Chemosphere 72 (2008) 1448–1454

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Chemosphere
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Re-evaluation of the conformational structure of sulfadiazine species using

NMR and ab initio DFT studies and its implication on sorption and degradation
Gerd Huschek a,c,*, Dirk Hollmann b,*, Nadine Kurowski a, Martin Kaupenjohann a, Harry Vereecken c
a
Institute of Ecology, Technische Universität Berlin, Salzufer 12, D-10587 Berlin, Germany
b
Leibniz-Institut für Katalyse e.V. an der Universität Rostock, Albert-Einstein-Strasse 29a, D-18059 Rostock, Mecklenburg-Vorpommern, Germany
c
Agrosphere Institute, ICG-4, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany

a r t i c l e i n f o a b s t r a c t

Article history: In the environment, the sorption and the degradation of organic pollutants are of increasing interest.
Received 3 January 2008 The investigation of the chemical structures provides a basis for the development of a suitable binding
Received in revised form 7 May 2008 model approach and for the mechanistic understanding of the chemical fate processes. The aim of this
Accepted 12 May 2008
study was the identification of different species of the antibiotic compound sulfadiazine (SDZ) using
Available online 3 July 2008 1
H and 13C NMR experiments and ab initio density functional theory (DFT) calculations. In the neutral,
aprotic solvent dimethylsulfoxide-d6 (DMSO-d6), a new sulfadiazine structure containing an O–H–N
Keywords:
hydrogen bond was identified. In the protic solvent water-d2 and in dependence on pH and the position
Sulfadiazine
NMR
of the amidogen hydrogen atom nine possible SDZ conformations were analyzed and five structures were
Structure elucidation identified. Good conformity between theory and calculation of 1H NMR was observed. Unfortunately, 13C
Antibiotic NMR is not sensitive enough for comparison and differentiation. In order to verify the identified struc-
Sulfonamide tures, additional NBO/NLMO (natural localized molecular orbital) analyses were conducted (calculation
of net atomic charges, bond polarity, atomic valence, and electron delocalization). Finally, conformation
optimizations were performed in order to investigate the stability of the SDZ species.
We showed that SDZ contains no S@O double bond, but that it has two S–O single bonds. Surprisingly,
negative charges were observed at the pyrimidine nitrogen atom. With these results, the known structure
of SDZ was revised. Studies of the geometrical structure and the torsion angles showed that SDZ is very
flexible and can be easily fitted to the sorbent.
These observations would explain the strong sorbance and hence the rapid formation of non-extract-
able residues in the environment because SDZ acts as a strong ligand. These results show that that the
sulfonamide hydrogen is important for the biological activity but the pyrimidine nitrogen and the sulfon-
amide oxygen is responsible for the sorbance in environment.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction benzenesulfonamide), is widely used for specific therapeutic and

Pharmaceutical antibiotics are very important as veterinary
drugs. Over the last decade, the extensive use of these chemical
substances in animal husbandry has caused considerable environ-
mental contamination (Thiele-Bruhn, 2003; Boxall et al., 2004).
Most antibiotics are quickly excreted from the body, either unal-
tered or as metabolites (Halling-Sørensen et al., 1998; Kumar
et al., 2005). In the environment, these substances can cause
microbial resistance of soil microorganisms (Thiele-Bruhn, 2005).
For instance Sulfadiazine (SDZ, 4-amino-N-(pyrimidin-2yl)-
* Corresponding authors. Address: IGV GmbH, Arthur-Scheunert-Allee 40/41,
14558 Nuthetal, OT Bergholz-Rehbrücke, Germany. Tel.: +49 3320089 131; fax: +49
3320089 251 (G. Huschek), tel.: +49 3811281 215; fax: +49 3811281 51215
(D. Hollmann).
E-mail addresses: g_huschek@igv-gmbh.de (G. Huschek), dirk.hollmann@
catalysis.de (D. Hollmann).
prophylactic purposes (Mitema et al., 2001). Unaltered SDZ and
its conjugates are excreted and enter the environmental compart-
ments (Lindberg et al., 2005; Sukul and Spiteller, 2006). In the
environment, the rapid formation of non-extractable residues of
SDZ has been identified as the predominant process in manured
soil (Heise et al., 2006; Thiele-Bruhn and Peters, 2007). Up to
95% of 14C-labeled SDZ was found to be transferred into the non-
extractable fractions (Kreuzig and Höltge, 2005). Hence, intensive
investigations on the mechanism why and how SDZ or other anti-
biotics are bonded and sorbed to the environment are of high
interest. Initial investigations on the sorption of sulfonamides in
soil have already be published by Thiele-Bruhn et al. (2004), Gao
and Pedersen, (2005) and Kahle and Stamm (2007).
Sulfonamides act as competitive inhibitors of the enzyme dihy-
dropteroate synthase (DHPS) in bacteria, and catalyzes the conver-
sion of 4-aminobenzoic acid (PABA) into an essential nutrient for
some bacteria (Roland et al., 1979). The sulfonamide group

