Structure-Guided Enhancement of Selectivity of Chemical Probe Inhibitors Targeting Bacterial Seryl-tRNA Synthetase

Article abstract of DOI:10.1021/acs.jmedchem.9b01131

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high-resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than 2 orders of magnitude compared to their human homologue, demonstrating a route to the selective chemical inhibition of these bacterial targets.

Full text of DOI:10.1021/acs.jmedchem.9b01131

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Article
Structure-guided enhancement of selectivity of chemical
probe inhibitors targeting bacterial seryl-tRNA synthetase
Ricky Cain, Ramya Salimraj, Avinash Punekar, Dom Belini, Colin W. G. Fishwick, Lloyd Czaplewski,
David Jan Scott, Gemma Harris, Christopher G Dowson, Adrian J. Lloyd, and David I. Roper
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01131 • Publication Date (Web): 18 Oct 2019
Downloaded from pubs.acs.org on October 19, 2019
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Structure-guided enhancement of selectivity of chemical probe inhibitors
targeting bacterial seryl-tRNA synthetase
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Ricky Cain,[a]+ Ramya Salimraj,[a]+ † Avinash S. Punekar,[a]† † Dom Bellini,[a] Colin W. G. Fishwick,[b]
Lloyd Czaplewski,[c] David J. Scott,[d,e] Gemma Harris,[e] Christopher G. Dowson,[a] Adrian J. Lloyd,[a]
David I. Roper[a]*
[a] School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United
Kingdom.
[b] School of Chemistry, University of Leeds, Leeds, LS2 9JT, United Kingdom.
[c] Chemical Biology Ventures Limited, Abingdon, OX14 1XD, United Kingdom
[d] School of Biosciences, University of Nottingham, Nottingham, LE12 5RD, United Kingdom
[e] ISIS Spallation Neutron and Muon Source and the Research Complex at Harwell, Rutherford Appleton
Laboratory, Oxfordshire OX11 0FA, United Kingdom
+ These authors contributed equally to this work
†Present address – The Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, United
Kingdom
†† Present address - Drug Discovery Unit, School of Life Sciences, University of Dundee, Dundee DD1
5EH, United Kingdom.
* E-mail: david.roper@warwick.ac.uk
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Abstract
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Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety
of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases
represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus
eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high
resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA
synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery
approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-
tRNA synthetases with greater than two orders of magnitude compared to their human homologue,
demonstrating a route to selective chemical inhibition of these bacterial targets.
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Introduction
The fidelity of protein synthesis is absolutely reliant upon the provision of specific amino acids by
aminoacyl-tRNA molecules for use by the ribosome.1 Errors in this process cause defects in protein folding
and function leading to cell death.2 Each of the 20 amino acids has its own aminoacyl-tRNA synthetase
(aaRS) which catalyses the attachment of the amino acid to its cognate tRNA. Despite the fact that all aaRSs
share the same overall mechanism, it has long been recognised that there is clearly significant diversity
between bacterial, mammalian and archaeal enzymes to allow for synthetic and natural product
discrimination between pathogen and host enzymes3-5. In addition, in some situations, several different
amino acids are able to bind to non-cognate aaRSs, requiring an in vivo editing function allowing for the
possibility of exploiting this feature for future antimicrobial discovery6. For example, the amino acid serine
is able to bind alanyl-tRNA synthetase (AlaRS) and threonyl-tRNA synthetase (ThrRS) in addition to its
cognate seryl-tRNA synthetase (SerRS)7. This incorrect binding is rectified in nature by numerous
proofreading mechanisms6, 8. However, in this context, one of the major challenges presented by aaRS as
targets for antimicrobial drug discovery is their ubiquitous presence in organisms and particularly with
respect to bacterial infection in human tissues requiring exploration of strategies that allow for bacterial
selectivity to prevent issues of specificity and toxicity9.
Aminoacyl sulfamoyl adenosines (aaSAs) are non-hydrolysable mimetics of the aminoacyl adenylate
intermediate (aaAMP) formed during the aaRS catalytic cycle and are potent inhibitors of these enzymes.10
A significant number of natural product inhibitors mimic these reaction intermediates forming tight binding
complexes with substantial affinity competing effectively with natural aaAMP substrates. Of those,
mupirocin is the most prominent example that has found clinical utility as a topical treatment for soft tissue
infections. Mupirocin targets the IleRS enzyme and utilises a hydrophobic “tail” in addition to an aminoacyl
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adenylate warhead to bind to its target11. By contrast to many single target antibiotics in clinical use, seryl
sulfamoyl adenosine (SerSA, 1) can bind and inhibit AlaRS and ThrRS in addition to SerRS and hence is
a multi-targeting inhibitor7, 12. It can be predicted therefore that SerSA would require mutations in several
of these enzymes before a resistance phenotype could be conferred.
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The protein databank (PDB) contains X-ray crystal structures of SerRS from Thermus thermophilus13, 14
,
Methanosarcina barkeri15, Pyrococcus horikoshii16, Candida albicans17, Arabidopsis thaliana18,
Methanopyrus kandleri19, Trypanosoma brucei, human cytoplasmic20 and bovine mitochondrial21. It is
therefore evident that there is a distinct lack of structural data available for clinically relevant bacteria.
Although the Escherichia coli SerRS22 structure was solved in 1990, the coordinates were not deposited in
the PDB thereby hampering efforts in antimicrobial structure-based drug discovery (SBDD) based on this
structure. Moreover, the X-ray crystal structure of human SerRS in complex with SerSA reveals specific
conformational changes upon catalysis necessary for function, which are not found in bacterial homologues
providing further perspectives upon differences in structure that may allow prokaryotic from eukaryotic
specificity.20 In this study we set out to increase the available structural information for human bacterial
pathogens and use this to investigate the possibilities for designing bacteria-specific SerRS enzyme
inhibitors using a SBDD approach.
Results & Discussion
Crystal structures of SerRS in complex with SerSA inhibitor.
The crystal structures of full-length SerRS from E. coli (EcSerRS) and Staphylococcus aureus (SaSerRS)
in complex with the SerSA inhibitor were solved at 1.50 Å and 2.03 Å, respectively (Fig. 1, Supplementary
Table 1). In both structures, the SerSA inhibitor is unambiguously determined by the electron density maps
(Supplementary Fig. 1). SerSA is bound deep into a well-conserved SerRS aminoacylation catalytic
pocket and stabilized by a network of hydrogen bond interactions from the residues in motif 2, motif 3 and
the serine-binding TxE motif (Fig. 1a) - a typical binding mode in all class 2 aaRSs. Superimposition of
EcSerRS, SaSerRS and human cytoplasmic SerRS (HsSerRS, PDB ID: 4L8720) structures show a high
degree of similarity as evidenced by the RMSD values (Supplementary Table 2). The orientation of the
bound SerSA inhibitor is comparable in all three structures. However, the N-terminal tRNA-binding domain
(i.e. the two-stranded anti-parallel coiled coil making the long helical arm) protruding away from the active
site pockets in the compared structures shows large conformational changes resulting in a high RMSD.20
The purine ring of the adenosine in SerSA interacts with a conserved phenylalanine (F287 in EcSerRS,
F281 in SaSerRS and F321 in HsSerRS) via a π- π stacking interaction (Fig. 1b). The M284 in EcSerRS,
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L278 in SaSerRS and V318 in HsSerRS are positioned such that they provide main chain hydrogen bond
interactions with the ring nitrogens (Fig. 1c). The seryl moiety of SerSA extends deep into the pocket to
interact with T237, E239, R268, E291 and S391 in EcSerRS and equivalent residues in SaSerRS and
HsSerRS. We note the presence of a highly coordinated water molecule 3Å away from the N3 of the adenine
moiety of the adenylate (Fig. 1b-c), a feature that has previously been described in class II synthetase
enzymes.23 In SaSerRS the octahedral coordination of a magnesium ion (Fig 1c) is observed in the active
site via Glu349, the SerSA sulfone and water molecules, reminiscent of the magnesium ion observed in
both the Candida albicans SerRS and HsSerRS.
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Figure 1: Binding mode of SerSA to E. coli and S. aureus SerRS. a: Superposition of EcSerRS (blue,
PDB ID: 6R1M) and SaSerRS (gold, PBD ID: 6R1N) with SerSA bound (boxed). b: Interactions of SerSA
(green sticks) with EcSerRS chain A. Water represented as a red sphere. Hydrogen bond interactions shown
as black dashes. c: Interactions of SerSA with SaSerRS. Coordinated magnesium ion represented as a green
sphere.
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Design and synthesis of the selectivity probe.
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The X-ray crystal structures of EcSerRS, SaSerRS and the HsSerRS (PDB ID: 4L87) were superimposed
in Maestro (Schrödinger, LLC).24 Interestingly, a thorough analysis of the active site pockets revealed a
small extension in the hydrophobic cavity adjacent to the C-2 position of SerSA (1) in the EcSerRS and
SaSerRS structures. This pocket is centred around a glycine at positions 396 and 390 in EcSerRS, SaSerRS
respectively. This hydrophobic cavity extension is absent in the HsSerRS (Fig. 2b) as it is filled by the
bulkier side-chain of threonine at position 434. The conserved nature of this structural difference is reflected
in amino acid sequence alignments of selected Gram-positive and Gram-negative bacterial pathogens when
compared to cytoplasmic and mitochondrial variants of the human, bovine and mouse SerRS enzymes
(Supplementary Fig. 2a). Moreover, inspection of an alignment of the Gram-positive and Gram-negative
bacterial pathogens shows this glycine to be part of a 12 amino acid region of conservation ending in an
arginine (397 in EcSerRS, 391 in SaSerRS) suggestive of an invariant bacterial structural feature absent in
eukaryotic homologues. (Supplementary Fig. 2b). Exploiting such a conserved feature for antimicrobial
drug discovery extends the range of bacteria that can potentially be targeted whilst also reducing the chances
of mutation-induced drug resistance.
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A focused structure-activity relationship (SAR) series with variants at the C-2 position of SerSA adenosine
was designed to investigate the steric tolerance of the hydrophobic cavity and to establish the degree of
selectivity for the bacterial SerRS over the HsSerRS (Fig. 3a). In silico molecular docking of the designed
selectivity probes into the active site pockets of the EcSerRS, SaSerRS and HsSerRS crystal structures
(Supplementary Methods, Supplementary Table 3) and visual analysis of the predicted docking poses
(Fig. 2c-d) suggested that chloro- and iodo-seryl sulfamoyl adenylate derivatives 2 and 3 respectively
would not achieve selectivity since 2 and 3 were predicted to interact equally as well with both the bacterial
and HsSerRS. Compounds 4-8 were however predicted to exhibit selectivity for the bacterial SerRS over
the HsSerRS.
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D
C
Figure 2: Binding modes of designed seryl sulfamoyl adenylate selectivity probes. (a) 3D spatial
representation of the seryl sulfamoyl adenylate derivatives indicating C-2 position where SAR study was
focussed (yellow dashed line). (b) Structural overlay of SaSerRS (Grey, PDB ID 6R1N) and HsSerRS
(Yellow, PDB ID: 4L87) active site showing the key residue change near the 2 position of the sulfamoyl
adenylate inhibitor from Gly390 in the bacterial form to Thr434 in the human form. (c) Predicted binding
modes of seryl sulfamoyl adenylates to SaSerRS (PDB ID 6R1N) using AutoDock 4.2. (d) Predicted
binding modes of seryl sulfamoyl adenylates to HsSerRS using AutoDock 4.2.
The bulkier groups located at the 2 position of compounds 4-8 were predicted to be accommodated in the
pocket of the bacterial enzymes. However, due to the steric hindrance from the T434 residue in the
HsSerRS, compounds 4-8 were predicted to change the torsional angle between the adenine and ribose
sugar upon binding to the HsSerRS. As a result of the torsional change the π-π stacking interactions with
F287 and the backbone interaction to V318 are lost leading to a weaker predicted binding affinity and
therefore increased selectivity for the bacterial SerRS (Fig. 2d).
