Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes

Article abstract of DOI:10.1016/j.carres.2016.11.017

Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC,

Full text of DOI:10.1016/j.carres.2016.11.017

Accepted Manuscript
Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and
sugar-1-phosphate transferase activities in Euglena gracilis membranes
Irina M. Ivanova, Sergey A. Nepogodiev, Gerhard Saalbach, Ellis C. O'Neill, Michael
D. Urbaniak, Michael A.J. Ferguson, Sudagar S. Gurcha, Gurdyal S. Besra, Robert A.
Field
PII:
S0008-6215(16)30512-2
10.1016/j.carres.2016.11.017
CAR 7300
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 30 October 2016
Revised Date: 16 November 2016
Accepted Date: 29 November 2016
Please cite this article as: I.M. Ivanova, S.A. Nepogodiev, G. Saalbach, E.C. O'Neill, M.D. Urbaniak,
M.A.J. Ferguson, S.S. Gurcha, G.S. Besra, R.A. Field, Fluorescent mannosides serve as acceptor
substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis
membranes, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.11.017.
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ACCEPTED MANUSCRIPT
Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and
sugar-1-phosphate transferase activities in Euglena gracilis membranes
Irina M. Ivanova, Sergey A. Nepogodiev, Gerhard Saalbach, Ellis C. O’Neill, Michael D. Urbaniak, Michael
A. J. Ferguson, Sudagar S. Gurcha, Gurdyal S. Besra and Robert A. Field
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mannosides serve as acceptor
Fluorescent
substrates
for
glycosyltransferase and sugar-1-phosphate transferase activities in Euglena
gracilis membranes
Irina M. Ivanova,a Sergey A. Nepogodiev,a Gerhard Saalbach,a Ellis C. O’Neill,a,b Michael D.
Urbaniak,c,d Michael A. J. Ferguson,d Sudagar S. Gurcha,e Gurdyal S. Besrae and Robert A. Fielda*
aDepartment of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4
7UH, UK
bPresent address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford,
OX1 3RB, UK
cBiomedical and Life Sciences, Lancaster University, Furness Building, Lancaster LA1 4YG, UK
dCollege of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK
eSchool of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Email: rob.field@jic.ac.uk
Abstract: Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were
fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The
resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass
spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes,
as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the
fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the
glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were
detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These
studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for
exploring carbohydrate active enzymes.
Key words: Euglena gracilis; glycosyltransferases; fluorescent glycans; N-acetylglucosamine-1-
phosphate transferase, enzyme assays.
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1
Introduction
The single-celled protozoan microalga Euglena has been the subject of scientific endeavour since it
was first observed by van Leeuwenhoek [1]. This flagellated, unicellular microorganism mainly
inhabits fresh water, although it is highly adaptable and it can survive under harsh environmental
conditions (e.g. low pH, high salinity, high energy ionising radiation) [2]. Euglena exhibits both
plant- and animal-like characteristics: it can live in light, sustaining itself autotrophically through
photosynthesis; or it can survive heterotrophically in the dark, using nutrients from the environment.
Despite being considered colloquially as a green alga, Euglena is classified in the phylum Euglenozoa
[3], which also includes the human parasites Trypanosoma brucei [4] and Leishmania major [5]. The
rich glycobiology of these parasites [6] prompted us to explore the capabilities of E. gracilis [7].
Reports of the characterisation of glycans from Euglena are limited to date, with most efforts focusing
on its crystalline and granular storage β-1,3-glucan paramylon [8]. While Euglena do not possess a
plant-like polysaccharide cell wall, undefined glycoproteins rich in xylose, mannose, glucose and
galactose are present on the outer membrane of their flagella [9]. No other detailed information is
currently available about glycoprotein, polysaccharide or GPI anchor structures from these organisms.
The presence in Euglena of a typical eukaryote N-glycan precursor glycolipid [Glc3Man9GlcNAc2-P-
P-dolichol] has been confirmed, which is in contrast to that found in the trypanosomes
[Man7-9GlcNAc2-P-P-dolichol] [10]. We recently reported on the transcriptome of Euglena gracilis
[11], which identified unexpected capacity for carbohydrate biochemistry. This organism possesses at
least 126 glycoside hydrolases (GHs) and 229 glycosyltransferases (GTs) [7, 12], under the growth
conditions employed for transcriptome analysis – numbers that rival the human CAZome, albeit with
a different balance and repertoire of predicted activities. In order to experimentally validate assigned
functions, suitable assays are needed. While a varied range of GT assays are available [13], the use of
radiolabelled sugar nucleotides remains prevalent. To simplify the assays and avoid the necessity for
radioactive material, we were drawn to explore more flexible fluorescence-based assays, employing
fluorescently labelled acceptor glycans [14]. In recent work, we have explored capillary
electrophoresis with laser-induced fluorescence (CE-LIF) detection as a high sensitivity, high
resolution method for glycosyltransferase analyses, for instance [15]. Herein, we report the
development of fluorescent coumarin derivatives, coupled with TLC or HPLC-MS analysis, for the
straightforward detection and characterisation of Euglena carbohydrate-active enzyme activities.
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2
Results and Discussion
The initial goal of these studies was to develop acceptor glycan substrates that could be used to assess
the expected eukaryotic N-linked glycan and GPI anchor pathway GTs that one might reasonably
expect to find in Euglena microsomal membranes. This led us to simple α-D-mannoside derivative 1
and its α-1,6-linked disaccharide counterpart 2 (Fig. 1), which incorporate fluorescent coumarin
residues and can be assembled by standard glycosylation techniques and copper-catalysed azide-
alkyne cycloaddition (CuAAC) click chemistry [16]. These studies would therefore benchmark
against our earlier work on related alkyl glycosides which, in assays with radiolabelled sugar
nucleotide donors, we have demonstrated serve as acceptor substrates for GTs in Trypanosome [17]
and mycobacterial membranes [18].
Fig. 1. Structure of fluorescent acceptors α-Man-HCT (1) and α-Man-(1,6)-α-Man-HCT (2) and their schematic
representations. HCT denotes fluorescent aglycone residue [(7-hydroxycoumarin-3-yl)-1H-1,2,3-triazlole-4-
ylbutlyl)].
2.1 Chemical synthesis of fluorescent coumarin-based α-D-mannopyranoside derivatives
Hexynyl α-D-mannopyranoside (6) was synthesised in two steps starting from α-mannopyranosyl
bromide 3 and 5-hexyn-1-ol (4) as outlined in Scheme 1. Glycosylation was performed by activation
of glycosyl bromide 3 with AgOTf in the presence of 4Å molecular sieves, which gave α-glycoside 5
in 85% yield. De-O-benzoylation of 5 afforded target α-D-mannopyranoside 6 in 92% yield. The
corresponding α-Man-(1→6)-Man disaccharide 9 was synthesised in three steps from mannoside 6 by
regioselective 6-O-tritylation followed by per-O-benzoylation to afford 7, which was isolated in 92%
yield. AgOTf-promoted glycosylation of trityl ether 7 with glycosyl bromide 3 gave α-linked
disaccharide 8 in an unoptimised 45% yield. De-O-benzoylation of 8 afforded target disaccharide 9 in
84% yield. The α-configuration of the newly formed inter-sugar linkages in 5 and 8 were confirmed
by the characteristic value of one bond C-H coupling of the anomeric carbon signal (1JC-H ~ 171 Hz)
in 1H-coupled 13C NMR spectra of α-mannosides [19].
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Scheme 1. Synthesis of hexynyl glycosides 7 and 9. Reagents and conditions: (a) AgOTf, CH2Cl2, –20→22 °C,
3.5 h; (b) 30 mM NaOMe in MeOH, RT, 4 h; (c) Ph3CCl, DMAP, Py, 37 °C, 40 h then BzCl, Py, RT, 1 h; (d)
AgOTf, CH2Cl2, –20→RT °C, 18 h; e) 10 mM NaOMe in MeOH, RT, 24 h.
The alkyne functional groups of 5-hexyn-1-ol 4, monosaccharide 6 and disaccharide 9 were used in
fluorogenic CuAAC reactions with known non-fluorescent 3-azido-7-hydroxycoumarin (10) [20],
generating the corresponding fluorescent adducts 1, 2 and 11, as outlined in Scheme 2. These
reactions were generally complete after 2 h at room temperature, resulting in respectable yields (70-
90%) of the required fluorescent adducts.1 The formation of 1,4-linked triazoles was confirmed by
1
the presence of the characteristic triazole signal δ 8.17-8.25 in H NMR and δ 122.3-124.2 in 13C
NMR spectra of compounds 1, 2 and 11.
