Biometals (2019) 32:627–640

https://doi.org/10.1007/s10534-019-00198-0 (0123456789().,-volV)
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The interaction of silver(II) complexes with biological

macromolecules and antioxidants
Katherine D. Trotter . Olawale Owojaiye . Stuart P. Meredith .
Pat E. Keating . Mark D. Spicer . John Reglinski . Corinne M. Spickett

Received: 15 February 2019 / Accepted: 30 April 2019 / Published online: 16 May 2019
Ó The Author(s) 2019

Abstract Silver is widely used for its antimicrobial them, as well as determining the reaction products.
properties, but microbial resistance to heavy metals is Spectrophotometric analysis showed that Ag2,6P was
increasing. Silver(II) compounds are more oxidizing rapidly reduced by the antioxidants glutathione,
and therefore have the potential to overcome resis- ascorbic acid and vitamin E; the unsaturated lipids
tance via extensive attack on cellular components, but arachidonic and linoleic acids, model carbohydrate b-
have traditionally been hard to stabilize for biological cyclodextrin, and protein cytochrome c also reacted
applications. Here, the high oxidation state cation was readily. Analysis of the reaction with glutathione by
stabilised using pyridinecarboxylate ligands, of which NMR and electrospray mass spectrometry confirmed
the 2,6-dicarboxypyridine Ag(II) complex (Ag2,6P) that the glutathione was oxidized to the disulfide form.
was found to have the best tractability. This complex Mass spectrometry also clearly showed the addition of
was found to be more stable in phosphate buffer than multiple oxygen atoms to the unsaturated fatty acids,
DMSO, allowing studies of its interaction with water suggesting a radical mechanism, and cross-linking of
soluble antioxidants and biological macromolecules, linoleic acid was observed. The seven hydroxyl groups
with the aim of demonstrating its potential to oxidize of b-cyclodextrin were found to be completely
oxidized to the corresponding carboxylates. Treatment
of cytochrome c with Ag2,6P led to protein aggrega-
Electronic supplementary material The online version of tion and fragmentation, and dose-dependent oxidative
this article (https://doi.org/10.1007/s10534-019-00198-0) con-
tains supplementary material, which is available to authorized damage was demonstrated by oxyblotting. Thus
users. Ag2,6P was found to be highly oxidizing to a wide
variety of polar and nonpolar biological molecules.
K. D. Trotter
O. Owojaiye
P. E. Keating

M. D. Spicer
J. Reglinski
Department of Pure & Applied Chemistry, Strathclyde Keywords Ag(II) 2,6-dicarboxypyridine

University, 295 Cathedral Street, Glasgow G1 1XL, UK Antimicrobial metal
Glutathione
Lipid
peroxidation
Oxidative stress
S. P. Meredith
C. M. Spickett
School of Life and Health Sciences, Aston University,
Aston Triangle, Birmingham B4 7ET, UK

C. M. Spickett (&)
Strathclyde Institute of Pharmacy and Biomedical
Sciences, Strathclyde University, 161 Cathedral Street,
Glasgow G4 0NR, UK
e-mail: [email protected]

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628 Biometals (2019) 32:627–640

Introduction Heiman et al. 2015; Vindigni and Surawicz 2015),

development of more powerful formulations of silver
The antiseptic and oligodynamic behaviour of silver with higher oxidizing potential would be desirable,
has been appreciated for more than 200 years (Marx especially for external use in disinfection and
and Barillo 2014; Mijnendonckx et al. 2013). How- cleansing.
ever, even in recent years its application as a biocide In addition to Ag0 and Ag?, silver also has two
has been limited to the use of a few relatively simple higher oxidation states that are potentially extremely
preparations (Azócar et al. 2014). Many medical items powerful oxidants: Ag2? (Ag2? ? e- ? Ag?;
such as catheters and prostheses are coated with the E0 = 2.0 V) and Ag3? (Ag3? ? e- ? Ag2?; Eo
elemental form of silver, which prevents colonization unknown) (Weast 1979). Biocidal silver compounds
of their surface by pathogens (Hetrick and Schoenfisch such as SSD are all based on compounds of silver in its
2006). The release of silver cations through slow less reactive, lower oxidation states (Ag0/Ag?). This
dissolution of silver halide is an effective method of choice, in part, was due to an inability to stabilize and
disinfecting water for domestic consumption (Sil- control silver in its higher oxidation states in early
vestry-Rodriguez et al. 2007). Since the late 1960s, synthetic studies. However, many of these issues have
silver sulfadiazine (SSD, or FlamazineÒ) has been been resolved and routes are available for the produc-
common in anti-bacterial preparations for the treat- tion of a wide range of silver(II) compounds and a
ment of burns (Atiyeh et al. 2007). Even this more limited range of silver(III) species (Levason and
compound is a simple preparation comprising an Spicer 1987). This opens the door for development of
antimicrobial cation and anion formulated together as novel, high oxidation state silver compounds for
a weakly bonded complex (Cook and Turner 1975). antimicrobial disinfection.
Although there has been robust interest in developing Increasing the redox potential of the silver agent is
new silver-based anti-infective agents such as silver an effective method of enhancing biocidal activity, as
nanoparticles (Ag/Ag2O), novel silver complexes and it limits the effectiveness of antioxidant defence.
silver impregnated fabrics (Ag0/Ag?) (Fromm 2013; Powerful oxidants such as silver(II) can be expected to
Konop et al. 2016; Le Ouay and Stellacci 2015; irreversibly chemically oxidize a wide range of
Simoncic and Klemencic 2016; Singh et al. 2015), functional (sulfhydryl, vicinal diols) and structural
these are mainly reformulations of silver in its components (unsaturated lipid, proteins, carbohy-
commonest Ag0/Ag? forms. drates) on the surface and inside the microbial cell.
The mechanisms of antimicrobial action of these However, a change in oxidation state not only
silver formulations are still not completely under- increases the redox potential, it also changes the
stood, but the consensus is that they can act on a preferred shape of the metal complex. Silver(I) has a
variety of targets, including interactions with bacterial marked preference for tetrahedral geometry, whereas
cell membranes, binding to and inhibition of thiol- d9 silver(II) predominantly adopts square planar
containing proteins, and release of reactive oxygen geometry. It is known that the geometry a metal
species through processes still to be fully elucidated complex adopts can affect its biological activity; for
(Konop et al. 2016). As Ag? is a moderate oxidizing example, the ability of platinum compounds to interact
agent (Ag? ? e- ? Ag; E0 = 0.80 V), only the most with DNA (Rosenberg et al. 1969) and the antimicro-
sensitive redox sites are affected by it. Although it has bial and anticancer activities of various metal com-
sometimes been suggested that the multimodal mech- plexes (Malik et al. 2018). Metal complexes can be
anism of Ag0/Ag? formulations make resistance less transported across membranes by passive and active
likely to develop, resistance to heavy metals in general mechanisms (Martinho et al. 2018), and it has been
is well known (Pal et al. 2014), and reports of reported that specific coordination structures may
resistance to silver compounds are on the increase occur during active transport; for example, in N-MBD
(Hanczvikkel et al. 2018; Panacek et al. 2018; Percival Cu?-ATPases Cu? adopts a trigonal planar form
et al. 2005). In view of the fact that antimicrobial (Arguello et al. 2012). Thus, biocides based on
resistance is a major problem worldwide and aggres- silver(II) could allow an enhanced oxidative attack
sive microbial species such as E. coli 0157, MRSA, and, depending on their geometry, might exert diverse
and C. difficile are on the increase (Brandt et al. 2014; effects on biological systems. There is a wide range of

