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Supported by Synthetic and Biosynthetic Studies on Abyssomicin C Sbusisiwe Z. Mbatha, a Nicholas R. Lees, a Matthew J. Byrne, b Li-Chen Han, a Paul R. Race, b Christine L. Willis a a School of Chemistry, University of Bristol, Cantock’s Close, Bristol, BS8 1TS. b School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD. AbyU Studies Proposed Biosynthesis 2 • Abyssomicin C (1) is a polyketide derived spirotetronate isolated from the marine microorganism Verrucosispora maris. • It is a potent inhibitor of Gram-positive bacteria such as MRSA (MIC = 4 μg/mL) and VRSA (MIC = 13 μg/mL). • It mimics chorismate and so inhibits the pABA/tetrahydrofolate biosynthetic pathway, making it an attractive target for novel antibiotics. 1 • In this project, we aim to elucidate the structures of key enzymes involved in the biosynthesis of 1 and investigate their use as biocatalysts in for the preparation of novel bioactive compounds. Background Conclusions and Future Work AbyU crystals Crystal structure of AbyU Active site of AbyU • Diels-Alder (DA) reactions are typically conducted in heat or Lewis acid-mediated reactions. • AbyU was isolated and characterised as a homodimer containing 8-stranded antiparallel β-barrels. • Protected tetronate 18 was synthesised in order to assess enzyme function of AbyU. • AbyU catalyses the [4+2] cycloaddition of substrate 18 to spirotetronate 19. Synthetic reference Synthetic reference 30 min incubation with AbyU Denatured enzyme AbyA5 Studies AbyA5 crystal Crystal structure of AbyA5 AbyA5 active site • Few enzymes have been reported for acetate elimination. 3,4 • DNA sequence of wildtype AbyA5 was transformed into E. coli for protein expression. • AbyA5 structure was determined and it retains an alpha/beta hydrolase catalytic triad, with serine, histidine and aspartic acid residues housed in the active site. • Tetronic acids R-10 and S-10 were synthesised from D-mannitol (7) in order to investigate the function and stereospecificity of AbyA5. 5 8 10 12 14 16 18 20 8 10 12 14 16 18 20 8 10 12 14 16 18 20 8 10 12 14 16 18 20 8 10 12 14 16 18 20 8 10 12 14 16 18 20 with AbyA5 with denatured AbyA5 8 10 12 14 16 18 20 8 10 12 14 16 18 20 without AbyA5 synthetic standard of 13 • Tetronic acids R-10 and S-10 were incubated separately with AbyA5 in pH 7.5 Tris-HCl buffer at 25 °C for 30 minutes. Only R-10 was converted to tetronate 13. • A standard of 13 was synthesised in 6 steps from commercially available lactone 11 via formation of the exo-methylene on tetronate 12. kcat = 1.8 ± 0.1 min-1 KM= 26.8 ± 8.1 μM kcat/KM = 0.07 ± 0.01 min-1 μM-1 (R)-10 μM • Enzymes AbyA5 and AbyU were successfully isolated and characterised using X-ray crystallography. • Incubation of tetronic acids R-10 and S-10 with AbyA5 confirmed the function and stereospecificity of the enzyme, with R-10 giving 13 exclusively. • AbyU efficiently catalyses the DA reaction of linear tetronate 18 to spirotetronate 19. • Computational studies reveal contortion of linear substrate 18 within the AbyU active site to facilitate the [4+2] cycloaddition. • Investigations into AbyU promiscuity and development into a biocatalyst are ongoing. Feeding studies to determine building blocks to 1. • Synthesis of tetronate 18 was conducted from commercially available alcohol 14 to give intermediate lactone 16 which was subsequently transformed to the linear tetronate 18. • Synthetic standard of spirotetronate 19 was synthesised by heat mediated cyclisation of 18. • Incubation of 18 with AbyU in pH 7.5 Tris-HCl buffer at 25 °C for 30 minutes gave 19. • The enzymatic reaction accelerates the transformation >40,000 times. 6 OH Br O H O O O O OMe O O O O O O MeO O Ph N O OH 15 14 16 17 18 19 3 steps 97% 2 steps 85% 2 steps 70% 2 steps 72% Thermal DA reaction 88% OH I 20 References 1. Angew. Chem. Int. Ed., 2004, 43, 2574 –2576. 2. Chembiochem, 2011, 12, 1401–1410. 3. Angew. Chem. Int. Ed., 2013, 52, 5785–5788. 4. Org. Lett., 2014, 16, 1578–1581. 5. Angew. Chem. Int. Ed., 2019,131,2327–233. 6. J. Am. Chem. Soc., 2016, 138, 6095−6098. 13 R-10 R-10 S-10 S-10 S-10
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Supported by
Synthetic and Biosynthetic Studies on Abyssomicin CSbusisiwe Z. Mbatha,a Nicholas R. Lees,a Matthew J. Byrne,b Li-Chen Han,a Paul R. Race,b Christine L. Willisa
aSchool of Chemistry, University of Bristol, Cantock’s Close, Bristol, BS8 1TS. bSchool of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD.
