Herein we report the efficient synthesis of phenolic peptides by coupling amino acid esters and phenolic acids in the presence of a series of promoters; we then evaluated their activities as both antioxidant and anti-proliferative agents. The attachment of different amino acid residues to a phenolic structure might modulate both their bioavailability by improving uptake through the cell membrane, and their biological properties. This might therefore supply valuable information for structure-activity relationship studies.

A plethora of coupling reagents has been reported to date for the activation of the carboxylic acid moiety in peptide bond formation[27]. Among them, a carbodiimide, a phosphonium salt and a uranium salt were selected herein, giving four different synthetic pathways (Scheme 1, Methods A1, A2, B and C). Methods A1 and A2 involve the use of PyBOP (benzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate) with different solvents and basic conditions (anhydrous CH₂Cl₂ and DIPEA for A1, and anhydrous DMF and Et₃N for A2), whereas EDC/HOBt 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride)/1-hydroxybenzotriazole) and HATU (1-(bis(dimethylamino)methylene)-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) were selected in methods B and C, respectively.

In this context, the chosen phenolic templates were 3,4-dihydroxyphenylacetic acid (DOPAC) 12 and its O-protected counterpart 1. The former is a naturally-occurring phenolic compound formed by monoamine oxidase-mediated dopamine metabolism[28], exhibiting relevant biological properties; for instance, sub-stoichiometric concentrations of DOPAC have proven to inhibit the fibrillation of α-synuclein, a critical step in the etiology of Parkinson’s disease[29]. A series of derivatives bearing the 1,2-methylenebenzene scaffold, present in compound 1, have been reported to exhibit different biological properties[30]. Herein, the four methodologies indicated above were applied to compound 1 together with amino acid ester hydrochlorides 2-6, derived from glycine, phenylalanine, valine, serine and tryptophan (Scheme 1). When the uranium salt HATU was used as the coupling reagent (Scheme 1, method C), the formation of the targeted peptide 7 [30]^b was observed by ¹H-NMR spectroscopy, but the reaction was not complete even after 60 h, and furthermore, a significant amount of side-products was also observed; for all these reasons, this procedure was discarded.

Replacement of HATU with the phosphonium salt PyPOP in CH₂Cl₂ (method A1) or with the combination of EDC and HOBt as an additive (method B) allowed for the isolation of 7, 9-11 with moderate to good yields (40-74%, or 33-79%, respectively), but the reaction times were still relatively long, especially for the latter conditions (22-60 h). Remarkably, replacement of anhydrous CH₂Cl₂ by dimethylformamide (DMF) as a solvent, but retaining PyBOP as the coupling reagent (method A2), led to a significant increase in the yield of peptides 7-11 (77%-quantitative), and a general decrease in the reaction time (24-28 h), as depicted in Scheme 1.

The same conditions were employed for DOPAC 12 and amino acid ester derivatives 2-6 (Scheme 2).

Regarding the formation of the peptidomimetic derived from glycine 13, its synthesis was attempted using the four synthetic methodologies indicated in Scheme 2. Analogously to the preparation of its O-protected counterpart 7, a high amount of side-products was observed in ¹H-NMR when Method C was selected; the use of HATU was therefore discarded. Methods A1, A2 and C led to the formation of 13 in 43%, quantitative, and 72% yields, respectively.

Methods A1 and B proceeded with moderate to good yields, but significant amounts of starting materials were observed even after 60 h of reaction. Remarkably, Method A2 has again proven to be the best synthetic choice, where reactions took place in shorter reaction times (15-38 h) giving rise to compounds 13-17 in excellent to quantitative yields (Scheme 2).

Considering the excellent pro-apoptopic properties against HL60 tumor cells observed by hydroxytyrosyl disulfide, and chalcogen-containing polyphenols, recently prepared in our research group[31], we envisioned the possibility of preparing amides derived from L-cystine, where the combination of a dimeric phenolic template, together with a disulfide tether, might reveal significant anti-proliferative activities. Thus, using the optimized conditions of Method A2 (PyBOP, Et₃N, DMF), coupling between O-methylidene-protected DOPAC 1 and L-cystine methyl ester dihydrochloride 18 took place in a successful manner, with peptidomimetic 19 being obtained with a yield of 83% (Scheme 3).

We also attempted the preparation of unprotected disulfide 21 by direct PyBOP-mediated coupling reaction between 12 and 18. Nevertheless, attempts to isolate compound 21 using this procedure were unsuccessful, due to the difficulty in the chromatographic separation of the target compound from side-products derived from the coupling reagent. In order to overcome this problem, the crude coupling reaction between 12 and 18 was conventionally acetylated to furnish tetra-O-acetyl derivative 20, in a 72% overall yield for the two steps (Scheme 4).