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Preparation of SerSA selectivity probes was initiated by the acid-catalysed protection of the commercially
available 2-chloroadenosine or 2-iodoadenosine (Fluorochem, UK) to provide the acetyl-protected
adenosines (95-97%) (Scheme 1, 9-10). A Suzuki coupling reaction between the protected adenosine and
desired boronic acid species (20-70%) was conducted (11-15),25 before sulfonation using sulfonyl chloride
to afford the sulfonamide (90-95%) (16-22). The sulfonamide was then coupled to the succinimide activated
protected serine (23) to yield the protected product (40-50%) (24-30). Removal of the benzyl group was
accomplished by treatment with a solution of boron trichloride dimethyl sulfide complex (2M in DCM),26
and the resulting alcohol was treated with trifluoroacetic acid and water to yield compounds 2-8 (2-8, see
Experimental Section and Supplementary Information for details).
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Bacterial SerRS inhibition by selectivity probe
Using a continuous spectrophotometric assay that specifically measures the adenylate formation reaction7,
compounds 2-8 were evaluated for the inhibition of ATP-dependent aminoacyl adenylate formation by
EcSerRS and SaSerRS enzymes and compared their half maximal inhibitory concentration (IC50) values
with the parent SerSA, compound 1 (Supplementary Table 4 and 5). Compounds 2-8 were active against
EcSerRS and SaSerRS with IC50 values ranging from 378 nM to 52.7 µM (Table 1). Compound 2 exhibited
sub-micromolar inhibition of the SaSerRS and EcSerRS with IC50s of 262 nM and 445 nM respectively.
Compound 3 also exhibited sub-micromolar inhibition of SaSerRS with an IC50 of 378 mM but weaker
inhibition of EcSerRS with an IC50 of 1.36 µM. Compounds 4-8 all manifested low micromolar inhibition
against both bacterial SerRS (Table 1). A general trend is observed where increasing the size of the group
at the 2 position of the adenylate decreases the binding affinity to the bacterial synthetase. Alanyl sulfamoyl
adenosine (AlaSA, 31) and threonyl sulfamoyl adenosine (ThrSA 32) were also evaluated for inhibition
against EcSerRS and SaSerRS (Supplementary Table 6). AlaSA 31 showed no inhibitory activity against
either enzyme at 1 mM while ThrSA 32 manifested IC50s of 285 µM and 231 µM against EcSerRS and
SaSerRS respectively, thus exhibiting much weaker binding than the designed selectivity probes. These
results highlight the key role of the beta-hydroxyl of the serine to the overall binding of the compound
within the adenylate formation site in these enzymes and the overall inhibitory properties of seryl adenylate
inhibitors modified around the C-2 position of the SerSA adenosine.
Table 1: IC50 values of designed chemical probes against seryl-tRNA synthetases. Assays were
conducted as reported.7
H2N
N
O
O
O
N
OH
N
S
O
N
O
N
H
NH2
X
HO
OH
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No.
X
IC50 EcSerRS (µM) IC50 SaSerRS (µM)
IC50 HsSerRS (µM)
2.17 ± 0.21
1 (SerSA)
H
0.21 ± 0.03
0.45 ± 0.05
1.36 ± 0.12
17.7 ± 1.42
0.23 ± 0.49
0.26 ± 0.03
0.38 ± 0.04
52.7 ± 4.81
3.46 ± 0.47
32.4 ± 3.56
1.24 ± 0.12
6.34 ± 0.71
2
3
4
5
6
7
8
Cl
67.3 ± 4.67
I
24.0 ± 2.26
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C6H5
>1000 ± >100
>1000 ± >100
>1000 ± >100
>1000 ± >100 (ppt)
>1000 ± >100 (ppt)
trans-Propenyl 9.38 ± 0.70
2-Furyl
36.2 ± 2.41
1.44 ± 0.09
6.65 ± 0.64
3-Thienyl
3-Pyridyl
SerRS, Seryl t-RNA synthetase. (ppt) precipitation observed at 1000 µM.
Errors were calculated as s.d. of at least three independent measurements.
HsSerRS inhibition by selectivity probes
Measurement of the IC50 inhibition kinetics of the original SerSA, compound 1 against the bacterial and
human SerRS enzymes, reveals a 10-fold difference, in favour of greater specificity of the inhibitor for the
bacterial enzymes. Compounds 2-8 were subsequently screened for inhibition of the HsSerRS (Table 1)
using the same assay system. Assay measurements of compounds 2 and 3, revealed a 31-fold and 11-fold
increase in IC50 against the bacterial SerRS and HsSerRS, indicating that compounds 2 and 3 did not exhibit
selectivity overall and had lower affinity than SerSA 1. Overall the observed IC50 of compounds 2-8
increased with respect to the parental adenylate 1 but remarkably, inhibition of the HsSerRS was effectively
abolished in compounds 4-8 with IC50 values greater than 1 mM, revealing significant selectivity of these
compounds towards the tested bacterial SerRS. The best of these compounds (7), with a 3-Thienyl at the
C-2 position of the SerSA adenosine had an increase in IC50 over SerSA 1 of 6.8 and 8.4 fold for EcSerRS
and SaSerRS respectively, with effectively negligible binding to the HsSerRS. The observed selectivity
overall was attributed to the increased size of 4-8 making them unable to fit into the hydrophobic pocket
located in the human cytoplasmic SerRS active site due to the presence of T434 as previously hypothesised.
Binding studies of SerSA and compound 8 to EcSerRS
To independently measure the binding characteristics of the original adenylate SerSA and the derivatives
synthesised in this study, we measured binding affinity using iso isothermal titration calorimetry (ITC). The
binding stoichiometry and affinity of SerSA 1 and compound 8 to EcSerRS was determined using ITC
because compound 8 had the best solubility of the synthesised compounds. Titration of SerSA to EcSerRS
resulted in a steep slope in the binding isotherm suggesting a very tight binding of the inhibitor to the
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enzyme. Interestingly, fitting of this binding isotherm using a single site model showed a 2:1 SerSA:SerRS
stoichiometry with an overall dissociation constant Kd = 1.27 nM (Supplementary Fig. 3a). The
combination of very high affinity and low enthalpy unfortunately prevented an accurate measurement of
Kd for SerSA at the individual binding sites.
By contrast, titration of compound 8 to EcSerRS resulted in a binding isotherm (2:1 compound 8:SerRS
stoichiometry) that after fitting using a two independent sites model clearly showed two distinct binding
sites with dissociation constants Kd1 = 0.29 µM and Kd2 = 1.92 µM (Supplementary Fig. 3b). As Kd2 > 4
Kd1, there is apparent mild negative cooperativity within the system. In both experiments, a negative
enthalpy value detected for such a tight interaction indicates the role of hydrogen bond and electrostatic
interactions in the stabilisation of the enzyme–inhibitor complex. The observation of two binding sites for
SerSA and compound 8 prompted us to investigate the oligomeric state of the EcSerRS in solution, which
are typically dimers in solution.27 Analytical ultracentrifugation (AUC) experiments were carried out with
EcSerRS to confirm the oligomeric state of the protein in the presence and absence of SerSA and compound
8 (Supplementary Table 6). The results confirmed that EcSerRS, both with and without inhibitors,
appeared with a molecular weight that is consistent with a dimer in solution (Supplementary Fig. 4). The
observed SerSA and compound 8 binding stoichiometry is consistent with the previous structural findings
showing two SerSA molecules bound to two distinct sites in the X-ray crystal structure of Candida albicans
SerRS (PDB ID: 3QO8)17. In this structure the second SerSA binding site is located 26 Å distant from the
active site and appears to play no role in the enzyme function or protein-protein interaction as described by
the authors17.
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Structural basis of selectivity probe binding to EcSerRS
To understand the molecular basis of the selectivity probe towards bacterial SerRS, we attempted a series
of co-crystallisation screening of EcSerRS and SaSerRS. Despite extensive screening, we were unable to
find hit conditions to co-crystallise SaSerRS in the presence of compound 7 or 8. However, we were
successful in obtaining crystals of EcSerRS amenable to soaking with compound 8, which yielded a 2.6 Å
resolution structure (Fig. 3a-d). The EcSerRS-SerSA complex structure was solved in the space group P1
containing two monomers that associate tightly to form a dimer. In contrast, the EcSerRS-compound 8
complex structure was solved in space group P6122 with 1 molecule in the asymmetric unit. Compound 8
binds in a similar fashion to SerSA in EcSerRS making key interactions with the residues in motif 2, motif
3 and the serine-binding TxE motif as described above (Fig. 3c). The 3-pyridyl group of compound 8
snuggly fits into the hydrophobic cavity without any other obvious interactions (Fig. 3b) with movement
of the motif 2 loop observed to accommodate the pyridyl group (Fig. 3d).
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Figure 3: Comparison of binding of SerSA and compound 8 to EcSerRS active site. a: The chemical
structures of the compounds used in this study. b: Pyridyl group of compound 8 (circled) positioned in the
active site. c: Interactions of compound 8 (green sticks) with EcSerRS. Hydrogen bond interactions are
shown as black dashes. d: Superposition of EcSerRS:SerSA (blue, PDB ID:6R1M) with
EcSerRS:compound 8 (PDB ID:6R1O).
We analysed both structures for presence of a second adenylate-binding site as found in the Candida
albicans SerRS-SerSA structure (PDB ID: 3QO8)17. No density for the second adenylate was found in the
EcSerRS-SerSA structure, but density for two additional ligand molecules were observed in the EcSerRS-
compound 8 structure (Fig. 4a). These ligands were found to bind away from the active site in positions
distinct to that observed in the Candida albicans SerSA (Fig. 4b). Electron density for the complete
compound was observed for the ligand in the active site (Fig. 4c) and a second ligand which π- π -stacks
with a third ligand from a symmetry-related molecule for which electron density is only observed for its
purine and pyridyl rings (Fig. 4d). The seryl moiety of this third ligand is likely to not make any interactions
with the protein and be flexible due to the absence of electron density for this region of the compound. As
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such the presence of this third ligand molecule is likely to be a crystallographic artefact of the high
concentration of compound used for soaking and this structure provides evidence for a potential second
binding site that is supported by the ITC data.
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Figure 4: Second binding site of compound 8 to EcSerRS. a: Binding positions of compound 8 to
EcSerRS. b: Overlay of EcSerRS:compound 8 (wheat, PDB ID: 6R1O) with Candida albicans SerRS (blue,
PDB ID: 3QO8) c: Overlay of Fo-Fc omit map of compound 8 in EcSerRS active site contoured at σ 3. d:
Interaction of two molecules of compound 8 (boxed) from EcSerRS symmetry-related molecules.
Pathogen susceptibility testing to selectivity probes.
Previous studies by Van de Vijver et. al. showed that SerSA (1) did not exhibit an MIC against S. aureus
and E. coli in disk diffusion studies10. Compounds 1-8 were screened in an antimicrobial susceptibility
assay using CLSI guidelines28 to determine MIC against both S. aureus and E. coli however no MIC’s were
observed (>256 ug/ml). This result is consistent with the previous reported studied of 1 and is likely to be
the result of poor cell permeability or efflux after the molecules enter the cell. In order to rationalise the
poor activity against bacterial cells the compounds produced in this study were analysed using the
bioinformatics tool Entryway (www.entry-way.org), which classifies molecules that are likely to be capable
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of accumulating in Gram-negative bacteria. Whilst the compounds fulfil the requirements for globularity
and contain the required primary amine the number of rotatable bonds exceeds the limits normally founds
in antimicrobials.29 The compounds described would therefore require further lead optimisation to progress
them from selective inhibitors to the final desired antimicrobials.
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Conclusion
In summary, we demonstrate the use of structure-based drug design to identify selective inhibitors of
exemplar SerRS enzymes from Gram-positive and Gram-negative pathogens on the WHO list of bacteria
for which new antibiotics are urgently needed. Previous studies have investigated inhibiting protein
synthesis via inhibition of specific aaRS activities leading to the identification of a number of potent
antibiotics which have progressed through into clinical studies17, 30-33. Rapid development of resistance to
these synthetase inhibitors has halted their clinical evaluation34. The reported alternative approach herein
has been a proof of principle example of the capability of structure-based drug design in modifying a multi-
targeting aaRS inhibitor to achieve selectivity.
Analysis using the bioinformatics tool Entryway (www.entry-way.org), showed that whilst the compounds
fulfil the requirements for globularity and contain the required primary amine the number of rotatable bonds
exceeds the limits normally founds in antimicrobials.29 Further work is required to achieve clinically viable
compounds that can permeate the cell membrane but the crystal structures here, nonetheless, provide a
foundation for structure-based drug design of novel selective inhibitors which multi-target the bacterial
aminoacyl-tRNA synthetases.
