Dual mechanism of zinc-proline catalyzed aldol reactions in water

Article abstract of DOI:10.1039/b600703a

The aldol reaction of acetone with aldehydes in aqueous medium under catalysis by zinc-proline (Zn(l-Pro)₂) and secondary amines such as proline, (2S,4R)-4-hydroxyproline (Hyp) and (S)-(+)-1-(2-pyrrolidinomethyl) pyrrolidine (PMP) is shown to proceed by an enamine mechanism, as evidenced by reductive trapping of the iminium intermediate, while the aldol reaction of dihydroxyacetone (DHA) under catalysis by zinc-proline and by general bases such as N-methylmorpholine (NMM) is shown to occur under rate-limiting deprotonation of the α-carbon and formation of an enolate intermediate. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b600703a

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Dual mechanism of zinc-proline catalyzed aldol reactions in water{
Jacob Kofoed, Tamis Darbre and Jean-Louis Reymond*
Received (in Cambridge, UK) 18th January 2006, Accepted 14th February 2006
First published as an Advance Article on the web 2nd March 2006
DOI: 10.1039/b600703a
alkylation at proline. While proline shows only negligible aldolase
activity with this ketone, zinc-proline is very active for aldolization
of DHA, which suggests that zinc-proline acts by an enolate
mechanism in this case.
The aldol reaction of acetone with aldehydes in aqueous
medium under catalysis by zinc-proline (Zn(L-Pro)2) and
secondary amines such as proline, (2S,4R)-4-hydroxyproline
(Hyp) and (S)-(+)-1-(2-pyrrolidinomethyl)pyrrolidine (PMP) is
shown to proceed by an enamine mechanism, as evidenced by
reductive trapping of the iminium intermediate, while the aldol
reaction of dihydroxyacetone (DHA) under catalysis by zinc-
proline and by general bases such as N-methylmorpholine
(NMM) is shown to occur under rate-limiting deprotonation of
the a-carbon and formation of an enolate intermediate.
Catalysis by various amines was investigated to provide further
evidence for type I and type II mechanisms (Fig. 1). The
aldolization of acetone with 1 to form 2 in aqueous buffer pH
8.5 was catalyzed by zinc-proline, proline, Hyp and PMP, all of
which are suitable for enamine formation. Hyp showed the highest
activity and was efficiently converted to the N-isopropyl derivative
in the presence of NaBH4. These data suggest that acetone
aldolization in water occurs only by an enamine (type I)
mechanism, in agreement with an earlier study of enamine
catalysis in water.11 By contrast, the reaction with DHA was
catalyzed by most of the amines, including tertiary amines such as
NMM not capable of enamine formation, consistent with an
enolate mechanism for DHA under general base catalysis. The rate
of aldolization was correlated with the pKa values of the amines,
giving an apparent b-value of 0.45 (Fig. 2). This b-value indicates
that a proton transfer step is rate-limiting in the reaction,
presumably the enolate forming deprotonation at the a-carbon.
The aldol reaction is a key carbon–carbon bond forming reaction
in organic synthesis. The reaction is catalyzed by enzymes using
either an enamine (type I) or enolate (type II) mechanism.1 The
type I mechanism has inspired the design of aldolase catalytic
antibodies2 and related biocatalysts,3 and the development of
amino acids as organocatalysts for a variety of aldol and enolate
type reactions.4,5 Recently we showed that zinc-proline catalyzes
aldol additions in aqueous medium,6 including the self-condensa-
tion of glycolaldehyde to form carbohydrates,7 a reaction of
possible prebiotic significance.8 Herein we report a mechanistic
study of aqueous aldolization catalysis, showing that zinc-proline
uses both enamine and enolate type mechanisms in a substrate
dependent manner.
The reactions of acetone and DHA with nitrobenzaldehyde 1 to
give the corresponding aldols 2 and 4 are both efficiently catalyzed
by zinc-proline and were investigated as model reactions in
aqueous buffer at pH 8.5 (Scheme 1). Zinc-proline is a water-
soluble complex of zinc stable under these mild basic conditions,
featuring both a Lewis-acidic metal center potentially capable of
stabilizing an enolate, and two metal-coordinated secondary
amines that could engage in enamine catalysis, as suggested
earlier.6b Formation of the enamine proceeds via the iminium
intermediate I, which should be trapped by reducing agents as has
been shown for various aldolase enzymes.9 Reaction of acetone
with zinc-proline in the presence of NaBH4 led to the quantitative
formation of N-isopropyl proline 3, implying that an iminium
intermediate is formed with zinc-proline and acetone. 3 was also
formed when proline was incubated with aqueous acetone and
NaBH4,10 although to a lesser extent, in agreement with the
modest aldolase activity of proline in aqueous conditions. On the
other hand the reaction of NaBH4 and DHA with zinc-proline or
proline itself only gave glycerol without any trace of reductive
Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, CH-3012, Berne, Switzerland.
E-mail: reymond@ioc.unibe.ch; Fax: +41 31 631 80 57;
Tel: +41 31 631 43 25
{ Electronic supplementary information (ESI) available: NMR spectra of
the N-alkylation products. See DOI: 10.1039/b600703a
