Formation of carbohydrate-functionalised polystyrene and glass slides and their analysis by MALDI-TOF MS

Article abstract of DOI:10.3762/bjoc.8.86

Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry

Full text of DOI:10.3762/bjoc.8.86

Formation of carbohydrate-functionalised
polystyrene and glass slides and their
analysis by MALDI-TOF MS
Martin J. Weissenborn1, Johannes W. Wehner2, Christopher J. Gray1,
Robert Šardzík1, Claire E. Eyers*1, Thisbe K. Lindhorst*2
and Sabine L. Flitsch*1
Full Research Paper
Open Access
Address:
School of Chemistry & Manchester Interdisciplinary Biocentre, The
Beilstein J. Org. Chem. 2012, 8, 753–762.
1
doi:10.3762/bjoc.8.86
University of Manchester, 131 Princess Street, Manchester, M1 7DN,
United Kingdom and 2Otto Diels Institute of Organic Chemistry,
Christiana Albertina University of Kiel, Otto-Hahn-Platz 3/4, 24098
Kiel, Germany
Received: 10 February 2012
Accepted: 02 May 2012
Published: 21 May 2012
Email:
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
Claire E. Eyers* - Claire.eyers@manchester.ac.uk;
Thisbe K. Lindhorst* - tklind@oc.uni-kiel.de;
Sabine L. Flitsch* - sabine.flitsch@manchester.ac.uk
Associate Editor: A. Kirschning
*
Corresponding author
© 2012 Weissenborn et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
carbohydrate array; conductive tape; MALDI-TOF MS; nonconductive
surface; trityl-mediated adhesion
Abstract
Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array
format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrome-
try analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an
internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic,
noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such
ligands.
Introduction
Microarrays have become valuable tools in the high-throughput environments [1]. The initial success with DNA microarrays
analysis of biological interactions and have promising applica- has prompted investigations into other biomolecular ligands,
tions for the development of diagnostic devices in clinical such as protein, peptide and carbohydrate arrays [2,3]. An
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Beilstein J. Org. Chem. 2012, 8, 753–762.
important aspect of this field is the immobilisation of such desorption/ionisation time-of-flight (MALDI-TOF) mass spec-
ligands on solid array surfaces, which can be polymers, such as trometry (MS) analysis (Figure 1B), which has been highly
polystyrene, or glass or gold, i.a. [4-7]. The challenge for successful on ligands immobilised on gold plates [10]. MALDI-
immobilisations is in the efficiency of coupling and analysis of TOF MS requires an electrically conducting surface and a
the attached ligands to ensure quality control.
matrix for analysis. The matrix is typically cocrystallised with
the sample, which can lead to irregular surfaces, which can be a
Noncovalent attachment of biomolecules to hydrophobic problem for reproducible analysis, especially when used in
surfaces has been used for a long time in ELISA assays and is array format as a high-throughput tool. To avoid the use of such
attractive because no coupling reagents are required. However, a matrix, we were interested in investigating trityl functionalisa-
it requires inherent hydrophobicity in the biomolecule or attach- tion, which has the potential for self-formation of a matrix (self-
ment of a hydrophobic tether. The latter has been used highly matrix) for MS analysis, at the same time as acting as a hydro-
successfully by the Feizi group as part of the neoglycolipid phobic tether [11-13]. However, polystyrene has only minimal
array technology [8]. More recently, two groups [7,9] have innate electrical conductivity and to our knowledge has never
reported the application of hydrophobic tethers for binding to been used successfully, unmodified, as a target for MALDI-
polystyrene slides for glycan analysis. Initially, simple alkyl TOF MS analysis. Based on previous work one predicts that
chains were used as tethers [9] but more recently, Wong and photoelectrons generated by UV laser irradiation are not dissip-
co-workers improved on this technology by using trityl-derived ated by the polymeric surface. These photoelectrons distort the
glycans, which are easily attached to glycans and were reported local electric field causing a significant loss in resolution of the
to bind strongly to polystyrene (Figure 1A) [7].
analyte ions and a nonlinear shift in the mass-to-charge ratio
14].
