Transketolase Catalyzed Synthesis of N-Aryl Hydroxamic Acids Inés Fúster Fernández,a Laurence Hecquet,b and Wolf-Dieter Fessnera,* a Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany E-mail: fessner@tu-darmstadt.de b Institut de Chimie de Clermont-Ferrand, CNRS Auvergne Clermont INP, Université Clermont Auverne, 63000, Clermont- Ferrand, France Manuscript received: September 6, 2021; Revised manuscript received: October 29, 2021; Version of record online: December 2, 2021 Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.202101100 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Abstract: Hydroxamic acids are metal-chelating compounds that show important biological activity including anti-tumor effects. We have recently engineered the transketolase from Geobacillus stearothermopilus (TKgst) to convert benzaldehyde as a non-natural acceptor substrate. Realizing the structural and electronic similarity to nitrosobenzene, we studied the TK-catalyzed conversion of nitrosoarenes to yield N-arylated hydroxamic acids. Here we demonstrate that wild-type and variants of this versatile TKgst enzyme indeed induce the rapid biocatalytic conversion of variously p-, m- and o-substituted nitrosoarenes to produce a variety of corresponding N-aryl hydroxamic acids via creation of a carbon-nitrogen instead of a carbon-carbon bond. Further structural modifications can be introduced by varying the donor component, such as hydroxypyruvate or pyruvate. Keywords: biocatalysis; C� N ligation; hydroxamic acid; iron(III) chelation; nitrosoarenes Introduction Hydroxamic acids (HA) are a well-studied class of compounds that is of importance in many different areas of life sciences.[1] Their O=C� N� OH function- ality can act as a bidentate ligand that forms strong chelate complexes with different metal ions. This chelating capacity confers high pharmacological versa- tility that includes anti-cancer, anti-inflammatory, anti- bacterial, anti-fungal, anti-malarial, anti-tubercular and anti-oxidant activities. HA can be utilized for combat- ing accidental metal poisoning by complexation, e. g. treatment with deferoxamine B in case of iron(III) overload. They also can interact with zinc(II) and nickel(II) centers, which are among the most common catalytically active metal cofactors in metalloenzymes such as histone deacetylases (HDAC), rendering HA drugs attractive for the treatment of various cancer types.[2] For example, the aryl HA-based HDAC inhibitor Abexinostat (Figure 1A) shows nanomolar in vitro activity and is used alone or in combination[3] against a broad array of cancers.[4] Several related antitumor drugs also carry a terminal HA functionality (1) derived from a carboxylic acid precursor (3; Fig- ure 1B). Although different synthesis strategies have been developed,[5] HAs (1) are usually synthesized by chemical acylation of hydroxylamine with an activated carboxylic acid derivative, which can also be effected enzymatically using lipase, nitrilase, or amidase catalysis.[6] Recently, it was reported that HAs (1) can also be accessed by direct amidation of aldehydes with nitroso compounds via N-heterocyclic carbene catalysis.[7] Interestingly, hydroxamates having an inverse con- stitution (2; Figure 1B), derived from coupling an N- alkylated/arylated hydroxylamine with formic acid (or related acyl units), are less common although these can be expected to offer very similar metal binding capacity. To the best of our knowledge, this type of HA (2) also seems not to have been tested yet for RESEARCH ARTICLE doi.org/10.1002/adsc.202101100 Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 612 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 612/621] 1 http://orcid.org/0000-0002-9787-0752 https://doi.org/10.1002/adsc.202101100 http://crossmark.crossref.org/dialog/?doi=10.1002%2Fadsc.202101100&domain=pdf&date_stamp=2021-12-02 biomedical applications. Here, we present an approach to the latter kind based on an enzymatic nucleophilic acyl transfer to nitrosoarene electrophiles catalyzed by transketolase (TK, EC 2.2.1.1). TK is a thiamine diphosphate (ThDP) dependent enzyme that creates asymmetric carbon-carbon bonds in vivo by reversibly transferring a two-carbon ketol unit among various sugars phosphates.[8] In vitro, hydroxypyruvate (HPA) is preferentially utilized as the nucleophilic component because its decarboxylation shifts the reaction equili- brium towards product generation.[9] As aldehyde electrophiles, various aldose sugars and α-hydroxylated aliphatics can replace the natural phosphorylated substrates,[10] while non-hydroxylated aliphatic or aromatic aldehydes require enzyme engineering to enhance TK activity and stereoselectivity.