Synthesis of Imidazolidin-4-ones via a Cytochrome P450-Catalyzed Intramolecular C-H Amination

Article abstract of DOI:10.1021/acscatal.6b02189

Expanding Nature's catalytic repertoire to include reactions important in synthetic chemistry opens new opportunities for biocatalysis. An intramolecular C-H amination route to imidazolidin-4-ones via α-functionalization of 2-aminoacetamides catalyzed by

Full text of DOI:10.1021/acscatal.6b02189

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Article
Synthesis of imidazolidin-4-ones via a cytochrome
P450-catalyzed intramolecular C-H amination
Xinkun Ren, Jack A. O'Hanlon, Melloney Morris, Jeremy Robertson, and Luet Lok Wong
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b02189 • Publication Date (Web): 01 Sep 2016
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Synthesis of imidazolidinꢀ4ꢀones via a cytochrome
P450ꢀcatalyzed intramolecular CꢀH amination
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Xinkun Ren,a,‡ Jack A. O’Hanlon,b,‡ Melloney Morris,c Jeremy Robertson b,* and Luet Lok
Wonga,*
a Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks
Road, Oxford, OX1 3QR, UK
b Department of Chemistry, University of Oxford, Chemistry Research laboratory, Mansfield
Road, Oxford, OX1 3TA, UK.
c Chemistry Department, 100/79, Syngenta, Jealott's Hill International Research Centre,
Bracknell, RG42 6EY, UK.
Keywords: P450, heme monooxygenases, CꢀH amination, protein engineering, CꢀH activation.
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ABSTRACT:
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Expanding Nature’s catalytic repertoire to include reactions important in synthetic chemistry
opens new opportunities for biocatalysis. An intramolecular C–H amination route to
imidazolidinꢀ4ꢀones via αꢀfunctionalization of 2ꢀaminoacetamides catalyzed by evolved variants
of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium has been developed. Screening
of a library of ca. 100 variants based on four template mutants with enhanced activity for the
oxidation of unnatural substrates, and preparative scale reactions in vitro and in vivo, show that
the enzymes give up to 98% isolated yield of cyclization products for diverse substrates. 2ꢀ
Aminoacetamides with oneꢀ and twoꢀ ring cyclic amines bearing substituents, and aliphatic,
alicyclic and substituted aromatic amides are cyclized. Regiodivergent C–H amination was
achieved at benzylic and nonꢀbenzylic positions in a tetrahydroisoquinolinyl substrate by the use
of different mutants. This C–H amination reaction offers a scalable route to imidazolidinꢀ4ꢀones
with varied functionalized substituents that may have desirable biological activity.
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INTRODUCTION
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Nitrogen heterocycles play vital roles in numerous drugs, driving the search for efficient and
selective methods for C–N bond formation. Lateꢀstage cyclization via oxidative amination is an
attractive strategic choice in this regard. The two main approaches utilize a variety of oxidants
(KMnO4, Hg(OAc)2, K3[Fe(CN)6], etc.) to create an electrophilic carbon center that is trapped by
nitrogenꢀbased nucleophiles,1 or an electrophilic nitrenoid intermediate formed by transition
metal catalysts (Rh, Ru, Mn, Co, Fe, etc.) reacts with electronꢀrich alkenes, heteroatoms, or C–H
bonds.2 These chemical routes require the presence of specific functional groups in the substrate
and, conversely, may also be relatively harsh and intolerant of sensitive functionality; although
control of enantioselectivity of C–N bond formation is possible, the regioselectivity of these
processes is determined by innate chemical reactivity trends in the substrate and is usually not
controllable by modifying the reagent.
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As an alternative to chemical reagents, enzymes are known that catalyze C–N bond formation.