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Biometals (2019) 32:627–640 629

simple ligands which can be used to stabilize solvent. The resulting solution was subjected imme-
silver(II), but the pyridinecarboxylates are an excel- diately to spectrophotometric analysis and the con-
lent initial choice, as they have been synthesized centration of Ag2,6P in solution calculated
previously and mostly form planar complexes (Drew retrospectively using molar extinction coefficient
et al. 1970; Drew et al. 1971; Fowles et al. 1968), obtained from the reference sample.
although the silver(II) complex with 2,6-dicar- Thus although all the experiments were carried out
boxypyridine has been reported to be octahedral in duplicate or triplicate, the difficulty of producing
(Drew et al. 1969). However, their ability to react completely dry complex meant that it was impossible
with biological molecules and cause oxidative damage to generate solutions containing exactly the same
has not been studied previously. Consequently, sil- concentration of reagents. Consequently, the data
ver(II) complexes were prepared using pyridinecar- shown are derived from representative experiments.
boxylates as ligands, and the stability of these
complexes was investigated. The aim of the study The stability of bis-(2,6-
was to determine the effectiveness of the silver(II) dicarboxypyridyl)silver(II) in solution
complexes for oxidizing biological antioxidants, lipids
and proteins. A solution of bis-(2,6-dicarboxypyridyl)silver(II) was
prepared either in phosphate buffer (0.1 M KH2PO4,
pH 7.0) at 4.47 mM or in DMSO at 18.4 mM. Aliquots
Experimental of solutions were transferred immediately to a cuvette
and the absorbance (400–1000 nm) was monitored
All reagents were obtained commercially. UV–Vis over a 2 h period.
spectra were recorded on an Agilent Technologies
Cary 60 UV–Vis spectrophotometer. NMR analysis Reaction of bis-(2,6-dicarboxypyridyl)silver(II)
was carried out on a Bruker AMX 400 operating at with glutathione
400 MHz for 1H. Solid reflectance spectra
(400–900 nm) were recorded on a Photonics CCD An 8.5 mM solution of Ag2,6P in phosphate buffer
array UV–Vis spectrophotometer. Silver(II) com- was prepared and 2.25 mL was transferred to a cuvette
plexes of 2-carboxypyridine, 2,3-dicarboxypyridine, and the visible spectrum (400–900 nm) recorded.
2,4-dicarboxypyridine, 2,5-dicarboxypyridine and Aliquots (20 lL) of reduced glutathione (GSH) solu-
2,6-dicarboxypyridine (Ag2,6P) as were prepared tion (84.3 mM) in phosphate buffer were added to the
using literature methods (Drew et al. 1970; Drew cuvette and the spectrum re-recorded after each
et al. 1971; Fowles et al. 1968). addition until the band (kmax 570 nm) attributed to
silver(II) disappeared (* 120 lL).
The protocol for handling Ag2,6P in solution To investigate the products of the reaction using
NMR, three solutions of reduced glutathione (6.1 mg
A reference sample of Ag2,6P was prepared using in 1 mL of D2O, 20 mmol) were treated with 8.2 mg
published methods (Fowles et al. 1968). The sample (18.5 mmol), 16.6 mg (36 mmol) or 24.4 mg
was subjected to elemental analysis (found: C 32.77, H (53 mmol) of Ag2,6P respectively. The solutions
2.68, N 5.85%: expected for Ag2,6P
4H2O: C 32.97, H were allowed to react overnight and then filtered into
2.77, N 5.49%), which confirmed the hydration state. a 5 mm NMR tube. 1H NMR spectra were obtained
This reference sample was used to calculate the molar using a Bruker AVANCE 3 spectrometer operating at
extinction coefficient of Ag2,6P in water (e570, 252/M/ 400.12 MHz. Samples were maintained at 300 K
cm; e890, 207/M/cm) and DMSO (e600, 88.6/M/cm). during spectral acquisition. The NMR spectra were
Ag2,6P was found to decompose slowly with the collected using a standard pulse sequence. The free
natural green/black colour giving way to a white induction decay was generated by a 3.13 ls pulse
product. Hence small batches of Ag2,6P were pre- width corresponding to a 30o pulse. Each data set (4 k
pared immediately prior to use to avoid problems scans; no water suppression) was collected in 32 k of
associated with degradation, and given amounts of memory. A 1 Hz line broadening function was applied
Ag2,6P were quickly dissolved in a given amount of

