Expression and initial characterization of WbbI, a putative d-Galf:α-d-Glc β-1,6-galactofuranosyltransferase from Escherichia coli K-12

Article abstract of DOI:10.1039/b609455d

Cloning of E. coli K-12 orf8 (wbbI) and over-expression of the corresponding enzyme as a maltose-binding fusion protein provided recombinant WbbI β-1,6-galactofuranosyltransferase activity. Challenged with synthetic acceptor analogues in the presence of U

Full text of DOI:10.1039/b609455d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Expression and initial characterization of WbbI, a putative D-Galf :a-D-Glc
b-1,6-galactofuranosyltransferase from Escherichia coli K-12
Corin Wing,a James C. Errey,b Balaram Mukhopadhyay,a John S. Blanchardb and Robert A. Field*a
Received 3rd July 2006, Accepted 6th September 2006
First published as an Advance Article on the web 21st September 2006
DOI: 10.1039/b609455d
Cloning of E. coli K-12 orf 8 (wbbI) and over-expression of the corresponding enzyme as a
maltose-binding fusion protein provided recombinant WbbI b-1,6-galactofuranosyltransferase activity.
Challenged with synthetic acceptor analogues in the presence of UDP-galactofuranose as a donor,
WbbI showed a modest preference for pyranoside acceptor substrates of the a-D-gluco-configuration
but it also possessed the ability to turn-over acceptor analogues.
Introduction
A wide range of pathogenic microorganisms produce macro-
molecules containing galactofuranose (Galf ): bacterial
O
antigens,1,2 fungal exopolysaccharides,3 glycolipids and cell
walls,2,4 protozoal glycoproteins and lipophosphoglycans.5–7
Galactofuranose has been recognized to play an important
structural role in Mycobacterium tuberculosis, where galactofura-
nosyltransferase (Galf T) activities are essential for cell viability.8
Sequence analysis of parasitic protozoa shows that the Leishmania
genome encodes at least 6 candidate UDP-Galf transferases and
that there are more than 20 related genes present in Trypanosoma
cruzi.9,10 Beverley et al. note1 that no candidate UDP-Galf
transferases have so far been reported in fungi, although they must
exist given the number of Galf -containing glycoconjugates known
in these organisms.11 The absence of galactofuranose in mammals9
suggests that Galf biosynthesis and metabolism has potential as a
target for new anti-microbial drugs, and/or that Galf -containing
structures might be suitable as anti-microbial vaccines.2
It was originally shown in a Salmonella cell-free system
that 5-membered ring galactofuranose-containing structures are
biosynthesized from 6-membered ring UDP-galactopyranose.12,13
Genetic and biochemical studies14,15 showed that Galf arises
through the action of a novel UDP-galactopyranose mutase16,17
that catalyzes the reversible ring-contraction of UDP-Galp to
UDP-Galf , the putative donor substrate of galactofuranosyltrans-
ferases. The gene encoding UDP-galactopyranose mutase, GLF,
was first identified by Reeves et al. in connection with studies
on the genetics and structure of the E. coli K-12 O antigen, the
repeat unit of which contains b-D-Galf (Fig. 1).1
Fig. 1 Structure of the oligosaccharide repeat unit of E. coli K-12 O
antigen,1 with the key galactofuranose residue in grey.
the corresponding enzyme would be involved in the transfer of
Galf onto an a-D-gluco-configured acceptor substrate to form
a b-1,6-linkage. To date, only the galactofuranosyltransferase
that catalyses the formation of alternating b-1,5- and b-1,6-
linkages in the biosynthesis of the mycobacterial cell wall has
been subject to detailed analysis.19,20 However, BLAST sequence
homology analysis of WbbI and the M. tuberculosis transferase
shows there to be no significant homology between them. Protein
sequence identity between the putative b-1,6-Galf transferase
from Streptococcus gordonii, WefE, and E. coli WbbI is 32%.21 It
should be noted, however, that the assignment of WefE as a b-1,6-
Galf transferase is solely based on its sequence homology to WbbI
and S. thermophilus Eps6N21 (62% identity to WefE), neither of
which have been experimentally characterized. Therefore, in order
to confirm its assignment as a b-1,6-galactofuranosyltransferase,
and to assess its suitability as a generic tool for enzymatic synthesis,
we have cloned E. coli wbbI, over-expressed the corresponding
WbbI protein in E. coli as a maltose binding protein (MBP) fusion
protein (WbbI-MBP) and conducted a preliminary investigation
of is synthetic capability.
