Selective detection of the carbohydrate-bound state of concanavalin A at the single molecule level

Article abstract of DOI:10.1021/ja964366g

The labeling of molecules with charge-transfer dyes, such as 5-(dimethylamino)-1-napthalenesulfonyl (dansyl) chloride, is a powerful tool for examining the solvent shell of attached substances. This investigation describes the synthesis and application of

Full text of DOI:10.1021/ja964366g

7676
J. Am. Chem. Soc. 1997, 119, 7676-7684
Selective Detection of the Carbohydrate-Bound State of
Concanvalin A at the Single Molecule Level
James J. La Clair
Contribution from the Department of Molecular Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed December 19, 1996. ReVised Manuscript ReceiVed June 5, 1997X
Abstract: The labeling of molecules with charge-transfer dyes, such as 5-(dimethylamino)-1-napthalenesulfonyl
(dansyl) chloride, is a powerful tool for examining the solvent shell of attached substances. This investigation describes
the synthesis and application of a new charge transfer label, based on trans-1-[p-(N,N-dimethylamino)phenyl]-trans-
4-(p-nitrophenyl)-1,3-butadiene (NND). Unlike many commonly used fluorophores, the quantum yield of NND
decreases over 4 orders of magnitude upon changing from nonpolar to polar environments. In addition, several
derivatives of NND undergo little photodecomposition and can be detected at the picomolar level in a confocal
fluorescence correlation spectrometer. In conjunction with recent detection of single molecules in solution, this
paper describes a method to discriminate between single free and carbohydrate-bound aggregates of the Jack Bean
lectin, concanavalin A (Con A). To this end, two derivatives of NND were constructed possessing an additional
functional handle. One derivative, alkenyne 4, was efficiently attached to the â-anomeric position of glucopyranosides.
Transients from single aggregates of this fluorophore were detected in solutions which contained both Con A and
maltoside 1, and not the corresponding glucoside 2. This result is in agreement with the known affinity of Con A
for R-glucopyranosides and not â-glucopyranosides. A full description of the synthesis of these dyes, their
solvochromatic properties, and the method used for single aggregate detection is provided herein.
Introduction
spectroscopy, the method cannot directly operate at the single
molecule level (i.e., 10-24 M). Amplification to the molecular
level can be accomplished by reducing the probed volume to
or below a femtoliter. Modern developments in optical imaging
now provide confocally-imaged pinholes which when placed
in the path of a diffraction-limited laser beam provide an
illuminated cylinder with a diameter of 400 nm, a length of 2
µm, and a volume of approximately 0.2 fL (1 fL ) 10-15 L).
In this cavity, the concentration of a single molecule now
corresponds to 8.3 nM, a value within the capacity of LIF. Using
this and other methods for generation of small volumes, single
fluorescent molecules have been detected in flowing and static
solutions.
Within the last decade, the development of improved electron
tunneling probes and optical methods has made it possible to
detect single molecules in vacuum, solution, and the solid state
and on surfaces.1 In contrast to classical spectroscopy which
monitors an average molecular ensemble, single molecule
detection (SMD) examines unique events within a population.
This ability enables one to examine minor species whose
function would normally be lost by inclusion within an average.
In solution, single molecules have been detected by monitoring
their laser-induced fluorescence (LIF).2,3 While the detection
limit of LIF is far superior to that of traditional fluorescence
Since its discovery in 1974,4 fluorescence correlation spec-
troscopy (FCS) has provided new insight into a wide variety of
investigations, including diffusion, aggregation, chemical reac-
tions, and conformational analysis.5 The principle of this
method is based on monitoring the fluctuation in fluorescence
intensity as molecules diffuse through a specified illuminated
X Abstract published in AdVance ACS Abstracts, August 1, 1997.
(1) (a) Moerner, W. E. Acc. Chem. Res. 1996, 29, 563. (b) Geva, E.;
Reilly, P. D.; Skinner, J. L. Acc. Chem. Res. 1996, 29, 579. (c) Monroe,
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N. A.; Parks, D. C. Acc. Chem. Res. 1996, 29, 591. (e) Xie, X. S. Acc.
Chem. Res. 1996, 29, 589. (f) Dickson, R. M.; Norris, D. J.; Rzeng, Y. L.;
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J. Anal. Chem. 1996, 68, 690. (m) Bopp, M. A.; Meixner, A. J.; Tarrach,
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(3) In addition to the fluorescence methods described in ref 2, single
molecules have been detected in solution electrochemically; see: (a) Bard,
A. J.; Fan, F.-R. F. Acc. Chem. Res. 1996, 29, 572. (b) Fan, F. R. F.; Kwak,
J. Bard, A. J. J. Am. Chem. Soc. 1996, 118, 9669.
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S0002-7863(96)04366-1 CCC: $14.00 © 1997 American Chemical Society

Carbohydrate-Bound State of ConcanaValin A
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7677
cavity. When a molecule or group of molecules pass into a
cavity tuned near their absorption maximum, they undergo
cycles of excitation and relaxation by emission of a second
photon. When these events are recorded in a time-dependent
matter, the quanta which belong to these molecules can be
determined through autocorrelation.6 In 1994, Rigler and Eigen
described a method for detecting single rhodamine-labeled DNA
molecules, based on application of confocal microscopy to FCS.7
Concurrently, Zare and colleagues devised a similar method for
detecting single molecules of YOYO intercalated DNA in real
time, without the need for autocorrelation.8 Since these
discoveries, Webb and Gratton have expanded the spectral
window to include the UV region through use of multiple photon
excitation.9 When used in conjunction with techniques for
concentrating or separating particles, such as electrical focusing
or optical tweezers, the detection limit of this method becomes
infinite.7,8 To date, FCS has been applied to a number of
biophysical investigations, including the study of protein folding,
neuroreceptor-binding, the motion of actin filaments, and
membrane dynamics.10 This investigation describes the first
application of single molecule FCS to monitor the interaction
between carbohydrates and proteins.
been used to determine the affinity of sialosides for influenza
hemagglutinin, various oligosaccharides for E-selectin, Salmo-
nella trisaccharide epitope for a monoclonal antibody Se 155-
4, and numerous C- and O-glycosides for lectins. Although
accurate, both methods rely on a comparison between the free
and bound states. To date, neither method is capable of
specifically detecting molecules in one state nor can either
method operate at the single molecule level. One question that
becomes important to the understanding of carbohydrate-
protein binding events is the role and mechanistic aspects of
aggregation before and after binding. Early on, it was recog-
nized that several of the carbohydrate binding proteins exist in
aggregated (dimeric, tetrameric, or polymeric) forms. Multi-
valent ligands, which are already polymeric, are biased toward
aggregated forms and therefore do not easily allow one to
examine this aggregation. Therefore, discovery of a method
which selectively detects only the carbohydrate-bound or free
state provides an ideal tool for this type of investigation. On
the basis of low affinities of these events, the design must
incorporate a means to detect at very low concentration, ideally
at the single molecule level.
One limitation to the development of confocal FCS and
further laser-based fluorometric techniques is the photophysical
and spectroscopic properties of the fluorescent molecule or tag.
