Boronic acid porphyrin receptor for ginsenoside sensing

Article abstract of DOI:10.1021/ol1019647

Ginsenoside detection was pursued through the design of a porphyrin receptor containing two boronic acid units. This receptor was found to undergo different degrees of fluorescence quenching with five ginsenoside guests and an acylated derivative. The tre

Full text of DOI:10.1021/ol1019647

ORGANIC
LETTERS
2010
Vol. 12, No. 21
4804-4807
Boronic Acid Porphyrin Receptor for
Ginsenoside Sensing
Amanda E. Hargrove,† Ryan N. Reyes,† Ian Riddington,† Eric V. Anslyn,*,† and
Jonathan L. Sessler*,†,‡
Department of Chemistry and Biochemistry, UniVersity of Texas at Austin, 1 UniVersity
Station A5300, Austin, Texas 78712-0165, United States, and Department of
Chemistry, Yonsei UniVersity, Seoul, 120-749 Korea
anslyn@austin.utexas.edu; sessler@mail.utexas.edu
Received August 18, 2010
ABSTRACT
Ginsenoside detection was pursued through the design of a porphyrin receptor containing two boronic acid units. This receptor was found
to undergo different degrees of fluorescence quenching with five ginsenoside guests and an acylated derivative. The trends in the 1:1 binding
constants, as well as ESI-HRMS analysis, support a binding mode in which the ginsenoside sugar units are bound to the boronic acid
groups, while the steroid core and porphyrin ring participate in hydrophobic interactions.
Ginsenosides represent a class of over 30 glycosylated
steroids that are believed to be responsible for the physi-
ological benefits of ginseng.1 Ginseng has been used in
medicinal practices for over 5000 years, particularly in China
and Korea, and remains one of the most widely taken herbs
in the world. However, the extent of its efficacy remains a
subject of current study.2 Nevertheless, ginsenosides have
been implicated as treatments in a variety of diseases.3
Ginsenosides contain a gonane steroid nucleus but differ in
the degree, position, and type of sugar substitution (Figure
1). The difference in the biological activity of individual
ginsenosides has been attributed to their respective glyco-
sylation patterns.1 This makes analysis of these patterns a
topic of chemical interest.
It is recognized that the ginsenoside composition of
ginseng is highly variable. It appears to depend on the
species, plant age, the part of the plant from which the sample
is taken, the season of the harvest, and the extraction
method.4 These variations represent a significant problem
(3) (a) Chen, L.-W.; Wang, Y.-Q.; Wei, L.-C.; Shi, M.; Chan, Y.-S.
CNS Neurol. Disord.: Drug Targets 2007, 6, 273. (b) Jovanovski, E.;
Jenkins, A.; Dias, A. G.; Peeva, V.; Sievenpiper, J.; Arnason, J. T.; Rahelic,
D.; Josse, R. G.; Vuksan, V. Am. J. Hypertens. 2010, 23, 469. (c) Xie,
J.-T.; Wang, C.-Z.; Zhang, B.; Mehendale, S. R.; Li, X.-L.; Sun, S.; Han,
A. H.; Du, W.; He, T.-C.; Yuan, C.-S. Biol. Pharm. Bull. 2009, 32, 1552.
(4) (a) Sugimoto, S.; Nakamura, S.; Matsuda, H.; Kitagawa, N.;
Yoshikawa, M. Chem. Pharm. Bull. 2009, 57, 283. (b) Lee, S. M.; Shon,
H. J.; Choi, C. S.; Hung, T. M.; Min, B. S.; Bae, K. Chem. Pharm. Bull.
2009, 57, 92.
† University of Texas at Austin.
‡ Yonsei University.
(1) Radad, K.; Gille, G.; Liu, L.; Rausch, W.-D. J. Pharmacol. Sci. 2006,
100, 175.
(2) Zhou, L. G.; Wu, J. Y. Nat. Prod. Rep. 2006, 23, 789.
10.1021/ol1019647  2010 American Chemical Society
Published on Web 09/22/2010

