Spectroscopic Studies of 6-Membered Lipoic Acid Derivative, 1,2,3-Trithiane-4-pentanoic Acid, and Its Characteristic Stereochemical Profiles

TL;DR

Scientists modified lipoic acid by adding a sulfur atom, creating a new compound with unique properties revealed through spectroscopy.

Contribution

A novel 6-membered trisulfur lipoic acid derivative was synthesized and characterized with distinct stereochemical and spectroscopic profiles.

Findings

The 6-membered trisulfur ring showed distinct 1H- and 13C-NMR profiles compared to five-membered dithiolane compounds.

Raman spectroscopy confirmed the unique chromophore profile of the trisulfur lipoic acid derivative.

TR-LA was successfully regenerated to lipoic acid with high yield and preserved stereochemistry.

Abstract

Inserting a sulfur atom into the 1,2-dithiolane ring of lipoic acid (LA racemate) is a promising approach for improving the diversity of lipoic acid (LA racemate). For this purpose, we prepared 1,2,3-trisulfur–lipoic acid derivatives (trisulfur lipoic acid (R-enantiomer, TR-LA, trisulfur lipoamide, racemate TLPA)) by the reaction of R-LA (lipoic acid R-enantiomer) or lipoamide (LPA) and H2S and performed precise stereochemical studies. As a result, the 6-membered-1,2,3-trisulfur ring (TR-LA and TLPA) showed a completely different profile from that of the five-membered dithiolane compounds (R-LA and LPA) in 1H- and 13C-NMR spectroscopy. Raman spectroscopy of TR-LA and TLPA showed a different profile to that of LA and LPA, which also indicates the uniqueness of the 6-membered 1,2,3-trisulfur ring chromophore. The regeneration of lipoic acid from 1,2,3-trisulfur lipoic acid (TR-LA) was achieved using a phosphine derivative and sodium cyanide while maintaining the stereochemistry of the chirality center, with an almost quantitative yield.

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Full text

1. Introduction

R-lipoic acid (R-LA) is an essential material that plays a significant role in energy metabolism, especially in glucose metabolism [1,2]. Furthermore, lipoic acid (R-LA) and dihydrolipoic acid (DHLA) play important roles in the antioxidant network system [3]. It is well known that the reduction potential of LA-DHLA is greater than that of oxidized glutathione (GSSG)-reduced glutathione (GSH) [4,5,6], suggesting that DHLA might reduce oxidized glutathione to its reduced form. Glutathione is well known to play an important role in keeping the physiological status of our body, and the depletion of glutathione induces severe cell dysfunction, including cell death [7]. The administration of lipoic acid (LA) is known to recover glutathione levels in cells that survive under severe conditions [8]. In this context, lipoic acid (LA) is considered the ultimate antioxidant molecule, positioned at the final stage in the antioxidant network (Figure 1) [9].

Recently, much attention has been paid to hydrogen sulfide (H_2_S), a gaseous molecule that plays a physiological role similar to that of nitric oxide (NO) and carbon monoxide (CO) [10,11,12]. H_2_S is a well-known toxic material and is used as a poison gas weapon. However, H_2_S also acts as a signal transduction material in collaboration with polysulfur compounds present in the cells [13,14]. H_2_S is also generated by the photochemical reaction of lipoic acid, although the amount of H_2_S generated is quite limited [15,16,17]. Recent LA photochemical studies have revealed the formation of glutathione trisulfide (GSSSG) in the photodecomposition of LA in the presence of GSSG [18]. This result suggests the possibility of cross-talk between H_2_S (generated in the reaction course) and GSSG (disulfide compounds) under certain reaction conditions. If the conversion of trisulfide compounds to disulfide compounds occurs with the aid of reducing materials while retaining the stereochemistry, this provides the possibility of a new antioxidant recycling system. To confirm this possibility, we used enantiopure lipoic acid (R-LA) and prepared its trisulfide derivative (TR-LA) based on a previous study [19]. We also assigned the NMR spectra and the carbon and proton positions of TR-LA. The reduction in TR-LA to R-LA was also examined, which clearly demonstrated the retention of the chirality center of the R-enantiomer. These findings might provide a new picture of the antioxidant network system involving H_2_S in the antioxidant network system in the cell.

