The Organic Chemistry of

Enzyme-Catalyzed Reactions
Chapter 12
Formylations,
Hydroxymethylations, and
Methylations

Tetrahydrofolate-dependent Enzymes
Transfer of one-carbon units
H2N

H
N
5 6

HN
O

N
H

pKaofacid=4.8

5,6,7,8tetrahydro
pteridine

can be oligomer of up
to 12 Glu residues

N
H

H
C N CHCH2CH2COO

10

pKaofacidis1.25
pamino
benzoicacid

COO

glutamate

12.1

tetrahydrofolate
named as polyglutamate derivatives of
tetrahydrofolate (H4PteGlun) [Pte - pteroate]

pteroate
ring

H2N

HN
O

H
N

5
N
H
12.2

NHR'
10

abbreviated structure for tetrahydrofolate

Folic Acid
(a vitamin for humans)
H2N

HN

N
N

O
N
H
12.3

CNH

CHCH2CH2COO
COO

Reduction of Folate to Tetrahydrofolate

Reactions catalyzed by dihydrofolate reductase
(DHFR)
+B

H
H2N

HN

..
N
R

N
O

NH2

HN

H2N

H O

12.3

dihydrofolate
reductase

H
N

NH2

N
O

NHR'

H
B+

HN

O
NH2

N
NHR'

N+
R

dihydrofolate
H O

..
N
R

NHR'

H2N

H
N

dihydrofolate
reductase

H2N

H
N

NH2

HN
O

N
H

NHR'

N+
R

12.1

Scheme 12.1

Generation of the Transferring Carbon Unit

Serine hydroxymethyltransferase-catalyzed formation of formaldehyde via
a proposed -cleavage mechanism. The asterisk indicates the carbon unit
that becomes the one-carbon unit transferred in tetrahydrofolate-dependent
enzymes.
B:

Lys
HN+

:NH2
OH

=O PO
3

COO
H
*

SeeScheme
8.39

* H

H
COO

OH

OH

=O PO
3

..
N
H

-cleavage

+N
H

N+
H
12.4

HN+
H2N

COO

=O

OH

3PO

+N
H

=O

OH

3PO

+N
H

pro-S hydrogen
added/removed
(retention of
configuration) H

Lys

HN+
OH

=O PO
3

COO

HN+

*
O CH2

HN+

COO

=O

+B

COO Lys
:NH2
HN+
OH

3PO

+N
H

Ordered mechanism: no conversion of Ser to H2C=O

unless tetrahydrofolate is bound

COO

H
HN+

OH

=O PO
3

+N
H

Scheme 12.2

Transfer of One-Carbon Units

Transfer at the formaldehyde oxidation state
(transfer HOCH2- group)
Serine hydroxymethyltransferase-catalyzed reaction of
formaldehyde and tetrahydrofolate to methylenetetrahydrofolate
H2N

HN
O
*
O C
B+

H
N
5..
N
H

H2N

HN
10

+B

OH

H2N

H NHR'

B:

H
N

H2N

H
N

HN

+
N

HN

N
O

* CH2 : NHR'
B

12.6

*C
H2
H

H2N
HN
+
NR'
H
:B

N -methylene H4Pte
5

12.5

Scheme 12.3

H2O

O *CH2

NHR'

H
N

H
N

N
H +NR'
O
*C
H2
12.8

12.7

N ,N -methylene H4Pte
5

10

Keq = 3.2 x 104 in favor of 12.7

N10-methylene H4Pte

The one-carbon unit can be transferred in 3 oxidation states

Transfer at the Formate Oxidation State (transfer formyl group)

Oxidation of N5,N10-methylenetetrahydrofolate to N5,N10methenyltetrahydrofolate catalyzed by methylenetetrahydrofolate
dehydrogenase and hydrolysis of N5,N10-methenyltetrahydrofolate to
N5,N10-methenyltetrahydrofolate cyclohydrolase
H2N

H
N

H2 N

N
O

NR'
H

H *
12.12

:B
H

N5,N10-methenyltetrahydrofolate cyclohydrolase

H
N
N
H

NR'

