A binuclear silver complex with L-buthionine sulfoximine: synthesis,spectroscopic characterization, DFT studies and antibacterial assays{

Fernando Rodrigues Goulart Bergamini,a Marcos Antonio Ferreira Jr.,a Raphael Enoque Ferraz de Paiva,a

Alexandre Ferreira Gomes,b Fabio Cesar Gozzo,b Andre Luiz Barboza Formiga,c Fabiana Cristina Andrade

Corbi,d Italo Odone Mazali,d Danilo Antonini Alves,e Marcelo Lancellottie and Pedro Paulo Corbi*a

Received 12th July 2012, Accepted 31st August 2012

DOI: 10.1039/c2ra21433d

A binuclear silver(I) complex with the amino acid L-buthionine sulfoximine (BSO) of composition

Ag2C8H16N2O3S was synthesized and characterized by chemical and spectroscopic measurements,

and DFT (density functional theory) studies. Solid-state 13C nuclear magnetic resonance (SSNMR)

and infrared vibrational spectroscopy (IR) analyses indicate the coordination of the nitrogen and

carboxylate groups of the amino acid moiety to one of the silver atoms, while coordination to the

second silver atom occurs through the nitrogen of the sulfoximine group. ESI-QTOF-MS

measurements show the maintenance of the binuclear structure in solution. DFT studies confirm the

proposed structure as a minimum of the potential energy surface (PES) with calculations of the

hessians showing no imaginary frequencies. Raman spectroscopic measurements of the [Ag2(BSO)]

complex led to the assignments of the Ag–N bonds. Biological assays of BSO and [Ag2(BSO)] were

performed by the well-diffusion method over Staphyloccocus aureus (Gram-positive), Escherichia coli

and Pseudomonas aeruginosa (Gram-negative) bacterial strains. The ligand was inactive under the

tested concentration (100 mg mL21). The [Ag2(BSO)] complex was active against the Gram-negative

and Gram-positive bacteria tested, with MIC values of 3.125 mg mL21.

Introduction

Silver compounds have been considered for centuries in the

treatment of infectious diseases.1 Although effective, the use of

silver salts in high concentrations for the treatment of skin

bacterial infections has led to toxic side effects. The fast and

uncontrolled release of the metal ion and its further accumula-

tion in the kidneys and liver are probably the most prominent

causes of silver intoxications.2 The clinical introduction of

sulfonamides in the 1930’s, in addition to the serendipitous

discovery of penicillin by Sir Alexander Fleming, can be

considered a historic event in the treatment of bacterial

infections. The discovery of the biological activities of such

compounds led to the development of new synthetic and semi-

synthetic organic antibacterial drugs.3–5 As a result, the synthesis

and biological applications of new silver-based compounds as

antibacterial agents were drastically reduced.1

Silver nitrate was reconsidered to for clinical use in the 1960’s,

when Moyer et al. proposed that a 0.5% aqueous silver nitrate

solution could be efficiently used on burns against the S. aureus

bacterial strain. According to the authors, the use of silver

nitrate in this concentration does not interfere in epidermal

proliferation.6 This resurgence was closely followed by the

discovery of the antibacterial activities of silver(I)-sulfadiazine in

1968.7 Silver(I)-sulfadiazine presents considerable antibacterial

activity against both Gram-negative and Gram-positive bacterial

strains with reduced adverse effects when compared to silver(I)

nitrate.7

In spite of the antibacterial effect of free sulfadiazine, the

antibacterial activity of silver(I)-sulfadiazine was reported to be

exclusively due to the silver ion, since only it was found inside the

bacterial cells. Sulfadiazine was considered responsible for the

controlled release of the metal ion.7–11

These results have stimulated the search for new active silver(I)

complexes, since silver(I) based antiseptic materials have a low

propensity to induce bacterial resistance in comparison to

common antibiotics.12 In addition, silver also presents low

toxicity when compared to other heavy metals used for the same

purpose, such as gold and platinum.12–14

Nowadays, some bacterial strains have been shown to be

resistant even to the most effective commercially available

aBioinorganic and Medicinal Chemistry Research Laboratory, Institute ofChemistry, University of Campinas—UNICAMP, P.O. Box 6154, 13083-970 Campinas, Sao Paulo, Brazil. E-mail: ppcorbi@iqm.unicamp.brbCoordination Chemistry Laboratory, Institute of Chemistry, University ofCampinas—UNICAMP, Campinas, Sao Paulo, BrazilcDalton Mass Spectrometry Laboratory, Institute of Chemistry, Universityof Campinas—UNICAMP, Campinas, Sao Paulo, BrazildSolid-State Chemistry Laboratory, Institute of Chemistry, University ofCampinas—UNICAMP, Campinas, Sao Paulo, BrazileLaboratory of Biotechnology, Institute of Biology, University ofCampinas—UNICAMP, Campinas, Sao Paulo, Brazil{ Electronic Supplementary Information (ESI) available. See DOI:10.1039/c2ra21433d