[
The attachment of glycans to the surface was generally
confirmed indirectly by lectin binding, which severely limits the Previous attempts to get around this issue have involved coating
ligands that can be interrogated to those that can be detected by polymer or glass surfaces with a thin membrane of conductive
carbohydrate-binding proteins.
material, such as gold, carbon or indium-tin oxide [15-17], or
the addition of electron-accepting additives, such as methyl
To overcome this limitation, we were interested in developing viologen dichloride hydrate [14]. The addition of electron-
label-free methods for ligand detection on these polystyrene accepting additives, however, did not completely suppress the
surfaces, and have investigated the use of matrix-assisted laser mass shifts observed during MALDI-TOF MS on low-conduc-
Figure 1: Carbohydrate arrays on polystyrene slides can be obtained by noncovalent immobilisation of tritylated saccharide derivatives. (A) Lectin-
mediated analysis of carbohydrate arrays [7]. (B) The new concept of label-free MALDI-TOF MS analysis by aluminium-backing of polystyrene or
glass slides.
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Beilstein J. Org. Chem. 2012, 8, 753–762.
tivity supports. Additionally, glass slides coated with conduc- acid 3 was coupled with either of the aminoethyl glycosides 4
tive material are expensive and limited in their utility [14].
[24-26] or the GlcNAc derivative 6 [27], which were prepared
according to literature procedures. For the coupling a combina-
In order to address these issues, we have investigated a simple tion of HBTU/DIPEA or HATU/DIPEA in dry DMF was
and cheap method to enable MALDI-TOF MS analysis on applied yielding 74% of 5 and 71% of 7.
minimally conductive supports by applying a commercially
available aluminium tape to the underside of glass and poly- The analysis of molecules on materials such as polystyrene by
styrene slides. This has allowed us to make standard micro- MALDI-TOF MS is difficult and irreproducible, largely due to
scope slides suitable for MALDI-TOF MS analysis.
their minimal electrical conductivity. To our knowledge,
successful MS analysis on such surfaces has not been reported.
In order to circumvent this issue, commercially available
Results and Discussion
The hydrophobic trityl tethers chosen for our studies consisted aluminium tape was applied to the underside of a polystyrene
of S-tritylated instead of N-tritylated groups, which were used microscope slide.
previously [7]. This followed the idea of “orthogonal” surface
functionalisation. Thus, a thiol group would make the tethers The tape significantly enhanced both the signal intensity and
generally compatible with other platforms in our laboratories, resolution of MALDI-TOF MS analysis of the Man–Trt com-
such as through the formation of SAMs on gold [18] or by pound 5 (Figure 2). Furthermore, we observed that analysis of
coupling into maleimide-functionalised surfaces in a chemose- the Man–Trt compound 5 could not only be performed in the
lective fashion [19].
absence of any additional matrix [11,12], but under these condi-
tions a modest increase in mass-spectrometric resolution was
For the initial studies two carbohydrate derivatives, 5 and 7, also observed. Such self-matrix properties are very convenient,
were synthesised. The α-D-mannoside 5 would be useful in a yielding more robust and reproducible analyses, and negating
bacterial adhesion inhibition assay against the bacterial lectin the need to search for “sweet spots”, as no crystal formation is
FimH [20,21]. The second glycoside 7 has been used previ- required, in contrast to conventional MALDI-TOF MS analysis.
ously for well-established enzymatic surface modifications [22].
Both these compounds can be synthesised by starting from Interestingly, both Na and K cation adducts of Man–Trt 5 and
commercially available 11-mercaptoundecanoic acid (1), which its disulfide 8 were observed (Scheme 2). The relative ratios of
is tritylated with 2 in a straightforward synthesis, in 98% yield, the monomer 5 and the disulfide 8 were found to be concentra-
following the procedure of Kovács et al. [23] (Scheme 1). The tion-dependent in the analysis on stainless steel (Supporting
Scheme 1: Synthesis of the tritylated compounds 3 [28], 5 and 7: (a) dichloromethane, 2.5 h, rt, 98%, (b) HBTU/DIPEA, dry DMF, overnight, rt, 74%,
(c) HATU/DIPEA, dry DMF, 3 h, rt, 71%.
