(E)-Ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate: A Modified Yamaguchi Reagent for Enantioselective Esterification, Thioesterification, Amidation, and Peptide Synthesis

Abstract

Here, the synthesis and applications of (E)-ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate as a racemization suppressing and easily recyclable version of the Yamaguchi reagent that can be used for amide and peptide synthesis are reported. We demonstrated its application in racemization-free esterification, thioesterification, amidation, and peptide bond formation. We successfully synthesized oligopeptides on the solid support in dimethylformamide as well as in solution (dichloromethane) by applying this coupling reagent. It is important to note that a mixed-anhydride-based method provides peptide-forming reactions as good as the current methods using built-in coupling reagents. Mechanism investigation, racemization suppression, and recyclability are also discussed.

Introduction

Esterification and amidation are extensively used in the synthesis of natural products, polymers, active pharmaceutical ingredients, and other biologically relevant molecules.^1,2 These reactions are usually achieved by activation of a carboxylic acid by a suitable coupling reagent. To date, many efficient coupling reagents have been developed, including carbodiimides, phosphonium, and uronium/aminium salts.³ Some other carboxylic acid activation methodologies have also been established, for example, acyl chlorides in the Schotten–Baumann reaction,⁴ azides in the Staudinger reaction,⁵ activated esters,⁶ metal-catalyzed condensation reactions,⁷ Lewis acid catalysis,⁸ etc.

Similarly, mixed-anhydride-mediated coupling reactions are also widespread.⁹ In this direction, Yamaguchi and co-workers reported a coupling reagent, 2,4,6-trichlorobenzoyl chloride (TCBCl or Yamaguchi reagent),¹⁰ for efficient synthesis of esters and lactones via in situ formation of mixed anhydrides in 1979. Later, Shiina and co-workers introduced the use of aromatic anhydrides, especially, 2-methyl-6-nitrobenzoic anhydride¹¹ as a better alternative to the Yamaguchi reagent. A catalytic amount of 4-dimethylaminopyridine (DMAP) was used with the Yamaguchi reagent as well as with the aromatic anhydrides to improve the regioselectivity.¹² In 2014, a modified Yamaguchi reagent was reported by Yoshinori et al. They introduced 2,4,6-trichlorobenzoyl-4-dimethylaminopyridinium chloride (TCB-DMAP)¹³ as the coupling reagent to avoid the use of often intractable acid chlorides and acid anhydrides, which hinders their use in the presence of sensitive functional and protecting groups. These reagents are capable of affording condensation reactions efficiently, but they cause a significant amount of racemization in case of chiral substrates. Thus, the yield of the desired products decreases and purification of the products becomes cumbersome. Most importantly, these reagents become useless for the stepwise synthesis of oligopeptides because of the accumulation of undesired epimerized analogues at each step. Usually, N-hydroxy amine reagents, for example, hydroxybenzotriazole (HOBt) and hydroxyazabenzotriazole (HOAt), are frequently used as additive with the coupling reagents¹⁴ to prevent the racemization during peptide synthesis. However, due to the explosive nature of HOBt and HOAt, recently, ethyl-2-hydroxyimino-2-cyanoacetate (oxyma) has been suggested as a racemization suppressant in diisopropylcarbodiimide¹⁵ and oxyma-based phosphate¹⁶-mediated peptide syntheses. Earlier, we also reported the utility of oxyma-based reagents for racemization-free peptide synthesis and various other organic transformations.¹⁷

We describe herein a differently modified Yamaguchi reagent, (E)-ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate (Scheme 1, TCBOXY, I), which can be efficiently used for esterification, amidation, and peptide synthesis without causing detectable racemization. To the best of our knowledge, to date, there is no report on the racemization-free amide and peptide synthesis using Yamaguchi and modified Yamaguchi reagents.

Scheme 1. Preparation of the Coupling Reagent, Ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate (TCBOXY, I).

Results and Discussion

Reagent I can readily be synthesized by reacting oxyma with TCBCl in the presence of Hunig’s base (N,N-diisopropylethylamine (DIPEA)) under nitrogen atmosphere and dry dichloromethane (DCM) at 0 °C for 2 h (Scheme 1). Simple aqueous workup and recrystallization from hexane results in I that can be directly used for coupling reactions. Reagent I does not degrade at room temperature (25 °C) and therefore it can be stored for an extended period. A time-dependent high-performance liquid chromatography (HPLC) study indicated no change of I until 2 months (Figures S4 and S5, Supporting Information).

Its coupling efficiency was first investigated by the reaction between benzoic acid and cyclohexylamine in the presence of DIPEA (Table 1). We observed only 30% of the desired product and 60% of the corresponding amide of 2,4,6-trichlorobenzoic acid (Figures S154–S156, Supporting Information). To avoid the formation of side products, we used a catalytic amount of DMAP, which acted as a selective acyl-transfer reagent.¹⁸ We screened the various amounts of DMAP using the same reaction in DCM (Table 1). We observed a 91% yield of the desired product with 0.3 equiv of DMAP. By increasing the amount of DMAP beyond 0.3 equiv, no notable improvement in the yield was observed (entries 1 and 2). But, when we decreased the same below 0.3 equiv, the yield dropped (entries 4 and 5). Next, we screened several solvents using reagent I (1 equiv) with the catalytic amount of DMAP (0.3 equiv). We found that DCM, CH₃CN, EtOAc, CHCl₃, and dimethylformamide (DMF) afforded very good yields (entries 3 and 7–10). However, there was no reaction in CH₃OH and H₂O (entries 13 and 14). Therefore, 0.3 equiv of DMAP and DCM as a solvent were accepted under optimized conditions (entry 3).

Table 1. Optimization of Reaction Conditions^a.

entry	solvents	DMAP (equiv)	yieldb (%)
1	DCM	0.8	91
2	DCM	0.4	91
3	DCM	0.3	91
4	DCM	0.2	80
5	DCM	0.1	65
6	DCM	0.0	30
7	CH3CN	0.3	80
8	EtOAc	0.3	72
9	CHCl3	0.3	75
10	DMF	0.3	85
11	THF	0.3	59
12	toulene	0.3	40
13	MeOH	0.3	0
14	H2O	0.3	0

^aReaction conditions: benzoic acid (122 mg, 1 mmol), TCBOXY (349 mg, 1 mmol), DIPEA (193 mg, 1.5 mmol), DMAP, cyclohexylamine (99 mg, 1 mmol) stirred at room temperature for 15 min.

^bIsolated yield.

Under the optimized conditions, we proceeded to investigate the scope of esterification using various carboxylic acids and alcohols by using I. Reactions worked well with the sterically hindered amino acids (Scheme 2, 2a–d) as well as with heterocyclic, aromatic, and aliphatic carboxylic acids (2e–j). The reaction also worked with secondary alcohol (2h) in very good yield. We also extended this protocol for thioesterification (2k–n). A broad range of carboxylic acids was tolerated, including those bearing electron-donating groups (2l and 2n) and an electron-withdrawing group (2m), with aromatic thiols bearing neutral, electron-donating, and electron-withdrawing substituents.

Scheme 2. Esterification, Thioesterification, and Amidation by Using I.

Reaction conditions: acid (1 mmol), I (1 mmol), DIPEA (1.5 mmol), DMAP (0.3 mmol), and alcohol, thiol, or amine (1.2 mmol), stirred at room temperature for 5–30 min. Isolated yields after column chromatographic purification are mentioned.

Further, we extended this protocol for amidation reactions. Interestingly, the reactions worked well with the aromatic carboxylic acids (Scheme 2, 2o–x), aliphatic carboxylic acids (2y–z), aromatic amines (2o), aliphatic amines (2p–w and 2z), and C-protected amino acids (2x and 2y).

We further explored the applicability of I for peptide synthesis in solution with various N-protected amino acids bearing various side chains (Scheme 3). The reactions worked well with the common N-protections, such as Bz (Scheme 3, 3a), Fmoc (3b to rac-3n), Boc (3o and 3p), Cbz (3q–s), and sterically hindered amino acids with good to excellent yields.

Scheme 3. Wide Scope of the Synthesis of Peptides in Solution Using I.

Reaction conditions: acid (1 mmol), I (1 mmol), DIPEA (1.5 mmol), DMAP (0.3 mmol), and amine (1.5 mmol), stirred at room temperature for 20–120 min. Isolated yields after column chromatographic purification are mentioned.

We further synthesized a tetrapeptide, Boc-Val-Val-Ile-Ala-OMe, the C-terminal segment of the amyloid β-peptide²⁰ (Figure 1a), in DCM solution following Boc-chemistry. The yield of the crude peptide (after precipitation with cold ether but before purification by HPLC) was 75%, while the purity was 99%, as determined by reversed-phase (RP)-HPLC analysis (Figures S195 and S196, Supporting Information), indicating cleanliness of the procedure. Later, we synthesized acyl carrier protein (ACP) (24–33) peptide segment (ACP, acyl carrier protein: Asp-Asn-Ala-Ser-Phe-Val-Glu-Asp-Leu-Gly-NH₂, Figure 1b) and the tropoelastin peptide segment ((Pro-Gly-Val-Gly-Val-)₂-NH₂, Figure 1c)²¹ by stepwise coupling of amino acids on the Rink Amide MBHA resin following Fmoc/t-Bu orthogonal protection strategy. The electrospray ionization (ESI)-mass spectrometry (MS) data and HPLC profiles of each segment during the synthesis of ACP (24–33) (Table S1 and Figures S201–S218, Supporting Information) indicate occurrences of neither incomplete coupling nor side reactions during the coupling steps. Considering the scale of the reaction sequences, yields (21% for ACP (24–33) and 30% for the elastin peptide) with respect to the resin loading were good (Figures S217, S218 and S199, S200). We further synthesized specific segments of gramicidin A, B, and C on Wang resin (Figure 1d). The general structure of the segments of gramicidin A, B, and C is Xaa-d-Leu-l-Trp-d-Leu-l-Trp-OH, where the side chain of Xaa varied (Figure 1d). We obtained these peptides also in good yield, i.e., 23, 28, and 26% of gramicidin A, B, and C segments, respectively, with respect to the resin loading after purification using RP-HPLC (Figures S219–S224, Supporting Information).

