Keywords

1 Introduction

Nowadays, an increasing number of new pharmaceuticals have a natural lead structure or are directly derived from nature [1–4]. Their often remarkable physiological activities are frequently linked to structures bearing a number of stereogenic centers. Consequently, asymmetric synthesis is becoming even more important both in industry and in academic research [5, 6].

For providing chiral intermediates, different possibilities of asymmetric catalysis have been established. Various examples of transition metal-catalyzed or organocatalytic reactions can be found in the literature [7–9]. Biocatalysis – the application of isolated enzymes or whole cells in biotransformations – has a long historical background, being already known in ancient Egypt and the Far East for the preparation of food and alcoholic beverages [8, 10]. Plants, fruit, and extracts thereof were used for these purposes in the past, but they still find applications in recent research [11–13]. For example, coconut water was investigated as a new biocatalytical system for organic synthesis with some very promising results (Fig. 1) [14].

Fig. 1
figure 1

Enzymatic reactions catalyzed by coconut juice (Cocos nucifera)

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Major advances in the knowledge of biochemical pathways and the establishment of computer-based predictions of three-dimensional structures of proteins led to the development of new microbiological methods, such as rational protein design or directed evolution, giving scientists the possibility to provide tailor-made biocatalysts [15–19]. These methodological works on the disclosure of new efficient biocatalysts are not explicitly mentioned in this review unless they were applied in natural product synthesis. Biotransformations do not always compete with known chemical syntheses, but rather complement the portfolio of catalytic methods in organic chemistry.

Enzymatic processes are basically considered to be environmentally benign due to the mild reaction conditions needed and the high enantio-, regio-, and chemoselectivity of the enzymes. The ecological effect of using water as a solvent is quite often counterbalanced by low solubility of the substrates, making organic solvent resistant enzymes necessary [20], or by problems in downstream-processing, in most cases extracting the product with huge amounts of organic solvents [21]. Nevertheless, a biotransformation can sometimes substitute heavy metal catalysts or raise immensely the efficiency of a process and thus deliver both ecological and economical advantages.

In general there are two principle possibilities using a biocatalyst in organic synthesis, namely as whole cells or as isolated enzymes – free or immobilized. The advantages and disadvantages of each can be intensively discussed, but the outcome of this consideration always depends on the whole system and the kind of application. There are numerous examples of both and thus there is no partitioning between whole cell biotransformation and isolated enzymes in this review.

Biocatalytic approaches to key building blocks in the synthesis of natural products and pharmaceuticals have been reviewed and summarized in various books in the last few years [22–48]. The variety of enzymes that were used is rising day by day, so this chapter is only meant to be instructive, not complete. The compilation is also restricted to the most frequently used enzyme classes, trying to give brief insights by selected examples of some older, but mainly recent, applications.

2 Lipases and Esterases in Organic Synthesis

Hydrolases are widely used enzymes in organic synthesis, with most applications concentrating on lipases and esterases. This chapter discloses the possibilities of asymmetric accesses to chiral building blocks for the synthesis of natural products using lipases and esterases. Other hydrolases, such as amidases or peptidases, are not dealt with explicitly.

Asymmetric synthesis with lipases and esterases can basically be performed by two different approaches – the desymmetrization of prochiral or meso compounds and the enzymatic kinetic resolution of racemic mixtures. The main bottleneck of kinetic resolutions, product yields of maximum 50%, can be overcome if an in situ racemization of the starting material is possible. In this case all starting material can theoretically be converted to the desired product [34].

2.1 Lipase-Catalyzed Desymmetrizations

Desymmetrization of prochiral or meso compounds is a powerful tool in organic synthesis, yielding key building blocks with high enantiopurity [49]. Therefore, chiral reagents or catalysts can be used to achieve the differentiation between enantiotopic groups. Some of the enantioselective enzymatic desymmetrizations using lipases that have been described in the literature represent interesting opportunities in natural product synthesis.

Enantiomerically pure cyclopropanes are a frequent motif in the structure of natural products. Their synthesis is often demanding and many approaches have been made [50, 51]. Porcine pancreatic lipase (PPL) was used for the stereoselective desymmetrization of a cyclopropane dibutanoate (Fig. 2). The asymmetric hydrolysis of the meso compound yielded the corresponding enantiopure alcohol almost quantitatively. The intermediate obtained was successfully applied in the total synthesis of dictyopterenes A and C, sexual pheromones of brown algae [52], and constanolactones (see below) [53].

Fig. 2
figure 2

Chemoenzymatic synthesis of dictyopterenes A and C

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Due to its structural complexity and interesting physiological activities as an antifungal agent and especially as an immunosuppressive drug, the macrolide rapamycin has been a target of many total syntheses [54–58]. In their approach, Ley and co-workers used a lipase-catalyzed desymmetrization of a meso diol (Fig. 3). In early studies, the selective acetylation of meso-2,4-dimethylpentane-1,5-diol was achieved by a PPL immobilized on celite with moderate yields and 92% enantiomeric excess (ee) [59]. Later, advances in the enzymatic desymmetrization led to the substitution of the catalyst with the also commercially available and inexpensive Lipase PS-30, providing the monoacetate in 83% yield and 99% ee [60, 61].

Fig. 3
figure 3

Enantioselective acylation catalyzed by porcine pancreatic lipase (PPL)

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2.2 Kinetic Resolutions with Lipases and Esterases

Kinetic resolutions in general are regularly applied in organic synthesis. Since enzymes are highly attractive for asymmetric synthesis, various types of biocatalysts have been used in enzymatic (dynamic) kinetic resolutions, but the focus will remain on lipase- and esterase-mediated resolutions as the most common tools in early steps of natural product syntheses.

