Chemical properties of N-chlorotaurine sodium, a key compound in the human defence system

Article abstract of DOI:10.1002/1521-4184(200212)335:9<411::AID-ARDP411>3.0.CO;2-D

N-Chlorotaurine (NCT) is known to play an important role in the human defence system. The already proved utility of the sodium salt as a disinfectant in human medicine suggested a thorough investigation of its chemical properties. Chlorine transfer to N-H groups (transhalogenation) and oxidation of thio and aromatic compounds represent its main reactions. Auto-chlorination causes disproportionation forming N,N-dichlorotaurine (NDCT) with K_d = [NDCT][taurine]/f_a[NCT]² aH⁺ = (4.5 ± 0.8) times; 10⁶, while the reaction with ammonium releasing NH₂Cl is characterised by K_NHC2 = [NH₂Cl][taurine]/[NCT][NH₄⁺] f_a² = 0.02 ± 0.004. The verified unique stability and low-level reactivity of NCT are considered essential for its function in the mammalian defence system and its practical applicability, which manifests itself in an optimal compromise between microbicidal activity and toxicity.

Full text of DOI:10.1002/1521-4184(200212)335:9<411::AID-ARDP411>3.0.CO;2-D

Arch. Pharm. Pharm. Med. Chem. 2002, 9, 411–421
Chemical Properties of N-Chlorotaurine Sodium 411
Waldemar Gottardi,
Markus Nagl
Chemical Properties of N-Chlorotaurine Sodium, a
Key Compound in the Human Defence System
Institute for Hygiene and
Social Medicine,
Medical Faculty,
University of Innsbruck,
Innsbruck, Austria
N-Chlorotaurine (NCT) is known to play an important role in the human defence sys-
tem.The already proved utility of the sodium salt as a disinfectant in human medicine
suggested a thorough investigation of its chemical properties. Chlorine transfer to
N-H groups (transhalogenation) and oxidation of thio and aromatic compounds rep-
resent its main reactions. Auto-chlorination causes disproportionation forming N,N-
dichlorotaurine (NDCT) with Kd = [NDCT][taurine]/fa[NCT]2 aH+ = (4.5 ± 0.8) × 106,
while the reaction with ammonium releasing NH2Cl is characterised by KNHCl2
=
[NH2Cl][taurine]/[NCT][NH+4] fa2 = 0.02 ± 0.004.The verified unique stability and low-
level reactivity of NCT are considered essential for its function in the mammalian de-
fence system and its practical applicability, which manifests itself in an optimal com-
promise between microbicidal activity and toxicity.
Keywords: N-Chloroamino acids; Transhalogenation; Antimicrobial agent; Endog-
enous long-lived oxidant; Non-toxic disinfectant
Received: January 7, 2002 [FP663]
the production of pro-inflammatory cytokines, and sec-
ondly a contribution to the inactivation of pathogens [2,
Introduction
Active chlorine compounds (containing –O–Cl or >N–Cl
functions) are known to act as oxidants with bactericidal
properties. While O-Cl compounds used in practice are
limited to salts of HOCl (hypochlorites), a variety of N-Cl
compounds are in use, chiefly N-chloroamides and -imi-
des, for instance chloroisocyanuric acids and their salts,
chloramine T, 1,3-dichloro-5,5-dimethylhydantoin. N-
Chloroalkylamines (e. g. CH3NHCl), on the other hand,
are of no importance in practice because of their un-
pleasant smell and low stability. Even less stable are the
N-chloroamino acids which, except N-chlorotaurine
(NCT), are available only in solution.
5, 6]. The first reference to NCTa was given by Stel-
maszynska and Zgliczynski [7] who produced the com-
pound from a mixture of taurine, hydrogen peroxide, so-
dium chloride, and myeloperoxidase and proved its iden-
tity by spectrophotometry, chromatography, and the
Na36Cl isotope technique. Further evidence has been
gained by 13C-NMR spectroscopy, showing that taurine
is transformed in aqueous solution to NCT by hypochlo-
rous acid [8]. However, this synthetic route failed be-
cause it was not possible to isolate the compound from
the aqueous solution.It finally succeeded in an alcoholic
system where the pure sodium salt could be prepared
[9].
However, the free N-chloroamino acids attracted in-
creasing interest when their role in the human defence
system was detected, where they act as vehicles for the
oxidation capacity of the highly cytotoxic hypochlorous
acid formed during the “oxidative burst” in granulocytes
and monocytes [1, 2]. NCT plays a major role in this re-
gard, on the one hand because taurine is present in rela-
tively high concentrations in these cells and, on the other
hand, because of its comparatively high stability [3, 4].