2. Results

2.1. Precise Structural Assignment of Trisulfur-R-lipoic Acid (TR-LA)

TR-LA was synthesized based on a previously reported method using racemic lipoic acid [19] with slight modification considering the lower melting point of R-LA than that of LA (racemate). Furthermore, in the previous work, only six carbon atom signals were reported (in CDCl_3_) despite the fact that eight carbon atoms are present in racemic TLA. To obtain further information on the precise carbon signal assignment, we measured the ^1^H-NMR, COSY, ^13^C-NMR, HMBC, and HSQC spectra of TR-LA in various solvents, including CDCl_3_, CD_3_OD, CD_3_COCD_3_, and CD_3_SOCD_3_ (DMSO-d6), at various temperatures. First, we investigated the temperature-dependent profiles of the ^1^H- and ^13^C-NMR spectra of TR-LA at variable temperatures (Figure 2). In the ^1^H-NMR spectrum, the methylene and methine protons became sharp and slightly shifted as the temperature increased to 333 K. This was also the case for the ^13^C-NMR spectrum of TR-LA at 333 K. Furthermore, new carbon peaks appeared as the measurement temperature increased. Similar results were obtained in the ^1^H- and ^13^C-NMR measurements at variable temperatures of trisulfur lipoamide (TLPA, Supplementary Materials Figure S1).

To obtain a precise assignment of the proton and carbon signals, we measured TR-LA at 333 K in DMSO-d6. The ^1^H-NMR spectrum of TR-LA at 333 K (in DMSO-d6) shows the peaks shown in Figure 3A, where the methylene protons of H-7 (ring) were split at 2.25 and 1.80 ppm, respectively. The H-6 and H-8 protons (ring) were shifted to a lower field at around 3.2–3.3 ppm. The branched side-chain protons appeared as multiplets at 1.47–1.56 ppm (H-3, H-4, and H-5) and as triplets at 2.20 ppm (H-2).

The precise assignment of TR-LA was carried out using two-dimensional COSY in DMSO-d6 at 333 K (Figure 3B). The two-dimensional COSY spectrum of TR-LA clarified the correlation between the protons. For example, in the case of H-2, the COSY spectrum depicts the binding of H-2 with H-3, H-3 with H-2, H-4, H-4 with H-3, H-5, H-5 with H-4, H-6, H-6 with H-5, H-7, H-7 with H-6, H-8, and H-8 with H-7.

The ^13^C-NMR spectrum of TR-LA was also obtained in DMSO-d6 at 333 K, and the carbon atoms at C_6_ and C_8_ were not clearly visible under normal measuring conditions (293 K). The higher temperature measurement of TR-LA makes it possible to show C_5_, C_6_, and C_8_ carbons weakly but clearly (Figure 3C). The previous ^13^C-NMR measurement of TLA carried out in CDCl_3_ at room temperature showed only six carbons at 24.4, 25.4, 33.9, 36.6, 48.7, and 180.1 ppm [19]. This observation is attributed to the low solubility of TR-LA in CDCl_3_ and the significantly longer T_1_ time of the ring carbons (especially at the C_5_, C_6_, and C_8_ positions) compared to the five-membered cyclic compound (LA). Therefore, the detection of C_5_, C_6_, and C_8_ carbons was difficult. Therefore, a precise assignment is necessary to obtain information on the structural characteristics of TR-LA. The carbon atom signals of C_5_, C_6_, and C_8_ were small and broad, even under higher temperature conditions, as shown in Figure 3C.

To obtain the precise relationship between the carbon and hydrogen atoms attached to the carbon position, we measured the two two-dimensional NMR spectra HMBC and HSQC in DMSO-d6 at 333 K (Figure 3D,E).

HMBC analysis clarified the carbon and proton relationships, as depicted in Figure 3D. The correlation between the branched C_2_, C_3_, C_4_, C_5_, and C_7_ carbons and the H-2, H-3, H-4, H-5, and H-8 protons is depicted on the right side of Figure 3D.

To obtain a precise relationship between the proton and carbon, we measured the HSQC, and the results are shown in Figure 3E, in which the CH_2_ relationship is shown in blue, and the CH and CH_3_ relationships are shown in green. Only the green color was observed at the C_6_ position CH, which was clearly observed at 3.15 ppm (H-6) and 47 ppm (C_6_). Other correlations between protons and carbons (H-2 and C_2_, H-3 and C_3_, H-4 and C_4_, H-5 and C_5_, H-7, and C_7_, and H-8 and C_8_) were also clearly identified by HSQC. The precise assignments of C_6_, H-6, C_8_, and H-8 were only attained by the use of HSQC (not COSY, HMBC).

Under almost similar measuring conditions, we obtained the ^1^H- and ^13^C-NMR spectra of TLPA (Supplementary Materials Figure S2A–E).

The ^1^H- and ^13^C-NMR spectral results are summarized in Table 1, including the spectral results of R-LA, LPA, TLPA (in DMSO-d6), and TLA (in CDCl_3_).

