Solution-phase synthesis of chiral O -acyl isodipeptides

Mirna El Khatib; Lilibeth Jauregui; Srinivasa R. Tala; Levan Khelashvili; Alan R. Katritzky

doi:10.1039/C1MD00130B

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DOI: 10.1039/C1MD00130B (Concise Article) Med. Chem. Commun., 2011, 2, 1087-1092

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Solution-phase synthesis of chiral O-acyl isodipeptides†

Mirna El Khatib ^a, Lilibeth Jauregui ^a, Srinivasa R. Tala ^a, Levan Khelashvili ^a and Alan R. Katritzky *^ab
^aCenter for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA. E-mail: katritzky@chem.ufl.edu
^bDepartment of Chemistry, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

Received 19th May 2011 , Accepted 16th August 2011

First published on 14th September 2011

Abstract

O-Acylation of N-Boc-protected-serine and -threonine with N-Pg-(α-aminoacyl)benzotriazoles afforded the chiral O-acylated isodipeptides at 23 °C in yields of 74–91%.

The synthesis of peptides and proteins is of great importance to the understanding of biological functions. The solid-phase synthesis of peptides with “difficult sequences” remains problematic due to low yields and purity.¹ In addition, intermolecular hydrophobic interactions in “difficult sequences” can promote aggregation in solution and hydrogen bond networks in resin-bound peptides can form extended structures such as β-sheets.^1b–c,2

Kiso et al.^1d demonstrated that the introduction of an O-acyl in place of an N-acyl residue within a peptide backbone significantly altered the secondary structure of native peptides. Furthermore, these “O-acyl isopeptides” or “click peptides”‡ are more hydrophilic, and easier to purify by HPLC.^1d He found that a subsequent O–N intramolecular acyl migration, triggered by change in pH, could rapidly generate a target natural peptide under physiological conditions (pH 7.4) (Fig. 1).^1c,3a This “O-acyl isopeptide method”³ has been used to develop new water-soluble taxoid prodrugs,⁴ HIV-1 protease inhibitors,⁵ the anti-tumor agent, paclitaxel,⁶ difficult sequence-containing peptides including Ac-Val-Val-Ser-Val-Val-NH₂,^1b,4,7 Alzeheimer's disease-related amyloid βpeptide (Aβ) 1–42,^4,7–13 and cyclic peptides.¹⁴


	Fig. 1 O-Acyl isopeptide methodology.

However, epimerization during the esterification step in the solid-phase synthesis of O-acyl isopeptides has remained a major problem.^1c,e,15 Based on the hypothesis that epimerization during esterification should be suppressed in solution due to the faster coupling rate as compared to that on a solid support, Kiso^1c,e synthesized O-acyl isodipeptides in three steps (Scheme 1): (i) protection of the carboxylic acid group in serine or threonine by benzyl esterification, (ii) O-acylation and (iii) deprotection using Pd/C. Treatment of Cbz-protected isodipeptides containing Cys and Met with Pd/C–H₂ failed, although catalytic hydrogen transfer (CTH) to Cys- and Met-containing protected isodipeptides gave 45% of the desired product.^1c


	Scheme 1 Synthetic scheme of “O-acyl isodipeptide unit”.

We now report an efficient single-step preparation of chiral O-acyl isodipeptides from serine and threonine. The use of N-acylbenzotriazoles are advantageous for N-, O-, C-, S- acylation,^16–18 especially where the corresponding acid chlorides are unstable or difficult to prepare. N-(Protected-α-aminoacyl)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.¹⁸

O-Acyl isoserinedipeptides 3a–h were prepared by O-acylation of Boc-protected serine 1a with various N-Pg-(α-aminoacyl)benzotriazoles 2 in the presence of diisopropylethylamine in CH₃CN at 23 °C for 12 h in yields of 74–90%. This proved to be the optimum condition under which neither epimerization of 3 nor hydrolysis of 2 occurred. The presence of water (MeCN/H₂O, 9 [thin space (1/6-em)] :1) in the reaction caused minimal hydrolysis of 2 (4%) but no epimerization was detected according to HPLC-MS analysis on 3b and (3b + 3b′). The O-acylated serines were characterized by NMR and elemental analysis (Table 1). HPLC analysis [chirobiotic T column (250 mm × 4.6 mm), detection at 254 nm, flow rate 2.5 mL min⁻¹, MeOH] on 3b (single peak, retention time 1.3 min) and (3b + 3b′) (two peaks, retention times, 1.3 min and 1.5 min) as well as HPLC-MS and (-)ESI-MS on 3b (Fig. 2) confirmed the absence of racemization in the targeted isodipeptides.