Scheme 2. Synthesis of fluorescently labelled compounds 1, 2 and 11 and their schematic representation.
Reagents and conditions: (a) NaAsc, CuSO4, MeOH-H2O (1:1), RT, 2 h; (b) NaAsc, CuSO4, MeOH, RT, 2 h.
1 The efficiency of all three CuAAC reactions was affected by the formation of traces of by-products with lower
Rf values; the identities of these by-products were not pursued.
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2.2 Properties of fluorescent coumarin-based α-D-mannopyranoside derivatives
As is to be expected for a phenolic compound, the fluorescence emission of 2 was pH-dependent, with
the intensity at pH 9 twice as large as the intensity in the range pH 3 to 6. The detection limit of 2 by
TLC visualised by UV irradiation at 365 nm was ca 160 pmol, and ca 100 nM when detected in
solution by fluorimeter. Euglena
2.3 Benchmarking against published radiochemical assays
Published results from radiolabelled assays used to investigate the biosynthesis of cell surface
glycoconjugates in Mycobacterium smegmati [18] and Trypanosoma brucei [17] were used as a
benchmark for our practical fluorescence-based assay. In the first series of experiments we targeted
α-1,6-ManT activities responsible for the biosynthesis of lipomannan in M. smegmatis; in the second
series we investigated GalTs involved in the decoration of the core GPI anchor structures in T. brucei.
Both lipomannan and GPI anchor structures have a common α-Man-1,6-α-Man disaccharide motif.
Therefore, it was reasonable to envisage α-Man-HCT (1) and α-Man-1,6-α-Man-HCT (2) as mimics
of intermediate structures involved in the biosynthesis of these glycoconjugates (Fig. 2). In essence,
compounds 1 and 2 were anticipated to serve as generic mannoside acceptors for the detection of
several glycosyltransferase activities, benchmarking our fluorescence-based assay as well as enabling
further investigation of glycoconjugates biosynthesis in Euglena gracilis.
Fig. 2. Schematic representation of structures of glycoconjugates from Mycobacterium smegmatis,
Trypanosoma brucei and Euglena gracilis. The red boxes indicate disaccharide fragments mimicked by the
synthetic acceptors 1 and 2.
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In preliminary studies (SI, Sections 2 and 3), we have demonstrated that fluorescence-based assays
can be used to detect glycosyltransferase activities in Mycobacterium smegmatis and Trypanosoma
brucei.
We showed that α-Man-1,6-α-Man-HCT (2) was active towards both α-1,6-ManT in Mycobacterium
smegmatis membrane preparations, as well as α- and β-GalTs in Trypanosoma brucei microsomal
preparations. Thus, incubation of 2 and GDP-Man with Mycobacterium smegmatis resulted in the
formation of fluorescent α-1,6-linked manno-trioside and -tetraoside (Fig. S3 in SI). In similar
experiments involving 2 and UDP-Gal in the presence of Trypanosoma brucei microsomal
preparations, we observed addition of one galactose residue to fluorescent acceptor 2, leading to the
formation of α- and β-linked fluorescent trisaccharide products (Fig. S7 in SI). The presence of a
fluorescent aglycone allowed quick access to information on the biotransformation reaction by TLC.
The fluorescent label facilitated product purification by HPLC and product identification by
enzymatic digestion followed by TLC. Most importantly, results of our fluorescence-based assays
were consistent with data obtained in analogous studies that employed radiolabelled assays [17-18].
Further details can be found in the Supplementary Information (section 2 and 3).
2.4 Mannosyltransferase activities in Euglena gracilis
Glycosyltransferases that participate in the biosynthesis of many, perhaps most, eukaryote
glycoconjugates are located in the ER and Golgi apparatus [21], presenting a major challenge for their
isolation in active form. In this study, our focus was simply on the detection of membrane-bound
glycosyltransferases in Euglena gracilis microsomal membranes.
2.4.1 Microsomal membranes as a source of glycosyltransferases
Euglena microsomal membranes were prepared following a literature procedure [22], from cells
cultured in the dark in media supplemented with glucose (this approach provides far more biomass to
work with than cells grown autotrophically in the light). After seven days, cells were harvested by
centrifugation, lysed by ultrasonication and the microsomal membranes were obtained by
ultracentrifugation over a sucrose gradient.
2.4.2 Fluorescence assays to probe Euglena gracilis mannosyltransferase activities
With microsomal membrane preps in hand, we established enzyme assays with fluorescent
compounds 1 and 2 as acceptor substrates and GDP-Man as donor substrate. To ensure the absence of
endogenous GDP-Man in the membrane preparation as well as to assess the potential enzymatic
degradation of our fluorescent acceptors, two control assays were included containing either no
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enzyme or no donor substrate (Fig. 3A and 3B, lanes 3 and 4). The formation of fluorescent
glycosylation products was evident from TLC analyses (Fig. 3A and 3B, lane 5).
Fig. 3. TLC analyses of fluorescence-based assays to assess mannosyltransferase activities in Euglena gracilis
microsomal membranes. (A) α-Man-HCT (1) as acceptor; (B) α-Man-1,6-α-Man-HCT (2) as acceptor. Assay
conditions: acceptor (2 mM) and donor GDP-Man (4 mM) were incubated for 24 h at 30 °C in HEPES/KOH (10
mM, pH 7.0) buffer supplemented with MgCl2 (10 mM), MnCl2 (10 mM), KCl (25 mM), glycerol (10%) in the
presence of E. gracilis microsomal membranes (150ꢀL, 195 ꢀg of total protein) in total reaction volume of 200
ꢀL, TLC plates were eluted with CHCl3:MeOH:H2O (10:6:1) and visualised using mid-wave length range UV
light. The components of each reaction mixture are shown below the corresponding TLC image and acceptors
and products are shown to the side of each TLC image.
TLC analysis of enzymatic reactions that involved α-Man-HCT (1) acceptor showed two distinct
fluorescent bands (Fig. 3A, lane 5) corresponding to starting acceptor substrate 1 and product 12. The
latter possessed a similar Rf value to synthetic α-Man-1,6-α-Man-HCT (2) standard. This suggested a
probable transfer of one mannose residue to acceptor substrate 1. In a similar sense, TLC analysis of
enzymatic reactions that involved α-Man-1,6-α-Man-HCT (2) acceptor also showed two distinct
fluorescent bands (Fig. 3B, lane 5): one corresponding to starting acceptor substrate 2 and another
more polar band, suggesting the formation of a trisaccharide product 13. Unexpectedly, products
fluorescence intensities visually indicated a higher enzymatic activity in the case of the simpler
substrate, monosaccharide 1, than of the disaccharide 2 In both sets of assays, only minimal in situ
hydrolysis of the fluorescent acceptors was evident, indicating the absence of appreciable α-
mannosidase activity.
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2.4.3 Purification of fluorescent products 12 and 13 by HPLC
In order to establish the structure of the above fluorescent products, they were first purified by reverse
phase and then by normal phase HPLC methods. The former enabled isolation of disaccharide 12 and
trisaccharide 13 as single HPLC peaks (Fig. 4A), while the latter resolved disaccharide products 12
and 13 into two peaks each, designated as 12a,b and 13a,b (Fig. 4B, red trace). The ratio of major
(12a) to minor (12b) product was ca 2:1.
Fig. 4. HPLC analyses of reaction mixtures obtained from incubation of fluorescent acceptors with GDP-Man in
the presence of Euglena gracilis microsomal membranes. UV chromatograms of mannosylated products from
α-Man-HCT (2) assay (red trace) and α-Man-1,6-α-Man-HCT (1) assay (blue trace). (A) Reverse phase
purification of disaccharide 12 and trisaccharide 13 products. HPLC conditions: Phenomenex C18(2) (250 mm
x 10 mm) chromatography column, mobile phase: water–CH3CN (0.1% TFA) (10 to 90 % in 36 min), flow rate
5 mL/min), UV detector at 347 nm. (B) Normal phase HPLC purification of disaccharide 12 and trisaccharide
13 products. HPLC conditions: Phenomenex LUNA NH2 (250 mm x 10 mm) chromatography column, mobile
phase: CH3CN-water (0.1% TFA) (10 to 80% in 32 min, flow rate 5 mL/min), UV detector at 347 nm.
