Primary-amine-specific lactamization of ω-amino acids by an artificial cyclotransferase based on [18] crown-6

Article abstract of DOI:10.1002/anie.200504027

Feet first: Carboxylic acids with a primary amino group in the farthest position from the carboxy group are specifically bound to an artificial cyclotransferase based on [18]crown-6. The amino group fits into the [18]crown-6 substrate-binding site to bring the carboxyl group close to the 2-ammonio-1,3,5-triazine reaction site for selective conversion into a lactam. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200504027

Communications
Enzyme Models
DOI: 10.1002/anie.200504027
Primary-Amine-Specific Lactamization of w-
Amino Acids by an Artificial Cyclotransferase
Based on [18]Crown-6**
Munetaka Kunishima,* Kazuhito Hioki,
Takahiro Moriya, Jun Morita, Tomoko Ikuta, and
Shohei Tani
In memory of Kiyoshi Tanaka
Enzyme models are of practical importance for the develop-
ment of efficient molecular catalysts for organic reactions and
of theoretical importance in elucidating mechanisms of
enzymatic reactions.[1] In comparison with hydrolase models,
an artificial acyltransferase that promotes the reverse reaction
of dehydrocondensation is particularly difficult to design.[2]
As a representative host compound, [18]crown-6 has received
much attention in biomimetic chemistry and, although
specific binding of ammonium ions to [18]crown-6 has been
extensively investigated,[3–5] application of the complex as an
artificial acyltransferase is very limited.[6,7] Among acyltrans-
ferase models, cyclotransferase models that catalyze intra-
molecular dehydrocondensation to lead to the formation of
lactams have not been developed. Herein, we report a novel
enzyme model of cyclotransferase,[8] which utilizes the
specific binding of a primary ammonium ion and [18]crown-
6. The artificial enzyme substrate specifically activates the
carboxylic acids that possess a primary amino group at the g-,
d-, or e-positions, thus leading to the formation of the
corresponding lactams.
The reaction system is outlined in Scheme 1. Methanol is
employed as the solvent as it dissolves the amino acid
substrates and stabilizes the complex of the primary ammo-
nium salt and the [18]crown-6.[9–11] A combination system of
triazines and tertiary amines which is available in alcohols was
attached to the [18]crown-6 and employed for the activation
of the substrate carboxyl groups.[12,13] When CDMTwas added
to a methanolic solution of 5-aminovaleric acid (3a) and
crown derivative 1, which can be compared to an apoenzyme,
[*] Prof. Dr. M. Kunishima, Dr. K. Hioki, T. Moriya, J. Morita, T. Ikuta,
Prof. Dr. S. Tani
Faculty of Pharmaceutical Sciences
Kobe Gakuin University
Nishi-ku, Kobe 651-2180 (Japan)
Fax: (+81)78-974-5689
E-mail: kunisima@pharm.kobegakuin.ac.jp
Prof. Dr. M. Kunishima
PRESTO, JST
Nishi-ku, Kobe 651-2180 (Japan)
[**] This workwas supported partially by a Grant-in-Aid for Scientific
Research (No. 14572025) from the Ministry of Education, Science,
Sports, and Culture, Japan.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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observed by prolonging the reac-
tion time to 2 h (run 2). The
selectivity slightly increased with
an excess of 3a and 3b (2.0 equiv
each; run 3). In the control reac-
tion using 5, which has the dehy-
drocondensing group attached to
an ethyl group instead of the
crown ether, both the selectivity
and the yield dropped (54:46,
50% yield; run 4). Prolongation
of the reaction time increased the
yield to 80%, whereas the selec-
tivity was completely lost (run 5).
Similar results were obtained
with 3c, which has an isopropyl
group at the nitrogen atom
(runs 6 and 7). The reaction
rates uniformly decreased when
either the methyl or isopropyl
group was introduced to the
terminal amino group, thus indi-
cating that the mere existence of
Table 1: Competitive lactamization between primary and secondary
5-aminovaleric acids (3a vs 3b or 3c).
Scheme 1. Substrate-specific lactamization by artificial cyclotransferase.
