Attachment of 1,5,9-triazacyclododecane and β-cyclodextrin to poly(ethylenimine) in proximity by site-directed functionalization

Article abstract of DOI:10.1006/bioo.1998.1088

A novel methodology for the site-directed introduction of a functional group in the vicinity of β-cyclodextrin (CD) on the backbone of branched poly(ethylenimine) (PEI) is developed. Site-directed or random attachment of 1,5,9-triazacyclododecane (TC) to CD-containing PEI followed by acetylation of the primary and secondary amines of the polymer backbone produced [TC- CD](SD)AcPEI or [TC-CD](Ran)AcPEI, respectively. By the addition of Ni(II), Cu(II), or Zn(II) ion, [M(II)TC-CD](SD)AcPEI or [M(II)TC-CD](Ran)AcPEI was obtained. The formation constant for the complexation of a hydrophobic ester revealed that the TC portion was indeed positioned in the vicinity of the CD cavity in [M(II)TC-CD](SD)AcPEI. Compared with [M(II)TC-CD](Ran)AcPEI, [M(II)TC-CD](SD)AcPEI appears to exert an extra stabilization effect (- ΔΔG°= 1.0-1.3 kcal/mol) on the complexed ester by hydrogen bonding between the metal-bound water of the polymer and the carbonyl group of the ester.

Full text of DOI:10.1006/bioo.1998.1088

BIOORGANIC CHEMISTRY 26, 51–60 (1998)
ARTICLE NO. BH981088
Attachment of 1,5,9-Triazacyclododecane and ͱ-Cyclodextrin
to Poly(ethylenimine) in Proximity by
Site-Directed Functionalization
Sung Min Kim, In Seok Hong, and Junghun Suh1
Department of Chemistry and Center for Molecular Catalysis, Seoul National University,
Seoul 151-742, Korea
Received January 13, 1998
A novel methodology for the site-directed introduction of a functional group in the
vicinity of ͱ-cyclodextrin (CD) on the backbone of branched poly(ethylenimine) (PEI) is
developed. Site-directed or random attachment of 1,5,9-triazacyclododecane (TC) to CD-
containing PEI followed by acetylation of the primary and secondary amines of the
polymer backbone produced [TC–CD]SDAcPEI or [TC–CD]RanAcPEI, respectively. By
the addition of Ni(II), Cu(II), or Zn(II) ion, [M(II)TC–CD]SDAcPEI or
[M(II)TC–CD]RanAcPEI was obtained. The formation constant for the complexation of a
hydrophobic ester revealed that the TC portion was indeed positioned in the vicinity
of the CD cavity in [M(II)TC–CD]SDAcPEI. Compared with [M(II)TC–CD]RanAcPEI,
[M(II)TC–CD]SDAcPEI appears to exert an extra stabilization effect (Ϫ⌬⌬GЊ ϭ 1.0–1.3
kcal/mol) on the complexed ester by hydrogen bonding between the metal-bound water
of the polymer and the carbonyl group of the ester.
1998 Academic Press
INTRODUCTION
Branched poly(ethylenimine) (PEI) has been intensively exploited as macromo-
lecular backbone of artificial enzymes (1). Among ca. 1400 amino groups of PEI
(Mr ca. 60,000), ca. 25% are primary amines, ca. 50% are secondary amines, and
the rest are tertiary amines representing branching points. One of the major obsta-
cles faced in design of artificial enzymes with synthetic macromolecules has been
lack of specific binding sites. In a previous study (2), we attached ͱ-cyclodextrin
(CD) to PEI as a binding site. Thus, the CD-containing PEI (CD–PEI) selectively
recognized esters with t-butylphenyl moieties resulting in acylation of amino groups
near the CD cavity as illustrated in I (2).
1 To whom correspondence should be addressed.
51
0045-2068/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.