0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2008.05.038
 G. Huschek et al. / Chemosphere 72 (2008) 1448–1454 1449

(-SO2NHR1) in SDZ is very important for biological activity. Li et al.
(2005) pointed out that the antimicrobial activity disappears, if the
hydrogen atom on the dissociate amidogen is replaced by other
atoms or groups. Hence, sulfadiazine is an excellent representative
for a biomedical active substance where the position of the hydro-
gen atom is essential for the binding mechanism and the antimi-
crobial effects. Relationships between antimicrobial activities of
sulfonamides and the physico-chemical property pKa highlight
the dominant role played of the degree of ionization in the activity
(Mengelers et al., 1997). In principle, sulfonamide (SDZ) can be
present as cationic, zwitterionic, uncharged or anionic species.
Based on the two pKa values of 1.8 (protonated form) and 6.4
(deprotonated form), uncharged SDZ exists from approximately
pH 4 until pH 8. At pH 7, the neutral and the anionic form are pres-
ent in almost equal proportions in the environment. Consequently,
these species of SDZ are most important for bonding in the
environment.
For SDZ, depending on the pH and position of the dissociate
amidogen hydrogen atom, nine possible SDZ conformations can
be described for the neutral (N), deprotonated (D), and protonated
(P) forms. These relevant structures are sketched in Fig. 1: P-1:
+
NHþ 3 cation; P-2: –OH S cation; P-3: O–H–Npyrimidine –NHþ 3 (Fig. 1), sample solutions were prepared. Sulfadiazine (20 mg)
cation; P-4: O–H–N–pyrimidine S+ cation; N-1: uncharged; N-2:
tautomeric imido O–H; N-3: zwitterionic; N-4: O–H–N-amidogen;
N-5: tautomeric imido O–H–N-pyrimidine; D: anionic. In past,
attention was mainly devoted to the N-1 structure. However, from
a chemical point of view, all other structures are possible.
For the development of binding approaches for SDZ in the envi-
ronment, the identification of the active SDZ structures present is
important. Neither experimental nor theoretical data is available
for the identification of water-solved SDZ species. Theoretically,
NMR spectroscopy could be useful for identifying chemical struc-
tures. Benoit and Preston (2000) used this method to identify atra-
zine structures in soil. Theoretical ab initio studies could
supplement these measurements. Additionally, calculations of
energetics, atomic charges, minimum energy structures, geometry,
and natural bond orbital (NBO) could indicate the electronic den-
sity distribution of each atom. Finally, by taking NBO results show-
ing the presence of S–O single bonds in consideration, realistic
Lewis structures can be determined (Stefan and Janoschek, 2000).
These systematic data, regarding the variation of molecular prop-
erties, are important for the chemical structure and could therefore
provide first insights into the still poorly understood chemical
bonding of SDZ complexes to soil.
In brief, the objective of this study is to investigate the chemical
structures of SDZ species using 1H and 13C NMR experiments in
dependence on pH and comparison with the calculated chemical
shifts of optimized structures in solvents. To verify these struc-
tures, further NBO/NLMO (Natural Localized Molecular Orbital)
analyses were carried out for example calculation of net atomic
charges, bond polarity, atomic valence, and electron delocalization.
Finally, to investigate the stability of the SDZ species, conformation
optimizations were performed.
2. Methods and materials
2.1. Sample preparation
Due to the zwitterion structure, SDZ can act as a weak organic
acid (Nesbitt and Sandmann, 1977) and as a weak Lewis base
(LB). Hence, two different pKa values exist. For the characterisation
and identification of the proposed resulting structures of SDZ
and 10 ll acetone were added to 2 ml deuterated water. To adjust
the samples to five different pH values, 20 ll of 2 N hydrogen chlo-
ride solution (pH 1.35), 20 ll of 2 N hydrogen chloride solution and
30 mg ammonium chloride (pH 2.90), 50 mg ammonium chloride
(pH 4.40), no additives (pH 5.30), 20 ll of a 15 N sodium hydroxide
solution (pH 12.00) were added. Due to the low solubility of SDZ in
water (20 mg l1), the excess SDZ was separated by filtration.
After 10 min ultrasonication and pH controlling, the suspension
was filtered into an NMR tube.
2.2. NMR
1
H and 13C NMR spectra were recorded on a Bruker AV 300 MHz
spectrometer. 13C NMR measurements were performed using stan-
dard procedure (30° pulse, no signal saturation). 1H and 13C NMR
chemical shifts were reported relative to added acetone
(2.22 ppm (1H) and 30.9 ppm (13C) in water-d2, 2.09 ppm (1H),
and 30.56 ppm (13C) in acetone-d6, and DMSO-d6 (1H) (Gottlieb
et al., 1997). Deutero solvents, NaSDZ, and SDZ were purchased
from Sigma–Aldrich and used without further purification.

