Synthesis and structure-activity studies of novel anhydrohexitol-based Leucyl-tRNA synthetase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.113021

Leucyl-tRNA synthetase (LeuRS) is a clinically validated target for the development of antimicrobials. This enzyme catalyzes the formation of charged tRNA^Leu molecules, an essential substrate for protein translation. In the first step of catalysis LeuRS activates leucine using ATP, forming a leucyl-adenylate intermediate. Bi-substrate inhibitors that mimic this chemically labile phosphoanhydride-linked nucleoside have proven to be potent inhibitors of different members of the aminoacyl-tRNA synthetase family but, to date, they have demonstrated poor antibacterial activity. We synthesized a small series of 1,5-anhydrohexitol-based analogues coupled to a variety of triazoles and performed detailed structure-activity relationship studies with bacterial LeuRS. In an in vitro assay, K_i^app values in the nanomolar range were demonstrated. Inhibitory activity differences between the compounds revealed that the polarity and size of the triazole substituents affect binding. X-ray crystallographic studies of N. gonorrhoeae LeuRS in complex with all the inhibitors highlighted the crucial interactions defining their relative enzyme inhibitory activities. We further examined their in vitro antimicrobial properties by screening against several bacterial and yeast strains. While only weak antibacterial activity against M. tuberculosis was detected, the extensive structural data which were obtained could make these LeuRS inhibitors a suitable starting point towards further antibiotic development.

Full text of DOI:10.1016/j.ejmech.2020.113021

Journal Pre-proof
Synthesis and Structure-Activity Studies of Novel Anhydrohexitol-based Leucyl-tRNA
Synthetase Inhibitors
Dries De Ruysscher, Luping Pang, Stijn M.G. Lenders, Davie Cappoen, Paul Cos, Jef
Rozenski, Sergei V. Strelkov, Stephen D. Weeks, Arthur Van Aerschot
PII:
S0223-5234(20)30993-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.113021
EJMECH 113021
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2020
Revised Date: 20 October 2020
Accepted Date: 10 November 2020
Please cite this article as: D. De Ruysscher, L. Pang, S.M.G. Lenders, D. Cappoen, P. Cos, J. Rozenski,
S.V. Strelkov, S.D. Weeks, A. Van Aerschot, Synthesis and Structure-Activity Studies of Novel
Anhydrohexitol-based Leucyl-tRNA Synthetase Inhibitors, European Journal of Medicinal Chemistry,
https://doi.org/10.1016/j.ejmech.2020.113021.
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Synthesis and Structure-Activity Studies of Novel Anhydrohexitol-based Leucyl-tRNA
Synthetase Inhibitors
Dries De Ruysschera,#, Luping Pang a,b,#, Stijn M.G. Lendersa, Davie Cappoenc, Paul Cosc, Jef
Rozenskia, Sergei V. Strelkovb, Stephen D. Weeksb, d, *, 1 and Arthur Van Aerschota,*,1
a
Medicinal Chemistry, Rega Institute for Medical Research, Department of Pharmaceutical and
Pharmacological Sciences, KU Leuven, Herestraat 49 – Box 1030, 3000 Leuven, Belgium
b
Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven,
Herestraat 49 – Box 822, 3000 Leuven, Belgium
c
Laboratory of Microbiology, Parasitology and Hygiene, Department of Pharmaceutical Sciences,
University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
d Present address: OrthogonX, Gaston Geenslaan 1, 3001 Leuven, Belgium
* Corresponding author.
E-mail address: arthur.vanaerschot@kuleuven.be (A. Van Aerschot);
sweeks@orthogontherapeutics.com (S.D. Weeks).
# These authors contributed equally to this work.
1 Both authors equally supervised and gave guidance to this work.
Abstract
Leucyl-tRNA synthetase (LeuRS) is a clinically validated target for the development of
antimicrobials. This enzyme catalyzes the formation of charged tRNALeu molecules, an essential
substrate for protein translation. In the first step of catalysis LeuRS activates leucine using ATP,
forming a leucyl-adenylate intermediate. Bi-substrate inhibitors that mimic this chemically labile
phosphoanhydride-linked nucleoside have proven to be potent inhibitors of different members of the
aminoacyl tRNA synthetase family but, too date, they have demonstrated poor antibacterial activity.
We synthesized a small series of 1,5-anhydrohexitol based analogues coupled to a variety of triazoles
and performed detailed structure-activity relationship studies with bacterial LeuRS. In an in vitro
assay, Kꢁꢀ ꢂꢂ values in the nanomolar range were demonstrated. Inhibitory activity differences between
the compounds revealed that the polarity and size of the triazole substituents affect binding. X-ray
crystallographic studies of N. gonorrhoeae LeuRS in complex with all the inhibitors highlighted the
crucial interactions defining their relative enzyme inhibitory activities. We further examined their in
vitro antimicrobial properties by screening against several bacterial and yeast strains. While only weak
antibacterial activity against M. tuberculosis was detected, the extensive structural data which were
obtained could make these LeuRS inhibitors a suitable starting point towards further antibiotic
development.
Keywords
Leucyl-tRNA synthetase – bisubstrate competitive inhibitors – protein-ligand co-crystal structures –
structure-activity relationship – enzyme inhibition
1

1. Introduction
The World Health Organization (WHO) stated in a recent report that by 2050 10 million people
would die every year as a result of the rapid expansion of antimicrobial resistance [1]. Hence, as part
of a global action plan, this agency has highlighted the importance of investment in the development
of new treatments [2]. One strategy to identify new antibiotic classes is to explore novel or
underexplored cellular targets. Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes, which
play a pivotal role in protein biosynthesis in all cells. Due to the wide evolutionary divergence of
aminoacyl-tRNA synthetases (aaRSs) in pathogenic organisms with respect to the human orthologues,
these enzymes are promising targets for the development of new selective antimicrobials [3,4].
Although various aaRSs have been targeted for drug design, only the IleRS inhibitor mupirocin [5]
(Figure 1A) and the LeuRS targeting compound AN2690 (tavaborole [6], Figure 1A) have made it to
the clinic, used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) and
onychomycosis, a fungal infection of the toenails, respectively.
The aminoacylation reaction of LeuRS proceeds in two steps [3]. Both steps occur within the
same catalytic site defined by a class I specific Rossmann fold containing the HIGH and KMSKS
signature motifs [7]. Besides its cognate substrate, LeuRS is also able to activate structurally similar
amino acids including isoleucine (Ile), methionine (Met), norvaline and norleucine with a frequency
exceeding the tolerable translational error by the cell [8-10]. Therefore, this enzyme has evolved a
proofreading function which can either hydrolyze the misactivated amino acid in the active site by pre-
transfer editing, or clear mischarged tRNA through post-transfer editing [11]. The latter is catalysed by
an additional editing domain which is inserted into the Rossmann fold via two β-strands [12]. For
bacterial LeuRS, the post-transfer editing is the dominant error-correction pathway.
The validated LeuRS inhibitor AN2690 targets the editing domain by forming a stable boron-
mediated tRNALeu-AN2690 adduct. It traps the 3’-end of the tRNALeu isoacceptor in a non-productive
state which prevents subsequent LeuRS catalytic turnover and consequently, disrupts protein synthesis
[6]. Generally, benzoxaborole-containing compounds display low toxicity and high specificity [13]
and importantly, tavaborole demonstrated no resistance after repeated exposure [14]. However, the
development of the antibacterial 3-aminomethyl analogue GSK2251052 (AN3365, Figure 1A), which
could evade the main efflux mechanism of multidrug-resistant (MDR) Gram-negative bacteria [15],
was terminated after evaluation in phase II clinical trials owing to the rapid development of resistance
[16]. This was a result of the appearance of cells carrying editing domain mutations that were highly
resistant to the compound without compromised viability. A similar appearance of IleRS mutants is
also the cause for the low- and high-level resistance in the case of mupirocin [17-20]. More recently,
another boron-containing molecule GSK3036656 (Figure 1A) was synthesized and preclinically
evaluated as a selective inhibitor against the editing domain of Mycobacterium tuberculosis (Mtb)
LeuRS rather than human cytoplasmic and mitochondrial orthologues. This compound demonstrated
in vitro activity against the Mtb strain H37Rv with a MIC value of 23.5 ng/mL and an IC50 value of
58.8 ng/mL against Mtb LeuRS in an aminoacylation assay [21]. It emerged as a promising lead
candidate and was selected for further clinical development.
As the editing domain is highly degenerate or fully lost in a number of LeuRSs from
Mycoplasma parasites [22], the canonical aminoacylation site in the catalytic domain is still popular
for targeted drug design. The majority efforts to target this site have concentrated on identifying non-
hydrolysable analogues of the natural aminoacyl-adenylate (aa-AMP) intermediate [15]. Compounds
like leucyl-sulfamate adenosine (LeuSA, Figure 1B) demonstrate high affinity but exhibit poor
selectivity and low in vivo antibacterial activity. As a consequence, in the late 1990s, research shifted
towards other scaffolds. Cubist Pharmaceuticals (since 2015 acquired by Merck & Co.) used the high-
affinity ligand LeuSA as a starting point to synthesize inhibitors with improved chemical stability and
bacterial selectivity [23]. The adenine group was replaced by substituted thiazoles which were directly
attached to the ribose ring [24]. However, despite their high affinity and selectivity towards bacterial
LeuRS, no antibacterial activity was reported. A similar approach gave rise to selective IleRS
inhibitors in which the sugar ring was connected with aryl-substituted tetrazoles via a two-carbon atom
linker (Figure 1B) [25]. The (phenoxyphenyl)tetrazole CB 432 was reported to possess good
2

antibacterial activity, but low in vivo bioavailability halted further exploration of this scaffold for
potential antibiotics. More recently, scientists at Oxford Drug Design have reported LeuRS inhibitors
that target the enzyme active site [26]. Synthesis of these gram-negative bacteria-selective N-leucinyl
benzenesulfonamides was based on previously reported benzenesulfonamide ThrRS inhibitors [27].
Similar N-leucinyl benzenesulfonamides further connected to an acylated thiourea scaffold were
synthesized which appeared to exhibit good inhibitory activity against Trypanosoma brucei LeuRS
[28, 29].
Figure 1. (A) Structures of mupirocin, tavaborole, AN3365 and GSK3036656; (B) (Left) High-
affinity but non-selective competitive active site inhibitor LeuSA, (Right) Cubist Pharmaceuticals
aryl-tetrazole IleRS inhibitors; (C) (Left) Cubist Pharmaceuticals derived LeuRS inhibitor, (Right)
Structure for the novel proposed competitive inhibitors targeting LeuRS.
We recently published the synthesis of IleRS inhibitors having good affinity [30] where we
substituted the ribose ring of CB 168 (Figure 1B) with a 1,5-anhydrohexitol ring with implantation of
the aliphatic chain at C2 and the isoleucyl-sulfamoyl chain at C5, respectively. The three-atom linker
between phenyltriazole and the pyran ring was proven to be essential for preserving good affinity for
IleRS. Unfortunately, no antibacterial activity was observed, probably resulting from the high polarity
of the IleRS inhibitors. We therefore envisioned synthesis of LeuRS inhibitors with the same scaffold
and implantation of both side chains on the ring, but with various substituted triazoles to improve
antibacterial activity (Figure 1C). Following synthesis of compounds 11a-k, their inhibition activity
and antibacterial activity were evaluated. Combined with high-resolution crystal structures of the
enzyme-ligand complexes, we were able to carry out a small but thorough SAR study to provide better
insight into the crucial interactions for the substituted triazole to obtain potent enzymatic inhibitory
activity.
3