[11,12] Because of its high structural similarity to benzal- dehyde, which was previously investigated by our group[13] for acyloin formation,[14] we reasoned that TK could be able to accept nitrosobenzene (5a) (and related nitrosoarenes) as an alternative electrophilic substrate (Scheme 1). Such an operation would give rise to the formation of N-arylhydroxamic acids via creation of carbon-nitrogen instead of carbon-carbon bonds. This method would be complementary to the electrophilic acylation of N-arylhydroxylamines, and yield HA structures (2) complementary to the conven- tional type (1) (Figure 1B). Here we demonstrate that suitable TK variants can indeed efficiently catalyze the nucleophilic acyl addition from HPA to nitrosoarenes, and illustrate the scope of HAs (2) accessible by this route. Results and Discussion Nitrosobenzene as a Transketolase Substrate We have recently developed the first thermostable TK from Geobacillus stearothermopilus (TKgst) as a cata- lyst with very promising properties for synthetic applications.[15] TKgst offers enhanced resistance to- wards unconventional media, extended catalyst life- time at elevated temperatures and considerable robust- ness to protein mutagenesis. We have engineered this TKgst by directed evolution for improved conversion of aliphatic and arylated aldehydes,[14] as well as for modified stereoselectivities.[16] The TKgst variant that gave the best performance for ketol addition to benzaldehyde contained the L382N/D470S mutations (short: N/S). The stability of this variant against thermal unfolding was measured by nanoDSF,[17] which showed its melting temperature to be only slightly lower than that of the wild-type enzyme (Tm (N/S): 73.9 °C, Tm (wild-type): 75.5 °C). For the first tests with commercial nitrosobenzene (5a) we employed this TKgst variant (N/S) in the presence of its cofactors ThDP and Mg2+ in triethanol- amine (TEA) buffer at pH 7.5 using HPA as the ketol donor (Scheme 1b). Because of the hydrophobic nature of nitrosobenzene and its low aqueous solubility, we used DMSO as co-solvent as successfully used for preparative conversion of benzaldehyde.[14] Because of the limited stability of HPA solutions,[18] substrates were added in portions with monitoring of the reaction progress by HPLC. To our delight, the reaction was extremely fast even with low enzyme quantities, and identical results were obtained when adding all reagents at once. After an acid/base extractive work-up followed by column chromatography over silica, the desired product was isolated and identified via NMR. Furthermore the TLC, HPLC and NMR comparison of the generated HA with an authentic sample of N- phenyl-N,2-dihydroxyacetamide 6a, which was pre- pared by chemical synthesis following a literature Figure 1. A) FDA approved arylated hydroxamate drug Abex- inostat for the treatment of follicular lymphoma; B) Synthesis of isomeric hydroxamates 1/2 from benzoic acids or anilines. M=metal ion; R=CH3, CH2OH. Scheme 1. Comparison of the general TKgst catalyzed reaction of benzaldehyde (A) versus nitrosoarenes (B) in the presence of HPA. Newly formed C� C bond is shown in green, new C� N bond in red. RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 613 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 613/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de procedure,[19] further confirmed the enzymatic method to be successful. Nitrosoarene Substrate Scope To probe the reaction scope, we produced a set of variously mono- and disubstituted nitrosoarenes (5a– p), which were generated from commercial anilines (4) by oxone oxidation according to a literature protocol.[20] Although several by-products were formed concomitantly such as nitro-, azo- and azoxybenzenes in different compositions,[21] these derivatives were not considered relevant for the enzymatic conversion of the nitroso components because of the higher chemical stability of the former. The nitrosoarene compounds were obtained with approximately 80–90% purity and, in view of their relatively high volatility, were used directly in the enzymatic reaction without further purification. With the set of nitroso compounds at hand (5b–p), we attempted to conduct the enzymatic synthesis of the corresponding HA under the same conditions as for the parent (Scheme 2). Monitoring conversion by HPLC analysis indicated that almost all nitroso substrates became converted to some degree except for 5p. Products were isolated by column chromatography for spectroscopic characterization. Expectedly, the reactivity of nitrosoarenes in the TK catalyzed ketol addition seemed to be controlled by both electronic and steric factors. The π-conjugated nitroso group becomes more electrophilic by electron withdrawing arene substituents, which raises reaction rates and product yields with a Hammett-like trend. Substitution in p-position is well tolerated, whereas m-/ o-substitution seem to impose increasing steric compli- cations to fit the active site substrate channel (e.g., HA yields for 6c>6d>6e). Yields with twofold substitu- tion were lower than the corresponding single sub- stitution patterns (e. g., HA yields for 6f<6c/6d). In aqueous solution, electron poor nitrosoarenes are also more susceptible to chemical redox processes, low- ering HA yields and rendering isolation from compet- ingly formed hydroxylamine, nitro-, azo- and azox- ybenzenes difficult. For example, in case of 5e, HPLC showed conversion of starting material to give