Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation

Cyclic peptides and peptoids were prepared using the thiol-ene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization

Straightforward synthesis of cyclic and bicyclic peptides

Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifluoroacetic acid treatment, if the maleimide is not protected, or upon deprotection of the maleimide. The combination of free and protected maleimide moieties and two orthogonally protected cysteines gives access to structurally different bicyclic peptides with isolated or fused cycles

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1 ISD11 ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide com- pounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1 ISD11 ISCU complex

Conjugation and cyclization of polyamides derivatized with protected maleimides

[cat] Aquest treball està dividit en dues parts diferenciades. A la primera part s’ha desenvolupat una metodologia per obtenir conjugats i cicles de poliamides (pèptids i peptoides) fent ús de reaccions tipus “click” sobre maleimides, com ara addicions de Michael o cicloaddicions de Diels-Alder o Huisgen. Les maleimides s’incorporen a les cadenes de poliamida protegides amb dimetilfurà, i es desprotegeixen escalfant. Combinant en una mateixa seqüència maleimides protegides i desprotegides, cisteïnes amb grups protectors ortogonals entre ells, o alquins, s’han sintetitzat conjugats, dobles conjugats, cicles, bicicles i conjugats de pèptids cíclics. En tots els casos la poliamida s’ha ciclat amb una addició de Michael, i les conjugacions s’han dut a terme amb addicions de Michael o cicloaddicions de Diels-Alder o Huisgen. Tanmateix s’han sintetitzat conjugats tipus pèptid cíclic – PNA amb potencial activitat biològica, però malauradament els resultats no han millorat els obtinguts amb altres compostos similars. A la segona part s’ha estudiat la interacció de l’AD2, una molècula petita, amb el loop GUAA de l’IRES del virus de la febre aftosa. Els estudis biològics in vitro han indicat que inhibeix més la traducció mediada per IRES que la cap-dependent, però malauradament no s’ha pogut determinar de forma inequívoca el lloc d'unió. Paral•lelament s’han realitzat estudis biofísics amb RNAs sintètics. La determinació de la variació de la temperatura de fusió dicroisme circular i la calorimetria no han proporcionat informació útil. Amb estudis per fluorescència amb RNAs marcats, en canvi, s’ha determinat la EC50, i s’ha observat que l’AD2 és afí pel loop desitjat, però ho és encara més per un bulge proper. La determinació per ESI de l’estequiometria dels complexos mostra que l’AD2 és promiscu, ja que diverses molècules d’AD2 s’uneixen a cada seqüència curta d’RNA assajada. En resum, doncs, l’AD2 interacciona amb el loop desitjat amb alta afinitat, però també interacciona, i de forma més afí, amb altres estructures d’RNA presents a l’IRES.[eng] This work is divided in 2 different parts: Part A: Cyclization and conjugation can confer on biomolecules better activity, resistance toward enzymatic degradation and better internalization properties. In order to develop a versatile methodology for polyamide conjugation and cyclization, several protected maleimide-containing monomers were synthesized and incorporated into polyamide sequences. Maleimide protection was accomplished by Diels-Alder reaction with 2,5-dimethylfuran. The resulting cycloadduct underwent a retro-Diels-Alder reaction upon heating. These monomers were included in peptide and peptoid sequences, and it was assessed in all cases that the maleimides obtained after deprotection were fully reactive. Double conjugates were obtained by incorporating both a free and a protected maleimide in the same sequence. These were peptides were selectively linked to two different biomolecules, namely a peptide-thiol or an oligonucleotide-diene. By assembling sequences containing both maleimide and cysteine, several cyclic polyamides were obtained. These could include almost all proteinogenic amino acids. By combining orthogonally-protected cysteines and free and protected maleimides, bicyclic peptides could also be synthesized. Cyclic peptide conjugates were synthesized, by taking advantage of the different protecting group schemes developed. These compounds could contain the conjugation spot within or outside the cycle, either at the C- or the N- terminus. All cyclizations were performed by Michael addition, and conjugations were performed by reactions such as Diels-Alder cycloaddition, Michael addition or Huisgen cycloaddition. Finally, a series of cyclic peptide – PNA conjugates were synthesized and tested as alternative splicing inducers. Unfortunately their activity did not surpass the one from previously tested conjugates. Part B: Foot-and-mouth disease virus (FMDV) promotes translation of its genetic material through a cellular mechanism, in which part of its RNA sequence, the IRES, plays a crucial role. FMDV IRES contains a GNRA loop, and AD2, a small molecule, is known to interact with these kind of loops. The initial goal of this part was to study the interaction between AD2 and the GUAA loop of the FMDV IRES. AD2 was synthesized, and in vitro assays were performed in order to assess its activity and binding site. By using a bicistronic RNA, a differential response was observed in the inhibition of the cap- and the IRES-dependent translation, assessing that AD2 was active. The binding site determination was attempted with the SHAPE assay, but no defined binding site could be established. At the same time a series of biophysical assays with synthetic RNA were performed. Thermal denaturation experiments and CD measurements, as well as isothermal titration calorimetry assays were carried out, but none of them afforded relevant data. However, EC50 values could be obtained by employing fluorescently-labeled RNA. These experiments showed that AD2 interacted with the desired loop, but also with a bulge nearby. The binding stoichiometry was determined by mass spectrometry. The masses of the different RNA-ligand complexes indicated high stoichiometries, that is, several AD2 molecules were interacting with relatively short RNA sequences. Considering the in vitro results, the EC50 values obtained in the fluorescence titrations and the observed stoichiometries, we could conclude that AD2 interacts with the desired loop with high affinity, but it also interacts, and even more strongly, with other RNA structures found in FMDV IRES

Straightforward synthesis of cyclic and bicyclic peptides

Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifluoroacetic acid treatment, if the maleimide is not protected, or upon deprotection of the maleimide. The combination of free and protected maleimide moieties and two orthogonally protected cysteines gives access to structurally different bicyclic peptides with isolated or fused cycles

Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation

Cyclic peptides and peptoids were prepared using the thiol-ene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization

Protected maleimide building blocks for the decoration of peptides, peptoids and peptide nucleic acids

Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were established in each case. The stability of the adducts generated by Michael-type maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection conditions was exploited to prepare a variety of conjugates from peptide and PNA scaffolds incorporating one free and one protected maleimide. The target molecules were synthesized by using two subsequent maleimide-involving click reactions separated by a maleimide deprotection step. Carrying out maleimide deprotection and conjugation simultaneously gave better results than performing the two reactions subsequently

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1 ISD11 ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide com- pounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1 ISD11 ISCU complex