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10372 | RSC Adv., 2012, 2, 10372–10379 This journal is � The Royal Society of Chemistry 2012

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antibiotics. For instance, there are meticillin-resistant Staphylococcus

aureus (MRSA), vancomycin-resistant Enterococcus faecium,

extended-spectrum b-lactamase-producing Enterobacteriaceae and

multi-resistant Pseudomonas aeruginosa and Acinetobacter bauman-

nii.15 The intrinsic or acquired resistance of some bacterial strains,

the latter closely related to the indiscriminate use of antibiotics, led to

the development of new antibacterial compounds to overcome

multiresistant bacterial strains.16–19 Combining bioactive com-

pounds and metal ions which exhibit bacterial activity is one of

the possible approaches against bacterial multiresistance.20,21 The

coordination of molecules such as antibacterial agents or aspecific

enzyme inhibitor compounds with metal ions such as silver and gold

is also desirable due to the possibility to target different bacterial

strains in different phases of bacterial growth.22,23

Two silver(I) complexes derived from N-acetyl-L-methionine

(L-Hacmet) and N-acetyl-DL-methionine (DL-Hacmet), {[Ag(L-

acmet)]}n and {[Ag2(D-acmet)(L-acmet)]}n, were reported to

possess a wide spectrum of antimicrobial activities against the

Gram-negative E. coli and P. aeruginosa bacterial strains (MIC

15.7 mg mL21), and the yeasts Candida albicans (MIC 15.7 mg

mL21) and Saccharomyces cerevisiae (MIC 31.3 mg mL21).24

Cuin et al. recently reported the synthesis of silver(I), gold(I) and

gold(III) complexes with 6-mercaptopurine (MP).25 The Ag(I)-

MP and Au(I)-MP complexes showed good activity against

Mycobacterium tuberculosis, responsible for tuberculosis. In

addition, our research group recently reported the synthesis,

characterization, DFT studies and preliminary antibacterial

assays of Ag(I)-N-acetyl-L-cysteine (Ag-NAC) and Ag(I)-

Nimesulide (Ag-NMS) complexes.26,27 In the case of the Ag-

NAC complex, coordination of the ligand to Ag(I) was through

the sulphur atom, whereas in the Ag-NMS complex coordination

of the ligand was through the nitrogen and oxygen atoms of the

sulfonamide group. Both compounds were tested as antibacterial

agents using the disc diffusion method, being shown to be active

against P. aeruginosa, E. coli and S. aureus bacterial strains.

Besides, the gold(I) complex with N-acetyl-L-cysteine was also

synthesized. Biological studies are in progress in order to

compare its antibacterial activities with the respective silver(I)

complex.28

L-Buthionine sulfoximine (BSO, C8H18N2O3S, M.W.

222.31 g mol21) is a specific and potent inhibitor of c-gluta-

milcysteine synthetase, which decreases the level of glutathione

in tumor cells.29–32 Glutathione is one of the major intracellular

antioxidants with multiple biological functions,33 but has the

negative aspect of also being related to the development of

resistance of several tumors to anti-cancer drugs.34–37 BSO is

used as an adjuvant in treatment with metallodrugs such as cis-

diamminodichloridoplatinum(II) (cisplatin).38 Besides its biolo-

gical properties, BSO is a polyfunctional ligand due to the

presence of two basic nitrogen atoms, the oxygen in the

sulfoximine group and a carboxylic moiety. Here, we describe

the synthesis of a new Ag(I) complex with L-buthionine

sulfoximine [Ag2(BSO)] in aqueous solution, and its full

characterization by elemental, ESI-QTOF mass spectrometric

and thermogravimetric (TG) analyses, infrared (IR), Raman and

solid-state 13C nuclear magnetic resonance (SSNMR) spectro-

scopy. Density functional theory (DFT) studies and antibacterial

activities of the [Ag2(BSO)] complex against Gram-positive and

Gram-negative pathogenic bacterial strains are also described.

