Family-wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of aminoacyl-tRNA synthetases

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Graphical Abstract:

Family wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of

aminoacyl-tRNA synthetases

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Baole Zhang , Steff De Graef , Manesh Nautiyal , Luping Pang , Bharat Gadakh , Matheus

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Froeyen , Lieve Van Mellaert , Sergei V. Strelkov , Stephen D. Weeks and Arthur Van

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Aerschot

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Family wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine

analogues as inhibitors of aminoacyl-tRNA synthetases

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Baole Zhang , Steff De Graef , Manesh Nautiyal , Luping Pang , Bharat Gadakh , Matheus

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Froeyen , Lieve Van Mellaert , Sergei V. Strelkov , Stephen D. Weeks and Arthur Van Aerschot

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Medicinal Chemistry, Rega Institute for Medical Research, Herestraat 49 box 1041, B-3000 Leuven,

Belgium

Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences,

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Herestraat 49 box 822, B-3000 Leuven, Belgium

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Laboratory Molecular Bacteriology, Rega Institute for Medical Research, Herestraat 49 box 1037, B-

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3000 Leuven, Belgium

* To whom correspondence should be addressed. Tel: +32 16 37 26 24

arthur.vanaerschot@kuleuven.be

; Fax: +32 16 33 73 40; Email:

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These authors contributed equally to this work.

∞ Both authors equally supervised and gave guidance to this work

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ABSTRACT

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Aminoacyl-tRNA synthetases (aaRS) are enzymes that precisely attach an amino acid to its cognate

tRNA. This process, which is essential for protein translation, is considered a viable target for the

development of novel antimicrobial agents, provided species selective inhibitors can be identified.

Aminoacyl-sulfamoyl adenosines (aaSA) are potent orthologue specific aaRS inhibitors that

demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work

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on substitution of the base moiety, we evaluated the effect of the N -position of the adenine by

synthesizing the corresponding 3-deazaadenosine analogues (aaS3DA). A typical organism has 20

different aaRS, which can be split into two distinct structural classes. We therefore coupled six

different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-

deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class

bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when

compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the

presence of a conserved water molecule which could have importance for base recognition within

class II enzymes, a property that can be explored in future drug design efforts.

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1. Introduction

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Infectious diseases are the second largest cause of mortality worldwide [1]. In 2015, an estimated

9.3 million people died as result of microbial infection, surpassing the number of deaths caused by

cancer [2]. Critically, our most important weapons against such pathogens have become less effective

because of the rise of antimicrobial resistance [3]. This has led to predictions that the deaths

associated to infectious diseases will dramatically increase if no immediate action is taken [4]. As part

of a global effort to curtail this crisis it has been recommended that new antimicrobials are developed.

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Most organisms provide numerous viable targets for new compounds, yet the majority are poorly

exploited. The aminoacyl-tRNA synthetase (aaRS) family is one such group that has recently

garnered more attention for the development of candidate small molecule inhibitors [5].

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Aminoacyl-tRNA synthetases are essential enzymes, found in all cellular life, that play a central

role in the translation of the genetic code [6]. They catalyze a two-step process that results in the

attachment of a specific amino acid (aa) to its cognate tRNA (Fig 1. A). In the first step an aaRS

forms a high affinity mixed phosphoanhydride, the aminoacyl-adenylate intermediate (aa-AMP; Fig 2.

a), from an amino acid and an ATP molecule, yielding pyrophosphate as a byproduct (Fig 1. A). In the

subsequent step the intermediate carbonyl undergoes a nucleophilic attack from a hydroxyl group

from the 3’-terminal ribose of the tRNA, yielding the aminoacylated tRNA product and AMP [7].

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A typical organism contains 20 different aaRS, one for each proteinogenic amino acid. Reflecting

on their evolutionary origins these can be divided, almost equally, into two distinct classes based on

the structure of the catalytic site (Fig 1. B). Class I representatives contain the βαβ Rao-Rossman fold

commonly observed in nucleotide binding proteins [8]. Sequence analysis has shown that within this

domain, all class I orthologues present a highly conserved HIGH and a KMSKS sequence (where

each letter corresponds to the amino acid one letter code) [4]. These two motifs, both involved in ATP

binding and activation, sandwich the primary sequence of the catalytic domain. The HIGH-motif is

found in the first α-helix of the common core structure, while the KMSKS sequence occurs in the C-

terminus of this domain in a loop that bridges the catalytic core to the anticodon binding domain. All

class II orthologues share a central six-stranded curved β-sheet surrounded by a variable number of

α-helices (Fig 1.B). Contrasting with the class I aaRS, analysis of class II sequences has identified

three cryptic motifs shared amongst members of this family [9]. The first motif is involved in homo-

dimerization, a common quaternary arrangement shared amongst class II aaRS, whereas the

remaining are necessary for ATP binding and formation of the intermediate [10].

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Targeting aaRS is advantageous as these enzymes meet various criteria for modern antibacterial

drug discovery [11]. Nature itself has already come up with aaRS inhibitors, such as ileRS inhibitor

mupirocin (Fig 2. c) and aspRS inhibitor microcin C (Fig 2. d). Unfortunately, as with other

antibacterial agents that act on a single enzyme target, aaRS inhibitors possess an intrinsic

resistance liability [12], and the occurrence of clinical isolates of S. aureus that are resistant to

mupirocin has been reported [13]. Randall et al. demonstrated that simultaneous targeting of two

aaRS enzymes overcomes the resistance liabilities associated with inhibitors acting against a single

enzyme [14]. Therefore, it is of considerable interest to identify new compounds that target different

orthologues within this family. The simplest approach would be to employ a general scaffold, where

simply changing the attached amino acid would expand the portfolio of target aaRS.

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Replicating biology, one approach to rationally design aaRS inhibitors is to mimic the aa-AMP

intermediate [15]. Most non-hydrolysable aa-AMP analogues reported so far are based on replacing

the labile acyl-phosphate linkage of the intermediate with stable bioisosteres such as alkylphosphites

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[16-18], esters [19-21], amides [19], hydroxamates [19-21], sulfamates [17, 18, 22-25], sulfamides

[17], N-alkoxysulfamides [26] and N-hydroxysulfamides [26]. Among these molecules the aminoacyl-

sulfamate (aaSA) analogues (Fig 2. b) have proven to be the most potent inhibitors, with improved

hydrolytic stability compared to aminoalkyl and aminoacyl-adenylate [27].

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However, while the aaSA scaffold has been useful for structural studies of aaRS, their potential as

lead compounds for drug development has been hampered by their lack of selectivity and in vivo

efficacy [27, 28]. One suggested approach to resolve this problem is to replicate the Trojan-horse

strategy employed by some of the natural aaRS antibiotics, attaching such active moieties to bacterial

uptake modules that are then released in the cytoplasm by host enzymatic processing [29].

Unfortunately, coupling of the aaSAs to such carriers is limited as the sulfamate group has been

reported to be hydrolytically unstable under certain chemical conditions. This is due to the nucleophilic

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attack of the N of the base on the 5’-leaving moiety resulting in a N , C5’-cycloadenosine (Fig 2. e)

[30]. To resolve this issue we have investigated employing alternative base moieties, with varying

success in the resultant inhibitory activity [31]. Building upon these efforts, we report here the

synthesis and evaluation of stable 3-deazaadenosine analogues (aaS3DA, Fig 2. f). To fully assess

the consequence of substituting this single atom in aaSA we have performed a comprehensive family

wide analysis of the compounds, synthesizing and evaluating aaS3DA analogues that target both

class I and class II aaRS representatives.

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2. Results

2.1. Synthesis of aminoacyl-sulfamoyl 3-deazaadenosine analogues

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The synthesis of 3-deazaadenosine 7 from cheap and readily available 3,4-diaminopyridine 1 is

shown in Scheme 1. Cyclization of both amines using triethyl orthoformate and formic acid to afford

compound 2 was followed by oxidation to the N-oxide 3. Chlorination with phosphorus oxychloride

gave 4 with concomitant reduction of the N-oxide [32]. The heterocyclic base 4 was further reacted

with tetra-O-acetyl ribofuranose using stannic chloride followed by deprotection of the acetyl moieties

using methanolic ammonia to give the 4-chloro nucleoside analogue 6 [33]. Nucleophilic displacement

of the chloride using ammonia in methanol at 65 °C did not afford the desired amine 7 while this

method works well for the 6-chlorinated purine. This problem was overcome by a nucleophilic

displacement with hydrazine followed by reduction with Raney Nickel to yield 7 [34].

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Selective sulfamoylation at the primary hydroxyl moiety required prior protection of both secondary

alcohol groups. Hereto, formation of the acetonide compound 8 was accomplished with p-

toluenesulfonic acid in a mixture of anhydrous acetone and 2 equiv. of 2, 2-dimethoxypropane [35].

Direct sulfamoylation of 8 with sulfamoyl chloride followed by aminoacylation led to the synthesis of

base-aminoacylated compound 10 (see supplementary file) [36, 37].

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Hence, following an established protocol, successful benzoylation of 8 afforded compound 11 [38].