AbyU Studies
Proposed Biosynthesis2
• Abyssomicin C (1) is a polyketide derived spirotetronate isolated from the marine microorganism Verrucosispora maris.• It is a potent inhibitor of Gram-positive bacteria such as MRSA (MIC = 4 μg/mL) and VRSA (MIC = 13 μg/mL).• It mimics chorismate and so inhibits the pABA/tetrahydrofolate biosynthetic pathway, making it an attractive target for novel antibiotics.1
• In this project, we aim to elucidate the structures of key enzymes involved in the biosynthesis of 1 and investigate their use as biocatalysts infor the preparation of novel bioactive compounds.
Background
Conclusions and Future Work
AbyU crystals Crystal structure of AbyU Active site of AbyU
• Diels-Alder (DA) reactions are typically conducted in heat or Lewis acid-mediated reactions.• AbyU was isolated and characterised as a homodimer containing 8-stranded antiparallel β-barrels.• Protected tetronate 18 was synthesised in order to assess enzyme function of AbyU.• AbyU catalyses the [4+2] cycloaddition of substrate 18 to spirotetronate 19.
Synthetic reference
Synthetic reference
30 min incubation with AbyU
Denaturedenzyme
AbyA5 Studies
AbyA5 crystal Crystal structure of AbyA5 AbyA5 active site
• Few enzymes have been reported for acetate elimination.3,4
• DNA sequence of wildtype AbyA5 was transformed into E. coli for protein expression.• AbyA5 structure was determined and it retains an alpha/beta hydrolase catalytic triad, with
serine, histidine and aspartic acid residues housed in the active site.
• Tetronic acids R-10 and S-10 were synthesised from D-mannitol (7) in order to investigate thefunction and stereospecificity of AbyA5.5
8 10 12 14 16 18 20
8 10 12 14 16 18 20
8 10 12 14 16 18 208 10 12 14 16 18 20
8 10 12 14 16 18 20
8 10 12 14 16 18 20
with AbyA5
with denatured AbyA5
8 10 12 14 16 18 20 8 10 12 14 16 18 20
without AbyA5
synthetic standard of 13
• Tetronic acids R-10 and S-10 were incubated separately with AbyA5 in pH 7.5 Tris-HCl buffer at 25°C for 30 minutes. Only R-10 was converted to tetronate 13.
• A standard of 13 was synthesised in 6 steps from commercially available lactone 11 via formationof the exo-methylene on tetronate 12.
kcat = 1.8 ± 0.1 min-1
KM = 26.8 ± 8.1 μMkcat/KM = 0.07 ± 0.01 min-1 μM-1
(R)-10 μM
• Enzymes AbyA5 and AbyU were successfully isolated and characterised using X-ray crystallography.• Incubation of tetronic acids R-10 and S-10 with AbyA5 confirmed the function and stereospecificity of the enzyme, with R-10 giving 13 exclusively.• AbyU efficiently catalyses the DA reaction of linear tetronate 18 to spirotetronate 19.• Computational studies reveal contortion of linear substrate 18 within the AbyU active site to facilitate the [4+2] cycloaddition.• Investigations into AbyU promiscuity and development into a biocatalyst are ongoing.
Feeding studies to determine building blocks to 1.
• Synthesis of tetronate 18 was conducted from commercially available alcohol 14 to giveintermediate lactone 16 which was subsequently transformed to the linear tetronate 18.
• Synthetic standard of spirotetronate 19 was synthesised by heat mediated cyclisation of 18.
• Incubation of 18 with AbyU in pH 7.5 Tris-HCl buffer at 25 °C for 30 minutes gave 19.• The enzymatic reaction accelerates the transformation >40,000 times.6
OHBr
O
H
O
O
O O
OMe
O
O
O O
O
O
MeO
OPhN
O
OH
1514 16 17
18
19
3 steps
97%
2 steps
85%
2 steps
70%
2 steps72%
ThermalDA reaction
88%
OHI
20
References1. Angew. Chem. Int. Ed., 2004, 43, 2574 –2576.2. Chembiochem, 2011, 12, 1401–1410. 3. Angew. Chem. Int. Ed., 2013, 52, 5785–5788. 4. Org. Lett., 2014, 16, 1578–1581. 5. Angew. Chem. Int. Ed., 2019,131,2327–233. 6. J. Am. Chem. Soc., 2016, 138, 6095−6098.
13
R-10
R-10
S-10
S-10
S-10
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