N N N N
O N O N O N O N
H2N S NH H2N S N H3N+ S N- H2N S N
O OH O O H

N-1 N-2 N-3 N-4

H N N N H N
O N O N O N O N
H2N S N H3N+ S NH H2N S+ NH H3N+ S N
O O OH O

N-5 P-1 P-2 P-3

H N N N
O N O N O- N
H2N S+ NH H2N S N H2N S2+ N
O O O-

P-4 D L-1

Fig. 1. Protonated (P), neutral (N), deprotonated (D) and Lewis (L) structures of sulfadiazine studied in this paper.
1450 G. Huschek et al. / Chemosphere 72 (2008) 1448–1454
Due to the solvents and concentration dependence, the NH-shifts
were not representative. The aniline protons form an AA’BB’-spin
system. The assignment of the 1H and 13C signals were assigned
HMQC NMR. All NMR spectra are found in the Supporting informa-
tion. The pH was measured with a pH-meter WT pH 330 and a pH-
electrode SenTix 41 pH 0–14 at 22 °C.
2.3. Theoretical section
In order to facilitate the interpretation of the NMR spectra,
density functional type quantum-chemical calculations were
performed using the Gaussian 03 software (Frisch et al., 2004).
For the calculations, the B3LYP/6-311G+(d) basis set was
employed, and the default convergence criteria of the G03 were
adopted. This set-up provided good results for hydrogen bonding
modeling (Musin and Mariam, 2006). X-ray crystallography data
was used as the start configuration for SDZ (Sin et al., 1974; Heren
et al., 2006). The 1H and 13C NMR chemical shifts of the different
SDZ species were calculated using the GIAO method with water
and DMSO as solvents in order to allow this data be compared with
the experimental NMR data. The elucidation of the SDZ species was
accompanied by calculation of net atomic charges, bond polarity,
atomic valence, and electron delocalization using NBO/NLMO
(natural localized molecular orbital) analysis (Glendening et al.,
1988).
The electronic density distribution in the SDZ molecule as a
consequence of intra- and interhydrogen bonding is essential for
binding model approaches to SDZ in the environment. Solvents
could influence the order of preference of different structures
(see Fig. 1), which is why conformations of the SDZ species were
optimized in the gas phase, in water and in dimethylsulfoxide.
The calculations were performed using the integral equation for-
malism polarized continuum model (IEFPCM) (Cancès et al., 1997
and Cancès and Mennucci, 2001). The optimized gas phase struc-
tures were used as starting point for these calculations.
3. Results and discussion
3.1. Structure identification using 1H and 13
C NMR measurements
Until now, no NMR studies have been performed at different pH
levels in deutero water for the identification of the structure of SDZ
species. We began our investigations with the analysis of the neu-
tral form of SDZ. Due to the slow intermolecular rate of exchange,
the polar and aprotic solvent dimethylsulfoxide (d6-DMSO) was
chosen (Table 1 and Fig. 2, for the atom-labeling scheme of SDZ
see Fig. 3).
The aniline hydrogen atoms (H25/H26) were observed at
6.01 ppm. Surprisingly, a broad signal appeared at 11.28 ppm. This
high shift did not correspond with the sulfonamide structure N-1
nor the imido tautomeric forms N-2. The NH proton of N-1 would
be observed around 7 ppm and the acidic OH proton of N-2 at
10 ppm (Pretsch et al., 2004). Shift over 11 ppm are well known
from hydrogen bonds which are formed in 1,3-diketons by
keto-enol tautomerism. When the hydrogen bonds in SDZ were
also taken into account, we assumed a new structure (N-5) was
formed which bears a hydrogen between the oxygen of the sulfon-
amide and the nitrogen of the pyrimidine ring.
In order to determine the present form, ab initio calculations
were performed (Table 1). Calculations of the proton H27 gave
the following results: 6.7 ppm for N-2 and 11.5 ppm for N-5. More-
over, other calculated shifts of N-5 showed good agreement with
the measured proton shifts.
In addition, we examined the 13C-spectra of SDZ in DMSO-d6
(Table 1). The 13C signals correspond exactly with the 13C signals
reported by Chang et al. (1975). In this group the chemical shifts
were assigned with the off-resonance and selective proton decou-
pling technique. Within the margin of error, the calculated 13C
shifts of N-5 conformed well to the experimental 13C shifts. Only
the calculated C7 shift (172.0 ppm) showed a slight variation of
14 ppm compare to the experimental C7 shift (157.1 ppm), which