Experimental section

Reagents and equipment

L-Buthionine sulfoximine (98%), potassium hydroxide and

silver(I) nitrate (AgNO3) (98%) were purchased from Sigma-

Aldrich Laboratories. Elemental analyses for carbon, hydrogen

and nitrogen were performed using a Perkin Elmer 2400 CHN

analyzer. Infrared spectra from 4000–400 cm21 of BSO and the

silver(I) complex [Ag2(BSO)] were measured using an ABB

Bomen MB Series FT-IR spectrophotometer; samples were

prepared as KBr pellets. The 1H solution-state nuclear magnetic

resonance (1H-RMN), 13C solution-state nuclear magnetic

resonance (13C-NMR), [1H-13C] heteronuclear single-quantum

correlation (HSQC), and [1H-13C] heteronuclear Multiple Bond

Correlation (HMBC) spectra of BSO were recorded on a

AVANCE III 400 MHz spectrometer, using a 5 mm probe at

303 K. The compound was analyzed in a deuterated water

solution. The 13C-{1H} solid-state nuclear magnetic resonance

(SSNMR) spectra of BSO and of the [Ag2(BSO)] complex were

recorded on a Bruker AVANCE II 400 MHz (9.395T) spectro-

meter operating at 100 MHz, using the combination of cross-

polarization, proton decoupling and magic angle spinning (CP/

MAS) at 10 kHz. The 1H radio-frequency field strength was set

to give a 90u pulse. Contact time and recycle delay were 4 ms and

1 s, respectively. Samples were analyzed at room temperature

and the chemical shifts were referenced to TMS.

The 15N NMR chemical shift of BSO was indirectly detected

in the solution-state by a heteronuclear [1H–15N] multiple bond

coherence (HMBC) experiment. The 1H–15N NMR data were

acquired on a Bruker AVANCE III 400 MHz spectrometer,

using a 5 mm probe at 303 K. The compound was analyzed in a

deuterated water solution. Due to the poor solubility and low

percentage of nitrogen in the [Ag2(BSO)] complex composition

the 15N NMR data for the complex could not be obtained either

in the solution or in the solid-state.

Electrospray ionization quadrupole time-of-flight mass spec-

trometry (ESI-QTOF-MS) measurements were carried out in a

Waters Synapt HDMS instrument (Manchester, UK). A sample

of [Ag2(BSO)] was solubilized in 50 : 50 H2O/MeCN (0.1%

formic acid v/v) at a concentration of ca. 4 mg mL21, then

further diluted 100-fold in the same solvent mixture and

immediately analyzed. Resulting solutions were directly infused

into the instruments ESI source at a flow rate of 15 mL min21.

Typical acquisition conditions were capillary voltage: 3 kV,

sampling cone voltage: 20 V, source temperature: 100 uC,

desolvation temperature: 200 uC, cone gas flow: 30 L h21,

desolvation gas flow: 900 L h21, trap and transfer collision

energies: 6 and 4 eV, respectively. ESI{ mass spectra (full scans)

and fragment ion spectra for quadrupole-isolated ions (QTOF-

MS/MS) were acquired in reflectron V-mode at a scan rate of 1

Hz. For fragment ion spectrum experiments by collision-induced

dissociation (argon as collision gas), the desired ion was isolated

in the mass-resolving quadrupole, and the collision energy of the

trap cell was increased until sufficient fragmentation was

observed. Prior to all analyses, the instrument was externally

calibrated with phosphoric acid oligomers (H3PO4 0.05% v/v in

50 : 50 H2O/MeCN) ranging from m/z 99 to 980. Thermal

analysis was performed on a SEIKO EXSTAR 6000 thermo-

analyzer to obtain simultaneous TGA/DTA in the following

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conditions: synthetic air, flow rate of 50 cm3 min21 and heating

rate of 10 uC min21, from 25 uC to 1100 uC.

The Raman spectrum was recorded using a Jobin-Yvon

T64000 single spectrometer system, equipped with a confocal

microscope and a nitrogen-cooled charge-coupled device (CCD)

detector. The spectrum was collected using a 633.0 nm (1.5 mW)

line of He/Ne laser at room temperature. The sample was

analyzed in the solid-state.

Synthesis

The silver(I) complex with BSO was synthesized by the reac-

tion of 2.0 mL of an aqueous solution of silver(I) nitrate (9.0 61024 mol) with 6.0 mL of a freshly prepared aqueous solution of

BSO containing 4.5 6 1024 mol of the ligand. The aqueous

AgNO3 solution was added to the BSO solution under magnetic

stirring at room temperature followed by the addition of 9.0 61024 mol of KOH. After 40 min of constant stirring, the white

solid obtained was vacuum-filtered, washed with cold water and

dried in a desiccator over P4O10. Elemental analysis led to the following

composition: Ag2C8H16N2O3S. Calcd. for Ag2C8H16N2O3S (%): C,

22.0; H, 3.70; N, 6.42; found (%): C, 22.9; H, 3.34; N, 6.60. The

[Ag2(BSO)] complex is insoluble in water, ethanol, methanol,

dimethylsulfoxide, acetonitrile, chloroform, acetone and hexane.

