Carbocyclic Substrate Analogues Reveal Kanosamine Biosynthesis Begins with the α-Anomer of Glucose 6-Phosphate

Abstract

NtdC is an NAD-dependent dehydrogenase that catalyzes the conversion of glucose 6-phosphate (G6P) to 3-oxoglucose 6-phosphate (3oG6P), the first step in kanosamine biosynthesis in Bacillus subtilis and other closely-related bacteria. The NtdC-catalyzed reaction is unusual because 3oG6P undergoes rapid ring opening, resulting in a 1,3-dicarbonyl compound that is inherently unstable due to enolate formation. We have reported the steady-state kinetic behavior of NtdC, but many questions remain about the nature of this reaction, including whether it is the α-anomer, β-anomer, or open-chain form that is the substrate for the enzyme. Here, we report the synthesis of carbocyclic G6P analogues by two routes, one based upon the Ferrier II rearrangement to generate the carbocycle and one based upon a Claisen rearrangement. We were able to synthesize both pseudo-anomers of carbaglucose 6-phosphate (C6P) using the Ferrier approach, and activity assays revealed that the pseudo-αanomer is a good substrate for NtdC, while the pseudo-β-anomer and the open-chain analogue, sorbitol 6-phosphate (S6P), are not substrates. A more efficient synthesis of α-C6P was achieved using the Claisen rearrangement approach, which allowed for a thorough evaluation of the NtdC-catalyzed oxidation of α-C6P. The requirement for the α-anomer indicates that NtdC and NtdA, the subsequent enzyme in the pathway, have co-evolved to recognize the α-anomer in order to avoid mutarotation between enzymatic steps.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue

“…8,15 We recently demonstrated a carbocyclic analogue of the α-anomer of G6P was a substrate for NtdC, and the resulting product, which is incapable of the ring opening and enolization shown in Figure 1B, can act as a substrate for NtdA. 16 This confirmed all previous observations and established that these two enzymes had co-evolved so that both process the α-anomer, avoiding the observed side reactions associated with ring opening that would be required for mutarotation.…”

“…21 Methyl tri-O-benzyl glucopyranoside 5 was synthesized easily in two steps from commercially available 4,6-O-benzylidene 3 as previously reported. 16 The resulting C6 hydroxyl was converted to triflate 6 in quantitative yield, followed by displacement with either diethyl methylphosphonate or diethyl difluoromethyl phosphonate to give the desired protected glucose phosphonate 7 or 8, respectively. It is important to note that excess methyl or difluoromethyl phosphonate was difficult to remove by flash column chromatography, as it co-elutes with the product and could be removed by rotary evaporation only at higher temperatures under high vacuum for extended periods of time.…”

“…We were able to synthesize the desired phosphonate analogues of G6P according to a published procedure with minor changes (Scheme ). Methyl tri- O -benzyl glucopyranoside 5 was synthesized easily in two steps from commercially available 4,6- O -benzylidene 3 as previously reported . The resulting C6 hydroxyl was converted to triflate 6 in quantitative yield, followed by displacement with either diethyl methylphosphonate or diethyl difluoromethyl phosphonate to give the desired protected glucose phosphonate 7 or 8 , respectively.…”

“…The first step in this pathway catalyzed by NtdC, a G6P 3-dehydrogenase, oxidizes the C3 hydroxyl of G6P to generate a unique 3-keto reducing sugar. This product, 3-oxo-G6P (3oG6P), is only one of very few examples of known biologically relevant 3-keto sugars. − In solution, G6P and 3oG6P rapidly undergo mutarotation, and therefore as a result, 3oG6P transiently exists in the open-chain form as a 1,3-dicarbonyl, which is then deprotonated at C2 under alkaline conditions to give the enolate form shown in Figure B, producing a strong, characteristic ultraviolet (UV) absorbance at 310 nm. , We recently demonstrated a carbocyclic analogue of the α-anomer of G6P was a substrate for NtdC, and the resulting product, which is incapable of the ring opening and enolization shown in Figure B, can act as a substrate for NtdA . This confirmed all previous observations and established that these two enzymes had co-evolved so that both process the α-anomer, avoiding the observed side reactions associated with ring opening that would be required for mutarotation.…”

Simultaneous Broadband Suppression of Homonuclear and Heteronuclear Couplings in 1H NMR Spectroscopy

“…These methods can lead to a single signal for each chemical site, but only if no other abundant NMR‐active nuclei such as 19 F and 31 P are present in the spin system. NMR measurements on compounds containing these particular nuclei are increasingly widespread due to their importance in pharmaceuticals [5–7] and biochemistry [8,9] . 19 F and 31 P have a natural abundance of 100 % and are spin‐$${{ 1/2 }}$$ , so in 1 H NMR their heteronuclear couplings cause multiplet structure in just the same way as homonuclear couplings.…”