Chemoenzymatic synthesis of differentially protected 3-deoxysugars

Article abstract of DOI:10.1038/nchem.504

3-Deoxysugars are important constituents of complex carbohydrates. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) is an essential component of lipopolysaccharides in Gram-negative bacteria, 2-keto-3-deoxy-D-glycero-D- galacto-nonulosonic acid (KDN) is widely found in carbohydrates of the bacterial cell wall and in lower vertebrates, and sialic acid is a common cap of mammalian glycoproteins. Although ready access to such sugars would benefit the creation of vaccine candidates, antibiotics and small-molecule drugs, their chemical synthesis is difficult. Here we present a simple chemoenzymatic method for preparing differentially protected 3-deoxysugar derivatives from readily available starting materials. It exploits the promiscuous aldolase activity of the enzyme macrophomate synthase (MPS) to add pyruvate enolate diastereoselectively to a wide range of structurally complex aldehydes. A short synthesis of KDN illustrates the utility of this approach. Enzyme promiscuity, which putatively fosters large functional leaps in natural evolution, has great promise as a source of synthetically useful catalytic transformations.

Full text of DOI:10.1038/nchem.504

ARTICLES
PUBLISHED ONLINE: 17 JANUARY 2010 | DOI: 10.1038/NCHEM.504
Chemoenzymatic synthesis of differentially
protected 3-deoxysugars
†
Dennis G. Gillingham‡, Pierre Stallforth‡ , Alexander Adibekian, Peter H. Seeberger † and
*
Donald Hilvert
*
3-Deoxysugars are important constituents of complex carbohydrates. For example, 2-keto-3-deoxy-D-manno-octulosonic acid
(KDO) is an essential component of lipopolysaccharides in Gram-negative bacteria, 2-keto-3-deoxy-D-glycero-D-galacto-
nonulosonic acid (KDN) is widely found in carbohydrates of the bacterial cell wall and in lower vertebrates, and sialic acid is
a common cap of mammalian glycoproteins. Although ready access to such sugars would benefit the creation of vaccine
candidates, antibiotics and small-molecule drugs, their chemical synthesis is difficult. Here we present a simple
chemoenzymatic method for preparing differentially protected 3-deoxysugar derivatives from readily available starting
materials. It exploits the promiscuous aldolase activity of the enzyme macrophomate synthase (MPS) to add pyruvate
enolate diastereoselectively to a wide range of structurally complex aldehydes. A short synthesis of KDN illustrates the
utility of this approach. Enzyme promiscuity, which putatively fosters large functional leaps in natural evolution, has great
promise as a source of synthetically useful catalytic transformations.
ecent mechanistic work on the putative Diels-Alderase macro-
The MPS-catalysed Cornforth reaction with differentially pro-
phomate synthase (MPS) revealed the enzyme to be a profi- tected trioses and tetroses such as 5, 8a and 8b (Fig. 1) confirmed
R
cient aldolase. It promotes the reaction of a variety of that the enzyme tolerates functionally complex substrates.
aromatic and aliphatic aldehydes with pyruvate enolate, that is gener- Racemic triose 5 was selected to examine the tolerance of MPS to
ated in situ by decarboxylation of oxaloacetate1. Of particular note isthe synthetically useful protecting groups before committing to more
enzyme’s remarkable tolerance to substrate size and shape. Moreover, complex substrate syntheses. It gave the triose aldol product 6,
although simple substrates are converted with only modest selectivities, which contains both a methoxymethyl ether (MOM) group at the
protected glyceraldehyde derivatives afford synthetically useful diaster- C5 hydroxyl and a selectively cleavable silyl protecting group at
eoselectivities, suggesting that MPS might provide a mild and practical the C6 hydroxyl. Strategic release of the latter allows selective cycli-
route to various 3-deoxysugars. Indeed, as shown here, a host of differ- zation to the 3-deoxysugar (see 6 to 7 in Fig. 2a). Both tetroses 8a
entially protected 3-deoxysugar derivatives is accessible through the and 8b were converted to product in good yield, with the
promiscuous MPS-catalysed pyruvate aldol reaction.
