BIOSYNTHESIS OFHETEROCYCLES

BIOSYNTHESIS OFHETEROCYCLES

From Isolation to Gene Cluster

PATRIZIA DIANAGIROLAMO CIRRINCIONEDipartimento di Scienze e Tecnologie Biologiche Chimiche eFarmaceutiche (STEBICEF) Università degli Studi di Palermo

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Diana, Patrizia.Biosynthesis of heterocycles: from the isolation to gene cluster/Patrizia Diana, Girolamo Cirrincione.

pages cm“A John Wiley & Sons, Inc., publication.”Includes bibliographical references and index.ISBN 978-1-118-02867-4 (cloth)1. Heterocyclic compounds–Synthesis. 2. Biosynthesis. I. Cirrincione, Girolamo. II. Title.QD400.5.S95D53 2015547’.59–dc23

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To Gabriella, Giovanni, and little Vincenzo.

CONTENTS

PREFACE xiiiACKNOWLEDGMENTS xv

1 Introduction 1

1.1 Natural Products: Primary and Secondary Metabolites, 41.2 Common Reactions in Secondary Metabolites, 6

1.2.1 Alkylations, 61.2.2 Wagner–Meerwein Rearrangements, 161.2.3 Aldol and Claisen Reactions, 171.2.4 Schiff Base Formation and Mannich Reactions, 231.2.5 Transaminations, 251.2.6 Decarboxylations, 261.2.7 Oxidation and Reduction Reactions, 311.2.8 Dehalogenation/Halogenation Reactions, 391.2.9 Glycosylation Reactions, 46References, 48

2 Techniques for Biosynthesis 51

2.1 Isotopic Labeling, 522.1.1 Stable Isotopes, 522.1.2 Radioactive Isotopes, 61

2.2 Gene Coding for Enzymes, 622.3 Combinatorial Biosynthesis, 63

References, 70

vii

viii CONTENTS

3 Three-Membered Heterocyclic Rings and Their Fused Derivatives 73

3.1 Aziridines and Azirines, 733.1.1 Azicemicins, 733.1.2 Miraziridine, 743.1.3 Maduropeptin, 753.1.4 Azinomycins, 793.1.5 Ficellomycin, 873.1.6 Mitomycins, 893.1.7 Azirinomycin and Related Azirines, 101

3.2 Oxiranes and Oxirenes, 1043.2.1 Fosfomycin, 1043.2.2 AK, HC, and AF toxins, 1113.2.3 Cerulenin, 1173.2.4 Polyhydroxyalkanoates, 1183.2.5 Epoxyrollins, 1183.2.6 Asperlactone, Aspyrone, Asperline, 1213.2.7 Tajixanthone, 1293.2.8 Cyclomarin, 1333.2.9 Cyclopenin, 1393.2.10 Ovalicin and Fumagillin, 1413.2.11 Methylenomycin A, 1433.2.12 Antibiotic LL-C10037, 1473.2.13 Manumycins, 1513.2.14 Scopolamine, 1643.2.15 Iridoid Glucosides, 1693.2.16 Cordiaquinone, 1723.2.17 Cyclizidine and Indolizomycin, 1723.2.18 Enediyne Antibiotics, 1753.2.19 Macrolides, 1953.2.20 Epothilones, 2253.2.21 Pimaricin, 2333.2.22 Hypothemycin, 2403.2.23 Radicicol and Monocillin I, 2433.2.24 Trichothecenes, 2483.2.25 Sporolides A and B, 255References, 258

4 Four-Membered Heterocyclic Rings and Their Fused Derivatives 277

4.1 Azetidine and Azetines, 2774.1.1 Azetidine-2-carboxylic acid, 2774.1.2 Polyoxins, 2804.1.3 Mugineic Acids, 2884.1.4 Tabtoxin and Tabtoxinine-β-lactam, 293

CONTENTS ix

4.1.5 Nocardicins, 2964.1.6 Thienamycin, 3034.1.7 Clavulanic Acid and Clavams, 3114.1.8 Penicillins and Cephalosporins, 319