Table 1 The preparation of serine-based O- acyl-isodipeptides 3a–h


Entry	O-acyl isodipeptides 3a–h	Yield (%)	mp/°C
1	Boc-L-Ser(Cbz-L-Ala)OH 3a	84	oil
2	Boc-L-Ser(Cbz-L-Phe)OH 3b	81	oil
3	Boc-L-Ser(Cbz-DL-Phe)OH(3b + 3b′)	81	60–62
4	Boc-L-Ser(Cbz-L-Trp)OH 3c	87	67–69
5	Boc-L-Ser(Cbz-L-Met)OH 3d	74	oil
6	Boc-L-Ser(Cbz-L-Val)OH 3e	90	oil
7	Boc-L-Ser(Cbz-Gly)OH 3f	87	57–58
8	Boc-L-Ser(Cbz-Cys(Bzl))OH 3g	85	oil
9	Boc-L-Ser(Boc-Gly)OH 3h	82	58–59


	Fig. 2 HPLC-MS and (-)ESI-MS analysis of 3b.

O-Acylated threonine based isodipeptides 4a–h were also prepared by O-acylation of Boc-protected threonine 1b with various N-Pg-(α-aminoacyl)benzotriazoles 2 in the presence of diisopropylethylamine in CH₃CN at room temperature in yields of 86–91% (Table 2). The synthesized compounds were characterized by NMR and elemental analysis.

Table 2 The preparation of O-isodipeptides 4a–h


Entry	O-acyl isodipeptides 4a–h	Yield (%)	mp/°C
1	Boc-L-Thr(Cbz-L-Ala)-OH 4a	91	oil
2	Boc-L-Thr(Cbz-L-Phe)-OH 4b	87	oil
3	Boc-L-Thr(Cbz-D,L-Phe)-OH (4b + 4b′)	87	60–62
4	Boc-L-Thr(Cbz-L-Trp)-OH 4c	91	89–90
5	Boc-L-Thr(Cbz-L-Met)-OH 4d	90	oil
6	Boc-L-Thr(Cbz-Gly)-OH 4e	86	54–58
7	Boc-L-Thr(Cbz-Cys(Bzl))-OH 4f	86	oil
8	Boc-L-Thr(Boc-Gly)-OH 4g	85	64–65
9	Boc-L-Thr(Boc-L-Phe)-OH 4h	90	47–50

HPLC analysis [chirobiotic T column (250 mm × 4.6 mm), detection at 254 nm, flow rate 0.5 mL min⁻¹, MeOH [thin space (1/6-em)] :H₂O, 4:1] on 4b (single peak, retention time 7.23 min) and (4b + 4b′) (two peaks, retention times, 6.54 min and 7.20 min) confirmed the absence of racemization in the desired isodipeptides.

Conclusions

In conclusion, the mild protocol reported herein enables the efficient preparation of optically pure O-acyl isopeptides from serine and threonine without protection of their carboxyl groups.

Materials and methods

General

Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. The NMR spectra were recorded in CDCl₃ with TMS for ¹H (300 MHz) and ¹³C (75 MHz) as an internal reference.

General procedure for the preparation of N-(Pg-α-aminoacyl)benzotriazoles 2

N-(Z-α-Aminoacyl)benzotriazoles 2. Thionyl chloride (0.6 mL, 8.00 mmol, 1.2 equiv) was added to a solution of 1H-benzotriazole (3.17 g, 26.67 mmol, 4 equiv) in methylene chloride to give a clear yellow solution that was stirred for 15 min at room temperature. The amino acid (6.67 mmol, 1 equiv) was then added to give a suspension which was stirred for 2.5 h at room temperature. The suspension was filtered, the filtrate evaporated, the residue dissolved in EtOAc and the solution was washed with a saturated solution of sodium carbonate. The organic portion was dried over anhyd MgSO₄, filtered, and dried to give the corresponding N-(Z-α-aminoacyl)benzotriazole 2.