2. Results
2.1. Synthesis of LeuRS inhibitors containing different substituted triazoles
Reagents and conditions: [A] (a) (1) CuI, allyl-MgCl, anh. THF, -30 °C, 15 min.; (2) 1, anh. THF, -30
°C, 1.5 h; (b) BH3·THF, anh. THF, rt, 1 h; (2) NaOH, H2O2, H2O, 0 °C to rt, overnight; (c) (1) TEA,
anh. DCM, 0 °C; (2) TsCl, anh. DCM, 0 °C to rt, 2 d; (d) NaN3, anh. DMF, 55 °C, overnight; (e)
PTSA·H2O, THF:H2O, 40 °C, 3 d; (f) PTSA·H2O, DMP, acetone, rt, overnight; (g) (1) chlorosulfonyl
isocyanate, HCOOH, 0 °C, 30 min.; (2) anh. MeCN, rt, 5 h; (3) 7, DMA, rt, overnight; (h) Boc-Leu-
OSu, Cs2CO3, anh. DMF, 0 °C to rt, 3 d; (i) CuI, TEA, respective alkyne, DMF, 50 °C, overnight; (j)
TFA:H2O, rt, 5 h; [B] (k) (1) propargylamine, acetic acid, anh. MeOH, rt, overnight; (2) NaBH4, 0 °C
to rt, 2 h; (l) TEA, propargylamine, anh. DCM, 0 °C to rt, overnight; (m) propargylamine, anh. DCM,
0 °C to rt, 3 h.
Scheme 1. [A] Procedure towards various leucine coupled triazoles 11; [B] Synthesis of non-
commercially available alkynes; [C] Cubist Pharmaceuticals-like inhibitor 15.
The envisaged target compounds 11a-k were based on the potent tetrazole IleRS inhibitors of
Cubist Pharmaceuticals (Figure 1B) [25]. The reported enzymatic activity of the latter ranged from 0.5
to 20 nM depending on the pathogen sources, and these compounds were endowed with high
selectivity for bacterial IleRS versus the human orthologue. LeuRS shares high structural similarity
with IleRS and both belong to the same aaRS Ia subclass. In addition, the inhibitory activity of LeuSA
(Kꢁꢀ ꢂꢂ = 0.14 nM) is substantially higher than the equivalent isoleucine coupled analogue for IleRS
4

(Kꢁꢀ ꢂꢂ = 1.92 nM) [31]. This improved affinity for LeuRS targeting compounds was also noted with
base modified inhibitors compared to those for IleRS [32], which further stimulated our choice for
LeuRS as the target for the synthesis of compounds 11a-k. These novel inhibitors are based on the CB
168 Cubist Pharmaceuticals compound (Figure 1B), but in which the ribose ring is replaced by a pyran
ring so that the implantation of both side chains is moved by one carbon atom. We previously reported
that for this particular chain arrangement, the optimal aliphatic linker between the six-membered ring
and the heterocycle is a C3 spacer [30].
The synthetic route commenced with commercially available allitol epoxide 1 (Scheme 1). The
Gilman-reagent of allylmagnesium chloride was made to open the epoxide regio- and stereoselectively
at the C2-position in a trans-diaxial manner [33]. The obtained alkene 2 underwent a hydroboration-
oxidation reaction, followed by selective tosylation of the resulting primary alcohol, which afforded
compound 5 upon subsequent in situ azide substitution allowing the coupling of a series of alkynes.
Hereto, the azide 5 was converted into the isopropylidene protected alcohol 7. Subsequent
sulfamoylation was followed by coupling leucine to the obtained sulfamate functional group, affording
compound 9. At this stage, we connected 11 different alkynes using conventional azide-alkyne click
chemistry, affording the protected compounds 10. The alkyne 12 as needed for 10h was made through
reductive amination, while alkynes 13 and 14 (respectively for synthesis of 10i and 10j) were
synthesized using standard chemistry. Finally, acidic removal of all protecting groups yielded the
desired compounds 11a-k. The Cubist Pharmaceuticals-like inhibitor 15 (Scheme 1C) was prepared
for comparative reasons, and its synthesis is analogous to the original isoleucine analogue [34].
2.2. In vitro enzymatic inhibitory activity
The ability of all new leucine coupled compounds 11a-k and 15 to inhibit LeuRS was verified
using a previously described in vitro aminoacylation assay, monitoring the effect on the transfer of the
14C-radiolabeled leucine to tRNALeu (Table 1 and Supplementary figure 1) [35]. Purified recombinant
E. coli LeuRS was employed with a total tRNA pool from the same pathogen. We herein opted for E.
coli LeuRS to carry out the enzymatic assay as many inhibitors before have been tested on this
specific enzyme, which allows to compare these different inhibitors for future studies.
Compared with the high-affinity non-hydrolysable Leu-AMP analogue LeuSA (Kꢁꢀ ꢂꢂ of 0.14
nM), all novel synthesized compounds demonstrated decreased inhibitory activity, but most of them
were still potent binders with Kꢁꢀ ꢂꢂ values in the lower nanomolar range. Despite compounds 11a-k
sharing a similar chemical scaffold, different substitutions connected with the triazole moiety showed
significant effects on the inhibitory potential. As shown in Table 1, the best compounds 11a and 11k
carried a phenyl substituent at C13 on the triazole ring (for carbon numbering, see structure in Table 1),
showing Kꢁꢀ ꢂꢂ values of 5.51 and 2.48 nM, respectively, whereas compound 11f with an additional
para-OCF3 modification on the phenyl ring or compound 11g substituting the phenyl with a 3-
pyridinyl moiety, showed 3 to 10-fold reduced activity. It suggested that the phenyl substituent is a
crucial element in determining the stronger LeuRS inhibition, while any electron-withdrawing
modification at this position displayed an opposite trend.
An attempt was made to investigate the effect of a spacer between the triazole and phenyl rings.
Consequently, each two to five-atom linker evaluated (compounds 11d and 11i-j) led to a significant
decrease in binding affinity. Furthermore, compounds 11c and 11e, carrying only an aliphatic chain on
the triazole moiety, likewise showed reduced inhibitory activity. In addition, it should be noted that
both compounds 11b and 11h carrying an amine which is protonated physiologically, completely lost
their inhibitory activity. Overall, we demonstrated that only 4-phenyltriazole modified compounds are
endowed with favorable inhibitory potential.
Cubist Pharmaceuticals designed a series of aryl-tetrazole IleRS inhibitors (Figure 1B), showing
excellent selectivity with strong inhibition against pathogenic bacteria but not the eukaryotic enzyme.
As compound 11k exhibited most potent inhibition against E. coli LeuRS, we synthesized compound
15, an analogue of the IleRS inhibitors while retaining the phenyl group connected with the
heterocycle and substituting the isoleucyl with a leucyl moiety (Scheme 1C). Surprisingly, a
5

significant 15-fold reduction of activity was observed for 15 compared to 11k. Two obvious
distinctions between these two compounds can be noted: one is the six-membered pyran ring in 11k
versus the canonical five-membered anhydroribitol for 15; the other one is the three-carbon linker
between pyran and triazole in 11k whereas a two-carbon linker was utilised for 15. It is well known
that mupirocin, an IleRS inhibitor, has a similar pyran ring in its chemical structure, and it has been
reported that this pyran ring is recognized analogously to the ribose moiety of IleSA [5]. Therefore, it
is reasonable to believe that the difference in activity between 11k and 15 is attributed to the length of
the carbon linker. This indicated a preference for a three-atom carbon linker for inhibitory activity
possibly due to the shape or size of the binding site.
Overall, by analyzing their structure-activity relationship, it can be postulated that the precise
length of the carbon linker and the chemical properties of the substituent on the triazole moiety are
essential features for the inhibitory activity of this compound family targeting LeuRS.
Table 1. Inhibitory constants (Kꢁꢀ ꢂꢂ) of the synthesized compounds on E. coli LeuRS.
Compounds
Ra
Kꢁꢀ ꢂꢂ (nM)d
5.51 ± 0.93
11a
2969.71 ± 148.85
52.36 ± 7.17
11b
11c
11d
11e
11f
108.35 ± 5.00
387.66 ± 19.44
17.83 ± 2.37
31.62 ± 2.60
11g
11h
1260.73 ± 92.11
31.28 ± 2.37
182.35 ± 4.20
11i
11j
2.48 ± 0.13
30.94 ± 4.95
0.14 ± 0.10
11k
15b
LeuSAc
aR represents the various substituents connected to the triazole ring.
b15 is the analogue of a Cubist Pharmaceuticals IleRS inhibitor but carrying a leucyl moiety.
6

cThe Kꢁꢀ ꢂꢂ value of high affinity inhibitor LeuSA was taken from our prior work [31].
dA concentration of 2.5 nM E. coli LeuRS was used in the aminoacylation assay. The corresponding
Kꢁꢀ ꢂꢂ values of the synthesized compounds were calculated using Greco-Hakala equation by
maintaining the input enzyme concentration as a constant. All values were normalized based on the
measured activity of the enzyme in the absence of inhibitor.
2.3. X-ray crystallographic studies of LeuRS-ligand bound complexes
To further elucidate the relative inhibitory activity and structural basis for the binding
mechanism of this new series of LeuRS inhibitors, X-ray crystallographic studies of the compounds in
complex with LeuRS were performed. N. gonorrhoeae LeuRS (Ng-LeuRS), a homologue to LeuRS
from E. coli (Ec-LeuRS), was successfully crystallized and soaked with the compounds. We have
previously reported that the overall sequence identity and similarity between these two enzymes is 57%
and 71%, respectively, whereas the active site, which is responsible for the catalysis of Leu-AMP
formation, share 92% identity and 100% similarity (Supplementary figure 2) [32]. Therefore,
structural information obtained from Ng-LeuRS complexes can be extrapolated to the inhibition
activity measured with E. coli LeuRS.
All Ng-LeuRS-ligand complexes were solved and determined at a resolution range between 1.9
and 2.6 Å (Supplementary table 1). Except for two low-affinity binders 11b and 11h, the remaining
compounds can be built unambiguously in the catalytic site according to the electron density map
(Figure 2). To gain further insight into the molecular mechanisms defining the inhibitory activity, we
split the synthesized leucyl-sulfamoyl analogue scaffold into four pharmacophoric regions: leucyl-
sulfamate moiety, the ring (six-member anhydrohexitol ring or 1,4-anhydroribitol for 15), carbon
linker (two-carbon or three-carbon linker for 15) and various substitutions connected to either the
triazole or tetrazole ring.
7

Figure 2. Omit maps of inhibitors bound to the active site of N. gonorrhoeae LeuRS. From left to
right, structures are shown in order of decreasing inhibitory activity with the exception of compound
15. The maps were calculated using phenix.polder and shown as a grey mesh framework contoured at
3.5 σ. All ligands are shown as a stick representation where the catalytic site of LeuRS backbone is
shown as a cartoon representation and colored in green. For compounds 11h and 11b, two different
conformations are observed in the active site of enzyme due to weak binding.
Superposition of the structures of LeuRS in complex with 11a-k by the catalytic core (mean
RMSD of 0.22 Å with residues surrounding the ligand within 6 Å), shows that all compounds share
very similar conformations (Figure 3A and Supplementary figure 3). The leucyl-sulfamate moiety is
bound in the same pocket as observed for LeuSA (Figure 3B and 3D). Briefly, the amino group of the
leucyl moiety forms H-bonds with Oδ2 of Asp80 and carbonyl oxygen in Phe41, while the carbonyl
oxygen of the leucyl moiety itself forms a H-bond with Nε of His547. The side chain of the leucyl
moiety is further recognized by a conserved hydrophobic pocket in all LeuRS structures, formed by
Met40, Phe41, Tyr43, Phe507, Tyr513, His547 and His551 residues. The sulfamate oxygen further
establishes either direct H-bonds with the main chain nitrogen of Tyr43 and Nε of His52 or via a water
bridge with Oε2 of Glu546 (Figure 3B). His52 is part of the class I signature HIGH motif, which in the
case of Ng-LeuRS has the sequence 49HMGH52. In further descriptions we will refer to any residues in
this region using the former nomenclature. The tetrahydropyran adopts a chair conformation where the
C1 atom is located in the same position as O4’ of ribose and the 3-hydroxyl and 4-hydroxyl groups are
superimposing with 2’- and 3’-hydroxyl of ribose in LeuSA, respectively. Therefore, the six-
membered anhydrohexitol retains the same interactions as seen for ribose (Figure 3B). Compound 15
mimics the selective IleRS inhibitor CB 168 produced by Cubist Pharmaceuticals while replacing
isoleucyl with a leucyl moiety. Comparing with LeuSA, the leucyl-sulfamoyl ribose moiety in 15 is
fully superposed with the equivalent group in LeuSA and makes the same active site interactions
observed for compounds 11a-k and LeuSA (Figure 3C and 3D). Therefore, the obvious distinction in
inhibitory activity of the synthesized compounds can be attributed to the linker and further
substitutions on either the triazole or tetrazole ring.
Figure 3. Comparison of compounds 11a-k bound in N. gonorrhoeae LeuRS complexes.
(A) Superposition of 11a-k (except for 11b and 11h) by aligning the active site of the protein. (B)
Interactions between leucyl-sulfamoyl anhydrohexitol moiety in 11k and LeuRS. (C) Interactions
between leucyl-sulfamoyl ribose moiety in 15 and LeuRS. Ligands and the interacting protein residues
are shown as stick representations. The backbone of LeuRS is shown as a cartoon view. H-bonds are
8

