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From nitriles to nitrogen heterocycles; chemoenzymatic approaches toward diverselysubstituted enantiopure building blocks

Vink, M.K.S.

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Citation for published version (APA):Vink, M. K. S. (2003). From nitriles to nitrogen heterocycles; chemoenzymatic approaches toward diverselysubstituted enantiopure building blocks.

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Download date: 13 Jun 2020

CHAPTERR 1

BIOSYNTHESISS OF NITRILES

1.11 General Introduction

Thiss thesis details novel approaches toward diversely functionalized enantiopure nitrogen

heterocycless through a combination of biocatalytic conversions of nitriles and N-acyliminium

ionn chemistry. Two types of enzyme systems have been applied: nitrile hydrolyzing enzymes

andd a hydroxynitrile lyase. This chapter wil l give a short overview on the biosynthesis of

nitriless and their presence in nature. In addition, the purpose and outline of the investigation

aree presented.

Naturallyy occurring nitriles1 comprise a diverse set of secondary metabolites in plants,

arthropods,, bacteria and fungi. They may serve as a natural defense against pathogens and

predators.. For instance, ricinine (1, figure 1) is a toxic alkaloid which occurs in the seeds and

leavess of the castor plant, Ricinus communis.2

Figuree 1 OMe e

„CN N

NN ^O Me e 1 1

CN N OH H

HO O

CN N

Onn the other hand, indole-3-acetonitrile (2) occurs in a number of Cruciferae as a precursor

forr indole-3-acetic acid, a plant growth hormone.3 The most extensively occurring class of

nitrile-containingg natural products comprises cyanogenic glycosides (O-glycosylated

cyanohydrins)) such as prunasin (3).4

Schem ee 1

C02H H

" ~ - ^ N H 2 2

4 4

R^-C N N

glucose e

" - ^ N O H H

5 5

atdoxime e dehydratase e

R-^,CN N

< < " v / /

,„glucose e

Wrosof f 6 6

R^ /CN N

7a a 7b b

Whereass the cyano function in ricinine is derived from the carboxylic acid via the amide,

mostt naturally occurring nitriles are believed to originate from amino acids (viz. 4, scheme 1)

1 1

BiosyntiiesisBiosyntiiesis ofNitriles

viavia an aldoxime (5).1 These may be proteinogenic amino acids (e.g. valine, isoleucine,

methionine,, phenylalanine or tryptophan), but also cyclopent-2-enylglycine or nicotinic acid.

Thee biotransformation of amino acids into nitriles can be divided into two pathways. The

firstt pathway involves formation of glucosinolates (6)5 and the second pathway leads to the

biosynthesiss of cyanogenic glycosides (8).6 These two routes generally involve different

aminoo acids and metabolic enzymes, but both compound classes serve as agents in the

naturall defense mechanism.

1.22 Glucosinolates

Thee first glucosinolates were isolated from mustard seeds early in the 1830s, and are

thereforee sometimes referred to as mustard oils. These compounds are present in

dicotyledonouss angiosperms, which include cabbage, broccoli and mustard plants.

Glucosinolatess have attracted much attention due to their high levels of toxicity, and they are

knownn to cause acute and chronic diseases. On the other hand, several glucosinolates and

theirr degradation products have proven to prevent gastric cancer.7 Several examples of

naturall glucosinolates are depicted in figure 2.

Figuree 2 OH H

« i ö b ^ ^ NN «-M'H 0 O 3 SO' NN e 0 3 SO' N e 0 3 S O ' N ^ H

99 10 11

Singrinn (9) was one of the first glucosinolates isolated form black mustard seeds, and it is one

off the major glucosinolates in cabbage leaves. One of the degradation products of

glucoraphaninn (10) has shown high bacteriostatic activity against Heliobacter pylori strains,

thee bacteria that are responsible for gastric infections that lead to ulcers and may even lead to

gastricc cancer.8 Glucobrassicin 11 is an intermediate in the biosynthesis of indole-3-acetic

acidd (4).

Thee first step in the biosynthesis of glucosinolates is the N-hydroxylation of an amino acid to

thee N-hydroxyamino acid (12, scheme 2). The amino acids that have been identified as

naturall starting materials for glucosinolates are alanine, methionine, valine, leucine,

isoleucine,, phenylalanine, tyrosine and tryptophan. Most of the time, the side chains of these

aminoo acids are elongated before N-hydroxylation takes place, allowing a wider variety of

glucosinolatess to be formed. N-hydroxylation is catalyzed by monooxygenase enzymes,

requiringg 02 and NADPH.