close to the maximum error of the GIAO method. A combination
of the 1H and 13C NMR showed that N-5 exists in DMSO.
3.2. 1H and 13
C shifts in water at different pH values
Based on the observations in DMSO we focused our attention on
the identification of chemical shifts for the proposed SDZ struc-
tures (Fig. 1) in water in dependence on the pH. A buffer was re-
quired to obtain the corresponding species of SDZ. The buffer and
the low solubility of SDZ in water at pH < 6 proved very problem-
atic for 13C NMR experiments. Hence, we could only observe 13C
signals for pH 1.35 and pH 12.00 (high solubility as anion and cat-
ion). In the other cases (pH 2.90, 4.40, 5.30), no 13C signals could be
determined even after scanning for several days.
In comparison to DMSO, the high intermolecular rate of
exchange in water resulted in the disappearance of OH and NH
protons. Only the CH protons of the aniline and the pyrimidine ring
were determined (Table 2). Moreover, the solvent effect of DMSO
and water ruled out a direct correlation between DMSO and water.
A compendium of the NMR spectra at different pH values has been
included in the Supplementary information Fig. 1.
pH 1.35: In contrast to the other pH values, a very high shift of
the aniline protons was measured at pH 1.35. The protons H18/21
and H19/20 were observed at 8.07 ppm and 7.51 ppm, respectively.
Compared to the 1H shifts at pH 2.90, the difference was +0.30 ppm
and +0.64 ppm. This is in accordance with decreased magnetic
shielding of the aniline protons by an ammonium group (R-NHþ 3 ).
This ammonium group militates in favour of the postulated struc-
tures P-1 and P-3. To support these structures P-1 and P-3, we per-
formed ab initio calculations of the 1H shifts (Supplementary
information Table 2). For both structures P-1 and P-3, the hydro-
gen shifts for H18/21 and H22/24 agreed with the calculated shifts.
A differentiation of P-1 and P-3 was determined for the H19/20
and H23 protons. For P-1 the calculated shifts were 7.8 ppm (H19/
20) and 6.9 ppm (H23). For P-3 the calculated shifts were 8.0/
7.7 ppm (H19/20) and 6.6 ppm (H23). The best agreement with the
experimental data (7.51 ppm for H19/20 and 7.05 ppm for H23)
was confirmed for P-1. In addition, due to the P@N double bond,
a high rotation barrier in P-3 was assumed (Jordan, 1982). This
was identified in the calculations of the H19 and H20 shifts, which
resulted in two different shifts. This suggests that one clear confor-
mation exists. However, in the experimental spectra only one
signal for H19/20 appeared. This observation favours the P-1 form.
Additionally, we compared the experimental 13C shifts (pH
1.35) with the calculated 13C shifts of P-1 and P-3 (see Supplemen-
tary information Table 1). Unfortunately, if the margin of error is
taken into account, it became clear that no clear differentiation is
possible for P-1 and P-3 using 13C measurements. The results of
further calculations (inclusion of important additional water mol-
ecules) were no better than those of the 13C NMR calculations. In
summary we propose that the protonated form P-1 exists at pH
1.35 which agrees with the known knowledge.
pH 2.90–5.30: The comparison of the proton spectra at pH 2.90,
4.40, 5.30, and 12.00 revealed that a gradual shielding of all pro-
tons occurred. The position of the protons H18/21 and H19/20 chan-
ged only slightly (7.7 ppm and 6.8 ppm) for all pH. This
means that no changes occurred at the aniline ring. In contrast,
proton H22/24 was found to be stabile up to pH 4.40 (8.46 ppm),
but at higher pH values the shift decreased to 8.28 ppm. A similar
observation was also made for H23. Unfortunately, because the buf-
fer signal of ammonium chloride overlapped with the proton signal
Table 1
Comparison of experimental and calculated 1H and 13
C NMR shifts of neutral and deprotonated species in DMSO-d6a
Species Experimental Calculated
SDZ Na-SDZ N-2 N-5 D-1 (Na)
H number H shift ppm H shift ppm H shift ppm H shift ppm H shift ppm
300 MHz DMSO 300 MHz DMSO (ref. TMS = 0) GIAO, (ref. TMS = 0) GIAO, (ref. TMS = 0) GIAO, DMSO
DMSO DMSO
H18/21 7.61 7.45 7.5/7.7 7.6 7.5/7.9
H19/20 6.56 6.44 6.6/6.8 6.7/6.7 6.6/6.6
H22/24 8.48 8.08 8.2/8.4 8.3/8.6 8.2/8.2
H23 7.00 6.34 6.6 6.7 6.1
H25/26 6.01 5.33 4.4/4.4 4.1 3.4/3.4
H27 11.27 – 6.7 11.5 –

