1,4-NADH Biomimetic Co-factors with Horse Liver Alcohol Dehydrogenase (HLADH), Utilizing [Cp*Rh(bpy)H](OTf) for Co-Factor Regeneration, Do in Fact, Produce Chiral Alcohols from Reactions with Achiral Ketones

In this Catalysts Comment Article, we will present our latest published results [...]

In his ACS Catalysis review article, Hollmann et al. [8] stated that our initial results on biomimetic NAD + co-factors, published in Angew. Chem. Int. Ed., in 2002 [5], were due to a contamination problem of the HLADH enzyme with natural NAD + (<5%), that we utilized in our studies. We then decided to publish a full account of our results, and describe our extensive control experiments, which clearly demonstrated the validity of our biomimetic NAD + and 1,4-NADH concept for HLADH enzyme recognition [1,5].
We describe, from the general procedure published in Reference 1, the biocatalytic reaction with the HLADH enzyme, NAD + biomimics 1 and 2, and the in situ formed [Cp*Rh(bpy)H](OTf). Moreover, the most decisive control experiments (1 and 2) are also described: The reported reaction conditions, such as the reactant ratio, reaction temperature, and the pre-catalyst amount, were obtained after numerous trial tests; however, the reaction conditions were not optimized. For reaction temperatures tested, from RT to 39 • C, while 30 • C exhibited the highest ee (85-99%) for the chiral alcohol products.
In his ACS Catalysis review article, Hollmann et al [8] stated that our initial results on biomimetic NAD + co-factors, published in Angew. Chem. Int. Ed., in 2002 [5], were due to a contamination problem of the HLADH enzyme with natural NAD + (<5%), that we utilized in our studies. We then decided to publish a full account of our results, and describe our extensive control experiments, which clearly demonstrated the validity of our biomimetic NAD + and 1,4-NADH concept for HLADH enzyme recognition [1,5].
We describe, from the general procedure published in Reference 1, the biocatalytic reaction with the HLADH enzyme, NAD + biomimics 1 and 2, and the in situ formed [Cp*Rh(bpy)H](OTf). Moreover, the most decisive control experiments (1 and 2) are also described: The reported reaction conditions, such as the reactant ratio, reaction temperature, and the precatalyst amount, were obtained after numerous trial tests; however, the reaction conditions were not optimized. For reaction temperatures tested, from RT to 39 °C, while 30 °C exhibited the highest ee (85-99%) for the chiral alcohol products.
Control experiment (2) [1] under the same conditions as described in the Control Experiment (1), but using twice the amounts of the ketone substrates, at room temperature, provided only racemic 2pentanol, without a measurable % ee, detected after 24 h. The yield was not determined. As a result of these control experiments, there appears to be no detectable NAD + in the HLADH used in our biomimetic reactions, and could be excluded as the responsible source for the obtained enantioselectivity in our biomimetic studies. That is, the recognition of the formed 1,4-NADH biomimics, 3 and 4, by HLADH, was clearly shown under the given conditions in our biomimetic system.
The Hollmann et al. Catalysts paper describes their results from a variety of ADH enzymes, which they contend does not provide chiral products from our biomimetic NAD + co-factors, 1 and 2, even after the ADH enzymes are extracted to remove ~<5% of natural NAD + . However, we show unequivocally that when we remove the biomimetic co-factors, 1 and 2, from the tandem biocatalysis reactions with HLADH, ketone substrates, and in situ formed [Cp*Rh(bpy)H](OTf) for biomimetic 1,4-NADH regeneration, we only see racemic alcohols, which to our mind, dictates that our samples of HLADH have ~ 0% natural NAD + .
Thus, we stand by our unequivocal control experiment results. Finally, we also noted many errors in Hollmann et al. Catalysts paper, Figure 1: We used the triflate salt, N-1-benzylnicotinamide triflate, and not the chloride salt; the Cl − anion has interfered with the formation of  Since the purchased HLADH enzyme contained sodium phosphate, and a very small amount of NAD + , per the description by Sigma (combined impurities, <5% by weight), a series of control experiments were performed to verify the possible influence of the stated NAD + impurity in HLADH, used in our biomimetic reduction reactions, for the synthesis of chiral alcohols.
