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Winner of the IUPAC Prize
for Young Chemists - 2003

Christoph Schaffrath wins one of the 5 IUPAC Prize for Young Chemists, for his Ph.D. thesis work entitled "Biosynthesis and Enzymology of Fluorometabolite Production in Streptomyces Cattleya."

Current address

StylaCats Ltd.
The Robert Robinson Laboratories
Department of Chemistry
University of Liverpool
Liverpool L69 7ZD, United Kingdom

E-mail: christophschaffrath@yahoo.co.uk

Academic degrees

  • Ph.D. in Bio-Organic Chemistry, University of St. Andrews, Scotland, Dec. 2002
  • Diplom Ingenieur Applied Chemistry and Biotechnology, University of Applied Science, Emden, Germany, Aug 1998

Ph.D. Thesis

Title Biosynthesis and Enzymology of Fluorometabolite Production in Streptomyces Cattleya.
Adviser Prof. David O'Hagan
Thesis Committee Prof. Jim Naismith, University of St. Andrews Scotland, UK; Prof. Tim Bugg, University of Warwick, England, UK

Essay

Organohalogens were once considered to be very rare or even bizarre compounds, as it was believed that the few early examples were not of natural origin but artifacts of the isolation process. With only 30 compounds isolated in 1968 [ref. 1], the reported number in the latest review in early 1999 [ref. 2] was 3200. Although there has been a vast increase in the number of isolated organohalogens, only 13 of these secondary metabolites contain a fluorine atom [ref. 3].

As fluorine is the most abundant halogen in the Earth's crust and ranks 13th in abundance of all of the elements, the small number of isolated natural organofluorine compounds is rather surprising. However, this can be explained by three characteristics of the fluoride ion. Fluoride exists mainly as the tightly bound form in minerals (e.g. fluorspar) making it highly insoluble in water and therefore restricting its bioavailability to living organisms. Secondly, due to its high heat of hydration, the fluoride ion is a poor nucleophile in aqueous solution thereby limiting its participation in displacement reactions. Finally, fluorine cannot be incorporated into organic compounds via the haloperoxidase [ref. 4] reaction since the redox potential required for the oxidation of fluoride is much greater than that generated by the reduction of hydrogen peroxide.

Therefore, the mechanism by which C-F bonds are formed in biological systems is of considerable interest. Additionally, an understanding of the mechanism of enzymatic C-F bond formation could provide new methods for stereospecific incorporation of fluorine into pharmaceutically important compounds.

The aim of my thesis was to investigate the biosynthesis of fluoroacetate and 4-fluorothreonine in the actinomycete Streptomyces cattleya (Scheme 1) using a combination of chemical synthesis, isotopic labelling experiments, enzymatic studies and protein purification. The way by which fluorine is inserted into these compounds was unknown before the research described in this thesis.

Scheme 1 Biosynthesis of 4-fluorothreonine and fluoroacetate in the bacterium S. cattleya when batch cultures are grown in the presence of fluoride ion [ref. 5].

Earlier work by Hamilton et al. [ref. 6] and Moss [ref. 7] had established that fluoroacetaldehyde is the direct precursor to fluoroacetate and that there was a biosynthetic relationship between fluoroacetate and 4-fluorothreonine. In order to investigate the role of fluoroacetaldehyde as the common precursor for both fluorometabolites and to demonstrate its direct involvement in 4-fluorothreonine biosynthesis, [1-2H]-fluoroacetaldehyde was efficiently synthesised in three steps and subsequently administered to resting cells of S. cattleya. Analysis of the experiment using GC-MS and 19F NMR showed a substantial incorporation (34 %) of single 2H into the C-3 and C-4 fragment of 4-fluorothreonine, clearly indicating fluoroacetaldehyde as the common intermediate in the biosynthesis of both fluorometabolites.


Scheme 2 Deuterium incorporation into 4-fluorothreonine from feeding studies using [1-2H]-fluoroacetaldehyde.

Since the aldehyde dehydrogenase enzyme responsible for the oxidation of fluoroacetaldehyde to fluoroacetate was already known [ref. 8], my subsequent goal was to identify the enzyme responsible for the transformation of fluoroacetaldehyde to 4-fluorothreonine. In a series of experiments using cell-free extract of S. cattleya, it was found that the second substrate for the formation of 4-fluorothreonine is L-threonine and that the reaction is strictly dependent on the co-factor pyridoxal phosphate (PLP). The enzyme was partially purified and mechanistic investigations were carried out using isotopically labelled precursors.

