The Organic Chemistry of Enzyme-Catalyzed Reactions

Chapter 12

Formylations, Hydroxymethylations, and

Methylations

Transfer of one-carbon unitscan be oligomer of up to 12 Glu residues

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

Tetrahydrofolate-dependent Enzymes

HN

N

NH

HN

NH

C

O

CHCH2CH2COO-

COO-

H2N

O

H6

glutamate5,6,7,8-tetrahydro-pteridine

pKa of acid is -1.25pKa of acid = 4.8

p-amino-benzoic acid

10

12.1

HN

5

5

10

abbreviated structure for tetrahydrofolate

12.2

HN

N

NH

HNH2N

O NHR'

pteroate ring

Folic Acid (a vitamin for humans)

HN

N

N

N

NH

CNHO

CHCH2CH2COO-

COO-

H2N

O

12.3

Reduction of Folate to Tetrahydrofolate

Scheme 12.1

dihydrofolate

Reactions catalyzed by dihydrofolate reductase (DHFR)

+BH

N

H H

NH2

O

R N

NH2

OH

R

NR

H O

NH2

B+

H

HN

N

N

N

NHR'

H2N

O

HN

N

N

HN

NHR'

H2N

O

HN

N

N

HN

NHR'

H2N

O

HN

N

NH

HN

NHR'

H2N

O

H

dihydrofolatereductase

+

12.1

..

12.3

+

N

H H

NH2

O

R

dihydrofolatereductase..

Lys

HN+

NH

OH

:NH2COO-

H

OH

HN+

NH

OH

OHB:

O CH2

HN+

NH

OH

HN+

NH

OH

HN+

NH

OH

COO-H

COO-

+B

COO- H

H

H

HN+

NH

OH

COO-HH Lys

:NH2

Lys

HN+

NH

OHH2N COO-

COO-H

+

=O3PO

=O3PO=O3PO=O3PO =O3PO

=O3PO =O3PO

*

*

*

+ +

++

See Scheme 8.39

..

+

12.4

Scheme 12.2

-cleavage

pro-S hydrogen added/removed

(retention of configuration)

Ordered mechanism: no conversion of Ser to H2C=O unless tetrahydrofolate is bound

Generation of the Transferring Carbon UnitSerine 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.

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

Scheme 12.3

N5-methylene H4Pte

N5,N10-methylene H4Pte

N10-methylene H4PteKeq = 3.2 x 104 in favor of 12.7

Transfer of One-Carbon Units

Serine hydroxymethyltransferase-catalyzed reaction of formaldehyde and tetrahydrofolate to methylenetetrahydrofolate

Transfer at the formaldehyde oxidation state (transfer HOCH2

- group)

HN

N

NH

HNH2N

O NHR'

HN

B+ H

O CH

H

N

N

HNH2N

O NHR'HCH2

OHB:+B

H

N

CH2

HN

NHR'O

H2NHNN

HN

N

N

HNH2N

O NR'CH2

:B

HB H

NR'CH2

O

H2NHN

NH

N

HN+

** +

:*

+

*

*

..

12.5

12.6

12.7

12.8

5

10

-H2O

Scheme 12.5

N5,N10-methenyltetrahydrofolate cyclohydrolase

N10-formyl H4PteN5-formyl H4Pte

Transfer at the Formate Oxidation State (transfer formyl group) Oxidation of N5,N10-methylenetetrahydrofolate to N5,N10-

methenyltetrahydrofolate catalyzed by methylenetetrahydrofolate dehydrogenase and hydrolysis of N5,N10-methenyltetrahydrofolate to

N5,N10-methenyltetrahydrofolate cyclohydrolase

NHN

NHNH2N

O NR'H

H

N

NH2

O

N

R

NR'

H

O

H2NHNN

HN

NHN

NHNH2N

O NR'

H O

:B

HO H

N

R

O

NH2

HH

H

:B

B+H

+BH

NR'O

H2NHN

NH

N

HN

H

OH

HN

N

N

HNH2N

O NHR'

Ob

*

+

*

*+

..

12.9

a*

b a

12.1212.11

12.10

*

N5-methyl H4Pte

Requires NADPH and FAD to make 12.13 from N5-methylene H4Pte

Transfer at the Methanol Oxidation State (transfer methyl group)

N

CH3 NHR'O

H2NHNN

HN

12.13

H5 6

Excludes [1,3]-hydride shift

Excludes tautomerization of N5-methylene H4Pte to 12.14

N

CH3NHR'O

H2NHNN

HN

12.14

N

CH2NHR'O

H2NHNN

HNH

N

CH2NHR'O

H2NHNN

HN

3H

Reaction Run Backwards with [6-3H]-12.13 Releases No 3H and Does Not Transfer 3H to Methyl

