Amino Proton Exchange Research Articles

Amino Proton Exchange Processes in Mononucleosides

Amino proton exchange with water could be a useful probe of nucleic acid structure and kinetics. This requires an understanding of exchange processes in the mononucleosides, which has been only partial up till now. We investigate these processes by nuclear magnetization transfer in cytidine, cAMP, cGMP, and endocyclic-methylated derivatives. From the effect of pH on the exchange rate, we disentangle the exchange processes which involve the neutral nucleoside or the nucleoside protonated on an endocyclic nitrogen, and those which operate by basic catalysis (proton transfer to OH- or to water) or by acid catalysis (proton transfer from hydronium). (Cyclic AMP) At basic pH, the amino proton transfers from the neutral nucleoside to OH-. Between pH 7 and pH 5, it transfers from the endocyclic-protonated nucleoside to water. Endocyclic protonation lowers the deprotonation pK of the amino group by about 9 units. This is confirmed by the effect of proton acceptors such as ammonia on amino proton exchange. Below p...

Structure and dynamics of interstrand guanine association in quadruplex telomeric DNA.

Exchanges of the amino and imino protons in guanine quartets of telomeric DNA have been time-resolved by laser Raman spectroscopy. The Raman dynamic probe has been applied to parallel and antiparallel quadruplexes formed by the telomeric repeat of Oxytricha nova, d(T4G4)4, and to the highly thermostable parallel quadruplex formed by d(G12). Time-dependent Raman spectra of the d(G12) quadruplex reveal two characteristic exchange reactions for guanine N2 amino groups. At 10 degrees C, the pseudo-first-order exchange rates are kN2H(10 degrees C) = 5.7 x 10(-3) and k'N2H'(10 degrees C) = 1.2 x 10(-2) min-1, assignable to Hoogsteen-hydrogen-bonded and non-hydrogen-bonded N2 protons, respectively. These measurements provide the first quantitative determination of two kinetically distinct N2 amino proton exchange reactions in the guanine quartet and demonstrate that amino group rotation about the C2-N2 bond is highly restricted in the quadruplex. No exchange of guanine N1 imino sites occurs in d(G12) at 10 degrees C, and N1 exchange remains slow even at 95 degrees C [kN1H(95 degrees C) = 2.7 x 10(-2) min-1], indicating severe suppression of imino exchange in guanine quartets. For both parallel and antiparallel quadruplexes of d(T4G4)4, proton exchange rates decrease in the order thymine N3 imino > guanine N2 amino > guanine N1 imino. The rapid exchange of thymine N3 imino sites indicates that thymine quartets are not stabilized in Oxytricha quadruplexes. The protium-->deuterium exchange experiments also establish new guanine Raman band assignments. Importantly, the 1603 cm-1 band is due to in-plane bending of N1-H, while the 1644 cm-1 band involves scissoring of the N2 amino group. Accordingly, the 1603 and 1644 cm-1 bands are potentially valuable markers of hydrogen-bonding interactions specific to guanine imino and amino sites, respectively. The present findings also show that guanine hydrogen bonding and exchange dynamics are not interrelated in a simple manner. Despite extraordinary retardation of N1 imino proton exchange, Raman markers suggest that Hoogsteen-type guanine-guanine hydrogen bonding (N1-H...O=C6) is comparable in strength to hydrogen bonding of N1-H with water and, surprisingly, much weaker than hydrogen bonding between N1-H and the cytosine N3 acceptor of Watson-Crick B DNA.

Premelting thermal fluctuational interbase hydrogen‐bond disrupted states of a B‐DNA guanine–cytosine base pair: Significance for amino and imino proton exchange

Modified self-consistent phonon theory when applied to the DNA double helix indicates the existence of fairly long-lived states in which single interbase H bonds are disrupted. One can then postulate a number of situations in which particular disrupted H bonds can enhance particular proton exchange. In this paper we postulate a number of such partially open states for a B-conformation GC base pair and calculate the probability of each of these states for a B-conformation poly(dG).poly(dC). We compare these probabilities to those probabilities needed to explain various observed proton exchange rates. We propose that, for a GC base pair in B conformation, there are two amino proton exchangeable states--a cytosine amino proton exchangeable state and a guanine amino proton exchangeable state; both require the disruption of only the corresponding interbase H bond. The imino proton exchange, however, requires the disruption of all three interbase H bonds and this defines a third open state. Our calculated probabilities for a GC base pair in these three states are in fair agreement with available experimental estimates from measurements of amino and imino proton exchange.