Subsequent reactions with sulfamoyl chloride and hydroxysuccinimide activated esters afforded

compounds 12 and 13a-f, respectively (scheme 2). Treatment with sodium methoxide gave

compounds 14a-f [39], and removal of the tert-butyloxycarbonyl and acetonide groups was

accomplished simultaneously by treating with TFA/water (5:2) to afford 15a-f [31]. For compounds

15b and 15c, the benzyl moieties were removed by treatment with hydrogen in methanol under

palladium catalysis right before the final deprotection. In total six aaS3DA compounds were

successfully produced. Three of these compounds (15b, 15e and 15f) target class I aaRS and the

remaining ones (15a, 15c and 15d) target class II enzymes. All compounds synthesized in this project

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were unambiguously characterized by H, C NMR spectroscopy and ESI-MS, except the

intermediates 13b-f and 14b-f that were only corroborated by ESI-MS.

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2.2. Inhibitory activity in E. coli S30 extracts

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The aaS3DA compounds were evaluated for their ability to inhibit aaRS catalyzed aminoacylation of

tRNA in whole cell extracts (Fig. 3). As a positive control, the comparative activity was assessed in

parallel with the canonical aaSA intermediate analogues. Reactions were performed using a S30

extract from the wild-type E. coli K-12 BW28357 strain preincubated with 2.5 µM of each compound.

In comparison to their aaSA equivalents, the six aaS3DA analogues showed a clear aaRS class

dependent difference in inhibitory activity (Fig. 3). For the class I aaRS (ileRS, leuRS and tyrRS), the

aaS3DA compounds demonstrated a similar reduction of the enzymatic activity as the adenosine

containing compounds (ileSA, leuSA and tyrSA). The amount of inhibition varied between the

different aaRS, from a reduction of almost 90% of the extract activity when preincubated with

leuS3DA to a significant 40-50% reduction in catalysis with ileS3DA and tyrS3DA. In contrast the

aaS3DA compounds demonstrated no, or severely limited, inhibitory activity when tested against the

corresponding class II enzymes (aspRS , glyRS and serRS), while the aaSA analogues showed

potencies equivalent to earlier reports [27].

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2.3. Measurement of in vitro inhibitory activity with purified E. coli aaRS

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To investigate further whether the observed difference in the class dependent inhibitory activity of the

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aaS3DA compounds was a result of substitution of the N atom in the base, rather than an effect of

extract mediated modification or aberrant binding of the compounds to other proteins in the clarified

lysate, the aaS3DA analogues were also evaluated with the corresponding isolated aaRS. Using

serial dilutions of the synthesised compounds comparative dose-response curves were generated for

each aaSA and aaS3DA pair (Fig. 4). These curves were then fitted using the Greco-Hakala equation

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for high affinity binders to determine the K_ifor each compound (Table 1).

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In the presence of 500 µM ATP all class I targeting aaS3DA compounds demonstrated sub-

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micromolar K_ivalues (Fig. 4 A and Table 1). Relative to aaSA the 3-deazaadenosine containing

compounds showed reduced inhibitory activity, but to varying degrees depending on the aaRS. In the

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worst case, with ileRS, substitution of the N resulted in a 110-fold increase in the K_i. For leuRS and

tyrRS the effect was less pronounced, where the aaS3DA analogues were 4.7 and 10.2 times less

active than the analogous aaSA, respectively. In contrast introduction of 3-deazaadenosine into the

aminoacyl-sulfamate scaffold had a more dramatic effect on the three compounds targeting class II

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aaRS, despite the aaSA equivalents demonstrating a range of K_ivalues similar to those seen for

the class I enzymes (Fig.4 B and Table 1). In particular, at the highest concentration tested (2 µM)

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glyS3DA showed no inhibitory activity against glyRS. Similarly, the aspS3DA and serS3DA

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compounds were also negatively affected, showing a respective increase in K_iof 486 and 2900-fold

when compared to aspSA and serSA (Table 1). The observed aaRS class dependent reduction of

inhibitory activity for the aaS3DA compounds, when compared to analogous aaSA, in this assay is in

accordance with the above results in cell extracts.

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2.4. Antibacterial activity measurements

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The obtained target compounds were evaluated for their in vitro antibacterial activity against Gram-

positive bacterial strains (Staphylococcus aureus ATCC 6538P, Staphylococcus epidermidis RP62A

and Kocuria rhizophila ATCC 9341 (formerly Sarcina lutea)), Gram-negative strains (Escherichia

coli NCIB 8743 and Pseudomonas aeruginosa PAO1) and one yeast species (Candida

albicans CO11) using the broth dilution method. Serial dilutions, from 100 µM to 0.39 µM, of each

aaS3DA compound were tested. Unfortunately, none of the compounds demonstrated growth

inhibition, even at the maximum concentration tested (data not shown), behaving similarly to what

was previously reported for the aaSA equivalents [40].

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2.5. Quantum calculation of the electrostatic potential of 3-deazaadenisone

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To assess the influence of the removal of the N -nitrogen on the electron distribution in 3-

deazaadenosine, quantum chemical calculations were performed for adenine and 3-deazaadenine

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using a N -methylated derivative as a substitute for the ribose (Fig. 5 A and B). The calculations

suggest that although the p-orbital electrons are similarly delocalised, there is a clear loss of

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electronegativity at the position where the N is substituted. In addition, the calculated structures show

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the N and both hydrogens to be coplanar with the purine ring, while for 3-deazaadenine the

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equivalent hydrogens are found out of plane. This illustrates that in adenine, the N has more sp2

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character, in contrast with a larger sp3 hybridization character seen for the N in 3-deazaadenine.

This change is reflected in a predicted rise in the pKa of this amine group and an increased

nucleophilicity for the 3-deaza congener. This observation is in good agreement with the observed

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reactivity of the N -group during primary coupling of the amino acid to the sulfamate, and the need to

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use an alternative approach employing the N -benzoyl protecting group (Scheme 2).

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2.6. Bioinformatic analysis

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To understand in more detail the apparent class based difference of aaS3DA activity we performed a

structure based alignment of all aaRS members in E. coli and the gram-positive bacterium S. aureus.

For some orthologues high resolution X-ray structures are available of the enzyme bound to their

respective aaSA. These permitted the identification of crucial residues involved in base recognition

that could be extrapolated to the other members in the alignment. In the case of the class I aaRS the

adenine moiety is sandwiched between the HIGH containing α-helix and a residue that is typically

non-polar, found in most class I aaRS 5-6 residues upstream of the conserved KMSKS loop (Fig. 5 C,

and Supplementary movie). The bulk of the interaction of the base with the active site appears to be

driven by Van der Waals forces. Only two polar interactions with the base are consistently observed in

different aaRS:aaSA complex structures, mediated by the interaction of the protein backbone atoms

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with the N and N position of adenine. Crucially the N -atom makes no clear interactions with the

protein supporting the described biochemical results.

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In stark contrast, the adenosine makes numerous interactions with active site residues in the class II

aaRS (Fig. 5 D and Supplementary movie). One face of the base makes a π/π interaction with a

conserved phenylalanine (or rarely tyrosine) residue, found in the class wide motif-2, while the

opposite face of this heterocycle interacts with a highly conserved arginine residue present in the

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structurally preserved α-helix of motif-3 via cation/π bonding. In addition to these weak interactions N ,

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N and N all make H-bond interactions with conserved features shared amongst all class II aaRS

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members. Both the N and N make polar interactions with backbone atoms of a poorly conserved

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residues in motif-2, similarly to that seen in the class I enzymes. In addition, the N -amine also

interacts with a highly conserved acidic residue within the same motif (Fig. 5 D and Supplementary

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movie). Crucially, the N interacts with the protein via a water molecule, typically positioned 3 Å away,

that is found capping the N-terminus of the conserved α-helix of motif-3 (Fig. 5 D and

Supplementary movie). This water is held in place by interactions with protein backbone amines and

a conserved side chain interaction.

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As the QM calculations suggest a loss of negative electrostatic potential in the 3-deazaadenosine

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compounds at the position of substitution, it is thus likely that the reduction of K_iobserved for the

class II targeting aaS3DA (Figs. 3 and 4) is principally a result of a loss of interaction with water. The

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absence of this interaction, further modified by steric interference of the water with the proton on C

could result in repositioning of the aaS3DA base in the active site such that the additional H-bond

interactions are also destabilised leading to the dramatic reduction in inhibitory activity observed for

these compounds.

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3. Discussion

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Non-hydrolysable aa-AMP analogues potently inhibit aaRS in vitro, with nanomolar range affinities,

yet they are devoid of antibacterial activity due to limited uptake [27, 28]. One approach to overcome

this problem is to extend the mimicry of natural compounds (e.g. Fig. 2 d), by coupling these

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enzymatic intermediate equivalents to an uptake module such as a peptide or a siderophore. To

simplify the synthesis of such complex molecules it is essential to reduce the number of reactive

groups present in the inhibitor without comprising activity or negatively affecting translocation of the

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compound into the cell. In this work we investigated the effect of replacement of the N of adenine in

the aaSA scaffold, a modification predicted to enhance chemical stability, on the activity of the

resultant compounds [35].

