**1. Introduction**

Pyrrolizidine alkaloids (PAs) are heterocyclic organic compounds synthesized by plants that are thought to act as defense compounds against herbivores [1]. Estimates indicate that approximately 6.000 plant species worldwide, representing 3% of all flowering plants, produce these secondary metabolites. In particular, members of the Asteraceae, Boraginaceae, Heliotropiaceae, Apocynaceae, and some genera of the Orchidaceae and the Fabaceae are PA producers [2]. Reported concentrations vary greatly, from trace amounts to up to 19% dry weight, and are considered to be dependent on a number of factors including the developmental stage, tissue type, environmental conditions, and extraction procedures [3].

PAs consist of a necine base esterified with a necic acid. The necine base typically includes pyrrolizidine, a bicyclic aliphatic hydrocarbon consisting of two fused five-membered rings with a nitrogen at the bridgehead [4] (Figure 1). Loline alkaloids may be formally considered as PAs since they also possess a pyrrolizidine system, although it contains an ether bridge linking carbon 2 (C-2) and carbon 7 (C-7). While *stricto sensu* PAs are exclusively formed in plants, lolines are synthesized by endophytic fungal symbionts of the genus *Epichloë* [5]. In addition, their biosynthesis is distinct from PAs [5–7]. Thus, lolines will be discussed only peripherally in this review.

**Figure 1.** Core structures and examples for pyrrolizidine, loline, indolizidine, quinolizidine, tropane and granatane alkaloids. In contrast to the other alkaloids pyrrolizidine alkaloids appear mainly as *N*-oxides, as shown for the example of senecionine-*N*-oxide.

The fused bicyclic system of PAs resembles indolizidine and quinolizidine alkaloids, which contain a five and a six-membered ring or two six-membered rings, respectively [8] (Figure 1). Tropane and granatane alkaloids also show a similar structure consisting of a five and a six-membered ring or two six-membered rings, respectively [9]. However, in contrast to necine bases, the rings of tropane and granatane alkaloids are bridged rather than fused. While several tropane and quinolizidine alkaloids including atropine (the racemic mixture of (±)-hyoscyamine) and sparteine are used in medicine [9], PAs are mainly known for their hepatotoxic and potentially carcinogenic properties [10]. Nevertheless, some PAs show interesting pharmacological properties that are currently under investigation (see Section 5.3) [10,11]. While tropane and quinolizidine alkaloids are usually present in plants in their free forms, PAs are mainly present as *N*-oxides (Figure 1), which are highly water-soluble and considered less toxic than the free PAs.

#### **2. Structural Diversity of Pyrrolizidine Alkaloids**

Within the combination of a set of necine bases (Figures 2 and 3) and a considerable number of necic acids (Figure 4), an enormous structural diversity of PAs can be obtained. This is further amplified by modifications, including *N*-oxidation of the tertiary nitrogen of the necine base, hydroxylation of the necine base and/or the necic acid, and acetylation of hydroxy groups of the acid moiety. Thus, it is not surprising that several hundreds of different PAs have already been identified and each year new variants are described.

## *2.1. Diversity of Necine Bases*

In addition to the pyrrolizidine ring system most necine bases possess a hydroxymethyl group at position 1 (Figure 2), which is a consequence of the biosynthetic pathway (see Section 3.1). Since 1-hydroxymethylpyrrolizidine contains two chiral centers, carbons C-1 and C-8, in total four compounds exist: The enantiomers (-)/(+)-trachelanthamidine and (-)/(+)-isoretronecanole (Figure 2B). Among them, (-)-trachelanthamidine and (-)-isoretronecanole are most frequently found, for instance, as the necine base of trachelanthamine (Figure 5C) and the nervosines [12] (Figure 5E), respectively. Examples for PAs containing (+)-trachelanthamidine and (+)-isoretronecanole are acetyllaburnine [13] (Figure 5G) and madhumidine A [14], respectively. The most frequent modification of saturated necine bases is hydroxylation at C-7. However, the positions C-2 and C-6 are also occasionally hydroxylated. Necine bases like (-)-platynecine, possessing hydroxy groups on C-7 and C-8, and (-)-rosmarinecine containing hydroxy groups on C-2, C7 and C-9 (Figure 2), are often esterified by dicarboxylic necic

acids to form macrocyclic PAs like platyphylline and rosmarinine (Figure 5A) [15]. In general, saturated PAs are considered as non-toxic [16].

**Figure 2.** Structures of necine bases. (**A**) Basic structures and numbering of atoms in necine bases. (**B**) Saturated necine bases. (**C**) 1,2-Desaturated necine bases. (**D**) Unusual necine bases.

