o Uso Antigo

o Variedade Estrutural

o Atividade Biológica

(Kutchan T. Plant Cell 7, p.1059, 1995)

• They are found mostly in plants

• Naturally-occurring compounds that contain nitrogen

• Many have heterocyclic rings as a part of their structure

• Many are physiologically active (often spectacularly)

• Many are used by native peoples for religious or medicinal

purposes.

• Many are basic (“alkaline”, due to an unshared pair on N)

• Those nitrogen compounds that are found in all organisms

(i.e., amino acids, nucleic acids, etc.) are not considered

alkaloids.

• Alkaloids are “secondary metabolites”, they are not

involved in primary metabolism.

HO

NMe

O

HO

morfina(sedativo)

US$ 340/g

MORPHINE

O

N CH3

OHMeO

..

contains nitrogen basic due to the

unshared pair

heterocyclic ring

Found only in the Opium Poppy - papaver somniferum

Plant source.

Most alkaloids

are found in

plants.

….. not ubiquitous.

This was the first

alkaloid discovered

(1804, Serturner).

A heroína é um

derivado Sintético

da morfina, portanto

não é considerada

como um

“Alcalóide”.

HOW ARE ALKALOIDS CLASSIFIED ?

Common classification schemes use either:

• The heterocyclic ring systems found as a

part of the compound’s structure

• The plant or plant family where they originate*

* The majority of alkaloids (>90%) are found in plants -

therefore, we will speak mostly about plants and their

biochemistry.

HETEROCYCLIC RING SYSTEMS LEARN THESE RINGS (plus the ones on the next page)

N

H

N

H

pyrrolidine pyrrole

N

H

N

piperidine pyridine

NN N

H

quinoline isoquinoline indole

N

H

dihydroindole

HETEROCYCLIC RING SYSTEMS LEARN THESE ALSO (cont)

N N

NH

N

quinolizidine pyrrolizidine tropane

benzylisoquinoline

N

N N

N

H

purine

C C N

-phenylethylamine

Some Examples of Classification

NH

N

OMe

OMeMeO

MeO

emetine

N

ON

CH3

CH3

CH3PO OH

O

H

psilocybin

-

+

N

N

CH3

H

nicotine

BY RING TYPE

N

N N

N

O

O

CH3

CH3

CH3

caffeine

Some Examples of Classification BY PLANT FAMILY :

N

O

O

OH

OH

N

H

MeO

MeO

MeO

O

OH

N CH3

MeOO

O

N CH3

O

H

H

OH

MeO

These alkaloids are found in Amaryllidaceae

daffodils

narcissus

lillies

etc

belladine

lycorine

tazettine

galanthamine

The other three

are biochemically

derived from

belladine.

“Amaryllis” Alkaloids

Some Examples of Classification BY PLANT OF ORIGIN

“Cinchona” Alkaloids

“Opium” Alkaloids

N

N

MeO

OH

O

N CH3

OHMeO

quinine

morphine

putrescina

N

Me

HMeN

H

O

NH2H2N

L-ornitina

CO2H NH2H2N

cadaverina

N

Me

HMeN

H

O

NH2H2N

L-lisina

NH2CO2HH2N

NICOTINE BIOSYNTHESIS >300 Feeding experiments in literature

Starts from L-ornithine

Symmetrical putrescine

Retention of

configuration

HNH

2NH

2

CO2H

HNH

2NH

2

HOrnithineDecarboxylase

HNH

2NH

H

CH3

NextSlide

N-methylation Putrescine N-Methyltransferase

NICOTINE BIOSYNTHESIS:

FORMATION OF PYRROLINIUM ION

Transamination with pyridoxal 5-phosphate

Cyclisation to pyrrolinium ion

Both mediated by N-Methylputrescine oxidase

“Half” of nicotine molecule is accounted for

NH

CH3

HH

HONH

CH3

HH

HNH

2

(from previous slide)

N+

CH3

HH

H

N

OH

O

H

H

HH+

N

R

HO

NH2

N

OH

O

NADPH

NICOTINE BIOSYNTHESIS:

REDUCTION OF NICOTINIC ACID

Nicotinic acid is reduced by NADPH

Dihydronicotinic acid: Zwitterion

NICOTINE BIOSYNTHESIS:

NICOTINIC ACID DECARBOXYLATION

Zwitterion of dihydronicotinic acid

N+

O

O

H

H

H

H

N

OH

O

H

Frompreviousslide

Loss of CO2 1,2-Dihydropyridine

NH

H

H

H

-CO2

N

N

CH3

H

H

H

NICOTINE BIOSYNTHESIS:

CARBON FRAMEWORK FORMATION

Combination of 1,2-dihydropyridine

And previously formed pyrrolinium ion

Addition occurs from rear face of ion

Basic carbon framework of nicotine

N

H

N+

CH3

HH

H

NICOTINE BIOSYNTHESIS:

COMPLETION OF BIOSYNTHESIS

Oxidation of dihydropyridine (NADP+)

i.e. Regeneration of NADPH

Completes biosynthesis of (S)-Nicotine

N

N

CH3

H

H

HN

N

CH3

H

NADP+

NICOTINE

Anti-herbivory

Causes addiction

Decreases appetite

Induced upon

wounding

Expensive to make

NICOTINE AND HERBIVORY N

NCH

3

H

NICOTINE AND HERBIVORY

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

N

NCH

3

H

ALCALÓIDES

PIRROLIZIDÍNICOS

Senecio spp

Invasoras de pastagens

Evitada pelos

mamíferos

Toxinas Hepáticas

PYRROLIZIDINE ALKALOIDS

NH2

NH2

NH2

NH2

NH2

NH

NH2 N

CH2OH

COOH

COOH

isoleucine

putrescine Homospermidine

trachelanthamidine

Senecionine N-oxide

(transport form)

N

O

OOH

OO

O

SYNTHETIC COMPARTMENTALIZATION

Root = Core Structure

Shoot = Modification

N

O

OOH

OO

O

N

O

OOH

OO

O

N

O

OOH

OO

O

N

O

OOH

OO

O

PYRROLIZIDINE ALKALOIDS OCCUR IN

MIXTURES.

N

O

OOH

OO

N

O

OOH

OO

OH

N

O

OOH

OO

O

N

O

OOH

OO

N

O

OOH

OO

O

Ácido Cáprico (C8)

g-coniceina Alanina

Brugmansia

ALCALÓIDES TROPÂNICOS

EXEMPLOS DE ALCALÓIDES

TROPÂNICOS

NH2

NH2

N Me

CH2

H

SCoA

O

N Me SCoA

O

*

*CO2H

NH2

NH2

*

ornitina [5-14C]

N Me

O

O

SCoA N Me

O

O

SCoA

OH

hidroxilase

N Me

O

O

SCoAH

NMe O

O

SCoA

NMe O

O

OMeN

Me O

OMe

OCOPh

NH2

NH2

COOH

NH2

COOH

CHO

N

COOH

HNH

COOH

CH2 C

CH3

O

N

CH2 C

CH3

O

N O

N

H

OH

CH3

CH3

CCH

2

O

C O

SCoA

ornithine

malonyl CoA

CH3

C OS

CoA

+

acetyl CoA

x2

NADH SAM

H2O -CO2

-CoASH

amine

oxidase

Mannich-1 (from glycolysis)

tropine

BIOSYNTHETIC PATH TO TROPINE

Mannich-2

BIOSYNTHETIC PATH TO (-)-TROPIC ACID

CH2

C

H

NH2

COOH

phenylalanine

CH2

C COOH

O

CH C COOH

COOH

O

CH C H

COOH

O

CH CH2

OH

COOH

(-)-tropic acid

NADH

TPP

-CO2

Biotin-COOH

PLP

B6

+

N

H

OH

CH3

(-)-hyoscyamine

tropine

ALCALÓIDES DERIVADOS DE

AMINOÁCIDOS AROMÁTICOS

Figure 1- Diversion of chorismic acid into the secondary metabolism by the enzymes anthranilate synthase (AS), chorismate mutase and isochorismate synthse (ICS)

