Synthesis and characterization of DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link

Short DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C(4) overhang at their 5'-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5'-end with O(4)-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert-butyldimethylsilyl-N(4)-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3'-direction from the resulting 3'-hydroxyl of the cross-link using protected nucleoside 5'-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

A pyrazolato-bridged dinuclear platinum(II) complex induces only minor distortions upon DNA-binding

The cytotoxic, pyrazolato-bridged dinuclear platinum(II) complex [(cis-{Pt(NH3)2})2(mu-OH)(mu-pz)]2+ (pz=pyrazolate) has been found to cross-link two adjacent guanines of a double-stranded DNA decamer without destabilizing the duplex and without changing the directionality of the helix axis. A 1H NMR study of the oligonucleotide d(CTCTG*G*TCTC)-d(GAGACCAGAG), cross-linked at the two G* guanines by [(cis-{Pt(NH3)2})2(mu-pz)]3+, and molecular dynamics simulations of the explicitly solvated duplex were performed to characterize the structural details of the adduct. The dinuclear platinum cross-link unwinds the helix by approximately 15 degrees , that is, to a similar extent as the widely used antitumor drug cisplatin, but, in contrast to the latter, induces no significant bend in the helix axis. The Watson-Crick base-pairing remains intact, and the melting temperature of the duplex is unaffected by the cross-link. The helical twist is considerably reduced between the two platinated bases, as becomes manifest in an unusually short sequential H1'-H1' distance. This unwinding also affects the sugar ring of the guanosine in the 3'-position to the cross-link, which presents an N<-->S equilibrium. This is the first cytotoxic platinum complex that has been successfully designed by envisioning the structural consequences of its binding to DNA.     

O(4)-Alkyl-2'-deoxythymidine cross-linked DNA to probe recognition and repair by O(6)-alkylguanine DNA alkyltransferases

DNA duplexes containing a directly opposed O(4)-2'-deoxythymidine-alkyl-O(4)-2'-deoxythymidine (O(4)-dT-alkyl-O(4)-dT) interstrand cross-link (ICL) have been prepared by the synthesis of cross-linked nucleoside dimers which were converted to phosphoramidites to produce site specific ICL. ICL duplexes containing alkyl chains of four and seven methylene groups were prepared and characterized by mass spectrometry and nuclease digests. Thermal denaturation experiments revealed four and seven methylene containing ICL increased the T(m) of the duplex with respect to the non-cross-linked control with an observed decrease in enthalpy based on thermodynamic analysis of the denaturation curves. Circular dichroism experiments on the ICL duplexes indicated minimal difference from B-form DNA structure. These ICL were used for DNA repair studies with O(6)-alkylguanine DNA alkyltransferase (AGT) proteins from human (hAGT) and E. coli (Ada-C and OGT), whose purpose is to remove O(6)-alkylguanine and in some cases O(4)-alkylthymine lesions. It has been previously shown that hAGT can repair O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine ICL. The O(4)-dT-alkyl-O(4)-dT ICL prepared in this study were found to evade repair by hAGT, OGT and Ada-C. Electromobility shift assay (EMSA) results indicated that the absence of any repair by hAGT was not a result of binding. OGT was the only AGT to show activity in the repair of oligonucleotides containing the mono-adducts O(4)-butyl-4-ol-2'-deoxythymidine and O(4)-heptyl-7-ol-2'-deoxythymidine. Binding experiments conducted with hAGT demonstrated that the protein bound O(4)-alkylthymine lesions with similar affinities to O(6)-methylguanine, which hAGT repairs efficiently, suggesting the lack of O(4)-alkylthymine repair by hAGT is not a function of recognition.     

Chemical synthesis of cross-link lesions found in nitrous acid treated DNA: a general method for the preparation of N2-substituted 2'-deoxyguanosines

Treatment of DNA with nitrous acid results in the formation of DNA-DNA cross-links. Two cross-link lesions have previously been isolated and their structures assigned based on spectroscopic data. The major lesion has been proposed to consist of two deoxyguanosine (dG) nucleosides sharing a common N2 atom (1), while the structure of the minor lesion has been proposed to consist of a common nitrogen atom linking C2 of a dG nucleoside to C6 of deoxyadenosine (2). The chemical synthesis of 1 and 2, utilizing a palladium-catalyzed coupling, is described herein. It is demonstrated that the spectroscopic properties of synthetic 1 are identical to that of lesion 1 obtained from nitrous acid cross-linked DNA, thus providing a proof of its structure. Comparison of the limited spectroscopic data available for lesion 2 originating from nitrous acid cross-linked DNA to synthetic 2 supports its structural assignment. The synthetic approach used for synthesis of 1 and 2 is shown to be a general method for the preparation of a variety of N2-substituted dG nucleosides in good yields.     