In the ^13^C-NMR spectra of TLA in CDCl_3_, two carbons were not assigned, and only six carbons were reported in the literature [19]; however, in DMSO-d6 at high temperature (333 K), we observed eight carbons. The carbon atom observed at δ 33.9 ppm (in CDCl_3_) corresponds to three overlapping carbons, which is attributed to the low solubility of TR-LA in CDCl_3_. These three carbons exhibited almost the same chemical shifts as those in TR-LA. In the cases of TR-LA and TLPA, the C_6_ position showed signals at 47.1 and 47.2 ppm, respectively, whereas in the cases of R-LA and LPA, the C_6_ position carbons were observed at 55.9 and 55.9 ppm, respectively. In TR-LA and TLPA, C_7_ positions showed signals at 32.6 and 32.6 ppm, respectively, while in the cases of R-LA and LPA, C_7_ positions showed 39.6 and 39.6 ppm, respectively. As shown in Table 1, the protons at the C_6_ position of TR-LA and TLPA had a higher magnetic field shift than those of the corresponding R-LA and LPA, respectively. Furthermore, the C_8_ position of TR-LA and TLPA showed signals at 34.4 and 34.4 ppm in DMSO-d6, while in the cases of R-LA and LPA, the C_6_ position carbons showed signals at 37.9 and 37.9 ppm. The order of the higher magnetic field shift of the ring carbons was C_6_ > C_7_ > C_8_. However, the carbons attached to the side chain did not show clear lower or higher magnetic field shifts in either case (less than 3 ppm, except for the C_3_ carbon of R-LA and TR-LA and the C_4_ carbon of LPA and TLPA). In the ^13^C-NMR spectrum, the carbon atoms of TR-LA on the ring shifted to a higher magnetic field than those of R-LA, especially in the case of the C_6_ carbon. The C_7_ carbon atom of TR-LA, adjacent to the C_6_ carbon, also shifted in a higher magnetic field than that of R-LA. In the case of LPA and TLPA, a similar higher magnetic field shift of the C_6_ and C_7_ carbons was observed. In the case of C_8_ carbon atoms of TR-LA and TLPA, higher magnetic field shifts were observed in both cases; however, the higher shift values (less than half the value shift) were lower than those of the C_6_ and C_7_ carbon atoms in both cases. These results clearly demonstrate that carbons consisting of six-membered trisulfur rings are shifted to a higher magnetic field than those consisting of five-membered rings (R-LA and LPA).

In the ^1^H-NMR spectra in DMSO-d6 at 333 K, the H-6 protons of TR-LA and TLPA showed a higher magnetic field than those of R-LA and LPA by approximately 0.4 ppm, respectively. In the case of H-7, one proton shifted to a higher magnetic field (ca. 0.2 ppm), while another proton shifted to a higher magnetic field of less than 0.1 ppm. In the case of C_8_ protons, a small lower magnetic field shift (0.1 ppm) was observed in both cases. Based on these results, it is reasonable to conclude that all these chemical shift changes observed in five- (LPA, R-LA) and six-membered rings (TR-LA, TLPA) might arise from the structural difference of these molecules.

2.2. Raman Spectroscopy

Raman spectra of R-LA, TR-LA, LPA, and TLPA measured in their solid states are shown in Figure 4. The carboxylic acid (R-LA and TR-LA) and amide (LPA and TLPA) compounds can be easily distinguished from one another. The amides have amide I and II bands at 1674 and 1585 cm^−1^ for LPA and 1670 and 1598 cm^−1^ for TLPA, respectively. They also have symmetric and antisymmetric N-H stretching bands (v_s_(NH_2_) and v_as_(NH_2_)) at 3340 and 3160 cm^−1^ for LPA and 3390 and 3160 cm^−1^ for TLPA, respectively. In contrast, carboxylic acids have only one peak in those ranges; that is, the C=O stretching band appeared at 1645 cm^−1^ for R-LA and 1650 cm^−1^ for TR-LA. To discuss the differences in the Raman spectra of the disulfide and trisulfide compounds, the observed spectra in the C-H, C-S, and S-S stretching regions (ν(C-H), ν(C-S), and ν(S-S)) were compared with the spectra calculated using density functional theory (DFT), and the assignments based on these calculations are also shown in Figure 4a,b. The optimized geometries are shown with the total electron energy in Figure 5. The Raman spectra in the ν(C-S) and ν(S-S) regions of lipoic acid and related molecules are known to be sensitive to molecular geometry and environmental conditions [20,21,22,23]. Figure 4c clearly shows that the spectra changed with the number of sulfur atoms. R-LA and LPA are disulfide compounds and have a single ν(S-S) band at 516 and 500 cm^−1^, respectively. TR-LA and TLPA are trisulfide compounds, and the ν(S-S) was split into symmetric and antisymmetric modes owing to the vibrational coupling of the two S-S stretching vibrations, which were observed at 508 and 490 cm^−1^ in TR-LA and 516 and 498 cm^−1^ (shoulder) in TLPA. Regarding the ν(C-S) bands, two bands of comparable intensities were observed at 636 and 678 cm^−1^ for R-LA and at 708 and 674 cm^−1^ for LPA. In contrast, one weak and one strong band were observed at 623 and 661 cm^−1^ in TR-LA and at 699 and 644 cm^−1^ in TLPA, respectively. Regarding the ν(C-H) region (Figure 4a), the Raman peaks in the 2800–2900 cm^−1^ range originate from the methylene groups in the side chain, and those in the 2900–3000 cm^−1^ range originate from the methine and methylene of the five- or six-membered ring. Therefore, differences in the Raman spectra of the carboxylic acids (R-LA and TR-LA) and the amides (LPA and TLPA) are observed in the former range, and those of the five-membered ring compounds (LPA and R-LA) and the six-membered ring compounds (TLPA and TR-LA) are observed in the latter range.