In fluorescence-based single molecule detection, a laser beam
tuned near the absorption maximum of the fluorophore is used
to initially provide high-lying rotational and vibrational states
which then undergo picosecond nonradiative decay to a low-
lying singlet state (S1). In doing so, molecules which contain
degrees of rotational freedom can adopt more than one singlet
state, such as twisted intramolecular charge transfer (TICT)
states.15 These states originate from internal rotation to
conformers where the orbitals of one portion of the molecule
are oriented orthogonally with the other. Observation of these
states was first seen in the fluorescence spectrum of p-(N,N-
dimethylamino)benzonitrile and soon thereafter attributed in-
ternal twisting about the dimethylamino group through analogy
to several locked and rotationally restricted derivatives, such
as those shown in Figure 1. Emission from these states is
typically sensitive to solvent polarity, low in energy and
intensity, and of short lifetime. In addition to formation of TICT
states, several fluorophores readily undergo spin-forbidden
relaxation from the S1 state to a long-lived triplet state (T1),
additionally decreasing in their fluorescence. For single mol-
ecule detection, the efficiency of a chromophore is measured
with its absorption cross-section (σ), its fluorescence quantum
yield (Φf), and its photodecomposition.
Recently, increased attention has been devoted to gaining a
better understanding of the biological significance of carbohy-
drate-protein recognition, due to the participation of these
events in a wide variety of disease-related processes including
cellular growth-development, fertilization, metastasis, and
inflammatory response, as well as bacterial and viral recogni-
tion.11 Low affinity, often with an association constant (Ka) as
low as 10-4 M-1, has been a major problem facing these
investigations. One solution to this problem has appeared
through enhancement of the binding by modification of one
partner. Several groups have reported dramatically increased
affinities of molecules possessing multiple (polyvalent) carbo-
hydrates.12 The affinity of these ligands and their monomeric
counterparts can be determined with techniques such as
fluorescence anisotropy or microcalorimetry.13,14 The advantage
of the latter is that it provides a complete energetic description,
including entropic and enthalpic terms. These methods have
(6) Autocorrelation was pioneered by Norbert Wiener. Information on
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1981; Vol. 4, 1984.
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G. S.; Kirmaier, C.; Abbas, S. Z.; Howard, S. C.; Steininger, C. N.; Welply,
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Kiessling, L. L. J. Org. Chem. 1996, 61, 534. (d) Shimura, K.; Kasai, K.
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Landschoot, A.; Loontiens, F. G.; De Brune, C. K. Eur. J. Biochem. 1980,
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M. Eur. J. Biochem. 1980, 103, 313.
(14) For studies using microcalorimetry see: (a) Ambrosino, R.; Barone,
G.; Castronuovo, G.; Ceccarini, C.; Cultrera, O.; Elia, V. Biochemistry 1987,
26, 3971. (b) Williams, B. A.; Chervenak, M. C.; Toone, E. J. J. Biol. Chem.
1992, 267, 22907. (c) Mandal, D. K.; Brewer, C. F. Biochemistry 1993,
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Chem. 1993, 268, 7668. (e) Mandal, D. K.; Kishore, N.; Brewer, C. F.
Biochemistry 1994, 33, 1149. (f) Toone, E. Curr. Opin. Struct. Biol. 1994,
4, 719. (g) Chervenak, M. C.; Toone, E. J. Biochemistry 1995, 34, 5685.
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J.; Baumann, W. NouV. J. Chim. 1979, 3, 443. (b) Rettig, W. Angew. Chem.,
Int. Ed. Engl. 1986, 25, 971. (c) Rulliere, C.; Grabowski, Z. R.; Dobkowski,
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7678 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
La Clair
Figure 1.
Figure 2.
In addition to photophysical considerations, fluorometric
detection of binding requires a substance which undergoes
significant modification of its absorption or emission maxima,
emission quantum yield, and/or excited state lifetime upon
binding. Unlike many commonly used fluorophores,16 the
quantum yield of trans-1-[p-(N,N-dimethylamino)phenyl]-trans-
4-(p-nitrophenyl)-1,3-butadiene (NND) decreases upon changing
from nonpolar to polar solvent systems as seen in the compari-
son of heptane (Φf ) 0.097) to methanol (Φf ) 4 × 10-6).17
In addition, the absorption and fluorescence maxima of these
materials are red shifted by a respective 130 and 109 nm over
the same interval. Even more remarkably, the quantum yield
of this material increased to 0.14, a 44% improvement over that
seen in the most nonpolar solvent, when embedded in a
phospholipid vesicle.17 This fact clearly shows that restriction
of the space for internal rotation results in a dramatic gain of
fluorescence. Similar findings have been seen in the insertion
of trans-stilbene into vesicle membranes.18 In this case, the
increased fluorescence quantum yield was attributed to inhibiting
photoisomerization, to cis-stilbene, which is known to compete
for the singlet excited state with fluorescence. Comparable
isomerizations have been seen in trans-1-[p-(N,N-dimethylami-
no)phenyl]-2-(p-nitrophenyl)ethylene (NNS).19 The isomeriza-
tion yield of this process (Φt-c ) 0.034 in toluene) as well as
the amount of crossing to the triplet state decreased with solvent
polarity, suggesting that nonradiative relaxation was the major
path back to the ground state in polar solvents. When
extensively photolyzed, NND in cyclohexane or toluene reached
a photostationary state containing a mixture of the initial
trans,trans-isomer (67%) and the corresponding cis,trans-
isomers.17 Inhibition of this isomerization may be one factor
contributing to the increased fluorescence seen in vesicles.
Alternatively, this fluorescence gain can be explained by
restricting the formation of TICT states. Although there is no
direct evidence for the presence of TICT states in NND, due to
the complicated nature associated with several positions for
rotation, the decreased fluorescence and red-shifted absorption
upon increasing solvent polarity are comparable with those of
substances known to have TICT states. Here, the goal is to
apply this fluorescence response to distinguish between the
environment surrounding a carbohydrate as it passes from saline
solution to the surface of a protein. Since many carbohydrate-
protein complexes exist in aggregated forms, the appended
fluorophore will respond not only to the surface interaction with
the protein but also the spatial considerations imparted by
inclusion in an aggregate. In addition, this aggregation increases
the number of fluorescent units per molecular entity, as each
aggregate can contain up to one ligand per protein. This
investigation chose to examine the lectin concanavalin A (Con
A) since its carbohydrate affinity has been determined,20 it is
known to exist as a tetramer at pH 7.2,21 and its binding pocket
has been reported to be more hydrophobic than predicted.22 The
first steps toward this experimentation required derivatization
of NND so that it could be attached to carbohydrates.
Results and Discussion
The investigation began with the synthesis of two analogs of
NND, compounds 3 and 4 (Figure 2), which contain a phenolic
handle for linkage to the anomeric center of a carbohydrate.
Prior to this work, Aykiyama reported that NND can be
converted to 3 by exposing its DMF solution to potassium tert-
butoxide in air.23 This procedure provided a 13% yield of 3
along with several alkynic products from oxidation of the
internal alkenes. The synthetic approach employed herein uses
(16) These quantum yields can be compared to other commonly used
fluorophores such as fluorescein (0.91 in 0.1 M NaOH) or rhodamine B
(0.70 in ethanol). Chen, R. F. Anal. Biochem. 1967, 20, 339.
(17) Shin, D. M.; Whitten, D. G. J. Phys. Chem. 1988, 92, 2945.
