Solid State Nuclear Magnetic Resonance, 1 (1992) 121-125
121
Elsevier Science Publishers B.V., Amsterdam
Proton relaxation NMR study of polycrystalline progesterone E.R. Andrew, K. Jurga ‘, J.M. Radomski ’ and E.C. Reynhardt
2
Departments of Physics and Radiology, University of Florida, Gainesville, FL 32611, USA
(Received 25 February 1992; accepted 10 March 1992)
Abstract
Polycrystalline progesterone (4-pregnene-3,20-dione, C,,H,,Oz) has been investigated by proton NMR methods between 80 and 3.50 K. A reduction in dipolar second moment is ascribed to methyl group reorientation. Minima in the spin-lattice relaxation time found in measurements at five frequencies from 7 to 200 MHz are attributed to reorientation of two of the three methyl groups in each molecule, characterized by activation energy E, = 10.9 + 0.8 kJ/mol and re = (2.3 + 0.2) x lo-r3 s. Additional relaxation at lower temperatures is attributed to reorientation of the third methyl group with E, approximately 3.4 kJ/mol. Measurements were also made of relaxation in the rotating frame. Keywords: progesterone;
proton NMR; relaxation; molecular dynamics; methyl group reorientation
Introduction Progesterone (4-pregnene-3,20-dione) is a hormone secreted by the female reproductive system which regulates the condition of the inner lining of the uterus. It is produced by the ovaries, placenta and adrenal glands. It plays a role in the menstrual process and also in pregnancy. Among other functions it inhibits ovulation during pregnancy and synthetic derivatives form the active agent in many contraceptive pills. Pure progesterone is a white powder at room temperature with melting point 401-402 K [l]. The molecular structure is shown in Fig. 1. X-ray studies have shown that the crystal structure is
Correspondence to: Professor E.R. Andrew, Department of Physics. University of Florida, Gainesville, FL 32611, USA. ’ On leave from Institute of Physics, A. Mickiewicz University, 60-780 Poznan, Poland. ’ On leave from Department of Physics, University of South Africa, P.O. Box 392, Pretoria, South Africa.
0926-2040/92/$05.00
orthorhombic, space group P2,2,2,, with cell dimensions a = 12.559 A, b = 13.798 A, c = 10.340 A [2]. Each unit cell contains four C,,HaOO, molecules. Progesterone is a steroid with the same fused group of four carbon rings as cholesterol and cortisone, both recently studied in the solid state by NMR [3,4].
a
6
Fig. 1. Molecular structure of progesterone. The carbon skeleton is shown without hydrogen atoms.
0 1992 - Elsevier Science Publishers B.V. All rights reserved
E.R. Andrew et al. /Solid
122
glass ampoules after evacuation for several days at room temperature. Measurements of Ti at 7, 14 and 25 MHz were made using a home-built, variable-frequency pulse spectrometer in conjunction with a Varian electromagnet and Nicolet 1180 signal averaging system. The temperature was controlled by a platinum thermometer and Muller bridge to an accuracy and stability of N 1 K. Ti was measured by saturation recovery following a train of sixteen 90” pulses. Measurements of T, at 30 and 200 MHz were made using a Bruker CXP 200 spectrometer. The magnetization was found to re-
NMR relaxation studies in the solid state provide insight into molecular dynamics of both intramolecular and intermolecular motions. In this investigation measurements were made of T, at several frequencies and also of T,, and second moment from 80 to 350 K.
Experimental Polycrystalline progresterone was supplied by Sigma, Product No. P-0130, Standard for Chromatography grade. Samples were sealed off in
3
4
5
6
7
8
1000/T
9
10
State Nucl. Magn. Reson. I (1992) 121-125
11
12
13
(K-l)
Fig. 2. Temperature dependences of the proton relaxation times T, and T,, for polycrystalline progesterone. MHz; A 25 MHz; v 14 MHz; 1-7 MHz; * TIP.
q
200 MHz; 030
E.R. Andrew et al. /Solid
State Nucl. Magn. Reson. I (1992) 121-125
cover exponentially within experimental error ( N 5%) at all temperatures. Measurements of TIP were made at 200 MHz in an rf magnetic field of 10 G [SJ. Proton second moments M, were obtained by numerical integration of Cw first derivative spectra recorded with a Spin-Lock spectrometer operating at 50 MHz. Corrections for finite modulation amplitude were made [6]. Less accurate but mutually consistent values of Mz were also obtained from the initial shape of the FID, assuming a Gaussian lineshape [7j. Errors in determination of TI were estimated to be N 5%, those in TIP N 10% and those in M2 N 10%.
