1. Mechanistic Study of Diketopiperazine Formation during Solid-Phase Peptide Synthesis of Tirzepatide
Jingyao Wang, Mark R Berglund, Timothy Braden, Matthew C Embry, Martin D Johnson, Stephen R Groskreutz, Fareed Bhasha Sayyed, Sergey Vladimirovich Tsukanov, Timothy D White, Ankur Jalan, Kevin D Seibert, Michael E Kopach ACS Omega. 2022 Dec 6;7(50):46809-46824. doi: 10.1021/acsomega.2c05915. eCollection 2022 Dec 20.
This study focused on investigating diketopiperazine (DKP) and the formation of associated double-amino-acid deletion impurities during linear solid-phase peptide synthesis (SPPS) of tirzepatide (TZP). We identified that the DKP formation primarily occurred during the Fmoc-deprotection reaction and post-coupling aging of the unstable Fmoc-Pro-Pro-Ser-resin active pharmaceutical ingredient (API) intermediate. Similar phenomena have also been observed for other TZP active pharmaceutical ingredient (API) intermediates that contain a penultimate proline amino acid, such as Fmoc-Ala-Pro-Pro-Pro-Ser-resin, Fmoc-Pro-Pro-Pro-Ser-resin, and Fmoc-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-resin, which are intermediates for both hybrid and linear synthesis approaches. During post-coupling aging, it is found that Fmoc deprotection can proceed in dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and acetonitrile (ACN) solvents without any piperidine addition. Density functional theory (DFT) calculations showed that a peptide that has a penultimate proline stabilizes the transition state through the C-H···π interaction during Fmoc decomposition, which causes those peptides to be more prone to cascade-deprotection reactions. Pseudo-reaction pathways are then proposed, and a corresponding macrokinetics model is developed to allow accurate prediction of the TZP peptide intermediate self-deprotection and DKP formation rate. Based on those studies, control strategies for minimizing DKP formation were further investigated and an alternative to Fmoc protection was identified (Bsmoc-protected amino acids), which eliminated the formation of the DKP byproducts. In addition, the use of oxyma additives and lower storage temperature was demonstrated to markedly improve the peptide intermediate stability to DKP degradation pathways.
2. Theragra chalcogramma Hydrolysates, Rich of Fragment Gly-Leu-Pro-Ser-Tyr-Thr, Ameliorate Alcohol-Induced Cognitive Impairment via Attenuating Neuroinflammation and Enhancing Neuronal Plasticity in Sprague-Dawley Rats
Defeng Xu, Mouming Zhao J Agric Food Chem. 2022 Oct 5;70(39):12513-12524. doi: 10.1021/acs.jafc.2c05163. Epub 2022 Sep 26.
Chronic alcohol abuse induces the cognitive deficits and is associated with low-grade inflammation and neurodegeneration. Currently, by virtue of the immunomodulatory and neuroprotective properties, nutrients represent a promising strategy to attenuate cognitive impairments. We previously prepared the hydrolysates from Theragra chalcogramma skin (TCH), and this study aims to evaluate the neuroprotection of TCH on alcohol-induced cognitive impairment (AICI) and to elucidate the associated mechanism. Behavioral results showed that TCH effectively ameliorated AICI and this amelioration was highly associated with the decrease of IL-1β and the increase of BDNF, CREB, and PSD95 in AICI rats (P < 0.05). Furthermore, TCH restored the histopathological impairment in hippocampus by reactivating extracellular signal-regulated kinase and suppressing Caspase-3 apoptosis signal pathways and modulating the abnormality of neurotransmitters acetylcholine and γ-aminobutyric acid(P < 0.05 or 0.01). Therefore, TCH exhibits potent attenuation of neuroinflammation and represents a potential ingredient for prevention of AICI.
3. Theragra chalcogramma Hydrolysates, Rich in Gly-Leu-Pro-Ser-Tyr-Thr, Exerts Anti-Photoaging Potential via Targeting MAPK and NF-κB Pathways in SD Rats
Defeng Xu, Mouming Zhao, Haisheng Lin, Caihong Li Mar Drugs. 2022 Apr 24;20(5):286. doi: 10.3390/md20050286.
Previous studies have revealed that excessive exposure to UV irradiation is the main cause of skin photoaging and the signaling pathways of MAPK and NF-κB are involved in this progression. The present study aims to investigate the anti-photoaging effects of low molecular weight hydrolysates from Theragra chalcogramma (TCH) and to clarify the underlying mechanism. The degradation of mechanical barrier functions in photoaged skin was substantially ameliorated after TCH administration; meanwhile, TCH significantly elevated the antioxidant capacity and suppressed the over-production of inflammatory cytokine IL-1β. Moreover, the histopathological deteriorations such as epidermal hyperplasia and dermal loss were significantly alleviated, along with the increase in procollagen type I content and decrease in MMP-1 activity (p < 0.05). Furthermore, TCH effectively blocked the MAPK and NF-κB signaling pathways through inhibition of the phosphorylation of p38, JNK, ERK, iκB, and p65 proteins. Collectively, these data indicate that TCH has potential as a novel ingredient for the development of anti-photoaging foods.
