research article enzymatic transformation and bonding of...

Research ArticleEnzymatic Transformation and Bonding of SulfonamideAntibiotics to Model Humic Substances

J Schwarz12 H Knicker3 G E Schaumann2 and S Thiele-Bruhn1

1Soil Science University of Trier Behringstraszlige 21 54286 Trier Germany2Institute for Environmental Sciences Group of Environmental and Soil Chemistry University of Koblenz-Landau Fortstraszlige 776829 Landau Germany3Institute of Natural Resources and Agrobiology of Seville CSIC Avenida Reina Mercedes 10 41012 Sevilla Spain

Correspondence should be addressed to S Thiele-Bruhn thieleuni-trierde

Received 1 December 2014 Accepted 29 January 2015

Academic Editor Chengshuai Liu

Copyright copy 2015 J Schwarz et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sulfonamides are consumed as pharmaceutical antibiotics and reach agricultural soils with excreta used as fertilizer Subsequentlynonextractable residues rapidly form in soil which has been researched in a couple of studies To further elucidate conditionsstrength and mechanisms of the fixation to soil humic substances three selected sulfonamides were investigated using thebiochemical oligomerization of substituted phenols as a model for the humification process Catechol guaiacol and vanillin wereenzymatically reacted using laccase from Trametes versicolor In the presence of the substituted phenols alone the concentrationof sulfonamides decreased This decrease was even more pronounced when additional laccase was present Upon the enzymaticoligomerization of the substituted phenols to a humic-like structure the sulfonamides were sorbed transformed sequesteredand nonextractable bound Sulfonamides were transformed depending on their molecular properties Fractions of differentbonding strength were determined using a sequential extraction procedure Isolated nonextractable products were analyzed bychromatographic spectroscopic and calorimetric methods to identify coupling and bonding mechanisms of the sulfonamidesDifferential scanning calorimetry measurements suggested cross-linking of such incorporated sulfonamides in humic oligomersNuclearmagnetic resonance spectroscopymeasurements showed clear differences between the vanillin-sulfapyridine oligomer andthe parent sulfapyridine indicating bound residue formation through covalent binding

1 Introduction

Sulfonamides are a group of synthetic pharmaceutical antibi-otics As a result of their prevalent consumption in humanand veterinarymedicine and subsequent excretion they reachagricultural soils via contaminated sewage sludge wastewater andmanure used for soil fertilization [1 2] Antibioticswere shown to persist in soil for months so that reapplicationof organic fertilizers leads to a plateau of residual concen-trations [3] Such long-term contamination of soils withbioactive antibiotics substantially increases the risk of theirtranslocation into adjacent environmental compartmentsand of effects on soil microorganisms [4 5] Following theapplication to soil the detectable concentration of sulfon-amides rapidly decreases within a few hours [6ndash8] whichprocess subsequently decelerates but further continues [9ndash11]

Nonextractable residues are formed that even resist harshextraction methods such as pressurized liquid extractionprocedures [12 13] whereas mineralization of the fixedresidues is low with lt2 [10 11] From an environmentalperspective it is highly relevant to knowwhether these nonex-tractable and persistent residues are physically entrappedparentmolecules whichwould exhibit antibiotic activity aftera possible remobilization or if covalent binding along withtransformation leads to the disintegration and inactivation ofthe sulfonamides

However soil as a heterogeneous matrix possessesnumerous disruptive factors that hamper the elucidation oftransformation pathways The analytical determination oforganic nitrogen compounds bound to soil humus is impededin particular due to the formation of various metabolites anda large number of different modes of binding which leads

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 829708 11 pageshttpdxdoiorg1011552015829708

2 Journal of Chemistry

Table 1 Physicochemical properties and structures of the selected sulfonamides

CompoundCAS numberMolecular weightMolar volume

FormulaStructure

Physicochemicalproperties

Sulfanilamide(SAA)63-74-11722 gmolminus11206 cm3

C6H8N2O2S

H2N

O

S

O

NH

H

log119870ow minus062pk1198861

19pk1198862

106H-Acceptor 4H-Donor 4

Sulfapyridine(SPY)144-83-22493 gmolminus11740 cm3

C11H11N3O2S

H2N

O

S

O

NN

H log119870ow 035pk1198861

29pk1198862


Sulfadimethoxine(SDT)122-11-23103 gmolminus12152 cm3

C12H14N4O4S

CH3

CH3

H2N

O

S

O

O

O

N

N

N

H

log119870ow 163pk1198861

24pk1198862


to low concentrations of each single compound [14 15] It isassumed that this is also valid for N-containing sulfonamidesas wellThese restrictions can be overcome usingwell-definedsubstituted phenolic compounds that represent special sitesand functionalities of humic substances These compoundsare major building blocks of humic molecules [16] and werefound to polymerize to humus-like substances [17] Reactingsuch substituted phenols with oxidoreductase enzymes orabiotically through pedogenic oxides hydroxides and clayminerals [18 19] serves as model to elucidate transformationreactions or modes of binding happening in soil uponhumification processes

Using that experimental approach Bialk et al [21 22]and Bialk and Pedersen [23] reported enzymatically cat-alyzed oxidative coupling of selected sulfonamides Theyfound that a catalyst is needed to gain a transformationof sulfonamides in the presence of phenolic substances[21] A significant direct oxidation of the sulfonamides sul-famethoxazole sulfamethazine and sulfapyridine occurredin the presence of acid birnessite while bound residueformation was even stronger by fungal peroxidase catalyzedcovalent cross coupling in the presence of o-phenols and26-dimethoxyphenols respectively [21] Similar results wereobtained when humic acid [23] or when a whole soil samplewas used [24] Also it was found that the amount of quinonesin soil that can take part in the nucleophilic addition is thelimiting factor in the bound residue formation of sulfon-amides [24] Binding mechanisms were identified as Michaeladdition leading to the formation of anilinohydroquinones

and anilinoquinones and possible further covalent linkageswhile the formation of a Schiff base (imine formation) wascontroversially discussed [21ndash23 25 26] Gulkowska et al[26 27] concluded that radical coupling reactions are mostlikely not relevant for the initial covalent bond formation

The findings of these reports are further completed byour study The aim was to (i) elucidate the reaction ofphenolic substances with sulfonamides in the absence of afurther catalyst and its dependence on the concentrationand reaction time and (ii) to determine the strength andtype of enzyme catalyzed immobilization with the help of asequential extraction procedure and analytical identificationof reaction products by using 15N CPMAS NMR and differ-ential scanning calorimetry (DSC) For this purpose threedifferently substituted phenols that is catechol guaiacol andvanillin were used as model substances to determine theimmobilization of three selected sulfonamides that is sul-fanilamide (SAA) sulfapyridine (SPY) and sulfadimethoxine(SDT) in the presence and absence of the enzyme laccasefrom Trametes versicolor taken as biocatalyst

2 Materials and Methods

21 Chemicals and Enzyme Three pharmaceutical sub-stances were selected from the group of sulfonamidesthat are composed of a core structure and different sub-stituents at the sulfonamide-N (Table 1) The SAA (4-aminobenzenesulfonamide) was obtained from Riedel-de

Journal of Chemistry 3

Table 2 Physicochemical properties and structures of the selectedphenolic model substances


