Efficient synthesis of a statin precursor in high space-time yield by a new aldehyde-tolerant aldolase identified from Lactobacillus brevis

Abstract

A novel 2-deoxyribose-5-phosphate aldolase (LbDERA) was identified from Lactobacillus brevis, with high activity, excellent thermostability and high tolerance against aldehyde substrates. The half-lives of LbDERA incubated in 300 mM acetaldehyde and chloroacetaldehyde were 37.3 and 198 min, respectively, which are 2-and 7-fold higher than those of EcDERA from Escherichia coli. The crystal structure of LbDERA determined at 1.95 Å resolution revealed a stable quaternary structure which might account for its excellent aldehyde tolerance. A single mutation, E78K, was introduced to LbDERA through a consensus sequence approach, resulting in significant improvements of both thermostability and aldehyde tolerance. According to the crystal structure of LbDERA E78K , two additional hydrogen bonds and one salt bridge were introduced compared with wild-type LbDERA. As a result of its high substrate tolerance, LbDERA E78K could efficiently catalyze a sequential aldol condensation with 0.7 M chloroacetaldehyde and 1.4 M acetaldehyde, affording a key chiral precursor of statins, IJ3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside, with an unprecedented space-time yield of 792.5 g L −1 d −1 and only 2.5 g L −1 of catalyst loading.

2‐Deoxyribose‐5‐phosphate aldolase from Thermotoga maritima in the synthesis of a statin side‐chain precursor: characterization, modeling and optimization

“…More than 80% of its initial activity was retained after 67 h of incubation at temperatures in the range between 20 and 40 °C, with highest residual activity at 20–25 °C (≈ 87%) (Fig. S4), rendering the DERA Tm more thermostable than the corresponding DERA from Lactobacillus brevis . It was found that the residual enzyme activity was practically constant (>80%) within the pH range from 5.4 to 8.0 and 24 h of incubation, which also is in accordance with previous investigations .…”

“…By using achiral substrates, such as chloroacetaldehyde and acetaldehyde, DERA catalyzes a sequential aldol addition that results in an enantiomerically pure lactol, (3 R ,5 R )‐6‐chloro‐2,4,6‐trideoxyhexose . This 2,4,6‐trideoxyhexose is a valuable chiral synthon for HMG‐CoA reductase inhibitors, collectively known as statins, and used for the production of cholesterol‐lowering drugs, such as atorvastatin and rosuvastatin .…”

“…US$20 billion worldwide sales, among which Pfizer's Lipitor (atorvastatin calcium) was the world's top‐selling drug with revenues in excess of US$10 billion per year . Therefore, the preparation of the 2,4,6‐trideoxyhexose has attracted immense effort because of its extremely high market price, and the requirement for high chemical and stereochemical purity at its two chiral centers . Because the chemical synthesis of statins is time‐consuming and consists of a number of synthetic steps that are carried out under very harsh conditions, the use of DERA for the synthesis of the statin side‐chains presents one of the most attractive and promising routes, due to its ability to introduce both stereocenters in a single step …”

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

“…EA01524.86509.1137 c Maximal activity at 200 mM(Kim et al 2009)  Paenibacillus sp . EA00124.565014562 c n.s.(Kim et al 2010)  Rhodococcus erythropolis 22.9725 e 4.8417 c Half-life = 64.4 min300 mM 25 °C(Kullartz and Pietruszka 2012)  Haemophilus influenza 23.67.5400.1470.42 b Maximal activity at 300 mM(Woo et al 2014)  Staphylococcus epidermidis 29.2n.s.67.1 c 11.3% retention of activity2 h 300 mM 25 °C(Fei et al 2015)  Lactobacillus brevis ECU8302n.s.6403.34102 b Half-life = 37.3 min300 mM 25 °C(Jiao et al 2015)Extremophiles (hyperthermophilic unless specified)  Aeropyrum pernix 24.56.5n.s.0.057**4.5 c **n.s.(Sakuraba et al 2003)  Thermococcus kodakaraensis 24.54950.81***285 b ***n.s.(Rashid et al 2004)  Pyrobaculum aerophilum 24.56n.s.0.0660.25 c 53% retention of activity20 h 300 mM 25 °C(Sakuraba et al 2007)  Thermotoga maritima 27.86.5n.s.0.021 c 46% retention of activity20 h 300 mM 25 °C(Sakuraba et al 2007)  Hyperthermus butylicus 26.45.5800.150.5 c 75% retention of activity8 h 300 mM 25 °C(Wang et al 2010)  Aciduliprofundum boonei 26.67800.12n.s.70% retention of activity4 h 250 mM 25 °C(Yin et al 201...…”

Synthesis of Heterocycles in Contemporary Medicinal Chemistry

“…This will trigger the continuous search for the more efficient, cheaper, and sustainable synthesis of statins [137] and in particular super-statins. This has already been reflected in some reports that have appeared in the literature since this review was written [138][139][140][141][142][143][144]. …”