Solution-Phase Synthesis of Chiral N-, O-, and S-Acyl Isopeptides

Abstract

A convenient synthesis of chiral N-, O-, and S-acyl monoiso- and diisopeptides from di- and tripeptides containing tryptophan, tyrosine, and cysteine units using benzotriazole is reported in solution phase.

Key words

amino acids - peptides - chirality - benzotriazole method - solution-phase synthesis

Solid-phase peptide synthesis (SPPS) has been used routinely for the synthesis of peptides and proteins. However, the synthesis of ‘difficult sequence’ containing peptides is still a challenge in peptide chemistry since these peptides are often obtained in low yield and purity by SPPS.[1] [2] [3] The difficult sequences are generally hydrophobic and prone to aggregation in solvent during chain elongation and final purification. This is attributed to inter/intramolecular hydrophobic interactions and hydrogen-bond networks formed among resin-bound peptide chains, resulting in the formation of extended secondary structures such as β-sheets.

Kiso and co-workers reported that 21% d-Val was detected during the synthesis of Boc-Thr(Fmoc-Val) via solid phase, while epimerization was completely avoided in the solution phase.[4] In addition, due to the presence of an additional amino group, N-, O-, or S-acyl isopeptides are generally hydrophilic, which is advantageous in effective purification by HPLC. The native peptides are then generated from the corresponding N-, O, or S-acyl isopeptide via an N-to-N,[5] O-to-N,[6] or S-to-N[7] [8] [9] intramolecular acyl migration reaction. The strategy facilitates the synthesis of peptides with ‘difficult sequences’. The O-acyl isopeptide method has already been used in various fields including peptide synthesis,[4a] ^, [10–14] ‘click peptide’ (‘switch peptide’) concept,[15] [16] [17] [18] macromolecules,[19] peptide localization,[20] protein splicing,[21] and proteomics.[22]

We now report the efficient single-step preparation of chiral N-, O, or S-acyl isopeptides incorporating tryptophan, tyrosine, and cysteine. N-Acylbenzotriazoles are advantageous for N-, O-, C-, and S- acylation,[23] especially where the corresponding acid chlorides are unstable or prone to racemization. N-[Protected (Pg)-α-aminoacyl]- and N-(Pg-dipeptidoyl)benzotriazoles have enabled fast preparations of biologically relevant peptides and peptide conjugates in high yields and purity, under mild reaction conditions, with full retention of the original chirality.[23] [24]

N-(Pg-α-aminoacyl)benzotriazoles 1a–e and N-(Pg-dipeptidoyl)benzotriazoles 1f–h were prepared following reported procedures[25] and were reacted with tryptophan, tryrosine, and cysteine to obtain the corresponding di- and tripeptides. These were reacted further with N-(Pg-α-aminoacyl)benzotriazoles and N-(Pg-dipeptidoyl)benzotriazoles to obtain mono- and diisotripeptides and -tetrapeptides.

Synthesis of Tryptophan Isopeptides

Benzotriazolides 1a–c were coupled with free tryptophan (2) at 0 to 20 °C in the presence of triethylamine in acetonitrile to give Cbz-protected dipeptides 3a–c (Table 1). These dipeptides 3a–c were N-acylated by (Cbz-protected-α-aminoacyl)benzotriazoles 1a,b,d in the presence of a base (Et₃N, DIPEA, K₂CO₃, or DBU) in acetonitrile to obtain protected monoisotripeptides 4a–d (Table 2). DBU gave better results than the other evaluated bases (Scheme [1]).

Table 1 Preparation of N-Protected Dipeptides 3a–c Containing a Tryptophan Unit

Product 3	Yield (%)	Mp (°C)	Lit. mp (°C)
Z-Gly-l-Trp-OH, 3a	79	139–141	142–143[26]
Z-l-Ala-l-Trp-OH, 3b	78	154–155	154–155[27]
Z-l-Val-l-Trp-OH, 3c	77	183–185	185–187[27]

Table 2 Preparation of N-Acyl Monoisotripeptides 4a–d

Product 4	Yield (%)	Mp (°C)
Z-Gly-l-Trp(Z-l-Phe)-OH, 4a	79	48–50
Z-l-Ala-l-Trp(Z-l-Phe)-OH, 4b	78	56–58
Z-l-Val-l-Trp(Z-Gly)-OH, 4c	75	54–56
Z-l-Val-l-Trp(Z-l-Ala)-OH, 4d	76	52–54

Scheme 1 Preparation of N-acyl monoisotripeptides 4a–d

Synthesis of Tyrosine Isopeptide

The benzotriazolide 1e was coupled with free tyrosine (5) at 0 to 20 °C in the presence of DBU in DMF to give Boc-protected dipeptide 6. The dipeptide 6 was O-acylated by N-(Pg-α-aminoacyl)benzotriazole 1b in the presence of triethylamine to obtain the protected monoisotripeptide 7 (Scheme [2]).

