Pyridoxal 5′-phosphate binding in lysine-modified PAMAM dendrimers: A biomimetic approach

Article abstract of DOI:10.1021/ol901299e

Image Persented (G:3-7)-dendri-PAMAM-(APO-Phe-Lys)_x (2, APO = aminopropanol, Phe = phenylalanine, Lys = lysine) were prepared and used in a binding study with pyridoxal 5′-phosphate. The results revealed a positive dendritic effect, and binding

Full text of DOI:10.1021/ol901299e

ORGANIC
LETTERS
2009
Vol. 11, No. 16
3526-3529
Pyridoxal 5′-Phosphate Binding in
Lysine-Modified PAMAM Dendrimers:
A Biomimetic Approach
Kuang-Chan Hsien,†,§ Hui-Ting Chen,‡,⊥ Yi-Chen Chen,† Yeh-Long Chen,†
Chi-Yu Lu,| and Chai-Lin Kao*,†,⊥
Department of Medicinal and Applied Chemistry, Department of Fragrance and
Cosmetic Science, School of Pharmacy, Department of Biochemistry, and Center of
Excellence for EnVironmental Medicine, Kaohsiung Medical UniVersity,
Kaohsiung 807, Taiwan
clkao@kmu.edu.tw
Received March 12, 2009
ABSTRACT
(G:3-7)-dendri-PAMAM-(APO-Phe-Lys)x (2, APO ) aminopropanol, Phe ) phenylalanine, Lys ) lysine) were prepared and used in a binding
study with pyridoxal 5′-phosphate. The results revealed a positive dendritic effect, and binding ability was found to vary with the environment.
(G:3-7)-dendri-PAMAM-(APO-Phe-Lys)x (2) demonstrated better binding ability at higher pH, and protonation of lysine was considered to
affect binding. The strongest binding affinity was Kb ) 254.3 mM-1 at pH 9, which was shown by (G:7)-dendri-PAMAM-(APO-Phe-Lys)490 (2e).
Pyridoxal 5′-phosphate (PLP) is the major active component
in six vitamin B6 metabolites and is crucial for the activities
of a variety of enzymes.1 PLP can regulate a number of
biological functions, including metabolism of amino acids,
replenishment of one carbon unit, and synthesis of amino
sugars. Moreover, PLP is also involved in the metabolism
of homocysteine2 and polyunsaturated fatty acids.3 In addi-
tion to its role in enzymatic reactions, levels of B6 and/or
PLP are associated with a variety of physiological responses,
such as calcium ion flux.4 In addition, PLP serves as either
a biomarker or regulator of different syndromes and dis-
eases;5 PLP has been shown to prevent progression of
diabetic nephropathy,6 to inhibit apoptosis,7 and to assist the
recovery from the damage caused by reperfusion after
(4) (a) Tardif, J.-C.; Carrier, M.; Kandzari, D. E.; Emery, R.; Cote, R.;
Heinonen, T.; Zettler, M.; Hasselblad, V.; Guertin, M.-C.; Harrington, R. A.
J. Thorac. CardioVasc. Surg. 2007, 133, 1604. (b) Alexander, J. H.; Emery,
R. E., Jr.; Carrier, M.; Ellis, S. J.; Mehta, R. H.; Hasselblad, V.; Menasche,
P.; Khalil, A.; Cota, R.; Bennet-Guerrero, E.; Mack, M. J.; Schuler, G.;
Harrington, R. A.; Tardif, J.-C. JAMA, J. Am. Med. Assoc. 2008, 299, 1777.
(5) (a) Yoshimura, T.; Goto, M. FEBS J. 2008, 275, 3527. (b) Dierkes,
J.; Weikert, C.; Klipstein-Grobusch, K.; Westphal, S.; Luley, C.; Mo¨hlig,
M.; Spranger, J.; Boeing, H. Am. J. Clin. Nutr. 2007, 86, 214. (c) Lima,
C. P.; Davis, S. R.; Mackey, A. D.; Scheer, J. B.; Williamson, J.; Gregory,
J. F., III. J. Nutr. 2006, 2141. (d) Chiang, E.-P.; Selhub, J.; Bagley, P. J.;
Dallal, G.; Roubenoff, R. Arthritis Res. Ther. 2005, 7, R1404.