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There is a clear precedent in the synthesis and application of deazapurines in medicinal chemistry

[41]. In particular compounds containing 3-deazadenosine have been successfully introduced into a

number of antibacterials. C. Shi et al reported the successful synthesis of 5’-O-[N-

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( biotinyl)sulfamoyl]-3-deazaadenosine, a compound that inhibits the biotin protein ligase birA, a

global regulator of fatty acid biosynthesis in mycobacterium [35]. Competitive affinity measurements

demonstrated that this compound had a similar K to the adenosine analog, and possessed

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equivalent antitubercular activity, with an MIC of 1.56 µM. In addition, the chemical stability of this

compound was also increased when compared to the adenine containing equivalent [30]. In contrast,

the same research group also reported 5’-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosine

analogues as bispecific compounds designed to block siderophore biosynthesis in Mycobacterium

tuberculosis by inhibiting the adenylating enzyme MbtA [42]. Unfortunately, the combination of 8-aza

and 3-deaza in the base at same time increased the apparent K by 30 fold, when compared to the

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equivalent conjugated adenine, and increased the MIC greater than 500 fold, despite the compound

demonstrating enhanced chemical stability. These discrepancies point to need for a thorough

evaluation of equivalent deazapurines to fully understand their SAR properties.

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To obtain a broader understanding of the implications of replacing the N of adenine in the potent

aaSA compounds we synthesized six aminoacyl-sulfamoyl-3-deazaadenosine analogues. As the

aaRS family is divided into two specific structural subclasses [10], we specifically created three

aaS3DA compounds targeting representatives from each group. For the class I aaRS we chose ileRS,

leuRS and tyrRS as targets for the aaS3DA analogues as the attached amino acid demonstrate low

solubility and thus could improve the drug-like qualities of the resultant products. For the class II

targeting aaS3DA we picked aspRS, serRS and glyRS as the attached amino acids extending the

chemical profile under evaluation from acidic and polar, and to the simple unbranched glycine. The

approach used varied from that previously reported [35], by direct ring closure of 3,4-diamino-pyridine

and subsequent chlorination of the N-oxide, overall providing a shorter and higher yielding route to

construct the 3-deaza adenine heterocycle. (Scheme 1).

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The activity of all six aaS3DA compounds were evaluated by determining their ability to prevent a

full catalytic cycle of each enzyme, measuring the final transfer of the activated amino acid to tRNA

(Fig.1 A). For completeness, these measurements were performed in an E. coli whole cell extract as

well as with the isolated enzymes. In the cell lysates the aaS3DA intermediate mimics targeting class

I aaRS demonstrated inhibitory activity similar to the parent aaSA molecule. With the purified

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enzymes there was a measurable increase in K_ifor all three class I aaS3DAs, with ileS3DA

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showing the largest increase in this value, over 100-fold, when compared to ileSA (Fig. 4 and Table

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1). Ultimately, the measured K_ivalues were still sub-micromolar, below the concentration tested in

the cell extracts, explaining the observed comparable potency of the class I targeting aaS3DA and

aaSA in this complex system. Surprisingly however, for class II targeted enzymes the inhibitory

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activity in cell extracts of the aaS3DA was almost completely lost upon removal of the N -position.

This class bias was further confirmed in assays with the purified aaRS, where all tested compounds

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demonstrated an increase in their Ki relative to the equivalent aaSA (Table 1). This raising of the

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K_i, varying from 500-fold for aspS3DA to 4000-fold for serS3DA, was significantly higher than that

observed for even the most affected class I targeting aaS3DA.

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To further understand how the aaRS class dependent selectivity of the 3-deazaadenosine

analogues is manifested we performed QM chemical calculations to determine changes to the

electrostatic potential of the base. Overall, the p-orbital electrons are similarly delocalised in 3-

deazaadenine and adenine, but there is a clear loss of electronegativity at the substituted 3-carbon

position in the former compound. This principle difference therefore likely dictates the observed aaRS

class specificity. A detailed structural and bioinformatics analysis of the two classes point to quite

distinct mechanisms of base recognition in terms of the whole heterocycle but, more importantly,

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points to a key difference between the enzyme groups in terms of identifiable N interactions (Fig. 5).

Specifically, the presence of a conserved water molecule at a position where it can engage in

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hydrogen bonding between the N of the aaS3D, is a conserved feature in the class II enzymes. At

present though how the loss of this single interaction results in a dramatic increase in the apparent K_i

of the aaS3DA analogues that target class II aaRS is not clear, and requires further biophysical

investigation.

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In addition to demonstrating that the aaS3D analogues can be successfully employed to inhibit

class I aaRS, the combined results could be useful in the future design of novel inhibitors. In particular

the single atom substitution performed here has highlighted key differences in the pharmacophores

between the two aaRS classes that could be exploited. Previously our group investigated the

importance of adenine in ileSA by replacing it with other natural nucleobases. Surprisingly,

substitution of this conserved purine with a pyrimidine resulted in an increase in inhibitory activity of

the synthesised compounds in an S30 extract [31]. This observation is in agreement with the results

of this study, which shows that class I tRNA aaRS make a minimal number of interactions with the

base, and suggests that this moiety can be readily replaced. This hypothesis is supported by

examination of the structure of mupirocin (Fig 2 C), a natural ileRS inhibitor, which presents a

conjugated system with an ester function in place of adenine. Diverging from this, the class II aaRS

offer multiple modes of interaction with the base, a property that could be explored for developing high

affinity compounds.

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4. Conclusion

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Large deviations of the standard adenine base moiety in both natural and synthetic active site

inhibitors, have been shown to be accommodated by various aaRS members. In contrast, our detailed

analysis has shown that even subtle changes of the base moiety can dramatically influence ligand

affinity for its respective target enzyme. SAR analysis suggests that the presence of a water molecule

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bridging the N -position between adenine and local residues in the active site of class II aaRS, could

point to specific recognition of this base in this enzyme class, highlighting the importance of the need

to fully understand the pharmacophore of these enzymes for further rational design of inhibitors.

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5. Experimental section

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5.1. Reagents and chemical analysis

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Reagents and solvents were purchased from commercial suppliers (Acros, Sigma-Aldrich, Bachem,

Novabiochem) and used as provided, unless indicated otherwise. DMF and THF were of analytical

grade and were stored over 4 Å molecular sieves. All other solvents used for reactions were analytical

grade and used as provided. Reactions were carried out in oven-dried glassware under a nitrogen

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atmosphere with stirring at room temperature, unless indicated otherwise. C-radiolabeled amino

acids and scintillation liquid were purchased from Perkin Elmer.

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H and C NMR spectra of the compounds dissolved in CDCl , CD OD, DMSO-d6 or D O were

3 3 2

recorded on a Bruker UltraShield Avance 300 MHz, 500 MHz or when needed at 600 MHz

spectrometer. The chemical shifts are expressed as δ values in parts per million (ppm), using the

1

13

1

13

residual solvent peaks (CDCl : H, 7.26 ppm; C, 77.16 ppm; DMSO: H, 2.50 ppm; C, 39.52 ppm;

3

1

13

D O: 1H, 4.79 ppm; CD3OD: H, 3.31 ppm; C, 49.00 ppm) as a reference. Coupling constants are

2

given in Hertz (Hz). The peak patterns are indicated by the following abbreviations: bs = broad singlet,

d = doublet, m = multiplet, q = quadruplet, s = singlet and t = triplet. High resolution mass spectra

were recorded on a quadrupole time-of-flight mass spectrometer (Q-Tof-2, Micromass, Manchester,

UK) equipped with a standard ESI interface; samples were infused in 2- propanol/H2O (1:1) at 3 mL

min-1. For TLC, precoated aluminum sheets were used (Merck, Silica gel 60 F254). The spots were

visualized by UV light at 254 nm. Chromatography was performed on ICN silica gel 60A 60-200. Final

products (acylated sulfamate nucleosides) were purified using a semi-prep PLRP-S 100 Å column

(7.5 × 300 mm) connected to a Merck-Hitachi L6200A Intelligent pump. Eluent compositions are

expressed as v/v. the characterization of the final compounds by NMR and mass are provided in the

supplementary file.

3

2

3

4

5.2. Chemical synthesis of the intermediates and final compounds

5.2.1. 1H-imidazo[4,5-c]pyridine (2)

3

5

6

A mixture of pyridine-3,4-diamine (2 g, 18.3 mmol) and triethyl orthoformate (50 mL) was refluxed for

about 3 h at 145 °C. After completion of the reaction monitored by TLC, 2 mL of formic acid was

9

ACCEPTED MANUSCRIPT

1

2

3

4

5

6

7

8

added and the mixture was refluxed at the same temperature for another 2 h. The solution was

evaporated to dryness at reduced pressure, and the residue was dissolved in methanol and stirred at

room temperature overnight in presence of charcoal. Following removal of the charcoal by vacuum

filtration through celite-545, the filtrate was evaporated to give the title compound 2 as an off-white

1

solid in quantitative yield without further purification. H NMR (300 MHz, DMSO-d6) δ 8.97 (d, J = 1.1

13

Hz, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.61 (dd, J = 5.6, 1.1 Hz, 1H); C NMR (75 MHz, DMSO) δ 144.35,

+

141.18 (2×C, overlapped), 139.72, 137.73, 109.46. HRMS (ESI): m/z calcd. for C H N [M+H] :

6

3

120.0556; found, 120.0556.