Scheme 2 Preparation of O-acyl monoisotripeptide 7

Synthesis of Cysteine Isopeptides

The benzotriazolides 1b–h were coupled with free cysteine (8) at 0 to 20 °C in the presence of triethylamine in acetonitrile to give N-protected di- and tripeptides 9a–f (Table 3, Scheme [3]). These di- and tripeptides 9a–f were S-acylated by N-(Pg-α-aminoacyl)benzotriazoles and dipeptidoylbenzotriazoles­ in the presence of potassium bicarbonate to obtain protected monoisotri-, -tetra-, and -pentapeptides 10a–h (Table 4, Scheme [3]).

Scheme 3 Preparation of O-acyl monoisotripeptides 10a–h

Table 3 Preparation of N-Protected Peptides 9a–f Containing a Cysteine Unit

Product 9	Yield (%)	Mp (°C)
Z-l-Ala-l-Cys-OH, 9a	96	170–171
Z-l-Val-l-Cys-OH, 9b	96	169–170
Z-l-Phe-l-Cys-OH, 9c	98	125–126
Z-l-Phe-Gly-l-Cys-OH, 9d	90	156–158
Z-l-Phe-l-Ala-l-Cys-OH, 9e	92	177–179
Z-l-Ala-l-Phe-l-Cys-OH, 9f	95	170–172

Table 4 Preparation of S-Acyl Peptides 10a–h

Product 10	Yield (%)	Mp (°C)
Z-l-Phe-l-Cys(Z-Gly)-OH, 10a	45a	171–173
Z-l-Val-l-Cys(Z-Gly)-OH, 10b	84	145–147
Z-l-Ala-l-Cys(Z-l-Phe)-OH, 10c	86	142–143
Z-l-Ala-l-Phe-l-Cys(Z-l-Ala)-OH, 10d	97	167–169
Z-l-Ala-l-Cys(Z-l-Ala-l-Phe)-OH, 10e	95	161–163
Z-l-Phe-Gly-l-Cys(Z-l-Ala)-OH, 10f	94	169–171
Z-l-Phe-l-Ala-l-Cys(Z-l-Ala-)-OH, 10g	96	170–171
Z-l-Phe-Gly-l-Cys(Z-l-Ala-l-Phe)-OH, 10h	98	146–148

^a Compound was isolated by extraction with EtOAc.

In summary, N-peptidoylbenzotriazoles are advantageous coupling reagents that (i) are sufficiently reactive to form amide bonds at ambient temperature; (ii) are stable enough to resist side reactions and can be stored in the crystalline state at room temperature; (iii) provide good yields without detectable racemization; (iv) are almost always crystalline; (v) are relatively insensitive to moisture and can be used in aqueous solution, and (vi) are inexpensive to prepare. Hence N-(Pg-α-aminoacyl)benzotriazole and N-(Pg-α-dipeptidoyl)benzotriazole reagents allow efficient peptide couplings to generate monoisopeptides via N-, O-, and S-acylation.

Commercial reagents were purchased from Sigma-Aldrich and were used without purification. Solvents were purified by distillation. Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl₃ or CD₃OD on Mercury or Gemini NMR spectrometers operating at 300 MHz for ¹H (with TMS as an internal standard) and 75 MHz for ¹³C. Elemental analyses were performed on a Carlo Erba-EA1108 instrument. Analytical TLC was performed on E. Merck silica gel 60 F254 plates and visualized by UV and KMnO₄ staining. Flash column chromatography was performed on E. Merck­ silica gel 60 (40–63 mm). Yields refer to chromatographically and spectroscopically pure compounds. Mass spectrometry was done with electrospray ionization (ESI).

Cbz-Protected Dipeptides 3a–c; General Procedure

To the respective N-(Pg-α-aminoacyl)benzotriazole 1a–c (0.5 mmol) in MeCN (10 mL) was added a solution of tryptophan (2; 102 mg, 0.5 mmol) and Et₃N (0.5 mL) in H₂O (3 mL). The reaction mixture was stirred for 8 h at 0 °C. The mixture was acidified by aq 1 M HCl and extracted with EtOAc (10 mL). The organic layer was washed with aq 1 M HCl (3 mL) and brine (5 mL), and dried (MgSO₄). After evaporation of solvent, the residue was triturated with Et₂O and the solid formed was filtered and dried under vacuum to give dipeptides 3a–c, respectively (Table 1).

[(Benzyloxy)carbonyl]glycyl-l-tryptophan (3a)

Yield: 0.3 g (79%); white solid; mp 139–141 °C (Lit.[26] mp 142–143 °C).

¹H NMR (DMSO-d ₆): δ = 12.65 (br s, 1 H), 10.87 (d, J = 2.4 Hz, 1 H), 8.08 (d, J = 7.7 Hz, 1 H), 7.53 (d, J = 7.8 Hz, 1 H), 7.48–7.22 (m, 7 H), 7.14 (s, 1 H), 7.07 (t, J = 7.5 Hz, 1 H), 6.99 (t, J = 7.4 Hz, 1 H), 5.03 (s, 2 H), 4.74–4.28 (m, 1 H), 3.77–3.52 (m, 2 H), 3.18 (dd, J = 14.7, 5.2 Hz, 1 H), 3.05 (dd, J = 14.6, 7.8 Hz, 1 H).