4.2 Oxetanes, 3414.2.1 Oxetanocins, 3414.2.2 Salinosporamides, 3424.2.3 Taxol, 352

4.3 Dithiethanes, 3634.3.1 Tropodithietic acid and Thiotropocin, 363References, 367

5 Five-Membered Heterocyclic Rings and Their Fused Derivatives 379

5.1 Pyrroles (Including Tetrapyrroles), 3795.1.1 2-Acetyl-1-pyrroline, 3795.1.2 Pyrrolnitrin, 3805.1.3 Broussonetines, 3855.1.4 Prodigiosin and Undecylprodigiosin, 3865.1.5 Anatoxin-a and Homoanatoxin-a, 4025.1.6 Nostopeptolides A, 4075.1.7 Pyrrolizidine Alkaloids, 4105.1.8 Toyocamycin and Sangivamycin, 4165.1.9 Tetrapyrroles, 420

5.2 Indoles, 4285.2.1 Indole-3-acetic acid and Glucobrassicin, 4285.2.2 Camalexin, 4395.2.3 Cyclomarazines, 4445.2.4 Rebeccamycin and Staurosporine, 4455.2.5 Paxilline, 455

5.3 Furans, 4605.3.1 Furanomycin, 4605.3.2 Xenofuranones A and B, 4625.3.3 Acyl α-L-Rhamnopyranosides and Rhamnosyllactones, 4635.3.4 Tuscolid and Tuscoron A and B, 4665.3.5 Tetronomycin and Tetronasin, 4695.3.6 Nonactin and Macrotetrolides, 4745.3.7 Furanonaphthoquinone I, 481

5.4 Thiophenes, 4885.5 Pyrazoles, 4895.6 Imidazoles, 490

5.6.1 Histidine, 4905.6.2 Amaranzole A, 4935.6.3 Oroidin, 493

x CONTENTS

5.6.4 Nikkomycins, 4935.6.5 Anosmine, 496

5.7 Thiazoles, 4975.7.1 Thiamin (Vitamin B1), 4975.7.2 Polypeptide Antibiotics, 5025.7.3 Barbamide, 5085.7.4 BE-10988, 5085.7.5 Pheomelanins, 510

5.8 Dithioles, 511References, 516

6 Six-Membered Rings, and Their Fused Derivatives 533

6.1 Pyridines and Piperidines, 5336.1.1 Pyridoxal 5′-phosphate, 5336.1.2 Nicotinamide Adenine Dinucleotide, 5366.1.3 Nicotine and Related Compounds, 5406.1.4 Tropane Alkaloids, 5426.1.5 Stenusine, 5436.1.6 Antidesmone, 5466.1.7 Quinolobactin, 5466.1.8 Pyridomycin, 5466.1.9 Lycopodine, 5506.1.10 Acridone Alkaloids, 5516.1.11 Benzylisoquinolines, 5516.1.12 Saframycins, 559

6.2 Pyrans, 5616.2.1 Lovastatin and Compactin, 5616.2.2 Bafilomycins and Concanamycin, 5676.2.3 Citrinin, 5716.2.4 Aminocoumarin Antibiotics, 5716.2.5 Flavonoids, 5776.2.6 Actinorhodin and Granaticin, 5816.2.7 Trichothecenes, 5826.2.8 Gilvocarcins, 582

6.3 Pyridazines, 5866.3.1 Kutznerides, 5866.3.2 Pyridazomycin, 5916.3.3 Azamerone, 591

6.4 Pyrimidines, 5926.4.1 Purine and Pyrimidine Nucleotides, 5926.4.2 Methylxanthines and Methyluric Acids, 6026.4.3 Cytokinins, 6066.4.4 Uridyl Peptide Antibiotics, 6076.4.5 Riboflavin, FMN, and FAD, 611

CONTENTS xi

6.5 Pyrazines, 6136.5.1 Alkyl and Methoxy Pyrazines, 6136.5.2 Pteridines, 6166.5.3 Epipolythiodioxopiperazines, 6176.5.4 Roquefortine C and Related Compounds, 621