9

0

1

5.2.2. 1H-imidazo[4,5-c]pyridine-5-oxide (3)

1

2

3

4

5

6

7

8

Compound 2 (2.5 g, 20.83 mmol) was dissolved in a mixture of 60 mL DCM + 30 mL of MeOH and

10.3 g of m-CPBA (41.67 mmol) dissolved in methanol was added dropwise at 0 °C. The reaction was

stirred at room temperature for 22 h, after which the mixture was concentrated in vacuum. Methanol

(30 mL) was added to the residue which was adsorbed on silica. The title compound 3 was isolated

1

via silica gel chromatography using EA/MeOH (65:35) in 80% yield. H NMR (300 MHz, DMSO-d6) δ

13

8.68 (d, J = 1.5 Hz, 1H), 8.39 (s, 1H), 8.07 (dd, J = 6.9, 1.8 Hz, 1H), 7.64 (d, J = 6.9 Hz, 1H). C NMR

(75 MHz, DMSO) δ 147.96, 137.85, 136.67, 133.51, 128.35, 111.77. HRMS (ESI): m/z calcd. for

+

C H N O [M+H] : 136.0505; found, 136.0510.

6

3

1

2

9

0

5.2.3. 4-chloro-1H-imidazo[4,5-c]pyridine (4)

2

1

2

3

4

5

6

Compound 2 (255 mg, 1.88 mmol) was dissolved in 10 mL POCl and stirred at 110 °C for 3 h until a

3

clear solution was obtained. Excess POCl was removed in vacuum and ice was added, followed by

3

adding aqueous ammonia until reaching a pH around 9 and an equivalent amount of MeOH was

added. Some precipitate formed and was filtered off, and the filtrate was adsorbed on silica and

purified by silica gel chromatography (EA with 5% MeOH) affording 4 in 83% yield. HRMS (ESI): m/z

+

calcd. for C H ClN [M+H] : 154.0166; found, 154.0171.

6

5

3

2

7

8

5.2.4. 4-chloro-1-(2’, 3’, 5’-tri-O-acetyl-β-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridine (5)

2

3

9

0

1

2

3

4

5

6

Compound 4 (231 mg, 1.51 mmol) and 1’,2’,3’,5’-tetra-O-acetyl-β-D-ribofuranose (720 mg, 2.26 mmol)

were dissolved in 10 mL of dry acetonitrile. A solution of SnCl (2.26 mL) was added and the reaction

4

mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with DCM (10 mL)

and then poured into 20 mL of an ice cooled saturated sodium bicarbonate solution under stirring and

the mixture was filtered by celite. Following separation of the layers, the organic phase was further

washed with brine and was co-evaporated with silica, and purified by silica gel chromatography to

1

afford compound 5 in 65% yield. H NMR (300 MHz, DMSO-d ) δ 8.74 (d, J = 1.0 Hz, 1H), 8.25 (dd, J

6

= 5.7, 1.1 Hz, 1H), 7.87 (dd, J = 5.7, 1.1 Hz, 1H), 6.41 (dd, J = 6.0, 1.0 Hz, 1H), 5.65 (t, J = 6.2 Hz,

1

0

ACCEPTED MANUSCRIPT

1

2

3

4

1H), 5.45 (dd, J = 6.3, 4.8 Hz, 1H), 4.50 – 4.41 (m, 1H), 4.39 (d, J = 4.2 Hz, 2H), 2.14 (d, J = 1.1 Hz,

1

3

3H), 2.08 (d, J = 1.1 Hz, 3H), 2.04 (d, J = 1.1 Hz, 3H). C NMR (75 MHz, DMSO) δ 170.14, 169.60,

169.39, 144.61, 141.80, 141.47, 139.15, 137.52, 107.51, 86.85, 79.94, 72.36, 69.65, 63.03, 20.66,

+

20.52, 20.32. HRMS (ESI): m/z calcd. for C H ClN O [M+H] : 412.0906; found, 412.0907.

17

19

3

7

5

6

5.2.5. 4-chloro-1 -β-D-ribofruanosyl-1H-imidazo[4,5-c]pyridine (6)

7

8

9

0

1

2

3

4

5

Compound 5 (1.635 g, 3.98 mmol) was dissolved in methanolic ammonia (7N, 25 mL) at 0 °C. The

reaction was stirred for 4 h while reaching room temperature. The reaction mixture was concentrated

in vacuum in presence of silica gel and the main compound was purified by silica gel chromatography

1

using EA/methanol (8:2) in 90% yield. H NMR (300 MHz, DMSO-d ) δ 8.71 (s, 1H), 8.17 (d, J = 5.6

6

Hz, 1H), 7.92 (d, J = 5.6 Hz, 1H), 5.94 (d, J = 6.3 Hz, 1H), 5.55 (d, J = 6.4 Hz, 1H), 5.28 (d, J = 4.6 Hz,

1H), 5.21 (t, J = 5.1 Hz, 1H), 4.43 – 4.29 (m, 1H), 4.14 (td, J = 4.8, 2.9 Hz, 1H), 4.03 (q, J = 3.5 Hz,

13

2H), 3.67 (td, J = 5.3, 3.4 Hz, 2H). C NMR (75 MHz, DMSO) δ 144.72, 141.23, 141.18, 139.34,

+

137.63, 107.90, 89.35, 86.25, 74.31, 70.26, 61.22. HRMS (ESI): m/z calcd. for C H ClN O [M+H] :

11

13

3

4

286.0589; found, 286.0591.

1

6

7

5.2.6. 3-deazaadenosine (7)

1

2

8

9

0

1

2

3

4

5

6

7

Compound 6 (700 mg, 2.46 mmol) was dissolved in 20 mL hydrazine hydrate (98% pure) and the

reaction was stirred at 120 °C for 4 h. TLC analysis indicated a new more polar product was formed.

The reaction was concentrated in vacuum and the residue was dissolved in 10 mL of water, followed

by adding 0.7 mL Raney Ni (50% in water) and stirring for 30 min. The catalyst was filtered off and

washed with water (10 mL), the filtrate was evaporated in presence of some silica gel and the title

1

compound was separated by silica gel chromatography in 87% yield. H NMR (300 MHz, DMSO-d ) δ

6

8.37 (s, 1H), 7.68 (d, J = 6.0 Hz, 1H), 7.02 (d, J = 6.1 Hz, 1H), 6.63 (s, 2H), 5.80 (d, J = 6.2 Hz, 1H),

5.51 (s, 1H), 5.24 (s, 1H), 5.13 (s, 1H), 4.32 (s, 1H), 4.10 (d, J = 4.8 Hz, 1H), 4.10 (d, J = 4.8 Hz, 1H),

+

3.98 (d, J = 3.4 Hz, 1H), 3.64 (s, 2H). HRMS (ESI): m/z calcd. for C H N O [M+H] : 267.1087;

11

15

4

found, 267.1089.

2

8

9

5.2.7. 3-deaza-2’,3’-O-isopropylidene-adenosine (8)

3

0

1

2

3

4

5

6

Compound 7 (300 mg, 1.13 mmol) was dissolved in 20 mL of dry acetone, followed by addition of 2 g

p-TSA (12 mmol) and 3 mL 2, 2-dimethoxypropane, and the reaction was stirred at room temperature

for 2 h. A precipitate was gradually formed after 2 h but disappeared when the temperature was

increased to 50 °C, and stirring was continued for another 2 h. Following completion of the reaction by

formation of a less polar product, the reaction was neutralized by adding saturated NaHCO , and

3

concentrated. The title compound 8 was purified by silica gel chromatography (EA/MeOH 9:1) in 81%

1

yield. H NMR (300 MHz, DMSO-d ) δ 8.73 (s, 1H), 8.19 (d, J = 5.6 Hz, 1H), 7.80 (d, J = 5.7 Hz, 1H),

6

1

ACCEPTED MANUSCRIPT

1

2

3

7.77 (s, 2H), 6.01 (d, J = 7.4 Hz, 1H), 4.60 (dd, J = 7.5, 4.7 Hz, 1H), 4.36 (d, J = 4.4 Hz, 2H), 4.32 –

+

4.24 (m, 2H), δ 1.62 (s, 3H), 1.39 (s, 3H). HRMS (ESI): m/z calcd. for C H N O [M+H] : 307.1401;

14

19

4

found, 307.1404.

4

5

5.2.8. 3-deaza-2’,3’-O-isopropylidene-5’-O-sulfamate-adenosine (9)

6

7

8

9

0

1

2

3

4

5

6

7

8

9

Chlorosulfonyl isocyanate (210 mg, 1.5 mmol) was taken into a 10 mL flask and after cooling to 0 °C

formic acid (70 mg, 1.5 mmol) was added and the mixture was allowed to stir for 5 min. The resulting

solid was dissolved in dry acetonitrile (2 mL) and the solution was cooled to 0 °C and stirred for

another 5 h gradually reaching room temperature Compound 8 (100 mg, 0.33 mmol) was dissolved in

10 mL of DMA and cooled to 0 °C, and the obtained sulfamoyl chloride was then added to the stirred

solution of compound 8 in DMA and the mixture was stirred overnight. After adding TEA (1 mL) the

reaction was stirred for 10 min, followed by adding 2 mL of methanol and further stirring for 15 min.

The reaction was concentrated and the residue was partitioned between EA and saturated NaHCO₃.