The E. coli K-12 rfb cluster,1,18 which codes for O antigen
biosynthesis, contains 11 open reading frames. Functions for
most of the corresponding proteins have been identified ei-
ther experimentally, or by homology to known enzymes from
other species. Nassau et al. identified orf 8 (otherwise known
as wbbI, b2034 or yefG)18 of the rfb cluster as a putative b-
1,6-galactofuranosyltransferase.14 Assuming that this is correct,
Results and discussion
aCentre for Carbohydrate Chemistry, School of Chemical Sciences and
Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K. E-mail:
r.a.field@uea.ac.uk
To obtain protein for analysis, the wbbI gene was cloned into
the pMAL-c2x vector and WbbI was expressed in E. coli BL21
(DE3) pLysS cells as a maltose-binding protein fusion. The fusion
bDepartment of Biochemistry, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, New York, 10461, USA
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protein was purified by affinity chromatography on an amylose
column, typically eluting with 3 mM maltose. The resulting
protein displayed an apparent molecular weight consistent with
that predicted for the MalE-WbbI fusion protein (ca. 80 KDa)
and was >90% pure, as judged by SDS-PAGE (Fig. 2). Material
of this quality, of which 15 mg was obtained per litre of cell culture,
was sufficiently clean for direct use in biotransformation studies.†
Scheme
1
Synthesis
of
authentic
disaccharide
product
Fig. 2 Expression and purification of WbbI-MBP fusion protein. From
left to right: (1) molecular weight markers; (2) cell pellet; (3) cell lysate;
(4) column flow through; (5) column wash; (6) eluted WbbI-MBP fusion
protein. (10% SDS-PAGE gel.)
Galf -b-(1→6)-Glcp-a-octyl 6. Reagents and conditions: (a) n-octanol,
BF3·Et2O, 47%; (b) trityl chloride, Py, 87%; (c) BnBr, NaH, DMF, 89%;
(d) 90% aq. TFA, CH2Cl2, 82%; (e) NIS, TfOH, CH2Cl2, 78%; (f) H2,
Pd/C, EtOH, 85%; (g) NaOMe, MeOH, quant.
WbbI is thought to be involved in the transfer of Galf onto
an a-D-glucose-based acceptor substrate to form a b-1,6-linkage.
We have recently reported a convenient chemoenzymatic synthesis
of UDP-Galf ;22 for convenience, it can also be made in situ
by the action of UDP-galactose mutase on commercial UDP-
Galp.15 The simplest possible saccharide acceptor substrate for
this enzyme is therefore an a-D-glucopyranoside. For convenient
assessment of WbbI action, TLC analysis in conjunction with octyl
glycoside acceptor substrates was employed.23 Hence, octyl a-D-
glucopyranoside 1 was selected as a model acceptor. In addition,
substrate specificity was evaluated with a number of stereoisomers
of octyl a-D-glucopyranoside (Glcp-a-octyl, 1), namely octyl b-
D-glucopyranoside (Glcp-b-octyl, 2), octyl a-D-mannopyranoside
(Manp-a-octyl, 3), octyl a-D-galactopyranoside (Galp-a-octyl,
4) and the structurally similar octyl 2-acetamido-2-deoxy-a-D-
glucopyranoside (GlcNAcp-a-octyl, 5). These compounds were
either commercially available (1 and 2) or were prepared essentially
as described in the literature (3,24 4,24 525). In addition, the putative
product of WbbI action on Glcp-a-octyl 1, namely octyl b-1,6-
D-galactofuranosyl-a-D-glucopyranoside [Galf -b-(1→6)-Glcp-a-
octyl, 6], was also chemically synthesized in order to validate
analytical protocols. Briefly, the synthesis of authentic disaccha-
ride product, 6, was achieved as follows (Scheme 1). Octyl a-D-
glucopyranoside 1 was prepared from D-glucose by BF3·Et2O-
promoted Fischer-type glycosylation in neat n-octanol; excess of
the latter was subsequently removed on a silica gel column using
gradient elution with CH2Cl2–MeOH. Selective protection of the
primary alcohol of compound 1 gave trityl ether 7, which was tri-
O-benzylated, giving 8, and subsequently treated with acid to give
the primary alcohol 9. Glycosylation of 9 with galactofuranosyl
phenylselenide donor 1026 using N-iodosuccinimide and triflic
acid afforded protected disaccharide 11, which was hydrogenated,
giving 12, and deacetylated to afford the required octyl disaccha-
ride 6.
