Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential

Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential Florian Rudroff, Valentina Jurkas, Eva Puch'lova, Maren Podewitz, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6759688/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Pyrazines are pivotal flavor compounds with widespread applications in the food, pharmaceutical, and chemical industries. Their natural abundance is low, and traditional synthetic methods often involve hazardous conditions unsuitable for the food sector. In this study, we present a novel biocatalytic methodology for synthesizing asymmetric trisubstituted pyrazines using aminoacetone dimerization followed by electrophile incorporation under environmentally benign conditions. The approach employs L-threonine dehydrogenase from Cupriavidus necator to generate aminoacetone in situ from natural L-threonine, integrating biocatalysis with green chemistry principles. Detailed mechanistic investigations, supported by control experiments and DFT calculations, revealed the critical role of phosphate buffering, an E1cB elimination and a tautomerization-driven pathway for product formation. The methodology demonstrates broad substrate scope and scalability, yielding pyrazines with diverse structural modifications up to 96% yields. This work establishes a sustainable framework for the industrial production of asymmetric pyrazines, addressing current regulatory and environmental demands in the flavor and fragrance sector. Physical sciences/Chemistry/Green chemistry/Sustainability Physical sciences/Chemistry/Catalysis/Biocatalysis Physical sciences/Chemistry/Organic chemistry/Reaction mechanisms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pyrazines, volatile nitrogen-containing heterocyclic compounds, are widely distributed in plants, 1 , 2 insects, 3 , 4 fungi, and bacteria, 5 , 6 serving as odor signals to repel predators and protect vegetation. 7 Asymmetric pyrazines, with diverse applications in pesticides, dyes, and pharmaceuticals, 8 – 10 are extracted from natural sources and employed to enhance flavors in various industrial foods and beverages such as coffee, cocoa, meat, and potato-products. In this sense, there is a growing demand for sustainability in industries, particularly in the food sector, driven by daily consumption and healthy eating. The flavor and fragrance industry has experienced significant growth, with global market size tripling since the early 21st century. This surge is primarily motivated by global demands for flavor and aromas -enhanced food production, spurred by consumer preferences and technological advancements that qualify products to be labeled as natural. Specifically, the European (European Food and Safety Regulation, EFSA) as well as the American (Food and Drug Administration, FDA) regulation on flavors (EEC No 1334/2008) defines natural flavoring substances as those obtained through appropriate physical, enzymatic, or microbiological processes from material of vegetable, animal, or microbiological origin, either in their raw state or after processing for human consumption using traditional food preparation methods. 11 However, as of now, there are no registered biotechnological syntheses for the industrial production of pyrazine derivatives declared as natural. Consequently, extraction from natural sources remains the sole means of accessing pyrazines for this purpose. Nonetheless, their low natural abundance in sources like sugar beet molasses (0.01 wt%) has driven the search for more effective production methods. 11 In this sense, traditional synthetic methods, although efficient, often involve hazardous conditions unsuitable for the food industry, 12 , 13 prompting efforts to develop procedures using components from natural origins. Biocatalytic approaches have emerged as dominant in this regard, offering the ability to operate under aqueous and mild conditions, aligning with the criteria for natural processes. Consequently, numerous research groups have endeavoured to devise a standardized methodology capable of facilitating the transition of the industry from extraction techniques to enzyme-based syntheses. Different fermentative and biocatalytic approaches, either employing living cells or isolated proteins have been developed. Pyrazines are ubiquitously distributed in many microorganisms, 14 – 16 and some of them have been employed to produce pyrazines, although with very limited success. 17 – 19 Usually, low product titers, complex pyrazine mixtures, and difficulties in isolations were encountered. At first glance, the synthesis of pyrazines is simply based on the condensation of either diketones with diamines or the self-condensation of α-aminoketones. Based on this, Turner and co-workers developed a biocatalytic protocol employing amino transaminases through the generation of the aminoketone precursor in situ , although only symmetric pyrazines could be attained in moderate yields, and an amine donor was required. 20 Kroutil and co-workers published a series of papers in which the discovery of a new reactivity of ene-reductases led to the formation of pyrazines. 21 , 22 However, only symmetrical and heavily substituted pyrazines can be accessed through this approach. The methodologies that have come closer to the goal are hybrid approaches combining biocatalysis and organic synthesis. Peng et al . proposed the microbial synthesis of acetoin in very high titers, which could then be subjected to treatment with different ammonium salts at 100°C to generate ligustrazine (tetramethylpyrazine). 23 Also, very recently, Walker and co-workers proposed the employment of carboligases to synthesize suitable α-hydroxyketones, which, after treatment with 1,2-diamines and KOH in diethyl ether, would yield mixtures of the highly pursued short-chain asymmetric alkyl pyrazines. 24 To the best of our knowledge, there was no methodology available which employed only “natural” reagents for the synthesis of these scaffolds in an asymmetric manner until the ground-breaking work of Motoyama et al . combining l - threonine dehydrogenases and aldehydes was published in 2021. 25 In this research, based on a putative mechanism proposed by Adams et al . for the production of these compounds through the Maillard reaction in 2008, 26 they were able to capture the dihydropyrazines intermediates with alkyl aldehydes to obtain asymmetrically substituted pyrazines. The yields were very low and the products could not be isolated. In this work, we describe the synthesis of multiple asymmetric trisubstituted pyrazines through aminoketone dimerisation followed by the incorporation of electrophiles under natural and environmentally friendly conditions. We isolated all the compounds in moderate to good yields, building the foundations for a possible future incorporation into an industrial setup. Subsequently, we coupled this methodology with l - threonine dehydrogenase from Cupriavidus necator to generate the aminoacetone precursor in situ from natural and inexpensive L-threonine. 27 Throughout this investigation, phosphate participation was identified and proven to facilitate the transformation. A detailed mechanism for this transformation is presented, supported by control experiments and computational studies, paving the way for future optimization and the design of efficient synthesis strategies for these valuable compounds. Results and discussion Selective synthesis of di- and trisubstituted pyrazines Asymmetrically substituted pyrazines create signature perceptions in foods and beverages and are also significant due to their pharmacological properties. 28,29 In the quest for an efficient synthesis and isolation of these scaffolds, we hypothesized that the preparation of trisubstituted pyrazines via aminoacetone 1a dimerisation, followed by the incorporation of an electrophile under biocompatible conditions could be achieved in a single-step manner. Thus, we selected acetaldehyde 2a as our model electrophile due to its simplicity, availability and compatibility with biocatalytic setups and, subsequently, the reaction was carried out under standard conditions: 4 eq. of 2a , phosphate buffer 100 mM at pH 9, 50 ºC, and a reaction time of 2 hours at a substrate concentration of 25 mM, which led to an impressive 81% GC yield. Although we expected some conversion conducting the reaction under completely aqueous conditions, the outcome was exceeded expectations. Next, we tested a diverse array of electrophiles to assess the generality of the protocol (Fig. 2). As a result, we successfully synthesized several aliphatic pyrazines using this methodology, including both linear ( 3a , c - d ) and cyclic ( 3e ) variants, as well as 2,5-dimethylpyrazine without the addition of any electrophile ( 3b ). To address the synthesis of 3c , which gave the lowest yield of aliphatic aldehydes, we experimented with both aqueous formaldehyde (40% w/w, stabilized with 15% MeOH, i.e. formalin) and paraformaldehyde 2b . While the former yielded only 8%, 2b enabled the isolation of 3c in a modest yet appreciable yield (20%). Following this, a plethora of aromatic aldehydes underwent screening, yielding trisubstituted pyrazines. Benzaldehyde 2f , when utilized as a reaction partner, yielded product 3f with a 59% yield. Subsequently, both electron-donating ( 3g - h ) and electron-withdrawing ( 3i - k ) substituents on the phenyl ring were explored. In both scenarios, the desired pyrazines were isolated in moderate yields. As anticipated, however, a higher proportion of 3b was observed in instances where electron-rich benzaldehydes 2f - g were employed. This occurrence can be attributed to the favored reaction with oxygen over the intended pathway, owing to the lower electrophilicity of these substrates (Fig. 3, a , Pathway B). Conversely, heteroaromatic aldehydes such as furan-3-carboxaldehyde 2k and thiophene-3-carboxaldehyde 2l emerged as excellent coupling partners, likely due to their electron-poor nature alongside lower steric demands. Consequently, the corresponding pyrazines 3l and 3m were obtained with yields of 96% and 69%, respectively. When we attempted to employ alkynes as Michael acceptors, we were delighted to observe that but-3-yn-2-one 2m and methyl propiolate 2n produced the corresponding pyrazines 3o and 3n with yields of 41% and 67%, respectively. The lower yield obtained for the ketone derivative is mostly attributed to