The Organic Chemistry of Enzyme-Catalyzed Reactions

Chapter 11

Aldol and Claisen Reactions and Retroreactions

Scheme 11.1

aldehyde or ketone

-hydroxyaldehyde or -ketone

Aldol Reactions

Generalized aldol reaction

R C

O

C

H

R'

H

O-

CR CHR'

R C CH2R'

O

R C

O

CH

R'

C

O

R

CH2R'

H Base+

11.1

:Base

condensation

retroreaction

11.2

H :Base

Scheme 11.2

Type I

Type II

AldolasesGeneral mechanisms for type I (pathway a) and

type II (pathway b) aldolases

b

a

R

O

X

Lys

NH

RX

H R'

O

His

NN

R

O

X

H

Zn2+

H R'

O

donor

acceptor

acceptor

:Schiff base mechanism

Metal ion catalyzed

Scheme 11.3

dihydroxyacetonephosphate (DHAP)

glyceraldehyde3- phosphate (G3P)

fructose 1,6-phosphate(FDP)

tagatose 1,6-diphosphate aldolase

(3S,4R)11.3 + 11.4 (3S,4S)11.5

Reaction Catalyzed by Fructose 1,6-Diphosphate Aldolase

CH2OPO3=

C

C

C

C

CH2OPO3=

O

HHO

OHH

H OH

CH2OPO3=

C

C

O

OHH

H

C

CH2OPO3=

H OH

CHO

=O3POOPO3

=

O

OH

OH

OH

=O3PO

aldol reaction

O

OH

retroreaction+

OPO3=

O

OH

11.3 11.4

H

11.5

11.311.4

11.5

+aldol reaction

retroreaction

Tagatose 1,6-Diphosphate

=O3POOPO3

=

O

OH

OH

OH

11.6

CH2OPO3=

C

C

C

C

CH2OPO3=

O

HHO

HHO

H OH

C4-epimer

DHAP in D2O in absence of G3P

pro-S

FDP Aldolase

CH2OPO3=

C

C

O

OHD

H

=O3PO

O

OH

11.7

HD

11.7

Figure 11.1

Therefore in the reverse reaction G3P is released faster than DHAP

Ordered Product ReleaseRate of incorporation of 14C into fructose 1,6-diphosphate

(FDP) from [14C]glyceraldehyde 3-phospate (G3P) and [14C]dihydroxyacetone phosphate (DHAP) catalyzed by

fructose 1,6-diphosphate aldolase

from [14C]DHAP

from [14C]G3P

into FDP

incorporation

Rate of 14C

Time

Scheme 11.4

DHAP is released last in retroreaction; therefore it binds first in forward reaction (ordered reaction)

Ordered Reaction of Fructose 1,6-Diphosphate Aldolase

E FDP EG-3-P

DHAPE DHAP + G-3-P

[14C]-G-3-PE + DHAP

[14C]DHAP

slow fast

(Principle of Microscopic Reversibility)

Scheme 11.5

Evidence for Schiff Base Mechanism

[18O]G3P alone releases no 18O

Evidence for the involvement of an active-site lysine residue in the reaction catalyzed by fructose 1,6-diphosphate aldolase

CH2OH

CH

CH2OH

NH (CH2)4 CHCOO-

NH3+

CH2OPO3=

C

CH2OH

CH2OPO3=

18O C NH

CH2OH

LysLysNH2

CH2OPO3=

C NH

CH2OH

Lys14

++

H141414

11.8

NaBH4 H3O+

Δ

-H218O

Scheme 11.6

pro-S

si

Overall Proposed Mechanism and Stereochemistry for Fructose 1,6-Diphosphate Aldolase

CH2OPO3=

C

CH2OH

CH2OPO3=

O

C NH

CHO H

H

Lys

NH2Lys

B:

CH2OPO3=

C NH

C

HO H

Lys+

+

CH2OPO3=

C NH

C

HO H

Lys+

DHAP

+

+

11.5

NH LysHO

H

O

OPO3=

=O3PO

NH

HO

OHHOH

B H

H

H

H

H

HO

Lys

OPO3=

=O3PO

HO

OHHO

H

H

H

O

H2O

COO-

O

NH2

NHNH2

COO-

-OOC

2 + 2 H2O

A-side P-side

11.10 11.11

Scheme 11.8 porphyrins (heme)

corrins (vitamin B12)

-aminolevulinate porphobilinogen

Contains M2+ (Zn2+ or Mg2+)

Reaction Catalyzed by Porphobilinogen Synthase

[14C] substrate + NaBH4 gives [14C] enzymeICH2COOH and ICH2CONH2 inactivate (2 different Cys)Requires a thiol reducing agent

Scheme 11.9

Proposed Mechanism (imine, then aldol) for the Reaction Catalyzed by Porphobilinogen Synthase

NH2

NH-Lys252

COO-

HLys252

NH2

NH2

O

COO-

NH2

O

COO-

NH

NH-Lys252

COO-

NH2

COO-

S-Cys223

S-Cys223

B:

NH

NH-Lys252

COO-

NH2

COO-

S-Cys223

NH

COO- COO-

H

NH2

S-Cys223

NH-Lys252

H-B

B:H

:B

NH

COO- COO-

NH2

S-Cys223

HNH

COO- COO-

NH2

S-Cys223

Tyr

Asp

Tyr

S S SH SH

Asp

Tyr

Asp

+ +

..

+

+

+

..

11.12 Asp

Tyr

+

Tyr

Asp

Tyr

Cys119

ZnB

Cys122

Cys132 Cys124

Asp

aldolreaction

inactiveenzyme

activeenzyme

11.13 11.14

11.1511.11

P-side A-side P-side

11.10

RSH

H2O

ZnA

H2O H2O

ZnA

ZnA

H2OH2O

ZnA

ZnA

ZnA

H2O

Scheme 11.10

Alternative Proposed Mechanism (aldol, then imine) for the Reaction Catalyzed by

Porphobilinogen Synthase

Lys252

NH2

NH2

O

COO-

11.16 11.1711.12

Asp

NH2

NH-Lys252

COO-

NH2

O

COO-

S-Cys223

P-side A-side

Tyr

aldolreaction

H

B:

NH2

NH-Lys252

COO-

NH2

O

COO-

S-Cys223

Asp

.. NH2

NH-Lys252

COO-

NH2

O

COO-

S-Cys223

+ +

TyrTyr

AspCys119

ZnB

Cys122

Cys132 Cys124

S S SH SHinactiveenzyme

activeenzyme

NH

COO- COO-

H

NH2

S-Cys223

NH-Lys252

H-B

B:H

:B

NH

COO- COO-

NH2

S-Cys223

HNH

COO- COO-

NH2

S-Cys223

Tyr

Asp

+ +

Tyr

Asp

Tyr

Asp 11.1511.11

11.10

P-side

H2O

ZnAZnA ZnA

RSH

H2O

ZnA

H2O H2O

ZnAZnA

citrateoxaloacetate

Isotope exchange and kinetics support a double displacement mechanism with a phosphoryl enzyme intermediate

Scheme 11.11

Retro-aldol Reactions

CO18O-

CH2

CHO COO-

CH2

COO-

COO-

C

CH2

COO-

OCH3C SCoA

O

+ Mg•ADP + Pi(18O)

+ fatty acidbiosynthesis

pro-Sarm

11.1911.18

+ Mg•ATP + CoASH11.20

retro-aldol

Reaction catalyzed by ATP citrate-lyase

can substitute for citrate and ATP

gives product in absence of citrate, ATP, CoASH

C

CH2

CHO COO-

CH2

COO-

OOPO3

=C

CH2

CHO COO-

CH2

COO-

11.22

OSCoA

11.21

Reasonable Intermediates

Scheme 11.12

Exchange between [14C]ADP and ATP in the absence of all other substrates --- enzyme phosphorylation