Most PAs contain a necine base possessing a double bond between C-1 and C-2 (Figure 2). Introducing that double bond eliminates the chiral centre at C-1, thus leaving only the stereocentre at C-8. Consequently, only two forms, (-)/(+)-supinidine, exist of the C-9 monohydroxalated derivatives and four, (-)/(+)-retronecine and (-)/(+)-heliotridine, of the C-7 and C-9 dihydroxylated compounds. Among them (+)-retronecine is the most frequently observed necine base in PAs.

In addition to the saturated and desaturated bases discussed above, necine bases of the otonecine type also exist. Otonecine is not a genuine bicyclus, but may act as such due to transannular interactions of the keto group and the tertiary amine (Figure 3A) [17]. These interactions are also likely for the reason that otonecine-type PAs are present in plants as free bases rather than *N*-oxides.

**Figure 3.** Otonecine and representative PAs. (**A**) Resonance structures of otonecine. (**B**) Structures of the otonecine-type PAs otosenine, florosenine, ligularidine, and doronine.

There are also several necine bases with unusual structures (Figure 2D). One of them is 1-aminopyrrolizidine, wherein the hydroxymethyl group is replaced by an amino group. This unusual necine base is found for instance in laburnamine, an alkaloid present in trace amounts in *Laburnum anagyroides* [18]. From the leaves of *Ehretia asperia,* ehretinine was isolated, which is very unusual since the 7-hydroxy group of its necine base, (1*R*,7*S*)-7-methylhexahydro-1*H*-pyrrolizin-1-ol, is esterified with 4-methylbenzoic acid and the typical hydroxymethyl group on C-1 is replaced by a methyl residue [19]. Similarly, *Senecio polypodioides* contains, besides sarracine *N*-oxide, also 7β-angeloyloxy-1-methylene-8α-pyrrolizidine, a PA with a methylene group instead of the typical hydroxymethyl residue on its necine base. The 7-hydroxy group of this PA is esterified with angelic acid [20]. In *Echium glomeratum*, PAs with a tricyclic ring were found. The 9-hydroxy group of the necine base was found to be esterified with angelic acid [21]. Another example is the necine base of tussilagine from *Tussilago farfara*, which possesses a carboxy group instead of the typical hydroxymethyl group (Figure 5H) [22].

#### *2.2. Diversity of Necic Acids*

While necine bases share a common structure, the necic acids show broad structural diversity. Some, particularly the smaller and simpler ones, are typical metabolites of plant metabolism, while others, particularly the monocarboxylic acids of the trachelanthic acid type and the dicarboxylic acids (Figure 4) are formed in specific, complex pathways.

Acetic acid (Figure 4A) is frequently observed in simple PAs, for instance 7-acetylretronecine present in *Onosma arenaria* [23] and acetyllaburnine present in *Vanda*, a genus of the Orchidaceae [13,24] (Figure 5G). Acetic acid may also esterify the second hydroxy group of the necine base in triangularine and lycopsymine-type PAs, such as 7-acetyl-9-sarracinoylretronecine present in *Alkanna tuberculata* [25] (Figure 5B) and uplandicine found in pollen of *Echium vulgare* (Figure 5C). In addition, acetic acid also frequently esterifies hydroxy groups of other necic acids in more complex PAs, for instance florosenine [26], ligularidine [27] (Figure 3B), or acetylerucifoline *N*-oxide [28]. In contrast to the frequently observed acetic acid, lactic acid has, so far, only been found in lactoidine, a PA of *Cynoglossum furcatum* [29].

C5 acids of the tiglic acid type (Figure 4A) are characteristic for the triangularine group of PAs (Figure 5B). They may esterify one or two hydroxy groups of the necine base. In the former case they may appear together with acetic acid or more complex necic acids, particularly branched C7 acids, which is seen for instance in the PAs echimidine [30] and heliosupine [31] (Figure 5F). In addition to esterifying necine bases directly, C5 acids may also esterify hydroxy groups of other

necic acids. Examples are scorpioidine, a PA of *Myosotis scorpioides* [32] (Figure 5F), and anadoline, a PA of *Symphytum orientale* [33,34]. Latifolic acid [35–37] and the closely related hackelic acid [38] are examples of cyclic C7 acids.

Aromatic systems are rarely present in necic acids except in PAs found in the Orchidaceae. Many of them, for instance benzoic acid, salicylic acid and *p*-coumaric acid, are simple aromatic acids present as primary or secondary metabolites in most plant species. However, some aromatic necine bases, particularly those found in the genera *Phalaenopsis* and *Liparis*, show a very complex structure, for instance the phalaenopsines [39,40] and the nervosines [41] (Figures 4B and 5E).

