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Conformational analysis of sisomicin and penta-N-di-O-acetylsisomicin using NMR spectroscopy and molecular mechanics calculations
Journal of Molecular Structure, 291 (1993) 191-195 Elsevier Science Publishers B.V., Amsterdam
191
Conformational analysis of sisomicin and penta-WdiO-acetylsisomicin using NMR spectroscopy and molecular mechanics calculations G.I. Enevaa, S.L. Spassova,’ and M.A. Haimovab aInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy 1113 Sofia (Bulgaria) bFaculty of Chem istry, University of Sofia, 1126 Sofia (Bulgaria)
of Sciences,
(Received 9 October 1992)
Abstract The conformations of sisomicin and penta-N-di-0-acetylsisomicin were investigated by NMR spectroscopy and molecular mechanics calculations. Experimental NOE data for aqueous solutions were in accordance with the computational data.
Introduction
Experimental
The biological importance of aminoglycoside antibiotics have stimulated interest in a detailed knowledge of their conformation in solution. We now report results concerning the preferred conformations of the aminoglycosides sisomicin (1) and 1,3,2’,6’,3”-penta-N-acetyl-2”,4”-di-O-acetylsisomicin (2) (Fig. l), based on NMR spectral studies and computational methods. The NMR data were acquired using 1D and 2D NMR techniques, with special emphasis on nuclear Overhauser effect @JOE) experiments (DNOE, NOESY). The computations permitted quantitative estimation of NOE, and their comparison with the experimental data yielded information about the conformational preference with respect to the glycosidic bonds in aqueous solution.
Samples of 1 (free base) and 2 were obtained as described previously [l]. The ‘H NMR spectra were obtained on a Bruker WM-250 spectrometer operating at 250.1 MHz. All spectra were measured on 0.1 molar solutions in DzO, at ambient temperature (x 300 K), pH 10 for 1 and pH 6 for 2, with internal standard acetone (2.22ppm). The ‘H NOE experiments were performed in the difference mode (DNOE), with a preirradiation time of 10 s. The two-dimensional NOESY plots were obtained on a Varian XL-300 spectrometer (299.94MHz for ‘H) using the standard Varian software. The calculations of the conformations with respect to the glycosidic bonds were carried out with the MM2(85)-based programme PCMODEL (88.0) using default parameters on a PC-AT computer. The energy minimizations were performed on different starting conformations with consecutive variation of the torsional angles 4, , &, $, and $*.
Correspondence to: Professor S.L. Spassov, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
0022-2860/93/%06.00 0 1993 Elsevier Science Publishers B.V. All rights reserved.
192
G.I. Eneva et al./J. Mol. Struct., 291 (1993) 191-195
TABLE 1 Data on the minimum energy conformations Conformer
of 1 and 2 from molecular mechanics calculations r (H l’-H4)
cp, (“)
(62(“)
la lb lc Id le 2a 2b
2c 2d 2e
& (kcalmol-‘)
-
16.6 17.7 20.9 15.5 10.4
113.5 113.5 114.0 113.2 113.9
2.27 2.27 2.33 2.33 2.23
- 37.3 - 37.4 -45.2 2.8 3.6
- 13.3 -8.9 - 25.0 38.7 39.2
113.8 113.9 114.0 115.6 115.9
2.22 2.19 2.43 2.21 2.23
0.0 0.3 0.5 2.2 3.3
-57.6 -52.1 -61.7 - 55.7 -25.7
- 18.3 - 13.6 16.7 -8.1 - 10.2
114.8 114.0 115.2 114.7 115.4
2.55 2.41 2.59 2.42 2.15
- 14.9
49.4 48.0 50.7 49.9 52.3
115.9 116.0 116.0 116.3 116.0
2.23 2.26 2.24 2.25 2.27
0.0 0.5 2.3 2.7 3.9
The chemical structure of 1 has been established by chemical and spectroscopic studies [2]. The complete ‘H and 13C NMR assignment and parameter determination of 1 and 2 was reported in an earlier paper [l]. The coupling constants found [l] for the deoxystreptamine and aminoglucose moieties indicate essentially undistorted 4C, chair conformations of the saturated six-membered rings. The half-chair ~onfo~ation of the dihydropyran residue in 1 and 2 is clearly reflected in the different vi&al couplings observed [l]. The deoxystreptamine residue in the 1’ position and the amino group in the 2’ position are cis to each other [2]; and one should be axial and the other equatorial. Empirical force field calculations performed on a model compound with the MMP2(85) force field [3,4] predicted the conformer with equatorial
-6.3 - 15.5 - 12.9 - 14.7
amino and axial deoxystreptamine residues to be the more stable one, in agreement with the calculated [S] vicinal coupling constant values fl]. The conformational preference with respect to the glycosidic linkages, which remained to be established, is important for understanding the chemical and biological behaviour of such molecules in solution. Investigations of that type by NMR and computational methods (HSEA) have been reported TABLE 2 Observed and calculated NOE data for the minimum energy conformations of compounds 1 and 2 Proton irrad.