two peaks in the absence of enzyme; addition of TK clearly triggered the formation of an additional peak. TLC analysis revealed the corresponding additional spot to turn red upon contact with FeCl3 stain (vide infra), proving the product to be the desired HA (6e) if only in much lower concentration than the para- and meta- isomers. The strongest electron withdrawing p-NO2 group also yielded mainly side products, but HPLC and TLC indicated formation of small amounts of HA (6m) that could not be isolated. Probably, these products would need optimized reaction conditions to enhance the amounts and facilitate isolation. In line with the difficult chemical reactivity of p-nitro-sub- stituted 6m, the electron poor p-CF3-substituted 6k gave a lower HA proportion. In comparison, the corresponding m-substituted 6 l is favoured despite aggravated steric effects, indicating a negative impact of too strong electron withdrawal on the enzymatic versus chemical reaction selectivity. Carboxylic acid substituents are only moderately resonance active groups but also improve substrate solubility. Both p- and m-substituted nitroso benzoates were converted and yielded the corresponding HA. Unfortunately, purification of the highly polar products 6n and 7n by column chromatography resulted in large loss of material while side products could not completely be removed. In contrast, strong electron donating groups retard the rate of the reaction such that the p-dimethylamino substituted 5p did not give any detectable HA product. This observation is in agreement with early kinetic studies using yeast TK and fructose 6-phosphate as ketol donor.[19] However, electronically different p-CH3 (5g) and p-Cl (5c) substitution both gave very similar results with our TKgst from HPLC analysis and isolated product yields. Also, both regioisomeric, m,p-substi- tuted chloro-nitrosotoluenes 5 i and 5j showed very similar yields. In comparison, the dichloro substituted 5f led to a significantly lower yield despite its similar steric situation, revealing the lower reaction selectivity induced by a deactivating substituent. Reaction Engineering The initially developed reaction conditions were in need of improvement to upgrade the product yields. Attempts for a continuous feeding strategy to avoid substrate evaporation and decomposition were not met with success, e.g., adding 50 mM of compound 5b dissolved in 2 mL of DMSO over 5h by means of a syringe pump, to a final reaction volume of 25 mL (at a rate of 0.4 ml/h, i. e. 20% of the total amount per hour), rather resulted in significant increase of by- products percentage and lower HA yield. Larger co- solvent concentration (10%) to aid the aqueous dissolution of the nitrosoarenes did not significantly improve the HA yields but in fact demanded higher work-up efforts for DMSO removal involving column chromatography for product purification. Ethanol (8%) Scheme 2. General synthesis of hydroxamic acids by ketol addition to nitrosoarenes catalyzed by TKgst. RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 614 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 614/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de had been previously used as co-solvent for yeast TK reactions,[19] but our results were unsatisfactory, possi- bly due to the fact that aqueous alcoholic solutions promote the substrate decomposition.[22] Therefore, various alternative co-solvents were tested at different concentrations and varying temper- atures (25–50 °C). To avoid material loss from evapo- ration of nitrosobenzene (5a) experiments were con- ducted with p-bromonitrosobenzene (5b), which showed very similar reactivity but is less volatile. Considering consumption rate of starting material and selectivity of HA formation, the best results from this solvent screen were obtained at 25 °C with DMF, acetonitrile, acetone and DMSO (Figure 2). The four best co-solvents were tested up to a final 20% concentration, taking advantage of the high tolerance of the thermostable TKgst towards organic solvents.[17,35] Here, acetone resulted in the best overall results because this co-solvent also allowed for a significantly simplified work-up (Table 1, method B). Although not tested on the entire range of substrates, the optimized protocol is likely to lead to improved HA yields for all less soluble starting materials. We also tested the relative rate advantage of using the mutant versus wild-type TKgst activities in the production of 6b. Reactions were performed in acetone (20%) as co-solvent at 25 °C in the presence of the TKgst wild-type or N/S variant (0.2 mg/mL) against a control reaction without enzyme, and product forma- tion was monitored by HPLC analysis. In the absence of enzyme, no HA formation was detectable. The N/S variant resulted to be almost 7-fold faster in producing the desired HA than the wild-type TKgst already within the first 5 minutes, which also led to less decom- position of the starting material. After 30 min the variant had reached almost complete conversion (98% conversion) while the wild-type enzyme had converted