The enantiomeric excess of substrate and product shifts during the course of a kinetic resolution, making the determination of the efficiency of the reaction dependent on the reaction time. Hence, two kinetic resolutions can only be compared at the same extent of conversion [45, 62]. For a better comparison of two reactions, equations for the calculation of the enantioselectivity were established [63, 64]. In most cases, the enantiomeric excess of both substrate and product is determined for greater accuracy, but the determination of one and the conversion can also be used for the calculation of the E value (enantioselectivity):

$$ E = \frac{{ln[1 - c(1 + e{e_p})]}}{{ln[1 - c(1 - e{e_p})]}};\quad E = \frac{{ln[(1 - c)(1 - e{e_s})]}}{{ln[(1 - c)(1 + e{e_s})]}};\quad E = \frac{{ln\left[ \displaystyle {\frac{{1 - e{e_s}}}{{1 + ({{e{e_s}} \mathord{\left/{\vphantom {{e{e_s}} {e{e_p})}}} \right.} {e{e_p})}}}}} \right]}}{{ln\left[ \displaystyle{\frac{{1 + e{e_s}}}{{1 + ({{e{e_s}} \mathord{\left/{\vphantom {{e{e_s}} {e{e_p})}}} \right.} {e{e_p})}}}}} \right]}} $$
(1)

E values above 100 are very inaccurate due to the logarithmic function, so that usually such high enantioselectivities are stated as E ≥ 100. In these calculations the E value increases with increasing enantioselectivity. For the enzymatic kinetic resolutions, only E values greater than 20 indicate a potentially useful application in organic synthesis.

However, this theoretical approach does not cover all side effects of applied biocatalysis. The calculation of E values for enzyme mixtures or in systems with inherent inhibitory effects must be handled with care. Nevertheless, calculating E from (1) usually provides a very useful tool for estimating the efficiency of a resolution system.

An example for the application of enzymatic kinetic resolutions with high E values in natural product synthesis is the chemoenzymatic synthesis of the northern half of epothilones (also see Sect. 4.1). Various lipases and esterases could be found with outstanding enantioselectivity (up to >100); among these were lipase B from Candida antarctica, a lipase from Burkholderia cepacia, a lipase from Pseudomonas sp., and a lipase from Streptomyces diastochromogenes, all affording the desired (S)-configurated alcohol with >99% enantiomeric excess (Fig. 4) [65].

Fig. 4
figure 4

Enzymatic kinetic resolution of epothilone precursors

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Allenes are versatile intermediates in organic synthesis and a variety of useful applications has been established [66]. A very interesting feature of allenes is their axial chirality along the cumulated diene system, so that optically active allenes were frequently used in asymmetric synthesis, in most cases being prepared from enantiomerically enriched precursors.

Since allenic systems are rather rare in nature, enzymatic approaches were hardly investigated due to a lack of known organisms metabolizing these structures. Recently, a mutant of a lipase from Pseudomonas aeruginosa (PAL) was generated by directed evolution, which was applied in the kinetic resolution of an allenic p-nitrophenolester [67]. Further investigations on the possibility of lipase-catalyzed kinetic resolutions of allenes with different lipases led to the application of PPL in the total synthesis of (−)-striatisporolide A, a fungal metabolite isolated from an Australian Penicillium striatisporum strain (Fig. 5) [68]. Before, neither a synthetic approach, nor the absolute configuration of this butenolide had been reported. Combining the enzymatic kinetic resolution with a palladium(II)-mediated racemization [69] could lead to a highly efficient dynamic kinetic resolution (DKR) of allenes.

Fig. 5
figure 5

Enzymatic kinetic resolution of primary allenic alcohols

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The complex tetramic acid lactam cylindramide was isolated from the marine sponge Halichondria cylindrata and is known to possess a significant cytotoxic activity against human B16 melanoma cells [70]. A chemoenzymatic access to the characteristical substituted bicyclo[3.3.0]octane moiety was recently established, using lipase PS (Amano) for an enzymatic kinetic resolution (Fig. 6) [71]. The racemic diol was generated by a transannular Pd-catalyzed ring-closure of cycloocta-1,5-diene and removal of the acetyl groups. The resolution step provided the (1S,3aR,4S,6aR)-configured diol in 44% yield with 98% ee. Side products, the monoacetate (8%) and the (1R,3aS,4R,6aS)-configured diacetate (38%), could easily be separated by chromatography.

Fig. 6
figure 6

Enzymatic kinetic resolution in the total synthesis of cyclindramide

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Psymberin (irciniastatin A), a cytotoxin of the pederin family, has been isolated from the marine sponges Psammocinia sp. and Ircinia ramosa. Its highly cytostatic activity against various human cancer cell lines made it an attractive target for total syntheses [72–74]. Lately, a chemoenzymatic approach by an esterase-catalyzed kinetic resolution towards psymberic acid, one of the main structural elements of psymberin, was disclosed [75]. A number of lipases and esterases was screened affording esterase BS3 from Bacillus subtilis and commercially available esterase 001 (Codexis) as suitable biocatalysts for enantioselective hydrolysis. Especially esterase 001 showed superior performance, yielding the protected psymberic acid with 98% ee (Fig. 7).

Fig. 7
figure 7

Chemoenzymatic synthesis of PMB-protected psymberic acid

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The importance of enantiomerically pure cyclopropane derivatives has already been pointed out (see Sect. 2.1) and many examples of lipase- or esterase-catalyzed kinetic resolutions of cyclopropanes can be found in the literature, but, unfortunately, high E values are scarce [76–84]. For instance, several lipases were screened for their ability to perform a kinetic resolution of cyclopropylmethanols [85].

