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Base-specific interaction of polymers containing adenine: effect of chiral spacer on the interaction with polynucleotide
REACTIVE
ELSEVIER
Reactive &
FUNCiONAL POLYMERS
Functional Polymers 37 (1998) 189-198
Base-specific interaction of polymers containing adenine: effect of chiral spacer on the interaction with polynucleotide Yoshiaki
Inaki *, Shigeki
Kamo, Mikiji Miyata
Department of Materials and Life Science, Osaka University, Yamuahoka 2-1, Suita, Osaka 565, Japan
Received 15 June 1997; revised version received 25 July 1997; accepted 26 July 1997
Abstract Polyethyleneimine derivatives of adenine with L- and D-serine as spacers were prepared. The polymer having D-Serhe spacer 2 gave a stable polymer complex with poly(uridylic acid) (poly U), but the polymer having L-serine spacer 1 gave an unstable polymer complex. The reason was concluded to be caused by the steric repulsion of L-serine units along the polymer chain when the polymer formed polymer complex with poly U. 0 1998 Elsevier Science B.V. All rights reserved. Keywords:
1.
Adenine; L-Serine; D-Serine; Polyethyleneimine;
Polynucleotide; Poly U; Interaction
Introduction
In recent years, considerable attention has been directed towards synthetic nucleic acid analogues in the hope of discovering new and more effective antisense compounds [ 11. Synthetic polymers containing nucleic acid bases have been prepared, and the interactions of these polymers were studied with DNA and RNA [2]. These synthetic polymers without ribose nor phosphodiester were found to interact with polynucleotides, DNA and RNA. One of these polymers is the polyethyleneimine derivative containing nucleic acid bases with DL-homoserine as spacer units [3]. These water-soluble polymers were found to interact with polynucleotide by specific hydrogen bonding between complementary nucleic acid bases. Since DNA is a chiral polymer, the chit-al unit in the synthetic nucleic acid model should influence *Correspondingauthor. Tel.: +81 (6) 879-7405, Fax: +81 (6) 879-7404, E-mail: [email protected] 1381-5148/98/$19.00 0 1998 Elsevier Science B.V. AU rights reserved PII S1381-5148(97)00118-l
the complex formation with DNA. This paper deals with the preparation of polyethyleneimine derivatives containing adenine with chiral spacer units 1 (PEI-L-Ser-Ade) and 2 (PEI-D-Ser-Ade) (Fig. l), and interaction of these polymers with poly(uridylic acid) 3 (poly U) to clear the effect of chiral unit on the polymer complex formation (Fig. 2).
9H2 FH2
Czq H VH H-N.&&Hz k=o +CH2k-CHlf
k=o fCH,-Ii-CH+
1: PEI-L-Ser-Ade
2: PEI-D-Ser-Ade
Fig. 1. Polyethyleneimine derivatives of adenine having L- and D-serines.
I! Inaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
AH2
kH2 O:i: H VH H-k,$H2
+
&=o +CH2-ti-CH+
1
3: Poly u
Polymer Complex
Fig. 2. Formation of polymer complex between the adenine-containing polymer and poly U.
2. Experimental 2.1. Preparation of polymers 2.1.1. Pentachlorophenyl3-(adenin-9-yl)propionate 7
To the suspension in water of 3-(adenin-9-yl)propionic acid 5 [4] (2.1 g, 10 mmol) prepared from adenine 4 and ethyl acrylate, NaOH (0.96 g, 24 mmol) and tetrabutylammonium hydrogen sulfate (3.4 g, 10 mmol) were added to give a clear solution. After evaporation of water, excess methanol was added to the residue, and insoluble NazS04 was removed by filtration. The filtrate was evaporated and the residue was extracted with chloroform. After evaporation of chloroform, yellowish crystal was washed with diethyl ether to give the tetrabutylammonium (TBA) salt 6 as white crystal. The obtained ammonium salt 6 was dissolved in chloroform (100 ml) and pentachlorophenyl trichloroacetate (PCP-TCA) (4.94 g, 12 mmol) was added to the solution. The product 7 was precipitated after 10 min stirring. Yield: 3.80 g (83.5%). IR (KBr, cm-‘): 3320, 3150, 1780, 1670, 1600, 1480, 1380, 1360, 1320, 1110,780,760,720, 650. ‘H NMR (270 MHz, DMSO-4) S 8.31 (lH, s, 2(8)-H), 8.16 (lH, S, 8(2)-H), 7.23 (2H, S, 6-NH2), 4.55 (2H, t, N-CH2), 3.54 (2H, t, CH2-CO). 2.1.2. 3-(Adenin-9-yl)propionyl-L-serine 9 L-Serine methyl ester hydrochloride 8 (1.5 g, 9.6 mmol) was dissolved in WV-dimethylformamide (80 ml) and dried under reduced pressure at room temperature. The activated ester 7 (3.6 g, 8.0 mmol) and triethylamine (1.4 ml, 9.6 mmol) were added to
the solution and the mixture was stirred overnight at room temperature. The reaction was followed by TLC and IR spectra. After the completion of the reaction, the solvent was removed under reduced pressure, and the residue was recrystallized with ethanol to give 3-(adenin-9-yl)propionyl-L-se&e methyl ester as white crystals. Yield: 1.9 g (85%). TLC: Rf = 0.15 (CHCls : MeOH = 5 : 1). IR (KBr, cm-‘): 3300, 3100, 1740, 1690, 1650, 1610, 1580, 1550, 1420,1340, 1220,1160,1140,1060, 1040,950,680, 640. ‘H NMR (270 MHz, DMSO-&) S 8.20 (lH, d, CO-NH), 8.13 (lH, s, 2(8)-H), 8.00 (lH, s, 8(2)-H), 7.18 (2H, S, 6-NH& 5.00 (lH, t, OH), 4.34 (3H, m, CH, N-CH2), 3.60 (5H, s and t, 0-CH3, CH2-0), 2.77 (2H, t, CH2-CO). The obtained methyl ester (1.9 g, 6.8 mmol) was hydrolyzed in 1 M NaOH (8 ml) at 0°C. After the reaction, pH was adjusted to 4 with 6 M HCl to give 3-(adenin-9-yl)propionyl-L-serine 9 as white precipitate. Yield: 1.6 g (87%). TLC: Rf = 0.13 (l-BuOH : CH&OOH : Hz0 = 5 : 2 : 3). [a]g - 1.72 (H20). IR (KBr, cm-‘): 3300, 1690, 1640, 1560, 1510, 1410, 1230, 1080, 1030, 700, 640. ‘H NMR (270 MHz, DMSO-4) S 12.5 (lH, br, COOH), 8.20 (lH, d, CO-NH), 8.14 (lH, s, 2(8)-H), 8.02 (lH, s, 8(2)-H), 7.17 (2H, S, 6-NH2), 5.0 (lH, br, OH), 4.34 (2H, t, N-CH2), 4.26 (1H t, CH), 3.60 (5H, d, CH2-0), 2.77 (2H, t, CH2-CO). 2.1.3. 3-(Adenin-9-yl)propionyl-D-serine This compound was also prepared according to the method used for the L-serine derivative. [a]? +1.74 (H20).
