Synthesis of screening substrates for the directed evolution of sialic acid aldolase: Towards tailored enzymes for the preparation of influenza a sialidase inhibitor analogues

Article abstract of DOI:10.1039/b501503k

The stereoselective synthesis of two epimeric screening substrates, (4R, 5R, 6R)- and (4S, 5R, 6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxy-hexanoic acid, for the directed evolution of sialic acid aldolase is described. The complementary methods relied on stereoselective indium-mediated additions of ethyl α-bromomethyl acrylate to functionalised aldehydes. With an α-hydroxy aldehyde, (2R, 3R)-2,3-dihydroxy-4-oxo butanoic acid dipropylamide, the addition was chelation controlled, and the syn product, (6R, 5R, 4S)-6-dipropylcarbamoyl-2-methylidene-4,5,6-trihydroxy-hexanoic acid ethyl ester, was obtained. In contrast, the stereochemical outcome of the addition to (2R, 3A)-N, N-dipropyl-2,3-O-isopropylidene-4-oxobutyramide was consistent with Felkin-Anh control, and the anti adduct, (4R, 5R, 6R)-6-dipropylcarbamoyl-2- methylidene-4-hydroxy-5,6-O-isopropylidene-hexanoic acid ethyl ester, was the major product. Ozonolysis and deprotection gave the screening substrates as mixtures of furanose and pyranose forms, in good yields. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b501503k

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A R T I C L E
Synthesis of screening substrates for the directed evolution of sialic
acid aldolase: towards tailored enzymes for the preparation of
influenza A sialidase inhibitor analogues†
Thomas Woodhall,a,b Gavin Williams,b,c Alan Berryb,c and Adam Nelson*a,b
a School of Chemistry, University of Leeds, Leeds, UK LS2 9JT
b Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK LS2 9JT
c School of Biochemistry and Microbiology, University of Leeds, Leeds, UK LS2 9JT
Received 31st January 2005, Accepted 15th March 2005
First published as an Advance Article on the web 12th April 2005
The stereoselective synthesis of two epimeric screening substrates, (4R, 5R, 6R)- and (4S, 5R, 6R)-6-dipropylcarba-
moyl-2-oxo-4,5,6-trihydroxy-hexanoic acid, for the directed evolution of sialic acid aldolase is described. The
complementary methods relied on stereoselective indium-mediated additions of ethyl a-bromomethyl acrylate to
functionalised aldehydes. With an a-hydroxy aldehyde, (2R, 3R)-2,3-dihydroxy-4-oxo butanoic acid dipropylamide,
the addition was chelation controlled, and the syn product, (6R, 5R, 4S)-6-dipropylcarbamoyl-2-methylidene-4,5,6-
trihydroxy-hexanoic acid ethyl ester, was obtained. In contrast, the stereochemical outcome of the addition to (2R,
3R)-N,N-dipropyl-2,3-O-isopropylidene-4-oxobutyramide was consistent with Felkin–Anh control, and the anti
adduct, (4R, 5R, 6R)-6-dipropylcarbamoyl-2-methylidene-4-hydroxy-5,6-O-isopropylidene-hexanoic acid ethyl ester,
was the major product. Ozonolysis and deprotection gave the screening substrates as mixtures of furanose and
pyranose forms, in good yields.
The sialic acid mimetics 1 and 2a are potent inhibitors of
Introduction
influenza sialidases.18,19 Indeed, Zanamivir, 1, inhibits influenza
High levels of catalytic efficiency, compatibility with aqueous
A and B sialidases with IC50 ≈ 5 nM, prevents viral replication
reaction conditions and low levels of side reactions have led to
in vitro and in vivo and is marketed as a drug for the treatment of
the widespread exploitation of enzymes in organic synthesis.1
influenza.19 Its derivative 2a is a selective inhibitor of influenza
Nonetheless, the narrow substrate specificity of many enzymes
limits their potential as general catalysts for synthetic organic
chemistry. In addition, Nature rarely provides complementary
enzymes for the preparation of all possible stereoisomeric
products. Directed evolution offers an opportunity to address
these deficiencies, and has huge potential for exploitation in
synthetic organic chemistry.2–4
A sialidase (IC50 for influenza A: 4 nM; and B: 4500 nM), and
has been prepared via a multi-step reaction sequence involving
the oxidative cleavage of the side chain of sialic acid.20,21An
Evolved enzymes may have broader or altered sub-
strate specificity,3,5 may catalyse reactions with modified lev-
els of stereoselectivity6,7 and may display altered physical
characteristics.8–12‡ For example, an enantioselective lipase for
the kinetic resolution of chiral esters,13 and a hydantoinase
with reversed enantioselectivity,14 have been generated using
directed evolution. In addition, an amine oxidase has been
evolved which can catalyse the deracemisation of a wide range of
chiral amines.15 To date, we have concentrated on the evolution
of aldolases which catalyse aldol reactions with a modified
stereochemical course:16 this approach enabled the preparation
of a diastereoisomeric product from the substrates accepted by
the wild-type enzyme.5,17
alternative approach for the preparation of sialic mimetics of
general structure 2 could involve an enzyme-catalysed aldol
condensation of an aldehyde 3 and pyruvate (→ 4), followed by
functional group manipulation (Scheme 1). Sialic acid aldolase
catalyses the reversible aldol condensation between pyruvate and
N-acetyl mannosamine,1 and is an ideal starting point for the
directed evolution of a suitable tailored enzyme. There are two
possible stereochemical outcomes from the aldol condensation
of pyruvate and the aldehyde 3 (→ anti- or syn-4), and, ideally,
complementary enzymes would be available for the synthesis of
either diastereoisomer.
† Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/ob/b5/b501503k/
‡ Enzymes with enhanced thermostability,8,9 and/or compatibility with
non-aqueous solvents have been evolved.10–12
Scheme 1
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
T h i s j o u r n a l i s
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 9 5 – 1 8 0 0
1 7 9 5
©

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mind, the b-amino esters 5, 6 and 7a–b, in which an aldehyde
could be unmasked by ozonolysis of the remaining alkene, were
prepared using a Michael addition of the lithium amide 8 as the
key step23 (for example, see Scheme 2). Unfortunately, attempted
conversion of the b-amino esters 6 and 7 into the corresponding
b-amino dipropylamides was unsuccessful.
An alternative approach involved the diester 9 in which the
enantiotopic carbonyl groups of meso tartaric acid have been
differentiated. It has previously been shown that the equatorial
ester of 9 is more susceptible to attack by the aluminium amide
14;24 following this procedure, we were able to isolate the amide
13 but in extremely low yield (6%).The diester 9 was found
Fig. 1
Scheme 2
Sialic acid aldolase has reasonably broad substrate specificity:
although only pyruvate is a competent donor, many six- and five-
carbon aldehydes are substrates.1,22 Condensations involving
shorter aldehydes are less promising: L- and D-erythrose and
threose react at between 0.3% and 5% of the rate of N-acetyl
mannosamine, and two- and three-carbon aldehydes are not
substrates. In this paper, we describe the preparation of screening
substrates for the evolution of enzymes able to accept the
aldehydes 3 efficiently. A possible screening strategy is shown
in Fig. 1. Although the aim was to generate enzymes for use
in synthetic chemistry, we chose to use the required products
4 of the condensation as screening substrates. Mutant enzymes
able to catalyse the required aldol condensation would also, of
course, be able to cleave the screening substrates 4: one of the
cleavage products, pyruvate, may be detected using a coupled
enzyme assay. This approach is technically much simpler than
detecting the required product of the synthetic reaction, and
might enable the evolution of complementary enzymes for the
preparation of either of the possible diastereomeric products
anti- or syn-4.
to be highly resistant to aminolysis under a range of reacti◦on
conditions: treatment either with neat dipropylamine at 80 C
in a sealed vessel or with an excess of Pr2NAlMe2 under a range
of reaction conditions§ returned only starting material.
The hydrolysis of the diester 9 (see Table 1 and Scheme 3) was
plagued by problems with epimerisation. Treatment of 9 with
potassium hydroxide in MeOH–water, and amide formation,
gave a mixture of the required amide 11 and the diequatorial
diamide 15 (entry 1, Table 1).¶ With lithium hydroperoxide in
THF–water, only the diamide 15 was obtained (entry 2). The
equatorial ester of 9 is more susceptible to hydrolysis (→ 17,
Scheme 4); however, epimerisation of the axial ester (→ 18)
was competitive with its hydrolysis, and once epimerisation had
occurred, hydrolysis to give 19 was rapid. With DBU in water,
epimerisation was minimised, and after amide formation, an
80 : 20 mixture of the required amide 11 and the diequatorial
diamide 15 was observed (entry 3).
