Kellie Hom

PHOTOCHEMICAL ADDITION OF AMINO ACIDS AND PEPTIDES TO DNA

Abstract— The quantum yields for photochemical addition of twenty of the amino acids commonly occurring in proteins to denatured calf thymus DNA have been determined in deoxygenated phosphate buffer at λ 254 nM and pH 7 using a fluorescamine assay technique. Fifteen were found to be reactive, with cysteine, lysine, phenylalanine, tryptophan and tyrosine being the most reactive. Alanine, aspartic acid, glutamic acid, serine and threonine were unreactive. Analogous quantum yields for a series of eighteen peptides of the form glycyl X (X being one of the commonly occurring amino acids) were also determined, along with the corresponding quantum yields for L‐alanyl‐L‐alanine, L‐alanyl‐L‐tryptophan, L‐seryl‐L‐seryl‐L‐serine, L‐threonyl‐L‐threonyl‐L‐threonine, and L‐cystine‐bis‐glycine. All of the peptides were found to be reactive. The modified amino acids Nε‐methyllysine, Nε, Nε, Nε‐trimethyllysine and Nε‐acetyllysine, all occurring in minor amounts in the histone group of chromosomal proteins, were also found to be reactive as was Nα‐acetyllysine. The quantum yields for photoaddition of a selected group of amino acids and peptides to denatured DNA and native DNA are compared. In some cases higher quantum yields for photoaddition to denatured DNA are observed while in other cases the reverse is true. The effect of oxygen on the quantum yields for photoaddition of selected peptides to DNA was examined. While for most systems studied the amount of reaction in aerated systems was less than in deoxygenated systems, in the case of glycyl‐L‐phenylalanine the reverse was true.

Photochemical addition of amino acids and peptides to polyuridylic acid.

Abstract— The quantum yields for photochemical addition of glycine and the L‐amino acids commonly occurring in proteins (excluding proline) to polyuridylic acid have been determined in deoxygenated phosphate buffer at pH 7, using a fluorescamine assay technique. All twenty amino acids were found to be reactive, with cysteine, tryptophan, phenylalanine, tyrosine, arginine, lysine and methionine being the most reactive. The analogous quantum yields for a series of eighteen dipeptides of the form glycyl X (X being one of the commonly occurring amino acids, including proline), of L‐alanyl‐L‐tryptophan, of the tripeptides L‐seryl‐L‐seryl‐L‐serine and L‐threonyl‐L‐threonyl‐L‐threonine, of the tetrapeptide L‐cystine‐bis‐glycine, and of the lysine derivative Nα‐acetyllysine were also measured. All were found to be reactive toward photoaddition to poly U.

Photochemical addition of amino acids and peptides to homopolyribonucleotides of the major DNA bases.

Abstract— The photochemical quantum yields for addition of glycine and the L‐amino acids commonly occurring in proteins (excluding proline) to polyadenylic acid, polycytidylic acid, polyguanylic acid and polyribothymidylic acid have been determined in deoxygenated phosphate buffer at Λ 254 nm and pH 7, using a fluorescamine assay technique. Polyadenylic acid was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine having the highest quantum yields. Polyguanylic acid reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Polycytidylic acid showed reactivity with fifteen amino acids with lysine, phenylalanine, cysteine, tyrosine and arginine having the greatest quantum yields. Polyribothymidylic acid, reactive with fifteen of nineteen amino acids surveyed, showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid.