For example, transaminases,3 ammonia lyases,4 the nitrating enzyme P450TxtE,5 and amino acid
dehydrogenases6 target oxidized or chemically activated carbon centers. Developing from
Breslow’s pioneering studies,7a,b engineered P450 enzymes and myoglobin have been shown to
activate azido groups in azidoformates and arylsulfonyl azides to effect nitrenoid formation and
intramolecular C–H insertion (Scheme 1A).7c,d Regioselectivity was achieved in the C–H
animation of 2,5ꢀdipropylbenzenesulfonylazide at either the benzylic or homobenzylic position
by different P450 mutants, with substrate binding overcoming the preference for insertion into
the weaker C–H bond (Scheme 1B).8
As part of a larger study on drug metabolism by mutant cytochrome P450BM3 (CYP102A1)
enzymes, we reported the high yield oxidative cyclization of lidocaine to an imidazolidinꢀ4ꢀone
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(Scheme 1C).9 The formation of an imidazolidinone, by Nꢀcyclization, rather than an
oxazolidinimine, by Oꢀcyclization, was confirmed spectroscopically and by comparison with a
sample prepared by condensation with acetaldehyde.10 This outcome accords with the instability
of oxazolidinimines in aqueous solution and their conversion into the isomeric imidazolidinones
by heating in pyridine.11 In the proposed reaction pathway (Scheme 2) an iminium intermediate
is formed by oxidation of an aminyl αꢀradical (pathway A), or via αꢀhydroxylation of the amine
(pathway B), and is then trapped by the amide nitrogen. The overall transformation constitutes an
intramolecular amination of an sp3 C–H bond.
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Lidocaine oxidation was screened with a library of ca. 100 variants of P450BM3 (Tables S1 &
S2). These were based on four mutants, A330P (AP), A191T/N239H/I259V/A276T/L353I
(KT2), I401P (IP), and F87A/H171L/Q307H/N319Y (KSK19), that possess increased oxidation
activity for a wide range of organic compounds,12 and were generated by adding mutations at
two or more of the active site residues Arg47, Tyr51, Ser72, Ala74, Val78, Phe81, Ala82, Phe87,
Thr88, Ala184, Leu188, Ala328, Pro329, Ile263, Glu267 and L437, to create diversity of
substrate pocket topology. The partition between lidocaine dealkylation (to MEGX, norlidocaine,
Scheme 2) and intramolecular C–H amination is controlled by the mutations, e.g. the
RP/FV/EV/F81W mutant gave 96% dealkylation while RT2/A330W only showed cyclization
activity.9 Therefore, the P450BM3 library appeared to encompass variants with an inherent bias
away from the trivial activity of dealkylation and towards C–H amination.
We envisaged the possibility of exploiting these P450 catalysts for engaging 2ꢀ
aminoacetamides derived from cyclic amines in this intramolecular C–H amination process
(Schemes 1D & 3). The construction of the soꢀformed imidazolidinꢀ4ꢀones would be of interest
as these compounds are 3D templates with polar groups, hydrogen bond acceptors and
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potentially donors within the core. Further structural and functional group diversity around these
cores may give compounds with varied biological activity. Such diversity covers more chemical
space, e.g. for drug discovery, but requires the cyclization catalyst to tolerate varied substituents
at both the amine and amide ends of the substrate. Furthermore, the reduced pyrroloꢀ and pyridoꢀ
imidazolone motifs are validated biologically active cores found in the nootropic analgesic dimiꢀ
racetam and the mannosidase I inhibitor kifunensine, respectively.
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EXPERIMENTAL METHODS
General methods. DNA and microbiological manipulations were carried out by standard
methods. Siteꢀdirected mutagenesis was carried out by a polymerase chain reaction (PCR)ꢀbased
method using the KOD Hot Start Polymerase kit from Merck Bioscience, UK. Heterologous
production of P450BM3 enzyme variants in Escherichia coli BL21 (DE3), and their purification
by anion exchange chromatography have been reported previously.12a, 12d Synthesis of the 2ꢀ
aminoacetamide substrates is reported in the Supporting Information.
Screening of 2-aminoacetamides for oxidation by the P450BM3 mutant library. Screening
reactions were carried out in 0.2 M phosphate buffer, pH 7.5, at an assay volume of 0.5 mL in 14
mL glass vials using a NADPH regeneration system. The P450BM3 mutant was added as a 10 ꢁM
stock in phosphate buffer, pH 7.5 (final concentration = 1 or 2 ꢁM), the substrate as a 100 mM
methanol or ethanol stock (final concentration = 1 or 2 mM), glucose dehydrogenase as a 2
units/ꢁL stock in 0.2 M phosphate buffer, pH 7.5 (final concentration = 2 units/mL), and glucose
as a 1 M stock in 0.2 M phosphate buffer, pH 7.5 (final concentration = 0.1 M). NADP+
monosodium salt was added as a 4 mM stock in 0.2 M phosphate buffer, pH 7.5 (final
concentration = 80 ꢁM), to initiate the reaction. The reaction mixtures were shaken at 200 rpm
for 16 h at ambient temperature. The aqueous phase was extracted with 300 ꢁL of ethyl acetate
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after adjusting the pH to 11 with 2 M KOH. The phases were separated by centrifugation at
14,300 g for 2 min, and the organic phase was analyzed by GC.