FormulaStructure


Hydroxyphenol derivate

Catechol(Cat)120-80-911011 gmolminus1862 cm3

C6H6O2OH

OH

log119870ow 088pk119886

945

H-Acceptor 2H-Donor 2

Methoxyphenol derivatives

Guaiacol(Gua)90-05-112410 gmolminus11118 cm3

C7H8O2OH

OCH3

log119870ow 132pk119886

100


Vanillin(Van)121-33-515220 gmolminus11235 cm3

C8H8O3OH

OCH3

O H

log119870ow 121pk119886

740


Haen (Seelze Germany) SDT (4-amino-N-[26-dimethoxy-4-pyrimidinyl] benzenesulfonamide) and SPY (4-amino-N-(2-pyridinyl) benzenesulfonamide) were obtained fromSigma-Aldrich (Steinheim Germany) All sulfonamides hada purity of ge99

Three phenolic compounds were used for the experi-ments Catechol (12-benzenediol) guaiacol (2-methoxyphe-nol) and vanillin (4-hydroxy-3-methoxybenzaldehyde) werepurchased from Fluka Biochemika (Buchs Switzerland) Allsubstances used were of pa quality (ge97) Structures andphysicochemical properties of the substituted phenols aresummarized in Table 2 These substituted phenols have beenchosen because they comprise different functional groupsand are typical fragments from soil humic and fulvic acids[16 28]

The native soil phenoloxidase laccase from T versi-color (CAS 80498-15-3 Enzyme Commission (EC) Number11032 268Umgminus1 Fluka Steinheim Germany) was usedas biocatalyst Laccase was used in this study sincemany phe-noloxidases naturally occur in soil [29] and cross coupling ofxenobiotics to soil humus in the presence of isolated fungalenzymes has been demonstrated via in situ model systems[19] One enzyme unit (U) corresponded to the amountof the laccase that transforms 1 120583mol 26-dimethoxyphenolin 1 minute at 25∘C and a pH of 50 The oxidationof 26-dimethoxyphenol to the colored product 353101584051015840-tetramethoxy-bicyclohexyl-252101584051015840-tetraene-441015840-dione was

photometrically measured at 468 nm using a Shimadzu UV1650-PC (Duisburg Germany) spectrophotometer

22 Model Experiments

Experiment 1 The influence of an increasing concentrationof the phenolic compound on the free dissolved concen-tration of the sulfonamides in the absence of enzyme wasinvestigated Stock solutions of the three individual sulfon-amides were prepared in citrate-phosphate-buffer (pH 45)To 05mL of each solution between 0 and 1mL of catecholor vanillin solution was added The reaction vials were filledwith buffer solution to a total volume of 2mL reachingfinal concentrations for the substituted phenols (catechol orvanillin) of 0 0007 0013 0020 0027 and 0033M and forthe sulfonamides of 03mM During the experiment timeranging from 1 h to 24 d samples were exposed to roomtemperature (24∘C plusmn 1) without shaking Tubes were wrappedwith parafilm retarding moisture loss while allowing for gasexchange Samples were kept in the dark and protected fromphotodegradation The concentration of the sulfonamideswas analyzed with HPLC

Experiment 2 To investigate the enzymatic transformation asulfonamide (1mM) and a phenolic substance (005M) weremixed in citrate-phosphate-buffer (pH 45) in 1 50 molarratio of sulfonamide and phenolic compound To this laccasefrom T versicolor was added to reach an enzyme concen-tration of 2DMPUmLminus1 in total This was done accordingto Wang et al [30] The samples were incubated for 15 h inthe dark at a temperature of 21∘C (plusmn1∘C) and agitated on ahorizontal shaker Samples with a sulfonamide concentrationof 1mM but without the addition of neither enzyme norphenolic compound served as controls All samples were pre-pared in triplicate and the further experimental conditions(temperature gas exchange and light exclusion) were similaras in Experiment 1 Samples were further analyzed using asequential extraction procedure

Experiment 3 A similar but slightly modified approach wasused to investigate the influence of the sulfonamides on thestructure of the enzymatically formed phenolic oligomerThemolar ratio of sulfonamide to catechol was changed to 1 25and a higher enzyme concentration (19DMPUmLminus1) wasnecessary to facilitate a longer incubation time of 3 d In thisexperiment a solid reaction product resulted after separationfrom the solution and air-dryingThis solid product was usedas received without further clean-up and analyzed by DSC

Experiment 4 For solid state NMR analysis large amountsof enzymatic transformation products of ca 500mg wererequired Preexperiments showed that citrate buffer was notsuitable for NMR (data not shown) Thus 05 g vanillin and250mg of laccase (23Umgminus1) were weighted into 100mLcentrifuge tubes To this 25mL of a 1mM SPY solution in aformate-formic acid-buffer (pH45)were added and vortexedfor 1min Subsequently additional 25mL of the SPY solutionwas added The reaction vessels were locked with joints andvortexed again Sample tubes were covered with parafilm and


samples were kept in the dark over night at 21∘C and wereagitated on a horizontal shaker After 24 h the solution wasseparated from the solid residue The residue was washedwith dilute formic acid and with 05MNaOHmethanol(0210 vv) The washing solutions were decanted and theremaining precipitate was air-dried The elemental composi-tion of selected samples was determined by dry combustionusing a VarioEl (Elementar Analysensysteme GmbH HanauGermany)

23 Sequential Extraction Procedure The binding strength ofthe sulfonamides to the phenolicmodel substances was deter-mined in the reaction solutions of the previously describedmodel experiments For that purpose a modified sequentialextraction procedure was used according to Kim et al [31]and Thiele et al [32] Briefly a four-step extraction schemewas applied to reaction solutions that contained enzymati-cally formed precipitates Step 1 the reaction solution wascentrifuged at 3940 g for 30min and the supernatant wasfiltered through a 045 120583m PTFE-Filter (Macherey-NagelDuren Germany) To prevent losses the filter was washedwith 1mL methanol Step 2 the remaining precipitate wasdissolved in 1mL citrate buffer (pH 17) and agitated on a vor-texer for 20 sec To separate the solution from the residue thesamples were centrifuged as described above This procedurewas repeated once and solutions were combined Step 3 tothe remaining solid 20120583LNaOH (05M) and 1mL methanolwere added the solution was vortexed and centrifuged Thesolution was separated from the solid residue using a pipetteStep 4 the remaining solid was completely dissolved by theaddition of 1mL dimethyl sulfoxide (DMSO) and directlytransferred to a vial forHPLC analyses A 1mL aliquot of eachextraction step was pipetted into HPLC vials

24 Chromatographic Spectroscopic andCalorimetric Analysis

241 High Performance Liquid Chromatography (HPLC)Analysis of Sulfonamides For HPLC analysis an HP 1050gradient elution system (Agilent Boblingen Germany)equipped with an Agilent diode array detector G1315B oper-ated at 260 nm was used A C18 250 times 46mm 100ndash5120583mreversed phase column (Macherey-Nagel Duren Germany)served as stationary phase The temperature of the columnwas kept at 22∘C As mobile phase 001M H

3

PO4

(A) andmethanol (B) were used at a flow rate of 1mL minminus1 Forgradient elution the composition of the solvents was changedas follows 0 to 3min 100 A 3 to 10min linear to 80 A and20 B 10 to 22min linear to 100 B 22 to 28min 100 B28 to 29min linear to 100 A and 29 to 30min 100 A Theinjection volumewas 10 120583L and quantificationwas done usingexternal standards Detection limits were 003 120583mol Lminus1 forSAA and below for SPY and SDT further details on detectionlimits and recovery rates have been published elsewhere [33]

