Aqueous aldol catalysis by a nicotine metabolite

Article abstract of DOI:10.1021/ja017774f

Nornicotine, an endogenous tobacco alkaloid and minor nicotine metabolite, can catalyze aldol reactions at physiological pH. Catalysis appears to be due to a covalent enamine mechanism, an unprecedented reaction with small organic molecule catalysts in aqueous buffer. Kinetic parameters for nornicotine as well as other related alkaloids were measured and demonstrate that both the pyrrolidine and pyridine rings are critical for optimal catalysis. Substrate compatibility of this catalyst and its implications in vivo are discussed. Copyright

Full text of DOI:10.1021/ja017774f

Published on Web 03/08/2002
Aqueous Aldol Catalysis by a Nicotine Metabolite
Tobin J. Dickerson and Kim D. Janda*
Department of Chemistry, The Scripps Research Institute, and The Skaggs Institute for Chemical Biology,
10550 North Torrey Pines Road, La Jolla, California 92037
Received December 14, 2001
The aldol reaction, in addition to being an effective method for
the formation of carbon-carbon bonds in organic synthesis, is also
a critical biological reaction in the context of metabolism. A great
deal of effort has been devoted to the discovery of stoichiometric
and catalytic methods that predictably and efficiently generate aldol
products with known stereochemistry.1 Biological methodologies,
Figure 1. Structures of nornicotine 1 and proline.
such as aldolase enzymes1,2 and catalytic antibodies,3 have also been
extensively developed for accomplishing aldol reactions with high
efficiency and selectivity.
Scheme 1. Synthesis of Nornicotine and Related Modified
Alkaloids
Recently, small organic molecules have been shown to serve as
enantioselective catalysts for chemical reactions. Indeed, compounds
such as proline will catalyze a variety of processes including the
aldol reaction.4,5 The proposed mechanism of this reaction is
reminiscent of a type I aldolase, that is, the amine catalyst activates
the aldol donor by forming an iminium ion which then is converted
to the corresponding enamine nucleophile. Interestingly, proline,
an essential building block of biomolecules, has not been shown
to act as an “aldolase” under physiological conditions.
The use of water has been studied in the context of aqueous
Mannich reactions and the total synthesis of compounds under
physiological conditions can be found in the literature of the late
1930s.6 Furthermore, although catalytic aqueous aldol reactions are
documented, all involve metal-based Lewis acid activation of the
acceptor and frequently use silyl enol ethers as aldol donors.7
However, to our knowledge only enzymes have been shown to
catalyze aldol reactions in water via an enamine-based mechanism.
Nornicotine 1 is a nicotine-related alkaloid that is found both
endogenous in tobacco and as a minor metabolite of nicotine in
vivo.8 Nicotine has been identified as the addictive constituent of
tobacco, while nornicotine has been found to be the only psycho-
active nicotine metabolite.9 Nornicotine has a longer half-life in
vivo relative to its parent compound, nicotine,10 and therefore heavy
smokers can have relatively constant plasma levels of nornicotine.
On the basis of our work in the treatment of nicotine addiction by
immunopharmacotherapy,11 we recognized the structural similarity
between nornicotine and proline (Figure 1). Herein, we demonstrate
that nornicotine, a biologically relevant constituent of tobacco, can
serve as an aldol catalyst. This represents the first example of a
small molecule organic catalyst for aldol reactions that operates
exclusively in aqua.
in aqueous phosphate buffer (pH 7.4, 12 h), the aldol addition
product, 4-hydroxy-4-(4-nitro-phenyl)-butan-2-one, was formed in
81% yield with no apparent dehydration or retro-aldolization of
the product.12,13 In the absence of catalyst, the aldol product was
formed in less than 6% yield.
To determine if the observed catalysis was due to a unique
property of nornicotine or if other substituted pyrrolidines could
also catalyze the aldol reaction in water, compounds 2a, 2b, and 3
were synthesized according to known procedures (Scheme 1).14 By
using acetone as the donor and 4-nitrobenzaldehyde as the acceptor,
the pseudo-first-order rate constant for each compound was
determined (Table 1). Surprisingly, proline and pyrrolidine exhibited
little difference as catalysts while the three pyridine-containing
alkaloids displayed the highest rate enhancement over background.
Furthermore, the nature of the aromatic ring clearly plays some
role in the efficiency of the catalyst as 2-phenylpyrrolidine (3)
showed acceleration over the uncatalyzed rate with a rate constant
comparable to that of 2-pyridyl(2′-pyrrolidine) (2a). Finally, the
combination of pyridine and pyrrolidine (1:1) did not serve as an
effective catalyst (entry 6, Table 1).
There are two distinct possibilities for the catalytic mechanism
of the observed aldol reaction, general base catalysis and covalent
catalysis via an enamine nucleophile. A series of experiments was
conducted to provide evidence for either of these mechanisms.
Initially, we studied the reaction of acetone with 4-nitrobenzal-
dehyde. According to the precedent shown with proline,4 we
