Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives

Abstract

Sugar coated: We recently developed methylcyclopropenes as low-molecular-weight tetrazine coupling partners. Here, we demonstrate that methylcyclopropenes can meet the stringent steric demands required for metabolic imaging of unnatural mannosamines on live cells. Using sequential azide-alkyne chemistry, we also demonstrate multicolor imaging of two different metabolically incorporated unnatural sugars.

Figure 1

Imaging glycans using cyclopropene-mannosamine derivatives. A) SKBR3 cell surfaces were stained by incubation with Ac₄ManNCyc (3; 100 μm) followed by reaction with tetrazine-Alexa Fluor 488 (10 μm). B) Staining was significantly lower when SKBR3 cells were incubated with 3 as well as tunicamycin (1.2 μm), a glycosylation inhibitor. C) Staining was also reduced when SKBR3 cells were incubated with 3 as well as of ManNAc (20 mm) as a competitor. Simultaneous imaging of 3 and Ac₄GalNAz incorporation. LS174T cells were incubated with of 3 (100 μm) and Ac₄GalNAz (100 μm) for 48 h. After incubation, cells were reacted sequentially with tetrazine-Alexa Fluor 488 (10 μm) and DIBO 647 (15 μm). Confocal microscopy revealed extensive surface staining in the D) 488 nm channel and E) 647 nm channel with good colocalization of surface staining (F). Controls revealed minimal cross staining of tetrazine-Alexa Fluor 488 with cells incubated only with Ac₄GalNAz and DIBO 647 with cells incubated only with 3 (see the Supporting information). Scale bars: 20 μm.

Scheme 1

A) Comparison of N-acyl substituents on unnatural mannosamine derivatives. The cyclopropene handle is similar in size to the commonly used azide handle. B) Synthesis of peracetylated Ac₄ManNCyc (3). a) 1.0 eq. NaOH, dioxane, sat. NaHCO₃, Boc₂O; b) pyridine, Ac₂O, 20% (2 steps); c) 20% TFA, CH₂Cl₂; d) CH₂Cl₂, Et₃N, DMAP, 2-methyl-2-cyclopropenyl-1-carbonyl chloride, 60% (2 steps).

Scheme 2

Cartoon outlining proposed fluorescent staining of glycans using tetrazine–cyclopropene chemistry. Ac₄ManNCyc (3) is incubated with live cells for 48 h. If the unnatural mannosamine derivative is processed by the cell, methylcyclopropenes will be displayed on the surface and tagged by fluorogenic tetrazine-AlexaFluor 488. This staining pattern will be visible by confocal microscopy.