2.3. Stereochemistry of TR-LA and Back Reaction of TR-LA to R-LA

In a previous report [19], the starting material used was LA (racemate), so it is not clear whether the stereochemistry was retained under the reaction conditions. The reaction mechanism is considered to start with the oxidation of the sulfur atom of the dithiolane ring, which affords two oxidized lipoic acids (β-lipoic acid and thiosulfinate). The nucleophilic attack of disodium sulfide on the dithiolane ring provides the ring-opened compound, which smoothly rearranges to form the corresponding sulfenic acid. An additional intramolecular nucleophilic attack of the thiosodium ion on the sulfenic acid moiety is used to provide TR-LA as the final product. If this type of reaction occurs during the reaction, the stereochemistry at the C_6_ position might remain unchanged. To clarify this point, we used R-LA as the starting material. If the C-S bond scission occurs during the reaction, the optical purity is lost in the final product (TR-LA). As a result, we obtained optically pure TR-LA [α] $[eqn]$ −47.9 (c 1.0, MeOH). This result suggests that the sulfur atom of the hydrogen sulfide used in the reaction might be incorporated into the center position of the trisulfide ring.

The reductive reaction of TR-LA to R-LA is interesting considering the physiological importance of R-LA. Under certain circumstances, this type of reaction may occur, indicating a new role of hydrogen sulfide in the physiological system. To confirm this, we carried out a reaction of TR-LA and triphenylphosphine (reducing agent) in MeOH. After the reaction, we isolated LA by medium-pressure liquid chromatography and measured the ^1^H-NMR along with the densitometer to obtain the optical purity of R-LA. Although the isolated yield is not satisfactory (ca. 30%), the optical rotation measurement of the obtained R-LA was [α] $[eqn]$ +100.0 (c 0.105, MeOH). The commercially available standard sample used in the reaction was [α] $[eqn]$ +114.8 (c 0.105, MeOH). From these experimental results, it can be concluded that more than 90% (93.65%) of the R-stereoisomer was retained in this reduction procedure. Although some side reactions might occur under the reaction conditions employed, more than 90% of the sulfur atom were eliminated from the central sulfur atom, suggesting the possibility of TR-LA to R-LA occurring in the cells. We also carried out the reaction of TR-LA in CDCl_3_ in the presence of NaCN, and new peaks corresponding to R-LA were observed after 5 days, as shown in Figure 6. This result also demonstrates that a reductive reaction occurred in the presence of ^−^CN. In sulfane sulfur metabolism, the transformation of toxic cyanide to nontoxic thiocyanide occurs in vitro and in vivo, and the R-LA/R-DHLA system is involved in this rhodanese reaction [24].

Furthermore, to confirm the optical purity, we measured the CD spectra of R-LA (A), where TR-LA reacted with PPh_3_(B) and TR-LA reacted with NaCN(C), and TR-LA(D) was performed under the same reaction conditions. The results shown in Figure 7 clearly demonstrate that TR-LA reacted with PPh_3_ or NaCN and was smoothly converted to R-LA. In contrast, TR-LA exhibited a profile that was completely different from that of R-LA. These results also support the retention of the chirality center at the six position during the synthesis of TR-LA and TR-LA reduction to R-LA.

