Thioctic acid amides: Convenient tethers for achieving low nonspecific protein binding to carbohydrates presented on gold surfaces

Article abstract of DOI:10.1039/b503843j

The thioctic acid amides of 2′-aminoethyl α-D-mannopyranoside and 2′-aminoethyl α-1,3-D-mannopyranosyl (α-1,6-D- mannopyranosyl)-α-D-mannopyranoside presented on both planar and nanoparticle gold surfaces give higher specific and lower non-specific protei

Full text of DOI:10.1039/b503843j

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Thioctic acid amides: convenient tethers for achieving low nonspecific
protein binding to carbohydrates presented on gold surfaces{
Rositsa Karamanska, Balaram Mukhopadhyay, David A. Russell* and Robert A. Field*
Received (in Cambridge, UK) 16th March 2005, Accepted 25th April 2005
First published as an Advance Article on the web 27th May 2005
DOI: 10.1039/b503843j
resonance (SPR) and UV-visible spectroscopy were used to
evaluate specific and non-specific protein recognition by these
surfaces.
The thioctic acid amides of 29-aminoethyl a-D-mannopyrano-
side and 29-aminoethyl a-1,3-D-mannopyranosyl (a-1,6-D-man-
nopyranosyl)-a-D-mannopyranoside presented on both planar
and nanoparticle gold surfaces give higher specific and lower
non-specific protein binding than the related 29-thioethyl
glycosides.
Mannoside SAMs were prepared by immersion of gold coated
SPR sensor chips (bare Au; Biacore SA) in 2 mM methanol/20%
water solution of thiols 1a or 2a, or thioctic-amides 1b or 2b. The
integrity of monolayers formed in this manner was determined by
cyclic voltammetry (ESI). Surfaces modified with thiols 1a and 2a
displayed characteristics similar to bare gold, indicating that a
poorly ordered monolayer, with significant ‘‘holes’’, had been
formed. In contrast, surfaces modified with thioctic-amides 1b and
2b showed a higher degree of insulation [reduced penetration of
ferricyanide in solution giving a decreased current response]
consistent with a more ordered surface.
The presentation of carbohydrates on planar gold sensor surfaces,1
colloidal gold particles2 and recently gold-based microarrays3 has
been the focus of considerable research interest for studies of
carbohydrate recognition. In moving from model studies to
biomedical applications, two considerations become critical viz.:
non-specific protein binding; and the stability of the modified
surface with respect to ligand desorption. Optimal presentation of
ligands on planar gold surfaces is generally achieved using PEG-
thiol-containing SAMs using the protocols developed by
Whitesides et al.4 However, less effort has gone into defining an
optimal linker/presentation system for carbohydrates on gold
nanoparticles. Recent studies from these5 and other6 labs have
aimed to develop glyconanoparticles for the colorimetric detection
of lectins. Such studies have often relied on the readily accessible
29-thioethyl glycosides, such as 1a, although short chain alka-
nethiols are known to form disordered SAMs.7 Therefore, we have
investigated other linker technologies that might prove convenient
for applications that use either planar or nanoparticulate gold
surfaces.
The higher affinity of Con A for trimannoside than for
mannoside has been previously established.10 However, the SPR
response obtained when Con A was passed over the mannoside 1b
surface was greater than that achieved from surfaces presenting the
corresponding trimannoside 2b (Fig. 2). Ligand density on surfaces
is well known to have a profound impact on carbohydrate
recognition selectivity and affinity.1c,4 Both the 1b and 2b surfaces
gave a substantially greater response to Con A than surfaces
modified with the corresponding thiols 1a and 2a, which is
presumably due to greater organisation of the monolayer, and
hence more optimal ligand presentation with the thioctic acid
derivatives. The ‘‘on’’ rate for binding to both trimannosides, 2a
and 2b, is higher than compared to the corresponding mannosides,
1a and 1b (Fig. 2). These observations are consistent with the
notion that recognition of the preferred trimannoside ligand is
prevented by sub-optimal ligand density on these particular
surfaces.11
Thioctic acid-based systems have been used to present a variety
of classes of molecule on gold surfaces,8 but to the best of our
knowledge they have not previously been used with carbohydrate
ligands. Naturally occurring thioctic acid (lipoic acid) is potentially
attractive for a number of reasons: the presence of two sulfur
atoms obviates the need to engage in noxious sulfur chemistry,
whilst also providing two potential points of attachment to gold.9
The alkyl chain length in thioctic acid would be expected to result
in a more ordered self-assembled monolayer than a simple
mercaptoethyl tether, a factor that may be reinforced by amide
coupling to sugar derivatives, which is also expected to give
enhanced order and hence stability.1c In the current study, a-D-
mannopyranoside and a-1,3-D-mannopyranosyl(a-1,6-D-manno-
pyranosyl)-a-D-mannopyranoside-based thiols (1a and 2a) and
thioctic-amides (1b and 2b) (Fig. 1) (see ESI) were presented on
both planar gold and on gold nanoparticles. Surface plasmon
Specific and non-specific binding of the trimannoside SAMs was
evaluated by SPR using multivalent lectins [concanavalin A (Con
A), Tetragonolubus purpureas lectin (TPL) and Ricinus communis
agglutinin 120 (RCA120)] as well as two ‘‘sticky’’ proteins
{ Electronic supplementary information (ESI) available: synthesis and
characterisation of all compounds; cyclic voltammograms of monolayers;
UV-vis. spectra of protein induced nanoparticle aggregation. See http://
www.rsc.org/suppdata/cc/b5/b503843j/
*d.russell@uea.ac.uk (David A. Russell)
r.a.field@uea.ac.uk (Robert A. Field)
Fig. 1 Structures of the 4 mannoside derivatives.
