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Repurposing non-heme iron hydroxylases to enable catalytic nitrile installation through an azido group assistance Madison Davidson, Meredith McNamee, Ruixi Fan, Yisong Guo, and Wei-chen Chang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13906 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019
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Journal of the American Chemical Society
Repurposing non-heme iron hydroxylases to enable catalytic nitrile installation through an azido group assistance Madison Davidson,a Meredith McNamee,a Ruixi Fan,b Yisong Guo,*,b Wei-chen Chang,*,a Department of Chemistry, North Carolina State University, Raleigh, NC 27695 b Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213 Supporting Information Placeholder a
ABSTRACT: Three mononuclear non-heme iron dependent enzymes, L-Ile 4-hydroxylase, L-Leu 5-hydroxylase and polyoxin dihydroxylase, are reported to catalyze the hydroxylation of Lisoleucine, L-leucine and L-α-amino--carbamoylhydroxyvaleric acid (ACV). In this study we showed that these enzymes can accommodate leucine isomers and catalyze regiospecific hydroxylation. Based on these results, as a proof-of-concept, we demonstrated that the outcome of the reaction can be redirected by installation of an assisting group within the substrate. Specifically, instead of canonical hydroxylation, these enzymes can catalyze non-native nitrile group installation when an azido group is introduced. The reaction is likely to proceed through C-H bond activation by an Fe(IV)-oxo species, followed by azido-directed C≡N bond formation. These results offer a unique opportunity to investigate and expand the reaction repertoire of Fe/2OG enzymes.
Amino acids are an important and widely used class of small molecules in chemical and biochemical applications. However, the reactive nature of the carboxylate and amine moieties of amino acids hampers the development of synthetic approaches to directly utilize these molecules as reaction substrates. In contrast, amino acids are common substrates for enzymatic transformations found in nature. For example, mononuclear non-heme iron and 2oxoglutarate dependent (Fe/2OG) enzymes have been reported to utilize amino acids as substrates to carry out regio- and stereospecific hydroxylation.1-4 In Fe/2OG enzymes, non-native reactivity is generally achieved by changing the coordinating ligand or amino acid residues around the iron center of the enzyme. For example, in the halogenase, SyrB2, replacement of a halide that is coordinated to the iron center with an azide altered the reaction outcome from halogenation to azidation.5 A second example is the structure-guided mutation study that converted SadA, a hydroxylase, into a halogenase.6 In contrast, for cytochrome P450 enzymes, non-native chemical transformations, such as C-C, C-N, or C-Si bond formations, have been achieved by using substrate analogs.7-10 Even though redirecting reaction outcomes using substrate analogs has been reported in Fe/2OG enzymes, e.g. replacing L-Thr with L-norvaline changes reactivity of SyrB2 from chlorination to hydroxylation,11 expanding Fe/2OG enzyme reaction repertoire and investigating plausible reaction pathways using a substrate-oriented approach has not been carefully elucidated. Inspired by the mechanistic understanding of Fe/2OG enzyme catalyzed hydroxylation,3, 12-13 herein, as a proof-of-concept, we used an azido group as the assisting group to divert the reaction outcome from hydroxylation to nitrile formation on an amino acid substrate in three Fe/2OG hydroxylases.