Incubation of WbbI-MBP fusion protein with n-octyl a-D-
glucopyranoside 1 as a putative acceptor and authentic UDP-Galf
as a donor substrate gave a new product as a function of time, as
judged by TLC (Fig. 3). Similar results were obtained regardless
of whether authentic UDP-Galf was used, or it was generated
in situ from UDP-Galp by the action of UDP-galactose mutase.
Reactions with UDP-Galp in the absence of UDP-galactose
mutase gave no product.‡
Fig. 3 TLC analysis of a time course of the E. coli WbbI-MBP
Galf transfer reaction using synthetic UDP-Galf as donor and n-octyl
a-D-glucopyranoside 1 as acceptor. (1) time = 0; (2) time = 1 h; (3) time =
3 h; (4) n-octyl a-D-glucopyranoside standard.
These results suggest that WbbI is a galactofuranosyltrans-
ferase which can use n-octyl a-D-glucopyranoside as an acceptor
substrate for the addition of galactofuranose from the donor
† Cleavage of the fusion protein could be effected with factor Xa, giving
WbbI as essentially a single species of molecular mass 38 236 Da (predicted
38 234 Da), as judged by electrospray ionization mass spectrometry.
‡ In enzyme activity analyses the intact WbbI-MBP fusion protein and the
cleaved re-purified WbbI behaved similarly.
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Table 1 Transformations and product characterization for the action of WbbI on prospective octyl glycoside acceptor substrates
Substrate
Product
Turnover
100%
1H NMR data
Mass spectrometry data
Galf -b-(1→6)-Glcp-a-octyl 6
H1: 4.75 ppm (3.6 Hz)
H1ꢀ: 4.88 ppm (1.2 Hz)
Calcd for C20H38O11 454
Found [M + Na+] 477.4
Galf -b-(1→6)-Glcp-b-octyl
Galf -b-(1→6)-Manp-a-octyl
Galf -b-(1→6)-Galp-a-octyl
<5%
50%
25%
—
—
H1: 4.69 ppm (1.2 Hz)
H1ꢀ: 4.85 ppm (1.2 Hz)
Calcd for C20H38O11 454
Found [M + Na+] 477.2
H1: 4.73 ppm (3.2 Hz)
H1ꢀ: 4.78 ppm (1.2 Hz)
Calcd for C20H38O11 454
Found [M + Na+] 477.2
Galf -b-(1→6)-GlcNAcp-a-octyl
75%
H1: 4.76 ppm (3.3 Hz)
H1ꢀ: 4.82 ppm (0.9 Hz)
Calcd for C22H40NO11 495
Found [M + Na+] 518.2
substrate UDP-galactofuranose. In order to confirm the structure
of the product formed, reactions were scaled up accordingly
and octyl disaccharide product was purified by C18 reverse
phase HPLC. 1H NMR spectroscopy and mass spectrometry
characterization showed the enzymatic product to be identical
to authentic octyl b-1,6-D-galactofuranosyl-a-D-glucopyranoside
[Galf -b-(1→6)-Glcp-a-octyl, 6] produced by chemical synthesis
(see Scheme 1 and Table 1).
Table 1 summarizes a preliminary assessment of the acceptor
substrate specificity of WbbI-MBP, based on challenging the
enzyme with a series of octyl monosaccharide acceptor analogues
(vide supra). Whilst the enzyme exhibits a strong preference for
a-configured acceptor sugars (1 > 5 > 3 > 4 ꢁ 2), it is less
fussy about the relative configuration elsewhere on the acceptor
sugar ring (1 > 3 > 4) and it will also tolerate replacement of
the C-2 hydroxyl groups by an acetamido group (1 vs. 5). This
suggests that WbbI-MBP is a potentially versatile biocatalyst for
in vitro synthesis of Galf -containing saccharides. For instance,
the Galf -b-(1→6)-Manp-a-octyl disaccharide arising from WbbI
action on Manp-a-octyl 3 represents a fragment analogue of the
phosphoglyceroglycolipid antigen of Paracoccidioides brasiliensis,
the causative agent of a systemic mycosis, which contains Galf -b-
1,6-Man-b-1,3-Man-b-1,2-myo-inositol.27
Conclusions
In summary, we have cloned E. coli K-12 wbbI and over-expressed
the corresponding protein as a maltose-binding fusion protein.
The fusion protein shows activity as a UDP-Galf -dependent
b-1,6-galactofuranosyltransferase capable of glycosylating octyl
a-D-glucopyranoside but not its b-anomer, in keeping with
the a-configuration of the natural oligosaccharide acceptor for
WbbI. The relaxed acceptor substrate specificity displayed by
WbbI in vitro identifies this enzyme as a potentially useful
biocatalyst for formation of Galf -b-1,6-Glyp units. However, given
that WbbI does not appear to require more than a single sugar
residue for acceptor recognition, this raises questions about its in
vivo specificity. Further kinetic analysis is ongoing to investigate
this point.