Deprotection of the phenolic ester motifs was a more difficult task than previously anticipated. Firstly, strongly basic conditions (NaOMe/MeOH) should be avoided, as they enhance significantly the catechol oxidation to o-quinones, and subsequently to a complex mixture of compounds[32]. Nevertheless, mild conditions (K₂CO₃/MeOH or NH₄OAc/MeOH), or even the use of lipases from C. Antarctica and P. cepacia for the deprotection of 20 failed, as extensive decomposition or a complex mixture of compounds was observed. To our delight, treatment with CsCO₃ in a 1:1 mixture of CH₂Cl₂-MeOH afforded unprotected dimeric disulfide 21 in a good yield (65%). To our knowledge, this is the first example of a L-cystine-containing phenolic peptidomimetic.

The transformation of compound 21 into the L-cysteine derivative 23 was accomplished in a straightforward redox pathway by treatment of 21 with (±)-dithiothreitol (Scheme 4), an inexpensive dithiol quite useful in the mild reduction of disulfide linkages[33]; derivative 23 was isolated with a 44% yield after chromatographic purification.

2.2. Biological evaluation

2.2.1. Antioxidant activity

The capacity of unprotected peptidomimetics 13-17, 21 and 23 to scavenge ROS (free radicals, H₂O₂ and alkyl hydroperoxides) has been evaluated; the results were compared with natural hydroxytyrosol (HT), an abundant phenolic compound in olives.[32] ^b This study is an important task for the preliminary evaluation of the compounds, as their inherent antioxidant capacity is responsible for many of their biological properties.

To evaluate the antiradical activity, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used [34]. DPPH is a commercially-available free radical widely used for the quantification of the free radical scavenging activity of an antioxidant agent. DPPH solutions exhibit a deep purple color, with a λ_max at 515 nm; the presence of an H-donor acting as a free radical scavenger reduces DPPH, and hence a decrease in the absorbance is observed. Quantification of the potency of an antioxidant is carried out by calculating EC₅₀, that is, the concentration of the antioxidant that scavenges 50% of the original DPPH. Therefore, there is an inverse relationship between EC₅₀ values and the potency of the antioxidant. Calculated EC₅₀ values in the DPPH assay for various compounds, along with that found for HT, are depicted in Figure 1. It can be observed that phenolic peptides 13-17 and 23 exhibit an antiradical activity similar to HT (EC₅₀ = 11.3 ± 1.3 µM), with EC₅₀ values ranging from 10.5-18.4 µM. The activity found for disulfide 21 is especially remarkable as it behaved as a potent antioxidant agent (EC₅₀ = 4.5 ± 0.2 µM), with a 2.5-fold increase in activity when compared with natural HT.

To determine H₂O₂ scavenging activity, we used the methodology developed by Bahorun et al.[35]; in this assay, the peroxidase-mediated oxidation of phenol red with H₂O₂ is followed spectrophotometrically at 610 nm. The percentage of scavenging exerted by the tested compounds at 1.0 mM concentration is depicted in Figure 2. Compounds 14-16 exhibited virtually the same scavenging activity (60.7-65.1%) as HT (68.8 ± 5.0%); derivatives 17 and 23 showed reduced activity compared to reference compound (34.5 ± 1.0, 54.0 ± 5.3%, respectively). Remarkably, glycine and L-cystine derivatives 13 and 21 behaved as more potent H₂O₂ scavengers (74.6 ± 2.3% and 77.7 ± 10.3%, respectively) than HT.

The inhibition of the first stages of the lipid peroxidation was also evaluated. For this purpose, linoleic acid was used as the lipid model, and AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was employed as a free radical initiator that affords alkyl peroxides by thermal degradation of linoleic acid [36]. The inhibition of the lipid peroxidation exerted by the tested compounds at 0.74 mM concentration is depicted in Figure 2. In this assay, HT behaved as a poor antioxidant agent (27.4 ± 8.6% inhibition). Remarkably, peptides derived from glycine 13 (47.7 ± 4.9%), L-phenylalanine 14 (54.8 ± 0.4%), L-serine 16 (45.2 ± 4.6%), L-tryptophan 17 (43.1 ± 11.7%) and L-cysteine 23 (53.9 ± 5.3%) turned out to be more efficient antioxidants for lipid protection than natural HT.

2.2.2. Anti-proliferative activity assay

Some natural polyphenols, like curcumin, ferulic acid, green tea polyphenols, or resveratrol, have been reported to exhibit chemopreventive and chemotherapeutic effects against cervical cancer [37], the second most common cancer worldwide [38]. This prompted us to assess the anti-proliferative effects of the synthesized peptidomimetics and to compare their activity with natural products like DOPAC and HT; in this study, three different cervical cancer cell lines were used: HeLa and CaSki, associated with the human papillomavirus (HPV), and ViBo, which is not associated with HPV.

The degree of necrosis involved in cell death, together with the selectivity against tumor cells compared to normal healthy human lymphocytes were also determined, a key property of chemotherapeutic agents.