Me Me
IDO
CO2H
NH2 L-isoleucine
Me
NH2
L-leucine
O
ACV
CO2H NH2
5
LdoA
CO2H
HO Me
2
O H 2N
Me
OH
1
CO2H
Me
4
Me
CO2H NH2
NH2 OH
O
PolL H 2N
O
4
3
OH
CO2H NH2
Figure 1. Hydroxylation catalyzed by IDO, LdoA and PolL. For this initial study, we focused on three enzymes, L-Ile 4hydroxylase (IDO), L-Leu 5-hydroxylase (LdoA) and polyoxin dihydroxylase (PolL), which are known to hydroxylate L-leucine isomers and L-α-amino--carbamoylhydroxyvaleric acid (ACV)1418 (Figure 1). During Fe/2OG enzyme catalyzed hydroxylation, the reaction is initiated by a reaction between Fe(II) center with O2 and 2OG to form an Fe(IV)-oxo (Fe(IV)=O) species, succinate and CO2. The reaction is followed by C-H bond activation, or so called hydrogen atom transfer (HAT), to produce an Fe(III)-OH intermediate and an substrate radical. Formation of a C-O bond though the hydroxyl rebound mechanism completes the hydroxylation and reforms the Fe(II) species (Scheme 1a).1-4 We reasoned that if the minimum substrate structural requirements to maintain efficient substrate binding were achieved, a functional group, such as N3, could be appended onto the substrate to interact with the intermediate, and redirect the reaction outcome. (Scheme 1b). We first established substrate flexibility and reactivities of these enzymes. IDO, LdoA and PolL were overexpressed and purified (see SI). To test the activity and the substrate promiscuity of these enzymes, an anaerobic reaction mixture containing enzyme (IDO, LdoA or PolL), Fe(II), 2OG and substrate (1, 2, or 3) with final concentrations of 110 µM enzyme, 100 µM Fe(II), 1.0 mM 2OG, and 0.5 mM of substrate was prepared. The reaction mixture was then exposed to air for 20 minutes to react with O2. Analysis by liquid chromatography coupled mass spectrometry (LC-MS) revealed that IDO and LdoA have a strong preference for catalyzing C4- or C5-hydroxylation (m/z: 132.1 148.1) of L-leucine isomers, respectively; with the exception that L-isoleucine (1) cannot be converted by LdoA. These results are consistent with previously reported IDO and LdoA reactivity.16-18 In addition, PolL shows high fidelity for C4-hydroxylation towards all tested leucine
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isomers with equal or better efficiency than those of IDO or LdoA under the current conditions (Figure 2 and S21a).
a D/N H
FeII H
b N3
H
H
R
O O O
(2OG)
H R
OH FeIII
FeII
R FeII
H N N
H
NC
H 2O N
N
OH
R
H H 2O
+ R H ,N2
H
H 2O
N OH
NH2
1
CO2H
4
N
ii-a
N2
CO2H Me
N3 4
R
FeII
R
R
N
Me Me
i-a
HO
i-b
ii-b
H
R N FeIV
H 2O
c
H
HO
N2
HO FeII
FeII succ
OH
N
OH
R succ
III
Fe
R
-OH rebound N N
E.T.
H
O
(succ)
H
N3
R
O
CO2
H
CO2-
O O FeIV
CO2-
R O FeIV H C-H activation N3
R
O2
CO2H Me
NC
NH2 CO2H HO N
NH2 CO2H
NH2
2
CO2H
Me
NH2 5 N3
5
3
CO2H NH2
6
CO2H
8 NH2 7 NH2 Scheme 1. a. A generally accepted mechanism of the Fe/2OG enzyme catalyzed hydroxylation. b. The possible pathways for azido-directed nitrile group formation. c. Substrates and products used in this study.
hydroxylases.13, 19 A reaction mixture containing 0.6 mM PolL, 0.54 mM Fe(II), 12 mM 2OG, and 3 mM 3 was prepared anaerobically. The reaction was initiated by mixing the solution containing the PoIL•Fe(II)•2OG•3 complex (the enzyme quaternary complex) with O2 saturated buffer at 5 oC. A rapid depletion of an absorption feature centered at ~ 520 nm was detected up to ~ 0.1 s, which was followed by an almost complete recovery of this spectral feature after ~ 2.0 s. Meanwhile, a spectral feature centered ~ 320 nm developed to a maximum within ~ 0.03 s and subsequently decayed (Figure 3). Compared with the presteady state kinetics reported on several characterized Fe/2OG enzymes, the former absorption feature (~520 nm) decay can be attributed to the kinetics of FeII-2OG metal-to-ligand charge transfer band, which reflects the rapid reaction of the enzyme quaternary complex with O2 (the depletion of the 520 nm band).13, 19-21 It is then followed by reformation of the PoIL•Fe(II)•2OG complex (the reformation of the 520 nm band). The latter absorption feature (~320 nm) change can be attributed to the kinetics of the Fe(IV)=O intermediate.19, 22 A kinetic simulation using a three step kinetic model (PoIL•Fe(II)•2OG•3 + O2 Fe(IV)=O PoIL•Fe(II)•product PoIL•Fe(II)•2OG•3) derived the following rate constants: k1 ~ 60 mM-1 s-1; k2 = 172 s-1; and k3 = 61 s-1. The same model has been used to delineate the pre-steady state kinetic behaviors of several Fe/2OG enzymes, such as TauD and SyrB2.13, 19, 23 Thus, PoIL catalyzed hydroxylation follows a similar mechanism as that of other Fe/2OG enzymes where an Fe(IV)=O species is used as the key intermediate to carry out HAT. Combined with the LC-MS results, these observations suggest that all three enzymes recognize the L-leucine isomers and can trigger HAT to afford the corresponding hydroxylated products. In addition, these enzymes are promiscuous toward substrates and may serve as good candidates to test the azide-assisted reaction outcome hypothesis.