¹³C NMR (DMSO-d ₆): δ = 174.2, 169.9, 157.4, 138.0, 137.0, 129.3, 128.7, 128.2, 124.6, 121.9, 119.4, 119.1, 112.3, 110.6, 66.4, 53.9, 44.2, 28.1.

Anal. Calcd for C₂₁H₂₁N₃O₅: C, 63.79; H, 5.35; N, 10.63. Found: C, 63.71; H, 5.226; N, 10.37.

Dipeptides 3b,c gave also physical and spectral data in conformity with the reported values.[26] [27]

Protected Monoisotripeptides 4a–d; General Procedure

To a precooled solution of tryptophan containing the appropriate peptide 3a–c (0.5 mmol) in MeCN (10 mL) and Et₃N (1.5 equiv) at 0 °C was added a solution of N-(Pg-α-aminoacyl)benzotriazole 1a,b, or d (0.5 mmol) in MeCN (3 mL). After completion of the reaction (8 h), the reaction mixture was acidified with aq 1 M HCl and then extracted with EtOAc (10 mL). The organic layer was washed with H₂O (10 mL) and dried (Na₂SO₄). Evaporation of the solvent gave the desired product 4a–d, respectively, which was recrystallized from EtOAc–hexanes (Table 2).

1-{[(Benzyloxy)carbonyl]-l-phenylalanyl}-N ^α-{[(benzyl­oxy)carbonyl]glycyl}-l-tryptophan (4a)

Yield: 0.52 g (79%); off-white solid; mp 48–50 °C.

¹H NMR (DMSO-d ₆): δ = 12.70 (br s, 1 H), 10.87 (s, 1 H), 8.09 (d, J = 7.8 Hz, 1 H), 7.66 (d, J = 8.6 Hz, 1 H), 7.54 (d, J = 7.8 Hz, 1 H), 7.49–7.24 (m, 16 H), 7.12–6.94 (m, 3 H), 5.04–4.80 (m, 4 H), 4.58–4.46 (m, 1 H), 4.28–4.14 (m, 1 H), 3.75–3.55 (m, 2 H), 3.27–2.98 (m, 4 H).

¹³C NMR (DMSO-d ₆): δ = 173.3, 173.2, 169.0, 156.5, 156.0, 137.9, 137.1, 137.0, 136.1, 129.1, 128.3, 128.3, 128.3, 128.2, 127.8, 127.7, 127.6, 127.5, 127.2, 123.7, 120.9, 118.4, 118.2, 111.4, 109.6, 65.5, 65.3, 55.5, 52.9, 43.3, 36.5, 27.2.

HRMS (–ESI-TOF): m/z [M – H]^– calcd for C₃₈H₃₆N₄O₈: 675.2460; found: 675.2477.

N ^α-{[(Benzyloxy)carbonyl]-l-alanyl}-1-{[(benzyloxy)carbonyl]-l-phenylalanyl}-l-tryptophan (4b)

Yield: 0.53 g (78%); off-white solid; mp 56–58 °C.

¹H NMR (DMSO-d ₆): δ = 12.69 (s, 1 H), 10.86 (s, 1 H), 8.04 (d, J = 7.7 Hz, 1 H), 7.65 (d, J = 8.4 Hz, 1 H), 7.53 (d, J = 7.9 Hz, 1 H), 7.39–7.19 (m, 18 H), 7.06 (t, J = 7.5 Hz, 1 H), 6.98 (t, J = 7.4 Hz, 1 H), 5.08–4.90 (m, 4 H), 4.49 (q, J = 7.0 Hz, 1 H), 4.28–4.04 (m, 2 H), 3.23–3.02 (m, 3 H), 2.91–2.71 (m, 1 H), 1.21 (t, J = 6.8 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 173.3, 173.2, 172.5, 156.0, 155.6, 137.9, 137.0, 136.0, 129.1, 128.3, 128.3, 128.2, 128.2, 127.8, 127.7, 127.6, 127.5, 127.2, 126.4, 123.7, 120.9, 118.4, 118.2, 111.3, 109.6, 65.4, 65.3, 55.5, 52.9, 49.9, 39.5, 36.5, 27.0, 18.2.

HRMS (–ESI-TOF): m/z [M – H]^– calcd for C₃₉H₃₈N₄O₈: 689.2617; found: 689.2637.

N ^α-{[(Benzyloxy)carbonyl]-l-valyl}-1-{[(benzyloxy)carbonyl]glycyl}-l-tryptophan (4c)

Yield: 0.46 g (75%); off-white solid; mp 54–56 °C.