Y. Inaki et al./Reactive
& Functional Polymers 37 (1998) 189-198
2.1.4. Poly-{N-[3-(adenin-9-yl)propionyl]-L-se& ethyleneimine} (1:PEI-L-Ser-Ade) Ade-L-Ser-OH 9 (0.80 g, 3.0 mmol) in chloroform (30 ml) was added to N,N-dimethylformamide dimetbyl acetal (DMM: 4.0 ml, 30 mmol) and stirred overnight at room temperature to give a clear solution. After evaporation of the solvent, chloroform was added to the residue to give oily product 10. To the oily product, chloroform (30 ml), pentachlorophenol (PCP) (2.5 g, 9.5 mmol) and N,N-dicyclohexylcarbodiimide (DCC) (0.62 g, 3.0 n-m-101)were added and stirred for 2 h at 0°C and for 12 h at room temperature. After the precipitated dicyclohexyl urea was removed by filtration, the filtrate was evaporated to give solid product, which was washed with methanol to give the activated ester 11. Yield: 0.79 g (43%). IR (KBr, cm-‘): 1790, 1640, 1580. The product was used for the next reaction without purification. The activated ester 11 (0.18 g, 0.29 mmol) in dry dimethyl sulfoxide (5 ml) was added polyethyleneimine (PEI, 0.020 g, 0.29 mmol) and imidazole (0.020 g, 0.29 mmol), and was stirred for 80 h at 60°C to give a clear solution. Polyethyleneimine (M.W.: 22000) used here was prepared from poly(2-ethyl-2-oxazoline) (Dow Chemical Japan) by hydrolysis [5]. The mixture was poured into excess acetone to give the polymer as precipitates. In order to remove the DMM protecting group, the polymer in water (5 ml) was reacted with 6 M HCl (0.2 ml) for 9 h at room temperature. The mixture was poured into excess acetone to give the polymer as precipitates, quantitatively. TLC: Rf = 0.00 (BuOH : CHsCOOH : Hz0 = 5 : 2 : 3). Degree of substitution was obtained by UV spectra to be 91 unit mol%. IR (KBr, cm-‘): 1640, 1540,1480, 1420,1250,1200,1060,800,720,650. ‘HNMR (270 MHz, DMSO-4) 6 8.20 (lH, br, CO-NH), 8.13 (lH, s, 2(8)-H), 8.08 (lH, s, 8(2)-H), 7.20 (2H, s, 6-NH& 4.33 (7H, br, N-CH2, CH, N-CH2CH2-N), 3.03.4 (4H, br, CHz-0, CHz-CO). 2.1.5. Poly-{N-[3-(adenin-9-yl)propionyl]-D-seryl ethyleneimine) (2: PEI-D-Ser-Ade) The same method as for the L-serine derivative was used for preparation of the D-Serine derivative. Degree of substitution was 95 unit mol%.
191
2.2. Interaction of the adenine-containing polymer with polynucleotides Interactions of the polyetbyleneimine derivatives with polynucleotide were measured with UV spectra (JASCO J-40A). Solvents used were water, Kolthoff buffer (50 n&I) and phosphate buffer (50 n&I), and temperatures were controlled at 20 and 5°C. UV spectra were measured at 1 h and 6 days after mixing of the synthetic polymer and the polynucleotide solutions. Absorbance of the solution was measured at 260 nm. A digital polarimeter used was JASCO DIP-370. ‘H-NMR spectra were recorded with JEOL GSX270. 3. Results and discussion 3.1. Preparation of the polymers Grafting of the nucleic acid bases onto synthetic polymers has been studied for various derivatives and various polymers [2,4]. The most satisfactory result was obtained by the reaction of the carboxylic derivatives of nucleic acid base and polyethyleneimine [6]. Therefore, the polyethyleneimine derivatives of adenine with serine were prepared according to Schemes 1 and 2. Using the activated ester method, it was not necessary to protect the 6-amino group of adenine. Since the reaction of the carboxyethyl derivative (5) to the activated ester (7) with pentachlorophenyl trichloroacetate (TCA-PCP) is an equilibrium reaction, it is necessary to use the solvent such as chloroform as precipitant in order to force the equilibrium to the product 7. Low solubility of the adenine derivatives 5 in chloroform was improved by preparation of tetrabutylammonium salt 6. IR spectrum at 1780 cm-’ indicated that the precipitated product from chloroform was the activated ester 7. The activated ester 7 was stable in solid state but was unstable in solution. Change of spectra with time was observed by ‘H-NMR spectra in dimethyl sulfoxide-4 (DMSO-&). The 6-amino-protected adenine derivative, however, was stable in solution (dimethyl sulfoxide). Therefore, the instability of 7 in solution is due to the activity of adenine base (N-3 of adenine) as intramolecular catalyst. L-Serine derivative of carboxyethyl adenine 9, and also D-serine derivative, were successfully prepared
E: Inaki et al./Reactive
192
& Functional Polymers 37 (1998) 189-198
Ethyl Acrylate NaOH, HCI t+H2 bH2COOH
kH2 &HP-C00 - TBA+ 6
5
4
+
tfH2
B
HCI H2N-GHC-0CH3 8
+2
NaOH, HCI
*
OH
kH2 bH2 O+ y
YH
H-N,;,CH~ 9: Ade-L-Ser-OH
&OOH
Scheme 1.
