Bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens

Article abstract of DOI:10.1021/bc100288c

Bishydrazides are versatile linkers for attaching glycans to substrates for lectin binding and pathogen detection schemes. The α,ω- bishydrazides of carboxymethylated hexa(ethylene glycol) (4) can be conjugated at one end to unprotected oligosaccharides, then attached onto carrier proteins, tethered onto activated carboxyl-terminated surfaces, or functionalized with a photoactive cross-linking agent for lithographic patterning. Glycoconjugates of bishydrazide 4 can also be converted into dithiocarbamates (DTCs) by treatment with CS₂ under mild conditions, for attachment onto gold substrates. The immobilized glycans serve as recognition elements for cell-surface lectins and enable the detection and capture of bacterial pathogens such as Pseudomonas aeruginosa by their adsorption onto micropatterned substrates. A detection limit of 10³ cfu/mL is demonstrated, using a recently introduced method based on optical pattern recognition.

Full text of DOI:10.1021/bc100288c

Bioconjugate Chem. 2010, 21, 2065–2075
2065
Bishydrazide Glycoconjugates for Lectin Recognition and Capture of
Bacterial Pathogens
Avijit Kumar Adak,† Alexei P. Leonov,† Ning Ding,‡ Jyothi Thundimadathil,§ Sumith Kularatne,† Philip S. Low,†
and Alexander Wei*,†
Department of Chemistry, 560 Oval Drive, Purdue University, West Lafayette, Indiana 47907-2084, United States. Received
June 30, 2010; Revised Manuscript Received September 11, 2010
Bishydrazides are versatile linkers for attaching glycans to substrates for lectin binding and pathogen detection
schemes. The R,ω-bishydrazides of carboxymethylated hexa(ethylene glycol) (4) can be conjugated at one end to
unprotected oligosaccharides, then attached onto carrier proteins, tethered onto activated carboxyl-terminated
surfaces, or functionalized with a photoactive cross-linking agent for lithographic patterning. Glycoconjugates of
bishydrazide 4 can also be converted into dithiocarbamates (DTCs) by treatment with CS2 under mild conditions,
for attachment onto gold substrates. The immobilized glycans serve as recognition elements for cell-surface lectins
and enable the detection and capture of bacterial pathogens such as Pseudomonas aeruginosa by their adsorption
onto micropatterned substrates. A detection limit of 103 cfu/mL is demonstrated, using a recently introduced
method based on optical pattern recognition.
can be retained in its native cyclized form. Traditional methods involve
covalent attachment to the free hemiacetal via reductive amination or
glycation, but while the conditions are mild, they sacrifice the reducing-
end sugar by generating an open-chain structure (25, 26). However,
weakly basic nucleophiles like aminooxy ethers (H2NO-R, MeHNO-
R) and hydrazides (H2NNHCO-R) have been shown to condense with
hemiacetals in aqueous solutions for efficient glycoconjugation (27-39).
Hydrazides have been widely used for bioconjugation onto aldehyde-
or carboxyl-presenting surfaces (25); conversely, hydrazides can also
be presented on substrates for the specific generation of carbohydrate
microarrays.
Here, we investigate R,ω-bishydrazides as bifunctional linkers for
conjugating underivatized glycans onto various substrates and present
several surface functionalization methods with application toward the
binding of lectins and pathogenic bacteria. Bishydrazides can be useful
as chelating agents or as supramolecular recognition motifs (40, 41),
but are most often applied as polymer or biopolymer cross-linking
agents (42-44). At first glance, bishydrazides appear to be an unlikely
choice for heterofunctionalization; however, we find that they can be
selectively monofunctionalized and are versatile linkers for glycocon-
jugate chemistry.
In this work, we use glycan-bishydrazide conjugates to target
specific lectins and adhesins found in Pseudomonas aeruginosa,
an opportunistic, Gram-negative bacteria often found in hospital
settings. Nosocomial Pseudomonas infections have become
increasingly drug-resistant and can be especially pernicious in
immunocompromised patients or those suffering from cystic
fibrosis, so there is a strong need to develop detection platforms
against this pathogen. Epidemiological studies suggest that the
risk of Pseudomonas infections in healthy individuals increases
withenvironmentalpathogencountsabove103 cfu/mL(45,46).We
aim to address this important public health concern by focusing
on glycan recognition as a mechanism for detecting virulent
pathogens such as Pseudomonas at densities as low as 103 cfu/
mL, the estimated threshold for nosocomial infection.
INTRODUCTION
Pathogenic microorganisms are an omnipresent threat to
global biosecurity, and effective biodetection strategies are
necessary to control the spread of infectious diseases. One viable
approach to pathogen detection is to develop diagnostic
technologies based on “immutable” recognition of ligands
presented on cell surfaces, a vital first step in the cycle of
infection. These recognition elements are often low molecular
weight molecules such as glycans, which are exploited by
pathogens for gaining entry into their host cells. Pathogens must
maintain this ligand affinity in order to remain virulent, as those
which undergo mutations that disable recognition can no longer
infect their target cells. Glycan-based strategies have proven to
be particularly successful in detecting bacteria that use lectins
for adhesion to host cell membranes (1-8). Furthermore, the
immutability of this functional recognition provides an important
advantage over antibody-based detection schemes, which are
susceptible to nonfunctional mutations that are often irrelevant
to pathogen virulence.
Glycoconjugates are also of considerable importance for high-
throughput diagnostics or screening of carbohydrate-binding
proteins associated with various biological functions or disease
states (9-12). Several different covalent chemistries have been
successfully implemented for surface functionalization, such as
thiol-ene and thiol-maleimide additions (13-16), Diels-Alder
or Cu-catalyzed Huisgen cycloadditions (17-19), nucleophilic
addition to epoxides and isothiocyanates (20, 21), amide bond
ligation (22, 23), and Staudinger ligation (24). However, many
of these methods require that glycans be in a protected form
prior to linker installation and may involve multiple synthetic
or purification steps.
Chemoselective coupling of bifunctional linkers to underivatized
glycans is an attractive option, particularly if the reducing-end sugar
* To whom correspondence should be addressed. E-mail: alexwei@
purdue.edu; Tel: (765) 494-5257; Fax: (765) 494-0239.
† Purdue University.
EXPERIMENTAL PROCEDURES
‡ Present address: Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, P.R. China.
§ Present address: Peptisyntha, Inc., Torrance, CA 90501.
General Procedures. All starting materials and reagents were
obtained from commercial sources and used as received unless
otherwise noted. Fuc(R1f2)Gal(ꢀ1f4)Glc (2′-fucosyllactose
10.1021/bc100288c  2010 American Chemical Society
Published on Web 10/06/2010

2066 Bioconjugate Chem., Vol. 21, No. 11, 2010
Adak et al.