242 Cross Polarization Magic Angle Spinning Solid StateNuclear Magnetic Resonance (15N CPMAS NMR) AnalysisThe 15N NMR spectra were acquired on a Bruker DMX

400 spectrometer (Bruker BiospinGmbH Rheinstetten Ger-many)with a frequency of 405MHz for 15N using zirconiumrotors of 7mmODwith Kel-F-caps that were spun at 45 kHzAfter a contact time of 1msec 15000 to 500000 scans with apulse delay of 1 to 10 sec were acquired A line broadening of100 to 300Hz was applied for the optimization of the signal-to-noise ratio About 500mg of solid oligomer material wasused without any further prepreparations Chemical shiftswere referenced to nitromethane (0 ppm [34])

243 Differential Scanning Calorimetry (DSC) AnalysisMeasurements of the enzymatic reacted catechol were doneusing DSC and according to Schaumann and LeBoeuf [35]Measurements were carried out with a DSC Q1000 andthe Universal Analysis V41B software for data analysis wasused (both TA Instruments Alzenau Germany) Of eachsample 3 to 5mg were weighed into a hermetically sealedaluminum pan An empty hermetic aluminum pan wasused as reference The whole measuring cell was purgedwith nitrogen at a flow of 50mL minminus1 to avoid oxidationHeat flow and temperature calibration were conducted withindium To reduce the influence of water samples were air-dried under light exclusion prior to DSC analysis Duringthe measurement the following temperature cycles were pro-grammed the sample was kept atminus50∘C isothermal for 2minThe temperature was increased with 10Kminminus1 to 110∘C andkept for 30min isothermal The cell was cooled again tominus50∘C and a second heating cycle was started with a rate of10 Kminminus1 to 200∘C The data from the second heating cyclein a temperature range from minus20∘C to 200∘C were evaluatedThe first derivative of the endothermic heat flow was plottedas function of temperature The glass transition-like steptransition [36] is indicated by peaks with themaximum at thestep transition temperature

3 Results and Discussion

31 Nonenzymatic Reaction of Substituted Phenols and Sul-fonamides The influence of the substituted phenols aloneon the sulfonamide concentration (03mM) without theaddition of a catalyzing enzyme was exemplarily investigatedfor catechol as a hydroxyphenol derivative and vanillin as amethoxyphenol derivative Varying the concentration of thesubstituted phenols over a range of 0 to 0033M resulted in achange in concentration for some of the sulfonamidesThis isshown for catechol in Figure 1

The concentration of free SPY decreased with increasingcatechol concentration and timeThe decrease was negligibleafter 1 h but increased during the time course of the exper-iment After 24 d and at catechol concentrations ge0020Mthe SPY concentration was below the limit of detectionAt each catechol concentration the decrease in the SPYconcentration (119888) during time (119905) was linear and followed azero order decay

119888 = 1198880

minus 119896 times 119905 (1)

Results of the model-fit are listed in Table 3 The rate coeffi-cient (119896) increased with increasing concentration of catechol


Table 3 Concentrations of sulfonamides and hydroxyphenol derivate reacted in the absence or presence of laccase and resulting ratecoefficients of the dissipation of sulfapyridine (SPY) sulfadimethoxine (SDT) and sulfanilamide (SAA)

sulfonamide 1198880

sulf (mmol Lminus1) 119888 phenol (mol Lminus1) Laccase (UmLminus1) Kinetic model 119896 (hminus1) 1198772 SE

SPY

0075 0 0

Zero order (1)

0003 0439 00080075 0007 0 0047 0910 00350075 0013 0 0093 0996 00600075 0020 0 0140 0950 00890075 0027 0 0144 0981 01250075 0033 0 0156 0998 0144

SDT 0075 0033 0 0131a

SAA 0075 0033 0 0003a

SPYb 100 0 48First order (2)

0113 0995 0033SDTb 100 0 48 429 10minus3 0882 0125SAAb 100 0 48 299 10minus4 0990 0034SPY 100 0050 2

First order (2)0517 0966 0032

SDT 100 0050 2 0490 0971 0060SAA 100 0050 2 0447 0982 0037aDegradation of SAA and SDT was measured only at one timepoint (5 d) It was assumed that degradation followed zero order kinetics as it was determinedfor SPYbData from [20]

0 0005 0010 0015 0020 0025 0030 0035

10

08

06

04

02

0

SPY (24d)SPY (5d)SDT (5d)

SPY (1h)SAA (5d)

cc 0

of su

lfona

mid

es

Concentration of catechol (mol Lminus1)

SPY (41h)

Figure 1 Changes of the sulfonamide concentration in dependenceon the catechol concentration The triangles indicate the datameasured at different reaction time for sulfapyridine and solidsymbols indicate the different sulfonamides measured after five days(SPY = triangles SAA = squares and SDT = circles) Error bars notshown are smaller than symbols

At a catechol concentration of 0007M the rate coefficientwas 0047 hminus1 and increased to 0156 hminus1 at a concentration of0033M The decrease of SDT within 5 d was similar or evensomewhat stronger compared to SPY and showed a likewisedependence on the concentration of catechol In contrastthe free concentration of SAA after 5 d was still 100 Therecovery rate of all three sulfonamides was also 100 when

incubated without catechol no hydrolysis andmdashas it wasintendedmdashno photodegradation occurred

In contrast to catechol the concentration of sulfonamidesdid not decline in the presence of vanillin during the timecourse of 24 d (not shown) This and the finding that thedecrease of the sulfonamides followed zero order kinetics ledto the interpretation that fractions of the substituted phenolsare abiotically oxidized whereby catechol but not vanillincan form a reactive quinone [37] The quinone can oxidizeother organic molecules by being reduced back to the parentcatecholThe results show that oxidationwas relevant for SPYand SDT but not for SAA indicating that the N-heterocycleof the sulfonamides (see Table 1) was involved in the reaction

32 Enzymatic Transformation of Sulfonamides Decliningsulfonamide concentrations were also determined whencombined with the enzyme laccase alone (Table 3)The enzy-matic transformation of the sulfonamides SPY SDT and SAAinto free dissolved degradation products has been previouslyreported [20] and several metabolites were proposed Theseare in part similar to those reported from photodegradation[38ndash41] and biodegradation in soil [42 43] In the studyof Schwarz et al [20] the metabolism through oxidasecatalyzed free radical coupling reactionswas reached at a highlaccase concentration (48UmLminus1) Yet Gulkowska et al [26]stated that radical reactions are not relevant at lower oxidaseactivities

The enzymatic reaction followed pseudo-first-orderkinetics as described by

119888 = 1198880

times 119890(minus119896times119905)

(2)

The calculated reaction-rate coefficients (119896) were lower com-pared to the coefficients determined for the transformationof the three sulfonamides with the substituted phenols alone


Table 4 Percentage of sulfonamides recovered through sequential extraction after 15 h of enzymatic transformation with laccase fromTrametes versicolor in the absence (control) and presence of substituted phenols (standard deviation in parentheses)

