Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins

Article abstract of DOI:10.1038/s41429-021-00408-3

Spectinomycin, an aminocyclitol antibiotic, is subject to inactivation by aminoglycoside modifying enzymes (AMEs) through adenylylation or phosphorylation of the 6-hydroxy group position. In this study, the effects of deoxygenation of the 2- and 6-hydroxy group positions on the spectinomycin actinamine ring are probed to evaluate their relationship to ribosomal binding and the antimicrobial activities of spectinomycin, semisynthetic aminomethyl spectinomycins (amSPCs), and spectinamides. To generate these analogs, an improved synthesis of 6-deoxyspectinomycin was developed using the Barton deoxygenation reaction. 6-Dehydrospectinamide was also synthesized from spectinamide 4 to evaluate the H-bond acceptor character on the C-6 position. All the synthesized analogs were tested for antibacterial activity against a panel of Gram (+) and Gram (?) pathogens, plus Mycobacterium tuberculosis. The molecular contribution of the 2- and 6-hydroxy group and the aryl functionalities of all analogs were examined by measuring inhibition of ribosomal translation and molecular dynamics experiments with MM/GBSA analysis. The results of this work indicate that the 6-hydroxy group, which is the primary target of AMEs, is a required motif for antimicrobial activity in current analogs. Removal of the 6-hydroxy group could be partially rescued by offsetting ribosomal binding contributions made by the aryl side chains found in the spectinamide and amSPCs. This study builds on the knowledge of the structure–activity relationships of spectinomycin analogs and is being used to aid the design of next-generation spectinomycins.

Full text of DOI:10.1038/s41429-021-00408-3

The Journal of Antibiotics (2021) 74:381–396
https://doi.org/10.1038/s41429-021-00408-3
ARTICLE
Synthesis, antibacterial action, and ribosome inhibition
of deoxyspectinomycins
1
1
Laura A. Wilt1 Jiuyu Liu1 Stephanie M. Reeve
Carl W. Thompson1 John M. Elmore1
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Suresh Dharuman
Dimitri Shcherbakov2 Robin B. Lee1 Erik C. Böttger2 Richard E. Lee
1
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Received: 12 September 2020 / Revised: 13 December 2020 / Accepted: 3 January 2021 / Published online: 27 January 2021
© The Author(s) 2021. This article is published with open access
Abstract
Spectinomycin, an aminocyclitol antibiotic, is subject to inactivation by aminoglycoside modifying enzymes (AMEs)
through adenylylation or phosphorylation of the 6-hydroxy group position. In this study, the effects of deoxygenation of the
2- and 6-hydroxy group positions on the spectinomycin actinamine ring are probed to evaluate their relationship to ribosomal
binding and the antimicrobial activities of spectinomycin, semisynthetic aminomethyl spectinomycins (amSPCs), and
spectinamides. To generate these analogs, an improved synthesis of 6-deoxyspectinomycin was developed using the Barton
deoxygenation reaction. 6-Dehydrospectinamide was also synthesized from spectinamide 4 to evaluate the H-bond acceptor
character on the C-6 position. All the synthesized analogs were tested for antibacterial activity against a panel of Gram (+)
and Gram (−) pathogens, plus Mycobacterium tuberculosis. The molecular contribution of the 2- and 6-hydroxy group and
the aryl functionalities of all analogs were examined by measuring inhibition of ribosomal translation and molecular
dynamics experiments with MM/GBSA analysis. The results of this work indicate that the 6-hydroxy group, which is the
primary target of AMEs, is a required motif for antimicrobial activity in current analogs. Removal of the 6-hydroxy group
could be partially rescued by offsetting ribosomal binding contributions made by the aryl side chains found in the
spectinamide and amSPCs. This study builds on the knowledge of the structure–activity relationships of spectinomycin
analogs and is being used to aid the design of next-generation spectinomycins.
Introduction
used to treat Neisseria gonorrheal infections [4] and is well
tolerated at a high dosage, lacking the ototoxicity com-
monly associated with aminoglycoside antibiotics [5].
However, spectinomycin is subject to native efflux [6, 7],
target modification [8], and enzymatic inactivation of the
drug [8, 9]. Therefore, to overcome innate resistance
mechanisms, we developed semisynthetic analogs of spec-
tinomycin called spectinamides [10] and aminomethyl
spectinomycins (amSPCs) [11]. The spectinamides have
excellent narrow spectrum antitubercular activity, and the
amSPCs have broad-spectrum activity against the common
respiratory tract, sexually transmitted and biothreat bacterial
pathogens (Fig. 1b, c) [12–14]. These analogs have
improved bacterial accumulation, with the spectinamides
overcoming efflux by the Rv1258c transporter found in
Mycobacterium tuberculosis which transports spectinomy-
cin [10]. While these analogs overcome the resistance from
efflux pumps, they are still subject to modification by
aminoglycoside modifying enzymes (AMEs) in non-
mycobacterial pathogens, which limits their development
for broader indications, particularly for the treatment of
Spectinomycin, an aminoinositol antibiotic [1, 2], is an
inhibitor of bacterial protein synthesis (IC50 = 0.4 μg ml−1
)
with moderate broad-spectrum activity against both Gram
(+) and Gram (−) bacteria (Fig. 1a). Spectinomycin binds
to the interface between the minor groove of helix-34 of the
16S ribosomal RNA and the RpsE protein to inhibit protein
translation [3]. Clinically, spectinomycin has been widely
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-021-00408-3) contains supplementary
material, which is available to authorized users.
* Richard E. Lee
Richard.Lee@stjude.org
1
Department of Chemical Biology and Therapeutics,
St. Jude Children’s Research Hospital, Memphis, TN, USA
2
Institut für Medizinische Mikrobiologie, Universität Zürich,
Zürich, Switzerland

382
S. Dharuman et al.
Fig. 1 a Structure of spectinomycin (1) with activity against
M. tuberculosis and ribosomal IC50 against M. smegmatis ribosomes.
b Structure of spectinamide 2 with activity against M. tuberculosis and
ribosomal IC50 against M. smegmatis ribosomes. c Structure of amSPC
3 with activity against S. pneumoniae and ribosomal IC50 against
M. smegmatis ribosomes
Fig. 2 Proposed analogs of deoxyspectinomycins to overcome aminoglycoside modifying enzyme resistance
Gram-negative infections [15, 16]. One strategy to alleviate
AME resistance is to remove the sites of inactivation found on
aminoglycosides [17]. Spectinomycin is susceptible to inac-
tivation by nucleotidyltransferases ANT(9) [18] and the
bifunctional enzyme ANT(3,9), as well as phosphorylation by
the APH(9) enzyme, which all modify the 6-hydroxy group
position of spectinomycin [15, 16, 19]. Previously, several
deoxyspectinomycins were synthesized by researchers at
Hoffmann-La Roche Inc. and were found to be micro-
biologically inactive [20–22]. However, it was unknown if the
inactivity is due to lack of ribosomal binding or poor accu-
mulation. As part of our systematic approach to develop the
structure–activity relationship (SAR) around the spectino-
mycin ring, we chose to reinvestigate the antimicrobial
properties of deoxyspectinomycin analogs and to evaluate
their ability to inhibit bacterial ribosomes, which has not been
previously reported.
6-deoxyspectinomycin using Barton’s radical deox-
ygenation reaction as the key step [23] and demonstrate an
efficient synthesis of 6- and 2-deoxyspectinamides and
6-deoxyaminomethyl spectinomycin (6-deoxy-amSPC)
analogs [21]. 6-epi-chlorospectinomycins and 6-
dehydrospectinamide were also synthesized to examine
hydrogen bonding acceptor character to the ribosome
(Fig. 3). The antimicrobial activity and cellular accumu-
lation of the analogs were determined, and the energetic
contribution of key binding interactions was examined
computationally. Results from these studies are combined
to provide a holistic picture of the value of the 2- and 6-
hydroxy groups to spectinomycin binding and anti-
microbial activity.
Material and methods
In this study, we examine whether modifications to the
2- and 6-hydroxy groups in spectinomycin, spectina-
mides, and amSPCs (Fig. 2) can be tolerated, how mod-
ification or removal of these groups affects antibacterial
potency, ribosome inhibition, and cellular accumulation,
and finally, computationally examine the relative
binding contributions of these motifs in the ribosome
binding site. We report an improved synthesis of
Chemicals, reagents, and instrumental
Spectinomycin dihydrochloride pentahydrate was purchased
from Waterstone Technology (catalog number 81249, CAS
number 22189-32-8, 95% in purity). All solvents used for
chromatography and liquid chromatography were purchased
from Aldrich. Flash column chromatography silica

