a short introduction to chemical biology and medicinal...
TRANSCRIPT
A Short Introduction to Chemical Biology and Medicinal ChemistryPart II Ben Davis [[email protected]] – 3 Lectures - Enzymes and Their Uses
Prior Knowledge Required
1. 1st year Biological Chemistry course: enzyme catalysis principles; Michaelis-Menten kinetics basicsof protein structure; rough idea of transcription/translation/DNA→mRNA→protein; one letter andthree letter codes for amino acids
2. 1st year Stereochemistry: resolution techniques3. 2nd year Organic Reaction Mechanisms II: enzyme mechanism aspects
Books:
“Enzyme Structure and Mechanism” Fersht;“An Introduction to Biotransformations in Organic Chemistry” Hanson;“Biotransformations in Organic Chemistry” Faber;“Biochemistry and Molecular Biology” Elliott & Elliott OCP 98 Foundations of Chemical Biology OCP 99 Carbohydrate Chemistry
Topics to be Covered
� Enzymes in Synthesis - prejudices; advantages & disadvantages; Enzyme Classifications andReactions;
� Serine Hydrolases are Really Acyl Transferases; Common Mechanisms and Diverse Mechanisms e.g.Serine Proteases, Metalloproteases & Carboxyproteases; Ribosomal peptide synthesis;
� Acyl transferases in synthesis; regioselective transformations; stereoselective transformations;resolution techniques; desymmetrization;
� Peptide ligation; protein ligation (enzymatic & native chemical); inteins.� Carbohydrate Processing Enzymes: Glycosidases and Glycosyltransferases� Protein Engineering; techniques and results; mutagenesis & chemical modification; introduction of
non-coded amino acids into proteins; examining the effects; Creating new catalysts: de novo enzymes,polyamino acid catalysts, engineered enzymes, catalytic antibodies; novel subtilisins.
� Sample Exam Questions
Enzyme classificationEnzyme commission 1955 (IUPAC)EC 1.2.3.4~20,000? existOnline directory http://www.expasy.ch/enzyme/
NumberClass Reaction Type
Classified Available
Usage
1. Oxidoreductases Redox: C-H, C-C, C=C oxygenation;(de)hydrogenases
~1000 ~100 25%
2. Transferases Transfer acyl, sugar, phosphoryl, methyl ~1000 ~100 10%3. Hydrolases Hydolyse/form esters, amides, lactones, lactams,
epoxides, nitriles, anhydrides, glycosides~1000 ~300 55%
4. Lyases Addition/elimination to C=X (X = C, N, O) ~300 ~50 5%5. Isomerases Racemization, epimerization ~150 ~10 3%6. Ligases Formation/cleavage of C-X (O, S, N, C) ~100 ~10 2%
Serine Hydrolases
O
O H N N H O
ASPHIS
SER
NH
R'O
O
O N N O
ASPHIS
SER
O
H
NH
O
O H N N
ASP
HIS
HO
H
O
O N N HO
ASP
HIS
SER
O
OH
RR'
R
ES TI-1
H
H
N
N
Oxy-anion hole
OSER
O
R
H
H
N
N
Oxy-anion hole
O
O H N N
ASPHIS
HNH
R'
OSER
O
R
H
H
N
N
Oxy-anion hole
EA EA
O
O N N O
ASP
HIS
SER
O
H
OH
R
TI-2
H
H
N
N
Oxy-anion hole
EPR
H
HH
EC 1.2.3.4
class
subclass(substrate or bond cleaved)
co-substrate
individual enzyme number
Metallo/Aspartylproteases
O
O
ASP
NH
R'O
R
HO
H
Zn++
O
O
ASP
NH
R'
O
R
HO
Zn++
H
O
O
ASP
O
R
HO
Zn++
NH2R'
EPTIES
O
O
ASP
NH
R'
O
R
HO
H
O
O
ASP
NH
R'O
R
HO
H
O
O
ASP
O
R
HO
NH2R'
EPTIES
O
O
ASP
H
O
O
ASP
H
O
O
ASP
H
NNH
N
NH
N
HN
The Ribosome
O
O
pep
tRNAA
NH
HN
N
N N
H2N
OO
OH
O
tRNAA
NH
NN
N N
H2N
OO
OH
O
H
O
O
pep
NN
N N
H2N
OO
OH
O
tRNAP tRNAP
tRNAA
NH
OH
O
peptRNAP
NN
N N
N
OO
OH
ON
NH
N
NO
O
OHO
O
H
H
NH
H
PO O
tRNAA
NH
H
O
O
OAc OAc
N3
α-CT PPL
H2O H2OO
O
OH OAc
N3
O
O
OAc OH
N3
OO O
OH
OMe
OH
HOOHOH
HOHO tBuOH O
O O
O
OMe
OH
HOOHOH
HOHO
O
O CCl3
O
PPL
N
OHHO OH
HO N
OH
HO OH
OO
O
CCl3
α-CT
pyridine
O
N
O
HO OH
OO
O
O
PPL, THF
O
N
O
HO OH
HO
O
α-CT
H2O
N
OH
HO OH
OOPLE
H2O
O
OH
HO
HO
OH
OOO
H
R
O
OH
HO
OH
OH
OR'
OO
O
OH
HO
HO
OH
OO
OO
H
OR'
OO
H
OO
O
OH
HO
HO
OH
OO
OO
H OR'
O
OH
HO
HO
OH
OOO
H
R
O
OH
HO
OH
OH
OR'
OO
H
OR'
O
OH
HO
HO
OH
HO
OO
R
HO
O
OR'
OO
H
OO
GLYCOSIDASES
O
OR+ H2O
O
OHROH+
Naturally, they catalyze the hydrolysis of the glycosidic bond, i.e., the split glycosides
To make glycosidic bonds i.e., to make there are TWO tactics:O
OR
1. Reverse the Equilibrium
O
OR+ H2O
O
OHROH+
remove this to drive equilibrium overto the left
lots of this to drive towards other side
2. Transglycosylation - i.e., We swop one group at the anomeric centre R' for another group R
O
OR'
O O
O
OH
O
OR
glycosyl cation enzyme-bound intermediate
H2O
ROHThis OR just acts as a leaving group as for all glycosidation reactionse.g, para-nitrophenol are good ones here
this OR' group is the one we want e.g., another monosaccharide
Prevent formation of this by trying to exclude H2O
N.B. This is also the mechanism for the equilibrium shown above at the top and in 1.