Figure 1.

Sequences of the synthesized long peptides: (a) Boc-VVIA-OMe, in solution; (b) DNASFVEDLG-NH₂; (c) PGVGVPGVGV-NH₂; and (d) segments of gramicidin A, B, and C using solid phase peptide synthesis strategy.

Epimerization during such syntheses is highly important for industry and academia, but we could not find any systematic study on the epimerization potential of such reagents. Therefore, we investigated the epimerization potential of the Yamaguchi and related reagents and compared it to that of I. We first synthesized dl-Fmoc-Phe-OBn (rac-2a) using the optimized protocol and passed through a chiral column. Two well-separated peaks, corresponding to the two enantiomers, were observed in HPLC profile (Figures 2 and S157–S159, Supporting Information) that were used as a reference. Next, we synthesized l-Fmoc-Phe-OBn (2a) using the Yamaguchi reagent in the absence of DMAP and in the presence of DMAP, the modified Yamaguchi reagent (TCB-DMAP), and I. The products were passed through the chiral column using the same eluant. Although all of the tested reagent combinations resulted in 12–24% epimerization, I did not cause any detectable epimerization (Figures 2 and S160–S166, Supporting Information). This result indicates that no racemization occurred during the coupling reaction using I, unlike other similar reagents. The above results also suggest that DMAP has no apparent role in racemization suppression.

Figure 2.

HPLC images to compare the epimerization caused by various coupling reagents.

We synthesized Fmoc-l-Ala-l-Leu-OMe (3g) and Fmoc-dl-Ala-l-Leu-OMe (3h) using I to investigate the associated racemization in peptide synthesis. The appearance of the single peak in the HPLC profile of 3g corresponds to the unique stereoisomeric product, whereas the presence of the twin peak in the same of 3h indicates the presence of two diastereomeric products (Figures 3 and S167–S171, Supporting Information). The ¹H and ¹³C NMR spectra of 3h and 3g were also compared (Figures 3 and S106–S111, Supporting Information). We found one singlet at δ = 3.72 ppm for the methoxy proton of 3g and two singlets at δ = 3.70 and 3.68 ppm for the methoxy proton of 3h in the ¹H NMR spectra. Similarly, in the ¹³C NMR spectra, we found two peaks at δ = 173.4 and 172.3 ppm corresponding to the two carbonyls of the amide and the ester groups of 3g, indicating the presence of the single diastereomer, whereas the presence of four peaks at δ = 173.4, 173.3, 172.5, and 172.4 ppm for those carbonyl carbons of 3h indicates the presence of two diastereomeric products (Figure 3). Therefore, it was inferred that no detectable racemization occurred during the synthesis of the mentioned dipeptides using I. Similarly, comparison of the HPLC profiles (Figures S172–S176, Supporting Information) of the l and dl forms of the Fmoc-Phe-Gly-OMe dipeptides (3n and rac-3n, respectively) leads to the same conclusion. Also, for all of the remaining l,l-dipeptides depicted in Scheme 3, single peaks corresponding to the only stereoisomeric products were noted, confirming the occurrence of no detectable racemization during peptide synthesis by I.

Figure 3.

Comparative study of racemization by HPLC, ¹H NMR, and ¹³C NMR of Fmoc-l-Ala-l-Leu-OMe (left panel) and Fmoc-dl-Ala-l-Leu-OMe (right panel).

Next, we synthesized a tripeptide Z-Gly-Phe-Val-OMe (Scheme 3, 3s and Figures S145–S147, S177 and S178, Supporting Information) in solution using I, determined the yield and the degree of racemization, and compared the results to those reported for popular coupling reagents, such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-ethylmethanaminium hexafluorophosphate N-oxide (HATU), 1-((dimethylamino)-(morpholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium hexafluorophosphate-3-oxide (HDMA), 1-((dimethylamino)(morpholino)methylene)-1H-benzotriazolium hexafluorophosphate-3-oxide (HDMB), and N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene] N-methylmethanaminium hexafluorophosphate N-oxide (HBTU).¹⁹ Although the yields were comparable, no racemization could be observed during the synthesis by using I unlike the other reagents (Table 2).

Table 2. Comparison of the Yield and Racemization of Z-Gly-Phe-Val-OMe Synthesized Using Various Coupling Reagents.

entry	coupling reagent	yield (%)	racemization (%)
1	HDMB	90	2.9
2	HDMA	90	0.7
3	HBTU	89	5.9
4	HATU	90	1.6
5	TCBOXY	90	n.d.a

^aNo racemization could be detected.

Next, we turned our attention to the mechanism elucidation. As suggested in the original article, Yamaguchi reagent, which is a sterically hindered benzoyl chloride, works via the formation of the mixed-anhydride intermediate¹⁰ (II, Scheme 4), followed by the nucleophilic attack of DMAP to the less hindered electrophilic center of II to generate the resonance-stabilized N-acyl pyridinium salt intermediate III.¹³ Such preference of DMAP results in the regioselectivity of the reaction. Further attack of the added nucleophile, e.g., alcohol or amine, leads to the final product. However, SantaLucia and Dhimitruka¹² suggested that aliphatic carboxylates should be more reactive toward the aliphatic moiety of the mixed anhydride II due to the steric factor. Thus, once II is formed, the less hindered carboxylate reacts with it to generate the relatively less hindered symmetrical anhydride intermediate (IV). They indeed demonstrated that the byproduct, 2,4,6-trichlorobenzoate anion, did not react with the Yamaguchi reagent to form 2,4,6-trichlorobenzoic anhydride, instead intermediate IV was generated exclusively.

Scheme 4. Plausible Mechanism of the Reaction Mediated by I.

Therefore, most probably in the TCBOXY-mediated reaction, the nucleophile generated by the deprotonation of the substrate carboxylic acid in the presence of DIPEA attacks the carbonyl carbon of I, forming the mixed anhydride II, and releases the resonance-stabilized oxyma anion. Then, another molecule of the already created carboxylate attacks the less hindered carbonyl of II to generate IV. DMAP attacks regioselectively at the less hindered carbon atom of either II or IV, forming the N-acyl pyridinium salt III. The released oxyma anion then attacks the carbonyl carbon of III, resulting in the formation of the intermediate V, which is the oxyma ester of the substrate carboxylic acid. V then undergoes nucleophilic substitution in a stereoselective fashion to produce the corresponding esters, thioesters, amides, and peptides. The intermediate V was isolated as a product in a similar reaction of a carboxylic acid (Fmoc-Ala-OH), I, the appropriate amount of DMAP and DIPEA, but devoid of the nucleophile. It was characterized by ¹H NMR and ¹³C NMR spectroscopies (Figures S6 and S7, Supporting Information).

Chemical waste generation and nonrecyclability are common problems for the majority of the popular peptide-coupling reagents. Therefore, a recent trend is to develop easily recyclable coupling reagents that are highly required for sustainability.²² We investigated the recyclability of I. We repeated the synthesis of 2v (Scheme 2) for that. After completion of the reaction, the product and byproducts, oxyma (a) and trichlorobenzoic acid (b) (Scheme 5 and Figures S148–S153, Supporting Information), were purified by eluting with specific eluents from a silica gel column. The recovered b was chlorinated with thionyl chloride by heating at 110–114 °C in toluene for 3 h and mixed with the recovered a in the presence of DIPEA to obtain I with 52% overall yield with respect to the initial I used. Alternatively, the recovered a and b were recombined by merely heating in the presence of silica gel under microwave irradiation²³ (Scheme 5)-based dehydration without using unhealthy thionyl chloride. By this way, we were able to recover byproducts and recombine to regenerate the coupling reagent easily.

Scheme 5. Recyclability of the Coupling Reagent, TCBOXY.

Conclusions

Yamaguchi reagent could solve a lot of practical problems, but to the best of our knowledge, could never be used as a peptide-coupling reagent, primarily because of the arduous acid chloride and significant racemization during condensation. We have developed a modified Yamaguchi reagent, TCBOXY (I), which can be efficiently used for the syntheses of esters, thioesters, amides, peptides in both solution and solid support with very good to excellent yield. The advantages of TCBOXY are as follows: (a) its preparation protocol is easy and involves single-step reaction; (b) it suppresses racemization during the coupling reaction and allows enantioselective syntheses; (c) only 2,4,6-trichlorobenzoic acid and oxyma are generated as byproducts, which are nontoxic and can be recovered and recycled to generate the same coupling reagent easily that can be used in the same pool; (d) operationally simple, as both of the byproducts are acidic in nature and just basic workup renders pure products. Thus, the described method for the synthesis of the mentioned compounds using this new reagent is a more eco-friendly and green process than the current alternatives discussed in Introduction.