Rfsults
The temperature dependence of the proton spin-k&ice relaxati times Tr for s&d progesterone are dispIayed on a logarithmic scale against inverse temperature in Fig. 2. This figure also shows the measured values of TIP. The temperature variation of the second moment A& is shown in Fig. 3. An initial fati at 100 K is followed by a slow decrease from 22 G* at
I33
120 K to 21 G2 at 300 K, the decrease being barely experimentally significant. Mscussion First we consider the second mm’ Par molecular solids with known crystal ~&UC&& experimental values of secoad moment may be compared with dues htah$ ushns tan,* theary of Van VI&C f8] tir &jid *#truj#&l
tion of the protans‘we &&S&B& lattice value of M2 is 28~3 G2 &8-I?& tiaily higher than th& vaiw led ( Wecondudethatthe&id~jsonQ~ at lower temperatures and t&t $6&U ,$0&j&* molecular motiob reduces G2 fowdat1ufK.TW reorientatiun of tbt three their C, axes, Aesrrming mob&zd we c-&&b @-1a of f&i2of 21.8 CP, in gwd a experimental valud. l%e sI0w 120 and 300 K to 21 G* expansion of the crystal lattice.
25
18 100
120
140
160
180
200
TEMPERATURE
220
240
260
280
300
(K)
Fig. 3. Temperature dependence of the second moment of poiycrystanine progesterone.
E.R. Andrew et al. /Solid State Nucl. Magn. Reson. I (1992) 121-125
124
The spin-lattice relaxation time T,, shown in Fig. 2, demonstrates a minimum at each measuring frequency, w0/(27r>, characteristic of the type first described by Bloembergen et al. [13] and subsequently analyzed in detail by Kubo and Tomita [14]. The data exhibit in an accurate manner the essential features of the BPP behaviour: (1) on the high temperature side of the minima where &r,” < 1, T, is independent of o0 (T, is the correlation time of the molecular motion), (2) on the low temperature side of the minima where air,’ Z+ 1, T, is proportional to w& and (3) at the minima TI is proportional to w,,. The data have been least-squares fitted by computer using the same parameters at all frequencies of measurements to the relaxation expression of Kubo and Tomita [14]:
T,1
-c
rc
0 c i 1 + U2T2
+
47, 1 + 4&,2
assuming that the correlation Arrhenius activation law
1
(1)
time 7c follows an
TV= 7. exp( EJRT)
(2)
For relaxation in the rotating frame the theoretical expression is [15,16]: 1 -=_ T 1P
37,
c
1 c 2 [ 1 +4m2r2
+
57, 0 E 1 + W2T2
+
27,
1 (3)
0 c 1 + 4w2r2
The best agreement, shown by the lines in Fig. 1, was obtained with C = (1.47 k 0.09) X lo9 se2, E, = 10.9 f 0.8 kJ/mol, r0 = (2.3 k 0.2) X lo-l3 s. Bearing in mind the wide range of measuring frequencies for TI and also the measurements of TIP and that the measurements were made on different instruments, the agreement of the least-squares fitted calculations with the data is excellent. Some detail of the relaxation mechanism may be obtained by consideration of the derived value of the relaxation constant C. For methyl group reorientation in the presence of rapid proton spin exchange, the value of C is given by [3,17]: 9 C=_.--.20
n
yW
N
b6
where N is the total number of protons in the molecule, n is the number of protons in the methyl groups contributing to the relaxation process and b is the interproton distance in the methyl group. For progesterone N is 30 and taking for b the value 1.80 A, we obtain values of C of 0.73, 1.46 and 2.18 X lo9 ss2 for relaxation by one, two and three methyl groups, respectively. Since the experimental value of C is 1.47 x lo9 sp2, it seems probable that just two of the three methyl groups in the molecule contribute to the main relaxation minima in solid progesterone and that these two groups have similar molecular hindrances. An examination of the molecule suggest that these two methyl groups might be those at positions 18 and 19 (see Fig. 1). This identification could be confirmed by isotopic substitution with CD, groups. Figure 2 shows that the experimental values of TI fall again at lower temperatures, for example, below 110 K for T, measurements at 200 MHz, suggesting an additional low temperature mechanism. An additional independent source of relaxation with E, = 3.4 kJ/mol was therefore included in the calculations to fit the lower temperature data. Measurements below the lower limit of our cryostat (77 K) would be of interest in order to observe a possible relaxation minimum at lower temperatures and to characterize the motion more accurately. This further motion may be attributed to the reorientation of the third methyl group at position 21 in the molecule (Fig. 1). This methyl group, projected well away from the steroid skeleton, may be less hindered than the other two.
