Hydroxylamine as an oxygen nucleophile: Substitution of sulfonamide by a hydroxyl group in benzothiazole-2-sulfonamides

Article abstract of DOI:10.1039/c2ob26929e

Benzothiazole-2-sulfonamides react with an excess of hydroxylamine in aqueous solutions to form 2-hydroxybenzothiazole, sulfur dioxide, and the corresponding amine. Mechanistic studies that employ a combination of structure-reactivity relationships, oxygen labeling experiments, and (in)direct detection of intermediates and products reveal that the reaction proceeds via oxygen attack, and that oxygen incorporated in the 2-hydroxybenzothiazole product derives from hydroxylamine. The reaction, which is performed under mild conditions, can be used as a deprotection method for cleavage of benzothiazole-2-sulfonyl-protected amino acids.

Full text of DOI:10.1039/c2ob26929e

Organic&
BiomolecularChemistry
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Volume 11 | Number 7 | 21 February 2013 | Pages 1073–1260
ISSN 1477-0520
PAPER
Jasmin Mecinović et al.
Hydroxylamine as an oxygen nucleophile: substitution of sulfonamide by a
hydroxyl group in benzothiazole-2-sulfonamides

Organic &
Biomolecular
Chemistry
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Hydroxylamine as an oxygen nucleophile: substitution
of sulfonamide by a hydroxyl group in benzothiazole-
2-sulfonamides†
Cite this: Org. Biomol. Chem., 2013, 11,
1103
Jos J. A. G. Kamps, Roman Belle and Jasmin Mecinović*
Benzothiazole-2-sulfonamides react with an excess of hydroxylamine in aqueous solutions to form
2-hydroxybenzothiazole, sulfur dioxide, and the corresponding amine. Mechanistic studies that employ a
combination of structure–reactivity relationships, oxygen labeling experiments, and (in)direct detection
of intermediates and products reveal that the reaction proceeds via oxygen attack, and that oxygen
incorporated in the 2-hydroxybenzothiazole product derives from hydroxylamine. The reaction, which is
performed under mild conditions, can be used as a deprotection method for cleavage of benzothiazole-
2-sulfonyl-protected amino acids.
Received 1st October 2012,
Accepted 26th November 2012
DOI: 10.1039/c2ob26929e
www.rsc.org/obc
aldehydes or ketones and hydroxylamine to form oximes. In
Introduction
addition, hydroxylamines react with α,β-unsaturated esters to
Hydroxylamine is an ambident α-effect nucleophile1 that exists form Michael adducts exclusively via the N-attack,3 and with
in aqueous solutions as a mixture of four species: neutral α-ketoacids to form amide bonds under mild reaction con-
(NH2–OH), zwitterionic (NH3 –O−), protonated (NH3 –OH), ditions.4 Reactions in which hydroxylamine reacts with electro-
and deprotonated (NH2–O−) (Scheme 1).2 A combination of philes via oxygen attack are rare. For instance, recent studies
structure–reactivity studies that examined the leaving abilities by Kirby, Nome and co-workers illustrated that hydroxylamines
of the alkoxy group from ethers, and the free energy for the react with phosphate esters via O-attack to form O-phosphory-
ionization of hydroxylamine employing the linear free energy lated intermediates that are further hydrolysed in the presence
relationship (LFER) demonstrated that the zwitterionic form of of an excess of hydroxylamine.5–7 Another reaction investigated
hydroxylamine represents about 20% of all the hydroxylamine in detail, employing the kinetic isotope effect, is the formation
+
+
in the aqueous solution at neutral pH.2
of O-acylhydroxylamine from p-nitrophenylacetate.8–10 Herein,
Hydroxylamine usually reacts with electrophiles through its we describe that hydroxylamine reacts with benzothiazole-2-
nitrogen atom. The most widely used reaction in organic sulfonamides in aqueous media in which hydroxylamine acts
chemistry employing hydroxylamine is the reaction between as an oxygen nucleophile.
