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Fluorescence Labeled Peptide Synthesis

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Fluorescently labeled peptides are produced by incorporating a fluorescent dye or fluorescent tag, such as FAM or FITC, directly into the peptide during synthesis. CD Formulation applies a variety of fluorescent labels, which can be incorporated into any desired position within the peptide. Most commonly, they are integrated into the N-terminus, or into the side chain of a cysteine (Cys) or lysine (Lys) residue, such as the C-terminus.

Introduction to Fluorescence Labeled Peptide

Fluorescent dyes typically find their niche nestled at the N-terminus, C-terminus, or even gracefully affixed to the side chains of particular amino acids—most notably cysteine and lysine. These markers have several advantages over other types of dyes. One of the most important is that they can be easily visualized using fluorescence microscopy or other fluorescence-sensitive techniques. Their capacity to emit light at precise wavelengths—activated by diverse light sources—imbues them with remarkable adaptability and uncanny accuracy in detection. Moreover, these fluorescent labels boast commendable stability and are largely devoid of toxicity, rendering them quintessential for the monitoring of biological processes, both in vitro and in vivo.

Fig. 1 Fluorescent labeling of peptides. Fig. 1 Strategies for fluorescent labeling of peptides. (Larsen JB, et al., 2021)

Explore Our Custom Fluorescence Labeled Peptide Synthesis Services

At CD Formulation, we delve deep into the intricate world of fluorescent peptide synthesis, crafting bespoke solutions tailored to your unique research endeavors. With a rich tapestry of experience spanning decades in peptide production, we present an expansive array of fluorescent peptides, each brimming with diverse modifications and exceptional purity levels.

Fluorescent tags can be incorporated into peptides at many positions and require different precursors to attach the dye. The three main functional groups used for fluorescent labeling in common peptide chains include:

Amino group (N-terminus, Lys).
Thiol group (Cys).
Carboxyl group (C-terminus, Asp, Glu).

Below, we provide a detailed list of commonly used fluorescent dyes and fluorescent tags and their attachment options:

Fluorophore Full Name	Short Name	Excitation /Emission (nm)
Anthranilyl, 2-aminobenzoyl	Abz	320 / 420
Fluorescein isothiocyanate	FITC	490 / 520
5-Carboxyfluorescein	5-FAM	492 / 517
6-Carboxyfluorescein	6-FAM	492 / 517
Carboxytetramethylrhodamine	TAMRA	543 / 572
5-(Dimethylamino)naphtalene-1-sulfonyl	Dansyl	335 / 493 494 / 518 494 / 519 502 / 517 550 / 564
5-[2-aminoethyl))amino]naphtalene-1-sulfonic acid	EDANS	340 / 490
1-(5-carboxypentyl)-3,3-dimethyl-2-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium chloride	Cy3	550 / 570
1-(5-carboxypentyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dienyl-1-yl)-3H-indolium chloride	Cy5 peptide	648 / 663
7-Methoxycoumarinyl-4-acetyl	Mca	320 / 393
Rhodamine B	Rhodamine B	550 / 580
N-Methyl-anthranilyl	N-Me-Abz	340 / 360
Aminomethylcoumarin Acetate	AMCA	350 / 450
DyLight 488	Dylight	493 / 518
Atto465	Atto	453 / 508
Tryptophan	Trp	280 / 360

Our Fluorescent Labeling Strategies

Amine-Reactive Labeling

Our main strategy for fluorescent labeling is based on the amine reactivity between amino acids in the peptide chain and the fluorophore. The vast majority of tags are designed for amino acid labeling. Thus, peptides can be labeled with an N-terminal free amine, via an internal sequence of amino acid side chains such as lysine or Dap, or at the C-terminus of the peptide via the side chain of a lysine residue.

Fig. 2 Peptide fluorescence labeling using amine-reactive. Fig. 2 Amine-reactive strategies for peptide fluorescence labeling. (CD Formulation)

Click Chemistry Labeling

Fluorescent dyes can be conjugated to peptides via click chemistry. Click chemistry involves cleaving two molecules against each other. The reaction involves an alkyne and a nitrogen-containing functional group (azide) and is catalyzed by a copper molecule. Click chemistry is a selective, efficient, rapid, quantitative conjugation method that is performed in water and is easy to set up.

Fig. 3 Peptide fluorescence labeling using click chemistry. Fig. 3 Click chemistry strategies for peptide fluorescence labeling. (CD Formulation)

Common Applications of Fluorescently Labeled Peptides

In vivo biomedical imaging.
Intracellular localization studies.
Protein interaction studies.
Development of novel disease models.
Biosensing.

Peptide Manufacturing & Analytical Services

In addition to peptide synthesis capabilities, CD Formulation combines flexible GMP manufacturing facilities with cutting-edge peptide analytical knowledge to provide a full range of quality control testing services to accelerate the commercialization of your products, including:

Peptide identification (ESI-MS).
Peptide Molecular weight determination.
Peptide sequencing.
Peptide quantification/peptide content determination.
Peptide purity and impurity analysis (HPLC/UV).
Amino acid sequence.
Amino acid composition determination.
Net peptide content.

Enantiomeric purity testing (GC/MS; LC).
Residual counterion testing (e.g. TFA).
Elemental analysis.
Residual solvent testing.
Water content testing (GC or KF).
Peptide solubility testing.
Peptide stability testing.

Optical rotation determination.
Bioburden testing(TAMC/TYMC).
Bacterial endotoxin testing.
Sterility testing.
Cytotoxicity testing.
Process/product related impurity testing.
Other pharmacopoeia testing.

Publication

Published Data

Technology: N-Terminal Fluorescent Labeling of Peptides

Journal: ACS Org Inorg Au.

IF: 5.0

Published: 2024

Results:

The authors evaluated the coupling efficiency of commonly used peptide synthesis reagents for N-terminal labeling with carboxylic acid-functionalized fluorophores such as 6-carboxyfluorescein, rhodamine B, and BODIPY-FL. The amino-terminal Cu(II) and Ni(II) binding site (ATCUN) peptide was used as a model system. Results show that conventional coupling strategies do not always yield optimal results, as reagents such as HATU, HBTU, and PyBOP were inefficient in coupling FAM, RhoB, and BODIPY-FL to the ATCUN core when a primary amine was present at the N-terminus. In contrast, an uncommon synthesis of activated pentafluorophenol (PFP) esters was the most efficient strategy when labeling primary amines on peptides, accelerated by microwave irradiation.

Fig. 4 The fluorophore-labeled ATCUN peptides employed. Fig. 4 Synthetic scheme of the fluorophore-labeled ATCUN peptides employed in this study. (Hintzen JCJ, et al., 2024)

CD Formulation is a trusted partner for peptide synthesis. Please don't hesitate to contact us if you are considering using fluorescence labeled peptides in your project. We look forward to cooperating with you.

References

Larsen JB, Taebnia N, Dolatshahi-Pirouz A, et al. Imaging therapeutic peptide transport across intestinal barriers. RSC Chem Biol. 2021 Jun 15;2(4):1115-1143.
Hintzen JCJ, Devrani S, Carrod AJ, et al. Fluorescence Labeling of Peptides: Finding the Optimal Protocol for Coupling Various Dyes to ATCUN-like Structures. ACS Org Inorg Au. 2024 Jun 4;4(5):517-525.

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