Experimental
All chemicals were purchased from Sigma-Aldrich. Escherichia
coli strain BL21 (DE3) pLysS cells and factor Xa were purchased
from Novagen. All restriction enzymes, T4 DNA ligase, amylose
resin and pMAL-c2X vector were obtained from New England
Biolabs. PCR primers and pCR-Blunt plasmid kit were obtained
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from Invitrogen. Pfu DNApolymerase was purchasedfrom Strata-
gene. The Klebsiella pneumoniae UDP-galactopyranose mutase
clone was kindly provided by Prof. Chris Whitfield, University of
Guelph, Canada.15
over 10 column volumes) in Buffer A. Protein was detected with
an on-line detector monitoring A280 and column fractions were
collected and analyzed by SDS-PAGE. Fractions containing the
ca. 80 kDa protein were pooled and dialyzed twice against 4 L of
20 mM HEPES (pH 7.5), 200 mM NaCl, 1 mM EDTA, pH 8 and
concentrated using a YM10 Amicon ultrafiltration membrane to
a final concentration of 20 mg mL−1 and stored in 50% glycerol at
−20 ◦C.
General methods
Solution pH values were measured at 25 ◦C with an Accumet
model 20 pH meter and Accumet combination electrode standard-
ized ◦at pH 7.0 and 4.0 or 10.0. Protein purification was performed
at 4 C using an FPLC system (Amersham-Pharmacia Biotech).
Protein concentration was determined using the bicinchoninic
acid (BCA) protein assay (Pierce) with bovine serum albumin
as a standard. Spectrophotometric assays were performed using
a UVIKON XL double beam UV-vis spectrophotometer (BIO-
Factor Xa cleavage of WbbI-MBP
Cleavage of the fusion protein was achieved by the addition of
400 U of Factor Xa to 50 mg of fusion protein during dialysis
against 4 L of 20 mM HEPES, 200 mM NaCl, 5 mM CaCl2 for
36 h at 4 ◦C, with the subsequent re-application of the protein to
the amylose column to effect removal of cleaved maltose binding
protein.
1
TEK Instruments). H NMR spectra were recorded on a Varian
Gemini spectrometer at 300 MHz and are referenced to residual
HOD at dH 4.75 ppm in D2O. ESI mass spectra were recorded on
an ABI QSTAR Pulsar QTOF spectrometer.
Donor and acceptor substrates
UDP-Galf was prepared as described previously.23 Octyl a-D-
glucopyranoside 1 and octyl b-D-glucopyranoside 2 were pur-
chased from Sigma-Aldrich. Octyl a-D-mannopyranoside 3,24
octyl a-D-galactopyranoside 424 and octyl 2-acetamido-2-deoxy-a-
D-glucopyranoside 525 were prepared essentially according to lit-
erature procedures. Authentic octyl b-D-galactofuranosyl-(1→6)-
a-D-glucopyranoside 6 was prepared as described below.
Cloning of wbbI, over-expression and purification of WbbI-MBP
The E. coli K-12 wbbI gene was cloned as described previously14
but due to problems in obtaining soluble protein the gene
was further sub-cloned. The gene was amplified by PCR using
the primers: 5-GATATAGAATTCATGTATTTTTTGAATCAT-
3 and 5-GAATTCGGATCCTTAGAGAGTTTTAAG-3, which
were designed to create EcoRI and BamHI restriction endonu-
clease sites (underlined) using the initial construct as a template.