Results and discussion
During the evaluation of arylsulfonamides as ligands for
binding to carbonic anhydrase11 and CS2 hydrolase,12 we
observed that an excess of hydroxylamine in buffered water
solutions caused an apparent degradation/reaction of benzo-
thiazole-2-sulfonamides.
Initially, we investigated the reaction between benzothia-
Scheme 1 Equilibria of hydroxylamine in aqueous solutions.2
zole-2-sulfonamide (BTA) and hydroxylamine. BTA is converted
to 2-hydroxybenzothiazole in the presence of 50 equiv. of
Institute for Molecules and Materials, Radboud University Nijmegen,
hydroxylamine in water at pH 9 in 100% yield after 3 hours at
Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
room temperature (Table 1, entry 1, and Fig. 1). The progress
E-mail: j.mecinovic@science.ru.nl
†Electronic supplementary information (ESI) available: Additional experimental
of the reaction was monitored by real-time 1H NMR spec-
troscopy (Fig. 1). The formation of 2-hydroxybenzothizole was
confirmed by doping experiments in which the authentic
information, including synthetic procedures, labeling experiments, NMR spectra
and LC-MS analyses. See DOI: 10.1039/c2ob26929e
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Table 1 Screening of HONH2 derivativesa
We then examined the reactivity of other simple hydroxyl-
amine analogues and derivatives. Ammonia, hydrazine, or
hydrogen peroxide did not react with BTA under standard reac-
tion conditions (Table 1, entries 4–6). To investigate whether
hydroxylamine reacts with BTA via its oxygen or nitrogen atom,
we tested both O- and N-substituted hydroxylamines as poten-
tial reagents for the substitution reaction (Table 1, entries
7–12). O-Methylhydroxylamine and hydroxylamine-O-sulfate
did not convert BTA to any observable product (2-hydroxyben-
zothiazole or potential 2-aminobenzothiazole), implying that
the unsubstituted oxygen atom is essential for the reactivity.
N-Methylhydroxylamine, however, reacted with BTA, although
more slowly than hydroxylamine, to form O-(benzothiaz-2-yl)-
N-methylhydroxylamine and 2-hydroxybenzothiazole. In con-
trast, N,N-dimethylhydroxylamine and trimethylammonium
oxide did not yield any product in the presence of BTA.
Because the reaction of BTA and hydroxylamine occurs in
slightly basic conditions, we performed control experiments
under the same conditions in the absence of hydroxylamine
Entry
Reagent (equiv.)
Time (h)
Conversionb (%)
1
2
3
4
HONH2 (50)
HONH2 (10)
HONH2 (100)
NH3 (665)
3
5
1
100
100
100
n.d.c
n.d.
n.d.
n.d.
n.d.
86d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
<10
48
48
48
48
48
24
48
48
48
48
48
48
48
5
6
H2NNH2 (50)
H2O2 (50)
7
8
9
10
11
12
13
14
15
16
CH3ONH2 (50)
H2NOSO3H (50)
HONHCH3 (50)
HON(CH3)2 (50)
ON(CH3)3 (50)
CH3ONHCH3 (50)
H2NCH2CH2OH (50)
NaOCl (10)
NaOHe
NaOHf
1
(Table 1, entries 15–16): no product was observed by H NMR
a Standard reaction conditions: BTA (20 mM), reagent (50 equiv., 1 M),
H2O, pH = 9–10. b Conversion determined by 1H NMR and LC-MS.
c n.d. = not detected. d A mixture of 2-hydroxybenzothiazole and
O-(benzothiaz-2-yl)-N-methylhydroxylamine. e pH ∼ 9. f pH ∼ 12.
at pH 9 and traces of the product at pH 12. Taken together,
these experiments suggest that an unsubstituted oxygen of
hydroxylamine is required for an efficient reaction with BTA,
hence proposing the mechanism that likely involves an initial
nucleophilic attack of the hydroxyl group.
