Synthesis of Natural products in organism in organism

了解一次代谢和二次代谢及其产物；

了解天然产物的生物合成方法；

Secondary Metabolism

The building blocks and construction mechanism

All organisms need to transform and interconvert avast number of organic compounds to enable themto live, grow, and reproduce. They need to providethemselves with energy in the form of ATP themselves with energy in the form of ATP

(Adenosine Triphosphate 三磷酸腺甙), and a supply of building blocks to construct their own tissues.

叶绿素

代谢图

Section 1. Primary and secondary metabolism：Primary metabolism

Despite the extremely varied characteristics of living organisms, the pathways for generally modifying

and synthesizing carbohydrates, proteins, fats, and nucleic acids are found to be essentially the same in all organisms, apart from minor variations. in all organisms, apart from minor variations.

These processes demonstrate the fundamentalunity of all living matter, and are collectivelydescribed as primary metabolism, withthe compounds involved in the pathways beingtermed primary metabolites： carbohydrates,

proteins, fats, and nucleic acids.

FIGURE Energy relationships between the pathways of catabolism（分解代谢） and anabolism（合成代谢）.

• Thus degradation of carbohydrates and sugars generally proceeds via the well characterized pathways known as glycolysis（糖酵解） and the Krebs克雷布斯循环,三羧酸循环(指机体糖代谢的一系列生化反应)/citric acid柠檬酸/tricarboxylic acid cycle, which release energy from the /tricarboxylic acid cycle, which release energy from the organic compounds by oxidative reactions. Oxidation of fatty acids from fats by the sequence called β-oxidation also provides energy.

Biosynthesis of natural products天然产物化学的生物合成

• The primary metabolism一次代谢CO2+叶绿素chlorophyl＋H2O

糖类glucide＋O2 代谢 赤藓糖－4－磷酸酯O2 代谢

三磷酸腺苷adenosine triphosphate（ATP）＋辅酶coenzyme I (NADPH)

丙酮酸（Pyruvic acid), 磷酸烯醇丙酮酸（PEP）

4（erythrose－4－phosphate核糖ribose

The main products of primary metabolism including 糖类sugars，蛋白质proteins，核酸nucleic acids and 脂质lipids that materials are necessary to living organisms for keeping their lives.

FIGURE Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation. 糖酵解划分：柠檬酸循环、氧化磷酸化

FIGURE The glycolytic pathway.

FIGURE The second phase of glycolysis. Carbon atoms are numbered to showtheir original positions in glucose.

赤藓糖－4－磷酸酯（erythrose－4－phosphate

cinnamic aldehyde

pyruvate丙酮酸酯

肉桂醛

Secondary metabolism二次代谢In contrast to these primary metabolic pathways,which synthesize, degrade, and generally interconvertcompounds commonly encountered in allorganisms, there also exists an area of metabolismconcerned with compounds which have a muchmore limited distribution in nature. Such compounds,more limited distribution in nature. Such compounds,called secondary metabolites, are found in only specific organisms, or

groups of organisms, and are an expression of the individuality of species.

It is this area of secondary metabolism that provides most of the pharmacologically active natural products.

• Secondary Metabolism1. 一次代谢产物在酶enzyme作用下，经过一系列不同的代谢过程，生成了

不同类型的物质，如生物碱alkaloids，萜类terpenes，黄酮flavenoids类，等等。

2. 此代谢对维持生命活动不起重要作用，称其为二次代谢过程。

3. 这些物质不是维持植物生命活动所必须，为二次代谢产物。3. 这些物质不是维持植物生命活动所必须，为二次代谢产物。

4. 二次代谢产物种类繁多，功能不同，是天然有机化学研究的主要内容和目标

Section 2. The building blocks • The building blocks for secondary metabolites are derived

from primary metabolism as indicated in Figure 2.1.the most important building blocks employed in the biosynthesis of secondary metabolites are derived from the intermediates acetyl coenzyme A （辅酶A）derived from the intermediates acetyl coenzyme A （辅酶A）(acetyl-CoA), shikimic acid（莽草酸）, mevalonic acid（甲戊二羟酸）,

and 1-deoxyxylulose（xylulose－木酮糖） 5-phosphate.