WGkNWOWk
7H2
O$
H
?H
CHC13
DCC, PCP
FH2 *
FH2
O’$
CH2
kH2
7H2
CHC13
H ?H
-
O+
I
I tOOH
COOH
H
?H
H-N+cH~
H-N+cH,
H-N+cH~
Loo
11
10
-i3
,-
9: Ade-L-Ser-OH
PEI , lmidazole
HCI *
DMSO ,5O"c
kH2 &HP
CH2 +42
O+
H PH 1 H-N,i,CH2
o’y
j
1: PEI-L-Ser-Ade Scheme 2.
PH
A=0
A=0 +CH2-I(S-CH2
,,
H-N,&.\\\cH2
+H2-I(I-CH2+
2: PEI-D-Ser-Ade
Cl5
Z Inaki et al. /Reactive
by the reaction of the activated ester 7 with L-serine methyl ester hydrochloride 8 in the presence of triethylamine (Scheme 2). After hydrolysis of the ester group, 6-amino group of 9 was protected with DMM (dimethylamino methyl) group to increase the solubility in organic solvent. Reaction of the serine derivatives of adenine with polyethyleneimine was carried out using the activated ester 11. The DMM protecting group in the polymer was removed with hydrochloric acid, followed by purification through reprecipitation. The contents of adenine unit in the obtained polymer were determined by UV spectra [4] to be 91 unit mol% for the L-serine derivative 1 and 95% for the D-serine derivative 2. 3.2. Interaction with polynucleotide in pH 7 at 20°C Formation of the polymer complex between polynucleotides is usually confirmed by UV spectra as hypochromicity that is caused by interaction of nucleic acid bases as chromophores. For the formation of polymer complex, freely orientated nucleic acid bases in the polynucleotide should change their orientation of nucleoside to the same direction, which causes stacking of the bases to give hypochromicity. Mixing curves in water (pH 7, 20°C) for L- or D-Serine derivatives with the complementary polynucleotide poly U are shown in Fig. 3, where absorbenties at 260 nm in UV spectra were plotted against nu-
cleic acid base molar ratio. Hypochromicity should be observed in the case where the polymer complex is formed between these polymers by hydrogen bonding interaction. Fig. 3, however, shows additivity of absorbance, that is, no hypochromicity was observed, suggesting absence of the hydrogen bonding interaction between adenine and uracil bases such as shown in Fig. 2. Interaction was also studied at 20°C in Kolthoff buffer (50 mM, KH2PO4, Na2B407) and in phosphate buffer (50 mM, KH2PO4, KzI-IPOd), but the hypochromicity was not observed under these conditions. For this reason, it is considered that a stable intramolecular interaction of adenine bases inhibited the intermolecular interaction of adenine units with complementary uracil units in polynucleotide. The intramolecular interaction may be the hydrophobic and/or hydrogen bonding interactions between the adenine units. 3.3. Interaction in pH 7 at 5°C Hydrogen bonding interaction between polynucleotides is known to be stable at low temperature. Fig. 4 shows the mixing curves at 5°C in water (pH 7) without buffer. Hypochromicity (about 11%) was observed for the D-serine derivative (Fig. 4b) at the base ratio of 2 : 1 (adenine : uracil), though no hypochromicity was observed for the L-serine derivative (Fig. 4a). These results suggest that the
b: PEbD-Ser-Ade
a: PEI-L-Ser-Ade ‘I
‘I 8
0.9
z 8
O.&d
z
193
& Functional Polymers 37 (1998) 189-198
-
3
e 8 2
after
-
0.7 -
.+...._ 0.6
,
I
0
0.5
after
lhr
-D-
. ......+.. ....
6 days
Unit Mol Fraction of Poly (U)
0.6 1
after
1 hr
after
6 days
I 0
0.5
Unit Mol Fraction of Poly (U)
Fig. 3. Mixing curves for PEI-L-Ser-Ade (a) and PEGD-Ser-Ade (b) with poly U in neutral aqueous solution at 20°C.
1
194
I: Inaki et al. /Reactive
& Functional Polymers 37 (1998) 189-198
a: PEI-L-Ser-Ade
$
b: PEI-D-Ser-Ade
0.9
s
e 8
0.8 e
2
after
lhr
0.7
V.,
0
0.5
1
Unit Mol Fraction of Poly (U)
,
0
0:5
;
Unit Mol Fraction of Poly (U)
Fig. 4. Mixing curves for PEI-L-Ser-Ade (a) and PEI-D,-Ser.-Ade (b) with poly U in neutral aqueous solution at 5°C.
