Molecular Imprinting of Carboxylic Acids Employing Novel Functional Macroporous Polymers

Article abstract of DOI:10.1021/jo982118s

Imprinted network polymers incorporating basic functional groups were developed to assess the binding and specificity of carboxylic acids. The binding affinities were determined using t-BOC-phenylalanine and 2-phenylbutyric acid as templates and substrates. Chiral selectivity for the enantiomers of t-BOC-phenylalanine was found for polymers incorporating adenine or 2-aminopyridine functionality. Chiral selectivity in the case of (R)-(-)-2-phenylbutyric acid was found for the polymer utilizing N-(2-aminoethyl) methacrylamide as the functional monomer. Optimization of binding was achieved by changing polymerization conditions (thermal versus photochemical polymerization, monomer:template ratio) for t-BOC-phenylalanine imprinted polymers employing the N-(2-aminopyridine) methacrylamide monomer.

Full text of DOI:10.1021/jo982118s

J . Org. Chem. 1999, 64, 4627-4634
4627
Molecu la r Im p r in tin g of Ca r boxylic Acid s Em p loyin g Novel
F u n ction a l Ma cr op or ou s P olym er s
David Spivak† and Kenneth J . Shea*
Department of Chemistry, University of California, Irvine, Irvine, California 92967-2025
Received October 20, 1998
Imprinted network polymers incorporating basic functional groups were developed to assess the
binding and specificity of carboxylic acids. The binding affinities were determined using t-BOC-
phenylalanine and 2-phenylbutyric acid as templates and substrates. Chiral selectivity for the
enantiomers of t-BOC-phenylalanine was found for polymers incorporating adenine or 2-aminopy-
ridine functionality. Chiral selectivity in the case of (R)-(-)-2-phenylbutyric acid was found for the
polymer utilizing N-(2-aminoethyl) methacrylamide as the functional monomer. Optimization of
binding was achieved by changing polymerization conditions (thermal versus photochemical
polymerization, monomer:template ratio) for t-BOC-phenylalanine imprinted polymers employing
the N-(2-aminopyridine) methacrylamide monomer.
Sch em e 1. Ou tlin e of th e Molecu la r Im p r in tin g
Str a tegy
In tr od u ction
Biological macromolecules, such as antibodies and
enzymes, benefit from a variety of functional groups to
effect recognition and catalytic properties. The rapidly
developing technique of molecular imprinting,1 which
provides polymeric artificial receptors and catalysts,
would also benefit from an expanded repertoire of
functional groups. The concept of molecular imprinting
is illustrated in Scheme 1. Functional monomers are
bound either covalently or noncovalently to a print
molecule or template. The resulting pre-polymer complex
is copolymerized with an excess of cross-linking monomer
in the presence of an equal volume of inert solvent
(porogen) and a free radical initiator. Thermal or photo-
chemical polymerization results in a highly cross-linked
insoluble polymer.2 Removal of the template leaves
cavities in the polymer that are complementary in size,
shape, and chemical functionality to the template mol-
ecule.
Two methods are available for formation of the pre-
polymer complex; one uses covalently bound templates,
and the other utilizes noncovalent interactions. Molecular
imprinting using noncovalent interactions has been
dominated by the use of the functional monomer meth-
acrylic acid.3,4 Despite its utility, this limits the choice
of templates to those that bind to carboxylate functional-
ity. To imprint templates that do not bind carboxylic
acids, new monomers needed to be synthesized. The goal
of this project was to design, synthesize, and survey the
binding potential of methacrylamide-based monomers
incorporating various carboxylate binding functional
groups. Some weakly basic monomers such as N-vi-
nylimidazole, 4-vinylpyridine, and 2-vinylpyridine have
been used as functional monomers for molecular imprint-
ing.5 Reports of methacrylamide-based monomers con-
taining amine functionality primarily dealt with catalysis
rather than binding by imprinted polymers.6 Six novel
monomers to create selective binding sites in polymers
for carboxylic acids by molecular imprinting are reported
in this paper.
† Present address: Department of Chemistry, Louisiana State
University.
(1) (a) Shea, K. J . Trends Polym. Sci. 1994, 2, 166. (b) Wulff, G.
Angew. Chem., Int. Ed. Engl. 1995, 34, 1812. (c) Mosbach, K.,
Ramstrom, O. Biotechnology 1996, 14, 163.
(2) (a) Guyot, A. Synthesis and Separations Using Functional
Polymers; Sherrington, D. C., Hodge, P., Eds.; J ohn Wiley & Sons: New
York, 1989; p 1. (b) Lloyd, L. L. J . Chromatogr. 1991, 544, 201. (c)
Guyot, A.; Bartholin, M. Prog. Polym. Sci. 1982, 8, 277.
(3) (a) Andersson, L.; Sellergren, B.; Mosbach, K. Tetrahedron Lett.
1984, 25, 5211. (b) Sellergren, B.; Ekberg, B.; Mosbach, K. J . Chro-
matogr. 1985, 347, 1. (c) Sellergren, B.; Lepisto, M.; Mosbach, K. J .
Am. Chem. Soc. 1988, 110, 5853. (d) Fischer, L.; Muller, R.; Ekberg,
B.; Mosbach, K. J . Am. Chem. Soc. 1991, 113, 9358. (e) Ramstrom, O.;
Nicholls, I. A.; Mosbach, K. Tetrahedron: Asymmetry 1994, 5, 649.
(4) (a) Shea, K. J .; Spivak, D. A.; Sellergren, B. J . Am. Chem. Soc.
1993, 115, 3368. (b) Spivak, D.; Gilmore, M. A.; Shea, K. J . J . Am.
Chem. Soc. 1997, 119, 4388.
Mon om er Design . Candidates for functional mono-
mers were generated from three areas: amino acids and
(5) (a) Leonhardt, A.; Mosbach, K. React. Polym. 1987, 6, 285. (b)
Kempe, M.; Fischer, L.; Mosbach, J . J . Mol. Recognit. 1993, 6, 25. (c)
Ramstrom, O.; Andersson, L. I.; Mosbach, K. J . Org. Chem. 1993, 58,
7562. (d) Sarhan, A.; El-Zahab, M. A. Makromol. Chem., Rapid
Commun. 1987, 8, 555. (e) Sarhan, A.; Ali, M. M.; Abdelaal, M. Y.
React. Polym. 1989, 11, 57.
(6) (a) Beach, J . V.; Shea, K. J . J . Am. Chem. Soc. 1994, 116, 379.
