An activated triple bond linker enables ‘click’ attachment of peptides to oligonucleotides on solid support

Abstract

A general procedure, based on a new activated alkyne linker, for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition (‘click’ reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into ‘clickable’ azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.

Synthesis and evaluation of stability of m3G-CAP analogues in serum-supplemented medium and cytosolic extract

“…The reaction was vortexed and then shaken in an eppendorf tube at rt for four days. The product was purified by RP-HPLC using triethylammonium acetate buffer pH 6 Stock solutions of compounds 1-3 in water were prepared so that 72 lL of each solution contained 0.07 ODU (at 254 nm). 72 lL of the respective stock solution of 1, 2 or 3, and 300 lL of cell medium or cytosolic extract (containing 1.3 mg/mL of total protein) were then mixed.…”

“…Modifications of the m 3 G-CAP structure should ensure long enough survival of the m 3 G-CAP part of the therapeutic construct so that it can pass biological media (blood) leave behind the cytosolic compartment and enter the cell nucleolus where therapeutic splice-switching or anti-gene action takes place. Here we present a study on the stability of m 3 G-CAP analogues possessing methylene modifications in the triphosphate bridge since these modifications may give improved enzymatic stability, should not introduce steric hindrance upon binding to snurportin and should not interfere with Cu catalyzed attachment 5,6 to the therapeutic splice-switching oligonucleotides.…”

Sequence-selective DNA recognition and enhanced cellular up-take by peptide–steroid conjugates

“…The PATA linker has been specifically developed for bioconjugation purposes as an active alkyne for preparation of peptide-oligonucleotide conjugates via CuAAC. 28 Functionalization of the steroid nucleus was performed by Steglich esterification, affording final scaffolds (1-3). In case of PATA as linker (4), the diamino derivative of deoxycholic acid proved necessary for the coupling of the linker, as esterification gave rise to byproducts due to the high reactivity of the alkyne.…”

“…However, reaction proceeds well with excess of copper ion complex and the copper ions can be readily removed after reaction by use of EDTA. 28 An excess of scaffold was also required for complete reaction of the peptide, the dipodal construct being favoured over the monopodal one under these conditions. The reaction was complete after 3 hours at room temperature and compatible with the presence of all unprotected amino acids in the sequence.…”

On-Resin Conjugation of Diene–Polyamides and Maleimides via Diels–Alder Cycloaddition

“…8,9 The addition of Cu(I)-stabilizing entities accelerates the reaction and prevents formation of oxidizing species 10 and has been key in the preparation of all types of conjugates linked through stable 1,2,3-triazoles. However, complete elimination of the metal catalyst may be difficult to achieve, 11,12 and optimization of reaction conditions may be highly demanding. 13,14 Suitably modified strained alkynes (such as cyclooctynes) react with azides in the absence of Cu(I), 15,16 in other words in biocompatible conditions, but the reaction gives a mixture of regioisomers.…”