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KIM, HONG, AND SUH
For designing effective artificial enzymes or receptors on synthetic macromole-
cules, it is necessary to develop a methodology to introduce an additional functional
element in the vicinity of the functional group already present on the molecular
backbone. In the case of biomimetic molecules based on CD–PEI, for instance,
site-directed functionalization can lead to cooperative action by the CD moiety and
the newly introduced functional element. Functionalization of CD–PEI can be also
regarded as preparation of new derivatives (3) of CD.
In this study, site-directed functionalization of CD–PEI was attempted with a
t-butylphenyl ester containing a precursor of 1,5,9-triazacyclododecane (TC) by
taking advantage of recognition of t-butylphenyl moiety by CD as illustrated in II.
TC was chosen as the additional functional group in view of catalytic activity of its
metal complexes (4).
EXPERIMENTAL PROCEDURES
Materials
4-t-Butylphenyl 5-(6,8-dioxo-1,5,9-triazacyclododec-1-yl)-5-oxopentanoate (1).
Glutaric acid (1.0 g, 7.6 mmol) was dissolved in 150 ml dimethyl formamide : methy-
lene chloride (1 : 5, v/v). 1,3-Dicyclohexylcarbodiimide (1.4 g, 6.8 mmol) and 4-t-

SITE-DIRECTED FUNCTIONALIZATION
53
butylphenol (0.92 g, 6.1 mmol) were added, and the solution was stirred for 2 h at
room temperature. After the solvent was evaporated under a reduced pressure,
1 N HCl aqueous solution (10 ml) was added, and oily 4-t-butylphenyl hydrogen
glutarate obtained through extraction with ethyl acetate was purified with silica gel
(1 : 3 ethyl acetate : n-hexane). 1H NMR (CDCl3) ͳ: 1.25–1.30 (s, 9H), 1.90–2.20 (m,
2H), 2.35–2.60 (t, 2H), 2.45–2.75 (t, 2H), 6.85–7.05 (d, 2H), 7.25–7.45 (d, 2H). 4-
t-Butylphenyl hydrogen glutarate (1.0 g, 4.8 mmol), 1,3-dicyclohexylcarbodiimide
(1.2 g, 5.8 mmol), and 4-dimethylaminopyridine (0.1 g) were dissolved in 150 ml
methylene chloride. 1,5,9-Triazacyclododecane-2,4-dione (1.1 g, 5.5 mmol) prepared
according to the literature (5) was added, and the solution was stirred at room
temperature for 2 h. After the solution was washed with aqueous 1 M NaHCO3 to
remove unreacted 4-t-butylphenyl hydrogen glutarate, the product was extracted
with methylene chloride. Compound 1 was purified with silica gel (10 : 1 methylene
chloride : methanol), and recrystallized from methylene chloride–n-hexane, mp
1
205–206ЊC. H NMR (DMSO-d6) ͳ: 1.26 (s, 9H), 1.70–1.85 (m, 2H), 2.30–2.40 (t,
2H), 2.52–2.62 (t, 2H), 2.95 (s, 2H), 2.98–3.34 (m, 12H), 6.97–7.04 (d, 2H), 7.37–7.45
(d, 2H), 7.90–8.02 (t, 2H). 13C NMR (DMSO-d6) ͳ: 171.8, 170.5, 167.1, 167.0, 148.2,
148.0, 126.1, 121.1, 47.0, 36.0, 35.7, 34.2, 32.9, 31.2, 30.8, 27.3, 26.6. MS (FAB,
glycerol) m/e 446 (M ϩ H). Anal. Calcd for C24H35N3O5 : C, 64.68; H, 7.92; N, 9.43.
Found: C, 64.35; H, 8.00; N, 9.38.
Phenyl 5-(6,8-Dioxo-1,5,9-triazacyclododec-1-yl)-5-oxopentanoate (2). This com-
pound was synthesized and purified as described above for 1 except that phenol
was used instead of t-butylphenol, mp 198–200ЊC. 1H NMR (DMSO-d6) ͳ: 1.65–1.90
(m, 2H), 2.30–2.50 (t, 2H), 2.52–2.68 (t, 2H), 2.95 (s, 2H), 2.98–3.34 (m, 12H),
6.97–7.55 (m, 5H), 7.90–8.02 (t, 2H). MS (FAB, glycerol) m/e 390 (M ϩ H). Anal.