RING OPENING PHOTOREACTIONS OF 5-BROMOURACIL AND 5-BROMO-2′-DEOXYURIDINE WITH SELECTED ALKYLAMINES

Abstract— Several studies in the literature indicate that histones (lysine rich proteins found associated with DNA in eukaryotic chromatin), as well as poly‐L‐lysine, can be photocross‐linked by ultraviolet (UV) light to DNA in which 5‐bromo‐2′‐deoxyuridine has been substituted for thymidne. To gain some insight into the possible nature of this cross‐linking, we have studied the photoreactions occurring in deoxygenated aqueous solutions containing 5‐bromouracil (I) (BrUra) or 5‐bromo‐2′‐deoxyuridine (III) (BrdUrd) and ethylamine, a lysine side chain analog. In the case of I this reaction produced the ring opened compound N‐(N′‐ethylcarbamoyl)‐3‐amino‐2‐bromoacrylamide (Ia). A small amount of N‐(N′‐ethylcarbamoyl)‐3‐ethylamino‐2‐bromoacrylamide (Ic) was also isolated. It was found that purified Ia, standing in the presence of ethylamine, was gradually converted to Ic in a dark reaction. The βandαanomers of N‐(N′‐ethylcarbamoyl)‐3‐(2′ deoxyribofuranos‐1′‐yl)amino‐2‐bromoacrylamide (IIIa and IIIb respectively) were isolated as products in the photoreaction of III with ethylamine; the α anomer was produced in a dark reaction from the β anomer. The identity of these anomers was established by comparison of their proton NMR spectra with those of the four corresponding α and β furanosyl and pyranosyl isomeric nucleosides of thymine, which are presented in the Appendix. A study was also made of the reaction of I with methylamine; a ring opened product analogous to In, viz. N‐(N′‐methylcarbamoyl)‐3‐amino‐2‐bromoacrylamide(IIa) was formed. A similar study with 5‐bromo‐1‐methyluracil produced N‐(N′‐methylcarbamoyl)‐3‐methylamino‐2‐bromoacrylamide (IIc) as a product. Likewise, the reaction of 5‐chlorouracil with ethylamine was studied and N‐(N′‐ ethylcarbamoyl)‐3‐amino‐2‐chloroacrylamide (Ie), which is analogous in structure to Ia, was found to be produced. Structural identifications were made through use of UV spectroscopy, high resolution 1H‐NMR spectroscopy, mass spectrometry and, in the case of Ia and IIa, 13C‐NMR spectroscopy. In the BrUra and BrdUrd reaction systems, described above, dehalogenation reactions accounted for a major portion of the products. The yields of ring opened products, determined at pH 10, ranged from a high of 10.3% in the BrUra‐ethylamine system to a low of 1.7% in the MeBrUra‐methylamine system.

Ring Opening Photoreactions of Cytosine and Uracil with Ethylamine

Abstract The photochemical reactions of cytosine (Cyt) and uracil (Ura) with ethylamine, an analog of the side chain of the amino acid lysine, have been studied. After irradiation of Cyt in aqueous ethylamine at λ = 254 nm, N-(N′-ethylcarbamoyl)-3-aminoacrylamidine (Ia) and N-(N′-ethylcarbamoyl)-3-ethylaminoacrylamidine (Ib) were isolated as products, while irradiation of Ura gave N-(N′-ethylcarbamoyl)-3-aminoacrylamide (IIa) and N-(N′-ethylcarbamoyl)-3-ethylaminoacrylamide (IIb) as products. Studies in which Ia and IIa were incubated with ethylamine at various pH values indicate that Ib and IIb are secondary products produced via thermal reactions of Ia and IIa with ethylamine. Heating of Ia and Ib leads to ring closure with the resultant formation of 1-ethylcytosine; small amounts of 1-ethyluracil are also produced. Heating of IIa and IIb produces 1-ethyluracil as the sole product. Spectroscopic properties were determined for each of these opened ring products, as well as for N-(N′-ethylcarbamoyl)-3-amino-2-methylacrylamidine (III) and N-(N′-ethylcarbamoyl)-3-amino-2-methylacrylamide (IV). Quantum yield measurements showed that Ia was formed with a Φ of 1.6 × 10−4 at pH 9.8, while Φ for formation of IIa was 7.2 × 10−4 at pH 11.5. A profile of the relative quantum yield for formation of Ia, determined as a function of pH, showed that the maximum quantum yield occurs at around pH 9.5; the analogous profile for IIa shows a maximum quantum yield at pH 11.3 and above. Acetone sensitization does not produce Ia in the Cyt–ethylamine system, which indicates that the known triplet state of Cyt is not involved in reactions leading to this opened ring product.

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) β-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae.