The assumed biological function of NCT is first the termi-
nation of the inflammatory process, since it decreases
The aim of this paper is to present the results of investi-
gations on the pure compound NCT-Na which includes
also reviewing its properties and comparing them with
some features observed for hypochlorite in the presence
of surplus taurine. Considering its function in innate im-
munity and its proposed use as a disinfectant in human
medicine, a thorough understanding of the pure com-
pound seems to be mandatory.
a
Because N-chlorotaurine and N,N-dichlorotaurine are strong
Correspondence: Waldemar Gottardi, Institute for Hygiene
and Social Medicine, Medical Faculty, University of Innsbruck,
Fritz-Pregl-Strasse 3, A 6010 Innsbruck, Austria. Phone:
+43 512 507 3430, Fax: +43 512 507 2870, e-mail:
waldemar.gottardi@uibk.ac.at.
acids, their salts are completely dissociated. The abbrevia-
tions NCT and NDCT, therefore, concern the anions ClHN–
CH2–CH2–SO–3 and Cl2N–CH2–CH2–SO–3, respectively, which
are responsible for the reactions quoted in this paper. The
solid sodium salt is specified by NCT-Na.

412 Gottardi and Nagl
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
not catalysed by moisture as could be observed with a
specimen thoroughly purified by re-crystallisation and
kept in an evacuated and sealed glass-bulb. After
3.5 years at room temperature without light protection
the oxidation capacity had completely vanished though
the crystals exhibited no macroscopic alteration. Since
taurine (by re-crystallisation from water) and cy-
anomethane-sulfonic acidb (by extraction with hot meth-
anol) could be isolated from the strongly acidic product,
a decomposition according Eq. (1) seems plausible (see
also “Discussion”).
Results
Properties of the sodium salt
NCT-Na forms column like spears (re-crystallised from
methanol/ethanol) which decompose at 130–135 °C.
Long-term tests reveal an outstanding stability com-
pared to other N-chloroamino acids which exist only in
solution and for a short time [9]. Upon storage at 2–4 °C
the salt loses only 10 % of its oxidation capacity during
one year, while the same decrease needs approximately
two years if it is kept at –20 °C (Figure 1a).
2 ClHNCH2CH2SO3Na → H3N+CH2CH2SO3– +
The loss of oxidation capacity is connected with the ap-
pearance of a nitrile band at 2265 cm–1.The reaction is
N≡C-CH2-SO3H + 2 NaCl
(1)
NCT-Na is readily soluble in water (1.4 g/mL) and dime-
thyl sulfoxide (0.43 g/mL), poorly soluble in alcohols
(methanol:0.04 g/mL, ethanol:0.0076 g/mL), and insol-
uble in apolar solvents. The aqueous solution of NCT,
too, is distinguished by an outstanding stability showing
a decomposition rate of only 0.43 %/day at room temper-
ature which declines to ≈0.03 %/day and 10 %/year, re-
spectively, if kept at 2–4 °C (Figure 1 b). These results
are derived from iodometrically assayed loss of oxidation
capacity at the equilibrium pH 8.2 (see below).Based on
the absorbance at 250 nm, Antelo et al. [11] reported a
decrease of less than 1 % after 100 hours (< 0.24 %/
day) at pH 9.31, however, without indicating the temper-
ature of storage.
The dimethyl sulfoxide solution, on the other hand,
turned out to be less stable with a decrease of oxidation
capacity amounting to ≈11 % per day at room tempera-
ture, which is in contrast to previous statements that
chloramines in aqueous solution would not react with
dimethyl sulfoxide [12, 13].
Spectra
IR spectrum:The introduction of one chlorine atom gives
rise to a considerable simplification compared to the
spectrum of taurine, mainly in the N-H region where
NCT-Na shows only one sharp band at 3260 cm–1 typical
for secondary amines (Figure 2). Both spectra were re-
corded with KBr pellets.
UV Spectrumc: NCT-Na displays one peak at 251 nm
which is characteristic for the chromophore NHCl (Fig-
Figure 1. Stability of NCT (mean values of three meas-
urements). (a) crystalline NCT-Na at room temperature
(-ᮀ-), 2–5 °C (-᭛-) and -20 °C (-᭺-); the difference be-
tween all curves was significant (mean values ± S.D.,
P < 0.01, t test). (b) 0.75 % (w/v) aqueous solution of
crude (-᭺-) and recrystallised NCT-Na (-᭝-) without buff-
er at 2–5 °C; P > 0.05 (t test). Variation coefficients
≤ 1.5 % (not shown).
b
A
reference sample of cyanomethane-sulfonic acid
(νCN = 2266 cm–1) was prepared from chloroacetonitrile and
sodium sulfite [10].
c
The UV spectrum of NCT in neutral and acidic solution was
first published by Zgliczynski and Stelmaszynska [14] who
found peaks at 250 and 300 nm which they assigned to NCT
and NDCT, respectively. The peak of NDCT at 206.6 nm,
however, was not detected.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
Chemical Properties of N-Chlorotaurine Sodium 413
Figure 2. IR spectra of taurine and N-chlorotaurine sodium, 4000–370 cm–1 (KBr pellets).