The PCR product was digested with EcoRI and BamHI and was
subjected to agarose (0.8%) gel electrophoresis. The amplified
DNA product was ligated into the pCR-Blunt plasmid and
transformed into One Shot TOP10 cells. Plasmid DNA isolated
from these cells was then digested with EcoRI and BamHI and
the purified insert was ligated into purified plasmid pMAL-c2x
previously linearized with the same restriction enzymes, yielding
an expression plasmid for E. coli WbbI with an N-terminal maltose
binding protein fusion tag. The plasmid (pMAL-c2x:wbbI) was
then isolated, sequenced, and used to transform E. coli BL21
(DE3) pLysS cells. Transformed cells were grown overnight in
50 mL of LB broth containing 100 lg mL−1 ampicillin and 34 lg
mL−1 of chloramphenicol. 1 L cultures were then inoculated to
an A600 of ∼0.05. The cells were grown at 37 ◦C to an A600 of
0.8. Cells were cooled for 1 h at 4 ◦C and protein expression was
subsequently induced by the addition of 0.5 mM isopropyl b-D-
thiogalactopyranoside (IPTG) and left to grow for a further 16 h at
20 ◦C. Expression of the protein was confirmed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
Octyl 6-O-trityl-a-D-glucopyranoside 7. A solution of octyl a-
glucoside 1 (4.0 g, 14 mmol) and trityl chloride (5.9 g, 21 mmol) in
pyridine (30 mL) was stirred at room temperature for 12 h. Solvent
was then removed in vacuo and the resulting residue was dissolved
in CH2Cl2 (50 mL) and washed successively with 1 M HCl (2 ×
50 mL), NaHCO3 solution (2 × 50 mL) and H2O (50 mL). The
organic extract was dried (Na2SO4) and concentrated to a syrup.
Purification of the crude product on silica gel column using EtOAc
afforded trityl ether 7 (6.4 g, 87%) as a light yellow gum. [a]2D3 +131
(c 1.0, CHCl3). 1H NMR (CDCl3) d: 7.46–7.21 (15H, ArH), 4.85
(d, 1H, J1,2 = 4.0 Hz, H-1), 3.77–3.67 (m, 2H, H-3, O–CH2), 3.53–
3.42 (m, 3H, H-2, H-4, H-6a), 3.42–3.32 (m, 2H, H-6b, O–CH2),
2.98 (m, 1H, H-5), 2.69 (bs, 1H, OH), 2.22 (bd, 2H, 2 OH), 1.63 (m,
2H, O–CH2–CH2), 1.34 (m, 10H, octyl-CH2), 0.93 (t, 3H, octyl-
CH3). 13C NMR (CDCl3) d: 143.8, 128.7, 128.0, 127.2 (ArC), 97.9
(C-1), 87.0, 74.9, 72.3, 71.7, 70.0, 68.3, 63.9 (C-6), 31.7, 29.4, 29.3,
29.1, 26.1, 22.5, 14.0. HRMS [M + NH4]+ calcd. for C33H46O6N
552.3325, found 552.3321.
Octyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside 9. To a solution
of trityl ether 7 (4.0 g, 7.5 mmol) in DMF (30 mL), was added NaH
(810 mg, 50% suspension in mineral oil) followed by BnBr (3.2 mL,
27 mmol) and the mixture was stirred at room temperature for 8 h.
Excess NaH was destroyed by careful addition of MeOH and
solvent was removed in vacuo. The resulting residue was dissolved
in CH2Cl2 (70 mL) and washed with H2O (3 × 70 mL); the organic
extract was dried (Na2SO4) and concentrated to a syrup. The
crude product was purified on a silica gel column using EtOAc–
n-hexane (1 : 5) to give octyl 2,3,4-tri-O-benzyl-6-O-trityl-a-D-
glucopyranoside 8 (5.4 g, 89%) as light yellow oil. An aqueous
solution of TFA (90% v/v, 10 mL) was added to a solution of
compound 8 (5.0 g, 6.2 mmol) in CH2Cl2 (30 mL) and the mixture
was stirred at room temperature for 30 min. The reaction mixture
All protein purification steps were carried out at 4 ◦C. The cell
pellet (30 g) was resuspended in 50 mL of 20 mM HEPES, 200 mM
NaCl, pH 8, containing two tablets of complete protease inhibitor
cocktail (Roche). Cells were disrupted on ice by sonication using
a Branson Sonifier 450. The suspension was then centrifuged
(10 000 g for 30 min) to remove cell debris. The supernatant was
then filtered using a 0.2 lm syringe filter. The filtered supernatant
was applied to a pre-equilibrated (Buffer A; 20 mM HEPES,
200 mM NaCl, pH 8) amylose column (3 × 15 cm). The column
was washed at 1 mL min−1 with 8 column volumes of the same