† Department of Medicinal and Applied Chemistry.
§ School of Pharmacy.
‡ Department of Fragrance and Cosmetic Science.
⊥ Center of Excellence for Environmental Medicine.
| Department of Biochemistry.
(1) (a) Toney, M. D. Arch. Biochem. Biophys. 2005, 279. (b) Zhao, Z.;
Hong, L.; Liu, H.-w. J. Am. Chem. Soc. 2005, 127, 7692.
(2) Troccaz, M.; Benattia, F.; Borchard, G.; Clark, A. J. Chem. BiodiVers.
2008, 5, 2372.
(3) Bordoni, A.; Cabrini, L.; Marchetti, M.; Danesi, F.; Bochicchio, D.;
Biagi, P. L.; Maranesi, M. Ann. Nutr. Metab. 2006, 50, 305.
(6) Nakamura, S.; Li, H.; Adijiang, A.; Pischeterieder, M.; Niwa, T.
Nephrol. Dial. Transplant 2007, 22, 2165.
10.1021/ol901299e CCC: $40.75
Published on Web 07/21/2009
 2009 American Chemical Society

ischemia.8 Although PLP is involved in a variety of
enzymatic reactions, the reaction mechanisms share common
features. In PLP-associated enzymes, PLP is usually bound
to lysine residues in proteins, forming a Schiff base, and
this species is known as an internal aldimine. Binding of
the holoenzyme to inbound substrate causes the formation
of an external aldimine, in which PLP forms a new imine
bond with the substrate and carries out the certain enzymatic
reactions. The exchange between internal and external
aldimine is important for the progress of enzymatic reactions
involving PLP. In addition, because of the aromaticity of
PLP, phenylalanine, tyrosine, and tryptophan residues can
improve the binding of PLP to protein.9
Dendrimers are globular and monospheric polymers that
are produced by an iterative process.10 A dendrimer thus
possesses a well-defined architecture with a high number of
end groups and tunable physical properties. These unique
characteristics make dendrimers attractive platforms for
execution of desired biological activities,11 including roles
as cell-membrane transporters12 and as carriers of drugs13
and imaging reagents.14 In addition, because dendrimer sizes
and shapes are similar to those of proteins, they are often
referred to as “artificial proteins”.15 Several studies have
examined dendrimers associated with pyridoxal or pyridox-
amine,16 and the studies revealed that noncovalent linkages
between cofactors and dendrimers are better than nonex-
changable covalent linkages in terms of biological activity.17
To achieve reversible binding, hydrophobic interactions are
widely used. Therefore, natural PLP has to be functionalized
with a hydrophobic chain, and extra modifications must be
made before PLP can be employed in the desired reactions.
Figure 1. Designed peptide 1 and the synthetic dendrimers 2.
In contrast, we suggest that a dendrimer which binds
unmodified PLP in a more natural manner may shorten the
effort of construction and improve catalytic activity.
(7) Ardestani, A.; Yazdanoarast, R.; Nejad, A. S. Toxicol. in Vitro 2008,
22, 968.
To achieve reversible binding, physical entrapment of
guest molecules by dendrimers might be desirable. Such a
construction is known as a “dendritic box”,18 in which a
variety of interaction forces are applied to prevent guest
molecules from escaping. However, release of the box
contents will be difficult if the box has a strong binding
affinity for the guest molecules; this system is therefore not
suitable for our purpose.19 Thus, we considered the dendritic
effect, which is another important feature of dendrimers.20
The dendritic effect is a complex phenomenon usually
demonstrated by dendrimers. Two major factors have been
proposed to contribute to the dendritic effect. One is a
clustering of reaction centers caused by the dense distribution
of functional groups in dendrimers. The other is the creation
of a nanoenvironment for preorganization of reaction sites;
as a consequence, the desired reaction is improved. Reymond
and colleagues have demonstrated the application of a
positive dendritic effect in the construction of enzyme
models.21 We postulated that a suitably designed dendrimer,
(8) Tadif, J.-C.; Carrier, M.; Kandzari, D. E.; Emery, R.; Cote, R.;
Heinonen, T.; Zettler, M.; Hasselblad, V.; Guertin, M.-C.; Harrington, R. A.
