Synthesis of carbohydrate-functionalized thiophene-capped cyclopenta[c]thiophene for concanavalin A recognition

Article abstract of DOI:10.1016/j.tetlet.2012.01.034

Synthetic route to two different carbohydrate-functionalized oligothiophenes comprising central cyclopenta[c]thiophene (CPT) moiety was demonstrated. Turbidimetric analysis revealed enhanced interaction for divalent α-mannose-functionalized terthiophene 7 with Con A. The extent of binding interaction for 7-Con A complex formation was determined from isothermal titration calorimetry (ITC) measurements and the binding constant found (K _ITC) 2.4(6) × 10⁵ M^-1.

Full text of DOI:10.1016/j.tetlet.2012.01.034

Tetrahedron Letters 53 (2012) 1464–1467
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of carbohydrate-functionalized thiophene-capped
cyclopenta[c]thiophene for concanavalin A recognition
⇑
⇑
Soumyajit Das, Santanu Mandal, Balaram Mukhopadhyay , Sanjio S. Zade
Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, P.O. BCKV Campus Main Office, Mohanpur 741 252, Nadia, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic route to two different carbohydrate-functionalized oligothiophenes comprising central cyclo-
Received 16 November 2011
Revised 8 January 2012
Accepted 9 January 2012
Available online 18 January 2012
penta[c]thiophene (CPT) moiety was demonstrated. Turbidimetric analysis revealed enhanced interaction
for divalent a-mannose-functionalized terthiophene 7 with Con A. The extent of binding interaction for
7-Con A complex formation was determined from isothermal titration calorimetry (ITC) measurements
and the binding constant found (KITC) 2.4(6) Â 105 MÀ1
.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cyclopenta[c]thiophene
oligothiophene
Carbohydrate
Concanavalin A
The synthesis of sugar-substituted polymeric materials for the
detection of lectin is of significant interest. Since monomeric
carbohydrate–protein interaction occurs with low binding
affinities; supramolecular carbohydrate-functionalized organic
compounds,1 metal nanoparticles,2 dendrimers3 or polymers4–9
have been exploited to study the carbohydrate–protein interac-
tions based on their intrinsic fluorescence quenching and polymer
aggregation through multivalent binding. However, their applica-
tions to aqueous sensing have gained very scant attention due to
strong interactions between hydrophobic backbones and aromatic
may be then eluted with a-methyl-D-mannoside which is known
to posses stronger Con A binding affinity.13
We have designed a new compound which has carbohydrate
units ‘clicked’ to the thiophene-capped central CPT unit. Carbohy-
drate-functionalization provides better water solubility which is a
prerequisite for studying lectin recognition; whereas the p-conju-
gation in oligothiophene would enable one to detect lectins by
fluorescence spectrometry, UV–vis spectrophotometry, or turbidi-
metric analysis. Herein, we describe a new synthetic route for
the synthesis of new carbohydrate-functionalized oligothiophenes
which can be used as a molecular probe to quantitatively detect
different lectins by changing the pendant sugar substitution. To
the best of our knowledge, except the report published recently
by Bäuerle and co-workers,11 no mannosyl-oligothiophene conju-
gate was reported for the recognition of lectins. Since Con A is a
p–p stacking, which severely restrict their water solubility. Carbo-
hydrate-functionalized non-ionic oligothiophenes are interesting
as they may interact in both solid and solution states using non-
covalent interactions such as van der Waals interactions, hydrogen
bonding, and
p–p A
stacking which facilitate self-assembly.10
recent report by Bäuerle and co-workers11 on the surface-tethered
carbohydrate substituted oligothiophene demonstrated efficient
sensing of Concanavalin A (Con A, Canavalia enisformis,12 is a less
toxic model protein which is usually studied for sugar–protein
interaction, isolated from jack bean) using turbidity and fluoromet-
ric analysis as experimental tools; although the measurement of
binding constants of such carbohydrate-functionalized oligothi-
ophenes with Con A is yet to be reported. Recognition of Con A is
important due to its carbohydrate binding specificity; as it can
bind some specific glycoproteins and the bound glycoprotein
well-known
a
-mannose binding lectin because of its selectivity
towards
a
-mannopyranosides;14 the divalent
a-mannosyl-oligoth-
iophene was exploited in sensing Con A which revealed remarkable
selectivity and binding affinity.