Sum of all fractions= recovery

Reaction solution= unreacted

Buffer pH 17= easily desorbable

NaOHmethanol= total desorbable DMSO = sequestered

SAAControl 98 (1) 98 (1) nd nd ndCatechol 84 (11) nd 26 (10) 43 (13) 15 (13)Guaiacol 58 (8) nd 4 (1) 53 (8) 1 (1)Vanillin 21 (7) nd 8 (1) 10 (7) 3 (1)

SPYControl 99 (01) 99 (01) nd nd ndCatechol 49 (10) nd nd 39 (10) 10 (2)Guaiacol 33 (4) nd 1 (0) 31 (4) 1 (0)Vanillin 27 (4) nd 6 (2) 17 (1) 4 (1)

SDTControl 98 (1) 98 (1) nd nd ndCatechol 94 (7) nd 48 (23) 45 (20) 1 (1)Guaiacol 66 (7) nd 15 (2) 18 (2) 34 (6)Vanillin 92 (3) 11 (2) 23 (9) 24 (4) 35 (11)nd not determined at a detection limit of 4120583molmLminus1

(Section 31 Table 3) The rate coefficients declined in thesame sequence SPY gt SDT gt SAA showing that the influenceof the sulfonamide molecular properties was similar

The decrease in sulfonamide concentration was muchstronger and substantially faster (see rate coefficients 119896in Table 3) when the sulfonamides were incubated in thepresence of both a phenolic compound and laccase Thesubstituted phenols act as mediators and thus enhance theeffect of natural laccase [44] The sum of the extractablefractions after 15 h is listed in Table 4 In the presence ofcatechol and guaiacol the total portion recovered declinedin the order SDT gt SAA gt SPY (with differences betweenrecoveries being significant at 119875 lt 005) In combinationwith vanillin however the sequence was SDT gt SPY ge SAA(with the recoveries of SPY and SAA being not significantlydifferent) Changes in color of the reaction solutions andthe precipitation of solid products clearly indicated thatphenolic oligomerswere formedThiswas further analyticallyconfirmed by pyrolysis field-ionization mass spectrometry[45] In the presence of such a phenolic oligomer polarchemicals can be immobilized with mechanisms rangingfrom reversible surface adsorption to the formation of crosscoupling products of the substituted phenols and the sulfon-amides respectively as it was reported for sulfonamides andother chemicals [21 31 44] Based on the findings of Bialket al [21] using autoclaved phenoloxidases the losses causedby sorption to the protein structure of the enzyme wereconsidered to be negligible while coefficients of sulfonamidesorption to the phenolic oligomers and polymers reach valuessimilar to those obtained in the presence of soil humic acids[45] In the control samples the spiked sulfonamides werealmost completely recovered despite a minor loss of 1 to 2of the spiked sulfonamides due to filtration (Table 4)

A sequential extraction procedure was carried out tofurther investigate the efficiency of the immobilization that

led to the decrease in sulfonamide concentrations Thefour extraction steps were meant to determine (i) the freedissolved sulfonamides (ii) the easily desorbed sulfonamideamounts (iii) the totally desorbable fraction and (iv) the ster-ically sequestered (entrapped) sulfonamides while the frac-tion not recovered with this sequential extraction procedurewas assumed to be covalently bound andor metabolized

The extractability of the sulfonamides varied with thechemical properties of the substituted phenols and amongthe different sulfonamides It was concluded that the immo-bilization of the sulfonamides was influenced by the typeof sulfonamide as well as the type of substituted phenolThe distribution over the four fractions of the sequentialextraction was essentially similar for SPY and SAAThe SAAand SPY were no longer detectable in the reaction solutionBetween 0 and 26 were easily desorbed with buffer solutionof pH 17 It is assumed that the extractability partly resultedfrom the cleavage of less stable bonds within the phenolicoligomer leading to a proteolytic reduction of the quinonemoiety [46] The extractability varied among the specificcombinations of the sulfonamides and phenolic compoundsA substantial influence of acidic hydrolysis as it has beenreported for the sulfonamides [47] was not reflected bythe in part high recoveries of the sulfonamides (Table 4)The percentage of SPY and SAA (31 to 53) extracted fromcatechol and guaiacol was clearly higher when methanolwas used This indicates that substantial parts of thesesulfonamides have been adsorbed to the phenolic oligomerIn a previous study it has been determined that the sorptionof SPY SDT and SAA to the vanillin oligomer yielded similarsorption coefficients (119870

119891

) as the sorption to humic acid [45]The DMSO extractable fraction of SAA and SPY opera-

tionally defined as the sequestered fractionwas small rangingfrom 1 to 15 Also the overall recovery of SAA and SPYby the sequential extraction scheme was well below the


0 minus100 minus200 minus300 minus400 minus500

minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH

minus100 minus200 minus300 minus400 minus500

(ppm)

R1

R2

(b)

Figure 2 15N CPMAS NMR spectra of (a) sulfapyridine and (b) of an enzymatic coupling product of sulfapyridine with a vanillin oligomer

recovery from control samples and declined in the ordercatechol gt guaiacol gt vanillin This sequence corresponds tofindings for N-containing sulfonated dyes [44] The electrondonor effect of methoxy substituents as in vanillin furtherenhances laccase activity due to a decreased redox potential[44] Vanillin showed an especially strong formation ofprecipitate in our study (data not shown) It is suggested thatthe sulfonamide fractions not recovered with the sequen-tial extraction procedure formed nonextractable residuesthat were covalently bound to the phenolic oligomer Thenonextractable fraction amounted to up to 73 and 79 forSPY and SAA in the presence of vanillin Correspondinglythe emergence of nonextractable residues has been reportedfor sulfonamides in soil reaching up to gt80 of the initialsulfonamide content [9ndash11 48] Since the DMSO extractablefraction was small it is concluded that nonextractability wasmainly due to the formation of covalently bound residuesthan to the sequestration (physical entrapment) with stronglyrate-limited back-diffusion in micropores as it was reportedby Muller et al [6] and Schmidt et al [10] The percentageof SPY recovered in the different fractions was mostly lowerthan for SAAThis indicates a stronger binding of SPY to thephenolic oligomers However it cannot be ruled out that theincomplete recovery of the sulfonamides by the extractionscheme was also due to the formation of metabolites Yetat the enzyme activities used in this experiment no relevantmetabolization of the sulfonamides is expected [21 26]Also additional signals from metabolites have not beendetermined in HPLC analysis (spectra not shown) Howeverthe identification of metabolites was not the aim of thisspecific experiment

Extractability was substantially different for SDT com-pared to SAA and SPY (Table 4) The percentage of SDTrecovered ranged between 66 and 94when it was combinedwith the three phenolic substances While the other sulfon-amides were no more detectable in the reaction solution 11of SDT was present in the reaction solution with vanillinOther thanwith SPY and SAA substantial percentages of SDTwere recovered as easily and total desorbable fraction which

was especially the case in the presence of catechol with 93of SDT recovered in the two desorbable fractions (Table 4)In the presence of guaiacol and vanillin large amounts ofSDT (34 to 35) were sequestered in the oligomeric structureof the precipitate This corresponds to findings of Thiele-Bruhn et al [49] who determined frommolecularmechanicscomputational chemistry modelling that SDT substantiallysorbs in voids of soil organicmatterThe different reactivity ofSDT was not related to a different speciation At pH 45 of thereaction solution the speciation of SDT was in between thatof SAA and SPY The speciation of the sulfonamides (+0ndash)was 029980 (SAA) 0896131 (SDT) and 259750(SPY) It is assumed that methoxyl groups attached to theN-heterocycle of SDT (Table 1) give the molecule a stericallyless flexible and thus less reactive shape This may also beindicated by the 12 to 18 times larger molar volume of SDTcompared to SAA and SPY (Table 1) Hence a much smallerfraction of SDT (6 to 34) was nonextractable

