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T. Gubica et al. / Carbohydrate Research 346 (2011) 1855–1859
recognizing the substrate. Almost in all cases, CDs were noncom-
petitive inhibitors of PAL. With increasing CD concentration, only
3.3. Analyses
TM-a-CD and TM-b-CD increased their inhibitory properties.
1H and 13C NMR spectra for CDCl3 solutions were recorded at
300 and 75 MHz, respectively, on a Varian VNMRS-300 spectrom-
eter. The 2D experiments (COSY, HSQC, and HMBC) were run using
standard Varian software. Optical rotation values were measured
on a Perkin–Elmer 241 polarimeter at a wavelength of 589 nm.
Molecular peaks in mass spectrometry were collected on an AMD
604 Intectra spectrometer.
Therefore, we can expect that TM-
a
-CD and TM-b-CD exhibited
definitely unfavourable interactions with PAL protein. On the
other hand, the specific dependences of 1=V0max values versus
DM-a-CD and DM-b-CD concentration can be caused by two dif-
ferent effects of these CDs on PAL. As mentioned above, DM-
a
-
CD and DM-b-CD form the strongest inclusion complexes with
Phe of all CDs examined; therefore, we can assume that in this
case there is the highest concentration of complexed Phe. With
3.4. Methods of kinetic data determination
an increasing concentration of DM-
of complexed to free CD molecules decreases. Probably host–
guest complexes of Phe with DM- -CD and DM-b-CD unfavour-
ably interact with PAL protein, whereas free DM- -CD and
DM-b-CD help PAL in molecular recognition of the substrate.
Therefore, probably for DM- -CD concentrations of and
a-CD and DM-b-CD, the ratio
Phe for measurements was incubated with a proper excess of
each CD derivative. Incubation was carried out overnight at room
temperature. A typical assay contained borate buffer at pH 8.8,
a
a
2.5 lL of 10 U/mL PAL, various amounts of the particular CD deriv-
a
2
ative (1, 2 or 3 mM), and various amounts of Phe (0.08, 0.12, 0.2,
0.32, 0.4 or 0.8 mM). Six assays of different concentrations of Phe
were applied per one experiment. The substrate concentration var-
ied between the experiments of one series, whereas the CD total
concentration remained unchanged. The CDs concentrations varied
between different experimental series. The concentration of CDs
was always in proper excess in comparison with substrate. Each
experiment was repeated at least three times. The increase of
absorbance was measured at a wavelength of 290.0 nm on a
1202 Shimadzu UV–vis spectrophotometer during the time of reac-
tion. The experiments were performed at room temperature
(20 °C). The Lineweaver–Burk plots were used to determine the
intercepts (inverse maximum velocity), and slopes (ratio of the
Michaelis constant per maximum velocity) using the least-squares
method.
3 mM, the activation of PAL was observed.
3. Experimental
3.1. Materials
Cyclomaltohexaose (
(phenylalanine ammonia-lyase from Rhodotorula glutinis, solution
in 60% glycerol, 10 U/mL, EC 4.3.1.5), -phenylalanine (Phe) were
a-CD) and cyclomaltoheptaose (b-CD), PAL
L
purchased from Sigma–Aldrich Chemie GmbH.
3.2. CD derivatives
3.2.1. Hexakis(2,3-di-O-methyl)cyclomaltohexaose (DM-a-CD)
The synthesis and full analytical data for DM-
a-CD have been
4. Conclusions
reported earlier.9
The aim of this paper was to evaluate the influence of native
and some methyl ether derivatives of CDs on the enzymatic
decomposition of Phe catalyzed by PAL. The oral administration
of microencapsulated PAL was considered as a very convenient
treatment in phenylketonuria, and CD derivatives were found as
the substances which preserved PAL activity during the process
of preparation of such PAL form.14 In the cited paper, hydroxypro-
pylcyclomaltooctaose and hydroxypropylcyclomaltoheptaose
served as efficient protectors of PAL. However, in our studies the
CDs behaved mostly as both competitive and noncompetitive
3.2.2. Heptakis(2,3-di-O-methyl)cyclomaltoheptaose (DM-b-CD)
The synthesis and full analytical data for DM-b-CD have been
reported earlier.9
3.2.3. Hexakis(2,3,6-tri-O-methyl)cyclomaltohexaose (TM-
The synthesis of TM-
-CD has been reported earlier.8 White
amorphous powder; yield 62%; +163.0° (c 1.5; CHCl3),
lit.36
+164° (c 1.1; CHCl3); Rf 0.50 (2:1 chloroform–acetone).
