Directed molecular recognition: Design and synthesis of neutral receptors for biotin to bind both its functional groups

Article abstract of DOI:10.1021/jo061013j

The neutral receptors 1 and 2 are designed and synthesized for the recognition of biotin, a biologically significant molecule, in chloroform to bind completely both of its functional groups simultaneously, i.e., cyclic urea and the carboxyl groups. The truncated receptor 3 binds only the cyclic urea moiety.

Full text of DOI:10.1021/jo061013j

Directed Molecular Recognition: Design and Synthesis of Neutral
Receptors for Biotin To Bind Both Its Functional Groups
Shyamaprosad Goswami* and Swapan Dey
Department of Chemistry, Bengal Engineering and Science UniVersity, Shibpur, Howrah-711 103,
West Bengal, India
spgoswamical@yahoo.com
ReceiVed May 17, 2006
The neutral receptors 1 and 2 are designed and synthesized for the recognition of biotin, a biologically
significant molecule, in chloroform to bind completely both of its functional groups simultaneously, i.e.,
cyclic urea and the carboxyl groups. The truncated receptor 3 binds only the cyclic urea moiety.
Introduction
substrates, e.g. binuclear substrates such as adenine,5a caffeine,5b
uric acid, and also small molecules such as urea,5c,d creatinine,
etc. has been our keen interest5 where we have effectively
complexed by hydrogen bonding and/or π-stacking forces as
demonstrated by the solubilization of these polar guest substrates
in less polar solvents such as chloroform. Biotin is an essential
vitamin (anti-egg white injury factor) that functions as an
indispensable co-enzyme in the mammalian metabolism in a
range of biocarboxylations related to crucial physiological
processes such as gluconeogenesis and fatty acid biosynthesis.6
The molecular recognition implies a pattern of recognition
process through a structurally well-defined set of intermolecular
interactions between the host and the guest substrates.1 Multiple
hydrogen bonds have a good logical path to achieve and direct
the molecular recognition process. The specificity in molecular
recognition is related to the strength and number of hydrogen
bonds.2 In this connection, we have already reported3 the
recognition of mono- and dicarboxylic acids,3a,b hydroxy acid
(tartaric acid),3b and amino acid,3c and also inhibition of
hydrogen bonding in molecular recognition.4 The directed
molecular recognition by design, synthesis, and binding studies
of specific receptors for various types of biologically important
(3) (a) Goswami, S. P.; Ghosh, K.; Dasgupta, S. J. Org. Chem. 2000,
65, 1907. (b) Goswami, S. P.; Ghosh, K.; Mukherjee, R. Tetrahedron 2001,
57, 4987. (c) Goswami, S. P.; Ghosh, K.; Mukherjee, R. J. Ind. Chem. Soc.
1999, 661. (d) Goswami, S. P.; Ghosh, K.; Dasgupta, S. Tetrahedron 1996,
52, 12223. (e) Alvarez-Rua, C.; Garc´ıa-Granda, S.; Goswami, S. P.;
Mukherjee, R.; Dey, S.; Claramunt, R. M.; Santa Mar´ıa, M. D.; Rozas, I.;
Jagerovic, N.; Alkorta, I.; Elguero, J. New J. Chem. 2004, 28, 700.
(4) Goswami, S. P.; Dey, S.; Maity, A. C.; Jana, S. Tetrahedron Lett.
2005, 46, 1315.
(5) (a) Goswami, S. P.; Hamilton, A. D.; Van Engen, D. J. Am. Chem.
Soc. 1989, 111, 3425. (b) Goswami, S. P.; Mahapatra, A. K.; Mukherjee,
R. J. Chem. Soc., Perkin Trans. 1 2001, 2717. (c) Goswami, S. P.;
Mukherjee, R. Tetrahedron Lett. 1997, 38, 1619. (d) Goswami, S. P.;
Mukherjee, R.; Roy, J. Org. Lett. 2005, 7, 1283.
* Address correspondence to this author. Fax: +91-033-668-2916.
(1) Lehn, J. M. Supramolecular Chemistry: Concepts and PerspectiVes;
VCH: Weinheim, Germany, 1995. (b) MacDonald J. C.; Whitesides, G.
M. Chem. ReV. 1994, 94, 2383.
(2) (a) Mazik, M.; Radunz, W.; Boese, R. J. Org. Chem. 2004, 69, 7448.
(b) Chou, H.-C.; Hsu, C.-H.; Cheng, Y.-M.; Cheng, C.-C.; Liu, H.-W.; Pu,
S.-C.; Chou, P.-T. J. Am. Chem. Soc. 2004, 126, 1650. (c) Hegde, V.; Hung,
C.-Y.; Madhukar, P.; Cunningham, R.; Hopfner, T.; Thummel, R. P. J. Am.
Chem. Soc. 1993, 115, 872. (d) Meyer, E. A.; Castellano, R. K.; Diederich,
F. Angew. Chem., Int. Ed. 2003, 42,1210.
10.1021/jo061013j CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/17/2006
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J. Org. Chem. 2006, 71, 7280-7287

Design and Synthesis of Neutral Receptors for Biotin
FIGURE 1. Possible modes of complexation: 1C (receptor 1 with biotin); 2C (receptor 2 with biotin), and 3C (truncated receptor 3 with biotin).
SCHEME 1. Synthesis of Receptors 1, 2, and 3a
a Reagents and conditions: (i) NaH, dry THF, 12 h, rt, 68%; (ii) 4 N KOH-EtOH (1:1); reflux, 6 h, 95%; (iii) acetic anhydride, dry CH2Cl2, 2 h, rt, 42%;
(iv) 2-amino-6-methylpyridine, isophthaloyl chloride, Et3N, dry CH2Cl2, 24 h, rt (isolated yield: 17% for receptor 1, 22% for receptor 2 and 26% for
receptor 3).