(b) Ohkubo, K.; Funakoshi, Y.; Urata, Y.; Hirota, S.; Usui, S.; Sagawa,
T. J . Chem. Soc., Chem. Commun. 1995, 2143.
10.1021/jo982118s CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/06/1999

4628 J . Org. Chem., Vol. 64, No. 13, 1999
Spivak and Shea
F igu r e 1. Selective receptors using the 2-aminopyridine9 or
guanidinium10 functionality to bind dicarboxylic acids.
proteins, nucleic acids, and designed small molecule
carboxylate receptors. The amino acids capable of binding
carboxylic acids are essentially those with ionizable side
chains.7 The guanidinium group of arginine in proteins
is known to bind strongly to carboxylate, as well as to
phosphate groups.8-13 Other carboxylate-binding amino
acid subgroups are the primary amine of lysine, the
secondary amine of imidazole on histidine, and finally,
heterodimers with the carboxylate group of aspartic and
glutamic acid. The DNA bases guanine and adenine form
the strongest association of all of the nucleotide bases
with butyric acid in chloroform.14 Previous molecular
imprinting experiments have shown that adenine binds
well to imprinted polymers containing carboxylate groups.4
Guanine, however, does not bind as well to these tem-
plated polymers. Designed synthetic small molecules that
bind carboxylic acids have been developed in a number
of laboratories including those of Hamilton,8-10 Lehn,11
Schmidtchen,12 and Anslyn.13 The majority of these
incorporate either a 2-aminopyridine substructure or a
guanidinium moiety embedded in their structures (Figure
1).
From a consideration of the examples above, functional
monomers 1-7 (Figure 2) were chosen for molecular
imprinting of carboxylates. N-(Diaminomethylene)-2-
methylprop-2-enamide (1) incorporates the guanidine
group; in addition, the rigid structure is expected to
minimize conformational flexibility (inherent in monomer
2) which will maintain the specific orientation of the
guanidinium group(s) originally positioned by the im-
printing process. This has been shown to positively
influence selective binding by molecularly imprinted
polymers.15 The design of the monomer N-(3-guanidino-
propyl) methacrylamide (2) mimics the amino acid argi-
nine, which is responsible for binding and catalysis in
many enzymes. Earlier work in designed polymer cata-
lysts utilized N-propyl and N-ethyl methacrylamides for
F igu r e 2. Chemical structures of functional monomers used
for the carboxylic acid imprinting study.
imprinting dicarboxylic acid templates.6 N-Propyl meth-
acrylamide (3) was commercially available from Kodak.
Use of the N-ethyl methacrylamide monomer (4) has been
previously reported for use in a catalytic imprinted
polymer; however, its carboxylate binding potential has
not been evaluated.
The nucleotide base adenine binds strongly with car-
boxylate-functionalized imprinted polymers.4 The meth-
acrylate-based adenine monomer 9-(â-methacryloyloxy-
ethyl) adenine (5) has been reported in the literature16
and used for the synthesis polyadenine methacrylate.
One of the major binding motifs of adenine is the
2-aminopyridine moiety, responsible for Watson-Crick
binding in DNA duplexes.4,17 As previously noted, Hamil-
ton has used this moiety successfully for creating car-
boxylate receptors.8-10 N-(2-Aminopyridine) methacryla-
mide (6) was predicted to take advantage of this binding.
Finally, methacrylic acid (7) has the potential to create
carboxylate heterodimers with target carboxylic acid
templates.
Ethylene glycol dimethacrylate (EGDMA) was chosen
as the principal cross-linking monomer. Although this
monomer was not chosen by a systematic process,
macroporous polymers prepared with EGDMA give stable,
robust materials that were suitable for use as stationary
phases in HPLC. In addition, they provide an average
polarity that permits optimization of chromatographic
retention with common organic elements.
(7) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Company:
New York, 1988.
(8) (a) Hamilton, A. D. Advances in Supramolecular Chemistry, Vol.
1; J AI Press Inc., 1990; pp 1-64. (b) Fan, E.; Van Arman, S. A.;
Kincaid, S.; Hamilton, A. D. J . Am. Chem. Soc. 1993, 115, 369. (c)
Vicent, C.; Fan, E.; Hamilton, A. D. Tetrahedron Lett. 1992, 4269.
(9) Garcia-Tellado, F.; Goswami, S.; Chang, S.; Geib, S. J .; Hamilton,
A. D. J . Am. Chem. Soc. 1990, 112, 7393.
(10) Dixon, R. P.; Geib, S. J .; Hamilton, A. D. J . Am. Chem. Soc.
1992, 114, 365.
(11) (a) Echavarren, A.; Galan, A.; Lehn, J .-M.; de Mendoza, J . J .
Am. Chem. Soc. 1989, 111, 4994. (b) Dietrich, B.; Fyles, D. L.; Fyles,
T. M.; Lehn, J .-M. Helv. Chim. Acta 1979, 2763. (c) Echavarren, A.;
Galan, A.; de Mendoza, J .; Salmeron, A.; Lehn, J .-M. Helv. Chim. Acta
1988, 685.
(12) (a) Schmidtchen, F. P. Tetrahedron Lett. 1989, 4493. (b) Muller,
G.; Riede, J .; Schmidtchen, F. P. Angew. Chem., Int. Ed. Engl. 1988,
27, 1516. (c) Kurzmeier, H.; Schmidtchen, F. P. J . Org. Chem. 1990,
55, 3749.
(13) Kneeland, D. M.; Ariga, K.; Lynch, V. M.; Huang, C.; Anslyn,
E. V. J . Am. Chem. Soc. 1993, 115, 10042.
Resu lts a n d Discu ssion
Syn th esis of F u n ction a l Mon om er s. N-(Diamino-
ethylene)-2-methylprop-2-enamide was synthesized by
condensation of guanine and methyl methacrylate. Guani-
dinium hydrochloride was desalted by passing through
ion-exchange resin prior to refluxing with methyl meth-
acrylate to give 1 as a pale yellow solid (Scheme 2).
Following the method of Lehn,11 S-ethyl thiourea was
reacted with N-(3-aminopropyl) methacrylamide to give
(14) (a) Lancelot, G. J . Am. Chem. Soc. 1977, 99, 7037. (b) Lancelot,
G. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4872.
(15) Wulff, G.; Gimpel, J . Makromol. Chem. 1982, 183, 2469.
(16) Kondo, K.; Iwasaki, H.; Ueda, N.; Takemoto, K.; Imoto, M.
Makromol. Chem. 1968, 120, 21.
(17) Watson, J . D.; Crick, F. H. C. Nature (London) 1953, 171, 737.

Molecular Imprinting of Carboxylic Acids
J . Org. Chem., Vol. 64, No. 13, 1999 4629
Sch em e 2a
Sch em e 4a
a
(a) DMF/NaOH/reflux/3 h. (b) NaH/DMF/rt/1 h. (c) 0 °C/
methacryloyl chloride/14 h. (d) CH2Cl2/Et3N/0 °C.
a
(a) EtOH/100 °C. (b) NH4OH (aq)/rt/16 h. (c) Dowex 1 × 8
anion exchange resin.