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins
383
Fig. 3 Proposed mechanism for
6-dehydrospectinamide 11 to
sustain resistance from AMEs
cartridges were obtained from Biotage Inc. Reactions were
monitored by thin-layer chromatography on pre-coated
Merch 60 F254 silica gel plates and visualized using UV
light (254 nm). IR (neat) spectra were obtained using a
Nicolet-iS5 FT-IR instrument. A Biotage FLASH column
6-Deoxy-3′-deoxy-3′-dihydro-3′(R)-[(pyridin-2-yl)
acetylamino]spectinomycin dihydrochloride (6)
Compound 6 was synthesized analogously as 5 from 17, as
a white solid. Yield: 0.017 g (95%); IRυmax (neat): cm−1
1
1
chromatography system was used to purify mixtures. H
3363, 2973, 1656,1561, 1467, 1166, 1064; H NMR (500
NMR spectra were recorded on a Varian INOVA-500
spectrometer or on a Bruker 400 MHz NMR spectrometer.
Chemical shifts (δ) are reported in parts per million relative
to the residual solvent peak or internal standard (tetra-
methylsilane), and coupling constants (J) are reported in
hertz (Hz). High-resolution mass spectra were recorded on a
Waters Xevo G2 QTOF LC–MS using ESI. Purity of
the products was confirmed by UPLC/MS (the Waters
Acquity). Spectral data for final compounds is given below,
and data for intermediates are provided in Supplementary
information.
MHz, D2O) δ 8.66 (d, J = 5.8 Hz, 1H, Ar-H), 8.47 (t, J =
8.0 Hz, 1H, Ar-H), 7.95–7.84 (m, 2H, Ar-H), 4.90 (s, 1H),
4.56 (s, 1H), 4.26 (t, J = 10.4 Hz, 1H), 4.17–3.96 (m, 5H),
3.37 (d, J = 11.6 Hz, 2H), 2.73 (s, 3H), 2.71 (s, 3H),
2.15 (dd, J = 10.6, 6.7 Hz, 1H), 1.83 (t, J = 13.5 Hz,
2H), 1.69 (d, J = 14.6 Hz, 1H), 1.18 (d, J = 6.0 Hz, 3H);
13C NMR (125 MHz, D2O) δ 169.0, 149.2, 146.9, 141.4,
128.3, 125.8, 93.2, 90.2, 68.0, 67.8, 65.3, 61.6, 58.7, 55.9,
52.3, 39.2, 34.1, 30.6, 30.5, 25.4, 19.8; HRMS (ESI)
m/z calcd for C21H33N4O6 [M + H]+, 437.2400; found,
437.2393.
Chemical synthesis
6-Deoxy-3′-dihydro-3′(R)-[(4-fluorobenzyl)aminomethyl]
spectinomycin trihydrochloride (7)
6-Deoxyspectinomycin dihydrochloride (5)
Compound 7 (0.011 g, 70%) was synthesized analogously
28
To a solution of cbz protected 6-deoxyspectinomycin 16
[20] (0.080 g, 0.136 mmol) in 1 M HCl in MeOH (3 ml) was
added Pd/C (30 mg) at room temperature and the reaction
mixture was hydrogenated for 30 min under H2 atmosphere
(H2 Balloon). Excess catalyst was filtered off and the eva-
poration, washing with acetone (5 ml) and EtOAc (5 ml)
gave pure product 5 as a white solid. Yield: 0.060 g, 81%;
1H NMR (500 MHz, D2O) δ 4.76 (s, 1H), 4.57 (s, 1H), 4.18
(t, J = 10.5 Hz, 1H), 4.07 (td, J = 11.3, 4.0 Hz, 1H), 3.92
(q, J = 6.6 Hz, 1H), 3.40–3.32 (m, 2H), 3.28 (s, 3H),
2.77–2.74 (m, 1H), 2.71 (s, 3H), 1.89–1.72 (m, 3H), 1.17
(d, J = 6.2 Hz, 3H); 13C NMR (125 MHz, D2O) δ 93.8,
93.4, 92.0, 68.4, 67.8, 65.2, 61.1, 59.0, 56.0, 41.5, 30.6,
30.0, 25.5, 19.6. HRMS (ESI) m/z calcd for C14H25N2O6
[M + H]+, 317.1713; found, 317.1712.
as 6 from 24, as a white solid. Yield: 0.011 g (70%); [α]D
+ 0.072 (c 0.15, CH3OH); MP: 211.1–211.8; IRυmax (neat):
cm−1 3358, 2974, 1603, 1513, 1460, 1163, 1081; 1H NMR
(500 MHz, D2O) δ 7.46–7.43 (m, 2H, Ar-H), 7.17–7.14 (m,
2H, Ar-H), 4.67 (s, 1H), 4.58 (s, 1H), 4.27 (d, J = 13.5 Hz,
1H), 4.20 (d, J = 13.6 Hz, 1H), 4.18–4.02 (m, 2H), 3.68
(dq, J = 12.3, 6.1 Hz, 1H), 3.47–3.26 (m, 3H), 3.13 (d, J =
13.7 Hz, 1H), 2.73 (s, 3H), 2.71 (s, 3H), 2.14 (dt, J = 8.8,
4.3 Hz, 1H), 1.88–1.63 (m, 3H), 1.14 (d, J = 5.9 Hz, 3H);
13C NMR (125 MHz, D2O) δ 163.2 (d, J = 247.0 Hz, C-F),
132.3, 132.2, 125.9, 116.3, 116.1, 93.4, 92.5, 72.2, 67.5,
67.4, 65.0, 61.2, 58.8, 55.9, 50.8, 48.9, 40.1, 30.5, 30.2,
25.4, 19.8; 19F NMR (D2O, 375 MHz) δ −112.0; HRMS
(ESI) m/z calcd for C22H35FN3O6 [M + H]+, 456.2510;
found, 456.2510.