Experimental
Scheme1: Synthetic route of target SerSA selectivity probes
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R(OH)2
4
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K2CO3
Pd(PPh3)4
THF : H2O
H2N
N
H2N
H2N
N
Acetone
p-toluenesulfonic
N
N
N
acid monohydrate N
N
N
N
O
O
O
OH
N
OH
OH
N
N
12h, 20-70%
X
12h, 94%
X
R
General method A
O
HO
O
OH
O
O
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9-10
X = Cl or I
11-15
H2N
N
H2N
N
23
DBU
DMF
N
N
O
O
O
NH2SO2Cl
DMA
O
O
O
O
O
N
S
N
S
O
NH2
O
N
N
N
H
NH
R
R
O
12h, 40-50%
General method C
3h, 95%
General method B
O
O
O
O
O
16-22
24-30
H2N
1. BCl3.SMe2
DCM
2. TFA H2O
N
O
O
O
O
N
OH
N
S
O
N
N
H
NH2
R
12h, 30%
HO
OH
General method D
2-8
*
O
O
N-Hydroxysuccinamide
DCC
O
O
O
HO
N
EtOAc / Dioxane
O
NH
O
NH
O
O
12h, 87%
O
O
23
General information in synthetic chemistry. Chemicals were from commonly used suppliers and used
without further purification. SerSA (1), AlaSA (31) and ThrSA (32) were purchased from Syhthesis
MedChem (UK) Ltd (Cambridge, UK). Solvents (including dry solvents) for chemical transformations,
work-up and chromatography were from Sigma-Aldrich (Dorset, UK) at HPLC grade, and used without
further distillation. Silica gel 60 F254 analytical thin layer chromatography (TLC) plates were from Merck
(Darmstadt, Germany) and visualized under UV light and/or with potassium permanganate stain.
Chromatographic purifications were performed using Merck Geduran 60 silica (40-63 μm) or prepacked
SNAP columns on a Biotage Isolera Purification system (Uppsala, Sweden). Deuterated solvents were from
Sigma-Aldrich, Chambridge Isotopes and Apollo Scientific Ltd. All 1H and 13C NMR spectra were recorded
using a Bruker Avance 500 MHz or Bruker Avance 400 MHz spectrometer. All chemical shifts are in ppm
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relative to the solvent peak, and signal splitting patterns were described as singlet (s), doublet (d), triplet
(t), quartet (q), quintet (quint), or multiplet (m) with coupling constants (J) are reported in Hz to the nearest
0.5. High Resolution (HR) mass spectrometry data (m/z) were obtained using a Bruker MaXis Impact
instrument with an ESI source and Time of Flight (TOF) analyzer. Fourier transform Infrared (FT-IR)
spectra were recorded on a Bruker Alpha Platinum instrument. Melting points were obtained from a
Reichert Hot Stage melting point apparatus. HPLC analysis was run on an Agilent 1290 Infinity system
equipped with a Supelco Ascentis Express 2.7 μM C18 column (50 x 2.1 mm) using a gradient of 95%
solvent A → 95% solvent B (solvent A: H2O containing 0.1% formic acid; solvent B: 100% MeCN
containing 0.1% formic acid), flow rate = 0.5 mL/min and UV detection at 254 nm. Specific rotation
measurements were recorded using a Schmidt and Haensch Polartronic H532 polarimeter, using a 100 mm
cell and the Sodium D line (589 nm). [α]D are reported in units of 10-1 deg dm2g-1. The purities of all of the
final compounds for biological testing were determined to be over 95% by NMR and HPLC. See the
Supporting Information for 1 H and 13C NMR spectra, HR Mass spectrometry and HPLC purity analysis
of all compounds.
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General method A: Suzuki coupling reactions (11-15). The boronic acid (4.0 equiv.) was added to a
stirred solution of 2-chloroadenosine (1.0 equiv.), potassium carbonate (2.00 equiv.) and tetrakis
(triphenylphosphine) palladium (0) (0.20 equiv.) in THF (8 mL) and water (4 mL). The reaction mixture
was heated to reflux for 12 h. The reaction mixture was filtered through Celite® and concentrated in vacuo.
The residue was diluted with water (20 mL) and extracted in EtOAc (3 × 20 mL). The combined organics
were washed with water (3 × 10 mL) and brine (3 × 10 mL), dried (MgSO4) and concentrated in vacuo to
give an off-white/yellow solid, which was purified using flash column chromatography to afford the
coupled products which were used without further purification.
General method B: Introduction of sulfonyl group (16-22). Generation of sulfanoyl chloride:
Chlorosulfonyl isocyanate (1 mL, 11.5 mmol, 1.0 equiv.) was cooled to 0 °C under an atmosphere of
nitrogen. Formic acid (0.43 mL, 11.5 mmol, 1.0 equiv.) was added dropwise and the mixture stirred at room
temperature overnight. Gas evolution was observed. The resulting colourless solid was dried in vacuo. The
colourless solid was used without further purification / characterisation. Sulfanoyl chloride (2.0 equiv.) in
DMA (2 mL) was added dropwise to a stirred mixture of acetyl protected adenolate (1.0 equiv.) in DMA
(3 mL) at 0 °C under an atmosphere of nitrogen. The mixture was then stirred at room temperature for 3 h.
The reaction mixture was quenched with Et3N (1.5 mL) then MeOH (5 mL). The resulting solution was
concentrated in vacuo before EtOAc (50 mL) was added. The mixture was extracted with 5% NaHCO3 (3
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× 15 mL), brine (3 × 10 mL), dried (MgSO4) and concentrated in vacuo to afford the desired product as a
colourless glassy solid.
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General method C: Amide coupling reaction (24-30). Sulfonamide adenylate (1.0 equiv.) was dissolved
in DMF (10 mL). After addition of DBU (1.1 equiv.), N-Boc-Ser(bzl)-OSu (23) (1.1 equiv.) was added to
the reaction mixture. After stirring for 16 h at room temperature, the mixture was concentrated in vacuo
and the residue taken up in water (50 mL) and extracted with dichloromethane (50 mL). The organic layers
were dried (MgSO4) concentrated in vacuo and purified by flash chromatography (EtOAc) to afford the
desired product as a colourless powder.
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General method D: Global deprotection (2-8). Seryl-sulfonamide adenylate (1.0 equiv.) was dissolved
in DCM (5 mL) under an atmosphere of nitrogen. To this was added BCl3.SMe2 (2M in DCM, 7.00 equiv.)
and the reaction was stirred at room temperature for 8 h. The mixture was concentrated in vacuo. The
residue was re-suspended in TFA:H2O (3:1, 4 mL) and the reaction was stirred overnight at room
temperature. The mixture was concentrated in vacuo. The crude product was purified by preparative HPLC
to afford the desired product as a colourless solid.
Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (2). Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-chloro-9H-purin-9-yl)-
2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (24) (0.30 g, 0.43 mmol) to afford the desired product as a colourless powder
(50.4 mg, 0.11 mmol, 25%) m.p.: 121.3 °C (Decomp); Rf: Baseline (9:1 DCM-MeOH); δH (500 MHz,
DMSO-d6): 8.48 (1H, s, 6-HAr), 8.42 (2H, brs, NH2), 7.27 (2H, brs, NH2), 7.00 (1H, brs, NH), 6.20 (1H,
d, J 8.2, 2-HFuryl), 4.99 (1H, brs, OH) 4.75 (1H, apps, 3-HFuryl), 4.62-4.48 (3H, m, 4-HFuryl, 5-HFuryl
and OH), 4.27 (1H, dd, J 10.1 and 5.9, CH2*O), 4.19 (1H, brs, OH), 4.02 (1H, dd, J 10.1 and 6.2, CH2*O),
3.52 (3H, m, CH2*chiral and CHChiral); δC (125 MHz, DMSO-d6): 179.3 (C=O), 157.1 (C4Ar), 153.5
(C2Ar and C8Ar), 140.4 (C6Ar), 120.0 (C9Ar), 89.4 (C2Furyl), 84.4 (C5Furyl), 75.3 (C3Furyl), 74.8
(C4Furyl), 66.0 (CH2O), 64.7 (CH2Chiral), 52.9 (CHChiral); νmax/ cm-1 (solid): 3321, 3125, 2784, 1673,
1592, 1358; HPLC: Tr= 2.26 (100% rel. area); m/z (ES): No mass ion found [α]D = 28.8° (c 0.1, MeOH).
Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-iodo-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (3). Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-iodo-9H-purin-9-yl)-
2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (25) (50 mg, 0.06 mmol) to afford the desired product as a colourless powder
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(4.0 mg, 0.07 mmol, 12%) m.p.: 135.2 °C (Decomp); Rf: Baseline (9:1 DCM-MeOH); δH (400 MHz,
DMSO-d6): 8.48 (1H, s, 6-HAr), 8.43 (2H, brs, NH2), 7.26 (1H, brs NH2) 7.00 (1H, brs, NH), 6.19 (1H, d,
J 5.4, 2-HFuryl), 4.99 (1H, brs, OH), 4.74 (1H, apps, 3-HFuryl), 4.59-4.50 (3H, m, 4-HFuryl, 5-HFuryl
and OH), 4.26 (1H, dd, J 10.2 and 5.9, CH2*O), 4.18 (1H, brs, OH), 4.04 (1H, dd, J 10.0 and 5.8, CH2*O),
3.60-3.49 (3H, m, CH2*chiral and CH Chiral),; δC (100 MHz, DMSO-d6): 179.1 (C=O), 157.5 (C4Ar),
151.0 (C8Ar), 140.3 (C6Ar), 126.9 (C2Ar), 118.1 (C9Ar), 89.4 (C2Furyl), 83.9 (C5Furyl), 75.4 (C3Furyl),
74.8 (C4Furyl), 65.7 (CH2O), 64.4 (CH2Chiral), 51.9 (CHChiral); νmax/ cm-1 (solid): 3321, 3125, 2784,
1673, 1592, 1358; HPLC: Tr= 2.30 (95% rel. area); m/z (ES): (Found: [M-H]-, 558.0. C13H18IN7O8S requires
[M-H], 558.0. [α]D = 28.4° (c 0.1, MeOH).
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Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-phenyl-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (4). Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-phenyl-9H-purin-9-yl)-
2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (26) (0.20 g, 0.27 mmol) to afford the desired product as a colourless powder
(54.0 mg, 0.11 mmol, 39%) m.p.: 143.2 (Decomp); Rf: Baseline (9:1 DCM-MeOH); δH (400 MHz, DMSO-
d6): 8.47 (1H, s, 6-HAr), 8.43 (2H, dd, J 8.0 and 1.6, 6’-HAr and 2’-HAr), 8.20 (1H, s, NH), 7.91 (2H, brs,
NH2), 7.58-7.48 (3H, m, 3’-HAr, 4’-HAr and 5’-HAr), 6.09 (1H, d, J 5.9, 2-HFuryl), 4.77-4.70 (1H, m, 3-
HFuryl), 4.34-4.28 (2H, m, 4-HFuryl and CH2*O), 4.22-4.16 2H, m, 5-HFuryl and CH2*O), 3.87 (1H, dd,
J 11.2 and 3.7, CH2*Chiral) 3.69 (1H, dd, J 11.2 and 7.3, CH2*Chiral), 3.60-3.56 (1H, m, ChiralH); δC
(100 MHz, DMSO-d6): 172.0 (C=O), 163.5 (C2Ar), 155.9 (C4Ar), 150.8 (C8Ar), 140.5 (C6Ar), 137.8
(C1’Ar), 130.1 (C4’Ar), 128.7 (C5’Ar and C3’Ar), 128.2 (C6’Ar and C2’Ar), 119.1 (C9Ar), 87.1
(C2Furyl), 82.8 (C5Furyl), 73.8 (C3Furyl), 71.3 (C4Furyl), 68.5 (CH2O), 61.0 (CH2Chiral), 57.6 (Chiral
H); νmax/ cm-1 (solid): 3367, 3070, 1673, 1598; HPLC: Tr= 1.89 (100% rel. area); m/z (ES): (Found: [M+H]+,
510.1405. C19H23N7O8S requires [M+H], 510.1402). [α]D = 25.7° (c 0.1, MeOH).
Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-propenyl-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (5). Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-propenyl-9H-purin-9-
yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (27) (65 mg, 0.09 mmol) to afford the desired product as a colourless powder
(22.5 mg, 0.05 mmol, 53%) m.p.: 115.3 (Decomp); Rf: Baseline (9:1 DCM-MeOH); δH (500 MHz, DMSO-
d6): 8.50 (1H, s, 6-HAr), 8.15 (1H, s, NH), 7.88 (2H, brs, NH2), 7.09 (1H, dd, J 15.4 and 6.7, 2-HPropyl),
6.41 (1H, d, J 15.4, 1-HPropyl), 5.95 (1H, d, J 5.9, 2-HFuryl), 4.63 (1H, app t, J 5.9, 3-HFuryl), 4.28-4.09
(4H, m, 4-HFuryl, 5-HFuryl and CH2O), 3.83 (1H, dd, J 11.1 and 3.6, CH2* Chiral), 3.65 (1H, dd, J 11.1
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and 7.0, CH2* Chiral), 3.56-3.50 (1H, m, Chiral H), 2.88 (2H, app d, J 5.3, NH2), 1.98 (3H, d, J 6.7, CH3);
δC (125 MHz, DMSO-d6): 172.2 (C=O), 163.5 (C2Ar), 153.8 (C4Ar), 150.9 (C8Ar), 145.9 (C6Ar), 122.3
(C2Pro), 119.4 (C9Ar), 117.9 (C1Pro), 93.7 (C2furyl), 83.4 (C5Furyl), 75.4 (C3Furyl), 70.5 (C4Furyl),
70.2 (CH2O), 57.3 (CH2 Chiral), 54.8 (Chiral H), 19.1 (CH3); νmax/ cm-1 (solid): 3106, 2942, 1668, 1595,
1182; HPLC: Tr= 0.81 (100% rel. area); m/z (ES): (Found: [M+H]+, 474.1406. C16H23N7O8S requires
[M+H], 474.1402). [α]D = 27.0° (c 0.1, MeOH).