L -phenylalanineL- tyrosine

L-tryptophan

anthranilic acid

isochorismic acid

prephenic acid

chorismic acid

o -succynylbenzoic acid

ASICSCM

indole acetic acidindole alkylaminescarbolinesindole alkaloids

phylloquinoneanthraquinones

lignin precursorsflavonoidscoumarinsanthocyanins

isoquinoline alkaloidsbetalaines

CO2H

OH

O CO2H

H2OC

OH

CO2H

OCO2H

NH2CO2H

OH

O CO2H

OH

NH

CCO2H

NH2

CNH2

CO2H

CNH2

CO2H

CO2H

CO2H

O

tirosina

transaminase

fenolase

H

HO

O

NH2HO

HO

TYDC

NH2HO

OH

O

HOO

L-tirosinaOH

O

NH2HO

ALCALÓIDES

BENZILISOQUINOLÍNICOS

O H

lofocerina

(Amaryllidaceae)

HO

HO

NH

NH2HO

HO

Mannichsintase

(S)-norcoclaurine

HO

HO

NH

HO

H

HO

O

NH2HO

HO

Mannich

6-OMT

(S)-reticulina

HO

MeO

NMe

MeO

HO

HO

MeO

NMe

HO

HO

HO

MeO

NMe

HO

HO

MeO

NH

HO

(S)-norcoclaurine

HO

HO

NH

HO

(S)-coclaurine (S)-N-Methyl-coclaurine

NMe

H

MeO

HO

HO

MeO

(S)-reticuline

NMe

MeO

HO

HO

MeO

NMe

H

MeO

HO

HO

MeO

1,2-dehydroreticulinium cation (R)-reticuline

NADP+

NADPH

morphinansaporphineprotoberberinesbenzophenantridines

(R)-reticulina

HO

MeO

NMe

MeO

HO

HO

MeO

NMe

MeO

HO

HO

MeO

NMe

MeO

HO

(S)-reticulina

redutase

MeO

HO

HO

MeO

NMe

MeO

HO

MeO

NMe

OH

MeO

O

MeO

NMe

O

-2H.

MeO

MeO

NMe

HO

OH

MeO

MeO

NMe

O

HO

MeO

NMe

O

MeO

MeO

NMe

O

HO

HO

NMe

O

HO

tebaína codeína morfina

Curares

NMe

OHO

Me2N

O

OH

OMe

MeO

H

H

(+)-tubocurarina

NMe

OHO

MeN

O

OH

OMe

MeO

H

H

(-)-curina

NMe

OHO

MeN

O

OH

OMe

MeO

H

H

(+)-(R,R)-isocondrodendrina

SÍNTESE DE BERBERINA EM

COPTIS JAPONICA E

BERBERIS WILSONIAE - OS

INTERMEDIÁRIOS (RETICULINA

E SCOULERINA) MOVIMENTAM-

SE ENTRE O CITOPLASMA E

VESÍCULAS ESPECÍFICAS

ALTERAÇÃO DO LOCAL DE

BIOSSÍNTESE

Endress, 1994

INTERIOR

EXTERIOR

ALCALÓIDES INDÓLICOS

ALCALÓIDES INDÓLICOS - DERIVADOS DO

L-TRIPTOFANO (1/4 DO TOTAL)

Pscilocybe

(cogumelo alucinogênico) Banisteriopsis caapi,

Virola, Mimosa

e Piptadenia peregrina

5-metoxi-N,N-dimetiltriptamina R=OMe

bufotenina R=OH

N,N-dimetiltriptamina R=H

NNMe2

H

R

psilocina

NN Me

OH

H

Resina de ucuúba como

fonte de rapé alucinogênico

e veneno para flechas

NN

R1

H

R3R2

NN

R1

H

R3

R2

Figure 1- Diversion of chorismic acid into the secondary metabolism by the enzymes anthranilate synthase (AS), chorismate mutase and isochorismate synthse (ICS)