6.6 Oxazines, 6226.6.1 Minimycin, 6226.6.2 Benzoxazinoids, 625

6.7 Dioxanes, 6266.7.1 Plakortolides, 6266.7.2 Alnumycin, 627References, 632

7 Seven-, Eight-Membered and Larger Heterocyclic Ringsand Their Fused Derivatives 649

7.1 Azepines, 6497.2 Oxepanes and Oxepines, 6577.3 Diazepines, Oxazepines, and Thiazepines, 6617.4 Diazocines, 6747.5 Oxocines, 6747.6 Erythromycin A, 6757.7 Tylosin, 6837.8 Zearalenone, 6907.9 Polyene Macrolide Antibiotics, 693

7.9.1 Nystatin and Amphotericin, 6947.9.2 Candicidin D, 705

7.10 Geldanamycin and Herbimycins, 7167.11 Rifamycins, 7247.12 Rapamycin, 738

References, 745

INDEX 757

PREFACE

This book, which is devoted to the biosynthesis of heterocycles, presents the isolationof heterocycles and their related sources, their structural determination, biosyntheticstudies on them, and, whenever available, the identification of the gene clusters. Italso reports several cases in which gene manipulations allowed the biosynthesis ofunnatural compounds generally used in medicinal chemistry.

The book is organized into seven chapters. In the introductory chapter, thesynthetic pathways of some natural products illustrating the basic common reactionsin secondary metabolites are described. In Chapter 2, methods and techniquesinvolved in the biosynthesis of heterocycles are dealt with. The subsequent fourchapters deal with three- to six-membered heterocycles starting from the naturalproducts to approach the preparation of unnatural heterocyclic compounds withparticular attention to bioactive molecules. In Chapter 7, seven- and eight-memberedheterocycles are treated, as well as larger ones, using the same approach as used inthe preceding four chapters.

Because of the incredibly large number of isolated heterocycles from naturalsources, a selection had to be made, choosing both those possessing biologicalactivity and those isolated in the past 15 years.

To the best of our knowledge, there are currently no books available with a specialemphasis on the biosynthesis of the whole range of heterocycles following a highlysystematic approach and also dealing with the identification of the gene clusters andthe use of biogenetic engineering to get unnatural compounds of pharmaceuticalinterest.

This book is primarily addressed to meet the requirements of graduate andpostgraduate students in biology, biochemistry, biotechnology, chemistry, andpharmacy. This book can also be a useful tool for teachers of the degree courses

xiii

xiv PREFACE

mentioned and for investigators and professionals (industry) working within thefields of medicinal, organic, and process chemistry. A secondary audience can beconstituted by biochemists, enzymologists, and microbiologists.

Finally, we have to mention the support given to us by our partners, to whom thisbook is dedicated and without whose continued patience and understanding this bookwould not have been possible.

Patrizia DianaGirolamo Cirrincione

Università degli Studi di PalermoPalermo, Italy

ACKNOWLEDGMENTS

The authors wish to express their appreciation to Dr. Barbara Parrino for thehelpful collaboration during the preparation of the manuscript and for coordination,and Vincenzo Cilibrasi, Gloria Di Vita, Simona Di Martino, Salviana Ullo, andMaria Ferraro for preparation of the figures.

xv

1INTRODUCTION

Throughout human history, natural products, compounds that are derived from naturalsources such as plants, animals, or microorganisms, have played a very important rolein health care and prevention of diseases. For example, some of the first records onthe use of natural products in medicine were written in cuneiform in Mesopotamia onclay tablets and date to approximately 2600 BC; Chinese herb guides document theuse of herbaceous plants as far back in time as 2000 BC; Egyptians have been foundto have documented the uses of various herbs in 1500 BC.

However, it’s only in the nineteenth century that scientists isolated active com-ponents from various medicinal plants. The first commercial pure natural productintroduced for therapeutic use is considered to be the narcotic morphine, in 1826.Natural products still play a very important role in modern medicine; in fact, they areincreasingly the primary sources in drug discovery.

