WO2019111268A1

WO2019111268A1 - Method for selective and sensitive quantification of hydrogen sulfide

Info

Publication number: WO2019111268A1
Application number: PCT/IN2018/000055
Authority: WO
Inventors: K. M. Muraleedharan; K Ramshad; C Lakshmi; T.t DIVYA
Original assignee: INDIAN INSTITUTE OF TECHNOLOGY MADRAS (IIT Madras); National Institute Of Technology Calicut
Priority date: 2017-12-05
Filing date: 2018-12-03
Publication date: 2019-06-13

Abstract

A method for selective and sensitive quantification of hydrogen sulfide (H₂S) using new triarylmethane dyes. The proposed method involves a two-step protocol in which the nucleophile is added to malachite green derivatives which lead to initial loss or decrease of its characteristic absorption. Subsequently, a second metal salt-mediated de-sulfuration step is adapted where selective regeneration of the original dye from its hydrogen sulfide (H₂S) adduct is allowed, which can be detected and quantified. The proposed method takes very shorter time period (less than a minute) therefore effective and useful in continuous monitoring of H₂S levels in biological and mammalian samples.

Description

METHOD FOR SELECTIVE AND SENSITIVE QUANTIFICATION OF

HYDROGEN SULFIDE TECHNICAL FIELD

[0001] Embodiments are generally related to the field of analytical chemistry. Embodiments are further related to methods for selective detection and quantification of hydrogen sulfide (H₂S) in biological samples or samples of environmental/industrial origin using triarylmethane dyes. Embodiments are furthermore related to detection and management of clinical conditions, including, but not limited to, sepsis and quantification of hydrogen sulfide in other environmental or industrial samples. Embodiments are more particularly related to methods for selective and sensitive quantification of hydrogen sulfide (H₂S) using new triaryl methane dyes.

BACKGROUND OF THE INVENTION [0002] Hydrogen sulfide is generally known for its foul smell and has recently been identified as a gasotrasmitter that regulates a number of physiological and pathological processes. It is one of the endogenous gasotransmitters like nitric oxide (E. Culotta et al. , Science, 1992, 258, 1862) and carbon monoxide (S.

Kourembanas et.al., P. Natl. Acad. Sci., 1995, 92, 1475).

[0003] The imbalance of hydrogen sulfide levels (either over-production or decreased levels) can lead to different problems in biological systems (P. Kamoun et al ., Nat. Rev. Drug. Disco ., 2007, 26, 243). During hypoxic pulmonary hypertension a decreased level of hydrogen sulfide in blood has been reported (T. Chaoshu et al., Biochem. Biophys. Res. Commun., 1997, 302, 810). Similarly, decrease in the level of hydrogen sulfide was also seen in the brain samples of patients with Alzheimer’s disease (H Kimura et al., Biochem. Biophys. Res. Commun., 2002, 293, 1485).

[0004] Over-production of cystathionine-p-synthase (CBS), the enzyme responsible for the production of hydrogen sulfide was found associated with cancer in colon and ovaries (P. K. Moore and M. Whiteman, Handbook of Experimental Pharmacology, 2015, 230, page 233; ISBN 978-3-319- 18143-1). Exogenous hydrogen sulfide at the same time was able to inhibit the growth of tumour cells (S. He et al, Biol chem., 2015, 396, 1247). In Down’s syndrome and diabetes conditions, over-production of hydrogen sulfide has been reported (P.

Kamoun et ai, Am. J. Med. Sci., 2003, 1 16, 310; B. Geng et ai, Biochem. Biophys. Res. Commun., 2009, 380, 153). So, in general, an imbalance in hydrogen sulfide level is seen in different pathological conditions.

[0005] In particular, Sepsis is a clinical condition in which the body’s reactions to microbial infection become uncontrolled, which in turn damages various organs. Clinical studies have shown that H₂S levels are elevated under Sepsis condition and therefore levels of H₂S can be used as a marker for monitoring the inflammatory reposes or the onset of Sepsis.

[0006] Development of efficient molecular probes that can selectively detect and quantify hydrogen sulfide in biological milieu could significantly impact the clinical outcome of sepsis treatment. Prior art probes known for reporting and sensing of hydrogen sulfide for biological samples have limited application due to limitations in sensitivity and chemical stability factor. Furthermore, such prior art probes are unable to provide required results in shorter detection time and selectivity. Such prior art probes can mainly be classified into following groups based on their mechanism of sensing such as, reduction of azide or nitro groups by hydrogen sulfide, nucleophilic addition of sulfide to the electrophilic centers in the probe, or metal displacement methods. In majority of such prior art probes, the interaction with hydrogen sulfide alters the photo-physical behavior of the probe thereby enabling detection.