receptor 8 were appended to the 5 and 15 porphyrin
substitution positions with the goal of allowing the simul-
taneous binding of the saccharide substituents on both sides
of the ginsenoside analyte (i.e., Figure 2).
The first step in the synthesis of receptor 8 involved the
formation of zinc(II) porphyrin 3. This was accomplished
using the traditional Lindsey procedure10 in three steps with
an overall 19% yield (Scheme 1). Porphyrin 3 contains cyano
groups as the latent amine functionalities. It also contains
phenolic units, which were designed to serve as attachment
sites for solubilizing groups. Substitution of the phenolic units
of 3 with triethyleneglycol chains11 to give 4, followed by
protection with tetrahydropyran, yielded intermediate 5 in
32% yield for two steps.12 Reduction of the cyano units was
accomplished with the use of LAH.13 Subsequent heating
in aqueous HCl served to oxidize any reduced macrocycle
and cleave the protecting groups. This step also removed
the zinc(II) ion from the porphyrin core. Recovered porphyrin
6, obtained in 62% yield, was then used in the reductive
amination of 2-formylphenylboronic acid (7).14 The final
product (8) was purified through reverse-phase chromatog-
raphy and isolated in quantities of ca. 20 mg (44% yield for
two steps).
The absorbance and fluorescence properties of 8 were
found to be consistent with those previously reported for free
base tetraphenylporphyrin macrocycles.10 The absorbance
intensity at the Soret band (λmax ) 420 nm) was found to be
linear with the concentration up to 3.5 µM in a 9:1 DMSO:
water solution buffered to pH 7.4. Efforts to study higher
concentrations were precluded due to the high absorptivity
of the receptor. The observed linearity was taken as evidence
of minimal aggregation at concentrations e3.5 µM in this
solvent mixture.
The effects of ginsenosides on the optical properties of 8
were then explored using a receptor concentration of 3 µM.
While little change was observed in the absorption spectrum,
the addition of ginsenosides led to significant quenching of
the emission of receptor 8. Fluorescence titration experiments
revealed that the degree of quenching varied as a function
of the ginsenoside structure (Figure 3).
The quenching curves were then fit to a 1:1 binding
equation to estimate the association constant of the underly-
ing interactions.15 The method used was based on a previous
report that allowed for the calculation of binding constants
using the change in absorbance relative to concentration of
Figure 1. Examples of ginsenoside structures. Glc ) ꢀ-D-glucose;
Xyl ) ꢀ-D-xylose; Rha ) R-L-rhamnose.
in terms of quality control.5 Normally, ginseng products are
analyzed through a time-consuming HPLC process.6 The
resulting inefficiency is providing a motivation to develop
improved methods for the analysis of ginsenoside compo-
nents. Despite recent efforts in this area,7 a simple optical
assay for ginsenosides has not been reported.
To address the need for improved ginsenoside sensing,
we designed and synthesized the boronic acid appended
porphyrin 8 (Scheme 1).8 This hybrid system was expected
to serve as a supramolecular receptor for ginsenosides.
Specifically, it was expected that the hydrophobic porphyrin
ring would interact with the steroid core of the ginsenosides
while also providing a large scaffold for the appendage of
sugar binding units. Boronic acids were chosen to serve this
latter purpose. These groups have been used in some of the
most successful synthetic saccharide receptors reported to
date and are known to form reversible covalent bonds with
1,2- and 1,3-cis-diols.9 The two boronic acid groups in
(5) Wills, R. B. H.; Stuart, D. L. Production of high quality Australian
ginseng: a report for the Rural Industries Research and DeVelopment
Corporation; RIRDC: Barton, A.C.T., 2001.
(10) Lindsey, J. S. Porphyrin Handb. 2000, 1, 45.
(11) (a) van Ameijde, J.; Liskamp, R. M. J. Org. Biomol. Chem. 2003,
1, 2661. (b) Wei, W. H.; Wang, Z.; Mizuno, T.; Cortez, C.; Fu, L.;
Sirisawad, M.; Naumovski, L.; Magda, D.; Sessler, J. L. Dalton Trans. 2006,
1934.
(6) Fuzzati, N. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2004,
812, 119.
(7) For recent reviews, see: (a) Jiang, Y.; David, B.; Tu, P.; Barbin, Y.
Anal. Chim. Acta 2010, 657, 9. (b) Yap, K. Y.; Chan, S. Y.; Weng Chan,
Y.; Sing Lim, C. Assay Drug DeV. Technol. 2005, 3, 683.
(8) For examples of other boronic acid-appended porphyrin receptors,
please see: (a) Imada, T.; Kijima, H.; Takeuchi, M.; Shinkai, S. Tetrahedron
1996, 52, 2817. (b) Sugasaki, A.; Ikeda, M.; Takeuchi, M.; Shinkai, S.
Angew. Chem., Int. Ed. 2000, 39, 3839. (c) Sugasaki, A.; Sugiyasu, K.;
Ikeda, M.; Takeuchi, M.; Shinkai, S. J. Am. Chem. Soc. 2001, 123, 10239.
(d) Hirata, O.; Kubo, Y.; Takeuchi, M.; Shinkai, S. Tetrahedron 2004, 60,
11211. (e) Zhang, C.; Suslick, K. S. J. Porphyrins Phthalocyanines 2005,
9, 659.
(12) Wuts, P. G. M.; Greene, T. W. Greene’s ProtectiVe Groups in
Organic Synthesis, 4th ed.; John Wiley & Sons, Inc.: Hoboken, 2007.
(13) Carcel, C. M.; Laha, J. K.; Loewe, R. S.; Thamyongkit, P.;
Schweikart, K. H.; Misra, V.; Bocian, D. F.; Lindsey, J. S. J. Org. Chem.
2004, 69, 6739.
(14) (a) Cabell, L. A.; Monahan, M.-K.; Anslyn, E. V. Tetrahedron Lett.
1999, 40, 7753. (b) Wiskur, S. L.; Lavigne, J. J.; Ait-Haddou, H.; Lynch,
V.; Chiu, Y. H.; Canary, J. W.; Anslyn, E. V. Org. Lett. 2001, 3, 1311.
(15) We presumed a 1:1 binding stoichiometry for the purpose of
estimating the overall strength of binding interactions. The low concentration
of receptor 8 required to prevent aggregation, particularly as compared to
the concentration of the ginsenoside guests, precluded efforts to carry out
continuous variation (Job plot) experiments.
(9) For recent reviews, see: (a) Mader, H. S.; Wolfbeis, O. S. Microchim.
Acta 2008, 162, 1. (b) Jin, S.; Cheng, Y.; Reid, S.; Li, M.; Wang, B. Med.
Res. ReV. 2010, 30, 171.
Org. Lett., Vol. 12, No. 21, 2010
4805