Synthesis of protected versions of the aldehydes 3
An obvious strategy for the synthesis of the screening substrates
4 would involve the diastereoselective addition of a pyruvate
equivalent to a protected version of an aldehyde 3. With this in
The amide 11 and the diamide 15 were difficult to separate,
and so we chose to purify the carboxylic acid 10 (66% yield)
§ The ester 9 (0.1–0.4 M in toluene or dichloromethane) was treated with
4 or 10 equivalents of Pr2NAlMe2 at room temperature or 40 ◦C.
¶ The configuration of the C2-symmetric diamide 15 was inferred from
the simplicity of its 500 MHz 1H NMR spectrum.
Scheme 3
Table 1 Synthetic transformations of the diester 9
Entry
Conditions
Product
Yielda (%)
1
2
3
1. KOH (3.6 eq.), 85 : 15 MeOH–H2O; 2. EDC, HOBt, Pr2NH, EtOAc
1. LiOOH (10 eq.), 75 : 25 THF–H2O; 2. EDC, HOBt, Pr2NH, EtOAc
1. DBU (2.1 eq.), H2O; 2. EDC, HOBt, Pr2NH, EtOAc
15b
15
23
90
49
11c
a Yield of purified product. b Analysis of the crude reaction mixture by 500 MHz 1H NMR spectroscopy revealed a mixture of the amide 11 and the
diamide 15. c Analysis of the crude reaction mixture by 500 MHz 1H NMR spectroscopy revealed an 80 : 20 mixture of the amide 11 and the diamide
15.
1 7 9 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 9 5 – 1 8 0 0

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after the hydrolysis step (Scheme 3); treatment of the acid 10
with dipropylamine, EDC and HOBt gave the required amide
11 (72%) and its diequatorial epimer 16 (4%). The relative
configurations of the amides 11 and 16 were deduced by careful
analysis of their 500 MHz 1H NMR spectra (for 11: J2,3
=
4.0 Hz; for 16: J2,3 = 10.1 Hz); the relative configuration of
11 was confirmed by X-ray crystallography (Fig. 2)* and the
observation of diagnostic NOEs (Fig. 3). Unfortunately, the
extremely hindered nature of the axial methoxycarbonyl group
of 11 prevented its reduction: treatment with a range of reagents
i
(LiBH4, Bu2AlH etc.) gave only recovered starting material.
Fig. 2
Treatment of the amide 11 with TFA–water did, however, give
the corresponding diol 12 in 64% yield, whose ester we were
unable to reduce using a range of reagents (LiBH4, iBu2AlH or
NaBH4).
The problems encountered in the preparation of a BDA-
protected version of the aldehyde 3 prompted us to prepare
the corresponding acetonide instead. The diol 20, prepared by
oxidative degradation of isoascorbic acid (24),25 was converted
into the corresponding acetonide 22 (Scheme 5).The c-lactones
20 and 22 were aminolysed to give the dimethylamides 21a and
23a and the dipropylamide 23b. The effect of the acetonide on
the reactivity of the c-lactones was remarkable: aminolysis with
dipropylamide gave a 5% yield of 21b (from 20) after 6 days, and
a 65% yield of 23b (from 22) after 3 days. Deprotection of 23b
(9 : 1 TFA–water) gave the triol 21b. Unfortunately, attempted
selective oxidation of the primary alcohol of 21b with TEMPO
was unsuccessful.
Fig. 3 Diagnostic NOEs for the amide 11.
Preparation of protected sialic acid mimetics
The alcohol 23b was converted into the corresponding aldehyde
(29) using a Swern oxidation, and was used immediately
in an indium-mediated allylation without purification.26–28
solution of the aldehyde 29 and ethyl a-bromomethyl acrylate
in THF–water was treated with indium powder. The required
A
* CCDC reference numbers 262317 and 262318. See http://www.rsc.
org/suppdata/ob/b5/b501503k/ for crystallographic data in CIF or
other electronic format.
Scheme 4
Scheme 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 9 5 – 1 8 0 0
1 7 9 7

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Scheme 6
addition products 25 and 26 (crude ratio: 25 : 26 77 : 23)
were isolated in 45% and 13% yield respectively (Scheme 6);
in addition, the lactones 27 and 28 were each obtained in ca.
1% yield.The relative configuration of the major product 25
the lactone 27 (Fig. 6).†† In addition, Swern oxidation of 25 gave
a mixture of the regioisomeric a,b-unsaturated esters 30 and 31.
In view of these observations, this strategy was not pursued.
Fig. 5
was determined by X-ray crystallography (Fig. 4)*, an outcome
which is consistent with Felkin–Anh-controlled attack29 on the
intermediate aldehyde 29 (Fig. 5).