[0007] Hydrogen sulfide is present in human blood in the concentration range of 10-100 mM (J. Chang et al., Curr. Opin. Chem. Biol., 2012 , 16, 595) and up to 600 mM in the brain (Z. Guo et al, Angew. Chem., Int. Ed., 2013, 52, 1688). Therefore, H₂S probes for biological applications should be able to respond to H₂S in the micro molar range. However there are only a few probes like Danzyl azide which satisfy this requirement (B. Wang et al., Angew. Chem. Int. Ed., 2011, 50, 9672) The Rhodamine based probes have a wider concentration range for detection compared to other probes but requires more response time (10-600 mM; C. J. Chang et al., J. Am. Chem. Soc., 2011, 133, 10078). The reaction takes more than 60 minutes for completion and only a 7 - fold enhancement in fluorescence is observed. Real-time analysis is possible only when the probe changes its photo- physical properties rapidly. Majority of prior art probes which use reducing power of H₂S or its nucleophilicity (other than sulfonyl azide derivatives) take longer duration (more than 30 minutes) to respond. (B. Wang et al, J. Fluoresc., 2014, 24, 1). Also, sulfonylderivarives (such as, dansyl azide) have low chemical stability which is a drawback. Furthermore, the emission intensity of the reduction product in this case is known to get enhanced upon binding with plasma proteins, which could bring error during H₂S quantifications. The signal output is faster in the case of metal displacement method (S. Wang et al, Analytica Chimica Acta , 2015, 879, 104) like CuS precipitation (J. Chang et al., Curr. Opin. Chem. Biol., 2012, 16, 595). However, this method is less selective compared to reaction-based probes.

[0008] Based on the foregoing aspects, the prior arts probes are unable to satisfy the requirements for continuous monitoring of H₂S with short detection time, high sensitivity, high selectivity, good chemical stability, water solubility and noninterference from other bio-molecules in the photo-physical output.

[0009] Based on the foregoing a need therefore exists for an improved method for selective and sensitive quantification of hydrogen sulfide (H₂S) using new triaryl methane dyes, as discussed in greater detail herein.

SUMMARY OF THE INVENTION

[0010] The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description. A full appreciation of the various aspects of the embodiments disclosed herein can be gained by taking the entire specification, claims, drawings, and abstract as a whole.

[0011] Therefore, one aspect of the disclosed embodiment is to provide method for selective detection and quantification of hydrogen sulfide in samples of biological origin such as blood plasma.

[0012] Another aspect of the disclosed embodiment is to provide a method for selective detection and quantification of hydrogen sulfide in samples of environmental origins including oils, etc.

[0013] Further aspect of the disclosed embodiment is to provide method for selective and sensitive quantification of hydrogen sulfide (H2S) using new triaryl methane dyes.

[0014] The aforementioned aspects and other objectives and advantages can now be achieved as described herein. A method for selective and sensitive quantification of hydrogen sulfide (H₂S) using new triaryl methane dyes, is disclosed herein. The proposed method involves a two-step protocol in which addition of this nucleophile to the dye leads to initial loss of its characteristic absorption. Since this addition step cannot discriminate ions such as, hydrogen sulfide, sulfite, thiosulfate etc.,., a second metal salt-mediated de-sulfiiration step is adapted where selective regeneration of the original dye from its hydrogen sulfide (H₂S) adduct is allowed, which can be detected and quantified. The proposed method takes very shorter time period (less than a minute altogether) therefore effective and useful in continuous monitoring of H₂S levels in different types of samples mentioned above.

[0015] The proposed invention pertains to the development of triarylmethane dyes (malachite green derivatives) for the selective detection and quantification of hydrogen sulfide in different types of analytes such as, for example, but not limited to, biological samples, samples of environmental origins such as oils, gases etc.

The proposed method can be effectively adapted for monitoring of H₂S levels in patients with Sepsis and could significantly help in the early detection and management of severe inflammatory responses. In an alternative embodiment of the proposed method, the proposed method may be effectively employed for detection and monitoring of H₂S in industrial processes such as for example, but not limited to, oil and coal fields, and handle health-and environmental issues.