Scheme 1.
Synthesis of Receptor 8a
a Compound 7 ) 2-formylphenyl boronic acid.
Figure 2. Proposed binding mode of ginsenosides to porphyrin 8
(the protopanaxtriol derivative Re is shown). This molecular model
was built in HyperChem 8.0.3 and the geometry optimized using
the MM+ force field (convergence at 0.002 kcal/(Å mol)). The
planarity of the porphyrin ring was moderately constrained based
on the reported crystal structures of similar compounds.18
Figure 3. Emission intensity of a solution of porphyrin 8 (3 µM)
following the addition of aliquots of solutions containing 8 (3 µM)
and one of the following ginsenosides: Rb1 (30.7 mM), Rb3 (23.1
mM), Rd (26.4 mM), Re (26.3 mM), or Rg1 (31.0 mM). The data
are plotted as the difference in intensity (F) relative to the initial
(0 mM) point of each titration (F(0)). Each point is the average of
4-5 measurements. All measurements were performed in 9:1
DMSO:50 mM HEPES pH 7.4 at 25 °C. Excitation wavelength:
420 nm. Emission wavelength: 655 nm.
the guest.16,17 This method was applied to fluorescence data
(see Supporting Information (SI)) with the change in emis-
sion (F(0) - F) relative to ginsenoside concentration being
used in these analyses.
Within the series of five ginsenosides studied here (Figure
1), a number of correlations between the calculated association
constants (Table 1) and the sugar substitution pattern can be
proposed. The protopanaxdiol derivatives (see Figure 1) were
considered first. Here, it was found that the tetrasaccharide
dervatives Rb1 and Rb3 displayed significantly higher binding
(16) Zhong, Z.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 124, 9014
(17) Hargrove, A. E.; Zhong, Z.; Sessler, J. L.; Anslyn, E. V. New
J. Chem. 2010, 34, 348
.
.
(18) Lin, Y. C.; Liou, J. C.; Lu, T. H.; Liao, F. L.; Chung, C. S. Acta
Crystallogr., Sect. E: Struct. Rep. Online 2003, E59, o969.
4806
Org. Lett., Vol. 12, No. 21, 2010