buffer and then eluted with a linear gradient of maltose (0–5 mM
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ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
,
was diluted with CH2Cl2 (20 mL) and washed successively with
H2O (50 mL), aqueous NaHCO3 solution (2 × 50 mL) and brine
(50 mL). The organic extract was dried (Na2SO4) and concentrated
to a syrup. The crude product was purified on a silica gel column
using EtOAc–n-hexane (1 : 3) to give compound 9 (2.9 g, 82%) as
a foam. [a]2D3 +103 (c 1.2, CHCl3). 1H NMR (CDCl3) d: 7.39–7.26
(m, 15H, ArH), 5.04–4.65 (6d, 6H, JAB = 10.8 Hz, 3CH2Ph), 4.71
(d, 1H, J1,2 = 3.6 Hz, H-1), 4.04 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-
(dd, 1H, J5 ,6a = 4.2 Hz, J6a ,6b = 12.0 Hz, H-6aꢀ), 4.32 (t, 1H, J3 ,4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J4 ,5 = 5.7 Hz, H-4 ), 4.19 (dd, 1H, J5 ,6b = 7.2 Hz, J6a ,6b = 12.0 Hz,
H-6bꢀ), 3.93 (bd, 1H, H-6a), 3.74–3.60 (m, 2H, H-6b, OCH2), 3.45
(t, 1H, J2,3, J3,4 = 5.7 Hz, H-3), 3.43 (m, 1H, OCH2), 3.38 (dd, 1H,
J
1,2, J2,3, H-2), 3.34–3.25 (m, 2H, H-4, H-5), 2.09, 2.08, 2.06, 2.02
(4s, 12H, 4 COCH3), 1.63 (m, 2H, O–CH2–CH2), 1.38 (m, 10H,
octyl-CH2), 0.96 (t, 3H, octyl-CH3). 13C NMR (CD3OD) d: 172.4,
172.0, 171.8, 171.4 (4 COCH3), 107.1 (C-1ꢀ), 100.0 (C-1), 82.5,
81.4, 78.0, 75.0, 73.4, 72.7, 71.8, 70.8, 69.2, 67.7, 63.8, 32.9, 30.5,
30.4, 30.3, 27.2, 23.6, 20.7, 20.6 (2), 20.5 (4 COCH3), 14.4. HRMS
[M + NH4]+ calcd. for C28H50O15N 640.3175, found 640.3181.
3), 3.80–3.66 (m, 4H, H-5, H-6a, H-6b, OCH2), 3.64 (t, 1H, J3,4
,
J4,5 = 10.2 Hz, H-4), 3.51 (dd, 1H, J1,2, J2,3, H-2), 3.42 (m, 1H,
OCH2), 2.28 (bs, 1H, OH), 1.61 (m, 2H, O–CH2–CH2), 1.33 (m,
10H, octyl-CH2), 0.92 (t, 3H, octyl-CH3). 13C NMR (CDCl3) d:
138.9, 138.4, 138.2, 128.6, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8,
127.6 (ArC), 96.8 (C-1), 81.9, 80.2, 75.6, 75.0, 73.1, 70.6, 68.2, 61.9
(C-6), 31.7, 29.3, 29.1, 26.1, 22.5 (2), 14.0. HRMS [M + NH4]+
calcd. for C35H50O6N 580.3638, found 580.3635.
Octyl b-D-galactofuranosyl-(1→6)-a-D-glucopyranoside 6. To
a solution of partially acetylated disaccharide 8 (1.0 g, 1.6 mmol)
in MeOH (20 mL) was added methanolic NaOMe (2 mL, 0.5 M);
the mixture was stirred at room temperature for 2 h. After
neutralization with Dowex 50 W × 8 (H+) resin, the mixture was
filtered through Celite and the filtrate was evaporated to afford
Octyl 2,3,5,6-tetra-O-acetyl-b-D-galactofuranosyl-(1→6)-2,3,4-
tri-O-benzyl-a-D-glucopyranoside 11. A mixture of primary al-
cohol 9 (2.0 g, 3.6 mmol), galactofuranosyl phenylselenide 1026
octyl disaccharide 9 (730 mg, quantitative) as a white powder. [a]D23
1
ꢀ
ꢀ
+43 (c 1.0, MeOH). H NMR (CD3OD) d: 4.88 (d, 1H, J1 ,2
=
1.2 Hz, H-1ꢀ), 4.75 (d, 1H, J1,2 = 3.6 Hz, H-1), 1.61 (m, 2H, O–
˚
(2.3 g, 4.6 mmol) and 4 A molecular sieves (3 g) in CH2Cl2 (30
mL) was stirred under nitrogen for 2 h. After cooling to 0 ◦C, NIS
(1.4 g, 6 mmol) was added followed by TfOH (53 lL, 0.6 mmol)
and the mixture was stirred at 0 ◦C for 45 min. After complete
consumption of the starting material, the mixture was diluted with
CH2Cl2 (20 mL), filtered through Celite and washed successively
with aqueous Na2S2O3 solution (2 × 50 mL), NaHCO3 solution
(2 × 50 mL) and brine (50 mL). The organic extract was dried
(Na2SO4) and concentrated to a syrup. The crude product was
purified on a silica gel column using EtOAc–n-hexane (1 : 3) to
give pure compound 7 (2.5 g, 78%) as a foam. [a]2D3 +78 (c 0.9,
CHCl3). 1H NMR (CDCl3) d: 7.39–7.25 (m, 15H, ArH), 5.39 (m,
CH2–CH2), 1.29 (m, 10H, octyl-CH2), 0.95 (t, 3H, octyl-CH3). 13
C
NMR (CD3OD) d: 111.8 (C-1ꢀ), 100.5 (C-1), 88.0, 82.1, 80.7, 75.3,
73.6, 73.1, 72.4, 72.2, 69.5, 67.9, 65.0, 33.0, 30.6, 30.5, 30.4, 27.3,
23.7, 14.4. HRMS [M + NH4]+ calcd. for C20H42O11N 472.2752,
found 472.2754.