[0016] The proposed method uses nucleophilicity of H₂S or its other ionic forms like HS or S (here referred as sulfide) which on addition to malachite green derivatives (such as, tri aryl methane dyes) cause a change in their photophysical properties. The selective detection of such sulfide can be achieved by including a second step in which the dye-H₂S adduct was made to react with metal salts (ZnCl₂, CdCl₂ etc). This regenerates the parent dye with precipitation of corresponding metal sulfide. Since the regeneration step is highly selective for sulfide, the recovery of photo-physical signal can be quantitatively correlated with sulfide concentration. The first step is instantaneous and the second metal- mediated dye regeneration require only about 50 seconds. Thus the detection and quantification can be completed in a minute to achieve fastest results of the method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying figures, in which like reference numerals refer to identical or functionally-similar elements throughout the separate views and which are incorporated in and form a part of the specification, further illustrate the present invention and, together with the detailed description of the invention, serve to explain the principles of the present invention.

[0018] FIG. 1 illustrates a schematic representation of a method for selective detection and quantification of hydrogen sulfide (H₂S) using triarylmethane dyes, in accordance with the disclosed embodiments; and

[0019] FIG. 2 illustrates a graphical representation illustrating the concentration of Sodium Sulfite, in accordance with the disclosed embodiments.

DETAILED DESCRIPTION

[0020] The particular values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.

[0021] The embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0023] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0024] FIG. 1 illustrates a schematic representation 100 of a method for selective detection and quantification of hydrogen sulfide (H₂S) in samples of biological or environmental origin using triarylmethane dyes, in accordance with the disclosed embodiments. The proposed method involves a two-step protocol in which the nucleophile is added to malachite green derivatives which lead to initial loss of its characteristic absorption. Note that this addition step do not discriminate ions such as, hydrogen sulfide, sulfite, thiosulfate etc. Subsequently, a second metal salt-mediated de-sulfuration step is adapted where selective regeneration of the original dye from its hydrogen sulfide (H2S) adduct is allowed, which can be detected and quantified. The proposed method takes very shorter time period (less than a minute) therefore effective and useful in continuous monitoring of H₂S levels in samples of biological and environmental origin.

[0025] Malachite green is a well-known compound in the area of dye chemistry. It has good absorption at 618 nm and has very good affinity towards nucleophilic compounds like sulfite, H₂S and thiosulfate. Absorbance of malachite green gets abolished in the presence of these nucleophiles due to adduct formation (F. Colmenero et.al., Food Additives and Contaminants , 2008, 25, 1167). Apart from its use in dye industry, it is generally known as a sensor for sulfiite and is used for the removal of sulfite from aquatic medium. Major emphasis of research has been on sulfite ion detection since it is a well-known food additive (C. Francisco et. al.,

Food Chemistry, 2009, 1 12, 487). Although, use of malachite green as a qualitative sensor for H₂S is also known (M. Mahjoub et al., Environ. Monit. Assess., 2015,

187, 248), complete quenching of colour requires about 25 equivalents of sulfur source.

[0026] The proposed invention pertains to the development of malachite green derivatives for the selective detection and quantification of hydrogen sulfide in different types of analytes such as, for example, but not limited to, biological samples, those of environmental origins and mammalian samples. The proposed method can be effectively adapted for monitoring of H₂S levels in patients with Sepsis and can significantly help in the early detection and management of severe inflammatory responses. In an alternative embodiment of the proposed method, the proposed method can be effectively employed for detection and safe handling of H₂S in industrial applications such as for example, but not limited to, oil and coal fields, and handle health-and environmental impacts.

[0027] New triarylmethane dyes (Scheme 1 ) which responds quantitatively to H₂S in shortest time. Initially, the selectivity and the sensitivity profiles of new derivatives (Compound 14 a-h, Scheme 1) with electron rich (14g, 14h etc) and electron deficient (14b, 14c, 14d, 14e etc) substituents were compared. Of these, electron-rich systems showed poor affinity towards sulfur nucleophile and required more than 30 equivalents of hydrogen sulfide for decolourization. Remarkably, derivatives with electron deficient substituents like, 14b, 14c, 14d and 14e required

2.5, 2, 4.5 and 5 equivalents respectively for complete de-colourization of the probe solution. The compound 14b was selected as representative example for detailed studies, which was prepared according to the protocol shown in Scheme 1.

(B Parvin et ai, Molecules, 2008, 13, 986).

Scheme 1. General procedure for the synthesis of triarylmethane dyes with the general structure 14.

