Tribolet R, Sigel H
Eur J Biochem. 1987 Mar 2;163(2):353-63. doi: 10.1111/j.1432-1033.1987.tb10807.x.
The concentration dependence of the chemical shifts of the protons H-2, H-8 and H-1' for 2'-, 3'- and 5'-AMP2- and of the protons H-2, H-7, H-8 and H-1' for tubercidin 5'-monophosphate (= 7-deaza-AMP2-; TuMP2-) has been measured in D2O at 27 degrees C to elucidate the self-association of the nucleoside monophosphates (NMPs). The results are consistent with the isodesmic model of indefinite non-cooperative stacking; the association constants for all four NMPs are very similar: K approximately 2 M-1. These 1H-NMR measurements and those on the dependence of the chemical shifts on the pD of the solutions indicate that the NMP2- species exist predominately in the anti conformation. Comparison of the shift data for 5'-TuMP and 5'-AMP shows that no hydrogen bonding between N-7 and -PO3H- occurs; hence, the previously observed and confirmed 'wrongway' chemical shift [Martin, R. B. (1985) Acc. Chem. Res 18, 32] connected with the deprotonation of the -PO3H- group most probably results from the anisotropic properties of the phosphate group which is in the anti conformation close to N-7. From the dependence between the chemical shift and the pD of the solutions the acidity constants were calculated for the four protonated NMPs, and for adenosine and D-ribose 5'-monophosphate. The measurements also allow an estimation of the first acidity constant of H3(5'-AMP)+ (pKDD3(AMP) = 0.9 and pKHH3(AMP) = 0.4). The values for pKHH2(NMP) and pKHH(NMP) were also determined from potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The agreement of the results obtained by the two methods is excellent. The position of the phosphate group at the ribose moiety and the presence of N-7 in the base moiety influence somewhat the acid-base properties of the mentioned NMPs. Measurements with 5'-AMP in 50% (v/v) aqueous dioxane show that lowering of the solvent polarity facilitates removal of the proton from the H+(N-1) site while the -PO2-3 group becomes more basic; this increases the pH range in which the monoprotonated H(5'-AMP)- species is stable and which is now also extended into the physiological pH region. Some consequences of this observation for biological systems are indicated.
为阐明核苷单磷酸（NMPs）的自缔合作用，在27℃下于重水中测量了2'-、3'-和5'-AMP2-中质子H-2、H-8和H-1'以及结核菌素5'-单磷酸（=7-脱氮腺苷-5'-单磷酸；TuMP2-）中质子H-2、H-7、H-8和H-1'的化学位移对浓度的依赖性。结果与无限非协同堆积的等键模型一致；所有四种NMPs的缔合常数非常相似：K约为2 M-1。这些1H-NMR测量以及化学位移对溶液pD的依赖性测量表明，NMP2-物种主要以反式构象存在。5'-TuMP和5'-AMP的位移数据比较表明，N-7与-PO3H-之间不存在氢键；因此，先前观察到并得到证实的与-PO3H-基团去质子化相关的“错误方向”化学位移[Martin, R. B. (1985) Acc. Chem. Res 18, 32]很可能是由于处于靠近N-7的反式构象的磷酸基团的各向异性性质所致。根据化学位移与溶液pD的依赖性，计算了四种质子化NMPs、腺苷和D-核糖5'-单磷酸的酸度常数。这些测量还允许估算H3(5'-AMP)+的第一酸度常数（pKDD3(AMP)=0.9和pKHH3(AMP)=0.4）。pKHH2(NMP)和pKHH(NMP)的值也通过在水溶液（I = 0.1 M，NaNO3；25℃）中的电位pH滴定法测定。两种方法所得结果的一致性非常好。磷酸基团在核糖部分的位置以及碱基部分中N-7的存在对上述NMPs的酸碱性质有一定影响。在50%（v/v）的二氧六环水溶液中对5'-AMP的测量表明，溶剂极性的降低有利于从H+(N-1)位点去除质子，而-PO2-3基团变得更具碱性；这增加了单质子化H(5'-AMP)-物种稳定的pH范围，并且现在该范围也扩展到了生理pH区域。指出了这一观察结果对生物系统的一些影响。