(4)
Acknowledgement This work was supported by NIH through grant P41 RR02278.
References 1 0. Dideberg and L. DuPont, J. A&. Crystallogr., 4 (1971) 80. 2 H. Campsteyn, L. DuPont and 0. Dideberg, Acfa Crystallogr. Sect. B, 28 (1972) 3032.
E.R. Andrew et al. /Solid
State Nucl. Magn. Reson. 1 (1992) 121-125
3 E.R. Andrew and B. Peplinska, Mol. Phys., 70 (1990) 505. 4 E.R. Andrew and M.F. Kempka, Proc. 10th ISMAR Meeting, Morzine Pl-11, 1989 International Society of Magnetic Resonance (ISMAR), Grenoble. 5 D.C. Look and I.J. Lowe, J. Chern. Phys., 44 (1966) 2995. 6 E.R. Andrew, Phys. Rec., 91 (1953) 425. 7 J. Jeneer and P. Brookaert, Phys. Rev., 157 (1967) 232. 8 J.H. Van Vleck. Phys. Rev., 74 (1948) 1168. 9 E.R. Andrew, Nuclear Magnetic Resonance, Cambridge University Press, Cambridge, 1955.
125
10 11 12 13 14 15 16 17
G.W. Smith, J. Chem. Phys., 42 (1965) 4229. G.W. Smith, J. Chem. Phys., 51 (1969) 3569. R.L. Hilt and P.S. Hubbard, Phys. Rec. A, 134 (1964) 392. N. Bloembergen, E.M. Purcell and R.V. Pound, Phys. Rev., 13 (1948) 679. R. Kubo and K. Tomita, J. Phys. Sot. Jpn., 9 (1954) 888. G.P. Jones, Phys. Rev., 148 (1966) 332. D.E. 0’ Reilly and T. Tsang, Phys. Rev., 157 (1967) 417. E.R. Andrew, W.S. Hinshaw, M.G. Hutchins and R.0.1. Sjoblom, Mol. Phys., 34 (1977) 1965.
121
Elsevier Science Publishers B.V., Amsterdam
Proton relaxation NMR study of polycrystalline progesterone E.R. Andrew, K. Jurga ‘, J.M. Radomski ’ and E.C. Reynhardt
2
Departments of Physics and Radiology, University of Florida, Gainesville, FL 32611, USA
(Received 25 February 1992; accepted 10 March 1992)
Abstract
Polycrystalline progesterone (4-pregnene-3,20-dione, C,,H,,Oz) has been investigated by proton NMR methods between 80 and 3.50 K. A reduction in dipolar second moment is ascribed to methyl group reorientation. Minima in the spin-lattice relaxation time found in measurements at five frequencies from 7 to 200 MHz are attributed to reorientation of two of the three methyl groups in each molecule, characterized by activation energy E, = 10.9 + 0.8 kJ/mol and re = (2.3 + 0.2) x lo-r3 s. Additional relaxation at lower temperatures is attributed to reorientation of the third methyl group with E, approximately 3.4 kJ/mol. Measurements were also made of relaxation in the rotating frame. Keywords: progesterone;
proton NMR; relaxation; molecular dynamics; methyl group reorientation
Introduction Progesterone (4-pregnene-3,20-dione) is a hormone secreted by the female reproductive system which regulates the condition of the inner lining of the uterus. It is produced by the ovaries, placenta and adrenal glands. It plays a role in the menstrual process and also in pregnancy. Among other functions it inhibits ovulation during pregnancy and synthetic derivatives form the active agent in many contraceptive pills. Pure progesterone is a white powder at room temperature with melting point 401-402 K [l]. The molecular structure is shown in Fig. 1. X-ray studies have shown that the crystal structure is
Correspondence to: Professor E.R. Andrew, Department of Physics. University of Florida, Gainesville, FL 32611, USA. ’ On leave from Institute of Physics, A. Mickiewicz University, 60-780 Poznan, Poland. ’ On leave from Department of Physics, University of South Africa, P.O. Box 392, Pretoria, South Africa.