matched case (that is, 8a) (ref. 9) affording very high diastereoselec-
A number of routes to 3-deoxysugars have been described2. Because tivity. The thioacetal group of aldehyde 8 serves as a useful handle
they generally involve circuitous, multistep procedures, direct addition for subsequent elongation and is readily accommodated at the
of pyruvate enolate to an appropriate aldose, known as the Cornforth enzyme active site. MPS appears to enforce stereoinduction regard-
synthesis3, would be an attractive alternative. However, this process less of the configuration of other stereocentres in the molecule. Thus
suffers from poor scope, low yields, irreproducibility and harsh reaction enantiomers 8a and 8b gave diastereomeric products with the new
conditions. A catalytic Ni2þ work-up has been developed to increase stereocentre in the same configuration. The stereocentres of the
reproducibility and yields4, but a general solution has proven elusive. products 9a and 9b are tentatively assigned the S configuration
This challenge has spurred the development of enzymatic processes5, based on analogy to stereoproofs in the triose1, pentose (see
such as those based on KDO aldolase6 or sialic acid aldolase7, which Supplementary Information) and hexose series (see below).
work well for unprotected sugars but are unproven with other sub-
Reactions with diverse pentoses further illustrate the scope of the
strates8. In contrast, an MPS-catalysed Cornforth reaction potentially MPS-catalysed pyruvate aldol reaction. Fully protected aldehydes of
offers dual advantages. First, unlike the present indirect chemical various pentoses were prepared by ozonolysis of the corresponding
methods2, unprotected pyruvate generated in situ serves as the nucleo- oximes (full details provided in Supplementary Information)10 with
phile. Second, in a departure from other enzymatic protocols8, the start- the goal of mapping the substrate range and protecting group toler-
ing aldehydes are differentially protected sugars, which yield products ance of the enzyme active site. Diisopropylidene-protected sugars
that can be selectively manipulated downstream in achemical synthesis. work efficiently in both the L- and D-sugar series (see, for
OH
OH
HO
HO
HO
HO
HO
HO
OH
OH
CO2H
OH
CO2H
OH
O
O
HO
AcHN
HO
HO
O
O
COOH
COOH
HO
HO
1, Sialic acid
HO
2, KDN
OH
4, gluco-KDO
OH
3, KDO
OH
Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland; †Present address: Department of Biomolecular Systems, Max-Planck-Institute of
Colloids and Interfaces, Am Mu¨hlenberg 1, 14476 Potsdam, Germany (P.S.), Institute of Chemistry and Biochemistry, Freie Universita¨t Berlin, Arnimallee 22,
14195 Berlin, Germany (P.H.S.); ‡These authors contributed equally to this work. e-mail: hilvert@org.chem.ethz.ch; peter.seeberger@mpikg.mpg.de
*
102
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.504
ARTICLES
The seemingly general preference of MPS for inducing the S con-
figuration regardless of substrate bias suggests a selective aldehyde
binding mode. The recently determined X-ray structure of MPS
containing a pyruvate molecule bound to the active site magnesium
ion12 enables rationalization of this preference. Figure 3a shows the
active site formed at the interface of two subunits. The wide entrance
leading to the deeply buried binding pocket is consistent with the
broad substrate tolerance observed. The preferred binding mode
of the aldehyde, shown in Fig. 3b,c, is most likely set by three
factors. First, proximity to the positively charged arginine residue
(Arg101) is needed to polarize the carbonyl group and stabilize
the incipient negative charge created in the aldol reaction. Second,
the aldehyde should be aligned antiperiplanar to the enolate, as is
preferred for open transition states in aldol reactions13. Last, the
aldehyde should be positioned such that its alkyl group is facing
the large entrance to the active site. The binding mode illustrated
in Fig. 3 meets all three of the binding conditions and predicts
the S stereochemistry of the product. Closed Zimmerman–
Traxler-type transition states14 were not considered as a synclinal
arrangement through a magnesium chelate would be sterically
crowded around the magnesium ion and because steric clashes
between the alkyl group of the aldehyde and a number of active
site residues would preclude the observed S stereoselectivity.
a
15 mM
O2C
O
OH
O
O
O
CO2
TEOCO
CO2Na
O
O
5 µM MPS
50 mM NaPi pH 7.5
5 mM MgCl2
OMOM
SiMe3
O
5
6, 40% yield, 3:2 d.r.
