TY - BOOK
T1 - On the Modular Combinatorial Synthesis of NHC Palladium Peptides and their use as Artificial Enzyme Mimics
T2 - Catalytic peptides as Nanomedicines towards treatment of neurodegenerative diseases
AU - Wolfram, Martin
PY - 2018
Y1 - 2018
N2 - A novel Lego® building block approach has been developed for facile incorporation of Nheterocyclic
carbenes (NHC) into peptide side-chains in conjunction with auxiliary ligands, such
as pyridine and triazoles. The methodology uses the CuAAC click reaction to incorporate NHC
precursors after or during peptide assembly followed by palladium complexation on solid
support. Various model catalysts were synthesized and subsequently tested for hydrolytic
activity on the nucleating peptide sequence in amyloid Aβ42 - (Aβ14-24 HQKLVFFAEDV) - to
investigate possible cleavage conditions on-bead and in solution. Based on the developed
sequential-modular peptide assembly strategy combinatorial one-bead two compound catalyst
libraries have been synthesized and probed toward their on-bead activity in hydrolyzing the
fluorescence quenched target sequence (Abz)-Pra-HQKLVFFAEDVY(NO2)G in aqueous
solution. Possible hit catalysts were indicated by the increased fluorescence of individual beads
and subjected to linker cleavage followed by single bead MS/MS analysis. The sequence
determination allowed re-synthesis of the supposed active catalysts, which were probed again
toward the substrate in various solution assays indicating condition dependent hydrolytic
activity.
Employing the evolved catalyst motifs towards the target substrate did not provide unambiguous
evidence for site-selective hydrolysis of the target substrate. The chosen sequence surprisingly
was found to exhibit high autoproteolytic activity in a wide concentration range (10 – 800 M)
under various buffer conditions from ambient temperature to 37 C. These unique observations
were repetitively made in a number of hydrolysis experiments in which any possible measures
were undertaken to exclude microbial and external proteases contaminations. The discovered
phenomenon is conceived a novel type of autoproteolytic cascade reaction.
Apparently, some of the developed Pd-NHC peptides are able to initiate the cascade of
amyloid autohydrolysis and can have an impact on the selectivity of the reaction. Independently
from the employed catalysts, a general amino endopeptidase cleavage was observed along the
hydrophobic core of the terminal modified amyloid Aβ14-24.The herein presented one-bead two
compound library methodology provides tools for the combinatorial finding and evolutionary
refinement of Pd-NHC peptide catalysts suitable for selective peptide cleavage in disease related
proteins.
En nyt Lego® byggestenstilgangen for facile inkorporering af N-heterocykliske carbener (NHC)
i peptidsidekæder sammen med supplerende ligander såsom pyridin og triazoler er blevet
udviklet. Metoden bruger CuAAC klik reaktion at indarbejde NHC forløber efter eller under
peptid samling Efterfulgt af palladium kompleksdannelse på fast støtte. Forskellige model
katalysatorer er blevet anvendt på Suzuki-koblingsreaktioner for at vurdere deres anvendelighed
i katalyse med reducerede katalysatorfyldninger under milde betingelser, og deres potentielle
anvendelse i sted-selektiv transformationer af biologiske interessante molekyler. Endvidere har
modellen katalysatorer testet for hydrolytisk aktivitet på kimdannende peptidsekvens i Amyloid-
β (1-42) for at undersøge mulige spaltningsbetingelser på vulst og i opløsning. Baseret på de
udviklede sekventiel-modulær peptid samling strategi kombinatoriske én-bead to sammensatte
katalysatorbiblioteker er blevet syntetiseret og undersøgt mod deres on-bead aktivitet i hydrolyse
af fluorescens quenchet målsekvens (Abz) A (PRA) HQKLVFFAEDVY (NO2) G i vandig
løsning. Mulige hit katalysatorer blev indikeret ved den forøgede fluorescens af individuelle
perler og underkastet linkerspaltning efterfulgt af enkelt vulst MSMS analyse. Sekvensen
bestemmelse tillod re-syntese af de formodede aktive katalysatorer, som igen blev probet mod
substratet i forskellige løsning assays indikerer betingelse afhængig hydrolytisk aktivitet.
Under anvendelse af de udviklede katalysatorer nærværende arbejde kunne ikke gives entydige
beviser for site-selektiv hydrolyse af mål-substratet da den valgte sekvens overraskende viste sig
at udvise høj autoproteolytisk aktivitet i et bredt koncentrationsområde (< 20 - 600 M) under
forskellige pufferbetingelser fra omgivelsestemperatur til 37 C som hurtig hydrolyse i
forskellige negative kontrolprøver assays foreslå.
Den heri præsenteres en perle/vulst to stofbibliotek metode giver værktøjer til den
kombinatoriske fund og evolutionær videreudvikling af Pd-NHC peptid katalysatorer egnede til
selektiv peptid spaltning i sygdomsrelaterede proteiner.