Scheme 1 Aqueous aldol reactions of zinc-proline and reductive
trapping of the iminium intermediate.
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Scheme 2 The umbelliferyl ether of DHA 5 is a fluorogenic probe for
enolization.
catalysts should also be apparent by measuring this deprotonation
directly. The umbelliferyl ether of DHA
5 should allow
measurement of the reaction by fluorescence since enolate
formation should be followed by b-elimination of umbelliferone
6 due to the strong leaving group ability of this fluorophore
(Scheme 2).12 Ketone 5 indeed liberated umbelliferone under the
action of zinc-proline and the various amine catalysts with an
apparent b-value of 0.55, also consistent with rate-limiting a-CH
deprotonation (Fig. 2B). The reactivity of the various catalysts
with ketone 5 was proportional to their aldolization reactivity
with DHA, providing further support for rate-limiting enolate
formation in the aldol reaction (Fig. 1B).
Fig. 1 Reactivity of zinc-proline and amines as catalysts for aldol
reactions of nitrobenzaldehyde 1 and deprotonation of probe 5 in aqueous
buffer pH 8.5. A) Aldol reaction with DHA (y-axis) and acetone (x-axis).
B) Aldol reaction with DHA (y-axis) as function of the rate with probe 5
(x-axis). Conditions for aldol reactions: 100 mM 1, 10 mM catalysta),
50% v/v aq. 20 mM bicine buffer pH 8.5, and 50% v/v 2 M DHA in
MeOH, or 50% v/v acetone. Formation of aldols 4 (t = 36 h) or 2 (t = 3 h)
was determined by RP-HPLC. Fluorogenic reaction of probe 5: 200 mL
assays in polypropylene 96-well plates were initiated by mixing 180 mL
20 mM catalyst in 20 mM aq. bicine buffer pH = 8.5 with 20 mL 1.0 mM 5
in buffer with 10% CH3CN. The fluorescence increase was measured at
The reaction of ketone 5 with NaBH4 and zinc-proline, proline,
or hydroxyproline only gave umbelliferone and the reduced
1,2-diol, with no detectable N-alkylation product as analyzed by
HPLC, suggesting that enolization of 5 occurs via the enolate only.
Nevertheless, the fluorogenic reaction of 5 was catalyzed by
aldolase antibody 38C2 (Km = 81 mM, kcat = 0.16 min21, kcat/kuncat
= 1570). The reaction was completely inhibited by acetyl acetone,
showing that the active site lysine residue was responsible for the
reaction. Antibody 38C2 also catalyzes aldol reactions with
a-hydroxy ketones very efficiently,13 and is probably using an
enamine mechanism with these substrates as well as with 5.
In summary, the formation of N-alkylation products with
acetone and zinc-proline, proline or Hyp together with the absence
of aldolization catalysis with tertiary amines, supports an enamine
(type I) mechanism of aldolization with acetone as substrate. By
contrast, the absence of N-alkylation of the same catalysts using
DHA and the occurrence of aldolization under general base
catalysis by various amines including tertiary amines indicate an
enolate (type II) mechanism. The mechanistic switch from type I to
type II from acetone to DHA is presumably caused by the OH
groups, which lower the pKa of the a-CH by inductive effect and
probably stabilize the enolate by intramolecular hydrogen
bonding, thereby facilitating enolate formation. Inductive effects
by the OH groups should also destabilize the iminium cation,
rendering enamine formation more difficult.
l
em = 460 ¡ 20 nm (lex = 360 ¡ 20 nm) using a Cytofluor II plate reader
from Perseptive Biosystems. a)The pH of all stock solutions were adjusted
to pH 8.5 with 0.1 M NaOH/HCl.
If deprotonation at the a-carbon is rate-limiting for the aldol
reaction of DHA, the aldolase reactivity of the different amine
Zinc-proline appears to be a particularly efficient catalyst for
both enamine and enolate type catalysis. This dual reactivity is
shared by PMP and in part by Hyp, both of which feature a
pyrrolidine with a relatively low pKa (Hyp: 9.73, PMP: 7.31, 10.94)
which can act as an enamine catalyst in one case and as a general
base in the other case. Addition of base to zinc-proline induces
Fig. 2 Brønsted plots: A) Rate of aldol reaction between DHA and
nitrobenzaldehyde (1) as a function of pKa values of the different amines
(b = 0.45, r2 = 0.75). B) Rate of reaction of probe 5 as a function of pKa
values of the different amines (b = 0.55, r2 = 0.88). Assay conditions are as
in Fig. 1.
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that the reactivity with probe 5 correlates with aldolase reactivity
for a variety of catalysts suggests that it might be useful for high-
throughput screening of aldolases under aqueous conditions.14
Applications of this screening method to discover aldolases from
combinatorial libraries of peptide dendrimers15 will be reported in
due course.
This work was supported by the University of Berne, the Swiss
National Science Foundation, the Marie Curie Training Network
IBAAC, and the COST Action D25.
Notes and references
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Scheme 3 Mechanisms of zinc-proline catalysis.
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The fluorogenic probe 5 reported here offers a simple means for
measuring catalysis of a-carbon deprotonation, and provides a
useful high-throughput screening assay for this process. The fact
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