[0028] The response from nitro-derivative 14b is nearly 15 times better than that of malachite green. Note that the scope of the proposed method is not however limited to 14 b, and any derivative in this series with one or more electron- withdrawing groups like CN, CF₃, N0₂ etc. on one or more of the aromatic rings with good balance of reactivity may be considered.

[0029] The proposed method uses nucleophilicity of H₂S which on addition to malachite green derivatives (such as, triarylmethane dyes) cause a change in their photo-physical properties. The selective detection of H₂S can be achieved by including a second step in which the dye-H₂S adduct was made to react with metal salts like ZnCl₂, CdCl₂ etc. This is not however limited to these salts but others which also promote desulfuration and regenerate the dye may also be considered. The second step thus regenerates the parent dye with precipitation of corresponding metal sulfide. Since the regeneration step is highly selective for sulfide, the recovery of photo-physical signal can be quantitatively correlated with sulfide concentration. The first step is instantaneous and the second metal mediated dye regeneration require only about 50 seconds. Thus the detection and quantification can be completed in a minute to achieve fastest results of the method.

[0030] FIG. 2 illustrates a graphical representation 200 of the change in absorbance in samples containing mixture of sulfite and sulfide when they are subjected to the present method for H₂S estimation. . These set of experiments involving mixture of H₂S and sulfite ions were conducted to estimate the concentration of sulfide accurately in presence an interfering ion like Sulfite. The concentrations of the probe and H₂S in all the samples were kept constant (40 mM and 50 mM respectively) and that of sulfite ions were varied systematically from 0-50 mM. In principle, the first step should show a reduction in absorbance of 14b due to reactions involving both H₂S and the sulfite, but the regeneration step should be specific for only H₂S-adduct. Since the concentration of H₂S used was same in all the experiments, the gain in absorbance on treatment with the metal salt should remain the same in all cases. As shown in FIG. 2, the first step indicated a progressive reduction of absorbance due to combined effects from both ions. However, on treatment with 200 mM CdCl₂ solution (second step), the regenerated absorbance remained the same as expected. The FIG. 2 given below depicts the absorbance values after step 1 and step 2 for each of the sulfite concentrations. As can be seen, there is good linearity for data points in each case (step 1 and 2). Moreover, their slopes are the same which proves that the absorbance gain is linearly related to the desulfuration of H₂S adducts without any influence from sulfite ions.

[0031] It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

CLAIMS I/We CLAIM:

1. A method for selective and sensitive quantification of hydrogen sulfide (H₂S) using new triaryl methane dyes, said method comprising:

a nucleophile is added to malachite green derivative which lead to initial loss of its characteristic absorption; and

adapting a second metal salt-mediated de-sulfuration step where selective regeneration of the original dye from its hydrogen sulfide (H₂S) adduct is allowed, which can be detected and quantified at very shorter time period (less than a minute) therefore effective and useful in continuous monitoring of H₂S levels in biological and mammalian samples.

2. The method of claim 1 wherein further comprising development of malachite green derivatives for the selective detection and quantification of hydrogen sulfide in at least one of the analytes: biological samples; environmental origins; and mammalian samples.

3. The method of claim 1 further comprising monitoring of H₂S levels in patients with Sepsis and can significantly help in the early detection and management of severe inflammatory responses.

4. The method of claim 1 further comprising detecting and safe handling of H₂S in industrial applications to handle health-and environmental impacts.

5. The method of claim 1 further comprising detecting and quantifying hydrogen sulfide using triarylmethane dyes wherein triarylmethane dye is initially made to react with the sample of interest which leads to loss or decrease of its intrinsic absorption due to covalent adduct formation with the sulfide.

6. The method of claim 1 further comprising adding aqueous solution of metal salts to selectively regenerate the chromophore from its H₂S adduct through metal sulfide formation wherein the regenerated absorbance can be used for hydrogen sulfide quantification.

7. The method of claim 6 wherein the metal salts can be ZnCI₂ or CdCl₂ or other such salts which promote the regeneration of the parent dye with concomitant desulfuration

8. The method of claim 1 further comprising using nucleophilicity of H₂S which on addition to triarylmethane dyes cause a change in their photo-physical properties.

9. The method of claim 1 further comprising making dye-H₂S adduct to react with metal salts which regenerates the parent dye with precipitation of corresponding metal sulfide wherein said method can be used for detection and quantification of Hydrogen sulfide with shorter time period (less than a minute).