q-Decoupled ‘H homonuclear shift-correlated nuclear magnetic reson~ce spectroscopy (COSYDEC) applied to steroids David N. Kirk and Harold C. Toms Medical Research Council Steroid Reference Queen Mary and Westfield College, London,

Collection, UK

Department

of Chemistry,

Problems of cross-peak overlap in repro-dimensional ‘H homonuc~ear shut-correlated (COSY) specrra of steroids can often be avoided by use of the ~~-decoltpled COS Y (COS YDEC) method. The selection of experimental parameters is discussed, and COS YDEC spectra are illustrated for 17a-oxa-o-homoandrosr4ene-3,17-dione (restololactone), testosterone, and 17a-hydroxyprogesterone. In a good case, a COSYDEC spectrum obtained at 250 MHz allows cross-peak recognition and assignment with facility comparuble to that avuilable only at 500 MHz for normal COSY spectra. (Steroids 56: 195-200, 1991)

Keywords: steroids; two-dimensional ‘H homonuclear COSY; COSYDEC; 17a-oxa-D-homoandrost-4-ene-3,17dione: ‘H spectral assignment; testosterone: ‘H spectral assignment; 17cu-hydroxyprogesterone: ‘H spectral assignment; ‘H NMR; NMR spectroscopy; w,-Decoupled ‘H COSY (COSYDEC) spectroscopy

rntroduction Two-dimensional ‘H homonuclear shift-correlated nuclear magnetic resonance (NMR) spectroscopy’ (COSY) at high field is the single most informative procedure for assigning the ‘H chemical shifts in steroids and similar molecules, especially when sample size is so small as to preclude use of the ‘H-‘3C heteronuclear correlation method.2A In an exceptionally good case, the COSY spectrum may afford a complete solution: when uncertainties remain, it may be necessary to resort to decoupling difference experiments or to nuclear Overhauser effect difference spectra to locate signals from particular protons, although more concentrated solutions are then required.‘-’ In a normal COSY-90 spectrum,8 with adequate digital resolution and absolute value display, off-diagonal cross-peaks comprise two-dimensional arrays of contours from all the connected transitions. The 7&-H/ 7/3-H cross-peak for a steroid, for example, is usually seen as a 4 x 2 array, showing the peak contours that result from the large axial/axial (6&7cu and 7a3j.3) and

Address reprint requests to Dr. David N. Kirk, Medical Research Council Steroid Reference Collection, apartment of Chemistry, Queen Mary and Westfield College, Mile End Road, London El 4NS, UK. Received January 17, 1990; accepted November 19, 1990.

0 1991 Buttetworth-Heinemann

geminal (7cu,7/3) couplings. These multiplicities are a valuable aid to recognition of cross-peaks and to spectral assignment. However, the complexity of a steroid ‘H spectrum, even at the highest frequency that is at present routinely available (500 MHz), often results in overlapping cross-peaks that defy first-order analysis. This problem can be especially acute near the diagonal, making full spectral assignments difficult or impossible without supporting use of the other and more demanding NMR techniques mentioned above. Resorting to the COSY-45 or COSY-60 alternatives’ may marginally improve the situation by asssisting with the recognition of cross-peaks from geminal pairs of protons, but rarely removes the problem of intractable crosspeak overlap. We find that this difficulty can often be avoided or minimized by use of the broad-band w,-decoupling (COSYDEC) technique proposed by Bax and Freeman’ and analyzed in detail by Ernst and co-workers,10

Experimental Testololactone, testosterone, and 17a-hydroxyprogesterone were from the Steroid Reference Collection (Curator, David N. Kirk). Spectra were recorded at 250 MHz on a Bruker AM250 spectrometer (Queen Mary and Westfield College), at 400 MHz on a Bruker WH-400 spectrometer (UniverSteroids,

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sity of London Intercollegiate Research Service), and at 500 MHz on a Bruker AM-500 spectrometer (National Institute for Medical Research, Mill Hill, London, UK). Data acquisition and processing were performed by the DISNMR program running on an ASPECT 3000 computer. Samples were dissolved in CDCl, (99.8%, GOSS Scientific Instruments Ltd, Ingatestone, Essex, UK) containing 0.03% Me,Si as internal standard. The pulse sequence and the evolution and mixing times are discussed in the text. For each transient, 2,048 data points were taken; t, was incremented up to 512 times, zero-filling to 1,024 points before Fourier transform for both COSY and COSYDEC spectra. A sine-bell window function was used on both dimensions.