Experimental Section

General Information

All of the reagents, except those mentioned, were procured from usual commercial sources. NMR experiments were performed on 600 and 400 MHz spectrometers using CDCl₃. Tetramethylsilane was used as an internal standard. Chemical shifts (δ) were indicated in parts per million. Spin–spin coupling constants (J) were indicated in hertz. The multiplicity of the signals was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Thin-layer chromatography using silica gel G254 was used to monitor the reactions. Column chromatography using silica gel (60–120 mesh) and EtOAc/hexane as eluent was used to purify the products. Melting points and Fourier transform infrared (FT-IR) spectra were recorded on a dedicated meting point apparatus and an FT-IR spectrometer, respectively. High-resolution mass spectrometry (HRMS) experiments were performed on a Micromass quad time-of-flight (Q-TOF) ESI-MS instrument and a Q-TOF liquid chromatography/MS system. HPLC analyses were performed with reversed-phase chromatographic columns and CHIRAL PAK AS-H (5 μm, 4.6 × 250 mm²) column attached to an UV detector. HPLC analyses were performed with HPLC-grade solvents.

Procedure for the Synthesis of the Coupling Reagent (E)-Ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate (TCBOXY, I)

DIPEA (129 mg, 1 equiv) was added to a solution of oxyma (142 mg, 1 equiv) in 2 mL of DCM under nitrogen. The temperature of the reaction mixture was decreased to 0 °C. Then, 2,4,6-trichlorobenzoyl chloride (243 mg, 1 equiv) was added dropwise. The mixture was then stirred at room temperature for another 2 h. After completion of the reaction, 10 mL of DCM was added to it and washed with 5% HCl (3 × 5 mL). Finally, the organic portion was collected, dried using anhydrous CaCl₂, and evaporated. The obtained solid mass was recrystallized with hexane. R_f: 0.50 (EtOAc/hexane, 1:9); yield 326 mg, 94%; white crystalline solid, mp 83–85 °C; ¹H NMR (600 MHz, CDCl₃): δ 7.47 (s, 2H), 4.54–4.53 (q, J = 7.2 Hz, 2H, CH₂), 1.47–1.44 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 158.7, 156.6, 138.6, 134.0, 132.8, 128.7, 127.9, 106.6, 65.0, 14.1; IR (KBr) 3076, 1806, 1732, 1577, 1375, 1220, 1150, 1087, 990 cm^–1; HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₂H₇C_l3N₂NaO₄ 370.9369, found 370.9367.

Procedure for the Synthesis of Esters, Thioesters, and Amides

TCBOXY (1 equiv) was added to a DCM (2 mL) solution of carboxylic acid (1 equiv), DMAP (0.3 equiv), and DIPEA (1.5 equiv). All of the components were mixed well for 3–5 min for preactivation, and then, alcohol, thiol, or amines (1.2 equiv) was added to it. Then, the mixture was stirred for 5–30 min at room temperature. After completion of the reaction, the whole mixture was diluted with 20 mL of ethyl acetate. The organic portions were collected, washed with 5% HCl (3 × 5 mL) and 5% NaHCO₃ (3 × 5 mL), and dried using anhydrous Na₂SO₄. Finally, Na₂SO₄ was filtered off and the solvent was evaporated to obtain the product, which was purified by column chromatography.

Procedure for the Peptide Synthesis

TCBOXY (1 equiv) was added to a solution of N-protected amino acid (1 equiv), DMAP (0.3 equiv), and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture was stirred for 5 min for preactivation, followed by the addition of methyl ester of amino acid (1.5 equiv) and DIPEA (1.5 equiv) in 1 mL of DCM. The reaction mixture was stirred at room temperature for 20–120 min. After completion of the reaction, the reaction mixture was diluted with 20 mL of ethyl acetate; the organic phase was washed with 5% HCl (3 × 5 mL), 5% NaHCO₃ (3 × 5 mL), and brine; and dried using anhydrous Na₂SO₄. Finally, Na₂SO₄ was filtered and the solvent was evaporated. The product was purified by silica gel column chromatography.

Solution-Phase Synthesis of Boc-VVIA-OMe

TCBOXY (1 equiv) was added to a solution of Boc-Ile-OH (1 equiv), DMAP (0.3 equiv), and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture was stirred for 5 min for preactivation. In another RB, methyl ester of alanine (1.5 equiv) was taken in DCM and DIPEA was added to it until basic pH was reached. Finally, this solution was added to the above solution and stirring was continued until completion of the reaction. Then, the reaction mixture was diluted by 20 mL of EtOAc and washed by 5% NaHCO₃ solution (2 × 5 mL) and 5% citric acid solution (2 × 5 mL). Finally, the combined organic layer was dried using anhydrous Na₂SO₄. The solid product (Boc-IA-OMe) was obtained after evaporation of EtOAc by a rotary vacuum evaporator.

In 50 mL of RB, solid product (Boc-IA-OMe) was taken and TFA/DCM (1:1) mixture was added and stirred up to 2.5 h. After that, TFA was evaporated by a rotary vacuum evaporator, the solution was washed three to four times with diethyl ether, and finally a white solid (IA-OMe) was obtained. After Boc deprotection, the resulting IA-OMe was coupled with Boc-V-OH following the procedure as mentioned earlier to obtain Boc-VIA-OMe. Another cycle of Boc-deprotection and coupling with Boc-V-OH resulted in white solid Boc-VVIA-OMe, which was characterized by reversed-phase HPLC, with a retention time 4 min on a linear gradient of 0–70% for 0–10 min and then 70–100% for 10–25 min CH₃CN in H₂O with 0.1% formic acid in a symmetry C8 analytical column. Low-resolution mass spectrometry (LRMS) (ESI) m/z: [M + H]⁺ calcd for C₂₅H₄₇N₄O₇ 515.3445, found 515.3521. The yield was 75% with respect to starting material Boc-isoleucine.

Solid-Phase Synthesis of PGVGVPGVGV-NH₂ and DNASFVEDLG-NH₂

Decapeptide was manually assembled stepwise on Fmoc Rink Amide MBHA resin by Fmoc/tert-butyl (t-Bu) protection strategy. Fmoc amino acids (1.5 equiv), TCBOXY (1 equiv), DMAP (0.3 equiv), and DIPEA (3 equiv) were kept for preactivation for 5 min. Then, amino acid coupling was performed for 2–4 h. Fmoc deprotection was carried out using TFA/DCM (1:1) mixture for 2.5 h. Purification of the peptide was carried out by preparative HPLC, and lyophilization afforded the final peptide.

Solid-Phase Synthesis of Segments of Gramicidin A, B, and C

The syntheses were carried out by stepwise coupling of amino acids on Wang resin, as mentioned before, by Fmoc/t-Bu protection strategy. Fmoc amino acids (1.5 equiv), TCBOXY (1 equiv), DMAP (0.3 equiv), and DIPEA (3 equiv) were kept for preactivation for 5 min. Then, amino acid coupling was performed for 2–4 h. Fmoc deprotection was carried out using TFA/DCM (1:1) mixture for 2.5 h. Purification of the peptide was carried out by preparative HPLC, and lyophilization afforded the final peptide.

Procedure to Identify (E)-ethyl-9-cyano-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,7-dioxa-4,8-diazadec-8-en-10-oate (Intermediate V, Scheme 4)

TCBOXY (1 equiv) was added to a solution of Fmoc-Ala-OH (1 equiv), DMAP (0.3 equiv), and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture was stirred for 30 min at room temperature. After 30 min, we observed one spot in TLC. The reaction mixture was diluted with 20 mL of ethyl acetate; the organic phase was washed with 5% HCl (3 × 5 mL), 5% NaHCO₃ (3 × 5 mL), and brine; and dried using anhydrous Na₂SO₄. Finally, Na₂SO₄ was filtered off and the solvent was evaporated to obtain the intermediate, which was purified by column chromatography. R_f: 0.50 (EtOAc/hexane, 2:8); yield 357 mg, 82%; white solid, mp 95–97 °C; ¹H NMR (600 MHz, CDCl₃): δ 7.77–7.76 (d, J = 7.8 Hz, 2H), 7.61–7.59 (t, J = 6.6 Hz, 2H), 7.41–7.39 (t, J = 7.2 Hz, 2H), 7.33–7.31 (t, J = 7.2 Hz, 2H), 5.38 (br s, 1H), 4.43–4.36 (m, 3H), 4.24–4.20 (m, 3H), 1.44–1.43 (d, J = 7.2 Hz, 3H), 1.29–1.28 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.3, 155.8, 144.0, 141.5, 127.9, 127.3, 125.3, 120.2, 67.2, 61.8, 49.9, 47.4, 19.0, 14.3; IR (KBr) 2925, 1747, 1692, 1534, 1450, 1260, 1027, 738 cm^–1.

Recyclability of the Coupling Reagent, TCBOXY

TCBOXY (1 equiv) was added to a solution of 2-picolinic acid (1 equiv), DMAP (0.3 equiv) and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture was stirred for 3–5 min for preactivation, followed by the addition of tert-butyl amine (1.2 equiv). The reaction mixture was stirred at room temperature for 45 min. After completion of the reaction, the reaction mixture was diluted with ethyl acetate and washed with 5% HCl solution (3 × 5 mL). The concentrated organic layer was directly purified by silica gel column chromatography. The product and byproducts a and b were purified by elution with specific eluents. In path a, the recovered b was chlorinated with thionyl chloride by heating at 110–114 °C in toluene for 3 h and mixed with the recovered oxyma (a) in the presence of DIPEA and I was obtained with 52% yield. In path b, we recovered I by recombination of byproducts a and b in the presence of silica gel under microwave irradiation with 35% yield. The yield of recyclable coupling reagent was calculated with respect to the initial amount of I used in the reaction. By this way, we were able to recover byproducts and recombine to regenerate the coupling reagent (I) very easily.