(18) Suddabay, B. R.; Brown, P. E.; Russel, J. C.; Whitten, D. G. J.
Am. Chem. Soc. 1985, 107, 5609.
(19) (a) Go¨rner, H.; Schulte-Frohlinde, D. J. Mol. Struct. 1982, 84, 227.
(b) Bent, D. V.; Schulte-Frohlinde, D. J. Phys. Chem. 1974, 78, 446. (c)
Go¨rner, H.; Schulte-Frohlinde, D. Ber. Bunsen-Ges. Phys. Chem. 1978, 82,
1102.
(20) (a) Goldstein, I. J.; Reichert, C. M.; Misaki, A. Ann. N.Y. Acad.
Sci. 1979, 234, 283. (b) Poretz, R. D.; Goldstein, I. J. Biochemistry 1970,
9, 2890.
(21) (a) Kalb, A. J.; Lustig, A. Biochim. Biophys. Acta 1968, 168, 336.
(b) McCubbin, W. D.; Kay, C. M. Biochem. Biophys. Res. Commun. 1971,
44, 101. (c) Huet, M. Eur. J. Biochem. 1975, 59, 627.
(22) Isbister, B. D.; St. Hilaire, P. M.; Toone, E. J. J. Am. Chem. Soc.
1995, 117, 12877.
(23) Akiyama, S.; Tajima, K.; Nakatsuji, S.; Nakashima, K.; Abiru, K.;
Watanabe, M. Bull. Chem. Soc. Jpn. 1995, 68, 2043.

Carbohydrate-Bound State of ConcanaValin A
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7679
Scheme 1a
which primarily operates through an SN2 displacement of a
glycosyl bromide by a phenolate.27 This method provided a
high degree of stereoinversion and was amenable to several
carbohydrate and phenolic units. Application of this method
to NND-OH (3) was complicated by the low solubility of 3 in
applicable solvents. At saturation in methylene chloride,
glycosylation of 3 was only 20% complete after 2 weeks. In
addition, purification of this material was complicated by the
presence of several unwanted side products which arose from
in situ acetate hydrolysis and further reaction with R-D-
glucopyranosyl bromide tetraacetate. In an attempt to improve
the efficiency of this process, attention was turned to construc-
tion alkyne 4, with the hope that it would be more soluble.
Application of the previously described Wittig protocol for
4 required construction of aldehyde 13. This material was
prepared in three steps from N,N-dimethylaniline (10) (Scheme
2). The sequence began by regioselectively introducing an
iodine atom onto the para-position of 10 through reaction with
iodine in an aqueous sodium bicarbonate buffer. Upon one
recrystallization from an ether-hexane mixture, iodide 11 could
be coupled to propargyl alcohol using a procedure described
by Takalo and Haenninen.28 The resulting alkynol 12 was then
oxidized with a slurry of MnO2 to the desired aldehyde 13.
Condensation of this aldehyde with the previously described
ylide of 8 provided alkenyne 4, as evidenced by the presence
a Conditions: (a) NaBH4, MeOH, THF, Et2O (5:5:1), 0 °C to rt,
97%. (b) CBr4, PPh3, CH2Cl2, 0 °C to rt, 86%. (c) P(OEt)3, DMF, 155
°C, 1.5 h, 99%. (d) (i) Add NaHMDS (2.2 equiv) in THF to crude 8
in DMF, 0 °C to rt, 1 h; (ii) add 9 in THF, -20 °C to rt, 6 h, 55%.
1
of a single 16 Hz vinylic coupling constant in its H-NMR
spectrum. Unlike NND-OH (3), alkenyne 4 was soluble in
methylene chloride up to 0.2 M and readily reacted with 2,3,4,6-
tetraacetoxy-R-D-glucopyranosyl bromide under the conditions
of Roy. Since partial hydrolysis of the C(6)-acetate often
occurred under these conditions, the crude material was directly
subjected to methanolysis, providing a single compound, 2.
Alternatively, buffering of the methanolysis reaction with
benzoic acid provided the readily recrystallized benzoate salt
of 2. Encouraged by this success, efforts were directed at
preparing a derivative which contained an R-glucopyranosidic
linkage for binding to Con A. This was accomplished by
appending maltose, as it already contained an R-glucopyranoside
and the association constant for the binding of maltose to Con
A was determined to microcalorimetrically to be 1.6 × 103
a direct construction, providing 3 and 4 in significantly higher
overall yield and without the need for tedious chromatographic
separation. This was accomplished through coupling of pho-
sponate 8 with the corresponding aldehydes 9 and 13 by means
of a Wadsworth-Horner-Emmons modified Wittig reaction.24
This disconnection was chosen on the basis the high trans-
stereoselectivity of this method and the fact that aldehyde 9 is
commercially-available and the other materials were obtained
in three steps. Phosphonate 8 was prepared through functional
manipulation of aldehyde 5. This sequence began by reducing
the carbonyl group in 5 with NaBH4 as described in Scheme 1.
The resulting alcohol 6 was then converted to bromide 7 using
the phosphonium salt method of Appel.25 In turn, this bromide
was displaced with triethyl phosphite at 150 °C in DMF,
providing phosphonate 8. This sequence was readily conducted
on a 10 g scale with an 83% overall yield. In accordance with
the Wadsworth-Horner-Emmons modified Wittig procedure,
the ylide of 8 was generated by the addition of 2.2 equiv of
NaHMDS in THF to the crude displacement mixture at 0 °C.
The production of a deep purple color upon surpassing addition
of the first equivalent of base provided a convenient internal
standard. Condensation of this dianion with p-(N,N-dimethyl-
amino)cinnamaldehyde (9) provided a 54% yield of NND-OH
(3).
M-1 14g
Under conditions previously described for construction
.
of 2, the benzoate of â-maltoside 1 was obtained in 73% yield
from 4 and the corresponding peracetylated R-pyranosyl bromide
of maltose. The free bases 1 and 2 were obtained by warming
solutions of their benzoate salts in 1,4-dioxane containing
powdered 4 Å molecular sieves.
The absorption and fluorescence spectra of 1 and 2 were
examined in solvents ranging from THF to methanol (see Table
1). These materials absorbed in two regions, one centered at
about 390-435 nm and the other between 280 and 320 nm.
The position and intensity of this absorption were nearly
identical for both 1 and 2. With the exception of methylene
chloride and chloroform, the position of the lower energy band
deviated only 3% from ∼400 nm and was on average 20-30
nm lower than that of 4. The fluorescence maxima were nearly
identical for 1 and 2, and their quantum yields in methanol were
36% and 39% less than those in THF, respectively. In addition,
fluorescence from maltoside 1 and glucoside 2 could be detected
up to 720 nm in THF, while no fluorescence was detected above
580 nm in methanol. The closest line of an argon laser to the
Numerous methods were examined for attaching 3 to the
anomeric center of carbohydrates.26 Unfortunately, NND-OH
(3) decomposed under the conditions generated during activation
of phenylthio, phenylselenyl, fluoro, and pentenyl glycosides.
Classical Konigs-Knorr coupling with R-D-glucopyranosyl
bromide tetraacetate resulted in either recovery or slow decom-
position of 3. Recently, Roy developed a phase transfer method
(24) Wadsworth, W. S. Org. React. 1977, 25, 73.