shown as black dashed lines and water molecules as red spheres. (D) Left panel: Superposition of
structures of 11k and LeuSA (PDB code: 6Q89) [32]; Middle panel: Superposition of the structures 15
and LeuSA; Right panel: Superposition of the determined structures for 11k and 15. The movement of
the KMSKS loop between the different structures was measured according to Cα of the Lys637
residue.
In comparison with LeuSA, replacement of adenine with a propyl-triazole decorated with
various phenyl modifications or aliphatic chains in the studied compounds, leads to disappearance of
some extensive interactions as noted between the adenine ring and LeuRS protein residues, which
explains the lower inhibitory activity observed for 11a-k relative to LeuSA (Figure 4, 5 and
Supplementary figure 4). In addition, to accommodate the binding of compounds 11a-k and 15, the
KMSKS signature motif with the attached leucine specific domain (LSD) in LeuRS is positioned 4.3
Å outwards from the active site. Furthermore, the HIGH motif His49 rotates away from the active site
compared to the LeuSA-bound structure (Figure 3D). This specific positioning of the KMSKS loop in
these structural complexes establishes an enlarged open binding cavity for the synthesized inhibitors
when compared to that seen when bound to LeuSA (Figure 3D and 4).
Figure 4. The binding pockets of compound 11k and LeuSA in the respective LeuRS complexes. The
protein is shown as surface representation and the ligands are shown as sticks.
To further investigate the forces defining the inhibitory activity, structural analysis of the
interactions between the protein and the various substituents in compounds 11a-k and 15 were
performed (Figure 5). All compounds adopt a stretched conformation where the substituted triazole
lies along the catalytic loop bearing the class I signature motif, 634KMSKS638, and stacking with the
side chain of His49, being the first residue of the HIGH motif. As shown in Figure 5, the three-carbon
atom linker apart from Van der Waals interactions does not make any specific interactions with the
protein but provides the appropriate length to allow the triazole connecting group to move towards the
binding cavity being positioned at the right side of adenine in the LeuSA complex structure. The N11
and N12 of the triazole heterocycle make one or two H-bonds with the backbone nitrogen of Val583.
In addition, a cation-π interaction is formed with the side chain of Lys634 (Figure 5). For compounds
11k, 11a, 11f and 11g, an additional aromatic ring is directly connected to the triazole moiety,
providing a supplementary π-π interaction with the imidazole of His49. Compounds 11a and 11k
display the same interactions which further correlates with their comparable activities (Table 1).
However, introduction of electron withdrawing substituents cause the aromatic ring to have weaker π-
π interaction, which is the rationale for the decreased activities of compounds 11f and 11g.
In the remaining compounds various linkers connect the phenyl and triazole moieties. However,
since the phenyl ring is moved away from His49, the contributing π-π interaction is lost, resulting in
dramatically reduced binding affinity (Figure 5 and Table 1). However, compound 11i demonstrated
comparable activity to 11g, which can be ascribed to the supplementary direct or indirect H-bond
interactions between the carbonyl oxygen of the amide linker in 11i and the backbone nitrogen of
Met50 and carbonyl oxygen of Val643, respectively. In contrast, despite having similar H-bonds of the
urea carbonyl oxygen for 11j, the terminal phenyl ring becomes more exposed to the solvent resulting
in increased entropic penalty and reduced binding capacity compared to 11i. The aliphatic chain in
compound 11c and phenylethyl in 11d are positioned parallel to the side chain of His49. Although
losing the π-π interaction, their improved inhibitory activity compared to 11j is likely the result of
9

hydrophobic interactions with the binding cavity. Moreover, introduction of a polar terminal hydroxyl
group as in 11e led to a 6-fold reduction in inhibitory activity versus 11c (Table 1).
In view of the reduced length of the two-carbon atom linker in compound 15, the binding mode
for the tetrazole and phenyl rings is significantly different from that seen in compound 11k. On
superimposing with the LeuSA-bound structure, we noted the dihedral angle (O4’-C1’-C1-C2) in 15 is
rotated 113.7° compared to the equivalent dihedral angle (O4’-C1’-N9-C8) in LeuSA. This results in
the tetrazole and phenyl group being positioned further at left in the pocket than the adenine base
(Figure 3D). The Chi1 dihedral angle of His49 is simultaneously rotated 123.4° towards the opposite
direction of the catalytic site to accommodate the binding of 15 and makes a π-π interaction between
its phenyl ring and the His49 imidazole. Furthermore, the N4 nitrogen H-bonds with the backbone
nitrogen of Gly51. Since compound 15 is rotated away from the binding cavity observed for
compound 11k, it only stacks with the HIGH motif and forms a weak cation-π contact with Lys634,
but loses potential hydrophobic interactions resulting in its lower binding affinity compared to 11k.
10

Figure 5. Analysis of protein-ligand interactions of the various inhibitor-bound N. gonorrhoeae
LeuRS structures. Except for compound 15, the remaining structures are shown in order of decreasing
inhibitory activity. For each complex, 3D protein-ligand interactions are shown in the upper panel
whereas the corresponding 2D interactions shown underneath. In the 3D interactions, protein
structures are shown as cartoon representations while protein residues and ligands are shown as stick
representations. All protein structures are colored in green. In 2D interactions, protein residues are
shown as circles with numbers inside. KMSKS signature motif is shown as a black box with the
starting residue in a magenta circle. Water molecules are shown as cyan filled circles. π-π stacking
interaction is colored in orange, cation-π interaction is colored in magenta and all the remaining
11

interacting residues in blue. All H-bonds are shown as black dashed lines. The protein-ligand
interactions are identified via the respective optimized structures in Schrödinger [36].
In the case of compounds 11b and 11h, electron density maps (Figure 2) clearly show the
unambiguous binding of the leucyl-sulfamoyl anhydrohexitol moiety. However, two conformations for
the aminomethyl-triazole part are observed for 11b, while no clear density is detected for the
substituent at the equivalent position in 11h, demonstrating unstable binding of this part of the
molecule for both compounds. Evaluating their chemical structure, it is worth noting that these two
compounds are protonated at physiological pH, distinguishing them from the other studied inhibitors.
To investigate the effect of a charged ligand, the relative electrostatic potential map was calculated for
the LeuRS-11b complex. As shown in Figure 6, the protonated aminomethyl-triazole is located in a
strongly positive charged cavity in LeuRS, leading to weak and flexible binding for this part of the
molecule. In 11h the benzylaminomethyl substituent on triazole is expected to lead to the same
electronic repulsion, but the additional benzyl moiety apparently causes increased flexibility and hence
lack of electron density is noted for this part of the molecule.
Taken together, the novel synthesized compounds demonstrated two distinct binding modes to
their target LeuRS protein indicating a significant effect of the length of the linker. Further structural
analysis for compounds 11a-k showed that the various substituents on the triazole moiety either
strongly contribute or reduce the inhibitory activity. Due to the positively charged binding pocket
beyond the triazole ring, non-charged substituents are preferred over both positively charged
examples. It can hence be postulated that a negative charged modification potentially could further
enhance the binding affinity of an inhibitor. In particular IleRS, belonging to the same subclass as
LeuRS, shares close homology and structural architecture in the active site region. The obtained data
therefore can be employed to interpret the results of the early efforts of Cubist Pharmaceuticals in
obtaining IleRS inhibitors and offer the possibility to design similar drugs binding to these enzymes.
Figure 6. Electrostatic potential mapping on the protein surface surrounding ligand 11b. The map was
calculated using APBS Electrostatics in Pymol in which the blue, white and red represent the
positively, neutral and negatively charged regions, respectively. Compound 11b is shown as a stick
representation and colored in yellow.
2.4. Antibacterial tests
Due to their potency against LeuRS, nine of the final synthesized 3-carbon atom linker
compounds were screened for their in vitro antimicrobial activity against Gram-negative bacterium
Escherichia coli, Gram-positive bacterium Staphylococcus aureus and yeast Candida albicans, using a
previously established resazurin assay [37]. Although the lengths of LeuRSs from these different
organisms vary considerably, comparison of the ligand binding site by sequence alignment clearly
shows that all the important interacting residues are highly conserved in all tested species
(Supplementary Figure 2). Only a small number of residues making backbone interactions are
variable. Therefore, all these different LeuRSs should all be targeted by the synthesized LeuRS
12

inhibitors. Unfortunately, no antimicrobial activity was observed at the maximum concentration tested
of 64 µM (data not shown).
These compounds were further evaluated for their in vitro activity against the Gram-positive
non-pathogenic strain Mycobacterium tuberculosis (Mtb) H37Ra (ATCC® 25177TM) using a REMA
assay (Table 2) [37]. While no anti-mycobacterial activity was observed for most of the compounds,
analogue 11a exhibited 86% inhibition at 128 µM providing a very modest IC50 value of 24.7 µM. In
contrast, the Cubist-like compound 15 exhibited a five-fold reduced antimycobacterial activity with an
IC50 of approximately 128 µM, which agrees with its decreased enzymatic activity comparing with
11a.
Table 2. In vitro anti-mycobacterial activity test. Moxifloxacin, a first-line anti-TB drug with IC50 =
0.25 ±0.13 µM, was used as a positive control.
Compounds
11a
% Inhibition at 128 µM
86.08
9.44
11b
4.26
11c
38.21
23.24
27.81
16.85
12.06
4.24
11d
11e
11f
11g
11h
11k
49.83
15
3. Discussion and conclusion
Aminoacyl-tRNA synthetases are validated targets for the development of antimicrobials [3,38].
However, to date only mupirocin, a competitive IleRS inhibitor, and tavaborole, an antifungal
molecule targeting the editing site of LeuRS, are commercially available (Figure 1A). Cubist
Pharmaceuticals previously envisioned synthesis of selective IleRS and LeuRS inhibitors (Figure 1B)
based on the high-affinity aminoacyl-sulfamate adenosine (aaSA) bio-isosteres (Figure 1B) of the
active site intermediate (aa-AMP) [24,25]. We recently embraced the aryl-tetrazole strategy by Cubist
Pharmaceuticals in combination with the tetrahydropyran ring of mupirocin. We therefore
functionalized the C2 position with phenyltriazoles/tetrazoles using different linkers, while having the
aminoacyl-sulfamate moiety in the same 1,4-cis arrangement as in mupirocin. Despite obtaining strong
enzymatic inhibition for the IleRS triazole inhibitors with a three-atom spacer, no antibacterial activity
was noted, which was attributed to the polarity of these molecules [30].
We now synthesized a series of LeuRS targeting compounds, having a C3 linker connecting the
anhydrohexitol and the triazole heterocycle, with the latter carrying various substituents of different
13