12.9

H2N

H
N

N -formyl H4Pte

+B

N
*

NR'

H2N

H
N

HN

HN

HN

HO

N+
R

10

..
N

H
NH2

H O

NH2

HN

HN

H2N

H
N

H
12.10

NR'
a
O
H
:B

N
O

B+

O NHR'

12.11

N5-formyl H4Pte

Scheme 12.5

Transfer at the Methanol

Oxidation State (transfer methyl group)
H2N

H
N
5 6

HN

N
O

CH3

NHR'

12.13

N5-methyl H4Pte

Requires NADPH and FAD to make 12.13 from

N5-methylene H4Pte

Reaction Run Backwards with [6-3H]-12.13 Releases

No 3H and Does Not Transfer 3H to Methyl
Excludes tautomerization of N5-methylene H4Pte to 12.14
H 2N

H
N

H2N
H

HN

HN

N
O

CH2

N
O

NHR'

H
N

CH3
12.14

H2N

N
HN

H
N
N

CH2

NHR'

3H

Excludes [1,3]-hydride shift

NHR'

Proposed Mechanism for the Reduction of

N5,N10-methylenetetrahydrofolate by
N5,N10-methylenetetrahydrofolate Reductase
FAD

NADPH
+H+

NADP+

FADH

H2N

HN

+
H2N

H
N
N

O HC
2

CH2 NHR'
12.6

FADH

Scheme 12.7

H2N

+
N

HN

H
N

+
NHR'

12.7

H
N
+
N

HN
O

CH3 NHR'

FAD
B

H2N

HN

H
N
N

CH3 NHR'
12.13

Proposed Alternative Hydride Mechanism for

N5,N10-Methylenetetrahydrofolate Reductase
NADPH
+
FAD +D

NADP+

H2 N
FAD

+
N

HN

D2O

H
N

CH2 NHR'
12.6

Scheme 12.8
H2 N

HN

H2N
HN

H
N
N

O HC
2

+
NHR'

12.7

H
N
N

CH2 NHR'
D

With [5-3H]-deazaFADH2, 3H transferred to

methyl group, consistent with this mechanism

Transfer of a Formyl Group

Proposed mechanism for glycinamide
ribonucleotide (GAR) transformylase
H2N

HN
O

H
N
N
H
H

O
=O PO
3

O
HO

..
NH2

HN

H2N

HN
O

NR'
O

H
B

=O

3PO

HN

HO

OH

12.17

12.16

GAR

H
N

H2N
HN

N
H

NR'

O
N H H
H

N
H

12.2

NHR'
H

O
:B

=O PO
3

B:
OH

H
N

HO

HN

OH

12.18

FGAR

Scheme 12.9

Third step in biosynthesis of purines

NH

Transfer at Formaldehyde Oxidation State

Reaction catalyzed by thymidylate synthase
(an anomalous transfer of a methylene group)
exchanges

O
5

HN

=O

3PO

O
O

CH3

HN
H2N

H
+

N
N

12.19

Scheme 12.10

=O PO
3

N
O

N
HO

HO

H
N
H

reduced
(normally CH2OH)

H2N

H
+

H
N

N
N

N
HO

HO
12.20

N
H

oxidized

Last step in de novo biosynthesis of thymidylate

C-5 H exchanges with solvent
inverse 2 isotope effect rehybridization of C-5 from sp2 sp3
inverse 2 isotope effect at C-6 also

Transfer of the C-6 Hydrogen of N5,N10Methylenetetrahydrofolate to the Methyl Group of

Thymidylate Catalyzed by Thymidylate Synthase
transferred to
O
H

HN
O

H2N
+

N
dRP

H
N
3H

HN
O

Scheme 12.11

C3HH2

HN

H2 N

O
R

N
dRP

N
HN

H
N

N
O

N
H

Chemical Model Study for Thymidylate

Synthase-catalyzed Exchange of the C-5
Hydrogen of 2-Deoxyuridine-5-monophosphate
C-5 2H washed out in base
O
D