0926-2040/92/$05.00
orthorhombic, space group P2,2,2,, with cell dimensions a = 12.559 A, b = 13.798 A, c = 10.340 A [2]. Each unit cell contains four C,,HaOO, molecules. Progesterone is a steroid with the same fused group of four carbon rings as cholesterol and cortisone, both recently studied in the solid state by NMR [3,4].
a
6
Fig. 1. Molecular structure of progesterone. The carbon skeleton is shown without hydrogen atoms.
0 1992 - Elsevier Science Publishers B.V. All rights reserved
E.R. Andrew et al. /Solid
122
glass ampoules after evacuation for several days at room temperature. Measurements of Ti at 7, 14 and 25 MHz were made using a home-built, variable-frequency pulse spectrometer in conjunction with a Varian electromagnet and Nicolet 1180 signal averaging system. The temperature was controlled by a platinum thermometer and Muller bridge to an accuracy and stability of N 1 K. Ti was measured by saturation recovery following a train of sixteen 90” pulses. Measurements of T, at 30 and 200 MHz were made using a Bruker CXP 200 spectrometer. The magnetization was found to re-
NMR relaxation studies in the solid state provide insight into molecular dynamics of both intramolecular and intermolecular motions. In this investigation measurements were made of T, at several frequencies and also of T,, and second moment from 80 to 350 K.
Experimental Polycrystalline progresterone was supplied by Sigma, Product No. P-0130, Standard for Chromatography grade. Samples were sealed off in
3
4
5
6
7
8
1000/T
9
10
State Nucl. Magn. Reson. I (1992) 121-125
11
12
13
(K-l)
Fig. 2. Temperature dependences of the proton relaxation times T, and T,, for polycrystalline progesterone. MHz; A 25 MHz; v 14 MHz; 1-7 MHz; * TIP.
q
200 MHz; 030
E.R. Andrew et al. /Solid
State Nucl. Magn. Reson. I (1992) 121-125
cover exponentially within experimental error ( N 5%) at all temperatures. Measurements of TIP were made at 200 MHz in an rf magnetic field of 10 G [SJ. Proton second moments M, were obtained by numerical integration of Cw first derivative spectra recorded with a Spin-Lock spectrometer operating at 50 MHz. Corrections for finite modulation amplitude were made [6]. Less accurate but mutually consistent values of Mz were also obtained from the initial shape of the FID, assuming a Gaussian lineshape [7j. Errors in determination of TI were estimated to be N 5%, those in TIP N 10% and those in M2 N 10%.
Rfsults
The temperature dependence of the proton spin-k&ice relaxati times Tr for s&d progesterone are dispIayed on a logarithmic scale against inverse temperature in Fig. 2. This figure also shows the measured values of TIP. The temperature variation of the second moment A& is shown in Fig. 3. An initial fati at 100 K is followed by a slow decrease from 22 G* at
I33
120 K to 21 G2 at 300 K, the decrease being barely experimentally significant. Mscussion First we consider the second mm’ Par molecular solids with known crystal ~&UC&& experimental values of secoad moment may be compared with dues htah$ ushns tan,* theary of Van VI&C f8] tir &jid *#truj#&l
tion of the protans‘we &&S&B& lattice value of M2 is 28~3 G2 &8-I?& tiaily higher than th& vaiw led ( Wecondudethatthe&id~jsonQ~ at lower temperatures and t&t $6&U ,$0&j&* molecular motiob reduces G2 fowdat1ufK.TW reorientatiun of tbt three their C, axes, Aesrrming mob&zd we c-&&b @-1a of f&i2of 21.8 CP, in gwd a experimental valud. l%e sI0w 120 and 300 K to 21 G* expansion of the crystal lattice.
25
18 100
120
140
160
180
200
TEMPERATURE
220
240
260
280
300
(K)
Fig. 3. Temperature dependence of the second moment of poiycrystanine progesterone.
