S1
Tween-20/ H2O Promoted Green Synthesis, Computational and Antibacterial
Activity of Amino Acid Substituted Methylene Bisphosphonates
S. Siva Prasada, S.H. Jayaprakasha, Ch. Syama sundara, P. Sreelakshmia, C.
Bhuvaneswarb,c, B. Vijaya Bhaskarb, W. Rajendrab and C. Suresh Reddya,*
a
b
Department of Chemistry, Sri Venkateswara University, Tirupati - 517 502, INDIA
Division of Molecular Biology, Department of Zoology, Sri Venkateswara University, Tirupati
– 517 502, INDIA
c
Department of Microbiology, Sri Venkateswara University, Tirupati - 517 502, INDIA
E-mail: csrsvu@gmail.com
Supplemental Materials
Biological Activity
Ciprofloxacin was used as standard antibacterial agent. Solutions of the test compounds
and reference drug were prepared in dimethyl sulfoxide (DMSO) at concentrations of 100, 50, 25,
12.5, 6.25, 3.125 and 1.5625 µg/ mL. Nine tubes were prepared in duplicate with the second set
being used as MIC reference controls (16-24 h visual). After sample preparation, the controls were
placed in a 37°C incubator and read for macroscopic growth (clear or turbid) the next day. Into
each tube, 0.8 mL of nutrient broth was pipetted (tubes 2–8), tube 1 (negative control) received
1.0 mL of nutrient broth and tube 9 (positive control) received 0.9 mL of nutrient. Tube 1, the
negative control, did not contain bacteria or antibiotic. The positive control, tube 9, received 0.9
mL of nutrient broth since it contained bacteria but not antibiotic. The test compound were
dissolved in DMSO (100 µg/ mL), 0.1 mL of increasing concentration of the prepared test
compounds which are serially diluted from tube 2 to tube 8 from highest (100 µg/ mL) to lowest
(1.5625 µg/ mL) concentration (tube 2-8 containing 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 µg/mL).
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After this process, each tube was inoculated with 0.1 mL of the bacterial suspension, concentration
of which corresponded to 0.5 McFarland scale (9 X 108 cells/ mL) and each bacterium was
incubated at 37 °C for 24 h. The final volume in each tube was 1.0 mL. The incubation chamber
was kept humid. At the end of the incubation period, MIC values were recorded as the lowest
concentration of the substance that gave no visible turbidity, i.e. no growth of inoculated bacteria.
The results were shown in Table S 1.
Table S 1: Results of antibacterial activity of 4a-j: MIC values in μg/ mL.
Gram -ve
Gram +ve
Compounds
Klebsiella
pneumonia
Pseudomonas
aeruginosa
Staphylococcus
aureus
Bacillus subtilis
4a
1.5625
3.125
3.125
1.5625
4b
>100
>100
50
25
4c
50
>100
>100
50
4d
1.5625
1.5625
1.5625
1.5625
4e
50
>100
>100
>100
4f
50
50
3.125
3.125
4g
>100
>100
25
25
4h
3.125
50
>100
>100
4i
50
50
25
50
4j
50
25
>100
>100
Ciprofloxacin
1
2
2
2
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Table S 2: Antibacterial activity of Compounds4a-j: Zone of Inhibition (mm).
