<p> Studies on the isolation and purification of Fraction I protein from various plants are described. Clear differences in the electrophoretic mobilities of Fr I from various species were observed. The genetic implication of observations on the electrophoretic mobilities of Fr I from wheat, rye and triticales are discussed. It is suggested that a non-chromosomal gene codes for Fr I. Conclusions are drawn from the fingerprint, N-terminal amino acid, and amino acid analysis studies. The presence of ribulose diphosphate carboxylase activity in Fr I protein is also investigated.</p> / Thesis / Doctor of Philosophy (PhD)

Thesis advisor: Abhishek Chatterjee / Directed evolution is a powerful technique to expand chemical space in biological systems. In particular, this method has been used to develop cellular machinery to enable genetic code expansion (GCE), the incorporation of unnatural amino acids (UAAs) into proteins during the translation process. GCE relies on evolving an aminoacyl tRNA synthetase (aaRS) and tRNA pair from a different domain of life to incorporate a UAA into proteins in their new host, as these evolutionarily distant pairs are less likely to be cross-reactive with host pairs. The aaRS and tRNA must meet a number of conditions to be useful for GCE: the pair must be orthogonal (non-cross-reactive) to the host’s native aaRS/tRNA pairs in order to ensure site-specific UAA incorporation; the aaRS must have an active site suited to accept the shape of the UAA; and the tRNA must cooperate with the host ribosome, elongation and release factors, and other translational machinery to efficiently incorporate the UAA into the protein. Numerous aaRS/tRNA pairs have been evolved to allow incorporation of diverse UAAs in bacteria due to the tractable nature of these organisms for directed evolution experiments. While an aaRS evolved in bacteria to charge a novel UAA can be used in eukaryotes, tRNAs cannot be evolved for GCE in bacteria and then used in eukaryotes because they will not have evolved in the presence of the correct translational machinery. It is necessary to evolve tRNAs directly in their host cells. Unfortunately for researchers working on GCE in mammalian cells, it is difficult to perform directed evolution on small gene products in these hosts. Transformation efficiency in mammalian cells is poor, and transient transfection yields heterogeneous DNA distribution to target cells, making selection based on performance of individual library members impossible. Viruses are an ideal DNA delivery vector for mammalian cells, as production of recombinant viruses allows control over library member generation, and viruses can be delivered with exquisite copy number control. The Chatterjee lab recently developed a platform, Virus-Assisted Directed Evolution of tRNAs (VADER), using adeno-associated virus (AAV) to evolve tRNAs for GCE directly in mammalian cells. While VADER is the first directed evolution platform that allows the evolution of small gene products in mammalian cells, its efficiency is limited by its continued reliance on transient transfection to deliver non-library DNA that is necessary for the production of rAAV. To overcome this limitation, baculovirus delivery vectors were developed to boost DNA delivery and AAV capsid production to improve virus production efficiency during selections. VADER allows the evolution of tRNAs to incorporate certain UAAs, but the technique relies on installing a UAA into the AAV capsid, which is sensitive to disruption caused by slight modifications in structure. To expand the scope of VADER to evolve tRNAs for UAAs that cannot be incorporated into the AAV capsid, an alternate selection handle (Assembly Activating Protein, or AAP) was deleted from the genome and provided in trans to incorporate 5-hydroxytryptophan (5HTP). Incorporating the UAA into this flexible protein allows UAA-dependent production of AAV and expands the scope of tRNAs that can be evolved in mammalian cells. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23843号 / 薬博第850号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 木村 郁夫, 教授 中山 和久, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