2.4.4 Structural characterisation of fluorescent products
LC-MS analyses confirmed the formation of disaccharide and trisaccharide products from acceptors 1
and 2 (Fig. S10 in SI). The nature of the glycosidic linkages was then analysed by digestion with jack
bean α-mannosidase [23], which showed that all products formed from 1 and 2 were α-linked
mannosides (Fig. S11 and S12 in SI). Sequential digestion of 12a,b and 13a,b was then conducted
with three different linkage-specific enzymes to assess the regioselectivity of the enzymatic
mannosylation reactions. These experiments employed Aspergillus saitoi α-1,2-mannosidase [17b],
Xanthomonas manihotis α-1,6-mannosidase and a Xanthomonas manihotis α-1,2/3-mannosidase [24]
(for full details, see Supplementary Information sections 4 and 5). These experiments indicated that
α-mannoside products derived from acceptor 1 were mainly α-1,2-linked, with only a minor
proportion being α-1,3-linked. The same sequential enzymatic digestions of products derived from
disaccharide acceptor 2 showed that solely α-1,2-mannosidic linkages have been formed. Based on
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these results, structures of disaccharide products 12a,b and trisaccharide products 13a,b can be
proposed (Fig. 5).
Fig. 5. Symbolic representation of mannosyltransferase-catalysed reaction products 12 and 13 and their
relationship with structures of N-glycan and GPI anchor precursors.
The characterisation of products 12a,b and 13a,b confirmed that α-Man-HCT (1) and α-Man-1,6-α-
Man-HCT (2) can act as acceptor substrates for α-1,2- and α-1,3-ManT present in E. gracilis. The
oligosaccharide sequences of products 12a,b and 13a,b shown in Fig. 5 have a close resemblance to
structures of fragments of N-glycans high mannose chain, thus indicating that the detected enzyme
activities are most likely involved in the construction of N-glycans in E. gracilis. However, the
possibility of the involvement of the observed α-1,2-ManT activity in GPI anchor biosynthesis cannot
be ruled out.
2.5 Exploration of other glycosyltransferases in Euglena gracilis
The detection of α-1,2-ManT and α-1,3-ManT activities in E. gracilis microsomal membranes
prompted us to look for other GT activities in these membrane preparations. Given that we achieved
different outcomes with monosaccharide acceptor 1 and disaccharide acceptor 2, we persisted with
both compounds.
2.5.1 Fluorescence assays to probe glycosyltransferase activities
In order to more broadly assess GT activities, acceptor substrates 1 and 2 were incubated with UDP-
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D
-Glc, UDP-D-GlcNAc, or GDP-L-Fuc, in the presence of E. gracilis microsomal membranes and the
formation of fluorescent products was monitored by TLC (Fig. 6).
Assays with α-Man-HCT (1) in the presence of UDP-Glc revealed two new fluorescent bands,
designated compounds 14 and 15 (Fig. 6A, lane 4). Judging by their Rf values, which were similar to
that one of disaccharide 2, faster moving compound 14 was tentatively assigned as a fluorescent
disaccharide, while the much more polar product 15 was initially assigned as an oligosaccharide
arising from the transfer of several glucose units. Assays with acceptor 1 and UDP-GlcNAc generated
a single dominant polar product 16 (Fig. 6A, lane 5), which ran similarly on TLC to polar UDP-Glc-
derived 15.
Assays with disaccharide acceptor 2 led to a TLC outcomes that were qualitatively similar to those of
monosaccharide acceptor 1. Thus, in the presence of UDP-Glc two fluorescent products 17 and 18
were detected whereas in the presence of UDP-GlcNAc TLC showed that only one product,
designated as compound 19, was formed (Fig. 6B, lanes 4 and 5). In reactions with UDP-Glc,
acceptor substrate turnover was demonstrably better in the case of disaccharide 2 compared to the
monosaccharide 1,in contrast to the mannosyltransferase activity described in Section 2.4 which
provided lower turnover of disaccharide than monosaccharide acceptor. In contrast assays employing
GDP-Fuc showed no turnover, in keeping with the anticipated lack of Fuc-Man linkages in
eukaryotes.
Fig. 6. TLC analyses of enzymatic assays involving incubation of acceptors 1 and 2 with a number of sugar
nucleotides in the presence of Euglena gracilis microsomal membranes. Fluorescent assay conditions: acceptor
(2 mM) and donor (4 mM) were incubated for 24 h at 30 °C in HEPES/KOH (10 mM, pH 7.0) buffer
supplemented with MgCl2 (10 mM), MnCl2 (10 mM), KCl (25 mM), glycerol (10%) in the presence of E.
gracilis microsomal membrane (150 ꢀL, 195 µg of total proteins) in total reaction volume of 200 ꢀL. TLC
plates were eluted with CHCl3:MeOH:H2O (10:8:2) and visualised using mid-wave length range UV light.
Components of each reaction mixture are shown below the corresponding TLC trace and products are labelled
on the side of the TLC image.
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2.5.2 Characterisation of the less polar fluorescent products 14 and 17 from reactions of α-Man-
HCT (1) and α-Man-1,6-α-Man-HCT (2) with UDP-Glc
In order to establish the nature of newly formed linkages in fluorescent products obtained from
incubations of α-Man-HCT acceptor (1) and α-Man-1,6-α-Man-HCT acceptor (2) with UDP-Glc and
UDP-GlcNAc, these products were purified by reverse phase HPLC and then subjected to LC-MS
analysis. Analysis of LC-MS data for disaccharide 14 and trisaccharide 17 confirmed the addition of
a single glucose residue to α-Man-HCT (1) and to α-Man-1,6-α-Man-HCT (2) (Fig. 7). The major
peaks observed in the MS spectrum of 14 was m/z of 626.12 while for 17 an m/z of 788.23 was
observed, corresponding to [M+H]+ signals of disaccharide and trisaccharide, respectively (Fig. 7B).
The tandem mass-spectrometry (MS2) fragmentation data for both compounds showed a sequential
loss of hexose units (m/z 162) and ultimate release of fluorescent HCT aglycone (11) (m/z 302.00)
(Fig. 7C). Since the glucosylated adducts were the minor products obtained for both acceptors,
specific details of the newly formed linkages were not pursued further.
Fig. 7. MS and MS2 analyses of products 14 (A, B) and 17 (C, D) obtained from enzymatic biotransformation
with UDP-Glc in the presence of Euglena gracilis microsomal membranes.
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2.5.3 Characterisation of the more polar fluorescent products 15 and 18 from reactions of α-Man-
HCT (1) and α-Man-1,6-α-Man-HCT (2) with UDP-Glc
As described in section 2.5.1, TLC mobilities of glucosylated 15 and 18 gave Rf values that suggested
the presence of more than one added sugar residue on acceptors 1 and 2. In contrast, LC-MS data
analyses revealed additions of no more than one sugar residue in each case, with a slightly higher
(+80) m/z than that calculated for the straightforward addition of a single glucose residue (Fig. 8). On
closer inspection of the MS and MS2 data, it became evident the masses observed for products 15 and
18 were consistent with the transfer of a glucose phosphate residue to the acceptor, which
immediately accounted for the unexpected polarity of these compounds. While it is formally possible
that a glucose residue and a phosphate residue may have been separately transferred to the acceptor,
the en bloc transfer of a sugar phosphate seemed more plausible on biosynthetic grounds. However, to
the best of our knowledge, no enzyme is known that transfers Glc-1-P en-bloc onto an acceptor
glycan; on the other hand, the corresponding transfer of GlcNAc-1-P is central to the generation of
mannose-6-phosphate in lysosomal targeting [25], which informed our thinking about the likely
structure of the polar products derived from α-Man-HCT (1) and α-Man-1,6-α-Man-HCT (2) with
UDP-GlcNAc.
Fig. 8. MS and MS2 analyses of products 15 (A, B) and 18 (C, D) obtained from enzymatic biotransformation
with UDP-Glc in the presence of Euglena gracilis microsomal membranes.