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Preparative scale enzymatic 2-aminoacetamide oxidation in vitro. Preparative scale
reactions were performed in phosphate buffer (0.2 M, pH 7.5) in a final volume of 200 mL. In a
typical reaction, to phosphate buffer (167 mL) in a 500 mL beaker was added sequentially
glucose in phosphate buffer (20 mL of a 1.0 M stock solution, 20 mmol, final glucose
concentration = 0.1 M), the P450BM3 mutant enzyme in phosphate buffer (5.0 mL of a 40 ꢁM
stock solution, 0.2 ꢁmol, final enzyme concentration = 1.0 ꢁM), the amide in methanol (4.0 mL
of a 0.1 M stock solution, 0.4 mmol, final substrate concentration = 2.0 mM), and glucose
dehydrogenase (GDH) in phosphate buffer (200 ꢁL of a 2 units ꢁL stock solution, 400 units,
final GDH concentration = 2 units/mL). NADP+ monosodium salt in phosphate buffer (4.0 mL of
a 4.0 mM stock solution, 16.0 ꢁmol, final NADP+ concentration = 80 ꢁM) was added to initiate
the reaction. The mixture was stirred at 500 rpm at room temperature for 2–6 h. The reaction
mixture was extracted with ethyl acetate (3 × 200 mL), the combined organics were dried over
MgSO4, filtered, and the solvent was removed by rotary evaporation. The products were purified
by silica gel chromatography, eluting with a mixture of petrol and ethyl acetate.
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Whole cell oxidation of 2-aminoacetamides. A single colony of E. coli BL21 (DE3)
harboring the plasmid containing the gene encoding the relevant P450BM3 mutant was inoculated
into 250 mL LB media containing 34 mg/L kanamycin and grown for 16 h at 37 °C with shaking
at 120 rpm. Protein production at 20 °C was induced by adding IPTG to 0.05 mM. After shaking
for a further 24 h, 200 mL of culture was taken for the in vivo reaction while 50 mL of culture
was used for enzyme quantitation. Cells were harvested from 200 mL of culture by
centrifugation at 9,250 g for 5 min at 4 °C, and resuspended in 200 mL E. coli minimal media
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(EMM). The substrate was added to a final concentration of 2.0 mM from a 100 mM methanol
stock, along with glucose to a final concentration of 100 mM, from a 1.0 M stock in phosphate
buffer. 600 ꢁL samples were taken in duplicate every 4 h and centrifuged, after which 500 ꢁL of
the supernatant was extracted with 300 ꢁL ethyl acetate or chloroform for GC analysis of the
soluble organics to determine the substrate conversion. More aliquots of substrates could be
added, typically to a total of 10 mM. The whole in vivo biotransformation mixture was extracted
with ethyl acetate and the combined organics were dried over MgSO4. Solvent was removed by
rotary evaporation after filtration. The crude residue was purified by silica gel column
chromatography to isolate the product using the same procedure as for the in vitro reactions.
Computation. For each diastereomer of 3, conformers were obtained in Spartan 14 following
a Monte Carlo search (MMFF). Each conformer was then submitted for an equilibrium geometry
calculation (B3LYP/6ꢀ31G**) with a water solvation model, and a Boltzmannꢀweighted Go was
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obtained from a thermodynamics calculation.13 The obtained ꢂGo
= 2.46 kcal mol–1
298K
corresponds to an ~63:1 ratio of exo- to endo- diastereomers at equilibrium.