Calcd for C20H27N3O5 : C, 61.67; H, 6.99; N, 10.79. Found: C, 61.50; H, 6.99; N, 10.72.
CD-PEI. PEI (Mr 50,000–60,000) purchased from Aldrich was purified by dialysis
(cutoff Mr 12,000) to remove fractions of small molecular weights. CD–PEI was
prepared as reported previously (2), and the content of CD in CD–PEI was 1.15
residue mol%.
[TC–CD]SDAcPEI. To an aqueous solution (500 ml) of 0.215 resM CD–PEI,
dimethyl sulfoxide (DMSO) (40 ml) was added and 1 (0.55 g, 1.24 mmol) dissolved
in 10 ml DMSO was added dropwise over the period of 3 h at 50ЊC. After the
mixture was stirred at 50ЊC for an additional 6 h, it was purified by dialysis against
40% aqueous ethanol (20 liters, twice), 0.1 M NaCl aqueous solution (20 liters,
three times), and water (20 liters, three times). The resulting [dioxo TC–CD]SDPEI
was concentrated to 10 ml at Ͻ50ЊC, and was dissolved in 150 ml DMSO. Sodium
borohydride (2.5 g, 0.066 mol) powder and methanesulfonic acid (2.0 ml, 0.031 mol)
solution in 5 ml DMSO were added alternately in several small portions over the
period of 3 h at room temperature (6). Then the reaction mixture was stirred for
an additional 10 h. After 1 N NaOH aqueous solution (30 ml) was added to the
solution, the mixture was stirred for 1 h. Then, 0.1 N HCl aqueous solution (30 ml)
was added and the resulting mixture was stirred for 1 h. The resulting [TC–
CD]SDPEI was purified by dialysis against an aqueous HCl solution (pH ca. 5, 20
liters, twice) and water (20 liters, four times). Whether the dioxo form of TC
(dioxoTC) in the PEI derivatives is completely reduced by the reduction procedure

54
KIM, HONG, AND SUH
(6) was not positively checked with [TC–CD]SDPEI or [TC–CD]RanPEI due to the
low content of TC. Instead, almost quantitative reduction of dioxoTC was confirmed
by NMR in the case of a PEI derivative in which the content of dioxoTC was 10
residue mol%. The volume of [TC–CD]SDPEI was adjusted to 150 ml. To the
75 ml solution of [TC–CD]SDPEI, NiCl2 (0.0546 M, 11.4 ml) was added, and the
solution was stirred for 2 h. Then acetic anhydride (15 ml, 0.16 mol) was added
dropwise with pH kept at 7.5–8.5 over the period of 3 h at room temperature. The
resulting PEI derivatives was purified by dialysis against 1% (w/v) NaCN aqueous
solution (20 liters, three times), water (20 liters, twice), 0.1 M NaCl aqueous solution
(20 liters, three times), and water (20 liters, four times) to obtain [TC–CD]SDAcPEI.
ICP analysis of [TC–CD]SDAcPEI indicated that more than 99% of Ni(II) ion was
removed. As discussed later in this article, the content of TC for each CD in
[TC–CD]SDAcPEI is close to 1.0.
[TC–CD]RanAcPEI. To a 95% (v/v) DMSO solution (500 ml) of 0.215 resM CD–
PEI, 2 (0.48 g, 1.24 mmol) dissolved in 5 ml DMSO was added, and the mixture was
stirred at 50ЊC for 6 h. The resulting [dioxoTC–CD]RanPEI was purified by dialysis.