Among the enzymes tested, Candida antarctica lipase B (CAL-B) (commercially available as Novozyme 435 or Chirazyme L-2) was the most efficient. For the kinetic resolution of the corresponding acetate in tetrahydrofuran at 60°C, a comparably high E value was achieved (E = 44). Hence, this enzyme was applied in the total synthesis of constanolactones A and B, marine oxylipins isolated from the red algae Constantinea simplex [86]. An enantiomerically enriched cyclopropylmethanol was submitted to enzymatic kinetic resolution after acetylation, giving the enantiopure (>98% ee) in good yield (76%) over the two steps (Fig. 8). Application of enzymatic kinetic resolution to other cyclopropylmethanol derivatives delivered the series with opposite cyclopropane configuration, yielding building blocks for the total synthesis of solandelactones [87], halicholactone, and neohalicholactone [88].

Fig. 8
figure 8

Enzymatic kinetic resolution of cyclopropylmethanols

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The kinetic resolution of 5-hydroxymethyl-2-cyclohexenone towards the total synthesis of penienone, a fungal metabolite of Penicillium sp. no. 13 [89], has been performed by using Lipase PS from B. cepacia for the acetylation of the (S)-enantiomer, leaving the desired (R)-alcohol. As a main problem inherent with enzymatic kinetic resolutions, the reaction yielded both products in a 1:1 mixture almost quantitatively. For prohibiting the loss of half of the starting material, the acetate was racemized by hydrolysis with another lipase (PPL), followed by an oxidation–reduction sequence yielding the racemic starting material for the kinetic resolution (Fig. 9).

Fig. 9
figure 9

Enzymatic kinetic resolution in the chemoenzymatic synthesis of (−)-penienone

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2.3 Dynamic Kinetic Resolutions with Lipases and Esterases

The enzymatic kinetic resolutions presented in Sect. 2.2 all clearly revealed the bottleneck of kinetic resolutions, a maximum yield of 50% for the desired enantiomer. In the total synthesis of penienone this problem was solved by choosing a rather inconvenient multistep racemization of the isolated remaining starting material.

In modern biotransformations, the insufficient yield is optimized by establishing an in situ racemization of the starting material, turning the system into a DKR [90–92]. Due to the high enantioselectivity of some enzymes, the number of applications of DKR is increasing; especially the utilization of the readily available lipases and esterases is most common. The racemization process can either be realized by biocatalysts or by chemocatalytic approaches. In some cases, the addition of base or acidification can shift an existing equilibrium towards racemization sufficiently. For catalyzed racemization, especially some ruthenium-complexes were proven to show high compatibility with enzymatic systems [93]. Other transition metals were also applied; for the DKR of allyl alcohols, an oxovanadium (V) catalyst was used in combination with a lipase to give high yields with excellent enantiomeric excesses [94].

The principle of kinetic resolutions (Fig. 10) can be explained by the different rates of conversion for the different enantiomers. In the special case of a DKR, the rate for the conversion of one enantiomer is many times higher (k R ≫ k S). Without the possibility of racemization of the starting material (k rac = 0), a kinetic resolution with a maximum yield of 50% is observed. As soon as k rac exceeds the rate for the conversion of the unfavored enantiomer (k rac ≥ k S), a DKR becomes efficient.

Fig. 10
figure 10

Principle of dynamic kinetic resolutions

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Bornscheuer and co-workers studied the lipase-catalyzed dynamic resolution of acyloins, using miscellaneous amine bases and ion-exchangers for racemization (Fig. 11) [95]. Unfortunately, acidic resins, e.g., Amberlyst 15, furnishing the best results for racemization, also deactivated the enzyme. As a solution, a two-compartment setup was established, separating ion exchanger and enzyme, the two vessels being connected by a pump loop. The enzymatic resolution of the acyloin and the racemization were thus carried out simultaneously furnishing a nice DKR with good yields and enantioselectivities. The enantiomerically enriched acyloins produced are important building blocks for natural products, e.g., epothilones [96].

Fig. 11
figure 11

Two-compartment enzymatic dynamic kinetic resolution of acyloins

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Enzymatic DKRs have also been applied in domino one-pot processes [97]. The combination of a lipase-catalyzed resolution with an intramolecular Diels–Alder reaction led to interesting building blocks for the synthesis of natural products such as compactin [98, 99] or forskolin [100–102]. A ruthenium catalyst is employed for the racemization of the slow reacting enantiomer of the starting material. The DKR with lipase B from C. antarctica delivered high enantiomeric excesses which could mainly be contained through the Diels–Alder reaction (Fig. 12).

Fig. 12
figure 12

Chemoenzymatic domino DKR and Diels–Alder reaction

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3 Enzymatic Reductions and Oxidations

In current research, oxidoreductases are second in the number of applications of enzymes in organic synthesis. The number of commercially available biocatalysts of this class has increased tremendously during the last few years and various screening kits for oxidation and reduction are sold. Many oxidoreductases are rather easy to handle, though, in contrast to hydrolases, they are dependent on cofactors [22].

3.1 Chiral Alcohols Through Enantioselective Reduction

The chemoenzymatic synthesis of chiral alcohols is a field of major interest within biocatalytic asymmetric conversions. A convenient access to secondary highly enantiomerically enriched alcohols is the usage of alcohol dehydrogenases (ADHs) (ketoreductases) for the stereoselective reduction of prochiral ketones. Here, as in many other cases in asymmetric catalysis, enzymes are not always only an alternative to chemical possibilities, but are rather complementary. Albeit biocatalysts might sometimes seem to be more environmentally friendly, asymmetric ketone reduction with Noyori’s ruthenium catalysts [8, 103] or by chiral oxazaborolidins (Corey-Bakshi-Shibata) [104] are known to be very efficient for many applications and do not leave much room for improvement for these. Yet there are also examples in which chemical asymmetric reduction fails or shows low selectivity [105].

Ketoreductases (KREDs) are dependent on nicotinamide cofactors NADH or NADPH. Due to the reaction mechanism, these rather costly cofactors are needed in stoichiometric amounts, disclosing an economic problem that has to be dealt with when using these enzymes. Many different possibilities for cofactor recycling have been established with three major approaches finding application in research and industry (Fig. 13). Further regeneration systems, such as electrochemical methods, are not discussed within this review [22–24, 37, 106–108].