The synthetic routes to the precursors and title compounds are
shown in Scheme 1. A simple synthetic approach was employed to
attach carbohydrate moiety to an oligothiophene scaffold using
‘click’ chemistry. Compound 1 was synthesized15 according to
the literature procedure, starting from diethyl malonate.16 Sono-
gashira coupling between acetonide protected hepta-1,6-diyne 1
and 2-bromothiophene provided compound 2 in a 70% yield. Treat-
ment of 2 with zirconocene equivalent, generated in situ from zir-
conocene dichloride and n-butyllithium, followed by quenching
with disulfur dichloride and further treatment with silica gel under
sunlight afforded 3 in a 27% yield.17 While doing the zirconocene-
⇑
Corresponding authors. Fax: +91 33 25873020.
E-mail addresses: mbalaram@iiserkol.ac.in (B. Mukhopadhyay), sanjiozade@ii-
serkol.ac.in (S.S. Zade).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.01.034

S. Das et al. / Tetrahedron Letters 53 (2012) 1464–1467
1465
O
O
HO
OH
O
O
i.Cp2ZrCl2, n-BuLi,S2Cl2
Pd(PPh3)4
ii. Silica gel/on-column
deprotectionunder sunlight
2-bromothiophene
CuI, DiPA
27%
70%
S
S
S
1
S
S
3
2
Propargyl bromide
85%
NaH, DMF
OR
O
OR
OR
RO
RO
RO
O
OR
OR
N
N N
N
O
O
N
N
CuI,CH3CN, DIPEA 70%
O
O
O
O
OAc
OAc
O
OAc
OAc
N3(H2C)2O
5
S
S
S
S
4
S
S
6
7
R= Ac
R= H
NaOMe, MeOH
89%
Scheme 1. Synthetic route to a-manno-TCPTT 7.
mediated reaction with 2 and subsequent quenching with disulfur
dichloride, we have observed the colour change from yellow to
brown-red to finally deep red. The red colour originated due to
the presence of 1,2-dithiin compound 11.18 As we were more inter-
ested to isolate the diol substituted terthiophene species, silica gel
was added to the reaction mixture and it was exposed to sunlight
for ꢀ6 h. Due to mild acidic nature, silica gel deprotected the ace-
tonide to diol whereas sunlight exposure photolytically extruded
the sulfur from 1,2-dithiin compound 11 or its deprotected form.
After that, volatile materials were evaporated and the silica slurry
85% yield. It was allowed to react with 2 equiv of known per-O-
acetylated azido-mannose derivative 520 in the presence of CuI
and DIPEA in acetonitrile to afford the bis-mannose derivative 6
in a 70% yield.21 To make it suitable for further protein–carbohy-
drate interaction experiments, the acetyl groups were removed
by using sodium methoxide in methanol to furnish the final com-
pound a
-manno-TCPTT 7 in an 89% yield.22
Applying the similar synthetic route, using known azido-galact-
ose derivative 8,23 the b-galacto-TCPTT 10 was also synthesized
(Scheme S1).
OR
RO
OR
OR
N
N
N
N
N
O
O
O
O
N
OR
O
RO
RO
OR
O
O
O
OAc
AcO
AcO
O
O(CH2)2N3
S
S
S
OAc
S
S
S
S
11
8
68%
9
10
R= Ac
R= H
NaOMe, MeOH
82%
was loaded on a 60–120 mesh silica gel column to perform column
chromatography. Sulfur impurities were removed by using 5%
ethyl acetate in hexane. Then the polarity was gradually increased
(up to 60–80% ethyl acetate in hexane); to isolate the desired
product, thiophene-capped cyclopenta[c]thiophenediol 3, from
the column. The diol 3 was soluble in dimethyl sulfoxide, dimeth-
ylformamide, acetone and partially soluble in tetrahydrofuran.