3) was purchased from V-laboratories, Inc., and GalNAc(ꢀ1f4)-
Gal(ꢀ1f4)Glc (pulmonary trisaccharide 2) was prepared by
multistep synthesis (see below). Bovine serum albumin (BSA),
galactose-binding lectin from Arachis hypogaea (peanut lectin),
antilectin rabbit IgG, and FITC-labeled antirabbit goat IgG were
purchased from Sigma and used as received. Pseudomonas
aeruginosa (PA01 strain) was obtained from ATCC and grown
overnight on agar plates (15 g/L of Bacto Agar, 8 g/mL of Difco
Nutrient Broth) at 37 °C. CS2, CH2Cl2, and CH3CN were freshly
distilled from CaH2 before use; anhydrous and anaerobic MeOH
and THF were passed through activated alumina and dipensed
from a solvent purification system (MBraun). Reactions were
carried out in oven-dried glassware under an inert atmosphere,
cleaned substrates were coated with BSA by soaking in a 1 wt
% solution in PBS for several hours at rt, followed by immersion
in a solution of photoactive glycoconjugate (20 mM in 2:1
MeOH/CH2Cl2) for 5 min. The dried films were covered by a
quartz photomask having linewidths of 10 or 15 µm and grating
periods of 20 µm (Advanced Reproductions Corp.), then
exposed to a short-wave ultraviolet lamp (Spectroline, ENF-
240C) operating at 254 nm for 25 min, followed by a rinse
with MeOH. Microcontact printing (see below) could also be
used for patterning photoactive glycoconjugates onto BSA-
coated glass slides.
Microcontact Printing (µCP). A freshly prepared poly(dim-
ethylsiloxane) (PDMS) stamp (grating period a ) 20 µm; line
width ) 15 µm) was lightly oxidized by a 15 s exposure in a
plasma cleaner, then coated with 30 µL of an aqueous solution
of 6-DTC for 2 min (see above for preparation of stock solution).
The PDMS stamp was dried in air after blotting excess solution,
then pressed lightly onto a roughened Au substrate (see below)
at an applied pressure of 6 kPa for 3 s, as measured by a
mechanical stamper developed in our laboratories. A brass plate
was placed at the top of the stamp for even distribution of
pressure, and the stamp remained in conformal contact with the
substrate for an additional 5 min.
Similar µCP conditions were used to pattern BSA glycocon-
jugates onto smooth Au substrates. A 30 µL aliquot of a 1 wt
% BSA-lactose hydrazide conjugate (6-BSA) in PBS was cast
onto a lightly oxidized PDMS stamp as described above and
allowed to sit for 2 min prior to blotting. The PDMS stamp
was dried in air, pressed lightly onto the substrate at an applied
pressure of 6-12 kPa for 3 s, and then allowed to remain in
conformal contact without added pressure for another 5 min.
The micropatterned substrate was washed sequentially with PBS,
PBS containing 0.05% Tween-20, and then deionized water.
The patterned substrate was further incubated with a 5% BSA
solution in PBS for 2 h, then washed as above prior to use.
Glycan-Directed Affinity Binding and Imaging. Micropat-
terned glycoconjugates were exposed to either soluble lectins
or to live bacterial cultures and visualized by immunofluorescent
staining (bound lectin) or darkfield microscopy (bound bacteria)
using an upright microscope (Olympus BH2-RFL-T3) equipped
with a darkfield condensor, a high-pressure Hg lamp and filter
set for FITC emission, and a DP70 camera for image acquisition.
Fluorescence imaging was performed either on glass or on
roughened Au substrates; the latter were prepared by immersing
clean Au substrates into a solution of AgNO3 (3.7 mM) and
hydroquinone (120 mM) in 1.3 M citric acid buffer (pH 3.5)
for 1.5 min, followed by a quick rinse with deionized water
and immersion in an aqueous HAuCl4 solution (0.5 mM) for
30 min. Roughened Au substrates were dried in air, prior to
use.
1
and solvents were freshly distilled from CaH2 prior to use. H
and 13C NMR spectra were recorded on a Bruker DRX 400
spectrometer operating at 400 and 100 MHz respectively.
Chemical shifts (δ) are reported in ppm and referenced to the
solvent used (CDCl3, δ 7.24 and 77.23; CD3OD, δ 3.31 and
49.15; D2O, δ 4.80), with coupling constants (J) reported in
Hz. Deionized water with a resistivity of >18 MΩ·cm was
obtained from an ultrafiltration system (Milli-Q, Millipore) and
passed through a 0.22 µm filter to remove particulate matter.
Silica gel chromatography was performed using ICN SiliTech
32-63 D; reverse-phase HPLC was performed using a Phe-
nomenex Synergi C-18 column (250 × 21.2 mm, 4 µM, 80 Å)
with UV detection at 214 nm, using an aqueous CH3CN solvent
gradient with a linear flow rate of 10 mL/min.
Neoglycoprotein Formation. In a typical experiment, a
freshly prepared solution of BSA (10 mg) in phosphate buffered
solution (PBS, pH 7.4, 200 µL) was treated with 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC ·HCl,
3 mg in 50 µL PBS) and gently stirred at rt for 1 h. The activated
protein solution was then treated with pulmonary trisaccharide-
hydrazide conjugate 7 (5.1 mg) in 200 µL PBS, then stirred for
another 2 h, followed by membrane dialysis and passage through
a 0.45 µm filter to remove aggregates and particulate matter.
These BSA glycoconjugates could be stored safely at 4 °C for
several months.
In Situ Dithiocarbamate (DTC) and Monolayer Formation
(47, 48). In a typical experiment, a 10 mM solution of
lactose-bishydrazide conjugate 6 in deaerated methanol (1.5
mg in 200 µL) was treated with 10 mM solutions of CS2 (200
µL) and Et3N (200 µL), then stirred at rt for 1 h in a capped
vial prior to use (see below). In situ DTC formation was
monitored by diluting aliquots of the reaction mixture in
deionized water, then measuring increases in characteristic
absorption peak intensities at 250 and 292 nm using a Cary-50
spectrophotometer (cell path length ) 1 cm).
DTC-anchored monolayers (DAMs) were produced from a
freshly prepared solution of 6-DTC, diluted 10-fold with
deionized water to a final concentration of 1 mM. A 30 µL
aliquot of this stock solution was spread across a lightly oxidized
PDMS stamp, then patterned onto Au substrates by microcontact
printing (see below). Lactose-terminated DAMs were treated
with a solution of peanut lectin (50 µg/mL) and visualized by
fluorescence immunostaining. The robustness of the patterned
DAMs was evaluated by measuring changes in relative emission
intensities over time, with exposure to 2-mercaptoethanol (10
or 100 µM) in PBS. Mean luminosity values and standard
deviations were taken from selected regions on the printed
substrate using Photoshop v.7.0.1 (Adobe Systems).
For affinity patterning with proteins, micropatterned substrates
were incubated with a dilute solution of lectin from Arachis
hypogaea (peanut lectin, 50 µg/mL in PBS) for 2 h at 4 °C.
The chip was washed with PBS, then treated with a solution of
antipeanut lectin rabbit IgG (1 µg/mL in PBS) for 2 h at 4 °C,
then washed again and treated with a solution of FITC-labeled
antirabbit goat IgG (1 µg/mL in PBS) for another 2 h at 4 °C.
The chip was washed with PBS, deionized water, and then dried
under a stream of air prior to fluorescence imaging.