3334 | Chem. Commun., 2005, 3334–3336
This journal is ß The Royal Society of Chemistry 2005

View Article Online
colour changes to blue.14 These colorimetric properties make it
possible to detect multivalent lectins following derivatisation of
the gold surface with appropriate carbohydrate ligands.5,6 In the
current study, gold nanoparticles were prepared by citrate
reduction of tetrachloroaurate, following the method of
Turkevich,15 and modified by interaction with 20 mM solutions
of thiols 1a or 2a, or thioctic-amides 1b or 2b. The nanoparticles
were purified by several centrifugation–washing steps and redis-
persed in Tris buffer, pH 7. Centrifugation and resuspension could
be repeated several times without observable loss of colloidal
stability; no observed colour change due to aggregation was
detected following storage for 4 months at 4 uC. The average
diameter of the particles prepared in this manner was determined
by light-scattering and TEM5 to be ca. 17 nm. UV-vis spectros-
copy showed that derivatisation of gold particles with monolayers
of mannosides 1a or 1b or trimannosides 2a or 2b resulted in only
a small shift in Amax (typically from ca. 520 to 524 nm), indicating
only a small change in the dielectric properties of the metal surface
upon self-assembly.
Fig. 2 SPR response curves for the binding of 250 mg/ml Con A to gold
sensor chips coated with thiols 1a (dash–dot) and 2a (dot), thioctic-amides
1b (solid) and 2b (dash).
(fibrinogen and cytochrome c).4 Surfaces prepared from trimanno-
side thiol 2a showed high non-specific binding to three of the
proteins tested (Fig. 3; Table 1). In spite of the different binding
affinities, molecular weights and isoelectric points of proteins
tested,12 surfaces prepared with thioctic-amide 2b showed low non-
specific binding in all cases; less than 60 RU in all cases cf. 3000 RU
response for the Con A (1 RU 5 1 pg mm22; the surface area of
the flow cell 5 1.2 mm2). Surfaces prepared with 2b appear to
show higher resistance to adsorption of fibrinogen and cytochrome
c than ethylene oxide-containing mixed SAMs.4 The lack of non-
specific binding13 to the thioctic-amide-based surface by a series of
different proteins suggests that protein adsorption on these
surfaces is dominated by the interfacial properties of the surface
and not by specific properties of the proteins concerned.
All four sets of red coloured gold nanoparticle conjugates
turned blue on addition of Con A due to particle aggregation
(ESI). For all four sets of particles, Amax shifted from ca. 525 nm to
longer wavelengths when the concentration of Con A was
increased from 0 to 20 mg/ml (Fig. 4A) (and ESI). Particles
derived from thioctic-amides 1b or 2b showed a more pronounced
response at lower concentrations of Con A than those derived
from thiols 1a or 2a. At 5 mg/ml Con A, formation of stable
aggregates from particles derived from thioctic-amides 1b and 2b
was essentially complete within 10 min; the resulting aggregates
were stable for at least 18 h. In contrast, particles derived from
mannoside-thiol 1a failed to respond to Con A at 5 mg/ml, whilst
particles derived from trimannoside-thiol 2a achieved maximum
aggregation within 18 h, i.e., much slower than the thioctic-amide-
derived particles (Fig. 4B). It is clear that the choice of tether
has a profound impact on particle response to Con A, with
thioctic-amide-based particles outperforming their thiol-derived
counterparts.