We were then interested in knowing whether other N-substi-
tuted hydroxylamine analogues that possess a free OH group
also exhibit reactivity towards BTA (Scheme 2). Butyloxime, pyri-
dine-N-oxide, and 2-hydroxypyridine were inert towards BTA.
Interestingly, hydroxyurea showed a comparable reactivity to
hydroxylamine, quantitatively producing 2-hydroxybenzothia-
zole in 6 hours at room temperature (also in 1 hour at 70 °C).
N-Methylhydroxyureas possessed a significant decrease in reac-
tivity whereas O-methylhydroxyurea was found to be inactive.
Acetohydroxamic acid reacted with BTA in 8 hours under
Fig. 1 The progress of the hydroxylamine-mediated substitution of a sulfona-
mide group from benzothiazole-2-sulfonamide (BTA) monitored by 1H NMR.
BTA in the presence of 50 equiv. of hydroxylamine at 25 °C after (A) 10 minutes;
○
●
represents
(B) 30 minutes; (C) 1 hour; (D) 3 hours.
2-hydroxybenzothiazole.
represents BTA and
sample of 2-hydroxybenzothiazole was added into the reaction
mixture after the completion of the reaction: no new signal
appeared in the 1H NMR spectrum. LC-MS analysis of the reac-
tion product also provided evidence of the formation of
2-hydroxybenzothiazole (retention time 13.94 minutes, M + H+
= 152.16). The completion times in the presence of 10 equiv.
and 100 equiv. of hydroxylamine were 5 h and 1 h, respectively
(Table 1, entries 2–3). The lack of solubility of BTA at pH 6–8
restricted us from checking the progress of the reaction at pH
values close to neutral. In addition, due to the explosive poten-
tial of hydroxylamine, reactions at higher temperatures were
not pursued.
Scheme 2 Analogues of HONH2 used in the standard substitution reaction.
(a) Conversion determined by 1H NMR and LC-MS; (b) n.d. = not detected;
(c) at 70 °C.
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Table 2 The effect of the leaving group
Entry
Substrate
Time
Conversiona (%)
1
2
3
4
5
6
7
8
9
SO3Na
SOMe
SO2Me
COOH
COOMe
CONH2
F
Cl
NH2
SMe
48 h
12
30 min
30 min
48 h
48 h
48 h
48 h
48 h
48 h
48 h
100
100
n.d.b
n.d.
n.d.
100
100
n.d.
n.d.
10
Scheme 3 The scope of the reaction. (a) Conversion determined by 1H NMR
a Conversion determined by 1H NMR and LC-MS. b n.d. = not detected.
and LC-MS; (b) n.d. = not detected.
very poor substrate for the reaction with hydroxylamine, yield-
ing only 12% of 2-hydroxybenzimidazole. The observed limited
reactivity of benzimidazole-2-sulfonamide relative to benzo-
thiazole-2-sulfonamide can be rationalized by a substantial
decrease in the electrophilic character of C-2 (nitrogen’s elec-
trons in 2p orbital overlap better than sulfur’s electrons in 3p
orbitals with carbon’s 2p orbital). Benzothiophene-2-sulfona-
mide, notably, did not react with hydroxylamine under stan-
dard conditions. Similarly, 4-carboxybenzenesulfonamide was
found to be inactive in the reaction with hydroxylamine.
Overall, these results indicate that the substrate requires
highly electrophilic C-2 and the nitrogen atom on the ortho
position to the sulfonamide group for the reaction to proceed.
We then investigated whether other functionalities posi-
tioned at C-2 of the benzothiazole ring allow the substitution
reaction (Table 2). Sodium benzothiazole-2-sulfonate afforded
2-hydroxybenzothiazole in poor yield, whereas sulfone and
sulfoxide quantitatively reacted with hydroxylamine to form
2-hydroxybenzothiazole in 30 minutes (Table 2, entries 1–3).