Several elegant total syntheses of this biologically important
molecule have been reported.7 Biotin contains a cyclic urea
linkage with a fused tetrahydrothiophene nucleus where both
the hydrogens at the ring junctions and the hydrogen attached
to the asymmetric center bearing the n-petanoic acid side chain
to the tetrahydrothiophene nucleus are all syn. The presence of
two such polar groups makes biotin notoriously insoluble in
chloroform. Thus it is an interesting and critical problem in the
molecular recognition study of biotin to bind both the polar
groups and thereby solubilize biotin itself in less polar organic
solvents. The crystal structure of biotin8 shows the carboxyl
group of one biotin molecule is intermolecularly hydrogen
bonded to the urea linkage of the other biotin molecule.
Receptors 1 and 2 are designed to bind both the functional
groups (urea and carboxyl moieties) of biotin by breaking these
self-intermolecular hydrogen bonds in the guest biotin itself to
form hetero-hydrogen bonds with the host receptors. In contrast
to amino acid, the carboxylic acid and urea moieties in biotin
do not exist as zwitterions because urea linkage is not as basic
as amino group. Wilcox and co-workers9 reported an elegant
Troger’s base receptor having two carboxyl groups that bind
biotin methyl ester (soluble in chloroform unlike biotin itself)
where the cyclic urea part in the biotin ester was only available
for binding and not the carboxyl group present in biotin.
Recently Claramunt et al. have studied the recognition of biotin
methyl ester using our previously reported receptor (3) along
with a new receptor.10 But there is no report yet in the literature
to our knowledge of a complete receptor for biotin itself where
both its important functional groups, the top urea part and the
carboxyl moiety, are involved in binding and it is still a critical
unsolved problem.
This paper describes the first model of tandem binding of
biotin. The present approach is to design neutral complete
receptors 1 and 2 that direct their own functional groups for
complexation with both moieties of the guest biotin compared
to the truncated partial diamide receptor 3, which binds only
with the urea part of biotin. The neutral triamide receptors (1
and 2) are soluble in common organic solvents such as
chloroform, dichloromethane, etc. and we have attempted to
solubilize biotin via complexation with the receptors in chlo-
roform, which is a common NMR solvent having comparable
polarity to that of the interior cavity of an enzyme.
(6) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry,
3rd ed.; Worth Publishers: New York, 2000.
(7) Confalone, P. N.; Pizzolato, G.; Lollar-Confalone, D.; Uskokovic,
M. R. J. Am. Chem. Soc. 1980, 102, 1954.
(8) (a) DeTitta, G. T.; Edmonds, J. W.; Stallings, W.; Donohue, J. J.
Am. Chem. Soc. 1976, 98, 1920. (b) DeTitta, G. T.; Blessing, R. H.; Moss,
G. R.; King, H. F.; Sukumaran, D. K.; Roskwitalski, R. L. J. Am. Chem.
Soc. 1994, 116, 6485.
(10) (a) Herranz, F.; Santa Mary´’a, M. D.; Claramunt, M. R. J. Org.
Chem. 2006, 71, 2944. (b) Rosa, M.; Claramunt, R. M.; Herranz, F.; Mary´’a,
M. D. S.; Pinilla, E.; Torres, M. R.; Elguero, J. Tetrahedron 2005, 61, 5089.
(c) Rosa, M.; Claramunt, R. M.; Herranz, F.; Mary´’a, M. D. S.; Jaime, C.;
Federicob, M. D.; Elguero, J. Biosens. Bioelectron. 2004, 20, 1242. (d)
Peikert, C.; Seeger, K.; Bhat, R. K.; Berger, S. Org. Biomol. Chem, 2004,
2, 1777.
(9) (a) Adrian, J. C., Jr.; Wilcox, C. S. J. Am. Chem. Soc. 1989, 111,
8055. (b) Rao, P.; Maitra, U. Supramol. Chem. 1998, 9, 325.
J. Org. Chem, Vol. 71, No. 19, 2006 7281

Goswami and Dey
FIGURE 2. 1H NMR spectra of (i) receptor 1 in CDCl3, (ii) receptor 1 with biotin in 2% d6-DMSO in CDCl3, and (iii) receptor 1 with ethyl ester
of biotin in CDCl3 (for a, b, c, d, e, and f refer to Figure 1).
SCHEME 2. Synthesis of Receptors 2 and 3a
The flexible cavity in the open but semirigid receptors 1 and
2 is specially designed based on the fact that their isophthaloyl
pyridine diamide moieties are both directed downward for the
complementary hydrogen bonding with the urea part5 (as also
shown by the truncated receptor 3) and the terminal pyridine
a Reagents and conditions: (i) compound 7, Et3N, dry CH2Cl2, 24 h, rt,
amide of the receptors 1 and 2 are free to bind the carboxyl
58%; (ii) 2-amino-6-methylpyridine, Et3N, dry CH2Cl2, 24 h, rt, 72%.
moiety of the guest biotin. The possible modes of complexation
are thus shown in 1C, 2C, and 3C, respectively (Figure 1).
SCHEME 3. Synthesis of Compound 4 (biotin ethyl ester)a
Hydrogen bonding points are thus complimentarily well ar-
ranged in the complexes of receptors 1 and 2. All the donor
and acceptor hydrogen bonding interactions between these
receptors and biotin can then be saturated.