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.     

Kinetic analysis of the stepwise formation of a long-range DNA interstrand cross-link by a dinuclear platinum antitumor complex: evidence for aquated intermediates and formation of both kinetically and thermodynamically controlled conformers.

Reported here is a detailed study of the kinetics and mechanism of formation of a 1,4 GG interstrand cross-link by [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(n)NH(2))](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of platinum antitumor complexes. The reaction of the self-complementary 12-mer duplex 5'-[d(ATATGTACATAT)(2)] with (15)N-1 has been studied at 298 K, pH 5.4, by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Initial electrostatic interactions with the duplex are observed for 1 and the monoaqua monochloro species (2). Aquation of 1 to yield 2 occurs with a pseudo-first-order rate constant of (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding to the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.47 +/- 0.06 M(-(1) s(-1). There is an electrostatic interaction between the unbound [PtN(3)Cl] group of 3 and the duplex, which is consistent with H-bonding interactions observed in the molecular model of the monofunctional (G/Cl) adduct. Closure of 3 to form the 1,4 GG interstrand cross-link (5) most likely proceeds via the aquated (G/H(2)O) intermediate (4) (pseudo-first-order rate constant = (3.62 +/- 0.04) x 10(-5) s(-1)) followed by closure of 4 to form 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 +/- 0.04) x 10(-5) s(-1). Closure is ca. 10-55-fold faster than that found for 1,2 GG intrastrand cross-link formation by the diaqua form of cisplatin. Changes in the (1)H and (15)N shifts of the interstrand cross-link 5 indicate that the initially formed conformer (5(i)) converts irreversibly into other product conformer(s) 5(f). The NMR data for 5(i) are consistent with a molecular model of the 1,4 GG interstrand cross-link on B-form DNA, which shows that the NH(2) protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH(2) environments indicative of interactions between the NH(2) protons and the DNA. HPLC characterization of the final product showed only one major product peak that was confirmed by ESI-FTICR mass spectroscopy to be a cross-linked adduct of (15)N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.     

Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link.

Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.     

Preparation of N3-thymidine–butylene–N3-thymidine interstrand cross-linked DNA via an orthogonal deprotection strategy

A DNA duplex containing an N3-thymidine–butylene–N3-thymidine interstrand cross-link (ICL) was prepared using an on-column orthogonal deprotection strategy to permit different nucleotide sequence composition around the cross-linked site. The conditions used to remove 5′-O-allyloxycarbonyl and 3′-O-tert-butyldimethylsilyl protective groups for various on-column oligonucleotide intermediates did not affect the cross-linked lesion. Efficient removal of these groups enabled successful coupling of 2′-deoxyphosphoramidites to produce the desired duplex with a 31% yield after deprotection and purification.     

2.4 A crystal structure of an oxaliplatin 1,2-d(GpG) intrastrand cross-link in a DNA dodecamer duplex

(1R,2R-Diaminocyclohexane)oxalatoplatinum(II) (oxaliplatin) is a third-generation platinum anticancer compound that produces the same type of inter- and intrastrand DNA cross-links as cisplatin. In combination with 5-fluorouracil, oxaliplatin has been recently approved in Europe, Asia, and Latin America for the treatment of metastatic colorectal cancer. We present here the crystal structure of an oxaliplatin adduct of a DNA dodecanucleotide duplex having the same sequence as that previously reported for cisplatin (Takahara, P. M.; Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J. Nature 1995, 377, 649-652). Pt-MAD data were used to solve this first X-ray structure of a platinated DNA duplex derived from an active platinum anticancer drug other than cisplatin. The overall geometry and crystal packing of the complex, refined to 2.4 A resolution, are similar to those of the cisplatin structure, despite the fact that the two molecules crystallize in different space groups. The platinum atom of the [Pt(R,R-DACH)](2+) moiety forms a 1,2-intrastrand cross-link between two adjacent guanosine residues in the sequence 5'-d(CCTCTGGTCTCC), bending the double helix by approximately 30 degrees toward the major groove. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice. The latter is positioned in the minor groove opposite the platinum cross-link. A novel feature of the present structure is the presence of a hydrogen bond between the pseudoequatorial NH hydrogen atom of the (R,R)-DACH ligand and the O6 atom of the 3'-G of the platinated d(GpG) lesion. This finding provides structural evidence for the importance of chirality in mediating the interaction between oxaliplatin and duplex DNA, calibrating previously published models used to explain the reactivity of enantiomerically pure vicinal diamine platinum complexes with DNA in solution. It also provides a new kind of chiral recognition between an enantiomerically pure metal complex and the DNA double helix.     

