o Uso Antigo - Moodle USP: e-Disciplinas · o Uso Antigo o Variedade Estrutural o Atividade...
Transcript of o Uso Antigo - Moodle USP: e-Disciplinas · o Uso Antigo o Variedade Estrutural o Atividade...
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
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)