Sch em e 3a
F igu r e 3. Carboxylic acid templates used for imprinting using
new monomers: t-BOC-phenylalanine (8) and 2-phenylbutyric
acid (9).
condensation of methacryloyl chloride with 2-aminopy-
ridine (Scheme 4).
P olym er s Im p r in ted w ith t-BOC-P h en yla la n in e.
The monomers were first tested for their ability to bind
a carboxylic acid in a molecularly imprinted polymer
using the template t-BOC-L-phenylalanine (8, Figure 3).
This template was chosen for the following reasons: (1)
the chiral center allows for evaluation of stereochemical
differentiation (one of the most difficult tasks in separa-
tion science); (2) there is a chromophore for UV detection;
(3) both enantiomers are commercially available; and (4)
t-BOC-L-phenylalanine is soluble in organic solvents and
may assist in solubilizing the functional monomers.
These results will also permit comparison with the
performance of imprinted polymers using methacrylic
acid and C-protected phenylalanine derivatives.20 The
polymerization mixtures were formulated using template
(2.9 mol %), functional monomer (2.9 mol %), ethylene
glycol dimethacrylate (92.7 mol %), and AIBN (1.5 mol
%) in porogen (40/60% v/v monomers/porogen). The
mixtures were polymerized photochemically to give a
highly cross-linked network polymer.2 The template,
t-BOC-L-Phe, was removed by Soxhlet extraction and
quantified by proton NMR using an internal standard
(the fraction of template removed is called the split ratio).
The conditions for preparation of the imprinted poly-
mers are shown in Table 1. Initially, DMF was used as
porogen for all polymers. Additional porogens were used
in subsequent studies based on binding studies described
in the following sections. As a result of the insolubility
of the functional monomer N-(3-guanidinopropyl) meth-
acrylamide, a modified formulation utilizing a 10-fold
(a) THF/0 °C/12 h. (b) HCl/EtO2/rt/3 h. (c) THF/Et3N/0 °C to
rt/24 h. (d) HCl/EtO2/rt/24 h. (e) EtOH/NaOEt/rt/1 h.
N-(3-guanidinopropyl) methacrylamide 2 (Scheme 2).
Because of sensitivity toward polymerization, the reaction
with N-(3-aminopropyl) methacrylamide (3) was carried
out at room temperature.
Synthesis of N-(aminoethyl) methacrylamide (Scheme
3) employed bis-protection of ethylenediamine and sub-
sequent mono-deprotection to give the intermediate N-(2-
aminoethyl) carbamic acid tert-butylester hydrochloride.18
Coupling of this intermediate with methacryloyl chloride
and deprotection give monomer 4. Synthesis of 9-(â-
methacryloyloxyethyl) adenine16 was accomplished by
alkylation with ethylene carbonate to give 9-(â-hydroxy-
ethyl) adenine. This was followed by treatment with
methacryloyl chloride to give monomer 5 (Scheme 4).
N-(2-Aminopyridine) methacrylamide was synthesized by
(18) Futao Wakui, Ph.D. Thesis, University of California at Irvine,
1991.

4630 J . Org. Chem., Vol. 64, No. 13, 1999
Ta ble 1. Con d ition s for th e P r ep a r a tion of Ea ch Im p r in ted P olym er
Spivak and Shea
split ratio
polymer
functional monomer
porogen
tempa
timeb
P(DOMPE)
P(GPM)
P(APM)
1 N-(diaminoethylene)-2-methylprop-2-enamide
2 N-(3-guanidinopropyl) methacrylamide
3 N-(3-aminopropyl)methacrylamide
4 N-(2-aminoethyl)methacrylamide
5 9-(â-methacryloyloxyethyl) adenine
6 N-(2-aminopyridine)methacrylamide
7 methacrylic acid
CHCl3
DMF
DMF
DMF
DMF
4.9 °C
5.8 °C
6.6 °C
5.8 °C
6.6 °C
5.7 °C
6.6 °C
10 h
8 h
6 h
8 h
6 h
8 h
6 h
>99%
85%
53%
61%
57%
P(AEM)
P(MAOA)
P(APYM)
P(MAA)
CHCl3
MeCN
>99%
83%
a
b
Temperature of polymerization. Time of polymerization.
Ta ble 2. Ch r om a togr a p h ic Con d ition s, Ca p a city F a ctor s, a n d Sep a r a tion F a ctor s for L a n d D t-BOC-p h en yla la n in e on
Colu m n s P a ck ed w ith th e In d ica ted Tem p la te P olym er sa
polymer
functional monomer
mobile phase
MeCN
96/4 MeCN/AcOH
CHCl3
96/4 MeCN/AcOH
MeCN
CHCl3
k′L
k′D
a
P(DOMPE)
P(GPM)
P(APM)
1 N-(diaminoethylene)-2-methylprop-2-enamide
2 N-(3-guanidinopropyl) methacrylamide
3 N-(3-aminopropyl)methacrylamide
4 N-(2-aminoethyl)methacrylamide
5 9-(â-methacryloyloxyethyl) adenine
6 N-(2-aminopyridine)methacrylamide
7 methacrylic acid
0.0
0.5
0.0
2.2
13.3
0.7
0.0
0.5
0.0
2.2
9.1
0.6
0.4
1.0
1.0
1.0
1.0
1.5
1.2
1.0
P(AEM)
P(MAOA)
P(APYM)
P(MAA)
MeCN
0.4
a
Flow rate ) 1 mL/min, injections were 5.0 mL of a 1.0 mM solution of L or D t-BOC-phenylalanine, UV detection at λ ) 257 nm. The
one exception was P(MAOA), which required a 10.0 mL injection of 10.0 mM substrates.
excess of t-BOC-L-phenylalanine was used (10/1 t-BOC-
L-phenylalanine/monomer) to dissolve the monomer in
the polymerization mixture.
The highest binding affinity for t-BOC-phenylalanine
was obtained by polymer P(AEM). This polymer incor-
porates the N-(2-aminoethyl) methacrylamide monomer
(4). An interesting result is that polymers incorporating
the N-(3-aminopropyl) methacrylamide monomer (3)
showed no affinity for the template, despite differing from
monomer 4 by only a single methylene group. This is
unusual because monomers 3 and 4 should provide the
same electrostatic interactions. It may be that the
diminished affinity of monomer 3 is due to intramolecular
chelation preventing interaction with the substrate. The
second largest binding affinity was observed with mono-
mer 2, which contains the guanidium moiety. N-(Diami-
noethylene)-2-methylprop-2-enamide (monomer 1) also
incorporates the guanidinium functionality; however,
t-BOC-phe imprinted polymers made using monomer 1
show little affinity for 8. The reason may be due to a
decrease in the inherent binding strength of the guani-
dinium moiety of monomer 1 from direct acylation to the
methacrylate moiety. P(MAOA) made with monomer 5
exhibited moderate binding affinity; P(MAA) and P(A-
PYM) provided minimal binding affinities.
