A biocatalyst for efficient synthesis of β-, γ-, and δ lactams!
In 2018, Frances Arnold was among three scientists who were awarded the Nobel Prize in Chemistry. In her Nobel Prize lecture Arnold states, “I wish to go beyond optimizing biological functions that are already known, and instead bring to life whole new chemistries. But how can one create enzymes that catalyze reactions invented by chemists?” To answer this question, Arnold’s lab developed a protein engineering process called directed evolution. Directed evolution is the process of altering amino acids at various sites of an enzyme to create or enhance a protein’s ability to carry out desired chemical reactions. In Arnold’s words, it is taking enzymes “where biology has never gone.” Evolved enzymes can subsequently be integrated into biosynthetic pathways of microorganisms, or cascade reactions to improve the synthesis of pharmaceuticals and other value-added compounds. Enzyme catalysts are also environmentally friendly, an advantage difficult to underscore given our planet’s warming climate.
Using the directed evolution approach described above, Arnold’s lab engineered cytochrome p450 monooxygenases capable of olefin cyclopropanation. Cytochrome p450s and other monooxygenases have since been engineered for nitrene insertion. The paper discussed in this article is work from Rudi Fasan’s lab, a former member of the Arnold lab and now a full professor at Rochester University. The paper, published in Nature Catalysis, reports on a myoglobin variant capable of nitrene insertion for biosynthesis of β-, γ-, and δ-lactam rings. The authors write, “The synthesis of cyclic amides (lactams), which are key structural motifs in many pharmaceuticals, agrochemicals, and other fine chemicals, has presented a major challenge.” Below is a summary of the studies undertaken by the Fasan group to address this challenge.
An H64V, V68A myoglobin variant (Mb*) that synthesizes γ-lactams was engineered. Since the groundbreaking report of their use in cyclopropanation reactions, p450s have been engineered for C-H amination through nitrene transfer. And while Ir and Ru-based organic catalysts have been employed to synthesize γ-lactams, no organic catalysts or biocatalysts have been available for making β- and δ- lactams. Well, not anymore! Using a dioxazolone substrate and an H64V myoglobin variant, the authors were able to synthesize a γ-lactam at 2% yield. Further engineering yielded a double mutant (H64V,V68A or Mb*) with 25x increase in yield and >99% e.e. of the γ-lactam! As a side note, dioxazolone substrates were chosen as substrates because they can be synthesized easily from abundant carboxylate starting materials. The figure below was obtained from the article.
The yield of lactams is enhanced by low pH and the use of acetonitrile as a co-solvent. To limit the acyclic amide byproduct generated in the reaction (presumably resulting from protonation of the nitrene intermediate), the authors ran the anaerobic reactions under alkaline conditions. Various organic solvents were tested as reaction co-solvents to further disfavor acyclic byproduct formation. Both approaches led to an increase in yield (from 50% to 75%), all while retaining excellent e.e. values.
The γ-lactam-generating biocatalyst has a broad substrate scope. Substitutions at the para, ortho, and meta positions of the aryl ring of the dioxazolone substrate were well-tolerated in general. However, replacing the aryl ring with heterocyclic substituents dropped yields and e.e. values in some instances.
Mb* can synthesize β- and δ-lactams from dioxazolone substrates with a similarly broad substrate scope. β-lactams could be synthesized at yields as high as 93% with excellent e.e. values (generally 99%). δ-lactams were also synthesized successfully but contained impurities comprising acyclic amides and β-lactams. However, by replacing the γ-C-H atom with oxygen, the authors were able to eliminate unwanted lactams and increase both the yield and e.e. of the reaction for δ-lactam biosynthesis.
Mechanistic studies reveal that the active site of Mb* plays a significant role in determining enantioselectivity. Mb* catalysis is expected to follow the general mechanism established for p450s: dioxazolone activation (accompanied by CO2 elimination) and nitrene formation is followed by an intramolecular hydrogen atom abstraction (HAA) of the β, γ, or δ C-H atoms. In the radical rebound step, an intramolecular cyclization yields β-, γ-, or δ-lactams. Kinetic isotope effect (KIE) studies revealed, interestingly, that irrespective of deuteration at pro-S or pro-R positions, a single product (the S enantiomer) was the dominant product. From this observation, it was surmised that the active site milieu was critical for enantioselective synthesis (i.e., preferential Si face attack). Additionally, density functional theory (DFT) calculations revealed that the radical rebound step (formation of the C-N bond) displayed a higher energy barrier (12.5 kcal/mol) compared to the HAA step (8 kcal/mol). As a result, the final step was concluded to be the rate-limiting step of the reaction in Mb*. The figure below was obtained from the article.
Mb* was used to synthesize an alkaloid and a pharmaceutical in fewer steps. A phenyl-substituted β-lactam generated by Mb* was used to synthesize (S,S)-(–)-homaline and (S)-dapoxetine. Incorporating Mb& into the synthetic route reduced the number of steps from 11 to 7 and 12 to 8, respectively. As an added advantage, an enantiodivergent variant of the Mb lactam biocatalyst was also engineered.