Gold nanoparticles are characterised by a discrete surface
plasmon absorption band in the visible electronic spectrum14
which is dependent on, amongst other parameters, the average
diameter of the particles. Hence gold nanoparticles characterised
by interparticle distances greater than the average particle diameter
appear red; when the interparticle distance in aggregates decreases
to less than approximately the average particle diameter, the
Fig. 3 Comparison of SPR responses after injection of 10 mg Con A (solid); 20 mg RCA120 (dash–dot–dot); 20 mg TPL (dash–dot); 20 mg fibrinogen (dot);
and 20 mg cytochrome c (dash) over gold surfaces modified with: (A) trimannoside 2a; (B) trimannoside 2b.
Table 1 SPR response (RU) of gold surfaces modified with 2a or 2b to specific and non-specific protein binding
Ligand \ Protein
Con A
RCA120
TPL
Fibrinogen
Cytochrome c
Thiol 2a
Thioctic-amide 2b
1634 (100%)
3000 (100%)
47 (3%)
4 (1.3%)
353 (22%)
20 (0.7%)
1105 (68%)
46 (1.5%)
434 (27%)
57 (1.9%)
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 3334–3336 | 3335

View Article Online
Fig. 4 Change in Amax of gold particles derivatised with 1a (open triangles), 1b (solid triangles), 2a (open squares), 2b (solid squares) with (A) 0–20 mg/ml
Con A and; (B) with 5 mg/ml Con A after 10 min, 1.5 h and 18 h. (The lines on both A and B are shown for line of sight only.)
2001, 40, 2257; (b) M. J. Hernaiz, J. M. de la Fuente, A. G. Barrientos
All four sets of particles were evaluated for non-specific
and S. Penades, Angew. Chem., Int. Ed., 2002, 41, 1554.
binding using the set of proteins used previously in SPR studies
3 (a) K. R. Love and P. H. Seeberger, Angew. Chem., Int. Ed., 2002, 41,
at 50 mg/ml. Non-specific particle aggregation was assessed by a
red shift of Amax (ESI). Mannoside-thiol 1a derivatised particles
showed a degree of non-specific binding to all proteins, whilst only
fibrinogen induced aggregation of particles derived from trimanno-
side 2a. Essentially no non-specific aggregation of particles derived
from thioctic-amides 1b and 2b was observed; specific binding to
Con A is substantially higher than non-specific binding to particles
derived with the thioctic-amide-based ligands. These results are
similar to those obtained for non-specific binding of the same
proteins to SAMs on the SPR chips. Further, the induced
aggregation of thioctic-amide 2b derivatised particles by 2.5 mg/ml
Con A in the presence of up to 50 mg/ml fibrinogen exhibited ,
10% competitive binding (ESI).
3583; (b) B. T. Houseman and M. Mrksich, Chem. Biol., 2002, 9, 443; (c)
M. D. Disney and P. H. Seeberger, Chem. Biol., 2004, 11, 1701; (d)
D. M. Ratner, E. W. Adams, J. Su, B. R. O’Keefe, M. Mrksich and
P. H. Seeberger, ChemBioChem, 2004, 5, 379.
4 (a) K. L. Prime and G. M. Whitesides, J. Am. Chem. Soc., 1993, 115,
10714; (b) M. Mrksich, J. R. Grunwell and G. M. Whitesides, J. Am.
Chem. Soc., 1995, 117, 1209; (c) P. Harder, M. Grunze, R. Dahint,
G. M. Whitesides and P. E. Laibinis, J. Phys. Chem., 1998, 102, 426; (d)
J. Lahri, L. Issacs, J. Tien and G. M. Whitesides, Anal. Chem., 1999, 71,
777.
5 D. C. Hone, A. H. Haines and D. A. Russell, Langmuir, 2003, 19, 7141.
6 (a) H. Otsuka, Y. Akiyama, Y. Nagasaki and K. Kataoka, J. Am.
Chem. Soc., 2001, 123, 8226; (b) C. C. Lin, Y.-C. Yeh, C.-Y. Yang,
G.-F. Chen, Y. C. Chen, Y.-C. Wu and C.-C. Chen, Chem. Commun.,
2003, 2920.
7 (a) M. Lewis, M. Tarlov and K. Carrou, J. Am. Chem. Soc., 1995, 117,
9574; (b) A. Ulman, Chem. Rev., 1996, 96, 1533.