Fig. 13
figure 13

Recycling of nicotinamide cofactors

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One possibility is the addition of a second substrate that itself is also a possible substrate for the KRED being used. Most isolated enzymes are quite robust to 2-propanol as a substrate, so that in some systems 2-propanol concentrations of more than 40% can be used, also bearing an improved solubility of the ketones for conversion. However, on a larger scale the acetone formed can show inhibitory effects. Removal of this by-product shifts the equilibrium of the reaction towards the desired products and prohibits a competitive inhibition [109, 110].

A further opportunity for cofactor regeneration is the addition of a second enzyme with a corresponding substrate to the reaction mixture. Often glucose dehydrogenase (GDH) is used for this purpose, converting glucose to gluconate by reducing the nicotinamide cofactor. Formate dehydrogenases (FDHs) also have a broad spectrum of applications, bringing the advantage of catalyzing the oxidation of formate to carbon dioxide, which by leaving the reaction mixture draws the equilibrium to the desired side.

There is quite a lively discussion going on about the advantages and disadvantages of the usage of whole cells for biocatalysis vs isolated enzymes [23, 24, 43]. Intact cells contain all enzymes and the cofactors needed for the biotransformation, so that the addition of a carbon source should be sufficient. But whole cell biotransformations also cause some problems. One aspect is the transport of the substrate through the cell membrane, which might be a great hindrance. Partial lysis of the cells by adding organic solvents to the reaction medium sometimes allows transport of the substrate into the cell, but also provides a possibility of loss of the cofactor from the inner cell. Conclusively it is hardly possible to tell whether whole cell biotransformation or isolated enzymes are the method of choice, leading to both methods being almost equally applied [23, 24].

Chiral secondary alcohols are main targets of biotransformations since they are very common intermediates in various natural product syntheses. Often, kinetic or DKRs using lipases are used to obtain these key building blocks. The asymmetric reduction of prochiral ketones is more than an alternative to this approach. A huge number of applications, in which KREDs are used for reduction, can be found in the literature [22–24, 37]. For example, (R)-2-bromo-(4-nitrophenyl)ethanol, a precursor to the β-blocker (R)-nifenalol, which was obtained by kinetic resolution in the past, was conveniently synthesized in a direct approach from the corresponding ketone using whole cells of Rhodotorula sp. AS2.2241 (Fig. 14). This ketone and a number of derivatives thereof were converted with good yields and excellent enantioselectivities [111].

Fig. 14
figure 14

Chemoenzymatic asymmetric synthesis of (R)-nifenalol with whole cells

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Whole cells were also used as biocatalysts in the biocatalytical synthesis of (R)-o-chloromandelates, highly valuable building blocks for biologically active substances, such as clopidogrel (Fig. 15) [112]. In this straightforward approach a versatile carbonyl reductase (SCR) was expressed in Escherichia coli and the cells fed with glucose for enabling the cofactor regeneration during the biotransformation. Methyl (R)-o-chloromandelate was isolated in enantiomerically pure form (>99% ee) in very high yield (89%).

Fig. 15
figure 15

Whole cell biocatalytic synthesis of methyl (R)-o-chloromandelate

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5-Hydroxyhept-6-enoates have been key intermediates in the synthesis of a variety of natural products, especially of the arachidonic acid metabolic pathway, including prostaglandins, leukotrienes, and isoprostanes [105, 113–118]. The usage of two enantiocomplementary enzymes allowed convenient access to both enantiomers via an ADH-catalyzed reduction of 5-oxo-hept-6-enoate. Alcohol dehydrogenase from Lactobacillus brevis (ADH-LB) furnished the (S)-enantiomer, Thermoanaerobacter sp. ADH (ADH-T) the (R)-enantiomer in excellent enantiomeric access respectively [119]. A cross-metathesis reaction followed by cyclopropanation led to the formal synthesis of constanolactones C and D (Fig. 16) [86, 120, 121].

Fig. 16
figure 16

(a) Enzymatic synthesis of building blocks for arachidonic acid metabolites. (b) Chemoenzymatic synthesis of constanolactones C/D

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Not only has the remarkable enantioselectivity of ADHs made them a very powerful tool in organic synthesis: Besides the excellent enantioselectivity of ADH from L. brevis already shown, the same enzyme’s regioselectivity has been exploited in the chemoenzymatic synthesis of statin side chains [122]. A series of 3,5-dioxocarboxylates (β,δ-diketo esters) were successfully reduced selectively in the 5-position using ADH-LB as biocatalyst. The enantiomeric purity of the 5-hydroxy compound obtained was excellent [123–125]. The second ketone was then either reduced by Prasad’s borohydride method [126] delivering the syn-dihydroxy building block, or according to Evans furnishing the anti-configured compound, respectively (Fig. 17) [127]. Furthermore, chiral building blocks for the synthesis of some δ-lactone containing natural products, such as goniothalamin, argentilactone, or callistatin A, could be accessed by this chemoenzymatic approach [125].

Fig. 17
figure 17

Ketoreductase-catalyzed chemoenzymatic synthesis of statin side chains

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Similar building blocks have also been obtained by a variety of other enzymatic transformations. 4-Chloro-3-oxobutanoate esters have been reduced with outstanding enantioselectivity by ADHs from Candida magnoliae in combination with cofactor regeneration by a GDH (from Bacillus megaterium) or by enzymatic reduction using whole cells of Geotrichum candidum [128, 129]. This process was improved to a large scale application. The combination with a halohydrin dehydrogenase step led to versatile compounds for organic synthesis. Statistical analysis of protein sequence activity relationships (ProSAR) and recombination-based directed evolution led to an optimized enzyme catalyzing the epoxide formation in a first step and the addition of a nucleophile in a second step, accepting alternative nucleophiles, such as CN− or N −3 (Fig. 18) [130–132].