J. Thorac. CardioVasc. Surg. 2007, 133, 1604.
(9) Bhavani, B. S.; Rajaram, V.; Bisht, S.; Kaul, P.; Prakash, V.; Murthy,
M. R. N.; Rao, N. A.; Savithri, H. S. FEBS Lett. 2008, 275, 4606.
(10) (a) Newkome, G. R.; Moorefield, C. N.; Vogtle, F. Dendrimers
and Dendrones; Wiley-VCH: Weinheim, 2001. (b) Tomalia, D. A.; Fre´chet,
J. M. J. Dendrimers and other dendritic polymers; Wiley & Sons Ltd:
Chichester, 2001. (c) Boas, U.; Christensen, J. B.; Heegard, P. M. H.
Dendrimers in medicine and biotechnology; RSC Publishing: Cambridge,
UK, 2006.
(11) (a) Lee, C. C.; MacKay, J. A.; Fre´chet, J. M. J.; Szoka, F. C. Nat.
Biotechnol. 2005, 23, 1517. (b) Svenson, S.; Tomalia, D. A. AdV. Drug.
DeliVery ReV. 2005, 57, 2106.
(12) (a) Yin, M.; Kuhlmann, C. R. W.; Sorokina, K.; Li, C.; Mihov,
G.; Pietrowski, E.; Koynov, K.; Klapper, M.; Luhmann, H. J.; Mullem, K.;
Weil, T. Biomacromolecules 2008, 9, 1381. (b) Tsutsumi, T.; Hirayama,
F.; Uekama, K.; Arima, H. J. Pharm. Sci. 2008, 97, 3022. (c) Venuganti,
V. V. K.; Perumal, O. P. Int. J. Pharm. 2008, 361, 230.
(13) (a) Thomas, T. P.; Shukla, R.; Kotlyer, A.; Liang, B.; Ye, J. Y.;
Norris, T. B.; Baker, J. R., Jr. Biomacromolecules 2008, 9, 603. (b) Yang,
H.; Lopina, S. T.; DiPersio, L. P.; Schmidt, S. P. J. Mater. Sci. Mater.
Med. 2008, 19, 1991. (c) Kolhatkar, R. B.; Swaan, P.; Ghandehari, H.
Pharm. Res. 2008, 25, 1723.
(14) Hamoudeh, M.; Kamleh, M. A.; Diab, R.; Fessi, H. AdV. Drug.
Del. ReV. 2008, 60, 1329.
(15) (a) Rudick, J. G.; Percec, V. Acc. Chem. Res. 2008, 41, 1641. (b)
Tomalia, D. A.; Huang, B.; Swanson, D. R.; Brothers, H. M., II; Klimash,
J. W. Tetrahedron 2003, 59, 3799. (c) Liang, D. L.; Aida, T. Chem.
Commun. 1996, 1523.
(18) (a) Naylor, A. N.; Goddard, W. A., III; Kiefer, G. E.; Tomalia,
D. A. J. Am. Chem. Soc. 1989, 111, 2339. (b) Jansen, J. F. G. A.; Brabander-
van den Berg, E. M. M; Meijer, E. W. Science 1994, 266, 1226.
(19) Liu, M.; Fre´chet, M. J. Pharm. Sci. Tech. Today 1999, 2, 393.
(20) (a) Zaupa, G.; Scrimin, P.; Prins, L. J. J. Am. Chem. Soc. 2008,
130, 5699. (b) Dahan, A.; Portnoy, M. J. Am. Chem. Soc. 2007, 130, 5860.
(21) Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004,
126, 15642.
(16) (a) Breslow, R.; Wei, S.; Kenesky, C. Tetrahedron 2007, 63, 6317.
(b) Liu, L.; Breslow, R. Bioorg. Med. Chem. 2004, 12, 3277. (c) Liu, L.;
Breslow, R. J. Am. Chem. Soc. 2003, 125, 12110.
(17) Liu, L.; Zhou, W.; Chruma, J.; Breslow, R. J. Am. Chem. Soc. 2004,
126, 8136.