L -phenylalanineL- tyrosine

L-tryptophan

anthranilic acid

isochorismic acid

prephenic acid

chorismic acid

o -succynylbenzoic acid

ASICSCM

indole acetic acidindole alkylaminescarbolinesindole alkaloids

phylloquinoneanthraquinones

lignin precursorsflavonoidscoumarinsanthocyanins

isoquinoline alkaloidsbetalaines

CO2H

OH

O CO2H

H2OC

OH

CO2H

OCO2H

NH2CO2H

OH

O CO2H

OH

NH

CCO2H

NH2

CNH2

CO2H

CNH2

CO2H

CO2H

CO2H

O

Pathways to L-tryptophan

OH

O

CO2-

CO2-

chorismate

H2N

O

CO2-

NH3+

L-glutamine

O

CO2-

CO2-

NH3+

anthranilate

O

PPO

HOOH

OP

5-phosphoribosyl-pyrphosphate

O

HO

OH

OP

CO2-

N

H

N-phosphoribosyl-anthranilate

N-phosphoribosyl-anthranilate

O

HO

OH

OP

-O2C

NH

-O2C

NH

O

OP

OH

HO

1-(o-carboxyphenylamino)-deoxyribulose phosphate

N

OP

OH

HO

H

Indole glycerolphosphate

N

-O2C NH3

+

H

OP

O H

OH

-O2C NH3+

OH L-tryptophan

D-glyceraldehyde3-phosphate

L-serine

ß-CARBOLINAS SIMPLES

NH

NH2

COOH

O NH

N

COOH

H+

NH

NH

COOH

NH

N

DescarboxilaçãoOxidativa

Reação do tipoMannich

Formação da Base deSchiff

Redução

Oxidação

NH

NH

R/S

Eleagnina

NH

N

Harmana NH

N

H3CO

Harmina

ácido lisérgico(fungo, Claviceps purpurea)

(Ipomeae sp)

N

H

NHO2C

(Catharanthus roseus)

vinblastina R=Mevincristina R= CHO

estricnina(Strychnos sp)

N

OO

N

N

OHN

HHO2C

N

R

N

CO2H

OCOMe

HO

N

H

NHO2C

ácido lisérgico - LSD(fungo, Claviceps purpurea)

(Ipomeae sp)