E.R. Andrew et al. /Solid State Nucl. Magn. Reson. I (1992) 121-125
124
The spin-lattice relaxation time T,, shown in Fig. 2, demonstrates a minimum at each measuring frequency, w0/(27r>, characteristic of the type first described by Bloembergen et al. [13] and subsequently analyzed in detail by Kubo and Tomita [14]. The data exhibit in an accurate manner the essential features of the BPP behaviour: (1) on the high temperature side of the minima where &r,” < 1, T, is independent of o0 (T, is the correlation time of the molecular motion), (2) on the low temperature side of the minima where air,’ Z+ 1, T, is proportional to w& and (3) at the minima TI is proportional to w,,. The data have been least-squares fitted by computer using the same parameters at all frequencies of measurements to the relaxation expression of Kubo and Tomita [14]:
T,1
-c
rc
0 c i 1 + U2T2
+
47, 1 + 4&,2
assuming that the correlation Arrhenius activation law
1
(1)
time 7c follows an
TV= 7. exp( EJRT)
(2)
For relaxation in the rotating frame the theoretical expression is [15,16]: 1 -=_ T 1P
37,
c
1 c 2 [ 1 +4m2r2
+
57, 0 E 1 + W2T2
+
27,
1 (3)
0 c 1 + 4w2r2
The best agreement, shown by the lines in Fig. 1, was obtained with C = (1.47 k 0.09) X lo9 se2, E, = 10.9 f 0.8 kJ/mol, r0 = (2.3 k 0.2) X lo-l3 s. Bearing in mind the wide range of measuring frequencies for TI and also the measurements of TIP and that the measurements were made on different instruments, the agreement of the least-squares fitted calculations with the data is excellent. Some detail of the relaxation mechanism may be obtained by consideration of the derived value of the relaxation constant C. For methyl group reorientation in the presence of rapid proton spin exchange, the value of C is given by [3,17]: 9 C=_.--.20
n
yW
N
b6
where N is the total number of protons in the molecule, n is the number of protons in the methyl groups contributing to the relaxation process and b is the interproton distance in the methyl group. For progesterone N is 30 and taking for b the value 1.80 A, we obtain values of C of 0.73, 1.46 and 2.18 X lo9 ss2 for relaxation by one, two and three methyl groups, respectively. Since the experimental value of C is 1.47 x lo9 sp2, it seems probable that just two of the three methyl groups in the molecule contribute to the main relaxation minima in solid progesterone and that these two groups have similar molecular hindrances. An examination of the molecule suggest that these two methyl groups might be those at positions 18 and 19 (see Fig. 1). This identification could be confirmed by isotopic substitution with CD, groups. Figure 2 shows that the experimental values of TI fall again at lower temperatures, for example, below 110 K for T, measurements at 200 MHz, suggesting an additional low temperature mechanism. An additional independent source of relaxation with E, = 3.4 kJ/mol was therefore included in the calculations to fit the lower temperature data. Measurements below the lower limit of our cryostat (77 K) would be of interest in order to observe a possible relaxation minimum at lower temperatures and to characterize the motion more accurately. This further motion may be attributed to the reorientation of the third methyl group at position 21 in the molecule (Fig. 1). This methyl group, projected well away from the steroid skeleton, may be less hindered than the other two.
(4)
Acknowledgement This work was supported by NIH through grant P41 RR02278.
References 1 0. Dideberg and L. DuPont, J. A&. Crystallogr., 4 (1971) 80. 2 H. Campsteyn, L. DuPont and 0. Dideberg, Acfa Crystallogr. Sect. B, 28 (1972) 3032.
E.R. Andrew et al. /Solid
State Nucl. Magn. Reson. 1 (1992) 121-125
3 E.R. Andrew and B. Peplinska, Mol. Phys., 70 (1990) 505. 4 E.R. Andrew and M.F. Kempka, Proc. 10th ISMAR Meeting, Morzine Pl-11, 1989 International Society of Magnetic Resonance (ISMAR), Grenoble. 5 D.C. Look and I.J. Lowe, J. Chern. Phys., 44 (1966) 2995. 6 E.R. Andrew, Phys. Rec., 91 (1953) 425. 7 J. Jeneer and P. Brookaert, Phys. Rev., 157 (1967) 232. 8 J.H. Van Vleck. Phys. Rev., 74 (1948) 1168. 9 E.R. Andrew, Nuclear Magnetic Resonance, Cambridge University Press, Cambridge, 1955.
125
10 11 12 13 14 15 16 17
G.W. Smith, J. Chem. Phys., 42 (1965) 4229. G.W. Smith, J. Chem. Phys., 51 (1969) 3569. R.L. Hilt and P.S. Hubbard, Phys. Rec. A, 134 (1964) 392. N. Bloembergen, E.M. Purcell and R.V. Pound, Phys. Rev., 13 (1948) 679. R. Kubo and K. Tomita, J. Phys. Sot. Jpn., 9 (1954) 888. G.P. Jones, Phys. Rev., 148 (1966) 332. D.E. 0’ Reilly and T. Tsang, Phys. Rev., 157 (1967) 417. E.R. Andrew, W.S. Hinshaw, M.G. Hutchins and R.0.1. Sjoblom, Mol. Phys., 34 (1977) 1965.
