Totarol is a naturally produced

diterpene Diterpenes are a class of chemical compounds composed of four isoprene units, often with the molecular formula C20H32. They are biosynthesized by plants, animals and fungi via the HMG-CoA reductase pathway, with geranylgeranyl pyrophosphate being ...

that is bioactive as totarol. It was first isolated by McDowell and Esterfield from the heartwood of ''

Podocarpus totara ''Podocarpus totara'' (; from the Maori-language ; the spelling "totara" is also common in English) is a species of podocarp tree endemic to New Zealand. It grows throughout the North Island and northeastern South Island in lowland, montane and ...

'', a conifer tree found in

New Zealand New Zealand ( mi, Aotearoa ) is an island country in the southwestern Pacific Ocean. It consists of two main landmasses—the North Island () and the South Island ()—and over 700 smaller islands. It is the sixth-largest island count ...

.Short, W. F.; Stromberg, H. ''J. Chem. Soc.'' 1937, 516-520 Podocarpus totara was investigated for unique molecules due to the tree's increased resistance to rotting. Recent studies have confirmed totarol's unique antimicrobial and

therapeutic A therapy or medical treatment (often abbreviated tx, Tx, or Tx) is the attempted remediation of a health problem, usually following a medical diagnosis. As a rule, each therapy has indications and contraindications. There are many different ...

properties. Consequently, totarol is a candidate for a new source of

drugs A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via inhalat ...

and has been the goal of numerous syntheses.

Occurrence

Although totarol was first isolated in ''

'', totarol has also been identified in numerous other species of

Podocarpaceae Podocarpaceae is a large family of mainly Southern Hemisphere conifers, known in English as podocarps, comprising about 156 species of evergreen trees and shrubs.James E. Eckenwalder. 2009. ''Conifers of the World''. Portland, Oregon: Timber Pre ...

and

Cupressaceae Cupressaceae is a conifer family, the cypress family, with worldwide distribution. The family includes 27–30 genera (17 monotypic), which include the junipers and redwoods, with about 130–140 species in total. They are monoecious, subdio ...

, with the majority found in the genus ''Podocarpus'' of the family Podocarpaceae and the subfamily Cupressoideae of the family Cupressaceae.Sharp, H.; Latif, Z.; Bartholomew, B.; Bright, C.; Jones, C. D.; Sarker, S. D.; Nash, R. "Totarol, totaradiol and ferruginol: three diterpenes from Thuja plicata (Cupressaceae)." ''Biochemical systematics and ecology'' 2001, ''29'', 215-217. Outside Podocarpus and Cupressoideae, totarol is rarely found in the plant kingdom.Le Metayer, P.; Schaeffer, P.; Adam, P.; Albrecht, P.; Rousse, S.; Duringer, ''P. Org. Geochem.'' 2008, ''39'', 658-675. However, totarol has recently been isolated in ''

Rosmarinus officinalis ''Salvia rosmarinus'' (), commonly known as rosemary, is a shrub with fragrant, evergreen, needle-like leaves and white, pink, purple, or blue flowers, native to the Mediterranean region. Until 2017, it was known by the scientific name ''Rosmari ...

'' (rosemary).Bendall, J. G.; Cambrie, R. C. ''Aust. J. Chem.'' 1995, ''48'', 883-917. The

gymnosperm The gymnosperms ( lit. revealed seeds) are a group of seed-producing plants that includes conifers, cycads, ''Ginkgo'', and gnetophytes, forming the clade Gymnospermae. The term ''gymnosperm'' comes from the composite word in el, γυμνό� ...

s that contain totarol are distributed worldwide but are concentrated in North America, the far-south regions of

South America South America is a continent entirely in the Western Hemisphere and mostly in the Southern Hemisphere, with a relatively small portion in the Northern Hemisphere at the northern tip of the continent. It can also be described as the southe ...