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630 Biometals (2019) 32:627–640

before Fourier transformation to reduce the effect of Reaction of bis-(2,6-dicarboxypyridyl)silver(II)

the baseline noise. with b-cyclodextrin

Reaction of bis-(2,6-dicarboxypyridyl)silver(II) The reaction with b-cyclodextrin (25 mmol/L) with

with ascorbic acid Ag2,6P was tested essentially as for ascorbic acid
described above, except that the reaction was carried
The reaction of bis-(2,6-dicarboxypyridyl)silver(II) out in a sample bottle and 200 lL aliquots of b-
(7.6 mM) with ascorbic acid (144 mM) was investi- cyclodextrin were added sequentially until the band at
gated essentially as described for glutathione. Aliquots 570 nm had been extinguished (after addition of
(20 lL) of the ascorbate solution were added to a 2.0 mL).
cuvette containing 2.25 mL of Ag2,6P and the spec- For analysis by mass spectrometry, aqueous solu-
trum re-recorded until the band (kmax 570 nm) disap- tions (2 mL) of b-cyclodextrin containing 100 mg
peared, which corresponded to the addition of 80 lL. (88 lmoles) were treated with Ag2,6P (200 mg,
0.44 mmol). The solutions were allowed to react
Reaction of bis-(2,6-dicarboxypyridyl)silver(II) overnight and then filtered before analysis as
with vitamin E (a-tocopherol) described below.

The visible spectrum (400–900 nm) of 2.25 mL of Electrospray mass spectrometric analysis of small
9.1 mM bis-(2,6-dicarboxypyridyl)silver(II) in molecules
DMSO was recorded before and after addition of
10 lL aliquots of 54.3 mM a-tocopherol in DMSO, Electrospray mass spectra were recorded using an
with loss of absorbance at 620 nm after addition of Agilent 6130 (dual source). Samples of glutathione,
70 lL. Due to the competition between the concurrent vitamin E, linoleic acid, and arachidonic acid prepared
reactions of Ag2,6P with a-tocopherol and Ag2,6P as described above were diluted in methanol, while b-
with DMSO, a definite end point cannot be given for cyclodextrin was diluted in 50:50 acetonitrile con-
the reaction of Ag2,6P with a-tocopherol (vide infra). taining 0.1% formic acid:water and introduced into the
instrument with an infusion rate of 0.2 mL/min using
Reaction of bis-(2,6-dicarboxypyridyl)silver(II) methanol. Spectra were acquired with the following
with linoleic and arachidonic acid parameters: ionization mode, MM-ES ? APCI, -ve
ionization; source temperature, 300 °C; Voltage,
A 2.25 mL aliquot of 19.5 mM bis-(2,6-dicar- 4000 V; Curtain gas flow rate, 12 L/min; m/z range
boxypyridyl)silver(II) in DMSO in a cuvette was 50–2000. Spectra were typically acquired for 30 s and
reacted sequentially with 10 lL aliquots of 91.1 mM averaged. The MS data were analysed using Agilent
sodium linoleate or 98.7 mM sodium arachidonate, Chemstation.
both prepared in DMSO. The reaction was monitored
by recording the visible spectrum until the band (kmax Reaction of bis-(2,6-dicarboxypyridyl)silver(II)
620 nm) had disappeared. No definite end point could with cytochrome-c
be given due to the competing reaction with DMSO
occurring. Aqueous solutions (2 mL) of cytochrome c (16.5 mg,
For analysis by mass spectrometry, 2 mL of 54 lmoles) were incubated with 20 mg (44 lmoles),
aqueous suspensions of the sodium salts of the fatty 40 mg (88 lmoles) or 60 mg (131 lmoles) of Ag2,6P.
acids (sodium linoleate; 16.5 mg, 54 lmoles or The solutions were allowed to react overnight and then
arachidonic acid; 16.5 mg, 50 lmoles) were reacted filtered before analysis as described below.
with 20 mg (45 lmoles), 40 mg (91 lmoles) or 60 mg
(136 lmoles) of Ag2,6P. The solutions were allowed Analysis of protein oxidation by oxyblotting
to react overnight and then filtered before analysis as for DNP-carbonyl adducts
described below.
Aliquots of the samples (10 lL; * 75 lg protein)
were resolved by SDS-PAGE with a 12% resolving gel

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Biometals (2019) 32:627–640 631