(S)-Benzyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanoate (2a)

White solid (386 mg, 81%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 107–109 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.78–7.76 (d, J = 7.8 Hz, 2H), 7.57–7.55 (t, J = 7.2 Hz, 2H), 7.42–7.39 (t, J = 7.8 Hz, 2H), 7.37–7.22 (m, 8H), 5.29 (br, 1H), 5.19–5.12 (m, 2H), 4.74–4.71 (m, 1H), 4.45–4.42 (m, 1H), 4.35–4.32 (m, 1H), 4.21–4.19 (t, J = 7.2 Hz, 1H), 3.15–3.08 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 171.5, 155.7, 144.0, 143.9, 141.5, 135.7, 135.2, 129.5, 128.8, 128.7, 127.9, 127.3, 127.2, 125.3, 125.2, 120.1, 67.5, 67.1, 55.0, 47.3, 38.3; FT-IR (KBr, cm^–1): 2923, 1719, 1504, 1482, 1349, 1260, 1150, 1012, 740, 697; LRMS (ESI) m/z: [M + H]⁺ calcd for C₃₁H₂₈NO₄ 478.2018, found 478.2002.

dl-(S)-Benzyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanoate (rac-2a)

White solid (382 mg, 80%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 107–109 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.78–7.76 (d, J = 7.8 Hz, 2H), 7.57–7.55 (t, J = 7.2 Hz, 2H), 7.42–7.39 (t, J = 7.8 Hz, 2H), 7.37–7.22 (m, 8H), 5.29 (br, 1H), 5.20–5.13 (m, 2H), 4.74–4.71 (m, 1H), 4.45–4.42 (m, 1H), 4.35–4.32 (m, 1H), 4.21–4.19 (t, J = 7.2 Hz, 1H), 3.15–3.08 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 171.5, 155.7, 143.9, 141.5, 135.7, 135.2, 129.6, 128.8, 127.9, 127.3, 127.2, 125.3, 125.2, 120.2, 67.5, 67.2, 55.0, 47.4, 38.4; FT-IR (KBr, cm^–1): 2923, 1719, 1504, 1482, 1349, 1260, 1150, 1012, 740, 697; LRMS (ESI) m/z: [M + H]⁺ calcd for C₃₁H₂₈NO₄ 478.2018, found 478.1996.

(S)-Benzyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoate (2b)

White solid (352 mg, 82%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 86–89 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.78–7.77 (d, J = 7.2 Hz, 2H), 7.61–7.60 (d, J = 7.2 Hz, 2H), 7.42–7.40 (t, J = 7.2 Hz, 2H), 7.36–7.31 (m, 3H), 5.35 (br, 1H), 5.23–5.15 (m, 2H), 4.41–4.36 (m, 3H), 4.25–4.22 (t, J = 7.2 Hz, 1H), 2.23–2.18 (m, 1H), 0.96–0.95 (d, J = 6.6 Hz, 3H), 0.88–0.87 (d, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 172.2, 156.4, 144.1, 143.9, 141.5, 135.5, 128.8, 128.7, 128.6, 127.9, 127.2, 125.3, 120.2, 67.3, 67.2, 59.2, 47.4, 31.5, 19.2, 17.7; FT-IR (KBr, cm^–1): 2925, 1744, 1689, 1530, 1449, 1270, 1156, 1030, 736, 696; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₇H₂₈NO₄ 430.2018, found 430.2018.

Benzyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropanoate (2c)

Yellow solid (324 mg, 78%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 68–70 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.78–7.76 (d, J = 7.8 Hz, 2H), 7.59–7.58 (d, J = 7.2 Hz, 2H), 7.42–7.39 (t, J = 7.2 Hz, 2H), 7.32–7.30 (t, J = 7.2 Hz, 3H), 5.45 (br, 1H), 5.18 (s, 2H), 4.35 (s, 2H), 4.19 (s, 1H), 1.59 (s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 174.6, 155.1, 144.1, 141.4, 135.7, 128.7, 128.4, 128.1, 127.8, 127.2, 125.2, 120.1, 67.4, 66.7, 56.7, 47.3, 25.2; FT-IR (KBr, cm^–1): 2987, 1736, 1693, 1537, 1450, 1386, 1277, 1143, 1107, 743, 696; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₆H₂₆NO₄ 416.1862, found 416.1864.

(2S, 3R)-4-Nitrophenyl-2-((tert-butoxycarbonyl)amino)-3-methylpentanoate (2d)

Yellow liquid (299 mg, 85%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (600 MHz, CDCl₃) δ 8.29–8.28 (d, J = 9.0 Hz, 2H), 7.30–7.29 (d, J = 9.0 Hz, 2H), 5.09 (br, 1H), 4.51–4.48 (q, 1H), 1.58–1.54 (m, 1H), 1.47 (s, 9H), 1.33–1.29 (m, 2H), 1.08–1.07 (d, J = 7.2 Hz, 3H), 1.01–0.99 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 170.6, 156.0, 155.3, 145.7, 126.4, 125.5, 122.6, 115.8, 80.8, 58.5, 38.0, 28.5, 25.4, 15.9, 11.8; FT-IR (KBr, cm^–1): 2973, 2879, 1765, 1691, 1583, 1367, 1162, 1045, 853, 629; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₇H₂₅N₂O₆ 353.1713, found 353.1716.

Prop-2-yn-1-yl-quinoline-2-carboxylate (2e)

Brown solid (188 mg, 89%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 104–107 °C; ¹H NMR (600 MHz, CDCl₃) δ 8.34–8.31 (t, J = 7.8 Hz, 2H), 8.23–8.22 (d, J = 8.4 Hz, 1H), 7.90–7.89 (d, J = 7.8 Hz, 1H), 7.82–7.79 (t, J = 7.8 Hz, 1H), 7.68–7.66 (t, J = 7.2 Hz, 1H), 5.08 (s, 2H), 2.56–2.55 (t, J = 2.4 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 164.8, 147.8, 147.4, 137.6, 131.0, 130.6, 129.7, 129.1, 127.8, 121.3, 77.5, 75.7, 53.7; FT-IR (KBr, cm^–1): 2924, 2854, 2127, 1716, 1461, 1372, 1209, 1134, 781, 624; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₃H₁₀NO₂ 212.0712, found 212.1075.

(E)-But-2-en-1-yl-2-naphthoate (2f)

Yellow liquid (206 mg, 91%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (600 MHz, CDCl₃) δ 8.62 (s, 1H), 8.08–8.07 (m, 1H), 7.96–7.95 (d, J = 7.8 Hz, 1H), 7.89–7.87 (d, J = 8.4 Hz, 2H), 7.60–7.53 (m, 2H), 5.95–5.90 (m, 1H), 5.79–5.75 (m, 1H), 4.83–4.82 (d, J = 6.6 Hz, 2H), 1.79–1.78 (d, J = 6.6 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.7, 135.6, 132.6, 131.6, 131.2, 129.5, 128.3, 128.2, 127.9, 127.7, 126.7, 125.4, 125.3, 65.9, 18.0; FT-IR (KBr, cm^–1): 3024, 2939, 1716, 1631, 1510, 1446, 1281, 1227, 1195, 1094, 963, 779, 474; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₅H₁₅O₂ 227.1072, found 227.1075.

Furan-2-ylmethyl-2-phenylacetate (2g)

Black liquid (189 mg, 87%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (600 MHz, CDCl₃) δ 7.40–7.39 (m, 1H), 7.32–7.24 (m, 5H), 6.37–6.32 (m, 2H), 5.06 (s, 2H), 3.62 (s, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.3, 149.4, 143.4, 133.8, 129.4, 128.7, 127.2, 110.8, 110.7, 58.5, 41.1; FT-IR (KBr, cm^–1): 3031, 2927, 1734, 1604, 1497, 1245, 1151, 700, 600; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₃H₁₃O₃ 217.0865, found 217.0863.

(1R, 2S, 5R)-2-Isopropyl-5-methylcyclohexyl Picolinate (2h)

Brown liquid (196 mg, 75%); R_f = 0.50 (EtOAc/hexane) 2:8; H NMR (600 MHz, CDCl₃) δ 8.79–8.78 (d, J = 4.2 Hz, 1H), 8.11–8.09 (d, J = 7.8 Hz, 1H), 7.47–7.46 (m, 1H), 5.03–4.99 (m, 1H), 1.96–1.91 (m, 1H), 1.72–1.52 (m, 4H), 1.26–1.07 (m, 4H), 0.90–0.87 (m, 6H), 0.77–0.75 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 164.8, 150.0, 148.6, 137.2, 126.9, 125.3, 76.1, 47.1, 40.9, 34.4, 31.7, 26.5, 23.6, 22.2, 20.9, 16.5; FT-IR (KBr, cm^–1): 2955, 2870, 1713, 1584, 1456, 1306, 1244, 1133, 1087, 748, 707, 619; LRMS (ESI) m/z: [M + H]⁺ calcd for C₁₆H₂₄NO₂ 262.1807, found 262.2001.

3-Bromophenyl-quinoline-2-carboxylate (2i)

Liquid (271 mg, 83%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (400 MHz, CDCl₃) δ 8.43–8.41 (d, J = 8.4 Hz, 1H), 8.32–8.29 (m, 2H), 7.94–7.92 (d, J = 8.4 Hz, 1H), 7.81–7.77 (t, J = 7.2 Hz, 1H), 7.71–7.68 (t, J = 7.2 Hz, 1H), 7.38–7.36 (m, 2H), 7.25–7.21 (t, J = 7.6 Hz, 1H), 7.17–7.14 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 156.7, 150.9, 147.1, 146.2, 138.9, 131.7, 130.9, 129.7, 128.0, 125.0, 123.6, 122.8, 121.5, 120.4, 119.0, 114.5; FT-IR (KBr, cm^–1): 2924, 1650, 1582, 1472, 1351, 1250, 1062, 994, 863, 770, 679.