**Figure 4.** Examples for necic acids. (**A**) Monocarboxylic aliphatic acids. (**B**) Monocarboxylic aromatic acids. (**C**) Dicarboxylic acids forming macrocyclic PAs. Adapted from Reference [17].

The dicarboxylic necic acids (Figure 4C) are a particularly interesting group because they form macrocyclic PAs, which are considered to be the most toxic. Necic acids of the monocrotalic acid type are a relatively small group; they form 11-membered rings. In contrast, senecic acid-like necic acids typically form 12-membered rings and represent a large group. The considerable diversity is obtained by modification of the senecic acid core structure by a number of reactions (Section 3.2.4). Interestingly, a few among them contain chlorine (Figures 3B and 4C), a modification rarely observed in plant metabolites.

#### *2.3. Linkage Patterns of Necine Bases with Necic Acids*

Based on the combination of necine bases and necic acids and their linkage patterns the PAs have been classified into five groups [42]. The first and largest group are senecionine-like PAs, which consist of necine bases of the retronecine (Figure 2C), platynecine, rosmarinecine (Figure 2B), or otonecine-type (Figure 3) and typically branched C10 dicarboxylic necic acids (Figure 4C) derived from two molecules of L-isoleucine (see Section 3.2.4), which together form 12-membered macrocyclic rings. An exception is the small sub-group of nemorensine-like PAs [43], which form 13-membered macrocycles (Figure 5A). Typically, the necine bases are esterified at their C-7 and C-9 hydroxy groups. PAs of this type are mainly found in the tribe Senecioneae and family Fabaceae [44].

The second group is represented by triangularine-type PAs, which are open-chain mono- or diesters of necine bases with the C5 acids tiglic, angelic, senecioic, and sarracinic acid (Figure 5B). These PAs are mainly present in Senecioneae and Boraginaceae [44].

The third type, the lycopsamine-like PAs are mainly found in Boraginaceae and Eupatorieae [44]. This type possesses branched C7 necic acids esterifying the C-9 hydroxy group (Figure 5C). A number of PAs represent a combination of group 2 and 3 since they also possess a C5 acid residue in addition to a C7 necic acid. The C5 acid residue can either be linked directly with the necine base or attached to a hydroxy group of the C7 acid (Figure 5F).

The fourth group are the 11-membered macrocyclic PAs of the monocrotaline type. Similar to senecionine-like PAs the hydroxy groups of C-7 and C-9 are esterified with dicarboxylic necic acids (Figure 5D). This group is found predominantly in Fabaceae [44].

Phalaenopsine and ipanguline-type PAs represent the fifth group, which is characterized by the presence of an aromatic acid (Figure 4B), esterifying the usually saturated necine base (Figure 5E). The acidic compound shows a high structural diversity and includes simple aromatic acids like benzoic, salicylic and *p*-coumaric acid, but also very complex ones like nervogenic acid. This is the only group of PAs that are frequently glycosylated. Members of this group are found in the Orchidaceae, Convolvulaceae, and in a few representatives of other tribes including the Boraginaceae [44].

In addition to these five groups, there are also very simple PAs consisting only of the necine base and a small acid residue, particularly acetate, as illustrated by the examples shown in Figure 5G. A number of PAs show unusual linkage patterns distinct from that of the five groups discussed above. In madurensine the hydroxy group of C-9 is bridged by the dicarboxylic acid intergerrinecic acid with a hydroxy group placed at C-6 rather than the usual C-7 hydroxyl [45]. This leads to a 13-membered macrocyclic ring (Figure 5H). The structure of laburnamine [46] matches that of PAs of the triangularine type. However, since its necine base (1*S*,8*R*)-1-aminopyrrolizidine (Figure 2D) possesses an amino group instead of the hydroxy group on C-9, a reaction with isovaleric acid yields an amide rather than an ester bond (Figure 5G). Tussilagine, a PA of *Tussilago farfara* (coltsfoot), is very special since its necine base possesses, instead of the typical hydroxymethyl residue, a carboxy group on C-1, which is esterified with methanol [22]. Anhydroplatynecine is devoid of any necic acid and the C-7 and C-9 hydroxy groups of platynecine (Figure 2B) instead combine together via an ether bridge. However, anhydroplatynecine is likely not a naturally-occurring PA, but is rather formed by heating of platynecine containing PAs during isolation [47]. Finally, it is worth mentioning that several plant species also contain unmodified necine bases in their free form or as *N*-oxides [48].

**Figure 5.** Linkage patterns of necic acids with necine bases. (**A**) Senecionine type. (**B**) Triangularine type. (**C**) Lycopsamine type. (**D**) Monocrotaline type. (**E**) Phalaenopsine/ipanguline type. (**F**) Compounds combining necic acids of triangularine and lycopsamine types. (**G**) Simple PAs. (**H**) PAs with unusual linkage patterns.

#### *2.4. Modification and Conjugation of Pyrrolizidine Alkaloids*

As discussed above, the astonishing diversity of PAs is achieved by hydroxylation and desaturation of necine bases and necic acids and their combination to PAs. Complete PAs might also be modified by hydroxylation, desaturation and epoxidation. The latter may be further metabolized to a diol or a chlorine-containing PA (see Section 3.2.4). In addition to these generally irreversible modifications, PAs can also be reversibly modified. By far the most frequently observed modification of this type is *N*-oxidation (Figure 1). In plants, the major fraction of PAs is present as *N*-oxides. Exceptions include seeds of several *Crotalaria* species [49] and leaves of *Crassocephalum crepidioides*, wherein the majority of the PAs are present in their basic form [50], and shoots of jacobine-chemotype plants of *Senecio jacobaea*, in which up to 50% might be present as tertiary PAs [51]. *N*-oxidation of the tertiary amine nitrogen changes the properties of a PA significantly. In contrast to basic tertiary amines, which are positively charged under physiological conditions, amine *N*-oxides are neutral and behave like very polar, highly water-soluble, salt-like compounds that are thought to be membrane impermeable. These characteristics might be important for their role in transport and storage of PAs. Accordingly, it was shown that PA transporters in membranes of plant cells have a higher affinity for PA *N*-oxides than for the tertiary amines [52].

In addition to *N*-oxidation, a number of PAs are also acetylated, particularly at hydroxy groups of the necic acid moiety. Examples are 7-acetylscorpionidine, the 7-*O*-acetylation product of scorpionidine (Figure 5F), and the otonecine-type PAs florosenine and ligularidine (Figure 3B), which are acetylated forms of otosenine and petasitenine, respectively.

While glycosylation is frequent among secondary metabolites, modifications of that type are rarely observed for PAs. Only among the PAs with aromatic necic acids some examples are known. They include thesinine-4 -*O*-α-L-glucoside present at high levels in borage seeds [53] (Section 6.1), thesinine-4'-*O*-α-L-rhamnoside found in *Lolium* species [54] (Section 6.4) and nervone PAs isolated from *Liparis nervosa* [41] (Figure 5E).

Other modifications are rarely seen in PAs.

#### **3. Biosynthesis of Pyrrolizidine Alkaloids**

Attempts at deciphering PA biosynthesis (Figure 6) date back to the early 1960s, when Nowacki and Byerrum performed their first feeding experiments with radiolabeled precursors [55,56]. Later, this work was continued by others, mainly the groups of Robins and Crout. Robins also introduced labeling with stable isotopes, particularly 13C, 2H and 15N, and subsequent analysis by NMR spectroscopy for analysis of PA biosynthesis [57–59]. This technique provided detailed information about the fate of single C and H atoms during biosynthesis of the necine bases and necic acids. In the late 1990s, the first biosynthetic enzyme, homospermidine synthase, catalyzing the first committed step in PA biosynthesis, was identified [2]. Its analysis in different plant species provided interesting data about PA evolution, or more precisely, homospermidine biosynthesis.

#### *3.1. Biosynthesis of Necine Bases*

Feeding of *Crotalaria spectabilis* plants, which produce monocrotaline, with 14C-labelled precursors showed that [14C]-ornithine was efficiently incorporated into monocrotaline, particularly into its necine base retronecine [56]. Studies with *Senecio isatideus* [60] and *Senecio douglasii* [61] confirmed that [ 14C]-ornithine is mainly incorporated into the necine base. Degradation studies in the latter study showed that approximately 25% of the incorporated radioactivity was present in carbon C-9 of the retronecine unit irrespective of whether [2-14C]-ornithine or [5-14C]-ornithine were fed, indicating that C-2 and C-5 of ornithine become equivalent during biosynthesis (at least for biosynthesis of the right-handed ring) and suggesting 1,4-diaminobutane (putrescine) as a symmetrical intermediate. Indeed, feeding of [1,4-14C2]-putrescine again yielded retronecine bearing approximately 25% of the radioactivity on C-9. Using *Senecio magnificus,* it was shown that arginine is also selectively incorporated

into the necine base part of senecionine [62]. Subsequently, Robins and Sweeney compared the incorporation efficiency of several compounds using *Senecio isatideus* and found that putrescine, spermidine, and spermine were more efficiently incorporated than arginine and ornithine [63] and that the two latter were only effectively incorporated when present in the L configuration [64].

