312 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 as-15-2.5 Hz if B falls in the trans region and the et al. [90], B-DNA can be either in a bl or a bll torsion angle y in the gauche region, i.e. when conformation, characterized by a ranging from 160- the molecular fragment H4-C4-C5-05'-P5 has a 220 and from 260-300, respectively. These ranges close to planar W-shaped conformation; for other con- fall within the sterically allowed region for e in dNA formations a much smaller value of the JH4,Ps-coupling for both C2 -endo and C3'-endo sugars [88].For will be found C2'-endo these ranges correspond to energy minima ate≈l80°ande≈288°[91 while for C3’endo 5.5. Determination of the e torsion angle sugar puckers only a small range of e values centered around 195 is energetically favorable. For RNA The JH3'P3-coupling describes the e to Mooren et al. [88 have shown that e has a narrower via the Karplus equation range, namely170°<≈E<≈280,forC2endo 3 JH3=153c0s2(+120°)-6.2cos(+120°) sugar puckers than for the usual C3"-endo sugar puckers,185<≈ε<≈280°. Also for rna a +1.5 (27) maximum tends to exist around e s 210. with these data in mind it is interesting to consider the Karplus Here the parametrization by Mooren et al. is used curve of the JH3'P3-coupling versus the torsion angle [88]. The torsion angle a can for steric reasons only e as shown in Fig. 6. As can be seen the two take values between approximately 170 and 300, expected regions, 160-220 and 260-300, have depending somewhat on the pucker of the sugar ring quite similar JH3'P3-coupling constants. Thus, deter- [88, 89]. The usual value for s in A-DNA, B-DNA and mination of the JH3'P3-coupling constant is of limited A-RNA is trans, but as has been shown by Schroeder value for determining the torsion angle a 360 Fig. 6. The Jc4,", Jcr,]and JHs'P3-coupling constants calculated as a function of the torsion angle e on the basis of their Karplus relations (see text)
as ,1.5–2.5 Hz if b falls in the trans region and the torsion angle g in the gauche þ region, i.e. when the molecular fragment H49–C49–C59–O59–P59 has a close to planar W-shaped conformation; for other conformations a much smaller value of the 4 JH49P59-coupling will be found. 5.5. Determination of the « torsion angle The 3 JH39P39-coupling describes the « torsion angle via the Karplus equation 3 JH39P39 ¼ 15:3 cos2 (« þ 1208) ¹ 6:2 cos(« þ 1208) þ 1:5 ð27Þ Here the parametrization by Mooren et al. is used [88]. The torsion angle « can for steric reasons only take values between approximately 1708 and 3008, depending somewhat on the pucker of the sugar ring [88,89]. The usual value for « in A-DNA, B-DNA and A-RNA is trans, but as has been shown by Schroeder et al. [90], B-DNA can be either in a BI or a BII conformation, characterized by « ranging from 160– 2208 and from 260–3008, respectively. These ranges fall within the sterically allowed region for « in DNA for both C29-endo and C39-endo sugars [88]. For C29-endo these ranges correspond to energy minima at « < 1808 and « < 2888 [91], while for C39-endo sugar puckers only a small range of « values centered around 1958 is energetically favorable. For RNA, Mooren et al. [88] have shown that « has a narrower range, namely 1708 , < « , < 2808, for C29-endo sugar puckers than for the usual C39-endo sugar puckers, 1858 , < « , < 2808. Also for RNA a maximum tends to exist around « < 2108. With these data in mind it is interesting to consider the Karplus curve of the 3 JH39P39-coupling versus the torsion angle « as shown in Fig. 6. As can be seen the two expected regions, 160–2208 and 260–3008, have quite similar 3 JH39P39-coupling constants. Thus, determination of the 3 JH39P39-coupling constant is of limited value for determining the torsion angle «. Fig. 6. The 3 JC49P39-, 3 JC29P39- and 3 J H39P39-coupling constants calculated as a function of the torsion angle « on the basis of their Karplus relations (see text). 312 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387