3. Discussion

The synthesis of six-membered trisulfur lipoic acid (TR-LA) was previously carried out using racemic lipoic acid (LA) [19]; however, we used naturally occurring R-LA as the starting material. This is because of the precise evaluation of the reaction course along with the examination of the stereochemistry in the reaction course. First, the ^13^C-NMR structure determination of TLA in a previous report [12] is not completely assigned; for example, the chemical shifts of six carbons were reported in TLA, although TLA has eight carbons. This may be due to the low solubility of TLA in CDCl_3_. First, we measured ^13^C-NMR in DMSO-d6 (polar solvent), and using HSQC and HMBC, we completely assigned the carbon signals along with the related proton signals, as shown in Table 1. The use of R-LA in the reaction indicated whether stereochemical changes may occur at C_6_. The optical rotation of R-LA was [α] $[eqn]$ +114.8 (c 0.105, MeOH), while that of TR-LA was [α] $[eqn]$ −47.9 (c 1.0, MeOH). No racemization occurred under the reaction conditions, suggesting that the reaction does not involve C-S cleavage at the C_6_ position.

The ^13^C-NMR spectrum of R-LA was smoothly measured under normal measuring conditions; however, in the case of TR-LA, the measurement was very difficult, especially for the carbon atoms (C_6_, C_7_, and C_8_) consisting of the six-membered ring chromophore. To obtain precise information on the TR-LA, we measured the NMR spectra (HMBC and HSQC, Supplementary Materials Figure S2D,E) at high temperatures. R-LA and TR-LA showed a clear difference in their NMR spectra. LA showed a clear coupling constant in ^1^H-NMR, whereas, in the case of TR-LA, the coupling constants of hydrogens attached to the C_6_, C_7_, and C_8_ carbons were difficult to decipher, even at high temperatures. This difference arises from the different conformational states of R-LA and TR-LA. To obtain information on the difference spectroscopically, we focused on Raman spectroscopy measurements. It is well known that C-S and S-S stretching modes are very weak in the IR spectrum [25]; however, they show strong bands in Raman spectroscopy [20,21,22,23]. RLA is a five-membered cyclic dithiolane ring compound, whereas TR-LA is a six-membered cyclic trithiane compound. The conformations are completely different, and the torsional angles due to S-S and S-S-S are also different, which affords different stable stereoisomers. Therefore, focusing on the ν(C-S) and ν(S-S) bands is quite meaningful for considering the stable stereoisomer of these compounds.

From the stem wood of Cassipourea guianensis, cassipoureamide A, bearing a 1,2-dithiolane compound, and B, bearing a 1,2,3-trithiane compound, were isolated [26]. The 1,2-dithiolane ring carbon atoms of cassipoureamide A showed clear peaks in the ^13^C-NMR spectrum, whereas the carbon atoms of the 1,2,3-trithiane ring of cassipoureamide B showed broad carbon peaks in the ^13^C-NMR spectrum. This observation is the same as that observed in the TR-LA and TLPA ^13^C-NMR spectra.

In the molecular calculation using Gaussian 16, one stable stereoisomer was calculated for R-LA and LPA. In the cases of TR-LA and TLPA, two conformers were obtained, and the energy difference between these two conformers was very small (ca. 3 kcal/mol) in both cases. Therefore, in both cases, the twisted conformers occupying C_5_ and C_8_ in the gauche conformation (conformer-2) were more stable than the chair conformers occupying C_5_ and C_8_ in the anti-conformation (conformer-1). According to a computational study of 1,2,3-trithiane, the calculated energy difference between the chair conformer and distorted 1,4-boat transition state was 10.59 kcal/mol [27]. However, the energy difference between the chair and boat foam is reported to be approximately 5.5 kcal/mol [28]. The calculated energy difference between the conformers of TR-LA and TLPA was less than 3 kcal/mol, and these values were lower than those of non-substituted 1,2,3-trithiane and cyclohexane. Considering that these energy differences are quite small, this indicates that two conformers of TR-LA and TLPA can exist at the same time under normal conditions, such as at room temperature. By increasing the temperature, the two conformers rapidly convert to each other to form an equilibrated state, which affords clear ^1^H- and ^13^C-NMR spectra. The methylene proton coupling became clear, as shown in Figure 2 and Figure 3A. It is also indicated that the slight shift of the proton signals from those at 300 K was observed at 333 K. In the ^13^C-NMR spectra, new carbon peaks were observed in TR-LA (TLPA) at 333 K. These results suggest that a rapid equilibrium might occur between the two conformers, resulting in the formation of a single conformer. Subsequently, one conformer in equilibrium provided clear peaks in the ^1^H- and ^13^C-NMR spectra at 333 K.

The UV spectra of R-LA and TR-LA were quite different; R-LA had an absorption band at approximately 320 nm owing to the torsional five-membered ring of the S-S bond (Figure 8a), whereas TR-LA did not show an absorption maximum at 300 nm (Figure 8b). TR-LA exhibited a peak maximum at approximately 263 nm, similar to that of linear disulfide compounds [15,16]. This difference also arises from the structural difference between R-LA and TR-LA. R-LA occupied a distorted structure owing to its S-S bond, whereas TR-LA does not have a distorted structure. Therefore, the absorbance in the UVA region observed in R-LA was not observed in TR-LA.