Scheme 12.7

Proposed Mechanism for the Reduction of N5,N10-methylenetetrahydrofolate by

N5,N10-methylenetetrahydrofolate Reductase

FAD

B

FADH-

BH

N

CH2

HN

NHR'O

H2NHNN

HN

N

N

HNH2N

O NHR'H2C

FADH-

BN

CH3 NHR'O

H2NHNN

HN

FAD

B

NADPH +H+ NADP+

+

+

+ N

CH3 NHR'O

H2NHNN

HN

+

12.13

12.6 12.7

Scheme 12.8

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

methyl group, consistent with this mechanism

Proposed Alternative Hydride Mechanism for N5,N10-Methylenetetrahydrofolate Reductase

12.7

FAD FADN

CH2

HN

NHR'O

H2NHNN

HN

N

N

HNH2N

O NHR'H2C

D-NADPH +D+ NADP+

+

+

12.6

N

CH2 NHR'O

H2NHNN

HN

D

D2O

Proposed mechanism for glycinamide ribonucleotide (GAR) transformylase

Scheme 12.9GAR FGAR

Transfer of a Formyl Group

O

=O3PO

HO OH

HNNH2

O

NR'O

H2NHN

NH

N

HN

HO

O

=O3PO

HO OH

HNN

O

NR'O

H2NHN

NH

N

HN

H O

B

H

H

H

B: O

=O3PO

HO OH

HNNH

O

12.16

NHR'O

H2NHN

NH

N

HN

H O

12.17

HB

12.2

H

12.18

:B

..

Third step in biosynthesis of purines

O

HO

HN

N=O3PO

O

H

HO

N

N

N

HN

N

H2N

HO

H

R

O

HO

HN

N=O3PO

O

CH3

HO

N

N

N

HN

NH

R

H2N

HO

12.19

++

12.20

5

6

Transfer at Formaldehyde Oxidation State

Scheme 12.10

exchanges reduced(normally CH2OH)

oxidized

C-5 H exchanges with solvent

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

Last step in de novo biosynthesis of thymidylate

inverse 2° isotope effect rehybridization of C-5 from sp2 sp3

inverse 2° isotope effect at C-6 also

Scheme 12.11

transferred to

Transfer of the C-6 Hydrogen of N5,N10-Methylenetetrahydrofolate to the Methyl Group of Thymidylate Catalyzed by Thymidylate Synthase

dRP

HN

N

O

H

HOHN

N

N

HN

N

H2N

O

3H

R dRP

HN

N

O

C3HH2

HO

HN

N

N

HN

NH

R

H2N

O

++

Me

MeN

N

O

D

HOMe

MeN

N

O

D

HOOH

H OH

Me

MeN

N

O

D

HOOH

H

Me

MeN

N

O

HOOH

H

Me

MeN

N

O

H

HO

12.21 12.22OH

OH

C-5 2H washed out in base

Scheme 12.12note: C-5 and C-6 are rehybridized to sp3

Thiols are more effective than hydroxide

Chemical Model Study for Thymidylate Synthase-catalyzed Exchange of the C-5

Hydrogen of 2-Deoxyuridine-5-monophosphate

Scheme 12.13structure identified

by X-ray

Inactivation of Thymidylate Synthase by 5-Fluoro-2-deoxyuridylate

MeN

N

O

F

HOO

HO

HN

N=O3PO

O

CH2

SO

Cys

FN

NH

N

HN

H2N

NH

C

Glu

O

O

12.24

O

HO

=O3PO

12.23

Scheme 12.14

Proposed Mechanism for the First Part of the Reaction Catalyzed by Thymidylate Synthase Based on

Inactivation Complex with 5-Fluoro-2-deoxyuridylate

O

HO

HN

N=O3PO

O

HO

H

SCys

HN

N

N

HN

CH2 NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HO

H

SCys

HN

N

N

HN

H2C NR

H2N

O

HHN

N

N

HN

CH2NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HO

H

SCys

BH B

..

B

Original Proposal

Scheme 12.15

[1,3]-H shift suprafacial

Not allowed by Woodward-Hoffman rulesShould have occurred with 5-F analogue, but does not

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

HN

N

N

HN

CH2 NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HO

H

SCys

B

12.25

HN

N

N

HN

CH3NHR

H2N

O

O

HO

HN

N=O3PO

O

HO

H

SCys

..