Shikimate pathway

Mevalonate pathway

G10H

SSS

TDC

tryptamine

10-hydroxygeraniol

strictosidine

tryptophan

secologanin

geraniol

geranyl-diphosphate

NH

NH2

COOH

O

NH

NH

O

HOGlu

H

H

CHO

CH3OOC

HOGlu

H

O-P-P OH

OH

NH

NH2

CH3COO

OH

BIOSYNTHESIS

of ALKOLOIDS

strictosidine hemiacetal

dialdehyde

- Glucose

+ H2O

OH -H2O+

+

4,21-dehydrocorynantheine aldehydecarbinolamine

20,21-dehydrocorynantheine aldehyde

4,21-dehydrogeissoschizine cathenamine

NH

CH3OOC

NH

O

H OGlu

H

H NH

CH3OOC

NH

O

H

H

HOH

NH

CH3OOC

NHC

OH

HH

H

H

ONH

CH3OOC

N

OH

H

H

HNH

CH3OOC

N

OH

H

H

H

NH

CH3OOC

N

OH

H

H

NH

CH3OOC

N

OH

H

H

NH

CH3OOC

N

O

H CH3

H

OH

strictosidine hemiacetal

carbinolaminedialdehydeenol

cathenamine

carbinolamine

+H2O

- H2O

OH

- Glucose

+ H2ONH

CH3OOC

NH

O

HOGlu

H

H NH

CH3OOC

NH

O

H

H

H

OH

NH

CH3OOC

NHC

OH

HH

H

H

ONH

CH3OOC

NH

C

OH

H

HOH

H

NH

CH3OOC

N

OH

H

H

NH

CH3OOC

N

O

HCH3

H

H

NH

CH3OOC

N

O

HCH3

H

OH

carbinolaminestrictosidine

NH

NH2+

O

CH3OOC

H

HOGlu

H

NH

N

O

CH3OOC

H

H

O

H3COOC

OGlc

Tipo Corinante

NH

N

O

H3COOC

Ajmalicina

NH

N

COOCH3

Aquamicina

Próximo slide

Diferentes Esqueletos dos Indóis

Diferentes Esqueletos dos Indóis

Tipo Aspidosperma

NH

Tabersonina

N

COOCH3

Tipo Iboga

N

COOCH3

Catarantina

FORMAÇÃO DOS ALCALÓIDES

CATHARANTINA E TABERSONINA

strictosidine

H

O

CH3OOC

H

HOGlu

N

H

NH N

H

N

CH3OOC

OH

H

H

4,21-dehydrogeissoschizine

N

N

CH2OHCH3OOC

H

(+)stemmadenine

N

H

N

COOCH3

H

catharanthine tabersonine

H

O

CH3OOC

H

CH3

N

H

N

H

ajmalicine

N

H

N

COOCH3

H

BIOSSÍNTESE DO ALCALÓIDE VINDOLINA

N

H

N

COOCH3

H

N

H

N

COOCH3

H

HO N

H

N

COOCH3

H

CH3O

N

H

N

COOCH3

H

CH3OH OH

N

N

COOCH3

H

CH3OH OH

CH3

N

N

COOCH3

H

CH3OH OH

CH3

OH

N

N

COOCH3

H

CH3OH OH

CH3

OOCOCH3

DAT

D17H NMT

MTH

OMTT11OH

vindoline

desacetylvindolinedesacetoxyvindoline 11-methoxy-2,16-dehydro-

16-tabersonine

11-methoxytabersonine11-hydroxytabersoninetabersonine

BIOSSÍNTESE DOS ALCALÓIDES BIS-

INDÓLICOS

+

catharanthine vindoline

iminium

vinblastine

anhydrovinblastine

N

N

CH3

OCOCH3

OH

COOCH3

N

H

N

R OH

CH3O

N

N

CH3

OCOCH3CH3O

OH

COOCH3

N

H

N

R

N

N

CH3

OCOCH3CH3O

OH

COOCH3

N

H

N

R

N

N

CH3

OCOCH3CH3O

OH

COOCH3

N

H

N

R

H

R=COOCH3

R=COOCH3

R=COOCH3

R=COOCH3

vacúolo

Citoplasma

SG

H+

ADP + PiATP

PEROXIDASE

ajmalicina + H+ ajmalicina[H+]

ajmalicina[H+]

SSS

serpentina

ajmalicina + H+precursor geral

strictosidona

secologanina

TDC

triptamina

triptamina

triptofâno

ESQUEMA DA REGULAÇÃO DA BIOSSÍNTESE

DE ALCALÓIDES

NH

N

H

H

CHO

NH

N

CHO

H

NH2

CHO

N

O

H

N

N

O

H

N

N

HO

H

H3CO

Corinanteal

Cinchonaminal

Cinchonidinona

Quinina

Biossíntese da Quinina

ALCALÓIDES IMIDAZÓLICOS

Pilocarpus sp.

ALCALÓIDES IMIDAZÓLICOS

São mais raros, poucos

exemplos

Encontrados em Rutaceae

Pilocarpus spp. e Casimiroa

edulis (sementes)

Mammilaria, Dolichotele

sphaerica (Cactaceae)

Algumas Facáceaes

Reconhecidos como alcalóides do

Jaborandi

NH

N

Núcleo Imidazol

DOLICOTELINE

Único com

biossíntese definida

HN

N NH2

COOH

- CO2

HN

N NH2

HOOC

NH2

Histidina Histamina

- NH3

L-Leucina

HOOC

Ácido Isovalérico

HN

N

N

O

Dolicotelina

Dolichotele sphaerica

Biossíntese de alcalóides imidazólicos (pilocarpina)

• Poucos dados experimentais

• Relação estrutural com o aminoácido L-histidina

• Nenhuma referência a respeito das enzimas envolvidas

na formação dos alcalóides imidazólicos em espécies de

Pilocarpus

Duas propostas biogenéticas

R. ROBINSON, The Structural Relationship of Natural Products (1955)

E. BROCHMANN-HANSSEN et al., Planta Medica (1975)

Raízes Folhas

Biossíntese de pilocarpina em Pilocarpus spp.