Formation of intrastrand cross-link products between cytosine and adenine from UV irradiation of d((Br)CA) and duplex DNA containing a 5-bromocytosine

Here, we showed that Pyrex-filtered UV light irradiation of d((Br)CA) gave rise to three types of intrastrand cross-link products, that is, d(C[5-N6]A), d(C[5-2]A), and d(C[5-8]A), where the C5 carbon atom of cytosine is covalently bonded to the N6 nitrogen atom, C2, and C8 carbon atoms of adenine, respectively. Furthermore, we demonstrated by LC-MS/MS that the UV irradiation of a 5-bromocytosine-containing duplex oligodeoxynucleotide (ODN) led to the formation of five cross-link products, that is, C[5-N6]A, C[5-2]A, C[5-8]A, A[2-5]C, and A[8-5]C, under both aerobic and anaerobic conditions. LC-MS/MS quantification results showed that the yields for the formation of these cross-link products are different. The presence of molecular oxygen reduces the yields for the formation of all cross-link products except A[2-5]C. To our knowledge, this is the first report about the formation of intrastrand cross-link products between cytosine and adenine in duplex DNA. The chemistry discovered here may facilitate the future preparations of oxidative cross-link lesion-bearing substrates for biochemical and biophysical studies.     

RNA-tethered phenyl azide photocrosslinking via a short-lived indiscriminant electrophile

Arylazide mediated photocrosslinking has been widely used to obtain structural constraints in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We establish a mechanistic framework for formation of adducts between photoactivated 3-hydroxyphenyl azide and RNA. Tethered to an internal site in an RNA duplex via a 2'-amido linkage, photolysis of the aryl azide yields a cross-strand cross-link. Analysis of the ability of reagents with diagnostic reactivities to intercept formation of this cross-strand cross-link supports the assignment that the photoactivated intermediate is the ketenimine or a ketenimine-derived ring expansion product. Neither the initially produced singlet nitrene nor the subsequently formed triplet nitrene contribute to cross-link formation. Argon matrix and time-resolved solution experiments show that photolysis of free 3-hydroxyphenyl azide releases (in 相似文献   

DNA interchain cross-links formed by acrolein and crotonaldehyde

Acrolein and higher alpha,beta-unsaturated aldehydes are bifunctional genotoxins. The deoxyguanosine adduct of acrolein, 3-(2-deoxy-beta-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (8-hydroxy-1,N(2)-propanodeoxyguanosine, 2a), is a major DNA adduct formed by acrolein. The potential for oligodeoxynucleotide duplexes containing 2a to form interchain cross-links was evaluated by HPLC, CZE, MALDI-TOF, and melting phenomena. Interchain cross-links represent one of the most serious types of damage in DNA since they are absolute blocks to replication. In oligodeoxynucleotides containing the sequence 5'-dC-2a, cross-linking occurred in a slow, reversible manner to the extent of approximately 50%. Enzymatic digestion to form 3-(2-deoxy-beta-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-(N(2)-2'-deoxyguanosinyl)pyrimido[1,2-a]purin-10(3H)one (5a) and reduction with NaCNBH(3) followed by enzymatic digestion to give 1,3-bis(2'-deoxyguanosin-N(2)-yl)propane (6a) established that cross-linking had occurred with the exocyclic amino group of deoxyguanosine. It is concluded that the cross-link is a mixture of imine and carbinolamine structures. With oligodeoxynucleotide duplexes containing the sequence 5'-2a-dC, cross-links were not detected by the techniques enumerated above. In addition, (15)N-(1)H HSQC and HSQC-filtered NOESY spectra carried out with a duplex having (15)N-labeling of the target amino group established unambiguously that a carbinolamine cross-link was not formed. The potential for interchain cross-link formation by the analogous crotonaldehyde adduct (2b) was evaluated in a 5'-dC-2b sequence. Cross-link formation was strongly dependent on the configuration of the methyl group at C6 of 2b. The 6R diastereomer of 2b formed a cross-link to the extent of 38%, whereas the 6S diastereomer cross-linked only 5%.     