The pathways for generally modifying and synthesizing carbohydrates, proteins,fats, and nucleic acids are found to be essentially the same in all organisms, except forminor variations. Metabolism encompasses a wide variety of reactions for buildingmolecules that are necessary to the life of the organism and for disruption of othersfor energy or secondary metabolites.

Primary metabolites are compounds that are essential for an organism’ssurvival, growth, and replication. Secondary metabolites, such as alkaloids, gly-cosides, flavonoids, and so on, which are biosynthetically derived from primary

Biosynthesis of Heterocycles: From Isolation to Gene Cluster, First Edition.Patrizia Diana and Girolamo Cirrincione.© 2015 John Wiley & Sons, Inc. Published 2015 by John Wiley & Sons, Inc.

1

2 INTRODUCTION

metabolites, are substances that are often present only in certain types of specializedcells, and are not directly involved in the normal growth, development, or reproduc-tion of an organism. They represent chemical adaptations to environmental stresses,or serve as defensive, protective, or offensive chemicals against microorganisms,insects, and higher herbivorous predators. They are sometimes considered as wasteor secretory products of metabolism and are of pharmaceutical importance.

The building blocks for secondary metabolites are derived from primarymetabolism. In fact, the biosynthesis of secondary metabolites is derived from thefundamental processes of photosynthesis, glycolysis, and the Krebs cycle to affordbiosynthetic intermediates, which, ultimately, results in the formation of secondarymetabolites also known as natural products. The most important building blocksemployed in the biosynthesis of secondary metabolites are those derived from theintermediates: acetyl-coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid, and1-deoxyxylulose-5-phosphate (Figure 1.1).

Acetyl-CoA is formed by the oxidative decarboxylation of the glycolytic pathwayproduct pyruvic acid. Shikimic acid is produced from a combination of phos-phoenolpyruvate, a glycolytic pathway intermediate, and erythrose 4-phosphate,obtained from the pentose phosphate pathway. Mevalonic acid is itself formed fromthree molecules of acetyl-CoA. Deoxyxylulose phosphate originates from a com-bination of pyruvic acid and glyceraldehyde-3-phosphate (GAP). Moreover, otherbuilding blocks based on amino acids (e.g., phenylalanine, tyrosine, tryptophan,lysine, ornithine) (Figure 1.2) are frequently employed in natural product synthesis(e.g., proteins, alkaloids, antibiotics). Though the number of building blocks islimited, the number of novel secondary metabolites formed is infinite.

Biosynthesis of secondary metabolites involves numerous different mechanismsand reactions that are enzymatically catalyzed using several common mechanismssuch as acylation, alkylation, decarboxylation, phosphorylation, hydride transfer,oxidation, elimination, reduction, condensation, rearrangement, and so on. Thebiosynthetic pathway may undergo changes due to natural causes (e.g., virusesor environmental changes) or unnatural causes (e.g., chemical or radiation) in anattempt to adapt or provide long life to the organism.

The elucidation of the biosynthetic pathway for the production of various metabo-lites has been extensively examined through the use of techniques that use isotopiclabeling (stable isotopes and radioactive isotopes). Initially, radiolabeled precursorswere introduced into plants and the resultant radioactive compounds were chemicallydegraded to identify the positions of the label. As the development of analytical instru-mentation advanced, the isotopically labeled natural products were analyzed by massspectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy instead ofchemical degradation.

The biosynthesis of each secondary metabolite is catalyzed by a number ofenzymes, usually encoded by a gene cluster. The disclosure of biosynthetic geneclusters has great potential for the identification of entire biosynthetic pathways forbioactive compounds of pharmaceutical interest.

Genome sequence analysis provides a source of the information necessary for pre-dicting the biosynthesis pathways for secondary metabolites because the sequence

INTRODUCTION 3

Primary Metabolism

CO2 + H2OPhotosynthesis O

OH

OH

OH

OH

HO

Glucose

Glycolysis

HO O

2−O3PO

OH

2−O3PO

CO2H

Phosphoenol Pyruvate

Erytrose-4-phosphate

HO OH

CO2H

OH

Shikimic acid

Aminoacids

OC

CO2H

Pyruvic acid

KrebsCycle

O

SCoA

Acetyl-CoA

CCO2H

O

CoAS

Malonyl-CoAHO CO2H

HO

Mevalonic acid

O

OH

OPO32−

OH

OPO32−

O

OH

Glyceraldehyde 3-phosphate

Deoxyxylulose5-phosphate

Figure 1.1 Building blocks employed in the biosynthesis of secondary metabolites.

analysis could reveal all the enzymes specific to each organism from their genes codedon the genome.

