1. Introduction
The detection of organic molecules that absorb at low wavelengths is a critical issue and a challenge for analytical chemistry. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle and is rarely used as a routine tool for structural analysis. To overcome this problem, chemical derivatization is often employed in order to conduct indirect spectrophotometric analysis. Therefore, depending on the reactivity and available functional groups of the analytes, we can choose from a variety of chemical or enzymatic reactions in order to bring absorption to longer wavelengths [
1].
Buthionine sulfoximine (BSO) is a specific γ-glutamylcysteine synthetase (γ-GCS) inhibitor that blocks a rate-limiting step in glutathione (GSH) biosynthesis by inhibiting the synthesis of glutamylcysteine, the first precursor of GSH [
2]. BSO is a chiral molecule and its racemate was first successfully synthesized in 1979 [
3]. The two isomers (D and L) were also prepared and investigated (
Figure 1), however
l-buthionine-(
S,R)-sulfoximine, contrary to the racemate and the D isomer, shows a high inhibition efficacy [
4]. More recently, a new improved synthetic route of the racemate, using mild and safe conditions, was also reported [
5].
The fact that BSO is capable of altering metabolism makes it a promising key molecule for cancer therapeutics, and its role in cell growth inhibition, apoptosis induction [
6], drug resistance reduction [
7], and as a cancer adjuvant therapeutic in ovarian clear cell carcinoma [
8], and others [
9] has already been investigated.
Nevertheless, BSO detection and quantification in biological assays is still limited, mainly due to its very low molar absorptivity and an absorption maximum below 200 nm [
10]. Therefore, its detection is difficult using high-performance liquid chromatography (HPLC) using a UV detector. BSO detection, along with isomers separation, was first reported in 1987 using HPLC after sample derivatization with
o-phthalaldehyde [
11]. However, this method has several limitations. Because of the instability of the
o-phthalaldehyde derivative at room temperature, each sample was prepared immediately prior to injection onto the HPLC column. Additionally, butyrophenone, used as the internal standard, required a fluorescence detector in addition to the UV detector. Another limitation of this method was the prolonged time of the analysis (>75 min/sample). Later, in 1993, an alternative method was developed. In this case, derivatization was performed using phenylisothiocyanate, which produces a more stable derivative [
12], but its high toxicity is a major drawback.
In this work, we developed a simple, safe, and fast methodology for visible detection of l-Buthionine sulfoximine (l-BSO) using a folate-targeted polyurea dendrimer nanoformulation.
3. Materials and Methods
3.1. Reagents and Materials
l-Buthionine-(
S,
R)-sulfoximine (
l-BSO) (≥97% purity) was obtained from Sigma-Aldrich. All chemicals and solvents were used as received without further purification. Polyurea (PURE) dendrimer generation four (PURE
G4) was synthesized following our reported supercritical-assisted polymerization [
21].
3.2. Synthesis of PUREG4-FA2
Folate-targeted polyurea dendrimer generation four (PURE
G4-FA
2,
Figure 2) was prepared by reacting PURE
G4 with activated folic acid succinic ester (FA-NHS) (
Scheme 2).
FA-NHS was synthesized following the literature [
22]. Typically, in a round bottom flask, 250.0 mg (0.566 mmol) of folic acid (FA) was dissolved in dimethylsulfoxide (DMSO) (2.75 mL). After the addition of 130.8 mg (1.137 mmol) of
N-hydroxysuccinimide (NHS), 128.5 mg (0.623 mmol) of dicyclocarbodiimide (DCC), and 0.15 mL (1.082 mmol) of triethylamine (TEA), the reaction was stirred at room temperature (RT) overnight in the dark. The product was precipitated and washed several times with diethyl ether. After drying under vacuum, FA-NHS was obtained as a yellow powder (263.4 mg) in 86.4% yield.