(Sambrook and Russell 2006) and then either stained cell membrane transport, in the case of Ag(II) their
with InstantBlue stain (Sigma-Aldrich, UK) or trans- potential may be limited by their lack of solubility.
ferred onto PVDF membrane for oxyblotting as In view of the high redox potential of silver(II)
described previously (Shacter 2000). After washing, (equation 2; E0 = 2.0 V NHE (Weast 1979)), the
the membrane was acidified with 2 N HCl and labelled silver (II) compounds were expected to be quite
with 10 mM dinitrophenylhydrazine (DNPH) (Sigma- reactive even to the extent of potentially oxidizing
Aldrich, UK) for 5 min. After further washing and water. Therefore, the first step was to investigate the
blocking the membrane was incubated in blocking lifetime of Ag2,6P in aqueous buffer and DMSO, by
buffer containing monoclonal primary antibodies monitoring the visible absorbance spectrum (Suppl.
rabbit anti-dinitrophenylhydrazone (anti-DNP) Fig. 1a). In aqueous solution a small but manageable
(D9656, Sigma-Aldrich, UK) at a working dilution degradation (* 10%) of Ag2,6P was observed over a
of 1:1000 overnight at 4 °C. The secondary antibody 2 h period. The profile of the degradation process was
was HRP-linked goat anti-mouse (6154, Sigma- linear within the lifetime of the experiment, suggest-
Aldrich, UK) antibody (working dilution 1:1000) for ing that decomposition does not occur via an SN2
2 h at room temperature. The membrane was washed displacement of the axial carboxylates by water or
again as described above and HRP-linked anti-mouse phosphate. In contrast, the stability of Ag2,6P in
was detected using enhanced chemiluminescence DMSO was poor, having a half life of only 25 min
(ECL kit 34078, Thermo Fisher Scientific, Hemel (Suppl. Fig. 1b). DMSO can be oxidized to dimethyl-
Hempstead, UK) according to the manufacturer’s sulphone (Me2SO2; E0 = 1.54 V vs. NHE) and the
instructions. The membrane was scanned using a silver(II) complex studied here is therefore theoreti-
G:BOX system (Syngene, Cambridge, UK) running cally capable of driving this reaction (Krtil et al.
the GeneSys software (Syngene, Cambridge, UK). 1996). Solid reflectance spectrophotometry indicated
that the solid and DMSO solution phase structures of
Ag2,6P are similar (kmax 600 nm), suggesting that
Results and discussion decomposition occurs via electron transfer rather than
ligand exchange. The reaction of the silver complex
Solubility and stability of silver(II) complexes with DMSO limits the interpretation of reactions with
other compounds carried out in this solvent, but in
Silver(II) complexes of 2-carboxypyridine (Ag2P), some cases there was no feasible alternative. To
2,3-dicarboxypyridine (Ag2,3P), 2,4-dicar- obviate problems with the slow decomposition of
boxypyridine (Ag2,4P), 2,5-dicarboxypyridine Ag2,6P in solution, fresh solutions were prepared
(Ag2,5P) and 2,6-dicarboxypyridine (Ag2,6P) were immediately before the start of each experiment.
prepared using literature methods (Drew et al. 1970;
Drew et al. 1971; Fowles et al. 1968). Ag2P, Ag2,3P, Reaction with antioxidants
Ag2,4P and Ag2,5P had limited solubility in both
water and DMSO, and were not studied further owing Biological systems utilize a number of species as co-
to the limited relevance to biological environments. In factors and reducing agents (e.g. glutathione, ascorbic
contrast, Ag2,6P was observed to be reasonably acid, a-tocopherol), and depletion or oxidation of
soluble in both water (* 10 mM) and DMSO antioxidants and structural biological molecules is an
(* 20 mM). The solubility profile of the compounds early stage in the stress leading to the toxic effects of
most likely arises from their solid state structures: oxidizing compounds (Halliwell and Gutteridge
Ag2P and Ag2,3P are planar species and prone to p- 1998). Consequently, the reactions of Ag2,6P with
stacking in the solid state (Fowles et al. 1968), which is these three antioxidants was investigated. The reaction
known to affect solubility detrimentally. In contrast, with an antioxidant can readily be inferred by
Ag2,6P adopts an octahedral geometry in the solid spectrophotometric titrations in which the stepwise
state and is unable to p-stack, which lowers the lattice reduction of the coloured Ag2,6P to its colourless
energy and promotes its solubility in polar solvents silver(I) product is observed. The reactions of glu-
(Drew et al. 1970). The structures are shown in Fig. 1. tathione and ascorbate were carried out in aqueous
Thus, despite the desirability of planar compounds for solution, whereas the reaction of a-tocopherol was

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632 Biometals (2019) 32:627–640

Fig. 1 Structures of the silver (II) complexes synthesized. 2,3-dicarboxypyridine (Ag2,3P), 2,4-dicarboxypyridine (Ag2,4P), 2,5-
dicarboxypyridine (Ag2,5P) and 2,6-dicarboxypyridine (Ag2,6P)

carried out in DMSO. Using the diminution of the Fig. 2), which clearly showed that treatment of GSH
band at 570 nm it was possible to titrate Ag2,6P with (m/z 306.1) with Ag2,6P resulted in conversion to
glutathione (Fig. 2), and the molar ratio at the end GSSG (m/z 611.1).
point was calculated to be slightly greater than 2:1 The reaction of Ag2,6P with ascorbic acid was
Ag2,6P:GSH. This would be generally consistent with monitored spectrophotometrically in the same way as
the 2-electron oxidation of GSH to GSSG, assuming that of glutathione, and it could clearly be seen that
that the Ag2,6P undergoes a 1-electron reduction to addition of ascorbic acid resulted in loss of absorbance
Ag?, as the appearance of metallic silver was not at 570 nm (Suppl. Fig. 3). The molar ratio at the end
observed. point was calculated to be approx. 2:1 Ag2,6P:ascor-
To investigate the nature of the oxidation in more bate, consistent with the 2-electron oxidation of
depth, the reaction of GSH was monitored using 1H- ascorbate to dehydroascorbate. However, this reaction
NMR. Figure 3 clearly demonstrates that Ag2,6P was not investigated further due to the lack of stability
oxidized GSH to the disulfide form (GSSG), with of dehydroascorbate in the presence of redox metals;
increasing Ag2,6P amounts correlating with increased metal-mediated ascorbic acid oxidation and redox
loss of the GSH triplet signals at * 2.95 ppm and cycling is a facile process involving low as well as
appearance of the GSSG pairs of doublets at * 3.05 high valent metals (Halliwell and Gutteridge 1998;
and 3.3 ppm. This finding was supported by negative Skov and Vonderschmitt 1975).
ion electrospray mass spectrometry analysis (Suppl