For affinity patterning with bacteria, Pseudomonas aeruginosa
(PA01 strain, ATCC) was cultured in NB broth containing
choline (0.2% w/v), the latter being necessary for the ectopic
expression of fucose-binding lectin PA-IIL (49). Micropatterned
substrates were incubated with suspensions of live Pseudomonas
for 1 h at room temperature, at concentrations ranging from
108 to 102 cfu/mL. After incubation, the chip was washed with
PBS and deionized water prior to darkfield imaging. The density
UV Photolithography. Glass substrates coated with a 50 nm
Au layer (1.2 × 1.2 cm2, Reichert) were cleaned with a piranha
solution (2 parts 18 M H2SO4, 1 part 30% H2O2) for 3 min,
then thoroughly rinsed with deionized water and dried under a
stream of argon. (Caution: Piranha solution is a strong oxidizing
agent, and should be handled with extreme care.) Freshly

Bishydrazide Glycoconjugates for Bacterial Detection
Bioconjugate Chem., Vol. 21, No. 11, 2010 2067
of live Pseudomonas was estimated by correlating turbidity
measurements (ca. 109 cfu/mL at O.D. 1) with colony counts
obtained by plating serial dilutions. Fluorescent staining of active
cultures (SYTO 9/propidium iodide (PI) stains, Invitrogen)
confirmed that >90% of the bacteria were alive prior to their
exposure to glycan-patterned substrates.
Darkfield images were subjected to fast Fourier transform
(FFT) for pattern demodulation to reveal characteristic peaks
in reciprocal lattice space (k ) 1/a) indicating pathogen capture,
according to a recently established protocol (50). Standard sizes
and magnification were established for each micropatterned
image, so that the units defining periodicity (in µm) were
correctly translated into reciprocal lattice units (in µm-1) in the
spectra derived from the FFT images. Signal-to-noise (S/N)
levels were determined as the ratio of the peak value at 0.05
µm-1 (after background subtraction) to the standard deviation
of background intensity; signal qualities were considered
acceptable above S/N ) 3.
Glycoconjugate-Lectin Binding Immunoassays. Each in-
cubation was performed for 2 h at rt, followed by rinsing three
times with PBS to remove excess proteins prior to the next step.
96-well microtiter plates were coated with 6-BSA conjugate (10
mg/mL), blocked with a 1% BSA solution to minimize
nonspecific protein adsorption, and then incubated with increas-
ing concentrations of peanut lectin in the presence or absence
of 100-fold excess lactose. Wells were then treated with
antilectin rabbit IgG (1 µg/mL), followed by antirabbit horserad-
ish peroxidase (HRP) IgG (1 µg/mL). The optical absorbances
of the wells were analyzed using a microplate reader (VER-
SAmax, Molecular Devices) following addition of the HRP
substrate (100 µL). Untreated and antilectin rabbit IgG treated
wells (without primary antibody) in PBS served as negative
controls.
For flow cytometry, carboxylic acid functionalized micro-
spheres were conjugated to lactose-bishydrazide 6 via standard
EDC coupling for 16 h at rt, followed by multiple washes with
PBS. The lactose-conjugated microspheres were dispersed in
PBS (1.2 mL) and split into several Eppendorf tubes, which
were incubated with increasing concentrations of peanut lectin
in the presence or absence of 100-fold excess lactose. After
incubating for 2 h at rt, tubes were rinsed and subjected to
sequential incubation with antilectin rabbit IgG and antirabbit
FITC IgG as described above. The microspheres were then
analyzed for fluorescent immunolabeling using a flow cytometer
(Cytomics F500, Beckman Coulter, 104 microspheres/sample).
Untreated and antilectin rabbit IgG (without primary antibody)
treated microspheres in PBS served as negative controls.
of hydrazine using toluene (3 × 10 mL), and finally dried in
vacuo to afford a light yellow oil (0.92 g, 98%). The resulting
bishydrazide could be used to prepare glycoconjugates without
1
further purification. H NMR (400 MHz, CDCl3) δ 8.61 (br s,
2H), 4.06 (s, 4H), 3.74-3.58 (m, 28H). 13C NMR (100 MHz,
CD3OD) δ 173.72, 171.15, 73.66, 72.10, 71.51, 71.44, 71.75,
62.12. MS (ESI) calcd for C16H34N4O9Na [M+Na]+ m/z 449.44;
found: m/z 449.02.
Heptanediol-Linked Bishydrazide (5). Heptane-1,7-diol (1.1 g,
7.56 mmol) was treated with ethyl bromoacetate (4.35 mL, 37.82
mmol) as described above, followed by workup and silica gel
purification (EtOAc/hexanes 1:2) to yield the pure diester as a
colorless oil (0.92 g, 36%). 1H NMR (400 MHz, CDCl3) δ 4.15
(t, 4H, J ) 6.7 Hz), 3.81 (s, 4H), 3.61 (t, 4H, J ) 6.1 Hz), 1.63
(quint, 4H, J ) 6.4 Hz), 1.53 (quint, 4H, J ) 6.7 Hz), 1.34 (3,
6H), 1.26 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 167.52,
66.54, 63.04, 32.77, 29.10, 28.48, 26.13, 25.87, 25.76.
Anhydrous hydrazine (0.185 mL, 5.91 mmol) was treated with
heptanediol-linked diester (0.36 g, 1.18 mmol) in methanol (10
mL), according to the reaction conditions described above. After
azeotropic removal of the volatiles using toluene, bishydrazide
5 was obtained as an oil (0.32 g, 92%) and could be used to
1
prepare glycoconjugates without further purification. H NMR
(400 MHz, CD3OD) δ 3.40 (t, 4H, J ) 6.4 Hz), 1.40 (quint,
4H, J ) 6.6 Hz), 1.25-1.21 (m, 10H). 13C NMR (100 MHz,
CD3OD) δ 173.73, 172.39, 62.94, 33.57, 30.38, 26.90. MS (ESI)
calcd for C11H24N4O4 [M]+ m/z 276.17; found 276.76.
Hexa(ethylene glycol)-Linked Lactose-Bishydrazide Conjugate
(6). A mixture of lactose (10 mg, 0.029 mmol) and bishydrazide
linker 4 (124.6 mg, 0.29 mmol) in NaOAc buffer (1 mL, pH
4.2) was stirred at 70 °C for 2 days. The reaction mixture was
evaporated under reduced pressure, then purified by reverse-
phase HPLC (1-30% aq CH3CN gradient over 60 min) to afford
6 as colorless oil (18.9 mg, 86%). 1H NMR (400 MHz, CD3OD)
δ 4.62 (s, 1H), 4.34 (d, 1H, J ) 7.5 Hz), 4.14 (d, 1H, J ) 8.6
Hz), 4.09-4.03 (m, 3H), 3.98 (dd, 1H, J ) 4.0, 12.0 Hz), 3.92
(dd, 1H, J ) 4.0, 8.0 Hz), 3.84-3.79 (m, 2H), 3.78-3.74 (m,
2H), 3.70-3.65 (m, 22H), 3.59-3.52 (m, 6H), 3.49-3.46 (m,
2H). 13C NMR (100 MHz, CD3OD) δ 172.32, 171.81, 105.10,
91.89, 80.45, 77.76, 77.11, 76.63, 74.81, 73.66, 73.53, 72.51,
72.14, 72.07, 71.54, 71.31, 70.75, 70.29, 62.48, 62.21, 62.12.
MS (ESI) calcd for C28H54N4O19Na [M+Na]+ m/z 773.32;
found: m/z 773.37.