Thus, a new and convenient approach to synthesize diol 3 has been
described.19 Alkylation of the sodium derivative of 3 with propar-
gyl bromide in dry dimethylformamide afforded compound 4 in an
The 7 exhibits absorption maximum peak at 360 nm and emis-
sion maximum peak at 428 nm in a 0.1 M Tris buffer solution (pH
7.5), which were ascribed to the * transition of the oligothioph-
p–p
ene backbone (Fig. 1). The observed Stokes shift 4413 cmÀ1 was
typical for oligothiophenes.24 Carbohydrate-functionalized oli-
gothiophenes with central CPT unit posses short emissive lifetimes
in the order of 0.16 ns and fluorescence quantum yield (UPL) of
0.134 (compared with the standard emission of quinine sulfate25
in 1 N H2SO4, UPL = 0.546). The low fluorescence quantum
yield of 7 may not accurately provide the binding constant of

1466
S. Das et al. / Tetrahedron Letters 53 (2012) 1464–1467
the binding affinity accurately. The divalent mannoside-containing
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
substrate 7 was further titrated against Con A using ITC measure-
ments. In the present Letter, a 1.08 mM solution of 7 in Tris buffer
at pH 7.5, containing 0.1 mM of CaCl2 and MnCl2, was titrated into
a solution of 0.03 mM Con A at 25 °C. The binding reaction was
exothermic. However, the heat of dilution of Con A or 7 at pH 7.5
was endothermic in nature. Prior to the simulation using single-
site binding model (Con A has a single binding site per monomeric
unit) the resultant heat of reaction of Con A versus 7 was corrected
for heat of dilution of Con A and 7. The integrated heat of reaction
was measured as a function of the glycooligothiophene–Con A mo-
lar ratio (Fig. 3).
Previous reports,26 mainly highlight the binding of carbohy-
drates to Con A in the pH range of 5.0–5.2 when Con A is a dimer.
However, there are a few reports,27 on binding studies of Con A at
pH ꢀ7.5 where Con A is in tetrameric form. The results obtained
show that at around the 16th injection that is, substrate to Con A
mole ratio of 6:1, the solution showed no more binding but only
heat of dilution of the substrate and protein. The fitting of the
experimental data using single-site binding model shows that
the binding constant (KITC) is 2.4(6) Â 105 MÀ1 with a binding en-
300
400
500
600
Wavelength [nm]
Figure 1. Normalized UV–vis (black line) and fluorescence spectra (blue line) of
mannose derivative 7 in 0.1 M Tris buffer (pH 7.5).
multivalent interactions for any change in fluorescence property
with different amount of lectins (see Supplementary data,
Fig. S1–S3). Therefore, as a preliminary characterization technique,
we used tubidimetric analysis as a tool to investigate the interac-
tion of Con A with 7 and determined the binding constant for pro-
tein–ligand multivalent interactions from isothermal titration
calorimetry (ITC) which is mainly used to measure affinity for
protein–protein and protein–ligand interactions.
thalpy (
D
H) of À2.3(2) kcal molÀ1 and T
D
S of 4.7 kcal molÀ1. ITC
data show that the binding affinity of 7 is higher than that of
methyl-
a
-mannopyranoside (Me-
a
-Man, KITC = 0.82 Â 104 MÀ1
)
and even closely matching with the affinity values of several other
di or oligosaccharides.27 The number of moles of 7 bound per Con A
tetramer (N) is 2.08(8) that is, 0.5 mol of 7 is bound per Con A
monomer which matches well in agreement with the literature
since compound 7 has two sugar units per mole and each mono-
mer of Con A can bind only one.27 Our results suggest a stronger
binding of 7 (two mannose conjugated oligothiophene unit) to
The interaction of 7 and 10 with Con A was investigated mea-
suring the changes in optical density (OD) (Fig. 2). When 20
l
l of
1 Â 10À7 M Con A was added into an 80
l
l of 10À6 M Tris buffer
solution (pH 7.5, containing 0.1 mM CaCl2 and 0.1 mM MnCl2) of
Con A as compared to methyl-a-mannopyranoside with an N value
7, the turbidimetric analysis revealed the increase in OD, indicating
of ꢀ0.5 per monomeric Con A which could be attributed to cross-
an efficient carbohydrate–Con A interaction. After 20 min, a-meth-
ylmannoside was added into this mixture solution and subsequent
OD measurement showed a decrease in the optical properties
which undoubtedly indicates the partial disaggregation of the car-
bohydrate–Con A complex.11 However, the
a-methylmannoside
Time (min)
10 20 30 40 50 60 70 80
-10
0
addition did not result in the complete recovery of optical proper-
ties; which may be attributed to a stronger carbohydrate–Con A
binding and the extent of binding affinity was further investigated
using ITC measurement. Additionally, charging of Con A into a Tris
buffer solution containing 10, under identical experimental condi-
tions, showed no change in the optical density as Con A carries no
binding sites for galactose.