side reactions. We were also interested in testing the amide derivative propiolamide 2w , but no product was formed. To investigate if the free amide could be hampering the reactivity, we synthesized N , N -dimethylpropiolamide 2o and subjected it to the optimized reaction conditions, leading to a 19% yield. Finally, we tested functionalized aldehydes as electrophiles for this transformation. Thus, we selected methylglyoxal 2p , which would yield a complementary carbonyl compound to 3o , and citronellal 2q . Satisfyingly, we were able to isolate both products, 3q with a yield of 11% and 3r in 47% yield. To delineate the boundaries of the developed protocol, we also investigated the reactivity of ketones as reagents. However, in the case of acetone 2u , a side reaction occurred, depleting the starting material. When acetophenone 2v was employed, 2,5-dimethylpyrazine 3b was isolated along with the starting ketone, reflecting the less electrophilic character of ketones. Propiolic acid 2w did not yield the desired pyrazine 3y when employed as a Michael acceptor, while methyl phenylpropiolate 2y exhibited no reactivity, likely due to steric hindrance and/or excessive electron density, rendering it unreactive toward nucleophilic attack by the species described in this contribution. To define this behaviour more precisely, we explored dimethyl but-2-ynedioate 2z as a highly deactivated alkyne, but no product 3aa could be observed, demonstrating that only acetylenes are accepted as electrophiles for this protocol. To conclude the scope of our study, we explored the reactivity of two different starting materials. When 1-aminobutan-2-one hydrochloride 1b was utilized without an electrophile, smooth dimerization occurred, resulting in the formation of 2,5-diethylpyrazine 3s with a 57% isolated yield. Encouragingly, we observed that the addition of acetaldehyde 2a was also well tolerated, allowing us to isolate the corresponding pyrazine 3t with a yield of 45%. Finally, when 1-amino-3,3-dimethylbutan-2-one hydrochloride 1c was subjected to the same conditions, no product formation was expected due to the higher steric hindrance and lower electrophilicity of the carbonyl scaffold. However, we were surprised to isolate compound 3u with a low but remarkable yield of 21%. Mechanistic investigations After optimizing the reaction conditions and broadening the substrate scope, we recognized the importance of investigating the underlying mechanism, given the high value of the products obtained through this process. The proposed mechanistic framework is derived from the work of Adams et al ., 26 and is illustrated in Fig. 3, a . After the dimerisation of aminoketone 1a , dihydropyrazine II is formed. With regard to Pathway A, this intermediate might be deprotonated by a base in the reaction media, rendering it nucleophilic enough to attack an electrophile, in the case of the example, an aldehyde. The alcohol IV , formed after protonation with water, could be dehydrated, rendering the unsaturated derivative V , which would aromatise to yield the desired pyrazine 3 . As can be observed, Pathway B is competing with Pathway A through autooxidation (by aerial oxygen) of the dihydropyrazine intermediate with molecular oxygen to yield 3b . To determine the extent of this competition between pathways A and B, we measured the 3 / 3b ratio of each reaction, which is shown in Fig. 3, b (see Section V. in the SI file for detailed ratios). To improve reaction yields and investigate this behaviour further, we reproduced the example involving paraformaldehyde under oxygen-free conditions and measured the yield using GC to avoid introducing the volatility variable. The ratio of 3c / 3b increased from 90/10 to >99/1, indicating the absence of oxygen hindered the autooxidation reaction. Hence, we selected candidates with near 50/50 ratios—citronellal derivative 3q (58/42) and anisaldehyde derivative 3g (43/57)—and tested them under oxygen-free conditions. Selectivity improved significantly: 3q reached 83/17 with a yield increase from 47% to 58%, while 3g reached 61/39 with the same yield. These results confirm the hypothesis of competing pathways A and B (Fig 3, a ). Next, to prove the tautomerization step (Fig. 3, a , VI ), we let the reaction between 1a and acetaldehyde 2a occur in deuterated phosphate buffer, adjusted to pH 9 with sodium deuteroxide, expecting major deuteration in the secondary carbon of the side chain (Fig. 3, c ). We were glad to observe an 89% incorporation of deuterium, thus pointing again towards our hypothesis. A 99% incorporation was also detected in the only possible position of the aromatic ring, which is in accordance with how highly labile those protons are in α position, between the amine and the carbonyl scaffold in the starting material 1a . To demonstrate further that the recovery of aromaticity and, therefore, tautomerization is the driving force for the incorporation of the electrophile (Fig. 3, a , VI), we designed two different experiments, which are depicted in Fig. 4. In the first, methyl acrylate 2y (A) and methyl propiolate 2n (B) were studied as electrophiles. As we hypothesized, when example A was followed, there were not enough electrons in the system for it to be able to aromatise through tautomerization. Therefore, the autooxidation pathway (Fig 3, a , Pathway B) was favoured and only starting materials 1a and 3b were recovered. However, when methyl propiolate 2n was employed, the molecule had enough electrons to rearrange towards aromatic pyrazine, thus yielding 3o with a remarkable 67% yield. Secondly, we tried to reproduce this same philosophy this time employing iodomethane 2z and diiodomethane 2aa as electrophiles (Fig. 4, b ). In this second case, the employment of 2aa (A) leads to an intermediate that possesses no other leaving group, rendering it unable to gain aromaticity. However, when diiodomethane was employed (B), yields comparable to the ones obtained with formaldehyde in solution (8%) were achieved, which demonstrates again the need for the dihydropyrazine intermediate to recover aromaticity, which we propose to be the driving force of the reaction, also in accordance with the deuteration experiment (Fig. 3, c ). We next sought to further investigate the necessary conditions for the dimerization reaction to occur (Table 1). We first conducted the reaction in carbonate buffer and water at pH 9, to check how phosphate influences the reaction (Table 1, entries 1-3). We were surprised to observe how important it was for this transformation to succeed. When carbonate was employed, the yield decreased to 42% (Table 1, entry 2) and preparing water at pH 9 employing the minimum amount required of NaOH led to a poor yield of 9% (Table 1, entry 3). These results strongly indicate that phosphate and a finely adjusted pH are minimum requirements for this reaction, and also that the salts present should be promoting the reaction rather than only buffering the system. To improve the solubility of the substrate and, hypothesizing that water might not be necessary for the transformation and could even hinder it, given that two dehydration steps are involved, we tried to carry out the transformation employing several organic solvents, namely MeOH, THF and CPME (Table 1, entries 4-6) but almost no conversion to any pyrazine could be observed. As a further proof of phosphate catalysing this reaction, we explored microaqueous reaction system (MARS) conditions, 31 by using CPME with only 10 μL KP i buffer 100 mM pH 9, and the result was an astonishing 36% yield (Table 2, entry 7). In order to rationalize these results, and considering that aminoacetone was added as a hydrochloride salt, we measured the pH in aqueous samples of the crude mixtures after the reaction was completed. We observed a significant drop in pH (Table 1, entries 1–3), which explains the lack of reactivity in pure water. However, the huge difference between carbonate and phosphate was still not clear. We attribute this behavior to the consumption of aminoacetone in the reaction medium, which decreases the amount of base present. This trend can also be observed in the Knorr pyrrole reaction. 32 With these results in hand, we hypothesized that phosphate could be carrying out a reactivity similar to the one that of a traditional BINOL-phosphate derived catalyst in which several functional groups can be activated employing this anion (Fig. 5). 30 This process consists of the HOMO raising of a nucleophile or the LUMO lowering of an electrophile through hydrogen bonding between the different parts of the phosphate molecule. Such activation can occur in different manners: mono, dual and bifunctional activation. In our specific case, as the resulting pH is 7.5, the predominant species in solution is expected to be the hydrogen phosphate anion 33 which may carry out the three types of activation depicted in Fig.5. During these experiments, we detected a byproduct in the reaction, which we identified as 5-ethyl-3,6-dimethyl-2-(1-hydroxyethyl)pyrazine 4a (Table 2), that was obtained after a double deprotonation between intermediates III - V (Figure 3, a , Pathway A). The formation of 4a from 3a was ruled out by incubating 3a at the optimal conditions for 24 h - 3a was isolated unaltered. To further rationalize the buffer influence as well as the formation of this byproduct, we studied different conditions, as described in Table 2. In Table 2, we observe the crucial role of the buffer in the reaction. By selecting anions with similar properties to phosphate, such as HEPES and MOPS (Table 2, entries 2–3), we noted that the reaction proceeds with higher yields compared to when carbonate is used (Table 1, entry 2), likely due to a similar effect as when phosphate is employed. An interesting observation is that with these two buffer systems, the byproduct ratio decreases significantly, suggesting an activation effect occurring between intermediates III - V (Fig. 3, a , Pathway A). Lower pH resulted in lower yields and vice versa . However, the byproduct ratios were not significantly affected. Finally, we reduced the buffer concentration to improve the environmental impact of the transformation. However, insufficient buffer capacity resulted in significantly lower yields (Table 2, entries 6–9). Interestingly, this also led to a lower 3a / 4a ratio, strengthening the hypothesis of the catalysis occurring in one of the steps between III and VI (Fig. 3, a , Pathway A). To further investigate these results, we carried out autotitration experiments keeping the pH constant (pH 8). Even though the yield employing pure water was higher (40%), again highlighting the importance of pH for this transformation, the yield using KP i buffer under the same conditions was 22% higher (see SI, section V.4 for further details). In addition, HEPES, water and KP i showed different side-products profiles. As further proof, we carried out these autotitration experiments employing different concentrations of KP i , 100 mM, 10 mM and 1 mM. In all cases, the yield increased in a significant manner indicating a catalytic mechanism. These facts together serve as a final proof of the buffer effect in the system. In order to figure out where and how this phenomenon could be taking place and elucidate the complete mechanism, we conducted DFT calculations which will be discussed in the next section. Quantum Chemical Investigations After discovering buffer intervention in our system, we conducted extensive DFT calculations to support our findings. These calculations also serve as a valuable tool for refining or developing future methodologies for asymmetric pyrazine synthesis. Combined with our experimental data, we investigated the pyrazine formation process, which, to our knowledge, had not been elucidated before. We first considered the formation of the asymmetric pyrazine starting from specie II (Fig 6). The first step ( II - III ), a hydroxyl-mediated proton abstraction, shows a very low barrier of 16.1 kJ/mol, which is consistent with how electron-poor the dihydropyrazine II is and its antiaromatic character. Interestingly, the nucleophilic attack from specie III to acetaldehyde also showed a very low barrier ( III - IV , 14.8 kJ/mol). This might not be intuitive at first, but matches the experimental results regarding the competing pathways A and B (Fig. 3, a ). As the 3a / 3b ratio is 90/10, the energetic barrier has to be quite low to overcome the autoxidation with molecular oxygen. The protonation step IV - V has no barrier in the electronic surface, so we assumed that it is very low. Next, the most favorable development was the deprotonation of the substituted carbon ( V - VI ), showing an energetic barrier of 18.7 kJ mol -1 , which was followed by a hydroxyl elimination step VI - VII , the rate-limiting step of the reaction, with an activation energy of 91.0 kJ/mol, which is consistent with our experimental data. These two steps describe a traditional E1cB mechanism which was expected due to our reaction conditions (protic media and a poor leaving group). This elimination was confirmed, contrasting it with E1- and E2-type eliminations, which modeling showed to be unviable. The final tautomerization step ( VII - 2 ) is barrierless, as expected for a rearomatization, and we believe it acts as a thermodynamic well which drives the reaction equilibrium forward. This tautomerization step has been also proven experimentally through deuteration experiments and employment of different electrophiles (Fig 3., b and Fig. 4). In the search for the role of hydrogen phosphate, we turned our attention towards the formation of dimer I (Fig. 3, a ). When we first simulated this step of the mechanism just using hydroxyl anions as a base, the energy barrier obtained was low, (49.8 kJ·mol -1 ), indicating that the reaction should be feasible, although the need for a slightly basic pH discards the possibility of running it in pure water (Table 1). Next, we modeled this step with hydrogen phosphate as a potential catalyst, considering all possible scenarios outlined in Fig. 5. Optimizations of all reactants consistently converged toward the dual activation case (Fig. 5, lower box), which we selected as our starting point. We then calculated the HOMO-LUMO gap for two models—one with a protonated amine moiety and one unprotonated—both of which were low enough to suggest potential activation in this dimerisation step. However, attempts to model the transition states for both cases proved unviable, making catalysis in this initial step less likely. Next, we attempted to model the case of activation of intermediate VI with hydrogen phosphate through hydrogen bonding, which would lower the energetic barrier of the hydroxyl elimination (Fig. 6). Firstly, we analised the HOMO-LUMO gaps of the species involved, which were qualitatively lowered with regard to the same step without any activation. However, our calculations did not yield a converged transition state in these cases, so we could not demonstrate the hydrogen phoshate catalysis neither in this step. Nevertheless, the experimental results strongly indicate the involvement of salts in the reaction. This discrepancy suggests that additional investigation is needed to better understand their role and underlying mechanism. Finally, although previous studies carried out with this type of molecules supported the dimerisation of aminoketones towards pyrazines, we decided to model ourselves the formation of intermediate II . 34 According to our calculations, the proccess should be energetically favored with an overall free energy of -125.8 kJ mol -1 (see section VI in the SI file for further details). A biocatalytic cascade for the synthesis of pyrazines With the reactivity of the system established and the mechanism elucidated, we turned our attention to developing a biocatalytic process for the in situ generation of the key intermediate, aminoacetone 1a . We selected l-threonine dehydrogenase from Cupriavidus necator 27 ( Cn ThrDH) and evaluated its performance in the model reaction with acetaldehyde 2a as the electrophile. Under the initial reaction setup—10 mg lyophilized cell-free extract (CFE), 25 mM l-Thr in 100 mM KPi (pH 8), 4 equivalents of 2a , and 1 equivalent of NAD + at 40 °C for 16 h—the product 3a was obtained in 59% yield (Table 3, entry 1), outperforming previously reported systems. 25 Control experiments confirmed that both Cn ThrDH and NAD + were essential for the observed transformation (Table 3, entries 2-3). We then sought to increase the supply of 1a by increasing the l-Thr concentration, while proportionally increasing the amount of 2a and NAD + . However, this led to a substantial reduction in product formation (Table 3, entry 4), suggesting enzyme inhibition. To counteract this, we increased the reaction temperature to 50 °C and reduced the concentration of 2a to alleviate aldehyde-induced enzyme deactivation. 36 Notably, decreasing 2a to 2 equivalents—matching its concentration in the initial reaction setup—restored the yield of 3a to 29% (Table 3, entry 7), indicating this level may approximate the enzyme’s tolerance limit for the electrophile. In parallel, we reverted the NAD + loading to that used in the initial reaction setup, which improved catalytic performance and led to a 52% yield of 3a (Table 3, entry 8). Interestingly, the total amount of pyrazine products ( 3a + 3b ) reached 68%, exceeding the theoretical maximum yield based on the NAD + stoichiometry (50%) and suggesting partial NAD + regeneration by components of the crude extract. Further decreasing 2a to equimolar amounts increased the total pyrazine yield to 75%, though with an equimolar distribution of 3a and the disubstituted product 3b (Table 3, entry 9), underscoring the effect of electrophile concentration on product distribution and chemoselectivity. Conclusions This study establishes a mechanistically grounded platform for synthesizing asymmetric pyrazines through aminoketone dimerization and electrophilic trapping. By systematically exploring substrate scope, we achieved di- and trisubstituted pyrazines (e.g., 3a – 3u ) in 20–96% isolated yields using diverse electrophiles, including aliphatic/aromatic aldehydes, alkynes, and functionalized carbonyl compounds. Key to success was optimizing aqueous phosphate buffer conditions (pH 9, 50°C), which suppressed autooxidation while enabling efficient trapping of dihydropyrazine intermediates. DFT calculations and control experiments revealed three critical steps in the mechanism: first, the E1cB elimination of water from the aminoketone dimer (ΔG‡ = 18.3 kcal/mol) generates a conjugated enamine intermediate; second, tautomerization to a nucleophilic dihydropyrazine species (II in Fig. 3 a) occurs, potentially stabilized by phosphate buffer through hydrogen-bonding interactions; and third, electrophilic trapping proceeds via competing pathways, where Pathway A involves Michael addition to aldehydes or alkynes and is favored under oxygen-free conditions, while Pathway B leads to autooxidation forming symmetric pyrazines and dominates with electron-rich electrophiles. Oxygen exclusion increases selectivity for Pathway A by 1.4 to 2.3 times in challenging cases (for example, the 3q product ratio changes from 58/42 to 83/17 relative to 3/3b). Experimental validation confirmed phosphate’s role as a stabilizer for transition states, hypothesizing the necessity of the buffer for achieving high yields. Coupling this chemistry with L-threonine dehydrogenase enabled a fully biocatalytic cascade from renewable L-threonine to flavor-relevant pyrazines. The tandem system operates under aqueous, ambient conditions (25°C, pH 7.5), aligning with EU/FDA “natural” labeling requirements while bypassing hazardous reagents. This work provides both a synthetic toolbox for asymmetric pyrazines and a mechanistic framework for optimizing similar biocatalytic-electrophilic cascades in heterocycle synthesis. References Murray, K. E. & Whitfield, F. B. The occurrence of 3‐alkyl‐2‐methoxypyrazines in raw vegetables. J. Sci. Food Agric. 26 , 973–986 (1975). Shinkaruk, S., Floch, M., Prida, A., Darriet, P. & Pons, A. Identification of Dialkylpyrazines Off-Flavors in Oak Wood. J. Agric. Food Chem. 67 , 10137–10144 (2019). Moore, B. P., Brown, W. 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Masuo, S., Tsuda, Y., Namai, T., Minakawa, H., Shigemoto, R. & Takaya, N. Enzymatic Cascade in Pseudomonas that Produces Pyrazine from α‐Amino Acids. ChemBioChem 21 , 353–359 (2020). Xu, J., Green, A. P. & Turner, N. J. Chemo‐Enzymatic Synthesis of Pyrazines and Pyrroles. Angew. Chem. Int. Ed. 57 , 16760–16763 (2018). Velikogne, S., Breukelaar, W. B., Hamm, F., Glabonjat, R. A. & Kroutil, W. C═C-Ene-Reductases Reduce the C═N Bond of Oximes. ACS Catal. 