33 NMR Characterization of Coupling Products To exem-plarily investigate the binding mechanisms of nonextractablesulfonamides vanillin and SPYwere enzymatically reacted toform an oligomeric substance This combination yielded anespecially large amount of precipitate with an assumably highamount of nonextractable SPY (cf Table 4) The elementalcomposition of the precipitate formed was 602 C 19 Nand 05 S Considering the elemental composition of laccasefrom T versicolor of 1513 N and 0496 S [50] and that ofSPY it was estimated from the NS and CN ratios that theprecipitate comprised about 10 of SPY

The reaction product was further analyzed using 15NCPMAS NMR The 15N NMR spectra of unaltered SPY(Figure 2(a)) and SPY in the vanillin oligomer (Figure 2(b))differed clearly The signal at minus306 ppm (Figure 2(a)) indi-cates an NH

2

-group most tentatively covalently bound to abenzene ring (aniline) This signal is missing in the spec-trum of the reaction product whereas a signal at minus260 ppmassignable to amide-N appeared (Figure 2(b) [51])The signalfrom aniline-Nwas shifted because the chemical surrounding


of the nitrogen changes upon a coupling to vanillin Theobserved shift of the signal from the aniline-N correspondswith the results of Berns et al [52] They reported thatwhen sulfadiazine was transformed to trimethoxybenzoyl-sulfadiazine which is a similar cross coupling product asassigned here the signal of the aniline-N was shifted fromminus309 ppm to minus251 ppm Hence the formation of a reactionproduct via the aromatic amino group is indicated Furtheridentification of the reaction product was not possible but itis assumed that it is most probably a structure as proposed byBerns et al [52] or Bialk et al [22] Bialk et al [22] identified ahydroxyphenol compound as a T versicolor laccase mediatedcross coupling product of SPY with protocatechuic acidIn our study the aniline-N was identified as the couplingmoiety of SPY Coupling to vanillin may have occurred intwo different ways First as determined by Bialk et al [22]aniline-N was bound to the aromatic ring in the parapositionof the methoxy substituent Alternatively a cross couplingto the aldehyde-function might have happened [52] Theformation of a Schiff base as it has been identified by Bialket al [21] and Gulkowska et al [25] for the oxidoreductasecatalyzed cross coupling products of sulfonamides is notindicated by our results A Schiff base with a C=N-doublebond of the imine engenders in the 15N CPMAS NMRspectra a chemical shift around minus60 ppm [51] that was notdetermined here

34 Differential Scanning Calorimetry (DSC) of the CatecholOligomer To characterize effects of the sulfonamides onthe structural properties of the phenolic oligomers DSCwas carried out on example of the catechol oligomer Glasstransitions express as steps in the heat flow thermogramof theDSC-measurements where the inflection indicates the steptransition [53] Glass transitions are common features of syn-thetic polymers and weak reversing glass transitions bettertermed as step transitions were also found for biopolymershumic substances and selected soil samples [35 36] For thecatechol oligomer we expected a step transition due to thehypothesized polymeric humic-like structure In Figure 3the first derivative of the endothermic heat flow is plottedand shows a maximum at the step transition temperaturein the range of 60 to 140∘C (indicated by arrows) Theunderlying processes were interpreted as step transitionsbecause they follow the same characteristics as the reversingtransitions described in Schaumann and LeBoeuf [35] Onthe basis of this interpretation the transition temperatureswere interpreted as indicators of matrix rigidity [54] Smallmolecules such as the sulfonamides or water can act asplasticizer which is indicated by a shift of the step transitiontemperature towards lower values An antiplasticizer effect ofsubstances within the molecule is indicated by a shift of thestep transition towards higher temperatures

The curves in Figure 3 were separated into four tem-perature ranges The first range starts at minus20∘C and endsat 20∘C while the second range goes from 20∘C to 60∘CDistinct peaks with maxima at temperatures from 39∘C to41∘C indicate structural changes uponheating in the catechol-core structure units that were identically found for allinvestigated samples Another transition occurred as a broad

minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat

Figure 3 Endothermic heat flow curves (first derivation) of differ-ential scanning analyses of catechol that was enzymatically reactedwith laccase in the presence and absence of sulfonamide antibiotics

peak in the third temperature range from 60∘C to 140∘CClear differences between the different combinations weredeterminedThemaximum for the catechol oligomerwithoutsulfonamide was located at 105∘C in combination with SPYthis maximumwas slightly shifted to 108∘CThe difference of3∘C is negligible and therefore we concluded that bound SPYhad no effect on the rigidity of the catechol oligomer WhenSAA was present the maximum was clearly shifted towardshigher temperatures (133∘C) This suggests a stabilization ofthe catechol oligomer through SAA We assume that theformation of bound residues occurred via both NH

2

-groupsmaking SAA a bridge within the oligomeric network of thesubstituted phenol It is suggested that a similar transfor-mation of the NH

2

-group occurred as it was determined by15N NMR for SPY and vanillin In combination with SDTthe transition temperature was reduced to 90∘C indicatinga plasticizer effect of SDT on the oligomeric structure Thiseffect is not related to unreacted SDT since no signal wasdetermined at the step transition temperature of pure SDTof 66∘C [55] Hence it is concluded that SDT was at leastreversibly adsorbed to the phenolic oligomer as it wasshown by the sequential extraction procedure (Section 32)The decomposition of structures is indicated by an endother-mic peak in the fourth range above 140∘C In this range thethermograms show a similar curve shape for all investigatedcatechol structures There is no clear distinction between thevarious combinations

4 Conclusions

The results presented show that sulfonamides are immo-bilized upon humification processes that were investigatedusing substituted phenols as model humic monomers andthe enzyme laccase The sulfonamides were especially trans-formed in the presence of enzymatically reacted substituted


phenols but also abiotic transformation was determined inthe presence of substituted phenols alone The mechanismsof immobilization varied from reversible adsorption to theformation of nonextractable bound residues most probablythrough cross coupling of SPY and vanillin via the aniline-N of the sulfonamide The extent of each mechanism variedwith the physicochemical properties of the sulfonamide andsubstituted phenol tested respectively On the one hand itmust be expected that the mechanisms determined in thesemodel experiments are valid for field soils as well sincephenolic and quinoid groups are also contained in largenumber in soil humic substances and oxidoreductases such aslaccase are widespread in the environment Covalently boundresidues were formed and based on nonextractability mightmake up 6 to 79 of the spiked amount of sulfonamidesWith no doubt the bound residues are less bioactive thanthe parent compound On the other hand the extent ofeach mechanism varied between model experiments Con-sequently the binding mechanisms and ecological relevanceof sulfonamide residues remaining in field soil may also varyupon the specific conditions at different sites