R o b i n s o n , R . ( 1 9 5 5 ) T h e S t r u c t u r a l R e l a t i o n s o f N a t u r a l P r o d u c t s . O x f o r d P r e s s , L o n d o n .

O NH

N

O

O O NH

N

O

O

H

O NH

N

OH

O

O NH

N

OH

O

H

O NH

N

O

NH

N

HOCH2

O

O NH

NO

O

O

H

SCoA

O

O

CH3

S CO2H

NH2H

L-metionina (11)

FOLHAS

PENTOSE FOSFATO

N

NH

NH2 H

HO2C

L-histidina (8)

NH

N

CO2H

O

ácido imidazol-pirúvico (9)

O NH

N

O

pilocarpidina (10)

O NH

N

O

(10)

O N

N

O

CH3

(1)

RAÍZES

Robinson (1955)The Structural Relations of Natural Products. Oxford Press.

TRANSLOCAÇÃO

?

+

SAM

NH2

CO2H

OH

NH2

CO2H

O

CO2H

NH

N

OO

NH

N

CO2HHO

2C

NH

N

HO2C

OHN

N

CO2HHO

2C

OHH

CH3

S CO2H

NH2H

N

NH

NH2 H

HO2C

L-histidina (8)

NH

N

OO

pilocarpidina (10)

O NH

N

O

(10)

O N

N

O

CH3

(1)

RAÍZES

NH

N

CO2H -

Brochmann-Hanssen et al. Planta Medica(1975) 28, 1-5.

L-treonina

?

TRANSLOCAÇÃO

PENTOSE FOSFATO

SAM

FOLHAS

L-metionina (11)

ácido trans-urocânico (12)

Preparação do padrão ácido imidazol-pirúvico

NH

N

NH2

CO2H

H

NH

N

OH

CO2H

H

NH

N

CO2H

H N2+

(b)(a)

L-histidina ácido 2-hidróxi-imidazol-propanóico

(a) H2SO4 10%/ NaNO2; 2h (45-50oC) (b) Coluna de troca iônica eluente NH4OH/ H2O (3: 1)

Karanewsky et al, (1988) J Med Chem, 31, 204- 212

NH

NO

CO2H

NH

N

OH

CO2H

H

(c) KMnO4/ H2O/ 72h (TA)

Corson, et al. (1932) Org Synth Coll, 1, 241- 245

(c)

ácido imidazol-pirúvico

Rendimento: 56%

Rendimento: 47 %

1) Não foi verificada a formação de nenhum produto com tempo de

retenção igual ao do ácido trans-urocânico nos ensaios feitos com

extrato protéico de folhas e raízes

2) No ensaio para transferases apenas o extrato protéico das raízes

levou a formação de um produto com o mesmo tempo de retenção do

ácido imidazol-pirúvico

Cromatograma obtido para o ensaio enzimático de

aminotransferases com extrato enzimático de raízes

Atividade enzimática do homogenato: 0,530 nKat/ mL

Atividade enzimática específica: 46,09 nKat/ mg

0,07

0,09

0,11

0,13

0,15

0,17

0,19

0,21

4 5 6 7 8 9 10

pH

Airv e

nz (

nK

ata

l/ m

L)

Caracterização da enzima

• Determinação do pH ótimo

1. Acetato (pH= 4,6)

2. HEPES (pH= 6,7)

3. MES (pH= 7,7)

4. TRIS (pH= 8,5)

5. TRIS (pH= 10)

1

2

3

4

5

Caracterização da enzima

• Cinética de reação

0 5 10 15 20 25 300,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

ativ

enz

(nkat/

ml)

conc L-histidina (mM)

• Saturação enzimática

Caracterização da enzima

0 2 4 6 8 10 12 14

0,0

0,1

0,2

0,3

ati

v e

nz (

nK

at/

mL

)

conc de L-histidina ( M)

• Dependência do cofator PLP

Caracterização da enzima

0,000

0,015

0,030

1 2

sequência (1) s/ PLP

sequência (2) c/ PLP

ativ

en

z (n

Kat/ m

L)