1’ 4 1”
OR CH, 4-
R’ 3 %
(H 1“H6)
-39.3 -38.4 - 39.6 -46.0 -38.8
Results and discussion
C&N
(A)
r
72 (“1
(A)
6 2 2”
“0 RO
RHN 0 RHN 1 -k_
OH 2
’ :
4
?J 0 3
Proton obs.
4 2 1’ 6 2” 1” 1’ 1”
NOE exp. (%)
11 13 9 12 16 8 8 7
14’ CH,NHR
NHR
Fig. 1. Structure of: sisomicin (l), R = H; 1,3,2’,6’,3”-pentaN-acetyl-2”,4”-di-O-acetylsisomicin (2), R = COCH,.
1’ 1” 2”
4 6 1”
11 11 6
Calculated NOE(%)
la
lb
lc
ld
le
10.0 13.3 11.0 9.7 17.3 8.0 12.5 8.9
10.0 13.4 11.0 9.5 17.2 7.9 12.5 8.9
10.0 13.4 11.5 10.6 17.3 6.9 12.5 8.9
10.0 13.4 11.4 9.6 17.3 8.1 12.5 9.0
9.7 13.2 10.9 9.6 17.4 8.2 12.4 9.1
2a
2b
2c
2d
2e
10.6 9.9 6.9
10.6 9.2 8.6
10.5 9.7 8.6
9.6 11.5 8.2
9.7 9.6 8.0
G.I. Enevaet al./J. Mol. Struct..291 (1993) 191-195
F2
193
(Pf’M)
e
a c9
i
Fig. 2. Two-dimensional NOESY contour plot for compound 1 in D,O at 300 MHz, indicating some of the NOE cross-peaks. The conventional 1H NMR spectrum is shown at the top, together with the signal assignment 121.
for some disaccharide fragments [6], trisaccharides [7], pseudotrehaloses [8] and kanamycin A [9]. The results of the conformational analysis using molecular mechanics calculations for 1 and 2 are presented in Table 1. For the five minimum energy conformations of each compound, the torsional angles 4,(Hl’-Cl’-0-C4), &(Hl”-Cl”-0-C6),
$, (Cl’-O-C4-H4) and @,(Cl”-O-C6-H6), as well as the valence angles r, (C I’-0-C4) and r2 (C 1“-OC6), are shown together with the distances r between the protons attached to the glycosidic and aglyconic carbon atoms. The calculated steric energy is given in relative values with respect to the lowest-energy conformation (I?,,).
194
G.I. Eneva et al.lJ. Mol. Struct.. 291 (1993) 191-195
Fig. 3. Lowest-energy
conformation
of sisomicin (I), as calculated by the molecular mechanics method.
The results of the difference NOE experiments for 1 and 2 are presented in Table 2 together with those obtained by calculation on the basis of the equation [lo]: NOEd
= rz6/2cr,;”
Here rds is the distance between observed proton (d) and irradiated proton (s), and rdj is the distance between proton d and each other protons in the molecule. Some of the experimental NOE interactions can also be qualitatively observed as crosspeaks on the two-dimensional NOESY contour plot (Fig. 2). An analysis of the NOES observed for 1 (Table 2) shows a strong inter-ring enhancement of the resonances of H4 (11%) and H6 (12%), when Hl’ and Hl” are irradiated, together with a strong intra-ring enhancement of the resonances of H2’ (13%) and H2” (16%), respectively. These data require that Hl’ and H4, as well as H 1” and H6, are in close proximity. As the distance between the protons H 1‘-H4 and H 1“-H6 is sensitive to changes around the glycosidic linkage, this interaction puts a serious restraint on the allowed 4-$ space. In accordance with this, we considered only the conformations with interproton distance < 2.5 A. The
distances were obtained from the geometries calculated by the MM method. The lowest-energy conformation of sisomicin (la) is shown in Fig. 3. As seen in Table 2, a rather good correlation between the experimental and computational NOE data was found, both for the inter- and intra-ring effects, in spite of the approximate character of the equation used [lo]. Comparison of the data for compounds 1 and 2 shows that the N,O-acetylation does not cause any significant changes in the inter-ring NOE (Table 2). In some cases differences were found for inter-ring effects: e.g., small enhancements (4%) of the signals of Hl and H3 upon irradiation of H5 were observed for 1, but not for 2. Probably the N,O-acetylation caused some ring deformations, which has been also assumed on the basis of the changes of the vicinal proton-proton couplings [l]. Conclusion
The present conformational
study of sisomicin derivative (2) indicates a relatively restricted conformation at both glycosidic linkages. This is reflected in the rather small variations of the torsional angles 4,) &, $, (1) and its penta-l\r-di-0-acetyl
195
G.I. Eneva et al./J. Mol. Struct., 291 (1993) 191-195
and ti2 observed for the calculated conformational minima falling within f 0.5 kcal mol-’ : la, b and c for compound 1, and 2a and b for compound 2. Acknowledgements
The authors are grateful to the National Foundation for Scientific Research (Contract No. X-82) for partial support of this work, and to Professor J. Sandstrom (Chemical Centre, University of Lund, Sweden) for making possible the NOESY spectra measurements. References 1 G.I. Eneva, S.L. Spassov, M.A. Haimova and J. SandStrom, Magn. Reson. Chem., 30 (1992) 841.