Zone of Inhibition (mm)
Compound
Gram -ve
Klebsiella
Pseudomonas
pneumonia
aeruginosa
50
100
50
100
µg/mL µg/mL µg/mL µg/mL
Gram +ve
Staphylococcus
Bacillus subtilis
aureus
50
100
50
100
µg/mL µg/mL µg/mL µg/mL
4a
33
36
18
25
25
30
21
28
4b
25
32
16
18
18
20
8
11
4c
26
32
14
16
20
29
10
10
4d
30
34
21
28
30
33
27
31
4e
26
31
17
17
0
21
2
4
4f
25
32
12
19
15
20
10
21
4g
26
29
11
20
22
24
4
12
4h
4i
25
28
30
31
14
10
19
18
25
17
26
28
18
12
20
24
4j
25
30
13
16
0
25
1
7
Gentamycin
18
25
33
24
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Table S 3: Hydrogen bonding interactions between protein and ligands 4a-j
Compound
4a
4b
4c
4d
4e
4f
4g
Interactions
Protein
ligand
Asp517NH1------------OS13
Asp517NH2------------ OS12
Asp517NH2------------ OS13
Arg1012NH2-----------OC24
Ser1021OG-------------OC6
Ser1021OG-------------OP12
Ser1028OG-------------OP15
Arg1033NH2-----------OP13
Arg1048NH1-----------OC24
Arg1048NH2----------- OC24
His1081NE2------------OC25
Asp510CO-----------HO21
Asp510CN----------HO21
Arg517NH2--------OC21
Arg517NH2--------OP24
Ser1021OG---------OP22
Asp1024OD2-------OD2
Asp508OD2-----------HN24
Lys581NZ------------OP21
Lys581NZ------------OP23
Lys581NZ------------OP19
Gly584N--------------OC32
Gln1095NE2---------OC34
Ser1098OG--------------OP24
Ser1112N-------------OC35
Arg1092NH1-----------OP10
Gln1095OE1------------HN14
Ser1098OG-------------OP10
Ser1098OG-------------OP11
Ser1112CO--------------HO2
Asp1116CO------------- HO2
Thr507CO---------------HO3
ARg517NH1-------------OC31
Distance
(Å)
3.3
2.7
2.8
3.2
3.0
3.2
3.2
3.0
3.0
3.2
3.2
2.5
3.2
3.1
3.3
2.8
2.2
2.6
3.1
3.0
3.1
2.9
2.8
3.1
3.3
3.2
2.4
3.2
2.8
2.6
2.4
2.0
3.2
Binding energy
(Kcal/ mol)
-6.8
-6.5
-5.8
-6.8
-7.1
-5.8
-6.1
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Compound
4h
4i
4j
Ciprofloxacin
Interactions
Protein
ligand
Arg517NH2--------------OP12
Arg517NH2------------- OP13
Glu541NE2--------------OP10
Ser1021OG--------------OP12
Asp508CO---------------OC9
ARg517NH1-------------OC31
Arg517NH2--------------OC27
Arg517NH2------------- OP15
Arg517NH2------------- OP17
Glu541NE2--------------OP20
Thr544Og1--------------OP16
Arg1092NH1-----------OP15
Gln1095OE1-----------ON22
Gln1095NE1-----------OC27
Ser1112N---------------OC27
Asp1116CO-------------OC7
Val1268CO--------------HN5
Arg517NH2-------------OC31
Asp510N-----------------O31
Asp510O-----------------HO31
Ser1021OG--------------OP19
Ser1021OG------------OP23
Asp1024OD2------------HN24
Tyr580CO---------HN25
Ser1028OG-------OC23
Arg1033NH2------OC23
His1081NE2-------OC22
Distance
(Å)
3.0
3.2
2.9
3.1
2.8
2.9
2.8
3.0
3.4
3.3
3.1
3.3
2.7
3.3
3.3
2.5
2.3
3.1
3.2
2.1
3.1
3.1
2.2
2.2
2.7
3.0
3.3
Binding energy
(Kcal/ mol)
-6.6
-6.2
-7.4
-6.8
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4j
4d
4h
4a
Ciprofloxacin
Figure S 1: Binding behavior of Compounds 4j, 4d, 4h, 4a and Ciprofloxacin with residues of
metal binding actives sites of Type IIA topoisomerase
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Figure S 2: 1H NMR Spectrum of compound 4a
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Figure S 3: 13C NMR Spectrum of compound 4a
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Figure S 4: 31P NMR Spectrum of compound 4a
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Figure S 5: 1H NMR Spectrum of compound 4d
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Figure S 6: 13C NMR Spectrum of compound 4d
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Figure S 7: 31P NMR Spectrum of compound 4d