Reduction of [dioxoTC–CD]RanPEI and the subsequent acetylation of [TC–
CD]RanPEI were carried out by the method described above for [TC–CD]SDAcPEI,
and [TC–CD]RanAcPEI thus obtained was purified by dialysis. As discussed later in
this article, the content of TC for each CD in [TC–CD]RanAcPEI is close to 1.0.
4-Nitrophenyl (4-t-butylphenyl)acetate (3). This compound was prepared ac-
cording to the literature (7).
Measurements
Estimation of formation constants for complexes of PEI derivatives formed with
Ni(II) ion was carried out by dialysis as reported previously (8). Reaction rates
for the hydrolysis of 3 in the presence of the PEI derivatives were measured
spectrophotometrically. Buffers (0.05 M) used were N-(2-hydroxyethyl)-1-pipera-
zineethanesulfonate (pH 7–8) and boric acid (pH 9.1). Distilled and deionized water
was used in dialysis and preparation of buffer solutions. ICP analysis of metal ions
was carried out with a Perkin–Elmer Plasma 40 or a Shimadzu ICP S-1000IV.
Spectrophotometric measurements were performed with a Beckman DU-68 UV/
vis spectrophotometer or a Jasco FP-777. pH was measured with a Dongwoo DP880
pH/ion meter. Temperature was controlled with a Fisher Scientific 71 circulator.
RESULTS AND DISCUSSION
Synthetic routes for various PEI derivatives containing both CD and TC
([TC–CD]SDPEI, [TC–CD]SDAcPEI, [TC–CD]RanPEI, and [TC–CD]RanAcPEI)2
are indicated in Scheme I. The content of CD in CD–PEI was 1.15 residue mol%.
2 The abbreviated names of the PEI derivatives indicate the nature of the pendants (TC, CD, Ac, or
M(II)) attached to PEI and whether TC is introduced in the vicinity of CD by site-directed or random
(SD or Ran, respectively) functionalization. For example, [Cu(II)TC–CD]SDAcPEI stands for the PEI
derivative containing CD and acetyl groups as well as the Cu(II) complex of TC introduced by site-
directed functionalization.

SITE-DIRECTED FUNCTIONALIZATION
55
SCHEME I
The key step for site-directed functionalization of CD–PEI is acylation of CD–PEI
with 1. Because of the limited solubility of 1 in water, it was inevitable to add an
organic solvent to the reaction mixture. Since complexation of CD with guest
compounds takes place even in 60% (v/v) DMSO–water (3b), 1 was added to a
solution of CD–PEI in 9% (v/v) DMSO–water in small portions. On the other
hand, 2 was added to a solution of CD–PEI in 95% (v/v) DMSO for random
functionalization of CD–PEI in view of the weaker affinity of CD toward phenyl
derivatives than toward t-butylphenyl derivatives and in 95% (v/v) DMSO than in
9% (v/v) DMSO–water. The molar amount of 1 or 2 added to CD–PEI was the
same as that of the CD moiety. TC was formed by reduction of its precursor (6).
Intermediate PEI derivatives as well as the final products were purified by repetitive
dialysis. To minimize complications due to amino groups of the PEI backbone, it
is desirable to block the amino groups. Acetylation of PEI derivatives with acetic
anhydride is known to acetylate the primary and secondary amino groups of PEI
(9). To prevent secondary amino groups of the TC moiety from acetylation by
acetic anhydride, the TC moiety was selectively protected with Ni(II) ion.
