Creation of a tailored aldolase for the parallel synthesis of sialic acid mimetics

Article abstract of DOI:10.1002/anie.200462733

(Chemical Equation Presented) Broadening substrate specificity: A modified form of sialic acid aldolase (E192N) exhibits a 640-fold switch in substrate specificity relative to the wild-type enzyme. Ozonolysis of the unsaturated amides 1, followed by an E192N-mediated step, was exploited in the parallel synthesis of 14 different sialic acid mimetics of general structure 2.

Full text of DOI:10.1002/anie.200462733

Angewandte
Chemie
Enzyme Engineering
Creation of a Tailored Aldolase for the Parallel
Synthesis of Sialic Acid Mimetics
Thomas Woodhall, Gavin Williams, Alan Berry, and
Adam Nelson*
The exquisite selectivity and efficiency of enzymes has served
as an inspiration to chemists for many years.[1] These
remarkable properties enable enzymes to guide the assembly
of complex products from mixtures of reactants present in low
concentrations (in the nm–mm range).[2] Indeed, high levels of
substrate specificity, and stereo- and chemoselectivity are
hallmarks of enzymatic catalysis.
The most useful catalysts to the synthetic chemist,
however, are those that are broadly applicable. Indeed,
Sharpless has commented on the synthetic virtues of asym-
metric dihydroxylation in terms of its remarkable scope: “It
[OsO4] reacts only with olefins and it reacts with all olefins
(slight poetic license here)”.[3] The substrate ranges of many
enzymes are rather more restricted which limits their utility in
chemical synthesis. The power of directed evolution has been
brought to bear on this problem[4] and has, for example, been
used to create an amine oxidase with broad substrate
specificity and high enantioselectivity[5] and aldolases that
[6,7]
ꢀ
modify the stereochemical course of C C bond formation.
Our challenging objective was to broaden the substrate
specificity of the carbon–carbon bond-forming enzyme, sialic
acid aldolase (N-acetylneuraminic acid aldolase), in a manner
sufficient for application in the parallel synthesis of sialic acid
mimetics. Sialic acid aldolase catalyses the reversible aldol
condensation between pyruvate and N-acetylmannosamine 3
to give sialic acid 4 (Scheme 1). Although a number of
hexoses, pentoses, and their analogues are substrates for this
enzyme, condensations that involve shorter aldehydes are less
promising: l- and d-erythrose and threose react at between
0.3 and 5% of the rate of N-acetylmannosamine, and two- and
three-carbon aldehydes are not substrates.[8] The substituted
dihydropyran 2 is an influenza A sialidase inhibitor[9] whose
activity was optimized from the first potent inhibitor of
influenza sialidases, zanamivir (1).[10] We decided, therefore,
to engineer an aldolase with sufficiently broad substrate
specificity to convert four-carbon aldehydes of general
structure 5 into the corresponding sialic acid mimetics 6
(Scheme 1).
[*] T. Woodhall, Dr. A. Nelson
School of Chemistry
University of Leeds, Leeds, LS29JT (UK)
Fax : (+44)113-233-6565
E-mail: a.s.nelson@leeds.ac.uk
T. Woodhall, Dr. G. Williams, Dr. A. Berry, Dr. A. Nelson
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS29JT (UK)
Dr. G. Williams, Dr. A. Berry
School of Biochemistry and Microbiology
University of Leeds, Leeds, LS29JT (UK)
Angew. Chem. Int. Ed. 2005, 44, 2109 –2112
DOI: 10.1002/anie.200462733
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2109