384
S. Dharuman et al.
6-Epi-chloro-6-deoxyspectinomycin dihydrochloride (8)
Compound 8 was synthesized from cbz-spectinomycin [24].
filtration, and the ethyl acetate phase was washed with water
and dried over Na2SO4. After removing solvent, the residue
was purified by C18 chromatography (H2O/CH3CN), and
200 mg (47%) white solid was obtained as 6-dehydro-cbz-
25. 6-Dehydro-cbz-25 (157 mg, 0.190 mmol) was mixed
with 10% Pd/C (20 mg) and 1 M HCl in MeOH (1 ml) in
MeOH (10 ml), the mixture was hydrogenated at room
temperature for 4 h. Pd/C was removed and then the filtrate
was dried under vacuum to yield 11 as a white solid. Yield:
0.080 g (73%); IRυmax (neat): cm−1 3254, 2923, 2850, 1651,
1552, 1495, 1168, 1067;1H NMR (400 MHz, D2O) δ 8.29
(d, J = 2.8 Hz, 1H), 8.02–7.93 (m, 1H), 7.75 (d, J = 8.9 Hz,
1H), 5.14–5.04 (m, 1H), 4.61–4.49 (m, 1H), 4.30–4.03 (m,
6H), 3.60 (dd, J = 11.2, 3.2 Hz, 1H), 3.51–3.35 (m, 1H),
2.94–2.84 (m, 6H), 2.03–1.85 (m, 1H), 1.88–1.71 (m, 1H),
1.29 (dd, J = 6.1, 4.2 Hz, 3H); 13C NMR (125 MHz, D2O) δ
169.6, 169.5, 154.9, 140.3, 133.5, 133.5, 129.2, 129.2,
129.0, 128.9, 94.1, 93.9, 93.2, 93.0, 92.2, 92.1, 90.2,
90.0, 89.8, 71.8, 70.8, 69.9, 68.4, 67.2, 64.7, 61.8, 60.1,
58.3, 57.8, 54.0, 52.2, 48.8, 38.2, 34.1, 30.8, 30.7, 30.2,
30.0, 19.8, 19.8. HRMS (ESI) m/z calcd for C21H31N4O8
[M + H]+, 467.2142; found, 467.2144.
1
Yield: 0.012 g (42%); H NMR (500 MHz, D2O) δ 4.93 (s,
1H), 4.75–4.73 (m, 1H); 4.66 (t, J = 10.6 Hz, 1H), 4.44 (dd,
J = 10.5, 3.5 Hz, 1H), 4.05–3.99 (m, 1H), 3.93–3.81 (m,
1H), 3.54 (dd, J = 11.0, 3.3 Hz, 1H), 2.89 (s, 3H),
2.84–2.83 (m, 1H), 2.81 (s, 3H), 1.95–1.80 (m, 2H), 1.26
(d, J = 6.3 Hz, 3H); 13C NMR (125 MHz, D2O) δ 93.8,
93.4, 91.7, 68.6, 66.5, 63.0, 61.6, 58.8, 56.8, 54.4, 41.5,
30.5, 30.2, 19.5. HRMS (ESI) m/z calcd for C14H24ClN2O6
[M + H]+, 351.1323; found, 351.1329.
2-Deoxyspectinomycin dihydrochloride (9)
Compound 9 [21] was synthesized analogously as 5 from
1
20, as a white solid. Yield: 0.010 g (80%) H NMR (500
MHz, D2O) δ 4.77 (s, 1H), 4.04 (t, J = 10.3 Hz, 1H),
3.96–3.88 (m, 2H), 3.74 (t, J = 10.2 Hz, 1H), 3.46 (t, J =
11.6 Hz, 1H), 3.22 (t, J = 10.2 Hz, 1H), 2.70 (s, 3H), 2.68
(s, 3H), 2.63–2.57 (m, 1H), 1.81–1.68 (m, 3H), 1.15 (d, J =
6.1 Hz, 3H); 13C NMR (125 MHz, D2O) δ 93.5, 93.4, 91.6,
69.9, 68.4, 68.2, 68.0, 57.3, 54.3, 41.5, 30.1, 29.7, 23.3,
19.6; HRMS (ESI) m/z calcd for C14H25N2O6 [M + H]+,
317.1713; found, 317.1713.
Molecular modeling
Mycobacterial RNA-RpsE protein complex generation
2-Deoxy-3′-deoxy-3′-dihydro-3′(R)-[(pyridin-2-yl)
acetylamino]spectinomycin dihydrochloride (10)
The molecular docking and molecular dynamics studies of
spectinomycin and spectinomycin analogs were performed
using a comparative model of the M. tuberculosis RNA-
RpsE protein complex prepared using the Escherichia coli
complex as a template. The Mycobacterial RpsE proteins
(M. tuberculosis and Mycobacterium smegmatis) have a
high degree of homology, with 89% sequence similarity.
The differences in the RpsE protein sequences lie outside
the spectinomycin binding site on the RpsE loop (Fig. 4).
Therefore, the M. tuberculosis RpsE protein sequence was
used to create a Mycobacterial RNA-RpsE protein complex
for molecular modeling of both species. The E. coli RpsE
protein structure (PDB ID: 4V56 (2QOU)) served as the
structural template to generate the model, 55% homology
[26]. The model was created using Schrödinger’s Prime
program [27, 28] and was validated in PROCHECK [29]
with 2.7% residues allowed and 0% disallowed (data not
shown). A sequence alignment of RpsE proteins from the
Mycobacteria and E. coli revealed two amino acid differ-
ences located in the RpsE protein loop in the spectinomycin
binding site: V55T and R61I (Fig. 4). Despite variations in
the RpsE protein, the rRNA sequence found in the specti-
nomycin binding site is highly conserved between Myco-
bacteria and E. coli. To reduce the computational cost of
subsequent molecular docking and molecular simulations,
the complex model was truncated to a 20 Å sphere around
Compound 10 was synthesized analogously as 6 from 21, as
a white solid. Yield: 0.010 g (79%); IRυmax (neat): cm−1
1
3350, 2974, 1648, 1564, 1466, 1168, 1063; H NMR (500
MHz, D2O) δ 8.66 (d, J = 5.9 Hz, 1H), 8.48 (t, J = 8.0 Hz,
1H), 8.05–7.81 (m, 2H), 4.93 (s, 1H), 4.27–3.89 (m, 6H),
3.77 (q, J = 9.9, 9.2 Hz, 1H), 3.50 (td, J = 11.5, 4.1 Hz,
1H), 3.25 (ddd, J = 14.1, 10.2, 4.3 Hz, 1H), 2.72 (s, 3H),
2.71 (s, 3H), 2.68–2.56 (m, 1H), 1.86–1.68 (m, 3H), 1.18
(d, J = 5.7 Hz, 3H); 13C NMR (125 MHz, D2O) δ 166.3,
146.5, 146.4, 144.4, 138.7, 125.7, 123.1, 90.3, 87.3, 67.4,
65.6, 65.3, 54.6, 51.4, 49.5, 36.5, 31.5, 27.4, 20.9, 17.1,
11.1; HRMS (ESI) m/z calcd for C21H33N4O6 [M + H]+,
437.2400; found, 437.2401.
6-Dehydro-3′-deoxy-3′-dihydro-3′(R)-[(5-hydroxypyridin-2-
yl)acetylamino]spectinomycin trihydrochloride (11)
To a mixture of dimethylsulfoxide (5 ml), dry benzene
(5 ml), dicyclohexyl carbodiimide (0.320 g, 1.560 mmol),
and pyridinium trifluoroacetate (0.050 g, 0.260 mmol) was
added 25 [25] (0.430 g, 0.520 mmol). The mixture was
stirred at 50 °C for 4 h. The reaction mixture was then
poured into 100 ml of ethyl acetate and 100 ml of water with
stirring, the insoluble dicyclohexyl urea was removed by

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins
385
Fig. 4 Alignment of E. coli, M.
tuberculosis, and M. smegmatis
RpsE protein. a The alignment
of the protein structures of the
RpsE protein from E. coli from
PDB ID 2QOU [26] (orange),
the homology model from M.
tuberculosis (blue), and M.
smegmatis (green) developed in
Schrödinger using Prime
[27, 28]. Residues that are
different in the RpsE protein
loop between species are shown
as sticks and are labeled. b The
sequence alignment of the RpsE
proteins [49, 50]. Black
highlights represent identical
residues, gray highlights
represent similar residues, and
the red boxes highlight the RpsE
protein loop
the spectinomycin binding site. The Mycobacterial RNA-
RpsE complex was protonated at pH 7.0 using PROPKA
and was energy minimized with the OPLS3 force field
[30, 31].
Glide standard precision molecular docking
All conformations generated from LigPrep were docked
using Schrödinger’s Glide program [33–35]. Ligand sam-
pling was flexible, and docked conformers were required to
match at least one hydrogen bonding interaction from the
constraints generated in the receptor grid. To soften the
potential for nonpolar parts of the ligands, van der Waals
(VDW) radii were scaled using a scaling factor of 0.80 for
ligand atoms with a partial charge < 0.15 (default Glide
settings). Post-docking minimization was performed on all
poses generated by Glide using the OPLS3 force field prior
to the assignment of Glide docking scores. Ligand poses
with the highest ranked Glide scores were used to evaluate
binding. The highest ranked analogs, 1 (spectinomycin), 2,
5, 6, 9, and 10, were advanced to molecular dynamics
simulations.
Ligand preparation
SMILES strings for spectinomycin and analogs were used to
render 3D conformations of the compounds in Schrödinger.
Minimum energy conformations of each analog were gener-
ated by LigPrep using the OPLS3 force field [30]. Specified
chiralities were retained and the ionization states at a target
pH 7.0 2.0 were generated using Epik [32].
Receptor grid generation
For the molecular docking of spectinomycin and analogs, a
receptor grid was generated using Schrödinger’s Glide
program [33–35]. A cubic box was positioned in the center
of the spectinomycin binding site. The inner box was set to
10 × 10 × 10 Å and the outer box was 30 × 30 × 30 Å. This
larger box was designed to permit the docking of specti-
namides and amSPCs in the binding site. The spectinomy-
cin binding site determined by X-ray crystallography (PDB
ID 2QOU) [26] revealed a series of hydrogen bond inter-
actions from the rRNA nucleotides. Therefore, hydrogen
bonding constraints were generated for residues G1064,
C1066, G1068, A1191, C1192, and G1193 for a data-
driven docking approach.
Molecular dynamics
All molecular dynamic simulations were performed using
AMBER18 [36] as previously described [25]. Briefly, the
ff14SB and OL3 force field was used for the RNA and
protein parameterization and the general Amber force
field was used for parameterization of the ligand [37–39].
Restrained electrostatic potential charges within the
ANTECHAMBER module in AMBER were applied to the
positively charged spectinomycin, 2, 5, 6, 9, and 10 ana-
logs. Sodium ions were applied to neutralize the charge of