When Ni(II), Cu(II), or Zn(II) ion was added ([M(II)]0 ϭ [CD]0) to [TC–
CD]SDAcPEI or [TC–CD]RanAcPEI at pH 7.00 or 7.57 and the mixture was dialyzed
for 1 week at 25ЊC, the metal ions were almost completely (92–99%) retained by
the PEI derivative as checked by ICP analysis. When the amount of Ni(II) or Cu(II)
added was raised up to four times of [CD]0 , about 1 eq (Ͼ90% of [CD]0) of the
metal ion was retained by the polymer even after three successive dialyses. Spectral
titration of [TC–CD]SDAcPEI or [TC–CD]RanAcPEI with Ni(II) (Fig. 1) also indi-
cated that the content of strong binding sites for Ni(II) ion agrees with the content
of the CD moiety within Ϯ5%. The visible spectrum for the Ni(II) complex of
1
1
[TC–CD]SDAcPEI (␭
ϭ 607 nm, pale green, ␧
ϭ 9.1 MϪ cmϪ ) or [TC–
max
max

56
KIM, HONG, AND SUH
FIG. 1. Plot of absorbance at 606 nm against [Ni(II)]0/[CD]0 for [TC–CD]SDAcPEI (᭺) or [TC–
3
CD]RanAcPEI (᭹) at 25ЊC and pH 8.00. [CD]0 was 5.89 ϫ 10Ϫ M.
1
1
CD]RanAcPEI (␭max ϭ 606 nm, ␧max ϭ 1.0 ϫ 101 MϪ cmϪ ) was similar to that of TC
1
1
(␭max ϭ 607 nm, ␧max ϭ 1.3 ϫ 101 MϪ cmϪ ) but was distinctly different from that3 of
1
1
PEI (␭max ϭ 543 nm, pale pink, ␧max ϭ 8.3 MϪ cmϪ ). This reveals that the primary
Ni(II) binding site of [TC–CD]SDAcPEI or [TC–CD]RanAcPEI is TC. It further indi-
cates that TC is successfully protected by Ni(II) ion during the acetylation of [TC–
CD]SDPEI or [TC–CD]RanPEI. Although the PEI backbone contains ethylenedi-
amineunitswhichcanact(10)asligandsformetalions, metalbindingbythebackbone
is suppressed upon acetylation of the primary and the secondary amines of PEI.
That TC is protected by Ni(II) ion during the acetylation step is confirmed again
by measurement of formation constants (Kf) for the Ni(II) complex of the TC
moiety and that of the PEI backbone. Estimation of the Kf values was carried out
by dialyzing Ni(II) complex of the PEI derivative with a competing ligand by a
method reported previously (8). Either nitrilodiacetic-3-propanoic acid (NDPA)
(log Kf ϭ 8.80 (11) for Ni(II)(NDPA) at pH 7.00 and 25ЊC) or nitrilotriacetic acid
(NTA) (log Kf ϭ 11.02 (11) for Ni(II)(NTA)2 at pH 7.00 and 25ЊC) was used as
the competing ligand, and the results obtained with CD–PEI and [TC–CD]SDPEI
are summarized in Table 1. When [Ni(II)]0 was four times greater than [TC]0 , the
limiting amount of Ni(II) ion that can be extracted by NDPA was 77 Ϯ 2% of the
Ni(II) ion added to [TC–CD]SDPEI. When NTA, a stronger chelating agent than
NDPA, was used as the competing ligand, all of the added Ni(II) ion was extracted
by NTA at large concentrations of NTA even when [Ni]0 ϭ 4[CD]0 . Thus, the
Ni(II) ion added in excess of [TC]0 was associated with a weaker Kf value than
that bound to the TC moiety. The TC moiety manifests about 10-fold greater affinity
for Ni(II) ion compared with the PEI backbone.4 Thus, Ni(II) can selectively block
at least 90% of the TC moiety during the acetylation stage.
3 The relative amount of Ni(II) ion added to PEI was 1.15 residue mol% and ␧max was based on [Ni(II)]0 .
4 The log Kf for [Ni(II)TC–CD]SDAcPEI (12.38) is greater than that (10.93) reported (12) for the
Ni(II) complex of TC. This may be attributed to favorable conformational and/or medium effects exerted
by [Ni(II)TC–CD]SDAcPEI.

