TETRAHEDRON Pergamon

Tetrahedron 57 (2001) 33693372

Long-range 13C 1H coupling constants (3JCH) of monensin sodium

Akito Nagatsu,p Rie Tanaka, Hajime Mizukami, Yukio Ogihara and Jinsaku Sakakibara
Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
Received 15 January 2001; accepted 21 February 2001

AbstractLong-range 13C 1H coupling constants (3JCH) of monensin sodium in CDCl3 and in CD3OD were measured by gs-J-HMBC experiments. The 3JCH of monensin sodium was almost the same both in CDCl3 and in CD3OD. The obtained 3JCH suggested a small difference in the conformation in solvents from that in crystals. q 2001 Elsevier Science Ltd. All rights reserved.

Monensin (Fig. 1) is a representative of naturally occurring polyether acid ionophores, and transports cations, especially the sodium ion, through a biological membrane.1 Monensin attracted much attention because of not only its complex structure but also the conformation when the molecule complexes cations. X-Ray crystal structure analysis was carried out on various salts of monensin and free acid,2 and extensive data regarding the conformations in the crystals were accumulated. We have prepared various monensin derivatives to obtain more potent ionophores3,4 and claried the conformations in the crystals.4 As the ionophores transport ions in the liquid phase and are always surrounded by polar and/or non-polar molecules, we are interested in the conformation in solvents. However, only limited information about the conformation of monensin in solvents has been reported.5 When the conformations of organic compounds in solvents are discussed, the data from NMR spectra such as NOE and/ or 3JHH are usually used. In the case of monensin, we can interpret only limited numbers of 3JHH, because of over31 11 B 13 C 15 32 17

lapping of the methylene and methine proton signals resonated at d 12 ppm. Even in such case, 3JCH should give us considerable information related to CXXH dihedral angles and should reveal the conformational changes in the molecule caused by cation complexation, solvents, or substituents on their functional groups. Some methods for measurements of 3JCH have been developed610 and applied to some molecules in order to determine the conformations11 or the congurations.12 In addition, the higherresolution FT-NMR machine with a highly efcient processing system has been quite popular for routine work during the last decade. Thus, the measurement of 3JCH is much more practical than before with these machines. Here, we report 3JCH of monensin sodium and the conformational difference between in solvents and in crystals based on the 3 JCH. 1. Results and discussion Some methods for measuring 3JXH were reported such as long-range 13C J resolved NMR,6 2D hetero half-ltered TOCSY (HETLOC)7 and other TOCSY-based methods,8 the methods based on the HMQC or HSQC pulse sequence,9 and the methods based on the HMBC pulse sequence.10 We chose the 2D version of gradient-selected HMBC method (gs-J-HMBC)10b,13 to determine 3JCH of monensin sodium. By the HMBC-based methods, we can theoretically detect all 3JCH in the molecule including those between a quaternary carbon and a proton and small 3JCH values. In addition, the gs-J-HMBC method should be a practical method for the researchers who are not extremely skillful in the operation of an NMR spectrometer. The present experiment was carried out with JEOL Lambda500 spectrometer. In order to determine the difference in conformation in polar and non-polar solvents, we performed gs-J-HMBC experiments both in CDCl3 and CD3OD.

O 9 O
5 29 28 MeO 3 27 A 7

34

OH CO2
1

Na+ HO
25

O D
20

O
E 23 36

35

HO

26

Figure 1. Chemical structure of monensin sodium. Keywords: monensin; long-range 13C 1H coupling constants; gs-J-HMBC; conformation. p Corresponding author. Tel./fax: 181-52-836-3437; e-mail: anagatsu@phar.nagoya-cu.ac.jp

00404020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0040-402 0(01)00217-4