Communications
Figure 1. Schematic diagrams of the active sites of sialic acid aldo-
lases: a) residues which have been shown by X-ray crystallography to
interact with C7-C9 of 4-oxosialic acid; b) representation of the active
site of an evolved enzyme in which a hydrophobic pocket has been
sculpted to accommodate the dipropylaminocarbonyl group of the
screening substrate.
Scheme 1. Directed evolution of an aldolase for application in the parallel
synthesis of sialic acid mimetics.
We used a semirational approach, starting with the
structure of the sialic acid aldolase from Haemophilus
influenzae, which has 35% identity and 59% similarity to
the corresponding E. coli protein. Analysis of the X-ray
crystallographic structure[11] of the aldolase in complex with
the inhibitor 4-oxosialic acid revealed three residues in
contact with the C7–C9 side chain. The corresponding
residues in the E. coli protein, Asp191, Glu192, and Ser208
were targeted separately by using saturation mutagenesis
(Figure 1a). As the aim of this investigation was to evolve an
aldolase with broad substrate specificity, we designed a
screening substrate 12 with relatively large R1 and R2
groups (nPr); it was hypothesized that an active site able to
accommodate this rather bulky substrate would also be able
to accept a wide range of smaller substrates. Furthermore, the
tolerance of the wild-type enzyme toward a wide range of C2-
substituted aldehydes[8] was expected to be preserved, as this
substituent (NHAc in N-acetylmannosamine) is solvent-
exposed.[11] A schematic diagram of the designed screening
substrate in complex with the mutant enzyme is shown in
Figure 1b.
The screening substrate 12 was prepared with the
synthetic sequence outlined in Scheme 2. The lactone 7,
readily available by oxidative cleavage of isoascorbic acid,[12]
was opened with dipropylamine to give the g-hydroxyamide 8.
Swern oxidation of 8 and indium-mediated coupling with
ethyl a-bromomethylacrylate (13)[13] gave the g-hydroxy-
amides 9 (anti/syn 77:33), an outcome consistent with Felkin–
Anh-controlled[14] attack on the intermediate aldehyde.
Cleavage of the acetonide and hydrolysis of the ester gave
the screening substrate 12.
Scheme 2. Synthesis of the screening substrate 12. Reagents and conditions: a) nPr2NH, MeOH; b) DMSO, (COCl)2, Et3N, CH2Cl2; c) In, 13,
THF/H2O (1:1); d) O3, MeOH, ꢀ788C then Me2S; e) TFA/H2O (1:1); f) Ba(OH)2, EtOH/H2O; g) (NH4)2SO4, H2O. DMSO=dimethyl sulfoxide;
TFA=trifluoroacetic acid.
2110
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 2109 –2112

Angewandte
Chemie
The library of enzymes produced by saturation muta-
genesis of residues 191, 192, and 208, in turn, were screened
for useful synthetic activity on the premise that mutant
enzymes able to cleave the screening substrate 12 would also
be able to catalyze the forward reaction.[6] Libraries of
proteins were screened in 96-well plates by analysis of
thermally treated crude-cell lysates in which His-tagged
mutant proteins had been overexpressed to ꢁ 40% of the
total protein content. A coupled enzyme assay was used in
which the cleavage of the screening substrate to generate
pyruvate was detected spectroscopically at 340 nm by the
lactate dehydrogenase catalyzed reduction of pyruvate, with
concomitant oxidation of NADH. In this way, a mutant
enzyme was identified that contains the Glu192!Asn
Scheme 3. Parallel synthesis of the sialic acid mimetics 16/17 (see
Table 2). Reagents and conditions: a) EDC, HOBt, R1R2NH, CH2Cl2;
b) TFA/H2O (9:1); c) O3, MeOH, ꢀ788C then Me2S; d) E192N
(2ꢀ10ꢀ2 mol%), pyruvate, buffer, (pH 7.4). EDC=1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride; HOBt=1-hydroxy-
benzotriazole hydrate.
mutation (E192N), for which substrate specificity [(kcat