S.S. Wijmenga, B N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 Fig 6 also gives JC4P3 and Jcpa as a function of narrower on can be made when these e as derived from the Karplus equation J-couplings are used together with the JH3'P3" IC2'P3'=8.0cos2o-34 cos d+0.5 (28) coupling. In addition, there is the JH2'P3-coupling, which has a low value when e is in the trans region, withφ= e for c4′andφ=e-120°forC2′. Here while its val 2.0-3.0 Hz when s is in the again the latest parametrization as given by Mooren et gauche region. The JH'P3-coupling can therefore al. [88] is used. As can be seen, Jc4'P3 and Jc2'p3' further confirm the presence of a gauche rotamer for e together define the a torsion angle quite well for the As for the torsion angle B, the additional trans range as well as for the gauche range. An even J-couplings make it also possible to determine the J45 50 H3-H5 H3’-H5 H4°-Hs 240 360 Y(dog) Fig. 7. The y torsion angle dependence of: ( A)The JH4'Hs- and JH4H5-coupling constants(calculated according to their Karplus relations(see text);(B)The H-H distances, d (3, 5), dA(3, 5), d (475)and d (475)
Fig. 6 also gives 3 JC49P39 and 3 JC29P39 as a function of « as derived from the Karplus equation 3 JC49=C29P39 ¼ 8:0 cos2 f ¹ 3:4 cos f þ 0:5 (28) with f ¼ « for C49 and f ¼ « ¹ 1208 for C29. Here again the latest parametrization as given by Mooren et al. [88] is used. As can be seen, 3 JC49P39 and 3 JC29P39 together define the « torsion angle quite well for the trans range as well as for the gauche range. An even narrower determination can be made when these J-couplings are used together with the 3 JH39P39- coupling. In addition, there is the 4 JH29P39-coupling, which has a low value when « is in the trans region, while its value is < 2.0–3.0 Hz when « is in the gauche region. The 4 JH29P39-coupling can therefore further confirm the presence of a gauche rotamer for «. As for the torsion angle b, the additional J-couplings make it also possible to determine the Fig. 7. The g torsion angle dependence of: (A) The 3 J H49H59- and 3 J H49H50-coupling constants (calculated according to their Karplus relations (see text); (B) The 1 H– 1 H distances, di(39;59), di(39;50), di(49;59) and di(49;50). S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 313
314 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 rotamer fractions; in the case when only one rotamer on the torsion angle y as determined by the general is populated it is, at least in principle, possible to ized JHH Karplus, given by Eqs. (12)and(13), and as determine the width of the libration motion shown in Fig. 7(A)[63]. In Table 4, these J-couplings are given for the y,y nd y rotamers at the usual 5.6. Torsion angle y and H5 and H5"stereospecific angles of 60, 180 and 290, respectively. As can be read off from this figure and seen in Table 4. when the torsion angle y is in its usual y domain both the Traditionally, the torsion angle y as well as the H5/ J4s-coupling and the 3J4's-coupling are rather H5 stereospecific assignment have been determined small, about 1. 1 and 2.5 Hz, respectively, while for from a combination of the J45,/couplings y the J45 -coupling is large(about 10. 2 Hz) and (Fig. 7(A), [63]), and the distances d (4 /3, 5/5") the J45-coupling is small (about 2.7 Hz), while the (Fig. 7(B), 621). With the availability of C labeled reverse holds for the y conformation. Assuming that compounds it is now possible to also utilize a number the least populated rotamers, gauche(t)and gauche(-), or this purpose have middle values for y of 180 and 290, it can be [49, 92]. Table 4 summarizes all the J-coupling con- derived using the Karplus equation that the fraction tants and distances involved, and gives their values gauche( +)can be calculated from [62] three main rotamers. As can be seen, a large number of