Fig. 18
figure 18

Enzymatic synthesis of ethyl (R)-4-cyano-3-hydroxybutyrate

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3.2 Reduction of C=C Bonds

The generation of stereogenic centers by asymmetric reduction of carbon–carbon double-bonds is a current topic in chemoenzymatic synthesis. Though enzymes of the old yellow enzyme (OYE) family were identified to perform alkene reduction and were characterized some years ago [133–135], applications of enoate reductases in natural product syntheses are still rare. Thus, potential applications are also shown in this chapter. With an increasing number of new enoate reductases, such as YqjM reductase from B. subtilis, more and more possible targets for biotransformations can be found.

Enzymes from the OYE family were reported to mediate efficiently the reduction of ketoisophorone. A subsequent regio- and stereoselective reduction of the thus produced levodione leads to actinol, both compounds being important building blocks for carotenoids. While early approaches used baker’s yeast in the fermentative production of levodione [136], more recent approaches used enzymes from different organisms. Besides OYE analogs from Candida macedoniensis, Saccharomyces carlsbergensis, and Saccharomyces cerevisiae, a newly discovered enoate reductase from Zymomonas mobilis was applied successfully [137, 138]. Wada et al. combined the OYE-mediated step with an enzymatic formation of actinol catalyzed by a levodione reductase from Corynebacterium aquaticum (Fig. 19) [139].

Fig. 19
figure 19

Enzymatic synthesis of carotenoid building blocks

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Some reductases isolated from tobacco (Nicotiana tabacum) were found to exhibit excellent enantioselectivities on the reduction of a number of α,β-unsaturated compounds [140, 141]. For example, reductase p44 catalyzed the asymmetric reduction of N-phenyl-2-methylmaleimide, yielding the enantiopure (R)-succinimide. Reductase p90 mediated the enantioselective hydrogenation of a number of methyl or ethyl substituted cyclopentenones and cyclohexanones (Fig. 20).

Fig. 20
figure 20

Enantioselective reductions catalyzed by reductases from N. tabacum

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3.3 Reductive Amination Towards Amino Acids

Unnatural amino acids in general, but especially α-amino acids being important intermediates for various biologically active compounds, have enjoyed great popularity as a growing field of interest during recent years [142]. Many enzymatic approaches to these important building blocks, including resolutions with l-amino acid acylase or d-amino acid oxidase, the application of transaminases, and reductive amination by amino acid dehydrogenases, were studied [143]. For the production of enantiopure α-amino acids, reductive amination was most successfully applied.

The significance of chiral unnatural amino acids to drug and natural product synthesis is shown in the example of the antihypertensive drug omapatrilat (Vanlev®), which is composed of no less than three amino acid derived intermediates [144–147]. Diverse biocatalytical approaches to L-6-oxonorleucine were made (Fig. 21). Two different enzymes were applied in reductive amination reactions to produce derivatives of the desired intermediate.

Fig. 21
figure 21

Enzymatic synthesis of unnatural amino acids as building blocks for omapatrilat. (a–c) Enzymatic approaches to 6-oxonorleucine analogs

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With a protected aldehyde function, allysine ethylene acetal was synthesized via reductive amination catalyzed by phenylalanine dehydrogenase (PheDH). For regeneration of the nicotinamide cofactor, FDH was used as described before (see Sect. 3.1). Another similar approach was taken in the synthesis of l-6-hydroxynorleucine. In this case, reductive amination was catalyzed by glutamate dehydrogenase (GluDH) delivering the desired (S)-configured amino acid. The synthesis of the same compound has also been achieved by an interesting resolution process. Racemic 6-hydroxynorleucine was submitted to kinetic resolution using a d-amino acid oxidase, generating an enantiomeric excess of >99% for the remaining enantiomer. The mixture was then converted completely to enantiopure l-hydroxynorleucine by reductive amination of 2-oxo-6-hydroxyhexanoic acid with GluDH. For the oxidation step, whole cells of Trigonopsis variabilis, bearing the oxidase and the catalase for the degradation of hydrogen peroxide to oxygen, were successfully used.

3.4 Cytochrome P450 Monooxygenases

Cytochrome P450 (CYP450) monooxygenases are very common oxidoreductases, frequently used in synthetic applications in research and industry. The versatility and potential as powerful industrial biocatalysts has been strengthened by important advances that have been made regarding mechanistic insights and optimization of the activity and high stereo- and regioselectivities of these hemoproteins recently [148–151].

Pravastatin, a 3-hydroxy-3-methyl glutaryl CoA reductase inhibitor applied as a therapeutic agent for hypercholesterolemia, can be synthesized by stereo- and regioselective hydroxylation of compactin by the soil microorganism Streptomyces sp. Y-110 (Fig. 22) [152]. The fermentative production of pravastatin has already been applied on an industrial scale by Sankyo Co. using different Streptomyces bacteria strains [153, 154].

Fig. 22
figure 22

Biocatalytic conversion of compactin to pravastatin

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Besides the stereo- and regioselective hydroxylations, CYP450 are also capable of performing regioselective oxidations to aldehydes or carboxylic acids. The sesquiterpene lactone artemisinin is highly effective against multidrug resistant Plasmodium falciparum, agent of malaria tropica. First, genes from the mevalonate pathway in S. cerevisiae were upregulated by means of metabolic engineering, increasing the production of farnesyl pyrophosphate, and the natural further metabolism to sterols was downregulated. Introduction of an amorphadiene synthase (ADS) from Artemisia annua led to the formation of amorpha-4,11-diene which could then be converted to artemisic acid, a precursor of artemisinin, via a three-step oxidation sequence catalyzed by a novel P450 from A. annua in S. cerevisiae (Fig. 23) [155].