Figure 3. UV spectra of NCT (cell length 0.10 cm). (a) NCT-Na in water (0.04438 M). (b) NCT-Na in 0.5 N H2SO4
(0.0219 M); overlay-spectra, scanning time 5 min.

414 Gottardi and Nagl
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
ure 3 a).The exact parameters are λmax = 250.9 nm and
ε = 397.4 L mol–1 cm–1, while Carr et al. [15] found
ε252 = 429 L mol–1 cm–1.The valley at 215 nm becomes
important for verifying the absence of NDCT or other im-
purities which manifests itself in a ratio of A250.9 : A215 > 4
which is the value for pure NCT. If disproportionation is
provoked by addition of acid (see below), overlay-spec-
tra with two isosbestic points at 240.0 and 285.5 nm can
be obtained (Figure 3 b). The two peaks at 206.6 and
302.1 nm are characteristic for NDCT. Because of these
specific differences, the actual concentrations of NCT
and NDCT during disproportionation can be assayed by
means of UV spectroscopy (see “Materials and
Methods”).
Mass spectrum: Using chemical ionisation the most im-
portant fragments were HCl+, ClH=CH2+, SO2+, and
NHCH2CH+2, while the molecular peak was not found.
This was possible using electrospray ionisation MS
which showed clearly the mass peaks of M+ and M+ – H
at 181.1 and 180.1 dalton.
13C-NMR spectrum:NCT-Na dissolved in D2O shows two
methylene singlets at 51.8 and 50.0 ppm (Bruker AM
300, operating at 75.47 MHz with dioxane as internal
standard), whereas Lin et al. [8] reported a pair of sin-
glets at 53.1 and 51.3 ppm, respectively, showing the
same difference of 1.8 ppm. Since the differences of the
absolute values can be attributed to the different operat-
ing parameters – Lin et al. [8] used an external standard
– there is no doubt that we are dealing with the same
compound in both cases.
Figure 4. Change of pH in fresh aqueous solutions of
NCT-Na. (a) equilibration of 5.85 (-᭺-), 16.7 (-᭝-), and
54.6 (-᭛-) mM in normal atmosphere. Each point repre-
sents a single measurement.P < 0.01 (ANOVA).(b) equi-
libration of 8.6 mM under nitrogen.
HPLC: In contrast to aqueous solution, a chromatogram
with only one peak, indicating absence of the reaction
products of disproportionation, was obtained in meth-
anolic solution.
also be another origin of pH decrease, probably the
following elimination reaction leading to an imine (See
also “Discussion”).
Hydrolysis
ClHN–CH2–CH2–SO3–
Upon dissolution in water an alkaline reaction arises im-
mediately which can be explained by hydrolysis
(Eq. (2)).
→ HN=CH–CH2–SO–3 + H+ + Cl–
(3)
The extrapolated initial pH of weighed samples of NCT-
Na dissolved in CO2-free H2O allowed an estimation of
the equilibrium constant of Eq. (2) which comes to pKb =
[NCTH+] aOH–/[NCT] = 7.9 ± 0.2 (25 °C).
ClHN–CH2–CH2–SO–3 + H2O
↔ ClH2N+–CH2–CH2–SO3– + OH–
(2)
The initial pH depends on the concentration and ranges
between approx.8.8 and 9.3 in 0.1 % and 1.0 % (w/v) so-
lution, respectively.However, the pH immediately begins
to decrease and settles at pH 7.1 and 8.2 within 0.2 and
5 h and remains constant for extended periods (Fig-
ure 4 a).This equilibration is a result of the acidifying ac-
tion of airborne CO2 and the proton consuming dispro-
portionation (see below).The main contribution to acidifi-
cation comes from CO2 as the course of pH under nitro-
gen flushing shows (Figure 4 b). However, there must
Disproportionation
NCT equilibrates in aqueous solution by an auto-chlorin-
ation forming NDCT and taurine, a reaction which con-
sumes protons (Eq. (4))
2 ClHNCH2CH2SO–3 + H+
↔ Cl2NCH2CH2SO–3 + H3N+CH2CH2SO–3
2 NCT + H+ ↔ NDCT + taurine
(4)

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
Chemical Properties of N-Chlorotaurine Sodium 415
The disproportionation constant was found to be
Kd = [NDCT][taurine]/ fa [NCT]2 aH+
= (4.5 ± 0.8) × 106 M–1
(5)
which was derived from measurements in the pH range
of 4.0–7.4.The equilibrium concentration of NCT, calcu-
lated with Kd over the whole pH range is shown in
Figure 5. Based on NCT solutions made from 0.14 M
NaOCl and 0.1 M taurine in the presence of 0.5 M
NaClO4, Antelo et al. [11] found a somewhat lower value
of Kd = (1.07 ± 0.15) × 106 M–1.