CDMT=2-chloro-4,6-dimethoxy-1,3,5-triazine, ES=enzyme–substrate,
EP=enzyme–product.
a reactive dehydrocondensing reagent 2 (similarly, compared
to holoenzyme; X = Cl) was generated by coupling 1 with
CDMT through the dimethylamino group. Upon introduction
of substrate 3a, the primary amino group in its ionized form
preferentially interacts with 2 to form a so-called ES complex.
In the resulting complex, the carboxylate ion of the substrate
is brought into proximity with the triazino group and attacks it
preferentially, thus giving an EP complex. Intramolecular
aminolysis of the resulting acyloxytriazine takes place quickly
to give d-valerolactam with concomitant liberation of 1, which
can be reused in the next catalytic cycle. Since the product 4a
lacks a free amino group, it no longer binds to the crown ring,
and thus there is no competitive binding of the product with
the catalyst to inhibit the next reaction.
First, to verify that the substrate-specific lactamization
takes place through such molecular recognition, we
attempted the reaction under stoichiometric conditions
using 2 (X = OTf; Tf = trifluoromethanesulfonyl). A compet-
itive reaction between 3a and N-methyl-5-aminovaleric acid
(3b) was performed. Since the primary amino group of 3a has
a much greater affinity for [18]crown-6 than the secondary
amino group of 3b,[11] 3a can be expected to react preferen-
tially (Table 1). When a mixture of 1.1 equivalents of both the
amino acids was treated with 1.0 equivalents of 2 in methanol
at room temperature for 10 min, 4a was obtained predom-
inantly (4a/4b = 92:8) in 89% yield (run 1). No significant
improvement in either the selectivity or the yield was
Entry Substrate[a] Condensing t [min]
Product[b]
Yield [%] Ratio
agent
(4a/4b or 4c)
1
3a vs 3b
(1.1:1.1)
3a vs 3b
(1.1:1.1)
3a vs 3b
(2.0:2.0)
3a vs 3b
(1.1:1.1)
3a vs 3b
(1.1:1.1)
3a vs 3c
(3.0:3.0)
3a vs 3c
(3.0:3.0)
3a vs 3b
(1.1:1.1)
2
10
120
10
89
92
92:8
2
2
90:10
94:6
3
2
100
50
4
5[c]
5[c]
2
10
54:46
51:49
94:6
5
120
20
80
6
100
88
7
5[c]
2
120
10
55:45
94:6
8[d]
93
[a] The molar ratios given in parenthesis are based on the amount of 2 or
5 (1.0 equiv). [b] Determined by HPLC. [c] Control. [d] Reaction was
carried out in the presence of 2-piperidone (4a; 0.85 equiv).
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a substituent at the amino group (rather than size or steric
of 4-aminobutyric acid (6), 3a, and 6-aminocaproic acid (7)
toward 2 at room temperature was estimated to be 1:1.5:2.1,
respectively, based on their competitive reactions, thus
indicating that the kinetically most unfavorable seven-mem-
bered e-caprolactam 9 was formed faster than five- or six-
membered lactams (8 and 4a). Since the order of observed
reactivity of the amino acids does not correlate with their
affinity for [18]crown-6,[17] the number of the methylene
groups that link the amino group and the carboxyl group
could be important. The selectivity of 9/8 was improved with
decreasing reaction temperature; the ratio of 9 increased up
to 85:15 (88% yield) at À408C [Eq. (1)].
hindrance) was the main factor in decreasing the reaction rate.
To confirm that the observed selectivity is attributable to
the supramolecular interaction between the protonated
primary amino group of 3a and the crown ether of 2, we
examined the effect of alkaline-metal ions on the selectivity
because the affinity of these cations toward [18]crown-6
correlates with the match of their ionic radii to the diameter
of the host.[10,14,15] Competitive reactions under the same
conditions as run 2 in Table 1 were conducted in the presence
of 2 equivalents of various alkaline-metal triflates. As shown
in Table 2, the lithium ion, which has the smallest binding
Table 2: Effect of alkali-metal ions on substrate selectivity.