Evidence of chiral selectivity, indicated by separation
factors (R) greater than 1.0, was found for P(MAOA) and,
to a lesser extent, P(APYM). These two polymers have
in common the 2-aminopyridine substructure. A possible
reason for the selectivity observed may be the directional
hydrogen bonding afforded by the 2-aminopyridine moi-
ety demonstrated by Hamilton.8,9 Directed hydrogen
bonding groups in a cleft or cavity have been shown to
lead to good orientation of a substrate with complemen-
tary functional groups.8,21 The selectivity of the polymers
may also benefit from this type of directional hydrogen
bonding. Specific binding by P(MAA) is not seen even
though directional bonds are anticipated to exist between
carboxylate groups. It is possible that the functional
groups of P(APYM) and P(MAOA) interact with the
carbamate moiety, as well as the carboxylate group, to
aid in discrimination of enantiomers, whereas P(MAA)
may not. P(AEM) showed very good binding potential;
Following removal of the template, the polymers were
ground to 25-38 µm particle size and slurry packed into
strainless steel chromatographic columns. Binding data
was recorded in the form of capacity factors (k′)20 for L
and D t-BOC-Phe as substrates to determine binding
affinities and enantiomeric discrimination. This measure
of binding is superior to the use retention time because
it is independent of column size or the amount of
stationary phase in the column. Chiral selectivity was
measured by the separation factor (R), taken as the ratio
of capacity factors for each enantiomer of t-BOC-Phe (i.e.,
R ) k′L/k′D). Table 2 summarizes the mobile phase
conditions and chromatographic results for each polymer.
The chromatographic data was used to evaluate both
the carboxylate binding potential and the molecular
specificity of the polymers. Two criteria were used to
evaluate binding strength: first, the elutropic strength
of the mobile phase and, second, the magnitude of the
capacity factor found in each mobile phase. Three mobile
phases with different degrees of elutropic strength were
employed to probe binding affinities. If the retention
volume of t-BOC-L-Phe using the mobile phase 96/4
MeCN/AcOH was not significantly above dead volume
(i.e., k′ ) 0), a less elutropic mobile phase, acetonitrile,
was evaluated next. Finally, chloroform was employed
as the mobile phase if no significant binding was found
in acetonitrile. The magnitude of the capacity factors
within each mobile phase group was used to fine tune
the order of binding affinities. Using these criteria, the
order of binding strength by the polymers for t-BOC-L-
phenylalanine was found to be P(AEM) > P(GPM) >
P(MAOA) > P(MAA) > P(APYM), P(DOMPE), P(APM).
(19) (a) Sellergren, B.; Ekberg, B.; Mosbach, K. J . Chromatogr. 1985,
347, 1. (b) Sellergren, B.; Lepisto, M.; Mosbach, K. J . Am. Chem. Soc.
1988, 110, 5853. (c) O’Shannessy, D. J .; Ekberg, B.; Andersson, L. I.;
Mosbach, K. J . Chromatogr. 1989, 470, 391. (d) Lepisto, M.; Sellergren,
B. J . Org. Chem. 1989, 54, 6010.
(20) The capacity factor (kl) is defined as: k′ ) [V(t) - V(0)}/V(0)]
where V(t) is the retention volume and V(0) is the dead volume or the
retention volume of a nonbinding substrate.
(21) (a) Zimmerman, S. C.; Wu, W.; Zeng, Z. J . Am. Chem. Soc. 1991,
113, 196. (b) Rebek, J .; Askew, B.; Ballester, P.; Buhr, C.; Costero, A.;
J ones, S.; Williams, K. J . Am. Chem. Soc. 1987, 109, 6866.

Molecular Imprinting of Carboxylic Acids
J . Org. Chem., Vol. 64, No. 13, 1999 4631
Ta ble 3. Con d ition s for Ea ch (R)-(-)-2-P h en ylbu tyr ic Acid Im p r in ted P olym er
polymer
monomer
porogen
tempa
timeb
split ratio
P2(GPM)
P2(AEM)
P2(MAOA)
2 N-(3-guanidinopropyl) methacrylamide
4 N-(2-aminoethyl)methacrylamide
5 9-(â-methacryloyloxyethyl) adenine
DMF
DMF
DMF
5.4 °C
5.4 °C
5.4 °C
8 h
8 h
8 h
91%
65.%
>99%
a
b
Temperature of polymerization. Time of polymerization.
Ta ble 4. Ch r om a togr a p h ic Con d ition s, Ca p a city F a ctor s, a n d Sep a r a tion F a ctor s for (R)- or (S)-2-P h en ylbu tyr ic Acid
on Colu m n s P a ck ed w ith th e In d ica ted P olym er sa
polymer
monomer
mobile phase
k′R
k′S
a
P2(GPM)
P2(AEM)
P2(MAOA)
2 N-(3-guanidinopropyl) methacrylamide
4 N-(2-aminoethyl)methacrylamide
5 9-(â-methacryloyloxyethyl) adenine
MeCN
MeCN
MeCN
0.1
0.8
0.1
0.1
0.7
0.1
1.0
1.1
1.0
a
Flow rate ) 1 mL/min, and injections were 5.0 mL of a 1.0 mM solution of R or S 2-phenylbutyric acid, UV detection at λ ) 208 nm.
however, under the protic chromatographic conditions,
the primary amine probably interacts via electrostatic
interactions that may be considered point charges and
lack directionality. P(GPM) can interact both by electro-
static interaction and by hydrogen bonding; however, the
latter can have multiple points of interaction that may
preclude any directional binding. Lastly, polymers
P(DOMPE) and P(APM) simply do not bind substrates
well enough to evaluate selectivity.
Chromatographic investigation of the polymers re-
vealed the following important observations. First, the
capacity factors in Table 4 show that the order of binding
strength for these copolymers is P2(AEM) > P2(GPM),
P2(MAOA). This is consistent with the order found for
the t-BOC-L-phenylalanine imprinted polymers. Thus the
binding potential of these polymers does appear to follow
a general trend. Next, the chiral selectivity of the
polymers was evaluated by comparing separation factor
(R) values. Both P2(MAOA) and P2(GPM) gave R values
of 1.0, indicating nonstereoselective binding. This result
is the same as for P(GPM); however, the previous R value
for P(MAOA) was 1.5. The chiral selectivity by P2(MAOA)
may not be reflected in the R values as a result of its low
binding affinity in this case. Without significant binding,
the imprinted polymers cannot display their potential
chiral selectivity. Thus, selectivity is to some extent a
function of binding strength.
