CN- scavenger: A leap towards development of a CN- antidote

Article abstract of DOI:10.1039/c1cc12668g

Compound 4 removes cyanide from water and human serum in the form of compound 7 (cyano-derivative of 4) and experiments show its suitability as an antidote of CN^- poisoning.

Full text of DOI:10.1039/c1cc12668g

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COMMUNICATION
CNÀ scavenger: a leap towards development of a CNÀ antidotew
Palwinder Singh* and Matinder Kaur
Received 6th May 2011, Accepted 20th June 2011
DOI: 10.1039/c1cc12668g
Compound 4 removes cyanide from water and human serum in
the form of compound 7 (cyano-derivative of 4) and experiments
show its suitability as an antidote of CNÀ poisoning.
Irrespective of the fact that they are integral components of
fauna and flora, cations as well as anions are dangerous once
their concentration exceeds a maximum limit and their selective
removal from the system becomes an issue of medicinal and
ecological importance.1 Among the anions of biotic significance,
Chart 1
cyanide is highly toxic when it exceeds its maximum permissible
level of 1.9 mM.2 It binds with Fe3+ of heme unit and retards the
À
HCO3 or may be CNÀ present in tap water caused the color
oxygen supply.3 The cellular respiration is affected due to the
binding of CNÀ with cytochrome c which blocks the electron
transport chain and hence the CNÀ poisoning causes ultimate
death.4 Practically no effective antidotes of CNÀ are available so
far5 which further aggravates the gravity of the issue. As per
the reports available,6 sensing of CNÀ is achieved either via
H-bonding or chemical reaction between the sensor and CNÀ.
However, these reports do not comment on the biological profiles
(toxicity etc.) and probable metabolites of CNÀ sensors and their
use as antidotes of CNÀ.
change when water was added to the ethanolic solution of
compound 4. A solution of compound 4 (10 mM in ethanol–
distilled water, 1 : 1) exhibited UV-vis absorption bands at 219,
249 and 403 nm (Fig. 1a). On incremental addition of (0.01–1
equiv, 0.1–10 mM) tetrabutylammonium cyanide (TBACN)/
NaCN to the above solution of compound 4, absorption at 219
and 403 nm decreased with increase at 249 nm. A weak band at
302 nm and a strong band at 520 nm emerged with three isosbestic
points at 276, 321 and 444 nm (Fig. 1a). Color of the solution
changed from light yellow to orange to red within a second. The
same change in color and absorption spectrum was observed when
compound 4 (10 mM) was added to a solution of NaCN in
ethanol–water. Job’s plot indicated 1 : 1 stoichiometry of 4ÁCNÀ
(Fig. S20) and Ka was 106 MÀ1. UV-vis spectra of the above
solution of 4 + CNÀ did not undergo any change with time
(Fig. S21) indicating that first order reaction (Fig. S22) between 4
and CNÀ is very fast, completed in a fraction of a second, a time
which is probably less than the poisoning time of CNÀ (seconds
to a few minutes). Further addition of CNÀ (up to 100 equiv) to a
solution of 4 + CNÀ resulted in a decrease in absorption intensity
at 520 nm (Fig. 1b). A solution of 4 + CNÀ gives a new spot in
TLC (thin layer chromatography) which after isolation showed a
Being used for detection and binding of CNÀ from living
systems, molecules similar to trimethoprim7/its metabolite (1–2,
Chart 1) were designed (3–4, Chart 1). Use of trimethoprim as
antibacterial agent and also the biological acceptance of
barbituric acid and pyrazole are the safeguards for non-toxicity
of the new compounds to living systems. Compounds 3 and 4
were built in quantitative yield by Knoevenagel condensation
between an equimolar mixture of pyrimidine/pyrazole and
syringaldehyde on irradiation under microwaves (SI, Scheme 1).
Observation of a change in color of a solution of compound
3/4 (in dry ethanol) from light yellow to orange on addition of
tap water, and no color change on addition of distilled water led
to systematic investigations on these compounds. Various anions
were added to the solution of compound 4 (10 mM in ethanol–
distilled water, 1 : 1) but color change from light yellow to orange
to red was observed only when minimum 0.1 mM solution of
NaCN was added, and color change from light yellow to orange
on addition of 1 mM solutions of Na2CO3 and NaHCO3
2À
(in ethanol–distilled water,1 : 1) (Fig. S19). It seems that CO3
,
Department of Chemistry, UGC Sponsored Centre for Advanced
Studies, Guru Nanak Dev University, Amritsar-143005, India.
E-mail: palwinder_singh_2000@yahoo.com; Fax: 91 183 2258819;
Tel: 91 183 2258802x3495
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc12668g
Fig. 1 (a) UV-vis absorption spectra of compound 4 (ethanol–water,
1 : 1 v/v) upon incremental addition of CNÀ (up to 1 equiv).