Fig. 6
An alternative approach would involve the chelation-
controlled30 addition of a carbon nucleophile to an analogue of
the aldehyde 29. The c,d-unsaturated amide 35 was synthesised
from the corresponding acid 34, prepared by protection of D-
ribonolactone, iodination (→ 33) and reductive fragmentation
(Scheme 7).31 Acetonide deprotection gave the corresponding
1,2-diol 36.
The a,b-dihydroxy aldehyde 39 was prepared by ozonolysis
of the corresponding alkene 36, and was used immediately
without purification (Scheme 8).A solution of the aldehyde 39
and ethyl a-bromomethyl acrylate in THF–water was treated
with indium powder,26–28 and the required addition product was
obtained in 43% yield (syn : anti 86 : 14) together with the
(syn) lactone 38 (7% yield). The high level of syn selectivity
observed is consistent with chelation-controlled addition30 to
the intermediate aldehyde 39 (Fig. 7). Recrystallisation gave the
Fig. 4
A strategy for controlling the configuration of the alcohol 26
would involve inversion of its epimer, 25, either directly or via
an oxidation–reduction sequence. However, mesylation of the
alcohol 25 triggered participation of the amide oxygen to give
†† This result indicated that the lactone 27 had stemmed from lactoni-
sation of the minor diastereomeric adduct (26) of the indium-mediated
allylation. The conversion of the alcohols 25 (J2,3 = 6.1 Hz) and 26
(J2,3 = 6.7 Hz) into a common compound demonstrated that 25 and
26 were C-4 epimers (and were, therefore, both cis acetonides) and that
epimerisation of the aldehyde 29 had not occurred under the conditions
of the allylation.
Scheme 7
1 7 9 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 9 5 – 1 8 0 0

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Table 2 Ratios of pyranose and furanose forms observed for the
compounds 41–43 and 45–47
Compound
Pyranose forms : furanose formsa
41
42
43
45b
46
47
20 : 0 : 40 : 40
30 : 0 : 40 : 30
15 : 10 : 45 : 30
55 : 0 : 30 : 15
85 : 5 : 10 : 0
80 : 10 : 5 : 5
Scheme 8
addition product 37 in 24% yield as a >98 : < 2 mixture of
a Determined ( 5%) by 500 MHz 1H NMR spectroscopy. b Initially, a
72 : 14 : 14 mixture of one pyranose and two furanose forms was
obtained, which equilibrated to the mixture shown in the Table.
diastereoisomers.
in methanol–water, cation exchange, and purification by ion
exchange chromatography, gave the sialic acid mimetics 43 and
47.
Fig. 7
The ketones 41–43, and 45–47 existed as mixture of pyranose
and furanose forms (see Table 2 and Scheme 9); confirmation
that all signals in the spectra of these compounds were due to in-
terchanging anomeric forms was provided by 500 MHz exchange
spectroscopy (EXSY) NMR experiments. NMR spectroscopic
details of each of the forms of 42, 43, 46 and 47 are summarised
in Table 3. The J3,4 values for the pyranose forms of the ketones
42 and 43 are consistent with an axial orientated C-4 substituent.
The J3,4 values for the pyranose forms of the ketones 46 and 47
are consistent with an equatorial orientated C-4 substituent.