P olym er s Im p r in ted w ith P h en ylbu tyr ic Acid . To
investigate whether the binding and selectivity results
for t-BOC-L-phenylalanine were general or template-
specific, three of the functional monomers were used to
imprint (R)-(-)-2-phenylbutyric acid (9, Figure 3). This
template incorporates a single carboxylate functionality
to test binding potential, one chiral center as a diagnostic
for stereoselectivity, and a UV chromophore for detection
in an HPLC assay. Polymers incorporating N-(aminoet-
hyl) methacrylamide and N-(3-guanidinopropyl) meth-
acrylamide were chosen to test the binding potential with
the new template because of their high binding affinity
in the case of t-BOC-L-Phe, and functional monomer 9-(â-
ethacryloyloxyethyl) adenine was chosen to test selectiv-
ity. The three new polymers, P2(AEM), P2(GPM), and
P2(MAOA), employing the functional monomers N-(2-
aminoethyl) methacrylamide, N-(3-guanidinopropyl) meth-
acrylamide, and 9-(â-methacryloyloxyethyl) adenine, re-
spectively, were prepared in a fashion similar to that for
the t-BOC-L-Phe imprinted polymers. The specific po-
lymerization conditions and split ratios for each of the
new imprinted polymers are shown in Table 3. It should
be noted in the case of P2(MAOA) that 20% of unreacted
functional monomer was recovered.
Effects of Ch a n gin g th e P olym er iza tion Con d i-
tion s on Bin d in g a n d Selectivity. Although the N-(2-
aminopyridine) methacrylamide monomer did not create
the highest affinity polymer for t-BOC-L-phenylalanine,
it did show positive results for chiral resolution. Further
investigation was done to determine if selectivity could
be enhanced and controlled by changing the polymeri-
zation parameters. Five imprinted polymers were pre-
pared, using the N-(2-aminopyridine) methacrylamide
monomer, with three objectives. First, the D enantiomer
of t-BOC-phe was imprinted to verify that chiral selectiv-
ity is not specific to the L enantiomer. Second, the binding
behavior of a thermally initiated polymer was compared
to a photochemically initiated polymer. Third, the ratio
of functionalized monomer to template was increased to
probe the effect on binding and selectivity. Nonspecific
binding was elucidated by control polymers made using
benzoic acid as a “generic” template to randomly distrib-
ute the 2-aminopyridine functionality in the polymer.
Columns were made with the polymers as in the
previous experiments and evaluated by HPLC. The
mobile phase for all columns was acetonitrile, which is
less elutropic than the 96/4 acetonitrile/acetic acid em-
ployed for the chromatography of t-BOC-phenylalanine
substrates on P(AEM) and P(GPM). No binding was
observed for (R)-(-)-2-phenylbutyric acid employing mo-
bile phases containing any portion of acetic acid for
columns containing the polymers P2(AEM), P2(GPM),
and P2(MAOA). Qualitatively, this indicates that binding
in the case of phenylbutyric acid is not as good as for
t-BOC-L-phenylalanine. The stronger binding affinities
observed for t-BOC-L-Phe imprinted polymers must be
due to binding contributions of the carbamate group in
addition to the carboxylic acid. Table 4 summarizes the
chromatographic data for the (R)-(-)-2-phenylbutyric acid
imprinted polymers.
The preparation of the polymers with a 1:1 ratio of
monomer to template followed the general formulation
and procedures outlined previously (and in the Experi-
mental Section). The preparation of polymers with a 2:1
ratio of monomer to template followed the general
formulation and procedures except that 5.8 mol %
template, 86.9 mol % EGDMA, and 5.8 mol % N-(2-
aminopyridine) methacrylamide were used. The porogen
was CHCl3, and the polymerization was carried out at
5.7 °C for 8 h for photopolymerized polymers and at 80
°C for 8 h for thermally initiated polymers. The split ratio

4632 J . Org. Chem., Vol. 64, No. 13, 1999
Spivak and Shea
Ta ble 5. Ch r om a togr a p h ic Con d ition s, Ca p a city F a ctor s, a n d Sep a r a tion F a ctor s for L a n d D t-BOC-P h e Su bstr a tes on
Colu m n s P a ck ed w ith th e In d ica ted P (AP YM) P olym er sa
entry
template
initiation method
ratio of 2apym/template
k′L
k′D
a (k′L/k′D)
a (k′D/k′L)
1
2
3
4
5
6
t-BOC-L-Phe
t-BOC-L-Phe
benzoic acid
benzoic acid
t-BOC-D-Phe
t-BOC-D-Phe
photo
thermal
photo
photo
photo
photo
1:1
1:1
1:1
2:1
1:1
2:1
0.7
0.7
0.5
0.7
0.7
1.0
0.6
0.7
0.5
0.7
0.8
1.3
1.2
1.0
1.0
1.0
1.0
1.0
1.2
1.4
a
Flow rate ) 1 mL/min; injections were 5.0 mL of a 1.0 mM solution of L or D t-BOC-phe, UV detection at λ ) 257 nm.
of all polymers was >99%. Chromatographic data was
obtained as before, and the results are shown in Table
5.
group directly acylated to the methacrylate group, a
significant difference from monomer 2, which has a three-
carbon linker in between. The adenine-functionalized
polymer P(MAOA) exhibited good binding affinity, and
significant but reduced binding was observed for poly-
mers incorporating N-(2-aminopyridine) methacrylamide
(e.g., P(APYM)), a monomer containing a carboxylate-
binding substructure of adenine.
Possibly more important than the binding potential of
these polymers is their ability to stereodifferentiate
enantiomers of t-BOC-phenylalanine. Only two polymers
exhibited enantioselective binding, P(MAOA) and P(A-
PYM). The order of enantioselectivity was P(MAOA) >
P(APYM), which might be attributed to a correlation with
binding affinity. Furthermore, these two polymers have
in common the 2-aminopyridine moiety, for which direc-
tional hydrogen-bond interactions have been demon-
strated for synthetic carboxylate receptors.8,21 This may
serve to fine tune selectivity in the polymer binding sites;
however, the origins of chiral selectivity are postulated
to arise from the specific three-dimensional positioning
of complementary functional groups within the binding
site. Such is the case for chiral selectivity for (R)-(-)-2-
phenylbutyric acid by P2(AEM), which contains a pri-
mary amine functional group that can be considered a
nondirectional point charge.
The effects of changing the polymerization conditions
on binding selectivity were explored for polymers made
using the N-(2-aminopyridine) methacrylamide monomer.