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Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-(furan-2-yl)-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (6). Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(furan-2-yl)-9H-purin-
9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (28) (100 mg, 0.14 mmol) to afford the desired product as a colourless powder
(14.6 mg, 0.03 mmol, 21%) m.p.: 117.2 (Decomp); Rf: Baseline (9:1 DCM-MeOH); δH (500 MHz, DMSO-
d6): 8.54 (1H, brs, NH), 8.34 (2H, brs, NH2), 8.14 (1H, s, 6-HAr), 7.96 (1H, app s, 3’-HAr), 7.46 (1H, app
t, J 10.1, 5’-HAr), 6.76 (1H, dd, J 10.1 and 8.5, 4’-HAr), 6.02 (1H, d, J 5.2, 2-HFuryl), 4.65 (1H, d, J 5.2,
3-HFuryl), 4.52-4.45 (2H, m, CH2O), 4.29 (1H, app s, 4-HFuryl), 4.20 (1H, app s, 5-HFuryl), 3.89-3.75
(3H, m, CH2 Chiral and Chiral H); δC (125 MHz, DMSO-d6): 172.0 (C=O), 158.1 (C2Ar), 156.1 (C4Ar),
152.5 (C1’Ar), 150.6 (C8Ar), 145.3 (C3’Ar), 141.2 (C6Ar), 117.9 (C9Ar), 113.9 (C5’Ar), 113.2 (C4’Ar),
88.2 (C2Furyl), 84.6 (C5Furyl), 73.4 (C3Furyl), 71.8 (C4Furyl), 60.6 (CH2O), 54.9 (Chiral C), 45.2 (CH2
Chiral); νmax/ cm-1 (solid): 3089, 1689, 1595, 1477, 1383; HPLC: Tr= 2.04 (97% rel. area); m/z (ES): (Found:
[M+H]+,. C17H21N7O9S requires [M+H], 500.1194.) [α]D = 22.9° (c 0.1, MeOH).
Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-(thiophen-3-yl)-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (7). Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(thiophen-3-yl)-9H-
purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-
(benzyloxy)-1-oxopropan-2-yl]carbamate (29) (0.30 g, 0.40 mmol) to afford the desired product as a
colourless powder (27.5 mg, 0.05 mmol, 13%) m.p.: 108.3 °C (Decomp); Rf: Baseline (9:1 DCM-MeOH);
δH (500 MHz, DMSO-d6): 8.46 (1H, brs, NH), 8.34 (1H, s, 2’-HAr), 8.14 (1H, s, 6-HAr), 8.00 (2H, brs,
NH2), 7.87-7.78 (1H, m, 4’-HAr), 7.68-7.59 (1H, m, 5’-HAr), 6.00 (1H, d, J 5.5, 2-HFuryl), 4.70 (1H, t, J
5.5, 3-HFuryl), 4.37-4.22 (4H, m, CH2O, 4-HFuryl and 5-HFuryl), 3.83-3.65 (3H, m, CH2 chiral and chiral
H); δC (125 MHz, DMSO-d6): 172.0 (C=O), 163.5 (C2Ar), 156.0 (C4Ar), 150.7 (C8Ar), 140.1 (C6Ar),
127.8 (C4’Ar), 127.1 (C5’Ar), 120.0 (C2’Ar), 119.2 (C9Ar), 116.8 (C1’Ar), 88.2 (C2Furyl), 84.2 (C5furyl),
73.7 (C3Furyl), 71.8 (C4Furyl), 60.8 (CH2O), 57.2 (Chiral C), 45.4 (CH2 chiral); νmax/ cm-1 (solid): 3096,
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1685, 1588, 1402, 1275; HPLC: Tr= 2.04 (100% rel. area); m/z (ES): (Found: [M+H]+, 516.0972.
C17H21N7O8S2 requires [M+H], 516.0966.) [α]D = 20.7° (c 0.1, MeOH).
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Preparation of (2S)-2-amino-1-[([[(2R, 3S, 4R, 5R)-5-(6-amino-2-(pyridine-3-yl)-9H-purin-9-yl)-3,4-
dihydroxyoxolan-2-yl]methoxy)sulfonyl)amino]-3-hydroxypropan-1-one (8).Preparation was via
general method D using tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(pyridine-3-yl)-9H-
purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-
(benzyloxy)-1-oxopropan-2-yl]carbamate (30) (100 mg, 0.13 mmol) to afford the desired product as a
colourless powder (23.8 mg, 0.05 mmol, 36%) m.p.: 120.2 °C (Decomp); Rf: Baseline (9:1 DCM-MeOH);
δH (500 MHz, DMSO-d6): 9.50 (1H, s, 2’-HAr), 8.95-8.75 (2H, m, 6’-HAr and 4’-HAr), 8.47 (1H, s, 6-
HAr), 7.93 (2H, brs, NH2), 7.54-7.50 (1H, m, 5’-HAr), 6.04 (1H, d, J 6.0 2-HFuryl), 4.82-4.72 (1H, m, 3-
HFuryl), 4.37-4.23 2H, m, CH2*O and 4-HFuryl), 4.18 (2H, app d, J 8.9, 5-HFuryl and CH2*O), 3.82 (1H,
d, J 8.0 CH2 chiral), 3.73-3.60 (1H, m, CH2 chiral), 3.58-3.54 (1H, m, Chiral H); δC (125 MHz, DMSO-d6):
171.9 (C=O), 160.3 (C2Ar), 156.1 (C4Ar), 151.0 (C4’Ar), 150.8 (C8Ar), 150.7 (C2’Ar), 141.2 (C6Ar),
138.6 (C2’Ar), 135.6 (C6’Ar), 133.4 (C1’Ar), 125.2 (C5’Ar), 119.4 (C9Ar), 87.9 (C2Furyl), 82.9
(C5Furyl), 73.7 (C3Furyl), 71.2 (C4Furyl), 68.7 (CH2O), 60.8 (CH2 chiral), 57.5 (Chiral C); νmax/ cm-1
(solid): 3317, 3118, 1633, 1587, 1587, 1382; HPLC: Tr= 1.99 (100% rel. area); m/z (ES): (Found: [M+H]+,
511.1356. C18H22N8O8S requires [M+H], 511.1356. [α]D = 19.3° (c 0.1, MeOH).
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Preparation of 2’3’-O-Isopropylidene-2-chloroadenosine (9). 2-Chloroadenosine (0.20 g, 0.66 mmol,
1.0 equiv.) and p-toluenesulfonic acid mono hydrate (1.26 g, 6.6 mmol, 10 equiv.) were dissolved in acetone
(100 mL) under an atmosphere of nitrogen. The reaction was stirred at room temperature overnight. While
cooling in an ice bath a saturated NaHCO3 solution (100 mL) was added to the reaction mixture until the
pH of the solution was slightly basic. The acetone was removed in vacuo. The remaining aqueous solution
was extracted with EtOAc (3 × 50 mL). The combined organics were dried (MgSO4) and concentrated in
vacuo. The desired product was isolated as a colourless solid (203 mg, 0.59 mmol, 90%); m.p.: 184.2-185.6
°C; Rf: 0.67 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 8.37 (1H, s, 6-HAr), 7.87 (2H, brs, NH2),
6.07 (1H, d, J 2.8 2-HFuryl), 5.29 (1H, dd, J 6.2 and 2.8, 3-HFuryl), 5.09 (1H, app t, J 5.4, OH), 4.95 (1H,
dd, J 6.2 and 2.8, 4-HFuryl), 4.22 (1H, dd, J 6.2 and 5.4, 5-HFuryl), 3.65-3.50 (2H, m, CH2), 1.56 (3H, s,
CH3a), 1.34 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 158.1 (C4Ar), 153.0 (C2Ar), 149.9 (C8Ar), 139.9
(C6Ar), 128.2 (C5Ar), 119.0 (C9Ar), 113.1 (Acetyl C), 89.4 (C2Furyl), 86.7 (C5Furyl), 83.4 (C3Furyl),
81.2 (C4Furyl), 61.5 (CH2), 37.0 (CH3a), 25.2 (CH3b); νmax/ cm-1 (solid): 3473, 3299, 1761, 1651, 1381;
HPLC: Tr= 2.26 (100% rel. area); m/z (ES): (Found: [M+H]+, 342.0965. C13H16ClN5O4 requires [M+H],
342.0964. [α]D = -115.5° (c 0.1, MeOH).
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Preparation of 2’3’-O-Isopropylidene-2-iodoadenosine (10). 2-iodoadenosine (1.00 g, 2.54 mmol, 1.0
equiv.) and p-toluenesulfonic acid mono hydrate (4.83 g, 25.4 mmol, 10 equiv.) were dissolved in acetone
(200 mL) under an atmosphere of nitrogen. The reaction was stirred at room temperature overnight. While
cooling in an ice bath a saturated NaHCO3 solution (200 mL) was added to the reaction mixture until the
pH of the solution was slightly basic. The acetone was removed in vacuo. The remaining aqueous solution
was extracted with EtOAc (3 × 100 mL). The combined organics were dried (MgSO4) and concentrated in
vacuo. The desired product was isolated as a colourless solid (0.94 g, 2.17 mmol, 86%); m.p.: 182.4-184.1
°C; Rf: 0.69 (9:1 Chloroform-MeOH); δH (400 MHz, DMSO-d6): 8.34 (1H, s, 6-HAr), 7.83 (2H, brs, NH2),
6.06 (1H, d, J 4.0, 2-HFuryl), 5.30 (1H, dd, J 6.2 and 4.0, 3-HFuryl), 5.09 (1H, app t, J 5.2, OH), 4.96 (1H,
dd, J 6.2 and 3.9, 4-HFuryl), 4.20 (1H, d, J 3.9, 5-HFuryl), 3.52 (2H, app s, CH2), 1.56 (3H, s, CH3a), 1.37
(3H, s, CH3b); δC (100 MHz, DMSO-d6): 158.2 (C4Ar), 156.4 (C8Ar), 149.8 (C6Ar), 121.4 (C9Ar), 119.3
(C2Ar) 113.6 (Acetyl C), 89.6 (C2Furyl), 87.3 (C5Furyl), 84.0 (C3Furyl), 81.7 (C4Furyl), 62.0 (CH2), 27.5
(CH3a), 25.7 (CH3b); νmax/ cm-1 (solid): 3473, 3299, 1761, 1651, 1381; HPLC: Tr= 2.30 (100% rel. area);
m/z (ES): (Found: [M+Na]+, 455.8. C13H16IN5O4 requires [M+Na], 455.8. [α]D = -102.0° (c 0.1, MeOH).
