N-(9-Xanthenyl)amino acid N-carboxyanhydrides in the solid-phase synthesis of a Lys-Lys-linked cyclic leucine-enkephalin.

The side-chain-protected heptapeptide Phe-Leu-Lys(F3Ac)-Lys(F3Ac)-Tyr(Cac)-Gly-Gly is assembled by stepwise chain elongation on a polystyrene resin, using inter alia N-(9-xanthenyl)-amino acid N-carboxyanhydrides. After HBr cleavage from the resin, cyclization via carbodiimide/N-hydroxysuccinimide is carried out in a yield of 24%. Cleavage of the 9-carbazolylcarbonyl group by hydrazinolysis, and alkaline hydrolysis of the trifluoroacetyl groups, affords cyclo (-Tyr-Gly-Gly-Phe-Leu-Lys-Lys-) in an over-all yield of 12%. The structure of the peptides is confirmed by mass spectrometry.

Solid phase synthesis of oligonucleotides on a crosslinked polyacrylmorpholide support.

A suitable support for the synthesis of oligonucleotides by the phosphodi- and triester approaches was prepared by treatment of commercially available crosslinked polyacrylmorpholide beads with piperazine. The resulting polymer was used as a support for the synthesis of the self complementary decanucleotide d(T-C-G-G-A-T-C-C-G-A). An analog of dDMTrt in which one of the methoxy groups had been replaced by a carboxymethyloxy group was anchored to the polymer by an amide addition of an excess of protected nucleotide using phosphodiester methodology. Polymer supported synthesis by the phosphotriester approach of the octanucleotide d(T-T-T-T-T-T-T-T) and the hexanucleotide d(G-C-C-C-A-T) is also described. In this case 5'-O-dimethoxytritylthymidine 3'-O-monophthalate was synthesized and linked to the piperazinylated polymer. For the chain extension(3' to 5') a very simple deprotection and coupling cycle was devised.

The use of omicron-phthalaldehyde for fluorescence detection in conventional amino acid analyzers. Sub-nanomole sensitivity in the analysis of phenylthiohydantoin-amino acids.

A conventional amino acid analyzer has been modified for fluorescence detection using omicron-phthalaldehyde. The original ninhydrin system is retained, and the amino acid analyzer can be used with either of the detection systems. The fluorescence detection is used for identification of PTH-amino acids after back hydrolysis, when nanomole amounts of proteins are sequenced. Impurities in the water and the chemicals used for buffer preparations are limiting factors for the sensitivity of the fluorescence detection system. A 100-fold increase in sensitivity above the original ninhydrin system is obtained.

An improved procedure for automated Edman degradation used for determination of the N-terminal amino acid sequence of human transcobalamin I and human intrinsic factor.

An improved procedure for automated Edman degradation is presented. Three programs are described, one with double cleavage and two with single cleavage. The programs presented are characterized by a reversed delivery scheme for buffer and phenyl isothiocyanate, and by reduced cleavage times. The modified procedures applied on automated Edman degradation of the vitamin B12-binding proteins human transcobalamin I and human intrinsic factor, containing approximately 390 and 350 amino residues respectively, gave the following N-terminal amino acid sequences: Human transcobalamin I Glu-Ile-Cys-Glu-Val-Ser-Glu-Glu-Asn-Tyr-Ile-Arg-Leu-Lys-Pro-Leu-Leu-Asn-Thr-Met-Ile-Gln-Ser-Asn-Tyr-Asn-?-Gly- Human intrinsic factor Ser-Thr-Gln-Thr-Gln-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Gln-Glu-Pro-Leu-Val-Asn-Gly-Ile-Gln-?-Leu-Met-Glu-Thr- The background accumulation seems to be related not only to the length of the polypeptide chain being degraded, but also to the content of serine (and possibly threonine). A possible N leads to O acyl shift during the cleavage is a tentative explanation. The programs here represented lead to a significant reduction in background compared to conventional programs and allowed considerable prolongation of the degradations.

Catalytic oxidation of two ribonucleosides in the 5'-position.

Guanosine and 7-beta-D-ribofuranosyltheophylline was oxidized to the corresponding nucleoside 5'-carboxylic acids by molecular oxygen and a platinum catalyst.