N-(Boc-α-Aminoacyl)benzotriazoles 2. Boc-protected amino acid (0.03 mol) was added to a solution of DCC (1 equiv) in methylene chloride under an atmosphere of nitrogen. After 30 min., BtH (1 equiv) was added and this was stirred for 12 h. The suspension was filtered on a bed of silica and celite, the filtrate evaporated, and the residue dissolved in EtOAc, then filtered on a bed of silica and celite and washed with a solution of saturated sodium carbonate, then with water and brine. The organic portion was dried over anhyd MgSO₄, filtered on a bed of silica, and dried to give the corresponding N-(Boc-α-aminoacyl)benzotriazole 2. ¹H NMR and mp of Boc-Gly-Bt 2h and Boc-L-Phe-Bt 2i matched that reported in the literature.^19a,b

(S)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-yl)carbamate (2a). white solid (90%); mp 115 °C (lit.^19c mp 113–115 °C); ¹H NMR (CDCl₃): δ 8.16 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.40–7.03 (m, 6H), 5.80–5.60 (m, 2H), 5.10–4.99 (m, 1H), 1.59 (d, J = 6.3 Hz, 3H); ¹³C NMR (CDCl₃): δ 172.2, 155.6, 145.9, 136.0, 131.0, 130.6, 128.4, 128.1, 126.4, 120.2, 114.3, 67.1, 50.5, 19.0.

(S)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate (2b). white solid (90%); mp 150–152 °C (lit.²⁰ mp 149.0–150.0 °C); ¹H NMR (CDCl₃): δ 8.23 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.32–7.23 (m, 7H), 7.14 (br s, 3H), 6.09 (d, J = 4.2 Hz, 1H), 5.57 (d, J = 6.6 Hz, 1H), 5.08 (s, 2H), 3.48 (d, J = 9.6 Hz, 1H), 3.24 (d, J = 7.8 Hz, 1H); ¹³C NMR (CDCl₃): δ 170.8, 155.7, 146.0, 135.9, 134.9, 131.0, 130.8, 129.2, 128.7, 128.5, 128.1, 127.4, 126.5, 120.4, 114.3, 67.2, 55.6, 38.8.

Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate (2b + 2b′). white solid (90%); mp 141–142 °C (lit.²¹ mp 141–142 °C); ¹H NMR (CDCl₃): δ 8.23 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.32–7.23 (m, 7H), 7.14 (br s, 3H), 6.09 (d, J = 4.2 Hz, 1H), 5.57 (d, J = 6.6 Hz, 1H), 5.08 (s, 2H), 3.48 (d, J = 9.6 Hz, 1H), 3.24 (d, J = 7.8 Hz, 1H); ¹³C NMR (CDCl₃): δ 170.8, 155.7, 146.0, 135.9, 134.9, 131.0, 130.8, 129.2, 128.7, 128.5, 128.1, 127.4, 126.5, 120.4, 114.3, 67.2, 55.6, 38.8.

(S)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (2c). yellow solid (80%); mp 101 °C (lit.¹⁹ mp 98–100 °C); ¹H NMR (CDCl₃): δ 8.23 (br s, 1H), 8.14–8.06 (m, 2H), 7.57 (t, J = 7.5 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.31–7.24 (m, 4H), 7.20 (s, 1H), 7.10–7.04 (m, 1H), 6.95–6.90 (m, 2H), 6.87–6.85 (m, 1H), 6.14–6.10 (m, 1H), 5.70 (d, J = 7.5 Hz, 1H), 5.03 (s, 2H), 3.58 (dd, J = 15, 4.5 Hz, 1H), 3.40 (dd, J = 14.9, 7.7 Hz, 1H); ¹³C NMR (CDCl₃): δ 171.1, 155.9, 145.8, 136.1, 131.0, 130.6, 128.4, 128.1, 127.0, 126.4, 123.2, 122.2, 120.2, 119.7, 118.3, 114.3, 111.2, 108.9, 67.2, 55.1, 28.7.