Measurement of enzyme activity
Assay mixtures contained 50 mM MOPS, 2 mM magnesium
chloride, 5 mM sodium dithionite pH 7.5, in addition to 10 mM
acceptor and 10 mM UDP-galactopyranose. Reactions were
initiated by the addition of 50 lM UDP-galactopyranose mutase15
and 125 lM galactofuranosyltransferase fusion protein in a total
volume of 100 lL and were maintained at 37 ◦C. Reactions were
terminated with 50 lL ethanol, followed by centrifugation at
10 000 rpm for 5 min. Reactions were monitored by TLC using
ꢀ
ꢀ
1H, H-5ꢀ), 5.09 (d, 1H, J2 ,3 = 2.1 Hz, H-2 ), 5.04 (s, 1H, H-1 ), 5.01
ꢀ
ꢀ
(dd, 1H, J2 ,3 , J3 ,4 = 6.0 Hz, H-3ꢀ), 5.05–4.59 (6H, JAB = 11.2 Hz,
ꢀ
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
3CH2Ph), 4.75 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.33 (dd, 1H, J5 ,6a
=
4.2 Hz, J6a ,6b = 12.0 Hz, H-6aꢀ), 4.28 (m, 1H, H-5ꢀ), 4.21 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1H, J5 ,6b = 7.5 Hz, J6a ,6b , H-6b ), 4.02 (t, 1H, J2,3, J3,4 = 9.0 Hz,
˚
ꢀ
glass-backed 10 cm by 10 cm, 250 lm, 60 A K6F silica gel TLC
ꢀ
ꢀ
ꢀ
ꢀ
H-3), 3.86 (m, 1H, H-6a), 3.80 (dd, 1H, J3 ,4 = 9.9 Hz, J5 ,6a , H-4 ),
3.69–3.63 (m, 2H, H-6b, OCH2), 3.58 (t, 1H, J3,4, H-4), 3.55–3.49
(m, 2H, H-2, H-5), 3.41 (m, 1H, OCH2), 2.13, 2.06, 2.04, 2.03 (4s,
12H, 4 COCH3), 1.64 (m, 2H, O–CH2–CH2), 1.28 (m, 10H, octyl-
CH2), 0.94 (t, 3H, octyl-CH3). 13C NMR (CDCl3) d: 170.5, 170.0,
169.9, 169.4 (4 COCH3), 138.8, 138.3 (2), 128.4, 128.3 (2), 127.9,
127.8, 127.7, 127.6, 127.5 (ArC), 105.7 (C-1ꢀ), 96.5 (C-1), 81.9,
81.0, 80.0, 79.5, 77.6, 76.2, 75.5, 74.9, 72.8, 69.7, 69.0, 68.0, 66.3,
62.6 (C-6), 31.6, 29.2, 29.1, 29.0, 26.0, 22.4, 20.5, 20.4, 20.3 (2),
13.8. HRMS [M + NH4]+ calcd. for C49H68O15N 910.4583, found
910.4586.
plates run in CHCl3–CH3OH–H2O (65 : 25 : 4.1) using a variation
of a literature method.28 Compounds were detected with an orcinol
solution (20 mg of orcinol dissolved in 10 mL of 70% sulfuric acid)
by heating at 100 ◦C. Spots containing sugars appeared pink-violet
on a white background. The detection limit was ca. 0.5 nmol (ca. 1
lg) of total sugar per spot. Octyl glycoside turnover was estimated
using a densitometer (SynGene Gel Analysis and Documentation
System).