productive boronic acid-diol interactions. However, reduced
binding due to the greater steric bulk of the AcRe derivative
(relative to Re) cannot be definitely ruled out.
Table 1. Summary of Results Obtained from the Best Fit
Curves of the Data Shown in Figure 3a
Additional support for the proposed binding mode was
provided by high resolution ESI-MS experiments. A solution
of receptor 8 in the presence of excess Re (1:1 chloroform:
methanol) was found to display strong peaks for the predicted
1:1 8:Re complex containing one or two sodium adducts (see
SI). Peaks corresponding to a 1:2 host:guest ratio were also
observed. This latter result highlights the possibility of
multiple host:guest stoichiometries, although it should be
noted that data obtained through this method do not neces-
sarily correspond to the compositions present in the solution.
guest
K (1:1), M-1
guest
K (1:1), M-1
Rb1
Rb3
Rd
2500 ( 350
2300 ( 470
550 ( 98
Re
Rg1
3900 ( 210
1200 ( 390
a The reported errors reflect the differences between the calculated curve
fits and the experimental data.
affinities than the trisaccharide derivative Rd. This result led to
the suggestion that the fourth sugar substituent of Rb1 and Rb3
plays a significant role in mediating the binding process, perhaps
by allowing for simultaneous interactions with both boronic acid
units (i.e., Figure 2).
The very similar binding affinities seen for Rb1 and Rb3 are
consistent with a lack of preference for xylose over glucose in
the case of 8, at least for the protopanaxdiol derivatives. This
result, which differs from the modest preference for xylose seen
for simple phenylboronic acid in aqueous medium,19 is thought
to reflect the importance of ancillary interactions, including
porphyrin-steroid associations.
The protopanaxtriol derivatives (Re and Rg1) were also
studied. As can be seen from Table 1, this class of
ginsenosides exhibits stronger binding affinities than similarly
substituted protopanaxdiol derivatives. We ascribe this
finding to an increased geometric complementarity with the
host porphyrin. The protopanaxtriol Re exhibited the stron-
gest binding affinity of the ginsenosides studied; however,
the general preference of phenylboronic acids for mannose
over glucose may also contribute to the high affinity observed
for this rhamnose (6-deoxy-L-mannose) derivative.19
To probe these binding interactions further, we sought to
protect the hydroxyl units of ginsenoside Re with acyl
groups. We expected the interaction of 8 with an acylated
Re derivative (AcRe) would be significantly reduced as
compared to the interaction with Re as the result of blocking
the proposed boronic acid-diol binding interactions. Fol-
lowing the reaction of Re with acetic anhydride in the
presence of pyridine, one major product fraction was isolated
through column chromatography. Proton and 13C NMR
analyses were consistent with a mixture of heavily acylated
derivatives. Low and high resolution ESI-MS revealed peaks
for only undeca- and dodecaacylated Re derivatives. An
average of the molecular weights of these two derivatives,
which differ by less than 3%, was used to estimate a yield
and prepare solutions of the AcRe mixture.
In conclusion, we designed and synthesized bisboronic acid-
functionalized porphyrin 8, which was used to detect success-
fully five ginsenoside derivatives through fluorescence spec-
troscopy. To the best of our knowledge, receptor 8 represents
the first small molecule optical sensor for ginsenosides. Further,
different signals were obtained for each ginsenoside through
fluorescence titrations with receptor 8. The determination of
1:1 host:guest binding constants revealed that the strongest
binding was obtained for ginsenosides with an increased number
of saccharide units and also with the protopanaxtriol (as opposed
to protopanaxdiol) substitution pattern. We propose a bifunc-
tional binding mode in which the two boronic acid units of
receptor 8 interact with the ginsenoside saccharide units, while
the porphyrin ring participates in hydrophobic interactions with
the steroid core of the guest. This binding mode was supported
by the finding that receptor 8 interacts with an acylated
derivative of Re (AcRe), albeit with a significantly reduced
binding affinity compared to Re. It is expected that the dual
binding mode of this receptor will allow for detection of
ginsenosides in the presence of common interferents, such as
oligosaccharides and starch found naturally in ginseng, as well
adulterants, such as sawdust, that are sometimes found in
commercial products.7b This study has served to demonstrate
that hybrid bisboronic acid-monoporphyrin receptors can
function as effective optical sensors for ginsenosides. This opens
the door for the use of these or other supramolecular approaches
to develop improved quality control methods for the ginseng
industry.
Acknowledgment. The authors gratefully acknowledge the
NSF-IGERT program, the National Science Foundation
(grant no. CHE-0716049 to E.V.A.), the National Institute
ofHealth(grantno.CA68682toJ.L.S.andno.1S10RR17263-1
to the Dept. of Chemistry and Biochemistry at UT-Austin),
and the Welch Foundation (grant no. F-1151 and F-1018 to
EVA and J.L.S., respectively). J.L.S. also thanks the Korean
government for support under the WCU (Word Class
University) Program (grant no. 1232-2008-000-10217-00).
The fluorescence titration and binding constant determination
with 8 and AcRe were then performed using the same procedure
used for the ginsenoside derivatives described above. While
greater quenching of receptor 8 emission was seen with AcRe
than Re, further analysis revealed a significantly lower binding
constant of 1600 ( 200 in the case of AcRe as compared to
Re (3900 ( 210). This value represents ca. 40% of that seen
for Re. Presumably, this reflects a reduction in the number of
Supporting Information Available: Experimental pro-
cedures and spectral characterization for new compounds,
optical spectra of 8, best fit curves, AcRe titration results,
and ESI-MS spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
(19) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291.
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