Species Experimental Calculated

SDZ Na-SDZ N-5 D-1 (Na)
C number C shift ppm C shift ppm C shift ppm C shift ppm
75.5 MHz DMSO 75.5 MHz DMSO (ref. TMS = 0) GIAO, DMSO (ref. TMS = 0) GIAO, DMSO
C1 124.7 133.6 131.1 146.4/139.5

G. Huschek et al. / Chemosphere 72 (2008) 1448–1454

C2/C6 129.7 128.2 133.8/134.1 131.1/136.4
C3/C5 112.0 111.8 115.8/117.7 116.2/115.0
C4 152.9 149.7 160.2 157.6
C7 157.1 164.5 172.0 171.3
C8/C10 158.1 157.1 165.9/160.6 160.9/162.9
C9 115.4 108.8 116.5 111.2
a
300.1 MHz for 1H and 75.5 MHz for 13C NMR, d in ppm, experimental shifts are determined with acetone as internal standard. Calculated 1H NMR shifts using ab initio calculations (B3LYP/6-311G+(d) basis set) in DMSO against
TMS as reference. For 13C calculations margin of error ±6 ppm.

Table 2
Comparison of experimental and calculated 1H NMR shifts of Sulfadiazine in D2O at different pHa

Species N-1 N-2 N-3 N-5 P-1 D-1

H H shift ppm H shift ppm H shift ppm H shift ppm H shift ppm H shift ppm (ref. H shift ppm (ref. H shift ppm (ref. H shift ppm (ref. H shift ppm (ref. H shift ppm (ref.
number pH 1.35 D20 pH 2.90 D20 pH 4.40 D20 pH 5.30 D20 pH 12.00 D20 TMS = 0) GIAO, TMS = 0) GIAO, TMS = 0) GIAO, TMS = 0) GIAO, TMS = 0) GIAO, TMS = 0) GIAO,
water water water water water water
H18/21 8.07 7.77 7.75 7.70 7.67 8.0/8.2 7.9/8.1 7.9/8.1 7.7/7.7 8.3/8.3 7.9/8.1
H19/20 7.51 6.87 6.83 6.82 6.84 7.1/7.1 7.0/7.2 7.6/7.6 6.8/6.8 7.8/7.8 7.0/7.0
H22/24 8.47 8.46 8.46 8.36 8.28 8.7/8.8 8.5/8.8 7.9/8.2 8.4/8.7 8.4/8.6 8.4/8.5
H23 7.05 7.07 n.d 6.92 6.79 7.3 7.0 6.2 6.8 6.9 6.7
H25/26 n.d. n.d. n.d. n.d. n.d. 4.8 4.9/4.9 6.1/6.3/ 6.4 4.6/4.6 6.3/6.4 6.5 4.4/4.4
H27 n.d. n.d. n.d. n.d. n.d. 8.4 7.4 (O12–H) – 12.2 (O12–H) 8.0 –
H28 n.d. n.d. n.d. n.d. n.d.
a
300.13 MHz, d in ppm, experimental shifts are determined with acetone as internal standard. Calculated 1H NMR shifts in ppm using ab initio calculations (B3LYP/6-311G+(d) basis set) in water against TMS as reference.