chiral spacer group of serine influences the polymer complex formation with polynucleotide. In Fig. 4b, time dependency of the interaction was observed: hypochromicity increased for 6 days after mixing. The time course of the interaction of PEI-D-Ser-Ade with poly U may be illustrated as Fig. 5. For 1 h after mixing of the polymer solutions, most of adenine bases in PEI-D-Ser-Ade are aggregated, and only small amounts of free adenine bases can form hydrogen bonding with uracil in poly U. The loose polymer complex like this may be unstable at 20°C. This loose polymer complex becomes a tight complex during 6 days by conformational change of PEI-D-Ser-Ade to give high hypochromicity value. The highest hypochromicity was observed at the base ratio of 2 : 1 (adenine : uracil), suggesting that about half of the adenine bases was in base pairing and the other half of the adenine bases was still in aggregation. In buffer solutions, however, hypochromicity was not observed even at 5°C. Intramolecular association of adenine bases may be accelerated by inorganic salt, which is known as salting-out. 3.4. Inter-action with polynucleotide in acidic solution (pH 3) at 5°C Solubility of adenine derivatives in water at neutral pH is generally very low. In acidic solution,
however, the adenine derivatives are soluble in water, because pK, value is around 4.5. At pH 3, the polymer having adenine is in extended conformation by electrostatic repulsion of the protonated adenine units. Therefore, no intramolecular association of adenine bases may be occurred at pH 3. Fig. 6b shows mixing curve for PEI-D-Ser-Ade with poly U at pH 3 and 5°C. High hypochromicity of 77% was observed at 1 : 1 base ratio immediately after mixing of the polymer solutions and also overnight. In Fig. 7, the formations of the polymer complexes at pH 3 are illustrated. At pH 3, PEI-D-Ser-Ade may exist in extended form by electrostatic repulsion between protonated adenine bases. The protonated PEI-D-Ser-Ade can easily approach to the anionic polymer of poly U by the electrostatic attractive forces. Therefore, both the extended conformations of PEI-D-Ser-Ade and the electrostatic attractive forces caused an acceleration of the base pairing between adenine and uracil. In the case of the L-serine derivative (Fig. 6a), the mixing curve shows low hypochromicity (43%) at the base ratio of 2 : 1 and small time dependency. Low hypochromicity and abnormal stoichiometry (2 : 1, adenine : uracil) for PEI-L-Ser-Ade indicated that only half of adenine bases formed hydrogen bondings. The dependence of interaction on chirality of the spacer group (L-serine) is illustrated in Fig. 7a. The L-serine polymer has the same advantageous
I
Poly
u PEI-D-Ser-Ade
I
PECD-Ser-Ade
Tight Complex (after 6 days)
d\ Poly u
-p-o_
0% I
Hd
Fig. 5. Illustrated time-course for polymer complex formation of PEGD-Ser-Ade with poly U.
Loose Complex (after 1 hr)
0,
‘Ip’o-
O-
Interaction
d
I: Inaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
196
a: PEI-L-Ser-Ade
b: PECD-Ser-Ade
1.2
‘.*, E c
1
% N z
0.8
8 c
0.6
B $ $
after 1 hr
-
0.4
0.2
-
lhr
..... I ..I
0 0
1
0.5
i 0
Unit Mel Fraction of Poly (U)
(>
.... ...
over night 1
0.5
Unit Mol Fraction of Poly (U)
Fig. 6. Mixing curves for PEI-L-Ser-Ade (a) and PEI-D-Ser-Ade (b) with poly U in acidic aqueous solution (pH 3) at 5°C. Table 1 Hypochromicity at 260 nm and stoichiometry of the polymer complex PEI-L-Ser-Ade Temperature: 5°C
PEI-D-Ser-Ade 20°C
5°C
20°C
Pure water (pH = 7)
Hypochromicity Stoichiometry
0%
0%
11% 2:l
0%
Kolthoff buffer
Hypochromicity Stoichiometry
0%
0%
2% 5:l
0%
Phosphate buffer
Hypochromicity Stoichiometry
Pure water (pH = 3)
Hypochromicity Stoichiometry
0%
43% 2:l
factors of the extended conformation and the electrostatic attractive forces as the D-serine polymer. In order to form the polymer complex, however, the adenine bases in the polymer should be situated in the same direction along the polyethyleneimine chain toward the uracil base of poly U. Since the righthanded helical structure is favored for the chiral poly U, the polymer complex may be the right-handed double helical structure. Therefore, the conformation may be allowed for the D-serine spacer (Fig. 7b), but not for L-serine spacer (Fig. 7a) because of the steric hindrance. The steric hindrance may be the repulsion between the hydroxymethyl units of L-serine,
0%
77% 1:l
and may occur only when the polymers exist in the right-handed double helical structure. As the summary shows in Table 1, the polyethyleneimine derivative of adenine having D-serine as a spacer (PEI-D-Ser-Ade) forms the stable polymer complex with poly U at base stoichiometry of 1 : 1 in acidic solution (pH 3) at 5°C. The L-serine derivative (PEI-L-Ser-Ade), however, forms less stable polymer complex at 2 : 1 base ratio under the same condition. These polymer complex is less stable in neutral pH solution, and no polymer complex is formed in buffer solution. Effect of the chiral spacer on the polymer complex formation was concluded to be caused by
Y. lnaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
197
198
Z Inaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
steric repulsion of the serine spacer units in the righthanded double helical structure. References [l] P. wittung, l?E. Nielsen, 0. Buchardt, M. Egholm, B. Norden, Nature 368 (1994) 561. [2] Y. Inaki, Prog. Polym. Sci. 17 (1992) 515.
[3] Y. Inaki, T. Wada, in: M. Kamachi, A. Nakamura (Eds.), New Macromolecular Architecture and Functions, SpringerVerlag, Berlin, 1996, p. 80. [4] K. Takemoto, Y. Inaki, in: K. Takemoto, Y. Inaki, R.M. Ottenbrite (Eds.), Functional Monomers and Polymers, Marcel Dekker, New York, NY, 1987, p. 149. [5] T. Saegusa, H. Ikeda, H. Fujii, Polymer. J. 3 (1972) 35. [6] C.G. Overberger, Y. Inaki, J. Polymer Sci. Polymer Chem. Ed. 17 (1979) 1739.
