1. Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein
Julien Tailhades, Ashish Sethi, Emma J Petrie, Paul R Gooley, Ross A Bathgate, John D Wade, Mohammed A Hossain Chemistry. 2016 Jan 18;22(3):1146-51. doi: 10.1002/chem.201503599. Epub 2015 Nov 27.
The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N(α) -deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca(2+) ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.
2. p-Nitrobenzyl protection for cysteine and selenocysteine: a more stable alternative to the acetamidomethyl group
Markus Muttenthaler, Yesica Garcia Ramos, Debby Feytens, Aline D de Araujo, Paul F Alewood Biopolymers. 2010;94(4):423-32. doi: 10.1002/bip.21502.
This study evaluated the acidic lability of the acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), and the p-nitrobenzyl (pNB) as protecting groups for cysteine and selenocysteine (Sec) during the tert-butyloxycarbonyl (Boc)-chemistry solid-phase peptide synthesis of oxytocin (OT). Two novel Sec building blocks (Nalpha-tert-butyloxycarbonyl-Se(acetamidomethyl)-L-selenocysteine (Boc-L-Sec(Acm)-OH) and Nalpha-tert-butyloxycarbonyl-S(4-nitrobenzyl)-L-selenocysteine (Boc-L-Sec(pNB)-OH)) were developed for this study. Six partially protected thio- and seleno-OT analogues were synthesized, purified, and exposed to neat trifluoroacetic acid (TFA) at temperatures of 25, 40, 50, and 60 degrees C for 1 h, and HF treatment at 0 degrees C for 1 h. Significant losses were observed for the Acm and Tacm group in TFA at temperatures greater than 25 degrees C and during HF treatment at 0 degrees C, whereas the pNB group remained intact. Removal of the pNB was achieved via reduction to the p-aminobenzyl group either with zinc in acetic acid in solution or via tin chloride in hydrochloric acid on solid support, followed by oxidative cleavage with iodine yielding the corresponding disulfide or diselenide bond. No major side reactions were observed. This study confirms the occasionally described Acm instability and underpins the development of the pNB group as an alternative for cysteine and Sec protection.
3. Synthesis and antiviral activity of peptide-oligonucleotide conjugates prepared by using N alpha-(bromoacetyl)peptides
K Arar, A M Aubertin, A C Roche, M Monsigny, R Mayer Bioconjug Chem. 1995 Sep-Oct;6(5):573-7. doi: 10.1021/bc00035a011.
Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression. In order to direct oligonucleotides to specific compartments within the cell, we have investigated the possibility of coupling them to a signal peptide Lys-Asp-Glu-Leu (KDEL). This sequence should be able to convey oligonucleotides to the endoplasmic reticulum and from there to the cytosol and the nucleus where their targets are located. On this basis we prepared peptide-oligonucleotide conjugates by coupling, in a single step, a Nalpha-bromoacetyl peptide with an oligonucleotide bearing a thiol group, through a thioether bond. This paper deals with the definition of the optimal pH and temperature conditions leading to an efficient synthesis of peptide-oligonucleotide conjugates: the reaction was quantitative at pH 7.5 within few hours. This method was first set up using a 5',3'-modified dodecanucleotide and a (bromoacetyl)pentapeptide as a conjugation model. Then a 5',3'-modified pentacosanucleotide, complementary to the translation initiation region of the gag mRNA of HIV, was coupled to a (bromoacetyl)dodecapeptide containing a KDEL signal sequence. The anti-HIV activity of the pentacosanucleotide was compared with that of pentacosanucleotide-dodecapeptide conjugates linked through either a thioether bond or a disulfide bridge. The conjugate with a thioether bond has a higher antiviral activity than the peptide-free oligonucleotide and the conjugate linked via a disulfide bond.
