Visual Detection of Cysteine and Homocysteine

Homocysteine is a risk factor for disorders including cardiovascular¹ and Alzheimer's disease.² Cysteine deficiency is involved in slowed growth, hair depigmentation, edema, lethargy, liver damage, muscle and fat loss, skin lesions, and weakness.³ The detection of important biological thiols including cysteine and homocysteine has been the focus of numerous research efforts.⁴ The majority of the reported methods are based on redox chemistry or derivatization with chromophores or fluorophores. The determination of specific thiols is often carried out in conjunction with HPLC or capillary electrophoresis separations or via immunoassays.⁴ Recent reports describe a need for much simpler methods that employ stable, nontoxic reagents^5a,b which are less sensitive to pH^5c and afford the requisite sensitivity^5d,6a as well as high selectivity.^6b

We have reported prior progress toward the colorimetric and fluorimetric detection of mono- and oligosaccharides.⁷ Our studies featured new functionalized xanthenes which we found arose in situ from ring-opened resorcinarenes and related materials.^7c Our interest in the title compounds derives initially from the interference of cysteine with known sialic acid determinations.⁸ Herein we report the use of xanthene dye 1⁹ for the efficient detection of cysteine and homocysteine.

The selective reaction of aldehydes with N-terminal cysteines to form thiazolidines has been used to label and immobilize peptides and proteins.¹⁰ Compound 1 was employed as an intermediate toward the synthesis of a fluorescent sensor for zinc.⁹ We reasoned that the reaction of the aldehyde moieties of 1 with cysteine or homocysteine would promote readily monitored colorimetric and fluorometric responses (Scheme 1).

Scheme 1.

We find that upon addition of l-cysteine or l-homocysteine (1.0 × 10⁻³ M) to a solution of 1 (1.0 × 10⁻⁶ M, H₂O, pH 9.5), a solution color change from bright yellow to brownish-orange is observed. Similar color changes are observed on C₁₈-bonded silica (Figure 1). UV–vis absorbance changes of cysteine-1 solutions, readily monitored in the 10⁻⁵–10⁻⁶ M cysteine concentration range, exhibit a 25 nm red shift.^11–13 Addition of 2a or 2b to solutions of 1 results in fluorescence quenching.¹³

Figure 1.

(Left) Color changes of solutions of 1 and various analytes. A, no analyte; B, l-cysteine; C, l-homocysteine; D, bovine serum albumin; E, l-glycine; and F, n-propylamine. (Right) Co-spots of 1 (1.0 × 10⁻³ M) with and without various analytes (1.0 × 10⁻³ M) under visible and UV light.

Respective solutions of 1 containing identical concentrations of 2a and 2b exhibit similar spectrophotometric changes (Figure 2). UV–vis spectra of solutions containing 1 and other common thiols (l-methionine, mercaptoethanol, glutathione), other amino acids (l-glutamine, l-serine, l-glycine, l-glutamic acid), and amines (d-glucosamine hydrochloride and n-propylamine (8.0 × 10⁻⁴ M, pH 9.5) confirm the selectivity of 1 for cysteine and homocysteine.¹³

Figure 2.

Absorbance vs concentration plots for l-cysteine (▲) or l-homocysteine (◯) in aqueous solutions of 1 (2.5 × 10⁻⁶ M) at pH 9.5. The figure highlights the similarity of the absorbance responses of 1 to 2a and 2b. An absorbance decrease is shown at 480 nm for 2a and 2b at 5.0 × 10⁻⁶ M concentrations, while the absorbance increase is shown at 505 nm for increasing concentrations of 2a and 2b from 10 × 10⁻⁶ to 40 × 10⁻⁶ M.

At most a 15% change in absorbance at 480 nm is observed in response to the aforementioned analytes. No wavelength shift occurs. Solutions containing 1 and bovine serum albumin or urease (8.0 × 10⁻⁴ M, pH 9.5) also exhibit relatively small absorbance (<15%) decreases and no λ_max shifts.¹³

The addition of l-cysteine to a sample of commercial human blood plasma (previously centrifuged at 3000g through a cellulose 3000 MW cutoff filter, the low-molecular-weight fraction is used for analysis), containing 1 and excess glutathione (1.0 mM), results in concentration-dependent UV–vis changes (Figure 3). Figure 4 shows a linear correlation between fluorescence emission intensity and healthy to abnormal homocysteine concentrations in plasma containing 1.^6a This demonstrates the potential utility of 1 toward calibrating and determining aminothiol concentrations in plasma samples in the presence of other biological thiols.

Figure 3.

UV–vis spectra of 1 (4 × 10⁻⁶ M) and l-cysteine (4.9 × 10⁻⁵−7.4 × 10⁻⁴ M) in deproteinized human plasma at room temperature containing 1.0 mM added glutathione (pH 9.5). Each spectrum is acquired 5 min after cysteine addition. As the concentration of l-cysteine increases, a red shift from 480 to 500 nm is observed.

Figure 4.

Fluorescence emission spectra of 1 (5.2 × 10⁻⁷ M) and l-homocysteine (2.9 × 10⁻⁶−2.5 10⁻³ M) in deproteinized human plasma excited at 460 nm (pH 9.5). (Inset) Fluorescence emission plotted vs [2b].

In conclusion, compound 1 can be used to readily detect l-cysteine and l-homocysteine in the range of their physiological levels.^6a Interference from amines, amino acids, and certain thiols and proteins is minimal. The methodology shows great promise for the fluorescence and UV–vis detection of aminothiols in plasma.