1

The organic layer was further washed with water and brine, and the title compound 9 was purified by

1

silica gel chromatography in 85% yield. H NMR (300 MHz, DMSO-d ) δ 8.29 (s, 1H), 7.70 (d, J = 5.8

6

Hz, 1H), 7.66 (s, 2H), 6.88 (d, J = 5.8 Hz, 1H), 6.26 (s, 2H), 6.13 (d, J = 3.5 Hz, 1H), 5.21 (dd, J = 6.5,

3.5 Hz, 1H), 4.99 (dd, J = 6.5, 3.3 Hz, 1H), 4.41 (q, J = 4.6 Hz, 1H), 4.15 (dd, J = 4.9, 2.0 Hz, 2H),

1

3

1.59 (s, 3H), 1.35 (s, 3H). C NMR (75 MHz, DMSO) δ 152.63, 141.17, 139.67, 137.40, 126.92,

114.36, 97.21, 90.05, 83.00, 82.10, 80.43, 67.94, 27.01, 25.31.

2

0

1

6

5.2.9. N -[N-(tert-butoxycarbonyl)glycyl]-3-deaza-2’,3’-O-isopropylidene-5’-O-sulfamate-adenosine (10)

2

3

2

3

4

5

6

7

8

9

0

1

Compound 9 (40 mg, 0.1 mmol) and 41 mg N-Boc-L-Gly-OSu were dissolved in 16 mL of dry DMF,

followed by adding 30 mg DBU and the reaction was stirred at room temperature for 17 h. The

reaction was concentrated and the residue was partitioned between EA and saturated NaHCO . The

3

organic layer was further washed with water and brine, evaporated at reduced vacuum and was

1

subjected to silica gel chromatography. The title compound 10 was isolated in 60% yield. H NMR

(300 MHz, DMSO-d ) δ 8.65 (s, 1H), 8.04 (s, 2H), 7.70 (d, J = 6.8 Hz, 1H), 7.25 (d, J = 6.8 Hz, 1H),

6

6.35 (t, J = 5.6 Hz, 1H), 6.20 (d, J = 3.4 Hz, 1H), 5.22 (dd, J = 6.2, 3.4 Hz, 1H), 5.01 (dd, J = 6.1, 2.1

Hz, 1H), 4.47 (d, J = 2.8 Hz, 1H), 4.10 – 3.98 (m, 2H), 3.92 (dd, J = 11.2, 4.2 Hz, 1H), 3.42 (d, J = 5.7

-

Hz, 2H), 1.58 (s, 3H), 1.36 (s, 9H), 1.34 (s, 3H). HRMS (ESI): m/z calcd. for C H N O S [M-H] :

21

29

6

9

541.1722; found, 541.1725.

3

2

3

4

5

6

7

The structure of compound 10 was confirmed by NMR and mass analysis, where in proton NMR the

exocyclic 6-amine (purine numbering) traditionally showing a signal at δ 6.0-6.5 ppm had

disappeared, while the newly introduced sulfamoyl amine signal of compound 9 remained and slightly

shifted from δ 7.7 ppm to δ 7.5 ppm. This undesired reaction at the base moiety is explained by

increased nucleophilicity of the heterocyclic amine in removing the 3-nitrogen of the adenine base,

establishing the need for base protection.

1

2

ACCEPTED MANUSCRIPT

1

2

6

5.2.10. N -benzoyl- 3-deaza-2’,3’-O-isopropylidene-adenosine (11)

3

4

5

6

7

8

9

0

1

2

Compound 8 (100 mg, 0.33 mmol) was dissolved in 10 mL dry pyridine, followed by adding TMSCl

(180 mg, 1.66 mmol) at 0 °C and the reaction was stirred at 0 °C for 4 h. After the starting compound

had disappeared on TLC analysis, 300 mg BzCl was added and the mixture was stirred for 3 h at

room temperature. The reaction was stopped by adding diluted ammonia (12%, 4 mL) at 0 ºC with

stirring. The mixture was extracted with DCM (50 mL) and further washed by brine. The organic layer

was concentrated in vacuum and the title compound 11 was purified by silica gel chromatography in

1

79% yield. H NMR (300 MHz, DMSO-d ) δ 10.73 (s, 1H), 8.56 (s, 1H), 8.23 (d, J = 5.6 Hz, 1H), 8.05

6

1

(d, J = 7.4 Hz, 3H), 7.68 (d, J = 5.7 Hz, 1H), 7.66 – 7.50 (m, 2H), 6.23 (d, J = 3.4 Hz, 1H), 5.24 (dd, J

= 6.3, 3.4 Hz, 1H), 5.17 (t, J = 5.1 Hz, 1H), 4.98 (dd, J = 6.2, 2.6 Hz, 1H), 4.26 (d, J = 3.1 Hz, 2H),

+

1.60 (s, 3H), 1.36 (s, 3H). HRMS (ESI): m/z calcd. for C H N O [M+H] : 411.1663; found, 411.1660.

21

23

4

5

1

3

4

6

5.2.11. N -benzoyl- 3-deaza-2’,3’-O-isopropylidene-5’-O-sulfamate-adenosine (12)

1

2

5

6

7

8

9

0

1

2

3

4

5

6

Compound 11 (100 mg, 0.25 mmol) was dissolved in 10 mL DMA followed by adding sulfamoyl

chloride (dissolved in MeCN, 0.4 mmol, prepared separately as described for compound 9), and the

reaction was stirred at room temperature overnight. After adding 0.5 mL of TEA a precipitate was

formed, which dissolved after adding 2 mL of MeOH and stirring was continued for another 20 min.

The reaction was concentrated in vacuum and the residue was dissolved in EA, washed with

saturated NaHCO , and the title compound 12 was purified by silica gel chromatography in 85% yield

3

1

(MeOH:EA, 2:98). H NMR (300 MHz, DMSO-d ) δ 10.76 (s, 1H), 8.54 (s, 1H), 8.24 (d, J = 5.6 Hz,

6

1H), 8.06 (d, J = 7.5 Hz, 2H), 7.76 – 7.46 (m, 6H), 6.31 (d, J = 3.5 Hz, 1H), 5.31 (dd, J = 6.4, 3.5 Hz,

1H), 5.04 (dd, J = 6.5, 3.2 Hz, 1H), 4.47 (q, J = 4.4 Hz, 1H), 4.18 (dd, J = 4.7, 2.6 Hz, 2H), 1.61 (s,

1

3

3H), 1.37 (s, 3H). C NMR (75 MHz, DMSO) δ 165.73, 144.42, 142.65, 140.78, 139.19, 135.13,

134.12, 132.00, 114.43, 105.95, 90.22, 83.00, 82.31, 80.46, 67.97, 27.01, 25.33. HRMS (ESI): m/z

+

calcd. for C H N O S [M+H] : 489.1391; found, 489.1391.

21

24

5

7

2

7

6

2

8

9

5.2.12. 5’-O-[N-(tert-butoxycarbonyl-glycyl)]sulfamoyl-N -benzoyl-3-deaza-2’,3’-O-isopropylidene-

adenosine (13a)

3

0

1

2

3

4

5

6

7

Compound 12 (70 mg, 0.143 mmol) and 58 mg of N-Boc-Gly-OSu were dissolved in 8 mL of dry DMF,

followed by adding 33 mg DBU and the reaction was stirred at room temperature overnight. The

reaction was concentrated in vacuum and the residue was dissolved in EA, and washed with

saturated NaHCO . The title compound 13a was purified by silica gel chromatography in 82% yield

3

(EA:hexane, 2:1). 1H NMR (300 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.60 (s, 1H), 8.22 (d, J = 5.5 Hz,

1H), 8.06 (d, J = 7.5 Hz, 2H), 7.96 (s, 1H), 7.77 – 7.46 (m, 3H), 6.33 (s, 1H), 6.23 (d, J = 3.6 Hz, 1H),

5.26 (s, 1H), 5.04 (d, J = 6.5 Hz, 1H), 4.44 (s, 1H), 4.12 – 3.87 (m, 2H), 1.60 (s, 3H), 1.35 (d, J = 2.4

-

Hz, 12H). HRMS (ESI): m/z calcd. for C H N O S [M-H] : 645.1984; found, 645.1984.

28

33

6

11

1

3

ACCEPTED MANUSCRIPT

1

6

2

3

5.2.13. 5’-O-[N-(tert-butoxycarbonyl-4-benzyloxy-L-tyrosyl)]sulfamoyl-N -benzoyl-3-deaza-2’,3’-O-

isopropylidene-adenosine (13b)

4

5

This compound was synthesized in analogy to 13a. Yield: 68%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 841.2872; found, 841.2867.

42

45

6

11

6

7

8

5.2.14. 5’-O-[N-(tert-butoxycarbonyl-O-benzyl-L-seryl)]sulfamoyl-N -benzoyl-3-deaza-2’,3’-O-

isopropylidene-adenosine (13c)

9

0

This compound was synthesized in analogy to 13a. Yield: 71%. HRMS (ESI): m/z calcd. for

+

1

C H N O S [M+H] : 767.2705; found, 767.2722.