¹H NMR (DMSO-d ₆): δ = 12.59 (br s, 1 H), 10.85 (s, 1 H), 8.15 (d, J = 7.5 Hz, 1 H), 7.53 (d, J = 7.8 Hz, 1 H), 7.38–7.22 (m, 12 H), 7.20–7.16 (m, 1 H), 7.06 (t, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.4 Hz, 1 H), 5.12–4.95 (m, 4 H), 4.49 (dd, J = 13.8, 7.3 Hz, 2 H), 4.09–3.54 (m, 2 H), 3.22–2.80 (m, 2 H), 1.95 (dd, J = 14.1, 7.4 Hz, 1 H), 0.82 (t, J = 7.2 Hz, 6 H).

¹³C NMR (DMSO-d ₆): δ = 173.2, 171.2, 159.4, 156.1, 137.1, 136.1, 128.4, 128.3, 127.8, 127.7, 123.6, 120.9, 118.4, 111.3, 109.6, 65.4, 59.9, 52.9, 30.5, 27.1, 19.2, 18.1.

HRMS (–ESI-TOF): m/z [M – H]^– calcd for C₃₄H₃₆N₄O₈: 627.2460; found: 627.2488.

1-{[(Benzyloxy)carbonyl]-l-alanyl}-N ^α-{[(benzyloxy)carbonyl]-l-valyl}-l-tryptophan (4d)

Yield: 0.48 g (76%); off-white solid; mp 52–54 °C.

¹H NMR (DMSO-d ₆): δ = 8.15 (s, 1 H), 7.85–7.72 (m, 2 H), 7.57 (dd, J = 10.7, 7.3 Hz, 1 H), 7.45–7.19 (m, 12 H), 7.09–6.92 (m, 2 H), 5.10–4.90 (m, 4 H), 4.78 (t, J = 6.4 Hz, 1 H), 4.27–4.12 (m, 1 H), 4.04 (dd, J = 7.2, 3.7 Hz, 1 H), 3.47–3.09 (m, 2 H), 2.10–1.90 (m, 1 H), 1.36 (t, J = 7.1 Hz, 3 H), 0.88 (d, J = 7.4 Hz, 3 H), 0.84 (d, J = 6.2 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 176.5, 174.9, 174.8, 173.9, 158.2, 138.1, 138.0, 137.8, 129.6, 129.5, 129.0, 128.8, 128.8, 127.1, 124.8, 122.5, 120.0, 119.4, 115.7, 112.5, 67.5, 61.9, 54.5, 50.8, 32.1, 28.6, 19.8, 18.8, 18.0.

HRMS (–ESI-TOF): m/z [M – H]^– calcd for C₃₅H₃₈N₄O₈: 641.2617; found: 641.2626.

(S)-3-(4-{[((Benzyloxy)carbonyl)-l-alanyl]oxy}phenyl)-2-{(S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanamido}propanoic Acid (7)

To Boc-Phe-Bt (1e; 183 mg, 0.5 mmol) in MeCN (10 mL) was added a solution of tyrosine (5; 91 mg, 0.5 mmol) and DBU (1.0 mmol) in DMF (5 mL). The reaction mixture was stirred for 6 h at 20 °C. The mixture was acidified with aq 2 M HCl and extracted with EtOAc­ (10 mL). The organic layer was washed with aq 2 M HCl (3 mL) and brine (5 mL), and dried (MgSO₄). The crude product 6 was treated with Z-Ala-Bt (1b; 109 mg, 0.34 mmol) in the presence of Et₃N (1.5 equiv) in MeCN–H₂O (7 mL:3 mL) at 0 °C. After completion of the reaction (6 h), the mixture was acidified with aq 4 M HCl. The solution was then extracted with EtOAc (10 mL), the EtOA­c layer was washed with H₂O (10 mL), and dried (Na₂SO₄). Evaporation of the solvent gave the desired product 7; yield: 0.41 g (65%); white solid; mp 171–173 °C.

¹H NMR (DMSO-d ₆): δ = 8.12 (d, J = 8.0 Hz, 1 H), 7.95 (d, J = 6.9 Hz, 1 H), 7.40–7.15 (m, 13 H), 6.98 (d, J = 8.3 Hz, 2 H), 6.88 (d, J = 8.6 Hz, 1 H), 5.07 (s, 2 H), 4.48 (s, 1 H), 4.32 (s, 1 H), 4.18 (s, 1 H), 3.13–2.89 (m, 3 H), 2.69 (t, J = 12.3 Hz, 1 H), 1.42 (d, J = 7.0 Hz, 3 H), 1.28 (s, 9 H).

¹³C NMR (DMSO-d ₆): δ = 172.7, 171.8, 171.7, 156.0, 155.2, 149.1, 138.2, 136.9, 135.1, 130.4, 129.2, 128.4, 128.0, 127.9, 127.8, 126.2, 121.2, 78.1, 65.6, 55.8, 53.3, 49.6, 37.4, 36.0, 28.1, 16.8.

HRMS (–ESI-TOF): m/z [M – H]^– for C₃₄H₃₉N₃O₉: 632.2614; found: 632.2599.