additional J-couplings has become avail 13.3-(34y+345y) f (29) able. We will consider below, for each parameter in turn, its conformational dependence, and then how The distances d( 3, 575)also depend on the torsion they can be used to determine the torsion angle y angle y Fig. 7(B). As can be seen, when the torsion Ind the H5/H5" stereospecific assignment angle y is in its usual y* domain the distance di(3, 5) The J45 -coupling and J45 -coupling depend is large and the distance d (3, 5)is small, for y both the distances are small while for the y conformation the reverse holds as for y(see Table 4) How pre cisely one has to know these distances to determine Overview of J-couplings and distances for the determination of the the torsion angle y or the fraction y has been dis- torsion angle y and for the stereospecific assignment of the H5 and H5 resonances cussed in detail in section 4 Together with the J-couplings these distances also provide the H5/H5 stereospecific assignment +52 +1.3 3J gauche(+), but in this case it is not possible to obtain 2J the stereospecific assignment from these J-couplings The stereospecific assignments of H5 and H5can HScs then be obtained from the two distances d (3,5) +2.5 and d (3 5"), since for ?t the latter is small while +102 +3.6 the former is large(see Table 4) In addition to the classical parameters discussed d45)° above, a number of heteronuclear J-couplings have become accessible. Firstly, we consider the JHSHsC3-couplings. Although no well established b The values are estimated on the basis of the known values of Karplus equation exists for the y torsion dependence these J-couplings for the y and y rotamers and the Bock of this J-coupling, a qualitative assessment of the tor sion angle y is still possible from the available data There is some uncertainty (see Ref [49 ). For y, the trans coupling JH5'C3is d Estimated values at torsion angles y of 60, 300 and 290 about +5.2 Hz and larger than the gauche coupling, corresponding to the y,y and 7" rotamers, respectively. JHsc3, which is about +1. 2-1.4 Hz. For 1, bot
rotamer fractions; in the case when only one rotamer is populated it is, at least in principle, possible to determine the width of the libration motion. 5.6. Torsion angle g and H59 and H50 stereospecific assignment Traditionally, the torsion angle g as well as the H59/ H50 stereospecific assignment have been determined from a combination of the 3 J4959/50-couplings (Fig. 7(A), [63]), and the distances di(49/39;59/50) (Fig. 7(B), [62]). With the availability of 13C labeled compounds it is now possible to also utilize a number of heteronuclear n JCH-couplings for this purpose [49,92]. Table 4 summarizes all the J-coupling constants and distances involved, and gives their values for the three main rotamers. As can be seen, a large number of additional J-couplings has become available. We will consider below, for each parameter in turn, its conformational dependence, and then how they can be used to determine the torsion angle g and the H59/H50 stereospecific assignment. The 3 J4959-coupling and 3 J4950-coupling depend on the torsion angle g as determined by the generalized 3 JHH Karplus, given by Eqs. (12) and (13), and as shown in Fig. 7(A) [63]. In Table 4, these J-couplings are given for the gþ, gt and g¹ rotamers at the usual angles of 608, 1808 and 2908, respectively. As can be read off from this figure and seen in Table 4, when the torsion angle g is in its usual g þ domain both the 3 J4959-coupling and the 3 J4950-coupling are rather small, about 1.1 and 2.5 Hz, respectively, while for gt the 3 J4959-coupling is large (about 10.2 Hz) and the 3 J4959-coupling is small (about 2.7 Hz), while the reverse holds for the g¹ conformation. Assuming that the least populated rotamers, gauche(t) and gauche( ¹ ), have middle values for g of 1808 and 2908, it can be derived using the Karplus equation that the fraction gauche( þ ) can be calculated from [62] fg þ ¼ 13:3 ¹ ( 3 J4959 þ 3 J4950) 9:7 (29) The distances di(39;59/50) also depend on the torsion angle g Fig. 7(B). As can be seen, when the torsion angle g is in its usual gþ domain the distance di(39;59) is large and the distance di(39;59) is small, for gt both the distances are small while for the g¹ conformation the reverse holds as for gþ (see Table 4) How precisely one has to know these distances to determine the torsion angle g or the fraction gþ has been discussed in detail in Section 4 (see also Ref. [62]). Together with the J-couplings these distances also provide the H59/H50 stereospecific assignment. For example, when both the 3 J4959-coupling and 3 J4950-coupling are small the g torsion angle is gauche(þ), but in this case it is not possible to obtain the stereospecific assignment from these J-couplings. The stereospecific assignments of H59 and H50 can then be obtained from the two distances di(39;59) and di(39;50), since for gþ the latter is small while the former is large (see Table 4). In addition to the classical parameters discussed above, a number of heteronuclear J-couplings have become accessible. Firstly, we consider the 3 JH59=H50C39-couplings. Although no well established Karplus equation exists for the g torsion dependence of this J-coupling, a qualitative assessment of the torsion angle g is still possible from the available data (see Ref. [49]). For gþ, the trans coupling 3 JH59C39 is about þ5.2 Hz and larger than the gauche coupling, 3 JH50C39, which is about þ1.2–1.4 Hz. For gt , both Table 4 Overview of J-couplings and distances for the determination of the torsion angle g and for the stereospecific assignment of the H59 and H50 resonances a gþ gt g¹ 3 J H59C39 þ5.2 þ1.3 þ1.3 3 J H50C39 þ1.3 þ1.3 þ5.2 2 J H59C49 ¹4.8 þ1.2 ¹4.8b 2 J H50C49 þ1.0 ¹4.2 ¹4.8b 2 J H49C59 þ3.5 ¹1.5 ¹1.5b 1 J H59C59 151c 151c 151c 1 J H50C59 141c 141c 141c 3 J 4959 d þ2.5 þ2.7 þ10.1 3 J 4950 d þ1.1 þ10.2 þ3.6 di(3959) d 3.6 2.4 2.7 di(3950) d 2.5 2.7 3.7 di(4959) d 2.4 2.4 3.0 di(4950) 2.4 3.0 2.5 a J-coupling values are given in Hz and distances are in A˚ . b The values are estimated on the basis of the known values of these J-couplings for the gþ and gt rotamers and the Bock projection rule [86] (see text). c There is some uncertainty considering the data in [49] that this relation holds for other torsion angle values than gþ. d Estimated values at torsion angles g of 608, 3008 and 2908 corresponding to the gþ, gt and g¹ rotamers, respectively. 314 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387
S.S. Wimenga, B NM van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 H5 and H5 are gauche with respect to C3, so that These additional heteronuclear J-couplings provide both JH5'c3 and JH5'C3are expected to be small, i.e. an alternative means for the determination of the tor 1.2-1.4 Hz(see Table 4,[491). Secondly, the sion angle y and the H5/H5 stereospecific assign- JHs C4 and 2JHsCA-couplings also monitor the ment. Their usage for determining the torsion angle torsion angle y and can provide stereospecific assign- y may or may not require stereospecific assignment of ment of the H5'and H5"protons [49, 92). For +, the the H5'and H5"resonances. For example, the JJH4'CS"- oupling JHs'C4'=-4.6 to-5.0 Hz, while JHs'CA is coupling depends only on the torsion angle y, and small positive, +0.3-1.4 Hz, for y, JHs'c4 and does not involve the H5/H5" protons. It is thus ideally JHs"C4 are +1.2 Hz and-4.2 Hz, respectively(see suited for the estimation of the torsion angle y, since it Table 4, [49, 921). Finally, JH4cs, can be estimated at does not require a knowledge of the stereospecific 3.5 Hz and -1.5 Hz for the y* and y states assignment of the H5 and H5"resonances. A positive respectively(see Table 4,[49,921). These variations value immediately ascertains y(see Table 4). On the in JHc are in accordance with the projection