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2.5.4 Identification of fluorescent polar products 16 and 19 obtained from incubation of α-Man-
HCT (1) and α-Man-1,6-α-Man-HCT (2) with UDP-GlcNAc
Purified fluorescent compounds 16 and 19, obtained from incubation of compounds 1 and 2 with
UDP-GlcNAc in the presence of E. gracilis microsomal membranes, were subjected to LC-MS
analysis. These analyses showed similar overall outcomes to those described above for fluorescent 15
and 18, derived from UDP-Glc (Fig. 9). Briefly, MS revealed m/z signals of 746.97 for 16 and m/z of
909.16 for 19, which corresponded to [M+H]+ molecular ions of phosphodiester-linked disaccharide
and trisaccharide, respectively (Fig. 9B1 and 10B2). The MS2 fragmentation data for both compounds
showed loss of GlcNAc, leaving phosphorylated versions of α-Man-HCT (1) and α-Man-1,6-α-Man-
HCT (2).
Fig. 9. LC-MS analyses of fluorescent products 16 (A, B) and 19 (C, D) obtained from enzymatic
biotransformationwith UDP-GlcNAc in the presence of Euglena gracilis microsomal membranes.
Ahead of scale up (vide infra), and in order to gain further evidence for phosphodiester formation, a
series of sequential digestions with alkaline phosphatase and TFA were conducted (see Fig. S17-S19
in SI). These experiments showed the lack of sensitivity of 15/18 (generated with UDP-Glc) and
16/19 (generated with UDP-GlcNAc) to alkaline phosphatase, unless the compounds were pre-treated
with TFA. This is in keeping with the proposed inter-sugar phosphodiester linkages.
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2.5.5 Confirmation of fluorescent product 19 structure by NMR spectroscopy
In order to substantiate the phosphodiester structures proposed above, an enzymatic reaction involving
UDP-GlcNAc and acceptor 2 in the presence of E. gracilis microsomal membranes was scaled up.
Under standard conditions (24 h at 30 °C, section 4.7) enzymatic conversion of acceptor 2 into
product 19 was estimated as 10%. On addition of an extra portion of E. gracilis microsomal
membranes after an initial 24 h at 30 °C and further incubation for 24 h, the isolated yield of 19 was
increased to 43%, amounting to 0.48 mg of material, which was subjected to detailed NMR analysis.
1
The H NMR spectrum of phosphodiester-linked 19 displayed all four coumarin signals in the
aromatic region and a singlet at δ 8.26 ppm for the 1,4-triazole ring proton. The signal at δ 5.48 ppm
was assigned to the anomeric proton of the GlcNAc residue based on 2D NMR analyses, which
1
showed a H-13C cross-peak in HSQC spectrum at δC 94.3 ppm (Fig. 10). The other two anomeric
1
signals of the mannose residues, hidden in H NMR spectra under the solvent peak, were identified
through the 1H‒13C HSQC correlation with H-1/C-1 at 5.42/93 ppm and 4.62/100 ppm (Fig. 10). The
signal of the anomeric proton of the GlcNAc residue in 19 appears as a characteristic doublet of
doublets as a result of both 3J coupling between H-1 and H-2 (J1,2 = 3.6 Hz) and 1J coupling between
H-1 and 31P (J1,P = 7.2 Hz). The small values for both coupling constants are as expected for GlcNAc
residue with α-configuration [26]. Only one signal can be found in 31P NMR spectrum of compound
19 with a chemical shift (–1.26 ppm) characteristic for phosphodiester linkages [27], 2D analysis of
1H-31P HMBC spectrum of 19 revealed cross-peaks at δ 5.48 ppm, which corresponds to H-1 of
GlcNAc, at 3.95 ppm, which corresponds to H-2 of GlcNAc, and at 4.14 ppm, which corresponds to
H-6 of Man' residue, therefore defining that the precise position of the phosphodiester linkage is as
shown in Fig. 11. High resolution MS (HRMS) confirmed the molecular formula of 19 as
C35H50N4O22P (SI, Section 8). These NMR and MS data confirmed that the sugar-phosphate
transferase enzyme present in E. gracilis microsomal membranes catalyses transfer of a GlcNAc-P
sugar residue from the UDP-GlcNAc to the 6’-hydroxyl of the mannose sugar residue of acceptor
substrate 2 to form 19, which possesses a phosphodiester-linkage (Fig. 11).
With both fluorescent monosaccharide 1 and disaccharide 2 acceptor substrates it was possible to
detect N-acetylglucosamine-1-phosphate transferase activity. Transcriptom analysis [7] for the
predicted E. gracilis GlcNAc-P-Tase using the amino acid sequence of the Homo sapiens gene
yielded a hit that is predicted to be a transmembrane protein consisting of 1140 amino acids (124
kDa). This putative enzyme shares 26% identity with the catalytic domain of the Homo sapiens
enzyme and 26% identity with the Dictyostelium discoideum enzyme.
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Fig. 10. HSQC NMR (25 °C, D2O) of compound 19.
Fig. 11. Structure of fluorescent phosphodiester-linked compound 19 as determined by NMR and HRMS.
Double arrows indicate 1H-31P couplings observed in the 1H–31P HMBC spectrum of 19.
3
Conclusions
In this study we set out to develop convenient assays for the analysis of carbohydrate-active enzymes
that rely upon fluorescent acceptor substrates, which may be used in place of established
radiochemical approaches that generally rely on isotope-labelled donor substrates. Our goal was
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initially to benchmark the fluorescent approach against our earlier radiochemical work with
glycosyltransferase activities found in mycobacterial and trypanosomal membranes.
The installation of a fluorophore on acetylenic alkyl mannosides was easily achieved through click
chemistry with a readily accessible azidocoumarin precursor, giving adducts that were straightforward
to analyse by TLC, by reverse phase and by normal phase HPLC, providing a convenient interface to
inline mass spectrometry analyses. The resulting fluorescent α-mannoside (1) and α-1,6-linked
dimannoside (2) proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial
membranes and α- and β-GalT activities in trypanosomal membranes, in keeping with data reported
previously from our radiochemical work [17-18].
Similar experiments with Euglena membranes detected the presence of α-1,3- and/or α-1,2-ManT
activities when α-mannoside (1) and α-1,6-linked dimannoside (2) were used as acceptor substrates.
Interestingly, monosaccharide (1) proved to be a more efficient acceptor than disaccharide (2),
although the latter was less promiscuous, accepting α-1,3-linkages only. Wider-ranging assays with
the same fluorescent acceptors demonstrated no turnover in the presence of GDP-L-Fuc, while UDP-
GlcNAc gave rise to a single very polar product and UDP-Glc generated both an apparent
glycosylation product as well as a much more polar compound. Detailed LC-MS analyses confirmed
the conventional glucosylation of acceptors 1 and 2 as a minor reaction, in addition to the production
of much more polar material that from MS analysis proved to be an unexpected glucosyl phosphate
diester of the acceptors, namely compounds 15 and 18. Where UDP-GlcNAc was employed as the
donor, only phosphodiester products were formed. In light of the efficiency of this latter reaction, the
product from disaccharide 2, compound 19, was isolated and further characterised by NMR
spectroscopy, confirming it to be the α-GlcNAc-1,6-phospho-α-Man-1,6-α-Man product.
Bioinformatic analysis confirmed the presence of a putative GlcNAc phosphotransferase in Euglena,
albeit one with low sequence identity to the established human and slime mould enzymes.
In summary, these studies illustrate the utility of fluorescent acceptors based on triazolyl-coumarin for
the TLC, HPLC and mass spectrometry analysis of glycosyltransferase activities in membrane
preparations from a number of species. They also confirmed the presence of ManTs and GlcNAc
phosphotransferase in Euglena membranes, along with the unexpected presence of a similar activity
that transfers glucose-1-phosphate en bloc to α-mannoside acceptors. These readily accessible
fluorescent acceptors open the way for wider analysis of the under-explored repertoire of
carbohydrate-active enzymes in bacterial and protozoal systems.
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4
Experimental
4.1 General methods
Chemicals were purchased as reagent grade and used without further purification. All moisture-
sensitive reactions were performed under a dry nitrogen atmosphere using oven-dried glassware.
Anhydrous solvents were purchased from Sigma Aldrich and CH2Cl2 was freshly distilled from
calcium hydride prior to use. Reactions were monitored by thin-layer chromatography (TLC)
aluminium plates (Silica Gel 60 F254, E. Merck) with indicated eluents. Compounds were visualised
under UV light (λ 254 nm) and by dipping in ethanol-sulphuric acid (95:5, v/v) followed by heating.