polarities (Figure 1C). We redirected our research focus on LeuRS because compounds sharing similar
scaffolds always displayed higher binding affinity with the target enzyme versus the equivalent
analogs targeting IleRS [31,32]. Following synthesis of eleven LeuRS-targeted analogues using click
chemistry (Scheme 1), their inhibitory constants were determined in an in vitro aminoacylation assay
using purified recombinant E. coli LeuRS (Table 1) [32]. In comparison with LeuSA, their inhibitory
activities clearly drop but most of them still inhibit in the lower nanomolar range with 11k as most
potent compound. However, some exceptions including compounds 11b and 11j showed a dramatic
decrease in their inhibitory profile. To obtain a molecular understanding of their binding modes and
establish a SAR study of these congeners with diverse polarity substitutions, N. gonorrhoeae LeuRS
was crystallized and the corresponding enzyme-inhibitor structures were determined providing
insights into the specific protein-ligand interactions.
All the new compounds clearly bind the canonical aminoacylation catalytic site of LeuRS.
Comparing with LeuSA, the leucyl-sulfamoyl and six-membered pyran ring are recognized in the
same manner as the equivalent leucyl-sulfamoyl ribose. Therefore, notable differences affecting the
potential inhibition activity are highly related to the protein residues surrounding the triazole
substitutions and the positioning of these triazole substitutions relative to each other and the adenine
base in LeuSA. As described in our previous work [32], the adenine base makes several H-bonds with
surrounding residues with the KMSKS-containing loop in a closed conformation. However, the long
aliphatic chains or aromatic rings coupled to triazole bind in an enlarged cavity with an open
conformation for the KMSKS motif (Figures 3 and 4). The substituted triazole in the synthesized
compounds is sitting along KMSKS loop and stacked with the side chain of His49 of the HIGH motif.
This binding mode of the synthesized inhibitors is very reminiscent of mupirocin binding to IleRS. It
has been reported that the KMSKS loop is highly dynamic and can move in and out to assist the
substrates binding during catalysis [39,40]. Therefore, the open conformation of the KMSKS loop in
these inhibitor-bound structures indicates that these compounds are likely binding in the apo state of
the enzyme. The loss of H-bonds as observed for the adenine congener provides a good rationale for
the relative lower activity for the triazole series.
Further comparing the different substitutions demonstrates that the aromatic ring directly
connected with triazole is essential for π-π and cation-π interactions explaining the good activity as
seen for 11a, 11f, 11g and 11k. The phenyl-functionalized triazole 11k proved to be the best LeuRS
inhibitor with a Kꢀꢁꢂꢂ of 2.48 nM, only one order of magnitude less active than the high-affinity
LeuSA bioisoster of the natural enzyme intermediate. Electron withdrawing substituents on this
phenyl moiety have a negative impact on the enzymatic activity as seen for inhibitors 11f and 11g. The
influence of an additional aliphatic chain on the aromatic ring is rather insignificant as noted for 11a
compared with 11k. Replacing the aromatic ring with an aliphatic chain as in 11c replicates the
binding mode of the former but loses stacking interaction leading to its 10-fold decreased activity, but
still inhibiting LeuRS with a Kꢁꢀ ꢂꢂ of 52 nM. This hints to similar binding as seen for mupirocin where
the fatty acid-mimicking chain runs into a hydrophobic pocket in the active site of IleRS [41]. The
activity drops when a more polar group is attached to the aliphatic chain as in 11e.
In contrast, adding various length and polarity of linkers between triazole and aromatic ring has
significant negative contribution for the binding affinity showing a 50 to 600-fold reduction. This is
likely caused by the loss of stacking interaction and the solvent repulsive effect due to the aromatic
benzene ring becoming exposed to the solvent. One exception is observed for compound 11i, which
appears to be complemented by the additional H-bonds generated with surrounding residues.
Surprisingly, compounds 11b and 11h showed three orders of magnitude decreased inhibitory activity
comparing with the most potent compound 11k, which can be explained by the positive charged amine
present in both former compounds. Electrostatics calculations of the protein shows that the binding
pocket of this series of compounds is also positively charged indicating electron repulsive effects for
these two compounds upon binding. Indeed, their electron density maps clearly suggest dynamic
behavior in the pocket (Figure 2). However, the aminomethyl-substituted triazole 11b was deliberately
chosen in the design phase due to a recent report in which broad-spectrum antibiotics were obtained
upon functionalization with this moiety. The rationale was that the aminomethyl moiety would
improve uptake into bacterial cells with the molecule obeying to the so-called eNTRy rules [42]. The
14

interesting finding in this work suggests that designing negatively charged modifications at the para-
position of the benzene ring in compound 11a or totally replacing benzene could probably lead to
improved LeuRS inhibitors as seen with the presence of the terminal carboxylic acid moiety in
mupirocin.
In an attempt to have a good comparison for our best analogue 11k, with the Cubist
Pharmaceuticals compounds, we generated 15 using the two-atom carbon linker and standard ribose
instead of pyran. Surprisingly, its activity is 15-fold lower than 11k suggesting that the two-atom
carbon linker is likely the determinant of the poorer activity. The X-ray crystallographic structure of
15-bound LeuRS supports this hypothesis. While positioning of the leucyl-sulfamoyl ribose group is
comparable to the equivalent moiety in 11k the phenyltetrazole entity does not occupy the same part
of the pocket as seen for 11k as the two-atom linker is too short to explore this cavity. This results in a
very different conformation of the phenyltetrazole group (Figure 3D).
Although demonstrating excellent in vitro activity against bacterial LeuRS, no broad-spectrum
antibacterial activity including Gram-positive and Gram-negative pathogens was observed for the
synthesized compounds which is likely the result of poor uptake. Interestingly, further
antimycobacterial tests showed that compound 11a exhibits the best albeit still weak activity with an
IC50 value of 24.7 µM which is better than the similar compound 11k indicating the aliphatic chain
attached at the para-position of the benzene ring is important for mycobacterial uptake. The observed
5-fold improved activity compared to Cubist-like compound 15 suggests that our LeuRS inhibitor
scaffold is however suited for further optimization. A potential solution for facilitating permeation is
to ensure active uptake by bacterial cells by conjugation of a peptidic uptake signal or siderophore
moiety. Such modifications are based on examples found in Nature as documented for the Trojan
horse compounds microcin C and its analogues [43] which make use of a peptidic uptake signal and
albomycin which contains a siderophore group for facilitated uptake [44]. Such strategies however
need to be tailored for the specific species targeted but can also be used for targeting of mycobacteria
as highlighted in a recent review [45].
In conclusion, we have uncovered inhibitor 11a as a good starting compound for further
optimization using the above proposed prodrug strategies in order to obtain antibacterial active
molecules targeting LeuRS.
4. Experimental section
4.1. Reagents and analytical procedures
Reagents and solvents were purchased from commercial suppliers (Acros, Sigma-Aldrich) and used as
provided, unless indicated otherwise. DMF, THF, DCM and MeOH 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 atmosphere with
stirring at room temperature, unless indicated otherwise. 14C-radiolabeled amino acids and scintillation
liquid were purchased from Perkin Elmer. E. coli tRNA was purchased from Roche.
13
1H and C NMR spectra of the compounds dissolved in CDCl3, CD3OD or DMSO-d6 were recorded
on a Bruker UltraShield Avance 300 MHz and 400 MHz or when needed on a 500 MHz and 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 (CDCl3: H, 7.26 ppm; C, 77.16 ppm; DMSO-d6: H, 2.50 ppm; C, 39.52
ppm; CD3OD: 1H, 3.31 ppm; 13C, 49.00 ppm) as a reference. Coupling constants are reported in Hertz
(Hz). The peak patterns are indicated by the following abbreviations: br s = 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 (SYNAPT G2 HDMS, Waters, Milford, US)
equipped with a standard ESI interface; samples were infused in acetonitrile/H2O (1:1) at 5 µL/min.
For TLC, precoated aluminum sheets were used (Merck, Silica gel 60 F254). The spots were
visualized by UV light at 254 nm or when needed with 5% H2SO4 in EtOH. Chromatography was
performed on ICN silica gel 60Ǻ 60-200. Eluent compositions are expressed as v/v. The
15

characterization of all intermediates and final compounds by NMR and mass spectrometry are
provided in the supplementary file.
4.2. Chemical synthesis and analysis of intermediates and final compounds
4.2.1. (4aR,7R,8S,8aS)-7-allyl-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol (2).
Copper(I) iodide (0.163 g, 0.85 mmol, 0.2 eq.) was put under an atmosphere of nitrogen and
anhydrous tetrahydrofuran (THF, 15.7 mL) was added. The suspension was cooled to -30 °C and
allylmagnesium chloride (10.7 mL, 5.0 eq., 2.0 M in THF) was added dropwise. Epoxide 1 (1.0 g,
4.27 mmol, 1.0 eq.) was dissolved in anhydrous THF (9.5 mL, 0.45 M) and was added dropwise to the
Gilman-like reagent. The mixture was stirred for 1.5 h at -30 °C. After completion, the reaction was
quenched with aqueous saturated ammonium chloride (50 mL) at -30 °C. The mixture was then stirred
for 30 min. at room temperature. The aqueous layer was extracted with ethyl acetate (3x). The
combined organic layers were washed with water and brine and dried over anhydrous MgSO4.
Following filtration, the solvent was removed under reduced pressure. The residue was purified by
silica gel chromatography (heptane:ethyl acetate 50:50) to give compound 2 (0.810 g, 69%) with Rf =
0.70 (heptane:ethyl acetate 50:50) as a colourless oil. 1H NMR (300 MHz, CDCl3): δ = 7.53 – 7.44 (m,
2H), 7.42 – 7.31 (m, 3H), 5.77 (m, 1H), 5.62 (s, 1H), 5.11 (m, 2H), 4.30 (dd, J = 10.2, 4.8 Hz, 1H),
4.04 (s, 1H), 3.98 (dd, J = 11.7, 2.8 Hz, 1H), 3.94 – 3.85 (m, 1H), 3.76 – 3.67 (m, 2H), 3.64 (d, J =
11.6 Hz, 1H), 2.43 – 2.17 (m, 3H), 1.92 (dd, J = 8.7, 6.4 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ
= 137.63, 136.31, 129.53, 128.64, 126.47, 117.42, 102.33, 77.95, 69.65, 68.73, 67.60, 66.11, 41.53,
34.48 ppm. HRMS (ESI): calcd. for C16H20O4Na [M + Na]+ 299.1254; found 299.1253.
4.2.2. (4aR,7R,8S,8aS)-7-(3-hydroxypropyl)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol (3).
Alkene 2 (0.266 g, 0.96 mmol, 1.0 eq.) was put under an atmosphere of nitrogen and anhydrous THF
(3.9 mL, 0.25 M) was added. Borane tetrahydrofuran complex (1.1 mL, 1.1 eq., 1.0 M in THF) was
added and the mixture was stirred for 1 h at room temperature. After completion of the reaction, the
solution was cooled to 0 °C. Aqueous sodium hydroxide (1.4 mL, 1.5 eq., 1.0 M) and aqueous
hydrogen peroxide (0.1 mL, 1.5 eq., 35 wt.%) were added dropwise. The mixture was allowed to
warm to room temperature and was stirred overnight at room temperature. The aqueous layer was
extracted with ethyl acetate (3x). The combined organic layers were washed with aqueous saturated
sodium bicarbonate and brine and dried over anhydrous MgSO4. After filtration, the solvent was
removed under reduced pressure. The residue was purified by silica gel chromatography
(heptane:ethyl acetate 50:50 to 0:100) to afford compound 3 (0.258 g, 91%) with Rf = 0.12
(heptane:ethyl acetate 50:50) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ = 7.45 (m, 2H), 7.35
(m, 3H), 5.64 (s, 1H), 4.98 (d, J = 3.5 Hz, 1H), 4.41 (t, J = 5.1 Hz, 1H), 4.13 (dd, J = 9.9, 4.8 Hz, 1H),
3.87 – 3.81 (m, 2H), 3.76 (td, J = 9.9, 4.9 Hz, 1H), 3.67 (dd, J = 9.4, 2.4 Hz, 1H), 3.62 (t, J = 10.1 Hz,
1H), 3.50 (d, J = 11.3 Hz, 1H), 3.41 (q, J = 5.7 Hz, 2H), 1.64 – 1.58 (m, 1H), 1.58 – 1.38 (m, 4H)
ppm. 13C NMR (101 MHz, DMSO-d6): δ = 138.49, 129.18, 128.38, 126.86, 101.47, 77.86, 68.96,
67.78, 67.15, 65.57, 61.16, 43.49, 31.30, 26.27 ppm. HRMS (ESI): calcd. for C16H22O5Na [M + Na]+
317.1360; found 317.1360.
4.2.3. (4aR,7R,8S,8aS)-7-(3-azidopropyl)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol (5).
Diol 3 (0.216 g, 0.73 mmol, 1.0 eq.) and triethylamine (0.2 mL, 1.1 mmol, 1.5 eq.) were put under
nitrogen and anhydrous dichloromethane (DCM, 1.8 mL) was added. The solution was then cooled to
-20 °C. p-Toluenesulfonyl chloride (TsCl; 0.140 g, 0.73 mmol, 1.0 eq.) was dissolved in anhydrous
DCM (1.8 mL) and was added dropwise, resulting in a final 0.2 M solution. The mixture was allowed
to warm to room temperature and was stirred overnight at room temperature. The reaction was
quenched in cold water. The aqueous layer was extracted with DCM (3x). The combined organic
layers were washed with water and brine and dried over anhydrous MgSO4. After filtration, the solvent
was removed under reduced pressure to yield crude compound 4. HRMS (ESI): calcd. for C23H29O7S
[M + H]+ 449.1628; found 449.1628.
Sodium azide (0.072 g, 1.1 mmol, 1.5 eq.) was placed in a round bottom flask under nitrogen. Crude
tosylate 4 was dissolved in anhydrous N,N-dimethylformamide (DMF, 3.7 mL, 0.2 M) and was added
16

