Biocatalytic synthesis of linear dinitriles
MeOH EtOH iPrOH
DMSO Sulfolan
THF DMC PPC DMF DMAc
0 10 20 30 40 50 60 70 80 90 100
rel. activity [%]
Cosolvent
10%
20%
30%
Figure 11: Relative activity of OxdA(C) in presence of water soluble cosolvents (for different volumetric percentages). The relative activity values correlate to a reference
activity assay with 2.5 Vol% DMSO as cosolvent.
MeOH EtOH iPrOH
DMSO Sulfolan
THF DMC PPC DMF DMAc
0 10 20 30 40 50 60 70 80 90 100 110 120
rel. acitivity [%]
Cosolvent
10%
20%
30%
Figure 12: Relative activity of OxdB in presence of water soluble cosolvents (for different volumetric percentages). The relative activity values correlate to a reference
activity assay with 2.5 Vol% DMSO as cosolvent.
MeOH EtOH iPrOH
DMSO Sulfolan
THF DMC PPC DMF DMAc
0 10 20 30 40 50 60 70 80 90 100 110 120 130
rel. activity [%]
Cosolvent
10%
20%
30%
Figure 13: Relative activity of OxdFG(N) in presence of water soluble cosolvents (for different volumetric percentages). The relative activity values correlate to a reference
activity assay with 2.5 Vol% DMSO as cosolvent.
MeOH EtOH iPrOH
DMSO Sulfolan
THF DMC PPC DMF DMAc
0 10 20 30 40 50 60 70 80 90 100
rel. activity [%]
Cosolvent
10%
20%
30%
Figure 14: Relative activity of OxdRE(N) in presence of water soluble cosolvents (for different volumetric percentages). The relative activity values correlate to a reference
activity assay with 2.5 Vol% DMSO as cosolvent.
Biocatalytic synthesis of linear dinitriles
MeOH EtOH iPrOH
DMSO Sulfolan
THF DMC PPC DMF DMAc
0 10 20 30 40 50 60 70 80 90 100
rel. activity [%]
Cosolvent
10%
20%
30%
Figure 15: Relative activity of OxdRG(N) in presence of water soluble cosolvents (for different volumetric percentages). The relative activity values correlate to a reference
activity assay with 2.5 Vol% DMSO as cosolvent.
The selected ten cosolvents ranged from polar, protic solvents like methanol, ethanol and 2-propanol over to polar, non-protic solvents like dimethyl sulfoxide (DMSO), sulfolan, tetrahydrofuran (THF), dimethyl carbonate (DMC), propylene carbonate (PPC), dimethylformamide (DMF) and dimethylacetamide (DMAc). As one can depict from the figures above, especially the whole-cell catalysts containing OxdA and OxdB showed high short-time tolerance against a broad selection of the ten cosolvents. For OxdFG, OxdRE and OxdRG, they only showed some tolerance against DMSO at 10 vol% and almost no tolerance against the other five cosolvents.
For OxdA, especially methanol and DMSO were tolerated quite well with levels of up to 20 vol%. The most promising results were obtained for OxdB. Every cosolvent, expect for THF, is short-termed tolerated with up to 20 vol%. Especially ethanol, 2-propanol and DMSO showed high potential for further investigation. Additionally, DMC was deemed to be further investigated. Based on these results, only OxdA and OxdB were further investigated.
Since most biotransformations require several hours to complete, a long-term stability study for the stability of the whole-cell catalysts against the cosolvents was necessary. The long-term study was conducted with 10 or 20 vol% of methanol or DMSO for OxdA and with 20 vol% of ethanol, 2-propanol and DMSO for OxdB. Additionally, 10 vol% of DMC were investigated for OxdB. The whole-cell suspension was incubated with the cosolvent and the standard activity assay was started after incubation times of 15, 30, 60, 120 and 180 minutes (Scheme 31, Scheme 32). The obtained activity values were set in relation to a reference activity assay in which 2.5 Vol% DMSO were used as cosolvent.
10% MeO H
20% MeO H
10% DMSO 20% DMSO 0
50 100 150 200
rel. activity [%]
Cosolvent
15 min 30 min 60 min 120 min 180 min
Scheme 31: Long-term stability study for OxdA(C).
20% EtOH 20% iPrOH 20% DMSO 10% DMC 0
50 100 150 200 250 300
rel. activity [%]
Cosolvent
15 min 30 min 60 min 120 min 180 min
Biocatalytic synthesis of linear dinitriles
The results were highly intriguing. While for OxdA the relative activity (and also overall activity) decreased slowly with DMSO over three hours, methanol led to a stronger deactivation over three hours. Moreover, DMSO seemed to increase the activity of the whole-cell catalyst compared to a reference experiment without DMSO. This may result from higher permeability of the cell membrane because of DMSO. However, this hypothesis would have to be confirmed by further experiments. Nevertheless, OxdA was stable enough in the presence of 20 vol% DMSO to continue with the studies of the α,ω-dialdoxime conversion.