386
S. Dharuman et al.
the system. The complex was solvated in a TIP3P octahe-
dron box with a 10 Å boundary. An initial energy mini-
mization of the solvated complex was performed with the
complex fixed for 1000 steps, followed by a second step
of energy minimization without restraints for 2500 steps.
The minimized complex was equilibrated to 300 K over
250,000 steps for 500 ps with weak restraints applied to the
complex (Fig. S1). The equilibrated structure was simulated
for 20 ns with the NPT ensemble at 300 K with terminal
residues harmonically restrained (Fig. S1). The energetic
contributions during the MD simulations were calculated by
the MM/GBSA [40] method using 1000 frames from the
last 2 ns of the MD ensemble. Lastly, the free energy con-
tributions to ligand binding were decomposed for analysis
using a per-residue basis [40].
M. tuberculosis H37Rv. Equal volume of bacterial strains of
5 × 105 CFU ml−1 was added to each well to give a final
drug concentration starting at 200 μg ml−1. Plates were
incubated for 18 h at 37 °C except M. tuberculosis H37Rv,
which was incubated for 7 days at 37 °C. The MIC for all
strains were determined as the lowest concentration to
visually inhibit bacterial growth and are reported as the
consensus of three independent experiments.
Whole-cell accumulation assay
Methods for this assay have been optimized from previously
published studies [42, 43]. E. coli (BW25113 for WT or
JW5503 for ΔtolC) is grown at 37 °C to mid-log phase
(OD600 of 0.6−0.7) in Mueller–Hinton broth. The bacteria
are pelleted and washed twice with PBS before being resus-
pended in 3.5 ml of PBS per 100 ml of cells cultured. Cells
are allowed to equilibrate for 10 min at 37 °C prior to drug-
ging. 1 ml of E. coli is incubated with a final concentration of
50 μM of compound (in DMSO) shaking at 37 °C for 10 min.
800 μl of treated cells is layered over supercooled (−78 °C)
silicone oil (9:1 AR20 and high-temperature silicone oil,
Sigma Aldrich) and pelleted to remove cells from free com-
pound. For lysis, the pellets were resuspended in 200 μl of
HPLC grade water and subjected to three freeze−thaw cycles
using liquid nitrogen and a 65 °C water bath. Cell debris was
separated from lysate by centrifugation, and 150 μl of extract
is recovered. The pellet is resuspended in the remaining 50 μl
of water, and once thoroughly resuspended, 100 μl of 5%
TCA was added. The cells are once again pelleted, and lysates
are combined. Lysates are pelleted at high speed for 10 min
and then filtered through a 0.22 μm filter before injection for
LC–MS/MS.
Samples are analyzed with a tandem Waters Acquity M
Class series UPLC system and Xevo G2 QTOF tandem MS/
MS with Zspray. 100 nl of extract was separated using a
Phenomenex Kinetex 2.6 μm XB-C18, 100 Å (300 μm ×
150 mm) column with solvent A, 0.1% formic acid in water,
and solvent B, 0.1% formic acid in acetonitrile. The inlet
method for these samples utilized a flow rate of 8 μl min−1
with the following gradient: 0−4 min, 99.9% solvent A and
0.1% solvent B; 4–5 min, 10% solvent A and 90% solvent
B; 5–6 min, 99.9% solvent A and 0.1% solvent B. Tandem
mass spectra were acquired with a cone voltage and colli-
sion energy optimized for each compound. High-resolution
spectra were calibrated by co-infusion of 2 ng ml−1 leucine
enkephalin lockspray (Waters). Data were quantified using
Waters MassLynx software where the AUC was determined
by integrating the corresponding daughter peak of the par-
ent compound. Concentrations of the unknown compounds
were determined by the linear fit of the corresponding
standards. Concentrations are reported as the average of
three biological replicates.
Ribosomal inhibition
Purified 70S M. smegmatis bacterial ribosomes were used in
translation reactions. Firefly luciferase mRNA was pro-
duced in vitro using T7 RNA polymerase. Translation
reactions were carried out as previously described [11]. The
IC50 values represent the drug concentration that inhibits
luciferase activity by 50%.
Bacterial strains and growth conditions
Strains were routinely grown with Mueller−Hinton broth at
37 °C with shaking at 225 rpm. Bacterial strains were
obtained through ATCC or BEI Resources or from academic
laboratories. The strains used were the following: Staphylo-
coccus aureus ATCC 29213, S. aureus NRS70, Enterococcus
faecalis ATCC 33186, E. faecium ATCC 19434, Strepto-
coccus pyogenes ATCC 700294, Streptococcus pneumoniae
R6, Acinetobacter baumannii ATCC 19606, Pseudomonas
aeruginosa ATCC 15692, Klebsiella pneumoniae ATCC
700603, Proteus mirabilis ATCC 25933, Stenotrophomonas
maltophilia ATCC 13637, Enterobacter cloacae ATCC
13047, Staphylococcus epidermidis ATCC 14990, E. coli K-
12, and E. coli JW5503 (ΔtolC). The M. tuberculosis H37Rv
strain was grown in Middlebrook 7H9 broth supplemented
with 10% albumin-dextrose complex, 0.05% (v/v) Tween
80 at pH 7.4, (7H9/ADG pH 7.4) with shaking at 225 rpm
at 37 °C.
Antibacterial susceptibility testing
Minimum inhibitory concentrations are determined fol-
lowing CLSI broth microdilution guidelines [41]. Briefly,
experimental compounds (in DMSO) and control specti-
nomycin (Sigma Aldrich, DMSO) are serially diluted,
twofold, across
a 96-well round bottom plate in
Mueller–Hinton II broth or 7H9/ADG pH 7.4 for

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins
387
Scheme 1 Synthesis of 6-deoxyspectinomycin
5
and 6-
over two steps; [iii] Pd/C-H2, MeOH, 81%; [iv] (a) NH4NO3/
CH3COOH, 2-picoline-borane complex, MeOH, (b) 2-Pyridylacetic
acid hydrochloride, HBTU, DMF, 25% over two steps; [v] Pd/C-H2,
MeOH, 95%
deoxyspectinamide 6. Reagents and conditions: [i] (a) 1,1′-Thio-
carbonyldiimidazole, DMAP, DCM, (b) Bu3Sn-H/AIBN, Toluene,
55% over two steps; [ii] (a) 1 M HCl in MeOH, (b) IBX, DMSO, 85%
Scheme 2 Synthesis of 2-
deoxyspectinomycin 9 and 2-
deoxyspectinamide 10. Reagents
and conditions: [i] (a) NaH,
BnBr, THF, (b) 1 M HCl in
MeOH, 65% over two steps; [ii]
IBX, DMSO; 86% [iii] Pd/C-H2,
MeOH, 80%; [iv] (a) NH4NO3/
CH3COOH, 2-picoline-borane
complex, MeOH, (b) 2-
Pyridylacetic acid
hydrochloride, HBTU, DMF,
34% over 2 steps; [v] Pd/C-H2,
1 M HCl in MeOH, 79%
Results and discussion
which then coupled with 2-pyridyl acetic acid to give com-
pound 17 [45, 46]. The newly generated stereochemistry
on the 3′(R) position was assigned using coupling constant
(J3′4′ax = 4.4 Hz; J3′4′eq = 2.3 Hz) [47] values between 3′ and
4′ hydrogens by synthesizing compound 16b (data available
in Supplementary Scheme 1, compound 16b). Finally,
catalytic hydrogenolysis of cbz product of 17 under Pd/C
gave 6-deoxyspectinamide 6.
Attempts to deoxygenation of C-2-hydroxy group using
the Barton imidazolylthiocarbamate intermediate failed
presumably due to the steric hindrance of C-2-hydroxy
group, we consequently followed literature procedure to
make 2-deoxyspectinomycin. The 2-deoxyderivative 18
was achieved from spectinomycin using the protocol of
Foley et al. (Scheme 2) [21]. Benzyl protection of 6-
hydroxy group followed by removal of acetonide group
yielded diol 19. Compound 9 was achieved by oxidation of
compound 19 to give compound 20 and followed by cbz
group deprotection. The 2-deoxyspectinamide 10 was syn-
thesized from 20 via 21 following similar protocol [45] as
used in 6.
Synthesis
Adapting literature procedures, compound 14 [22, 44] was
synthesized from spectinomycin via catalytic hydrogena-
tion of keto group, cbz protection of amines, and diace-
tonide protection diol. Deoxygenation at C-6 position in
compound 14 was achieved efficiently by following Bar-
ton’s radical deoxygenation [23] method (Scheme 1).
Thus, selective installation of imidazolyl thiocarbamate
group on C-6-hydroxy group was carried out using 1,1′-
thiocarbonyldimimidazole in the presence of catalytic
amount of DMAP and treatment of thiocarbamate with
tributyltin hydride and AIBN gave deoxygenated product
15 in good yields. An acetonide group was removed under
1 M HCl in methanol to give diol and selective oxidation
of 4′-hydroxy group with IBX yielded cbz protected 6-
deoxyspectinomycin 16. Lastly, compound 16 was con-
verted to 6-deoxyspectinomycin [20] 5 by removal cbz group
under catalytic hydrogenation (Scheme 1). To achieve 6-
deoxyspectinamide 6, compound 16 underwent reductive
amination reaction with ammonium nitrate and 2-picoline-
borane complex to provide 4′R-amino-6-deoxyspectinomycin,
6-Deoxy-amSPC 7 was synthesized from amSPC inter-
mediate 22 [11]. First, the benzyl amine group in 22 [11] was
protected as tert-butyl carbamate derivative and then the