It is slightly soluble in a mixture of water and acetonitrile

(50 : 50 v/v). The composition of the complex has a molar ratio of

2 : 1 metal/ligand. No single crystals of the complex were

obtained, even after several attempts, in order to perform an

X-ray structure determination.

Molecular modeling

Geometry optimizations were carried out using the GAMESS

software39 with a convergence criterion of 1026 a.u. in a

conjugate gradient algorithm. The LANL2DZ40 effective core

potential was used for silver and the atomic 6-31G(d) basis set41

for all other atoms. Density functional theory (DFT) calcula-

tions were performed for BSO and [Ag2(BSO)] using the

B3LYP42 gradient-corrected hybrid functional to solve the

Kohn–Sham equations with a 1025 convergence criterion for

the density charge. For the BSO zwitterions and [Ag2(BSO)]

complex the polarizable continuum model (PCM)43 was used to

simulate the effect of water in the geometry optimization. The

final geometries were confirmed as a minimum of the potential

energy surface (PES) with calculation of the hessians. The

harmonic vibrational frequencies and intensities were calculated

at the same level of theory with the analytical evaluation of the

second derivatives of the energy as a function of the atomic

coordinates. The calculated intensities were used to generate the

theoretical spectra. Frequencies were scaled by a factor of

0.9614, as recommended by Scott and Radom.44 Simulated

vibrational spectra were obtained from the sum of the

Lorentzian functions with 20 cm21 half-bandwidths using the

software MOLDEN 4.7.45 Raman intensities for the [Ag2(BSO)]

complex were simulated by the numerical differentiation

procedure applying an electric field of 2 6 1023 a.u., as

previously reported.46 Frequencies were scaled by a factor of

1.0013, as recommended by Scott and Radom, for low

frequencies.44

Biological assays

Six pathogenic bacterial strains, E. coli ATCC 25922, P.

aeruginosa ATCC 27853, P. aeruginosa 31NM, S. aureus

ATCC 25923, S. aureus BEC9393 and S. aureus Rib1 were

selected. Stock solutions (10.0 mg mL21) of BSO and AgNO3 in

water, and also a 2.0 mL water suspension containing 20.0 mg of

the [Ag2(BSO)] complex were prepared before the experiment.

Sufficient inocula of the bacterial strains were added to a 24

multiwell plate until the turbidity equaled 0.5 McFarland (y1.5

6 1028 CFU mL21). Then, 100 mL of the BSO and AgNO3

solutions and 100 mL of the [Ag2(BSO)] suspension were added

to the plates. The negative control was obtained by leaving one

of the wells of each bacterial strain with no addition of the

considered compounds. The minimal inhibitory concentration

(MIC) of the [Ag2(BSO)] complex was estimated as recom-

mended by the Clinical and Laboratory Standards Institute

(CLSI).47 In this case, the [Ag2(BSO)] complex was submitted to

serial dilutions (1 : 1) in a 96 multiwell plate with 100 mL of each

compound/dilution. Samples were transferred to the plates

containing the respective bacterial strain (0.5 McFarland) in

seven decreasing concentrations.

Results and discussion

Infrared spectroscopic data

The [Ag2(BSO)] infrared (IR) spectrum was analyzed in

comparison to that of free BSO. The IR spectra of BSO and

[Ag2(BSO)] are provided in Fig. 1.

Two bands were related to the asymmetrical and symmetrical

NH2 stretching bands of the amino group and were observed in

the BSO spectrum at 3230 cm21 and 3144 cm21, respectively.

The d(NH2) band is observed in the free ligand spectrum at

1516 cm21. Also, the IR spectrum of the ligand shows a weak

band with a maximum at 2095 cm21, which is assigned to the

combination of the asymmetrical NH3+ bending vibration and

the torsional oscillation of the NH3+ group.48 The asymmetrical

and symmetrical stretching modes of the carboxylate group are

observed at 1618 cm21 and 1447 cm21, respectively. The energy

Fig. 1 Infrared spectra of (A) BSO and (B) the [Ag2(BSO)] complex.

10374 | RSC Adv., 2012, 2, 10372–10379 This journal is � The Royal Society of Chemistry 2012

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difference (D) between nas(COO2) and nsym(COO2), which is

used as the parameter to evaluate coordination modes of the

carboxylate group,49,50 is 171 cm21. Moreover, another band

corresponding to NH3+ is also observed in the IR spectrum of

the ligand at 1582 cm21, which reinforces the existence of the

ligand in its zwitterionic form. Characteristic sulfoximine

(HNLSLO) bands are observed in the BSO spectrum at

1210 cm21, and 1012 cm21, and are assigned as the NLS and

SLO stretching vibrations, respectively.51 The combination of the

N–H bending with the SLO and NLS stretching modes is

observed at 1139 cm21. The other characteristic bands for BSO

are the asymmetric and symmetric stretching bands due to the

CH2 group, which are observed at 2962 and 2930 cm21,

respectively.

