum of c-di-GMP, the nucleotide content of the reaction mixture was evaluated by reverse-phase HPLC, as previously published 54. For IC50 determination, the initial velocity was considered. Molecular Modeling Molecular docking was carried out by means of Molegro Virtual Docker (MVD) software (® CLCbio). Ligands were built and energy minimized by using the PRODRG server

55

. Flexible

torsions were automatically detected by MVD, and manually checked for consistency. The threedimensional structures of PleD (PDB: 1W25) and RocR (PDB: 3SY8) were prepared by automatically assigning bond orders and hybridization, and adding explicit hydrogens, charges and Tripos atom types. Missing heavy atoms were fixed by modeling them, using Modeler v.9.8 and PyMod 56, 57. A search space of 15Å radius was used for docking. Whenever possible, guanine rings of c-di-GMP were taken as pharmacophoric groups for template-based dockings. In this way, if an atom of the ligand matches a group definition, it is rewarded by using a weighted score (w) that depends on its distance to the group centers, by using the following Gaussian function for each 30 ACS Paragon Plus Environment

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center: w = ω*exp(-d2/r02)

(1)

where d is the distance (Å) from the position of the atom to the center in the group. ω is a weight factor for the template group, and r0 is a distance parameter, specifying a characteristic distance for the template group. ω and r0 were set at ω = 2; r0 = 1.80. An overall normalization of the similarity score term, to balance it with other scoring terms, is then applied for each atom. Grid-based MolDock score

58

with a grid resolution of 0.30 Å was used as scoring function for docking.

MolDock SE was used as docking algorithm. For each ligand, ten runs were defined. Similar poses (RMSD < 1.2 Å) were clustered, and the best scoring one was taken as representative. Other docking parameters were fixed at their default values. After docking, energy optimization of hydrogen bonds was performed. Biofilm formation assays Biofilm formation assays were performed on P. aeruginosa PAO1 and E. coli MG1655 strains as previously described

59, 60

, with few modifications. P. aeruginosa PAO1 and E. coli MG1655 cells

were grown for 14 hours at 37°C in M9 minimal medium supplemented with 0.4% (w/v) glucose [2.5% (v/v) Luria-Bertani broth was also added to the medium for E. coli cultures]. Then cells were diluted to an absorbance at 600 nm wavelength (A600) in fresh medium, and 200 µL of the diluted cultures were aliquoted in 96-well polystyrene microtiter plates. Media were supplemented with DMSO alone (as a control) or with compounds DCI058 or DCI061 dissolved in DMSO at the concentrations indicated in Figure S-1. After 16 hours of incubation at 30°C, the A600 of planktonic cells was recorded and planktonic cells were removed, wells were washed twice with 200 µL PBS and air-dried for 20 minutes. Cells bound to the wells were stained with 200 µL of 1% (w/v) crystal violet (CV) before solubilisation with 200 µL ethanol, and A595 measurement. Biofilm formation was estimated as Adhesion Units by normalizing the A595 value of the CV solution to the A600 value of the planktonic culture. In Figure S-1 the average of eight independent experiments is reported with SD. 31 ACS Paragon Plus Environment


