Identification of Intermediates and Enzymes Involved in the Early Steps of Artemisinin Biosynthesis inArtemisia annua

Abstract

IntroductionArtemisia annua L. (sweet wormwood), a member of the Asteraceae family has been used for many years in the treatment of malaria. The active compound responsible for its pharmacological action is the sesquiterpene lactone endoperoxide artemisinin (Fig. 1). Based on this secondary plant metabolite, several synthetic derivatives such as artemether, arteether, artesunic acid and artelinic acid have been produced, which are effective against multidrug-resistant Plasmodium falciparum strains, the organism responsible for malariaBecause chemical synthesis of artemisinin is an expensive multistep process, the plant remains the only commercial source of the drug. However, this compound is present in the leaves and the flowers in only small amounts ranging from 0.01 % to 0.8 % of dry weight [3]. AbstractAn important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the ± so far unknown ± amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic acid.

An Efficient Chemoenzymatic Synthesis of Dihydroartemisinic Aldehyde

“…The identity of the minor product was determined to be (11 R )‐ 4 based on 1 H‐NMR data and comparison with an authentic sample of 4 (Figures S26, S27) 17a. The structure of the major aldehyde was deduced to be the (11 S )‐ 4 epimer based on its diagnostic 1 H‐NMR signals at δ H 5.27 (s, H2), 1.64 (s, 3‐Me), 1.07 (d, J= 6.8 Hz, 11‐Me), and 0.87 ppm (d, J= 6.5 Hz, 7‐Me), which are complementary to those of (11 R )‐ 4 at δ H 5.13 (s, H2), 1.64 (s, 3‐Me), 1.06 (d, J= 6.9 Hz, 11‐Me), and 0.87 ppm (d, J= 6.5 Hz, 7‐Me; Figure 1).…”

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering

“…For example, limonene is extensively used as a scent compound in cosmetic products (Brokl et al 2013). The potential anticancer and anti-inflammatory compound zerumbone from shampoo ginger is a subject of interest in pharmacological studies (Bertea et al 2005;Yu and Utsumi 2009).…”

Research to Improve Artemisinin Production for use in the Preparation of Anti-Malarial Drugs

“…The chromatograms obtained with these two extracts were very similar, indicating that most (if not all) of A. annua terpenoids are present in the trichomes ( Figure 5). In both cases, artemisinic alcohol, artemisinic aldehyde, artemisinic acid, dihydroartemisinic alcohol, dihydroartemisinic aldehyde, dihydroartemisinic acid and a series of olefinic terpenes were detected (Bertea et al 2005). Artemisinic acid, dihydroartemisinic acid and the sesquiterpene olefins have been reported before as constituents of A. annua (Bouwmeester et al 1999a;Wallaart et al 1999b) but this was the first time artemisinic alcohol, artemisinic aldehyde, dihydroartemisinic alcohol and dihydroartemisinic aldehyde have been identified in A. annua.…”

“…The structures of all isolated or synthesized compounds were confirmed using NMR and MS (Bertea et al 2005). Subsequently, we looked for these compounds in extracts of A. annua leaves as well as in extracts of isolated trichomes.…”

“…A biotechnological approach could be another way to increase artemisinin content in A. annua. Because chemical synthesis of artemisinin is an (Bertea et al 2005) expensive multistep process, the plant remains the only commercial source of the drug. However, this compound is present in the leaves and the flowers in only small amounts ranging from 0.01 to 0.8 % of dry weight (Abdin et al 2003).…”