N-Protected Di- and Tripeptides 9a–f; General Procedure

To the corresponding N-protected aminoacyl- and dipeptidoylbenzotriazole 1b–h (0.5 mmol) in MeCN (10 mL) was added a solution of cysteine (8; 61 mg, 0.5 mmol) and Et₃N (0.5 mL) in H₂O (3 mL). The reaction mixture was stirred for 4 h at 0 °C. The mixture was acidified with aq 4 M HCl and extracted with EtOAc (10 mL). The organic layer was washed with aq 4 M HCl (3 mL) and brine (5 mL), and dried (Na₂SO₄). After evaporation of the solvent, the residue was triturated with Et₂O–hexanes (1:1) and the solid formed was filtered and dried under vacuum to give the respective dipeptides 9a–c and tripeptides 9d–f (Table 3).

[(Benzyloxy)carbonyl]-l-alanyl-l-cysteine (9a)

Yield: 0.31 g (97%); white solid; mp 170–171 °C.

¹H NMR (DMSO-d ₆): δ = 8.67 (d, J = 7.9 Hz, 1 H), 7.92–7.81 (m, 1 H), 7.81–7.65 (m, 5 H), 5.52–5.35 (m, 2 H), 4.99–4.86 (m, 1 H), 4.61–4.45 (m, 1 H), 3.65–3.33 (m, 2 H), 1.65 (d, J = 7.1 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 173.1, 172.2, 156.1, 137.4, 128.8, 128.2, 128.2, 65.9, 51.9, 50.4, 31.1, 18.7.

Anal. Calcd for C₁₄H₁₈N₂O₅S: C, 51.52; H, 5.56; N, 8.58. Found: C, 51.83; H, 5.55; N,9.10.

[(Benzyloxy)carbonyl]-l-valyl-l-cysteine (9b)

Yield: 0.17 g (96%); white solid; mp 169–170 °C.

¹H NMR (DMSO-d ₆): δ =12.84 (s, 1 H), 8.16 (d, J = 7.7 Hz, 1 H), 7.45–7.20 (m, 6 H), 5.04 (s, 2 H), 4.57–4.27 (m, 1 H), 3.94 (dd, J = 8.9, 6.8 Hz, 1 H), 2.92–2.71 (m, 2 H), 2.43 (t, J = 8.5 Hz, 1 H), 2.09–1.86 (m, 1 H), 0.89 (d, J = 6.7 Hz, 3 H), 0.85 (d, J = 6.6 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 171.4, 156.1, 137.1, 128.3, 127.8, 127.6, 65.4, 60.0, 54.3, 30.3, 25.5, 19.2, 18.1.

Anal. Calcd for C₁₆H₂₂N₂O₅S: C, 54.22; H, 6.26; N, 7.90. Found: C, 54.26; H, 6.37; N, 7.82.

[(Benzyloxy)carbonyl]-l-phenylalanyl-l-cysteine (9c)

Yield: 0.39 g (98%); white solid; mp 125–126 °C.

¹H NMR (DMSO-d ₆): δ = 12.95 (br s, 1 H), 8.48 (d, J = 7.7 Hz, 1 H), 7.48 (d, J = 8.8 Hz, 1 H), 7.38–7.10 (m, 10 H), 5.00–4.82 (m, 2 H), 4.61–4.47 (m, 1 H), 4.39–4.24 (m, 1 H), 3.21 (dd, J = 13.7, 4.7 Hz, 1 H), 3.12–2.95 (m, 2 H), 2.74 (dd, J = 13.8, 10.9 Hz, 1 H).

¹³C NMR (DMSO-d ₆): δ = 171.8, 171.8, 155.8, 138.1, 137.0, 129.2, 128.3, 128.0, 127.7, 127.4, 126.3, 65.2, 56.0, 51.6, 37.5.

Anal. Calcd for C₂₀H₂₂N₂O₅S: C, 59.69, H, 5.51; N, 6.96. Found: C,60.10; H,5.50; N,6.83.

[(Benzyloxy)carbonyl]-l-phenylalanylglycyl-l-cysteine (9d)

Yield: 0.4 g (90%); white solid; mp 156–158 °C.

¹H NMR (DMSO-d ₆): δ = 12.90 (s, 1 H), 8.39 (t, J = 5.8 Hz, 1 H), 8.07 (d, J = 7.9 Hz, 1 H), 7.58 (d, J = 8.5 Hz, 1 H), 7.40–7.10 (m, 10 H), 4.97 (d, J = 12.9 Hz, 1 H), 4.92 (d, J = 11.9 Hz, 1 H) 4.53–4.40 (m, 1 H), 4.36–4.21 (m, 1 H), 3.90–3.71 (m, 2 H), 3.04 (dd, J = 14.0, 4.0 Hz, 1 H), 2.95–2.72 (m, 2 H), 2.43 (t, J = 8.5 Hz, 1 H), 1.36 (s, 1 H).

¹³C NMR (DMSO-d ₆): δ = 172.0, 171.7, 168.8, 156.0, 138.2, 137.0, 129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 65.3, 56.2, 54.3, 42.0 37.3, 25.7.

Anal. Calcd for C₂₂H₂₅N₃O₆S: C, 57.50; H, 5.48; N, 9.14. Found: C, 57.28; H, 5.48; N, 9.00.