388
S. Dharuman et al.
Scheme 3 Synthesis of 6-deoxy-
amSPC 7. Reagents and
conditions: [i] (a) (Boc)2O,
Et3N, MeOH, (b) 1,1′-
Thiocarbonyldiimidazole,
DMAP, DCM, 71% overs two
steps; [ii] (a) Bu3Sn-H/AIBN,
Toluene, (b) TFA, DCM, 41%
over two steps; [iii] Pd/C-H2, 1
M HCl in MeOH, 70%
Scheme 4 Synthesis of 6-
dehydrospectinamide 11.
Reagents and conditions: [i] (a)
pyridinium trifluoroacetate,
dicyclohexyl carbodiimide,
DMSO, 50 °C, (b) 10% Pd/C-
H2, 0.1 M HCl in MeOH, 73%
6-imidazolylthiocarbamate derivative 23 was synthesized
using 1,1′-thiocarbonyl diimidazole. Deoxygenation at C-6
position was then achieved efficiently by following Barton’s
radical deoxygenation [23] method as used in compound 15
to give deoxygenated product 24 in moderate yield. Finally,
deoxy compound 7 was achieved by removing tert-butoxy
carbonyl group using trifluoroacetic acid and benzyloxy car-
bonyl group deprotection under hydrogenolysis condition
(Scheme 3). The 6-epi-chloro compound 8 was synthesized
following literature protocol [24]. The synthesis of 6-
dehydrospectinamide 11 was accomplished from cbz pro-
tected 25 [25] via selective oxidation of C-6-hydroxy group
by Pfitzner–Moffatt method and followed hydrogenolytic
deprotection of cbz groups (Scheme 4).
binding inhibition (Ribo IC50 > 60 μg ml−1) compared to
spectinomycin 1 (Ribo IC50 0.36 μg ml−1). The molecular
docking of 5 and 9 suggests that the hydrogen bonding
interactions from the 6- and 2-hydroxy groups are crucial
for ribosomal binding inhibition (Fig. S2A, B). Substitution
of the hydroxy group at the 6-position on spectinomycin
was tested to determine if the loss in activity could be
rescued while avoiding AME activity. However, addition of
a chlorine at the 6-position (8) maintained poor activity
(Table 1) and disrupted the majority of hydrogen bonding
interactions involved with spectinomycin binding
(Fig. S2C). Therefore, the hydroxy group at the 6-position
of spectinomycin forms hydrogen bonding interactions that
are crucial to maintain ribosomal inhibition.
The 6-deoxyspectinamide 6 showed improved ribosomal
inhibition compared to 5, (Ribo IC50 = 9.50 μg ml−1) sug-
gesting the aryl side chain could partially rescue inhibitor
activity. The aryl side chain extends from the spectinomycin
binding site and interacts directly with the RpsE protein
loop through VDW interactions (Fig. S3). Similarly, 6-
deoxy-amSPC 7 had improved ribosomal inhibition (Ribo
IC50 = 23.3 μg ml−1) from the contribution of the aryl side
chain (Fig. S4). This feature may be used in part to com-
pensate for the loss of hydrogen bonding interaction with
the spectinomycin core and the bacterial ribosome, a phe-
nomenon that could not be observed in 5 as it lacks the aryl
side chain. 2-Deoxyspectinamide 10 also showed improved
ribosomal inhibition (Ribo IC50 = 10.2 μg ml−1) compared
to the 2-deoxyspectinomycin 9 (Ribo IC50 > 60 μg ml−1).
From the molecular docking of 10, the aryl side chain is
also shown to gain interactions with the RpsE protein
backbone, providing further evidence that aryl side chain
rescues ribosomal binding (Fig. S3).
Antibacterial susceptibility and ribosomal activity of
deoxy analogs
The antibacterial susceptibilities of the synthesized deoxy
analogs were tested against a panel of Gram (+) and Gram
(−) pathogens, plus M. tuberculosis (abbreviated in Table 1,
full data available in Supplementary Table 1). All deox-
yspectinomycin analogs lacked MIC activity in line with
previous reports [20]. Deoxyspectinamides 6 and 10 and
deoxy-amSPC 7 had some limited MIC activity against M.
tuberculosis and S. pneumoniae, weaker than their corre-
sponding parent spectinamide 2 and amSPC 3, consistent
with their reduced ribosomal binding affinities described
subsequently.
The ability of the analogs to inhibit ribosomal
translation was performed with M. smegmatis ribosomes in
a cell-free translation assay [11] (Table 1). The 6- and
2-deoxyspectinomycins, 5 and 9, show poor ribosomal

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins
389
Table 1 Antibacterial susceptibility (MIC, μg ml−1) and ribosomal affinity IC50 (μg ml−1) studies of synthesized compounds
Compounds
M. tuberculosis H37Rv S. pneumoniae R6
E. coli K-12 MIC E. coli cellular
Ribosome translation
MIC (µg ml−1
)
MIC (μg ml−1
)
(µg ml−1
)
accumulation (µM)
inhibition IC50 (μg ml−1
)
50
12.5
25
1.1 0.1
0.36
Spectinomycin (1)
1.6
12.5
200
5.4 0.8
0.89
2
6.25
3.13
50
40 + 1
0.74
3
1.6
12.5
100
5.9 + 0.8
0.84
4
>200
>200
>200
2.3 + 0.4
>60
5
50
25
>200
10.0 + 0.6
9.50
6

390
S. Dharuman et al.
Table 1 (continued)
Compounds
M. tuberculosis H37Rv S. pneumoniae R6
E. coli K-12 MIC E. coli cellular
Ribosome translation
MIC (µg ml−1
)
MIC (μg ml−1
)
(µg ml−1
)
accumulation (µM)
inhibition IC50 (μg ml−1
)
100
25
>200
47 + 1
23.3
7
>200
>200
>200
1.29 0.06
54.7
8
>200
>200
>200
<0.4
>60
9
200
50
>200
5.6 0.5
10.2
10
100
>200
>200
1.77 0.06
34.7
11
In the case of the 6-dehydrospectinamide 11, we
observed an increase in ribosomal binding affinity com-
pared to other 6-deoxy analogs (Ribo IC50 = 34.7 μg ml−1).
The molecular docking studies of the parent compound, 4,
and the 11 analog reveal the carbonyl at the 6-position
docked with a similar predicted affinity (Fig. S5). The exact
molecular entity of 11 is not known in solution state at this
stage as it likely exists in equilibrium with its hydrated form
as indicated by its 13C NMR (Fig. 3). Therefore, we also
docked the 12 (gem-diol-11) (Fig. S5). This analog exhib-
ited two hydrogen bond interactions with G1064 and
G1193, but loses the hydrogen bonding interaction with the
backbone of Arg60 and has a weaker docking score com-
pared to 4 and 11 (Fig. S5). These results point to reason

