Reversal of the enantioselectivity in aldol addition over immobilized di- and tripeptides: Studies under continuous flow conditions

Article abstract of DOI:10.1039/c4ra07188c

Heterogeneous asymmetric direct aldol reactions between aldehydes (2-nitrobenzaldehyde, 2-methylpropanal) and acetone catalyzed by polystyrene resin (PS) supported di- and tripeptides H-Pro-Pro-, H-Pro-Pro-Pro-, H-Pro-Glu(OH)-, H-Pro-Pro-Glu(OH)-, H-Pro-Asp(OH)-, H-Pro-Pro-Asp(OH)-, H-Ser-Glu(OH)-, H-Ser-Ser-Glu(OH)-, H-Val-Glu(OH)-, H-Val-Val-Glu(OH)-MBHA-PS, were studied under identical experimental conditions at room temperature in a continuous-flow fixed-bed reactor (CFBR) system. In the asymmetric aldol reactions reversal of enantioselectivity was observed on H-Pro-Pro-Glu(OH)- and H-Pro-Pro-Asp(OH)-MBHA-PS-supported catalysts (ee 42-67% S) as compared to the H-Pro-Glu(OH)- and H-Pro-Asp(OH)-MBHA-PS-supported catalyst (ee 28-82% R). In the case of H-Pro-Pro- and H-Pro-Pro-Pro-MBHA-PS-supported catalysts reversed enantioselectivity was observed by using the benzoic acid additive (12% S) as compared to the H-Pro-MBHA-PS catalyst (25% R). The stability of the catalysts in the flow system was consistent with the heterogeneous character of the reaction, as was the linear behavior obtained using mixtures of l- and d-enantiomers of the supported H-Pro-MBHA-PS catalyst. The enamine character of the reaction intermediates was supported by ESI-MS measurements. Based on these and the computed structure of the peptides, the conformation of the intermediate adducts is held responsible for chiral induction, therefore for the enantioselectivity inversion observed in these reactions.

Full text of DOI:10.1039/c4ra07188c

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Reversal of the Enantioselectivity in Aldol Addition over Immobilized Di-
and Tripeptides: Studies under Continuous Flow Conditions
1
1
2
3
1,3
András Gurka, Imre Bucsi, Lenke Kovács, György Szőllősi * and Mihály Bartók *
1
2
Department of Organic Chemistry, University of Szeged, Dóm tér 8, Hꢀ6720 Szeged,
Hungary.
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös utca 6, Hꢀ6720 Szeged,
Hungary.
3
MTAꢀSZTE Stereochemistry Research Group, Dóm tér 8, Hꢀ6720 Szeged, Hungary. Eꢀmail:
bartok@chem.uꢀszeged.hu, szollosi@chem.uꢀszeged.hu
Abstract
Heterogeneous asymmetric direct aldol reactions between aldehydes (2ꢀnitrobenzaldehyde, 2ꢀ
methylpropanal) and acetone catalyzed by polystyrene resin (PS) supported diꢀ and tripeptides
HꢀProꢀProꢀ, HꢀProꢀProꢀProꢀ, HꢀProꢀGlu(OH)ꢀ, HꢀProꢀProꢀGlu(OH)ꢀ, HꢀProꢀAsp(OH)ꢀ, Hꢀ
ProꢀProꢀAsp(OH)ꢀ, HꢀSerꢀGlu(OH)ꢀ, HꢀSerꢀSerꢀGlu(OH)ꢀ, HꢀValꢀGlu(OH)ꢀ, HꢀValꢀValꢀ
Glu(OH)ꢀMBHAꢀPS, were studied under identical experimental conditions at room
temperature in a continuousꢀflow fixedꢀbed reactor (CFBR) system. In the asymmetric aldol
reactions reversal of enantioselectivity was observed on HꢀProꢀProꢀGlu(OH)ꢀ and HꢀProꢀProꢀ
Asp(OH)ꢀMBHAꢀPSꢀsupported catalyst (ee 42–67% S) as compared to the HꢀProꢀGlu(OH)ꢀ
and HꢀProꢀAsp(OH)ꢀMBHAꢀPSꢀsupported catalyst (ee 28–82% R). In case of HꢀProꢀProꢀ and
HꢀProꢀProꢀProꢀMBHAꢀPSꢀsupported catalyst reversed enantioselectivity was observed by
using benzoic acid additive (12% S) as compared to the HꢀProꢀMBHAꢀPS catalyst (25% R).
The stability of the catalysts in the flow system was consistent with the heterogeneous
character of the reaction, similarly as was the linear behavior obtained using mixtures of
Lꢀ
and ꢀenantiomers of the supported HꢀProꢀMBHAꢀPS catalyst. The enamine character of the
D
reaction intermediates was supported by ESIꢀMS measurements. Based on these and the
computed structure of the peptides, the conformation of the intermediate adducts is held
responsible for chiral induction, therefore for the enantioselectivity inversion observed in
these reactions.
1

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1. Introduction
Asymmetric organocatalysis, in general, and stereoselective catalytic formation of CꢀC bonds,
1
,2
in particular, has become an important method in the production of chiral compounds. One
of the intensively studied reactions utilized for the formation of the CꢀC bonds is the
1
ꢀ6
asymmetric aldol addition. Its significance is emphasized in numerous monographs.
Advantages of the heterogeneous catalytic procedures put the investigation of asymmetric
aldol reactions over insoluble solid catalysts in the forefront of catalytic research. Results
2,7ꢀ11
obtained in such studies have been published continuously.
We were among the first to
initiate studies on heterogeneous catalytic asymmetric aldol reactions, our first report was
12
13ꢀ16
published in 2003, and we have published on a regular basis ever since.
A widely used group of chiral catalysts, especially in asymmetric aldol additions is
Lꢀ
proline (Pro, P) and its synthetic derivatives. In addition, considerable progress has been made
in the use of peptideꢀtype organocatalysts. Some short peptides (in particular tripeptides) have
17ꢀ19
proved to act as especially active and enantioselective catalysts.
Asymmetric syntheses
using chiral organocatalysts usually lead to the production of important products of high
optical purity. An unfortunate property of these catalysts is their low activity requiring
reaction times that may range from a few hours to a few days and the relatively high amount
of catalysts (up to 30 mol %) needed to obtain reasonable yields. These are among the most
important obstacles preventing industrial applications of the numerous organocatalytic
methods developed up to now. Moreover, the industry prefers continuousꢀflow fixedꢀbed
(CFBR) operating methods, which allow process intensification and have several other
advantages, such as saving space, time and energy, possibility of increased reactivities and
11
selectivities and easy scaleꢀup of processes. These methods have only recently been applied
for carrying out asymmetric aldol reactions, so far only 5 publications have reported different
20ꢀ24
variants of such procedures.
Recently we have reported the application of polystyrene resin immobilized proline
terminated diꢀ and tripeptides in asymmetric catalytic direct aldol reactions of aldehydes with
ketones. Our study revealed that reversal of the enantioselectivity is obtained by increasing
16
the number of the terminal proline units. Accordingly, we have shown that both enantiomers
of an aldol reaction may be prepared by using polystyrene resin immobilized proline
terminated peptide of appropriate length. Moreover, the immobilized peptides may be used in
continuous flow systems. In continuation of our research on the use of heterogeneous chiral
organocatalysts we have studied the use of immobilized diꢀ and tripeptides in the direct
asymmetric aldol reaction of aldehydes and acetone under flow conditions using fixedꢀbed
2

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reactor. The aim of this study was to ascertain that the phenomenon observed in batch system,
i.e. the inversion of the enantioselectivity by increasing the peptide length, occurs also in a
flow systems where the reactions are carried out under markedly different conditions (i.e.
catalyst/reactant ratio can reach high values). Besides the immobilized proline terminated
peptides reported in our previous publication, other amino acid terminated supported diꢀ and
tripeptides were also prepared and used under identical reaction conditions, to test the role of
the terminal proline units in the enantioselectivity inversion. The following resinꢀsupported
diꢀ and tripeptides were used as catalysts: PꢀNHꢀR, PPꢀNHꢀR, PPPꢀNHꢀR, PDꢀNHꢀR, PPDꢀ
NHꢀR, PEꢀNHꢀR, PPEꢀNHꢀR, SEꢀNHꢀR, SSEꢀNHꢀR, VEꢀNHꢀR and VVEꢀNHꢀR (Fig. 1).
Fig. 1 Organocatalysts used in these experiments for aldol reactions (R = MBHAꢀpolystyrene
resin).
2. Experimental section
2.1. Preparation and characterization of resin-bonded peptides
The immobilized peptide catalysts were synthetized by a solidꢀphase technique using Fmocꢀ
16
strategy. Solidꢀphase synthesis was carried out manually on pꢀmethylbenzhydrylamine
polystyrene resin (MBHAꢀPS resin purchased from Bachem GmbH) with standard
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methodology. The catalyst loading was calculated from CHNꢀanalysis. IR spectra of the
prepared materials were collected on a BioꢀRad Digilab Division FTSꢀ65A/896 FTꢀIR
−1
spectrometer operated in diffuse reflectance mode (DRIFT) between 4000 and 400 cm at 2
−1
cm resolution by averaging 256 scans. The resinꢀbonded peptides were also characterized by
HPLC analysis of the peptides cleaved from the resins and by electrospray ionization mass
16
spectrometry (ESIꢀMS) according to published methods.
2.2. General procedure for the direct aldol reactions
The aldehydes: 2ꢀnitrobenzaldehyde (2-NBA) and 2ꢀmethylpropanal (2-MPA) were
purchased from SigmaꢀAldrich Co. 2-NBA was purified by crystallization, whereas 2-MPA
was distilled before use. Acetone of analytical purity was also purchased from SigmaꢀAldrich
Co. and used as received.
2.2.1. Reactions in continuous flow system using fixed-bed reactors (CFBR, Fig 2). In the
tubular catalyst cartridge of 4 mm inner diameter and 50 mm length the given amount of
catalyst (or its mixture with silica gel) was placed. The catalyst was washed for 0.5 h with 0.1
−
1
mL min flow of acetone. A solution of the aldehyde and additive (when used) in acetone
ꢀ1
was fed continuously with a HPLC pump (flow rate 0.1 mL min ) over the catalyst bed. The
effluent of the reactor was sampled (150 µL) at 30 and 60 min. The samples were diluted to
500 ꢁL with acetone and these samples were directly analyzed by chiral gas chromatography.
Few experiments were also carried out for longer times (5 h) and the yields of the aldol
addition products were determined following purification of the aldol products by flash
chromatography.
The aldol reactions using mixtures of chiral catalysts were conducted over
LꢀProꢀ
MBHAꢀPS (PꢀNHꢀR) and ꢀProꢀMBHAꢀPS catalysts and their mixtures in various ratios. The
D
reactions were carried out in closed glass batch reactors. The given amount of immobilized
catalyst (containing 10 mol% of amino acid compared to the aldehyde) was suspended in
acetone followed by addition of the given amount of the corresponding aldehyde.
4

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Fig. 2 Continuousꢀflow aldol reaction in fixedꢀbed microreactor.
The reaction mixture was stirred at rt. After the specified reaction time the catalyst was
removed by filtration and products were analyzed by GC.
2.2.2. Analytical procedures. Products were identified by their mass spectra using Agilent
Techn. 6890N GCꢀ5973 inert MSD and HPꢀ1MS 60 m × 0.25 mm i.d. capillary column.
Quantitative analysis including enantiomeric separation was performed using GC equipped
with flame ionization detector (FID): Agilent Techn. 6890N GCꢀFID and CyclosilꢀB 30 m ×
0.25 mm i.d. chiral capillary column. Analysis conditions and retention times of the aldol
16
products were given in our previous publication. Enantiomeric excesses (ee) were calculated
using the formula ee (%) = │[R]–[S]│/([R]+[S]) × 100, where [R] and [S] are the
concentrations of the aldol product enantiomers. The reproducibility of the reactions was
±3%. The absolute configurations of the aldol products were assigned by analogy with
previously reported results and data obtained using ꢀ and ꢀproline as catalysts.
L
D
Isolated yields were determined following purification of the aldol products by flash
chromatography on silica gel using hexane/ethyl acetate 7/3 (2ꢀNBA) or hexane/ethyl acetate
1
13
3
/1 (2ꢀMPA) mixtures as eluent. The purified products were characterized by their H and C
NMR spectra recorded on a Bruker AVANCE DRX 400 NMR instrument (for spectra see
1
Supporting information). 4-Hydroxy-4-(2-nitrophenyl)-butan-2-one: pale yellow oil, H
NMR (400 MHz, CDCl
3
) δ (ppm): 7.94 (1H, d, J = 8.1 Hz, ArH), 7.88 (1H, d, J = 8.1, ArH),
7
3
.65 (1H, tr, J = 7.6 Hz, ArH), 7.42 (1H, tr, J = 7.1 Hz, ArH), 5.66 (1H, dd, J = 9.6, 2.0 Hz),
13
3
.10 (1H, dd, J = 17.6, 2.0 Hz), 2.72 (1H, dd, J = 17.6, 9.6 Hz), 2.22 (3H, s, CH ); C NMR
(
100 MHz, CDCl ) δ (ppm): 208.6, 147.2, 138.4, 133.7, 128.2, 124.4, 65.6, 51.0, 30.4. 4-
3
1
Hydroxy-5-methylhexan-2-one: pale yellow oil, H NMR (400 MHz, CDCl ) δ (ppm): 3.77
3
(1H, m, J = 9.6, 5.5, 3.0 Hz), 2.45ꢀ2.60 (2H, m), 2.15 (3H, s, CH
3
CO), 1.64 (1H, m), 0.85ꢀ
1
3
0
3
.90 (6H, 2 d, J = 9.6 Hz, C(CH ) ); C NMR (100 MHz, CDCl ) δ (ppm): 210.1, 72.2, 46.9,
3 2 3
3.0, 30.7, 18.2, 17.6 (where s singlet, d doublet, tr triplet, dd double doublet, m multiplet).
5

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The transformations of the peptide catalysts were followed by ESIꢀionꢀtrapꢀMS
measurements on a AGILENT 1100 LCꢀMSD TRAP SL ionꢀtrap MS instrument operated
under positive ion and auto MSꢀMS mode using the following parameters: ESI: capillary
ꢀ1
(
2
needle) voltage = 3.5 kV, capillary exit voltage = 136 V, drying gas (N ) = 9 L min , drying
gas temperature = 623 K, nebulizer gas = 40 psi; ionꢀtrap: scan range = 80ꢀ350 m/z, max.
accumulation time = 300 ms, fragmentation amplitude = 1.5 V, fragmentation time = 40 ms.
ꢀ1
ꢀ
Solvent: methanol/0.1% AcOH; flow rate: 0.5 mL min ; concentration of sample: 0.1 ꢀmol L
1
;
injected volume 1.5 ꢀL.
3. Results and Discussion
Catalysts having been used to date in asymmetric aldol reactions using the CFBR method are
shown in Fig. 3. The main objective of these studies, worthy of being described as pioneering,
was to confirm the applicability of catalysts shown in Fig. 3 earlier proven to be active in the
batch system for the purpose of efficient synthesis of chiral βꢀhydroxy ketones. Using
catalysts with the necessary activity and stability, enantioselectivities exceeding even 90%
20ꢀ24
could be occasionally achieved in the reactions of various aldehydes and ketones.
the catalysts having outstanding activities are ProꢀProꢀAspꢀbased tripeptide organocatalysts
Among
1
7,19,25
developed by Wennemers et al.
The remarkable novelty of our research lies in the
recognition and its experimental confirmation that (S)ꢀamino acids, easily and economically
prepared from proteins, and their derivatives can act as catalysts of chiral building blocks not
only of (R)ꢀ but also of (S)ꢀconfiguration. On this basis, making use of the justified and
26
attested successful combination of enamine catalysis with the flow technique – in
16
accordance with batch measurements – we performed experiments using Pro terminated diꢀ
and tripeptides followed by testing immobilized peptides containing other amino acids. The
catalysts studied are presented in Fig. 1.
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Fig. 3 Organocatalysts studied under continuous flow conditions in direct asymmetric aldol
2
0ꢀ24
reaction
(PS = polystyrene, TG = TentaGel)
3.1. Aldol reactions in CFBR condition
The MBHAꢀPSꢀimmobilized peptide catalysts were synthesized according to published
1
2,16
method.
Immobilization of the amino acids was confirmed by IR spectroscopy and by
HPLC and ESIꢀMS analysis of the peptides obtained by cleavage from the resins. The IR
spectra of the prepared materials were compared with the spectra of the HCl x MBHAꢀPS
1
6
resin, as described earlier. The appearance of vibrational bands characteristic of amino acids
showed the immobilization of these over the resin. Furthermore, the cleavage of the peptides
from the resins using HF and analysis of the obtained compounds by HPLC and ESIꢀMS
confirmed the presence of the desired peptides on the surface in 80ꢀ95% purity.
Results of aldol additions using the immobilized peptides are summarized in Table 1.
It was expedient to start our studies on aldol reactions with Pꢀ, PPꢀ and PPPꢀNHꢀR catalysts,
since proline had been the basic chiral component of the catalysts used in the already
published experiments. Furthermore, no flowꢀtype experiments with such catalysts had been
described prior to our present study. According to these data, the catalysts containing only
proline units exhibited satisfactory activities in the reaction of 2ꢀNBA under the given
experimental conditions. Their enantioselectivities, however, proved to be moderate. Most
important in the reaction carried out over all three catalysts, the reaction yielded the aldol
addition product of (R)ꢀconfiguration in excess (entries 1–3).
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Next, the investigation of diꢀ and tripeptide catalysts containing proline, aspartic acid
and glutamic acid was undertaken. Experiments using PEꢀ, PPEꢀ, PDꢀ and PPDꢀNHꢀR
catalysts were performed under completely identical experimental conditions. However, due
to the solventꢀdependent, varied swellability of the resins, the peptide catalysts immobilized
on the resins would occasionally yield nonꢀreproducible experimental data. Consequently,
immobilized peptide catalysts mixed with silica gel were used for experiments on aldol
reactions. The catalysts exhibited moderate ee and satisfactory activities even without
optimization (entries 4–7). The most interesting observation is, however, the reversal of the ee
in the reactions of tripeptide containing catalysts as compared to the reactions of dipeptide
catalysts. Namely in case of tripeptide catalysts (S)ꢀproduct formed, while in the presence of
immobilized dipeptides (R)ꢀproduct was obtained. Accordingly, these results showed that the
phenomenon observed earlier in batch system was also detected in continuous flow system, in
which the catalyst/aldehyde ratio in the reactor is significantly different. To the best of our
knowledge, results obtained using immobilized diꢀ and tripeptide catalysts under identical
continuous flow conditions, have not been described in the literature. Optimization of the
individual reactions was not among our goals; nevertheless, the reversal of enantioselection
was significant.
Further experiments were carried out using diꢀ and tripeptide catalysts lacking proline.
As shown in Table 1, SEꢀ, SSEꢀ, VEꢀ and VVEꢀNHꢀR catalysts, which were not examined
until now, were less active in the aldol reaction of 2ꢀNBA, but at the same time afforded good
ee values (52–66%) and produced the (R)ꢀconfiguration product in excess (entries 8–11).
Table 1. Direct aldol reaction using supported diꢀ and tripeptides under CFBR
a
conditions
b
c
d
Entry
Catalyst
mg
Aldehyde Conversion Selectivity
Ee
mmol
0.22
%
%
%
1
2
3
4
5
6
PꢀNHꢀR
250
250
240
250
250
250
2ꢀNBA
2ꢀNBA
2ꢀNBA
2ꢀNBA
2ꢀNBA
2ꢀNBA
31
89
13 R
PPꢀNHꢀR
PPPꢀNHꢀR
PEꢀNHꢀR
PPEꢀNHꢀR
PDꢀNHꢀR
0.19
0.14
0.095
0.085
0.1
24
37
63
83
95
95
96
13 R
7 R
76
28 R
28 S
24 R
e
e
70; 63
87; 82
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7
8
9
1
1
1
1
1
PPDꢀNHꢀR
SEꢀNHꢀR
SSEꢀNHꢀR
VEꢀNHꢀR
VVEꢀNHꢀR
PEꢀNHꢀR
PPEꢀNHꢀR
PDꢀNHꢀR
PPDꢀNHꢀR
250
300
300
300
300
250
250
250
250
0.07
0.125
0.12
0.125
0.11
0.095
0.085
0.1
2ꢀNBA
2ꢀNBA
2ꢀNBA
2ꢀNBA
2ꢀNBA
2ꢀMPA
2ꢀMPA
2ꢀMPA
2ꢀMPA
78
10
2
96
97
94
96
94
99
99
98
98
42 S
52 R
60 R
54 R
66 R
82 R
35 S
80 R
67 S
0
1
2
3
4
5
14
2
7
3
e
18; 10
13
1
0.07
a
2
CFBR = continuousꢀflow fixedꢀbed reactor, immobilized catalyst/SiO : 1/1.
b
c
d
e
2ꢀNBA = 2ꢀnitrobenzaldehyde, 2ꢀMPA = 2ꢀmethylpropanal.
dehydration of the aldol addition product also occurred.
enantiomeric excess and the configuration of the excess enantiomer.
isolated yields of the aldol product following experiments prolonged to 5 h time on stream
under otherwise identical conditions.
This observation is in agreement with results obtained in experiments using immobilized
21,27ꢀ30
chiral primary amine catalysts, a group of material studied scarcely before.
In addition
to the aromatic aldehyde 2ꢀNBA, the experimental results of the aldol reaction of an aliphatic
aldehyde, 2ꢀMPA over PDꢀ, PPDꢀ, PEꢀ and PPEꢀNHꢀR catalysts are also presented in Table 1
(
entries 12–15). These catalysts exhibited lower activities in the transformation of 2ꢀMPA as
compared to 2ꢀNBA, but gave significantly higher and also reversed enantioselectivities (80ꢀ
2% (R), 35ꢀ67% (S)).
The positive effect of acids – among them, benzoic acid (BA) – on the efficiency of
8
31,32
the aldol reaction has been reported.
These observations led us to examine the behavior of
the less active and enantioselective Pꢀ, PPꢀ and PPPꢀNHꢀR catalysts in the presence of BA. To
this end, the reaction between 2ꢀNBA and acetone was studied in the presence of each of the
three catalysts, and the effect of BA on conversion and enantioselection at various
temperatures was investigated. The experimental data shown in Fig. 4 allow the following
conclusions: (i) naturally, conversion increases with temperature on all three catalysts; (ii) it is
remarkable, however, that conversion in the presence of BA is significantly higher and its
increase by the effect of temperature is faster than in the reactions without BA; (iii) the most
prominent novelty is the difference in ee brought about by the presence of BA: on PꢀNHꢀR
catalyst the (R)ꢀproduct is formed in a higher ee both in the presence and absence of BA,
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whereas on the diꢀ and tripeptide catalysts the product of reverse configuration, i.e. the (S)ꢀ
product is formed in higher ee in the presence of BA.
1
0

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Fig. 4 Effect of the temperature on the conversion (open symbols) and enantioselectivity
closed symbols) of the aldol addition of 2ꢀNBA and acetone in the absence ( ) and
presence of benzoic acid (BA) ( ) using a) Pꢀ, b) PPꢀ, c) PPPꢀNHꢀR supported catalysts
under flow conditions. Reagents and conditions: 2ꢀNBA (0.026 mmol mL ), acetone (25
(
ꢀ,ꢁ
,ꢀ
ꢀ1
ꢀ1
ꢀ1
mL), BA (0.04 mmol mL ), catalyst (250 mg, 0.9 mmol/g), flow rate 0.1 mL min , Conv. =
conversion of 2ꢀNBA.
To our best knowledge, no similar observation has been published in the field of asymmetric
direct aldol reactions and studies on this interesting phenomenon are carried out presently in
our laboratory and will be reported in due course. One may assume a conformational change
of the flexible diꢀand tripeptide due to protonation with benzoic acid.
3.2. ESI-MS measurements
ESIꢀMS and MS2 spectra of the filtrates of aldol reactions over ProꢀGlu(OH)ꢀNH and
2
2
ProꢀProꢀGlu(OH)ꢀNH peptides on the one hand and over derivatives of the same peptides
immobilized on MBHAꢀPS resin (PEꢀNHꢀR and PPEꢀNHꢀR) on the other hand were
recorded. The experimental data obtained in these studies allowed to propose the reaction
pathways outlined in Scheme 1, which are consistent with certain partꢀsteps of the reaction
3
3ꢀ35
mechanism suggested in earlier mechanistic studies.
1
1

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Pro-Glu(OH)-NH2 (M1)
Pro-Pro-Glu(OH)-NH2 (M2)
M2 + H]+ m/z 341
M2 + Na]+ m/z 363
M1 + H]+ m/z 244
M1 + Na]+ m/z 266
+
acetone
+ acetone
HO
Pro-Glu(OH)-NH2
HO
Pro-Pro-Glu(OH)-NH2
+
+
+
+
m/z 302 (H ), m/z 324 (Na )
H2O
m/z 399 (H ), m/z 421 (Na )
-
- H2O
Pro-Glu(OH)-NH2
Pro-Pro-Glu(OH)-NH2
+
+
+
+
m/z 284 (H ), m/z 306 (Na )
m/z 381 (H ), m/z 403 (Na )
O
+
O
+
Ar
Ar
Ar
Ar
HO
HO
Pro-Pro-Glu(OH)-NH2
Pro-Glu(OH)-NH2
+
+
+
+
m/z 435 (H ), m/z 457 (Na )
H2O
m/z 532 (H ), m/z 554 (Na )
+
+ H2O
Ar
Ar
OH
Pro-Glu(OH)-NH2
OH
HO
HO
Pro-Pro-Glu(OH)-NH2
+
+
+
+
m/z 453 (H ), m/z 475 (Na )
m/z 550 (H ), m/z 572 (Na )
-
catalyst
- catalyst
Ar
Ar
OH
O
OH
O
Scheme 1. Hypothetic intermediates in asymmetric aldol reaction and species consistent with
the results of the ESIꢀMS measurements (underlined).
According to results of these measurements we concluded: (i) the aldol reaction takes place
on surface active centers of immobilized peptide catalysts, because neither the peptides, nor
their fragmented products (leached amino acids) can be detected in the solution; (ii) the
experimentally identified intermediates suggested the validity of the soꢀcalled enamine
mechanism for these catalysts as well; (iii) regrettably, due to their less stable nature, the
presence of intermediates that also include the aldehyde cannot be convincingly verified
under the experimental conditions applied.
3.3. Stability of the catalysts and reactions with mixtures of L- and D-Pro-NH-R catalysts
One of the most important characteristic of heterogeneous catalysts which may be studied
easily in continuous flow system is the stability of the catalysts during reaction. Deactivation
may be caused either by leaching of the catalytically active species or by poisoning of the
active centers by irreversible reaction with one of the reactants or products. The stability of
the heterogeneous catalysts developed for asymmetric aldol additions was seldom tested in a
20ꢀ22
continuous flow system.
Results obtained during reactions of 8 h in stream using catalysts
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PDꢀNHꢀR and PPDꢀNHꢀR are presented in Fig. 5. Both catalysts were found to be stable
following 8 h use, though the tripeptide containing material lost some activity during the first
few hours of use.
Fig. 5 Stability of PDꢀ and PPDꢀNHꢀR catalysts during time on stream in the asymmetric
2
aldol addition of acetone to 2ꢀNBA. Reagents and conditions: 300 mg catalyst + SiO (1/1), 2ꢀ
ꢀ1
ꢀ1
NBA (0.0326 mmol mL ) in acetone, flow rate 0.1 mL min , TON (turn over number)
number of transformations over one active site during the given time.
Both materials provided the same conversions at 8 h, which confirmed the successful
immobilization of the corresponding amino acids in each preparation step. The
enantioselectivity was also found constant during time on stream following a short induction
period. The turn over number obtained in 8 h was similar (~7.5) as obtained in batch reactor
considering the product obtained in two reactions of 4 h. Thus these catalysts may be used in
continuous flow system providing the similar productivity as in batch mode and sparing the
workꢀup procedure necessary in the latter case.
One of the methods for studying the reaction mechanism of asymmetric reactions is to
determine the relationship between the chirality of the catalyst and the ee of the product
formed. Nonꢀlinear relationship may indicate the involvement of diastereomeric intermediates
36
with different reactivity of the heteroꢀ and homochiral complexes. There has been
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significant progress in studying the phenomenon in amino acid catalyzed direct aldol
3
7ꢀ38
reactions,
to our best knowledge, however, data on supported peptide catalysts have not
been published. Thus, in our efforts to obtain further information on the mechanism of the
asymmetric aldol reaction taking place on the supported peptide catalysts experiments using
mixtures of resins having bonded Lꢀ and Dꢀproline on their surface (LꢀPꢀNHꢀR + DꢀPꢀNHꢀR)
were carried out. Data obtained in these experiments are summarized in Fig. 6, which showed
a linear behavior in the reaction of 2ꢀNBA and acetone as a function of the composition of the
catalyst mixture.
Fig. 6 Effect of LꢀPꢀNHꢀR and DꢀPꢀNHꢀR catalyst mixture composition on the ee obtained in
the aldol reaction of 2ꢀNBA with acetone (reaction condition: batch reactor, 2ꢀNBA 0.2
mmol, acetone 1 mL, catalyst 0.02 mmol, rt, reaction time 22 h).
This clearly linear behavior is consistent with the heterogeneous nature of the reaction, as
under homogeneous catalytic systems low solubility and aggregation of the amino acid used
15,36
as catalyst cause deviations from the linearity.
4. Interpretation and conclusion
Our studies on the direct asymmetric aldol reaction between 2ꢀNBA or 2ꢀMPA and acetone
over peptideꢀtype catalysts performed using a CFBR procedure led to the following
conclusions: (i) under the given experimental conditions, in the presence of dipeptideꢀNHꢀRꢀ
type catalysts (PPꢀ, PEꢀ, PDꢀ, SEꢀ, VEꢀNHꢀR) and PꢀNHꢀR βꢀhydroxy ketones of (R)
absolute configuration are formed; (ii) (R)ꢀproducts are also formed on tripeptide catalysts
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containing only proline (PPPꢀNHꢀR) and on those containing no proline (SSEꢀ and VVEꢀNHꢀ
R); (iii) catalysts containing proline and either aspartic acid or glutamic acid (PPDꢀNHꢀR,
PPEꢀNHꢀR) promote the formation of (S)ꢀproducts in excess; (iv) according to additional new
experimental observation, in the presence of benzoic acid reversal of enantioselection takes
place on catalysts containing only proline diꢀ and tripeptides. Namely, on PꢀNHꢀR the (R)ꢀ
product, whereas on PPꢀNHꢀR and PPPꢀNHꢀR catalysts the (S)ꢀproduct is formed in larger
amount; (v) prolonged experiments over two catalysts showed good stability of the
immobilized peptides both as concerns their activity and the enantioselectivity of the
products; (vi) the results of ESIꢀMS as well as those of studies on mixtures of the Lꢀ and DꢀPꢀ
NHꢀR catalysts supported the enamine mechanism and the heterogeneous character of the
asymmetric direct aldol reaction occurring on the peptides bonded to the resin support.
The stereochemistry of organocatalyzed asymmetric reactions is usually determined by
1
ꢀ5
the absolute configuration of the chiral catalyst. According to numerous experimental
observations, however, other factors may also play decisive roles in determining the sense of
14
enantioselection, i.e. the absolute configuration of the product. Results so far obtained in
asymmetric aldol reactions catalyzed by peptide catalysts highlight the importance of the
threeꢀdimensional position of the functional groups of catalysts and the conformation of
17,19
intermediates leading to chiral induction.
Based on the hypotheses, computation and experimental data reported to date, the
interpretation of the above can be summarized as follows. The conformations of the diꢀ and
16
tripeptide catalysts studied, as optimized according to our calculations, are shown in Fig. 7.
1
7,19
16
In accordance with results earlier published,
as well as with our own calculations, the
PPDꢀ and PPEꢀNHꢀR tripeptides have a turnꢀlike structure, in which the secondary amine of
17
proline is in close proximity of the carboxylic acid group of aspartic acid and glutamic acid.
The higher activity and the stronger effect on ee of PPD as compared to PPE can be explained
by the difference between the “close proximities” in the two tripeptides. The Pꢀ, PPꢀ and PPPꢀ
NHꢀR catalysts contain no carboxylic group, and in the case of the SSEꢀ and VVEꢀNHꢀR
catalysts the distance between the primary amine and the carboxyl groups is not optimal.
Therefore, not only activity and ee will be lower, but the sense of enantioselection may also
be unchanged as compared with the immobilized dipeptides.
Based on these new experimental data, it is reasonable to assume that the presence of
the enamineꢀtype intermediate plays a determinant role on supported catalysts under
continuousꢀflow conditions as well. The probable intermediates detected by our
measurements suggest similar mechanisms for the formation of (S) and (R) products, because
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the formation of hydroxylamine and the pertinent enamineꢀtype intermediates in the presence
of acetone can be assumed on both diꢀ and tripeptide catalysts (Scheme 1). Taking into
consideration these new experimental results, the cause of the reversal of enantioselectivity
observed on catalysts PPDꢀNHꢀR and PPEꢀNHꢀR as compared the other catalysts used in this
study is the divergent structures of the intermediate adducts formed between the enamine of
the chiral catalysts and the aldehyde. Consequently, catalysts containing Glu and Asp
exhibited similar behavior as regards the inversion of the enantioselectivities.
To sum up, in the asymmetric aldol addition catalyzed by polystyreneꢀsupported diꢀ
and tripeptides containing proline and aspartic or glutamic acid reversal of enantioselectivity
was observed in the CFBR conditions, too. The determinant role of the conformation of the
intermediate adducts is held responsible for chiral induction. These results point our attention
to further tasks such as (i) optimizations (including the preparation of the catalysts) in order to
increase the activities and enantioselectivities of these catalysts in flow conditions, (ii)
studying further chiral catalysts in the aldol reaction and other asymmetric reactions in flow
conditions, too. The experimental data reported above highlight the practical significance of
the possibility that further research in this promising direction may permit the development of
procedures to synthesize chiral building blocks with (R) or (S) absolute configuration using
the same cheap chiral source, i.e. (S)ꢀamino acids.
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Fig. 7 Computed conformations of immobilized peptides.
Acknowledgements
Financial support by the Hungarian National Science Foundation (OTKA Grant K 109278) is
highly appreciated. This research was realized in the frames of TÁMOPꢀ4.2.2.Aꢀ11/1/KONVꢀ
2012ꢀ0047 project. The research was also supported in the framework of TÁMOP 4.2.4. A/2ꢀ
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11ꢀ1ꢀ2012ꢀ0001 „National Excellence Program – Elaborating and operating an inland student
and researcher personal support system” key project. The project was subsidized by the
European Union and coꢀfinanced by the European Social Fund.
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Table of contents entry
Manuscript title: Reversal of the Enantioselectivity in Aldol Addition over Immobilized
Di- and Tripeptides: Studies under Continuous Flow Conditions
Authors: András Gurka, Imre Bucsi, Lenke Kovács, György Szőllősi* and Mihály Bartók*
The reversal of the enantioselectivity in the heterogeneous asymmetric
direct aldol reactions obtained over resin supported diꢀ versus
tripeptides was studied in continuousꢀflow system.