Control experiment (2) [1] under the same conditions as described in the Control Experiment (1), but using twice the amounts of the ketone substrates, at room temperature, provided only racemic 2pentanol, without a measurable % ee, detected after 24 h. The yield was not determined. As a result of these control experiments, there appears to be no detectable NAD + in the HLADH used in our biomimetic reactions, and could be excluded as the responsible source for the obtained enantioselectivity in our biomimetic studies. That is, the recognition of the formed 1,4-NADH biomimics, 3 and 4, by HLADH, was clearly shown under the given conditions in our biomimetic system.
The Hollmann et al. Catalysts paper describes their results from a variety of ADH enzymes, which they contend does not provide chiral products from our biomimetic NAD + co-factors, 1 and 2, even after the ADH enzymes are extracted to remove ~<5% of natural NAD + . However, we show unequivocally that when we remove the biomimetic co-factors, 1 and 2, from the tandem biocatalysis reactions with HLADH, ketone substrates, and in situ formed [Cp*Rh(bpy)H](OTf) for biomimetic 1,4-NADH regeneration, we only see racemic alcohols, which to our mind, dictates that our samples of HLADH have ~ 0% natural NAD + .
Thus, we stand by our unequivocal control experiment results. Finally, we also noted many errors in Hollmann et al. Catalysts paper, Figure 1: We used the triflate salt, N-1-benzylnicotinamide triflate, and not the chloride salt; the Cl − anion has interfered with the formation of  Since the purchased HLADH enzyme contained sodium phosphate, and a very small amount of NAD + , per the description by Sigma (combined impurities, <5% by weight), a series of control experiments were performed to verify the possible influence of the stated NAD + impurity in HLADH, used in our biomimetic reduction reactions, for the synthesis of chiral alcohols.
Control experiment (2) [1] under the same conditions as described in the Control Experiment (1), but using twice the amounts of the ketone substrates, at room temperature, provided only racemic 2pentanol, without a measurable % ee, detected after 24 h. The yield was not determined. As a result of these control experiments, there appears to be no detectable NAD + in the HLADH used in our biomimetic reactions, and could be excluded as the responsible source for the obtained enantioselectivity in our biomimetic studies. That is, the recognition of the formed 1,4-NADH biomimics, 3 and 4, by HLADH, was clearly shown under the given conditions in our biomimetic system.
The Hollmann et al. Catalysts paper describes their results from a variety of ADH enzymes, which they contend does not provide chiral products from our biomimetic NAD + co-factors, 1 and 2, even after the ADH enzymes are extracted to remove ~<5% of natural NAD + . However, we show unequivocally that when we remove the biomimetic co-factors, 1 and 2, from the tandem biocatalysis reactions with HLADH, ketone substrates, and in situ formed [Cp*Rh(bpy)H](OTf) for biomimetic 1,4-NADH regeneration, we only see racemic alcohols, which to our mind, dictates that our samples of HLADH have ~ 0% natural NAD + .
Thus, we stand by our unequivocal control experiment results. Finally, we also noted many errors in Hollmann et al. Catalysts paper, Figure 1: We used the triflate salt, N-1-benzylnicotinamide triflate, and not the chloride salt; the Cl − anion has interfered with the formation of  Since the purchased HLADH enzyme contained sodium phosphate, and a very small amount of NAD + , per the description by Sigma (combined impurities, <5% by weight), a series of control experiments were performed to verify the possible influence of the stated NAD + impurity in HLADH, used in our biomimetic reduction reactions, for the synthesis of chiral alcohols.
