Synthesis and Evaluation of Non‐Hydrolyzable Phospho‐Lysine Peptide Mimics

Abstract

The intrinsic lability of the phosphoramidate P−N bond in phosphorylated histidine (pHis), arginine (pHis) and lysine (pLys) residues is a significant challenge for the investigation of these post‐translational modifications (PTMs), which gained attention rather recently. While stable mimics of pHis and pArg have contributed to study protein substrate interactions or to generate antibodies for enrichment as well as detection, no such analogue has been reported yet for pLys. This work reports the synthesis and evaluation of two pLys mimics, a phosphonate and a phosphate derivative, which can easily be incorporated into peptides using standard fluorenyl‐methyloxycarbonyl‐ (Fmoc‐)based solid‐phase peptide synthesis (SPPS). In order to compare the biophysical properties of natural pLys with our synthetic mimics, the pKa values of pLys and analogues were determined in titration experiments applying nuclear magnetic resonance (NMR) spectroscopy in small model peptides. These results were used to compute electrostatic potential (ESP) surfaces obtained after molecular geometry optimization. These findings indicate the potential of the designed non‐hydrolyzable, phosphonate‐based mimic for pLys in various proteomic approaches.

Design and synthesis of fosmidomycin analogs containing aza‐linkers and their biological activity evaluation

“…The α ‐amino acids used as the Michael donors come from two sources: some are commercially available, including glycine ( 3a ), sarcosine ( 3b ), d ‐/ l ‐alanine ( 3m / 3n ), and d ‐/ l ‐proline ( 3o / 3p ); others are N ‐substituted glycines ( 3c–l ), which were synthesized through the nucleophilic substitution of tert ‐butyl bromoacetate with various amines ( 1c – l ) in tetrahydrofuran (THF) in the presence of Et 3 N followed by removal of the t ‐butyl protection group by CF 3 CO 2 H. Dibenzyl vinylphosphonate ( 6 ) was obtained by conversion of vinylphosphonic acid ( 4 ) to vinylphosphonic dichloride ( 5 ) with oxaloyl chloride, followed by reacting with benzyl alcohol (Scheme 1A). 31 The aza‐Michael addition of glycines ( 3a – l ) to 6 in a 1,4‐dioxane/H 2 O mixed solvent with NaOH as base afforded the intermediate β ‐aminophosphonates ( 7a – l ), 32 which condensed with O ‐benzylhydroxylamine and O ‐benzyl‐ N ‐methylhydroxylamine in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDCI), 1‐hydroxybenzotriazloe (HOBt), and N , N ‐diisopropylethylamine (DIEA) to give the corresponding intermediates 8a – l and 9a – l , respectively. The benzyl protecting groups were removed simultaneously from the phosphonate and hydroxamate moiety by catalytic hydrogenation, and after neutralization with equimolar NaOH, products 10a – l and 11a – l were obtained in 60%–76% overall yields.…”

“…RESULTS AND DISCUSSION3.1 ChemistryThe principal reaction for synthesis of the aza-linker FOS analogs is the aza-Michael addition of ⊍-amino acids to dibenzyl vinylphosphonate (Scheme 1B). The ⊍-amino acids used as the Michael donors come from two sources: some are commercially available, including glycine (3a), sarcosine (3b), D-/L-alanine (3m/3n), and D-/L-proline (3o/3p); others are N-substituted glycines (3c-l), which were synthesized through the nucleophilic substitution of tert-butyl bromoacetate with various amines (1c-l) in tetrahydrofuran (THF) in the presence of Et 3 N followed by removal of the t-butyl protection group by CF 3 CO 2 H. Dibenzyl vinylphosphonate(6) was obtained by conversion of vinylphosphonic acid (4) to vinylphosphonic dichloride (5) with oxaloyl chloride, followed by reacting with benzyl alcohol (Scheme 1A) 31. The aza-Michael addition of glycines (3a-l) to 6 in a1,4-dioxane/H 2 O mixed solvent with NaOH as base afforded the www.soci.org X Wu et al wileyonlinelibrary.com/journal/ps © 2023 Society of Chemical Industry.…”

Staudinger Ligation and Reactions – From Bioorthogonal Labeling to Next‐Generation Biopharmaceuticals

“…[38,40] This reaction was further explored to chemically synthesize naturally occurring labile phosphorylations, more specifically, phospholysine (pLys) [41] and phosphocysteine (pCys) [42] peptides (Scheme 7). Taken together with a recent publication where the Staudinger phosphite reaction was used to produce stable, non-hydrolyzable phospholysine mimics, [43] the toolbox to study these rare and poorly understood phosphorylation sites is greatly expanded. [44] The chemoselective Staudinger-phosphonite reaction, first reported in 2011, [33b] follows the same principle as the Staudinger-phosphite reaction, starting this time with a nucleophilic attack of the trivalent phosphorus of a phosphonite 27 to the azide 28.…”

“…This reaction was further explored to chemically synthesize naturally occurring labile phosphorylations, more specifically, phospholysine (pLys) [41] and phosphocysteine (pCys) [42] peptides (Scheme 7). Taken together with a recent publication where the Staudinger phosphite reaction was used to produce stable, non‐hydrolyzable phospholysine mimics, [43] the toolbox to study these rare and poorly understood phosphorylation sites is greatly expanded [44] …”