Amphiphilic Iodine(III) Reagents for the Lipophilization of Peptides in Water

Abstract

We report the functionalization of cysteine residues with lipophilic alkynes bearing a silyl group or an alkyl chain using amphiphilic ethynylbenziodoxolone reagents (EBXs). The reactions were carried out in buffer (pH 6 to 9), without organic co-solvent or removal of oxygen, either at 37 °C or room temperature. The transformation led to a significant increase of peptide lipophilicity and worked for aromatic thiols, homocysteine, cysteine, and peptides containing 4 to 18 amino acids. His₆ -Cys-Ubiquitin was also alkynylated under physiological conditions. Under acidic conditions, the thioalkynes were converted into thioesters, which could be cleaved in the presence of hydroxylamine.

Keywords: amphiphilic reagents; hypervalent iodine; lipidation; lipopeptide; ubiquitin.

Figure 1

a) Chemical methods for stable natural lipidation. b) Reported reagents for non‐natural lipidation. c) EBX reagents for cysteine labeling. d) This work: amphiphilic reagents for non‐natural lipidation under physiological conditions. DMPA=2,2‐dimethoxy‐2‐phenylacetophenone, NMP=N‐methyl‐2‐pyrrolidone, BIPHEPHOS=6,6′‐[(3,3′‐di‐tert‐butyl‐5,5′‐dimethoxy‐1,1′‐biphenyl‐2,2′‐diyl)bis(oxy)]bis(di‐benzo[d,f][1,3,2]dioxaphosphepin), TIPS=triisopropylsilyl, TMS=trimethylsilyl.

Scheme 1

Synthesis of TIPS‐EBX‐SO₃M (4 a) and C₁₄H₂₉‐EBX‐SO₃M (4 b). a) NaIO₄ (1.05 equiv), 30 % aq. AcOH (v/v), reflux, 4 h; b) TMSOTf (3.0 equiv), pyridine (6.0 equiv), DCE, 40 °C, 22 h; c) BF₃⋅Et₂O (3.0 equiv), pyridine (1.1 equiv), CH₃CN, rt, 24 h.

Scheme 2

Optimized conditions for the reaction of 4 b with glutathione (6). HPLC‐MS yield is indicated. [a] Isolated yield. [b] Calibrated yield.

Scheme 3

Scope of the alkynylation reaction for a) non‐peptides, b) tetra‐ and hexa‐peptides and c) larger peptides. All the reactions were performed in 0.5 to 64.0 μmol scale at 10 mM concentration. Freshly prepared buffer was used without removing oxygen. Yields: relative ratio based on reverse phase HPLC‐MS chromatogram unless stated otherwise. [a] Isolated yield. [b] Reactions were performed in 100 mM PB buffer at pH 8.0 at rt. [c] Calibrated yield.

Scheme 4

Alkynylation of peptides 22–24 and His₆‐Cys‐ubiquitin (25). Reaction conditions: for 22 a and 23 a: 4 a (1.5 equiv), 10 mM Tris pH 7.4, 37 °C, for 22 b and 23 b: 4 b (1.2 equiv), 200 mM Tris buffer pH 8.0, rt. [a] Isolated yield. [b] Calibrated yield. [c] 4 equiv and [d] 6 equiv of 4 a was used in 10 mM Tris pH 7.4 at 37 °C, 5 mM. [e] Reaction was performed at 300 μM concentration.

Scheme 5

a) Access to thioesters from unprotected peptides in one pot via thioalkynes and b) Cleavage of the thioesters. See Supporting Information for detailed reaction conditions. [a] VBXs remain untouched under these reaction conditions. [b] Isolated yield. [c] Calibrated yield.