1451
1452 G. Huschek et al. / Chemosphere 72 (2008) 1448–1454

11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 ppm
1.0

2.0

2.0

1.0

2.0

1.9
Fig. 2. 1H NMR spectra of sulfadiazine in DMSO-d6.

Table 3
Comparison of DESE [kJ mol1] for SDZ species of optimized structures using DFT/
B3LYP/ 311G(d) of G03, N-1 (for N-2–N-5) and P-4 (for P-1–P-3) normalized to zeroa

kcal/mol N-1 N-2 N-3 N-5 P-1 P-2 P-3 P-4

Gas phase 0 +117 +284 +79 +26 +23 +29 0
(0) (+1.2) (+2.9) (+0.8) (+1.1) (+1.0) (+1,2) (0)
Water 0 +32 +83 +92 +5 +38 +9 0
(0) (+0.3) (+0.8) (+0.9) (+0.2) (+0.9) (+0.3) (0)
a
Gas phase: 1154,883 kJ mol1; water: 1154,921 kJ mol1; values in paren-
theses are in eV.

By comparing the experimental 13C shifts (pH 12.00) with the

Fig. 3. Atom-labelling scheme of SDZ.
of H23, a shift for H23 could not be detected at pH 4.40. However,
because of the unchanged chemical shift of H22/24 at pH 2.90 and
pH 4.40, we assume that the chemical shift of H23 also remained
unchanged.
A comparison of the chemical shifts at pH 1.35 and pH 2.90 re-
vealed that only the chemical shifts of the aniline changed. This
means that the chemical environment of the protons of the pyrim-
idine ring remained unchanged. This would therefore eliminate
structure N-3. At pH 1.35 we succeeded in confirming the presence
of form P-1. At pH 2.90 the analogue neutral form N-1 was ob-
served. Unfortunately, ab initio calculation of N-1, but also the con-
templable forms N-2 and N-5 showed good conformity with the
observed chemical shifts. By comparing all of the calculated neu-
tral structures, we recognized that the calculated values for N-1,
N-2, and N-5 were very close together. This could also be seen in
the calculated stability of these complexes (see Chapter 3.3 and
Table 3). No exact structure could be assigned to the experimental
data at pH 2.90 and pH 4.40. Under these conditions (water and
room temperature) no rotation barrier existed and these forms
were quickly transformed into each other. Therefore we could
not no determination of the exact species could be conducted.
We suppose that between pH 2.90 and pH 5.50 a state of equilib-
rium exists between N-1 and the imido tautomeric forms N-2
and N-5 which expand the knowledge about SDZ structures.
pH 12.00: At pH 12.00 we assume that only one species, D is
present. The calculated 1H chemical shifts of D showed excellent
conformity with the measured chemical shifts.
calculated 13C shifts of D a good conformity was observed (see Sup-
plementary information). Only small variations were determined
for the C-1 and C-5 (+8 ppm). To prove the D form, additional 1H
and 13C measurements of sodium sulfadiazine (NaSDZ) were done
in DMSO-d6 (Table 1). In consideration of the margin of error of the
GIAO method, a good conformity of the measured and calculated
chemical shifts of D is given. Therefore at pH 12.00, the form D
exists which agrees with the known knowledge, too.
3.3. Molecular properties and relative energies
All structures shown in Fig. 1 could be optimized except for
structure N-4. In addition to G03 calculations, Vienna ab initio
simulation package (VASP) calculations were used to confirm the
optimum of these conformations (Kresse and Hafner, 1993 and
Kresse and Furthmüller, 1996). The energetic, geometrical optimi-
zations showed that the energetic global minimum of the N-1
structure, the pharmaceutically active substance, is the preferred
energetic form. Hence, in order to compare the energy of the differ-
ent structures for neutral species in gas phase or water, respec-
tively, the computed energy of N-1 was normalized to zero, as
shown in Table 3. In general, the optimized structures of the SDZ
species exhibited planar aniline and pyrimidine rings, and the
geometry around the sulfur atom was distorted from the ideal tet-
rahedral symmetry. The gauche conformation about the S–N bond,
indicated by the torsional angle C1–S–N–C7, is important for the
energy minimum of the SDZ forms. The most relevant bond lengths
and bond angles of the optimized structures are listed in Table 5 in
the Supplementary information, together with the experimental
data on the crystal structures of SDZ. Two different dihedral angles
of 76° (Sin et al., 1974) and 59.6° (Heren et al., 2006) were reported
between the planes through the benzene ring and the pyrimidine