1H NMR (400 MHz, DMSO-
d6) δ (ppm): 8.64 (1H, s), 7.63 (2H, d,
J = 8.0 Hz), 6.64 (2H, d,
J = 8.0 Hz), 4.49 (2H, s), 4.28 (1H, s), 2.54 (4H, s), 2.29 (1H, s), 2.03 (1H, s), 1.93 (1H, s).
Next, FA-NHS was conjugated with PUREG4 (via NH2 surface groups) to obtain PUREG4-FA2. In a 25 mL bottom round flask, 100 mg (0.0127 mmol) of PUREG4 was dissolved in 5.0 mL of DMSO. To this solution, 13.68 mg (0.0254 mmol) of FA-NHS and 6.9 μL (0.051 mmol) of TEA were added. The reaction was stirred at RT overnight in the dark. Next, TEA excess was removed on the rotary evaporator and diethyl ether was added. The obtained precipitate was dried under vacuum and PUREG4-FA2 was obtained as yellow oil in 93.9% yield. By NMR, it was found that two molecules of folic acid had been conjugated to the surface of PUREG4. 1H NMR (400 MHz, D2O) δ (ppm): 8.64 (2H, s), 7.70 (4H, bs), 6.86 (4H, d, J = 8.0 Hz), 4.61 (2H, s), 3.54–3.00 (180H, m), 2.96–2.40 (462H, m).
3.3. Encapsulation of l-BSO in PUREG4-FA2
In a vial, 78.0 mg (8.90 µmol) of PURE
G4-FA
2 was dissolved in 20 mL of ethanol. Then, 21.6 mg (0.0972 mmol) of
l-BSO was added and the mixture was vigorously stirred. The encapsulation occurred at RT overnight, in the dark, for 24 h. Afterwards, no
l-BSO on suspension was observed and the product was purified by dialysis for 30 min (MWCO 100–500 Da). After evaporation of the solution, the product was dried under vacuum and characterized by
1H NMR. The amount of
l-BSO loaded into the dendrimer (
[email protected]G4-FA
2) was determined by
1H NMR.
1H NMR (300 MHz, D
2O) δ (ppm): 8.60 (s, 2H), 7.63 (br, 4H), 6.78 (br, 4H), 3.62 (s, 16H), 3.44–3.07 (m, 180H), 3.06–2.40 (m, 462H), 1.76 (m, 32H), 1.45 (q,
J = 6.0 Hz, 30H), 0.92 (t,
J = 6.0 Hz, 48H).
3.4. l-BSO Release Profile
l-BSO release studies were performed at 37 °C in sodium phosphate buffer medium (PBS, pH 7.4). First, 6.3 mg of
l[email protected]G4-FA
2 were dispersed in 1 mL of medium and placed in a SnakeSkin™ dialysis membrane (MWCO 3500 Da). The dialysis bag was then immersed in 60 mL of release medium and kept in at constant temperature with stirring. Samples (1 mL) were periodically collected and replaced by the same volume of fresh medium. The amount of
l-BSO released was determined by UV-Vis spectroscopy. The release of
l-BSO from the dendrimers was obtained in triplicate.
3.5. Quantification of BSO by UV-Vis Spectroscopy
Using a modified protocol [
18],
l-BSO was derivatized in order to be detected by UV-Vis spectroscopy.
l-BSO quantification was performed by adding to the samples 300 µL of catechol 2.25 mM in PBS, followed by 300 µL of sodium periodate 6.75 mM in PBS. After 60 sec, the absorption of the
l-BSO derivative (503 nm) was measured in a PerkinElmer Lambda 25 UV-Vis Spectrometer with a slit width of 5 nm at a scan rate of 240 nm min
−1 at 25 °C. For the calibration curve, standard solutions of
l-BSO were prepared in the concentration range of 0.1–150 µM in PBS and processed using the same protocol. A good correlation coefficient (R
2 = 0.997) was obtained (see
Figure S1 in Supporting Information).