(E)-Prop-2-yn-1-yl 3-(4-methoxyphenyl)acrylate (2j)

Liquid (197 mg, 91%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (400 MHz, CDCl₃) δ 7.64–7.60 (d, J = 16 Hz, 1H), 7.41–7.39 (d, J = 8.8 Hz, 2H), 6.84–6.82 (d, J = 8.8 Hz, 2H), 6.28–6.24 (d, J = 16 Hz, 1H), 4.75–4.74 (d, J = 2.4 Hz, 2H), 3.75 (s, 3H), 2.52–2.50 (t, J = 2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 161.5, 145.5, 129.9, 126.8, 114.3, 78.0, 74.9, 55.3, 51.8; FT-IR (KBr, cm^–1): 3290, 2937, 2839, 2128, 1714, 1633, 1512, 1424, 1252, 1156, 1031, 828, 551; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₃H₁₃O₃ 217.0865, found 217.0863.

(S)-S-Phenyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanethioate (2k)

Solid (392 mg, 91%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 127–129 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.75–7.74 (d, J = 7.2 Hz, 3H), 7.62–7.59 (t, J = 7.2 Hz, 2H), 7.40–7.36 (m, 5H), 7.31–7.28 (t, J = 7.2 Hz, 3H), 5.28 (br, 1H), 4.53–4.50 (m, 1H), 4.47–4.40 (m, 2H), 4.25–4.23 (t, J = 6.6 Hz, 1H), 2.36–2.30 (m, 1H), 1.01–1.00 (d, J = 6.6 Hz, 3H), 0.92–0.91 (d, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 199.0, 156.4, 144.0, 143.9, 141.6, 134.8, 129.8, 129.5, 128.0, 127.3, 127.2, 125.3, 120.2, 67.4, 65.9, 47.5, 31.4, 19.7, 17.1; FT-IR (KBr, cm^–1): 2925, 1726, 1674, 1527, 1450, 1225, 1104, 1009, 741, 689; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₆H₂₆NO₃S 432.1633, found 432.1620.

S-p-Tolyl-4-methylbenzothioate (2l)

Solid (225 mg, 93%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 121–123 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (d, J = 7.8 Hz, 2H), 7.39–7.38 (d, J = 7.8 Hz, 2H), 7.28–7.25 (t, J = 7.2 Hz, 4H), 2.42 (s, 3H), 2.40 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 190.4, 144.7, 139.9, 135.3, 134.3, 130.3, 129.6, 127.7, 124.1, 21.9, 21.6; FT-IR (KBr, cm^–1): 2920, 1666, 1602, 1401, 1204, 1172, 904, 811, 717, 624; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₅H₁₅OS 243.0844, found 243.0843.

S-p-Tolyl-2-nitrobenzothioate (2m)

Brown solid (235 mg, 86%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 112–115 °C; ¹H NMR (600 MHz, CDCl₃) δ 8.09–8.08 (d, J = 7.8 Hz, 1H), 7.74–7.72 (t, J = 7.8 Hz, 1H), 7.68–7.64 (m, 2H), 7.46–7.44 (d, J = 8.4 Hz, 2H), 7.28–7.27 (d, J = 7.8 Hz, 2H), 2.40 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 190.9, 146.3, 140.7, 135.0, 134.9, 133.8, 131.7, 130.5, 128.6, 124.9, 123.2, 21.6; FT-IR (KBr, cm^–1): 2879, 1694, 1532, 1350, 1204, 1104, 904, 618; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₄H₁₂NO₃S 274.0538, found 274.0534.

S-(4-Nitrophenyl)-4-methylbenzothioate (2n)

White solid (224 mg, 82%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 118–120 °C; ¹H NMR (600 MHz, CDCl₃) δ 8.29–8.28 (d, J = 8.4 Hz, 2H), 7.92–7.91 (d, J = 8.4 Hz, 2H), 7.71–7.70 (d, J = 9.0 Hz, 2H), 7.32–7.31 (d, J = 7.8 Hz, 2H), 2.45 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 187.7, 148.4, 145.6, 136.6, 135.6, 133.6, 129.8, 127.9, 124.1, 22.0; FT-IR (KBr, cm^–1): 2923, 2852, 1673, 1598, 1518, 1345, 1261, 1025, 904, 811, 742, 636; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₄H₁₂NO₃S 274.0538, found 274.0533.

2-Nitro-N-phenylbenzamide (2o)

Brown solid (191 mg, 79%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 140–143 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.03–8.01 (d, J = 8.0 Hz, 1H), 7.67–7.63 (t, J = 7.2 Hz, 1H), 7.56–7.54 (d, J = 7.6 Hz, 4H), 7.33–7.29 (t, J = 7.6 Hz, 2H), 7.15–7.11 (t, J = 7.6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 146.2, 137.8, 134.0, 133.0, 130.6, 129.1, 128.9, 125.0, 124.5, 120.6, 120.5; FT-IR (KBr, cm^–1): 2924, 2854, 1655, 1599, 1442, 1346, 1258, 856, 788, 588; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₃H₁₁N₂O₃ 243.0770, found 243.0769.

N-Cyclopropyl-4-methoxybenzamide (2p)

White solid (157 mg, 82%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 123–125 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.72–7.70 (d, J = 8.4 Hz, 2H), 6.86 (br s, 1H), 6.81–6.79 (d, J = 8.8 Hz, 2H), 3.76 (s, 3H), 2.83–2.78 (m, 1H), 0.76–0.71 (m, 2H), 0.59–0.55 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 168.7, 162.1, 128.9, 126.8, 113.6, 55.4, 23.2, 6.6; FT-IR (KBr, cm^–1): 3005, 2839, 2058, 1624, 1504, 1324, 1311, 1176, 958, 845, 770; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₁H₁₄NO₂ 192.1025, found 192.1026.

4-Methyl-N-(prop-2-yn-1-yl)benzamide (2q)

White solid (147 mg, 85%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 117–119 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.70–7.68 (d, J = 8.4 Hz, 2H), 7.19–7.17 (d, J = 8.0 Hz, 2H), 6.82 (br s, 1H), 4.21–4.19 (q, 2H), 2.36 (s, 3H), 2.25–2.23 (t, J = 2.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 167.4, 142.3, 131.0, 129.3, 127.2, 79.9, 71.7, 29.8, 21.6; FT-IR (KBr, cm^–1): 2926, 1919, 1639, 1545, 1416, 1309, 1258, 1122, 840, 751, 654, 565; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₁H₁₂NO₂ 174.0919, found 174.0914.

N-Butylquinoline-2-carboxamide (2r)

Brown liquid (198 mg, 87%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (600 MHz, CDCl₃) δ 8.31–8.28 (m, 2H), 8.10–8.09 (d, J = 8.4 Hz, 1H), 7.87–7.85 (d, J = 7.8 Hz, 1H), 7.76–7.74 (t, J = 7.2 Hz, 1H), 7.61–7.59 (t, J = 7.2 Hz, 1H), 3.55–3.51 (m, 2H), 1.70–1.65 (m, 2H), 1.49–1.42 (m, 2H), 0.98–0.96 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 164.5, 150.0, 146.5, 137.5, 130.1, 129.7, 129.3, 127.9, 127.8, 118.9, 39.4, 31.9, 20.3, 13.9; FT-IR (KBr, cm^–1): 2926, 2856, 1643, 1547, 1369, 1133, 856, 725, 587; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₄H₁₇N₂O 229.1341, found 229.1340.

N-Benzylbenzamide (2s)

White solid (192 mg, 91%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 80–82 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.79–7.78 (d, J = 7.2 Hz, 2H), 7.51–7.48 (t, J = 7.2 Hz, 1H), 7.43–7.41 (t, J = 7.2 Hz, 2H), 7.35–7.34 (d, J = 4.2 Hz, 4H), 7.32–7.28 (m, 1H), 6.59 (br, 1H), 4.64–4.63 (d, J = 6.0 Hz, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 167.9, 138.3, 134.3, 131.7, 128.8, 128.7, 128.0, 127.7, 127.2, 44.2; FT-IR (KBr, cm^–1): 2954, 1637, 1552, 1416, 1316, 1260, 985, 805, 725, 695, 461; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₄H₁₄NO 212.1075, found 212.1075.

N-Cyclohexylbenzamide (2t)

Light yellow solid (189 mg, 93%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 152–154 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.75–7.74 (d, J = 7.2 Hz, 2H), 7.49–7.46 (t, J = 7.2 Hz, 1H), 7.43–7.40 (t, J = 7.8 Hz, 2H), 6.02 (br s, 1H), 4.00–3.94 (m, 1H), 2.04–2.01 (m, 2H), 1.76–1.73 (m, 3H), 1.66–1.64 (m, 1H), 1.45–1.38 (m, 2H), 1.26–1.18 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 167.5, 134.8, 131.4, 128.5, 126.9, 48.9, 33.0, 32.9, 25.5, 25.0; FT-IR (KBr, cm^–1): 2927, 2850, 1624, 1574, 1452, 1261, 1082, 700; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₃H₁₈NO 204.1388, found 204.1395.

