We synthesized four optical isomers [_D-(ApA), A_DpA_L, A_LpA_D, _L-(ApA)] of adenylyl-(3’-5’)-adenosine (ApA) and investigated the chemical and helical structures of the dimers by means of enzymatic digestion, circular dichroism (CD) and UV melting experiments. The results of enzymatic digestion experiments with nuclease P1, snake venom phosphodiesterase (SVPD) and RNase T₂ confirmed the chemical structures of the dimers. It is known that _D-(ApA) and _L-(ApA) form right- and left-handed helical structures, respectively [P. O. P. Ts’o et al. Biochemistry, 9, 3499-3514 (1970)]. The CD spectra of the heterochiral dimers suggested that A_LpA_D has a right-handed helical sense whereas A_DpA_L has a left-handed helical sense. This result was also confirmed by UV melting experiments of the triple helices formed by the dimers with _D-poly(U), which showed that the thermal stability of _D-(ApA)•2poly(U) and A_LpA_D•2poly(U) is much higher than that of _L-(ApA)•2poly(U) and A_DpA_L•2poly(U). Thus, the propensity of A_LpA_D to form the right-handed helical structure is similar to that of _D-(ApA), whereas _L-(ApA) and A_DpA_L have the similar propensity of resisting the formation of the right-handed helical structure. These results indicate that the chirality of the 3’-end residue is the primary factor for determining the helical sense of ApA. On the basis of the above results, the chemical evolution of RNA and the origin of the homochirality of RNA were discussed.

The homochirality of biological amino acids (L-form) or sugars (D-form) might be established before the origin of life. It has been considered that D-amino acids and L-sugars were eliminated on the primitive earth. Therefore, the presence and function of D-amino acids in living organism have not been studied except for D-amino acids in the cell wall of micro-organism. However, D-amino acids were recently found in various living higher organisms in the form of free amino acids, peptides, and proteins. This review deals with the recent advances in the various studies of D-amino acids in higher organism.

  Asymmetric automultiplication of chiral compounds by asymmetric autocatalysis is realized for the first time where a chiral product acts as a chiral catalyst for its own production. We disclosed chiral 3-pyridyl alkanol, 3-quinolyl alkanol, and 5-pyrimidyl alkanols operate as asymmetric autocatalysts in the enantioselective additions of i-Pr₂Zn to pyridine-3-carbaldehyde, quinoline-3-carbaldehyde and, pyrimidine-5-carbaldehyde, respectively. Especially, practically perfect asymmetric autocatalysis (＞99%, ＞99.5% ee) is attained using 2-alkynyl-5-pyrimidyl alkanol as an asymmetric autocatalyst in the enantioselective addition of diisopropylzinc to corresponding pyrimidine-5-carbaldehyde. In addition, asymmetric autocatalysis with amplification of ee is realized. Asymmetric autocatalyst with very low ee enhances its ee significantly up to ＞99.5% ee during the asymmetric autocatalysis without the assistance of any other chiral auxiliaries.

  Moreover, various chiral compounds with very low ee act as chiral initiators in the reaction of pyrimidine-5-carbaldehyde and diisopropylzinc to give 5-pyrimidyl alkanol with high ee in combination of asymmetric autocatalysis. Amino acids and [6]helicene with very low ee which are produced by asymmtric photolysis and photosynthesis using circularly polarized light (CPL) serve as chiral initiators of asymmetric autocatalysis, and 5-pyrimidyl alkanol with high ee is obtained. Inorganic chiral crystals such as quartz and sodium chlorate also work as chiral initiators. These results correlate for the first time the proposed origins of chirality of organic compounds such as CPL and quartz with the chirality of organic compounds with very high ee.

  Of archaea, methane-producing organisms do not possess D-enantiomers of amino acids. However, other archaea contain D-serine and D-aspartic acid. In order to confirm the presence of D-amino acids in archaea, we cultivated the hyperthermophile cells at 95℃, and searched for enzyme activities of D-amino acid production. Since a D-amino acid-dehydrogenase activity and an alanine-racemase activity were detected, we partially purified these enzymes. The optimum temperature of D-amino acid dehydrogenase was found to be 80℃, and the optimum pH was between 6.5 to 9.0. The enzyme activity was higher with D-proline as the substrate than with D-alanine. Alanine racemase activity was shown with both of the D- and L-alanine. In addition, the racemase showed some activity with L-serine, although the activity was less than 40% of that with L-alanine. The optimum temperature of the racemase was 40℃ with L-alanine as substrate, but no temperature dependency of the enzyme was observed with D-alanine.