SITE-DIRECTED FUNCTIONALIZATION
57
TABLE 1
Formation Constants for the Ni(II) Complex of PEI Derivatives Measured with
Various [Ni(II)]0 at pH 7.00 and 25ЊCa
PEI derivative
[Ni(II)]0/[CD]0
Competing ligand
log Kf
CD–PEI
1
1
4
4
NDPA
NTA
NTA
11.48 Ϯ 0.22
12.38 Ϯ 0.06b
12.00 Ϯ 0.08c
11.38 Ϯ 0.06d
[Ni(II)TC–CD]SDPEI
[Ni(II)TC–CD]SDPEI
[Ni(II)TC–CD]SDPEI
NDPA
4
a [CD]0 ϭ (0.5–1.0) ϫ 10Ϫ M.
b Represents metal affinity of TC portion.
c Represents average metal affinity of both TC portion and PEI backbone.
d Represents metal affinity of PEI backbone.
By adding metal ions such as Ni(II), Cu(II), or Zn(II) ion to [TC–CD]SDAcPEI
or [TC–CD]RanAcPEI ([M(II)]0 ϭ [TC]0), [M(II)TC–CD]SDAcPEI or [M(II)TC–
CD]RanAcPEI was obtained. That the TC group is positioned near the CD moiety
in [M(II)TC–CD]SDPEI was confirmed by measuring kinetics of deacylation of 3
promoted by these PEI derivatives. Kinetics of reactions catalyzed by PEI deriva-
tives follow the scheme of Eq. [1] which is analogous to Michaelis–Menten scheme
(1). In the present study, C0 (the initially added concentration of the catalyst) is
expressed in terms of the concentration of CD moieties. Under the conditions of
C0 ӷ [CS], pseudo-first-order reactions were observed as expected from Eq. [1].
Saturation kinetic behavior was observed for the plot of k0 (pseudo-first-order rate
constant) against C0 as illustrated in Fig. 2. The values of kcat and 1/Km estimated
from the kinetic data are summarized in Fig. 3.
5
FIG. 2. Pseudo-first-order rate constants obtained for hydrolysis of 3(5.00 ϫ 10Ϫ M) in the presence
of [Cu(II)TC–CD]SDAcPEI (᭜), [Cu(II)TC–CD]RanAcPEI (ᮀ), [TC–CD]SDAcPEI (᭺), or acetylated
CD–PEI (close to those marked by ᭺) at 25ЊC and pH 7.57.

58
KIM, HONG, AND SUH
FIG. 3. Values of 1/Km and kcat for the hydrolysis of 3 catalyzed by [M(II)TC–CD]SDAcPEI and
[M(II)TC–CD]RanAcPEI at 25ЊC in the presence of 5.9% (v/v) CH3CN. pH is 7.57 unless noted otherwise.
4
5
Kinetic data were obtained with C0 ϭ 2–25 ϫ 10Ϫ M and S0 ϭ 5 ϫ 10Ϫ M. At pH 9.11, kcat is 1.09 ϫ
10Ϫ and 2.74 ϫ 10Ϫ sϪ for [Cu(II))TC–CD]SDAcPEI and [Cu(II)TC–CD]RanAcPEI, respectively.
2
2
1
Relative standard deviations are 10–20% for 1/Km and 5–10% for kcat
.
K
k
cat
m
C ϩ S s CS —Ǟ C ϩ products
[1]
Deacylation of 3 is very slow in the presence of [TC–CD]SDAcPEI or acetylated
CD–PEI (Fig. 2). This excludes the possibility of nucleophilic attack at 3 by the
hydroxyl groups of CD or the tertiary amino groups of the PEI backbone in the
deacylation by [M(II)TC–CD]SDAcPEI or [M(II)TC–CD]RanAcPEI.
As indicated in Fig. 3, kcat for [M(II)TC–CD]SDAcPEI is smaller than that for
[M(II)TC–CD]RanAcPEI by 3.9–4.7 times, whereas 1/Km for [M(II)TC–

SITE-DIRECTED FUNCTIONALIZATION
59
CD]SDAcPEI is greater than that for [M(II)TC–CD]RanAcPEI by 5.8–8.7 times.