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Beilstein J. Org. Chem. 2012, 8, 753–762.
Figure 2: Comparison of the polystyrene and glass surfaces with and without aluminium backing by matrix-free MALDI-TOF MS analysis. Each spot
contains 15 nmol Man–Trt (5): (A) polystyrene without aluminium; (B) glass without aluminium; (C) polystyrene with aluminium and (D) glass with
aluminium. The peaks at m/z of 688.3, 704.3, 867.5 and 883.4 correspond to [5 + Na]+, [5 + K]+, [8 + Na]+, and [8 + K]+, respectively.
Scheme 2: The Man–Trt compound 5 forms the disulfide 8 during UV ionisation in MALDI-TOF MS analysis.
756

Beilstein J. Org. Chem. 2012, 8, 753–762.
Information File 1). It was observed that at 15 nmol, almost observed with the polystyrene slides, the resolution and signal
exclusively the disulfide 8 was detected. Conversely at 50 pmol intensity of MALDI-TOF MS analysis (Figure 2) was dramati-
only K+ and Na+ adducts of Man–Trt 5 were found.
cally improved following application of the aluminium tape to
the back of the glass slides.
We were interested to see whether the addition of the
aluminium tape would also enhance signals on surfaces other Next, the limit of detection of MS analysis of aluminium-
than polystyrene, and we therefore assessed the influence of the backed polystyrene and glass slides was compared (Figure 3),
conductive tape on analysis using glass slides, which are widely using dilutions of the analyte Man–Trt 5 from 15 nmol to
used in protein-, peptide- and glycoarrays [2,29,30]. As 50 pmol. The resolution between the two supports was compar-
Figure 3: Limit-of-detection analysis of 5 on aluminium-backed glass and polystyrene slides. Both systems showed the MALDI-TOF MS detection
limit at 0.5 nmol. The peaks at m/z of 688.3, 704.3, 867.4 and 883.4 correspond to [5 + Na]+, [5 + K]+, [8 + Na]+, and [8 + K]+, respectively.
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Beilstein J. Org. Chem. 2012, 8, 753–762.
able and analysis showed that Man–Trt 5 could still be detected We were intrigued by the role of the aluminium backing and
at a concentration of 0.5 nmol for aluminium-backed poly- decided to investigate it in more detail. The aluminium backing
styrene and glass slides.
was applied in three different ways: First, the entire underside
of the slide was covered as in the previous experiments. Second,
After application of trityl samples, the polystyrene and glass only a narrow strip of tape was applied to the back of the slide,
slides were gently washed with 1 μL of water (washing proce- making sure that the strip was in contact with the frame of the
dure 1). Subsequent analysis by MALDI-TOF MS showed no slide adapter (Figure 5). This configuration should still allow
noticeable change to the prewashed samples, confirming the for efficient dissipation of any produced photoelectrons. Spots
trityl-group-mediated noncovalent adhesion of the ligands to the were analysed directly over and also next to the strip. The
surfaces. On the other hand, much more rigorous washing under results showed similar intensity and resolution as for the fully
running distilled water (washing procedure 2) caused a signifi- aluminium-backed polystyrene slide (Supporting Information
cant reduction in signal on the polystyrene slide (Figure 4). File 1, Figures S2 and S3). Third, only a small aluminium rect-
Analysis of the glass slide, however, showed only a slight angle was attached to the back of the polystyrene slide, this time
decrease in signal intensity even after three rigorous washes. making sure that there was no electrical contact to the slide
Figure 4: Comparison of MALDI-TOF MS spectra on the aluminium-backed polystyrene and glass slides after washing. (A) At 7.5 nmol following
washing procedure 1 and (B) at 15 nmol following washing procedure 2.