Photochemical exchange reactions of thymine, uracil and their nucleosides with selected amino acids

The photoinduced exchange reactions of thymine with lysine at basic pH, using 254 nm light, have been studied. Three products have been isolated, namely, 6‐amino‐2‐(1‐thyminyl)hexanoic acid (Ia), 2‐amino‐6‐(1‐thyminyl)hexanoic acid (IIa) and 1‐amino‐5‐(1‐thyminyl)pentane (IIIa). Compound IIIa was shown to be a secondary product, produced by photochemical decarboxylation of Ia. Photochemical reaction of thymine with glycine and alanine at basic pH led, respectively, to formation of 2‐(1‐thyminyl)acetic acid (Ic) and 2‐(1‐thyminyl)propionic acid (Id). Compounds Ic and Id underwent photolysis to produce the decarboxylated secondary products 1‐methylthymine and 1‐ethylthymine, respectively. Thymidine reacts photochemically with glycine and alanine to produce the same products.

Photoexchange products of cytosine and 5-methylcytosine with Nα-acetyl-l-lysine and l-lysine

Abstract The photoinduced exchange reactions of cytosine ( Ia ) and 5-methylcytosine ( IIa ) with N α-acetyl- l -lysine, a derivative of the common amino acid l -lysine, were studied. These reactions of Ia and IIa at pH 7.5 produce, respectively, 2- N -acetylamino-6-(1-cytosinyl)hexanoic acid ( Ib ) and 2- N -acetylamino-6-(1-(5-methylcytosinyl))hexanoic acid ( IIb ) as major final products. In addition, small amounts of the corresponding deamination products were formed in the 5-methylcytosine- N α-acetyl- l -lysine and cytosine- N α-acetyl- l -lysine systems, namely 2- N -acetylamino-6-(1-thyminyl)-hexanoic acid and 2- N -acetylamino-6-(1-uracilyl)hexanoic acid. The compounds Ib and IIb were deacetylated by acid hydrolysis to yield the corresponding lysine products: 2-amino-6-(1-cytosinyl)hexanoic acid ( Ic ) and 2-amino-6-(1-(5-methylcytosinyl))hexanoic acid ( IIc ). The compound Ic was identified as a product in the photoreaction of cytosine with l -lysine at near neutral pH, while IIc is found as a product in the corresponding reaction of 5-methylcytosine. The occurrence of the above photoexchange reactions at, pH values near those found in physiological systems could have biological implications; in particular, our observations suggest that cytosine and 5-methylcytosine residues, contained in DNA, might react with the ϵ-amino groups of lysine residues in proteins upon UV irradiation of nucleosomes and other DNA-protein complexes under physiological conditions.

MIXED PRODUCTS OF THYMINE AND CYSTEINE PRODUCED BY DIRECT AND ACETONE‐SENSITIZED PHOTOREACTIONS

Abstract. The nature of the major mixed products of the photoreaction of thymine with L‐cysteine has been restudied. Our results, from application of recent advances in separation and spectroscopic technology to this problem, allow extension and require major revision of the published descriptions of the products produced in this system. We have found that the previously described major mixed photoproduct in the system, 5‐S‐L‐cysteinyl‐5, 6‐dihydrothymine, can be readily separated into diasteromers (Ia and Ib) that have quite different chromatographic and NMR spectral properties. Confirming previously published work, 5‐S‐L‐cysteinylmethyluracil (III) was also produced. Major modifications in the published descriptions of other mixed photoproducts produced in the photoreaction of thymine with cysteine are the following. While the product 5‐S‐L‐cysteinylmethyl‐5, 6‐dihydrouracil (II) is indeed produced, it does not correspond in its properties to the compound previously reported as this substance. Our experimental findings also indicate that observations, previously interpreted to imply formation of the disulfide 5‐S, S‐L‐cysteinyl‐5, 6‐dihydrothymine (IV), can instead be explained in terms of the properties of the β‐carbamoylsulfide functionality contained in Ia and Ib. Indeed, both diastereomers of the addition product of L‐cysteine with N‐ethylmaleimide, S‐(l‐ethyl‐2, 5‐dioxopyrrol‐idin‐3‐yl)‐L‐cysteine (VI), as well as 5‐carbamoyl‐4‐thiapentanoic acid (VII), exhibit properties assumed in the previous work to be specific to disulfide linkages; each of these compounds contains a β‐carbamoylsulfide functionality. Acetone photosensitization of reaction in the thymine‐cysteine system also yielded Ia, Ib, II and III as products. Again, no evidence was found for formation of IV.