RESULTS AND DISCUSSION
Selected synthetic 2ꢀaminoacetamides bearing cyclic amino moieties were screened in vitro for
oxidation by the enzyme library (Scheme 3) using glucose dehydrogenase/glucose to regenerate
the NADPH cofactor. At the end of each reaction, the organicꢀsoluble extract was analyzed by
gas chromatography. Reactions that showed high substrate conversion and product selectivity
were scaled up (50 – 100 mg) in vitro for product purification and characterization. Pleasingly
5,5ꢀ and 5,6ꢀbicyclic imidazolidinꢀ4ꢀones were formed from lidocaineꢀlike 2ꢀaminoacetamides
with pyrrolidinyl (Scheme 3, entry 1, Fig. S1, Table S3) and piperidinyl (entry 2, Fig. S2, Table
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S4) groups (all characterization data are in the SI). The 2ꢀaminoacetamides 1 and 2 are
challenging substrates, only the RP/FV/EV (for 1) and RT2/AP/A184I (for 2) mutants showed
>90% conversion (total turnover number, TTN ~2,000) and isolated yields for reactions in vitro,
although more mutants had TTN >300 (0.33 mol% catalyst loading). The reactions were also
readily carried out in wholeꢀcells in shake flasks where substrates were added in 2 mM aliquots
up to 10 mM total concentration. The results suggest that 2ꢀaminoacetamides and imidazolidinꢀ
4ꢀones readily cross the E. coli cell wall. Higher product concentrations are likely to be feasible
at higher cell densities and more efficient mass transport, e.g. in a bioreactor vessel.
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The functional group tolerance of the P450BM3 mutants at both the amine and amide ends of 2ꢀ
aminoacetamides was then explored. Introduction of substituents to the cyclic amine had no
adverse effect on turnover activity or chemoselectivity for C–H amination. The methylꢀLꢀ
prolinate derivative (entry 3) was converted into >97% of the corresponding cyclization product
by, for example, the RT2/F81W mutant (Fig. S3, Table S5). Interestingly the presence of a
substituent on the pyrrolidine ring increased the number of mutants showing high conversion and
TTN (Table S5). The 1D NOE spectra (Fig. S24) showed that 3 was generated solely as the
(5S,7aR)ꢀdiastereoisomer by all mutants within the library. Calculations showed that this was the
thermodynamically more stable diastereoisomer by 2.46 kcal mol–1, suggesting rapid
equilibration of the N,Nꢀacetal center likely occurred during the reaction and isolation procedure.
This is consistent with the observation that the unsubstituted compounds 1 and 2, as well as the
products from the other aminoacetamides in Scheme 3, were obtained in racemic form.
Electron withdrawing (entry 4) and electron donating substituents (entry 5) were introduced at
the 4ꢀposition in place of the 2,6ꢀdimethyl substituents on the phenyl group of 2ꢀ
pyrrolidinylacetamides. The most selective mutants KSK19/A82M/E267F and KSK19/QP/FV
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provided the cyclized amination products with 84% and 95% conversion and 67% and 88%
isolated yield of 4 and 5, respectively (Fig. S4 & S5). Notably, selectivity for the cyclization
product 4 was reduced for some variants (as low as 33% for variants giving >60% conversion,
Table S6) but remained high, often >85%, for 5 (Table S7). Reduced nucleophilicity of the
amide nitrogen in the trifluoromethyl substrate presumably allows other pathways to compete
more effectively with cyclization. Products from these other pathways were not isolated.
The mutant library also oxidized the 2ꢀphenylethylamide derivative with up to 79% conversion
and 70% isolated yield for the cyclized product (entry 6). Selectivity for the cyclization product
was higher than for 4 and as high as that for the lidocaine reaction (Fig. S6, Table S8),
supporting the importance of the nucleophilicity of the amide nitrogen. These results
demonstrate that substitutions on the aromatic ring and different amine groups are tolerated by
the enzyme. Entries 7 and 8 show that thioamides are also successful substrates for this reaction
and the N,Nꢀacetals were formed with high conversion (Fig. S7 & S8, Table S9 & S10).
The conversion of aliphatic and alicyclic amides to cyclization products 9 and 10 illustrates
that an Nꢀaryl substituent at the amide end is not required for 2ꢀaminoacetamide binding or for
achieving a binding orientation that facilitates αꢀamine functionalization and cyclization. For
these reactions, high conversions were found although with some mutants there was some loss of
selectivity for cyclization (Fig. S9 & S10, Tables S11 & S12). The RP/FV/EV/V78F mutant
converted 93% of the cyclohexyl amine derivative (entry 9) and gave 66% isolated yield of the
cyclization product while the RP/FV/EV/L188Q mutant showed 72% conversion of the pentyl
substituted 2ꢀaminoacetamide (entry 10) to the corresponding N,Nꢀacetal in 55% isolated yield.