around half of the starting material (57% conversion). This proves the significant advantage of using the engineered variant even at very low enzyme concen- trations. Figure 2. Co-solvent screen (10%) for conversion of 5b (50 mM) at 25 °C. Assay solution (250 μL) contained Li-HPA (50 mM), ThDP (2.4 mM), MgCl2 (9 mM), triethanolamine (TEA) buffer (50 mM, pH 7.5) and TKgst N/S (0.1 mg). Table 1. Enzymatic synthesis of HA from nitrosoarenes 5. Nitroso arene Aryl substitution[a] Acyl residue HA product Reaction conditions[b] Yield (%)[c] 5a H CH2OH 6a A/B 41/50 5b 4-Br CH2OH 6b A/B 28/54 5c 4-Cl CH2OH 6c A 41 5d 3-Cl CH2OH 6d A 20 5e 2-Cl CH2OH 6e A n.i. 5 f 3,4-Cl2 CH2OH 6 f A 10 5g 4-CH3 CH2OH 6g A 49 5h 3-CH3 CH2OH 6h A 29 5 i 4-Cl, 3-CH3 CH2OH 6 i A 37 5 j 3-Cl, 4-CH3 CH2OH 6 j A 32 5k 4-CF3 CH2OH 6k A 9 5 l 3-CF3 CH2OH 6 l A 17 5m 4-NO2 CH2OH 6m A n.i. 5n 4-COOH CH2OH 6n A n.i. 5o 3-COOH CH2OH 6o A n.i. 5p 4-N(Me)2 CH2OH 6p A 0 5a H CH3 7a A 5 5n 4-COOH CH3 7n A n.i. [a] Substituent positions relative to the nitroso group. [b] Conditions A: co-solvent DMSO; B: co-solvent acetone, [c] isolated yields; n.i.: product formation verified by iron complex formation but compound was not isolated. RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 615 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 615/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de Chelate Complex Formation with Iron (III) Ions The most striking property of the HA is their ability to chelate iron (III) ions (Scheme 3). Particularly, in alkaline media the dissociable HA (pKa~9) favors a stable hexacoordinate, fully 3:1 liganded structure.[23] Thus, we tested the iron complex formation as a method for an unequivocal identification of the HA formed and for monitoring the HA synthesis reaction. Mixing equal quantities of reaction mixture or pure product solution with 0.5% aqueous FeCl3 indeed immediately gave an intense red-violet color for all HA samples in the product array (Figure 3A). The utility of such a test could also be demonstrated for detection of the HA array upon TLC analysis (Fig- ure 3B). Expanding the Scope to Other Nucleophiles Because TK was reported to be highly specific for the ketol transfer reaction,[12,24] we recently engineered TKgst variants by directed in vitro evolution that were able to accept pyruvate (PA) as an analogous non- hydroxylated donor substrate.[25] We decided to probe the capacity of those variants to also convert nitro- soarenes, considering the apparent high kinetic reac- tivity of the latter and the sensitive product detection principle based on the iron (III) staining. For an evaluation we applied the TKgst variant H102L/H474S (short: L/S) that had shown best overall properties.[25] We first proved by nanoDSF analysis that the engineered TKgst variant had maintained the high stability against thermal unfolding (Tm (wild- type): 75.5 °C and Tm (L/S): 75.6 °C), which correlates with co-solvent tolerance.[17] From the first test conducted with 5a indeed formation of the unsubstituted HA 7a could be detected and verified by the ferric chloride staining of the TLC sheet (Scheme 4). For a preparative scale synthesis significant larger enzyme quantities were required for conversion of pyruvate with the (L/S) variant relative to HPA conversion using the (N/S) variant. At least 5-fold amounts of (L/S) enzyme had to be applied and the isolated yield of 7a turned out to be rather low (Table 1). The identity of the enzymati- cally formed HA was confirmed against an authentic standard prepared by chemical synthesis of N-phenyl- N-hydroxy-acetamide according to a literature procedure.[26] Interestingly, HPLC analysis of the negative control revealed that there was a small chemical background formation of 7a also in the absence of enzyme, although much smaller than with the (L/S) variant. This phenomenon was also later observed for nitrosoarenes bearing electron-withdraw- ing substituents and may be initiated by a redox reaction involving PA, but not with the electron deficient HPA substrate. With the aim to evaluate, and further broaden the application scope of the enzymatic acyl transfer from pyruvate, the remaining substrate array 5b–l was tested for conversion by TKgst (L/S) with PA as donor. Product formation was monitored via HPLC and verified by TLC staining with FeCl3 as before. Due to the rather low efficiency of these conversions, the resulting HA products 7b–k were not isolated and characterized. However, the relative Rf migration pattern in RP-HPLC is directly comparable to that of the product array obtained from the HPA reaction, except for an offset due to the slightly lower polarity expected from the lack of one hydroxyl group (Fig- ure 4). Overall, this initial screening confirms that at least the TKgst (L/S) variant is capable not only to efficiently Scheme 3. Colored chelate complex formation between HA and FeCl3. Figure 3. (A) 96-Well microtiter plate for analysis of chelate complex formation. Top: MeOH solution of purified HA array (6a–l) compared to 7a from chemical synthesis; center: 0.5% aqueous