However, the gene information is not always described in a comprehensive mannerand the related information is not always integrated. The database BIoSynthesis clus-ters CUrated and InTegrated (DoBISCUIT) integrates the latest literature informationand provides standardized gene/module/domain descriptions related to the geneclusters [1].

4 INTRODUCTION

COH

NH2

O

l-Phenylalanine

COH

NH2

O

HO

l-Tyrosine

NH

H2N

l-Tryptophan

H2N COH

NH2

O

C

O

OH

l-Lysine

H2NC

NH2

O

OH

l-Ornitine

Figure 1.2 Building blocks based on amino acids.

The explanation of the biosynthetic pathway may also be possible through molecu-lar biology techniques that use mutants. The use of tandem analytical instrumentation(e.g., GC/MS (gas chromatography/mass spectrometry), NMR/MS, LC/MS (liquidchromatography/mass spectrometry)) has improved the identifications of primary andsecondary metabolites.

1.1 NATURAL PRODUCTS: PRIMARY AND SECONDARYMETABOLITES

Primary metabolites can originate from fundamental processes: photosynthesis, gly-colysis, and the citric acid cycle (Krebs cycle). They represent biosynthetic interme-diates useful as building blocks for the synthesis of secondary metabolites. The lattercan be synthesized through a combination of various building blocks (Figure 1.3):

1. a single carbon atom (C1), usually in the form of a methyl group, obtained froml-methionine;

2. a two-carbon unit (C2), an acetyl group, derived from acetyl-CoA or from themore active malonyl-CoA;

3. a branched chain (C5), the isoprene moiety, formed from mevalonic acid ormethylerythritol phosphate;

4. a phenylpropyl moiety (C6C3) and a (C6C2N) fragment, both originating froml-phenylalanine or l-tyrosine;

5. an indole C2N group, obtained from l-tryptophan;

6. C4N and C5N portions, generated from l-ornithine and l-lysine, respectively.

Secondary metabolites can be synthesized by combining several building blocksof the same type, or by using a mixture of different building blocks.

NATURAL PRODUCTS: PRIMARY AND SECONDARY METABOLITES 5

S CO2H

NH2

L-Methionine

(a) CH3 (C1)

(b) SCoA

O

or SCoA

O

CO2H

C C (C2)

)

(c)HO2C

OH

HO

Mevalonic acid

or OPO32-

OH

HO

OH

Methylerythritolphosphate

(C 5

(d) CO2H

NH2

L-Phenylalanine

orCO2H

NH2HO

L-Tyrosine

N(C6C2N)

(C6C3)

(e)

NH

CO2H

NH2

L-Tryptophan

NH

N (Indole C2N)

(f)

H2NCO2H

NH2

L-Ornithine

N (C4N)

H2N CO2H

NH2L-Lysine

N (C5N)

Acetyl CoA

Isoprene unit

Malonyl CoA

Figure 1.3 (a–f) Biosynthetic intermediates useful as building blocks for the synthesis ofsecondary metabolites.

6 INTRODUCTION

Some examples of secondary metabolites are antibiotics, alkaloids, anthraquinones,coumarines, flavonoids, xanthones, and terpenoids.

1.2 COMMON REACTIONS IN SECONDARY METABOLITES

The building blocks used in the biosynthesis of secondary products are assembledthrough biochemical reactions and catalyzed by enzymes, including alkylation reac-tions (nucleophilic substitutions and electrophilic additions); Wagner–Meerweinrearrangements; aldol and Claisen reactions; Schiff base (SB) formation andMannich reactions; transaminations, decarboxylations, oxidation, and reductionreactions (hydrogenation/dehydrogenation reactions); monooxygenase and dioxy-genase reactions; Baeyer–Villiger reactions; oxidative deamination reactions;dehalogenation–halogenation reactions; and glycosylations.