NeissLock: a method for labeling unmodified endogenous proteins in mild conditions
As chemical biology becomes increasingly translational, its tools and strategies must adhere to higher standards of resolution to ensure success and longevity in the clinic. As a result, labeling approaches often incorporate avenues for spatial and temporal control for in vivo scenarios. In this review, I focus on summarizing a labeling strategy designed to do exactly that. NeissLock is a method for targeting endogenous proteins through proximity-induced conjugation. Initially reported by the Howarth lab using ornithine decarboxylase (ODC) and ornithine decarboxylase antienzyme (OAZ) complex as a model system, NeissLock enables covalent linkage between binding and target proteins.
1. Self-processing module (SPM) of Neisseria meningitidis has autoproteolysis capabilities: SPM is a key component of NeissLock. It displays Ca2+-induced autoproteolysis at an Asp-Pro dipeptide site. Autoproteolysis cleaves between the Asp and Pro residues generating an aspartic anhydride in the process.
2. SPM can be repurposed for tagging endogenous proteins: SPM is fused to the C-terminal of a binding protein. Upon exposure to Ca2+ (0.5 mM and above), SPM autopreotolysis releases an SPM-free binding protein with a C-terminal anhydride. When in proximity to the “activated” binding protein, a nucleophilic Lys in the target protein reacts with the C-terminal anhydride of the binding protein to form a covalent bond between the two (NeissLock). In vitro reactions with OAZ-SPM and ODC showed calcium-induced ligation of OAZ and ODC. Additionally, that affinity is crucial for NeissLock was demonstrated by showing selective reaction of ODC to OAZ in reactions of OAZ-SPM with cell lysate (Figure source is the article).
3. A computational analysis software NeissDist was created to predict NeissLock sites in protein-protein complexes: A software called NeissDist was designed to identify nucleophilic amines close to the anhydride of a binding for protein protein-protein complexes in the PDB. It should be noted, however, that the authors report that the anhydride reacts with nucleophiles other than amines, including thiols, amides, and hydroxyls, implicating Cys, Asn, Gln, and Tyr in NeissLock-mediated conjugation of protein-protein complexes.
4. NeissLock is efficient at neutral pH and is compatible with different buffers: The authors were concerned that the high pKa of amines might necessitate higher pH conditions for NeissLock. However, reactions were efficient between pH 6.5-8.5. Reactions were also shown to be efficient in HEPES and TBS buffers. Phosphate-based buffers are not recommended due to precipitation upon calcium addition.
5. NeissLock works for cell surface labeling: SPM-tagged TGF-α is shown to efficiently label sEGFR in A431 cells. Although the authors observed significant staining with an SPM-tagged TGF-α variant that should be non-reactive, this could be abolished with an R42A mutation in TGF-α that weakens its affinity to EGFR, demonstrating once-again that NeissLock is a proximity-dependent conjugation process.
The advantage for spatiotemporal control in this technology is that no reaction occurs until reactivity is initiated by addition of calcium. Additionally, no conjugation occurs without proximity, conferring spatial resolution on the NeissLock approach. This advantages are crucial for applications in imaging and diagnostics. Limitations include possibly having to remove nucleophilic residues near the anhydride to minimize self-reaction, and relative ambiguity regarding the conjugation sites due to anhydride reactivity with different nucleophilic residues.
Engineering radical acyl transferases from benzaldehyde lyase
Looking for a paper that combines your passion for enzyme engineering and photoredox catalysis? Look no further! In this paper, the authors report a “dual-catalyst system” that combines the organophotoredox catalyst eosin Y with a ThDP (thiamine diphosphate) enzyme that they engineered to function as a radical acyl transferase. Key take aways:
1. Two mutations, A480L and T481L, were made in ThDP-dependent enzyme benzaldehyde lyase (BAL) to introduce radical acyl transferase (RAT) capabilities. Using eosin Y as a photoredox catalyst, the authors show that 4-methoxybenzaldehyde and an aromatic N-(acyloxy)phthalamide can be fused by the engineered RAT to synthesize alpha-chiral ketones!
2. Different aromatic aldehydes and phthalimide derivatives were tested to survey the substrate scope. The authors demonstrate that with a few exceptions the high yield and enantioselectivity observed for the control reaction are retained! Seriously, the numbers are just beautiful.
3. To elucidate the mechanism of their engineered enzyme, the authors performed EPR spectroscopy and quenching (TEMPO) experiments alongside computational studies. Their investigations revealed that following green light-mediated oxidation of eosin Y, the Breslow intermediate (common to all ThDP-dependent enzymes as well as synthetic N-heterocylic carbene catalysts) undergoes a single electron transfer that converts it into a ketyl. N-(acyloxy)phthalamide radicals are also generated through interaction with an eosin Y radical anion in a subsequent step.
4. Based on their findings (listed above) the authors conclude that an enantioselective radical-radical cross coupling reaction between the ketyl and prochiral N-(acyloxy)phthalamide radicals leads to the formation of alpha-chiral ketones.
Limitations: Yields and enantiopurity do dwindle for some reactions, but are still decent. The authors also report that the reaction must be done in an inert atmosphere (N2) and requires green light.
Read the article here!