In the [Ag2(BSO)] spectrum, asymmetrical carboxylate

stretching appears at 1582 cm21, while the symmetrical band is

observed at 1390 cm21. So, in this case, the energy difference (D)

between the nas(COO–) and nsym(COO–) is 192 cm21. According

to the literature, the larger D value for the complex when

compared to the ligand suggests a monodentate coordination of

the carboxylate group to the metal.50

In the [Ag2(BSO)] spectrum, a broad band in the region 3550–

3100 cm21 is observed. This band can be assigned to the

hydrogen bonds between the hydration water molecules and the

NH2 group of the [Ag2(BSO)] complex. Hydrogen bonds lead to

a poor resolution of the asymmetrical and symmetrical N–H

stretching bands. As reported in the literature, the shifting of the

N–H stretching mode in the spectrum of the complex, when

compared to the spectrum of the ligand can be attributed to the

coordination of the amino group of BSO to Ag(I). Moreover, the

band referring to the combination of the asymmetrical NH3+

bending vibration and the torsional oscillation of the NH3+

group, observed for BSO, is not present in the [Ag2(BSO)]

spectrum, which is also indicative of silver(I) coordination to

BSO through the nitrogen of the amino group.

The SLN and SLO stretching bands in the [Ag2(BSO)]

spectrum are observed at 1203 cm21 and at 1012 cm21,

respectively. The shifting of the SLN band suggests the

coordination of BSO to the second silver ion through the

nitrogen of the sulfoximine. The SLN–H bending vibration band

is not present in the [Ag2(BSO)] spectrum, which also indicates

that the other silver ion is coordinated to the nitrogen of the

sulfoximine group. The characteristic bands for the asymmetric

and symmetric stretching modes of the CH2 group are observed

in the same region as for free BSO.

13C and 15N NMR spectroscopic measurements

The structure of BSO with the hydrogen and carbon atoms

numbered is shown in Fig. 2. The solution-state 1H-NMR and13C-NMR for free BSO are presented in the ESI (#1).{ For a

more accurate assignment of the hydrogen and carbon atoms,

the DEPT135, [1H-13C]-HSQC, [1H-13C]-HMBC and [1H-15N]-

HMBC NMR data for the ligand in D2O were also obtained.

The [1H-15N]-HMBC, [1H-13C]-HMBC and DEPT 135 spectra

are shown in the ESI (#2).{ The [1H-13C]-HSQC spectrum is

presented in the ESI (#3).{As observed in the [1H-13C] HSQC spectrum, the triplet at

0.86 ppm in the 1H-NMR spectrum couples with the carbon at

12.7 ppm in the 13C-NMR spectrum. The high field signals in

both the 1H-NMR and 13C-NMR spectra can be respectively

attributed to the hydrogen atoms (H-8) and the carbon atom (C-

8) of the methyl group. The high field carbons at 20.9 ppm and

23.6 ppm can be assigned, respectively, as C-7 and C-6. The C-7

atom couples with the sextet at 1.39 ppm which can be attributed

to the H-7 hydrogen atoms. On the other hand, the C-6 atom

couples with the quintet at 1.70 ppm, which is assigned to the

H-6 hydrogen atoms. With a chemical shift similar to C-6, the

C-3 carbon atom is assigned at 23.6 ppm and couples with

the quartet of the H-3 hydrogen atoms at 2.43 ppm. Due to the

direct bond of the C-4 to the sulfoximine chiral center, the

hydrogen atoms H-4a and H-4b are no longer equivalent and

appear in the spectrum as two quartet signals, in the range 3.27–

3.36 ppm.

The C-2 and C-5 atoms in the 13C-NMR spectrum appear,

respectively, at 53.0 ppm and 53.4 ppm. The C-2 couples with the

quartet at 3.80 ppm. The position of C-2 can be explained by the

direct bonding of this atom to the primary amine. The C-5

carbon couples with a quartet signal at 3.20 ppm which is

assigned to H-5. Finally, the signal that appears at 172.9 ppm is

assigned to carbon C-1.

The [1H-13C]-HMBC and [1H-15N]-HMBC spectra confirm

the HSQC assignment. The DEPT-135 NMR analysis of BSO

confirms C-7, C-6, C-5, C-4 and C-3 as methylenes (CH2).

The NMR spectra of the [Ag2(BSO)] complex were analyzed in

comparison to that of BSO. Due to the low solubility of the

[Ag2(BSO)] complex in both polar and non-polar solvents, the

solid-state nuclear magnetic resonance technique (SSNMR) was

applied. The [Ag2(BSO)] complex and the free ligand 13C-{1H}

SSNMR spectra are provided in Fig. 3, with the respective

carbon assignments.