1.2.1 Alkylations

The alkylation reactions are classified, based on the character of the alkylating agent,into nucleophilic substitutions and electrophilic additions. Natural alkylating agentsare S-adenosyl-L-methionine (SAM) and dimethylallyl diphosphate (DMAPP).

In nucleophilic substitutions, SAM is commonly used as methyl donor innumerous methylation reactions. The 3-amino-3-carboxypropyl (acp) group of SAMcan also be transferred to different acceptor molecules. SAM-dependent acp-transferreactions are relatively rare compared to methyl-transfer ones.

The positively charged sulfonium ion in SAM makes the three carbon atoms thatare bonded to the sulfur atom prone to attack by nucleophiles. When the alkyl acceptoris a heteroatom (most commonly O, N), the methyl- or the acp-transfer reactionsoccur via simple nucleophilic mechanism (SN2): O-methyl or O-acp and N-methyl orN-acp linkages may be generated using hydroxyl and amino functions as nucleophiles(Figure 1.4). Some examples of O-methylation in the presence of SAM as the donormethyl group are depicted in Figure 1.5.

In the biosynthesis pathway for 3-alkyl-2-methoxypyrazines (MPs) – an impor-tant group of natural flavor constituents of some foods and raw vegetables includinggrapes – the methylation of 3-alkyl-2-hydroxypyrazines (HPs) is mediated by theVitis vinifera genes O-methyltransferase proteins (VvOMTs). These genes encodethe SAM-dependent O-methyltransferases, which have the ability to methylateHPs, which are the putative final intermediates in MP production. As a prod-ucts of this reaction, 3-alkyl-2-MP and S-adenosylhomocysteine are generated(Figure 1.5a) [2].

Mycophenolic acid (MPA) is being used as an immunosuppressant in patientsundergoing kidney, heart, and liver transplants. The final step in the biosynthesis ofMPA involves the transfer of a methyl group from SAM to the demethylmycophenolicacid (Figure 1.5b) [3].

The last step of the biosynthetic pathway of Khellin and Visnagin (coronaryvasodilators and spasmolytic agents) involves a methylation of 5,7-dihydroxy- and

COMMON REACTIONS IN SECONDARY METABOLITES 7

R XH..

+

H3CS-Ad

COOHH2N

(SAM)(X = O, NH)

SN2

SN2

R X CH3

H+

COOH

SAd

H2N

(SAH)

−H+

R X CH3

(X = O, NH)

R XH..

+

H3CS-Ad

COOHH2N

(SAM)(X = O, NH)

R X CH2

H+

−H+

X CH2

(X = O, NH)

H2NCOOH

H3C S Ad

(MTA)

H2NCOOH

R

Figure 1.4 O- and N-alkylation using SAM.

5-hydroxy-furochromone, respectively, in presence of SAM as a methyl donorgroup. Also, the furocoumarine xanthotoxol generates xanthoxin as a result of SAMmethylation (Figure 1.5c) [4].

The alkaloid anhalonine was generated by methylation of the correspo-nding 1,2,3,4-tetrahydro-6,7-dimetoxy-8-hydroxy-1-methylisoquinoline (anhaloni-dine); Kreysigine, a benzocyclohepta-isoquinoline alkaloid, was obtained by themethylation of 1,10-dihydroxy-2,11,12-trimethoxy-4,5,6,6a,7,8-hexahydrobenzo[6,7]cyclohepta [1,2,3-ij]isoquinoline (floramultine alkaloid) (Figure 1.5d).