/
KM(12))/(kcat/KM(sialic acid))] had been switched 640-fold
(Table 1). The kcat/KM value of the E192N mutant toward
the screening substrate 12 is 50-fold higher than that of the
wild-type enzyme; indeed, cleavage of substrate 12 by the
1
16/17h–m, determined by H NMR
spectroscopy, were low (19–55%).
After purification, the tert-butyl
amide 16h/17h was obtained in
35% yield over two steps from
15h as a 45:55 mixture of epimers.
Remarkably, the enantiomeric
aldehyde ent-15c, prepared analo-
gously from d-lyxose,[17] was also a
Table 1: Characterization of the kinetics of the wild-type and E192N aldolases.
Sialic Acid
KM
[mm]
12
KM
[mm]
k
cat=KMð12Þ
kcat
kcat/KM
kcat
kcat/KM
k
cat=KMðsialic
acidÞ
[minꢀ1
]
[minꢀ1 mmꢀ1
]
[minꢀ1
]
[minꢀ1 mmꢀ1
]
WT
260ꢂ6
4.4ꢂ0.3 59ꢂ4
4.4ꢂ0.6
74ꢂ4 11ꢂ1
130ꢂ3
6.9ꢂ1.0
0.12
77
E192N 170ꢂ10 38ꢂ5
0.39ꢂ0.04 340ꢂ30
mutant enzyme was six times more efficient than the wild-
type enzyme-catalyzed cleavage of sialic acid!
The synthetic utility of the evolved E192N enzyme was
investigated with the crude aldehydes 18 generated by
ozonolysis of the corre-
substrate for the evolved aldolase (Table 2, entry 14). After
7 days, the enzymatic reaction was worked up, and the
enantiomeric mimetic ent-16c/ent-17c was obtained in 32%
yield. The catalysis of the aldol reaction was not as efficient in
sponding g,d-unsaturated
amides 15, prepared in
parallel from the known
g,d-unsaturated acid 14[15]
(Scheme 3 and Table 2).
With the tertiary amide-
Table 2: Synthetic utility of the mutant sialic acid aldolase E192N.
Entry Alkene 15
R1
R2 Yield[a] 15
Products
t [days] 16:17[b] Yield 16/17[c,d]
[%]
[%]
1
2
3
4
5
6
7
15a
15b
15c
15d
15e
15 f
15g
Et
nPr
nPr
nBu
ꢀ
Et
61
73[e]
78
88
66
16/17a
16/17b
16/17c
16/17d
16/17e
16/17 f
16/17g
3
3
3
3
3
3
3
82:18
82:18
82:18
82:18
82:18
79:21
82:18
37
42
42
66
48
55
47
Me
nPr
nBu
containing
aldehydes
18a–g (Table 2, entries 1–7), the enzymatic
reactions reached completion well within
3 days, and, after purification by ion-
exchange chromatography, the sialic acid
mimetics 16/17a–g were obtained in 37–
66% yield over the two steps from the
corresponding g,d-unsaturated amides 15a–
g. In each case, the products 16/17 were
obtained as ꢁ 80:20 mixtures of epimers
(16/17) which is consistent with thermody-
namic stereochemical control.[16]
ꢀ
ꢀ
(CH2)4
ꢀ
(CH2)5
59
68
ꢀ
ꢀ
(CH2)2O(CH2)2
8
9
10
11
12
13
15h
15i
15j
15k
15l
15m
tBu
nPent
cHex
H
79
81
90
67
74
68
16/17h
16/17i
16/17j
16/17k
16/17l
16/17m
14
14
14
14
14
14
45:55
70:30
60:40
60:40
60:40
60:40
35 (55)
(13)[f]
(29)[f]
(19)[f]
(30)[f]
(35)[f]
H
H
H
H
H
Ph
(R)-CHMeBn
(S)-CHMeBn
14
ent-15c
nPr
nPr
78[g]
ent-16c/ent-17c
7
64:36
32[h]
The enzymatic reactions of the secon-
dary amide substrates 15h–m were less
efficient (Table 2, entries 8–13). In each
case, the required products were observed
after 14 days by analysis of crude reaction
mixtures by MS and 1H NMR spectroscopy
at 500 MHz. The yields of the products
[a] Yield of purified product over two steps from the acid 14. [b] Determined by integration of the
1H NMR spectrum (500 MHz). [c] Yield of purified product over two steps from the corresponding
alkene 15; (yields in parentheses were determined by integration of the 1H NMR spectrum (500 MHz) of
the crude reaction mixture). [d] Products were obtained as mixtures of the two pyranose and the two
furanose forms: for 16, ꢁ75:8:8:7 and for 17, ꢁ13:7:44:36. [e] 53:47 mixture of rotomers. [f] The ratio
of the two pyranose and two furanose forms was not determined. [g] The enantiomeric acid ent-14 was
used. [h] The enantiomeric starting material ent-15c (R1 =R2 =nPr) was used.
Angew. Chem. Int. Ed. 2005, 44, 2109 –2112
www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2111