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Table 1. Assignments of 13C and 1H in the spectra of monensin sodium in CDCl3 and in CD3OD Position In CDCl3 (ppm) In CD3OD (ppm) Position In CDCl3 (ppm) In CD3OD (ppm)

dC
1 (C) 2 (CH) 3 (CH) 4 (CH) 5 (CH) 6 (CH) 7 (CH) 8 (CH2) 9 (C) 10 (CH2) 11 (CH2) 12 (C) 13 (CH) 14 (CH2) 15 (CH2) 16 (C) 17 (CH) 18 (CH) 181.2 45.0 83.0 37.4 68.3 34.8 70.4 33.5 106.9 39.2 33.2 85.2 82.5 27.2 29.8 85.8 84.9 34.3

dH
2.47 3.12 1.99 3.97 2.15 3.82 1.61, 1.84 1.63, 1.94 1.92, 2.11 3.47 1.50, 1.71 1.39, 2.23 3.87 2.18

dC
183.3 46.0 83.9 38.7 69.1 36.1 71.6 34.4 108.4 40.2 34.0 86.8 83.2 28.1 31.3 87.0 85.9 35.7

dH
2.46 3.12 2.07 3.96 2.02 3.89 1.66, 1.95 1.76, 1.98 1.82, 1.93 3.60 1.53, 1.87 1.47, 2.33 4.00 2.30 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 (CH2) (CH) (CH) (CH) (CH2) (CH) (C) (CH2) (CH3) (CH3) (CH3) (CH3) (CH3) (CH2) (CH3) (CH3) (CH3) (CH3)

dC
33.2 76.4 74.4 31.8 35.6 36.5 98.3 64.8 16.7 57.9 11.0 10.5 27.4 30.6 8.2 14.5 16.8 16.0

dH
1.46, 1.63 4.33 3.76 1.31 1.25, 1.41 1.38 3.22, 3.91 1.17 3.31 1.11 0.87 1.44 1.44, 1.51 0.87 0.83 0.73 0.78

dC
34.3 77.5 76.2 32.9 36.9 37.4 99.2 65.8 16.8 58.3 11.5 11.0 28.1 31.1 8.3 14.9 17.0 16.5

dH
1.61, 2.21 4.42 3.85 1.42 1.38, 1.41 1.55 3.30, 3.91 1.17 3.37 1.10 0.94 1.48 1.49, 1.71 0.93 0.93 0.83 0.85

Before starting the gs-J-HMBC experiment, we checked the assignment of all 1H and 13C of monensin sodium in both solvents by measuring 1H and 13C NMR, HH COSY, HMQC, and HMBC (2,3JCH8.3 Hz) spectra (Table 1). The assignment in CDCl3 was identical with the reported data.14 The 3JCH of monensin sodium both in CD3OD and CDCl3 are indicated in Table 2. From the experiment in CDCl3, 43 3 JCH out of 84 CXXH in the molecule were obtained, and 45 from that in CD3OD. The other 3JCH were not determined probably due to too small 3JCH, too low signal-to-noise ratio of the cross peaks, and/or overlapping of the cross peaks of 3JCH with those of 2JCH. 3JCH in CD3OD and CDCl3 were almost the same in most of the positions. This fact indicated that the conformations of monensin sodium in polar and nonpolar solvent are almost the same and the solvents had no essential inuence on the conformation. In Table 2, the dihedral angles obtained from X-ray crystal structure analysis2d and some 3JCH calculated from the dihedral angles are also indicated. The calculated 3JCH were obtained from the Karplus equations: 3 JCH 5:7 cos2 w 2 0:6 cos w 1 0:5 for COCH,15 and 3 JCH 3:6 cos 2w 2 1:0 cos w 1 4:3 for CCCH16 having no oxygenated carbons on the dihedral intersection. As the Karplus equations for CCCH with the oxygenated carbon(s) on the dihedral intersection have not been established, the calculated 3JCH were not indicated for those systems. The calculated 3JCH around C-23 are found to be different from the data obtained in our experiments as shown in Table 2. The E ring was known to be an exact chair form in the crystal. So 3JC21H23a and 3JC25H23a were expected to be large (.6 Hz) and 3JC21H23b and 3JC25H23b to be small (,3 Hz), but these 3JCH obtained from our experiment were medium (around 4 Hz), except for 3 JC25H23a. The vicinal couplings of H-23a/H-22 and H-23b/H-22 were reported as 3.5 and 11 Hz,5a respectively, which supported the exact chair form of the E ring. However, those of H-23a,b and H-24 were reported unclear and we could not conrm JH23a,bH22 and JH23a,bH24. Thus, it