The position of the equilibrium is influenced by the de-
gree of acidity. The results of kinetic measurements are
shown inTable 1, which reveals a second order reaction:
rate = –d[NCT]/dt = kr [NCT]2 dm3 mol–1 min–1
The observed rate constants are pH dependent and fol-
low the regression line of Eq. (6).
log kr = (3.83 ± 0.14) – (0.52 ± 0.03) × pH
(6)
Since they increase substantially with acidity, the partici-
pation of the protonated form NCTH+ as the real chlorin-
ating agent (Eq. (7)) seems plausible.
NCT + NCTH+ ↔ NDCT + taurine
(7)
The same mechanism was also presumed for the chlo-
rine transfer from inorganic chloramine to amino acids
where the active form is NH3Cl+ [16].
Figure 5. Disproportionation of NCT. (a) Calculated equi-
librium concentrations of NCT (—) and NDCT (- - -) as well
as reaction rate (᭺) in dependence on pH. (b) Measured
disproportionation of 2 % (w/v) NCT in 0.1 M phosphate
at room temperature (actual pH: 7.00 → 7.35).
From the observed second order rate constants the half-
reaction times for the disproportionation of NCT solu-
tions can be estimated with t0.5 = k–r 1 a–1 [17] where a
denotes the initial concentration.Table 1 shows impres-
sively that the t0.5 values decrease with acidity and in-
crease with dilution, ranging from 0.3 s (1 % [w/v] NCT,
pH 0.3) to 8.0 h (0.1 % [w/v] NCT, pH 7.8).
Table 1. Kinetic data of disproportionation at 25.0 °C.
pH Rate constant kr
Half-reaction timea
1.0% NCT 0.1 % NCT
rb
dm3 mol–1 min–1
However, it takes much longer to completely establish
the disproportionation equilibrium, mainly in a neutral or
weakly alkaline medium. Thus, at an initial pH of 7.00
(0.1 M phosphate, final pH 7.35) the equilibrium was at-
tained after approx. 2 h with 2 % NCT (Figure 5b) while
t0.5 comes to 8 min.
0.3
2.5
2.9
3.1
3.7
4.7
6.5
7.1
7.8
3600c
886d
177d
108d
0.3 s
1.2 s
6.2 s
10.1 s
17.4 s
46.4 s
4.8 min 49 min
12 min
48 min
3 s
12 s
1.0 min
1.7 min
2.9 min
7.6 min
0.97
0.999
0.997
0.987
0.9994
1
0.9998
0.999
0.998
62.1d
23.5d
3.8e
As a result of disproportionation, aqueous solutions of
NCT at pH < 8 are always mixtures of the three com-
pounds NCT, NDCT, and taurine. However, at pH ≈ 8.2,
which is the equilibrium pH of pure and not buffered 1 %
(w/v) NCT solution equilibration proceeds very slowly
and even after one day at room temperature virtually no
NDCT could be detected. In freshly prepared weakly al-
kaline solutions, therefore, the portion of the reaction
products NDCT and taurine can be neglected.
1.5e
2.0 h
8.0 h
0.38e
a
Calculated for 1 % and 0.1 % (w/v) NCT solution (0.055
resp. 0.0055 M) after t0.5 = 1/kr[NCT] [17];
correlation coefficient (goodness of fit of linear regres-
sion:time versus 1/[NCT] plot for second order kinetics);
0.5 M HClO4;
b
c
d
e
Reaction Eq. (4) is reversible as the disproportionated
mixture comproportionates if the pH is raised to neutral
0.5 M citrate;
0.5 M phosphate.

416 Gottardi and Nagl
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
or weakly alkaline values. It becomes important at
pH 6–8 while it is very slow in a more alkaline environ-
ment. Probably it is a two stage reaction with a primary
hydrolysis step forming HOCl, which is the actual agent
which transfers the halogen to taurine, a reaction which
increases acidity:
Cl2NCH2CH2SO–3 + H2O
↔ ClHNCH2CH2SO–3 + HOCl
HOCl + H3N+CH2CH2SO–3
↔ ClHNCH2CH2SO–3 + H3O+
Because of the inductive effect of the second chlorine at-
om in NDCT, the hydrolytic formation of HOCl by splitting
off Cl+ seems to be plausible.
However, it was not possible to establish a kinetic pattern
for comproportionation as clear as that shown for dispro-
portionation.
Oxidation of thio compounds (sulfhydryls,
thioether)
Compounds containing SH groups like cysteine are im-
mediately oxidised, upon which disulfides (Eq. (8)) and
sulfur-oxygen acids (Eq. (9)) are formed:
2 R–SH + NCT → R–SS–R + taurine + Cl–
R–SH + n NCT + n H2O
(8)
Figure 6. Reaction of NCT with thio-compounds. (a)
Temporal decrease of oxidation capacity of 100 mM
NCT in the presence of 50 mM cysteine at pH 7 and dif-
ferent modes of mixing (addition drop by drop):-᭡- addi-
tion of cysteine to NCT; -᭿- addition of NCT to cysteine.