Entry
CF3SO3M (2 equiv)
Product[a]
Ratio (4a/4b)
pKa[b]
<0.5
4.38
6.20
4.55
Yield [%]
1
2
3
4
CF3SO3Li
CF3SO3Na
CF3SO3K
CF3SO3Cs
96
93
69
84
90:10
84:16
55:45
85:15
[a] Determined by HPLC. [b] The pKa values in MeOH/benzene (8:2) are
cited from reference [15].
In conclusion, we have succeeded in developing a novel
artificial enzyme based on [18]crown-6, which is capable of
converting w-amino acids into their cyclized lactams in a
substrate-specific manner. The catalyst can recognize the
distance and the degree of substitution of the amino group
farthest from the carboxyl group undergoing reaction.
Furthermore, although reaction yield and selectivity are
sometimes inversely related in organic reactions, both
increase in the present system because the reaction rate is
enhanced by the formation of the ES complex, which, in turn,
is based on molecular recognition. Finally, the present system
offers a new concept for circumventing product inhibition.
constant (pKa = < 0.5),[15] was found to have almost no effect
on selectivity, whereas the potassium ion, well known to have
the highest affinity for the crown ring, nearly abolished the
selectivity and generated a lower yield (runs 1 and 3). The
moderate decrease in the selectivity for 4a was observed with
sodium and cesium salts. The extent to which alkaline-metal
ions lowered selectivity correlated very well with their affinity
toward [18]crown-6, and we therefore conclude that the
observed substrate selectivity for 3a resulted from the
formation of a host–guest complex of the primary ammonium
group within the crown ring.
In general, reactions using host compounds suffer from
product inhibition because the products retain a high affinity
for the host.[1b] In the present case, the primary amino group
of 3a, which is recognized by the crown at the initial stage of
the reaction, disappears as it is converted into the amide
nitrogen atom of 4a, which should no longer bind to 2. In fact,
when the competitive reaction was carried out in the presence
of 4a, no decrease in selectivity was observed (Table 1, run 8).
Thus, properties intrinsic to this reaction circumvent the
problem of product inhibition and, in principle, this benefit
applies to all reactions that modify the guest moiety to one
with significantly less affinity for the host.[16]
It should be noted that the substrate-specific lactamiza-
tion proceeds under catalytic conditions. The competitive
reaction between 3a and 3b was performed using a combi-
nation of a catalytic amount of 2 (0.2 equiv) and CDMT
(1.0 equiv). To neutralize HCl generated during the reaction,
N-cyclohexylmorpholine was slowly added by a syringe pump
over a period of 5 h. As a result, 4a and 4b were obtained in a
ratio of 96:4 (yield = 78%). The control reaction using 5
instead of 2 under the same conditions resulted in a low
selectivity (4a/4b = 53:47, 70% yield).
Experimental Section
General procedure for competitive lactamization using a stoichio-
metric amount of dehydrocondensing agent (2 or 5): A solution of 2
(95.1 mg, 0.142 mmol) in MeOH was added at room temperature to a
stirred solution of 3a (18.3 mg, 0.157 mmol) and 3b (20.5 mg,
0.157 mmol) in MeOH (the total amount of MeOH = 26 mL). The
reaction mixture was stirred at room temperature, and the products
were quantified by HPLC at a definite time (1,3-dimethyl-3,4,5,6-
tetrahydro-2(1H)-pyrimidinone was used as the internal standard).
General procedure for competitive lactamization under the
catalytic system of
0.029 mmol) and CDMT (20.0 mg, 0.114 mmol) in MeOH at room
temperature was added to stirred solution of 3a (18.3 mg,
2 and CDMT: A solution of 2 (19.0 mg,
a
0.157 mmol) and 3b (20.5 mg, 0.157 mmol) in MeOH. N-cyclohex-
ylmorpholine (0.142 mmol, 0.6 mL of 0.237m) dissolved in MeOH
was added over a period of 5 h by a syringe pump (the total amount of
MeOH = 8.5 mL). The reaction mixture was stirred at room temper-
ature, and the products were quantified by HPLC at a definite time.
Received: November 12, 2005
Published online: January 17, 2006
Interestingly, the chain length between the amino group
and carboxyl group can be discriminated by 2. The reactivity
Keywords: condensation · crown compounds · enzyme models ·
lactams · transferases
.
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