Keywords: D-amino acids, bifunctional reagent, diastereomers l-fluoro-2,4-dinitrophenyl-5-L-alanine amide has been synthesized in high yield (76%) from 1,5-difluoro-2,4-dinitrobenzene and L-AIa-NH2. This compound contains a reactive fluorine atom which can be used for the reaction with a mixture of L-and D-amino acids. The resulting diastereomers which are obtained in quantitative yield can be separated and estimated by HPLC. With the five amino acids studied (Ala, Asp, Glu, Met and Phe), L-diastereomers were eluted from the reverse-phase column before D-diastereomers. This behavior can be explained by a stronger i ntramolecular hydrogen bonding in the latter diastereomer. When artificial mixtures of the five amino acids containing known proportions of L-and D-isomers were derivatized with the reagent and the reaction products analyzed by HPLC, it was possible to determine the relative content of each isomer in a nanomole range.

Ultraviolet thermal denaturation studies substantiate our earlier hypothesis that substitution of a L-nucleotide residue for a D-nucleotide within a DNA duplex permits a stable structure in which all bases are paired through Watson-Crick hydrogen bonds (Damha, M. J., Giannaris, P. A., Marfey, P., & Reid, L. S. (1991) Tetrahedron Lett. 32, 2573-2576). This conclusion is also evident from the NMR work of Blommers et al. [Blommers, M. J. J., et al. (1994) Biochemistry (following paper in this issue)]. Our thermal denaturation studies indicate that, while weakening the interaction with target DNA and RNA, these substitutions allow for excellent cooperative binding. When the target is single-stranded DNA, the melting temperature of the complex is lowered by 4-5 degrees C per L-dU incorporation and by 0.4-2.6 degrees C when an internal D-dC is replaced by L-dC (1 M NaCl). When the target is RNA, the depression of Tm is also greater for L-dU substitutions (5-8 degrees C) than for L-dC substitutions (2-4 degrees C). The depressions of Tm caused by introducing A/C and G/T mismatches at the same positions were significantly greater. L/D-DNA chimeras were found to activate RNase H cleavage when hybridized to RNA. Furthermore, the stability of chimeric L/D-DNA against degradation by various commercial phosphodiesterases was found to be significant, as was their stability against digestion in human serum. These experiments establish that L/D-DNA chimeras serve as excellent models of antisense oligonucleotides.

No abstract

L-and D-amino acids were treated with fluorodinitrobenzene to form DNP-derivatives. The latter were esterified with methanol in the presence of dry HCI gas to the corresponding methyl esters. Treatment of the ester mixture with carboxypeptidase-Y resulted in the hydrolysis of only L-esters. All D-esters and some L-esters were unaffected, The produced DNP-L-amino acids could be separated from DNP-D-amino acid methyl esters by high performance Iiquid chromatography aIIowing quantitation of the D-isomers, When a test mixture consisting of AIa, Asp, Glu, Met and Phe, present in different proportions as 1,-and D-isomers, was subjected to the above procedure, all D-amino acids could be quantilated. The method allows detection of D-amino acids in the nanomole range.

SummaryHamster platelets have been separated from their plasmatic enviroment by means of gel filtration. This method of separation gave an excellent yield of platelets free of non-adsorbed plasma proteins.Gel filtered platelets (GFP), and platelets washed by repeated centrifugations and resuspensions (CP) were compared to each other and to platelets left undisturbed in their native plasma (PRP). Only slight morphological differences between the three platelet preparations could be detected by phase microscopy. In the concentration range of .28–.71 μM ADP, GFP, resuspended in PPP, showed the same degree of aggregation as did PRP. At lower concentrations of ADP, GFP aggregated to a lesser degree than did PRP. CP exhibited aggregation comparable to PRP only at higher concentrations of ADP (.71–1.42 (μM ADP). GFP also supported clot retraction to the same extent as did PRP.It would appear that gel filtration provides a new, gentle and rapid method for separation, from plasma, of platelets with activity very similar to that of platelets in PRP.