Acetyl-CoAs formed by oxidative decarboxylation of the glycolytic pathway product pyruvic acid.

It is also produced by the β-oxidationof fatty acids, effectively reversing the

process by which fatty acids are themselves synthesized from acetyl-CoA.

The shikimate pathway leads to a variety of phenols, cinnamic acid derivatives, lignans, and alkaloids。

Mevalonic acid 甲戊二羟酸is itself formed from three molecules

Shikimic acid is produced from a combination of phosphoenolpyruvate磷酸烯醇丙酮酸酯),a glycolytic pathway intermediate, and erythrose （赤藓糖，四碳糖） 4-phosphate from the pentose 戊糖 phosphate pathway.

is itself formed from three molecules of acetyl-CoA, but the mevalonate pathway channelsacetate into a different series of compounds thandoes the acetate pathway。

• Deoxyxylulose phosphate arises from a combination of two glycolytic（糖分解） pathway intermediates, namely pyruvic acid and glyceraldehyde（甘油醛） 3-phosphate.

• The mevalonate甲戊二羟酸酯and deoxyxylulose phosphate pathways are together responsible for the biosynthesis of a vast array of terpenoid and steroid metabolites.

• In addition to acetyl-CoA, shikimic acid, mevalonic acid, • In addition to acetyl-CoA, shikimic acid, mevalonic acid, and deoxyxylulose phosphate, other building blocks based on amino acids are frequently employed in natural product synthesis.

building blocksC1: The simplest of the building blocks is composed of a single carbon atom, usually in the form of a methyl group, and most frequently it is attached to oxygen or nitrogen, but occasionally to carbon. It is derived from the S –methyl of L-methionine（蛋氨酸,甲硫氨酸）. The methylenedioxy group(OCH2O) is also an example of a C1 unit.

O

O

OMeOMe

OMeOMe

C2: A two-carbon unit may be supplied by acetyl-CoA. This could be a simple acetyl group, as in an ester, but more frequently it forms part of a long alkyl chain (as in a fatty acid) or may be part of an aromatic system (e.g. phenols). Of particular relevance is that in the latter examples, acetyl-CoA is first converted into the more reactive malonyl-CoA before its incorporation.

• C5: The branched-chain C5 ‘isoprene’ unit is a feature of compounds formed from mevalonate or deoxyxylulose phosphate. Mevalonate itselfis the product from three acetyl-CoA molecules, but only five of mevalonate’s six carbons are used, the carboxyl group being lost. The alternative precursor deoxyxylulose phosphate, a straight-chain sugar derivative, undergoes a skeletal rearrangement to form the branchedchain isoprene unit.

• • C6C3: This refers to a phenylpropyl unit and is obtained from the carbon skeleton of either L-phenylalanine or L-tyrosine 酪氨酸, two of the shikimate-derived aromatic amino acids. This, of course, requires loss of the amino group. The C3 side-chain may be saturated or unsaturated, and may be oxygenated. Sometimes the sidechain is cleaved, removing one or two carbons.

Thus, C6C2 and C6C1 units represent modified shortened forms of the C6C3 system.

C6C2N: Again, this building block is formedfrom either L-phenylalanine or L-tyrosine, L-tyrosinebeing by far the more common. In theelaboration of this unit, the carboxyl carbon ofthe amino acid is removed.

• indole.C2N: The third of the aromatic aminoacids is L-tryptophan色氨酸. This indole-containingsystem can undergo decarboxylation in a similarway to L-phenylalanine and L-tyrosine soproviding the remainder of the skeleton as anindole.C2N unit.

• C4N: The C4N unit is usually found as a heterocyclic Pyrrolidine（吡咯烷）system and is produced from the non-protein amino acid L-ornithine（鸟氨酸）. In marked contrast to the C6C2N and indole.C2N units described above, ornithine supplies not itsα-amino nitrogen, but the δ-amino nitrogen. The carboxylic acid function and the α-amino nitrogen are both lost.

• C5N: This is produced in exactly the same way• C5N: This is produced in exactly the same wayas the C4N unit, but using L-lysine （赖氨酸） as precursor.The ε-amino nitrogen is retained, and the unittends to be found as a piperidine （哌啶）ring system.

