1-(2,5-dimethyl-4-alkylphenyl)-propanone (4b) To a mixture of propionyl chloride (0.51 mL, 5.88 mmol) and AlCl3 (780 mg, 5.88 mmol) in CH2Cl2 (25 mL) was added a solution of 4-(2,5-dimethylphenyl)butanoic acid (4a, 377 mg, 1.96 mmol) in CH2Cl2 (5 ml) at 0 °C. The reaction mixture was stirred for 1 h at the same temperature and poured into cold HCl solution (40 g ice and 10 mL of conc. HCl). The organic material was extracted by addition of CH2Cl2 (25 mL) and the aqueous layer was extracted with another portion of CH2Cl2 (50 mL). The combined organic layer was washed with brine (50 mL), dried with anhydrous MgSO4, passed through 3 cm silica gel pad, and evaporated under reduced pressure. The crude product was dissolved in oxalyl chloride (3 mL) and stirred overnight. After removal extra oxalyl chloride by evaporation under reduced pressure, the organic material was dissolved in CH2Cl2 (30 mL). To this solution, benzyl amine (0.257 mL, 2.35 mmol) and DIEA (1.7 mL, 9.8 mmol) were added and stirred 3 h. The organic layer was washed with brine (30 mL), dried with anhydrous MgSO4, evaporated under reduced pressure and purified by flash column chromatography using 33% EtOAc in Hexanes to give 4b as a pale-yellow solid.

1-(2,5-dimethyl-4-alkylphenyl)−1-hydroxypropan-2-one oxime (4c) To a solution of 4b (0.51 g, 1.50 mmol) and isoamyl nitrite (0.24 mL, 1.80 mmol) in MeOH (10 mL) at 45 °C was added dropwise to concentrated HC1 (0.25 mL) and the resulting mixture was stirred for 3 h at the same temperature. After cooling to room temperature, the organic material was partitioned into EtOAc (40 mL) and H2O (40 mL). The organic layer was washed with brine (40 mL), dried with anhydrous MgSO4, passed through 3 cm silica gel pad, and evaporated under reduced pressure. The residue was dissolved in MeOH (15 mL) and NaBH4 (50 mg) was added under ice bath. After stirring for 30 min, the organic material was partitioned into EtOAc (40 mL) and HCl solution (0.1 M, 30 mL). The organic layer was washed with saturated NaHCO3 (30 mL) and with brine (30 mL), dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography using 66% EtOAc in hexanes to give 4c as a white solid.

1-(2,5-dimethyl-4-alkylphenyl)−1-hydroxypropan-2-one (4) To a solution of 4c (0.30 g, 0.81 mmol) in DMF (2 mL) was added glyoxylic acid (60% water solution, 5 mL). After stirring for 5 h, the organic material was partitioned into EtOAc (40 mL) and H2O (40 mL). The organic layer was washed with brine (2 × 30 mL), dried with anhydrous MgSO4, evaporated under reduced pressure, and the residue was purified by flash column chromatography using 50% EtOAc in hexanes to give 4 as a pale-yellow oil.

H-[Op]-Gly-OMe (6a). To a solution of 1, (150 mg, 0.66 mmol) in anhydrous CH3CN (5 mL) were added GlyOMe·HCl (248 mg, 1.98 mmol), and DIEA (0.52 mL, 2.97 mmol). The reaction mixture was stirred overnight at room temperature, evaporated under reduced pressure and partitioned into CH2Cl2 (20 mL) and H2O (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO4, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6a as a pale-yellow oil.

H-[Mop]-Gly-OMe (6b). To a solution of 2, (200 mg, 0.63 mmol) in anhydrous CH3CN (8 mL) were added GlyOMe·HCl (237 mg, 1.89 mmol), DIEA (0.49 mL, 2.84 mmol) and tetrabutylammonium iodide (25 mg). The reaction mixture was refluxed for overnight, cooled to room temperature, evaporated under reduced pressure, and partitioned into CH2Cl2 (20 mL) and H2O (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO4, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6b as a colorless oil.

H-[Obz]-Gly-OMe (6c). To a solution of 3, (123 mg, 0.39 mmol) in anhydrous MeOH (20 mL) was added GlyOMe·HCl (242 mg, 1.93 mmol). The reaction mixture was refluxed for 24 h, cooled to room temperature, evaporated under reduced pressure, and partitioned into CH2Cl2 (20 mL) and H2O (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO4, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6c as a pale-yellow oil.

H-[Dmp]-Gly-OMe (6d). To a solution of 4, (200 mg, 0.57 mmol) in anhydrous toluene (20 mL) was added GlyOMe·HCl (215 mg, 1.71 mmol). The reaction mixture was refluxed for 5 h, cooled to room temperature, and partitioned into EtOAc (20 mL) and H2O (40 mL). The organic layer was washed with brine (40 mL), dried with anhydrous MgSO4, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6d as a colorless oil.

