TY - JOUR
AU - Kokotos,, George
AB - At present, great interest exists in relatively small molecular weight compounds that can interact with DNA in a site-specific manner. A rapid and successful way of monitoring such interactions was achieved by reversed-phase HPLC based on DNA peak size exclusion (1). Phenylalanine (Phe), a biogenic small molecule and an essential amino acid in humans, has been a diagnostic tool for detecting phenylketonouria, a metabolic inherited disease. On the basis of the charge and shape of Phe, we tried to measure in vitro the possible interaction of Phe with DNA by using calf thymus DNA. We also attempted a preliminary approach regarding a Phe mechanism of action on DNA by using compounds structurally related to the known antitumor antibiotic doxorubicin. DNA binding was evaluated according to the method of Karikas et al. (1). Calf thymus DNA was purchased from Sigma Chemical Co., and doxorubicin was from Pharmacia. HPLC-grade solvents were obtained from Lab Scan. A Hewlett-Packard HPLC series 1050 and a Lichrospher RP-18 (250 × 4 mm, 5 μm) were used. DNA solutions (0.1 g/L) were prepared in H2O and kept at 4 °C. Doxorubicin (0.5 g/L) was used as a typical intercalating agent with major binding (100%) capability. l-Phe (Serva) and molecules related to l-Phe structure such as l-alanine (Serva), benzyloxycarbonyl-l-Phe, benzyloxycarbonyl-asparagine, as well as C6H5CH2CH(NH2)CH2NH2 · 2HCl, were dissolved in H2O (each at 0.5 g/L). The column was equilibrated with a H2O:methanol (80:20, by vol) solution. Test samples and DNA solutions at an equivolume ratio were then introduced into the sample loop (20 μL) without incubation. The flow rate was maintained at 1 mL/min, and the free DNA eluted from the column in ∼1 min. After the appearance of DNA peak, the column was later washed with methanol for 20 min to elute the sample mixture. DNA binding is expressed as a percentage of DNA peak exclusion. All samples were tested in triplicate. Phe and doxorubicin were tested at three final concentrations (0.1, 0.25, and 0.5 g/L) vs DNA at 0.05 g/L. The results of our experiments are presented in Tables 1 and 2. Table 1. Effect of doxorubicin (Doxo) and l-Phe on DNA. Compounds (g/L) . % DNA peak size exclusion . DNA (0.05)+ Doxo (0.1) 45.8 ± 5.41 DNA (0.05)+ Doxo (0.25) 100 DNA (0.05)+ Doxo (0.5) 100 DNA (0.05)+ Phe (0.1) 9.3 ± 3.41 DNA (0.05)+ Phe (0.25) 31.6 ± 8.51 DNA (0.05)+ Phe (0.5) 62.5 ± 9.11 Compounds (g/L) . % DNA peak size exclusion . DNA (0.05)+ Doxo (0.1) 45.8 ± 5.41 DNA (0.05)+ Doxo (0.25) 100 DNA (0.05)+ Doxo (0.5) 100 DNA (0.05)+ Phe (0.1) 9.3 ± 3.41 DNA (0.05)+ Phe (0.25) 31.6 ± 8.51 DNA (0.05)+ Phe (0.5) 62.5 ± 9.11 1 Mean ± SD (n = 3). Open in new tab Table 1. Effect of doxorubicin (Doxo) and l-Phe on DNA. Compounds (g/L) . % DNA peak size exclusion . DNA (0.05)+ Doxo (0.1) 45.8 ± 5.41 DNA (0.05)+ Doxo (0.25) 100 DNA (0.05)+ Doxo (0.5) 100 DNA (0.05)+ Phe (0.1) 9.3 ± 3.41 DNA (0.05)+ Phe (0.25) 31.6 ± 8.51 DNA (0.05)+ Phe (0.5) 62.5 ± 9.11 Compounds (g/L) . % DNA peak size exclusion . DNA (0.05)+ Doxo (0.1) 45.8 ± 5.41 DNA (0.05)+ Doxo (0.25) 100 DNA (0.05)+ Doxo (0.5) 100 DNA (0.05)+ Phe (0.1) 9.3 ± 3.41 DNA (0.05)+ Phe (0.25) 31.6 ± 8.51 DNA (0.05)+ Phe (0.5) 62.5 ± 9.11 1 Mean ± SD (n = 3). Open in new tab Table 2. Effect of Phe and related compounds on DNA. Compounds (0.25 g/L) . % DNA peak size exclusion . 1. Doxorubicin · HCl 100 2. l-Alanine, CH3CH(NH2)COOH 12.3 ± 4.11 3. Benzyloxycarbonyl-l-Phe, C6H5CH2CH- (NHCOOCH2C6H5)COOH 0 4. Benzyloxycarbonyl-Asp, H2NCOCH2CH- (NHCOOCH2C6H5)COOH 0 5. l-Phenylalanine, C6H5CH2CH(NH2)COOH 31.6 ± 8.51 6. C6H5CH2CH(NH2)CH2NH2 · 2HCl 100 Compounds (0.25 g/L) . % DNA peak size exclusion . 1. Doxorubicin · HCl 100 2. l-Alanine, CH3CH(NH2)COOH 12.3 ± 4.11 3. Benzyloxycarbonyl-l-Phe, C6H5CH2CH- (NHCOOCH2C6H5)COOH 0 4. Benzyloxycarbonyl-Asp, H2NCOCH2CH- (NHCOOCH2C6H5)COOH 0 5. l-Phenylalanine, C6H5CH2CH(NH2)COOH 31.6 ± 8.51 6. C6H5CH2CH(NH2)CH2NH2 · 2HCl 100 1 Mean ± SD (n = 3). Compounds 3, 4, and 6 have been synthesized previously according to Kokotos and Constantinou [4]. Open in new tab Table 2. Effect of Phe and related compounds on DNA. Compounds (0.25 g/L) . % DNA peak size exclusion . 