is likely that the conformation of the E ring is a more or less distorted chair form or the E ring moves in the solvents. There are two other positions showing the much larger 3JCH than those expected from the crystal data. One is 3JC20H17 (5.4 Hz in CDCl3 and 5.1 Hz in CD3OD), which indicated that the dihedral angle was not around 908. This data suggested the conformational change of the D ring. The other is 3JC11H13, which can be expected to be medium (around 23 Hz) from the dihedral angle in the crystal, although the 3JCH was not calculated. The value of 3 JC11H13 (7.4 Hz) in the solvents was large enough to suggest that the dihedral angle was around 0 or 1808 and that the bond between the B and C rings might rotate or might be a different conformation from the crystal. The 3JC4H2, 3JC5H3, and 3JC29H3 in the chain part at C1C5 seemed smaller than those deduced from the corresponding dihedral angles in the crystal data. However, 3JCH with oxygenated carbon(s) on the dihedral intersection frequently showed smaller values relative to those without oxygenated carbon(s).17 So the 3JCH values of the chain part were possible to indicate the same dihedral angles as crystals. The 3 JHH values (Table 3) of the chain part exhibited that H2H3 and H4H5 were anti and H3H4 was gauche and 2JC3H2 (Table 4) suggested H2O3 was gauche.12 These data indicated that the conformation of C1C5 in the solvents should be identical with that in the crystal. In conclusion, we have demonstrated the measurement of 3 JCH of monensin, a complex but well-known molecule, and claried that the conformations of monensin in nonpolar and aprotic CDCl3 and in polar and protic CD3OD were identical. At the same time, the 3JCH together with 3 JHH and 2JCH suggested that the conformation in solvents might be a little different from that in crystals not at chain part but at the rings, although the relation between 3JCH and dihedral angles was not sufciently established yet and it may be risky to discuss the conformation based on the 3JCH. The obtained 3JCH should constitute a basic data set to compare the conformation of monensin with those of the

A. Nagatsu et al. / Tetrahedron 57 (2001) 33693372 Table 2. 3JCH of monensin sodium in CDCl3 and CD3OD obtained from gs-J-HMBC experiment and calculated from X-ray crystal structure analysis CXXH CDCl3 C1C2C3H3 C2C3C4H4 C3C4C5H5 C4C3C2H2 C4C5C6H6 C5C4C3H3 C5C6C4H7 C6C5C4H4 C6C7C8H8a C6C7C8H8b C7C6C5H5 C8C7C6H6 C8C9C10H10a C8C9C10H10b C9OC5H5 C9C8C7H7 C9C10C11H11a C9C10C11H11b C10C9C8H8a C10C9C8H8b C11C12C13H13 C12C11C10H10a C12C11C10H10b C12C13C14H14a C12C13C14H14b C13C12C11H11a C13C12C11H11b C13C14C15H15a C13C14C15H15b C15C14C13H13 C15C16C17H17 C15C16C32H32a C15C16C32H32b C16C15C14H14a C16C15C14H14b C16OC13H13 C16C17C18H18 C17C16C32H32a C17C16C32H32b C17C16C15H15a C17C16C15H15b C17OC20H20
a b c d 3

3371

JCH (Hz) CD3OD 1.9 Nda Nda 1.3 Nda 1.2 4.5 Nda 4.2 Nda 1.1 Nda Ndb Ndb 1.7 7.6 Ndb 4.5 Ndb 1.0 Nda Ndb Ndb Ndb Ndb Nda 6.1 3.2 4.1 Nda Ndb 2.6 1.9 Ndb Ndb 2.6 Ndb Nda 3.7 Nda 4.3 2.2 Calcd