[(Benzyloxy)carbonyl]-l-phenylalanyl-l-alanyl-l-cysteine (9e)

Yield: 0.43 g (92%); white solid; mp 177–179 °C.

¹H NMR (DMSO-d ₆): δ = 12.93 (br s, 1 H), 8.21 (d, J = 7.9 Hz, 1 H), 7.97 (d, J = 8.9 Hz, 1 H), 7.43 (d, J = 7.5 Hz, 1 H), 7.39–7.16 (m, 10 H), 5.04 (d, J = 12.6 Hz, 1 H), 4.98 (d, J = 12.4 Hz, 1 H), 4.56 (dd, J = 9.1, 4.6 Hz, 1 H), 4.43 (dd, J = 7.0, 4.4 Hz, 1 H), 4.06–3.94 (m, 1 H), 3.07 (dd, J = 13.6, 4.1 Hz, 1 H), 2.94–2.72 (m, 3 H), 1.19 (dd, J = 15.6, 8.3 Hz, 1 H), 1.12 (d, J = 7.1 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 172.3, 171.3, 170.9, 155.7, 137.6, 136.9, 129.3, 128.3, 128.0, 127.8, 127.7, 126.2, 65.4, 54.4, 53.5, 50.2, 37.2, 25.5, 18.0.

Anal. Calcd for C₂₃H₂₇N₃O₆S: C, 58.34; H, 5.75; N, 8.87. Found: C, 57.96; H, 5.81; N, 8.90.

[(Benzyloxy)carbonyl]-l-alanyl-l-phenylalanyl-l-cysteine (9f)

Yield: 0.44 g (95%); white solid; mp 170–172 °C.

¹H NMR (DMSO-d ₆): δ = 12.93 (s, 1 H), 8.22 (d, J = 7.9 Hz, 1 H), 7.97 (d, J = 8.9 Hz, 1 H), 7.43 (d, J = 7.4 Hz, 1 H), 7.40–7.15 (m, 10 H), 5.04 (d, J = 12.6 Hz, 1 H), 4.98 (d, J = 12.4 Hz, 1 H), 4.56 (dd, J = 9.2, 4.6 Hz, 1 H), 4.43 (dd, J = 7.1, 4.4 Hz, 1 H), 4.06–3.95 (m, 1 H), 3.07 (dd, J = 13.8, 4.2 Hz, 1 H), 2.92–2.73 (m, 2 H), 2.44 (d, J = 8.7 Hz, 1 H), 1.12 (d, J = 7.2 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 172.3, 171.3, 170.9, 155.7, 137.6, 136.9, 129.3, 128.3, 128.0, 127.8, 127.7, 126.2, 65.4, 54.4, 53.5, 50.2, 37.2, 25.5, 18.0.

Anal. Calcd for C₂₃H₂₇N₃O₆S: C, 58.34, H, 5.75; N, 8.87. Found: C, 57.96; H, 5.81; N, 8.90.

S-Acyl Peptides 10a–h; General Procedure

To a precooled solution of cysteine containing the appropriate peptide 9a–f (0.5 mmol) in MeCN–H₂O (7 mL:3 mL) at 0 °C was added a solution of N-acylbenzotriazole or N-(Pg-α-aminoacyl)benzotriazole 1b–h (0.5 mmol) in MeCN (3 mL) with stirring followed by addition of KHCO₃ (0.14 g) for 10 min in four installments. After additional stirring for 2–3 h at 0 to 10 °C, the reaction mixture was acidified with aq 4 M HCl. The solution was then extracted with EtOAc (10 mL), the EtOAc layer was washed with H₂O (10 mL), and dried (Na₂SO₄). Evaporation of the solvent gave the respective desired product 10a–h, which was recrystallized from EtOAc–hexanes­ (Table 4).

N-{[(Benzyloxy)carbonyl]-l-phenylalanyl}-S-{[(benzyloxy)carbonyl]glycyl}-l-cysteine (10a)

Yield: 0.3 g (45%); white solid; mp 171–173 °C.

¹H NMR (DMSO-d ₆): δ = 13.00 (s, 1 H), 8.48 (d, J = 6.6 Hz, 1 H), 8.03 (t, J = 5.6 Hz, 1 H), 7.49 (d, J = 8.9 Hz, 1 H), 7.41–7.13 (m, 15 H), 5.14–4.82 (m, 4 H), 4.38–4.29 (m, 2 H), 3.95 (d, J = 6.1 Hz, 2 H), 3.27–2.91 (m, 3 H), 2.77–2.68 (m, 1 H).

¹³C NMR (DMSO-d ₆): δ = 198.4, 171.7, 171.4, 156.5, 155.8, 138.1, 137.0, 136.7, 129.2, 128.5, 128.4, 128.3, 128.0, 127.9, 127.7, 127.6, 127.4, 65.8, 65.2, 56.0, 51.7, 50.4, 37.5, 29.1.

HRMS (–ESI-TOF): m/z [M – H]^– calcd for C₃₀H₃₁N₃O₈S : 592.1759; found: 592.1746.