Fig. 23
figure 23

Fermentative production of artemisinic acid

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3.5 Baeyer–Villiger Oxidations

Among the most popular oxidative biotransformations, Baeyer–Villiger monooxygenases (BVMOs) belong to the main fields of research. Nowadays, manifold enzymes catalyzing the Baeyer–Villiger oxidation are expressed in common recombinant organisms, such as E. coli or S. cerevisiae. The mechanism of the enzymatic Baeyer–Villiger oxidation involves NADPH and flavin (FAD) as cofactors and was originally proposed by Walsh et al. based on data obtained from cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (Fig. 24) [156]. In a first step, enzyme-bound flavin is reduced, followed by the addition of oxygen yielding a hydroperoxide anion. Reaction with the ketone substrate gives a Criegee intermediate, which is then converted into the product under dissociation of water. The cofactor FAD is recovered via oxidation with NADP+.

Fig. 24
figure 24

Mechanism of flavin-dependent Baeyer–Villiger monooxygenases

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Enzymatic Baeyer–Villiger oxidations have been studied for a long time; some very useful applications in natural product synthesis date back more than two decades. For example, prochiral (3R,5S)-4-hydroxy-3,5-dimethylcyclohexanone was successfully oxidized using a CHMO from Acinetobacter sp. NCIMB 9871. In this case the seven-membered ring formed rearranges spontaneously into the thermodynamically more stable γ-lactone. The rearranged lactone has been used in the synthesis of natural products such as tirandamycin or calyculin A (Fig. 25) [157–159].

Fig. 25
figure 25

BVMO-catalyzed synthesis of a precursor to calyculin A and tirandamycin

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As most enzymes, BVMOs feature excellent enantio- and regioselectivities, making them very versatile tools for asymmetric synthesis. Bicyclic ketones such as functionalized norboranones have been resolved to the corresponding lactones with good regioselectivities and enantiomeric excesses of more than 95% [160]. Whole cells of Pseudomonas putida or isolated 2,5-/3,6-diketocamphane 1,2-monooxygenase (2,5-/3,6-DKCMO) were used for the biotransformation. Conversion into an acetal yielded a potential advanced intermediate to azadirachtin, a potent antifeedant and growth regulator (Fig. 26) [161–163].

Fig. 26
figure 26

Chemoenzymatic approach to azadirachtin

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The biocatalytical formation of γ-butyrolactones through Baeyer–Villiger oxidation is catalyzed by a number of different monooxygenases, yielding precursors to various natural products [29, 38]. Best enantioselectivities were obtained for the synthesis of precursors for lignans, which are known for their antileukemic, antiviral, antifungal, and antineoplastic activities [164–166]. For instance, building blocks for (+)-hinokinin and (+)-schizandrin have been generated with excellent enantioselectivities using CHMOs from Xanthobacter sp. ZL5 and Brachymonas petroleovorans respectively (Fig. 27).

Fig. 27
figure 27

Cyclohexanone monooxygenase-mediated synthesis of lignan building blocks

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More recently, the formal total syntheses of some bioactive natural products bearing a tetrahydrofurane moiety were achieved by enzymatic Baeyer–Villiger oxidation using cyclopentanone monooxygenases (CPMOs) from Comamonas sp. NCIMB 9872, overexpressed in E. coli, as a whole cell biotransformation [167]. The substrate, an oxabicycloketone, was successfully converted to the corresponding heterobicyclic lactone (Fig. 28) in 70% yield with an enantiomeric excess of 95%. This access to the chiral intermediate for several naturally occurring compounds, including (+)-trans-kumausyne and (+)-showdomycin, represents the first application of a whole cell biotransformation using recombinant E. coli with CPMO in natural product synthesis.

Fig. 28
figure 28

Enzymatic Baeyer–Villiger oxidation in the synthesis of bioactive compounds

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3.6 Cyclohexadienediols by Fermentation

There are only a few reactions known in which aromatic compounds suffer a permanent loss of aromaticity and at the same time the new stereogenic centers are formed selectively. The enzymatic oxidation is one of the few examples leading to exceptionally results: the reaction in eukaryotic systems is mainly catalyzed by cyctochrome-type monooxygenases, leading to epoxides. Furthermore, in prokaryotic systems, the processing of arenes by dioxygenases is well known [168], and especially cis-cyclohexadienediols (cis-CHDs) are generated by fermentation with recombinant E. coli strains. They have found broad application in several natural product and drug syntheses [28, 169–174].

For example, the fermentation of (2-bromoethyl)-benzene with recombinant E. coli furnished excellent yields of the corresponding cis-diol, enantiopure 3-(2-bromoethyl)-benzene-1,2-diol. The latter was used as a building block in the total synthesis of (+)-codeine (Fig. 29). Besides a Mitsunobu inversion of one of the stereogenic centers, two successive Heck cyclizations led to the enantiomer of the natural product [173]. Slight modifications of the reaction sequence, generating an epoxide intermediate, also furnished access to the naturally occurring enantiomer (−)-codeine [28].

Fig. 29
figure 29

Chemoenzymatic synthesis of (+)-codeine

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Versatile epoxyquinol intermediates were conveniently accessed via metabolites derived from the enzymatic dihydroxylation of bromobenzene [172]. The chemoenzymatic approach led to shortened total syntheses of several epoxyquinols derived from bromoxone, such as (+)-hexacyclinol, a possible lead target for new antimalarial agents of fungal origin (Fig. 30).

Fig. 30
figure 30

Chemoenzymatic access to epoxyquinols

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Various approaches to the antiviral agent oseltamivir (Tamiflu®), which is one of the most effective drugs against avian influenza (H5N1) and the new influenza A (H1N1), have recently been published starting from biotechnologically provided cis-CHDs [175–177]. Hudlicky et al. started from ethyl benzoate, performing the oxidative biotransformation with a recombinant E. coli strain (Fig. 31).