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins
391
interactions between the ligand, rRNA nucleotides, RpsE
loop, and associated water molecules. Removing or mod-
ifying the 2- and 6-hydroxy groups on spectinomycin and
analogs had large effect on the binding and antibacterial
activity (Table 1). To examine the molecular basis of these
effects, the docked conformations of spectinomycin 2, 5, 6,
9, and 10 analogs were subjected to a 20 ns molecular
dynamic simulation (Figs. 6 and 7). The docking pose of
spectinomycin into the M. tuberculosis rRNA/RpsE com-
plex homology model was compared to the E. coli X-ray
crystal structure (PDB ID: 2QOU [26]) to ensure the
binding mode was not altered between species. The ligand
RMSD between the two structures was 0.65 Å (data not
shown). The final spectinomycin binding mode following a
20 ns MD simulation is shown (Fig. 6). Like the X-ray
crystal structure, the nucleotides in the spectinomycin
binding pocket participate in several key hydrogen bonding
interactions. The 2-hydroxy group coordinates with a water
molecule and interacts with A1191, while the 6-hydroxy
group has one binding interaction with G1064.
In the simulation, 6-deoxyspectinomycin 5 lost the 6-
hydroxy group-mediated hydrogen bonding interaction with
the G1064 nucleotide (Fig. 6b) and resulted in a 1.65 Å
RMSD shift in the pocket compared to spectinomycin
(Fig. 6d). The free energy of binding for spectinomycin and
analogs was calculated using MM/GBSA in Amber18 [40]
(Table 2). The MM/GBSA method calculates binding free
energies using molecular mechanics at low computational
cost [40, 48]. Furthermore, GBSA is accurate and robust in
ranking binding affinities for structure-based drug dis-
covery. Removal of the 6-hydroxyl group resulted in a
decrease of 8.6 kcal mol−1 in binding energy compared to
spectinomycin and is reflective of the poor IC50. Removal of
the 2-hydroxy in 9 results in the loss of two hydrogen
bonding interactions in the pocket, i.e., a water molecule
and the A1191 nucleotide (Fig. 6c). Despite the remaining
hydrogen bonding partners, 9 produces a larger shift in the
pocket with a RMSD of 2.81 Å (Fig. 6d). This shift results
in a 6.3 kcal mol−1 decrease in the binding energy compared
to spectinomycin (Table 2). While both analogs had poor
binding affinities, loss of the 2-hydroxy group was more
favorable than the 6-hydroxy group for the spectinomycin
core.
Fig. 5 Whole-cell accumulation assay tested per compound in wild-
type E. coli. (BW25113) and Δtolc (JW5503) strains. *Not determined
for the weak binding (Ribo IC50 = 34.7 μg ml−1) for 11
compared to 4 (Ribo IC50 = 0.84 μg ml−1) is due to 12
(gem-diol-11) being the dominant binding isoform.
Cellular accumulation
To examine if altered cellular accumulation contributes to
reduced MIC activity of deoxyspectinomycin analogs, the
concentration of all analogs within E. coli cells was mea-
sured and compared to their corresponding spectinomycin
family parent (Fig. 5 and Table S3) [43]. These experiments
indicated a substantial difference in the accumulation
between the different families, spectinomycins (1, 5, 8, 9;
range <0.4 to 1.2 μM), spectinamides (2, 4, 6, 10; range
<5.4 to 10 μM), and amSPCs (3, 7; 40 and 47 μM, respec-
tively). When examining matched pairs within families (1
vs 5 and 9; 2 vs 6 and 10; 3 vs 7), deoxygenation led to a
slight increase in accumulation, with the most pronounced
effect for 6-deoxy analogs. The increased accumulation is,
however, modest and unlikely to impact MIC levels. The
pattern of uptake was generally mirrored in the E. coli
ΔtolC strain. However, these data suggest that the early
spectinamide, 2, is subjected to efflux by tolC. The deple-
tion of the efflux pump results in a 15-fold increase in
intracellular accumulation, which matches the trends in
susceptibility where knocking out the efflux pump reduces
the MIC of 2 from 200 to 50 μg ml−1. Overall, these results
indicate that altered accumulation does not affect the anti-
microbial potency among the deoxyspectinomycin analogs.
Molecular dynamics simulations
Spectinomycin binding is mediated through a complex
pattern of hydrogen bond interactions between the rRNA
nucleotides and the ligand [26]. A better understanding of
the contributions of the 2- and 6-hydroxy group positions
on the spectinomycin scaffold is crucial for the development
of novel analogs that overcome inactivation by AMEs. We
chose to examine these interactions using molecular
dynamics simulations, rather than the rigid body docking
used earlier in this paper. These are computationally intense
experiments that account for dynamically changing
Interactions driving spectinomycin binding are also
observed in the spectinamide 2-bound complex, with the
addition of a hydrogen bond between the aryl chain on
spectinamide and G1193 (Fig. 7a). The aryl chain also gains
VDW interactions on the RpsE protein loop. Binding of 2
did not perturb the spectinomycin binding pocket, with a
RMSD value of 0.75 Å (Fig. 7d). However, binding of 6,
the corresponding 6-deoxy analog, shifted 2.47 Å from 2 in
the pocket and reduced the calculated ΔG of binding by
16.0 kcal mol−1 (Table 2). Despite the loss of one hydrogen

392
S. Dharuman et al.
Fig. 6 Snapshot from 20 ns molecular dynamic simulation of specti-
nomycin analogs. The binding mode of a spectinomycin, b 5, and c 9
from 20 ns MD simulations. The protein is shown as a gray ribbon
with nucleotides and waters interacting with the ligand are labeled.
The hydrogen bonding interactions are depicted as dashed lines. d An
overlay of spectinomycin (orange), 5 (cyan), and 9 (purple) from the
20 ns MD simulations
bond interaction with G1064, 6 maintains VDW interaction
RpsE protein loop (Fig. 7b), improving the IC50 compared
to the 6-deoxyspectinomycin analog 5. Additionally, 6
gains a hydrogen bonding interaction between G1193
phosphate backbone and the spectinomycin scaffold
because of the large shift in the binding pocket (Fig. 7b). In
contrast, the 2-deoxyspectinamide analog 10 remains in the
spectinomycin binding pocket with a RMSD value of 1.28
Å compared to 2 (Fig. 7d). Losing the A1191 hydrogen
bond is alleviated through interactions from the aryl chain
to G1193 and VDW interactions with Val56 on the RpsE
protein loop (Fig. 7c). Binding of 10 reduced the ΔG of
binding by 5.3 kcal mol−1 compared to 2, suggesting the
loss of the 2-hydroxy group is more favorable than the 6-
hydroxy group as observed with deoxyspectinomycin ana-
logs. However, despite loss of the hydroxy groups, both
deoxyspectinamide analogs recover binding interactions
and rescue activity via additional protein–ligand interac-
tions on the RpsE protein loop.
each nucleotide and amino acid in the binding pocket was
compared between the analogs (Fig. 8). From the specti-
nomycin MD simulation, G1064, C1066, and G1068 have
the strongest interaction in the binding pocket, with A1191,
C1192, and G1193 being the weakest (Fig. 8 and Table S2).
When the 6-hydroxy group is removed, the hydrogen
bonding interaction from G1064 is lost and results in a 1.8
kcal mol−1 decrease in free energy (Fig. 8). The loss of the
6-hydroxy group indirectly effected nucleotide binding
interactions with 5, 0.82 kcal mol−1 loss for C1066 and
0.45 kcal mol−1 loss for G1068. In contrast, the removal of
the 2-hydroxy group did not produce these large changes in
the free energy of binding (Fig. 8). Despite the loss of
A1191 hydrogen bonding interactions, the C1066 had the
largest loss in free energy of 2.33 kcal mol−1. This trend
was observed for the deoxyspectinamide analogs compared
to the parent 2 compound. The 6-deoxy 6 analog had a
decrease in G1064 free energy by 2.29 kcal mol−1, as the
hydrogen bond interaction is lost as observed for 5. Addi-
tionally, the binding free energy for C1192 decreased, 3.96
kcal mol−1, which was not observed for 5. This change in
C1192 binding energy may be a result of the large shift in
the binding pocket. The aryl side chain increased binding
The calculated ΔG of binding for the analogs correlates
to the measured ribosomal IC50 data; however, the energetic
contribution for each nucleotide is unknown from this
analysis. Therefore, the free energy decomposition [40] of