(S)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-4-(methylthio)-1-oxobutan-2-yl)carbamate (2d). beige solid (85%); mp 108–109 °C (lit.¹⁹ mp 108–109 °C); ¹H NMR (CDCl₃): δ 8.15 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.30–7.24 (m, 5H), 5.83–5.79 (m, 1H), 5.67–5.65 (m, 1H), 5.03 (s, 2H), 2.59 (t, J = 7.1 Hz, 2H), 2.35 (br s, 1H), 2.10–2.03 (m, 1H), 1.97 (s, 3H);¹³C NMR (CDCl₃): δ 171.2, 155.7, 130.9, 146.0, 130.8, 128.4, 128.5, 128.3, 126.6, 120.4, 114.3, 67.4, 54.2, 32.4, 30.0, 15.4; Anal. Calcd for C₁₉H₂₀N₄O₃S: C, 59.36; H, 5.24; N, 14.57. Found: C, 59.69; H, 5.23; N, 14.54.

(S)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (2e). white powder (87%); mp 107 °C (lit.²⁰ mp 73–74 °C); ¹H NMR (CDCl₃): δ 8.28 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.68 (dd, J = 8.0, 7.4 Hz, 1H), 7.54 (dd, J = 8.1, 7.3 Hz, 1H), 7.37 (br s, 5H), 5.78–5.74 (m, 1H), 5.57 (d, J = 9.2 Hz, 1H), 5.14 (s, 2H), 2.61–2.43 (m, 1H), 1.13 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H); ¹³C NMR (CDCl₃): δ 171.5, 156.2, 146.0, 136.0, 131.0, 130.7, 128.5, 128.2, 126.5, 120.3, 114.3, 67.3, 59.4, 31.6, 19.7, 17.0.

(S)-Benzyl (2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)carbamate (2f). white solid (90%); mp 110–111 °C (lit.²² mp 106–108 °C); ¹H NMR (CDCl₃): δ 8.24 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.39–7.34 (m, 5H), 5.61 (br s, 1H), 5.19 (s, 2H), 5.09 (d, J = 5.7 Hz, 2H); ¹³C NMR (CDCl₃): δ 168.3, 156.4, 145.9, 136.0, 130.8, 128.5, 128.2, 128.1, 126.5, 120.3, 114.0, 67.4, 44.8.

(R)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(benzylthio)-1-oxopropan-2-yl)carbamate (2g). yellow oil (87%); ¹H NMR (CDCl₃): δ 8.31 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H), 7.74 (t, J = 8.1 Hz, 1H), 7.60 (t, J = 8.1 Hz, 1H), 7.47– 7.17 (m, 10H), 6.03 (br s, 2H), 5.22 (br s, 2H), 3.79 (br s, 2H), 3.28–2.89 (m, 2H); ¹³C NMR (CDCl₃): δ 169.8, 155.7, 145.9, 136.0, 135.9, 130.8, 128.8, 128.4, 128.2, 128.1, 127.1, 126.6, 125.8, 120.3, 114.3, 67.4, 53.7, 36.1, 33.6; Anal. Calcd for C₂₄H₂₂N₄O₃S: C, 64.56; H, 4.51; N 12.55. Found: C, 64,26; H, 4.93; N 12.90.

General procedure for the preparation of O-acyl isodipeptides 3 and 4

DIPEA (0.44 mL, 3 equiv) was added to a solution of Boc-L-SerOH or Boc-L-ThrOH (0.49 mmol, 1 equiv) in MeCN (15 mL). The appropriate N-(Pg-α-aminoacyl)benzotriazole 2 (0.49 mmol, 1 equiv) dissolved in MeCN (5 mL) was added to the clear solution and the mixture was stirred for 12 h at room temperature. Complete reaction was judged by the disappearance of starting material. The solution was acidified with 1N HCl, evaporated; the residue was dissolved in EtOAc and washed with 1N HCl. The organic portion was dried over anhyd Na₂SO₄, filtered and dried to give the corresponding O-acyl isodipeptide. All samples were then freeze-dried and fully characterized.

(R)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)propanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3a). prepared from 2a. colorless oil (84%); ¹H NMR (CDCl₃): δ 10.2 (br s, 1H), 7.27–7.15 (m, 5H), 5.62–5.49 (m, 1H), 5.25–4.96 (m, 2H), 4.63–4.29 (m, 4H), 4.10–3.98 (m, 1H), 1. 36 (s, 9H); ¹³C NMR (CDCl₃): δ 172.8, 172.4, 156.1, 155.6, 136.0, 128.4, 128.1, 80.6, 67.1, 64.9, 52.7, 49.7, 28.2, 18.1; Anal. Calcd for C₁₉H₂₆N₂O₈: C, 55.60; H, 6.39; N, 6.83. Found: C, 55.31; H, 6.41; N, 6.70.