Marked dependence on carrier-ligand bulk but not on carrier-ligand chirality of the duplex versus single-strand forms of a DNA oligonucleotide with a series of G-Pt(II)-G intrastrand cross-links modeling cisplatin-DNA adducts

The N7-Pt-N7 adjacent G,G intrastrand DNA cross-link responsible for cisplatin anticancer activity is dynamic, promotes local "melting" in long DNA, and converts many oligomer duplexes to single strands. For 5'-d(A1T2G3G4G5T6A7C8C9C10A11T12)-3' (G3), treatment of the (G3)2 duplex with five pairs of [LPt(H2O)2]2+ enantiomers (L = an asymmetric diamine) formed mixtures of LPt-G3 products (1 Pt per strand) cross-linked at G3,G4 or at G4,G5 in all cases. L chirality exerted little influence. For primary diamines L with bulk on chelate ring carbons (e.g., 1,2-diaminocyclohexane), the duplex was converted completely into single strands (G3,G4 coils and G4,G5 hairpins), exactly mirroring results for cisplatin, which lacks bulk. In sharp contrast, for secondary diamines L with bulk on chelate ring nitrogens (e.g., 2,2'-bipiperidine, Bip), unexpectedly stable duplexes having two platinated strands (even a unique G3,G4/G4,G5 heteroduplex) were formed. After enzymatic digestion of BipPt-G3 duplexes, the conformation of the relatively nondynamic G,G units was shown to be head-to-head (HH) by HPLC/mass spectrometric characterization. Because the HH conformation dominates at the G,G lesion in duplex DNA and in the BipPt-G3 duplexes, the stabilization of the duplex form only when the L nitrogen adducts possess bulk suggests that H-bonding interactions of the Pt-NH groups with the flanking DNA lead to local melting and to destabilization of oligomer duplexes. The marked dependence of adduct properties on L bulk and the minimal dependence on L chirality underscore the need for future exploration of the roles of the L periphery in affecting anticancer activity.     

NMR determination of the conformation of a trimethylene interstrand cross-link in an oligodeoxynucleotide duplex containing a 5'-d(GpC) motif

Malondialdehyde interstrand cross-links in DNA show strong preference for 5'-d(CpG) sequences. The cross-links are unstable and a trimethylene cross-link has been used as a surrogate for structural studies. A previous structural study of the 5'-d(CpG) cross-link in the sequence 5'-d(AGGCGCCT), where G is the modified nucleotide, by NMR spectroscopy and molecular dynamics using a simulated annealing protocol showed the guanine residues and the tether lay approximately in a plane such that the trimethylene tether and probably the malondialdehyde tether, as well, could be accommodated without major disruptions of duplex structure [Dooley et al. J. Am Chem. Soc. 2001, 123, 1730-1739]. The trimethylene cross-link has now been studied in a GpC motif using the reverse sequence. The structure lacks the planarity seen with the 5'-d(CpG) sequence and is skewed about the trimethylene cross-link. Melting studies indicate that the trimethylene cross-link is thermodynamically less stable in the GpC motif than in the 5-d(CpG). Furthermore, lack of planarity of the GpC cross-link precludes making an isosteric replacement of the trimethylene tether by malondialdehyde. A similar argument can be used to explain the 5'-d(CpG) preference for interchain cross-linking by acrolein.     

Analytical characterisation of glutardialdehyde cross-linking products in gelatine-gum arabic complex coacervates

Encapsulates having shells of cross-linked mixtures of proteins and polysaccharides are widely used in the food and pharmaceutical industry for controlled release of actives and flavour compounds. In order to be able to predict the behaviour and the release characteristics of the microcapsules, a better understanding of the nature and extent of the cross-linking reaction is needed. Several analytical techniques were applied for the characterisation of glutardialdehyde (GDA) cross-linked encapsulates made of gelatine and gum arabic. To allow the use of sensitive, high-resolution methods such as chromatography and mass spectrometry, the sample first had to be hydrolysed. In this way, a mixture of amino acids, small peptides and the cross-link moieties was obtained. High-resolution liquid chromatography coupled to high-resolution mass spectrometry (HPLC-MS) was applied to detect possible cross-link markers through a comparison of HPLC-MS mass-chromatograms obtained for cross-linked and non-cross-linked coacervates. HPLC-MS/MS was used to identify the species responsible for the differences. Cross-linking occurred between GDA molecules and lysine and hydroxylysine epsilon-amino groups, and up to eight cross-link products of different nature could be identified. They included pyridinium ions and Schiff bases, and also unreacted GDA condensation products. Next, based on the insight gained in the possible chemical structures present in the cross-link markers, methods for selective labelling of these functionalities were employed to allow easier detection of related reaction products. Both liquid chromatography (LC) and gas chromatography (GC) were used in these experiments. Unfortunately, these approaches failed to detect new cross-link markers, most likely as a result of the low levels at which these are present.     