In vitro anti-proliferative activity

Anti-proliferative activity against human cervical cancer cell lines (HeLa, CaSki, and ViBo) was determined after 24 h of incubation of the tested compounds (13-16, 20, 21, 23, HT and DOPAC) by crystal violet staining[39], and using MeOH or DMSO as solvent vehicles. Compound 17 could not be tested due to the lack of solubility under the experimental conditions. The inhibitory effect of title compounds on the proliferation of the tumor cell lines was observed to occur in a dose-dependent manner; calculated IC₅₀ values are depicted in Table 1.

Natural phenolic compounds HT and DOPAC behaved as moderate antitumor agents, with EC₅₀ values ranging from 227.8-765.0 µM (Table 1). In general, synthetic peptides 13-16 exhibited reduced activity compared to HT and DOPAC. O-Acetylated peptide 20, bearing a disulfide linkage behaved as a slightly better antitumor agent (IC₅₀ 199.7-229.6 µM) than HT. Similar results were obtained for unprotected L-cysteine derivative 23. Remarkably, symmetrical fully unprotected compound 21, derived from L-cystine, was found to be the strongest anti-proliferative agent (108.2-121.8 µM), with a 2-7-fold increase in activity in comparison with natural DOPAC and HT. This result indicates that combining a dimeric phenolic template with a disulfide linkage results in a substantial enhancement of the anti-proliferative properties. A similar behavior was found for hydroxytyrosyl disulfide against HL60 tumor cells.

Evaluation of cell necrosis in tumor and non-tumor cells

Cell death can occur via two different pathways: apoptosis (a programmed cell death) or necrosis (an unordered and accidental form of cell death) [40] . As the desired cellular death in a chemotherapy treatment is apoptosis, measurement of the degree of necrosis is an important concern when developing antitumor agents. In order to determine if a necrotic process is involved in the anti-proliferative activity, the lactate dehydrogenase (LDH) assay was carried out. The amount of LDH released to the supernatant is a measure of the loss of membrane integrity, and thus, an indication that a necrotic process takes place. Triton X-100, a non-ionic polyethoxylated surfactant detergent was used as a reference compound to induce cellular lysis [41]. The three tumor cell lines were treated with Triton X-100 in three independent experiments, and released LDH was adjusted to 100%. Cervical tumor cell cultures were also stimulated with the tested compounds at their IC₅₀ concentrations, and the release of LDH was compared to the control groups (Figure 3A-C). The same experiments were also undertaken when studying human lymphocytes instead of human tumor cell lines (Figure 3D).

Regarding tumor cells, the compound provoking the highest degree of necrosis was the L-valine derivative 15 (37%, 22% and 37% for HeLa, CaSki and ViBo, respectively). The rest of the peptides exhibited an exceptionally low ratio of necrosis; HT, which exhibited a good IC₅₀ value, turned out to be involved to a significant extent in necrosis when considering tumor cells death (35% and 37% for HeLa and ViBo cells, respectively). It is worth mentioning the behavior found for compounds 21 (5% and 4% of necrosis against HeLa and ViBo cells) and 23 (3%, 1% and < 0.5% of necrosis against the three cervical cultures). These data strongly suggest that when these compounds exert their cytotoxic activity against the tumor cell lines studied, they do not act via necrosis but probably via an apoptotic pathway.

Concerning human lymphocytes, the worst compound in terms of necrosis was L-phenylalanine derivative 14. Remarkably, O-unprotected Sulphur-containing peptidomimetics 21 and 23 were found to exhibit a necrotic activity that was even lower than the corresponding solvent used as a vehicle, which could indicate that they do not provoke the death of lymphocytes by necrosis, but even a moderate activation of such cells.

Evaluation of anti-proliferative activity on non-tumor cells

One of the major problems associated with conventional chemotherapeutic treatments is the lack of selectivity of the cytotoxic agents, leading to the appearance of a series of undesirable side-effects; such side effects are predominantly due to the suppression of the host immune system by affecting normal lymphocytes. It is therefore crucial to assess the selectivity of cytotoxic agents towards non-tumor cells. Herein we carried out the study of the anti-proliferative effect of the tested compounds towards peripheral blood lymphocytes. In this assay, an enriched lymphocyte population (ELP) from a normal blood donor is labelled with carboxyfluorescein succinimidyl ester (CFSE), a fluorescent dye widely used in monitoring cell division, and stimulated with phytohemagglutinin (PHA) and/or treated with the tested compounds or with the pure vehicle. After 72 h, cells were harvested and the anti-proliferative activity was determined by flow cytometry[42]; results are depicted in Figure 4. Untreated control proliferating cells were around 87% under normal conditions, and after treatment with the vehicle, roughly 83% (MeOH) and 80% (DMSO). Whereas, peptides 15, 21 and 23 displayed a remarkable selectivity against tumor cell lines, as normal human lymphocytes were practically unaffected, as shown in Figure 4 (i.e. only 2-9% loss of lymphocyte proliferation was observed). These results strongly contrast with the natural HT and DOPAC, which, although active against tumor cells, also elicited a significant decrease in lymphocyte proliferation (roughly 24% and 26%, respectively). Moreover, compound 20, the tetra-O-acetylated derivative of 21 exhibited a decreased selectivity, as it inflicted a ~ 25% decrease in lymphocyte proliferation. This observation suggests the importance of free phenolic OH’s for exhibiting a good selectivity over normal healthy cells.