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Beilstein J. Org. Chem. 2012, 8, 753–762.
frame (Supporting Information File 1, Figures S4 and S5). In 2-Aminoethyl α-D-mannopyranoside (4) [24-26] and 2-amino-
this last case very poor signals, both in resolution and intensity, ethyl 2-acetamido-2-deoxy-β-D-glucopyranoside (6) [27] were
were observed, which were analogous to the non-aluminium- prepared according to the literature. Reactions were monitored
backed polystyrene slide. Thus, contact of the tape to the slide by thin-layer chromatography using silica gel 60 GF254 on
adapter frame appears to be essential for good signal intensity, aluminium foil (Merck) with detection by UV light and char-
but it is not necessary to cover the slide fully.
ring with sulfuric acid in EtOH (10%). Preparative MPLC was
performed on a Büchi apparatus using a LiChroprep Si 60
(
40–60 mm, Merck) column for normal-phase silica-gel chro-
matography. Analytical HPLC was performed on a Merck
Hitachi LaChrom L-7000 series apparatus with a LiChrospher
1
00 RP-8 (5 μm, Merck) column. 1H and 13C NMR spectra
were recorded on a Bruker DRX-500 spectrometer. NMR
spectra were calibrated with respect to the solvent peak. 2D
NMR techniques (COSY, HSQC, HMBC) were used for full
assignment of the spectra. ESI-MS measurements were
performed on a Mariner ESI-TOF 5280 instrument (Applied
Biosystems). High-resolution mass spectra (HRMS) were
Figure 5: Photo of the aluminium strip on the back of the polystyrene
support.
Given that the MS analysis on the polystyrene was successful, obtained with the Waters Micromass LCT-TOF mass spectro-
we attempted an enzymatic galactosylation of GlcNAc–Trt 7 meter. MALDI-TOF mass spectra were recorded on a Bruker
using bovine β-(1→4)-galactosyltransferase (β-(1→4)-GalT, Biflex-III 19 kV instrument with Cl-CCA (4-chloro-α-cyano-
EC 2.4.1.38) on the polystyrene slides. This enzymatic trans- cinnamic acid) or DHB (2,5-dihydroxybenzoic acid) as matrix.
formation has proven to be a very reproducible and robust reac- Optical rotation was measured on a Perkin-Elmer polarimeter
tion, which appears to proceed to completion on gold arrays 341 (Na-D-line: 589 nm, length of cell 1 dm). IR spectra were
[
31] and is routinely performed in our laboratory. After treat- recorded on a Perkin-Elmer Paragon 1000 FTIR spectrometer.
ment of slides containing GlcNAc–Trt 7 with the enzyme by For sample preparation a Golden Gate diamond ATR unit with
using previously reported procedures [31], followed by washing a sapphire stamp was used.
procedure 1, neither product nor starting material could be
observed by MALDI-TOF MS analysis. In fluorescence- 11-Tritylsulfanylundecanoic acid (3) [28]
assisted studies this problem is mostly overcome by blocking Chlorotriphenylmethane (2, 2.53 g, 9.07 mmol) was dissolved
with nonfluorescent milk proteins or BSA; however, in in dichloromethane. 11-Mercaptoundecanoic acid (1, 2.00 g,
MALDI-TOF MS analysis the blocking proteins would also be 9.15 mmol) dissolved in dichloromethane (60 mL) was added
ionised and consequently quench the signal [32].
dropwise over 1 h. The reaction mixture was stirred for 1.5 h at
ambient temperature until TLC (cyclohexane/ethyl acetate 3:1)
indicated no further conversion. The reaction mixture was
Conclusion
Successful MALDI-TOF MS analysis on minimally conductive washed with H2O (50 mL), the organic layer was dried over
surfaces was achieved by application of aluminium tape. Poly- MgSO4, and the solvent was removed under reduced pressure.
styrene and glass surfaces were spotted with the analyte The crude product was purified by MPLC (100 g silica column,
Man–Trt 5 and were analysed over a range of concentrations A: cyclohexane, B: ethyl acetate, A: 90% → 40%, 120 min)
and after washing. This new technique enables the direct yielding 3 (4.10 g, 8.90 mmol, 98%) as a colourless solid.
analysis of any noncovalent glycoarray on glass and poly-
styrene. So far, our attempts to study enzymatic reactions on the Rf 0.61 (methanol/dichloromethane 3:18); mp 80–82 °C; HPLC
modified polystyrene surface have been unsuccessful and will tR 7.31 min (A = water, B = methanol, A: 20%, 10 min,
require further investigation.