An example of N-methylation in presence of SAM as the methyl group donoris provided by the caffeine biosynthetic pathway involving three SAM-dependentmethylation steps (Figure 1.6) [5]. The methylation reactions are catalyzed byN-methyltransferases (CaXMT1, CaMXMT1, and CaDXMT1), which, respec-tively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into

8 INTRODUCTION

(a) 3-Alkyl-2-hydroxypyrazines

N

N R

OH

SAM SAH

N

N R

O

R = Isopropyl, isobuthyl

(b) Demethylmycophenolic acid

O

O

HO

R

SAM SAH

R = CH2-CH=CH(CH3)-(CH2)2-COOH

O

O

O

R

Mycophenolic acid

(c) Furocoumarins

O O

O

R1

R

R = R1 = OMe KhellinR = OMe; R1= H Visnagin

;O O

O

O

Xanthotoxin

(d) Alkaloids

NH

O

O

O8

Anhalonine

;NH

H

O

HOO

O

O

OH OH

10

Kreysigine

1

Figure 1.5 Examples of SAM O-methylations. (a) 3-Alkyl-2-hydroxypyrazine, (b)mycophenolic acid, (c) furocoumarins (khellin, visnagin, and xanthotoxin), and (d) alkaloids(anhalonine and kreysigine).

COMMON REACTIONS IN SECONDARY METABOLITES 9

SAM SAHHN

NH

N

N

RibO

O

Xanthosine

HN

NH

N

N

RibO

O

7-Methylxanthosine

HN

NH

N

N

O

O

7-Methylxanthine

SAM

SAH

HN

N N

N

O

O

Theobromine

SAMSAH

N

N N

N

O

O

Caffeine

Figure 1.6 Examples of SAM N-methylations: biosynthesis of caffeine.

3,7-dimethylxanthine (theobromine), and the latter into 1,3,7-trimethylxanthine(caffeine). Further examples of natural compounds N-methylated by SAMare lophocerine and galanthamine (alkaloid derivative isolated from snow-drop Galanthus nivalis L.) (Figure 1.7). N-methylation reactions catalyzed bySAM-dependent methyltransferases are also involved in the assembly of nonri-bosomal peptides (NRPs) [6]. For instance, in the biosynthesis of lyngbyatoxin,an embedded SAM-dependent methyltransferase domain mediates the methy-lation of the free amine of the NRPS (nonribosomal peptide synthetase) [7].A further case of N-methylation is provided by the biosynthesis of saframycinA (an antibiotic with antitumor activity produced by Pseudomonas fluorescensA2-2) [8].

An example of O-amino-carboxy-propylation is provided by the biosynthesis ofnocardicin A (β-lactam antibiotic produced by the actinomycete Nocardia uniformis)(Section 4.1.5) (Figure 1.8).

Examples of N-amino-carboxy-propylation are mainly observed in RNA modi-fications, such as 3-(3-amino-3-carboxypropyl)uridine [9] or 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine [10, 11]. Another N-ACP-transfer reaction was alsoobserved in the biosynthesis of 2-(3-amino-3-carboxypropyl)-isoxazolin-5-one (neu-rotoxic amino acid from Lathyrus odoratus) (Figure 1.8) [12].

In SAM-dependent alkylation reactions, when the methyl acceptors are carbonatoms, the enzymatic reaction mechanisms are more complicated and depend onthe electronic properties of the acceptor molecules. The generation of C-alkyl link-ages requires the formation of a nucleophilic carbon. An interesting SAM-dependent

10 INTRODUCTION

N

O

O

N-Methyl-trasfer

Lophocerine

O

ON

OH

Galanthamine

NH

N

HN

O

Lyngbyatoxin

N

O

O

O

O

O

NO

NH

OO

Saframycin A

OH

CN

Figure 1.7 Further examples of natural compounds N-methylated by SAM: lophocerine,galanthamine, lyngbyatoxi, and saframycin A.

C-methylation reaction is the methylation of the C-5 position of cytosine in DNA. Inthis case, the carbon C-5 of cytosine cannot directly act as a nucleophile. The elec-tron withdrawal by N-3 and the carbonyl, however, makes the C-5—C-6 double bondelectron deficient and prone to attack by nucleophiles in a reaction that is similar to aMichael reaction. In DNA methyltransferases (DNMTs), this nucleophile is the thio-late from a Cys residue. The addition product is nucleophilic and reacts with SAM viaan SN2-like mechanism to capture the methyl group. The resulting intermediate theneliminates the Cys of DNMT to give the methylated cytosine product (Figure 1.9).The methylation of C-5 of cytosine is an example of converting an electron-deficientmethyl acceptor to a nucleophile for the methyl-transfer reaction by addition of anactive site Cys thiolate.

