Studies on the biosynthesis of the various subunits of neomycins

This thesis is concerned with the elucidation of the mechanism of the formation of the various subunits of neomycins B and C. The first target was to study the epimerization process at C-5 of D-glucose during its incorporation into the neosamine B ring of neomycin B. For this purpose an unambiguous, new method for the preparation of [5-3H] glucose was developed and using this labelled species it was found that most of the tritium (90% ) from the 5-position of the glucose was lost during conversion into the neosamine B moiety. These results suggest that the epimeric centre at C-5 of the neosamine B ring is produced through an enolization process involving a carbonyl group at either C-4 or C-6. The involvement of C-4 was eliminated by demonstrating that the 3H at C-4 of [3,4-3H] D-glucose was incorporated intact into neosamine B. On the bases of these results a reaction sequence for the epimerization process is suggested. The second problem investigated in this thesis is regarding the pathway for the biosynthesis of the 2-deoxystreptamine ring of neomycins. Four compounds expected to be involved in this pathway were prepared and used for the incorporation experiments. These compounds are [6-3H]6-deoxy-5-keto-D-glucose, [5,6-3H]6-deoxy-D-glucose, 2-deoxyscylloinosose and viboquercitol. The results show that 6-deoxysugar derivatives are incorporated into 2-deoxystreptamine suggests that the elimination of the unwanted C-6 hydroxyl group of D-glucose occurs prior to or during the cyclization process. In order to shed further light on the biosynthesis of 2-deoxystreptamine, we investigated whether C-3, C-4 and C-5 are involved in this process by using [3-3H]; [4-3H]; [5-3H] D-glucose. The results from these experiments showed that the 3H atoms at C-4 and C-5 of D-glucose are lost during the biosynthesis of 2-deoxystreptamine. On the other hand, the C-3 tritium of D-glucose was retained at C-5 of 2-deoxystreptamine. The loss of C-4 tritium of D-glucose implies that C-4 of D-glucose must be involved in the biosynthesis of 2-deoxystreptamine either by conversion into a carbonyl group which is necessary for the enhancement of the acidity of C-5 hydrogen during the elimination of the unwanted -OH group at C-6 or formation of a C-4 - C-5 endiol intermediate. The elimination of the C-5 tritium of D-glucose is in accord with the notion that C-5 of D-glucose is converted to the C-3 amino group of 2-deoxystreptamine via a carbonyl intermediate. The above results were used to suggest detailed reaction sequence for the biosynthesis of 2-deoxystreptamine. (D73650/87)