(25) Appel, R. Angew. Chem. Int. Ed. Engl. 1975, 14, 801.
(26) (a) Paulsen, H. Chem. Soc. ReV. 1989, 61, 1257. (b) Hale, K. J.;
Richardson, A. C. In The Chemistry of Natural Products; Thompson, R.
H., Ed.; Chapman and Hall: New York, 1985; pp 1-59. (c) Schmidt, R.
R. In ComprehensiVe Organic Synthesis; Trost, B. M., Fleming, I., Eds.;
Permagon Press: Oxford, 1991; Vol. 6, pp 33-64. (d) Toshima, K.; Tatsuta,
K. Chem. ReV. 1993, 93, 1503.
(27) (a) Roy, R.; Tropper, F. D. Synth. Commun. 1990, 20, 2097. (b)
Roy, R.; Tropper, F. D.; Romanowska, A.; Letellier, M.; Cousineau, L.;
Meunier, S. J.; Boratynski, J. Glycoconjugate J. 1991, 8, 75. (c) Roy, R.;
Tropper, F. D. Can. J. Chem. 1991, 69, 817.
(28) Takalo, H.; Kankare, J.; Haenninen, E. Acta. Chim. Scand. 1988,
B42, 448.

7680 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
La Clair
Scheme 2a
a Conditions: (a) (i) iodine, NaHCO3, H2O, 12-15 °C, 10 min; (ii) then rt, 1 h, 86%. (b) Propargyl alcohol, Cl2Pd(PPh3)2 (1.4 mol %), CuI (1.4
mol %), Et3N, rt, 18 h, 89%. (b) MnO2, CH2Cl2, rt, 4 h, 89%. (d) (i) Add NaHMDS (2.2 equiv) in THF to crude 8 in DMF, 0 °C to rt, 1 h; (ii) add
13 in THF, -20 °C to rt, 6 h, 56%. (e) (i) R-D-Maltopyranosyl bromide heptaacetate, N-benzyltriethylammonium chloride, 1 M NaOH, CH2Cl2, rt,
12 h; ii. NaOCH3, CH3OH, rt, 1 h; (iii) PhCO2H; (iv) Powdered 4 Å molecular sieves, 1,4-dioxane, 70 °C, 2 h; 69%. (f) (i) R-D-Glucopyranosyl
bromide tetraacetate, N-benzyltriethylammonium chloride, 1 M NaOH, CH2Cl2, rt, 12 h; (ii) NaOCH3, CH3OH, Ph, rt, 1 h; (iii) PhCO2H; (iv)
Powdered 4 Å molecular sieves, 1,4-dioxane, 70 °C, 2.5 h; 71%.
Table 1. Absorption Maxima, Extinction Coefficients (ꢀ), Fluorescence Maxima, and Quantum Yields (Φf) for Compounds 1 and 2
â-glucoside 1
â-maltoside 2
â-glucoside 1
â-maltoside 2
solvent
THF
Et
λA (max), nm ꢀ, 104 cm-1 M-1 λA (max), nm ꢀ, 104 cm-1 M-1 λf (max), nm
Φf
λf (max), nm
Φf
37.4
358-427
298
428
314
419
359-431
402
301
406
3.23
4.04
3.41
3.54
3.52
4.85
3.41
3.23
4.52
3.54
4.52
4.06
381-419
3.97
4.13
610
478
519
0.000 068
0.000 094
0.000 088
602
482
0.000 073
0.000 097
298
CHCl3
39.1
CH2Cl2
acetone
DMF
41.4
42.2
43.8
531
492
495
0.000 082
0.000 071
0.000 075
375-421
406
302
410
306
401
300
4.89
3.32
3.23
4.64
3.97
4.63
4.07
488
480
434
476
0.000 061
0.000 073
0.000 082
0.000 094
DMSO
45.0
472
499
0.000 11
304
366-421
299
acetonitrile 46.0
isobutanol 49.0
l-butanol
0.000 063
495
0.000039
397
301
4.02
4.21
405
300
4.13
4.25
551
0.000 099
548
0.000096
361-452
282
356-419
298
367-422
296
3.89
4.21
3.01
2.82
3.07
3.75
376-425
297
401
299
398
4.05
4.32
3.12
2.96
3.31
3.76
556
550
541
0.000 1
542
537
499
0.000 11
0.000 055
0.000 027
ethanol
51.9
55.5
0.000 053
0.000 027
methanol
absorption maxima of 1 and 2 was at 457 nm. At this excitation
wavelength, the emission from 1 and 2 was ∼25% of that at
400 nm in THF. Fluorescence from 1 and 2 in media
established for monosaccharide binding to the tetrameric form
of Con A (i.e., 0.05 M in PIPES (pH 7.2), 10 mM in MnCl2,
10 mM in CaCl2, and 1 M in NaCl) could not be detected in a
SLM-Aminco fluorimeter, even at saturation or at its absorption
maximum.
Confocal fluorescence correlation spectrometry was measured
using the spectrometer described by Eigen and Rigler.7 Samples
of 1-4 were examined in several organic solvents by placing
a droplet of the appropriate solution in a ∼20 µL conical gold
well and bringing the droplet in contact with a thin microscope
slide which hung from a water immersible microscope objective
by a drop of water.29 Excitation was provided by passing the
457 nm line of an argon ion laser through the objective at 0.5

Carbohydrate-Bound State of ConcanaValin A
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7681
Figure 3. Real time and autocorrelated traces demonstrating carbohydrate binding to Con A. Samples were in a buffer which was 10 mM in
MnCl2, 10 mM in CaCl2, 0.05 M in PIPES (pH 7.2), and 1 M in NaCl, irradiated with a laser tuned to 457 nm (0.5 mW) and collected through an
Omega Optics 545 cutoff filter. For each sample, the upper trace indicates the frequency of emitted photons (kHz) and shows transients from
aggregates in real time. The lower trace provides the autocorrelation function, where the number of particles inside the volume element is given by
1/g2 as t f 0. The midpoint of the autocorrelated curve provides an approximation of the diffusion time. (A) 51 µM 1, (B) 12 µM 2, (C) 51 µM
1 with 170 µM Con A, (D) 12 µM 2 with 170 µM Con A.
Table 2: Behavior of Compounds 1-4 in the Eigen-Rigler
mW. Aqueous solutions were more easily sampled by directly
Confocal Fluorescence Correlation Spectrometera
hanging the droplet (approximately 20 µL) from the microscope
photostability,
% deviation
slide. The resulting fluorescence were collected through the
detection limit, nM
same objective by harvesting the emitted photons through a
dichroic mirror, followed by filtering with an Omega Optics
545 nm cutoff filter, and counting with a SPAD detector.