X-Ray2d (deg)

CXXH CDCl3

JCH (Hz) CD3OD Calcd 0.9d 7.1d

X-Ray2d (deg)

1.6 Nda Nda 1.0 Nda 1.6 4.2 Nda 3.6 Nda 1.6 Nda Ndb Ndb 1.7 7.4 2.1 5.7 3.8 Nda 7.4 Ndb Ndb Nda Nda 4.4 2.8 ,1.0 4.3 0.9 3.4 1.2 Nda Nda Nda 2.4 Ndb Nda Nda Ndc 6.3 ,1.0

2.0c 0.7d 6.5d

7.4d 0.7d

2.3d 4.5d

4.1d 1.2d 0.7c

2.4c

274.8 168.5 71.6 61.5 64.1 252.4 174.8 261.3 69.1 2174.5 259.2 2172.6 279.9 32.6 255.6 164.9 84.4 2146.2 253.5 61.6 266.2 154.3 288.2 33.2 292.0 2134.0 219.3 114.2 2131.0 86.0 161.9 233.2 75.4 128.3 2102.3 75.1 281.0 2165.6 257.0 2112.6 4.5 122.0

C17C18C19H19a C17C18C19H19b C18C19C20H20 C19C18C17H17 C19C20C21H21 C20OC17H17 C20C19C18H18 C20C21C22H22 C21C20C19H19a C21C20C19H19b C21C22C23H23a C21C22C23H23b C22C21C20H20 C22C23C24H24 C23C22C21H21 C24C23C22H22 C24C25C26H26a C24C25C26H26b C25OC21H21 C25C24C23H23a C25C24C23H23b C26C25C24H24 C27C2C3H3 C28OC3H3 C29C4C3H3 C29C4C5H5 C30C6C7H7 C30C6C5H5 C31C12C11H11a C31C12C11H11b C31C12C13H13 C32C16C15H15a C32C16C15H15b C32C16C17H17 C34C18C17H17 C34C18C19H19a C34C18C19H19b C35C22C21H21 C35C22C23H23a C35C22C23H23b C36C24C23H23a C36C24C23H23b

Nda Ndb Nda 1.4 4.5 5.4 6.2 Nda 4.9 Nda 3.6 Nda Nda Ndb 1.3 Nda Nda Nda Nda 6.1 3.0 Nda 3.2 5.7 4.8 3.2 1.3 4.5 Nda Nda 1.2 5.3 Nda 3.8 6.6 Nda 3.9 0.7 Ndb Ndb Ndb Ndb

Nda 6.6 Nda 1.7 4.7 5.1 5.8 2.8 5.0 Nda 3.2 3.6 Ndb Ndb 1.7 Nda 1.0 Nda 1.6 4.3 3.5 3.8 2.8 5.7 4.4 Ndb 1.1 Ndb 7.1 Nda 1.3 6.0 3.8 Ndb 6.4 Nda 4.3 1.1 Ndb Ndb Ndb Ndb

0.5c 7.3d

8.9d 1.3d 2.5d 1.4d 1.3c 8.9d 1.6d 4.9c

8.1d 5.4d 1.9d 2.4d 2.0d 2.0d

75.7 2151.4 293.6 75.0 2180.0 84.6 152.7 255.6 228.5 96.2 175.4 268.0 68.0 55.3 268.5 66.5 50.6 256.5 265.0 175.8 264.3 257.7 48.8 21.9 2180.0 256.3 49.3 176.4 28.0 106.7 60.8 18.3 135.5 267.6 2164.3 2161.5 228.6 54.6 261.1 55.5 260.0 59.9

Not determined because of too small s/n values including too small 3JCH values. Not determined because of overlapping of the cross peaks. Calculated with Karplus equations 3 JCH 5:7 cos 2 w 2 0:6 cos w 1 0:5 where w are the corresponding dihedral angles. Calculated with Karplus equations 3 JCH 3:6 cos 2w 2 1:0 cos w 1 4:3 where w are the corresponding dihedral angles.