To Rationalize Stability of 5-F Adduct

Scheme 12.16

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

HN

N

N

HN

CH2 NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HO

H

SCys

B

HN

N

N

HN

CH2 NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HOS

Cys

BH

HN

N

NH

HN

CH2

NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HOS

Cys

CH3

O

HO

HN

N=O3PO

O

HOS

Cys

HN

N

N

HN

NHR

H2N

O

CH3

O

HO

HN

N=O3PO

O

HO

12.25

SCys

12.26

:

..

when F, it is stable

Precedence for Elimination Mechanism

Scheme 12.17

Model study for the formation of the C-5 exo-methylene intermediate proposed in the reaction catalyzed by thymidylate synthase

Me

MeN

N

O

CH2

HO

NO2O

Me

MeN

N

O

CH2

HO

NO2O

OH Me

MeN

N

O

CH2

HOOH

Me

MeN

N

O

CH2OH

HOOH

Me

MeN

N

O

CH2OH

HO

12.27

OH

OH

Enzymatic Intermediate Trapped with -Mercaptoethanol

Scheme 12.18isolated

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

O

HO

HN

N=O3PO

O

HOS

Cys O

HO

HN

N=O3PO

O

HOS

Cys O

HO

HN

N=O3PO

O

HO

12.28

SCys

12.26

SOH S

OHS

OH

Scheme 12.19

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

Alternative to Hydride Transfer

12.29

HN

N

NH

HN

CH2

NHR

H2N

O

H

O

HO

HN

N=O3PO

O

HOS

Cys

HN

N

NH

HN

CH2

NHR

H2N

O

H

O

HO

HN

N=O3PO

HO

SET SET

12.30

SCys

O

HN

N

N

HN

CH3

NHR

H2N

O

O

HO

HN

N=O3PO

HOS

Cys

OH

HN

N

N

HN

NHR

H2N

O

CH3

O

HO

HN

N=O3PO

HOS

Cys

O

CH3

O

HO

HN

N=O3PO

O

HOS

Cys

12.26

-H+

Transfer at the Methanol Oxidation State

Scheme 12.20

Reaction catalyzed by the cobalamin-independent methionine synthase

N

HNH2N

O

HN

N

CH3 NHR

HS COO-

NH3+

NH

HNH2N

O

HN

NCH3S

NHR

COO-

NH3+

+

12.31

+

12.32

Two different forms of methionine synthase:• one transfers CH3 directly from N5-methyl H4PteGlu

• one first transfers CH3 to a cobalt complex (cobalamin)

Scheme 12.21

SN2

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

Enzyme requires Zn2+ (coordinates to the thiol S)

Model Study for the Reaction Catalyzed by the Cobalamin-independent Methionine Synthase

N

HN

HN

O

HN

N

CH3

CH3

S COO-

NH3+

O

CH3

H3C

12.33

+N

HN

HN

O

HN

N

CH3

CH3O

CH3 CH3S COO-

NH3+

+Na+

corrin ring

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

Cobalamin-dependent Methionine Synthase

N

NH3C

H3C

OHO

O

CH2OHHP-OO

O

HN

CH3N

CH3

N

CONH2

NN

H3C

H3CH2NOC

H3CH3C

H2NOCCH3

CONH2

CH3

CONH2

H3C

CH3

CONH2

12.34

H

H

O

C

A

Co+

B

D

Scheme 12.22

retention of Me configuration

Cleland notation

(A) Reaction Catalyzed by Cobalamin-dependent Methionine Synthase

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

E cob(I)alamin

CH3-H4PteGlu

E CH3-cobalamin

HCys Met

E cobalamin

HCys

E CH3-cob(I)alamin

Met

E cobalamin

H4PteGlu

H4PteGluCH3-H4PteGlu

A

B Enzyme reactionpathway

Scheme 12.23

Model Study for the Methylation of Cob(I)alamin during the Reaction Catalyzed by the

Cobalamin-dependent Methionine Synthase

N

HN

HN

O

HN

N

CH3

CH3O

CH3

H3C

12.33

N

HN

HN

O

HN

N

CH3

CH3O

CH3

cob(I)alamin methylcobalamin

12.35

O

HO

CH2 A

OH

O

O

HO

A

OH

S-OOC

H3N

CH3P

O

O-

OP

O

O-

O=O3P

12.36

CH3S

COO-

H3N

PPi + Pi

methionineadenosyltransferase

S-Adenosylmethionine (SAM)-DependentTransfer of CH3

Scheme 12.24

rare attack at C-5

ATP SAM

more common methylating agent

Proposed mechanism for the synthesis of S-adenosylmethionine catalyzed by methionine adenosyltransferase

Scheme 12.25

With chiral CH3 group gives inversion of stereochemistry

Generalized Reaction Catalyzed by S-adenosylmethionine-dependent

Methyltransferases

O

HO

A

OH

S-OOC

H3N

CH3

XRH :B

XRCH3 O

HO

A

OH

S-OOC

H3N

12.36

+

12.37

SN2

Scheme 12.26

indolylpyruvate indolmycin

inversion of stereochemistry

Stereochemistry of Methylation of Indolylpyruvate in the Biosynthesis of Indolmycin

NH

O

COO-

HH

:B

NH

O

COO-

H

S

adenosyl

COO-

H3N D T

H

NH

O

COO-

HD

TH

NH

H

DT

H

N

O

H

12.39

B:

12.38

O

NHCH3