(R)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-3-phenylpropanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3b). prepared from 2b. yellow transparent oil (81%); ¹H NMR (CDCl₃): δ 10.8 (bs, 1H), 7.31–7.18 (m, 10H), 7.11–7.05 (m, 2H), 5.53–5.44 (m, 1H), 5.10–4.97 (m, 2H), 4.64–4.55 (m, 2H), 4.41–4.32 (m, 2H), 3.04–2.98 (m, 1H), 1.42 (br s, 9H); ¹³C NMR (CDCl₃): δ 172.6, 171.2, 156.0, 135.5, 129.3, 129.1, 128.6, 128.5, 128.4, 128.1, 128.0, 127.2, 80.6, 67.2, 65.2, 54.9, 52.5, 38.0, 28.2; Anal. Calcd for C₂₅H₃₀N₂O₈: C, 61.72; H, 6.22; N, 5.76. Found: C, 61.79; H, 6.34; N, 5.42.

3-(((S)-2-(((Benzyloxy)carbonyl)amino)-3-phenylpropanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3b + 3b′). prepared from (2b + 2b′). colorless solid (81%); mp 60–62 °C; ¹H NMR (CDCl₃): δ 8.85 (br s, 1H), 7.33–7.10 (m, 10H), 7.05–6.95 (m, 2H), 5.59–5.41 (m, 1H), 5.03 (bs, 2H), 4.64–4.17 (m, 3H), 3.03 (bs, 2H), 1.41 (bs, 9H), 1.23–1.18 (m, 3H) ¹³C NMR (CDCl₃): δ 175.2, 172.5, 171.1, 156.0, 155.5, 135.9, 135.5, 129.1, 128.5, 128.4, 128.0, 127.1, 80.5, 67.1, 65.2, 60.5, 54.9, 53.6, 52.5, 38.8, 37.9, 37.7, 29.6, 28.2, Anal. Calcd for C₂₅H₃₀N₂O₈: C, 61.72; H, 6.22; N, 5.76. Found: C, 61.79; H, 6.35; N, 5.42.

(S)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-3-(1H-indol-3-yl)propanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3c). prepared from 2c. yellow gel (85%); ¹H NMR (CDCl₃): δ 8.37–8.31 (m, 1H), 7.92 (br s, 1H), 7.55–7.42 (m, 1H), 7.25–7.04 (m, 7H), 6.90–6.81 (m, 1H), 5.50–5.44 (m, 1H), 5.12–4.98 (m, 2H), 4.70–4.65 (m, 1H), 4.54 (br s, 1H), 4.38–4.28 (m, 1H), 3.31–3.21 (m, 2H), 1.44 (br s, 9H); ¹³C NMR (CDCl₃): δ 175.9, 172.8, 171.6, 156.1, 155.7, 136.1, 128.5, 128.1, 127.3, 123.0, 122.0, 120.0, 118.3, 111.4, 109.3, 80.7, 67.2, 65.0, 54.7, 52.7, 28.3; Anal. Calcd for C₂₇H₃₁N₃O₈: C, 61.71; H, 5.95; N, 8.00. Found: C, 61.96; H, 6.00; N, 7.61.

(R)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-4-(methylthio)butanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3d). prepared from 2d. yellow oil (74%); ¹H NMR (CDCl₃): δ 9.40 (bs, 1H), 7.27–7.12 (m, 5H), 5.72–5.62 (m, 1H), 5.08–4.97 (m, 2H), 4.57–4.42 (m, 3H), 2.49–2.41 (m, 2H), 2.22–1.84 (m, 5H), 1.36 (br s, 9H); ¹³C NMR (CDCl₃): δ 175.7, 172.7, 171.4, 156.2, 155.5, 135.8, 128.4, 128.1, 80.6, 67.2, 65.1, 53.2, 52.7, 31.5, 29.7, 28.2, 15.2; Anal. Calcd for C₂₁H₃₀N₂O₈S: C, 53.60; H, 6.43; N, 5.95. Found: C, 53.94; H, 6.56; N, 5.52.

(S)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-3-methylbutanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3e). prepared from 2e. yellow oil (90%); ¹H NMR (CDCl₃): δ 9.54 (br s, 1 H), 7.34–7.22 (m, 5H), 5.80–5.51 (m, 1H), 5.12–5.00 (m, 2H), 4.62–4.02 (m, 3H), 2.13–2.00 (m, 1H), 1.39 (br s, 9H), 0.96–0.83 (m, 6H); ¹³C NMR (CDCl₃): δ 172.5, 171.4, 156.5, 155.5, 135.9, 128.4, 128.1, 80.5, 67.2, 64.7, 59.1, 52.7, 31.0, 28.2, 18.9, 17.4; Anal. Calcd for C₂₁H₃₀N₂O₈: C 57.52; H 6.90; N 6.39. Found: C: 57.79, H 6.69, N 6.45.

(S)-3-(2-(((Benzyloxy)carbonyl)amino)acetoxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3f). prepared from 2f. colorless solid (87%); mp 57–58 °C; ¹H NMR (CDCl₃): δ 10.18 (br s, 1H), 7.13–7.11 (m, 5H), 5.62–5.47 (m, 1H), 4.90 (br s, 2H), 4.41–4.17 (m, 2H), 3.73 (br s, 2H), 1.23 (br s, 9H); ¹³C NMR (CDCl₃): δ 172.5, 169.7, 156.7, 155.5, 135.9, 128.4, 128.0, 80.5, 67.2, 65.0, 52.5, 42.5, 28.2; Anal. Calcd for C₁₈H₂₄N₂O₈: C, 54.54; H, 6.10; N, 7.07. Found: C, 54.72; H, 6.04; N, 6.73.

(S)-3-(((R)-2-(((Benzyloxy)carbonyl)amino)-3-(benzylthio)propanoyl)oxy)-2-((tert-butoxycarbonyl)amino)propanoic acid (3g). prepared from 2g. yellow gel (85%); ¹H NMR (CDCl₃): δ 10.9 (br s, 1H), 7.49–7.16 (m, 11H), 5.81–5.71 (m, 1H), 5.09–4.98 (m, 2H), 4.61–4.32 (m, 3H), 3.66 (s, 2H), 2.79–2.73 (m, 2H), 1.36 (br s, 9H); ¹³C NMR (CDCl₃): δ 172.3, 170.1, 156.0, 155.4, 137.2, 135.7, 128.8, 128.4, 128.0, 127.1, 80.4, 67.2, 65.2, 53.3, 52.5, 36.1, 33.1, 28.1; Anal. Calcd for C₂₆H₃₂N₂O₈S: C, 58.63; H, 6.06; N, 5.26. Found: C, 58.95; H, 6.08; N, 5.12.

(S)-2-((Tert-butoxycarbonyl)amino)-3-(2-((tert-butoxycarbonyl)amino)acetoxy)propanoic acid (3h). prepared from 2h. white microcrystals (82%); mp 58–59 °C; ¹H NMR (CDCl₃): δ 7.38–7.28 (s, 2H), 5.59–5.20 (m, 1H), 4.58–4.45 (m, 3H), 3.86 (d, J = 4.2 Hz, 2H), 1.38 (s,18H); ¹³C NMR (CDCl₃): δ 172.3, 170.1, 156.1, 155.6, 126.1, 114.9, 82.1, 80.5, 65.1, 52.7, 43.9, 42.3, 28.3; Anal. Calcd for C₁₅H₂₆N₂O₈: C, 49.72; H, 7.23; N, 7.73. Found: C, 50.41; H, 7.29; N, 8.16.

(2S)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)propanoyl)oxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4a). prepared from 2a. colorless oil (91%); ¹H NMR (CDCl₃): δ 9.73 (bs, 1H), 7.13–7.10 (m, 6H), 5.56–5.53 (m, 1H), 5.28 (br s, 1H), 4.89 (br s, 2H), 4.31–3.82 (m,2H); 1.24 (s, 9H), 1.11–1.05 (m, 6H); ¹³C NMR (CDCl₃): δ 173.8, 173.2, 171.8, 156.1, 136.0, 128.4, 128.1, 80.3, 71.9, 67.0, 58.2, 57.0, 49.6, 28.2, 18.0, 16.6; Anal. Calcd for C₂₀H₂₈N₂O₈: C, 56.60; H, 6.65; N, 6.60. Found: C, 56.21; H, 6.55; N, 6.40.

