Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

Abstract

The rise of CuI-catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in-house preparation of a set of five Fmoc azido amino acids: β-azido l-alanine and d-alanine, γ-azido l-homoalanine, δ-azido l-ornithine and ω-azido l-lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user-friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3-5, 20, 25, 26, 30 and 43-47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Keywords: alanine; azide elimination; azido amino acid; homoalanine; lysine; ornithine; synthesis.

Scheme 1

Reagents, conditions and yields: (a) 10% Pd/C, H₂, methanol, RT, overnight then Fmoc‐OSu, NaHCO₃, water and dioxane, 0 °C for 1 h, then RT overnight (74% over two steps); (b) CBr₄, PPh₃, DCM 0 °C for 1 h, then RT overnight (84%); (c) Trimethylsilyl azide, hexamethylphosphoramide, 70 °C overnight; (d) 10% Pd/C, H₂, methanol, RT overnight, then Boc₂O, NaHCO₃, water and dioxane, 0 °C for 1 h, then RT overnight (86% over two steps); (e) Boc₂O, NaHCO₃, water and dioxane, 0 °C for 1 h, followed at RT overnight, then C₄H₉Br, K₂CO₃, benzyl triethylammonium chloride, N,N‐dimethyl acetamide, 60 °C overnight (72% over two steps for 8, 67% over two steps for 9); (f) MsCl, TEA, DCM, 0 °C for 0.5 h, then NaN₃, DMSO, 70 °C overnight (16% for 10, 47% for 11 over two steps, 21% for 10, 50% for 12 over two steps); (g) MsCl, pyridine, DCM, 0 °C for 0.5 h, then NaN₃ DMSO, 70 °C overnight (26% for 10, 46% for 11 over two steps); (h) CBr₄, PPh₃, DCM, 0 °C for 1 h, then RT overnight (68%); (i) NaN₃, DMSO, 70 °C overnight (40% for 10, 41% for 11 over two steps); (j) TFA, DCM, water, RT overnight, then NaHCO₃ and Fmoc‐OSu, dioxane and water, 0 °C for 1 h, then RT overnight (86% for 14 over two steps, 86% for 15 over two steps); (k) 2‐chlorotrityl chloride resin, DIPEA, DMF, 2 h, then piperidine, DMF (75%); (l) l‐Val‐OBn·TsOH, DIPEA, PyBroP, DMF RT overnight (74%); (m) 20, DIPEA, PyBroP, DMF, RT overnight (70%); (n) TFA, DCM, 2 h, 2‐chlorotrityl chloride resin, DIPEA, DMF, 2 h, then piperidine, DMF (67%); (o) NaN₃, DMSO, 70 °C overnight.

Scheme 2

Reagents, conditions and yields: (a) Na₂CO₃, Fmoc‐OSu, dioxane and water, 0 °C for 1 h, then RT overnight (87% for 25, 93% for 26); (b) PhI(OAc)₂, CH₃CN, ethyl acetate and water at RT overnight (75% for 27, 56% for 28); (c) TfN₃, NaHCO₃, CuSO₄·5H₂O, water and methanol at RT overnight (80% for 14, 92% for 29); (d) Na₂CO₃, Boc₂O, dioxane and water 0 °C 1 h then RT overnight (73%); (e) PhI(OAc)₂, CH₃CN, ethyl acetate and water at RT overnight (75%); (f) TfN₃, TEA, CuSO₄·5H₂O, water and methanol at RT overnight; (g) TFA, DCM, 2 h at RT; (h) NaHCO₃, Fmoc‐OSu, dioxane and water, 0 °C for 1 h, then RT overnight (37% over three steps).

Scheme 3

Reagents, conditions and yields: (a) CuAc₂·H₂O, water, then Boc₂O in acetone overnight (90% for 35, 92% for 36); (b) 8‐hydroxyquinoline, water, 4 h (90% for 37, 88% for 38); (c) Fmoc‐OSu, NaHCO₃, water and dioxane, 0 °C for 0.5 h, then RT overnight (93% for 39, 95% for 40); (d) TFA, DCM, 2 h at RT (e) TfN₃, NaHCO₃, CuSO₄·5H₂O, water and methanol at RT overnight (92% for 41 over two steps, 89% for 42 over two steps).

Scheme 4

Reagents, conditions and yields: (a) 20% piperidine/DMF; (b) (i) Fmoc‐l‐Phe, HBTU, DIPEA, DMF, 2 × 2 h at RT (ii) 20% piperidine/DMF; (c) (i) Fmoc‐l‐Phe or Fmoc‐l‐Val, HBTU, DIPEA, DMF, 2 × 2 h at RT (ii) 20% piperidine/DMF; (d) 14, 29, 41 or 42, HBTU, DIPEA, DMF, 2 × 2 h at RT (ii) 20% piperidine/DMF; (e) Ac₂O, DIPEA, DMF, 15 min at RT; (f) 95% TFA/water, 1 h at RT. Yields (after HPLC purification) 69% for 43, 76% for 44, 80% for 45, 77% for 46 and 71% for 47. In the case of long couplings (5 and 18 h) with 14, the yield for 44 was only 17% accompanied by the presence of 48 in 32% yield.

Figure 1

HPLC trace of the purification of the crude tripeptide 44. The major isolated products (44 and 48) are labeled with compound numbers.