ELSEVIER
Reactive &
FUNCiONAL POLYMERS
Functional Polymers 37 (1998) 189-198
Base-specific interaction of polymers containing adenine: effect of chiral spacer on the interaction with polynucleotide Yoshiaki
Inaki *, Shigeki
Kamo, Mikiji Miyata
Department of Materials and Life Science, Osaka University, Yamuahoka 2-1, Suita, Osaka 565, Japan
Received 15 June 1997; revised version received 25 July 1997; accepted 26 July 1997
Abstract Polyethyleneimine derivatives of adenine with L- and D-serine as spacers were prepared. The polymer having D-Serhe spacer 2 gave a stable polymer complex with poly(uridylic acid) (poly U), but the polymer having L-serine spacer 1 gave an unstable polymer complex. The reason was concluded to be caused by the steric repulsion of L-serine units along the polymer chain when the polymer formed polymer complex with poly U. 0 1998 Elsevier Science B.V. All rights reserved. Keywords:
1.
Adenine; L-Serine; D-Serine; Polyethyleneimine;
Polynucleotide; Poly U; Interaction
Introduction
In recent years, considerable attention has been directed towards synthetic nucleic acid analogues in the hope of discovering new and more effective antisense compounds [ 11. Synthetic polymers containing nucleic acid bases have been prepared, and the interactions of these polymers were studied with DNA and RNA [2]. These synthetic polymers without ribose nor phosphodiester were found to interact with polynucleotides, DNA and RNA. One of these polymers is the polyethyleneimine derivative containing nucleic acid bases with DL-homoserine as spacer units [3]. These water-soluble polymers were found to interact with polynucleotide by specific hydrogen bonding between complementary nucleic acid bases. Since DNA is a chiral polymer, the chit-al unit in the synthetic nucleic acid model should influence *Correspondingauthor. Tel.: +81 (6) 879-7405, Fax: +81 (6) 879-7404, E-mail: [email protected] 1381-5148/98/$19.00 0 1998 Elsevier Science B.V. AU rights reserved PII S1381-5148(97)00118-l
the complex formation with DNA. This paper deals with the preparation of polyethyleneimine derivatives containing adenine with chiral spacer units 1 (PEI-L-Ser-Ade) and 2 (PEI-D-Ser-Ade) (Fig. l), and interaction of these polymers with poly(uridylic acid) 3 (poly U) to clear the effect of chiral unit on the polymer complex formation (Fig. 2).
9H2 FH2
Czq H VH H-N.&&Hz k=o +CH2k-CHlf
k=o fCH,-Ii-CH+
1: PEI-L-Ser-Ade
2: PEI-D-Ser-Ade
Fig. 1. Polyethyleneimine derivatives of adenine having L- and D-serines.
I! Inaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
AH2
kH2 O:i: H VH H-k,$H2
+
&=o +CH2-ti-CH+
1
3: Poly u
Polymer Complex
Fig. 2. Formation of polymer complex between the adenine-containing polymer and poly U.
2. Experimental 2.1. Preparation of polymers 2.1.1. Pentachlorophenyl3-(adenin-9-yl)propionate 7
To the suspension in water of 3-(adenin-9-yl)propionic acid 5 [4] (2.1 g, 10 mmol) prepared from adenine 4 and ethyl acrylate, NaOH (0.96 g, 24 mmol) and tetrabutylammonium hydrogen sulfate (3.4 g, 10 mmol) were added to give a clear solution. After evaporation of water, excess methanol was added to the residue, and insoluble NazS04 was removed by filtration. The filtrate was evaporated and the residue was extracted with chloroform. After evaporation of chloroform, yellowish crystal was washed with diethyl ether to give the tetrabutylammonium (TBA) salt 6 as white crystal. The obtained ammonium salt 6 was dissolved in chloroform (100 ml) and pentachlorophenyl trichloroacetate (PCP-TCA) (4.94 g, 12 mmol) was added to the solution. The product 7 was precipitated after 10 min stirring. Yield: 3.80 g (83.5%). IR (KBr, cm-‘): 3320, 3150, 1780, 1670, 1600, 1480, 1380, 1360, 1320, 1110,780,760,720, 650. ‘H NMR (270 MHz, DMSO-4) S 8.31 (lH, s, 2(8)-H), 8.16 (lH, S, 8(2)-H), 7.23 (2H, S, 6-NH2), 4.55 (2H, t, N-CH2), 3.54 (2H, t, CH2-CO). 2.1.2. 3-(Adenin-9-yl)propionyl-L-serine 9 L-Serine methyl ester hydrochloride 8 (1.5 g, 9.6 mmol) was dissolved in WV-dimethylformamide (80 ml) and dried under reduced pressure at room temperature. The activated ester 7 (3.6 g, 8.0 mmol) and triethylamine (1.4 ml, 9.6 mmol) were added to
the solution and the mixture was stirred overnight at room temperature. The reaction was followed by TLC and IR spectra. After the completion of the reaction, the solvent was removed under reduced pressure, and the residue was recrystallized with ethanol to give 3-(adenin-9-yl)propionyl-L-se&e methyl ester as white crystals. Yield: 1.9 g (85%). TLC: Rf = 0.15 (CHCls : MeOH = 5 : 1). IR (KBr, cm-‘): 3300, 3100, 1740, 1690, 1650, 1610, 1580, 1550, 1420,1340, 1220,1160,1140,1060, 1040,950,680, 640. ‘H NMR (270 MHz, DMSO-&) S 8.20 (lH, d, CO-NH), 8.13 (lH, s, 2(8)-H), 8.00 (lH, s, 8(2)-H), 7.18 (2H, S, 6-NH& 5.00 (lH, t, OH), 4.34 (3H, m, CH, N-CH2), 3.60 (5H, s and t, 0-CH3, CH2-0), 2.77 (2H, t, CH2-CO). The obtained methyl ester (1.9 g, 6.8 mmol) was hydrolyzed in 1 M NaOH (8 ml) at 0°C. After the reaction, pH was adjusted to 4 with 6 M HCl to give 3-(adenin-9-yl)propionyl-L-serine 9 as white precipitate. Yield: 1.6 g (87%). TLC: Rf = 0.13 (l-BuOH : CH&OOH : Hz0 = 5 : 2 : 3). [a]g - 1.72 (H20). IR (KBr, cm-‘): 3300, 1690, 1640, 1560, 1510, 1410, 1230, 1080, 1030, 700, 640. ‘H NMR (270 MHz, DMSO-4) S 12.5 (lH, br, COOH), 8.20 (lH, d, CO-NH), 8.14 (lH, s, 2(8)-H), 8.02 (lH, s, 8(2)-H), 7.17 (2H, S, 6-NH2), 5.0 (lH, br, OH), 4.34 (2H, t, N-CH2), 4.26 (1H t, CH), 3.60 (5H, d, CH2-0), 2.77 (2H, t, CH2-CO). 2.1.3. 3-(Adenin-9-yl)propionyl-D-serine This compound was also prepared according to the method used for the L-serine derivative. [a]? +1.74 (H20).