N-Benzyl-2-naphthamide (2u)

Brown solid (232 mg, 89%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 138–140 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H), 7.88–7.85 (m, 4H), 7.59–7.50 (m, 2H), 7.40–7.29 (m, 5H), 6.86 (br s, 1H), 4.70–4.68 (d, J = 5.6 Hz, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 167.7, 138.4, 134.8, 132.7, 131.7, 129.1, 128.9, 128.5, 128.0, 127.8, 127.7, 127.6, 127.5, 126.8, 123.8, 44.3; FT-IR (KBr, cm^–1): 2923, 2853, 1637, 1547, 1414, 1320, 1264, 1146, 1048, 835, 780, 694, 478; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₈H₁₆NO 262.1232, found 262.1235.

N-(tert-Butyl)picolinamide (2v)

Brown liquid (153 mg, 86%); R_f = 0.50 (EtOAc/hexane) 2:8; ¹H NMR (400 MHz, CDCl₃) δ 8.51–8.50 (d, J = 4.8 Hz, 1H), 8.18–8.16 (d, J = 11.0 Hz, 1H), 8.01 (br s, 1H), 7.84–7.80 (t, J = 7.6 Hz, 1H), 7.41–7.37 (m, 1H), 1.49 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 163.6, 151.0, 147.9, 137.9, 126.0, 121.9, 51.0, 28.9; FT-IR (KBr, cm^–1): 2966, 1679, 1522, 1461, 1365, 1231, 998, 751, 621; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₀H₁₅N₂O 179.1184, found 179.1188.

N-(tert-Butyl)-2-naphthamide (2w)

White solid (182 mg, 80%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 162–164 °C; ¹H NMR (600 MHz, CDCl₃) δ 8.21 (s, 1H), 7.90–7.89 (d, J = 7.8 Hz, 1H), 7.86–7.84 (t, J = 6.6 Hz, 2H), 7.79–7.77 (m, 1H), 7.55–7.51 (m, 2H), 6.23 (br s, 1H), 1.52 (s, 9H); ¹³C NMR (150 MHz, CDCl₃) δ 167.2, 134.7, 133.3, 132.8, 129.0, 128.5, 127.8, 127.6, 127.1, 126.8, 123.8, 51.9, 29.1; FT-IR (KBr, cm^–1): 2963, 1638, 1571, 1452, 1318, 834, 474; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₅H₁₈NO 228.1388, found 228.1384.

(S)-Methyl-2-(4-bromobenzamido)propanoate (2x)

White solid (239 mg, 84%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 104–106 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.64–7.62 (d, J = 8.4 Hz, 2H), 7.51–7.50 (d, J = 8.4 Hz, 2H), 6.99 (br s, 1H), 4.76–4.72 (m, 1H), 3.75 (s, 3H), 1.48–1.47 (d, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.8, 166.1, 132.8, 131.9, 128.9, 126.6, 52.8, 48.7, 18.6; FT-IR (KBr, cm^–1): 2958, 2853, 1918, 1748, 1637, 1531, 1462, 1365, 1224, 1170, 845, 762, 620, 552; LRMS (ESI) m/z: [M + H]⁺ calcd for C₁₁H₁₃BrNO₃ 286.0079, found 286.9795.

(S)-Methyl-3-phenyl-2-pivalamidopropanoate (2y)

White solid (213 mg, 81%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 88–90 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.30–7.25 (m, 3H), 7.09–7.07 (d, J = 7.2 Hz, 2H), 6.06 (br s, 1H), 4.88–4.85 (m, 1H), 3.74 (s, 3H), 3.19–3.08 (m, 2H), 1.15 (s, 9H); ¹³C NMR (150 MHz, CDCl₃) δ 178.0, 172.4, 136.0, 129.4, 128.6, 127.2, 53.0, 52.4, 38.7, 37.8, 27.4; FT-IR (KBr, cm^–1): 2973, 1754, 1731, 1634, 1531, 1202, 1038, 754, 702; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₅H₂₂NO₃ 264.1600, found 264.1608.

N-Cyclohexyl-2-phenylacetamide (2z)

Light yellow solid (197 mg, 91%); R_f = 0.50 (EtOAc/hexane) 2:8; mp 131 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.37–7.34 (t, J = 7.2 Hz, 2H), 7.31–7.24 (m, 3H), 5.24 (br s, 1H), 3.78–3.72 (m, 1H), 3.55 (s, 2H), 1.84–1.81 (m, 2H), 1.62–1.55 (m, 3H), 1.35–1.25 (m, 2H), 1.12–0.97 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 170.2, 135.3, 129.4, 128.9, 127.2, 48.3, 43.9, 32.9, 25.5, 24.8; FT-IR (KBr, cm^–1): 2966, 1679, 1522, 1461, 1365, 1231, 998, 751, 621; LRMS (ESI) m/z: [M + H]⁺ calcd for C₁₄H₂₀NO 218.1545, found 218.1625.

(S)-Methyl-2-(2-benzamido-2-methylpropanamido)-3-methylbutanoate (3a)

White solid (256 mg, 80%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 109–111 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.78–7.77 (d, J = 7.8 Hz, 2H), 7.51–7.49 (t, J = 7.2 Hz, 1H), 7.44–7.42 (t, J = 7.2 Hz, 2H), 7.01 (br, 1H), 6.91 (br, 1H), 4.56–4.54 (m, 1H), 3.73 (s, 3H), 2.24–2.18 (m, 1H), 1.73–1.71 (d, J = 8.4 Hz, 6H), 0.97–0.90 (dd, J = 7.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 174.7, 172.6, 167.4, 134.9, 131.9, 128.8, 127.1, 57.9, 57.6, 52.4, 31.5, 25.7, 19.2; FT-IR (KBr, cm^–1): 2960, 1745, 1655, 1532, 1314, 1194, 1021, 694; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₇H₂₅N₂O₄ 321.1814, found 321.1813.

(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-3-phenylpropanoate (3b)

White solid (420 mg, 89%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 185–187 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.77–7.76 (d, J = 7.8 Hz, 2H), 7.55–7.52 (t, J = 7.8 Hz, 2H), 7.41–7.39 (t, J = 7.2 Hz, 2H), 7.31–7.19 (m, 8H), 6.38 (br, 1H), 5.42 (br, 1H), 4.51–4.49 (t, J = 7.2 Hz, 1H), 4.44–4.41 (m, 2H), 4.33–4.30 (t, J = 6.6 Hz, 1H), 4.19–4.17 (t, J = 7.2 Hz, 1H), 3.71 (s, 3H), 3.12–3.03 (m, 2H), 1.35–1.33 (d, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.0, 170.5, 156.1, 143.8, 141.5, 136.4, 129.6, 128.9, 127.9, 127.3, 125.2, 120.2, 67.3, 56.2, 52.7, 48.4, 47.3, 38.8, 18.5; FT-IR (KBr, cm^–1): 2923, 2852, 1737, 1696, 1649, 1538, 1105, 619; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₈H₂₉N₂O₅ 473.2076, found 473.2076.

(R)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-3-methylbutanoate (3c)

White solid (396 mg, 85%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 130–132 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.77–7.75 (d, J = 7.8 Hz, 2H), 7.58–7.57 (d, J = 6.6 Hz, 2H), 7.41–7.38 (t, J = 7.2 Hz, 2H), 7.32–7.29 (t, J = 6.6 Hz, 2H), 6.52 (br, 1H), 5.30 (br, 1H), 4.54–4.52 (m, 1H), 4.43–4.37 (m, 2H), 4.26–4.20 (m, 2H), 3.73 (s, 3H), 2.18–2.15 (m, 1H), 1.69–1.63 (m, 2H), 1.58–1.53 (m, 1H), 0.96–0.94 (m, 6H), 0.91–0.88 (m, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 172.4, 172.3, 156.4, 143.4, 141.4, 127.9, 127.2, 125.2, 120.1, 67.2, 57.3, 53.7, 52.3, 47.3, 41.5, 31.4, 24.8, 23.1, 22.2, 19.1, 17.9; FT-IR (KBr, cm^–1): 3257, 2962, 1724, 1649, 1535, 1252, 743, 619; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₇H₃₅N₂O₅ 467.2546, found 467.2537.

(S)-Methyl-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropanamido)propanoate (3d)

Yellowish liquid (365 mg, 89%); R_f = 0.50 (EtOAc/hexane) 4:6; ¹H NMR (600 MHz, CDCl₃) δ 7.79–7.76 (d, J = 7.8 Hz, 2H), 7.62–7.61 (d, J = 6.6 Hz, 2H), 7.43–7.39 (m, 2H), 7.35–7.31 (m, 2H), 7.09 (br, 1H), 6.04 (br, 1H), 4.43 (s, 2H), 4.22–4.21 (m, 1H), 4.10–4.07 (m, 1H), 3.73 (s, 3H), 1.50–1.38 (m, 9H); ¹³C NMR (150 MHz, CDCl₃) δ 174.2, 173.4, 155.1, 143.8, 141.3, 127.7, 127.1, 125.0, 120.0, 66.6, 56.8, 52.4, 48.3, 47.2, 25.5, 18.1; FT-IR (KBr, cm^–1): 2984, 1732, 1652, 1255, 739, 620; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₃H₂₇N₂O₅ 411.1920, found 411.1929.