Other electron-withdrawing groups, such as carboxylic acid,
ester, and amide, surprisingly, did not furnish any detectable
product (Table 2, entries 4–6). 2-Fluorobenzothiazole and
2-chlorobenzothiazole both reacted with hydroxylamine, but
the reaction proceeded at a much slower rate compared to BTA
(Table 2, entries 7–8).
standard conditions (3 hours at 70 °C), while N-methylaceto-
hydroxamic acid and N-hydroxysuccinimide produced only traces
of 2-hydroxybenzothiazole. Boc-protected hydroxylamine was
observed to be less reactive than hydroxylamine; the conver-
sion was completed in 48 hours. Collectively, these results are
in agreement with those employing substituted hydroxyl-
amines (Table 1), demonstrating that there has to be an NH
group adjacent to the free OH group in hydroxylamine in order
to achieve an enhanced reactivity of the reagent.
The scope of the reaction in the presence of hydroxylamine
was then investigated. Modifications of the heteroaromatic
group as well as of the sulfonamide functionality of BTA were
considered (Scheme 3). Readily available N-alkyl BTAs13 were
quantitatively converted to 2-hydroxybenzothiazole within
6 hours under standard reaction conditions. Importantly,
unlike BTA, which only contains exchangeable hydrogens on
the amino side chain (i.e. NH2), reaction between N-alkylated-
BTA derivatives and hydroxylamine provided evidence that
1
alkylamine is the product of the reaction. Real time H NMR
analysis clearly showed the disappearance of the alkyl signals
from the starting material, and the appearance of new signals
that correspond to the alkylamine product (doping/enhance-
ment experiments ultimately proved their existence, see ESI†).
Thiazole-2-sulfonamide in the presence of 50 equiv. of
hydroxylamine afforded only traces of 2-hydroxythiazole
(<10%). The observed difference in the reactivity between thia-
zole-2-sulfonamide and benzothiazole-2-sulfonamide can be
explained by the fact that the benzothiazole ring retains the
aromatic character upon nucleophilic attack by hydroxylamine,
whereas the thiazole ring becomes dearomatic. The reaction
between 6-ethoxy-BTA and hydroxylamine took longer than
that with BTA (40% conversion in 48 hours), whereas 5-chloro-
BTA quantitatively reacted with hydroxylamine in only
30 minutes. These results indicate that the electrophilic char-
acter of the C-2 position of BTA is perturbed in the presence of
electron-donating or electron-withdrawing groups, hence,
decreasing or increasing the rate of conversion relative to BTA.
Interestingly, benzimidazole-2-sulfonamide was found to be a
The benzothiazole-2-sulfonyl (Bts) group is a known N-pro-
tecting group in organic chemistry. Current deprotection
methods, which have been used for the removal of the Bts
group from N-protected amino acids, include 50% H3PO2,14
Zn/HOAc–EtOH,14 Al–Hg/ether–water,14 thiophenol/base,15,16
and 4-methoxythiophenol/DIEA.17 Using our method, cleavage
of the Bts-protected glycine, alanine or phenylalanine in the
presence of 50 equiv. of hydroxylamine afforded unprotected
amino acids in quantitative yield in 6 hours (Scheme 4, for the
real-time NMR analyses see ESI†).
To investigate the mechanism of the hydroxylamine-
mediated substitution of the sulfonamide group in BTA by a
hydroxyl group, we performed labeling experiments. The
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formation of diimide (HNvNH) intermediate, as previously
reported for the reaction between phosphate esters and hydroxyl-
amine,7 we performed the reaction under standard conditions,
1
but in the presence of 2 equiv. of fumarate. Careful H NMR
analysis showed that fumarate is converted to succinate, there-
fore indicating that diimide is an intermediate of the reaction.
Control experiments using base only (without hydroxylamine) or
hydroxyurea instead of hydroxylamine illustrated that diimide is
only formed when hydroxylamine is used as a reagent (Fig. 3).