Hexa(ethylene glycol)-Linked Pulmonary Trisaccharide-Bishy-
drazide Conjugate (7). This compound was prepared according
to the procedure above, starting from pulmonary trisaccharide
2 (32 mg, 0.058 mmol) and bishydrazide linker 4 (60.0 mg,
0.14 mmol). Purification by RP-HPLC (1-30% aq CH3CN
gradient over 60 min) yielded glycoconjugate 7 as a light-yellow
oil (32 mg, 58%). 1H NMR (400 MHz, CD3OD) δ 4.61 (d, 1H,
J ) 8.3 Hz), 4.32 (d, 1H, J ) 7.6 Hz), 4.14 (d, 1H, J ) 5.5
Hz), 4.08-3.97 (m, 10H), 3.89-3.47 (m, 36H), 2.05 (s, 3H).
13C NMR (100 MHz, CD3OD) δ 180.31, 175.05, 105.05, 104.44,
93.98, 78.18, 76.80, 76.23, 75.98, 74.69, 74.14, 73.57, 73.07,
72.60, 71.44, 69.53, 62.61, 61.72, 55.05, 24.15, 23.18. MS (ESI)
calcd for C36H67N5O24Na [M+Na]+ m/z 976.40; found: m/z
976.18.
Hexa(ethylene glycol)-Linked Bishydrazide (4). A solution
of hexa(ethylene glycol) (5.0 g, 17.70 mmol) in dry THF (50
mL) at room temperature was treated portionwise with potas-
sium tert-butoxide (9.92 g, 88.54 mmol) at rt, then stirred for
30 min. The reaction mixture was treated dropwise with ethyl
bromoacetate (20 mL, 170 mmol), then stirred at reflux for 20 h.
The reaction mixture was cooled to rt, filtered and concentrated,
and then purified by silica gel chromatography (EtOAc/hexanes
1:1) to yield pure diethyl ester as a colorless oil (5.5 g, 68%).
1H NMR (400 MHz, CDCl3) δ 4.62 (s, 4H), 4.17 (q, 4H, J )
7.2 Hz), 3.71-3.69 (m, 4H), 3.66-3.63 (m, 8H), 3.61-3.59
(m, 12H), 1.23 (t, 6H, J ) 7.2 Hz). 13C NMR (100 MHz, CDCl3)
δ 170.01, 167.44, 71.01, 70.85, 70.63, 68.88, 68.36, 61.60,
60.79, 14.16. MS (ESI) calcd for C20H38O11[M]+ m/z 454.24;
found: m/z 454.98.
Hexa(ethylene glycol)-Linked 2′-Fucosyllactose-Bishydrazide
Conjugate (8). This compound was prepared according to the
procedure above, starting from commercially available 2′-
fucosyllactose (2.5 mg, 5.1 µmol) and bishydrazide linker 4
(21.8 mg, 51 µmol). Purification by RP-HPLC (1-40% aq
CH3CN gradient over 60 min) yielded glycoconjugate 8 as a
colorless oil (2.9 mg, 51%). 1H NMR (300 MHz, D2O) δ 5.17
(d, 1H, J ) 3.1 Hz), 4.38 (d, 1H, J ) 7.7 Hz), 4.10 (m, 1H),
4.02-3.98 (m, 4H), 3.82 (m, 1H), 3.75-3.71 (m, 2H),
3.67-3.43 (m, 41H), 3.29 (m, 1H), 3.23 (m, 1H), 1.09 (d, 1.7H,
The hexa(ethylene glycol)-linked diester (1.0 g, 2.2 mmol)
was dissolved in MeOH (20 mL) passed through an activated
alumina column (MBraun) and treated with anhydrous hydrazine
(0.34 mL, 11.0 mmol). The reaction mixture was stirred at rt
for 24 h, then concentrated followed by the azeotropic removal

2068 Bioconjugate Chem., Vol. 21, No. 11, 2010
Adak et al.
J ) 6.6 Hz), 1.03 (d, 1.3H, J ) 6.6 Hz). MS (ESI) calcd for
C34H64N4O23Na [M+Na]+ m/z 919.38; found: m/z 919.70.
4.91 (d, 2H, J ) 11.2 Hz), 4.87 (d, 1H, J ) 8.6 Hz), 4.82 (d,
2H, J ) 9.3 Hz), 4.78 (d, 2H, J ) 10.6 Hz), 4.75 (d, 1H, J )
11.2 Hz), 4.65 (d, 2H, J ) 11.2 Hz), 4.61 (d, 2H, J ) 8.6 Hz),
4.57 (d, 1H, J ) 4.0 Hz), 4.49 (d, 1H, J ) 7.8 Hz), 4.42 (d,
1H, J ) 10.2 Hz), 4.39 (d, 1H, J ) 6.4 Hz), 4.34 (d, 1H, J )
12.0 Hz), 4.24 (d, 2H, J ) 12.0 Hz), 4.20 (d, 1H, J ) 10.6
Hz), 4.07 (dd, 1H, J ) 5.8, 11.2 Hz), 4.00 (d, 1H, J ) 4.0 Hz),
3.95 (t, 1H, J ) 12.0 Hz), 3.84 (t, 1H, J ) 7.4 Hz), 3.77 (dd,
1H, J ) 4.0, 10.0 Hz), 3.70 (d, 1H, J ) 9.8 Hz), 3.65-3.62
(m, 1H), 3.55 (t, 1H, J ) 8.9 Hz), 3.50 (d, 1H, J ) 7.5 Hz),
3.46 (d, 1H, J ) 8.0 Hz), 3.42 (dd, 1H, J ) 2.6, 8.0 Hz),
3.37-3.32 (m, 2H), 2.19 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H),
1.60 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.72, 170.31,
170.22, 169.91, 138.79, 138.53, 138.40, 138.34, 138.22, 137.44,
137.25, 129.04, 128.94, 128.71, 128.32, 128.19, 127.93, 127.87,
127.73, 127.53, 127.34, 127.07, 103.32, 102.76 (2C), 82.79,
81.95, 80.78, 76.10, 75.58, 75.33, 74.45, 73.51, 73.38, 72.07,
71.19, 70.96, 68.75, 68.31, 66.71, 61.35, 51.01, 23.41, 21.08,
20.87. MS (ESI) 1324.25 [M+Na]+. MS (MALDI-TOF) calcd
for C75H83NO19Na [M+Na]+ m/z 1324.5456; found: m/z
1324.5573.
Heptanediol-Linked Lactose-Bishydrazide Conjugate (9). This
compound was similarly prepared as that described above,
starting from lactose (50 mg, 0.146 mmol) and 1,7-heptanediol
bishydrazide 5 (201 mg, 0.736 mmol). Purification by RP-HPLC
(1-50% aq CH3CN gradient over 45 min) yielded glycocon-
jugate 9 as a light-yellow oil (54 mg, 62%). 1H NMR (400 MHz,
CD3OD) δ 4.30 (d, 1H, J ) 7.6 Hz), (d, 1H, J ) 8.2 Hz), (d,
1H, J ) 4.4 Hz), 3.97 (s, 2H), 3.83-3.77 (m, 2H), 3.67-3.59
(m, 4H), 3.54-3.45 (m, 4H), 3.41-3.35 (m, 2H), 3.22-3.19
(m, 1H), 1.92-1.69 (m, 14H). 13C NMR (100 MHz, CD3OD)
δ 181.97, 173.86, 103.51, 89.97, 78.93, 76.39, 75.95, 75.52,
73.09, 71.55, 70.84, 69.18, 61.66, 61.01, 60.84, 23.98, 20.65,
20.44, 20.37. MS (ESI) calcd for C23H45N4O14 [M+H]+ m/z
601.28; found: m/z 601.09.
