DOI: 10.1002/chem.201404497

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& Chiral Amplification

A Closer Look at Spontaneous Mirror Symmetry Breaking in Aldol Reactions Guillem Valero, Josep M. Rib, and Albert Moyano*[a] In memory of Carlos F. Barbas III

mirror-symmetry-breaking process, which originates at very low conversions (i.e., strongly depends on events taking place at the very first stages of the process). The reaction is not autocatalytic in the aldol product, which rules out a simple Frank-type reaction network as the source of the observed symmetry breaking. On the other hand, the isolation and characterisation of a double-aldol adduct suggested a reaction network that involved both indirect autocatalysis and indirect mutual inhibition between the enantiomers of the reaction product.

Abstract: The aldol reaction between acetone and 4-nitrobenzaldehyde run in the nominal absence of any enantioselective catalyst was monitored by chiral HPLC with the aid of an internal standard. The collected data show the presence of a detectable initial enantiomeric excess of the aldol product in the early stages of the reaction in about 50 % of the experiments. Only a small fraction of the reaction contained the non-racemic aldol product after 24 h. This temporary emergence of natural optical activity could be the signature of a coupled reaction network that leads to a spontaneous

Introduction It has long been recognised that understanding the origins of terrestrial biological homochirality (the fact that only one enantiomeric form of amino acids or carbohydrates can be found in living organisms) is an essential pre-requisite of any theory dealing with the origins of life on Earth.[1] In this context, physical and chemical processes that take place with spontaneous mirror symmetry breaking (SMSB) are usually invoked as a possible source of the homochirality of biomolecules.[2] The possibility of chemical reactions that start from achiral reagents, and can lead to an essentially homochiral (i.e., enantiomerically homogeneous) product by amplification of a very tiny enantiomeric imbalance was theoretically predicted by Frank in 1953.[3] In this seminal paper, Frank proposed that SMSB would be readily achieved for a reaction network composed of an irreversible asymmetric autocatalysis (i.e., a reaction in which the chiral product L (or D) enantioselectively catalyses its own formation from an achiral compound, M) coupled with an irreversible mutual inhibition reaction between the product enantiomers L and D (Scheme 1). The original Frank model predicts that a homochiral stationary state (i.e., a state in which all of the molecules of the chiral product have the same absolute

Scheme 1. Frank model for SMSB in chemical reactions.

configuration) will eventually be reached if the concentration of the initial achiral reagent(s) M is held constant. Later, generalisation of the Frank model showed that in any (not necessarily irreversible) reaction network in which the two enantiomers of a chiral autocatalytic species are enantioselectively generated from achiral reactants and react in a heterochiral fashion to give a catalytically inactive product, a critical concentration value of the achiral reactants can always be found. Above this concentration the racemic stationary state becomes unstable, so a very small enantiomeric excess (ee) of the chiral product (that may arise spontaneously by random fluctuations around the racemic composition) will drive the system to a thermodynamically more stable non-racemic (but not necessarily homochiral) stationary state.[4, 5] The confinement of a Frank-type reaction network in a closed system (i.e., a system that can only exchange thermal energy with its surroundings, so that the starting achiral material M is consumed and the inhibition product P remains in the system) will inevitably lead to an equilibrium racemic state,[5, 6] because of the restrictions imposed by the principle of micro-

[a] G. Valero, Prof. Dr. J. M. Rib, Prof. Dr. A. Moyano Departament de Qumica Orgnica Universitat de Barcelona, Facultat de Qumica C. Mart I Franqus 1-11, 08028-Barcelona, Catalonia (Spain) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404497. Chem. Eur. J. 2014, 20, 1 – 15

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Full Paper scopic reversibility.[7] However, in closed systems with quasiirreversible linear enantioselective autocatalysis and a fast exergonic mutual inhibition step (closed Frank-type systems), a kinetically controlled transient non-racemic state can also be reached, in which the system is resilient towards racemisation, and can occur much later than the time necessary for complete conversion of the starting material M.[8] Considerable attention has also been paid to so-called recycled systems.[9] These can be built from reaction networks that take place in systems closed to matter flows but exhibit a specific exchange of energy between their surroundings and some chemical species of the reaction network. In this way, chiral photo- or mechano-stationary states can be achieved by coupling of one reaction to an external source of energy. As another example, a reversible Frank-type reaction network that takes place in a closed system can give rise to a non-racemic stationary state because of the contra-thermodynamic recycling of the inhibition product P to the achiral starting product(s) M, brought about by coupling to an external irreversible reaction (Scheme 2). Notably, in recycled systems linear enan-

the principle of microscopic reversibility determine that in either open-flow or closed-system conditions a racemic final state will always be reached.[6, 7] However, it has recently been shown that the mirror symmetry of the final stationary state can be broken in a closed limited enantioselectivity system in which both autocatalytic reactions are spatially separated at different temperatures in two compartments between which matter can be exchanged by means of a continuous internal flow.[12] We can conclude that, in principle, several reaction schemes can be devised that convert enantioselective autocatalysis into a bifurcating SMSB system by coupling with other reactions in a non-linear fashion. In contrast to this, experimental evidence that can give support to a SMSB chemical process taking place in a homogeneous phase (“absolute asymmetric synthesis”) is still very scarce.[13] In fact, the first clear-cut example of a reaction that showed both highly enantioselective autocatalysis and strong chirality amplification was reported by Soai and co-workers in 1995.[14] In an improved version of this process, the chiral pyrimidyl alcohol obtained by the addition of diisopropyl zinc to a 2-(tertbutylethynyl)-pyrimidyne-5-carboxaldehyde could amplify the extremely tiny ee of the product (  0.00005 % ee) to almost enantiomeric purity (57 % ee in the first batch, 99 % ee in the second batch, > 99.5 % ee in the third batch) in a sequential batch reaction protocol (Scheme 3).[15] The ability of the Soai reaction to amplify the statistical fluctuation between the ratio of the two enantiomers in a nominally racemic mixture was dramatically demonstrated in 2003, when it was shown that in 1:3.7 toluene/diethyl ether enantiomerically enriched pyrimidyl alcohols were obtained with an approximately stochastic distribution of the two enantiomers

Scheme 2. Recycling model for SMSB in a reversible, Frank-type reaction network taking place in a system closed to matter flow.

tioselective autocatalysis and mutual inhibition can be replaced by unidirectional reaction fluxes, which convert the minor enantiomer into the major enantiomer, a process that is also achieved by recycling of the products to the starting materials and is driven by an external source of chemical free Scheme 3. Asymmetric autocatalysis with chirality amplification in the Soai reaction. energy.[10] It is worth noting that in the Frank model it is assumed that (Scheme 4).[16] However, it is worth noting that under the initial the heterochiral interaction between opposite enantiomers of the product is favoured with respect to a homochiral interacreaction conditions developed by Soai (only toluene as the tion. This is the more usual situation for chiral compounds, solvent) a non-stochastic distribution of enantiomers was obgauged from the relatively low number of compounds that served[17] and it took several years to develop experimental crystallise as racemic mixtures of enantiopure crystals (racemic conditions that could reproducibly lead to a stochastic conglomerates),[11] although this is not a necessary condition, distribution of products. In the presence of cryptochiral compounds, in which the exemplified by the “limited enantioselectivity” model first poschirality is only due to isotopic substitution at carbon or ited by Avetisov and Goldanskii.[2d] In this model, a reversible oxygen atoms, the stochastic behaviour of the SMSB in the non-enantioselective autocatalysis substitutes the mutual inSoai reaction becomes totally deterministic, so that the absohibition of the Frank reaction network, so SMSB is mathematilute configuration of the major enantiomer of the chiral alcocally possible, but the thermodynamic constraints imposed by &

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Scheme 4. SMSB in the Soai reaction.

hol can be correlated with the absolute configuration of the isotopically chiral compound added as a “chiral inductor”.[18] Several experimental[19] and theoretical[20] studies have elucidated important mechanistic features of the Soai reaction, but a complete explanation of the dynamics of this process is still lacking.[21] In any case, it is clear that kinetic models derived from the classical Frank-type scenario (Scheme 1) can account for the unique experimental features of the Soai reaction,[8, 22] for instance the need to maintain a low concentration of the catalyst/product in each step of the sequential batch procedure to achieve detectable ee values from nominally achiral conditions.[8] The Soai reaction stands out as a paradigm for absolute asymmetric synthesis in the absence of external chiral polarisations, but its role as a model chemical process in the emergence of biological homochirality has been rightly questioned given that it involves highly sensitive organometallic compounds, which are incompatible with the aqueous conditions of prebiotic chemistry on Earth.[2e] The first fully organic example of asymmetric autocatalysis was disclosed by Mauksch and co-workers in 2007; these authors demonstrated that the chiral Mannich adduct from the reaction between acetone (1) and the (p-methoxyphenyl)imine of ethyl glyoxylate (2) was able to replicate itself with moderate yield and good enantioselectivity.[23] Similar results for enantioselective organo-autocatalysis of the Mannich reaction of 2 with cyclohexanone[24] or isobutyraldehyde[25] were subsequently reported by other authors. The observation of SMSB in the asymmetric organo-autocatalytic Mannich reaction was also reported by Mauksch, Tsogoeva and co-workers in 2007.[26] Starting from nominally achiral starting conditions, the reaction between 1 (used as a solvent) and 2 (c = 0.25 m) afforded the optically active Mannich adduct 3 (up to 9.5 % ee, determined by HPLC) with variable yields (up to 36 %) after 2–8 d at room temperature (Scheme 5). In the same paper, evidence for SMSB in the aldol reaction between 1 (used as a solvent) and 4-nitrobenzaldehyde (4) was also given (Scheme 6). Depending on the concentration of aldehyde 4 (c = 0.50–5.80 m), variable amounts of DMSO (0– 20.5 % v/v) were also present to ensure the solubilisation of 4. After stirring for 10 d at room temperature, non-racemic aldol adduct 5 was obtained with variable enantiomeric purities (up to 50.8 % ee, determined by HPLC) but in very low yields (0.6–11 %).[26] Chem. Eur. J. 2014, 20, 1 – 15

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Scheme 5. SMSB in the Mannich reaction.

Scheme 6. SMSB in the aldol reaction.

In another set of experiments subsequently performed by the same research group under similar, but somewhat more uniform, conditions (1 as solvent, DMSO (2.6–3.3 % v/v), [4] = 1.33 m), enantioenriched aldol product 5 was again consistently obtained (reaction yields were not reported), but the enantiomeric purities were considerably lower (0.9–14.3 % ee, determined by HPLC after purification by TLC). Although in this second set of experiments the distribution of the absolute configurations of the product was more random than in the preceding case, a clear predominance of the S-configured aldol adduct 5 was again observed (seven-times enantioenriched (S)-5 versus two-times enantioenriched (R)-5) if only experiments that gave enantioselectivities higher than 2 % ee were considered.[27] The spontaneous emergence of chirality in aldol and Mannich reactions, which are much less exergonic than the addition of alkylzinc reagents to aldehydes, therefore take place under reversible conditions (long reaction times, low yields), cannot be easily rationalised within the framework of the Frank reaction network (because linear autocatalysis alone, in the absence of strong mutual inhibition between opposite enantiomers of the chiral product, cannot lead to asymmetric amplification).[8] The situation was originally explained as arising from a closed reaction network, in which linear asymmetric autocatalysis by the chiral aldol or Mannich adduct is followed by endergonic formation of labile heterochiral dimers, then re3

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Full Paper cycling to the achiral reagents.[23, 26–28] However, as discussed above, SMSB in a recycled system can only take place by coupling of the recycling reaction to an irreversible process that maintains the whole system out of the equilibrium,[10b] a requisite that is not easily fulfilled under the typical conditions of aldol or Mannich reactions. On the other hand, in view of the importance of mirror symmetry breaking in reversible organic reactions closely related to key biochemical processes, further understanding of this rather overlooked and strongly counterintuitive phenomenon is necessary. We report the results of a re-examination of the SMSB in aldol reactions, for which we have followed the evolution of both the yield and enantiomeric purity of aldol adduct 5 by HPLC and isolated and identified a previously unnoticed side product that may play a significant role in the observed chirality amplification.

Three main conclusions were drawn from these results: 1) The formation of aldol adduct 5 in the absence of any nominal catalyst was clearly observed, although the approximate yields were much lower than those initially reported by Mauksch et al. in 2007.[26] Note, no yields were given in the 2010 report.[27] 2) Although the majority of the experiments led to the formation of essentially racemic 5, in two instances the ee determined by HPLC was higher than 2 % (2.4 and 15 % ee, respectively). 3) Contrary to the previous results of Mauksch et al., which showed a statistical bias towards the S-aldol adduct,[26, 27] we observed that SMSB in the aldol reaction led to the predominant formation of the R enantiomer. However, this observation was not statistically meaningful due to the small number of experiments that gave rise to non-racemic aldol adduct 5.

Results and Discussion In view of the long reaction times, we reasoned that it would be very convenient to develop an alternative experiment that would allow us to monitor both the yield and enantiomeric composition of the aldol reaction during the first hours of the reaction. We decided to perform the aldol reaction in the presence of a suitable internal standard, to determine the composition of the reaction mixture by chiral HPLC analysis. After some experimentation, we found that commercially available 2,7-dimethoxynaphthalene (6) was highly convenient for this purpose because it was totally soluble in the reaction medium, was unlikely to affect the reaction and presented a relatively small absorptivity at l = 265 nm (the wavelength at which 5 exhibits the maximum absorption). By means of a diode-array UV/Vis detector, we examined four different wavelengths (l = 250, 260, 270 and 280 nm) and by analysis of four samples that contained different 6/5 molar ratios, we found, for all wavelengths, a good linear relationship with the corresponding peak-area ratios (we used a racemic sample of 5 with a chiral HPLC column (Chiralpak IC), therefore we summed the areas of the two peaks that corresponded to each enantiomer of 5). Figure 1 shows the calibration graph obtained at l = 250 nm (see the Supporting Information for the results at other wavelengths). Next, we ran a series of experiments under the same conditions that we had used for the “preparative” reactions

Assessment of SMSB in the aldol reaction between 1 and 4 in the nominal absence of any chiral catalyst (Mauksch and Tsogoeva’s conditions) Firstly, we checked the reproducibility of SMSB in the aldol reaction between 1 and 4 by following the experimental procedure reported by Mauksch et al.[26, 27] Thus, we performed several aldol reactions in which a solution of 4 in acetone (c = 1.29 m) that contained DMSO (5 % v/v), typically at a 1.0 mmol scale, was magnetically stirred in a tightly closed reaction flask (to prevent evaporation of 1). After stirring for 10 d at room temperature, 1H NMR analysis of the reaction mixture showed the formation of aldol adduct 5 with an estimated yield of 1– 2 % (Scheme 7). After evaporation of the solvent, a portion (5–

Scheme 7. SMSB in the aldol reaction (present results).

10 % w/w) of the crude reaction mixture was purified by preparative TLC on a single plate, to avoid self-disproportionation of the enantiomers in the non-racemic mixture.[29] The band that corresponded to the aldol adduct was carefully removed from the TLC plate, extracted with ethyl acetate, and concentrated to afford pure 5, the enantiomeric composition of which was established by chiral HPLC analysis by comparison to a racemic standard obtained by a racemic proline-catalysed reaction between 1 and 4. &

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Figure 1. HPLC calibration graph for aldol adduct 5 with 6 as an internal standard.

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Scheme 8. Aldol reaction between 1 and 4 with 6 as an internal standard.

described above: 4 in solution in 1 (c = 1.29 m) and DMSO (5 % v/v), scale = 1.0 mmol, in the presence of 6 (5 mol %), stirred at room temperature in a septum-capped flask (Scheme 8). Aliquots were removed from the reaction mixture at regular intervals and directly analysed by chiral HPLC. In all instances, we observed the formation of aldol product 5. The yield increased very slowly and linearly with time and typical values (peak areas measured at l = 250 nm) were 0.6 % after 10 d and 0.8 % after 17 d (consistent with our previous estimation by 1 H NMR spectroscopy). A representative graph is shown in Figure 2 (see the Supporting Information for more details).

Figure 3. HPLC monitoring of SMSB in the aldol reaction.

Table 1. SMSB in the aldol reaction in the presence of co-solvents. Entry

Co-solvent ([% v/v])

Results[a]

1 2 3 4 5 6 7

– DMSO (20) hexane (20) toluene (20) dichloromethane (20) water (1) 2-propanol (10)

1  S, 1  rac 1S 1  rac 1  rac 1  R, 6  rac 1  rac 1R

[a] The predominant enantiomer of aldol adduct 5 after 4 h, according to HPLC analysis, is indicated. We consider the aldol product to be racemic (rac) if the difference between the absolute ee value and the standard deviation is less than 2 %.

DMSO (5 % v/v), [4] maintained at 1.29 m) on the outcome of the reaction (Table 1). In the absence of DMSO as a co-solvent (Table 1, entry 1) we found in one experiment the predominant formation of (S)-5 (8.9  3.1 % ee after 4 h, 0.011  0.001 % yield), but in another experiment no significant SMSB could be detected. In addition, we ran a parallel series of experiments with different co-solvents (Table 1, entries 2–7). In an acetone/dichloromethane solvent mixture (1:1 v/v), the R enantiomer of the aldol product 5 formed after 4 h (0.017  0.003 % yield) was clearly predominant in the reaction mixture (45  6.1 % ee). However, in a subsequent series of six independent experiments performed under the same conditions, the aldol product formed after the first 4 h of the reaction was essentially racemic (Table 1, entry 5). In summary, SMSB can be observed in media other than acetone/ DMSO (5 % v/v), but only in a small fraction of the experiments (4 instances in a total of 14 experiments). After a total of 39 experiments, we obtained the following results:

Figure 2. HPLC monitoring of the formation of aldol adduct 5.

However, it is worth noting that in some experiments higher yields of aldol product 5 were observed (5 % yield after 15 d, measured both by HPLC and 1H NMR spectroscopy). A parallel experiment run without internal standard 6 also showed the formation of 5 with a similar reaction rate (estimated by 1H NMR spectroscopy), thus any possibility of catalysis of the reaction by 6 was discarded. From measurement of the area ratios of the two peaks that correspond to each enantiomer of 5, we determined the enantiomeric composition of each aliquot. A representative example (for experiments that take place with SMSB) is shown in Figure 3 (see the Supporting Information for more examples). The measured enantiomeric composition is slightly different for each wavelength, but the standard deviation is much lower than the mean value. The highest ee value (30 % ee of (R)-5) corresponds to the first measurement (1 h after the addition of 4), when the yield of 5 is about 0.01 %. This ee value drops rapidly with time and the aldol product is essentially racemic 24 h after the beginning of the reaction (0.05 % yield). In an independent set of experiments, we investigated the effect of changes in the standard solvent composition (1 with Chem. Eur. J. 2014, 20, 1 – 15

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1) In 22 instances, no SMSB was observed, and from the beginning of the reaction essentially racemic (< 2 % ee) aldol adduct 5 was obtained. In these reactions, the yields of rac5 after 4 h were 0.00–0.013 % and 0.006–0.064 % after 24 h. In one isolated example run in the presence of CH2Cl2, a 0.232 % yield of rac-5 was measured after 24 h. 2) In 15 instances, non-racemic (R)-5 was initially formed (ee > 2 % after 4 h, yield = 0.002–0.022 %). In these experiments, the conversion and ee were monitored again after 24 h and 5

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Full Paper we found that the yield of 5 had uniformly increased (0.008–0.282 %) and that the aldol adduct 5 was racemic in 7 of these 15 experiments. Partial racemisation was observed in another 7 experiments and, in one instance, a small increase of the enantiomeric purity (from 38.8 to 43.7 % ee) was observed. In another two experiments, the enantiomeric excess of 5 measured after 24 h was also greater than 40 % ee. It is worth noting here that in all of these three experiments, the yield of 5 after 24 h was either equal to, or somewhat higher (less than 1.7 times), than that observed after 4 h. 3) In one instance, non-racemic (S)-5 was initially formed (7 % ee after 4 h, 0.003 % yield). In this case, after 24 h the yield of 5 had increased to 0.008 %, but the product was essentially racemic. 4) In one experiment (run in the absence of DMSO), the aldol adduct 5 (0.004 % yield) was racemic after 4 h, but after 24 h the predominant formation of (S)-5 (0.019 % yield, 6.9 % ee) was observed.

Table 2. SMSB in aldol reactions performed in other laboratories.

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Laboratory 1 (ee [%])

Laboratory 2 (ee [%])

13 rac 17 rac

8 rac 5 rac

purity had diminished to 5.1 % ee. In the presence of d-diethyl tartrate similar yields of 5 were recorded (0.005 % after 4 h and 0.064 % after 24 h), but the product was racemic. The reaction does not seem to be affected by the presence of this nonracemic chiral diol. The rapid drop of the enantiomeric purity of 5 with time (after 24 h, only racemic aldol was present in the majority of the experiments) could be due to racemisation of the aldol product under the reaction conditions. To test this hypothesis, we prepared a dilute solution (1.55  10 4 m) of (R)-5 (26 % ee, prepared by mixing 73 % ee (R)-5[30] with racemic product) in 1, that also contained DMSO (5 % v/v) and naphthalene 6 (800 mol %)—to mimic the typical reaction composition after 4 h—and a catalytic amount of aldehyde 4 (5 mol %), to see if its presence was necessary in any reaction leading to racemic product from enantioenriched 5, and we monitored the evolution of the ee of the aldol product 5. No significant drop of enantiomeric purity was observed in the first 25 h (Figure 4).

A series of experiments were performed under the same conditions with aldehydes other than 4. When 2-nitrobenzaldehyde was used, we observed that the formation of the aldol adduct took place with a similar reaction rate, but we could not find HPLC conditions to obtain a reliable measure of the enantiomeric purity of the aldol adduct. On the other hand, the use of 4-cyanobenzaldehyde always led to the formation of racemic aldol product, even in the first few hours of the reaction. To make sure that the enantiomeric composition of 5 measured in situ was not an artefact of the analytical method, in one independent experiment we measured a 7 % ee (of the R enantiomer) after 95 h (0.092 % yield, estimated by HPLC). At this point, we stopped the reaction by using the workup previously used for the “preparative” experiments. Aldol adduct 5 isolated from this crude reaction mixture after purification by column chromatography on silica gel showed essentially the same enantiomeric composition (8 % ee, (R)-5). We also investigated if the predominance of the R enantiomer observed in the reactions that led to the formation of non-racemic aldol product was due to an unidentified chiral impurity present in our laboratory. We performed two independent sets of experiments in two independent laboratories located in other parts of the building and used the glassware, magnetic stirrers, magnetic stir bars, solvents and reagents located in these laboratories (and from a different commercial source in the case of reagent 4). We monitored the enantiomeric composition of 5 by HPLC after 6 h (Table 2). Of a total of eight experiments, SMSB was observed in four cases (two from each laboratory), and (R)-5 was always the major enantiomer. We also performed two parallel experiments under the standard conditions, with the addition of either l- or d-diethyl tartrate (1 mol %). For the experiment run in the presence of l-diethyl tartrate, aldol product 5 formed after the first 4 h of the reaction (0.003 % yield) with 14.1 % ee (R enantiomer). After 24 h, the yield had increased to 0.059 % and the enantiomeric &

Experiment 1 2 3 4

Figure 4. HPLC monitoring of the stereochemical stability of 5 under the reaction conditions.

This sample was stored at room temperature in the laboratory for 3 months after which time a 24 % ee of 5 was determined by HPLC. Aldol adduct 5 is, therefore, stereochemically stable under the reaction conditions and the drop in enantiomeric purity can be explained by the assumption that any process that gives rise to non-racemic aldol product is only operative during the very first stages of the reaction, whereas racemic 5 is subsequently formed by a non-enantioselective process. We conclude that the results obtained from these monitoring experiments show that, in the nominal absence of any chiral catalyst, SMSB initially takes place in about 50 % of the reactions and non-racemic aldol product 5 is present in the reaction mixture after the first 4 h of the reaction. This symmetry-breaking process is only operative at very low conversions 6

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Full Paper (in general less than 0.02 % yield of aldol 5 is recorded after the first 4 h of reaction), and the subsequent slow, but continuous, formation of racemic aldol product leads to a rapid drop of the enantiomeric purity of 5. Only in a smaller fraction of the reactions (about 30 %) is the ee greater than 2 % after 24 h. In the reactions that give high ee values (40–50 % ee) for aldol adduct 5 after 24 h, the reaction that gives rise to the racemic aldol product after initial formation of the enantiomerically enriched product is very slow. A bias for the preferential formation of (R)-5 is observed. The “preparative” experiments (see Scheme 8 above) show that after 10 d the fraction of reactions that display non-racemic aldol adduct 5 is less than 20 %.

Bis(aldol) adducts as side products in the reaction between 1 and 4 During the course of monitoring the composition of the reaction mixture by 1H NMR spectroscopy after stirring for 10 d at room temperature in the “preparative” aldol reactions described in Scheme 8, we identified the aldol adduct 5 as the main product, although minor quantities of other products were apparent. During repeated preparative TLC purifications, we observed that, in addition to aldol product 5, a more polar, strongly UV-active product was subsequently eluted. We isolated this compound, which was formed in very small amounts and, after the extracts from several experiments were combined, we obtained about 4 mg of this byproduct. 1H NMR spectroscopic analysis of this sample suggested that this compound was in fact an inseparable diastereomeric mixture of the double-aldol addition product, namely 1,5-dihydroxy-1,5bis-(4-nitrophenyl)pentan-3-one (7), probably as an anti/syn mixture). Additional evidence for this species was subsequently obtained by HRMS. We set out to obtain this double-aldol adduct by an independent synthesis. However, a literature search revealed that the formation of bis(aldol) adducts of 1 from direct aldol reactions is an extremely inefficient process.[33] In their seminal studies of enantioselective aldol reactions catalysed by l-proline thioamides, Gryko and Lipinski[34] observed that the reaction of highly electrophilic pentafluorobenzaldehyde or 2-chlorobenzaldehyde with 1 (neat) afforded the desired aldol products, along with variable quantities (up to 40 % in highly concentrated solutions) of the double-aldol products (as anti/syn mixtures). On the other hand, the low yield of bis(aldol) byproduct 7 from the organocatalytic aldol reactions between 1 and 4 has been observed in several instances,[35, 36] but has never been adequately characterised. In light of these precedents, we set out to prepare enantiomerically enriched anti-7 by modification of the method reported by Gryko and Lipinski.[34] After considerable experimentation, we found that the best conditions involved treatment of 1 (1 equiv) with 4 (2 equiv) in the presence of the proline-derived thioamide catalyst 8 (20 mol %) and almost neat conditions (CH2Cl2 was added in the minimum amount required to obtain a suspension that could be efficiently stirred). In this way, by using (S,R)-8 (prepared from (S)-proline and (R)-(1-phenyl)ethylamine) as a catalyst, we obtained an inseparable mixture of (R,R)-anti-7 and syn-7 in 13 % total yield after 3 d at room temperature and chromatographic purification. The anti/ syn ratio was estimated as 3:1 (by 13C NMR spectroscopy) and chiral HPLC analysis determined an 80 % ee for anti-7 (Scheme 9). The use of the enantiomeric catalyst (R,S)-8 under the same reaction conditions allowed us to obtain enantiomerically enriched (S,S)-anti-7 (78 % ee), also in a 3:1 anti/syn mixture with syn-7. On the other hand, as described in the Experimental Section, racemic anti-7 was prepared (1:1 syn/anti) by using the conditions described by Peng and co-workers (pyrrolidine (20 mol %), phenol (20 mol %), neat).[35] With both enantiomerically enriched and racemic anti-7 in hand, we ran an aldol reaction under the same conditions as

Searching for asymmetric autocatalysis in the aldol reaction between 1 and 4 The phenomenon of asymmetric autocatalysis is a necessary (but not sufficient) condition for SMSB in a chemical reaction.[2d, 4b, 7b, 31, 32] As we discussed in the Introduction section, experimental evidence for asymmetric autocatalysis in the Mannich reaction was first disclosed by Mauksch, Tsogoeva and coworkers in 2007,[23] and was subsequently corroborated by other authors.[24, 25] On the other hand, it is notable that no analogous experiments for asymmetric autocatalysis in the aldol reaction can be found in the literature, besides a brief mention in the initial report by Mauksch et al.[23] It can be seen from Figure 2 above, in a typical experiment the rate of formation of aldol adduct 5 is constant in the first 400 h, thus the typical kinetic signature of an autocatalytic process, which calls for a sigmoidal dynamic profile,[32b] is clearly absent. We therefore set out to monitor the autocatalytic effect of optically active 5 in the reaction between 1 and 4 by HPLC. Aldol product (S)-5 with 73 % ee was prepared by the d-proline-catalysed reaction under the conditions described by List et al.[30] We next repeated the “analytical” aldol reactions described above, under the same conditions noted in Scheme 8, but in the presence of variable amounts (0.10–20 mol %) of enantioenriched aldol product (S)-5. The reaction was followed by HPLC, and the 6/5 ratio (after correction with respect to the initial concentration of 5) was monitored. Both the yield and ee values of the aldol product remained unchanged (within experimental error) during the first 30 h of the reaction. In an experiment in which the initial amount of enantioenriched (S)-5 was very low (0.15 mol %), we observed that after 10 d the yield of 5 had risen to only 0.20 %, with a concomitant drop of the measured enantiomeric purity (from the initial 73 % ee to 58 % ee). Thus, the newly formed aldol adduct 5 appeared to be essentially racemic. In addition, the use of enantiomerically enriched aldol adduct (from the proline-catalysed reaction of 1 with 4-cyanobenzaldehyde)[30] as an autocatalyst mimic in our benchmark process did not show any significant difference relative to a blank reaction. These data indicated that aldol adduct 5 is neither an asymmetric nor a non-enantioselective autocatalyst. Chem. Eur. J. 2014, 20, 1 – 15

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Scheme 9. Enantioselective preparation of the double-aldol adduct 7. Figure 5. HPLC monitoring of SMSB in the aldol reaction in the presence of 7. Yield [%] (^); ee [%] (*).

our “preparative” autocatalytic experiments (see Scheme 7 above), but on a much larger scale (3.3 mmol). After stirring for 28 d at room temperature and chromatographic purification of the crude reaction mixture (column chromatography, followed by preparative TLC), we obtained 7 (0.014 mmol, 0.8 % yield) in an anti/syn ratio of approximately 1:2. Together with chiral HPLC analysis, we could establish a maximum 4 % ee for anti-7, which suggested that under these conditions 7 is either formed in a non-enantioselective fashion or that it has racemised during the course of the reaction. Because the yields of mono-aldol adducts in our experiments, extrapolated to 28 d, typically fall in the 2–5 % yield range, the formation of the double-aldol 7 in such relatively large amounts is certainly surprising. We set out to investigate if this product could play a role in the symmetry-breaking process. First, we assessed the stability of the double-aldol product under the reaction conditions. We prepared a solution of 7 (1:1 syn/anti, 78 % ee of (S,S)-anti-7) in 1 (c = 0.064 m), in the presence of 6 as an internal standard (c = 0.071 m) and monitored the formation of mono-aldol product 5 by HPLC. After stirring for 25 h at room temperature, the yield of 5 (relative to the reaction of 7 with 1) was only 1 %, with a very small ee of (S)-5 (2 % ee). This indicates that the retro-aldol reaction of 7 is very slow, particularly for the anti isomer. Next, we performed an aldol reaction with our typical conditions [a solution of 4 in 1 (c = 1.291 m) that contained DMSO (5 % v/v) and internal standard 6 (5.1 mol %)], but in the presence of 7 (10 mol %, 2:1 anti/syn, 78 % ee of (S,S)-anti-7) as a catalyst (Scheme 10), and monitored both the yield and enantiomeric purity of the aldol product 5 by chiral HPLC (Figure 5)

The results of this experiment show that: 1) the yield of aldol product 5 after 24 h is 0.05 %, comparable to that previously obtained in several reactions run in the absence of any catalyst; 2) the ee of the initially formed aldol adduct (before 0.75 h) was high (  50 % ee) and diminished with time (30 % ee after 5 h, 13 % ee after 24 h), which indicated that after the initial stages of the reaction the newly formed aldol product had a very low ee (1.6 % ee calculated for the aldol product generated between 0.75 and 24 h); 3) the major enantiomer of 5 had the R configuration, as in the majority of our previous experiments. It should be noted that we used a mixture enriched in the S,S enantiomer of anti-7, therefore the newly formed, non-racemic aldol product 5 does not come from the retro-aldol reaction of 7, which, as we have seen, is very slow (and gives rise to (S)-5 with very low enantiomeric purity).

Thus, although 7 does not appear to have any catalytic effect on the aldol reaction, it seems that it may play a role in the symmetry breaking of the process. We also tested the effect of the initial presence of both the enantioenriched mono-aldol product (S)-5 (0.02 mol %, 68 % ee) and bis(aldol) product 7 (4.1 mol %, 2:1 anti/syn, 78 % ee for (S,S)-anti-7) in the aldol reaction (Scheme 11 and Figure 6). In this case, although we could detect an increase of the yield of 5 to 0.4 % after the first 24 h (when the reaction was run without initial addition of 5 or 7 we had observed a maximum 0.3 % yield of 5 after the first 24 h), the ee of 5 decreased exponentially, again indicative that the newly formed product was essentially racemic. In a similar experiment run in the presence of enantioenriched mono-aldol adduct (R)-5 (0.04 mol %, 73 % ee) and double-aldol product 7 (10 mol %, 3:1 anti/syn, 80 % ee for (R,R)-anti-7) the yield of mono-aldol product 5 was lower (0.12 % after 80 h), although higher than observed in a blank reaction (in which both 5 and 7 were not initially present in the reaction mixture), which afforded 0.03 % yield of 5 after 80 h. Again, we observed that the newly formed product was essentially racemic (we calculated a maximum 2 % ee for the aldol product 5 formed in the first 24 h). Scheme 10. Study of the catalytic role of 7 in the aldol reaction between acetone and 4.

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Scheme 13. Study of the effect of the double-aldol mimic anti-9 in the aldol reaction between 1 and 4.

Scheme 11. Study of the catalytic role of bis(aldol) product 7 in the presence of 5 in the aldol reaction between 1 and 4.

Figure 7. HPLC monitoring of SMSB in the aldol reaction in the presence of anti-9. Yield [%] (^); ee [%] (*). Figure 6. HPLC monitoring of SMSB in the aldol reaction in the presence of both 5 and 7. Yield [%] (^); ee [%] (*).

yield and the enantiomeric purity of the aldol adduct 5 were monitored with the aid of internal standard 6 (Scheme 13 and Figure 7). The results of this experiment were compared with those shown in Scheme 10 above, in which case a mixture of anti-7 and syn-7 were used. We observed an increase in the yield of aldol product 5 (0.10 % yield after 1 h, 0.53 % yield after 71 h), and, interestingly, the initial ee of (R)-5 was also high (50 % ee in the first control experiment). In this case, the ee values of the product decreased with time, which indicated the production of racemic aldol product after the first stages of the reaction, but still showed a sizable enantiomeric purity after 71 h (10.6  0.1 % ee). In another experiment run under similar conditions but with (S,S)-anti-9 (90 % ee, > 20:1 anti/syn) as a catalyst (5 mol %), we did not observe this increase in yield, but the initially formed aldol product was again non-racemic (0.01 % yield and 28 % ee after 1 h). Note that in this case the presence of enantioenriched aldol product (R)-5 cannot be due to decomposition of the double-aldol product, nor to the presence of traces of catalyst 8 (which, in any case, would produce (S)-5). Finally, symmetry breaking was again observed (0.05 % yield and about 20 % ee in favour of (R)-5 after 1 h) when the aldol reaction was run in the presence of the achiral syn-isomer of 9 (4.0 mol %, > 20:1 syn/anti). In all of the experiments performed in the presence of either 7 or 9 we observed the initial formation of non-racemic aldol product, thus we conclude that bis(aldol) adducts probably play a role in SMSB, a process that only takes place in the very first stages of the

Scheme 12. Enantioselective preparation of the double-aldol adduct 9.

Building upon the observation made by Gryko and Lipinski that the syn and anti isomers of the double-aldol adduct from the reaction between 1 and 2-chlorobenzaldehyde were separable by column chromatography,[34] we prepared the previously unknown double-aldol product 9 by reaction between 1 and 2-bromobenzaldehyde (10, 2.0 equiv), catalysed by proline thioamide derivative (S,R)-8 (Scheme 12). We were pleased to find that, as expected, the syn and anti diastereomers of 9 were separable by chromatography and that (R,R)-anti-9 was obtained with 90 % ee. In a similar way, but with (R,S)-8 as the catalyst, (S,S)-anti-9 was obtained with 90 % ee. We then investigated the possible role of anti-9 as a catalyst mimic[25] in the aldol reaction between acetone 1 and 4; the Chem. Eur. J. 2014, 20, 1 – 15

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work by playing a role similar to that of mutual inhibition in a Frank-type model.[39] Taking these three points into account, we constructed the coupled reaction network depicted in Scheme 14.

A new reaction network for the aldol reaction, which involves indirect autocatalysis and limited enantioselectivity, and leads to kinetically controlled SMSB in a closed system In light of the above results that concern the bis(aldol) product 7, we decided to look for a mechanistic model that could relate its formation under the actual reaction conditions with the observation of SMSB. On one hand, and bearing in mind that a hyper-competitive mechanism in which the autocatalytic step has a reaction order greater than one could lead to chirality amplification without the need for direct mutual inhibition between the product enantiomers,[31, 37] we envisaged that anti-7 could act as a stereoselective catalyst for the aldol reaction between 1 and 4, whereas syn-7 would play the role of non-enantioselective catalyst for the aldol addition. Firstly, as depicted in Figure 8, if we assume that the oxygenated functional groups of the bis(aldol) product interact with both the aldehyde and enol form of 1, an extension of

Scheme 14. Fully reversible coupled reaction network leading to SMSB in the formation of aldol adduct 5.

The reaction network is composed of reversible reactions in a closed system and the equilibrium constants of the individual steps obey the relationships necessary to ensure the fulfilment of the principle of microscopic reversibility.[7, 10] We were pleased to find that assuming moderately exergonic energy values for C C bond formation in the aldol-type reaction steps, together with chemically reasonable values for the individual rate constants, simple numerical simulations (COPASI, version 4.8)[40] showed that significant chirality amplifications could be readily achieved. Subsequently, we submitted the differential rate equations of our model to numerical integration with the Mathematica programme package. After some trials, we confirmed that the model could reproduce, for a variety of reaction parameters, the essential features of the product outcomes of our experiments, and yielded a kinetically controlled SMSB for the aldol reaction. For the simulation example illustrated in Figure 9, assuming fast formation of the aldol product 5 and the bis(aldol) adducts anti-7 and syn-7 in the first few seconds of the reaction, the plot of ee (starting from 10 6 % ee of 5, much lower than expected from statistical fluctuations around the initial ideal racemic composition)[41] versus time exhibited an abrupt early (approximately after 4 s from the beginning of the reaction) increase of the ee to measurable values, followed (after  1 h of reaction) by the preferential formation of racemic aldol product (Figure 9 a). Also, when this same set of reaction parameters was modified by imposition of an initial non-zero concentration of the double-aldol adducts 7, the system exhibited a greater facility for SMSB, be-

Figure 8. Possible catalysis of the aldol reaction by the bis(aldol) adducts.

Tsogoeva’s model for the autocatalytic Mannich reaction[23] shows that the R,R enantiomer of anti-7 will preferentially lead to the formation of (R)-5, whereas (S,S)-anti-7 will catalyse the formation of (S)-5. In a similar way, achiral syn-7 would lead to a pair of enantiomeric ternary complexes (not shown) that would result in the non-enantioselective formation of aldol adduct 5. Secondly, we hypothesised that the formation of the bis(aldol) product 7 would be extremely slow in homogeneous solution due to the very low concentration of 5, thus its formation would have taken place mainly in the very first stages of the process when dissolution of the solid aldehyde 4 may lead to very high concentration gradients so that in these initial stages a fast equilibrium between the bis(aldol) adduct 7 and the aldol monomer 5 could occur. In this way, catalysis by 7 is kinetically equivalent to second-order autocatalysis by 5. Thirdly, building on our recent analysis of SMSB in the limited enantioselectivity model,[38] we reasoned that non-enantioselective catalysis by the meso bis(aldol) product syn-7 should greatly enhance the overall non-linearity of the reaction net-

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Full Paper product and does not support the hypothesis that a Franktype reaction network may give rise to SMSB. Careful analyses of the compounds formed in the reaction led to identification of double-aldol adduct 7 (mixture of syn and anti diastereomers) as the main byproduct of the reaction. Independent asymmetric synthesis of 7 (as well as of the corresponding double-aldol adduct 9, from acetone and 2-bromobenzaldehyde) fully confirmed the assigned structure. In a set of experiments performed in the presence of either 7 or 9, the initial formation of non-racemic aldol adduct 5 was always observed. These observations, together with the fact that the non-catalysed formation of 7 is very unlikely to happen under homogeneous conditions in view of the very low concentrations of 5, led us to conclude that: 1) the presence of non-racemic aldol adduct 5, initially with a relatively high ee that decreases after several hours of reaction, could be explained by SMSB taking place during the initial stages of the reaction (i.e., during the dissolution of 4), followed by the slow but continuous formation of racemic aldol product; 2) this initial SMSB could be due to a coupled reaction network, which cannot involve asymmetric autocatalysis or strong direct mutual inhibition by aldol adduct 5, and in which the double-aldol adduct 7 plays a key role. We have been able to devise a novel reaction network that involves both indirect autocatalysis and indirect mutual inhibition between the enantiomers of the reaction product through the intermediacy of the double-aldol product 7, which (upon numerical integration with the Mathematica programme) was able to reproduce the essential features of the reaction outcomes of our experiments, and yielded a kinetically controlled SMSB for the aldol reaction. This reaction network is composed of reversible reactions that take place in a closed system (in thermal equilibrium with the surroundings) and preserves the principle of microscopic reversibility. In summary, the present results support the idea that the Soai reaction is not the sole example of SMSB in a chemical transformation and that this phenomenon can also arise in other processes that take place under conditions much more similar to those of several biochemical reaction cycles.

Figure 9. Numerical simulation of the reaction network shown in Scheme 14 a) in the initial absence of bis(aldol) adducts 7, and b) in the presence of a non-zero initial concentration of bis(aldol) adducts 7.

cause the initial increase of the ee from practically zero to more than 90 % ee takes place much earlier (before 0.1 s from the beginning of the reaction; see Figure 9 b). This would account for the essential role of either the bis(aldol) product 7 or the bis(aldol) mimic 9 in the autocatalytic evolution of the ee. Of course, in these simulations the stereochemical outcome of the reaction always correlates with the absolute configuration of the enantiomer that is arbitrarily assumed to be initially in excess. The non-stochastic behaviour of SMSB in our experiments (i.e., the predominant formation of (R)-5) could then be explained by assuming that the initial enantiomeric excess that can be amplified by the system has a sign determined by the unavoidable presence of a trace amount of a chiral non-racemic compound, which may have a very small chiral effect. This is a characteristic feature of coupled reaction networks that lead to SMSB[8] and has been experimentally demonstrated for the Soai reaction.[17, 18]

Conclusion Experimental Section

When a solution of 4 in 1 (c = 1.29 m) that contained DMSO (5 % v/v) was stirred for 10 d at room temperature, the formation of enantiomerically enriched aldol product 5 (up to 15 % ee of (R)-5) took place in some experiments, partially corroborating previous reports by Mauksch, Tsogoeva and co-workers.[26, 27] When this process was monitored by chiral HPLC with the aid of an internal standard, the initially formed aldol product was often found to be non-racemic and in about 50 % of the experiments performed the ee of the aldol product 5, measured after 4 h, was greater than 2 %. However, only a small fraction of the reactions still contained non-racemic aldol product after 24 h. A clear predominance for (R)-5 was observed in a set of more than 45 experiments. Chiral HPLC monitoring of this reaction failed to reveal any autocatalytic role of the aldol Chem. Eur. J. 2014, 20, 1 – 15

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General Chemicals and solvents were purchased either analytically pure from commercial suppliers or purified by standard techniques. DMSO was purchased from Sigma–Aldrich (A.C.S. spectrophotometric grade) and 1 was purchased from Sigma–Aldrich (Chromasolv Plus for HPLC grade, unless otherwise specified). Silica gel plates (Merck 60 F254) were used for TLC analysis and compounds were visualised by irradiation with UV light and/or by treatment with a solution of p-anisaldehyde (23 mL) in concd H2SO4 (35 mL), acetic acid (10 mL) and ethanol (900 mL), followed by heating. For TLC purification of the double-aldol adducts, Analtech Uniplate 500 mm silica gel G plates with UV 254, glass-backed 20  20 layers were used. Flash chromatography was performed by using Merck 60 silica gel (particle size = 0.040–0.063 mm). 1H and 13C NMR spectra were recorded with a Varian VNMRS500, Varian Mercury 400, or

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Full Paper Varian Inova 300 spectrometer. Chemical shifts (d) are given in ppm relative to tetramethylsilane (TMS) and coupling constants (J) are given in Hz. The spectra were recorded in CDCl3 at room temperature. TMS served as internal standard (d = 0 ppm) for 1H NMR spectroscopy and CDCl3 was used as internal standard (d = 77.0 ppm) for 13C NMR spectroscopy. HRMS was performed with a Bruker MicrOTOF spectrometer.

General procedure for the preparation of double-aldol adducts (adapted from reference [34]) Proline thioamide derivative (S,R)-8[34] (20 mg, 0.085 mmol, 0.20 mol equiv) was added to a small flask equipped with a magnetic stirrer bar (for reactions with solid aldehyde 4 dry CH2Cl2 (0.5 mL) was added), then 1 (25 mg, 0.43 mmol) was added via syringe. The desired aldehyde (0.861 mmol) was added in one portion and the reaction mixture was stirred at rt for 16–72 h. A saturated aqueous solution of ammonium chloride (10 mL) was added and the solution was extracted with ethyl acetate (3  10 mL). The combined organic layers were dried over MgSO4 and the solvent was removed in vacuo to afford an oily residue, which was purified by column chromatography on silica gel to separate the double-aldol adduct from the less-polar mono-aldol adduct. The fractions that contained the double-aldol adduct were combined and the solvent was evaporated under reduced pressure. The desired adduct was further purified by preparative TLC to eliminate traces of the mono-aldol adduct.

General procedure for the “preparative” aldol reaction between 1 and 4: adapted from references [26] and [27] In a typical experiment, 4 (159.0 mg, 1.05 mmol) was added to a dry round-bottomed flask equipped with a magnetic stirrer bar and 95:5 1/DMSO (0.82 mL, [4] = 1.29 m) was added in one portion, with stirring, to give an initially clear and homogeneous solution. The flask was sealed with a septum and an inert argon atmosphere was introduced. The septum cap was covered with Parafilm and the reaction was stirred at rt. After 10 d, the septum was removed and deionised water (5 mL) was added. The mixture was extracted with CH2Cl2 (3  10 mL). The combined organic extracts were dried with MgSO4, filtered and the solvent was evaporated under vacuum. The yield of aldol product 5 was estimated by 1H NMR spectroscopic analysis of a sample by measuring the relative areas of the signals that corresponded to the CHO aldehyde proton and the CH O aldol proton. A portion of the crude reaction product (5–10 mg) was purified by preparative TLC (95:5 hexane/ethyl acetate; 5 extracted from the silica with ethyl acetate) and the enantiomeric purity of 5 was determined by chiral HPLC analysis.

Compounds anti-7 and syn-7: Yellow solid; 13 % (72 h reaction time); d.r. = 3:1 anti/syn (inseparable); 63 % ee (anti-7); HPLC (Chiralpak IC column, 1 mLmin 1, hexane/IPA 90:10, l = 250–270 nm): retention time (tR) = 63 (syn-7 < M + > (S,S)-anti-7), 70 min ((R,R)anti-7) or (Chiralpak IC column, 1 mLmin 1, gradient = 0–45 min, hexane/IPA 95:5; 45–120 min, hexane/IPA 90:10, l = 250–270 nm): tR = 98 (syn-7 < M + > (S,S)-anti-7), 106 min ((R,R)-anti-7); 1H NMR (400 MHz, CDCl3, TMSint): d = 8.21 (d, J = 8.7 Hz, 4 H), 7.54 (d, J = 8.7 Hz, 4 H), 5.35–5.32 (m, 2 H), 3.31 (br s; syn-OH), 3.29 (br s; antiOH), 2.98–2.82 ppm (m, 4 H); 13C NMR (100 MHz, CDCl3): d = 209.4 (syn), 209.3 (anti), 149.8, 147.6, 126.5, 124.0, 69.2* (syn), 69.1* (anti), 52.0 (anti), 51.9 ppm (syn); HRMS: m/z calcd for C17H15N2O7: 359.0885 [M H] ; found: 359.0890. * Relative areas were used to calculate the d.r.

Compound 5: Yellow solid; HPLC (Chiralpak IC column, 1 mL min 1, hexane/isopropyl alcohol (IPA) 95:5, l = 270 nm): retention time (tR) = 38 (S), 41 min (R); 1H NMR (300 MHz, CDCl3, TMSint): d = 8.22 (d, J = 8.9 Hz, 2 H), 7.54 (d, J = 8.9 Hz, 2 H), 5.27 (dd, J1 = 7.5 Hz, J2 = 4. 4 Hz, 1 H), 3.57 (br s, 1 H), 2.87 (s, 1 H), 2.84 (d, J = 4.4 Hz, 1 H), 2.23 ppm (s, 3 H).

To prepare the racemic compound, pyrrolidine (0.20 equiv) and phenol (0.20 equiv) were used instead of proline thioamide 8 as catalyst and co-catalyst, respectively, under the same reaction conditions.[35] In this way, we obtained a 1:1 mixture of rac-anti-7 and syn-7 after extensive purification by preparative TLC.

General procedure for the “analytical” aldol reaction between 1 and 4

Compound anti-9: Colourless solid; 22 % (24 h reaction time) after chromatographic purification; 90 % ee; HPLC (Chiralpak IA column, 1 mL min 1, hexane/IPA 90:10, l = 250–270 nm): tR = 36 ((S,S)-9), 43 min ((R,R)-9); 1H NMR (400 MHz, CDCl3, TMSint): d = 7.62 (dd, J = 7.8, 1.7 Hz, 2 H), 7.51 (dd, J = 8.0, 1.2 Hz, 2 H), 7.35 (td, J = 7.6, 1.2 Hz, 2 H), 7.14 (td, J = 7.7, 1.7 Hz, 2 H), 5.51 (dd, JAX = 9.7 Hz, JBX = 2.3 Hz, 2 H), 3.42 (s, 2 H; OH), 2.99 (dd, JAB = 17.2 Hz, JBX = 2.3 Hz, 2 H), 2.76 ppm (dd, JAB = 17.2 Hz, JAX = 9.7 Hz, 2 H); 13C NMR (100 MHz, CDCl3): d = 210.9, 141.6, 132.8, 129.2, 128.1, 127.4, 121.3, 69.0, 50.1, 32.1, 29.9 ppm; HRMS: m/z calculated for C17H15Br2O2 : 408.9433 [M H2O] + ; found: 408.9443.

In a typical experiment, 4 (159.00 mg, 1.052 mmol), internal standard 6 (9.90 mg, 0.053 mmol), and (when necessary) the corresponding additives (0.01–20 mol %) were added to a round-bottomed flask equipped with a magnetic stirrer bar, then 95:5 1/ DMSO (0.82 mL, [4] = 1.29 m) was added in one portion, with stirring, to give an initially clear and homogeneous solution. The flask was sealed with a septum and an inert argon atmosphere was introduced. The septum cap was covered with Parafilm and the reaction was stirred at rt. Small samples (  50 mL) of the reaction mixture were collected at intervals by using a syringe equipped with a long needle. The solvent from the crude sample was quickly removed with pressurised air and the oily residue was dissolved in the minimum amount of CH2Cl2, followed by the addition of 4:1 hexane/IPA, then the solution was filtered. Finally, the filtrate was analysed by chiral-phase HPLC. A UV diode-array detector was used to determine both the yield and ee of the product. For each injection, the peak-area ratios were measured at l = 250, 260, 270 and 280 nm, whixh allowed a mean value and standard deviation to be determined for each magnitude. In some cases, the remainder of the crude product was purified by preparative TLC and the ee was determined by chiral HPLC to validate the analysis of the reaction mixture.

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Compound syn-9: Colourless solid; 17 % (24 h reaction time) after chromatographic purification; HPLC (Chiralpak IA column, 1 mL min 1, hexane/IPA 90:10, l = 250–270 nm): tR = 45 min; 1 H NMR (400 MHz, CDCl3, TMSint): d = 7.62 (dd, J = 7.8, 1.7 Hz, 2 H), 7.52 (dd, J = 8.0, 1.2 Hz, 2 H), 7.36 (td, J = 7.5, 1.2 Hz, 2 H), 7.15 (td, J = 7.7, 1.7 Hz, 2 H), 5.53 (dd, JAX = 9.7 Hz, JBX = 2.3 Hz, 2 H), 3.30 (s, 2 H; OH), 3.06 (dd, JAB = 17.2 Hz, JBX = 2.3 Hz, 2 H), 2.71 ppm (dd, JAB = 17.2 Hz, JAX = 9.7 Hz, 2 H); 13C NMR (100 MHz, CDCl3): d = 210.9, 141.6, 132.8, 129.3, 128.1, 127.4, 121.4, 69.1, 50.2, 29.9 ppm; HRMS: m/z calcd for C17H15Br2O2 : 408.9433 [M H2O] + ; found: 408.9443.

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Full Paper Numerical simulations Simulations were performed by numerical integration of the differential rate equations according to rate-equation theory as applied in chemical kinetics. The concentration units are mol L 1 (m) and the rate constants have the appropriate units to yield rate values in units of mol s 1. Numerical integration was performed with the Mathematica programme package, and was run between 0 and 1  1012 s. The results were monitored and verified to ensure that the total system mass remained constant over time. The suppression of “computational noise” in our simulations, which arises from round-off errors, was effected by setting a high numerical precision of the input parameters (500 significant decimal digits) and exact number representations of the reaction rates and initial concentration values. The fluctuations of chirality able to convert the racemic output to a chiral one were simulated by using an initial ee of 5 of 10 6 %, much lower than that expected for statistical fluctuations about the ideal racemic composition at the very small initial concentration of 5 (2  10 15 m).[38, 41] The actual numerical simulation of the reaction network shown in Scheme 14, which led to the results shown in Figure 9, was performed by assuming the intermediate reversible formation of binary complexes between 7 and 4, discussed in more detail in the Supporting Information. The Mathematica input notebook for the simulation shown in Figure 9 b is provided in the Supporting Information.

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Acknowledgements G.V. thanks the Spanish Ministry of Economy and Competitivity (MINECO) for a FPI predoctoral fellowship. Financial support from MINECO (Project CTQ2013–47401-C2–1-P) and participation in the COST Action CM0905 (ORCA) are also gratefully acknowledged.

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Keywords: aldol reaction · autocatalysis · biological homochirality · mirror symmetry breaking · organocatalysis

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Received: July 21, 2014 Published online on && &&, 0000

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Full Paper

FULL PAPER & Chiral Amplification

Mirror, mirror on the wall! HPLC monitoring of the aldol reaction between 4-nitrobenzaldehyde and acetone (see figure) in the absence of a chiral catalyst reveals that spontaneous mirror symmetry breaking takes place initially in about 50 % of the reactions. A novel coupled reaction network that involves indirect autocatalysis and does not require heterochiral inhibition in the aldol product can explain this behavior.

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G. Valero, J. M. Rib, A. Moyano* && – && A Closer Look at Spontaneous Mirror Symmetry Breaking in Aldol Reactions

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