MeN
O

N
Me
12.21

OH

OH

MeN

N
Me

Scheme 12.12

H
OH

H
D

MeN
N
Me

H
OH

OH

MeN
O

N
Me

H
OH

12.22

note: C-5 and C-6 are

rehybridized to sp3

Thiols are more effective than hydroxide

MeN
N
Me

Inactivation of Thymidylate Synthase

by 5-Fluoro-2-deoxyuridylate
H2N
N
HN
O

O
F

MeN
=O

3PO

NH

O
F

N
CH2

HN
=O

3PO

NH
N

S
Cys

C
HO

HO
12.23

Scheme 12.13

12.24

structure identified
by X-ray

Glu

Proposed Mechanism for the First Part of the Reaction

Catalyzed by Thymidylate Synthase Based on
Inactivation Complex with 5-Fluoro-2-deoxyuridylate
H2N

H
N

..
N

HN
O
O

3PO

N
O

H
S

O
B

=O PO
3

HN
N

O
O

CH2

Cys

Scheme 12.14

HO

CH2
H

HN

=O PO
3

N
O

Cys

HO

N
O

NHR

H
S

HN

N
O

H
N

H2N

HN

NR
H

HN

=O

H2C

H
N

H2N

HO

H
S

Cys

NHR
B

Original Proposal
Highly unlikely [1,3]-H shift mechanism for reduction
of the substrate catalyzed by thymidylate synthase
H
N

H2N
HN

=O

3PO

HO

[1,3]-H shift
suprafacial

HN

NHR

12.25

Cys

CH3

=O

3PO

O
O

HO

NHR

HN

B
S

N
O

H
N

CH2

HN
O

H2N

..
N

H
N

H
S

Cys

Scheme 12.15

Not allowed by Woodward-Hoffman rules

Should have occurred with 5-F analogue, but does not

To Rationalize Stability of 5-F Adduct

Proposed mechanism for the second part of the
reaction catalyzed by thymidylate synthase
H
N

H2N

HN
O

CH2
H

HN

=O

3PO

:B
N

HO

NHR

HN

H
S

Cys

CH2

..
N
H

Cys

B
O

=O PO
3

Scheme 12.16

HO

H
S

Cys

12.26

O
CH3

HN

=O PO
3

N
O

HO

CH2

HN

O NHR

HO

12.25

H
N

NHR

=O PO
3

HN

N
OO

when F, it
is stable

H2N
H

HN

N
O

H
N

H2N

H
Cys

CH3

HN

=O PO
3

N
O

HO

H
S

Cys

H2N

HN

H
N
N

NHR

Precedence for Elimination

Model study for the Mechanism
formation of the C-5 exomethylene intermediate proposed in the
reaction catalyzed by thymidylate synthase
O
CH2 O

MeN
O

N
Me
12.27

NO2

H
OH

CH2 O

MeN
O

N
Me

H
OH

NO2

CH2

MeN
O

OH

N
Me

H
OH

O
CH2OH

MeN
N

Me

H
OH

Scheme 12.17

CH2OH

MeN
O

N
Me

Enzymatic Intermediate Trapped with

-Mercaptoethanol
Trapping of the proposed C-5 exo-methylene
intermediate during catalytic turnover of
thymidylate synthase
O
S

HN

=O

3PO

N
O

HO

O
HN

OH

H
S

Cys

12.26

=O

OH

3PO

N
O

HO

HN

S
H
S

Cys

=O

3PO

S
N

O
O

HO

H
S

12.28

isolated

Scheme 12.18

Cys

OH

Alternative to Hydride Transfer

Alternative proposed electron transfer mechanism for the
reduction of the exo-methylene intermediate in the reaction
catalyzed by thymidylate synthase
H
N