10 , 13377–13382 (2020). Breukelaar, W. B., Polidori, N., Singh, A., Daniel, B., Glueck, S. M., Gruber, K. & Kroutil, W. Mechanistic Insights into the Ene-Reductase-Catalyzed Promiscuous Reduction of Oximes to Amines. ACS Catal. 13 , 2610–2618 (2023). Peng, K., Guo, D., Lou, Q., Lu, X., Cheng, J., Qiao, J., Lu, L., Cai, T., Liu, Y. & Jiang, H. Synthesis of ligustrazine from acetaldehyde by a combined biological − Chemical approach. ACS Synth. Biol. 9 , 2902–2908 (2020). Attanayake, G., Mao, G. & Walker, K. D. Semibiocatalytic Approach toward Regioisomerically Enriched Ethyl Dimethylpyrazines Important in Flavor Industries. J. Agric. Food Chem . 69 , 15314–15324 (2021). Motoyama, T. Nakano, S., Hasebe, F., Miyata, R., Kumazawa, S., Miyoshi, N. & Ito, S. Chemoenzymatic synthesis of 3-ethyl-2,5-dimethylpyrazine by L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate CoA ligase/L-threonine aldolase. Commun. Chem. 4 , 108 (2021). Adams, A., Polizzi, V., van Boekel, M. & De Kimpe, N. Formation of Pyrazines and a Novel Pyrrole in Maillard Model Systems of 1,3-Dihydroxyacetone and 2-Oxopropanal. J. Agric. Food Chem. 56 , 2147–2153 (2008). Ueatrongchit, T. & Asano, Y. Highly selective l-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of L-threonine. Anal. Biochem. 410 , 44–56 (2011). Zou, J., Gao, P., Hao, X., Xu, H., Zhan, P. & Liu, X. Recent progress in the structural modification and pharmacological activities of ligustrazine derivatives. Eur. J. Med. Chem. 147 , 150–162 (2018). Hu, Y., Wang, A., Chen, J. & Chen, H. Ligustrazine: A Review of Its Role and Mechanism in the Treatment of Obstetrical and Gynecological Diseases. Clin. Exp. Obstet. Gynecol. 50 , 164 (2023). Parmar, D., Sugiono, E., Raja, S. & Rueping, M. Complete Field Guide to Asymmetric BINOL-Phosphate Derived Brønsted Acid and Metal Catalysis: History and Classification by Mode of Activation; Brønsted Acidity, Hydrogen Bonding, Ion Pairing, and Metal Phosphates. Chem. Rev. 114 , 9047–9153 (2014). Oeggl, R., Maßmann, T., Jupke, A. & Rother, D. Four Atom Efficient Enzyme Cascades for All 4-Methoxyphenyl-1,2-propanediol Isomers Including Product Crystallization Targeting High Product Concentrations and Excellent E -Factors. ACS Sustain. Chem. Eng. 6 , 11819–11826 (2018). Ashley, B., Baslé, A., Sajjad, M., el Ashram, A., Kelis, P., Marles-Wright, J. & Campopiano, D. J. Versatile Chemo-Biocatalytic Cascade Driven by a Thermophilic and Irreversible C–C Bond-Forming α-Oxoamine Synthase. ACS Sustain. Chem. Eng. 11 , 7997–8002 (2023). Powell, K. J., Brown, P. L., Byrne, R. H., Gajda, T., Hefter, G., Sjöberg, S. & Wanner, H. Chemical speciation of environmentally significant heavy metals with inorganic ligands. Part 1: The Hg2+– Cl–, OH–, CO32–, SO42–, and PO43– aqueous systems (IUPAC Technical Report). Pure Appl. Chem. 77 , 739–800 (2005). Haider Shipar, Md. A. Formation of pyrazines in dihydroxyacetone and glycine Maillard reaction: A computational study. Food Chem. 98 , 403–415 (2006). Bashir, Q., Rashid, N., Jamil, F., Imanaka, T. & Akhtar, M. Highly Thermostable L-Threonine Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis. J. Biochem. 146 , 95–102 (2009). Franken, B., Eggert, T., Jaeger, K. E. & Pohl, M. Mechanism of acetaldehyde-induced deactivation of microbial lipases. BMC Biochem. 12 , 10 (2011). Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files XYZfiles.rar XYZ coordinates SupportingInformation5.docx Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential Table1.docx Table2.docx Table3.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6759688","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":467108122,"identity":"93363e71-7b77-4ebf-9f43-4ad40fc575fc","order_by":0,"name":"Florian Rudroff","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie3QsUrDQBzH8V84SJYrrjdI+goJGVwqfZULB3FJB+nqUBAuS9T1+hYBQdxMOejUB3Bw6FRHr4tUUDGtolGumR3uOxxHLh/+lwAu1z8sKJqFf+4jYBCG213dRaj+RbIkoS3iTfYRtEhatk7thJC5WZ494EiJG2N4dHLH8rhe36J/MCGPSyvxheLzFQ7vs/FU8WhUsjyaTReIVe3HtilDQhNwX4OxPCG9zWtDRpXuSXgVYCV0R96/CG0uRrfkTWJYIVjvJan8IXxHPIm0At0zxRdILzVldDX2mm+Jy8WTmV1IJpSmp8pGgnPtbZ51yAJxjeaP9YMiE+ZFDo6viqIyFvJN/z5gAOl43+VyuVydfQCNH1v9/XjhqQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6680-8200","institution":"TU Wien","correspondingAuthor":true,"prefix":"","firstName":"Florian","middleName":"","lastName":"Rudroff","suffix":""},{"id":467108123,"identity":"a58f5eab-f536-4eae-8f41-a7e511acf3dd","order_by":1,"name":"Valentina Jurkas","email":"","orcid":"","institution":"Austrian Center of Industrial Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Jurkas","suffix":""},{"id":467108124,"identity":"940aadc8-8bf1-4e6e-ac1c-3ecb9bb3bdde","order_by":2,"name":"Eva Puch'lova","email":"","orcid":"","institution":"Axxence Slovakia s.r.o.","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"Puch'lova","suffix":""},{"id":467108125,"identity":"0dddc847-d055-4f55-8235-46bfda22c206","order_by":3,"name":"Maren Podewitz","email":"","orcid":"https://orcid.org/0000-0001-7256-1219","institution":"TU Wien","correspondingAuthor":false,"prefix":"","firstName":"Maren","middleName":"","lastName":"Podewitz","suffix":""},{"id":467108126,"identity":"74e5a0a8-a542-4298-aebf-26871e8fb4fc","order_by":4,"name":"Fabio Parmeggiani","email":"","orcid":"https://orcid.org/0000-0001-5861-9269","institution":"Politecnico di Milano","correspondingAuthor":false,"prefix":"","firstName":"Fabio","middleName":"","lastName":"Parmeggiani","suffix":""},{"id":467108127,"identity":"7eeb37de-4550-4cc9-a35c-adb1b8580cb1","order_by":5,"name":"Margit Winkler","email":"","orcid":"","institution":"Austrian Center of Industrial Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Margit","middleName":"","lastName":"Winkler","suffix":""},{"id":467108128,"identity":"7a279140-239f-46dc-abfe-b8532ec46b62","order_by":6,"name":"Peter Both","email":"","orcid":"","institution":"Axxence Slovakia s.r.o.","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Both","suffix":""},{"id":467108129,"identity":"09ca8ed1-6bef-4587-9d06-40adb5a9735e","order_by":7,"name":"Peter Siska","email":"","orcid":"","institution":"Axxence Slovakia s.r.o.","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Siska","suffix":""}],"badges":[],"createdAt":"2025-05-27 13:01:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6759688/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6759688/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86000625,"identity":"a810ab95-3e88-42e6-a9f4-8c9535202eda","added_by":"auto","created_at":"2025-07-04 07:29:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the biobased synthesis of pyrazines.\u003c/strong\u003e In this work, starting from the simple starting material aminoacetone we gained an understanding of the transformation, thus leading to enhanced results that were further rationalized through control experiments and DFT calculations.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/caa9d131a08bfbd87d28b0f9.png"},{"id":86000619,"identity":"ac5bc2af-da00-45cb-8f74-c26b2d982212","added_by":"auto","created_at":"2025-07-04 07:29:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScope of the pyrazine formation reaction.\u003c/strong\u003e By simplifying the approach and starting from aminoketones, a broad substrate scope could be accessed, greatly varying the nature of the electrophiles employed, yielding the different target pyrazines in moderate to good isolated yields.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/f75125ae04e06ef63c62ca74.png"},{"id":86000616,"identity":"aa96e4d8-015c-47e4-85c2-7c9e9b28f8fd","added_by":"auto","created_at":"2025-07-04 07:29:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic investigations. a\u003c/strong\u003e, Adapted mechanistic proposal done by Adams \u003cem\u003eet al\u003c/em\u003e.,\u003csup\u003e26\u003c/sup\u003e used as a benchmark for this study, highlighting the two possible reaction pathways for the target transformation. Molecular oxygen triggers autooxidation of intermediate \u003cstrong\u003eII\u003c/strong\u003e, leading to the depletion of the substrate through the formation of pyrazine \u003cstrong\u003e3b\u003c/strong\u003e. \u003cstrong\u003eb\u003c/strong\u003e, Relationship between pathways A and B for each substrate of the scope, depicted as a percentage of pathway A. c, Representation of the deuteration experiment. Optimized conditions were applied, and a deuterated buffer was employed.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/50a5a82c179cc8882458a55d.png"},{"id":86001159,"identity":"8f494c14-4095-485c-b24d-45e2755d9fe4","added_by":"auto","created_at":"2025-07-04 07:37:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTautomerization experiments. \u003c/strong\u003eThe aromatic character of the final pyrazine product is critical to drive the equilibria towards the products and make the incorporation of the electrophile predominate over the oxidation pathway. Therefore, experiments with methyl acrylate \u003cstrong\u003e2y\u003c/strong\u003e, methyl propiolate \u003cstrong\u003e2n\u003c/strong\u003e, iodomethane \u003cstrong\u003e2z\u003c/strong\u003e and diiodomethane \u003cstrong\u003e2aa\u003c/strong\u003ewere carried out to reveal this effect and prove that a tautomerization step is neccesary. This hypothesis is further supported by the deuteration experiments shown in Fig. 3, \u003cstrong\u003ec.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/c57b39bea67cbaa7ab994523.png"},{"id":86000626,"identity":"5173eabf-7270-4ef3-8d9d-27670aa6de2f","added_by":"auto","created_at":"2025-07-04 07:29:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBINOL-phosphate catalysis’ types of activation. \u003c/strong\u003eFigure adapted from Rueping and co-workers\u003csup\u003e30\u003c/sup\u003e depicting the different types of phosphate-mediated activations. In the lower box, a hypothetical visual description of how this effect could take part in the transformation described in this manuscript.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/5608d567259286803dd590db.png"},{"id":86000622,"identity":"c58d58dd-4776-4060-b7b7-62d7dc0df8c7","added_by":"auto","created_at":"2025-07-04 07:29:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFree energy reaction profile describing the formation of asymmetric pyrazines. \u003c/strong\u003eThe DFT calculations have been carried out employing ωB97x-D4/def2-TZVP in implict water, described by conductor-like polarizable continuum model (CPCM). To obtain free energies (given in kJ mol\u003csup\u003e-1\u003c/sup\u003e) thermodynamic corrections have been added at 323.15 K, which is the temperature of the reaction. Explicit solvent molecules (water) have been added in cases in which the species modelled need stabilisation.