(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-2-phenylacetate (3e)

White solid (459 mg, 86%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 176–178 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.75 (d, J = 7.2 Hz, 2H), 7.54–7.51 (t, J = 7.2 Hz, 2H), 7.41–7.38 (t, J = 7.2 Hz, 2H), 7.30–7.25 (m, 10H), 7.18–7.17 (m, 2H), 6.78 (br, 1H), 5.46–5.45 (d, J = 6.6 Hz, 1H), 5.37 (br, 1H), 4.52 (br, 1H), 4.42–4.37 (m, 1H), 4.32–4.31 (d, J = 6.6 Hz, 1H), 4.18–4.16 (t, J = 7.2 Hz, 1H), 3.68 (s, 3H), 3.18–3.01 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 170.8, 170.4, 156.1, 144.0, 141.5, 136.1, 129.1, 128.9, 128.8, 127.9, 127.4, 127.3, 125.2, 120.2, 67.3, 56.8, 56.1, 53.0, 47.3, 38.8; FT-IR (KBr, cm^–1): 2921, 1739, 1690, 1654, 1535, 1283, 730; LRMS (ESI) m/z: [M + H]⁺ calcd for C₃₃H₃₁N₂O₅ 535.2233, found 535.2211.

(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-2-phenylacetate (3f)

White solid (423 mg, 87%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 190–192 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.75 (d, J = 7.2 Hz, 2H), 7.57–7.56 (t, J = 7.2 Hz, 2H), 7.41–7.38 (m, 2H), 7.33–7.29 (m, 7H), 6.79 (br, 1H), 5.54–5.52 (d, J = 7.2 Hz, 1H), 5.40 (br, 1H), 4.41–4.38 (t, J = 7.8 Hz, 1H), 4.35–4.32 (t, J = 6.6 Hz, 1H), 4.21–4.18 (t, J = 7.2 Hz, 1H), 4.07–4.06 (t, J = 7.2 Hz, 1H), 3.73 (s, 3H), 2.17–2.13 (m, 1H), 1.01–0.96 (dd, J = 6.6 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 171.2, 170.9, 156.6, 144.1, 141.5, 136.0, 129.3, 128.9, 127.9, 127.5, 127.3, 125.3, 120.2, 67.3, 60.4, 56.8, 53.1, 47.3, 31.6, 18.1; FT-IR (KBr, cm^–1): 3064, 2957, 1738, 1691, 1653, 1536, 1450, 1389, 1289, 1174, 1034, 732, 647; LRMS (ESI) m/z: [M + H]⁺ calcd for C₂₉H₃₁N₂O₅ 487.2233, found 487.2135.

(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-methylpentanoate (3g)

White solid (390 mg, 89%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 126–128 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.77–7.75 (d, J = 7.8 Hz, 2H), 7.59–7.58 (t, J = 7.2 Hz, 2H), 7.41–7.39 (t, J = 7.2 Hz, 2H), 7.32–7.30 (t, J = 7.2 Hz, 2H), 6.41 (br, 1H), 5.45 (br, 1H), 4.64–4.59 (m, 1H), 4.39–4.38 (d, J = 6.6 Hz, 2H), 4.32–4.26 (m, 1H), 4.22–4.20 (t, J = 7.2 Hz, 1H), 3.73 (s, 3H), 1.67–1.61 (m, 2H), 1.56–1.53 (m, 1H), 1.41–1.40 (d, J = 7.2 Hz, 3H), 0.91–0.89 (t, J = 7.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 173.4, 172.3, 157.0, 144.0, 141.5, 127.9, 127.2, 125.2, 120.2, 67.1, 54.6, 52.2, 47.3, 41.4, 24.8, 23.0, 17.6; FT-IR (KBr, cm^–1): 2955, 1744, 1691, 1653, 1537, 1448, 1258, 738; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₅H₃₁N₂O₅ 439.2233, found 439.2226.

dl-(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-methylpentanoate (3h)

White solid (372 mg, 85%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 126–128 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.74 (d, J = 7.2 Hz, 2H), 7.59–7.58 (d, J = 7.8 Hz, 2H), 7.40–7.37 (t, J = 7.2 Hz, 2H), 7.31–7.28 (t, J = 7.2 Hz, 2H), 6.91 (br, 1H), 6.77 (br, 1H), 5.75–5.72 (t, J = 7.8 Hz, 1H), 4.64–4.60 (m, 1H), 4.40–4.34 (m, 3H), 4.24–4.20 (m, 1H), 3.71, 3.68 (s, 3H), 1.67–1.62 (m, 2H), 1.58–1.53 (m, 1H), 1.42–1.40 (d, J = 7.2 Hz, 3H), 0.91–0.88 (m, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 173.4, 173.3, 172.5, 172.4, 156.8, 156.2, 143.8, 141.4, 127.9, 127.2, 125.2, 120.1, 67.3, 52.5, 52.4, 50.9, 47.2, 41.4, 25.0, 22.9, 22.0, 21.9, 19.1; FT-IR (KBr, cm^–1): 2955, 1744, 1691, 1653, 1537, 1448, 1258, 738; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₅H₃₁N₂O₅ 439.2233, found 439.2200.

(S)-Methyl-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-3-phenylpropanoate (3i)

White solid (403 mg, 88%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 175–178 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.77–7.75 (d, J = 7.6 Hz, 2H), 7.59–7.57 (d, J = 7.2 Hz, 2H), 7.42–7.38 (t, J = 7.2 Hz, 2H), 7.32–7.28 (t, J = 7.6 Hz, 2H), 7.23–7.19 (m, 3H), 7.08–7.06 (d, J = 7.2 Hz, 2H), 6.59 (br, 1H), 5.57 (br, 1H), 4.91–4.86 (m, 1H), 4.39–4.37 (d, J = 7.2 Hz, 2H), 4.22–4.19 (t, J = 7.2 Hz, 1H), 3.88–3.85 (t, J = 8.0 Hz, 1H), 3.71 (s, 3H), 3.16–3.06 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 168.7, 156.5, 143.9, 141.5, 135.7, 128.8, 127.9, 127.4, 127.3, 125.2, 120.2, 67.2, 60.2, 53.3, 47.1, 44.4, 37.8; FT-IR (KBr, cm^–1): 2951, 1734, 1668, 1525, 1449, 1216, 1047, 741; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₇H₂₇N₂O₅ 459.1920, found 459.1921.

Methyl-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropanamido)acetate (3j)

Yellowish solid (337 mg, 85%); R_f = 0.50 (EtOAc/hexane) 4:6; ¹H NMR (600 MHz, CDCl₃) δ 7.77–7.76 (d, J = 7.2 Hz, 2H), 7.59–7.58 (d, J = 7.2 Hz, 2H), 7.41–7.39 (t, J = 7.2 Hz, 2H), 7.33–7.30 (t, J = 7.2 Hz, 2H), 6.73 (br, 1H), 5.30 (br, 1H), 4.45 (s, 2H), 4.21–4.19 (t, J = 6.6 Hz, 1H), 4.02 (s, 2H), 3.74 (s, 3H), 1.53 (s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 175.0, 170.4, 155.1, 143.8, 141.3, 127.7, 127.0, 125.0, 120.0, 66.5, 60.4, 52.2, 47.2, 41.4, 25.3; FT-IR (KBr, cm^–1): 2925, 2854, 1717, 1664, 1524, 1450, 1260, 1090, 739; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₂H₂₅N₂O₅ 397.1763, found 397.1765.

Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-3-hydroxypropanoate (3k)

White solid (444 mg, 91%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 191–193 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.75 (d, J = 7.2 Hz, 2H), 7.53–7.51 (t, J = 7.2 Hz, 2H), 7.41–7.38 (t, J = 7.2 Hz, 2H), 7.31–7.28 (t, J = 7.2 Hz, 5H), 7.19–7.18 (d, J = 6.6 Hz, 2H), 6.84 (br, 1H), 5.45 (br, 1H), 4.58 (s, 1H), 4.46–4.38 (m, 2H), 4.33–4.30 (t, J = 7.2 Hz, 1H), 4.18–4.15 (t, J = 7.2 Hz, 1H), 3.90 (s, 2H), 3.73 (s, 3H), 3.10–3.09 (d, J = 6.0 Hz, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 171.5, 170.6, 156.4, 143.8, 141.5, 136.3, 129.5, 128.9, 128.0, 127.3, 127.2, 125.3, 125.2, 120.2, 67.5, 63.0, 56.4, 55.1, 53.0, 47.2, 38.6; FT-IR (KBr, cm^–1): 2925, 1733, 1662, 1542, 1450, 1292, 738, 699; LRMS (ESI) m/z: [M + H]⁺ calcd for C₂₈H₂₉N₂O₆ 489.2026, found 489.2002.

(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-3-phenylpropanoate (3l)

White solid (488 mg, 89%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 171–173 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.77–7.76 (d, J = 7.2 Hz, 2H), 7.54–7.51 (t, J = 7.8 Hz, 2H), 7.42–7.39 (t, J = 7.8 Hz, 2H), 7.32–7.24 (m, 6H), 7.19–7.18 (d, J = 7.2 Hz, 2H), 6.25 (br, 1H), 5.31 (br, 1H), 4.79–4.76 (m, 1H), 4.43–4.41 (m, 2H), 4.29 (br, 1H), 4.19–4.17 (t, J = 6.6 Hz, 1H), 3.67 (s, 3H), 3.09–3.00 (m, 4H); ¹³C NMR (150 MHz, CDCl₃) δ 171.5, 170.5, 155.8, 143.9, 141.5, 135.7, 129.6, 129.4, 128.9, 128.7, 128.0, 127.3, 127.2, 125.2, 120.2, 67.3, 56.1, 53.5, 52.5, 47.3, 38.5, 38.1; FT-IR (KBr, cm^–1): 2924, 1738, 1697, 1644, 1535, 1444, 1257, 1033, 738, 698; LRMS (ESI) m/z: [M + H]⁺ calcd for C₃₄H₃₃N₂O₅ 549.2389, found 549.2388.

(S)-Methyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propanoate (3m)

White solid (360 mg, 91%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 141–143 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.77–7.76 (d, J = 7.2 Hz, 2H), 7.60–7.58 (d, J = 7.2 Hz, 2H), 7.42–7.39 (t, J = 7.2 Hz, 2H), 7.33–7.31 (t, J = 7.2 Hz, 2H), 6.43 (br, 1H), 5.35 (br, 1H), 4.59–4.56 (t, J = 7.2 Hz, 1H), 4.41 (br, 2H), 4.23–4.21 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H), 1.42–1.41 (d, J = 6.6 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 173.3, 172.0, 156.1, 144.0, 141.5, 127.9, 127.3, 125.2, 120.2, 67.3, 52.7, 50.6, 48.3, 47.3, 19.0, 18.5; FT-IR (KBr, cm^–1): 2927, 1741, 1688, 1650, 1530, 1451, 1259, 1050, 758; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₂H₂₅N₂O₅ 397.1763, found 397.1754.

Methyl-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)acetate (3n)

White solid (408 mg, 89%); R_f = 0.40 (EtOAc/hexane) 4:6; mp 143–146 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.75 (d, J = 7.2 Hz, 2H), 7.54–7.51 (m, 2H), 7.41–7.38 (t, J = 7.2 Hz, 2H), 7.31–7.28 (m, 4H), 7.25–7.20 (m, 3H), 6.41 (br, 1H), 5.41 (br, 1H), 4.48–4.33 (m, 3H), 4.19–4.17 (t, J = 7.2 Hz, 1H), 4.05–3.91 (m, 2H), 3.72 (s, 3H), 3.10 (s, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 171.3, 170.0, 156.2, 143.9, 141.5, 136.5, 129.5, 128.9, 127.9, 127.3, 127.2, 125.2, 120.2, 67.3, 56.2, 52.6, 47.3, 41.4, 38.6; FT-IR (KBr, cm^–1): 3299, 2921, 1749, 1692, 1649, 1540, 1439, 1260, 1032, 739; LRMS (ESI) m/z: [M + H]⁺ calcd for C₂₇H₂₇N₂O₅ 459.1920, found 459.1876.

dl-Methyl-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)acetate (rac-3n)

White solid (398 mg, 87%); R_f = 0.40 (EtOAc/hexane) 4:6; mp 143–146 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.75 (d, J = 7.2 Hz, 2H), 7.54–7.51 (m, 2H), 7.41–7.38 (t, J = 7.2 Hz, 2H), 7.31–7.28 (m, 4H), 7.25–7.20 (m, 3H), 6.37 (br, 1H), 5.39 (br, 1H), 4.48–4.33 (m, 3H), 4.19–4.17 (t, J = 7.2 Hz, 1H), 4.04–3.91 (m, 2H), 3.72 (s, 3H), 3.10 (s, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 171.3, 170.0, 156.2, 143.9, 141.5, 136.5, 129.5, 128.9, 127.9, 127.3, 127.2, 125.2, 120.2, 67.3, 56.2, 52.6, 47.3, 41.4, 38.6; FT-IR (KBr, cm^–1): 3299, 2921, 1749, 1692, 1649, 1540, 1439, 1260, 1032, 739; LRMS (ESI) m/z: [M + H]⁺ calcd for C₂₇H₂₇N₂O₅ 459.1920, found 459.1874.

(S)-Methyl-2-((S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-2-phenylacetate (3o)

White solid (288 mg, 70%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 126–128 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.32 (s, 3H), 7.28–7.11 (m, 7H), 6.93 (br, 1H), 5.48 (br, 1H), 4.98 (br, 1H), 4.42 (br, 1H), 3.69 (s, 3H), 3.12–3.00 (m, 2H), 1.40 (s, 9H); ¹³C NMR (150 MHz, CDCl₃) δ 171.1, 170.9, 155.6, 136.6, 136.4, 129.6, 129.5, 129.1, 129.0, 128.9, 128.7, 127.4, 127.1, 80.5, 56.7, 55.7, 53.0, 38.3, 28.4; FT-IR (KBr, cm^–1): 2979, 1750, 1648, 1535, 1269, 1168, 1017, 699; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₃H₂₉N₂O₅ 413.2076, found 413.2077.

(S)-Methyl-2-((S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-4-methylpentanoate (3p)

White solid (290 mg, 74%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 86–88 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.30–7.28 (t, J = 7.2 Hz, 2H), 7.25–7.20 (m, 3H), 6.28 (br, 1H), 5.02(br, 1H), 4.57–4.55 (t, J = 8.4 Hz, 1H), 4.35–4.34 (d, J = 6.0 Hz, 1H), 3.69 (s, 3H), 3.07–3.06 (m, 2H), 1.60–1.55 (m, 2H), 1.49–1.45 (m, 1H), 1.41 (s, 9H), 0.91–0.85 (m, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 173.1, 171.3, 155.6, 136.7, 129.5, 129.4, 128.7, 128.6, 127.0, 80.3, 55.7, 52.4, 50.9, 41.6, 38.2, 28.4, 24.8, 22.8; FT-IR (KBr, cm^–1): 2959, 2870, 1751, 1691, 1510, 1367, 1172, 702; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₁H₃₃N₂O₅ 393.2389, found 393.2389.

(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)propanamido)-3-phenylpropanoate (3q)

White solid (330 mg, 86%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 99–101 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.34–7.31 (m, 5H), 7.27–7.18 (m, 3H), 7.10–7.08 (d, J = 6.8 Hz, 2H), 6.80 (br, 1H), 5.56 (br, 1H), 5.12–5.03 (m, 2H), 4.87–4.82 (m, 1H), 4.28 (br, 1H), 3.69 (s, 3H), 3.15–3.02 (m, 2H), 1.32–1.31 (d, J = 10.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.2, 171.9, 156.0, 136.3, 135.8, 129.4, 128.6, 128.3, 128.1, 127.2, 67.1, 53.4, 52.5, 50.5, 37.9, 18.6; FT-IR (KBr, cm^–1): 2925, 1757, 1691, 1537, 1451, 1265, 1072, 734, 695; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₁H₂₅N₂O₅ 385.1763, found 385.1758.

(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-3-methylbutanoate (3r)

White solid (342 mg, 83%); R_f = 0.50 (EtOAc/hexane) 4:6; mp 103–105 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.36–7.18 (m, 10H), 6.33 (br, 1H), 5.39 (br, 1H), 5.11–5.06 (m, 2H), 4.46–4.43 (m, 2H), 3.68 (s, 3H), 3.08–3.04 (m, 2H), 2.10–2.06 (m, 1H), 0.85–0.71 (m, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 171.9, 170.9, 156.1, 136.5, 136.3, 129.5, 129.4, 129.0, 128.9, 128.7, 128.4, 128.3, 128.2, 127.3, 127.2, 67.3, 57.5, 56.4, 52.3, 38.5, 31.4, 19.0, 17.9; FT-IR (KBr, cm^–1): 2956, 1741, 1655, 1528, 1286, 750, 699; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₃H₂₉N₂O₅ 413.2076, found 413.2081.

Methyl-8-benzyl-11-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (3s)

White solid (422 mg, 90%); R_f = 0.40 (EtOAc/hexane) 4:6; mp 101 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.32–7.30 (m, 5H), 7.21–7.13 (m, 5H), 5.87 (br, 1H), 5.08 (s, 2H), 4.82 (br, 1H), 4.42–4.40 (m, 1H), 3.89–3.80 (m, 1H), 3.65 (s, 3H), 3.06–3.00 (m, 2H), 2.07–2.04 (m, 1H), 0.83–0.82 (d, J = 7.2 Hz, 3H), 0.80–0.79 (d, J = 6.6 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 172.0, 171.2, 169.5, 156.8, 136.5, 136.4, 129.5, 128.7, 128.3, 128.2, 127.1, 67.3, 57.6, 54.6, 52.2, 44.5, 38.5, 31.2, 19.0, 18.0; FT-IR (KBr, cm^–1): 3406, 1728, 1677, 1651, 1646, 763, 543; HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₅H₃₂N₃O₆ 470.2291, found 470.2295.

Recovered 2,4,6-Trichlorobenzoic Acid (Scheme 4)

White solid; R_f = 0.40 (EtOAc/hexane) 4:6; mp 162–164 °C; ¹H NMR (600 MHz, CDCl₃) δ 9.98 (br s, 1H), 7.39 (s, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 169.5, 136.9, 132.8, 131.3, 128.4; FT-IR (KBr, cm^–1): 3449, 1717, 1579, 1390, 1281, 1126, 849; LRMS (ESI) m/z: [M – H]⁺ calcd for C₆H₃Cl₃O₂ 222.9121, found 222.9214.

Recovered (E)-Ethyl-2-cyano-2-(hydroxyimino)acetate (Scheme 4)

Yellow solid; R_f = 0.40 (EtOAc/hexane) 4:6; mp 130–134 °C; ¹H NMR (600 MHz, CDCl₃) δ 4.41–4.37 (q, 2H), 1.37–1.35 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 159.0, 126.1, 107.9, 63.8, 14.0; FT-IR (KBr, cm^–1): 1729, 1433, 1314, 1071, 849, 767; LRMS (ESI) m/z: [M – H]⁺ calcd for C₄H₆N₂O₃ 141.0300, found 141.0510.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00732.

• Copies of the HPLC profiles; HPLC spectra for racemization study; and copies of characterization spectra for all of the synthesized compounds (PDF)

The authors declare no competing financial interest.