From a physiological standpoint, the oxidation of lipoic acid might proceed to various oxidized small molecules (degradation), and the recovery of oxidized lipoic acid or short-chained lipoic acid by metabolism was not known [29]. The intervention of hydrogen sulfide in the oxidized decomposition of lipoic acid is important for understanding the physiological role of hydrogen sulfide. Furthermore, hydrogen sulfide produced by the photochemical reaction of LA reacts with the oxidized form of glutathione (GSSG) to afford glutathione trisulfide (GSSSG). GSSSG reacts with the reduced form of glutathione (GSH) to afford GSSG [18]. In other words, this observation provides the possibility that hydrogen sulfide might intervene in the antioxidant network shown in Figure 1. Hydrogen sulfide is a well-known toxic gas; however, recent studies have provided new possibilities for hydrogen sulfide at low concentrations, especially as a gaseous signal transduction product [30,31,32,33,34]. Hydrogen sulfide derivatives, such as persulfide, have received much attention for their physiological roles in cells [35,36,37]. The chemical and biochemical reactivity of this type of molecule is a topic in the field of activated sulfur science [38,39,40,41,42]. Our study revealed a novel possibility that oxidized lipoic acid can be reduced with the aid of a reducing agent to regenerate lipoic acid while retaining its stereochemistry. A similar reaction might occur under certain physiological conditions. It is also important to indicate the physiological properties of the 1,2,3-trithiane ring system because some natural products have been isolated from various sources. For example, 1,2,3-trithiane-5-carboxylic acid was isolated from raw asparagus shoots [43]. From the New Zealand ascidian Aplidium sp., the 1,2,3-trithiane derivative was isolated and shown activity against leukemia cells [44]. From New Zealand ascridians, enantiomeric 1,2,3-trithiane-containing alkaloids and two 1,3-dithiane alkaloids were isolated [45]. Furthermore, Lissoclinotoxin A, an antibiotic, was isolated from L. perforatum [46]. From a chemical and biological standpoint, further studies on these compounds are necessary.

4. Materials and Methods

4.1. Reagents and Apparatus

R-LA was purchased from Tokyo Chemical Industry (Tokyo, Japan) and used without further purification. Lipoamide and Oxone^®^ were purchased from Sigma Aldrich (Saint Louis, MO, USA) and used without further purification. Sodium sulfide nonahydrate was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan), and used without further purification. All other reagents were purchased from FUJIFILM Wako Chemical Industries (Osaka, Japan) and used without further purification. NMR spectroscopy was performed using a Bruker (Billerica, MA, USA) Advance DPX™ III 400 spectrometer. J values were recorded in Hertz, and the abbreviations used were s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad). Chemical shifts are expressed in δ values relative to the internal TMS standard. Medium-pressure liquid chromatography was performed using a Yamazen Corporation (Osaka, Japan) EPCLC-AI-580S instrument. Optical rotations were measured using a JASCO (Tokyo, Japan) P-2300 polarimeter in MeOH. UV spectra were measured using a JASCO V-650 spectrometer in MeOH. The micro-Raman system was constructed using an upright microscope (Leica, DM IRE2, Wetzlar, Germany) in a backscattering configuration. The excitation light from the Nd/YAG laser (532 nm, 50 mW, DL-532-50) reflected by a beam splitter was applied to the sample using an objective lens (Olympus (Tokyo, Japan), PL FLUOTAR, 100×/NA = 0.90). CD spectra were measured in CH_3_OH (1 cm quartz cell) at 298 K using a JASCO J-820 spectropolarimeter. Melting points were measured using a Yanaco (Kyoto, Japan) Model MP-J3 micro-melting point apparatus and were uncorrected. IR spectra were recorded using a SHIMADZU IRSpirit-X FTIR Spectrometer (Kyoto, Japan). High-resolution ESI-TOF mass spectra were recorded on a Brucker Daltonics micrOTOF II mass spectrometer.

