Locking the GFP Fluorophore to Enhance Its Emission Intensity

Abstract

The Green Fluorescent Protein (GFP) and its analogues have been widely used as fluorescent biomarkers in cell biology. Yet, the chromophore responsible for the fluorescence of the GFP is not emissive when isolated in solution, outside the protein environment. The most accepted explanation is that the quenching of the fluorescence results from the rotation of the aryl-alkene bond and from the Z/E isomerization. Over the years, many efforts have been performed to block these torsional rotations, mimicking the environment inside the protein β-barrel, to restore the emission intensity. Molecule rigidification through chemical modifications or complexation, or through crystallization, is one of the strategies used. This review presents an overview of the strategies developed to achieve highly emissive GFP chromophore by hindering the torsional rotations.

Keywords: Z/E isomerization; aggregation-induced emission enhancement; difluoroborate; fluorescence; green fluorescent protein.

Figure 1

p-hydroxybenzylidene-imidazolidinone chromophore responsible for the emission of the GFP.

Scheme 1

Erlenmeyer–Plöchl azlactone synthesis of a GFPc analogue.

Scheme 2

Synthesis of GFPc analogues via Knoevenagel condensation.

Figure 2

GFPc analogues with multiple phenyl substituents [15].

Figure 3

GFPc analogues presenting AIEE properties [10,11].

Figure 4

GFPc analogues presenting AIEE properties, combined with ACQ for some of them [16,17].

Figure 5

GFPc analogue emitting in the solid state, at different wavelengths depending on the polymorph [19].

Figure 6

GFPc analogues with large aromatic substituents [20].

Figure 7

GFPc analogues without Z /E isomerization [21].

Figure 8

GFPc analogues demonstrating an increased emission intensity in the presence of a deep cavity cavitand [22].

Figure 9

GFPc analogues used as selective fluorescence turn-on molecular probes for RNA and HSA [23].

Figure 10

GFPc analogues with enhanced emission intensity in the presence of HSA [24].

Figure 11

GFPc analogue with enhanced emission when encapsulated in NaCh vesicles [25].

Figure 12

GFPc analogue isolated and attached to a β-cyclodextrin [12].

Figure 13

GFPc analogues used to study the supramolecular interaction with cyclodextrin [27].

Figure 14

GFPc analogues that can be conjugated with the TMV channel [28].

Figure 15

GFPc analogues used as molecular probe for imaging β-amyloids and lysosomes [29].

Figure 16

GFPc analogues linked to copolymer [30].

Figure 17

GFPc-copolymer with strong fluorescence after self-assembly into micelles [31,32].

Figure 18

GFPc analogues used in the preparation of porous organic frameworks [33].

Figure 19

GFPc analogue used as linker in the preparation of a green-fluorescent metal–organic framework [34].

Figure 20

GFPc analogues demonstrating that the intramolecular hydrogen bond increases the emission intensity in solution [35].

Figure 21

GFPc analogues with enhanced emission intensity due to the intramolecular hydrogen bond [36].

Figure 22

GFPc analogues presenting a hydrogen bond and/or a cyclic substituent [37].

Figure 23

GFPc analogues with a benzimidazole ring substituted with nitrogen, and intramolecular hydrogen bond [38,39].

Figure 24

GFPc analogues with a boron complex, demonstrating higher emission intensity than the hydrogen-bonded precursors [9].

Figure 25

GFPc analogue with the Z/E isomerization blocked by intramolecular interaction [41].

Figure 26

GFPc analogues locked with intramolecular interaction, used to study the effect of the cyclic substituents [40,44].

Figure 27

GFPc analogues with a boron complex blocking the Z/E isomerization [45].

Figure 28

GFPc analogues with a more conjugated backbone [46].

Figure 29

GFPc analogues with solvent-dependent quantum yield (31a) or not (31b,c) [47].

Figure 30

GFPc analogues with an increased emission intensity when complexed with Zn (II) [41,48].

Figure 31

GFPc analogues for the detection of metallic cations [13,49].

Figure 32

GFPc analogues with a chelating pocket selective for cobalt cations [6].

Figure 33

GFPc analogues locked with palladium [50].

Figure 34

GFPc analogues with isomerization restricted by a cyclic pattern [51].

Figure 35

GFPc analogues with their isomerization hampered by benzylidene substituents [26].

Figure 36

GFPc analogues structurally unconstrained presenting high quantum yields in solution [52].