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/82ca4df654fe269b38e1ef00.png"},{"id":86001161,"identity":"4832088b-b5ea-48ff-93cc-7420b702ac17","added_by":"auto","created_at":"2025-07-04 07:37:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1463888,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/459940e7-808b-4d2f-817e-eecdc69ce03f.pdf"},{"id":86000621,"identity":"6487505c-6984-442a-b4b3-4b42ca3651e9","added_by":"auto","created_at":"2025-07-04 07:29:07","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18094,"visible":true,"origin":"","legend":"XYZ coordinates","description":"","filename":"XYZfiles.rar","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/a8c5eacf90d75fb2d1b36658.rar"},{"id":86000623,"identity":"a62dcebd-d91d-4069-8c56-293a84afa0f3","added_by":"auto","created_at":"2025-07-04 07:29:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19024205,"visible":true,"origin":"","legend":"Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential","description":"","filename":"SupportingInformation5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/b8927429eff1b33a1925e68b.docx"},{"id":86000624,"identity":"e3b6663f-ce33-4a2a-b423-0dfef2c1808c","added_by":"auto","created_at":"2025-07-04 07:29:12","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24075,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/1d03956d9d20a3b4090a7215.docx"},{"id":86000620,"identity":"b204b45b-6317-4fd3-afc5-a3d9fc683f55","added_by":"auto","created_at":"2025-07-04 07:29:06","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":26072,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/5d18a92f3725cdccdfde623d.docx"},{"id":86001160,"identity":"9908e60d-9b95-452a-b93f-faed2b0526ac","added_by":"auto","created_at":"2025-07-04 07:37:06","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":27057,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6759688/v1/989fdb8734ed790872b8520e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePyrazines, volatile nitrogen-containing heterocyclic compounds, are widely distributed in plants,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e insects,\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e fungi, and bacteria,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e serving as odor signals to repel predators and protect vegetation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Asymmetric pyrazines, with diverse applications in pesticides, dyes, and pharmaceuticals,\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e are extracted from natural sources and employed to enhance flavors in various industrial foods and beverages such as coffee, cocoa, meat, and potato-products. In this sense, there is a growing demand for sustainability in industries, particularly in the food sector, driven by daily consumption and healthy eating. The flavor and fragrance industry has experienced significant growth, with global market size tripling since the early 21st century. This surge is primarily motivated by global demands for flavor and aromas -enhanced food production, spurred by consumer preferences and technological advancements that qualify products to be labeled as natural. Specifically, the European (European Food and Safety Regulation, EFSA) as well as the American (Food and Drug Administration, FDA) regulation on flavors (EEC No 1334/2008) defines natural flavoring substances as those obtained through appropriate physical, enzymatic, or microbiological processes from material of vegetable, animal, or microbiological origin, either in their raw state or after processing for human consumption using traditional food preparation methods.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e However, as of now, there are no registered biotechnological syntheses for the industrial production of pyrazine derivatives declared as natural. Consequently, extraction from natural sources remains the sole means of accessing pyrazines for this purpose. Nonetheless, their low natural abundance in sources like sugar beet molasses (0.01 wt%) has driven the search for more effective production methods. \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this sense, traditional synthetic methods, although efficient, often involve hazardous conditions unsuitable for the food industry,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e prompting efforts to develop procedures using components from natural origins. Biocatalytic approaches have emerged as dominant in this regard, offering the ability to operate under aqueous and mild conditions, aligning with the criteria for natural processes. Consequently, numerous research groups have endeavoured to devise a standardized methodology capable of facilitating the transition of the industry from extraction techniques to enzyme-based syntheses. Different fermentative and biocatalytic approaches, either employing living cells or isolated proteins have been developed. Pyrazines are ubiquitously distributed in many microorganisms,\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and some of them have been employed to produce pyrazines, although with very limited success.\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Usually, low product titers, complex pyrazine mixtures, and difficulties in isolations were encountered.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt first glance, the synthesis of pyrazines is simply based on the condensation of either diketones with diamines or the self-condensation of α-aminoketones. Based on this, Turner and co-workers developed a biocatalytic protocol employing amino transaminases through the generation of the aminoketone precursor \u003cem\u003ein situ\u003c/em\u003e, although only symmetric pyrazines could be attained in moderate yields, and an amine donor was required.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Kroutil and co-workers published a series of papers in which the discovery of a new reactivity of ene-reductases led to the formation of pyrazines.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e However, only symmetrical and heavily substituted pyrazines can be accessed through this approach.\u003c/p\u003e \u003cp\u003eThe methodologies that have come closer to the goal are hybrid approaches combining biocatalysis and organic synthesis. Peng \u003cem\u003eet al\u003c/em\u003e. proposed the microbial synthesis of acetoin in very high titers, which could then be subjected to treatment with different ammonium salts at 100\u0026deg;C to generate ligustrazine (tetramethylpyrazine).\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Also, very recently, Walker and co-workers proposed the employment of carboligases to synthesize suitable α-hydroxyketones, which, after treatment with 1,2-diamines and KOH in diethyl ether, would yield mixtures of the highly pursued short-chain asymmetric alkyl pyrazines.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e To the best of our knowledge, there was no methodology available which employed only \u0026ldquo;natural\u0026rdquo; reagents for the synthesis of these scaffolds in an asymmetric manner until the ground-breaking work of Motoyama \u003cem\u003eet al\u003c/em\u003e. combining \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e- threonine dehydrogenases and aldehydes was published in 2021.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In this research, based on a putative mechanism proposed by Adams \u003cem\u003eet al\u003c/em\u003e. for the production of these compounds through the Maillard reaction in 2008,\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e they were able to capture the dihydropyrazines intermediates with alkyl aldehydes to obtain asymmetrically substituted pyrazines. The yields were very low and the products could not be isolated.\u003c/p\u003e \u003cp\u003eIn this work, we describe the synthesis of multiple asymmetric trisubstituted pyrazines through aminoketone dimerisation followed by the incorporation of electrophiles under natural and environmentally friendly conditions. We isolated all the compounds in moderate to good yields, building the foundations for a possible future incorporation into an industrial setup. Subsequently, we coupled this methodology with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e- threonine dehydrogenase from \u003cem\u003eCupriavidus necator\u003c/em\u003e to generate the aminoacetone precursor \u003cem\u003ein situ\u003c/em\u003e from natural and inexpensive L-threonine.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Throughout this investigation, phosphate participation was identified and proven to facilitate the transformation. A detailed mechanism for this transformation is presented, supported by control experiments and computational studies, paving the way for future optimization and the design of efficient synthesis strategies for these valuable compounds.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eSelective synthesis of di- and trisubstituted pyrazines\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAsymmetrically substituted pyrazines create signature perceptions in foods and beverages and are also significant due to their pharmacological properties.\u003csup\u003e28,29\u003c/sup\u003e In the quest for an efficient synthesis and isolation of these scaffolds, we hypothesized that the preparation of trisubstituted pyrazines \u003cem\u003evia\u003c/em\u003e aminoacetone \u003cstrong\u003e1a\u003c/strong\u003e dimerisation, followed by the incorporation of an electrophile under biocompatible conditions could be achieved in a single-step manner. Thus, we selected acetaldehyde \u003cstrong\u003e2a\u003c/strong\u003e as our model electrophile due to its simplicity, availability and compatibility with biocatalytic setups and, subsequently, the reaction was carried out under standard conditions:\u0026nbsp;4 eq. of \u003cstrong\u003e2a\u003c/strong\u003e, phosphate buffer 100 mM at pH 9, 50 \u0026ordm;C, and a reaction time of 2 hours at a substrate concentration of 25 mM, which led to an impressive 81% GC yield.