36

41

6

11

1

6

1

2

3

5.2.15. 5’-O-[N-(tert-butoxycarbonyl-O-tert-butyl-L-aspartyl)]sulfamoyl-N -benzoyl-3-deaza-2’,3’-O-

isopropylidene-adenosine (13d)

1

4

5

This compound was synthesized in analogy to 13a. Yield: 85%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 759.2665; found, 759.2673.

34

43

6

12

1

6

1

7

8

5.2.16. 5’-O-[N-(tert-butoxycarbonyl-L-leucyl)]sulfamoyl-N -benzoyl-3-deaza-2’,3’-O-isopropylidene-

adenosine (13e)

1

2

9

0

This compound was synthesized in analogy to 13a. Yield: 87%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 701.2610; found, 701.2598.

32

41

6

10

2

1

6

2

3

5.2.17. 5’-O-[N-(tert-butoxycarbonyl-L-isoleucyl)]sulfamoyl-N -benzoyl-3-deaza-2’,3’-O-

isopropylidene-adenosine (13f)

2

4

5

This compound was synthesized in analogy to 13a. Yield: 89%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 701.2610; found, 701.2612.

32

41

6

10

2

6

7

5.2.18. 5’-O-[N-(tert-butoxycarbonyl-glycyl)]sulfamoyl-3-deaza-2’,3’-O-isopropylidene-adenosine (14a)

2

3

8

9

0

1

2

3

4

A solution of compound 13a in methanol (10 mL) was treated with 2 drops of 30% sodium methoxide

in methanol and the solution was refluxed for 2.5 h when according to TLC the reaction was

completed. The mixture was concentrated and adsorbed on silica at reduced vacuum. The title

1

compound 14a was purified by silica gel chromatography (MeOH:EA, 18:82) in 90% yield. H NMR

(300 MHz, DMSO-d ) δ 8.50 (s, 1H), 7.70 (d, J = 6.2 Hz, 1H), 7.19 – 7.01 (m, 3H), 6.33 (s, 0H), 6.13

6

(d, J = 3.5 Hz, 1H), 5.19 (d, J = 4.1 Hz, 1H), 5.05 – 4.93 (m, 1H), 4.42 (s, 1H), 4.07 – 3.96 (m, 2H),

13

3.91 (dd, J = 11.2, 4.5 Hz, 1H), 1.58 (s, 3H), 1.35 (d, 12H). C NMR (75 MHz, DMSO) δ 173.74,

1

4

ACCEPTED MANUSCRIPT

1

2

3

155.59, 151.04, 141.03, 138.22, 126.73, 113.67, 98.10, 90.89, 83.35, 83.21, 81.24, 77.62, 66.79,

-

45.83, 45.76, 28.39, 27.04, 25.28. HRMS (ESI): m/z calcd. for C H N O S [M-H] : 541.1722; found,

21

29

6

9

541.1725.

4

5

6

5.2.19. 5’-O-[N-(tert-butoxycarbonyl-4-benzyloxy-L-tyrosyl)]sulfamoyl-3-deaza-2’,3’-O-isopropylidene-

adenosine (14b)

7

8

This compound was synthesized in analogy to 14a. Yield: 91%. HRMS (ESI): m/z calcd. for

+

C H N O S [M+H] : 739.2756; found, 739.2774.

35

43

6

10

9

1

0

1

5.2.20. 5’-O-[N-(tert-butoxycarbonyl-O-benzyl-L-seryl)]sulfamoyl-3-deaza-2’,3’-O-isopropylidene-

adenosine (14c)

1

2

3

This compound was synthesized in analogy to 14a. Yield: 89%. HRMS (ESI): m/z calcd. for

+

C H N O S [M+H] : 663.2443; found, 663.2452.

29

39

6

10

1

4

1

5

6

5.2.21. 5’-O-[N-(tert-butoxycarbonyl-O-tert-butyl-L-aspartyl)]sulfamoyl-3-deaza-2’,3’-O-isopropylidene-

adenosine (14d)

1

7

8

This compound was synthesized in analogy to 14a. Yield: 89%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 655.2403; found, 655.2396.

27

39

6

11

1

9

2

0

1

5.2.22. 5’-O-[N-(tert-butoxycarbonyl-L-leucyl)]sulfamoyl-3-deaza-2’,3’-O-isopropylidene-adenosine

(14e)

2

3

This compound was synthesized in analogy to 14a. Yield: 93%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 597.2348; found, 597.2347.

25

37

6

9

2

4

2

5

6

5.2.23. 5’-O-[N-(tert-butoxycarbonyl-L-isoleucyl)]sulfamoyl-3-deaza-2’,3’-O-isopropylidene-adenosine

(14f)

2

7

8

This compound was synthesized in analogy to 14a. Yield: 94%. HRMS (ESI): m/z calcd. for

-

C H N O S [M-H] : 597.2348; found, 597.2349.

25

37

6

9

2

9

5.2.24. 5’-O-(N-glycyl)sulfamoyl-3-deaza-adenosine (15a)

3

0

1

2

3

4

Compound 14a (60 mg, 0.11 mmol) was dissolved in a solvent mixture of TFA and H O (7 mL, 5:2 v/v)

2

and the reaction mixture was stirred for 40 min. After reaction, the volatiles were evaporated under

reduced pressure, followed by co-evaporation with EtOH, and once more with EtOH + 1 mL Et N to

3

neutralize any remaining acid. The title compound 15a was obtained by RP-HPLC as a white solid in

1

60% yield. H NMR (300 MHz, deuterium oxide) δ 8.38 (s, 1H), 7.55 (d, J = 7.0 Hz, 1H), 7.14 (d, J =

1

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3

4

5

7.1 Hz, 1H), 5.95 (d, J = 5.8 Hz, 1H), 4.55 – 4.29 (m, 6H), 3.61 (s, 1H), 3.11 (q, J = 7.3 Hz, 1H), 1.19

13

(t, J = 7.3 Hz, 1.5H) (δ 3.11 and 1.19 belonging to triethylamine). C NMR (75 MHz, D O) δ 172.48,

2

148.16, 142.46, 139.03, 130.30, 125.84, 99.29, 88.92, 82.59, 73.96, 69.73, 67.94, 46.34, 42.44, 7.88

+

(δ 46.34 and 7.88 belonging to triethylamine). HRMS (ESI): m/z calcd. for C H N O S [M+H] :

13

19

6

7

403.1030; found, 403.1032.

6

7

5.2.25. 5’-O-(N-L-tyrosyl)sulfamoyl-3-deaza-adenosine (15b)

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

To a solution of compound 14b (130 mg, 0.176 mmol) in methanol (10 mL) was added 69 mg Pd/C

under argon, after which the argon was changed to hydrogen and the mixture was stirred at room

temperature overnight. TLC analysis indicated the reaction to be completed, and the Pd/C was filtered

and the filtrate was concentrated. The residue was adsorbed on silica and was purified by

chromatography (MeOH/EA, 1:9) in 57% yield. Mass for the intermediate: HRMS (ESI): m/z calcd for

1

2

+

C H N O S [M+H] : 649.2286; found, 649.2282. The obtained compound (65 mg, 0.1 mmol) was

28

37

6

10

dissolved in a solvent mixture of TFA and H O (7 mL, 5:2 v/v) and the reaction mixture was stirred for

2

30 min. The volatiles were evaporated under reduced pressure followed by co-evaporation with EtOH,

and once more with EtOH + 1 mL Et N to neutralize any remaining acid. The title compound 15b was

3

1

obtained by RP-HPLC as a white solid in 42% yield. H NMR (300 MHz, deuterium oxide) δ 8.21 (s,

1H), 7.65 (d, J = 6.2 Hz, 1H), 7.04 – 6.89 (m, 3H), 6.62 (d, J = 8.3 Hz, 2H), 5.91 (d, J = 6.0 Hz, 1H),

4.49 (t, J = 5.3 Hz, 1H), 4.33 (s, 2H), 4.20 (d, J = 6.2 Hz, 1H), 3.75 (t, J = 6.4 Hz, 1H), 3.11 (q, J = 7.4

13

Hz, 2H,), 2.96 – 2.76 (m, 2H), 1.19 (t, J = 7.3 Hz, 3H) (δ 3.11 and 1.19 belonging to triethylamine).

C

NMR (75 MHz, D O) δ 176.48, 154.42, 148.24, 140.91, 139.12, 138.08, 130.38, 126.36, 122.29,

2

115.14, 98.90, 88.59, 82.24, 73.54, 69.76, 68.02, 56.68, 46.36, 36.49, 7.90 (δ 46.36 and 7.90

+

belonging to triethylamine). HRMS (ESI): m/z calcd. for C H N O S [M+H] : 509.1449; found,

20

25

6

8

509.1450.