(b) Temporal decrease of oxidation capacity of each
150 mM NCT (-ᮀ-) and CAT (-᭝-) in the presence of
50 mM methionine at pH 7.0 (0.2 M phosphate buffer).
Each point represents the mean ± S.D. of three inde-
pendent experiments; P < 0.01 (t test).
→ R–SOnH + n taurine + n Cl–
n = 1,2,3
(9)
Plausible intermediates are unstable S-Cl compounds
which react either with another thiol molecule or with
water:
NCT + R–SH + H2O → taurine + [R–S–Cl] + OH–
+ R–SH → R–SS–R + H+ + Cl–
[R–S–Cl]
Ά
+ H2O → R–SOH + H+ + Cl–
molar ratio also the mode of mixing the reactants has a
great influence. Figure 6a reveals a drastic difference in
the decrease of oxidation capacity depending upon
whether the NCT solution was added slowly (drop by
drop) to the cysteine solution or if it was done in the re-
verse order. The first, very fast breakdown of oxidation
capacity can be attributed to Eq. (8) which correlates
largely with the amount of precipitating cystine.
R–SOH + NCT + H2O → R–SO2H + taurine + Cl–
R–SO2H + NCT + H2O → R–SO3H + taurine + Cl–
Taking into account both the decrease of oxidation ca-
pacity and the amount of precipitated insoluble cystine
reveals that NCT reacts with cysteine already at room
temperature in both directions at a high rate with the re-
sult that at least four reaction products, disulfides, sulfen-
ic, sulfinic, and sulfonic acids are possible with a ratio of
NCT to cysteine ranging from 1 : 2 to 3 : 1. Besides the
Thioethers like methionine predominantly interact only
with one NCT molecule at pH 7 which is consistent with
formation of the corresponding sulphoxide [18].

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
NCT + CH3–S–R + H2O
Chemical Properties of N-Chlorotaurine Sodium 417
The reaction is accompanied with the formation of the
deep blue color of indophenol created by the secondary
decomposition product NH2Cl (emerging at the reaction
of NH3 with NCT, see Eqs.(3), (15), and (11)) which com-
bines with phenol (Berthelot reaction) [19]. CAT reacts
much faster under the same conditions, however, with-
out producing the blue colour of the Berthelot reaction.
→ taurine + CH3–SO–R + Cl–
R = CH2CH(NH2)COOH
(10)
The immediate decrease of oxidation capacity shown in
Figure 6b uncovers a stoichiometric transformation with
NCT according to Eq. (10). For chloramine T (CAT)
which was used for comparison as a more reactive oxi-
dant, this stoichiometric feature is not shown and the oxi-
dation might go towards a sulfone. There follows a sec-
ond reaction with a substantially slower rate for both
agents which can be attributed to the typical decomposi-
tion of N-chloroaminocarbonic acids (see “Discussion”
and Figure 7d).
Another aromatic compound susceptible to halogenation,
the amino acid histidine, reacts very fast with NCT and
with a less pronounced difference to CAT (Figure 7 b).
Chlorination of N-H compounds (transhalo-
genation)
The first reference to transhalogenation was from Lin et
al. [8] who proved the chlorine transfer from NCT to ser-
ine. In contrast to more reactive oxidants like hypochlo-
rite, NCT equilibrates to an appreciable amount only with
Chlorination of aromatic C-H compounds
Phenol, which is known to be susceptible to substitution
by halogens, reacts with NCT very slowly (Figure 7a).
Figure 7.Reaction of NCT (-ᮀ-) and CAT (-᭝-) with phenol (a), histidine (b), ammonium chloride (c), and glycine (d).All
reactants equivalent to 1 % (w/v) NCT (55 mM), pH 7 (0.05–0.1 M phosphate buffer). Each point represents a single
measurement.The difference between NCT and CAT was significant (P < 0.01 for a, c, and d; P = 0.0104 for b; t test).

418 Gottardi and Nagl
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
NH3, organic amines and amino acids, but not with
amides and imides.
(i) Isolation of the N-chloro derivatives of α-aminocar-
bonic acids (which represent the majority of biologically
important amino acids) has not been possible until now
because of their inclination to decomposed. A plausible
explanation for the divergence in stability between the α-
and β-amino acids has its roots in the initial step of dis-
integration of N-chloroamino acids which concerns
elimination of HCl leading to an unstable imine as shown
in Eq. (13) for an N-chloro-α-amino acid [20].