Table 3. Determined HH coupling constants of monensin sodium (Hz) In CDCl3 J 23 J 34 J 45 J 56 J 67 J 68a J 78a J 78b J 1314a J 1314b J 1718 J 1819a J 1819b J 19a20 J 19b20 J 2021 J 2122
a

In CD3OD 10.1 2.3 11.5 2.3 2.3 4.3 4.1 4.9 10.4 3.6 0.0 6.5 7.2 9.9 4.1 9.9

derivatives. Investigation of 3JCH of dicarboxylic monensin derivatives3 we prepared is now in progress. 2. Experimental Monensin sodium recrystallized from n-hexaneEt2O (40 mg) was dissolved in CDCl3 or CD3OD (0.55 ml) and packed in the NMR tube (f 5 mm). The gs-J-HMBC experiments were carried out with JEOL JMN Lambda-500 spectrometer at 268C. The spectra were recorded with a data size of 4K (F2)128 (F1) points for the spectral width of 2000 Hz (1H) and 22600 Hz (13C) with a 200 ms constant time. Ten gs-J-HMBC spectra in CDCl3 were acquired with 84 scans (ca. 8 h) and those in CD3OD with 80 scans (ca. 7.5 h) per increment with varying the pulse interval time (D) every 20 ms within 20200 ms. Two-fold zero-lling was conducted for F1 dimension and the digital resolution in F2 were 0.49 Hz. The signal amplitudes were determined

10.2 2.4 11.0 2.6 3.9 3.4 4.4 3.4 5.0 10.6 3.5 0.0 6.3 7.0 10.2 4.1 9.8

W-coupling.

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A. Nagatsu et al. / Tetrahedron 57 (2001) 33693372

Table 4. Determined 2JCH of monensin sodium in CDCl3 and CD3OD by gs-J-HMBC experiment
2

JCH (Hz) In CD3OD 5.9 6.3 3.1 3.0 2.9 6.7 ,1.0 2.7 3.6 2.6 2.6 3.5 C16C15H15a or b C16C17H17 C18C17H17 C34C18H18 C18C19H19a C18C19H19b C20C19H19a C20C19H19b C21C20H20 C20C21H21 C22C23H23a or b C24C23H23a or b C25C26H26a or b C33C32H32a C33C32H32b In CDCl3 2.4 4.6 1.2 2.3 2.1 3.3 5.8 3.2 3.8 3.5 3.9 3.8 2.1

JCH (Hz) In CD3OD 4.7 1.9

In CDCl3 C1C2H2 C3C2H2 C27C2H2 C2C3H3 C4C5H5 C7C6H6 C30C6H6 C6C7H7 C8C7H7 C7C8H8a C7C8H8b C9C8H8a or b C9C10H10a or b C12C13H13 C15C14H14a or b C14C15H15a or b 6.1 6.3 2.4 2.6 6.7 4.6 1.8 5.2 1.1 2.2 3.0 2.3 1.2

4.2 4.2 1.4 4.3 4.0

from peak heights recorded using the peak-picking program of the JEOL JMN Lambda-500 data processing software. The obtained data were tted to a sine curve by the leastsquares method. In order to assign the 1H of monensin sodium more clearly, the 1H NMR spectra were also recorded with JEOL Eclipse 800 spectrometer. Acknowledgements We thank Mr H. Utsumi and Mr N. Esumi of JEOL, and Ms S. Kato of this faculty for kind advice on measurement of gs-J-HMBC. We also thank Dr K. Suenaga and Professor D. Uemura of Faculty of Science, Nagoya University, for the measurement of the 1H NMR spectra with 800 MHz spectrometer. This work was nancially supported in part by a Grant-in-Aid for Scientic Research and the Hi-tech Research Center Project from the Ministry of Education, Sports, and Culture of Japan. References
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