2

5

6

5.2.26. 5’-O-(N-L-seryl)sulfamoyl-3-deaza-adenosine (15c)

2

3

7

8

9

0

1

2

3

4

5

6

7

A solution of compound 14c (93 mg, 0.15 mmol) in methanol (10 mL) was processed in analogy with

15b. Purification by silica gel chromatography (MeOH/EA, 1:9) afforded the debenzylated

intermediate in 69% yield (54 mg, 0.09 mmol) which was processed in analogy with 15a. The title

1

compound 15c was separated by RP-HPLC as a white solid in 54% yield. H NMR (300 MHz,

deuterium oxide) δ 8.27 (s, 1H), 7.47 (d, J = 6.8 Hz, 1H), 6.98 (d, J = 6.8 Hz, 1H), 5.86 (d, J = 5.9 Hz,

1H), 4.45 (dd, J = 5.9, 4.9 Hz, 1H), 4.33 (dt, J = 9.7, 3.5 Hz, 4H), 3.88 (m, 3H), 3.09 (q, J = 7.3 Hz,

13

4H), 1.17 (t, J = 7.3 Hz, 6H) (δ 3.09 and 1.17 belonging to triethylamine). C NMR (75 MHz, D O) δ

2

173.02, 148.24, 142.11, 138.52, 131.66, 125.50, 117.95, 114.08, 98.97, 88.86, 82.50, 73.98, 69.73,

68.09, 60.06, 56.71, 46.32, 7.87 (δ 46.32 and 7.87 belonging to triethylamine). HRMS (ESI): m/z

-

calcd for C H N O S [M-H] : 431.0990; found, 431.0990. Mass for the intermediate: HRMS (ESI):

14

19

6

8

+

m/z calcd. for C H N O S [M+H] : 573.1973; found, 573.1982.

22

33

6

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5.2.27. 5’-O-(N-L-aspartyl)sulfamoyl-3-deaza-adenosine (15d)

3

4

5

6

7

8

9

0

Compound 14d (90 mg, 0.137 mmol) was processed in analogy with 15a. The title compound 15d

1

was separated by RP-HPLC as a white solid in 53% yield. H NMR (300 MHz, deuterium oxide) δ

8.31 (d, J = 1.2 Hz, 1H), 7.46 (dd, J = 7.1, 1.2 Hz, 1H), 7.04 (dd, J = 7.1, 1.2 Hz, 1H), 5.86 (dd, J = 6.2,

1.2 Hz, 1H), 4.46 – 4.26 (m, 5H), 3.92 (ddd, J = 6.1, 4.7, 1.2 Hz, 1H), 3.10 (q, J = 7.4, 3H), 2.71 (td, J

13

= 4.7, 1.2 Hz, 2H), 1.18 (td, J = 7.3, 1.2 Hz, 4H) (δ 3.10 and 1.18 belonging to triethylamine).

C

NMR (75 MHz, D O) δ 176.56, 174.79, 147.84, 142.62, 138.77, 129.88, 125.57, 99.17, 88.86, 82.84,

2

74.05, 69.81, 68.12, 52.61, 46.34, 36.26, 7.89 (δ 46.34 and 7.89 belonging to triethylamine). HRMS

-

1

(ESI): m/z calcd. for C H N O S [M-H] : 459.0940; found, 459.0945.

15

19

6

9

1

2

5.2.28. 5’-O-(N-L-leucyl)sulfamoyl-3-deaza-adenosine (15e)

1

3

4

5

6

7

8

Compound 14e (60 mg, 0.1 mmol) was deprotected analogously to 14d. The title compound 15e was

1

separated by RP-HPLC as a white solid in 70% yield. H NMR (300 MHz, deuterium oxide) δ 8.20 (s,

1H), 7.55 (d, J = 6.2 Hz, 1H), 6.91 (d, J = 6.2 Hz, 1H), 5.85 (d, J = 6.0 Hz, 1H), 4.50 – 4.26 (m, 5H),

13

3.65 (d, J = 6.6 Hz, 1H), 1.69 – 1.36 (m, 3H), 0.73 (d, 6H). C NMR (75 MHz, D O) δ 176.38, 150.14,

2

141.05, 137.95, 137.80, 125.99, 98.86, 88.60, 82.37, 73.60, 69.79, 67.98, 53.91, 40.02, 23.65, 21.44,

-

20.54. HRMS (ESI): m/z calcd. for C H N O S [M-H] : 457.1510; found, 457.1507.

17

25

6

7

1

2

9

0

5.2.29. 5’-O-(N-L-isoleucyl)sulfamoyl-3-deaza-adenosine (15f)

2

1

2

3

4

5

6

7

Compound 14f (60 mg, 0.1 mmol) was deprotected analogously to 14d. The title compound 15f was

1

separated by RP-HPLC as a white solid in 75% yield. H NMR (300 MHz, deuterium oxide) δ 8.20 (s,

1H), 6.91 (d, J = 6.2 Hz, 1H), 5.85 (d, J = 6.1 Hz, 1H), 4.56 – 4.18 (m, 5H), 3.59 (d, J = 4.1 Hz, 1H),

1.94 – 1.75 (m, 1H), 1.29 (ddd, J = 12.6, 7.5, 4.8 Hz, 1H), 1.04 (ddd, J = 13.4, 9.4, 7.1 Hz, 1H), 0.82

13

(d, J = 7.0 Hz, 3H), 0.67 (t, J = 7.4 Hz, 3H). C NMR (75 MHz, D O) δ 175.12, 150.25, 140.99,

2

138.14, 137.97, 126.03, 99.66, 98.85, 88.54, 82.34, 73.55, 69.77, 67.98, 59.81, 36.15, 23.73, 14.13,

-

10.51. HRMS (ESI): m/z calcd. for C H N O S [M-H] : 457.1510; found, 457.1505.

17

25

6

7

2

8

9

5.3. E. coli S30 cell whole extracts inhibition assays

3

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1

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3

4

5

10 µL of an E. coli K-12 BW28357 strain glycerol stock was used to inoculate 5 mL of LB-medium and

shaken at 180 rpm and 37 °C, overnight. 50 µL of this pre-culture was used to inoculate 50 mL LB-

medium and grow the culture until the absorbance at 600 nm of 0.5. Cells where then centrifuged at

3000 g for 10 min and the media discarded. The cell pellet was resuspended in 40 mL buffer

containing 20 mM Tris.HCl, 10 mM MgCl , 100 mM KCl, pH 8.0. The cell suspension was centrifuged

2

again at 3000 g. This procedure was repeated twice. The pellet was the resuspended in 1 mL of the

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same buffer containing in addition 1 mM DTT and kept at 0 °C. Subsequently, the cells were

sonicated for 10 s and left at 0 °C for 10 min. This procedure was repeated 5-8 times. The lysate was

centrifuged at 15,000 g for 30 min at +4 °C using a bench-top centrifuge.

4

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6

7

8

9

0

1

2

3

For the tRNA aminoacylation a 20 µl reaction was prepared. First, 1 µl of the inhibitor (at a stock

concentration of 50 µM) or water was added to 3 µl of the E. coli extract and incubated for 5 min. Next,

16 µl of the following aminoacylation mixture was added: Tris.HCl (30 mM, pH 8.0), DTT (1 mM), E.

14

coli tRNA (5 g/L), ATP (3 mM), KCl (30 mM), MgCl (8 mM), and the specified C-radiolabeled amino

2

acid (40 µM, see supplementary file). The reaction products were precipitated in cold 10% TCA on

Whatman 3 MM paper, 5 min after the aminoacylation mixture was added. The aminoacylation

reaction was carried out at room temperature. After thorough washing with cold 10% TCA, the papers

were washed twice with acetone and dried on a heating plate. Following the addition of scintillation

liquid (12 mL), the amount of radionuclide incorporation was determined using a Tri-card 2300 TR

liquid scintillation counter.

1

4

5.4. Cloning, expression and purification of E. coli aminoacyl-tRNA synthetases

1

2

5

6

7

8

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1

2

3

4

5

6

7

8

Sequences encoding for aspRS, ileRS, leuRS, glyRS, serRS and tyrRS were amplified from

genomic DNA isolated from the E. coli B strain derivative E. coli BL21 (DE3). Sequences for the

forward and reverse primer can be found in the supplementary file. The coding sequences for aspRS,

ileRS, leuRS and tyrRS were cloned into pETRUK (an in-house sumo fusion plasmid containing a pI

modified sumo sequence). SerRS and glyRS were cloned into pETHSUK, a derivative of pETHSUL

containing an additional KpnI cleavage site in the MCS. In the case of glyRS, which is a hetero-

tetramer composed of two different monomers, the encoding genes (glyQ and glyS) were cloned

separately into the pETHSUK vector. For all constructs, except aspRS, cloning was performed such

that upon expression and purification the final cleaved product contained no non-native amino acids.

In the specific case of aspRS, initial trials showed that the sumo hydrolase failed to cleave the sumo-

aspRS fusion at the start methionine therefore an additional glycine spacer was placed between the

terminal sumo residue and the first residue of aspRS. Overexpression of all constructs was carried

out in the E. coli Rosetta 2 (DE3) pLysS strain. Further, culturing, cell harvesting and isolation of

proteins is detailed in the supplementary section.

2

9

5.5. tRNA purification

3

0

1

2

3

4

5

6

7

BL21 (DE3) E.coli cells were grown overnight in 50 mL LB media at 30°C. 25 mL of the preculture

was transferred to 2L LB media and grown at 37°C until an absorbance at 600nm of 0.6 was

reached at which point the cells were harvested by centrifugation at 7000g at 4°C. Total RNA was

extracted using guanidinium thiocyanate-phenol-chloroform method [43]. The RNA was precipitated

using 3 volumes of isopropanol and stored at -20°C until further workup. The precipitated total RNA

was resuspended using 200 mM TRIS pH 9 and incubate for 1 h at 37 °C to deacylate all tRNA prior

to loading onto a two 5 mL Hitrap Q HP columns (GE Lifesciences). The column was washed with 5

CV of buffer A (20 mM TRIS pH 7.5, 10 mM MgCl , 300 mM NaCl) followed by elution of the RNA

2

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ACCEPTED MANUSCRIPT

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2

3

using a gradient from 0-55 % Buffer B (20 mM TRIS pH 7.5, 10 mM MgCl , 1 M NaCl) over 20 CV.