Y. Inaki et al./Reactive
& Functional Polymers 37 (1998) 189-198
2.1.4. Poly-{N-[3-(adenin-9-yl)propionyl]-L-se& ethyleneimine} (1:PEI-L-Ser-Ade) Ade-L-Ser-OH 9 (0.80 g, 3.0 mmol) in chloroform (30 ml) was added to N,N-dimethylformamide dimetbyl acetal (DMM: 4.0 ml, 30 mmol) and stirred overnight at room temperature to give a clear solution. After evaporation of the solvent, chloroform was added to the residue to give oily product 10. To the oily product, chloroform (30 ml), pentachlorophenol (PCP) (2.5 g, 9.5 mmol) and N,N-dicyclohexylcarbodiimide (DCC) (0.62 g, 3.0 n-m-101)were added and stirred for 2 h at 0°C and for 12 h at room temperature. After the precipitated dicyclohexyl urea was removed by filtration, the filtrate was evaporated to give solid product, which was washed with methanol to give the activated ester 11. Yield: 0.79 g (43%). IR (KBr, cm-‘): 1790, 1640, 1580. The product was used for the next reaction without purification. The activated ester 11 (0.18 g, 0.29 mmol) in dry dimethyl sulfoxide (5 ml) was added polyethyleneimine (PEI, 0.020 g, 0.29 mmol) and imidazole (0.020 g, 0.29 mmol), and was stirred for 80 h at 60°C to give a clear solution. Polyethyleneimine (M.W.: 22000) used here was prepared from poly(2-ethyl-2-oxazoline) (Dow Chemical Japan) by hydrolysis [5]. The mixture was poured into excess acetone to give the polymer as precipitates. In order to remove the DMM protecting group, the polymer in water (5 ml) was reacted with 6 M HCl (0.2 ml) for 9 h at room temperature. The mixture was poured into excess acetone to give the polymer as precipitates, quantitatively. TLC: Rf = 0.00 (BuOH : CHsCOOH : Hz0 = 5 : 2 : 3). Degree of substitution was obtained by UV spectra to be 91 unit mol%. IR (KBr, cm-‘): 1640, 1540,1480, 1420,1250,1200,1060,800,720,650. ‘HNMR (270 MHz, DMSO-4) 6 8.20 (lH, br, CO-NH), 8.13 (lH, s, 2(8)-H), 8.08 (lH, s, 8(2)-H), 7.20 (2H, s, 6-NH& 4.33 (7H, br, N-CH2, CH, N-CH2CH2-N), 3.03.4 (4H, br, CHz-0, CHz-CO). 2.1.5. Poly-{N-[3-(adenin-9-yl)propionyl]-D-seryl ethyleneimine) (2: PEI-D-Ser-Ade) The same method as for the L-serine derivative was used for preparation of the D-Serine derivative. Degree of substitution was 95 unit mol%.
191
2.2. Interaction of the adenine-containing polymer with polynucleotides Interactions of the polyetbyleneimine derivatives with polynucleotide were measured with UV spectra (JASCO J-40A). Solvents used were water, Kolthoff buffer (50 n&I) and phosphate buffer (50 n&I), and temperatures were controlled at 20 and 5°C. UV spectra were measured at 1 h and 6 days after mixing of the synthetic polymer and the polynucleotide solutions. Absorbance of the solution was measured at 260 nm. A digital polarimeter used was JASCO DIP-370. ‘H-NMR spectra were recorded with JEOL GSX270. 3. Results and discussion 3.1. Preparation of the polymers Grafting of the nucleic acid bases onto synthetic polymers has been studied for various derivatives and various polymers [2,4]. The most satisfactory result was obtained by the reaction of the carboxylic derivatives of nucleic acid base and polyethyleneimine [6]. Therefore, the polyethyleneimine derivatives of adenine with serine were prepared according to Schemes 1 and 2. Using the activated ester method, it was not necessary to protect the 6-amino group of adenine. Since the reaction of the carboxyethyl derivative (5) to the activated ester (7) with pentachlorophenyl trichloroacetate (TCA-PCP) is an equilibrium reaction, it is necessary to use the solvent such as chloroform as precipitant in order to force the equilibrium to the product 7. Low solubility of the adenine derivatives 5 in chloroform was improved by preparation of tetrabutylammonium salt 6. IR spectrum at 1780 cm-’ indicated that the precipitated product from chloroform was the activated ester 7. The activated ester 7 was stable in solid state but was unstable in solution. Change of spectra with time was observed by ‘H-NMR spectra in dimethyl sulfoxide-4 (DMSO-&). The 6-amino-protected adenine derivative, however, was stable in solution (dimethyl sulfoxide). Therefore, the instability of 7 in solution is due to the activity of adenine base (N-3 of adenine) as intramolecular catalyst. L-Serine derivative of carboxyethyl adenine 9, and also D-serine derivative, were successfully prepared
E: Inaki et al./Reactive
192
& Functional Polymers 37 (1998) 189-198
Ethyl Acrylate NaOH, HCI t+H2 bH2COOH
kH2 &HP-C00 - TBA+ 6
5
4
+
tfH2
B
HCI H2N-GHC-0CH3 8
+2
NaOH, HCI
*
OH
kH2 bH2 O+ y
YH
H-N,;,CH~ 9: Ade-L-Ser-OH
&OOH
Scheme 1.