While these experiments established L-arginine and L-ornithine as precursors and putrescine as an intermediate, they did not allow a more comprehensive investigation since the fate of the individual C and H atoms could not be followed during biosynthesis. This problem was overcome by introduction of stable isotope labeled precursors and NMR analysis of the obtained products in combination with an improved plant feeding technique. Previously, plants were mainly fed hydroponically or as cut shoots, which resulted in incorporation rates significantly below 1%. In contrast, by absorption of aqueous solutions of the precursors directly into the xylems of freshly rooted cuttings through stem punctures, incorporation rates of up to 5% could be obtained [63]. Feeding of both [1,4-13C2]-putrescine and [1-13C]-putrescine gave enriched 13C signals for C-3, C-5, C-8 and C-9 (Figure 7A), confirming that both rings originate from putrescine [57,65].

**Figure 6.** Biosynthesis of PAs. The polyamines putrescine and spermidine are derived from the basic amino acid arginine. Subsequently, homospermidine synthase (HSS) exchanges the 1,3-diamonopropane residue of spermidine by putrescine, which releases 1,3-diaminopropane and forms symmetric homospermidine. Oxidation of homospermidine, likely by copper-dependent diamine oxidases, to 4,4´-iminodibutanal initiates cyclization to pyrrolizidine-1-carbaldehyde, which is reduced, likely by an alcohol dehydrogenase, to 1-hydroxymethylpyrrolizidine. Desaturation and hydroxylation by unknown enzymes form retronecine, which is acylated with an activated necic acid, for instance with senecyl-CoA2 as in the example shown above. Acylation might be catalyzed by an acyltransferase of the BAHD family. PA *N*-oxides, which are believed to be the primary products of PA biosynthesis, may be reduced to the free tertiary amine.

*Molecules* **2019**, *24*, 498

Because approximately 1.1% of the natural occurring carbon is the 13C isotope, a 13C label is easily obscured, particularly if the incorporation efficiency is moderate to low. Thus, further studies made use of double labeled precursors, where the 13C-13C double label can be sensitively detected by 13C-NMR spectroscopy as doublet around the natural abundance signal. Feeding of [2,3-13C2]-putrescine gave a pair of doublets for C-1 and C-2 with a coupling constant *J* of 34 Hz and a second doublet pair for C-6 and C-7 with a coupling constant *J* of 70 Hz [65] (Figure 7B). Finally, feeding *Senecio isatideus* with [1,2-13C2]-putrescine gave rise to four pairs of doublets, namely C-1/C-9, C-2/C-3, C-5/C6 and C-7/C-8, with four different coupling constants (Figure 7C). To investigate which C-N bond remains intact during retronecine biosynthesis [1-15N,1-13C], double labeled putrescine was synthesized and fed to *Senecio vulgaris* [66,67] or *Senecio isatideus* [58,65]. Analysis of the necine base showed an equal amount of retronecine with a 15N and a 13C label at C-3 and retronecine with a 15N and a 13C label at C-5 (Figure 7D). The observation that both variants appeared at the same level confirmed the presence of a symmetric C4-N-C4 compound in retronecine biosynthesis. A similar series of experiments was also performed using *Senecio pleistocephalus*, which forms rosmarinine as the sole PA. Rosamarinine consists of senecic acid and the saturated necine base rosmarinecine, which has, in addition to the typical 7 and 9-hydroxy groups, an additional hydroxy group on C-2 (Figure 2B). Additionally, within this experimental system, the same result was obtained that a symmetric C4-N-C4 intermediate is involved in necine base biosynthesis [68].

**Figure 7.** Investigation of necine base biosynthesis by feeding of stable isotope labeled precursors. Feeding of (**A**) [1,4-13C2]-putrescine and [1-13C]-putrescine, (**B**) [2,3-13C2]-putrescine, (**C**) [1,2-13C2]-putrescine, (**D**) [1-15N, 1-13C]-putrescine and (**E**) [1,9-13C2]-homospermidine. Red dots symbolize 13C labels, a red N symbolizes a 15N label. 13C-13C double labels are marked with bonds in red. Please mind that the retronecine structures shown in (A), (B), and (C) are composite representations of all labeled species present.

#### 3.1.1. Homospermidine Synthase

The most obvious candidate for the symmetric C4-N-C4 intermediate was homospermidine, which is known to be present in a number of plant species, particularly in such producing PAs. Initial experiments with radiolabeled homospermidine showed that this compound was incorporated into the necine base part of retrorsine in feeding experiments with *Senecio isatideus* [69]. To investigate whether the C4-N-C4 unit stays intact during biosynthesis 13C labels were placed on the most distal carbons (Figure 7E). Feeding of [1,9-13C2]-homospermidine to *Senecio pleistocarpus* revealed enrichment of the label on C-8 and C-9 of the isolated retronecine [70], confirming that homospermidine is incorporated intact into necine bases.