2-Deoxy-3,4,6-tri-O-acetyl-2-trichloroacetamido-ꢀ-D-galactopy-
ranosyl-(1f4)-2,4,6-tri-O-benzyl-ꢀ-D-galactopyranosyl-(1f4)-
1,2,3,6-tetra-O-benzyl-D-glucopyranose (12). D-Galactosamine
thiophenyl glycoside 10 (51) (36.8 mg, 0.066 mmol) and lactose
derivative 11 (52) (43.0 mg, 0.044 mmol) were stirred under
argon in dry CH2Cl2 (2 mL) for 30 min at rt in the presence of
freshly activated, powdered 4 Å molecular sieves. The reaction
mixture was cooled to -70 °C, then treated with N-iodosuc-
cinimide (NIS, 17.8 mg, 0.079 mmol) and a catalytic amount
of triflic acid (TfOH, 2.0 mg, 0.0132 mmol). The reaction
mixture was slowly warmed to 0 °C, then stirred for an
additional 4 h at rt. The reaction mixture was then neutralized
with a few drops of triethylamine and filtered over a pad of
Celite with rinsing by CH2Cl2. The mixture was washed with a
10% Na2SO3 solution followed by water, and the aqueous layer
was extracted twice with CH2Cl2. The organic phases were
collected and dried over anhydrous Na2SO4 and concentrated
then purified by silica gel chromatography (EtOAc/hexanes 2:3)
2-Acetamido-2-deoxy-ꢀ-D-galactopyranosyl-(1f4)-ꢀ-D-galac-
topyranosyl-(1f4)-D-glucopyranose (2). A solution of triacetate
13 (100 mg, 0.076 mmol) in dry methanol (5.0 mL) was treated
at rt with a catalytic amount of NaOMe in methanol (1 M, 32
µL). The reaction mixture was stirred for 1 h at rt and then
treated with Dowex 50WX8 (H+) resin (500 mg), filtered, and
concentrated to dryness. The partially deprotected trisaccharide
was dissolved in MeOH (5 mL) and treated with a catalytic
amount of Pd(OH)2 on charcoal (80 mg, 20% on active carbon),
purged with H2, and stirred under an H2 atmosphere for 12 h.
The catalyst was filtered through a pad of Celite, and the filtrate
was concentrated to yield pulmonary trisaccharide 2, as an off-
white solid (38 mg, 90% over two steps). 1H NMR (400 MHz,
CD3OD) δ 4.61 (dd, 1H, J ) 2.9, 8.4 Hz), 4.48 (d, 1H, J ) 7.2
Hz), 4.33 (d, 1H, J ) 7.7 Hz), 4.00 (d, 1H, J ) 1.7 Hz),
3.90-3.82 (m, 4H), 3.79-3.75 (m, 2H), 3.71-3.64 (m, 2H),
3.62-3.58 (m, 4H), 3.51-3.46 (m, 4H), 3.42-3.39 (m, 1H),
2.02 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 175.12, 105.12,
104.43, 99.25, 93.69, 80.93, 78.29, 76.82, 76.49, 76.27, 76.00,
74.68, 74.38, 72.59, 69.91, 62.63, 61.95, 61.62, 55.27, 23.15.
MS (ESI) calcd for C20H35NO16Na [M+Na]+ m/z 568.47; found:
m/z 568.11.
1
to afford trisaccharide 12 as a yellow oil (52.2 mg, 84%). H
NMR (400 MHz, CDCl3) δ 7.44-7.25 (m, 35H, ArH), 6.23 (d,
1H, J ) 10 Hz, NH), 5.26 (d, 1H, J ) 2.8 Hz), 4.97 (d, 2H, J
) 12.0 Hz), 4.94 (d, 2H, J ) 12.0 Hz), 4.88 (d, 2H, J ) 8.0
Hz), 4.85 (d, 2H, J ) 8.0 Hz), 4.81 (d, 2H, J ) 9.6 Hz), 4.78
(d, 2H, J ) 8.0 Hz), 4.71 (d, 2H, J ) 8.0 Hz), 4.66 (d, 2H, J
) 12.0 Hz), 4.51 (d, 1H, J ) 8.0 Hz), 4.49 (d, 1H, J ) 8.0
Hz), 4.44 (d, 2H, J ) 12.0 Hz), 4.13-3.98 (m, 5H), 3.90 (dd,
2H, J ) 6.0, 11.2 Hz), 3.74-3.67 (m, 2H), 3.62-3.59 (m, 1H),
3.56 (d, 2H, J ) 9.2 Hz), 3.49 (t, 1H, J ) 7.6 Hz), 3.36 (d, 1H,
J ) 9.6 Hz), 2.09 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 170.49, 170.44, 170.30, 162.00, 139.15,
138.66, 137.84, 137.56, 129.00, 128.88, 128.57, 128.42, 128.30,
128.16, 128.00, 127.94, 127.83, 127.70, 127.59, 102.77, 99.51,
92.53, 82.53, 81.76, 82.53, 81.76, 76.11, 75.43, 75.06, 74.32,
73.88, 71.29, 70.64, 69.95, 68.20, 66.31, 60.87, 53.43, 20.87,
20.78, 20.68. MS (MALDI-TOF) calcd for C75H80Cl3NO19Na
[M+Na]+ m/z 1426.4287; found: m/z 1426.4573.
Fluorescein-Labeled 2′-Fucosyllactose Bishydrazide Conjugate
(14). A solution of glycoconjugate 8 (2.2 mg, 2.4 µmol) and
fluorescein isothiocyanate (FITC, 1.14 mg, 2.9 µmol) in
anhydrous methanol was treated with N,N-diisopropylethylamine
(1.2 µL, 9.8 µmol) and stirred for 10 h at rt. The reaction mixture
was concentrated and the residue was purified by RP-HPLC
(1-30% aq CH3CN gradient over 45 min) to yield FITC
conjugate 14 as a greenish-yellow solid (2.1 mg, 67%). 1H NMR
(400 MHz, CD3OD) δ 8.18 (d, 1H, J ) 8.9 Hz), 7.50 (d, 1H,
J ) 2.0 Hz), 7.36 (d, 1H, J ) 8.8), 4.21 (br s, 2H), 4.14 (d, 1H,
J ) 9.4 Hz), 4.05 (d, 1H, J ) 9.3 Hz), 3.79-3.78 (m, 4H),
3.74-3.62 (m, 37H), 3.52-3.50 (m, 2H), 3.45-3.42 (m, 1H),
3.24-3.23 (m, 2H), 3.21-3.17 (m, 2H), 3.12-3.09 (m, 1H),
2.00 (3, 3H). MS (MALDI) calcd for C55H76N5O28S [M+H]+
m/z 1290.43; found: m/z 1290.27.