Maybe more than two intermediates

Enz PO3=ATP + Enz ADP +

[14C]ADP

ATP phosphorylation of ATP citrate-lyase and exchange with [14C]ADP

Scheme 11.13

Phosphoryl Enzyme Isolated with [32P]ATP

ATP phosphorylation of ATP citrate-lyase

[32P]-phosphoenzyme + ADP[γ-32P]ATP + Enz

[14C]citrate + [32P]phosphoenzyme [14C]enzyme + [32P]PO43-

CoASH

CH3 SCoA

O

-OOC

O

COO-

oxaloacetate

Scheme 11.14

may not form

Mechanism Proposed for the Reaction Catalyzed by ATP Citrate-lyase

Enz X- Enz X PO3=

Enz X PO3=

O

-O

COO-

OH-OOC

Enz X-

O

C OPO3=

COO-

OH-OOC

O

CXEnz + Enz X-

CH2 CSCoA

O- -OOC

COO-

O

BH

11.22

Pi

+

citryl enzyme

11.19

11.20

11.18

retroaldolreaction

aldolcondensation

+

-OOC OH

COO-

O

C SCoA

COO-

O-OOCH

:B

CH3 CSCoA

O

phosphoryl enzyme

+ ADPATP +

11.21

(X = His)

CoAS

Scheme 11.15

Possible Alternative Mechanism Proposed for the Reaction Catalyzed by

ATP Citrate-lyase

Enz X-

Enz X CH3

OO

CXEnz

11.19-OOC O

COO-

H:B

-OOC

COO-

OO-

CXEnzCH2 H B

+

-S[3H]CoA

[3H]CoASH

+

citryl enzyme

11.20

CH3 CSCoA

O

Whenever there are at least two steps in a mechanism, consider reversing two of the consecutive steps

Scheme 11.16

C5

ketoseC5

aldose

C3

aldoseC7

ketose

Requires thiamin diphosphate (TDP) and Mg2+

Reaction Catalyzed by Transketolase

CHO

CH OH

C OHH

C OHH

CH2OPO3=

C OHH

C OHH

CH2OPO3=

C O

H2C OH

CHO

CH OH

CH2OPO3=

C HHO

C OHH

C

C O

H2C OH

OHH

C OHH

CH2OPO3=

transketolase+ +

11.2411.23 11.2611.25

Scheme 11.17

equivalent

TDP provides a way of stabilizing a carbanion at the carbonyl

Mechanism Proposed for Transketolase

CH2OH

C O

C HHO

C OHH

C

C O

H2C OH

OHH

C OHH

CH2OPO3=

NS

RR'

C OHH

C

NS

C O

CH2OHR

R'

CHOH2C OH

CH O H

C

N SR

R'

CHOH2C OH

C

C OHH

C

OH

OHH

CH2OPO3=

OHH

CH2OPO3=

C

OHH

H OH

CH2OPO3=

N SR

R'

C OHOH2C

C OHH

C

C

OHH

H OH

CH2OPO3=

11.24

C

11.23

HHO

11.26

11.25

H

:B

B

H aldol

BH

TDP

:B

Scheme 11.18

Transketolase in Organic Synthesis

Synthesis of substituted D-threo-trihydroxylated ketones catalyzed by transketolase

-OOCOH

O

RH

OH

O

R

OH

+

OH11.28

transketolaseTDPMg2+

OH

O

11.27

11.29

+ CO2

Scheme 11.19

Substrate and product are in the same oxidation state

Reaction Catalyzed by Dehydroquinate Synthase

O

HO

-OOC

OHOPO3

=

OHHO

-OOC

OHO

OH

11.30 11.31

6

53

7

Requires NAD+

Scheme 11.20

double bond activates elimination

This mechanism suggests the enzyme is capable of catalyzing 5 different reactions!