(2S)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-3-phenylpropanoyl)oxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4b). prepared from 2b. colorless oil (87%); ¹H NMR (CDCl₃): δ 9.86 (bs, 1H), 7.31–7.13 (m, 10 H), 7.13 (br s, 2H), 5.42 (br s, 1H), 5.10–4.99 (m, 2H), 4.79–4.40 (m, 2H), 3.03 (d, J = 6.0 Hz, 2H), 1.46 (s, 9H), 1.28–1.18 (m, 3H); ¹³C NMR (CDCl₃): δ 173.4, 170.6, 156.1, 135.7, 129.2, 128.5, 128.0, 127.0, 80.3, 72.2, 67.1, 56.8, 54.8, 38.1, 28.2, 16.6; Anal. Calcd for C₂₆H₃₂N₂O₈: C, 62.39; H, 6.44; N, 5.60. Found: C, 61.99; H, 6.44; N, 5.53.

(2S)-3-((2-(((Benzyloxy)carbonyl)amino)-3-phenylpropanoyl)oxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4b + 4b′). prepared from (2b + 2b′). yellow solid (87%); mp 60–62 °C; ¹H NMR (CDCl₃): δ 9.56 (br s, 1H), 7.38–7.19 (m, 10H), 7.08 (br s, 2H), 5.36–5.30 (m, 2H), 5.01 (br s, 2H), 4.64–4.44 (m, 2H), 3.13–2.99 (m, 3H), 1.42 (br s, 9H), 1.19 (d, J = 22.8 Hz, 3H); ¹³C NMR (CDCl₃): δ 175.7, 173.5, 170.8, 156.0, 136.0, 135.6, 129.3, 128.5, 128.4, 128.1, 127.0, 80.4, 72.3, 67.0, 56.8, 54.6, 38.1, 37.7, 28.2, 16.7; Anal. Calcd for C₂₆H₃₂N₂O₈: C, 62.39; H, 6.44; N, 5.60. Found: C, 62.46; H, 6.50; N, 5.59.

(2S)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-3-(1H-indol-3-yl)propanoyl)oxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4c). prepared from 2c. yellow oil (90%); ¹H NMR (CDCl₃): δ 8.38–8.15 (m, 1H), 7.54 (d, J = 5.4 Hz, 1H), 7.42–6.90 (m, 12H), 5.52–5.39 (m, 2H), 5.10–4.96 (m, 3H), 3.22 (s, 2H), 1.45 (s, 9H), 1.25–1.14 (m, 3H); ¹³C NMR (CDCl₃): δ 172.7, 170.9, 156.1, 136.0, 128.5, 128.1, 127.5, 122.9, 122.3, 119.7, 118.6, 111.2, 109.6, 80.5, 72.0, 67.2, 56.8, 54.6, 28.3, 27.7, 16.5; Anal. Calcd for C₂₈H₃₃N₃O₈: C, 62.33; H, 6.16; N, 7.79. Found: C, 62.34; H, 6.30; N, 7.60.

(2R)-3-(((S)-2-(((Benzyloxy)carbonyl)amino)-4-(methylthio)butanoyl)oxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4d). prepared from 2d. yellow oil (90%); ¹H NMR (CDCl₃): δ 8.84 (br s, 1H), 8.58 (br s, 1H), 7.34–7.27 (m, 5H), 5.86–5.83 (m, 1H), 5.71 (t, J = 8.9 Hz, 1H), 5.52 (br s, 1H), 5.10 (br s 2H), 4.63–4.38 (m, 1H), 2.54–2.47 (m, 2H), 2.05–1.91 (m, 4H), 1.38 (s, 9H), 1.28–1.27 (m, 3H); ¹³C NMR (CDCl₃): δ 173.6, 170.7, 156.3, 156.1, 135.9, 128.4, 128.1, 80.4, 72.1, 67.2, 56.8, 53.2, 31.5, 29.8, 28.2, 16.7, 15.4; Anal. Calcd for C₂₂H₃₂N₂O₈S: C, 54.53; H, 6.66; N, 5.78. Found: C, 54.87; H, 6.77; N, 5.76.