2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-ꢀ-D-galactopyranosyl-
(1f4)-2,4,6-tri-O-benzyl-ꢀ-D-galactopyranosyl-(1f4)-1,2,3,6-
tetra-O-benzyl-D-glucopyranose (13). A solution of trichloro-
acetamide 12 (50 mg, 0.036 mmol) in dry benzene (1 mL) was
treated with tributyltin hydride (52.4 mg, 0.18 mmol) and 2,2′-
azobis(isobutyronitrile) (AIBN, 5.86 mg, 0.036 mmol). The
mixture was degassed for 15 min with argon, then heated to
reflux for 1 h, cooled to rt, and poured into an aq solution of
NaHCO3, and extracted with CH2Cl2. The combined extracts
were washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. Purification of the residue
by silica gel chromatography (EtOAc/hexanes 1:1) afforded
PhotoactiVe Glycan-Bishydrazide Conjugates (15-17). In a
typical reaction, a solution of pulmonary trisaccharide-
bishydrazide conjugate 7 (11 mg, 11.5 µmol) and N-5-azido-
2-nitrobenzoyloxy-succinimide (ANB-NOS, 5.3 mg, 17.3 µmol)
in anhydrous DMF (1 mL) was treated with N,N-diisopropyl-
ethylamine (2.0 µL, 12.7 µmol) and stirred for 12 h at rt,
protected from light. The reaction mixture was concentrated,
and the residue was purified by RP-HPLC (1-30% aq CH3CN
gradient over 45 min) to afford ANS conjugate 16 as a brown
1
acetamide 13 as a yellow oil (32.5 mg, 70%). H NMR (400
MHz, CDCl3) δ 7.53-7.23 (35H, ArH), 5.47 (d, 1H, J ) 8.9
Hz, NH), 5.32 (d, 1H, J ) 2.7 Hz), 4.97 (d, 1H, J ) 8.0 Hz),

Bishydrazide Glycoconjugates for Bacterial Detection
Bioconjugate Chem., Vol. 21, No. 11, 2010 2069
Scheme 1. Synthesis of Glycan-Bishydrazide Conjugates
Scheme 2. Synthesis of Pulmonary Trisaccharidea
a TCA ) trichloroacetyl.
1
solid (8.4 mg, 63%). H NMR (400 MHz, CD3OD) δ 8.08 (s,
cated that the bishydrazides form ꢀ-glycoside linkages, in accord
with earlier reports (54-57). It should be noted that the
formation of bishydrazide glycoconjugates 7 and 8 is not always
complete after 48 h under these conditions; on the other hand,
the formation of glycan dimers is negligible, circumventing the
need for heterobifunctional linkers.
Glycan-bishydrazide conjugates 7 and 8 were prepared using
GalNAc(ꢀ1f4)-Gal(ꢀ1f4)Glc (2) and 2′-fucosyllactose (Fuc-
(R1f2)Gal(ꢀ1f4)Glc, 3), with the former obtained through
multistep organic synthesis (see below). Trisaccharide 2 is a
common substructure in the glycans presented on the epithelial
lining of the pulmonary tract and is a high-affinity ligand for
adhesins presented on the pili of virulent Pseudomonas (58, 59).
Fucose-presenting glycans such as 3 have been shown to be
potent ligands for the lectin PA-IIL, also expressed by
Pseudomonas (60, 61). These glycans presented us an op-
portunity to evaluate the bishydrazide glycoconjugate chemistry
in the context of bacterial pathogen detection.
Briefly, pulmonary trisaccharide 2 was synthesized by the
coupling of N-trichloroacetyl-protected GalNAc donor 10 (51)
with lactose-derived acceptor 11 (52) and N-iodosuccinimide-
triflic acid activation conditions, yielding the desired ꢀ-glycoside
12 in 86% yield (Scheme 2, see Experimental Procedures section
for details). Reductive dechlorination with tributyltin hydride,
followed by methanolic deacetylation and global debenzylation,
yielded trisaccharide 2 in excellent overall yields.
1H), 7.89 (br s, 1H), 7.69 (d, 1H, J ) 8.0 Hz), 7.41 (d, 1H, J
) 7.9 Hz), 7.14 (t, 1H, J ) 7.9 Hz), 6.53-6.51 (m, 3H), 5.20
(s, 1H), 4.53 (s, 1H), 4.43 (d, 1H, J ) 8.0 Hz), 4.17-4.13 (m,
2H), 4.06-4.05 (m, 2H), 4.03-4.01 (m, 2H), 3.77-3.57 (m,
35H), 3.53-3.48 (m, 4H), 1.16 (d, 1H, J ) 6.4 Hz), 1.10 (d,
1H, J ) 7.4 Hz). MS (ESI) calcd for C43H70N9O27Na [M+H]+
m/z 1144.43; found: m/z 1144.87.
RESULTS AND DISCUSSION
Synthesis and Reactivity of Bishydrazide Glycoconjugates.
The preparation of bishydrazide cross-linkers have been previ-
ously reported in the literature, but with little detail regarding
their purification (53). We found bis(carboxymethyl) esters to
be practical intermediates for bishydrazide formation, but
hydrazinolysis should be performed under strictly anhydrous
conditions to avoid the generation of byproducts during glyco-
conjugate formation. Bishydrazide linkers 4 and 5 were
synthesized from their corresponding R,ω-diols by base-
promoted carboxymethylation with ethyl bromoacetate in THF,
followed by treatment with excess hydrazine in anhydrous
methanol under an inert atmosphere. The bishydrazides prepared
in this manner could be collected in nearly quantitative yields
simply by removing all volatiles by azeotropic drying with
toluene, then used to generate glycoconjugates without further
purification.
Glycan-bishydrazide conjugates were prepared by adapting
the conditions reported by Shin and co-workers (Scheme
1) (20, 21). After an extensive evaluation of reaction conditions
using hexa(ethylene glycol)-linked bishydrazide 4 and com-
mercial-grade lactose, we determined that the highest yields were
obtained by treating 1 equiv of glycan in AcOH/NaOAc buffer
(pH 4.2) with 10 equiv of bishydrazide linker at 70 °C for 48 h.
The glycoconjugates were readily separated from the excess
linker by preparative reverse-phase HPLC using an aqueous
CH3CN gradient. 13C NMR analysis of lactose-bishydrazide
conjugate 6 indicated the anomeric carbons to have the expected
chemical shifts for O,O- and N,O-acetals (δc 105.10 and 91.89
ppm, respectively), confirming the pyranosidic nature of the
The glycan-bishydrazide conjugates react readily with
standard labeling and bioconjugation reagents. Fluorescent
glycoconjugate 14 was generated by treatment of 2′-fucosyl-
lactose-bishydrazide 8 with fluorescein isothiocyanate (FITC),
whereas photoreactive glycoconjugates 15-17 were produced
by coupling bishydrazide-linked glycans 6-8 with N-(5 azido-
2-nitrobenzoyloxy)succinimide (ANB-NOS) (Figure 1). In both
of these cases, hydrazide coupling was most efficient in
anhydrous polar solvents such as DMF and MeOH, using N,N-
diisopropylethylamine as the base (see Experimental Procedures
section for details). Neoglycoproteins could also be formed in
phosphate-buffered saline (PBS) solutions at physiological pH
using typical EDC coupling conditions (39), and stored at 4 °C
for several months.
1
reducing-end sugar. H NMR coupling constant analysis indi-

2070 Bioconjugate Chem., Vol. 21, No. 11, 2010
Adak et al.
Figure 1. Glycan-bishydrazide conjugates labeled with fluorescent or
photoreactive chromophores. (EG)6 ) -O(CH2CH2O)6-. 15: R1, R2
) H. 16: R1 ) ꢀ-D-GalNAc, R2 ) H. 17: R1 ) H, R2 ) R-L-Fuc.