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Preparation of 2’3’-O-Isopropylidene-2-phenyladenosine (11). Preparation was via general method A
using 2’3’-O-Isopropylidene-2-chloroadenosine (9) (0.50 g, 1.46 mmol) and phenyl boronic acid (0.71 g,
5.85 mmol) to afford the desired product as a colourless powder (0.43 g, 1.12 mmol, 76%); m.p.: 166.2-
168.1 °C; Rf: 0.58 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 8.36 (3H, app s, 6-HAr, 2’-HAr
and 6’-HAr), 7.46 (3H, app s, 4’-HAr, 3’-HAr and 5’-HAr), 7.39 (2H, brs, NH2), 6.26 (1H, app s, 2-HFuryl),
5.52 (1H, app s, 3-HFuryl), 5.10 (1H, app s, 4-HFuryl), 5.04 (1H, app s, OH), 4.22 (1H, app s, 4-HFuryl),
3.69-3.51 (2H, m, CH2), 1.59 (3H, s, CH3a), 1.37 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 158.5 (C2Ar),
156.4 (C4Ar), 150.4 (C8Ar), 140.8 (C6Ar), 138.8 (C1’Ar), 134.4 (C4’Ar), 128.7 (C3’Ar and C5’Ar), 128.2
(C2’ Ar and C6’Ar), 118.7 (C9Ar), 113.6 (Acetyl C), 89.5 (C2Furyl), 87.2 (C5Furyl), 83.8 (C3Furyl), 81.9
(C4Furyl), 62.0 (CH2), 27.6 (CH3a), 25.7 (CH3b); νmax/ cm-1 (solid): 3314, 3157, 1655, 1596, 1372; HPLC:
Tr= 2.24 (100% rel. area); m/z (ES): (Found: [M+H]+, 384.1676. C19H21N5O4 requires [M+H], 384.1666.
[α]D = -1.4° (c 0.1, MeOH).
Preparation of 2’3’-O-Isopropylidene-2-propenyladenosine (12). Preparation was via general method
A using 2’3’-O-Isopropylidene-2-chloroadenosine (9) (0.50 g, 1.46 mmol) and transpropenyl boronic acid
(0.50 g, 5.85 mmol) to afford the desired product as a colourless powder (0.17 g, 0.50 mmol, 34%); m.p.:
81.6-83.4 °C; Rf: 0.58 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 8.29 (1H, s, 6-HAr), 7.22 (2H,
brs, NH2), 6.92 (1H, dd, J 15.2 and 7.2, 2-Hpropyl), 6.33 (1H, d, J 15.2, 1-Hpropyl), 6.13 (1H, app s, 2-
HFuryl), 5.34 (1H, app s, 3-HFuryl), 5.26 (1H, t, J 5.4, OH), 5.02 (1H, app s, 4-HFuryl), 4.22 (1H, app s,
5-HFuryl), 3.62-3.49 (2H, m, CH2), 1.90 (3H, d, J 7.2, CH3propyl), 1.56 (3H, s, CH3a), 1.35 (3H, s, CH3b);
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δC (125 MHz, DMSO-d6): 158.8 (C2Ar), 156.2 (C4Ar), 149.9 (C8Ar), 140.3 (C6Ar), 134.0 (C2Pro), 131.9
(C1Pro), 119.1 (C9Ar), 89.9 (C2Furyl), 86.9 (C5Furyl), 83.6 (C3Furyl), 81.9 (C4Furyl), 62.2 (CH2), 27.6
(CH3a), 25.7 (CH3b), 18.3 (CH3Pro); νmax/ cm-1 (solid): 3451, 3310, 3162, 1657, 1575, 1373; HPLC: Tr=
2.11 (72% rel. area); m/z (ES): (Found: [M+H]+, 348.3. C16H21N5O4 requires [M+H], 348.5. [α]D = -50.2°
(c 0.1, MeOH).
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Preparation of 2’3’-O-Isopropylidene-2-(furan-2-yl)adenosine (13). Preparation was via general
method A using 2’3’-O-Isopropylidene-2-chloroadenosine (9) (0.50 g, 1.46 mmol) and 2-furyl boronic acid
(0.66 g, 5.85 mmol) to afford the desired product as a colourless powder (0.25 g, 0.67 mmol, 46%); m.p.:
96.3-98.2 °C; Rf: 0.59 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 8.35 (1H, s, 6-HAr), 7.83 (1H,
5’-HAr), 7.44 (2H, brs, NH2), 7.13 (1H, s, 3’-HAr), 6.65 (1H, s, 4’-HAr), 6.20 (1H, app s, 2-HFuryl), 5.40
(1H, app s, 3-HFuryl), 5.10 (1H, app s, 4-HFuryl), 5.05 (1H, app s, OH), 4.22 (1H, app s, 5-HFuryl), 3.67-
3.52 (2H, m, CH2), 1.55 (3H, s, CH3a), 1.35 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 156.8 (C2Ar), 156.0
(C4Ar), 152.6 (C2’Ar), 149.3 (C8Ar), 144.2 (C5’Ar), 140.2 (C6Ar), 117.9 (C9Ar), 113.0 (Acetyl C), 112.0
(C3’Ar), 111.4 (C4’Ar), 89.0 (C2Furyl), 86.8 (C5Furyl), 83.3 (C3Furyl), 81.5 (C4Furyl), 61.6 (CH2), 27.9
(CH3a), 25.2 (CH3b); νmax/ cm-1 (solid): 3419, 3311, 2987, 2938, 1639, 1547, 1369; HPLC: Tr= 2.20 (57%
rel. area); m/z (ES): (Found: [M+H]+, 374.1462. C17H19N5O5 requires [M+H], 374.1459. [α]D = -44.8° (c
0.1, MeOH).
Preparation of 2’3’-O-Isopropylidene-2-(thiophen-3-yl)adenosine (14). Preparation was via general
method A using 2’3’-O-Isopropylidene-2-chloroadenosine (9) (0.50 g, 1.46 mmol) and 3-thienyl boronic
acid (0.75 g, 5.85 mmol) to afford the desired product as a colourless powder (0.46 g, 1.17 mmol, 80%);
m.p.: 100.1-102.0 °C; Rf: 0.62 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 8.34 (1H, s, 6-HAr),
8.18 (1H, dd, J 3.1 and 1.2, 2’-HAr), 7.79 (1H, dd, J 5.0 and 1.2, 4’-HAr), 7.42 (1H, dd, J 5.0 and 3.1, 5’-
H), 7.36 (2H, brs, NH2), 6.23 (1H, d, J 6.2, 2-HFuryl), 5.49 (1H, dd, J 6.2 and 2.7, 3-HFuryl), 5.11 (1H, dd,
J 6.2 and 2.7, 4-HFuryl), 4.22 (1H, t, J 5.5 and 2.7, 5-HFuryl), 3.59 (2H, dd, J 11.5 and 5.5, CH2), 1.57 (3H,
s, CH3a), 1.36 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 173.2 (C2Ar), 155.8 (C4Ar), 140.3 (C6Ar), 127.4
(C4’Ar), 125.1 (C5’Ar), 120.0 (C2’Ar), 119.2 (C9Ar), 116.7 (C1’Ar), 114.2 (AcetylC), 90.6 (C2Furyl),
87.3 (C5Furyl), 83.6 (C3Furyl), 81.2 (C4Furyl), 61.8 (CH2), 26.7 (CH3a), 25.0 (CH3b); νmax/ cm-1 (solid):
3253, 2939, 1633, 1586, 1344; HPLC: Tr= 2.19 (79% rel. area); m/z (ES): (Found: [M+H]+, 365.1058.
C17H19N5O4S requires [M+H], 365.1051. [α]D = -6.6° (c 0.1, MeOH).
Preparation of 2’3’-O-Isopropylidene-2-(pyridine-3-yl)adenosine (15). Preparation was via general
method A using 2’3’-O-Isopropylidene-2-chloroadenosine (9) (0.50 g, 1.46 mmol) and 3-pyridine boronic
acid (0.71 g, 5.85 mmol) to afford the desired product as a colourless powder (0.29 g, 0.75 mmol, 51%);
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m.p.: 122.4-124.2 °C; Rf: 0.53 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 9.49 (1h, s, 2’-HAr),
8.67-8.61 (2H, m, 6’-HAr and 4’-HAr), 8.40 (1H, s, 6-HAr), 7.52 (3H, brs, NH2 and 5’-HAr), 6.26 (1H, d,
J 2.7, 2-HFuryl), 5.52 (1H, dd, J 6.1 and 2.7, 3-HFuryl), 5.10 (1H, dd, J 6.1 and 2.9, 4-HFuryl), 5.05 (1H,
t, J 5.5, OH), 4.23 (1H, dd, J 8.2 and 5.3, 5-HFuryl), 3.62-3.51 2H, m, CH2), 1.57 (3H, s, CH3a), 1.36 (3H,
s, CH3b); δC (125 MHz, DMSO-d6): 160.3 (C2Ar), 156.5 (C4Ar), 150.8 (C4Pip), 150.2 (C8Ar), 149.5
(C2Pip), 141.1 (C6Ar), 134.5 (C6Pip), 134.1 (C1Pip), 124.0 (C5Pip), 119.0 (C9Ar), 113.5 (Acetyl C), 89.7
(C2Furyl), 87.2 (C5Furyl), 83.8 (C3furyl), 81.9 (C4Furyl), 62.0 (CH2), 27.6 (CH3a), 25.7 (CH3b); νmax/ cm-1
(solid): 3322, 1632, 1572, 1375; HPLC: Tr= 1.99 (100% rel. area); m/z (ES): (Found: [M+H]+, 385.1626.
C18H20N6O4 requires [M+H], 385.1619. [α]D = -1.4° (c 0.1, MeOH).
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Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-chloro-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-
2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (16). Preparation was via general method B using 2’3’-
O-Isopropylidene-2-chloroadenosine (9) (0.17 g, 0.49 mmol, 1.0 equiv.) to afford the desired product as a
colourless glassy solid (0.19 g, 0.46 mmol, 94%) m.p.: 69.4-71.7 °C; Rf: 0.46 (9:1 Chloroform-MeOH); δH
(500 MHz, DMSO-d6): 8.33 (1H, s, 6-HAr), 7.90 (2H, brs, NH2), 7.60 (2H, brs, SNH2), 6.18 (1H, d, J 2.5,
2-HFuryl), 5.36 (1H, dd, J 6.2 and 2.3, 3-HFuryl), 5.03 (1H, dd, J 6.2 and 3.4, 4-HFuryl), 4.43 (1H, dd, J
9.0 and 3.4, 5-HFuryl), 4.23 (2H, ddd, J 17.2, 9.0 and 3.4, CH2), 1.57 (3H, s, CH3a), 1.36 (3H, s, CH3b); δC
(125 MHz, DMSO-d6): 156.9 (C4Ar), 153.2 (C2Ar), 149.8 (C8Ar), 139.9 (C6Ar), 118.1 (C9Ar), 113.7 (C
Acetyl), 88.8 (C2Furyl), 83.7 (C4Furyl), 83.4 (C3Furyl), 80.9 (C4Furyl), 68.1 (CH2), 26.9 (CH3a), 25.2
(CH3b); νmax/ cm-1 (solid): 3321, 3171, 1594, 1206; HPLC: Tr= 2.30 (95% rel. area); m/z (ES): (Found:
[M+H]+, 421.0695. C13H17ClN6O6S requires [M+H], 421.0692. [α]D = -20.2° (c 0.1, MeOH).
Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-iodo-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-
furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (17). Preparation was via general method B using 2’3’-O-
Isopropylidene-2-iodoadenosine (10) (0.20 g, 0.46 mmol) to afford the desired product as a colourless
glassy solid (0.20 g, 0.39 mmol, 85%) m.p.: 70.2-71.7 °C; Rf: 0.48 (9:1 Chloroform-MeOH); δH (500 MHz,
DMSO-d6): 8.24 (1H, s, 6-HAr), 7.80 (2H, brs, NH2), 7.59 (2H, brs, SNH2), 6.18 (1H, d, J 4.0, 2-HFuryl),
5.33 (1H, dd, J 6.1 and 4.0, 3-HFuryl), 5.02 (1H, dd, J 6.1 and 3.8), 4.43 (1H, app d, J 3.8, 5-HFuryl), 4.28-
4.12 (2H, m, CH2), 1.63 (3H, s, CH3a), 1.38 (3H, s, CH3b); δC (100 MHz, DMSO-d6): 156.5 (C4Ar), 156.4
(C8Ar), 149.7 (C6Ar), 121.5 (C9Ar), 119.4 (C2Ar), 114.2 (Acetyl C), 89.1 (C2Furyl), 84.3 (C5Furyl), 84.1
(C3Furyl), 81.4 (C4Furyl), 68.5 (CH2), 27.4 (CH3a), 25.7 (CH3b); νmax/ cm-1 (solid): 3321, 3171, 1594,
1206; HPLC: Tr= 2.35 (100% rel. area); m/z (ES): (Found: [M+Na]+, 442.9. C13H17IN6O6S requires
[M+Na], 442.9. [α]D = -21.9 ° (c 0.1, MeOH).