A proposed mechanism of hydroxylamine-mediated substi-
tution of benzothiazole-2-sulfonamides is based on the follow-
ing observations: (i) hydroxylamine and N-methylhydroxylamine,
Scheme 4 Deprotection of amino acids. (a) Conversion determined by 1H
NMR.
oxygen atom in 2-hydroxybenzothiazole product could be
incorporated from hydroxylamine or from water. We per-
formed reactions in H2O and H218O and analyzed products by
LC-MS (Fig. 2). LC-MS analyses revealed that the product in
both cases has the same molecular mass (m/z 152.16), demon-
strating that there is no incorporation (<5%) of oxygen in the
product from water. When O-labeled H18ONH2 (∼65% 18O) was
used for the cleavage reaction, about 65% of 2-hydroxyben-
zothiazole product had incorporated the heavy oxygen atom
(Fig. 2). These data suggest that the oxygen atom in the
product derives from hydroxylamine, and not from water. Also,
these experiments provided evidence about the intermediate
of the cleavage reaction (see ESI†). In agreement with labeling
experiments, hydroxylamine quantitatively reacts with BTA
to afford 2-hydroxybenzothiazole under argon, as well as in
the dark, suggesting that there is no dioxygen-mediated mech-
anism or photochemical pathway involved in the cleavage.
1H NMR and LC-MS analyses provided evidence that products
Fig. 3 Indirect detection of diimide; diimide reduces fumaric acid to succinic
of the hydroxylamine-mediated reaction of benzothiazole-2- acid. Peaks at 6.3 ppm represent the fumaric acid protons, while peaks at
2.3 ppm represent the succinic acid protons. (A) BTA substitution reaction in the
sulfonamides are 2-hydroxybenzothiazole and alkylamine.
presence of fumaric acid and using hydroxyurea as a reagent; (B) BTA, base and
Using new fuchsine, a colorimetric reagent for the detection
fumaric acid; (C) hydroxylamine, base and fumaric acid; (D) BTA substitution
of sulfur dioxide,18,19 we additionally proved that sulfur dioxide
reaction in the presence of fumaric acid using hydroxylamine as a reagent; (E)
is the remaining product of the reaction (see ESI†). To test the
BTA substitution reaction mixture in the presence of fumaric acid, which was
possibility that the conversion of hydroxylamine results in the after completion (as in D) doped with sodium succinate.
Fig. 2 LC-MS+ spectra of (A) standard substitution of BTA using HONH2 in H216O; (B) standard substitution of BTA using HONH2 in H218O; (C) standard substitution
of BTA using H18ONH2 in H216O.
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2-hydroxybenzothiazole, sulfur dioxide, and the corresponding
amine via the mechanism in which hydroxylamine acts as an
oxygen nucleophile. The reaction reported here thus represents
one of the very few examples in which hydroxylamine reacts
with electrophiles via its oxygen atom. The utility of the reac-
tion was exemplified by the efficient cleavage of benzothiazole-
2-sulfonyl-protected amino acids: hydroxylamine-mediated
cleavage, thus, represents a mild and alternative method to
the currently existing methods for the deprotection of the Bts
group.
Experimental
Standard substitution reaction of BTA and analogues
4.3 mg (0.020 mmol, 1 equiv.) of benzothiazole-2-sulfonamide
(BTA) was added to water (1 mL). To this suspension were
added the reagent (1.0 mmol, 50 equiv.) and 8 μL of NaOH
(50% in H2O) to adjust the pH to 9–10. When the reaction
mixture became clear and colorless after shaking, it was trans-
ferred to an NMR tube and analysed at specific time points by
1H NMR and additionally by LC-MS. For 1H NMR analysis,
D2O was used as a solvent, whereas H2O was used to analyse
the sample by LC-MS.
Scheme 5 Proposed mechanism of the substitution reaction.