Preparation of the screening substrates
The synthesis of the screening substrates 43 and 47 was
completed by deprotection of the diastereomeric esters 25 and 26
(Scheme 9). Ozonolysis of 25, followed by work-up with aqueous
hydrogen peroxide solution, gave the required ketone 40 and
the lactone by-product 48 (26%). Presumably, the a-keto ester
must have been cleaved under the conditions of the work-up
(49 arrows) to give the by-product, whose formation could be
avoided with a reductive work-up with dimethyl sulfide: under
these conditions, the required ketone 40 was obtained in 81%
yield. Similarly, ozonolysis of 26, and reductive work-up, gave
the corresponding ketone 44. Acetonide hydrolysis of 40 and
44 gave the diols 41 and 45. The diol 45 was also prepared
more directly by ozonolysis of the a,b-unsaturated ester 37
(>98% yield). Treatment of 41 and 45 with barium hydroxide
Summary
The synthesis of the diastereoisomeric screening substrates
43 and 47 was described. The routes were amenable to the
synthesis of each substrate on >500 mg scale: the screening
substrate 43 was prepared in 9 steps and 7% overall yield
from D-isoascorbic acid, and its epimer 47 was prepared in 10
Scheme 9
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 9 5 – 1 8 0 0
1 7 9 9

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Table 3 NMR spectroscopic details of 42, 43, 46 and 47 (recorded in D2O)
d/ppm
C-2
d/ppm
H-4
d/ppm
H-5
d/ppm
H-6
J/Hz
H3a–H3b
J/Hz
H3–H4
J/Hz
H4–H5
J/Hz
H5–H6
Compound
Form
d/ppm H-3
42
fur(1)
fur(2)
pyr
pyr(1)
pyr(2)
fur(1)
fur(2)
pyr(1)
pyr(2)
fur
pyr(1)
pyr(2)
fur(1)
fur(2)
96.7
104.7
104.3
1.88, 1.82
2.31, 1.77
2.11, 2.03
2.15, 2.11
2.54, 1.90
2.64, 2.09
2.42, 2.39
2.11, 2.11
2.50, 1.84
2.37, 2.27
2.23, 1.84
2.60a, 1.68
2.60a, 2.34
2.60a, 2.17
3.95a
4.30a
4.30a
4.20
4.16
4.52
4.57
3.92
3.84
4.40
3.95
3.79
4.55a
4.55a
3.64
3.95a
3.84
3.86
3.93
4.25
4.10
3.62
3.60
4.12
3.64
3.64a
4.16
4.10
4.70
4.38
4.31
4.95
4.93
4.46
4.54
4.56
4.45
4.78
4.61
4.33
4.80
4.87
15.0
14.1
14.1
15.0
14.1
14.5
15.0
13.1
12.8
14.5
13.3
12.8
15.0
14.5
3.4, 3.4
7.3, 2.6
6.8, 5.6
3.4, 3.4
5.1, 2.6
6.9, 2.6
5.8, 6.4
5.1, 11.7
5.1, 12.0
0, 4.7
3.2
9.9
5.6
6.8
9.8
9.0
7.3
6.8
9.4
9.4
9.0
9.4
9.4
9.0
9.0
b
3.9
3.4
3.0
2.6
3.9
9.4
9.4
2.8
9.4
9.4
3.4
3.4
43
95.5
b
103.2
102.9
46
47
97.6
b
b
96.4
5.1, 11.5
5.1, 12.0
b, 5.6
b
b
b
b, 0
a Part of a multiplet centred on this chemical shift. b Not determined.
steps and 10% overall yield from D-ribonolactone. Furthermore,
both screening substrates may also be prepared in 5 steps
from a common precursor, 35, derived from D-ribonolactone.
The complementarity of the stereoselective syntheses of 43
and 47 stems from alternative anti- and syn-selective indium-
mediated additions26–28 of ethyl a-bromomethyl acrylate to the
functionalised aldehydes 29 and 36. It was possible to switch
between Felkin–Anh29 and chelation control,30 allowing the
synthesis of either diastereomeric series at will. The application
of the screening substrates 43 and 47 in the directed evolution
of tailored aldolases for the synthesis of analogues of influenza
A sialidase inhibitors will be described elsewhere.32
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Crystal structure determination of the dipropylamide 11
Crystal data. C17H31NO7, M = 361.43, monoclinic, a =
◦
◦
◦
˚
˚
8.7774(4) A, a = 90 , b = 8.3066(4) A, b = 96.2110(17) , c =
3
˚
˚
13.4961(8) A, c = 90 , U = 978.23(9) A , T = 150(2) K, space
group P21, Z = 2, l(Mo–Ka) = 0.094 mm−1, 10308 reflections
measured, 3748 unique (Rint = 0.0739) which were used in all
calculations. The final wR (F2) was 0.1247 (all data).*
Crystal structure determination of the dipropylamide 25
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3663.
Crystal data. C19H33NO6, M = 371.46, orthorhombic, a =
◦
◦
˚
˚
5.73880(10) A, a = 90 , b = 9.49090(10) A, b = 90 , c =
◦
3
˚
˚
38.2430(8) A, c = 90 , U = 2082.96(6) A , T = 100(2) K,
space group P212121, Z = 4, l(Mo–Ka) = 0.087 mm−1, 16140
reflections measured, 4085 unique (Rint = 0.0973) which were
used in all calculations. The final wR (F2) was 0.1138 (all data).*
Acknowledgements
We thank the BBSRC, EPSRC and the Wellcome Trust for
funding, and the EPSRC Mass Spectrometry Service, Swansea,
for accurate mass determinations.
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