Autocorrelation was provided online with an ALV-card attached
to a PC. The fluorescence fluctuation from compounds 1-4
deviated within 5% in most solvents, with the exception of
chloroform and methanol (Table 2). The enhanced decomposi-
tion in these solvents can be attributed to slow cleavage of the
anomeric center by methanolysis or trace acidity often found
in chloroform. In the confocal FCS spectrometer, autocorre-
lation from samples of 3 and 4 was readily detected at the
picomolar level in heptane and decreased to nearly the micro-
molar level with increased polarity, as reflected by the loss of
quantum efficiency. Autocorrelation from 1 and 2 could be
detected at a 10-fold lower concentration in THF than in
methanol. Samples of 1 and 2 in the buffer commonly used
for binding of carbohydrates to Con A did not autocorrelate at
any concentration, nor were transients detected as shown in
solvent
1
2
3
4
1
5
2
3
4
heptane
THF
CHCl3
acetone
DMF
0.002
0.011
1
1
2
9
3
2
4
2
2
70
74
62
79
58
14
0.28
14
49
12
0.85
20
61
240
93
1
8
4
1
2
2
9
1
13
4
2
3
2
2
5
4
2
3
7
350 165
acetonitrile 240 230 220
ethanol
methanol
170 120
510 290 160
62
380
a Excitation was provided at 455 nm (0.5 mW), and the fluorescence
was collected through a 545 nm cutoff filter.
Figure 3 (traces A and B). Samples of 51 µM 1, 12 µM 2, and
a blank fluoresced with a fluctuation of about 5.0, 6.8, and 4.3
kHz, respectively. As shown in the upper portion of trace C,
transients from the diffusion of single molecules containing the
fluorophore into the volume element were observed in a solution
which contained 170 µM Con A and 51 µM 1 at pH 7.2. The
number of molecules in the cavity is N ) 0.83, as given by N
) 1/g2, and therefore the concentration of the bound material
in this cavity (volume 2 × 10-15 L) is 0.69 nM.30 On the basis
of the reported affinity of Con A for R-glucopyranosides and
lack of â-glucopyranosides, the transients seen originate from
(29) The absorption cross-sectional area for alkenyne 4 at 457 nm is
maximally 3 × 10-16 cm2. A laser beam with a single line power of 0.5
mW provides a photon density of 1024 photons/(cm2 s). Within this beam,
the singlet excitation rate of this fluorophore would be 3 × 10-8 s-1, as
given by the product of its cross-sectional area and photon density.

7682 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
La Clair
22.4 mmol) with NaBH4 (3.46 g, 91.5 mmol) in a 5:5:1 mixture of
methanol, ether, and THF (77 mL). This was accomplished by adding
NaBH4 to the solution of 5 at 0 °C over 30 min and then warming
over 2 h to room temperature (rt). The reaction was quenched with
10% aqueous HCl (until the pH was approximately 6), poured onto 80
mL of brine (80 mL), extracted with 200 mL of CH2Cl2 (3×), dried
with Na2SO4, and concentrated. The crude product was used directly
for the next step. Pure material could be obtained through flash
chromatography (SiO2, 50% ethyl acetate/hexanes), yielding 3.67 g
the complex of 1 with Con A. This was verified by the fact
that transients due to aggregates were not detected in samples
of 1 without Con A (trace A) or 2 with and without the addition
of Con A (traces B and D, respectively).31 Upon complexation,
the weight about the fluorescent label dramatically increases
from a formula weight of 632 to approximately 100 000. This
increase would therefore be translated into a slower diffusion
time upon binding, as the diffusion is dependent on the size of
a material. As given by autocorrelation, the midpoint of descent
in the autocorrelated curve is an approximation of the size about
the fluorescent moiety. The diffusion times of 1 and 2 ranged
between 0.04 and 0.06 ms in various solvents, while that
attributed to complexes of 2 with Con A was approximately 19
ms, as seen in Figure 3. Similar increases in diffusion time
have been seen in the complexation of a BODIPY-labeled DNA
primer to M13-DNA.7
1
(97%) of 6: mp 79.7-81.3 °C; Rf ) 0.37; H NMR (CDCl3) δ 10.60
(s, 1H), 8.06 (d, J ) 8.8 Hz, 1H), 7.14 (s, 1H), 6.94 (d, J ) 8.8 Hz,
1H), 4.73 (d, J ) 5.5 Hz, 2H), 1.97 (t, J ) 5.5 Hz, 1H); 13C NMR
(CDCl3) δ 63.7, 116.9, 117.9, 125.2, 151.7, 155.3; IR (CHCl3) 3459,
2356, 1620, 1579, 1520, 1475 cm-1. Anal. Calcd for C7H7NO4: C
49.71, H 4.17, N 8.28. Found: C 50.13, H 4.30, N 8.20.
m-Hydroxy-p-nitrobenzyl Bromide (7). Carbon tetrabromide (2.86
g, 8.63 mmol) was added over 30 min to a solution of 6 (∼1.22 g,
∼7.22 mol) and PPh3 (2.45 g, 9.35 mmol) in 25 mL of dry CH2Cl2 at
0 °C. The reaction was warmed to rt over 1.5 h and allowed to stand
for an additional 1 h, at which point the mixture was poured onto water,
extracted with 100 mL of CH2Cl2 (2×), dried with Na2SO4, and
concentrated. Pure material was obtained by flash chromatography
(SiO2, 33% ethyl acetate/hexanes) yielding 1.42 g (86%) of 7: mp
Conclusion
This investigation describes a new scheme for monitoring
interactions between carbohydrates and proteins. Although the
study so far is limited to the interaction between Con A and a
â-maltopyranoside, the method provides selective detection of
carbohydrate-bound lectin. Combined with the fact that this
fluorescent tag responds not only to solvent polarity but also to
confinement, the method should be applicable to a wide variety
of events where the label encounters restriction upon binding.
Investigations into the reason for this fluorescence enhancement
and application of this method to study aggregation of these
complexes are currently underway.
1
68.9-70.2 °C; Rf ) 0.58; H NMR (CDCl3) δ 10.58 (s, 1H), 8.07 (d,
J ) 8.6 Hz, 1H), 7.16 (d, J ) 1.9 Hz, 1H), 6.99 (dd, J ) 1.9, 8.8 Hz,
1H), 4.39 (s, 2H); 13C NMR (CDCl3) δ 30.6, 120.1, 120.8, 125.7, 147.8,
155.2; IR (CHCl3) 2354, 1622, 1584, 1479, 1328, 1258, 1159, 968,
885, 844, 652 cm-1. Anal. Calcd for C7H6NO3Br: C 36.23, H 2.61,
N 6.04. Found: C 36.10, H 2.69, N 5.92.
Diethyl (m-Hydroxy-p-nitrobenzyl)phosphonate (8). A mixture
of 7 (0.91 g, 3.98 mmol) and triethyl phosphite (0.87 mL, 5.09 mmol)
was refluxed (bath temperature 155 °C) in 2.5 mL of dry DMF for 1.5
h. Pure material was obtained through flash chromatography (SiO2,
25% ethyl acetate/hexanes), yielding 1.14 g (99%) of 8: mp 59.3-
62.1 °C, Rf ) 0.09; 1H NMR (CDCl3) δ 10.56 (s, 1H), 8.01 (d, J ) 8.7
Hz, 1H), 7.04 (dd, J ) 2.2 Hz, JH-P ) 2.2 Hz, 1H), 6.91 (dd, J ) 2.2,
8.7 Hz, JH-P ) 2.2 Hz, 1H), 4.04 (qd J ) 7.1 Hz, JH-P ) 8.1 Hz, 2H),
3.04 (d, JH-P ) 22.5 Hz, 2H), 1.25 (q, J ) 7.1 Hz, 3H); 13C NMR
(CDCl3) δ 16.2 (d, JC-P ) 5.3 Hz), 34.2 (d, JC-P ) 136.7 Hz), 62.4 (d,
JC-P ) 6.5 Hz), 120.7 (d, JC-P ) 8.3 Hz), 121.8 (d, JC-P ) 6.0 Hz),
Experimental Section
Chemical Synthesis. All reactions were conducted under an argon
atmosphere in rigorously dried glassware and were magnetically-stirred
with a Teflon-coated stir bar, unless otherwise indicated. Reagents
were added to reaction vessels Via a cannula or dry syringe. Anhydrous
tetrahydrofuran (THF) was distilled from sodium-benzophenone ketyl.