Processes of base-pair opening and proton exchange in Z-DNA

Using proton magnetic resonance, we have investigated imino and amino proton exchange in the Z form of the four oligomers d(Cbr8GCGCbr8G), d(CGm5CGCG), d(CG)6, and d(CG)12. In the latter two oligomers, all five exchangeable protons have been assigned. We find that proton acceptors such as NH3 or the basic form of Tris enhance imino proton exchange. The base-pair lifetime can then be obtained by extrapolation of the exchange time to infinite concentration of proton acceptor. For d(CG)6 and d(CG)12, the values are ca. 3.5 ms at 80 degrees C and ca. 130 ms at 35 degrees C. The latter value is about 65 times longer than in the same oligomers in the B form. The activation energy of base-pair opening, 80 kJ/mol, is the same in the Z and the B forms of d(CG)12. At 5 degrees C, the base-pair lifetime is about 3 s, much smaller than the time constant of the Z to B transition, to which it is therefore unrelated. The base-pair dissociation constant at 35 degrees C, 0.5 X 10(-6), is 5 times smaller than for the same oligomers in the B form. In the absence of added catalyst, at pH 7, the exchange time of the imino proton is 30 min at 5 degrees C. That of both cytidine amino protons, assigned by NOE, is about 50 min. The longest proton exchange time, ca. 330 min, is assigned unambiguously to the guanosine amino protons. Thus assigned and interpreted in terms of exchange chemistry rather than structural kinetics, the exchange times do not support earlier models of Z-DNA internal motions.

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.

Contrasting observations on buffer catalysis of guanosine amino proton exchange.

The two amino protons of 3', 5'-cyclic guanosine monophosphate are shown to differ drastically in their solvent exchange properties: One is rapidly exchanging and sensitive to buffer catalysis; the other slow and insensitive. This observation accounts for the marked contrast between stopped-flow and NMR observations on buffer catalysis of amino proton exchange in guanosine monophosphates. The amino protons of guanine compounds traverse a "fast" solvent exchange position through the process of amino rotation, which together with kinetic considerations and comparative data on adenine and cytosine compounds, supports proposals of solvent exchange mediated by events at the guanine (N-3) site, rather than the (N-7) site. Exchange does not conform to rate expressions used by different workers for amino proton exchange.

A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions.

The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.

General acid-base catalysis in nucleobase amino proton exchange: cytidine.

A useful property of DMSO solvent has been exploited to reveal a new catalytic route for cytidine amino proton exchange, relevant to exchange in the macromolecular state, but hidden in aqueous solution. Additional exchange mechanisms in aqueous monomeric cytidine (and adenosine) are obscured by the formation of a fast-exchanging endocyclic-protonated intermediate, which dominates the kinetics. Endocyclic nucleobase protonation could be circumvented in the presence of buffer conjugate acid by the use of DMSO/water solvent, permitting the first unequivocal observation buffer acid-catalyzed exchange from the neutral, unprotonated nucleobase, i.e., general acid catalysis. Because buffer ionization is greatly reduced in DMSO through anion desolvation, nucleobase protonation is suppressed in the presence of buffer acid. Evidence is presented to describe this catalytic route as one involving hydrogen bond formation between the buffer acid and the endocyclic protonation site, C(N-3). Since this same configuration is found in Watson-Crick hydrogen bonding, experiments are presented to demonstrate faster cytidine amino proton exchange with the formation of the G-C base pair in DMSO. The importance of this mechanism in past aqueous monomer studies and in the interpretation of macromolecular (DNA) hydrogen exchange is discussed.

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the “closed” macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.

Proton exchange and base-pair kinetics of poly(rA) · poly(rU) and poly(rI) · poly(rC)

Proton exchange of poly(rA) · poly(rU) and poly(rI) · poly(rC) has been studied by nuclear magnetic resonance line broadening and saturation transfer from H 20. Five exchangeable peaks are observed. They are assigned to the imino, amino and 2′-OH ribose protons. The aromatic spectrum is also assigned. Contrary to previous observations, we find that the exchange of the imino proton is strongly buffer sensitive. This property is used to derive the base-pair lifetime, which is in the range of milliseconds at 27 °C, 100 times smaller than published values. The enthalpy for the base-opening reaction (−86 kJ/mol) and the insensitivity of the reaction to magnesium suggest that the open state involves a small number of base-pairs. The similarities in the exchange from the two duplexes indicate that the same open state is responsible for exchange of purine and pyrimidine imino protons. For the lifetime of the open state and for the base-pair dissociation constant, we obtain only lower limits. At 27 °C they are three microseconds and 10 −3, respectively. The analysis that yields the much larger values published previously is based on the assumption that amino protons exchange only from open base-pairs. But theory and preliminary experiments indicate that it may occur from the closed duplex. The exchange of amino protons is slower than that of the imino protons. Exchange of the 2′-OH protons from the duplexes is much slower than from single-stranded poly(rU), and it is accelerated by magnesium. This could indicate hydrogen-bonding to backbone phosphate. Discrepancies between our results and those of previous studies are discussed.