Fluorescent products were purified on semi-preparative TLC on pre-coated silica gel aluminium
plates (Silica Gel 1000 UV254, Analtech). Flash chromatography was performed on a Biotage Isolera
MPLC system using pre-packed silica gel cartridges. Nuclear magnetic resonance spectra were
recorded on a Bruker Avance III 400 NMR or Bruker Avance 800 spectrometer at 298 K. Chemical
shifts (δ) are reported in parts per million (ppm) with respect to internal tetramethylsilane (TMS) in
CDCl3 and residual HOD signal in D2O. NMR signal assignments were made with the aid of COSY
and HSQC experiments. The mode of atom numbering of HCT aglycone used for NMR assignments
is as shown for compound 11 below.
Optical rotations were measured at 20 °C in 1 mL cell in the stated solvent using a Perkin-Elmer 341
polarimeter equipped with a sodium lamp. For HRMS, the samples were diluted into 50%
methanol/0.1% formic acid and infused into a Synapt G2-Si mass spectrometer (Waters, Manchester,
UK) at 5-10 µl min-1 using a Harvard Apparatus syringe pump. The mass spectrometer was
controlled by Masslynx 4.1 software (Waters). It was operated in resolution and positive ion mode
and calibrated using sodium formate. The sample was analysed for 2 min with 1 s MS scan time over
the range of 50-1200 m/z with 3.5 kV capillary voltage, 40 V cone voltage, 120°C cone temperature.
Leu-enkephalin peptide (1 ng µl-1, Waters) was infused at 10 µl min-1 as a lock mass (m/z 556.2766)
and measured every 10 s. Spectra were generated in Masslynx 4.1 by combining a number of scans,
and peaks were centred using automatic peak detection with lock mass correction. For elemental
composition prediction the spectrum elemental composition tool in the Masslynx 4.1 software was
used.
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Euglena gracilis var saccharophila Klebs (strain 1224/7) was supplied by culture collection of alga
and protozoa (CCAP). The original EG:JM medium was prepared according to recipes from CCAP
(www.ccap.ac.uk). HPLC was carried out using a Dionex HPLC system. Total protein concentration
was determined using commercially available BradfordUltra, that was purchased from Expedion [28],
calibrated against a BSA standard curve. cOmplete™, Mini EDTA-free Protease Inhibitor cocktail
was purchased from Sigma Aldrich. Jack bean α-mannosidase were obtained from Sigma Aldrich,
Xanthomonas manihotis α-1,6-mannosidase from New England Biolabs and Aspergillus saitoi α-1,2-
mannosidase from Prozyme. Purification of fluorescent products was carried out using a Dionex
HPLC system with semi-prep normal phase HPLC Phenomenex Luna NH2 (250×10 mm) and or C18
reverse phase column (Phenomenex Luna C18(2) 100 Å, 250×10 mm) and visualised using UV
detector at 347 nm. LC-MS was carried out using a Thermo Finnigan Surveyor HPLC system, with
either reverse phase (C18) or normal phase (NH2) columns, equipped with a UV detector or an LCQ
Deca XP plus (ion trap) MS detector (Thermo Fisher Scientific Inc., Hemel Hempstead, UK) . The
following HPLC conditions were used for characterisation of compounds 14-19: Kinetex C18 column
(50 mm×2.1 mm, 2.6 ꢀm), 0.1% aq. TFA-CH3CN (10 to 90 % over 18 min), flow rate 0.3 mL/min),
selected ion monitoring mode with UV detection at 347 nm. TLC separations were performed at room
temperature on aluminium-backed silica gel 60 F254 or glass plated silica gel 60Å TLC plates.
Samples from enzymatic transformations were applied onto a TLC plate in 2 ꢀL aliquots and dried
with a hairdryer between applications. The TLC plates were eluted with either CHCl3/MeOH/H2O
(10:6:1) or (10:8:2) solvent mixture, air dried and directly visualised with the gel imager (Synoptics
2.0 MP) and processed with the GENE SYS ver 1.2.5.0 program.
4.2 Chemical synthesis of non-fluorescent and fluorescent α-
4.2.1 Hexyn-5-yl 2,3,4,6-tetra-O-benzoyl-α- -mannopyranoside (5)
To a solution of 2,3,4,6-tetra-O-benzoyl-α- -mannopyranosyl bromide [29] (3) (11.7 g, 17.9 mmol)
D-mannopyranoside derivatives
D
D
and 5-hexyn-1-ol (4) (2.1 g, 21.5 mmol) in anhydrous CH2Cl2 (40 mL) containing 4 Å mol. sieves
cooled at -20 °C, a solution of AgOTf (6.4 g, 25.1 mmol) in anhydrous toluene (160 mL) was added.
The reaction mixture was allowed to warm to room temperature and stirred for 3.5 hours. The
reaction mixture was neutralised with Et3N (2.0 mL), filtered through Celite and concentrated under
reduced pressure. The residue was purified by flash column chromatography (hexane/EtOAc 7:3) to
give protected hexynyl mannoside 5 as a colourless amorphous solid (10.2 g, 85%). Rf 0.6
(hexane/EtOAc, 6:4); [α]D - 55.0 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 8.11-8.05 (4H, m,
Ar), 7.98-7.96 (2H, m, Ar), 7.85-7.83 (2H, m, Ar), 7.61-7.24 (12H, m, Ar), 6.12 (1H, t, J3,4 ≈ J4,5 = 8
Hz, H-4), 5.93 (1H, dd, J2,3 = 3.4 Hz, J3,4 = 8.0 Hz, H-3), 5.71 (1H, dd, J1,2 = 1.6 Hz, J2,3 = 3.4 Hz, H-
2), 5.10 (1H, d, J1,2 = 1.6 Hz, H-1), 4.70 (1H, dd, J5,6a = 2.5 Hz, J6a,6b = 12.1 Hz, H-6a), 4.50 (1H, dd,
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J
5,6b = 4.5 Hz, J6a,6b = 12.1 Hz, H-6b), 4.45-4.41 (1H, m, H-5); 3.90-3.84 (1H, m, OCHaHb), 3.66-3.58
(1H, m, OCHaHb), 2.30-2.26 (2H, m, OCH2CH2CH2CH2CCH), 2.00 (1H, t, J = 2.6 Hz
OCH2CH2CH2CH2CCH), 1.86-1.81 (2H, m, OCH2CH2CH2CH2CCH), 1.72-1.66 (2H, m,
OCH2CH2CH2CH2CCH); 13C NMR (100.6 MHz, CDCl3): δ 166.1-165.4 (C=O), 133.4-128.3
(aromatic C), 98.4 (JC-H = 171.9 Hz, C-1), 84.0, 70.6 (C-2), 70.1 (C-3), 68.9 (C-4), 68.8, 68.1, 67.0
(C-5), 62.9 (C-6), 28.4, 25.0, 18.2; HRMS (ESI) m/z calcd for C40H36O10Na+ (M+Na]+): 699.2201,
found 699.2202.
4.2.2 Hexyn-5-yl α-D-mannopyranoside (6)
A solution of protected hexynyl mannoside 5 (3.10 g, 4.8 mmol) in absolute MeOH (60 mL) was
treated with 1 M NaOMe in MeOH (1.9 mL, 1.9 mmol). The solution was kept at room temperature
for 4 h, neutralised with ion-exchange resin (Amberlite IR-120 H+) and concentrated under reduced
pressure. The resulting residue was re-dissolved in water (50 mL) and the aqueous phase was washed
with dichloromethane (3 x 50 mL). The aqueous extract was evaporated to give unprotected hexynyl
mannoside 6 as a colourless syrup (1.1 g, 92%). Rf 0.2 (CH2Cl2/MeOH, 9:1); [α]D +72.0 (c 1.0,
1
CHCl3); H NMR (400 MHz, D2O): δ 4.87 (1H, d, J1,2 = 1.7 Hz, H-1), 3.94 (1H, dd, J1,2 = 1.7 Hz, J2,3
= 3.4 Hz, H-2), 3.89 (1H, dd, J5,6a = 1.8 Hz, J6a,6b = 12.4 Hz, H-6a), 3.81-3.73 (3H, m, H-3, H-6b,
OCHaHb), 3.68-3.55 (3H, m, H-4, H-5, OCHaHb), 2.28-2.23 (3H, m, OCH2CH2CH2CH2CCH,
OCH2CH2CH2CH2CCH), 1.77-1.68 (2H, m, OCH2CH2CH2CH2CCH), 1.66-1.56 (2H, m,
OCH2CH2CH2CH2CCH); 13C NMR (100.6 MHz, D2O): δ 98.4 (C-1), 84.2, 71.5 (C-5), 69.4 (C-3),
68.8 (C-2), 67.9, 66.1, 65.5 (C-4), 59.7 (C-6), 26.5, 23.3, 16.1; HRMS (ESI) m/z calcd for
C12H20O6Na+ (M+Na]+): 283.1152, found 283.1147.