Results and discussion The visible effect of the COSYDEC technique is to collapse the normal two-dimensional form of each cross-peak to give instead an essentially one-dimensional array of contours for each of the protons (or equivalent sets) in the molecule, parallel to the w2 dimension. Adjacent rows of peaks in a COSYDEC spectrum overlap only if the chemical shift difference is less than the residual widths of contours in the o, dimension, and cross-peaks in two such adjacent rows will overlap only if the protons concerned chance to be spin-coupled to the same third proton or to other protons that are themselves very similar in chemical shift. In practice, it is normally possible to distinguish easily between adjacent rows of cross-peaks separated by more than 0.02 ppm. Finer resolution may be possible by recognition of correlations with proton signals elsewhere on the chemical shift scale. In two-dimensional NMR, a two-pulse sequence is applied: T _~

2 Prepare ++--

ti

~-

Evolve +t

7T

2

~ Mix *-

FID (tJ Acquire ---+

The period t, is a variable delay that is incremented by the sampling delay. A series of free induction decays (FID) is obtained, one for each distinct value of t, . Each FID is an oscillation and can be Fourier-transformed with respect to t, , giving a series of frequencydomain spectra. Each point set (e.g., all the first points of the digital data) also constitutes an oscillation and can be Fourier-transformed with respect to t, , giving a two-dimensional spectrum with two frequency axes. The basic pulse sequence for COSYDEC is FID (7r/2) t,/2 - (7T)7, - t,/2 (p) --7,--e----+

Prepare

Evolve

Mix

Acquire

The phases of the radio frequency pulses and receiver are cycled according to the scheme of Bax and Freeman.9 Decoupling in o, is achieved by fixing the evolution 196

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period (7,; the time between the 7r/2 preparation pulse and the /3 mixing pulse-see below). A t, evolution under chemical shift terms only is introduced by applying a movable pulse at a time t,/2 after the preparation pulse. A 7~pulse leaves the spin-spin coupling terms invariant” in the case of weak coupling; in the case of strong coupling, it gives rise to spurious peaks at the average of the two shifts involved9 and/or on the other side of the diagonal. We note examples of this phenomenon below. Its effects are reduced at higher frequencies. Another feature of COSYDEC spectra is that some off-diagonal cross-peaks can be weak or even absent on one side of the diagonal, although rarely absent on both sides. This problem is likely to be serious with a rr/2 flip angle (p) (COSYDEC-90), so we prefer to use a 7r/3 flip angle (COSYDEC-60) despite a tendency toward emphasis of on-diagonal peaks by this procedure. Resolution of COSYDEC-60 spectra is found to be comparable with that of COSYDEC-90 in spite of all signals having mixed phase (both sine and cosine contributions). The good resolution may be due to the sine and cosine contributions being very unequal, so each cross-peak is dominated by one phase or the other, but generally not both. The other experimental parameter that can be adjusted to maximize cross-peaks (and minimize diagonal peaks) is the length of the fixed evolution period 7,. Amplitude factors are most favorable when 7, = (2n + 1)/25 where n is an integer. While it is not possible to suggest a value of r, that will always show all cross-peaks, due to the wide range of J values present in any reasonably large molecule, it is found that a value of 0.2s is satisfactory in many cases. If necessary, a second experiment at the complementary value of 0.3s could be run: this would strongly favor all those cross-peaks missing at 7, = 0.2s but would of course double the experiment time. As with COSY, it is possible to incorporate various filters into the COSYDEC pulse sequence to further simplify spectra, but we find that the basic experiment gives excellent results for steroids. One of the important advantages of the COSYDEC method is that relatively weak couplings often show up very strongly if they have favorable amplitude factors.’ In most steroids, for example, there are weak couplings between the 18-methyl protons and the 12a-proton, and between the 19-methyl protons and the la-proton. Both are usually clearly visible in the COSYDEC spectrum (even at 250 MHz), although they may be not seen or may be heavily overlapped in the COSY spectrum. These entry points for the full analysis of proton signals for rings A and C are of the utmost value. In the course of analyzing ‘H NMR spectra for a wide variety of steroids,]* we have increasingly found COSYDEC spectra to be very helpful, either to supplement the information available from normal COSY spectra or as an alternative method. Figures 1, 3, and 4 represent three cases (see Figure 2 for structures) in

COS YDEC spectra

of steroids:

Figure 2 Structures of testololactone 17~hydroxyprogesterone (4).