OMe
15 mM
O2C
b
O
EtS
SEt
OH
C4
O
O
O
C6
CO2
C1
CO Na
C2
O
2
EtS
O
5 µM MPS
50 mM NaPi pH 7.5
5 mM MgCl2
O
SEt
8a (S,S)
8b (R,R)
9a, >19:1 d.r., 77 % yield, (4S,5S,6S)
9b, 4:1 d.r., 75 % yield, (4S,5R,6R)
Figure 1 | MPS-catalysed synthesis of representative 3-deoxysugars.
a, Triose aldehyde 5 is readily converted to the differentially protected
3-deoxysugar derivative 6. b, Both enantiomers of thioacetal tetrose 8 are
accepted as substrates by MPS. The reactions proceed with good to
excellent diastereoselectivity for the mismatched (R,R) and matched (S,S)
isomers, respectively. (TEOC: trimethylsilylethoxy-carbonate,
d.r.: diastereomeric ratio).
The promiscuous MPS-catalysed reaction between oxaloacetate and
protected aldoses is both efficient and stereoselective, providing ready
access to natural and unnatural derivatives of valuable higher sugars
such as KDN and sialic acid. The enzyme’s broad substrate tolerance
and its use of pyruvate enolate rather than a surrogate provide an
attractive complement to existing methods for preparing differentially
protected sugars. Such molecules represent unique building blocks for
oligosaccharide assembly and their rapid synthesis should facilitate the
development of synthetic vaccines and carbohydrate-based antibiotics.
Because the stereochemical preferences of MPS favour formation of
4S-configured alcohols, discovery and/or engineering of new
enzymes for the Cornforth-type synthesis of 3-deoxysugars like
KDO that are R-configured at this position represents a challenge for
the future. In this context, design through evolution offers one possible
example, 10a and 10b in Table 1). In contrast to 8, where the mis-
matched substrate enantiomer gave only a 4:1 diastereomeric ratio
(see 8b to 9b in Fig. 1), the homologated sugar 10 led to the for-
mation of products 16a, 16b and 16c in high diastereoselectivity
irrespective of the aldehyde configuration. The configuration of
the new stereocentre for 16a, 16b, and 17 was rigorously established
as S by comparison to a known compound (see Supplementary
Fig. S2), and the others (16c, 19, 20 and 21) are assigned by
analogy. MPS tolerates ether, acetal, thioacetal, allyl, benzyl, silyl
and ester protecting groups, as well as combinations thereof, in dif-
ferentially protected sugar aldehydes (Table 1). In the cases where
the aldehyde starting material was stable enough to be purified
(for example 8a and 8b in Fig. 1 and 13a and 13b in Table 1) the
enzymatic reaction was clean. The moderate yields of 30–65%
seen for substrates 10–12 in Table 1 can likely be ascribed to the
impurity and instability of the aldehydes used directly following
oxime ozonolysis (see Supplementary Information for a full discus-
sion). Positioning of an orthogonal protecting group at the C3
hydroxyl (aldehyde numbering) in pentoses 13a–c and hexose
15b allows strategic control of ring closure to the 3-deoxysugar
derivative (see, for example, Fig. 2b). Although the enzyme active
site can accommodate sterically bulky and hydrophobic protecting
groups, the reaction yield suffers when polar groups are close to
the aldehyde centre. For example, the peracetylated aldehyde
derived from D-arabinose was not converted by MPS. Substrates
of low solubility in aqueous buffer, including multiply benzylated
or silylated aldehydes, were similarly ineffective, producing no
detectable product.
strategy to identify enantiocomplementary MPS mutants15,16
.
a
OH
O
OMOM
OH
TBAF, THF
87%
O
HO
TEOCO
CO2Na
HO2C
OMOM
6
7
OH
OH
O
b
O
RP-HPLC
O
CO2Na
O
O
pH 2,
44% from 13c
O
CO2H
OP
OH
O
O
19c
22
MPS also provides access to C9 sugar derivatives from the corre-
sponding hexoses (substrates 14 and 15a–b in Table 1).