Results and Discussion
The combinatorial approach for the synthesis of all the desired
receptors 1, 2, and 3 has been successfully carried out by the
one-step high-dilution reaction followed by preparative chro-
matography (Scheme 1). 2-(N-Pivaloylamino)-6-hydroxymeth-
ylpyridine and 2-(N-pivaloylamino)-6-hydroxymethylpyridine
are coupled in the presence of sodium hydride in dry THF at
a Reagents and conditions: (i) biotin, ethanol, two drops concentrated
H2SO4, 24 h, reflux, 90%.
room temperature to prepare compound 5.11 Compound 5 on
hydrolysis under reflux with 4 N KOH ethanol-water (1:1)
solution produces 6. Then 7 is obtained from 6 by controlled
acetylation with acetic anhydride in dichloromethane (2 h of
stirring). Finally receptors 1, 2, and 3 are isolated from the
reactions of 7, 2-amino-6-methylpyridine, and isophthaloyl
chloride by high dilution technique.
synthesized by coupling isophthaloyl chloride with 2-aminopi-
coline (Scheme 2). From the NMR studies, large δ shifts of the
key binding protons are observed when all the receptors undergo
1:1 complexation respectively with biotin in 2% d6-DMSO in
CDCl3. The two pyridine amide protons which are flanked by
isophthaloyl spacer in unsymmetrical receptor 1 do not dif-
ferentiate and appear at the same position (δ 8.94 ppm). But
on complexation with the urea linkage of biotin, these are shifted
and distinctly differentiated. The formation of a tight complex
The coupling of isophthaloyl chloride and compound 7 also
produced the receptor 2. Similarly the receptor 3 was simply
(11) Papadopoulon, M. V.; Goswami, S. P.; Hamilton, A. D. J.
Heterocyclic Chem. 1995, 32, 2675.
7282 J. Org. Chem., Vol. 71, No. 19, 2006

Design and Synthesis of Neutral Receptors for Biotin
FIGURE 3. 1H NMR spectra of (i) receptor 2 in CDCl3, (ii) receptor 2 with biotin in 2% d6-DMSO in CDCl3, and (iii) receptor 2 with ethyl ester
of biotin in CDCl3 (for a, b, c, e, and f refer to Figure 1).
FIGURE 4. 1H NMR spectra of (i) receptor 3 in CDCl3, (ii) receptor 3 with biotin in 2% d6-DMSO in CDCl3, and (iii) receptor 3 with ethyl ester
of biotin in CDCl3 (for a, b, e, and f refer to Figure 1).
resolves the amide protons of receptor 1 as shown by the
appearance of the amide peaks at two different positions, at δ
10.45 and 10.24 ppm (∆δ 1.51 ppm and ∆δ 1.30 ppm),
respectively. The remaining terminal pyridine amide proton is
shifted from δ 8.11 to 9.20 ppm (∆δ 1.09 ppm) on complexation
with the carboxylic acid group of biotin. So all three pyridine
amide protons of receptor 1 are easily discriminated on
complexation due to differential hydrogen bonding. The biotin
urea protons are also actually different though it does not resolve
in NMR itself (δ 5.98 ppm), but on complexation with receptor
1, the peaks are more prominent to have distinguishable
chemical shifts (δ 6.02 ppm and δ 5.90 ppm) (Table 1). In the
case of biotin ethyl ester, complexation with receptor 1 in CDCl3,
the first two isophthaloyl amide protons have shifted their
positions from δ 8.94 to δ 10.12 and δ10.18 (∆δ 1.18 ppm
and ∆δ 1.24 ppm) respectively but the third terminal pyridine
amide proton of the receptor does not change its position. Biotin
ureido protons (δ 5.26 ppm and δ 4.98 ppm) remain in the same
position (δ 5.18 ppm and δ 5.08 ppm). These results suggest
the hydrogen bonded complex structure of receptor 1 with biotin
J. Org. Chem, Vol. 71, No. 19, 2006 7283

Goswami and Dey
TABLE 1. Chemical Shifts of Different Amide and Peri Protons of Receptors 1, 2, and 3 and Their Changes on Complexation with Biotin and
Ethyl Ester of Biotin
complex 1C
complex 2C
complex 3C
a
b
c
peri d
a
b
peri c
a
peri b
receptor itself
with biotin
with biotin ethyl ester
8.94
10.24
10.12
8.94
10.45
10.18
8.11
9.20
8.33
8.56
8.70
8.68
9.16
10.24
9.94
8.33
9.30
8.37
8.61
8.66
8.67
8.81
10.08
9.99
8.56
8.67
8.66
is 1C. Though final confirmation can only be obtained by single-
crystal X-ray analysis, we have not yet been able to grow good
quality crystals of the complex. A dummy titration was
performed taking d6-DMSO as a guest substrate to the receptors.
There was no DMSO-induced chemical shift of key protons
observed in any case. In the case of receptor 2, two identical
pyridine amide protons between the isophthaloyl spacer and
pyridine rings appeared at the same place (δ 9.16 ppm) and
shifted to δ 10.24 ppm (∆δ 1.08 ppm) on the event of a 1:1
complex formation with the urea part of biotin. The other two
terminal pyridine acetamide protons, which appeared at δ 8.33
ppm, have shifted to δ 9.30 ppm (∆δ 0.97 ppm) on complex-
ation with the carboxyl group of biotin. So this supports structure
2C for the complex of receptor 2 with biotin. An experiment
of complexation of biotin ethyl ester with receptor 2 has been
performed in CDCl3. Here only the amide protons nearer to the
isophthaloyl group have changed their peak position from δ
9.16 ppm to δ 9.94 ppm (∆δ 0.78 ppm), but the other two do
not change their position with biotin ethyl ester which also
reaffirms the complex structure 2C of receptor 2 with biotin.