4.2.3 Hexyn-5-yl 2,3,4-tri-O-benzoyl-6-O-trityl-α-D-mannopyranoside (7)
To a solution of unprotected hexynyl mannoside 6 (1.2 g, 4.4 mmol) in anhydrous pyridine (15 mL)
triphenylmethyl chloride (1.8 g, 6.6 mmol) and 4-dimethylaminopyridine (108 mg, 0.88 mmol) were
added. The reaction mixture was heated at 37 °C for 40 h, diluted with pyridine (45 mL) and cooled
down to 0 °C prior to benzoyl chloride (51.0 mL, 44.2 mmol) addition. The mixture was stirred for 1
h at room temperature and ice-cold water was carefully added. The product was extracted with
CH2Cl2 (3 x 75 mL), the organic extracts were combined and washed with ice-cold 1M HCl (3 x 75
mL), saturated aqueous NaHCO3 solution (4 x 75 mL), dried (MgSO4), filtered and concentrated
under reduced pressure. The obtained residue was purified by flash column chromatography
(hexane/EtOAc 7:3) to give ester protected trityl ether 7 as a colourless amorphous solid (3.3 g, 92%).
1
Rf 0.77 (hexane/EtOAc, 6:4); [α]D -95.0 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 8.16-8.10
(2H, m, Ar), 7.85-7.82 (2H, m, Ar), 7.75-7.73 (2H, m, Ar), 7.50-7.40 (10H, m, Ar), 7.33-7.24 (6H, m,
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Ar), 7.17-7.07 (8H, m, Ar), 6.02 (1H, t, J = 10.2, H-4), 5.78 (1H, dd, J2,3 = 3.4 Hz, J3,4 = 10.2 Hz, H-
3), 5.67 (1H, dd, J1,2 = 1.6 Hz, J2,3 = 3.4 Hz, H-2), 5.12 (1H, d, J1,2 = 1.6 Hz, H-1), 4.20-4.16 (1H, m,
H-5), 3.93-3.87 (1H, m, OCHaHb), 3.65-3.59 (1H, m, OCHaHb), 3.38 (1H, dd, J5,6a = 2.2 Hz, J6a,6b
=
10.5 Hz, H-6a), 3.28 (1H, dd, J5,6b = 4.8 Hz, J6a,6b = 10.5 Hz, H-6b), 2.30-2.26 (2H, m,
OCH2CH2CH2CH2CCH), 1.97 (1H, t, J = 2.6 Hz, OCH2CH2CH2CH2CCH), 1.86-1.81 (2H, m,
OCH2CH2CH2CH2CCH), 1.74-1.66 (2H, m, OCH2CH2CH2CH2CCH); 13C NMR (100.6 MHz,
CDCl3): δ 165.7-165.1 (C=O), 146.8-126.8 (aromatic C), 97.5 (C-1), 84.0, 70.9 (C-2), 70.6 (C-3),
70.9 (C-5), 68.7, 67.7, 67.0 (C-4), 62.3 (C-6), 28.4, 25.1, 18.2; HRMS (ESI) m/z calcd for
C52H46O9Na+ (M+Na]+): 837.3034, found 837.3029.
4.2.4 6-O-Hexyn-5-yl
mannopyranoside (8)
(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-2,3,4-tri-O-benzoyl-α-D-
To a solution of esterified trityl ether 7 (3.7 g, 4.5 mmol) and mannosyl bromide 3 (4.5 g, 6.8 mmol)
in anhydrous CH2Cl2 containing 4 Å mol. sieves, at -20 °C, a solution of AgOTf (1.6 g, 6.4 mmol) in
anhydrous toluene (110 mL) was added. The reaction mixture was allowed to warm to room
temperature and stirred for 18 h, neutralised with Et3N (3.0 mL), filtered through Celite and
concentrated under reduced pressure. The resulting residue was purified by flash column
chromatography (hexane/EtOAc 7:3) to give protected hexynyl disaccharide 8 as a colourless
amorphous solid (2.3 g, 45%). Rf 0.6 (hexane/EtOAc, 1:1); [α]D - 54.0 (c 1.0, CHCl3); 1H NMR (400
MHz, CDCl3): δ 8.17-8.15 (2H, m, Ar), 8.04-7.98 (8H, m, Ar), 7.87-7.84 (4H, m, Ar), 7.58-7.48 (6H,
m, Ar), 7.44-7.34 (10H, m, Ar), 7.30-7.21 (5H, m, Ar), 6.11-6.06 (2H, m, H-4, H-4’), 6.00 (1H, dd,
J2’,3’ = 3.2 Hz, J3’,4’ = 10.1 Hz, H-3’), 5.94 (1H, dd, J2,3 = 3.3 Hz, J3,4=10.1 Hz, H-3), 5.78 (1H, dd,
J1’,2’ = 1.5 Hz, J2’,3’ = 3.2 Hz, H-2’), 5.75 (1H, dd, J1,2 = 1.6 Hz, J2,3 = 3.3 Hz, H-2), 5.15 (1H, d, J1’,2’
=
1.5 Hz, H-1’), 5.12 (1H, d, J1,2 = 1.6 Hz, H-1), 4.51 (1H, dd, J5’,6a’ = 2.4 Hz, J6a’,6b’ = 12.2 Hz, H-6’a),
4.44-4.37 (2H, m, H-5’ H-5), 4.30 (1H, dd, J5’,6a’ = 4.2 Hz, J6a’,6b’ = 12.2 Hz, H-6’b), 4.13 (1H, dd, J5,6a
= 5.4 Hz, J6a,6b = 10.9 Hz, H-6b), 4.00-3.95 (1H, m, OCHaHb), 3.78 (1H, dd, J5a,6b = 2.0 Hz, J6a,6b
=
10.8 Hz, H-6a), 3.70-3.64 (1H, m, OCHaHb), 2.35-2.31 (2H, m, OCH2CH2CH2CH2CCH), 1.98 (1H, t,
J = 2.6 Hz, OCH2CH2CH2CH2CCH), 1.95-1.87 (2H, m, OCH2CH2CH2CH2CCH), 1.80-1.72 (2H, m,
OCH2CH2CH2CH2CCH); 13C NMR (100.6 MHz, CDCl3): δ 166.0-165.1 (C=O), 133.5-132.9
(aromatic C), 130.1-128.3 (aromatic C), 97.8 (C-1), 97.7 (C-1’), 84.1, 70.6 (C-2), 70.3 (C-2’), 70.2
(C-3; 3’), 69.6 (C-5), 68.9 (C-5’), 68.7, 68.1, 67.0 (C-6), 66.7 (C-4), 62.3 (C-6’), 28.5, 25.1, 18.2;
HRMS (ESI) m/z calcd for C67H58O18Na+ (M+Na]+): 1173.3515, found 1173.3517.