Communications
[12] N. Cohen, B. L. Banner, A. J. Laurenzano, L. Carozza, Org.
Synth. 1985, 63, 127 – 135.
[13] a) T. H. Chan, C. J. Li, J. Chem. Soc. Chem. Commun. 1992, 747 –
748; b) M. D. Chappell, R. L. Halcomb, Org. Lett. 2000, 2, 2003 –
2005; c) C.-J. Li, T.-H. Chan, Tetrahedron 1999, 55, 11149 –
11176.
this case, however, as thermodynamic equilibration between
the epimeric products was not complete (Table 2, compare
the 82:18 ratio of epimers, entry 3, with the 64:36 ratio of
epimers, entry 14).
In summary, we have engineered an aldolase that was
exploited in the parallel synthesis of sialic acid mimetics. The
mutant enzyme was most efficient in catalysis of the synthesis
of the tertiary amides 16a–g/17a–g, presumably a conse-
quence of the screening assay used; it is well known that “you
get what you screen for”[18] and our assay would have selected
for enzymes able to accept a range of sterically varied tertiary
amides. The novel enzyme might be used to catalyze the
interconversion of dynamic combinatorial libraries,[19] for
example, in the discovery of functional sialic acid mimetics
such as paramyxovirus sialidase inhibitors.[20]
[14] a) M. Chꢀrest, H. Felkin, N. Prudent, Tetrahedron Lett. 1968,
2199 – 2204; b) N. T. Anh, Top. Curr. Chem. 1980, 88, 145 – 162.
[15] V. Jaeger, B. Haefele, Synthesis 1987, 801 – 806.
[16] C. H. Lin, T. Sugai, R. L. Halcomb, Y. Ichikawa, C. H. Wong, J.
Am. Chem. Soc. 1992, 114, 10138 – 10145.
[17] B. V. Lao, S. Lahiri, J. Carbohydr. Chem. 1996, 15, 975 – 984.
[18] F. H. Arnold, Acc. Chem. Res. 1998, 31, 125 – 131.
[19] a) R. J. Lins, S. L. Flitsch, N. J. Turner, E. Irving, S. A. Brown,
Angew. Chem. 2002, 114, 3555 – 3557; Angew. Chem. Int. Ed.
2002, 41, 3405 – 3407; b) R. J. Lins, S. L. Flitsch, N. J. Turner, E.
Irving, S. A. Brown, Tetrahedron 2004, 60, 771 – 780.
[20] For the application of dihydropyran carboxamides similar to 2 in
the inhibition of paramyxovirus sialidase, see: P. Chand, Y. S.
Babu, S. R. Rowland, T.-H. Lin, PCT Int. Appl. 2002, WO
2002076971.
Received: November 26, 2004
Published online: March 2, 2005
Keywords: aldol reactions · biotransformations · enzyme
.
catalysis · mutagenesis · protein engineering
[1] For example, see: W. J. Albery, J. R. Knowles, Angew. Chem.
1977, 89, 295 – 304; Angew. Chem. Int. Ed. Engl. 1977, 16, 285 –
293.
[2] X. Li, D. R. Liu, Angew. Chem. 2004, 116, 4956 – 4979; Angew.
Chem. Int. Ed. 2004, 43, 4848 – 4870.
[3] For a transcript of his Nobel lecture, see: K. B. Sharpless, Angew.
Chem. 2002, 114, 2126 – 2135; Angew. Chem. Int. Ed. 2002, 41,
2024 – 2032.
[4] a) M. Alexeeva, R. Carr, N. J. Turner, Org. Biomol. Chem. 2003,
1, 4133 – 4137; b) K. A. Powell, S. W. Ramer, S. B. del Cardayre,
W. P. C. Stemmer, M. B. Tobin, P. F. Longchamp, G. W. Huis-
man, Angew. Chem. 2001, 113, 4068 – 4080; Angew. Chem. Int.
Ed. 2001, 40, 3948 – 3959; c) M. T. Reetz, K.-E. Jaeger, Top. Curr.
Chem. 1999, 200, 31 – 57; d) K.-E. Jaeger, T. Eggert, Curr. Opin.
Biotechnol. 2004, 15, 305 – 313.
[5] R. Carr, M. Alexeeva, A. Enright, T. S. C. Eve, M. J. Dawson,
N. J. Turner, Angew. Chem. 2003, 115, 4955 – 4958; Angew.
Chem. Int. Ed. 2003, 42, 4807 – 4810.
[6] G. J. Williams, S. Domann, A. Nelson, A. Berry, Proc. Natl.
Acad. Sci. USA 2003, 100, 3143 – 3148.
[7] M. Wada, C.-C. Hsu, D. Franke, M. Mitchell, A. Heine, I. Wilson,
C.-H. Wong, Bioorg. Med. Chem. 2003, 11, 2091 – 2098.
[8] C.-H. Wong, G. M. Whitesides, Enzymes in Synthetic Organic
Chemistry, Pergamon, Oxford, 1994.
[9] a) P. W. Smith, S. L. Sollis, P. D. Howes, P. C. Cherry, I. D.
Starkey, K. N. Cobley, H. Weston, J. Scicinski, A. Merritt, A.
Whittington, P. Wyatt, N. Taylor, D. Green, R. Bethell, S. Madar,
R. J. Fenton, P. J. Morley, T. Pateman, A. Beresford, J. Med.
Chem. 1998, 41, 787 – 797; b) N. R. Taylor, A. Cleasby, O. Singh,
T. Skarzynski, A. J. Wonacott, P. W. Smith, S. L. Sollis, P. D.
Howes, P. C. Cherry, R. Bethell, P. Colman, J. Varghese, J. Med.
Chem. 1998, 41, 798 – 807.
[10] M. von Itzstein, W.-Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason,
B. Jin, T. van Phan, M. L. Smythe, H. F. White, S. W. Oliver,
P. M. Colman, J. N. Varghese, D. M. Ryan, J. M. Woods, R. C.
Bethell, V. J. Hotham, J. M. Cameron, C. R. Penn, Nature 1993,
363, 418 – 423.
[11] J. A. R. G. Barbosa, B. J. Smith, R. DeGori, H. C. Ooi, S. M.
Macuccio, E. M. Campi, W. R. Jackson, R. Brossmer, M.
Sommer, M. C. Lawrence, J. Mol. Biol. 2000, 303, 405 – 421.
2112
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 2109 –2112