At the same time, Bötcher et al. partially purified an enzyme with homospermidine synthase (HSS) activity from root cultures of *Eupatorium cannabinum* [71]. Enzymatic assays were performed by addition of [14C]-labeled putrescine to the partially purified enzyme. The pH optimum was 9 and the enzymatic reaction was strictly dependent on the presence of NAD+. The enzyme showed high selectivity for putrescine and NAD+, and accepted neither NADP+ as co-substrate nor any of the other tested amines including 1,3-diaminopropane, cadaverine, and pyrroline as substrate. The Michaelis-Menten constants (KM) for putrescine and NAD<sup>+</sup> were 13.5 μM and 3 μM, respectively. In contrast to NAD+, its reduced form, NADH, acted even at low concentrations (inhibition constant Ki: 2 μM) as a strong inhibitor indicating that the NADH formed in the first step of the reaction, the formation of an imine intermediate, remains bound to the enzyme and serves as electron donor for the second step, the reduction of the imine intermediate to the secondary amine. The enzymatic activity was also inhibited by 1,3-diaminopropane, spermidine, and homospermidine with Ki values of 6.3, 94 and 950 μM, respectively. While it was believed that the partially purified enzyme utilized two molecules of putrescine for formation of one molecule of homospermidine, later work with HSS purified to homogeneity showed that the enzyme instead uses putrescine and spermidine to produce homospermidine and 1,3-diaminopropane as by-products [2]. The reason for the initially wrong conclusion was that spermidine was added at all steps during enzyme purification since it was found that this compound preserves enzyme activity. Consequently, spermidine was also present in the partially purified enzyme and thus in the enzymatic reaction at sufficient concentrations allowing transfer of the radiolabeled putrescine moiety to spermidine. Thus, the first step of PA biosynthesis is transfer of the 1,4-diaminobutan part of spermidine to a specific lysine residue of HSS accompanied by release of 1,3-diaminopropane and reduction of NAD+ to NADH, which remains bound to HSS (Figure 8). Subsequently, putrescine reacts with the HSS-bound 1,4-diaminobutane moiety under formation of an imine intermediate and regeneration of the lysine-NH2. Finally, the imine is reduced by the HSS-bound NADH to homospermidine and released from the regenerated HSS/NAD<sup>+</sup> complex [72].

**Figure 8.** Mechanism of homospermidine formation by HSS. The amino group of a lysine residue of HSS reacts with spermidine, which releases 1,3-diaminopropane and reduces HSS-bound NAD<sup>+</sup> to NADH. Next, the residue is transferred to putrescine, forming an imine intermediate, which is reduced by the HSS-bound NADH to release homospermidine and recycle the HSS/NAD<sup>+</sup> complex. Adapted from [72].

Purification of HSS from *Senecio vernalis* to homogeneity allowed identification of the protein and corresponding gene. After protease treatment, four fragments were obtained that were microsequenced to give short peptide sequences. Database searches revealed close homology to deoxyhypusine synthase (DHS) [2]. DHS catalyzes the NAD+-dependent transfer of an amino-butyl moiety from spermidine to a specific lysine side chain of the precursor for the eukaryotic initiation factor 5A (eIF5A), forming the amino acid deoxyhypusine. This reaction is one of the most specific post-translational modifications known [73,74] and similar to the reaction proposed for HSS except that elF5A is replaced by spermidine (Figure 8). Subsequent PCR with redundant primers yielded the first sequences, which were completed by 3´ and 5´-RACE allowing subsequent for a cloning of the complete *Senecio vernalis* HSS coding sequence. Heterologous expression of the cloned gene as His(6)-tagged fusion protein in *E. coli* and in vitro enzymatic assays confirmed that the enzyme for homospermidine synthesis was obtained and that putrescine and spermidine are required as substrates. With respect to that, it is worth mentioning that this may also explain the considerable incorporation of radiolabeled spermidine into homospermidine previously observed in feeding experiments using *Senecio isatideus* [63].