Org. Lett., Vol. 11, No. 16, 2009
3527

with sufficient numbers of surface functional groups, would
be able to bind PLP to form natural aldimine moieties. The
polyamidoamine (PAMAM) dendrimer is widely used, and
in this molecule, both the tertiary amine and the secondary
amide can be either hydrogen-bond donors or acceptors. This
feature, along with the amide skeleton, makes the PAMAM
dendrimer a good platform with which to demonstrate protein
function.22 Furthermore, a recent study indicated that the
conformation and density of PAMAM dendrimers were
flexible depending on their surroundings.23 These charac-
teristics make the PAMAM dendrimer similar to enzymes;
therefore, the PAMAM dendrimer was used as a core
structure in this study. Lysine-containing peptides (1, Figure
1) were introduced onto the surface of a PAMAM dendrimers
in which lysine was anticipated to play the same role as in
PLP-associated enzymes. In addition, we integrated a phe-
nylalanine residue to increase the binding affinity of PLP
using π-π interaction.
appeared, and the peaks at 325 and 388 nm disappeared.24
Thereafter, (G:3-7)-dendri-PAMAM-(APO-Phe-Lys)x (2)
were titrated with different concentrations of PLP at three
different pH values. The difference spectra of PLP and (G:
3-7)-dendri-PAMAM-(APO-Phe-Lys)x (2) versus PLP were
used to determine binding affinities (Figure 2).
At pH 5, neither a growth nor decline in absorption was
identified in difference spectra, except in those of (G:4)-
dendri-PAMAM-(APO-Phe-Lys)59 (2b). The difference spec-
trum of dendrimer 2b at pH 5 clearly showed a reduction of
intensity at 388 nm but enhancement of the peak at 325 nm,
and no change in intensity was found between 420 and 450
nm. This implied that the addition of (G:4)-dendri-PAMAM-
(APO-Phe-Lys)59 did not cause the formation of bound PLP
at pH 5; rather, it only contributed to the equivalent of two
free PLP moieties. Presumably, the low pH environment led
to protonation of lysine residues of (G:3-7)-dendri-PAMAM-
(APO-Phe-Lys)x (2) and, consequently, prevented them from
forming imine bonds with PLP.
At pH 7 and 9, an obvious decrease in absorption at 388
nm accompanied by a rise in absorption around 430 nm was
observed. This finding was the first evidence of the existence
of bound PLP. A Scatchard-Klotz plot was applied to
determine the binding of PLP and (G:3-7)-dendri-PAMAM-
(APO-Phe-Lys)x (2). The results are presented in Figure 3
Figure 2. Difference spectrum of PLP and (G:7)-dendri-PAMAM-
(APO-Phe-Lys)490 2e with dendrimer vs PLP at pH 9. The
concentration of dendrimer 2e was 0.07 mM, and the concentration
of PLP was varied from 0 to 0.014 mM.
The detailed procedure for synthesizing (G:3-7)-dendri-
PAMAM-(APO-Phe-Lys)x (2a-e, APO ) aminopropanol,
Phe ) phenylalanine, Lys ) lysine) is reported in the
Supporting Information. The products were identified by
NMR and MALDI-mass spectrometry. The degrees of
substitution of the modified PAMAM dendrimer were 32,
59, 119, 253, and 490 for 2a-e, respectively, and the
polydispersities are narrow. The PDI values are 1.05, 1.12,
1.09, 1.07, and 1.14 for 2a-e, respectively. To examine the
UV spectra of free and bound PLP, free lysine was titrated
with different concentrations of PLP, and UV spectra were
recorded and individually analyzed at pH 5, 7, and 9. The
UV spectrum of free PLP showed two absorption maxima
at 325 and 388 nm. Upon mixing with lysine, a peak at 422
nm, which is correlated with the presence of aldimine,
Figure 3. Scatchard-Klotz plots of 2a-e at pH 7 and 9.
and Table 1. The linearity of the plot indicated that only
one type of binding site existed. This fact is consistent with
our original assumption that the PLPs were assumed to bind
to lysine residues. In addition to (G:6)-dendri-PAMAM-
(APO-Phe-Lys)253 (2d), four other generations of dendrimers
showed better binding ability in basic than in neutral solution,
except for the binding of (G:7)-dendri-PAMAM-(APO-Phe-
Lys)490 (2e). This finding indicates that the binding behavior
of dendrimer 2 was affected by the environment. Remark-
ably, (G:6)-dendri-PAMAM-(APO-Phe-Lys)253 (2d) exhib-
ited relatively low binding affinity and binding number. A
(22) Boas, U.; Christensen, J. B.; Heegaard, P. M. H. J. Mater. Chem.