Control experiment (2) [1] under the same conditions as described in the Control Experiment (1), but using twice the amounts of the ketone substrates, at room temperature, provided only racemic 2pentanol, without a measurable % ee, detected after 24 h. The yield was not determined. As a result of these control experiments, there appears to be no detectable NAD + in the HLADH used in our biomimetic reactions, and could be excluded as the responsible source for the obtained enantioselectivity in our biomimetic studies. That is, the recognition of the formed 1,4-NADH biomimics, 3 and 4, by HLADH, was clearly shown under the given conditions in our biomimetic system.
The Hollmann et al. Catalysts paper describes their results from a variety of ADH enzymes, which they contend does not provide chiral products from our biomimetic NAD + co-factors, 1 and 2, even after the ADH enzymes are extracted to remove ~<5% of natural NAD + . However, we show unequivocally that when we remove the biomimetic co-factors, 1 and 2, from the tandem biocatalysis reactions with HLADH, ketone substrates, and in situ formed [Cp*Rh(bpy)H](OTf) for biomimetic 1,4-NADH regeneration, we only see racemic alcohols, which to our mind, dictates that our samples of HLADH have ~ 0% natural NAD + .
Thus, we stand by our unequivocal control experiment results. Finally, we also noted many errors in Hollmann et al. Catalysts paper, Figure 1: We used the triflate salt, N-1-benzylnicotinamide triflate, and not the chloride salt; the Cl − anion has interfered with the formation of Since the purchased HLADH enzyme contained sodium phosphate, and a very small amount of NAD + , per the description by Sigma (combined impurities, <5% by weight), a series of control experiments were performed to verify the possible influence of the stated NAD + impurity in HLADH, used in our biomimetic reduction reactions, for the synthesis of chiral alcohols.
Control experiment (2) [1] under the same conditions as described in the Control Experiment (1), but using twice the amounts of the ketone substrates, at room temperature, provided only racemic 2-pentanol, without a measurable % ee, detected after 24 h. The yield was not determined. As a result of these control experiments, there appears to be no detectable NAD + in the HLADH used in our biomimetic reactions, and could be excluded as the responsible source for the obtained enantioselectivity in our biomimetic studies. That is, the recognition of the formed 1,4-NADH biomimics, 3 and 4, by HLADH, was clearly shown under the given conditions in our biomimetic system.
The Hollmann et al. Catalysts paper describes their results from a variety of ADH enzymes, which they contend does not provide chiral products from our biomimetic NAD + co-factors, 1 and 2, even after the ADH enzymes are extracted to remove~<5% of natural NAD + . However, we show unequivocally that when we remove the biomimetic co-factors, 1 and 2, from the tandem biocatalysis reactions with HLADH, ketone substrates, and in situ formed [Cp*Rh(bpy)H](OTf) for biomimetic 1,4-NADH regeneration, we only see racemic alcohols, which to our mind, dictates that our samples of HLADH have~0% natural NAD + . Thus, we stand by our unequivocal control experiment results. Finally, we also noted many errors in Hollmann et al. Catalysts paper, Figure 1: We used the triflate salt, N-1-benzylnicotinamide triflate, and not the chloride salt; the Cl − anion has interfered with the formation of [Cp*Rh(bpy)H](OTf) [1]. Furthermore, in their Catalysts paper, Figure 1B, the NMN we utilized, as a totally water soluble biomimetic co-factor, was a methyl ester, β-1,4-dihydronicotinamide-5 -ribose methylphosphate, 4, and was not shown; only R = H, PO 3 2− . Again, lack of details, appears to negate some of their results, if they do not use the exact same co-factors and counterions, as we used, or a similar batch of HLADH, in their comparison experiments. Incidentally, biomimetic co-factor, 4, a difficult to prepare compound, could be more easily synthesized with our procedure, as shown in reference 4 of our Inorg. Chem. 2001 paper [4], also not referenced in the Hollmann et al. Catalysts paper; it is essential to cap the phosphate group as a methyl ester, in order to avoid reaction with the precatalyst, [Cp*Rh(bpy)(H 2 O)](OTf) 2 .

Conflicts of Interest:
The authors declare no conflict of interest.