NMR study of slowly exchanging imino protons in yeast tRNAasp.

We have monitored the exchange of imino and amino protons by NMR after quick transfer of yeast tRNAAsp in 2H2O solvent. When the concentration of exchange-catalyzing buffer is not too high, one imino proton exchanges considerably more slowly than any other (e.g., 100 hr versus 4 hr for the second-slowest imino proton at 18 degrees C in 15 mM Mg). This provides excellent conditions for identification, by the nuclear Overhauser effect, of the slowest exchanging proton, which we show to be the imino proton of the U-8 . A-14 reverse Hoogsteen tertiary-structure base pair; other slowly exchanging protons are identified as imino protons from A . U-11 and G . psi-13. In preliminary experiments, we find that the exchange of these protons is catalyzed by cacodylate or Tris buffer. The lifetimes of two other imino protons, ca. 10 min at 28 degrees C, are buffer independent. Slowly exchanging amino protons have also been observed. Correlation with the exchange of the uracil-8 imino proton suggests that they may be from adenine-14.

Exceptional characteristics of amino proton exchange in guanosine compounds.

Amino 1H NMR line width as a measure of amino proton exchange in guanosine compounds is completely unaffected by the addition of ca. 1 M tris(hydroxymethyl)-aminomethane, imidazole, 2-(N-morpholino)ethanesulfonic acid, glycine, or cacodylate, all shown to be effective buffer catalysts in adenosine and cytidine proton exchange. Line broadening, seen only with phosphate and acetate, is established by intermolecular interactions, as well as by amino to water proton exchange. This absence of buffer catalysis of exchange is accounted for by the relatively small implied effect of G(N-7) protonation on amino acidity, based on similar observations with 7-methylguanosine as a model for endocyclic protonation. The requirement for diffusion-controlled proton transfer in buffer catalysis is achieved by nucleobase protonation in adenine and cytosine, but not in guanine.

Isotopic exchange reactions of amines. 2. Amino-amide proton exchange in the potassium methylamide-methylamine system

The kinetics of the methylamine-potassium methylamide amino proton exchange reaction were studied by /sup 1/H NMR as a function of temperature and catalyst concentration. Rates were determined by analysis of the line shapes of both the amino and methyl peaks. The exchange kinetics cannot be described by a single bimolecular rate constant based on solvent and stoichiometric amide concentration. By using published data for the dissociation constant of potassium methylamide in methylamine, we calculated bimolecular rate constants for two cataytically active species in solution: free methylamide ions and potassium methylamide ion pairs. These are at 25/sup 0/C as follows: k/sub 2/(free ion) = 1.42 x 10/sup 9/ L.mol/sup -1/.s/sup -1/, k/sub 2/(ion pair) = 1.04 x 10/sup 8/ L.mol/sup -1/.s/sup -1/, with activation parameters ..delta..H double dagger = 12.8 and 10.3 kJ.mol/sup -1/ and ..delta..S double dagger = -27 and -57 J.mol/sup -1/.K/sup -1/, respectively. The NMR spectrum of anhydrous methylamine was recorded as a function of temperature over the range -82 to +75/sup 0/C. Values of 46.7 and 7.0/sub 4/ Hz, respectively, were found for the /sup 14/N-H and H-N-C-H spin coupling constants in methylamine. In addition the spin lifetimes of the methylamine /sup 14/N yielded an activation energymore » for quadrupole relaxation, a parameter characterizing rotational diffusion in the liquid phase, of 8.7 kJ.mol/sup -1/. 8 figures, 3 tables.« less

Rhodium(III) complexes containing unidentate amines

Convenient syntheses of [Rh(NRH2)5X]2+(R = Me, Et, Prn, Bun, or Bui; X = Cl or Br), trans-[Rh(NR′H2)4Cl2]+(R′= Pri, cyclohexyl, or Ph), and trans-[Rh(pyr)4Cl2]+(pyr = pyrrolidine) are reported and [Rh(NRH2)5Cl]2+ is shown to be a convenient starting material for the synthesis of trans-[Rh(NRH2)4Cl2]+. Polarographic half-wave reduction potentials are reported together with physical and spectroscopic properties. The rate of exchange of amino-protons in trans-[Rh(NMeH2)4Cl2]+ and the rate constants and activation parameters for the substitution of chloride by bromide in [Rh(NRH2)5Cl]2+ and trans-[Rh(NRH2)4Cl2]+(R = Me, Et, or Prn) have been determined and are compared with data on related complexes of RhIII, CoIII, and CrIII.