to the sodium azide. The mixture was stirred overnight at 55 °C after which the solution was cooled to
room temperature and water was added. The aqueous layer was extracted with ethyl acetate (3x). The
combined organic layers were washed with water and brine and dried over anhydrous MgSO4. After
filtration, the solvent was removed under reduced pressure. The residue was purified by silica gel
chromatography (heptane:ethyl acetate 50:50) to obtain compound 5 (0.218 g, 93%) with Rf = 0.58
(heptane:ethyl acetate 50:50) as a solid. 1H NMR (400 MHz, CDCl3): δ = 7.50 – 7.46 (m, 2H), 7.40 –
7.35 (m, 3H), 5.63 (s, 1H), 4.30 (dd, J = 10.3, 5.0 Hz, 1H), 4.02 (dd, J = 11.7, 2.6 Hz, 2H), 3.92 (td, J
= 10.1, 4.9 Hz, 1H), 3.74 – 3.67 (m, 2H), 3.62 (d, J = 11.7 Hz, 1H), 3.39 – 3.27 (m, 2H), 2.35 (s, 1H),
1.86 – 1.80 (m, 1H), 1.78 – 1.50 (m, 4H) ppm. 13C NMR (101 MHz, CDCl3): δ = 137.27, 129.26,
128.36, 126.14, 102.06, 77.72, 69.29, 68.85, 67.41, 65.90, 51.36, 41.47, 27.31, 26.78 ppm. HRMS
(ESI): calcd. for C16H21N3O4Na [M + Na]+ 342.1424; found 342.1426.
4.2.4. ((3aS,4R,7R,7aS)-7-(3-azidopropyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-
yl)methanol (7).
Azide 5 (1.1 g, 3.44 mmol, 1.0 eq.) and p-toluenesulfonic acid monohydrate (PTSA; 0.655 g, 3.44
mmol, 1.0 eq.) were put under an atmosphere of nitrogen and were dissolved in a 1:1 mixture of
THF:water (23 mL : 23 mL, 0.075 M). The mixture was stirred for 3 days at 40 °C, after which the
solvents were removed under reduced pressure to give crude compound 6 as a transparent oil. HRMS
(ESI): calcd. for C9H17N3O4Na [M + Na]+ 254.1111; found 254.1111.
The obtained residue and an additional quantity of p-toluenesulfonic acid monohydrate (2.247 g, 11.8
mmol, 3.4 eq.) were dissolved in acetone (140 mL, 0.025 M). 2,2-Dimethoxypropane (DMP; 2.7 mL,
21.8 mmol, 6.3 eq.) was added and the mixture was stirred overnight at room temperature under
nitrogen, after which the reaction was quenched with aqueous saturated sodium bicarbonate. Acetone
was removed under reduced pressure and the remaining aqueous layer was extracted with ethyl acetate
(3x). The combined organic layers were washed with water and brine and dried over anhydrous
MgSO4. Following filtration, the solvent was removed under reduced pressure and the residue was
purified by silica gel chromatography (heptane:ethyl acetate 50:50) to afford compound 7 (0.866 g,
93%) with Rf = 0.37 (heptane:ethyl acetate 50:50) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ =
4.11 (dd, J = 4.9, 2.4 Hz, 1H), 3.90 (dd, J = 9.1, 5.1 Hz, 1H), 3.86 – 3.75 (m, 2H), 3.67 (dd, J = 11.7,
1.3 Hz, 1H), 3.59 (m, 1H), 3.43 – 3.36 (m, 1H), 3.33 (m, 2H), 2.20 – 2.12 (m, 1H), 1.98 (m, 1H), 1.80
– 1.52 (m, 4H), 1.50 (s, 3H), 1.37 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ = 109.26, 78.71, 76.29,
70.56, 65.96, 63.58, 51.63, 36.74, 28.47, 27.77, 27.13, 26.54 ppm. HRMS (ESI): calcd. for
C12H21N3O4Na [M + Na]+ 294.1424; found 294.1423.
4.2.5. ((3aS,4R,7R,7aS)-7-(3-azidopropyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-
yl)methyl sulfamate (8).
A flask was charged with nitrogen and was cooled to 0 °C. Chlorosulfonyl isocyanate (CSI; 0.9 mL,
10.7 mmol, 3.1 eq.) was added and then formic acid (0.4 mL, 10.8 mmol, 3.1 eq.) was added
dropwise. The mixture was stirred for 30 min. at 0 °C. The resulting white solid was dissolved in
anhydrous acetonitrile (10.7 mL, 1.0 M) and the solution was stirred for 5 h at room temperature.
Alcohol 7 (0.935 g, 3.45 mmol, 1.0 eq.) was dissolved in N,N-dimethylacetamide (DMA; 17 mL) and
was added dropwise to sulfamoyl chloride. The mixture was stirred overnight at room temperature,
after which triethylamine (7.2 mL, 52 mmol, 15 eq.) was added and the mixture was stirred for 10
min. at room temperature. Methanol (MeOH, 7.0 mL, 172 mmol, 50 eq.) was then added, resulting in
a transparent solution and the mixture was stirred for another 15 min. at room temperature. The
solvents were removed under reduced pressure, and the residue was dissolved in aqueous saturated
sodium bicarbonate. The aqueous layer was extracted with ethyl acetate (3x). The combined organic
layers were washed with water and brine and dried over anhydrous MgSO4. After filtration, the solvent
was removed under reduced pressure and the residue was purified by silica gel chromatography
(heptane:ethyl acetate 50:50) yielding compound 8 (0.964 g, 80%) with Rf = 0.33 (heptane:ethyl
acetate 50:50) as a white oil. 1H NMR (300 MHz, CDCl3): δ = 5.14 (s, 2H), 4.39 (m, 1H), 4.30 – 4.19
(m, 1H), 4.18 – 4.11 (m, 1H), 3.89 (dd, J = 9.3, 4.7 Hz, 1H), 3.82 – 3.65 (m, 2H), 3.56 (dd, J = 7.6, 5.9
Hz, 1H), 3.32 (t, J = 5.5 Hz, 2H), 1.98 (m, 1H), 1.85 – 1.45 (m, 8H), 1.36 (s, 3H) ppm. 13C NMR (75
17

MHz, CDCl3): δ = 109.71, 76.73, 76.12, 70.95, 69.79, 66.31, 51.58, 36.49, 28.48, 27.73, 27.11, 26.47
ppm. HRMS (ESI): calcd. for C12H23N4O6S [M + H]+ 351.1333; found 351.1338.
4.2.6. ((3aS,4R,7R,7aS)-7-(3-azidopropyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-
yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate (9).
Cesium carbonate (2.69 g, 8.25 mmol, 3.0 eq.) was placed in a round bottom flask charged with
nitrogen and was cooled to 0 °C. Compound 8 (0.964 g, 2.75 mmol, 1.0 eq.) and t-butyloxycarbonyl-
L-leucine hydroxysuccinimide ester (1.807 g, 5.50 mmol, 2.0 eq.) were dissolved in anhydrous DMF
(90 mL, 0.03 M) and were added. The mixture was gradually warmed to room temperature and was
stirred for 3 days at room temperature. The solvent was then removed under reduced pressure and the
residue was suspended in a 9:1 mixture of DCM:MeOH. The suspension was filtered over celite, and
the solvent was removed under reduced pressure. The obtained residue was purified by silica gel
chromatography (DCM:MeOH 95:5 to 90:10) to afford the leucine coupled compound 9 (1.551 g,
100%) with Rf = 0.07 (DCM:MeOH 90:10) as a yellow oil. 1H NMR (300 MHz, CD3OD): δ = 4.39
(dd, J = 10.8, 1.6 Hz, 1H), 4.29 – 4.16 (m, 2H), 4.10 (t, J = 7.4 Hz, 1H), 3.93 (dd, J = 9.0, 5.0 Hz, 1H),
3.70 (m, 2H), 3.63 – 3.53 (m, 1H), 3.37 (t, J = 6.5 Hz, 2H), 2.01 – 1.90 (m, 1H), 1.79 – 1.42 (m, 22H),
1.36 (s, 3H), 1.01 – 0.91 (m, 7H) ppm. 13C NMR (75 MHz, CD3OD): δ = 108.53, 75.69, 75.57, 71.11,
69.18, 65.24, 50.75, 40.29, 35.21, 27.04, 26.84, 26.81, 26.08, 24.78, 24.23, 21.77, 20.19 ppm. HRMS
(ESI): calcd. for C23H40N5O9S [M - H]- 562.2552; found 562.2558.
4.2.7. ((3aS,4R,7R,7aS)-2,2-dimethyl-7-(3-(4-(4-pentylphenyl)-1H-1,2,3-triazol-1-
yl)propyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate
(10a).
Azide 9 (0.150 g, 0.27 mmol, 1.0 eq.), copper(I) iodide (0.005 g, 0.03 mmol, 0.1 eq.) and
triethylamine (0.1 mL, 0.8 mmol, 3.0 eq.) were dissolved in DMF (1.3 mL, 0.2 M) and the solution
was put under an atmosphere of nitrogen. 1-Ethynyl-4-pentylbenzene (0.1 mL, 0.53 mmol, 2.0 eq.)
was added and the mixture was stirred overnight at 50 °C. The solvent was then removed under
reduced pressure and the residue was purified by silica gel chromatography (DCM:MeOH 100:0 to
95:5) to give triazole 10a (0.196 g, 100%) as a yellow oil. HRMS (ESI): calcd. for C36H26N5O9S [M -
H]- 734.3804; found 734.3838.
4.2.8. ((3aS,4R,7R,7aS)-7-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)propyl)-2,2-dimethyltetrahydro-
4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate (10b).
The same procedure as for 10a was followed making use of 0.03 mL of propargylamine. Purification
by silica gel chromatography was performed using DCM:MeOH 95:5 to 90:10 as the eluents affording
triazole 10b (0.082 g, 50%) with Rf = 0.24 (DCM:MeOH 90:10) as a yellow oil. HRMS (ESI): calcd.
for C26H45N6O9S [M - H]- 617.2974; found 617.2955.
4.2.9. ((3aS,4R,7R,7aS)-2,2-dimethyl-7-(3-(4-octyl-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-4H-
[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate (10c).
The same procedure as for 10a was followed making use of 0.1 mL of 1-decyne. Purification by silica
gel chromatography was performed using DCM:MeOH 100:0 to 95:5 as the eluents giving triazole 10c
(0.187 g, 100%) with Rf = 0.35 (DCM:MeOH 90:10) as a yellow oil. HRMS (ESI): calcd. for
C33H58N5O9S [M - H]- 700.3960; found 700.3973.
4.2.10. ((3aS,4R,7R,7aS)-2,2-dimethyl-7-(3-(4-phenethyl-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-4H-
[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate (10d).
The same procedure as for 10a was followed making use of 0.08 mL of 3-butyn-1-ylbenzene.
Purification by silica gel chromatography was performed using DCM:MeOH 100:0 to 95:5 as the
eluents yielding triazole 10d (0.185 g, 100%) with Rf = 0.43 (DCM:MeOH 90:10) as a yellow oil.
HRMS (ESI): calcd. for C33H50N5O9S [M - H]- 692.3334; found 692.3337.
18