It was found that the discovered enzyme, threonine transaldolase, can only accept L-threonine as substrate, thereby representing a new class of aldolase enzyme not previously described. All known threonine aldolases have a specificity for glycine and use this solely with acetaldehyde to produce threonine. Furthermore, an earlier proposal that 4-fluorothreonine is derived from glycine and fluoroacetaldehyde5 was disproved with the discovery of the threonine transaldolase. On the basis of these studies, a minimal mechanism for the action of the enzyme was proposed, which is outlined in the Scheme below.


Scheme 3 Proposed pyridoxal phosphate (PLP) catalysed mechanism for the enzymatic formation of 4-fluorothreonine from fluoroacetaldehyde and L-threonine by threonine transaldolase.

Clearly, the main objective of my PhD was to discover the enzyme responsible for the formation of a C-F bond. Although several research groups have been working on this challenge for more than 40 years, there has been little progress made and proposed mechanisms for biological fluorination are all speculative. During the course of my PhD, I found that cell-free extract of S. cattleya, when incubated with S-adenosyl methionine (SAM) and fluoride ion, was able to synthesise a previously unobserved organofluorine compound, which we proposed to be 5'-fluoro-5'-deoxyadenosine, generated by nucleophilic attack of the fluoride ion on the C-5' carbon of SAM (Scheme 4).


Scheme 4 Proposed mechanism for the formation of the identified organofluorine compound.

However, isolation by semi-preparative HPLC determined the structure to be 5'-fluoro-5'-deoxyinosine, which was found to be a shunt product biosynthesised by the action of a deaminase on the initial fluorination product, 5'-fluoro-5'-deoxyadeosine. The production of 5'-fluoro-5'-deoxyadenosine by the fluorinase enzyme present in S. cattleya was confirmed by incubating cell-free extract with synthetic 5'-fluoro-5'-deoxyadenosine, which led to the accumulation of fluoroacetate and 4-fluorothreonine.

The enzyme was efficiently purified to homogeneity from the wildtype strain in 4 steps using ammonium sulfate precipitation, hydrophobic interaction chromatography, gel-filtration chromatography and anion exchange chromatography. The enzyme was found to be a hexamer with the molecular mass of approximately 180-190 kDa.

As a potential application of the fluorinase enzyme, positron emission tomography was investigated in collaboration with GlaxoSmithKline and it was shown that partially purified enzyme was capable of generating 5'-[18F]-fluoro-5'-deoxyadeosine after incubation with SAM and an aqueous solution of [18F]HF. The enzymatic radiofluorination of an organic molecule is a novel route to the incorporation of 18F and could have potential applications in the future.

In summary, my research gave a major insight into the biosynthesis of fluoroacetate and 4-fluorothreonine in the bacterium S. cattleya. Fluoroacetaldehyde was identified as the common precursor of both fluorometabolites and the enzyme responsible for the biotransformation of fluoroacetaldehyde to 4-fluorothreonine was identified and isolated.

The isolation and purification of the fluorinase enzyme represents my greatest achievement. The enzyme is the first of its class and represents a major breakthrough in the unknown area of enzymatic fluorination. The fluorinase enzyme now provides a system with which to study the enzymatic syntheses of organofluorine compounds at a mechanistic level and also opens the prospects of biotransformation routes to the important group of fluorinated pharmaceutical compounds.

References
1. G.W. Gribble, J. Chem. Educ., 1994, 71, 907.
2. G.W. Gribble, Chem. Soc. Rev., 1999, 28, 335.
3. M. Sanada, T. Miyano, S. Iwadare, J.M. Williamson, B.H. Arison, J.L. Smith, A.W. Douglas, J.M. Liesch and E. Inamine, J. Antibiotics, 1986, 39, 259.
4. S.L. Neidleman and J. Geigert, 'Biohalogenation: Principles, basic roles and applications', Ellis Horwood Ltd, Chichester, 1986.
5. M. Sanada, T. Miyano, S. Iwadare, J. M. Williamson, B. H. Arison, J. L.Smith, A. W. Douglas, J. M. Liesch and E. Inamine, J. Antibiotics, 1986, 39, 259.
6. J.T.G. Hamilton, C.D. Murphy, M.R. Amin, D. O'Hagan and D.B. Harper, J. Chem. Soc., Perkin Trans. 1, 1998, 759.
7. S.J. Moss, PhD Thesis, 1999, University of Durham.
8. C.D. Murphy, S.J. Moss and D. O'Hagan, Appl. Environ. Microbiol., 2001, 40, 4919.


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