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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

Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates. / Ph. D.

It is important to evaluate periodic fluctuations in environment or climate recorded through time to better understand the nature of Earth's history as well as to develop ideas about what the future may hold. There exist numerous proxies by which these environmental patterns can be demonstrated and analyzed through various time scales; from sequence stratigraphic bundles of transgressive-regressive cycles that demonstrate eustatic changes in global sea level, to the geochemical composition of a skeleton that records fluctuations in ocean temperature through the life of the biomineralizing organism. This study examines some of the methods by which we can analyze environmental fluctuations recorded at different time scales. The first project examines the methods by which extrabasinal orbital forcing (i.e. Milankovitch cycles) can be tested in the rock record. In order to distinguish these patterns, computer generated carbonate rock records were simulated with the resulting outcrops tested using common methods. These simulations were built upon eustatic sea level fluctuations with periods similar to what has been demonstrated in the rock record, as well as maintaining the many factors that affect the resultant rock composition such as tectonics, subsidence, and erosion. The result demonstrated that substantially large sea level fluctuations, such as those that occur when the planet is in an icehouse condition, are necessary to produce recognizable and preservable patterns that are otherwise overwhelmed by other depositional factors. The second project examines the temporal distribution of the bivalve Semele casali from Ubatuba Bay, Brazil by using amino acid racemization (AAR) calibrated with ¹⁴C radiometric dates. This data set is one of the largest ever compiled and demonstrates that surficial shell assemblages in the area have very long residence times extending back in time 10,000 years. The area has had very little change in sea level and the AAR ratios which are highly temperature dependent could be calibrated across sites varying from 10 to 53 meters in water depth. Long time scales of dated shells provide us with an opportunity to study climate fluctuations such as El Niño southern oscillation. The third project describes a newly developed method for estimating growth rates in organisms using closely related species from similar environments statistically analyzed for error using a jackknife corrected parametric bootstrap. As geochemical analyses get more precise while using less material, data can be collected through the skeleton of a biomineralizing organism, thus revealing information about environmental shifts at scales shorter than a year. For such studies, the rate of growth of an organism has substantial effects on the interpretation of results, and such rates of growth are difficult to ascertain, particularly in fossilized specimens. This method removes the need for direct measures of growth rates and even the most conservative estimates of growth rates are useful in constraining the age ranges of geochemical intra-skeletal studies, thus elucidating the likely time period under analysis. This study assesses the methods by which periodic environmental fluctuations at greatly varying time scales can be used to evaluate our understanding of earth processes using rigorous quantitative strategies. / Ph. D.

Functional amyloids found throughout nature have demonstrated that amyloid fibers are potential industrial biomaterials. This work introduces a new 'template plus adder' cooperative mechanism for the spontaneous self-assembly of micrometer sized amyloid fibers. A short hydrophobic template peptide induces a conformation change within a highly α-helical adder protein to form β-sheets that continue to assemble into micrometer sized amyloid fibers. This study utilizes a variety of proteins that have template or adder characteristics which suggests that this mechanism may be employed throughout nature. Depending on the amino acid composition of the proteins used the mixtures form amyloid fibers of a cylindrical (~10 μm diameter, ~2 GPa Young's modulus) or tape (5-10 μm height, 10-20 μm width and 100-200 MPa Young's modulus) morphology. Processing conditions are altered to manipulate the morphology and structural characteristics of the fibers. Spectroscopy is utilized to identify certain amino acid groups that contribute to the self-assembly process. Aliphatic amino acids (A, I, V and L) are responsible for initiating conformation change of the adder proteins to assemble into amyloid tapes. Additional polyglutamine segments (Q-blocks) within the protein mixtures will form Q hydrogen bonds to reinforce the amyloid structure and form a cylindrical fiber of higher modulus. Atomic force microscopy is utilized to delineate the self-assembly of amyloid tapes and cylindrical fibers from protofibrils (15-30 nm width) to fibers (10-20 μm width) spanning three orders of magnitude. The aliphatic amino acid content of the adder proteins' α-helices is a good predictor of high density β-sheet formation within the protein mixture. Thus, it is possible to predict the propensity of a protein to undergo conformation change into amyloid structures. Finally, Escherichia coli is genetically engineered to express a template protein which self-assembles into large amyloid fibers when combined with extracellular myoglobin, an adder protein. The goal of this thesis is to produce, manipulate and characterize the self-assembly of large amyloid fibers for their potential industrial biomaterial applications. The techniques used throughout this study outline various methods to design and engineer amyloid fibers of a tailored modulus and morphology. Furthermore, the mechanisms described here may offer some insight into naturally occurring amyloid forming systems. / Ph. D.

Ten cows were used in 4 x 4 Latin squares with an extra period for estimation of carry-over effects to study plasma amino acid responses to jugular infusion of amino acids at early, mid, and late lactation. Methionine (M), methionine + lysine (ML), and methionine + lysine + phenylalanine (MLP) in a balance with glutamic acid (C) were infused at 25% of the amino acid content of pretreatment milk via the jugular vein. Rations were formulated at 16, 15, and 14% crude protein and 15, 18, and 21% crude fiber for 30, 120, and 240 day lactational groups. Carry-over effects were present only in arterial blood at 240 days. Differences in milk production were not significant, although production increased on M, ML, and MLP at 30 days. Plasma amino acid responses of nonruminants were utilized to aid in evaluation. Arterial and venous plasma amino acid concentrations were lowest on ML at 30 days, while amino acid uptake was significantly increased by M. At 120 days plasma responses were inconclusive and non-significant. The amino acid uptake trend followed that observed at 30 days. At 240 days plasma amino acid concentrations were lowest with ML, while uptake was increased on MLP. Essential amino acids were ranked in potential orders of limitation by amino acid extraction and utilization for milk protein by the mammary gland. Order of limitation differed between calculation methods but was similar for all three stage.s of lactation. Orders of limitation were as follows: Amino acid extraction - methionine~ lysine, arginine, leucine, phenylalanine, isoleucine, threonine, valine, and histidine. Amino acid utilization - phenylalanine, threonine or histidine, valine, leucine, methionine, lysine, isoleucine, and arginine. Since there was no substantial change in order of limitation with infusion, either protein intakes were above requirements or amino acids not infused are candidates for limitation. However, among amino acids infused, the parameters observed suggested lysine or methionine at 30 days, methionine at 120 days, and no apparent choice at 240 days. / Master of Science