  After some doubt, it has now been confirmed that D-amino acids occur naturally in mammalian tissues. In this review, I describe our work with D-aspartate. Immunohistochemical staining reveals that D-aspartate is found in specific cells at distinct periods during the development of rat brain, adrenal, pineal, and pituitary glands, and testis. D-Aspartate appears to be synthesized by the pituitary gland and testis and then secreted into the vascular system which transports it to tissues such as the adrenal and pineal glands, which take it up using the L-Glu transporter. The D-aspartate synthesized by the anterior lobe of the pituitary gland may stimulate prolactin-producing cells to secrete more prolactin in an autocrine and paracrine fashion. In the testis, D-aspartate produced inside the seminiferous tubules may act in a paracrine fashion on Leydig cells that reside outside the tubules to increase their testosterone production by stimulating their expression of Steroidogenic Acute Regulatory protein gene. D-Aspartate in the pineal gland, apparently primarily derived from outside the tissue, suppresses the melatonin secretion by the parenchymal cells (pinealocytes). Studies with cultured mammalian cell lines reveal that intracellular D-aspartate concentrations appears to change during the cell cycle and can be regulated by the cell density of the culture. These studies also showed that mammalian cells contain all the molecular components needed to regulate D-aspartate homeostasis, as they can biosynthesize, release, take up, and degrade D-aspartate. D-Aspartate thus appears to function in the mammalian body as a novel type of a messenger.

  _D-Amino acid oxidase (DAO) catalyzes oxidative deamination of _D-amino acids (steroisomers of naturally occurring _L-amino acids), producing 2-oxo acids, hydrogen peroxide and ammonia. In higher animals DAO is mainly present in the kidney, liver and brain. However, since its substrates (_D-amino acids) are considered very rare in higher animals, the physiological role of DAO has been enigmatic since its discovery in 1935. We are interested in why we have such a curious enzyme in our body.

  We believed that mutant animals having dysfunctions in DAO would be useful for the elucidation of the physiological function of this enzyme. We chose mice as our experimental animals due to their small size, short generation time, clear genetic background, and their close relationship to humans. A screening method for mutant mice possessing reduced levels of DAO activity was devised. Mutant mice lacking DAO activity were isolated and their strain was established.

  Genetic cross experiments between the mutant mice and normal mice indicated that mice have one gene encoding DAO and that it is expressed in the kidney and brain. The DAO gene was transmitted through the Mendelian manner. The cross experiments revealed a gene dosage effect on DAO activity: mice carrying two normal DAO alleles have twice the DAO activity than mice carrying one normal and one mutant DAO allele. The DAO gene was mapped at 65 cM on the chromosome 5 by the linkage analysis. Fluorescence in situ hybridization mapped this gene at E3-F on chromosome 5.

  The nucleotide sequence analysis of the cloned DAO cDNA revealed the presence of a nucleotide substitution in the middle of the coding region of mutant DAO. This nucleotide substitution caused a substitution of amino acid residue of DAO. The transfection and expression of the mutant and normalized DAO cDNA in cells in culture verified that the nucleotide substitution was the real cause of the loss of DAO activity.

  From the nutritional experiments using the mutant and normal mice, DAO was shown to be the most important and indispensable enzyme in the utilization of _D-amino acids in the body.

  Large amounts of amino acids were detected in the urine of the mutant mice. Most abundant alanine was mainly _D-isomer and was determined to originate from the cell walls of intestinal bacteria. Abundant methionine was also mostly _D-isomer and determined to derive from _DL-methionine supplemented in the diet of the mice. Serine was mostly _D-isomer and a portion of the _D-serine was indicated to be synthesized in the body. These _D-amino acids were not observed in the urine of normal mice. That means that they are constantly metabolized by DAO in normal mice. Therefore, we conclude that the metabolism of endogenous and exogenous _D-amino acids is the physiological role of DAO.

  Several free D-amino acids, which were thought not to exist in eukaryotes, have recently been detected in various aquatic invertebrates. In particular, free D-alanine was found in large amount (3-50 mmol/g wet wt.) in the tissues of several crustaceans and bivalve mollusks. Under high salinity stress, these animals largely accumulate D- and L-alanine irrespective of species, together with several other non-essential L-amino acids of which increases are species dependent. In these species, D- alanine is proven to be one of the major compatible osmolytes responsible for the intracellular isosmotic regulation or cell volume regulation. D-Alanine is also accumulated with L-alanine and inorganic ions in the tissues of Japanese mitten crab Eriocheir japonicus during gonadal maturation in the river and during spawning downstream migration toward the sea.

  Under hypoxia stress, red swamp crayfish Procambarus clarkii in creased D- and L-alanine in muscle and hepatopancreas in addition to the in crease of lactate. The in crease is much higher in seawater than in freshwater. Thus, D- and L-alanine are possible to be anaerobic end products during prolonged hypoxia of this species and other invertebrates.

  Alanine racemase [EC 5.1.1.1] has been proven to catalyze the interconversion of D- and L-alanine in crustaceans and bivalve mollusks and purified from two crustacean species and a mollusk. The enzyme isolated from the muscle of black tiger prawn Penaeus monodon is a dimer having molecular mass of 90 kDa. Several partial amino acid sequences of peptide fragments obtained from the isolated enzyme showed positive homologies from 52 to 76% (identity from 31 to 45%) with bacterial counterparts and a catalytic tyrosine residue of the bacterial enzyme was also retained in the prawn one, indicating that alanine racemase gene is well conserved from bacteria to invertebrates.