2
H. Reimann, D.J. Cooper, A.K. Mallams, R.S. Jaret, A. Yehaskel, M. Kugelman, H.F. Vemay and D. Schumacher, J. Org. Chem., 39 (1974) 1451. 3 U. Burkert and N.L. Allinger, Molecular Mechanics, ACS Monograph 177, American Chemical Society, Washington, DC, 1982. 4 T. Liljefors, J. Tai, S. Li and N.L. Allinger, J. Comput. Chem., 8 (1987) 1051. 5 C.A.G. Haasnoot, F.A.M. De Leeuw and C. Altona, Tetrahedron, 36 (1980) 2783. 6 K. Bock, H. Lonn and T. Peters, Carbohydr. Res., 198 (1990) 375. 7 K. Bock, T. Hvidt, J. Marino-Albemas and V. VerezBencomo, Carbohydr. Res., 200 (1990) 33. 8 K. Bock, J. Guzman, J.O. Duus, S. Ogawa and S. Yokoi, Carbohydr. Res., 209 (1991) 51. 9 K. Bock, Pure Appl. Chem., 55 (1983) 605. 10 H. Thogersen, R.U. Lemieux, K. Bock and B. Meyer, Can. J. Chem., 60 (1982) 44.
191
Conformational analysis of sisomicin and penta-WdiO-acetylsisomicin using NMR spectroscopy and molecular mechanics calculations G.I. Enevaa, S.L. Spassova,’ and M.A. Haimovab aInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy 1113 Sofia (Bulgaria) bFaculty of Chem istry, University of Sofia, 1126 Sofia (Bulgaria)
of Sciences,
(Received 9 October 1992)
Abstract The conformations of sisomicin and penta-N-di-0-acetylsisomicin were investigated by NMR spectroscopy and molecular mechanics calculations. Experimental NOE data for aqueous solutions were in accordance with the computational data.
Introduction
Experimental
The biological importance of aminoglycoside antibiotics have stimulated interest in a detailed knowledge of their conformation in solution. We now report results concerning the preferred conformations of the aminoglycosides sisomicin (1) and 1,3,2’,6’,3”-penta-N-acetyl-2”,4”-di-O-acetylsisomicin (2) (Fig. l), based on NMR spectral studies and computational methods. The NMR data were acquired using 1D and 2D NMR techniques, with special emphasis on nuclear Overhauser effect @JOE) experiments (DNOE, NOESY). The computations permitted quantitative estimation of NOE, and their comparison with the experimental data yielded information about the conformational preference with respect to the glycosidic bonds in aqueous solution.
Samples of 1 (free base) and 2 were obtained as described previously [l]. The ‘H NMR spectra were obtained on a Bruker WM-250 spectrometer operating at 250.1 MHz. All spectra were measured on 0.1 molar solutions in DzO, at ambient temperature (x 300 K), pH 10 for 1 and pH 6 for 2, with internal standard acetone (2.22ppm). The ‘H NOE experiments were performed in the difference mode (DNOE), with a preirradiation time of 10 s. The two-dimensional NOESY plots were obtained on a Varian XL-300 spectrometer (299.94MHz for ‘H) using the standard Varian software. The calculations of the conformations with respect to the glycosidic bonds were carried out with the MM2(85)-based programme PCMODEL (88.0) using default parameters on a PC-AT computer. The energy minimizations were performed on different starting conformations with consecutive variation of the torsional angles 4, , &, $, and $*.
