Fluorescence Studies: A9 Peptide, Functionalized with a Fluorogenic Probe, Interacts with Its Receptor Model HER2-DIVMP.

pubs.acs.org/acsmedchemlett Letter Fluorescence Studies: A9 Peptide, Functionalized with a Fluorogenic Probe, Interacts with Its Receptor Model HER2-DIVMP Valentina Verdoliva, Giuseppe Digilio, Ivana Miletto, Michele Saviano, and Stefania De Luca* Cite This: ACS Med. Chem. Lett. 2022, 13, 807−811 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: A recently developed synthetic protocol allowed for the functionalization of the active peptide A9 with a fluorogenic probe, which is useful for studying biomolecular interactions. Essentially, a nucleophilic attack on a halo-substituted benzofurazan is selectively performed by a cysteine sulfhydryl group. The process is assisted by the basic catalysis of activated zeolites (4 Å molecular sieves) and promoted by microwave irradiation. Fluorescence studies revealed that a donor− acceptor pair within the peptide sequence was introduced, thus allowing a deeper investigation on the interaction process between the peptide ligand and its receptor fragment. The obtained results allowed us to come full circle for all the currently understood structural determinants that were found to be involved in the binding process. KEYWORDS: chemoselective S-conjugation, zeolite as basic catalyst, microwave activation, fluorescent peptide, fluorescence binding studies, FRET ver the past 20 years, fluorescence spectroscopy has been widely employed in the field of biological chemistry, since it provides a quite rapid information about the interaction among biomolecules. FRET (fluorescence resonance energy transfer) has also been implemented, allowing further characterizations that concern the supersolved optical measurements of intra- and intermolecular distances within the molecular target.1 Fluorescent tags based on substituted 2,1,3-benzoxadiazole (benzofurazan) have been long used to label proteins and peptides for biological assays.2−7 Recently, we have introduced a new method for the bioconjugation of cysteine containing peptides with benzofurazan halogenides as fluorescent tags.8 Such a procedure relies on the selective S-alkylation of the cystein thiol group by benzofurazan halogenides, promoted by the mild basic catalysis of activated zeolites (4 Å molecular sieves). We have shown that this reaction is very chemoselective, and it can be performed even in the presence of other unprotected nucleophilic groups (Met, Trp, Thr, His, Lys) without compromising the yield. The high chemoselectivity for the cysteine sulfur atom over other potentially competing nucleophiles was confirmed in a number of model peptides by fluorescence and NMR spectroscopy.8 In this report, we describe the application of our synthetic method to functionalize the bioactive peptide called A9 with the ionic 7-sulfobenzofurazan as the fluorescent tag.9−11 Peptide A9 is a nine-amino acid peptide that binds specifically to the extracellular domain IV of the HER2 receptor with a nanomolar dissociation constant. The design, synthesis, and validation of this peptide was previously described, as well as O © 2022 American Chemical Society its application as a radiolabeled tracer for molecular imaging of HER2.12−15 The A9 sequence was rationally designed by analyzing the X-ray structure of the bimolecular complex between the extracellular region of the HER2 receptor and the antigen-binding fragment (Fab) of Herceptin, an antibody specific for HER2. The A9 sequence represents the minimal sequence derived from Herceptin Fab making closer contacts with the HER2 extracellular domain. In the same study, the minimal binding region of the receptor was identified as well. Such a region, called HER2-DIVMP (Domain IV mimicking peptide), was chemically synthesized and validated as a synthetic model of the Herceptin binding domain. It consists in a modified fragment of the receptor HER2 that was previously proved as fully representative of the receptor domain IV binding properties, as well as easy to obtain and implement in reproducible screening of ligands.16 As the plain A9 peptide does not contain cysteine, we have elongated the N-terminus of A9 with a N-acetyl-cysteine residue in order to perform an S-conjugation with 4-chloro-7sulfo-benzofurazan (Cl-Sbf). The key feature of the synthetic protocol consists in using activated molecular sieves (MS) as the basic catalyst to activate the thiol function for nucleophilic Received: January 17, 2022 Accepted: April 4, 2022 Published: April 11, 2022 807 https://doi.org/10.1021/acsmedchemlett.2c00026 ACS Med. Chem. Lett. 2022, 13, 807−811 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett substitution.17,18 Peptide A10 (i.e., the N-terminus elongated A9 peptide, Scheme 1) was dissolved in DMF, under an argon Letter alkylation sites (especially aspartic acid and tryptophan) were detected. Next, the binding affinity of the fluorescent peptide ligand toward its receptor model HER2-DIVMP was studied by means of fluorescence spectroscopy titrations. As the method we have optimized uses excitation at 280 nm, we first investigated the emission spectrum of the ligand A10-Sbf upon excitation at such wavelength. As shown in Figure 2A, Scheme 1. Incorporation of Sulfo-Benzofurazan in Peptide A10 atmosphere, and added with an excess of 1.2 equiv of 4-chloro7-sulfobenzofurazan. Molecular sieves (4 Å MS activated at 280 °C for 4 h under vacuum, then allowed to cool to room temperature) were finally added to the reaction mixture. This was irradiated with microwaves and stirred for 5 min at 40 °C in a microwave source apparatus. (Scheme 1). The nucleophilic substitution of the cysteine sulfhydryl occurred selectively and with a good yield (85%), even in the presence of potentially competing nucleophilic groups, such as the carboxylic function of aspartic acid and the indolic nitrogen of tryptophan. The chemical structure of the final conjugate (compound A10-Sbf) was unambiguously confirmed by NMR spectroscopy. All 1H NMR resonances were assigned according to the sequence specific method. The expected short distances between the cysteine Hα/Hβ protons and the benzofurazan H5 ring proton yielded clear NOE peaks in 2D-NOESY spectra (Figure 1). No NOE distances connecting the benzofurazan ring protons with side chain protons of potentially competing Figure 2. Emission fluorescence spectra of A10-Sbf upon excitation at 280 nm (panel A) and of a generic peptide conjugated to a sulfobenzofurazan moiety upon excitation at 280 and 380 nm (panel B). two emission peaks can be found: one at λmax = 354 nm related to the tryptophan residue, and second one at λmax = 533 related to the sulfobenzofurazan fluorescent tag. It is worth noting that the latter emission requires excitation at 380 nm, while it should be negligible upon excitation at 280 nm. In addition, it was demonstrated by a control peptide AcCGTVANH2−Sbf, which contains a sulfobenzofurazan group conjugated to cysteine (similarly to A10-Sbf) but lacks of any tryptopha (...truncated)