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins
393
Fig. 7 Modeled binding modes of spectinamide analogs from 20 ns
molecular dynamic simulation in AMBER18. The binding mode of
a 2, b 6, and c 10 from 20 ns MD simulations. The protein is rendered
as a gray ribbon with nucleotides and waters interacting with the
ligand shown as sticks. The dashed lines represent hydrogen bonding
interactions. d An overlay of spectinomycin (yellow), 2 (purple), 6
(orange), and 10 (green) from the 20 ns MD simulations
Table 2 Binding free energy
onto the Mycobacterial RNA/
RpsE complex determined using
the MM/GBSA method in
Amber18
a
a
a
b
Compound
EVDW (kcal−1 mol−1
)
Enonpolar (kcal−1 mol−1
)
ΔGtotal (kcal−1 mol−1
)
Ribosomal IC50 (μg ml−1
)
Spectinomycin −38.9 2.70
−5.41 0.05
−5.21 0.05
−5.34 0.05
−7.29 0.06
−6.86 0.05
−7.34 0.06
−78.5 2.32
−69.9 2.92
−72.2 2.21
−97.6 2.78
−81.6 2.49
−92.3 2.27
0.36
>60
>60
0.89
9.50
10.2
5
−36.9 2.60
−39.3 2.55
−57.2 2.98
−49.8 2.76
−56.3 2.91
9
2
6
10
aValues represent an average over the 1000 MD frames standard deviation
bTested with M. smegmatis
interactions with G1193, 1.13 kcal mol−1. The 2-deoxy
analog 10 maintained similar binding free energies com-
pared to the parent compound 2. This is not surprising, as
10 did not perturb the binding pocket, RMSD value of 1.28
Å. The loss of the A1191 binding interaction in the pocket
resulted in a 1.16 kcal mol−1 decrease in energy, as well as
indirect effect on C1066, 1.58 kcal mol−1. The aryl chains
in the spectinamide series did increase binding interactions
through the RpsE protein loop, as observed in the increase
of Val56 free energy (Fig. 8).
However, loss of the 6-hydroxy group in 6 greatly impacted
the free energy landscape of the binding pocket. Binding
free energies decreased for G1064, A1191, and C1192 and
increased with G1068 and G1193 (Fig. 8). This increase in
energy is a result of shifting the spectinamide out of the
pocket to maintain contacts between aryl side chain and the
RpsE protein. This compensatory mechanism shown for
both 6 and 10 suggests that the aryl side chain on these
analogs promote favorable binding interactions, i.e., Val56
(Fig. 8). Structure-based drug discovery of the aryl chain on
deoxyspectinamides to increase protein–ligand interactions
may be a viable method to overcome the energetic penalties
for removing the 2- or 6-hydroxy groups to avoid AME
activity.
From the decomposition of the free energy of binding,
loss of the 2-hydroxy group in 9 and 10 did not greatly
perturb the binding free energy of the nucleotides in the
pocket except for a decrease in C1066 and A1191 (Fig. 8).