3.1. General procedures

General procedures concerning NMR, MS, TLC visualization and UV-Vis spectroscopy can be found in references [31,43,44].

3.2. Determination of the antioxidant activity

3.2.1. DPPH method

The antiradical activity of the phenolic peptides (DPPH method) was measured using the procedure reported by Prior et al [34].

3.2.2. H₂O₂ scavenging activity

The H₂O₂ scavenging activity of the peptides was measured using the procedure reported by Bahorun et al. [35].

3.2.3. Lipid peroxidation assay (ferric thiocyanate method, FTC)

Inhibition of the lipid perxoxidation was measured using the ferric thiocyanate method (FTC) [36].

3.3. General procedures for anti-proliferation assays

3.3.1. Cell culture

General procedures for the cell culture of HeLa, CaSki and ViBo cell lines can be found in references [45,46].

3.3.2. Cell proliferation assay

Assays were performed as reported in reference [47]. (IC₅₀) values were determined after 24 h using crystal violet staining [39].

3.3.3. Cell necrosis study

The necrotic activity was determined as reported in reference [48].

3.3.4. Carboxyfluorescein succinimidyl ester (CSFE) labeling assay

Assays involving carboxyfluorescein were carried out as reported in reference [48].

3.3.5. Statistical analysis

For antioxidant assays, all tests were run in triplicates for each experimental condition. Values are expressed as the confidence interval, which was calculated for n=3, P = 0.95 using the Student’s t-distribution.

For anti-proliferative assays, the median and standard deviation (SD) for six measurements were calculated. Statistical analysis of differences was performed by analysis of variance (ANOVA) using SPSS 10.0 for Windows. A p-value < 0.05 (Tukey’s t-test) was considered to be significant.

3.3.6 General procedures for the preparation of peptides derived from (3,4-methylenedioxy) phenylacetic acid 1

Method A1. To a solution of 1 (50 mg, 0.28 mmol) in anhydrous CH₂Cl₂ (1.5 mL), PyBOP (166 mg, 0.32 mmol, 1.15 equiv.), the corresponding amino acid ester hydrochloride 2, 4 or 5 (0.28 mmol, 1.0 equiv.) and DIPEA (0.83 mL, 4.86 mmol, 17.4 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 24-48 h. After that, the solvent was removed under reduced pressure, and the residue was purified as indicated in each case.

Method A2. To a solution of 1 (50 mg, 0.28 mmol) in anhydrous DMF (4.0 mL) at 0ºC, PyBOP (144 mg, 0.28 mmol, 1.0 equiv.), the corresponding amino acid ester hydrochloride 2-6 (0.28 mmol, 1.0 equiv.) and Et₃N (0.16 mL, 1.11 mmol, 4.0 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 15-38 h. Thereafter, the solvent was eliminated under reduced pressure, and the residue was purified as indicated in each case.

Method B. To a solution of 1 (50 mg, 0.28 mmol) in anhydrous CH₂Cl₂ (3.0 mL), HOBt (41 mg, 0.31 mmol, 1.1 equiv.), EDC (64 mg, 0.33 mmol, 1.2 equiv.), the corresponding amino acid ester hydrochloride 2, 5 or 6 (0.31 mmol, 1.1 equiv.) and Et₃N (0.06 mL, 0.42 mmol, 1.5 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 60 h. Then, the solution was diluted with EtOAc (15 mL) and washed with H₂O (1 x 15 mL), and the aqueous phase was further washed with EtOAc (3 x 10 mL). The combined organic fractions were washed with sat. aq. NaHCO₃ (1x10 mL) and brine (1x10 mL), dried over MgSO₄ and filtrated; the filtrate was concentrated to dryness and the residue was purified by column chromatography using the eluent indicated in each case.

Method C. To a solution of 1 (50 mg, 0.28 mmol) in anhydrous CH₂Cl₂ (2.0 mL), HATU (116 mg, 0.31 mmol, 1.1 equiv.), glycine ethyl ester hydrochloride 2 (41 mg, 0.29 mmol, 1.05 equiv.) and DIPEA (0.10 mL, 0.58 mmol, 2.1 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 72 h. Thereafter, the crude reaction was washed with 1M HCl (1 x 10 mL), saturated aqueous NaHCO₃ (1 x 10 mL) and brine (1 x 10 mL). The organic phase was dried over MgSO₄, filtered and the filtrate was concentrated to dryness.