Figure 3. (A) Affinity and lactose competition assay of peanut lectin
binding to microspheres conjugated with lactose-bishydrazide 6 using
flow immunocytometry. (B) ELISA and competition assay of peanut
lectin binding to immobilized 6-BSA.
Lectin and Bacterial Binding by Bishydrazide Glycoconju-
gates. Bishydrazide glycoconjugates can be directly attached
onto surface-active substrates for the selective capture of lectins
and subsequent binding immunoassays. For example, lactose-
bishydrazide 6 was attached onto NHS-activated latex micro-
spheres in order to measure the binding affinity of peanut lectin
by flow immunocytometry (Kd ) 50 nM), using a FITC-labeled
secondary antibody for fluorescence detection (Figure 3A and
Supporting Information Figure S1). Selective binding was
established by a competition assay against 1 mM lactose, which
increased Kd by 10-fold. However, in some cases the use of
carrier proteins such as BSA is more convenient for surface
functionalization by simple physisorption. An ELISA of 6-BSA
conjugate indicates its affinity for peanut lectin to be the same
as that measured for lactose-conjugated microspheres.
Glycoconjugates can also be covalently tethered onto protein
layers and other organic substrates by photo-cross-linking, which
provides opportunities to create glycan microarrays and micro-
patterns by photolithography (71-75). Au substrates coated with
a monolayer of BSA were immersed in a solution of lactose-
bishydrazide-ANB conjugate 15, then dried in air and exposed
to UV irradiation through a photomask. The intermediate
nitrenes that are generated upon UV irradiation (76) presumably
insert into the amino acid side chains of the immobilized BSA,
resulting in a patterned array of glycans after washing away
the unexposed ligands. Incubation with peanut lectin followed
by fluorescence immunostaining revealed well-defined line
patterns, defined by the photomask features (Figure 4).
A similar approach was used to generate photopatterned
arrays of the pulmonary trisaccharide and 2′-fucosyllactose on
BSA-coated substrates, using glycan-bishydrazide-ANB con-
jugates 16 and 17, respectively. These micropatterned substrates
were exposed to the opportunistic pathogen Pseudomonas
aeruginosa at 106-108 cfu/mL, then washed and imaged by
optical darkfield microscopy to visualize patterns of glycan-
immobilized bacteria in a label-free manner (Figure 5 and
Supporting Information Figures S2 and S3). Specificity for 16
and 17 was confirmed by introducing Pseudomonas to control
substrates patterned with lactose-bishydrazide conjugate, which
resulted in no apparent binding. Furthermore, only live Pseudomo-
Figure 2. (A) In situ DTC formation starting from lactose-bishydrazide
conjugate 6; (EG)6 ) O(CH2CH2O)6. (B) UV absorption spectra of
6-DTC in dilute aqueous solution, taken during in situ DTC formation.
No further increases in peak intensities were observed after 60 min.
The hydrazide-terminated glycoconjugates are also amenable
to in situ dithiocarbamate (DTC) formation, a recently estab-
lished method for anchoring amine-terminated ligands onto
metal surfaces (47, 48, 62-69). DTCs are conveniently prepared
by the addition of nucleophilic amines to CS2 in polar organic
or aqueous media and exhibit a strong binding affinity for gold
and several types of inorganic oxides. Bishydrazide-DTCs can
be prepared in a similar fashion: in this work; an equimolar
mixture of lactose-bishydrazide conjugate 6, CS2, and Et3N in
methanol resulted in essentially quantitative formation of DTC
triethylammonium salt within 1 h under ambient conditions (6-
DTC, Figure 2). The reaction time was determined by using
UV absorption spectroscopy to monitor hydrazide-DTC forma-
tion based on increases in characteristic peak intensities at 250
and 292 nm (70).

Bishydrazide Glycoconjugates for Bacterial Detection
Bioconjugate Chem., Vol. 21, No. 11, 2010 2071
Scheme 3a
a (A,B) Glycan-patterned slides by microcontact printing of BSA
glycoconjugates onto NHS-activated glass substrates, followed by a
blocking step. (C) Patterned capture slides were exposed to Pseudomo-
nas at variable concentrations, then imaged under darkfield condtions.
(D) Image processing by FFT analysis produced reciprocal lattice peaks
at k ) (1/a in spectral format; central peak (k ) 0) scales with spatially
averaged intensity of original image; noise from nonspecific binding
is dispersed throughout k-space.
Figure 4. Fluorescence immunostaining of peanut lectin bound to
lactose-bishydrazide-ANB conjugate 15, photopatterned onto a BSA-
coated substrate by UV irradiation (λ ) 254 nm) through a quartz mask.
allows the linear array of scattering signals to be analyzed as
reciprocal lattice peaks, enabling much lower limits of detection
(50). These demodulated signals are largely separated from
background noise and can be presented with much greater
contrast than the original darkfield images (Figure 6A-H,
insets). As a result, we find that slides patterned with 7-BSA
can detect Pseudomonas capture in a label-free manner, with
acceptable signal-to-noise (S/N) ratios down to 103 cfu/mL
(Figure 6F).
Interestingly, we observe that our Pseudomonas cultures have
a much lower avidity for 2′-fucosyllactose 8 than for pulmonary
trisaccharide 7, which are recognized by adhesins that are
constitutively presented at the tips of bacterial pili (82). Whereas
affinity for 7-BSA is robust and minimally affected by the
presence of competing GalNAc or lactose up to 100 mM
(Supporting Information, Figure S5), affinity for 8-BSA is
observed only when the bacteria has been cultured in the
presence of choline (49). It has been shown that the expression
of the fucose-binding lectin PA-IIL is regulated by quorum
sensing circuitry (las and rhl), which are activated in an
environmentally dependent (paracrinal) fashion (83, 84).
Presentation of Glycan-Bishydrazides on Metal Surfaces
by in Situ Dithiocarbamate Formation. Biosensor applications
are often performed with gold substrates, like those based on
surface plasmon resonance (SPR) and quartz crystal microbal-
ance (QCM) (85, 86). For short-term biosensing studies, it is
often appropriate to employ chemisorptive ligands such as
ω-functionalized alkanethiols, which are well-known to form
self-assembled monolayers (SAMs) on gold surfaces (87).
However, alkanethiol-based SAMs can have limited stability
when exposed to even mildly oxidizing conditions (88), so it is
worth considering other types of chemisorptive ligands that can
support robust surface functionalization under variable environ-
ments or in physiological settings.
DTC-anchored monolayers (DAMs) are useful alternatives
to thiol-based SAMs, as they have been shown to have greater
resistance to surface desorption or oxidative degradation (47).
Although earlier studies involving in situ DTC and DAM
formationhavebeenconductedwithalkyl-anddialkylamines(47,48,
62-69), hydrazide-DTCs can be formed just as easily and may
also serve as chemisorptive ligands. Lactose-bishydrazide 6
was thus converted into the corresponding DTC (Figure 2), then
diluted in water and deposited onto a PDMS stamp for pattern
transfer onto a chemically roughened Au surface by µCP. The
Figure 5. Capture of Pseudomonas on BSA-coated substrates with
photopatterned glycan-bishydrazide-ANB conjugate, imaged by dark-
field microscopy. Bacterial capture mediated by 2′-fucosyllactose
conjugate 17 at 108 cfu/mL; grating period a ) 20 µm. Additional
images in Supporting Information (Figures S2-S4).
nas were captured by the trisaccharide ligands; exposing the
bacteria to UV irradiation for 2 h prior to their introduction
resulted again in no observable pattern (Supporting Information
Figure S4). We note that the patterns formed by bacterial
adhesion at lower pathogen densities can be well below
saturation levels; however, our pathogen detection method only
requires a fill factor of a few percent for pattern recognition, as
will be demonstrated shortly.