0.15
0.10
(a)
0.05
0.00
-0.05
-0.10
-0.15
The significant interaction of Con A with 7 is quite apparent
from turbidimetric analysis and it is thus important to measure
0.05
(b)
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
α-methylmannoside addition
0.04
0.03
0.02
7 + Con A
7 + Con A + -methylmannoside
α
10 + ConA
0.01
0.00
0
1
2
3
4
5
6
7
8
Molar Ratio
0
5
10
15
20
25
30
35
time [min]
Figure 3. (a) Raw ITC data, and (b) corrected integrated heat of reaction fitted with
a single-site binding model. Conditions: Con A (0.03 mM) with 7 (1.08 mM); both in
0.1 M Tris buffer at pH 7.5 with 0.1 mM each of CaCl2 and MnCl2 at 25 °C; 19
Figure 2. Turbidimetric analysis in a 0.1 M Tris buffer of pH 7.5, containing 0.1 mM
CaCl2 and 0.1 mM MnCl2, at 405 nm.
injections, 2 ll each of 7.

S. Das et al. / Tetrahedron Letters 53 (2012) 1464–1467
1467
13. Campbell, K. P.; MacLennan, D. H. J. Biol. Chem. 1981, 256, 4626.
14. Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.; Wong, C. H. Chem. Rev. 1998,
98, 833.
linked complex formation between the monovalent lectin (Con A)
and the multivalent carbohydrate.
In conclusion, an efficient synthetic approach was demon-
strated for the synthesis of diol functionalized thiophene-capped
CPT 3 which was in turn converted to carbohydrate-functionalized
oligothiophenes 7 and 10 following a few steps. Using this
synthetic approach, synthesized terthiophene 4 can be modified
to different sugar-functionalized derivatives which may be
exploited in various carbohydrate–lectin binding studies
depending on carbohydrate substitution. Compound 7 showed
specific interaction with Con A while compound 10 was non-
responsive as evident from turbidimetry analysis which stimulated
us to measure the extent of binding interaction of 7 with Con A and
hence, ITC experiment was employed. The enhanced binding
affinities shown by 7 for Con A may be credited to the multivalent
effect.27 Although of moderate affinity; this is the first demonstra-
tion where ITC was used to measure the binding affinity for
15. Llerena, D.; Buisine, O.; Aubert, C.; Malacria, M. Tetrahedron 1998, 54, 9373.
16. (a) Eglinton, G.; Galbraith, A. R. J. Chem. Soc. 1959, 889; (b) Kim, S. H.; Kim, Y.
H.; Cho, H. N.; Kwon, S. K.; Kim, H. K.; Choi, S. K. Macromolecules 1996, 29, 5422;
(c) Das, S.; Bedi, A.; Krishna, G. R.; Reddy, C. M.; Zade, S. S. Org. Biomol. Chem.
2011, 9, 6963.
17. Synthesis of 3: To Cp2ZrCl2 (1.37 g, 4.7 mmol) in 40 mL of dry THF was added n-
BuLi (6 mL, 9.4 mmol, 1.6 M in hexane) at À78 °C under nitrogen. After the
solution was stirred for about 10 min, 2 (1.52 g, 4.3 mmol) in 5 mL of dry THF
was added dropwise. The resulting solution was allowed to warm to room
temperature, and stirred for an additional 2 h while the colour of solution
changed from pale yellow to dark brown-red. The solution was again cooled
back to 0 °C and S2Cl2 (0.35 mL, 4.3 mmol) was added via syringe while the
colour of solution turned deep red, and left stirred overnight. Silica gel (2 g of
60–120 mesh) was put into reaction mixture and left for 5–6 h under sunlight.