A word of warning is also necessary. Some natural products have been produced by processes in which a fundamental rearrangement of the carbonskeleton has occurred. This is especially common with structures derived from isoprene units, and it obviously disguises some of the original buildingblocks from immediate recognition. The same is true if one or more carbon atoms are removed by oxidation reactions.

THE CONSTRUCTION MECHANISMS

Natural product molecules are biosynthesized by a sequence of reactions which, with very few exceptions, are catalysed by enzymes.

Alkylation Reactions: NucleophilicSubstitution

• The C1 methyl building unit is supplied from L-methionine蛋氨酸 and is introduced by a nucleophilic substitution reaction. In nature, the leaving group is enhanced by converting L-methionine into S –adenosylmethionine （腺苷甲硫氨酸SAM)

nucleophilic substitution (SN2) type mechanism

Dimethylallyl diphosphate (DMAPP)nucleophilic substitution (SN2) type mechanism

Alkylation Reactions: ElectrophilicAddition

Wagner–Meerwein Rearrangementsoriginating from C5 isoprene units.Rearrangements in chemical reactions involving

carbocation intermediates, e.g. SN1 and E1 reactions, are not uncommon, and typically consist of 1,2- shifts of hydride, methyl, or alkyl groups.

Aldol and Claisen Reactions

In most cases, the biological reactionsinvolve coenzyme A esters, e.g. acetyl-CoA

Claisen reactions involving acetyl-CoA are made even more favourable by first convertingacetyl-CoA into malonyl-Co丙二酸辅酶 by a carboxylation HCO3−) using ATP and the

coenzyme reaction with CO2(as bicarbonate), biotin. ATP and CO2 (as bicarbonate,HCO3−) form the mixed anhydride, which carboxylates the coenzyme in a biotin（维生素

H–enzyme complex.

Bio-claisen pathway

Chemical Claisen Reaction

β-Oxidation of fatty acids

The reverse Claisen reaction is a prominentfeature of the β-oxidation sequence for thecatabolic degradation of fatty acids .

Schiff Base Formation and theMannich Reaction

Formation of C−N bonds is frequently achieved bycondensation reactions between amines and aldehydesor ketones. A typical nucleophilic additionis followed by elimination of water to give animine or Schiff base [Figure 2.12(a)].

Of almost equal importance is the reversal of this process, i.e. the hydrolysis of imines to amines and aldehydes/ketones [Figure 2.12(b)].

The imine亚胺 so produced,or more likely its protonated form the iminium ion, can then act as an electrophile in a Mannich reaction [Figure 2.12(c)]. The nucleophile might be provided by an enolate anion, or in many examples by a suitably activated centre in an aromatic ring system.

It should be appreciated that the Mannich-like addition reaction in Figure 2.12(c) is little different from nucleophilic addition to a carbonyl group.

Indeed, the imine/iminium ion is merely acting as the nitrogen analogue of a carbonyl/protonated carbonyl. To take this analogy further, protons on carbon adjacent to an imine group will be acidic as are those α to a carbonyl group, and the isomerization to the enamine shown in Figure 2.13 is analogous to keto–enol tautomerism互变现象. Just as two carbonyl compounds can react via an aldol reaction, so can two imine systems, and this is indicated in Figure 2.13.

TransaminationTransamination is the exchange of the amino group from an amino acid to a keto a

cid, and provides the most common process for the introduction of nitrogen into amino acids, and for the removal of nitrogen from them.

Reductive amination of the Krebs cycle intermediate 2-oxoglutaric acid to glutamic acid (Figure 2.14) is responsible for the initial incorporation of nitrogen, a reaction which involves imine formation and subsequent reduction. Transamination

This reaction is dependent on the coenzyme pyridoxal维生素B phosphate (PLP) and features a Schiff base/imine intermediate (aldimine) with the aldehyde group of PLP (Figure 2.14). The α-hydrogen of the original amino acid is now made considerably more acidic and is removed, leading to the ketimine by a reprotonation process which also restores the aromaticity in the pyridine ring. The keto acid is then liberated by hydrolysis of the Schiff base function, which generates pyridoxamine phosphate. The remainder of the sequence is now a reversal of this process, and transfers the amine function from pyridoxamine phosphate to another keto acid.