(2S)-3-(2-(((Benzyloxy)carbonyl)amino)acetoxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4e). prepared from 2f. colorless solid (86%); mp 54–57 °C; ¹H NMR (CDCl₃): δ 7.33–7.31 (m, 5H), 7.01 (bs, 1H), 5.62–5.40 (m, 2H), 5.10 (s, 2H), 3.90 (d, J = 5.7 Hz, 2H), 1.45 (br s, 9H), 1.29 (d, J = 6.9 Hz, 3H); ¹³C NMR (CDCl₃): δ 173.3, 169.2, 156.5, 156.0, 136.0, 128.5, 128.1, 80.5, 72.0, 67.2, 56.8, 42.6, 28.2, 16.8; HRMSm/z for for C₁₉H₂₆N₂O₈Na [M + Na]⁺ calcd 433.1581, found 433.1597

(2S)-3-(((R)-2-(((Benzyloxy)carbonyl)amino)-3-(benzylthio)propanoyl)oxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4f). prepared from 2g. yellow gel (86%); ¹H NMR (CDCl₃): δ 9.05 (br s, 1H), 7.50–7.09 (m, 11H), 5.83–5.78 (m, 1H), 5.44–5.39 (m, 1H), 5.08–4.96 (m, 2H), 4.56–4.44 (m, 1H), 3.62 (s, 2H), 2.82–2.50 (m, 2H), 1.39 (br s, 9H), 1.20 (d, J = 6.3 Hz, 3H); ¹³C NMR (CDCl₃): δ 173.2, 169.5, 156.0, 137.4, 135.8, 128.8, 128.4, 128.0, 127.1, 80.3, 72.4, 67.1, 56.7, 53.3, 36.2, 33.0, 28.2, 16.6; HRMSm/z for for C₂₇H₃₄N₂O₈SNa [M + Na]⁺ calcd. 569.1928, found 569.1939.

(2S,3S)-2-((Tert-butoxycarbonyl)amino)-3-(2-((tert-butoxycarbonyl)amino)acetoxy)butanoic acid (4g). prepared from 2h. white microcrystals (85%); mp 64–65 °C; ¹H NMR (CDCl₃): δ 7.89 (s, 2H), 5.58–5.38 (m, 3H), 4.47–4.45 (m, 1H), 3.84 (s, 2H), 1.43 and 1.42 (overlapped s, 18H), 1.31 (d, J = 5.7 Hz, 3H);¹³C NMR (CDCl₃): δ 173.2, 169.7, 156.6, 156.1, 81.8, 80.4, 72.0, 56.9, 43.6, 28.3, 16.8; Anal. Calcd for C₁₆H₂₈N₂O₈: C, 51.05; H, 7.50; N, 7.44. Found: C, 50.72; H, 7.64; N, 7.41.

(2S,3S)-2-((Tert-butoxycarbonyl)amino)-3-(((S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanoyl)oxy)butanoic acid (4h). prepared from 2i. white microcrystals (90%); mp 47–50 °C; ¹H NMR (CDCl₃): δ 7.07–7.01 (m, 5H), 6.97–6.95 (m, 2H), 5.21 (s, 1H), 4.92–4.80 (m, 3H), 4.31–4.25 (m, 2H), 2.83 (s, 1H), 1.28 (s, 9H), 1.21 (s, 9H), 1.06–1.05 (m, 3H); ¹³C NMR (CDCl₃): δ 171.0, 156.1, 155.5, 129.2, 128.6, 127.1, 80.4, 72.1, 56.8, 54.4, 38.1, 28.3, 16.6; Anal. Calcd for C₂₃H₃₄N₂O₈: C, 59.21; H, 7.35; N, 6.00. Found: C, 59.62; H, 7.92; N, 6.54.

Acknowledgements

We thank the University of Florida, The Kenan Foundation and King Abdulaziz University, Jeddah, Saudi Arabia for financial support. We thank Dr C. D. Hall for helpful discussions.

Notes and references

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Footnotes

† Electronic Supplementary Information (ESI) available: ¹H and ¹³ C NMR for compounds 2a–g, 3a–h, 4a–h. See DOI: 10.1039/c1md00130b/

‡ Kiso denotes O-acyl peptides as “click peptides” because of their easy conversion to target native peptides under physiological conditions.^1d

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