4.2.11. ((3aS,4R,7R,7aS)-7-(3-(4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-yl)propyl)-2,2-
dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate
(10e).
The procedure as for 10a was followed making use of 0.06 mL of 5-hexyn-1-ol. Purification by silica
gel chromatography was performed using DCM:MeOH 100:0 to 90:10 as the eluents affording triazole
10e (0.176 g, 100%) with Rf = 0.18 (DCM:MeOH 90:10) as a yellow oil. HRMS (ESI): calcd. for
C29H50N5O10S [M - H]- 660.3284; found 660.3297.
4.2.12. ((3aS,4R,7R,7aS)-2,2-dimethyl-7-(3-(4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-
yl)propyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate
(10f).
The procedure as for 10a was followed making use of 0.08 mL of 1-ethynyl-4-
(trifluoromethoxy)benzene. Purification by silica gel chromatography was performed using
DCM:MeOH 100:0 to 95:5 as the eluents giving triazole 10f (0.200 g, 100%) with Rf = 0.53
(DCM:MeOH 90:10) as a yellow oil. HRMS (ESI): calcd. for C32H45N5O10SF3 [M - H]- 748.2844;
found 748.2850.
4.2.13. ((3aS,4R,7R,7aS)-2,2-dimethyl-7-(3-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-
4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate (10g).
The procedure as for 10a was followed making use of 0.055 g of 3-ethynylpyridine. Purification by
silica gel chromatography was performed using DCM:MeOH 100:0 to 95:5 as the eluents yielding
triazole 10g (0.139 g, 78%) with Rf = 0.23 (DCM:MeOH 90:10) as a yellow solid. HRMS (ESI):
calcd. for C30H45N6O9S [M - H]- 665.2974; found 665.2994.
4.2.14. ((3aS,4R,7R,7aS)-7-(3-(4-((benzylamino)methyl)-1H-1,2,3-triazol-1-yl)propyl)-2,2-
dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate
(10h).
The procedure as for 10a was followed making use of 0.144 g of N-benzylprop-2-yn-1-amine (0.992
mmol, 3.7 eq.). Purification by silica gel chromatography was performed using DCM:MeOH 100:0 to
95:5 as the eluents affording triazole 10h (0.189 g, 100%) with Rf = 0.42 (DCM:MeOH 90:10) as a
brown oil. HRMS (ESI): calcd. for C33H51N6O9S [M - H]- 707.3443; found 707.3458.
4.2.15. ((3aS,4R,7R,7aS)-7-(3-(4-(benzamidomethyl)-1H-1,2,3-triazol-1-yl)propyl)-2,2-
dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate
(10i).
The same procedure as for 10a was followed making use of 0.085 g of N-(2-propyn-1-yl)benzamide.
Purification by silica gel chromatography was performed using DCM:MeOH 100:0 to 95:5 as the
eluents giving triazole 10i (0.192 g, 100%) as a yellow oil. HRMS (ESI): calcd. for C33H49N6O10S [M -
H]- 721.3236; found 721.3248.
4.2.16. ((3aS,4R,7R,7aS)-7-(3-(4-((3-benzylureido)methyl)-1H-1,2,3-triazol-1-yl)propyl)-2,2-
dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate
(10j).
The same procedure as for 10a was followed making use of 0.100 g of 1-benzyl-3-(prop-2-yn-1-
yl)urea. Purification by silica gel chromatography was performed using DCM:MeOH 95:5 to 90:10 as
the eluents yielding triazole 10j (0.142 g, 71%) as a yellow oil. HRMS (ESI): calcd. for C34H52N7O10S
[M - H]- 750.3502; found 750.3521.
4.2.17. ((3aS,4R,7R,7aS)-2,2-dimethyl-7-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-4H-
[1,3]dioxolo[4,5-c]pyran-4-yl)methyl ((tert-butoxycarbonyl)leucyl)sulfamate (10k).
The same procedure as for 10a was followed making use of 0.1 mL of phenylacetylene. Purification
by silica gel chromatography was performed using DCM:MeOH 95:5 to 90:10 as the eluents affording
19

triazole 10k (0.098 g, 55%) as a yellow solid. HRMS (ESI): calcd. for C31H46N5O9S [M - H]-
664.3021; found 664.3011.
4.2.18. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-(4-pentylphenyl)-1H-1,2,3-triazol-1-
yl)propyl)tetrahydro-2H-pyran-2-yl)methyl leucylsulfamate (11a).
Triazole 10a (0.196 g, 0.27 mmol, 1.0 eq.) was placed under nitrogen and an 1:1 mixture of
trifluoroacetic acid (TFA):water (0.5 mL:0.5 mL) was added. The mixture was stirred for 5 h at room
temperature, after which more water was added and the reaction mixture was lyophilized. The residue
was purified by silica gel chromatography (DCM:MeOH 95:5 to 90:10) to provide the desired final
compound 11a (0.159 g, 83%) with Rf = 0.11 (DCM:MeOH 90:10) as a white solid. 1H NMR (300
MHz, CD3OD): δ = 8.32 (s, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 4.46 (t, J = 6.9 Hz,
2H), 4.37 (d, J = 9.5 Hz, 1H), 4.24 (dd, J = 10.8, 5.5 Hz, 1H), 3.93 – 3.84 (m, 2H), 3.83 – 3.70 (m,
2H), 3.62 (dd, J = 9.9, 3.0 Hz, 1H), 3.56 (d, J = 11.4 Hz, 1H), 2.62 (t, J = 7.6 Hz, 2H), 2.13 – 1.93 (m,
2H), 1.92 – 1.47 (m, 7H), 1.47 – 1.20 (m, 5H), 0.98 (t, J = 6.0 Hz, 6H), 0.90 (t, J = 6.8 Hz, 3H) ppm.
13C NMR (75 MHz, CD3OD): δ = 173.91, 147.26, 142.69, 128.28, 127.42, 125.00, 120.34, 74.44,
69.87, 69.33, 64.26, 64.03, 53.65, 49.65, 41.46, 40.06, 34.91, 30.84, 30.50, 27.69, 25.10, 23.87, 21.81,
21.50, 20.46, 12.66 ppm. HRMS (ESI): calcd. for C28H44N5O7S [M - H]- 594.2967; found 594.2972.
4.2.19. ((2R,3S,4S,5R)-5-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)propyl)-3,4-dihydroxytetrahydro-
2H-pyran-2-yl)methyl leucylsulfamate (11b).
In analogy, triazole 10b (0.082 g, 0.13 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH:triethylamine 90:10:0 to 80:20:0 to 70:30:1 as the eluents
affording final compound 11b (0.038 g, 60%) as a yellow solid. 1H NMR (300 MHz, D2O): δ = 7.90 –
7.76 (m, 1H), 4.34 (t, J = 6.5 Hz, 2H), 4.21 – 4.04 (m, 2H), 3.93 (m, 1H), 3.84 – 3.62 (m, 4H), 3.56
(m, 1H), 3.46 (m, 2H), 3.22 (s, 2H), 1.82 (m, 2H), 1.67 (m, 1H), 1.41 (m, 5H), 0.82 (t, J = 5.2 Hz, 6H)
ppm. 13C NMR (75 MHz, D2O): δ = 123.57, 74.00, 69.76, 68.69, 64.33, 64.06, 54.55, 50.10, 41.69,
40.44, 34.90, 27.04, 24.72, 23.98, 21.88, 21.03 ppm. HRMS (ESI): calcd. for C18H35N6O7S [M + H]+
479.2282; found 479.2279.
4.2.20. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-octyl-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-2H-pyran-
2-yl)methyl leucylsulfamate (11c).
In analogy, triazole 10c (0.187 g, 0.27 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 95:5 to 90:10 as the eluents yielding final compound 11c (0.131
g, 88%) with Rf = 0.07 (DCM:MeOH 90:10) as a colourless solid. 1H NMR (300 MHz, CD3OD): δ =
7.80 (s, 1H), 4.39 (dd + t, 3H), 4.24 (dd, J = 10.7, 5.3 Hz, 1H), 3.95 – 3.83 (m, 2H), 3.82 – 3.70 (m,
2H), 3.65 – 3.49 (m, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.11 – 1.48 (m, 9H), 1.40 – 1.24 (m, 11H), 1.00 (t, J
= 6.0 Hz, 6H), 0.90 (t, J = 6.7 Hz, 3H) ppm. 13C NMR (75 MHz, CD3OD): δ = 173.72, 147.57, 121.52,
74.44, 69.85, 69.33, 64.23, 64.00, 53.65, 49.45, 41.45, 40.07, 31.24, 28.84, 28.65, 28.57, 28.52, 27.67,
25.07, 24.58, 23.89, 21.93, 21.50, 20.47, 12.69 ppm. HRMS (ESI): calcd. for C25H46N5O7S [M - H]-
560.3123; found 560.3115.
4.2.21. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-phenethyl-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-2H-
pyran-2-yl)methyl leucylsulfamate (11d).
In analogy, triazole 10d (0.185 g, 0.27 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 95:5 to 90:10 as the eluents resulting in final compound 11d
1
(0.122 g, 83%) with Rf = 0.05 (DCM:MeOH 90:10) as a colourless solid. H NMR (300 MHz,
CD3OD): δ = 7.65 (s, 1H), 7.31 – 7.08 (m, 5H), 4.42 – 4.17 (m, 4H), 3.92 – 3.68 (m, 4H), 3.64 – 3.47
(m, 2H), 3.05 – 2.88 (m, 4H), 2.04 – 1.63 (m, 6H), 1.51 (m, 1H), 1.34 (m, 1H), 1.06 – 0.89 (m, 6H)
ppm. 13C NMR (75 MHz, CD3OD): δ = 173.51, 146.63, 140.62, 127.81, 127.71, 125.46, 121.85, 74.42,
69.84, 69.45, 64.21, 63.97, 53.56, 49.41, 41.46, 40.05, 34.83, 27.64, 26.56, 24.97, 23.88, 21.52, 20.45
ppm. HRMS (ESI): calcd. for C25H38N5O7S [M - H]- 552.2497; found 552.2503.
20