Glycan-bishydrazide conjugates could also be patterned onto
substrates by microcontact printing (µCP), a well-established
“soft lithography” technique based on microfabricated poly-
dimethylsiloxane (PDMS) stamps for the physical transfer of
organic or biomolecular ligands, including glycans (77-81).
Stamps imprinted with 20 µm gratings were coated with
solutions of 7-BSA or 8-BSA in phosphate buffer, then dried
in air and placed in conformal contact with activated glass slides
under lightly applied pressure (see Scheme 3 and Experimental
Procedures section). The substrates were blocked then exposed
for 1 h to Pseudomonas at 102-108 cfu/mL, then evaluated for
evidence of patterned pathogen capture (Figure 6). On average,
we find the pattern transfer of 7-BSA by µCP to be more
consistent than that produced by photolithography, and remark-
ably reliable for mediating bacterial adhesion at low pathogen
densities.
Visual inspection of the patterned glycan slides can indicate
Pseudomonas capture at a concentration of 105 cfu/mL (Figure
6D). However, image processing by fast Fourier transform

2072 Bioconjugate Chem., Vol. 21, No. 11, 2010
Adak et al.
Figure 6. Capture of live Pseudomonas on glass substrates patterned with BSA-glycan bishydrazide conjugates using µCP, as imaged by darkfield
microscopy. (A-G) Substrates patterned with pulmonary trisaccharide conjugate 7-BSA after a 1-h exposure to Pseudomonas, at concentrations
ranging from 108 to 102 cfu/mL. Pattern contrast correlates with signal-to-noise (S/N) ratio defined by reciprocal lattice peak produced by FFT (k
) 0.05 µm-1). (H) Substrate patterned with 7-BSA without exposure to bacteria (control). (I) Pseudomonas capture mediated by 2′-fucosyllactose
conjugate 8-BSA, in the presence of choline (108 cfu/mL).
Figure 7. (A) Fluorescence immunostaining of peanut lectin bound to lactose conjugate 6-DTC, presented as DAMs on Au substrates by µCP. (B)
Stability profile of hydrazide-DTC patterns on roughened Au in PBS containing ME (10 or 100 µM), based on changes in relative luminosity.
patterned substrates were blocked by BSA and incubated with
peanut lectin, whose binding to lactose-terminated DAMs was
visualized by immunofluorescent staining (Figure 7). It is
important to note that Au substrates are often incompatible with
fluorescence imaging due to their propensity to quench emission;
however, radiative decay rates can be accelerated on roughened
Au substrates by coupling with local plasmon modes and may
even produce a net gain in emission (89).
As expected, the oriented presentation of 6-DTC on Au
surfaces is well-suited for the immobilization of multivalent
lectins, and patterned substrates can be soaked in PBS for
extended periods without concern for degradation. To further
establish whether glycan-bishydrazide DAMs are capable of
providing robust support as carbohydrate microarrays for protein
screening or pathogen binding, we investigated their stability
in the presence of 2-mercaptoethanol (ME), a polar thiol that is
frequently used to displace alkanethiol-based SAMs from Au
surfaces (90). This displacement assay is important in the context
of physiological sensing, as biogenic thiols such as cysteine and
glutathione can also be considered as competing adsorbates. The
immunofluorescent patterns on roughened Au substrates were
thus exposed to 10 µM or 100 µM ME in PBS at room
temperature for 2 days, with monitoring of selected regions at
different exposure times by fluorescence microscopy (Supporting

Bishydrazide Glycoconjugates for Bacterial Detection
Bioconjugate Chem., Vol. 21, No. 11, 2010 2073
(5) Liu, L.-H., Dietsch, H., Schurtenberger, P., and Yan, M. (2009)
Photoinitiated coupling of unmodified monosaccharides to iron
oxide nanoparticles for sensing proteins and bacteria. Biocon-
jugate Chem. 20, 1349–1355.
(6) Disney, M. D., and Seeberger, P. H. (2004) The use of
carbohydrate microarrays to study carbohydrate-cell interactions
and to detect pathogens. Chem. Biol. 11, 1701–1707.
(7) Ngundi, M. M., Kulagina, N. V., Anderson, G. P., and Taitt,
C. R. (2006) Nonantibody-based recognition: alternative mol-
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524.
Information, Figure S6). Both conditions produced very gradual
decay profiles, with an estimated halflife of over 80 h (Figure
7B). It should be mentioned that displacement assays with
millimolar levels of ME could not be performed in this case,
as this was sufficient to cause degradation of the roughened
Au substrates themselves. Nevertheless, exposure of 6-DTC
ligands to ME at these levels has minimal detrimental effect
on DAM stability and merits further investigation. A direct
comparison between SAMs and DAMs is in progress and will
be reported in due course.
(8) Kulkarni, A. A., Weiss, A. A., and Iyer, S. S. (2010) Glycan-
based high-affinity ligands for toxins and pathogen receptors.
Med. Res. ReV. 30, 327–393.
(9) Wu, C.-Y., Liang, P.-H., and Wong, C.-H. (2009) New
development of glycan arrays. Org. Biomol. Chem. 7, 2247–
2254.
CONCLUSIONS
R,ω-Bishydrazides are useful and versatile linkers for attach-
ing glycans onto substrates for bacterial pathogen detection and
lectin profiling. Unprotected glycans are conjugated straight-
forwardly to bishydrazides with retention of the native pyranose
conformation at the reducing end, and can be further conjugated
to amine-reactive substrates, carrier proteins, and to metal
surfaces by in situ DTC formation. With respect to the latter,
glycan-bishydrazide-DTCs form robust attachments to rough-
ened gold substrates and are able to withstand displacement by
competing thiols under physiologically relevant conditions.
Glycan-bishydrazide conjugates can be subsequently employed
in microfabrication schemes involving photolithography or
PDMS-based microcontact printing for the facile detection of
glycan-binding proteins and pathogens, as demonstrated by the
patterned adsorption of peanut lectin and Pseudomonas. We
anticipate using this methodology to support real-time biosensing
applications involving protein biomarker or pathogen detection.
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ACKNOWLEDGMENT
The authors gratefully acknowledge financial support from
the National Institutes of Health (GM-069862), the National
Science Foundation (CHE-0243496), the Department of Defense
(W911SR-08-C-0001) administered through the U.S. Army
RDECOM (Edgewood Contracting Division), and the Center
for Sensing Science and Technology at Purdue University. We
thank David Lyvers and Ron Reifenberger for their contributions
toward FFT analysis, and Youngsoon Kim and James Newton
for helpful discussions and assistance with microcontact printing,
bacteria culture, and quantitative bacterial cell culture analysis.
Supporting Information Available: Representative data
from flow immunocytometry (Figure S1); control experiments
for specificity of pathogen capture (Figures S2-S5); additional
immunofluorescence images of lectins bound to DTC-anchored
glycan bishydrazides on Au substrate (Figure S6); H and 13C
1
NMR spectra of all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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