a-CD)
a
½ ꢁ
a 2D0
½ ꢁ
a 2D2
1H NMR (300 MHz, CDCl3): d 5.05 (d, 6H, JH-1,H-2 3.3 Hz, 6H-1);
3.86–3.74 (m, 12H, 6H-6a,6H-5); 3.71–3.68 (m, 6H, 6H-6b);
3.64 (s, 18H, 6 ꢃ C-3–OCH3); 3.61–3.50 (m, 12H, 6H-4,6H-3);
3.49 (s, 18H, 6 ꢃ C-2–OCH3); 3.40 (s, 18H, 6 ꢃ C-6–OCH3); 3.17
(dd, 6H, JH-2,H-3 9.3 Hz, 6H-2); 13C NMR (75 MHz, CDCl3): d
100.34 (C-1); 82.67 (C-4); 82.40 (C-2); 81.42 (C-3); 71.66 (C-
6); 71.42 (C-5); 62.02 (C-3–OCH3); 59.20 (C-6–OCH3); 58.04 (C-
2–OCH3). ESIMS: calcd for C54H96O30, m/z 1225.3; found, m/z
1248.9 [M+Na]+.
inhibitors. Only for selected concentrations of b-CD and DM-a-
CD, were they PAL activators; therefore, only these compounds of
all CD derivatives considered in this study can serve as protectors
of PAL. Nevertheless, the rest of the CDs under investigation, that
is,
a-CD, TM-a-CD, DM-b-CD and TM-b-CD cannot be considered
as the additives to PAL-based drugs towards phenylketonuria.
Although the results obtained are mostly atypical for classical
inhibitors or activators, it should be stressed that CDs played a tri-
ple role in these cases. CDs can affect the concentrations of both
substrate and the product of biotransformation under investiga-
tion, and also they can interact with the enzyme protein.
3.2.4. Heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose (TM-b-
CD)
The synthesis of TM-b-CD has been reported earlier.8 Froth;
yield 79%; ½a 2D0
ꢁ
+144.6° (c 1.0; CHCl3), lit.37
[a
]
D
+158° (c 1.4;
Acknowledgment
CHCl3); Rf 0.62 (10:1 chloroform–MeOH). 1H NMR (300 MHz,
CDCl3): d 5.13 (d, 7H, JH-1,H-2 3.3 Hz, 7H-1); 3.88–3.80 (m, 14H,
7H-6a,7H-5); 3.65 (s, 21H, 7 ꢃ C-3–OCH3); 3.62–3.48 (m, 21H,
7H-4,7H-6b,7H-3); 3.51 (s, 21H, 7 ꢃ C-2–OCH3); 3.39 (s, 21H,
7 ꢃ C-6–OCH3); 3.19 (dd, 7H, JH-2,H-3 9.6 Hz, 7H-2); 13C NMR
(75 MHz, CDCl3): d 99.16 (C-1); 82.25 (C-2); 81.96 (C-3); 80.49
(C-4); 71.61 (C-6); 71.12 (C-5); 61.65 (C-3–OCH3); 59.16 (C-6–
Financial support from the University of Warsaw (BST 153123)
ane the Medical University of Warsaw (FW 28/N/2011) is grate-
fully acknowledged.
References
OCH3); 58.71 (C-2–OCH3). ESIMS: calcd for C63H112O35
, m/z
1. de Paula, W. X.; Denadai, A. M. L.; Santoro, M. M.; Braga, A. N. G.; Santos, R. A. S.;
1429.6; found, m/z 1452.0 [M+Na]+.
Sinisterra, R. D. Int. J. Pharm. 2011, 404, 116–123.