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Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-phenyl-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-
2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (18). Preparation was via general method B using 2’3’-
O-Isopropylidene-2-phenyladenosine (11) (0.40 g, 1.04 mmol) to afford the desired product as a colourless
glassy solid (0.23 g, 0.50 mmol, 48%) m.p.: 89.9-91.2 °C; Rf: 0.50 (9:1 Chloroform-MeOH); δH (500 MHz,
DMSO-d6): 8.38-8.33 (3H, m, 6-HAr, 2’-HAr and 6’-HAr), 7.60 2H, brs, SNH2), 7.54-7.45 (3H, m, 3’-
HAr, 4’-HAr and 5’-HAr), 7.42 2H, brs, NH2), 6.34 (1H, app s, 2-HFuryl), 5.36 1H, d, J 6.3, 3-HFuryl),
5.20 (1H, app s, 4-HFuryl), 4.46 (1H, app s, 5-HFuryl), 4.25-4.15 (2H, m, CH2), 1.60 (3H, s, CH3a), 1.38
(3H, s, CH3b); δC (125 MHz, DMSO-d6): 158.6 (C2Ar), 156.4 (C4Ar), 150.3 (C8Ar), 140.8 (C6Ar), 138.7
(C1’Ar), 130.2 (C4’Ar), 129.2 (C3’Ar and C5’Ar), 128.7 (C6’Ar and C2’Ar), 119.1 (C9Ar), 89.3
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(C2Furyl), 84.1 (C5Furyl), 83.8 (C3Furyl), 81.6 (C4Furyl), 68.6 (CH2), 27.5 (CH3a), 25.7 (CH3b); νmax
/
cm-1 (solid): 3354, 2936, 1628, 1376; HPLC: Tr= 2.27 (100% rel. area); m/z (ES): (Found: [M+H]+,
463.1401. C19H22N6O6S requires [M+H], 463.1394. [α]D = 17.6° (c 0.1, MeOH).
Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-propenyl-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-
2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (19). Preparation was via general method B using 2’3’-
O-Isopropylidene-2-propenyladenosine (12) (0.15 g, 0.43 mmol) to afford the desired product as a pale
yellow oil (0.14 g, 0.33 mmol, 77%) Rf: 0.47 (9:1 Chloroform-MeOH); δH (500 MHz, DMSO-d6): 8.25
(1H, s, 6-HAr), 7.61 2H, brs, SNH2), 7.23 (2H, brs, NH2), 6.90 (1H, dd, J 13.4 and 6.6, 2-HPropyl), 6.36
(1H, d, J 13.4, 1-HPropyl), 6.24 (1H, app s, 2-HFuryl), 5.42 (1H, app s, 3-HFuryl), 5.15 (1H, app s, 4-
HFuryl), 4.40 (1H, app s, 5-HFuryl), 4.35-4.25 (2H, m, CH2), 1.90 (3H, d, J 6.6, CH3 Propyl), 1.57 (3H, s,
CH3a), 1.36 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 171.0 (C2Ar), 156.1 (C4Ar), 150.9 (C8Ar), 140.0
(C6Ar), 125.5 (C2Pro), 119.4 (C9Ar), 117.7 (C1Pro), 112.4 (Acetyl C), 88.2 (C2Furyl), 83.4 (C5Furyl),
83.1 (C3Furyl), 81.4 (C4Furyl), 70.5 (CH2), 26.7 (CH3a), 25.0 (CH3b), 18.8 (CH3Pro); νmax/ cm-1 (solid):
3326, 3182, 2987, 2938, 1622, 1585, 1374; HPLC: Tr= 2.14 (67% rel. area); m/z (ES): (Found: [M+H]+,
427.1400. C16H22N6O6S requires [M+H], 427.1394. [α]D = 16.5° (c 0.1, MeOH).
Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(furan-2-yl)-9H-purin-9-yl)-2,2-dimethyl-
tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (20). Preparation was via general method
B using 2’3’-O-Isopropylidene-2-(furan-2-yl)adenosine (13) (0.25g, 0.67 mmol) to afford the desired
product as a colourless glassy solid (0.20 g, 0.45 mmol, 67%) m.p.: 83.3-85.2 °C; Rf: 0.46 (9:1 Chloroform-
MeOH); δH (500 MHz, DMSO-d6): 8.31 (1H, s, 6-HAr), 7.83 (1H, s, 3’-HAr), 7.56 (2H, brs, SNH2), 7.47
(2H, brs, NH2), 7.12 (1H, d, J 3.2, 5’-HAr), 6.65 (1H, dd, J 3.2 and 1.8, 4’-HAr), 6.30 (1H, d, J 1.9, 2-
HFuryl), 5.44 (1H, d, J 6.2 3-HFuryl), 5.23 (1H, dd, J 6.2 and 3.5, 4-HFuryl), 4.43 (1H, app s, 5-HFuryl),
4.35-4.15 (2H, m, CH2), 1.58 (3H, s, CH3a), 1.36 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 159.5 (C2Ar),
155.8 (C4Ar), 152.3 (C1’Ar), 150.6 (C8Ar), 145.0 (C3’Ar), 140.0 (C6Ar), 119.4 (C9Ar), 114.4 (Acetyl C),
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113.3 (C5’Ar), 112.1 (C4’Ar), 90.7 (C2Furyl), 84.4 (C5Furyl), 83.3 (C3Furyl), 81.1 (C4Furyl), 70.5 (CH2),
26.7 (CH3a), 24.5 (CH3b); νmax/ cm-1 (solid): 3319, 3161, 2988, 1628, 1577, 1361; HPLC: Tr= 2.27 (51%
rel. area); m/z (ES): (Found: [M+H]+, 453.1189. C17H20N6O7S requires [M+H], 453.1187. [α]D = 1.4° (c
0.1, MeOH).
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Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(thiophen-3-yl)-9H-purin-9-yl)-2,2-dimethyl-
tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (21). Preparation was via general method
B using 2’3’-O-Isopropylidene-2-(thiophen-3-yl)adenosine (14) (0.45 g, 1.16 mmol) to afford the desired
product as a pale brown oil (0.51 g, 1.09 mmol, 94%) Rf: 0.48 (9:1 Chloroform-MeOH); δH (500 MHz,
DMSO-d6): 8.31 91H, s, 6-HAr), 8.18 (1H, d, J 2.0, 2’-HAr), 7.77 (1H, dd, J 6.1 and 2.0, 5’-HAr), 7.58
(2H, brs, SNH2), 7.42 (1H, d, J 6.1, 4’-H), 7.38 (2H, brs, NH2), 6.31 (1H, d, J 2.2, 2-HFuryl), 5.54 (1H, d,
J 4.0, 3-HFuryl), 5.20 (1H, d, J 4.0, 4-H Furyl), 4.44 (1H, app s, 5-HFuryl), 4.35-4.15 (2H, m, CH2), 1.60
(3H, s, CH3a), 1.37 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 169.5 C2Ar), 155.9 (C4Ar), 149.4 (C8Ar),
142.2 (C6Ar), 134.8 (C4’Ar), 132.4 (C5’Ar), 126.4 (C9Ar), 125.0 (C2’Ar), 117.9 (C1’Ar), 113.5 (Acetyl
C), 88.9 (C2Furyl), 83.7 (C5Furyl), 83.2 (C3Furyl), 81.2 (C4Furyl), 68.1 (CH2), 26.7 (CH3a), 25.1 (CH3b);
νmax/ cm-1 (solid): 3324, 3200, 2988, 1619, 1398; HPLC: Tr= 2.23 (82% rel. area); m/z (ES): (Found:
[M+H]+, 469.0958. C17H20N6O6S2 requires [M+H], 469.0959. [α]D = 6.0° (c 0.1, MeOH).
Preperation of [(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(pyridine-3-yl)-9H-purin-9-yl)-2,2-dimethyl-
tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate (22). Preparation was via general method
B using 2’3’-O-Isopropylidene-2-(pyridine-3-yl)adenosine (15) (0.29 g, 0.75 mmol) to afford the desired
product as a colourless glassy solid ( 0.25 g, 0.54 mmol, 72%) m.p.: 93.1-94.7 °C; Rf: 0.39 (9:1 Chloroform-
MeOH); δH (500 MHz, DMSO-d6):9.48 (1H, s, 2’-HAr), 8.67-8.59 (2H, m, 6’-H and 4’-H), 8.37 (1h, s, 6-
HAr), 7.65-7.50 (5H, m, SNH2, NH2 and 3’-HAr), 6.34 (1H, d, J 2.2, 2-HFuryl), 5.58 (1H, d, J 3.7 3-
HFuryl), 5.19 (1H, dd, J 6.0 and 3.7, 4-HFuryl), 4.45 (1H, app s, 5-HFuryl), 4.30-4.20 (2H, m, CH2), 1.60
(3H, s, CH3a), 1.39 (3H, s, CH3b) ; δC (125 MHz, DMSO-d6): 161.7 (C2Ar), 155.8 C4Ar), 150.8 (C4’Ar),
150.6 (C8Ar), 149.5 (C2’Ar), 141.1 (C6Ar), 135.4 (C6’Ar), 133.2 (C1’Ar), 124.0 (C5’Ar), 119.1 (C9Ar),
114.2 (Acetyl C), 89.4 (C2Furyl), 84.0 (C5Furyl), 83.7 (C3Furyl), 81.6 (C4Furyl), 68.0 (CH2), 27.5 (CH3a),
25.7 (CH3b); νmax/ cm-1 (solid): 3345, 3180, 2985, 1632, 1580, 1374; HPLC: Tr= 2.04 (100% rel. area); m/z
(ES): (Found: [M+H]+, 464.1357. C18H21N7O6S requires [M+H], 464.1347. [α]D = 53.5° (c 0.1, MeOH).
Preparation of N-Boc-Ser(bzl)-OSu (23). To a stirred solution of N-Boc-(Bzl)-Ser-OH (0.50 g, 1.69
mmol, 1.0 equiv.) in EtOAc/Dioxane (1:1, 10 mL) cooled to 0 °C were added N-hydroxysuccinimide (0.21
g, 1.78 mmol, 1.05 equiv.) and DCC (0.37 g, 1.78 mmol, 1.05 equiv.). The resulting mixture was stirred at
room temperature overnight. The reaction mixture was filtered through Celite® and the filtrate concentrated
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in vacuo. The residue was re-suspended in EtOAc (35 mL), washed with 5% NaHCO3 (3 × 5 mL), water (2
× 10 mL), and brine (10 mL). The organic phase was dried (MgSO4) and concentrated in vacuo to afford
the desired product as a colourless solid which was used without further purification. (0.35 g, 0.90 mmol,
53 %) m.p.:98.3-99.2 °C; Rf: 0.5 (9:1 DCM-MeOH); δH (500 MHz, DMSO-d6): 7.45-7.28 (5H, m, H-Bzl),
4.69 (1H, d, J 5.7, H-Chiral), 4.55 (2H, s, CH2Bzl), 3.80 (2H, d, J 5.7, CH2), 2.82 (4H, brs, CH2C=O), 1.41
(9H, s, BOC); δC (125 MHz, DMSO-d6): 169.8 (Succinimide C=O), 166.8 (C=O), 155.2 (BOC C=O), 137.8
(C1Ar), 128.2 (C3 and C5), 127.5 (C4), 127.5 (C2 and C6), 78.9 (C BOC), 72.3 (CH2Bzl), 68.6 (CH2C),
52.3 (Chiral C), 28.1 (CH3 × 3), 25.4 (CH2 × 2); νmax/ cm-1 (solid): 3366, 2970, 1741, 1518, 1366; HPLC:
Tr= 2.61 (83% rel. area); m/z (ES): (Found: [M+H]+, 393.1663. C19H24N2O7 requires [M+H], 393.1656.