Diisopropylidene-protected glucosamine aldehyde 14 was syn-
thesized and subjected to MPS and oxaloacetate. Although the con-
version was only 50%, stereoselectivity was excellent. Aldehydes 15a
and 15b, which were synthesized in four steps from D-mannose,
were also good substrates in the aldol reaction. The products of
these reactions, a-keto acids 21a and 21b, are in the same stereoiso-
meric series as KDN. Indeed, acidic deprotection of 21a afforded a
c
OH
O
O
OAc
OH
CO2H
OH
10% TFA
OH
O
HO
HO
O
CO2Na
O
O
HO
2, KDN
21a
product identical to a commercial sample of KDN as judged by Figure 2 | Selective deprotection of 3-deoxysugar derivatives provides
1
high-resolution mass spectrometry and H NMR spectroscopy (see strategic control of ring-closure. a,b, Acid-induced deprotection/cyclization
Fig. 2c and Supplementary Information). The short (six steps of 6 or 19c affords novel, differentially protected building blocks 7 (a) and
overall) synthesis of KDN, a valuable member of the higher sugar 22 (b), respectively. c, Global deprotection of compound 21 yields KDN, a
family11, from D-mannose once again demonstrates the preference of widely distributed deaminated neuraminic acid derivative. (RP-HPLC ¼
MPS for S stereoinduction in the carbon–carbon bond-forming step. reverse phase HPLC).
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.504
ARTICLES
Table 1 | Synthesis of acyclic epi-KDO, KDN and related derivatives.
O
O2C
OR4 OR2
OH
C4
O
O
CO2
5 µM MPS
C1
CO2Na
R5
C8
C6
OR3
C2
R
H
50 mM NaPi pH 7.5
5 mM MgCl2
OR1
10-15
16-21
Aldehyde
RCHO
Product (configuration)
Yield* (%)
Diastereomeric ratio†
CHO
10a, L-arabino
10b, D-arabino
10c, D-xylo
16a (4S,5S,6S,7S)
16b (4S,5R,6R,7R)
16c (4S,5S,6S,7R)
49
65
44
.19:1
.19:1
8:1
O
O
O
O
CHO
MOMO
MOMO
11
17 (4S,5S,6S,7S)
18 (4S,5R,6R,7R)
38
31
.19:1
.19:1
O
O
CHO
AcO
12
AcO
O
O
CHO
OP
O
13a
13b
13c
19a (4S,5R,6S,7R,8R) P ¼ Allyl
19b (4S,5R,6S,7R,8R) P ¼ Bn
82
84
44‡
.19:1
.19:1
.19:1
O
19c (4S,5R,6S,7R,8R) P ¼ TEOC
O
14
20 (4S,5S,6S,7R,8R)
50
.19:1
O
CHO
O
NHAc
O
O
OP
CHO
15a
15b
21a (4S,5R,6R,7R,8R) P ¼ Ac
21b (4S,5R,6R,7R,8R) P ¼ Bn
39
28
8:1
8:1
O
O
O
O
*Yields represent HPLC purified product after two steps (ozonolysis and aldol, see Supplementary Information for details). † . 19:1 means the minor diastereomer was not observed in the 300 MHz 1H NMR.
‡Yield is of product obtained after acidic cyclization.
a
c
b
Mg
O
Arg101
O
O
O
EtS
SEt
Me
O
O
Me
Figure 3 | Structural basis for MPS promiscuity. a, View into the MPS active site12 with the S,S-thioaldehyde substrate 4a docked proximal to the
magnesium-bound pyruvate enolate (substrate carbons are shown in teal, magnesium in magenta). The wide entrance to the binding pocket can readily
accommodate a wide range of structurally complex aldehydes. b, The dimensions of the binding pocket adjacent to the magnesium-bound pyruvate dictate
the preferred orientation of the aldehyde substrate. Placement of the bulky alkyl substituent in the entry chamber allows the carbonyl group to penetrate
deeply into the active site and contact Arg101, which can orient and activate it for attack by the nearby enolate. The modelled antiperiplanar arrangement of
the substrates rationalizes the observed formation of the S-configured alcohol in the course of the reaction. A synclinal arrangement of aldehyde and enolate
is precluded by clashes with the protein backbone. c, Stick representation of the interactions illustrated in b.