Correspondence to: Professor S.L. Spassov, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
0022-2860/93/%06.00 0 1993 Elsevier Science Publishers B.V. All rights reserved.
192
G.I. Eneva et al./J. Mol. Struct., 291 (1993) 191-195
TABLE 1 Data on the minimum energy conformations Conformer
of 1 and 2 from molecular mechanics calculations r (H l’-H4)
cp, (“)
(62(“)
la lb lc Id le 2a 2b
2c 2d 2e
& (kcalmol-‘)
-
16.6 17.7 20.9 15.5 10.4
113.5 113.5 114.0 113.2 113.9
2.27 2.27 2.33 2.33 2.23
- 37.3 - 37.4 -45.2 2.8 3.6
- 13.3 -8.9 - 25.0 38.7 39.2
113.8 113.9 114.0 115.6 115.9
2.22 2.19 2.43 2.21 2.23
0.0 0.3 0.5 2.2 3.3
-57.6 -52.1 -61.7 - 55.7 -25.7
- 18.3 - 13.6 16.7 -8.1 - 10.2
114.8 114.0 115.2 114.7 115.4
2.55 2.41 2.59 2.42 2.15
- 14.9
49.4 48.0 50.7 49.9 52.3
115.9 116.0 116.0 116.3 116.0
2.23 2.26 2.24 2.25 2.27
0.0 0.5 2.3 2.7 3.9
The chemical structure of 1 has been established by chemical and spectroscopic studies [2]. The complete ‘H and 13C NMR assignment and parameter determination of 1 and 2 was reported in an earlier paper [l]. The coupling constants found [l] for the deoxystreptamine and aminoglucose moieties indicate essentially undistorted 4C, chair conformations of the saturated six-membered rings. The half-chair ~onfo~ation of the dihydropyran residue in 1 and 2 is clearly reflected in the different vi&al couplings observed [l]. The deoxystreptamine residue in the 1’ position and the amino group in the 2’ position are cis to each other [2]; and one should be axial and the other equatorial. Empirical force field calculations performed on a model compound with the MMP2(85) force field [3,4] predicted the conformer with equatorial
-6.3 - 15.5 - 12.9 - 14.7
amino and axial deoxystreptamine residues to be the more stable one, in agreement with the calculated [S] vicinal coupling constant values fl]. The conformational preference with respect to the glycosidic linkages, which remained to be established, is important for understanding the chemical and biological behaviour of such molecules in solution. Investigations of that type by NMR and computational methods (HSEA) have been reported TABLE 2 Observed and calculated NOE data for the minimum energy conformations of compounds 1 and 2 Proton irrad.
1’ 4 1”
OR CH, 4-
R’ 3 %
(H 1“H6)
-39.3 -38.4 - 39.6 -46.0 -38.8
Results and discussion
C&N
(A)
r
72 (“1
(A)
6 2 2”
“0 RO
RHN 0 RHN 1 -k_
OH 2
’ :
4
?J 0 3
Proton obs.
4 2 1’ 6 2” 1” 1’ 1”
NOE exp. (%)
11 13 9 12 16 8 8 7
14’ CH,NHR
NHR
Fig. 1. Structure of: sisomicin (l), R = H; 1,3,2’,6’,3”-pentaN-acetyl-2”,4”-di-O-acetylsisomicin (2), R = COCH,.
1’ 1” 2”
4 6 1”
11 11 6
Calculated NOE(%)
la
lb
lc
ld
le
10.0 13.3 11.0 9.7 17.3 8.0 12.5 8.9
10.0 13.4 11.0 9.5 17.2 7.9 12.5 8.9
10.0 13.4 11.5 10.6 17.3 6.9 12.5 8.9
10.0 13.4 11.4 9.6 17.3 8.1 12.5 9.0
9.7 13.2 10.9 9.6 17.4 8.2 12.4 9.1
2a
2b
2c
2d
2e
10.6 9.9 6.9
10.6 9.2 8.6
10.5 9.7 8.6
9.6 11.5 8.2
9.7 9.6 8.0
G.I. Enevaet al./J. Mol. Struct..291 (1993) 191-195
F2
193
(Pf’M)
e
a c9
i
Fig. 2. Two-dimensional NOESY contour plot for compound 1 in D,O at 300 MHz, indicating some of the NOE cross-peaks. The conventional 1H NMR spectrum is shown at the top, together with the signal assignment 121.
for some disaccharide fragments [6], trisaccharides [7], pseudotrehaloses [8] and kanamycin A [9]. The results of the conformational analysis using molecular mechanics calculations for 1 and 2 are presented in Table 1. For the five minimum energy conformations of each compound, the torsional angles 4,(Hl’-Cl’-0-C4), &(Hl”-Cl”-0-C6),
$, (Cl’-O-C4-H4) and @,(Cl”-O-C6-H6), as well as the valence angles r, (C I’-0-C4) and r2 (C 1“-OC6), are shown together with the distances r between the protons attached to the glycosidic and aglyconic carbon atoms. The calculated steric energy is given in relative values with respect to the lowest-energy conformation (I?,,).