FeCl3; bottom: both solutions equally mixed. (B) TLC plate spotted with the same HA array after development with cyclohexane/ethyl acetate (1:4) and staining with FeCl3. Scheme 4. General reaction of nitrosoarenes (5a–p) with PA catalyzed by TKgst to yield hydroxamic acids (7a–p). RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 616 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 616/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de accept nitrosoarenes as electrophilic substrates but also PA as a donor substrate analogue. Possibly, the yield could be further improved by optimization of the reaction conditions. However, it seems obvious that in order to truly expand the reaction scope towards a combination of two non-native substrate components, that is, for both aromatic electrophiles and PA (or other 2-oxoacids different from HPA), novel engineered TK variants are required. The use of nitrosoarenes as benzaldehyde surrogates coupled to a novel colorimet- ric screening procedure based on the formation of HA- iron chelate complex offers a promising outlook. Chemical Side Product Formation During the reaction engineering we also tested con- tinuous addition of the nitroso compound via syringe pump, however, this resulted in the rise of side product formation. Closer analysis revealed the unexpected formation of N-(4-bromophenyl)formamide (9b, ca. 3%). The non-enzymatic generation of formanilide 9p had been previously observed by reductive formylation of 5p with glyoxylic acid (GA; Scheme 5),[27] which plausibly occurs via the corresponding HA precursor (8p).[28] Indeed, control reactions with 5a/5b and GA in the absence of enzyme catalysis cleanly gave the HA products 8a/8b in good yields (70/79%).[27] However, in our case 9b was only formed in the presence of TKgst and HPA. The mechanism is still unclear, because no potential intermediate 8b was observed and the formation of GA from HPA is yet unknown. To summarize our observations for non-enzymatic HA formation from nitrosoarenes with ketoacid com- ponents, GA reacted spontaneously, PA produced small amounts of HA very slowly, whereas HPA did not produce any HA in the absence of enzyme even upon extended incubation times. Also, no HA formed in the absence of a donor substrate. This confirms that HA formation is strongly structure dependent and that TK catalysis is an exclusive opportunity for conversion of HPA. Conclusion Reflecting the high structural and electronic similarity of nitrosobenzene and benzaldehyde, wild-type and variants of the versatile TKgst enzyme were shown to accept nitrosoarenes as alternative electrophilic sub- strate for rapid conversion to the corresponding N-aryl HA. This reaction is analogous to the acyloin carboligation[13] but rather is to be classified as an N- acyl condensation. The TKgst (N/S) variant, which had been engineered for improved conversion of benzalde- hyde, expectedly also showed about 7-fold higher initial rates with nitrosoarenes as compared to the wild-type enzyme. Among the tested set of variously p-, m- and o- mono- and disubstituted nitrosoarenes (5a–p), electron withdrawing substitution accelerated the reactivity, whereas the electron donating p-dimethylamino com- pound 5p was not converted. Fifteen different N-aryl HA could be prepared by this method. Further structural modifications in the HA structure could be induced by varying the donor component. The accept- ance of PA as alternative donor by the engineered (L/ S) variant further widens the scope of this biocatalytic process, albeit at the cost of reduced rates and product yields. From a co-solvent screen, acetone proved particularly beneficial in view of a significantly simplified work-up and highest overall yields. Un- equivocal and sensitive product detection was per- Figure 4. (A) HPLC profile of the HA array obtained from HPA as donor. (B) HPLC profile of the HA array obtained from pyruvate (PA) as donor. Conditions: RP-18 column, aq. MeCN, λ=254 nm. Scheme 5. Spontaneous non-enzymatic condensation of nitro- sarenes with glyoxylic acid (GA). Subsequent formation of formanilide (9) can occur under acidic conditions. RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 617 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 617/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de formed via the deeply colored iron(III) complexation, which possibly could be exploited for development of a sensitive colorimetric activity assay. For HA having the inverse constitution 3 such chelating capacity with various metal ions has been shown to offer a broad scope of pharmacological activities. Interestingly, HA with less common type 4 constitution seem not to have been studied yet for related biomedical applications, possibly because the genotoxicity of nitrosarenes and their potential for cancer generation was speculated to be due to their metabolic conversion by transketolase,[19,29] heart mitochondria[30] or rat liver preparations.