Cyclization of the tetrahydroisoquinoline (THIQ) derivative (entries 11 & 12) to a tricyclic
compound further highlights the tolerance of the P450BM3 mutants for variations in the amine
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moiety and the ability to control the regioselectivity of C–H amination (Fig. S11, Table S13).
The RP/H171L mutant showed 91% conversion with 86% regioselectivity for C–N bond
formation at the benzylic position (entry 11) while RP/IA/EV showed 94% conversion with 77%
selectivity for the nonꢀbenzylic position (entry 12). Mutants demonstrating nearꢀcomplete shift
between the two regioisomers were found from in vitro screening of the library. The IP/QP
mutant showed 95% selectivity for 11 while RP/F81W gave 94% 12 although the conversions
under screening conditions were low (~25%). Encouragingly, however, the conversion in both
reactions was increased to >95% in wholeꢀcell reactions without loss of chemoꢀ or regioꢀ
selectivity (IP/QP mutant, TTN = 800 for whole cells vs. 110 in vitro for 11; RP/F81W mutant,
TTN = 1,720 vs. 125 in vitro for 12), leading to isolated yields of >90% for each product. If an
organic compound such as 2ꢀaminoacetamides can cross the E. coli cell wall, large increases in
total turnover for in vivo reactions over in vitro conditions are possible because the cytoplasmic
enzyme is protected from high concentrations of organics and diffusion of products into the
external medium reduces product inhibition.
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This is the first report of selective direct C–H heterofunctionalization in simple THIQ
derivatives. In addition to the many bondꢀforming processes at the 1ꢀposition, a small number of
nonꢀselective 1ꢀ/3ꢀoxyfunctionalizations are known.14 Selective C–C bond forming processes at
the nonꢀbenzylic 3ꢀposition have been reported recently via two strategies: (1) for carbon
substituents capable of supporting a negative charge (malonyl, cyano, nitroalkyl), enrichment of
the fraction of 3ꢀderivative can be achieved by thermal equilibration;15 (2) the 3ꢀC–H bond in Nꢀ
(benzoxazolꢀ2ꢀyl)THIQ is more accessible to bulky Ir(I) catalysts that achieve direct alkylation
at that position with terminal alkenes.16 Both processes require high temperatures and offer
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limited scope; in contrast, the selective formation of compound 12 occurs under benign reaction
conditions.
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The activity profile of the mutant library (Table S3 to S13) suggests that the active site
residues F81, A82, F87, I263, E267 and A330 have important effects on 2ꢀaminoacetamide
cyclization via C–H amination. However, the base mutants with their enhanced activity for
unnatural substrate oxidation are required because the single site mutants such as F87A, etc. have
very low activity. The data showed that the mutants RT2/F81W, RT2/A330P/A184I,
RP/F87V/E267V, RP/I263A/E267V and KSK19/A82M/E267F (see Scheme 3), would have
established the possibility of intramolecular C–H amination for all the tested 2ꢀaminoacetamides.
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CONCLUSION
In summary, by screening variants of P450BM3 for activity on 2ꢀaminoacetamides we have
developed a scalable enzymatic C–H amination process for the straightforward synthesis of
bicyclic imidazolidinꢀ4ꢀones under mild conditions for both 5,5ꢀ and 5,6ꢀ fused bicyclic systems.
Enzymes offer advantages over transition metal catalysts because they operate under mild
conditions in aqueous solvent, are highly active, essentially inexhaustible, and can be evolved to
be highly selective for the desired product. The increased conversion for in vivo C–H amination
over reactions in vitro adds to the versatility of the system. The enzymes accept aromatic and
aliphatic amides as well as substituents on the amine ring that also, by thermodynamic
stereochemical relay, establish defined stereochemistry at the newlyꢀformed ring junction.
Cyclization of the tetrahydroisoquinoline derivative (entries 11 and 12, Scheme 3) to a tricyclic
compound further highlights the tolerance of P450BM3 mutants for variations in the amine moiety
and the possibility of controlled siteꢀselective C–H amination. Imidazolidinꢀ4ꢀones are latent
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iminium ions, being amenable to Lewis acid mediated ringꢀopening with subsequent nucleophilic
attack offering new routes for αꢀfunctionalization of tertiary amines.17 Ongoing engineering
studies are directed at identifying P450BM3 variants that will hydroxylate the imidazolidinꢀ4ꢀone
products in a second step to introduce additional functional diversity and, potentially, biological
activity.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge.