Spectroscopical data of the new compounds prepared herein can be found in https://idus.us.es/xmlui/handle/11441/53604 (researchrepository of Seville University, Doctoral Thesis of Azucena Marset-Castro).

3.3.7. Ethyl 2-[2’-(3”,4”-methylenedioxyphenyl)acetamido] acetate (7) [30]^b

Method A1. Glycine ethyl ester hydrochloride 2 was used (39 mg, 0.28 mmol), and the reaction proceeded for 48 h. Then, the solvent was removed under reduced pressure, and the residue was purified by column chromatography (hexane→1:2 hexane-EtOAc) to give compound 7: 51 mg, 69%.

Method A2. Glycine ethyl ester hydrochloride 2 was used (39 mg, 0.28 mmol), and the reaction proceeded for 24 h. Thereafter, the crude reaction was washed with 1M HCl (1 x 10 mL), sat. aq. NaHCO₃ (1 x 10 mL) and brine (1 x 10 mL). The organic layer was dried over MgSO₄, filtered and the filtrate was concentrated to dryness to give compound 7: 65 mg, 89%.

Method B. Glycine ethyl ester hydrochloride 2 was used (43 mg, 0.31 mmol), and the reaction proceeded for 48 h. Column chromatography (hexane→1:2 hexane-EtOAc) yielded compound 7: 58 mg, 79%.

Method C. Glycine ethyl ester hydrochloride 2 was used (41 mg, 0.29 mmol), and the reaction proceeded for 72 h. After removal of the solvent, the crude product was analyzed by ¹H-NMR.

R_F 0.62 (1:2 hexane-EtOAc); ¹H-NMR (300 MHz, CDCl₃) δ 6.79-6.76 (m, 2H, H-2’’, H-5’’), 6.71 (dd, 1H, J_{2’’,6’’}=1.7 Hz, J_{5’’,6’’}=7.8 Hz, H-6’’), 6.03 (brs, 1H, NH), 5.94 (s, 2H, O-CH₂-O), 4.17 (q, 2H, J_H,H=7.2 Hz, CH₂-CH₃), 3.98 (d, 2H, J_2,NH=5.3 Hz, H-2), 3.51 (s, 2H, H-2’), 1.25 (t, 3H, CH₂-CH₃); ¹³C-NMR (75.5 MHz, CDCl₃) δ 171.3, 169.9 (C-1’, C-1), 148.2 (C-3’’), 147.1 (C-4’’), 128.1 (C-1’’), 122.8 (C-6’’), 109.9 (C-2’’), 108.7 (C-5’’), 101.2 (O-CH₂-O), 61.6 (CH₂-CH₃), 43.1 (C-2’), 41.6 (C-2), 14.2 (CH₂-CH₃); CI-MS m/z 265 ([M]⁺, 57%); HRCI-MS calculated for C₁₃H₁₅NO₅ ([M]⁺): 265.0950, found: 265.0946.

3.3.8. Ethyl (S)-2-[2’-(3”,4”-methylenedioxyphenyl)acetamido]-3-phenylpropanoate (8)

Method A2. L-Phenylalanine ethyl ester hydrochloride 3 was used (64 mg, 0.28 mmol), and the reaction proceeded for 38 h. Column chromatography (hexane→1:1 hexane-EtOAc) yielded 8: 76 mg, 77%.$[α]_{D}^{26}$-5 (c 0.94, MeOH); R_F 0.48 (1:2 hexane-EtOAc 1:2); ¹H-NMR (300 MHz, CD₃OD) δ 7.28-7.16 (m, 3H, Ar-H), 7.15-7.10 (m, 2H, Ar-H), 6.70 (d, 1H, J_{5’’,6’’}=7.8 Hz, H-5’’), 6.67 (d, 1H, J_{2’’,6’’}=1.3 Hz, H-2’’), 6.63 (dd, 1H, H-6’’), 5.91 (s, 2H, O-CH₂-O), 4.64 (dd, 1H, J_2,3a=5.7 Hz, J_2,3b=8.8 Hz, H-2), 4.13 (q, 2H, J_H,H=7.2 Hz, CH₂-CH₃), 3.39 (s, 2H, H-2’), 3.14 (dd, 1H, J_3a,3b=13.9 Hz, H-3a), 2.96 (dd, 1H, H-3b), 1.20 (t, 3H, CH₂-CH₃); ¹³C-NMR (75.5 MHz, CD₃OD) δ 173.9, 173.0 (C-1’, C-1), 149.2 (C-3’’), 148.0 (C-4’’), 138.0 (Ar-C), 130.2 (x2) (2Ar-C), 130.1 (x2) (2Ar-C), 129.5 (C-1’’), 127.9 (Ar-C), 123.4 (C-6’’), 110.4 (C-2’’), 109.1 (C-5’’), 102.3 (O-CH₂-O), 62.4 (CH₂-CH₃), 55.3 (C-2), 43.1 (C-2’), 38.3 (C-3), 13.4 (CH₂-CH₃); CI-MS m/z 355 ([M]⁺, 52%); HRCI-MS calculated for C₂₀H₂₁NO₅ ([M]⁺): 355.1420, found: 355.1408.