Methylene chloride, methanol, 1,4-dioxane, and N,N-dimethylforma-
mide (DMF) were purchased dry from Aldrich. Materials reacted under
anhydrous conditions were dried extensively with toluene azeotrope
prior to use. Thin layer chromatography (TLC) on Merck silica gel
DC 60 plates was routinely used to monitor all reactions. TLC plates
were developed by staining with iodine absorbed on silica gel. All Rf
values were collected from runs in 33% ethyl acetate/hexane. Melting
points were measured on a Bu¨chi 520 and are uncorrected. Infrared
spectra (IR) were collected on a Perkin-Elmer Paragon 1000 PC FT-
IR spectrometer. Samples were prepared on sodium chloride (NaCl)
plates, neat or in a chloroform smear. UV-vis and fluorescence spectra
were measured on a Perkin-Elmer Lambda 17 UV-vis spectrometer,
Perkin-Elmer LS-5B luminescence spectrometer, and SLM-Aminco
SPF-500C. 1H-NMR and 13C-NMR spectra were obtained at 300 and
75 MHz, respectively, on a Bruker MSL300. Chemical shifts (δ) are
given in parts per million and coupling constants (J) in hertz.
Microanalyses were obtained from Beller Microanalytisches Labor
(Go¨ttingen, Germany). Standard flash chromatography was performed
on Merck 9395 silica gel using a gradient from hexane to the solvent
listed. Samples of 1 and 2 were recrystallized three times from spectral
grade methanol at -20 °C to ensure purity.
125.0 (d, JC-P ) 2.4 Hz), 143.2 (d, JC-P ) 8.8 Hz), 154.9 (d, JC-P
)
3.8 Hz); IR (CHCl3) 3850, 3444, 2985, 1623, 1587, 1520, 1480, 1443
cm-1. Anal. Calcd for C11H16NO6P C 45.68, H 5.58, N 4.84. Found:
C 45.60, H 5.74, N 4.83.
trans-1-[p-(N,N-Dimethylamino)phenyl]-trans-4-(m-hydroxy-p-ni-
trophenyl)-1,3-butadiene (3). Sodium bis(trimethylsilyl)amide (0.99
mL, 1.0 M in THF) was added to the crude solution of phosphonate 8
(143.6 mg, 0.453 mmol in 0.8 mL of DMF) from the above step at 0
°C. Thirty minutes later, the solution was warmed to room temperature
and kept there for 20 min, at which point, the contents were recooled
to -20 °C and reacted with a solution of (N,N-dimethylamino)-
cinnamaldehyde (74.2 mg, 0.430 mmol) in 4 mL of THF. After 8 h at
ambient temperature, 15 mL of ice cold brine was added. The pH
was adjusted to 7 with dilute HCl, and the crude product was obtained
by repetitive extraction with 10% THF in CH2Cl2, drying with Na2-
SO4, and concentration. Flash chromatography (33% CHCl3/hexane)
and recrystallization from 10:1 heptane/THF provided 302.7 mg (55%)
1
of 3: mp 216.3-217.2 °C; Rf ) 0.43; H NMR (CDCl3) δ 10.74 (s,
1H), 7.99 (d, J ) 8.7 Hz, 1H), 7.24 (d, J ) 8.7 Hz, 2H), 7.08 (dd, J
) 7.8, 15.5 Hz, 1H), 7.03 (dd, J ) 7.8, 16.9 Hz, 1H), 7.02 (d, J )
16.9 Hz, 1H), 6.75 (d, J ) 7.8 Hz, 2H), 6.74 (s, 1H), 6.65 (d, J ) 8.7
Hz, 2H), 6.47 (d, J ) 15.4 Hz, 1H), 2.98 (s, 6H); 13C NMR (DMSO-
d6): δ 30.6, 112.3, 115.9, 117.0, 124.1, 124.6, 125.8, 127.4, 128.0,
133.9, 135.2, 136.7, 145.6, 150.6, 153.6; IR (trace CHCl3) 3850, 3741,
2357, 2169, 1574, 1470, 1219, 962, 944, 772, 674 cm-1. Anal. Calcd
for C18H18N2O3: C 69.66, H 5.85, N 9.03. Found: C 69.72, H 5.97,
N 9.06.
m-Hydroxy-p-nitrobenzyl Alcohol (6). The synthesis of phospho-
nate 8 was accomplished in three operations from commercial aldehyde
5. This functional conversion began by reducing aldehyde 5 (3.74 g,
(30) The concentration provided is a description of the Con A-maltoside
1 complex which undergoes fluorescence. The total concentration of the
Con A-maltoside 1 complex is much larger and requires knowledge of its
quantum yield. Since there is no direct means to determine this yield, the
affinity of this interaction cannot be calculated with this method.
(31) Transients were also not detected in samples with concentrations
of 2 ranging from 0.012 µM up to the point where the background was to
large for the detector (∼200 µM) with and without Con A.
N,N-Dimethyl-p-iodoaniline (11). Resublimed iodine (4.28 g, 16.8
mmol) was added in small portions over 45 min to a mixture of N,N-
dimethylaniline (10) (2.37 mL, 18.7 mmol) and NaHCO3 (2.35 g, 27.9
mmol) in 16 mL of water between 12 and 15 °C. Ten minutes after

Carbohydrate-Bound State of ConcanaValin A
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7683
complete addition, the mixture was warmed to rt. This mixture was
diluted with 500 mL of ether and the organic phase extracted
consecutively with 50 mL of water, 100 mL of sodium thiosulfate,
and 100 mL of water (2×). Afterward, the crude product was dried
with Na2SO4, concentrated, and recrystallized from 10:1 hexane/ether
of 1 M NaOCH3 in methanol. After 1 h, 330 mg of benzoic acid was
added followed by 1.2 g of NaHCO3, 15 min later. The residual
NaHCO3 was filtered off, and the excess methanol was removed by
rotary evaporation. Flash chromatography (SiO2, 10:1:1 ethyl acetate/
methanol/toluene) and recrystallization from methanol afforded 510.2
mg (86%) of a 1:1 mixture of 2 and its benzoate salt. The pure amine
was obtained by heating this material for 2 h in dry 1,4-dioxane
containing 1.3 g of 4 Å molecular sieves. The crude material was
obtained by filtration aided by extensive washing with methanol and
concentration. Flash chromatography (SiO2, 10:1:1 ethyl acetate/
methanol/toluene) and recrystallization from methanol (3×) yielded
375.2 mg (71%) of pure 2: mp ) 177.9-182.5 °C; Rf ) 0.28 (10:1:1
1
to yield 3.98 g (86 %) of 11: mp 63.5-66.1 °C; Rf ) 0.66; H NMR
(CDCl3) δ 7.45 (d, J ) 8.9 Hz, 2H), 6.46 (d, J ) 8.9 Hz, 2H), 2.90 (s,
6H); 13C NMR (CDCl3) δ 40.2, 114.8, 137.3, 149.9.