Abbreviations

Cat CatecholGua GuaiacolVan VanillinSAA SulfanilamideSDT SulfadimethoxineSPY SulfapyridineDMSO Dimethyl sulfoxideDSC Differential scanning calorimetryHPLC High performance liquid

chromatography15N CPMAS NMR Solid state cross polarization magic

angle spinning nitrogen-15 nuclearmagnetic resonance

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

J Schwarz and S Thiele-Bruhn contributed equally to thiswork H Knicker did the work on 15N CPMAS NMRwhile differential scanning calorimetry was done by G ESchaumann

Acknowledgment

This project was funded by the German Research Foundation(DFGTh 6784-1 and DFGTh 6784-2)

References

[1] A B Boxall L A Fogg P A Blackwell P Kay E J Pembertonand A Croxford ldquoVeterinary medicines in the environmentrdquoReviews of environmental contamination and toxicology vol 180pp 1ndash91 2004

[2] K-R Kim G Owens S-I Kwon K-H So D-B Lee andY S Ok ldquoOccurrence and environmental fate of veterinaryantibiotics in the terrestrial environmentrdquo Water Air and SoilPollution vol 214 no 1ndash4 pp 163ndash174 2011

[3] G Hamscher H T Pawelzick H Hoper andH Nau ldquoDifferentbehavior of tetracyclines and sulfonamides in sandy soilsafter repeated fertilization with liquid manurerdquo EnvironmentalToxicology and Chemistry vol 24 no 4 pp 861ndash868 2005

[4] H Schmitt H Haapakangas and P Van Beelen ldquoEffects ofantibiotics on soil microorganisms time and nutrients influ-ence pollution-induced community tolerancerdquo Soil Biology andBiochemistry vol 37 no 10 pp 1882ndash1892 2005

[5] P Sukul and M Spiteller ldquoSulfonamides in the environment asveterinary drugsrdquoReviews of Environmental Contamination andToxicology vol 187 pp 67ndash101 2006

[6] T Muller I Rosendahl A Focks J Siemens J Klasmeier andM Matthies ldquoShort-term extractability of sulfadiazine afterapplication to soilsrdquo Environmental Pollution vol 172 pp 180ndash185 2013

[7] Q Wang M Guo and S R Yates ldquoDegradation kinetics ofmanure-derived sulfadimethoxine in amended soilrdquo Journal ofAgricultural and Food Chemistry vol 54 no 1 pp 157ndash1632006

[8] S Thiele-Bruhn and D Peters ldquoPhotodegradation of phar-maceutical antibiotics on slurry and soil surfacesrdquo Landbau-forschung Volkenrode vol 57 no 1 pp 13ndash23 2007

[9] T Junge K C Meyer K Ciecielski A Adams A Schaffer andB Schmidt ldquoCharacterization of non-extractable 14C- and13C-sulfadiazine residues in soil including simultaneous amend-ment of pig manurerdquo Journal of Environmental Science andHealth Part B Pesticides Food Contaminants and AgriculturalWastes vol 46 no 2 pp 137ndash149 2011

[10] B Schmidt J Ebert M Lamshoft et al ldquoFate in soil of 14C-sulfadiazine residues contained in the manure of young pigstreated with a veterinary antibioticrdquo Journal of EnvironmentalScience and Health Part B Pesticides Food Contaminants andAgricultural Wastes vol 43 no 1 pp 8ndash20 2008

[11] R Kreuzig and S Holtge ldquoInvestigations on the fate of sulfa-diazine in manured soil laboratory experiments and Test PlotStudiesrdquo Environmental Toxicology and Chemistry vol 24 no4 pp 771ndash776 2005

[12] K Stoob H P Singer S Stettler N Hartmann S RMuller andC H Stamm ldquoExhaustive extraction of sulfonamide antibioticsfromaged agricultural soils using pressurized liquid extractionrdquoJournal of Chromatography A vol 1128 no 1-2 pp 1ndash9 2006

[13] M Forster V Laabs M Lamshoft T Putz and W AmelungldquoAnalysis of aged sulfadiazine residues in soils using microwaveextraction and liquid chromatography tandem mass spectrom-etryrdquo Analytical and Bioanalytical Chemistry vol 391 no 3 pp1029ndash1038 2008

[14] H-R Schulten andM Schnitzer ldquoThe chemistry of soil organicnitrogena reviewrdquo Biology and Fertility of Soils vol 26 no 1 pp1ndash15 1998


[15] N Senesi B Xing and P M Huang Biophysico-ChemicalProcesses Involving Natural Nonliving Organic Matter in Envi-ronmental Systems John Wiley amp Sons Hoboken NJ USA2009

[16] J M Bracewell K Haider S R Larter and H R SchultenldquoThermal degradation relevant to structural studies of humicsubstancesrdquo in Humic Substances II In Search of Structure MH B Hayes P MacCarthy R L Malcolm and R S Swift Edspp 181ndash222 John Wiley amp Sons New York NY USA 1989

[17] J M Suflita and J-M Bollag ldquoPolymerization of phenolic-compounds by a soil-enzyme complexrdquo Soil Science Society ofAmerica Journal vol 45 pp 297ndash302 1981

[18] S B Haderlein and R P Schwarzenbach ldquoEnvironmentalprocesses influencing the rate of abiotic reduction of nitroaro-matic compounds in the subsurfacerdquo in Biodegradation ofNitroaromatic Compounds J C Spain Ed pp 199ndash225 PlenumPress New York NY USA 1995

[19] J M Bollag ldquoDecontaminating soil with enzymes an in situmethod using phenolic and anilinic compoundsrdquo Environmen-tal Science and Technology vol 26 no 10 pp 1876ndash1881 1992

[20] J Schwarz M-O Aust and S Thiele-Bruhn ldquoMetabolitesfrom fungal laccase-catalysed transformation of sulfonamidesrdquoChemosphere vol 81 no 11 pp 1469ndash1476 2010

[21] H M Bialk A J Simpson and J A Pedersen ldquoCross-couplingof sulfonamide antimicrobial agents with model humic con-stituentsrdquo Environmental Science and Technology vol 39 no 12pp 4463ndash4473 2005

[22] H M Bialk C Hedman A Castillo and J A PedersenldquoLaccase-mediated Michael addition of 15N-sulfapyridine to amodel humic constituentrdquo Environmental Science and Technol-ogy vol 41 no 10 pp 3593ndash3600 2007

[23] H M Bialk and J A Pedersen ldquoNMR investigation ofenzymatic coupling of sulfonamide antimicrobials with humicsubstancesrdquo Environmental Science and Technology vol 42 no1 pp 106ndash112 2008

[24] A Gulkowska B Thalmann J Hollender and M KraussldquoNonextractable residue formation of sulfonamide antimicro-bials new insights from soil incubation experimentsrdquo Chemo-sphere vol 107 pp 366ndash372 2014

[25] A Gulkowska M Krauss D Rentsch and J Hollender ldquoReac-tions of a sulfonamide antimicrobial with model humic con-stituents assessing pathways and stability of covalent bondingrdquoEnvironmental Science and Technology vol 46 no 4 pp 2102ndash2111 2012

[26] A Gulkowska M Sander J Hollender and M Krauss ldquoCova-lent binding of sulfamethazine to natural and synthetic humicacids assessing laccase catalysis and covalent bond stabilityrdquoEnvironmental Science and Technology vol 47 no 13 pp 6916ndash6924 2013