2 2

ChapterChapter 1

Schemee 2

C0 2H H

V NH 2 2 — 7 7 NADPH+O2 2

C0 2 H H

N N H H 7-^ 7-^

NADPH+02 2

C0 2 H H

OH H 13 3

C0 2 +H 2 0 0

"NOH H

Althoughh most reports designate cytochrome P450 monooxygenase enzymes to catalyze this

process,99 evidence has been presented that also non-heme flavin dependent

monooxygenases100 or peroxidases11 may be involved in the hydroxylation of the amino

moiety.. In principle, each type of amino acid is converted by a different enzyme, since these

biocatalystss are highly selective. In the same step and catalyzed by the same enzymes,

oxidativee decarboxylation generates the aldoxime (5) via the N,N-dihydroxyamino acid (13).

Furtherr transformation of the aldoxime to the glucosinolate is a multi-step process, involving

variouss intermediates and enzymes that are not completely undisputed. The conversions are

believedd to follow the route depicted in scheme 3.5

Schemee 3

vNOH H

,glucose e

,osof f

P450 0

PAPS S

„ ,© . . © , 0 0

14a a

OH H

14b b

,, glucose

, 0 H H

17 7

Cys s

UDPG G

H J N ^ C O J H H

.OH H

15 5

SH H

, 0 H H

16 6

First,, another cytochrome P450 monooxygenase12 catalyzed oxidation of the aldoxime (5) to a

nitril ee oxide (14a) or an ari-nitro compound (14b) occurs. This intermediate has not been

full yy identified, but it is believed to act as the acceptor for an S-donor, which in most cases

appearss to be a cystein residue, generating an S-alkylthiohydroximate (15). Cleavage of the

aminoo acid residue by p-elimination generates the thiohydroximate (16) that upon reaction

withh uridine diphosphate glucose (UDPG) forms a desulfoglucosinolate (17). This reaction is

catalyzedd by thiohydroximate glucosyltransferase enzymes, which are highly selective for

thiohydroximates,, but display a broad tolerance toward both aliphatic and aromatic side

chains.. The final step in the formation of the glucone moiety, is sulfonation of the

desulfoglucosinolatee with 3'-phosphoadenosine-5'-phosphosulfate (PAPS), catalyzed by a

desulfoglucosinolatee sulfotransferase.

3 3

BiosynthesisBiosynthesis of Nitrites

Besidess side chain elongation at the start of the biosynthesis of glucosinolates, side chain

modificationn contributes to the differentiation from the original amino acids. This allows that

overr 120 different glucosinolates have been identified.50 Side chain modification includes

oxidation,, desaturation, hydroxylation and methoxylation.

Althoughh glucosinolates are stable, water-soluble compounds, upon tissue disruption they

comee in contact with myrosinase13 which hydrolyzes the glucosinolates to isothiocyanates,

thiocyanatess or nitriles (scheme 4).14 This process serves as a defense mechanism against

externall attacks.

Schemee 4

^glucose e

RAN,osof f myrosinase e SH H

F A - 0 5 0 0 .6 6

R-N=C=S S

-<< R-S-C5N

R-C=N N

Whichh of these degradation products are formed depends on the pH, temperature, additives

andd the presence of additional enzymes. Isothiocyanate formation, via a Lossen

rearrangementt (equation 1), is the most readily understood degradation pathway of a

hydrolyzedd glucosinolate, and it is known that this rearrangement predominantly takes

placee at pH values above 5.

Equationn 1

R ^ V 5 S 3 3 R-N=C=S S

Thee mechanism of thiocyanate formation is unknown, and only three glucosinolates appear

too form these degradation products. It is proposed that the rearrangement already takes

placee before myrosinase hydrolysis via a non-enzymatic process.

Schemee 5

acidicc pH

'' e S+HSO4 4

?HH © R A N , oso 3 3

Fe2++ or cysteine e

H2S+HS04 4

R-CSN N

Althoughh the exact mechanism is not entirely clear, degradation of glucosinolates to nitriles

iss thought to be an acid induced process (scheme 5). Furthermore, this pathway can even

havee a significant contribution at pH 7.4, when promoted by the presence of Fe2+ or

cysteine.15 5

4 4

ChapterChapter 1

1.33 Cyanogenic Glycosides

Cyanogenicc glycosides1-6 occur in over 2500 plant species, but also in fungi, bacteria and a

numberr of animals. Just as glucosinolates, cyanogenic glycosides are derived from amino

acids,, which are limited to phenylalanine, tyrosine, valine, isoleucine, cyclopent-2-

enylglycinee and nicotinic acid. These amino acids are, analogous to the biosynthesis of

glucosinolates,, first transformed into the aldoxime via the N-hydroxyamino acid (vide supra).