The physical meanings of kcat and Km for the Michaelis–Menten scheme are not
straightforward, especially when stable intermediates are involved in (13). It is not
likely that any covalent intermediates accumulate in the hydrolysis of nitrophenyl
esters investigated in the present study with the PEI derivatives. In enzymatic
reactions, conversion of the Michaelis complex to intermediates or products is very
fast and the Michaelis complex is not in equilibrium with the reactants. For the
hydrolysis of 3 by the PEI derivatives, the catalytic step is much slower than the
enzymatic processes. Then, the CS complex of Eq. [1] could be in equilibrium with
C and S and, therefore, 1/Km might be taken as a measure of formation constant
of the most stable complex formed between the polymer and 3. The greater 1/Km
for [M(II)TC–CD]SDAcPEI compared with [M(II)TC–CD]RanAcPEI may be taken
to indicate that an extra binding force is present in the complex formed between
3 and [M(II)TC–CD]SDAcPEI. Similar values of 1/Km were observed for [Cu(I-
I)TC–CD]SDAcPEI at pH 7.57 and 9.11. Thus, the extra binding interaction is not
affected by the pH change. On the other hand, the smaller kcat for [M(II)TC–
CD]SDAcPEI compared with [M(II)TC–CD]RanAcPEI indicates that the bound
ester is hydrolyzed more slowly by the PEI derivatives prepared by the site-directed
functionalization.
The stronger but less productive binding of 3 by [M(II)TC–CD]SDAcPEI com-
pared with [M(II)TC–CD]RanAcPEI suggests the binding mode of III. Interaction
of the metal-bound water molecule of TC complex with the carbonyl group of the
bound ester facilitates the complexation. On the other hand, the assembly of III
may sterically protect the ester linkage from attack by nucleophiles. Similar 1/Km
values for [Cu(II)TC–CD]SDAcPEI at pH 7.57 and 9.11 are also consistent with
III. When the central metal ion of III has additional coordination sites, they may
be occupied by water. For [Cu(II)TC–CD]SDAcPEI, the extra water ligand may
change its ionization state upon raising pH from 7.57 to 9.11. Even if this happens,
it would exert only secondary effects on binding of the ester.
Unlike 1/Km , kcat values for both [Cu(II)TC–CD]SDAcPEI and [Cu(II)TC–
CD]RanAcPEI are 11 and 6 times, respectively, greater at pH 9.11 than at pH 7.57.
This suggests that hydroxo ligand attached to the metal center may be the nucleo-
phile attacking the bound ester. Mechanism of the catalyzed hydrolysis, however,
is not certain at present.
The difference in 1/Km between [M(II)TC–CD]SDAcPEI and [M(II)TC–
CD]RanAcPEI corresponds to a decrease in the free energy for complexation of
1.0–1.3 kcal/mol (Ϫ⌬⌬Gf). Hydrogen bond energies in an enzyme–substrate com-
plex have been estimated by mutagenesis (14). Fersht and co-workers estimated
0.5–1.5 kcal/mol as the energy for hydrogen bond between a good hydrogen-bond
donor on the enzyme and an uncharged group on the substrate. The value of 1.0–1.3
kcal/mol for Ϫ⌬⌬Gf estimated in this study agrees with the existence of one extra
hydrogen bonding in III which is absent in [M(II)TC–CD]RanAcPEI complexed
with 3.
Introduction of functional groups in planned positions on backbones of synthetic
polymers is important for design of polymer derivatives performing various func-
tions. The novel methodology reported here can be extended to construction of

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KIM, HONG, AND SUH
functional sites comprising several groups on synthetic polymers as well as design
of multiply functionalized derivatives of CD.
ACKNOWLEDGMENTS
This work was supported by grants from Basic Science Research Institute Program (1997), Ministry
of Education and from Korea Science and Engineering Foundation through Center for Molecular
Catalysis at Seoul National University.