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Real Time RT-PCR analyses Total RNA was extracted from 1 mL of P. aeruginosa PAO1 cultures grown at 37°C to an A600 of 2.0 in M9 minimal medium supplemented with 0.4% (w/v) glucose and DMSO or 200 µM DCI058 or DCI061 (both compounds were dissolved in DMSO). Bacterial cultures were mixed with 2 mL of RNA Protect Bacteria Reagent (Qiagen), and cell lysis was performed as recommended by the manufacturer. RNA was purified by using RNeasy mini columns (Qiagen), including the on-column DNase I digestion step described by the manufacturer. In addition, the eluted RNA was treated for 1 hour at 37°C with TURBO DNase (0.1 units per µg of RNA; Ambion). DNase I was removed with the RNeasy Column Purification Kit (Qiagen). RNA integrity was monitored by agarose gel electrophoresis, and the absence of contaminant chromosomal DNA was verified by PCR performed with oligonucleotides FW16SRT (5’-GAGAGTTTGATCCTGGCTCAG-3’) and RV16SRT (5’-CTACGGCTACCTTGTTACGA-3’). cDNA synthesis was performed from 1 µg of total purified RNA by using random hexamer primers and the iScript Reverse Transcription Supermix for RT-qPCR kit (BioRad), according to manufacturer’s instructions. The resulting cDNAs were quantified by spectrophotometric analysis. Real-time PCRs were performed using the iTaq™ Universal SYBR® Green Supermix (BioRad) Gene-specific primers employed in this analysis were designed using the Primer-Blast software (www.ncbi.nlm.nih.gov/tools/primer-blast) to avoid unspecific amplification of P. aeruginosa DNA. The cdrA and pctC cDNAs were amplified with oligonucleotide pairs FWcdrART (5’-CGAAAGCTGGTAGGGAAGGG-3’) and RVcdrART

(5’-GTCGAAGCCCTTCCAGTTGA-3’),

and

FWpctCRT

(5’-

TCGGCATCATGCTTCTCCTC-3’) and RVpctCRT (5’-TCGATTCGTGGATACGCTGC-3’), respectively. The cdrA and pctC genes have been selected for this analysis because they were previously demonstrated to be positively and negatively controlled by c-di-GMP, respectively 61, 62. A preliminary analysis was performed on serial cDNA dilutions to identify the amount of cDNA showing the highest dynamic range in gene expression (5 ng of cDNA per reaction), and the most efficient primers concentration (300 nM). The reaction procedure involved incubation at 95°C for 1 32 ACS Paragon Plus Environment

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minute and 40 cycles of amplification at 95°C for 10 seconds and 60°C for 45 seconds. Fluorescence was registered in the last 15 seconds of the 60°C step. 16S ribosomal RNA was chosen as an internal control (housekeeping gene amplified with oligonucleotides FW16SRT and RV16SRT) to normalize the real-time PCR data in each single run, and to calculate the relative fold change in gene expression by using the 2∆∆Ct method. The analysis was performed in duplicate on three technical replicates. ASSOCIATED CONTENT Figures SI-1, SI-2, and SI-3

reaction Scheme SI-1, Tables SI-1 and SI-2 and Supplemental

Methods are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Serena Rinaldo: [email protected]; Phone: +39.06.49910713. Fax: +39064440062 Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, 00185 Rome, Italy Author Contributions *,§

S.F., I.T., L.C. and F.C. contributed equally to this work

ACKNOWLEDGEMENTS This work was supported by MIUR of Italy [20094BJ9R7 to F.C and L.C., and RBFR10LHD1 to S.R. and G.R.], Sapienza University of Rome to A.P. (C26A149EC4); Fondo di Ricerca di Ateneo (FAR 2011/12 STI000044) University of Camerino to R.P.; Italian Cystic Fibrosis Research Foundation (FFC 13/2011; www.fibrosicisticaricerca.it) to L.L. The plasmids expressing the RocR and WspR genes are kind gifts of H. Sondermann (USA). We are grateful to Dr. Daniel J. Wilson for critically reading the manuscript.



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Scheme 1.

Scheme 2.


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Scheme 3.

Scheme 4.

Scheme 5.



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Page 44 of 52

Chart 1. Three different scaffolds used to synthetize potential inhibitors of DGCs and/or PDEs.


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Figure 1: Panel A) PleD and RocR inhibition assay at various concentrations of compound DCI061. Each point of the concentration response curve represents the mean value and standard deviation of three independent experiments. The line is the best-fit curve generated by the software Prism, using the log-dose vs response equation. Y-axis intercept represents the 100% residual activity obtained in the absence of inhibitor. Data fit allowed to extrapolate the IC50 for PleD and RocR, being 17.5±1.1 µM and 66.3±1.3 µM, respectively. Panel B) DGC residual activity of WspR and YfiNHAMP-GGDEF observed in the presence of excess DCI061 (100 µM).