Kirk and Toms

(11, testosterone

(21, and

which severe problems of cross-peak overlap in normal COSY spectra have been resolved by the COSYDEC method. Selected from the many that we have run, they illustrate both the possible advantages of COSYDEC over COSY spectra and the residual problems. For testololactone (17a-oxa-D-homoandrost-4-ene3,17-dione (Figure 2, l),the normal COSY spectrum, even at 400 MHz (Figure 1A) shows overlapping crosspeaks in several regions. At 250 MHz, the overlap was enough to render analysis of the COSY spectrum quite intractable. In contrast, the 250-MHz COSYDEC spectrum (Figure 1B) allowed recognition of sufficient aligned sets of cross-peaks to permit a full assignment. A subsequent COSYDEC spectrum at 500 MHz (Figure 1C) achieved complete resolution, making the assignment task first order. Notable among the coupling interactions revealed even by Figure IB (250 MHz) are the typically very strong la, 19 and 12cz,18 cross-peaks. A suitably modified COSY experiment would, in principle, provide the same information; however, in this case, the 12a, 18 cross-peaks lie so close to the 1la, 1Ifi and 1lp,12a cross-peaks as to render analysis difficult except by the COSYDEC method. Other overlapping clusters of cross-peaks, notably those relating to the la, 7p, 12& and 15a proton signals, are sufficiently resolved in the 250-MHz COSYDEC spectrum of testololactone to allow full analysis with greater facility than by use of the 400-MHz COSY spectrum. Two complicating features of COSYDEC spectra

Figure 1 Spectra of testololactone (Figure 2, 1). excluding only the 4-H signal (65.76) and its cross-peaks. (A) COSY, 400 MHz. (B) COSYDEC-60,250 MHz, showing ‘H assignments of rows of cross-peaks decoupled in the O, dimension (-----, artifacts associated with the strong 16~H/16/3-H’ coupling). (C) COSYDEC-60, 500 MHz, showing the improved resolution available if required.

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are evident in Figure lB, but, once recognized, can be ignored. One is the appearance of strong artifacts associated with the signals from tightly coupled pairs of protons of similar chemical shifts. The 16~~and 16p proton signals from testololactone show this effect more strongly than any others in our experience to date. The main artifacts form a separate row of peaks exactly midway between those in the 16~ and 16p rows, and with virtually identical contours. Integration of that part of the one-dimensional spectrum showed that there were only two contributing protons. Weaker artifacts associated with the 16a/l6@ coupling also appear at the corresponding position on the other side of the diagonal. The other complicating features are the artifacts (ridges) in the o, dimension associated with the strong methyl proton singlets at 61.13 (19-H,) and al.37 (18H3). In a normal COSY spectrum, these could be eliminated by symmetrization,‘3 which is, of course, not possible with COSYDEC spectra. These ridges can be reduced by using weighted mean &-ridge subtraction,14 a procedure applicable to all nonsymmetric, two-dimensional spectra. By careful inspection, significant sets of cross-peaks lying close to these t, ridges can be easily observed. In the present case (Figure 1B). they include the 9,l la, 9,1 l/3, and 7a,7/3 cross-peaks. Figures 3 and 4 illustrate sections of the COSYDEC spectra of testosterone (Figure 2,2; 400 MHz) and 17ahydroxypregn-4-ene-3,20-dione (17cr-hydroxyprogesterone; Figure 2, 3; 250 MHz), respectively. Both spectra were acquired to aid resolution of areas of cross-peak overlap in the corresponding COSY spectra, which are illustrated for comparison. These COSYDEC spectra are of COSYDEC-90 type, and clearly illustrate the ready separation of neighboring rows of contours. The testosterone spectrum exemplifies the imbalance of cross-peaks on each side of the diagonal that can arise with COSYDEC-90, and shows a few mainly weak artifacts, as discussed above. Once recognized, this was not a serious impediment to spectral assignment. The COSYDEC-90 spectrum of 17a-hydroxyprogesterone (Figure 4) was chosen to show how even regions of intractable overlap very close to the diagonal in the COSY spectrum can yield to the COSYDEC method. Full ‘H assignments of all three compounds, obtained by analysis of the COSYDEC spectra, are presented in Table 1. Chemical shifts for testosterone correspond quite closely to those reported’ for ldehydrotestosterone (17P-hydroxyandrosta-1,4-dien3-one), and are included in Table 1 for comparison. The only appreciable differences, apart from the signals for protons at C-l and C-2, are for those at C-l 1 and at the 8@position which, in testosterone, do not experience

Figure 3 Parts of the spectra of testosterone (Figure 2, 2). (A) COSY, 400 MHz. (B) COSYDEC80.400 MHz, after subtraction of t, ridges. (C) Same as panel B, but before subtraction oft, ridges.