Hemoglobin-superoxide dismutase-chemical linkages that create a dual-function protein

Chemical reagents were designed to cross-link and connect hemoglobin and superoxide dismutase, combining the oxygen transport and superoxide-removal capabilities of the red cell in a dual-function protein. Reaction of 1 with thiol-protected hemoglobin followed by reduction produces cross-linked hemoglobin with a free thiol on the cross-link. Reaction of SOD with 5 produces a cross-linked protein with a maleimide on the cross-link. Addition of the hemoglobin-thiol to the SOD-maleimide produces a protein with the desired dual properties. Hemoglobin's oxygenation cooperativity is lowered as a result of being in the conjugate, while SOD's activity is equal to that of the native protein.     

Competitive reactions of interstrand and intrastrand DNA-Pt adducts: A dinuclear-platinum complex preferentially forms a 1,4-interstrand cross-link rather than a 1,2 intrastrand cross-link on binding to a GG 14-mer duplex

A study of the kinetics and mechanism of the reaction between the dinuclear Pt complex [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1) and the 14-mer duplex 5'-d(ATACATG(7)G(8)TACATA)-3'.5'-d(TATG(25)TACCATG(18)TAT)-3' is reported. [(1)H,(15)N]-HSQC NMR was used to follow the reaction at 298 K, pH 5.4. The product is primarily the 5'-5' 1,4-interstrand cross-link between G(8) and G(18) bases and exists in two conformational forms. No evidence for the possible 1,2-intrastrand G(7)G(8) adduct was seen, confirming the preferential formation of interstrand cross-links by these dinuclear complexes. An initial electrostatic association of (15)N-1 with the duplex is indicated by changes in its (1)H/(15)N chemical shifts, followed by aquation of 1 to form the monoaqua monochloro species 2, with a rate constant of 4.00+/-0.03x10(-5) s(-1). Monofunctional binding to the duplex occurs primarily at G(8), the 3' base of the nucleophilic GG grouping, with a rate constant of 1.5+/-0.7 M(-1) s(-1). Changes in the (1)H/(15)N shifts indicate there is an electrostatic interaction between the unbound (PtN(3)Cl) group of the monofunctional adduct and the duplex. No peaks for a transient aquated monofunctional species are seen and closure of 3 to form the 1,4-G(8)G(18) interstrand cross-link (5) was treated as direct, with a rate constant of 4.47+/-0.06x10(-5) s(-1). The G(8)G(18) cross-link was confirmed from analysis of the NOESY NMR spectrum of the final product. Structural perturbations for the 1,4-interstrand cross-link extend over approximately four base-pairs and are similar to those found for a 1,4-interstrand cross-link with a shorter 8-mer -GTAC- sequence. A major distortion was evident for the 5'T (T(17)) adjacent to the platinated G(18), consistent with the findings from the use of chemical probes to investigate the conformation of 1,4-interstrand cross-links.     

Di-carboxylic acid cross-linking interactions improves thermal stability and mechanical strength of reconstituted type I collagen

This study emphasizes, cross-linking potential of a simple di-carboxylic acid, namely, oxalic acid with type I collagen for the preparation of collagen based biomaterial for clinical applications. Further the study discusses the characteristics features of the cross-linked material in comparison with the standard cross-linker. In addition, the study also demonstrates the role of ionic interactions in providing the thermal stability and tensile strength to the cross-linked biopolymer material. Type I collagen from rat tail tendon treated with oxalic acid at optimized concentrations provided a biopolymer material without changing the triple helical pattern of collagen (CD spectrum) and also with 6–7 fold increase in tensile strength than native collagen. FTIR spectral details demonstrate the ionic interactions between collagen and oxalic acid. Thermal stability analyses of oxalic acid cross-linked biopolymer revealed, high thermal stability compared to materials of glutaraldehyde cross-linked. The results of the study suggest oxalic acid as a suitable cross-linker for collagen and it cross-link with collagen through ionic interactions.