Product purification and analysis
Octyl 2,3,5,6-tetra-O-acetyl-b-D-galactofuranosyl-(1→6)-a-D-
glucopyranoside 12. To a solution of protected disaccharide
11 (2.0 g, 2.2 mmol) in EtOH (25 mL) was added 10% Pd/C
(70 mg); the mixture was stirred at room temperature under a
positive hydrogen pressure for 48 h. After filtration through Celite,
the filtrate was evaporated and dried under vacuum. Column
chromatography of the crude product with EtOAc as eluent
afforded partially esterified disaccharide 12 (1.2 g, 85%) as a foam.
[a]2D3 +48 (c 1.1, MeOH). 1H NMR (CD3OD) d: 5.36 (m, 1H, H-5ꢀ),
Octyl glycoside (ca. 8 mg, 27 lmol) and UDP galactopyranose
(40 mg, 65 lmol) were dissolved in buffer (20 mL, 50 mM TRIS,
10 mM sodium dithionate and 2 mM MgCl2, pH 8.0). Purified
recombinant Klebsiella pneumoniae UDP-galactopyranose mutase
(4 mg, 93 nmol)15 and purified UDP-galactofuranosyltransferase
fusion protein (10 mg, 125 nmol) was added, the solution was
flushed with nitrogen and the reaction was stirred at 30 ◦C
for 16 h. The reaction mixture was passed through an Amicon
YM10 spin filter (13 000 rpm, 4 ◦C). A proportion of the flow
through was applied to a C18 Phenomenex Luna 5 l column
(4.6 mm × 250 mm) and eluted with a gradient from 0.5% to
5.14 (s, 1H, H-1ꢀ), 5.03 (d, 1H, J2 ,3 = 1.8 Hz, H-2ꢀ), 4.98 (dd, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
J
2 ,3 , J3 ,4 = 5.7 Hz, H-3ꢀ), 4.76 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.32
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99.5% acetonitrile in water over 25 column volumes at a flow rate
of 1 mL min−1. UDP galactopyranose eluted at the beginning of
the gradient, whilst octyl disaccharide typically eluted at ca. 50%
acetonitrile. Relevant samples were combined and concentrated in
vacuo to give the required compounds, which were subjected to
NMR and mass spectrometry analysis (Table 1).
10 K. Zhang, T. Barron, S. J. Turco and S. M. Beverley, Mol. Biochem.
Parasitol., 2004, 136, 11–23.
11 Although no galactofuranosyltransferase genes have been identified
in fungi, the UDP-galactose mutase from Aspergillus fumigatus has
recently been cloned: H. Bakker, B. Kleczka, R. Gerardy-Schahn and
F. H. Routier, Biol. Chem., 2005, 386, 657–661.
12 M. Sarvas and H. Nikaido, J. Bacteriol., 1971, 105, 1063–1072.
13 H. Nikaido and M. Sarvas, J. Bacteriol., 1971, 105, 1073–1082.
14 P. M. Nassau, S. L. Martin, R. E. Brown, A. Weston, D. Monsey, M. R.
McNeil and K. Duncan, J. Bacteriol., 1996, 178, 1047–1052.
15 R. Koplin, J.-R. Brisson and C. Whitfield, J. Biol. Chem., 1997, 272,
4121–4128.
16 D. A. R. Sanders, A. G. Staines, S. A. McMahon, M. R. McNeil, C.
Whitfield and J. H. Naismith, Nat. Struct. Biol., 2001, 8, 858–863.
17 M. Soltero-Higgin, E. E. Carlson, T. D. Gruber and L. L. Kiessling,
Nat. Struct. Biol., 2004, 11, 539–543.
Acknowledgements
This work was supported by the EPSRC and the Weston Founda-
tion. We are indebted to the EPSRC Mass Spectrometry Service
Centre, Swansea, for invaluable support. We thank Dr Steve
Martin (GSK, Stevenage, UK) for helpful discussions during the
early phases of this project and Prof. Chris Whitfield, University of
Guelph, Canada, who kindly provided the UDP-galactopyranose
mutase clone.15
18 F. R. Blattner, G. Plunkett, C. A. Bloch, N. T. Perna, V. Burland, M.
Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J.
Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B.
Mau and Y. Shao, Science, 1997, 277, 1453–1474.
19 L. Kremer, L. G. Dover, C. Morehouse, P. Hitchini, M. Everett, H. R.
Morris, A. Dell, P. J. Brennan, M. R. McNeil, C. Flaherty, K. Duncan
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20 N. L. Rose, G. C. Completo, S.-J. Lin, M. McNeil, M. M. Palcic and
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