[31] Studies in this direction are currently pursued. Experimental Section Materials and Methods. Commercial solvents and reagents were purchased from global vendors and used without further purification, except for 3-chloroaniline, 3-methylaniline, 3- chloro-4-methyl-aniline, 2-chloroaniline and 3-(trifluorometh- yl)-aniline, which were distilled prior to use. HPA was synthesized as the lithium salt according to a literature procedure.[32] All reactions were performed under argon atmos- phere. Melting points were recorded on a Stuart SMP10 apparatus. Column chromatography was performed on Merck 60 silica gel (0.063–0.200 mesh; Millipore); analytical TLC was performed on Merck silica gel plates 60 GF254 using p- anisaldehyde stain or iron (III) chloride stain for detection. HPLC analysis was performed on a Shimadzu LC-20AT device with SPD-M20A detector, using a Waters XBridge C18 column (3×150 mm) with particle size of 3.5 μm. Mobile phase consisted of a mixture of acetonitrile-water with 0.1% of formic acid using a gradient starting from 10% of MeCN to 95% with a flow rate of 0.5 mLmin� 1. UV detection was set at 254 nm for the HA and 320 nm for the nitroso compounds. NMR spectra were recorded on Bruker AR-300 and DRX-500 spectrometers, and are reported relative to the deuterated solvent peaks. Chemical shifts δ are given in ppm and coupling constants J in Hz. Mass spectra were recorded with an Impact II, Fa Bruker Daltonik spectrometer (ESI) and a Finnigan MAT 95 spectrom- eter (EI). Melting points of the TK variants were measured using a Prometheus NT.48 instrument from NanoTemper Technologies. General procedure for nitrosoarenes synthesis.[20] To a solution of the corresponding aniline in DCM was added dropwise a solution of Oxone (K2SO5×K2SO4×KHSO4) (2– 4 eq.) in deionized water. The mixture was stirred under argon atmosphere at room temperature with TLC or HPLC monitor- ing. Once consumption of starting material was complete, satd NaHCO3 solution (80 mL) was added slowly and the aq. phase extracted three times with DCM. The combined organic layers were consecutively washed with 1 M HCl (40 mL) and brine (50 mL), then dried over MgSO4. The solvent was removed at room temperature under vacuum. For spectroscopic character- ization of individual nitrosoarenes see the Supplementary Material. Expression and purification of TKgst variants. The enzyme variants L382N/D470S and H102 L/H474S were produced and purified as previously reported.[14,25] General procedure for preparative scale enzymatic synthesis (Method B: acetone optimized protocol). ThDP (28 mg, 2.4 mM) and MgCl2 ·6 H2O (48 mg, 9.4 mM) were dissolved in TEA buffer (17 mL, 50 mM) and the pH was carefully adjusted to 7.45. Lyophilized TKgst enzyme (10 mg of the mutant L382N/D470S for HPA and 50 mg H102L/H474S for PA) was added and the mixture incubated at 25 °C for 30 min. Then, Li- HPA (152 mg, 50 mM) dissolved in 3 mL of TEA buffer (50 mM, pH 7.5), followed by 3 mL of acetone, and a solution of the corresponding nitroso compound (50 mM) in 2 mL of acetone were added. The total volume was adjusted to 25 mL with 20% of acetone as co-solvent. Reactions were stirred at 25 °C and conversion monitored by TLC and HPLC. After consumption of starting material, pH was basified (pH 10–12) by addition of 6 M NaOH and the reaction mixture was extracted three times with ethyl acetate. The aqueous phase was then acidified to pH 4–5 by means of 6 M HCl, and extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over MgSO4, and the solvent was removed under reduced pressure. No further purification was performed. (Method A). In exploratory preparative reactions with EtOH and DMSO as co-solvents, the total volume was also 25 mL but adding 8% of EtOH or DMSO (2 mL) instead of acetone. Reactions were performed at 50 °C. Following extractive work- up, the crude material was dry loaded on a silica column and separated using CH/EA (1:1) as eluent. N-Phenyl-N,2-Dihydroxyacetamide (6a). Compound 6a was isolated as a colorless solid (104 mg, 50% yield); mp 65–66 °C (lit. [19] 64.5–65.5 °C). 1H NMR (500 MHz, CD3OD): δ=7.67 (d, 3J=8.4 Hz, 2H, o,o’-H), 7.38 (t, 3J=8.0 Hz, 2H, m,m’-H), 7.19 (t, 3J=7.5 Hz, 1H, p-H), 4.46 (s, 2H); 13C NMR (126 MHz, MeOD): δ=173.28 142.53, 129.63, 126.61, 121.43, 61.57. MS (EI): m/z calcd for [C8H9NO3]+ 167, found 167, 149, 119, and 109 (base peak). The spectroscopic properties are in accordance with literature data.[33] N-(4-Bromophenyl)-N,2-dihydroxyacetamide (6b). Com- pound 6b was isolated as a slightly tan solid (130 mg, 54% yield); mp 128–129 °C. 1H NMR (300 MHz, CD3OD): δ=7.66 (dt, 3J=9.1 Hz, 2H, o,o’-H), 7.52 (dt, 3J=9.1 Hz, 2H, m,m’-H), 4.47 (s, 2H); 13C NMR (75 MHz, MeOD): δ=173.53, 141.86, 132.59, 122.66, 61.65. HRMS (ESI)+: m/z calcd for [C8H8BrNO3]+H+ = 245.9760, found 245.9759. N-(4-Chlorophenyl)-N,2-dihydroxyacetamide (6c). Com- pound 6c was isolated as slightly tan solid (102 mg, 41% yield); mp 123–124 °C (lit. [19] 124–125 °C). 1H NMR (300 MHz, CD3OD): δ=7.71 (dt, J=9.1 Hz, 2H, o,o’-H), 7.38 (dt, J=9.1 Hz, 2H, m,m’-H), 4.48 (s, 2H); 13C NMR (75 MHz, CD3OD): δ=173.52, 141.37, 131.41, 129.58, 122.45, 61.62. MS (EI): m/z calcd for [C8H8ClNO3]+ =201, found 201, 185, 153, 143 (base peak), 125. N-(3-Chlorophenyl)-N,2-dihydroxyacetamide (6d). Com- pound 6d was isolated as a slightly tan solid (48 mg, 20% yield); mp 110–111 °C. 1H NMR (300 MHz, CD3OD): δ=7.81 (t, 4J=2.1 Hz, 1H), 7.67 (dd, 3J=8.5, 4J=2.1 Hz, 1H), 7.35 (t, RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 618 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 618/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de 3J=8.2 Hz, 1H), 7.17 (dd, 3J=8.1, 2.0 Hz, 1H, p-H), 4.49 (s, 2H); 13C NMR (75 MHz, CD3OD): δ=173.77, 143.83, 135.25, 130.91, 125.98, 120.59, 118.76, 61.75. MS (ESI)+: m/z calcd for [C8H8ClNO3]+H+ =202.03, found 202.03. N-(3,4-Dichlorophenyl)-N,2-dihydroxyacetamide (6 f). Com- pound 6f was isolated as an amber solid (30 mg, 10% yield); mp 126–127 °C. 1H NMR (500 MHz, CD3OD): δ=7.87 (d, 4J= 2.5 Hz, 1H), 7.58 (dd, 3J=8.9, 4J =2.5 Hz, 1H), 7.40 (d, 3J= 8.9 Hz, 1H), 4.37 (s, 2H); 13C NMR (126 MHz, CD3OD): δ= 173.92, 142.35, 133.19, 131.37, 128.87, 121.92, 119.84, 61.76. MS (EI): m/z calcd for [C9H10ClNO3]+ =235, found 235, 219, 187, 177 (base peak), 161, 124. N-(p-Tolyl)-N,2-Dihydroxyacetamide (6g). Compound 6g was isolated as a colorless solid (110 mg, 49% yield); mp 120– 121 °C (lit. [19] 119–119.5 °C). 1H NMR (300 MHz, CD3OD): δ=7.52 (d, 3J=8.0 Hz, 2H, o,o’-H), 7.21 (d, 3J=8 Hz, 2H, m,m’-H), 4.45 (s, 2H), 2.34 (s, 3H); 13C NMR (75 MHz, CD3OD): δ=173.09, 140.00, 130.16, 121.87, 61.47, 20.96. HRMS (ESI)+: m/z calcd for [C9H11NO3]+H+ =181.0812, found 181.0812. N-(m-Tolyl)-N,2-Dihydroxyacetamide (6h). Compound 6h was isolated as a slightly tan solid (66 mg, 29% yield); mp 92– 93 °C. 1H NMR (300 MHz, CD3OD): δ=7.57–7.39 (m, 2H), 7.25 (td, J=7.9, 2.4 Hz, 1H), 7.03 (d, J=7.6 Hz, 1H), 4.83 (s, 2H), 4.44 (s, 2H), 2.35 (s, 3H); 13C NMR (75 MHz, CD3OD): δ=173.21, 142.41, 139.70, 129.50, 127.47, 122.36, 119.00, 61.55, 21.53. MS (EI): m/z calcd for [C9H11NO3]+ =181, found 181, 165, 134, 123 (base peak), 106. N-(4-Chloro-3-methylphenyl)-N,2-dihydroxyacetamide (6 i). Compound 6 i was isolated as a colorless solid (100 mg, 37% yield); mp 143–144 °C. 1H NMR (300 MHz, CD3OD): δ=7.77 (d, 4J=2.2 Hz, 1H), 7.55 (dd, 3J=8.4, 4J=2.3 Hz, 1H), 7.29 (d, 3J=8.4 Hz, 1H), 4.47 (s, 2H), 2.36 (s, 3H); 13C NMR (75 MHz, CD3OD): δ=173.48, 141.56, 134.99, 133.88, 131.85, 121.36, 119.35, 61.61, 19.48. HRMS (ESI)+: m/z calcd for [C9H10ClNO3]+H+ =216.0422, found 216.0423. N-(3-Chloro-4-methylphenyl)-N,2-dihydroxyacetamide (6 j). Compound 6j was isolated as a slightly tan solid (87 mg, 32% yield); mp 125–126 °C. 1H NMR (500 MHz, CD3OD): δ=7.64 (d, 4J=2.7 Hz, 1H), 7.51 (dd, 3J=8.8, 4J=2.7 Hz, 1H), 7.34 (d, 3J=8.8 Hz, 1H), 4.45 (s, 2H), 2.37 (s, 3H); 13C NMR (126 MHz, CD3OD): δ=173.77, 141.61, 137.68, 130.33, 123.84, 120.52, 61.94, 20.60. MS (EI): m/z calcd for [C9H10ClNO3]+ =215, found 215, 199, 147 (base peak), 140. N-(4-(Trifluoromethyl)phenyl)-N,2-dihydroxyacetamide (6k). Compound 6k was isolated as a pale pink solid (27 mg, 9% yield); mp 110–112 °C (lit. [19] 112–114 °C). 1H NMR (300 MHz, CD3OD): δ=7.85 (d, 3J=8.6 Hz, 2H, m,m’-H), 7.56 (d, 3J=8.7 Hz, 2H, o,o’-H), 4.41 (s, 2H); 13C NMR (75 MHz, CD3OD): δ=174.08, 145.75, 126.77, 123.83, 120.05, 114.34, 61.85. MS (EI): m/z calcd for [C9H8F3NO3]+H+ =236, found 236, 219, 187, 178 (base peak), 159. N-(3-(Trifluoromethyl)phenyl)-N,2-dihydroxyacetamide (6 l). Compound 6 l was isolated as a colorless solid (50 mg, 17% yield); mp 90–91 °C (lit. [19] 90.5–91.5 °C). 1H NMR (300 MHz, CD3OD): δ=7.98 (s, 1H), 7.89 (d, 3J=8.3 Hz, 1H), 7.46 (t, 3J=8.0 Hz, 1H), 7.34 (d, 3J=7.8 Hz, 1H), 4.40 (s, 2H); 13C NMR (75 MHz, MeOD): δ=174.01, 143.33, 132.16, 131.73, 130.55, 127.25, 123.63, 122.38, 122.32, 61.76. MS (ESI)+: m/z calcd for [C9H8F3NO3]+H+ = 236.05, found 236.05. N-Phenyl-N-Hydroxyacetamide (7a). Sodium pyruvate (152 mg, 50 mM) was used as substrate instead of Li-HPA. Compound 7a was isolated as a slightly tan solid (10 mg, 5% yield); mp 65–66 °C (lit. 66–67 °C). 1H NMR (300 MHz, CD3OD): δ=7.46 (d, 3J=7.8 Hz, 2H, o,o’-H), 7.28 (t, 3J= 7.8 Hz, 2H, m,m’-H), 7.19–7.03 (m, 1H, p-H), 2.15 (s, 3H); 13C NMR (75 MHz, CD3OD): δ=174.89, 142.63, 129.66, 127.26, 122.94, 22.05. All properties were in agreement with literature data and with those of an authentic sample prepared by chemical synthesis.[26] General procedure for preparative scale chemical synthesis of HA with glyoxylic acid. Glyoxylic acid monohydrate (115 mg, 50 mM) was dissolved in 20 mL of TEA buffer 50 mM and the pH was adjusted to 7.45. Then, 3 mL of acetone were added and the mixture was shortly stirred. Afterwards, the nitroso compound (50 mM) dissolved in 2 mL of acetone is added. The total volume was adjusted to 25 mL and 20% of acetone as co-solvent. Reactions were stirred at 25 °C and monitored by TLC and HPLC. After consumption of starting material, pH was basified (pH 10–12) by addition of 6 M NaOH and reaction mixture was extracted twice with ethyl acetate. The aqueous phase was then acidified to pH 4–5 by addition of 6 M HCl, and extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over MgSO4, and the solvent was removed under reduced pressure. No further purification was performed. N-Phenyl-N-Hydroxyformamide (8a). Compound 8a was isolated as a cream color solid (136 mg, 79% yield); mp 66– 68 °C (lit. 67–69 °C). 