Fig. 31
figure 31

Chemoenzymatic synthesis of oseltamivir

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The oxidation of benzenes to trans-cyclohexadienediols (trans-CHDs) has not been reported yet. Nevertheless, the fermentative production of trans-CHDs with recombinant E. coli strains has also been established and the value as a building block for natural product synthesis was demonstrated [178–180]. Starting from chorismate, trans-CHDs are metabolites derived from the shikimate–chorismate pathway in bacteria, plants, and fungi towards the biosynthesis of the iron chelator enterobactin (Fig. 32). Isochorismate synthase (EntC) and isochorismatase (EntB) catalyze the degradation to 2,3-trans-CHD or 3,4-trans-CHD respectively (Fig. 30). Using methods of metabolic pathway engineering, E. coli isochorismate synthase and isochorismatase were overexpressed in E. coli strains with a deficiency of entA, encoding 2,3-dihydroxybenzoate synthase (EntA).

Fig. 32
figure 32

Biosynthesis of trans-cyclohexadienediols

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Microbially produced (2S,3S)-trans-dihydroxy-2,3-dihydrobenzoic acid was used in the synthesis of ent-streptol, ent-senepoxide, and iso-crotepoxide (Fig. 33). The short and efficient synthesis of these biologically active compounds included the esterification of the carboxylic acid and protection of the diol moiety, delivering control of the regio- and stereoselectivity of the following epoxidation or dihydroxylation steps [178, 180].

Fig. 33
figure 33

Natural product syntheses starting from 2,3-trans-CHD

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Recently, (2S,3S)-dihydroxy-2,3-dihydrobenzoic acid was isolated from the fermentation broth of a recombinant E. coli strain and the methyl ester was used in an interesting cyclopropanation sequence [181] yielding dicyclopropane building blocks that are potentially useful for the synthesis of oligocyclopropane natural products [51] such as the antifungal agent FR-900848 and the cholesteryl ester transfer protein inhibitor U-106305 (Fig. 34). For the formation of the second cyclopropane moiety, treatment with diazomethane led to a 1,3-dipolar cycloaddition to a five-membered heterocycle which furnished the desired second cyclopropane by photo induced nitrogen elimination.

Fig. 34
figure 34

(a) Synthesis of dicyclopropane building blocks via [2+3]-dipolar cycloaddition. (b) Oligocyclopropane-containing natural products

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4 C–C-Bond Formations

Enzymatic reactions forming new carbon–carbon bonds are a further important field of biotransformations in natural product synthesis. The construction of new, often complex carbon frameworks or their decomposition is performed by nature under catalysis of a set of enzymes. For organic chemists some of these enzymes, belonging to the enzyme class of lyases, such as aldolases, decarboxylases, hydroxynitrile lyases (HNLs), or benzaldehyde lyases (BALs), have been proven to represent versatile amendments to their synthetic toolbox.

4.1 Enzymatic Aldol Reactions

Aldol reactions enjoy great recognition as a useful tool for the synthesis of building blocks in natural product and drug synthesis [42, 182]. The stereochemistry of the stereogenic centers formed can be controlled by various means. Besides chiral auxiliaries, catalytic methods with chiral Lewis acids, organocatalysts, or catalytic antibodies were established for stereochemical control [183–187].

In nature, most aldolases are rooted in the sugar metabolic cycle and accept highly functionalized substrates for the aldol reaction. Nevertheless, the scope of enzymatic aldol reactions is limited, since aldolases strictly distinguish between the acceptor and the donor, yielding almost exclusively one product, and is furthermore restricted to only a few different possible natural donors. According to the donor molecules, aldolases are grouped in dihydroxyacetone phosphate-, phosphoenolpyruvate- or pyruvate-, acetaldehyde-, and glycine-dependent aldolases [41].

Fructose 1,6-biphosphate aldolase from rabbit muscle in nature reversibly catalyzes the addition of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde 3-phosphate. The tolerance of this DHAP-dependent enzyme towards various aldehyde acceptors made it a versatile tool in the synthesis of monosaccharides and sugar analogs [188], but also of alkaloids [189] and other natural products. For example, the enzyme-mediated aldol reaction of DHAP and an aldehyde is a key step in the total synthesis of the microbial elicitor (−)-syringolide 2 (Fig. 35a) [190].

Fig. 35
figure 35

Synthetic applications of (a) DHAP-, (b) pyruvate-, and (c) glycine-dependent aldolases

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A convenient chemoenzymatic access to sialic acid mimetics as important inhibitors of influenza sialidases has been established by Nelson et al. (Fig. 35b) [191, 192]. Application of a pyruvate-dependent sialic acid aldolase improved by directed evolution disclosed a new route to the core structure of important pharmaceuticals, such as zanamivir (Relenza®).

Glycine-dependent threonine aldolases have been used to synthesize a number of γ-halogenated and long-chain β-hydroxy-α-amino acids. For d-threonine aldolase syn-selectivity was observed exclusively. Further chemical conversion yielded the 2-amino-1,3-diols, potential precursors for the synthesis of short-chain sphingosine-derivatives (Fig. 35c) [193].

To date, 2-deoxy-d-ribose 5-phosphate aldolase (DERA) is the only acetaldehyde-dependent aldolase being applied in organic synthesis. Thus the stereoselectivity of DERA is significant, all known enzymes from different organisms showing the same preferences, limiting the field of application to syntheses in which specifically the DERA-catalyzed enantiomer is needed.

In comparison to other aldolases, DERA has a rather broad substrate range. DERA-catalyzed aldol reactions were used to get an access to key intermediates for epothilones (Fig. 36) [194]. According to retrosynthetic analysis, both fragments of the molecule could be obtained from aldol building blocks, and two out of seven stereocenters were established enzymatically. For the southern part of epothilone A, (S)-3-hydroxy-2-methoxypropanal was successfully converted with acetaldehyde, with the primary open chain aldol product forming the lactol. The aldehyde needed as starting material for the northern part was generated in situ from the acetal: a DERA-catalyzed kinetic resolution led to conversion of the (R)-enantiomer in the aldol reaction, only.