Then volatile materials were evaporated and the product was column
chromatographed on 60–120 mesh silica gel using 5–10% EtOAc/hexane first
and then 60–80% EtOAc/hexane. Yellow solid mixed with some amount of red
solid was isolated when solvent polarity was increased. These two compounds
were almost inseparable. So, the two isolated solid compounds were re-
dissolved in THF and exposed to sunlight. Another column chromatography
was performed to isolate the desired pure diol as a yellow solid (400 mg,
yield = 27%). 1H NMR (400 MHz, DMSO-d6): d 7.54 (d, 2H, J = 5.0 Hz), 7.17 (d,
2H, J = 3.2 Hz), 7.11 (dd, 2H, J = 3.6 Hz, 4.8 Hz), 4.79 (t, 2H, J = 5.5 Hz), 3.42 (d,
4H, J = 5.0 Hz), 2.64 (s, 4H).
any carbohydrate-functionalized oligothiophene and Con
interaction.
A
Acknowledgments
18. Schroth, W.; Billig, F.; Reinhold, G. Angew. Chem., Int. Ed. Engl. 1967, 6, 698.
19. Otsubo, T.; Ueno, S.; Takimiya, K.; Aso, Y. Chem. Lett. 2004, 33, 1154.
20. Hayes, W.; Osborn, H. M. I.; Osborne, S. D.; Rastall, R. A.; Romagnoli, B.
Tetrahedron 2003, 59, 7983.
21. Synthesis of 6: To a solution of compound 4 (500 mg, 1.2 mmol) and 5 (1.2 g,
2.9 mmol) in anhydrous acetonitrile (20 ml), CuI (95 mg, 0.5 mmol) was added
We gratefully acknowledge the financial support from DST,
India. We thank Dr. Arindam Mukherjee and Mr. Sudipta Bhatta-
charyya for helpful discussions and assistance with the ITC exper-
iment. S.M. acknowledges CSIR for senior research fellowship.
followed by DIPEA (260 lL, 1.5 mmol) and the reaction mixture was stirred at
room temperature for 5 h till the completion of the reaction was confirmed by
checking TLC (hexane–EtOAc: 1:1). The solution was diluted with water
(20 mL) and solution of NH4Cl (10 mL). The aqueous layer was extracted by
EtOAc (2 Â 20 mL), the combined organic layer was washed with brine solution
(20 mL), dried over Na2SO4, and evaporated to obtain the crude product. It was
then purified by flash chromatography using hexane–EtOAc: 1:1 to get the
pure compound 6 (1.0 g, yield = 70%) as yellowish amorphous mass. 1H NMR
(500 MHz, CDCl3): d 7.67 (s, 2H), 7.22 (d, 2H, J = 5.0 Hz), 7.08 (d, 2H, J = 3.0 Hz),
7.01 (dd, 2H, J = 3.5 Hz, 5.0 Hz), 5.26 (t, 2H, J = 8.5 Hz), 5.21 (d, 2H, J = 2.0 Hz),
5.20 (dd, 2H, J = 2.0 Hz, 8.5 Hz), 5.18 (s, 2H), 4.80 (s, 2H), 4.66 (s, 4H), 4.56 (m,
4H), 4.20 (dd, 2H, J = 5.5 Hz, 12.5 Hz), 4.09 (m, 2H), 4.03 (dd, 2H, J = 2.5 Hz,
12.5 Hz), 3.87 (m, 2H), 3.56 (s, 4H), 2.75 (s, 4H), 2.13 (s, 6H), 2.09 (s, 6H), 2.03
(s, 6H), 1.97 (s, 6H).
Supplementary data
Supplementary data (detailed experimental procedures, charac-
terization data and NMR spectra) associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2012.01.034.
References and notes
1. Singh, Y.; Dolphin, G. T.; Razkin, J.; Dumy, P. ChemBioChem 2006, 7, 1298.
2. Manea, F.; Bindoli, C.; Fallarini, S.; Lombardi, G.; Polito, L.; Lay, L.; Bonomi, R.;
Mancinand, F.; Scrimin, P. Adv. Mater. 2008, 20, 4348.