1H NMR (300 MHz, CDCl3): δ=9.38 (br s, 1H), 8.37 (s, 1H), 7.34–7.09 (m, 5H); 13C NMR (75 MHz, CDCl3): δ=155.54, 138.07, 129.49, 127.06, 118.92. HRMS (ESI)+: m/z calcd for [C7H7NO2]+H+ =138.0549, found 138.0548. All properties were in agreement with literature data.[26] N-(4-Bromophenyl)-N-Hydroxyformamide (8b). Compound 8b was isolated as cream crystals (190 mg, 70% yield); mp 123–124 (lit. [34] 126 °C). 1H NMR (500 MHz, CD3OD; mixture of Z/E rotamers, ratio 1.1:0.9): δ=8.64 (s, 2H), 7.78– 7.23 (m, 8H). 13C NMR (126 MHz, MeOD): δ=163.16 (H1’- E), 158.65 (H1-Z), 140.84 (H2, H2’), 133.47 (H3’-E), 132.70 (H3-Z), 121.33 (H4, H4’). MS (EI): m/z calculated for [C7H6BrNO2]+ =215, found 215, 199, 187, 171 (base peak). Chemical synthesis of N-phenyl-N,2-dihydroxyacetamide (6a). Adapting a literature procedure,[19] a suspension of phenylhydroxylamine (150 mg, 1.4 mmol) in 3 mL of anhy- drous ether was stirred and cooled in an ice bath. To this suspension N,N’-diisopropylcarbodiimide (0.3 mL, 0.2 g, 1.6 mmol) in 0.5 mL of anhydrous ether was added, followed by the addition of glycolic acid (0.1 g, 1.6 mmol) of in 0.4 mL of anhydrous DMF over a 10 min period. The ice bath was removed and stirring was continued for 80 min until TLC showed complete conversion of the starting material. Then, 3 mL of n-butanol were added and the mixture was stirred for 15 min. The reaction mixture was extracted twice with 1 mL of RESEARCH ARTICLE asc.wiley-vch.de Adv. Synth. Catal. 2022, 364, 612–621 © 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH 619 Wiley VCH Montag, 31.01.2022 2203 / 226373 [S. 619/621] 1 16154169, 2022, 3, D ow nloaded from https://advanced.onlinelibrary.w iley.com /doi/10.1002/adsc.202101100 by T echnische U niversitat D arm stadt, W iley O nline L ibrary on [18/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://asc.wiley-vch.de 1 N NaOH, followed by 1.2 mL of water. The combined aqueous extracts was washed with 1.2 mL of ether and the pH was adjusted to 6 with a 4 N HCl. The suspension was extracted with 5 mL of ethyl acetate, and the organic portion washed with 0.6 mL of H2O and 0.6 mL of brine. Afterwards, the organic layer was dried over MgSO4 and the solvent was removed under reduced pressure. Compound 6a was obtained as yellow needles (80 mg, 35% yield). 1H NMR (300 MHz, CD3OD): δ= 7.72–7.62 (m, 2H, o,o’-H), 7.44–7.31 (m, 2H, m,m’-H), 7.25– 7.13 (m, 1H, p-H), 4.47 (s, 2H). 13C NMR (75 MHz, CD3OD): δ=173.27, 142.50, 129.65, 126.71, 121.66, 61.59. Chemical synthesis of N-phenyl-N-hydroxyacetamide (7a). Following a literature procedure,[26] to glacial acetic acid (20 ml) containing nitrosobenzene (0.7 g, 6.7 mmol) was added slowly 10 mL of an aqueous solution of sodium pyruvate (1.8 g, 16.7 mmol) with neutral pH. The mixture was stirred at room temperature until TLC and/or HPLC showed complete con- sumption of the nitrosobenzene. Afterwards 1 mL of NH4HCO3 (16.7 mmol) was added and the solvent was removed under reduced pressure. The residue was dissolved in 1 M NaOH (80 mL) and the resulting solution was extracted three times with Et2O. The pH of the aqueous layer was adjusted to 5.5 with 5 M H3PO4, followed by extraction with Et2O. The combined organic layers were dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude product was recrystallized from a mixture of benzene/hexane (1:1). Compound 7a was obtained as colorless crystals (405 mg, 41% yield); mp 66–67 (lit. 66–67 °C). 1H NMR (300 MHz, CD3OD): δ=7.57 (d, 3J=7.9 Hz, 2H, o,o’-H), 7.37 (t, 3J=7.7 Hz, 2H, m,m’-H), 7.21 (t, 3J=7.6 Hz, 1H, p-H), 2.25 (s, 3H); 13C NMR (75 MHz, CD3OD): δ=172.52, 142.28, 129.33, 126.69, 122.62, 21.76. HRMS (EI)+: m/z calcd for [C8H9NO2]+ =151.0628, found 151.0629. The spectroscopic properties were in agreement with literature data.[26] N-(4-Bromophenyl)-Formamide (9b). Compound 9b was isolated as a side product from the enzymatic reaction from 5b to 6b via slow addition of 5a with a syringe pump. Substance 9b was obtained as a pale orange solid (7 mg, 3% yield). 1H NMR (500 MHz, CD3OD; mixture of Z/E rotamers, 4.2:1:0 ratio) δ=8.71 (s, 0.3H, H1’-E), 8.28 (s, 1.2H, H1-Z), 7.60–7.39 (m, 6.0H, H3-, H4-Z, H3’-E), 7.14–7.08 (m, 0.6H, H4-E). 13C NMR (126 MHz, CD3OD) δ=164.55 (H1’-E), 161.58 (H1-Z), 138.28 (H2-Z), 133.61 (H3’-E), 132.94 (H3-Z), 122.70 (H4-Z), 121.20 (H4’-E), 117.82 (H5-Z). MS (EI): m/z calcd for [C7H6BrNO]+ = 199; found 199, 171, 143. The spectroscopic properties were in agreement with literature data.[35] Acknowledgements This work was supported by The German Federal Ministry of Education and Research (BMBF) under Grant Agreement No 031B0595 and the initiative ERA CoBioTech. ERA CoBioTech has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 722361. Open access funding enabled and organized by Projekt DEAL. References [1] a) E. M. F. Muri, M. J. Nieto, R. D. Sindelar, J. S. Williamson, Curr. Med. 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