104
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.504
ARTICLES
7. Williams, G. J., Woodhall, T., Farnsworth, L. M., Nelson, A. & Berry, A. Creation
of a pair of stereochemically complementary biocatalysts. J. Am. Chem. Soc. 128,
16238–16247 (2006).
8. Fong, S., Machajewski, T. D., Mak, C. C. & Wong, C. H. Directed evolution of
D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient
synthesis of D- and L-sugars. Chem. Biol. 7, 873–883 (2000).
9. Masamune, S., Choy, W., Petersen, J. S. and Sita, L. R. Double asymmetric
synthesis and a new strategy for stereochemical control in organic synthesis.
Angew. Chem. Int. Ed. 24, 1–76 (1985).
10. Weitz, D. J. & Bednarski, M. D. Synthesis of acyclic sugar aldehydes by
ozonolysis of oximes. J. Org. Chem. 54, 4957–4959 (1989).
11. Inoue, S. & Kitajima, K. KDN (deaminated neuraminic acid): Dreamful past and
exciting future of the newest member of the sialic acid family. Glycoconjugate J.
23, 277–290 (2006).
Methods
Buffers were prepared with ultrapure water. All chemicals were purchased from
Sigma-Aldrich, Fluka or Acros and used as received. Macrophomate synthase was
produced as previously described1. For MPS-catalysed reactions a buffer of 50 mM
NaPi, 5 mM MgCl2 at pH 7.5 was used. The aldol reaction for substrate 8b is
described here as an example. Aldehyde 8b (37 mg, 0.14 mmol) was dissolved in a
minimum amount of acetonitrile (MeCN) and transferred quantitatively to a 50 ml
round-bottom flask (note: in cases where the substrate forms large oil bubbles the
yields are low unless the product is removed with a pipette, redissolved in additional
MeCN and added again to the reaction mixture). Enough buffer was then added
such that the final oxaloacetate concentration (including enzyme solution) was
15 mM (13.1 ml). MPS (925 ml of a 56 mM stock solution to give a 5 mM final
[MPS]) was then added by pipette. Oxaloacetate was added in four portions
(4 Â 6.9 mg, 0.21 mmol) as a solid powder over 4 h, and the reaction was shaken (or
stirred slowly) for an additional 1 h. After 5 h in total the mixture was frozen and the
water was removed by lyophilization. The crude residue was suspended in methanol
and stirred for 15 minutes. The resulting suspension was filtered (with washings)
12. Ose, T. et al. Insight into a natural Diels-Alder reaction from the structure of
macrophomate synthase. Nature 422, 185–189 (2003).
13. Denmark, S. E. & Henke, B. R. Investigations on transition-state geometry in the
aldol condensation. J. Am. Chem. Soc. 113, 2177–2194 (1991).
14. Zimmerman, H. E. & Traxler, M. D. The stereochemistry of the Ivanov and
Reformatsky reactions. I. J. Am. Chem. Soc. 79, 1920–1923 (1957).
15. Ja¨ckel, C., Kast, P. & Hilvert, D. Protein design by directed evolution. Ann. Rev.
Biophys. 37, 153–173 (2008).
16. Mugford, P. F., Wagner, U. G., Jiang, Y., Faber, K. & Kazlauskas, R. J.
Enantiocomplementary enzymes: Classification, molecular basis for their
enantiopreference, and prospects for mirror-image biotransformations. Angew.
Chem. Int. Ed. 47, 8782–8793 (2008).
1
and the methanol was evaporated from the filtrate. H NMR of the crude
product was used to assess the diastereoselectivity by integrating the C3 methylene
proton peaks at 2.88 and 3.08 ppm. The product was then purified by reversed-phase
high-performance liquid chromotography under neutral conditions to prevent
elimination. A linear gradient of eluant from 5% to 40% CH3CN in water was used
and the product 9b (40 mg, 0.11 mmol, 76%) was obtained as a white powder
after lyophilization.
Received 21 April 2009; accepted 27 November 2009;
published online 17 January 2010
Acknowledgements
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Additional information
The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://npg.nature.
com/reprintsandpermissions/. Correspondence and requests for materials should be addressed
to P.H.S. and D.H.
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