4.2.22. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-
yl)propyl)tetrahydro-2H-pyran-2-yl)methyl leucylsulfamate (11e).
In analogy, triazole 10e (0.176 g, 0.27 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 90:10 to 80:20 as the eluents giving final compound 11e (0.076 g,
55%) with Rf = 0.04 (DCM:MeOH 80:20) as a colourless solid. 1H NMR (300 MHz, CD3OD): δ =
7.82 (s, 1H), 4.41 (t, J = 6.9 Hz, 2H), 4.32 (d, J = 9.9 Hz, 1H), 4.20 (dd, J = 10.7, 5.4 Hz, 1H), 3.88
(m, 2H), 3.76 (dd, J = 9.1, 4.3 Hz, 1H), 3.68 (dd, J = 8.1, 5.0 Hz, 1H), 3.58 (m, 4H), 2.74 (t, J = 7.4
Hz, 2H), 2.11 – 1.89 (m, 2H), 1.89 – 1.51 (m, 9H), 1.44 – 1.29 (m, 1H), 1.01 (t, J = 6.1 Hz, 6H) ppm.
13C NMR (75 MHz, CD3OD): δ = 174.27, 121.56, 74.51, 69.87, 68.83, 64.24, 63.98, 60.87, 53.83,
49.43, 41.46, 40.17, 31.31, 27.65, 25.14, 25.06, 24.33, 23.91, 21.45, 20.49 ppm. HRMS (ESI): calcd.
for C21H38N5O8S [M - H]- 520.2446; found 520.2448.
4.2.23. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-
yl)propyl)tetrahydro-2H-pyran-2-yl)methyl leucylsulfamate (11f).
In analogy, triazole 10f (0.200 g, 0.27 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 95:5 to 90:10 as the eluents providing final compound 11f (0.145
g, 89%) with Rf = 0.05 (DCM:MeOH 90:10) as a white solid. 1H NMR (300 MHz, CD3OD): δ = 8.45
(s, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 4.49 (t, J = 6.9 Hz, 2H), 4.37 (dd, J = 10.7,
1.3 Hz, 1H), 4.25 (dd, J = 10.8, 5.5 Hz, 1H), 3.94 – 3.84 (m, 2H), 3.83 – 3.71 (m, 2H), 3.66 – 3.53 (m,
2H), 2.16 – 1.93 (m, 2H), 1.88 – 1.74 (m, J = 11.2, 6.3 Hz, 3H), 1.74 – 1.55 (m, J = 14.2, 13.7, 7.1 Hz,
2H), 1.51 – 1.37 (m, J = 13.5, 7.8 Hz, 1H), 0.98 (t, J = 5.9 Hz, 6H) ppm. 13C NMR (75 MHz, CD3OD):
δ = 173.79, 148.50, 145.79, 129.30, 126.66, 121.89, 121.10, 120.78, 118.50, 74.46, 69.87, 69.36,
64.28, 64.01, 53.66, 49.74, 41.47, 40.06, 27.65, 25.10, 23.87, 21.46, 20.45 ppm. HRMS (ESI): calcd.
for C24H33N5O8SF3 [M - H]- 608.2007; found 608.2017.
4.2.24. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-
2H-pyran-2-yl)methyl leucylsulfamate (11g).
In analogy, triazole 10g (0.139 g, 0.21 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 90:10 to 80:20 as the eluents yielding final compound 11g (0.089
g, 81%) as a colourless solid. 1H NMR (300 MHz, CD3OD): δ = 8.61 (s, 1H), 8.37 (d, J = 7.6 Hz, 1H),
7.61 (s, 1H), 4.52 (t, J = 6.6 Hz, 2H), 4.34 (dd, J = 10.4 Hz, 1H), 4.21 (dd, J = 10.1, 5.0 Hz, 1H), 3.95
– 3.83 (m, 2H), 3.83 – 3.66 (m, 2H), 3.66 – 3.50 (m, 2H), 2.17 – 1.94 (m, 2H), 1.90 – 1.73 (m, 3H),
1.71 – 1.53 (m, 2H), 1.51 – 1.36 (m, 1H), 0.98 (t, J = 6.0 Hz, 6H) ppm. 13C NMR (75 MHz, CD3OD):
δ = 174.44, 147.13, 145.01, 143.45, 133.93, 121.76, 74.53, 69.86, 68.89, 64.29, 63.98, 53.86, 49.84,
41.48, 40.17, 27.62, 25.08, 23.91, 21.46, 20.51 ppm. HRMS (ESI): calcd. for C22H33N6O7S [M - H]-
525.2137; found 525.2139.
4.2.25. ((2R,3S,4S,5R)-5-(3-(4-((benzylamino)methyl)-1H-1,2,3-triazol-1-yl)propyl)-3,4-
dihydroxytetrahydro-2H-pyran-2-yl)methyl leucylsulfamate (11h).
In analogy, triazole 10h (0.189 g, 0.27 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 95:5 to 85:15 to 75:25 as the eluents affording final compound
1
11h (0.076 g, 50%) with Rf = 0.04 (DCM:MeOH 90:10) as a yellow solid. H NMR (300 MHz,
CD3OD): δ = 8.23 (s, 1H), 7.62 – 7.26 (m, 6H), 4.56 – 4.23 (m, 8H), 3.86 (m, 2H), 3.68 (m, 2H), 3.62
– 3.47 (m, 2H), 2.08 – 1.93 (m, 2H), 1.89 – 1.59 (m, 4H), 1.51 – 1.31 (m, 2H), 1.05 – 0.94 (m, 6H)
ppm. 13C NMR (75 MHz, CD3OD): δ = 174.43, 130.56, 129.58, 128.97, 128.56, 74.38, 69.64, 68.64,
63.82, 63.74, 53.67, 50.18, 49.87, 41.68, 40.72, 40.13, 27.29, 24.66, 23.91, 21.46, 20.42 ppm. HRMS
(ESI): calcd. for C25H39N6O7S [M - H]- 567.2606; found 567.2611.
4.2.26. ((2R,3S,4S,5R)-5-(3-(4-(benzamidomethyl)-1H-1,2,3-triazol-1-yl)propyl)-3,4-
dihydroxytetrahydro-2H-pyran-2-yl)methyl leucylsulfamate (11i).
In analogy, triazole 10i (0.192 g, 0.27 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 95:5 to 90:10 to 85:15 as the eluents giving final compound 11i
21

1
(0.095 g, 61%) with Rf = 0.01 (DCM:MeOH 90:10) as a colourless solid. H NMR (300 MHz,
CD3OD): δ = 8.99 (s, 1H), 8.22 – 7.75 (m, 3H), 7.59 – 7.39 (m, 3H), 4.79 – 4.09 (m, 6H), 3.95 – 3.80
(m, 2H), 3.79 – 3.65 (m, 2H), 3.63 – 3.46 (m, 2H), 2.09 – 1.88 (m, 2H), 1.88 – 1.60 (m, 4H), 1.59 –
1.26 (m, 2H), 0.99 (t, J = 5.6 Hz, 6H) ppm. 13C NMR (75 MHz, CD3OD): δ = 174.15, 131.11, 127.87,
126.74, 74.43, 69.78, 69.04, 64.14, 63.96, 53.76, 49.67, 41.46, 40.12, 34.54, 27.56, 25.00, 23.89,
21.47, 20.45 ppm. HRMS (ESI): calcd. for C25H37N6O8S [M - H]- 581.2399; found 581.2404.
4.2.27. ((2R,3S,4S,5R)-5-(3-(4-((3-benzylureido)methyl)-1H-1,2,3-triazol-1-yl)propyl)-3,4-
dihydroxytetrahydro-2H-pyran-2-yl)methyl leucylsulfamate (11j).
In analogy, triazole 10j (0.142 g, 0.19 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 90:10 to 85:15 as the eluents providing final compound 11j
1
(0.095 g, 82%) with Rf = 0.01 (DCM:MeOH 90:10) as a colourless solid. H NMR (300 MHz,
CD3OD): δ = 7.91 (s, 1H), 7.42 – 7.14 (m, 5H), 4.52 – 4.16 (m, 8H), 3.93 – 3.80 (m, 2H), 3.80 – 3.65
(m, 2H), 3.64 – 3.46 (m, 2H), 2.08 – 1.85 (m, 2H), 1.73 (m, 4H), 1.58 – 1.43 (m, 1H), 1.35 (s, 1H),
0.99 (t, J = 5.9 Hz, 6H) ppm. 13C NMR (75 MHz, CD3OD): δ = 173.96, 139.56, 127.75, 126.51,
126.26, 74.37, 69.75, 69.11, 64.06, 63.97, 53.69, 49.66, 43.08, 41.52, 40.09, 34.83, 27.54, 24.99,
23.88, 21.49, 20.46 ppm. HRMS (ESI): calcd. for C26H40N7O8S [M - H]- 610.2664; found 610.2672.
4.2.28. ((2R,3S,4S,5R)-3,4-dihydroxy-5-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)propyl)tetrahydro-2H-
pyran-2-yl)methyl leucylsulfamate (11k).
In analogy, triazole 10k (0.098 g, 0.15 mmol, 1.0 eq.) was deprotected and purified by silica gel
chromatography using DCM:MeOH 90:10 to 85:15 as the eluents providing final compound 11k
(0.077 g, 100%) with Rf = 0.10 (DCM:MeOH 90:10) as a colourless solid.1H NMR (300 MHz,
CD3OD): δ = 8.38 (s, 1H), 7.87 – 7.79 (m, 2H), 7.50 – 7.39 (m, 2H), 7.39 – 7.30 (m, 1H), 4.48 (t, J =
7.1 Hz, 2H), 4.34 (dd, J = 10.9, 1.9 Hz, 1H), 4.21 (dd, J = 10.9, 5.5 Hz, 1H), 3.93 – 3.85 (m, 2H), 3.82
– 3.74 (m, 1H), 3.70 – 3.53 (m, 3H), 3.37 (s, 1H), 2.13 – 1.93 (m, 2H), 1.88 – 1.74 (m, 3H), 1.70 –
1.54 (m, 2H), 1.50 – 1.38 (m, 1H), 1.29 (s, 1H), 0.99 (t, J = 6.6 Hz, 6H) ppm. 13C NMR (75 MHz,
CD3OD): δ = 174.84, 147.18, 130.06, 128.23, 127.58, 125.00, 120.59, 74.54, 69.92, 68.88, 64.33,
64.00, 53.94, 49.66, 41.44, 40.28, 27.66, 25.13, 23.92, 21.43, 20.48 ppm. HRMS (ESI): calcd. for
C23H35N5O7S [M - H]- 524.2184; found 524.2194.
4.2.29. N-benzylprop-2-yn-1-amine (12).
An oven-dried flask was put under an atmosphere of nitrogen and was loaded with benzaldehyde (0.2
mL, 2.4 mmol, 1.0 eq.), which was dissolved in anhydrous MeOH (9.4 mL, 0.25 M). Propargylamine
(0.2 mL, 2.5 mmol, 1.1 eq.) and acetic acid (0.1 mL) were subsequently added and the reaction
mixture was stirred overnight at room temperature. The solution was cooled to 0 °C and sodium
borohydride (0.267 g, 7.1 mmol, 3.0 eq.) was carefully added in portions. The mixture was stirred for
2 h at room temperature, whereafter the reaction was quenched with aqueous sodium hydroxide (1.0
M) at 0 °C. The aqueous layer was extracted with ethyl acetate (3x). The combined organic layers
were washed with water and brine and dried over anhydrous MgSO4. Following filtration, the solvent
was removed under reduced pressure and the residue was purified by silica gel chromatography
(heptane:ethyl acetate 25:75 to 0:100) yielding compound 12 (0.144 g, 42%) with Rf = 0.66 (ethyl
acetate) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ = 7.28 (m, 8H), 3.86 – 3.76 (m, 2H), 3.38 (m,
2H), 2.24 (m, 1H), 1.80 (br s, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ = 139.70, 128.77, 127.50,
82.38, 71.92, 52.58, 37.64 ppm.
4.2.30. N-(prop-2-yn-1-yl)benzamide (13).
An oven-dried flask was put under an atmosphere of nitrogen and was loaded with benzoyl chloride
(0.5 mL, 4.5 mmol, 1.25 eq.), which was dissolved in anhydrous DCM (10 mL). Triethylamine (1.0
mL, 7.3 mmol, 2.0 eq.) and propargylamine (0.2 mL, 3.6 mmol, 1.0 eq.), dissolved in anhydrous DCM
(5.0 mL), were subsequently added, resulting in a final 0.25 M solution, and the reaction mixture was
stirred overnight at room temperature. After completion, water was added and the aqueous layer was
22