2006, 16, 3785.
(23) Liu, Y.; Bryantsev, V. S.; Diallo, M. S.; Goddard, W. A., III. J. Am.
Chem. Soc. 2009, 131, 2798.
(24) The extinction coefficient is 3052.5, 3083, and 3094.2 M-1 cm-1
at pH 5, 7, and 9.
3528
Org. Lett., Vol. 11, No. 16, 2009

tions. We thus believe that the binding behavior of (G:3-7)-
dendri-PAMAM-(APO-Phe-Lys)x (2) results from coopera-
tion of the PAMAM dendrimer and the anchoring ability of
synthetic peptide 1.
Table 1. Binding Constant (Kb) and Binding Number (n) of 2 at
pH 7 and 9
dendrimer
pH
Kb (mM-1
)
n
In summary, this study has demonstrated for the first time
that peptide-conjugated PAMAM dendrimers can bind
natural PLP via imines bonds to form a natural aldimine.
Furthermore, we have shown that the pH environment is
critical with respect to binding affinity. In acidic solution,
no obvious binding was seen, and the binding affinity is
higher at high pH. The binding ability showed a positive
trend with increasing dendrimer complexity, except in the
case of (G:6)-dendri-PAMAM-(APO-Phe-Lys)253 (2d). The
highest binding constant measured was 254.3 mM-1 with
dendrimer (G:7)-dendri-PAMAM-(APO-Phe-Lys)490 (2e) at
pH 9. Because only one type of binding site existed, and as
no obvious binding was seen with unmodified (G:3-7)-
dendri-PAMAM-(NH2)x, the lysine residue in (G:3-7)-
dendri-PAMAM-(APO-Phe-Lys)x (2) is suggested to serve
as the binding site for PLP. The positive relationship between
the size of the dendrimer and the stability of the imine bond
may assist in the development of new carrier systems for
medical applications. Moreover, this discovery sets the stage
for a future investigation of the application of peptide-
conjugated PAMAMs as artificial enzymes demonstrating
useful biological activities.
2a
7
9
7
9
7
9
7
9
7
9
0.37
1.34
0.15
0.31
6.49
242.01
14.33
7.79
0.78
254.30
12.5
16.3
35.5
39.1
2.1
20.7
77.5
67.6
129.9
56.2
2b
2c
2d
2e
detailed examination of the UV/vis spectra of this dendrimer
indicated no obvious isosbestic point. Presumably, one or
more species intervene between the exchange of the internal
aldimine and external aldimine of 2d. Therefore, the binding
constant is likely to be underestimated, despite high cor-
relation coefficients shown by Scatchard-Klotz analysis (R2
) 0.98 at pH 7 and 0.97 at pH 9).
Moreover, the (G:7)-dendri-PAMAM-(APO-Phe-Lys)490
(2e) demonstrated a higher binding number and a stronger
binding constant than did any of the lower generation
analogues. This result confirms our hypothesis that increases
in the number of end groups and dendrimer size improves
binding ability. The higher generation of dendrimer structure
increases the area density of branching and improves
preorganization, which results in stronger binding affinity.
In comparison, when either free peptide 1 or (G:3-7)-dendri-
PAMAM-(NH2)x (X ) 32, 64, 128, 256, or 512 for G2-7,
respectively) was analyzed in the same manner, a peak
between 420 and 440 was observed in the UV/vis spectra.
However, Scatchard-Klotz analysis yielded no reliable
statistical result because of a low correlation coefficient
(Table S2, Supporting Information). Therefore, we conclude
that no obvious binding exists between PLP and either
peptide 1 or free PAMAM dendrimer at the tested concentra-
Acknowledgment. We are grateful to the National Science
Council of the Republic of China (NSC 96-2113-M-037-
007-MY2 and NSC 96-2323-B-037-003). The authors are
also grateful to the Center for Resources, Research and
Development (Kaohsiung Medical University) for instru-
mental support of this work.
Supporting Information Available: Detailed synthetic
procedure for dendrimer 2, characterization spectra for
synthetic compounds, and additional UV spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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3529