Decarboxylation ReactionsMany pathways to natural products involve steps which remove portions of the carbon

skeleton.

Oxidation and Reduction Reactions

DehydrogenasesDehydrogenases remove two hydrogen atoms from the substrate, passing them to a

suitable coenzyme acceptor. The coenzyme system involvedcan generally be related to the functional group being oxidized in the substrate. Thus if

the oxidation process is

• then a pyridine nucleotide, nicotinamide 烟碱adenine 腺嘌呤dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+), tends to be utilized as hydrogen acceptor. One hydrogen from the substrate (that bonded to carbon) is transferred as hydride to the coenzyme, and the other, as a proton, is passed to the medium (Figure 2.16).

OxidasesOxidases also remove hydrogen from a substrate, but pass these atoms to

molecular oxygen or to hydrogen peroxide, in both cases forming water.Oxidases using hydrogen peroxide are termed peroxidases. Mechanisms of

action vary and need not be considered here. Important transformationsin secondary metabolism include the oxidation of ortho- and para-quinols to

quinones (Figure 2.18), and the peroxidase-induced phenolicoxidative coupling processes (see page 28).

Mono-oxygenases单加氧酶

With monooxygenases,the second oxygen atom from O2 is reduced to water by an appropriate hydrogendonor, e.g. NADH, NADPH, or ascorbic acid

(vitamin C).

Aromatic hydroxylation catalysed by monooxygenases (including cytochrome细胞色素P-450 systems) probably involves arene芳烃 oxide (epoxide) intermediates

(Figure 2.20). A high proportion of these hydrogen atoms is subsequently retained in the product, even though enolization allows some loss of this hydrogen. This migration is known as the NIH shift, having been originally observed at the National Institute of Health, Bethesda, MD, USA.

DioxygenasesDioxygenases introduce both atoms from molecular oxygen into the substrate, and are frequently

involved in the cleavage of bonds, includin aromatic rings. Cyclic peroxides are likely to be intermediates (Figure 2.22). Oxidative cleavage of aromatic rings typically employs catechol (1,2-dihydroxy) or quinol (1,4-dihydroxy) substrates, and in the case of catechols, cleavage may be between or adjacent to the two hydroxyls, giving products containing aldehyde and/or carboxylic acid functionalities (Figure 2.22).

Some dioxygenases utilize two acceptor substrates and incorporate one oxygen atom into each. Thus, 2-oxoglutarate-dependent dioxygenases

hydroxylate one substrate, whilst also transforming 2-oxoglutarate into succinate with the release of CO2 (Figure 2.23).

Amine OxidasesIn addition to the oxidizing enzymes outlined above, those which

transform an amine into an aldehyde, the amine oxidases, are frequently involved in metabolic pathways. These include monoamine oxidases and diamine oxidases.

Monoamine oxidases utilize a flavin nucleotide,typically FAD, and molecular oxygen, and involve initial dehydrogenation to an imine, followed by hydrolysis to the to an imine, followed by hydrolysis to the aldehyde and ammonia(Figure 2.24).

Baeyer–Villiger Oxidationsthe Baeyer–Villiger oxidation, yields an ester. For comparable ketone →

ester conversions known to occur in biochemistry, cytochrome-P-450- or FAD-dependent enzymes requiring NADPH and O2 appear to be involved. This leads to formation of a peroxy–enzyme complex and a mechanism similar to that for the chemical Baeyer–Villiger oxidation may thus operate. The oxygen atom introduced thus originates from O2.

Phenolic Oxidative CouplingMany natural products are produced by the coupling of two or more phenolic

systems, in a process readily rationalized by means of free radical reactions. The reactions can be broughtabout by oxidase enzymes, including peroxidase and laccase(漆酶） systems, known to be radical generators.

Glycosylation ReactionsThe widespread occurrence of glycosides and polysaccharides requires

processes for attachingsugar units to a suitable atom of an aglycone to give a glycoside, or to another sugar giving a polysaccharide. Linkages tend to be through oxygen, although they are not restricted to oxygen, since S -, N -, and C-glycosides are well known.

The agent for glycosylation is a uridine diphosphosugar, e.g. UDP glucose.