1.2 mL/min); 1H NMR (500 MHz, CD3OD, 300 K) δ 7.38 (mc,
6
2
2
H, Haryl,Trt), 7.25 (mc, 6H, Haryl,Trt), 7.19 (mc, 3H, Haryl,Trt),
.18 (t, 3J = 7.6 Hz, 2H, HO(O)CCH2CH2), 2.11 (t, 3J = 7.4 Hz,
H, CH2STrt), 1.59 (q, 3J = 7.9 Hz, 3J = 7.0 Hz, 2H,
Experimental
General experimental section for the
saccharide synthesis
HO(O)CCH2CH2), 1.37–1.09 (m, 14H, CH2CH2CH2) ppm;
Commercially available starting materials and reagents were 13C NMR (125 MHz, CD3OD, 300 K) δ 179.3 (C(O)OH),
used without further purification. Reactions requiring dry condi- 146.5 (3 Caryl,Trt), 130.8 (6 CHaryl,Trt), 128.8 (6 CHaryl,Trt),
tions were performed under an atmosphere of nitrogen. Anhy- 127.6 (3 CHaryl,Trt), 67.6 (Cquart,Trt), 36.5 (HO(O)CCH2CH2),
drous DMF was purchased.
32.9 (CH2STrt), 30.5, 30.4, 30.4, 30.3, 30.1, 30.0, 29.7 (7
759

Beilstein J. Org. Chem. 2012, 8, 753–762.
CH2CH2CH2), 26.9 (HN(O)CCH2CH2) ppm; MALDI-TOF MS (HN(O)CCH2CH2) ppm; MALDI-TOF MS (DHB) m/z: 688.11
(
(
DHB) m/z: 483.13 [M + Na]+, 499.10 [M + K]+; HRMS–ESI [M + Na]+, 704.08 [M + K]+; HRMS–ESI (m/z): [M + Na]+
m/z): [M + Na]+ calcd for C30H36NNaO2S, 483.2328; found, calcd for C40H54N2NaO7S, 729.3544; found, 729.3506; IR
4
1
6
83.2343; IR (ATR–IR) : 3384, 3189, 3056, 2923, 2850, (ATR–IR) : 3293, 2923, 2852, 1645, 1548, 1488, 1443, 1253,
651, 1594, 1489, 1444, 1419, 1032, 770, 740, 695, 674, 1132, 1057, 1031, 975, 810, 741, 697, 676, 616 cm−1.
21 cm−1.
2-((11-Tritylsulfanylundecanoyl)amino)ethyl 2-acet-
2-((11-Tritylsulfanylundecanoyl)amino)ethyl α-D-
amido-2-deoxy-β-D-glucopyranoside (7)
mannopyranoside (5)
11-Tritylsulfanylundecanoic acid (3, 18.2 mg, 39.5 μmol) and
1
1-Tritylsulfanylundecanoic acid (3, 750 mg, 1.63 mmol) and HATU (30.0 mg, 79.0 μmol) were dried for 1 h under vacuum.
HBTU (743 mg, 1.96 mmol) were dried for 2 h under vacuum, Then, dry DMF (2 mL) and DIPEA (7.00 μL, 40.9 μmol) were
and then dry DMF (5 mL) and DIPEA (400 μL, 2.33 mmol) added, and the mixture was stirred for 20 min under a nitrogen
were added, and the mixture was stirred for 20 min under a atmosphere at ambient temperature. Simultaneously, in a
nitrogen atmosphere at ambient temperature. Simultaneously, in different reaction vessel 2-aminoethyl 2-acetamido-2-deoxy-β-
a different reaction vessel aminoethyl mannoside 4 (438 mg, D-glucopyranoside (6, 11.5 mg, 43.5 μmol) was dried for 1 h
1
.96 mmol) was dried for 2 h under vacuum, and then dissolved under vacuum and dissolved in dry DMF (1 mL), and then
in dry DMF (5 mL), and DIPEA (160 μL, 931 μmol) was DIPEA (7.00 μL, 40.9 μmol) was added. The mixture was
added. The mixture was stirred for 20 min under a nitrogen stirred for 20 min under a nitrogen atmosphere at ambient
atmosphere at ambient temperature. The reaction mixture with temperature. The solution of 6 in dry DMF was added to the
the preactivated 11-tritylsulfanylundecanoic acid (3) was cooled preactivated 11-tritylsulfanylundecanoic acid (3) and it was
to 0 °C, the solution of mannoside 4 was added and the stirred under a nitrogen atmosphere at ambient temperature for
resulting mixture was stirred under a nitrogen atmosphere at 3 h. All volatile compounds were removed under reduced pres-
ambient temperature overnight. All volatile compounds were sure and the crude product was subjected to MPLC (50 g silica
removed under reduced pressure and the crude product was column, A: dichloromethane, B: methanol, A: 99% → 85%,
subjected to MPLC (150 g silica column, A: dichloromethane, 180 min) yielding 7 (19.7 mg, 27.9 μmol, 71%) as a colourless
B: methanol, A: 99% → 90%, 120 min) and another round of lyophylisate.