2

The tRNA containing fractions were combined and the tRNA was precipitated using 3 volumes of

isopropanol and stored at -20°C. Prior to use, the tRNA was resuspended in assay buffer.

4

5

5.6. Purified aaRS inhibition assays

6

7

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1

2

3

4

5

6

7

8

To examine the inhibitory effect of the aaSA and aaS3DA compounds we performed a radiolabel

transfer assay using purified E. coli aaRS [31]. Briefly, either 50 nM ileRS, 0.5 nM tyrRS, 2.5 nM

leuRS, 2 nM aspRS, 2 nM serRS or 2 nM glyRS in 20 mM Tris, 100 mM KCl, 10 mM MgCl , 5 mM β-

2

mercaptoethanol, pH 7.5 was pre-incubated with the compound, at different concentrations, at 37°C in

1

4

1

the presence of 50 µM of the appropriate C-labeled amino acid, 2 mg/ml tRNA and 0.5 mg/ml

inorganic pyrophosphatase. After 10 min, pre-warmed ATP was added to the mixture at a final

concentration of 500 µM. The reaction was quenched by addition of 0.2 M sodium acetate pH 4, 0.1%

N-lauroylsarcosine and 5 mM unlabeled amino acid (2 mM for tyrosine). 20 µL was spotted on 3MM

Whatmann paper, precipitated using cold 10% TCA, washed twice with 10 % TCA and once with

acetone and dried. Addition of scintillation liquid was followed by measurement of the radio activity

using scintillation counter. The linear zone of enzyme activity was determined for each aaRS. The

quench time point was picked within this zone and with approximately 50% of total RNA

aminoacylation. Copeland pointed out [44] that when the

lower than the used enzyme concentration the Michaelis-Menten equation is no longer valid.

Therefore, the was determined using the Greco-Hakala equation:

of the compound approaches or is

1

2

9

0

2

1

2

3

The fractional activity is determined in the presence of a range of inhibitors (Io). The

the parameters of the equation.

and

are

2

4

5

5.7. MIC determination

2

3

6

7

8

9

0

All compounds were screened for their in vitro antibacterial activity against representative Gram-

positive, Gram-negative strains or yeast, by means of standard twofold serial dilution method using

LB media. The 50% minimum inhibitory concentration (MIC ) is defined as the minimum

50

concentration of the compound required to give 50% inhibition of bacterial growth after incubation at

37 for 18-24 h.

3

1

2

5.8. Quantum calculations

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4

5

Calculations of the electrostatic potential were done by Gamess program (version released on Aug 18,

2016), and graphics produced by Molden version 5.7. Structures were first energy optimized at radial

distribution functions RHF/6-31G** basis set, five basic colours used at the potential contour values:

red -0.1, yellow -0.05, green 0.000, light blue 0.05, blue 0.1 (Left: 9-methyladenine, right: 3-deaza-9-

9

methyladenine). To simulate nucleosides, the bases were capped with a methyl group at N .

6

7

5.9. Bioinformatic analysis

8

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1

2

3

4

5

6

7

8

9

0

X-ray crystallographic structures of various E. coli aaRS were identified in the Protein Databank

(PDB). For the enzymes where no coordinates were available, structures were downloaded from the

E. coli template based protein structure prediction dataset [45]. The coordinates were split into two

groups based on the aaRS class type. The structures in each group were aligned by structural

superposition of the catalytic core using UCSF chimera [46]. Homologous aaRS sequences, identified

in the genome of the methicillin resistant Staphylococcus aureus subsp. aureus HO 5096 0412, were

added to the two structure-based alignments using the MAFFT online server [47]. In case of the class

I alignment no homologue of glnRS was identified in the S. aureus genome as this species utilizes the

1

2

gln

Glu-tRNA amidotransferase to generate the correct aminoacylated tRNA. The resultant alignments

we further manually modified using Jalview [48]. Also for the class I alignment the leuRS specific

insert, that is uniquely found between the end of the core catalytic domain and the start of the KMSKS

loop in this aaRS, was removed. Sequence logos of identified core regions were generated using the

WebLogo 3 server [49].

2

1

2

Supplementary data

2

3

4

Supplementary Data related to this article can be found on online.

2

5

Acknowledgement

2

3

6

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0

1

The authors thank the China Scholarship Council for providing scholarships to Baole Zhang and

Luping Pang, the Belgian FWO for providing a SB Fellowship of the Research Foundation - Flanders

to Steff De Graef and KU Leuven for financial support. Mass spectrometry was made possible by the

support of the Hercules Foundation of the Flemish Government [20100225E7]. We are also indebted

to Luc Baudemprez for help with NMR spectra measurement and Jef Rozenski for providing MS

analysis. (Manesh mentioned to me that maybe his grand should be mentioned as well)

3

2

Funding

3

4

This work was supported by the Research Fund Flanders [Fonds voor Wetenschappelijk Onderzoek,

G077814N to S.S. and A.V., G0A4616N to S.W. and A.V., 12A3916N to B.G., G077814N to M.N.

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1S53516N to S. D. G.]; and the KU Leuven Research Fund [3M14022 to S.W and A.V.]; and the

Chinese Scholarship Council [to Z.B. and LP].

3

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6

Conflict interest

No conflicts of interest are declared.

7

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Figures and Legends:

Figure 1. Activity and architecture of aaRS family members.

Figure 2. Chemical structures of aminoacyl-adenylate (a), aminoacyl-sulfamoyl adenosines (b), natural

aaRS inhibitors (c and d), cyclized adenosine (e) and aminoacyl-sulfamoyl 3-deazaadenosines (f).

Scheme 1. Synthesis of 3-deazaadenosine.

Scheme 2. Synthesis of aminoacyl-sulfamoyl aaS3DA derivatives.

Figure 3. Comparative inhibitory activity of the aaSA (black bars) and the aaS3DA analogues (grey bars)

in E. coli K-12 BW28357 strain S30 extracts.

app

i

Table 1. K

of aaSA and the aaS3DA analogues, where aa is the corresponding amino acid.

Figure 4. Dose response curves of purified E. coli aaRS in the presence of aaSA (black circles, black

line) or aaS3DA (grey boxes, grey dashed line).

Figure 5. Computational chemistry and bioinformatics analyses.

ACCEPTED MANUSCRIPT

Figure 1. Activity and architecture of aaRS family members. (A) The two step reaction mechanism shared

amongst all aaRS. (B) Architecture of the core catalytic domain of class I (upper) and class II (bottom)

aaRS enzymes. Cartoon and surface representation of residues 1-222 of class I tyrosyl-tRNA synthetase

(

PDB:3TS1) and class II glycyl-tRNA synthetase α-subunit (PDB: 3UFG). For clarity only the monomeric

subunit of each aaRS is shown.

ACCEPTED MANUSCRIPT

Figure 2. Chemical structures of aminoacyl-adenylate (a), aminoacyl-sulfamoyl adenosines (b), natural

aaRS inhibitors (c and d), cyclized adenosine (e) and aminoacyl-sulfamoyl 3-deazaadenosines (f).

ACCEPTED MANUSCRIPT

Scheme 1. Synthesis of 3-deazaadenosine. Reagents and conditions: (i) triethyl orthoformate, formic

acid, 145 ℃, 6 h; (ii) m-CPBA, DCM/methanol (2:1), room temperature overnight; (iii) POCl , 120 ℃, 3 h;

3

(

iv) 1’,2’,3’,5’-tetra-O-acetyl-β-D-ribofuranose, SnCl , dry acetonitrile, room temperature 24 h; (v) 7 N

4

ammonia in methanol, 0 ℃ to rt., 1 h; (vi) a) hydrazine hydrate, 120 ℃, 6-8 h b) Raney Ni, 120 ℃, 30 min;

viii) p-TSA, dry acetone, DMP, rt, 3h.

(

ACCEPTED MANUSCRIPT

Scheme 2. Synthesis of aminoacyl-sulfamoyl aaS3DA derivatives. Regents and conditions: (i) a) TMSCl,

b) BzCl, c) 12% aqueous ammonia, 0 to r.t, 6-8 h; (ii) NH SO Cl, acetonitrile, room temperature

2

overnight; (iii) N-Boc-aa-(tBu/Bn)-OSu, DBU, dry DMF, room temperature overnight; (iv) sodium

methoxide, methanol, reflux, 2 h; (v) TFA/H

temperature overnight.

2 2

O, 5:2 (v/v), 3h; (vi) Pd/C, methanol, H atm. room

Article Doi

Family-wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of aminoacyl-tRNA synthetases

DOI: 10.1016/j.ejmech.2018.02.013

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Article abstract of DOI:10.1016/j.ejmech.2018.02.013

Full text of DOI:10.1016/j.ejmech.2018.02.013

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