REFERENCES
1. (a) Klotz, I. M. (1987) in Enzyme Mechanisms (Page, M. I., and Williams, A., Eds.), Chap. 2, Royal
Society of Chemistry, London. (b) Suh, J., Cho, Y., and Lee, K. J. (1991) J. Am. Chem. Soc. 113,
4198. (c) Suh, J. (1992) Acc. Chem. Res. 25, 273. (d) Suh, J. (1996) in Polymeric Materials Encyclopedia
(Salamone, J. C., Ed.), p. 4210, CRC Press, Boca Raton.
2. Suh, J., Lee, S. H., and Zoh, K. D. (1992) J. Am. Chem. Soc. 114, 7916.
3. (a) Tabushi, I., Shimizu, N., Sugimoto, T., Shiozuka, M., and Yamamura, K. (1977) J. Am. Chem.
Soc. 99, 7100. (b) Breslow, R., and Czarniecke, M. F. (1978) J. Am. Chem. Soc. 100, 7771. (c)
Breslow, R., Bovy, P., and Hersh, C. L. (1980) J. Am. Chem. Soc. 102, 215. (d) Akkaya, E. U., and
Czarnik, A. W. (1988) J. Am. Chem. Soc. 110, 8553. (e) Nakamura, M., Ikeda, T., Nakamura, A.,
Ikeda, H., Ueno, A., and Toda, F. (1995) Chem. Lett. 343. (f) Breslow, R., and Schmuck, C. (1996)
J. Am. Chem. Soc. 118, 6601. (g) Breslow, R., and Zhang, X. (1996) J. Am. Chem. Soc. 118, 11678.
(h) Breslow, R., and Zhang, B. (1997) J. Am. Chem. Soc. 119, 1676.
4. (a) Kimura, E., Shionoya, M., Hoshino, A., Ikeda, T., and Yamada, Y. (1992) J. Am. Chem. Soc.
114, 10134. (b) Kimura, E., Nakamura, I., Koike, T., Shinoya, M., Kodama, Y., Ikeda, T., and Shiro,
M. (1994) J. Am. Chem. Soc. 116, 4764.
5. Jankowski, K. J., and Parker, D. (1989) J. Chem. Soc. Perkin Trans. I 2079.
6. Kreevoy, M. M., and Wann, S. R. (1981) J. Org. Chem. 46, 2579.
7. Suh, J., Hah, S. S., and Lee, S. H. (1997) Bioorg. Chem. 25, 63.
8. (a) Suh, J., and Park, H. S. (1997) J. Polym. Sci. A: Polym. Chem. 35, 1197, (b) Suh, J., Lee, S. H.,
Kim, S. M., and Hah, S. S. (1997) Bioorg. Chem. 25, 221. (c) Suh, J., and Lee, S. H. (1998) J. Org.
Chem. 63, 1519.
9. Zoh, K. D., Lee, S. H., and Suh, J. (1994) Bioorg. Chem. 22, 242 and references therein.
10. (a) Takagishi, T., Okuda, S., Kuroki, N., and Kozuka, J. (1985) J. Polym. Sci. Pol. Chem. Ed. 23,
2109. (b) Kobayashi, S., Hiroshi, K., Rokunoh, M., and Saegusa, T. (1987) Marcomolecules 20, 1496.
11. Martell, A. E., and Smith, R. M. (1974–1982) Critical Stability Constants. Vols. 1–5. Plenum,
New York.
12. Izatt, R. M., Pawlak, K., and Bradshaw, J. S. (1991) Chem. Rev. 91, 1721.
13. Bender, M. L., and Kezdy, F. J. (1965) Annu. Rev. Biochem. 34, 49.
´
14. Fersht, A. R., Shi, J-P., Knill-Jones, J., Lowe, D. M. Wilkinson, A. J., Biow, D. M.; Brick, P., Carter,
P., Waye, M. M. Y., and Winter, G. (1985) Nature 314(21), 235.