F. J. Leeper, Abstracts 60-69

60
K. Ichinose, M. Kodera, F. J. Leeper and A. R. Battersby
"Proof that the Biosynthesis of Vitamin B₁₂ Involves a Reduction Step in an Anaerobic as well as an Aerobic Organism"
J. Chem. Soc., Chem. Commun.,1993, 515-517. Full Text

Labelling experiments with ²H and ³H prove that as cobyrinic acid is biosynthesised by the anaerobic bacterium Propionibacterium shermanii, its H-19 is derived from H_R (and not H_S) at C-4 of a reduced nicotinamide cofactor, so demonstrating that the biosynthetic pathway involves a reduction step.

61
K. Ichinose, F. J. Leeper and A. R. Battersby
"Preparation of [4R-³H]NADH, [4R-³H]NADPH and the Corresponding 4S-Isomers All with Substantial Specific Activities"
J. Chem. Soc., Perkin Trans. 1, 1993, 1213-1216. Full Text

Methods are developed for the enzymic preparation of [4R-³H]NADH, [4R-³H]NADPH and the corresponding 4S-isomers, all stereospecifically labelled and of specific activities amply high enough for sensitive labelling experiments. Sodium borotritiide provides the tritium for all four cofactors.

62
F. J. Leeper
"Evidence for a Spirocyclic Intermediate in the Formation of Urogen III by Cosynthetase"
in The Biosynthesis of the Tetrapyrrole Pigments, 1994, J. Wiley & Sons, Chichester, p. 111-123. Full Text

In the course of the cyclization of the linear tetrapyrrole hydroxymethylbilane to uroporphyrinogen III, catalysed by uroporphyrinogen III synthase (cosynthase), ring D of the bilane becomes inverted. Many different mechanisms have been proposed for this transformation but the most economical is one involving a spirocyclic pyrrolenine. Synthesis of a spirolactam, and other compounds closely related to the spirocyclic pyrrolenine, has shown that such compounds are not impossibly strained. The spirolactam is a powerful inhibitor of the enzyme, which suggests it does resemble an intermediate in the enzymic process. In the synthetic procedure to make an ester of the spirolactam the two products obtained were initially thought to be conformational isomers. However, molecular mechanics calculations on a model of the spirolactam predicted that several low energy conformations should exist and that the energy barriers for their interconversion are all lower than 32 kJ/mol. Reinvestigation revealed that one of the two products is in fact a macrocyclic dimer with a 28-membered ring. On the basis of the predicted preferred conformations of the spirolactam and of uroporphyrinogen III, a detailed three-dimensional mechanism is proposed, along with a rationalization of how the rearrangement of ring D may be directed by the enzyme.

63
F. J. Leeper, S. E. Shaw and P. Satish
"The Biosynthesis of Cyclizidine: Incorporation of Singly and Doubly Labelled Precursors"
Can. J. Chem., 1994, 72, 131-141. Full Text

Incorporation of isotopically labelled forms of CH₃CO₂Na and CH₃CH₂CO₂Na into the indolizidine alkaloid cyclizidine, produced by Streptomyces species NCIB 11649, shows that the oxygen attached to C-2 is derived intact from acetate and that the cyclopropyl ring is derived from a single intact propionate unit. However, the level and stereochemistry of the incorporation of deuteriated sodium propionate indicates that it undergoes unexpected modification during incorporation into the cyclopropyl ring.

64
W. M. Stark, C. J. Hawker, G. J. Hart, A. Philippides, P. M. Petersen, J. D. Lewis, F. J. Leeper and A. R. Battersby
"Biosynthesis of Porphyrins and Related Macrocycles, Part 40. Synthesis of a Spiro-Lactam Related to the Proposed Spiro-Intermediate for Porphyrin Biosynthesis: Inhibition of Cosynthetase"
J. Chem. Soc., Perkin Trans. 1,1993, 2875-2892. Full Text

Routes are developed for synthesis of the tripyrrolic macrocyclic spiro-lactam 39. A minor product from the synthesis, thought earlier to be an atropisomer, has been shown by molecular mechanics calculations and re-investigation to be a dimer. The octa-acid derived from 39 closely resembles the spiro-pyrrolenine proposed as a biosynthetic intermediate for uroporphyrinogen III. This octa-acid acts as a strong inhibitor of cosynthetase (uroporphyrinogen III synthase) whilst other similar systems which lack some of its functionality do not. These results strongly support the view that the spiro system is indeed the biosynthetic intermediate for formation of uroporphyrinogen III from hydroxymethylbilane.