Since HSS catalyzes the first committed step in PA biosynthesis, thereby linking primary metabolism with PA biosynthesis, it is also interesting to study its evolution in order to deduce how PA biosynthesis was established in plants [72]. Due to 70 to 90% sequence homology—depending on the species—it was suggested that HSS evolved through gene duplication from DHS [75]. HSS has been recruited from DHS independently in different plant families at least eight times: Once in the Apocynaceae, Boraginaceae, Convolvulaceae, Fabaceae, Orchidaceae, and Poaceae, and twice in the Asteraceae [76–78]. The major difference lies in the biochemical properties of the two enzymes. DHS accepts eIF5A (lys) as well as putrescine, though with far lower efficiency, as substrate and can therefore catalyze the formation of both eIF5A(dhp) and homospermidine, the latter at a very low rate. In contrast, HSS has lost the ability to bind eIF5A, hence can only produce homospermidine [2,79,80]. This change in substrate specificity was possible because binding of putrescine occurs within the active site of DHS, while binding of eIF5A(lys) happens at the surface of the enzyme [75]. Reimann et al. [78] compared the rates of non-synonymous to synonymous mutations in HSS and DHS and found higher rates in HSS, suggesting higher selection pressure on DHS. The same study also found that it is not possible to distinguish between DHS and HSS solely by sequence data since there are no differing characteristic patterns. Despite the similar biochemical properties and sequences, the expression levels of both genes clearly differ. A study of the expression patterns of HSS and DHS in *Senecio vernalis* (Asteraceae, Senecioneae) revealed that DHS is expressed in all plant tissues in an almost constant manner throughout plant development. In contrast, HSS expression was found to be restricted to root cells, particularly to endodermis and cortex parenchyma cells [81]. In *Eupatorium cannabium* (Asteraceae, Eupatorieae) HSS expression was also found in the cortex parenchyma cells but not in the endodermis. In addition, HSS expression was shut down when the flower buds opened [82]. In contrast, in *Phalaenopsis* (Orchidaceae) HSS is expressed in the tips of aerial roots and in young flower buds [83]. In the Boraginaceae different HSS expression patterns were observed. In *Heliotropium indicum*, HSS was expressed exclusively in non-specialized cells of the lower epidermis of young leaves and shoots while in *Symphytum officinale* HSS expression was detected in the cells of the root endodermis and in leaves underneath developing inflorescences. In *Cynoglossum officinale* HSS expression was only observed in roots. In young roots, its expression was limited to cells of the endodermis, while in later developmental stages cells of the pericycle, it also showed HSS expression [84].

One theory regarding establishment of secondary metabolic pathways suggests that changes in gene function lead to subfunctionalization and a subsequent duplication event to two genes with different but complementary subfunctions, preserving the original enzyme function [75]. This complements an early suggestion by [85] that, prior to a gene duplication leading to novel protein function, the original gene was bifunctional. This theory is also supported by the bifunctionality of DHS, when considering this model for the explanation of HSS evolution. Furthermore, gene regulation and therefore gene expression patterns, might vary for the two genes, resulting from gene duplication [86]. This suggestion finds support in the varying expression patterns of DHS and HSS described above.

While extensive research on the recruitment of HSS has been conducted, there is still little knowledge about the evolution of the entire PA biosynthetic pathway, as it cannot be explained by the presence of homospermidine alone. Introduction of HSS into non-PA producing plants only results in formation of homospermidine, rather than of PAs or any precursors downstream of homospermidine [87].

#### 3.1.2. Copper-Dependent Diamine Oxidases and Cyclization of the Dialdehyde

Early evidence for involvement of a diamine oxidase in the incorporation of homospermidine into necine bases was provided by incubating homospermidine in vitro with a diamine oxidase fraction prepared from pea. Reduction of the reaction products by sodium borohydride and subsequent analysis by GC revealed that mainly trachelanthamidine and a small amount (approximately 5%) of isoretronecanol were obtained. In another experiment, reduction of the reaction products was performed with liver alcohol dehydrogenase, which again mainly yielded trachelanthamidine and little isoretronecanol [88]. However, the stereochemical properties of the obtained products were not investigated. Subsequent studies confirmed these initial findings and suggested involvement of a copper-dependent diamine oxidase since treatment of *Senecio vulgaris* and *Heliotropium indicum* with 2-hydroxyethylhydrazine (HEH), a potent diamine oxidase inhibitor, caused homospermidine accumulation and impeded PA biosynthesis [71,89].