MPLC (125 g silica column, A: ethyl acetate, B: methanol, A:
9
9% → 90%, 120 min) yielding 5 (808 mg, 1.21 mmol, 74%) as Rf 0.21 (methanol/dichloromethane, 3:18); HPLC tR 5.44 min
a colourless foam. (A = water, B = methanol, A: 20%, 10 min, 1.2 mL/min);
1.6 (c 0.1, methanol); 1H NMR (500 MHz, CD3OD,
Rf 0.16 (methanol/dichloromethane, 1:9); HPLC tR = 5.49 min 300 K) δ 7.39 (mc, 6H, Haryl,Trt), 7.28 (mc, 6H, Haryl,Trt), 7.21
−
(
A = water, B = methanol, A: 20%, 10 min, 1.2 mL/min); (mc, 3H, Haryl,Trt), 4.39 (d, 3J = 8.4 Hz, 1H, H1GlcNAc), 3.88
23.7 (c 0.5, MeOH); 1H NMR (500 MHz, CD3OD, (dd, 2J = 11.8 Hz, 3J = 2.2 Hz, 1H, H6aGlcNAc), 3.82 (ddd, 2J =
00 K) δ 7.38 (mc, 6H, Haryl,Trt), 7.27 (mc, 6H, Haryl,Trt), 7.20 10.6 Hz, 3J = 6.7 Hz, 3J = 4.5 Hz, 1H, OCHHCH2NH), 3.67
+
3
(
dt, 3J = 7.3 Hz, 4J = 1.3 Hz, 3H, Haryl,Trt), 4.76 (d, 3J = 1.7 Hz, (dd, 2J = 11.8 Hz, 3J = 5.8 Hz, 1H, H6bGlcNAc), 3.67–3.59 (m,
1
3
3
1
H, H1Man), 3.83 (dd, 2J = 11.6 Hz, 3J = 2.3 Hz, 1H, H6aMan), 2H, H2GlcNAc, OCHHCH2NH), 3.43 (dd, 3J = 10.4 Hz, 3J =
.80 (dd, 3J = 1.7 Hz, 3J = 3.3 Hz, 1H, H2Man), 3.77–3.67 (m, 8.3 Hz, 1H, H3GlcNAc), 3.40–3.36 (m, 1H, OCH2CHHNH),
H, OCHHCH2NH, H3Man, H6bMan), 3.60 (dd~t, 3J = 9.5 Hz, 3.40–3.26 (m, 3H, OCH2CHHNH, H4GlcNAc, H5GlcNAc), 2.18
H, H4Man), 3.56–3.50 (m, 2H, H5Man, OCHHCH2NH), 3.41 (t, 3J = 7.6 Hz, 2H, HN(O)CCH2CH2), 2.12 (t, 3J = 7.4 Hz, 2H,
(
ddd, 2J = 14.0 Hz, 3J = 6.3 Hz, 3J = 4.6 Hz, 1H, CH2 STrt), 1.98 (s, 3H, NHAc), 1.59 (m, 2H,
OCH2CHHNH), 3.35 (ddd, 2J = 14.0 Hz, 3J = 6.7 Hz, 3J = 4.7 HN(O)CCH2CH2), 1.38–1.10 (m, 14H, 7 CH2CH2CH2) ppm;
Hz, 1H, OCH2CHHNH), 2.19 (t, 3J = 7.6 Hz, 2H, 13C NMR (125 MHz, CD3OD, 300 K) δ 176.4 (HNC(O)CH2),
HN(O)CCH2CH2), 2.12 (t, 3J = 7.4 Hz, 2H, CH2STrt), 1.59 (q, 173.9 (HNC(O)CH3), 146.5 (3 Caryl,Trt), 130.8 (6 CHaryl,Trt),
3
J = 7.6 Hz, 3J = 7.3 Hz, 2H, HN(O)CCH2CH2), 1.38–1.10 (m, 128.8 (6 CHaryl,Trt), 127.7 (3 CHaryl,Trt), 102.9 (C1GlcNAc), 78.0
1
3
4H, 7 CH2CH2CH2) ppm; 13C NMR (125 MHz, CD3OD, (C5GlcNAc), 76.1 (C3GlcNAc), 72.1 (C4GlcNAc), 69.2