3.3.9. Ethyl (S)-3-methyl-2-[2’-(3”,4”-methylenedioxyphenyl) acetamido] butanoate (9)

Method A1. L-Valine ethyl ester hydrochloride 4 was used (50 mg, 0.28 mmol), and the reaction proceeded for 44 h. Column chromatography (hexane→2:1 hexane-EtOAc) yielded 9: 34 mg, 40%.

Method A2. L-Valine ethyl ester hydrochloride 4 was used (50 mg, 0.28 mmol), and the reaction proceeded for 17 h. Column chromatography (hexane→1:2 hexane-EtOAc) yielded compound 9: 81 mg, 95%. $[α]_{D}^{24}$ +9 (c 0.98, CH₂Cl₂); R_F 0.54 (1:2 hexane-EtOAc); CI-MS m/z 307 ([M]⁺, 64%); HRCI-MS m/z calcd for C₁₆H₂₁NO₅ ([M]⁺): 307.1420, found: 307.1424.

3.3.10. Ethyl (S)-3-Hydroxy-2-[2’-(3”,4”-methylenedioxyphenyl) acetamido]propanoate (10)

Method A1. L-Serine ethyl ester hydrochloride 5 was used (47 mg, 0.28 mmol), and the reaction proceeded for 30 h. Column chromatography (hexane→1:3 hexane-EtOAc) resulted in compound 10: 61 mg, 74%.

Method A2. L-Serine ethyl ester hydrochloride was used (47 mg, 0.28 mmol), and the reaction proceeded for 19 h. Column chromatography (hexane→1:3 hexane-EtOAc) yielded compound 10: 65 mg, 80%.

Method B. L-Serine ethyl ester hydrochloride was used (52 mg, 0.31 mmol), and the reaction proceeded for 60 h. Liquid-liquid extractions, as described in the general procedure, yielded pure 10: 45 mg, 55%. $[α]_{D}^{23}$ +3 (c 1.22, MeOH); R_F 0.64 (1:2 hexane-EtOAc); HRCI-MS calcd for C₁₄H₁₇NO₆ ([M]⁺): 295.1056, found: 295.1049.

3.3.11. Methyl (S)-3-(1”’H-indol-3”’-yl)-2-[2’-(3”,4”-methylenedioxyphenyl) acetamido]propanoate (11)

Method A2. L-tryptophan methyl ester hydrochloride 6 was used (71 mg, 0.28 mmol), and the reaction proceeded for 15 h. Column chromatography (hexane→1:7 hexane-EtOAc) resulted in compound 11: 106 mg, quant.

Method B. L-tryptophan methyl ester hydrochloride 6 was used (78 mg, 0.31 mmol), and the reaction proceeded for 60 h. Column chromatography (hexane→1:2 hexane-EtOAc) yielded compound 11: 35 mg, 33%. $[α]_{D}^{24}$ +28 (c 1.10, (CH₃)₂CO); R_F 0.55 (1:2 hexane-EtOAc); CI-MS 381 ([M]⁺, 21%); HRCI-MS calcd for C₂₁H₂₁N₂O₅ ([M+H]⁺): 381.1450, found: 381.1436.

3.3.12. General procedures for the preparation of peptides derived from (3,4-dihydroxyphenyl)acetic acid 12 (DOPAC)

Method A1. To a solution of DOPAC (50 mg, 0.30 mmol) in anhydrous CH₂Cl₂ (1.5 mL), PyBOP (178 mg, 0.34 mmol, 1.15 equiv.), glycine ethyl ester hydrochloride 2 (42 mg, 0.30 mmol, 1.0 equiv.) and DIPEA (0.89 mL, 5.20 mmol, 17.4 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature during 48 h. Then, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (CH₂Cl₂→10:1 CH₂Cl₂-MeOH).

Method A2. To a solution of DOPAC (50 mg, 0.30 mmol) in anhydrous DMF (4.0 mL) at 0ºC, PyBOP (155 mg, 0.30 mmol, 1.0 equiv.), the corresponding amino acid ester hydrochlorides 2-6 (0.30 mmol, 1.0 equiv.) and Et₃N (0.17 mL, 1.19 mmol, 4.0 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 16-38 h. Thereafter, the solvent was eliminated under reduced pressure, and the residue was purified by column chromatography using the eluant indicated in each case.