East Asia East Asia is the eastern region of Asia, which is defined in both geographical and ethno-cultural terms. The modern states of East Asia include China, Japan, Mongolia, North Korea, South Korea, and Taiwan. China, North Korea, South Korea and ...

and

East Africa East Africa, Eastern Africa, or East of Africa, is the eastern subregion of the African continent. In the United Nations Statistics Division scheme of geographic regions, 10-11-(16*) territories make up Eastern Africa: Due to the historical ...

.Farjon, A. ''World Checklist and Bibliography of Conifers''. 2nd Ed.; Royal Botanic Gardens: Kew, 2001, p. 212.

Biological activity

Antimicrobial activity

Totarol motivates research in drug discovery due to its ability to inhibit numerous microorganisms. Totarol exhibits antimicrobial properties in numerous species including

gram-positive bacteria In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. Gram-positive bact ...

, acid-fast bacteria,

nematode The nematodes ( or grc-gre, Νηματώδη; la, Nematoda) or roundworms constitute the phylum Nematoda (also called Nemathelminthes), with plant-Parasitism, parasitic nematodes also known as eelworms. They are a diverse animal phylum inhab ...

s, parasitic protozoans, crustaceous foulers (Table 1). In addition to inhibiting microorganisms by itself, totarol exhibits inhibitory synergy with currently used antimicrobial drugs: totarol potentiates isonicotinic acid hydrazide against various Mycobacteria.;Mossa, J. S.; El-Feraly, F. S.; Muhammad, ''I. Phytotherapy Research.'' 2004, ''18'', 934-937. methicillin against ''

Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb) is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' has an unusual, waxy coating on its c ...

'' and ''

Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often positive ...

'';Gibbons, S. ''Phytochemistry Reviews.'' 2005, ''4'', 63-78. and anacardic acidKubo, I.; Muroi, H.; Himejima, M. J. Nat. Prod. 1992, ''55'', 1436-1440. and erythromycinSmith, E. C.; Kaatz, G. W.; Seo, S. M.; Wareham, N.; Williamson, E. A.; Gibbons, ''S. Antimicrob. Agents Chemother.'' 2007, ''51'', 4480-4483. against ''Staphylococcus aureus''. In nature, totarol is a key player in gymnosperm's defense against harmful microbes: gymnosperms that produce totarol are resistant to rotting. Table 1. Antibacterial activity of totarol against microorganisms

Mechanism of antimicrobial inhibition

Although totarol exhibits antimicrobial properties, the mode of action is unclear and various methods of inhibitory action have been proposed. In ''Staphylococcus aureus'' strains resistant to

penicillin Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...

via creation of

penicillin binding protein Penicillin-binding proteins (PBPs) are a group of proteins that are characterized by their affinity for and binding of penicillin. They are a normal constituent of many bacteria; the name just reflects the way by which the protein was discove ...

2’ (PBP2’), totarol may inhibit the synthesis of PBP2’. Totarol may inhibit effluxing ''Staphylococcus aureus'' strains through inhibition of MsrA, although it is unclear if MsrA is an efflux pump. Totarol may also gain its antibacterial properties by inhibiting bacterial respiratory transportHaraguchi, H.; Ishikawa, H.; Sakai, S.; Ying, B. P.; Kubo, I. ''Experientia''. 1996, ''52'', 573-576. but this is very unlikely because totarol is also effective against

anaerobic organism An anaerobic organism or anaerobe is any organism that does not require molecular oxygen for growth. It may react negatively or even die if free oxygen is present. In contrast, an aerobic organism (aerobe) is an organism that requires an oxygenate ...

s.Shapiro, S.; Guggenheim, B. ''Quant. Struct.-Act. Relat.'' 1998, ''17'', 327-337. Recently totarol was also hypothesized to inhibit gram-positive and acid-fast bacteria via inhibition of FtsZ protein, which forms the Z-ring, a polymer necessary for efficient bacterial cell

cytokinesis Cytokinesis () is the part of the cell division process during which the cytoplasm of a single eukaryotic cell divides into two daughter cells. Cytoplasmic division begins during or after the late stages of nuclear division in mitosis and meios ...