The ureido protons of biotin on complexation with receptor 2
(δ 5.98 ppm) have also exhibited more downfield shift (δ 5.89
ppm and δ 5.78 ppm, respectively), whereas the ureido protons
of ethyl biotin shifted more downfield (δ 5.18 ppm to δ 6.40
ppm and δ 5.08 ppm to δ 5.99 ppm). Now receptor 3, containing
only the isophthaloyl pyridine diamide groups, can bind either
the urea part or the carboxyl group of biotin. But only the urea
part is bound and not the carboxyl group of biotin as proved
by the NMR experiments. In NMR titration with biotin, only
one pyridine amide proton is shifted from δ 8.81 ppm to δ 10.08
ppm (∆δ 1.27 ppm) on complexation with biotin and no other
different amide proton peak has been found. An interesting result
was observed here that biotin shows a significant upfield
chemical shift from δ 5.98 ppm to δ 5.71 and 5.67 ppm when
it was complexed with receptor 3. Two ureido protons at δ 5.26
ppm and δ 4.98 ppm shifted downfield to δ 7.21 ppm and δ
6.83 ppm on complexation with the same receptor. In all cases,
the peak of the amide protons of biotin and biotin ethyl ester
has changed position toward downfield. From these observa-
tions, it is concluded that only the urea linkage of biotin is
incorporated into the cavity but not that of the carboxyl group.
No evidence of proton transfer to the receptor from the biotin
-CO2H group is found in the solution phase. The intermolecular
hydrogen bonding of carboxyl and ureido part of biotin is
obviously broken by the interactions in heterocomplexation. The
peri protons of the isophthaloyl moiety in receptors 1, 2, and 3
furnish significant downfield shifts (from δ 8.56 to 8.70 ppm,
∆δ 0.14 ppm; δ 8.61 to 8.66 ppm, ∆δ 0.05 ppm; δ 8.56 to
8.67 ppm, ∆δ 0.11 ppm, respectively) on complexation with
biotin and similar shifts are observed in the case of complexation
with biotin ethyl ester. Interestingly, in biotin as well as biotin
ethyl ester, the urea protons do not resolve in the NMR spectra,
but they do resolve on complexation with all the receptors
undergoing differential chemical shifts.
FIGURE 5. (a) Titration curves of receptors 1, 2, and 3 with biotin in
2% d6-DMSO-CDCl3.14 (b) Job plots for the determination of the
stoichiometry of the complexes. (c) Titration curves of receptors 1, 2,
and 3 with biotin ethyl ester (compound 4) in CDCl3.
7284 J. Org. Chem., Vol. 71, No. 19, 2006

Design and Synthesis of Neutral Receptors for Biotin
TABLE 2. Association Constants (Ka) of Receptors 1, 2, and 3 Respectively with Biotin and Biotin Ethyl Ester (compound 4)
UV-Vis method
Ka (M-1) (CHCl3)
NMR method
free energy change
∆G (kcal mol-1
Ka (M-1) (2% d6-DMSO-CDCl3)
)
with biotin
ethyl ester
with biotin
ethyl ester
with biotin
ethyl ester
receptors
with biotin
with biotin
with biotin
receptor 1
receptor 2
receptor 3
4.51((0.01) × 104
2.23((0.01) × 104
5.23((0.01) × 103
3.33((0.01) × 104
2.24((0.01) × 103
1.95((0.01) × 103
1.3((0.01) × 104
9.0((0.1) × 103
2.4((0.2) × 103
1.74((0.01) × 103
8.84((0.1) × 102
3.18((0.2) × 102
-5.60
-5.39
-4.60
-4.41
-4.01
-3.41
In electronic spectra, receptor 1 (7.843 × 10-5 mL-1
)
exhibited absorption maxima at λmax 286 nm. Addition of biotin
solution to receptor 1 shows a decrease in absorption intensity.
Similar results were found when the UV titrations were carried
out taking receptors 2 (8.902 × 10-5 mL-1) and 3 (1.734 ×
10-5 mL-1), respectively. Receptors 2 and 3 have shown the
absorption maxima at λmax 282 and 288 nm in the ground state.
However, we do not find any λ-shift (blue or red) on dilution
of the complexes. The gradually decreasing absorbance values
(λmax ) 286, 282, and 288 nm, respectively) on dilution of the
1:1 complex between the receptors and biotin have been shown
in all three cases in their UV spectra.12
We have also studied the IR spectra of biotin and receptors
1, 2, and 3 along with their 1:1 complexes with biotin,
respectively. The biotin carbonyl group (νCdO) mainly appears
at 1705 cm-1. On complexation of biotin with receptor 2, the
peak is shifted to 1700 cm-1. In the case of receptor 3, in
complex formation with biotin a big hump is found around 1686
cm-1 for biotin carbonyl. A similar observation is found for
receptor 1 in biotin on complexation where a broad peak for
biotin carbonyl around 1680 cm-1 appears in the complex. The
shift to lower wavenumbers of carbonyl in biotin on complex-
ation with receptors 1, 2, and 3, respectively, proves the
involvement of carbonyl oxygen of biotin in the formation of
hydrogen bond to form the complexes with receptors 1, 2, and
3, respectively.
The energy minimized structure (CPK model)13 of the
complexes has also shown that the biotin molecule has been
incorporated in the cavity of receptors 1 and 2, respectively,
and thus supports the compatible donor-acceptor points for
favorable binding.12
Determination of Stoichiometry. From the NMR titration,
the graphs [G]/[H] vs ∆δ are plotted which show parallel straight
lines with respect to the X-axis after 1:1 complexation, i.e.,
[G]/[H] ) 1, for the above three receptors with biotin and biotin
ethyl ester, respectively (Figure 5a,c). Figure 5b represents the
Job plots (XH vs ∆δXH) of the three receptors drawn from
another NMR experiment, which clearly indicates the (1:1)
stoichiometry of the complexes.