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4.2.5 Hexyn-5-yl α-D-mannopyranosyl-(1→6)-α-D-mannopyranoside (9)
A solution of protected hexynyl disaccharide 8 (880 mg, 0.8 mmol) in absolute MeOH (60 mL) was
treated with 1M NaOMe in MeOH (0.5 mL, 0.54 mmol). The solution was kept at room temperature
for 24 h, neutralised with ion-exchange resin (Amberlite IR-120 H+) and concentrated under reduced
pressure. The resulting residue was purified by flash column chromatography (CH3CN/H2O/NH3
6:3:1) to give unprotected hexynyl disaccharide 9 as a colourless amorphous solid (323 mg, 84%). Rf
0.66 (CH3CN/H2O/NH3 6:3:1); [α]D + 80.0 (c 1.0, MeOH); 1H NMR (400 MHz, D2O): δ 4.91 (1H, d,
J1’,2’ = 1.7 Hz, H-1’), 4.87 (1H, d, J1,2 = 1.2 Hz, H-1), 4.00 (1H, dd, J1’,2’ = 1.7 Hz, J2’,3’=3.4 Hz, H-2’),
4.00-3.95 (2H, m, H-2; H-6b’), 3.92 (1H, dd, J5’,6a’ = 1.6 Hz, J6a’,6b’ = 11.8 Hz, H-6a’), 3.85 (1H, dd,
J2,3 = 3.4 Hz, J3,4 = 9.0 Hz, H-3), 3.81-3.65 (8H, m, H-4’, H-4, H-6a, H-6b, OCHaHb, H-3’, H-5’, H-
5), 3.62-3.57 (1H, m, OCHaHb), 2.39 (1H, t, J = 2.6 Hz, OCH2CH2CH2CH2CCH), 2.29-2.25 (2H, m,
OCH2CH2CH2CH2CCH), 1.78-1.71 (2H, m, OCH2CH2CH2CH2CCH), 1.67-1.58 (2H, m,
OCH2CH2CH2CH2CCH); 13C NMR (100.6 MHz, D2O): δ 99.8, 99.4, 85.8, 72.6 (C-5), 70.9 (C-3, 3’),
70.0 (C-2), 69.9 (C-2’), 67.3, 67.3, 66.7 (C-4), 66.6 (C-4’), 65.7 (C-6), 60.9 (C-6’), 27.6, 24.5, 17.2;
HRMS (ESI) m/z calcd for C18H30O11Na+ (M+Na]+): 445.1680, found 445.1677.
4.2.6 4-(1-(7-Hydroxy-coumarin-3-yl)-1H-1,2,3-triazol-4-yl)-)butyl α-D-mannopyranoside (1)
A solution of hexynyl mannoside 6 (17 mg, 0.06 mmol) and 3-azido-7-hydroxy coumarin (10) (16
mg, 0.05 mmol) in MeOH/H2O (1:1) (1 mL) was treated with 1 M aq. CuSO4 (10 µL) and 1 M aq.
NaAsc (25 µL). The reaction mixture was allowed to stir at room temperature for 2 h and
concentrated under reduced pressure. The resulting residue was purified by semi-prep TLC
(CH2Cl2/MeOH/H2O, 80:20:3) to give click adduct 1 as a yellow amorphous solid (25 mg, 84%). Rf
1
0.62 (CH2Cl2/MeOH/H2O, 10:8:2); H NMR (400 MHz, CD3OD) δ 8.23 (1H, s, H-f), 8.17 (1H, s, H-
j), 7.36 (1H, d, Jc,d = 8.8 Hz, H-c), 6.60 (1H, dd, Jc,d = 8.8, Jd,e = 2.2 Hz, H-d), 6.46 (d, Jd,e = 2.2 Hz,
H-e), 4.64 (1H, d J1,2 = 1.5 Hz, H-1), 3.76-3.67 (3H, m, H-2, H-6a, OCHaHb), 3.62-3.59 (2H, m, H-3,
H-6b), 3.51-3.35 (3H, m, H-4, H-5, OCHaHb), 2.72 (2H, t, J = 7.5 Hz, OCH2CH2CH2CH2), 1.78-1.71
(2H, m, OCH2CH2CH2CH2), 1.65-1.57 (2H, m, OCH2CH2CH2CH2); 13C NMR (100.6 MHz, CD3OD):
δ 159.3, 158.0, 138.5 131.3, 124.2, 119.5, 104.7, 101.4 (C-1), 74.7 (C-5), 72.6 (C-3), 72.3 (C-2), 68.7
(C-4), 68.2 (C-6), 30.0, 27.3, 26.0; HRMS (ESI) m/z calcd for C21H25N3O9Na+ ([M+Na]+): 486.1483,
found 486.1476.
4.2.7 4-(1-(7-Hydroxy-coumarin-3-yl)-1H-1,2,3-triazol-4-yl)-)butyl α-
-mannopyranoside (2)
D-mannopyranosyl-(1→6)-α-
D
A solution of hexynyl disaccharide 9 (15 mg, 0.04 mmol) and 3-azido-7-hydroxycoumarin (10) (7.2
mg, 0.04 mmol) in MeOH/H2O (1:1) (1 mL) was treated with 1 M aq. CuSO4 (10 µL) and 1 M aq.
21

ACCEPTED MANUSCRIPT
NaAsc (25 µL). The reaction mixture was allowed to stir at room temperature for 2 h and
concentrated under reduced pressure. The resulting residue was purified by semi-prep TLC
(CH2Cl2/MeOH/H2O, 80:20:3) to give click adduct 2 as a yellow amorphous solid (20 mg, 90%). Rf
0.37 (CH2Cl2/MeOH/H2O, 10:8:2); 1H NMR (400 MHz, CD3OD): δ 8.37 (1H, s, H-f), 8.25 (1H, s, H-
j), 7.52 (1H, d, Jc,d = 8.7 Hz, H-c), 6.78 (1H, dd, Jc,d = 8.7, Jd,e = 2.3 Hz, H-d), 6.71 (1H, d, Jd,e = 2.3
Hz, H-e), 4.72 (1H, d, J1’,2’ = 1.7 Hz, H-1’), 4.63 (1H, d, J1,2 = 1.5 Hz, H-1), 3.81-3.50 (14H, m, H-6a,
H-6b, H-2’, H-2; H-3, H-3’, OCHaHb, H-6’a, H-6’b, H-5’, H-5, H-4’, H-4), 3.41-3.36 (1H, m,
OCHaHb), 2.73 (2H, t, J = 7.5 Hz, OCH2CH2CH2CH2), 1.77-1.70 (2H, m, OCH2CH2CH2CH2), 1.64-
13
1.56 (2H, m, OCH2CH2CH2CH2); C NMR (100.6 MHz, D2O): δ 157.8, 156.8, 138.6, 131.8, 124.3,
115.9, 103.4, 101.6 (C-1), 101.3 (C-1’), 74.8, 74.4, 73.2, 72.8, 72.6, 72.2, 68.7 (H-4, H-4’), 68.6
(OCHaHb), 68.2 (C-6), 67.5, 62.9 (C-6’), 30.0, 27.4, 26.0; HRMS (ESI) m/z calcd for C27H35N3O14Na+
([M+Na]+): 648.2011, found 648.2002.
4.2.8 4-(1-(7-Hydroxy-coumarin-3-yl)-1H-1,2,3-triazol-4-yl)-)butan-1-ol (11)
A solution of 5-hexynol (25 mg, 0.1 mmol) and 3-azido-7-hydroxycoumarin (19 mg, 0.1 mmol) in
MeOH (0.8 mL) was treated with 1 M aq. CuSO4 (10 µL) and 1 M aq. sodium ascorbate (25 µL). The
reaction mixture was allowed to stir at room temperature for 2 h and concentrated under reduced
pressure. The resulting residue was purified by semi-prep TLC (CH2Cl2/MeOH/H2O, 80:20:3) to give
click adduct 2.11 as a yellow amorphous solid (29 mg, 70%). Rf 0.84 (CH2Cl2/MeOH/H2O, 10:8:2);
1H NMR (400 MHz, MeOD): δ 8.37 (1H, s, H-f), 8.24 (1H, s, H-j), 7.54 (1H, d, Jc,d= 8.6 Hz, H-c),
6.80 (1H, dd, Jc,d=8.6, Jd,e=2.3 Hz, H-d), 6.72 (d, Jd,e=2.3 Hz, H-e), 3.51 (2H, t, J=6.5 Hz, CH2-
triazole), 2.72 (2H, t, J=7.5 Hz, HO-CH2), 1.74-1.66 (2H, m, CH2), 1.56-1.49 (2H, m, CH2); 13C NMR
(100.6 MHz, CD3OD): δ 137.1, 131.9, 122.3, 115.6, 103.4, 33.1, 26.9, 26.0; HRMS (ESI) m/z calcd
+
for C15H16N3O4 ([M+H]+): 302.1135 found 302.1139.