(b) Addition of CNÀ (45 equiv) to solution of 4 + CNÀ resulted in
decrease in absorbance at 520 nm.
c
9122 Chem. Commun., 2011, 47, 9122–9124
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Similar change in the color of the solution and absorption spectra
of compound 4 was observed as in neutral medium indicating its
suitability for working under acidic conditions (usual pH of major
part of digestive system).9 A solution of compound 4 in basic
medium (pH 8, pH range of intestinal part of digestive system)9
showed absorption bands at 219, 249, 369 and 520 nm. The
absorption band at 520 nm was probably due to abstraction of
phenolic H as in the presence of CNÀ. However, addition of CNÀ
to the basic solution of compound 4 intensified the absorption
band at 520 nm (Fig. S31, S32) which means this compound is
detecting CNÀ in alkaline medium also. Compound 7 was isolated
from both experiments (in acidic and basic media).
The UV-vis spectrum of compound 4 in presence of other
2À
anionsÀlike FÀ, ÀOAc, H2PO4À, ClÀ, BrÀ except CO3 and
HCO3 did not undergo any change indicating the selectivity of
compound 4 for CNÀ over other anions (Fig. S19, S33, S34).
2À
Addition of CO3 (up to 10 equiv, 100 mM, no change on further
addition of CO32À) to a solution of 4 (10 mM) in ethanol–water
resulted in an increase in absorbance at 520 nm. It is apparent
from Fig. 1 and Fig. SI35 that increase in absorbance of 4 at
520 nm is much higher when CNÀ (1 equiv only) was added in
comparison to the addition of CO32À (1/10 equiv). This might
be due to early attainment of equilibrium between 4 and 5
Scheme 1 Capturing of CNÀ by compound 4.
new absorption band at 590 nm (Fig. S23). It seems that initial
deprotonation of phenolic H from compound 4 changed the
conjugation of p-electrons to form species 5 (tautomeric forms,
5a and 5b) (Scheme 1). Now abstraction of H from OH/NH
group on the pyrazole moiety changed 5 to compound 4 and
a decrease in absorption band at 520 nm took place until
equilibrium was attained between 4 and 5. Reaction of CNÀ at
carbonyl carbon (1,2-addition8) of the a,b-unsaturated carbonyl
system forms 6 which on protonation, loss of water and reduction
ultimately gave compound 7. Isolation of compound 7 from a
solution of 4 + CNÀ and an absorption band at 590 nm in its
UV-vis spectrum (Fig. S23) supported the route of CNÀ action as
proposed in Scheme 1. NMR titrations also supported the mode
of action of CNÀ as the signal of OH (d 6.24) disappeared on
addition of CNÀ (Fig. S24, S25). No response of compound 8
(Chart 2) to CNÀ (Fig. S26) also indicated that action of CNÀ
started from the phenolic moiety of compound 4. Moreover,
addition of CNÀ (1 equiv) to compound 9 (Chart 2) in
water–ethanol resulted in an increase in absorption at 520 nm
which is B8% only of what was observed in the case of
compound 4 (Fig. S27). This indicated the role of OCH3
groups in the abstraction of phenolic H (probably H-bond of
OH with O–CH3 increases the acidity of phenolic OH).
2À
(Scheme 1) during the addition of CO3 while in presence of
CNÀ, equilibrium is more to the forward direction. This is also
supported by the increased absorbance at 520 nm on additioÀn
2À
of CNÀ to the solution of 4 + CO3 and 4 + HCO3
2À
À
(Fig. S35, S36). Ka for 4ÁCO3 and 4ÁHCO3 was 0.3 Â
106 MÀ1 and 1.6 Â 104 MÀ1, respectively (Fig. S37 and S38)
which were considerably less than that of 4ÁCNÀ indicating
better interactions of 4 with CNÀ. Based on this observation,
the UV-vis spectrum of compound 4 in tap water–ethanol
(1 : 1) was recorded. Irrespective of the initial color change
of compound 4 in tap water and the appearance of a
weak absorption band at 520 nm, addition of CNÀ further
2À
À
intensified this band indicating that CO3
and HCO3
present in natural water (here ground water through tube-well)
did not interfere much in the detection/capturing of CNÀ by
compound 4 (Fig. S139a).
Practicability of compound 4 was evident from its competitive
binding with CNÀ in presence of other anions. Solution of
compound 4 did not undergo color change even in presence of
100 equiv of various anions. But addition of CNÀ to these solutions
made the color change from light yellow to orange. The UV-vis
spectrum of a solution of compound 4 + anions (other than CNÀ)
exhibited characteristic absorption bands of compound 4 but an
To rule out if it is OHÀ, generated from hydrolysis of NaCN,
that is responsible for color change and absorption spectra of
compound 4 in aqueous medium, all these experiments were also
carried out in dry THF, dry ACN and dry CHCl3 using TBACN
(Fig. S28, S29). The same observations as in aqueous medium
indicated the participation of CNÀ only in changing the color of
compound 4.
To look into the behaviour of compound 4 towards CNÀ in
acidic and basic media, incremental addition of CNÀ was made to
the solution of compound 4 (10 mM, ethanol–distilled water, 1 : 1)
at pH 2–7 (Fig. S30) (4 is stable under aqueous, acidic conditions).