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COS YDEC spectra

of steroids:

Kirk and Toms

Table 1 ‘H dataa for testololactone (1). testosterone (2). l-dehydrotestosterone (3). 17whydroxyprogesterone (41, and progesterone (5) H lff

1P 2a 26 4 :; 7ff 76 8 9 Ilcu 116 12a 120 14 15ff 156 16~~ 166 17 18 19 21

1

2

1.74 2.07 2.35 2.43 5.76 2.34 2.40 1.11 2.05 1.41 1.17 1.82 1.34 1.68 2.01 1.44 2.01 1.57 2.59 2.70 1.36 1.18 -

2.35 2.42 5.73 2.29 2.40 1 .oo 1.85 1.56 0.93 1.60 1.43 1.10 1.86 0.98 1.62 1.31 2.09 1.48 3.65 0.80 1.19 -

3

1 6.23 6.08 2.36 2.47 1.01 1.96 1.67 1.04 1.77 1.68 1.09 1.87 0.95 1.61 1.33 2.07 1.47 3.64 0.82 1.24 -

4

5

2.30 2.40 5.74 2.36 2.40 1.12 1.87 1.60 0.99 1.67 1.44 1.70 1.44 1.73 1.85 1.37 1.60 2.69 0.77 1.19 2.28

2.34 2.44 5.73 2.28 2.41 1.065 1.87 1.57 0.99 1.65 1.46 1.46 2.08 1.18 1.72 1.27 1.68 2.19 2.55 0.67 1.20 2.13

Note: See Figure 2 for structures 1, 2, and 4. a For solutions in CD& relative to internal Me,% Spectrometer frequencies: 1, 500 MHz; 2,400 MHz; 3, 400 MHz5; 4, 250 MHz; 5, 300 MHz.14

the a-face of ring D. We also note that the usual relative chemical shifts of 12a-H and 12/?-H (a,,, > 6,$ are reversed in the presence of 17a-OH, an effect also found for 17a-acetoxy-6a-methylprogesterone.’ In conclusion, we recommend the COSYDEC method for routine use whenever normal COSY spectra cannot be fully analyzed because of cross-peak overlap. 2.8

2.6

2.4

2.2

2.0

I.8

1.6

I.1

1.2

1.c

0.8

0.6

Figure 4 Parts of the spectra of 17a-hydroxyprogesterone ure 2,4). (A) COSY, 250 MHz. (B) COSYDEC-90, 250 MHz.

,*sl

(Fig-

References 1.

2.

the deshielding effect of A’ unsaturation. The 17a-hydroxy group in 17a-hydroxyprogesterone has very marked effects on neighboring protons. Those at the 14a and 16/3 positions are strongly deshielded by the 17cu-hydroxyl group by comparison with their chemical shifts in progesterone” (Table 1). The effect at the 14a position is similar in nature to that caused by the 17aacetoxy group in 17a-acetoxy-6a-methylpregn-4-ene3,20-dione,‘ where the 14a-H assignment has been suggested as being in error. is However, our original findings are confirmed by re-examination of the spectral data (Dr. J. K. M. Sanders, personal communication). The deshielding effect of 17cy-hydroxyl or 17a-acetoxyl is clearly an effect of spatial proximity to 14a-H, across

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6.

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Barrett MW, Farrant RD, Kirk DN, Mersh JD, Sanders JKM, Duax WL (1982). The solution conformation of 17a-acetoxy6a-methylprogesterone (‘medroxy progesterone acetate’): use of circular dichroism, nuclear Overhauser difference, and twodimensional J-spectroscopy. J Chem Sot Perkin Tram

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Shaw D (1976). Fourier Transform NMR Spectroscopy. Elsevier, Amsterdam, pp. 336-338. Kirk DN, Toms HC, Douglas C, White KA, Smith KE, Latif S, Hubbard RWP (1990). A survey of the high-field ‘H NMR spectra of the steroid hormones, their hydroxylated derivatives, and related compounds. J Chem Sot Perkin Trams 2~1567-1594.

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Baumann R, Kumar A, Ernst RR, Wuthrich K (1981). Improvement of 2D nOe and 2D correlated spectra by triangular multiplication. .I Magn Reson 44~76-83. Klevitt RE (1985). Improving two-dimensional NMR spectra by t, ridge subtraction. J Magn Reson 62551-555. Wong TC, Rutar V (1984). Homonuclear decoupling in heteronuclear chemical shift correlation spectroscopy. 1. Study of progesterone. J Am Chem Sot 106~7380-7384.