3-[p-(N,N-dimethylamino)phenyl]prop-2-ynol (12). A mixture of
iodide 11 (309.7 mg, 1.25 mmol), Cl2Pd(PPh3)2 (6.4 mg, 0.0091 mmol),
and CuI (9.5 mg, 0.0499 mmol), in 1.2 mL of triethylamine was
degassed by a rapid bubbling of argon. After 30 min, 2-propyn-1-ol
(0.087 ml, 1.37 mmol) was added via microliter syringe. Three hours
later, a second batch of Cl2Pd(PPh3)2 (6.4 mg) and CuI (9.5 mg) was
added, and the reaction went to completion within 18 h. A strict
maintenance of an argon atmosphere was crucial to the yield of this
manipulation. The crude solution was filtered through 20 g of silica
gel with ethyl acetate and concentrated. Pure material was obtained
through flash chromatography (SiO2, 25% ethyl acetate/hexanes),
yielding 195.2 mg (89%) of 12: mp 51.2-53.7 °C; Rf ) 0.31; 1H NMR
(CDCl3) δ 7.30 (d, J ) 8.9 Hz, 2H), 6.60 (d, J ) 8.9 Hz, 2H), 4.45 (d,
J ) 4.1 Hz, 2H), 2.95 (s, 6H); 13C NMR (CDCl3) δ 40.1, 51.8, 85.1,
88.7, 109.5, 111.8, 132.8, 150.3; IR (trace CHCl3) 3355, 2860, 1608,
1520, 1445, 1360, 1225, 1190, 1024, 955, 818 cm-1. Anal. Calcd for
C11H13NO: C 75.40, H 7.48, N 7.99. Found: C 75.33, H 7.42, N
7.96.
1
ethyl acetate/methanol/toluene); H NMR (CDCl3) δ 7.72 (d, J ) 8.4
Hz, 1H), 7.44 (d, J ) 1.4 Hz, 2H), 7.20 (d, J ) 8.7 Hz, 1H), 7.17 (dd
J ) 1.4, 8.4 Hz, 1H), 6.85 (d, J ) 16.2 Hz, 1H), 6.62 (d, J ) 8.7 Hz,
2H), 6.60 (d, J ) 16.0 Hz, 2H), 5.03 (d, J ) 7.3 Hz, 2H), 3.86 (dd, J
) 2.1, 12.0 Hz, 2H), 3.67-3.57 (m, 2H), 3.50-3.37 (m, 3H), 3.28-
3.22 (m, 1H), 2.90 (s, 6H); 13C (CD3OD) δ 40.6, 63.1, 71.8, 75.2, 78.4,
79.0, 88.0, 97.4, 103.2, 111.4, 113.4, 115.1, 116.5, 121.1, 127.0, 134.0,
139.1, 144.7, 152.4; HRMS (FAB) (C24H26N2O8) found 470.1689, found
470.1699.
Maltopyranoside 1. A saturated solution of HBr in HOAc (1.5 mL)
was added to an 18:1 mixture of â/R peracetylated maltose in 0.4 mL
of HOAc at 0 °C. The mixture was warmed to 15 °C over 30 min and
kept there for an additional 30 min, at which point it was concentrated
to dryness by warming to 40 °C at 10 mmHg (generated by a water
aspirator pump). N-Benzyltriethylammonium chloride (106.0 mg, 0.32
mmol) was added to a suspension of 4 (0.0987 mg, 0.32 mmol) and
the above maltopyranosyl bromide (891.0 mg, 1.27 mmol) in 8 mL of
CH2Cl2 and 8 mL of 1 M NaOH. After 12 h, the reaction mixture was
diluted with 4 mL of cyclohexane and filtered. The filter paper was
dried, washed extensively with methanol (∼100 mL), and concentrated
to 80 mL. This solution was treated with 1.2 mL of a 1 M methanolic
solution of NaOCH3 for 1 h. The reaction was buffered by addition of
380 mg of benzoic acid. The crude product was obtained by filtering,
washing extensively with methanol, and concentrating. Flash chro-
matography (SiO2, 10:1:1 ethyl acetate/methanol/toluene to methanol)
and recrystallization from methanol yielded the benzoate salt of 1. The
pure amine was obtained by heating the above material for 2 h in 5
mL of dry 1,4-dioxane containing 0.8 g of 4 Å molecular sieves. The
crude material was obtained by filtration aided by extensive washing
with methanol and concentration. Recrystallization from methanol (3×)
yielded 139.7 mg (69%) of pure 1: mp ) 229.2-232.7 °C; Rf ) 0.54
(CH3OH); 1H NMR (CDCl3) δ 7.72 (d, J ) 8.6 Hz, 1H), 7.42 (d, J )
1.2 Hz, 2H), 7.20 (d, J ) 8.7 Hz, 1H), 7.17 (dd J ) 1.2, 8.6 Hz, 1H),
6.66 (d, J ) 16.2 Hz, 1H), 6.62 (d, J ) 8.7 Hz, 2H), 6.60 (d, J ) 16.2
Hz, 2H), 5.13 (d, J ) 3.7 Hz, 2H), 5.07 (d, J ) 7.6 Hz, 2H), 3.88 (dd,
J ) 12.2 Hz, 2H), 3.79-3.41 (m, 8H), 3.37 (dd, J ) 3.7, 9.7 Hz, 1H),
3.35-3.21 (m, 2H), 2.90 (s, 6H); 13C (DMSO-d6) δ 39.6, 61.5, 62.4,
67.1, 67.7, 71.2, 73.5, 73.6, 74.0, 74.4, 76.6, 77.0, 80.5, 87.3, 101.8,
102.1, 112.3, 113.9, 115.1, 120.2, 125.5, 125.8, 128.9, 129.9, 132.9,
133.1, 137.1, 143.2. Anal. Calcd for C30H36N2O13: C 56.96, H 5.74,
N 4.43. Found: C 56.89, H 5.62, N 4.41.
[3-p-(N,N-dimethylamino)phenyl]prop-2-ynal (13). Activated MnO2
(1.148 g, 13.2 mmol) was added to a solution of 12 (421.5 mg, 2.41
mol) in 10 mL of CH2Cl2 at rt. After 4 h, the reaction was purified
directly by flash chromatography (SiO2, 25% ethyl acetate/hexanes),
yielding 371.8 mg (89%) of 13: mp 81.4-82.3 °C; Rf ) 0.49; 1H NMR
(CDCl3) δ 9.33 (s, 1H), 7.44 (d, J ) 9.0 Hz, 2H), 6.60 (d, J ) 9.0 Hz,
2H), 3.01 (s, 6H); 13C NMR (CDCl3) δ 39.8, 96.0, 99.9, 104.9, 111.5,
135.2, 152.1, 176.2; IR (trace CHCl3) 2149, 1643, 1595, 1380, 1190,
981 cm-1
. Anal. Calcd for C11H11NO: C 76.28, H 6.40, N 8.09.
