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DOI: 10.1055/s-0042-1751397
Challenges and Strategies for Synthesizing Glutamyl Hydrazide Containing Peptides
Funding for this work was provided by Clark University. N.S.M. acknowledges a Gustaf H. Carlson Summer Research Fellowship. S.I. acknowledges a summer fellowship provided through Clark University’s Global Scholars program. J.P.M. acknowledges a summer fellowship from PCI Synthesis Inc.
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 acidsSupporting Information
- Supporting information for this article is available online at https://doi.org/10.1055/s-0042-1751397.
- Supporting Information
Publication History
Received: 03 November 2022
Accepted after revision: 23 November 2022
Article published online:
22 December 2022
© 2022. Thieme. All rights reserved
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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: Rf = 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: Rf = 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.