8 See for example: (a) Y. Miura, S. Kimura, Y. Imanishi and J. Umemura,
Langmuir, 1998, 14, 6935; (b) Y. Dong and C. Shannon, Anal. Chem.,
2000, 72, 2371; (c) J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo
and G. M. Whitesides, Chem. Rev., 2005, 105, 1103.
In conclusion, the thioctic-amide system serves as an accessible
and effective method for carbohydrate presentation on gold
surfaces in a manner that limits non-specific protein binding whilst
enhancing specific binding of the target lectin.
9 B. Garcia, M. Salome, L. Lemelle, J.-L. Bridot, P. Gillet, P. Perriat,
S. Roux and O. Tillement, Chem. Commun., 2005, 369.
10 (a) D. K. Mandal, N. Kishore and C. F. Brewer, Biochemistry, 1994, 33,
1149. Crystallographic studies have revealed the structural basis for high
affinity binding of trimannoside by Con A – ; J. H. Naismith and
R. A. Field, J. Biol. Chem., 1996, 271, 972.
This work was supported by the EPSRC Crime Prevention
Programme (GR/S64134/01) and the RCUK Basic Technologies
Programme (GR/S79268/01). We gratefully acknowledge the
EPSRC Mass Spectrometry Service Centre, University of Wales,
Swansea, for invaluable support.
11 A linear response of carbohydrate-presenting surfaces to lectins is
typically only seen at ligand coverage (, 5%) (ref. 1b).
Rositsa Karamanska, Balaram Mukhopadhyay, David A. Russell* and
Robert A. Field*
Centre for Carbohydrate Chemistry, School of Chemical Sciences and
Pharmacy, University of East Anglia, Norwich, UK NR4 7TJ.
E-mail: d.russell@uea.ac.uk; r.a.field@uea.ac.uk; Fax: +44-1603-59203;
Tel: +44-01603 593145
12 RCA120 m.w. 120 kDa, isoelectric point 7.5–7.9, binds a-D-galactose;
TPL: isoelectric point 7.3, affinity for a-D-fucose; Fibrinogen: isoelectric
point 5.5, a structurally complex, flexible molecule of approximately
340 kDa; Cytochrome c: isoelectric point 9.1–9.5, m.w. 12.4 kDa,
displays a significant level of non-specific adsorption to surfaces
presenting carboxylic acid groups: M. Collinson, E. F. Bowden and
M. J. Tarlov, Langmuir, 1992, 8, 1247; J. Lahiri, G. D. Fate,
S. B. Ungash and J. T. Groves, J. Am. Chem. Soc., 1996, 118, 2347.
13 Non-specifically adsorbed proteins could be removed from both surfaces
by washing with 0.1 M HCl, so regenerating functional surfaces.
Surfaces prepared with thioctic-amide 2b retained 73% of their binding
capacity after six cycles of Con A injection–regeneration and storage at
4 uC for three months whilst those prepared from thiol 2a lost specific
binding to Con A after similar treatment and storage of the sensor chip.
14 (a) J. L. Coffer, J. R. Shapley and H. G. Drickamer, J. Am. Chem. Soc.,
1990, 112, 3736; (b) R. Elghanian, J. J. Storhoff, R. C. Mucic,
R. L. Letsinger and C. A. Mirkin, Science, 1997, 277, 1078.
15 B. V. Enustun and J. Turkevich, J. Am. Chem. Soc., 1963, 85, 3317.
Notes and references
1 (a) D. J. Revell, J. R. Knight, D. J. Blyth, A. H. Haines and
D. A. Russell, Langmuir, 1998, 14, 4517; (b) E. A. Smith, W. D. Thomas,
L. L. Kiessling and R. M. Corn, J. Am. Chem. Soc., 2003, 125, 6140; (c)
N. Noran, L. Yan, H. Isobe, G. M. Whitesides and D. Kahne, Proc.
Natl. Acad. Sci. U. S. A., 1999, 96, 11782; (d) R. Yonzon, E. Jeoung,
S. Zou, G. C. Schatz, M. Mrksich and R. P. V. Duyne, J. Am. Chem.
Soc., 2004, 126, 12669.
2 (a) J. M. de la Fuente, A. G. Barrientos, T. C. Rojas, J. Rojo,
J. U. Canada, A. Fernandez and S. Penades, Angew. Chem., Int. Ed.,
3336 | Chem. Commun., 2005, 3334–3336
This journal is ß The Royal Society of Chemistry 2005