Synthesis of γ-Hydroxy-α-amino Acid Derivatives by Enzymatic Tandem Aldol Addition–Transamination Reactions

Moreno, Carlos J.; Hernández, Karel; Charnok, Simon J.; Gittings, Samantha; Bolte, Michael; Joglar, Jesús; Bujons, Jordi; Parella, Teodor; Clapés, Pere

doi:10.1021/acscatal.1c00210

“…Sufficiently hydrophobic products were isolated as the free amino acid, while others required protection with fluorenylmethoxycarbonyl (Fmoc) to increase hydrophobicity, simultaneously adding a handle commonly used in solid phase peptide synthesis. Bespoke manipulations, such as lactonization with the γ-hydroxy group, can also be employed to facilitate isolation (14).…”

Section: Resultsmentioning

confidence: 99%

“…While nature employs side chain hydroxylation to tune bioactivity, these nsAAs are virtually absent from medicinal chemistry (13) because they require multistep synthesis (12). This challenge extends to biocatalysis, where an elegant multi-enzyme cascade was required to access γ-hydroxy nsAAs (14). Beyond their use in pharmaceuticals, nsAAs can be enabling for a host of synthetic and chemical biology applications (15,16).…”

Section: One-sentence Summarymentioning

confidence: 99%

Biocatalytic Synthesis of Non-Standard Amino Acids by a Decarboxylative Aldol Reaction

Buller¹,

Ellis²,

Campbell³

et al. 2021

Preprint

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The formation of carbon-carbon bonds lies at the heart of organic chemistry, but relatively few C-C bond forming enzymes have found their way into the biocatalysis toolbox. We report that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is very efficient in a whole-cell biocatalysis format and readily crystallizes. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and empowers future studies. The utility of UstD2.0 was demonstrated via the stereoselective gram-scale syntheses of non-standard amino acids.

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“…While nature employs side chain hydroxylation to tune bioactivity, these nsAAs are virtually absent from medicinal chemistry 14 because they require multistep synthesis 13 . This challenge extends to biocatalysis, where an elegant multi-enzyme cascade was required to access γ-hydroxy nsAAs 15 . Beyond their use in pharmaceuticals, nsAAs can be enabling for a host of synthetic and chemical biology applications 16,17 .…”

Section: Main Textmentioning

confidence: 99%

Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

Buller

¹

,

Ellis

²

,

Campbell

³

et al. 2021

Preprint

0

View full text Add to dashboard Cite

Enzymes are renowned for their catalytic efficiency and selectivity, but relatively few carbon-carbon bond forming enzymes have found their way into the biocatalysis toolbox. While engineering can overcome the challenges associated with C-C bond formation for some enzyme systems, the broader synthetic potential of biocatalysis is hindered by the lack of high-quality C-C bond forming transformations. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is efficient in a whole-cell biocatalysis format, which circumvents the need for enzyme purification, thereby facilitating its use in traditional organic settings. This new, highly stereoselective enzyme represents a unique expansion of the biosynthetic toolbox. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and the molecular basis for the remarkably promiscuity of this catalyst. Taking inspiration from the versatile reactivity of enamines in organic synthesis, we hypothesize that the enamine intermediate of UstD can be engineered to react with electrophiles other than aldehydes. The advent of structural information enabled by engineering of UstD2.0 provides a foundation for probing the unique mechanism of UstD and will guide efforts to expand the reactivity of this unique enzyme.

show abstract

“…While nature employs side chain hydroxylation to tune bioactivity, these nsAAs are virtually absent from medicinal chemistry (13) because they require multistep synthesis (12). This challenge extends to biocatalysis, where an elegant multi-enzyme cascade was required to access γ-hydroxy nsAAs (14). Beyond their use in pharmaceuticals, nsAAs can be enabling for a host of synthetic and chemical biology applications (15,16).…”

mentioning

confidence: 99%

Biocatalytic Synthesis of Non-Standard Amino Acids by a Decarboxylative Aldol Reaction

Ellis¹,

Campbell²,

Kumar³

et al. 2021

Preprint

View full text Add to dashboard Cite

The formation of carbon-carbon bonds lies at the heart of organic chemistry, but relatively few C-C bond forming enzymes have found their way into the biocatalysis toolbox. We report that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is very efficient in a whole-cell biocatalysis format and readily crystallizes. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and empowers future studies. The utility of UstD2.0 was demonstrated via the stereoselective gram-scale syntheses of non-standard amino acids.

show abstract

Synthesis of γ-Hydroxy-α-amino Acid Derivatives by Enzymatic Tandem Aldol Addition–Transamination Reactions

Cited by 33 publications

References 64 publications

Biocatalytic Synthesis of Non-Standard Amino Acids by a Decarboxylative Aldol Reaction

Biocatalytic Synthesis of Non-Standard Amino Acids by a Decarboxylative Aldol Reaction

Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

Biocatalytic Synthesis of Non-Standard Amino Acids by a Decarboxylative Aldol Reaction

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