Fig. 2 Spectrophotometric
analysis of the reaction
between Ag2,6P and
glutathione. The titration of
8.5 mM Ag2,6P
(18.7 lmoles in 2.2 mL) in
0.1 M KH2PO4, pH 7.0 with
glutathione (GSH). The
glutathione (84.3 mM) was
added in 20 uL aliquots (a
total of 6) and the
corresponding lmoles of
GSH are indicated on the
right-hand side of the traces

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Biometals (2019) 32:627–640 633

Fig. 3 1H-NMR analysis of

the reaction between Ag2,6P
and glutathione. 27 lmols
of glutathione (GSH) was
reacted with increasing
amounts of Ag2,6P as
indicated on the spectra. The
spectrum of commercial
glutathione disulfide is
shown at the bottom for
comparison

The reaction of a-tocopherol with Ag2,6P in ESI-mass spectrometry was used to monitor the
DMSO was also discernable using spectrophotometry reaction of Ag2,6P with the unsaturated fatty acid
(Suppl. Fig. 4). However, due to the competing linoleic acid (Fig. 4). A small amount of adventitious
interaction of Ag2,6P with DMSO discussed above, oxidation of the control sample is apparent in Fig. 4a,
it was not possible to obtain an accurate end point for but the major signal is the native fatty acid at m/z 279
the titration. ([M–H]-). There was also a strong signal at m/z 325,
which was identified as the formate adduct of linoleic
ESI–MS analysis of the reaction with fatty acids acid ([M ? CHO2]-). At the lower treatment concen-
tration (Fig. 4b) the signal of the native lipid was
The unsaturated fatty acids linoleic acid and arachi- greatly reduced and the major signal was at m/z 311,
donic acid were used as models to investigate the corresponding to the addition of O2 (? 32 Da). There
ability of Ag2,6P to oxidize lipid. Preliminary studies was also evidence of addition of a single oxygen atom
by spectrophotometric analysis indicated that reac- at m/z 295, and another signal at m/z 293, which was
tions occurred, but the reactions were so fast that most probably due to loss of water from the species at
kinetic analysis was not possible; moreover the m/z 311, suggesting that it may be a bis-hydroxide
experiments were carried out in DMSO as the fatty rather than a hydroperoxide (Spickett and Pitt 2015).
acid salts were sparingly soluble in aqueous solution, Interestingly, a peak was observed at m/z 557, which
and therefore were limited by the issues with this was consistent with the formation of a cross-linked
solvent mentioned above. The focus of these studies dimer of linoleic acid (loss of 3H but singly charged)
was therefore the analysis of oxidation products of the and there were also dimers containing 2, 3 and 4
biomolecules. additional oxygens. Crosslinking of oxidized fatty
acyl chains under highly oxidizing conditions has been

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634 Biometals (2019) 32:627–640

Fig. 4 The reaction of

linoleic acid with Ag2,6P
studied by ESI-mass
spectrometry in negative ion
mode. a Untreated linoleic
acid. b 29 mM Linoleic acid
treated with 37 mM Ag2,6P
and c 29 mM linoleic acid
treated with 110 mM
Ag2,6P

reported previously (Muizebelt and Nielen 1996; of the lipid to small, non-ionized breakdown products,
Schroter et al. 2016; Tolvanen et al. 2008). At the but otherwise the oxidation pattern was comparable.
higher treatment concentration the native lipid was Treatment of arachidonic acid with Ag2,6P also
further depleted, probably resulting from degradation clearly showed the occurrence of oxidation (Fig. 5).
As with linoleic acid, there was some adventitious

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Biometals (2019) 32:627–640 635

Fig. 5 The reaction of

arachidonic acid with
Ag2,6P studied by negative
ion ESI-mass spectrometry.
a ESI-mass spectra of
untreated arachidonic acid.
b arachidonic acid (27 mM)
treated with Ag26P
(22.5 mM) and
c arachidonic acid (27 mM)
treated with Ag26P
(68 mM). All samples were
diluted equivalently in
methanol prior to infusion
into the instrument. The
signal at m/z 349 is probably
a formate adduct, while the
one at 333 appears to be a
contaminant

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636 Biometals (2019) 32:627–640

oxidation in the untreated sample, reflecting the previously (Fraschini and Vignon 2000). Spectropho-
susceptibility of this polyunsaturated fatty acid to tometric titrations in aqueous solution (400–900 nm)
autoxidation (Fig. 5a), but even at the low treatment clearly showed evidence of a reaction of Ag2,6P with
concentration (Fig. 5b) the incorporation of oxygen b-cyclodextrin (Fig. 6). An end point was obtained at
was increased, with signals at m/z 319 (1 O), m/z 335 approximately 3:1 Ag2,6P: b-CD, which suggests that
(2 O), m/z 351 (3 O) and m/z 367 (4 O). A strong signal multiple oxidations might be occurring. Analysis by
at 349.2 was also observed, corresponding to mass spectrometry also suggested that oxidation had
? 46 Da; in view of its appearance only in treated occurred (Fig. 7). Figure 7a shows untreated b-cy-
samples, this is likely to be a dehydration product clodextrin, which was the major species in the sample
following the addition of 4 oxygens such as an at m/z 1133.1 and therefore 100% relative intensity. As
epoxyisoprostane, which are known as relatively the samples were prepared in solvent containing
stable products of arachidonic acid (Spickett and Pitt formate, there was also a significant formate adduct
2015). At the higher treatment concentration the at 1179.0. Treatment of the b-cyclodextrin with a
native signal at m/z/303 was almost abolished 5-fold excess of Ag2,6-P led to free 2,6-picolinate
(Fig. 5c), and the fatty acid was more highly oxidized presenting the strongest signal (100%) at m/z 166.1
with the strongest signal at m/z 335.1 and all other (data not shown), while the b-cyclodextrin signal was
products at higher masses and levels of oxidation. In substantially depleted and a signal at m/z 1231.1
contrast, there was no evidence of dimers of arachi- appeared, corresponding to the oxidation of all of the
donic acid analogous to those observed with linoleic hydroxyl groups into carboxylic acid to form a hepta-
acid, which should have occurred at m/z 605 (data not carboxylate b-cyclodextrin (plus 7 9 14 Da). A 2,6-
shown), although the ions that appeared between m/z dicarboxypyridine adduct of cyclodextrin at m/z
515–553 were not identified. Comparing the reactions 1300.3 was also observed, probably reflecting the high
of linoleic and arachidonic acids with Ag2,6P, it was level of the free ligand present in the sample after
clear that higher concentrations of the silver were reduction of Ag2,6-P by the carbohydrate. In contrast
required to deplete the more unsaturated fatty acid, to the treatment of b-cyclodextrin with HOCl reported
reflecting its greater capacity for oxidative previously [20], there was no clear evidence of
modification. intermediate oxidation products, such as tris and
hexakis carboxylate species, presumably owing to the
Reaction with the carbohydrate beta-cyclodextrin highly oxidizing nature of the Ag2? complex.