Method B. To a solution of DOPAC (50 mg, 0.30 mmol) in anhydrous CH₂Cl₂ (3.0 mL), HOBt (44 mg, 0.33 mmol, 1.1 equiv.), EDC (68 mg, 0.36 mmol, 1.2 equiv.), glycine ethyl ester hydrochloride 2 (46mg, 0.33 mmol, 1.1 equiv.) and Et₃N (0.06 mL, 0.42 mmol, 1.5 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 60 h. Then, the solution was diluted with EtOAc (15 mL) and washed with H₂O (1 x 15 mL), and the aqueous phase was further washed with EtOAc (3 x 10 mL). The combined organic fractions were washed with 1M HCl (1 x 10 mL), sat. aq. NaHCO₃ (1 x 10 mL) and brine (1x10 mL), dried over MgSO₄ and filtered; the filtrate was then concentrated to dryness and the residue was purified by column chromatography (CH₂Cl₂→10:1 CH₂Cl₂-MeOH).

Method C. To a solution of DOPAC (50 mg, 0.30 mmol) in anhydrous CH₂Cl₂ (2.0 mL), HATU (124 mg, 0.33 mmol, 1.1 equiv.), glycine ethyl ester hydrochloride 2 (44 mg, 0.31 mmol, 1.05 equiv.) and DIPEA (0.11 mL, 0.62 mmol, 2.1 equiv.) were added under N₂. The corresponding mixture was stirred at room temperature for 72 h. Thereafter, the crude reaction was washed with 1M HCl (1x10 mL), sat. aq. NaHCO₃ (1x10 mL) and brine (1x10 mL). The organic phase was dried over MgSO₄, filtered and the filtrate was concentrated to dryness.

3.3.13. Ethyl 2-[2’-(3”,4”-Dihydroxyphenyl)acetamido]acetate (13)

Method A1. Glycine ethyl ester hydrochloride 2 was used (42 mg, 0.30 mmol). Chromatographic purification afforded 13: 32 mg, 43%.

Method A2. Glycine ethyl ester hydrochloride 2 was used (42 mg, 0.30 mmol), and the reaction proceeded for 22 h. Column chromatography (CH₂Cl₂→10:1 CH₂Cl₂-MeOH) gave 13: 76 mg, quant.

Method B. Glycine ethyl ester hydrochloride 2 was used (46 mg, 0.33 mmol). Column chromatography gave 13: 54 mg, 72%.

Method C. Glycine ethyl ester hydrochloride 2 was used (44 mg, 0.31 mmol). After removal of the solvent, the crude product was analyzed by ¹H-NMR.

R_f 0.81 (40:1 CH₂Cl₂-MeOH); HRLSI-MS calcd for C₁₂H₁₅NNaO₅ ([M + Na]⁺: 276.0848, found: 276.0842.

3.3.14. Ethyl (S)-2-[2’-(3”,4”-dihydroxyphenyl)acetamido]-3-phenylpropanoate (14)

Method A2. L-Phenylalanine ethyl ester hydrochloride 3 was used (68 mg, 0.30 mmol), and the reaction proceeded for 38 h. Column chromatography (hexane→1:4 hexane-EtOAc) afforded 14: 102 mg, quant. $[α]_{D}^{21}$-4 (c 0.95, MeOH); R_F 0.40 (10:1 CH₂Cl₂-MeOH ); HRCI-MS calcd for C₁₉H₂₂NO₅ ([M+H]⁺): 344.1498, found: 344.1503.

3.3.15. Ethyl (S)-2-[2’-(3”,4”-dihydroxyphenyl)acetamido]-3-methylbutanoate (15)

Method A2. L-Valine ethyl ester hydrochloride 4 was used (54 mg, 0.30 mmol), and the reaction proceeded for 16 h. Column chromatography (hexane→1:2 hexane-EtOAc) yielded 15. Yield: 84 mg, 96%. $[α]_{D}^{25}$-6 (c 0.71, DMSO); R_F 0.38 (10:1 CH₂Cl₂-MeOH); HRCI-MS calcd for C₁₅H₂₁NO₅ ([M]⁺): 295.1420, found: 295.1428; calcd for C₁₅H₂₂NO₅ ([M + H]⁺): 296.1498, found: 296.1488.

3.3.16. Ethyl (S)-2-[2’-(3”,4”-Dihydroxyphenyl)acetamido]-3-hydroxypropanoate (16)

Method A2. L-Serine ethyl ester hydrochloride 5 was used (50 mg, 0.30 mmol), and the reaction proceeded for 38 h. Column chromatography (hexane→1:2 hexane-EtOAc) afforded 16: 84 mg, quant.; $[α]_{D}^{25}$-6 (c 1.02, MeOH); R_F 0.39 (1:2 hexane-EtOAc); HRCI-MS calcd for C₁₃H₁₇NO₆ ([M]⁺): 283.1056, found: 283.1054.

3.3.17. Methyl (S)-2-[2’-(3”,4”-dihydroxyphenyl)acetamido]-3-(1”’ H-indol-3”’-yl)propanoate (17)

Method A2. L-Tryptophan methyl ester hydrochloride 6 was used (76 mg, 0.30 mmol), and the reaction proceeded for 38 h. Column chromatography (hexane→1:7 hexane-EtOAc) yielded 17: 110 mg, quant. $[α]_{D}^{23}$+4 (c 1.15, DMF); R_F 0.33 (1:2 hexane-EtOAc); HRLSI-MS calcd for C₂₀H₂₀N₂NaO₅ ([M + Na]⁺): 391.1270, found: 391.1276.