Imidazole catalysis of amino proton exchange in 2',3'-cyclic adenosine monophosphate. General exchange mechanism

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTImidazole catalysis of amino proton exchange in 2',3'-cyclic adenosine monophosphate. General exchange mechanismBruce McConnellCite this: Biochemistry 1974, 13, 22, 4516–4523Publication Date (Print):October 1, 1974Publication History Published online1 May 2002Published inissue 1 October 1974https://doi.org/10.1021/bi00719a007RIGHTS & PERMISSIONSArticle Views62Altmetric-Citations34LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (920 KB) Get e-Alerts Get e-Alerts

Coenzyme Interactions: VI. PROPERTIES OF THE PYRIMIDINE MOIETY OF THIAMINE

Abstract A discrete proton magnetic resonance signal is observed for the amino protons of the pyrimidine moiety of thiamine in water from pH 0 to pH 9; the integrated area of this signal is indicative of only two protons, confirming that the NH2 group is not the site of protonation of the aminopyrimidine moiety. The chemical shift of the (C-4') —NH2 signal is pH dependent, changing approximately 1.6 ppm over the range pH 0 to pH 7. A plot of chemical shift versus pH describes a curve with an inflection point at pH 5, corresponding to the pK1 for protonation of the pyrimidine moiety. The chemical shifts of all the other signals in the spectrum of thiamine are affected much less by pH changes, but all, like the (C-4')—NH2 signal, show a linear dependence on the fraction of thiamine in the protonated form. In addition, the line width of the amino signal is pH dependent, and a detailed study of changes in line width under various conditions suggest three mechanisms of exchange of the NH2 protons: (a) specific acidic catalysis, K30°h+ = 6.8 x 102 m-1 sec-1; (b) general acid-base catalysis, K30°thiamine = 1.8 x 102 m-1 sec-1; K30°acetate = 3.3 x 102 m-1 sec-1; (c) specific basic catalysis, K30°oh- = 1.6 x 108 m-1 sec -1. The energy of activation of the amino proton exchange was found to be similar for specific catalysis by H+ and OH-, and general acid-base catalysis by thiamine and by acetate; Ea = 12.5 to 12.9 Kcal per mole. The changes in width of the water signal were qualitatively similar to those of the thiamine (C-4')—NH2 signal. H-D exchange at the C-2 atom of the thiazolium ring proceeds approximately 10 times faster in the case of thiamine than in the case of oxythiamine, over the range of pH 3 to 6. The pK1 value for thiamine is lowered approximately 0.5 unit by complex formation with tryptophol in 50% methanol-water. Determination of apparent association constant for the 1:1 complex from potentiometric titration data (ΔpK) agreed with the value calculated from proton magnetic resonance data (Δδ), Kassoc = 1.5 m-1.

Proton Magnetic Resonance Spectrum of 2-Chloroaniline-15N. Long-range Coupling Constants to Amino Protons from all Ring Protons

The p.m.r. spectra of 2-chloroaniline and 2-chloroaniline-15N in benzene and toluene solutions are analyzed at 60 and 100 MHz under conditions where the amino protons are exchanging intermolecularly and also where they are not exchanging. The spectrum of 2-chloroaniline-H15ND in the absence of amino proton exchange is observed between −48 and 80 °C. In all situations fast rotation about the C—N bond evidently occurs. Long-range couplings of the amino protons to all ring protons are observed and their signs are determined. The nonplanarity of the amino group is discussed in terms of the long-range couplings, of the one-bond 15N–H couplings, and of the two-bond H–H couplings in the amino group. The signs and magnitudes of the coupling constants between the ring protons and 15N are also determined.

The exchange of amino-protons in trifluoroacetamide in perdeuterioacetone: 19F nuclear magnetic resonance study

19 F n.m.r. spectroscopy is more useful than 1H n.m.r. in the study of the exchange of hydrogen between trifluoroacetamide and (CD3)2CO.