Materials and methods, substrate synthesis, enzyme activity screening method, in vitro and in
vivo substrate conversion methods, oligonucleotides for mutagenesis, list of mutants, tables of
substrate conversion and product selectivity for screened mutants, product distribution analysis
by GC, NMR and MS data for substrates and products (PDF)
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail for L.L.W.: luet.wong@chem.ox.ac.uk; for J.R.: jeremy.robertson@chem.ox.ac.uk
Author Contributions
‡Xinkun Ren and Jack O’Hanlon contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
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This research is supported by the Biotechnology and Biological Sciences Research Council, UK
(BB/L024381/1). X. Ren was supported by the China Oxford Scholarship Fund and J. O'Hanlon
by Syngenta and the Engineering and Physical Sciences Research Council, UK (EP/L505031/1).
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9483–9493; (c) Eiermann, B.; Edlund, P. O.; Tjernberg, A.; Dalén, P.; Dahl, M.ꢀL.; Bertilsson,
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15. (a) Shirakawa, E.; Yoneda, T.; Moriya, K.; Ota, K.; Uchiyama, N.; Nishikawa, R.; Hayashi,
T. Chem. Lett. 2011, 40, 1041–1043; (b) Ma, L.; Chen, W.; Seidel, D. J. Am. Chem. Soc. 2012,
134, 15305–15308; (c) Kang, Y.; Chen, W.; Breugst, M.; Seidel, D. J. Org. Chem. 2015, 80,
9628–9640.
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17. O’Hanlon, J.; Robertson, J.; Wong, L. L. unpublished work.
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Scheme 1. Enzyme-catalyzed C–H amination
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N
Ar NH
O
N
Ar NH
O
N
Ar
N
O
cyclization
P FeIV=O
− e
A
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HO
PFeIV−OH Ar NH
N
−CH3CHO
Ar NH
O
N
Ar NH HN
O
B
O
dealkylation
(→ MEGX)
Ar = 2,6-xylyl
Scheme 2. Proposed mechanism of P450-catalyzed amination of lidocaine
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R
R
NH
P450BM3
N
X
7
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X
Glucose
NADP+, GDH
N
N
( )1,2
( )1,2
X = O, S; R = aryl, alkyl
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F3C
MeO
H
N
N
N
N
O
N
N
O
O
O
O
O
N
N
N
N
N
N
COOMe
1, 99%, RP/FV/EV,
1980, 97% yield
6, 79%, KSK19/AM/E267F,
3, 100%, RT2/F81W,
1000, 98% yield
4, 84%, KSK19/AM/E267F,
5, 95%, KSK19/QP/FV,
2, 94%, RT2/AP/A184I,
370, 70% yield
350, 67% yield
460, 88% yield
1890, 92% yield
1,2,3,4-tetrahydroisoquinolinyl
N
N
N
N
N
N
N
S
O
O
S
O
O
N
N
N
N
N
7, 95%, RP/FV/EV
1900, 92% yield
8, 84%, KSK19/QP/FV, 9, 93%, RP/FV/EV/V78F, 10, 72%, RP/FV/EV/L188Q,
690, 66% yield 350, 66% yield 290, 55% yield
11, 97%, RP/H171L,
420, 81% yield
Benzylic position
12, 94%, RP/IA/EV,
360, 70% yield
Non-benzylic position
¶
The data are given as: % substrate conversion, the P450BM3 mutant, total turnover number
(TTN), isolated yield for reactions in vitro. TTN is the concentration of cyclization product
formed per unit enzyme concentration. Where conversion exceeds ca. 95%, the enzyme is
capable of further substrate conversion and the actual TTN is higher than the value given, e.g. for
1 under unꢀoptimized conditions a TTN of 7,600 (76% conversion, 10 mM substrate, 1 ꢁM
enzyme) was observed.
Scheme 3. P450BM3-catalyzed intramolecular C–H amination of 2-aminoacetamides to
imidazolidin-4-ones ¶
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