According to the experimental data, the chemical shift of C-2

in the free ligand spectrum and in the complex spectrum are

observed at 53.8 ppm and at 56.6 ppm, respectively, with a Dd

(d complex˜ d ligand) of 2.8 ppm. Moreover, the C-6, C-3 and

C-5 signals are observed, respectively, at 23.1 ppm, 26.3 ppm and

56.0 ppm in the free ligand spectrum while for the complex the

same carbon atoms appear at 28.9 ppm, 33.7 ppm and 61.6 ppm.

The Dd of the C-6, C-3 and C-5 carbon atoms are DdC-6 =

5.8 ppm, DdC-3 = 7.4 ppm and DdC-5 = 5.6 ppm. As suggested by

the IR data, the NMR data reinforces the coordination of BSO

to one of the silver atoms through the nitrogen of the

sulfoximine. This coordination would lead to changes in the

chemical shift of the carbon C-4. In our case, the DdC-4 was

0.7 ppm.

In the 13C SSNMR spectra of BSO and the [Ag2(BSO)]

complex, C-1 appears as two signals. This phenomenon can be

explained due to the existence of polymorphism for both BSO

and the [Ag2(BSO)] complex in the solid state.52 The chemical

shift for C-1 is observed at 176.6 ppm for the free ligand and at

Fig. 2 The schematic structure of BSO with the carbon and hydrogen

atoms numbered.

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178.7 ppm for the complex (DdC-1 = 2.1 ppm). No substantial

difference was observed in the chemical shifts of the C-7 and C-8

carbon atoms in the complex when compared to the free ligand

(DdC-7 = 1.0 ppm; DdC-8 = 20.1 ppm). The carbon assignments for

BSO and for the [Ag2(BSO)] complex with their respective Dd are

listed in Table 1. According to the NMR data, we suggest that one

of the silver atoms is coordinated to the nitrogen of the amino

group and the oxygen of the carboxylate group, while the other

silver atom is coordinated to the nitrogen of the sulfoximine group.

Thermal analysis

Thermogravimetric (TGA) data for the [Ag2(BSO)] complex is

presented in the ESI (#4).{ Ligand decomposition starts at

170 uC and occurs in two steps, leading to the formation of the

residue of the thermal treatment at 550 uC. Calcd. for loss of

BSO (%) 48.5; found (%) 53.6. The residue represents the

formation of metallic silver. Anal. Calcd. for Ag0 (%) 47.7; found

(%) 43.0. Water content (3.44%) is lost until 100 uC. The presence

of water is most probably due to absorption from the

environment during sample handling before the experiment.

Raman spectroscopic measurements

In order to confirm nitrogen coordination of the sulfoximine and

of the amino groups to the silver atoms, the Raman spectrum of

[Ag2(BSO)] was obtained. The spectrum is provided in the ESI

(#5).{ As observed, the [Ag2(BSO)] spectrum presents two broad

bands with their maxima at 484 cm21 and 668 cm21. According

to the literature, these bands can be assigned to the Ag–N

stretching modes from Ag–N(SO) and Ag–N(H2), respectively.53

Mass spectrometry measurements

The Ag2C8H16N2O3S composition was also confirmed by ESI-

QTOF-MS measurements. The [Ag2(BSO)] spectrum, presented

in Fig. 4, shows the presence of the monoprotonated ion

[Ag2BSO2H]+ at m/z 436.91, as well as the presence of the

[AgBSO]+ ion at m/z 329.00. The spectrum also shows the

presence of the [Ag(BSO)2]+ (m/z 553.11), [Ag2(BSO)2+H]+ (m/z

659.34) and [Ag3(BSO)222H]+ (m/z 764.86) ions. The experi-

mental isotopic pattern for [Ag2BSO2H]+ was compared to the

expected isotopic pattern considering the proposed composition

and was shown to be in good agreement to the latter with an

error of 22.0 ppm (calcd. m/z 434.9062, exp. m/z 434.9053).

To further investigate the structure of the observed ions, the

[Ag2BSO2H]+ and [AgBSO]+ ions, as well as the [BSO+H]+ ion,

were analyzed by ion fragmentation MS/MS spectrometry

(Fig. 5). The energy for the fragmentation of the monoproto-

nated ions was 14 eV for both [BSO+H]+ and [AgBSO]+ and

20 eV for [Ag2BSO2H]+. The [Ag2BSO2H]+ MS/MS spectrum

shows a signal at 216.82 m/z, which corresponds to the

monoprotonated species minus a C8H16N2O3S fragment. This

fragment can be attributed to one BSO ligand with the absence

of two hydrogen atoms. The Ag+ ion is also observed in the

spectrum at 106.90 m/z.