To investigate the effects of Ag2,6P on carbohydrates, Reaction with cytochrome c

b-cyclodextrin was used as a model, as it can readily be
observed by mass spectrometry ([M-H]- at m/z 1133), Many adverse effects of oxidizing antimicrobial
and moreover its oxidation by HOCl has been studied agents are mediated by protein oxidation and

Fig. 6 Spectrophotometric 12.0

analysis of the reaction of b-
cyclodextrin with Ag2,6P. 10.0
7.6 mM Ag2,6P (17.2 lmol
[Ag26P] mmol/L

in 2.2 mL) in 0.1 M 8.0

KH2PO4, pH 7.0 was
titrated against b- 6.0
cyclodextrin (b-CD). The b-
cyclodextrin (25.6 mM) was 4.0
added in 200 lL aliquots
(9 10). The mole ratio at the 2.0
end point was calculated to
be 1:3 Ag2,6P: b-CD 0.0
mol:mol
0.0 2.0 4.0 6.0 8.0 10.0 12.0
[cyclodextrin] mmol/L

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Biometals (2019) 32:627–640 637

Fig. 7 Electrospray mass

spectrometry analysis of the
reaction of Ag2,6P with b-
cyclodextrin a the negative
ion ESI-mass spectra of b-
cyclodextrin, also showing a
formate adduct at ? 46 Da;
b b-cyclodextrin (100 mg,
88 lmoles) treated with a
5-fold excess of Ag2,6P
(200 mg, 0.44 mmol); and
c structure of b-cyclodextrin
together with its formula.
Note that the y-axis scale is
relative signal intensity,
where in a the native b-
cyclodextrin is the major
species and therefore 100%,
whereas in b 2,6-picolinate
at m/z 166.1 was the major
species at 100%

disruption of cell signaling, so the effect of Ag2,6P on coomassie blue; then labeling of carbonyl groups with
cytochrome C as a model ubiquitous protein was DNPH followed by western blotting with antibody to
studied. Preliminary studies by spectrophotometry the DNP-adduct (commonly known as oxy-blotting)
showed that Ag2,6P was consumed by relatively small was carried out. Figure 8 shows that even low levels of
amounts of protein (data not shown), which is Ag2,6P resulted in loss of the cytochrome c band at
consistent with the presence of multiple oxidation 12.3 kDa and appearance of high molecular weight
sites on the polypeptides. To confirm the oxidative aggregates that were retained at the top of the
action of Ag2,6P on the protein, the formation of resolving gel, while higher concentrations led to more
protein carbonyls was investigated, as these are well- aggregates and additionally some degradation prod-
established oxidation products (Domingues et al. ucts observed at the bottom of the gel (Fig. 8a).
2013; Shacter 2000). Carbonyl formation may occur Oxyblotting for oxidative damage to the protein
by oxidative deamination of lysines, or radical attack showed the presence of increased carbonyls with
and fragmentation of various other residues (Davies increasing severity of the Ag2,6P treatment, initially
2016). The samples were first separated on denaturing in the cytochrome c band but this was lost at higher
polyacrylamide gels and visualized by staining with Ag2,6P concentrations and staining of the high

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638 Biometals (2019) 32:627–640

Fig. 8 The effect of Ag2,6P treatment on Cytochrome c. blue-stained 12% SDS-PAGE reducing gel. b Oxyblot of a
Protein (8.25 mg/mL) was treated with 0, 63, 126, 189 or comparable gel using an anti-DNPH primary antibody to
252 lM Ag2,6P overnight. Molecular weights markers (MWM) determine the formation of carbonyl groups on cytochrome c
are shown on the lefthandside with numbers in kDa; the as a marker of oxidative damage
molecular weight of horse heart Cytc is 12,384 Da. a Coomassie

molecular weight aggregates predominated (Fig. 8b). intermediate action via oxidation of the solvents H2O
This clearly confirmed that extensive protein oxida- or DMSO, leading to production of hydroxyl radicals
tion occurred following Ag2,6P treatment of the (HO
) or other radical species.
cytochrome c. The reactivity of Ag2,6P with a range of different
biomolecules suggests that it is likely to have signif-
icant toxicity to bacterial cells and therefore good
Concluding remarks biocidal potential. Its solubility in the organic solvent
DMSO implies that it may be able to penetrate lipid
Although planar metal complexes have been reported membranes and thus gain access to the intracellular
to be useful in terms of their ability to penetrate cell environment, as lipophilicity is known to be a factor in
membranes, it is clear that for Ag(II) dicar- biological transport of metal complexes (Oldfield et al.
boxypyridine complexes, the planar ones have lower 2007). The reaction with beta-cyclodextrin is impor-
aqueous solubility compared to one with octahedral tant and similar reactions could contribute to microbial
geometry, namely Ag(II) 2,6-dicarboxypyridine toxicity by altering carbohydrates in the cell wall or on
(Ag2,6P). This compound has satisfactory stability the surface of microbial cells.
in aqueous solution and was found to oxidize a variety However, Ag(II) complexes remain challenging to
of biomolecules. It was shown to react readily with the work with, owing to their limited stability in relevant
thiol-containing antioxidant glutathione, as well as solvents or even in pure form at room temperature.
with ascorbic acid (vitamin C) and a-tocopherol Although the study has shown that it should be
(vitamin E). It caused extensive oxidation of polyun- possible to design improved biocides based on silver
saturated fatty acids as well as cross-linking of the in a ?2 formal oxidation state, further effort is
chains, and induced carbonyl formation, a common required to design new ligands that support and target
marker of oxidative damage, on the small model this moiety better.
protein cytochrome c. Together, these effects suggest
a one-electron or free radical mechanism of action, Acknowledgements SM and CMS acknowledge BSSRC and
Mologic for the Industrial CASE Award BB/J012939/1. CMS
although it is not clear whether it involves direct
and JR acknowledge support from the UK Engineering and
reaction of the metal ion with the biomolecules, or