extracted with DCM (3x). The combined organic layers were washed with water and brine and dried
over anhydrous MgSO4. After filtration, the solvent was removed under reduced pressure and the
residue was purified by silica gel chromatography (heptane:ethyl acetate 75:25 → 50:50) resulting in
compound 13 (0.553 g, 96%) with Rf = 0.52 (heptane:ethyl acetate 50:50) as a white solid. 1H NMR
(300 MHz, CDCl3): δ = 7.87 – 7.70 (m, 2H), 7.57 – 7.37 (m, 3H), 6.33 (s, 1H), 4.33 – 4.18 (m, 2H),
2.32 – 2.24 (m, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ = 132.07, 128.93, 127.29, 72.19, 30.09 ppm.
HRMS (ESI): calcd. for C10H10NO [M + H]+ 160.0757; found 160.0752.
4.2.31. 1-benzyl-3-(prop-2-yn-1-yl)urea (14).
An oven-dried flask was put under an atmosphere of nitrogen and was loaded with benzyl isocyanate
(0.2 mL, 1.6 mmol, 1.0 eq.), which was dissolved in anhydrous DCM (3.3 mL). Propargylamine (0.1
mL, 1.8 mmol, 1.1 eq.), dissolved in anhydrous DCM (3.3 mL), was added dropwise at 0 °C, resulting
in a final 0.25 M solution, and the reaction mixture was stirred for 3 h at room temperature after
slowly warming the solution to ambient temperature. A white solid 14 (0.305 g, 100%) was formed,
which was filtered and dried under vacuum. 1H NMR (300 MHz, DMSO-d6): δ = 7.38 – 7.16 (m, 5H),
6.49 (t, J = 5.8 Hz, 1H), 6.28 (t, J = 5.6 Hz, 1H), 4.21 (d, J = 6.0 Hz, 2H), 3.81 (dd, J = 5.8, 2.4 Hz,
2H), 3.07 (t, J = 2.4 Hz, 1H) ppm. 13C NMR (75 MHz, DMSO-d6): δ = 157.65, 140.80, 128.33,
127.17, 126.71, 82.64, 72.62, 43.12, 29.05 ppm. HRMS (ESI): calcd. for C11H13N2O [M + H]+
189.1022; found 189.1023.
4.2.32. 2-deoxy-4,5-dihydroxy-1-[5-phenyltetrazole]-D-allo-heptitol-7-N-(leucyl)-sulfamate (15).
1H NMR (300 MHz, CD3OD): δ = 8.08-8.16 (m, 2H), 7.48-7.57 (m, 3H), 4.81-4.88 (m, 2H), 4.11-4.24
(m, 3H), 3.99-4.07 (m, 1H), 3.77-3.89 (m, 2H), 3.60-3.68 (m, 1H), 2.36-2.53 (m, 1H), 2.18-2.32 (m,
1H), 1.74-1.87 (m, 2H), 1.58-1.69 (m, 1H), 0.96-1.03 (m, 6H) ppm. 13C NMR (75 MHz, CD3OD): δ =
129.75, 128.31, 126.98, 126.02, 81.79, 79.04, 74.27, 71.31, 68.13, 53.87, 49.61, 40.32, 32.38, 23.96,
21.42, 20.42 ppm. HRMS (ESI): calcd. for C20H29N6O7S [M-H]- 497.1824; found: 497.1815.
4.3. Cloning, expression and purification of E. coli and N. gonorrhoeae LeuRSs
Cloning, expression and purification of LeuRS from E. coli were performed as previously
described [31]. The encoding sequence of N. gonorrhoeae LeuRS was amplified by a one-step PCR
from the genomic DNA of bacterial strain Neisseria gonorrhoeae ATCC 49226. This reaction was
carried out by using the high-fidelity Q5 DNA polymerase (New England Biolabs) with forward
primer 5’-gcgaacagattggtggtggtATGCAAGAACATTACCAGCCCG-3’ and reverse primer 5’-
ttgttagcagaagcttaTTAGACGACGATGTTCACCAGT-3’, where the small letters represent the adaptor
complementary to the plasmid, while the capital letters represent the annealing sequences and the
underlined parts correspond to a glycine residue which was introduced before the start codon of
LeuRS to facilitate the cleavage of the SUMO tag during purification. The PCR product was
visualized and separated in agarose gel, followed by gel extraction and in-fusion cloning with
linearized pETRUK vector containing a pI enhanced SUMO tag as described [31]. The assembled
plasmid pETRUK-Ng-LeuRS was first propagated in E. coli NEB5α and then confirmed by DNA
sequencing (LGC genomics).
Ng-LeuRS construct was transformed into E. coli Rosetta (DE3) pLysS expression host. The
selected colony was inoculated into 2 mL LB medium containing 100 μg/mL ampicillin and 30 μg/mL
chloramphenicol and incubated at 37 with 250 rpm for 8 h. Then the pre-culture was transferred into
2 L ZYP-5052 auto-induction medium [46] supplemented with a final concentration of 1 mM ZnSO4
(Sigma Aldrich) and 1 mL of antifoam SE-15 (Sigma Aldrich). The culture was grown overnight at 24
°C and when the OD600nm reached 4.0, temperature was decreased to 18 °C. After another 24 h of
growth, cells were harvested by centrifugation at 4 °C and cell pellets were resuspended in cation
exchange buffer A (CEXA) containing 25 mM Hepes pH 8, 200 mM NaCl, 5 mM β-mercaptoethanol
(β-ME) and frozen in -80 °C.
For protein purification of Ng-LeuRS, cell pellet was thawed and further diluted using CEXA in a
final 8:1 v/w (CEXA: weight of pellet) ratio supplemented with 100 U cryonase (Takara) and 10 mM
23

MgCl2. Cells were lysed by sonication on ice and then clarified by centrifugation at 18,000 g at 4 °C
for 30 min. The supernatant was loaded onto 5 mL Hitrap SP HP column (GE Healthcare Life
Sciences). SUMO tag fused LeuRS protein bound onto the column and was eluted by applying a linear
gradient 0-50% cation exchange buffer B (CEXB) comprising of 25 mM Hepes pH 8, 1000 mM NaCl,
5 mM β-ME. Corresponding protein fractions were collected and SUMO hydrolase was added in a 1:
250 (m/m) ratio to remove SUMO tag. Due to the presence of additional glycine in front of the target
protein LeuRS, the SUMO tag was more flexible and could be easily cleaved in 10 min on ice. The
cleaved protein was dialyzed against 1 L buffer (20 mM Tris pH 7, 10% w/v glycerol and 5 mM β-
ME) overnight at 4 °C and further dialyzed in the same fresh buffer for another 2 h to remove all salts.
Then the protein was loaded onto HiTrap SP HP column to remove the SUMO tag and contaminants,
followed by applying on anion exchange Hitrap Q HP column (GE Healthcare Life Sciences). After
collecting the fractions from anion exchange chromatography, the protein was concentrated to 30
mg/mL using a concentrator (10 KDa, 50 mL, Millipore) by centrifugation at 4 °C, 4000 rpm and then
flash frozen in liquid nitrogen and stored in -80 °C.
4.4. In vitro inhibitory activity determination with purified E. coli LeuRS
The inhibitory effects of synthesized compounds 11a-11k and 15 were evaluated by a
radiolabeled aminoacyl transfer assay as described in our prior work [31,32]. Briefly, 2.5 mM E. coli
LeuRS in 20 mM Tris pH 7.5, 100 mM KCl, 10 mM MgCl2, 5 mM β-ME and 5% (v/v) DMSO was
preincubated with serial dilutions of different compounds at 37 °C for 10 min in the presence of 50
μM 14C-labeled leucine, 2 mg/mL E. coli total tRNA pool (Roche), and 0.25 mg/mL inorganic
pyrophosphatase. A final concentration of 500 μM ATP was added to the mixture to start the reaction.
After 4 min, the reaction was quenched by adding 4 μL buffer containing 0.2 M sodium acetate pH4,
0.1% (w/v) N-lauroylsarcosine and 5 mM unlabeled leucine. 20 μL aliquot was spotted on a 3MM
Whatmann paper and precipitated utilizing 10% (w/v) cold trichloroacetic acid (TCA). All papers
were washed twice with 10% (w/v) TCA and once with acetone at 10-minute intervals and dried in air.
Each paper was transferred into a vial and soaked into 12 mL scintillation liquid. Subsequent
measurements were performed by a scintillation counter. The apparent inhibition constants (Kꢁꢀ ꢂꢂ) of
all LeuRS inhibitors were determined by applying Greco-Hakala equation [47].
4.5. Crystallization and X-ray data collection for N. gonorrhoeae LeuRS
The crystallization procedure of N. gonorrhoeae LeuRS was carried out using hanging drop
vapor diffusion method as previously described [32]. A solution of 10 mg/mL Ng-LeuRS in
crystallization buffer (10 mM Tris pH 7, 100 mM NaCl, 2.5 mM β-ME) was mixed with reservoir
solution (20% w/v PEG 3350, 100 mM bis-tris propane pH 8.5 and 100 mM MgCl2) and crystal seeds
from earlier optimizations in a 0.75:1.0:0.25 (v/v/v) ratio. Suitable crystals were soaked with 2 mM of
each compound prepared in an equivalent reservoir solution supplemented with 22% (v/v) ethylene
glycol for 2 h. These crystals were subsequently mounted in cryo-loops and flash frozen in liquid
nitrogen. Diffraction data were collected at 100 K at Synchrotron ESRF beamline Massif-3 (Grenoble,
France) and Synchrotron Soleil beamline Proxima 1 and 2 (Paris, France).
4.6. Structure determination of N. gonorrhoeae LeuRS in complex with synthesized inhibitors
Collected datasets were processed using autoPROC processing pipeline [48]. The initial phase of
Ng-LeuRS complexed with corresponding inhibitor was solved by molecular replacement in Phenix
[49] using our published structure of Ng-LeuRS in complex with LeuSA (PDB code: 6Q89) as the
start searching model by removing all the solvent and ligand LeuSA [32]. The resulting model was
refined in Phenix.refinement and manually built in COOT [50]. All ligand restraints were generated by
Grade Web Server (http://grade.globalphasing.org). The crystallographic data collection and
corresponding refinement statistics are shown in Supplementary table 1. LeuRS-inhibitor interactions
were analyzed in Schrödiger Maestro Software.
All the structures were deposited in PDB and the related accession codes are as follows: 6YKK,
6YKL, 6YKN, 6YKO, 6YKQ, 6YKS, 6YKT, 6YKU, 6YKV, 6YKW, 6YKX, 7A0P.
24

4.7. Antimicrobial screening
Bacteria are cultured in MHB (Mueller Hinton Broth) and maintained on TSA (Tryptone Soy
Agar). All cultures and assays are conducted at 37°C. Mycobacterium tuberculosis is cultured in 7H9
+ 10% OADC. Compound stock solutions are prepared in 100% DMSO at 20 mM or mg/mL. The
compounds are serially pre-diluted (2-fold or 4-fold) in DMSO followed by a further (intermediate)
dilution in demineralized water to assure a final in-test DMSO concentration of <1%. Assays are
performed in sterile 96-well microtiter plates, each well containing 10 µL of the watery compound
dilutions together with 190 µL of bacteria inoculum (5x103 CFU/ml). Bacterial growth is compared to
untreated-control wells (100% cell growth) and medium-control wells (0% cell growth). After 17 h
incubation, bacterial viability is assessed fluorimetrically after addition of 20 µL resazurin per well.
After 1/2 h at 37 °C, fluorescence is measured (λex 550 nm, λem 590 nm). The results are expressed as
% reduction in bacterial growth/viability compared to control wells and IC50 (50% inhibitory
concentration) is determined. M. tuberculosis H37Ra ATCC 2517, S. aureus ATCC6538 and E. coli
ATCC8739 are used. The compounds are tested at 5 concentrations (64 - 16 - 4 - 1 and 0.25 µM or
mg/ml). The compound is classified inactive when the IC50 is higher than 15 µM (or µg/ml). Between
15 and 5 µM (or µg/ml), the compound is regarded as weakly active. Only when the IC50 is lower than
5 µM (or µg/ml), the compound is classified as active and is evaluated further. Standard bacterial
reference compounds include doxycyclin (IC50 S. aureus 0.11 ± 0.06 µM; E.coli 0.73 ± 0.38 µM) and
the first line anti-TB drug isoniazid (0.1 ± 0.07 µM) and moxifloxacine (0.25 ± 0.13 µM). The IC50 is
determined using an extended dose range (2-fold compound dilutions) still with a highest
concentration of 128 µM. The IC50 against C. albicans was determined by a 4-fold serial dilution of
the test compounds in RPMI with a 1% DMSO background. The concentration used for the test
compounds ranged from 64.0 µM to 3.9 x 10-3µM. As a positive control substance, miconazole was
selected at 0.25 µM.
4.8. Electrostatic surface potential calculation
Electrostatic surface potential of Ng-LeuRS was calculated using APBS electrostatic tool in
Pymol. The protein molecule was prepared by adding hydrogens and missing side chain atoms,
assigning partial charges and radii using the default setting in the program pdb2pqr. The linearized
Poisson-Boltzmann equation was applied. Parameters of the calculation were: protein and water
dielectric constants, 2 and 78, respectively; 150 mM ionic strength; size of the 3D grid, 257×225×
225. Finally, the result was displayed together with the protein structure, color-coded according to the
electrostatic potential (±5 kT/e). Negatively charged regions are presented in red, neutral regions in
white and positively charged regions in blue.
4.9. Bioinformatic analysis of LeuRS protein sequences
Sequence alignment of N. gonorrhoeae LeuRS (Uniprot B4RNT1), E. coli LeuRS (Uniprot
P07813), S. aureus LeuRS (Uniprot Q2FXH2), M. tuberculosis LeuRS (Uniprot P9WFV1) and C.
albicans cytosolic LeuRS (Uniprot A0A1D8PS12) and mitochrondrial LeuRS2 (Uniprot
A0A1D8PQ56) were performed in Jalview [51] by using Muscle web server.
Funding
This work was supported by the Research Fund Flanders [Fonds voor Wetenschappelijk Onderzoek,
1109117N to D.D.R., 12N5918N to D.C., G077814N to S.S. and A.V., G0A4616N to S.W. and
A.V.]; the KU Leuven Research Fund [3M14022 to S.W. and A.V.]; and the Chinese Scholarship
Council [to L.P.]. Mass spectrometry was made possible by the support of the Hercules Foundation of
the Flemish Government [20100225-7].
Conflicts of interest
25

No conflicts of interest are declared.
Acknowledgement
The authors thank the Belgian FWO for providing a PhD Fellowship of the Research Foundation -
Flanders to Dries De Ruysscher and a postdoctoral mandate to Davie Cappoen, and the China
Scholarship Council for providing a scholarship to Luping Pang and KU Leuven and FWO-Flanders
for financial support.
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