Chiral interaction in Gly-capped N-terminal motif of 3(10)-helix and domino-type induction in helix sense

Chiral interaction of helical peptide with chiral molecule, and concomitant induction in its helix sense have been demonstrated in optically inactive nonapeptide (1) possessing Gly at its N-terminus: H-Gly-(Delta(Z)Phe-Aib)(4)-OCH(3) (1: Delta(Z)Phe = Z-dehydrophenylalanine; Aib = alpha-aminoisobutyric acid). Spectroscopic measurements [mainly nuclear magnetic resonance (NMR) and circular diochroism (CD)] as well as theoretical simulation have been carried out for that purpose. Peptide 1 in the 3(10)-helix tends to adopt preferentially a right-handed screw sense by chiral Boc-L-amino acid (Boc: t-butoxycarbonyl). Induction in the helix sense through the noncovalent chiral domino effect should be derived primarily from the complex supported by the three-point coordination on the N-terminal sequence. Thus the 3(10)-helical terminus consisting of only alpha-amino acid residues enables chiral recognition of the Boc-amino acid molecule, leading to modulation of the original chain asymmetry. Dynamics in the helix-sense induction also have been discussed on the basis of a low-temperature NMR study. Furthermore, the inversion of induced helix sense has been achieved through solvent effects.     

Terminal effect of chiral residue on helical screw sense in achiral peptides

To understand the terminal effect of chiral residue for determining a helical screw sense, we adopted five kinds of peptides I – V containing N‐ and/or C‐terminal chiral Leu residue(s): Boc–L ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( I ), Boc–(Aib–ΔPhe)₂–L ‐Leu–OMe ( II ), Boc–L ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( III ), Boc–D ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( IV ), and Boc–D ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( V ). The segment –(Aib–ΔPhe)₂– was used for a backbone composed of two “enantiomeric” (left‐/right‐handed) helices. Actually, this could be confirmed by ¹H‐nmr [nuclear Overhauser effect (NOE) and solvent accessibility of NH resonances] and CD spectroscopy on Boc–(Aib–ΔPhe)₂–Aib–OMe, which took a left‐/right‐handed 3₁₀‐helix. Peptides I – V were also found to take 3₁₀‐type helical conformations in CDCl₃, from difference NOE measurement and solvent accessibility of NH resonances. Chloroform, acetonitrile, methanol, and tetrahydrofuran were used for CD measurement. The CD spectra of peptides I – III in all solvents showed marked exciton couplets with a positive peak at longer wavelengths, indicating that their main chains prefer a left‐handed screw sense over a right‐handed one. Peptide V in all solvents showed exciton couplets with a negative peak at longer wavelengths, indicating it prefers a right‐handed screw sense. Peptide IV in chloroform showed a nonsplit type CD pattern having only a small negative signal around 280 nm, meaning that left‐ and right‐handed helices should exist with almost the same content. In the other solvents, peptide IV showed exciton couplets with a negative peak at longer wavelengths, corresponding to a right‐handed screw sense. From conformational energy calculation and the above ¹H‐nmr studies, an N‐ or C‐terminal L ‐Leu residue in the lowest energy left‐handed 3₁₀‐helical conformation was found to take an irregular conformation that deviates from a left‐handed helix. The positional effect of the L ‐residue on helical screw sense was discussed based on CD data of peptides I – V and of Boc–(L ‐Leu–ΔPhe)_n–L ‐Leu–OMe (n = 2 and 3). © 1999 John Wiley & Sons, Inc. Biopoly 49: 551–564, 1999     

Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis

Monoclonal antibody MI315 was produced against hamster tooth germ homogenate by in vitro immunization. It was found that MI315 reacted with enamel matrix, ameloblasts, and bone matrix at an early stage of osteogenesis. Decalcified tissues of rat femurs and mandibles were examined with MI315 using indirect immunofluorescence. In endochondral ossification of femurs, immunoreactivity was found in bone extracellular matrix (ECM) deposited on the surface of the cartilage core of primary spongiosa, but not in the cartilage core itself. In intramembranous ossification of 0-day-old rat mandibles, intense immunofluorescence was detected in bone ECM and a few young osteocytes, but not in osteoblasts. Immunoreactivity in bone ECM of 2-day-old rats decreased and almost disappeared from bone ECM of 4-day-old rats. Although in nondecalcified sections of 0-day-old rats, negligible immunofluorescence was detected in bone ECM which showed positive staining in decalcified tissues, the immunostaining appeared after decalcification using ethylenediaminetetraacetic acid (EDTA). These results indicate that a substance(s), which had a common epitope with an enamel-derived protein(s), existed in immature bone ECM of both endochondral and intramembranous ossification, and that it might be masked by bone mineral. Monoclonal antibody MI315 is a useful tool to investigate the time- and position-specific changes in osteogenesis and amelogenesis.     

Identification of TMCO2 as an acrosome‐associated protein during rat spermiogenesis

We isolated the transmembrane and coiled‐coil domains 2 (Tmco2) gene using a polymerase chain reaction‐based subtraction technique. Tmco2 is predominantly expressed in rat testes starting from 4 weeks of age. Rat TMCO2 consists of 187 amino acids with a predicted molecular mass of 20.6 kDa. When expressed in COS7 cells, TMCO2 was found as vesicle‐like structures in the cytoplasm, whereas TMCO2ΔTM lacking the transmembrane (TM) region was found diffused in the cytoplasm. These results suggest that the TM region in TMCO2 is essential for its specificity of localization. Immunocytochemical analyzes indicated that rat TMCO2 was localized as small semiluminate bodies or cap‐like structures in the vicinity of round spermatid nuclei and as curved lines associated with nuclei of elongated spermatids and caput epididymal spermatozoa. However, it was detected in only a small part of cauda epididymal spermatozoa. Double immunolabeling of the spermatids and spermatozoa with the anti‐TMCO2 antibody and the monoclonal anti‐MN7 antibody showed that TMCO2 was predominantly associated with the inner acrosomal membrane in spermatids and caput epididymal spermatozoa. Our findings suggest that TMCO2 might be involved in the process of acrosome biogenesis, especially binding of acrosome to a nucleus, during spermiogenesis.     

Divergent synthesis of kinase inhibitor derivatives,leading to discovery of selective Gck inhibitors

We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors.