Development of an organo- and enzyme-catalysed one-pot, sequential three-component reaction

Article abstract of DOI:10.1016/j.tet.2012.02.010

A one-pot, three-component process is described which involves both organo- and enzyme-catalysed carbon-carbon bond-forming steps. In the first step, an organocatalyst catalyses the aldol reaction between acetaldehyde and a glyoxylamide. After dilution with additional aqueous buffer, and addition of pyruvate and an aldolase enzyme variant, a second aldol reaction occurs to yield a final product. Crucially, it was possible to develop a reaction in which both the organo- and enzyme-catalysed reactions could be performed in the same aqueous buffer system. The reaction described is the first example of a one-pot, three-component reaction in which the two carbon-carbon bond-forming processes are catalysed using the combination of an organocatalyst and an enzyme.

Full text of DOI:10.1016/j.tet.2012.02.010

Tetrahedron 68 (2012) 7719e7722
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Development of an organo- and enzyme-catalysed one-pot, sequential
three-component reaction
,
,
,
d
,
a,b,
Angela Kinnell a b, Thomas Harman a b, Matilda Bingham c, Alan Berry b , Adam Nelson
*
*
a School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK
b Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
c Merck Sharp and Dohme, Newhouse, Motherwell, ML1 5SH, UK
d Institute for Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A one-pot, three-component process is described which involves both organo- and enzyme-catalysed
carbonecarbon bond-forming steps. In the first step, an organocatalyst catalyses the aldol reaction be-
tween acetaldehyde and a glyoxylamide. After dilution with additional aqueous buffer, and addition of
pyruvate and an aldolase enzyme variant, a second aldol reaction occurs to yield a final product. Cru-
cially, it was possible to develop a reaction in which both the organo- and enzyme-catalysed reactions
could be performed in the same aqueous buffer system. The reaction described is the first example of
a one-pot, three-component reaction in which the two carbonecarbon bond-forming processes are
catalysed using the combination of an organocatalyst and an enzyme.
Received 15 November 2011
Received in revised form 20 January 2012
Accepted 6 February 2012
Available online 19 February 2012
Keywords:
Aldolase
Three-component reaction
Organocatalysis
Directed evolution
Biocatalysis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
1. O3 then
Cascade reactions can enable the highly efficient synthesis of
complex organic molecules from multiple components, reducing
the number of work-up and purification steps required.1 Many
combinations of catalyst types have been exploited in bicatalytic
processes including pairs of organocatalysts;2a,b pairs of enzy-
mes;2c,d organometallic and organo-catalysts;2e,f and organome-
tallic catalysts and enzymes.2g In addition, an aldolase variant has
been used to catalyse two sequential aldol reactions to yield
a precursor of the statin side-chain.3 The combination of organo-
catalysts and enzymes is, however, rare.4 Here, we report the first
example of a three-component reaction in which two carbon-
ecarbon bond-forming steps are catalysed using the specific
combination of an organocatalyst and an enzyme.
We have previouslyexploited directed evolution in the discovery
of aldolases with modified, synthetically-useful properties.5 Wild
type N-acetylneuraminic acid lyase (NAL) catalyses the reversible
aldol condensation betweenpyruvateand N-acetylmannosamine to
give N-acetylneuraminic acid. However, we discovered that the
E192N variant of NAL can accept a range of alternative aldehydes 2;
thus, ozonolysis of the alkenes 1 (/2), and condensation with py-
ruvate, gave the aldol products 3 (Scheme 1).5c We have also used
OH
OH
OH O
OH
Me2S
R2N
R2N
O
O
1a; R = Pr
1b; R = Bu
2a
2b
; R = Pr
; R = Bu
; R2 = (CH2)5
; R2 = (CH2)2O(CH2)2
1c; R2 = (CH2)5
1d; R2 = (CH2)2O(CH2)2
2c
2d
2.pyruvate,
NAL
(E192Nvariant)
O
OH
O
CO2H
R2N
5
4
cis or
trans
HO
OH
3a
3b
; R = Pr, 42%; cis:trans 82:18
; R = Bu, 66%; cis:trans 82:18
3c
; R2 = (CH2)5, 55%; cis:trans 79:21
3d
; R2 = (CH2)2O(CH2)2, 47%; cis:trans 82:18
Scheme 1. Aldolase reaction catalysed by an aldolase variant.
* Corresponding authors. E-mail address: a.berry@leeds.ac.uk (A. Berry).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.02.010

7720
A. Kinnell et al. / Tetrahedron 68 (2012) 7719e7722
Table 1
directed evolution to create stereochemically complementary al-
dolases that enable the stereoselective synthesis of both (4R)- and
(4S)-configured products 3.5d
Synthesis of the potential enzyme substrates 12 (see Scheme 3)
Substrate
Yield, 9/%
Yield, 10/%
Yield, 11b/%
Yield, 12/%
[anti/syna]
[cis/transa]
[cis/transa]
A drawback of our chemoenzymatic synthesis of the aldol
products 3 was that the synthesis of the alkenes 1 was rather
lengthy (six steps from ribonolactone).5c,6 We envisaged, however,
that it might be possible to develop a three-component synthesis of
similar products (such as 7) in a single pot (Scheme 2). Thus, it was
hoped that organocatalysed condensation7 between a glyox-
ylamide 4 and acetaldehyde would yield an aldol product 6. The
structure of complexes between the E192N NAL variant, pyruvate
8a
8b
8c
8d
91c
46
30
60
24 [70:30]
12 [80:20]
33 [68:32]
18 [74:26]
59 [95:5]
62 [95:5]
27 [88:12]
49 [78:22]
>98 [95:5]
>98 [95:5]
>98 [90:10]
83 [78:22]
a
Determined by 500 MHz 1H NMR spectroscopic analysis of the isolated products.
Column chromatography gave mixtures that were enriched in the cis anomers.
b
The relative configuration of the products was determined by analysis of diagnostic
geminal coupling constants.
c
Yield from the isolated glyoxylamide derived from 8a.
and a substrate analogue suggested that a hydroxyl group
a to the
aldehyde may not be essential for binding.8 Accordingly, it was
hoped that the aldol products 6 would be viable substrates for the
E192N variant, enabling the synthesis of the final products 7 by
enzymic addition of pyruvate. The condensation of the aldehyde 4,
acetaldehyde and pyruvate (5), to give 7, would constitute the first
example of a three-component reaction in which two carbon-
ecarbon bond-forming steps were catalysed using the specific
combination of an organocatalyst and an enzyme.
Ozonolysis of the fumaric amides 8, and allylation by treatment
with allyltrimethylsilane and titanium tetrachloride, gave the racemic
homoallylic alcohols 9. Ozonolysis of the homoallylic alcohols 9, and
indium-mediated condensationwithethyl a-(bromomethyl)acrylate,
gave the diols 10 with modest anti diastereoselectivity.9 Finally, ozo-
nolysis of the diastereomeric mixtures of the diols 10 gave the di-
astereomeric esters 11, which were at least partially separable by
column chromatography. Ester hydrolysis and cation exchange, gave
the ammonium salts 12. The relative configuration of the major di-
astereomers of the diols 10 was determined by careful analysis of
diagnostic geminal coupling constants in their derivatives 11.
O
organo-/
enzyme-
OH
CO2H
O
O
O
O
R2N
R2N
CO2H
catalysed
cascade
A coupled enzyme assay was used to assess the efficiency of
aldolase-catalysed cleavage of the substrates.5b In this assay, aldolase-
catalysed cleavage to yield pyruvate would be followed by lactate
dehydrogenase-catalysed reduction; the concomitant oxidation of
NADH being detected spectroscopically by monitoring the change in
absorption at 340 nm. The steady state kinetic parameters for the
cleavage of 3aed and 12aed by the E192N NAL variant are presented
inTable 2. Crucially, thealdols12aedwereallsubstratesfortheE192N
NAL variant, albeit with up to w10-fold reduced kcat/KM relative to the
substrates 3aed, which bear an additional hydroxyl group at C-5.
O
OH
7
4
5
organo-
catalyst
enzyme
OH O
R2N
O
6
Scheme 2. Overview of the proposed organo- and enzyme-catalysed cascade.
2. Development of a viable three-component reaction
Table 2
Steady state kinetic parameters for enzyme-catalysed cleavage by the E192N NAL
variant
2.1. Synthesis and evaluation of potential products of
aldolase-catalysed reactions
Substrate
R2
kcat/minꢀ1
KM/mM
kcat/KM/minꢀ1 mMꢀ1
3aa
3ba
3ca
Pr
Bu
200ꢁ2
61ꢁ2
0.10ꢁ0.01
0.11ꢁ0.02
0.34ꢁ0.06
0.70ꢁ0.20
0.39ꢁ0.04
0.25ꢁ0.04
0.57ꢁ0.02
3.4ꢁ0.2
1960
580
850
230
340
360
167
22
We first determined whether the compounds 7 were viable
substrates for retro-aldol reactions catalysed by variants of NAL.
The synthesis of the ammonium salts, 12, of the carboxylic acids, 7,
is described in Scheme 3 and Table 1.
(CH2)5
(CH2)2O(CH2)2
Pr
Bu
(CH2)5
330ꢁ6
160ꢁ8
130ꢁ3
90ꢁ6
3da
12a
12b
12c
12d
95ꢁ1
(CH2)2O(CH2)2
73ꢁ2
a
See Ref. 5b.
2.2. Effect of removal of an
a-hydroxyl group on the
stereoselectivity of aldolase-catalysed reactions
The effect of removing the a-hydroxyl group from the aldehyde
substrate 2a on the stereoselectivity of aldolase-catalysed reactions
was investigated. Both enantiomers of 9a were prepared using an
established chiral relay approach:10 reaction of 13 with dipropyl-
amine, and deprotection, gave the corresponding homoallylic alco-
hols 9a, which were ozonolysed to give the aldehydes 6a (Scheme 4).
The stereoselectivity of the aldolase-catalysed condensations of
three substratesdthe aldehyde 2a, and the enantiomeric aldehydes
(R)- and (S)-6adwas determined by 1H NMR spectroscopy in
deuterated sodium phosphate buffer (pH 7.4) (Table 3). We in-
vestigated the stereocontrol exerted by the E192N variant, as well
as two stereochemically complementary variants that we had
previously discovered using directed evolution: E192N/T167G
and E182N/T167V/S208V.5d As expected, the condensation of the
Scheme 3. Synthesis of the potential enzyme substrates 12 (see Table 1).

A. Kinnell et al. / Tetrahedron 68 (2012) 7719e7722
7721
With an organocatalysed aldol reaction in hand that operated
under appropriately buffered conditions, we investigated the effect of
variousadditivesontheactivityoftheE192Nenzyme.Ineachcase, we
assayed enzyme activity by monitoring condensation of the aldehyde
2a and pyruvate to give 3a (Scheme 1): the formation of 3a was
assayed by periodic cleavage and condensation with thiobarbituric
acid.12 At the concentrations of propionaldehyde and the glyox-
ylaldehyde 4a needed for aldol condensation, the enzyme was still
fully active. However, in the presence of the concentration of the
organocatalyst 16 that had corresponded to a 10 mol % loading (see
Scheme 5), the activity of the enzyme was very significantly impaired.
We decided, therefore, to develop a one-pot, three-component
process in which the reaction mixture was diluted after the orga-
nocatalysed step (Scheme 6). In order to gain insight into the
stereoselectivity of the overall process, we determined the enan-
tiomeric excess of both the intermediate aldehyde 6a (R¼Pr) and
the final product 7a. Thus, 6a and 7a were converted into the diol 17
(by reduction with NaBH4) and the lactone 18 (by oxidative cleav-
Scheme 4. Investigation of the stereoselectivity of reactions of aldehydes 9a catalysed
by NAL variants. The enantiomeric substrate (S)-9a was also prepared and studied. The
stereoselectivity of the aldolase-catalysed condensation was determined in pH 7.4
deuterated sodium phosphate buffer; the structures 6a and 14a are drawn in pro-
tonated form for convenience.
Table 3
Kinetic and thermodynamic stereoselectivity of aldol reactions catalysed by NAL variants
Enzyme
Substrate and product
2a/3a cis/transa [re/sib]
(R)-6a/14a cis/transa [re/sib]
(S)-6a/14a cis/transa [re/sib]
Thermodynamic ratioc
E192N
E192N/T167G
69:31
74:26
74:26
79:21 [79:21]
>95:<5 [>95:<5]
>13:<87 [>13:<87]
58:42 [42:58]
60:40 [40:60]
29:71 [71:29]
62:38 [62:38]
58:42 [58:42]
63:27 [63:27]
E182N/T167V/S208V
a
Determined by integration of the 500 MHz 1H NMR spectrum.
Kinetic facial control in the attack on the respective aldehyde.
Ratio after equilibration. After 1 week, a second aliquot of enzyme was added and the reaction incubated for a second week.
b
c
aldehyde 2a with pyruvate was highly stereoselective with the
age, lactonisation and dehydration), respectively (Fig.1). The diol 17
was found to be essentially racemic (<5% ee), and, at completion,
the lactone 18 was also racemic (<5% ee).
stereochemically
complementary
variants
E192N/T167G
(/>95:<5 cis/trans-3a) and E182N/T167V/S208V (/<13:>87 cis/
trans-3a). Unfortunately, stereocontrol in the condensations of the
aldehydes (R)- and (S)-6a was modest in all of the aldolase-
1. 5 mol% 16
catalysed condensations. Evidently, the
a-hydroxyl group in the
pH 7.4 buffer
OH
CO2H
O
aldehyde 2a (corresponding to the hydroxyl group at C-5 in 3a)
plays a crucial role in stereocontrol.
2. 2.5-fold dilution
with buffer
O
O
O
R1R2N
R1R2N
3.sodiumpyruvate
NAL
cis or
trans
O
2.3. Identification of reaction medium in which both organo-
and enzyme catalysis are possible
OH
(E192N variant)
35 °C
7a, R1,R2 = Pr
7c, R1,R2 = (CH2)5
7e, R1 = Me, R2 = Pr
7f, R1,R2 = Me
4
We investigated the possibility of performing an organocatalysed
aldol condensation under the buffered conditions needed for enzy-
mic catalysis. The diamine 16 has been developed as an organo-
catalyst for asymmetric aldol reactions performed in water.11
Remarkably, we found that, with 10 mol % 16, organocatalysed al-
dol condensation was possible under buffered conditions, and after
reduction, the diols 15 were obtained (Scheme 5); in contrast, with
just 1% 16 at pH 7.0, no product was detected The diastereoselectivity
of the condensation was modest, and varied significantly with pH.
The relative configuration of the diols was determined by analysis of
diagnostic coupling constants in the corresponding acetonides.
N
H
N(C10H21 2
)
7g, R1, R2 = Et
16
Scheme 6. Organo- and enzyme-catalysed condensation of the glyoxylamides 4, ac-
etaldehyde and pyruvate to give products 7 (see Table 4).
Fig. 1. Derivatives used to determine the enantioselectivity of the one-pot process: the
diol 17 and the lactone 18 were derived from the aldehyde intermediate 6a and the
product 7a, respectively.
3. Determination of the scope of the one-pot, three-
component reaction
Our investigations into the scope of the one-pot, three-compo-
nent reaction are summarised in Scheme 6 and Table 4. The
Scheme 5. Study of the organocatalysed condensation of 3a and propionaldehyde
(10 equiv) in aqueous buffer.

7722
A. Kinnell et al. / Tetrahedron 68 (2012) 7719e7722
glyoxylamides 4 were reacted with acetaldehyde (10 equiv) in the
presence of 5 mol % 16 in pH 7.4 buffer for 20 h. Given the lack of
enantioselectivity in the organocatalysed step, and the poor dia-
stereoselectivity of the aldolase-catalysed reactions of the alde-
hydes 6, we made no attempt to ensure that the aldolase-catalysed
reactions proceeded under kinetic control. Thus, after w2.5-fold
dilution with buffer, and addition of sodium pyruvate and the
E192N variant, the reaction mixture was incubated for 70 h at 35 ꢂC.
The yield and stereoselectivity of the process was determined for
a range of substrates (Table 4). The reaction was successful with
a range of glyoxylamides 4, and 40e51% yield of products were
obtained. In common with similar aldolase-catalysed reactions
with extended reaction times,5c,d it is likely that the diaster-
eoselectivity of the one-pot reactions was thermodynamically
controlled. Our studies demonstrate the value of the specific
combination of organo- and enzymic catalysis in three-component
reactions leading to heterocyclic products.
purification by preparative HPLC (gradient elution: 0:100/30:701%
TFA in H2Oe1% TFA in MeCN) over 30 min gave the product 7g
(13.2 mg, 51%; cis/trans 61:39) as a colourless film, nmax/cmꢀ1 (liquid
film) 3369, 2979,1761,1627,1462 and 1202 cmꢀ1
; dH (500 MHz, D2O)
5.13(0.20H,dd, J9.2and3.4Hz,6-Htrans(maj)), 5.07(0.20H,dd, J6.4and
4.2 Hz, 6-Htrans(min)), 4.84 (0.53H, dd, J 11.7 and 1.6 Hz, 6-Hcis(maj)),
4.56e4.51 (0.28H, m, 6-Hcis(min) and 4-Htrans(min)), 4.26(0.20H, br s, 4-
Htrans(maj)), 4.14e4.04 (0.53H, m, 4-Hcis(maj)), 3.94e3.84 (0.08H, m, 4-
Hcis(min)) 3.40e3.06 (2H, m, NCH2), 2.15e1.48 (2H, m, H-5 and H-3),
1.12e1.02 (3H, m, CH3), 0.96 (3H, t, J 6.8 Hz, CH3); dC (100 MHz, D2O,
cis(maj) only) 163.2 (amide or 1-C), 67.2 and 62.7 (6-C and 4-C), 42.5,
14.2, 39.1 and 35.3 (5-C, 3-C and NCH2),13.4 and 11.7 (CH3); m/z (ESꢀ)
260.1 (100%, [MꢀH]ꢀ); HRMS (EI) MꢀHꢀ, found 260.1152. C11H20NO6
requires 260.1140. Correlation of the 1H NMR spectrum with that of
the acid 7a allowed the identification of the anomers of the cis and
trans diastereoisomers. The ratio of the species were determined by
the integration of the following signals: 5.13 ppm (trans(maj)),
5.07 ppm (trans(min)), 4.14e4.04 ppm (cis(maj)), 3.94e3.84 ppm
(cis(min)). Analysis by 500 MHz 1H NMR spectroscopy revealed that
a 61:39 mixture of cisand trans diastereomers was present and that
the cis isomer existed as an 87:13 mixture of anomers and the trans
isomer existed as a 50:50 mixture of anomers.
Table 4
One-pot, three-component reactions (see Scheme 6)
Substrate
R1,R2
Yielda 7/%
cis/transb
4a
4c
4e
4f
Pr,Pr
40
48
48
43
51
78:22c
66:34
57:43
71:29
61:39
e(CH2)5e
Me,Pr
Me,Me
Et,Et
Acknowledgements
4g
We thank EPSRC, BBSRC and Merck Sharp and Dohme for funding,
and Nicole Timms and Adam Daniels for helpful discussions.
a
Based on the limiting reactant 4. Products were purified by preparative HPLC.
Determined by integration of the 500 MHz 1H NMR spectrum of the purified
b
product.
c
Supplementary data
The product had <5% ee.
Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2012.02.010.
4. Summary
In summary, we have shown that the combination of organo-
and enzymic catalysis may be exploited in one-pot reactions with
two carbonecarbon bond formation steps. Crucially, it was possible
to identify buffered aqueous conditions under which both of the
catalysed steps were possible. A range of glyoxylamides 3 were
condensed with both acetaldehyde and pyruvate to give the cor-
responding heterocyclic products 7. Unfortunately, in this specific
case, the stereoselectivity of the overall process was poor, both
because the organocatalysed step was not enantioselective, and
because the aldolase-catalysed reactions of the intermediate alde-
References and notes
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Huang, Y.; Walji, A. M.; Larsen, C. H.; MacMillan, D. W. C. J. Am. Chem. Soc. 2005,
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hydes
6 (lacking an a-hydroxy group) were poorly diaster-
eoselective. Nonetheless, this study demonstrated the possibility of
combining organo- and enzymatic catalysis in bicatalytic chemis-
try: the combination of organo- and enzymatic catalysis is power-
ful, and is likely to find further application in one-pot reactions
involving two or more carbonecarbon bond-forming steps.
€
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5. Experimental section
5.1. General
6. Woodhall, T.; Williams, G.; Berry, A.; Nelson, A. Org. Biomol. Chem. 2005, 3,
1795e1800.
The glyoxamide 3g (R1]R2]Et) (13.5 mg, 95.5
solved in buffer (308 L, pH 7.4, 20 mM potassium phosphate), and
was added to the diamine 16 (1.81 mg, 4.76 mol). Acetaldehyde
(54 L, 0.955 mmol) was added and reaction mixture was stirred at
room temperature for 20 h, diluted with a pH-adjusted solution (pH
7.4) of sodium pyruvate (105 mg, 0.955 mmol) in buffer (115 L, pH
7.4, 20 mM potassium phosphate), the E192N aldolase variant
mmol) was dis-
7. For similar crossed organocatalysed aldol reactions, see: Kano, T.; Noishiki, A.;
Sakamoto, R.; Maruoka, K. Chem. Commun. 2011, 10626e10628.
8. The a-hydroxyl group interacts with Asp191 in three of the four subunits, but
does not interact directly with the protein in the fourth subunit: Campeotto, I.;
Bolt, A. H.; Harman, T. A.; Dennis, C.; Trinh, C. H.; Phillips, S. E.; Nelson, A.;
Pearson, A. R.; Berry, A. J. Mol. Biol. 2010, 404, 56e69.
9. For examples of indium-mediated allylation reactions, see: (a) Chan, T. H.; Li, C.
J. J. Chem. Soc., Chem. Commun. 1992, 747e748; (b) Chappell, M. D.; Halcomb, R.
L. Org. Lett. 2000, 2, 2003e2005.
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D. Org. Biomol. Chem. 2004, 2, 3608e3617.
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12. (a) Warren, L. Methods Enzymol. 1963, 6, 463e465; (b) Warren, L. J. Biol. Chem.
1959, 234, 1971e1975.
m
m
m
m
(416 mL, 6.98 mg/mL in pH 7.4, 20 mM potassium phosphate) added,
and incubated at 35 ꢂC for 70 h. The pH of the reaction mixture was
lowered to pH 2 byaddition ofan aqueous solution of2 M formicacid,
and after 5 min, raised to pH 7 by addition of an aqueous solution of
2 M NH4OH. The reaction mixture was filtered through Celite, and