FIGURE 6. Probable secondary hydrogen bonding interactions
between the two flexible pyridine amide parts of receptor 2.
the δ shifts of all the NH protons of receptors 1, 2, and 3 (Table
2), respectively. These Ka values from the NMR (plotted ∆δ
vs ∆δ/[G]) are found to be less compared to those obtained
from the UV methods (plotted 1/[G] vs Io/∆I). In NMR, a higher
concentration of the individual dimer may give rise to weaker
forces of attraction resulting in smaller Ka values. The host and
guest are more free in lower concentrations as used in UV and
consequently the effective concentration of the 1:1 complex
dimer is relatively higher in the UV studies. DMSO-d6 used as
a cosolvent in NMR studies is a polar solvent, which competes
as a H-bond acceptor. So Ka values found by UV are generally
higher than those determined by NMR.
From the NMR analysis, receptor 1 shows large chemical
shift changes as well as higher Ka values with biotin compared
to those of receptor 2. This may be due to the flexibility of the
two aliphatic chains of receptor 2 which may come closer by
secondary hydrogen bonding interactions and thus possibly
create hindrance to the carboxyl group of biotin for its entry
into the binding region (Figure 6).17 However, the complexation
of receptor 2 and biotin shows that the hetero-hydrogen bonding
interactions are stronger than self-hydrogen bonding.
Receptor 3 has the lowest binding constant in the series as it
binds only the cyclic urea part of biotin. These observed results
with receptors 1, 2, and 3 are significant in the molecular
recognition studies of biotin. The terminal pyridine amide of
the receptor should be acetylamino and preferably not pivaloyl-
amino in this case for binding biotin carboxyl group due to
possible steric inhibition of the hydrogen bond by bulky
pivaloylamide.
Quantification of Association Constants. The association
constants (Ka) for the 1:1 complexes with the guest biotin have
been determined by both the UV (in CHCl3)15 and NMR
methods16 (in 2% d6-DMSO-CDCl3) respectively at T ) 298
K. By the NMR method, Ka values have been calculated from
The binding constants of receptors 1, 2, and 3 with biotin
and its ethyl ester (compound 4) are shown in Table 2. Biotin
ethyl ester binds weaker with receptors 1 and 2 compared to
biotin, which suggests the formation of more hydrogen bonds
of these receptors with biotin compared to biotin ethyl ester.
The truncated receptor 3 also binds biotin ethyl ester with its
ureido moiety.
(12) See the Supporting Information.
(13) PCMODEL Serena Software 93.
(14) Titration curve of Figure 3a,c was drawn taking the shifts of N-Ha
proton of receptor 1, N-Ha proton of receptor 2, and N-Ha proton of
receptor 3 as a function of molar concentration of biotin.
(15) For association constant (Ka) calculations see: (a) Connors, K. A.
Binding Constants; The Measurement of Molecular Complex Stability;
Wiley-Interscience: New York, 1987. (b) Liao, J.-H.; Chen, C.-T.; Chou,
H.-C.; Cheng, C.-C.; Chou, P.-T.; Fang, J.-M.; Slanina, Z.; Chow, T. J.
Org. Lett. 2002, 4, 3107.
(16) (a) By Foster-Fyfe analysis of the titration data at 25 °C. Foster,
F.; Fyfe, C. A. Prog. Nucl. Magn. Reson. Spectrosc. 1969, 4, 1. (b) Fielding,
L. Tetrahedron 2000, 56, 6151.
(17) Mazik, M.; Blaser, D.; Boese, R. Tetrahedron 1999, 55, 12771.
J. Org. Chem, Vol. 71, No. 19, 2006 7285

Goswami and Dey
eluant: 230 mg, yield 42%, semisolid. 1H NMR (CDCl3; 300 MHz):
δ 8.53 (br s, 1H), 8.11 (d, 1H, J ) 8.2 Hz), 7.71 (t, 1H, J ) 7.9
Hz), 7.45 (t, 1H, J ) 7.7 Hz), 7.20 (d, 1H, J ) 7.5 Hz), 6.80 (d,
1H, J ) 7.8 Hz), 6.42 (d, 1H, J ) 8.2 Hz), 4.60 (s, 4H), 4.57 (s,
2H), 2.20 (s, 3H). 13C(CDCl3, 125 MHz): δ 169.3, 158.5, 156.7,
156.4, 151.3, 139.4, 138.9, 118.0, 113.1, 111.9, 108.1, 73.9, 73.4,
25.0. MS(ESI) (m/z, %): 295 (M + Na+, 80%), 273 (MH+), 139
(100). FT-IR (KBr, νmax): 3336, 1682, 1539, 1456, 1369, 1302,
Conclusion
We thus report here the design and synthesis of receptors 1
and 2 for tandem binding of biotin. The receptors are aimed at
binding simultaneously both functional groups of biotin. The
binding results when compared with those of the truncated
receptor 3 suggest that both receptors 1 and 2 bind stronger
than 3 supporting the higher hydrogen bonding capability of 1
and 2 with biotin. Moreover the binding ability of 1 is more
than that of 2 with biotin, which may be partially due to steric
reasons and also due to secondary hydrogen bonding interaction.
The energy minimization study also reveals the involvement
of maximum hydrogen bonding in the cases of receptors 1 and
2 with biotin as shown in their complexes 1C and 2C
respectively. The corresponding truncated receptor 3 acts as a
partial receptor binding only the urea part and not the carboxyl
moiety of biotin as shown in 3C. The binding of biotin ethyl
ester (compound 4) with all these receptors 1, 2, and 3 is also
compared.
1158, 1103, 793 cm-1
.
Synthesis of Receptors 1, 2, and 3. To dichloromethane (30.0
mL) was added isophthaloyl chloride (40 mg, 0.19 mmol) with
stirring. Compound 7 (100 mg, 0.36 mmol) and 2-amino-6-
methylpyridine (40 mg, 0.36 mmol) were each dissolved separately
in dry dichloromethane (15.0 mL). Then they were added dropwise
to the isophthaloyl chloride from a high dilution dropping funnel.
