Photoactivatable Janelia Fluor Dyes via Methyl Carbamate Caging

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Solution Overview

Problem

Current small molecule fluorophores, such as Janelia Fluor dyes, face challenges with solubility and reactivity due to large and hydrophobic caging groups, and existing photocaging strategies are incompatible with fully N-alkylated rhodamine dyes like JF549 and JF646, limiting their use in live-cell imaging and self-labeling tag systems.

Innovation Solution

Development of photoactivatable derivatives of Janelia Fluor dyes, specifically JF549 and JF646, through a caging strategy involving treatment with oxalyl chloride and diazomethane, which forms a photoactive fluorophore that decarboxylates upon photoinduction, maintaining brightness and enabling live-cell labeling and imaging.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If large and hydrophobic caging groups are used to protect fluorophores, then photostability is improved, but solubility and reactivity with self-labeling tag proteins deteriorate

Engineering Contradiction:

ImprovephotostabilityVSAvoidsolubility and reactivity

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The caging group is divided into two separate components: a small hydrophilic photolabile group (e.g., methyl carbamate) and a separate protecting group strategy. This segmentation allows the fluorophore to maintain solubility and reactivity while still providing photostability through the methyl carbamate caging mechanism.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The chemical structure of the caging group is changed from large hydrophobic groups to small hydrophilic methyl carbamate groups. This parameter change in molecular size and polarity directly improves solubility and reactivity while maintaining the photoactivatable function.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If classic photocaging strategies are used, then fluorophore protection is achieved, but compatibility with fully N-alkylated rhodamine dyes like JF549 and JF646 deteriorates

Engineering Contradiction:

Improvefluorophore protectionVSAvoidcompatibility with N-alkylated rhodamine dyes

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

Instead of using traditional N-acyl caging groups on the rhodamine nitrogen, the invention applies methyl carbamate caging at a different position on the fluorophore structure. This inverted approach to caging location enables compatibility with N-alkylated rhodamine dyes while maintaining photoactivatable protection.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The methyl carbamate group serves as an intermediary caging mechanism that bridges the gap between the need for fluorophore protection and the structural constraints of N-alkylated rhodamine dyes. This intermediary approach allows both requirements to be satisfied simultaneously.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Adaptability or versatility

If photoactivatable derivatives are developed through oxalyl chloride and diazomethane treatment, then photoactivation capability is improved, but manufacturing complexity increases

Engineering Contradiction:

Improvephotoactivation capabilityVSAvoidmanufacturing complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The methyl carbamate caging is incorporated into the fluorophore synthesis pathway as a preliminary action during standard manufacturing steps. By integrating the photoactivatable group formation into existing synthesis protocols using oxalyl chloride and diazomethane treatment, the additional complexity is minimized and the photoactivation capability is built-in from the start.

Inventive Principle:

Principle #10Preliminary action

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The photoactivatable Janelia Fluor dyes retain superior brightness and photostability, facilitating improved single-particle tracking and localization microscopy, and are compatible with existing live-cell labeling strategies, enhancing imaging capabilities in live cells.

Implementation Method 1

the photoactive fluorophore is decarboxylated by photoinduction

Methodology Applied

Scientific EffectPhotoinduced decarboxylation: Photodissociation

Implementation Method 2

small molecule fluorophores are important tools for advanced imaging experiments. These fluorophores, which are brighter than fluorescent proteins

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS11958976B2Photoactive fluorophores and methods of in vivo labeling

Publication Date: 2024.04.16 HOWARD HUGHES MEDICAL INST

AI summary

Provided are a photoactive fluorophore, a photoactive ligand, and a photoactive complex. The photoactive fluorophore includes a photoactivatable derivative of an azetidine-containing Janelia-Fluor dye. The photoactive ligand includes a photoactive fluorophore and a protein tag. The photoactive complex includes a photoactive ligand conjugated to a protein. Also provided are methods of in vivo labeling with and photoactivation of the photoactive fluorophore, ligand, and complex.