Challenges and Strategies for Synthesizing Glutamyl Hydrazide Containing Peptides

Abstract

Herein, we detail several specific challenges that hinder the effective synthesis of glutamyl hydrazide containing peptides, and we describe a synthetic strategy to work around these challenges. Glutamyl hydrazide is an unnatural amino acid residue that bears an acyl hydrazide functional group on its side chain. This family of compounds has the potential to provide potent and selective inhibitor molecules for several families of enzymes. During peptide synthesis, however, these side chains—even in protected form—can derail the synthesis by initiating undesired side reactions. Avoiding these side reactions is critical for enabling effective access to this family of compounds.

Key words

hydrazides - glutamyl hydrazides - peptides - inhibitors - cyclization - unnatural amino acids

Publication History

Received: 03 November 2022

Accepted after revision: 23 November 2022

Article published online:
22 December 2022

• References and Notes

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• 18 Amide 15 and Cyclized Byproduct 16 Cbz-Glu(NHNHBoc)-OH (13) (328 mg, 0.83 mmol, 1 equiv), DCM (0.8 mL, 1 M), isopropylamine (0.28 mL, 3.4 mmol, 4 equiv), HOBt hydrate (254 mg, 1.66 mmol, 2 equiv), and EDC hydrochloride (318 mg, 1.66 mmol, 2 equiv) were stirred for 3 h, diluted with DCM (35 mL), and washed with a mixture of 1 M sodium bisulfate and brine (1:1, 30 mL). The aqueous phase was extracted with DCM (3 × 20 mL) and the combined organic phase was washed with 10% sodium carbonate (20 mL) and dried with sodium sulfate. The volatiles were removed under reduced pressure and the crude material was purified by silica gel chromatography (3:1 EtOAc/hexane to pure EtOAc) to give cyclized byproduct 16 (200 mg, 0.535 mmol, 64% yield) and amide product 15 (107 mg, 0.245 mmol, 30% yield). Data for Amide 15 TLC: R_f = 0.17 (DCM/EtOAc, 1:1; CAM stained). IR (crystalline solid scanned via ATR probe): 3283, 2976, 1641, 1535, 1244, 1159, 1052 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 9.17 (s, 1 H, NH), 7.42–7.29 (m, 5 H, Ar), 6.79 (s, 1 H, NH), 6.76–6.62 (m, 1 H, NH), 6.06–5.97 (m, 1 H, NH), 5.11 (s, 2 H, Bn), 4.76 (br s, 1 H, α), 4.00 (oct, J = 6.7 Hz, 1 H, iPr), 2.42–2.23 (m, 2 H, γ), 2.07–1.87 (m, 2 H, β), 1.48 (s, 9 H, Boc), 1.15 (d, J = 6.5 Hz, 3 H, CH₃), 1.13 (d, J = 6.5 Hz, 3 H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 173.7, 170.4, 157.8, 156.1, 136.1, 128.7, 128.4, 128.1, 82.1, 67.4, 52.8, 41.5, 33.3, 31.2, 28.3, 22.7. LRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₃₃N₄O₆H: 437.24; found: 437.2. Data for Cyclized Byproduct 16 TLC: R_f = 0.69 (EtOAc/DCM, 1:1; CAM stained). IR (crystalline solid scanned via ATR probe): 3314, 2981, 1705, 1513, 1456, 1393, 1369, 1333, 1313, 1246, 1159, 1047, 980, 862, 733, 698 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 7.53–7.28 (m, 5 H, Ar), 6.85 (s, 0.4 H, NNH), 6.78 (s, 0.6 H, NNH), 5.84 (br s, 0.4 H, CbzNH), 5.73 (d, J = 5.3 Hz, 0.6 H, CbzNH), 5.18–5.06 (m, 2 H, Bn), 4.65–4.38 (m, 1 H, α), 3.06–2.62 (m, 2 H, γ), 2.50–2.32 (br s, 1 H, β), 2.14–1.89 (m, 1 H, β), 1.46 (s, 9 H, Boc). ¹³C NMR (100 MHz, CDCl₃): δ = 170.0, 169.5, 169.1, 156.3, 154.1, 136.1, 128.7, 128.4, 128.3, 128.2, 82.8, 67.4, 52.9, 31.2, 31.1, 28.2, 24.3, 24.2. HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₈H₂₄N₃O₆: 378.1660; found: 378.1660.
• 19 Amide 18 and Cyclized Byproduct 19 Fmoc-Glu(NHNHBoc)-OH (14) (69.5 mg, 0.144 mmol, 1 equiv), DCM (1.5 mL, 0.1 M), HOBt hydrate (44 mg, 0.29 mmol, 2 equiv), Hünig’s base (0.04 mL, 0.3 mmol, 2 equiv), 17 (55 mg, 0.29 mmol, 2 equiv), and EDC (55 mg, 0.29 mmol, 2.0 equiv) were stirred for 12 h, diluted with additional DCM (30 mL), washed with 5% Na₂CO₃ (40 mL), 1 M NaHSO₄ (30 mL), and water (30 mL), and dried with Na₂SO₄. The volatiles were removed under reduced pressure to give a crude product mixture (55 mg). Analysis was performed by LCMS via integrating UV signals (which predominantly come from the Fmoc groups, which are analogous in compounds 18 and 19). Data for Amide 18 Elutes at 5.1 min. LRMS (ESI+): m/z [M + H]⁺ calcd for C₃₂H₄₃N₄O₈: 611.38; found: 611.2; m/z [M + Na]⁺ calcd for C₃₂H₄₂N₄O₈Na: 633.29; found: 633.3. Data for Cyclized Byproduct 19 Elutes at 3.1 min. LRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₅H₂₇N₃O₆Na: 488.18; found: 488.1.