Fig. 36
figure 36

DERA mediated access to key synthons in the synthesis of epothilone A

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Since the product of the aldol reaction still contains an aldehyde function, it is readily available for further reactions. Wong and co-workers very elegantly used a DERA mutant (Ser238Asp) for a sequential one-pot aldol condensation towards the synthesis of atorvastatin (Fig. 37) [195], a process also being applied by DSM [196]. When using wild-type DERA, the choice of residues in the acceptor aldehyde was limited to rather smaller negatively charged groups. The chosen mutation in the hydrophilic binding pocket preserved its nature, but went along with a 2.5-fold improvement in enzyme activity. Furthermore, the mutant presented a highly improved tolerance towards unnatural substrates. The performance in sequential aldol additions was also increased compared to the wild-type enzyme.

Fig. 37
figure 37

Application of DERA in sequential one-pot reactions

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4.2 Cyanohydrins by Hydroxynitrile Lyases

Native HNLs from bitter almonds (Prunus amygdalus), cassava (Manihot esculenta), millet (Sorghum bicolor), and flax (Linum usitatissimum) were repeatedly used in the synthesis of chiral cyanohydrins [39, 41, 197]. Cyanohydrins are versatile building blocks in natural product synthesis, giving organic chemists the possibility of introducing all kinds of functional groups (Fig. 38) [198].

Fig. 38
figure 38

Cyanohydrins as building blocks for organic synthesis

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Effenberger and co-workers reported the biotransformation of pivaldehyde-derivatives to the corresponding cyanohydrins using wild-type PaHNL [199]. In this context, a precursor to (R)-pantolactone could be obtained with a yield of 84% and 89% ee. Since (R)-pantolactone is the most important building block in the synthesis of vitamin B5, further efforts were made for optimization. Recently, an acid stable almond HNL isoenzyme was generated by semirational design and applied in the synthesis described earlier, showing full conversion of hydroxypivalaldehyde to the corresponding cyanohydrin with an enantiomeric excess of 97% (Fig. 39) [200].

Fig. 39
figure 39

Chemoenzymatic synthesis of (R)-pantolactone

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A large number of the most important modern insecticides are derived from cyanohydrins. Especially some esters formed from enantiopure cyanohydrins and chrysanthemum acid derivatives are known to be very potent [201]. Nowadays, the formation of 3-phenoxybenzaldehyde cyanohydrin is performed in a biocatalytic industrial process using MeHNL or HbHNL isolated from rubber trees (Hevea brasiliensis) yielding precursors for cypermethrin or deltamethrin, two pyrethroids (Fig. 40) [21, 39, 41, 202].

Fig. 40
figure 40

Application of HNL in large-scale production of insecticides

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Another application of HNLs can be found in the chemoenzymatic synthesis of the broadband antibiotic thiamphenicol and its fluorinated derivative florfenicol [203]. The conversion of 4-methylsulfanyl-benzaldehyde to the mandelonitrile was catalyzed by a novel enzyme from a Chinese almond (Prunus communis L. var. dulcis Borkh). A concentrated powder from the kernels was prepared and a mixture of the crude meal, aldehyde, and HCN was stirred in isopropyl ether at room temperature for 12 h, yielding the cyanohydrin with 99% ee after recrystallization. The building block formed was then successfully applied in the total synthesis of thiamphenicol and florfenicol (Fig. 41).

Fig. 41
figure 41

Chemoenzymatic synthesis of broadband antibiotics using hydroxynitrile lyases

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4.3 Biocatalytic Formation of Acyloins

Enantiopure, bifunctional acyloins (α-hydroxy ketones) are versatile intermediates in natural product synthesis (also see Sect. 2.3, Fig. 11). In nature, the formation of α-hydroxy ketones is efficiently catalyzed by thiamine diphosphate-dependent enzymes: transketolases, decarboxylases, and other lyases, such as BALs. A great portfolio of biotransformations, especially with benzaldehyde derivatives as starting materials, were realized [204].

A very short and efficient chemoenzymatic synthesis of (−)-ephedrine can be achieved by a decarboxylase-mediated reaction of benzaldehyde with pyruvate [205–207]. The formation of the acyloin intermediate is catalyzed by pyruvate decarboxylase (PDC) with excellent enantioselectivity (Fig. 42).

Fig. 42
figure 42

Chemoenzymatic synthesis of (−)-ephedrine

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2,3-Dioxygenated aryl propanones are very frequently used chiral building blocks for the synthesis of different biologically active compounds. They were synthons in the synthetic approach to 1,4-benzodioxane lignans, such as 5′-methoxyhydno-carpin or cytoxazone, or for the production of aryl isoserines, which are precursors to the side-chain of paclitaxel (Taxol®). A new access to these compounds was established by an enzymatic approach using BAL as the acyloin forming biocatalyst (Fig. 43) [208].

Fig. 43
figure 43

(a) Benzaldehyde lyase-catalyzed acyloin formation. (b) Application of acyloins in natural product synthesis

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5 Conclusion

Nature with its complexity has provided a cornucopia of tools for improvements in twenty-first century natural science research. With new insights into metabolic pathways, chemists can adopt an increasing amount of evolutionary innovation and apply this knowledge to drug and natural product synthesis.

The usage of microorganisms or isolated enzymes thereof is gaining more and more in popularity, leading to an enormous variety of new enzymes being found and optimized for commercialization. Especially in asymmetric synthesis, enzymes can substitute or complement existing methods, often providing ecological advantages due to greater efficiency or less toxicity of the catalyst (enzyme vs transition metal). Thus, the number of applications of enzyme-mediated synthesis has risen enormously during the last few years and the trend does not seem to slow down. Biocatalysis is a main part of white biotechnology and in combination with the usage of renewable primary products should lead to a more sustainable chemical and pharmaceutical industry.