194
G.I. Eneva et al.lJ. Mol. Struct.. 291 (1993) 191-195
Fig. 3. Lowest-energy
conformation
of sisomicin (I), as calculated by the molecular mechanics method.
The results of the difference NOE experiments for 1 and 2 are presented in Table 2 together with those obtained by calculation on the basis of the equation [lo]: NOEd
= rz6/2cr,;”
Here rds is the distance between observed proton (d) and irradiated proton (s), and rdj is the distance between proton d and each other protons in the molecule. Some of the experimental NOE interactions can also be qualitatively observed as crosspeaks on the two-dimensional NOESY contour plot (Fig. 2). An analysis of the NOES observed for 1 (Table 2) shows a strong inter-ring enhancement of the resonances of H4 (11%) and H6 (12%), when Hl’ and Hl” are irradiated, together with a strong intra-ring enhancement of the resonances of H2’ (13%) and H2” (16%), respectively. These data require that Hl’ and H4, as well as H 1” and H6, are in close proximity. As the distance between the protons H 1‘-H4 and H 1“-H6 is sensitive to changes around the glycosidic linkage, this interaction puts a serious restraint on the allowed 4-$ space. In accordance with this, we considered only the conformations with interproton distance < 2.5 A. The
distances were obtained from the geometries calculated by the MM method. The lowest-energy conformation of sisomicin (la) is shown in Fig. 3. As seen in Table 2, a rather good correlation between the experimental and computational NOE data was found, both for the inter- and intra-ring effects, in spite of the approximate character of the equation used [lo]. Comparison of the data for compounds 1 and 2 shows that the N,O-acetylation does not cause any significant changes in the inter-ring NOE (Table 2). In some cases differences were found for inter-ring effects: e.g., small enhancements (4%) of the signals of Hl and H3 upon irradiation of H5 were observed for 1, but not for 2. Probably the N,O-acetylation caused some ring deformations, which has been also assumed on the basis of the changes of the vicinal proton-proton couplings [l]. Conclusion
The present conformational
study of sisomicin derivative (2) indicates a relatively restricted conformation at both glycosidic linkages. This is reflected in the rather small variations of the torsional angles 4,) &, $, (1) and its penta-l\r-di-0-acetyl
195
G.I. Eneva et al./J. Mol. Struct., 291 (1993) 191-195
and ti2 observed for the calculated conformational minima falling within f 0.5 kcal mol-’ : la, b and c for compound 1, and 2a and b for compound 2. Acknowledgements
The authors are grateful to the National Foundation for Scientific Research (Contract No. X-82) for partial support of this work, and to Professor J. Sandstrom (Chemical Centre, University of Lund, Sweden) for making possible the NOESY spectra measurements. References 1 G.I. Eneva, S.L. Spassov, M.A. Haimova and J. SandStrom, Magn. Reson. Chem., 30 (1992) 841.
2
H. Reimann, D.J. Cooper, A.K. Mallams, R.S. Jaret, A. Yehaskel, M. Kugelman, H.F. Vemay and D. Schumacher, J. Org. Chem., 39 (1974) 1451. 3 U. Burkert and N.L. Allinger, Molecular Mechanics, ACS Monograph 177, American Chemical Society, Washington, DC, 1982. 4 T. Liljefors, J. Tai, S. Li and N.L. Allinger, J. Comput. Chem., 8 (1987) 1051. 5 C.A.G. Haasnoot, F.A.M. De Leeuw and C. Altona, Tetrahedron, 36 (1980) 2783. 6 K. Bock, H. Lonn and T. Peters, Carbohydr. Res., 198 (1990) 375. 7 K. Bock, T. Hvidt, J. Marino-Albemas and V. VerezBencomo, Carbohydr. Res., 200 (1990) 33. 8 K. Bock, J. Guzman, J.O. Duus, S. Ogawa and S. Yokoi, Carbohydr. Res., 209 (1991) 51. 9 K. Bock, Pure Appl. Chem., 55 (1983) 605. 10 H. Thogersen, R.U. Lemieux, K. Bock and B. Meyer, Can. J. Chem., 60 (1982) 44.