Figure 3. SF-Abs of PoIL reaction with 3. Left: Change in absorbance at various times after mixing the PoIL•Fe(II)•2OG•3 with O2. The spectra were obtained by subtracting the spectrum at 0.002 s. Right: Kinetic traces used to follow the kinetics of the Fe(IV)=O intermediate (320 nm) and the quaternary complex (520 nm). Simulations are shown in black.
Figure 2. LC-MS chromatograms of IDO, LdoA and PolL catalyzed regiospecific hydroxylation when 1-3 were used. IDO and PolL catalyze C4-hydroxylation and LdoA catalyzes C5hydroxylation. All peaks are detected at the corresponding hydroxylation m/z values. Next, we carried out kinetics studies using stopped-flow absorption spectroscopy to elucidate the reaction intermediates and to illustrate that the hydroxylation carried out by PolL follows a similar reaction mechanism as that of other Fe/2OG
Based on the specific HAT site preference of IDO, LdoA, and PolL, mechanistic probes with site-specifically incorporated azide group were designed. Namely, the probes (4 and 6) having an azido group appended next to C4 and C5 were prepared. Comparing to the established hydroxylation profile, we anticipate that IDO, LdoA and PolL can employ 4 and 6 as the substrates. During the IDO and PolL reactions with 4, we envision that the reaction will likely proceed via C4-HAT, and followed by hydroxylation. The Fe(II) center would then engage with the azido group to form the corresponding Fe(IV)-nitrene intermediate. A Fe(IV)-nitrene intermediate has been recently reported in P450 enzyme catalyzed C-N formation.24 Due to its high reactivity, the nitrene species would rearrange to an oxime or an iminol and then be converted to the corresponding nitrile 5 (pathway i, scheme 1b). Alternatively,
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Journal of the American Chemical Society the substrate radical can be stabilized by the azido group and further converted to a carbocation species via an electron transfer step. In this pathway, the cationic species can be used as the key intermediate to trigger nitrile formation (pathway ii, scheme 1b). For LdoA, similar pathways can be envisioned where 6 is converted to 7 through C5-HAT, respectively. To test the hypothesis that a substrate analog can enable a designed reaction outcome, enzymatic assays using 4 or 6 as the substrate were carried out under analogous conditions as described above. LC-MS analysis of the PolL catalyzed reaction with 4 showed a peak with the corresponding m/z value of the predicted nitrile product (m/z: 145.1 115.1, Figure 4a). Meanwhile, no hydroxylation could be detected. When 2OG or O2 was absent, this putative nitrile product was not observed. To validate this result, the nitrile product (5) was prepared synthetically and showed the same retention time and isotope distribution as the product obtained from the PolL reaction. Compound 4 was also tested with IDO and LdoA. No obvious product formation, i.e. hydroxylation or nitrile formation, was detected. Combined with the C4-hydroxylation when 1-3 were used, PolL catalyzed nitirile formation likely initiates from C4-HAT. When LdoA was used, no reactivity was due to the replacement of C5, the preferred C-H activation site, by the N3. Following the same hypothesis, one would expect that LdoA can convert 6 to the corresponding 7 via C5-HAT, and PolL and IDO will produce the corresponding C4-hydroxylated product. Indeed, when 6 was tested with LdoA, the major product detected has an identical retention time and m/z value (m/z = 129.1) as the synthetic nitrile 7 (Figure 4b). In contrast, the PolL reaction with 6 revealed a possible hydroxylated product (m/z: 159.1 171.1, Figure 4c). No obvious hydroxylation or nitrile formation was detected when IDO was employed. Combining with the regiospecific hydroxylation results, it is likely that nitrile (5 and 7) formation catalyzed by PoIL and LdoA is initiated by C4-H and C5H bond activation, respectively. The reaction is then followed by the azido group directed nitrile formation. In contrast, IDO is unable to employ either 4 or 6 as a substrate. Compared to the steady state kinetics of LdoA catalyzed nitrile formation (KM = 1.30 ± 0.32 mM, kcat = 1.90 ± 0.18 min-1), the observed kcat for nitrile formation is ~ 2 fold higher for PolL (KM = 1.49 ± 0.65 mM, kcat = 4.08 ± 0.74 min-1, Figure S22). Under the current conditions, PolL and LdoA can catalyze nitrile formation of 4 and 6 with total turnover numbers of ~ 150 and 180, respectively.