NH2Cl-Formation: Contrary to the N-chloroamino acids
which are characterised by nearly the same UV absorp-
tion peak at ≈250 nm, the corresponding peak of NH2Cl
at 242 nm [16] diverges from NCT (λmax = 250.9 nm)
enabling a photometric assessment of the equilibration
with NH+4:
HClN–CH2–CH2–SO–3 + NH4+
↔ NH2Cl + H3N+–CH2–CH2–SO–3
NCT + NH+4 ↔ NH2Cl + taurine
(11)
(13)
Based on the inductive effect of the carboxylic group, the
elimination is favoured for the NHCl function in α-posi-
tion, while in a β-amino acid it is weakened by the inter-
vening CH2 group.
The equilibrium constant for this halogen transfer comes
at 25 °C to
KNH2Cl = [NH2Cl][taurine]/[NCT][NH+4] fa2
(ii) The fact that the N-chloro derivative of the β-amino
acid taurine is easily accessible, while it is not possible
with β-alanine – all attempts of its synthesis resulted in
non-oxidising and non-definable material – is more diffi-
cult to understand. Molecular models of NCT-Na and N-
chloro-β-alanine sodium do not explain this divergence.
The key, however, might be the difference in acidity of the
–COOH and the –SO3H groups which comes to nearly
six powers of ten in the case of their methyl derivatives
(CH3COOH: pKa = 4.75; CH3SO3H: pKa = –1.20 [22]).
= 0.02 ± 0.004
(12)
The NH2Cl equilibrium concentrations calculated by
Eq. (12) disclose that 0.1 % (1.0 %, 3.0 %) NCT produc-
es 1.1 mM (3.1 mM, 4.7 mM) NH2Cl in the presence of
0.1 % (18.7 mM) ammonium chloride. The according
values were 2.4 mM (8.7 mM, 14.7 mM) NH2Cl for
1.0 % (187 mM) ammonium chloride.Upon this reaction,
there is virtually no loss of oxidation capacity in the pres-
ence of NCT, which is contrary to CAT (Figure 7 c).
Reaction with glycine:There is a significant decrease of
oxidation capacity in the presence of glycine (Fig-
ure 7 d), indicating an equilibration to the unstable N-
chloroglycine:
NCT + H2N–CH2–COOH + H+
→ taurine + ClHN–CH2–COOH
Therefore, it seems plausible that the sodium salt of N-
chloro-β-alanine does not only exist in the form A – which
dominates in NCT-Na according to the IR spectrum – but
also in the tautomeric form C which can be formed by a
rearrangement via the cyclic transition state B.
The significantly faster decrease in the presence of CAT
points to a considerably increased degree of conversion
to N-chloroglycine.
Discussion
By detaching chloride from structure C (formation of
NaCl by an intramolecular redox reaction) a nitrene
would remain whose trend to undergo transmutation is
known. The non-oxidising products emerging from the
reaction of CAT with β-alanine as well as the evidence of
chloride therein at least support this explanation.
The unique status of N-chlorotaurine
It is surprising that NCT-Na represents until now the sole
N-chloroamino acid which has been isolated as a pure
compound.Its outstanding stability compared to other N-
chloroamino acids can be quoted as a main explanation
for this feature.However, also the impossibility of prepar-
ing the sodium salt of N-chloro-β-alanine (Gottardi, un-
published) is difficult to understand all the more the com-
pound exhibits a stability in aqueous solution which sug-
gests its isolation is feasible [20].
Based on these considerations, the outstanding stability
of NCT can be reduced to its structure as a β-aminosul-
fonic acid.However, this assertion concerns only its salts
because the free acid does not exist on grounds of rapid
disproportionation.
As reasons for these discrepancies can be quoted items
of structure, (i) the position of the amino function (α- or
β-amino acid), and (ii) the nature of the acid function
(carbonic or sulfonic acid).
d
This assertion concerns only the free N-chloroamino acids,
while the N-chloro derivatives of diverse esters of α-amino-
isobutyric acid turned out to be sufficiently stable to be
synthesised [21].

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
Chemical Properties of N-Chlorotaurine Sodium 419
Another feature illustrating the unique status of NCT
concerns the feasibility of its synthesis, which was not
possible for N-chloro-γ-aminopropanesulfonic acid, the
homologue of NCT (Gottardi, unpublished). Obviously,
the successful and probably simultaneously occurring
exchange of sodium and chlorine for two hydrogen at-
oms during the one-stage synthesis of NCT-Na
(Eq. (14)) from chloramine-T and taurine in ethanolic so-
lution needs a special structural condition which is not
present in γ-aminopropanesulfonic acid.
For this halogen transfer between two amine com-
pounds which is characterised by retention of oxidation
capacity, the term “transhalogenation” has been
introduced [24] to distinguish it from other halogena-
tions, e. g.of C–H and S–H compounds, where oxidation
capacity is lost.