Addition was continued for 1 h with stirring under nitrogen
atmosphere. The reaction was prolonged for 12 h. Receptors 1, 2,
and 3 were isolated through preparative TLC, using methanol (5%)
in dichloromethane.
Biotin: 1H NMR (d6-DMSO; 500 MHz): δ 5.98 (br s, 2H), 4.46
(t, 1H, J ) 5.1 Hz), 4.28-4.26 (m, 1H), 3.15 (m, 2H), 2.88 (dd,
2H, J ) 5.0, 5.0 Hz), 2.74 (d, 2H, J ) 12.70 Hz), 2.28 (t, 2H, J )
7.4 Hz), 1.66-1.62 (m, 2H), 1.45 (t, 1H, J ) 7.6 Hz).
The chiral recognition of biotin by a specific chiral receptor
is underway along with the development of its fluorescent
receptor which will be reported in due course.
N-[6-(6-Acetylaminopyridin-2-ylmethoxymethyl)pyridin-2-
yl]-N′-(6-methylpyridin-2-yl)isophthalamide (Receptor 1). Mp
1
86-87 °C. Isolated yield 17%. H NMR (CDCl3; 500 MHz): δ
Experimental Section
8.94 (br s, 2H), 8.56 (s, 1H), 8.30 (d, 1H, J ) 8.2 Hz), 8.19 (d,
1H, J ) 8.2 Hz), 8.16 (d, 2H, J ) 7.7 Hz), 8.11 (br s, 1H), 7.79 (t,
1H, J ) 7.8 Hz), 7.72 (d, 1H, J ) 7.8 Hz), 7.69-7.64 (m, 3H),
7.24 (d, 1H, J ) 7.4 Hz), 7.18 (d, 1H, J ) 7.4 Hz), 6.96 (d, 1H,
J ) 7.4 Hz), 4.64 (s, 2H), 4.62 (s, 2H), 2.46 (s, 3H), 2.16 (s, 3H).
13C NMR (CDCl3; 125 MHz): δ 169.2, 165.1, 165.1, 157.3, 156.7,
156.4, 151.4, 151.4, 151.2, 139.6, 139.5, 139.3, 135.3, 135.1, 131.5,
131.4, 129.8, 126.3, 120.1, 118.3, 117.9, 113.5, 113.3, 111.6, 73.5,
73.4, 25.0, 24.3. MS (FAB) (m/z, %): 533 (M + Na+, 20), 511
(MH+, 100), 467 (18), 439 (30), 242 (80), 154 (80). FT-IR (KBr,
ν): 2360, 1682, 1577, 1540, 1455, 1303, 797 cm-1. Anal. Calcd
for C28H26N6O4 (510.56): C, 65.87; H, 5.13; N, 16.46; O, 12.53.
Found: C, 65.89; H, 5.14; N, 16.46.
Preparation of Ethyl Ester of Biotin (compound 4). Com-
mercially available D(+)-biotin (250 mg) was added into dry ethanol
(10 mL) in a round-bottomed flask. Concentrated H2SO4 (two drops)
was added to it and refluxed (12 h). After the usual workup, an
off-white solid material was isolated (yield 79%; 220.0 mg). Mp
1
80-81 °C. H NMR(CDCl3; 500 MHz): δ 5.26 (br s, 1H), 5.20
(br s, 1H), 4.52 (t, 1H, J ) 6.2 Hz), 4.33 (t, 1H, J ) 5.9 Hz), 4.13
(q, 2H, J ) 7.1 Hz), 3.17 (m, 2H), 2.93 (dd, 1H, J ) 5.0, 4.9 Hz),
2.74 (d, 2H, J ) 12.83 Hz), 2.32 (t, 2H, J ) 7.3), 1.69-1.66 (m,
2H), 1.49-1.44 (m, 2H), 1.25 (t, 3H, J ) 7.11 Hz).
Preparation of N-{6-[6-(2,2-Dimethylpropionylamino)pyri-
din-2-ylmethoxymethyl]pyridin-2-yl}-2,2-dimethylpropiona-
mide (5). To a solution of 2-(N-pivalylamino)-6-hydroxymethylpy-
ridine (500 mg, 2.4 mmol) in dry tetrahydrofuran was added sodium
hydride (173 mg) and the solution was stirred under nitrogen
atmosphere for 1 h. The solution of 2-(N-pivalylamino)-6-bromo-
methylpyridine (650 mg, 2.4 mmol) in dry tetrahydrofuran was
added dropwise to the reaction mixture under nitrogen atmosphere
with stirring for 12 h. Tetrahydrofuran was removed under vacuum
and dichloromethane was added to the solid. The organic layer was
washed with brine solution and dried over anhydrous sodium sulfate.
Then the solvent was stripped off and the residue was purified by
column chromatography (using 60-120 silica gel) by using 1%
methanol and chloroform to yield a brownish white semisolid 5 as
reported earlier (255 mg, yield 64%).11
1
Complex of Receptor 1 with Biotin. H NMR (CDCl3; 500
MHz): δ 10.46 (br s, 1H), 10.24 (br s, 1H), 9.20 (br s, 1H), 8.70
(s, 1H), 8.32 (d, 1H, J ) 8.2 Hz), 8.26 (t, 2H, J ) 7.2 Hz), 8.22
(d, 1H, J ) 8.1 Hz), 8.11 (d, 1H, J ) 7.8 Hz), 7.79 (t, 1H, J ) 7.9
Hz), 7.74-7.70 (m, 2H), 7.64 (t, 1H, J ) 7.7 Hz), 7.26 (d, 1H, J
) 7.4 Hz), 7.18 (d, 1H, J ) 7.4 Hz), 6.99 (d, 1H, J ) 7.4 Hz),
6.02 (br s, 1H), 5.90 (br s, 1H), 4.69 (s, 2H), 4.64 (s, 2H), 4.49-
4.47 (m, 1H), 4.28-4.26 (m, 1H), 2.88 (dd, 2H, J ) 5.0, 5.0 Hz),
2.53 (s, 3H), 2.46 (d, 1H, J ) 4.4 Hz), 2.29 (t, 2H, J ) 7.4 Hz),
2.18 (s, 3H), 1.66-1.58 (m, 2H), 1.49-1.41 (m, 2H), 0.87 (t, 2H,
J ) 6.8 Hz).