rule other hand, although JH5'/Hs"C3 and JH5'/HS CA also (1871, see also discussion in Section 5.3). For exam- depend on the torsion angle y, their unambiguous ple, for y H5 is oriented gauche with respect to O4 se requires the stereospecific assignment of the giving rise to a negative value for JHs'c3', while H5"is H5 and H5"resonances; the same applies to JHH trans with respect to o4 so that JHs'c3 is positive couplings, JHSH4' and JH5H4', discussed previously The easily measurable JHs'Ic-couplings have been Finally, the JHs'C5'-and JHs'cs-couplings seem no discussed in Section 5. 1. They seem to depend mainly to depend on the torsion angle y, but they can provi on the stereospecificity, with the larger value for stereospecific assignment of the H5 and H5"reso- lHs'c5(see Table 4,[49]) nances, since JH5'C5> JH5C5[49]. Another aspect JHI'C8/6 270 360 Fig.8. The JHI'cae and JHIcz-coupling constants calculated as a function of the torsion angle x on the basis of their Karplus relations(see
H59 and H50 are gauche with respect to C39, so that both 3 JH59C39 and 3 JH50C39 are expected to be small, i.e. < 1.2–1.4 Hz (see Table 4, [49]). Secondly, the 2 JH59C49 and 2 JH50C49-couplings also monitor the torsion angle g and can provide stereospecific assignment of the H59 and H50 protons [49,92]. For g þ , the coupling 2 JH59C49 ¼ ¹ 4.6 to ¹5.0 Hz, while 2 JH50C49 is small positive, þ0.3–1.4 Hz; for gt , 2 JH59C49 and 2 JH50C49 are þ1.2 Hz and ¹4.2 Hz, respectively (see Table 4, [49,92]). Finally, 2 JH49C59 can be estimated at 3.5 Hz and ¹1.5 Hz for the gþ and gt /g¹ states, respectively (see Table 4, [49,92]). These variations in 2 JHC are in accordance with the projection rule ([87], see also discussion in Section 5.3). For example, for gþ H59 is oriented gauche with respect to O49 giving rise to a negative value for 2 JH59C39, while H50 is trans with respect to O49 so that 2 JH50C39 is positive. The easily measurable 1 JH59/50C-couplings have been discussed in Section 5.1. They seem to depend mainly on the stereospecificity, with the larger value for 1 JH59C59 (see Table 4, [49]). These additional heteronuclear J-couplings provide an alternative means for the determination of the torsion angle g and the H59/H50 stereospecific assignment. Their usage for determining the torsion angle g may or may not require stereospecific assignment of the H59 and H50 resonances. For example, the 2 JH49C59- coupling depends only on the torsion angle g, and does not involve the H59/H50 protons. It is thus ideally suited for the estimation of the torsion angle g, since it does not require a knowledge of the stereospecific assignment of the H59 and H50 resonances. A positive value immediately ascertains gþ (see Table 4). On the other hand, although 3 JH59/H50C39 and 2 JH59/H50C49 also depend on the torsion angle g, their unambiguous use requires the stereospecific assignment of the H59 and H50 resonances; the same applies to 3 JHHcouplings, 3 JH59H49 and 3 JH50H49, discussed previously. Finally, the 1 JH59C59- and 1 JH50C59-couplings seem not to depend on the torsion angle g, but they can provide stereospecific assignment of the H59 and H50 resonances, since 1 JH59C59 . 1 JH50C59 [49]. Another aspect Fig. 8. The 3 J H19C6/8- and 3 J H19C2/4-coupling constants calculated as a function of the torsion angle x on the basis of their Karplus relations (see text). S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 315