4.1.1. Synthesis of Trisulfur-R-lipoic Acid (TR-LA)

(R)-lipoic acid (2.016 g, 10 mmol) was dissolved in 75% ethanol solution, and the solution was maintained at 273 K. Oxone^®^ (3.403 g, 10.19 mmol) was slowly added to the solution to maintain the temperature, and the solution was stirred for 2 h after the addition. After the reaction, the inorganic material produced was subjected to celite filtration. The filtrate was collected in an Erlenmeyer flask, to which a solution (14 mL) containing sodium sulfide nonahydrate (5.813 g, 24.2 mmol) was added slowly. In this procedure, the solution was kept at pH 6–8 using 3 mol/L sulfuric acid by checking the pH with pH test paper. Subsequently, 3 mol/L sulfuric acid was added to lower the solution pH below 1.5. Ethyl acetate (45 mL) and water were added to the reaction mixture, and the ethyl acetate layer was collected. The water layer was further extracted with successive ethyl acetate (20 mL) twice. The combined ethyl acetate layer was evaporated to afford an oily residue, to which ethyl acetate solution (20 mL) was added, and the solution was dried using disodium sulfate. After filtering the disodium sulfate, the filtrate was evaporated in vacuo at 303 K to afford crude trisulfur lipoic acid (1.576 g). The crude trisulfur lipoic acid was dissolved in MeOH, and separation was performed by medium-pressure liquid chromatography using gradient conditions from MeOH/H_2_O = 50:50 (15 min) to MeOH/H_2_O = 70:30 (10 min). Trisulfur lipoic acid (0.4253 g) was obtained after 80 min. for the injection, along with the starting material ((R)-lipoic acid 0.1878 g) for 50 min. Rf = 0.22 (R-TLC, MeOH/H_2_O = 7:3 v/v);mp: 56.9–57.8 °C;IR: 2923, 1710, 1652, 1457, 1404, 1270, 1252, 1185, 1129, 923, 883, 749, 598 cm^−1;^CD (MeOH, c 0.545 mmol/L) λ_ext_ 212 nm (Δε −6.42), 257 nm (Δε −0.78), 272 nm (Δε −0.34), 312 nm (Δε +0.32);ESI-HRMS calcd for C_8_H_13_O_2_S_3_ m/z [M-H]^−^: 237.0078. Found: 237.0080.

4.1.2. Synthesis of Trisulfur Lipoamide (TLPA)

d,l-lipoamide (LPA, 300 mg) was dissolved in 85% dimethylformamide solution (55 mL) and stirred at 273 K for 1 h. Oxone^®^ (150 mg) was added to the reaction mixture three times at 10 min intervals. The progress of the reaction was monitored by TLC, and the pH of the reaction mixture was monitored using pH test paper. It was stirred for 4 h. One-third of disodium sulfide 9 hydrate (0.35 g total) was added three times at 10 min intervals, keeping the solution pH range at 5–11 and adding 3 mol/L sulfuric acid to keep the pH range. The progress of the reaction was monitored by TLC, and the reaction was continued for 1 h at pH 9.0. Distilled water (54 mL) was added to the reaction mixture, and the mixture was extracted twice with methylene chloride (15 mL). The combined methylene chloride solution was dried using disodium sulfate and stored for three days. The solution was filtered, and the filtrate was evaporated in vacuo at room temperature (below 303 K) to afford an oily residue. Distilled water (24 mL) was added to the residue, and the solution was evaporated in vacuo at low temperature to afford a solid. The solid was filtered, washed with distilled water, and dried under vacuum to afford Trisulfur d,l-lipoamide (0.090 g). Recrystallization of the trisulfur d,l-lipoamide (TLPA, 0.090 g) in MeOH-H_2_O afforded pure TLPA (0.046 g) as a white solid.

4.1.3. NMR Spectral Analyses of TR-LA and TLPA

^1^H- and ^13^C-NMR spectra of TR-LA, R-LA, TLPA, and LPA were measured using a Bruker Advance DPX™ III 400 spectrometer using various solvents at 300 K. To obtain the precise assignment of each proton and carbon atom, NMR spectra measurements were carried out in DMSO-d6 by increasing the temperature to 333 K. Under these conditions, HSQC, HMBC, and COSY spectra of TR-LA and TLPA (Supplementary Materials Figure S2A–E) were measured to obtain the precise assignment of each carbon and proton.

4.1.4. Reaction of TR-LA with Triphenylphosphine

According to the literature [47], the reaction was modified as follows: TR-LA (119 mg) in methylene chloride (CH_2_Cl_2_, 10 mL) was added to triphenylphosphine (131 mg) slowly. The reaction was monitored by TLC for 2 h and then stirred for 18 h at room temperature. After the reaction, the reaction mixture was evaporated in vacuo at room temperature to afford a solid residue, which was analyzed by ^1^H-NMR to confirm the presence of R-LA. The reaction mixture was subjected to medium-pressure liquid chromatography at a gradient interval of 0–3 min. (water: MeOH = 70:30), 11–19 min. (water: MeOH = 60:40), 27–35 min. (water: MeOH = 50:50), and 43–53 min. (water: MeOH = 0:100). The flow rate was 8 mL/min, and the sample detection was performed using a UV detector at 254 nm. R-LA was obtained at around 40 min range, and the fractions were collected. After evaporating the collected fractions, R-LA was obtained (31.2 mg, 30% yield). The optical purity was measured to be [α] $[eqn]$ +100.0 (c 0.105, MeOH). The standard sample under the same conditions was [α] $[eqn]$ +114.8. From these two results, the optical purity of the sample was calculated to be 87.30% (R-LA 93.65, S-LA 6.35%). Synthetic: CD (MeOH, c 0.582 mmol/L) λ_ext_ 216 nm (Δε −2.22), 237 nm (Δε −0.51), 313 nm (Δε −0.24), 365 nm (Δε +0.12);[Purchased: CD (MeOH, c 0.727 mmol/L) λ_ext_ 214 nm (Δε −2.52), 236 nm (Δε −0.53), 316 nm (Δε −0.22), 364 nm (Δε +0.15)];ESI-HRMS calcd for C_8_H_13_O_2_S_2_ m/z [M-H]^−^: 205.0357. Found: 205.0363.