Fig. 2 (a) UV-vis spectrum of proteinaceous human serum (lower red
trace), on addition of compound 4 (second lower purple trace) and
further addition of CNÀ. (b) UV-vis spectrum of non-proteinaceous
human serum (lower red trace), additon of compound 4 (second lower
purple) and further addition of CNÀ.
Chart 2
c
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intense new band at 520 nm appeared on addition of CNÀ
indicating competitive binding of 4 with CNÀ (Fig. S40).
As the concentration of Fe3+ (0.01 mM) in the human body10 is
B5 times the lethal concentration of CNÀ (41.9 mM), it was
worth checking the behaviour of 4 towards CNÀ in presence of
Fe3+. Addition of 15 equiv of Fe3+ (w.r.t. CNÀ) to a solution of
4 + CNÀ did not affect the color of the solution and the
absorption spectrum as well but further addition of Fe3+ changed
the color of the solution from orange to light yellow and an
absorption band at 520 nm disappeared (Fig. S139b). Therefore,
irrespective of the strong affinity of CNÀ for Fe3+, CNÀ preferred
compound 4 in the presence of even 15 equiv of Fe3+. Moreover,
addition of Mg2+, Cu2+, Zn2+, Co2+ and Ni2+ did not disturb
the color and the absorption spectrum of a solution of 4 + CNÀ.
Scheme 2 Hydrolysis of compound 7 to compound 10.
human serum were recorded. Non-toxicity of compound 4 was
apparent from its higher LC50 value at various cell lines of
human cancer (data not shown here) as well as its similarity to
trimethoprim.7b,12 Under reaction conditions of 4 and CNÀ, 3/4
did not react with cysteine (analogue of GSH).13 Hydrolysis of
compound 7 under basic conditions (usual pH in lower part of
digestive system) provided compound 10 (Scheme 2) indicating the
conversion of CN group to COOH and hence the safe removal of
CNÀ at the end.
Compound 4 did not respond to the biological cations like Fe3+
,
Mg2+, Cu2+, Zn2+, Co2+ and Ni2+. Solutions of compounds
3a and 3b in the same solvent (ethanol–water, 1 : 1) also gave
similar responses to CNÀ (SI, Fig. S41–50).
Therefore, a simple synthesis which could be scaled up to
procure tons of compounds provided compounds 3 and 4 in
which the appreciable acidity of phenolic OH helped in the
detection and binding of CNÀ in presence of other anions and
even 15 equiv of Fe3+ also (biological target of CNÀ). All the
experiments carried out here with compound 4 indicated the
suitability of this compound as an antidote of CNÀ. Study in
living systems is underway.
To check further the working of compound 4 under biological
conditions and the affinity of CNÀ for compound 4 vs. biological
Fe3+ (Fe3+ in porphyrin), human serum (diluted in water) was
used as medium for performing the experiments.
Proteinaceous blood sample (sample I)11 showed absorption
bands at 215 nm and 412 nm (Fig. 2a). Addition of 20 mL of
compound 4 (10 mM) to the above solution of sample I turned the
colorless solution light yellow. The UV-vis spectrum of this
solution exhibited usual absorption bands of compound 4 and a
new band at 520 nm (Fig. 2a). To rule out if the absorption band
at 520 nm is due to the interactions of compound 4 with
some proteins in the blood sample, a non-proteinaceous sample
(sample II)11 of blood serum was prepared. Sample II showed
absorption bands at 227 nm and 271 nm (Fig. 2b). Addition of
20 mL of compound 4 (10 mM) to a solution of sample II turned
the colorless solution light yellow and the UV-vis spectrum of this
solution also exhibited an absorption band at 520 nm (Fig. 2b).
The absorption band at 520 nm seems to be due to the presence of
Financial assistance from CSIR, DST New Delhi is gratefully
acknowledged. MK thanks UGC, New Delhi for SRF.
Notes and references
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CNÀ/CO32À/HCO3 in blood itself and this band gets intensified
À
on incremental addition (1 mM to 5 mM) of CNÀ along with color
change of the solution from light yellow to orange to red
(Fig. 2a,b). Addition of CNÀ to a solution of sample I + 4 also
led to an increase in the absorption intensity at 250 nm and
360 nm with concomitant decrease at 228 and 299 nm (Fig. 2a).
Alternatively, addition of compound 4 (10 mM) to a solution of
sample I + CNÀ (10 mM CNÀ) resulted in change in color and the
appearance of an absorption band at 520 nm. Compound 7 was
also isolated from this solution showing the scavenging of CNÀ
from blood serum by compound 4.
It was ensured that treatment of CNÀ containing water sample
and human serum with compound 4 completely removes CNÀ
from the medium. The aqueous part, after extraction of solution
of 4 + CNÀ (10 mM compound 4 and 10 mM NaCN) with ethyl
acetate, did not show color change on addition of compound 4
which otherwise could have been there if CNÀ had been present in
the aqueous part. The aqueous part, left after extraction of
solution of NaCN (in water–ethanol) with ethyl acetate, gave a
color change on addition of compound 4 indicating that CNÀ was
not extracted with ethyl acetate only but it is compound 7 which is
carrying cyanide from the aqueous solution (Fig. S51). The same
observations of complete removal of CNÀ by compound 4 from
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