N-{[(Benzyloxy)carbonyl]-l-valyl}-S-{[(benzyloxy)carbonyl]glycyl}-l-cysteine (10b)

Yield: 0.45 g (84%); white solid; mp 145–147 °C.

¹H NMR (DMSO-d ₆): δ = 8.32 (d, J = 7.8 Hz, 1 H), 7.99 (t, J = 6.1 Hz, 1 H), 7.46–7.19 (m, 11 H), 5.14–4.96 (m, 4 H), 4.40–4.22 (m, 1 H), 3.94 (d, J = 6.4 Hz, 3 H), 3.34 (dd, J = 13.5, 5.3 Hz, 2 H), 3.10 (dd, J = 13.5, 8.4 Hz, 1 H), 2.02–1.95 (m, 1 H), 0.87(d, J = 6 Hz, 3 H), 0.83 (d, J = 6 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 198.4, 171.5, 171.3, 156.5, 156.1, 137.1, 136.8, 128.4, 128.4, 127.9, 127.8, 127.6, 65.9, 65.5, 59.9, 51.7, 50.4, 30.6, 29.1, 19.2, 17.9.

Anal. Calcd for C₂₆H₃₁N₃O₈S: C, 57.24; H, 5.73; N, 7.70. Found: C, 57.0; H, 5.78; N, 7.68.

N-{[(Benzyloxy)carbonyl)]-l-alanyl}-S-{[(benzyloxy)carbonyl]-l-phenylalanyl}-l-cysteine (10c)

Yield: 0.51 g (86%); white solid; mp 142–143 °C.

¹H NMR (DMSO-d ₆): δ = 8.24 (d, J = 8.1 Hz, 1 H), 8.15 (d, J = 7.1 Hz, 1 H), 7.44 (d, J = 8.3 Hz, 1 H), 7.37–7.20 (m, 15 H), 5.08–4.94 (m, 4 H), 4.43–4.34 (m, 2 H), 4.17–4.06 (m, 1 H), 3.36 (dd, J = 13.6, 5.5 Hz, 1 H), 3.18–3.05 (m, 2 H), 2.81 (dd, J = 13.9, 11.1 Hz, 1 H), 1.24 (d, J = 7.1 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 200.6, 172.6, 171.6, 156.0, 155.6, 137.4, 137.0, 136.8, 129.2, 128.4, 128.3, 128.2, 127.8, 127.8, 127.4, 126.5, 65.6, 65.5, 62.7, 51.5, 50.0, 36.5, 29.7, 18.3.

Anal. Calcd for C₃₁H₃₃N₃O₈S: C, 61.27; H, 5.47; N, 6.91. Found: C, 60.88; H, 5.35; N, 7.20.

S-{[(Benzyloxy)carbonyl]-l-alanyl}-N-{[(benzyloxy)carbonyl]-l-alanyl-l-phenylalanyl}-l-cysteine (10d)

Yield: 0.65 g (97%); white solid; mp 173–175 °C.

¹H NMR (DMSO-d ₆): δ = 12.92 (s, 1 H), 8.60 (d, J = 7.9 Hz, 1 H), 8.20 (d, J = 8.0 Hz, 1 H), 7.42–7.15 (m, 17 H), 5.14–4.88 (m, 4 H), 4.58 (s, 1 H), 4.42–4.24 (m, 1 H), 4.20–3.95 (m, 2 H), 3.29 (s, 1 H), 3.19–3.00 (m, 2 H), 2.88 (dd, J = 14.1, 10.1 Hz, 1 H), 1.21 (d, J = 6.9 Hz, 3 H), 1.19 (d, J = 6.6 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 199.8, 172.8, 172.5, 171.5, 155.6, 155.5, 137.1, 137.0, 136.9, 129.1, 128.3, 128.2, 128.1, 127.8, 127.7, 126.5, 65.4, 65.4, 60.4, 51.5, 49.9, 36.5, 29.4, 18.3, 17.9.

Anal. Calcd for C₃₄H₃₈N₄O₉S: C, 60.16; H, 5.64; N, 8.25. Found: C, 59.88; H, 5.66; N, 8.21.

N-{[(Benzyloxy)carbonyl]-l-alanyl}-S-[(benzyloxy)carbonyl]-l-alanyl-l-phenylalanyl-l-cysteine (10e)

Yield: 0.64 g (95%); white solid; mp 161–163 °C.

¹H NMR (DMSO-d ₆): δ = 12.92 (s, 1 H), 8.60 (d, J = 7.9 Hz, 1 H), 8.20 (d, J = 8.0 Hz, 1 H), 7.44–7.15 (m, 17 H), 5.10–4.91 (m, 4 H), 4.61–4.58 (m, 1 H), 4.38–4.26 (m, 1 H), 4.16–3.99 (m, 2 H), 3.32–3.29 (m, 1 H), 3.16–3.02 (m, 2 H), 2.88 (dd, J = 14.1, 10.1 Hz, 1 H), 1.21 (d, J = 6.9 Hz, 3 H), 1.19 (d, J = 6.3 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 199.8, 172.8, 172.5, 171.5, 155.6, 155.5, 137.0, 129.1, 128.3, 128.2, 128.2, 127.8, 127.7, 126.5, 65.4, 60.4, 51.5, 49.9, 36.5, 29.4, 18.3, 17.9.