EXAMPLES OF GLYCOSIDASE REACTIONS
O
OHHO
HOHO
β-galactosidase O
OHHO
HOHO OHH2O
O
OH
OHO
HOOR
β
galactose
O
OH
HOHO
HOOR
doesn't touch this one because α-glucose
O
OHHO
HOHO
O
OH
OHO
HOOR
NHBoc
COOHHO
β-galactosidaseO
OHHO
HOHO
O NHBoc
COOH
Transglycosylation
Hydrolysis
O
OHHO
HOHO NHBoc
COOHHO
β-galactosidase
L-serine derivative
O
OHHO
HOHO
O NHBoc
COOH
Equilibrium Reversal
OH
Because normally it only cleaves β - when we reverse things it only makes β
O
OP
- O O
O-
O
OP
- O O
O
GLY
CO
SY
LTR
AN
SF
ER
AS
ES
1 or
2O
B
OH
HO
Su
gar
Nu
cleo
tid
e
O
OH
HO H
OH
O
Gly
cosy
l Acc
epto
r
Gly
cosy
ltra
nsf
eras
e
OO
OH
O HO
HO
Gly
cosi
de
- OP
- O O
O 1 or
2O
B
OH
HO
Nu
cleo
tid
e
B =
the
appr
opria
te
pyrim
idin
e or
pur
ine
base
(t
his
depe
nds
on th
e G
ly-T
Lelo
ir T
ype
Non
-Lel
oir
Typ
eG
lyco
syl d
onor
is
a S
ugar
Pho
spha
te in
stea
d an
d so
the
leav
ing
grou
p is
just
pho
spha
te io
n
glyc
osyl
don
or
Su
gar
Ph
osp
hat
e
- OP
- O O
O-
Ph
osp
hat
e
Gly
cosy
l Acc
epto
rG
lyco
sid
e
Gly
cosy
ltra
nsf
eras
e
O
OP O
OO
NP O
OO
OH
OH
HO
HO
NH
O
O
OO
OH
OH OH
HO
H
B
OO
OH
OH O
H
HO
O
OH
HO
OH
OH
OP O
OO
NP O
OO
NH
O
O
+
α
β
man
nose
β(1
,4)
gala
ctos
yltr
ansf
eras
e
Res
idue
in a
ctiv
e si
te th
at
acts
as
a ge
nera
l bas
e
Mn2+
A M
n2+ io
n in
the
activ
e si
te a
ids
the
loss
of t
he
leav
ing
grou
p
O
HO
OH
O
HO
OH
O
OP
- O O
O-
O
OP
- O O
O
GLY
CO
SY
LTR
AN
SF
ER
AS
ES
1 or
2O
B
OH
HO
Su
gar
Nu
cleo
tid
e
O
OH
HO H
OH
O
Gly
cosy
l Acc
epto
r
Gly
cosy
ltra
nsf
eras
e
OO
OH
O HO
HO
Gly
cosi
de
- OP
- O O
O 1 or
2O
B
OH
HO
Nu
cleo
tid
e
B =
the
appr
opria
te
pyrim
idin
e or
pur
ine
base
(t
his
depe
nds
on th
e G
ly-T
Lelo
ir T
ype
Non
-Lel
oir
Typ
eG
lyco
syl d
onor
is
a S
ugar
Pho
spha
te in
stea
d an
d so
the
leav
ing
grou
p is
just
pho
spha
te io
n
glyc
osyl
don
or
Su
gar
Ph
osp
hat
e
- OP
- O O
O-
Ph
osp
hat
e
Gly
cosy
l Acc
epto
rG
lyco
sid
e
Gly
cosy
ltra
nsf
eras
e
O
OP O
OO
NP O
OO
OH
OH
HO
HO
NH
O
O
OO
OH
OH OH
HO
H
B
OO
OH
OH O
H
HO
O
OH
HO
OH
OH
OP O
OO
NP O
OO
NH
O
O
+
α
β
man
nose
β(1
,4)
gala
ctos
yltr
ansf
eras
e
Res
idue
in a
ctiv
e si
te th
at
acts
as
a ge
nera
l bas
e
Mn2+
A M
n2+ io
n in
the
activ
e si
te a
ids
the
loss
of t
he
leav
ing
grou
p
O
HO
OH
O
HO
OH