.Jaiswal, R.; Beuria, T. K.; Mohan, R.; Mahajan, S. K.; Panda, D. ''Biochemistry''. 2007, ''46'', 4211-4220. Totarol may also function by disrupting the structural integrity of the

phospholipid bilayer The lipid bilayer (or phospholipid bilayer) is a thin polar membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many vir ...

of bacteria by weakening Van der Waals interactions with its phenolic group,Micol, V.; Mateo, C. R.; Shapiro, S.; Aranda, F. J.; Villalain, ''J. Biochim. Biophys. Acta-Biomembranes''. 2001, ''1511'', 281-290.Mateo, C. R.; Prieto, M.; Micol, V.; Shapiro, S.; Villalain, ''J. Biochim. Biophys. Acta-Biomembranes''. 2000, ''1509'', 167-175.Bernabeu, A.; Shapiro, S.; Villalain, ''J. Chem. Phys. Lipids''. 2002, ''119'', 33-39. which also results in bacterial cells unable to synthesize ATP.Evans, G. B.; Furneaux, R. H.; Gainsford, G. J.; Murphy, M. P. ''Bioinorganic & Medicinal Chemistry''. 2000, ''8'', 1663-1675. Motivation for totarol functioning via disruption of membrane structure is due to its high phospholipid/water partition coefficient. However, totarol's partitioning capability was only observed at concentrations 10 to 100 fold higher than required for antibacterial activity. Thus it is unlikely that totarol is an uncoupler of bacterial respiration at the low levels observed in antimicrobial studies.

Traditional medicine

The use of Podocarpus totara extract in Maori medicines for treatment of

fever Fever, also referred to as pyrexia, is defined as having a body temperature, temperature above the human body temperature, normal range due to an increase in the body's temperature Human body temperature#Fever, set point. There is not a single ...

asthma Asthma is a long-term inflammatory disease of the airways of the lungs. It is characterized by variable and recurring symptoms, reversible airflow obstruction, and easily triggered bronchospasms. Symptoms include episodes of wheezing, cou ...

cough A cough is a sudden expulsion of air through the large breathing passages that can help clear them of fluids, irritants, foreign particles and microbes. As a protective reflex, coughing can be repetitive with the cough reflex following three pha ...

cholera Cholera is an infection of the small intestine by some strains of the bacterium ''Vibrio cholerae''. Symptoms may range from none, to mild, to severe. The classic symptom is large amounts of watery diarrhea that lasts a few days. Vomiting and ...

distemper Distemper may refer to: Illness *A viral infection **Canine distemper, a disease of dogs **Feline distemper, a disease of cats ** Phocine distemper, a disease of seals *A bacterial infection **Equine distemper, or Strangles, a bacterial infectio ...

, chest complaints and

venereal disease Sexually transmitted infections (STIs), also referred to as sexually transmitted diseases (STDs) and the older term venereal diseases, are infections that are spread by sexual activity, especially vaginal intercourse, anal sex, and oral se ...

dates back to over 100 years.Abdillahi H.S.; Stafford G.I.; Finnie J.F.; Staden J.V. ''South African Journal of Botany''. 2009, In Press. Despite totarol's antimicrobial potential, its commercial use is currently limited to cosmetic purposes. For totarol to be used clinically, its mode of action needs to be clearly defined.

Biochemical properties

Totarol decreases the plasma levels of estrogensMinami, T.; Iwamoto, M.; Ohtsu, H.; Ohishi, H.; Tanaka, R.; Yoshitake, A. ''Planta Med.'' 2002, ''68'', 742-745. and can also effectively reduce pathogenic hepatic cells ''

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

''.Lee, M. K.; Yang, H.; Yoon, J. S.; Jeong, E. J.; Kim, D. Y.; Ha, N. R.; Sung, S. H.; Kim, Y. C. ''Arch. Pharmacal Res''. 2008, ''31'', 866-871. Totarol's anti-cancer activity is hypothesized to be due to the natural product's ability to form an ''o''-quinone methide ''in vivo''.Water, R. W.; Pettus, T. R. ''Tetrahedron''. 2002, ''58'', 5367-5405. Totarol also prevents cells from undergoing oxidative stress ''in vitro'' by acting as a hydrogen donor to peroxy radicals or reacting with other peroxy radicals to terminate undesirable radical reactions.