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Biometals (2019) 32:627–640 639

Physical Sciences Research Council (EPSRC) funded ‘‘Aston molecular structure of bis(pyridine-2,3-dicarboxylato)sil-
Multidisciplinary Research for Antimicrobial Resistance: The ver(II) dihydrate. J Chem Soc A. https://doi.org/10.1039/
AMR4AMR project’’ Grant No. EP/M02735X/1. j19710002959
Fowles GWA, Matthews RW, Walton RA (1968) Studies on co-
Open Access This article is distributed under the terms of the ordination compounds of silver(II). Part I. Magnetic and
Creative Commons Attribution 4.0 International License (http:// spectral properties of complexes with pyridine carboxylic
creativecommons.org/licenses/by/4.0/), which permits unre- acids. J Chem Soc A 25:124. https://doi.org/10.1039/
stricted use, distribution, and reproduction in any medium, j19680001108
provided you give appropriate credit to the original Fraschini C, Vignon MR (2000) Selective oxidation of primary
author(s) and the source, provide a link to the Creative Com- alcohol groups of beta-cyclodextrin mediated by 2,2,6,6-
mons license, and indicate if changes were made. tetramethylpiperidine-1-oxyl radical (TEMPO). Carbo-
hydr Res 328:585–589. https://doi.org/10.1016/s0008-
6215(00)00129-4
Data availability The original data associated with this Fromm KM (2013) Silver coordination compounds with
project are available at https://doi.org/10.17036/researchdata. antimicrobial properties. Appl Organomet Chem
aston.ac.uk.00000418. 27:683–687. https://doi.org/10.1002/aoc.3047
Halliwell B, Gutteridge JMC (1998) Free radicals in biology and
medicine, 5th edn. Oxford University Press, Oxford
Hanczvikkel A, Fuzi M, Ungvari E, Toth A (2018) Transmis-
References sible silver resistance readily evolves in high-risk clone
isolates of Klebsiella Pneumoniae. Acta Microbiol Imm H
Arguello JM, Raimunda D, Gonzalez-Guerrero M (2012) Metal 65:387–403. https://doi.org/10.1556/030.65.2018.031
transport across biomembranes: emerging models for a Heiman KE, Mody RK, Johnson SD, Griffin PM, Gould LH
distinct chemistry. J Biol Chem 287:13510–13517. https:// (2015) Escherichia coli O157 outbreaks in the United
doi.org/10.1074/jbc.R111.319343 States, 2003-2012. Emerg Infect Dis 21:1293–1301.
Atiyeh BS, Costagliola M, Hayek SN, Dibo SA (2007) Effect of https://doi.org/10.3201/eid2108.141364
silver on burn wound infection control and healing: review Hetrick EM, Schoenfisch MH (2006) Reducing implant-related
of the literature. Burns 33:139–148. https://doi.org/10. infections: active release strategies. Chem Soc Rev
1016/j.burns.2006.06.010 35:780–789. https://doi.org/10.1039/b515219b
Azócar MI, Gómez G, Levı́n P, Paez M, Muñoz H, Dinamarca N Konop M, Damps T, Misicka A, Rudnicka L (2016) Certain
(2014) Review: antibacterial behavior of carboxylate sil- aspects of silver and silver nanoparticles in wound care: a
ver(I) complexes. J Coord Chem 67:3840–3853. https:// minireview. J Nanomater. https://doi.org/10.1155/2016/
doi.org/10.1080/00958972.2014.974582 7614753
Brandt C, Makarewicz O, Fischer T, Stein C, Pfeifer Y, Werner Krtil P, Kavan L, Hoskovcová I, Kratochvilová K (1996)
G, Pletz MW (2014) The bigger picture: the history of Anodic oxidation of dimethyl sulfoxide based electrolyte
antibiotics and antimicrobial resistance displayed by solutions: an in situ FTIR study. J Appl Electrochem
scientometric data. Int J Antimicrob Agents 44:424–430. 26:523–527. https://doi.org/10.1007/bf01021976
https://doi.org/10.1016/j.ijantimicag.2014.08.001 Le Ouay B, Stellacci F (2015) Antibacterial activity of silver
Cook DS, Turner MF (1975) Crystal and molecular structure of nanoparticles: a surface science insight. Nano Today
silver sulphadiazine (N1-pyrimidin-2-ylsulphanilamide). 10:339–354. https://doi.org/10.1016/j.nantod.2015.04.002
J Chem Soc, Perkin Trans 2:1021–1025. https://doi.org/10. Levason W, Spicer MD (1987) The chemistry of copper and silver
1039/P29750001021 in their higher oxidation-states. Coord Chem Rev 76:45–120.
Davies MJ (2016) Protein oxidation and peroxidation. Biochem https://doi.org/10.1016/0010-8545(87)85002-6
J 473:805–825. https://doi.org/10.1042/BJ20151227 Malik MA, Dar OA, Gull P, Wani MY, Hashmi AA (2018)
Domingues RM, Domingues P, Melo T, Perez-Sala D, Reis A, Heterocyclic Schiff base transition metal complexes in
Spickett CM (2013) Lipoxidation adducts with peptides antimicrobial and anticancer chemotherapy. MedChem-
and proteins: deleterious modifications or signaling Comm 9:409–436. https://doi.org/10.1039/c7md00526a
mechanisms? J Proteom 92:110–131. https://doi.org/10. Martinho N, Santos TCB, Florindo HF, Silva LC (2018) Cis-
1016/j.jprot.2013.06.004 platin-membrane interactions and their influence on plat-
Drew MGB, Fowles GWA, Matthews RW, Walton RA (1969) inum complexes activity and toxicity. Front Physiol
Silver(II) Bis(pyridine-2,6-dicarboxylate) monohydrate a 9:1898. https://doi.org/10.3389/fphys.2018.01898
novel six-coordinate structure. J Am Chem Soc Marx DE, Barillo DJ (2014) Silver in medicine: the basic sci-
91:7769–7771. https://doi.org/10.1021/ja50001a056 ence. Burns 40(Suppl 1):S9–S18. https://doi.org/10.1016/j.
Drew MGB, Matthews RW, Walton RA (1970) Studies on burns.2014.09.010
coordination compounds of silver 3. Crystal structure of Mijnendonckx K, Leys N, Mahillon J, Silver S, Van Houdt R
silver(2)-Bis(Pyridine-2,3-dicarboxylate) dihydrate. Inorg (2013) Antimicrobial silver: uses, toxicity and potential for
Nucl Chem Lett 6:277. https://doi.org/10.1016/0020- resistance. Biometals 26:609–621. https://doi.org/10.1007/
1650(70)80231-8 s10534-013-9645-z
Drew MGB, Matthews RW, Walton RA (1971) Studies on co- Muizebelt WJ, Nielen MWF (1996) Oxidative crosslinking of
ordination complexes of silver(II). Part VI. Crystal and unsaturated fatty acids studied with mass spectrometry.
J Mass Spectrom 31:545–554