1. Doxorubicin · HCl 100 2. l-Alanine, CH3CH(NH2)COOH 12.3 ± 4.11 3. Benzyloxycarbonyl-l-Phe, C6H5CH2CH- (NHCOOCH2C6H5)COOH 0 4. Benzyloxycarbonyl-Asp, H2NCOCH2CH- (NHCOOCH2C6H5)COOH 0 5. l-Phenylalanine, C6H5CH2CH(NH2)COOH 31.6 ± 8.51 6. C6H5CH2CH(NH2)CH2NH2 · 2HCl 100 Compounds (0.25 g/L) . % DNA peak size exclusion . 1. Doxorubicin · HCl 100 2. l-Alanine, CH3CH(NH2)COOH 12.3 ± 4.11 3. Benzyloxycarbonyl-l-Phe, C6H5CH2CH- (NHCOOCH2C6H5)COOH 0 4. Benzyloxycarbonyl-Asp, H2NCOCH2CH- (NHCOOCH2C6H5)COOH 0 5. l-Phenylalanine, C6H5CH2CH(NH2)COOH 31.6 ± 8.51 6. C6H5CH2CH(NH2)CH2NH2 · 2HCl 100 1 Mean ± SD (n = 3). Compounds 3, 4, and 6 have been synthesized previously according to Kokotos and Constantinou [4]. Open in new tab As is well known, the association of cationic polyamines with negatively charged DNA induces important structural changes in DNA. Thus, spermidine and spermine can cause DNA to condense and aggregate, phenomena that have just recently been observed chromatographically (1). The amino acid l-alanine, which does not contain the aromatic ring of the Phe side chain because of the presence of the amino group, exhibited a measurable percentage of DNA peak exclusion. No DNA molecular effect was observed with benzyloxycarbonyl-l-Phe, which carries a protected amino group, and benzyloxycarbonyl-l-asparagine, a molecule structurally related to phenylacetyl-glutamine, a metabolic product of Phe. The Phe derivative, C6H5CH2CH(NH2)CH2NH2 · 2HCl, in which the carboxyl group of Phe has been replaced by an additional amino group, at 0.25 g/L showed a considerable increase of DNA peak exclusion over that caused by Phe (100% and 31.6%, respectively). This increased effect on DNA is directly related to the presence of two amino groups because DNA possesses negative charges. The latter is in full agreement with previous data (1). The potency of the phenomenon attributed to ionic interactions is increased, when the number of amino groups increases (Table 2). Furthermore, the ratio of DNA:ligand determines the potency of the observed DNA peak exclusion (Table 1). Thus, we suggest that interaction of l-Phe with DNA involves its amino group (ionic interaction) as well as its aromatic ring (partial intercalation). This interaction of the aromatic group of Phe with the base-pairs of DNA is reinforced by recent findings of Burley (2), who showed that two Phe residues of TATA box binding protein interact with the T-A basepair by Van der Waals contacts. The observed DNA peak size exclusion in the case of doxorubicin, which contains only one ionized sugar amino group, is additional compared with Phe and related compounds (Table 1). This is probably due to extra binding mechanisms because polyaromatic cations are known to intercalate to DNA by inserting and stacking between the base pairs at the double-helix. Furthermore, hydrogen bonding and strand breakage could also play a substantial role in this molecular multiple interaction (3)(4). We suggest this simple chromatographic technique can serve as a prescreen assay to study DNA interactions with important small molecule biomarkers. References 1 Karikas GA, Constantinou V, Kokotos G. An HPLC method for the measurement of polyamines and lipidic amines binding to DNA. J Liq Chromatogr 1997 ; 20 : 1789 -1796. Crossref Search ADS 2 Burley SK. The TATA box binding protein. Curr Opin Struct Biol 1996 ; 6 : 69 -75. Crossref Search ADS PubMed 3 Silverman RB. The organic chemistry of drug design and drug action 1992 : 242 -243 Academic Press San Diego. . 4 Kokotos G, Constantinou V. Modified amino acids and peptides. J Chem Res 1992 ; (Part 2) : 3117 -3132. © 1998 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Direct Molecular Interaction of Phenylalanine with DNA Measured by Reversed-Phase HPLC
JF - Clinical Chemistry
DO - 10.1093/clinchem/44.1.178
DA - 1998-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/direct-molecular-interaction-of-phenylalanine-with-dna-measured-by-FC8fSIXScO
SP - 178
EP - 179
VL - 44
IS - 1
DP - DeepDyve
ER -