S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 is that these extra J-couplings make it possible both to 5.8. Measurement of homo-and heteronuclear determine the torsion angle y and obtain the stereo- J-coupling constants specific assignment of the H5 and H5"protons, from various combinations of these J-couplings. In addi We discuss here the various methods that are avail- tion, NoE or distance data may also be available, able for measuring J-coupling constants in nucleic which can be used as an extra source of information. acids. A large number of (novel) approaches exist Table 4 can be consulted to assess which particular for the determination of J-couplings, the majority of combination of J-coupling and/or NoE data is which have been tested and applied to the determina- required. For example, it can be gleaned from Table tion of J-couplings in proteins( for reviews see, for 4 that for the most common situation, namely that of example, Refs. [93, 94]). The application to nucleic n, various different combinations of J-couplings acids has been more limited. Here we discuss the suffice to both establish the torsion angle and obtain established and new approaches as they pertain to stereospecific assignment: (1)(J45I5, JH5H5'C4), their application in the determination of J-coupling (2)(J45/5, JHS/H5'C3),(3)(JH4'C5, JH5'H5'C4 ), constants in nucleic acid (4)(JH4'C5, JHS/H5C3),(5)(JHS/H5'C4, JHS/5C3), J-couplings from the shape of the signal; (2) Determi (6)(JH4'C5, JH5H5C5) nation of J-coupling constants with the ECOSY principle; (3)Determination of J-coupling constants from signal intensities 5.7. x torsion angle and JHc sugar-to-base 5.8.1. Determination of -couplings fi The three-bond couplings JHI'CI and HI'CAn the signal convey information about the glycosidic torsion These methods apply generally well for small angle x Ippel et al. [49 have derived a new molecules where the line width is smaller than the parametrization for the JHI' C& Karplus equation J-coupling. The two main factors that complicate the determination are (i) the complexity of the 3JHI'C68=4.5 cos(x-60)-06 cos(x-60%)+0.1 multiplet pattern and (i) the J-coupling to the line (30) width ratio, JILw. The higher the complexity of the multiplet pattern and the smaller the value of JIlw the and for the JHI'cAn Karplus equation they derive more advanced methods are required Direct measurement of the J-couplings from either ID NMR spectra or from 2D COSY in-phase or c24=47cos2(x-60°)+2.3cos(x-609)+0.1 anti-phase multiplets works well only when the (1) J-couplings are larger than or equal to the line width, otherwise corrections have to be applied. Kim and As can be seen from Fig. 8, the combined use of Prestegard [95] have proposed a simple and straight JHI c8 and JHI'C4L makes it possible to discriminate forward method for correcting for the line width in a between the anti- and the syn-range for the torsion doublet. Unfortunately, in nucleic acids no such angle x. The JHI'C8/-coupling has a value of about simple doublets occur, except on the HI'resonance 4.5 Hz in both the xanti and the xsn domain. On the in RNAs which only couples to H2. This JHI'H2" other hand, the JE has quite different coupling is for RNAs the main JHH-coupling usec values in the xanti and the xsyn domains, 2.0 Hz and in practice, to determine the sugar puckering state 6.0 Hz, respectively. Thus, in a qualitative sense, The more complex J-coupling patterns generally when JHI C4/ JHI'C8/ the glycosidic torsion need to be simulated in order to obtain quantitative angle is in the anti domain, while for values for the -couplings involved. Programs such as 3JHIC4nJHIC the glycosidic torsion angle is in r example, Sphinx/Linsha [96], can be used to the syn domain. We note that Davies and co-workers extract the JHH-couplings in a ribose ring by simula [82] also derived a correlation between the glycosidic tion of the multiplet patterns in a(H, H) DQF torsion angle x and the one-bond coupling JHI'CI COSY spectrum. More qualitative or less detailed