394
S. Dharuman et al.
Fig. 8 Decomposition of free
energy of binding in the
spectinomycin binding pocket.
The total ΔG of binding for each
nucleotide interacting with
spectinomycin 1 (dark red), 5
(dark orange), 9 (dark blue), 2
(light red), 6 (light orange), and
10 (light blue) in the binding
pocket. The ΔG is an average
over the 20 ns MD ensemble and
the error bars represent the
standard deviation
Conclusions
small increases in cellular accumulation and was not found to
affect the antimicrobial potency.
Aminoglycoside resistance mediated by AME activity remains
a major hurdle in the field of antibiotic drug discovery.
This study sought to determine how removal or modification
of the AME targeted motifs in spectinomycin, and the related
spectinamides and amSPCs, affected ribosomal potency,
antibacterial activity, and cellular accumulation. We used an
efficient method for the deoxygenation of spectinomycin
enabling the synthesis of deoxyspectinamides and amSPCs.
This method involves formation of imidazolylthiocarbamate
and followed by deoxygenation reaction with tributyltin
hydride, as used in classical “Barton-McCombie” deox-
ygenation method. The deoxyspectinomycin analogs had poor
antibacterial activity and ribosomal binding affinity, as did the
6-dehydrospectinamide 11 and the epi-chloro spectinomycin 8
also synthesized as comparators. Molecular dynamic simula-
tions followed by energy decomposition of the individual
nucleotides involved in spectinomycin binding revealed the 6-
hydroxy group has a larger effect on binding compared to the
2-hydroxy group consistent with in vitro protein translational
inhibition data. These results experimentally validate our
modeling approach, which allow for key binding site residues
to be targeted in the design of future spectinomycin analogs.
Other important spectinomycin binding/structure–activity
information can be gleaned from examining the testing data in
this study, including an evaluation of the contribution the aryl
side chains make to spectinamide and amSPC binding. In this
case, the loss of binding affinity caused by deoxygenation of
the spectinomycin ring affinity is demonstrated to be partially
rescued by the additional interactions between the RpsE pro-
tein and the aryl side chains extending from the deox-
yspectinamide and deoxy-amSPC analogs. Deoxygenation
decreases the hydrophilicity of the spectinomycins, a goal of
our program, as more lipophilic analogs are expected to have
higher permeability in mammalian and bacteria cells. How-
ever, deoxygenation of the analogs in this study led to only
This study builds the on the knowledge of the SAR of
spectinomycin analogs and is being used to aid the design of
next-generation spectinomycins. Analogs that maintain a non-
AME modifiable hydrogen bond donor at the 6-position to
interact with G1064 in combination with aryl side chains
modifications are being explored in subsequent studies. Such
analogs may be successful new class of antibiotics targeting
the helix-34 ribosomal binding site, overcoming AME resis-
tance, if their overall binding affinities can be increased and
their cellular permeability optimized.
Acknowledgements This research was supported by NIH grants
(R01AI090810, R01AI136803, and T32AI106700) and ALSAC, St
Jude’s Children Research Hospital. The content is solely the respon-
sibility of the authors and does not necessarily represent the official
views of the National Institutes of Health. ECB and DS thank Tanja
Viloria for expert technical assistance.
Compliance with ethical standards
Conflict of interest The authors declare that they have no coflict of
interest.
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References
a dual-specificity aminoglycoside adenylyltransferase from Sal-
monella enterica. J Biol Chem. 2018;293:11481–90.
20. Foley L, Lin JT, Weigele M. Spectinomycin chemistry. II.) 9-
Deoxy-4(R)-dihydrospectinomycin and 9-deoxyspectinomycin. J
Antibiot. 1978;31:979–84.
21. Foley L, Lin JT, Weigele M. Preparation of 7-deoxyspectinomycin
and 7-deoxy-8-epi-4(R)-dihydrospectinomycin. J Antibiot. 1979;32:
418–9.
1. Mason DJ, Dietz A, Smith RM. Actinospectacin, a new antibiotic.
I. Discovery and biological properties. Antibiot Chemother.
1961;11:118–22.
2. Bergy ME, Eble TE, Herr RR. Actinospectacin, a new antibiotic. IV.
Isolation, purification, and chemical properties. Antibiot Chemother.
1961;11:661–4.
22. Rosenbrook W, Carney RE. Spectinomycin modification. I. 7-Epi-
9-deoxy-4(R)-dihydrospectinomycin. J Antibiot. 1975;28:953–9.
23. Barton DHR, McCombie SW. A new method for the deox-
ygenation of secondary alcohols. J Chem Soc. 1975:1574–85.
https://doi.org/10.1039/P19750001574.
24. Carney RE, Rosenbrook W. Spectinomycin modification. III
Chloro-deoxy analogs. J Antibiot. 1977;30:960–4.
25. Liu J, Bruhn DF, Lee RB, Zheng Z, Janusic T, Scherbakov D, et al.
Structure–activity relationships of spectinamide antituberculosis
agents: a dissection of ribosomal inhibition and native efflux avoid-
ance contributions. ACS Infect Dis. 2017;3:72–88.
26. Borovinskaya MA, Pai RD, Zhang W, Schuwirth BS, Holton JM,
Hirokawa G, et al. Structural basis for aminoglycoside inhibition of
bacterial ribosome recycling. Nat Struct Mol Biol. 2007;14:727–32.
27. Jacobson MP, Friesner RA, Xiang Z, Honig B. On the role of the
crystal environment in determining protein side-chain conforma-
tions. J Mol Biol. 2002;320:597–608.
28. Jacobson MP, Pincus DL, Rapp CS, Day TJ, Honig B, Shaw DE,
et al. A hierarchical approach to all-atom protein loop prediction.
Proteins. 2004;55:351–67.
29. Pontius J, Richelle J, Wodak SJ. Deviations from standard atomic
volumes as a quality measure for protein crystal structures. J Mol
Biol. 1996;264:121–36.
30. Harder E, Damm W, Maple J, Wu C, Reboul M, Xiang JY, et al.
OPLS3: a force field providing broad coverage of drug-like small
molecules and proteins. J Chem Theory Comput. 2016;12:281–96.
31. Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman W.
Protein and ligand preparation: parameters, protocols, and influ-
ence on virtual screening enrichments. J Comput Aided Mol Des.
2013;27:221–34.
32. Greenwood JR, Calkins D, Sullivan AP, Shelley JC. Towards the
comprehensive, rapid, and accurate prediction of the favorable
tautomeric states of drug-like molecules in aqueous solution. J
Comput Aided Mol Des. 2010;24:591–604.
33. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ,
Mainz DT, et al. Glide: a new approach for rapid, accurate
docking and scoring. 1. method and assessment of docking
accuracy. J Medicinal Chem. 2004;47:1739–49.
34. Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR,
Halgren TA, et al. Extra precision glide: docking and scoring
incorporating a model of hydrophobic enclosure for protein
−ligand complexes. J Med Chem. 2006;49:6177–96.
35. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL,
Pollard WT, et al. Glide: a new approach for rapid, accurate
docking and scoring. 2. Enrichment factors in database screening.
J Med Chem. 2004;47:1750–9.
36. Case DA, Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TE,
III, Cruzeiro VWD, et al. AMBER. San Francisco: University of
California; 2018.
37. Aytenfisu AH, Spasic A, Grossfield A, Stern HA, Mathews DH.
Revised RNA dihedral parameters for the amber force field improve
RNA molecular dynamics. J Chem Theory Comput. 2017;13:900–15.
38. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA.
Development and testing of a general Amber force field. J Comput
Chem. 2004;25:1157–74.
3. Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ,
Wimberly BT, Ramakrishnan V. Functional insights from the
structure of the 30S ribosomal subunit and its interactions with
antibiotics. Nature. 2000;407:340–8.
4. Laird SM, Taylor G. Treatment of gonorrhoea with actinos-
pectacin. Br J Vener Dis. 1962;38:60–3.
5. Fernandes PB, Vojtko CM, Bower RR, Weisz J. Spenolimycin, a
new spectinomycin-type antibiotic. III. Biological properties. J
Antibiot. 1984;37:1525–7.
6. Balganesh M, Dinesh N, Sharma S, Kuruppath S, Nair AV, Sharma
U. Efflux pumps of Mycobacterium tuberculosis play a significant
role in antituberculosis activity of potential drug candidates.
Antimicrob Agents Chemother. 2012;56:2643–51.
7. Ramon-Garcia S, Martin C, De Rossi E, Ainsa JA. Contribution of
the Rv2333c efflux pump (the Stp protein) from Mycobacterium
tuberculosis to intrinsic antibiotic resistance in Mycobacterium
bovis BCG. J Antimicrob Chemother. 2007;59:544–7.
8. Galimand M, Gerbaud G, Courvalin P. Spectinomycin Resistance
in Neisseria spp. Due to mutations in 16S rRNA. Antimicrob
Agents Chemother. 2000;44:1365–6.
9. Sandvang D. Novel streptomycin and spectinomycin resistance gene
as a gene cassette within a class 1 integron isolated from Escherichia
coli. Antimicrob Agents Chemother. 1999;43:3036–8.
10. Lee RE, Hurdle JG, Liu J, Bruhn DF, Matt T, Scherman MS,
et al. Spectinamides:
a new class of semisynthetic anti-
tuberculosis agents that overcome native drug efflux. Nat Med.
2014;20:152–8.
11. Bruhn DF, Waidyarachchi SL, Madhura DB, Shcherbakov D,
Zheng Z, Liu J, et al. Aminomethyl spectinomycins as therapeutics
for drug-resistant respiratory tract and sexually transmitted bacterial
infections. Sci Transl Med. 2015;7:288ra275.
12. Iverson A, Meyer CJ, Vogel P, Waidyarachchi S, Das N,
Bruhn DF, et al. Efficacy of aminomethyl spectinomycins against
complex upper respiratory tract bacterial infections. Antimicrob
Agents Chemother. 2019;63:e02096–18.
13. Scarff JM, Waidyarachchi SL, Meyer CJ, Lane DJ, Chai W,
Lemmon MM, et al. Aminomethyl spectinomycins: a novel anti-
bacterial chemotype for biothreat pathogens. J Antibiot. 2019;72:
693–701.
14. Butler MM, Waidyarachchi SL, Connolly KL, Jerse AE, Chai W,
Lee RE, et al. Aminomethyl spectinomycins as therapeutics for
drug-resistant gonorrhea and chlamydia coinfections. Antimicrob
Agents Chemother. 2018;62:e00325–18.
15. Ramirez MS, Tolmasky ME. Aminoglycoside modifying
enzymes. Drug Resist Updat. 2010;13:151–71.
16. Suter TM, Viswanathan VK, Cianciotto NP. Isolation of a gene
encoding a novel spectinomycin phosphotransferase from Legionella
pneumophila. Antimicrob Agents Chemother. 1997;41:1385–8.
17. Zárate SG, De la Cruz Claure ML, Benito-Arenas R, Revuelta J,
Santana AG, Bastida A. Overcoming aminoglycoside enzymatic
resistance: design of novel antibiotics and inhibitors. Molecules.
2018;23:284.
18. Kanchugal PS, Selmer M. Structural recognition of spectinomycin
by resistance enzyme ANT(9) from Enterococcus faecalis. Anti-
microb Agents Chemother. 2020;64:e00371–20.
39. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE,
Simmerling C. ff14SB: improving the accuracy of protein side
19. Stern AL, Van der Verren SE, Kanchugal PS, Nasvall J,
Gutierrez-de-Teran H, Selmer M. Structural mechanism of AadA,

396
S. Dharuman et al.
chain and backbone parameters from ff99SB. J Chem Theory
Comput. 2015;11:3696–713.
45. Maier R, Woitun E, Reuter A, Reuter W, Wetzel B. Modification
of spectinomycin 1. Synthesis of 4-aminospectinomycins. J
Antibiot. 1981;34:16–21.
46. Woitun E, Maier R, Wetzel B, Reuter W, Lechner U. Modification
of spectinomycin 2. Derivatives of 4-dihydro-4-deoxy-4(R)-ami-
nospectinomycin. J Antibiot. 1981;34:22–7.
47. Maier R, Woitun E, Reuter A, Reuter W, Wetzel B. Modification
of spectinomycin. 1. Synthesis of 4-aminospectinomycins. J
Antibiot. 1981;34:16–21.
48. Hou T, Wang J, Li Y, Wang W. Assessing the performance of the
MM/PBSA and MM/GBSA methods. 1. The accuracy of binding
free energy calculations based on molecular dynamics simula-
tions. J Chem Inf Model. 2011;51:69–82.
40. Miller BR, McGee TD, Swails JM, Homeyer N, Gohlke H, Roitberg
AE. MMPBSA.py: an efficient program for end-state free energy
calculations. J Chem Theory Comput. 2012;8:3314–21.
41. Weinstein MP. Performance standards for antimicrobial suscept-
ibility testing. 30th ed. Clinical and Laboratory Standards Institute;
2020. p. 332.
42. Richter MF, Drown BS, Riley AP, Garcia A, Shirai T, Svec RL,
et al. Predictive compound accumulation rules yield a broad-
spectrum antibiotic. Nature. 2017;545:299–304.
43. Wallace MJ, Dharuman S, Fernando DM, Reeve SM, Gee CT,
Yao J, et al. Discovery and characterization of the antimetabolite
action of thioacetamide-linked 1,2,3-triazoles as disruptors of
cysteine biosynthesis in gram-negative bacteria. ACS Infect Dis.
2020;6:467–78.
49. Notredame C, Higgins DG, Heringa J. T-coffee: a novel method
for fast and accurate multiple sequence alignment. J Mol Biol.
2000;302:205–17.
44. Rosenbrook W, Carney RE, Egan RS, Stanaszek RS, Cirovic M,
Nishinaga T, et al. Spectinomycin modification. II. 7-Epi-
sepctinomycin. J Antibiot. 1975;28:960–4.
50. Robert X, Gouet P. Deciphering key features in protein structures
with the new ENDscript server. Nucleic Acids Res. 2014;42:
W320–4.