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Figure 2: PleD inhibition assay at various concentrations of compound DCI058. Each point of the concentration response curve represents the mean value and standard deviation of three independent experiments. The line is the best-fit curve generated by the software Prism, using the log-dose vs response equation. Y-axis intercept represent the 100% residual activity obtained in the absence of inhibitor. Data fit allowed to extrapolate the IC50 for PleD, being 25.5±1.2 µM.


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Figure 3. Predicted binding mode of DCI061 to the I-site of PleD. A) Two mutually intercalated DCI061 moieties (white and pink sticks, respectively) are represented bound at the interface between the DGC and D2 domains of PleD (grey ribbons and sticks). Residues are labeled according to PDB Code: 1W25. Polar interactions are shown in yellow. B) Structural superposition between two DCI061 compounds and two c-di-GMPs (cyan and slate sticks, respectively), as found in PDB Code: 1W25.



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Figure 4. Predicted binding mode of DCI061 to the active site of RocR. A) DCI061 (white sticks) binds in an extended conformation into the active site of RocR. Residues are labeled according to PDB Code: 3SY8. Polar interactions are shown in yellow. B) Structural superposition between DCI061 and c-di-GMP (slate sticks). The latter has been modelled into the active site of RocR on the basis of the binding mode observed in the close homolog BlrP1 (PDB Code: 3GG1).


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Figure 5. Proposed mechanism of action of inhibitors characterized in the present study. On the left (blue box) representation of the general catalytic mechanism of GGDEF and EAL domains, and of the conformational rearrangement taking place during non-competitive product inhibition of GGDEF containing proteins. On the right, effect and binding site of inhibitors DCI061 and DCI058 on GGDEF and EAL containing proteins (i.e. PleD and RocR). While DCI061 inhibits both PleD and RocR, DCI058 fails to inhibit RocR, but it is still able to undergo dimerization and bind to the I-site of GGDEF domain.



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Table 1. C-di-GMP-based molecules synthetized and assayed in the present work. The residual enzymatic activity of PleD or RocR has been measured in the presence of 100 µM compound and 100 µM GTP or 30 µM c-di-GMP as substrate, respectively. Values are the % of the observed activity as compared to the activity in the absence of potential inhibitors (100% of activity) and correspond to the mean of at least two independent experiments ± standard deviation. % of residual activity has been extrapolated as the difference between the 100% activity and the % of the observed one. Residual activity lower than 30% has been considered significant and it has been discussed in the text.


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Scaffold

Name

A

Structural formula

PleD

RocR

DCI015

105±10

113±11

A

DCI016

97±4.9

79±2.1

A

DCI021

n.d.

n.d.

A

DCI028

63±7.8

88±8

B

DCI091

82±1.9

163±3.8

B

DCI059

89±6

77±4.5

C

DCI061

12.5±1.8

41±2.2

C

DCI072

106±10

64±4



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Table 2. C-di-GMP analogues synthetized and assayed in the present work, as modifications of compound DCI061. Values have been obtained as reported in Table 1. Linker PleD

RocR

n=3

32±4.2

96±2.7

DCI134

n=3

96±16.2

70±0.1

C

DCI136

n=3

109±10

75±1

C

DCI070

n=3

97±2.4

93±2.3

C

DCI096

n=4

87±3.1

92±3.3

C

DCI095

n=4

98±3.2

128±4.5

C

DCI135

n=2

104±6.2

77±4.6

C

DCI133

n=2

92±2.4

86±2.1

Scaffold

Name

C

DCI058

C

Structural formula


Synthesis of Triazole-Linked Analogues of c-di ... - ACS Publications

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