22. Synthesis of 7: Methanolic NaOMe (0.5 N, 2 mL) was added to a solution of
compound 6 (500 mg, 0.4 mmol) in dry MeOH (20 mL) and the solution was
allowed to stir at room temperature till TLC (CH2Cl2–MeOH: 4:1) showed
complete conversion of the starting material to a slower moving component
(ꢀ4 h). The solution was neutralized with DOWEX 50 W H+ resin, filtered and
evaporated to afford pure compound 7 (325 mg, yield = 89%) as light yellow
powder. 1H NMR (500 MHz, DMSO-d6): d 8.04 (s, 2H), 7.53 (dd, 2H, J = 1.0 Hz,
5.0 Hz), 7.17 (dd, 2H, J = 1.0 Hz, 3.5 Hz), 7.10 (dd, 2H, J = 3.5 Hz, 5.0 Hz), 4.61 (d,
2H, J = 1.0 Hz), 4.57–4.48 (m, 8H), 3.94 (m, 4H), 3.53 (m, 4H), 3.48 (br s, 4H),
3.42–3.33 (m, 8H), 3.15 (m, 2H), 2.67 (br s, 4H).
3. Zanini, D.; Roy, R. J. Am. Chem. Soc. 1997, 119, 2088.
4. (a) Disney, M. D.; Zheng, J.; Swager, T. M.; Seeberger, P. H. J. Am. Chem. Soc.
2004, 126, 13343; (b) Kim, I. B.; Wilson, J. N.; Bunz, U. H. F. Chem. Commun.
2005, 1273; (c) Kelly, T. L. M.; Lam, C. W.; Wolf, M. O. Bioconjugate Chem. 2006,
17, 575; (d) Phillips, R. L.; Kim, I. B.; Tolbert, L. M.; Bunz, U. H. F. J. Am. Chem. Soc.
2008, 130, 6952; (e) Phillips, R. L.; Kim, I. B.; Carson, B. E.; Tidbeck, B.; Bai, Y.;
Lowary, T. L.; Tolbert, L. M.; Bunz, U. H. F. Macromolecules 2008, 41, 7316.
5. Xue, C.; Jog, S. P.; Murthy, P.; Liu, H. Biomacromolecules 2006, 7, 2470.
6. Xue, C.; Luo, F. T.; Liu, H. Macromolecules 2007, 40, 6863.
23. Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc.
2002, 124, 14397.
24. Zhang, X.; Matzger, A. J. Org. Chem. 2003, 68, 9813.
7. Xue, C.; Velayudham, S.; Johnson, S.; Saha, R.; Smith, A.; Brewer, W.; Murthy, P.;
Bagley, S. T.; Liu, H. Chem. Eur. J. 2009, 15, 2289.
8. Takasu, A.; Iso, K.; Dohmae, T.; Hirabayashi, T. Biomacromolecules 2006, 7, 411.
9. Otsuka, I.; Hongo, T.; Nakade, H.; Narumi, A.; Sakai, R.; Satoh, T.; Kaga, H.;
Kakuchi, T. Macromolecules 2007, 40, 8930.
10. Schmid, S.; Mena-Osteritz, E.; Kopyshev, A.; Bäuerle, P. Org. Lett. 2009, 11, 5098.
11. Schmid, S.; Mishra, A.; Bäuerle, P. Chem. Commun. 2011, 47, 1324.
12. Sharon, N.; Lis, H. Science 1972, 177, 949.
25. Eaton, D. F. Pure Appl. Chem. 1988, 60, 1107.
26. Dam, T. K.; Brewer, C. F. Chem. Rev. 2002, 102, 387.
27. (a) Mandal, D. K.; Kishore, N.; Brewer, C. F. Biochemistry 1994, 33, 1149; (b)
Casas-Solvas, J. M.; Ortiz-Salmerón, E.; García-Fuentes, L.; Vargas-Berenguel, A.
Org. Biomol. Chem. 2008, 6, 4230.