A mechanistic approach could imply a nucleophilic at-
tack of the free electron pair of the amino group of the ac-
ceptor molecule R1-NH2 towards the chlorine atom of the
protonated donor molecule R2–NH2Cl+:
CH3–C6H5–SO2NClNa + H3N+C2H4SO3–
→ NCT–Na + CH3–C6H5–SO2NH2
(14)
Routes of decomposition of NCT
– Aqueous solution: There are two categories of reac-
tions occurring in aqueous solution, with retention of and
with loss of oxidation capacity.To the first belong hydroly-
sis (Eq. (2)) and disproportionation (Eq. (4)), to the lat-
ter spontaneous decomposition launched by elimination
of HCl forming an imine and subsequent hydrolysis to an
aldehyde and ammonia. In case of NCT decomposition
to sulphoacetaldehyde (Eq. (3) and Eq. (15)) has al-
ready been proved to be a possible route for mammalian
taurine metabolism [23].
The facility of transhalogenations seems to be funda-
mental for the operation of the immune system. During
an oxidative burst numerous reactions take place when
oxidation capacity in the form of HOCl is produced which
is absorbed immediately by amino compounds present
in the intra- and extracellular domain.NCT, because of its
superior stability and the high concentration of taurine, is
an important partner for transhalogenation reactions.
HN=CH–CH2–SO–3 + H2O
A special case of transhalogenation concerns dispropor-
tionation at pH < 8 which causes the presence of taurine
and NDCT at concentrations varying with pH and the
time elapsed since dissolution.
→ O=CH–CH2–SO–3 + NH3
(15)
The iodometric assessment of stability of NCT-Na
(Figure 1 b) complies with such an elimination because
aldehydes do not oxidise iodide. Furthermore, the very
sensitive Berthelot reaction (see above) originating from
a cascade including Eq. (3), Eq. (15), and Eq. (11) may
serve as an evidence for the development of NH3.
NCT as a weak oxidant
The presented reactions reveal NCT to be an agent
which is much less oxidising than CAT. This difference
was also observed in a study on the consumption of both
oxidants by organic material like human plasma and fetal
calf serum [25]. Based on another publication [26]
which deals with the interaction of peptone with a series
of halogen compounds (including several N-chloro and
N-bromo compounds as well as iodine, bromine and hy-
pochlorite), the following ranking of active chlorine com-
pounds has been established: hypochlorite > trichloro-
isocyanuric acid > 1,3-dichlorohydantoin > chloramineT.
According to all of these results, NCT represents the
weakest oxidising active chlorine compound accessible
for use in practice.
– Solid NCT-Na: A somewhat differing decomposition
mechanism can be discussed, probably a disproportion-
ation in the solid state according to Eq. (16) (with the so-
dium salts of NDCT and taurine as intermediates) which
precedes the formation of cyanomethanesulfonic acid
by elimination of two molecules of HCl from NDCT-Na
(see the overall reaction Eq. (1)).
2 ClHNCH2CH2SO3Na
→ Cl2NCH2CH2SO3Na + H2NCH2CH2SO3Na (16)
Transhalogenation
Because N–Cl bonds are easily formed and split, the
equilibrium (Eq. (17)) is established already at room
temperature with considerable speed.
N-chlorotaurine sodium – a novel disinfectant
in human medicine
The unique stability and low reactivity of NCT prove to be
the requirements for its function in the mammalian de-
R1–NHCl + R2–NH2 → R1–NH2 + R2–NHCl
(17)

420 Gottardi and Nagl
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
E302.4 = E2 = [NCT] × ε1 + [NDCT] × ε2
E250.9 = E1 = [NCT] × ε3 + [NDCT] × ε4
which can be transformed to:
fence system as a long-lived oxidant [1]. Moreover,
these features are fundamental for the optimal compro-
mise between microbicidal activity and low toxicity dem-
onstrated in previous studies concerning the in vitro
bactericidal [5, 9, 24], fungicidal [27], and virucidal
activity [28], as well as its outstanding tolerability as an
antiseptic [29–31]. Not least because of its availability in
the form of the sodium salt NCT can be noted as a prom-
ising new disinfectant in human medicine.
[NCT] = (E1 – ε4 × E2/ε2)/(ε3 – ε1 × ε4/ε2)
[NDCT] = (E2 – ε1 × E1/ε3)/(ε2 – ε1 × ε4/ε3)
The molar absorption coefficients were measured with a Lamb-
da 20 UV/VIS Photometer from Perkin Elmer.To avoid any dis-
proportionation, NCT-Na was dissolved in aqueous 0.01 M
Na2HPO4 solution which was adjusted with NaOH to pH 9.7.
Since taurine shows no absorption at >250 nm, a NCT-Na
sample completely disproportionated in 2N H2SO4 could be
used as a NDCT standard.