Anal. Calcd for C₃₄H₃₈N₄O₉S: C, 60.16; H, 5.64; N, 8.25. Found: C, 60.23; H, 5.82; N, 8.31.

S-{[(Benzyloxy)carbonyl]-l-alanyl}-N-[(benzyloxy)carbonyl]-l-phenylalanylglycyl-l-cysteine (10f)

Yield: 0.61 g (94%); white solid; mp 169–171 °C.

¹H NMR (DMSO-d ₆): δ = 12.95 (s, 1 H), 8.34–8.19 (m, 2 H), 8.05 (d, J = 7.4 Hz, 1 H), 7.52 (d, J = 8.5 Hz, 1 H), 7.40–7.14 (m, 15 H), 5.11–4.84 (m, 4 H), 4.45–4.12 (m, 3 H), 3.83–3.63 (m, 2 H), 3.30–3.23 (m, 1 H), 3.12–2.96 (m, 2 H), 2.73 (dd, J = 13.8, 10.7 Hz, 1 H), 1.24 (d, J = 7.2 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 201.6, 171.8, 171.4, 168.7, 155.9, 155.8, 138.2, 137.0, 129.2, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.4, 126.2, 65.8, 65.2, 56.7, 56.2, 51.5, 41.7, 37.4, 29.5, 17.3.

Anal. Calcd for C₃₃H₃₆N₄O₉S: C, 59.63; H, 5.46; N, 8.43. Found: C, 59.24; H, 5.55; N, 8.42.

S-{[(Benzyloxy)carbonyl]-l-alanyl}-N-[(benzyloxy)carbonyl]-l-phenylalanyl-l-alanyl-l-cysteine (10g)

Yield: 0.66 g (96%); white solid; mp 170–171 °C.

¹H NMR (DMSO-d ₆): δ = 12.92 (br s, 1 H), 8.60 (d, J = 7.9 Hz, 1 H), 8.20 (d, J = 8.1 Hz, 1 H), 7.48–7.16 (m, 17 H), 5.09–4.92 (m, 4 H), 4.65–4.52 (m, 1 H), 4.38–4.26 (m, 1 H), 4.16–4.00 (m, 2 H), 3.31–3.29 (m, 1 H), 3.16–3.02 (m, 2 H), 2.88 (dd, J = 14.1, 10.1 Hz, 1 H) 1.21 (d, J = 6.8 Hz, 3 H), 1.19 (d, J = 6.6 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 199.8, 172.8, 172.5, 171.5, 155.6, 155.5, 137.0, 129.1, 128.3, 128.2, 128.2, 127.8, 127.7, 126.5, 65.4, 65.4, 60.4, 49.9, 49.8, 36.5, 29.4, 18.3, 17.9.

Anal. Calcd for C₃₄H₃₈N₄O₉S: C, 60.16; H, 5.64; N, 8.25. Found: C, 60.35; H, 5.67; N, 8.19.

S-[(Benzyloxy)carbonyl]-l-alanyl-l-phenylalanyl-N-[(benzyl­oxy)carbonyl]-l-phenylalanylglycyl-l-cysteine (10h)

Yield: 0.79 g (98%); white solid; mp 146–148 °C.

¹H NMR (DMSO-d ₆): δ = 8.64 (d, J = 8.0 Hz, 1 H), 8.32 (t, J = 5.7 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1 H), 7.56 (d, J = 8.7 Hz, 1 H), 7.42–7.15 (m, 21 H), 5.07–4.88 (m, 4 H), 4.65–4.55 (m, 1 H), 4.42–4.26 (m, 2 H), 4.16–4.04 (m, 1 H), 3.85–3.72 (m, 2 H), 3.40–3.29 (m, 1 H), 3.16–3.03 (m, 3 H), 2.96–2.84 (m, 1 H), 2.77 (dd, J = 13.1, 10.0 Hz, 1 H), 1.21 (d, J = 7.2 Hz, 3 H).

¹³C NMR (DMSO-d ₆): δ = 199.9, 172.8, 171.4, 168.6, 155.9, 137.1, 137.0, 129.2, 129.1, 128.3, 128.3, 128.2, 128.0, 127.8, 127.7, 127.4, 126.2, 65.2, 60.4, 56.2, 51.7, 49.9, 41.7, 36.5, 29.8, 17.9.

HRMS (–ESI-TOF): m/z [M – H]^– calcd for C₄₂H₄₅N₅O₁₀S: 810.2814; found: 810.2822.

Acknowledgment

We thank the University of Florida and the Kenan Foundation for financial support. This paper was also funded in part by generous support from King Abdulaziz University, under grant No. D-006/431. The authors, therefore, acknowledge the technical and financial support of KAU. We also thank Dr. C. D. Hall for useful suggestions and English checking.

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