WGkNWOWk
7H2
O$
H
?H
CHC13
DCC, PCP
FH2 *
FH2
O’$
CH2
kH2
7H2
CHC13
H ?H
-
O+
I
I tOOH
COOH
H
?H
H-N+cH~
H-N+cH,
H-N+cH~
Loo
11
10
-i3
,-
9: Ade-L-Ser-OH
PEI , lmidazole
HCI *
DMSO ,5O"c
kH2 &HP
CH2 +42
O+
H PH 1 H-N,i,CH2
o’y
j
1: PEI-L-Ser-Ade Scheme 2.
PH
A=0
A=0 +CH2-I(S-CH2
,,
H-N,&.\\\cH2
+H2-I(I-CH2+
2: PEI-D-Ser-Ade
Cl5
Z Inaki et al. /Reactive
by the reaction of the activated ester 7 with L-serine methyl ester hydrochloride 8 in the presence of triethylamine (Scheme 2). After hydrolysis of the ester group, 6-amino group of 9 was protected with DMM (dimethylamino methyl) group to increase the solubility in organic solvent. Reaction of the serine derivatives of adenine with polyethyleneimine was carried out using the activated ester 11. The DMM protecting group in the polymer was removed with hydrochloric acid, followed by purification through reprecipitation. The contents of adenine unit in the obtained polymer were determined by UV spectra [4] to be 91 unit mol% for the L-serine derivative 1 and 95% for the D-serine derivative 2. 3.2. Interaction with polynucleotide in pH 7 at 20°C Formation of the polymer complex between polynucleotides is usually confirmed by UV spectra as hypochromicity that is caused by interaction of nucleic acid bases as chromophores. For the formation of polymer complex, freely orientated nucleic acid bases in the polynucleotide should change their orientation of nucleoside to the same direction, which causes stacking of the bases to give hypochromicity. Mixing curves in water (pH 7, 20°C) for L- or D-Serine derivatives with the complementary polynucleotide poly U are shown in Fig. 3, where absorbenties at 260 nm in UV spectra were plotted against nu-
cleic acid base molar ratio. Hypochromicity should be observed in the case where the polymer complex is formed between these polymers by hydrogen bonding interaction. Fig. 3, however, shows additivity of absorbance, that is, no hypochromicity was observed, suggesting absence of the hydrogen bonding interaction between adenine and uracil bases such as shown in Fig. 2. Interaction was also studied at 20°C in Kolthoff buffer (50 mM, KH2PO4, Na2B407) and in phosphate buffer (50 mM, KH2PO4, KzI-IPOd), but the hypochromicity was not observed under these conditions. For this reason, it is considered that a stable intramolecular interaction of adenine bases inhibited the intermolecular interaction of adenine units with complementary uracil units in polynucleotide. The intramolecular interaction may be the hydrophobic and/or hydrogen bonding interactions between the adenine units. 3.3. Interaction in pH 7 at 5°C Hydrogen bonding interaction between polynucleotides is known to be stable at low temperature. Fig. 4 shows the mixing curves at 5°C in water (pH 7) without buffer. Hypochromicity (about 11%) was observed for the D-serine derivative (Fig. 4b) at the base ratio of 2 : 1 (adenine : uracil), though no hypochromicity was observed for the L-serine derivative (Fig. 4a). These results suggest that the
b: PEbD-Ser-Ade
a: PEI-L-Ser-Ade ‘I
‘I 8
0.9
z 8
O.&d
z
193
& Functional Polymers 37 (1998) 189-198
-
3
e 8 2
after
-
0.7 -
.+...._ 0.6
,
I
0
0.5
after
lhr
-D-
. ......+.. ....
6 days
Unit Mol Fraction of Poly (U)
0.6 1
after
1 hr
after
6 days
I 0
0.5
Unit Mol Fraction of Poly (U)
Fig. 3. Mixing curves for PEI-L-Ser-Ade (a) and PEGD-Ser-Ade (b) with poly U in neutral aqueous solution at 20°C.
1
194
I: Inaki et al. /Reactive
& Functional Polymers 37 (1998) 189-198
a: PEI-L-Ser-Ade
$
b: PEI-D-Ser-Ade
0.9
s
e 8
0.8 e
2
after
lhr
0.7
V.,
0
0.5
1
Unit Mol Fraction of Poly (U)
,
0
0:5
;
Unit Mol Fraction of Poly (U)
Fig. 4. Mixing curves for PEI-L-Ser-Ade (a) and PEI-D,-Ser.-Ade (b) with poly U in neutral aqueous solution at 5°C.