There are two known examples of acp transfer to carbon atoms, namely diph-thamide and wybutosine (characterized by a tricyclic 1H-imidazo[1,2-α]purine corewith a large side chain) biosyntheses.

The biosynthesis of diphthamide was proposed to involve three steps, with the firstone being the acp transfer from SAM to the C-2 of the imidazole ring (Figure 1.10).The proposed reaction mechanism involves an electron transfer event from the[4FE-4S] cluster, which leads to the breaking of C—S bond and consequentlygenerates the acp radical. The latter is added to imidazole ring, and then a hydrogenatom is eliminated to give the desired product. The formation of the acp radical is

COMMON REACTIONS IN SECONDARY METABOLITES 11

OH

NH

HON

O N

H O

HO2C

OH

Nocardicin E

+

(a) O-acp-transfer

S Ad

H2NCO2H O

NH

HON

O N

H O

HO2C

OH

CO2HH2N

+ MeS-Ad

(b) N-acp-transfer

O

OHOH

HO

N

N

O

O

NH2

HO2C

acp-uridine

O

OHOH

HO

N

O

O

CO2H

NH2

acp-pseudouridine

O N

O

CO2H

NH2

2-(3-Amino-3-carboxy-(propyl)-isoxazolin-5-one

Nocardicin A

Figure 1.8 acp-transfer reactions. (a) O-amino-carboxy-propylation (nocardicin A) and(b) N-amino-carboxy-propylation (3-(3-amino-3carboxypropyl)uridine), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, and 2-(3-amino-3-carboxypropyl)-isoxazolin-5-one).

12 INTRODUCTION

N

NO

NH2

R

(−)S-DNMT N

NO

NH2

R

S

DNMT

N

NO

NH2

R

S

DNMT

+

S

CO2H

Ad

H2N

(SAM)

N

NO

NH2

R

S

DNMT

HB−N

NO

NH2

R

+S-DNMT

Cytosine

5

Figure 1.9 acp C-methylation of cytosine.

supported by the detection of 2-aminobutyrate and homocysteine sulfinic acid whenthe imidazole substrate was not present in the reaction (Figure 1.10b) [13].

In the proposed biosynthesis pathway of wybutosine, the acp-transfer step is cat-alyzed by Tyw2, which has similarity to methyltransferases that catalyze nucleophilicmethyl-transfer reactions (Figure 1.11).

DMAPP may act as an alkylating agent (isopropene unit) via an SN2 nucleophilicdisplacement in which the diphosphate is the leaving group. In some cases, DMAPPmay ionize first to the resonance-stabilized allylic carbocation, and thus an SN1 reac-tion occurs on the C-activated position (Figure 1.12).

The initial step in cytokinin (adenine derivatives with an isoprenoid side chain)biosynthesis is N-prenylation of adenosine 5-phosphate, a reaction catalyzed byadenosine phosphate-isopentenyltransferases (PTs). PTs catalyze the isopropeneunit transfer reaction to an acceptor (adenosine monophosphate, AMP) which servesas a nucleophile. The latter is alkylated by DMAPP to form, by an SN2-nucleophilicdisplacement reaction, a prenylated AMP and pyrophosphate (PP) as products[14, 15].

A further example of DMAPP alkylation is the N-prenylation of tryptophan in thebiosynthesis of the cyclic peptides cyclomarin and cyclomarazine (diketopiperazinedipeptides) (Section 3.2.8) (Figure 1.13) [16].

Electrophilic additions occur frequently in the biosynthesis of steroids and ter-penoids. The electrophile in such reactions is a positively charged or a positivelypolarized carbon atom, which often adds to an unsaturated (electron-rich) partner,usually an alkene, and leads to the formation of a saturated product. In most cases,biochemical pathways have evolved in such a way that electrophilic addition reactions