H2N

HN

NHR

CH2

HN
=O

3PO

O
O
HO

Cys

CH2

HN

H
=O

12.26

3PO

O
O
HO

12.29

H
S

Cys

=O

3PO

Cys

NHR

SET
H+

CH3

HN

H
S

NHR
CH3

O
HO

HN

HN

H
N

H2N

NHR

SET

HN

N
H

H
N

H2N

HN

N
H

H
N

H2N

=O

3PO

N
O

H
S

Cys

HO

12.30

Scheme 12.19

CH3

HN
=O

3PO

O
O
HO

H
Cys

Transfer at the Methanol Oxidation

State

Two different forms of methionine synthase:

one transfers CH3 directly from N5-methyl H4PteGlu
one first transfers CH3 to a cobalt complex (cobalamin)

Reaction catalyzed by the cobalamin-independent

methionine synthase
H2N

H
N

HS

HN

N
O

CH3

COO

NHR

Scheme 12.20

NH3+
12.31

H2N

H
N
+

HN
O

N
H

COO

CH3S

NH3+
NHR

12.32

Model Study for the Reaction Catalyzed by the

Cobalamin-independent Methionine Synthase
H
N
O

H
N

Na+

CH3

HN

N
O H3C

CH3
CH3

H
N

COO
NH3+

SN 2

N
HN

H
N

CH3

N
O

CH3S

CH3

COO
NH3+

CH3

12.33

increases leaving
group ability
(model for protonated
N5-Me-H4PteGlu)

Scheme 12.21

Enzyme requires Zn2+ (coordinates to the thiol S)

Cobalamin-dependent Methionine Synthase

CONH2
CH3 CH3

CONH2

CH3
H2NOC
A
H3C
N B
N
corrin
H3C
Co+
CONH2
H
CH
H2NOC
N
N
3
D
H3C
C
CH3

HN

H3C

H3C

H3C

CONH2

H3C
H
O

ring

HO
O

O
P

H
O

CH2OH

12.34

methylcobalamin
from the methylation of cob(I)alamin by N5-MeH4PteGlu

(A) Reaction Catalyzed by Cobalamin-dependent

Methionine Synthase
(B) Cleland Diagram for the Reaction Catalyzed by
Cobalamin-dependent Methionine Synthase
HCys
E
A

Met

CH3cobalamin

H4PteGlu

CH3H4PteGlu

HCys

cob(I)alamin

retention of Me
configuration

CH3H4PteGlu

Met

Cleland
notation

H4PteGlu

B
E

cobalamin

Scheme 12.22

CH3cob(I)alamin

cobalamin

Enzyme reaction
pathway

Model Study for the Methylation of Cob(I)alamin

during the Reaction Catalyzed by the
Cobalamin-dependent Methionine Synthase
H
N
O

cob(I)alamin

H
N

N
HN

H
N

CH3

N
O H3 C

methylcobalamin

CH3
CH3

12.33

Scheme 12.23

N
HN

H
N

N
O

CH3

CH3
CH3

S-Adenosylmethionine (SAM)-Dependent
Transfer of CH3
Proposed mechanism for the synthesis of S-adenosylmethionine
catalyzed by methionine adenosyltransferase
COO
H3N
O
=O

3P O P O

CH3

rare attack at C-5

O
P

CH2

O
HO

OH
12.35

ATP

Scheme 12.24

A methionine
adenosyltransferase
PPi+Pi

OOC

CH3
S

O
H3N
HO

OH

12.36

SAM
more common methylating agent

Generalized Reaction Catalyzed by

S-adenosylmethionine-dependent
Methyltransferases

H
R
OOC

Scheme 12.25

:B

X
CH3

OOC

O
H3N
HO

OH

12.36

SN2

CH3

O
H3N
HO

OH

12.37

With chiral CH3 group gives inversion of stereochemistry

Stereochemistry of Methylation of Indolylpyruvate

in the Biosynthesis of Indolmycin

inversion of
stereochemistry

COO
H3 N

D
S
adenosyl

:B
H

T B:

H
H

12.38

indolylpyruvate

Scheme 12.26

D
O

COO

COO

COO
N
H

N
H

N
H

D
O

NHCH3
N

N
H
12.39

indolmycin