Polymers imprinted with the template t-BOC-L-pheny-
lalanine were both photochemically and thermally initi-
ated. Binding affinity was found to be the same for both
photochemically and thermally initiated polymers. Ste-
reoselectivity, however, was only observed for photo-
chemically initiated polymers and not for thermally
initiated polymers. This is in agreement with previous
studies that suggest molecular recognition in imprinted
polymers is enhanced at lower temperatures. It was also
found that an increase in selectivity was found for
polymer formulated with an increased monomer-to-
template ratio. Doubling the ratio of monomer to tem-
plate increased the nonspecific background binding of
both imprinted and control polymers; however, there was
a 14% net increase in selectivity by t-BOC-D-phenylala-
nine imprinted polymer for its own template. Future
improvements for separation using these and other
imprinted polymers should consider optimization of the
monomer to template ratio.
In Table 5, entries 1 and 5 show that both the L and D
t-BOC-phe imprinted polymers are selective for their own
enantiomer, and the magnitude of the R value for both
is the same (1.2). Thus, chiral selectivity is equally
obtained by either enantiomer. Comparison of entries 1
and 2 shows that photoinitiation results in polymers with
higher R values than thermal initiation (1.2 vs 1.0). This
is expected because there should be a stronger pre-
polymer complex at 5 °C compared to that found at 80
°C,22 which is ultimately responsible for formation of the
specific binding sites within the polymer. Increasing the
ratio of functionalized monomer to template (t-BOC-D-
phe) from 1:1 to 2:1 for entries 5 and 6 reveals an increase
in both binding affinity (k′) and selectivity (R). This may
be an indication that the increase in k′ may be respon-
sible, at least in part, for the increase in R. For the control
polymers (entries 3 and 4), doubling the ratio of func-
tional monomer to template (benzoic acid) showed only
an increase in binding affinity (k′); however, there was
no increase in stereochemical bias imparted to these
polymers (R ) 1.0), as expected. For previous work with
methacrylic acid functionalized imprinted polymers, a
ratio of 12/1 functional monomer/template is generally
used with good results.4 Thus there remains the potential
to achieve even higher selectivity by using larger ratios
of functional monomer to template.
Su m m a r y a n d Con clu sion
Functionalized methacrylamide monomers for imprint-
ing carboxylic acids were designed, synthesized, and
copolymerized with a large excess of ethylene glycol
dimethacrylate in the presence of carboxylic acid tem-
plates. The binding and selectivity of the resulting
imprinted polymers were evaluated chromatographically.
The highest binding affinities were found for polymers
incorporating primary amine monomer 4, followed by
polymers incorporating guanidine-functionalized mono-
mer 2. The proximity of the functional group to the
polymerizable group of monomers 2 and 4 proved to be
important for monomer design. For example, monomer
4 exhibited the best binding affinity of all the monomers,
whereas monomer 3 showed worst binding affinity, even
though these two differ by only one methylene unit.
Similarly, of the two guanidinium functionalized poly-
mers investigated, monomer 2 showed the second highest
binding affinity, whereas monomer 1 did not exhibit any
binding potential at all. Monomer 1 has the guanidinium
Exp er im en ta l Section
Gen er a l. UV irradiation experiments utilized a Hanovia
medium-pressure mercury arc lamp. Thin-layer chromatog-
raphy separations were conducted on precoated plates of silica
with a 0.025 mm thickness containing PF 254 indicator. Flash
columns were packed with 230-400 mesh silica gel. Ethylene
(22) (a) Sellergren, B.; Shea, K. J . J . Chromatogr. 1993, 635, 31. (b)
Sellergren, B.; Lepisto, M.; Mosbach, K. J . Am. Chem. Soc. 1988, 110,
5853. (c) O’Shannessy, D. J .; Ekberg, B.; Andersson, L. I.; Mosbach,
K. J . Chromatogr. 1989, 470, 391.

Molecular Imprinting of Carboxylic Acids
J . Org. Chem., Vol. 64, No. 13, 1999 4633
glycol dimethacrylate (EGDMA, Polysciences) was first washed
twice with aqueous 1 M NaOH and once with aqueous
saturated NaCl solution to remove inhibitor and further dried
with anhydrous MgSO4. The monomer was distilled under
reduced pressure (10 mmHg, 60 °C). Methacrylic acid (MAA,
Aldrich) was distilled over CaCl2 (10 mmHg, 80 °C). AIBN
(Fluka) was recrystallized from methanol.
All solvents were obtained from commercial suppliers and
purified prior to use. THF and diethyl ether were dried over
Na-benzophenone ketyl and distilled. Benzene and acetoni-
trile were dried by refluxing over CaH2 and then distilled.
DMF was dried over 4 Å molecular sieves and distilled under
reduced pressure. Unless otherwise stated, all reactions were
conducted in oven-dried (160 °C) glassware under a positive
nitrogen atmosphere.
added to H2O (50 mL) containing phenolphthalein (1.0 mg).
The mixture was titrated with 1.0 M NaOH to establish HCl
concentration. The HCl/EtO2 solution was diluted with enough
dry diethyl ether to make a 2.0 M solution. To a portion of
this solution (600 mL, 1.20 mole) was added 1,2-ethanebis-
carbamic acid bis-tert-butylester (31.3 g, 0.12 mole). The
heterogeneous mixture was stirred for 3 h. The precipitate was
filtered and washed thoroughly with dry ether to provide a
white solid. This solid was recrystallized from acetonitrile to
give white, platelike crystals (8.28 g, 35% yield, mp 149-150
°C). 1H NMR (250 MHz, CD3OD) δ 3.24 (t, 2H, J ) 6.1 Hz,
CH2 - NH3+), 2.94 (t, 2H, J ) 6.0 Hz, CONH - CH2), 1.36 (s,
9H, C(CH3)3). 13C NMR (125.8 MHz, CD3OD) δ 159.0, 81.0,
41.3, 39.4, 29.0. IR (KBr pellet) 3375, 2978, 2902, 1697, 636
cm-1
.
Ch r om a t ogr a p h ic E xp er im en t s. The polymers were
ground by mortar and pestle or by mechanical mill (J anke &
Kunkel IKA WERK grinding mill type A 10 S, 20 000 rpm with
water circulating temperature control) and sized using U.S.A.
Standard Testing Sieves (ASTME.-11 specification). The par-
ticles (25-38 µm size range, unless otherwise indicated) were
slurry packed in stainless steel chromatographic columns
(length 100 mm, i.d. 4.6 mm). Approximately 0.5-0.8 g of
material is necessary to pack a column of this size. Once slurry
packed, the columns were then washed on line (in addition to
previous Soxhlet extraction) with acetonitrile or 7/3 acetoni-
trile/water until a stable baseline was obtained. HPLC analy-
ses were performed isocratically at room temperature; flow
rate, mobile phase, and substrate conditions are indicated in
the text for each experiment. The void volume was determined
by injecting a small amount of an inert substance, namely
acetone, acetonitrile or sodium nitrate; of the three, the void
volume marker utilized was that with the smallest retention
volume.