3.3.18. Methyl (R,R)-3,3’-Dithiobis{2-[2”-(3”’,4”’-methylenedioxyphenyl) acetamido]propanoate} (19)

To a suspension of L-cystine dimethyl ester dihydrochloride 18 (102 mg, 0.03 mmol) in DMF (4.0 mL) at 0ºC, Et₃N (0.33 mL, 2.40 mmol), (3,4-methylenedioxy)phenylacetic acid 1 (108 mg, 0.60 mmol) and PyBOP (312 mg, 0.60 mmol) were added under N₂. The corresponding mixture was kept under stirring at room temperature and in the dark for 7 h. Then, the reaction was concentrated to dryness and the residue was purified by column chromatography (CH₂Cl₂→5:1 CH₂Cl₂-MeOH) to give 19 as a white solid. Yield: 147 mg, 83%; $[α]_{D}^{28}$ +77 (c 1.0, CH₂Cl₂); R_F 0.44 (20:1 CH₂Cl₂-MeOH); m.p. 133-135 ºC; CI-MS m/z 593 ([M + H]⁺, 4%); HRCI-MS calcd for C₂₆H₂₉N₂O₁₀S₂ ([M + H]⁺): 593.1264, found: 593.1245.

3.3.19. Methyl (R,R)-3,3’-Dithiobis{2-[2’’-(3’’’,4’’’-diacetoxyphenyl)acetamido]propanoate} (20)

To a suspension of L-cystine dimethyl ester dihydrochloride 18 (102 mg, 0.30 mmol) in DMF (4.0 mL), Et₃N (0.33 mL, 2.40 mmol), DOPAC (101 mg, 0.60 mmol) and PyBOP (312 mg, 0.60 mmol) were added under N₂. The corresponding mixture was stirred at room temperature in the dark for 12 h. Then, the solvent was removed under reduced pressure and the residue was conventionally acetylated with a 1:1 Ac₂O-Py mixture (2.0 mL) for 6.5 h. Then, the excess of Ac₂O was hydrolyzed with crushed ice, and concentrated to dryness; the residue was purified by column chromatography (CH₂Cl₂→60:1 CH₂Cl₂-MeOH) to give 20. Yield: 158 mg, 72%; $[α]_{D}^{25}$ +1 (c 1.0, CH₂Cl₂); R_F 0.64 (10:1 CH₂Cl₂-MeOH); LSI-MS m/z 759 ([M + Na]⁺, 41%); HRLSI-MS calcd for C₃₂H₃₆N₂NaO₁₄S₂ ([M + Na]⁺): 759.1506, found: 759.1502.

3.3.20. Methyl (R,R)-3,3’-Dithiobis{2-[2’’-(3’’’,4’’’-dihydroxyphenyl)acetamido]propanoate} (21)

To a solution of 20 (96 mg, 0.13 mmol) in a 1:1 CH₂Cl₂-MeOH mixture (2.0 mL) CsCO₃ was added (10 mg, 0.031 mmol), and the corresponding mixture was stirred at room temperature in the dark under inert atmosphere for 30 min. Then, the crude reaction was neutralized using Amberlite IR-120(H⁺) resin, filtered, washed with MeOH, and the filtrate was concentrated to dryness. The residue was purified by column chromatography (CH₂Cl₂→20:1 CH₂Cl₂-MeOH) to give 21 as a yellowish syrup. Yield: 48 mg, 65%; $[α]_{D}^{26}$ +28 (c 1.0, MeOH); R_F 0.38 (10:1 CH₂Cl₂-MeOH); LSI-MS m/z 591 ([M + Na]⁺, 11%); HRLSI-MS calcd for C₂₄H₂₈N₂NaO₁₀S₂ ([M + Na]⁺): 591.1083, found: 591.1058.

3.3.21. Methyl (R)-2-[2’-(3’’,4’’-dihydroxyphenyl)acetamido]-3-mercaptopropanoate (23) [49]

To a solution of 21 (43 mg, 0.076 mmol) in an anhydrous 1:1 CH₂Cl₂-MeOH mixture (2.0 mL) (±)-dithiothreitol was added (12 mg, 0.076 mmol). The corresponding mixture was stirred at room temperature in the dark under inert atmosphere for 2 h. Then it was concentrated to dryness and the residue was purified by column chromatography (hexane→1:10 hexane-EtOAc) to give 23 as a syrup. Yield: 19 mg, 44%; $[α]_{D}^{25}$-15 (c 1.2, MeOH); R_F 0.48 (1:5 1:10 hexane-EtOAc); HRLSI-MS calcd for C₁₂H₁₅NNaO₅S ([M + Na]⁺): 308.0569, found: 308.0569.