Molecular modeling

The geometries of BSO and [Ag2(BSO)] were obtained by

theoretical calculations using density functional theory (DFT).

Fig. 3 The 13C-SSNMR spectra of (A) [Ag2(BSO)] and (B) BSO.

Table 1 13C NMR assignments for BSO and for the [Ag2(BSO)]complex with the respective Dd

d (ppm) (BSO) d (ppm) (Ag2BSO) Dd (ppm)

C-1 176.6 178.7 2.1C-2 53.8 56.6 2.8C-3 26.3 33.7 7.4C-4 52.6 53.3 0.7C-5 56.0 61.6 5.6C-6 23.1 28.9 5.8C-7 22.1 23.1 1.0C-8 14.5 14.4 20.1

Fig. 4 Mass spectra for the [Ag2(BSO)] complex. (A) The ESI(+)-

QTOF mass spectrum from m/z 150 to 800. The term BSO2H refers to

the BSO ligand minus one hydrogen (C8H17N2O3S, 221.0960 Da). (B)

The isotope pattern comparison for the [Ag2BSO2H]+ ion of m/z 434.90.

The mass error was 22.0 ppm for [Ag2BSO2H]+ ([C8H17Ag2N2O3S]+,

calcd. m/z 434.9062, exp. m/z 434.9053), considering the monoisotopic

ion of the composition.

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The BSO molecule presents a zwitterionic structure. This

structure was optimized and its equilibrium geometry was

confirmed by vibrational analysis. The bond distances found

for the BSO structure are comparable to those previously

reported in the literature for an optimized structure using

DFT.54 The theoretical IR spectrum was obtained by calculation

of the hessians, showing no imaginary frequencies, and

compared to the experimental spectrum, which also confirms

the BSO optimized structure in the solid state. For the

calculations, the polarizable continuum model (PCM) was

employed to account for the effect of water in the description

of the zwitterionic forms. For comparative purposes, the

[Ag2(BSO)] complex structure was optimized at the same level

of theory.

As observed by elemental and thermal analysis, the complex

presents a 2 : 1 metal/ligand ratio, which was also observed by

the ESI-QTOF-MS analysis. The spectroscopic techniques

indicate the coordination of one silver(I) atom through the

nitrogen atom of the amino group and a monodentate

carboxylate, while the other silver(I) ion appears coordinated

to the sulfoximine moiety. So, in the case of the [Ag2(BSO)]

complex, the most stable structure is obtained considering the

coordination number two for the first silver(I) ion, and

coordination number one to the second silver(I) ion, which is

bonded to the nitrogen of the sulfoximine group. Silver(I) ions

can adopt diverse coordination numbers, passing through

coordination numbers one (monodentate) and two to high

coordination numbers and geometries, as reported in the

literature for various silver compounds.55–58 These data permit

us to propose a possible structure for the [Ag2(BSO)] complex,

which was also confirmed as a minimum of PES by calculation

of the hessians.

The optimized structure for the [Ag2(BSO)] complex is

presented in Fig. 6. The calculated Ag–NH2 and Ag–O distances

were 2.075 A and 2.032 A, respectively, while the H2N–Ag–O

angle was 83.0u. The Ag–N and Ag–O distances for the silver

atom bonded to the nitrogen atom of the sulfoximine group are

1.992 A and 2.679 A. The N–Ag–O angle was 83.1u. Detailed

bond distances, angles and dihedrals are reported in the ESI

(#6).{

Fig. 5 The fragment ion mass spectrum (collision-induced dissociation)

for the (A) monoprotonated BSO ligand, [BSO+H]+ of m/z 223.11. The

collision energy of the trap cell was 14 eV. The term BSO refers to the

neutral BSO ligand (C8H18N2O3S, 222.1038 Da). (B) The fragment ion

mass spectrum (collision-induced dissociation) for the [AgBSO]+ ion of

m/z 329.00. The collision energy of the trap cell was 14 eV. The term BSO

refers to the neutral BSO ligand (C8H18N2O3S, 222.1038 Da). (C) The

fragment ion mass spectrum (collision-induced dissociation) for the

[Ag2BSO2H]+ ion of m/z 436.91. The collision energy of the trap cell was

20 eV. The term [BSO22H] refers to the BSO ligand minus two

hydrogens (C8H16N2O3S, 220.0882 Da).

Fig. 6 The [Ag2(BSO)] complex optimized structure obtained by

B3LYP/DFT using LANL2DZ(Ag) and 6-31(d,p). The PCM model

was used to simulate the water effect on the geometric optimizations.