chiral spacer group of serine influences the polymer complex formation with polynucleotide. In Fig. 4b, time dependency of the interaction was observed: hypochromicity increased for 6 days after mixing. The time course of the interaction of PEI-D-Ser-Ade with poly U may be illustrated as Fig. 5. For 1 h after mixing of the polymer solutions, most of adenine bases in PEI-D-Ser-Ade are aggregated, and only small amounts of free adenine bases can form hydrogen bonding with uracil in poly U. The loose polymer complex like this may be unstable at 20°C. This loose polymer complex becomes a tight complex during 6 days by conformational change of PEI-D-Ser-Ade to give high hypochromicity value. The highest hypochromicity was observed at the base ratio of 2 : 1 (adenine : uracil), suggesting that about half of the adenine bases was in base pairing and the other half of the adenine bases was still in aggregation. In buffer solutions, however, hypochromicity was not observed even at 5°C. Intramolecular association of adenine bases may be accelerated by inorganic salt, which is known as salting-out. 3.4. Inter-action with polynucleotide in acidic solution (pH 3) at 5°C Solubility of adenine derivatives in water at neutral pH is generally very low. In acidic solution,
however, the adenine derivatives are soluble in water, because pK, value is around 4.5. At pH 3, the polymer having adenine is in extended conformation by electrostatic repulsion of the protonated adenine units. Therefore, no intramolecular association of adenine bases may be occurred at pH 3. Fig. 6b shows mixing curve for PEI-D-Ser-Ade with poly U at pH 3 and 5°C. High hypochromicity of 77% was observed at 1 : 1 base ratio immediately after mixing of the polymer solutions and also overnight. In Fig. 7, the formations of the polymer complexes at pH 3 are illustrated. At pH 3, PEI-D-Ser-Ade may exist in extended form by electrostatic repulsion between protonated adenine bases. The protonated PEI-D-Ser-Ade can easily approach to the anionic polymer of poly U by the electrostatic attractive forces. Therefore, both the extended conformations of PEI-D-Ser-Ade and the electrostatic attractive forces caused an acceleration of the base pairing between adenine and uracil. In the case of the L-serine derivative (Fig. 6a), the mixing curve shows low hypochromicity (43%) at the base ratio of 2 : 1 and small time dependency. Low hypochromicity and abnormal stoichiometry (2 : 1, adenine : uracil) for PEI-L-Ser-Ade indicated that only half of adenine bases formed hydrogen bondings. The dependence of interaction on chirality of the spacer group (L-serine) is illustrated in Fig. 7a. The L-serine polymer has the same advantageous
I
Poly
u PEI-D-Ser-Ade
I
PECD-Ser-Ade
Tight Complex (after 6 days)
d\ Poly u
-p-o_
0% I
Hd
Fig. 5. Illustrated time-course for polymer complex formation of PEGD-Ser-Ade with poly U.
Loose Complex (after 1 hr)
0,
‘Ip’o-
O-
Interaction
d
I: Inaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
196
a: PEI-L-Ser-Ade
b: PECD-Ser-Ade
1.2
‘.*, E c
1
% N z
0.8
8 c
0.6
B $ $
after 1 hr
-
0.4
0.2
-
lhr
..... I ..I
0 0
1
0.5
i 0
Unit Mel Fraction of Poly (U)
(>
.... ...
over night 1
0.5
Unit Mol Fraction of Poly (U)
Fig. 6. Mixing curves for PEI-L-Ser-Ade (a) and PEI-D-Ser-Ade (b) with poly U in acidic aqueous solution (pH 3) at 5°C. Table 1 Hypochromicity at 260 nm and stoichiometry of the polymer complex PEI-L-Ser-Ade Temperature: 5°C
PEI-D-Ser-Ade 20°C
5°C
20°C
Pure water (pH = 7)
Hypochromicity Stoichiometry
0%
0%
11% 2:l
0%
Kolthoff buffer
Hypochromicity Stoichiometry
0%
0%
2% 5:l
0%
Phosphate buffer
Hypochromicity Stoichiometry
Pure water (pH = 3)
Hypochromicity Stoichiometry
0%
43% 2:l
factors of the extended conformation and the electrostatic attractive forces as the D-serine polymer. In order to form the polymer complex, however, the adenine bases in the polymer should be situated in the same direction along the polyethyleneimine chain toward the uracil base of poly U. Since the righthanded helical structure is favored for the chiral poly U, the polymer complex may be the right-handed double helical structure. Therefore, the conformation may be allowed for the D-serine spacer (Fig. 7b), but not for L-serine spacer (Fig. 7a) because of the steric hindrance. The steric hindrance may be the repulsion between the hydroxymethyl units of L-serine,
0%
77% 1:l
and may occur only when the polymers exist in the right-handed double helical structure. As the summary shows in Table 1, the polyethyleneimine derivative of adenine having D-serine as a spacer (PEI-D-Ser-Ade) forms the stable polymer complex with poly U at base stoichiometry of 1 : 1 in acidic solution (pH 3) at 5°C. The L-serine derivative (PEI-L-Ser-Ade), however, forms less stable polymer complex at 2 : 1 base ratio under the same condition. These polymer complex is less stable in neutral pH solution, and no polymer complex is formed in buffer solution. Effect of the chiral spacer on the polymer complex formation was concluded to be caused by
Y. lnaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
197
198
Z Inaki et al. /Reactive & Functional Polymers 37 (1998) 189-198
steric repulsion of the serine spacer units in the righthanded double helical structure. References [l] P. wittung, l?E. Nielsen, 0. Buchardt, M. Egholm, B. Norden, Nature 368 (1994) 561. [2] Y. Inaki, Prog. Polym. Sci. 17 (1992) 515.
[3] Y. Inaki, T. Wada, in: M. Kamachi, A. Nakamura (Eds.), New Macromolecular Architecture and Functions, SpringerVerlag, Berlin, 1996, p. 80. [4] K. Takemoto, Y. Inaki, in: K. Takemoto, Y. Inaki, R.M. Ottenbrite (Eds.), Functional Monomers and Polymers, Marcel Dekker, New York, NY, 1987, p. 149. [5] T. Saegusa, H. Ikeda, H. Fujii, Polymer. J. 3 (1972) 35. [6] C.G. Overberger, Y. Inaki, J. Polymer Sci. Polymer Chem. Ed. 17 (1979) 1739.