Biosynthesis

Totarol is a precursor to the formation of nagilactones that possess

antifungal An antifungal medication, also known as an antimycotic medication, is a pharmaceutical fungicide or fungistatic used to treat and prevent mycosis such as athlete's foot, ringworm, candidiasis (thrush), serious systemic infections such as crypto ...

properties not possessed by totarol.Kubo, I.; Muroi, H.; Kubo, A. ''J. Agric. Food Chem.'' 1993, ''41'', 2447-2450. Consequently, gymnosperms that produce totarol and nagilactones are able to defend themselves against bacteria and fungi. The biosynthesis of totarol was difficult to determine. The main reason for the challenge in determining how the secondary metabolite is produced is because totarol does not follow the isoprene rule: the isopropyl group of totarol is in the “wrong” place at C14. Initially, it was hypothesized that totarol and the “normal” diterpene ferruginol, also found in Podocarpaceae, were derived by a precursor ''2'' that would be dehydrated and have its isopropyl group migrate to produce totarol 1 and ferruginol 3 (Scheme 1).Short, W. F.; Wang, H. ''J. Chem. Soc.'' 1951, 2979-2987. This hypothesis was motivated by the well known santonin-desmotroposantonin rearrangement of steroid dienones into aromatic compounds. It is now accepted that totarol is synthesized biologically from ferruginol. Geranyl geranyl pyrophosphate 4 undergoes typical diterpene cyclization to form (−)-abietadiene 5, which is oxidized to form ferruginol 3, which proceeds through a spiro intermediate to form totarol (Scheme 2).

Synthesis

Totarol has been the subject of numerous syntheses. The first total synthesis of totarol (Scheme 3)Barltrop, J. A.; Rogers, N. A. ''J. Chem. Soc.'' 1958, 2566-2572. utilized 6 and the alkyne 7 to yield 8 which was converted to the corresponding

ketone In organic chemistry, a ketone is a functional group with the structure R–C(=O)–R', where R and R' can be a variety of carbon-containing substituents. Ketones contain a carbonyl group –C(=O)– (which contains a carbon-oxygen double bo ...

9 via

hydrogenation Hydrogenation is a chemical reaction between molecular hydrogen (H2) and another compound or element, usually in the presence of a Catalysis, catalyst such as nickel, palladium or platinum. The process is commonly employed to redox, reduce or S ...

followed by

cyclization A cyclic compound (or ring compound) is a term for a compound in the field of chemistry in which one or more series of atoms in the compound is connected to form a ring. Rings may vary in size from three to many atoms, and include examples where al ...

with

polyphosphoric acid A phosphoric acid, in the general sense, is a phosphorus oxoacid in which each phosphorus (P) atom is in the oxidation state +5, and is bonded to four oxygen (O) atoms, one of them through a double bond, arranged as the corners of a tetrahedron. ...

. 9 was subsequently converted to 10 and another ketone that were inseparable by

chromatography In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...

. The synthesis was finalized by treatment with

N-Bromosuccinimide ''N''-Bromosuccinimide or NBS is a chemical reagent used in radical substitution, electrophilic addition, and electrophilic substitution reactions in organic chemistry. NBS can be a convenient source of Br•, the bromine radical. Preparation ...

followed by debromination to yield (±)-totarol. The main downfall of this synthesis was that in multiple steps, complete conversion of reactant to products was not observed and undesirable side products were often not separable by chromatography. However, since this was the first total synthesis of (±)-totarol, it is notable.

