Cell‐Penetrating Peptides Containing Fluorescent d‐Cysteines

Abstract

A series of fluorescent d-cysteines (Cys) has been synthesized and their optical properties were studied. The key synthetic step is the highly diastereoselective 1,4-conjugate addition of aryl thiols to a chiral bicyclic dehydroalanine recently developed by our group. This reaction is fast at room temperature and proceeds with total chemo- and stereoselectivity. The Michael adducts were easily transformed into the corresponding amino acids to study their optical properties and, in some selected cases, into the corresponding N-Fmoc-d-cysteine derivatives to be used in solid-phase peptide synthesis (SPPS). To further demonstrate the utility of these non-natural Cys-derived fluorescent amino acids, the coumaryl and dansyl derivatives were incorporated into cell-penetrating peptide sequences through standard SPPS and their optical properties were studied in different cell lines. The internalization of these fluorescent peptides was monitored by fluorescence microscopy.

Light-Emitting Probes for Labeling Peptides

“…Non-fluorescent amino acids can became fluorescent by structural modifications, allowing their use as probes that minimally disturb peptide structure and function. 4 , 8 , 9 Non-natural FlAAs have been developed for insertion at specific positions of the peptide sequence without altering its biological function, both using solid-phase peptide synthesis and through genetic encoding. 4 For example, cyanotryptophans have been synthesized to improve the fluorescent properties of Trp by the insertion of cyanide (CN − ), an electron-withdrawing substituent.…”

Elusive Dehydroalanine Derivatives with Enhanced Reactivity

“…More recently, we developed an improved version of these chiral Dha/Dhb derivatives through lactonisation of the first‐generation scaffolds, yielding chiral bicyclic structures with reduced conformational flexibility and superior reactivity and diastereoinducing properties with thiols as nucleophiles . This methodology allowed the synthesis of cell‐penetrating peptides containing fluorescent d ‐cysteine components . Unfortunately, such Dha/Dhb scaffolds were unsuitable for introducing any other nucleophiles besides thiols, due to their limited reactivity.…”

Fluorescent amino acids as versatile building blocks for chemical biology

“…The simplicity of some reactions and the availability of properly derivatized fluorescent building blocks has also facilitated the preparation of collections of FlAAs. For example, using Michael addition, a small library of D-cysteine FlAAs (18) with thiol-containing fluorophores was generated 66 , and maleimide-4-aminophthalimide 67 (19) was coupled to L-cysteine. Furthermore, the Fukuyama-Mitsunobu reaction was used to couple 6-acyl-2-naphthylamine to L-serine to produce 3-(6-acetylnaphthalen-2-ylamino)-2aminopropanoic acid (ANAP) (20), a building block with remarkable optical properties (λem.= 490 nm, quantum yield ~50%) that is widely used for fluorescent labelling of proteins 68 .…”