When the reagent exists as a hydrochloride salt, more
equivalents of base were used to adjust the pH.
unlike O-methylhydroxylamine, react with BTA, (ii) the reaction
does not occur in the absence of hydroxylamine under basic
conditions, (iii) the nitrogen atom at the ortho position to the
sulfonamide group is likely necessary for the reaction, (iv) the
oxygen atom incorporated in the product derives from hydroxyl-
amine, and does not derive from water, and (v) diimide is an
intermediate of the reaction. These observations propose a
mechanism in which hydroxylamine via its N–O oxygen atom
attacks the electrophilic C-2 atom of the substrate, and con-
comitantly, the ortho nitrogen atom of the heteroaromatic ring
abstracts the proton from the zwitterionic hydroxylamine
(Scheme 5). Deprotonation and donation of lone-pair electrons
from the nitrogen to the five-membered ring causes the elimi-
nation of the sulfonamide group (via the formation of sulfur
dioxide and alkylamine) and the formation of an unstable ben-
zothiazole-O-hydroxylamine intermediate. The intermediate
further reacts with excess of hydroxylamine to afford 2-hydro-
xybenzothiazole product (or its tautomeric keto form) and
hydroxyhydrazine. There has been some discrepancy between
experimental and computational data in the literature about
the existence of hydroxyhydrazine.20,21 Although we do not
have direct evidence for the formation of hydroxyhydrazine, its
potential existence is in agreement with the report by Kirby
et al.,7 in which hydroxyhydrazine dehydrates to form diimide.
Diimide can further react with another molecule of diimide to
form hydrazine and nitrogen.22
Detection of 2-hydroxybenzothiazole
A standard substitution reaction of BTA was performed using
59 μL of HONH2 (1.0 mmol, 50 equiv., 50% in H2O) as a
reagent. The progress of the reaction was monitored by
1H NMR. For doping/enhancement experiment, the solution
was treated with 3.0 mg (0.020 mmol, 1 equiv.) of 2-hydroxy-
benzothiazole after the reaction was fully completed. The
resulting solution was again recorded by 1H NMR, which
showed that no new peaks appeared.
Detection of amine
A standard substitution reaction of BTA was performed, using
0.020 mmol of N-alkyl substituted BTA and 59 μL of HONH2
(1.0 mmol, 50% in H2O) as a reagent. The progress of the reac-
1
tion was monitored using H NMR. For doping/enhancement,
0.020 mmol (1 equiv.) of the corresponding amine was added
after the reaction was completed. This final solution was
recorded by 1H NMR, showing that no new peaks were formed.
Detection of SO2
The color reagent was prepared by dissolving 24.1 mg of new
fuchsine in 1 mL of EtOH. To this dark pink solution were
added 58 mL of H2O and 2.75 mL of concentrated H2SO4. The
mixture was briefly shaken before 250 μL of 37–41% form-
aldehyde was added. After the brown clear solution was formed,
it was left for 10 minutes before it was used to color the
samples.
Conclusions
In summary, we have demonstrated that hydroxylamine
A standard BTA substitution reaction was performed using
mediates the conversion of benzothiazole-2-sulfonamides to HONH2 as a reagent. The mixture was stirred for 24 hours,
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followed by 1H NMR and LC-MS analyses, showing that no
starting material was left in the reaction mixture. A control
experiment was started at the same time using 4.3 mg
(0.020 mmol, 1 equiv.) of benzothiazole-2-sulfonamide in
1 mL of D2O and 8 μL of NaOH (50% in H2O). A sample
(50 μL) was then added to the colour reagent (950 μL) and the
mixture was recorded after a few minutes by UV-Vis spec-
troscopy at 587 nm.
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18O-labeling experiments
To a suspension of BTA (0.43 mg, 2.0 μmol, 1 equiv.) in 100 μL
of H218O were added 5.9 μL of HONH2 (50% in H2O, 100 μmol,
50 equiv.) and NaOH (1 μL, 50% in H2O). Similarly, to a sus-
pension of BTA (0.43 mg, 2.0 μmol, 1 equiv.) in H216O (100 μL)
were added H18ONH2·HCl (7 mg, 100 μmol, 50 equiv.) and
NaOH (2 μL, 50% in H2O). The incorporation of oxygen atom
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To a white suspension of 4.3 mg (0.020 mmol, 1 equiv.) of ben-
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financial support.
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