H-[Nve]-Gly-OMe (6e). To a solution of 5 (515 mg, 2.44 mmol) in MeOH (20 mL), were added GlyOMe·HCl (459 mg, 3.66 mmol), sodium bicarbonate (348 mg, 4.15 mmol). The reaction mixture was stirred for 4 h, cooled to 0 °C. After portionwise addition of NaBH4 (6.0 mmol, 227 mg), the mixture was stirred for 30 min and poured into HCl solution (0.1 N, 50 mL). The pH of water layer was adjusted to 4 ~ 6 by dropwise addition of NaOH (1 M), and the organic material was extracted with CH2Cl2 (50 mL). The aqueous layer was extracted with another portion of CH2Cl2 (50 mL). The combined organic layer was dried with anhydrous MgSO4, evaporated under reduced pressure and purified by flash column chromatography using 50% EtOAc in hexanes to give 6e as a colorless oil.

Acylation of 6a-e using symmetric anhydrides (7a-e). The symmetric anhydride of Boc-Phe was prepared in situ by mixing Boc-Phe (860 mg, 4 mmol) with EDCI (0.422 g, 2.2 mmol) in CH2Cl2 (10 ml, 0.2 M) for 5 min. To the solution of symmetric anhydride was added 6a-e (1 mmol), and the reaction mixture was stirred for 24 h at room temperature. After dilution with CH2Cl2 (50 ml), the organic layer was washed with brine (50 mL), dried with anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography to obtain an Hcnb-linked dipeptide (7a-e).

Methyl (E)−4-(2-(dimethylamino)vinyl)−3,5-dinitrobenzoate (13). To a stirred mixture of 4-Methyl-3,5-dinitrobenzoic acid (12) (2.0 g, 8.8 mmol) in anhydrous toluene (29 mL) was added DMF dimethyl acetal (3.2 mL, 24 mmol) dropwise at ambient temperature. The resulting mixture was heated to reflux in an oil bath and became dark red in color. After 16 h, the reaction was cooled to room temperature and the solvent removed under reduced pressure by co-evaporation with MeOH. The crude residue was recrystallized from hot ethanol to afford enamine 13 as a lustrous green solid which became red upon grinding.

Methyl 4-formyl-3,5-dinitrobenzoate (14). To a mixture of 13 (1.08 g, 3.66 mmol) and RuCl3 solution (0.1 M in H2O, 1.2 mL, 0.13 mmol) in CH3CN-H2O (6:1, 37 mL) was added NaIO4 (2.0 g, 9.1 mmol) in portions at rt. The color quickly dissipated, and solids formed. The reaction continued until TLC analysis showed consumption of starting material (typically 10–30 min). After stirring for 30 min, the solids were removed by vacuum filtration and washed with CH2Cl2. The filtrate was partitioned between H2O and CH2Cl2 and the aqueous layer was extracted twice with CH2Cl2. The combined organic extracts were washed with brine and sat. aq. Na2S2O4, dried over Na2SO4, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (30% EA/Hex) to afford 14 as an off-white solid.

Methyl 4-formyl-3-hydroxy-5-nitrobenzoate (15). A solution of 14 (678 mg, 2.67 mmol) in anhydrous DMF (5.1 mL) was added dropwise to a mixture of acetaldoxime [mixture of isomers] (325 µL, 5.33 mmol) and K2CO3 (811 mg, 5.87 mmol) in anhydrous DMF (5.1 mL) dropwise at rt. The reaction mixture immediately turned bright red purple upon mixing, then brownish green after stirring overnight. After 21 h, H2O (10 mL) was added to the reaction and the resulting solution extracted 5x with Et2O. The aqueous layer was then acidified to pH ~ 1–2 with concentrated HCl and extracted 4x with CH2Cl2. The combined organic extracts were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (25% EA/Hex) to afford 15 as a bright yellow solid.

4-Formyl-3-hydroxy-5-nitrobenzoic acid (16). Solid Ba(OH)2∙8 H2O (1.97 g, 6.24 mmol) was added in portions to a solution of 15 (468 mg, 2.08 mmol) in MeOH (23 mL) at rt. Following reaction completion (TLC), the mixture was concentrated under reduced pressure and the crude residue was partitioned between EtOAc and H2O. The aqueous layer was extracted twice with EtOAc, acidified to pH ~ 1–2 with concentrated HCl and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to afford 16 (379 mg, 86%) as a brown solid.

General procedure for solution-phase reductive amination (17a-c). HCl-AA-OtBu (0.666 mmol) was added to a solution of 15 (200.0 mg, 0.666 mmol) and DIEA (244 µL, 1.40 mmol) in CH3CN (9.5 mL). The reaction was stirred at rt for 45 min. Volatiles were removed and the residue was dissolved in MeOH (3.3 m). NaBH4 (75.6 mg, 2.00 mmol) was added, and the reaction was stirred for 15 min. Volatiles were removed and the residue was purified by flash column chromatography (20–45% EtOAc/Hex) to yield 17a-d.

General procedure for solution-phase acylation (18a-c). A mixture of Fmoc-AA-OH (0.29 mmol) and DIPC (22.5 µL, 0.14 mmol) was preactivated in dry CH2Cl2: DMF (1:1) (0.6 mL) for 5 min. 17a-c (0.13 mmol) as a solution in CH2Cl2 (2.1 mL) was added and the reaction was stirred for 2 h. The volatiles were removed and then dissolved in methanol (1.0 mL) and a solution of ammonium acetate (80.9 mg, 1.05 mmol) in water (0.3 mL) was added and the reaction was stirred overnight. Volatiles were removed and the residues were washed with water and extracted with ethyl acetate three times, concentrated, and then purified by flash column chromatography (30–40% EA/Hex).