Figure 4. LC-MS chromatograms of IDO, LdoA and PolL reaction with 4 and 6. (a) and (b) PolL and LdoA can catalyze nitrile 5 and 7 formation. (c) PolL is able to catalyze hydroxylation using 6. All analytes are detected using selected ion monitoring. To probe the possible reaction pathway, the oxime analog (8) was prepared. We reasoned that if an oxime is an intermediate, one should expect to observe a formation of the nitrile (5) when 8 is incubated with PolL. A similar observation has been demonstrated in a heme-containing enzyme, aldoxime dehydratase, and a Reiskecluster containing enzyme, toluene dioxygenase, where incubating the proposed oxime intermediates with enzymes produced the corresponding nitriles.25-28 In our case, no nitrile product (5) formation can be detected, even after 60 minute incubation with 8 (Figure S20). The competition experiment using 8 and 2 suggests that 8 binds to the active site of PolL and prevents hydroxylation of 2 (Figure S21b). Even though it is still possible that 8 binds to PoIL at different orientation than L-leucine isomers or 4, these results imply that the pathway involving an oxime as the intermediate is unlikely to operate in the PolL catalyzed nitrile formation and is different from the proposed mechanism where the nitrile is generated through an oxime intermediate.25-26 In PolL and LdoA reaction, the pathway involving an iminol or a cationic specie is likely to be utilized. In summary, these results represent the first example of using an assisting group approach to alter the reaction outcome of Fe/2OG enzyme catalysis from hydroxylation to a designed non-native reaction outcome. Through initial studies, we propose that nitrile formation likely initiates from regioselective C-H bond activation by an Fe(IV)-oxo species. Next, the reaction may proceed through hydroxylation. Followed by N2 departure, the nitrene species rearranges to an iminol intermediate, which further converts to the nitrile (pathway i-b, scheme 1b). Alternatively, subsequent to HAT, the cation intermediate can be produced.29 The reaction is followed by deprotonation and N2 elimination to complete nitrile formation (pathway ii-a, scheme 1b). The second pathway is analogous to the Schmidt reaction.30-31 The pathway involving an oxime intermediate25-26 is unlikely to operate in PolL catalyzed nitrile formation. This work reveals the plausibility to use the assisting group approach to redirect reaction outcome. It also underscores the importance of employing mechanistic understanding to
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redesign substrates in order to enhance the generality of Fe/2OG enzymes as potential biocatalysts.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental methods and Figures S1-22.
AUTHOR INFORMATION Corresponding Author *
[email protected] *
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by North Carolina State University and Carnegie Mellon University. M.M. acknowledges Undergraduate Research Summer Grant at North Carolina State University.
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28. Oinuma, K.; Hashimoto, Y.; Konishi, K.; Goda, M.; Noguchi, T.; Higashibata, H.; Kobayashi, M., Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23. Sequencing, gene expression, purification, and characterization. J. Biol. Chem. 2003, 278 (32), 29600-8. 29. Chang, W.-c.; Dey, M.; Liu, P.; Mansoorabadi, S. O.; Moon, S. J.; Zhao, Z. K.; Drennan, C. L.; Liu, H.-w., Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction. Nature 2013, 496 (7443), 114-8. 30. Rokade, B. V.; Prabhu, K. R., Chemoselective Schmidt reaction mediated by triflic acid: selective synthesis of nitriles from aldehydes. J. Org. Chem. 2012, 77 (12), 5364-70. 31. Lamani, M.; Devadig, P.; Prabhu, K. R., A non-metal catalysed oxidation of primary azides to nitriles at ambient temperature. Org. Biomol. Chem. 2012, 10 (14), 2753-9.
C
H
R
hydroxylation
R
C
Fe/2OG enzyme
C
N3
H
nitrile installation
OH non-native reactivity
R
assistant group
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