123
640 Biometals (2019) 32:627–640

Oldfield SP, Hall MD, Platts JA (2007) Calculation of Simoncic B, Klemencic D (2016) Preparation and performance
lipophilicity of a large, diverse dataset of anticancer plat- of silver as an antimicrobial agent for textiles: a review.
inum complexes and the relation to cellular uptake. J Med Text Res J 86:210–223. https://doi.org/10.1177/
Chem 50:5227–5237. https://doi.org/10.1021/jm0708275 0040517515586157
Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson Singh R, Shedbalkar UU, Wadhwani SA, Chopade BA (2015)
DGJ (2014) BacMet: antibacterial biocide and metal Bacteriagenic silver nanoparticles: synthesis, mechanism,
resistance genes database. Nucleic Acids Res 42:D737– and applications. Appl Microbiol. Biot 99:4579–4593.
D743. https://doi.org/10.1093/nar/gkt1252 https://doi.org/10.1007/s00253-015-6622-1
Panacek A et al (2018) Bacterial resistance to silver nanopar- Skov KA, Vonderschmitt DJ (1975) Kinetics of iron and copper
ticles and how to overcome it. Nat Nanotechnol 13:65. catalysis of ascorbate oxidation. Bioinorg Chem
https://doi.org/10.1038/s41565-017-0013-y 4:199–213. https://doi.org/10.1016/S0006-
Percival SL, Bowler PG, Russell D (2005) Bacterial resistance 3061(00)80103-9
to silver in wound care. J Hosp Infect 60:1–7. https://doi. Spickett CM, Pitt AR (2015) Oxidative lipidomics coming of
org/10.1016/j.jhin.2004.11.014 age: advances in analysis of oxidized phospholipids in
Rosenberg B, VanCamp L, Trosko JE, Mansour VH (1969) physiology and pathology. Antioxid Redox Sign
Platinum compounds: a new class of potent antitumour 22:1646–1666. https://doi.org/10.1089/ars.2014.6098
agents. Nature 222:385–386 Tolvanen P, Maki-Arvela P, Eranen K, Warna J, Holmbom B,
Sambrook J, Russell D (2006) SDS-polyacrylamide gel elec- Salmi T, Murzin DY (2008) Thermal polymerisation and
trophoresis of proteins. Cold Spring Harb Protoc 3:18–47 autoxidation of technical grade linoleic acid. J Am Oil
Schroter J et al (2016) Unexpected products of the hypochlorous Chem Soc 85:567–572. https://doi.org/10.1007/s11746-
acid-induced oxidation of oleic acid: a study using high 008-1229-7
performance thin-layer chromatography-electrospray ion- Vindigni SM, Surawicz CM (2015) C. difficile infection:
ization mass spectrometry. J Chromatogr A 1439:89–96. changing epidemiology and management paradigms. Clin
https://doi.org/10.1016/j.chroma.2015.11.059 Transl Gastroen 25:124. https://doi.org/10.1038/ctg.2015.
Shacter E (2000) Quantification and significance of protein 24
oxidation in biological samples. Drug Metab Rev Weast RC (1979) Handbook of chemistry and physics, 60th edn.
32:307–326. https://doi.org/10.1081/Dmr-100102336 CRC Press, Florida
Silvestry-Rodriguez N, Sicairos-Ruelas EE, Gerba CP, Bright
KR (2007) Silver as a disinfectant. Reviews of environ-
Publisher’s Note Springer Nature remains neutral with
mental contamination and toxicology. Springer, New
regard to jurisdictional claims in published maps and
York, NY, pp 23–45. https://doi.org/10.1007/978-0-387-
institutional affiliations.
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