ring for SDZ structures based on X-ray crystallographic data.
 G. Huschek et al. / Chemosphere 72 (2008) 1448–1454
Differences of DESE among structures within the gas phase and
between the gas phase and water as a solvent arose because of the
rotation barriers about the S–N bond in the molecule. For water-
solved structures, the rotation about the S–N bond was restricted
as a result of the surrounding water molecules. By comparison of
bond lengths, the best agreement was achieved for N-1 and D with
the reported experimental crystal data. No experimental data was
available for the cation.
Depending on the position of the hydrogen atom of the amido-
gen group two tautomeric imido forms N-2 and N-5 (with an addi-
tional hydrogen bond of 1.787 Å) between O13H27 and N17 of
pyrimidine) were obtained from our calculations using G03. The
O13H27 bond was confirmed by different start positions of H27. Tau-
tomerization to N-2 and N-5 was achieved by the formal migration
of the hydrogen atom to the oxygen atom. Imido tautomeric forms
of sulfanilamide derivates were described by 13C- NMR measure-
ments (Bult, 1983) but no structures were reported for these forms.
In solutions where tautomerization was possible, a chemical
equilibrium of N-1 to N-2 could be reached, because the tautomer-
ization barrier only amounted to 32 kJ mol1 and 92 kJ mol1 for
N-5, respectively. It is possible that the mechanism, whereby a
high tautomerization barrier maintains the tautomeric transforma-
tion, is far from a state of equilibrium. The exact ratio of the tauto-
mer concentration depends on several factors, including
temperature, solvent, and pH. N-1, N-2, and N-5 forms were iden-
tified by NMR measurements.
The protonated form P-4 was the most energetically stable form
in the gas phase. The DESE of this structure compared to the next
cation form P-2 was about 23 kJ mol1. The deprotonated form D
was computed using DFT calculations. The species D of SDZ is
not stable in solution and reacted spontaneously with H3O+ to
N-1 and with other cations such as Ag+, Cu2+, and Ca2+. In these
drugs, the structure D acts as ligand.
The protonated form P-4 was the most energetically stable form
in the gas phase. This form made it possible to explain, the LC-MS/
MS observations regarding SDZ mass fragments made by Pfeifer
et al. (2005) and Lamshöft et al. (2007). Studies on the SDZ struc-
tures using positive (protonated) LC-ESI/MS/MS measurements
revealed two key mass fragments (m/z = 159 and 92) in the MS/
MS spectrum. Both positive fragment structures had no additional
hydrogen atom on the amino group. Pfeifer et al. (2005) and Lam-
shöft et al. (2007) indicated a possible modification of the pyrimi-
dine moiety but they could not explain how these fragments are
formed [starting from the P-1 form]. If we assume the P-4 struc-
ture, the mass fragment m/z = 159 is caused by the [M+H]+ ion
(m/z = 251) losing the [NH–C4H3N2–H] fragment (m/z = 95), by
means of the cleavage of the S–N bond and the O13–H28N17 bond.
The dihedral angle about rotational S–N bond with 145.3° is
responsible for the P-4 structure. This angle is stabilized by the H
bond between O13 and N17–H28 of the pyrimidine ring and will
be lost in Q2 of MS. The S–N (S–C1) bond is broken by the rotation
energy of the pyrimidine ring. The hydrogen bond bridge O13–H28–
N17 was confirmed using different start positions of H28.
In order to understand a binding approach to sorbat and sorbent
in the environment, it is important to understand the electronic
structure of a molecule. Thus, the calculation of Lewis structures
and the characterization of Lewis orbitals and their occupancies
were performed by NBO/NLMO (natural bond/natural localized
molecular orbital) analysis. Nine SDZ species were investigated in
terms of net atomic charge, bond polarity, and electron delocaliza-
tion (see Supplementary information Table 4).
The collected results of the natural population analysis (NPA)
(see Supplementary information Table 3) indicates a remarkably
high positive natural net atomic charge of about 2.2 on sulfur
and of about 0.8 to 1.0 on both oxygen atoms for all SDZ forms.
This means that both oxygen atoms bear one negative charge and
1453
sulfur bears two positive charges. This agrees with the results for
sulfurous and sulfuric acid molecules (Stefan and Janoschek,
2000). Hence, the calculated resonance structures for all forms