Conclusions
Using the pure crystalline sodium salt we were able to
elucidate the behaviour of NCT, both apart from and in
the presence of reactants. In the first case we could (a)
ascribe the outstanding stability to its structure as a
β-aminosulfonic acid and (b) attribute a weakly alkaline
environment necessary to keep an NCT solution free
from the reaction products of disproportionation. The
main feature concerning reactions might be the ease of
transhalogenation which enables an equilibration with
proteinaceous N–H compounds already at room
temperature, a feature important in view of the role of N-
chloro compounds in the human defence system.The re-
actions established for NCT can be transposed to the
bulk of N-chloro-α-amino acids emerging (besides NCT)
during the oxidative burst which, however, can not be in-
vestigated in substance because of their instability.
The concentration of the NCT and NDCT solution was verified
by iodometric titration.
302.4 nm: εNCT = ε1 = 22.4 L mol–1 cm–1,
εNDCT = ε2 = 332.9 L mol–1 cm–1
250.9 nm: εNCT = ε3 = 397.4 L mol–1 cm–1,
εNDCT = ε4 = 220.1 L mol–1 cm–1
Estimation of pKb
From Eq. (2) follow the mass balances
c(NCT) = [NCTH+] + [NCT] = C
and
[OH–] = [NCTH+]
which allow the definition
pKb = [NCTH+] aOH–/[NCT] = [OH–]2/(C-[OH–])
2
2
≈ KW /C(aH+)2f a
Acknowledgements
The course of pH was scanned for ten min when weighed sam-
ples of NCT-Na (50–200 mg) were dissolved at zero time in
10.0 mL portions of stirred distilled water at 25.0 °C.The pH at
zero time was estimated by graphic extrapolation.
This study was supported by the Austrian Science Fund
(grant P15240-MED) and by the Jubilee Research Fund
of the Austrian National Bank (grant 8366). We thank
Prof. J. Schantl, Institute for Organic Chemistry, for help-
ful contributions and Magdalena Hagleitner for excellent
technical assistance.
The pKb was 7.9 ± 0.2 (mean value ± standard deviation,
N = 19).
Evaluation of the disproportionation constant
From Eq. (4) follows [NDCT] = [taurine], and Eq. (5) can be re-
arranged to
Experimental part
Chemicals
Kd = [NDCT]2/fa[NCT]2aH+.
N-Chlorotaurine sodium (NCT-Na) was purchased from Gatt-
Koller GmbH, 6067 Absam, Austria. N-Chloro-4-toluenesulfon-
amide-sodium (CAT) and buffers were from Merck, while the
amino acids were from Sigma. All reagents were of the highest
available purity.All concentrations in percent are weight per vol-
ume (w/v).
A solution of 0.0058 mol/L NCT-Na (0.63 g in 600 mL) contain-
ing 0.05 M phosphate buffer was divided in 6 parts which were
adjusted to initial pH between 4.0 and 7.4 and kept in the dark
at room temperature. Using the UV spectrum the progress of
disproportionation (see above) was monitored for several days
until no further change could be observed. From the final
pH values between 5.24 and 7.45 and the photometrically as-
sessed equilibrium concentrations of NCT and NDCT the dis-
proportionation constant was found to be Kd = 4.5 ± 0.8 × 106;
N = 6
Assessment of oxidation capacity
Iodometric titrations were performed with 0.100 M thiosulfate
at pH 2–3 (acetic acid) using the automatic titration assembly
TIM900 from Radiometer, Copenhagen.
Photometric analysis of aqueous NCT, NDCT, and mixtures
thereof
Kinetic measurements
The pH dependence of disproportionation was investigated in
the pH range of 0.3–7.8 using 0.5 M of HClO4 (pH 0.3), citrate
buffer (pH 2.5–4.7), or phosphate buffer (pH 6.5–7.8). One
The absorptions at 302.4 nm (λmax of NDCT) and 250.9 nm
(λmax of NCT) measured with a 1-cm cuvette were:

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 411–421
Chemical Properties of N-Chlorotaurine Sodium 421
[14] J. M. Zgliczynski, T. Stelmaszynska, J. Domanski, W. Os-
compartment of a double chamber cell (each 0.437 cm) was
filled with 1 mL of a NCT-Na solution (6.0–9.0 × 10–3 M) in wa-
ter and the other with 1 mL of the 1 M buffer solution. After
10 min at 25.0 °C in the thermostatted cell holder the first scan
was made. It was used for the calculation of the [NCT] at t = 0.
Then the contents of both compartments were mixed and
scanned at appropriate times (max 60 min).The NCT concen-
trations in the resulting NCT/NDCT mixtures were calculated
as described above. Since the plots of 1/[NCT]i against ti were
linear for the different pH values, the disproportionation reac-
tion is of second order with slopes indicating the observed rate
constants (Table 1).
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[17] H. E. Avery, Basic Reaction Kinetics and Mechanisms,
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[18] D. S. Mahadevappa, S. Ananda, N. M. M. Gowda, K. S.
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