Total enantioselective synthesis

The first total enantioselective synthesis of totarol was achieved in 1979 (Scheme 4).Matsumoto, T.; Suetsugu, A. ''Bull. Chem. Soc. Jpn.'' 1979, ''52'', 1450-1453. The key step in the synthesis is the formation of 13 via a Wittig reaction between 11 and 12. This same cyclization can also be achieved via a Friedel-Crafts alkylation and cyclization.Das, S.; Bhattacharyya, S.; Mukherjee, D. ''Tetrahedron''. 1992, ''48'', 9101-9110. Subsequent hydrogenation of 13 followed by intramolecular cyclization with aluminium chloride forms the B ring and totarylmethyl ether which is demethylated by boron tribromide to yield totarol. A more recent organic synthesis of totarol was achieved by utilizing 14, a lamdane diterpene named zamoranic acid (Scheme 5).Marcos, I. S.; Cubillo, M. A.; Moro, R. F.; Diez, D.; Basabe, P.; Sanz, F.; Urones, J. G. ''Tetrahedron Lett.'' 2003, ''44'', 8831-8835. The addition of the isopropyl group in the chemical synthesis was achieved with complete stereoselectivity. Acetylation to yield 15 required high temperatures due to the steric hindrance of the isopropyl group. Cis-hydroxylation followed by cleavage with H₅IO₆ yielded a diol that was acylated in pyridine and oxidized to give 16. The key step in the synthesis was the cyclization of ring C: 16 was treated with SmI₂ to yield totarane diastereomers which were separated by column chromatography. The desired diastereomer was treated with ''p''-TsOH in benzene to yield 17. The synthesis was completed by a halogenation-dehydrogenation sequence and subsequent bromination to yield 18 and ring aromatization with elimination via a lithium complex.

Total chemoenzymatic synthesis

Chemoenzymatic synthesis of totarol has also been achieved with high yield (41.8%) (Scheme 6).Miyake, T.; Kigoshi, H.; Akita, H. ''Tetrahedron: Asymmetry''. 2007, ''18'', 2915-2922. A racemic beta-keto ester 19 undergoes lipase-assisted resolution to yield chiral alcohol 20. Treatment of 20 with 10% HCl and p-TsOH gives ''αβ''-unsaturated ketone 21. A Michael addition with the anion obtained from the reaction of methyl 5-methyl-3-oxohexanoate 13 with NaOMe gives a 2:1 diastereomeric mixture of 22 which is hydrolyzed to yield 23 which is brominated and debrominated to yield totarol.

Other uses

Totarol may also be used as an indicator for the quality of juniper berry based spirits. Juniper berries that contain

diterpenoid Diterpenes are a class of chemical compounds composed of four isoprene units, often with the molecular formula C20H32. They are biosynthesized by plants, animals and fungi via the HMG-CoA reductase pathway, with geranylgeranyl pyrophosphate being ...

s including totarol are used for the aromatization and production of some gins. Consequently, totarol can aid in the characterization of different types of gin or commercial brands, vouching for the authenticity and quality of the product.Vichi, S.; Aumatell, M. R.; Buxaderas, S.; Lopez-Tamarnes, ''E. Analytica Chimica Acta''. 2008, ''628'', 222-229. Totarol has been found on the posterior tibia of ''

Frieseomelitta silvestrii ''Frieseomelitta silvestrii'' is a stingless bees species in the genus ''Frieseomelitta'' found in Brazil. '' Frieseomelitta silvestrii languida'' is a subspecies making use of resin and of the chemical compound totarol Totarol is a naturally ...

languida'', a species of stingless bees from Brazil. ''Frieseomelitta silvestrii languida'' collect resin to create a protective barrier around the opening of their nest to ward off insects from settling near the nest's entrance.Patricio, E.; Cruz-Lopez, L.; Maile, R.; Tentschert, J.; Jones, G. R.; Morgan, E. D. ''Journal of Insect Physiology''. 2002, ''48'', 249-254. "The propolis of stingless bees: terpenes from the tibia of three Frieseomelitta species" The presence of totarol can aid in the determination of this bee species.

References

{{reflist Terpeno-phenolic compounds Diterpenes Phenanthrenes Phenols Isopropyl compounds