[27] A Gulkowska J Hollender and M Krauss ldquoCovalent bondingof sulfonamide antimicrobials with organic matter dominanceof nucleophilic addition over radical couplingrdquo in Proceedingsof the SETAC Europe 20th Annual Meeting Sevilla Spain May2010

[28] F J Gonzalez-Vila F Martin C Saiz-Jimenez and H H NimzldquoThermofractography of humic substancesrdquo Journal of ThermalAnalysis vol 15 no 2 pp 279ndash284 1979

[29] P Baldrian ldquoFungal laccasesmdashoccurrence and propertiesrdquoFEMS Microbiology Reviews vol 30 no 2 pp 215ndash242 2006

[30] C-J Wang S Thiele and J-M Bollag ldquoInteraction of 246-trinitrotoluene (TNT) and 4-amino-26-dinitrotoluene with

humic monomers in the presence of oxidative enzymesrdquoArchives of Environmental Contamination and Toxicology vol42 no 1 pp 1ndash8 2002

[31] J-E Kim E Fernandes and J-M Bollag ldquoEnzymatic couplingof the herbicide bentazon with humus monomers and char-acterization of reaction productsrdquo Environmental Science andTechnology vol 31 no 8 pp 2392ndash2398 1997

[32] S Thiele E Fernandes and J M Bollag ldquoEnzymatic transfor-mation and binding of labeled 246-trinitrotoluene to humicsubstances during an anaerobicaerobic incubationrdquo Journal ofEnvironmental Quality vol 31 no 2 pp 437ndash444 2002

[33] S Thiele-Bruhn and M-O Aust ldquoEffects of pig slurry onthe sorption of sulfonamide antibiotics in soilrdquo Archives ofEnvironmental Contamination and Toxicology vol 47 no 1 pp31ndash39 2004

[34] H Knicker D Bruns-Nagel O Drzyzga E Von Low andK Steinbach ldquoCharacterization of 15N-TNT residues after ananaerobicaerobic treatment of soilmolasses mixtures by solid-state 15N NMR spectroscopy 1 Determination and optimiza-tion of relevantNMR spectroscopic parametersrdquoEnvironmentalScience and Technology vol 33 no 2 pp 343ndash349 1999

[35] G E Schaumann and E J LeBoeuf ldquoGlass transitions in peattheir relevance and the impact of waterrdquo Environmental Scienceand Technology vol 39 no 3 pp 800ndash806 2005

[36] J Hurrass and G E Schaumann ldquoIs glassiness a commoncharacteristic of soil organic matterrdquo Environmental Scienceand Technology vol 39 no 24 pp 9534ndash9540 2005

[37] H R Christen Grundlagen der Organischen Chemie Salle ampSauerlander Frankfurt Germany 6th edition 1985

[38] A L Boreen W A Arnold and K McNeill ldquoTriplet-sensitizedphotodegradation of sulfa drugs containing six-memberedheterocyclic groups identification of an SO

2

extrusion photo-productrdquo Environmental Science and Technology vol 39 no 10pp 3630ndash3638 2005

[39] A L BoreenW A Arnold and KMcNeill ldquoPhotodegradationof pharmaceuticals in the aquatic environment a reviewrdquoAquatic Sciences vol 65 no 4 pp 320ndash341 2003

[40] A L Boreen W A Arnold and K McNeill ldquoPhotochemicalfate of sulfa drugs in then aquatic environment sulfa drugscontaining five-membered heterocyclic groupsrdquo EnvironmentalScience and Technology vol 38 no 14 pp 3933ndash3940 2004

[41] P Sukul M Lamshoft S Zuhlke and M Spiteller ldquoPhotolysisof 14C-sulfadiazine in water andmanurerdquo Chemosphere vol 71no 4 pp 717ndash725 2008

[42] W Tappe M Herbst D Hofmann et al ldquoDegradation of sulfa-diazine by Microbacterium lacus strain SDZm4 isolated fromlysimeters previously manured with slurry from sulfadiazine-medicated pigsrdquo Applied and Environmental Microbiology vol79 no 8 pp 2572ndash2577 2013

[43] M J Garcıa-Galan M Silvia Dıaz-Cruz and D BarceloldquoIdentification and determination of metabolites and degra-dation products of sulfonamide antibioticsrdquo TrACmdashTrends inAnalytical Chemistry vol 27 no 11 pp 1008ndash1022 2008

[44] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[45] J Schwarz SThiele-BruhnK-U Eckhardt andH-R SchultenldquoSorption of sulfonamide antibiotics to soil organic sorbentsbatch experiments with model compounds and computational


chemistryrdquo ISRN Soil Science vol 2012 Article ID 159189 10pages 2012

[46] P S Guin S Das and P CMandal ldquoElectrochemical reductionof quinones in different media a reviewrdquo International Journalof Electrochemistry vol 2011 Article ID 816202 22 pages 2011

[47] S-F Yang C-F Lin A Yu-Chen Lin and P-K Andy HongldquoSorption and biodegradation of sulfonamide antibiotics byactivated sludge experimental assessment using batch dataobtained under aerobic conditionsrdquoWater Research vol 45 no11 pp 3389ndash3397 2011

[48] M Forster V Laabs M Lamshoft et al ldquoSequestration ofmanure-applied sulfadiazine residues in soilsrdquo EnvironmentalScience and Technology vol 43 no 6 pp 1824ndash1830 2009

[49] S Thiele-Bruhn T Seibicke H R Schulten and P LeinweberldquoSorption of sulfonamide pharmaceutical antibiotics on wholesoils and particle-size fractionsrdquo Journal of EnvironmentalQuality vol 33 no 4 pp 1331ndash1342 2004

[50] G Fahraeus and B Reinhammar ldquoLarge scale production andpurification of laccase from cultures of the fungus Polyporusversicolor and some properties of laccase Ardquo Acta ChemicaScandinavica vol 21 no 9 pp 2367ndash2378 1967

[51] H Knicker G Almendros F J Gonzalez-Vila F Martin andH-D Ludemann ldquo13C- and 15N-NMR spectroscopic examina-tion of the transformation of organic nitrogen in plant biomassduring thermal treatmentrdquo Soil Biology and Biochemistry vol28 no 8 pp 1053ndash1060 1996

[52] A E Berns H Philipp and H Lewandowski ldquoElucidating thereaction of sulfadiazine with soil humic acid with 15N-CPMAS-NMR spectroscopyrdquo in Proceedings of the EUROSOIL Soil-Society-Environment Conference p 9 European GeosciencesUnion Vienna Austria 2008

[53] E J LeBoeuf and L Zhang ldquoThermal analysis for advancedcharacterization of natural nonliving organic materialsrdquoBiophysico-Chemical Processes Involving Natural NonlivingOrganic Matter in Environmental Systems pp 783ndash836 2009

[54] G E Schaumann E J LeBoeuf R Delapp and J HurraszligldquoThermomechanical analysis of air-dried whole soil samplesrdquoThermochimica Acta vol 436 no 1-2 pp 83ndash89 2005

[55] E Fukuoka M Makita and S Yamamura ldquoGlassy state ofpharmaceuticals III Thermal properties and stability of glassypharmaceuticals and their binary glass systemsrdquo Chemical andPharmaceutical Bulletin vol 37 no 4 pp 1047ndash1050 1989

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Table 1 Physicochemical properties and structures of the selected sulfonamides