is that these extra J-couplings make it possible both to determine the torsion angle g and obtain the stereospecific assignment of the H59 and H50 protons, from various combinations of these J-couplings. In addition, NOE or distance data may also be available, which can be used as an extra source of information. Table 4 can be consulted to assess which particular combination of J-coupling and/or NOE data is required. For example, it can be gleaned from Table 4 that for the most common situation, namely that of gþ, various different combinations of J-couplings suffice to both establish the torsion angle and obtain stereospecific assignment: (1) (3 J4959/50, 2 JH59/H50C49), (2) (3 J4959/50, 3 JH59/H50C39), (3) (2 JH49C59, 2 JH59/H50C49), (4) (2 JH49C59, 3 JH59/H50C39), (5) (2 JH59/H50C49, 3 JH59/H50C39), (6) (2 JH49C59, 1 JH59/H50C59). 5.7. x torsion angle and 3 JHC sugar-to-base The three-bond couplings 3 JH19C8/6 and 3 JH19C4/2 convey information about the glycosidic torsion angle x. Ippel et al. [49] have derived a new parametrization for the 3 JH19C8/6 Karplus equation: 3 JH19C6=8 ¼ 4:5 cos2 (x ¹ 608) ¹ 0:6 cos(x ¹ 608) þ 0:1 (30) and for the 3 JH19C4/2 Karplus equation they derive 3 JH19C2=4 ¼ 4:7 cos2 (x ¹ 608) þ 2:3 cos(x ¹ 608) þ 0:1 (31) As can be seen from Fig. 8, the combined use of 3 JH19C8=6 and 3 JH19C4/2 makes it possible to discriminate between the anti- and the syn-range for the torsion angle x. The 3 JH19C8/6-coupling has a value of about 4.5 Hz in both the xanti and the xsyn domain. On the other hand, the 3 JH19C4/2-coupling has quite different values in the xanti and the xsyn domains, 2.0 Hz and 6.0 Hz, respectively. Thus, in a qualitative sense, when 3 JH19C4/2 , 3 JH19C8/6 the glycosidic torsion angle is in the anti domain, while for 3 JH19C4=2.3 JH19C8/6 the glycosidic torsion angle is in the syn domain. We note that Davies and co-workers [82] also derived a correlation between the glycosidic torsion angle x and the one-bond coupling 1 JH19C19. 5.8. Measurement of homo- and heteronuclear J-coupling constants We discuss here the various methods that are available for measuring J-coupling constants in nucleic acids. A large number of (novel) approaches exist for the determination of J-couplings, the majority of which have been tested and applied to the determination of J-couplings in proteins (for reviews see, for example, Refs. [93,94]). The application to nucleic acids has been more limited. Here we discuss the established and new approaches as they pertain to their application in the determination of J-coupling constants in nucleic acids: (1) Determination of J-couplings from the shape of the signal; (2) Determination of J-coupling constants with the E.COSY principle; (3) Determination of J-coupling constants from signal intensities. 5.8.1. Determination of J-couplings from the shape of the signal These methods apply generally well for small molecules where the line width is smaller than the J-coupling. The two main factors that complicate the determination are (i) the complexity of the multiplet pattern and (ii) the J-coupling to the line width ratio, J/LW. The higher the complexity of the multiplet pattern and the smaller the value of J/LW the more advanced methods are required. Direct measurement of the J-couplings from either 1D NMR spectra or from 2D COSY in-phase or anti-phase multiplets works well only when the J-couplings are larger than or equal to the line width, otherwise corrections have to be applied. Kim and Prestegard [95] have proposed a simple and straightforward method for correcting for the line width in a doublet. Unfortunately, in nucleic acids no such simple doublets occur, except on the H19 resonance in RNAs which only couples to H29. This 3 JH19H29- coupling is for RNAs the main 3 JHH-coupling used in practice, to determine the sugar puckering state. The more complex J-coupling patterns generally need to be simulated in order to obtain quantitative values for the J-couplings involved. Programs such as, for example, Sphinx/Linsha [96], can be used to extract the 3 JHH-couplings in a ribose ring by simulation of the multiplet patterns in a (1 H, 1 H) DQF– COSY spectrum. More qualitative or less detailed 316 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387