65
R. A. Vishwakarma, S. Balachandran, A. I. D. Alanine, N. P. J. Stamford, F. Kiuchi, F. J. Leeper and A. R. Battersby
"Biosynthesis of Porphyrins and Related Macrocycles. Part 41. Fate of Oxygen Atoms as Precorrin-2 Carrying Eight Labelled Carboxyl Groups (¹³C¹⁸O₂H) is Enzymatically Converted into Cobyrinic Acid"
J. Chem. Soc., Perkin Trans. 1,1993, 2893-2899. Full Text

5-Amino[1,4-¹³C₂]laevulinic acid and 5-amino[1-¹³C]laevulinic acid are synthesised and all three ¹⁶O atoms of the latter are exchanged for ¹⁸O. The ¹³C,¹⁸O-labelled material is then converted in vitro into precorrin-2 by the combined action of four genetically overproduced enzymes. The product is isolated in its aromatised form, sirohydrochlorin and ¹³C-NMR shows that all 8 carboxyl groups retain both oxygen atoms throughout the biosynthesis. A cell-free enzyme preparation from Propionibacterium shermanii converts the ¹³C,¹⁸O-labelled sirohydrochlorin via precorrin-2 into cobyrinic acid, a late precursor of vitamin B₁₂. 13C-NMR proves that 6 carboxyl groups of cobyrinic acid (b-g, inclusive) retain both oxygen atoms whereas the a-carboxyl group undergoes specific loss of one labelled oxygen atom.

66
A. I. D. Alanine, K. Ichinose, D. Thibaut, L. Debussche, N. P. J. Stamford, F. J. Leeper, F. Blanche and A. R. Battersby
"Biosynthesis of Vitamin B₁₂: Use of Specific ¹³C-Labelling for Structural Studies on Factor IV"
J. Chem. Soc., Chem. Commun.,1994, 193-196. Full Text

[1,10,20-¹³C₃]Uro'gen III is unambiguously synthesised for enzymic conversion into precorrin-4 isolated after aerial oxidation as two epimers of Factor IV, the ¹³C NMR spectra of which rigorously confirm the presence of a C-1 acetyl group in precorrin-4; attachment of the fourth methyl group at C-17 of precorrin-4 is also confirmed by related ¹³C-labelling experiments.

67
K. E. Holt, F. J. Leeper and S. Handa
"Synthesis of beta-1-Homonojirimycin and beta-1-Homomannojirimycin using the Enzyme Aldolase"
J. Chem. Soc., Perkin Trans.1,1994, 231-234. Full Text

The four stereoisomers of the four-carbon azido sugar 11 have been stereoselectively synthesised by a route involving Sharpless epoxidation and all are found to be substrates for rabbit muscle fructose 1,6-bisphosphate aldolase, giving (after treatment with phosphatase) 6-azido-6-deoxyheptuloses 14; hydrogenation of 14a and 14b gave beta-1-homomannojirimycin 15a and beta-1-homonojirimycin 15b with high selectivity.

68
S. Balachandran, R. A. Vishwakarma, S. M. Monaghan, A. Prelle, N. P. J. Stamford, F. J. Leeper and A. R. Battersby
"Biosynthesis of Porphyrins and Related Macrocycles, Part 42. Pulse Labelling Experiments Concerning the Timing of Cobalt Insertion During Vitamin B₁₂ Biosynthesis"
J. Chem. Soc., Perkin Trans. 1,1994, 487-491. Full Text

Pulse labelling experiments using ⁶⁰Co²⁺ and [methyl-¹³C,¹⁴C]-S-adenosyl-L-methionine have established that in the anaerobic organism Propionibacterium shermanii cobalt is inserted into the macrocycle after the second and before the fourth C-methylation step.

69
I. M. Bell, C. Abell and F. J. Leeper
"Design and Synthesis of Transition State Analogues for a Cationic Cyclisation"
J. Chem. Soc., Perkin Trans.1,1994, 1997-2006. Full Text

Transition-state analogues based upon the 6-(hydroxymethyl)-13- azagona-1,3,5(10),8-tetraene structure have been designed and synthesized as part of a programme to elicit antibodies capable of catalysing cationic cyclisations. Methodology for conjugating such analogues to proteins has also been developed.

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