In the study of structurally restricted cyclic β-amino acids and peptides, methanopyrrolidine-5-carboxylic acids (MetPyr-5-acids), or 5-syn-carboxy-2-azabicyclo[2.1.1] hexanes, and derivatives were investigated. MetPyr-5-acids are a series of highly conformationally constrained β-proline derivatives, which belong to a novel category of β-amino acids utilized as building blocks for the synthesis of β-peptides. These β-peptides lack the backbone hydrogen bonds necessary for folding in the usual manner. Substituents and functional groups in this ring system were envisioned to impact the folding properties and functionalities of the corresponding β-peptides. In the present study, the analogues of MetPyr-5-acids with C6- substitutions were prepared, and the folding properties of their peptides were explored. To introduce different functionalities at C6 in MetPyr-5-acids, 6-syn-hydroxymethyl substituted derivatives were synthesized and were used as key intermediates. In the synthesis of this core structure, the major steps in their preparation included the Michael addition of benzyloxymethyl allyl amine to 3-butynone, followed by UV light irradiation of the diene to afford 5-acetyl-6-benzyloxymethyl-2-azabicyclo[2.1.1]hexane. Haloform (Br2/NaOH) oxidation of the acetyl group leads to the 6-substituted MetPyr-5-acid. Resolution of the racemate was achieved either by resolving (±)-6-syn-benzyloxymethyl-MetPyr-5-acid via a classical crystallization resolution method using (S)-(-)-α-methylbenzylamine, or by chiral preparative HPLC separation of (±)-6-syn-benzyloxymethyl-MetPyr-5-acid methyl ester. The absolute stereochemistry was confirmed by X-ray crystallography of a derivative. Novel analogs with a range of functionalities incorporated at the C6 position in MetPyr-5-acid were synthesized from 6-syn-hydroxymethyl-MetPyr-5-acid methyl ester, and include hydrophilic groups such as hydroxyl, amino, methyl ether, and hydrophobic groups, such as substituted phenyl groups and triazole. From the protected C6-substituted analogs of MetPyr-5-acids, peptide oligomers of C6-benzyloxymethyl-2,4-methanopyrrolidine-b-amino acid were prepared up to the length of octomer in high yields. This series of oligomers were characterized by circular dichroism (CD) and indicated enhanced order of folding uniformity for the tetramer and up, with increasing ordered folding for longer oligomers. The octomer exhibited minimal solvent effects, and was stable with increasing temperature up to 80 °C. Analysis by NMR of the iso-butyric amide capped monomer indicated a mixture of cis/trans conformation favoring the cis conformation. This was slightly different from the C6 unsubstituted iso-butyric amide derivative, which favored the trans conformation. For the dipeptide, the C6-benzyloxymethyl substitution increased the percentage of cis conformation of the dipeptide amide bond, but the major peptide had the trans conformation. This demonstrated that C6 substitutions could shift the cis/trans equilibrium towards the cis conformation. Longer oligomers showed ordered secondary folding structure as demonstrated by the increase in ellipticity per amino acid unit, but was too complicated to be determined by NMR analysis. Both the CD patterns and molecular model calculation predicted that the longer oligomers (tetramer and above) favor the trans conformation. This preference was driven by the backbone dipole effect. II. SYNTHESIS OF 2,2-DISUBSTITUTED PYRROLIDINE-3-CARBOXYLIC ACIDS Due to the perceived steric influence of 2,2-disubstitution in the pyrrolidine-3-carboxylic acid, it is believed that the adjacent amide/peptide bonds should result in a trans amide bond conformation. Because of the difficulty in introducing disubstitution at the hindered C2 position, the synthesis of such derivatives has not been successful. For this reason a new method was introduced to prepare novel derivatives, at the N- and C- termini of protected 2,2-dimethyl pyrrolidine-3-carboxylic acid, i.e., benzyloxycarbonyl protected 2,2-dimethylpyrrolidine-3-carboxylate. This procedure included the Michael addition of 2-nitropropane to dimethyl fumarate, followed by ring closure of the amino ester derived from reduction of the nitro ester providing the pyrrolidinone. Reduction of the pyrrolidinone to the pyrrolidine with borane finished 2,2-dimethylpyrrolidine-3-carboxylate in moderate overall yield. A preliminary set of two amides, iso-butyric amide and 3,5-dichlorobenzamide of this 2,2-dimethylpyrrolidine-3-carboxylate, were also prepared. NMR analysis of this pyrrolidine derivative suggested the amide bonds adopted the trans conformation. It was concluded that steric bulk of the 2,2-disubstitution favorably influenced the trans amide conformation. This demonstrated that trans amide conformation control of a β-proline amide was possible. / Chemistry