Thiss conversion is, in this pathway, exclusively performed by a cytochrome P450

monooxygenase166 and no evidence has been found that other monooxygenase enzymes may

bee involved.

Afterr aldoxime formation, the biosynthesis of cyanogenic glycosides deviates from that of

glucosinolates.. This time, the aldoxime is dehydrated to form the corresponding nitrile. This

conversionn can be catalyzed by another cytochrome P450, or by an aldoxime dehydratase.17

Dehydrationn of the aldoxime by the aldoxime dehydratase is not a part of the cyanogenic

glycosidee pathway, since the nitrile is subsequently hydrolyzed by nitrile hydrolyzing

enzymess (see chapter 2). The nitrile that is produced by the cytochrome P450 is successively

oxidizedd by the same enzyme to the corresponding cyanohydrin (19, scheme 6). The last step

iss glycosylation of the hydroxyl, a conversion that is catalyzed by a p-glucosyltransferase.18

Schemee 6

P450 0 R ^ C N N

Cyanogenicc glycosides serve their purpose in the defense mechanism of their host against

externall attack by predators. Upon tissue disruption, the cyanogenic glycoside releases the

cyanohydrinn which in turn easily degrades to the aldehyde and the highly toxic HCN

(schemee 7).

o o III +HCN

R-^H H

Thiss process is enzyme mediated, and the responsible enzymes are hydroxynirrile lyases (see chapterr 4).

.. 9'UTSy' „.glucose OHH transferase O TT i

RR CN R ^ C N 199 8

Schemee 7 ^ g l u c o s ee Q H

II — T RR CN R " ^ N

5 5

BiosynthesisBiosynthesis ofNitriles

1.44 Other Nitrile Containing Natural Products

Somee other classes of nitril e containing natural products are cyanolipids (esters of

hydroxynitriles),, a- and f3-amino nitriles (e.g. saframycin, figure 3) a,p-unsaturated nitriles

(e.g.(e.g. ehretioside), aromatic nitriles (e.g. mycalisine A) and calyculins (e.g. calyculin A).1

Figuree 3

MeO O

saframycin n OMe e

"glucose e

ehretiosidee A 1 : R1 = OH, R2 = 02CCH=C(CH3)2, R3 = OH

ehretiosidee A2: R1 = OH, R2 = OH, R3 = 02CCH=C(CH3)2

ehretiosidee A3: R1 = 02CCH=C(CH3)2, R2 = OH, R3 = OH

OHH O

N H , , CN N

NN N '

* * # # MeO^^ 'OH

mycalisinee A

OHH OH OMe calyculinn A

a-Aminoo nitriles are widely present in nature and often show antibiotic and antitumor

activity.. (J-Amino nitriles are less common than a-amino nitriles, and some of these

compoundss also possess antibiotic activity. The ehretiosides were isolated from the stem

barkk of Ehretia philipinensis that is used as an anti-inflammatory remedy by inhabitants of the

Phillipines.. Mycalisine A was isolated from a marine sponge and inhibits the cell division of

fertilizedd starfish eggs. The most biologically relevant unsaturated nitriles are the calyculins,

whichh are isolated from marine sponges. They are potent and selective inhibitors of protein

phosphatasess and exhibit antitumor activity.

1.55 Purpose and Outline of the Investigation

Inn this project, we were mainly interested in the application of the nitrile group in the

preparationn of novel, synthetically versatile heterocyclic compounds. During this study, we

combinedd biological transformations with synthetic organic chemistry, a combination that

provedd to be quite powerful.

6 6

ChapterChapter 1

Chapterr 2 details the enzymatic hydrolysis of nitriles. After a short overview of the

applicationn of nitrile hydrolyzing enzymes in organic synthesis, the results that have been

obtainedd with the Rhodococcus erythropolis NCIMB 11540 bacterium wil l be shown.

Inn chapter 3 the first follow-up chemistry is presented, showing the preparation and

ff unctionalization of 4-hydroxypiperidin-2-ones.

Thee application of a hydroxynitrile lyase is demonstrated in chapter 4, as well as the further

transformationn of the obtained product to substituted 5-hydroxypiperidin-2-ones.

Additionally,, a novel chemical transformation is presented, leading to bicyclic JV,N-acetals.

Chapterr 5 describes the synthesis of morpholinones from cyanohydrins.

Inn chapter 6, the chemistry described in chapters 3 and 4 is applied in the (partial)

preparationn of two natural compounds, i.e. febrifugine and pseudoconhydrine.

1.66 References

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8 8