{[(2-Meth yl-1-oxo-2-p r op en yl)a m in o]eth yl}-ca r ba m ic
Acid ter t-Bu tylester . A solution containing (2-aminoethyl)-
carbamic acid tert-butylester hydrochloride (5.96 g, 30.0 mmol),
THF (200 mL), and triethylamine (12.7 mL, 90.0 mmol) was
stirred at 0 °C for 1 h. To this solution (at 0 °C) was added
dropwise a solution of methacryloyl chloride (2.96 mL, 30.3
mmol) in THF (50 mL). The mixture was stirred at room
temperature for 24 h. The salts were filtered and washed
thoroughly with THF, and the combined extracts were con-
centrated. The residue was triturated with hexane to provide
a white powder (6.61 g, 96% yield). This product could be
further purified by flash chromatography using hexane/diethyl
ether as eluant (mp 82.5-83.5 °C). 1H NMR (250 MHz, CDCl3)
δ 7.08 (br s, 1H, RCONH), 5.76 (s, 1H, vinyl), 5.52 (br s, 1H,
OCONH), 5.32 (s, 1H, vinyl), 3.40 (m, 2H, RCONH-CH2), 3.33
(m, 2H, OCONH-CH2), 1.96 (s, 3H, CdC-CH3), 1.43 (s, 9H,
C(CH3)3). 13C NMR (125.8 MHz, CDCl3) δ 169.5, 157.9, 140.1,
120.5, 80.2, 42.1, 40.5, 28.9, 19.1. FTIR (KBr pellet) 3361, 3327,
1685, 1651, 1618, 980, 667 cm-1. HRMS (CI) calcd for
Gen er a l P olym er F or m u la tion a n d P r oced u r es. The
following procedure is general for all carboxylate-binding
polymers unless otherwise indicated. The specific conditions
(time of polymerization, temperature, etc.) for each polymer
are indicated in the text. In a 20 mL scintillation vial is added
a solution of template (0.6486 mmol, 2.9 mol %) dissolved in
porogen (6.0 mL), functionalized monomer (0.6486 mmol, 2.9
mol %), ethylene glycol dimethacrylate (20.0 mmol, 92.7 mol
%), and AIBN (0.3 mmol, 1.5 mol %). The solution was purged
with nitrogen for 5 min at room temperature to remove oxygen,
and the vial was sealed with a screw-on cap. Polymerization
was initiated photochemically by a standard laboratory UV
light source (Hanovia medium-pressure mercury arc lamp) at
5 °C and allowed to proceed for 6-10 h. The polymerization
tubes were turned one-quarter turn every 15 min for the first
hour of polymerization, then every hour for the duration.
Afterward, the polymers were removed, crushed, and Soxhlet
extracted in 80/20 methanol/triethylamine overnight. The
splitting yield was determined by proton NMR of the extract
using an exact amount of methylene chloride or benzene as
an internal standard. Following preparation, the polymers
were sized, packed into stainless steel chromatographic col-
umns, and used for HPLC experiments as described in the
previous section.
C
11H20N2O3 229.1551, found 229.1549.
N-(2-Am in oeth yl) Meth a cr yla m id e. A solution contain-
ing {[(2-methyl-1-oxo-2-propenyl)amino]ethyl}-carbamic acid
tert-butylester (5.17 g, 22.6 mmol), CH2Cl2 (57 mL), and 4 M
HCl/Et2O (57 mL, 0.23 mole) was stirred at room temperature
for 24 h. The hygroscopic salt was filtered and washed with
diethyl ether to afford a white solid. This solid was added to
0.39 M ethanolic sodium ethoxide (59 mL, 23.0 mmol), and
the mixture was stirred for 1 h. The salt was filtered and
washed with ethanol. The combined filtrates were evaporated
(keeping water bath below 25 °C), and the resulting oily
residue was extracted with benzene or acetonitrile, dried over
magnesium sulfate, and then concentrated to yield a clear,
light yellow oil (2.58 g, 89% yield). Note, this compound is quite
1
unstable with regard to polymerization. H NMR (250 MHz,
CDCl3) δ 6.88 (br s, 1H, CONH), 5.72 (t, 1H, J ) 0.9 Hz, vinyl),
5.32 (quin, 1H, J ) 1.4 Hz, vinyl), 3.34 (q, 2H, J ) 5.9 Hz,
CONH-CH2), 2.85 (t, 2H, J ) 6.0 Hz, CH2-NH2), 1.97 (t, 3H,
J ) 1.2 Hz, NH2). 13C NMR (125.8 MHz, CDCl3) δ 169.3, 140.4,
119.8, 42.7, 19.1. FTIR (neat) 2927, 2256 (br), 1655, 1612, 933
cm-1
.
N-(3-Am in op r op yl) Meth a cr yla m id e. N-(3-Aminopropyl)
methacrylamide hydrochloride (Kodak, 2.91 g, 0.0163 mmol)
was added to 0.324 M ethanolic sodium ethoxide (55 mL,
0.0178 mole). The mixture was allowed to stir for 30 min and
then filtered. The filtrate was concentrated at room temper-
ature, and the residue was triturated with 40 mL of benzene
using a sonicator (Branson 2200) at room temperature. This
was filtered through Whatman filter paper, and the superna-
tant was concentrated at room temperature, leaving a light
brown oil (1.80 g, 78% yield). 1H NMR (300 MHz, CDCl3) δ
7.30 (br s, 1H, CONH), 5.61 (s, 1 H, CdCH), 5.20 (s, 1H, Cd
CH), 3.31 (q, 2H, J ) 6.2 Hz, CONH-CH2), 2.72 (t, 2H, J )
6.2 Hz, NH2-CH2), 1.85 (s, 3H, CH3), 1.56 (quin, 2H, J ) 6.2,
CH2-CH2-CH2), 1.41 (s, 2H, NH2). 13C NMR (75.4 MHz,
CDCl3) δ 168.2, 139.8, 119.0, 40.1, 38.2, 31.6, 18.5. IR (neat)
Syn th esis of F u n ction a l Mon om er s. 1,2-Eth a n ebis-
ca r ba m ic Acid Bis-ter t-bu tylester . To a solution of 1,2-
diaminoethane (6.11 g, 102 mmol) in THF (200 mL) at 0 °C
was added dropwise a mixture of di-tert-butyl dicarbonate (44.6
g, 204 mmol) and THF (40 mL). The heterogeneous mixture
was then heated at reflux for 12 h, after which time a clear
solution was obtained. Removal of the solvent in vacuo
provided a white solid (26.1 g, 99%) that could be recrystallized
1
from benzene to give long needles (mp 139-140 °C). H NMR
(250 MHz, CDCl3) δ 5.11 (br s, 2H, CONH), 3.23 (M, 4H,
CONH-CH2), 1.44 (s, 18H, C(CH3)3). 13C NMR (125.8 MHz,
CDCl3) δ 157.0, 79.9, 41.4, 29.0. IR (KBr pellet) 3373, 2983,
2937, 1685, 985, 871, 642 cm-1. HRMS (CI) calcd for C12H24N2O4
260.1736, found 260.1733.