Found: C 76.42, H 6.41, N 8.07. Alternatively, large-scale preparations
can be purified by recrystallization from 20:1 heptane/THF.
1-[p-(N,N-dimethylamino)phenyl]-trans-4-(p-nitrophenyl)-1-buten-
3-yne (4). Sodium bis(trimethylsilyl)amide (6.27 mL, 1.0 M in THF,
6.27 mmol) was added to the crude solution of the phosphonate 8
(∼852.0 mg, ∼2.95 mmol) in 2.0 mL of DMF at 0 °C. A dramatic
color change (light yellow to deep magenta) occurred upon exceeding
the first equivalent of base. This internal standardization was routinely
used to ensure proper addition of base. The solution was warmed to
rt after 30 min at 0 °C and kept there for 20 min, at which point it was
cooled to -20 °C and aldehyde 6 (310.0 mg, 1.79 mmol) in 5 mL of
THF was added Via cannula. After 8 h at rt, 5 mL of water was added,
the pH was adjusted to 7 with dilute HCl, and then the solution was
further diluted with 10 mL of brine. Crude product was obtained by
extraction with 40 mL of 10% THF in CH2Cl2 (3×), dried with Na2-
SO4, and concentrated. Flash chromatography (SiO2, 33% CHCl3/
hexanes) and recrystallization from heptane/THF (10:1) yielded 308.2
1
mg (56%) of 4: mp 185.6-186.9 °C; Rf ) 0.28; H NMR (CDCl3) δ
Absorption and Fluorescence Measurements. Samples of 1 and
2 were prepared by dissolving between 0.2 and 3.8 mg in a 5 mL
volumetric flask using the appropriate solvent. Absorption spectra were
run in quartz cuvettes with a width of 0.5 cm. Fluorescence intensities
were compared at a standard concentration of 10 µM, and the quantum
10.65 (s, 1H), 8.02 (d, J ) 8.8 Hz, 1H), 7.34 (d, J ) 8.9 Hz, 2H), 7.07
(d, J ) 1.6 Hz, 1H), 6.99 (dd J ) 1.6, 8.9 Hz, 1H), 6.84 (d, J ) 16.0
Hz, 1H), 6.62 (d, J ) 8.9 Hz, 2H), 6.55 (d, J ) 16.0 Hz, 2H), 2.98 (s,
6H); 13C NMR (DMSO-d6): δ 39.6, 87.4, 96.6, 108.9, 111.2, 113.8,
116.6, 117.0, 125.7, 132.6, 135.2, 136.8, 143.7, 150.5, 153.1; IR (trace
CHCl3) 3850, 3741, 2357, 2169, 1574, 1470, 1219, 962, 944, 772, 674
yields were standardized against 0.70 for rhodamine B in ethanol.16
A
list of the absorption maxima, extinction coefficients, fluorescence
maxima, and quantum yields provided is an average of three repetitions
of the above; the data deviated within 4%. Several samples were diluted
10-fold (to 1 µM) and their fluorescence spectra retaken to ensure that
the data were not enhanced by aggregation.
Concanavalin A Binding Studies. Concanavalin A, type VI, was
purchased from Sigma (Lot 105H9567) and used as is. Water used
for FCS studies was distilled twice following deionization. MnCl2 and
CaCl2 were purchased from Sigma, molecular biology grade. The
buffer was prepared with 0.05 M PIPES (pH 7.2), 10 mM MnCl2, 10
mM CaCl2, and 1 M NaCl. Stock solutions of glucosides 1 and 2 were
prepared at 0.24 and 0.12 mM in the above buffer by first dissolving
in 100 µL of ethanol and then diluting with buffer to 10 mL. These
cm-1
. Anal. Calcd for C18H16N2O3: C 70.12, H 5.23, N 9.09.
Found: C 70.22, H 5.49, N 9.07.
Glucopyranoside 2. N-Benzyltriethylammonium chloride (254.8
mg, 1.12 mmol) was added to a suspension of 4 (345.1 mg, 1.12 mmol)
and 2,3,4,6-tetraacetoxy-R-D-glucopyranosyl bromide (922.0 mg, 2.24
mmol) in 4 mL of CH2Cl2 and 10 mL of 1 M NaOH. After 12 h, the
reaction mixture was diluted with 100 mL of ethyl acetate and 20 mL
of water and extracted. The aqueous phase was further extracted with
50 mL of ethyl acetate (2×), and the combined organic layers were
washed with 10 mL of water (2×). Crude material was obtained by
drying with Na2SO4 and concentrating. This material was dissolved
in 10 mL of methanol and 6 mL of benzene and treated with 0.25 mL

7684 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
La Clair
samples were warmed to 45 °C prior to usage to ensure complete
solvation. A 0.5 mM stock solution of Con A was prepared in each
buffer and verified by comparison to the reported ꢀ ) 1.14 cm2/mg at
280 nm.32 Samples were filtered through a Cameo 25 GAS syringe
filter immediately prior to use to remove any interfering particles. The
stability of Con A to the conditions was verified by electrophoresis
upon completion of the measurements.
Samples containing 51 µM 1 and 12 µM 2 were prepared with a
gradient of Con A from 0 µM, to 0.5 µM, 5.0 µM to 50 mM to 170
µM. These samples were stored at room temperature for 12 h prior to
measurement. A single droplet of these solutions (∼20 µL) was hung
from fresh microscope slides and its fluorescence observed over 30 s.
Repetition with three different preparations provided intensities of
fluctuation within 3% of the original run. Signals due to aggregates
were detected in a solution with 50 µM 1 and 170 µM Con A. Signals
were not detected in samples of 2 with 170 µM Con A nor in the
presence of 1 mM Con A.
FCS studies were conducted by excitation with the 457 nm line of
an argon laser (Lexel Argon Ion, Waldbroon, Germany) at 0.5 mW
which was focused through a water-immersion microscope objective
(Zeiss Plan Neofluar 40 × 0.9), providing a volume element of
approximately 2 × 10-16 L. Fluorescent molecules were excited for
0.1-50 ms as they passed through this volume element, as given by
diffusion coefficients. The fluorescence was collected by the same
objective, and light scattering was blocked with a dichroic mirror and
passed through a 545 cutoff filter (Omega Optics) and a pinhole in
image space. Fluorescence was detected by a SPAD (EG & G-Chemie,
Steinheim, Germany), and signal autocorrelation was carried out by a
PC with a digital autocorrelator card (ALV-5000 Fa. Peters, Langen,
Germany).
Acknowledgment. I especially thank Manfred Eigen and
Richard Lerner for their gracious support and encouragement
while at the Max-Planck-Institute for Biophysical Chemistry
(MPI-BPC), Petra Schwille (MPI-BPC) for assistance with
collection of the FCS data, Hansjorg Eibl (MPI-BPC) for use
of his laboratories for the synthetic portion of this project, and
Kim Janda (TSRI) for use of his SLM-Aminico fluorimeter.
Special thanks is given to Mary-Gabriela Cotenescu for as-
sistance with preparation of this document. This paper is
dedicated to Gilbert Stork on the occasion of his 75th birthday.
(32) Wang, J. L.; Becker, J. W.; Reeke, J. N.; Edelman, G. M. Proc.
Natl. Acad. Sci. U.S.A. 1971, 68, 1130.
JA964366G