1
Complex of Receptor 1 with the Ethyl Ester of Biotin. H
NMR (CDCl3; 500 MHz): δ 10.18 (br s, 1H), 10.12 (br s, 1H),
8.68 (s, 1H), 8.35 (br s, 1H), 8.32 (d, 1H, J ) 8.3 Hz), 8.24 (d,
1H, J ) 8.2 Hz), 8.20 (d, 2H, J ) 7.4 Hz), 8.10 (d, 1H, J ) 7.9
Hz), 7.77 (t, 1H, J ) 7.9 Hz), 7.70-7.66 (m, 2H), 7.64 (t, 1H, J
) 7.7 Hz), 7.17 (d, 1H, J ) 7.4 Hz), 7.10 (d, 1H, J ) 7.5 Hz),
6.96 (d, 1H, J ) 7.4 Hz), 5.18 (br s, 1H), 5.08 (br s, 1H), 4.59 (s,
4H), 4.53-4.50 (m, 1H), 4.32-4.30 (m,1H), 4.12 (q, 2H, J ) 7.1
Hz), 3.18-3.14 (m, 1H), 2.92 (dd, 1H, J ) 5.0, 5.0 Hz), 2.73 (d,
1H, J ) 12.8 Hz), 2.46 (s, 3H), 2.31-2.27 (m, 2H), 2.12 (s, 3H),
1.69-1.66 (m, 2H), 1.49-1.38 (m, 2H), 1.25 (t, 3H, J ) 7.0 Hz),
0.88 (t, 2H, J ) 6.7 Hz).
N,N′-Bis[6-(6-acetylaminopyridin-2-ylmethoxymethyl)pyridin-
2-yl]isophthalamide (receptor 2). Mp 167-69 °C; isolated yield
22%. 1H NMR (CDCl3; 500 MHz) δ 9.17 (br s, 2H), 8.61 (s, 1H),
8.34 (br s, 2H), 8.29 (d, 2H, J ) 8.2 Hz), 8.19 (d, 2H, J ) 7.7 Hz),
8.08 (d, 2H, J ) 7.7 Hz), 7.78 (t, 2H, J ) 7.8 Hz), 7.68 (t, 1H, J
) 7.9 Hz), 7.64 (d, 2H, J ) 7.7 Hz), 7.21 (d, 2H, J ) 7.4 Hz),
7.14 (d, 2H, J ) 7.4 Hz), 4.60 (s, 4H), 4.59 (s, 4H), 2.15 (s, 6H).
6-{[(6-Amino-2-pyridyl)methoxy]methyl}-2-pyridinamine (6).
Compound 5 (0.5 g, 1.25 mmol) was dissolved in 1:1 ethanol-
water (10 mL) and 4 N KOH and the solution was refluxed for 6
h. Then the solvent was removed and water was added to the residue
and extracted with ethyl acetate. The organic layer was separated
and dried over anhydrous sodium sulfate. The diamino compound
6 was obtained through short column chromatography with 3%
methanol in chloroform. A deep brown solid material 6 was isolated
1
(260 mg, yield 90%, mp-85 °C). H NMR(CDCl3; 500 MHz): δ
7.45 (t, 2H, J ) 7.77 Hz), 6.83 (d, 2H, J ) 7.32), 6.40 (d, 2H, J
) 8.10 Hz), 4.57 (s, 4H), 4.25 (br s, 4H).
N-[6-(6-Aminopyridin-2-ylmethoxymethyl)pyridin-2-yl]aceta-
mide (7). Compound 6 (550 mg, 2.0 mmol) was taken in dry
dichloromethane (5 mL) and 0.5 equiv of acetic anhydride (1 mmol)
was added to it. Then the solution was stirred at room temperature
for 2 h. Compound 7 was isolated through column chromatography,
using 100-200 silica gel and 1% methanol in chloroform as
7286 J. Org. Chem., Vol. 71, No. 19, 2006

Design and Synthesis of Neutral Receptors for Biotin
13C NMR (CDCl3; 125 MHz): δ 169.7, 156.6, 156.4, 151.9, 151.5,
139.4, 134.8, 132.1, 131.9, 129.7, 126.5, 119.8, 118.2, 117.8, 113.8,
113.2, 111.8, 73.5, 24.9. MS (ESI) (m/z, %): 697 (M + Na+, 15),
675 (MH+, 100), 525 (20), 476 (30). FT-IR (KBr, ν): 2926, 2360,
1680, 1601,1578, 1536, 1455, 1302, 1235, 796, 721 cm-1. Anal.
Calcd for C36H34N8O6 (674.72): C, 64.09; H, 5.08; N, 16.61; O,
14.23. Found: C, 64.10; H, 5.08; N, 16.62.
5.0, 4.8 Hz), 2.74 (d, 1H, J ) 12.8 Hz), 2.52 (s, 6H), 2.30 (t, 2H,
J ) 6.5 Hz), 1.69-1.62 (m, 2H), 1.46 (t, 3H, J ) 6.9 Hz).