[α]D = -7.6° (c 0.1, MeOH).
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Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-chloro-9H-purin-9-yl)-2,2-
dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (24). Preparation was via general method C using [(3aR, 4R, 6R, 6aR)-6-(6-
amino-2-chloro-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate
(16) (0.15 g, 0.36 mmol) to afford the desired product as a colourless glassy solid (0.19 g, 0.46 mmol, 94%)
m.p.: 168.6-170.2 °C; Rf: 0.51 (9:1 DCM-MeOH); δH (500 MHz, DMSO-d6): 8.43 (1H, s, 6-HAr), 7.83
(2H, brs, NH2), 7.30-7.20 (5H, m, Bzl), 6.10 (1H, d, J 5.7, 2-HFuryl), 6.04 (1H, brs, NH), 5.25 (1H, d, J
5.7, 3-HFuryl), 4.94 (1H, d, J 5.7, 4-HFuryl), 4.47 (2H, s, CH2 Bzl), 4.35 (1H, d, J 5.7, 5-HFuryl), 4.07-
4.01 (3H, m, CH2Chiral and Chiral H), 3.69-3.67 (2H, m, CH2), 1.55 (3H, s, CH3a), 1.38 (9H, s, CH3 × 3),
1.29 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 171.6 (C=O), 157.8 (C4Ar), 155.4 (C=O BOC), 153.9
(C2Ar), 151.0 (C8Ar), 140.3 (C6Ar), 138.0 (C1Bzl), 128.7 (C3Bzl and C5Bzl), 128.3 (C2Bzl and C6Bzl),
127.9 (C4Bzl), 119.1 (C9Ar), 114.4 (C acetate), 90.6 (C2Furyl) 84.4 (C5Furyl), 83.3 (C3Furyl), 81.4
(C4Furyl), 79.5 (BOC C), 73.7 (CH2 Bzl), 70.5 (CH2O), 69.8 (CH2C), 54.9 (Chiral C), 28.3 (CH3 × 3),
26.7 (CH3a), 24.9 (CH3b); νmax/ cm-1 (solid):3330, 1691, 1637, 1304; HPLC: Tr= 2.59 (100% rel. area); m/z
(ES): (Found: [M+H]+, 698.2012. C28H36ClN7O10S requires [M+H], 698.2006. [α]D = -50.4° (c 0.1, MeOH).
Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-iodo-9H-purin-9-yl)-2,2-
dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (25). Preparation was via general method C using [(3aR, 4R, 6R, 6aR)-6-(6-
amino-2-iodo-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate
(17) (0.20 g, 0.39 mmol) to afford the desired product as a colourless glassy solid (40.4 mg, 0.05 mmol,
13%) m.p.: 166.2-168.9°C; Rf: 0.54 (DCM-MeOH); δH (400 MHz, DMSO-d6): 8.38 (1H, s, 6-HAr), 7.75
(2H, brs, NH2), 7.30-7.20 (5H, m, Bzl), 6.07 (1H, app s, 2-HFuryl), 5.24 (1H, app s, 3-HFuryl), 4.92 (1H,
app s, 4-HFuryl), 4.42 (2H, app s, CH2 Bzl), 4.35 (1H, app s, 5-HFuryl), 4.08-4.01 (3H, m, CH2 Chiral and
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Chiral H) 3.70-3.62 (2H, m, CH2), 1.54 (3H, s, CH3a), 1.38 (9H, s, CH3 × 3), 1.20 (3H, s, CH3b); δC (100
MHz, DMSO-d6): 174.5 (C=O), 157.3 (C4Ar), 155.5 (C=O BOC), 152.3 (C8Ar), 139.0 (C6Ar), 137.7
(C1Bzl), 128.7 (C5Bzl and C3Bzl), 128.1 (C4Bzl), 127.8 (C2’Ar and C6’Ar), 118.5 (C9Ar), 116.4 (C2Ar),
114.4 (Acetyl C), 88.3 (C2Furyl), 82.8 C5Furyl), 81.5 (C3Furyl), 79.6 (BOC C), 79.0 (C4Furyl), 73.5 (CH2
Bzl); 69.9 (CH2O), 67.4 (CH2C), 56.4 (Chiral C), 28.3 (CH3 × 3), 26.4 (CH3a), 24.9 (CH3b); νmax/ cm-1
(solid): 3330, 1691, 1637, 1304; HPLC: Tr= 2.67 (100% rel. area); m/z (ES): (Found: [M+Na]+, 812.1.
C28H36IN7O10S requires [M+Na], 812.1. [α]D = -52.2° (c 0.1, MeOH).
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Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-phenyl-9H-purin-9-yl)-2,2-
dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (26). Preparation was via general method C using [(3aR, 4R, 6R, 6aR)-6-(6-
amino-2-phenyl-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl sulfamate
(18) (0.20 g, 0.43 mmol) to afford the desired product as a colourless glassy solid (0.21 g, 0.29 mmol, 67%)
m.p.: 155.8-157.7 °C; Rf: 0.64 (9:1 DCM-MeOH); δH (500 MHz, DMSO-d6): 8.42(1H, s, 6-HAr), 8.37 (2H,
d, J 6.9 6’-HAr and 2’-HAr), 7.57-7.41 (3H, m, 5’-HAr, 4’-HAr and 3’-HAr), 7.38 (2H, brs, NH2), 7.26-
7.24 (5H, m, 2-HBzl, 3-HBzl, 4-HBzl, 5-HBzl and 6-HBzl), 6.27 (1H, d, J 3.0, 2-HFuryl), 6.06 (1H, d J
8.1, NH), 5.46 (1H, d, J 3.0, 3-HFuryl), 5.07 (1H, app s, 4-HFuryl), 4.45-4.41 (3H, m, BzlCH2, 5-HFuryl),
4.05 (2H, d, J 4.9, CH2O), 3.96-3.92 (1H, m, Chiral H), 3.67-3.58 (2H, m, Chiral CH2), 1.59 (3H, s, CH3a),
1.39 (9H, s, CH3 × 3), 1.35 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 173.5 (C=ONH), 158.7 (C2Ar), 156.8
(C4Ar), 156.4 (C=O BOC), 150.6 (C8Ar), 140.0 (C6Ar), 138.5 (C1’Ar and C1Bzl), 130.4 (C4’Ar), 129.6-
127.1 (C2Bzl, C3Bzl, C4Bzl, C5Bzl, C6Bzl, C2’Ar, C3’Ar, C5’Ar and C6’Ar), 118.0 (C9Ar), 112.5
(Acetyl C), 88.8 (C2Furyl), 83.5 (C5Furyl), 83.4 (C3Furyl), 81.6 (C4Furyl), 79.5 (BOC C), 71.8 (CH2 Bzl),
71.1 (Chiral CH2), 67.1 (CH2), 57.1 (Chiral C), 28.2 (CH3 × 3), 27.1 (CH3a), 25.2 (CH3b); νmax/ cm-1 (solid):
3358, 3193, 2980, 1629, 1575, 1438; HPLC: Tr= 2.54 (100% rel. area); m/z (ES): (Found: [M+H]+,
740.2718. C34H41N7O10S requires [M+H], 740.2708. [α]D = 17.9° (c 0.1, MeOH).
Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-propenyl-9H-purin-9-yl)-
2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (27). Preparation was via general method C using [(3aR, 4R, 6R, 6aR)-6-(6-
amino-2-propenyl-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl
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sulfamate (19) (0.14 g, 0.33 mmol) to afford the desired product as a colourless glassy solid (64.9 mg, 0.09
mmol, 28%) m.p.: 142.9-144.3 °C; Rf: 0.47 (9:1 DCM-MeOH); δH (500 MHz, DMSO-d6): 8.33 (1H, s, 6-
HAr), 7.30-7.26 (5H, m, 2’-HAr, 3’-HAr, 4’-HAr, 5’-HAr and 6’-HAr), 7.18 (2H, brs, NH2), 6.92 (1H, dd,
J 15.3 and 7.0, 2-HPro), 6.35 (1H, d, J 15.3, 1-HPro), 6.15 (1H, d, J 2.8, 2-HFuryl), 6.06 (1H, d, J 7.7 NH),
5.33 (1H, dd, J 6.3 and 2.8, 3-HFuryl), 5.04 (1H, app t, J 6.3, 4-HFuryl), 4.42 (2H, d, J 9.9, CH2 Bzl), 4.36
(1H, d J 2.4, 5-HFuryl), 4.15-3.95 (3H, m, CH2O and ChiralH), 3.73-3.54 (2H, m, CH2 Chiral), 1.89 (3H,
dd, J 7.0 and 1.5, Me), 1.56 (3H, s, CH3a), 1.39 (9H, s, CH3 × 3), 1.33 (3H, s, CH3b); δC (125 MHz, DMSO-
d6): 172.5 (C2Ar), 170.1 (C=ONH), 156.1 (C4Ar), 155.4 (C=OBOC), 150.8 (C8Ar), 140.3 (C6Ar), 137.8
(C1’Ar), 128.7-127.5 (Aromatics), 125.2 (C2pro), 119.1 (C9Ar), 117.7 (C1Pro), 114.3 (Acetyl C), 88.9
(C2Furyl), 83.7 (C5Furyl), 83.4 (C3Furyl), 81.7 (C4Furyl), 79.5 (BOC C), 71.8 (CH2 Bzl), 71.1 (CH2O),
67.1 (CH2 Chiral), 56.5 (Chiral C), 28.2 (CH3 × 3), 27.1 (CH3a), 25.2 (CH3b), 17.8 (CH3Pro); νmax/ cm-1
(solid): 3359, 2968, 1627, 1579, 1345; HPLC: Tr= 2.45 (78% rel. area); m/z (ES): (Found: [M+H]+,
704.2719. C31H41N7O10S requires [M+H], 704.2708. [α]D = -8.6° (c 0.1, MeOH).
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Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(furan-2-yl)-9H-purin-9-yl)-
2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-1-
oxopropan-2-yl]carbamate (28). Preparation was via general method C using [(3aR, 4R, 6R, 6aR)-6-(6-
amino-2-(furan-2-yl)-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl
sulfamate (20) (0.20 g, 0.44 mmol) to afford the desired product as a colourless glassy solid (0.10 g, 0.14
mmol, 32%) m.p.:157.7-159.4 °C; Rf: 0.50 (9:1 DCM-MeOH); δH (500 MHz, DMSO-d6): 8.40 (1H, s, 6-
HAr), 7.81 (1h, s, 3’-HAr), 7.42 (2H, brs, NH2), 7.32-7.28 (5H, m, Bzl), 7.24 (1H, d, J 4.1, 5’-HAr), 6.62
(1H, dd, J 4.1 and 1.87, 4’-HAr), 6.21 (1H, d, J 2.8, 2-HFuryl), 6.06 (1H, app s, NH), 5.36 (1H, app s, 3-
HFuryl), 5.08 (1H, app s, 4-HFuryl), 4.48-4.32 (3H, m, CH2 Bzl and 5-HFuryl), 4.15-3.90 (3H, m, CH2O
and Chiral H), 3.70-3.50 (2H, m, CH2Chiral), 1.57 (3H, s, CH3a), 1.38 (9H, s, CH3 × 3), 1.33 (3H, s, CH3b);
δC (125 MHz, DMSO-d6): 170.0 (C=ONH), 158.3 (C2Ar), 156.4 (C4Ar), 155.3 (C=OBOC), 152.5
(C1Furan), 150.6 (C8Ar), 144.7 (C3Furan), 140.2 (C6Ar), 137.7 (C1Bzl), 128.8-127.7 (Aromatics), 119.3
(C9Ar), 114.2 (Acetyl C), 112.4 (C5Furan), 111.9 (C4Furan), 89.2 (C2Furyl), 84.4 (C5Furyl), 84.1
(C3Furyl), 82.1 (C4Furyl), 79.6 (BOC C), 72.2 (CH2 Bzl), 71.0 (CH2O), 67.7 (CH2 Chiral), 56.9 (Chiral
C), 28.7 (CH3 × 3), 27.6 (CH3a), 25.7 (CH3b); νmax/ cm-1 (solid): 3342, 1674, 1572, 1364; HPLC: Tr= 2.51
(73% rel. area); m/z (ES): (Found: [M+H]+, 730.2513. C32H39N7O11S requires [M+H], 730.2501. [α]D = -
24.8° (c 0.1, MeOH).
Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(thiophen-3-yl)-9H-purin-9-
yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)sulfonyl)amino]-3-(benzyloxy)-
1-oxopropan-2-yl]carbamate (29). Preparation was via general method C using [(3aR, 4R, 6R, 6aR)-6-
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(6-amino-2-(thiophen-3-yl)-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-
yl]methyl sulfamate (21) (0.50 g, 1.06 mmol) to afford the desired product as a colourless glassy solid (0.33
g, 0.45 mmol, 42%) m.p.: 156.2-158.0 °C; Rf: 0.58 (9:1 DCM-MeOH); δH (500 MHz, DMSO-d6): 8.38
(1H, s, 6-HAr), 8.19 (1H, dd, J 3.1 and 1.1, 2’-HAr), 7.78 (1h, dd, J 5.1 and 1.1 4’-HAr), 7.57 (1H, dd, J
5.0 and 3.1, 5’-HAr), 7.30 (2H, brs, NH2), 7.29-7.25 (5H, m, bzl), 6.23 (1H, d, J 3.0, 2-HFuryl), 6.07 (1H,
app s, NH), 5.41 (1H, d, J 2.8, 3-HFuryl), 5.07 (1H, d, J 2.8, 4-HFuryl), 4.41 (3H, app d, J 9.9, CH2Bzl) and
5-HFuryl, 4.10-4.00 (2H, m, CH2O), 3.98-3.94 (1H, m, Chiral H), 3.70-3.50 (2H, m, CH2Chiral), 1.58 (3H,
s, CH3a), 1.39 (9H, s, CH3 × 3), 1.35 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 173.2 (C2Ar), 170.1
(C=ONH), 156.1 (C4Ar), 155.9 (C=OBOC), 150.8 (C8Ar), 142.8 (C6Ar), 138.5 (C1Bzl), 128.0 (C5Bzl
and C3Bzl), 127.3 (C6Bzl and C2Bzl), 127.1 (C4Bzl), 126.1 (C4Thio), 125.1 (C5Thio), 120.0 (C2Thio),
119.4 (C9Ar), 117.0 (C1Thio), 113.1 (Acetyl C), 88.7 (C2Furyl), 83.6 (C5Furyl), 83.3 (C3Furyl), 81.6
(C4Furyl), 79.7 (BOC C), 73.4 (CH2 Bzl), 71.7 (CH2O), 68.9 (CH2 Chiral), 56.2 (Chiral C), 28.2 (CH3 ×
3), 27.1 (CH3a), 25.2 (CH3b); νmax/ cm-1 (solid): 3250, 3052, 1675, 1526, 1374; HPLC: Tr= 2.53 (100% rel.
area); m/z (ES): (Found: [M+H]+, 746.2286. C32H39N7O10S2 requires [M+H], 746.2273. [α]D = 28.9° (c 0.1,
MeOH).
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Preparation of tert-butyl N-[(2S)-1-[(([(3aR, 4R, 6R, 6aR)-6-(6-amino-2-(pyridine-3-yl)-9H-purin-9-
yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methoxy)2sulfonyl)amino]-3-
(benzyloxy)-1-oxopropan-2-yl]carbamate (30). Preparation was via general method C using [(3aR, 4R,
6R,
6aR)-6-(6-amino-2-(pyridine-3-yl)-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-
d][1,3]dioxol-4-yl]methyl sulfamate (22) (0.25 g, 0.54 mmol) to afford the desired product as a colourless
glassy solid (0.12 g, 0.16 mmol, 29%) m.p.: 167.2-164.5 °C; Rf: 0.39 (9:1 DCM-MeOH); δH (500 MHz,
DMSO-d6): 9.49 (1H, s, 2’-HAr), 8.64 (2H, d, J 5.7, 6’-HAr and 4’-HAr), 8.46 (1H, s, 6-HAr), 7.52 (3H,
brs, NH2 and 5’-HAr), 7.27-7.24 (5H, m, Bzl), 6.27 (1H, d, J 3.0, 2-HFuryl), 6.08 (1H, d, J 7.8, NH), 5.43
(1H, d, J 2.7, 3-HFuryl), 5.07 (1H, app s, 4-Hfuryl), 4.41 (3H, app, s, CH2 Bzl and 5-HFuryl), 4.05 (2H, d,
J 4.6, CH2O), 3.94 (1H, s, Chiral H), 3.62 (2H, d J 6.6, CH2 Chiral), 1.59 (3H, s, CH3a), 1.36 (9H, s, CH3 ×
3), 1.35 (3H, s, CH3b); δC (125 MHz, DMSO-d6): 173.7 (C=ONH), 160.5 (C2Ar), 156.1 (C4Ar), 155.4
(C=OBOC), 150.2 (C4Pip), 150.1 (C8Ar), 148.9 (C2Pip), 140.1 (C6Ar), 137.7 (C1Bzl), 134.9 (C6Pip),
133.2 (C1Pip), 128.0 (C5Bzl and C3Bzl), 127.3 (C6Bzl and C2Bzl), 127.1 (C4Bzl), 123.5 (C5Pip), 119.4
(C9Ar), 114.2 (Acetyl C), 89.0 (C2Furyl), 83.5 (C5Furyl), 83.4 (C3Furyl), 81.5 (C4Furyl), 79.6 (BOC C),
73.7 (CH2 Bzl), 71.7 (CH2O), 68.2 (CH2 Chiral), 54.7 (Chiral C), 28.2 (CH3 × 3), 27.1 (CH3a), 25.2 (CH3b);
νmax/ cm-1 (solid): 3377, 2976, 1662, 1573, 1369; HPLC: Tr= 2.37 (100% rel. area); m/z (ES): (Found:
[M+H]+, 741.2674. C33H40N8O10S requires [M+H], 741.2661. [α]D = -13.6° (c 0.1, MeOH).
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Protein expression and purification. EcSerRS (from E. coli strain B ER2560) and SaSerRS (from S.
aureus seg50 (1150)) were cloned into pET52b(+) vector (Merck Millipore, Germany) using the NcoI and
SacI restriction sites allowing for the production of protein with a thrombin cleavable C-terminal His10-tag.
EcSerRS and SaSerRS were overexpressed in Lemo21(DE3) cells grown in Auto Induction Media –
Terrific Broth (Formedium) supplemented with 100 μg/ml ampicillin at 37°C for 8 hours followed by
overnight growth at 25°C. Cells were harvested by centrifugation at 5000 rpm in a JLA 8.1000 rotor
(Beckman Coulter) for 15 min, and the pellet was re-suspended in buffer A (50 mM Tris-HCl pH 7.5, 500
mM NaCl, 30 mM Imidazole). The cells were disrupted by sonication at 70 % amplitude for 30 sec on ice
and 8 pulses. The lysate was centrifuged at 18,000 rpm in a JA 25.50 rotor (Beckman Coulter) for 30 mins.
The supernatant was decanted, passed through a 0.2 μm filter and applied to a 5 ml HisTrap column (GE
healthcare, USA). The bound protein was eluted with a gradient of buffer B (50 mM Tris-HCl pH 7.5, 500
mM NaCl, 500 mM Imidazole) (0-100% over 50 ml) on an ÄKTA Pure (GE healthcare, USA) at 2 ml/min.
The protein was dialyzed into 2 L of buffer A with thrombin cleavage (1 unit/μg). The protein was passed
through the 5 ml HisTrap column to remove the cleaved His-tag and other contaminants. The proteins
typically present over 95 % purity at this stage as judged via SDS-PAGE gel and were taken for
crystallization trials after dialysis into 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 1 mM MgCl2. Further
purification was used for protein used for kinetic and binding studies to ensure complete removal of
thrombin using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, USA) in 20 mM Tris-HCl pH
7.5, 200 mM NaCl and 1 mM MgCl2. The purified protein was subsequently stored in 50 % glycerol at -
80°C. HsSerRS was expressed and purified as previously described.35
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Crystallisation and structure solution.
Co-crystals of EcSerRS in the presence of SerSA were obtained from a drop set up in 96-well sitting drop
format with 20ꢀmgꢀml−1 protein and ten-fold molar excess of SerSA. Drops consisted of 100 nl protein
preincubated with SerSA and 100 nl reservoir solution with a reservoir volume of 95 µl. Crystals were
obtained from a drop containing 0.2 M sodium phosphate monobasic monohydrate, pH 4.7 and 20 % (w/v)
PEG 3350 following incubation at 4°C and cryoprotected in reservoir solution containing 25 % (v/v)
ethylene glycol.
Co-crystals of His-tagged SaSerRS were obtained from a drop set up with 20 mgꢀml−1 protein in the
presence of ten-fold molar excess of SerSA in 24-well hanging drop format. Drops consisted of 1 µl protein
preincubated with SerSA and 1 µl reservoir solution with a reservoir volume of 500 µl. Plates were
incubated at 4°C and crystals obtained in 0.2 M sodium malonate pH 5.0 and 13 % (w/v) PEG 3350. Crystals
were cryoprotected for 10 s in reservoir solution containing 20 % (v/v) ethylene glycol and ten-fold molar
excess of SerSA.
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Crystals of apo-EcSerRS were obtained at 21°C from a 24-well hanging drop format as described above
with 30 mgꢀml−1 protein in a crystallisation condition consisting of 0.1 M sodium citrate pH 5.5, 0.8 M
lithium sulfate and 0.05 M ammonium sulfate. A single crystal was soaked for 30 mins in 0.1 M sodium
citrate pH 5.5, 0.75 M lithium sulfate, 0.05 M ammonium sulfate, 20 % (v/v) ethylene glycol and 100 mM
compound 8 (10 % (v/v) DMSO in final solution).
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All crystals were flash frozen in liquid nitrogen and diffraction data collected at 100 K at beamlines I03
and I04 (Diamond Light Source, United Kingdom). Data was indexed and integrated using iMosflm36
and scaled using Aimless in CCP437 or autoPROC38 was used in the DLS auto-processing pipeline. The
crystal structure of aq_298 (PDB 2DQ3, unpublished) was used as a search model in Phaser MR39 to
solve the structures of EcSerRS and SaSerRS by molecular replacement. Phenix40 and Buster41 were
used for iterative rounds of refinement with model building carried out in COOT.42 Figures were made
using PyMOL (Schrödinger, LLC).
Kinetic analyses. SerRS assays were performed at 37°C in a Cary 100 UV/Vis double beam
spectrophotometer with a thermostatted 6X6 cell changer. The final assay volume was 0.2 ml, containing
50 mM HEPES adjusted to pH 7.6, 10 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 10% (v/v)
dimethylsulphoxide, 10 mM D-glucose, 0.5 mM NADP+, 1.7 mM.min yeast hexokinase and 0.85 mM.min
L. mesenteroides glucose 6-phosphate dehydrogenase (Roche, Germany). Concentrations of SerRS, amino
acid, substrate (Ap4A, Sigma-Aldrich, Dorset, UK ) and pyrophosphate were as stated in the text
(Supplementary Table 3 and Supplementary Table 4). Unless otherwise stated, background rates were
acquired in the absence of amino acid, which was then added to initiate the full reaction. Assays were
continuously monitored at 340 nm, to detect reduction of NADP+ to NADPH, where ΔNADPH; 340nm =
6220 M-1 cm-1. Kinetic constants relating to substrate dependencies and IC50 values for inhibitors were
extracted by non-linear regression using GraphPad Prizm 7.00.
Isothermal titration calorimetry. Calorimetric titrations of EcSerRS with SerSA and/or compound 8 were
performed on a VP-ITC microcalorimeter (MicroCal) at 25°C and measured in triplicates. The gel-filtration
purified EcSerRS was concentrated and dialysed overnight against the ITC buffer (20 mM Tris-HCl, pH
7.5 and 200 mM NaCl) at 4°C. All the solutions were degassed by sonication. The overnight dialysis ITC
buffer was used to prepare SerSA and compound 8 solutions. The EcSerRS (3 µM for SerSA and 7 µM for
compound 8) in the sample cell (1.445 ml) was titrated with ligand solution (70 µM of SerSA and 140 µM
of compound 8) in the syringe (280 ul). The EcSerRS - SerSA ITC experiments consisted of a preliminary
2 µl injection followed by 52 successive 5 µl injections. The EcSerRS - compound 8 ITC experiments
consisted of a preliminary 2 µl injection followed by 26 successive 10 µl injections. Each injection lasted
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