00 K) δ 176.5 (C(O)NH), 146.5 (3 Caryl,Trt), 130.8 (6 (OCH2CH2NH), 67.3 (Cquart,Trt), 62.8 (C6GlcNAc), 57.3
CHaryl,Trt), 128.8 (6 CHaryl,Trt), 127.7 (3 CHaryl,Trt), 101.7 (C2GlcNAc), 40.6 (OCH2CH2NH), 37.1 (HN(O)CCH2CH2),
(
(
C1Man), 74.8 (C5Man), 72.6 (C3Man), 72.1 (C2Man), 68.6 32.9 (CH2STrt), 30.5, 30.4, 30.4, 30.3, 30.1, 30.0, 29.7 (7
C4Man), 67.3 (Cquart,Trt), 67.3 (OCH2CH2NH), 62.9 (C6Man), CH2CH2CH2), 27.0 (HN(O)CCH2CH2), 23.0 (HNC(O)CH3)
4
3
0.2 (OCH2CH2NH), 37.1 (HN(O)CCH2CH2), 32.9 (CH2STrt), ppm; MALDI-TOF MS (DHB) m/z: 729.48 [M + Na]+, 745.45
0.5, 30.4, 30.4, 30.3, 30.1, 30.0, 29.7 (7 CH2CH2CH2), 27.0 [M + K]+; HRMS–ESI (m/z): [M + Na]+ calcd for
760

Beilstein J. Org. Chem. 2012, 8, 753–762.
C40H54N2NaO7S, 729.3544; found, 729.350; IR (ATR–IR) : Acknowledgements
3
270, 2924, 2852, 1640, 1549, 1488, 1443, 1373, 1156, 1109, We thank Dr Stephan Mohr and Dr Jeremy Hawkes for
1
080, 1033, 896, 742, 698, 616 cm−1.
providing us with the polystyrene slides; and Dr Niels-Chris-
tian Reichardt, Javier Calvo Martinez, Dr Sonia Serna and Dr
Antonio Sanchez-Ruiz for help with the initial MALDI-TOF
MS analysis. This work was supported by the Royal Society
Array washing
Washing procedure 1
Distilled water (1 μL) was spotted over the dried analyte spot (Wolfson Award to SLF) and the European Commission’s
and was subsequently drawn back up with the pipette. This was Seventh Framework Programme (FP7), which funded the
repeated three times. The slides were then allowed to dry under EuroGlycoArrays ITN (MJW) and GlycoBioM (RS, SLF,
atmospheric conditions.
CEE). JWW was supported by the Evonik Foundation and CJG
by a BBSRC-funded doctoral training award.
Washing procedure 2
The MALDI target slide was washed with cool distilled water References
(
making sure the spot was not directly under the tap) for 6 s at a
1. Cao, B.; Li, R.; Xiong, S.; Yao, F.; Liu, X.; Wang, M.; Feng, L.; Wang, L.
Appl. Environ. Microbiol. 2011, 77, 8219–8225.
doi:10.1128/AEM.05914-11
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