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Fatty aldehyde bisulfite adducts as a purification handle in ionizable lipid synthesis.

ABSTRACT: Rapid access to ALC-0315, a crucial component of the formulated Pfizer Covid vaccine, was obtained by employing solid adduct formation and filtration after an oxidation step in place of the standard chromatographic separation, allowing for a more scalable synthesis. Impurities were removed by formation of this fatty aldehyde bisulfite adduct at the penultimate step and by performing the final reductive amination directly with the fatty aldehyde bisulfite adduct. This eliminates chromatographic separations for all prepared aldehyde containing intermediates. Along with ALC-0315, FTT5 and SM-102 ionizable lipids were prepared utilizing this strategy. This work paves the way for more sustainable access to these critical ionizable lipids that would de-risk the world supply of important vaccines and medicines in the future.

SUBMITTER: Atwood G

PROVIDER: S-EPMC11332587 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Fatty aldehyde bisulfite adducts as a purification handle in ionizable lipid synthesis.

Atwood Graham G Purbiya Sona S Reid Cassandra C Smith Brandon B Kaur Kuljit K Wicks Drew D Gaudet Peter P MacLeod K Cory KC Vincent-Rocan Jean-Francois JF

RSC advances 20240819 36

Rapid access to ALC-0315, a crucial component of the formulated Pfizer Covid vaccine, was obtained by employing solid adduct formation and filtration after an oxidation step in place of the standard chromatographic separation, allowing for a more scalable synthesis. Impurities were removed by formation of this fatty aldehyde bisulfite adduct at the penultimate step and by performing the final reductive amination directly with the fatty aldehyde bisulfite adduct. This eliminates chromatographic s ...[more]

PMID: 39161429

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Project description:To facilitate the movement of retinoids through the visual cycle and to limit nonspecific chemical reaction, multiple mechanisms are utilized to handle these molecules when not contained within the binding pocket of opsin. Vitamin A aldehyde is sequestered by reversible Schiff base formation with phosphatidylethanolamine (PE) and subsequently undergoes NADPH-dependent reduction. Otherwise inefficient handling of retinaldehyde can lead to the formation of fluorescent di-retinal compounds within the outer segments of photoreceptor cells. These bisretinoid fluorophores initiate photooxidative processes having adverse consequences for retina. Various carrier proteins confer water solubility and maintain the 11-cis-retinoid configuration. Mechanisms for sequestration of retinoid include the formation of a reversible Schiff base between retinaldehyde and taurine (A1-taurine, A1T), the most abundant amino acid in photoreceptor cells. Here we have undertaken to examine the effects of taurine depletion using the transport inhibitors guanidinoethyl sulfonate (GES) and β-alanine. Oral treatment of BALB/cJ mice with β-alanine reduced ocular A1T and the mice exhibited significantly lower scotopic and photopic a-wave amplitudes. As a secondary effect of retinal degeneration, A1T was not detected and taurine was significantly reduced in mice carrying a P23H opsin mutation. The thinning of ONL that is indicative of reduced photoreceptor cell viability in albino Abca4-/- mice was more pronounced in β-alanine treated mice. Treatment of agouti and albino Abca4-/- mice with β-alanine and GES was associated with reduced bisretinoid measured chromatographically. Consistent with a reduction in carbonyl scavenging activity by taurine, methylglyoxal-adducts were also increased in the presence of β-alanine. Taken together these findings support the postulate that A1T serves as a reservoir of vitamin A aldehyde, with diminished A1T explaining reduced photoreceptor light-sensitivity, accentuated ONL thinning in Abca4-/- mice and attenuated bisretinoid formation.

Project description:Methods: We performed mRNA sequence analysis by deep sequencing, in triplicate, using Illumina HiSeq to assess the impact of PHLPP1 knockdown in regulating gene expression in RAW 264.7 cells treated with/without OxLDL. The transcriptome libraries were constructed using the NEB adapters and were sequenced on at 150 nucleotide read length using the paired-end chemistry. The raw reads were subjected to Adapter and low-quality reads removal by Trimmomatic -0.36v. The raw reads were subjected to contamination [structural RNA / low complexity sequences] removal by mapping with bowtie 2-2.2.1. The decontaminated data set was mapped to the mouse genome. Reads mapping to gene list were counted using feature count module of sub reads package. The read counts were normalized in DESeq2-3.5, and subject to differential expression analysis. Differentially expressed genes were selected based on log2-ratio change with p-value <0.05 (Student’s t-test, unpaired). Hierarchical clustering was performed with the programs Cluster (uncentered correlation; average linkage clustering) and Treeview. Results: To gain a deeper understanding of the role of PHLPP1 in lipid metabolism in macrophages, we performed mRNA Seq analysis to assess the gene expression changes in a mouse macrophage cell line. The experiment has been carried out in triplicates. Alignment of the reads showed 97% alignment to the reference mouse genome. Identification of the differentially expressed genes were then carried out by DESeq2 version 1.2.10 with stringent criteria (FDR <0.05% with 2 fold up and down regulation) which reveals in control treated vs. control (121-down, 396-Up), Test vs. Control - (326-down, 569 Up) and Test treated vs. control treated (1314-down, 1328 up) of DEG to be altered in expression respectively. Inference: RNA-seq analysis revealed that PHLPP1 knockdown parallel with OxLDL treatment resulted in diminished expression profile of cholesterol biosynthesis and lipid metabolism genes.

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Fatty aldehyde bisulfite adducts as a purification handle in ionizable lipid synthesis.

Publications

Fatty aldehyde bisulfite adducts as a purification handle in ionizable lipid synthesis.

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