Monodisperse Chemical Oligophosphorylation of Peptides via Protected Oligophosphorimidazolide Reagents

Abstract

Protein poly- and oligophosphorylation are recently discovered post-translational modifications that remain poorly characterized due to (1) the difficulty of extracting endogenously polyphosphorylated species without degradation and (2) the absence of synthetic and analytical tools to prepare and characterize poly- and oligophosphorylated species in biochemical contexts. Herein, we report a methodology for the selective oligophosphorylation of peptides with monodisperse phosphate chain lengths (P_n=3-6). A library of oligophosphorimidazolide (oligoP-imidazolide) reagents featuring benzyl and o-nitrophenylethyl protecting groups was synthesized in moderate-to-good yields (65-93 %). These oligoP-imidazolide reagents enabled the selective and simultaneous conjugation of multiple phosphate units to phosphoryl nucleophiles, circumventing tedious iterative processes. The generalizability of this approach is illustrated by a substrate scope study that includes several biologically relevant phosphopeptide sequences, culminating in the synthesis of >60 examples of peptide oligophosphates (P_n=2-6). Moreover, we report the preparation of oligoP-diimidazolides (P_n=3-5) and discuss their application in generating unique condensed phosphate-peptide conjugates. We also demonstrate that human phospho-ubiquitin (pS65-Ub) is amenable to functionalization by our reagents. Overall, we envision the methods described here will enable future studies that characterize these newly discovered but poorly understood phosphorylation modes.

Keywords: Anions; Peptides and Proteins; Phosphorylation; Post-translational Modifications; Reagents.

Figure 1

A) Discovery of different phosphorylation modes; P=phosphoryl group. (B) Reported chemical methods for preparing peptide pyro‐ and triphosphates. (C) Summary of present work with new oligophosphorylation reagents; R=OBn, O‐NPE, im.

Scheme 1

Initial attempts to synthesize oligophosphorylated peptides from model substrate p‐Pep1 with (A) reagent 1 b, (B) diamidophosphate, and (C) reagents 6–9. Conditions: i) 3 equiv. 1 b, 8 equiv. ZnCl₂, 9 : 1 DMA/H₂O, 1.5 h, 45 °C. ii) 365 nm LED, 0.1 M aq. NH₄HCO₃. iii) 8 equiv. diamidophosphate, 2 equiv. MgCl₂, 2 equiv. imidazole, pH 5.5, 20 h, −20 °C. iv) 8 equiv. NaNO₂, pH 3, 8 h, −20 °C. (*RP‐HPLC yield).

Scheme 2

Synthesis of benzyl‐ and o‐nitrophenylethyl substituted oligoP‐imidazolide (A) diphosphorylation, (B) triphosphorylation, (C) tetraphosphorylation, and (D) pentaphosphorylation reagents. See Supporting Information for details regarding synthesis and characterization.

Figure 2

(A) ³¹P{¹H} and ³¹P NMR spectra and (B) CID MS/MS of isolated benzyl‐pentaphosphopeptide Bn‐p₅‐Pep1.

Figure 3

(A) Summary Scheme of oligoP‐peptides included in substrate scope. (B) Peptide oligophosphorylation with photocaged reagents. (C) Synthesis of peptide triphosphates with benzyl‐protected 2 a. (D) Substrate scope table. *Yields for peptide triphosphates reported over two steps carried out in a one‐pot synthesis.

Figure 4

(A) Synthesis of oligoP‐diimidazolide reagents. (B) Reactivity of 3 c and 4 c with phosphopeptide p‐Pep1. (C, inset) High‐resolution ESI mass spectrum of Pep1‐p₆‐Pep1. Conditions: i) 8 equiv. 3 c, 9 : 1 DMA/H₂O, 40 min, 45 °C. ii) 8 equiv. 4 c, 9 : 1 DMA/H₂O, 1 h, 45 °C. (*RP‐HPLC yield). ^aConversion to im‐p₄‐Pep1 was determined by quantifying the hydrolysis product p₄‐Pep1 as a proxy.

Figure 5

(A) Triphosphorylation of pS65‐Ub with 3 b and subsequent photodeprotection. (B) Overlaid deconvoluted (ESI+) mass spectra of pS65‐Ub, NPE‐p₄S65‐Ub, and p₄S65‐Ub. Peak labels: (•) signals of the expected products, (▵) species resulting from the dehydration of said products, and (◊) signals assigned as the adducts to metal ions (Na⁺, K⁺, and Fe³⁺).