I.J. Galpin

Applications of immobilised phenylboronic acids as supports for group-specific ligands in the affinity chromatography of enzymes

Several different aminophenylboronic acid (APBA)-agarose matrices have been compared. Nucleotides are able to bind to these matrices in the presence of either NaCl or MgCl2. NAD+ and FAD are more tightly bound to these columns when compared with other nucleotides. The properties of NAD+ and NADP+ complexes with immobilised phenylboronic acids have been compared using pure 6-phosphogluconate and alcohol dehydrogenases. The NADP+-dependent enzyme bound to the NADP+ complex. Conversely the NAD+-dependent enzyme was only retarded by the NAD+-immobilise APBA complex Glucose-6-phosphate dehydrogenase from yeast has been partially purified on NADP+ complexed to immobilized boronic acid-agarose columns. The desorption of bound enzymes to nucleotide-phenyl boronate columns can be effected by salt gradients, the addition of diols; monosaccharides, sorbitol, cofactor competition, by lowering the pH of the eluent or by specific retardation. Purification factors of 14-fold were achieved for yeast glucose-6-phosphate dehydrogenase by choosing the appropriate concentration of presaturating NADP+. The enzyme was selectively retarded and did not require specific elution. Similar experiments enabled yeast hexokinase to be purified by presaturation of APBA-agarose with ATP. Elution of the enzyme was dependent upon the concentration of ATP used to presaturate the column. The enzyme was simply retarded by the column and required no (specific) eluent.

Peptides—XXXII: The use of phenyl esters in peptide synthesis

Abstract Phenyl esters of N-terminal amino-protected peptides are valuable intermediates in synthesis of polypeptides. The phenyl ester function is stable during the customary manipulations of chain extension, and it can be removed selectively by treatment with one equivalent of hydrogen peroxide at pH 10.5 in a variety of solvents such as 80% acetone, dimethylfonnamide, hexamethylphosphoramide or trifluoroethanol. The method has always been satisfactory providing that dimethylsulphide is used as a scavenger.

Proteinase inhibitors protect Leishmania amazonensis amastigotes from destruction by amino acid esters.

Lysosomotropic amino acid esters and amides kill Leishmania amazonensis amastigotes by a mechanism which probably involves enzymatic hydrolysis of the compounds and rapid accumulation of less permeant amino acid within the parasites. We show here that, in agreement with this model, the proteinase inhibitors antipain and chymostatin prevented the killing of intracellular and isolated parasites by L-leucine methyl ester (Leu-OMe). Survival of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by tetrazolium (MTT) reduction. Near maximal protection of intracellular parasites was obtained after 24 h incubation of macrophage cultures with 50 micrograms ml-1 antipain or chymostatin. Incubation for greater than 1 h with chymostatin or greater than 4 h with antipain alone resulted in loss of viability of the parasites. Protective activity was only slightly diminished by 20 h chase of isolated parasites in inhibitor-free medium. Two synthetic chymostatin analogues, Z-Val-Phe-Sc and Z-Ile-Phe-Sc, protected isolated amastigotes at 4 or 10 micrograms ml-1. With the exception of Trp-NH2, the toxicity of which was only minimally inhibited, antipain and chymostatin also prevented parasite destruction by other amino acid derivatives. Finally, in concentration-dependent fashion, the inhibitors reduced the accumulation of [3H]leucine in isolated amastigotes incubated with [3H]Leu-OMe. Since uptake of labelled ester was unaffected, we postulate that protection involves inhibition of the parasite enzymes which hydrolyse the amino acid derivatives.

Gel filtration of protected peptides on Sephadex G-50 in hexamethylphosphoramide containing 5% water

Gel permeation chromatography on G-50 and G-75 Sephadex gels, using 5% water in hexamethylphosphoramide (phosphoric trisdimethylamide) has been applied to the purification of large protected peptides which are outside the molecular weight range of the Sephadex LH-20-dimethylformamide system.

Solution synthesis of n-methylated peptides by the diphenyl phosphinic mixed anhydride procedure.

Abstract A proton n.m.r. method has been used to monitor racemisation during the coupling of N -methylated amino acids by the diphenylphosphinic mixed anhydride procedure. No racemisation was observed when urethane protection was employed, but use of the benzoyl group led to extensive racemisation. The reagent was subsequently used for the assembly of several extensively N -methylated peptides.

Peptides—XXXVI: Synthesis of the 27–37 fragment of a lysozyme analogue

Abstract Combination of the protected peptide fragments 1–16, 17–26 and 27–37 to yield the 1–37 portion of a lysozyme analogue is described. The fragments were combined using DCCI with the addition of HONSu, and the products purified mainly by gel filtration.

THE ACTIVATION OF N‐HYDROXY COMPOUNDS BY μ‐OXO‐BIS(TRIS(DIMETHYLAMINO)PHOSPHONIUM)‐BIS‐TETRAFLUOROBORATE

Abstract A study is made of the effect of 1-hydroxybenzotriazole on μ-oxo-bis-[tris(dimethylamino)-phosphonium]-bis-tetrafluoroborate 1 during amide bond formation. The reagent 1 is used to activate phenylhydroxamic acid towards a Lossen-type rearrangement and bring about Beckmann rearrangement of ketoximes under mild conditions. Both syn - and anti -benzaldoxime give benzonitrile by elimination.

Synthesis of two linear octapeptide fragments of cyclosporin by Stepwise and frangment condensation stratigies

The Octapeptide Z-(Me)Leu-Val-(Me)Leu-Ala-D-Ala-(Me)Leu-(Me)Leu-(Me)Val-OBu was prepared by stepwise elongation starting from H-(Me)Val-OBut. Diphenylphosphinic mixed anhydrides were preferentially used throughout as they gave high yields of optically homogeneous products in this extensively N-methylated peptide. The above peptide and the octapeptide Z-Ala-D-Ala-(Me)Leu-(Me)Leu-(Me)Val-(Me)Thr(But)-Abu-Sar-OBut were also prepared by the fragment consensation approach employing a variety of coupling methods. Ultimately, it was clear that the stepwise assembly gave the highest yield, and most homogeneous product.

Synthetic studies of cyclosporin analogues

Abstract The syntheses of eleven analogues of Cyclosporin are described; the analogues which contain (Me)Thr, (Me)Ser, Hyp and Dab at position-1 and Abu, Nva, Nle and Thr at position-2 were prepared by stepwise assembly of the undecapeptide fragments followed by cyclisation with a variety of reagents. The highest yields were obtained using the Castro reagent, and in the best case, (Hyp 1 ,Abu 2 ) cyclosporin, a yield of 65% was obtained.

Peptides-XXXXV: Synthesis of the 118–129 fragment of a lysozyme analogue

Abstract The synthesis of the (118–129) fragment of a Lysozyme analogue was achieved by the fragment coupling approach. The fragments were assembled using the DCCI/HONSu method and the (118–122), (123–126) and (127–129) subfragments were each built up in a stepwise manner. At several stages the diphenylphosphinic mixed anhydride method was found to be superior to the pivalic mixed anhydride method.