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough we expected some conversion conducting the reaction under completely aqueous conditions, the outcome was exceeded expectations. Next, we tested a diverse array of electrophiles to assess the generality of the protocol (Fig. 2). As a result, we successfully synthesized several aliphatic pyrazines using this methodology, including both linear (\u003cstrong\u003e3a\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and cyclic (\u003cstrong\u003e3e\u003c/strong\u003e) variants, as well as 2,5-dimethylpyrazine without the addition of any electrophile (\u003cstrong\u003e3b\u003c/strong\u003e). To address the synthesis of \u003cstrong\u003e3c\u003c/strong\u003e, which gave the lowest yield of aliphatic aldehydes, we experimented with both aqueous formaldehyde (40% w/w, stabilized with 15% MeOH, i.e. formalin) and paraformaldehyde \u003cstrong\u003e2b\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWhile the former yielded only 8%, \u003cstrong\u003e2b\u003c/strong\u003e enabled the isolation of \u003cstrong\u003e3c\u003c/strong\u003e in a modest yet appreciable yield (20%). Following this, a plethora of aromatic aldehydes underwent screening, yielding trisubstituted pyrazines. Benzaldehyde \u003cstrong\u003e2f\u003c/strong\u003e, when utilized as a reaction partner, yielded product \u003cstrong\u003e3f\u003c/strong\u003e with a 59% yield. Subsequently, both electron-donating (\u003cstrong\u003e3g\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e) and electron-withdrawing (\u003cstrong\u003e3i\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e) substituents on the phenyl ring were explored. In both scenarios, the desired pyrazines were isolated in moderate yields. As anticipated, however, a higher proportion of \u003cstrong\u003e3b\u003c/strong\u003e was observed in instances where electron-rich benzaldehydes \u003cstrong\u003e2f\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e were employed. This occurrence can be attributed to the favored reaction with oxygen over the intended pathway, owing to the lower electrophilicity of these substrates (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e, Pathway B). Conversely, heteroaromatic aldehydes such as furan-3-carboxaldehyde \u003cstrong\u003e2k\u003c/strong\u003e and thiophene-3-carboxaldehyde \u003cstrong\u003e2l\u003c/strong\u003e emerged as excellent coupling partners, likely due to their electron-poor nature alongside lower steric demands. Consequently, the corresponding pyrazines \u003cstrong\u003e3l\u003c/strong\u003e and \u003cstrong\u003e3m\u003c/strong\u003e were obtained with yields of 96% and 69%, respectively. When we attempted to employ alkynes as Michael acceptors, we were delighted to observe that but-3-yn-2-one \u003cstrong\u003e2m\u003c/strong\u003e and methyl propiolate \u003cstrong\u003e2n\u003c/strong\u003e produced the corresponding pyrazines \u003cstrong\u003e3o\u003c/strong\u003e and \u003cstrong\u003e3n\u003c/strong\u003e with yields of 41% and 67%, respectively. The lower yield obtained for the ketone derivative is mostly attributed to side reactions. We were also interested in testing the amide derivative propiolamide \u003cstrong\u003e2w\u003c/strong\u003e, but no product was formed. To investigate if the free amide could be hampering the reactivity, we synthesized \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylpropiolamide \u003cstrong\u003e2o\u003c/strong\u003e and subjected it to the optimized reaction conditions, leading to a 19% yield. Finally, we tested functionalized aldehydes as electrophiles for this transformation. Thus, we selected methylglyoxal \u003cstrong\u003e2p\u003c/strong\u003e, which would yield a complementary carbonyl compound to \u003cstrong\u003e3o\u003c/strong\u003e, and citronellal \u003cstrong\u003e2q\u003c/strong\u003e. Satisfyingly, we were able to isolate both products, \u003cstrong\u003e3q\u003c/strong\u003e with a yield of 11% and \u003cstrong\u003e3r\u003c/strong\u003e in 47% yield. To delineate the boundaries of the developed protocol, we also investigated the reactivity of ketones as reagents. However, in the case of acetone \u003cstrong\u003e2u\u003c/strong\u003e, a side reaction occurred, depleting the starting material. When acetophenone \u003cstrong\u003e2v\u003c/strong\u003e was employed, 2,5-dimethylpyrazine \u003cstrong\u003e3b\u003c/strong\u003e was isolated along with the starting ketone, reflecting the less electrophilic character of ketones. Propiolic acid \u003cstrong\u003e2w\u003c/strong\u003e did not yield the desired pyrazine \u003cstrong\u003e3y\u003c/strong\u003e when employed as a Michael acceptor, while methyl phenylpropiolate \u003cstrong\u003e2y\u003c/strong\u003e exhibited no reactivity, likely due to steric hindrance and/or excessive electron density, rendering it unreactive toward nucleophilic attack by the species described in this contribution. To define this behaviour more precisely, we explored dimethyl but-2-ynedioate \u003cstrong\u003e2z\u003c/strong\u003e as a highly deactivated alkyne, but no product \u003cstrong\u003e3aa\u003c/strong\u003e could be observed, demonstrating that only acetylenes are accepted as electrophiles for this protocol. To conclude the scope of our study, we explored the reactivity of two different starting materials. When 1-aminobutan-2-one hydrochloride \u003cstrong\u003e1b\u003c/strong\u003e was utilized without an electrophile, smooth dimerization occurred, resulting in the formation of 2,5-diethylpyrazine \u003cstrong\u003e3s\u0026nbsp;\u003c/strong\u003ewith a 57% isolated yield. Encouragingly, we observed that the addition of acetaldehyde \u003cstrong\u003e2a\u003c/strong\u003e was also well tolerated, allowing us to isolate the corresponding pyrazine \u003cstrong\u003e3t\u003c/strong\u003e with a yield of 45%. Finally, when 1-amino-3,3-dimethylbutan-2-one hydrochloride \u003cstrong\u003e1c\u003c/strong\u003e was subjected to the same conditions, no product formation was expected due to the higher steric hindrance and lower electrophilicity of the carbonyl scaffold. However, we were surprised to isolate compound \u003cstrong\u003e3u\u0026nbsp;\u003c/strong\u003ewith a low but remarkable yield of 21%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanistic investigations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter optimizing the reaction conditions and broadening the substrate scope, we recognized the importance of investigating the underlying mechanism, given the high value of the products obtained through this process. The proposed mechanistic framework is derived from the work of Adams \u003cem\u003eet al\u003c/em\u003e.,\u003csup\u003e26\u003c/sup\u003e and is illustrated in Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e. After the dimerisation of aminoketone \u003cstrong\u003e1a\u003c/strong\u003e, dihydropyrazine \u003cstrong\u003eII\u003c/strong\u003e is formed. With regard to Pathway A, this intermediate might be deprotonated by a base in the reaction media, rendering it nucleophilic enough to attack an electrophile, in the case of the example, an aldehyde. The alcohol \u003cstrong\u003eIV\u003c/strong\u003e, formed after protonation with water, could be dehydrated, rendering the unsaturated derivative \u003cstrong\u003eV\u003c/strong\u003e, which would aromatise to yield the desired pyrazine \u003cstrong\u003e3\u003c/strong\u003e. As can be observed, Pathway B is competing with Pathway A through autooxidation (by aerial oxygen) of the dihydropyrazine intermediate with molecular oxygen to yield \u003cstrong\u003e3b\u003c/strong\u003e. To determine the extent of this competition between pathways A and B, we measured the \u003cstrong\u003e3\u003c/strong\u003e/\u003cstrong\u003e3b\u003c/strong\u003e ratio of each reaction, which is shown in Fig. 3, \u003cstrong\u003eb\u0026nbsp;\u003c/strong\u003e(see Section V. in the SI file for detailed ratios). To improve reaction yields and investigate this behaviour further, we reproduced the example involving paraformaldehyde under oxygen-free conditions and measured the yield using GC to avoid introducing the volatility variable. The ratio of \u003cstrong\u003e3c\u003c/strong\u003e/\u003cstrong\u003e3b\u003c/strong\u003e increased from 90/10 to \u0026gt;99/1, indicating the absence of oxygen hindered the autooxidation reaction. Hence, we selected candidates with near 50/50 ratios\u0026mdash;citronellal derivative \u003cstrong\u003e3q\u003c/strong\u003e (58/42) and anisaldehyde derivative \u003cstrong\u003e3g\u003c/strong\u003e (43/57)\u0026mdash;and tested them under oxygen-free conditions. Selectivity improved significantly: \u003cstrong\u003e3q\u003c/strong\u003e reached 83/17 with a yield increase from 47% to 58%, while \u003cstrong\u003e3g\u003c/strong\u003e reached 61/39 with the same yield. These results confirm the hypothesis of competing pathways A and B (Fig 3, \u003cstrong\u003ea\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eNext, to prove the tautomerization step (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eVI\u003c/strong\u003e), we let the reaction between \u003cstrong\u003e1a\u003c/strong\u003e and acetaldehyde \u003cstrong\u003e2a\u003c/strong\u003e occur in deuterated phosphate buffer, adjusted to pH 9 with sodium deuteroxide, expecting major deuteration in the secondary carbon of the side chain (Fig. 3, \u003cstrong\u003ec\u003c/strong\u003e). We were glad to observe an 89% incorporation of deuterium, thus pointing again towards our hypothesis. A 99% incorporation was also detected in the only possible position of the aromatic ring, which is in accordance with how highly labile those protons are in \u0026alpha; position, between the amine and the carbonyl scaffold in the starting material \u003cstrong\u003e1a\u003c/strong\u003e. To demonstrate further that the recovery of aromaticity and, therefore, tautomerization is the driving force for the incorporation of the electrophile (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e, VI), we designed two different experiments, which are depicted in Fig. 4. In the first, methyl acrylate \u003cstrong\u003e2y\u003c/strong\u003e (A) and methyl propiolate \u003cstrong\u003e2n\u003c/strong\u003e (B) were studied as electrophiles. As we hypothesized, when example A was followed, there were not enough electrons in the system for it to be able to aromatise through tautomerization. Therefore, the autooxidation pathway (Fig 3, \u003cstrong\u003ea\u003c/strong\u003e, Pathway B) was favoured and only starting materials \u003cstrong\u003e1a\u003c/strong\u003e and \u003cstrong\u003e3b\u003c/strong\u003e were recovered. However, when methyl propiolate \u003cstrong\u003e2n\u003c/strong\u003e was employed, the molecule had enough electrons to rearrange towards aromatic pyrazine, thus yielding \u003cstrong\u003e3o\u003c/strong\u003e with a remarkable 67% yield. Secondly, we tried to reproduce this same philosophy this time employing iodomethane \u003cstrong\u003e2z\u003c/strong\u003e and diiodomethane \u003cstrong\u003e2aa\u003c/strong\u003e as electrophiles (Fig. 4, \u003cstrong\u003eb\u003c/strong\u003e). In this second case, the employment of \u003cstrong\u003e2aa\u003c/strong\u003e (A) leads to an intermediate that possesses no other leaving group, rendering it unable to gain aromaticity. However, when diiodomethane was employed (B), yields comparable to the ones obtained with formaldehyde in solution (8%) were achieved, which demonstrates again the need for the dihydropyrazine intermediate to recover aromaticity, which we propose to be the driving force of the reaction, also in accordance with the deuteration experiment (Fig. 3, \u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe next sought to further investigate the necessary conditions for the dimerization reaction to occur (Table 1). We first conducted the reaction in carbonate buffer and water at pH 9, to check how phosphate influences the reaction (Table 1, entries 1-3). We were surprised to observe how important it was for this transformation to succeed. When carbonate was employed, the yield decreased to 42% (Table 1, entry 2) and preparing water at pH 9 employing the minimum amount required of NaOH led to a poor yield of 9% (Table 1, entry 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results strongly indicate that phosphate and a finely adjusted pH are minimum requirements for this reaction, and also that the salts present should be promoting the reaction rather than only buffering the system. To improve the solubility of the substrate and, hypothesizing that water might not be necessary for the transformation and could even hinder it, given that two dehydration steps are involved, we tried to carry out the transformation employing several organic solvents, namely MeOH, THF and CPME (Table 1, entries 4-6) but almost no conversion to any pyrazine could be observed.\u003c/p\u003e\n\u003cp\u003eAs a further proof of phosphate catalysing this reaction, we explored microaqueous reaction system (MARS) conditions,\u003csup\u003e31\u003c/sup\u003e by using CPME with only 10 \u0026mu;L KP\u003csub\u003ei\u003c/sub\u003e buffer 100 mM pH 9, and the result was an astonishing 36% yield (Table 2, entry 7). In order to rationalize these results, and considering that aminoacetone was added as a hydrochloride salt, we measured the pH in aqueous samples of the crude mixtures after the reaction was completed. We observed a significant drop in pH (Table 1, entries 1\u0026ndash;3), which explains the lack of reactivity in pure water. However, the huge difference between carbonate and phosphate was still not clear. We attribute this behavior to the consumption of aminoacetone in the reaction medium, which decreases the amount of base present. This trend can also be observed in the Knorr pyrrole reaction.\u003csup\u003e32\u003c/sup\u003e With these results in hand, we hypothesized that phosphate could be carrying out a reactivity similar to the one that of a traditional BINOL-phosphate derived catalyst in which several functional groups can be activated employing this anion (Fig. 5).\u003csup\u003e30\u003c/sup\u003e This process consists of the HOMO raising of a nucleophile or the LUMO lowering of an electrophile through hydrogen bonding between the different parts of the phosphate molecule. Such activation can occur in different manners: mono, dual and bifunctional activation. In our specific case, as the resulting pH is 7.5, the predominant species in solution is expected to be the hydrogen phosphate anion\u003csup\u003e33\u003c/sup\u003e which may carry out the three types of activation depicted in Fig.5. During these experiments, we detected a byproduct in the reaction, which we identified as 5-ethyl-3,6-dimethyl-2-(1-hydroxyethyl)pyrazine \u003cstrong\u003e4a\u0026nbsp;\u003c/strong\u003e(Table 2), that was obtained after a double deprotonation between intermediates \u003cstrong\u003eIII\u003c/strong\u003e-\u003cstrong\u003eV\u003c/strong\u003e (Figure 3, \u003cstrong\u003ea\u003c/strong\u003e, Pathway A). The formation of \u003cstrong\u003e4a\u003c/strong\u003e from \u003cstrong\u003e3a\u003c/strong\u003e was ruled out by incubating \u003cstrong\u003e3a\u003c/strong\u003e at the optimal conditions for 24 h - \u003cstrong\u003e3a\u003c/strong\u003e was isolated unaltered. To further rationalize the buffer influence as well as the formation of this byproduct, we studied different conditions, as described in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Table 2, we observe the crucial role of the buffer in the reaction. By selecting anions with similar properties to phosphate, such as HEPES and MOPS (Table 2, entries 2\u0026ndash;3), we noted that the reaction proceeds with higher yields compared to when carbonate is used (Table 1, entry 2), likely due to a similar effect as when phosphate is employed. An interesting observation is that with these two buffer systems, the byproduct ratio decreases significantly, suggesting an activation effect occurring between intermediates \u003cstrong\u003eIII\u003c/strong\u003e-\u003cstrong\u003eV\u003c/strong\u003e (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e, Pathway A). Lower pH resulted in lower yields and \u003cem\u003evice versa\u003c/em\u003e. However, the byproduct ratios were not significantly affected. Finally, we reduced the buffer concentration to improve the environmental impact of the transformation. However, insufficient buffer capacity resulted in significantly lower yields (Table 2, entries 6\u0026ndash;9). Interestingly, this also led to a lower \u003cstrong\u003e3a\u003c/strong\u003e/\u003cstrong\u003e4a\u003c/strong\u003e ratio, strengthening the hypothesis of the catalysis occurring in one of the steps between \u003cstrong\u003eIII\u003c/strong\u003e and \u003cstrong\u003eVI\u003c/strong\u003e (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e, Pathway A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further investigate these results, we carried out autotitration experiments keeping the pH constant (pH 8). Even though the yield employing pure water was higher (40%), again highlighting the importance of pH for this transformation, the yield using KP\u003csub\u003ei\u003c/sub\u003e buffer under the same conditions was 22% higher (see SI, section V.4 for further details). In addition, HEPES, water and KP\u003csub\u003ei\u0026nbsp;\u003c/sub\u003eshowed different side-products profiles. As further proof, we carried out these autotitration experiments employing different concentrations of KP\u003csub\u003ei\u003c/sub\u003e, 100 mM, 10 mM and 1 mM. In all cases, the yield increased in a significant manner indicating a catalytic mechanism. These facts together serve as a final proof of the buffer effect in the system. In order to figure out where and how this phenomenon could be taking place and elucidate the complete mechanism, we conducted DFT calculations which will be discussed in the next section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantum Chemical Investigations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter discovering buffer intervention in our system, we conducted extensive DFT calculations to support our findings. These calculations also serve as a valuable tool for refining or developing future methodologies for asymmetric pyrazine synthesis. Combined with our experimental data, we investigated the pyrazine formation process, which, to our knowledge, had not been elucidated before. We first considered the formation of the asymmetric pyrazine starting from specie \u003cstrong\u003eII\u003c/strong\u003e (Fig 6). The first step (\u003cstrong\u003eII\u003c/strong\u003e-\u003cstrong\u003eIII\u003c/strong\u003e), a hydroxyl-mediated proton abstraction, shows a very low barrier of 16.1 kJ/mol, which is consistent with how electron-poor the dihydropyrazine \u003cstrong\u003eII\u003c/strong\u003e is and its antiaromatic character. Interestingly, the nucleophilic attack from specie \u003cstrong\u003eIII\u003c/strong\u003e to acetaldehyde also showed a very low barrier (\u003cstrong\u003eIII\u003c/strong\u003e-\u003cstrong\u003eIV\u003c/strong\u003e, 14.8 kJ/mol). This might not be intuitive at first, but matches the experimental results regarding the competing pathways A and B (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e). As the \u003cstrong\u003e3a\u003c/strong\u003e/\u003cstrong\u003e3b\u003c/strong\u003e ratio is 90/10, the energetic barrier has to be quite low to overcome the autoxidation with molecular oxygen. The protonation step \u003cstrong\u003eIV\u003c/strong\u003e-\u003cstrong\u003eV\u003c/strong\u003e has no barrier in the electronic surface, so we assumed that it is very low. Next, the most favorable development was the deprotonation of the substituted carbon (\u003cstrong\u003eV\u003c/strong\u003e-\u003cstrong\u003eVI\u003c/strong\u003e), showing an energetic barrier of 18.7 kJ mol\u003csup\u003e-1\u003c/sup\u003e, which was followed by a hydroxyl elimination step \u003cstrong\u003eVI\u003c/strong\u003e-\u003cstrong\u003eVII\u003c/strong\u003e, the rate-limiting step of the reaction, with an activation energy of 91.0 kJ/mol, which is consistent with our experimental data. These two steps describe a traditional E1cB mechanism which was expected due to our reaction conditions (protic media and a poor leaving group). This elimination was confirmed, contrasting it with E1- and E2-type eliminations, which modeling showed to be unviable. The final tautomerization step (\u003cstrong\u003eVII\u003c/strong\u003e-\u003cstrong\u003e2\u003c/strong\u003e) is barrierless, as expected for a rearomatization, and we believe it acts as a thermodynamic well which drives the reaction equilibrium forward. This tautomerization step has been also proven experimentally through deuteration experiments and employment of different electrophiles (Fig 3., \u003cstrong\u003eb\u0026nbsp;\u003c/strong\u003eand Fig. 4). In the search for the role of hydrogen phosphate, we turned our attention towards the formation of dimer \u003cstrong\u003eI\u003c/strong\u003e (Fig. 3, \u003cstrong\u003ea\u003c/strong\u003e). When