FormulaStructure


Sulfanilamide(SAA)63-74-11722 gmolminus11206 cm3

C6H8N2O2S

H2N

O

S

O

NH

H

log119870ow minus062pk1198861

19pk1198862


Sulfapyridine(SPY)144-83-22493 gmolminus11740 cm3

C11H11N3O2S

H2N

O

S

O

NN

H log119870ow 035pk1198861

29pk1198862


Sulfadimethoxine(SDT)122-11-23103 gmolminus12152 cm3

C12H14N4O4S

CH3

CH3

H2N

O

S

O

O

O

N

N

N

H

log119870ow 163pk1198861

24pk1198862


to low concentrations of each single compound [14 15] It isassumed that this is also valid for N-containing sulfonamidesas wellThese restrictions can be overcome usingwell-definedsubstituted phenolic compounds that represent special sitesand functionalities of humic substances These compoundsare major building blocks of humic molecules [16] and werefound to polymerize to humus-like substances [17] Reactingsuch substituted phenols with oxidoreductase enzymes orabiotically through pedogenic oxides hydroxides and clayminerals [18 19] serves as model to elucidate transformationreactions or modes of binding happening in soil uponhumification processes

Using that experimental approach Bialk et al [21 22]and Bialk and Pedersen [23] reported enzymatically cat-alyzed oxidative coupling of selected sulfonamides Theyfound that a catalyst is needed to gain a transformationof sulfonamides in the presence of phenolic substances[21] A significant direct oxidation of the sulfonamides sul-famethoxazole sulfamethazine and sulfapyridine occurredin the presence of acid birnessite while bound residueformation was even stronger by fungal peroxidase catalyzedcovalent cross coupling in the presence of o-phenols and26-dimethoxyphenols respectively [21] Similar results wereobtained when humic acid [23] or when a whole soil samplewas used [24] Also it was found that the amount of quinonesin soil that can take part in the nucleophilic addition is thelimiting factor in the bound residue formation of sulfon-amides [24] Binding mechanisms were identified as Michaeladdition leading to the formation of anilinohydroquinones

and anilinoquinones and possible further covalent linkageswhile the formation of a Schiff base (imine formation) wascontroversially discussed [21ndash23 25 26] Gulkowska et al[26 27] concluded that radical coupling reactions are mostlikely not relevant for the initial covalent bond formation

The findings of these reports are further completed byour study The aim was to (i) elucidate the reaction ofphenolic substances with sulfonamides in the absence of afurther catalyst and its dependence on the concentrationand reaction time and (ii) to determine the strength andtype of enzyme catalyzed immobilization with the help of asequential extraction procedure and analytical identificationof reaction products by using 15N CPMAS NMR and differ-ential scanning calorimetry (DSC) For this purpose threedifferently substituted phenols that is catechol guaiacol andvanillin were used as model substances to determine theimmobilization of three selected sulfonamides that is sul-fanilamide (SAA) sulfapyridine (SPY) and sulfadimethoxine(SDT) in the presence and absence of the enzyme laccasefrom Trametes versicolor taken as biocatalyst

2 Materials and Methods

21 Chemicals and Enzyme Three pharmaceutical sub-stances were selected from the group of sulfonamidesthat are composed of a core structure and different sub-stituents at the sulfonamide-N (Table 1) The SAA (4-aminobenzenesulfonamide) was obtained from Riedel-de




FormulaStructure




C6H6O2OH

OH

log119870ow 088pk119886

945




C7H8O2OH

OCH3

log119870ow 132pk119886

100



C8H8O3OH

OCH3

O H

log119870ow 121pk119886

740
















3

PO4








119888 = 1198880

minus 119896 times 119905 (1)




sulfonamide 1198880


SPY

0075 0 0

Zero order (1)

0003 0439 00080075 0007 0 0047 0910 00350075 0013 0 0093 0996 00600075 0020 0 0140 0950 00890075 0027 0 0144 0981 01250075 0033 0 0156 0998 0144

SDT 0075 0033 0 0131a

SAA 0075 0033 0 0003a



First order (2)0517 0966 0032


0 0005 0010 0015 0020 0025 0030 0035

10

08

06

04

02

0


SPY (1h)SAA (5d)

cc 0

of su

lfona

mid

es


SPY (41h)







119888 = 1198880


(2)
















119891





minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH


(ppm)

R1

R2

(b)







2






minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




FormulaStructure




C6H6O2OH

OH

log119870ow 088pk119886

945




C7H8O2OH

OCH3

log119870ow 132pk119886

100



C8H8O3OH

OCH3

O H

log119870ow 121pk119886

740
















3

PO4








119888 = 1198880

minus 119896 times 119905 (1)




sulfonamide 1198880


SPY

0075 0 0

Zero order (1)

0003 0439 00080075 0007 0 0047 0910 00350075 0013 0 0093 0996 00600075 0020 0 0140 0950 00890075 0027 0 0144 0981 01250075 0033 0 0156 0998 0144

SDT 0075 0033 0 0131a

SAA 0075 0033 0 0003a



First order (2)0517 0966 0032


0 0005 0010 0015 0020 0025 0030 0035

10

08

06

04

02

0


SPY (1h)SAA (5d)

cc 0

of su

lfona

mid

es


SPY (41h)







119888 = 1198880


(2)
















119891





minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH


(ppm)

R1

R2

(b)







2






minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






3

PO4








119888 = 1198880

minus 119896 times 119905 (1)




sulfonamide 1198880


SPY

0075 0 0

Zero order (1)

0003 0439 00080075 0007 0 0047 0910 00350075 0013 0 0093 0996 00600075 0020 0 0140 0950 00890075 0027 0 0144 0981 01250075 0033 0 0156 0998 0144

SDT 0075 0033 0 0131a

SAA 0075 0033 0 0003a



First order (2)0517 0966 0032


0 0005 0010 0015 0020 0025 0030 0035

10

08

06

04

02

0


SPY (1h)SAA (5d)

cc 0

of su

lfona

mid

es


SPY (41h)







119888 = 1198880


(2)
















119891





minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH


(ppm)

R1

R2

(b)







2






minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



sulfonamide 1198880


SPY

0075 0 0

Zero order (1)

0003 0439 00080075 0007 0 0047 0910 00350075 0013 0 0093 0996 00600075 0020 0 0140 0950 00890075 0027 0 0144 0981 01250075 0033 0 0156 0998 0144

SDT 0075 0033 0 0131a

SAA 0075 0033 0 0003a



First order (2)0517 0966 0032


0 0005 0010 0015 0020 0025 0030 0035

10

08

06

04

02

0


SPY (1h)SAA (5d)

cc 0

of su

lfona

mid

es


SPY (41h)







119888 = 1198880


(2)
















119891





minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH


(ppm)

R1

R2

(b)







2






minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















119891





minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH


(ppm)

R1

R2

(b)







2






minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



minus134

minus183 minus

197

minus219

minus306

minus435

NH

HS

NO

O

H

N

(ppm)

(a)

minus260

NH


(ppm)

R1

R2

(b)







2






minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





minus20 0 20 40 60 80 100 120 140 160 180 200

Firs

t der

ivat

ion

heat

flow

(W(

g min

))

Temperature (∘C)

CatSPYCatSDT

CatSAACat



2


2


4 Conclusions




Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Abbreviations








Acknowledgment


References









































2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



























2






























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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