Originally Proposed Mechanism for Dehydroquinate Synthase

O

HO

-OOC

OHOPO3

=

O

HO

-OOC

OHO

OH

HH

:B

O

HO

-OOC

OHOPO3

=

O

H

:B

O

HO

-OOC

OH

O

HO

-OOC

O

OH

OH

H

B:

O

HO

-OOC

O

OH

11.31

11.30

H

BH

11.32

-Pi

11.33

BH

11.34

NAD

11.35

aldol reaction

3 4 5

6

NAD+ NADH

NAD+

NAD+H

isolated

Model Study to Show that the Last 2 Steps are Really Nonenzymatic

OH

-O2C

OHO

OHO

OH

-O2C

OH

OHO

OH

-O2C

O

OH

NO2

CHONO2

hυ spontaneous

protected

11.34

11.31

HO

-OOC

OH

OH

PH O

O--O

HO

-OOC

OH

OH

PH

O

O-

-O

HO

-OOC

OH

O

PH

O

O-

-O

HO

-OOC

OH

OHP

H

O

O-

-OHO

-OOC

OH

OH

11.40PH

11.36

O

O--O

11.37 11.38

11.39

exchanges in D2O with enzyme

does not exchange (but is oxidized)

does not exchange and is not oxidized

exchanges and is oxidized

Therefore the phosphate group removes the proton from the ring (self-catalyzed)

To Show Elimination Step Also Is Nonenzymatic

NAD+

cyclohexane - blocks ring cleavage phosphonate - not

a leaving group

Scheme 11.21

-H of thioesters is 100 times more acidic than

that of esters

Generalized Claisen reaction

RX C

O

C

H

R'

H

O-

CRX CHR'

R"X C R'

O

RX C

O

CH

R'

C

O

CH2R'

XR":Base

condensation

retroreaction

11.41X = O or S

Claisen Reactions

C-C bond forms with inversion of stereochemistry (aldol reactions go with retention)

Scheme 11.22

retro-Claisen

ICH2CONH2 labels a Cys

Reaction Catalyzed by Thiolase

R SCoAR SCoA SCoA

O OO O

11.46

++ CoASH

[14C]acetyl-CoA gives acetylated enzyme

Scheme 11.23

presumably via enolate

Mechanism Proposed for Thiolase

S

H

B:

R SCoA

O

CH2B:

S R

O

SCoA

O

H

S-

R SCoA

O O

- CoAS- Claisen

retro-Claisen+CoAS-

Scheme 11.24

aldol

Reaction Catalyzed by Kynureninase

NH2

NH2

O COOH

NH2

COH

O

CH3 NH2

COOH

Ph COOH

HO NH2H H

PLP+

(2S,4R)

11.48 11.49

11.50

PhCHO

retro-Claisen

NH2

NH2

O COOH

NH2

O

NH

H COO-

B:

B:

NH2

O

NH

HCOO-

OH

OHO

NH2

NH

COO-

NH2

COOH

NH

COO-B+

H

NH

COO-B+

HH :B

L Ala

NH

OH=O3PO

NH2

O

NH

COO-

NH

OH=O3PO

B

H

NH

OH=O3PO

NH

OH=O3PO

NH

OH=O3PO

+

NH

COO-

+

NH

OH=O3PO

+

NH

+

OH=O3PO

11.51

E•PLP

NH

COO-

+

11.52

+

NH

OH=O3PO

+

11.53

11.5411.5511.5611.57

11.58

11.48

E•PLP+

(lmax = 420 nm)(lmax = 494 nm) (lmax = 330 nm)

(lmax = 330 nm)(lmax = 330 nm)(lmax = 330 nm)(lmax = 500 nm)

(lmax = 420 nm)

11.49

..

..

retro-Claisen

Scheme 11.25

or PhCHOre face addition (behind)

Mechanism Proposed for Kynureninase

4R

Only the 4R isomer is a substrate;therefore water adds to re-face

Evidence for Intermediate 11.54

COOH

HO NH2H H

NH2

11.59

Ki = 70 nM

(300-fold lower than Km of substrate)

TS‡ Analogue Based on 11.59

SCOOH

O NH2O H

NH2

11.60