AB - A novel Lego® building block approach has been developed for facile incorporation of Nheterocyclic
carbenes (NHC) into peptide side-chains in conjunction with auxiliary ligands, such
as pyridine and triazoles. The methodology uses the CuAAC click reaction to incorporate NHC
precursors after or during peptide assembly followed by palladium complexation on solid
support. Various model catalysts were synthesized and subsequently tested for hydrolytic
activity on the nucleating peptide sequence in amyloid Aβ42 - (Aβ14-24 HQKLVFFAEDV) - to
investigate possible cleavage conditions on-bead and in solution. Based on the developed
sequential-modular peptide assembly strategy combinatorial one-bead two compound catalyst
libraries have been synthesized and probed toward their on-bead activity in hydrolyzing the
fluorescence quenched target sequence (Abz)-Pra-HQKLVFFAEDVY(NO2)G in aqueous
solution. Possible hit catalysts were indicated by the increased fluorescence of individual beads
and subjected to linker cleavage followed by single bead MS/MS analysis. The sequence
determination allowed re-synthesis of the supposed active catalysts, which were probed again
toward the substrate in various solution assays indicating condition dependent hydrolytic
activity.
Employing the evolved catalyst motifs towards the target substrate did not provide unambiguous
evidence for site-selective hydrolysis of the target substrate. The chosen sequence surprisingly
was found to exhibit high autoproteolytic activity in a wide concentration range (10 – 800 M)
under various buffer conditions from ambient temperature to 37 C. These unique observations
were repetitively made in a number of hydrolysis experiments in which any possible measures
were undertaken to exclude microbial and external proteases contaminations. The discovered
phenomenon is conceived a novel type of autoproteolytic cascade reaction.
Apparently, some of the developed Pd-NHC peptides are able to initiate the cascade of
amyloid autohydrolysis and can have an impact on the selectivity of the reaction. Independently
from the employed catalysts, a general amino endopeptidase cleavage was observed along the
hydrophobic core of the terminal modified amyloid Aβ14-24.The herein presented one-bead two
compound library methodology provides tools for the combinatorial finding and evolutionary
refinement of Pd-NHC peptide catalysts suitable for selective peptide cleavage in disease related
proteins.
En nyt Lego® byggestenstilgangen for facile inkorporering af N-heterocykliske carbener (NHC)
i peptidsidekæder sammen med supplerende ligander såsom pyridin og triazoler er blevet
udviklet. Metoden bruger CuAAC klik reaktion at indarbejde NHC forløber efter eller under
peptid samling Efterfulgt af palladium kompleksdannelse på fast støtte. Forskellige model
katalysatorer er blevet anvendt på Suzuki-koblingsreaktioner for at vurdere deres anvendelighed
i katalyse med reducerede katalysatorfyldninger under milde betingelser, og deres potentielle
anvendelse i sted-selektiv transformationer af biologiske interessante molekyler. Endvidere har
modellen katalysatorer testet for hydrolytisk aktivitet på kimdannende peptidsekvens i Amyloid-
β (1-42) for at undersøge mulige spaltningsbetingelser på vulst og i opløsning. Baseret på de
udviklede sekventiel-modulær peptid samling strategi kombinatoriske én-bead to sammensatte
katalysatorbiblioteker er blevet syntetiseret og undersøgt mod deres on-bead aktivitet i hydrolyse
af fluorescens quenchet målsekvens (Abz) A (PRA) HQKLVFFAEDVY (NO2) G i vandig
løsning. Mulige hit katalysatorer blev indikeret ved den forøgede fluorescens af individuelle
perler og underkastet linkerspaltning efterfulgt af enkelt vulst MSMS analyse. Sekvensen
bestemmelse tillod re-syntese af de formodede aktive katalysatorer, som igen blev probet mod
substratet i forskellige løsning assays indikerer betingelse afhængig hydrolytisk aktivitet.
Under anvendelse af de udviklede katalysatorer nærværende arbejde kunne ikke gives entydige
beviser for site-selektiv hydrolyse af mål-substratet da den valgte sekvens overraskende viste sig
at udvise høj autoproteolytisk aktivitet i et bredt koncentrationsområde (< 20 - 600 M) under
forskellige pufferbetingelser fra omgivelsestemperatur til 37 C som hurtig hydrolyse i
forskellige negative kontrolprøver assays foreslå.
Den heri præsenteres en perle/vulst to stofbibliotek metode giver værktøjer til den
kombinatoriske fund og evolutionær videreudvikling af Pd-NHC peptid katalysatorer egnede til
selektiv peptid spaltning i sygdomsrelaterede proteiner.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01011919679&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - On the Modular Combinatorial Synthesis of NHC Palladium Peptides and their use as Artificial Enzyme Mimics
PB - Department of Chemistry, Faculty of Science, University of Copenhagen
ER -