were strongly delocalized and indicated a strong electron defi-
ciency on sulfur. Formulas without multiple S@O bonds were
therefore favoured according to the valence Lewis net charge. Thus,
under conservation of the octet rule on sulfur and the Lewis struc-
tures of SDZ should be described by ionic S–O bonds, structure of L-
1 (Fig. 1), instead of covalent p-bonds. As reported by Stefan and
Janoschek (2000), the traditionally assumed violation of the octet
rule on the sulfur atom of SDZ conflicts with the charge distribu-
tion of sulfur and oxygen and the low total occupancy of 0.16 for
the natural 3d orbital of sulfur.
Surprisingly, in addition to the negative charge on oxygen, neg-
ative charges were also calculated for the nitrogen atoms N16 and
N17 of the pyrimidine ring (0.5) and 0.92 for N14 of D. The neg-
ative charges are important for the environmental fate of SDZ. On
the basis of the NBO analysis with a great number of lone pair orbi-
tals (LP) and donated electronic charges in antibonding non-Lewis
orbitals, it can be hypothesized that neutral and deprotonated
forms act as a Lewis base in solution. Lewis bases can form new
covalent coordinative bonds by donating a pair of electrons.
If this happens, SDZ acts as a strong ligand. The best examples
have been shown in sulfadiazine complexes with silver (de Wit
et al., 1983), copper (Hossain et al., 2006), zinc (Yuan et al.,
2001), cobalt (Ajibade et al., 2006), mercury (Garcia-Raso et al.,
1997), platinum (Pasini et al., 1983), and rhodium (Katsaros and
Anagnostopoulou, 2002). Furthermore we investigated the sterical
properties of the SDZ molecule, especially the dihedral angle of the
pyrimidine ring to the S–N bond, in dependence on the properties
of the sorbent. We observed a very flexible S–N bond of 53.6 to
152.4°. SDZ can easily adopt to heavy metal cations in solution or
to bounded organic matter.
All of these observations can be used to explain the strong sorp-
tion on soil. We suggest that ion–ion salt bridges and metal com-
plexes are responsible for the non-extractable fractions of SDZ in
the environment.
4. Conclusions
1
H und 13C NMR measurements combined with ab initio calcu-
lations has been shown to be efficient for the analysis of different
SDZ species. In dimethylsulfoxide (aprotic polar solvent), a new
from (N-5) of SDZ was identified, which contains a O–H–N hydro-
gen bond. Compared to DMSO, in water (protic solvent), at pH 2–4
an equilibrium of the tautomeric forms N-1, N-2 and N-5 is
assumed. This is reflected in the similar stability of these confor-
mations. Under acid conditions (pH 1), these forms were converted
into one stable form, namely P-1. Under basic conditions, the form
D was identified. In all solvents, the measured and calculated 1H
chemical shifts conformed very well. Assignment based on calcu-
lated 13C signals was restricted. Due to low sensitivity, these shifts
provided only limited information on existing forms.
The analysis of the electronic properties of SDZ point out nega-
tive charges for oxygen (1), and surprisingly about 0.5 for the
nitrogen atoms of the pyrimidine ring. A positive charge of +2
was calculated for sulfur means that two S–O single bonds exist.
Due to these properties we predict that SDZ can act as a strong li-
gand and Lewis base. In addition we observed a very flexible rota-
tion of the S–N bond, which allowed SDZ to easily adopt to heavy
metal cations or to bonded organic matter. This would explain the
rapid formation of non-extractable residues and the strong sor-
bance of SDZ in the environment.
We want to emphasize that the sulfonamide nitrogen is impor-
tant for the biological activity but the pyrimidine hydrogen and the
1454 G. Huschek et al. / Chemosphere 72 (2008) 1448–1454
sulfonamide oxygen is responsible for the sorbance in environ-
ment. Future research will focus on the revision of analogous sul-
fonamide structures.
Acknowledgements
The authors gratefully acknowledge the financial support pro-
vided by the German Research Foundation (DFG – Deutsche Fors-
chungsgemeinschaft) for Research Unit 566 Veterinary Medicines
in Soil. We would also like to thank Dr. A. Koch for his excellent
technical and analytical support.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.chemosphere.2008.05.038.
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