Fig. 7 Simulated infrared spectra of (A) BSO and (C) [Ag2(BSO)]. The

experimental spectra of (B) BSO and (D) [Ag2(BSO)] are presented for

comparison.

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The simulated and experimental IR spectra of BSO and

[Ag2(BSO)] are presented in Fig. 7. The [Ag2(BSO)] IR spectrum

is in good agreement with the experimental spectrum. These

spectra were used to confirm the experimental assignments. The

simulated vibrational spectrum for BSO shows asymmetric

amino group stretching nas(H–N–H) at 3461 cm21 with the

symmetric stretching nsym(H–N–H) observed at 3356 cm21. The

characteristic combination of the asymmetrical NH3+ bend

vibration and the torsional oscillation of the NH3+ group appear

at 2562 cm21. The difference between the simulated and

experimental values for this combination band is larger than

usual, but it can be attributed to intermolecular interactions in

the solid state.50 The n(C–O) appears as a combination mode

with d(NH2) at 1345 cm21. The asymmetric n(C–O) appears at

1699 cm21 whereas the asymmetric d(NH2) appears at

1582 cm21. A combination mode encompassing the sulfoximine

S–N–H bending and the SLO and SLN stretchings appears at

1128 cm21. The d(S–N–H) of the sulfoximine also contributes to

the band at 1072 cm21 whereas the SLN stretching is observed at

913 cm21.

In the simulated [Ag2(BSO)] spectrum, the asymmetric and

symmetric stretching modes nas(H–N–H) and nsym(H–N–H)

appear at 3300 cm21 and 3186 cm21, respectively. These data

reinforce the loss of the hydrogen atom of the NH3+ group and

coordination of the NH2 group to Ag(I).

The bands attributed to the combination mode concerning the

sulfoximine SLO and SLN stretchings appears at 997 cm21 and

1104 cm21. When compared to the free BSO spectrum, these

bands are shifted 224 cm21 and 284 cm1, respectively.

Moreover, the nas(C–O) band is observed at 1660 cm21, being

shifted 239 cm21 when compared to the free ligand.

The calculated values for the stretching modes in the Raman

spectrum, assigned as N–Ag from silver(I)-amine and silver(I)-

sulfoximine are presented in the ESI (#5).{ As observed, the Ag–

N stretching bands are in good agreement with the experimental

data and reinforce the band assignments.

Antibacterial studies

Antibiogram assays were carried out in order to evaluate the

antibacterial activities of the Ag-BSO complex. The activities of

the complex against the considered bacterial strains were

confirmed by MIC values, with concentrations ranging 3.125–

100.0 mg mL21. The results obtained show promising antibacter-

ial activity of the Ag-BSO complex against Gram-negative

bacteria, being comparable to the inhibitory effect of the

standard antibiotic chloramphenicol, used as positive control.

The Ag-BSO complex was less active than the standard

antibiotic vancomycin in the MIC assays for Gram-positive S.

aureus BEC9393, S. aureus Rib 1 and S. aureus ATCC 25923.

The free BSO did not exhibit antibacterial activity under the

same experimental conditions. Antibiotic sensitivity profiles of

the bacterial strains are listed in Table 2. The observed results

show promising potential of application of the Ag-BSO complex

as a cream in skin infections in the case of severe burns, due to

small number of effective antibiotics against some specific Gram-

negative bacterial strains.

Conclusions

A silver(I) complex with BSO was synthesized and structurally

characterized. Elemental, themogravimetric and ESI-QTOF-MS

analyses show a 2 : 1 metal/ligand composition, with the

molecular formula [Ag2(C8H16N2O3S)]. The 13C CP/MAS

SSNMR, IR and Raman spectroscopic data suggest the

coordination of BSO to one silver atom through the amino

and carboxylate groups of the amino acid moiety, and also the

coordination of the ligand to another silver atom through

the nitrogen of the sulfoximine group. DFT studies support the

proposed geometry. Biological studies revealed that the complex

is effective against all the tested bacteria, being more effective

against the tested Gram-negative bacterial strains. Further

studies are intended in order to evaluate the possible mechanism

of action of the [Ag2(BSO)] complex.

Acknowledgements

This study was supported by grants from the Brazilian Agencies

FAPESP (Sao Paulo State Research Foundation, Brazil—proc.

2006/55367-2, 2008/57805-2, 2012/08230-2 and 2009/54066—

Laboratory of Advanced Optical Spectroscopy, LMEOA/IQ—

UNICAMP), CAPES and CNPq (Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico, Brazil—proc.

573672/2008-3; 472468/2010-3 and 472067/2010-9). The authors

are also grateful to MSc. Helen Graci C. de Meneses for her

valuable contribution in the DFT studies and to Professor Carol

H. Collins for the English revision of the manuscript.

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