attempted to perform the reaction in anhydrous DMSO/acetone (4:
1) with 30 mol % 1. Interestingly, no aldol addition was observed,
even after extended reaction times, varied amounts of 1, and
elevated temperatures. Furthermore, catalysis was not observed in
any common organic solvents including chloroform, benzene,
acetonitrile, THF, and DMF. However, by performing the reaction
1
Using 2D-ROESY H NMR, the chemical exchange of acetone
for the corresponding enamine was observed. Additionally, upon
mixing nornicotine and acetone in buffer, we were able to trap
the putative enamine species as the corresponding amine using
NaCNBH3, indicating the possibility of an aqueous enamine
mechanism. Attempts at trapping the corresponding enamine inter-
mediate between proline and acetone in water were not successful,
* Address correspondence to this author. E-mail: kdjanda@scripps.edu.
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J. AM. CHEM. SOC. 2002, 124, 3220-3221
10.1021/ja017774f CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Pseudo-First-Order Rate Constants for the Reaction of
Acetone with 4-Nitrobenzaldehyde in the Presence of Amine
Catalysts (30 mol %) in Phosphate Buffera
catalyst. However, the ability of nornicotine to catalyze the aldol
reaction and other biologically significant enamine processes in ViVo
is highly relevant. Furthermore, to our knowledge, this demonstrates
the only known example of a metabolite capable of serving as a
catalyst.
entry
catalyst
k )
obs (min-1
1
2
3
4
5
6
7
8
9
1
2a
2b
3
10.1 × 10-3
8.2 × 10-3
9.4 × 10-3
6.4 × 10-3
2.4 × 10-3
1.3 × 10-3
1.8 × 10-3
0.7 × 10-3
0.7 × 10-3
Acknowledgment. The authors thank Dale Boger, Albert
Eschenmoser, Jonathan McDunn, and Paul Wentworth, Jr., for
helpful discussions as well as The Skaggs Institute for Chemical
Biology for financial support.
pyrrolidine
pyrrolidine + pyridine (1:1)
proline
nicotine
background
Supporting Information Available: Experimental procedures for
the preparation of all compounds and conditions for kinetic experiments
and measurement of order of nornicotine (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
a Kinetic assays were performed in aqueous buffer (200 mM sodium
phosphate, pH 8.0) at 37 °C with 10% DMSO to enhance substrate
solubility. The reaction was followed by monitoring generation of the aldol
addition product by reversed-phase HPLC. The assay was started by addition
of aldehyde (1-8 mM in DMSO) to a mixture of the catalyst (2.4 mM)
and acetone (240 mM) in the aqueous buffer system. Rate constants were
calculated by using linear regression analysis.
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suggesting that the nornicotine-derived enamine is stabilized in some
fashion to prevent hydrolysis relative to the proline-derived enamine.
A general base mechanism could also be invoked; however, nicotine
would be expected to catalyze the reaction at a comparable rate to
nornicotine due to their similarities in pKa. Yet, when nicotine was
used as a catalyst, no rate acceleration was observed. The order of
nornicotine was determined to be unity, implying the involvement
of one nornicotine molecule in the transition state of the rate-
determining step. An examination of the pH rate profile of this
reaction showed that above pH 9.5, catalysis of the reaction by
nornicotine becomes minimal as general base catalysis due to
hydroxide dominates. However, between pH 7.5 and 8, optimal
rate acceleration (kcat/kbkgd) was observed with over twenty turnovers
at 2.5 mol % 1.
Substrate specificity was briefly examined and it was found that
activated aldehyde acceptors are critical for the reaction to proceed.
Thus, aldehydes having suitable water solubility and electronic
activation, as with 4-nitrobenzaldehyde and 2-chlorobenzaldehyde,
are acceptors. This observation can be explained in the context of
the proposed mechanism. If the hydrolysis of the enamine inter-
mediate is significantly faster than carbon-carbon bond formation,
no product will be formed. We have also looked at a small set of
donors, simple ketones such as hydroxyacetone and cyclopentanone,
that were found to be competent reaction partners with 4-nitro-
benzaldehyde. Included in this list was also pyruvate, a biologically
relevant compound in metabolism. The compilation of these
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enamine-based processes. This could have significant implications
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reversed-phase HPLC.
We have demonstrated that nornicotine can catalyze aldol
reactions in water. Our findings suggest that nornicotine could
catalyze in vivo aldol reactions as maximum rate acceleration is
observed near physiological pH. The proposed mechanism will
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appears that the catalysis occurs via an enamine nucleophile,
contrary to the known rapid hydrolysis of enamines in water.15 At
this juncture, nornicotine would not be synthetically useful as a
(13) Both isomers of nornicotine were tested for enantioselective catalysis and
a slight amount of asymmetric induction (∼20% ee) was observed with
either enantiomer (data not shown).
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