Oxidation of amine precursors followed by cyclisation is a common theme in alkaloid biosynthesis. In tropane biosynthesis *N*-methylputrescine is oxidized by methylputrescine oxidase to 4-methylaminobutanal, which cyclizes spontaneously to the *N*-methyl-Δ1-pyrrolinium cation (Figure 9) [9]. This is similar to the first steps proposed for conversion of the dialdehyde 4,4 -iminodibutanal to the pyrrolium cation (Figure 10). However, biosynthesis of necine bases continues with reaction of the remaining aldehyde group for closure of the second ring via a Mannich-type reaction mechanism, which leads ultimately to pyrrolizidine-1-carbaldehyde. While cyclization in tropane alkaloid biosynthesis might be spontaneous, this is rather unlikely for necine base synthesis. Spontaneous cyclization of the dialdehyde and subsequent reduction would result in a mixture of the four saturated 1-hydroxymethylpyrrolizidines (±)-trachelanthamidine and (±)-isoretronecanol. After desaturation, a mixture of (±)-supinidine would be obtained and subsequent C-7 hydroxylation would, depending on the specificity of the hydroxylase, lead to a mixture of (+)-retronecine and (-)-heliotridine or (-)-retronecine and (+)-heliotridine. However, such mixtures are usually not observed in PA-producing plants. In contrast, plants usually contain PAs with necine bases of only a specific stereochemical configuration. For instance, *Senecio jacobaea* and *Senecio aquaticus* contain, apart from otonecine-type PAs, only senecionine-like, jacobine-like and erucifoline-like PAs, which are all (+)-retronecine-type PAs [90]. *Crassoceopahlum crepidioides* contains only jacobine, a PA of the (+)-retronecine type [50]. *Lolium perenne* contains only the (-)-isoretronecanol type PAs *Z*- and *E*-thesinine and its rhamnosides [91]. *Borago officinalis* contains only alkaloids of the (-)-isoretronecanol, (-)-supinine and (+)-retronecine-type [92], which have the same stereoconfiguration of the C-8 hydrogen. *Heliotropium europaeum* contains only (+)-heliotridine-type PAs [93]. The observed stereochemical specificity argues clearly against spontaneous cyclisation and suggests an enzymatic mechanism. Since enzyme-catalyzed cyclization must immediately follow oxidative deamination it is tempting to speculate that the copper-dependent diamine oxidase might also either support stereospecific cyclization of the dialdehyde or act in a protein complex with a second enzyme that catalyzes cyclization.

**Figure 9.** Cyclization in tropane alkaloid biosynthesis. The precursor *N*-methylputrescine is oxidized by methylputrescine oxidase to 4-methylaminobutanal, which cyclizes spontaneously by reaction of the amino group with the aldehyde. Adapted from Reference [9].

**Figure 10.** Cyclization in PA biosynthesis. Homospermidine is likely oxidized by a copper-dependent diamine oxidase to 4,4 -iminodibutanal, which can be inhibited by the synthetic compound 2-hydroxyethylhydrazine. The reaction product cyclizes first to the 1-(4-oxobutyl)-3,4-dihydro-2*H*-pyrrolium cation and further to pyrrolizidine-1-carbaldedye in a Mannich-type reaction. However, as indicated, spontaneous cyclization would lead to a mixture of the different stereomers and thus mixtures of necine bases would be obtained, arguing for enzyme-catalyzed cyclization.

#### 3.1.3. Further Downstream Reactions

After cyclization the formed pyrrolizidine-1-carbaldehyde is reduced to the alcohol. This might be catalyzed by an alcohol dehydrogenase (ADH), since Robins showed that ADH can in principle reduce the carbaldehyde to the alcohol [88]. Additional evidence comes from a detailed stereochemical study of necine base formation. Feeding of *Senecio isatideus* with deuterium labeled [1,1,4,4-2H4]-putrescine and hydrolysis of the obtained PA yielded retronecine that retained three deuterium atoms on the right handed ring: Two at C-3 and one at C-9. Importantly, the latter was in the *S* position, which is the stereochemistry expected for an ADH-catalyzed reduction of an carbaldehyde [59].

The sequence of retronecine base interconversions was studied by Birecka and Catalfamo, using pulse-chase experiments [94]. *Heliotropium spathulatum* was used for this study since this species produces (-)-trachelanthamidine, (-)-supinidine and (-)-retronecine containing PAs. The plants were treated with [14C]-carbon dioxide for 2 h prior quenching incorporation with unlabeled carbon dioxide. Samples were taken after 12 h, 24 h and 48 h and the specific activity of the necine bases analyzed. The activity of (-)-trachelanthamidine increased first, which was followed by an increase of the activity of (-)-supinidine and finally (-)-retronecine. This suggests that (-)-trachelanthamidine is first dehydrogenated at the C-1/C-2 bond to (-)-supinidine, which is subsequently hydroxylated at C-7 to (-)-retronecine. However, the involved enzymes remain elusive in addition to whether the free necine bases are modified or the, at least partially, esterified PAs.

#### *3.2. Biosynthesis of Necic Acids*

While necine bases are synthesized by a common pathway, different necic acids are formed by distinct modes. A number of acids found in PAs are normally present in plants. This includes, for instance, acetic acid, benzoic acid, and *p*-coumaric acid. These acids and their activated forms, the coenzyme A thioesters, are formed by common metabolic pathways of primary metabolism, which will not be discussed here.