4.3 Preparation of Euglena gracilis cells
As described previously [7], an axenic culture of Euglena gracilis var saccharophila Klebs (strain
1224/7a), was grown in the dark at 30 °C, with shaking (200 rpm), in modified Euglena gracilis plus
Jaworski’s medium (1 x EG plus 1 x JM medium) supplemented with glucose (15 g/L) for 7 days
(refer to the Supplementary Information for full details). Dark grown culture after seven days was
harvested by centrifugation (800 g for 5 min), washed twice with deionised water and once in HEPES
buffer (10 mM, pH 7.0 HEPES/KOH, 25 mM KCl). The harvested cells were re-suspended in HEPES
buffer and de-flagellated by the method of cold-shock [30] that required incubation on ice for 2 h and
the collection of de-flagellated cells by centrifugation (800 g for 5 min).
22

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4.4 Isolation of microsomal membranes from Euglena gracilis
The isolation of Euglena microsomal membranes was conducted according to published procedures
[22]. All isolation steps described were carried out at 0 to 4 °C. Dark-grown de-flagellated cells were
re-suspended in Euglena lysis buffer [20 mM, pH 7.0 HEPES/KOH, 50 mM NaCl, protease inhibitors
(1 tablet per 100 mL) and ribonuclease A (1 mg)] and disrupted by three 15 s bursts of ultrasonic
waves over three consecutive cycles. Residual cell debris was removed by centrifugation (300 g for 3
min) and supernatant was centrifuged at 4200 g for 30 min to remove mitochondria. The supernatant
was layered onto a 1.5 M sucrose cushion in 20 mM HEPES buffer (pH 7.0) and centrifuged at 15,000
g for 45 min. The supernatant was removed and layered over a step gradient consisted of 1.3 M, 1.0
M, 0.8 M and 0.5 M sucrose in 20 mM HEPES buffer (pH 7.0). The separation of microsomal
fraction was achieved by centrifugation at 150,000 g for 3 h. Microsomal-enriched fractions were
collected, re-suspended in 20 mM HEPES buffer (pH 7.0) and centrifuged at 150000 g for 2 h. The
pellets were re-homogenised in 20 mM HEPES buffer (pH 7.0) and stored at -80 °C; under these
conditions membrane enzymes remained active for up to a year.
4.5 Fluorescence-based assays to probe glycosyltransferase activities in Euglena gracilis
Assays were initiated by the addition of 150 ꢀL of freshly thawed E. gracilis microsomal membranes
(195 ꢀg protein) to a solution of 4 mM donor (GDP-D-Man, GDP-L-Fuc, UDP-D-Glc, or UDP-D-
GlcNAc) and 2 mM acceptor (1 or 2) in 50 ꢀL reaction buffer (40 mM HEPES/KOH, 100 mM KCl,
40 mM MgCl2, 40 mM MnCl2, 40% glycerol, pH 7.0). After incubation for 24 h at 30 °C reactions
were stopped by addition of CHCl3:MeOH (1:1, v/v), microsomal membranes were centrifuged
(16000 g for 5 min), then repeatedly washed with CHCl3:MeOH:H2O (10:6:1) and centrifuged.
Combined supernatants were dried under a gentle stream of air. The residue was re-dissolved in
deionised water and passed through 0.45 ꢀm PPTE filter, the filtrate was collected and the sample was
freeze-dried. Control assays without donor were performed in parallel in the same conditions.
4.6 Exo-glycosidase digestion, phosphate ester and phosphodiester linkage analysis
Jack bean α-mannosidase and green coffee beans α-galactosidase were obtained from Sigma Aldrich,
β-galactosidase from Calbiochem, Xanthomonas manihotis α-1,6-mannosidase from New England
Biolabs and Aspergillus saitoi α-1,2-mannosidase from Prozyme. Protocols for their use can be found
in the SI, along with details of acid and phosphatase reactions used to assess phosphate esters/diesters.
23

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4.7 Enzymatic synthesis of 4-(1-(7-hydroxy-coumarin-3-yl)-1H-1,2,3-triazol-4-yl)-butyl 6-(2-
acetamido-2-deoxy-α-
D-glucopyranosyl hydrogen phosphate)-α-D-mannopyranosyl-(1→6)-α-
D-mannopyranoside (19)
A large scale reaction was performed to generate sufficient material for structural characterisation by
NMR spectroscopy. In this enzymatic transformation acceptor 19 (0.8 mg, 1.28 ꢀ mol) and donor
(UDP-GlcNAc) (3.2 mg, 4.9 ꢀmol) were dried out and re-dissolved in 150 ꢀL of stock reaction buffer
(40 mM, pH 7.0 HEPES/KOH, 100 mM KCl, 40 mM MgCl2, 40 mM MnCl2, 40% glycerol) followed
by the addition of 450 ꢀL of E. gracilis microsomal membranes (585 ꢀg protein) in a total volume of
600 ꢀL. After incubation for 24 h at 30 °C the reaction was supplemented with another portion of E.
gracilis microsomal membranes (150 ꢀL, 195 ꢀg of membrane proteins) and incubated for a further
24 h at 30 °C. The reaction was stopped by addition of CHCl3/MeOH (1 mL). The denatured E.
gracilis microsomal membranes were removed by several centrifugations (14000 rpm for 5 min) and
washed with CHCl3/MeOH:H2O (10:6:1) (3 x 1 mL). The washings were combined and solvents dried
under gentle stream of air. The residue was re-dissolved in deionised water and passed through a 0.45
ꢀm PPTE filter, the filtrate was collected and the sample was freeze-dried. The residue was then
purified by reverse phase HPLC to give compound 20 (0.48 mg, 43%). 1H NMR (800 MHz, D2O): δ
8.43 (1H, s, H-f), 8.26 (1H, s, H-j), 7.73 (1H, d, Jc,d = 8.6 Hz, H-c), 7.06 (1H, dd, Jc,d = 8.6, Jd,e =2.2
Hz, H-d), 7.01 (bd, H-e), 5.48 (1H, dd J1,2 = 3.6 Hz, J1-P = 7.2 Hz, H-1), 4.13 (2H, m, H-6a’ and H-
6b’), 3.95 (4H, m, H-2’’, H-6a, H-6b, H-2’), 3.83-3.54 (m, sugar signals, OCHaHb, OCHaHb), 2.86
(2H, t, JH-H = 7.2 Hz, OCH2CH2CH2CH2), 2.07 (3H, s, CH3), 1.87-1.78 (2H, m, OCH2CH2CH2CH2),
31
1.72-1.70 (2H, m, OCH2CH2CH2CH2); P NMR (100.6 MHz, D2O): δ 1.23; HRMS (ESI) m/z calcd
for C35H50N4O22P+ ([M+H]+): calcd 909.2649, found, 909.2648.
Acknowledgements
These studies were supported by the UK BBSRC Institute Strategic Programme on Understanding and
Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation. We thank Martin
Rejzek for help with HPLC analyses and Sakonwan Kuhaudomlarp for assistance with protein
sequence analysis.
5
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Fluorescent mannosides serve as acceptor substrates for glycosyltransferase
and sugar-1-phosphate transferase activities in Euglena gracilis membranes
Irina M. Ivanova,a Sergey A. Nepogodiev,a Gerhard Saalbach,a Ellis C. O’Neill,a,b Michael D.
Urbaniak,c,d Michael A. J. Ferguson,d Sudagar S. Gurcha,e Gurdyal S. Besrae and Robert A. Fielda*
Highlights
•
•
•
•
Assays for the analysis of carbohydrate-active enzymes that rely upon fluorescent
acceptor substrates are set out. 113/125
New assays are validated by benchmarking against radiochemical work with known
glycosyltransferase activities. 110/153
The installation of a fluorophore on acceptor substrates was easily achieved through
click chemistry. 101
Fluorescence assays are used to discover GTs activities in Euglina gracilis microsomal
membranes. 97

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Suggested manuscript reviewers
Professor Laurent Legentil
Ecole Nationale Supérieure de Chimie de Rennes
CNRS, UMR 6226, Université europeenne de Bretagne
France
Laurent.legentil@ensc-rennes.fr
Professor David Jakeman
College of Pharmacy
Dalhousie University
Halifax, NS
B3H 4R2
Canada
david.jakeman@dal.ca
Professor Antony Fairbanks
Department of Chemistry
University of Canterbury
Private Bag 4800, Christchurch
New Zealand
antony.fairbanks@canterbury.ac.nz