we first simulated this step of the mechanism just using hydroxyl anions as a base, the energy barrier obtained was low, (49.8 kJ\u0026middot;mol\u003csup\u003e-1\u003c/sup\u003e), indicating that the reaction should be feasible, although the need for a slightly basic pH discards the possibility of running it in pure water (Table 1). Next, we modeled this step with hydrogen phosphate as a potential catalyst, considering all possible scenarios outlined in Fig. 5. Optimizations of all reactants consistently converged toward the dual activation case (Fig. 5, lower box), which we selected as our starting point. We then calculated the HOMO-LUMO gap for two models\u0026mdash;one with a protonated amine moiety and one unprotonated\u0026mdash;both of which were low enough to suggest potential activation in this dimerisation step. However, attempts to model the transition states for both cases proved unviable, making catalysis in this initial step less likely. Next, we attempted to model the case of activation of intermediate \u003cstrong\u003eVI\u003c/strong\u003e with hydrogen phosphate through hydrogen bonding, which would lower the energetic barrier of the hydroxyl elimination (Fig. 6). Firstly, we analised the HOMO-LUMO gaps of the species involved, which were qualitatively lowered with regard to the same step without any activation. However, our calculations did not yield a converged transition state in these cases, so we could not demonstrate the hydrogen phoshate catalysis neither in this step. Nevertheless, the experimental results strongly indicate the involvement of salts in the reaction. This discrepancy suggests that additional investigation is needed to better understand their role and underlying mechanism. Finally, although previous studies carried out with this type of molecules supported the dimerisation of aminoketones towards pyrazines, we decided to model ourselves the formation of intermediate \u003cstrong\u003eII\u003c/strong\u003e.\u003csup\u003e34\u0026nbsp;\u003c/sup\u003eAccording to our calculations, the proccess should be energetically favored with an overall free energy of -125.8 kJ mol\u003csup\u003e-1\u003c/sup\u003e (see section VI in the SI file for further details).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA biocatalytic cascade for the synthesis of pyrazines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith the reactivity of the system established and the mechanism elucidated, we turned our attention to developing a biocatalytic process for the \u003cem\u003ein situ\u003c/em\u003e generation of the key intermediate, aminoacetone \u003cstrong\u003e1a\u003c/strong\u003e. We selected l-threonine dehydrogenase from \u003cem\u003eCupriavidus\u003c/em\u003e \u003cem\u003enecator\u003c/em\u003e\u003csup\u003e27\u003c/sup\u003e (\u003cem\u003eCn\u003c/em\u003eThrDH) and evaluated its performance in the model reaction with acetaldehyde \u003cstrong\u003e2a\u003c/strong\u003e as the electrophile.\u0026nbsp;Under the initial reaction setup\u0026mdash;10 mg lyophilized cell-free extract (CFE), 25 mM l-Thr in 100 mM KPi (pH 8), 4 equivalents of \u003cstrong\u003e2a\u003c/strong\u003e, and 1 equivalent of NAD\u003csup\u003e+\u003c/sup\u003e at 40 \u0026deg;C for 16 h\u0026mdash;the product \u003cstrong\u003e3a\u003c/strong\u003e was obtained in 59% yield (Table 3, entry 1), outperforming previously reported systems.\u003csup\u003e25\u003c/sup\u003e Control experiments confirmed that both \u003cem\u003eCn\u003c/em\u003eThrDH and NAD\u003csup\u003e+\u003c/sup\u003e were essential for the observed transformation (Table 3, entries 2-3). We then sought to increase the supply of \u003cstrong\u003e1a\u003c/strong\u003e by increasing the l-Thr concentration, while proportionally increasing the amount of \u003cstrong\u003e2a\u003c/strong\u003e and NAD\u003csup\u003e+\u003c/sup\u003e. However, this led to a substantial reduction in product formation (Table 3, entry 4), suggesting enzyme inhibition. To counteract this, we increased the reaction temperature to 50 \u0026deg;C and reduced the concentration of \u003cstrong\u003e2a\u003c/strong\u003e to alleviate aldehyde-induced enzyme deactivation.\u003csup\u003e36\u003c/sup\u003e Notably, decreasing \u003cstrong\u003e2a\u003c/strong\u003e to 2 equivalents\u0026mdash;matching its concentration in the initial reaction setup\u0026mdash;restored the yield of \u003cstrong\u003e3a\u003c/strong\u003e to 29% (Table 3, entry 7), indicating this level may approximate the enzyme\u0026rsquo;s tolerance limit for the electrophile. In parallel, we reverted the NAD\u003csup\u003e+\u003c/sup\u003e loading to that used in the initial reaction setup, which improved catalytic performance and led to a 52% yield of \u003cstrong\u003e3a\u003c/strong\u003e (Table 3, entry 8). Interestingly, the total amount of pyrazine products (\u003cstrong\u003e3a\u003c/strong\u003e + \u003cstrong\u003e3b\u003c/strong\u003e) reached 68%, exceeding the theoretical maximum yield based on the NAD\u003csup\u003e+\u003c/sup\u003e stoichiometry (50%) and suggesting partial NAD\u003csup\u003e+\u003c/sup\u003e regeneration by components of the crude extract. Further decreasing \u003cstrong\u003e2a\u003c/strong\u003e to equimolar amounts increased the total pyrazine yield to 75%, though with an equimolar distribution of \u003cstrong\u003e3a\u003c/strong\u003e and the disubstituted product \u003cstrong\u003e3b\u003c/strong\u003e (Table 3, entry 9), underscoring the effect of electrophile concentration on product distribution and chemoselectivity.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study establishes a mechanistically grounded platform for synthesizing asymmetric pyrazines through aminoketone dimerization and electrophilic trapping. By systematically exploring substrate scope, we achieved di- and trisubstituted pyrazines (e.g., \u003cb\u003e3a\u003c/b\u003e\u0026ndash;\u003cb\u003e3u\u003c/b\u003e) in 20\u0026ndash;96% isolated yields using diverse electrophiles, including aliphatic/aromatic aldehydes, alkynes, and functionalized carbonyl compounds. Key to success was optimizing aqueous phosphate buffer conditions (pH 9, 50\u0026deg;C), which suppressed autooxidation while enabling efficient trapping of dihydropyrazine intermediates. DFT calculations and control experiments revealed three critical steps in the mechanism: first, the E1cB elimination of water from the aminoketone dimer (ΔG\u0026Dagger; = 18.3 kcal/mol) generates a conjugated enamine intermediate; second, tautomerization to a nucleophilic dihydropyrazine species (II in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) occurs, potentially stabilized by phosphate buffer through hydrogen-bonding interactions; and third, electrophilic trapping proceeds via competing pathways, where Pathway A involves Michael addition to aldehydes or alkynes and is favored under oxygen-free conditions, while Pathway B leads to autooxidation forming symmetric pyrazines and dominates with electron-rich electrophiles. Oxygen exclusion increases selectivity for Pathway A by 1.4 to 2.3 times in challenging cases (for example, the 3q product ratio changes from 58/42 to 83/17 relative to 3/3b). Experimental validation confirmed phosphate\u0026rsquo;s role as a stabilizer for transition states, hypothesizing the necessity of the buffer for achieving high yields.\u003c/p\u003e \u003cp\u003eCoupling this chemistry with L-threonine dehydrogenase enabled a fully biocatalytic cascade from renewable L-threonine to flavor-relevant pyrazines. The tandem system operates under aqueous, ambient conditions (25\u0026deg;C, pH 7.5), aligning with EU/FDA \u0026ldquo;natural\u0026rdquo; labeling requirements while bypassing hazardous reagents. This work provides both a synthetic toolbox for asymmetric pyrazines and a mechanistic framework for optimizing similar biocatalytic-electrophilic cascades in heterocycle synthesis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMurray, K. 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Mechanism of acetaldehyde-induced deactivation of microbial lipases. \u003cem\u003eBMC Biochem.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 10 (2011).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6759688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6759688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePyrazines are pivotal flavor compounds with widespread applications in the food, pharmaceutical, and chemical industries. Their natural abundance is low, and traditional synthetic methods often involve hazardous conditions unsuitable for the food sector. In this study, we present a novel biocatalytic methodology for synthesizing asymmetric trisubstituted pyrazines using aminoacetone dimerization followed by electrophile incorporation under environmentally benign conditions. The approach employs L-threonine dehydrogenase from Cupriavidus necator to generate aminoacetone in situ from natural L-threonine, integrating biocatalysis with green chemistry principles. Detailed mechanistic investigations, supported by control experiments and DFT calculations, revealed the critical role of phosphate buffering, an E1cB elimination and a tautomerization-driven pathway for product formation. The methodology demonstrates broad substrate scope and scalability, yielding pyrazines with diverse structural modifications up to 96% yields. This work establishes a sustainable framework for the industrial production of asymmetric pyrazines, addressing current regulatory and environmental demands in the flavor and fragrance sector.\u003c/p\u003e","manuscriptTitle":"Biocatalytic Synthesis of Asymmetric Pyrazines: Mechanistic Insights and Industrial Potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 07:29:00","doi":"10.21203/rs.3.rs-6759688/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"19c0c1a3-d029-4efd-8c08-df47ed4b34ab","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49595224,"name":"Physical sciences/Chemistry/Green chemistry/Sustainability"},{"id":49595225,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":49595226,"name":"Physical sciences/Chemistry/Organic chemistry/Reaction mechanisms"}],"tags":[],"updatedAt":"2025-07-04T07:29:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-04 07:29:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6759688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6759688","identity":"rs-6759688","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}