4.1.5. Reaction with TR-LA with Sodium Cyanide (NaCN)

According to the literature [48], the reaction was modified as described below. To a solution of TR-LA (115 mg, 0.4824 mmol) in H_2_O (1 mL), 1 mol/L NaOH solution (0.72 mL, 0.72 mmol) was added. After stirring for 1 h, NaCN (20 mg, 0.4020 mmol) was added slowly to the combined solution, and the reaction mixture was stirred for 9 h at room temperature. After the reaction, the pH of the reaction mixture was adjusted to the pH 4–5 range by the use of 1 mol/L HCl solution at 0 °C. The reaction mixture was extracted three times with 5 mL of ethyl acetate (total 15 mL). The extract was washed twice with 1 mL of water (total 2 mL) and 1 mL of brine. After washing, the extract was dried using disodium sulfate (Na_2_SO_4_). After filtration, the organic layer was evaporated in vacuo at room temperature. The residue was purified using MPLC (same conditions described in 4.2.1) to obtain R-LA as a pale-yellow solid (16.4 mg, 0.0795 mmol). The optical purity of the isolated sample was measured using a JASCO P-2300 polarimeter, which showed [α] $[eqn]$ +116.2 (c 0.16, MeOH), and the stereochemistry was retained throughout the reaction. Synthetic: CD (MeOH, c 0.485 mmol/L) λ_ext_ 215 nm (Δε −3.40), 234 nm (Δε −0.73), 314 nm (Δε −0.31), 364 nm (Δε +0.21);ESI-HRMS calcd for C_8_H_13_O_2_S_2_ m/z [M-H]^−^: 205.0357. Found: 205.0361.

4.1.6. Microscopic Raman Spectroscopy of R-LA, TR-LA, LPA, and TLPA

The Raman spectra of all samples were obtained using a micro-Raman system under the following conditions. The excitation light from the Nd/YAG laser (532 nm, 50 mW, DL-532-50) was reflected by a beam splitter and directed onto the sample using an objective lens (Olympus, PL FLUOTAR, 100×/NA = 0.90). The scattered light was collected using the same objective lens, transmitted through the same beam splitter, and filtered using a long-pass filter to remove Rayleigh scattering. Raman scattering was guided to a spectrometer (Princeton Instruments (Trenton, NJ, USA), Acton SP2800, grating: 1200 g/mm, 500 nm blaze) through an optical fiber and detected by a cooled CCD detector (Princeton Instruments, Pixis256, 1024 × 256 pixels) for spectral measurements.

4.1.7. Molecular Calculation of R-LA, TR-LA, LPA, and TLPA

Molecular calculations were performed for an isolated molecule in the gas phase using Gaussian16 on the supercomputer of ACCMS, Kyoto University. The structural optimization and vibration analysis in the ground state of the molecule were calculated based on density functional theory (CAM-B3LYP), which considers long-range interactions and the basis function of 6-311++g (d, p).

5. Conclusions

R-LA is an important antioxidant molecule present in the cells; however, the amount is quite limited. The oxidative degradation of lipoic acid affects the physiological state of our body; therefore, the recovery of lipoic acid is important. R-LA also plays a key role in the antioxidant network system due to its very high reducing ability; it is sometimes called the ultimate antioxidant. In this study, we found that the interaction of hydrogen sulfide and lipoic acid might occur in vivo. Oxidized lipoic acid reacts with hydrogen sulfide to afford TR-LA, which is reduced to R-LA by a reducing reagent, such as triphenylphosphine or cyanide ion. Furthermore, the use of poly-lipoic acid systems as a possible H_2_S donor molecule has recently gained attention for potential medical applications in heart disease [49], chronic kidney disease (CKD) [50], and cancer therapy [51]. It is unclear whether these studies are strongly connected with the antioxidant network system in cells; however, these studies and our results provide new potential uses for TLA and TR-LA in physiological and medical fields.