Regarding OxdB, even more promising results were obtained. While 2-propanol and DMC led to a rather fast deactivation of the whole-cell catalyst, ethanol seemed to be without any negative effect on the relative activity of the whole-cell catalyst. However, one has to carefully consider that this is relative activity in comparison to a reference experiment without any cosolvent. The absolute activity of the whole-cell catalyst slowly decreased during the three hours of the experiment. The best result was obtained with DMSO. DMSO activated the whole-cell catalyst, which is in agreement with the results for OxdA.
Additionally, the relative activity of the whole-cell catalyst increased over time in the presence of DMSO: This correlates with a long-term stable, absolute activity. By reasons unknown, DMSO seems to stabilize the whole-cell catalyst.
Based on the encouraging results for the stability of the whole-cell catalyst harboring OxdA and OxdB in presence of DMSO, biotransformations on analytical scale with the eight different α,ω-dialdoxime substrates (C3-C10) were conducted (Figure 17). The concentrations of the substrates ranged from 3.0 mM to 75 mM in order to get an insight into the impact of substrate concentrations on the activity and reaction course . Due to the low solubility of the α,ω-dialdoximes in most all organic media, a solvent screening including correction factors for the extraction of the α,ω-dialdoximes and α,ω-dinitriles were determined. The most suitable solvent for extraction of both, α,ω-dialdoxime and α,ω-dinitrile, was found to be 2-methyltetrahydrofuran.
Interestingly, a very strong dependency on the carbon chain length of the substrate and the conversion by the Oxds could be observed. The C3-dialdoxime, whose dinitrile malononitrile is a well-researched compound that is broadly applied in the chemical industry[122], was not accepted at all by OxdA or OxdB. This is in good agreement with the result of the attempted desymmetrization of a prochiral 1,3-dialdoxime in the investigation of the enantioselective, biocatalytic nitrile synthesis (chapter 3).
Regarding the C4 and C5-dioximes, both Oxds did only marginally produce the α,ω-dinitrile, but instead seemed to accumulate an unkown intermediate (Scheme 33, Figure 16). This tendency was also observed in the preparative scale experiments that were conducted with both OxdA and OxdB. The consumption of the substrate was accompanied by an increasing peak in the GC chromatograms that was located directly between the peaks of the α,ω-dialdoxime and the α,ω-dinitrile. Since the C6 dioxime and the higher analogues are converted towards the α,ω-dinitrile with the same appearing intermediate peak, we postulate that this unknown intermediate may indeed be the monodehydrated species that bears one aldoxime and one nitrile moiety. As a consequence, OxdA and OxdB only seem to be able to dehydrate α,ω-dialdoximes with a chainlength of up to five atoms only once. This phenomen should be rationalized by docking studies in the near future.
Scheme 33: Attempted synthesis of succino- and glutaronitrile by biocatalytic dehydration.
Figure 16: GC-chromatograms illustrating the formation of the postulated mononitrile-monoaldoxime intermediate in the biocatalytic dehydration of α,ω-dialdoximes.
A: Reference chromatogram of pure adiponitrile and adipaldehyde dioxime; B: Overlay of
Biocatalytic synthesis of linear dinitriles
The best results for the conversion of an α,ω-dialdoxime into its α,ω-dinitrile was obtained with the C6 dioxime, adipaldehyde dioxime. Both OxdA and OxdB showed the highest activity for this substrate, reaching up to 46 mU/mgBWW for OxdA and 169 mU/mgBWW for OxdB. It should be mentioned that the calculated activity values correspond to the formation of one molecule of α,ω-dinitrile out of one molecule of α,ω-dialdoxime, including two dehydration steps. The activity values peaked at substrate concentration of 12.5 mM and did only slight decrease at higher concentrations, showing great promise for preparative biotransformations with substrate concentrations of 75 mM and higher. To the great delight of the author, adipaldehyde dioxime is the most interesting substrate since its dinitrile adiponitrile is the precursor of hexamethylenediamine (HMDA), the most used α,ω-diamine for the synthesis of polyamides, in this case of Nylon 6,6.
Interestinly, the substrate preferences of the Oxds seemed to differ once the higher analogues of the α,ω-dialdoximes were investigated. While OxdB seemed to accept the C7 dioxime almost as good as the C6 dioxime, the activity values of the C8-C10 dioximes were drastically lower (Figure 17). In contrast to these results, OxdA seemed to have a higher affinity towards the α,ω-dialdoximes with longer carbon chains. The usage of the C7-C9 dioximes led to almost the same activity values of the C6 dioxime, but seemingly led to a mediocre substrate inhibition at elevated concentration. The C10 dioxime was the only α,ω-dialdoxime that led to increasing activity values even at 75 mM concentration.
Noteworthly, the C7-C10 dioxime showed slight precipitation of the substrate at elevated concentrations, but this did not negatively impact the activity values. In its reported crystal structure, OxdA is known to have a very big hydrophobic pocket in its active site, which may be the reason why longer chain α,ω-dialdoximes are so well accepted. Unfortunately, no crystal structure has so far been reported for OxdB, which could help to understand the substrate preference of this enzyme.
Figure 17: Activity values of OxdA and OxdB in mU/mgBWW for the C3-C10 dioximes.
BWW = Bio wet weight; U-values calculated according to the conversion of one molecule of dialdoximes to one molecule of dinitrile.
Biocatalytic synthesis of linear dinitriles