3295 (br), 2931, 2867, 1658, 1612, 925 cm-1
.
N-(Dia m in oeth ylen e)-2-m eth ylp r op -2-en a m id e. Guani-
dine hydrochloride (1.51 g, 15.8 mmol) was dissolved in
methanol and converted into its free base via anion-exchange
resin (Amberlite IRA-900, chloride form). The free base was
(2-Am in oeth yl)-ca r ba m ic Acid ter t-Bu tylester Hyd r o-
ch lor id e. Dry diethyl ether (500 mL) was saturated with HCl
gas at 0 °C for 30 min. An aliquot of the solution (10 mL) was

4634 J . Org. Chem., Vol. 64, No. 13, 1999
Spivak and Shea
dried by rotary evaporation and redissolved in 10 mL of
ethanol. Methyl methacrylate (1.60 g, 16.0 mmol) was added,
and the mixture was refluxed at 100 °C for 4 h. The solvent
was evaporated, and the residue was recrystallized from
ethanol/diethyl ether to give a pale yellow solid (0.72 g, 5.7
mmol, 36% yield, mp 171-176 °C). 1H NMR (300 MHz, DMSO-
d6) δ 5.56 (d, 1H, CdCH), 5.01 (br s, 1H, CdCH), 3.38 (br s,
5H, -NH-), 1.71 (s, 3H, -CH3). 13C NMR (75.4 MHz, DMSO-
d6) δ 173.03, 158.85, 144.51, 118.32, 20.33. FTIR (KBr pellet)
3401-3154 (br), 1654, 667, 597 cm-1. HRMS (CI) calcd for
C5H10N3O 128.0824, found 128.0818.
removed by vacuum distillation. The residual oil was placed
on a high-vacuum line and allowed to crystallize. The solid
was pulverized and dried in a desiccator to give 76.0 g (0.41
mol, 82% yield) of a pale yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ 9.06 (s, br, 2H, -NH2), 8.94 (s, br, 2H, -NH2),
3.13 (q, 2H, -CH2-), 1.17 (t, 3H, -CH3). 13C NMR (75.4 MHz,
DMSO-d6) δ 170.32, 25.26, 14.54.
N-(3-Gu a n id in op r op yl) Meth a cr yla m id e. In a 2 mL vial
with lid were placed N-(3-aminopropyl) methacrylamide (0.89
g, 5.0 mmol), S-ethylthiourea hydrobromide (1.11 g, 6.0 mmol),
and 1.0 mL of concentrated ammonium hydroxide solution. A
stirbar was added, the vial was sealed tightly (stench!), and
the mixture was shaken until all solids were dissolved. The
mixture was then stirred for 16 h at room temperature. The
vial was then opened, and the ethylmercaptan was removed
by bubbling in nitrogen. The remaining solution was evapo-
rated to dryness under high vacuum. Purification was ac-
complished in two parts. First, the mixed salts to be separated
were converted to the free amines by passing down a column
of Dowex 1 × 8 hydroxide form strongly basic anion-exchange
resin. The base solution was evaporated to dryness under high
vacuum, and purification was obtained by iterative flash
chromatography using methanol/acetic acid as eluant. A final
passing through the anion-exchange resin gave the hydroxide
as counterion to form a white oily solid (0.201 g, 20% yield).
1H NMR (300 MHz, DMSO-d6) δ 5.61 (s, 1H, -CRdCH), 5.37
(s, 1H, -CRdCH), 4.76 (s, 1H, -NH), 3.25 (t, 2H, (H2N)2C-
NH-CH2-), 3.14 (t, 2H, -CONH-CH2-), 1.84 (s, 3H, -CH3),
1.75 (t, 2H, -CH2-CH2-CH2-). 13C NMR (75.4 MHz, DMSO-
d6) δ 171.97, 156.75, 139.04, 121.00, 38.62, 36.62, 27.52, 17.67.
FTIR (KBr pellet) 3644-3178 (br), 1658, 863, 667 cm-1. HRMS
(FAB+) calcd for C8H17N4O 185.1402, found 185.1402.
N-(2-Am in op yr id in e) Meth a cr yla m id e. A solution of
2-aminopyridine (7.2 g, 81.9 mmol) and triethylamine (5.7 mL,
81.9 mmol) in methylene chloride (25 mL) in a 100 mL round-
bottom flask was brought to 0 °C. Methacryloyl chloride (4.0
mL, 41.0 mmol) was added dropwise over 10 min, and the
solution was allowed to come to room temperature and stirred
for 40 min. Water (25 mL) was added, and the mixture was
stirred for an additional 30 min. The organic layer was
removed, and the aqueous layer was extracted twice with
chloroform. The organics were combined, dried over sodium
sulfate, and evaporated in vacuo. Column purification (elu-
ant: methylene chloride/methanol 92.5/2.5), and recrystalli-
zation in hexanes gave 1.58 g (23.7%) of a white crystalline
solid. 1H NMR (300 MHz, CDCl3) δ 8.33 (br s, 1H, dNH-),
8.29 (m, 1H, dCH-Nd), 8.26 (m, 1H, dN-CNdCH-), 7.72
(t, 1H, N2CdCH-CH)), 7.05 (t, 1H, dN-CH-CHd), 5.88 (s,
1H, H3C-CRdCH), 5.52 (s, 1H, H3C-CRdCH), 2.07 (s, 3H,
-CH3). 13C NMR (75.4 MHz, CDCl3) δ 166.92, 151.57, 147.68,
140.26, 138.35, 120.85, 119.69, 114.28, 18.60. FTIR (KBr pellet)
3235-2923 (br), 1674, 1627, 1600-1430 (br), 779, 736 cm-1
HRMS (EI) calcd for C9H10N2O 162.0793, found 162.0790.
.
S-Eth ylth iou r ea Hyd r obr om id e. A mixture of powdered
thiourea (38.6 g, 0.5 mole), ethyl bromide (63.3 g, 0.58 mole),
and 50 mL of absolute ethanol was placed in a 500 mL round-
bottomed flask equipped with a condenser. The mixture was
warmed on a water bath (55-65 °C) for 3 h, with occasional
shaking. During this time, all of the thiourea dissolved. The
reflux condenser was replaced by one set for downward
distillation, and the ethanol and excess ethyl bromide were
Ack n ow led gm en t. We are grateful to the NIH for
financial support of this work.
Su p p or t in g In for m a t ion Ava ila b le: 13C and 1H NMR
spectra for all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O982118S