1
Complex of Receptor 3 with Ethyl Ester of Biotin. H NMR
(CDCl3; 500 MHz): δ 9.99 (br s, 2H), 8.66 (s, 1H), 8.23 (d, 2H, J
) 8.3 Hz), 8.18 (dd, 2H, J ) 1.7, 1.7 Hz), 7.67 (t, 2H, J ) 7.8
Hz), 7.62 (t, 1H, J ) 7.7 Hz), 7.21 (br s, 1H), 6.94 (d, 2H, J ) 7.4
Hz), 6.83 (br s, 1H), 4.65-4.62 (m, 1H), 4.43-4.41 (m, 1H), 4.09
(q, 2H, J ) 7.2 Hz), 3.21-3.16 (m, 1H), 2.95 (dd, 2H, J ) 5.1,
5.1 Hz), 2.76 (d, 1H, J ) 12.5 Hz), 2.47 (s, 6H), 2.26 (t, 2H, J )
7.1 Hz), 1.62-1.57 (m, 2H), 1.48-1.40 (m, 3H), 1.23 (t, 3H, J )
7.1 Hz).
Experimental Procedure for Scheme 2: Synthesis of Recep-
tors 2 and 3. Isophthaloyl chloride (70 mg, 0.34 mmol) was taken
in dichloromethane (10 mL). Compound 7 (200 mg; 0.68 mmol)
mixed with 0.25 mL of triethylamine was also dissolved in dry
dichloromethane (15.0 mL) and taken in a dropping funnel. Then
it was added dropwise to the isophthaloyl chloride solution and
addition was continued for 1 h with stirring under nitrogen
atmosphere. The reaction was prolonged for 12 h. Receptor 2 was
isolated through preparative TLC, using methanol (5%) in dichlo-
romethane.
1
Complex of Receptor 2 with Biotin. H NMR (2% d6-DMSO
+ CDCl3; 500 MHz): δ 10.25 (br s, 2H), 9.30 (br s, 2H), 8.66 (s,
1H), 8.31 (d, 2H, J ) 8.2 Hz), 8.22 (d, 2H, J ) 7.7 Hz), 8.10 (d,
2H, J ) 7.4 Hz), 7.80 (t, 2H, J ) 7.9 Hz), 7.70 (t, 2H, J ) 7.8
Hz), 7.64 (t, 1H, J ) 7.7 Hz), 7.26 (d, 2H, J ) 7.4 Hz), 7.18 (d,
2H, J ) 7.4 Hz), 5.89 (br s, 1H), 5.79 (br s, 1H), 4.66 (s, 4H),
4.62 (s, 4H), 4.48-4.45 (m, 1H), 4.27-4.25(m, 1H), 3.19-3.18
(m, 1H), 2.87 (dd, 2H, J ) 5.0, 5.0 Hz), 2.73 (d, 1H, J ) 12.7
Hz), 2.29 (t, 2H, J ) 7.3 Hz), 2.18 (s, 6H), 1.67-1.61 (m, 3H),
1.48-1.42 (m, 2H).
1
Complex of Receptor 2 with Ethyl Ester of Biotin. H NMR
(CDCl3; 500 MHz): δ 9.94 (br s, 2H), 8.67 (s, 1H), 8.37 (br s,
2H), 8.32 (d, 2H, J ) 8.2 Hz), 8.20 (d, 2H, J ) 8.9 Hz), 8.10 (d,
2H, J ) 7.6 Hz), 7.78 (t, 2H, J ) 7.8 Hz), 7.71-7.61 (m, 3H),
7.18 (d, 2H, J ) 7.4 Hz), 7.11 (d, 2H, J ) 7.4 Hz), 6.40 (br s, 1H),
5.99 (br s, 1H), 4.58 (s, 4H), 4.56 (s, 4H), 4.44-4.42 (m, 1H),
4.19-4.17 (m, 1H), 4.10 (q, 2H, J ) 6.8 Hz), 3.10-3.08 (m, 1H),
2.82 (dd, 2H, J ) 5.0, 4.9 Hz), 2.70 (d, 1H, J ) 12.7 Hz), 2.46-
2.44 (m, 2H), 2.31-2.26(m, 2H), 2.23 (t, 2H, J ) 7.5 Hz), 2.19 (s,
3H), 2.15 (s, 3H), 1.60-1.55 (m, 2H), 1.26 (t, 2H, J ) 6.0 Hz).
N,N′-Bis(6-methylpyridin-2-yl)isophthalamide (receptor 3).
Isolated yield 26%. 1H NMR (CDCl3; 500 MHz): δ 8.81 (brs, 2H),
8.56 (s, 1H), 8.21 (d, 2H, J ) 7.7 Hz), 8.17 (d, 2H, J ) 7.7 Hz),
7.70-7.64 (m, 3H), 6.97 (d, 2H, J ) 7.5 Hz), 2.49 (s, 6H).
The same procedure was followed to prepare receptor 3 taking
2-amino-6-methylpyridine instead of compound 7.
Acknowledgment. We thank DST, DST-FIST, and CSIR
[Project No. 01(1913)/04/EMR-II], Government of India, for
financial support and appreciate mass spectral help from
Professor Thomas Schrader, University of Marburg, Germany.
Supporting Information Available: NMR spectra, IR spectra,
UV titration spectra of receptors and the complexes and the
association constants calculation curves (both NMR and UV
method), and energy minimized structures. This material is available
free of charge via the Internet at http://pubs.acs.org.
1
Complex of Receptor 3 with Biotin. H NMR (CDCl3; 500
MHz): δ 10.08 (br s, 2H), 8.67 (s, 1H), 8.26-8.22 (m, 4H), 7.69
(t, 2H, J ) 7.8 Hz), 7.64 (t, 1H, J ) 7.7 Hz), 6.97 (d, 2H, J ) 7.4
Hz), 5.71 (br s, 1H), 5.67 (br s, 1H), 4.52 (t, 1H, J ) 6.3 Hz), 4.34
(t, 1H, J ) 5.5 Hz), 3.16 (t, 1H, J ) 6.7 Hz), 2.92 (dd, 2H, J )
JO061013J
J. Org. Chem, Vol. 71, No. 19, 2006 7287